Community Diversity and Functional Capabilities of Benzene-Degrading Enrichment Cultures by Sarah Elizabeth McRae A thesis submitted in conformity with the requirements for the degree of Master of Applied Science Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto © Copyright by Sarah McRae (2015) Community Diversity and Functional Capabilities of Benzene- Degrading Enrichment Cultures Sarah McRae Master of Applied Science Department of Chemical Engineering and Applied Chemistry University of Toronto 2015 Abstract Benzene is prevalent, toxic, and persistent in anaerobic environments. Anaerobic benzene degradation has been proven, but is not well understood. The goals of this research were to determine the community composition of anaerobic nitrate-reducing benzene-degrading enrichments and propose likely metabolic roles of organisms present. Highly diverse communities were present in the enrichments, with multiple organisms capable of carrying out similar roles. A metagenome assembly was generated from a nitrate-reducing, benzene- degrading enrichment culture. Further evidence for fermenting Peptococcaceae as the initiator of benzene degradation was established. The potential for benzene degradation to be linked to other terminal electron acceptors (i.e., CO2) was confirmed, further supporting that the initial benzene degradation steps are carried out in a fermenting syntroph. An understanding of the functions of different organisms can inform culturing techniques to increase culture growth and benzene degradation rates. Genes present in the metagenome could be used as biomarkers for benzene degradation. ii Acknowledgments I would like to thank the many people who have encouraged, helped and taught me so much during this project. My advisor, Elizabeth Edwards has provided crucial advice, support and taught me so much over the past two and half years. Lab mates in Edwards’ Laboratory have been essential sources of information, technical support, and enthusiasm. Cheryl Devine and Fei Luo were especially helpful throughout and gave advice on nearly every aspect of this project, and I want to thank them for it. This project builds on their work, and the work of other former EdLab students who created and maintained these cultures over the years, and whose research guided me. I also want to thank Shuiquan Tang and Olivia Molenda, from whom I learned sequence analysis and metagenome assembly techniques. Xiaoming Liang taught me key techniques at the beginning. Other members of EdLab, both past and current, have given me advice, shared discussions and been generally wonderful. I thank Luz Puentas Jacome, Po Hsiang (Tommy) Wang, Nigel Guilford, Kirill Krivushin, Line Lomheim, Ivy Yang, Mabel Ting Wong, Torsten Meyer, Sam Huang, Mahbod Hajighasemi, Wendy Han Zhou, and Chris Tran. Endang Susilawati, our wonderful BioZone manager always provided support. BioZone has been a fantastic environment, full of people who are full of bright ideas and always willing to help, whether with advice, technical support, or reinforcement. I especially want to thank Kayla Nemr for her support and editing of this thesis. Financial support for this project was provided by BEEM (Bioproducts + Enzymes from Environmental Metagenomes) and the University of Toronto. As always, I could not have done this without the love and encouragement of my wonderful family: Marie-Thérèse and Malcolm, Daniel, Jeremy and Michelle, Clare, and Rebecca. You have cheered me on from all corners of the globe, given me confidence to challenge myself and kept telling me I could do this. Thank you for everything. iii Table of Contents ACKNOWLEDGMENTS III TABLE OF CONTENTS IV LIST OF TABLES VII LIST OF FIGURES VIII LIST OF ABBREVIATIONS X LIST OF APPENDICES XI CHAPTER 1 LITERATURE REVIEW 1 1.1 INTRODUCTION 1 1.2 REMEDIATION OPTIONS 2 1.3 ANAEROBIC BIOREMEDIATION OF BENZENE 4 1.3.1 Methylation 5 1.3.2 Hydroxylation 6 1.3.3 Carboxylation 7 1.3.4 Central benzoate metabolism pathway 9 1.3.5 Syntrophy 10 1.3.6 Organisms implicated in benzene degradation 10 1.3.7 Metagenome assembly 11 CHAPTER 2 BACKGROUND 12 2.1 RATIONALE FOR RESEARCH 12 2.2 HYPOTHESES 12 2.3 OBJECTIVES 13 2.4 APPROACH 13 2.5 ENRICHMENT CULTURES USED IN THIS STUDY 13 CHAPTER 3 METHODS 17 3.1 CULTURE MAINTENANCE 17 3.2 CULTURE SET-UP 17 3.3 GAS CHROMATOGRAPHY 18 3.4 ION CHROMATOGRAPHY 19 3.5 DNA EXTRACTION AND SEQUENCING FOR METAGENOMIC ANALYSIS 19 3.6 PYROTAG SEQUENCING 20 iv CHAPTER 4 PYROTAG SEQUENCING TO DETERMINE COMMUNITY COMPOSITION 21 4.1 INTRODUCTION 21 4.2 RESULTS AND DISCUSSION 23 4.2.1 Alpha diversity of samples 23 4.2.2 Relative abundances of various OTUs 24 4.2.3 Relatedness of samples 29 4.2.4 Phylogenetic grouping of OTUs 30 4.2.5 Comparison to other benzene-degrading enrichment cultures 33 4.2.6 Comparison of OTUs to culture parameters 34 4.2.7 Multiple OTUs of a single phylogeny 37 4.3 KEY POINTS 39 CHAPTER 5 METAGENOMIC ANALYSIS OF A NITRATE-REDUCING BENZENE-DEGRADING CULTURE 40 5.1 INTRODUCTION 40 5.2 RESULTS AND DISCUSSION 41 5.2.1 ABySS Assembly 42 5.2.2 ALLPATHS-LS Assembly 45 5.2.3 Assessment of assemblies 50 5.2.4 Multiple Peptococcaceae present in the enrichment? 51 5.2.5 Identification of putative anaerobic benzene carboxylase operon 52 5.2.6 Identification of other enzymes in the anaerobic benzene degradation pathway 55 5.3 KEY POINTS 56 CHAPTER 6 METHANOGENIC CAPABILITY OF NITRATE-REDUCING BENZENE-DEGRADING CULTURE 57 6.1 INTRODUCTION 57 6.2 RESULTS 59 6.3 DISCUSSION 67 6.4 KEY POINTS 67 CHAPTER 7 CONCLUSIONS AND ENGINEERING SIGNIFICANCE 70 7.1 A HIGHLY DIVERSE COMMUNITY WITH FUNCTIONAL REDUNDANCY IS PRESENT 70 7.2 THE PUTATIVE ANAEROBIC BENZENE CARBOXYLASE “ABC” IS RESPONSIBLE FOR THE INITIATION OF BENZENE DEGRADATION 71 7.3 NITRATE-REDUCING BENZENE DEGRADING ENRICHMENTS CAN BE SWITCHED TO METHANOGENESIS 71 v 7.4 FUTURE DIRECTIONS SUGGESTED 72 7.5 ENGINEERING SIGNIFICANCE AND CONTRIBUTIONS OF THIS RESEARCH 73 REFERENCES 74 APPENDIX 1: ASSESSMENT OF GENOME COMPLETENESS 90 APPENDIX 2: DEGRADATION OF MONOCHLOROBENZENE 96 APPENDIX 3: ATTEMPTED ANAMMOX ENRICHMENT 105 APPENDIX 4: MICROSCOPIC TECHNIQUES TO EXAMINE COMMUNITY 109 APPENDIX 5: LOCATION OF SEQUENCE DATA FILES 119 vi List of Tables Table 3.1: Experimental set-up for testing the potential of CO as a terminal electron 2 18 acceptor in nitrate-reducing benzene-degrading enrichments Table 4.1: 18 OTUs common to all nitrate-reducing benzene-degrading enrichments 28 tested, with percent abundances in all samples Table 4.2: Groups present during benzene degradation with different electron acceptors, compared to the presence or absence at greater than one percent 33 abundance in pyrotag sequencing data Table 4.3: Percent abundances of methanogenic OTUs in consortia 34 Table 4.4: Metadata used in analysis of Pyrotag sequencing data 35 Table 5.1: Scaffolds that contain a 16S rRNA sequence identified from 46 ALLPATHS-LG assembly using digitally normalized data Table 5.2: Functions ascribed to genera present in assembly 48 Table 5.3: Proportions of genes putatively identified by myRAST 50 Table 5.4: Proportions of genes from single-copy gene set putatively identified by 50 myRAST Table 5.5: Putative annotation of protein encoding genes on Scaffold 559. The four 53 subunits of the abc operon are present Table 5.6: Presence of genes encoding enzymes responsible for different stages of 55 anaerobic benzene degradation in selected scaffolds vii List of Figures Figure 1.1: Anaerobic toluene degradation pathway, with metabolite and genes 6 involved shown Figure 1.2: Anaerobic phenol degradation pathway 7 Figure 1.3: Anaerobic carboxylation of benzene to benzoate 8 Figure 1.4: Anaerobic benzoyl-CoA degradation pathway 9 Figure 2.1: Approximate lineage of Cartwright enrichment cultures 15 Figure 2.2: Approximate lineage of Swamp enrichment cultures 16 Figure 4.1: Approximate lineage and benzene degradation rates of cultures submitted 22 for pyrotag sequencing Figure 4.2: Alpha diversity of pyrotag samples 23 Figure 4.3: Pyrotag sequencing results showing percent abundances of different OTUs 24 in a selection of cultures Figure 4.4: Venn diagram showing the number of OTUs at greater than one percent 27 abundance that are shared between, or unique to, different cultures sampled Figure 4.5: Bootstrap tree using UPGMA (Unweighted Pair Group Method with 29 Arithmetic mean) values to show clustering of samples Figure 4.6: Phylogenetic tree of Beta- and Gammaproteobacteria showing pyrotag sequences in blue, earlier sequences from these cultures in red, and root sequence in 31 purple Figure 4.7: Phylogenetic tree of all groups outside Beta- and Gammaproteobacteria showing pyrotag sequences in blue, earlier sequences from these cultures in red, and 32 root sequence in purple Figure 4.8: NMDS showing clustering of all pyrotag OTUs and the correlation of 36 benzene degradation rate with OTUs Figure 4.9: NMDS showing clustering of pyrotag OTUs present at greater than one 37 percent abundance in any of the enrichments, and the correlation with generation Figure 4.10: Geneious alignment of Peptococcaceae OTUs 38 Figure 5.1:a) ABySS assembly using four paired end libraries (k96, c20) 43 Figure 5.1:b) ABySS assembly using one paired end library (k96, c20) 43 Figure 5.1:c) ABySS assembly using one paired end library (k96, c10) 44 viii Figure 5.1:d) ABySS assembly using one paired end library (k96, c100) 44 Figure 5.2a) Scaffold 29 shows 69% pairwise identity to Thermincola potens genome 51 Figure 5.2b) Scaffold 39 shows 26% pairwise identity to Thermincola potens genome 51 Figure 6.1: Nitrate-reducing benzene degrading culture only (without nitrate). Replicate 59 A Figure 6.2: Nitrate-reducing benzene degrading culture only (without nitrate). Replicate 60 B Figure 6.3: Nitrate-reducing benzene degrading culture only (without nitrate).