Microbial Diversity and Cellulosic Capacity in Municipal Waste Sites By
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Microbial diversity and cellulosic capacity in municipal waste sites by Rebecca Co A thesis presented to the University of Waterloo in fulfilment of the thesis requirement for the degree of Master of Science in Biology Waterloo, Ontario, Canada, 2019 © Rebecca Co 2019 Author’s Declaration This thesis consists of material all of which I authored or co-authored: see Statement of Contributions included in the thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Statement of Contributions In Chapter 2, the sampling and DNA extraction and sequencing of samples (Section 2.2.1 - 2.2.2) were carried out by Dr. Aneisha Collins-Fairclough and Dr. Melessa Ellis. The work described in Section 2.2.3 Metagenomic pipeline and onwards was done by the thesis’s author. Sections 2.2.1 Sample collection – 2.2.4 16S rRNA gene community profile were previously published in Widespread antibiotic, biocide, and metal resistance in microbial communities inhabiting a municipal waste environment and anthropogenically impacted river by Aneisha M. Collins- Fairclough, Rebecca Co, Melessa C. Ellis, and Laura A. Hug. 2018. mSphere: e00346-18. The writing and analyses incorporated into this chapter are by the thesis's author. iii Abstract Cellulose is the most abundant organic compound found on earth. Cellulose’s recalcitrance to hydrolysis is a major limitation to improving the efficiency of industrial applications. The biofuel, pulp and paper, agriculture, and textile industries employ mechanical and chemical methods of breaking down cellulose. Enzymatic methods are attractive choices for industry due to their selectivity in their mode of action and high product yields. However, cellulases are not as economic as mechanical means of degrading cellulose, and few cellulases are optimized for large scales. Investigating the cellulolytic microbiome and functional potential of municipal waste sites, which house large amounts of paper waste, can identify novel cellulose degraders robust for industrial applications. The microbial diversity and metabolic potential in landfills have not been well studied. In this thesis, the cellulose degradation capacity was investigated at two municipal waste sites (MWS). First, the microbial composition and the cellulose degradation capacity of a leachate pond from a dump in Jamaica and the river adjacent to the dump were assessed using metagenomics. The diversity of metagenome-assembled metagenomes (MAGs) was greater in the leachate compared to the river, with thirteen high-quality MAGs identified across seven phyla, including Bacteroidetes, Proteobacteria and Firmicutes. In contrast, two high-quality MAGs, both members of the Proteobacteria, were reconstructed from the river metagenome. A MAG assigned to the candidate phylum CPR2 is the first candidate phylum radiation MAG to be reported from a landfill. The metagenomes were screened for genes belonging to glycosyl hydrolase (GH) families containing cellulases as a measure of cellulolytic potential at the sites. Beta-glucosidases were detected at both sites. In the metagenomes, the taxonomic affiliation of most potential cellulases in the leachate metagenome were to the Bacteroidetes, Firmicutes, Actinobacteria, Spirochaetes, iv and Tenericutes, whereas Bacteroidetes and Proteobacteria cellulases were most abundant in the river metagenome. The microbial composition of the leachate and river did not overlap based on read mapping, suggesting no contamination of the river by the leachate at the times and sites sampled. Secondly, the cellulolytic microbial diversity was also analyzed in six metagenomes from a landfill in Southern Ontario. The samples included a composite leachate cistern (CLC), three leachate wells, and one groundwater well. Twelve GH families containing cellulases were detected across the six metagenomes, with genes from GH3 and GH5 being the most prevalent. Beta-glucosidases and endocellulases were detected across all sites, but exocellulases were only detected in some of the leachate sites and the groundwater well. A large number of hypothetical proteins and non- specifically annotated proteins were also detected across all sites, which likely represent novel carbohydrate-modifying enzymes. The majority of the potential cellulase genes across the six sites were affiliated with the Bacteroidetes and Firmicutes. Thirdly, the potential cellulolytic capacity established from the metagenomes from the Ontario landfill was confirmed by enrichment cultivations of leachate biomass grown in synthetic leachate amended with cellulose. Several isolates from the enrichment cultures showed carboxymethylcellulose and cellobiose degradation capacities, signifying endocellulase and beta- glucosidase activities. Results from 16S rRNA gene amplicon sequencing of copy-paper, cardboard, newsprint, and filter paper-enriched cultures showed enrichment of exact sequence variants assigned to Paenibacillus, Cytophaga, and Proteiniphilum bacteria over time. The research in this thesis represents the first connections between the cellulolytic potential and relevant taxonomic groups in MWS to cellulose degradation by isolates enriched from landfill leachate. v Acknowledgements I would like to thank Dr. Laura Hug for accepting me as her first graduate student, teaching me metagenomics in spurts, providing feedback in the darkest hours, enriching many aspects of my graduate experience, and trusting me. I couldn’t have asked for more. Thank you to my committee members: Dr. Josh Neufeld for letting me use the equipment in his lab for library preparation and sequencing, providing robust feedback, and wonderful conversations. Thank you to Dr. Andrew Doxey for his valuable feedback in the bioinformatic side of my project. Thank you to Dr. Maria Strack for strengthening my project through new and refreshing viewpoints. I would also like to thank Dr. Aneisha Collins-Fairclough for sharing the Riverton Dump data with us – our collaboration was a pleasure. Thank you to the Joint Genome Institute for sequencing our southern Ontario landfill samples. Thank you to the engineers and consulting company at the Ontario landfill for their help and supervision in sampling. Thank you to Dr. Kelly Wrighton and Richard Wolfe from Colorado State University for allowing us to use their in-house metagenomic pipeline in processing the Jamaican datasets. Thank you to Michael Mansfield for showing me how to navigate and bin in Anvi’o, sharing your wisdom, and your continual offers to help me. Thank you to Katja Engel who I’ve been incredibly grateful for her expertise and unyielding patience with her guidance in 16S rRNA gene amplicon sequencing and library preparation. Thank you to Ching-Lung Huang with her relentless cheerfulness for supporting me while we were preparing to sequence our cultures. Thank you to Dr. Trevor Charles for allowing me to use the spectrophotometer in his lab. Thank you to Karen Miinch for providing bacterial cultures from the American Type Culture Collection. Thank you to both Karen Miinch and Susanne Vesely for so ever kindly providing me with useful materials to support my lab work as our lab started up. Thank vi you to Mishi Groh for allowing me to have some Congo red dye used for staining. Thank you to previous and current Hug Lab members: Veronica, Angus, Alex, Nikhil, and Lisa, for sample collection, script sharing, support, and encouragement. You are a wonderful bunch. I would also like to thank Jackson Tsuji for his offer to help me in MetAnnotate (although I never ended up using it in my analyses) and sharing his valuable insights in bioinformatics and metagenomics. Lastly, thank you to my family for all their love and support. I would also like to thank the following people for lending their ears and shoulders, always looking out for me, and sharing laughs: Barb B., Susanne, Karen, Joseph, Max, Donya, Emilie, Vivian, Barb K., and Bruce. Thank you to the amazing and generous people in the Biology Department. I am truly grateful from the bottom of my heart for all of you when the going got tough. vii Table of Contents Author’s Declaration ....................................................................................................................... ii Statement of Contributions ............................................................................................................ iii Abstract .......................................................................................................................................... iv Acknowledgements ........................................................................................................................ vi Table of Contents ......................................................................................................................... viii List of Figures ................................................................................................................................ xi List of Tables ................................................................................................................................ xii List of Abbreviations ................................................................................................................... xiii Chapter 1: Introduction .............................................................................................................1 1.1 Cellulose and cellulases .......................................................................................................1