Chapter 5. Bacteria/microbubble Interaction And Surface

Transcript

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The print copy, usually available in the University Library, may contain corrections made by hand, which have been requested by the supervisor.   A Study of Rhamnolipid Microbubble Dispersion for Bioremediation Applications Dispersion Properties and Bacteria/Surfactant/Contaminant Interactions             Wanhua Feng                 A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, The University of Auckland, 2010.     Abstract This thesis presents a study on the production of microbubble dispersions from rhamnolipid biosurfactant and the mechanism and factors that impact upon the effectiveness of the dispersion for improving bioremediation efficiency, with a specific focus on the use of microbubble dispersion as a carrier for contaminant-degrading bacteria within a bioremediation scenario. Microbubble dispersion is a suspension of a large number of minute spherical gas bubbles encapsulated in a soapy liquid film in an aqueous surfactant solution. Microbubble dispersion has promising potential for enhancing in situ bioremediation owing to its advantages over air sparging, bioventing and surfactant injection. Characterisation studies investigated the stability, size distribution and gas hold-up properties of the rhamnolipid microbubble dispersion. Drainage experiments and contact angle measurements were performed to identify factors that influence P.putida and R.erythropolis adhesion to microbubble dispersion. Fluorescence microscopy was used to investigate the interaction of P.putida and R.erythropolis and hexadecane, a model NAPLcontaminant on the microbubble surface. The LW-AB surface thermodynamic model was applied to quantify the interaction energy to better understand the bacteria/microbubble and bacteria/contaminant/microbubble interactions. The findings of the characterisation studies support that rhamnolipid microbubble dispersion had comparable properties to the synthetic surfactants reported in this study and in the literature. The stability of the rhamnolipid microbubble dispersion, prepared at rhamnolipid concentrations of 500 mg/L, 1,000 mg/L and 4,000 mg/L, was in the range from 385 to 546 seconds. The gas hold-up in the dispersion was fairly constant ranging from 67% to 72%, and majority of the microbubbles were in the size range of 20 μm to 140 μm. The bacterial drainage experiments, coupled with surface free energy calculation by the LWAB model, showed that rhamnolipid microbubble dispersion was more effective in delivering hydrophilic P.putida than hydrophobic R.erythropolis. Bacterial cell surface hydrophobicity and rhamnolipid concentration were demonstrated as two key factors to control when making the microbubble dispersion an effective bacterial carrier. Furthermore, fluorescence microscopic images revealed that rhamnolipid microbubble dispersion might potentially ii      overcome the problem of limited contaminant bioavailability by improving P.putida and R.erythropolis bacterial contact with hexadecane immobilised at the microbubble surface. iii      Acknowledgements Many people have supported me in various ways throughout my years at the University of Auckland. I would like to express my gratitude to all those helped making this thesis project successful. I am very grateful to the Auckland University Scholarship for providing the financial support for this PhD study. I would also like to express my sincere appreciation to my supervisor, Dr. Naresh Singhal for providing important resources and technical advice for this project. Special thanks go to Dr. Simon Swift, my co-supervisor, who has not only provided the technical guidance with great expertise and enthusiasm, but also the inspiration and encouragement to help me through the difficult times. Furthermore, I would also like to thank the other research committee members, Professor Bruce Melville and Dr. Elizabeth Fassman, for their time, assistance and thoughtful discussion and the external examiners, for their knowledge and insight in this research area and valuable suggestions and comments. Thanks also to the staff at the University of Auckland, especially Abel Francis for providing the laboratory instruments and materials and Catherine Hobbs for her expertise and assistance in scanning electron microscopy work. To Philip Nel, a big thank you for keeping the computer running smoothly during my study; to Mags Woo for such practical help in the use of required resources; to Laura Liang from the Department of Chemical Engineering and David Jenkinson from the School of Geography for their assistance in particle size analysis; and to Shane Crump of the Tamaki Campus for helping me with the viscosity measurement. A big thank you to you all for all the help provided. I would also like to thank other lecturers from the Department of Civil and Environmental Engineering that have given me support during the course of study; they are Dr Tam Lakin, Dr John St Geoge, Dr Theuns Hennings, and Dr Judith Wong. My gratitude also goes to all fellow postgraduates students who have made my graduate study enjoyable. Sincere thanks go to Yantao Song who so generously shared her lunch with me and more importantly her knowledge on experimental methods; to Maria Rowe, for her help with putting the bacteria in incubator during weekends. Special thanks are extended to Grace Jho, Anthea Johnson, Anuradha Permathilaka, Claudia Kayser, Sasha Jattansingh, iv      Avery Gottfried, Roy Elloit, Victoria Melville, Katherine Heays, Farhan Shams, Janine Louie, Benedic Uy, Emily Voyde, and Buddhika Gunawardana. My family and friends here in New Zealand have given me continuous support. Special thanks go to my aunties Fanny Lee, Auntie Chong, Alan Cheung, Cecilia Chan, Raymond Chan, Gareth and Yantao’s husband Andrew. I am also very grateful to the colleagues from MWH Ltd. To Mayurie Gunatilaka, Amy Clore, Garrett Hall, Sonja Bury, Rohan Naidu, Wendy Chan, Alex and Gabi, thank you for your understanding and support. Finally and most importantly to my parents in China, my rock of support, thank you. I offer my heartfelt gratitude for your never-ending support, love and understanding; you are the backbone of all my successes. This thesis is dedicated to my father Jingxin Feng and my mother Jianqun Huang. v      Table of Contents Abstract ................................................................................................................................................... ii  Acknowledgements ................................................................................................................................ iv  List of Figures ......................................................................................................................................... xi  List of Tables ........................................................................................................................................ xiv  List of Acronyms ................................................................................................................................... xvi  Chapter 1. General Introduction ............................................................................................................. 1  1.1. Research Objectives ..................................................................................................................... 5  1.2. Thesis Hypotheses ........................................................................................................................ 5  1.3. Thesis Outline ............................................................................................................................... 6  Chapter 2. Literature Review .................................................................................................................. 8  2.1. Introduction ................................................................................................................................. 8  2.2. Surfactant and Biosurfactant ....................................................................................................... 8  2.2.1. Biosurfactant ......................................................................................................................... 9  2.2.2. Rhamnolipid ........................................................................................................................ 12  2.3. Characterisation of Microbubble Dispersion ............................................................................. 13  2.3.1. Generation of Microbubble Dispersion .............................................................................. 13  2.3.2. Characterisation Techniques .............................................................................................. 14  2.3.3. Properties ............................................................................................................................ 17  2.3.4. Knowledge Gap ................................................................................................................... 23  2.4. Environmental Applications ....................................................................................................... 24  2.4.1. Flow Characteristics of Microbubble Dispersion in Porous Media ..................................... 24  2.4.2. Bacteria Mobility Control .................................................................................................... 25  2.4.3. Contaminant Mobilisation .................................................................................................. 26  2.4.4. In situ Biodegradation ......................................................................................................... 27  2.4.5. The Knowledge Gap ............................................................................................................ 28  2.5. Bacterial Adhesion to Surfaces/Interfaces ................................................................................. 30  2.5.1. Bacterial Cell Surface Characteristics .................................................................................. 31  2.5.2. Physicochemical Characteristics of  Surfaces/Interfaces .................................................... 35  2.5.3. Environmental Factors ........................................................................................................ 36  2.6. Theoretical Models for Bacterial Adhesion................................................................................ 38  2.7. Surface Thermodynamic Approach to Bacterial Adhesion ........................................................ 40  vi      2.7.1. Two Schools of Thought ...................................................................................................... 40  2.7.2. The LW‐AB Theory .............................................................................................................. 41  2.7.3. Surface Free Energy Calculation Based on LW‐AB Approach ............................................. 44  Chapter 3. Materials and Experimental Methods ................................................................................. 45  3.1. Materials .................................................................................................................................... 45  3.1.1. Surfactants .......................................................................................................................... 45  3.1.2. Bacterial Culture Media ...................................................................................................... 45  3.1.3. Other Chemicals .................................................................................................................. 46  3.2. Generation of Microbubble Dispersion ..................................................................................... 46  3.3. Characterisation Studies for Microbubble Dispersion ............................................................... 47  3.3.1. Viscosity Measurement....................................................................................................... 47  3.3.2. Stability (Half‐life) Measurement........................................................................................ 48  3.3.3. Gas Hold‐up Measurement ................................................................................................. 50  3.3.4. Size Distribution Measurement .......................................................................................... 50  3.3.5. Visualisation of Microbubble Evolution .............................................................................. 51  3.4. Bacteria Culture and Preparation .............................................................................................. 52  3.4.1. Gram‐Staining ..................................................................................................................... 52  3.4.2. Bacteria Strains ................................................................................................................... 52  3.4.3. Growth of Pseudomonas putida 852 for Microbubble Dispersion Experiments ................ 53  3.4.4. Growth of Rhodococcus erythropolis 3586 for Microbubble Dispersion Experiments ....... 54  3.4.5. Bacteria Preparation for Microbubble Dispersion Experiments ......................................... 54  3.5. Adsorption of Surfactant on Bacteria ........................................................................................ 55  3.6. Bacterial Survival Test ................................................................................................................ 55  3.7. Bacterial Cell Surface Hydrophobicity ........................................................................................ 56  3.7.1. Microbial Adhesion to Hydrocarbons (MATH) Assay .......................................................... 56  3.7.2. Contact Angle Measurement .............................................................................................. 56  3.8. Bacterial Cell Drainage from Microbubble Dispersion ............................................................... 58  3.9. Surfactant Analysis Procedure ................................................................................................... 60  3.9.1. Rhamnolipid Surfactant ...................................................................................................... 60  3.9.2. Tergitol 15‐S‐12 ................................................................................................................... 60  3.10. Magnesium Analytical Procedure ............................................................................................ 61  3.11. Polyacrylamide Gel Electrophoresis (PAGE) Analysis of Lipopolysaccharide .......................... 62  3.12. Microscopy Examination .......................................................................................................... 62  3.12.1. Fluorescence Microscopy.................................................................................................. 62  vii      3.12.2. Scanning Electron Microscopy .......................................................................................... 63  3.12.3. Cryo–Scanning Electron Microscopy ................................................................................. 63  3.13. Treatment of Data and Statistical Analysis Procedure ............................................................ 64  Chapter 4. Characterisation and Drainage Mechanism of Microbubble Dispersion ............................ 65  4.1. Introduction ............................................................................................................................... 65  4.2. Preliminary Testing .................................................................................................................... 66  4.2.1. Mixing Speed and Duration ................................................................................................ 66  4.2.2. Rhamnolipid Concentration ................................................................................................ 68  4.2.3. The Apparatus ..................................................................................................................... 70  4.2.4. Summary ............................................................................................................................. 71  4.3. Microbubble Dispersion Stability ............................................................................................... 72  4.3.1. Effect of Rhamnolipid Concentration on Microbubble Stability ......................................... 73  4.3.2. Effect of pH on Microbubble Dispersion Stability ............................................................... 76  4.3.3. Effect of Electrolyte Concentration on Microbubble Dispersion Stability .......................... 78  4.4. Liquid Drainage Model ............................................................................................................... 79  4.5. Gas Hold‐Up Measurement ....................................................................................................... 85  4.6. Size Distribution ......................................................................................................................... 86  4.6.1. Particle Size Analyser .......................................................................................................... 87  4.6.2. Image Processing Technique ............................................................................................... 89  4.6.3. Comparison Between Particle Size Analyser and Image Processing Technique ................. 91  4.6.4. Change of Microbubble Size Distribution with Time .......................................................... 93  4.7. Comparison with Synthetic Surfactants ..................................................................................... 97  4.8. Effect of Bacteria Addition ......................................................................................................... 98  4.9. Drainage Mechanism ................................................................................................................. 99  4.10. Chapter Summary .................................................................................................................. 104  Chapter 5. Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction ........................................................................................................................................... 106  5.1. Introduction ............................................................................................................................. 106  5.2. Bacterial Adhesion to Microbubble Dispersion ....................................................................... 107  5.2.1. Effect of Rhamnolipid Concentrations .............................................................................. 107  5.2.2. Effect of Salt Concentrations ............................................................................................ 109  5.2.3. Effect of Bacterial Cell Surface Properties ........................................................................ 110  5.3. Bacteria/Surfactant Interaction ............................................................................................... 113  5.3.1. Adsorption of Rhamnolipid to R.erythropolis ................................................................... 114  viii      5.3.2. Adsorption of Rhamnolipid onto P.putida ........................................................................ 116  5.3.3. Adsorption of Tergitol onto Bacterial Cells ....................................................................... 120  5.4. Microbial Adhesion to Hydrocarbon Assay for Cell Surface Hydrophobicity Measurement ... 121  5.5. Contact Angle Measurement ................................................................................................... 123  5.5.1. Bacterial Lawn Preparation ............................................................................................... 123  5.5.2. Contact Angles as A Function of Time .............................................................................. 124  5.5.3. Background Contact Angle Measurement on Filter Paper ............................................... 126  5.5.4. Contact Angle Results ....................................................................................................... 128  5.5.5. Discussion of Contact Angle Measurement ...................................................................... 130  5.6. Bacterial Cell Surface Thermodynamics and Hydrophobicity with Surfactant Present ........... 131  5.6.1. Lifshitz‐van der Waals Surface Tension Component ........................................................ 131  5.6.2. Electron‐donor (γ‐) and Electron‐acceptor (γ+) Parameters .............................................. 134  5.6.3. Bacterial Cell Surface Tension ........................................................................................... 136  5.6.4. Bacterial Cell Surface Hydrophobicity ............................................................................... 137  5.6.5. Discussion of Bacterial Cell Surface Hydrophobicity ......................................................... 139  5.7. Surface Thermodynamic Modelling of Bacteria/Microbubble Interaction ............................. 142  5.7.1. Model Assumptions .......................................................................................................... 142  5.7.2. Surface Tension Parameters of Rhamnolipid Solutions .................................................... 143  5.7.3. Approach A to Determine Surface Tension Parameters of Microbubble ......................... 144  5.7.4. Approach B to Determine Surface Tension Parameters of Microbubble ......................... 145  5.7.5. Summary of Surface Tension Parameters ......................................................................... 146  5.7.6. Surface Thermodynamics to Predict Bacteria – Microbubble Interaction ....................... 147  5.7.7. Discussion on Microbubble Dispersion as Bacterial Carrier ............................................. 153  5.8. Chapter Summary .................................................................................................................... 155  Chapter 6. Visualisation of Bacteria/Contaminant/Microbubble Interaction and Surface  Thermodynamic Modelling of the Interaction .................................................................................... 157  6.1. Introduction ............................................................................................................................. 157  6.2. Visualisation of Microbubble/Contaminant Interaction .......................................................... 158  6.3. Visualisation of Microbubble/Contaminant/Bacteria Interaction ........................................... 160  6.4. Surface Thermodynamic Modelling of Microbubble – Contaminant – Bacteria Interaction .. 162  6.4.1. Model Assumptions .......................................................................................................... 162  6.4.2. Surface Free Energy of Interaction ................................................................................... 164  6.5. Implication for Bioremediation ................................................................................................ 165  6.5.1. Microbubbles as Biodegradation Facilitator ..................................................................... 165  ix      6.5.2. Selection of Surfactant and Contaminant‐degrading Bacteria ......................................... 166  6.6. Chapter Summary .................................................................................................................... 168  Chapter 7. Conclusions and Recommendation for Future Research .................................................. 170  7.1. General Summary .................................................................................................................... 170  7.2. Conclusions and Implications for Bioremediation ................................................................... 170  7.3. Significant Contributions .......................................................................................................... 173  7.4. Recommendations for Future Research .................................................................................. 175  References .......................................................................................................................................... 176  Appendix I       MATLAB Command ........................................................................................................ I  Appendix II  P.putida Growth Curve ................................................................................................. II  Appendix III  Stability Test Data ........................................................................................................ III  Appendix IV  Viscosity Test Data ....................................................................................................... IV  Appendix V  Dissociation of Surfactants ........................................................................................... V  Appendix VI  Gas Hold‐up Data ......................................................................................................... VI  Appendix VII  Particle Size Analysis Data .......................................................................................... VII  Appendix VIII  Image Size Analysis Data ............................................................................................ VIII  Appendix IX  MATH Assay Data ......................................................................................................... IX  Appendix X  Contact Angle Measurement Data ............................................................................... X  Appendix XI  Mycolic Acid Contact Angle Measurement Data ......................................................... XI  x      List of Figures Figure 2‐1. A schematic of adsorption of surfactants at the air‐water interface and formation of  micelles in water. .................................................................................................................................... 9  Figure 2‐2. Rhamnolipid structure.. ...................................................................................................... 12  Figure 2‐3. Schematic illustration of potential benefits offered by microbubble dispersion. .............. 29  Figure 2‐4. Bacterial cell envelope structure. ....................................................................................... 32  Figure 2‐5. A schematic of contact angle of a liquid droplet on a solid surface. .................................. 43  Figure 3‐1. Setups used for making microbubble dispersion. .............................................................. 47  Figure 3‐2. Illustration of the measurement of liquid drainage from microbubble dispersion. .......... 48  Figure 3‐3. Procedure used to determine half‐life value. ..................................................................... 49  Figure 3‐4. Pictures illustrating the image processing procedure.. ...................................................... 51  Figure 3‐5. Screenshot of the contact angle analysis procedure. ......................................................... 58  Figure 3‐6. Serial dilution method for estimating cell concentration. .................................................. 59    Figure 3‐7. Standard curve for rhamnolipid spectrophotometry analysis. ......................................... 60   Figure 3‐8. Standard curve for tergitol spetrophotometry analysis. ................................................... 61  Figure 4‐1. Liquid drainage from microbubble dispersion generated from 20,000 mg/L rhamnolipid  solution under different mixing speeds and durations.. ...................................................................... 67   Figure 4‐2. Liquid drainage from microbubble dispersion created from rhamnolipid solution.. ........ 69  Figure 4‐3. Microbubble dispersion apparatus.. ................................................................................... 70  Figure 4‐4. Drainage curves with different apparatus configuration.. ................................................. 71  Figure 4‐5. Microbubble dispersion produced from rhamnolipid biosurfactant using Setup 1 at 8000  rpm for 3 minutes.. ............................................................................................................................... 72  Figure 4‐6. Drainage behaviour of microbubble dispersions with rhamnolipid surfactant at surfactant  concentrations of 500 mg/L, 1,000 mg/L, and 4,000 mg/L................................................................... 74  Figure 4‐7. Drainage behavior of microbubble dispersions at pH 6, pH 7 and pH 8 at surfactant  concentration of 1,000 mg/L.. .............................................................................................................. 77  Figure 4‐8. Dissociation of carboxyl head group of rhamnolipid. ......................................................... 78  Figure 4‐9. Plots of liquid drainage data and original model output for rhamnolipid microbubble  dispersion. ............................................................................................................................................. 81  Figure 4‐10. Correlation between fitted and measured half‐life (A) and maximum drained liquid (B)  from the rhamnolipid microbubble dispersions.. ................................................................................. 84  Figure 4‐11. Percentage of microbubble size distribution measured by particle size analyser.. ......... 88   Figure 4‐12. Cumulative distribution of microbubble diameter measured by particule size analyser.  .............................................................................................................................................................. 88  Figure 4‐13. Comparison of microbubble diameter at different percentiles measured by particle size  analyser. ................................................................................................................................................ 89  Figure 4‐14. Microbubble size distribution at pH 7 obtained by image processing technique. ........... 91  Figure 4‐15. Comparison between particle size analyser and image processing techniques for size  distribution of microbubble dispersions at surfactant concentration of 1,000 mg/L at pH 7. ............. 92  Figure 4‐16. Evolution of microbubble with time for microbubble dispersion made with 500 mg/L  rhamnolipid solution at pH 7. ............................................................................................................... 94  xi      Figure 4‐17. Evolution of microbubble with time for microbubble dispersion made with 1,000 mg/L  rhamnolipid solution at pH 7. ............................................................................................................... 95  Figure 4‐18. Evolution of microbubble with time for microbubble dispersion made with 4,000 mg/L  rhamnolipid solution at pH 7. ............................................................................................................... 96   Figure 4‐19. Typical drainage curve ................................................................................................... 100  Figure 4‐20. Photomicrographs of the microbubble dispersion at different times. ........................... 102  Figure 4‐21. Variation of foam volume fraction, liquid holdup and total volume with time for  microbubble dispersion formed with 500 mg/L rhamnolipid concentration at pH 6. ....................... 103  Figure 5‐1. Effect of rhamnolipid concentrations on percentage retention of P.putida (A) and  R.erythropolis (B) in microbubble dispersion at pH 7 at various drainage times. .............................. 108  Figure 5‐2. Effect of salt concentrations on percentage retention of P.putida in microbubble  dispersion at 1,000 mg/L rhamnolipid concentration and pH 7 at various drainage times.. ............. 110  Figure 5‐3. Effect of bacterial cell surface properties on bacteria retention (%) in microbubble  dispersion at 1000 mg/L rhamnolipid concentration at pH7.. ............................................................ 111  Figure 5‐4. Cryo‐SEM images of microbubble dispersion containing P.putida with EPS. The dispersion  was produced from 1,000 mg/L of rhamnolipid solution. .................................................................. 112  Figure 5‐5. Adsorption of rhamnolipid on R.erythropolis showing the equilibrium rhamnolipid  concentration after mixing of R.erythropolis with rhamnolipid solution ........................................... 114  Figure 5‐6. Adsorption coefficient of rhamnolipid on R.erythropolis. ................................................ 115  Figure 5‐7. Adsorption of rhamnolipid on P.putida showing the equilibrium rhamnolipid  concentration after mixing of P.putida with rhamnolipid solution .................................................... 116  Figure 5‐8. Mg2+ concentration in supernatant after mixing of bacteria with surfactant  at various  surfactant concentrations. .................................................................................................................. 118  Figure 5‐9. SDS‐PAGE gel showing LPS in P.putida‐rhamnolipid supernatant.. .................................. 119  Figure 5‐10. R.erythropolis and P.putida adherence to hexadecane. ................................................. 122  Figure 5‐11. SEM images of bacterial lawns prepared for contact angle measurement. .................. 124  Figure 5‐12. Water contact angle on bacterial lawn surface as a function of time. ........................... 125  Figure 5‐13. Images of water droplets on bacterial lawn. .................................................................. 126  Figure 5‐14. Contact angle measurement on filter paper rinsed with surfactant solutions.   ........... 127  Figure 5‐15. Lifshitz‐van der Waals surface tension component (γLW) of bacterial cell surface as a  function of surfactant concentration. ................................................................................................. 132  Figure 5‐16. Electron donor (A) and electron acceptor (B) surface tension parameters of bacterial cell  surface as a function of surfactant concentration. ............................................................................. 135  Figure 5‐17. Surface tension of the bacterial cell surface as a function of surfactant concentrations.  ............................................................................................................................................................ 136  Figure 5‐18. Free energy of aggregation of bacterial cells (∆Gbwb) in aqueous solution (∆Gbwb) as a  function of surfactant concentrations. ............................................................................................... 137  Figure 5‐19. Fluorescence images of bacteria cells in aqueous solution. ........................................... 139  Figure 5‐20. Correlation between the degree of hydrophobicity and surface free energy components..  ............................................................................................................................................................ 141  Figure 5‐21. Surface tension parameters as a function of rhamnolipid concentration in mg/L in log  scale. ................................................................................................................................................... 144  Figure 5‐22. Schematic presentation of bacteria and bubble surface interaction. ............................ 145  xii      Figure 5‐23. Comparison of bacterial adhesion to microbubble dispersion at drainage time of 480  seconds and calculated surface free energy of adhesion (∆Gadh) between (A) P.putida and  microbubble and (B) R.erythropolis and microbubble as a function of rhamnolipid concentration.  149  Figure 5‐24. Microscopy images of P.putida interaction with microbubble made from 1000 mg/L  rhamnolipid at pH 7.. .......................................................................................................................... 150  Figure 5‐25. Comparison of bacterial adhesion to microbubble at drainage time of 480 seconds and  calculated surface free energy of bacterial adhesion with microbubble (∆Gadh) at rhamnolipid  concentration of 1000 mg/L. .............................................................................................................. 151  Figure 5‐26. Microscopy images of R.erythropolis interaction with microbubble made from 1000  mg/L rhamnolipid.. .............................................................................................................................. 153  Figure 5‐27. Surface free energy of bacterial interaction with various types of surfaces. ................. 154  Figure 6‐1. Microbubble with globules of n‐hexadecane adhered to the surface. ............................ 159  Figure 6‐2. A schematic of microbubble structure showing the hydrophobic region. ....................... 159  Figure 6‐3. Fluorescence photomicrographs of P.putida cells interacting with n‐hexadecane attached  to microbubble surfaces. .................................................................................................................... 160  Figure 6‐4. Fluorescence photomicrographs of R.erythropolis cells interacting with n‐hexadecane  attached to microbubble surfaces.. .................................................................................................... 161  Figure 6‐5. Schematic of modelled interactions ................................................................................. 163  Figure 6‐6. Surface free energy of interactions for microbubble/hexadecane, hexadecane/P.putida  and hexadecane/R.erythropolis. ......................................................................................................... 164  xiii      List of Tables Table 2‐1. Types of biosurfactants and their characteristics ................................................................ 10  Table 2‐2. Summary of microbubble dispersion characterisation studies. .......................................... 15  Table 2‐3. Comparison of microbubble dispersion to conventional foams .......................................... 23  Table 2‐4. Surface tension data of five commonly used diagnostic liquids. ......................................... 43  Table 3‐1. Surfactant properties and supplier information. ................................................................. 45  Table 3‐2. A list of chemicals used in the study. ................................................................................... 46  Table 3‐3. Comparison of properties of Pseudomonas putida 852 and Rhodococcus erythropolis 3586.  .............................................................................................................................................................. 54  Table 3‐4. A summary of surface tension parameters of the diagnostic liquids. ................................. 57  Table 4‐1. Stability and gas hold‐up measurements (mean ± 1 S.D.) for various mixing speeds and  durations at 20,000 mg/L rhamnolipid concentration. ........................................................................ 67  Table 4‐2. Stability and gas hold‐up measurements (mean ± 1 S.D.) for various rhamnolipid  concentrations. ..................................................................................................................................... 69  Table 4‐3. Comparison of microbubble dispersion properties (mean ± 1 S.D.) at different apparatus  configurations. ...................................................................................................................................... 71  Table 4‐4. Summary of half‐life (T1/2, mean±1 S.D.) and final drained liquid volume (Vmax,  mean±1 .S.D) values. ........................................................................................................................... 75  Table 4‐5. Summary of effects of surfactant concentration, solution pH and NaCl concentrations on  stability. ................................................................................................................................................. 78  Table 4‐6. Comparison of observed and fitted half‐life (T1/2) and final drained liquid volume (Vmax)  values. ................................................................................................................................................... 83  Table 4‐7. Summary of Paired‐sample T‐test results for half‐life. ........................................................ 85  Table 4‐8. Summary of Paired‐sample T‐test results for drained volume. ........................................... 85  Table 4‐9. Gas hold‐up measurement (mean ± 1 S.D.) at different surfactant types and  concentrations. ..................................................................................................................................... 86  Table 4‐10. Gas hold‐up measurement (mean ± 1 S.D.) at different salt concentrations at 1,000  mg/L rhamnolipid. ................................................................................................................................. 86  Table 4‐11. Mean bubble diameter with 1 standard deviation under various experimental conditions.  .............................................................................................................................................................. 90  Table 4‐12. Summary of bubble diameters (mean ± 1 S.D.) in μm at selected times. ....................... 97  Table 4‐13. Stability (mean ± 1 S.D.) comparison. .............................................................................. 97  Table 4‐14. Gas hold‐up (mean ± 1 S.D.) comparison. ........................................................................ 98  Table 4‐15. Comparison of rhamnolipid microbubble dispersion with synthetic surfactants. ............ 98  Table 4‐16. Effect of bacterial cells addition on rhamnolipid microbubble dispersion stability and gas  hold‐up (mean ± 1 S.D.) at pH 7. ......................................................................................................... 99  Table 4‐17. Change of bubble size with time for microbubble dispersion produced from 500 mg/L  rhamnolipid solution. .......................................................................................................................... 102  Table 5‐1. P.putida survival rate (mean and 1 standard deviation) after rhamnolipid treatment. .... 120  Table 5‐2. Contact angles (θ/o; mean and 1 standard deviation) for P.putida and R.erythropolis. ... 128  xiv      Table 5‐3. Contact angles (θ/o; mean and 1 standard deviation) for washed P.putida and unwashed  P.putida (with EPS present) at 1000 mg/L rhamnolipid concentration. ............................................. 129  Table 5‐4. A summary of surface tension parameters of rhamnolipid solutions. .............................. 144  Table 5‐5. Surface tension parameters of various materials. ............................................................. 146  Table 5‐6. Surface Tension Parameters of Bacteria. ........................................................................... 147  Table 5‐7. Comparison of surface free energy of interaction (∆Gadh) calculated using Approach A and  B. ......................................................................................................................................................... 147  Table 6‐1. Summary of surface tension parameters of various materials. ......................................... 163  Table 6‐2. Comparison of surface free energy of adhesion of bacteria to hexadecane at different  rhamnolipid concentration. ................................................................................................................ 165  Table 6‐3. Summary of surface tension parameters of selected bacteria, surfactant and contaminants.  ............................................................................................................................................................ 167  xv      List of Acronyms AB Acid-Base ANOVA Analysis of Variance AO Acridine Orange AOT Dioctyl sodium sulfosuccinate BH Bushnell Hass DLVO Derjaguin, Landau, Verwey & Overbeek DTMAB Dodecyltrimethyl Ammonium Bromide DNAPL Dense Nonaqueous-Phase Liquid EPS Extracellular Polymeric Substance FITC Fluorescein Isothiocyanate Flame-AAS Flame-Atomic Absorption Spectrometry HTAB Hexadecyltrimethylammonium Bromide HTAC Hexadecyltrimethylammonium Chloride LDS Lithium Dodecyl Sulphate LNAPL Light Non-Aqueous Phase Liquid LPS Lipopolysaccharide LW Lifshitz-van der Waals MATH Microbial Adhesion to Hydrocarbon NAPL Non-Aqueous Phase Liquid PAGE Polyacrylamide Gel Electrophoresis PUM Phosphate Urea Mg SDBS Sodium Dodecylbenzenesulphonate SDS Sodium Dodecylsulfate SEM Scanning Electron Microscopy TCE Trichloroethylene TTAB Tetradecyltrimethyl Ammonium Bromide xvi    Chapter 1 – General Introduction  Chapter 1. General Introduction Bioremediation is a rapidly developing technology for the clean-up of a range of ubiquitous pollutants present in our environment. It is a process whereby living organisms, particularly microorganisms, are used to degrade or transform hazardous contaminants to benign products. Bioremediation is performed using ex situ as well as in situ techniques for solid and liquid wastes (Philp et al., 2005). There have been many successful applications of bioremediation for the clean-up of landfills, hazardous organic and heavy metal waste sites and oil spills (Gadd, 2000; Philp et al., 2005). The technology offers benefits of being effective, economical and eco-friendly when compared to chemical and physical methods, especially when it can be carried out in situ (Mohan et al., 2006; Paul et al., 2005). Nevertheless, a commonly cited drawback of bioremediation is that relatively long treatment times are needed and many factors have been found to hinder the rate of biodegradation, including: 1. the availability of molecular oxygen, as a majority of organic pollutants are degraded faster in an aerobic environment (Ripley et al., 2002; Romantschuk et al., 2000); 2. the absence of specific contaminant degrading bacteria (Atlas & Cerniglia, 1995; Romantschuk et al., 2000; Scullion, 2006; van Hamme, 2004; Vogel, 1996); 3. low bioavailability of contaminants (Abalos et al., 2004; Lamoureux & Brownawell, 1999; Paul et al., 2005; Providenti et al., 1995); 4. a physically heterogeneous subsurface environment (Scullion, 2006); and 5. a lack of trace nutrients (Ripley et al., 2002; Romantschuk et al., 2000). Among these factors, researchers have identified two principle causes for the slow degradation: the absence of bacteria with the necessary metabolic capacity and the limited bioavailability of the contaminant (Abalos et al., 2004; Atlas & Cerniglia, 1995; Makkar & Rockne, 2003). Microorganisms capable of metabolizing organic pollutants are often widespread in the terrestrial environment. The native microbial consortia can often be enriched for bioremediation by the introduction of naturally occurring organic chemicals such as petroleum hydrocarbons (van Hamme, 2004). However, when the degradation pathway for 1    Chapter 1 – General Introduction  xenobiotic compounds (e.g., polychlorinated biphenyls and dioxins) are absent from indigenous microbial population, the microbes need to evolve suitable pathways (Atlas & Cerniglia, 1995; Scullion, 2006; van Hamme, 2004; Vogel, 1996). From a soil restoration point of view, this evolution process is inefficient and slow and the introduction of exogenous inoculants with the required degradation gene clusters and/or metabolic capacities into contaminated soils can expedite the remediation process. In such cases, the transport of microbial inoculants to the contaminated subsurface can have a pronounced effect on the level of enhancement, but the delivery of microorganisms to the contaminated areas of the soil remains an engineering challenge (Bai et al., 1997; Fang & Logan, 1999). In the subsurface, soil acts as an efficient filter and inoculated bacteria often penetrate only a few centimetres from source point (Edmonds, 1976; Harvey et al., 1989; Jackson et al., 1994; Madsen & Alexander, 1982). The soil surfaces intercept bacterial transport via a combination of high collision frequencies and high attachment probabilities between bacteria and soil grains (Li & Logan, 1999). A travelling bacterium would be expected to collide more than 500 times with soil surfaces during its transport over 1 meter under typical environmental conditions (Li & Logan, 1999; Martin et al., 1992). As a result, the soil reduces the bacteria concentration by several orders of magnitude within 10 to 100 cm from the injection well (Li & Logan, 1999). The growth of retained bacteria surrounding the source point can subsequently obstruct the transport of more bacteria into deeper soils. Therefore the challenge is to introduce a homogeneous distribution of bacteria to the contaminated subsurface. The mere presence of contaminant-degrading microorganisms in subsurface is not sufficient to enhance degradation if the contaminant has low bioavailability as a result of the physical and chemical nature of the interaction between contaminant and soil (Haferburg & Kothe, 2007; Scullion, 2006). Hydrophobic organic contaminants, such as polycyclic aromatic hydrocarbons and polychlorinated biphenyls, are characterized by low water solubility and strong adsorption to soil (Lamoureux & Brownawell, 1999; Makkar & Rockne, 2003; Stucki & Alexander, 1986). Contaminants present in the form of non-aqueous phase liquid (NAPL) also demonstrate limited bioavailability. The NAPL resides in the subsurface as a continuous pool of bulk and undiluted liquid that consists of a mixture of tens or even hundreds of compounds (De Blanc et al., 1996). Crude oil is an example of an NAPL contaminant. The NAPL has limited contact area with the bacterial population and tends to persist in the subsurface due to low bioavailability (De Blanc et al., 1996; Sharmin et al., 2006). 2    Chapter 1 – General Introduction  Bioavailability is defined as the “amount of contaminant present that can be readily taken up by living organisms, e.g., microbial cells” (Maier, 2000). For the biodegradation of such organic chemicals to occur in soil systems, bioavailability needs to be enhanced via desorption or dissolution (Guerin & Boyd, 1992 ; Noordman et al., 2002; van Hamme, 2004; Volkering et al., 1998). Although some studies suggest that sorbed compounds can be directly taken up by direct adhesion of microbes to the substrate particles (Calvillo & Alexander, 1996; Stelmack et al., 1999), contaminant bioavailability has been cited as a major limiting factor in the bioremediation process of soils having adequate microbial degradation capacities (Bramwell & Laha, 2000; Johnsen et al., 2005; Paul et al., 2005; Stucki & Alexander, 1986; Tiehm, 1994; Volkering et al., 1992). Several in situ treatment technologies (such as air-sparging, bioventing and injection of a surfactant solution) have been developed to address some of the above limitations. Airsparging technology has been used since the mid-1980s primarily for aquifer remediation of volatile organic contaminants (Bass & Brown, 1997). Air is injected into the aquifer below the contaminant plume. Buoyant forces move the air upwards through the contaminated zone, and the volatile contaminants are then carried in the air flow. Concurrently, oxygen in the air will dissolve in groundwater, enhancing the in situ aerobic biodegradation activity. Similar to air-sparging, bioventing involves introducing air into the subsurface to stimulate aerobic biodegradation in unsaturated soils. The technology has been shown to be cost-effective for the degradation of absorbed hydrocarbon fuels (Balba et al., 1998; Hoeppel et al., 1991). However, neither of these technologies increases solubilisation of contaminants in situ. Surfactants are surface-active agents and have been shown to enhance the rate of remediation by increasing contaminant bioavailability after injection as a solution into the subsurface (Bai et al., 1998; Brown et al., 1999; McCray et al., 2001; Noordman & Janssen, 2002; Ron & Rosenberg, 2002; Tiehm, 1994; Tsai et al., 2009). Surfactant systems often exhibit complex behaviour when interacting with contaminant (Paria, 2008), but mobilisation by lowering interfacial tension and micellar solubilisation have been accepted as the main mechanisms responsible for improving organic contaminant bioavailability (Brown et al., 1994; Paria, 2008). Surfactants that are fabricated from petroleum origin are synthetic surfactants. Natural surfactants such as bio-surfactants secreted by bacteria, are biodegradable and environmentally more compatible than synthetic surfactants. There are many types of biosurfactants. Rhamnolipids are one of the most studied, and also one of the few commercially available biosurfactants. Rhamnolipids enhance the bioavailability of 3    Chapter 1 – General Introduction  contaminants in soils and allow a faster degradation of these compounds (Nitschke et al., 2005; Rahman et al., 2003). Surfactant foam technology has emerged as an approach for accelerating in situ bioremediation by combining surfactant and air injection (Park et al., 2009; Singh et al., 2007; Wang & Mulligan, 2004a). Surfactant foam, including microbubble dispersion technology, has been well studied for various applications in floatation/separation processes, enhanced aeration systems and improved oil recovery operations (Jauregi & Varley, 1999; Oliveira et al., 2004b; Save & Pangarkar, 1994; Sebba, 1971). Microbubble dispersion is commonly generated by intensive mixing of surfactant solution. It is also called colloidal gas aphrons (Jauregi et al., 2000; Sebba, 1985) or microbubble suspension (Choi et al., 2009; Park et al., 2009). Research on the application of microbubble dispersion for bioremediation purposes has gained momentum in recent years owing to its promising advantages over air sparging, bioventing and surfactant solution application. There have been two main streams of research for microbubble dispersion. Characterisation studies have focussed on the types of surfactants, dispersion stability, gas hold-up and size distribution. Application studies have used column experiments to understand the mobilisation and/or biodegradation of contaminants in soil matrices with microbubble dispersions. The underlying assumption is that microbubble dispersion can be an effective technology for enhancing bioremediation because it has combined the benefits of air sparging, bioventing and surfactant application. It has been found that the dispersions can either facilitate, retard, or have no effects on the removal of contaminants in the column studies (Choi et al., 2009; Jenkins et al., 1993; Michelsen et al., 1983; Park et al., 2009; Ripley et al., 2002; Rothmel et al., 1998). There have been no explanations offered for the inconsistency observed in the current literature. More than that, to date, most studies have a primary focus on chemical surfactants as foaming agent. There are only a few recent studies making use of natural surfactant for microbubble dispersion applications. Consequently, research on which factors and how they can affect the effective application of microbubble dispersion is needed, particularly in the case of biosurfactant (e.g., rhamnolipid) application. 4    Chapter 1 – General Introduction  1.1. Research Objectives The aim of this study was to better understand the factors and mechanisms that influence the interactions among bacteria, surfactant and contaminant within the microbubble dispersion, thereby optimising the microbubble dispersion for effective bioremediation applications. Specifically the study has the following objectives: 1. To develop the best possible generator configuration in order to provide improved dispersion stability; 2. To investigate microbubble dispersion properties including drainage/stability, size distribution and gas hold-up under various environmental conditions, such as pH, ionic strength and surfactant concentration; 3. To develop and improve a drainage model specific to microbubble dispersion because drainage models in the literature have yet to provide a satisfactory explanation to the drainage behaviour observed in microbubble dispersion; 4. To investigate the interactions between bacteria and a rhamnolipid microbubble dispersion under the influence of various process parameters such as rhamnolipid surfactant concentration, salt and bacterial cell surface hydrophobicity; 5. To investigate the interaction between bacteria and contaminant at the microbubble surface; and 6. To quantify the interaction between bacteria, contaminant and microbubble dispersion using contact angle measurement and the LW-AB surface thermodynamic approach. 1.2. Thesis Hypotheses The hypotheses contained in this thesis include: 1. Bacterial cell surface hydrophobicity is the dominant factor in influencing the effectiveness of a microbubble dispersion as a bacteria-carrier. 2. The bacterial cell surface hydrophobicity is affected by surfactant, which makes hydrophilic cells more hydrophobic and vice versa. 3. Microbubble dispersions are able to promote direct contact between bacteria and contaminant within the dispersion. 5    Chapter 1 – General Introduction  1.3. Thesis Outline Chapter 1 provides a brief overview of the in situ bioremediation technologies and the background on the increasing interest in microbubble dispersion in bioremediation process. It also presents the research objectives and approach adopted in this study Chapter 2 presents a literature review to define current knowledge of the processes of generating microbubble dispersion, the types of surfactants used and their properties, and the characteristics and factors affecting dispersion properties (e.g. stability and quality). A number of bench and field scale experiments from the current literature are reviewed and discussed to illustrate the beneficial use of microbubble dispersion in subsurface contaminant clean-ups. Factors influencing bacterial interaction with surfaces and thermodynamic model for characterising the interactions are also discussed. Knowledge gaps in the existing literature are identified to support the research objectives of this thesis. Chapter 3 describes the experimental setup and procedures and analysis methodology. The chapter starts with a description of some of the general experimental procedures, followed by methods that are specific to each stage of the study. Chapter 4 presents the results and discussion from the characterisation studies of microbubble dispersion. A drainage mechanism unique to the microbubble dispersion is established and a previous drainage model is improved to better describe the drainage behaviour observed in this study. Chapter 5 examines the factors and mechanisms that can impact upon the effectiveness of the microbubble dispersion as a carrier for contaminant-degrading bacteria within a bioremediation scenario. In this chapter, different methods including bacterial drainage experiment, fluorescence microscopy, contact angle measurement and surface thermodynamic modelling are utilised. Drainage experiments with two model bacterial species were performed. The effect of surfactant interaction on bacteria surface hydrophobicity and the consequent effect on adhesion to microbubbles are quantified using contact angle measurement and surface thermodynamic model. Fluorescence microscopy images and cryo-scanning electron micrographs are used to visualise the bacteriamicrobubble interactions. 6    Chapter 1 – General Introduction  Chapter 6 visually demonstrates the bacteria-contaminant-microbubble interactions using fluorescence microscopy images, coupled with surface thermodynamic model. The implications for the bioremediation process are discussed. Chapter 7 concludes the thesis with a summary of findings and implication for in situ bioremediation and offers recommendations for future research 7    Chapter 2 – Literature Review  Chapter 2. Literature Review 2.1. Introduction This chapter provides a comprehensive literature review on studies relating to microbubble dispersion. Firstly there is a brief discussion on the properties of surfactants and the rhamnolipid biosurfactant in particular. The commonly available rhamnolipid biosurfactant is used to make microbubble dispersion in this study. Secondly characterisation studies on microbubble dispersion are reviewed in order to understand the general characteristics of the dispersions. Thirdly, experiments on the use of microbubble dispersion in contaminant cleanups are presented to identify knowledge gaps in the study of microbubble dispersion. Fourthly, factors influencing bacterial interaction with interfaces are presented. Lastly, a thermodynamic model for describing bacterial adhesion to microbubble dispersion is introduced. 2.2. Surfactant and Biosurfactant Surfactants are surface active agents that possess both hydrophobic and hydrophilic groups. The hydrophilic head group can be anionic, cationic, zwitterionic (both positive and negative change on the same molecule), or non-ionic. The hydrophobic tail usually consists of linear, branched or unsaturated alkyl chains of 8 to 20 carbons in length that have low solubility in water. Their amphiphilic nature enables the surfactants to adsorb to and alter the conditions prevailing at surface boundaries or interfaces (Brown & Jaffé, 2001; Myers, 1988). The driving force for surfactants to adsorb at interfaces is to minimise the free energy of the phase boundary and thus of the whole system. For liquids excess free energy exists at the phase boundary (such as the air-water interface) and it is measured as surface tension. When surfactants are placed in the liquids, the surfactant molecules accumulate at the air-liquid interface to reduce the surface tension. The surface tension will cease to decrease once the surfactant concentration reaches a well-defined concentration known as the Critical Micelle Concentration, i.e., CMC (Myers, 1990). Above the CMC, the surfactant molecules aggregate to form micelles, vesicles, and lamellae to minimise the free energy of the entire system. Different surfactants have different CMC values. 8    Chapter 2 – Literature Review  Figure 22-1 schemaatically dem monstrates thhe adsorptio on of surfacctants at thee air-water interface i and form mation of micelles m in water. w The hhydrophilicc head group p interacts ffavourably with the surrounnding water molecules while w the hyydrophobic tail portion n, due to thee tail’s disru uption of the surrrounding hyydrogen bo onds conneccting waterr molecules, orients itsself away from f the water. For non-poolar solven nt, it is thee exposure of the hyd drophilic hhead groupss to the surrounnding solvent that is energeticall e ly unfavourrable. In th his case, a reverse micelle is formed.. Figure 2-1. A schem matic of adsorption of surfaactants at thee air-water in nterface and fformation of micelles in waater (Myers, 1990). 1 Due too their surrface activee propertiees, surfactaants are ab ble to incrrease hydrrocarbon contaminant bioavailability by y: 1. redducing the interfacial i tension t betw ween oil ph hase and aqu ueous phasee, thereby releasing r orgganic contaaminant sorb bed to soil particles orr trapped in n pore spacees or from pools p of noon-aqueous phase p liquid ds (NAPLs)) (Johnson et e al., 1999; West & Haarwell, 1992 2); and 2. thee formation of micelless, which proomote hydro ocarbon parrtitioning innto the hydrrophobic miicellar coree, resulting in higher apparent aqueous so olubility off the hydro ocarbons (C Cameotra & Bollag, 200 03; Lee et aal., 2005; Makkar M & Ro ockne, 20033; Noordman n, 1999). 2.2.1. B Biosurfactaant In recennt years, biosurfactant b ts, i.e. surffactants syn nthesized by y microorgganisms, haave been used ass alternativees to chemiccally manuffactured surrfactants du ue to increaasing enviro onmental concernn. The most importan nt advantagge of biosu urfactants when comp mpared to synthetic s counterrparts is theeir environm mental com mpatibility, due to theiir low ecoloogical toxiccity and biodegrradable natuure (Abaloss et al., 20004; Nitsch hke et al., 2005). 2 Manny of the synthetic s 9    Chapter 2 – Literature Review  surfactants are non-biodegradable, resulting in secondary contamination once injected into the subsurface, with some inhibiting bacterial growth (Hartmann, 1966; Rothmel et al., 1998; Tiehm, 1994). Biosurfactants are have been utilised in a great variety of applications including enhanced oil recovery, bioremediation of organic pollutants and heavy metal contaminated sites, and in the health care and food processing industry. Other developing areas of biosurfactant use are in cosmetic formulation, foods and pharmaceutical production (Banat et al., 2000; Makkar & Cameotra, 2002; Nitschke et al., 2005). Biosurfactants are synthesized by a variety of microorganisms, such as those from bacterial genera of Pseudomonas, Rhodococcus, Mycobacterium, and Acinetobacter (Desai & Banat, 1997). Biosurfactants are classified broadly based on their major structural features (Table 21). The major groups include (1) glycolipids, (2) lipoproteins, (3) phospholipids, neutral lipids, and fatty acids, and (4) polymeric biosurfactants (Maier et al., 2003). Table 2-1. Types of biosurfactants and their characteristics (Maier et al., 2003; Ron & Rosenberg, 2002). Biosurfactant group Characteristics Glycolipids - Lipoproteins - Phospholipids, neutral lipids and fatty acids Polymeric biosurfactants - Representative biosurfactant (microorganism) Carbohydrate groups attached to long- Rhamnolipid chain aliphatic acid or hydroxy-aliphatic (Pseudomonas aeruginosa) acid groups Molecular weights ranging from 500 to 1500 Contains a protein moiety attached to a Surfactin (Bacillus subtilis) fatty acid Molecular weights ranging from 1000 to 1500 Lipid-containing molecules forming Phospholipid (Thiobacillus bacterial cell surface structure thiooxidans) Carbohydrate or protein based polymers Alasan (Acinetobacter containing lipids radioresistens) Molecular weight ranging from 50,000 to greater than 1,000,000 The natural roles of biosurfactants are varied, due to the wide variety of microorganisms involved and the different chemical structures and surface properties of the surfactant. The following natural roles have been suggested and demonstrated in the literature. 1. Increasing the bioavailability of hydrophobic substrates – Hydrophobic substrates, due to their low water solubility, tend to be bound to surfaces and have limited availability to biodegrading microorganisms (Lamoureux & Brownawell, 1999; van 10    Chapter 2 – Literature Review  Hamme, 2004). Low-molecular-weight biosurfactants, for example, can increase the apparent solubility of water-immiscible hydrocarbons (Maier et al., 2003; Ron & Rosenberg, 2002). The presence of rhamnolipid at 500mg/L, a concentration that is about 10 times of its CMC, accelerated the biodegradation of total petroleum hydrocarbon from 32% to 61% at 10 days of incubation (Abalos et al., 2004). The biodegradation of PAHs, in particular, has increased from 9% at control condition to 44% (Abalos et al., 2004). However, increasing apparent solubility does not necessarily result in an increased biodegradation because studies have shown that the fraction of the contaminants associated with the micellar phase may not be directly bioavailable (Cort et al., 2002; Guha & Jaffé, 1996). 2. Regulating the attachment/detachment of microorganisms to and from surfaces – Another suggested function of biosurfactants is to enhance adhesion of microorganisms to insoluble substrates (Neu, 1996). During the growth phase, for example, Acinetobacter calcoaceticus RAG-1 accumulate biosurfactants on the cell surface with the hydrophobic end oriented towards the environment to facilitate adhesion onto insoluble hydrocarbons (Neu, 1996; Rosenberg & Mitchell, 1985). When the carbon substrate is depleted, the bacteria release the biosurfactants, making the cells hydrophilic and dispersed to search for new habitat (Rosenberg & Mitchell, 1985). 3. Binding of heavy metals – A mono-rhamnolipid biosurfactant produced by Pseudomonas aeruginosa ATCC 9027 has been shown to desorb soil-bound heavy metals such as cadmium, lead and zinc by forming rhamnolipid-metal complexation (Herman et al., 1995). Another study reported that rhamnolipid eliminated cadmium toxicity when added at a concentration 10-fold greater than cadmium (Sandrin et al., 2000). It is suggested that rhamnolipid reduces metal toxicity through a combination of rhamnolipid complexation of cadmium and rhamnolipid interaction with the cell surface. 4. Biofilm development – An increase in cell surface hydrophobicity by biosurfactants could increase the adhesiveness of the bacteria to a level which is critical for initial micro-colony formation in biofilms (Pamp & Tolker-Nielsen, 2007). Recent studies have indicated that biosurfactants play a role both in maintaining water channels 11    Chapter 2 – Literature Review  between multicellula m r structuress in biofilmss (Davey ett al., 2003) and in disp persal of cells from m biofilms (P Pamp & Tollker-Nielsen n, 2007; Yaasuhiko et al al., 2005). 2.2.2. R Rhamnolipiid One off the mostt widely sttudied biossurfactants is rhamno olipid, a rhhamnose-containing glycolippid surfactaant that is prrimarily prooduced by Pseudomona P as aeruginoosa (Desai & Banat, 1997; N Nitschke ett al., 2005). Rhamnoliipids demonstrate a good potentitial for com mmercial exploitaation due too high produ uction yieldds. Also, theey can be prroduced usiing relativelly cheap substrattes such ass carbohyd drates, vegeetable oils or even waste w from the food industry (Nitschkke et al., 20005). Rhamnoolipids are produced p ass mixtures iin various proportions. p . Mono-rham mnolipids (R1) ( and di-rham mnolipids (R R2) are th he two moost abundan nt forms of o rhamnoliipids produ uced by Pseudom monas aeruginosa du uring broth culture co ontaining glucose or gglycerol (S SoberónChávezz et al., 20005). R1 hass a single rhhamnose grroup whereas R2 conttains two rh hamnose groups jjoined to eaach other with w an etherr bridge. Th he hydrophiilic head grooup of rham mnolipid molecules consistss of the rham mnose and free carbox xyl groups, whereas the he hydropho obic tails are twoo hydrocarbon chains. General stru ructures of the t rhamnolipid compoounds (R1 and R2) are depiicted in Figgure 2-2 (Maaier et al., 22003). Rham mnolipids haave side-chaain lengths from C4 to C14 depending on how they are pproduced (Soberón-Ch hávez et aal., 2005). Due to protonaation of the carboxyl grroups, rham mnolipids beehave as anionic surfacctants at pH H greater than 4.0, with theeir pKa varrying betw ween 4.3 to 5.6, depen ndent on thhe structuree of the rhamnoolipid moleccule (Champ pion et al., 1995; Lebró ón-Paler et al., 2006). Figu ure 2-2. Rham mnolipid stru ucture. R = H for R1 and R = rhamnosee for R2; m aand n values vary. v 12    Chapter 2 – Literature Review  Rhamnolipids substantially reduce the surface tension of water from 72 mN/m to below 30 mN/m and the interfacial tension of water/hexadecane from 43 mN/m to less than 1 mN/m (Abalos et al., 2001; Nitschke et al., 2005; Parra et al., 1989; Syldatk et al., 1985). Rhamnolipid surfactants have been found to enhance apparent solubility of contaminants such as petroleum hydrocarbons, polycyclic aromatic hydrocarbons, chlorinated hydrocarbons and heavy metals, thereby improving bioavailability and allowing a faster degradation of these compounds (Abalos et al., 2004; Mulligan, 2005; Noordman & Janssen, 2002). For example, rhamnolipids increase the mobility of hexadecane and phenanthrene in contaminated soils more efficiently than synthetic surfactant such as SDS (Rahman et al., 2003) Rhamnolipids were chosen for this study because: 1) Rhamnolipids have proven abillity to increase contaminant bioavailabity as well as rate of biodegradation; 2) Rhamnolipids are the most studied biosurfactants, hence a large amount of published information is available; 3) Rhamnolipids are commercially available; and 4) To the authors knowledge, studies on microbubble dispersion have not been undertaken with rhamnolipids. 2.3. Characterisation of Microbubble Dispersion 2.3.1. Generation of Microbubble Dispersion Microbubble dispersions were first developed by Sebba (1971). These dispersions are referred to as colloidal gas aphrons because the bubbles are of colloidal dimensions (Sebba, 1985). The microbubble dispersion is essentially a suspension of a large number of minute spherical gas bubbles encapsulated in a soapy liquid film in an aqueous surfactant solution. A number of different generation methods are available for producing a microbubble dispersion. A. The Venturi Method The venturi method produces microbubbles by mixing gas through a fine orifice with a stream of rapidly flowing surfactant solution at a venturi throat. It is the original method 13    Chapter 2 – Literature Review  developed by Sebba (1971) to make colloidal gas aphron. By recycling a few times it was observed that the system entrained up to 65% of gas in the surfactant solution, and produced gas aphrons about 25 microns or greater in diameters (Sebba, 1985). However the need for high power input and recycling to form a stable dispersion is unfavourable for large-scale production (Sebba, 1985). B. Spinning Disk Method The spinning disk method is most commonly used among researchers. It makes microbubble dispersion by intensive stirring of surfactant solution with a spinning disk connected through a shaft to an electric motor in a baffled container (Sebba, 1985; 1987). Rotation of the disk at speeds above 4,500 rpm generates vigorous waves on the solution surface that hit the baffles, re-enter the surfactant solution along with trapped air, and subsequently break into minute bubbles that upon stabilisation by the surfactant molecules form the microbubble dispersion. The method makes several litres of microbubbles in less than a minute, and is suitable for scale up application (Sebba, 1985). Modifications to the basic method have been made: Chaphalkar et al. (1993) wrapped the container with cooling coil to allow temperature control during production; Save and Pangarkar (1994) used a propeller instead of a disc to increase aeration; and Wan et al. (2001) enclosed the apparatus with a stainless steel chamber to generate microbubbles under specific pressure and gas composition. C. Sonication It has been reported that microbubbles can be produced using sonication, where a mixture of surfactant solution is agitated with a sonicator probe. To produce stable microbubbles a combination of a water soluble surfactant and a solid hydrophobic surfactant is required (Singhal et al., 1993; Wan et al., 2001). The freshly made microbubble dispersion also required separation from the remaining solid surfactants prior to application. An advantage of using sonication is that microbubble dispersion can be made quickly. However, this method does not work with all surfactant types, nor does it suit mass production of microbubbles. 2.3.2. Characterisation Techniques A microbubble dispersion is a dynamic system and undergoes continuous change. A number of studies have characterised microbubble dispersion, reporting properties that are distinctive from conventional foams. In this section characterisation techniques are examined and the known properties of various microbubble dispersions are summarised in Table 2-2. 14    Chapter 2 – Literature Review  Table 2-2. Summary of microbubble dispersion characterisation studies. Surfactant Cationic (c) HTAC TTAB Anionic (a) AOT AOT Non-ionic (n) Tween 20 Saponins Various EHDA(c); SDBS(a) BDHA(c); SDBS(a); AOT(a); LUX flakes (n) Generation Method Stability Measurement Bubble Size Distribution Air Holdup Application Spinning disk at 6,000 rpm Half-life of 171s --- Gas-liquid mass Dai and Deng transfer (2003) Spinning disk at 6,500 rpm Creaming rate Microscopic image analysis of silici sol fixed bubbles; 4 – 100m with 26.6% less than 25m Average diameter of 35m with shell thickness of 0.75m 73% Separation of sulphur crystals Spinning disk at 8,000 rpm Half-life of 30 – 930s depending on surfactant and electrolyte concentrations --- --- 12% - 59% Protein recovery Jauregi et al. (1997) Microscopic image analysis; mean diameter of 32– 67m --- Spinning disk at 4,000 rpm Spinning disk --- Particle size analyzer --- Half-life of 170 – 720s varied with concentration Particle size analyzer; 30 – 300m Venturi throat --- --- 50% - 65% Precipitation & Ciriello et al. flotation (1982) Spinning disk at 6,000 rpm Half-life of 510s for BDHA; 540s for SDBS; 510 for AOT; 738s for LUX Half-life from 144 – 486s depending on process parameters DV fraction time; 360s for HTAB; 720s for NaDBS --- --- 65% --- 34% - 73% Solvent extraction Matsushita et al. (1992) Particle size analyzer; diameter range 44–176m Particle size analyzer; diameter range 30 – 300m Half-life from 210 – --490s --- Wastewater treatment Roy et al. (1992) --- Wastewater treatment Chaphalkar (1993) --- Wastewater treatment Save et al. (1994) Microscopic observation; stable for 4 – 6 weeks Microscopic observation; stable for >6 weeks --- Ultrasound contrast agent Wheatley and Singhal (1995) --- Subsurface remediation Wan et al. (2001) --- Separation Yan et al. (2005) Spinning disk at 8,000 rpm CTMAB(c); DTMAB(c); Spinning disk at CPB(c); SDS(a); >4,000 rpm SDBS(a); Tergitol(n) HTAB(c); NaDBS(a) Spinning disk at 8,000 rpm HTAB(c); SDBS(a); Tergitol(n) Spinning disk at 8,000 rpm DTAC(c); CTAC(c); CPC(c); DMDSAC(c); SDBS(a); SLS(a) Mixture of Tween(n) & Span 60 (n) Spinning disk at 8,000 rpm Mixture of SDS(a) & Span 60 (n) Enclosed chamber with spinning disk at 13,000 rpm Spinning disk at 6,000 to 8,000 rpm HTAB(c); SDS(a); Tween 80(n) Sonication Half-life varied from 141 to of 141 – 525s Particle size analyzer; mean diameter 4.5m Particle size analyzer; mean diameter 0.7 – 20m ---   15    Reference Amiri and Woodburn (1990) Protein recovery Jauregi et al. (2000) Bioreactor gas Bredwell and transfer Worden (1998) 50% - 70% Soil remediation Kommalapati (1996) Wastewater treatment Subramaniam et al. (1990) Chapter 2 – Literature Review  A. Stability Measurement The stability of aqueous foam is controlled by three fundamental bubble-collapsing mechanisms: the rapid hydrodynamic drainage of liquid between bubbles; disproportionation between two bubbles; and lamellar rupture, whereby the bubbles coalesce or collapse (Jacobi et al., 1956; Myers, 1990; Yan et al., 2005). Disproportionation is a coalescence mechanism whereby gas diffuses from smaller bubbles with higher pressure to larger bubbles with lower pressure, causing the larger bubbles to expand at the expense of the smaller bubbles. It has been reasoned that since no perceptible breakdown of bubbles takes place prior to the majority of liquid being drained from the dispersion, the liquid drainage rate is the principle parameter determining microbubble stability (Yan et al., 2005). Sebba (1971; 1987) described the stability of a microbubble dispersion in terms of its half-life, which is the time taken to drain half of initial liquid volume. The half-life method is the most commonly used measure of microbubble stability, and is simply estimated using a measuring cylinder (Jauregi & Varley, 1999). Freshly prepared microbubbles are transferred to the measuring cylinder and the volume of the drained liquid below the dispersion is measured at various times. The basic set up may be modified to maintain a constant temperature using a water jacket (Yan et al., 2005). B. Size Distribution Measurement A range of different techniques have been used to measure the size distribution of microbubbles. Light microscopy coupled with image analysis is the most commonly used approach. Some researchers have proposed that despite their relative stability, microbubbles undergo changes with time through the disproportionation mechanism (Cheng & Lemlich, 1983; Dai & Deng, 2003). It is especially so for bubbles smaller than 25 m diameter, many of which are found to vanish quickly due to extremely high internal pressure (Dai & Deng, 2003; Sebba, 1971). To eliminate the time effect, Cheng and Lemlich (1983) cryo-froze the bubbles in liquid nitrogen and made slices of the frozen bubbles for observation under a microscope. In another study Dai and Deng (2003) stabilised the microbubbles using silicic sol solution to form a thin but tight silicic gel film that enwrapped the bubbles. However this technique is effective only for surfactants of cationic type because reaction with the negatively charged silicic sol solution is required to form the film (Dai & Deng, 2003). In another study, Basu and Malpani (2001) took microphotographs of microbubbles at the end of the outlet tube of the microbubble generator with a stereo-zoom microscope. This 16    Chapter 2 – Literature Review  technique not only captures bubbles smaller than 25 mm before they vanish, but also works for all surfactant types. Other techniques such as the laser-scattering method with the use of particle size analyser are also commonly employed (Chaphalkar et al., 1993; Kommalapati et al., 1996; Roy et al., 1992; Wan et al., 2001) and is claimed to be a direct and reliable measurement of bubble sizes at a particular time (Chaphalkar et al., 1993). 2.3.3. Properties A. Microbubble Structure It is claimed that the structure of microbubbles is different from conventional bubbles that are simply surrounded by surfactant monolayer. Each microbubble, as per the structure proposed by Sebba (1987), is encapsulated in a soapy shell by three surfactant layers (see Table 2-3). The presence of the ordered layers of surfactant film retards coalescence of the microbubbles, thereby increasing their stability (Sebba, 1987). Although most researchers have assumed that microbubbles have this structure, only a limited number of studies have been reported in support of this. For example, using the rise-velocity of microbubbles Amiri and Woodburn (1990) estimated that the dispersion made from tetradecyltrimethyl ammonium bromide (TTAB) contained bubbles of 35 m diameter with a 0.75 m thick interface consisting of consecutive layers of surfactant molecules. Bredwell and Worden (1998) used a mass-transfer method to show that microbubbles made from Tween 20 surfactant had an interface thickness of between 0.2 and 0.3 m. The finite interface thickness implies a presence of multiple surfactant layers since the thickness of a monolayer of surfactant molecules would be much less. More recently, Jauregi et al. (2000) employed transmission electron microscopy and Xray diffraction techniques to estimate the average interfacial thickness of sodium bis(2ethylhexyl) sulfosuccinate (AOT) surfactant microbubbles as 0.96 m and reveal the existence of multilayer arrangement of surfactant molecules at the bubble interface. B. Stability The stability of microbubble dispersion refers to its ability to resist bubble breakdown. In bioremediation applications it is desirable to optimise the stability of the microbubble dispersion to harness the beneficial properties of subsurface penetration, nutrient delivery and contaminant mobilisation. Stability is influenced by many factors such as the surfactant type, surfactant concentration, electrolyte concentration, solution pH, stirring speed and duration, and pressure. Matsushita et al. (1992) compared the half-life of microbubble dispersion 17    Chapter 2 – Literature Review  generated from cationic (CTMAB, DTMAB), anionic (SDS, SDBS), and non-ionic (Tergitol) surfactants for similar surfactant concentrations and stirring speed. The ionic surfactants, except DTMAB, gave half-lives of 5 to 7 minutes, while the non-ionic surfactant gave values of less than 3 minutes. The greater stability of ionic surfactant microbubble dispersion has also been previously reported (Chaphalkar et al., 1993; Jauregi et al., 1997; Longe, 1989; Save & Pangarkar, 1994; Yan et al., 2005). However the magnitude of this effect is specific to the surfactant structure. It was shown that a change of surfactant structure from CTMAB to DTMAB, whereby the hydrophobic chain was reduced from C16 to C12, caused a decrease in the half-life by almost 50% to 2.9 minutes (Matsushita et al., 1992). Importantly, it has been shown that a mixture of water soluble and solid hydrophobic surfactants can form microbubbles that are stable for days (Singhal et al., 1993; Wan et al., 2001). It has been suggested that such a combination forms a solid-condensed monolayer at the bubble interface, which reduces gas diffusion and the surface tension (Wan et al., 2001; Wang et al., 1996). Jauregi et al. (2000; 1997) experimented with an anionic AOT surfactant and found that stability of a microbubble dispersion depends mainly on surfactant and electrolyte concentrations. At a mixing speed of 7,700 rpm, the dispersion half-life increased from 60 to 758 seconds when the surfactant concentration was raised from 0.1mM to 61mM (Jauregi et al., 1997). Similar findings are supported by many other researchers (Amiri & Woodburn, 1990; Chaphalkar et al., 1993; Matsushita et al., 1992; Oliveira et al., 2004b; Yan et al., 2005). The increased surfactant concentration, either at the microbubble interface or in bulk liquid phase, leads to greater electrostatic repulsion between adjacent bubbles, enhancing dispersion stability (Jauregi et al., 1997). Additionally, Yan et al. (2005) suggest that higher surfactant concentrations increase microbubble surface viscosity and elasticity producing an adhesive-like bonding that decreases the drainage rate. However, increasing the surfactant concentration above its CMC has a negligible effect on stability (Yan et al., 2005). At surfactant concentrations below its CMC, there are insufficient surfactant molecules to stabilise the large interfacial area of the microbubbles. Hence, raising the concentration stabilises the microbubbles, however, when this is above its CMC the adsorption of surfactant at the microbubble surface reaches saturation and the further addition of surfactant molecules causes them to remain suspended in the liquid phase surrounding the microbubbles (Yan et al., 2005). On a related note, increasing the surfactant concentration has shown to result in higher apparent viscosity of the dispersion, but the increment is small when the surfactant concentration is above CMC (Oliveira et al., 2004b). 18    Chapter 2 – Literature Review  Increasing the electrolyte concentration reduces dispersion stability (Amiri & Woodburn, 1990; Jauregi et al., 1997; Save & Pangarkar, 1994). An addition of 0.07M NaCl reduced the stability (measured as half-life) of an AOT surfactant microbubble dispersion from 375 to 133 seconds; and doubling the salt dosage decreased the stability further to 90 seconds (Jauregi et al., 1997). The addition of a salt compresses the electrical double layer and decreases the stability by affecting the electrostatic repulsion between adjacent bubbles (Jauregi et al., 1997; Myers, 1990; Save & Pangarkar, 1994; Sebba, 1987). The reported effects of pH on dispersion stability appear contradictory. While several studies have reported no significant effect of pH on dispersion stability (Jauregi et al., 1997; Save & Pangarkar, 1994; Subramaniam et al., 1990), Amiri and Woodburn (1990) observed that as pH was made neutral starting with an acidic value, the stability of a TTAB surfactant microbubble dispersion increased, but then dropped sharply when the pH was made alkaline. The observed effect of pH was reported to be attributed to changes in ionic strength brought by pH variation (Jauregi et al., 1997). Other process parameters such as stirring speed, mixing duration and applied pressure have variable effects on stability. In general, increasing the stirring speed results in greater stability, but the effect of stirring duration is not clear-cut. In contradiction to other studies (Jauregi et al., 1997; Matsushita et al., 1992), Save and Pangarkar (1994) reported that the half-life of microbubble dispersion fluctuated for stirring time of 1 to 5 minutes, but remained constant for stirring times above 5 minutes. Wan et al. (2001) made microbubbles under pressure and observed that increasing the pressure had an adverse effect on stability. C. Size Distribution Microbubbles are usually between 25 to 100 m in diameter (Sebba, 1985; 1987). It is observed that bubbles smaller than 25 m diameter disintegrate in under a minute due to rapid gas diffusion (Sebba, 1987). Thus a microbubble dispersion that is more than a few minutes old consists primarily of bubbles of 25 m and above in diameter, however, the size of bubbles between 10th and 50th percentiles remains fairly constant with time (Kommalapati et al., 1996; Roy et al., 1992). Using image analysis, Jauregi et al. (2000) reported the mean bubble diameter to be between 32.4 and 66.5 m, depending on the experimental conditions. A wider size range of 30 to 300 m was reported when particle size analyser was employed for the measurement, supporting the notion that in practice the bubble diameter exceeds 30 19    Chapter 2 – Literature Review  m (Chaphalkar et al., 1993; Kommalapati et al., 1996; Roy et al., 1992). However, by measuring the diameter immediately after production, researchers have observed comparatively smaller bubbles with mean diameters varying between 14 to 92 m, depending on surfactant type and mixing conditions (Basu & Malpani, 2001). Dai and Deng (2003) used a flow stabilisation procedure and showed that almost 27% of the HTAC microbubbles were smaller than 25 m diameter and the mean diameter of bubbles within the dispersion was about 55 m. Similar to stability, the size distribution is also influenced by factors such as stirring speed, surfactant type and concentration, and the presence of electrolyte. Higher stirring speed produces smaller sized microbubbles, for example, by increasing stirring speed from 4,000 to 8,000 rpm, the bubble mean diameter was reduced from 75 m to 22 m (Basu & Malpani, 2001). Some studies suggested that non-ionic surfactants produce smaller microbubbles than their ionic counterparts, as the bubbles generated from non-ionic surfactants are more easily compressed (Chaphalkar et al., 1993; Kommalapati et al., 1996). A combination of different surfactants may also yield smaller sized microbubbles. Wan et al. (2001) reported that a combination of aqueous hydrophilic SDS and solid hydrophobic Span 60 surfactants yielded microbubbles in the 0.7 to 20 m size range. Furthermore, increasing surfactant concentration also reduces the microbubble size, and the effect is common to both ionic and non-ionic surfactants (Chaphalkar et al., 1993; Kommalapati et al., 1996). While Kommalapati et al. (1996) observed little effect on the bubble size when the increase of surfactant concentration was raised above its CMC, other studies suggested that this effect is specific to the surfactant used (Chaphalkar et al., 1993). D. Gas Hold-Up The gas hold-up measures the amount of gas entrained in the microbubble dispersion, and can play a critical role during bioremediation enhancement as one of the functions of microbubbles is to deliver oxygen to enhance aerobic biodegradation activity. It is defined as the ratio of the total gas volume to the total dispersion volume. The gas volume is equivalent to the difference between the total dispersion volume and the final drained liquid volume from the dispersion. In general, microbubble dispersion displays a gas hold-up of as much as 50% to 70% (Ciriello et al., 1982; Sebba, 1987; Subramaniam et al., 1990). Below 65% gas hold-up the 20    Chapter 2 – Literature Review  microbubbles are spherical in shape, exhibit colloidal properties, and flow like water (Oliveira et al., 2004b; Sebba, 1987; Subramaniam et al., 1990). As the gas hold-up increases above the critical value of 65%, the bubbles become distorted from the spherical shape, forming a packing structure that results in a sharp increase in the apparent viscosity of the dispersion (Oliveira et al., 2004b; Subramaniam et al., 1990). For application purpose, the gas content should be kept below the critical value to enable smooth subsurface injection (Oliveira et al., 2004b). However, this may require careful control as gas hold-up of 50% to 60% generally results in higher dispersion stability (Jauregi et al., 1997). Just as the other properties of microbubble dispersion, the gas hold-up value is affected by operational parameters. The gas hold-up increases with stirring speed and mixing duration (Jauregi et al., 1997; Matsushita et al., 1992). For example, Matsushita et al. (1992) reported that the air content of a dispersion increased from 34% to 70% on increasing the stirring speed – from 5,000 to 6,500 rpm, but no further with continued increase in speed to 8,500 rpm. Also, the air content increased steadily from 60% to 73% with an increase in the stirring duration from 0.5 to 5 minutes. In general, gas hold-up also increases with surfactant concentration, reaching a maximum at the surfactant’s CMC (Jauregi et al., 1997; Kommalapati et al., 1996; Matsushita et al., 1992). However the type of surfactant, ionic or non-ionic, has no effect on the gas hold-up (Matsushita et al., 1992). Increasing the electrolyte concentration decreases gas hold-up (Jauregi et al., 1997). E. Drainage Mechanism Liquid drainage is the dominant parameter determining microbubble stability (Sebba, 1987; Yan et al., 2005). Since microbubble dispersion resembles emulsion, it has been suggested that its drainage mechanism differs from conventional foam (Sebba, 1987; Save and Pangarkar, 1994), with the latter typically exhibiting an exponential decrease in drainage with time (Ross, 1943; Jacobi, 1956). Microbubble dispersion demonstrates a typical “S”-shaped drainage profile, with an increase in the rate of drainage at initial times followed by a decrease in the rate of drainage at later times (Yan et al., 2008; Yan et al., 2005). Currently in the literature, the drainage of microbubble dispersion has been described in terms of microbubbles rising as per the Stroke’s velocity (Amiri & Woodburn, 1990) or a two-stage process consisting of an initial stage during which liquid drains under gravity followed by a stage in which foam breaks down due to thinning of films between bubbles (Save & Pangarkar, 1994; Yan et al., 2005). The drainage equation based on the Stroke’s 21    Chapter 2 – Literature Review  velocity describes a drainage profile similar to an exponentially decreasing profile for conventional foams, and so fails to address the “S”-shaped drainage curve typical of microbubble dispersion. Although the equation developed by Yan et al. (2005) agrees well to the “S”-shaped drainage profile, its conceptual model describes a two-stage drainage process that is similar to the drainage mechanisms proposed for conventional wet foams (Indrawati & Narsimhan, 2008; Koehler et al., 2000; Ross, 1943). Consequently, these above mentioned models fail to explain the “S”-shaped drainage profile observed in microbubble dispersion. F. Comparison with Conventional Foams Microbubble dispersion displays colloidal properties due to their micron-sized bubbles (Sebba, 1987). The colloidal character along with the unique surfactant arrangement at the microbubble surface distinguishes the dispersions from conventional foams. The microbubble dispersion provides a large interfacial area per unit volume, exhibit relatively high stability and flow like aqueous solutions (Jauregi et al., 1997; Save & Pangarkar, 1994; Sebba, 1971). On standing, microbubble dispersion will undergo creaming and transition into a conventional foam (Sebba, 1987). Table 2-3 compares the distinguishing features of microbubble dispersion and conventional foam (Barnes & Gentle, 2005; Jauregi & Varley, 1999; Myers, 1990; Sebba, 1987; Wang & Mulligan, 2004a). The structure of the microbubble was first proposed by Sebba (1987), under the name colloidal gas aphron, and was supported by several studies (Amiri & Woodburn, 1990; Bredwell & Worden, 1998; Jauregi et al., 2000). The microbubble is surrounded by a soapy film that is formed by three surfactant layers. This marks the key difference between the microbubble and a conventional bubble, which is surrounded by a surfactant monolayer. The multi-layer surfactant film arrangement of the microbubble delays coalescence, thus giving the dispersion its remarkable stability (Sebba, 1985; Sebba, 1987). 22    Chapter 2 – Literature Review  Tablle 2-3. Comparison of miccrobubble disspersion to co onventional fo foams Featuress Formatioon obubble dispeersion Micro Intensive stirring s above 4,500 rpm Conventioonal Foam Air spparging No coaleescence if keppt stirred 10 to 100μm 50% to 70% Flows like waater; can be puumped easily without w collapsse Agittation acceleraates foam breaak-up Varied; usuually >1mm Up too 99% Difficu ult to transportt from one loccation to anoother Structurre Stabilityy Size Gas Hold d-up Flow Prooperty 2.3.4. K Knowledge Gap Existingg studies onn microbubb ble dispersiion have a primary foccus on synth thetic surfacctants as the foam ming agentt. Studies with w microbbubble disp persion prep pared from natural surrfactants have beeen limited. Such stud dies may pootentially have h a signiificant impaact as stud dies with dispersiions of sapoonin, a plan nt-based nattural surfactant show that t these arre more staable than those oof synthetic surfactant SDS (Kom mmalapati et e al., 1996). Biosurfacctants, due to their biologiccal and ennvironmentaal compatibbility (Maieer et al., 2003), 2 mayy be preferrable to synthetiic surfactannts in mak king microbbubble disp persion for bioremediiation appliications. Therefoore, there iss a need to investigatee if microbu ubble dispersion madee with biosu urfactant such ass rhamnolippid possessiing similar properties to the disp persions maade from synthetic s surfactaants. Microbuubble dispeersion prop perties, inclluding stab bility, bubble size andd gas hold-up, are importaant parametters with reespect to itss applicatio on in biorem mediation. SStable micrrobubble dispersiion allows better b subsu urface penettration and contaminan c nt mobilisatiion. Smaller bubble size maakes the disppersion easier to movee through a porous soill. Microbubbble dispersion with a high ggas hold-upp ratio can deliver d moree air/oxygen to enhancce aerobic ddegradation n activity in the subsurface.. As summ marised in above sections, previous studiess have sho own that i thhe stability, bubble sizee and gas hoold-up (Chaaphalkar surfactaant type/struucture can influence 23    Chapter 2 – Literature Review  et al., 1993; Kommalapati et al., 1996; Matsushita et al., 1992; Wan et al., 2001). Since little characterisation research has been done with biosurfactant, it is important to investigate if biosurfactant produces microbubble dispersion with properties suitable for bioremediation application. The conceptual models for the drainage of microbubble dispersion in the current literature do not provide a satisfactory explanation for the observed differences between the drainage curves for microbubble dispersion and conventional foam, as discussed in previous section. Since liquid drainage is the dominant parameter determining microbubble stability, it is important to improve our understanding on the drainage mechanism for microbubble dispersion and provide a satisfactory model to better describe its drainage behaviour. 2.4. Environmental Applications Microbubble dispersions have been shown to enhance the bioremediation of hydrocarbon contaminated soils (Jauregi & Varley, 1999; Wang & Mulligan, 2004a). It has been suggested that microbubble dispersion can: (1) improve the sweep efficiency and mobility-control of the injected surfactant-laden fluids in porous media; (2) deliver bacterial inoculants in soil matrices, thereby enhancing the biodegradation potential of the contaminated soils; (3) increase contaminant availability; and hence (4) accelerate in situ biodegradation of persistent organic contaminants. Selected studies are discussed in the following section. 2.4.1. Flow Characteristics of Microbubble Dispersion in Porous Media Experiences with surfactant solution application and air sparging during subsurface remediation operations showed that the injected fluids usually channel through a limited number of preferential pathways of the least resistance to flow, such as fractures and macropores that are commonly found in the heterogeneous subsurface environment (Hirasaki, 1998; Mamun et al., 2002; Wan et al., 2001). Channelling can divert the treatment fluids away from the remediation zone as the permeability in the contaminated zone can be expected to be low due to the trapped contaminants within the pore throats of soil grains, consequently leading to low sweep efficiency (i.e., the percentage of the total pore volume that is passed through by the injected fluids) across the contaminant-rich zone (Enzien et al., 1995; Hirasaki et al., 1997; Oliveira et al., 2004a). 24    Chapter 2 – Literature Review  Microbubble dispersion may eliminate channelling and so achieve better sweep efficiency in porous medium by increasing the fluid viscosity while reducing interfacial tension (Enzien et al., 1995; Hirasaki, 1998; Huang & Chang, 2000; Longe et al., 1995; Mamun et al., 2002; Mulligan & Eftekhari, 2003; Oliveira et al., 2004b; Sebba, 1987; Wang & Mulligan, 2004a). Additionally, the buoyant rising of the microbubbles during groundwater flow results in enhanced contact with regions of low permeability (Wan et al., 2001). Others (Park et al., 2009) reported that microbubbles penetrate into low permeablility region as an alternative passage route due to blockage in the high permeability region caused by pressure build-up. An effect of both bubble size and soil properties on microbubble transport has been reported. Wan et al. (2001) reported a decrease in the number of microbubbles remaining in the effluent from 100% to 30% when coarse sand particles were replaced by fine sand. The number of microbubbles recovered in the effluent was increased, however, with longer pumping duration when 1 pore volume injection was increased to 3 pore volume injection. The transport of large microbubbles was severely limited in fine soils because of straining and capture during the buoyant rise, a trend that has also been observed for colloid transport in groundwater (Bradford et al., 2002; Wan et al., 2001). 2.4.2. Bacteria Mobility Control A homogeneous distribution of a contaminant-degrading microbial population in the contaminated subsurface can greatly speed up bioremediation process, but as discussed previously in Section 1, it can be difficult to transport and disperse microorganisms within the contaminated soil. Microbubble dispersion can serve as an effective carrier for contaminant-degrading bacteria due to the dispersion’s improved sweep efficiency and large air-water interfacial area. Since bacteria and macromolecules tend to associate with air-water interface (Dahlbäck et al., 1980; Hermansson & Dahlback, 1983; Hermansson et al., 1982; Marshall, 1980; Powelson & Mills, 1996; Wan & Wilson, 1994; Wan et al., 1994), a larger interfacial area not only improves the attachment of bacteria to the dispersion, but also provides greater mass transfer of air/oxygen in the subsurface. High sweep efficiencies help to achieve a homogenous distribution of contaminant-degrading bacteria in the subsurface. While some studies showed that bacteria are retained by air bubbles trapped in the soil matrix (Schäfer et al., 1998b; Wan et al., 1994), the movement of bubbles can facilitate bacterial transport in a porous medium (Rothmel et al., 1998). The passage of small bubbles has been shown to be effective in detaching bacteria from surfaces (Gómez-Suarez et al., 2001; Pitt et 25    Chapter 2 – Literature Review  al., 1993; Sharma et al., 2005). Jackson et al. (1998) also reported that that the use of microbubble dispersion made from anionic sodium dodecylbenzene sulfonate (DDBS) as a bacterial carrier increased the total number of Pseudomonas pseudoalcaligenes bacteria being transported through a soil column by at least 2 or 3 orders of magnitude when compared to either a surfactant solution or water, respectively. The authors concluded that microbubble dispersion can transport bacteria more efficiently than conventional surfactant solutions or water (Jackson et al., 1998). Rothmel et al. (1998) conducted bacterial injection studies into sand columns using water, surfactant solution and microbubble dispersion respectively. The bacteria strain tested was ENV 435, an environmental isolate close to Pseudomonas cepacia. The researchers reported that microbubble dispersions made from Steol CS-330, an anionic surfactant, was the most effective carrier with 85% of the bacteria migrating through a soil column, while the use of surfactant solution and water reduced the migration to 61% and 28%, respectively. When using non-ionic Tergitol 15-S-12 under equal experimental conditions, the percentage of ENV 435 migrating through the column dropped to 59%, similar to the result (62%) using just the Tergitol solution. A second anionic surfactant, Biosoft D-40, gave migration of just 20% of ENV 435 through the column with microbubble dispersion, which was significantly lower than using the Biosoft D-40 solution (62%) (Rothmel et al., 1998).. Furthermore, it is reported that microbubbles are effective carrier for hydrophobic cells such as Actinomyces naeslundii over hydrophilic cells such as Streptococcus oralis when the microbubbles were passed through a parallel plate flow chamber where bacterial cells had been attached to (Sharma et al., 2005). These results suggest that the combination of surfactant and bacterial species can affect the effectiveness of microbubble dispersion as a bacterial carrier. 2.4.3. Contaminant Mobilisation The low bioavailability of contaminants trapped in porous soil media, including NAPLs, oily wastes, and sorbed organic pollutants is a primary reason for their slow degradation by microorganisms. Microbubble dispersion may enhance the contaminant bioavailability through processes of desorption or dissolution. This treatment method is also cost effective and has been reported to require fewer pore volumes compared to surfactant solutions to achieve similar amount of contaminant removal (Wang & Mulligan, 2004b). In bench-scale column studies comparing the use of microbubble dispersion with surfactant solution for mobilising and dispersing trichloroethylene (TCE), a dense nonaqueous-phase 26    Chapter 2 – Literature Review  liquid (DNAPL), the DNAPL pool placed near the bottom of the sand column was mobilised to the mid-section of the column by flushing with 4 pore volumes of microbubble dispersion of the anionic surfactant Steol CS-330, but the TCE pool treated with surfactant solution remained at the bottom (Rothmel et al., 1998). In another study, Roy et al. (1995a) compared the performance of microbubble dispersion of anionic surfactant sodium dodecylsulfate (SDS) with surfactant solution while flushing automatic transmission fluid, a light non-aqueous phase liquid (LNAPL), from soil columns. The microbubble dispersion was found to be 10% more effective than surfactant flushing when washing the LNAPL under the downflow condition. Similarly, in a different study involving the removal of oily waste, waste recovery using microbubble dispersion was 9% higher than that from surfactant solution (Roy et al., 1994). Microbubble dispersion has also been used to mobilise sorbed contaminants from soils. Huang and Chang (2000) investigated the efficiency of surfactant foam flushing in removing n-pentadecane from a contaminated glass-bead column. Their results showed that the foam treatment removed up to 3 times more pentadecane than surfactant flushing. There are, however, also studies in which the use of microbubble dispersion did not increase contaminant removal. Kommalapati et al. (1998) reported that the recovery of hexachlorobenzene from soil column using microbubble dispersion made with a natural surfactant Saponin was 20% lower than conventional solution flushing. Roy et al. (1995b) performed soil flushing experiments for naphthalene removal using Tergitol surfactant in the form of microbubble dispersion and conventional solution. For all the three different Tergitol concentrations tested, the conventional solution flushing had showed better removal efficiency (>15%) than using microbubble dispersion. Since both hexachlorobenzene and naphthalene are volatile, it is possible that a portion of the contaminant was lost in the gaseous phase, of which its contaminant concentration was not measured. 2.4.4. In situ Biodegradation Microbubble dispersion has the potential to improve homogenous distribution of bacteria and to mobilise contaminants to increase their bioavailability. In addition, it has been found that the dispersions can deliver oxygen or air to create aerobic environments efficient for biological degradation (Choi et al., 2009; Jenkins et al., 1993; Michelsen et al., 1983; Park et al., 2009; Ripley et al., 2002; Rothmel et al., 1998). The oxygen mass transfer is greatly enhanced by using microbubbles compared to oxygenated solutions (Bredwell & Worden, 27    Chapter 2 – Literature Review  1998; Dai et al., 2004; Jenkins et al., 1993; Wallis et al., 1986) due to the large gas-liquid interfacial area associated with the micron-sized bubbles (Dai et al., 2004). The above improved conditions may promote in situ biodegradation of contaminant. Biodegradation here refers to the transformation of a harmful substance into benign products, resulting in the removal of the harmful substance. In a pioneer study, Michelsen et al. (1983) examined the degradation of phenol in a saturated sand matrix by injecting microbubble dispersion formed from a mixture of surfactant, Pseudomonas putida bacteria, and nutrients. More than 60% of the phenol was biodegraded after 24 hours with a single application of the microbubble dispersion. Stabnikova et al. (1996) applied a biosurfactant foam mixture containing microorganisms to crude oil contaminated soil at 1-week intervals, and reported that 89% of the oil was degraded after 35 days of treatment, 43% higher than the untreated control. In a similar experiment with n-hexadecane as a model hydrocarbon, Ripley et al. (2002; 2000) observed 40% higher removal efficiency using an oxygenated bioactive foam treatment compared to the non-foam control. In another study, Rothmel et al. (1998) reported that pulsed injection of bacteria-enriched microbubble dispersion had resulted in 95–99% degradation of residual TCE in a soil column after a total of 3 pore volumes and an aqueous column retention time of 1 hour. More recently, the bench-scale study carried out by Park et al. (2009) demonstrated that saponin microbubble suspension speed up the biodegradation of phenanthrene in a sand/clay column due to the supply of oxygen by the microbubbles. In addition for being effective on delivering oxygen, Choi et al. (2009) reported that the saponin microbubble suspension was more efficient than the saponin solution in transporting Burkholderia cepacia through a sand column. 2.4.5. The Knowledge Gap Microbubble dispersion has in general demonstrated the potential to speed up in situ bioremediation. Figure 2-3 summarises the potential benefits offered by microbubble dispersion, as suggested by studies in the literature. The dispersion is capable of immobilising all three states of matter, the gaseous (air or oxygen), liquid (surfactant solution) and solid (bacteria and contaminant) within the dispersion (Ripley et al., 2002), which offers the potential that other systems, such as air sparging and surfactant solution injection, may be lacking towards enhancing bioremediation. 28    Chapter 2 – Literature Review  F Figure 2-3. Schematic S illu ustration of pootential beneefits offered by b microbubbble dispersion n. It has bbeen suggessted that miicrobubble dispersion increase baacteria transsport by promoting adhesioon of the conntaminant-d degrading b acteria to th he bubble in nterface andd that the dispersion enhancee contaminnant bioavaailability bby facilitatiing contact between the bacteeria and contaminants (Jackkson et al., 1998; Rothhmel et al., 1998). Therre is howevver little evid dence to verify thhese mechaanisms. Macrosscopic bacteerial transport with m microbubble dispersion using soil columns has h been studied with the aim a to show w that the ddispersion is an effectiive carrier ffor bacteriaa. While some oof the colum mn studies demonstratted improveed bacteriall transport using micrrobubble dispersiion (Choi et al., 200 09; Jacksonn et al., 1998; 1 Rothmel et al.,, 1998), th here are contraddictory resullts (Rothmeel et al., 19998). Conseequently, th he statemennt that micrrobubble dispersiion is an efffective carrrier is debaatable. Reasons for su uch inconsisstency are not n well understood. It is possible p that surfactannt type can n affect thee attachmennt of bacteria onto microbuubble dispersion and so o affect its effectiveness as a bacterial carrierr. Furthermore, it is reportedd that the attachment a of o bacteria to microbub bbles is afffected by baacterial celll surface 29    Chapter 2 – Literature Review  hydrophobicity (Sharma et al., 2005). Surfactants have been shown to adsorb to abiotic surfaces, changing their surface properties (Bai et al., 1997; Bridgett et al., 1992; Brown & Jaffé, 2001; Li & Logan, 1999; Noordman et al., 1998). Surfactants can similarly affect the surface properties of bacterial cells, thereby affecting the bacterial interaction with surrounding surfaces. However the combined effects of surfactant and bacterial cell surface properties on bacterial attachment to microbubble have not been studied. In order to predict whether microbubble dispersion is an effective carrier of bacteria, it is important to understand the interaction between bacteria and microbubble and factors affecting the interaction. The study may therefore provide a range of factors that contribute to make microbubble dispersion an effective bacterial carrier, and also provide insight into the mechanisms for enhanced bioremediation. Since many factors and processes can affect bacterial adhesion to bubble surfaces, a review of existing literature on bacterial adhesion to surfaces/interfaces is presented in the following sections. 2.5. Bacterial Adhesion to Surfaces/Interfaces Bacterial adhesion begins with the transport of bacterial cells towards a substratum surface by different types of mechanism which may include Brownian motion, fluid dynamic forces, or the intrinsic mobility of a bacterium (An & Friedman, 1998; Bos et al., 1999; Chen & Zhu, 2005; Marshall, 1986). Brownian motion can account for random contacts with surface by small bacteria that have an effective radius of less than 1 µm in quiescent conditions. Fluid dynamic forces can result from temperature gradient, gravitation, or mixing, which generate turbulent currents that provide the main means for transporting bacteria over a long-range (>150 nm) to a surface/interface. Motile bacteria would response to nutrient gradient present on a surface / interface by chemotaxis (Marshall, 1986). However, this type of movement becomes insignificant in turbulent flow conditions, which can be generated during intensive mixing at the making of the microbubble dispersion. Once the bacterial cells are in close proximity to a surface, bacterial adhesion may occur through a process involving two stages. The initial adhesion is an instantaneous attraction of bacteria to a surface and is a reversible interaction, where the bacteria can be removed from the surface by the shear force of water turbulence or Brownian motion. The reversible adhesion is governed by non-specific physicochemical interactions such as the van der Waals, electrostatic and hydrophobic interactions, which are dependent on the physic-chemical 30    Chapter 2 – Literature Review  properties of the interacting surfaces, the intervening liquid phase and the bacteria themselves (An & Friedman, 1998; Bai et al., 1997; Bayoudh et al., 2009; Chen & Zhu, 2005; Hamadi & Latrache, 2008; Marshall, 1986). In the second stage, the adhesion may become irreversible over time and the bacteria no longer exhibit Brownian motion and cannot be removed by a moderate shear force (An & Friedman, 1998; Bowen et al., 2001; Marshall, 1986). The irreversible adhesion involves specific-receptor-ligand bond interaction, which is the result of forming physical bonding of the bacterial cells to the surface via cell surface structures, including pili, fimbriae, fibrils, and extracellular polymeric substances (Bos et al., 1999; Lamba et al., 2000). As shown in the above discussion, bacterial adhesion involves complex mechanisms, and numerous studies have been undertaken to understand the governing factors. The initial stage of adhesion process is of major concern to this research. Some of the physicochemical interactions and surface properties that may affect the initial adhesion are presented in the following sections. 2.5.1. Bacterial Cell Surface Characteristics The bacterial cell surface is where the interactions between the bacteria and their surrounding environment take place. Thus the cell surface properties greatly affect the bacterial attachment to surfaces. Studies have shown that cell surface lipopolysaccharide, surface protein, surface charge and cell surface hydrophobicity are some of the cell characteristics that can significantly influence the process of bacterial adhesion (Dahlbäck et al., 1980; Dickinson, 2006; Hermansson et al., 1982; Prescott et al., 2005; Ubbink & Schär-Zammaretti, 2007). Bacteria are distinguished as either Gram-negative or Gram-positive, corresponding to the different bacterial cell wall architecture (Beveridge, 1981; Prescott et al., 2005). For both types of bacteria, it is essentially the biomolecules decorating the cell wall that determine the surface properties of the bacteria and thus their interaction with the surrounding environment (Ubbink & Schär-Zammaretti, 2007). A. Gram-negative Cells Gram-negative cells have an inner and outer membrane sandwiching a thin 1 to 2 nm layer of peptidoglycan in the periplasmic space, as illustrated in Figure 2–4a. Generally, the cell 31    Chapter 2 – Literature Review  enveloppe of Gram--negative baacteria is strructurally and a chemicaally more coomplex than n that of gram-poositive baccteria. The outer mem mbrane is covalently c bound to this peptid doglycan networkk through lipoprotein n. The outter membraane lipid bilayer b connsists primarily of phosphoolipid and protein p on the t inner layyer and lipo opolysacchaaride (LPS)) and protein n on the outer laayer. The LP PS moleculee extends innto the surro ounding meedium and th therefore it plays p an importaant role on bacterial b interaction w with surfacess (Al-Tahhaan et al., 20000; Hermansson et al., 19882). Fig gure 2-4. Bactterial cell env velope structu ure. B. Graam-positivee Cells Gram-ppositive cellls have a sin ngle plasmaa membran ne that is clo osely linkedd to a well-defined, rigid ouuter cell wall of 15 to 30 3 nm thickkness. The relatively r th hick cell waall is compo osed of a 32    Chapter 2 – Literature Review  dense and covalently stabilised network of peptidoglycan (see Figure 2-4b). Other polymers linked to the peptidoglycan network include teichoic acids and various sugar or amino-sugar residues. The surface proteins anchored to the peptidoglycan network are primarily responsible for cell adhesion to surfaces (Prescott et al., 2005). C. Surface Appendages A variety of surface appendages may extend beyond the cell surface into the surrounding environment. Flagella and fimbriae are commonly formed on the cells of bacteria. Flagella are responsible for mobility and for overcoming potential energy barriers by propelling the cells to the substratum (van Oss, 2006). The primary role of fimbriae, which are filaments of up to 100 nm long, is to facilitate specific binding of infectious bacteria, for example, to adhere to specific host tissue cells (Dickinson, 2006). D. Cell Surface Charge Bacteria are typically negatively charged in aqueous suspensions due to the dissociation of the carboxylic groups in the cell surface molecules (Marshall, 1980). The charge is found to be asymmetrically distributed and may increase with increasing pH because of the increased dissociation of the carboxylic groups (Bayer & Sloyer, 1990; Dickinson, 2006; Sonnenfeld et al., 1985). In addition, bacterial species, growth medium and cell surface structure can also affect surface charge (Merritt & An, 2000). Isoelectric point, zeta potential and electrophoretic mobility are commonly used to characterise the surface charge. A large number of studies have been devoted to understand the role of surface charge on bacterial adhesion, yet no consistent correlation between charge and adhesion has been established (Dickinson, 2006). For example, one study reported that enhanced charge negativity of Staphylococcus epidermidis correlated directly to increased adhesion to negatively charged substrata, but was inversely correlated for Escherichia coli (Gilbert et al., 1991). While the difference in bacterial species may be a factor in causing the inconsistency, Rijnaarts et al. (Rijnaarts et al., 1995b) demonstrated that isoelectric point is a better measure than electrophoretic mobility for predicting bacterial adhesion. They found that bacteria with an isoelectric point ≤ 2.8 adhered less to both glass and Teflon surfaces, and bacteria with an isoelectric point ≥ 3.2 adhered in large amounts onto Teflon surface and in slightly lower amounts onto glass surface. 33    Chapter 2 – Literature Review  E. Cell Surface Hydrophobicity Hydrophobicity and hydrophilicity are relative descriptions with reference to the interaction with the hydrogen bonds of water molecules. Hydrophilic molecules are able to dissolve in water because they form hydrogen bonds with water molecules. Typical hydrophilic molecules include proteins, carbohydrates and salts. In contrast, molecules that do not dissolve in water are said to be hydrophobic, but it does not mean that these molecules are pushed away from water; it simply means that they cannot form hydrogen bonds with water. Therefore the water molecules are more structured near a hydrophilic surface than when they are near a hydrophobic surface (An & Friedman, 1998). The cell surface hydrophobicity is said to relate to acid-base nature (i.e., electron accepting-electron donating characteristics) of the cell surface because of the hydrogen bonding with water molecules (Bellon-Fontaine et al., 1996; van Oss, 1995). Cell surface hydrophobicity measures the macroscopic cell property that constitutes contributions from various surface biomolecules including lipopolysaccharide and surface protein and surface appendages (Dickinson, 2006). Cell surface hydrophobicity is recognised as the dominant factor to explain non-specific interactions including the adhesion to nonbiological surfaces that are common in both environmental and engineering processes (Chen & Strevett, 2001; Dahlbäck et al., 1980; Rosenberg, 1984; Southam et al., 2001; van Loosdrecht et al., 1987; Wan et al., 1994). Studies have shown that surfactants can absorb onto bacterial cell walls and change cell surface hydrophobicity (Brown & Ai Nuaimi, 2005; Pijanowska et al., 2007; Yuan et al., 2007; Zhong et al., 2008). Adsorption studies of surfactants on a Pseudomonas aeruginosa strain revealed that the adsorption kinetics followed the second-order law and the cells after adsorption had enhanced hydrophobicity by showing increased adhesion to hydrophobic surfaces (Yuan et al., 2007). These researchers also showed that, using Microbial Adhesion to Hydrocarbon (MATH) assays, rhamnolipid surfactants increased cell hydrophobicity at low surfactant concentrations (Zhong et al., 2007). A study looking at the bacterial aggregation in wastewater sludge showed that addition of SDS or CTAB surfactants at low concentration facilitated bacterial aggregation, but at high concentration, the surfactants promoted deflocculation (Malik et al., 2005), implying surfactants have modified the cell surface properties. The adsorption of nonionic polyoxyethylene onto cell surface of a Sphingomonas sp. was shown to follow a multi-interaction isotherm, where the first 34    Chapter 2 – Literature Review  interaction represents monolayer adsorption showing plateaus near the surfactant’s CMC and the second interaction describes lateral interaction between sorbed surfactant molecules and the surface aggregates (Brown & Ai Nuaimi, 2005). By adopting surface free energy calculations, Brown and Jaffé (Brown & Jaffé, 2006) demonstrated that cell surface hydrophobicity of the Sphingomonas sp increased with the polyoxyethylene surfactants adsorption, and its magnitude of change was influenced by the surfactant chain length, with one surfactant type (C18E10) making the hydrophilic cells hydrophobic. 2.5.2. Physicochemical Characteristics of Surfaces/Interfaces A. Surface Charge The surface charge of a substratum surface also influences the bacterial adhesion process. As discussed previously, bacterial cell surfaces are generally negatively charged (Marshall, 1980). Electrostatic forces of attraction will act between particles if they carry charges of opposite sign. Conversely, electrostatic forces of repulsion will act between particles with the same charge. Since both bacterial cells as well as most natural and man-made surfaces are negatively charged at neutral pH in an aqueous environment, the electrostatic interactions are generally repulsive (Rijnaarts et al., 1995a). Studies have shown that initial adhesion of negatively charged bacterial cells is promoted on positively charged surfaces (Harkes et al., 1991; Pereni et al., 2006; van der Mei et al., 1992), due to the attractive electrostatic forces. Although these positively charged surfaces increase initial bacterial adhesion, it has also been reported that the positive surface charge impedes bacterial surface growth due to an extremely strong binding of the bacteria to the surface, that prevented elongation and subsequent division of the bacteria (Gottenbos et al., 1999). B. Hydrophilicity / Hydrophobicity Depending on the hydrophobicity of both bacterial and interacting surfaces/interfaces, the tendency for bacteria to adhere to the surfaces varies. Some researchers have shown that hydrophobic materials are more prone to bacterial adhesion for both hydrophobic and hydrophilic bacteria than hydrophilic materials because the hydrophobic interactions have been shown to predominate over other interactions (Abbasnezhad et al., 2008; An & Friedman, 1998; Bowen et al., 2001; Wan & Wilson, 1994; Wan et al., 1994). For example, van Loosdrecht et al (1990) examined the adhesion of a variety of bacteria with different cell surface properties to a hydrophilic glass surface and a hydrophobic polystyrene surface. It 35    Chapter 2 – Literature Review  was shown that hydrophobicity was the dominant force in determining adhesion, where adhesion typically decreases with decreasing hydrophobicity of either the bacterial surface or the interacting surface. However, some hydrophilic surfaces have also demonstrated the ability to promote the adhesion of hydrophilic bacteria (Lamba et al., 2000; Otto et al., 1999). C. Hyper-hydrophobicity of Air-water Interface The air side of the air-water interface is the most hydrophobic surface known (van Oss et al., 2005). Theoretical calculations have demonstrated that the air-water interface is about 30% more hydrophobic than surfaces such as octane or Teflon (van Oss et al., 2005). The hyperhydrophobicity of the air-water interface attracts macro-molecules or colloidal particles by hydrophobic attraction. In nature this manifests on the accumulation of various organic molecules, microbes, and particles at the water surface of lakes and oceans (Norkrabn, 1980; Parker & Hatcher, 1974). It has been known for a long time that bacteria tend to accumulate at the air-water interface (Dahlbäck et al., 1980; Hermansson & Dahlback, 1983; Hermansson et al., 1982; Marshall, 1980; Powelson & Mills, 1996; Wan & Wilson, 1994). Ripley et al. (2002) observed that bacterial cells preferentially sorb onto gas bubbles, and that hydrophobic cells demonstrate a stronger affinity than hydrophilic cells due to stronger hydrophobic attraction. Similarly in column experiments by Wan et al. (1994), at least 52 % of the total cells injected to a soil column was sorbed onto the air-water interface and the proportion of sorbed bacteria was shown to increase with hydrophobic cells and larger air-water interface. Furthermore, bacterial sorption was found to be irreversible for both hydrophilic and hydrophobic strains because of capillary forces resulting from surface tension of the bubble interface (Wan et al., 1994). 2.5.3. Environmental Factors Various environmental factors such as electrolyte concentration, solution pH and the presence of surface active compounds affect bacterial adhesion by modifying surface properties of both the bacteria and the interacting surface or by altering the strength of attractive/repulsive physiochemical interactions involved in initial adhesion. 36    Chapter 2 – Literature Review  A. Electrolyte Concentration Concentrations of electrolytes (e.g., KCl, NaCl) in the aqueous phase can influence electrostatic interaction. In low electrolyte conditions, a large energy barrier due to the repulsive electrostatic forces prevents bacterial adhesion to surface (Rijnaarts et al., 1995a). In conditions of increasing ionic strength in an aqueous medium, the repulsive energy decreases while at the same time van der Waal’s attraction predominates, thus facilitating bacterial adhesion (Abbott et al., 1983; An & Friedman, 1998; Marshall, 1986; Morisaki & Tabuchi, 2009; Otto et al., 1999). However, others have reported no apparent correlation between bacterial adhesion and electrolyte concentration (Camesano & Logan, 2000; McEldowney & Fletcher, 1986). B. pH The influence of pH on bacterial adhesion varies with bacterial species, types of substratum and ionic strength (McEldowney & Fletcher, 1986; McWhirter et al., 2002). For example, investigation of the effects of pH using atomic force microscopy showed that the repulsive force between bacterial cells and a silicon nitride surface increased with pH (Camesano & Logan, 2000). In contrast, it was reported that bacteria-metal adhesion forces were maximal when pH was close to the isoelectric point of the bacterial cells, and with increasing pH (from 3 to 7) the adhesion force decreases (Sheng et al., 2008). However, the adhesion of bacterial cells to glass surface has been shown to be independent of the changes in pH at a low ionic strength of 0.05 mol/L (Abbott et al., 1983). C. The Presence of Surface-Active Compounds Several studies have shown that adsorption of surface-active compounds (e.g., surfactant and protein) from aqueous media to abiotic surfaces and bacterial cells changes the surface properties (Bai et al., 1997; Bridgett et al., 1992; Brown & Jaffé, 2001; Li & Logan, 1999; Noordman et al., 1998), which can influence microbial adhesion. For example, the addition of surface-active chemicals such as Pluronic F68 and serum reduced the adhesion of cultured insect cells to gas bubbles (Wu et al., 1997) and it was suggested that the surfactant reduces the bubble surface hydrophobicity by lowering its surface tension. Ducker et al. (1994) has shown using atomic force measurement that the attractive hydrophobic force between a colloidal particle and an air-water interface diminished when surfactant SDS was added to the aqueous medium. In another study, coating substrata surfaces with proteins was found to 37    Chapter 2 – Literature Review  decrease the surface hydrophobicity, leading to an inhibition of bacterial adhesion to the surfaces (Fletcher & Marshall, 1982). 2.6. Theoretical Models for Bacterial Adhesion The process of bacterial adhesion is generally explained by DLVO theory (named after its proposers Derjaguin, Landau, Verwey, and Overbeek) and by the surface thermodynamic approach (An & Friedman, 1998; Bos et al., 1999; Otto et al., 1999). The DLVO theory was originally developed to describe the behaviour of colloids in the presence of electrolytes (Derjaguin & Landau, 1941; Verwey & Overbeek, 1948). It describes bacterial adhesion as a model of physiochemical interactions between rigid and spherical particles of the same charge (Hermansson, 1999; Marshall et al., 1971). According to this theory, adhesion depends on the separation distance of the interacting surfaces and the balance between attractive van der Waals and generally repulsive electrostatic forces. At a long distance (> 10 nm), the attractive van der Waals forces dominate over repulsive electrostatic forces, bringing the bacterial cell close to a surface until a secondary minimum of energy is reached. As the separation distance decreases, repulsive electrostatic forces develop an energy barrier that impedes a closer contact between the bacteria and the surface. If the bacteria can overcome this barrier of energy and reach a primary minimum of energy, they will be strongly attached to the surface as predicted by the theory. An extended DLVO approach was developed to include effects of hydrophobic interaction occurring during the adhesion process (Camesano & Logan, 2000; Hermansson, 1999; Otto et al., 1999). However, it has been reported that these DLVO models fail to accurately explain experimental results of bacterial adhesion studies, which can be due to the assumptions made in the model that spherical and uniformly charged colloidal particles are used to represent bacteria, which are in reality living entities with heterogeneous cell walls and possessing a much lower surface charge than that ascribed to them (Azeredo et al., 1999; Bos et al., 1999; Otto et al., 1999; Sjollema et al., 1990; Vigeant & Ford, 1997). In the surface thermodynamic approach, the interactions involved in bacterial adhesion in aqueous media are predicted as a function of interfacial free energies of the cell and the interacting surface as well as the medium (Bos et al., 1999; Otto et al., 1999). The balance between these interfacial free energies of the system is the free energy of adhesion. The free energy balance is important to predict from a physicochemical perspective whether or not 38    Chapter 2 – Literature Review  microbial adhesion will occur. Like all systems in nature, the system of bacterial cells and surfaces will have a tendency to obtain a state of minimal free energy so that the overall system is stable. Therefore adhesion is facilitated if the free energy of adhesion is decreased. This approach has proven merits for describing bacterial adhesion in aqueous medium (Bos et al., 1999; Busscher et al., 1984; Chen & Strevett, 2001; Grasso et al., 1996; Neu & Marshall, 1990). However, some have reported that the theoretical predictions made by the thermodynamic model are not always in accordance with experimental results, which may be due to the presence of local attractive electrostatic interactions, or the difficulty of determining accurate values of the parameters of the three components involved in the adhesion process (Bellon-Fontaine et al., 1990; Hermansson, 1999; Pratt-Terpstra et al., 1988). Neither of these models perfectly accounts for cell adhesion experimental results, and research continues to improve the models for prediction of microbial adhesion. Nevertheless the thermodynamic approach is considered to have greater merit than the DLVO theory due to the following reasons:  It has been shown that bacterial cells have much lower surface charges than that ascribed to them as smooth colloidal particles in the DLVO theory (Morisaki & Tabuchi, 2009).  Unlike an inert particle, the bacterial surface is heterogeneous comprises surface molecules and structures extending out into the aqueous medium. Some cell surface structures (e.g., appendages) can extend beyond the interaction potential minima predicted by DLVO theory (Hermansson, 1999). These cell surface constituents are important factor for bacterial adhesion, but have not been accounted for in the DLVO theory. The differences in cell wall constitution are reflected in the cell surface thermodynamic parameters (Ryoo & Choi, 1999). In fact, changes in cell surface thermodynamics with changes in cell surface constituents as a result of physiological state and environmental conditions have been reported (Chen & Strevett, 2003; Grasso et al., 1996; Stenstrom, 1989).  At very close separation distances (often taken to be 0.157 nm), which represents the distance at which adhesion or physical contact occurs, electrostatic interaction can be neglected due to the induced charge balance caused by overlapping double layers 39    Chapter 2 – Literature Review  (Chen & Strevett, 2001; Israelachvili, 1992). Since electrostatic interaction can be neglected at such scales, thermodynamic approach is more appropriate for describing the adhesion of bacteria to a surface (Liu et al., 2007; Sharma & Hanumantha Rao, 2002). Consequently the surface thermodynamic approach will be applied in this study. The following section describes the thermodynamic approach in greater detail. 2.7. Surface Thermodynamic Approach to Bacterial Adhesion 2.7.1. Two Schools of Thought The surface thermodynamic approach to bacterial adhesion is generally based on the surface free energies of the bacterial cell, the interacting surface and the aqueous phase, as briefly discussed in previous section. The determination of surface free energies is commonly based on the input of contact angle (see Figure 2-5) of pure liquids on the solid surface in concern according to the Young’s equation (Bos et al., 1999): [1] where is the known surface tension of the liquid, θ is the contact angle, free energy of the solid and is the surface is the solid/liquid interfacial energy. When contact angles on microbial lawns are measured, the subscript s is replaced by m. The equation cannot be solved for the two unknowns and without additional assumptions, which have lead to the development of many theoretical and semi-empirical approaches in the literature trying to evaluate these unknowns (Bellon-Fontaine et al., 1990). After two centuries of scientific work, although some other approaches are still developing, two major theories have come into existence to tackle this problem (Bellon-Fontaine et al., 1990; Della Volpe et al., 2004; Sharma & Hanumantha Rao, 2002). These two approaches are (i) the surface tension component or Lifshitz-van der Waals/acid-base theory (LW-AB), proposed by Fowkes (1987) and further developed by van Oss, Chaudhury and Good (van Oss, 1993), and (ii) the equation of state approach proposed by Neumann, Kwok, and others (Neumann & Spelt, 1996). Both approaches share similar basic assumptions, i.e., validity and applicability of Young’s equation and constancy of the interfacial tensions during contact angle experiments. Criticisms on these two theories have been raised by various workers, and clarifications have also been provided by the proponent authors (Della Volpe et al., 2004). 40    Chapter 2 – Literature Review  For the LW-AB theory, for example, a large number of criticisms have been coming from the supporters of the equation of state theory, such as Kwok (1999; 1998). In his several papers, Kwok (1999; 1998) performed a critical review on the LW-AB approach and concluded that the theory was flawed. For example, one of Kwok’s major criticisms is that the LW-AB theory is impossible to calculate in a coherent and precise way the components of the solids, or the interfacial energies of the liquids, and in the same paper he reported that the LW-AB approach yielded inconsistent solid surface tensions parameters for fluorocarbon, polystyrene, and poly(methyl methacrylate) solid surfaces (Kwok, 1999). However more recently, Della Volpe et al. (2004) provided an extensive analysis, comparison and discussion on the two approaches and proved that the results of the surface tension parameters obtained from the LW-AB and the equation of state approaches are consistent if the LW-AB theory is correctly applied, using wide and well-obtained sets of the reference materials. The LW-AB approach is used in this study because it has been successfully applied by many researchers and there is a large amount of published information on this approach. It is also important to appreciate that the LW-AB theory is a semi-empirical approximation to a twocentury old problem, and has its limitations. The theory assumes that:  The Young’s equation is valid for solving surface free energies;  Contact angle equilibrium is attained;  The bacterial cells are rigid entities from where their surface tension parameters can be measured. This is a common assumption used in modelling theory involving bacteria such as the DLVO theory, where bacterial cells are assumed to perform like rigid colloidal particles; and  The surface tension data of the diagnostic solvents are accurate. In addition, the successful application of the LW-AB approach depends greatly on the correct choice of the solvents (i.e., the reference materials) used for the contact angle measurement. The selection of solvents is discussed in Section 2.7.2. 2.7.2. The LW-AB Theory Based on the LW-AB theory (van Oss, 2006), the surface tension  is seen as the sum of a Lifshitz-van der Waals apolar component  LW and a Lewis acid-base polar component  41    AB : Chapter 2 – Literature Review     LW   AB [2] The LW component accounts for the generally attractive LW interaction arising from the electrodynamic energy between neutral surfaces. The AB component is manifested in either attractive (“hydrophobic”) or repulsive (“hydration pressure”) interaction. It originates from the electron-acceptor-electron-donor interactions between polar moieties in aqueous medium such as water. The acid-base polar component can be further divided into an electron donor   and an electron acceptor   subcomponent:  AB  2     [3] The solid/liquid interfacial energy is the given by:   sl   s   l  2  sLW  lLW   s l   s l  [4] where subscript s denotes solid and l denotes liquid. Combining Equation [4] with the Young’s equation [1], a relation between the measured contact angle and the solid and liquid surface free energy components can be obtained:  (1  cos  ) l  2  sLW  lLW   s  l   s  l  [5] The contact angle measurement method is discussed in detail in Section 3.7.2. Contact angle describes the shape of a liquid droplet placed on a solid surface. As illustrated in Figure 2-5, the contact angle is the angle between a tangent line at the edge of droplet and the solid surface. 42    Chapter 2 – Literature Review  Figure 2-5. A schem matic of contaact angle of a liquid dropleet on a solid ssurface. In orderr to determine the threee surface fr free energy components (  LW ,   and   ) off a solid, three eqquations arre required.. Thereforee contact an ngle measu urements ussing three different d diagnosstic liquids (i.e. ( a triplet liquid) witth known su urface tension componnents are req quired. Studies have show wn using onee of the folllowing tripllet liquid co ombination will yield the t most accuratee solutions to the equaations (Dellla Volpe ett al., 2004; van der Meei et al., 19 998; van Oss, 20006). They are: a  Water – Foormamide – Methyleneiiodide  Water – Foormamide – 1-Bromonaaphthalene  Water – Glycerol – Meethyleneioddide  Bromonaphhthalene Water – Glycerol – 1-B  oxide – Metthyleneiodid de Water – Dimethylsulfo  methylsulfo oxide – 1-Brromonaphth halene Water– Dim a 1998; vvan Oss, 20 006) are The surrface tensioon data of these liquiids (van deer Mei et al., summarrised in Tabble 2-4 below. Tablle 2-4. Surfacce tension datta of five com mmonly used diagnostic d liqquids. Diagnoostic liquid Water Dimethhylsulfoxidee Formam mide Glycerool 1-Brom monaphthaleene   LW  AB   72 2.8 50 0.8 58 8.0 64 6 44 4.4 21.8 27.15 39.0 34 44.4 51.0 0 19.0 30 0 255.5 0 399.6 577.4 0 25.5 0.01 2.3 3.92 0 43    Chapter 2 – Literature Review  2.7.3. Surface Free Energy Calculation Based on LW-AB Approach The free energy of adhesion (∆ between a bacterial cell (b) and a surface (s) immersed in a liquid medium (l) is the balance of the interfacial free energies between the interacting surfaces and is expressed as ∆ in which [6] , , and are the surface-bacterium, surface-liquid, and bacterium-liquid interfacial free energies, respectively. By incorporating the LW-AB approach (Equation 4) with Equation 6, the free energy of adhesion can be expressed in LW-AB surface tension components according to    LW  LW   LW  LW   LW  LW   LW b l s l b s l   Gadh  2                b   s   l   l  b   s   l   b  s   b  s  l  [7]     In theory, the adhesion is energetically favourable if ∆ adhesion is energetically unfavourable when ∆ 0 , while is negative (∆ is positive (∆ 0 . Equation 7 can be modified to calculate bacterial cell surface hydrophobicity, which is expressed as the free energy of aggregation of bacterial cells immersed in aqueous solution (Bellon-Fontaine et al., 1996; van der Mei et al., 1998; van Oss, 1995). For bacteria (b) immersed in water (w), the free energy of aggregation (∆Gbwb) can be calculated according to:  Gbwb  2(  bLW   wLW ) 2  4  b  b   w  w   b  w   b  w  [8] If the interaction between two bacterial cells is stronger than the interaction between the cell with water, by definition, the cells are considered to be hydrophobic since ∆Gbwb<0. In other words, it is energetically favourable to form aggregate in the aqueous solution for hydrophobic cells. Conversely, for hydrophilic cells, due to their preference for aqueous phase, would have ∆Gbwb > 0, indicating that it is more energetically favourable for the cells to stay dispersed in water.   44    Chapter 3 – Materials and Experimental Methods  Chapter 3. Materials and Experimental Methods 3.1. Materials 3.1.1. Surfactants One biosurfactant and two chemical surfactants were used in experiments. The biosurfactant under investigation was rhamnolipid. It is produced by Pseudomonas aeruginosa and is commercially available from Jeneil Biosurfactant Company (Saukville, WI, USA). The Jeneil product JBR 425 is a mixture of mono-rhamnolipid (R1; α-L-rhamnopyranosyl-βhydroxydecanoyl-β-hydroxydecanoate) and dirhamnolipid (R2; 2-o-α-L-rhamnopyranosylα-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate), supplied as a 25% aqueous solution. The two synthetic surfactants used were Tergitol® 15-S-12, a secondary alcohol ethoxylate surfactant which is non-ionic in nature, and sodium dodecyl sulfate (SDS) which is anionic surfactant. Table 3–1 provides a summary of the surfactant properties and supplier information. Table 3-1. Surfactant properties and supplier information. References for rhamnolipid properties include (Clifford et al., 2007; Zhang & Miller, 1994). Reference for tergitol properties is (UnionCarbide, 1996) . Reference for SDS properties is (Sigma-Aldrich, 2009). Surfactant Molecular Weight (g/mol) CMC1 Chemical Charge (mg/L) Formula C26H48O9 (R1) 504 - 650 C32H58O13 (R2) Tergitol 15- NonC11-15H23-31-O738 104 S-12 ionic (CH2CH2O)12H CH3(CH2)11SDS Anionic 2304 288 OSO3Na Note: 1. Critical micelle concentration in water at 25 oC. Rhamnolipid Anionic 57.7 Source Purity Jeneil Biosurfactant Co. 25% solution 100% liquid >98.5% powder Sigma – Aldrich Sigma – Aldrich 3.1.2. Bacterial Culture Media Bushnell Hass (BH) broth (Sigma) is used for culturing Pseudomonas putida with D-glucose as the added carbon source (BDH AnalaR®). The BH broth contains potassium nitrate as a nitrogen source, while monopotassium phosphate and diammonium hydrogen phosphate provide buffering capacity. It also provides trace elements such as magnesium, calcium and iron necessary for bacterial growth. BH agar in BH broth with D-glucose solidified with 1.5% 45    Chapter 3 – Materials and Experimental Methods  w/v BD DifcoTM agar is used to grow Rhodococcus erythropolis. Nutrient agar (Fort Richard, NZ) is a general purpose growth medium used for the maintenance of bacterial cultures. Tryptone Soy Broth (TSB; BD DifcoTM) is a general purpose culture medium used for overnight growth of seed cultures. 3.1.3. Other Chemicals A list of chemicals that have been used in the experiments is shown in Table 3–2 below. Table 3-2. A list of chemicals used in the study. Chemical 1-Bromonaphthalene Acridine orange Dipotassium phosphate Formamide Hydrochloric acid Magnesium sulphate heptahydrate n-Hexadecane Nile red Potassium dihydrogen phosphate Sodium chloride Sodium hydroxide Urea Source Sigma Sigma-Aldrich Sigma Merck LabServ Sigma Purpose Diagnostic liquid for contact angle measurement Fluorescent dye for bacterial cells Buffer for hydrophobicity assay Diagnostic liquid for contact angle measurement Adjusting pH Buffer for hydrophobicity assay Sigma Sigma Sigma Hydrophobicity assay Fluorescent dye for hydrocarbon contaminant Buffer for hydrophobicity assay Scharlau LabServ Scharlau Adjusting electrolyte concentration Adjusting pH Buffer for hydrophobicity assay 3.2. Generation of Microbubble Dispersion Microbubble dispersion was made following the method described by Sebba (1985). Surfactant solution was mixed intensively with a spinning disk connected through a shaft to an electric motor in a 2L baffled container. The container was filled with one litre of surfactant solution for all tests. The electric motor has both running time and mixing speed controls. Two setups were tested in order to obtain a generator configuration that provides improved stability for the microbubble dispersion. Setup number one uses 2 baffles and a flat disk. Setup number two uses 4 baffles and a propeller aiming to intensify the mixing. Figure 3–1 illustrates the two different setups used. 46    Chapter 3 – Materials and Experimental Metthods  Figure 3-1.. Setups used d for making microbubble dispersion. In addiition to thhe generato or configuraation, mixiing speed and duratiion and su urfactant concenttration weree also testeed to identiify suitable ranges for these proccess parameeters for further testing. Thhe preliminaary test resuults identifi fied the mix xing speed and duratio on to be used inn further tessting were 8,000 8 rpm and 3 minu utes, respecctively. Theese conditio ons were determiined based on o the stability and air quality prop perties of th he microbubbble disperssion (see Sectionn 4.2). Unleess stated otherwise, o aall microbu ubble dispeersions weree made with these conditioons at room m temperatu ure (20 oC ± 5 oC) an nd atmospheeric pressurre using settup 1 to ensure constant poower input into the syystem. The surfactant solution cooncentration ns tested a 4,000 m mg/L, respecctively. Varriations in soolution pH,, sodium were 5000 mg/L, 1,000 mg/L and chloridee and bacterrial concenttration are inndicated wh here approp priate. 3.3. Ch haracteriisation Stu udies for Microbub bble Disp persion 3.3.1. V Viscosity Measuremen M nt Solutionn viscosity affects foaam stability (Germick et al., 1994 4). The visccosity of su urfactant solutionn for all expperimental conditions ttested was measured using u a Rheeometer (PH HYSICA UDS 2000, Paar Phyysica) at a constant c tem mperature off 25 oC. 47    Chapter 3 – Materials and Experimental Metthods  3.3.2. S Stability (H Half-life) Meeasuremen nt As disccussed in thhe literature review secction (Sectio on 2.3.3), various v proccess parameeters can affect stability of o microbu ubble dispeersion. Foaam stability is usuaally inferreed from experim mental meassurements of o liquid ddrainage and d bubble collapse. Seeveral experrimental techniquues such ass static drainage, surfaace decay, half-life h of foam f and hhalf-life of drainage d have beeen employyed for the investigatioon of conventional foaam stabilityy. For micrrobubble dispersiion, since no n perceptib ble breakdoown of bub bbles takes place priorr to the majjority of liquid bbeing draineed from the dispersion,, its stabilitty is measurred in termss of liquid drainage d (Yan ett al., 2005). The most commonly c used experiimental metthod in the literature measures m the halff-life (T1/2) of liquid drrainage as thhe time tak ken for half of the liquiid to drain from f the dispersiion (Jauregii et al., 199 97; Sebba, 11985). To determine d th he half-life, 100 mL meeasuring cylinderrs were fillled with freeshly made microbubb ble dispersio on to monittor liquid drainage. d Volumee of the draiined liquid (VL) was reecorded with h time. The final volum me of draineed liquid (Vmax) w was recordeed after thee foam had completely y collapsed. Each test was repliccated six times. T The processs, together with w the vaarious param meters recorrded duringg the experiiment, is illustratted in Figurre 3-2. Figure 3-2. Illu ustration of the t measurem ment of liquid d drainage fro om microbubbble dispersio on. 48    Chapter 3 – Materials and Experimental Metthods  The liquuid drainagge volume (VL) was plootted againsst time and the time coorresponding g to half 1 of the ffinal drained liquid (i.ee., Vmax ) w was identifiied from thee graph. Figgure 3–3 illustrates 2 this metthod. Figure 3-3. Procedurre used to determine half-llife value. In addiition to the liquid draainage profiile, monitorring changees within thhe dispersion/foam phase aallows for a better undeerstanding oof drainage mechanism m associatedd with micrrobubble dispersiion. The voolume of the dispersionn/foam phaase (VF) wass recorded w with time from f the 100 mL L cylinder as a shown in n Figure 3-22. Foam vollume fractio on is quantiified as the ratio of the foam m volume (VF) to total volume (VF + VL), as in Equation [9]. Foam vvolume fracttion = VF VF  VL [9] Liquid hold-up meeasures the volume fraaction of liquid retaineed within th the dispersion/foam phase aand the aveerage liquid d holdup vaalue can bee approximaated as the ratio of th he liquid volumee in the disppersion/foam m phase (Vmmax – VL) to the final drrained liquidd volume (Vmax), as in Equaation [10]. Liquid hhold-up = Vmax  VL Vmax [10] 49    Chapter 3 – Materials and Experimental Methods  3.3.3. Gas Hold-up Measurement The gas hold-up measures the amount of gas entrained in the microbubble dispersion. Unlike foam volume fraction, which evolves with time as bubbles collapse, the gas hold-up captures the initial amount of gas contained within the dispersion when it is made. It is defined as the ratio of the gas volume (Vg) to the initial dispersion volume, which is 100 mL in this case. The gas volume is equivalent to the difference between the dispersion volume (100 mL) and the final liquid volume (Vmax) after bubble collapse as shown in Figure 3-2, and the gas holdup can be expressed as: Gas hold-up = Vg 100 = 100  Vmax 100 [11] 3.3.4. Size Distribution Measurement Two commonly used experimental techniques were used in this study to measure bubble size distribution. One approach used particle size analyser; another used an image processing technique. The particle size analyser technique is relatively straight forward when compared to image processing method. Size distribution measurement was performed by dynamic light scattering using a Malvern Hydro 2000 SM particle size analyser (Malvern Instruments, UK). Freshly prepared microbubble dispersion (10 mL) was added to the sample dispersion unit attached to the analyser. The analysis was performed twice for each experimental condition. For the image processing technique, photomicrographs of microbubbles were taken following methods described in Section 3.3.5. The original image was difficult to work with using the MATLAB image processing tool box because the bubbles shared the same colour with the background and the built-in commands failed to distinguish the two apart. Photoshop pretreatment of the photomicrographs allowed the images to be loaded to MATLAB for analysis. A representative example is shown in Figure 3-4. The original image was in grey scale with the bubbles and the background having the same colour (Figure 3-4 a). The image was treated with Photoshop using the sharpen tool and the background colour was converted from grey to black using the magic wand tool (Figure 3–4 b). 50    Chapter 3 – Materials and Experimental Metthods  Figurre 3-4. Picturres illustratin ng the image p processing prrocedure. Piccture a is the original grey y scale microph hotograph. Piicture b is a grey g scale imaage with a black backgrou und after worrking with Ph hotoshop softw ware. Picture c is a black and a white bin nary image th hat allows thee labelling of aan object bassed on connectivvity of the wh hite pixels. Piccture d illustrrates the labeelled bubbles in colour. The pree-treated im mage was loaaded into M MATLAB an nd converteed from grey eyscale to bllack and white im mage (Figuure 3–4c). This T step coonverts the intensity im mage (the ggreyscale) to o binary image ((the black and a white), and allowss the differeentiation off an object ((white pixels) from the bacckground (black ( pixeels) using MATLAB command ds. Each buubble, whiich was identifieed as a grooup of conn nected whitee pixels, waas labelled as a uniquee object. Th he black and whhite image was then ‘coloured’ to allow visualisation v n of the laabelled objjects, as illustratted in Figurre 3–4d. Th he area of eaach object (i.e., ( bubblee) was counnted in term ms of the numberr of pixels and was converted c tto metric units u using the scale obtained from fr the microsccopy camerra. Finally, the diametter for each h bubble waas calculateed from thee bubble area. Foor the non--spherical bubbles, b theeir diameteer was estim mated as 2 times the average distancee between the t polygon n centre andd each side angle. App pendix I shoows the com mmands used in MATLAB for image processing p pprocedures. 3.3.5. V Visualisatioon of Micro obubble Evvolution Photom micrograph images off microbubbbles throug gh time prrovide visuual supportt to the formulaation of draainage proccesses in thhe dispersio on. A smalll sample oof freshly prepared p microbuubbles was placed in a cavity glaass slide un nder a light microscopee (Nikon ECLIPSE E600) w with 4 × maagnification n. A digital camera (Niikon Digitall Sight DS-U U1) attacheed to the microsccope was used u to ob btain photoographs imm mediately after the m microbubblees were produceed and at 3 minutes m inteervals up too 30 minutess. 51    Chapter 3 – Materials and Experimental Methods  3.4. Bacteria Culture and Preparation 3.4.1. Gram-Staining Gram-staining is the most widely employed staining method in microbiology to differentiate bacteria into two classes: Gram-negative and Gram-positive. It is used in this study to check for any contamination in bacteria culture. The Gram-staining was performed following the procedure by Prescott et al. (2005) A drop of bacterial culture (20 µl) was heat-fixed on glass microscope slide to form a smear. In the first step of the Gram-staining procedure, the smear was stained with crystal violet dye (0.4% w/v) for 30 seconds and then rinsed with water for 2 seconds. It was then treated with an iodine solution (1% w/v potassium iodide) for 1 minute. The iodine solution functions as a mordant, which increases the interaction between the cell and the dye so that the cell is stained more strongly. After rinsing with water, the smear was decolourised by washing with acetone for 20 seconds and then rinsed with water. This step generates the differential aspect of the Gram stain; Gram–positive bacteria retain the crystal violet, whereas Gram-negative bacteria lose their crystal violet and become colourless. Finally, the smear was counterstained with safranin (0.5% w/v) for 30 to 60 seconds. This step colours Gram-negative bacteria pink to red and leaves Gram-positive bacteria dark purple. After rinsing with water and drying, the slide was ready for examination under oil immersion light microscope with 100× objective lens. Gram-negative bacteria will be pink or red while Gram-positive bacteria will be dark purple in colour. Microscopic observation also allows differentiation on shape from bacilli (rod-shaped) to cocci (spherical). 3.4.2. Bacteria Strains Two bacterial strains isolated from petroleum contaminated soils were used during early stage of the study. The two strains were identified as the Gram-positive rod Pseudomonas putida named DS4 and a Nostocoida species (a bacterium closely related to Rhodococcus species) named DS3. They were given as a kind gift of Associate Professor Gillian Lewis of the School of Biological Science at the University of Auckland. Pseudomonas species and Rhodococcus species are often isolated from hydrocarbon-contaminated sites and hydrocarbon-degrading cultures (van Hamme & Ward, 2001). Due to problems such as slime formation and slow growth encountered when growing the two wild strains during preliminary experimental stages, the two strains were abandoned and replaced with two well 52    Chapter 3 – Materials and Experimental Methods  characterised laboratory strains Pseudomonas putida 852 (CDC KC1074) and Rhodococcus erythropolis 3586 (DSM 43066) from New Zealand Reference Culture Collection. Experiments involving these two strains of bacteria were performed either in a physical containment level 1 (PC1) or a level 2 (PC2) laboratory in compliance with Ministry of Agriculture and Forestry requirements. Seed stocks of the bacterial strains were stored at -80oC. Seed stocks for storage were prepared by mixing 1 volume of an overnight culture of the bacterium grown in TSB with 1 volume of autoclave sterilised 50% volume/volume glycerol in water. 3.4.3. Growth of Pseudomonas putida 852 for Microbubble Dispersion Experiments Pseudomonas putida (P.putida) are Gram-negative rod-shaped bacteria. They are aerobic bacteria commonly found in moist environments, such as soil and water. They are motile in aqueous media and grow optimally at a temperature of 25-30 oC. The bacteria have the ability to grow on and break down many petroleum pollutants including aromatic hydrocarbons (Otenio et al., 2005). P.putida 852 is used as model bacteria in this study to represent Gramnegative bioremediation organisms of the Pseudomonas family and other species such as the Proteobacteria (Riser-Roberts, 1998). To prepare a working culture, a loopful of the -80oC seed stock was taken from the vial, plated onto nutrient agar, and incubated at 30 oC for 24 hours, after which visible colonies were formed. The agar plate was subsequently stored in a refrigerator in a PC2 laboratory. The plate was kept up to 3 weeks; fresh plates were streaked using a single colony from the old plate. Colony morphology was used to verify that the plate contained only one colony type and to spot contaminant colonies. Table 3–3 summarises the morphological properties of the bacteria colonies and cells. To prepare bacterial culture for experiments, a colony of P.putida 852 was taken from the agar plate using an inoculating loop and placed in 10 mL of aqueous Bushnell Hass (BH) broth with 2% glucose as the sole carbon source in a screw-capped 50 mL polypropylene tube. The bacteria were grown in a shaker incubator (Innova 4330, New Brunswick Scientific) at 200 rpm and 30 oC overnight, after which 1 mL of the broth culture was transferred to 100 mL of fresh BH broth with 2% glucose in a 500 mL Erlenmeyer flask. The culture was grown in a shaker incubator at 200 rpm and 30 oC overnight until the stationary phase was reached. The stationary phase of P.putida 852 was previously determined from growth curve 53    Chapter 3 – Materials and Experimental Methods  experiment (see Appendix II for growth curve). The cells were harvested by centrifugation at10,000×g for 10 minutes (Sorval RC6 Centrifuge, F14S-6×250y rotor). The supernatant was decanted leaving the pellet for experiment. Table 3-3. Comparison of properties of Pseudomonas putida 852 and Rhodococcus erythropolis 3586. Gram-staining Cell Morphology Cell Size Colony Morphology Pseudomonas putida 852 Gram-negative Rods, cells shown in single or pairs 1×2 μm Greenish yellow, slimy and smooth looking, with characteristic smell Rhodococcus erythropolis 3586 Gram-positive Cocci, cells shown in pairs or clumps 0.5 μm Creamy white, waxy, rough 3.4.4. Growth of Rhodococcus erythropolis 3586 for Microbubble Dispersion Experiments Rhodococcus erythropolis (R.erythropolis) are Gram-positive aerobic bacteria that are capable of degrading a wide range of hydrocarbon pollutants (Aoshima et al., 2006; van Hamme & Ward, 2001). R.erythropolis 3586 is used as model bacteria to represent Grampositive bioremediation organisms such as Actinobacteria and Mycobacteria (Hamamura et al., 2006; Quatrini et al., 2008). The stock agar plate culture of R.erythropolis 3586 was prepared following the same procedure as P.putida 852 in Section 3.4.3. Table 3–4 provides a summary of their properties. To prepare bacterial culture for experiment, a single colony of R.erythropolis 3586 was streak plated on agar plates prepared from BH and glucose media. The plate was incubated at 30 oC for 48 hours until visible colonies were formed. At least 5 plates were prepared each time in order to harvest sufficient cells for an experiment. The cells were harvested using a moistened swap and re-suspended in saline solution to a concentration of 5.4×107 cfu/mL for further treatment. 3.4.5. Bacteria Preparation for Microbubble Dispersion Experiments Unless otherwise stated, both the P.putida 852 and R.erythropolis 3586 cells were washed twice in 0.85% (w/v) saline to remove soluble extracellular polymeric substance (EPS) (Nielsen & Jahn, 1999). After centrifugation (8,000×g, 10 minutes), the pellet was resuspended in surfactant solution (concentration ranging from 500, 1,000 and 4,000 mg/L) or 54    Chapter 3 – Materials and Experimental Methods  saline as indicated. The bacterial suspension was then adjusted to an absorbance of 0.3 at 600 nm (1cm path length, Novaspec II visible spectrophotometer, Biochrom Ltd, Cambridge, U.K.), giving final bacterial concentration of approximately 6×107 and 5.4×107 cfu/mL for P.putida 852 and R.erythropolis 3586, respectively. 3.5. Adsorption of Surfactant on Bacteria To quantify any adsorption of surfactant onto bacterial cells, 40 mL of bacterial suspension in surfactant concentration ranging from 500, 1,000 and 4,000 mg/L prepared following the procedure in Section 3.4.5 was placed into a 50-mL V-bottom tube (Sarstedt 62.547.252). The bacterial concentrations tested were 6×107, 1.2×108 and 2.4×108 cfu/mL for P.putida 852 and 5.4×107, 1.1×108 and 2.2×108 cfu/mL for R.erythropolis 3586. The experiments were carried out at pH 7. The V-bottom tubes were incubated at 30 oC while shaking at 100 rpm (innova 4330 incubator shaker, New Brunswick Scientific) for 1.5 hours. Other studies have shown that adsorption achieved equilibrium within 1.5 hours (Yuan et al., 2007) and that surfactants were not degraded by the bacteria in 1.5 hours (Zhong et al., 2007). Blanks were run in parallel to determine the adsorption on the flask walls. After 1.5 hours, the bacteria and the aqueous solution were separated by centrifugation at (4000 rpm) 3220×g (Eppendorf Centrifuge 5810R, Germany). The supernatant was then filtered through 0.20 μm filter (Sartorius 16534K), and the filtrate was analysed for the amount of surfactant remaining after the adsorption test (method see Section 3.8). 3.6. Bacterial Survival Test Bacterial survival test was carried out for P.putida to examine if the release of lipopolysaccharide from the cell surface has a detrimental effect on the cell viability. At the end of adsorption test (before centrifugation; see Section 3.5), 10 µL of the P.putida and rhamnolipid mixture was plated on nutrient agar plates for six samples and inoculated overnight at 30 oC. The cell count (cfu/mL) was compared with control condition where saline (0.85% v/w) was used instead of the rhamnolipid solution. 55    Chapter 3 – Materials and Experimental Methods  3.7. Bacterial Cell Surface Hydrophobicity 3.7.1. Microbial Adhesion to Hydrocarbons (MATH) Assay One of the most frequently used experimental methods to study bacterial cell surface hydrophobicity is the microbial adhesion to hydrocarbons (MATH) assay. The MATH assay followed the procedures developed by Rosenberg (1984). Bacteria were harvested by centrifugation (8000×g, 10 minutes) and resuspended in phosphate/urea/Mg (PUM) buffer (pH 7.1, 22.2g K2HPO4.3H2O, 7.26g KH2PO4, 1.8g urea, 0.2g MgSO4.7H2O and distilled water to 1000 mL). The initial absorbance (Optical Density or O.D. at 400 nm) of the bacterial suspension was taken. Then 0.2 mL n-hexadecane of was added to 1.2 mL of the suspension. Following pre-incubation at 30 oC for 10 minutes, the mixture was uniformly mixed for 2 minutes. The mixing creates micro-droplets of the hydrocarbon phase, which serves as a hydrophobic substratum for bacteria to adhere to. After allowing 15 minutes for the aqueous and the hydrocarbon phases to separate, the lower aqueous phase is carefully removed with a Pasteur pipette and its turbidity was measured at 400 nm. Hydrophobicity is expressed as the percentage of adherence to hydrocarbon that is calculated as [1 – (O.D.400nm of aqueous phase / O.D.400nm of initial cell suspension)] × 100%. The MATH method is based on a simple rationale that hydrophilic bacteria would remain in aqueous solution and hydrophobic bacteria would adhere to hydrophobic hydrocarbon phase. However, water contact angle measurements on bacterial lawns have recently become more accepted for the measurement of the intrinsic bacterial cell surface hydrophobicity. 3.7.2. Contact Angle Measurement Contact angle measurement provides a means to quantify the hydrophobicity of the bacterial cell surface and has become more popular despite requiring relatively more complicated procedures than MATH assay. A bacteria lawn is needed for contact angle measurement. The bacterial lawn is a confluent layer of bacterial cells collected on filter paper following the procedures by Busscher et al. (1984). Briefly, 40 mL of the bacterial suspension (prepared following Section 3.4.5) was filtered through a 0.45 μm (pore size) Millipore membrane filter (MF-MilliporeTM) mounted on a Büchner funnel to deposit a uniform lawn of approximately 2×106 cells per mm2. The membrane filter is made from biologically inert mixtures of cellulose acetate and cellulose 56    Chapter 3 – Materials and Experimental Methods  nitrate. The lawns were then mounted on a glass slide with double-sided tape and allowed to air-dry in a laminar flow cabinet for 45 minutes (van der Mei et al., 1998). The lawns have to be air-dried to reach the “plateau” contact angle state and the drying usually takes 30-60 minutes (Busscher et al., 1984; van der Mei et al., 1998). Scanning electron microscopy of the bacterial lawn was undertaken to confirm a homogenous deposition of bacterial cells. Bacterial contact angles were measured by placing a droplet (~ 1 µL) of selected diagnostic liquid on a bacterial lawn using a gas tight syringe (Hamilton GAS TIGHT®) on a goniometer (KSV Instrument). Ultra pure water (MilliQ), formamide (Merck) and 1-bromonaphthalene (Sigma-Aldrich) were used as the diagnostic liquids. These are the most commonly used diagnostic liquids combination in the literature (Bos et al., 1999) and their surface tension parameters are available from van Oss (2006). Table 3–4 provides a summary of the surface tension properties of the diagnostic liquids. Table 3-4. A summary of surface tension parameters of the diagnostic liquids. Liquid Water Formamide 1-bromonaphthalene  72.8 58.0 44.4  LW 21.8 39.0 44.4  AB   51.0 19.0 0 25.5 39.6 0 25.5 2.3 0 The angle between the droplet and the lawn was recorded using a digital imaging goniometer (KSV Instrument CAM 101 with an accuracy of ± 0.1o). The value of the angle was obtained by curve fitting analysis using the CAM software (KSV Instrument). Figure 3–5 shows a screen shot of the contact angle analysis. Six independent measurements were taken for each test. 57    Chapter 3 – Materials and Experimental Metthods  Figure 3-5. Screensh hot of the con ntact angle an nalysis proced dure. The im mage shows a ddroplet of dia agnostic liquid on bacterial lawn. Contacct angles werre obtained by y fitting tangents (purple lines) to the droplet d (show wn inside bluee rectangle) using u the You ung/Laplace method. m The fitted left andd right angles were higghlighted in blue. b The surrface free ennergy comp ponents, nonn-polar Lifshitz-van derr Waals (LW W, γLW) com mponent and pollar electron--donor (γ-) and electronn-acceptor (γ+) parameeters of a bbacterial surrface are related to the contaact angles (θ) ( on the bbacterial law wn by Equaation [5] whhich is expressed as (van der Mei et al., 1998; van Oss, 1995)):  l 1  ccos   2  bLW  lLW  2  b  l  2  b  l [5] in whicch subscriptts l and b stand for ddiagnostic liquid l and bacteria, b annd γl is the surface tension of the diaggnostic liquiid. The assuumptions rellated to the use of Equuation [5] haave been discusseed in Sectioon 2.7. 3.8. Baacterial Cell C Drain nage from m Microbu ubble Disp persion A bacteerial cell drrainage expeeriment waas performed d to investiigate bacterrial interactiion with microbuubble disperrsion underr static drainnage conditiion. Bacteriaa suspensioons (Section n 3.4.5) werre allowed to t acclimatee to the surrfactant solu ution for at least 1 hour prioor to making g microbubbble dispersiion. A bacteeria suspenssion of 100 mL was 58    Chapter 3 – Materials and Experimental Metthods  6 added to 900 mL of o surfactan nt solution, ggiving apprroximately 6×10 6 bacteeria per mL.. After 3 t bacteria--enriched microbubble m e dispersionn was left to drain in minutess of intensivve mixing, the the mixxer. Duplicaate samples (1.5 mL eaach) were removed r aseptically froom the foam m phase and draained liquidd phase resp pectively att every 2 minutes. m Thee samples w were kept in sterile eppendoorf tubes. To T prevent bacterial b conntamination n, the generator was dissinfected with w 70% alcohol and air-drieed in a lamiinar flow caabinet beforre each expeeriment. In orderr to determiine the num mber of bactteria presentt in each sam mple phase , serial dilu ution and viable pplate count were carrieed out. The steps weree illustrated in Figure 33-6. Serial dilutions d were peerformed inn 96-well plaates (Starsteedt, 82.1581.001) using g saline sollution. 0.1 mL m from the 10-33, 10-4 and 10-5 dilution ns was platted onto nutrient agar plates in duuplicate. Th he plates were inncubated at 30 oC overn night for baacterial colo onies to form. The coloonies were counted manually and the bacteria b con ncentration iin each sam mple was callculated froom the colon ny count results. Fiigure 3-6. Serrial dilution m method for esstimating celll concentratioon. The esttimated celll concentraation was then comb bined with previously determined d liquid drainagge data to deetermine thee number off cells in eaach phase with w referencce to drainaage time. The perrcentage off cells retaiined in the foam phasse, i.e., the microbubbble dispersiion, was expresssed as the nuumber of ceells remainiing in the foam fo phase over the tottal number of cells. Mass bbalance calcculation of the cells w was perform med to checck the conssistency of the cell count ddata. The enntire experim ment was rrepeated tw wice for each h experimenntal conditiion. The experim ment was peerformed in a PC2 laborratory. 59    Chapter 3 – Materials and Experimental Metthods  3.9. Su urfactant Analysis Procedurre 3.9.1. R Rhamnolipiid Surfacta ant Rhamnoolipid surfaactant conccentration w was measurred before and after tthe adsorpttion test (Sectionn 3.5). Thhe measurement was performed d using a spectrophottometry tecchnique. Samplees were centtrifuged (32 220×g, 15 m minutes) an nd filtered (S Sartorius; 00.22 μm) to remove biomasss that mayy interfere with absorrbance read ding. Quartz cuvette was used for the measureement. Abbsorbance was meassured at a wavelen ngth of 2260 nm using u a spectropphotometerr (Shimadzu UV-VIS S Spectroph hotometer; UVmini 11240, Japaan). The wavelenngth correspponded to the desirablee wavelength at which rhamnolipiid absorbs the t most light annd was identtified by scaanning the ssample overr the waveleength rangee of 190 nm m to 1100 nm. Figgure 3–7 shhows the staandard curvve obtained for rhamno olipid showiing the linear range up to 20000 mg/L. Dilution D waas required ffor rhamnollipid concen ntration highher than 200 00 mg/L before tthe measureement was taken. t Figu ure 3-7. Stand dard curve foor rhamnolipiid spectropho otometry anaalysis. 3.9.2. T Tergitol 15--S-12 Tergitol surfactantt concentrattion was meeasured beffore and after the adsor orption test (Section 3.5). Thhe measurem ment was performed ussing the speectrophotom metry techniique. Samples from the adssorption tessts (Section n 3.5) werre centrifug ged (3220× ×g, 15 minnutes) and filtered 60    Chapter 3 – Materials and Experimental Metthods  (Sartoriius; 0.22 μm m) to remov ve biomass that may interfere witth absorbannce reading. Quartz cuvette was used for f the meassurement. A Absorbance was measurred at a wavvelength off 225 nm using a spectrophootometer (S Shimadzu U UV-VIS Sp pectrophotom meter; UVm mini 1240, Japan). The waavelength was w the desiirable waveelength at which w tergittol absorbs the most liight and was ideentified by scanning th he sample oover the wavelength range r of 1990 nm to 1100 nm. Figure 33–8 shows the t standard d curve obtaained for terrgitol showiing the lineaar range up to 5000 mg/L. F Figure 3-8. Sta andard curvee for tergitol spetrophotom metry analysiis. 3.10. M Magnesiu um Analyttical Proccedure Flame-aatomic absoorption specctrometry (fflame–AAS S) was emplloyed to dettermine thee soluble magnessium presennt in supeernatant aftter the adssorption teests (Sectioon 3.5). After A the adsorption tests, 20 mL of th he supernataant sampless were nitric-acid presserved to av void lost due to aadsorption on o the container or ionn-exchange with glass container (A Ali & Abou ul-enein, 2006). C Concentrateed nitric aciid was addeed to the sam mples at an n acid volum me to waterr volume ratio off 1.5mL/L. The T AAS in nstrument w was Varian SpectrAA S and a the cathhode lamp used u was Varian calcium/m magnesium hollow h cathhode lamp.. Default method m from m the Flam me-AAS p no. 85-100009-00) waas loaded forr the analyssis. Analytiical Methodds (Varian; publication 61    Chapter 3 – Materials and Experimental Methods  3.11. Polyacrylamide Gel Electrophoresis (PAGE) Analysis of Lipopolysaccharide Polyacrylamide Gel Electrophoresis (PAGE) analysis was performed to semi-quantify the lipopolysaccharide (LPS) in the supernatant prepared from the P.putida and rhamnolipid adsorption tests (Section 3.5). The PAGE analysis followed the protocol published in the NuPAGE® Technical Guide (Manual part no. IM-1001) by Invitrogen for the NuPAGE® kit set. Each supernatant (1 mL) was lyophilised (SPD111V SpeedVac, ThermoSavant) and resuspended in 75 µl milliQ water. Then 25 µl of 4x NuPAGE Lithium Dodecyl Sulphate (LDS) sample buffer (Invitrogen, NP007) was added to each sample and preparations were heated in a water bath at 100oC for 5 minutes before cooling on ice. 20 µl of each sample was loaded to a NuPAGE Novex 4-12% Bis-Tris minigel (Invitrogen, NP0321BOX). On each gel, one well was loaded with 5 µg of Salmonella LPS (Sigma, L6011) in 20 µl 1x NuPAGE LDS sample buffer as a marker. Gels were run in 1x NuPAGE MES buffer (Invitrogen, NP002) at 200V for 35 minutes. LPS in the gel was stained using the PlusOne Silver staining kit (GE Healthcare, 17-1150-01) using the protocol for mini slab gels. 3.12. Microscopy Examination 3.12.1. Fluorescence Microscopy Visualisation of bacteria-microbubble interaction and bacteria-contaminant-microbubble interaction can be made using fluorescence microscopy (Nikon ECLIPSE E600, Japan). Bacteria and model hydrocarbon contaminant were labelled with fluorescent stains to enhance the visualisation. Both P.putida 852 and R.erythropolis 3586 cells were labelled with fluorescent stain acridine orange (AO; excitation λ 502 nm, emission λ 526 nm). AO is one of the most widely used fluorescent dyes. It is cell-permeable and interacts with nucleic acid (DNA and RNA) of the bacterial cell (Maier et al., 2009). Since it does not sorb to cell surface, AO stain does not affect cell surface property. It has been commonly applied with the use of fluorescence microscopy to study cell aggregation (Korber et al., 1994; Monier & Lindow, 2004). AO solution was prepared by dissolving pure AO in ultra-pure MilliQ water to a concentration of 0.5 mg/mL. The AO solution was mixed with bacterial suspension (prepared following procedures in Section 3.4) at a ratio of 1:5 (i.e., 1 mL of AO solution to 5 mL of cell 62    Chapter 3 – Materials and Experimental Methods  suspension). The mixture was vortexed and incubated for 2 minutes at 21 oC. The stained cell suspension was then added to the surfactant solution to make microbubble dispersion (see Section 3.7). A sample of the dispersion was transferred to a cavity glass slide protected with coverslip for observation under the fluorescence microscope under the FITC (fluorescein isothiocyanate) spectrum. Model hydrocarbon contaminant hexadecane was stained with Nile Red (excitation λ 515530 nm, emission λ 525-605 nm). Nile Red is a hydrophobic fluorescent dye that has been used to study bacterial interaction with organic compounds and the aggregation of organic particles in aqueous solution, and the staining has not been reported to affect the behaviour of the organic compounds or the bacteria (Auty et al., 2001; Ly et al., 2006; Sutter et al., 2007). A stock solution was prepared by dissolving Nile Red in hexadecane to make a concentration of 4 mg/mL. Prior to microscopy examination, 1 mL of hexadecane was vigorously mixed with 0.5 mL of the stock Nile Red for 1 minute. 10 μl of the stained hexadecane was added to 1 mL of freshly made microbubble dispersion (with or without stained bacterial cells) and the mixture was vortexed for 10 seconds at room temperature. A sample of the dispersion was transferred to a cavity glass slide protected with coverslip for observation under the fluorescence microscope under FITC spectrum. 3.12.2. Scanning Electron Microscopy Scanning electron microscopy (SEM) was carried out to verify whether homogenous surface of a bacteria lawn was formed. The bacterial lawn was gold sputtered (Polaron SC 7640 Sputter Coater) to form a very thin and conductive metal coating on the samples immediately prior to SEM observation. The samples were then examined under the SEM (Philips XL30S FEG, Netherlands). 3.12.3. Cryo–Scanning Electron Microscopy Cryo-scanning electron microscopy (cryo-SEM) was performed to investigate the presence of extracellular polymeric substance (EPS) from bacteria within microbubble dispersion. Freshly made microbubble dispersion was cryo-frozen in liquid nitrogen before transferring to the cryo unit (Gattan Alto 2500, England) connecting to the SEM (Philips XL30S FEG, Netherlands). The cryo unit includes a fracture stage and a sputter coater. Within the unit, the frozen sample was fractured to expose a cross-section of the microbubble dispersion, and the fractured surface was etched for 40 minutes by sublimation to remove ice crystal from the 63    Chapter 3 – Materials and Experimental Methods  surface before being sputtered with gold at -120 oC. Subsequently, the treated sample was moved into the viewing chamber for examination. 3.13. Treatment of Data and Statistical Analysis Procedure Experimental results were generally expressed in the form of mean ± 1 standard deviation of the dataset obtained from repeated runs of the experiments. It was assumed that the dataset was normally distributed and that one standard deviation represented a confidence interval of about 68%. Unless otherwise stated, analysis of variance (ANOVA) statistical tests were carried out to assess the significance of difference between data sets by comparing for equality of means with a confidence level of 95%. If the p-value is less than 0.05, the data sets are significantly different. If p>0.05, they are not significantly different. The lower the p-value, the higher is the confidence that the data sets are different. The ANOVA tests were performed using statistical program SPSS (version 18.0, USA). The normality of the data was tested with Shapiro-Wilk test and quantile-quantile plot using the SPSS program. 64    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  Chapter 4. Characterisation and Drainage Mechanism of Microbubble Dispersion 4.1. Introduction Literature review in Section 2.3 has identified that the characterisation study on microbubble dispersion has been limited on synthetic surfactants, although natural surfactants have demonstrated promising potential for bioremediation applications. In this chapter, characterisation studies of microbubble dispersion made from rhamnolipid are performed. Preliminary tests are carried out to establish desirable apparatus set-up and mixing conditions to generate microbubble dispersions. Properties of the dispersion – stability, size distribution and gas hold-up are investigated under various environmental conditions. In particular, stability is the focus of the characterisation study because it is a critical property with implications for bioremediation applications. Stable microbubbles will travel further in subsurface and reach deeper into contaminated zones (Wan et al., 2001). To help understand whether rhamnolipid biosurfactant delivers properties comparable to chemical surfactants, two chemical surfactants (tergitol and SDS) are used in some tests for comparison with rhamnolipid, and the results are also compared with findings in the literature. Tergitol surfactant is chosen because it has been used in a number of studies to make microbubble dispersion and tested to be biodegradable and biocompatible with a variety of contaminant-degrading bacteria (Matsushita et al., 1992; Rothmel et al., 1998; Wang & Mulligan, 2004a). SDS is chosen because it has been commonly used for making microbubble dispersion and was anioic in nature like rhamnolipid (Wan et al., 2001; Yan et al., 2005). However, SDS is not biocompatible with bacteria (Rothmel et al., 1998), and is a common reagent used to disrupt or lyse bacterial cell membrane. As discussed in Section 2.3, the drainage models for microbubble dispersion in the literature appear to be incomplete. An improved drainage mechanism and model are proposed using the drainage data obtained from the characterisation study of the rhamnolipid microbubble dispersion in this study to fill the gap in current understanding. The following objectives are achieved in this chapter. 65    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  1. To develop the best possible generator configuration in order to provide improved dispersion stability; 2. To investigate microbubble dispersion properties including drainage/stability, gas hold-up and size distribution under various environmental conditions, such as pH, ionic strength and surfactant concentration; 3. To develop and improve drainage model specific to microbubble dispersion because drainage models in the literature have yet to provide a satisfactory explanation to the drainage behaviour observed in microbubble dispersion. 4.2. Preliminary Testing 4.2.1. Mixing Speed and Duration Studies have shown that mixing speed and duration can affect the properties of microbubble dispersion. Sebba (1987) found that microbubble dispersion was formed at mixing speed greater than 4,000 rpm. The commonly used mixing speeds quoted in the literature range from 6,000 rpm to 13,000 rpm, with 8,000 rpm being the most popular choice (see Section 2.3.2). Studies of the contribution of mixing duration have not yielded consistent conclusions. While some report that mixing duration does not have significant influence on increasing dispersion properties (Jauregi et al., 1997), others have shown that dispersion stability fluctuates for stirring times of 1 to 5 minutes (Save & Pangarkar, 1994). Preliminary tests were therefore performed to identify the suitable mixing speed and duration for this study (see Appendix III for experimental data). Microbubble dispersion was made using setup 1 with a flat disk and two baffles (see Section 3.2 or 4.2.3). The tests were first performed with a high surfactant concentration at 20,000 mg/L rhamnolipid to eliminate any effects resulting from lack of surfactant molecules. The liquid drainage curves were shown in Figure 4-1 and the stability and gas hold-up values derived from Figure 4-1 were summarised in Table 4-1. 66    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figurre 4-1. Liquid d drainage fro om microbub bble dispersio on generated from 20,000 mg/L rhamn nolipid solution n under differrent mixing speeds and du urations. The conditions are mixing speeed of 8000 rpm r for 3 minutess ( ); mixin ng speed of 8000 8 rpm for 6 minutes ( ( ); mixing speed of 100000 rpm for 3 minutes ); mixing m speed of 10000 rpm m for 6 minuttes ( ). Tablle 4-1. Stabilitty and gas ho old-up measu urements (meean ± 1 S.D.)) for various m mixing speed ds and duratio ons at 20,000 mg/L rhamn nolipid concen ntration. Mixing Speed M d (rpm) 8,000 8,000 10,000 10,000 Duratioon (minutees) 3 6 3 6 Half-life (seeconds) 51 13 ± 16 49 98 ± 13 49 96 ± 13 45 50 ± 11 Gas hold--up (%) 70% ± 0% % 70% ± 1.44% 71% ± 0.77% 71% ± 0.77% The shaape of the drainage sh hown in Fiigure 4-1 does not varry much wi with varying g mixing speed aand durationn. The resullts in Tablee 4-1 indicaate that incrreasing the mixing speeed from 8,000 rppm to 10,0000 rpm sign nificantly deecreased staability (P<0 0.05). Hencee, the mixin ng speed of 8,0000 rpm is prreferred oveer 10,000 rppm. Increassing mixing duration fr from 3 minu utes to 6 minutess results in a significan nt reduction in stability (P<0.05). Therefore T m mixing durattion of 3 minutess is preferreed. It was allso observeed that at mixing m speed d of 10,000 rpm the vo olume of microbuubble dispersion becam me stabilizedd after abou ut 1.5 minuttes and furthher mixing resulted in heatting up thee dispersion n. The exceessive heatt may havee a detrimeental effectt on the dispersiion stabilityy. 67    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  Neither the mixing speed nor duration has significant influence on gas hold-up values (P>0.05). Overall, a mixing speed of 8,000 rpm for a duration of 3 minutes is a suitable combination for making microbubble dispersion and is used for subsequent experiments. 4.2.2. Rhamnolipid Concentration A wide range of rhamnolipid concentrations, varying from 300 mg/L to 20,000 mg/L, were tested to identify a suitable range for further testing. The tests were performed using setup 1 at mixing speed of 8,000 rpm for 3 minutes. Rhamnolipid solutions at higher concentrations would be less economical in field-scale remediation, and so it is desirable to produce microbubble dispersions with relatively low rhamnolipid concentrations that do not compromise the dispersion’s properties. The liquid drainage curves were shown in Figure 4-2 and the stability and gas hold-up values derived from the drainage curves were summarized in Table 4-2. Under equal mixing conditions, the microbubble dispersion created at a rhamnolipid concentration of 300 mg/L demonstrated a stability that is significantly lower than those at higher rhamnolipid concentrations (P<0.05), and the dispersion did not display the characteristic thickness as produced with rhamnolipid concentrations 500 mg/L or greater. Furthermore, it was observed that at 300 mg/L the microbubbles rose to the dispersion surface at a much faster rate than those from higher rhamnolipid concentrations, leaving a large volume of liquid phase at the bottom. The rapid ascent of the microbubbles indicated that the dispersion was very unstable. 68    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Fiigure 4-2. Liq quid drainagee from microb bubble dispersion created d from rhamnnolipid solution. Rham ), 10,000 mnolipid concentrations weere 300 mg/L L( ), 500 mg/L m ( ), 1,000 1 mg/L ( 0 mg/L ( ) an nd 20,000 mg g/L ( ). Taable 4-2. Stab bility and gass hold-up meaasurements (mean ( ± 1 S.D.) for variouus rhamnolip pid cconcentration ns. Rh hamnolipid Con ncentration (mg/L) H Half-life ((seconds) Gaas Hold-up (%) 300 5000 1,000 10,000 20,000 0 227 ± 13 3 410 ± 15 46 66 ± 14 454 ± 13 513 ± 16 1 66% ± 0% % 69% ± 0.7% 71% % ± 0.7% 70% ± 1.4% % 70% ± 0% 0 Increasiing rhamnoolipid conceentration froom 300 mg g/L to 500 mg/L and above significantly increaseed the dispersion stabiility (P<0.005), but the increasing the concenntration from m 1,000 mg/L too 10,000 mgg/L did not have a signnificant effecct on the staability (P>00.05). The gass hold-up vaalues increaased significcantly when n the rhamn nolipid conccentration in ncreased from 3000 mg/L to 500 mg/L and above (P<0.05), but b for conccentrations between 50 00 mg/L and 20,000 mg/L, the t gas hold d-up values stayed constant (P>0.0 05). Based on the preeliminary characterisa c ation resultts of the microbubbl m le dispersio ons, the rhamnoolipid conceentrations selected forr further tessting were 500 mg/L (low), 1,00 00 mg/L (medium m) and 4,0000 mg/L (high). 69    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  4.2.3. T The Apparaatus Setup 1 was the original o mix xer configurration used to generatee microbubbble dispersiion. The setup w was based on o the original apparattus describeed in the eaarly literatuure (Rothmeel et al., 1998; S Sebba, 19877). Other researchers m modified thee original seetup to 1) rreplace the flat disk with a propeller annd 2) use 4 baffles innstead of 2 (Jauregi et al., 1997). Schematiccs of the setups w were shownn in Figure 4-3. Both setups were tested to determine tthe one thaat would producee more staable microb bubble disppersion. Thee test was carried ouut for 1,00 00 mg/L rhamnoolipid conceentration at a mixing sppeed of 8,00 00 rpm for 3 minutes. Figure 4-3. Microbu ubble dispersion apparatu us. Apparatuss based upon the setups deescribed by A. A Sebba (11987), termed d setup 1 and B. Jauregi ett al., (1997), termed t setup 2, were testeed in this stud dy. Liquid drainage cuurves are sh hown in Figgure 4-4. Summary S off the stabiliity and gas hold-up m the drainaage curves aare presenteed in Table 4-3. values dderived from 70    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figure 44-4. Drainagee curves with h different ap pparatus conffiguration. Th he two setupss are setup 1 with w flat diisk and 2 bafffles ( ); seetup 2 with prropeller and 4 baffles ( ). Results in Figure 4–4 and Taable 4–3 shhow that setup 1 produ uced signifi ficantly morre stable (P<0.055) microbubbble disperrsions whenn compared d to setup 2. 2 It was oobserved th hat more vigorouus waves weere generateed with setuup 2, which could havee resulted inn higher gass volume being eentrained wiithin the disspersion. H However, thee gas hold-u up results sshow that th here was no signiificantly diffference in gas g hold-upp values betw ween the tw wo setups (PP>0.05). Tablee 4-3. Comparrison of microbubble disp persion propeerties (mean ± 1 S.D.) at ddifferent app paratus cconfiguration ns. Rhamnolipiid cconcentratioon (mg/L) 1,000 Setup 1 Hallf-life G Gas hold-up p (sec) (%) 466 ± 14 71% ± 0.7% % Halff-life (seec) 325 ± 24 Setup2 G Gas hold-up p (%) 73% ± 0.7% % Summary 4.2.4. S As a reesult of thee preliminaary testing, the follow wing mixing g conditionns and rham mnolipid concenttrations are selected forr further stuudy: - Setuup 1 with a flat disk and two bafflees; - Mixxing at 8,000 rpm for 3 minutes; annd - Rhaamnolipid concentration ns of 500 m mg/L, 1,000 mg/L and 4,000 4 mg/L . 71    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  4.3. M Microbubb ble Disperrsion Stab bility The miicrobubble dispersion resembless an emulssion, wheree the gas microbubbles (the disperseed phase) arre dispersed d in the surffactant contaaining wateer phase (thee continuou us phase). Unlike conventional foams, th he dispersioon flows likee water, as shown s in Fiigure 4–5. Figure 44-5. Microbub bble dispersio on produced from rhamn nolipid biosurrfactant usingg Setup 1 at 8000 8 rpm for 3 minutes. m A, microbubble m d dispersion is thick, but B, it flows like water. Studies in the literrature have reported thhat the stability of miccrobubble ddispersion iss mainly governeed by liquidd drainage because b no perceptiblee breakdown of bubblees takes plaace prior to the m majority off liquid bein ng drained from the diispersion (Y Yan et al., 2005). Thrree main processs parameterss, namely th he surfactannt and electtrolyte conccentrations and pH, haave been shown tto have signnificant effeects on dispeersion stabiility. In geneeral, it is reeported thaat increasingg the surfaactant conceentration raaises the dispersion stabilityy (Chaphalkkar et al., 19 993; Jauregii et al., 1997; Matsushita et al., 19992), but increasing the elecctrolyte conncentration has h an adveerse effect on o stability. The reporteed effects of o pH on microbuubble stabillity are inco onsistent, w with some studies s show wing no eff ffect (Jaureg gi et al., 1997; S Save & Panggarkar, 199 94; Subramaaniam et al., 1990) and d others repporting a strong, but complexx, dependeence on pH H (Amiri & Woodburn, 1990). For exampple, the dispersion stabilityy of cationic surfactantt tetradecylltrimethyl ammonium a bromide (T TTAB) increeased as pH wass raised from m acidic to neutral connditions, butt then sharply decreaseed when the pH was made allkaline (Am miri & Woodburn, 19900). Reasonss for such in nconsistent behaviour have h not been addequately diiscussed in the t literaturre. The efffects of surffactant conccentration, pH and eleectrolyte concentrationn on the stability of microbuubble dispeersion creatted from rhhamnolipid d biosurfactant were innvestigated d in this 72    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  section. The experimental results were fitted with the modified liquid drainage model (the solid line) which will be described in detail in the next section. 4.3.1. Effect of Rhamnolipid Concentration on Microbubble Stability Drainage experiments were carried out to investigate the effect of rhamnolipid concentration on microbubble stability at pH 6, 7 and 8. The drainage curves of rhamnolipid microbubble dispersions produced from rhamnolipid concentrations of 500, 1,000 and 4,000 mg/L at pH 6, 7, or 8 are shown in Figure 4–6, and the half-life values and final liquid-drainage volumes derived from the drainage curves are summarized in Table 4–4. Drainage model (solid line) using Equation [13] (Section 4.4) was also fitted in Figure 4-6. The model is discussed in detail in Section 4.4. No stable dispersions were produced at pH 5. For all concentrations tested, complete foam collapse occurred over approximately 2 hours. The values reported are the mean of six measurements. The liquid drainage curves in Figure 4–6 follow an “S”-shaped profile, with an increase in the rate of drainage at initial times followed by a decrease in the rate of drainage as time progresses. This observation is consistent with findings reported by others (Yan et al., 2005). 73    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  355 Liquid D rainage (m L) 300 255 200 155 100 5 H6 pH 0 0 2000 400 (A) 600 800 Drainage Time (seeconds) D 10000 1200 35 Liquid D rain age (m l) 30 25 20 15 10 5 pH 7 0 0 2000 400 (B) 600 800 ( Drainage Time (sec) 10000 1200 35 Liqu id D ra in ag e (m L) 30 25 20 15 10 5 pH H8 0 0 (C) 2000 400 600 800 Drainage Time (sseconds) D 1000 12000 Figuree 4-6. Drainagge behaviourr of microbub bble dispersio ons with rham mnolipid surffactant at surrfactant concentrrations of 5000 mg/L ( ), 1,000 mg/L ( ), and 4,0 000 mg/L ( ) at pH 6 (A A), pH 7 (B), and pH 8 (C) resp pectively. Soliid lines repre sent drainagee model output using Equuation 13. 74    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  Table 4-4. Summary of half-life (T1/2, mean±1 S.D.) and final drained liquid volume (Vmax, mean±1 .S.D) values. Rhamnolipid surfactant concentration (mg/L) 500 1,000 4,000 500 1,000 4,000 500 1,000 4,000 pH T1/2 (seconds) Vmax (mL) Solution viscosity (cP) 6 6 6 7 7 7 8 8 8 457 ± 12 495 ± 19 546 ± 13 405 ± 13 461 ± 15 519 ± 18 385 ± 13 447 ± 19 503 ± 11 29 ± 0 28 ± 1 31 ± 1 33 ± 2 29 ± 1 31 ± 1 32 ± 2 32 ± 1 31 ± 1 1.52 ± 0.00 1.62 ± 0.01 2.03 ± 0.01 1.59 ± 0.01 1.76 ± 0.01 1.96 ± 0.00 1.61 ± 0.01 1.77 ± 0.01 1.93 ± 0.01 Increasing rhamnolipid concentration from 500 mg/L to 4,000 mg/L, as shown in Table 4–4, significantly increased (P<0.05) the microbubble dispersion stability from 457 to 546 seconds at pH 6, from 405 to 519 at pH 7, and from 385 to 503 at pH 8. These increases corresponded to percentage changes of 19%, 28% and 31% at pH of 6, 7 and 8, respectively. The effect of pH will be discussed in Section 4.3.2. Rhamnolipid concentration did not have a significant effect on the final drained liquid volume. The drained liquid volume was directly related to the gas hold-up values, which will be discussed in greater detail in Section 4.4. The result that surfactant concentration has a significant effect on stability is consistent with findings present in the literature. The increased stability at higher rhamnolipid concentrations can be ascribed to several processes: 1. Increase in liquid viscosity from the presence of more surfactant molecules in solution, resulting in greater viscous drag that retards hydrodynamic film drainage between bubbles (Myers, 1990). The surfactant solution viscosity was measured using a Rheometer as described in Section 3.3.1. The mean value of the measurements is shown in Table 4–4. The experimental data is included in Appendix IV. In general, the results show that surfactant solution viscosity increases with surfactant concentration (P<0.05), but pH does not have a significant effect on the solution viscosity (P>0.05). 75    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  2. Higher concentrations of surfactant molecules at bubble surfaces (i.e. the lamellar walls) that increase surface viscosity and elasticity (Aveyard et al., 1999; Yan et al., 2005), thereby enhancing bubble integrity by improving the mechanical strength of lamellar walls. The stronger lamellar walls are able to slow down expansion of the bubbles (Myers, 1990), which in turn reduces liquid drainage. And, 3. A decrease in bubble coalescence due to larger electrostatic repulsion between bubbles caused by the increase in rhamnolipid concentration (Chen et al., 1988; Jauregi et al., 1997). Rhamnolipid is anionic due to the dissociation of the carboxyl group in the hydrophilic head portion. At higher rhamnolipid concentration, there is higher electrostatic repulsion between microbubbles (Jauregi et al., 1997). The repulsion prevents bubbles from approaching each other, minimising coalescence. It is likely that the combination of the above three processes resulting from the increase in rhamnolipid concentration contributes to the increased stability of the microbubble dispersion. 4.3.2. Effect of pH on Microbubble Dispersion Stability The effect of surfactant concentration on the dispersion stability (Table 4–4; Figure 4–6) was more pronounced at higher pH (change by 31%) than at lower pH (change by 19%), indicating that the change in pH affected dispersion stability. This was also demonstrated in the drainage curves in Figure 4–6, where the drainage curves moved further apart at higher pH. To illustrate the influence of pH in greater detail, the drainage behaviour of microbubble dispersions at 1,000 mg/L rhamnolipid and tergitol solutions for pH 6 to 8 was plotted in Figure 4–7, and their corresponding half-lives were presented in Table 4–5. The stability of the dispersion, described by the half-life, for the anionic surfactant rhamnolipid decreased by about 10% for a pH increase from 6 to 8 (P<0.05), while that for the non-ionic surfactant tergitol remained unaffected, maintaining a half-life of approximately 390 seconds (P>0.05). The trend of decreased dispersion stability with increasing pH from 6 to 8 was also observed for other rhamnolipid concentrations (see Table 4–5). 76    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  300 D ra in a g e V o lu m e (m L ) 255 200 155 100 5 0 0 200 400 600 800 1000 1200 1400 Draiinage Time (Seconds) ( Figgure 4-7. Draiinage behavio or of microbu ubble disperssions at pH 6,, pH 7 and pH H 8 at surfactant concen ntration of 1,0000 mg/L. Th he surfactantss tested are rh hamnolipid at a pH6 ( ); rrhamnolipid at pH 7 ( ); rrhamnolipid at pH 8 ( ); tergitol at pH H 6 ( ); terg gitol at pH 7 ( ); tergitol at pH 8 ( ). ) Lines represent the fittted output ussing Equation n 13. The sollution pH has h been rep ported to haave a signifi ficant influence on stabbility of disp persions of the cationic surfactant s TTAB T (Am miri & Wo oodburn, 1990), whille the stab bility of dispersiions of AO OT, an anio onic surfacttant, was unaffected u for f pH channging from m 4 to 8 (Jauregi et al., 19997). The diffferent behaaviour of AOT A and rhaamnolipid ddispersions, both of which aare anionic surfactants, s resulted froom the diffeerences in th heir degreess of ionization. The dissociaation constaants (pKa) of o rhamnoliipid was 5..6 (Mulligan n, 2005) annd for AOT was 2.9 (Jauregi & Varleyy, 1996). When pH waas equal to pKa, half of o the surfaactant conceentration becamee ionized duue to dissociation of thhe surfactantt. In the 4 to t 8 pH rangge, the disssociation of AOT T increased marginally y from 93% to 100%, while w in thee 6 to 8 pH H range rham mnolipid dissociaation changged from 70% to 100% %, explaining g the insenssitivity in A AOT stabilitty to the change in pH. Calcculations off the percenttage dissociiation are av vailable in A Appendix V. V 77    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figure 4-8. Dissociation D oof carboxyl heead group of rhamnolipidd. The aniionisation of o rhamnolip pid was priincipally du ue to the disssociation oof its carbox xyl head group (Champion et e al., 1995)), as shownn in Figure 4-8. 4 As the dissociation d n increased with pH, the proocess causeed greater electrostatiic repulsion between adjacent ionized caarboxyls, increasiing the effective size of the head ggroup of thee rhamnolip pid moleculees (Champion et al., 1995) aand reducinng the concentration off rhamnolip pid moleculles accumuulated at thee bubble surface,, thereby loowering the viscosity aand elasticitty of the bu ubble surfacce. The deccrease in surface viscosity and a elasticity promoteed the expansion of gaas bubbles, causing th he liquid betweenn adjacent bubbles to o be squeezzed out at faster rates. These cchanges resulted in decreassing in the dispersion d sttability. Thee absence of o an effect of pH increease on the stability of tergiitol dispersiions (see Figure 4–7) aalso supportted this hyp pothesis. Teergitol being g a nonionic suurfactant didd not underg go greater ioonization att higher pH. Tablee 4-5. Summaary of effects of surfactantt concentration, solution pH p and NaCll concentratio ons on stability. Surfactan nt C Concentrattion NaC Cl Concenttration (mg/ g/L) T1/2 (secoonds) pH 6 pH 7 pH p 8 Rham mnolipid 5000 mg/L 0 457 ± 12 2 405 ± 13 385 5 ± 13 Rham mnolipid 1,0000 mg/L 0 495 ± 19 9 461 ± 15 447 7 ± 19 Rham mnolipid 4,0000 mg/L 0 546 ± 13 519 ± 18 503 3 ± 11 Terrgitol 1,0000 mg/L 0 391 ± 5 383 ± 8 39 90 ± 6 Rham mnolipid 1,0000 mg/L 1,0000 440 ± 16 6 456 ± 16 46 60 ± 2 Rham mnolipid 1,0000 mg/L 3,0000 429 ± 19 9 472 ± 14 446 6 ± 30 4.3.3. E Effect of Eleectrolyte Concentrati C ion on Micrrobubble Dispersion D SStability Increasiing salt cooncentration n had a siggnificant efffect (P<0.0 05) on deccreasing dispersion stabilityy at low pH H (see Tablee 4–5 abov e). At pH 6, 6 increasing salt conceentration frrom 0 to 78    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  1,000 mg/L reduced the half-life from 495 to 440 seconds, an 11% reduction, and increasing the salt concentration further to 3,000 mg/L lowered the stability to 429 seconds for microbubble dispersion made with 1,000 mg/L. At pH 7 and 8, however, the microbubble dispersion stability was unaffected (P>0.05) by the increase in salt concentration; the observed changes in stability of 0.2% to 4% at the higher pH were all within one standard deviation of each other. Although decreases in dispersion stability with increasing electrolyte concentrations have been previously reported (Amiri & Woodburn, 1990; Jauregi et al., 1997; Save & Pangarkar, 1994), the effect of electrolytes on the stability of rhamnolipid microbubbles appeared to be generally small and significant only at low pH values. The observed decrease in stability at pH 6 with increasing salt concentration can be attributed to lowered electrostatic repulsion between adjacent bubble surfaces due to a compression of the double layer caused by the increase in ionic concentration of the solution (Jauregi et al., 1997; Myers, 1990). However, at pH 7 and 8 this effect is not observed as increasing the pH negates the effects of increasing the salt concentration. This is likely to be attributed to the dissociation of the rhamnolipid’s carboxyl head group as shown in Figure 4-8. At higher pH, greater ionization of the rhamnolipid’s carboxyl group increases the electrostatic repulsion between rhamnolipid molecules, counteracting the lowering of repulsion caused by the higher electrolyte concentration. Additionally, the Na+ from the added salt shields against the effects of negative charge of the carboxyl head group, preventing an increase in the effective size of the head group at higher pH (Bai et al., 1998), which could lead to reduced packing of the rhamnolipid molecules at the bubble surface as discussed in previous section. The concentration of rhamnolipid molecules at the bubble surface is therefore maintained, and the combination of the above interactions stabilizes the microbubble dispersion. 4.4. Liquid Drainage Model Several models are available to describe liquid drainage from conventional foams (Germick et al., 1994; Narsimhan & Ruckenstein, 1986; Wang & Narsimhan, 2004). However, these models do not appear to apply to the microbubble dispersion due to the observations that dispersion resembles emulsion and demonstrates different drainage behaviour to conventional foams, as discussed in Section 2.3. Two different drainage equations have been presented in the literature to describe the drainage from microbubble dispersion. One equation was developed based on Stroke’s law 79    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  (Amiri & Woodburn, 1990). This equation describes a drainage profile similar to an exponentially decreasing profile for conventional foams, and so fails to address the “S”shaped drainage curve typical of microbubble dispersion. The other equation proposed by Yan et al. (2005) fits well to the “S”-shaped drainage profile, although its conceptual model appears to be incomplete as it describes a two-stage drainage process that is similar to the drainage mechanisms proposed for conventional wet foams (Indrawati & Narsimhan, 2008; Koehler et al., 2000; Ross, 1943). The equation proposed by Yan et al. (2005) is considered to be a better candidate since it produces an “S”-shaped profile and so it is selected for further study in this thesis. It is expressed as Vt  Vmax tn K n  tn [12] where Vt and Vmax are the volume of drained liquid at time t and the final drained liquid volume respectively, K is the half-life for liquid drainage and n describes the sigmoid character of the drainage curve. Figure 4–9 (A) demonstrates an output from the original model (dashed line) by Yan et al. (2005) fitted to the measured values (triangle symbol) and (B) shows the residuals as the difference between the measured and the fitted values for microbubble dispersion created from 500 mg/L of rhamnolipid surfactant at pH 6. The original model consistently overestimates the drainage of liquid from rhamnolipid microbubble dispersions (Vmax and fitted half-life) used in this study. This is clearly shown in the residuals plot in Figure 4-9 that the modified model is better than the original model because the residuals of the modified model are closer to zero. 80    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figu ure 4-9. Plotss of liquid dra ainage data aand original model m output for rhamnolilipid microbu ubble disp persion (500 mg/L m rhamno olipid concen ntration at pH H 6). (A): liqu uid drainage curve ( sym mbol represen nts experimental data; da ashed line is ooutput from the t original model; m solid liine is output from the modifiied model). (B B): residual values v betweeen the predictted and the measured m draainage data (o original modeel represented d by ; mod dified model by b ). 81    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  Equation [12] has a form similar to the Monod kinetic equation for microbial growth. One of the modifications of Monod kinetic has been to include an inhibitory term to account for the effect of substrate inhibition on bacterial growth (Luong, 1987). To correct the deficiency in the original drainage model, a modification is proposed by borrowing the concept from the   a a n inhibitory model for Monod kinetics by including an inhibitory term  t K to give the following expression: Vt  Vmax tn 2( K n  t n  t a / K a  n ) [13] The arrangement of a and n has been made to ensure that the equation is dimensionally correct. Parameter K in Equation [13] represents the half-life (t1/2) of drainage, which is the time when half of the liquid has been drained from the microbubble dispersion, i.e, 1 t1 / 2  K when VK  2 Vmax [14] To ensure that the definition of K remains correct in the modified model, the value 2 is added to the denominator as shown in the following derivation steps. At t = K, by substituting K for t in Equation [13], it becomes V K  Vmax Kn 2( K n  K n  K a / K a  n ) [15] Equation 5-4 can be simplified to V K  Vmax Kn 2( K n  K n  K n ) [16] Further simplifying of Equation [16] yields the condition in Equation [14], i.e., 1 VK  Vmax 2 , which is consistent with the original definition. On differentiating Equation [13] the rate of liquid drainage is obtained as:      dVt t n 1 K n a  n  t a / K a  n  Vmax 2 dt 2 K n  t n  t a / K an  [17] 82    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  The modified model (solid line) and residuals for 500 mg/L rhamnolipid concentration at pH 6 is included in Figure 4–9 for comparison with the original model. The half-life and drained liquid volume values of the experimental data and model output are summarised and compared in Table 4–6. Both Figure 4–9 and Table 4–6 indicate that the modified model gives better fit to the measured values as well as more accurate prediction of half-life and drained liquid volume parameters than the original model. Table 4-6. Comparison of observed and fitted half-life (T1/2) and final drained liquid volume (Vmax) values. Experimental data Rhamnolipid concentration (mg/L) pH 500 1,000 4,000 500 1,000 4,000 500 1,000 4,000 6 6 6 7 7 7 8 8 8 T1/2 (sec) Vmax (mL) 457 ± 12 495 ± 19 546 ± 13 405 ± 13 461 ± 15 519 ± 18 385 ± 13 447 ± 19 503 ± 11 29 ± 0 28 ± 1 31 ± 1 33 ± 2 29 ± 1 31 ± 1 32 ± 2 32 ± 1 31 ± 1 Original model output T1/2 Vmax n (sec) (mL) T1/2 (sec) Vmax (mL) n a 485 578 669 484 506 697 473 557 638 481 478 537 387 505 529 387 432 473 31 25 31 33 34 31 33 31 28 1.51 1.63 1.56 1.24 1.51 1.57 1.33 1.41 1.58 1.10 1.28 1.21 0.95 1.01 1.22 1.06 1.11 1.24 31 32 39 41 32 42 39 40 44 2.09 2.08 1.93 1.57 2.05 1.82 1.68 1.76 1.51 Modified model output The values of n in the original model are found to depend mainly on the surfactant concentration (Yan et al., 2005). The parameters n and a in the modified model also correspond well to the surfactant concentration at all pH values tested. In general, the parameters increase with increasing surfactant concentration as shown in Table 4–6. To better assess the degree of fitness of the models, the equations for the line of best fit of the half-life and drained volume values were also shown in Figure 4 – 10. A gradient closer to 1 indicates a closer fit as the 1:1 slope line has a gradient of 1. Since the gradient of the line of best fit through the modified model is closer to 1 when compared to the original model, it shows that the modified model is better than the original model. 83    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figure 44-10. Correlattion between fitted and m measured halff-life (A) and maximum drrained liquid (B) from the rh hamnolipid microbubble m dispersions. d T The open ( d ( ) symbols rep present ) and solid diamond outputs from originaal and modifiied models, reespectively. The T dashed lines representt line of best fit of the data. d The sollid line is the 1:1 slope linee. Paired-ssample T-test at a sig gnificance level of 0..05 was alsso performeed to comp pare the significcance of diffference beetween the measured values v and the model output. A P-value below 00.05 is geneerally consid dered that thhere is statistically sign nificant diffference betw ween the 84    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  two data sets; but if the P-value is greater than 0.05, it indicates no difference between the groups. The analysis shows that while the fitted output from the original model is significantly different from the measured data in terms of both half-life and drained liquid volume values (P<0.05), the modified model is not significantly different from the measured data (P>0.05). Output from the statistical tests is summarised in Table 4–7 for half-life and Table 4–8 for drained liquid volume. Table 4-7. Summary of Paired-sample T-test results for half-life. Pair 1 Experiment – Original Model Pair 2 Experiment – Modified Model 95% Confidence Interval of the Difference Lower Upper Mean Standard Deviation Significance (2-tailed) -80.000 47.801 -110.371 -49.629 P<0.0001 12.917 29.540 -5.852 31.686 P=0.158 Table 4-8. Summary of Paired-sample T-test results for drained volume. Pair 1 Experiment – Original Model Pair 2 Experiment – Modified Model 95% Confidence Interval of the Difference Lower Upper Mean Standard Deviation Significance (2-tailed) -6.250 3.467 -8.453 -4.047 P<0.0001 1.083 2.746 -0.661 2.828 P=0.199 4.5. Gas Hold-Up Measurement The gas hold-up measures the amount of gas entrained in microbubble dispersions. It can be calculated from the final drained liquid volume as discussed in Section 3.3.3. The gas hold-up values measured with different surfactant types (rhamnolipid and tergitol) and concentrations are shown in Table 4–9 (experimental data in Appendix VI). The gas hold-up values under the influence of electrolyte concentrations are shown in Table 4–10. The gas hold-up results represent the average of at least 4 independent measurements. 85    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  Table 4-9. Gas hold-up measurement (mean ± 1 S.D.) at different surfactant types and concentrations. Surfactant Type Concentration (mg/L) pH 6 pH 7 pH 8 Rhamnolipid 500 c 71%±0% 67%±2% 68%±2% Tergitol 1,000 4,000 1,000 72%±1% 71%±1% 68%±1% 69%±1% 69%±1% 69%±1% 69%±1% 70%±1% 70%±1% Table 4-10. Gas hold-up measurement (mean ± 1 S.D.) at different salt concentrations at 1,000 mg/L rhamnolipid. Salt Concentration (mg/L) 0 1,000 3,000 pH 6 72% ± 1% 70% ± 1% 67% ± 4% pH 7 71% ± 1% 69% ± 1% 69% ± 1% pH 8 68% ± 1% 66% ± 2% 70% ± 1% As shown in Table 4–9, the gas hold-up did not vary significantly (P>0.05) with the surfactant types and concentration. The gas hold-up values in Table 4-9 were comparable with literature values in the range of 50% to 70% (Ciriello et al., 1982; Sebba, 1987; Subramaniam et al., 1990). Others too have noted that the type of surfactants did not affect the gas hold-up (Matsushita et al., 1992). The surfactant concentrations used in this study were well above the CMC and would have little effect on gas hold-up (Kommalapati et al., 1996). However, increasing salt concentration significantly affects the gas hold-up (P<0.05) for all pH levels tested. It has been reported that increasing electrolyte concentration causes a decrease in gas hold-up (Jauregi et al., 1997). The decrease in gas hold-up for pH 6 and 7 in this study is consistent with the study reported in the literature (Jauregi et al., 1997). The largest observed change from 72% to 67% occurs at pH 6 when the salt concentration is increased from 0 to 3,000 mg/L. But the effect at pH 8 is undefined, where the gas hold-up decreases with increasing salt concentration from 0 to 1,000 mg/L; but the gas hold-up increases with increasing salt concentration from 1,000 mg/L to 3,000 mg/L. 4.6. Size Distribution Microbubble size distribution was measured using two techniques. One involved a particle size analyser; another used image processing analysis. For soil bioremediation application, it is generally desirable to produce small sized bubbles so that they can be easily transported through soil systems. 86    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  4.6.1. Particle Size Analyser Size distribution measurement was undertaken using Malvern particle size analyser. The results presented here are the average of two independent measurements. Experimental data from the experiments are available in Appendix VII. Figures 4–11 and 4–12 present the percentage distribution and cumulative distribution of microbubble diameter representing three different experimental conditions at constant surfactant concentration of 1,000 mg/L at pH 7. Figure 4–13 shows the comparison of bubble size at 3 different percentiles. Three types of surfactant mixtures were tested. They were the anionic rhamnolipid surfactant, rhamnolipid with 1,000 mg/L of NaCl and tergitol surfactant. The bubble diameter distribution shown in Figure 4-11 is normally distributed, with most of the bubbles (>95%) in the size range of 26 µm to 182 µm. The mean bubble diameter calculated for the rhamnolipid microbubbles is 83 ± 2 µm. For microbubbles created from rhamnolipid with NaCl present, the mean is 76 ± 6 µm. For tergitol microbubbles, it is 77 ± 1 µm. The mean diameters are similar to the median values (i.e., 50%ile) as shown in Figure 4–13, supporting the normal distribution observed in Figure 4–11. The addition of salt to the rhamnolipid microbubble dispersion and using tergitol surfactant have no significant effect on the size distribution and percentile values of the rhamnolipid microbubble (P>0.05), as shown in Figures 4-12 and 4-13. 87    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figu ure 4-11. Percentage of microbubble siize distributio on measured by particle ssize analyser. The microb bubble disperrsion was mad de with surfaactant concen ntration of 1,0 000 mg/L at ppH 7. The con nditions are rhamnolipid surfactant ( ); rhamnollipid surfacta ant with 1000 0 mg/L NaCl ( ); and terrgitol su urfactant ( ). Figure 4-12. Cumulaative distribu ution of microobubble diam meter measured by particuule size analy yser. The is for microbubble disp persion was made m with surrfactant concentration of 1,000 1 mg/L aat pH 7. rhamnolipid d; is forr rhamnolipid d with 1,000 mg/L m of NaCl; and iis for tergitoll. 88    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figurre 4-13. Comp parison of miicrobubble diiameter at different perceentiles measurred by particcle size analysser. The micrrobubble disp persion was m made with surrfactant concentration of 11,000 mg/L at a pH 7. The coonditions are rhamnolipid d surfactant ( ); rhamno olipid with 1000 mg/L NaC Cl ( ); and tergitol urfactant ( ). su The sizze range fouund in this study s is smaaller than th he range off 30 µm to 3300 µm rep ported in the literrature usingg particle sizze analyserr. But the mean m or med dian diameteers are mucch larger than thhe values observed o by y others (B Basu & Maalpani, 2001 1; Dai & D Deng, 2003 3) using microphhotography and imagee processingg analysis. It is known n that diffeerent methods yield differennt size distrribution of the microbbubbles duee to their transient chaange in bub bble size (Dai & Deng, 20003). The parrticle size aanalyser pro ovides a quiick and eassy way to sttudy the microbuubble size characteristics, but itt gives resu ults that are generallyy larger thaan other methodds due to the t relativeely long prrocessing time t and the dilutionn occurring g during measureements. Nevertheless N s, the resuults demonstrate thaat rhamnoolipid micrrobubble dispersiions have siimilar size distribution d to those maade from terrgitol surfacctant. 4.6.2. Im mage Processing Tech hnique Despitee being a fairly quick and a easy waay to measu ure microbu ubble size ddistribution, particle size anaalyser tendss to over-esstimate bubbble size. A microscopiic image prrocessing teechnique was also employedd to perform m the size m measuremen nt (see Sectiion 3.3.4). T The reported values shown in Table 4–11 4 were the t mean bbubble diam meter estimaated for vaarious experrimental 89    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  conditions using the microscopy technique (see Appendix VIII for experimental data). The values are the mean and 1 standard deviation based on at least 100 bubbles. Table 4-11. Mean bubble diameter with 1 standard deviation under various experimental conditions. Surfactant Concentration (mg/L) Rhamnolipid 500 Rhamnolipid 1,000 Rhamnolipid 4,000 Tergitol 1,000 Mean Bubble Diameter (μm) pH 6 pH 7 pH 8 70 ± 3 69 ± 4 70 ±2 67 ± 2 71 ± 1 63 ± 5 64 ± 4 61 ± 6 65 ± 2 68 ± 4 70 ± 7 67 ± 4 The mean diameter of rhamnolipid microbubble dispersion obtained by image processing analysis varied between 63 to 71 μm. The rhamnolipid concentration had a significant effect on the bubble size (P<0.05), where increasing rhamnolipid concentration caused a decrease in bubble diameter. However, the effect was only significant between 500 mg/L and 4,000 mg/L; the step increase from 500 mg/L to 1,000 mg/L or from 1,000 mg/L to 4,000 mg/L did not have significant effect on bubble diameter (P>0.05). The mean bubble size of tergitol microbubbles was not significantly different to that of rhamnolipid microbubbles (P>0.05), which was consistent with what was found using particle size analyser. The change in pH did not have a significant effect on the mean bubble diameter. At pH 7, the majority of the rhamnolipid microbubbles were between 20 μm to 140 μm in diameter (Figure 4–14). This is a narrower range compared to the results given by particle size analyzer in Section 4.6.1. By comparing the cumulative distribution at the 90% mark in graphs of Figure 4–14, it shows that increasing rhamnolipid concentration from 500 mg/L to 4,000 mg/L reduced the corresponding bubble size from 120 μm to 90 μm (see A to C). Tergitol microbubbles had a size distribution similar to the rhamnolipid. 90    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figurre 4-14. Microobubble size distribution at pH 7 obtaiined by imag ge processing technique. A is for rhamnollipid at 500 mg/L; m B is forr rhamnolipid d at 1,000 mg g/L; C is for rhamnolipid at 4,000 mg//L; and D is for tergitol at 1,000 mg/L.. Arrows indiicate the corrresponding axxis. 4.6.3. C Comparison n Between Particle Sizze Analyser and Imag ge Processin ing Techniq que Comparrison betweeen the two o techniquees was mad de for rhamnolipid andd for tergito ol under 1000 m mg/L surfacctant conceentration att pH 7. Fiigure 4-15 presents tthe comparrison on percentage cumulaative distribu ution of thee bubble sizee. 91    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figu ure 4-15. Com mparison betw ween particlee size analyseer and image processing teechniques forr size distributtion of microbubble dispeersions at surffactant conceentration of 1,000 1 mg/L att pH 7. The conditions are (A) rrhamnolipid using particlle size analyseer ( ), rham mnolipid using image proccessing appro oach ( ); and d (B) tergitol using particlle size analyseer ( ), tergittol using ima age processingg approach ( ). The com mparison inn Figure 4-1 15 shows thhat the rangee of microbubble size m measured by b image processsing approacch is smaller than that at by particlle size analyser as shoown by the shift of curve too the right of the scale, which iss consistent with what was reportted in the literature (Dai & Deng, 20003). The diifference inn size rangee is caused by the conntinuous ch hange in microbuubble size with w time an nd the differrent measurrement time between thhe two techn niques. The imaage processsing techniq que measurees the micro obubble sizze immediattely after fo ormation by takinng microscoopy snapsho ots of the m microbubbles, therefore it will captture the presence of 92    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  smaller microbubbles before disproportionation occurs (as discussed in Section 2.3.2). However when using particle size analyser, it takes between half to one minute of processing time before measurement starts. During this time span, the microbubbles undergo disproportionation, causing the bubbles to expand at the disappearance of smaller bubbles. Since image processing approach is able to capture bubble size right after creation, it gives a more accurate representation of bubble size distribution at its formation and so it is considered a better method for measuring microbubble size when compared to particle size analyser. 4.6.4. Change of Microbubble Size Distribution with Time The microbubble dispersion is an energetically unstable system and its bubble size distribution varies with time (Sebba, 1987). The study of how microbubble size evolves with time is fundamental to the understanding of drainage process taking place in microbubble dispersion. Photomicrographs of microbubbles were taken at 3 minutes intervals up to 30 minutes during the drainage process. The photographs were analysed using the MatLab image analysis tool (see Section 3.34). Figures 4–16, 4–17 and 4–18 present the evolution of microbubbles under static drainage for microbubble dispersions generated from rhamnolipid concentrations of 500 mg/L, 1,000 mg/L and 4,000 mg/L at pH 7. 93    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figu ure 4-16. Evolution of microbubble witth time for microbubble dispersion maade with 500 mg/L m rhamnollipid solution n at pH 7. 94    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figurre 4-17. Evolu ution of micrrobubble with h time for miccrobubble dispersion madde with 1,000 mg/L rhamnollipid solution n at pH 7. 95    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figurre 4-18. Evolu ution of micrrobubble with h time for miccrobubble dispersion madde with 4,000 mg/L rhamnollipid solution n at pH 7. The phootomicrograaphs reveal that bubblee sizes grow w with time. The changge of bubblle size is caused by an interpplay of a nu umber of prrocesses, in ncluding creeaming, dispproportionaation and liquid ddrainage. These T processes are ddiscussed in i full in Section 4.99, where drainage d mechannism for miccrobubble dispersion d iss proposed. In addittion, the change of mean bubble ddiameters fo or different rhamnolipiid concentraations at selectedd times wass summarised in Tablee 4–12. Thee results suggest that m microbubbles made 96    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  from lower rhamnolipid concentration tend to expand quicker than those made from higher concentration. This corresponds to the results in stability measurement where microbubble dispersion created from lower rhamnolipid concentration would have lower stability due to faster liquid drainage. The expansion of bubble size and liquid drainage are correlated - as more liquid drains away the average bubble size becomes larger, which in turn leads to increased liquid drainage (Vera & Durian, 2002; Wang & Narsimhan, 2004). Table 4-12. Summary of bubble diameters (mean ± 1 S.D.) in μm at selected times. Rhamnolipid concentration (mg/L) 500 1,000 4,000 Bubble diameters (μm) at selected times (seconds) 0 360 1,080 1,620 69 ± 4 92 ± 9 239 ± 24 410 ± 20 71 ± 1 91 ± 3 187 ± 12 316 ± 13 61 ± 6 98 ± 8 152 ± 18 294 ± 10 4.7. Comparison with Synthetic Surfactants Properties of rhamnolipid microbubble dispersions were compared with two chemical surfactants (tergitol and SDS) under the same mixing conditions. Both tergitol and SDS are commonly used in making microbubble dispersion. The measured half-life and gas hold-up are summarised and presented in Table 4 –13 to Table 4–14. Table 4-13. Stability (mean ± 1 S.D.) comparison. Surfactant Rhamnolipid Type Concentration 500 1,000 4,000 (mg/L) pH 6 457 ± 12 495 ± 19 546 ± 13 pH 7 405 ± 13 461 ± 15 519 ± 18 pH 8 385 ± 13 447 ± 19 503 ± 11 SDS Tergitol 500 1,000 4,000 1,000 346 ± 13 341 ± 11 364 ± 9 358 ± 12 345 ± 16 361 ± 19 417 ± 16 459 ± 17 450 ± 15 391 ± 5 383 ± 8 390 ± 6 The half-life values of rhamnolipid microbubble dispersion are in the range between 385 to 546 seconds, which are greater than both SDS (between 341 and 459 seconds) and tergitol (around 390 seconds). However, the gas hold-up values shown in Table 4–13 are similar among the surfactants, which are consistent with what is reported in Section 4.5 that surfactant type had no significant effect on gas hold-up values. 97    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  Table 4-14. Gas hold-up (mean ± 1 S.D.) comparison. Surfactant Type Concentration (mg/L) pH 6 pH 7 pH 8 Rhamnolipid 500 71%±0%c 67%±2% 68%±2% 1,000 SDS 4,000 500 1,000 Tergitol 4,000 1,000 72%±1% 69%±1% 70%±1% 70%±1% 72%±1% 69%±1% 71%±1% 69%±1% 71%±1% 69%±2% 70%±0% 70%±1% 68%±1% 69%±1% 70%±1% 70%±1% 69%±1% 70%±1% In addition, the measured stability, gas hold-up and size distribution are compared with findings reported in the literature as shown in Table 4–15. Range of values is presented. The references selected are reported to use the mixing speed at 8,000 rpm. Table 4-15. Comparison of rhamnolipid microbubble dispersion with synthetic surfactants. Surfactant Stability Gas Hold-up (seconds) (%) Rhamnolipid 385 – 546 67% - 72% Tergitol 383 – 391 69% - 70% SDS 341 – 459 69% - 72% AOT 30 – 930 12% - 59% Tergitol N.D. N.D. SDS 330 – 535 HTAB 141 – 525 N.D. Tween 80 413 – 498 Saponins 170 – 720 50% - 70% N.D. means it is not determined. Size Distribution (μm) 20 – 140 20 – 130 N.D. N.D. 30 – 300 Source This study This study This study Jauregi et al. (1997) Chaphalkar et al. (1993) N.D. Yan et al. (2005) 30 – 300 Kommalapati et al. (1996) Table 4–15 shows that rhamnolipid microbubble dispersions have properties similar to the surfactants reported in the literature. It demonstrates that rhamnolipid biosurfactant is a potential alternative to synthetic surfactants for making microbubble dispersion. 4.8. Effect of Bacteria Addition An important potential application of the microbubble dispersion is as a carrier of contaminant-degrading bacteria into a contaminated soil. For this reason it is desirable that the addition of bacteria cells does not lower the stability of microbubble dispersion. The effect of adding bacteria P.putida and R.erythropolis on microbubble dispersion stability and gas hold-up is summarised in Table 4-16. The mean and standard deviation are based on at least six measurements. 98    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  Table 4-16. Effect of bacterial cells addition on rhamnolipid microbubble dispersion stability and gas hold-up (mean ± 1 S.D.) at pH 7. Stability (seconds) Rhamnolipid Concentration (mg/L) Without Bacteria P.putida R.erythropolis Gas Hold-up (%) 500 1,000 4,000 500 1,000 4,000 405 ± 13 417 ± 20 N.D. 461 ± 16 483 ± 36 470 ± 17 519 ± 19 493 ± 36 N.D. 68% ± 2% 69% ± 1% N.D. 71% ± 1% 69% ± 0% 69% ± 1% 69% ± 1% 69% ± 2% N.D. N.D. means it is not determined. The addition of bacteria (P. putida or R.erythropolis) has no significant effect (P>0.05) on the dispersion stability and gas hold-up values at all rhamnolipid concentrations. The results are consistent with the findings of Park et al. (2009), where the addition of bacteria Burkholderia cepacia hardly affected the drainage characteristics of microbubble dispersion made from saponin, a plant surfactant. It is possible that the lipopolysaccharides at the outer membrane of the Gram-negative P.putida could act as a surfactant (Maier et al., 2003), however the number of cells added here is not sufficient to have significant effect on the dispersion properties. 4.9. Drainage Mechanism A microbubble dispersion resembles an emulsion and it has been suggested that their drainage mechanism differs from conventional foams, the latter typically exhibiting an exponential decrease in drainage with time (Jacobi et al., 1956; Ross, 1943; Save & Pangarkar, 1994; Sebba, 1987). The drainage of microbubble dispersions has been described in terms of microbubbles rising as per the Stroke’s velocity (Amiri & Woodburn, 1990) or a two-stage process consisting of an initial stage during which liquid drains under gravity followed by a stage in which foam breaks down due to thinning of films between bubbles (Save & Pangarkar, 1994; Yan et al., 2005). However, the two-stage process is similar to the drainage mechanisms proposed for conventional wet foams (Indrawati & Narsimhan, 2008; Koehler et al., 2000; Ross, 1943), and it does not provide satisfactory explanation to the “S” shape drainage curves observed both in this study and from previous studies (Jauregi et al., 1997; Save & Pangarkar, 1994; Yan et al., 2008; Yan et al., 2005). Consequently, the models existing in the literature do not provide an adequate representation of the drainage mechanism of microbubble dispersions. 99    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Based oon drainage data from rhamnolipidd microbub bble dispersiion and the improved drainage d model derived inn Section 4.4, 4 this seection prop poses a draainage mecchanism un nique to microbuubble dispeersion. A typ pical “S”-shhaped drain nage curve and a the draiinage rate, obtained o as the time derivaative of thee experimenntal drainag ge data aree shown inn Figure 4– –19. The mental data are shown in symbolss, while thee solid line represents ffitted modeel output experim from Eqquations [133] and [17]. Figurre 4-19. Typiccal drainage curve (trianggle symbol, for experim mental data; ssolid line for model output)) and rate of drainage d (astterisk symboll, for expeerimental datta; solid line ffor model outtput) for rhamn nolipid disperrsions (500 mg/L m surfactan nt concentration at pH 7). Arrows indi dicate corresp ponding axis. Symbols a, b, c and d denotee the corresp onding placeements of pho otomicrograpphs from Figu ure 4–20. Based oon the obserrved rate off drainage inn Figure 4– –19, three ph hases can bee identified – in the first phaase the draiinage rate in ncreases rappidly with time, t while in the secoond phase th here is a sharp ddecrease in the drainaage rate witth time, an nd finally in the thirdd phase thee rate of drainagge becomes relatively sm mall. Liquid drainage occcurs invariably in foam m and the raate of drain nage is influuenced by th he initial distribuution of the liquid betw ween the fiilm and plaateau border (Narsimhhan & Ruck kenstein, 1986; W Wang & Naarsimhan, 2004). 2 Durinng the firstt phase, the drainage i s a combin nation of liquid flow due to gravity through thhe plateau boarder and a the upw pward cream ming of p in aqueous a foaams (Koehleer et al., microbuubbles, whiile liquid drrainage is a common process 2000), non-stirred dispersions, like emuulsions expeerience the additional effect of creaming 100    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  during which bubbles rise to the top driven by buoyancy forces resulting from a density difference between the liquid and gaseous phases (Chanamai & McClements, 2000b; Indrawati & Narsimhan, 2008). Also, in microbubble dispersions, larger bubbles increase in size with time due to disproportionation, whereby the gas diffuses from the smaller bubbles (with greater internal gas pressure) into larger bubbles (with lower internal gas pressure) (Sebba, 1987). The growth of bubble size and the liquid drainage are correlated (Vera & Durian, 2002; Wang & Narsimhan, 2004). As more liquid drains away the average bubble size becomes larger, which in turn leads to increased liquid drainage. Initially, as shown in Figure 4-20a and Table 4-17, the microbubbles are spherical in shape with a mean diameter of 69 µm. Over time, the mean bubble size increases to 96 µm due to disproportionation as demonstrated by an expansion of the larger bubbles and the disappearance of smaller bubbles in Figure 4-20a to b, and seen via the increase in 90 percentile bubble size in Table 4-16. As the creaming velocity increases with bubble size (Chanamai & McClements, 2000b; Lau & Dickinson, 2007), the increase in bubble size causes the creaming velocity (i.e., buoyancy velocity) to increase with time during Phase I – a phenomenon that has also been reported by others (Jeelani et al., 2005). Creaming is hindered at higher emulsion concentrations (Chanamai & McClements, 2000a; Lau & Dickinson, 2007), and bubble expansion over time impedes the upward migration of other bubbles from the underlying region. Over time the liquid holdup and foam volume fraction gradually decreased, but no breakdown of microbubbles was observed and the total volume remained unchanged (Figure 4-21). 101    Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Figure 44-20. Photomiicrographs off the microbu ubble disperssion at differeent times. Phooto a represeents T = 0 second; b representss T = 360 seco onds; c repreesents T = 108 80 seconds; and d represennts T = 1620 seconds. Table 4-17. Chan nge of bubblee size with tim me for microb bubble disperrsion produceed from 500 mg/L m rham mnolipid solu ution. Time (secon nd) 0 3600 10800 16200 Meean±1 S.D. (µm) 69 ± 4 92 ± 9 2 ± 24 239 4 ± 20 410 10%iile (µm m) 28 29 70 94 Median M (µm) 67 80 240 488 102    90%ille (µm) 110 176 377 592 Refeerence in Figu ure 4–20 a b c d Chapter 4 – Characteerisation and d Drainage M Mechanism o of Microbubb ble Dispersioon  Fiigure 4-21. Variation V of fo oam volume ffraction, liquiid holdup and total volum me with time for f symbol microb bubble disperrsion formed with 500 mgg/L rhamnolip pid concentra ation at pH 66. The symbol repressents foam voolume fraction n. The symbol represents liquid d holdup. Annd the repreesents total vo olume. The seccond phasee starts when the disppersion losees its collo oidal characcter due to o bubble expansiion and behaves simiilarly to coonventional wet foam.. Without ccontinuous stirring colloidaal gas aphroons will eveentually sepparate into conventiona c al foam (Seebba, 1987). Yan et al. (20005) state thaat the formaation of connventional foam f occurss when 90% % of the totaal liquid has beeen removedd from the foam. How wever, Figure 4-20b shows thatt the bubblles have expandeed significaantly and thee larger bubbbles are cro owded and distorted att the end of Phase I, indicatiing that the microbubb ble dispersioon has lost its colloidaal characterr. In this reg gion the dominaant drainagee mechanism m is gravity--driven liqu uid flow through a plateeau border network (Indraw wati & Narssimhan, 200 08; Vera & Durian, 20 002). From m Figure 4-119 the timeescale of plateau boarder draainage (~10 000 sec), whhich domin nates Phasess I and II, iss about 3 to o 4 times larger thhan that forr creaming (~300 sec),, suggesting g that plateaau boarder ddrainage wiill occur over a ssubstantiallly longer peeriod of tim me than creaaming. The continuouss decrease in i liquid content due to liquuid drainagee, as shownn by the graadual decreaase in the liiquid holdup p within the foam m phase in Figure 4-21, causes a steady dro op in the ratte of drainaage. This prrocess is accomppanied by thhe expansion n of bubbless and the th hinning of fiilms (see Figgure 4-20b and c). 103    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  The bubbles have changed from spherical to polyhedral in shape. During Phase II the mean bubble size increases from 92 µm to 239 µm (Table 4-17), but, as in Phase I no perceptible bubble collapse was observed and the total volume remained unchanged (see Figure 4-21). The end of Phase II at about 1,000 seconds is accompanied by a flattering of the decrease in liquid holdup with less than 10% of the liquid being retained in the foam phase (see Figure 419). The half-life of microbubble dispersion from liquid drainage data is estimated as 457 seconds. The timescale of microbubble stability (~2 × 457 seconds) is therefore consistent with the timescale for removal of the majority of liquid (~1,000 seconds) by the end of Phase II, at which time the foam converts from a wet to dry foam, initiating the third phase. In Phase III the drainage of liquid is primarily from lamellae (films) under the influence of plateau boarder suction, which is a much slower process (Koehler et al., 2000; Yan et al., 2005). As shown in Figure 4-20c and d the bubbles are polyhedral in shape and continue to expand in conjunction with the thinning of films. The bubbles now have an average size at least 3 times larger than those in Phase I (Table 4-17). Since the timescale of film drainage is much smaller than that of plateau border drainage (Narsimhan, 1991), the film drainage will attain equilibrium over a short time interval (Wang & Narsimhan, 2004). This is evident by the flattening of liquid drainage profile and drainage rate in Figure 4-19 and stabilized liquid holdup within the foam in Figure 4-21. With the removal of more than 90% of the liquid from the foam and the thinning of the films, bubbles coalesce due to rupturing of films and eventually the foam phase starts to collapse, and is manifested as a gradual decrease in total volume (Figure 4-21) after about 1,000 seconds. A complete collapse of foam was observed about 2 hours after the start of experiment. The equation developed for the rate of drainage (Equation 17 in Section 4.4) fitted the observed data well in all three phases. 4.10. Chapter Summary Desirable mixing conditions for making rhamnolipid microbubble dispersions have been identified. The mixing apparatus consisted of a flat disk and two baffles. The preferred speed was 8,000 rpm for 3 minutes. The characterisation studies showed that rhamnolipid makes microbubble dispersions with properties similar to the synthetic surfactants reported in this study and in the literature. The stability of the rhamnolipid microbubble dispersion, prepared at rhamnolipid concentrations of 500 mg/L, 1,000 mg/L and 4,000 mg/L at pH 6 to 8, was in the range from 385 to 546 104    Chapter 4 – Characterisation and Drainage Mechanism of Microbubble Dispersion  seconds. The gas hold-up in the dispersion was fairly constant ranging from 67% to 72%, and majority of the microbubbles was in the size range of 20 μm to 140 μm. The stability of rhamnolipid microbubble dispersion was a function of factors such as surfactant concentration, pH, and salt and bacterial concentrations due to their influence on liquid viscosity and viscous drag, mechanical properties of lamellar walls, and bubble coalescence. Increasing the rhamnolipid concentration enhanced dispersion stability. The gas hold-up did not vary significantly with changing rhamnolipid concentration, but was affected by electrolyte concentration. Increasing rhamnolipid concentration had a significant effect on reducing the mean microbubble diameter. Adding bacteria when making the microbubble dispersion had no significant effect on its stability and gas hold-up. An improved drainage model for microbubble dispersion was presented in this study. The drainage of microbubble dispersion was best described by three distinct phases instead of the two phases previously assumed in literature. Initially, the drainage rate increased with time due to a combination of upflow migration of bubbles and downward liquid drainage under gravity. Following this phase, dispersion behaviour was similar to conventional wet foam. Here the drainage rate decreased with time and was dominated by liquid flow under gravity. Eventually, the foam became water deficient and started to behave like dry foam, where the drainage rate was small due to slow liquid release from lamella under capillarity suction. The modified drainage equation (Equation 13 in Section 4.4) provided better fit to the experimental results, and the derived drainage rate equation (Equation 17 in Section 4.4) corresponded well to the proposed three-phase drainage mechanism. 105    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  Chapter 5. Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the Interaction 5.1. Introduction It has been suggested that microbubble dispersion increases bacteria transport through soil by promoting adhesion of the contaminant-degrading bacteria to the bubble interface within a mobile aqueous phase (Jackson et al., 1998; Rothmel et al., 1998). Ripley et al. (2002) proposed that the bubble interface works like a “dynamic trapping surface of bacteria” due to hydrophobic attraction. While some studies demonstrated improved bacterial transport through soil columns using microbubble dispersion (Choi et al., 2009; Jackson et al., 1998; Rothmel et al., 1998), there are contradictory results (Rothmel et al., 1998). As discussed in the literature review Section 2.4.5, Choi et al. (2009) reported that saponin microbubble suspension was more efficient than the saponin solution in transporting Burkholderia cepacia through a sand column. A study by Rothmel et al. (1998) showed that microbubble dispersion made with Steol CS-330 surfactant substantially improved the transport of ENV 435, a variant of Burkholderia (Pseudomonas) cepacia, through a sand column in comparison to surfactant solution application, but the transport of ENV 435 was either unaffected or even inhibited when using microbubble dispersion produced from surfactants Tergitol 15-S-12 or Biosoft D-40, respectively. These findings indicate that surfactant may have direct impact on the effectiveness of microbubble dispersion as a bacterial carrier. Furthermore, it is reported that the attachment of bacteria to microbubbles is affected by bacterial cell surface hydrophobicity (Sharma et al., 2005). Surfactants have been shown to adsorb to abiotic surface and change its surface properties (e.g., hydrophobicity) (Bai et al., 1997; Bridgett et al., 1992; Brown & Jaffé, 2001; Li & Logan, 1999; Noordman et al., 1998). Surfactants can similarly affect the surface hydrophobicity of bacterial cells, thereby affecting the bacterial interaction with microbubble dispersion. However, the understanding of the effects of surfactant on bacterial cell surface is limited and to my knowledge no study addressing the factors that can influence bacterial attachment to microbubble dispersion has been published. To better understand the factors and mechanisms affecting microbubble dispersion as a carrier of bacteria, there are two key objectives in this chapter: 106    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  1. To investigate the interactions between bacteria and a rhamnolipid microbubble dispersion under the influence of various process parameters such as rhamnolipid surfactant concentration, salt and bacterial cell surface hydrophobicity. 2. To quantify the interaction between the bacteria and microbubble dispersion using contact angle measurement and the LW-AB surface thermodynamic model. In this thesis it is hypothesised that cell surface hydrophobicity is the dominant factor in influencing bacterial adhesion to microbubble dispersion. The adsorption of amphiphilic surfactant molecules makes hydrophilic cells more hydrophobic and hydrophobic cells more hydrophilic. Therefore the desired effectiveness of microbubble dispersions would be optimised if bacterial cell surface properties and surfactant are matched. Bacterial drainage experiments were performed to study the bacterial adhesion to microbubble dispersion under a range of testing conditions. Bacterial cell surface hydrophobicity was quantified and applied to the LW-AB surface thermodynamic model to estimate the surface free energy of the bacteria-microbubble interaction. Fluorescence microscopy was also employed to visually examine the bacteria-microbubble interaction. The findings from this study can be applied to optimize microbubble dispersion to be an effective bacterial carrier for bioremediation application. 5.2. Bacterial Adhesion to Microbubble Dispersion One of the foremost conditions in making a microbubble dispersion an effective bacterial carrier is to maximise the bacterial adhesion to the microbubble dispersion. In this thesis, bacterial drainage experiments were performed to investigate factors that affect the adhesion of P.putida and R.erythropolis cells to rhamnolipid microbubble dispersion. The drainage experiment is designed to quantify the separation of bacterial population in the microbubble and liquid phases by enumeration of bacteria in each phase. 5.2.1. Effect of Rhamnolipid Concentrations Effect of rhamnolipid concentrations on P.putida and R.erythropolis cells adhesion to microbubble dispersion was investigated. The rhamnolipid concentrations tested were 500 mg/L, 1,000 mg/L and 4,000 mg/L. These tests were carried out at pH 7 without the presence of salt. The procedure of the drainage experiment is explained in Section 3.7. The bacterial adhesion was expressed as the percentage of cells attached to the microbubble phase. Figure 107    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  5-1 preesents the mean m and sttandard devviation of th he percentaage bacteriaal cells adh hesion to microbuubble dispeersion with drainage tiime. The values v weree calculatedd from 4 reeplicated measureements. Fiigure 5-1. Efffect of rhamn nolipid conceentrations on percentage retention of P P.putida (A) and R.errythropolis (B B) in microbu ubble dispersiion at pH 7 at various dra ainage times. The rhamnolipid concen ntrations aree rhamnolipid d 500 mg/L ( ); rhamnollipid 1000 mg g/L ( ); andd rhamnolipiid 4000 mg/L ( ). Vertical b ar representss 1 standard deviation. Under aall tested coonditions, th he amount oof cells (botth P.putida and R.erythhropolis) ad dhered to microbuubble disperrsion decreaases graduaally with draainage time,, as shown iin Figure 5--1 A and 108    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  B. Cells are being washed away from the foam phase by gravity-driven liquid flow through the plateau border network of the microbubbles. Increasing rhamnolipid concentration has a significant effect on keeping more P.putida cells in the foam phase (P<0.05). For example, at 50% liquid drainage of approximately 480seconds, 24% more cells are retained in the dispersion made with 4,000 mg/L rhamnolipid compared to 1,000 mg/L. However, increasing rhamnolipid concentration yielded no significantly positive effect on retaining more R.erythropolis cells in the foam phase (P=0.547). It has been shown in Section 4.3 that increasing rhamnolipid concentration improved microbubble stability and that the presence of bacterial cells would not affect the stability. If the improved stability is the main factor in retaining more P.putida cells in the dispersion, the same effect would be observed for R.erythropolis cells. However, increasing rhamnolipid concentration has no significant effect on R.erythropolis cells adhesion to microbubbles. It is likely that the different cell surface properties of P.putida and R.erythropolis and their interaction with rhamnolipid could explain to the observed difference. These factors will be explored in detail in later sections. 5.2.2. Effect of Salt Concentrations Effect of salt concentrations on P.putida cells adhesion to microbubble dispersion was investigated. Sodium chloride (NaCl) was added to make up a concentration of 1,000 mg/L and 3,000 mg/L at constant rhamnolipid concentration of 1,000 mg/L. The tests were carried out at pH 7. Figure 5-2 presents the mean and standard deviation of the percentage P.putida cells adhesion to microbubble dispersion under the influence of NaCl. The values were calculated from 4 replicated measurements. 109    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figure 55-2. Effect of salt s concentrations on perrcentage reten ntion of P.putida in microobubble dispeersion at 1,000 mgg/L rhamnoliipid concentrration and pH H 7 at variouss drainage tim mes. The NaC Cl concentrattions are 0 mg/L ( ); 1000 mg/L m ( ); and 3000 mg/L ( ). Vertica al bar represents 1 standaard deviation. It is shhown in Figure 5-2 that the peercentage P.putida P ceells adheredd to micro obubbles decreassed with draainage timee. Increasingg NaCl con ncentration from zero to 1,000 mg/L m and further to 3,000 mgg/L has no significant s eeffect on P.p putida cellss adhesion ((P=0.124). It is knoown that inncreasing saalt concentraation lowerrs electrostaatic repulsioon between bacteria and bubbble surfacee due to com mpression o f electrostattic double layer (Jaureggi et al., 1997), and so wouuld affect bacterial b atttachment iff electrostattic interactiion plays a major rolee in the adhesioon process (An & Friedman, 20000; Huysm man & Verrstraete, 19993). In thiis study howeveer, the preseence of salt has no signnificant efffect on the amount a of ccells adhereed to the dispersiions, indicaating that ellectrostatic interaction has no sign nificant inffluence on bacterial b adhesioon to the disspersions. 5.2.3. E Effect of Baacterial Celll Surface P Properties As disccussed in Seection 2.6.2,, bacterial ccell surface hydrophobiicity plays aan importan nt role in the inteeraction bettween bacteeria and airr-water inteerface, wherre hydrophoobic bacterria show greater adhesion too air-water interface i thaan hydrophiilic cells (W Wan et al., 11994). The presence p of surfaactant, howeever, can afffect the inteeraction (Ducker et al., 1994). Thhe results in n Section 110    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  5.2.1 aalso suggest that bactterial cell ssurface pro operties wh hen interactting with different d rhamnoolipid conceentration cou uld affect thhe amount of o bacteria adhered a to thhe dispersio on. In this section thee effect of bacterial ceell surface hydrophobicity on baacterial adhesion to nvestigatedd at rhamnollipid concen ntration of 11,000 mg/L at pH 7 microbuubble dispersion was in by com mparing hyydrophilic P.putida w with hydro ophobic R..erythropoliis. The efffect of extracelllular polym meric substaances (EPS)) on P.putid da was also examined ssince EPS has h been shown to influence bacterial cell surfacee hydropho obicity (San nin et al., 20003). To reetain the EPS onn P.putida, the t cells weere not washhed with NaCl N (0.85% % w/v). Figuure 5-3 pressents the mean aand standarrd deviation n of the ppercentage bacterial b ceells adhesioon to micrrobubble dispersiion under the t differen nt cell surfa face propertties. The vaalues were calculated from 4 replicatted measureements. Figure 55-3. Effect of bacterial celll surface prop perties on bacteria retentiion (%) in miicrobubble dispersion at 1000 mg/L rham mnolipid con ncentration att pH7. The ba acteria are washed P.putidda ( ); unw washed P.pu utida with EP PS ( ); wash hed R.erythrop opolis ( ).Veertical bar represents 1 staandard devia ation. Figure 5–3 shows that the am mount of cellls adhered to the micrrobubble disspersion is affected by the cell surface properties. P.putida cells with EPS preseent demonsstrated significantly greater adhesion to t microbub bble disperrsion than the t washed cells (P<00.05). Hydrrophobic R.erythrropolis cellls showed significantly s y less adheesion to miccrobubbles when comp pared to P.putida da (P<0.05). At 480 secconds (whicch is about half-life h of the dispersiion), less th han 10% 111    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  of the R R.erythropollis bacteria are retainedd in the foam m phase, wh hich is signiificantly low wer than P.putida da with apprroximately 47% 4 remainned and P.p putida EPS with w 53% re remained. After A 600 secondss, there are virtually v no o R.erythroppolis cells leeft in the foaam phase. The inccreased bactterial adhesiion in microobubble disspersion witth P.putida EPS presen nt can be explained by a nettwork of EP PS formed at the micrrobubble su urface, as shhown in Fig gure 5-4 below. The networrk of EPS trraps more bbacterial cells in the diispersion. FFigure 5–4 A shows the rham mnolipid microbubble m e surface w without P.pu utida presen nt and B reeveals the P.putida P cells wiith EPS on microbubblle surface. F Figure 5–4 C and D reeveal a 3-diimensional network of EPS after the frozen f rham mnolipid soolution was sublimated d. The subllimation pro ocess in obubble surfface. SEM evvaporates anny ice crystal formed oon the micro Figure 5-4. Cryo-SE EM images off microbubblle dispersion containing P.putida P with E EPS. The disspersion was prooduced from 1,000 mg/L of o rhamnolipiid solution. Im mage A show ws microbubbble dispersion n without P.putidda present; im mage B showss P.putida witth EPS on miicrobubble su urface; and im mages C and D show the 3-d dimentinal neetwork of EP PS after sublim mation. The liteerature preddicts that mo ore hydrophhobic bacterria will be attracted to tthe bubble interface i due to strong hydrrophobic atttraction whhen comparred with hy ydrophilic ccells (Ripley et al., 2002; W Wan et al., 1994). 1 How wever, it wass not expeccted that mo ore hydrophhobic R.erythropolis cells weere washed out when compared wiith hydroph hilic P.putid da bacteria. 112    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  Further studies were therefore carried out to better understand why there was less R.erythropolis cells adhered to microbubble dispersion than P.putida bacteria. It has been discussed in Section 2.5 that the tendency for bacteria to attach to any surface depends on the properties of the interacting surfaces, the liquid phase and the bacteria themselves (Bai et al., 1997). Various studies have shown that surfactants absorb to substratum and bacterial surfaces to change their surface properties, such as hydrophobicity, and so their interaction with surrounding environment (Bai et al., 1997; Bridgett et al., 1992; Brown & Jaffé, 2001; Li & Logan, 1999; Noordman et al., 1998). It is therefore hypothesised that rhamnolipid interacts and alters the cell surface hydrophobicity of P.putida and R.erythropolis cells by making hydrophilic cells more hydrophobic and vice versa, thereby changing their interaction with microbubble surfaces. Studies on bacteria-surfactant interaction were then carried out to investigate the hypothesis. A surface thermodynamic model based on LW-AB approach was used to quantify the influence of surfactant on bacterial cell surface hydrophobicity and the interaction between bacteria and microbubble and to help explain the observed discrepancy in bacterial adhesion to microbubble dispersion. The results of these studies were discussed in the following sections. 5.3. Bacteria/Surfactant Interaction Rhamnolipid surfactants can potentially influence surface properties of not just the bubble surfaces but also bacteria cells and therefore their tendency to attach to microbubbles. Surfactants adsorb to air-water interface to form a surfactant microlayer that predominate the surface properties of the air-water interface (Dahlbäck et al., 1980; Marshall, 1980; Schäfer et al., 1998a). For example, it has been shown that the adsorption of sodium dodecylsulfate surfactant at the hydrophilic air-water interfaces render them hydrophilic (Ducker et al., 1994). There is however limited understanding on how surfactants affect bacterial cell surface hydrophobicity. The adsorption of nonionic polyoxyethylene surfactants onto surface of a Sphingomonas sp. was shown to increase with increasing aqueous surfactant concentration above its CMC (Brown & Ai Nuaimi, 2005). Brown and Jaffé (2006) continued to show that the hydrophilic Sphingomonas sp became hydrophobic with the adsorption of polyoxyethylene surfactant C18E10 at a concentration four times its CMC. However, others 113    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  showedd that rhamnnolipid adso orbed to Pseeudomonass aeruginosa a cells folloowing secon nd-order adsorption kineticss at a concentration b elow its CM MC level, and a increassed the celll surface hydrophhobicity (Yuuan et al., 2007; 2 Zhongg et al., 200 07). While these t studiees showed in ncreased hydrophhobicity wiith surfactan nt adsorptioon onto hyd drophilic ceells, the effeect on hydrrophobic bacteriaa has not beeen well investigated. Adsorpttion tests were w perform med to inveestigate how w rhamnolip pid interactss with P.putida and R.erythrropolis cellls. Tergitol was also teested to find out wheth her the inteeraction of the t nonionic suurfactant wiith bacterial cells was th the same as the ionic rh hamnolipid.. Also Terg gitol was selectedd over SDS because Teergitol is bioocompatiblee with bacteeria but SDSS is not. 5.3.1. A Adsorption of Rhamno olipid to R..erythropollis Adsorpttion tests were perfformed to examine whether w rh hamnolipid would ad dsorb to R.erythrropolis cellls. Rhamnolipid concenntration waas measured d following method in Section 3.8.1. Innterference on absorbaance from bllank R.eryth hropolis sup pernatant w was found to o be 0.07 and waas subtracteed to correect for the true rham mnolipid con ncentrationn. The rham mnolipid concenttration in suupernatant is i presentedd in Figure 5-5. Averaage concentr trations with h 1 S.D. based oon 2 indepenndent measu urements weere shown. Figgure 5-5. Adssorption of rh hamnolipid oon R.erythropo olis showing the t equilibriuum rhamnoliipid concenttration after mixing of R.eerythropolis w with rhamnollipid solution (concentratiions shown in n x-axis). R.erythrropolis concen ntrations aree at 5.4×107 cffu/mL ( ), 1.1×108 cfu/m mL ( ) and 22.2×108 cfu/m mL ( ). Verrtical bar rep presents 1 sta andard deviattion. 114    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figure 55-5 shows that t the amo ount of rham mnolipid reemained in the t supernat atant is less than the amountt added, inddicating thatt rhamnolippid was adso orbed to R.eerythropoliss cells. The amount of rham mnolipid adssorbed increeases with iincreased co oncentration n of R.erythhropolis cellls due to the preesence of increased i absorbent. a F Figure 5-6 presents the t adsorpttion coefficcient of rhamnoolipid on R.erythropo R lis cells veersus equillibrium rhaamnolipid cconcentratio on. The adsorption coefficcient show ws the amoount of rh hamnolipid adsorbed per unit mass m of R.erythrropolis cells at equilibrrium rhamnnolipid conccentration. Figuree 5-6. Normallised adsorption of rhamn nolipid on R.eerythropolis. R.erythropolis R s concentrations are 5..4×107 cfu/mL L ( ), 1.1×1008 cfu/mL ( ) and 2.2×10 08 cfu/mL ( ). The norrmalised addsorption vaalues increassed with rhaamnolipid concentratio c on at R.erythropolis cell conncentrationss of 5.4×10 07 cfu/mL and 1.1×10 08 cfu/mL as a shown iin Figure 5-6. 5 The increasee in adsorpttion is posssible when ssurfactant is present in n abundancee with the bacterial b cells beeing the liimiting facttor, and iss due to laateral surfactant adsorrption onto o sorbed surfactaant monolayyer at high surfactant cconcentratio on (Brown & Ai Nuaim mi, 2005). In I lower microbiial concenttrations, rh hamnolipid adsorption n takes plaace in muultilayers, but b this mechannism is nott used at higher h miccrobial conccentrations. At high R R.erythropo olis cell concenttration of 22.2×108 cfu u/mL, due to the abu undance of absorbent,, the adsorrption is stabiliseed as monollayer adsorp ption predom minates. 115    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  5.3.2. A Adsorption of Rhamno olipid ontoo P.putida Adsorpttion tests were w perform med to exam mine wheth her rhamnolipid adsorbbs to P.putid da cells. Interferrence on abbsorbance frrom blank P P.putida su upernatant was w found tto be 0.09 and a was subtractted to correct for the true rhamnollipid concen ntration. Thee rhamnolippid concentrration in supernaatant is pressented in Fiigure 5-7. M Mean concentrations with w 1 standaard deviatio on based on 2 inddependent measuremen m nts were shoown. Figure 5-7. Adsorpttion of rhamn nolipid on P.pputida showin ng the equilib brium rhamnnolipid concen ntration affter mixing off P.putida witth rhamnolip pid solution (cconcentration ns shown in xx-axis). P.putiida concentrations aree at 6×107 cfu u/mL ( ), 1. 2×108 cfu/mL L ( ) and 2.4×108 cfu/mL L ( ).Verticcal bar representts 1 standard deviation. A peculliar trend was w observed d after mixiing rhamnollipid with P.putida, P whhere the abssorbance readingg in the supeernatant inccreased at thhe end of th he adsorptio on test. It aappears that there is more rhhamnolipid than what was initiallly added in the supernatant, as shhown in Fig gure 5-7. For exaample, at P.pputida cell concentratiion of 6×10 07 cfu/mL, the t absorban ance reading g returns a rhamnnolipid conncentration of o 704 mg/L /L, which iss 204 mg/L more thann the initiallly added concenttration (5000 mg/L). Furthermore F e, Figure 5–7 5 shows that the aamount of “extra” rhamnoolipid in suupernatant increases with bacteerial cell concentratio c on and thee initial rhamnoolipid conceentration. It appeaars that rham mnolipid was w being seecreted by bacteria b during the adsoorption testt, adding to the rhamnolipiid concentrration. How wever, it iss unlikely that P.putidda would produce rhamnoolipid underr the adsorrption expeerimental co onditions because therre was no suitable 116    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  growth ingredients present that would support rhamnolipid production by P.putida (Martínez-Toledo et al., 2006). There were only the bacterial cells and rhamnolipid molecules present in the adsorption test. Attempts had been made to measure rhamnolipid concentrations using HPLC method. However, the rhamnolipid concentrations obtained by the HPLC method had high uncertainty due to the adsorption of the rhamnolipid surfactant to the HPLC column. It has been shown that rhamnolipid biosurfactant can release lipopolysaccharide (LPS) from the cell surface of Pseudomonas spp. through chelation with Mg2+, a metal that is crucial for maintaining strong LPS bond with cell surface (Al-Tahhan et al., 2000). The weakening of LPS-cell surface interaction results in the release of LPS to aqueous medium. Their study also found that the amount of LPS released was dependent on rhamnolipid concentration (AlTahhan et al., 2000). Since P.putida bacteria are Gram-negative with LPS present on the cell surface, it is possible that the rhamnolipid formed complexation with Mg2+, causing the release of LPS from the P.putida 852 cell surface into the aqueous solution. If this is the case, after removing the cells by centrifugation, the soluble LPS remaining in the supernatant will contribute to an increase in the absorbance reading, which appears to be an increase in rhamnolipid concentration. On the other hand, the Gram-positive R.erythropolis bacteria do not have LPS on the cell envelop (see Section 2.5.1) and it is not expected to release Mg2+ during the adsorption test. To test this hypothesis, the supernatant from the adsorption tests was tested for the presence of LPS and Mg2+. Mg2+ concentration in the supernatant was measured using flame-AAS. The method is discussed in Section 3.9. The results presented here (Figure 5–8) are for adsorption test with P.putida concentration of 6×107 cfu/mL and that with R.erythropolis concentration of 5.4×107 cfu/mL. The Mg2+ present in the bacteria blank was also measured. The Mg2+ concentration detected in P.putida blank was 0.13 mg/L and that in R.erythropolis blank was 0.16 mg/L. The Mg2+ concentrations presented in Figure 5–8 are corrected values with the background concentration deducted. The results represent the mean of three measurements by flame-AAS. 117    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figuree 5-8. Mg2+ cooncentration in supernataant after mixiing of bacteriia (P.putida aat 6×107 cfu/m mL and 7 R.eryythropolis at 5.4×10 5 cfu/m mL) with surffactant (Rham mnolipid and Tergitol) at vvarious surfa actant concenttrations. Thee surfactant concentration ns are 500 mg g/L ( ), 1000 0 mg/L ( ) and 4000 mg g/L ( ). As show wn in Figurre 5–8, with h rhamnolippid present, Mg2+ was reeleased from m P.putida,, and the amountt of Mg2++ released into the solution increased i with w increaasing rham mnolipid concenttration. At 500 mg/L rhamnolipiid, about 0.17 0 mg/L of Mg2+ w was detected d in the supernaatant. As the rhamn nolipid conncentration increased to 4,000 mg/L, the Mg2+ 2 concenttration jumpped to almo ost 1.5 mg/L L. The releease of Mg2+ indicates the releasee of LPS from P P.putida celll surface. The T higher the concentration of Mg M 2+ the m more LPS would w be expecteed in the sollution. The result in Figgure 5–8 is consistent with w what w was reported d by AlTahhann et al. (20000). R.erythropolis is ggram-positiv ve bacteria and does nnot have LP PS on its cell walll and so it is not expeccted to havee LPS releaased. This iss consistent with the neegligible concenttration of M Mg2+ detecteed in the suupernatant. Tergitol su urfactant is nnon-ionic in n nature and no complexattion with Mg M 2+ is exppected. Thiis is also consistent c w with the neegligible concenttration of Mg M 2+ detected in the suppernatant wiith tergitol present. p The Mgg2+ results indicate thaat LPS willl be releaseed from thee P.putida cell surfacee during rhamnoolipid treatm ment. LPS in the suppernatants was w semi-q quantified ffrom the P.putidaP rhamnoolipid superrnatant usin ng SDS-PAG GE analysis (Section 3.11). 3 Figur ure 5-9 pressents the results oof the PAGE E analysis. 118    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figure 55-9. SDS-PAG GE gel showin ng LPS in P.pputida-rhamn nolipid supernatant. Lanee 1, purified LPS L from supernatantt from: P. puttida broth Salmoneella; lane 2, rh hamnolipid at a 1000 mg/L;; lanes 3-10, concentrated c (lane 3)); P. putida trreated with sa aline only (lan ne 4); P. putid da treated wiith rhamnolippid at 500 mg g/L (lane 5), 10000 mg/L (lane 6) and 4000 mg/L (lane 77); P. putida trreated with tergitol at 5000 mg/L (lane 8), 1000 mg/L (lane 9)) and 4000 mg/L m (lane 10)). PAGE analysis off concentrated supernattants taken from P. pu utida culture res incubateed in the presencce and absennce of rham mnolipid andd tergitol id dentified sub bstantial am mounts of LP PS were releasedd by the rhaamnolipid treatment, t aas shown in n Figure 5-9 9. The PAG GE banding patterns shown for supernaatants of P.p putida cellss treated witth rhamnoliipid (lane 5 to7) are siimilar to those ddemonstrated for P.aerruginosa (A Al-Tahhan et e al., 2000 0) and compprise a stro ong core LPS baand (arroweed in Figuree 5-9) and nnumerous other o bands reflecting minor LPS species and suurface proteeins releassed by rhaamnolipid treatment. For otherr supernataants, no characteeristic LPS S bands were w appareent. These results arre consistennt with th he Mg2+ concenttration meassured in sup pernatant annd support the t hypothesis that rham mnolipid caauses the release of LPS from m P.putida. Survivaal test was performed p to t examine if the releaase of LPS has h a detrim mental effecct on the viabilityy of P.putiida cell. Concentratioon of P.puttida cells after a the addsorption teests was comparred with conntrol condition withoutt rhamnolip pid present (see ( Sectionn 3.6). The viability v is repreesented byy the survival rate w which is th he ratio off the cell cconcentratio on with rhamnoolipid presennt against th he cell conccentration un nder controll condition. The surrvival rate (mean and 1 standard ddeviation off six samples) is summaarised in Taable 5-1. Despitee the releasee of LPS frrom P.putidda cell surfface, survival test show ws that on average, a 119    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  100% of the P.putida cells at 500 mg/L rhamnolipid were alive after the adsorption tests. Increasing the rhamnolipid concentration did not have a significant effect on the survival rate (P>0.05). However, the relatively large uncertainty of the survival rate at 4,000 mg/L rhamnolipid level suggests that rhamnolipid at such a high concentration could impact on the survival rate. Table 5-1. P.putida survival rate (mean and 1 standard deviation) after rhamnolipid treatment. Rhamnolipid Concentration (mg/L) 500 1,000 4,000 Survival Rate (%) 100% ± 13% 75% ± 20% 70% ± 21% 5.3.3. Adsorption of Tergitol onto Bacterial Cells Rhamnolipid is an anionic biosurfactant. To study if the interaction of the non-ionic surfactant with bacterial cells was the same as the ionic rhamnolipid, the non-ionic synthetic surfactant tergitol was used for adsorption tests on R.erythropolis and P.putida bacteria. The same procedure was carried out following the adsorption test method in Section 3.5. A standard curve for tergitol spectrophotometry analysis at 225 nm has been developed (Section 3.8.2). The absorbance signal at 225 nm is the highest for tergitol. However at the same time, interference from bacteria blank was significant at the short wavelength of 225 nm. It is caused by the absorbance by cell surface substances (Chen & Wang, 2001; Mayer et al., 1999). At a tergitol concentration of 5,000 mg/L, for example, the absorbance is 0.163. At 500 mg/L, the signal drops to just 0.018. But the absorbance from blank samples of R.erythropolis was 0.133 and that from P.putida was 0.402. The high absorbance from the bacteria blank has interfered with the tergitol measurement. After subtracting the reading from bacteria blank, only one condition yielded positive tergitol reading. Similary, attempts had been made to measure the tergitol concentrations using HPLC method. However, the tergitol concentrations obtained by the HPLC method had high uncertainty due to the adsorption of the surfactant to the HPLC column. After mixing R.erythropolis cells (5.4×107 cfu/mL) with 4,000 mg/L of tergitol, about 2,900 mg/L of tergitol was found to remain in supernatant, which means that 28% of the tergitol was adsorbed to R.erythropolis cells. For lower tergitol concentrations, the adsorption was not sensitive enough to be detected due to the interference from bacteria blank. Other studies 120    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  have demonstrated that non-ionic polyoxyethylene surfactant adsorbs to bacterial cells and the adsorption increases with surfactant concentration (Brown & Ai Nuaimi, 2005; Brown & Jaffé, 2006). Since tergitol surfactant has similar chemical structure to polyoxyethylene surfactant, it is reasonable to assume that tergitol surfactants adsorb onto the cell surface of both R.erythropolis and P.putida. 5.4. Microbial Adhesion to Hydrocarbon Assay for Cell Surface Hydrophobicity Measurement Experiments in previous section demonstrate that surfactants interact with bacterial cells and the interaction can result in adsorption of surfactants onto the cell surface or cause the release of cell surface LPS, depending on the types of surfactant and bacteria involved. The change in cell surface composition can cause a change in cell surface hydrophobicity. There are two commonly used assays for measuring bacterial cell surface hydrophobicity. They are the microbial adhesion to hydrocarbon (MATH) assay and contact angle measurement on a bacterial lawn. MATH assays have been used extensively to measure bacterial cell surface hydrophobicity. The assay was first developed by Rosenberg (1984) based on the simple rationale that hydrophobic organisms would partition to the hydrophobic hydrocarbon phase while hydrophilic organisms would remain in aqueous phase. Aqueous bacterial suspension is mixed with test hydrocarbon (e.g. n-hexadecane) and the absorbance readings of the aqueous phase before and after mixing are measured (see Section 3.8.1 for method). The experimental data is shown in Appendix IX. The hydrophobicity results are presented as a percentage of cells partitioned to the hydrocarbon phase. The higher the percentage of partition represents the higher the hydrophobicity of the bacterial cell surface. Figure 5-10 shows the MATH results for R.erythropolis and P.putida with and without the presence of rhamnolipid. 121    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figure 5-10. R.erytthropolis and P.putida adh herence to hex xadecane. Rh hamnolipid cooncentrations are 0 mg/L and 50 mg/L L (x-axis). Bacteria tested are R.erythro opolis at 5.4×107 cfu/mL ( ) and P.pu utida at 7 6 6×10 cfu/mL L ( ). Verticcal bar represents 1 standard deviationn. Withouut rhamnollipid preseent, the reesults reprresent intrinsic bacteerial cell surface hydrophhobicity. Figure 5–10 shows thaat without rhamnolipid present, about 95% % of the R.erythrropolis cells adhered to o hexadecanne, indicatin ng that the cells c are verry hydropho obic. On the otheer hand, lesss than 7% of o P.putida bbacteria adh hered to hex xadecane annd so compaaratively their cell surface iss very hydro ophilic. mg/L, the rhamnolipid r d concentraation is justt above its CMC of 440 mg/L (Z Zhang & At 50 m Miller, 1994), but well below w the rhamnnolipid conccentrations investigateed in the ad dsorption tests (rrefer to Secctions 5.3.1 and 5.3.22). The ad ddition of 50 5 mg/L oof rhamnoliipid has negligibble effect on o the cell hydrophobbicity of R.erythropol R lis as show wn in Figurre 5–10. Howeveer, the absoorbance of the aqueouus phase waas not meaasured for P P.putida du ue to the formation of stablle emulsion n with rham mnolipid su urfactant in the aqueouus phase. Previous P studies reported thhat stable em mulsion forrms at surfaactant concentration abbove the CM MC and deters aaccurate MA ATH measu urement (Guuellil et al., 1998; Zhon ng et al., 20008). Increasiing the rham mnolipid co oncentrationn to 500 mg g/L and 1,00 00 mg/L deecreases the amount of R.eryythropolis cells c adherin ng to hexaddecane, indiicating a deecrease in ccell hydroph hobicity. The exxperimentall variation at 1,000 mg/L rhaamnolipid concentratio c on is due to the 122    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  interference of absorbance in the aqueous phase by emulsion formation. At 2,000 mg/L rhamnolipid concentration, the formation of stable emulsion deters accurate measurement. In addition to the formation of emulsion with surfactant present, bacterial partition to hydrocarbon represents an interplay of all physcio-chemical and structural factors rather than just the hydrophobic interaction (van der Mei et al., 1998). Furthermore the MATH results are presented in relative terms, which cannot be used directly in model calculations such as the thermodynamics model. An alternative method based on contact angle measurement was used for further study. 5.5. Contact Angle Measurement Contact angle measurement provides an improved and direct method to MATH assay to determine bacterial cell surface hydrophobicity. The theoretical background of the contact angle method is discussed in Section 2.7 and its experimental procedure is detailed in Section 3.8.2. In this section, contact angle measurement was carried out to assess the effect of surfactant adsorption on cell surface hydrophobicity. 5.5.1. Bacterial Lawn Preparation Preparing a homogenous bacterial lawn is an important step towards obtaining consistent and accurate contact angle values. A bacterial lawn is formed by depositing bacterial cells on 0.45 μm (pore size) nitrocellulous filter membrane via vacuum suction. A sufficient number of cells are needed to form a uniform lawn surface. Tests were undertaken to determine the minimum volume of bacterial suspension needed for making a uniform lawn. Scanning electron microscopy (SEM) was employed to examine the bacterial lawn (method refers to Section 3.12.2). Figure 5-11 presents the SEM images of bacterial lawn prepared using various cell numbers. SEM images A and B of Figure 5–11 reveal the presence of cavities on the bacterial lawn when there were insufficient bacteria present. Image A shows the lawn surface formed using 10 mL of P.putida suspension at about 6×107 cfu/mL, which contains more voids on the surface when compared to the lawn surface formed with 20 mL of the same P.putida suspension (Figure 5–11B). 123    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figure 5-11. SEM im mages of bacterial lawns p prepared for contact angle measuremeent. A, P.putid da lawn with 10 mL of cells at 6×107 cfu//mL; B, P.pu utida lawn witth 20 mL of cells at 6×107 cfu/mL; C, P.putida P lawn w with 40 mL off cells at 6×10 07 cfu/mL; B, R.erythropollis lawn with 40 mL of cellls at 5.4×107 cfu/mL. c By incrreasing the volume v of bacterial b suuspension to o 40 mL, im mages C andd D of Figu ure 5–11 show thhat uniform mly depositeed bacteria lawn is fo ormed, expo osing a hom mogenous layer of bacteriaal cells for contact c anglle measurem ment. Imagee C shows the t lawn sur urface made with 40 mL of w washed P.putida cells at cell conccentration of 6×107 cfu u/mL. Imagee D shows the t lawn surface made withh 40 mL off washed R R.erythropollis cells at cell concenntration of 5.4×107 cfu/mL. 5.5.2. C Contact Angles as A Function F off Time The baccterial lawnns prepared from 40 mL L of bacterial suspensio on were useed for contaact angle measureement. It is known that the contacct angles off all dropletss on the baccterial lawn n slightly decreassed as a funcction of tim me (Busscheer et al., 198 84). To ensu ure consisteency of the method, nvestigate thhe effect off time on contact angle meassurement were made att regular inttervals to in unction of tiime is presented in the variiation of coontact anglee. Contact aangles of water as a fu Figure 55–12. It was observed that the air--dried bacteerial lawn ad dsorbed thee liquid, cau using the droplet to spread out o and thereeby the graddual decreasse in contacct angle. 124    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figure 55-12. Water contact c anglee on bacteriall lawn surfacee as a function of time. Thhe bacterial la awns are P.putida a lawn ( ) and R.eryth hropolis lawn n( ). To maiintain the method consistency, contact ang gle was taken at tim me zero forr all the measureements reported in thiis study. It is also a common c prractice in thhe literaturee to use contact angle readdings at tim me zero for bbacterial laawn measurrement (Bussscher et all., 1984; Daffoncchio et al., 1995; Ham madi et al., 2008). Bussscher et all. (1984) reeported thatt contact angles aat 2, 4, and 6 seconds for f the samee bacteria on o separate filters f oftenn differed markedly, m but the values obtaained at tim me zero werre comparab ble within experimenta e al error. Preeviously, contact angle at tiime zero was w obtainedd by linear extrapolation, howeveer in this sttudy the me zero andd beyond. Figure 5– digital ggoniometer was set up to capture the contact angle at tim 13 show ws the imagges of dropleets on bacteerial lawn caaptured by the t goniomeeter at time zero. 125    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figure 5--13. Images oof water drop plets on bacterial lawn. 5.5.3. B Background d Contact Angle A Meassurement on o Filter Pa aper In the uunlikely eveent of a non-uniform ddeposition of bacteriall cells, diaggnostic liqu uids may interactt with the exposed e filtter membraane, skewin ng the conttact angle vvalues for bacterial b surface.. Also, if surfactant s can accumullate on the filter mem mbrane, the residual su urfactant may aff ffect the conntact angle reading. Too confirm that t the residual surfacctant did no ot cause significcantly adverrse effects on the conntact angle measuremeent, surfacttant solutions were rinsed through thhe filter membrane w without baccterial lawn n present aand the efffects of ngle readinngs establisshed the surfactaant concenttrations weere measurred. These contact an backgroound valuess of the filtter membranne. Six ind dependent measuremen m nts were tak ken. The mean annd 1 standarrd deviation n of the meaasurements are presented in Figuree 5-14. 126    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Fiigure 5-14. Coontact angle measuremen nt on filter paper rinsed wiith surfactannt solutions. A – rhamn nolipid surfactant. B – terrgitol surfactaant. The conttact angles arre water conttact angle ( bromon naphthalene contact c anglee ( ); and formamide contact c angle ( stan ndard deviattion. ); 1- ). Verttical bar reprresents 1 The conntact anglees on filter membrane did not ex xhibit a trend of eitheer an increaasing or decreassing contact angle with increasing surfactant concentratio c on, as show wn Figure 5– –14. The mean vvalues remaiined fairly constant wiith the chan nge of surfaactant conccentration, but b there was higgh variabiliity between n the individdual valuess as shown by the stanndard deviaation on Figure 55–14. It is known k that contact anggle measureement is sen nsitive to suurface hetero ogeneity (Busschher et al., 1984). Since the meaasurement was w made on a filterr membranee which contains numerous micro-cav vities, it m may have been b that th he heterogeeneity of th he filter membraane surfacee attributed d to the higgh variability in the measured vvalues. Thiis result 127    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  illustrates the importance of forming a smooth bacterial lawn surface without voids for obtaining consistent contact angle values. The high variation was not seen for contact angles measured on bacterial lawns, suggesting that smooth bacterial lawn surfaces were formed (Section 5.5.4). 5.5.4. Contact Angle Results Contact angles of water, 1-bromonaphthalene and formamide droplets on lawns of P.putida and R.erythropolis are presented in Table 5–2. The lawns were made from 40 mL of the bacterial suspension as indicated in Section 5.5.1. The mean contact angle and 1 standard deviation of six independent measurements are shown (see Appendix X for experimental values). Table 5-2. Contact angles (θ/o; mean and 1 standard deviation) for P.putida and R.erythropolis. Surfactant treatment (mg/L) θWater θ1-Bromonaphthalene θFormamide P.putida R.erythropolis P.putida R.erythropolis P.putida R.erythropolis 34.7±1.0 95.7±1.0 47.9±1.1 41.6±1.4 46.6±0.9 68.2±3.5 36.8±2.6 35.0±0.5 43.7±1.8 92.0±2.8 87.7±1.4 79.5±1.1 53.1±4.7 54.1±4.4 61.4±4.7 37.7±1.0 40.4±1.2 38.9±3.7 46.5±0.6 46.1±1.0 49.1±0.7 66.6±0.8 66.5±4.0 62.7±3.2 36.7±0.5 37.9±1.3 44.0±1.3 94.3±4.6 92.9±4.2 84.7±1.8 34.2±1.4 32.4±0.6 29.3±1.1 39.9±4.2 41.1±1.6 41.6±1.7 38.2±5.6 35.4±3.2 37.6±1.7 68.9±0.9 68.9±0.7 65.9±1.7 No surfactant Saline alone Rhamnolipid 500 1000 4000 Tergitol 500 1000 4000 The contact angle values in Table 5-2 show that P.putida was significantly different from R.erythropolis for all three testing liquids and the surfactant conditions tested (P<0.05). The values of R.erythropolis were generally higher than P.putida, except for 1-bromonaphthalene angles with tergitol surfactant. Surfactant type had a significant effect on water contact angles for R.erythropolis (P<0.05), but did not have an impact on the water contact angles for P.putida (P>0.05). Surfactant concentration had a significant effect on water contact angles for both P.putida and R.erythropolis (P<0.05), In general, the angles increased with increasing surfactant 128    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  concentration for P.putida, but decreased with increasing surfactant concentration in the case of R.erythropolis. The 1-bromonaphthalene angles for P.putida increased slightly with increasing surfactant concentration (P<0.05), but the effect was not significant between the pairs of salinerhamnolipid 500 mg/L and rhamnolipid 500 mg/L-rhamnolipid 1,000 mg/L (P>0.05). However for R.erythropolis, the effect of surfactant concentration did not have a significant effect on the 1-bromonaphthalene angles (P>0.05). Increasing rhamnolipid concentration from zero to 1,000 mg/L had no significant effect on the formamide contact angles of P.putida (P>0.05), but increasing the concentration further to 4,000 mg/L greatly increased the contact angle (P<0.05). It was an opposite case with tergitol surfactant, where increasing the concentration from zero to 500 mg/L had significantly reduced the contact angle (P<0.05). However, further increase in concentration yielded no effect (P>0.05). The surfactant type and concentration for R.erythropolis did not have a measurable effect on the formamide contact angles (P>0.05). The standard deviation of the contact angles ranged from 0.5o to 5.6o, with majority of the standard deviations less than 2o. This range of variability is similar to the standard deviation (varying from 0o to 5o) reported by Hamadi and Latrache (2008). The contact angles for P.putida cells with EPS present were also measured after treatment with 1,000 mg/L rhamnolipid. The values are summarized and compared with the P.putida without EPS present in Table 5–3. Table 5-3. Contact angles (θ/o; mean and 1 standard deviation) for washed P.putida and unwashed P.putida (with EPS present) at 1000 mg/L rhamnolipid concentration. P.putida P.putida with EPS θwater 35.0±0.5 39.2±4.2 θ1-Bromonaphthalene 54.1±4.4 54.1±1.7 θFormamide 46.1±1.0 48.9±2.9 The water contact angle of unwashed P.putida cells with EPS is slightly higher than that of washed P.putida cells (P<0.05), indicating that the cells are more hydrophobic with EPS present. But the formamide contact angle of P.putida cells with EPS is not significantly different from that of washed P.putida cells (P=0.064). This is the same for 1bromonaphthalene contact angle that there is no difference between the two (P=0.971). 129    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  5.5.5. Discussion of Contact Angle Measurement Water contact angle correlates well to bacterial cell surface hydrophobicity (Bos et al., 1999; Daffonchio et al., 1995). Bacteria with water contact angle less than 45o are considered to be hydrophilic, while they are hydrophobic if the angle is greater than 45o (Daffonchio et al., 1995; Grotenhuis et al., 1992). Without surfactant present, water contact angle of P.putida is 34.7o, which is much less than the water contact angle of R.erythropolis at 95.7o. Therefore, P.putida bacteria are hydrophilic and R.erythropolis bacteria are hydrophobic. These results are consistent MATH assay observation that R.erythropolis bacteria are more hydrophobic than P.putida. With increasing rhamnolipid concentration, the water contact angle of P.putida increases, indicating that the cell surface hydrophobicity increases. At 4000 mg/L rhamnolipid concentration, the water contact angle is 43.7o, about 10o higher than the contact angle without surfactant present, but the value is still less than 45o. The addition of rhamnolipid increases P.putida hydrophobicity, but has not made the cells hydrophobic. A similar trend is observed for tergitol surfactant. For the hydrophobic R.erythropolis bacteria, an opposite trend to the hydrophilic P.putida is observed. With increasing rhamnolipid concentration, the water contact angle decreases to 79.5 o at 4000 mg/L rhamnolipid concentration. Since the value is still well above 45o, the R.erythropolis cells are still very hydrophobic. Tergitol surfactant also reduces surface hydrophobicity of R.erythropolis. The formamide contact angle differs substantially between P.putida and R.erythropolis. R.erythropolis cells have higher formamide contact angle than P.putida cells, a trend similar to the water contact angles. Since both water and formamide are polar liquids, their contact angles tend to differentiate between strains and species (van der Mei et al., 1998). However the formamide contact angle does not conform to a definite trend with increasing surfactant concentration. By examining the formamide contact angles reported in the literature, it is clear that these values do not necessary reflect bacterial cell surface hydrophobicity (Hamadi & Latrache, 2008). The 1-bromonaphthalene contact angles for P.putida increases with increasing rhamnolipid concentration, but decreases with increasing tergitol concentration. The values for R.erythropolis also do not follow a consistent pattern. Overall the 1-bromonaphthalene 130    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  contact angles vary between 29o to 61o, which are consistent with the range reported in the literature (van der Mei et al., 1998). 1-bromonaphthalene is an apolar liquid with zero   and   and so it probes the LW interactions only (van Oss, 2006). It has been reported that contact angles using apolar liquid such as the 1-bromonaphthalene do not conform to the cell surface hydrophobicity characteristics (Hamadi et al., 2008; van der Mei et al., 1998). In summary, the results (Section 5.5.4) suggest that the presence of surfactant changes bacterial cell surface hydrophobicity, making hydrophilic cells more hydrophobic and vice versa, and the magnitude of change increases with increasing surfactant concentration. 5.6. Bacterial Cell Surface Thermodynamics and Hydrophobicity with Surfactant Present As discussed in Section 2.5.1 and 2.7.2, the bacterial cell surface hydrophobicity is originated from the acid–base nature of the cell surface and the acid–base property is related to surface tension parameters  LW ,   and   (Bellon-Fontaine et al., 1996; van Oss, 1995), a study of these surface tension parameters provides an in-depth and also quantitative understanding of the changes on bacterial cell surface hydrophobicity with surfactant present (Brown & Jaffé, 2006). Measured mean contact angles (see Table 5–2) and surface tension parameters of the diagnostic liquids were used to solve the Young’s equation following the LW-AB approach (Equation 5 in Section 2.7.2). The assumption for using the LW-AB approach has also been discussed in Section 2.7. The equations were solved using MatLab to give bacterial cell surface thermodynamic parameters  LW ,   and   . 5.6.1. Lifshitz-van der Waals Surface Tension Component The Lifshitz-van der Waals (LW) surface tension values (γLW) for P.putida and R.erythropolis as a function of surfactant type and concentration are shown in Figure 5-15. Increasing rhamnolipid and tergitol concentrations had a significant effect on the γLW values for P.putida (P<0.05), but had no effects on γLW values for R.erythropolis (P>0.05). The significance of the effect is represented by symbols a-j in the figure, where points with different symbols are significantly different and points with the same symbols are not significantly different. 131    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  W Figu ure 5-15. Lifsshitz-van der Waals surfacce tension com mponent (γLW ) of bacteriaal cell surfacee as a function of surfactaant concentration. The baccteria and surrfactant are P.putida P withh rhamnolipid d( ); olipid ( ); and R.erythrropolis with teergitol P.puttida with tergitol ( ); R.erythropolis with rhamno ( ).. Vertical barr represents 1 standard deeviation. Sym mbols a-j represent level off significance, where poin nts with differrent symbols are significaantly different and points with w the sam me symbols are not signiificantly diffeerent. In the aabsence of surfactant γLW for P.pputida and R.erythropo R olis are 30.99±0.5 and 33.9±0.6 3 W mJ/m2, respectivelyy, and are within w the tyypical rangee (mean γLW of 36.5±33.5 mJ/m2 ) reported for bactteria (Brownn & Jaffé, 2006). 2 Bacteerial cell su urfaces are composed c oof biopolym mers such as lipoppolysaccharrides (gram--negative baacteria) and d proteins, which w havee a γLW in th he range of 26.0 to 45.6 mJ//m2 (van Osss, 2006). Additioon of surfacctant producces distinctt changes in n γLW valuees, dependinng on the bacterial b L strain aand surfactaant type, as shown in F Figure 5-14 4. For gram m-negative P P.putida, γLW drops significcantly (P<0..05) as rham mnolipid cooncentration n increases from 0 to 1000 mg/L L, where after furrther addition to 4000 mg/L yieldded no signiificant effecct (P>0.05). Tergitol su urfactant displayss an oppossite trend on the γLW of P.putida a to rhamn nolipid. Adssorption off tergitol significcantly increaases γLW wiith tergitol cconcentratio on increasin ng from 0 too 4000 mg/L L (P=0). Unlike P.putida, γLW values of the gram m-positive R.erythropolis do nott vary significantly factant adsorrption. (P>0.055) with surfa 132    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  These opposing trends in γLW demonstrated in Figure 5-15 can be attributed to bacterial cell surface characteristics. P.putida is gram-negative bacterium with a complex outer membrane structure consisting primarily lipopolysaccharide (LPS) (Prescott et al., 2005). R.erythropolis is gram-positive bacterium with relatively thick and simple cell wall structure (Prescott et al., 2005). R.erythropolis cell wall contains mycolic acids which are linked to cell hydrophobicity (Bendinger et al., 1993; Sokolovská et al., 2003; Sutcliffe, 1998). Rhamnolipid releases LPS from the cell surface of Pseudomonas aeruginosa (Al-Tahhan et al., 2000), where it was suggested that release followed complexation of Mg2+, which is critical in stabilizing LPS at the membrane surface, by the anionic rhamnolipid (Al-Tahhan et al., 2000). PAGE analysis of concentrated supernatants taken from P.putida cultures incubated in the presence and absence of rhamnolipid and tergitol identified substantial amounts of LPS were released by the rhamnolipid treatment (Figure 5-9). The presence of LPS in the supernatant following incubation of P.putida with rhamnolipid correlates with the rhamnolipid-dependent release of Mg2+ (Figure 5-8). The γLW of LPS is around 39.3 to 42.3 mJ/m2 (van Oss, 2006), which is higher than rhamnolipid with γLW found to be 37.2 mJ/m2 (Chen, 2004), the removal of the LPS may contribute to the decrease in γLW value of the P.putida cell surface (Brown & Jaffé, 2006). The further reduction of γLW at higher rhamnolipid concentration is possibly due to more LPS being removed with increasing rhamnolipid concentration. Survival tests (Section 5.3.2) confirmed that the P.putida cells were viable after the rhamnolipid treatment. Complexation of Mg2+ does not occur with the nonionic surfactant tergitol, and therefore neither LPS nor Mg2+ are released from P.putida cells (Figures 5-8 and 5-9). Tergitol contains ethylene oxide units that have a γLW in the range of 43.0 to 45.0 mJ/m2 (van Oss, 2006), which is higher than LPS. Therefore it is suggested that the adsorption of tergitol onto P.putida cell surface increases the γLW value. Adsorption of rhamnolipid and tergitol onto R.erythropolis cell surface does not have a significant effect on γLW value. The γLW measured for pure mycolic acid isolated from Mycobacterium is 36.6±0.3 mJ/m2 (see Appendix XI for contact angle values), which is similar to that of rhamnolipid and so there is no significant changes. Tergitol may have higher γLW than mycolic acid, but it does not seem to affect the R.erythropolis cell surface γLW value. 133    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  5.6.2. Electron-donor (γ-) and Electron-acceptor (γ+) Parameters The values of electron-donor (γ-) and electron-acceptor (γ+) parameters for P.putida and R.erythropolis as a function of surfactant type and concentration are shown in Figure 5-16. It shows that increasing rhamnolipid and tergitol concentration produce different changes in the cell surface properties of P.putida and R.erythropolis. In the absence of surfactant, γ– and γ+ for P.putida are 57.0±2.3 and 0.2±0.1 mJ/m2, respectively and those for R.erythropolis are 0.5±0.2 and 0.2±0.1 mJ/m2, as shown in Figure 5-16. The values obtained are consistent with those reported by others (Sharma & Hanumantha Rao, 2002; van der Mei et al., 1998): γ– ranging from 55.8 to 66.8 mJ/m2 for Pseudomonas spp. and 4.5 to 28.0 mJ/m2 for Rhodococcus spp.; and γ+ ranging from 0.2 to 1.37 mJ/m2 for Pseudomonas spp. and 0 to 20.2 mJ/m2 for Rhodococcus spp.. The high γ– and low γ+ values of P.putida are typical of the electron-donating characteristic of hydrophilic bacteria; whereas the low γ– and low γ+ values seen for R.erythropolis are typical of hydrophobic bacteria (Bos & Busscher, 1999; van der Mei et al., 1998; van Oss, 1995). 134    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figuree 5-16. Electrron donor (A)) and electron n acceptor (B B) surface ten nsion parametters of bacterrial cell surrface as a fun nction of surfa actant concen ntration. Thee bacteria and d surfactant aare P.putida with w rh hamnolipid ( ); P.putid da with tergittol ( ); R.eerythropolis with w rhamnoliipid ( ); and a R.erythrropolis with teergitol ( ). ) Symbols a-jj represent leevel of significcance, wheree points with different symb bols are signifficantly different and poin nts with the same s symbolss are not signnificantly diffferent. In the ppresence of increasing i surfactant s c oncentrations Figure 5-16 shows tthat for P.putida, γ– decreasses and γ+ inncreases, bu ut the γ+ vallues remain ns negligibly y small wheen comparin ng to γ–. A signiificant reduuction in γ– is only seeen for P.pu utida when rhamnolippid concentrration is increaseed to 4,0000 mg/L. A significant nt increase in γ+ occu urs betweenn 0 and 50 00 mg/L rhamnoolipid (p<00.05), but further inncrease off rhamnolip pid concenntration do oes not significcantly influeence γ+ valu ues. With teergitol preseent, the redu uction in γ– is greater th han with 135    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  rhamnoolipid, and is significaant at loweer concentraations. The increase inn γ+ is sim milar for tergitol and rhamnnolipid over the 0-1,0000 mg/L rang ge, but is less pronouncced at higheer levels of terggitol. Convversely, fo or R.erythrropolis, γ– increases with inccreasing su urfactant concenttration, while γ+ decreaases with sur urfactant con ncentration. 5.6.3. B Bacterial Ceell Surface Tension Bacteriaal cell surfface tension n is the sum m of Lifshiitz-van der Waals andd acid–base surface tension componennts accordin ng to Equatiion 2 in Seection 2.7.2 2. The valuues of bacteerial cell surface tension (γγ) for P.putida and R R.erythropollis as a fun nction of ssurfactant ty ype and concenttration are shown in Figure F 5-177. It shows that the ceell surface ttension of P.putida P significcantly increeases in thee presence of tergitoll. Other co ombinationss of surfacttant and bacteriaal cell do noo bring sign nificant chaanges to celll surface teension. Thee trend exhib bited by R.erythrropolis witth surfactaant treatmeent is dom minated by the sizeabble γLW vaalues in comparrison to the combined γ– and γ+ vvalues. For P.putida P with rhamnollipid treatm ment, the decreasse in γLW is balanced b by y the large γ – value. Figure 5-17. Surface tension of the bacterial ccell surface as a a function of o surfactantt concentratio ons. The bacteriaa and surfactant are P.puttida with rham mnolipid ( ); P.putida with tergitol ( ); R.eryythropolis ).Symbolss a-e represennt level of sig with rhaamnolipid ( ); and R.errythropolis wiith tergitol ( gnificance, where p points with diifferent symb bols are signifficantly different and poin nts with the ssame symbolss are not signiificantly diffeerent. 136    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  5.6.4. B Bacterial Ceell Surface Hydrophoobicity The freee energy of o aggregatiion betweenn bacterial cells in water (∆Gbwbb) is a quaantitative expresssion of the cell c surface hydrophiliccity or hydrrophobicity (van Oss, 22006). The equation e (Equation 8) for caalculating ∆Gbwb has beeen discusseed in Section 2.7.3. It iss expressed d as:  L Gbwb  2(  bLW   wLW ) 2  4  b  b   w  w   b  w   b  w  [8] If the innteraction between b the two cells iss stronger th han the interaction of eeach cell wiith water ∆Gbwb < 0, the cellls will aggreegate and arre considereed hydrophobic. Conveersely if ∆G Gbwb > 0, the cellls are hydroophilic. Thee calculatedd ∆Gbwb vallues for P.p putida and R R.erythropo olis as a functionn of surfactaant type and d concentraation are sho own in Figu ure 5-18. Figu ure 5-18. Freee energy of ag ggregation off bacterial cellls (∆Gbwb) in n aqueous soluution (∆Gbwb) as a function n of surfactan nt concentrattions. The baccteria and su urfactant are P.putida withh rhamnolipid ( ); P.putida with tergitoll ( ); R.eryythropolis witth rhamnolipid ( d R.erythropollis with tergittol ( ). );and Symbolss a-k represen nt level of sig gnificance, wh here points with w different symbols are significantly different and points with w the same symbols are not significan ntly differentt. Withouut surfactannt treatmentt, ∆Gbwb off R.erythro opolis is leess than -8 0 mJ/m2 which w is markeddly lower thhan P.putida a (~45 mJ/m m2) as show wn in Figurre 5-18. Thhe calculated ∆Gbwb values correspondd well to the t observeed water co ontact anglle measurem ements on the two bacteriaal lawns; R.erythropoliss is hydrophhobic and P.putida P is hydrophilic. h . Also, fluorrescence images in Figure 5–19 visuaally demonsstrate that without w surffactant pressent, P.putiida cells 137    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  were dispersed in water (Figure 5–19A) and R.erythropolis cells formed aggregates (Figure 5–19C). The surfactant treatment brings two distinct changes to the bacteria. The ∆Gbwb of P.putida decreases with increasing surfactant concentration, indicating an increase in hydrophobicity and the change is more significant with tergitol surfactant than rhamnolipid. The rhamnolipid surfactant at the range of concentrations has not made the P.putida cell surface hydrophobic since ∆Gbwb is still positive, and therefore is not energetically favorable for cell aggregates to form. This is shown in Figure 5–19B that no cell aggregates is formed. Conversely for R.erythropolis, ∆Gbwb increases with surfactant concentration, indicating an increase in hydrophilicity and the effect is more pronounced with rhamnolipid surfactant. However the increase in hydrophilicity is not pronounced enough to make the cells hydrophilic as ∆Gbwb is still negative at 4,000 mg/L rhamnolipid. There are still R.erythropolis cell aggregates as shown in Figure 5-19D. 138    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figuree 5-19. Fluoreescence images of bacteriaa cells in aqueous solution n. A: P.putidaa without surfactant present iin water; B: P.putida P at 4,000 mg/L off rhamnolipid d solution; C: R.erythropoolis without su urfactant present in water; and D: R.erythropolis at 4,0000 mg/L of rhamnolipid r solution. s Scalle bar equals to 5 μm. Fluorescen nt dye is acrid dine orange. 5.6.5. D Discussion of o Bacteria al Cell Surfa face Hydrop phobicity The meeasurement of water co ontact angle (Table 5-2) and hydro ophobicity ccalculationss (Figure 5-18) ddemonstratee that surffactant treaatment can n modify bacterial b ceell hydroph hobicity, dependiing on surffactant type, concentraation and th he bacterial surface. A consistent trend is observeed that withh increasin ng surfactannt concentrration, hydrrophilic celll surfaces become more hyydrophobic and hydrop phobic cell surfaces beecome moree hydrophiliic, and the extent e of change is surfactannt dependen nt. The finddings presen nted here are consistent nt with otherr reports drophilic bacteria b inncreases th heir cell demonsstrating thaat surfactaant treatmeent of hyd hydrophhobicity (Brown & Jafffé, 2006; S Simões et al., 2008; Yu uan et al., 22007; Zhon ng et al., 2008). The resultt in Figuree 5-18 furtther revealss that hydrrophobic ccells becom me more hydrophhilic follow wing surfacctant treatm ment and is i consisten nt with thee observatiion that 139    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  hydrophobic abiotic surfaces become more hydrophilic with surfactant treatment (Chen, 2004; Liu et al., 2006; Sharma & Hanumantha Rao, 2002). The extent of change observed in cell hydrophobicity/hydrophilicity is dependent on surfactant concentration. As shown in Figure 5-18, an increase in rhamnolipid and tergitol surfactant concentration leads to an increase in cell hydrophobicity for P.putida and an increase in cell hydrophilicity for R.erythropolis. Our results here support the findings of Simões et al. (2008), who showed that increasing CTAB surfactant concentration decreased the surface free energy of cell aggregation (∆Gbwb) of hydrophilic Pseudomonas fluorescens bacteria, corresponding to an increase in cell hydrophobicity. However, our results contradict the findings of Zhong et al. (2008). Although these researchers had used a rhamnolipid compound that was similar to the JBR 425 rhamnolipid used in this study, they reported that hydrophilic P.aeruginosa cell hydrophobicity increased with mono-rhamnolipid concentration up to 64 mg/L and with di-rhamnolipid concentration up to 82 mg/L; whereupon higher rhamnolipid concentrations either stabilized or decreased cell hydrophobicity. To explain this finding it was argued that at high rhamnolipid concentrations the adsorption sites on the cell surface tend to be saturated and multilayer adsorption or the accumulation of hemimicelles occurs, resulting in the stabilization or decrease of cell hydrophobicity. However, this argument contradicts the work of Brown and Ai Nuaimi (2005) and Brown and Jaffé (2006), which demonstrated that multilayer adsorption of surfactant CxEy at high concentrations on a cell surface makes cells more hydrophobic. The findings reported by Zhong et al. (2008) rely upon results obtained using the microbial adhesion to hydrocarbon (MATH) assay, whereas those reported by others relied upon contact angle measurements (Brown & Jaffé, 2006; Simões et al., 2008). At high rhamnolipid concentrations emulsification has been observed during the MATH assay (see Section 6.4), and the emulsion formation interferes with the MATH assay results, giving stabilized or decreased values. The results show that contact angle measurements are more reliable, especially at high concentrations of surfactant. Surfactant treatment may affect cell surface properties by adsorption at the cell surface and/or by the removal of surface exposed molecules (e.g. LPS) from the cell surface (Al-Tahhan et al., 2000; Brown & Jaffé, 2006; Zhong et al., 2008). Rhamnolipid treatment with P.putida (as shown in this study) and P.aeruginosa (Al-Tahhan et al., 2000) releases LPS from the cell surface, which in combination with rhamnolipid adsorption may consequently result in the increase in hydrophobicity. With tergitol surfactant, it is likely that the hydrophilic end of the 140    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  surfactaant molecuules interaccts with thhe hydroph hilic surfacee of P.puttida, expossing the hydrophhobic end of o the surfaactant moleecules at th he surface (Neu, ( 1996)). For hydrrophobic R.erythrropolis cellls, the reverse orientattion is prop posed, wherre the hydroophobic end of the surfactaant moleculles interactss with the hydrophobiic cell surfface, exposiing the hyd drophilic end to tthe surrounddings. The obsserved channges in the chemical c annd moleculaar composittion of the ccell surface brought about bby surfactannt treatment is manifestted as a chaange in the cell c surface tension parrameters γLW, γ– and γ+, ass shown in Figure 5-115 and Figu ure 5-16. These T param meters dem monstrate differennt extents of o change with w increasiing surfactaant concenttration. Figuure 5-20 sh hows the correlattion betweeen these surface tensionn parameterrs and surfaace free eneergy of agg gregation (∆Gbwb)). A proporrtional correelation exissts in electron-donor (γγ–) and hyddrophobicity y, where decreassing electron-donor co orresponds tto increasin ng hydrophobicity. Buut no correllation is found iin γLW andd γ+. The correlation aanalysis co onfirms the observatioon that γ– plays p an importaant role in predicting p ceell hydrophoobicity. Figure 55-20. Correlaation between n the degree oof hydrophob bicity and surrface free eneergy componeents. The surface free energy components c are a Lifshitz-vvan der Waalss (γLW; ), electron donorr (γ+; ), and d electron accceptor (γ-; ). This finnding is coonsistent with the findiings of van n der Mei et e al. (19988) and Ham madi and Latradcche (2008) that t bacteriia with a hiigh ∆Gbwb have h high electron e donnor values and that bacteriaa with low ∆G ∆ bwb are weak w electroon donors. The T results also a confirm m the reportts of van 141    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  Oss (1995) that electron donor and electron acceptor interactions are the origin of microbial cell surface hydrophobicity. For biological surfaces γ– is indicative of the material’s hydrophobicity as γ+ is, in most cases, close to zero (Chen, 2004; Grasso et al., 1996; van der Mei et al., 1998). Consistent with this finding, the calculated γ+ in this study is either close to zero or insignificantly small when compared to γ–; and the changes in γ– match well to the hydrophobicity of the cell surface. Surfactant treatment decreases γ– of hydrophilic P.putida cells, corresponding to an increase in hydrophobicity; for hydrophobic R.erythropolis cells, the reversed trend is observed. 5.7. Surface Thermodynamic Modelling of Bacteria/Microbubble Interaction 5.7.1. Model Assumptions The surface thermodynamic model based on LW-AB approach has been discussed in Section 2.7. The surface free energy of adhesion ∆ of the bacteria (b)/microbubble (s) interaction in a liquid medium (l) can be calculated from Equation [7], which has been previously presented in Section 2.7.3. It reads:    LW  LW   LW  LW   LW  LW   LW b l s l b s l   Gadh  2                b   s   l   l  b   s   l   b  s   b  s  l  [7]     To apply this model to bacteria/microbubble interaction, it is assumed that:  The surface tension components obtained from LW-AB theory are accurate. The validity of the theory has been discussed in Section 2.7.  The electrostatic interaction can be neglected when measuring bacterial interaction with a hydrophobic surface due to the predominant effect of the hydrophobic interaction (Busscher et al., 1984; Chen & Strevett, 2001; Grasso et al., 1996). The hydrophobic energies in aqueous media have been shown to be up to two orders of magnitude greater than that from electrostatic interactions (van Oss et al., 1988).  The electrostatic repulsion can be overcome by hydrodynamic forces that are generated from intensive mixing at 8,000 rpm. The hydrodynamic force brings the bacterial cell close to the microbubble surface. As the cell is brought close to the 142    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  surface, the double layers become superimposed. At this separation distance, which is at the point of physical contact that may lead to adhesion, the superimposition of the double layers neutralises the electrostatic charges, making the electrostatic interaction relatively insignificant in comparison to the energies of the LW-AB interaction (Chen & Strevett, 2001). 5.7.2. Surface Tension Parameters of Rhamnolipid Solutions Surface tension parameters of a rhamnolipid solution vary with rhamnolipid concentration, and these values have been published in the literature (Chen, 2004; Chen et al., 2004), and are used in this study to develop a relationship which will provide an estimation of surface tension parameters of rhamnolipid concentrations at 500 mg/L, 1,000 mg/L and 4,000 mg/L. By fitting trend lines to the surface tension values using Microsoft Excel as shown in Figure 5–21, it is possible to obtain the relationship between the surface tension parameters with rhamnolipid concentrations. While the values of γLW and γ– increase with rhamnolipid concentration, the value of γ+ decreases with increasing rhamnolipid concentration. For both γLW and γ–, the data fits well to a logarithm relationship since the R-square values are close to 1. For γ+, the data indicate that the value of γ+ reaches saturation at about 5 mJ/m2 as rhamnolipid concentration increases beyond 1,000 mg/L. The trend that γ+ decreases to a constant value with increasing rhamnolipid concentration is also observed in Figure 5–16 (B). The high γ– and low γ+ demonstrated in Figure 5-21 at high rhamnolipid concentration of 250,000 mg/L is consistent with the typical characteristics of high γ– and low γ+ exhibited by the cell surface of Pseudomonas species from which the rhamnolipid compound is harvested (Desai & Banat, 1997; Nitschke et al., 2005; van der Mei et al., 1998). 143    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figure 55-21. Surfacee tension para ameters as a ffunction of rh hamnolipid concentration c n in mg/L in log l scale. W T surface teension param The meters are γLW ( ); γ–( ); and γ+( ). Using tthe relationsship found in Figure 55–21, the su urface tensiion parametters at rham mnolipid concenttrations of 500 mg/L L, 1,000 m mg/L and 4,000 mg//L can be estimated. Linear interpollation basedd on the log garithm scaale is used to t estimate the γ+ valuues. The ressults are summarrised in Tabble 5–4. Table 5-4. 5 A summa ary of surfacee tension para ameters of rh hamnolipid soolutions. Rhamnoliipid Surfface tension n parameters (mJ/m2) concentration γ LW γ+ γ– (mg/L)) 500 225.6 8.0 28.4 1,000 226.9 5.6 28.9 4,000 229.5 5.3 30.0 5.7.3. A Approach A to Determ mine Surfacce Tension Parameterrs of Microobubble As prevviously disscussed in Section 2.77, the surfface free en nergy of innteraction between b bacteriaa and microobubbles (∆ ∆Gadh) can bbe calculateed through the t surface tension parrameters of the iinteracting entities. Th he parameteers for bacteerial cells were w obtainned through h contact angle m measuremennt. The valuees for rhamnnolipid solu ution were estimated e frrom values reported r in the lliterature. To T determine the surfacce tension parameters p of microbuubble surfacce, there are two different appproaches as a illustratedd in Figure 5–22. 144    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Approaach A assum mes that thee presence of rhamnollipid does not n change the surfacee tension propertiies of the aiir-water interface and sso the bacteeria interactt directly w with a pure air-water a interfacce. In this caase, the surfface 2 in Eqquation 7 is equivalent to the air suurface as illlustrated in Figurre 5–22A. Figurre 5-22. Schem matic presenttation of bactteria and bub bble surface interaction. Illlustration A and B represeents Approacch A and B (seee text), respectively. 5.7.4. A Approach B to Determ mine Surfacce Tension Parameterrs of Microobubble Approaach B considders that thee bacteria innteract with h a layer of rhamnolipid r d surfactantt that are absorbeed at the microbubble m surface, ass illustrated in Figure 5–22B. It iis assumed that the rhamnoolipid moleecules are closely paacked to form f the layer, l sincee the rham mnolipid concenttrations used in this stu udy were w well above th he surfactan nt’s CMC. T The surfacee tension parametters of the rhamnolipid d layer havve been prev viously evaaluated (Cheen, 2004) and a will be usedd in Approacch B calculaation. 145    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  5.7.5. Summary of Surface Tension Parameters Surface tension parameters of various materials and bacterial cell surface are needed for surface thermodynamic modelling of the bacterial interaction with microbubbles. These data are summarised in Table 5–5 and Table 5–6. The modelled interaction energy between bacteria and microbubble (∆Gadh) will indicate if the bacterial adhesion is energetically favourable. In theory, the interaction is favourable when ∆ adhesion is energetically unfavourable when ∆ 0. On the other hand, the 0. Furthermore, a theoretical assessment of whether the rhamnolipid microbubble dispersion is capable to perform as a carrier of bacteria within various soil environments (e.g., sand, clay or organic rich soil) can be made using surface free energy calculation of the bacteria/soil surface interaction in comparison with the bacteria/microbubble interaction. For example, if the bacteria/microbubble ∆Gadh is more negative than the bacteria/soil ∆Gadh, it indicates that, in theory, the microbubble dispersion is an effective carrier of the bacteria because it is energetically more favorable for the bacteria to attach to the microbubble than to the soil surface. On the other hand, if the bacteria/soil ∆Gadh is more negative than the bacteria/microbubble ∆Gadh, the microbubble dispersion is not an effective carrier of the bacteria. The surface tension parameters of various soil surfaces have been obtained from the literature, and are summarised in Table 5-5. The soil surfaces assessed include silica sand, kaoliniteclay, smectite-clay and peat for an organic rich soil. Table 5-5. Surface tension parameters of various materials. Material a watera aira rhamnolipid filmb rhamnolipid solution (500 mg/L)c rhamnolipid solution (1,000 mg/L)c rhamnolipid solution (4,000 mg/L)c silica sandd Kaolinite-claye Smectite-claye Peat (organic soil)f Surface tension parameters (mJ/m2) γLW γ+ γ– 21.8 25.5 25.5 0 0 0 37.2 5.3 33.1 25.6 8.0 28.4 26.9 5.6 28.9 29.5 5.3 30.0 22.7 1.57 15.4 41 0.7 30 10.9 0.4 44.6 20.5 1.2 23.3 Data adapted from van Oss (2006). b Data adapted from Chen (2004). c Data estimated from literature (Section 5.8.2). d Data adapted from Chen & Zhu (2005). e Data adapted from Wu (2001). f Data adapted from Michel et al. (2001). 146    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  Table 5-6. Surface Tension Parameters of Bacteria. Bacteria a Rhamnolipid concentration (mg/L) P.putida P.putida P.putida P.putida P.putida with EPS R.erythropolis R.erythropolis R.erythropolis R.erythropolis 0 500 1,000 4,000 Surface tension parametersa (mJ/m2) LW γ γ+ γ– 30.9 0.21 57.0 29.3 0.44 52.9 27.1 0.70 55.9 24.9 1.12 46.5 Cell surface hydrophobicity (mJ/m2) 46.0 39.0 41.1 28.4 1,000 27.9 0.50 52.8 39.2 0 500 1,000 4,000 33.9 35.6 34.4 0.22 0.08 0.02 0.5 1.5 3.6 34.7 0.01 8.8 -80.6 -74.1 -62.0 -41.2 The surface tension parameters (mean values shown) were obtained from previous calculations (Section 5.6). 5.7.6. Surface Thermodynamics to Predict Bacteria – Microbubble Interaction A. Approach Comparison Two approaches were evaluated to obtain an accurate representation of the bacteriamicrobubble interaction. Approach A assumes that microbubble surface can be approximated as pure air-water interface and bacteria interacts with the pure air-water interface immersed in rhamnolipid solution. The surface tension parameters of the microbubble surface are equivalent to those of the air-water interface. Approach B assumes that the microbubble surface comprises a rhamnolipid film due to the arrangement of multiple layers of surfactant reported in the literature. Therefore the surface tension parameters of the microbubble surface are equivalent to those of a rhamnolipid film. The surface free energy of interaction calculated from both approaches are shown and compared in Table 5-7. Table 5-7. Comparison of surface free energy of interaction (∆Gadh) calculated using Approach A and B. Rhamnolipid ∆Gadh (mJ/m2) concentration Approach A Approach B (mg/L) 500 -11.3 5.4 P.putida 1000 -11.5 5.1 P.putida 4000 -17.6 3.2 P.putida P.putida with EPS 1000 -14.0 4.9 1000 -40.6 -5.0 R.erythropolis Bacteria The results show that using Approach A, the ∆Gadh for both P.putida and R.erythropolis is negative, indicating that all of these interactions are energetically favorable. The ∆Gadh values 147    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  of Approach B are more positive than Approach A, indicating less energetically favorable interaction. Approach B assumes that the bacteria interact with the rhamnolipid film encapsulating the microbubble surface, and is considered a more accurate representation of the experimental conditions than Approach A. The reasons are two-fold. Firstly, previous studies have shown that the microbubble surface is surrounded with multiple layers of surfactant molecules to stabilize the bubble (Jauregi et al., 2000). Other researchers (Dahlbäck et al., 1980; Marshall, 1980; Schäfer et al., 1998a) reported that macromolecules such as lipids and surfactants tend to accumulate at surfaces to form a conditioned film that dictates the surface properties to the original surfaces. Since rhamnolipid is used in concentrations well above its CMC in this study, it is more accurate to assume that the bacteria interact with a rhamnolipid layer instead of a pure air-water interface. Secondly, the accumulation of surfactants at air-water interface reduces its hydrophobicity and so its tendency to attract macromolecules and colloidal particles (Ducker et al., 1994; Preuss & Butt, 1998). The ∆Gadh values using Approach A are negative for all rhamnolipid concentrations tested, indicating that the addition of surfactant does not affect the attraction. This observation does not seem to be consistent with what was reported in the literature. Approach B is therefore considered to provide a more accurate representation of the bacteriamicrobubble interaction than Approach A. The surface free energy of interaction calculation in the following sections is based on Approach B. B. ∆Gadh and Rhamnolipid Concentration Bacterial drainage experiments have demonstrated that the adhesion of hydrophilic P.putida cells to microbubble dispersion increased with rhamnolipid concentration (Figure 5-1). Also, it has been shown previously that P.putida cell surface hydrophobicity increased with increasing rhamnolipid concentration (Figure 5-18). The increase in P.putida cell surface hydrophobicity can result in increased adhesion to microbubble surfaces since cell hydrophobicity has been recognized as the dominant factor influencing bacterial adhesion (Chen & Strevett, 2001; Dahlbäck et al., 1980; Grasso et al., 1996; van Loosdrecht et al., 1987). To examine the linkage between bacteria adhesion and cell surface hydrophobicity at various rhamnolipid concentration, percentage adhesion of bacteria to microbubble dispersion at drainage time of 480 seconds (approximately at the half-life of the dispersion) (Ripley et al., 2000) was compared with calculated surface free energy of the interaction in Figure 5–23. 148    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figure 55-23. Comparrison of bacteerial adhesion n to microbub bble dispersio on at drainagge time of 480 0 seconds and caalculated surfface free enerrgy of adhesioon (∆Gadh) beetween (A) P.p putida and m microbubble and a (B) R.eerythropolis and microbub bble as a funcction of rham mnolipid conceentration. Coolumn represents perceentage bacteriial adhesion. Triangle sym mbol represen nts surface frree energy of interaction. Arrow A indicatees the corresp ponding axis.. Vertical barrs represent 1 standard deeviation. It is shhown in Figure F 5-23 3 (A) that the P.putiida-microbu ubble surfaace free en nergy of interacttion (∆Gadh) decreased d with increeasing rham mnolipid co oncentrationn. The ∆Gadh value a decreassed from 5.44 mJ/m2 at 500 5 mg/L rhhamnolipid to 5.1 mJ/m m2 at 1000 m mg/L (P<0.05), and further to 3.2 mJJ/m2 at 400 00 mg/L rhhamnolipid (P<0.05). The perceentage adheesion of 149    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  P.putida da, on the otther hand, increased i siignificantly y with rham mnolipid conncentration (already discusseed in Sectioon 5.2.1). The deccrease in ∆G ∆ adh, which h indicates that the adh hesion is en nergeticallyy more favo orable at higher rhamnolipiid concentration, corrrelates to the increaase in P.pputida adheesion to microbuubble dispeersion. Thee degree off change however h is not necesssarily prop portional (Busschher et al., 1984). By y definitionn, bacterial adhesion to a surfacce is energ getically favorabble if ∆Gadhh<0. Althou ugh the calcculated ∆Gadh n P.putida aand microbubble is a between positivee, the valuees are not far f from zerro and these values arre an averagge for the P.putida P populattion. Not alll of the cellls have the same interaaction energ gy as somee may posseess more negative values annd some may y have morre positive values. v Furtthermore, thhe intensivee mixing at 80000 rpm generaates hydrod dynamic forrce that is likely to prov vide the suppplementary y energy nd microbub bble feasiblle (Busscheer & van that maade the conttact betweeen the bacteerial cell an der Meii, 2006; Gjaaltema et al., 1995). Thhe adhesion of P.putida a cells to miicrobubble surfaces under fl fluorescencee microscop py is shown in Figure 5–24. Figu ure 5-24. Miccroscopy ima ages of P.putidda interaction n with microb bubble madee from 1000 mg/L m rhamnoolipid at pH 7. 7 Cells were stained s with fluorescent acridine a orange dye. Scalee bar equals to 20 μm. The ∆G Gadh values of R.erythrropolis-miccrobubble in nteraction, as shown iin Figure 5-23 (B), increaseed (becoming more po ositive) witth increasin ng rhamnoliipid concenntration. Th he ∆Gadh value inncreased frrom -7.7 mJ/m m 2 at 5000 mg/L rh hamnolipid to -5.0 mJJ/m2 at 100 00 mg/L 150    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  (P<0.055), and furtther to -2.2 2 mJ/m2 at 4000 mg/L L rhamnolip pid (P<0.055). The inccrease in ∆Gadh suggests thhat R.eryth hropolis addhesion is energeticallly less faavorable att higher rhamnoolipid conceentrations. However H thee percentag ge adhesion of R.erythrropolis did not n vary significcantly with rhamnolipid concentrration (alreeady discusssed in Secction 5.2.1)). Other factors are likely too affect the interaction and are discussed in deetail in the ffollowing section. C. ∆Gadh and Cell Surfacce Propertiies Cell suurface propeerties have been show wn to influ uence bacteerial adhesioon to micrrobubble dispersiion (see Seection 5.2.3). P.putida cells with extracellular polymeri ric substancce (EPS) present showed grreater percen ntage of addhesion than n washed ceells withoutt EPS, as sh hown in dhesion of hhydrophobiic R.erythro opolis cellss was show wn to be Figure 5-3. Howeever, the ad significcantly less than hydro ophilic P.puutida cells, which as discussed iin Section 5.2.3 is differennt from thatt reported by y Wan et a l. (Wan et al., a 1994) and a Ripley eet al. (Ripleey et al., 2002). T To examinee the influence of surfa face free eneergy, Figure 5–25 pressents and co ompares the surfface free ennergy of th he bacteriall-microbubb ble interaction (∆Gadh) together with w the percentage bacteriial adhesion n at drainagge time of 480 second ds at 1000 mg/L rham mnolipid concenttration. Figurre 5-25. Com mparison of ba acterial adhession to micro obubble at drainage time oof 480 second ds and caalculated surfface free enerrgy of bacteriial adhesion with w microbu ubble (∆Gadh) at rhamnolip pid concentrration of 10000 mg/L. Colu umn represen nts percentagee retention. Triangle T symbbol representts surface free en nergy of interraction. Arrow indicates t he correspon nding axis. Veertical bars reepresent 1 sta andard deviation. 151    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  The ∆Gadh value of unwashed hydrophilic P.putida cells with EPS present (4.7 mJ/m2) is not significantly different from the washed cells without EPS (5.1 mJ/m2). As discussed in Section 5.2.3, the significant increase in percentage P.putida adhesion with EPS present was attributed to the presence of an EPS network at the microbubble surface that keeps more bacterial cells in the dispersion. The ∆Gadh for hydrophobic R.erythropolis cells is much more negative than that for P.putida, as shown in Figure 5–25. This calculated value matches with the findings from the literature that hydrophobic bacteria are more energetically favorable to adhere to air-water interface than hydrophilic bacteria (Ripley et al., 2002; Wan et al., 1994). However, the adhesion result indicates that less R.erythropolis cells are retained in microbubble dispersion than P.putida, which contradicts to the predicted results of the surface free energy calculation. It is also shown in Section 5.7.6B above that the calculated surface free energy of interaction does not explain the R.erythropolis adhesion. The combined effect of R.erythropolis cell surface hydrophobicity and liquid drainage from the dispersion may offer an explanation to the observed difference. Despite the increase in hydrophilicity by rhamnolipid adsorption on R.erythropolis cells, the cell surface is still so hydrophobic that it is more energetically favorable for the cells to adhere to itself to form aggregates (∆Gbwb being -61.4 mJ/m2) than to adhere to microbubbles (∆Gadh being -5.0 mJ/m2). The cell aggregates drain faster under gravity due to increased mass than dispersed cells. This is shown in Figure 5-26 that aggregates of R.erythropolis cells are draining away from the microbubble surface. When compared with P.putida, the aggregation energy of the hydrophilic P.putida cells is at least seven times more positive than the adhesion energy with microbubble, indicating that it is more energetically favorable for P.putida cells to adhere to microbubble than forming aggregates, therefore there is higher probability for the P.putida to adhere to microbubbles as compared to R.erythropolis. 152    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  Figuree 5-26. Microoscopy imagess of R.erythrop opolis interacttion with miccrobubble maade from 1000 mg/L rrhamnolipid. Cells stained d with fluoresscent acridinee orange dye. Scale bar eqquals to 40 μm m. 5.7.7. D Discussion on o Microbu ubble Dispeersion as Bacterial B Ca arrier The abbove resultss demonstrrate that rhhamnolipid microbubb ble disperssion was better b at deliveriing hydrophhilic P.putid da than hydrrophobic R..erythropoliis bacteria, as supporteed by the significcantly greatter adhesion of P.puttida cells to t microbub bble disperrsion. Surfaace free energy calculationn also show ws that it is energettically morre favorablee for hydrrophobic R.erythrropolis cells to form ag ggregates thhan to adherre to the rhaamnolipid m microbubblee surface, but it iss energetically more fav vourable foor P.putida cells c to adhere to the m microbubblee surface than forrming cell aggregates. a The surrface free ennergy theory can be appplied to asssess how well the micrrobubble dispersion may peerform as a carrier of P.putida annd R.erythrropolis with hin a soil ennvironmentt from a theoretiical perspeective. Fourr types off soil surfaaces are seelected forr compariso on with microbuubble dispeersion. Theey are the silica sand d, kaolinite--clay, smecctite-clay an nd peat. Kaoliniite and smeectite are tw wo main grooups of min nerals found d in clay (M Murray, 200 00). The surface tension prooperties of a clay surfaace are depeendent on th he composittion of the minerals m T surface properties of kaolinitee and smecttite minerall groups or compponents in the clay. The are usedd to represeent two typees of clay suurfaces. Peaat is an accu umulation oof partially decayed vegetatiion matter and is used u to reppresent soil surface properties p with rich organic componnents (Michhel et al., 2001). 2 The surface free energy off bacteria innteraction with w the 153    Chapter 5 – Bacteriaa/Microbubb ble Interactioon and Surfacce Thermodyynamic Mod elling of the  Interaction  various soil surfaces is calculaated and coompared witth the bacteeria–microbuubble interaaction at mg/L rhamnoolipid conceentration in Figure 5-27 7. 1000 m Figure 5-27. Surfacee free energy of bacterial iinteraction with w various ty ypes of surfaaces. The surffaces are ); smectite-clay s microbubble ( ); silica sand ( ); kaolinitee-clay ( ( ); and peaat (×). Vertical bars representt 1 standard deviation. It is shhown in Figure 5-27 that for P.pputida the interaction n energy w with microbu ubble is significcantly differrent from th hose with saand, kaoliniite-clay, sm mectite-clay and peat (P P<0.05). Howeveer an oppossite trend iss observed for R.eryth hropolis, wh here by thee interaction n energy with miicrobubble is i significan ntly higher tthan those with w soil surrfaces. In theorry, if the surrface free en nergy of baacterial interraction with h microbubbble is smalleer (more negative) than the interaction energy withh soil surfacces, it is eneergetically m more favourrable for the bactteria to adhhere to the microbubble m e than to ad dsorb onto th he soil surfface. The caalculated interacttion energy values in Figure F 5-277 suggest th hat the miccrobubble ddispersion (aat 1,000 mg/L rhhamnolipid)) is a betterr carrier of P P.putida thaan R.erythro opolis throuugh sand, kaoliniteclay, ssmectite-claay and peaat soil typpes. The preferential p attachmennt of P.pu utida to microbuubbles allow ws the transsport of P.pputida cells with traveling microbbubbles furtther into the subssurface sincce soil adsorrption was a key factorr in determining the moovement of bacteria in a soil (Stenstrom m, 1989). This result iss consistent with the en nhanced trannsport obseerved for a Burkhholderia (ppreviously known k as P Pseudomona as) sp. (EN NV 345) thr hrough sand d-packed 154    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  column using Steol-surfactant made microbubble dispersion (Rothmel et al., 1998). However, the rhamnolipid microbubble dispersion is not effective for the delivery of R.erythropolis because adsorption of R.erythropolis cells on the four soil type surfaces is expected as it is energetically more favorable than microbubble surface. This is consistent with what have been reported by other studies that hydrophobic bacteria adhered better to soil particles including quartz sand, sandy loam and clay mixture when compared to hydrophilic bacteria (Huysman & Verstraete, 1993; Park et al., 2010; Stenstrom, 1989). It can be concluded based on experimental result and calculated interaction energy values that a rhamnolipid microbubble dispersion is more effective at delivering hydrophilic P.putida than hydrophobic R.erythropolis bacteria. 5.8. Chapter Summary This chapter examines the bacteria–microbubble interaction with an aim to understand the factors and mechanisms that can affect the adhesion of bacteria to microbubble dispersion and therefore its effectiveness as a carrier for contaminant-degrading bacteria. Bacterial drainage experiments were carried out under a range of testing conditions. The LW-AB surface thermodynamic model was used to quantify the surface free energy of the bacteriamicrobubble interaction. It is shown that increasing rhamnolipid concentration increases the adhesion of hydrophilic P.putida cells to microbubble dispersion, even though rhamnolipid adhesion onto P.putida cells could not be measured. The increase in rhamnolipid concentration from 500 mg/L to 4,000 mg/L brings about an increase in P.putida cell surface hydrophobicity, as shown by contact angle measurement and the LW-AB model calculation. The more hydrophobic P.putida cells at higher rhamnolipid concentration are energetically more likely to adhere to microbubble surface when compared to the cells at lower rhamnolipid concentration. Also, unwashed P.putida cells with EPS present are found to adhere more to microbubble dispersion when compared to washed cells without EPS. Scanning electron micrographs revealed an EPS network covering the microbubble surface that was proposed to trap bacterial cells in the dispersion. The adhesion of hydrophobic R.erythropolis cells to microbubble dispersion however was significantly less than P.putida cells, and rhamnolipid concentration did not increase R.erythropolis cells adhesion. The increase in rhamnolipid concentration results in a decrease 155    Chapter 5 – Bacteria/Microbubble Interaction and Surface Thermodynamic Modelling of the  Interaction  in R.erythropolis cell surface hydrophobicity, i.e., the cells becoming more hydrophilic. The increase in hydrophilicity makes the adhesion interaction less energetically favourable, but it is not enough to account for the significantly low adhesion for R.erythropolis. Surface thermodynamic model shows that it is energetically more favourable for R.erythropolis cells to form aggregates than to adhere to microbubble surface. The aggregates drain away faster from the microbubble surface with drainage liquid due to increased mass than dispersed cells. The addition of 1,000 mg/L and 3,000 mg/L salt showed no significant effect on the amount of P.putida cells adhered to the rhamnolipid microbubble dispersion. This finding indicates that electrostatic interactions have no significant influence on bacterial adhesion to the dispersions. The surface free energy theory is also used to investigate how well the rhamnolipid microbubble dispersion may perform as a carrier of P.putida and R.erythropolis within a soil environment from a theoretical perspective. It is shown that the rhamnolipid microbubble dispersion is a better carrier for delivering P.putida through various soils including sand, kaolinite-clay, smectite-clay and peat due to energetically favourable bacterial interaction with the microbubble. But according to the calculation, it is more energetically favourable for R.erythropolis to adsorb onto the soil surface than microbubble. The experimental findings, coupled with surface free energy calculation, have shown that rhamnolipid microbubble dispersion is more effective in delivering hydrophilic P.putida than hydrophobic R.erythropolis bacteria. Bacterial cell surface hydrophobicity and rhamnolipid concentration are demonstrated as two key factors in making the microbubble dispersion an effective bacterial carrier. Since increasing surfactant concentration changes bacterial cell surface hydrophobicity, by making hydrophilic P.putida more hydrophobic and hydrophobic R.erythropolis more hydrophilic, it is important to consider the compatibility of the type and concentration surfactant and bacteria in terms of surface hydrophobicity. Surface thermodynamic modelling can be applied to predict the adhesion of bacteria to microbubble dispersion. 156    Chapter 6 – Visualisation of Bacteria/Contaminant/Microbubble Interaction and Surface  Thermodynamic Modelling of the Interaction  Chapter 6. Visualisation of Bacteria/Contaminant/Microbubble Interaction and Surface Thermodynamic Modelling of the Interaction 6.1. Introduction The absence of contaminant-degrading bacteria and the limited bioavailability of the contaminant in subsurface have been identified as the two principle causes for slow biodegradation, as discussed in Chapter 1. Chapter 5 has addressed the use of microbubble dispersion as a carrier of contaminant-degrading bacteria by studying the interaction between rhamnolipid microbubble dispersion and two model bacterial strains. The findings suggest that bacterial cell surface hydrophobicity and its interaction with rhamnolipid are the key considerations for making the microbubble dispersion an effective bacterial carrier. This chapter looks at how rhamnolipid microbubble dispersion may improve contaminant bioavailability by studying the interaction involving microbubble dispersion, a model contaminant and the model bacteria. Microbubble dispersion is capable of mobilising and dispersing non-aqueous-phase-liquid (NAPL) waste (see Section 2.4.3). It is proposed that the presence of multiple layers of surfactant encapsulating the microbubble surface acts like a two dimensional micelle that provides a region where hydrophobic substances may be attracted (Sebba, 1987). Tiny oil drops have been shown to adhere to the microbubble surface and be transported to the surface by the rising bubbles during floatation application (Sebba, 1987). For bioremediation application, we may hypothesise that a microbubble dispersion improves contaminant bioavailability by promoting direct contact between the contaminant and contaminant-degrading bacteria immobilised at the microbubble surface. Column experiments have been used to demonstrate improved mobilisation of NAPL contaminant by microbubble dispersion (Rothmel et al., 1998; Roy et al., 1995a; Roy et al., 1994), but there is no direct evidence showing how the microbubble dispersion increases contact between contaminant and bacteria. The key objective of this chapter is to investigate the interaction between bacteria and the contaminant at the microbubble surface using fluorescence microscopy to support the 157    Chapter 6 – Visualisation of Bacteria/Contaminant/Microbubble Interaction and Surface  Thermodynamic Modelling of the Interaction  hypothesis. Hexadecane is used as a model NAPL contaminant (Noordman et al., 2000). Hexadecane is an apolar hydrocarbon that does not dissolve in water. The interaction of hexadecane with two model bacterial strains P.putida and R.erythropolis in rhamnolipid microbubble dispersion was examined. The LW-AB surface thermodynamic model was used to provide a theoretical explanation of the interaction between microbubble dispersion, hexadecane and bacteria. 6.2. Visualisation of Microbubble/Contaminant Interaction To verify the hypothesis that a microbubble dispersion promotes direct contact between the contaminant and contaminant-degrading bacteria immobilised at the microbubble surface, the first step is to show that adhesion of hexadecane to microbubble surface is possible. Photomicrographs of microbubble/hexadecane interaction may provide visual evidence to support this. Microbubble dispersion made from 1,000 mg/L rhamnolipid solution was mixed with hexadecane that was stained with fluorescent dye, and samples of the mixture were examined under fluorescence microscopy. The method is discussed in Section 3.12.1. Figure 6-1 shows the fluorescence photomicrographs of microbubble/hexadecane interaction. Small globules of hexadecane can be observed to adhere to the microbubble surface, as shown in Figure 6-1. The adhesion of hexadecane to microbubbles is consistent with the observation by Sebba (1987) that tiny oil droplets tend to adhere to microbubble surface. Since the microbubble surface comprises of multiple layers of surfactant, the orientation of the surfactant molecules creates a hydrophobic region (Sebba, 1987), as illustrated in Figure 6-2. It is possible that the hexadecane adhere to the hydrophobic region as it is energetically favourable. 158    Chapter 6 – Visualisaation of Bactteria/Contam minant/Micro obubble Inte eraction and  Surface  dynamic Mo odelling of the Interactionn  Thermod Figure 66-1. Microbub bble with glob bules of n-hexxadecane adh hered to the surface. s Scalee bar equals to t 80 µm. Figure 6-22. A schematic of microbu ubble structu ure showing th he hydrophobbic region. 159    Chapter 6 – Visualisaation of Bactteria/Contam minant/Micro obubble Inte eraction and  Surface  dynamic Mo odelling of the Interactionn  Thermod These pphotomicroggraphs reveeal a possib le explanation to whatt was report rted in the literature that microbubble dispersions enhanced mobilisatio on of contaminant in ssoil column ns when comparred to surfacctant solution (Rothmeel et al., 19 998; Roy et al., 1995a;; Roy et al.., 1994). As the m microbubblles pass thro ough pore sspace, the bu ubble surface picks upp contaminaants such as hexaadecane whhich then beecome physsically attacched to the microbubbbles and trav vel with the movving bubblees, resulting in increaseed mobilisattion of the contaminant c t. 6.3. Viisualisatioon of Miccrobubblee/Contam minant/Baccteria Intteraction The finndings that (1) ( the mod del contaminnant hexadeecane adherres to the m microbubblee surface (Figure 6-1) and (22) that bactteria can addhere to miccrobubble dispersion d ((Chapter 5) support the hyppothesis thaat microbu ubble disperrsion can improve co ontaminant bioavailab bility by providinng contact between the contaminnant and con ntaminant-d degrading bbacteria. To provide direct eevidence shoowing the in nteraction oof bacteria and a hexadeccane in micrrobubble dispersion, fluoresccence photoomicrograph hs of the m microbubble//contaminan nt/bacteria m mixture were taken and are shown in Figure F 6-3 and a 6-4. Both Fiigure 6-3 and a 6-4 reveeal that baccterial cellss adhere to a layer of hexadecanee that is attachedd to the microbubble surface. s P.pputida cellss adhere at the surface of the hex xadecane with thhe cells staaying in th he aqueous phase (Fig gure 6-3). In contrary ry to P.puttida, the R.erythrropolis cells penetrate the hexadeccane surfacee as shown in Figure 6 -4. Figuree 6-3. Fluorescence photom micrographs oof P.putida ceells interactin ng with n-hexxadecane atta ached to microbu ubble surfaces. Microbubb bles were mad de from 1000 0 mg/L rhamn nolipid. P.puttida cells werre stained with fluoorescent dye Aridine Orange. Hexadeccane was staiined with fluo orescent dye N Nile Red. A, scale bar equ uals to 40 µm.. B, scale bar equals to 16 µm. 160    Chapter 6 – Visualisaation of Bactteria/Contam minant/Micro obubble Inte eraction and  Surface  dynamic Mo odelling of the Interactionn  Thermod Figure 66-4. Fluoresceence photomiicrographs off R.erythropollis cells intera acting with nn-hexadecane attached to microobubble surfaaces. Microbu ubbles were m made from 10 000 mg/L rha amnolipid. R.eerythropolis cells c were stained d with fluorescent dye Arid dine Orange.. Hexadecanee was stained with fluoresccent dye Nile Red. A, scale barr equals to 400 µm. B, scalee bar equals to t 16 µm. These pphotomicroographs sho ow that thee microbub bble surfacee provides a platform m where bacteriaa are broughht into contaact with conntaminant. The T improv ved contamin inant bioavaailability to the bbacteria, couupled with the releasee of oxygen n or air from m the core oof the micrrobubble (Ripleyy et al., 20022), provide a possible eexplanation n to the enhaanced in situu biodegrad dation of contaminant in soiil columns reported inn the literature (Michelsen et al., 1983; Mullligan & Eftekhaari, 2003; Ripley et al., 2000; Rothhmel et al., 1998). Moreovver, these images i rev veal that thhe two typees of bacteeria have ddifferent modes m of adhesioon with hexxadecane, where w P.putiida cells staay at the aq queous sidee of the hex xadecane interfacce but R.ery rythropolis cells are w within the hexadecane h phase. Ussing phase contrast microsccopy, otherss have obseerved similaar behaviou ur, where Rhodococcuss sp. strain F9-D79 were abbsorbed intto crude oiil and the Pseudomon nas sp. straain JA5-B445 localised d in the aqueouss phase adjjacent to th he oil-waterr interface (van ( Hamm me, 2004). P P.putida ceells have hydrophhilic surfacee, whereas the t R.erythrropolis cells are highly y hydrophobbic (Section n 5.6). It is posssible that the hydrop phobic R.errythropolis cells can assimilatee to the similarly s hydrophhobic hexaddecane liqu uid due to hhydrophobicc attraction.. For the hyydrophilic P.putida P cells, thhe energy of o attraction n keeps thee cells at th he hexadeccane surfacee but is no ot strong enough to pull the cells acrosss the waterr-hexadecan ne interface,, therefore tthe cells staay in the aqueouss phase. Surrface free en nergy calcuulation is used to provid de support tto this explaanation. 161    Chapter 6 – Visualisation of Bacteria/Contaminant/Microbubble Interaction and Surface  Thermodynamic Modelling of the Interaction  6.4. Surface Thermodynamic Modelling of Microbubble – Contaminant – Bacteria Interaction 6.4.1. Model Assumptions The behaviour of bacteria and hexadecane at the microbubble surface can be explained using the LW-AB surface thermodynamic model. The LW-AB model has been used in Section 5 to calculate the bacterial interaction energy with microbubble and soil surfaces. The same equation (Equation 7) can be used to calculate the interaction energy between 1) hexadecane and microbubble and 2) bacteria and hexadecane. Equation 7 states that:    LW  LW   LW  LW   LW  LW   LW 1 3 2 3 1 2 3  Gadh  2                                3 1 2 3 3 1 2 3 1 2 1 2       [7] where subscript 1 represents microbubble surface or bacteria cell; 2 represents hexadecane; and 3 represent the aqueous medium in which the interaction occurs. As discussed in Section 2.7, the value of Gadh is a quantitative expression of the likelihood for the adhesion to occur. By definition, the adhesion is energetically favourable when Gadh <0. If Gadh >0, the adhesion is unlikely to occur since it is energetically unfavourable. The assumptions listed in Section 5.7 also apply to this model. For microbubble-hexadecane interaction, the two interacting surfaces are microbubble (subscript 1) whose surface tension parameters are represented by a rhamnolipid film (see Section 5.7), hexadecane (subscript 2), and the medium is rhamnolipid solution (subscript 3) at 1000 mg/L. For microbubble-bacteria-hexadecane interaction, the two interacting surfaces are bacteria (subscript 1) and hexadecane (subscript 2) and the medium is rhamnolipid solution (subscript 3) at 1000 mg/L. These modelled interactions are schematically illustrated in Figure 6-5. 162    Chapter 6 – Visualisaation of Bactteria/Contam minant/Micro obubble Inte eraction and  Surface  dynamic Mo odelling of the Interactionn  Thermod Figu ure 6-5. Schem matic of mod delled interactions There thhree interacctions modeelled are thhe microbub bble/hexadecane interac action, mark ked A in Figure 6-5; the hexadecanee/P.putida interaction n, marked B in Figgure 6-5; and a the hexadeccane/R.erythhropolis intteraction, m marked C in Figure 6-5. Table 66–1 providdes a summ mary of thee surface tension paraameters useed for the surface thermoddynamic calculation. Surface tensiion parametters of rham mnolipid sollution at 100 00 mg/L are usedd because thhe experiments were ccarried out with w microb bubble dispeersion creatted from 1000 m mg/L rhamnoolipid solutiion. Tab ble 6-1. Summ mary of surfacce tension parameters of various v materrials. Maaterial a Hexadecaanea Rhamnollipid filmb Rhamnollipid solutio on (1000 mgg/L)c P.putidad R.erythroopolisd S Surface tenssion param meters (mJ/m /m2) γ LW γ+ γ– 277.5 0 0 377.2 5.3 333.1 266.9 9.9 228.9 277.1 344.4 0.70 0.02 555.9 3.6 Data addapted from vaan Oss (2006)). b Data adaptted from Chen n (2004). c Daata estimated ffrom literaturee (Section 5.7.2). d D Data obtained from previou us calculationss (Section 5.6)). 163    Chapter 6 – Visualisaation of Bactteria/Contam minant/Micro obubble Inte eraction and  Surface  dynamic Mo odelling of the Interactionn  Thermod 6.4.2. S Surface Freee Energy of o Interactiion The callculated surrface free en nergy of intteraction ( Gadh ) of th he three inteeractions illlustrated in Secttion 6.4.1 is i presented in Figurre 6-6. Thee interaction n energy iis negative for the microbuubble-hexaddecane, hex xadecane-P..putida and d hexadecan ne-R.erythro ropolis interractions, with hexadecane-R R.erythropollis having thhe most neg gative valuee. Figuree 6-6. Surfacee free energy of interactio ns for microb bubble/hexad decane, hexaddecane/P.putiida and hexaadecane/R.eryythropolis. Veertical bar reepresents 1 standard deviaation. The neggative Gadh suggests that all of the three interactions i s are energeetically fav vourable. This is consistent with w what was w observeed in Figuree 6-1 that gllobules of hhexadecane adhered onto m microbubblee surface, and with Figure 6--3 and Fig gure 6-4 tthat P.putiida and R.erythrropolis cells adhered to o hexadecanne. Moreovver, the comparison c of the  Gadh valu ues betweeen hexadeccane/P.putida and hexadeccane/R.erythhropolis pro ovides an exxplanation to t the differrence in thee mode of ad dhesion. The G adh resultingg from the adhesion a off R.erythrop polis to hex xadecane (-554.3 mJ/m2) is four times m more negatiive than thaat of P.puttida (-11.5 mJ/m2), indicating thaat the attraaction to hexadeccane with R.erythropo R olis cells iss much stro onger than that with P P.putida ceells. The strong aattraction causes the R.erythropol R lis cells to penetrate th he hexadecaane-water interface i and rem main in the energeticall e ly favourablle apolar heexadecane phase. p In com mparison, since s the calculatted interacttion energy y between the P.putid da cells and hexadecaane is weaaker, the 164    Chapter 6 – Visualisation of Bacteria/Contaminant/Microbubble Interaction and Surface  Thermodynamic Modelling of the Interaction  attraction keeps the cells at the water-hexadecane interface within the aqueous phase but it is not strong enough to favour the penetration of the hydrophilic cells through the waterhexadecane interface. Several studies report that surfactants can inhibit biodegradation of hydrophobic contaminant by reducing bacterial adhesion to the contaminant (Deschenes et al., 1995; Efroymson & Alexander, 1991; Stelmack et al., 1999). The presence of either Triton X-100 or Dowfax 8390 surfactant at 0.5 CMC inhibited adhesion of a Mycobacterium strain and a Pseudomonas strain to NAPLs (Stelmack et al., 1999). Our findings demonstrate that rhamnolipid surfactant at 1000 mg/L concentration, which is well above the CMC, does not inhibit adhesion of bacteria to hexadecane, a model NAPL contaminant. Increasing the rhamnolipid concentration to 4,000 mg/L has not inhibited adhesion of the bacteria to hexadecane, as predicted by the calculated Gadh values in Table 6-2. Table 6-2. Comparison of surface free energy of adhesion of bacteria to hexadecane at different rhamnolipid concentration. Bacteria P.putida R.erythropolis Surface Free Energy of Interaction ( Gadh ; mJ/m2) at Different Rhamnolipid Concentration 500 mg/L 1,000 mg/L 4,000 mg/L -9.7 -11.5 -13.4 -59.3 -54.3 -44.8 For P.putida, increasing the rhamnolipid concentration has a positive effect on making the Gadh values more negative as shown in Table 6-2, indicative that the adhesion is energetically more favourable at higher rhamnolipid concentration. But for R.erythropolis, a reverse trend is observed where increasing the rhamnolipid concentration makes the adhesion less energetically favourable. 6.5. Implication for Bioremediation 6.5.1. Microbubbles as Biodegradation Facilitator It was hypothesised that microbubble dispersion can improve contaminant bioavailability by immobilising the bacteria and contaminant at the bubble surface, providing contact between the contaminant and contaminant-degrading bacteria. Fluorescence photomicrographs provide visual evidence showing the adhesion of model NAPL contaminant hexadecane at 165    Chapter 6 – Visualisation of Bacteria/Contaminant/Microbubble Interaction and Surface  Thermodynamic Modelling of the Interaction  the microbubble surfaces in the form of small globules (Section 6.2) and the adhesion of contaminant-degrading bacteria P.putida and R.erythropolis to the hexadecane at the microbubble surface (Section 6.3). NAPL contaminant has limited bioavailability because of its presence as a continuous pool of bulk liquid mixture and so it is difficult for bacteria to access the contaminant (Corapcioglu et al., 2000). When hexadecane adheres to the microbubble surface in the form of small globules, the small globules of hexadecane present an increased surface area for bacterial access. At the same time, the microbubbles facilitate bacterial adhesion to hexadecane by localising the bacteria and contaminant at the bubble surface. The gradual release of air or oxygen from the microbubble further enhances the rate of biodegradation at the bubble surface (Choi et al., 2009; Park et al., 2009; Ripley et al., 2002). These findings provide direct evidence to explain the enhanced in situ biodegradation observed in the current literature for microbubble dispersion. They also verify our hypothesis that microbubble dispersion can improve contaminant bioavailability by providing bacterial contact with the contaminant. 6.5.2. Selection of Surfactant and Contaminant-degrading Bacteria While some studies reported that addition of surfactant inhibited bacterial adhesion to NAPL contaminant (Deschenes et al., 1995; Efroymson & Alexander, 1991; Stelmack et al., 1999), our findings show that rhamnolipid at 1,000 mg/L concentration did not cause inhibition of the adhesion of P.putida bacteria to hexadecane. Although the addition of 1,000 mg/L rhamnolipid concentration made the adhesion of R.erythropolis to hexadecane less energetically favourable, the surface free energy value remained negative and therefore the adhesion was still favourable as evident by Figure 6-4. The mode of adhesion appears to depend on the bacterial cell surface hydrophobicity, with hydrophobic bacteria R.erythropolis assimilated in the hexadecane phase while hydrophilic P.putida stayed in the aqueous phase adjacent to the water-hexadecane interface. The different mode of adhesion may have an impact on the biodegradation efficiency (van Hamme, 2004). It is important to consider bacterial cell surface hydrophobicity when choosing a bacterial strain for bioremediation. Since our findings also suggest that surfactant can change bacterial cell surface hydrophobicity (Chapter 5), in addition to being bio-compatible, selecting a surfactant that has complimentary surface properties with the bacterial strain is an important 166    Chapter 6 – Visualisation of Bacteria/Contaminant/Microbubble Interaction and Surface  Thermodynamic Modelling of the Interaction  consideration. Therefore when designing a bioremediation scheme, one can consider a tailored approach by selecting bacteria and surfactant that are compatible with the contaminant hydrophobicity as well as the soil environment to accelerate the biodegradation process. The selection can be made using the LW-AB surface free energy calculation based on the surface tension parameters (γLW, γ+ and γ–) of the bacteria, surfactant and contaminant. To illustrate this idea, surface tension parameters of selected bacteria, surfactant and contaminants is summarised in Table 6-3. Table 6-3. Summary of surface tension parameters of selected bacteria, surfactant and contaminants. Material Bacteria Pseudomonas putida 852 Rodococcus erythropolis 3586 Escherichia coli HB 101 Escherichia coli JM 109 Pseudomonas fluorescens Pseudomonas putida Streptococcus mitis BA Staphylococcus epidermidis Contaminant Hexadecane Naphthalene (solid) Surfactant Rhamnolipid Linear polyoxyethylene Soil d silica sand Kaolinite-claye Smectite-claye Peat (organic soil)f Surface tension parameters (mJ/m2) LW γ γ+ γ– Source 30.9 0.21 57.0 This study This study 33.9 0.22 0.5 39.1 39.6 36.5 34.8 35.8 26 0.59 0.56 1.29 0.62 5.8 6 59.0 57.3 56.9 55.4 3.8 49 Chen et al. (2003) Chen et al. (2003) Chen et al. (2003) Chen et al. (2003) van der Mei et al. (1998) van der Mei et al. (1998) 27.5 42.7 0 0 0 1.36 van Oss (2006) van Oss (2006) 37.2 43.0-45.9 5.3 0 22.7 41 10.9 20.5 1.57 0.7 0.4 1.2 33.1 Chen (2004) 58.5-64.0 van Oss (2006) 15.4 30 44.6 23.3 Chen & Zhu (2005) Wu (2001) Wu (2001) Michel et al. (2001) Surface free energy calculation shows that the interaction energy Gadh for S.epidermidis and hexadecane, for example, is about 27 mJ/m2, which predicts that the bacteria may not be suitable for bioremediation of hexadecane. However, P.fluorescens is a promising candidate for the degradation of hexadecane (with Gadh of -16 mJ/m2) and naphthalene (with Gadh of -10 mJ/m2) as the negative interaction energy indicating an energetically favourable interaction. 167    Chapter 6 – Visualisation of Bacteria/Contaminant/Microbubble Interaction and Surface  Thermodynamic Modelling of the Interaction  6.6. Chapter Summary Column studies in the literature report that microbubble dispersion enhances contaminant mobilisation and increases the rate of biodegradation. However there is no direct evidence to explain the improved performance by microbubble dispersion. We hypothesised that microbubble dispersion can improve contaminant bioavailability by immobilising contaminant and contaminant-degrading bacteria at the bubble surface, providing contact between the contaminant and bacteria. Fluorescence microscopy was employed to obtain visual evidence to support the hypothesis. Fluorescence photomicrographs reveal that hexadecane adheres to the rhamnolipid microbubble surface in the form of small globules. Contaminant-degrading bacteria P.putida and R.erythropolis are also shown to adhere to the hexadecane that is immobilised to the rhamnolipid microbubble surface. The mode of adhesion is dependent on the bacterial cell surface hydrophobicity, with hydrophobic R.erythropolis assimilated in the hexadecane phase while hydrophilic P.putida remained in the aqueous phase adjacent to the water-hexadecane interface. The observation shows that the rhamnolipid microbubble dispersion improves bacterial contact with NAPL contaminant and offers an explanation for improved contaminant bioavailability. LW-AB surface thermodynamic modelling was used to explain the observed adhesion and the different mode of adhesion to hexadecane by P.putida and R.erythropolis. The calculation shows that both the adhesion of hexadecane to microbubble surface and the bacterial adhesion to hexadecane are energetically favourable since the interaction energy Gadh is negative. The Gadh values also predict that the attraction to hexadecane with R.erythropolis cells is much stronger than that with P.putida cells, which explains the observed difference in the localisation of P.putida and R.erythropolis in hexadecane. Rhamnolipid microbubble dispersion can potentially overcome the problem of limited contaminant bioavailability by improving contact for NAPL contaminant and the contaminant-degrading bacteria. However, the effectiveness is affected by the bacterial cell surface hydrophobicity as predicted by the LW-AB surface free energy model. Consequently a tailored approach is proposed whereby a complimentary selection of bacteria and surfactant is matched with the contaminant hydrophobicity that aims to speed up the bioremediation process. The LW-AB surface thermodynamic modelling based on the surface tension 168    Chapter 6 – Visualisation of Bacteria/Contaminant/Microbubble Interaction and Surface  Thermodynamic Modelling of the Interaction  parameters (γLW, γ+ and γ–) of the bacteria, surfactant and contaminant can be used to assist the selection process. 169    Chapter 7 – Conclusions and Recommendation for Future Research  Chapter 7. Conclusions and Recommendation for Future Research 7.1. General Summary This thesis investigated the production of microbubble dispersion from rhamnolipid biosurfactant and examined the mechanism and factors that impact upon the effectiveness of the dispersion for improving bioremediation efficiency, with a specific focus on the use of microbubble dispersion as a carrier for contaminant-degrading bacteria within a bioremediation scenario. Characterisation studies investigated the stability, size distribution and gas hold-up properties of the rhamnolipid microbubble dispersion. Drainage experiments and contact angle measurements were performed to identify factors that influence P.putida and R.erythropolis adhesion to microbubble dispersion. Fluorescence microscopy was used to investigate the interaction of P.putida and R.erythropolis and hexadecane, a model NAPLcontaminant on the microbubble surface. The LW-AB surface thermodynamic model was applied to quantify the interaction energy to better explain the bacteria-microbubble and bacteria-contaminant-microbubble interactions. 7.2. Conclusions and Implications for Bioremediation Overall, the findings from this thesis support that:  Rhamnolipid biosurfactant can be used to make microbubble dispersions that have comparable properties to synthetic surfactants;  The rhamnolipid microbubble dispersion is better suited for the transport of P.putida and is more effective at increased rhamnolipid concentration; but it is not suited for R.erythropolis delivery; and  The dispersion promotes P.putida and R.erythropolis bacterial contact with hexadecane at the microbubble surface, thereby increasing the contaminant bioavailability. Specifically, there are six main objectives (see Section 1.1) contained in this thesis. The conclusions drawn for each objective and their implications on bioremediation are discussed. 170    Chapter 7 – Conclusions and Recommendation for Future Research  The first objective was to develop the best possible generator configuration to make stable microbubble dispersion. The desirable mixing apparatus consist of a flat disk and two baffles, and the optimum mixing speed is 8,000 rpm at a mixing time of 3 minutes. However, these mixing conditions are designed for this study. For actual field-scale applications and benchtop studies that require continuous supply of microbubble dispersion, prolonged mixing at duration much greater than 3 minutes would be needed. It is important to implement temperature control to the mixing apparatus to avoid excessive heating from the prolonged mixing. The second objective was to investigate microbubble dispersion properties including drainage/stability, size distribution and gas hold-up under various environmental conditions, such as pH, ionic strength and surfactant concentration. The characterisation studies reveal that rhamnolipid makes microbubble dispersion with properties similar to synthetic surfactants used in this study as well as those reported in the literature. Stability (i.e., half-life) of the rhamnolipid microbubble ranges from 385 seconds to 546 seconds. Gas hold-up is fairly constant ranging from 67% to 72%. The majority of the microbubbles fall in the size range of 20 μm to 140 μm. These results suggest that rhamnolipid biosurfactant is a good alternative to synthetic surfactants for making microbubble dispersion. The stability of microbubble dispersion is an important consideration for bioremediation application, and depends on factors such as surfactant concentration, pH, and salt and bacterial concentrations. These parameters may be optimized to suit field-specific applications. For example, relatively stable rhamnolipid microbubble dispersion can be produced at pH between 6 to 8. The use of rhamnolipid may not be suitable for acidic or alkaline soils beyond those pH values. Increasing the rhamnolipid concentration from 1000 to 4000 mg/L increases the dispersion stability by 13%, but the 3-fold increase in rhamnolipid use will multiply the bioremediation cost. The addition of salt of up to 3000 mg/L and the presence of bacteria do not impact on the stability. Another important field property is the soil type that can impact on the dispersion stability (Wan et al., 2001). For example, it is expected that the dispersion is more stable in sandy soil than clay soils due to the larger pore size in sandy soils. 171    Chapter 7 – Conclusions and Recommendation for Future Research  The third objective was to develop and improve a drainage model specific to microbubble dispersion. The proposed drainage model consists of three distinct drainage phases and is best described by Equation 13 (Section 4.4). The improved drainage model may be integrated with transport models such as the colloid transport model (Wan et al., 2001) for microbubble dispersion through porous medium. This will provide better prediction of how far the dispersion travels in subsurface. The fourth objective was to investigate the interactions between bacteria and a rhamnolipid microbubble dispersion under the influence of various process parameters such as rhamnolipid concentration, salt and bacterial cell surface hydrophobicity. Rhamnolipid concentration and cell surface hydrophobicity were demonstrated as two important factors to control when making the microbubble dispersion an effective bacterial carrier (Sections 5.6 and 5.7). The rhamnolipid microbubble dispersion is more effective in delivering hydrophilic P.putida than hydrophobic R.erythropolis bacteria. Increasing the rhamnolipid concentration changes bacterial cell surface hydrophobicity, for example, by making hydrophilic P.putida more hydrophobic and hydrophobic R.erythropolis more hydrophilic. For P.putida in particular, the rhamnolipid treatment releases LPS from the P.putida cell surface, which may contribute to the increase in hydrophobicity. The increase in cell hydrophobicity makes the adhesion of P.putida to microbubble energetically more favourable at higher rhamnolipid concentration. But increasing rhamnolipid concentration does not have positive effect on increasing the adhesion of R.erythropolis cells due to their preference to form cell aggregates. The hydrophobicity of the bacteria and its compatibility with rhamnolipid are important considerations when choosing rhamnolipid microbubble dispersion as a delivery agent. The fifth objective was to investigate the interaction between bacteria and contaminant at the microbubble surface. It is found that the rhamnolipid microbubble dispersion provides direct contact between bacteria (P.putida or R.erythropolis) and hexadecane immobilised at the microbubble surface. The mode of contact is dependent on the bacterial cell surface hydrophobicity, with hydrophobic R.erythropolis assimilated in the hexadecane phase while hydrophilic P.putida stayed at the water-hexadecane interface. Therefore, in addition to the compatibility with rhamnolipid microbubble dispersion, the selection of bacteria needs to match with the contaminant hydrophobicity to improve the contaminant bioavailability. The LW-AB model using surface tension parameters (γLW, γ+ and γ–) of the bacteria, surfactant and contaminant can be used to assist the selection process. In this study, P.putida is 172    Chapter 7 – Conclusions and Recommendation for Future Research  considered a better candidate since it is compatible with both rhamnolipid microbubble dispersion and the NAPL contaminant hexadecane. The sixth objective was to quantify the interaction between bacteria, contaminant and microbubble dispersion using the LW-AB surface thermodynamic approach. The LW-AB model was successfully applied to explain the interactions from a theoretical surface free energy perspective. An energetically favourable interaction, demonstrated as the increased P.putida adhesion with microbubble dispersion at high rhamnolipid concentration, has lower (more negative) surface free energy than a less favourable interaction, such as the lower P.putida adhesion at lower rhamnolipid concentration (Section 5.7.6). The model also predicts that soil surface properties can affect how bacteria interact with microbubble dispersion. For example, the rhamnolipid microbubble dispersion is predicted to be a better carrier for delivering P.putida through soils such as sand, kaolinite-clay, smectite-clay and peat due to energetically favourable bacterial interaction with the microbubble, but it is not suited for delivering R.erythropolis because it is more energetically favourable for R.erythropolis to adsorb onto these soil surfaces than adhering to the microbubble. The LWAB surface thermodynamic model is useful for predicting the bacterial interaction with microbubble dispersion and contaminant, and can be used as a tool to assess their compatibility in terms of surface hydrophobicity. 7.3. Significant Contributions 1. At the start of the thesis, microbubble dispersion was described with a two-stage drainage process consisting of an initial stage during which liquid drains under gravity followed by a stage in which foam breaks down due to thinning of films between bubbles (Yan et al., 2005). The two-stage model fails to acknowledge the emulsionlike uniqueness of the microbubble dispersion and so it fails to explain the ‘S’- shaped drainage profile associated with the dispersion. A three-phase drainage mechanism unique to microbubble dispersion was proposed with supporting evidence in the thesis (see Section 4.9). The first phase describes a creaming process, where the liquid drains due to a combination of upflow migration of bubbles and downward liquid drainage under gravity. Following this phase, the drainage is dominated by liquid flow under gravity. Eventually, after the removal of about 90% of the liquid, the drainage is from liquid released from lamella under capillarity suction. The proposed drainage 173    Chapter 7 – Conclusions and Recommendation for Future Research  mechanism completes the explanation to the unique drainage profile of microbubble dispersion. A modified drainage equation is proposed to provide a better fit between the experimental results and the 3-stage drainage mechanism. 2. Surfactants, due to their surface active feature, tend to associate with surfaces or interfaces. However the importance of how surfactant affects the bacteriacontaminant-microbubble interaction within the dispersion has not received much attention. The thesis has used different methods, including bacterial drainage experiments, fluorescence microscopy, contact angle measurement and surface thermodynamic modelling, to investigate the interactions with surfactant present and substantial progress has been made. Findings from this thesis show that surfactant changes bacterial cell surface hydrophobicity by making hydrophilic cells more hydrophobic and vice versa. Surfactants modify the cell surface hydrophobicity through adsorption or releasing cell surface components such as EPS. This change in the cell hydrophobicity is mainly attributed to a change in the electron-donating property of the cell surface resulting from the surfactant interaction. These findings have not been reported in the literature before. Moreover, the finding that the bacteriacontaminant-microbubble interaction is dependant mainly on surfactant concentration and bacterial cell hydrophobicity allows us to improve existing bioremediation approach. By factoring surface hydrophobicity in the selection of the right bacterial species and surfactant match with the contaminant, the problem of inhibiting bacterial contact with contaminant with surfactant present may be eliminated. The best way to achieve this is using surface thermodynamic model with known or estimated surface tension parameters γLW, γ¯ and γ+. 3. This study shows that microbubble dispersions can localise contaminant and bacteria on microbubble surface using fluorescence microscopy and is supported by surface thermodynamic calculations. The efficiency of bioremediation can be constrained by the lack of contaminant-degrading bacteria and limited contaminant bioavailability (Abalos et al., 2004; Atlas & Cerniglia, 1995; Makkar & Rockne, 2003). These fluorescence photomicrographs provide visual evidence that microbubble dispersions may increase bacterial contact with NAPL contaminant at the microbubble surface. 174    Chapter 7 – Conclusions and Recommendation for Future Research  7.4. Recommendations for Future Research A number of recommendations for future research can be made based on the research results from this thesis. 1. The drainage equation may be incorporated to models describing microbubble transport through porous medium. The current drainage model does not take into account the effect of stability of microbubble dispersion on the transport of the microbubble. Since it has been observed that unstable microbubbles would collapse and regenerate as they travel through soil column (Huang & Chang, 2000; Jeong & Corapcioglu, 2003; Jeong & Yavuz Corapcioglu, 2001), incorporating the draining equation allows a more accurate prediction and description of the movement of microbubble dispersion in subsurface. 2. Surfactant type and concentration were shown to have profound effect on bacterial cell surface hydrophobicity. Surfactants can modify the cell surface hydrophobicity through adsorption or releasing cell surface components such as EPS. Since surfactants are ubiquitously present in the environment (Brown & Jaffé, 2006), determining how surfactants interact with bacteria can be of high importance in the biomedical, environmental and industrial research areas (Cloete, 2003). 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Applied Microbiology and Biotechnology, 77(2), 447-455. 191      Appendix I MATLAB Command   I    %Bubble size image analysis-MatLab Command History I=imread('Rhamno4k1.jpg'); level=graythresh(I); level I2=im2bw(I,level); whos I2 [labeled,numObjects]=bwlabel(bw,9); [labeled,numObjects]=bwlabel(I2,8); numObjects RGB_labeled=label2rgb(labeled,@spring,'c','shuffle'); imshow(RGB_labeled) bubbledata=regionprops(labeled,'basic') bubblearea=[bubbledata.Area]; whos bubblearea bubblearea clear, close all I=imread('Rhamno05k1.jpg'); whos I I2=im2bw(I, 0.8); figure,imshow(I),figure,imshow(I2) clear, close all I=imread('Rhamno05k1.jpg'); level=graythresh(I); level bw=im2bw(I,level); figure,imshow(I),figure,imshow(bw) whos bw [labeled,numObjects]=bwlabel(bw,8); numObjects RGB_labeled=label2rgb(labeled,@spring,'c','shuffle'); imshow(RGB_labeled) clear, close all I=imread('Rhamno05k1.jpg'); level=graythresh(I); level bw=im2bw(I,level); figure,imshow(I),figure,imshow(bw) [labeled,numObjects]=bwlabel(bw,8); numObjects RGB_labeled=label2rgb(labeled,@spring,'c','shuffle'); imshow(RGB_labeled) bubbledata=regionprops(labeled,'basic') bubblearea=[bubbledata.Area]; whos bubblearea bubblearea clear, close all   1   Appendix II P.putida Growth Curve   II    Time Elapsed O.D. 600 0 0.005 35 0.008 95 0.012 155 0.028 335 0.330 370 0.433 395 0.493 425 0.583 455 0.694 485 0.770 515 0.903 545 0.988 575 1.080 1480 1.097 P.Putida Growth Curve 1.2 1.0 0.8 O.D. at 600nm Bacterial growth curve P.putida 852 Bh+Glucose Time 2-Nov 10:20AM 10:55AM 11:55AM 12:55PM 3:55PM 4:30PM 4:55PM 5:25PM 5:55PM 6:25PM 6:55PM 7:25PM 7:55PM 3-Nov 11:00AM 0.6 0.4 0.2 0.0 0 200 400 1 600 800 1000 1200 Time Elapsed (minutes) 1400 1600   Appendix III Stability Test Data III    Preliminary Stability Tests Conditions: 10,000 mg/L of rhamnolipid Mixing at 8,000 rpm for 3 minutes Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Observation Foam volume stablised at 2 min Results Run #1 Volume mL Percentage Time (minutes) 10 33.33% 5 11 36.67% 6 12 40.00% 6 13 43.33% 6 14 46.67% 7 15 50.00% 7 16 53.33% 8 17 56.67% 8 18 60.00% 8 19 63.33% 9 20 66.67% 9 21 70.00% 10 22 73.33% 10 23 76.67% 11 24 80.00% 11 25 83.33% 12 26 86.67% 13 27 90.00% 14 28 93.33% 15 29 96.67% 18 Total volum Time in seconds Percentage Drainage 353 33.33% 374 36.67% 396 40.00% 418 43.33% 442 46.67% 463 50.00% 486 53.33% 511 56.67% 534 60.00% 559 63.33% 589 66.67% 612 70.00% 642 73.33% 675 76.67% 708 80.00% 748 83.33% 789 86.67% 848 90.00% 932 93.33% 1117 96.67% 30 Conditions: 10,000 mg/L of rhamnolipid Mixing at 8,000 rpm for 6 minutes Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Observation Foam volume stablised at 2 min Results Volume Run #1 mL Percentage Time (minutes) 10 33.33% 11 36.67% 12 40.00% 6 13 43.33% 6 14 46.67% 7 15 50.00% 7 16 53.33% 7 17 56.67% 8 18 60.00% 8 19 63.33% 9 20 66.67% 9 21 70.00% 10 22 73.33% 10 23 76.67% 11 24 80.00% 11 25 83.33% 12 26 86.67% 14 27 90.00% 15 28 93.33% 17 29 96.67% Total vol 53 14 36 58 22 43 6 31 54 19 49 12 42 15 48 28 9 8 32 37 Duplicate Time (minutes) 5 5 6 6 7 7 7 8 8 9 9 9 10 11 11 12 13 14 15 18 38 57 17 41 5 25 49 15 37 4 35 58 27 3 37 16 0 1 37 58 30 Time in seconds 25 43 7 32 57 18 44 10 37 9 35 11 44 24 5 14 33 0 0 385 403 427 452 477 498 524 550 577 609 635 671 704 744 845 914 1053 0 Percentage Drainage 33.33% 36.67% 40.00% 43.33% 46.67% 50.00% 53.33% 56.67% 60.00% 63.33% 66.67% 70.00% 73.33% 76.67% 80.00% 83.33% 86.67% 90.00% 93.33% 96.67% 30 30 1 Average Time in seconds Time in seconds 338 345.5 357 365.5 377 386.5 401 409.5 425 433.5 445 454 469 477.5 495 503 517 525.5 544 551.5 575 582 598 605 627 634.5 663 669 697 702.5 736 742 780 784.5 841 844.5 937 934.5 1138 1127.5 30 Duplicate Time (minutes) 6 7 7 7 8 8 9 9 10 10 11 11 12 12 14 15 17 40 5 30 53 17 43 9 35 1 32 1 34 10 51 33 49 56 Average Time in seconds Time in seconds 0 0 0 0 400 392.5 425 414 450 438.5 473 462.5 497 487 523 510.5 549 536.5 575 562.5 601 589 632 620.5 661 648 694 682.5 730 717 771 757.5 873 859 949 931.5 1076 1064.5 0 0 30 Conditions: 20,000 mg/L of rhamnolipid Mixing at 10,000 rpm for 3 minutes Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Observation Foam volume stablised at 1 min Results Volume Run #1 mL Percentage Time (minutes) 10 33.33% 5 11 36.67% 6 12 40.00% 6 13 43.33% 6 14 46.67% 7 15 50.00% 7 16 53.33% 7 17 56.67% 8 18 60.00% 8 19 63.33% 9 20 66.67% 9 21 70.00% 10 22 73.33% 11 23 76.67% 11 24 80.00% 12 25 83.33% 13 26 86.67% 15 27 90.00% 20 28 93.33% Total volum Time in seconds Percentage Drainage 348 33.33% 369 36.67% 389 40.00% 413 43.33% 437 46.67% 457 50.00% 479 53.33% 504 56.67% 531 60.00% 558 63.33% 590 66.67% 621 70.00% 660 73.33% 703 76.67% 755 80.00% 827 83.33% 938 86.67% 1239 90.00% 0 93.33% 30 Conditions: 20,000 mg/L of rhamnolipid Mixing at 10,000 rpm for 6 minutes Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Observation Foam volume stablised at 1 min Results Volume Run #1 mL Percentage Time (minutes) 10 33.33% 6 11 36.67% 6 12 40.00% 6 13 43.33% 7 14 46.67% 7 15 50.00% 8 16 53.33% 8 17 56.67% 8 18 60.00% 9 19 63.33% 9 20 66.67% 10 21 70.00% 10 22 73.33% 11 23 76.67% 12 24 80.00% 12 25 83.33% 13 26 86.67% 15 27 90.00% 17 28 93.33% 24 Total volum 48 9 29 53 17 37 59 24 51 18 50 21 0 43 35 47 38 39 Duplicate Time (minutes) 5 5 6 6 7 7 7 8 8 9 9 10 10 11 12 13 16 24 35 53 14 37 3 22 45 8 37 6 37 12 48 34 32 59 23 27 30 9 29 54 17 43 6 32 56 25 53 23 56 32 10 59 53 21 25 17 Time in seconds Percentage Drainage 369 33.33% 389 36.67% 414 40.00% 437 43.33% 463 46.67% 486 50.00% 512 53.33% 536 56.67% 565 60.00% 593 63.33% 623 66.67% 656 70.00% 692 73.33% 730 76.67% 779 80.00% 833 83.33% 921 86.67% 1045 90.00% 1457 93.33% 30 30 2 Average Time in seconds Time in seconds 335 341.5 353 361 374 381.5 397 405 423 430 442 449.5 465 472 488 496 517 524 546 552 577 583.5 612 616.5 648 654 694 698.5 752 753.5 839 833 983 960.5 1467 1353 0 0 30 Duplicate Time (minutes) 6 6 7 7 8 8 8 9 9 10 10 11 11 12 13 14 15 17 24 30 49 14 36 4 25 49 17 45 11 44 15 50 28 19 9 24 16 1 Average Time in seconds Time in seconds 390 379.5 409 399 434 424 456 446.5 484 473.5 505 495.5 529 520.5 557 546.5 585 575 611 602 644 633.5 675 665.5 710 701 748 739 799 789 849 841 924 922.5 1036 1040.5 1441 1449 Conditions: 20,000 mg/L of rhamnolipid Mixing at 8,000 rpm for 6 minutes Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Observation Foam volume stablised at 2.5 min Results Volume Run #1 mL Percentage Time (minutes) 10 33.33% 6 11 36.67% 6 12 40.00% 6 13 43.33% 7 14 46.67% 7 15 50.00% 8 16 53.33% 8 17 56.67% 8 18 60.00% 9 19 63.33% 9 20 66.67% 10 21 70.00% 10 22 73.33% 11 23 76.67% 11 24 80.00% 12 25 83.33% 13 26 86.67% 13 27 90.00% 14 28 93.33% 16 29 96.67% 18 Total volum Time in seconds Percentage Drainage 371 33.33% 394 36.67% 416 40.00% 440 43.33% 464 46.67% 488 50.00% 511 53.33% 536 56.67% 561 60.00% 586 63.33% 614 66.67% 641 70.00% 672 73.33% 705 76.67% 744 80.00% 784 83.33% 830 86.67% 886 90.00% 976 93.33% 1121 96.67% 30 Conditions: 20,000 mg/L of rhamnolipid Mixing at 8,000 rpm for 3 minutes Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Observation Foam volume stablised at 2.5 min Results Volume Run #1 mL Percentage Time (minutes) 10 33.33% 6 11 36.67% 6 12 40.00% 7 13 43.33% 7 14 46.67% 7 15 50.00% 8 16 53.33% 8 17 56.67% 9 18 60.00% 9 19 63.33% 10 20 66.67% 10 21 70.00% 10 22 73.33% 11 23 76.67% 11 24 80.00% 12 25 83.33% 13 26 86.67% 13 27 90.00% 14 28 93.33% 16 29 96.67% 18 Total volum 11 34 56 20 44 8 31 56 21 46 14 41 12 45 24 4 50 46 16 41 Duplicate Time (minutes) 6 6 7 7 8 8 8 9 9 10 10 11 11 12 12 13 14 15 16 19 30 54 15 39 3 27 52 11 42 6 35 4 33 7 45 26 12 18 50 34 Average Time in seconds Time in seconds 390 380.5 414 404 435 425.5 459 449.5 483 473.5 507 497.5 532 521.5 551 543.5 582 571.5 606 596 635 624.5 664 652.5 693 682.5 727 716 765 754.5 806 795 852 841 918 902 1010 993 1174 1147.5 Duplicate Time (minutes) 6 7 7 7 8 8 9 9 9 10 10 11 11 12 13 13 14 15 17 20 45 7 31 55 20 44 7 33 59 25 58 20 52 26 5 47 36 41 12 27 Average Time in seconds Time in seconds 405 394.5 427 417 451 440 475 464.5 500 489 524 513 547 536 573 561.5 599 587 625 612.5 658 644 680 669.5 712 699 746 732.5 785 770 827 812 876 857 941 917.5 1032 1003 1227 1166.5 30 24 47 9 34 58 22 45 10 35 0 30 59 26 59 35 17 58 54 14 26 Time in seconds Percentage Drainage 384 33.33% 407 36.67% 429 40.00% 454 43.33% 478 46.67% 502 50.00% 525 53.33% 550 56.67% 575 60.00% 600 63.33% 630 66.67% 659 70.00% 686 73.33% 719 76.67% 755 80.00% 797 83.33% 838 86.67% 894 90.00% 974 93.33% 1106 96.67% 30 30 3 Conditions: 100 mg/L of rhamnolipid Mixing at 8,000 rpm for 3 minutes Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Before After pH 6.27 6.14 Temperature 22.6oC 23.8oC Observation Foam volume did not stabilise Results Volume mL Conditions: Run #1 Duplicate Percentage DTime (minuTime in seconds Percentage DrainagTime (minutes) 10 29.41% 1:54 114 29.41% 11 32.35% 2:07 127 32.35% 12 35.29% 2:22 142 35.29% 13 38.24% 2:35 155 38.24% 14 41.18% 2:50 170 41.18% 15 44.12% 3:04 184 44.12% 16 47.06% 3:20 200 47.06% 17 50.00% 3:38 218 50.00% 18 52.94% 3:55 235 52.94% 19 55.88% 4:15 255 55.88% 20 58.82% 4:32 272 58.82% 21 61.76% 4:56 296 61.76% 22 64.71% 5:17 317 64.71% 23 67.65% 5:45 345 67.65% 24 70.59% 6:14 374 70.59% 25 73.53% 6:42 402 73.53% 26.57 78.15% 478 78.15% 27.52 80.94% 581 80.94% 28 82.35% 774 82.35% Total volum 34 34 Time in seconds 2:07 2:18 2:33 2:47 3:03 3:19 3:38 3:56 4:15 4:37 4:58 5:20 5:47 6:15 6:49 7:31 127 138 153 167 183 199 218 236 255 277 298 320 347 375 409 451 520 640 800 Average Time in seconds 120.5 132.5 147.5 161 176.5 191.5 209 227 245 266 285 308 332 360 391.5 426.5 499 610.5 787 34 100 mg/L of rhamnolipid Mixing at 8,000 rpm for 3 minutes Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Before After pH 6.27 6.14 Temperature 22.6oC 23.8oC Observation Foam volume did not stabilise Results Volume mL Run #1 Duplicate Percentage DTime (minuTime in seconds Percentage DrainagTime (minutes) 10 29.41% 1:54 114 29.41% 11 32.35% 2:07 127 32.35% 12 35.29% 2:22 142 35.29% 13 38.24% 2:35 155 38.24% 14 41.18% 2:50 170 41.18% 15 44.12% 3:04 184 44.12% 16 47.06% 3:20 200 47.06% 17 50.00% 3:38 218 50.00% 18 52.94% 3:55 235 52.94% 19 55.88% 4:15 255 55.88% 20 58.82% 4:32 272 58.82% 21 61.76% 4:56 296 61.76% 22 64.71% 5:17 317 64.71% 23 67.65% 5:45 345 67.65% 24 70.59% 6:14 374 70.59% 25 73.53% 6:42 402 73.53% 26.57 78.15% 478 78.15% 27.52 80.94% 581 80.94% 28 82.35% 774 82.35% Total volum 34 34 4 Time in seconds 2:07 2:18 2:33 2:47 3:03 3:19 3:38 3:56 4:15 4:37 4:58 5:20 5:47 6:15 6:49 7:31 127 138 153 167 183 199 218 236 255 277 298 320 347 375 409 451 520 640 800 Average Time in seconds 120.5 132.5 147.5 161 176.5 191.5 209 227 245 266 285 308 332 360 391.5 426.5 499 610.5 787 34 Conditions: 1,000 mg/L of rhamnolipid Mixing at 8,000 rpm for 3 minutes with setup 1 Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Before After pH 7.37 7.34 Temperature 21.8oC 22.3oC Observation Foam volume stabilised at 1min 40sec Results Volume mL 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Run #1 Duplicate Percentage DTime (minuTime in seconds Percentage DrainagTime (minutes) Time in seconds 33.33% 5:35 335 34.48% 5:40 36.67% 5:56 356 37.93% 6:03 40.00% 6:23 383 41.38% 6:26 43.33% 6:49 409 44.83% 6:52 46.67% 7:19 439 48.28% 7:20 50.00% 7:44 464 51.72% 7:46 53.33% 8:12 492 55.17% 8:13 56.67% 8:40 520 58.62% 8:39 60.00% 9:11 551 62.07% 9:10 63.33% 9:43 583 65.52% 9:43 66.67% 10:15 615 68.97% 10:16 70.00% 10:53 653 72.41% 10:56 73.33% 11:33 693 75.86% 11:36 76.67% 12:18 738 79.31% 12:28 80.00% 13:06 786 82.76% 13:21 83.33% 13:56 836 86.21% 14:18 86.67% 15:13 913 89.66% 15:58 90.00% 16:30 990 93.10% 18:19 93.33% 19:47 1187 96.55% 26:22:00 96.67% 1905 100.00% 100.00% 103.45% Total volum 30 Conditions: 1,000 mg/L of rhamnolipid Mixing at 8,000 rpm for 3 minutes with propeller Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Observation Foam volume stablised at 2 min Results Volume mL 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Run #1 Percentage DTime (minutes) 37.04% 4 40.74% 4 44.44% 4 48.15% 5 51.85% 5 55.56% 6 59.26% 6 62.96% 6 66.67% 7 70.37% 7 74.07% 8 77.78% 8 81.48% 9 85.19% 10 88.89% 11 92.59% 12 96.30% 19 Total volum 340 363 386 412 440 466 493 519 550 583 616 656 696 748 801 858 958 1099 1582 average 337.5 359.5 384.5 410.5 439.5 465 492.5 519.5 550.5 583 615.5 654.5 694.5 743 793.5 847 935.5 1044.5 1384.5 3 4 4 4 5 5 5 6 6 7 7 8 8 9 10 11 14 53 7 27 46 8 31 50 15 41 9 36 12 51 36 32 35 16 29 16 39 56 18 42 4 25 51 17 46 16 54 33 26 27 58 51 Duplicate Time in seconds Percentage Drainage Time (minutes) 256 35.71% 279 39.29% 296 42.86% 318 46.43% 342 50.00% 364 53.57% 385 57.14% 411 60.71% 437 64.29% 466 67.86% 496 71.43% 534 75.00% 573 78.57% 626 82.14% 687 85.71% 778 89.29% 1191 92.86% 27 28 5 Time in seconds 233 247 267 286 308 331 350 375 401 429 456 492 531 576 632 695 856 Conditions: 2,000 mg/L of rhamnolipid Mixing at 8,000 rpm for 3 minutes Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Before After pH 7.3 o Temperature 22.4 C Observation Foam volume stabilised at 1min 16sec Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Run #1 Duplicate Percentage DTime Time in seconds Percentage DrainagTime 6.90% 1:30 90 6.90% 10.34% 2:09 129 10.34% 13.79% 2:43 163 13.79% 17.24% 3:23 203 17.24% 20.69% 4:02 242 20.69% 24.14% 4:32 272 24.14% 27.59% 5:04 304 27.59% 31.03% 5:26 326 31.03% 34.48% 5:44 344 34.48% 37.93% 6:10 370 37.93% 41.38% 6:33 393 41.38% 44.83% 7:00 420 44.83% 48.28% 7:27 447 48.28% 51.72% 7:52 472 51.72% 55.17% 8:18 498 55.17% 58.62% 8:49 529 58.62% 62.07% 9:19 559 62.07% 65.52% 9:54 594 65.52% 68.97% 10:26 626 68.97% 72.41% 11:08 668 72.41% 75.86% 11:53 713 75.86% 79.31% 12:45 765 79.31% 82.76% 13:44 824 82.76% 86.21% 14:47 887 86.21% 89.66% 16:57 1017 89.66% 93.10% 21:43 1303 93.10% 96.55% 96.55% 100.00% 100.00% Total volum Conditions: 29 Time in seconds 1:19 2:00 2:41 3:16 3:51 4:17 4:42 5:06 5:29 5:54 6:17 6:41 7:08 7:34 8:00 8:29 9:00 9:33 10:07 10:47 11:33 12:26 13:27 14:39 17:13 22:30 79 120 161 196 231 257 282 306 329 354 377 401 428 454 480 509 540 573 607 647 693 746 807 879 1033 1350 29 4,000 mg/L of rhamnolipid (high surfactant concentration) Mixing at 8,000 rpm for 3 minutes Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Before After pH 7.28 7.24 o 21.6 oC Temperature 20.5 C Observation Foam volume stabilised at 1min 46sec Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Run #1 Duplicate Percentage DTime Time in seconds Percentage DrainagTime 6.45% 1:23 83 6.45% 9.68% 2:01 121 9.68% 12.90% 2:34 154 12.90% 16.13% 3:14 194 16.13% 19.35% 3:57 237 19.35% 22.58% 4:30 270 22.58% 25.81% 5:01 301 25.81% 29.03% 5:25 325 29.03% 32.26% 5:45 345 32.26% 35.48% 6:10 370 35.48% 38.71% 6:36 396 38.71% 41.94% 7:00 421 41.94% 45.16% 7:27 447 45.16% 48.39% 7:54 474 48.39% 51.61% 8:19 499 51.61% 54.84% 8:45 525 54.84% 58.06% 9:14 554 58.06% 61.29% 9:43 583 61.29% 64.52% 10:11 611 64.52% 67.74% 10:47 647 67.74% 70.97% 11:21 681 70.97% 74.19% 11:57 717 74.19% 77.42% 12:37 757 77.42% 80.65% 13:14 794 80.65% 83.87% 14:14 854 83.87% 87.10% 15:15 915 87.10% 90.32% 16:38 998 90.32% 93.55% 19:24 1164 93.55% 96.77% 27:07:00 1627 96.77% 100.00% 100.00% Total volum 31 Time in seconds 1:00 1:42 2:19 2:58 3:37 4:04 4:30 4:53 5:20 5:45 6:09 6:32 7:01 7:25 7:50 8:16 8:46 9:15 9:44 10:19 10:52 11:29 12:08 12:45 13:40 14:42 16:05:00 18:29 26:48:00 31 6 60 102 139 178 217 244 270 293 320 345 369 392 421 445 470 496 526 555 584 619 652 689 728 765 820 882 965 1109 1608 Conditions: 1,000 mg/L of rhamnolipid with 1,000 mg/l of NaCl Mixing at 8,000 rpm for 3 minutes Run #1 + Duplicate 100ml of foam in measuring cylinder Total volume taken after 2 and a half hours Before After pH 6.6 6.67 o o 17.4 C Temperature 18.4 C Observation Foam volume stabilised at 1min 17sec Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Run #1 Duplicate Percentage DTime Time in seconds Percentage DrainagTime 7.02% 1:22 82 6.90% 10.53% 2:16 136 10.34% 14.04% 2:53 173 13.79% 17.54% 3:35 215 17.24% 21.05% 4:11 251 20.69% 24.56% 24.14% 28.07% 5:01 301 27.59% 31.58% 5:26 326 31.03% 35.09% 5:52 352 34.48% 38.60% 6:17 377 37.93% 42.11% 6:44 404 41.38% 45.61% 7:11 431 44.83% 49.12% 7:41 461 48.28% 52.63% 8:06 486 51.72% 56.14% 8:33 513 55.17% 59.65% 9:02 542 58.62% 63.16% 9:32 572 62.07% 66.67% 10:09 609 65.52% 70.18% 10:47 647 68.97% 73.68% 11:26 686 72.41% 77.19% 12:12 732 75.86% 80.70% 13:07 787 79.31% 84.21% 14:11 851 82.76% 87.72% 15:29 929 86.21% 91.23% 18:08 1088 89.66% 94.74% 26:32 1592 93.10% Total volum 28.5 Time in seconds 29 7 1:35 2:09 2:44 3:16 3:52 4:23 95 129 164 196 232 263 5:07 5:25 5:52 6:20 6:41 7:14 7:36 8:04 8:31 9:01 9:33 10:02 10:46 11:24 12:22 13:10 14:08 15:59 19:18 307 325 352 380 401 434 456 484 511 541 573 602 646 702 742 790 848 959 1158 Stability test for low surfactant concentration Conditions: 500 mg/L of rhamnolipid Mixing at 8,000 rpm for 3 minutes 100ml of foam in measuring cylinder pH 5 pH 6 Run# pH Temperature oC 1 Before 5.11 22.4 Run# Adjustment After foamed 6.08 6.14 22.4 22.7 pH Temperature oC 2 Before 6.14 22.9 Run# Adjustment After foamed 6.01 6.01 23 23.1 Observation Foam volume stabilised at 1min 20s Observation Foam volume not stabilised at 1min 25s Results Volume mL Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Total volu Duplicate Run #1 % Drainage Time Time (s) % Drainage Time Time (s) 7.14% 1.29 89 6.90% 1.47 107 10.71% 2.14 134 10.34% 2.20 140 14.29% 2.54 174 13.79% 2.55 175 17.86% 3.33 213 17.24% 3.22 202 21.43% 4.07 247 20.69% 3.49 229 25.00% 4.35 275 24.14% 4.19 259 28.57% 5.00 300 27.59% 4.43 283 32.14% 5.25 325 31.03% 5.02 302 35.71% 5.50 350 34.48% 5.17 317 39.29% 6.15 375 37.93% 5.43 343 42.86% 6.39 399 41.38% 6.10 370 46.43% 7.05 425 44.83% 6.40 400 50.00% 7.35 455 48.28% 7.03 423 53.57% 8.04 484 51.72% 7.32 452 57.14% 8.33 513 55.17% 7.57 477 60.71% 9.01 541 58.62% 8.28 508 64.29% 9.37 577 62.07% 8.57 537 67.86% 10.11 611 65.52% 9.31 571 71.43% 10.47 647 68.97% 10.04 604 75.00% 11.32 692 72.41% 10.43 643 78.57% 12.20 740 75.86% 11.19 679 82.14% 13.19 799 79.31% 12.05 725 85.71% 14.25 865 82.76% 13.02 782 89.29% 15.55 955 86.21% 13.58 838 92.86% 19.10 1150 89.66% 15.21 921 96.43% 26.41 1601 93.10% 17.37 1057 96.55% 22.24 1344 28 29 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Total volu % Drainage 6.90% 10.34% 13.79% 17.24% 20.69% 24.14% 27.59% 31.03% 34.48% 37.93% 41.38% 44.83% 48.28% 51.72% 55.17% 58.62% 62.07% 65.52% 68.97% 72.41% 75.86% 79.31% 82.76% 86.21% 89.66% 93.10% 96.55% pH Temperature oC Run #2 Duplicate Time Time (s) % Drainag Time Time (s) 1.23 83 6.90% 1.45 105 2.05 125 10.34% 2.27 147 2.44 164 13.79% 3.07 187 3.28 208 17.24% 3.40 220 4.00 240 20.69% 4.02 242 4.27 267 24.14% 4.27 267 4.53 293 27.59% 4.52 292 5.18 318 31.03% 5.12 312 5.49 349 34.48% 5.31 331 6.14 374 37.93% 5.55 355 6.39 399 41.38% 6.23 383 7.06 426 44.83% 6.51 411 7.37 457 48.28% 7.19 439 8.00 480 51.72% 7.45 465 8.31 511 55.17% 8.11 491 8.57 537 58.62% 8.42 522 9.34 574 62.07% 9.13 553 10.08 608 65.52% 9.47 587 10.41 641 68.97% 10.20 620 11.22 682 72.41% 11.02 662 12.09 729 75.86% 11.44 704 13.00 780 79.31% 12.31 751 13.59 839 82.76% 13.27 807 14.58 898 86.21% 14.23 863 16.55 1015 89.66% 15.50 950 19.58 1198 93.10% 18.36 1116 29.58 1798 96.55% 23.21 1401 29 29.00 8 3 Before 7.15 20.7 Adjustment After foamed 6 6.06 20.7 20.8 Observation Foam volume stabilised at 2min 20s Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Run #1 Duplicate % Drainage Time Time (s) % Drainag Time Time (s) 6.90% 1.42 102 6.67% 1.55 115 10.34% 2.24 144 10.00% 2.33 153 13.79% 3.07 187 13.33% 3.05 185 17.24% 3.43 223 16.67% 3.31 211 20.69% 4.16 256 20.00% 3.59 239 24.14% 4.45 285 23.33% 4.27 267 27.59% 5.07 307 26.67% 4.50 290 31.03% 5.34 334 30.00% 5.08 308 34.48% 5.56 356 33.33% 37.93% 6.25 385 36.67% 5.51 351 41.38% 6.51 411 40.00% 6.20 380 44.83% 7.16 436 43.33% 6.47 407 48.28% 7.44 464 46.67% 7.14 434 51.72% 8.11 491 50.00% 7.41 461 55.17% 8.41 521 53.33% 8.08 488 58.62% 9.09 549 56.67% 8.39 519 62.07% 9.41 581 60.00% 9.09 549 65.52% 10.16 616 63.33% 9.40 580 68.97% 10.50 650 66.67% 10.16 616 72.41% 11.36 696 70.00% 10.49 649 75.86% 12.18 738 73.33% 11.32 692 79.31% 13.12 792 76.67% 12.19 739 82.76% 14.14 854 80.00% 13.09 789 86.21% 15.24 924 83.33% 14.18 858 89.66% 17.39 1059 86.67% 15.45 945 90.00% 18.19 1099 29 30 pH 7 Run# pH Temperature oC 1 Before 7.09 23.1 Run# Adjustment After foamed 7.08 23.8 pH Temperature oC 2 Before 7.01 23.5 Run# Adjustment After foamed 7.02 23.5 pH Temperature oC 3 Before 7.00 23.1 Adjustment After foamed 6.97 23.5 Observation Foam volume not stabilised at 3min Observation Foam volume stabilised at 2min 25s Observation Foam volume stabilised at 1min 30s Results Volume mL Results Volume mL Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Total volu Run #1 Duplicate % Drainage Time Time (s) % Drainage Time Time (s) 6.25% 0.39 39 5.88% 0.48 48 9.38% 1.12 72 8.82% 1.16 76 12.50% 1.43 103 11.76% 1.44 104 15.63% 2.09 129 14.71% 1.59 119 18.75% 2.38 158 17.65% 2.26 146 21.88% 2.56 176 20.59% 2.45 165 25.00% 3.16 196 23.53% 3.01 181 28.13% 3.40 220 26.47% 3.19 199 31.25% 3.58 238 29.41% 3.34 214 34.38% 4.21 261 32.35% 3.49 229 37.50% 4.41 281 35.29% 4.10 250 40.63% 5.02 302 38.24% 4.32 272 43.75% 5.25 325 41.18% 4.52 292 46.88% 5.45 345 44.12% 5.14 314 50.00% 6.06 366 47.06% 5.33 333 53.13% 6.28 388 50.00% 5.55 355 56.25% 6.55 415 52.94% 6.18 378 59.38% 7.21 441 55.88% 6.42 402 62.50% 58.82% 7.04 424 65.63% 8.15 495 61.76% 7.31 451 68.75% 8.45 525 64.71% 8.02 482 71.88% 9.20 560 67.65% 8.28 508 75.00% 9.55 595 70.59% 8.55 535 78.13% 10.34 634 73.53% 9.28 568 81.25% 11.18 678 76.47% 10.04 604 84.38% 12.11 731 79.41% 10.43 643 87.50% 13.17 797 82.35% 11.27 687 90.63% 15.12 912 85.29% 12.21 741 93.75% 21.06 1266 88.24% 13.25 805 91.18% 15.09 909 32 34 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Total volu % Drainage 6.25% 9.38% 12.50% 15.63% 18.75% 21.88% 25.00% 28.13% 31.25% 34.38% 37.50% 40.63% 43.75% 46.88% 50.00% 53.13% 56.25% 59.38% 62.50% 65.63% 68.75% 71.88% 75.00% 78.13% 81.25% 84.38% 87.50% 90.63% 32 Run #2 Duplicate Time Time (s) % Drainag Time Time (s) 0.41 41 5.71% 0.38 38 1.19 79 8.57% 1.01 61 1.57 117 11.43% 1.20 80 2.24 144 14.29% 1.35 95 2.53 173 17.14% 1.57 117 3.21 201 20.00% 2.15 135 3.39 219 22.86% 2.34 154 3.59 239 25.71% 2.57 177 4.24 264 28.57% 3.06 186 4.45 285 31.43% 3.23 203 5.06 306 34.29% 3.46 226 5.29 329 37.14% 4.12 252 5.54 354 40.00% 4.31 271 6.16 376 42.86% 4.52 292 6.39 399 45.71% 5.12 312 7.07 427 48.57% 5.35 335 7.28 448 51.43% 5.57 357 7.57 477 54.29% 6.20 380 8.23 503 57.14% 6.48 408 8.51 531 60.00% 7.06 426 9.27 567 62.86% 7.35 455 10.02 602 65.71% 8.00 480 10.39 639 68.57% 8.29 509 11.22 682 71.43% 9.01 541 12.14 734 74.29% 9.33 573 13.13 793 77.14% 10.05 605 14.42 882 80.00% 10.42 642 16.52 1012 82.86% 11.28 688 85.71% 12.15 735 88.57% 13.15 795 91.43% 14.27 867 94.29% 16.29 989 35 9 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Run #3 Duplicate % Drainage Time Time (s) % Drainag Time Time (s) 6.45% 0.45 45 6.06% 1.07 67 9.68% 1.26 86 9.09% 1.31 91 12.90% 2.01 121 12.12% 1.57 117 16.13% 2.36 156 15.15% 2.23 143 19.35% 3.07 187 18.18% 2.42 162 22.58% 3.33 213 21.21% 3.04 184 25.81% 3.53 233 24.24% 3.24 204 29.03% 4.18 258 27.27% 3.38 218 32.26% 4.42 282 30.30% 3.57 237 35.48% 5.03 303 33.33% 4.20 260 38.71% 5.25 325 36.36% 4.43 283 41.94% 5.48 348 39.39% 5.06 306 45.16% 6.15 375 42.42% 5.27 327 48.39% 6.39 399 45.45% 5.49 349 51.61% 7.00 420 48.48% 6.10 370 54.84% 7.26 446 51.52% 6.35 395 58.06% 7.54 474 54.55% 7.00 420 61.29% 8.22 502 57.58% 7.24 444 64.52% 8.51 531 60.61% 7.49 469 67.74% 9.27 567 63.64% 8.18 498 70.97% 10.01 601 66.67% 8.47 527 74.19% 10.40 640 69.70% 9.17 557 77.42% 11.22 682 72.73% 9.52 592 80.65% 12.04 724 75.76% 10.30 630 83.87% 13.07 787 78.79% 11.05 665 87.10% 14.17 857 81.82% 11.56 716 90.32% 15.58 958 84.85% 12.47 767 93.55% 19.34 1174 87.88% 13.58 838 90.91% 15.21 921 93.94% 18.02 1082 Total volum 31 33 pH7 Run# pH Temperature oC 4 Before 7.09 23.1 Run# Adjustment After foamed 7.08 23.8 pH Temperature oC 5 Before 7.15 20.8 Run# Adjustment After foamed 7 7.01 20.8 21.8 pH Temperature oC 6 Before 6.99 21.3 Adjustment After foamed 6.97 21.2 Observation Foam volume not stabilised at 2min 40s Observation Foam volume stabilised at 2min Observation Foam volume not stabilised at 3min Results Volume mL Results Volume mL Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Total volu Duplicate Run #4 % Drainage Time Time (s) % Drainage Time Time (s) 5.26% 5.26% 7.89% 7.89% 0.30 30 10.53% 0.32 32 10.53% 0.43 43 13.16% 13.16% 0.54 54 15.79% 1.11 71 15.79% 1.14 74 18.42% 1.24 84 18.42% 1.29 89 21.05% 1.39 99 21.05% 1.47 107 23.68% 1.55 115 23.68% 1.56 116 26.32% 2.08 128 26.32% 1.63 123 28.95% 2.24 144 28.95% 2.21 141 31.58% 2.36 156 31.58% 2.36 156 34.21% 2.52 172 34.21% 2.53 173 36.84% 3.09 189 36.84% 3.09 189 39.47% 3.25 205 39.47% 3.14 194 42.11% 3.39 219 42.11% 3.31 211 44.74% 3.56 236 44.74% 3.48 228 47.37% 4.11 251 47.37% 4.03 243 50.00% 4.30 270 50.00% 4.26 266 52.63% 4.47 287 52.63% 4.45 285 55.26% 5.10 310 55.26% 5.07 307 57.89% 5.32 332 57.89% 5.28 328 60.53% 5.54 354 60.53% 5.51 351 63.16% 6.16 376 63.16% 6.16 376 65.79% 6.39 399 65.79% 6.39 399 68.42% 7.03 423 68.42% 7.07 427 71.05% 7.31 451 71.05% 7.33 453 73.68% 8.00 480 73.68% 8.03 483 76.32% 8.31 511 76.32% 8.34 514 78.95% 9.02 542 78.95% 9.05 545 81.58% 9.36 576 81.58% 9.44 584 84.21% 10.16 616 84.21% 10.33 633 86.84% 11.02 662 86.84% 11.17 677 89.47% 12.00 720 89.47% 12.24 744 92.11% 13.37 817 92.11% 13.58 838 94.74% 15.34 934 38 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Total volu % Drainage 6.45% 9.68% 12.90% 16.13% 19.35% 22.58% 25.81% 29.03% 32.26% 35.48% 38.71% 41.94% 45.16% 48.39% 51.61% 54.84% 58.06% 61.29% 64.52% 67.74% 70.97% 74.19% 77.42% 80.65% 83.87% 87.10% 90.32% 93.55% Run #1 Duplicate Time Time (s) % Drainag Time Time (s) 1.04 64 6.06% 1.05 65 1.40 100 9.09% 1.32 92 2.13 133 12.12% 1.58 118 2.45 165 15.15% 2.22 142 3.16 196 18.18% 2.48 168 3.43 223 21.21% 3.11 191 4.06 246 24.24% 3.33 213 4.29 269 27.27% 3.47 227 4.49 289 30.30% 4.04 244 5.12 312 33.33% 4.32 272 5.34 334 36.36% 4.54 294 5.55 355 39.39% 5.16 316 6.22 382 42.42% 5.40 340 6.44 404 45.45% 6.04 364 7.08 428 48.48% 6.28 388 7.32 452 51.52% 6.52 412 7.58 478 54.55% 7.16 436 8.26 506 57.58% 7.40 460 8.52 532 60.61% 8.08 488 9.24 564 63.64% 8.37 517 9.57 597 66.67% 9.05 545 10.33 633 69.70% 9.39 579 11.11 671 72.73% 10.15 615 11.46 706 75.76% 10.44 644 12.35 755 78.79% 11.25 685 13.27 807 81.82% 12.10 730 14.36 876 84.85% 13.03 783 16.16 976 87.88% 14.00 840 90.91% 15.20 920 93.94% 17.22 1042 31 33 38 10 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Run #2 Duplicate % Drainage Time Time (s) % Drainag Time Time (s) 6.25% 1.03 63 5.88% 0 9.38% 1.31 91 8.82% 1.32 92 12.50% 2.02 122 11.76% 1.58 118 15.63% 2.36 156 14.71% 2.22 142 18.75% 3.05 185 17.65% 2.48 168 21.88% 3.30 210 20.59% 3.11 191 25.00% 3.51 231 23.53% 3.33 213 28.13% 4.15 255 26.47% 3.47 227 31.25% 4.37 277 29.41% 4.04 244 34.38% 5.02 302 32.35% 4.32 272 37.50% 5.23 323 35.29% 4.54 294 40.63% 5.45 345 38.24% 5.16 316 43.75% 6.10 370 41.18% 5.40 340 46.88% 6.33 393 44.12% 6.04 364 50.00% 6.55 415 47.06% 6.28 388 53.13% 7.20 440 50.00% 6.52 412 56.25% 7.45 465 52.94% 7.16 436 59.38% 8.14 494 55.88% 7.40 460 62.50% 8.39 519 58.82% 8.08 488 65.63% 9.14 554 61.76% 8.37 517 68.75% 9.44 584 64.71% 9.05 545 71.88% 10.18 618 67.65% 9.39 579 75.00% 10.52 652 70.59% 10.15 615 78.13% 11.31 691 73.53% 10.44 644 81.25% 12.20 740 76.47% 11.25 685 84.38% 13.10 790 79.41% 12.10 730 87.50% 14.15 855 82.35% 13.03 783 90.63% 15.48 948 85.29% 14.00 840 93.75% 18.30 1110 88.24% 15.20 920 91.18% 17.22 1042 Total volum 32 34 pH 8 Run# pH Temperature oC 1 Before 6.07 22.9 Run# Adjustment After foamed 7.93 7.35 21.4 22.5 2 pH Temperature oC Before 7.35 22.5 Run# Adjustment After foamed 8.11 7.75 22.7 23.6 3 pH Temperature oC Before 7.75 23.6 Adjustment After foamed 8.01 7.78 23 23.8 Observation Foam volume stabilised at 1min 30s Observation Foam volume not stabilised at 1min 10s Observation Foam volume stabilised at 1min 27s Results Volume mL Results Volume mL Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Total volu Duplicate Run #1 % Drainage Time Time (s) % Drainage Time Time (s) 6.45% 0.42 42 5.88% 0.53 53 9.68% 1.19 79 8.82% 1.26 86 12.90% 1.55 115 11.76% 1.53 113 16.13% 2.30 150 14.71% 2.20 140 19.35% 3.00 180 17.65% 2.40 160 22.58% 3.27 207 20.59% 3.02 182 25.81% 3.49 229 23.53% 3.22 202 29.03% 4.14 254 26.47% 3.37 217 32.26% 4.35 275 29.41% 3.54 234 35.48% 4.56 296 32.35% 4.20 260 38.71% 5.19 319 35.29% 4.41 281 41.94% 5.43 343 38.24% 5.07 307 45.16% 6.07 367 41.18% 5.30 330 48.39% 6.30 390 44.12% 5.51 351 51.61% 6.54 414 47.06% 6.13 373 54.84% 7.17 437 50.00% 6.37 397 58.06% 7.44 464 52.94% 7.00 420 61.29% 8.11 491 55.88% 7.25 445 64.52% 8.38 518 58.82% 7.48 468 67.74% 9.10 550 61.76% 8.16 496 70.97% 9.42 582 64.71% 8.44 524 74.19% 10.17 617 67.65% 9.14 554 77.42% 10.53 653 70.59% 9.44 584 80.65% 11.33 693 73.53% 10.15 615 83.87% 12.23 743 76.47% 10.51 651 87.10% 13.13 793 79.41% 11.28 688 90.32% 14.25 865 82.35% 12.11 731 93.55% 16.27 987 85.29% 13.04 784 96.77% 20.04 1204 88.24% 13.55 835 91.18% 15.13 913 94.12% 17.17 1037 31 34 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Total volu % Drainage 6.90% 10.34% 13.79% 17.24% 20.69% 24.14% 27.59% 31.03% 34.48% 37.93% 41.38% 44.83% 48.28% 51.72% 55.17% 58.62% 62.07% 65.52% 68.97% 72.41% 75.86% 79.31% 82.76% 86.21% 89.66% 93.10% 96.55% Run #2 Duplicate Time Time (s) % Drainag Time Time (s) 0.56 56 6.45% 1.06 66 1.34 94 9.68% 1.39 99 2.09 129 12.90% 2.13 133 2.43 163 16.13% 2.38 158 3.17 197 19.35% 2.54 174 3.40 220 22.58% 3.21 201 3.59 239 25.81% 3.39 219 4.21 261 29.03% 3.55 235 4.42 282 32.26% 4.08 248 5.05 305 35.48% 4.30 270 5.26 326 38.71% 4.52 292 5.48 348 41.94% 5.16 316 6.13 373 45.16% 5.38 338 6.38 398 48.39% 6.00 360 7.00 420 51.61% 6.21 381 7.24 444 54.84% 6.46 406 7.53 473 58.06% 7.10 430 8.21 501 61.29% 7.38 458 8.49 529 64.52% 8.03 483 9.24 564 67.74% 8.31 511 10.00 600 70.97% 9.04 544 10.42 642 74.19% 9.40 580 11.30 690 77.42% 10.12 612 12.20 740 80.65% 10.57 657 13.37 817 83.87% 11.46 706 15.29 929 87.10% 12.49 769 19.21 1161 90.32% 14.11 851 16.21 981 29 31 11 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Run #3 Duplicate % Drainage Time Time (s) % Drainag Time Time (s) 6.45% 0.50 50 5.88% 0.54 54 9.68% 1.23 83 8.82% 1.28 88 12.90% 1.54 114 11.76% 1.52 112 16.13% 2.25 145 14.71% 2.13 133 19.35% 2.53 173 17.65% 2.30 150 22.58% 3.18 198 20.59% 2.56 176 25.81% 3.39 219 23.53% 3.10 190 29.03% 4.03 243 26.47% 3.25 205 32.26% 4.22 262 29.41% 3.39 219 35.48% 4.43 283 32.35% 4.01 241 38.71% 5.04 304 35.29% 4.22 262 41.94% 5.26 326 38.24% 4.41 281 45.16% 5.50 350 41.18% 5.05 305 48.39% 6.13 373 44.12% 5.24 324 51.61% 6.33 393 47.06% 5.48 348 54.84% 6.57 417 50.00% 6.10 370 58.06% 7.23 443 52.94% 6.33 393 61.29% 7.51 471 55.88% 6.55 415 64.52% 8.15 495 58.82% 7.20 440 67.74% 8.45 525 61.76% 7.46 466 70.97% 9.17 557 64.71% 8.13 493 74.19% 9.53 593 67.65% 8.41 521 77.42% 10.28 628 70.59% 9.13 553 80.65% 11.04 664 73.53% 9.45 585 83.87% 11.57 717 76.47% 10.19 619 87.10% 12.50 770 79.41% 10.58 658 90.32% 14.09 849 82.35% 11.40 700 93.55% 16.18 978 85.29% 12.32 752 96.77% 23.06 1386 88.24% 13.30 810 91.18% 14.59 899 94.12% 17.28 1048 Total volum 31 34 Stability test for mediun surfactant concentration Conditions: 1,000 mg/L of rhamnolipid Mixing at 8,000 rpm for 3 minutes 100ml of foam in measuring cylinder pH 5 pH 6 Run# 1 Run# Foam volume stabilised at 2min Observation Foam volume stabilised at 1min 40s Observation Foam volume stabilised at 1min 30s Results Volume Run #1 Duplicate mL % Drainage Time Time (s) % Drainage Time Time (s) 2 6.67% 1.24 84 6.25% 1.32 92 3 10.00% 2.06 126 9.38% 2.03 123 4 13.33% 2.44 164 12.50% 2.38 158 5 16.67% 3.22 202 15.63% 3.10 190 6 20.00% 3.57 237 18.75% 3.34 214 7 23.33% 4.25 265 21.88% 4.05 245 8 26.67% 4.51 291 25.00% 4.25 265 9 30.00% 5.18 318 28.13% 4.44 284 10 33.33% 5.39 339 31.25% 4.59 299 11 36.67% 6.05 365 34.38% 5.23 323 12 40.00% 6.28 388 37.50% 5.47 347 13 43.33% 6.52 412 40.63% 6.12 372 14 46.67% 7.21 441 43.75% 6.36 396 15 50.00% 7.47 467 46.88% 7.00 420 16 53.33% 8.11 491 50.00% 7.28 448 17 56.67% 8.40 520 53.13% 7.52 472 18 60.00% 9.10 550 56.25% 8.19 499 19 63.33% 9.40 580 59.38% 8.49 529 20 66.67% 10.13 613 62.50% 9.15 555 21 70.00% 10.50 650 65.63% 9.49 589 22 73.33% 11.27 687 68.75% 10.19 619 23 76.67% 12.06 726 71.88% 10.53 653 24 80.00% 12.56 776 75.00% 11.27 687 25 83.33% 13.45 825 78.13% 12.08 728 26 86.67% 14.56 896 81.25% 12.53 773 27 90.00% 16.31 991 84.38% 13.48 828 28 93.33% 19.37 1177 87.50% 14.50 890 29 90.63% 16.12 972 30 93.75% 18.58 1138 Results Volume Run #2 Duplicate mL % Drainage Time Time (s) % Drainag Time Time (s) 2 7.14% 1.50 110 6.90% 2.07 127 3 10.71% 2.33 153 10.34% 2.42 162 4 14.29% 3.15 195 13.79% 3.14 194 5 17.86% 3.58 238 17.24% 3.45 225 6 21.43% 4.29 269 20.69% 4.08 248 7 25.00% 4.57 297 24.14% 4.38 278 8 28.57% 5.20 320 27.59% 5.03 303 9 32.14% 5.45 345 31.03% 5.16 316 10 35.71% 6.09 369 34.48% 5.34 334 11 39.29% 6.36 396 37.93% 5.57 357 12 42.86% 6.59 419 41.38% 6.26 386 13 46.43% 7.27 447 44.83% 6.52 412 14 50.00% 7.55 475 48.28% 7.18 438 15 53.57% 8.24 504 51.72% 7.45 465 16 57.14% 8.53 533 55.17% 8.10 490 17 60.71% 9.23 563 58.62% 8.39 519 18 64.29% 9.59 599 62.07% 9.11 551 19 67.86% 10.35 635 65.52% 9.42 582 20 71.43% 11.14 674 68.97% 10.15 615 21 75.00% 12.05 725 72.41% 10.50 650 22 78.57% 12.57 777 75.86% 11.30 690 23 82.14% 14.11 851 79.31% 12.15 735 24 85.71% 15.34 934 82.76% 13.06 786 25 89.29% 17.51 1071 86.21% 14.02 842 26 92.86% 24.20 1460 89.66% 15.30 930 27 93.10% 17.18 1038 28 96.55% 22.25 1345 29 Results Volume mL 30 32 Total vol Adjustment After foamed 6.03 6.25 23.2 23.5 3 Observation pH Temperature oC Before 6.26 23.3 Run# Before 6.84 23.9 Total vol Adjustment After foamed 6.06 6.14 23.9 23.5 2 pH Temperature oC 28 Before pH 6.25 Temperature oC 23.5 29 12 Adjustment After foamed 5.95 6.13 23.8 23.2 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Run #3 Duplicate % Drainage Time Time (s) % Drainag Time Time (s) 6.90% 1.46 106 7.14% 2.08 128 10.34% 2.26 146 10.71% 2.42 162 13.79% 3.05 185 14.29% 3.21 201 17.24% 3.44 224 17.86% 3.46 226 20.69% 4.18 258 21.43% 4.10 250 24.14% 4.46 286 25.00% 4.36 276 27.59% 5.12 312 28.57% 5.04 304 31.03% 5.38 338 32.14% 5.20 320 34.48% 6.01 361 35.71% 5.36 336 37.93% 6.25 385 39.29% 6.01 361 41.38% 6.50 410 42.86% 6.28 388 44.83% 7.19 439 46.43% 6.58 418 48.28% 7.46 466 50.00% 7.23 443 51.72% 8.17 497 53.57% 7.54 474 55.17% 8.46 526 57.14% 8.21 501 58.62% 9.16 556 60.71% 8.54 534 62.07% 9.52 592 64.29% 9.29 569 65.52% 10.33 633 67.86% 10.01 601 68.97% 11.10 670 71.43% 10.42 642 72.41% 12.00 720 75.00% 11.25 685 75.86% 12.58 778 78.57% 12.18 738 79.31% 14.13 853 82.14% 13.18 798 82.76% 15.50 950 85.71% 14.42 882 86.21% 18.27 1107 89.29% 16.45 1005 89.66% 25.36 1536 92.86% 23.11 1391 Total volum 29 28 Repeat pH 6 Run# Repeat Run# 1 Adjustment After foamed 6 6.12 20.1 20.3 Repeat Run# 1 Observation Foam volume stabilised at 2min 50sec Observation Foam volume stabilised at 1min 48sec Observation Foam volume stabilised at 1min 52sec Results Volume Run #1 Duplicate mL % Drainage Time Time (s) % Drainage Time Time (s) 2 6.06% 0.44 44 6.25% 1.09 69 3 9.09% 1.18 78 9.38% 1.44 104 4 12.12% 1.52 112 12.50% 2.15 135 5 15.15% 2.24 144 15.63% 2.40 160 6 18.18% 2.57 177 18.75% 3.11 191 7 21.21% 3.31 211 21.88% 3.42 222 8 24.24% 4.03 243 25.00% 4.04 244 9 27.27% 4.24 264 28.13% 4.22 262 10 30.30% 4.37 277 31.25% 4.39 279 11 33.33% 4.59 299 34.38% 5.06 306 12 36.36% 5.24 324 37.50% 5.31 331 13 39.39% 5.47 347 40.63% 5.55 355 14 42.42% 6.10 370 43.75% 6.18 378 15 45.45% 6.34 394 46.88% 6.44 404 16 48.48% 6.56 416 50.00% 7.08 428 17 51.52% 7.22 442 53.13% 7.33 453 18 54.55% 7.47 467 56.25% 8.00 480 19 57.58% 8.13 493 59.38% 8.27 507 20 60.61% 8.39 519 62.50% 8.53 533 21 63.64% 9.07 547 65.63% 9.23 563 22 66.67% 9.35 575 68.75% 9.53 593 23 69.70% 10.07 607 71.88% 10.27 627 24 72.73% 10.38 638 75.00% 11.02 662 25 75.76% 11.13 673 78.13% 11.38 698 26 78.79% 11.52 712 81.25% 12.21 741 27 81.82% 12.36 756 84.38% 13.12 792 28 84.85% 13.31 811 87.50% 14.16 856 29 87.88% 14.42 882 90.63% 15.43 943 30 90.91% 16.39 999 93.75% 18.42 1122 31 93.94% 21.36 1296 Total vol 33 32 Results Volume Run #1 Duplicate mL % Drainage Time Time (s) % Drainag Time Time (s) 2 7.41% 1.56 116 7.41% 2.08 148 3 11.11% 2.40 160 11.11% 2.52 192 4 14.81% 3.31 211 14.81% 3.35 235 5 18.52% 4.08 248 18.52% 4.09 269 6 22.22% 4.52 292 22.22% 4.38 298 7 25.93% 5.30 330 25.93% 5.08 328 8 29.63% 5.59 359 29.63% 5.33 353 9 33.33% 6.22 382 33.33% 5.54 374 10 37.04% 6.45 405 37.04% 6.14 394 11 40.74% 7.13 433 40.74% 6.44 424 12 44.44% 7.44 464 44.44% 7.15 455 13 48.15% 8.15 495 48.15% 7.48 488 14 51.85% 8.47 527 51.85% 8.21 521 15 55.56% 9.18 558 55.56% 8.51 551 16 59.26% 9.53 593 59.26% 9.28 588 17 62.96% 10.29 629 62.96% 10.06 626 18 66.67% 11.08 668 66.67% 10.43 663 19 70.37% 11.51 711 70.37% 11.25 705 20 74.07% 12.35 755 74.07% 12.12 752 21 77.78% 13.29 809 77.78% 13.03 803 22 81.48% 14.32 872 81.48% 14.09 869 23 85.19% 15.52 952 85.19% 15.24 944 24 88.89% 17.42 1062 88.89% 17.11 1051 25 92.59% 21.35 1295 92.59% 20.29 1249 26 27 28 29 30 31 Total vol 27 27 Results Volume mL pH Temperature oC Before 7.3 16.6 Adjustment After foamed 6.02 6.04 16.5 16.9 2 pH Temperature oC Before 7.3 20 13 Before pH 6.04 Temperature oC 16.7 Adjustment After foamed 6.07 16.9 Run #2 Duplicate % Drainage Time Time (s) % Drainag Time Time (s) 2 7.14% 1.43 103 7.14% 2.29 149 3 10.71% 2.30 150 10.71% 3.09 189 4 14.29% 3.13 193 14.29% 3.52 232 5 17.86% 3.55 235 17.86% 4.23 263 6 21.43% 4.33 273 21.43% 4.55 295 7 25.00% 5.13 313 25.00% 5.26 326 8 28.57% 5.49 349 28.57% 5.52 352 9 32.14% 6.13 373 32.14% 6.12 372 10 35.71% 6.31 391 35.71% 6.35 395 11 39.29% 6.57 417 39.29% 7.05 425 12 42.86% 7.29 449 42.86% 7.36 456 13 46.43% 7.57 477 46.43% 8.06 486 14 50.00% 8.27 507 50.00% 8.36 516 15 53.57% 8.58 538 53.57% 9.06 546 16 57.14% 9.27 567 57.14% 9.37 577 17 60.71% 9.59 599 60.71% 10.09 609 18 64.29% 10.36 636 64.29% 10.48 648 19 67.86% 11.12 672 67.86% 11.21 681 20 71.43% 11.54 714 71.43% 12.02 722 21 75.00% 12.34 754 75.00% 12.45 765 22 78.57% 13.20 800 78.57% 13.35 815 23 82.14% 14.13 853 82.14% 14.26 866 24 85.71% 15.19 919 85.71% 15.28 928 25 89.29% 16.46 1006 89.29% 16.41 1001 26 92.86% 18.44 1124 92.86% 18.35 1115 27 96.43% 23.04 1384 96.43% 21.53 1313 28 29 30 31 28 28 Total volum pH 7 Run# 1 Run# Foam volume not stabilised at 1min 30s Observation Foam volume stabilised at 1min 18s Observation Foam volume stabilised at 1min 20s Results Volume Run #1 Duplicate mL % Drainage Time Time (s) % Drainage Time Time (s) 2 6.90% 1.22 82 6.67% 2.01 121 3 10.34% 2.11 131 10.00% 2.42 162 4 13.79% 2.56 176 13.33% 3.17 197 5 17.24% 3.34 214 16.67% 3.45 225 6 20.69% 4.12 252 20.00% 4.11 251 7 24.14% 4.41 281 23.33% 4.37 277 8 27.59% 5.08 308 26.67% 4.57 297 9 31.03% 5.34 334 30.00% 5.18 318 10 34.48% 5.58 358 33.33% 5.35 335 11 37.93% 6.24 384 36.67% 5.59 359 12 41.38% 6.48 408 40.00% 6.26 386 13 44.83% 7.16 436 43.33% 6.55 415 14 48.28% 7.45 465 46.67% 7.21 441 15 51.72% 8.14 494 50.00% 7.48 468 16 55.17% 8.40 520 53.33% 8.14 494 17 58.62% 9.09 549 56.67% 8.45 525 18 62.07% 9.45 585 60.00% 9.11 551 19 65.52% 10.18 618 63.33% 9.44 584 20 68.97% 10.51 651 66.67% 10.17 617 21 72.41% 11.35 695 70.00% 10.52 652 22 75.86% 12.25 745 73.33% 11.29 689 23 79.31% 13.22 802 76.67% 12.15 735 24 82.76% 14.23 863 80.00% 13.01 781 25 86.21% 15.37 937 83.33% 13.59 839 26 89.66% 18.03 1083 86.67% 15.04 904 27 93.10% 23.36 1416 90.00% 16.42 1002 28 93.33% 19.46 1186 29 Results Volume Run #2 Duplicate mL % Drainage Time Time (s) % Drainag Time Time (s) 2 6.90% 1.35 95 6.67% 1.50 110 3 10.34% 2.18 138 10.00% 2.33 153 4 13.79% 2.57 177 13.33% 3.09 189 5 17.24% 3.38 218 16.67% 3.36 216 6 20.69% 4.11 251 20.00% 4.02 242 7 24.14% 4.35 275 23.33% 4.28 268 8 27.59% 5.00 300 26.67% 4.51 291 9 31.03% 5.26 326 30.00% 5.14 314 10 34.48% 5.47 347 33.33% 5.29 329 11 37.93% 6.15 375 36.67% 5.54 354 12 41.38% 6.37 397 40.00% 6.21 381 13 44.83% 7.03 423 43.33% 6.47 407 14 48.28% 7.31 451 46.67% 7.19 439 15 51.72% 7.58 478 50.00% 7.45 465 16 55.17% 8.25 505 53.33% 8.11 491 17 58.62% 8.52 532 56.67% 8.40 520 18 62.07% 9.27 567 60.00% 9.12 552 19 65.52% 10.00 600 63.33% 9.44 584 20 68.97% 10.33 633 66.67% 10.16 616 21 72.41% 11.18 678 70.00% 10.59 659 22 75.86% 12.03 723 73.33% 11.36 696 23 79.31% 12.59 779 76.67% 12.22 742 24 82.76% 14.08 848 80.00% 13.18 798 25 86.21% 15.23 923 83.33% 14.22 862 26 89.66% 17.47 1067 86.67% 15.56 956 27 93.10% 24.35 1475 90.00% 18.21 1101 28 93.33% 24.54 1494 29 30 Results Volume mL 29 Adjustment After foamed 7.00 22.4 3 Observation pH Temperature oC Before 6.98 21.8 Run# Before 7.35 26.1 Total vol Adjustment After foamed 7.00 6.98 26 21.8 2 pH Temperature oC Before pH 7.00 Temperature oC 23.5 30 Adjustment After foamed 6.97 24.1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Run #3 Duplicate % Drainage Time Time (s) % Drainag Time Time (s) 6.90% 1.22 82 6.90% 1.53 113 10.34% 2.02 122 10.34% 2.26 146 13.79% 2.42 162 13.79% 2.59 179 17.24% 3.21 201 17.24% 3.25 205 20.69% 3.55 235 20.69% 3.53 233 24.14% 4.20 260 24.14% 4.16 256 27.59% 4.45 285 27.59% 4.41 281 31.03% 5.10 310 31.03% 4.57 297 34.48% 5.34 334 34.48% 5.15 315 37.93% 6.02 362 37.93% 5.43 343 41.38% 6.24 384 41.38% 6.09 369 44.83% 6.49 409 44.83% 6.35 395 48.28% 7.16 436 48.28% 7.01 421 51.72% 7.43 463 51.72% 7.28 448 55.17% 8.09 489 55.17% 7.58 478 58.62% 8.36 516 58.62% 8.28 508 62.07% 9.08 548 62.07% 9.01 541 65.52% 9.42 582 65.52% 9.31 571 68.97% 10.11 611 68.97% 10.05 605 72.41% 10.56 656 72.41% 10.41 641 75.86% 11.34 694 75.86% 11.28 688 79.31% 12.25 745 79.31% 12.18 738 82.76% 13.25 805 82.76% 13.10 790 86.21% 14.31 871 86.21% 14.26 866 89.66% 16.27 987 89.66% 16.20 980 93.10% 20.15 1215 93.10% 19.57 1197 Total volum Total vol 29 30 14 29 29 pH 8 Run# 1 Run# Observation Foam volume stabilised at 1min 12s Observation Foam volume not stabilised at 1min 24s Observation Results Run #1 Duplicate Volume mL % Drainage Time Time (s) % Drainage Time Time (s) 2 6.45% 1.17 77 6.06% 1.28 88 3 9.68% 1.55 115 9.09% 2.00 120 4 12.90% 2.35 155 12.12% 2.34 154 5 16.13% 3.13 193 15.15% 3.00 180 6 19.35% 3.46 226 18.18% 3.28 208 7 22.58% 4.13 253 21.21% 3.54 234 8 25.81% 4.37 277 24.24% 4.22 262 9 29.03% 5.04 304 27.27% 4.38 278 10 32.26% 5.25 325 30.30% 4.55 295 11 35.48% 5.52 352 33.33% 5.22 322 12 38.71% 6.15 375 36.36% 5.49 349 13 41.94% 6.40 400 39.39% 6.16 376 14 45.16% 7.07 427 42.42% 6.41 401 15 48.39% 7.33 453 45.45% 7.06 426 16 51.61% 7.59 479 48.48% 7.31 451 17 54.84% 8.24 504 51.52% 7.57 477 18 58.06% 8.53 533 54.55% 8.22 502 19 61.29% 9.23 563 57.58% 8.51 531 20 64.52% 9.52 592 60.61% 9.20 560 21 67.74% 10.26 626 63.64% 9.47 587 22 70.97% 11.02 662 66.67% 10.19 619 23 74.19% 11.42 702 69.70% 10.57 657 24 77.42% 12.23 743 72.73% 11.26 686 25 80.65% 12.59 779 75.76% 12.04 724 26 83.87% 14.00 840 78.79% 12.45 765 27 87.10% 14.59 899 81.82% 13.35 815 28 90.32% 16.38 998 84.85% 14.36 876 29 93.55% 19.27 1167 87.88% 15.41 941 30 90.91% 17.10 1030 31 93.94% 20.17 1217 32 Results Volume Run #2 Duplicate mL % Drainage Time Time (s) % Drainag Time Time (s) 2 6.45% 1.12 72 6.25% 1.26 86 3 9.68% 1.47 107 9.38% 2.01 121 4 12.90% 2.26 146 12.50% 2.36 156 5 16.13% 3.04 184 15.63% 3.02 182 6 19.35% 3.38 218 18.75% 3.29 209 7 22.58% 4.06 246 21.88% 3.56 236 8 25.81% 4.30 270 25.00% 4.19 259 9 29.03% 4.54 294 28.13% 4.36 276 10 32.26% 5.21 321 31.25% 4.59 299 11 35.48% 5.42 342 34.38% 5.22 322 12 38.71% 6.07 367 37.50% 5.48 348 13 41.94% 6.34 394 40.63% 6.14 374 14 45.16% 7.06 426 43.75% 6.38 398 15 48.39% 7.28 448 46.88% 7.04 424 16 51.61% 7.53 473 50.00% 7.31 451 17 54.84% 8.20 500 53.13% 7.57 477 18 58.06% 8.49 529 56.25% 8.24 504 19 61.29% 9.18 558 59.38% 8.51 531 20 64.52% 9.45 585 62.50% 9.22 562 21 67.74% 10.20 620 65.63% 9.49 589 22 70.97% 10.55 655 68.75% 10.21 621 23 74.19% 11.35 695 71.88% 10.56 656 24 77.42% 12.15 735 75.00% 11.34 694 25 80.65% 12.55 775 78.13% 12.16 736 26 83.87% 13.52 832 81.25% 12.57 777 27 87.10% 14.55 895 84.38% 13.51 831 28 90.32% 16.24 984 87.50% 14.56 896 29 93.55% 19.07 1147 90.63% 16.25 985 30 93.75% 19.02 1142 31 Results 31 pH Temperature oC 33 Before 7.91 21.7 Run# Before 7.34 20.4 Total vol Adjustment After foamed 8.02 8.02 20.6 21.6 2 pH Temperature oC Total vol Adjustment After foamed 8.04 7.93 21.7 22.1 31 pH Temperature oC 32 15 3 Before 7.90 21.2 Adjustment After foamed 8.03 7.78 21.2 8.02 21.7 Foam volume stabilised at 2min Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Run #3 Duplicate % Drainage Time Time (s) % Drainag Time Time (s) 6.45% 1.00 60 6.06% 1.21 81 9.68% 1.34 94 9.09% 1.53 113 12.90% 2.11 131 12.12% 2.21 141 16.13% 2.45 165 15.15% 2.45 165 19.35% 3.15 195 18.18% 3.11 191 22.58% 3.42 222 21.21% 3.35 215 25.81% 4.05 245 24.24% 4.00 240 29.03% 4.33 273 27.27% 4.21 261 32.26% 4.52 292 30.30% 4.34 274 35.48% 5.17 317 33.33% 4.58 298 38.71% 5.37 337 36.36% 5.21 321 41.94% 6.01 361 39.39% 5.45 345 45.16% 6.26 386 42.42% 6.07 367 48.39% 6.50 410 45.45% 6.29 389 51.61% 7.14 434 48.48% 6.52 412 54.84% 7.37 457 51.52% 7.17 437 58.06% 8.02 482 54.55% 7.41 461 61.29% 8.29 509 57.58% 8.07 487 64.52% 8.55 535 60.61% 8.35 515 67.74% 9.25 565 63.64% 9.00 540 70.97% 9.59 599 66.67% 9.27 567 74.19% 10.33 633 69.70% 9.56 596 77.42% 11.08 668 72.73% 10.30 630 80.65% 11.41 701 75.76% 11.00 660 83.87% 12.30 750 78.79% 11.38 698 87.10% 13.22 802 81.82% 12.20 740 90.32% 14.31 871 84.85% 13.10 790 93.55% 16.22 982 87.88% 14.16 856 96.77% 21.30 1290 90.91% 15.55 955 93.94% 19.09 1149 Total volum 31 33 Stability test with salt Conditions: Mixing at 8,000 rpm for 3 minutes 100ml of foam in measuring cylinder pH 6 Run# 1 Run# Adjustme After foamed 6 6.04 20.2 20.4 Before pH 6.11 Temperature oC 20.2 Observation Foam volume stabilised at 2min 11sec Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Run #1 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 6.67% 1.19 79 6.45% 1.42 102 10.00% 1.59 119 9.68% 2.22 142 13.33% 2.35 155 12.90% 2.59 179 16.67% 3.10 190 16.13% 3.32 212 20.00% 3.48 228 19.35% 4.00 240 23.33% 4.26 266 22.58% 4.29 269 26.67% 4.57 297 25.81% 4.50 290 30.00% 5.12 312 29.03% 5.09 309 33.33% 5.30 330 32.26% 5.29 329 36.67% 5.51 351 35.48% 5.52 352 40.00% 6.16 376 38.71% 6.18 378 43.33% 6.41 401 41.94% 6.43 403 46.67% 7.05 425 45.16% 7.08 428 50.00% 7.31 451 48.39% 7.33 453 53.33% 7.55 475 51.61% 7.59 479 56.67% 8.21 501 54.84% 8.23 503 60.00% 8.48 528 58.06% 8.51 531 63.33% 9.17 557 61.29% 9.18 558 66.67% 9.47 587 64.52% 9.45 585 70.00% 10.17 617 67.74% 10.15 615 73.33% 10.50 650 70.97% 10.48 648 76.67% 11.27 687 74.19% 11.21 681 80.00% 12.05 725 77.42% 11.55 715 83.33% 12.48 768 80.65% 12.32 752 86.67% 13.39 819 83.87% 13.17 797 90.00% 14.49 889 87.10% 14.10 850 93.33% 16.40 1000 90.32% 15.17 917 96.67% 22.17 1337 93.55% 16.44 1004 96.77% 19.46 1186 Total volum 30 31 2 Run# AdjustmenAfter foamed 6.09 21.1 3 pH Temperature oC Before 6.07 20.8 Observation Foam volume stabilised at 2min 27sec Observation Foam volume stabilised at 2min 50sec Results Run #2 Volume Duplicate mL % DrainageTime Time (s) % Drainag Time Time (s) 2 6.67% 1.18 78 6.45% 1.40 100 3 10.00% 1.49 109 9.68% 2.15 135 4 13.33% 2.27 147 12.90% 2.45 165 5 16.67% 2.59 179 16.13% 3.19 199 6 20.00% 3.33 213 19.35% 3.45 225 7 23.33% 4.06 246 22.58% 4.13 253 8 26.67% 4.30 270 25.81% 4.34 274 9 30.00% 4.51 291 29.03% 4.53 293 10 33.33% 5.12 312 32.26% 5.08 308 11 36.67% 5.36 336 35.48% 5.34 334 12 40.00% 6.01 361 38.71% 5.59 359 13 43.33% 6.24 384 41.94% 6.24 384 14 46.67% 6.47 407 45.16% 6.48 408 15 50.00% 7.10 430 48.39% 7.11 431 16 53.33% 7.34 454 51.61% 7.35 455 17 56.67% 8.00 480 54.84% 7.59 479 18 60.00% 8.24 504 58.06% 8.24 504 19 63.33% 8.49 529 61.29% 8.49 529 20 66.67% 9.15 555 64.52% 9.15 555 21 70.00% 9.43 583 67.74% 9.44 584 22 73.33% 10.13 613 70.97% 10.14 614 23 76.67% 10.45 645 74.19% 10.44 644 24 80.00% 11.19 679 77.42% 11.15 675 25 83.33% 11.59 719 80.65% 11.53 713 26 86.67% 12.41 761 83.87% 12.34 754 27 90.00% 13.35 815 87.10% 13.22 802 28 93.33% 15.00 900 90.32% 14.24 864 29 96.67% 17.42 1062 93.55% 15.57 957 30 96.77% 18.55 1135 Results Run #3 Volume Duplicate mL % Drainag Time Time (s) % Drainag Time Time (s) 2 7.14% 1.29 89 6.90% 1.38 98 3 10.71% 2.13 133 10.34% 2.16 136 4 14.29% 2.50 170 13.79% 2.50 170 5 17.86% 3.28 208 17.24% 3.17 197 6 21.43% 3.58 238 20.69% 3.45 225 7 25.00% 4.24 264 24.14% 4.14 254 8 28.57% 4.48 288 27.59% 4.35 275 9 32.14% 5.09 309 31.03% 4.52 292 10 35.71% 5.35 335 34.48% 5.12 312 11 39.29% 5.58 358 37.93% 5.37 337 12 42.86% 6.21 381 41.38% 6.00 360 13 46.43% 6.44 404 44.83% 6.28 388 14 50.00% 7.10 430 48.28% 6.51 411 15 53.57% 7.35 455 51.72% 7.16 436 16 57.14% 8.00 480 55.17% 7.41 461 17 60.71% 8.24 504 58.62% 8.08 488 18 64.29% 8.55 535 62.07% 8.35 515 19 67.86% 9.23 563 65.52% 9.04 544 20 71.43% 9.51 591 68.97% 9.30 570 21 75.00% 10.25 625 72.41% 10.02 602 22 78.57% 11.00 660 75.86% 10.36 636 23 82.14% 11.42 702 79.31% 11.14 674 24 85.71% 12.28 748 82.76% 11.52 712 25 89.29% 13.19 799 86.21% 12.40 760 26 92.86% 14.40 880 89.66% 13.44 824 27 96.43% 17.20 1040 93.10% 15.17 917 28 96.55% 19.22 1162 Total volu 30 pH Temperature oC 31 16 Before 6.05 19.8 Total vol Adjustme After foamed 6.06 20.1 28 29 pH 7 Run# 1 Run# Adjustme After foamed 6.99 21.9 Before pH 6.98 Temperature oC 22.9 Observation Foam volume not stabilised at 1min 25 sec Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Run #1 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 6.67% 1.17 77 6.45% 1.47 107 10.00% 2.02 122 9.68% 2.20 140 13.33% 2.41 161 12.90% 2.59 179 16.67% 3.21 201 16.13% 3.25 205 20.00% 3.55 235 19.35% 3.52 232 23.33% 4.24 264 22.58% 4.21 261 26.67% 4.49 289 25.81% 4.46 286 30.00% 5.13 313 29.03% 5.02 302 33.33% 5.34 334 32.26% 5.22 322 36.67% 6.01 361 35.48% 5.48 348 40.00% 6.24 384 38.71% 6.13 373 43.33% 6.48 408 41.94% 6.39 399 46.67% 7.14 434 45.16% 7.05 425 50.00% 7.41 461 48.39% 7.30 450 53.33% 8.04 484 51.61% 7.54 474 56.67% 8.30 510 54.84% 8.22 502 60.00% 8.59 539 58.06% 8.49 529 63.33% 9.29 569 61.29% 9.17 557 66.67% 9.58 598 64.52% 9.46 586 70.00% 10.34 634 67.74% 10.16 616 73.33% 11.10 670 70.97% 10.51 651 76.67% 11.53 713 74.19% 11.30 690 80.00% 12.36 756 77.42% 12.07 727 83.33% 13.24 804 80.65% 12.55 775 86.67% 14.35 875 83.87% 13.49 829 90.00% 16.08 968 87.10% 15.02 902 93.33% 20.01 1201 90.32% 16.53 1013 93.55% 21.02 1262 Total volum 30 31 2 Run# AdjustmenAfter foamed 7.09 23 3 pH Temperature oC Before 7.07 22.7 Observation Foam volume stabilised at 1min 28s Observation Foam volume stabilised at 1min 35s Results Volume Run #2 Duplicate mL % DrainageTime Time (s) % Drainag Time Time (s) 2 6.67% 1.17 77 6.45% 1.40 100 3 10.00% 2.01 121 9.68% 2.14 134 4 13.33% 2.33 153 12.90% 2.53 173 5 16.67% 3.13 193 16.13% 3.20 200 6 20.00% 3.47 227 19.35% 3.34 214 7 23.33% 4.13 253 22.58% 4.08 248 8 26.67% 4.35 275 25.81% 4.31 271 9 30.00% 5.01 301 29.03% 4.47 287 10 33.33% 5.26 326 32.26% 5.04 304 11 36.67% 5.50 350 35.48% 5.31 331 12 40.00% 6.11 371 38.71% 5.55 355 13 43.33% 6.35 395 41.94% 6.19 379 14 46.67% 7.03 423 45.16% 6.45 405 15 50.00% 7.27 447 48.39% 7.09 429 16 53.33% 7.51 471 51.61% 7.35 455 17 56.67% 8.17 497 54.84% 8.01 481 18 60.00% 8.44 524 58.06% 8.26 506 19 63.33% 9.13 553 61.29% 8.55 535 20 66.67% 9.44 584 64.52% 9.24 564 21 70.00% 10.18 618 67.74% 9.53 593 22 73.33% 10.55 655 70.97% 10.26 626 23 76.67% 11.37 697 74.19% 11.03 663 24 80.00% 12.22 742 77.42% 11.42 702 25 83.33% 13.12 792 80.65% 12.23 743 26 86.67% 14.22 862 83.87% 13.21 801 27 90.00% 16.04 964 87.10% 14.29 869 28 93.33% 20.01 1201 90.32% 16.24 984 29 93.55% 20.35 1235 30 31 32 33 Total volu 30 31 Results Run #3 Volume Duplicate mL % Drainag Time Time (s) % Drainag Time Time (s) 2 6.45% 1.19 79 6.25% 1.32 92 3 9.68% 2.00 120 9.38% 2.13 133 4 12.90% 2.38 158 12.50% 2.43 163 5 16.13% 3.11 191 15.63% 3.14 194 6 19.35% 3.47 227 18.75% 3.35 215 7 22.58% 4.12 252 21.88% 4.02 242 8 25.81% 4.35 275 25.00% 4.21 261 9 29.03% 5.05 305 28.13% 4.42 282 10 32.26% 5.24 324 31.25% 4.58 298 11 35.48% 5.50 350 34.38% 5.25 325 12 38.71% 6.13 373 37.50% 5.51 351 13 41.94% 6.37 397 40.63% 6.17 377 14 45.16% 7.01 421 43.75% 6.39 399 15 48.39% 7.25 445 46.88% 7.04 424 16 51.61% 7.50 470 50.00% 7.28 448 17 54.84% 8.16 496 53.13% 7.54 474 18 58.06% 8.42 522 56.25% 8.19 499 19 61.29% 9.11 551 59.38% 8.47 527 20 64.52% 9.39 579 62.50% 9.11 551 21 67.74% 10.13 613 65.63% 9.39 579 22 70.97% 10.45 645 68.75% 10.13 613 23 74.19% 11.21 681 71.88% 10.44 644 24 77.42% 11.59 719 75.00% 11.16 676 25 80.65% 12.38 758 78.13% 11.56 716 26 83.87% 13.31 811 81.25% 12.36 756 27 87.10% 14.31 871 84.38% 13.23 803 28 90.32% 16.05 965 87.50% 14.24 864 29 93.55% 18.43 1123 90.63% 15.38 938 30 93.75% 17.31 1051 31 32 17 pH Temperature oC Before 7.02 21.7 Total vol Adjustme After foamed 7.07 22.4 31 32 pH 8 Run# 1 Run# Adjustme After foamed 8.1 8 21.5 21.5 Before pH 7.5 o Temperature C 21.5 Observation Foam volume not stabilised at 1min 30 sec Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 pH Temperature oC Run #1 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 6.25% 1.17 77 6.06% 9.38% 1.58 118 9.09% 1.58 118 12.50% 2.40 160 12.12% 2.37 157 15.63% 3.05 185 15.15% 3.15 195 18.75% 3.33 213 18.18% 3.52 232 21.88% 4.10 250 21.21% 4.23 263 25.00% 4.39 279 24.24% 4.57 297 28.13% 5.00 300 27.27% 5.19 319 31.25% 5.14 314 30.30% 5.52 352 34.38% 5.36 336 33.33% 6.13 373 37.50% 6.01 361 36.36% 6.35 395 40.63% 6.25 385 39.39% 7.01 421 43.75% 6.50 410 42.42% 7.24 444 46.88% 7.15 435 45.45% 7.48 468 50.00% 7.38 458 48.48% 8.13 493 53.13% 8.05 485 51.52% 8.39 519 56.25% 8.31 511 54.55% 9.06 546 59.38% 8.56 536 57.58% 9.36 576 62.50% 9.26 566 60.61% 10.01 601 65.63% 9.54 594 63.64% 10.31 631 68.75% 10.24 624 66.67% 11.00 660 71.88% 10.56 656 69.70% 11.31 691 75.00% 11.31 691 72.73% 12.02 722 78.13% 12.16 736 75.76% 12.35 755 81.25% 12.52 772 78.79% 13.10 790 84.38% 13.47 827 81.82% 13.50 830 87.50% 14.48 888 84.85% 14.30 870 90.63% 16.18 978 87.88% 15.21 921 93.75% 20.11 1211 90.91% 16.16 976 93.94% 17.17 1037 96.97% 18.51 1131 Total volum 32 33 2 Before 8 21.5 AdjustmenAfter foamed 7.8 21.5 Observation Foam volume stabilised at 1min 42s Results Volume Run #2 Duplicate mL % DrainageTime Time (s) % Drainag Time Time (s) 2 5.71% 5.71% 3 8.57% 1.13 73 8.57% 1.14 74 4 11.43% 1.51 111 11.43% 1.46 106 5 14.29% 2.24 144 14.29% 2.12 132 6 17.14% 2.56 176 17.14% 2.46 166 7 20.00% 3.30 210 20.00% 3.15 195 8 22.86% 4.05 245 22.86% 3.44 224 9 25.71% 4.23 263 25.71% 4.06 246 10 28.57% 4.34 274 28.57% 4.38 278 11 31.43% 4.58 298 31.43% 5.00 300 12 34.29% 5.25 325 34.29% 5.25 325 13 37.14% 5.50 350 37.14% 5.48 348 14 40.00% 6.14 374 40.00% 6.15 375 15 42.86% 6.38 398 42.86% 6.38 398 16 45.71% 7.02 422 45.71% 7.03 423 17 48.57% 7.27 447 48.57% 7.28 448 18 51.43% 7.52 472 51.43% 7.54 474 19 54.29% 8.17 497 54.29% 8.22 502 20 57.14% 8.44 524 57.14% 8.48 528 21 60.00% 9.09 549 60.00% 9.14 554 22 62.86% 9.37 577 62.86% 9.44 584 23 65.71% 10.04 604 65.71% 10.13 613 24 68.57% 10.36 636 68.57% 10.43 643 25 71.43% 11.07 667 71.43% 11.13 673 26 74.29% 11.37 697 74.29% 11.44 704 27 77.14% 12.12 732 77.14% 12.21 741 28 80.00% 12.53 773 80.00% 13.00 780 29 82.86% 13.28 808 82.86% 13.36 816 30 85.71% 14.23 863 85.71% 14.17 857 31 88.57% 15.28 928 88.57% 15.04 904 32 91.43% 17.00 1020 91.43% 15.55 955 33 94.29% 19.48 1188 94.29% 17.08 1028 34 97.14% 18.37 1117 35 35 Total volu 18 Stability test with salt Conditions: pH 6 1,000 mg/L of rhamnolipid and 3000 mg/L NaCl Mixing at 8,000 rpm for 3 minutes 100ml of foam in measuring cylinder Run# 1 Run# AdjustmenAfter foamed 6 6.07 21.1 20.9 pH o Temperature C Before 7 21.1 Observation Foam volume not stabilised at 3min Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 pH Temperature oC Run #1 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 5.71% 0.44 44 5.88% 0.59 59 8.57% 1.14 74 8.82% 1.26 86 11.43% 1.41 101 11.76% 1.56 116 14.29% 2.15 135 14.71% 2.24 144 17.14% 2.45 165 17.65% 2.50 170 20.00% 3.07 187 20.59% 3.17 197 22.86% 3.29 209 23.53% 3.37 217 25.71% 3.52 232 26.47% 3.56 236 28.57% 4.12 252 29.41% 4.13 253 31.43% 4.37 277 32.35% 4.39 279 35.29% 5.06 306 37.14% 5.18 318 38.24% 5.28 328 40.00% 5.40 340 41.18% 5.50 350 42.86% 6.02 362 44.12% 6.12 372 45.71% 6.22 382 47.06% 6.36 396 48.57% 6.45 405 50.00% 7.00 420 51.43% 7.09 429 52.94% 7.25 445 54.29% 7.34 454 55.88% 7.51 471 57.14% 7.56 476 58.82% 8.17 497 60.00% 8.23 503 61.76% 8.40 520 62.86% 8.51 531 64.71% 9.11 551 65.71% 9.17 557 67.65% 9.38 578 68.57% 9.44 584 70.59% 10.08 608 71.43% 10.08 608 73.53% 10.36 636 74.29% 10.39 639 76.47% 11.10 670 77.14% 11.09 669 79.41% 11.44 704 80.00% 11.42 702 82.35% 12.21 741 82.86% 12.15 735 85.29% 13.05 785 85.71% 12.59 779 88.24% 13.55 835 88.57% 13.46 826 91.18% 15.10 910 91.43% 14.49 889 94.12% 17.32 1052 94.29% 16.31 991 Total volum 35 34 2 Before 6.08 20.9 Run# AdjustmenAfter foamed 6 6.02 21 20.6 Observation Foam volume not stabilised at 3min Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 pH Temperature oC Run #2 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 5.56% 0.44 44 5.48% 0.56 56 8.33% 1.11 71 8.22% 1.22 82 11.11% 1.44 104 10.96% 1.49 109 13.89% 2.15 135 13.70% 2.12 132 16.67% 2.48 168 16.44% 2.35 155 19.44% 3.12 192 19.18% 2.57 177 22.22% 3.35 215 21.92% 3.18 198 25.00% 3.57 237 24.66% 3.35 215 27.78% 4.23 263 27.40% 3.54 234 30.56% 4.48 288 30.14% 4.18 258 33.33% 5.09 309 32.88% 4.45 285 36.11% 5.31 331 35.62% 5.10 310 38.89% 5.56 356 38.36% 5.33 333 41.67% 6.18 378 41.10% 5.56 356 44.44% 6.39 399 43.84% 6.19 379 47.22% 7.03 423 46.58% 6.43 403 50.00% 7.26 446 49.32% 7.06 426 52.78% 7.54 474 52.05% 7.33 453 55.56% 8.16 496 54.79% 7.56 476 58.33% 8.43 523 57.53% 8.20 500 61.11% 9.10 550 60.27% 8.48 528 63.89% 9.36 576 63.01% 9.15 555 66.67% 10.03 603 65.75% 9.40 580 69.44% 10.28 628 68.49% 10.07 607 72.22% 10.58 658 71.23% 10.38 638 75.00% 11.30 690 73.97% 11.09 669 77.78% 12.01 721 76.71% 11.44 704 80.56% 12.39 759 79.45% 12.16 736 83.33% 13.16 796 82.19% 12.56 776 86.11% 14.06 846 84.93% 13.40 820 88.89% 15.09 909 87.67% 14.35 875 91.67% 16.47 1007 90.41% 15.47 947 93.15% 18.02 1082 Total volum 36 36.50 19 3 Before 6.20 20.6 AdjustmenAfter foamed 6 6.11 20.8 21.4 Observation Foam volume stabilised at 2min 45sec Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Run #3 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 7.14% 1.35 95 7.14% 1.56 116 10.71% 2.18 138 10.71% 2.29 149 14.29% 2.53 173 14.29% 3.04 184 17.86% 3.31 211 17.86% 3.31 211 21.43% 4.03 243 21.43% 3.54 234 25.00% 4.50 290 25.00% 4.21 261 28.57% 5.12 312 28.57% 4.41 281 32.14% 5.34 334 32.14% 5.02 302 35.71% 5.57 357 35.71% 5.16 316 39.29% 6.18 378 39.29% 5.40 340 42.86% 6.44 404 42.86% 6.04 364 46.43% 7.07 427 46.43% 6.28 388 50.00% 7.28 448 50.00% 6.51 411 53.57% 7.52 472 53.57% 7.18 438 57.14% 8.19 499 57.14% 7.39 459 60.71% 8.48 528 60.71% 8.06 486 64.29% 9.15 555 64.29% 8.34 514 67.86% 9.44 584 67.86% 9.01 541 71.43% 10.19 619 71.43% 9.29 569 75.00% 10.56 656 75.00% 10.01 601 78.57% 11.40 700 78.57% 10.38 638 82.14% 12.30 750 82.14% 11.16 676 85.71% 13.19 799 85.71% 11.59 719 89.29% 14.51 891 89.29% 12.55 775 92.86% 17.27 1047 92.86% 14.12 852 96.43% 16.47 1007 Total volu 28 28 pH 7 Run# 1 Run# AdjustmenAfter foamed 7.01 6.96 19.9 20.7 pH o Temperature C Before 6.78 19.1 Observation Foam volume not stabilised at 2min Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 pH o Temperature C Run #1 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 6.67% 1.33 93 6.45% 1.48 108 10.00% 2.14 134 9.68% 2.29 149 13.33% 2.58 178 12.90% 3.08 188 16.67% 3.40 220 16.13% 3.40 220 20.00% 4.14 254 19.35% 4.06 246 23.33% 4.42 282 22.58% 4.35 275 26.67% 5.08 308 25.81% 4.56 296 30.00% 5.31 331 29.03% 5.18 318 33.33% 5.56 356 32.26% 5.36 336 36.67% 6.20 380 35.48% 6.02 362 40.00% 6.45 405 38.71% 6.26 386 43.33% 7.10 430 41.94% 6.54 414 46.67% 7.38 458 45.16% 7.18 438 50.00% 8.03 483 48.39% 7.45 465 53.33% 8.27 507 51.61% 8.08 488 56.67% 8.52 532 54.84% 8.36 516 60.00% 9.20 560 58.06% 9.03 543 63.33% 9.49 589 61.29% 9.30 570 66.67% 10.16 616 64.52% 10.00 600 70.00% 10.47 647 67.74% 10.26 626 73.33% 11.21 681 70.97% 10.58 658 76.67% 11.59 719 74.19% 11.36 696 80.00% 12.37 757 77.42% 12.08 728 83.33% 13.13 793 80.65% 12.50 770 86.67% 14.06 846 83.87% 13.29 809 90.00% 15.02 902 87.10% 14.28 868 93.33% 16.29 989 90.32% 15.37 937 93.55% 17.19 1039 Total volum 30 31 2 Before 6.94 20.8 Run# AdjustmenAfter foamed 7.01 6.97 20.9 21.1 pH o Temperature C 3 Before 6.94 20.8 AdjustmenAfter foamed 7 6.99 21 21.3 Observation Foam volume stabilised at 2min Observation Foam volume stabilised at 1min 53s Results Volume mL Results Volume mL Run #2 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 6.45% 1.37 97 6.45% 1.42 102 9.68% 2.18 138 9.68% 2.19 139 12.90% 2.58 178 12.90% 3.00 180 16.13% 3.35 215 16.13% 3.25 205 19.35% 4.10 250 19.35% 3.51 231 22.58% 4.36 276 22.58% 4.22 262 25.81% 5.00 300 25.81% 4.44 284 29.03% 5.25 325 29.03% 5.05 305 32.26% 5.49 349 32.26% 5.21 321 35.48% 6.14 374 35.48% 5.45 345 38.71% 6.35 395 38.71% 6.13 373 41.94% 7.01 421 41.94% 6.39 399 45.16% 7.25 445 45.16% 7.04 424 48.39% 7.49 469 48.39% 7.27 447 51.61% 8.13 493 51.61% 7.54 474 54.84% 8.36 516 54.84% 8.19 499 58.06% 9.04 544 58.06% 8.46 526 61.29% 9.32 572 61.29% 9.14 554 64.52% 9.57 597 64.52% 9.39 579 67.74% 10.30 630 67.74% 10.08 608 70.97% 11.00 660 70.97% 10.44 644 74.19% 11.36 696 74.19% 11.17 677 77.42% 12.09 729 77.42% 11.51 711 80.65% 12.45 765 80.65% 12.27 747 83.87% 13.34 814 83.87% 13.18 798 87.10% 14.26 866 87.10% 14.18 858 90.32% 15.39 939 90.32% 15.35 935 93.55% 17.46 1066 93.55% 17.53 1073 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Total volum 31 31 20 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Run #3 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 6.25% 1.19 79 6.25% 1.43 103 9.38% 2.00 120 9.38% 2.20 140 12.50% 2.38 158 12.50% 3.03 183 15.63% 3.11 191 15.63% 3.30 210 18.75% 3.47 227 18.75% 4.03 243 21.88% 4.12 252 21.88% 4.25 265 25.00% 4.35 275 28.13% 5.05 305 28.13% 5.06 306 31.25% 5.24 324 31.25% 5.28 328 34.38% 5.50 350 34.38% 5.52 352 37.50% 6.13 373 37.50% 6.19 379 40.63% 6.37 397 40.63% 6.46 406 43.75% 7.01 421 43.75% 7.12 432 46.88% 7.25 445 46.88% 7.35 455 50.00% 7.50 470 50.00% 8.02 482 53.13% 8.16 496 53.13% 8.29 509 56.25% 8.42 522 56.25% 8.53 533 59.38% 9.11 551 59.38% 9.21 561 62.50% 9.39 579 62.50% 9.48 588 65.63% 10.13 613 65.63% 10.18 618 68.75% 10.45 645 68.75% 10.52 652 71.88% 11.21 681 71.88% 11.22 682 75.00% 11.59 719 75.00% 11.57 717 78.13% 12.38 758 78.13% 12.37 757 81.25% 13.31 811 81.25% 13.16 796 84.38% 14.31 871 84.38% 14.08 848 87.50% 16.05 965 87.50% 15.05 905 90.63% 18.43 1123 90.63% 16.35 995 Total volu 32 32 pH 8 Run# 1 Run# AdjustmenAfter foamed 8.2 7.9 22 23 pH o Temperature C Before 7.6 22 Observation Foam volume not stabilised at 1min 18sec Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 pH o Temperature C Duplicate Run #1 % Drainag Time Time (s) % Drainag Time Time (s) 6.67% 1.19 79 6.90% 10.00% 1.54 114 10.34% 2.28 148 13.33% 2.34 154 13.79% 3.09 189 16.67% 3.08 188 17.24% 3.41 221 20.00% 3.40 220 20.69% 4.15 255 23.33% 4.09 249 24.14% 4.45 285 26.67% 4.40 280 27.59% 5.16 316 30.00% 4.59 299 31.03% 5.42 342 33.33% 34.48% 6.07 367 36.67% 5.33 333 37.93% 6.30 390 40.00% 5.57 357 41.38% 6.51 411 43.33% 6.19 379 44.83% 7.13 433 46.67% 6.42 402 48.28% 7.38 458 50.00% 7.03 423 51.72% 8.02 482 53.33% 7.29 449 55.17% 8.25 505 56.67% 7.54 474 58.62% 8.51 531 60.00% 8.16 496 62.07% 9.18 558 63.33% 8.42 522 65.52% 9.47 587 66.67% 9.10 550 68.97% 10.16 616 70.00% 9.42 582 72.41% 10.45 645 73.33% 10.12 612 75.86% 11.15 675 76.67% 10.41 641 79.31% 11.52 712 80.00% 11.27 687 82.76% 12.30 750 83.33% 12.12 732 86.21% 13.12 792 86.67% 13.05 785 89.66% 14.01 841 90.00% 14.06 846 93.10% 14.55 895 93.33% 16.12 972 96.55% 16.18 978 Total volum 30 29 2 Before 7.9 23 AdjustmenAfter foamed 7.9 23 Observation Foam volume stabilised at 2min Results Volume mL Run #2 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 2 6.67% 1.11 71 6.67% 1.27 87 3 10.00% 1.41 101 10.00% 2.27 147 4 13.33% 2.29 149 13.33% 3.06 186 5 16.67% 2.58 178 16.67% 3.36 216 6 20.00% 3.34 214 20.00% 4.10 250 7 23.33% 4.06 246 23.33% 4.43 283 8 26.67% 4.30 270 26.67% 5.09 309 9 30.00% 4.49 289 30.00% 5.33 333 10 33.33% 5.04 304 33.33% 5.59 359 11 36.67% 5.22 322 36.67% 6.19 379 12 40.00% 5.44 344 40.00% 6.40 400 13 43.33% 6.11 371 43.33% 7.05 425 14 46.67% 6.34 394 46.67% 7.29 449 15 50.00% 6.57 417 50.00% 7.53 473 16 53.33% 7.21 441 53.33% 8.16 496 17 56.67% 7.46 466 56.67% 8.42 522 18 60.00% 8.12 492 60.00% 9.08 548 19 63.33% 8.40 520 63.33% 9.34 574 20 66.67% 9.11 551 66.67% 10.02 602 21 70.00% 9.37 577 70.00% 10.31 631 22 73.33% 10.11 611 73.33% 11.03 663 23 76.67% 10.45 645 76.67% 11.36 696 24 80.00% 11.24 684 80.00% 12.14 734 25 83.33% 12.16 736 83.33% 12.57 777 26 86.67% 13.26 806 86.67% 13.43 823 27 90.00% 15.09 909 90.00% 14.38 878 28 93.33% 18.31 1111 93.33% 16.05 965 29 18.20 1100 30 31 32 33 Total volum 30 30 21 Stability test with bacterial cells Conditions: 500 mg/L of rhamnolipid and washed P.putida Mixing at 8,000 rpm for 3 minutes 100ml of foam in measuring cylinder pH 7 Run# 1 pH Temperature oC Before 7 22 Observation Results Run# Adjustme After foamed 2 pH Temperature oC Before 7 22 Foam volume stabilised at 1min 20sec Observation Foam volume stabilised at 1min 30s Volume mL Results Volume mL Duplicate Run #1 % Drainag Time Time (s) % Drainag Time Time (s) 2 6.67% 0.51 51 6.25% 0.58 58 3 10.00% 1.29 89 9.38% 1.39 99 4 13.33% 2.06 126 12.50% 2.14 134 5 16.67% 2.34 154 15.63% 2.43 163 6 20.00% 3.09 189 18.75% 3.16 196 7 23.33% 3.44 224 21.88% 3.43 223 8 26.67% 4.25 265 25.00% 4.15 255 9 30.00% 4.38 278 28.13% 4.36 276 10 33.33% 4.42 282 31.25% 5.04 304 11 36.67% 5.05 305 34.38% 5.27 327 12 40.00% 5.27 327 37.50% 5.47 347 13 43.33% 5.53 353 40.63% 6.09 369 14 46.67% 6.19 379 43.75% 6.32 392 15 50.00% 6.44 404 46.88% 6.57 417 16 53.33% 7.09 429 50.00% 7.23 443 17 56.67% 7.35 455 53.13% 7.49 469 18 60.00% 8.05 485 56.25% 8.18 498 19 63.33% 8.35 515 59.38% 8.44 524 20 66.67% 9.04 544 62.50% 9.15 555 21 70.00% 9.37 577 65.63% 9.45 585 22 73.33% 10.13 613 68.75% 10.18 618 23 76.67% 10.56 656 71.88% 10.52 652 24 80.00% 11.40 700 75.00% 11.31 691 25 83.33% 12.35 755 78.13% 12.09 729 26 86.67% 13.36 816 81.25% 12.54 774 27 90.00% 14.59 899 84.38% 13.42 822 28 93.33% 17.18 1038 87.50% 14.32 872 29 90.63% 15.54 954 30 93.75% 17.06 1026 31 96.88% 19.17 1157 30 32 Total volum 22 Adjustme After foamed Run #2 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 2 6.45% 0.48 48 6.25% 0.47 47 3 9.68% 1.20 80 9.38% 1.36 96 4 12.90% 1.59 119 12.50% 2.07 127 5 16.13% 2.28 148 15.63% 2.34 154 6 19.35% 3.02 182 18.75% 3.04 184 7 22.58% 3.31 211 21.88% 3.32 212 8 25.81% 4.02 242 25.00% 3.58 238 9 29.03% 28.13% 4.22 262 10 32.26% 4.29 269 31.25% 4.50 290 11 35.48% 4.51 291 34.38% 5.06 306 12 38.71% 5.16 316 37.50% 5.28 328 13 41.94% 5.38 338 40.63% 5.50 350 14 45.16% 6.03 363 43.75% 6.16 376 15 48.39% 6.27 387 46.88% 6.40 400 16 51.61% 6.52 412 50.00% 7.03 423 17 54.84% 7.19 439 53.13% 7.30 450 18 58.06% 7.46 466 56.25% 7.57 477 19 61.29% 8.13 493 59.38% 8.26 506 20 64.52% 8.40 520 62.50% 8.53 533 21 67.74% 9.14 554 65.63% 9.24 564 22 70.97% 9.45 585 68.75% 9.59 599 23 74.19% 10.22 622 71.88% 10.31 631 24 77.42% 11.01 661 75.00% 11.10 670 25 80.65% 11.50 710 78.13% 11.49 709 26 83.87% 12.43 763 81.25% 12.31 751 27 87.10% 13.44 824 84.38% 13.17 797 28 90.32% 15.24 924 87.50% 14.11 851 29 93.55% 17.58 1078 90.63% 15.15 915 30 96.77% 93.75% 16.39 999 31 100.00% 96.88% 18.31 1111 Total volum 31 32 Conditions: 1,000 mg/L of rhamnolipid and washed P.putida Run# 1 pH o Temperature C Before 7 22.5 Observation Results Run# Adjustme After foamed 2 pH Temperature oC Before 7 22.5 Foam volume stabilised at 1min 25sec Observation Foam volume stabilised at 1min 20s Volume mL Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Duplicate Run #1 % Drainag Time Time (s) % Drainag Time Time (s) 6.45% 0.55 55 6.45% 1.24 84 9.68% 1.34 94 9.68% 2.16 136 12.90% 2.12 132 12.90% 2.58 178 16.13% 2.50 170 16.13% 3.32 212 19.35% 3.24 204 19.35% 4.12 252 22.58% 4.02 242 22.58% 4.41 281 25.81% 4.31 271 25.81% 5.14 314 29.03% 4.54 294 29.03% 5.37 337 32.26% 5.10 310 32.26% 6.07 367 35.48% 5.33 333 35.48% 6.34 394 38.71% 5.58 358 38.71% 6.58 418 41.94% 6.26 386 41.94% 7.24 444 45.16% 6.52 412 45.16% 7.50 470 48.39% 7.15 435 48.39% 8.17 497 51.61% 7.44 464 51.61% 8.42 522 54.84% 8.10 490 54.84% 9.11 551 58.06% 8.39 519 58.06% 9.40 580 61.29% 9.07 547 61.29% 10.12 612 64.52% 9.38 578 64.52% 10.42 642 67.74% 10.13 613 67.74% 11.14 674 70.97% 10.48 648 70.97% 11.48 708 74.19% 11.29 689 74.19% 12.25 745 77.42% 12.10 730 77.42% 13.06 786 80.65% 13.03 783 80.65% 13.45 825 83.87% 14.00 840 83.87% 14.30 870 87.10% 15.10 910 87.10% 15.21 921 90.32% 17.34 1054 90.32% 16.26 986 93.55% 17.50 1070 96.77% 20.01 1201 Total volum 31 31 Adjustme After foamed 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Run #2 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 6.45% 1.01 61 6.45% 1.37 97 9.68% 1.40 100 9.68% 2.31 151 12.90% 2.17 137 12.90% 3.06 186 16.13% 2.54 174 16.13% 3.41 221 19.35% 3.27 207 19.35% 4.20 260 22.58% 4.08 248 22.58% 4.53 293 25.81% 4.41 281 25.81% 5.25 325 29.03% 29.03% 5.50 350 32.26% 5.13 313 32.26% 6.21 381 35.48% 5.38 338 35.48% 6.46 406 38.71% 6.06 366 38.71% 7.10 430 41.94% 6.31 391 41.94% 7.35 455 45.16% 6.59 419 45.16% 8.01 481 48.39% 7.22 442 48.39% 8.32 512 51.61% 7.48 468 51.61% 8.57 537 54.84% 8.15 495 54.84% 9.26 566 58.06% 8.46 526 58.06% 9.55 595 61.29% 9.16 556 61.29% 10.26 626 64.52% 9.52 592 64.52% 10.59 659 67.74% 10.22 622 67.74% 11.32 692 70.97% 10.57 657 70.97% 12.06 726 74.19% 11.38 698 74.19% 12.45 765 77.42% 12.18 738 77.42% 13.27 807 80.65% 13.11 791 80.65% 14.08 848 83.87% 14.09 849 83.87% 14.55 895 87.10% 15.11 911 87.10% 15.58 958 90.32% 17.11 1031 90.32% 16.59 1019 93.55% 20.19 1219 93.55% 18.42 1122 Total volum 23 31 31 Conditions: 4,000 mg/L of rhamnolipid and washed P.putida Run# 1 pH o Temperature C Before 7 21 Observation Results Run# Adjustme After foamed 2 pH Temperature oC Before 7 21 Foam volume stabilised at 1min 20sec Observation Foam volume stabilised at 1min 22s Volume mL Results Volume mL Run #1 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 6.67% 1.25 85 6.90% 1.51 111 10.00% 2.08 128 10.34% 2.48 168 13.33% 2.49 169 13.79% 3.32 212 16.67% 3.28 208 17.24% 4.04 244 20.00% 4.02 242 20.69% 4.47 287 23.33% 4.35 275 24.14% 5.18 318 26.67% 5.03 303 27.59% 5.51 351 30.00% 5.26 326 31.03% 6.18 378 33.33% 5.44 344 34.48% 6.47 407 36.67% 6.07 367 37.93% 7.12 432 40.00% 6.34 394 41.38% 7.37 457 43.33% 7.00 420 44.83% 8.02 482 46.67% 7.26 446 48.28% 8.27 507 50.00% 7.50 470 51.72% 8.56 536 53.33% 8.18 498 55.17% 9.23 563 56.67% 8.44 524 58.62% 9.48 588 60.00% 9.13 553 62.07% 10.19 619 63.33% 9.41 581 65.52% 10.49 649 66.67% 10.13 613 68.97% 11.20 680 70.00% 10.47 647 72.41% 11.52 712 73.33% 11.23 683 75.86% 12.26 746 76.67% 12.00 720 79.31% 13.04 784 80.00% 11.42 702 82.76% 13.44 824 83.33% 13.37 817 86.21% 14.28 868 86.67% 14.34 874 89.66% 15.20 920 90.00% 16.02 962 93.10% 16.20 980 93.33% 18.43 1123 96.55% 17.47 1067 20.08 1208 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Total volum 30 29 24 Adjustme After foamed Run #2 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 7.14% 1.53 113 6.67% 1.28 88 10.71% 2.58 178 10.00% 2.09 129 14.29% 3.42 222 13.33% 2.52 172 17.86% 4.18 258 16.67% 3.24 204 21.43% 5.00 300 20.00% 3.52 232 25.00% 5.32 332 23.33% 4.26 266 28.57% 6.08 368 26.67% 4.56 296 32.14% 6.40 400 30.00% 5.14 314 35.71% 7.05 425 33.33% 5.31 331 39.29% 7.28 448 36.67% 5.54 354 42.86% 7.55 475 40.00% 6.20 380 46.43% 8.20 500 43.33% 6.47 407 50.00% 8.47 527 46.67% 7.10 430 53.57% 9.14 554 50.00% 7.35 455 57.14% 9.42 582 53.33% 8.01 481 60.71% 10.12 612 56.67% 8.30 510 64.29% 10.44 644 60.00% 8.56 536 67.86% 11.13 673 63.33% 9.25 565 71.43% 11.49 709 66.67% 9.56 596 75.00% 13.03 783 70.00% 10.26 626 78.57% 13.45 825 73.33% 11.03 663 82.14% 14.35 875 76.67% 11.42 702 85.71% 15.34 934 80.00% 12.25 745 89.29% 16.49 1009 83.33% 13.18 798 92.86% 18.49 1129 86.67% 14.12 852 90.00% 15.40 940 93.33% 18.16 1096 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Total volum 28 30 Stability test with bacterial cells Conditions: 1,000 mg/L of rhamnolipid and washed R.erythropolis Mixing at 8,000 rpm for 3 minutes 100ml of foam in measuring cylinder pH 7 1000mg/l Rhl Run# 1 Before pH 7.1 Temperature oC 23 Run# 2 Adjustment After foamed Observation Foam volume stabilised at 1min 40sec Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Duplicate Avearge Run #1 % Drainage Time Time (s) % Drainag Time Time (s) 6.45% 1.01 61 6.45% 1.24 84 145 9.68% 1.40 100 9.68% 2.16 136 236 12.90% 2.17 137 12.90% 2.58 178 315 16.13% 2.54 174 16.13% 3.32 212 386 19.35% 3.27 207 19.35% 4.12 252 459 22.58% 4.08 248 22.58% 4.41 281 529 25.81% 4.41 281 25.81% 5.14 314 595 29.03% 4.57 297 29.03% 5.37 337 634 32.26% 5.13 313 32.26% 6.07 367 680 35.48% 5.38 338 35.48% 6.34 394 732 38.71% 6.06 366 38.71% 6.58 418 784 41.94% 6.31 391 41.94% 7.24 444 835 45.16% 6.59 419 45.16% 7.50 470 889 48.39% 7.22 442 48.39% 8.17 497 939 51.61% 7.48 468 51.61% 8.42 522 990 54.84% 8.15 495 54.84% 9.11 551 1046 58.06% 8.46 526 58.06% 9.40 580 1106 61.29% 9.16 556 61.29% 10.12 612 1168 64.52% 9.52 592 64.52% 10.42 642 1234 67.74% 10.22 622 67.74% 11.14 674 1296 70.97% 10.57 657 70.97% 11.48 708 1365 74.19% 11.38 698 74.19% 12.25 745 1443 77.42% 12.18 738 77.42% 13.06 786 1524 80.65% 13.11 791 80.65% 13.45 825 1616 83.87% 14.09 849 83.87% 14.30 870 1719 87.10% 15.11 911 87.10% 15.21 921 1832 90.32% 17.11 1031 90.32% 16.26 986 2017 20.19 1219 93.55% 17.50 1070 2289 96.77% 20.01 1201 1201 Total volum 31 31 pH Temperature oC Before 7 23.3 Observation Foam volume stabilised at 1min30sec Results Volume mL 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Total volume 25 Adjustment After foamed Run #1 Duplicate % Drainag Time Time (s) % Drainag Time Time (s) 6.67% 1.28 88 6.90% 1.28 117 10.00% 2.09 129 10.34% 2.09 183 13.33% 2.52 172 13.79% 2.52 244 16.67% 3.24 204 17.24% 3.24 295 20.00% 3.52 232 20.69% 3.52 346 23.33% 4.26 266 24.14% 4.26 398 26.67% 4.56 296 27.59% 4.56 446 30.00% 5.14 314 31.03% 5.14 474 33.33% 5.31 331 34.48% 5.31 506 36.67% 5.54 354 37.93% 5.54 543 40.00% 6.20 380 41.38% 6.20 582 43.33% 6.47 407 44.83% 6.47 621 46.67% 7.10 430 48.28% 7.10 660 50.00% 7.35 455 51.72% 7.35 697 53.33% 8.01 481 55.17% 8.01 736 56.67% 8.30 510 58.62% 8.30 778 60.00% 8.56 536 62.07% 8.56 821 63.33% 9.25 565 65.52% 9.25 867 66.67% 9.56 596 68.97% 9.56 915 70.00% 10.26 626 72.41% 10.26 961 73.33% 11.03 663 75.86% 11.03 1014 76.67% 11.42 702 79.31% 11.42 1073 80.00% 12.25 745 82.76% 12.25 1135 83.33% 13.18 798 86.21% 13.18 1207 86.67% 14.12 852 89.66% 14.12 1286 93.10% 15.40 1832 96.55% 18.16 2017 30 29 Stability test for high surfactant concentration Conditions: 4,000 mg/L of rhamnolipid (high surfactant concentration) Mixing at 8,000 rpm for 3 minutes 100ml of foam in measuring cylinder pH 6 Run# 1 Before pH 6.99 Temperature oC 17.4 Run# Adjustment After foamed 6.01 6.05 17.7 17.3 Observation Foam volume stabilised at 2min 16s Results Run #1 Volume mL % Drainage Time 2 6.45% 3 9.68% 4 12.90% 5 16.13% 6 19.35% 7 22.58% 8 25.81% 9 29.03% 10 32.26% 11 35.48% 12 38.71% 13 41.94% 14 45.16% 15 48.39% 16 51.61% 17 54.84% 18 58.06% 19 61.29% 20 64.52% 21 67.74% 22 70.97% 23 74.19% 24 77.42% 25 80.65% 26 83.87% 27 87.10% 28 90.32% 29 30 31 32 Total volu 31 1.49 2.39 3.24 4.11 4.53 5.24 5.51 6.21 6.46 7.15 7.43 8.08 8.42 9.09 9.38 10.08 10.39 11.13 11.45 12.23 13.02 13.44 14.28 15.17 16.14 17.25 19.05 pH Temperature oC Time (s) 109 159 204 251 293 324 351 381 406 435 463 488 522 549 578 608 639 673 705 743 782 824 868 917 974 1045 1145 Duplicate % Drainage Time Time (s) 6.25% 2.08 128 9.38% 2.49 169 12.50% 3.35 215 15.63% 4.05 245 18.75% 4.37 277 21.88% 5.09 309 25.00% 5.36 336 28.13% 5.56 356 31.25% 6.22 382 34.38% 6.50 410 37.50% 7.20 440 40.63% 7.53 473 43.75% 8.18 498 46.88% 8.48 528 50.00% 9.17 557 53.13% 9.51 591 56.25% 10.20 620 59.38% 10.51 651 62.50% 11.22 682 65.63% 11.57 717 68.75% 12.34 754 71.88% 13.14 794 75.00% 13.52 832 78.13% 14.40 880 81.25% 15.29 929 84.38% 16.33 993 87.50% 17.51 1071 32 2 Befor Adjustment 6.06 17.8 Run# After foamed 6.05 18.3 Observation Foam volume stabilised at 2min 3s Results Volum Run #2 mL % Drainage Time 2 6.67% 3 10.00% 4 13.33% 5 16.67% 6 20.00% 7 23.33% 8 26.67% 9 30.00% 10 33.33% 11 36.67% 12 40.00% 13 43.33% 14 46.67% 15 50.00% 16 53.33% 17 56.67% 18 60.00% 19 63.33% 20 66.67% 21 70.00% 22 73.33% 23 76.67% 24 80.00% 25 83.33% 26 86.67% 27 90.00% 28 93.33% 29 30 31 Total 1.30 2.23 3.11 3.58 4.38 5.08 5.35 5.59 6.27 6.55 7.21 7.49 8.20 8.47 9.14 9.42 10.15 10.50 11.20 12.00 12.43 13.30 14.17 15.02 16.20 17.51 20.34 30 pH Temperature oC Time (s) 90 143 191 238 278 308 335 359 387 415 441 469 500 527 554 582 615 650 680 720 763 810 857 902 980 1071 1234 Duplicate % Drainage Time Time (s) 6.45% 2.05 125 9.68% 2.50 170 12.90% 3.29 209 16.13% 3.56 236 19.35% 4.28 268 22.58% 4.59 299 25.81% 5.25 325 29.03% 5.47 347 32.26% 6.05 365 35.48% 6.35 395 38.71% 7.04 424 41.94% 7.31 451 45.16% 8.02 482 48.39% 8.30 510 51.61% 8.59 539 54.84% 9.29 569 58.06% 10.00 600 61.29% 10.32 632 64.52% 11.04 664 67.74% 11.39 699 70.97% 12.16 736 74.19% 12.55 775 77.42% 13.42 822 80.65% 14.28 868 83.87% 15.23 923 87.10% 16.46 1006 90.32% 18.28 1108 31 26 3 Befo Adjustment 6.05 18 After foamed 6.07 18.3 Observation Foam volume stabilised at 2min 20sec Results Volum Run #3 mL % Drainage Time 2 6.67% 3 10.00% 4 13.33% 5 16.67% 6 20.00% 7 23.33% 8 26.67% 9 30.00% 10 33.33% 11 36.67% 12 40.00% 13 43.33% 14 46.67% 15 50.00% 16 53.33% 17 56.67% 18 60.00% 19 63.33% 20 66.67% 21 70.00% 22 73.33% 23 76.67% 24 80.00% 25 83.33% 26 86.67% 27 90.00% 28 93.33% 29 30 31 32 Total 30 1.44 2.34 3.20 4.11 4.52 5.22 5.56 6.21 6.48 7.13 7.39 8.07 8.38 9.02 9.31 10.02 10.35 11.09 11.39 12.20 12.58 13.43 14.29 15.13 16.22 17.48 20.16 Duplicate Time (s) % Drainage Time Time (s) 104 6.45% 2.13 133 154 9.68% 2.55 175 200 12.90% 3.41 221 251 16.13% 4.10 250 292 19.35% 4.44 284 322 22.58% 5.11 311 356 25.81% 5.36 336 381 29.03% 5.58 358 408 32.26% 6.23 383 433 35.48% 6.51 411 459 38.71% 7.22 442 487 41.94% 7.50 470 518 45.16% 8.19 499 542 48.39% 8.44 524 571 51.61% 9.14 554 602 54.84% 9.45 585 635 58.06% 10.16 616 669 61.29% 10.48 648 699 64.52% 11.18 678 740 67.74% 11.57 717 778 70.97% 12.36 756 823 74.19% 13.08 788 869 77.42% 13.48 828 913 80.65% 14.38 878 982 83.87% 15.32 932 1068 87.10% 16.44 1004 1216 90.32% 18.20 1100 93.55% 21.12 1272 31 pH 7 Run# 1 Before pH 7.31 Temperature oC 19.1 Run# Adjustment After foamed 7.00 6.98 19.3 19.7 Observation Foam volume stabilised at 2min 30s Results Run #1 Volume mL % Drainage Time 2 6.25% 3 9.38% 4 12.50% 5 15.63% 6 18.75% 7 21.88% 8 25.00% 9 28.13% 10 31.25% 11 34.38% 12 37.50% 13 40.63% 14 43.75% 15 46.88% 16 50.00% 17 53.13% 18 56.25% 19 59.38% 20 62.50% 21 65.63% 22 68.75% 23 71.88% 24 75.00% 25 78.13% 26 81.25% 27 84.38% 28 87.50% 29 90.63% 30 93.75% 31 Total volu 32 1.19 2.00 2.43 3.22 3.59 4.24 4.54 5.18 5.44 6.11 6.36 7.01 7.30 7.56 8.21 8.46 9.14 9.44 10.11 10.44 11.16 11.52 12.27 13.02 13.46 14.32 15.30 16.54 19.08 Time (s) 79 120 163 202 239 264 294 318 344 371 396 421 450 476 501 526 554 584 611 644 676 712 747 782 826 872 930 1014 1148 Duplicate % Drainage Time Time (s) 6.06% 1.35 95 9.09% 2.09 129 12.12% 2.49 169 15.15% 3.16 196 18.18% 3.46 226 21.21% 4.13 253 24.24% 4.40 280 27.27% 4.56 296 30.30% 5.18 318 33.33% 5.46 346 36.36% 6.14 374 39.39% 6.42 402 42.42% 7.07 427 45.45% 7.33 453 48.48% 7.59 479 51.52% 8.27 507 54.55% 8.55 535 57.58% 9.22 562 60.61% 9.51 591 63.64% 10.21 621 66.67% 10.52 652 69.70% 11.25 685 72.73% 11.59 719 75.76% 12.33 753 78.79% 13.13 793 81.82% 13.59 839 84.85% 14.49 889 87.88% 15.46 946 90.91% 17.05 1025 93.94% 19.11 1151 33 pH Temperature oC 2 Befor Adjustment 6.99 17.4 Observation Foam volume stabilised at 2min Results Volum Run #2 mL % Drainage Time 2 6.67% 3 10.00% 4 13.33% 5 16.67% 6 20.00% 7 23.33% 8 26.67% 9 30.00% 10 33.33% 11 36.67% 12 40.00% 13 43.33% 14 46.67% 15 50.00% 16 53.33% 17 56.67% 18 60.00% 19 63.33% 20 66.67% 21 70.00% 22 73.33% 23 76.67% 24 80.00% 25 83.33% 26 86.67% 27 90.00% 28 93.33% 29 30 Total Run# After foamed 6.97 18 1.48 2.34 3.19 4.03 4.45 5.14 5.46 6.10 6.38 7.06 7.31 7.59 8.28 8.56 9.22 9.51 10.21 10.55 11.22 12.01 12.36 13.18 13.59 14.39 15.36 16.40 18.09 30 pH Temperature oC Time (s) 108 154 199 243 285 314 346 370 398 426 451 479 508 536 562 591 621 655 682 721 756 798 839 879 936 1000 1089 Duplicate % Drainage Time Time (s) 6.45% 2.03 123 9.68% 2.44 164 12.90% 3.29 209 16.13% 3.59 239 19.35% 4.29 269 22.58% 5.07 307 25.81% 5.30 330 29.03% 5.48 348 32.26% 6.11 371 35.48% 6.41 401 38.71% 7.10 430 41.94% 7.39 459 45.16% 8.08 488 48.39% 8.34 514 51.61% 9.03 543 54.84% 9.31 571 58.06% 10.00 600 61.29% 10.34 634 64.52% 11.01 661 67.74% 11.29 689 70.97% 12.06 726 74.19% 12.43 763 77.42% 13.20 800 80.65% 14.01 841 83.87% 14.49 889 87.10% 15.41 941 90.32% 16.44 1004 93.55% 18.14 1094 31 27 3 Befo Adjustment 7.00 17 After foamed 6.97 24.1 Observation Foam volume stabilised at 1min 20s Results Run #3 Volum mL % Drainage Time 2 6.67% 3 10.00% 4 13.33% 5 16.67% 6 20.00% 7 23.33% 8 26.67% 9 30.00% 10 33.33% 11 36.67% 12 40.00% 13 43.33% 14 46.67% 15 50.00% 16 53.33% 17 56.67% 18 60.00% 19 63.33% 20 66.67% 21 70.00% 22 73.33% 23 76.67% 24 80.00% 25 83.33% 26 86.67% 27 90.00% 28 93.33% 29 Total 30 1.50 2.41 3.26 4.10 4.48 5.18 5.43 6.09 6.37 7.05 7.31 8.00 8.30 8.57 9.26 9.55 10.27 10.59 11.33 12.13 12.56 13.43 14.31 15.24 16.44 18.27 22.11 Duplicate Time (s) % Drainage Time Time (s) 110 6.45% 2.02 122 161 9.68% 2.44 164 206 12.90% 3.28 208 250 16.13% 3.57 237 288 19.35% 4.31 271 318 22.58% 4.58 298 343 25.81% 5.26 326 369 29.03% 5.47 347 397 32.26% 6.06 366 425 35.48% 6.36 396 451 38.71% 7.07 427 480 41.94% 7.34 454 510 45.16% 8.03 483 537 48.39% 8.29 509 566 51.61% 8.58 538 595 54.84% 9.28 568 627 58.06% 9.58 598 659 61.29% 10.27 627 693 64.52% 10.58 658 733 67.74% 11.32 692 776 70.97% 12.08 728 823 74.19% 12.45 765 871 77.42% 13.30 810 924 80.65% 14.08 848 1004 83.87% 15.03 903 1107 87.10% 16.04 964 1331 90.32% 17.29 1049 93.55% 19.17 1157 31 pH 8 Run# 1 Before pH 7.28 Temperature oC 17.2 Run# Adjustment After foamed 8.00 7.99 17.3 17.6 Observation Foam volume stabilised at 1min 34s Results Run #1 Volume mL % Drainage Time 2 6.67% 3 10.00% 4 13.33% 5 16.67% 6 20.00% 7 23.33% 8 26.67% 9 30.00% 10 33.33% 11 36.67% 12 40.00% 13 43.33% 14 46.67% 15 50.00% 16 53.33% 17 56.67% 18 60.00% 19 63.33% 20 66.67% 21 70.00% 22 73.33% 23 76.67% 24 80.00% 25 83.33% 26 86.67% 27 90.00% 28 93.33% 29 30 31 Total volu 30 1.44 2.31 3.13 3.58 4.38 5.08 5.30 5.59 6.25 6.49 7.15 7.43 8.11 8.37 9.02 9.34 10.03 10.33 11.07 11.45 12.20 13.10 13.55 14.43 15.53 17.18 19.55 Time (s) 104 151 193 238 278 308 330 359 385 409 435 463 491 517 542 574 603 633 667 705 740 790 835 883 953 1038 1195 Duplicate % Drainage Time Time (s) 6.45% 2.08 128 9.68% 2.50 170 12.90% 3.28 208 16.13% 3.59 239 19.35% 4.28 268 22.58% 5.01 301 25.81% 5.22 322 29.03% 5.42 342 32.26% 6.03 363 35.48% 6.30 390 38.71% 7.00 420 41.94% 7.28 448 45.16% 7.56 476 48.39% 8.21 501 51.61% 8.48 528 54.84% 9.19 559 58.06% 9.47 587 61.29% 10.16 616 64.52% 10.46 646 67.74% 11.21 681 70.97% 11.58 718 74.19% 12.36 756 77.42% 13.16 796 80.65% 14.05 845 83.87% 15.01 901 87.10% 16.13 973 90.32% 17.54 1074 93.55% 20.55 1255 31 pH Temperature oC 2 Befor Adjustment After foamed 7.81 8.01 8.00 18.5 18.5 18.9 Observation Foam volume stabilised at 1min 37sec Results Volum Run #2 mL % Drainage Time 2 6.45% 3 9.68% 4 12.90% 5 16.13% 6 19.35% 7 22.58% 8 25.81% 9 29.03% 10 32.26% 11 35.48% 12 38.71% 13 41.94% 14 45.16% 15 48.39% 16 51.61% 17 54.84% 18 58.06% 19 61.29% 20 64.52% 21 67.74% 22 70.97% 23 74.19% 24 77.42% 25 80.65% 26 83.87% 27 87.10% 28 90.32% 29 93.55% 30 Total 1.01 1.49 2.32 3.07 3.44 4.26 4.59 5.24 5.38 6.02 6.29 6.54 7.22 7.49 8.12 8.41 9.09 9.39 10.10 10.44 11.19 11.59 12.41 13.31 14.27 15.46 17.54 21.13 31 Run# pH Temperature oC Time (s) 71 119 162 197 234 276 309 334 348 372 399 424 452 479 502 531 559 589 620 654 689 729 771 821 877 956 1084 1283 Duplicate % Drainage Time Time (s) 6.45% 2.02 122 9.68% 2.41 161 12.90% 3.19 199 16.13% 3.55 235 19.35% 4.23 263 22.58% 4.52 292 25.81% 5.17 317 29.03% 5.36 336 32.26% 5.52 352 35.48% 6.21 381 38.71% 6.50 410 41.94% 7.17 437 45.16% 7.44 464 48.39% 8.09 489 51.61% 8.37 517 54.84% 9.05 545 58.06% 9.35 575 61.29% 10.06 606 64.52% 10.34 634 67.74% 11.12 672 70.97% 11.47 707 74.19% 12.24 744 77.42% 13.07 787 80.65% 13.49 829 83.87% 14.50 890 87.10% 16.06 966 90.32% 17.51 1071 93.55% 21.10 1270 31 28 3 Befo Adjustment 7.98 19 After foamed 7.98 19 Observation Foam volume stabilised at 1min 40s Results Run #3 Volum mL % Drainage Time 2 6.67% 3 10.00% 4 13.33% 5 16.67% 6 20.00% 7 23.33% 8 26.67% 9 30.00% 10 33.33% 11 36.67% 12 40.00% 13 43.33% 14 46.67% 15 50.00% 16 53.33% 17 56.67% 18 60.00% 19 63.33% 20 66.67% 21 70.00% 22 73.33% 23 76.67% 24 80.00% 25 83.33% 26 86.67% 27 90.00% 28 93.33% 29 Total 30 1.26 2.14 2.57 3.34 4.14 4.53 5.24 5.47 6.00 6.27 6.54 7.22 7.49 8.17 8.46 9.17 9.49 10.19 10.54 11.34 12.14 13.00 13.52 14.56 16.24 18.41 24.09 Duplicate Time (s) % Drainage Time Time (s) 86 6.67% 2.05 125 134 10.00% 2.45 165 177 13.33% 3.25 205 214 16.67% 3.57 237 254 20.00% 4.26 266 293 23.33% 4.55 295 324 26.67% 5.22 322 347 30.00% 5.40 340 360 33.33% 5.58 358 387 36.67% 6.26 386 414 40.00% 6.54 414 442 43.33% 7.21 441 469 46.67% 7.48 468 497 50.00% 8.16 496 526 53.33% 8.43 523 557 56.67% 9.11 551 589 60.00% 9.44 584 619 63.33% 10.11 611 654 66.67% 10.43 643 694 70.00% 11.22 682 734 73.33% 12.02 722 780 76.67% 12.41 761 832 80.00% 13.28 808 896 83.33% 14.20 860 984 86.67% 15.30 930 1121 90.00% 17.04 1024 1449 93.33% 19.28 1168 30 Stability test with salt 4,000 mg/L of rhamnolipid and 1000 mg/L NaCl Mixing at 8,000 rpm for 3 minutes 100ml of foam in measuring cylinder pH 6 Run# 1 Before Adjustme After foamed pH 6.05 6 6.05 Temperature oC 18.3 18.4 18.4 Conditions: Run# 2 Before Adjustme After foamed pH 6.05 6.04 Temperature oC 18 17.7 Run# 3 Before Adjustme After foamed pH 6.04 6.05 Temperature oC 17.6 17.8 Observation Foam volume stabilised at 2min 5sec Observation Foam volume stabilised at 1min 40sec Observation Foam volume stabilised at 1min 44sec Results Volume Run #1 Duplicate mL % Drainag Time Time (s) % Drainag Time Time (s) 2 6.67% 1.51 111 6.45% 2.13 133 3 10.00% 2.39 159 9.68% 2.58 178 4 13.33% 3.22 202 12.90% 3.38 218 5 16.67% 4.07 247 16.13% 4.07 247 6 20.00% 4.45 285 19.35% 4.35 275 7 23.33% 5.12 312 22.58% 5.05 305 8 26.67% 5.41 341 25.81% 5.31 331 9 30.00% 6.06 366 29.03% 5.51 351 10 33.33% 6.35 395 32.26% 6.17 377 11 36.67% 6.59 419 35.48% 6.42 402 12 40.00% 7.25 445 38.71% 7.09 429 13 43.33% 7.52 472 41.94% 7.37 457 14 46.67% 8.22 502 45.16% 8.04 484 15 50.00% 8.45 525 48.39% 8.30 510 16 53.33% 9.14 554 51.61% 8.59 539 17 56.67% 9.41 581 54.84% 9.26 566 18 60.00% 10.15 615 58.06% 10.00 600 19 63.33% 10.46 646 61.29% 10.31 631 20 66.67% 11.16 676 64.52% 10.58 658 21 70.00% 11.55 715 67.74% 11.32 692 22 73.33% 12.35 755 70.97% 12.08 728 23 76.67% 13.18 798 74.19% 12.48 768 24 80.00% 14.05 845 77.42% 13.28 808 25 83.33% 14.52 892 80.65% 14.17 857 26 86.67% 16.07 967 83.87% 15.15 915 27 90.00% 17.43 1063 87.10% 16.27 987 28 93.33% 21.17 1277 90.32% 18.13 1093 29 93.55% 21.58 1318 Results Run #2 Volume Duplicate mL % Drainag Time Time (s) % Drainag Time Time (s) 2 6.67% 2.15 135 6.45% 2.37 157 3 10.00% 3.02 182 9.68% 3.13 193 4 13.33% 3.51 231 12.90% 3.54 234 5 16.67% 4.32 272 16.13% 4.29 269 6 20.00% 5.12 312 19.35% 4.59 299 7 23.33% 5.41 341 22.58% 5.31 331 8 26.67% 6.09 369 25.81% 5.52 352 9 30.00% 6.37 397 29.03% 6.14 374 10 33.33% 7.01 421 32.26% 6.34 394 11 36.67% 7.29 449 35.48% 7.03 423 12 40.00% 7.55 475 38.71% 7.30 450 13 43.33% 8.23 503 41.94% 8.02 482 14 46.67% 8.53 533 45.16% 8.30 510 15 50.00% 9.24 564 48.39% 9.00 540 16 53.33% 9.55 595 51.61% 9.29 569 17 56.67% 10.21 621 54.84% 9.59 599 18 60.00% 10.58 658 58.06% 10.32 632 19 63.33% 11.34 694 61.29% 11.05 665 20 66.67% 12.08 728 64.52% 11.42 702 21 70.00% 12.54 774 67.74% 12.20 740 22 73.33% 13.43 823 70.97% 13.02 782 23 76.67% 14.38 878 74.19% 13.48 828 24 80.00% 15.37 937 77.42% 14.46 886 25 83.33% 16.48 1008 80.65% 15.52 952 26 86.67% 18.55 1135 83.87% 17.24 1044 27 90.00% 22.42 1362 87.10% 19.50 1190 28 Results Volume Run #3 Duplicate mL % Drainag Time Time (s) % Drainag Time Time (s) 2 6.67% 1.49 109 6.45% 2.16 136 3 10.00% 2.42 162 9.68% 2.58 178 4 13.33% 3.26 206 12.90% 3.41 221 5 16.67% 4.12 252 16.13% 4.18 258 6 20.00% 4.51 291 19.35% 4.43 283 7 23.33% 5.24 324 22.58% 5.12 312 8 26.67% 5.51 351 25.81% 5.40 340 9 30.00% 6.20 380 29.03% 6.00 360 10 33.33% 6.48 408 32.26% 6.25 385 11 36.67% 7.12 432 35.48% 6.52 412 12 40.00% 7.40 460 38.71% 7.20 440 13 43.33% 8.08 488 41.94% 7.48 468 14 46.67% 8.38 518 45.16% 8.18 498 15 50.00% 9.05 545 48.39% 8.48 528 16 53.33% 9.34 574 51.61% 9.14 554 17 56.67% 10.03 603 54.84% 9.46 586 18 60.00% 10.35 635 58.06% 10.20 620 19 63.33% 11.09 669 61.29% 10.49 649 20 66.67% 11.45 705 64.52% 11.25 685 21 70.00% 12.22 742 67.74% 12.02 722 22 73.33% 13.03 783 70.97% 12.40 760 23 76.67% 13.49 829 74.19% 13.24 804 24 80.00% 14.41 881 77.42% 14.11 851 25 83.33% 15.34 934 80.65% 15.10 910 26 86.67% 16.50 1010 83.87% 16.16 976 27 90.00% 18.37 1117 87.10% 17.50 1070 28 93.33% 22.32 1352 90.32% 20.21 1221 Total vol 30 31 Total vo 30 31.00 29 Total v 30 31 pH 7 Run# 1 Before pH 7.84 Temperature oC 19 Observation Results Run# 2 Before Adjustme After foamed pH 7.02 7.06 Temperature oC 18.8 19 Adjustme After foamed 7 7.04 18.9 19.5 Foam volume not stabilised at 1min 20sec Adding 1 g of NaCl decreased pH from 7.84 to 7.6 Volume Run #1 Duplicate mL % Drainag Time Time (s) % Drainag Time Time (s) 2 6.67% 1.09 79 6.56% 2.09 129 3 10.00% 2.01 131 9.84% 2.46 166 4 13.33% 2.44 174 13.11% 3.24 204 5 16.67% 3.23 213 16.39% 3.56 236 6 20.00% 3.55 245 19.67% 4.27 267 7 23.33% 4.36 286 22.95% 4.56 296 8 26.67% 5.11 321 26.23% 5.20 320 9 30.00% 5.32 342 29.51% 5.39 339 10 33.33% 5.49 359 32.79% 6.00 360 11 36.67% 6.14 384 36.07% 6.28 388 12 40.00% 6.41 411 39.34% 6.57 417 13 43.33% 7.08 438 42.62% 7.24 444 14 46.67% 7.36 466 45.90% 7.49 469 15 50.00% 7.58 488 49.18% 8.14 494 16 53.33% 8.27 517 52.46% 8.43 523 17 56.67% 8.53 543 55.74% 9.13 553 18 60.00% 9.22 572 59.02% 9.43 583 19 63.33% 9.52 602 62.30% 10.10 610 20 66.67% 10.25 635 65.57% 10.41 641 21 70.00% 10.56 666 68.85% 11.11 671 22 73.33% 11.32 702 72.13% 11.51 711 23 76.67% 12.13 743 75.41% 12.26 746 24 80.00% 12.55 785 78.69% 13.06 786 25 83.33% 13.37 827 81.97% 13.57 837 26 86.67% 14.41 891 85.25% 14.51 891 27 90.00% 16.03 973 88.52% 16.06 966 28 93.33% 18.05 1095 91.80% 18.01 1081 29 96.67% 24.01 1451 95.08% 21.43 1303 30 Total vol 30 30.5 Run# 3 Before Adjustme After foamed pH 7.05 7.18 Temperature oC 19.2 19.8 Observation Foam volume stabilised at 1min 27s Observation Foam volume stabilised at 1min 34s Results Run #2 Volume Duplicate mL % Drainag Time Time (s) % Drainag Time Time (s) 2 6.45% 1.28 88 6.67% 2.05 125 3 9.68% 2.14 134 10.00% 2.47 167 4 12.90% 2.59 179 13.33% 3.28 208 5 16.13% 3.40 220 16.67% 4.02 242 6 19.35% 4.14 254 20.00% 4.28 268 7 22.58% 4.53 293 23.33% 4.57 297 8 25.81% 5.20 320 26.67% 5.21 321 9 29.03% 5.45 345 30.00% 5.40 340 10 32.26% 6.06 366 33.33% 6.02 362 11 35.48% 6.29 389 36.67% 6.28 388 12 38.71% 6.57 417 40.00% 6.57 417 13 41.94% 7.24 444 43.33% 7.25 445 14 45.16% 7.50 470 46.67% 7.52 472 15 48.39% 8.16 496 50.00% 8.19 499 16 51.61% 8.41 521 53.33% 8.45 525 17 54.84% 9.09 549 56.67% 9.15 555 18 58.06% 9.37 577 60.00% 9.43 583 19 61.29% 10.05 605 63.33% 10.13 613 20 64.52% 10.38 638 66.67% 10.46 646 21 67.74% 11.09 669 70.00% 11.15 675 22 70.97% 11.42 702 73.33% 11.52 712 23 74.19% 12.17 737 76.67% 12.28 748 24 77.42% 12.57 777 80.00% 13.08 788 25 80.65% 13.43 823 83.33% 13.55 835 26 83.87% 14.28 868 86.67% 14.49 889 27 87.10% 15.33 933 90.00% 15.53 953 28 90.32% 16.53 1013 93.33% 17.40 1060 29 93.55% 19.01 1141 96.67% 20.44 1244 30 31 32 33 Total vo 31 30 Results Volume Run #3 Duplicate mL % Drainag Time Time (s) % Drainag Time Time (s) 2 6.67% 1.14 74 6.90% 2.10 130 3 10.00% 2.03 123 10.34% 2.42 162 4 13.33% 2.44 164 13.79% 3.25 205 5 16.67% 3.23 203 17.24% 3.55 235 6 20.00% 3.59 239 20.69% 4.21 261 7 23.33% 4.39 279 24.14% 4.52 292 8 26.67% 5.06 306 27.59% 5.15 315 9 30.00% 5.29 329 31.03% 5.32 332 10 33.33% 5.45 345 34.48% 5.51 351 11 36.67% 6.10 370 37.93% 6.17 377 12 40.00% 6.37 397 41.38% 6.45 405 13 43.33% 7.02 422 44.83% 7.12 432 14 46.67% 7.29 449 48.28% 7.38 458 15 50.00% 7.55 475 51.72% 8.03 483 16 53.33% 8.20 500 55.17% 8.31 511 17 56.67% 8.47 527 58.62% 8.58 538 18 60.00% 9.15 555 62.07% 9.28 568 19 63.33% 9.45 585 65.52% 9.56 596 20 66.67% 10.17 617 68.97% 10.30 630 21 70.00% 10.47 647 72.41% 11.04 664 22 73.33% 11.24 684 75.86% 11.43 703 23 76.67% 12.02 722 79.31% 12.22 742 24 80.00% 12.42 762 82.76% 13.09 789 25 83.33% 13.35 815 86.21% 14.06 846 26 86.67% 14.32 872 89.66% 15.22 922 27 90.00% 15.53 953 93.10% 17.21 1041 28 93.33% 18.13 1093 96.55% 22.14 1334 29 96.67% 23.59 1439 30 31 32 30 Total v 30 29 Stability test with Tergitol Conditions: 1,000 mg/L of Tergitol 15-s-12 Mixing at 8,000 rpm for 3 minute 100ml of foam in measuring cylinder pH 6 Run# 1 Before Adjustment After foamed pH 6.64 6.06 6.12 Temperature oC 18.4 18.5 19 pH Temperature oC 2 Before Adjustment After foamed 6.16 5.92 6.04 17.5 17.5 18.5 Observation Foam volume not stabilised at 1min 58sec Observation Foam volume not stabilised at 1min 54sec Results Run #1 Duplicate Volume mL % Drainage Time Time (s) % Drainage Time Time (s) 2 6.67% 1.06 66 6.45% 1.30 90 3 10.00% 1.46 106 9.68% 1.59 119 4 13.33% 2.15 135 12.90% 2.28 148 5 16.67% 2.46 166 16.13% 2.53 173 6 20.00% 3.12 192 19.35% 3.18 198 7 23.33% 3.44 224 22.58% 3.40 220 8 26.67% 4.09 249 25.81% 3.59 239 9 30.00% 4.28 268 29.03% 4.13 253 10 33.33% 4.43 283 32.26% 4.33 273 11 36.67% 5.02 302 35.48% 4.53 293 12 40.00% 5.25 325 38.71% 5.16 316 13 43.33% 5.45 345 41.94% 5.37 337 14 46.67% 6.06 366 45.16% 5.58 358 15 50.00% 6.26 386 48.39% 6.19 379 16 53.33% 6.46 406 51.61% 6.41 401 17 56.67% 7.08 428 54.84% 7.03 423 18 60.00% 7.30 450 58.06% 7.25 445 19 63.33% 7.52 472 61.29% 7.48 468 20 66.67% 8.19 499 64.52% 8.11 491 21 70.00% 8.42 522 67.74% 8.33 513 22 73.33% 9.11 551 70.97% 8.59 539 23 76.67% 9.42 582 74.19% 9.28 568 24 80.00% 10.12 612 77.42% 9.57 597 25 83.33% 10.54 654 80.65% 10.32 632 26 86.67% 11.38 698 83.87% 11.13 673 27 90.00% 12.34 754 87.10% 11.49 709 28 93.33% 14.10 850 90.32% 12.52 772 29 96.67% 17.40 1060 93.55% 14.22 862 30 96.77% 17.20 1040 Results Volume Duplicate Run #2 mL % Drainage Time Time (s) % Drainag Time Time (s) 2 6.67% 1.12 72 6.45% 1.33 93 3 10.00% 1.47 107 9.68% 2.08 128 4 13.33% 2.18 138 12.90% 2.36 156 5 16.67% 2.46 166 16.13% 3.01 181 6 20.00% 3.15 195 19.35% 3.24 204 7 23.33% 3.42 222 22.58% 3.48 228 8 26.67% 4.06 246 25.81% 4.08 248 9 30.00% 4.25 265 29.03% 4.21 261 10 33.33% 4.45 285 32.26% 4.37 277 11 36.67% 5.04 304 35.48% 5.00 300 12 40.00% 5.26 326 38.71% 5.22 322 13 43.33% 5.47 347 41.94% 5.44 344 14 46.67% 6.07 367 45.16% 6.05 365 15 50.00% 6.29 389 48.39% 6.24 384 16 53.33% 6.50 410 51.61% 6.45 405 17 56.67% 7.12 432 54.84% 7.07 427 18 60.00% 7.34 454 58.06% 7.29 449 19 63.33% 7.55 475 61.29% 7.52 472 20 66.67% 8.23 503 64.52% 8.15 495 21 70.00% 8.46 526 67.74% 8.38 518 22 73.33% 9.12 552 70.97% 9.06 546 23 76.67% 9.42 582 74.19% 9.34 574 24 80.00% 10.16 616 77.42% 10.03 603 25 83.33% 10.55 655 80.65% 10.38 638 26 86.67% 11.40 700 83.87% 11.16 676 27 90.00% 12.36 756 87.10% 12.02 722 28 93.33% 14.12 852 90.32% 13.12 792 29 96.67% 18.15 1095 93.55% 15.00 900 29 93.55% 18.59 1139 Total vo 30 Run# 31 Total vo 31 30 31 pH 7 Run# 1 Before Adjustment After foamed pH 5.5 6.96 7.12 Temperature oC 19.9 19.9 20 pH Temperature oC 2 Before Adjustment After foamed 6.95 7.06 19.8 20.1 Observation Foam volume not stabilised at 2min Observation Foam volume not stabilised at 1min 49sec Results Duplicate Run #1 Volume mL % Drainage Time Time (s) % Drainage Time Time (s) 2 6.67% 1.06 66 6.67% 1.32 92 3 10.00% 1.46 106 10.00% 2.01 121 4 13.33% 2.16 136 13.33% 2.33 153 5 16.67% 2.44 164 16.67% 2.55 175 6 20.00% 3.14 194 20.00% 3.18 198 7 23.33% 3.43 223 23.33% 3.44 224 8 26.67% 4.09 249 26.67% 4.00 240 9 30.00% 4.26 266 30.00% 4.16 256 10 33.33% 4.42 282 33.33% 4.29 269 11 36.67% 5.00 300 36.67% 4.50 290 12 40.00% 5.20 320 40.00% 5.12 312 13 43.33% 5.41 341 43.33% 5.33 333 14 46.67% 6.02 362 46.67% 5.54 354 15 50.00% 6.22 382 50.00% 6.13 373 16 53.33% 6.43 403 53.33% 6.34 394 17 56.67% 7.02 422 56.67% 6.57 417 18 60.00% 7.25 445 60.00% 7.16 436 19 63.33% 7.45 465 63.33% 7.39 459 20 66.67% 8.11 491 66.67% 8.03 483 21 70.00% 8.34 514 70.00% 8.26 506 22 73.33% 9.00 540 73.33% 8.54 534 23 76.67% 9.28 568 76.67% 9.24 564 24 80.00% 10.00 600 80.00% 9.55 595 25 83.33% 10.39 639 83.33% 10.34 634 26 86.67% 11.24 684 86.67% 11.22 682 27 90.00% 12.22 742 90.00% 12.25 745 28 93.33% 14.08 848 93.33% 14.13 853 29 96.67% 18.01 1081 96.67% 18.43 1123 Results Volume Run #2 Duplicate mL % Drainage Time Time (s) % Drainag Time Time (s) 2 6.67% 1.10 70 6.45% 1.36 96 3 10.00% 1.48 108 9.68% 2.06 126 4 13.33% 2.21 141 12.90% 2.38 158 5 16.67% 2.52 172 16.13% 3.00 180 6 20.00% 3.15 195 19.35% 3.22 202 7 23.33% 3.42 222 22.58% 3.44 224 8 26.67% 4.08 248 25.81% 4.04 244 9 30.00% 4.29 269 29.03% 4.20 260 10 33.33% 4.44 284 32.26% 4.34 274 11 36.67% 5.04 304 35.48% 4.55 295 12 40.00% 5.23 323 38.71% 5.18 318 13 43.33% 5.45 345 41.94% 5.39 339 14 46.67% 6.06 366 45.16% 6.00 360 15 50.00% 6.27 387 48.39% 6.20 380 16 53.33% 6.48 408 51.61% 6.42 402 17 56.67% 7.11 431 54.84% 7.03 423 18 60.00% 7.34 454 58.06% 7.26 446 19 63.33% 7.57 477 61.29% 7.47 467 20 66.67% 8.23 503 64.52% 8.12 492 21 70.00% 8.51 531 67.74% 8.36 516 22 73.33% 9.19 559 70.97% 9.04 544 23 76.67% 9.54 594 74.19% 9.31 571 24 80.00% 10.31 631 77.42% 10.04 604 25 83.33% 11.20 680 80.65% 10.41 641 26 86.67% 12.22 742 83.87% 11.24 684 27 90.00% 14.00 840 87.10% 12.18 738 28 93.33% 18.22 1102 90.32% 13.43 823 29 93.55% 16.38 998 Total vo 30 Run# 30 Total vo 32 30 31 pH 8 Run# 1 Before Adjustment After foamed pH 6.78 7.98 7.99 Temperature oC 17.7 17.9 17.9 pH Temperature oC 2 Before Adjustment After foamed 7.64 7.99 19.4 19.4 Observation Foam volume not stabilised at 1min 40sec Observation Foam volume not stabilised at 1min 46sec Results Run #1 Duplicate Volume mL % Drainage Time Time (s) % Drainage Time Time (s) 2 6.90% 1.15 75 6.67% 1.41 101 3 10.34% 1.50 110 10.00% 2.11 131 4 13.79% 2.21 141 13.33% 2.43 163 5 17.24% 2.50 170 16.67% 3.07 187 6 20.69% 3.18 198 20.00% 3.31 211 7 24.14% 3.50 230 23.33% 3.53 233 8 27.59% 4.22 262 26.67% 4.13 253 9 31.03% 4.37 277 30.00% 4.29 269 10 34.48% 4.51 291 33.33% 4.44 284 11 37.93% 5.10 310 36.67% 5.06 306 12 41.38% 5.33 333 40.00% 5.30 330 13 44.83% 5.55 355 43.33% 5.52 352 14 48.28% 6.16 376 46.67% 6.14 374 15 51.72% 6.38 398 50.00% 6.37 397 16 55.17% 7.01 421 53.33% 6.59 419 17 58.62% 7.25 445 56.67% 7.21 441 18 62.07% 7.47 467 60.00% 7.45 465 19 65.52% 8.13 493 63.33% 8.10 490 20 68.97% 8.39 519 66.67% 8.35 515 21 72.41% 9.07 547 70.00% 9.02 542 22 75.86% 9.35 575 73.33% 9.33 573 23 79.31% 10.14 614 76.67% 10.03 603 24 82.76% 10.54 654 80.00% 10.38 638 25 86.21% 11.45 705 83.33% 11.18 678 26 89.66% 12.41 761 86.67% 12.06 726 27 93.10% 14.17 857 90.00% 13.05 785 28 96.55% 18.17 1097 93.33% 14.49 889 29 96.67% 18.25 1105 Results Volume Run #2 Duplicate mL % Drainage Time Time (s) % Drainag Time Time (s) 2 6.67% 1.07 67 6.45% 1.31 91 3 10.00% 1.41 101 9.68% 2.01 121 4 13.33% 2.11 131 12.90% 2.31 151 5 16.67% 2.39 159 16.13% 2.57 177 6 20.00% 3.09 189 19.35% 3.21 201 7 23.33% 3.42 222 22.58% 3.45 225 8 26.67% 4.06 246 25.81% 4.04 244 9 30.00% 4.25 265 29.03% 4.17 257 10 33.33% 4.38 278 32.26% 4.32 272 11 36.67% 4.58 298 35.48% 4.54 294 12 40.00% 5.19 319 38.71% 5.17 317 13 43.33% 5.40 340 41.94% 5.40 340 14 46.67% 6.02 362 45.16% 6.01 361 15 50.00% 6.22 382 48.39% 6.22 382 16 53.33% 6.43 403 51.61% 6.42 402 17 56.67% 7.06 426 54.84% 7.04 424 18 60.00% 7.29 449 58.06% 7.27 447 19 63.33% 7.53 473 61.29% 7.50 470 20 66.67% 8.19 499 64.52% 8.15 495 21 70.00% 8.45 525 67.74% 8.39 519 22 73.33% 9.13 553 70.97% 9.06 546 23 76.67% 9.44 584 74.19% 9.38 578 24 80.00% 10.20 620 77.42% 10.10 610 25 83.33% 11.04 664 80.65% 10.47 647 26 86.67% 11.53 713 83.87% 11.28 688 27 90.00% 13.08 788 87.10% 12.23 743 28 93.33% 15.26 926 90.32% 13.45 825 29 96.67% 24.01 1441 93.55% 16.14 974 30 96.77% 24.04 1444 Total vo 29 Run# 30 Total vo 33 30 31 Foam collapse Surfactant Concentration (m/L) Salt (mg/L) pH Temperature oC Rhamnolipid 500 0 7.2 24.2 Liquid Drainage Time Total volume Foam Volume Foam Volume Fraction Liquid Hold-up ml seconds ml ml 2 61 100 98 0.98 0.93220339 3 99 100 97 0.97 0.898305085 4 130 100 96 0.96 0.86440678 5 159 100 95 0.95 0.830508475 6 190 100 94 0.94 0.796610169 7 217 100 93 0.93 0.762711864 8 240 100 92 0.92 0.728813559 9 270 100 91 0.91 0.694915254 10 292 100 90 0.9 0.661016949 11 315 100 89 0.89 0.627118644 12 342 100 88 0.88 0.593220339 13 366 100 87 0.87 0.559322034 14 392 100 86 0.86 0.525423729 15 417 100 85 0.85 0.491525424 16 447 100 84 0.84 0.457627119 17 474 100 83 0.83 0.423728814 18 504 100 82 0.82 0.389830508 19 536 100 81 0.81 0.355932203 20 565 100 80 0.8 0.322033898 21 607 100 79 0.79 0.288135593 22 645 100 78 0.78 0.254237288 23 695 100 77 0.77 0.220338983 24 753 100 76 0.76 0.186440678 25 814 100 75 0.75 0.152542373 26 904 100 74 0.74 0.118644068 27 1064 100 73 0.73 0.084745763 27.5 1200 99 71.5 0.715 0.06779661 27.7 1320 99 71.3 0.713 0.061016949 27.9 1440 99 71.1 0.711 0.054237288 28 1560 98 70 0.7 0.050847458 28.1 1680 98 69.9 0.699 0.047457627 28.1 1800 98 69.9 0.699 0.047457627 Liquid Drainage Time Total volume Foam Volume Foam Volume Fraction Liquid Hold-up ml seconds ml ml 2 62 100 98 0.98 0.93220339 3 98 100 97 0.97 0.898305085 4 129 100 96 0.96 0.86440678 5 161 100 95 0.95 0.830508475 6 198 100 94 0.94 0.796610169 7 221 100 93 0.93 0.762711864 8 247 100 92 0.92 0.728813559 9 268 100 91 0.91 0.694915254 10 291 100 90 0.9 0.661016949 11 313 100 89 0.89 0.627118644 12 338 100 88 0.88 0.593220339 13 364 100 87 0.87 0.559322034 14 390 100 86 0.86 0.525423729 15 414 100 85 0.85 0.491525424 16 441 100 84 0.84 0.457627119 17 469 100 83 0.83 0.423728814 18 499 100 82 0.82 0.389830508 19 530 100 81 0.81 0.355932203 20 567 100 80 0.8 0.322033898 21 598 100 79 0.79 0.288135593 22 641 100 78 0.78 0.254237288 23 686 100 77 0.77 0.220338983 24 737 100 76 0.76 0.186440678 25 809 100 75 0.75 0.152542373 26 900 100 74 0.74 0.118644068 27 1048 99 72 0.72 0.084745763 27.5 1200 99 71.5 0.715 0.06779661 27.8 1320 98 70.2 0.702 0.057627119 27.9 1440 97 69.1 0.691 0.054237288 28 1560 96 68 0.68 0.050847458 28.1 1680 95 66.9 0.669 0.047457627 28.2 1800 94 65.8 0.658 0.044067797 34 Average Time Foam Volume Fraction Liquid Hold-up Foam Volu Liquid Volu Total Volum seconds 61.5 98 93.22033898 98 2 100 98.5 97 89.83050847 97 3 100 129.5 96 86.44067797 96 4 100 160 95 83.05084746 95 5 100 194 94 79.66101695 94 6 100 219 93 76.27118644 93 7 100 243.5 92 72.88135593 92 8 100 269 91 69.49152542 91 9 100 291.5 90 66.10169492 90 10 100 314 89 62.71186441 89 11 100 340 88 59.3220339 88 12 100 365 87 55.93220339 87 13 100 391 86 52.54237288 86 14 100 415.5 85 49.15254237 85 15 100 444 84 45.76271186 84 16 100 471.5 83 42.37288136 83 17 100 501.5 82 38.98305085 82 18 100 533 81 35.59322034 81 19 100 566 80 32.20338983 80 20 100 602.5 79 28.81355932 79 21 100 643 78 25.42372881 78 22 100 690.5 77 22.03389831 77 23 100 745 76 18.6440678 76 24 100 811.5 75 15.25423729 75 25 100 902 74 11.86440678 74 26 100 1056 72.5 8.474576271 72.5 27 99.5 1200 71.5 6.779661017 71.5 27.5 99 1320 70.75 5.93220339 70.75 27.75 98.5 1440 70.1 5.423728814 70.1 27.9 98 1560 69 5.084745763 69 28 97 1680 68.4 4.745762712 68.4 28.1 96.5 1800 67.85 4.576271186 67.85 28.15 96 Foam collapse Surfactant Concentration (m/L) Salt (mg/L) pH Temperature oC Rhamnolipid 1000 0 7 25 Liquid Drainage Time Total volume Foam Volume Foam Volume Fraction Liquid Hold-up ml seconds ml ml 2 62 100 98 0.98 0.93220339 3 93 100 97 0.97 0.898305085 4 125 100 96 0.96 0.86440678 5 151 100 95 0.95 0.830508475 6 177 100 94 0.94 0.796610169 7 207 100 93 0.93 0.762711864 8 238 100 92 0.92 0.728813559 9 260 100 91 0.91 0.694915254 10 281 100 90 0.9 0.661016949 11 307 100 89 0.89 0.627118644 12 332 100 88 0.88 0.593220339 13 354 100 87 0.87 0.559322034 14 380 100 86 0.86 0.525423729 15 404 100 85 0.85 0.491525424 16 430 99 83 0.83 0.457627119 17 458 99 82 0.82 0.423728814 18 485 99 81 0.81 0.389830508 19 515 99 80 0.8 0.355932203 20 543 99 79 0.79 0.322033898 21 581 99 78 0.78 0.288135593 22 615 99 77 0.77 0.254237288 23 662 98 75 0.75 0.220338983 24 709 98 74 0.74 0.186440678 25 763 97 72 0.72 0.152542373 26 848 97 71 0.71 0.118644068 27 981 96 69 0.69 0.084745763 27.5 1124 95 67.5 0.675 0.06779661 27.6 1200 95 67.4 0.674 0.06440678 28 1320 95 67 0.67 0.050847458 28.1 1440 94 65.9 0.659 0.047457627 28.2 1560 93 64.8 0.648 0.044067797 28.3 1680 91 62.7 0.627 0.040677966 28.3 1800 90 61.7 0.617 0.040677966 28.4 1920 90 61.6 0.616 0.037288136 28.5 2100 89 60.5 0.605 0.033898305 28.5 2280 87 58.5 0.585 0.033898305 28.5 2400 85 56.5 0.565 0.033898305 28.6 2700 82 53.4 0.534 0.030508475 28.6 3000 78 49.4 0.494 0.030508475 Liquid Drainage Time Total volume Foam Volume Foam Volume Fraction Liquid Hold-up ml seconds ml ml 2 61 100 98 0.98 0.93220339 3 90 100 97 0.97 0.898305085 4 121 100 96 0.96 0.86440678 5 150 100 95 0.95 0.830508475 6 179 100 94 0.94 0.796610169 7 209 100 93 0.93 0.762711864 8 239 100 92 0.92 0.728813559 9 261 100 91 0.91 0.694915254 10 276 100 90 0.9 0.661016949 11 299 100 89 0.89 0.627118644 12 323 100 88 0.88 0.593220339 13 347 100 87 0.87 0.559322034 14 372 100 86 0.86 0.525423729 15 397 100 85 0.85 0.491525424 16 419 100 84 0.84 0.457627119 17 447 100 83 0.83 0.423728814 18 475 99 81 0.81 0.389830508 19 504 99 80 0.8 0.355932203 20 536 99 79 0.79 0.322033898 21 567 99 78 0.78 0.288135593 22 601 99 77 0.77 0.254237288 23 639 99 76 0.76 0.220338983 24 682 99 75 0.75 0.186440678 25 732 98 73 0.73 0.152542373 26 801 97 71 0.71 0.118644068 27 879 97 70 0.7 0.084745763 28 1015 96 68 0.68 0.050847458 28.5 1200 94 65.5 0.655 0.033898305 28.8 1320 93 64.2 0.642 0.023728814 28.9 1440 91 62.1 0.621 0.020338983 29 1560 90 61 0.61 0.016949153 29.2 1680 88 58.8 0.588 0.010169492 29.3 1800 85 55.7 0.557 0.006779661 29.3 1920 84 54.7 0.547 0.006779661 29.3 2100 83 53.7 0.537 0.006779661 29.3 2280 80 50.7 0.507 0.006779661 29.4 2400 79 49.6 0.496 0.003389831 29.4 2700 75 45.6 0.456 0.003389831 29.5 3000 72 42.5 0.425 0 30 35 Average Time Foam Volume Fraction Liquid Hold-up seconds 61.5 0.98 0.93220339 91.5 0.97 0.898305085 123 0.96 0.86440678 150.5 0.95 0.830508475 178 0.94 0.796610169 208 0.93 0.762711864 238.5 0.92 0.728813559 260.5 0.91 0.694915254 278.5 0.9 0.661016949 303 0.89 0.627118644 327.5 0.88 0.593220339 350.5 0.87 0.559322034 376 0.86 0.525423729 400.5 0.85 0.491525424 424.5 0.835 0.457627119 452.5 0.825 0.423728814 480 0.81 0.389830508 509.5 0.8 0.355932203 539.5 0.79 0.322033898 574 0.78 0.288135593 608 0.77 0.254237288 650.5 0.755 0.220338983 695.5 0.745 0.186440678 747.5 0.725 0.152542373 824.5 0.71 0.118644068 930 0.695 0.084745763 1069.5 0.6775 0.059322034 1200 0.6645 0.049152542 1320 0.656 0.037288136 1440 0.64 0.033898305 1560 0.629 0.030508475 1680 0.6075 0.025423729 1800 0.587 0.023728814 Foam collapse Surfactant Concentration (m/L) Salt (mg/L) pH Temperature oC Rhamnolipid 4000 0 7 24.6 Liquid Drainage Time Total volume Foam Volume Foam Volume Fraction Liquid Hold-up ml seconds ml ml 2 74 100 98 0.98 0.931034483 3 125 100 97 0.97 0.896551724 4 142 100 96 0.96 0.862068966 5 190 100 95 0.95 0.827586207 6 215 100 94 0.94 0.793103448 7 248 100 93 0.93 0.75862069 8 275 100 92 0.92 0.724137931 9 303 100 91 0.91 0.689655172 10 327 100 90 0.9 0.655172414 11 356 100 89 0.89 0.620689655 12 380 100 88 0.88 0.586206897 13 407 100 87 0.87 0.551724138 14 430 100 86 0.86 0.517241379 15 456 100 85 0.85 0.482758621 16 484 100 84 0.84 0.448275862 17 506 99 82 0.82 0.413793103 18 544 99 81 0.81 0.379310345 19 568 99 80 0.8 0.344827586 20 601 98 78 0.78 0.310344828 21 637 98 77 0.77 0.275862069 22 677 98 76 0.76 0.24137931 23 714 97 74 0.74 0.206896552 24 769 97 73 0.73 0.172413793 25 833 97 72 0.72 0.137931034 26 919 96 70 0.7 0.103448276 27 1097 96 69 0.69 0.068965517 27.5 1130 95 67.5 0.675 0.051724138 27.5 1249 94 66 0.66 0.034482759 28 1643 93 64.9 0.649 0.031034483 28.1 1780 92 63.9 0.639 0.031034483 28.1 2019 90 61.8 0.618 0.027586207 28.2 2119 89 60.8 0.608 0.027586207 28.2 2314 87 0.024137931 28.3 2400 85 0.024137931 28.3 2700 80 0.017241379 28.5 3000 77 Liquid Drainage Time Total volume Foam Volume Foam Volume Fraction Liquid Hold-up ml seconds ml ml 2 79 100 98 0.98 0.931034483 3 127 100 97 0.97 0.896551724 4 162 100 96 0.96 0.862068966 5 195 100 95 0.95 0.827586207 6 229 100 94 0.94 0.793103448 7 256 100 93 0.93 0.75862069 8 283 100 92 0.92 0.724137931 9 308 100 91 0.91 0.689655172 10 325 100 90 0.9 0.655172414 11 252 100 89 0.89 0.620689655 12 377 100 88 0.88 0.586206897 13 399 100 87 0.87 0.551724138 14 425 100 86 0.86 0.517241379 15 452 100 85 0.85 0.482758621 16 476 100 84 0.84 0.448275862 17 502 100 83 0.83 0.413793103 18 530 99 81 0.81 0.379310345 19 558 99 80 0.8 0.344827586 20 590 99 79 0.79 0.310344828 21 620 98 77 0.77 0.275862069 22 657 98 76 0.76 0.24137931 23 698 98 75 0.75 0.206896552 24 745 98 74 0.74 0.172413793 25 801 98 73 0.73 0.137931034 26 866 98 72 0.72 0.103448276 27 976 97 70 0.7 0.068965517 27.5 1090 96 68.5 0.685 0.051724138 27.5 1122 95 67.5 0.675 0.051724138 28 1280 93 65 0.65 0.034482759 28.2 1351 93 64.8 0.648 0.027586207 28.5 1619 91 62.5 0.625 0.017241379 28.5 1758 90 61.5 0.615 0.017241379 28.6 1870 89 60.4 0.604 0.013793103 28.7 2008 87 58.3 0.583 0.010344828 28.8 2144 85 56.2 0.562 0.006896552 28.8 2365 82 53.2 0.532 0.006896552 28.8 2400 82 53.2 0.532 0.006896552 28.9 2700 77 48.1 0.481 0.003448276 28.9 3000 74 45.1 0.451 0.003448276 36 Average Time Foam Volume Fraction Liquid Hold-up seconds 76.5 0.98 0.931034483 126 0.97 0.896551724 152 0.96 0.862068966 192.5 0.95 0.827586207 222 0.94 0.793103448 252 0.93 0.75862069 279 0.92 0.724137931 305.5 0.91 0.689655172 326 0.9 0.655172414 304 0.89 0.620689655 378.5 0.88 0.586206897 403 0.87 0.551724138 427.5 0.86 0.517241379 454 0.85 0.482758621 480 0.84 0.448275862 504 0.825 0.413793103 537 0.81 0.379310345 563 0.8 0.344827586 595.5 0.785 0.310344828 628.5 0.77 0.275862069 667 0.76 0.24137931 706 0.745 0.206896552 757 0.735 0.172413793 817 0.725 0.137931034 892.5 0.71 0.103448276 1036.5 0.695 0.068965517 1110 0.68 0.051724138 1185.5 0.6675 0.043103448 1461.5 0.6495 0.032758621 1565.5 0.6435 0.029310345 1819 0.6215 0.022413793 1938.5 0.6115 0.022413793 2092 0.018965517 2204 0.017241379 2422 0.012068966 2682.5 Foam collapse Surfactant Concentration (m/L) Salt (mg/L) pH Temperature oC Tergitol 1000 0 7.1 24.8 Liquid Drainage Time Total volume Foam Volume Foam Volume Fraction Liquid Hold-up ml seconds ml ml 2 43 100 98 0.98 0.935483871 3 66 100 97 0.97 0.903225806 4 90 100 96 0.96 0.870967742 5 116 100 95 0.95 0.838709677 6 142 100 94 0.94 0.806451613 7 163 100 93 0.93 0.774193548 8 182 100 92 0.92 0.741935484 9 197 100 91 0.91 0.709677419 10 211 100 90 0.9 0.677419355 11 229 100 89 0.89 0.64516129 12 248 100 88 0.88 0.612903226 13 265 100 87 0.87 0.580645161 14 283 100 86 0.86 0.548387097 15 300 100 85 0.85 0.516129032 16 317 100 84 0.84 0.483870968 17 332 100 83 0.83 0.451612903 18 350 100 82 0.82 0.419354839 19 367 100 81 0.81 0.387096774 20 387 100 80 0.8 0.35483871 21 403 100 79 0.79 0.322580645 22 421 99 77 0.77 0.290322581 23 444 99 76 0.76 0.258064516 24 466 98 74 0.74 0.225806452 25 490 98 73 0.73 0.193548387 26 515 98 72 0.72 0.161290323 27 544 97 70 0.7 0.129032258 28 588 95 67 0.67 0.096774194 29 645 90 61 0.61 0.064516129 29.3 680 85 55.7 0.557 0.05483871 29.5 726 80 50.5 0.505 0.048387097 29.7 765 77 47.3 0.473 0.041935484 29.8 799 73 43.2 0.432 0.038709677 30 860 68 38 0.38 0.032258065 30.1 918 63 32.9 0.329 0.029032258 30.1 971 60 29.9 0.299 0.029032258 30.1 1011 58 27.9 0.279 0.029032258 30.1 1082 53 22.9 0.229 0.029032258 30.1 1191 52 21.9 0.219 0.029032258 30.2 1306 48 17.8 0.178 0.025806452 30.2 1574 47 16.8 0.168 0.025806452 30.3 1734 45 14.7 0.147 0.022580645 30.5 1997 44 13.5 0.135 0.016129032 31 Liquid Drainage Time Total volume Foam Volume Foam Volume Fraction Liquid Hold-up ml seconds ml ml 2 56 100 98 0.98 0.935483871 3 87 100 97 0.97 0.903225806 4 112 100 96 0.96 0.870967742 5 131 100 95 0.95 0.838709677 6 152 100 94 0.94 0.806451613 7 177 100 93 0.93 0.774193548 8 197 100 92 0.92 0.741935484 9 214 100 91 0.91 0.709677419 10 229 100 90 0.9 0.677419355 11 250 100 89 0.89 0.64516129 12 266 100 88 0.88 0.612903226 13 283 100 87 0.87 0.580645161 14 301 100 86 0.86 0.548387097 15 315 100 85 0.85 0.516129032 16 330 100 84 0.84 0.483870968 17 350 100 83 0.83 0.451612903 18 368 100 82 0.82 0.419354839 19 385 100 81 0.81 0.387096774 20 405 100 80 0.8 0.35483871 21 425 99 78 0.78 0.322580645 22 448 99 77 0.77 0.290322581 23 470 99 76 0.76 0.258064516 24 494 99 75 0.75 0.225806452 25 524 98 73 0.73 0.193548387 26 563 98 72 0.72 0.161290323 27 612 97 70 0.7 0.129032258 27.5 649 95 67.5 0.675 0.112903226 27.7 683 90 62.3 0.623 0.106451613 28 721 87 59 0.59 0.096774194 28 747 82 54 0.54 0.096774194 28.1 777 80 51.9 0.519 0.093548387 28.1 818 74 45.9 0.459 0.093548387 28.2 866 68 39.8 0.398 0.090322581 28.3 902 64 35.7 0.357 0.087096774 28.5 1004 57 28.5 0.285 0.080645161 28.6 1067 54 25.4 0.254 0.077419355 28.8 1225 50 21.2 0.212 0.070967742 28.8 1386 49 20.2 0.202 0.070967742 28.9 1533 47 18.1 0.181 0.067741935 29 1764 45 16 0.16 0.064516129 37 SDS Microbubble Dispersion SDS @ 500 mg/L SDS @ 1000 mg/L SDS @ 4000 mg/L Time (seconds) Time (seconds) Time (seconds) Volume (mL) ph=6 ph=7 ph=8 ph=6 ph=7 ph=8 ph=6 ph=7 ph=8 2 68 64 79 137 127 115 72 100 89 3 102 98 106 165 161 155 109 128 126 4 130 121 128 195 193 192 139 150 150 5 150 147 155 217 222 220 157 175 172 6 171 171 169 239 246 249 185 196 196 7 191 196 190 259 254 278 208 212 219 8 214 216 214 285 299 298 249 235 241 9 233 231 234 305 325 319 252 258 263 10 259 250 261 330 361 345 274 280 285 11 274 269 277 348 382 365 295 294 306 12 289 265 295 365 399 386 315 310 326 13 307 304 310 383 419 406 336 325 340 14 323 323 327 401 436 427 358 345 361 15 346 341 364 417 459 450 380 365 382 16 363 361 361 436 477 472 403 386 404 17 382 382 378 458 498 495 426 405 429 18 405 400 398 475 518 521 452 426 450 19 429 423 418 500 540 546 481 450 479 20 450 447 441 523 567 576 510 479 508 21 477 470 462 546 591 603 540 507 544 22 513 500 486 569 615 637 580 535 585 23 547 531 510 602 645 671 630 578 638 24 592 565 541 640 676 712 683 623 710 25 667 602 574 677 714 766 763 695 828 26 755 660 617 731 753 837 878 828 1104 27 959 730 662 796 806 969 1387 1224 1578 28 943 733 908 900 1764 2534 29 2576 884 1527 1064 30 1253 1862 31 Liquid V 30 29 30 30 31 30 28 30 31 38   Appendix IV Viscosity Test Data IV    Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: 20Mar‐Rhamnolipid 8 1000pH6run1 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 12:29 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.492 1.00E+03 0 1 4 4 Meas. Pts. Meas. Pt. Duration 30 s Measuring Profile:   Shear Rate d(gamma)/dt = 90 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque [1/s] [Pa] [Pa∙s] [1/min] [µNm] 1 90 0.146 0.00162 15 4.79 2 90 0.144 0.00162 15 4.71 3 90 0.144 0.0016 15 4.71 4 90 0.144 0.0016 15 4.72 1 Status [] Dy_100% Dy_100% Dy_100% Dy_100% Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: 20Mar‐Rhamnolipid 9 500pH6run1 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 12:39 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.664 1.00E+03 0 1 4 4 Meas. Pts. Meas. Pt. Duration 30 s Measuring Profile:   Shear Rate d(gamma)/dt = 90 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque [1/s] [Pa] [Pa∙s] [1/min] [µNm] 1 90 0.136 0.00152 15 4.46 2 90 0.137 0.00152 15 4.47 3 90 0.137 0.00152 15 4.47 4 90 0.137 0.00152 15 4.48 2 Status [] Dy_100% Dy_100% Dy_100% Dy_100% Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: 20Mar‐Rhamnolipid 7 4000pH6S5 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 12:19 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.49 1.00E+03 0 1 6 6 Meas. Pts. Meas. Pt. Duration 30 s Measuring Profile:   Shear Rate d(gamma)/dt = 90 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque [1/s] [Pa] [Pa∙s] [1/min] [µNm] 1 90 0.181 0.00201 15 5.92 2 90 0.183 0.00203 15 5.99 3 90 0.183 0.00203 15 5.98 4 90 0.182 0.00203 15 5.97 5 90 0.182 0.00202 15 5.95 6 90 0.181 0.00202 15 5.94 3 Status [] Dy_100% Dy_100% Dy_100% Dy_100% Dy_100% Dy_100% Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: 20Mar‐Tergitol 1 1000pH6run1 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 13:53 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.511 1.00E+03 0 1 3 3 Meas. Pts. Meas. Pt. Duration 30 s Measuring Profile:   Shear Rate d(gamma)/dt = 90 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque [1/s] [Pa] [Pa∙s] [1/min] [µNm] 1 90 0.175 0.00194 15 5.71 2 90 0.175 0.00194 15 5.72 3 90 0.175 0.00194 15 5.72 4 Status [] Dy_100% Dy_100% Dy_100% Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: Measuring Profile:   Shear Rate 20Mar‐Rhamnolipid 5 1000pH7S3 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 12:02 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.479 1.00E+03 0 1 12 12 Meas. Pts. Meas. Pt. Duration 30 s d(gamma)/dt = 100 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque [1/s] [Pa] [Pa∙s] [1/min] [µNm] 1 100 0.176 0.00176 16.7 5.75 2 100 0.175 0.00175 16.7 5.74 3 100 0.172 0.00172 16.7 5.64 4 100 0.176 0.00176 16.7 5.76 5 100 0.173 0.00173 16.7 5.66 6 100 0.175 0.00175 16.7 5.72 7 100 0.174 0.00174 16.7 5.68 8 100 0.175 0.00175 16.7 5.73 5 Status [] Dy_100% Dy_100% Dy_100% Dy_100% Dy_100% Dy_100% Dy_100% Dy_100% Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: 20Mar‐Rhamnolipid 16 500pH7run1 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 13:37 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.634 1.00E+03 0 1 4 4 Meas. Pts. Meas. Pt. Duration 30 s Measuring Profile:   Shear Rate d(gamma)/dt = 90 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque Status [1/s] [Pa] [Pa∙s] [1/min] [µNm] [] 1 90 0.142 0.00157 15 4.63 Dy_100% 2 90 0.144 0.0016 15 4.7 Dy_100% 3 90 0.143 0.00159 15 4.68 Dy_100% 4 90 0.143 0.00158 15 4.66 Dy_100% 6 Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: 20Mar‐Rhamnolipid 12 4000pH7run1 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 13:06 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.802 1.00E+03 0 1 4 4 Meas. Pts. Meas. Pt. Duration 30 s Measuring Profile:   Shear Rate d(gamma)/dt = 90 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque [1/s] [Pa] [Pa∙s] [1/min] [µNm] 1 90 0.177 0.00196 15 5.78 2 90 0.176 0.00196 15 5.78 3 90 0.176 0.00196 15 5.77 4 90 0.176 0.00196 15 5.76 7 Status [] Dy_100% Dy_100% Dy_100% Dy_100% Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: 20Mar-Tergitol 28 1000pH7run4 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 16:20 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.53 1.00E+03 0 1 3 3 Meas. Pts. Meas. Pt. Duration 30 s Measuring Profile:   Shear Rate d(gamma)/dt = 90 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque [1/s] [Pa] [Pa∙s] [1/min] [µNm] 1 90 0.173 0.00193 15 5.68 2 90 0.175 0.00194 15 5.71 3 90 0.174 0.00193 15 5.7 8 Status [] Dy_100% Dy_100% Dy_100% Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: 20Mar‐Rhamnolipid 21 1000pH8run2 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 14:40 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.532 1.00E+03 0 1 3 3 Meas. Pts. Meas. Pt. Duration 30 s Measuring Profile:   Shear Rate d(gamma)/dt = 90 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque [1/s] [Pa] [Pa∙s] [1/min] [µNm] 1 90 0.161 0.00179 15 5.27 2 90 0.159 0.00177 15 5.22 3 90 0.159 0.00177 15 5.22 9 Status [] Dy_100% Dy_100% Dy_100% Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: 20Mar‐Rhamnolipid 13 500pH8run1 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 13:13 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.56 1.00E+03 0 1 4 4 Meas. Pts. Meas. Pt. Duration 30 s Measuring Profile:   Shear Rate d(gamma)/dt = 90 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque [1/s] [Pa] [Pa∙s] [1/min] [µNm] 1 90 0.144 0.0016 15 4.73 2 90 0.145 0.00161 15 4.74 3 90 0.145 0.00161 15 4.74 4 90 0.145 0.00161 15 4.74 10 Status [] Dy_100% Dy_100% Dy_100% Dy_100% Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: 20Mar‐Rhamnolipid 3 4000pH8run1 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 14:09 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.397 1.00E+03 0 1 4 4 Meas. Pts. Meas. Pt. Duration 30 s Measuring Profile:   Shear Rate d(gamma)/dt = 90 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque Status [1/s] [Pa] [Pa∙s] [1/min] [µNm] [] 1 90 0.174 0.00194 15 5.7 Dy_100% 2 90 0.174 0.00194 15 5.7 Dy_100% 3 90 0.174 0.00193 15 5.69 Dy_100% 4 90 0.173 0.00193 15 5.68 Dy_100% 11 Data Series Information Name: Sample: Number of Intervals: Application: Device: Measuring Date/Time: Measuring System: Accessories: Calculating Constants: ‐ Csr: ‐ Css: ‐ Start Delay Time [s]: ‐ Substance Density [rho]: ‐ Measurement Type: Interval: Number of Data Points: Time Setting: 20Mar-Tergitol 19 1000pH8run3 1 US200/16 V2.50 21000297‐33025, Login: ANNIE MC200 SN310529; FW3.04 20/03/09; 14:26 MK 22   (50mm,1°) TU=TEK 150P 5.999391 30.55775 13.491 1.00E+03 0 1 3 3 Meas. Pts. Meas. Pt. Duration 30 s Measuring Profile:   Shear Rate d(gamma)/dt = 90 1/s Meas. Pts. Shear Rate Shear StresViscosity Speed Torque [1/s] [Pa] [Pa∙s] [1/min] [µNm] 1 90 0.174 0.00194 15 5.71 2 90 0.175 0.00194 15 5.72 3 90 0.174 0.00194 15 5.71 12 Status [] Dy_100% Dy_100% Dy_100%   Appendix V Dissociation of Surfactants V    Dissociation of Surfactants  Use formula for calculating the % ionisation of a compound at a particular pH from its pKa is   % Ionisation = 100 1 + 10 ( ch arg e ( pH − pKa ))   where charge = + 1 for base and ‐1 for acid.   Surfactant  Rhamnolipid  Sodium dioctyl  sulfosuccinate (AOT)    pKa  5.6  2.9  Charge  Anionic; ‐1  Anionic; ‐1  For rhamnolipid at pH6, % Ionisation =  At pH 8, % Ionisation =  100 = 99.6%.  1 + 10 −(8−5.6 ) For AOT at pH 4, % Ionisation =  At pH 8, % Ionisation =  100 = 71.5%.   1 + 10 −( 6−5.6 ) 100 = 92.6%.  1 + 10 −( 4− 2.9 ) 100 = 99.9%.  1 + 10 −(8− 2.9 ) 1   Appendix VI Gas Hold-up Data VI    Gas hold-up ε= Vg Vd = rhamnolipid concentration (mg/L) pH 500 1,000 4,000 500 1,000 4,000 500 1,000 4,000 6 6 6 7 7 7 8 8 8 tergitol concentration (mg/L) pH 1,000 1,000 1,000 6 7 8 V d − Vl Vd Rhamnolipid NaCl concentration concentration (mg/L) (mg/L) 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 0 0 0 1,000 1,000 1,000 3,000 3,000 3,000 liquid volume gas volume (V g ) gas hold-up (ε) Std dispersion volume (V d ) (V l ) 100 29 71 71 100 28 72 72 100 31 69 69 100 33 67 67 100 29 71 71 100 31 69 69 100 32 68 68 100 32 68 68 100 31 69 69 0 1 1 2 1 1 2 1 1 liquid volume dispersion volume (V d ) (V l ) gas volume (V g ) gas hold-up (ε) Std 100 31 69 69 100 30 70 70 100 30 70 70 1 1 1 pH 6 7 8 6 7 8 6 7 8 dispersion liquid volume volume (V d ) (V l ) 100 100 100 100 100 100 100 100 100 1 gas volume (V g ) 28 29 32 30 31 34 33 31 30 72 71 68 70 69 66 67 69 70 gas holdup (ε) Std 72 71 68 70 69 66 67 69 70 1 1 1 1 1 2 4 1 1   Appendix VII Particle Size Analysis Data VII    The University of Auckland School of Geography & Environmental Science Malvern Particle Analysis Results Residual Sample Name Measurement date and time % Sample Name micro bubbles micro bubbles micro bubbles bubbles 1 Bubbles 3min 2 Bubbles 3min 3 Bubbules 2 Bubbles 3 min 4 Bubbles 3 min 5 bubbles 3 Bubbles 6 min 1 bubbles 6 min 2 Bubbles 4 bubbles 6 min 3 bubbles 6 min 4 bubbles 5 bubbles 3min 6 bubbles 3min 7 Bubbles 6 bubbles 3 min a bubbles 3 min b bubbles 7 bubbles 3 min c bubbles 3 min d D[4,3] Volume Percentiles (micron) Weighted d (0.05) d (0.1) d (0.16) d (0.25) d (0.5) d (0.75) Mean (micron) d (0.84) d (0.9) Measurement date and time Residual D [4, 3] - Volume d (0.05) d (0.1) d (0.16) d (0.25) d (0.5) d (0.75) d (0.84) d (0.9) Thursday, 13 October 2005 9:43:29 a.m. 0.451 162.277 51.8 60.8 69.4 80.9 112.9 158.0 186.0 217.6 Thursday, 13 October 2005 9:45:10 a.m. 0.229 146.828 38.6 46.8 54.8 65.8 98.1 148.5 182.7 224.7 Thursday, 13 October 2005 9:47:42 a.m. 0.221 142.948 36.4 43.9 51.2 61.2 91.2 139.8 174.8 221.2 Thursday, 13 October 2005 10:10:23 a.m. 0.32 125.826 33.9 42.6 50.7 61.6 92.6 140.6 173.0 211.3 Thursday, 13 October 2005 10:15:39 a.m. 0.316 147.593 35.1 45.1 54.8 68.0 106.5 164.9 202.7 245.1 Thursday, 13 October 2005 10:21:43 a.m. 0.183 158.314 34.1 44.2 54.2 68.3 111.1 179.7 225.5 278.0 Thursday, 13 October 2005 10:27:53 a.m. 0.568 76.275 45.9 50.7 54.9 60.2 73.4 89.3 97.8 105.7 Thursday, 13 October 2005 10:34:06 a.m. 0.276 145.691 34.7 44.9 54.8 68.2 106.7 163.7 199.6 238.6 Thursday, 13 October 2005 10:40:32 a.m. 0.307 142.734 32.4 42.2 52.1 66.0 108.6 175.2 217.3 262.3 Thursday, 13 October 2005 10:44:54 a.m. 0.604 81.595 44.0 49.3 54.3 60.7 77.3 98.1 109.3 120.0 Thursday, 13 October 2005 10:50:50 a.m. 0.209 144.941 32.0 41.6 51.5 65.5 108.6 176.2 219.1 264.9 Thursday, 13 October 2005 10:56:13 a.m. 0.263 144.161 32.0 41.7 51.7 65.7 108.5 174.8 216.6 261.4 Thursday, 13 October 2005 11:05:30 a.m. 0.608 74.462 44.7 49.3 53.5 58.7 71.6 87.3 95.6 103.5 Thursday, 13 October 2005 11:12:36 a.m. 0.26 127.261 31.4 41.0 50.7 64.3 105.5 167.6 205.4 244.0 Thursday, 13 October 2005 11:18:17 a.m. 0.2 154.681 33.9 44.0 54.0 68.1 110.7 178.2 222.6 272.6 Thursday, 13 October 2005 11:24:14 a.m. 0.628 73.282 43.8 48.4 52.5 57.6 70.4 86.0 94.2 102.0 Thursday, 13 October 2005 11:29:56 a.m. 0.289 139.676 34.2 43.9 53.4 66.2 103.0 157.6 191.8 228.7 Thursday, 13 October 2005 11:35:11 a.m. 0.256 147.25 32.8 42.8 52.8 66.8 109.4 175.2 217.0 262.3 Thursday, 13 October 2005 11:42:55 a.m. 0.616 69.248 39.1 43.6 47.7 52.9 66.0 82.3 91.1 99.3 Thursday, 13 October 2005 11:47:54 a.m. 0.332 156.272 39.4 49.0 58.0 70.0 104.3 156.2 191.0 233.8 Thursday, 13 October 2005 11:53:16 a.m. 0.205 153.314 34.4 44.6 54.7 68.7 110.5 176.0 219.2 268.1 Thursday, 13 October 2005 11:58:35 a.m. 0.712 71.194 40.2 44.8 49.0 54.4 67.9 84.7 93.6 102.1 Thursday, 13 October 2005 12:05:36 p.m. 0.481 143.149 34.0 44.1 53.9 67.3 106.4 165.3 202.8 243.7 Thursday, 13 October 2005 12:11:30 p.m. 0.291 158.706 34.3 44.6 54.8 69.0 111.9 180.4 226.0 278.2 1 Half Phi Particle size distribution (% per micron size interval) d (0.95) 0.02 3.9 7.8 15.6 31 44 62.5 88 125 177 250 350 500 710 1000 1410 to to to to to to to to to to to to to to to to 3.9 7.8 15.6 31 44 62.5 88 125 177 250 350 500 710 1000 1410 2000 d (0.95) Result Between User Sizes 291.5 0.0 0.0 0.0 0.1 1.9 9.1 19.6 27.4 23.4 11.7 2.2 0.0 0.7 1.6 1.5 0.7 332.4 0.0 0.1 0.3 1.5 6.3 14.1 20.6 22.7 17.3 9.2 3.2 0.9 0.8 1.2 1.2 0.6 399.7 0.0 0.1 0.3 2.0 7.7 16.1 21.5 21.6 15.1 7.5 2.6 1.1 1.2 1.4 1.2 0.6 285.4 0.0 0.0 0.7 3.1 7.2 14.8 20.9 22.0 16.1 8.5 3.3 1.3 0.7 0.6 0.5 0.2 323.1 0.0 0.0 0.4 3.0 5.9 11.8 17.5 21.1 18.7 12.1 5.3 1.5 0.5 0.8 0.9 0.5 376.8 0.0 0.0 0.4 3.4 6.1 11.4 16.1 19.2 17.8 12.9 6.9 2.7 0.8 0.8 0.9 0.6 116.1 0.0 0.0 0.0 0.0 3.5 25.7 44.1 24.2 2.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 305.4 0.0 0.0 0.4 3.2 5.9 11.6 17.3 21.3 19.1 12.4 5.2 1.2 0.2 0.7 0.9 0.5 332.0 0.0 0.0 0.4 4.0 6.7 11.7 16.0 19.0 17.8 13.2 7.1 2.7 0.5 0.3 0.4 0.3 133.8 0.0 0.0 0.0 0.1 5.0 22.6 36.5 28.0 7.7 0.1 0.0 0.0 0.0 0.0 0.0 0.0 336.6 0.0 0.0 0.4 4.2 6.8 11.7 15.8 18.7 17.6 13.2 7.2 2.7 0.5 0.3 0.5 0.4 332.0 0.0 0.0 0.4 4.2 6.7 11.6 15.9 18.9 17.8 13.1 7.0 2.6 0.5 0.3 0.5 0.3 113.8 0.0 0.0 0.0 0.0 4.4 27.9 43.6 22.0 2.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 298.5 0.0 0.0 0.4 4.4 7.0 12.0 16.3 19.4 18.1 13.1 6.8 2.3 0.2 0.0 0.0 0.0 361.5 0.0 0.0 0.4 3.4 6.2 11.4 16.1 19.3 17.9 13.0 6.9 2.7 0.7 0.6 0.8 0.5 112.3 0.0 0.0 0.0 0.0 5.1 29.3 43.1 20.7 1.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 290.3 0.0 0.0 0.4 3.3 6.3 12.3 18.0 21.5 18.7 11.7 4.7 1.0 0.1 0.6 0.8 0.5 335.2 0.0 0.0 0.4 3.9 6.5 11.5 16.0 19.2 18.0 13.2 6.9 2.5 0.5 0.4 0.7 0.4 110.2 0.0 0.0 0.0 0.5 10.1 32.9 37.8 17.2 1.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 369.4 0.0 0.0 0.5 1.7 5.0 12.1 19.4 23.3 19.0 10.4 3.4 0.8 0.9 1.4 1.4 0.7 356.7 0.0 0.0 0.4 3.3 6.0 11.3 16.3 19.7 18.2 12.9 6.6 2.5 0.8 0.7 0.8 0.5 113.2 0.0 0.0 0.0 0.3 8.7 31.1 38.6 19.2 2.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 312.2 0.0 0.0 0.4 3.4 6.1 11.8 17.2 20.7 18.6 12.4 5.6 1.7 0.3 0.5 0.7 0.4 375.6 0.0 0.0 0.4 3.3 6.0 11.2 16.0 19.3 18.0 13.1 7.0 2.8 0.8 0.8 0.9 0.6 Particle size analysis Sample Name RHL1 RHL1 RHL1 RHL1- Average RHL2 RHL2 RHL2 - Average RHL3 RHL3 RHL3- Average RHL4 RHL4 RHL4 RHL4 - Average RHL5 RHL5 RHL5 - Average RHL6 RHL6 RHL6 RHL6 - Average RHL7 RHL7 RHL7 RHL7 - Average RHL8 RHL8 RHL8 RHL8 - Average RHL9 RHL9 RHL9 RHL9 - Average RHL10 RHL10 RHL10 RHL10 - Average RHL11 RHL11 RHL11 RHL11- Average d (0.1) d (0.5) d (0.9) 42.083 43.379 44.488 43.218 44.218 43.167 43.686 43.906 42.434 43.261 45.135 51.429 47.697 49.464 49.932 47.644 48.797 40.875 40.916 41.741 41.156 49.973 50.094 47.961 49.314 40.444 40.84 43.701 41.504 48.153 44.401 47.666 46.754 43.583 43.379 42.433 43.129 48.916 41.584 44.285 44.962 78.37 85.138 91.469 84.44 81.893 82.413 82.145 72.409 79.151 75.389 74.789 84.684 80.166 82.428 77.461 78.013 77.721 76.267 79.295 84.379 79.722 83.577 85.884 81.95 83.783 75.34 79.628 89.113 80.781 85.72 89.681 94.323 89.641 90.961 91.887 88.411 90.383 80.252 78.199 83.603 80.619 135.436 162.663 185.228 159.501 140.939 148.077 144.369 117.378 141.155 129.199 120.769 136.627 130.965 133.873 119.277 124.873 122.091 131.921 144.529 165.964 146.37 137.225 141.605 135.849 138.295 129.253 146.966 177.374 149.821 159.908 212.084 195.396 187.716 194.457 196.296 179.04 189.626 128.356 140.581 149.976 139.209 Result Between User Sizes (µm) 4.365 5.012 5.754 6.607 7.586 8.71 10 11.482 13.183 15.136 17.378 19.953 22.909 26.303 30.2 34.674 39.811 45.709 52.481 0 0 0 0.014607 0.127533 0.180408 0.217893 0.226761 0.204098 0.163609 0.144805 0.216454 0.474392 1.018972 1.944431 3.284226 5.002072 6.953903 8.894653 0 0 0 0.011561 0.101491 0.149818 0.190252 0.212377 0.213254 0.201334 0.206339 0.280919 0.501689 0.951054 1.710406 2.815774 4.25052 5.912608 7.616814 0 0 0 0.00895 0.078911 0.12038 0.15971 0.1895 0.207974 0.220737 0.250615 0.337431 0.541787 0.929934 1.571088 2.501375 3.718743 5.154359 6.671004 0 0 0 0.011706 0.102645 0.150202 0.189285 0.209546 0.208442 0.195227 0.200587 0.278268 0.505956 0.966653 1.741975 2.867125 4.323778 6.006957 7.72749 0 0 0 0.01234 0.109597 0.175941 0.230854 0.259021 0.250576 0.208469 0.161262 0.169443 0.324936 0.735792 1.512467 2.71719 4.34671 6.2888 8.318274 0 0 0 0.012436 0.109538 0.165759 0.212034 0.235838 0.23196 0.207819 0.19452 0.250063 0.45953 0.91674 1.714567 2.896523 4.446169 6.250231 8.100039 0 0 0 0.012388 0.109567 0.17085 0.221444 0.247429 0.241268 0.208144 0.177891 0.209753 0.392233 0.826266 1.613517 2.806856 4.39644 6.269515 8.209156 0 0 0 0 0 0 0 0 0 0 0 0 0.056441 0.616393 1.741356 3.582522 6.040736 8.813109 11.39016 0 0 0.012438 0.071384 0.135505 0.185503 0.217958 0.216489 0.178689 0.121969 0.08849 0.151692 0.410435 0.965714 1.908313 3.263502 4.983789 6.914173 8.804178 0 0 0.004925 0.038543 0.065711 0.093236 0.109041 0.108123 0.089547 0.060294 0.046765 0.066981 0.2463 0.783542 1.826728 3.422597 5.512332 7.863638 10.09717 0 0 0 0 0 0 0 0 0 0 0 0 0.036489 0.478857 1.486808 3.154797 5.449169 8.13137 10.75811 0 0 0 0 0 0 0 0 0 0 0 0 0 0.022344 0.456269 1.528375 3.29837 5.703665 8.458393 0 0 0 0 0 0 0 0 0 0 0 0 0.040937 0.259504 1.093733 2.408518 4.375793 6.811663 9.389167 0 0 0 0 0 0 0 0 0 0 0 0 0.018272 0.145383 0.772224 1.969055 3.83698 6.257668 8.923788 0 0 0 0 0 0 0 0 0 0 0 0 0 0.007846 0.367561 1.612695 3.885378 7.088311 10.67238 0 0 0 0 0 0 0 0 0 0 0 0 0 0.077207 0.984958 2.42167 4.619229 7.321229 10.12305 0 0 0 0 0 0 0 0 0 0 0 0 0 0.042527 0.67626 2.017183 4.252303 7.20477 10.39771 0 0 0.012613 0.072097 0.1345 0.183344 0.216016 0.218176 0.19005 0.151466 0.147312 0.251644 0.563501 1.180548 2.189339 3.607768 5.380183 7.34242 9.235312 0 0 0 0.012512 0.109142 0.153364 0.18751 0.202951 0.201335 0.198159 0.231687 0.362061 0.672336 1.24407 2.152667 3.416935 4.996086 6.75751 8.485762 0 0 0 0.010539 0.092157 0.132058 0.166881 0.189998 0.203321 0.219099 0.268987 0.401896 0.687138 1.19312 1.985596 3.084784 4.463864 6.020551 7.582548 0 0 0.000348 0.036581 0.110616 0.156564 0.190173 0.203652 0.198263 0.189563 0.215999 0.338532 0.640992 1.205913 2.109201 3.369829 4.946711 6.706827 8.434541 0 0 0 0 0 0 0 0 0 0 0 0 0 0.055248 0.731211 1.878428 3.707928 6.074002 8.688452 0 0 0 0 0 0 0 0 0 0 0 0 0.034636 0.209361 0.85332 1.901129 3.525309 5.637488 8.027988 0 0 0 0 0 0 0 0 0 0 0 0 0.037642 0.390407 1.10018 2.3496 4.159632 6.428035 8.873804 0 0 0 0 0 0 0 0 0 0 0 0 0.031603 0.206329 0.900426 2.041842 3.797826 6.046501 8.530067 0 0 0 0.013171 0.11572 0.171891 0.21465 0.231998 0.221874 0.198883 0.205445 0.313307 0.622778 1.235918 2.245995 3.677309 5.476861 7.476145 9.403722 0 0 0 0.010724 0.094201 0.139707 0.177918 0.200736 0.208832 0.215428 0.25662 0.39016 0.697707 1.260556 2.154852 3.400966 4.959524 6.699004 8.404846 0 0 0 0.005974 0.066706 0.106168 0.149239 0.184875 0.210039 0.231928 0.272223 0.371628 0.592292 1.002655 1.674622 2.645258 3.91075 5.395877 6.952475 0 0 0 0.010367 0.091423 0.139632 0.180691 0.205732 0.21365 0.215386 0.244772 0.358362 0.637593 1.166376 2.025156 3.241178 4.782378 6.523675 8.253681 0 0 0 0 0 0 0 0 0 0 0 0 0.02225 0.219574 1.141965 2.395037 4.139836 6.170674 8.240159 0 0 0.018574 0.101752 0.153266 0.185279 0.193412 0.167816 0.118801 0.018996 0 0.071846 0.371909 0.87266 1.693541 2.825719 4.220622 5.750506 7.228792 0 0 0.009382 0.072944 0.123589 0.17446 0.205395 0.207695 0.177077 0.122137 0.070094 0.072446 0.201718 0.539257 1.16546 2.122805 3.407784 4.941727 6.57054 0 0 0.00853 0.05985 0.091318 0.120051 0.132951 0.12515 0.098615 0.05017 0.015139 0.054221 0.189306 0.559667 1.324419 2.449878 3.922407 5.620981 7.346522 0 0 0.014681 0.080173 0.118994 0.1464 0.160431 0.15624 0.139437 0.128616 0.15779 0.2762 0.547301 1.028793 1.771546 2.78289 4.030848 5.423397 6.81734 0 0 0.011729 0.076194 0.106229 0.135065 0.151353 0.154052 0.149097 0.153311 0.198437 0.329059 0.603679 1.075122 1.791298 2.760378 3.955443 5.294163 6.645943 0 0 0.011613 0.076863 0.109828 0.142499 0.162795 0.169415 0.168127 0.175785 0.225315 0.363408 0.652098 1.148004 1.902775 2.925667 4.187573 5.598977 7.017459 0 0 0.01208 0.078963 0.110955 0.141425 0.158205 0.159883 0.15223 0.152567 0.193849 0.322889 0.601026 1.083973 1.821873 2.822978 4.057955 5.438845 6.826914 0 0 0 0 0 0 0 0 0 0 0 0 0 0.060967 0.808816 2.082075 4.108604 6.707566 9.524362 0 0 0.017884 0.099871 0.166592 0.212637 0.23562 0.222691 0.176145 0.118841 0.096744 0.184205 0.478266 1.073883 2.053697 3.432803 5.154932 7.059374 8.895524 0 0 0.01277 0.07361 0.142151 0.19501 0.231842 0.236469 0.20515 0.15032 0.105251 0.132159 0.318284 0.759746 1.550348 2.73427 4.295634 6.121796 8.004601 0 0 0.008344 0.061883 0.100126 0.136489 0.155892 0.152953 0.127101 0.089922 0.066505 0.108396 0.256003 0.645893 1.462195 2.751636 4.519401 6.62959 8.808188 2 Result Between User Sample Name Sizes 60.256 RHL1 10.53058 RHL1 9.133813 RHL1 8.091239 RHL1- Average 9.25188 RHL2 10.14159 RHL2 9.73496 RHL2 - Average 9.938278 RHL3 13.20743 RHL3 10.36462 RHL3- Average 11.78602 RHL4 12.79389 RHL4 11.09952 RHL4 11.64841 RHL4 - Average 11.37396 RHL5 13.77195 RHL5 12.47607 RHL5 - Average 13.12401 RHL6 10.7625 RHL6 9.929574 RHL6 8.944632 RHL6 - Average 9.878902 RHL7 11.11184 RHL7 10.34925 RHL7 11.09451 RHL7 - Average 10.85188 RHL8 10.94616 RHL8 9.827213 RHL8 8.38986 RHL8 - Average 9.721077 RHL9 10.03627 RHL9 8.454845 RHL9 8.097342 RHL9 - Average 8.862803 RHL10 8.051127 RHL10 7.861299 RHL10 8.279362 RHL10 - Average 8.063929 RHL11 12.03252 RHL11 10.38011 RHL11 9.68336 RHL11- Average 10.69864 (µm) 69.183 11.54886 10.20531 9.200752 10.3183 11.41918 10.86225 11.1407 13.78713 11.30097 12.5441 13.72599 13.0418 13.0786 13.0602 15.4434 13.80844 14.62592 11.62534 10.82578 9.88174 10.77762 12.81777 12.14134 12.61143 12.52351 11.78903 10.70658 9.481597 10.65907 11.22765 9.234324 9.288479 9.916824 8.950265 8.773829 9.203878 8.975991 13.63265 11.23136 10.86289 11.90902 79.433 11.73951 10.64575 9.834126 10.73982 11.89775 11.27141 11.58465 12.95352 11.42999 12.19154 13.28981 13.82312 13.33389 13.5785 15.14003 13.77398 14.457 11.63968 11.00203 10.2376 10.95978 13.40944 13.01355 13.06499 13.16266 11.74139 10.87632 10.0558 10.89116 11.5968 9.45976 9.968154 10.34157 9.397948 9.267364 9.665034 9.443448 13.91332 11.28106 11.32938 12.1744 91.201 11.02937 10.35864 9.876508 10.42141 11.44452 10.85889 11.15144 10.90201 10.70881 10.80618 11.55355 13.20794 12.31746 12.7627 12.94125 12.34865 12.64495 10.77003 10.40148 9.936545 10.36932 12.70985 12.71508 12.30123 12.57539 10.77654 10.28656 10.00176 10.35498 11.0674 9.114785 10.02096 10.06772 9.330152 9.268766 9.588157 9.395692 12.76072 10.50386 10.97296 11.41319 104.713 9.512421 9.378757 9.318268 9.403456 10.10788 9.672651 9.891166 8.102835 9.247477 8.672611 8.89812 11.31317 10.22769 10.77043 9.510382 9.84841 9.679396 9.140578 9.102389 9.015453 9.086245 10.84179 11.25748 10.44096 10.84674 9.039805 9.020692 9.321303 9.127179 9.736444 8.280805 9.448706 9.155318 8.765758 8.782921 8.983654 8.844111 10.43521 9.020667 9.835012 9.761388 120.226 7.471115 7.892498 8.263237 7.874652 8.141162 7.937528 8.036511 5.183458 7.331417 6.265427 5.906204 8.591 7.549071 8.070035 5.857958 6.858937 6.358447 7.055513 7.347812 7.64315 7.348495 8.275888 8.970762 7.93484 8.39383 6.863135 7.320634 8.140834 7.441808 7.88773 7.137482 8.373306 7.799506 7.810223 7.896597 7.955624 7.88748 7.47583 7.121436 8.132587 7.583652 138.038 5.227259 6.111059 6.854192 6.066901 5.862895 5.909674 5.894305 2.767814 5.241249 3.981918 3.286479 5.652334 4.805093 5.228714 2.892624 4.03769 3.465157 4.837284 5.382748 6.004206 5.409012 5.457033 6.260065 5.206603 5.641234 4.599597 5.42389 6.621926 5.547696 5.808682 5.848428 6.954598 6.203903 6.581917 6.716131 6.626277 6.641444 4.615931 5.07853 6.11184 5.24886 158.489 3.025677 4.344325 5.330785 4.22687 3.74077 3.938493 3.819878 0.855089 3.319112 2.13319 1.050357 3.03421 2.314867 2.674538 0.808247 1.300492 1.05437 2.592375 3.54124 4.372485 3.499735 3.093675 3.752992 2.930213 3.25896 2.244248 3.650867 5.024235 3.641694 3.869332 4.617973 5.442449 4.643251 5.27012 5.423688 5.200889 5.298226 1.76986 3.223931 4.128175 3.082184 181.97 0.846392 2.761244 3.840213 2.493624 1.422589 2.243697 1.865015 0 1.760292 0.847229 0 0.76949 0.345621 0.557556 0 0 0 0.500409 1.988299 2.894852 1.798296 1.14744 1.350252 1.075923 1.191205 0.174432 2.147012 3.501299 1.937842 2.245821 3.528318 3.984083 3.25274 3.978553 4.125909 3.797511 3.967336 0.071571 1.723556 2.380888 1.363333 208.93 0 1.54618 2.571561 1.366633 0 0.981816 0.473464 0 0.664133 0.335361 0 0 0 0 0 0 0 0 0.884124 1.739368 0.872464 0 0 0 0 0 1.067461 2.241857 1.104793 1.100877 2.666298 2.757793 2.17499 2.852118 2.975903 2.581472 2.803159 0 0.681884 1.073912 0.587336 239.883 0 0.71002 1.571106 0.759111 0 0.176328 0.087893 0 0.001706 0.000957 0 0 0 0 0 0 0 0 0.208008 0.91856 0.374501 0 0 0 0 0 0.392453 1.283641 0.556715 0.402348 2.005309 1.795494 1.40105 1.921576 2.013786 1.592564 1.842564 0 0.071608 0.218157 0.093198 3 275.423 0 0.247455 0.887251 0.379639 0 0.008535 0.003987 0 0 0 0 0 0 0 0 0 0 0 0.00243 0.4351 0.14752 0 0 0 0 0 0.008538 0.662112 0.227903 0.092347 1.527706 1.129657 0.916569 1.233473 1.294944 0.880395 1.136858 0 0.00228 0.00143 0.004288 316.228 0 0.067121 0.473797 0.179645 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.209699 0.068788 0 0 0 0 0 0 0.319506 0.103239 0.043042 1.172584 0.706608 0.640748 0.758449 0.793575 0.423946 0.655876 0 0 0 0 363.078 0 0.071972 0.276518 0.11638 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.157858 0.0531 0 0 0 0 0 0 0.188926 0.064212 0.133722 0.902427 0.474335 0.503478 0.468353 0.483423 0.151591 0.37259 0 0 0 0 416.869 0 0.161921 0.215671 0.125947 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.189973 0.062988 0 0 0 0 0 0 0.183527 0.060894 0.264906 0.682305 0.361738 0.436379 0.307575 0.305639 0.031445 0.214027 0 0 0 0 478.63 0 0.255315 0.215194 0.156261 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.22818 0.076762 0 0 0 0 0 0 0.22099 0.073248 0.368606 0.499874 0.310685 0.392837 0.221555 0.213004 0 0.14344 0 0 0 0 549.541 0 0.295035 0.222543 0.174459 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.238061 0.077331 0 0 0 0 0 0 0.248592 0.083221 0.414966 0.348557 0.271572 0.345733 0.171334 0.144805 0 0.103555 0 0 0 0 630.957 0 0.274779 0.188479 0.149918 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.170027 0.060554 0 0 0 0 0 0 0.21673 0.401821 0.22625 0.284909 0.067866 0 0.063738 0 0 0 0 724.436 831.764 0 0 0.159591 0.053008 0.10606 0 0.097926 0.011323 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.079969 0 0.024282 0 0 0 0 0 0 0 0 0 0 0 0 0 0.122798 0 0 0 0 0.140821 0 0 0 0 0 0 0 0.009926 0 0 0 0 0 0 0 0 0 954.993 1096.478 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   Appendix VIII Image Size Analysis Data VIII    Image analysis bubble diameter 500 mg/l Rhamnolipid at pH 7 4000 mg/l Rhamnolipid at pH 7 1000 mg/l Rhamnolipid at pH 7 Continue Continue Area (pixel2) Diameter (um) Area (pixel2) Diameter (um) Area (pixel2) Diameter (um) Area(pixel2) Diameter (um) Area(pixel2) Diameter (um) 18 23 150 67 21 25 16 22 147 66 18 23 150 67 23 26 16 22 155 68 20 25 151 67 26 28 16 22 155 68 21 25 154 68 27 28 17 23 157 69 26 28 157 69 29 30 17 23 159 69 27 28 159 69 30 30 18 23 163 70 27 28 159 69 34 32 18 23 164 70 30 30 161 70 34 32 19 24 164 70 30 30 163 70 37 33 20 25 164 70 30 30 168 71 38 34 20 25 164 70 31 31 168 71 49 38 21 25 164 70 31 31 170 72 52 40 21 25 167 71 32 31 171 72 56 41 22 26 168 71 33 32 173 72 58 42 22 26 170 72 33 32 174 72 62 43 22 26 176 73 33 32 177 73 68 45 23 26 177 73 33 32 178 73 70 46 24 27 181 74 33 32 180 74 75 47 24 27 184 74 34 32 181 74 76 48 24 27 188 75 34 32 181 74 77 48 24 27 191 76 36 33 182 74 78 48 25 27 191 76 37 33 187 75 84 50 25 27 193 76 37 33 190 76 90 52 26 28 193 76 39 34 191 76 90 52 26 28 195 77 40 35 191 76 92 53 27 28 200 78 40 35 194 76 93 53 27 28 202 78 40 35 195 77 97 54 27 28 203 78 40 35 197 77 98 54 27 28 203 78 42 36 198 77 108 57 27 28 203 78 44 36 199 77 109 57 27 28 204 78 45 37 199 77 114 59 29 30 208 79 45 37 199 77 116 59 29 30 209 79 47 38 199 77 122 61 30 30 211 80 47 38 200 78 129 62 32 31 213 80 47 38 202 78 133 63 32 31 215 80 51 39 203 78 147 66 33 32 221 82 53 40 204 78 164 70 34 32 221 82 53 40 208 79 166 71 37 33 223 82 53 40 209 79 168 71 37 33 223 82 53 40 211 80 169 71 37 33 226 82 54 40 213 80 177 73 38 34 226 82 54 40 213 80 179 73 38 34 227 83 56 41 214 80 179 73 39 34 229 83 56 41 217 81 179 73 41 35 231 83 57 41 218 81 179 73 41 35 232 84 57 41 219 81 181 74 43 36 235 84 58 42 219 81 183 74 49 38 236 84 59 42 220 81 189 75 49 38 237 84 61 43 220 81 205 79 49 38 239 85 62 43 221 82 206 79 50 39 241 85 62 43 222 82 207 79 50 39 241 85 62 43 223 82 210 79 50 39 242 85 63 44 224 82 218 81 53 40 245 86 64 44 225 82 219 81 54 40 249 87 64 44 232 84 224 82 54 40 252 87 66 45 232 84 225 82 54 40 255 88 69 46 232 84 229 83 54 40 255 88 71 46 238 85 231 83 55 41 256 88 74 47 239 85 232 84 56 41 256 88 75 47 239 85 234 84 57 41 260 88 75 47 242 85 242 85 59 42 261 89 77 48 243 85 250 87 59 42 263 89 77 48 244 86 258 88 60 42 265 89 77 48 246 86 259 88 60 42 267 90 78 48 249 87 261 89 60 42 267 90 79 49 253 87 270 90 61 43 269 90 80 49 256 88 272 90 62 43 273 91 80 49 256 88 272 90 62 43 274 91 82 50 257 88 278 91 63 44 274 91 83 50 258 88 283 92 64 44 281 92 1 Area (pixel2) 84 84 85 85 86 87 87 87 88 90 90 91 92 92 93 94 95 95 95 98 98 99 100 101 103 105 107 109 109 110 112 115 116 116 117 118 118 119 119 121 122 124 124 126 128 129 132 132 133 133 134 134 137 137 137 138 139 141 141 142 143 145 145 146 146 147 147 500 mg/l Rhamnolipid at pH 7 4000 mg/l Rhamnolipid at pH 7 1000 mg/l Rhamnolipid at pH 7 Continue Continue Diameter (um) Area (pixel2) Diameter (um) Area (pixel2) Diameter (um) Area(pixel2) Diameter (um) Area(pixel2) Diameter (um) 50 262 89 285 93 65 44 284 92 50 263 89 286 93 66 45 287 93 51 268 90 287 93 66 45 288 93 51 269 90 292 94 66 45 290 93 51 278 91 298 95 67 45 291 94 51 279 92 302 95 68 45 297 95 51 281 92 305 96 69 46 297 95 51 281 92 327 99 70 46 298 95 51 297 95 327 99 70 46 300 95 52 301 95 328 99 72 47 305 96 52 302 95 340 101 73 47 305 96 52 307 96 342 101 73 47 307 96 53 309 96 347 102 74 47 308 96 53 309 96 348 102 74 47 314 97 53 331 100 354 103 75 47 316 97 53 331 100 360 104 77 48 317 98 53 334 100 361 104 77 48 327 99 53 336 101 372 106 78 48 331 100 53 345 102 373 106 79 49 332 100 54 348 102 376 106 81 49 334 100 54 351 103 381 107 81 49 337 101 55 351 103 384 107 82 50 344 102 55 352 103 391 108 82 50 351 103 55 353 103 391 108 83 50 353 103 56 355 103 397 109 85 51 356 103 56 358 104 428 113 87 51 356 103 57 362 104 430 114 88 51 356 103 57 372 106 430 114 91 52 358 104 57 377 106 434 114 92 53 361 104 58 377 106 436 115 96 54 370 105 58 380 107 437 115 96 54 370 105 59 387 108 469 119 96 54 370 105 59 391 108 476 120 97 54 379 107 59 396 109 476 120 99 55 384 107 59 414 112 504 123 99 55 388 108 60 417 112 506 123 100 55 389 108 60 424 113 509 124 100 55 391 108 60 430 114 515 124 102 55 398 109 60 432 114 518 125 104 56 400 110 60 445 116 520 125 104 56 401 110 61 452 117 525 126 105 56 401 110 61 474 119 528 126 107 57 407 111 61 476 120 530 126 108 57 407 111 62 574 131 535 127 110 58 413 111 62 579 132 547 128 112 58 416 112 62 633 138 561 130 113 58 425 113 63 653 140 564 130 114 59 441 115 63 582 132 117 59 442 115 63 602 135 118 60 449 116 63 603 135 118 60 460 118 63 639 139 118 60 468 119 63 664 141 122 61 480 120 64 701 145 122 61 484 121 64 721 147 125 61 485 121 64 732 148 125 61 489 121 64 791 154 126 62 497 122 65 860 161 129 62 506 123 65 873 162 129 62 511 124 65 1023 175 133 63 519 125 65 1035 176 134 63 533 127 66 1103 182 137 64 569 131 66 1255 194 138 64 575 131 66 140 65 578 132 66 141 65 589 133 66 142 65 625 137 66 145 66 707 146 66 146 66 707 146 768 152 2 Hexagon size analysis Hexagon 1 2 3 4 5 6 7 8 Radius 1 Radius 2 Mean 454.42 449.86 452.14 508.96 580.69 544.825 684.58 706.84 695.71 531.59 515.98 523.785 504.51 591.4 547.955 397.04 258.94 327.99 89.3 106.64 97.97 93.43 77.87 85.65 Mean 409.5031 10ile 94.274 90ile 592.2815 3   Appendix IX MATH Assay Data IX    MATH Hydrophobicity Assay R.erythropolis Hydrophobicity Assay Initial O.D.   Rhamnolipid Concentration (mg/l) 1 1.002 2 0.921 3 Average 0.86 0.928 1 0.046 0.046 0.113 0.599 1.013 95.04% 95.04% 87.82% 35.43% 0.00% 1 0.923 2 0.904 3 Average 0.914 0.914 7.37% 0.00% 2 0.842 1.148 O.D.  0 50 500 1000 2000 2 0.044 0.052 0.136 0.487 1.182 95.26% 94.39% 85.34% 47.50% 0.00% 3 0.045 0.052 0.122 0.746 1.103 95.15% 94.39% 86.85% 19.58% 0.00% Average Std 95.15% 0.1% 94.61% 0.4% 86.67% 1.2% 34.17% 14.0% 0.00% 0.0% P.Putida 852 Hydrophobicity Initial O.D.   Rhamnolipid Concentration (mg/l) O.D.  0 50 1 0.846 1.546 7.84% 0.00% 1 3 0.866 1.149 Average Std 5.23% 6.81% 0.00% 0.00% 1.4% 0.0%   Appendix X Contact Angle Measurement Data X    Testing Liquid Treatment SALINE Background Rhamnolipid 500 mg/L Background Rhamnolipid 1000 mg/L Background Rhamnolipid 4000 mg/L Background WATER Time [s] Sample CA[L] CA[R] CA[M] Tilt 18.636 17.688 18.162 11.412 10.506 10.959 8.539 8.938 8.739 0 1.022 1.993 2 0 1.021 2.003 19.004 12.189 9.339 18.226 11.48 8.857 18.615 11.834 9.098 0 0 0 2.659 2.632 2.24 0.201 0.128 0.088 0.581 0.362 0.201 5.706 5.556 4.291 3 0 1.021 2.002 19.304 12.497 9.736 19.298 12.544 9.43 19.301 12.521 9.583 0 0 0 2.633 2.592 2.351 0.226 0.144 0.095 0.619 0.379 0.206 5.604 5.317 4.34 4 0 1.021 20.153 15.388 19.873 15.012 20.013 15.2 0 0 2.149 2.121 0.18 0.137 0.332 0.247 3.719 3.608 1 0 1.042 2.013 17.238 12.259 10.33 15.748 11.177 9.387 16.493 11.718 9.859 0 0 0 2.574 2.512 2.302 0.17 0.116 0.091 0.475 0.308 0.2 5.413 5.095 4.247 2 0 1.021 1.993 18.436 12.919 10.177 17.297 12.184 9.881 17.866 12.552 10.029 0 0 0 2.645 2.594 2.451 0.186 0.128 0.097 0.539 0.354 0.232 5.656 5.371 4.713 3 0 1.032 2.043 18.119 12.588 10.107 17.33 11.624 8.868 17.724 12.106 9.487 0 0 0 2.645 2.558 2.397 0.181 0.119 0.091 0.517 0.318 0.225 5.581 5.186 4.712 4 0 1.031 1.993 23.625 21.526 20.137 22.79 20.883 19.55 23.208 21.204 19.843 0 0 0 2.468 2.462 2.448 0.226 0.204 0.189 0.567 0.506 0.466 4.957 4.884 4.829 1 0 1.031 2.002 18.061 11.643 10.759 18.614 12.135 10.204 18.337 11.889 10.481 0 0 0 2.312 2.232 1.959 0.169 0.107 0.085 0.369 0.213 0.132 4.293 3.928 3.056 2 0 1.022 2.013 18.202 13.602 10.336 17.559 13.173 10.169 17.881 13.388 10.253 0 0 0 2.422 2.372 2.229 0.172 0.128 0.092 0.416 0.288 0.184 4.726 4.449 3.93 3 0 1.032 2.003 18.226 11.585 10.466 18.279 11.204 10.19 18.253 11.394 10.328 0 0 0 2.323 2.226 1.927 0.17 0.103 0.084 0.372 0.206 0.121 4.317 3.926 2.92 4 0 1.041 2.023 21.919 15.551 10.982 20.855 14.578 10.389 21.387 15.064 10.685 0 0 0 2.342 2.311 2.189 0.199 0.138 0.096 0.451 0.303 0.186 4.457 4.28 3.818 1 0 1.031 2.013 19.938 14.241 10.72 19.574 13.602 10.101 19.756 13.922 10.411 0 0 0 2.505 2.464 2.4 0.197 0.136 0.101 0.507 0.336 0.23 5.057 4.83 4.527 2 0 1.022 2.023 20.701 13.248 9.647 20.719 13.598 9.57 20.71 13.423 9.609 0 0 0 2.488 2.399 2.147 0.205 0.124 0.085 0.514 0.294 0.157 4.969 4.573 3.642 3 0 1.032 2.013 19.389 12.336 11.051 19.756 11.907 9.667 19.573 12.122 10.359 0 0 0 2.396 2.362 2.104 0.184 0.111 0.09 0.428 0.253 0.19 4.588 4.423 3.853 4 0 1.031 2.013 23.702 14.803 10.864 21.552 13.515 10.049 22.627 14.159 10.456 0 0 0 2.45 2.41 2.164 0.227 0.141 0.101 0.589 0.355 0.264 5.047 4.823 4.381 1 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 2.672 0.196 0.568 5.733 2.574 0.116 0.316 5.344 2.152 0.081 0.16 3.811 1 Testing Liquid Treatment Tergitol 500 mg/L Background Tergitol 1000 mg/L Background Tergitol 4000 mg/L Background WATER Time [s] Sample CA[L] CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] 1 0 1.011 2.013 22.09 17.152 12.89 22.018 17.143 12.85 22.054 17.148 12.87 0 0 0 2.538 2.506 2.442 0.228 0.173 0.129 0.595 0.441 0.308 5.214 5.011 4.736 2 0 1.021 2.033 16.303 10.776 9.954 15.837 10.639 9.915 16.07 10.708 9.934 0 0 0 2.511 2.328 1.859 0.16 0.102 0.077 0.413 0.22 0.107 5.04 4.264 2.733 3 0 1.011 2.033 20.681 12.517 10.404 20.433 12.336 10.256 20.557 12.427 10.33 0 0 0 2.512 2.429 2.113 0.208 0.123 0.091 0.533 0.289 0.163 5.085 4.658 3.533 4 0 1.012 2.033 20.554 13.366 10.046 20.048 13.226 9.776 20.301 13.296 9.911 0 0 0 2.176 2.133 1.819 0.185 0.12 0.081 0.355 0.216 0.108 3.842 3.602 2.661 1 0 1.021 2.003 17.026 11.219 9.729 16.12 10.441 9.451 16.573 10.83 9.59 0 0 0 2.732 2.674 2.308 0.18 0.119 0.088 0.558 0.353 0.186 6.021 5.768 4.15 2 0 0.951 1.953 17.26 12.186 10.099 17.191 11.818 9.197 17.225 12.002 9.648 0 0 0 2.754 2.688 2.502 0.192 0.128 0.096 0.591 0.371 0.249 6.077 5.696 5.023 3 0 1.012 2.033 17.257 11.184 9.663 16.772 10.577 9.365 17.014 10.881 9.514 0 0 0 2.698 2.607 2.265 0.18 0.111 0.089 0.537 0.309 0.18 5.827 5.378 4.027 4 0 1.041 2.033 24.641 15.453 10.072 24.507 15.419 10.173 24.574 15.436 10.122 -0.204 -0.204 -0.204 2.143 2.08 1.772 0.217 0.133 0.078 0.408 0.234 0.097 3.767 3.48 2.492 1 0 1.001 2.032 16.751 11.149 10.176 16.193 10.782 9.809 16.472 10.965 9.992 0 0 0 2.772 2.342 2.057 0.175 0.102 0.086 0.545 0.227 0.144 6.083 4.342 3.346 2 0 0.921 1.953 19.549 15.485 11.679 19.624 15.54 11.565 19.587 15.513 11.622 0 0 0 2.241 2.211 2.159 0.18 0.141 0.105 0.364 0.276 0.195 4.037 3.889 3.695 3 0 0.921 1.953 18.395 14.735 11.054 17.869 14.264 10.669 18.132 14.499 10.862 0 0 0 2.276 2.241 2.17 0.168 0.132 0.096 0.352 0.267 0.182 4.161 4.001 3.73 4 0 1.021 2.013 19.293 15.262 13.217 18.205 14.279 11.06 18.749 14.77 12.138 0 0 0 2.289 2.272 2.185 0.178 0.14 0.102 0.386 0.307 0.213 4.278 4.245 3.932 2 Testing Liquid Treatment SALINE Background Rhamnolipid 500 mg/L Background Rhamnolipid 1000 mg/L Background Rhamnolipid 4000 mg/L Background Sample 1-Bromonaphthalene Time [s] CA[L] CA[R] CA[M] Tilt 1 0 12.598 11.762 12.18 1.022 9.732 10.191 9.962 2.003 9.535 9.557 9.546 0 0 0 2 0 1 2 18.515 18.962 18.739 0 2.042 0.114 0.211 3.303 3 0 1.022 1.993 11.728 7.221 7.613 10.209 7.003 7.109 10.969 7.112 7.361 0 0 0 3.05 2.658 2.496 0.123 0.093 0.076 0.498 0.243 0.191 7.534 5.536 4.955 4 0 1.031 2.013 21.203 13.199 10.836 18.981 11.641 10.524 20.092 12.42 10.68 0 0 0 1.946 1.879 1.694 0.126 0.086 0.073 0.209 0.129 0.083 3.037 2.838 2.257 1 0 1.021 2.013 17.294 10.442 7.78 16.606 9.875 7.802 16.95 10.158 7.791 -0.306 -0.306 -0.306 2.032 1.999 1.829 0.14 0.084 0.064 0.242 0.145 0.084 3.365 3.282 2.651 2 0 1.031 2.013 17.315 5.568 9.345 16.586 9.128 8.086 16.95 7.348 8.715 0 0 0 2.088 2.296 1.807 0.14 0.086 0.064 0.245 0.192 0.09 3.473 4.18 2.705 3 0 1.012 2.033 19.357 12.758 6.657 18.153 11.786 8.834 18.755 12.272 7.745 0 0 0 1.967 1.911 1.919 0.148 0.095 0.076 0.236 0.145 0.108 3.135 2.963 2.907 4 0 1.031 2.033 17.861 14.135 11.154 18.255 14.05 11.661 18.058 14.092 11.408 -1.226 -1.226 -1.226 2.113 2.069 1.824 0.153 0.116 0.098 0.287 0.217 0.142 3.629 3.513 2.802 1 0 1.031 2.012 16.622 11.349 8.782 16.037 11.092 8.275 16.33 11.22 8.529 -0.306 -0.306 -0.306 2.001 1.9 1.688 0.131 0.087 0.066 0.218 0.13 0.074 3.236 2.897 2.246 2 0 1.021 2.032 17.151 11.639 9.731 17.451 11.265 9.594 17.301 11.452 9.663 0 0 0 2.03 1.953 1.718 0.146 0.092 0.068 0.242 0.138 0.079 3.305 3.004 2.317 3 0 1.031 2.013 24.475 18.156 15.867 22.852 17.26 15.548 23.664 17.708 15.707 -0.102 -0.102 -0.102 1.785 1.728 1.602 0.176 0.129 0.107 0.243 0.162 0.112 2.693 2.464 2.072 4 0 1.031 2.023 13.74 9.919 8.426 14.041 10.692 9.292 13.891 10.305 8.859 -0.409 -0.409 -0.409 1.884 1.811 1.721 0.106 0.079 0.065 0.153 0.109 0.084 2.833 2.679 2.473 1 0 1.022 2.003 18.261 14.092 13.604 17.599 14.531 14.347 17.93 14.311 13.976 -0.306 -0.306 -0.306 1.803 1.648 1.438 0.135 0.107 0.087 0.183 0.12 0.098 2.662 2.225 1.943 2 0 0.991 2.013 20.151 15.536 13.831 19.747 15.286 13.758 19.949 15.411 13.795 0 0 0 1.848 1.685 1.504 0.149 0.107 0.087 0.207 0.122 0.078 2.755 2.267 1.792 3 0 1.022 2.003 20.448 16.044 15.431 20.071 15.695 14.593 20.259 15.87 15.012 0 0 0 1.864 1.741 1.57 0.154 0.116 0.097 0.216 0.142 0.099 2.797 2.434 1.993 4 0 1.032 2.013 20.933 15.792 14.13 20.961 15.826 13.853 20.947 15.809 13.992 0 0 0 1.929 1.786 1.589 0.166 0.117 0.093 0.249 0.148 0.093 3.004 2.538 2.001 3 L[mm] H[mm] Vol[ml] A[mm2] 2.372 0.097 0.235 4.496 1.954 0.074 0.116 3.017 1.733 0.071 0.083 2.359 Testing Liquid Treatment Tergitol 500 mg/L Background Tergitol 1000 mg/L Background Tergitol 4000 mg/L Background 1-Bromonaphthalene Time [s] CA[L] CA[R] Sample CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] 1 0 1.031 2.003 16.13 12.133 11.735 15.553 10.916 10.16 15.842 11.524 10.948 0 0 0 1.983 1.79 1.502 0.124 0.083 0.065 0.199 0.113 0.063 3.141 2.618 1.847 2 0 0.992 1.993 15.365 12.136 9.872 15.36 11.574 9.657 15.362 11.855 9.765 -0.306 -0.306 -0.306 1.99 1.801 1.529 0.121 0.084 0.064 0.198 0.115 0.063 3.184 2.623 1.913 3 0 1.031 2.013 20.045 10.267 8.707 20.433 10.153 8.153 20.239 10.21 8.43 -0.511 -0.511 -0.511 1.9 1.736 1.425 0.156 0.075 0.054 0.231 0.095 0.053 2.925 2.449 1.765 4 0 1.021 2.023 14.589 10.491 9.07 14.542 10.319 9.051 14.566 10.405 9.06 -0.306 -0.306 -0.306 1.973 1.778 1.49 0.116 0.078 0.06 0.186 0.101 0.052 3.121 2.533 1.758 1 0 1.032 2.003 20.173 9.846 7.934 19.851 9.691 8.044 20.012 9.769 7.989 0 0 0 2.002 1.921 1.678 0.161 0.077 0.058 0.262 0.111 0.065 3.223 2.879 2.238 2 0 1.032 2.003 19.431 10.223 8.542 19.616 10.457 8.548 19.524 10.34 8.545 -0.511 -0.511 -0.511 2.026 1.945 1.703 0.158 0.082 0.061 0.268 0.127 0.074 3.322 3.016 2.335 3 0 1.032 2.013 12.765 9.1 7.661 12.538 8.872 7.463 12.652 8.986 7.562 -0.306 -0.306 -0.306 2.092 1.92 1.666 0.107 0.074 0.058 0.193 0.117 0.063 3.498 3.032 2.205 4 0 1.031 2.013 12.431 9.292 6.101 11.904 8.465 6.087 12.167 8.878 6.094 -0.306 -0.306 -0.306 2.021 1.826 1.6 0.101 0.068 0.05 0.173 0.113 0.049 3.316 2.953 2.043 1 0 1.041 2.013 18.927 9.949 8.297 18.755 9.83 7.943 18.841 9.889 8.12 -0.306 -0.306 -0.306 2.071 2.002 1.771 0.157 0.084 0.066 0.277 0.138 0.082 3.468 3.22 2.5 2 0 1.011 2.032 19.191 10.629 9.621 19.065 10.638 9.296 19.128 10.634 9.459 -0.306 -0.306 -0.306 2.042 1.961 1.701 0.158 0.087 0.068 0.271 0.136 0.082 3.378 3.076 2.358 3 0 1.031 2.003 13.642 10.024 8.274 13.108 9.491 7.801 13.375 9.757 8.037 0 0 0 2.084 1.902 1.632 0.112 0.078 0.06 0.195 0.115 0.066 3.434 2.897 2.159 4 0 1.021 2.003 14.507 11.493 9.822 12.145 10.269 8.846 13.326 10.881 9.334 0 0 0 2.09 1.872 1.639 0.109 0.081 0.065 0.214 0.12 0.084 3.673 2.849 2.364 4 Testing Liquid Treatment SALINE Background Rhamnolipid 500 mg/L Background Rhamnolipid 1000 mg/L Background Rhamnolipid 4000 mg/L Background Glycerol Time [s] Sample CA[L] CA[R] CA[M] Tilt 135.186 133.814 134.5 130.628 130.049 130.339 127.9 127.202 127.551 0 1.032 2.003 2 0 0.992 2.023 140.372 133.154 125.616 139.676 132.698 127.544 140.024 132.926 126.58 0 0 0 1.032 1.115 1.176 0.953 0.925 0.908 0.991 0.986 0.984 4.147 4.05 3.987 3 0 1.031 2.003 138.815 135.345 133.357 138.279 135.054 132.965 138.547 135.199 133.161 -0.306 -0.306 -0.306 1.088 1.129 1.154 0.97 0.956 0.947 1.082 1.079 1.077 4.375 4.326 4.295 4 0 1.041 2.013 134.348 130.8 128.81 133.42 130.37 127.995 133.884 130.585 128.402 0 0 0 1.157 1.196 1.223 0.955 0.941 0.931 1.101 1.098 1.096 4.362 4.318 4.286 1 0 1.042 2.013 138.731 124.893 117.639 137.688 124.336 117.11 138.209 124.615 117.375 0 0 0 1.062 1.222 1.305 0.956 0.897 0.859 1.02 1.005 0.994 4.208 4.016 3.92 2 0 1.032 2.013 138.828 126.438 119.982 138.088 126.009 119.372 138.458 126.224 119.677 0 0 0 1.062 1.206 1.284 0.957 0.905 0.871 1.023 1.012 1.003 4.218 4.05 3.963 3 0 1.031 2.002 138.008 123.768 115.192 136.749 124.145 116.427 137.379 123.956 115.809 0 0 0 1.054 1.205 1.292 0.939 0.885 0.85 0.971 0.961 0.953 4.064 3.894 3.808 4 0 0.982 2.003 136.345 119.731 116.888 135.326 118.283 116.381 135.836 119.007 116.634 -0.102 -0.102 -0.102 1.104 1.284 1.328 0.952 0.895 0.861 1.045 1.039 1.021 4.244 4.063 3.983 1 0 1.042 2.033 138.325 123.428 115.903 137.226 122.819 115.331 137.775 123.124 115.617 0 0 0 1.045 1.216 1.299 0.94 0.875 0.836 0.966 0.951 0.939 4.056 3.856 3.762 2 0 1.021 2.002 137.405 123.372 116.16 136.455 122.8 115.569 136.93 123.086 115.864 0 0 0 1.09 1.252 1.335 0.955 0.895 0.855 1.043 1.028 1.015 4.251 4.06 3.961 3 0 1.022 1.993 129.545 120.16 113.58 128.745 119.501 113.054 129.145 119.83 113.317 0 0 0 1.2 1.308 1.382 0.932 0.886 0.851 1.074 1.06 1.048 4.241 4.111 4.029 4 0 1.032 2.023 146.389 124.736 116.503 145.023 124.029 115.846 145.706 124.383 116.175 0 0 0 1.002 1.257 1.351 0.998 0.909 0.867 1.09 1.07 1.056 4.488 4.18 4.069 1 0 1.031 2.043 130.561 118.584 112.081 129.724 118.135 111.669 130.143 118.36 111.875 0 0 0 1.156 1.294 1.367 0.922 0.865 0.83 1.009 0.996 0.986 4.083 3.933 3.86 2 0 1.031 2.003 141.309 121.269 113.478 140.231 120.469 112.814 140.77 120.869 113.146 0 0 0 1.032 1.269 1.356 0.967 0.879 0.839 1.023 1.006 0.995 4.249 3.981 3.892 3 0 1.031 2.003 137.238 125.22 120.622 136.75 125.214 119.829 136.994 125.217 120.226 0 0 0 1.095 1.231 1.293 0.962 0.914 0.886 1.062 1.052 1.044 4.304 4.147 4.074 4 0 1.031 2.003 141.439 139.15 133.816 143.529 135.554 131.386 142.484 137.352 132.601 0 0 0 1.007 1.086 1.144 0.988 0.949 0.933 1.048 1.03 1.033 4.35 4.218 4.171 5 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.083 0.918 0.948 3.963 1.132 0.899 0.944 3.905 1.163 0.888 0.941 3.87 1 Testing Liquid Treatment Tergitol 500 mg/L Background Glycerol Time [s] Sample CA[L] CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] 1 0 1.031 2.003 130.329 114.984 106.107 129.429 114.193 105.486 129.879 114.589 105.797 0 0 0 1.132 1.309 1.405 0.903 0.827 0.78 0.945 0.93 0.917 3.909 3.729 3.646 2 0 1.032 2.003 127.664 115.26 106.859 126.897 114.697 106.384 127.28 114.978 106.621 0 0 0 1.184 1.329 1.424 0.905 0.844 0.799 0.991 0.982 0.971 4.005 3.87 3.791 3 0 1.032 2.003 119.833 107.79 99.506 119.372 107.524 99.579 119.602 107.657 99.543 0 0 0 1.254 1.387 1.474 0.855 0.79 0.745 0.94 0.922 0.907 3.796 3.668 3.603 1 0 1.032 2.023 124.535 114.075 107.364 124.042 113.701 106.741 124.288 113.888 107.052 0 0 0 1.26 1.384 1.464 0.911 0.858 0.822 1.075 1.066 1.06 4.192 4.078 4.021 2 0 1.001 2.003 127.177 112.328 102.6 126.881 112.063 102.46 127.029 112.195 102.53 -0.306 -0.306 -0.306 1.19 1.366 1.478 0.905 0.833 0.781 0.999 0.991 0.984 4.023 3.874 3.811 3 0 1.031 2.003 125.706 114.936 107.849 125.861 115.295 108.326 125.784 115.115 108.088 -0.306 -0.306 -0.306 1.174 1.295 1.374 0.881 0.828 0.79 0.929 0.919 0.91 3.824 3.704 3.638 4 0 1.032 2.003 124.322 111.987 103.14 123.989 111.676 102.97 124.156 111.832 103.055 -0.204 -0.204 -0.204 1.245 1.393 1.499 0.903 0.844 0.797 1.043 1.04 1.039 4.109 4 3.953 1 0 1.021 2.003 132.606 114.415 104.566 132.109 113.434 103.08 132.357 113.925 103.823 -0.306 -0.306 -0.306 1.124 1.34 1.45 0.926 0.835 0.778 0.994 0.975 0.956 4.069 3.845 3.743 2 0 1.022 2.003 123.793 107.571 97.482 122.981 107.004 97.032 123.387 107.288 97.257 0 0 0 1.205 1.386 1.498 0.872 0.79 0.735 0.936 0.918 0.906 3.818 3.657 3.598 3 0 1.032 2.013 138.841 114.586 103.698 137.797 113.926 102.935 138.319 114.256 103.317 0 0 0 1.021 1.301 1.423 0.932 0.822 0.763 0.927 0.91 0.896 3.956 3.675 3.583 4 0 1.031 2.013 135.072 114.134 102.811 134.177 113.437 102.597 134.624 113.785 102.704 0 0 0 1.103 1.347 1.473 0.937 0.841 0.781 1.004 0.991 0.978 4.12 3.885 3.797 4 Tergitol 1000 mg/L Background Tergitol 4000 mg/L Background 6 Testing Liquid Treatment SALINE Background Rhamnolipid 500 mg/L Background Rhamnolipid 1000 mg/L Background Rhamnolipid 4000 mg/L Background Sample Formamide Time [s] CA[L] CA[R] CA[M] Tilt 1 0 27.099 26.441 26.77 0.872 26.737 26.102 26.419 1.863 25.83 25.29 25.56 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 2.432 0.261 0.633 4.862 2.413 0.253 0.61 4.791 2.414 0.247 0.589 4.767 2 0 1.002 2.023 25.986 24.905 24.203 25.58 25.086 24.534 25.783 24.996 24.368 0.102 0.102 0.102 2.397 2.402 2.407 0.252 0.245 0.242 0.596 0.581 0.574 4.729 4.73 4.749 3 0 1.001 2.023 26.644 26.168 25.393 25.665 25.426 24.615 26.155 25.797 25.004 -0.306 -0.306 -0.306 2.414 2.405 2.397 0.255 0.248 0.243 0.625 0.599 0.582 4.845 4.774 4.748 4 0 1.032 2.043 27.337 26.415 25.65 26.95 25.864 25.455 27.144 26.14 25.553 0 0 0 2.344 2.34 2.336 0.257 0.248 0.242 0.578 0.558 0.542 4.525 4.504 4.482 1 0 1.031 2.023 26.698 25.918 25.269 25.888 25.111 24.53 26.293 25.515 24.899 0 0 0 2.42 2.425 2.428 0.256 0.249 0.243 0.617 0.602 0.59 4.818 4.828 4.829 2 0 0.961 1.973 26.123 25.33 24.707 25.326 24.186 23.82 25.724 24.758 24.264 0 0 0 2.384 2.391 2.391 0.248 0.239 0.234 0.582 0.563 0.551 4.68 4.694 4.687 3 0 1.002 2.003 25.396 24.213 23.699 25.126 23.939 23.428 25.261 24.076 23.563 0 0 0 2.361 2.365 2.371 0.243 0.232 0.227 0.549 0.527 0.517 4.549 4.556 4.562 4 0 0.991 1.993 27.357 25.796 25.353 26.619 25.272 24.806 26.988 25.534 25.079 0 0 0 2.387 2.412 2.427 0.258 0.246 0.241 0.607 0.59 0.581 4.697 4.774 4.795 1 0 1.021 2.023 28.347 24.754 23.918 27.922 24.473 23.696 28.134 24.614 23.807 -0.511 -0.511 -0.511 2.338 2.35 2.35 0.269 0.235 0.227 0.612 0.54 0.519 4.557 4.551 4.529 2 0 1.012 2.023 26.431 25.341 24.724 26.078 25.072 24.478 26.255 25.206 24.601 -0.409 -0.409 -0.409 2.385 2.387 2.387 0.253 0.242 0.236 0.599 0.575 0.557 4.706 4.694 4.676 3 0 0.991 2.013 27.186 25.659 25.36 26.336 25.141 24.008 26.761 25.4 24.684 0 0 0 2.411 2.42 2.418 0.259 0.25 0.243 0.624 0.603 0.589 4.803 4.82 4.825 4 0 1.011 2.012 25.5 23.745 22.816 25.62 23.265 22.355 25.56 23.505 22.585 -0.306 -0.306 -0.306 2.375 2.387 2.39 0.247 0.235 0.228 0.575 0.554 0.538 4.646 4.698 4.707 1 0 1.051 2.013 26.97 24.855 22.619 26.786 24.452 22.825 26.878 24.654 22.722 0 0 0 2.286 2.3 2.312 0.251 0.231 0.221 0.538 0.495 0.474 4.313 4.309 4.345 2 0 1.012 2.003 27.695 25.087 24.019 26.719 24.602 23.848 27.207 24.845 23.934 -0.204 -0.204 -0.204 2.331 2.35 2.348 0.248 0.234 0.226 0.567 0.538 0.514 4.496 4.543 4.512 3 0 1.031 2.043 26.491 23.675 23.313 25.493 23.378 22.449 25.992 23.527 22.881 -0.306 -0.306 -0.306 2.277 2.264 2.266 0.243 0.216 0.208 0.528 0.459 0.447 4.315 4.201 4.213 4 0 1.052 2.033 26.582 24.238 23.514 25.473 24.451 23.553 26.027 24.345 23.534 -0.102 -0.102 -0.102 2.252 2.267 2.268 0.239 0.225 0.216 0.509 0.475 0.458 4.221 4.208 4.204 7 Testing Liquid Treatment Tergitol 500 mg/L Background Tergitol 1000 mg/L Background Tergitol 4000 mg/L Background Formamide Time [s] CA[L] Sample CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] 1 0 1.021 2.002 27.293 25.561 24.81 26.694 25.079 24.302 26.993 25.32 24.556 0 0 0 2.421 2.431 2.435 0.263 0.252 0.244 0.634 0.611 0.587 4.824 4.863 4.839 2 0 1.012 2.003 26.445 24.641 23.643 25.753 24.076 23.084 26.099 24.359 23.363 0 0 0 2.456 2.476 2.48 0.258 0.246 0.238 0.637 0.617 0.595 4.946 5.017 5.016 3 0 1.032 2.043 25.733 24.619 23.951 25.072 24.047 23.222 25.403 24.333 23.587 0 0 0 2.457 2.456 2.456 0.253 0.243 0.235 0.623 0.598 0.579 4.942 4.933 4.92 4 0 1.042 2.033 25.178 24.262 23.641 25.1 24.262 23.668 25.139 24.262 23.655 0 0 0 2.433 2.435 2.432 0.248 0.239 0.232 0.598 0.573 0.559 4.841 4.817 4.811 1 0 1.012 2.013 26.762 20.2 18.72 26.418 23.648 23.044 26.59 21.924 20.882 0 0 0 2.444 2.562 2.593 0.253 0.241 0.234 0.625 1.28 2.509 4.896 7.834 11.665 2 0 1.001 2.022 25.059 24.107 23.596 24.425 23.68 23.328 24.742 23.894 23.462 0 0 0 2.437 2.438 2.435 0.244 0.236 0.23 0.594 0.57 0.559 4.863 4.84 4.834 3 0 1.022 2.003 24.814 24.036 23.361 24.179 23.319 22.754 24.496 23.677 23.057 0 0 0 2.419 2.418 2.414 0.242 0.233 0.227 0.578 0.561 0.539 4.791 4.787 4.746 4 0 1.001 2.013 25.118 23.757 23.21 24.814 24.036 23.361 24.966 23.897 23.286 0 0 0 2.442 2.438 2.436 0.245 0.23 0.224 0.609 0.568 0.553 4.948 4.885 4.883 1 0 1.042 2.043 26.283 24.563 23.674 25.658 24.229 23.181 25.971 24.396 23.428 -0.306 -0.306 -0.306 2.503 2.505 2.501 0.26 0.245 0.235 0.682 0.638 0.612 5.18 5.152 5.122 2 0 1.012 2.033 26.195 24.214 23.101 25.253 23.317 22.173 25.724 23.765 22.637 -0.204 -0.204 -0.204 2.474 2.483 2.49 0.256 0.238 0.228 0.658 0.611 0.588 5.075 5.068 5.087 3 0 1.031 2.003 25.236 24.116 23.457 24.837 23.658 22.798 25.037 23.887 23.127 0 0 0 2.488 2.489 2.483 0.247 0.237 0.229 0.626 0.605 0.584 5.042 5.057 5.035 4 0 1.021 2.003 25.104 23.996 23.255 24.601 23.25 22.595 24.853 23.623 22.925 0 0 0 2.478 2.486 2.489 0.246 0.234 0.228 0.617 0.593 0.577 5.01 5.025 5.024 8 Testing Liquid Treatment SALINE P.putida Rhamnolipid 500 mg/L P.putida Rhamnolipid 1000 mg/L P.putida WATER Sample Time [s] CA[L] CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] 1 0 36.3 35.856 36.078 -0.409 2.204 0.33 0.672 4.19 1.031 35.926 35.402 35.664 -0.409 2.21 0.326 0.667 4.198 2.023 35.28 34.844 35.062 -0.409 2.211 0.321 0.659 4.202 2 0 1.031 2.003 36.907 36.368 35.799 34.88 34.357 33.666 35.894 35.362 34.732 0 0 0 2.239 2.244 2.248 0.324 0.32 0.315 0.688 0.682 0.672 4.317 4.328 4.33 3 0 1.031 2.023 34.082 33.549 33.003 33.958 33.337 32.831 34.02 33.443 32.917 0 0 0 2.263 2.269 2.269 0.313 0.309 0.304 0.663 0.657 0.648 4.334 4.344 4.345 4 0 1.031 2.002 35.151 34.559 33.989 35.403 34.907 34.395 35.277 34.733 34.192 -0.511 -0.511 -0.511 2.215 2.223 2.227 0.323 0.32 0.315 0.664 0.658 0.65 4.211 4.225 4.227 5 0 1.022 2.003 37.875 37.332 36.797 36.953 36.437 35.923 37.414 36.884 36.36 -0.511 -0.511 -0.511 2.104 2.111 2.113 0.332 0.326 0.322 0.621 0.614 0.606 3.873 3.877 3.876 6 0 1.031 2.013 35.648 35.054 34.606 32.235 31.934 31.406 33.942 33.494 33.006 0 0 0 2.289 2.295 2.298 0.325 0.322 0.317 0.767 0.748 0.746 4.684 4.655 4.678 1 0 1.011 2.023 43.594 38.818 37.741 43.101 38.652 37.68 43.348 38.735 37.71 0 0 0 4.607 4.828 4.883 0.815 0.741 0.722 7.352 7.29 7.278 18.911 20.148 20.483 2 0 1.011 2.023 42.636 39.12 38.407 42.285 38.05 37.309 42.46 38.585 37.858 0 0 0 4.495 4.693 4.726 0.811 0.735 0.718 6.884 6.821 6.8 18.031 19.126 19.337 3 0 1.022 2.043 39.765 36.683 35.623 37.764 34.11 33.389 38.764 35.396 34.506 0 0 0 4.838 5.036 5.076 0.762 0.704 0.689 7.605 7.64 7.529 20.487 21.8 21.961 4 0 1.011 2.033 42.462 36.713 35.694 41.662 36.26 35.127 42.062 36.487 35.41 0 0 0 4.522 4.805 4.859 0.79 0.698 0.68 6.829 6.777 6.718 18.149 19.765 20.049 1 0 1.021 2.033 44.832 40.058 38.165 41.983 37.596 35.839 43.408 38.827 37.002 0 0 0 4.58 4.824 4.904 0.838 0.744 0.714 7.623 7.389 7.349 19.095 20.254 20.774 2 0 1.022 1.993 42.094 36.517 35.045 41.114 35.356 33.614 41.604 35.937 34.33 0 0 0 4.763 5.096 5.195 0.819 0.724 0.696 7.818 7.927 7.923 20.03 22.196 22.869 3 0 1.021 2.032 40.308 35.565 33.986 39.116 34.038 32.693 39.712 34.801 33.34 0 0 0 4.869 5.193 5.276 0.787 0.702 0.678 7.867 8.009 8.014 20.708 22.892 23.518 4 0 1.022 1.993 42.41 38.618 37.233 41.128 37.039 35.697 41.769 37.829 36.465 0 0 0 4.854 5.088 5.174 0.822 0.758 0.735 8.251 8.336 8.342 20.833 22.35 22.903 9 Testing Liquid Treatment Rhamnolipid 1000 mg/L Unwashed P.putida Rhamnolipid 4000 mg/L P.putida WATER Sample Time [s] CA[L] CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] 1 0 1.032 1.993 44.654 42.268 41.652 45.397 41.671 40.997 45.025 41.969 41.325 0 0 0 4.557 4.639 4.658 0.85 0.801 0.789 7.494 7.275 7.217 18.705 19.028 19.106 2 0 1.001 2.013 62.786 59.644 58.088 61.17 58.265 56.773 61.978 58.954 57.43 0 0 0 3.923 4.009 4.053 1.08 1.045 1.02 7.454 7.432 7.369 16 16.262 16.365 3 0 1.011 2.013 48.127 43.353 41.84 48.1 43.353 41.84 48.113 43.353 41.84 0 0 0 4.509 4.684 4.72 0.892 0.827 0.802 8.415 8.39 8.219 19.585 20.609 20.733 4 0 1.002 2.003 47.396 42.49 41.814 47.468 43.458 42.41 47.432 42.974 42.112 0 0 0 4.594 4.826 4.858 0.934 0.876 0.857 8.355 8.605 8.501 19.407 20.833 20.944 5 0 1.002 2.013 51.586 50.191 49.405 50.788 49.27 48.346 51.187 49.73 48.876 0 0 0 4.233 4.279 4.297 0.93 0.899 0.878 7.18 7.098 6.96 16.94 17.102 17.073 6 0 1.011 2.023 50.574 49.021 48.038 49.876 48.222 47.213 50.225 48.621 47.626 -0.1 -0.1 -0.1 4.29 4.34 4.356 0.928 0.895 0.873 7.35 7.253 7.119 17.325 17.49 17.48 10 Testing Liquid Treatment SALINE P.putida Rhamnolipid 500 mg/L P.putida Rhamnolipid 1000 mg/L P.putida WATER (Run number 2) Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 35.575 35.483 35.529 1.022 31.721 31.138 31.429 1.993 30.054 29.385 29.719 L[mm] 0 0 0 2.49 2.592 2.641 H[mm] Vol[ml] A[mm2] 0.365 0.936 5.293 0.333 0.926 5.646 0.319 0.923 5.829 2 0 1.022 2.003 33.648 29.479 27.826 33.332 28.417 26.694 33.49 28.948 27.26 0 0 0 2.542 2.63 2.684 0.351 0.314 0.299 0.935 0.894 0.892 5.472 5.77 5.984 3 0 1.031 2.002 33.623 29.136 26.993 33.608 28.743 26.651 33.615 28.939 26.822 0 0 0 2.555 2.657 2.714 0.355 0.318 0.298 0.96 0.917 0.897 5.544 5.866 6.065 4 0 1.031 2.003 35.05 29.71 27.075 35.181 29.491 26.911 35.115 29.601 26.993 0 0 0 2.539 2.651 2.717 0.372 0.326 0.303 0.99 0.941 0.91 5.509 5.868 6.084 5 0 1.031 2.002 35.931 31.479 29.07 35.757 30.79 28.226 35.844 31.135 28.648 0 0 0 2.534 2.645 2.71 0.379 0.342 0.321 1.007 0.985 0.974 5.508 5.88 6.136 6 0 1.032 2.003 34.813 30.352 28.058 34.819 29.915 27.633 34.816 30.134 27.846 0 0 0 2.548 2.665 2.736 0.37 0.333 0.314 0.991 0.974 0.954 5.54 5.943 6.176 7 0 1.032 2.003 34.785 30.604 28.262 35.247 30.494 28.017 35.016 30.549 28.139 0 0 0 2.525 2.634 2.709 0.368 0.336 0.315 0.97 0.952 0.945 5.443 5.806 6.078 8 0 1.021 2.003 34.513 29.622 27.283 34.944 29.329 27.019 34.728 29.475 27.151 0 0 0 2.531 2.638 2.704 0.368 0.324 0.303 0.977 0.928 0.903 5.484 5.823 6.031 1 0 1.021 2.003 35.395 33.41 33.014 36.249 34.53 34.257 35.822 33.97 33.635 0 0 0 2.56 2.611 2.614 0.373 0.36 0.354 1.024 1.025 1.014 5.616 5.799 5.803 2 0 1.032 2.003 37.875 31.661 31.229 37.638 30.973 30.566 37.756 31.317 30.897 0 0 0 2.467 2.654 2.661 0.388 0.347 0.342 0.984 1.009 0.996 5.276 5.938 5.946 3 0 1.011 2.033 37.403 36.386 35.833 35.189 34.165 33.504 36.296 35.276 34.668 0 0 0 2.569 2.583 2.584 0.378 0.37 0.362 1.06 1.049 1.032 5.709 5.755 5.755 4 0 1.021 1.993 36.256 35.102 34.142 36.743 35.849 34.611 36.499 35.476 34.376 0 0 0 2.516 2.524 2.532 0.371 0.36 0.351 0.984 0.962 0.941 5.429 5.443 5.452 5 0 1.021 1.993 40.961 33.561 33.144 41.074 35.142 34.513 41.017 34.352 33.829 0 0 0 2.375 2.545 2.545 0.409 0.352 0.346 0.967 0.956 0.943 4.977 5.517 5.516 6 0 1.011 2.043 32.714 30.872 30.084 33.564 31.531 30.573 33.139 31.202 30.329 0 0 0 2.63 2.646 2.649 0.36 0.343 0.333 1.032 0.992 0.965 5.864 5.896 5.89 1 0 1.022 2.003 34.471 30.882 29.891 33.964 30.497 29.463 34.218 30.69 29.677 0 0 0 2.589 2.699 2.702 0.361 0.333 0.321 1.001 1.002 0.972 5.679 6.08 6.081 2 0 1.032 2.003 35.662 33.158 31.925 35.373 33.55 31.795 35.517 33.354 31.86 0 0 0 2.559 2.601 2.616 0.375 0.36 0.347 1.017 1.003 0.971 5.592 5.729 5.748 3 0 1.021 2.043 32.625 30.447 29.159 33.393 30.912 29.707 33.009 30.68 29.433 0 0 0 2.573 2.61 2.615 0.354 0.337 0.321 0.97 0.935 0.9 5.624 5.708 5.707 4 0 1.032 2.003 34.741 31.476 29.798 34.731 31.348 29.807 34.736 31.412 29.803 0 0 0 2.545 2.602 2.602 0.364 0.332 0.314 0.98 0.925 0.876 5.525 5.662 5.634 5 0 1.022 2.003 36.042 34.937 33.907 35.166 34.295 33.3 35.604 34.616 33.603 0 0 0 2.509 2.533 2.536 0.369 0.356 0.346 0.963 0.953 0.93 5.386 5.464 5.462 6 0 1.042 2.013 35.547 34.486 33.68 33.86 33.081 31.709 34.704 33.783 32.695 0 0 0 2.549 2.566 2.567 0.351 0.341 0.329 0.961 0.943 0.914 5.535 5.569 5.561 11 Testing Liquid Treatment Rhamnolipid 1000 mg/L Unwashed P.putida Rhamnolipid 4000 mg/L P.putida WATER (Run number 2) Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 44.573 44.48 44.526 1.022 35.985 35.989 35.987 2.043 35.134 34.439 34.787 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 2.343 0.426 0.993 4.913 2.594 0.344 0.995 5.702 2.619 0.34 1 5.802 2 0 1.022 2.033 36.972 31.587 30.739 35.402 30.257 29.758 36.187 30.922 30.249 0 0 0 2.589 2.806 2.838 0.363 0.319 0.309 1.038 1.071 1.058 5.735 6.547 6.637 3 0 1.011 2.032 35.482 27.289 26.057 35.353 26.573 25.301 35.417 26.931 25.679 0 0 0 2.507 2.796 2.853 0.361 0.286 0.273 0.944 0.932 0.937 5.361 6.408 6.655 4 0 1.032 2.013 41.039 23.041 20.458 40.679 20.541 18.189 40.859 21.791 19.324 0 0 0 2.415 3.12 3.277 0.413 0.264 0.234 1.012 1.1 1.077 5.145 8.052 8.742 1 0 1.021 2.002 43.621 41.968 40.108 43.175 41.493 39.671 43.398 41.731 39.89 0 0 0 2.373 2.381 2.39 0.443 0.425 0.405 1.05 1.01 0.964 5.067 5.046 5.018 2 0 1.022 2.003 43.521 41.899 40.075 44.197 42.554 40.641 43.859 42.227 40.358 0 0 0 2.355 2.363 2.372 0.436 0.418 0.399 1.022 0.985 0.937 4.984 4.968 4.931 3 0 1.031 2.003 41.805 35.573 34.099 40.28 34.566 32.948 41.042 35.069 33.523 0 0 0 2.469 2.617 2.626 0.421 0.38 0.361 1.09 1.084 1.035 5.406 5.868 5.853 4 0 1.022 2.003 43.017 41.389 39.717 43.418 41.73 40.089 43.218 41.56 39.903 0 0 0 2.372 2.381 2.385 0.428 0.411 0.392 1.016 0.98 0.935 5.023 5.01 4.972 5 0 1.021 1.993 44.248 42.748 41.012 43.877 42.315 40.685 44.063 42.531 40.849 0 0 0 2.358 2.368 2.373 0.441 0.423 0.404 1.032 0.998 0.954 5.001 4.992 4.957 6 0 1.021 2.043 47.146 45.686 44.051 46.382 45.044 43.308 46.764 45.365 43.68 0 0 0 2.323 2.329 2.335 0.459 0.441 0.422 1.056 1.018 0.976 4.941 4.91 4.875 12 Testing Liquid Treatment SALINE P.putida Rhamnolipid 500 mg/L P.putida Rhamnolipid 1000 mg/L P.putida 1-Bromonaphthalene Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 50.061 49.121 49.591 1.001 48.739 47.669 48.204 2.013 48.716 47.644 48.18 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 3.353 0.698 3.376 10.463 3.38 0.68 3.339 10.528 3.382 0.681 3.336 10.527 2 0 1.012 2.023 49.166 49.113 48.728 48.591 48.479 48.312 48.879 48.796 48.52 0 0 0 3.329 3.33 3.335 0.684 0.684 0.684 3.252 3.249 3.254 10.261 10.26 10.284 3 0 1.001 2.013 47.562 47.471 47.302 46.16 45.923 45.884 46.861 46.697 46.593 0 0 0 3.322 3.326 3.339 0.664 0.662 0.661 3.135 3.136 3.14 10.156 10.172 10.215 4 0 1.012 2.023 47.327 47.106 46.569 48.018 47.742 47.65 47.672 47.424 47.109 -0.3 -0.3 -0.3 3.356 3.363 3.376 0.673 0.67 0.668 3.258 3.249 3.268 10.38 10.392 10.462 5 0 1.012 2.023 47.84 47.862 47.828 47.784 47.485 47.423 47.812 47.674 47.626 0 0 0 3.423 3.425 3.427 0.683 0.68 0.68 3.425 3.424 3.424 10.754 10.771 10.777 6 0 1.011 2.023 47.236 46.926 46.917 46.51 46.235 46.069 46.873 46.58 46.493 0 0 0 3.358 3.36 3.359 0.661 0.656 0.656 3.174 3.163 3.156 10.3 10.306 10.297 1 0 1.011 2.013 61.302 52.059 48.649 60.815 51.457 47.974 61.058 51.758 48.312 -0.3 -0.3 -0.3 3.031 3.265 3.36 0.816 0.723 0.687 3.348 3.343 3.34 9.444 10.128 10.47 2 0 1.012 2.013 54.097 49.325 46.928 53.421 48.715 46.381 53.759 49.02 46.654 0 0 0 3.197 3.322 3.391 0.737 0.69 0.666 3.262 3.261 3.266 9.816 10.241 10.51 3 0 1.002 2.013 49.411 45.623 43.57 48.79 44.928 42.843 49.101 45.275 43.206 0 0 0 3.377 3.502 3.572 0.7 0.657 0.634 3.422 3.437 3.44 10.581 11.084 11.38 4 0 1.012 2.023 50.928 45.642 43.338 50.424 45.336 43.209 50.676 45.489 43.274 0 0 0 3.285 3.434 3.504 0.703 0.647 0.622 3.26 3.248 3.249 10.107 10.655 10.95 5 0 1.002 2.013 51.712 43.658 42.046 50.515 44.218 42.062 51.114 43.938 42.054 0 0 0 3.338 3.535 3.6 0.713 0.659 0.633 3.439 3.461 3.451 10.463 11.22 11.496 1 0 1.011 2.013 50.328 47.317 46.166 52.34 47.755 46.088 51.334 47.536 46.127 -0.4 -0.4 -0.4 3.427 3.551 3.601 0.72 0.686 0.668 3.711 3.738 3.748 11.037 11.52 11.738 2 0 1.002 2.013 52.497 50.201 48.3 53.197 49.65 47.804 52.847 49.925 48.052 0 0 0 3.221 3.301 3.352 0.735 0.694 0.674 3.302 3.258 3.234 9.94 10.172 10.323 3 0 1.011 2.023 62.199 49.756 46.776 61.75 49.054 45.899 61.974 49.405 46.337 0 0 0 3.1 3.453 3.547 0.846 0.716 0.681 3.63 3.674 3.666 9.927 11.082 11.442 4 0 1.002 2.013 52.182 46.541 44.796 51.587 45.999 44.091 51.884 46.27 44.444 0 0 0 3.306 3.469 3.522 0.731 0.669 0.649 3.456 3.431 3.433 10.368 10.937 11.172 5 0 1.011 2.013 52.872 48.162 46.738 52.005 47.463 45.99 52.438 47.812 46.364 0 0 0 3.243 3.369 3.41 0.727 0.68 0.662 3.31 3.305 3.289 10.021 10.457 10.602 13 Testing Liquid Treatment Rhamnolipid 1000 mg/L Unwashed P.putida Rhamnolipid 4000 mg/L P.putida 1-Bromonaphthalene Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 56.167 55.35 55.759 1.012 56.106 55.635 55.87 2.043 56.229 55.654 55.941 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.707 0.405 0.516 2.836 1.706 0.403 0.515 2.832 1.706 0.403 0.515 2.833 2 0 1.031 2.012 55.954 55.943 55.982 55.554 55.522 55.602 55.754 55.732 55.792 0 0 0 1.689 1.689 1.689 0.401 0.401 0.401 0.5 0.501 0.5 2.776 2.779 2.777 3 0 1.021 1.992 54.516 54.534 54.408 54.821 54.776 54.746 54.669 54.655 54.577 0 0 0 1.719 1.72 1.719 0.397 0.398 0.397 0.513 0.512 0.512 2.851 2.849 2.848 4 0 1.021 2.003 51.76 51.821 51.807 51.362 51.324 51.31 51.561 51.572 51.558 0 0 0 1.731 1.73 1.73 0.375 0.375 0.375 0.485 0.486 0.485 2.82 2.822 2.821 5 0 1.022 2.003 53.069 53.136 53.063 52.807 52.848 52.763 52.938 52.992 52.913 0 0 0 1.739 1.739 1.739 0.389 0.389 0.389 0.511 0.51 0.511 2.882 2.879 2.882 6 0 1.022 2.003 54.153 54.116 54.024 53.779 53.813 53.663 53.966 53.964 53.843 0 0 0 1.716 1.716 1.716 0.389 0.389 0.389 0.499 0.5 0.5 2.82 2.821 2.823 7 0 1.032 2.003 53.375 53.379 53.373 53.31 53.24 53.337 53.342 53.31 53.355 0 0 0 1.722 1.722 1.722 0.388 0.388 0.388 0.502 0.501 0.5 2.837 2.835 2.833 1 0 1.002 2.003 56.3 56.046 55.777 55.664 55.203 55.039 55.982 55.624 55.408 0 0 0 3.233 3.237 3.242 0.774 0.766 0.76 3.542 3.514 3.508 10.207 10.192 10.207 2 0 1.012 2.023 60.804 55.43 54.861 59.619 54.471 53.932 60.211 54.95 54.397 0 0 0 3.176 3.348 3.365 0.843 0.791 0.783 3.771 3.864 3.857 10.28 10.88 10.928 3 0 1.011 2.023 56.024 54.717 53.871 55.979 54.185 53.146 56.002 54.451 53.509 0 0 0 3.212 3.261 3.264 0.783 0.753 0.742 3.519 3.484 3.442 10.115 10.235 10.213 4 0 1.011 2.013 66.951 65.947 65.673 65.509 64.862 64.642 66.23 65.405 65.158 0 0 0 3.03 3.057 3.07 0.905 0.892 0.885 3.786 3.773 3.778 9.947 9.987 10.024 5 0 1.012 2.013 65.254 64.155 63.568 64.045 63.533 63.064 64.649 63.844 63.316 0 0 0 2.99 3.024 3.045 0.883 0.86 0.85 3.563 3.535 3.538 9.596 9.636 9.689 6 0 1.011 2.023 65.915 64.133 63.214 65.112 63.64 63.036 65.513 63.886 63.125 0 0 0 2.984 3.019 3.033 0.864 0.852 0.845 3.494 3.493 3.482 9.5 9.576 9.596 14 Testing Liquid Treatment SALINE P.putida Rhamnolipid 500 mg/L P.putida Glycerol Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 101.955 101.124 101.539 1.031 99.678 98.366 99.022 2.013 97.763 96.819 97.291 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.339 0.721 0.743 3.16 1.372 0.706 0.745 3.158 1.396 0.695 0.746 3.161 2 0 1.032 2.013 101.906 100.038 96.81 101.56 99.589 96.457 101.733 99.813 96.634 0 0 0 1.322 1.363 1.402 0.732 0.708 0.691 0.74 0.741 0.743 3.154 3.149 3.151 3 0 1.032 2.033 104.582 100.274 97.086 104.65 99.668 96.683 104.616 99.971 96.885 0 0 0 1.437 1.501 1.542 0.799 0.766 0.747 0.973 0.972 0.974 3.792 3.773 3.774 4 0 1.021 2.002 102.285 98.759 96.081 101.473 97.802 95.345 101.879 98.28 95.713 0 0 0 1.458 1.506 1.537 0.771 0.748 0.735 0.942 0.942 0.942 3.699 3.693 3.693 5 0 1.011 2.042 101.98 99.861 97.688 101.288 99.239 97.292 101.634 99.55 97.49 0 0 0 1.459 1.492 1.518 0.772 0.755 0.743 0.941 0.942 0.943 3.698 3.695 3.696 6 0 1.011 2.002 101.893 99.239 97.045 102.461 99.165 96.988 102.177 99.202 97.017 -0.409 -0.409 -0.409 1.447 1.487 1.513 0.77 0.749 0.737 0.929 0.927 0.927 3.669 3.655 3.652 1 0 1.012 2.013 107.277 102.381 98.247 108.373 102.201 98.313 107.825 102.291 98.28 0 0 0 2.537 2.824 2.914 1.554 1.534 1.497 6.18 7.068 7.072 13.09 14.193 14.161 2 0 1.001 2.012 101.098 96.739 95.022 101.49 97.164 94.871 101.294 96.951 94.946 0 0 0 3.045 3.157 3.216 1.69 1.644 1.622 9.016 9.045 9.068 16.697 16.681 16.697 3 0 1.011 2.013 100.259 96.755 94.228 101.23 97.287 94.013 100.744 97.021 94.121 0 0 0 3.067 3.172 3.243 1.69 1.634 1.597 9.054 9.065 9.038 16.735 16.705 16.658 4 0 1.011 2.023 91.995 90.899 90.238 96.945 94.266 92.941 94.47 92.583 91.589 0 0 0 3.1 3.152 3.177 1.556 1.526 1.511 8.341 8.165 8.083 15.795 15.566 15.465 1 0 1.001 2.003 98.948 95.255 93.338 99.942 96.589 94.201 99.445 95.922 93.769 0 0 0 3.297 3.395 3.455 1.786 1.741 1.715 10.996 11.01 11.029 19.032 19.009 19.021 2 0 1.002 2.013 103.604 98.624 95.948 103.425 98.859 96.427 103.514 98.741 96.188 0 0 0 2.681 2.79 2.847 1.493 1.446 1.421 6.283 6.292 6.291 13.14 13.103 13.089 3 0 1.011 2.022 100.206 97.625 94.788 100.946 97.796 94.891 100.576 97.711 94.839 0 0 0 3.079 3.174 3.245 1.732 1.671 1.64 9.366 9.339 9.367 17.127 17.046 17.061 0 1.002 2.003 101.076 99.339 97.967 100.689 99.086 97.577 100.882 99.212 97.772 0 0 0 2.895 2.954 2.994 1.555 1.524 1.505 7.45 7.451 7.466 14.688 14.668 14.678 5 Rhamnolipid 1000 mg/L P.putida 4 5 15 Testing Liquid Treatment Rhamnolipid 1000 mg/L Unwashed P.putida Rhamnolipid 4000 mg/L P.putida Glycerol Sample Time [s] CA[L] CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] 1 0 1.001 2.013 106.06 97.496 93.699 106.725 98.637 94.896 106.392 98.067 94.298 0 0 0 2.982 3.219 3.311 1.798 1.681 1.652 9.718 9.71 9.757 17.668 17.497 17.53 2 0 1.001 2.013 103.973 99.984 97.75 103.982 99.715 97.922 103.978 99.85 97.836 0 0 0 2.802 2.941 2.994 1.685 1.593 1.571 7.889 7.803 7.796 15.351 15.144 15.113 3 0 1.001 2.013 102.196 98.971 95.943 102.076 98.49 96.013 102.136 98.731 95.978 0 0 0 3.148 3.255 3.325 1.784 1.731 1.701 10.242 10.249 10.279 18.198 18.146 18.157 4 0 1.002 2.013 99.603 97.861 96.03 100.308 98.356 96.128 99.955 98.108 96.079 0 0 0 2.688 2.758 2.806 1.479 1.436 1.413 6.098 6.09 6.091 12.855 12.82 12.81 5 6 16 Testing Liquid Treatment SALINE Rhamnolipid 500 mg/L Rhamnolipid 1000 mg/L Formamide Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 45.958 45.043 45.5 1.002 36.233 35.696 35.965 2.033 30.801 30.941 30.871 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 2.129 0.41 0.79 4.119 2.267 0.341 0.725 4.412 2.356 0.302 0.687 4.65 2 0 1.021 2.003 47.756 38.976 33.816 47.105 38.354 33.164 47.43 38.665 33.49 0 0 0 2.075 2.219 2.329 0.421 0.36 0.322 0.773 0.742 0.725 3.97 4.301 4.605 3 0 1.031 2.003 47.784 39.787 34.038 47.143 39.019 33.279 47.464 39.403 33.659 0 0 0 2.079 2.204 2.308 0.42 0.365 0.322 0.775 0.739 0.709 3.981 4.252 4.524 4 0 1.031 2.003 46.015 39.9 35.427 45.466 39.228 35.015 45.741 39.564 35.221 -0.409 -0.409 -0.409 2.121 2.242 2.34 0.412 0.372 0.342 0.79 0.791 0.784 4.103 4.428 4.707 5 0 1.001 2.003 46.662 40.215 36.207 45.63 39.505 35.518 46.146 39.86 35.862 -0.306 -0.306 -0.306 2.131 2.228 2.308 0.417 0.369 0.339 0.813 0.776 0.76 4.165 4.369 4.583 6 0 1.032 2.013 47.733 41.806 37.527 47.186 41.28 37.055 47.46 41.543 37.291 -0.306 -0.306 -0.306 2.119 2.222 2.303 0.429 0.386 0.354 0.825 0.807 0.79 4.144 4.38 4.595 1 0 1.021 2.003 48.152 46.494 44.335 46.652 44.991 42.797 47.402 45.742 43.566 0 0 0 1.692 1.715 1.745 0.612 0.599 0.584 0.842 0.839 0.838 3.504 3.515 3.541 2 0 1.022 2.003 46.927 44.9 42.798 46.798 44.38 42.024 46.862 44.64 42.411 0 0 0 1.685 1.714 1.742 0.602 0.588 0.574 0.816 0.815 0.815 3.438 3.461 3.487 3 0 1.021 2.003 46.325 43.678 41.076 46.475 43.584 41.138 46.4 43.631 41.107 0 0 0 1.685 1.712 1.739 0.596 0.576 0.559 0.806 0.793 0.787 3.417 3.411 3.424 4 0 1.032 2.003 47.28 45.428 42.913 46.101 44.603 42.09 46.69 45.015 42.501 0 0 0 1.682 1.709 1.743 0.602 0.587 0.571 0.814 0.812 0.814 3.432 3.449 3.487 5 0 1.031 2.002 46.469 44.449 42.068 45.857 43.817 41.523 46.163 44.133 41.795 0 0 0 1.699 1.726 1.755 0.599 0.585 0.57 0.824 0.821 0.818 3.468 3.485 3.506 0 1.031 2.003 45.109 43.729 41.47 46.286 44.219 41.605 45.698 43.974 41.537 0 0 0 1.702 1.721 1.747 0.602 0.585 0.564 0.828 0.817 0.801 3.481 3.471 3.463 0 1.032 2.003 42.583 37.964 34.578 41.814 37.373 33.603 42.199 37.669 34.09 0 0 0 1.694 1.72 1.75 0.561 0.524 0.497 0.751 0.709 0.686 3.301 3.238 3.229 1 0 1.021 2.002 76.769 72.052 67.837 75.888 71.356 67.437 76.328 71.704 67.637 0 0 0 1.682 1.723 1.765 0.597 0.566 0.538 0.804 0.783 0.764 3.409 3.4 3.405 2 0 1.021 2.003 76.359 69.878 64.582 76.06 69.706 64.176 76.209 69.792 64.379 0 0 0 1.681 1.727 1.774 0.594 0.548 0.51 0.798 0.752 0.723 3.395 3.337 3.334 3 0 1.021 1.992 76.923 70.72 65.363 76.304 70.217 64.977 76.614 70.468 65.17 0 0 0 1.684 1.727 1.771 0.599 0.555 0.517 0.812 0.765 0.733 3.429 3.364 3.351 4 0 1.031 2.003 77.57 73.394 70.073 76.957 72.953 69.72 77.264 73.174 69.896 0 0 0 1.682 1.715 1.748 0.604 0.575 0.552 0.818 0.793 0.778 3.441 3.414 3.414 5 0 1.032 2.013 75.777 69.738 64.884 75.677 69.501 64.668 75.727 69.619 64.776 0 0 0 1.696 1.735 1.776 0.596 0.549 0.513 0.813 0.762 0.728 3.439 3.367 3.346 6 0 1.031 2.003 74.599 68.598 63.509 74.193 68.182 62.748 74.396 68.39 63.128 0 0 0 1.711 1.754 1.8 0.586 0.543 0.506 0.81 0.764 0.735 3.445 3.393 3.395 17 Testing Liquid Treatment Rhamnolipid 1000 mg/L Unwashed Rhamnolipid 4000 mg/L Formamide Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 75.739 75.579 75.659 1.022 71.96 71.745 71.852 2.053 68.69 68.394 68.542 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.706 0.597 0.825 3.476 1.754 0.575 0.824 3.519 1.8 0.555 0.825 3.571 2 0 1.021 2.003 76.41 74.745 71.09 75.985 74.339 70.655 76.197 74.542 70.872 0 0 0 1.672 1.717 1.764 0.628 0.59 0.568 0.829 0.821 0.819 3.456 3.476 3.518 3 0 1.022 1.993 79.062 75.361 71.718 78.626 74.887 71.395 78.844 75.124 71.557 0 0 0 1.677 1.724 1.772 0.617 0.597 0.575 0.838 0.838 0.839 3.482 3.521 3.569 4 0 1.032 2.003 80.435 27.572 21.226 79.282 26.303 19.783 79.859 26.937 20.505 0 0 0 1.662 2.663 2.907 0.622 0.288 0.227 0.834 0.849 0.808 3.464 5.877 6.867 5 0 1.031 2.003 84.195 26.713 18.535 83.353 26.15 17.726 83.774 26.431 18.131 0 0 0 1.609 2.712 3.087 0.656 0.281 0.211 0.838 0.86 0.829 3.442 6.048 7.607 1 0 1.021 1.993 80.661 77.151 73.182 79.998 76.611 72.596 80.33 76.881 72.889 0 0 0 1.67 1.705 1.748 0.63 0.605 0.577 0.857 0.841 0.827 3.521 3.511 3.518 2 0 1.032 2.013 79.573 77.539 74.264 78.657 76.718 73.629 79.115 77.128 73.947 0 0 0 1.7 1.728 1.767 0.627 0.614 0.595 0.878 0.878 0.875 3.59 3.611 3.641 3 0 1.032 2.003 78.682 75.801 72.791 78.401 75.807 72.889 78.541 75.804 72.84 0 0 0 1.708 1.744 1.784 0.625 0.607 0.586 0.88 0.877 0.875 3.6 3.623 3.657 4 0 1.031 2.003 78.686 76.097 72.864 78.812 76.384 73.034 78.749 76.24 72.949 0 0 0 1.7 1.735 1.779 0.626 0.609 0.589 0.872 0.872 0.872 3.578 3.603 3.645 5 0 1.031 2.003 79.039 76.898 73.642 78.315 76.194 73.203 78.677 76.546 73.423 0 0 0 1.706 1.737 1.777 0.627 0.613 0.595 0.883 0.882 0.881 3.606 3.629 3.661 6 0 1.021 2.003 80.543 78.858 76.392 79.882 78.134 75.839 80.213 78.496 76.116 0 0 0 1.687 1.71 1.742 0.633 0.621 0.605 0.878 0.876 0.875 3.583 3.595 3.618 18 Testing Liquid WATER Treatment Sample Time [s] CA[L] CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] SALINE 1 0 36.3 35.856 36.078 -0.409 2.204 0.33 0.672 4.19 P.putida 1.031 35.926 35.402 35.664 -0.409 2.21 0.326 0.667 4.198 2.023 35.28 34.844 35.062 -0.409 2.211 0.321 0.659 4.202 Tergitol 500 mg/L P.putida 2 0 1.031 2.003 36.907 36.368 35.799 34.88 34.357 33.666 35.894 35.362 34.732 0 0 0 2.239 2.244 2.248 0.324 0.32 0.315 0.688 0.682 0.672 4.317 4.328 4.33 3 0 1.031 2.023 34.082 33.549 33.003 33.958 33.337 32.831 34.02 33.443 32.917 0 0 0 2.263 2.269 2.269 0.313 0.309 0.304 0.663 0.657 0.648 4.334 4.344 4.345 4 0 1.031 2.002 35.151 34.559 33.989 35.403 34.907 34.395 35.277 34.733 34.192 -0.511 -0.511 -0.511 2.215 2.223 2.227 0.323 0.32 0.315 0.664 0.658 0.65 4.211 4.225 4.227 5 0 1.022 2.003 37.875 37.332 36.797 36.953 36.437 35.923 37.414 36.884 36.36 -0.511 -0.511 -0.511 2.104 2.111 2.113 0.332 0.326 0.322 0.621 0.614 0.606 3.873 3.877 3.876 6 0 1.031 2.013 35.648 35.054 34.606 32.235 31.934 31.406 33.942 33.494 33.006 0 0 0 2.289 2.295 2.298 0.325 0.322 0.317 0.767 0.748 0.746 4.684 4.655 4.678 1 0 1.002 2.023 36.631 36.383 35.989 36.082 35.767 35.464 36.357 36.075 35.726 0 0 0 2.205 2.207 2.207 0.335 0.332 0.329 0.677 0.669 0.662 4.19 4.182 4.174 2 0 1.032 2.013 36.324 36.001 35.523 36.25 35.678 35.472 36.287 35.839 35.497 -0.306 -0.306 -0.306 2.197 2.201 2.201 0.332 0.328 0.325 0.671 0.665 0.657 4.166 4.174 4.162 3 0 1.031 2.013 37.67 37.369 37.067 37.033 36.7 36.521 37.351 37.034 36.794 -0.306 -0.306 -0.306 2.21 2.213 2.214 0.346 0.343 0.34 0.71 0.705 0.698 4.248 4.252 4.244 4 0 1.041 2.013 37.466 36.913 36.564 37.446 37.02 36.74 37.456 36.967 36.652 -0.511 -0.511 -0.511 2.2 2.202 2.203 0.342 0.339 0.335 0.694 0.688 0.682 4.194 4.197 4.193 5 0 1.042 2.033 36.347 35.901 35.551 36.642 36.206 35.963 36.494 36.053 35.757 -0.306 -0.306 -0.306 2.275 2.276 2.277 0.352 0.349 0.346 0.755 0.75 0.743 4.469 4.473 4.469 6 0 1.042 2.023 36.819 36.482 36.138 36.254 35.854 35.607 36.537 36.168 35.873 0 0 0 2.159 2.163 2.164 0.33 0.327 0.324 0.641 0.633 0.627 4.027 4.019 4.016 7 0 1.042 2.013 36.432 35.882 35.788 36.056 35.689 35.37 36.244 35.785 35.579 0 0 0 2.169 2.171 2.173 0.329 0.324 0.322 0.64 0.635 0.627 4.044 4.049 4.037 8 0 1.041 2.013 37.216 36.945 36.042 36.573 36.231 35.761 36.894 36.588 35.901 -0.102 -0.102 -0.102 2.212 2.214 2.222 0.338 0.334 0.331 0.694 0.687 0.682 4.235 4.23 4.241 19 Testing Liquid WATER Treatment Sample Time [s] CA[L] CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] Tergitol 1 0 38.807 38.776 38.792 -0.409 2.157 0.354 0.689 4.071 1000 mg/L 1.022 36.381 36.716 36.548 -0.409 2.16 0.337 0.653 4.042 P.putida 2.003 33.414 34.14 33.777 -0.409 2.162 0.313 0.603 3.996 2 0 1.032 2.003 39.838 39.667 39.382 39.435 38.989 38.64 39.636 39.328 39.011 0 0 0 2.183 2.184 2.188 0.362 0.358 0.354 0.718 0.714 0.71 4.162 4.169 4.176 3 0 1.021 2.002 37.135 36.342 36.183 37.253 36.038 35.509 37.194 36.19 35.846 -0.511 -0.511 -0.511 2.196 2.205 2.207 0.34 0.337 0.335 0.686 0.685 0.684 4.171 4.208 4.22 4 0 1.031 2.003 37.863 37.12 36.989 37.774 37.165 36.909 37.818 37.142 36.949 0 0 0 2.201 2.208 2.208 0.351 0.348 0.344 0.705 0.701 0.693 4.202 4.216 4.206 5 0 1.031 2.013 36.913 36.629 36.246 35.687 35.402 35.066 36.3 36.015 35.656 0 0 0 2.295 2.297 2.298 0.34 0.337 0.334 0.751 0.742 0.738 4.531 4.524 4.53 1 0 1.041 2.023 43.886 43.499 43.237 44.099 43.852 43.565 43.992 43.676 43.401 -0.102 -0.102 -0.102 2.091 2.093 2.094 0.391 0.388 0.385 0.723 0.72 0.715 3.941 3.944 3.94 2 0 1.031 2.013 43.238 43.001 42.721 42.805 42.616 42.37 43.021 42.808 42.546 -0.306 -0.306 -0.306 2.126 2.128 2.13 0.388 0.385 0.382 0.743 0.737 0.733 4.056 4.053 4.053 3 0 1.021 2.023 42.146 42.991 39.543 42.776 42.223 40.386 42.461 42.607 39.964 -0.306 -0.306 -0.306 2.173 2.176 2.223 0.391 0.382 0.373 0.779 0.773 0.775 4.217 4.222 4.346 4 0 0.991 2.013 43.108 42.802 42.599 43.225 43.029 42.756 43.166 42.916 42.678 0 0 0 2.114 2.116 2.116 0.391 0.387 0.385 0.732 0.725 0.723 4.003 3.999 4.002 5 0 1.032 2.003 46.128 45.712 45.357 45.295 44.308 43.716 45.712 45.01 44.536 -0.204 -0.204 -0.204 2.044 2.045 2.046 0.403 0.396 0.391 0.718 0.71 0.704 3.83 3.831 3.829 6 0 1.021 2.013 45.485 45.074 44.937 45.249 45.023 44.792 45.367 45.049 44.864 -0.306 -0.306 -0.306 2.09 2.091 2.092 0.41 0.407 0.404 0.759 0.753 0.749 3.985 3.983 3.98 7 0 1.022 2.003 43.809 43.578 43.406 44.888 44.621 44.42 44.348 44.1 43.913 0 0 0 2.12 2.122 2.123 0.402 0.398 0.395 0.763 0.759 0.755 4.066 4.065 4.064 8 0 1.041 2.012 44.963 44.771 44.435 44.841 44.511 44.165 44.902 44.641 44.3 0 0 0 2.11 2.113 2.113 0.404 0.4 0.397 0.761 0.755 0.749 4.034 4.032 4.026 9 0 1.001 2.023 44.353 44.019 43.832 44.091 43.656 43.412 44.222 43.838 43.622 0 0 0 2.09 2.094 2.095 0.391 0.388 0.385 0.723 0.714 0.709 3.937 3.93 3.925 6 Tergitol 4000 mg/L P.putida 20 Testing Liquid 1-Bromonaphthalene Treatment Sample Time [s] CA[L] CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] SALINE 1 0 61.854 61.97 61.912 -0.204 1.558 0.421 0.457 2.5 P.putida 1.022 61.406 61.5 61.453 -0.204 1.561 0.419 0.457 2.503 2.013 61.449 61.262 61.356 -0.204 1.562 0.418 0.457 2.504 Tergitol 500 mg/L P.putida 2 0 1.041 2.023 62.461 62.366 62.283 62.209 62.124 61.935 62.335 62.245 62.109 0 0 0 1.554 1.554 1.556 0.422 0.421 0.42 0.455 0.455 0.456 2.489 2.49 2.495 3 0 1.041 2.023 61.549 61.712 61.257 61.048 61.233 61.282 61.299 61.473 61.27 -0.204 -0.204 -0.204 1.555 1.554 1.556 0.416 0.415 0.414 0.45 0.449 0.449 2.481 2.477 2.479 4 0 1.002 2.023 61.4 61.278 61.074 60.501 60.349 60.362 60.95 60.813 60.718 -0.409 -0.409 -0.409 1.556 1.557 1.557 0.413 0.412 0.411 0.448 0.447 0.446 2.477 2.477 2.477 5 0 1.031 2.013 62.644 62.177 62.28 62.213 62.378 62.119 62.428 62.278 62.199 0 0 0 1.56 1.562 1.563 0.423 0.423 0.421 0.462 0.463 0.462 2.514 2.518 2.517 6 0 1.032 2.023 60.879 60.626 60.569 60.711 59.936 59.971 60.795 60.281 60.27 -0.102 -0.102 -0.102 1.571 1.575 1.575 0.414 0.414 0.413 0.456 0.457 0.457 2.517 2.522 2.522 1 0 1.031 2.023 34.941 33.051 32.091 34.616 32.506 31.737 34.778 32.778 31.914 0 0 0 1.987 2.002 2.002 0.276 0.261 0.255 0.455 0.434 0.422 3.352 3.365 3.352 2 0 1.031 2.023 34.327 33.758 33.708 33.075 32.718 32.814 33.701 33.238 33.261 -0.102 -0.102 -0.102 1.985 1.991 1.994 0.272 0.269 0.269 0.45 0.448 0.448 3.355 3.372 3.373 3 0 1.031 2.033 33.914 33.732 33.605 33.911 33.805 33.56 33.913 33.768 33.583 -0.306 -0.306 -0.306 1.959 1.959 1.959 0.269 0.267 0.267 0.432 0.43 0.429 3.26 3.26 3.262 4 0 1.031 2.012 36.649 32.599 32.63 36.713 31.886 31.95 36.681 32.242 32.29 -0.102 -0.102 -0.102 1.894 1.994 1.996 0.284 0.258 0.258 0.427 0.431 0.431 3.089 3.359 3.362 5 0 1.031 2.012 33.401 33.436 33.525 33.163 33.061 33.081 33.282 33.249 33.303 -0.306 -0.306 -0.306 1.944 1.947 1.945 0.263 0.262 0.262 0.415 0.417 0.417 3.204 3.216 3.215 6 0 1.021 2.002 32.995 32.91 32.878 32.261 32.186 32.066 32.628 32.548 32.472 -0.306 -0.306 -0.306 2 2.001 2 0.259 0.259 0.259 0.438 0.437 0.436 3.383 3.386 3.383 7 0 1.022 2.003 33.644 33.262 33.085 33.587 32.916 32.931 33.616 33.089 33.008 -0.306 -0.306 -0.306 1.992 2.004 2.007 0.27 0.269 0.268 0.449 0.453 0.452 3.364 3.408 3.413 8 0 1.032 2.013 34.063 30.712 29.89 33.394 30.547 30.024 33.728 30.63 29.957 -0.409 -0.409 -0.409 1.963 1.997 1.989 0.275 0.249 0.241 0.444 0.414 0.397 3.294 3.347 3.309 21 Testing Liquid 1-Bromonaphthalene Treatment Sample Time [s] CA[L] CA[R] CA[M] Tilt Tergitol 1 0 32.468 32.173 32.321 1000 mg/L 1.022 32.408 32.251 32.33 P.putida 1.993 32.469 32.074 32.272 Tergitol 4000 mg/L P.putida 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.973 0.256 0.413 3.27 1.974 0.256 0.412 3.266 1.97 0.256 0.413 3.269 2 0 1.012 2.003 32.574 31.984 32.105 33.134 32.827 32.809 32.854 32.405 32.457 -0.306 -0.306 -0.306 1.982 1.985 1.984 0.262 0.258 0.258 0.431 0.426 0.426 3.321 3.324 3.323 3 0 1.022 2.003 32.147 32.108 31.975 32.006 31.642 31.771 32.076 31.875 31.873 -0.306 -0.306 -0.306 1.965 1.967 1.969 0.253 0.251 0.252 0.41 0.409 0.408 3.255 3.264 3.263 4 0 1.031 2.013 32.88 31.946 32.04 32.953 31.543 31.474 32.916 31.744 31.757 -0.409 -0.409 -0.409 2.054 2.065 2.065 0.268 0.259 0.259 0.477 0.467 0.466 3.57 3.592 3.595 5 0 1.042 2.023 31.335 31.18 31.258 31.235 30.929 31.004 31.285 31.054 31.131 0 0 0 2.007 2.008 2.009 0.253 0.252 0.252 0.42 0.419 0.42 3.364 3.372 3.374 6 0 1.031 2.013 32.88 31.946 32.04 32.953 31.543 31.474 32.916 31.744 31.757 -0.409 -0.409 -0.409 2.054 2.065 2.065 0.268 0.259 0.259 0.477 0.467 0.466 3.57 3.592 3.595 1 0 1.042 2.023 29.109 27.644 27.01 28.929 27.312 26.681 29.019 27.478 26.846 -0.306 -0.306 -0.306 2.056 2.082 2.078 0.239 0.229 0.223 0.422 0.413 0.403 3.521 3.593 3.576 2 0 1.032 2.013 29.626 27.973 28.136 29.627 28.435 28.373 29.626 28.204 28.254 0 0 0 2.078 2.122 2.125 0.246 0.238 0.238 0.441 0.443 0.441 3.595 3.719 3.718 3 0 1.032 2.013 29.442 29.115 29.118 29.466 29.007 28.943 29.454 29.061 29.031 -0.306 -0.306 -0.306 2.102 2.109 2.108 0.246 0.244 0.243 0.454 0.453 0.452 3.679 3.7 3.698 4 0 1.022 2.003 29.671 29.28 29.253 28.861 28.331 28.36 29.266 28.806 28.806 -0.409 -0.409 -0.409 2.047 2.067 2.067 0.241 0.238 0.238 0.426 0.429 0.428 3.517 3.576 3.572 5 0 1.041 2.023 28.441 29.064 28.739 26.467 28.662 28.479 27.454 28.863 28.609 -0.613 -0.613 -0.613 2.059 2.045 2.046 0.239 0.232 0.23 0.463 0.411 0.406 3.739 3.493 3.485 6 0 1.042 2.023 30.833 28.516 27.981 30.606 28.021 27.621 30.719 28.269 27.801 -0.409 -0.409 -0.409 2.072 2.132 2.133 0.255 0.24 0.235 0.46 0.456 0.448 3.605 3.781 3.775 7 8 9 22 Testing Liquid Glycerol Treatment Sample Time [s] CA[L] CA[R] CA[M] Tilt SALINE 1 0 101.955 101.124 101.539 P.putida 1.031 99.678 98.366 99.022 2.013 97.763 96.819 97.291 Tergitol 500 mg/L P.putida 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.339 0.721 0.743 3.16 1.372 0.706 0.745 3.158 1.396 0.695 0.746 3.161 2 0 1.032 2.013 101.906 100.038 96.81 101.56 99.589 96.457 101.733 99.813 96.634 0 0 0 1.322 1.363 1.402 0.732 0.708 0.691 0.74 0.741 0.743 3.154 3.149 3.151 3 0 1.032 2.033 104.582 100.274 97.086 104.65 99.668 96.683 104.616 99.971 96.885 0 0 0 1.437 1.501 1.542 0.799 0.766 0.747 0.973 0.972 0.974 3.792 3.773 3.774 4 0 1.021 2.002 102.285 98.759 96.081 101.473 97.802 95.345 101.879 98.28 95.713 0 0 0 1.458 1.506 1.537 0.771 0.748 0.735 0.942 0.942 0.942 3.699 3.693 3.693 5 0 1.011 2.042 101.98 99.861 97.688 101.288 99.239 97.292 101.634 99.55 97.49 0 0 0 1.459 1.492 1.518 0.772 0.755 0.743 0.941 0.942 0.943 3.698 3.695 3.696 6 0 1.011 2.002 101.893 99.239 97.045 102.461 99.165 96.988 102.177 99.202 97.017 -0.409 -0.409 -0.409 1.447 1.487 1.513 0.77 0.749 0.737 0.929 0.927 0.927 3.669 3.655 3.652 1 0 1.041 2.023 92.142 64.299 55.748 91.623 63.467 54.592 91.882 63.883 55.17 0 0 0 1.578 1.958 2.103 0.728 0.563 0.506 0.958 0.969 0.977 3.733 4.057 4.325 2 0 1.032 2.023 72.991 58.515 52.171 72.93 58.068 51.114 72.96 58.291 51.643 0 0 0 1.806 2.023 2.136 0.606 0.515 0.474 0.925 0.93 0.932 3.782 4.097 4.326 3 0 1.032 2.013 88.379 62.518 55.556 87.627 62.181 55.089 88.003 62.35 55.322 0 0 0 1.6 1.937 2.047 0.689 0.538 0.49 0.904 0.901 0.894 3.604 3.902 4.083 4 0 1.032 2.013 70.983 57.61 52.009 71.755 59.767 54.158 71.369 58.689 53.084 0 0 0 1.8 1.971 2.063 0.596 0.526 0.487 0.896 0.916 0.914 3.715 4.001 4.17 5 6 7 8 23 Testing Liquid Glycerol Treatment Sample Time [s] CA[L] CA[R] CA[M] Tilt Tergitol 1 0 73.839 73.862 73.85 1000 mg/L 1.021 58.049 57.896 57.972 P.putida 2.043 51.74 51.495 51.618 Tergitol 4000 mg/L P.putida 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.787 0.611 0.918 3.75 2.003 0.514 0.904 4.017 2.107 0.471 0.896 4.209 2 0 1.032 2.023 76.019 59.631 52.602 75.631 59.307 52.164 75.825 59.469 52.383 0 0 0 1.79 2.018 2.132 0.632 0.531 0.484 0.961 0.952 0.949 3.842 4.122 4.341 3 0 1.021 2.003 69.32 57.175 51.056 69.111 57.094 50.958 69.216 57.135 51.007 0 0 0 1.861 2.034 2.135 0.588 0.512 0.47 0.936 0.927 0.917 3.865 4.11 4.299 4 0 1.022 2.023 66.6 56.063 50.336 66.316 55.886 50.125 66.458 55.975 50.23 0 0 0 1.915 2.069 2.165 0.574 0.508 0.469 0.956 0.948 0.943 3.971 4.21 4.407 5 0 1.031 2.013 78.98 60.313 53.014 78.509 59.872 52.318 78.745 60.093 52.666 0 0 0 1.727 1.972 2.088 0.64 0.526 0.477 0.923 0.904 0.896 3.711 3.965 4.169 6 0 1.031 2.003 73.568 61.092 55.185 73.275 60.855 54.831 73.421 60.974 55.008 0 0 0 1.872 2.054 2.15 0.629 0.55 0.508 1.037 1.031 1.024 4.077 4.315 4.488 1 0 1.042 2.023 69.117 59.455 54.327 67.769 57.769 52.782 68.443 58.612 53.554 0 0 0 1.926 2.074 2.159 0.588 0.523 0.49 1.002 0.999 0.997 4.075 4.306 4.474 2 0 1.031 2.013 69.333 59.01 53.934 69.128 58.947 53.963 69.23 58.978 53.948 -0.306 -0.306 -0.306 1.881 2.03 2.112 0.592 0.528 0.494 0.966 0.963 0.96 3.947 4.164 4.316 3 0 1.021 2.003 69.778 57.981 52.532 69.603 57.787 52.383 69.691 57.884 52.458 0 0 0 1.823 1.985 2.07 0.58 0.508 0.471 0.887 0.876 0.867 3.723 3.938 4.089 4 0 1.032 2.023 64.143 55.597 50.956 64.022 55.561 50.727 64.083 55.579 50.842 0 0 0 1.913 2.038 2.115 0.55 0.496 0.464 0.903 0.896 0.893 3.869 4.07 4.226 5 0 1.022 2.033 67.777 57.472 51.975 67.441 57.153 51.618 67.609 57.312 51.797 0 0 0 1.888 2.04 2.13 0.578 0.514 0.477 0.941 0.937 0.932 3.907 4.139 4.312 6 7 8 9 24 Testing Liquid Formamide Treatment Sample Time [s] CA[L] CA[R] CA[M] Tilt SALINE 1 0 45.958 45.043 45.5 P.putida 1.002 36.233 35.696 35.965 2.033 30.801 30.941 30.871 Tergitol 500 mg/L P.putida 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 2.129 0.41 0.79 4.119 2.267 0.341 0.725 4.412 2.356 0.302 0.687 4.65 2 0 1.021 2.003 47.756 38.976 33.816 47.105 38.354 33.164 47.43 38.665 33.49 0 0 0 2.075 2.219 2.329 0.421 0.36 0.322 0.773 0.742 0.725 3.97 4.301 4.605 3 0 1.031 2.003 47.784 39.787 34.038 47.143 39.019 33.279 47.464 39.403 33.659 0 0 0 2.079 2.204 2.308 0.42 0.365 0.322 0.775 0.739 0.709 3.981 4.252 4.524 4 0 1.031 2.003 46.015 39.9 35.427 45.466 39.228 35.015 45.741 39.564 35.221 -0.409 -0.409 -0.409 2.121 2.242 2.34 0.412 0.372 0.342 0.79 0.791 0.784 4.103 4.428 4.707 5 0 1.001 2.003 46.662 40.215 36.207 45.63 39.505 35.518 46.146 39.86 35.862 -0.306 -0.306 -0.306 2.131 2.228 2.308 0.417 0.369 0.339 0.813 0.776 0.76 4.165 4.369 4.583 6 0 1.032 2.013 47.733 41.806 37.527 47.186 41.28 37.055 47.46 41.543 37.291 -0.306 -0.306 -0.306 2.119 2.222 2.303 0.429 0.386 0.354 0.825 0.807 0.79 4.144 4.38 4.595 1 0 1.021 2.003 37.649 24.869 20.393 37.473 23.867 18.251 37.561 24.368 19.322 0 0 0 2.259 2.575 2.742 0.356 0.257 0.216 0.753 0.696 0.709 4.416 5.426 6.319 2 0 1.031 2.013 36.492 27.095 22.695 35.427 26.197 21.732 35.959 26.646 22.213 0 0 0 2.169 2.352 2.44 0.325 0.259 0.224 0.638 0.584 0.553 4.053 4.564 4.898 3 0 1.022 2.003 45.993 32.161 23.731 46.371 33.223 26.308 46.182 32.692 25.02 0 0 0 1.918 2.126 2.307 0.389 0.298 0.244 0.601 0.561 0.607 3.371 3.874 4.74 4 0 1.022 2.003 33.444 25.613 21.485 33.089 25.085 20.745 33.266 25.349 21.115 0 0 0 2.127 2.266 2.375 0.295 0.235 0.202 0.551 0.49 0.465 3.839 4.204 4.564 5 6 7 8 25 Testing Liquid Formamide Treatment Sample Time [s] CA[L] CA[R] CA[M] Tilt Tergitol 1 0 39.231 38.891 39.061 1000 mg/L 1.021 22.59 21.919 22.255 P.putida 2.003 17.442 16.574 17.008 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 2.106 0.346 0.638 3.869 2.373 0.215 0.493 4.575 2.54 0.174 0.459 5.187 2 0 1.032 2.013 35.516 25.716 20.682 32.21 23.308 18.484 33.863 24.512 19.583 -0.613 -0.613 -0.613 2.182 2.342 2.454 0.322 0.246 0.202 0.873 0.736 0.647 4.874 5.293 5.636 3 0 1.031 2.013 33.352 19.516 15.473 33.331 19.564 15.308 33.342 19.54 15.391 -0.919 -0.919 -0.919 2.195 2.447 2.58 0.304 0.193 0.157 0.611 0.482 0.447 4.106 4.873 5.415 4 0 1.021 1.993 48.598 29.871 22.756 48.707 29.575 22.623 48.653 29.723 22.69 -0.204 -0.204 -0.204 2.013 2.286 2.443 0.42 0.281 0.226 0.729 0.609 0.555 3.769 4.38 4.875 1 0 1.031 2.003 38.908 28.301 23.705 38.441 27.924 23.206 38.675 28.112 23.456 0 0 0 2.284 2.561 2.757 0.361 0.286 0.25 0.79 0.775 0.79 4.53 5.417 6.185 2 0 1.031 2.003 36.216 24.745 19.677 35.546 24.149 19.003 35.881 24.447 19.34 0 0 0 2.267 2.535 2.704 0.335 0.251 0.206 0.718 0.66 0.618 4.409 5.256 5.884 3 0 1.031 2.003 38.83 29.549 24.477 38.007 28.932 23.64 38.419 29.24 24.058 0 0 0 2.083 2.264 2.388 0.339 0.275 0.237 0.611 0.58 0.551 3.784 4.284 4.674 4 0 1.022 1.993 39.784 29.851 24.318 38.932 29.258 24.271 39.358 29.554 24.294 0 0 0 2.052 2.226 2.332 0.341 0.272 0.232 0.601 0.551 0.509 3.696 4.128 4.422 5 0 1.021 1.993 35.742 27.145 22.472 35.392 27.005 21.726 35.567 27.075 22.099 0 0 0 2.178 2.34 2.481 0.322 0.256 0.221 0.633 0.578 0.561 4.062 4.518 5.014 5 6 Tergitol 4000 mg/L P.putida 26 Testing Liquid Treatment SALINE R.erythropolis Rhamnolipid 500 mg/L R.erythropolis WATER Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0.02 94.561 93.937 94.249 0.972 92.672 91.56 92.116 2.003 87.079 86.369 86.724 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.605 0.78 1.081 4.044 1.627 0.758 1.061 3.997 1.652 0.707 0.981 3.806 2 0 0.971 1.963 97.598 94.487 88.57 96.478 93.598 87.902 97.038 94.042 88.236 0 0 0 1.573 1.595 1.622 0.783 0.757 0.707 1.067 1.034 0.955 4.011 3.928 3.734 3 0 0.971 1.963 95.327 91.069 85.647 94.613 90.469 85.247 94.97 90.769 85.447 0 0 0 1.598 1.621 1.648 0.774 0.737 0.691 1.068 1.012 0.944 4.013 3.875 3.716 4 0 1.072 2.033 96.648 93.554 88.171 96.319 93.126 87.89 96.484 93.34 88.031 0 0 0 1.578 1.605 1.631 0.791 0.758 0.71 1.079 1.043 0.967 4.041 3.949 3.766 5 0 1.072 2.033 95.625 89.853 84.558 95.597 90.331 85.078 95.611 90.092 84.818 0 0 0 1.592 1.624 1.654 0.778 0.733 0.69 1.07 1.006 0.945 4.019 3.861 3.72 6 0 1.012 1.983 96.616 95.1 87.207 95.171 93.422 86.062 95.893 94.261 86.635 0 0 0 1.592 1.608 1.648 0.786 0.773 0.703 1.087 1.076 0.971 4.061 4.033 3.78 1 0.03 0.871 1.853 90.98 88.003 84.328 91.812 87.802 83.78 91.396 87.902 84.054 0 0 0 1.651 1.698 1.73 0.765 0.728 0.686 1.098 1.074 1.025 4.089 4.044 3.944 2 3.845 4.827 13.039 95.912 90.236 85.042 95.092 89.294 84.505 95.502 89.765 84.774 0 0 0 1.599 1.671 1.722 0.779 0.747 0.703 1.082 1.082 1.044 4.048 4.054 3.982 3 15.092 16.043 17.084 94.161 89.487 84.093 93.525 88.83 83.455 93.843 89.159 83.774 0 0 0 1.615 1.674 1.719 0.763 0.73 0.68 1.066 1.056 1 4.008 3.994 3.882 4 26.328 27.349 28.331 92.48 87.527 83.067 90.213 85.509 80.796 91.346 86.518 81.931 0 0 0 1.641 1.7 1.74 0.756 0.72 0.673 1.075 1.059 1.007 4.031 4.007 3.91 5 30.334 31.365 38.385 88.129 81.876 76.616 87.95 80.133 73.57 88.039 81.005 75.093 0 0 0 1.774 1.868 1.946 0.763 0.721 0.674 1.229 1.233 1.22 4.422 4.477 4.518 6 7 8 27 Testing Liquid Treatment Rhamnolipid 1000 mg/L R.erythropolis Rhamnolipid 4000 mg/L R.erythropolis WATER Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 88.07 87.781 87.925 1.001 85.566 85.845 85.705 2.033 83.197 83.413 83.305 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.687 0.731 1.064 4.016 1.714 0.716 1.06 4.017 1.738 0.696 1.044 3.991 2 0 1.001 2.033 85.715 84.875 83.579 85.622 84.247 82.042 85.668 84.561 82.81 0 0 0 1.705 1.724 1.74 0.732 0.72 0.697 1.072 1.07 1.045 4.04 4.041 3.994 3 0 1.001 2.033 88.159 86.231 83.301 84.958 84.201 81.368 86.559 85.216 82.334 0 0 0 1.681 1.703 1.724 0.74 0.727 0.698 1.083 1.067 1.028 4.059 4.028 3.947 4 0 1.001 2.033 89.097 86.824 82.516 88.629 86.195 81.975 88.863 86.51 82.245 0 0 0 1.667 1.696 1.728 0.732 0.719 0.689 1.049 1.049 1.014 3.974 3.983 3.916 5 0 1.001 2.033 89.517 87.231 83.346 89.236 86.771 83.193 89.377 87.001 83.269 0 0 0 1.66 1.691 1.722 0.733 0.719 0.689 1.045 1.046 1.014 3.964 3.973 3.914 6 0 1.012 2.003 87.823 85.255 82.078 87.781 85.215 81.861 87.802 85.235 81.969 0 0 0 1.704 1.736 1.767 0.731 0.717 0.691 1.087 1.085 1.062 4.074 4.082 4.046 1 0 1.052 2.013 80.175 77.909 75.527 78.313 76.239 73.788 79.244 77.074 74.657 0 0 0 1.831 1.861 1.89 0.694 0.679 0.66 1.131 1.127 1.117 4.237 4.252 4.257 2 0 1.052 2.013 80.522 78.198 74.661 79.795 77.474 73.998 80.159 77.836 74.329 0 0 0 1.822 1.853 1.879 0.692 0.674 0.643 1.116 1.113 1.071 4.199 4.217 4.152 3 0 1.052 2.013 81.875 79.106 76.182 81.432 78.806 75.909 81.654 78.956 76.046 0 0 0 1.801 1.836 1.863 0.7 0.683 0.659 1.114 1.112 1.088 4.181 4.201 4.172 4 0 1.022 2.013 79.929 76.678 72.619 79.346 76.16 72.058 79.637 76.419 72.338 0 0 0 1.763 1.805 1.834 0.671 0.65 0.613 1.013 1.008 0.961 3.936 3.956 3.885 5 0 1.052 2.013 78.567 75.685 70.222 78.307 75.497 70.117 78.437 75.591 70.17 0 0 0 1.855 1.886 1.909 0.691 0.667 0.617 1.144 1.124 1.036 4.282 4.266 4.116 6 0 1.052 2.013 79.112 73.629 68.843 78.431 73.101 68.563 78.771 73.365 68.703 0 0 0 1.858 1.891 1.914 0.688 0.642 0.598 1.146 1.076 1.004 4.288 4.176 4.061 7 0 1.022 2.013 79.385 76.247 71.343 78.956 75.602 70.914 79.171 75.924 71.129 0 0 0 1.765 1.805 1.837 0.667 0.644 0.601 1.002 0.997 0.939 3.912 3.933 3.844 8 0 1.022 2.013 79.247 76.525 72.02 78.434 75.738 71.164 78.84 76.131 71.592 0 0 0 1.777 1.813 1.845 0.669 0.65 0.608 1.02 1.018 0.963 3.961 3.983 3.901 9 0 1.022 2.013 78.896 74.901 69.736 77.722 73.482 67.578 78.309 74.191 68.657 0 0 0 1.782 1.833 1.883 0.662 0.638 0.593 1.013 1.01 0.972 3.95 3.985 3.964 28 Testing Liquid Treatment SALINE R.erythropolis Rhamnolipid 500 mg/L R.erythropolis 1-Bromonaphthalene Sample Time [s] CA[L] CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] 1 0 43.109 43.641 43.375 -0.306 1.855 0.327 0.481 3.069 0.991 39.598 39.991 39.794 -0.306 1.853 0.298 0.434 3.002 2.003 35.947 36.288 36.117 -0.306 1.855 0.271 0.392 2.955 2 0 1.041 2.003 44.479 42.276 40.25 41.412 39.177 37.233 42.945 40.727 38.741 0 0 0 1.968 1.968 1.971 0.33 0.311 0.295 0.556 0.523 0.493 3.445 3.406 3.374 3 0 1.031 2.003 41.539 38.509 35.686 41.562 38.378 35.599 41.551 38.443 35.642 -0.306 -0.306 -0.306 1.886 1.886 1.887 0.322 0.296 0.273 0.485 0.445 0.408 3.144 3.095 3.05 4 0 0.992 2.003 40.154 38.108 36.277 38.985 36.846 35.078 39.57 37.477 35.678 -0.306 -0.306 -0.306 1.907 1.907 1.907 0.31 0.293 0.279 0.48 0.453 0.429 3.196 3.169 3.139 5 0 1.021 1.992 41.145 36.789 33.267 40.49 36.155 32.512 40.817 36.472 32.889 -0.306 -0.306 -0.306 1.893 1.891 1.89 0.313 0.277 0.247 0.478 0.42 0.372 3.154 3.08 3.023 6 0 1.031 1.993 41.901 39.297 37.02 40.896 38.21 36.095 41.399 38.754 36.558 0 0 0 1.926 1.925 1.926 0.327 0.304 0.287 0.513 0.474 0.446 3.272 3.221 3.191 1 0 1.022 2.003 34.122 27.305 22.912 35.475 28.303 23.962 34.799 27.804 23.437 0 0 0 1.852 1.845 1.839 0.26 0.206 0.173 0.374 0.289 0.24 2.928 2.815 2.758 2 0 1.032 2.023 38.885 27.076 21.405 38.102 26.461 20.887 38.493 26.769 21.146 0 0 0 1.808 1.781 1.774 0.328 0.193 0.153 0.442 0.252 0.195 2.937 2.616 2.542 3 0 1.031 2.013 36.916 25.748 21.549 36.919 25.898 21.406 36.917 25.823 21.477 -0.102 -0.102 -0.102 1.856 1.844 1.819 0.277 0.189 0.149 0.402 0.266 0.206 2.964 2.791 2.678 4 0 1.022 2.003 35.77 27.194 21.258 35.02 26.096 20.719 35.395 26.645 20.989 -0.204 -0.204 -0.204 1.825 1.818 1.822 0.265 0.194 0.156 0.37 0.268 0.214 2.859 2.742 2.706 5 0 1.022 1.993 32.194 27.036 22.659 31.143 25.269 21.475 31.668 26.153 22.067 0 0 0 1.818 1.813 1.812 0.237 0.192 0.163 0.326 0.264 0.22 2.793 2.733 2.683 6 0 1.021 2.002 30.84 23.816 18.465 30.888 23.682 18.344 30.864 23.749 18.405 0 0 0 1.809 1.805 1.794 0.232 0.178 0.138 0.31 0.234 0.178 2.74 2.655 2.589 7 8 29 Testing Liquid Treatment Rhamnolipid 1000 mg/L R.erythropolis Rhamnolipid 4000 mg/L R.erythropolis 1-Bromonaphthalene Sample Time [s] CA[L] CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] 1 0 36.837 39.972 38.404 -0.409 1.988 0.314 0.535 3.488 1.001 35.648 38.625 37.136 -0.409 2.016 0.306 0.533 3.554 2.003 35.092 37.572 36.332 -0.409 2.035 0.299 0.527 3.585 2 0 1.022 1.993 40.942 39.693 38.806 40.923 39.286 38.326 40.932 39.49 38.566 0 0 0 1.882 1.903 1.92 0.313 0.307 0.3 0.468 0.468 0.464 3.109 3.16 3.19 3 0 1.021 2.003 40.998 39.913 39.042 41.192 39.859 38.982 41.095 39.886 39.012 -0.306 -0.306 -0.306 1.917 1.935 1.95 0.322 0.314 0.309 0.502 0.498 0.496 3.237 3.275 3.312 4 0 1.021 2.003 41.226 40.054 38.728 41.093 39.476 38.621 41.159 39.765 38.674 0 0 0 1.895 1.919 1.94 0.318 0.309 0.304 0.48 0.481 0.48 3.15 3.216 3.26 5 0 0.991 2.013 41.564 40.708 39.766 41.073 40.56 39.006 41.318 40.634 39.386 0 0 0 1.897 1.903 1.92 0.32 0.312 0.307 0.486 0.478 0.477 3.165 3.171 3.214 6 0 1.032 2.013 39.387 37.773 36.241 39.749 38.065 36.86 39.568 37.919 36.551 -0.306 -0.306 -0.306 1.894 1.895 1.895 0.31 0.296 0.286 0.469 0.447 0.43 3.144 3.119 3.097 1 0 1.031 2.013 40.135 27.257 21.241 38.916 30.283 25.085 39.525 28.77 23.163 0 0 0 1.672 1.722 1.74 0.271 0.214 0.174 0.319 0.332 0.386 2.442 2.854 3.457 2 0 1.031 2.012 34.546 28.322 23.385 32.956 26.902 22.193 33.751 27.612 22.789 -0.204 -0.204 -0.204 1.656 1.656 1.656 0.23 0.187 0.154 0.268 0.216 0.178 2.356 2.299 2.268 3 0 1.042 2.023 45.016 33.801 28.97 45.381 33.675 28.798 45.198 33.738 28.884 0 0 0 1.734 1.73 1.731 0.33 0.243 0.209 0.421 0.299 0.255 2.723 2.539 2.491 4 0 1.031 2.002 39.207 34.22 31.466 39.048 33.799 30.728 39.127 34.009 31.097 0 0 0 1.746 1.743 1.742 0.285 0.245 0.222 0.361 0.307 0.276 2.654 2.581 2.541 5 0 1.031 2.023 38.003 32.734 28.976 36.804 31.266 27.329 37.404 32 28.153 0 0 0 1.714 1.711 1.708 0.256 0.214 0.184 0.318 0.263 0.225 2.536 2.461 2.411 6 0 1.031 1.992 38.774 36.775 34.263 38.214 36.159 32.901 38.494 36.467 33.582 0 0 0 1.85 1.859 1.885 0.287 0.272 0.255 0.414 0.392 0.381 2.968 2.953 3.021 7 8 9 30 Testing Liquid Treatment SALINE R.erythropolis Rhamnolipid 500 mg/L R.erythropolis Glycerol Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 103.193 102.314 102.754 1.022 100.468 100.82 100.644 2.013 99.367 98.802 99.085 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.501 0.8 1.044 3.966 1.531 0.785 1.04 3.949 1.553 0.777 1.046 3.962 2 0 1.032 2.003 103.892 100.501 99.062 103.372 100.553 99.417 103.632 100.527 99.239 0 0 0 1.496 1.546 1.563 0.811 0.786 0.777 1.058 1.06 1.06 4.004 3.999 3.997 3 0 1.032 2.013 102.206 101.94 101.072 102.502 101.385 100.207 102.354 101.663 100.639 0 0 0 1.513 1.532 1.545 0.803 0.793 0.787 1.06 1.063 1.062 4.004 4.01 4.005 4 0 1.031 2.023 104.423 102.644 101.269 103.206 102.039 100.07 103.814 102.342 100.67 -0.204 -0.204 -0.204 1.478 1.496 1.515 0.797 0.785 0.778 1.017 1.012 1.014 3.9 3.881 3.883 5 0 1.031 2.012 103.229 102.223 101.254 105.652 103.568 102.329 104.44 102.896 101.791 0 0 0 1.453 1.487 1.499 0.797 0.781 0.776 0.993 0.997 0.996 3.841 3.844 3.838 6 0 1.032 2.003 102.612 100.218 100.088 102.754 100.646 100.562 102.683 100.432 100.325 0 0 0 1.45 1.485 1.49 0.782 0.766 0.761 0.952 0.956 0.953 3.729 3.733 3.725 1 0 1.032 2.003 104.955 101.377 100.779 105.728 101.605 100.859 105.342 101.491 100.819 0 0 0 1.467 1.497 1.505 0.813 0.787 0.784 1.037 1.008 1.009 3.957 3.869 3.872 2 0.01 1.031 2.003 103.207 102.207 101.7 105.321 104.721 104.208 104.264 103.464 102.954 0 0 0 1.478 1.485 1.491 0.799 0.796 0.794 1.024 1.022 1.022 3.919 3.912 3.91 3 0 1.042 2.013 106.635 102.617 102.276 105.85 103.295 103.013 106.243 102.956 102.645 0 0 0 1.437 1.478 1.482 0.807 0.793 0.791 0.997 1.003 1.004 3.859 3.862 3.864 4 0 1.031 2.012 107.824 102.984 102.193 104.75 101.62 100.988 106.287 102.302 101.59 0 0 0 1.442 1.497 1.506 0.81 0.796 0.794 1.011 1.028 1.032 3.895 3.924 3.932 5 0 0.991 2.033 102.944 102.146 101.593 102.675 102.611 102.024 102.809 102.379 101.808 0 0 0 1.454 1.456 1.459 0.79 0.785 0.783 0.971 0.961 0.959 3.779 3.754 3.746 6 7 8 31 Testing Liquid Treatment Rhamnolipid 1000 mg/L R.erythropolis Glycerol Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 102.668 102.465 102.567 1.031 97.964 98.233 98.099 2.013 96.668 97.061 96.865 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.487 0.78 0.993 3.833 1.541 0.76 1 3.844 1.557 0.754 1.001 3.845 2 0 1.031 2.003 101.12 98.779 98.056 100.136 98.701 98.066 100.628 98.74 98.061 0 0 0 1.54 1.561 1.568 0.786 0.776 0.772 1.055 1.053 1.049 3.986 3.978 3.968 3 0 1.022 1.993 101.691 98.637 98.322 102.063 97.642 96.94 101.877 98.139 97.631 0 0 0 1.51 1.557 1.567 0.799 0.777 0.769 1.045 1.044 1.043 3.966 3.956 3.952 4 0 1.032 2.003 101.826 99.156 98.387 102.285 100.237 99.391 102.056 99.697 98.889 0 0 0 1.512 1.539 1.551 0.782 0.77 0.764 1.023 1.023 1.023 3.909 3.905 3.904 1 0 0.991 2.013 108.931 96.044 94.033 106.573 95.29 94.272 107.752 95.667 94.153 0 0 0 1.411 1.562 1.579 0.805 0.748 0.736 0.974 0.991 0.983 3.806 3.818 3.799 2 0 1.022 2.003 106.978 100.486 98.038 103.178 97.585 96.157 105.078 99.036 97.098 0 0 0 1.411 1.478 1.511 0.777 0.751 0.738 0.918 0.921 0.927 3.647 3.64 3.654 3 0 0.991 2.023 100.697 99.003 98.56 101.764 100.118 99.279 101.23 99.56 98.919 0 0 0 1.48 1.496 1.504 0.773 0.763 0.757 0.965 0.959 0.955 3.757 3.739 3.728 4 0 0.991 2.012 102.869 99.535 98.438 106.092 101.013 100.018 104.48 100.274 99.228 0 0 0 1.445 1.492 1.505 0.791 0.776 0.769 0.973 0.98 0.979 3.791 3.795 3.79 5 0 0.982 2.013 111.717 108.434 110.555 112.104 110.016 110.569 111.911 109.225 110.562 -0.306 -0.306 -0.306 1.376 1.391 1.382 0.784 0.775 0.771 0.929 0.917 0.909 3.7 3.657 3.638 5 6 Rhamnolipid 4000 mg/L R.erythropolis 6 7 8 9 32 Testing Liquid Treatment SALINE R.erythropolis Formamide Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0.02 68.273 67.63 67.952 0.991 59.083 58.678 58.881 2.033 57.383 57.276 57.33 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.816 0.56 0.844 3.628 1.874 0.487 0.752 3.537 1.877 0.472 0.728 3.499 2 0 0.991 2.043 65.973 62.29 61.555 66.013 61.862 61.069 65.993 62.076 61.312 0 0 0 1.862 1.911 1.914 0.547 0.521 0.514 0.86 0.851 0.84 3.718 3.773 3.759 3 0 1.052 2.043 65.797 60.883 59.213 65.655 61.478 59.703 65.726 61.18 59.458 0 0 0 1.868 1.911 1.915 0.547 0.514 0.499 0.864 0.836 0.81 3.735 3.747 3.707 4 0 1.022 2.043 73.314 60.741 57.477 73.161 58.613 56.391 73.237 59.677 56.934 0 0 0 1.786 1.913 1.938 0.613 0.506 0.489 0.915 0.827 0.804 3.742 3.742 3.739 1 0 1.012 2.023 66.001 65.626 65.23 65.29 64.999 64.472 65.646 65.312 64.851 0 0 0 1.857 1.862 1.865 0.543 0.541 0.537 0.85 0.849 0.843 3.694 3.699 3.692 2 0 1.052 2.023 67.204 66.664 65.675 65.673 65.085 64.336 66.439 65.874 65.006 0 0 0 1.83 1.835 1.842 0.539 0.532 0.525 0.822 0.816 0.808 3.605 3.6 3.595 3 0 1.012 2.104 67.043 66.448 65.538 66.414 65.791 64.953 66.728 66.119 65.246 0 0 0 1.834 1.838 1.846 0.548 0.54 0.533 0.837 0.83 0.822 3.638 3.629 3.624 4 0 0.982 1.963 67.978 67.785 67.032 67.059 66.662 66.074 67.519 67.223 66.553 0 0 0 1.823 1.827 1.835 0.545 0.541 0.537 0.83 0.826 0.825 3.611 3.607 3.618 5 0 1.052 2.023 67.204 66.664 65.675 65.673 65.085 64.336 66.439 65.874 65.006 0 0 0 1.83 1.835 1.842 0.539 0.532 0.525 0.822 0.816 0.808 3.605 3.6 3.595 6 0 1.011 2.032 67.421 64.192 63.697 66.188 64.069 63.503 66.805 64.13 63.6 0 0 0 1.838 1.89 1.895 0.545 0.531 0.526 0.838 0.853 0.85 3.646 3.745 3.748 5 6 Rhamnolipid 500 mg/L R.erythropolis 7 8 33 Testing Liquid Treatment Rhamnolipid 1000 mg/L R.erythropolis Formamide Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 68.48 67.124 67.802 1.012 65.524 64.134 64.829 2.043 63.094 62.21 62.652 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.796 0.545 0.805 3.527 1.825 0.524 0.791 3.538 1.851 0.509 0.782 3.557 2 0 1.042 2.003 67.69 64.435 60.172 66.573 63.324 59.246 67.131 63.879 59.709 0 0 0 1.817 1.849 1.894 0.547 0.524 0.494 0.825 0.807 0.787 3.593 3.601 3.636 3 0 1.011 2.043 59.439 56.366 52.996 59.709 56.366 52.996 59.574 56.366 52.996 0 0 0 2.031 2.07 2.114 0.513 0.479 0.46 1.029 0.999 0.991 4.368 4.416 4.503 4 0 1.001 2.013 69.959 67.467 65.41 69.752 66.977 64.925 69.855 67.222 65.168 0 0 0 1.767 1.792 1.814 0.559 0.54 0.523 0.805 0.79 0.78 3.492 3.491 3.5 5 0 1.012 2.003 71.219 67.649 65.157 64.914 59.891 55.036 68.067 63.77 60.097 0 0 0 1.815 1.872 1.944 0.58 0.558 0.543 1.002 1.237 1.866 4.003 4.615 6.018 1 0.01 1.312 1.993 61.595 57.108 54.072 64.041 59.137 55.568 62.818 58.123 54.82 -0.204 0 0 1.833 1.884 1.92 0.517 0.483 0.458 0.787 0.761 0.736 3.547 3.579 3.594 2 0 1.021 2.033 66.002 60.158 56.273 62.971 56.896 53.891 64.487 58.527 55.082 0 0 0 1.726 1.787 1.821 0.506 0.466 0.441 0.692 0.673 0.646 3.221 3.276 3.274 3 0 1.041 2.033 66.701 61.179 57.33 63.883 58.153 55.006 65.292 59.666 56.168 0 0 0 1.726 1.787 1.821 0.515 0.477 0.451 0.706 0.689 0.661 3.248 3.302 3.3 4 0 1.011 2.003 61.302 54.127 50.539 54.996 48.241 44.38 58.149 51.184 47.46 0 0 0 1.935 2.046 2.099 0.505 0.458 0.433 0.957 0.934 0.969 4.103 4.278 4.516 6 Rhamnolipid 4000 mg/L R.erythropolis 5 6 7 8 9 34 Testing Liquid Treatment SALINE R.erythropolis Rhamnolipid 500 mg/L R.erythropolis Water Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0.02 94.561 93.937 94.249 0.972 92.672 91.56 92.116 2.003 87.079 86.369 86.724 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.605 0.78 1.081 4.044 1.627 0.758 1.061 3.997 1.652 0.707 0.981 3.806 2 0 0.971 1.963 97.598 94.487 88.57 96.478 93.598 87.902 97.038 94.042 88.236 0 0 0 1.573 1.595 1.622 0.783 0.757 0.707 1.067 1.034 0.955 4.011 3.928 3.734 3 0 0.971 1.963 95.327 91.069 85.647 94.613 90.469 85.247 94.97 90.769 85.447 0 0 0 1.598 1.621 1.648 0.774 0.737 0.691 1.068 1.012 0.944 4.013 3.875 3.716 4 0 1.072 2.033 96.648 93.554 88.171 96.319 93.126 87.89 96.484 93.34 88.031 0 0 0 1.578 1.605 1.631 0.791 0.758 0.71 1.079 1.043 0.967 4.041 3.949 3.766 5 0 1.072 2.033 95.625 89.853 84.558 95.597 90.331 85.078 95.611 90.092 84.818 0 0 0 1.592 1.624 1.654 0.778 0.733 0.69 1.07 1.006 0.945 4.019 3.861 3.72 6 0 1.012 1.983 96.616 95.1 87.207 95.171 93.422 86.062 95.893 94.261 86.635 0 0 0 1.592 1.608 1.648 0.786 0.773 0.703 1.087 1.076 0.971 4.061 4.033 3.78 1 0 1.022 2.033 77.452 75.132 71.76 77.436 75.014 71.674 77.444 75.073 71.717 0 0 0 1.804 1.835 1.863 0.661 0.644 0.615 1.03 1.025 0.994 4.002 4.017 3.982 2 0 1.022 2.033 75.511 72.377 68.444 75.322 72.141 68.22 75.416 72.259 68.332 0 0 0 1.832 1.867 1.891 0.646 0.623 0.583 1.027 1.013 0.956 4.019 4.024 3.94 3 0 1.022 2.033 75.933 72.709 70.356 76.935 73.506 70.98 76.434 73.108 70.668 0 0 0 1.831 1.86 1.893 0.662 0.634 0.615 1.059 1.027 1.019 4.084 4.044 4.066 4 0 1.022 2.013 79.229 76.292 71.792 79.191 76.158 71.593 79.21 76.225 71.692 0 0 0 1.773 1.811 1.844 0.667 0.648 0.609 1.015 1.011 0.962 3.947 3.966 3.898 5 0 1.022 2.013 79.247 76.525 72.02 78.434 75.738 71.164 78.84 76.131 71.592 0 0 0 1.777 1.813 1.845 0.669 0.65 0.608 1.02 1.018 0.963 3.961 3.983 3.901 6 0 1.022 2.013 79.385 76.247 71.343 78.956 75.602 70.914 79.171 75.924 71.129 0 0 0 1.765 1.805 1.837 0.667 0.644 0.601 1.002 0.997 0.939 3.912 3.933 3.844 7 0 1.022 2.013 79.929 76.678 72.619 79.346 76.16 72.058 79.637 76.419 72.338 0 0 0 1.763 1.805 1.834 0.671 0.65 0.613 1.013 1.008 0.961 3.936 3.956 3.885 8 0 1.022 2.013 78.896 74.901 69.736 77.722 73.482 67.578 78.309 74.191 68.657 0 0 0 1.782 1.833 1.883 0.662 0.638 0.593 1.013 1.01 0.972 3.95 3.985 3.964 35 Testing Liquid Treatment Rhamnolipid 1000 mg/L R.erythropolis Rhamnolipid 4000 mg/L R.erythropolis Water Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 88.07 87.781 87.925 1.001 85.566 85.845 85.705 2.033 83.197 83.413 83.305 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.687 0.731 1.064 4.016 1.714 0.716 1.06 4.017 1.738 0.696 1.044 3.991 2 0 1.001 2.033 85.715 84.875 83.579 85.622 84.247 82.042 85.668 84.561 82.81 0 0 0 1.705 1.724 1.74 0.732 0.72 0.697 1.072 1.07 1.045 4.04 4.041 3.994 3 0 1.001 2.033 88.159 86.231 83.301 84.958 84.201 81.368 86.559 85.216 82.334 0 0 0 1.681 1.703 1.724 0.74 0.727 0.698 1.083 1.067 1.028 4.059 4.028 3.947 4 0 1.001 2.033 89.097 86.824 82.516 88.629 86.195 81.975 88.863 86.51 82.245 0 0 0 1.667 1.696 1.728 0.732 0.719 0.689 1.049 1.049 1.014 3.974 3.983 3.916 5 0 1.001 2.033 89.517 87.231 83.346 89.236 86.771 83.193 89.377 87.001 83.269 0 0 0 1.66 1.691 1.722 0.733 0.719 0.689 1.045 1.046 1.014 3.964 3.973 3.914 6 0 1.012 2.003 87.823 85.255 82.078 87.781 85.215 81.861 87.802 85.235 81.969 0 0 0 1.704 1.736 1.767 0.731 0.717 0.691 1.087 1.085 1.062 4.074 4.082 4.046 1 0.03 0.871 1.853 90.98 88.003 84.328 91.812 87.802 83.78 91.396 87.902 84.054 0 0 0 1.651 1.698 1.73 0.765 0.728 0.686 1.098 1.074 1.025 4.089 4.044 3.944 2 3.845 4.827 13.039 95.912 90.236 85.042 95.092 89.294 84.505 95.502 89.765 84.774 0 0 0 1.599 1.671 1.722 0.779 0.747 0.703 1.082 1.082 1.044 4.048 4.054 3.982 3 15.092 16.043 17.084 94.161 89.487 84.093 93.525 88.83 83.455 93.843 89.159 83.774 0 0 0 1.615 1.674 1.719 0.763 0.73 0.68 1.066 1.056 1 4.008 3.994 3.882 4 26.328 27.349 28.331 92.48 87.527 83.067 90.213 85.509 80.796 91.346 86.518 81.931 0 0 0 1.641 1.7 1.74 0.756 0.72 0.673 1.075 1.059 1.007 4.031 4.007 3.91 5 30.334 31.365 38.385 88.129 81.876 76.616 87.95 80.133 73.57 88.039 81.005 75.093 0 0 0 1.774 1.868 1.946 0.763 0.721 0.674 1.229 1.233 1.22 4.422 4.477 4.518 6 7 8 9 36 Testing Liquid Treatment SALINE R.erythropolis Tergitol 500 mg/L R.erythropolis WATER Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 111.935 112.794 112.365 1.021 110.717 109.552 110.135 2.013 106.674 105.767 106.221 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.373 0.897 1.104 4.185 1.389 0.874 1.069 4.079 1.411 0.836 1.008 3.897 2 0 1.011 2.033 105.895 94.212 87.305 106.015 95.643 88.194 105.955 94.927 87.75 0 0 0 1.514 1.644 1.731 0.886 0.813 0.766 1.202 1.192 1.175 4.375 4.316 4.286 3 0 1.031 2.003 116.201 115.162 109.142 116.006 114.731 109.508 116.103 114.946 109.325 0 0 0 1.333 1.347 1.378 0.909 0.884 0.844 1.095 1.067 1.002 4.187 4.101 3.896 4 0 1.031 2.013 117.235 114.368 110.739 117.629 113.148 109.961 117.432 113.758 110.35 0 0 0 1.32 1.348 1.366 0.909 0.888 0.846 1.094 1.068 0.998 4.195 4.099 3.89 5 0 1.001 2.023 114.541 111.917 108.012 113.928 111.653 107.379 114.235 111.785 107.696 0 0 0 1.329 1.347 1.368 0.886 0.862 0.823 1.043 1.009 0.946 4.042 3.933 3.742 6 0 1.032 2.013 115.919 112.586 108.771 114.731 111.675 107.343 115.325 112.13 108.057 -0.204 0 0 1.335 1.356 1.376 0.9 0.878 0.837 1.084 1.049 0.981 4.154 4.04 3.837 1 0.02 0.921 1.962 95.713 89.808 84.695 94.575 89.24 83.891 95.144 89.524 84.293 0 0 0 1.663 1.692 1.732 0.805 0.749 0.702 1.207 1.111 1.052 4.354 4.127 4.006 2 0 1.002 2.033 93.661 91.598 83.127 93.137 90.974 83.087 93.399 91.286 83.107 0 0 0 1.605 1.623 1.661 0.765 0.745 0.677 1.053 1.03 0.925 3.974 3.919 3.675 3 0.02 0.921 1.962 91.463 88.114 82.586 91.679 88.118 82.232 91.571 88.116 82.409 0 0 0 1.62 1.641 1.666 0.764 0.731 0.672 1.057 1.011 0.919 3.985 3.875 3.665 4 0.02 0.921 1.962 91.142 86.743 81.952 93.49 88.215 82.117 92.316 87.479 82.034 0 0 0 1.605 1.632 1.661 0.775 0.725 0.677 1.069 0.991 0.918 4.012 3.824 3.658 5 0 1.011 2.003 96.59 86.191 77.451 96.074 85.644 77.089 96.332 85.918 77.27 0 0 0 1.555 1.66 1.729 0.78 0.699 0.634 1.033 0.973 0.906 3.926 3.79 3.674 6 0 1.011 2.003 87.847 78.655 72.075 87.064 78.053 71.529 87.455 78.354 71.802 0 0 0 1.607 1.676 1.714 0.694 0.627 0.575 0.914 0.848 0.783 3.63 3.504 3.39 7 0 1.011 2.003 101.799 91.993 84.055 100.752 90.817 82.943 101.276 91.405 83.499 0 0 0 1.371 1.43 1.476 0.749 0.676 0.613 0.81 0.728 0.666 3.347 3.108 2.947 8 37 Testing Liquid Treatment Tergitol 1000 mg/L R.erythropolis Tergitol 4000 mg/L R.erythropolis WATER Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 91.955 91.812 91.884 1.041 85.786 85.358 85.572 2.033 80.45 80.165 80.307 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.682 0.772 1.153 4.225 1.709 0.711 1.046 3.981 1.743 0.664 0.982 3.854 2 0 1.041 2.033 98.233 91.847 86.208 96.721 89.961 84.608 97.477 90.904 85.408 0 0 0 1.607 1.636 1.665 0.82 0.759 0.703 1.175 1.069 0.983 4.278 4.016 3.816 3 0 1.041 2.033 98.968 92.792 87.621 98.624 92.532 87.262 98.796 92.662 87.442 0 0 0 1.598 1.622 1.646 0.822 0.763 0.715 1.173 1.066 0.989 4.276 4.009 3.824 4 0 1.031 2.032 90.655 88.426 81.932 90.356 88.078 81.712 90.505 88.252 81.822 0 0 0 1.719 1.737 1.763 0.774 0.752 0.691 1.194 1.165 1.056 4.327 4.265 4.031 5 0 1.031 2.032 89.386 85.552 78.986 88.452 85.071 78.745 88.919 85.311 78.866 0 0 0 1.735 1.752 1.784 0.769 0.731 0.671 1.196 1.127 1.029 4.335 4.183 3.986 6 0 1.031 2.032 89.786 83.646 77.59 89.426 83.476 77.538 89.606 83.561 77.564 0 0 0 1.733 1.76 1.792 0.762 0.707 0.656 1.184 1.089 1.008 4.308 4.102 3.944 1 0 1.011 2.023 85.603 79.639 72.186 85.979 79.768 72.236 85.791 79.704 72.211 0 0 0 1.76 1.794 1.834 0.729 0.673 0.607 1.138 1.05 0.951 4.214 4.038 3.864 2 0 1.011 2.023 83.303 75.016 69.148 83.015 74.681 68.686 83.159 74.849 68.917 0 0 0 1.788 1.82 1.857 0.716 0.632 0.575 1.135 0.989 0.91 4.22 3.929 3.81 3 0 1.011 2.023 87.069 79.826 72.895 86.222 79.031 71.909 86.645 79.429 72.402 0 0 0 1.736 1.77 1.81 0.728 0.66 0.594 1.11 1.003 0.909 4.14 3.92 3.753 4 0 1.011 2.023 83.425 75.097 68.771 83.237 74.944 68.397 83.331 75.02 68.584 0 0 0 1.781 1.814 1.855 0.713 0.634 0.574 1.121 0.988 0.905 4.185 3.921 3.798 5 0 1.011 2.023 84.515 77.87 71.048 83.942 77.365 70.702 84.228 77.617 70.875 0 0 0 1.786 1.822 1.859 0.73 0.669 0.606 1.163 1.064 0.969 4.279 4.088 3.928 6 7 38 Testing Liquid Treatment SALINE R.erythropolis Tergitol 500 mg/L R.erythropolis 1-Bromonaphthalene Sample Time [s] CA[L] CA[R] CA[M] Tilt L[mm] H[mm] Vol[ml] A[mm2] 1 0 43.109 43.641 43.375 -0.306 1.855 0.327 0.481 3.069 0.991 39.598 39.991 39.794 -0.306 1.853 0.298 0.434 3.002 2.003 35.947 36.288 36.117 -0.306 1.855 0.271 0.392 2.955 2 0 1.041 2.003 44.479 42.276 40.25 41.412 39.177 37.233 42.945 40.727 38.741 0 0 0 1.968 1.968 1.971 0.33 0.311 0.295 0.556 0.523 0.493 3.445 3.406 3.374 3 0 1.031 2.003 41.539 38.509 35.686 41.562 38.378 35.599 41.551 38.443 35.642 -0.306 -0.306 -0.306 1.886 1.886 1.887 0.322 0.296 0.273 0.485 0.445 0.408 3.144 3.095 3.05 4 0 0.992 2.003 40.154 38.108 36.277 38.985 36.846 35.078 39.57 37.477 35.678 -0.306 -0.306 -0.306 1.907 1.907 1.907 0.31 0.293 0.279 0.48 0.453 0.429 3.196 3.169 3.139 5 0 1.021 1.992 41.145 36.789 33.267 40.49 36.155 32.512 40.817 36.472 32.889 -0.306 -0.306 -0.306 1.893 1.891 1.89 0.313 0.277 0.247 0.478 0.42 0.372 3.154 3.08 3.023 6 0 1.031 1.993 41.901 39.297 37.02 40.896 38.21 36.095 41.399 38.754 36.558 0 0 0 1.926 1.925 1.926 0.327 0.304 0.287 0.513 0.474 0.446 3.272 3.221 3.191 1 0 1.031 2.003 41.477 37.203 35.051 40.555 37.407 35.754 41.016 37.305 35.402 -0.409 -0.409 -0.409 1.929 1.942 1.94 0.313 0.294 0.278 0.502 0.467 0.44 3.275 3.256 3.225 2 0 1.031 2.013 41.748 33.937 30.294 42.435 34.591 30.848 42.092 34.264 30.571 0 0 0 1.692 1.685 1.68 0.294 0.236 0.209 0.356 0.277 0.243 2.537 2.41 2.358 3 0 1.011 2.043 45.472 42.996 42.443 45.077 42.449 41.969 45.275 42.723 42.206 0 0 0 1.906 1.92 1.92 0.337 0.33 0.326 0.531 0.519 0.512 3.253 3.266 3.256 4 0 1.031 2.023 34.998 29.209 26.24 37.368 29.403 26.411 36.183 29.306 26.326 -0.511 -0.511 -0.511 1.908 1.897 1.893 0.276 0.221 0.197 0.427 0.332 0.294 3.137 2.997 2.948 5 0 1.021 2.003 36.651 29.966 29.109 33.539 29.155 27.18 35.095 29.561 28.144 -0.306 -0.306 -0.306 1.965 1.984 1.97 0.289 0.257 0.237 0.504 0.452 0.427 3.467 3.478 3.467 6 7 8 39 Testing Liquid Treatment Tergitol 1000 mg/L R.erythropolis Tergitol 4000 mg/L R.erythropolis 1-Bromonaphthalene Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 41.158 39.994 40.576 1.042 32.146 32.148 32.147 2.023 28.304 26.479 27.392 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.783 0.314 0.42 2.833 1.806 0.264 0.345 2.774 1.823 0.23 0.331 2.888 2 0 1.032 2.013 40.914 38.78 37.072 40.215 38.408 36.613 40.564 38.594 36.843 0 0 0 1.747 1.745 1.745 0.296 0.28 0.266 0.379 0.357 0.338 2.688 2.651 2.626 3 0 1.031 2.012 40.83 36.251 33.521 40.343 35.865 33.121 40.587 36.058 33.321 0 0 0 1.756 1.752 1.753 0.294 0.261 0.24 0.381 0.332 0.303 2.706 2.633 2.593 4 0 1.031 2.003 42.355 39.854 37.462 41.507 38.979 36.567 41.931 39.416 37.015 -0.409 -0.409 -0.409 1.753 1.754 1.754 0.307 0.286 0.268 0.402 0.374 0.349 2.742 2.703 2.671 5 0 1.031 2.023 43.945 38.36 35.064 43.739 38.156 34.77 43.842 38.258 34.917 0 0 0 1.739 1.733 1.729 0.319 0.276 0.25 0.408 0.344 0.31 2.71 2.602 2.554 6 0 1.021 2.012 39.348 37.465 35.5 39.098 37.326 35.804 39.223 37.396 35.652 -0.204 -0.204 -0.204 1.813 1.815 1.819 0.299 0.287 0.277 0.413 0.395 0.379 2.885 2.865 2.849 1 0 1.032 2.053 41.179 32.033 27.322 40.43 30.889 26.264 40.805 31.461 26.793 0 0 0 1.723 1.719 1.725 0.289 0.226 0.198 0.362 0.278 0.247 2.609 2.504 2.51 2 0 1.021 1.993 43.734 41.02 38.662 43.575 40.554 38.292 43.654 40.787 38.477 0 0 0 1.73 1.727 1.725 0.304 0.282 0.265 0.388 0.356 0.334 2.663 2.61 2.577 3 0 1.021 2.043 39.896 32.657 29.152 37.808 31.083 27.948 38.852 31.87 28.55 0 0 0 1.764 1.766 1.768 0.281 0.232 0.207 0.373 0.302 0.267 2.731 2.644 2.604 4 0 1.021 1.993 42.937 35.325 32.178 42.781 35.136 31.853 42.859 35.23 32.015 0 0 0 1.704 1.699 1.696 0.304 0.247 0.223 0.373 0.295 0.264 2.586 2.464 2.419 5 0 1.021 2.002 42.049 40.592 39.467 41.768 40.435 39.236 41.908 40.514 39.351 0 0 0 1.729 1.73 1.729 0.303 0.293 0.284 0.381 0.367 0.354 2.649 2.631 2.611 6 0 1.021 1.993 41.522 36.359 32.703 41.144 35.965 32.133 41.333 36.162 32.418 0 0 0 1.776 1.776 1.775 0.297 0.258 0.231 0.396 0.341 0.302 2.773 2.698 2.652 7 0 1.021 1.993 40.075 39.161 37.53 39.476 36.783 34.88 39.775 37.972 36.205 0 0 0 1.87 1.868 1.868 0.295 0.273 0.263 0.436 0.41 0.391 3.038 3.014 2.994 40 Testing Liquid Treatment SALINE R.erythropolis Tergitol 500 mg/L R.erythropolis Glycerol Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 103.193 102.314 102.754 1.022 100.468 100.82 100.644 2.013 99.367 98.802 99.085 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.501 0.8 1.044 3.966 1.531 0.785 1.04 3.949 1.553 0.777 1.046 3.962 2 0 1.032 2.003 103.892 100.501 99.062 103.372 100.553 99.417 103.632 100.527 99.239 0 0 0 1.496 1.546 1.563 0.811 0.786 0.777 1.058 1.06 1.06 4.004 3.999 3.997 3 0 1.032 2.013 102.206 101.94 101.072 102.502 101.385 100.207 102.354 101.663 100.639 0 0 0 1.513 1.532 1.545 0.803 0.793 0.787 1.06 1.063 1.062 4.004 4.01 4.005 4 0 1.031 2.023 104.423 102.644 101.269 103.206 102.039 100.07 103.814 102.342 100.67 -0.204 -0.204 -0.204 1.478 1.496 1.515 0.797 0.785 0.778 1.017 1.012 1.014 3.9 3.881 3.883 5 0 1.031 2.012 103.229 102.223 101.254 105.652 103.568 102.329 104.44 102.896 101.791 0 0 0 1.453 1.487 1.499 0.797 0.781 0.776 0.993 0.997 0.996 3.841 3.844 3.838 6 0 1.032 2.003 102.612 100.218 100.088 102.754 100.646 100.562 102.683 100.432 100.325 0 0 0 1.45 1.485 1.49 0.782 0.766 0.761 0.952 0.956 0.953 3.729 3.733 3.725 1 0 1.022 2.013 96.74 93.789 93.088 97.336 94.785 93.921 97.038 94.287 93.505 0 0 0 1.574 1.611 1.625 0.759 0.744 0.738 1.032 1.035 1.039 3.924 3.932 3.946 2 0 1.032 2.013 111 94.008 95.395 108.355 93.989 94.285 109.678 93.998 94.84 0 0 0 1.401 1.572 1.568 0.82 0.747 0.744 0.999 0.991 0.988 3.88 3.817 3.812 3 0 1.032 2.023 99.73 97.533 96.255 98.768 97.488 96.81 99.249 97.511 96.533 0 0 0 1.626 1.651 1.659 0.809 0.793 0.79 1.196 1.189 1.187 4.331 4.313 4.309 4 0 1.032 2.013 97.629 97.224 96.972 99.497 98.469 97.992 98.563 97.846 97.482 0 0 0 1.568 1.579 1.582 0.773 0.767 0.763 1.059 1.056 1.051 3.993 3.986 3.973 5 6 7 8 41 Testing Liquid Treatment Tergitol 1000 mg/L R.erythropolis Tergitol 4000 mg/L R.erythropolis Glycerol Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 93.358 88.477 90.918 1.031 93.282 87.4 90.341 2.003 93.559 87.883 90.721 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.561 0.682 0.879 3.532 1.566 0.681 0.887 3.555 1.567 0.68 0.885 3.55 2 0 1.031 2.013 92.046 87.408 84.067 90.481 86.393 84.785 91.264 86.9 84.426 0 0 0 1.608 1.678 1.692 0.714 0.681 0.678 0.968 0.974 0.97 3.766 3.803 3.798 3 0 1.022 2.003 100.449 96.359 94.895 99.928 94.783 93.416 100.189 95.571 94.155 -0.204 -0.204 -0.204 1.514 1.576 1.587 0.788 0.737 0.733 1.024 0.994 0.992 3.909 3.826 3.822 4 0 1.031 2.013 101.067 99.509 97.632 102.783 101.318 99.101 101.925 100.413 98.367 0 0 0 1.482 1.5 1.524 0.792 0.783 0.775 1.003 1.001 1.006 3.86 3.85 3.858 5 0 1.031 2.003 103.177 101.997 101.133 103.752 103.873 103.531 103.464 102.935 102.332 0 0 0 1.421 1.425 1.43 0.799 0.79 0.783 0.947 0.938 0.931 3.724 3.696 3.675 6 0 1.031 2.003 100.819 98.868 99.212 103.168 100.786 99.394 101.993 99.827 99.303 0 0 0 1.474 1.5 1.507 0.771 0.757 0.749 0.963 0.959 0.951 3.755 3.74 3.718 1 0 1.021 2.003 79.946 78.164 78.397 83.54 79.174 78.398 81.743 78.669 78.397 0 0 0 1.641 1.661 1.661 0.629 0.609 0.605 0.841 0.815 0.808 3.469 3.418 3.402 2 0 1.032 2.003 76.274 71.968 72.795 76.728 75.597 75.464 76.501 73.783 74.13 0 0 0 1.629 1.64 1.64 0.544 0.544 0.543 0.693 0.696 0.694 3.113 3.132 3.125 3 0 1.031 2.002 64.918 64.474 61.96 62.057 58.179 55.127 63.487 61.326 58.543 0 0 0 2.008 2.043 2.053 0.557 0.491 0.483 1.013 0.953 0.946 4.218 4.194 4.217 4 0 1.031 2.002 98.382 96.802 96.366 93.389 94.021 93.571 95.885 95.411 94.969 0 0 0 1.616 1.628 1.635 0.763 0.746 0.74 1.093 1.07 1.067 4.078 4.023 4.017 5 6 7 42 Testing Liquid Treatment SALINE R.erythropolis Formamide Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0.02 68.273 67.63 67.952 0.991 59.083 58.678 58.881 2.033 57.383 57.276 57.33 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.816 0.56 0.844 3.628 1.874 0.487 0.752 3.537 1.877 0.472 0.728 3.499 2 0 0.991 2.043 65.973 62.29 61.555 66.013 61.862 61.069 65.993 62.076 61.312 0 0 0 1.862 1.911 1.914 0.547 0.521 0.514 0.86 0.851 0.84 3.718 3.773 3.759 3 0 1.052 2.043 65.797 60.883 59.213 65.655 61.478 59.703 65.726 61.18 59.458 0 0 0 1.868 1.911 1.915 0.547 0.514 0.499 0.864 0.836 0.81 3.735 3.747 3.707 4 0 1.022 2.043 73.314 60.741 57.477 73.161 58.613 56.391 73.237 59.677 56.934 0 0 0 1.786 1.913 1.938 0.613 0.506 0.489 0.915 0.827 0.804 3.742 3.742 3.739 1 0 1.022 2.003 69.636 65.835 62.765 70.568 66.529 63.47 70.102 66.182 63.118 0 0 0 1.71 1.735 1.754 0.543 0.514 0.493 0.734 0.701 0.679 3.281 3.239 3.219 2 0 1.032 2.023 68.143 64.821 62.802 67.767 64.809 62.811 67.955 64.815 62.806 0 0 0 1.744 1.767 1.779 0.537 0.519 0.504 0.747 0.726 0.71 3.345 3.329 3.314 3 0 1.011 2.053 70.079 66.729 64.225 69.608 66.448 63.953 69.843 66.588 64.089 0 0 0 1.722 1.741 1.756 0.548 0.524 0.505 0.751 0.721 0.701 3.329 3.289 3.266 4 0 1.001 1.993 68.619 66.791 65.166 69.169 66.921 65.349 68.894 66.856 65.258 0 0 0 1.735 1.742 1.744 0.54 0.523 0.51 0.746 0.721 0.701 3.333 3.289 3.25 5 0 1.032 2.033 70.059 68.546 67.215 69.829 67.877 66.221 69.944 68.211 66.718 0 0 0 1.72 1.726 1.727 0.554 0.538 0.524 0.755 0.732 0.711 3.335 3.293 3.25 5 6 Tergitol 500 mg/L R.erythropolis 6 7 8 43 Testing Liquid Treatment Tergitol 1000 mg/L R.erythropolis Formamide Sample Time [s] CA[L] CA[R] CA[M] Tilt 1 0 68.344 69.558 68.951 0.952 66.507 67.859 67.183 1.973 65.251 66.438 65.845 0 0 0 L[mm] H[mm] Vol[ml] A[mm2] 1.747 0.545 0.765 3.388 1.75 0.527 0.735 3.327 1.751 0.51 0.709 3.276 2 0 1.051 2.053 61.204 59.607 57.934 63.436 61.122 58.927 62.32 60.364 58.43 0 0 0 1.842 1.849 1.856 0.521 0.504 0.489 0.799 0.765 0.741 3.583 3.526 3.49 3 0 1.032 2.033 70.059 68.546 67.215 69.829 67.877 66.221 69.944 68.211 66.718 0 0 0 1.72 1.726 1.727 0.554 0.538 0.524 0.755 0.732 0.711 3.335 3.293 3.25 4 0 1.062 2.033 68.448 67.679 62.702 68.084 67.309 59.831 68.266 67.494 61.266 0 0 0 1.746 1.749 1.828 0.537 0.53 0.5 0.75 0.739 0.758 3.356 3.333 3.49 5 0 1.012 1.983 68.835 65.377 65.377 68.485 64.84 64.84 68.66 65.109 65.109 0 0 0 1.748 1.776 1.776 0.545 0.52 0.52 0.763 0.741 0.741 3.382 3.373 3.373 1 0 1.062 2.023 68.448 67.679 67.679 68.084 67.309 67.309 68.266 67.494 67.494 0 0 0 1.746 1.749 1.749 0.537 0.53 0.53 0.75 0.739 0.739 3.356 3.333 3.333 2 0 1.021 2.043 67.767 66.926 66.926 66.009 65.309 65.309 66.888 66.117 66.117 0 0 0 1.771 1.773 1.773 0.537 0.531 0.531 0.769 0.759 0.759 3.425 3.407 3.407 3 0 1.022 2.053 67.47 67.132 67.132 69.138 68.809 68.809 68.304 67.97 67.97 0 0 0 1.694 1.695 1.695 0.52 0.517 0.517 0.685 0.682 0.682 3.16 3.153 3.153 4 0 1.022 2.013 67.799 67.284 67.284 67.864 67.21 67.21 67.832 67.247 67.247 0 0 0 1.746 1.748 1.748 0.536 0.531 0.531 0.746 0.737 0.737 3.346 3.328 3.328 5 0 1.001 2.002 65.339 63.838 63.838 64.258 61.75 61.75 64.798 62.794 62.794 0 0 0 1.838 1.856 1.856 0.528 0.516 0.516 0.806 0.801 0.801 3.585 3.603 3.603 6 0 1.042 2.023 64.544 61.029 61.029 64.054 60.519 60.519 64.299 60.774 60.774 0 0 0 1.849 1.879 1.879 0.532 0.507 0.507 0.817 0.795 0.795 3.619 3.624 3.624 6 Tergitol 4000 mg/L R.erythropolis 7 44 Testing Liquid Treatment SALINE R.erythropolis Tergitol 500 mg/L R.erythropolis Water Sample 1 0.02 0.972 2.003 94.561 92.672 87.079 93.937 91.56 86.369 94.249 92.116 86.724 0 0 0 1.605 1.627 1.652 0.78 0.758 0.707 1.081 1.061 0.981 4.044 3.997 3.806 2 0 0.971 1.963 97.598 94.487 88.57 96.478 93.598 87.902 97.038 94.042 88.236 0 0 0 1.573 1.595 1.622 0.783 0.757 0.707 1.067 1.034 0.955 4.011 3.928 3.734 3 0 0.971 1.963 95.327 91.069 85.647 94.613 90.469 85.247 94.97 90.769 85.447 0 0 0 1.598 1.621 1.648 0.774 0.737 0.691 1.068 1.012 0.944 4.013 3.875 3.716 4 0 1.072 2.033 96.648 93.554 88.171 96.319 93.126 87.89 96.484 93.34 88.031 0 0 0 1.578 1.605 1.631 0.791 0.758 0.71 1.079 1.043 0.967 4.041 3.949 3.766 5 0 1.072 2.033 95.625 89.853 84.558 95.597 90.331 85.078 95.611 90.092 84.818 0 0 0 1.592 1.624 1.654 0.778 0.733 0.69 1.07 1.006 0.945 4.019 3.861 3.72 6 0 1.012 1.983 96.616 95.1 87.207 95.171 93.422 86.062 95.893 94.261 86.635 0 0 0 1.592 1.608 1.648 0.786 0.773 0.703 1.087 1.076 0.971 4.061 4.033 3.78 1 0.02 0.921 1.962 95.713 89.808 84.695 94.575 89.24 83.891 95.144 89.524 84.293 0 0 0 1.663 1.692 1.732 0.805 0.749 0.702 1.207 1.111 1.052 4.354 4.127 4.006 2 0 1.002 2.033 93.661 91.598 83.127 93.137 90.974 83.087 93.399 91.286 83.107 0 0 0 1.605 1.623 1.661 0.765 0.745 0.677 1.053 1.03 0.925 3.974 3.919 3.675 3 0.02 0.921 1.962 91.463 88.114 82.586 91.679 88.118 82.232 91.571 88.116 82.409 0 0 0 1.62 1.641 1.666 0.764 0.731 0.672 1.057 1.011 0.919 3.985 3.875 3.665 4 0.02 0.921 1.962 91.142 86.743 81.952 93.49 88.215 82.117 92.316 87.479 82.034 0 0 0 1.605 1.632 1.661 0.775 0.725 0.677 1.069 0.991 0.918 4.012 3.824 3.658 5 0 1.011 2.003 96.59 86.191 77.451 96.074 85.644 77.089 96.332 85.918 77.27 0 0 0 1.555 1.66 1.729 0.78 0.699 0.634 1.033 0.973 0.906 3.926 3.79 3.674 6 0 1.011 2.003 87.847 78.655 72.075 87.064 78.053 71.529 87.455 78.354 71.802 0 0 0 1.607 1.676 1.714 0.694 0.627 0.575 0.914 0.848 0.783 3.63 3.504 3.39 7 0 1.011 2.003 101.799 91.993 84.055 100.752 90.817 82.943 101.276 91.405 83.499 0 0 0 1.371 1.43 1.476 0.749 0.676 0.613 0.81 0.728 0.666 3.347 3.108 2.947 8 45 Testing Liquid Treatment Tergitol 1000 mg/L R.erythropolis Tergitol 4000 mg/L R.erythropolis Water Sample 1 0 1.041 2.033 91.955 85.786 80.45 91.812 85.358 80.165 91.884 85.572 80.307 0 0 0 1.682 1.709 1.743 0.772 0.711 0.664 1.153 1.046 0.982 4.225 3.981 3.854 2 0 1.041 2.033 98.233 91.847 86.208 96.721 89.961 84.608 97.477 90.904 85.408 0 0 0 1.607 1.636 1.665 0.82 0.759 0.703 1.175 1.069 0.983 4.278 4.016 3.816 3 0 1.041 2.033 98.968 92.792 87.621 98.624 92.532 87.262 98.796 92.662 87.442 0 0 0 1.598 1.622 1.646 0.822 0.763 0.715 1.173 1.066 0.989 4.276 4.009 3.824 4 0 1.031 2.032 90.655 88.426 81.932 90.356 88.078 81.712 90.505 88.252 81.822 0 0 0 1.719 1.737 1.763 0.774 0.752 0.691 1.194 1.165 1.056 4.327 4.265 4.031 5 0 1.031 2.032 89.386 85.552 78.986 88.452 85.071 78.745 88.919 85.311 78.866 0 0 0 1.735 1.752 1.784 0.769 0.731 0.671 1.196 1.127 1.029 4.335 4.183 3.986 6 0 1.031 2.032 89.786 83.646 77.59 89.426 83.476 77.538 89.606 83.561 77.564 0 0 0 1.733 1.76 1.792 0.762 0.707 0.656 1.184 1.089 1.008 4.308 4.102 3.944 1 0 1.011 2.023 85.603 79.639 72.186 85.979 79.768 72.236 85.791 79.704 72.211 0 0 0 1.76 1.794 1.834 0.729 0.673 0.607 1.138 1.05 0.951 4.214 4.038 3.864 2 0 1.011 2.023 83.303 75.016 69.148 83.015 74.681 68.686 83.159 74.849 68.917 0 0 0 1.788 1.82 1.857 0.716 0.632 0.575 1.135 0.989 0.91 4.22 3.929 3.81 3 0 1.011 2.023 87.069 79.826 72.895 86.222 79.031 71.909 86.645 79.429 72.402 0 0 0 1.736 1.77 1.81 0.728 0.66 0.594 1.11 1.003 0.909 4.14 3.92 3.753 4 0 1.011 2.023 83.425 75.097 68.771 83.237 74.944 68.397 83.331 75.02 68.584 0 0 0 1.781 1.814 1.855 0.713 0.634 0.574 1.121 0.988 0.905 4.185 3.921 3.798 5 0 1.011 2.023 84.515 77.87 71.048 83.942 77.365 70.702 84.228 77.617 70.875 0 0 0 1.786 1.822 1.859 0.73 0.669 0.606 1.163 1.064 0.969 4.279 4.088 3.928 6 7 46   Appendix XI Mycolic Acid Contact Angle Measurement Data XI    Mycolic acid contact angle measurement Contact angles (θ/ o) of pure mycolic acid from Mycobacterium Probe Liquids Contact angles (θ/ o) Water Formamide 1-Bromonaphthalene 95.8±2.4 77.3±3.4 35.7±0.8 1 24