Formulation of microbial cocktails for biodegradation of btex

FORMULATION OF MICROBIAL COCKTAILS FOR BIODEGRADATION OF BTEX KARTHIGA NAGARAJAN NATIONAL UNIVERSITY OF SINGAPORE 2011 FORMULATION OF MICROBIAL COCKTAILS FOR BIODEGRADATION OF BTEX KARTHIGA NAGARAJAN (B. Tech., Anna University, India) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS It gives me immense pleasure to thank everyone who has been supportive of my efforts in this research study. Firstly, I would like to express my heartfelt gratitude to my supervisor Associate Professor Loh Kai-Chee, for his guidance, support and encouragement throughout my doctoral study. He is one of the very few supervisors who believed in their students and provided them with the freedom to explore new ideas. It indeed helped me to identify my strengths and weaknesses as a researcher. I owe my deepest gratitude to Associate Professor Sanjay Swarup for introducing me to the exciting field of molecular biology. He had been constantly supportive and encouraging of my several attempts to develop the genome based methodology. Special thanks to the research students of Small Molecule Biology Lab (SMBL), Ms Wei Ling, Dr Dennis, Ms Cui Jing, and Ms June for helping me to learn the ropes of genomic work. I would like to thank my former lab-mate Dr Cao Bin who patiently taught me the art of being friendly with bacteria. My warm thanks to my former and current lab-mates Satyen, Prashant, Linh, Duong, Bulbul and Jia-Jia for making our research lab a wonderful place to work. I am deeply grateful to lab officers Mdm Chow Pek, Ms Novel, Ms Alyssa, Mr Wee Siong and Ms Yanfang for their administrative support. I hereby record my sincere thanks to Mr Max Kong (Service Engineer at Perkin Elmer) for being a good friend and sharing his expertise in the operation and troubleshooting of Gas Chromatography. ii I am thankful to NUS for the research scholarship provided to me. I express my sincere thanks to the Department of Chemical and Biomolecular Engineering, NUS for providing me with a wonderful opportunity to serve as a Teaching Assistant. I thank my dear friends Ranjith, Thehazh, Gayathri and Anita for their emotional support and constant care forever. I am eternally thankful to my husband Mr Vivek for his love and support. I wish to acknowledge my extended family, sisters, brothers in-law, nieces and nephews for their unconditional love. I hereby pay my tribute to my parents, Mr Nagarajan and Mrs Anna Nagamani who always put my happiness to first, provided all the best things I could wish for, never gave up on me and always stood by me during critical times. I wish to dedicate my doctoral thesis to their hard work in putting up together a great family. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ii  TABLE OF CONTENTS iv  SUMMARY ix  LIST OF TABLES xii  LIST OF FIGURES xiv  LIST OF ABBREVIATIONS AND SYMBOLS xx  1  INTRODUCTION 1  1.1  Research background and motivations 1  1.2  Research objectives 5  1.3  Thesis organization 6  2  LITERATURE REVIEW 8  2.1  BTEX 8  2.2  BTEX remediation techniques 10  2.3  BTEX biodegradation research 11  2.3.1  BTEX degraders 12  2.3.2  Metabolic pathway 14  2.3.3  Biodegradation kinetics modeling 17  2.3.4  Microbial and substrate interaction 20  2.3.5  Bioreactor design 23  iv 2.3.6  Molecular biology approaches 3  MATERIALS AND METHODS 28  34  3.1  Hydrocarbons 34  3.2  Bacterial culture 34  3.3  Primers 34  3.4  Probes 35  3.5  Bacteria storage conditions 36  3.5.1  Glycerol stock 36  3.5.2  Agar slant 36  3.6  Bacterial culture medium 37  3.7  BTEX biodegradation 37  3.8  Cell concentration 38  3.9  BTEX concentration 38  3.10  Intermediates analysis 39  3.10.1  UV spectrum analysis 39  3.10.2  Extraction of intermediates 39  3.11  Molecular biology tools for species differentiation 40  3.11.1  Extraction of genomic DNA 40  3.11.2  DNA concentration 40  3.11.3  Polymerase chain reaction 41  3.11.4  Agarose gel electrophoresis 41  v 3.11.5  PCR product purification for sequencing 42  3.11.6  Nucleotide sequencing 42  3.11.7  TaqMan® based quantitative real-time PCR 42  4  DEVELOPMENT OF HEURISTICS TO CONCOCT P. PUTIDA F1 AND P. STUTZERI OX1 4.1  Introduction 44  44  4.1.1  Motivation 44  4.1.2  Research objective 44  4.2  Research Approach 45  4.3  Results and Discussions 48  4.4  4.3.1  Biodegradation characteristics of P. putida F1 48  4.3.2  Biodegradation characteristics of P. stutzeri OX1 61  4.3.3  Heuristics 76  4.3.4  Proof of concept 79  Conclusions 88  5  MICROBIAL POTENTIAL CHARACTERIZATION OF P. PUTIDA F1 AND P. STUTZERI OX1 5.1  5.2  Introduction 90  90  5.1.1  Motivation 90  5.1.2  Research objective 92  Research approach 92  vi Results and Discussions 5.3  95  5.3.1  Identification of marker sequence 95  5.3.2  Species-specific primer and probe design 98  5.3.3  Quantification of two Pseudomonas species 102  5.3.4  Validation of mixed culture 113  5.4  Conclusions 6  BTEX BIODEGRADATION BY MIXED CULTURE 6.1  Introduction 120  121  121  6.1.1  Motivation 121  6.1.2  Research objective 121  6.2  Research approach 122  6.3  Results and Discussion 125  6.3.1  Microbial growth – substrate utilization model 125  6.3.2  Concoction of Pseudomonas species 131  6.3.3  Mixed culture biodegradation 137  6.3.4  Biodegradation potential of synthetic consortia 149  6.4  Conclusions 163  7  CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE 165  WORK 7.1  Conclusions 165  7.2  Recommendations for future work 168  vii 7.2.1  BTEX biodegradation by constructed microbial community 168  7.2.2  Design and operation of a immobilized bioreactor for BTEX biodegradation 169  7.2.3  170  Metabolomic and Proteomic analysis of co-culture REFERENCES 172  LIST OF PUBLICATIONS AND PRESENTATIONS 201  viii SUMMARY Mono-aromatic group of hydrocarbons namely Benzene, Toluene, Ethyl benzene and o-Xylene (BTEX) are the most commonly cited environmental pollutants found in groundwater due to oil spillage and effluents from petroleum refineries. Conventional physico-chemical methods used for BTEX removal are either difficult to execute or expensive and invariably release secondary pollutants. On the other hand, BTEX biodegradation by a mixed community of micro-organisms offers a promising approach in terms of cost-effectiveness and elimination of secondary pollution. In this research, we aimed at developing a concoction of micro-organisms for effective biodegradation of BTEX and also to comprehend the microbial interactions through the analysis of population demographics. Two bacterial strains namely Pseudomonas putida F1 and Pseudomonas stutzeri OX1 were chosen to formulate synthetic consortia based on their ability to biodegrade mono-aromatic compounds. Pure culture biodegradation studies using single and mixed substrates revealed complementary biodegradation potentials of the two Pseudomonas species. 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Microbiol. 54(6): 1498-1503. 200 LIST OF PUBLICATIONS AND PRESENTATIONS 1. Cao B, Nagarajan K, Loh KC, (2009) Biodegradation of aromatic compounds: current status and oppurtunities for biomolecular approaches. Appl. Microbiol. Biotechnol. 85: 207-228. 2. Nagarajan K, Loh KC, Swarup S, Bioinformatics and molecular biology approach for quantification of closely related bacteria. (Submitted to Applied and Environmental Microbiology Journal). 3. Biswas A, Reuben S, Nagarajan K, Umashankar S, Swarup S, EnSePT: a pipeline for comparison of whole and partial genomes for unique biomarker discovery. (In preparation). 4. Nagarajan K, Loh KC, BTEX biodegradation by a co-culture of Pseudomonas putida F1 and Pseudomonas stutzeri OX1 (In preparation for Biodegradation Journal). 5. Nagarajan K, Loh KC, Formulation of microbial consortium for BTEX biodegradation (In preparation for Biodegradation Journal). 6. Formulation of microbial consortium for BTEX Degradation, ChemBiotech’08, National University of Singapore, Singapore, December 2008. 7. Formulation of microbial consortium for BTEX Degradation, AIChE Annual meeting, Philadelphia, Pennsylvania, USA, November 2008. 8. Concoction of Bacteria for Biodegradation of BTEX, AIChE Annual Meeting, Salt Lake City, Utah, USA, November 2007. 201 [...]... degradation profile and b) Cell growth profile 143  xviii Figure 6-11: BTEX- a biodegradation by concurrent inoculation of P putida F1 and P stutzeri OX1 a) Substrate degradation profile and b) Cell growth profile 145  Figure 6-12: Performance of pure and mixed culture combinations grown in a) BTEX- a b) BTEX- b c) BTEX- c 146  Figure 6-13: Biodegradation of BTEX- a by mixed... Substrate biodegradation profile and b) Cell growth profile (Error bars indicate standard deviation from mean of 6 replicates) 151  Figure 6-14: Biodegradation of BTEX- b by mixed culture a) Substrate biodegradation profile and b) Cell growth profile (Error bars indicate standard deviation from mean of 6 replicates) 152  Figure 6-15: Biodegradation of BTEX- c by mixed culture a) Substrate biodegradation. .. to the advantages offered by biodegradation and the need for a better understanding and wiser application of the biodegradation process, extensive research on BTEX biodegradation have been conducted Much of these focus on the isolation of potential micro-organisms, elucidation of biodegradation pathways, mathematical modeling of biodegradation kinetics, comprehension of substrate and microbial 11 interactions,... mg/L) biodegradation when present alone and with BTE by P stutzeri OX1 75  Figure 4-34: Cometabolic removal of ethyl benzene in BTEX by P stutzeri OX1 75  Figure 4-35: Comparison of BTEX biodegradation by consortium A, B and C 82  Figure 4-36: Comparison of growth of P putida F1 and P stutzeri OX1 in control flasks (Formulations A, B and C) 82  Figure 4-37: Comparison of BTEX biodegradation. .. the microbial community through comparisons of proteome of pure cultures with that of synthetic consortia xi LIST OF TABLES Table 2-1: Significance of BTEX pollutants 9  Table 2-2: Diversity of BTEX degrading micro-organisms 13  Table 2-3: Novel bioremediation techniques for BTEX biodegradation 26  Table 3-1: Primers for DNA amplification 35  Table 3-2: Probes for quantitative real-time PCR 36  Table... discharge Biodegradation is therefore a promising method in terms of elimination of secondary treatment processes and improving cost effectiveness The importance of BTEX biodegradation is evident from the increasing contributions of various research groups over the past 20 years (Figure 1-1) Hitherto, biodegradation studies on BTEX have emphasized the description of metabolic pathways, analysis of catabolic... Substrate biodegradation profile and b) Cell growth profile (Error bars indicate standard deviation from mean of 6 replicates) 153  Figure 6-16: Comparison of benzene biodegradation by pure and mixed cultures a) BTEX- a b) BTEX- b and c) BTEX- c 158  Figure 6-17: Comparison of toluene biodegradation by pure and mixed cultures 159  Figure 6-18: Comparison of ethyl benzene biodegradation by pure... and mixed cultures a) BTEX- a b) BTEX- b and c) BTEX- c 160  Figure 6-19: Comparison of o-xylene biodegradation by pure and mixed cultures a) BTEX- a b) BTEX- b and c) BTEX- c 161  xix LIST OF ABBREVIATIONS AND SYMBOLS  Lag time  Specific growth rate  Standard deviation A260 Absorbance at 260 nm A260/230 Ratio of absorbance at 260 nm to absorbance at 230 nm A260/280 Ratio of absorbance at 260... deviation from mean of 6 replicates) 119  Figure 6-1: Research approach for mixed culture biodegradation study 124  Figure 6-2: Modified Gompertz model fit (dotted lines) for growth profile of P putida F1 (PpF1) and P stutzeri OX1 (PsOX1) in BTEX 128  Figure 6-3: Modified Richards model fit (dotted lines) for growth profile of P putida F1 (PpF1) and P stutzeri OX1 (PsOX1) in BTEX 128 ... evaluation of biodegradation capacities of pure culture or consortium isolated from contaminated sites With regards to the latter, most of the research contribution (Figure 1-2) had been towards the evaluation of biodegradation characteristics of isolated consortia with little focus on concocting an efficient microbial consortium 2 Figure 1-1: Growth in BTEX biodegradation research (Based on the number of . FORMULATION OF MICROBIAL COCKTAILS FOR BIODEGRADATION OF BTEX KARTHIGA NAGARAJAN NATIONAL UNIVERSITY OF SINGAPORE 2011 FORMULATION OF MICROBIAL COCKTAILS FOR BIODEGRADATION. responses of members of the microbial community through comparisons of proteome of pure cultures with that of synthetic consortia. xii LIST OF TABLES Table 2-1: Significance of BTEX pollutants. combinations grown in a) BTEX- a b) BTEX- b c) BTEX- c 146  Figure 6-13: Biodegradation of BTEX- a by mixed culture a) Substrate biodegradation profile and b) Cell growth profile (Error bars indicate