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Friedline, Skyler Ellis

University of British Columbia

Linking the genomic potential of uncultivated microbes to their in situ metabolic functions remains a critical challenge in the field of microbial ecology. This knowledge gap is especially relevant to our understanding of methanogenic microbial communities inhabiting engineered anaerobic digestion (AD) bioreactor environments. For instance, one of the major methane-yielding pathways in AD is mediated by the coupling of syntrophic acetate oxidation (SAO) and hydrogenotrophic methanogenesis. Our understanding of the stoichiometry and enzymes involved in this relationship continues to develop, yet we still lack data to support many relevant metabolic processes, or even a complete census of the microorganisms capable of carrying out SAO. So far, very few SAO bacteria have been isolated, and conventional multi-omic methods have been unable to elucidate their in situ physiologies due in part to their low abundance in AD environments. To bridge this knowledge gap, this thesis proposes a novel application of molecular techniques to quantitatively resolve microbial activity in uncultivated consortia. The combination of metabolic profiling via protein-based stable isotope probing (protein-SIP) and bio-orthogonal non-canonical amino acid tagging (BONCAT) with fluorescence-activated cell sorting (FACS) was used to study the flux of acetate through anaerobic digestion microcosms seeded with digestate from a full-scale biogas facility. As a result, the proteome of a novel SAO bacteria was highly enriched and yielded sufficient expression information to describe its metabolic pathway for acetate oxidation. This study marks the first application of microliter processing of trace samples in one pot (μPOTS) to microbial communities, and generated the first known proteomic profile of the ‘oxidative glycine pathway’ in a putative SAOB. This work demonstrates that BONCAT-FACS coupled with protein-SIP can be used to generate activity-targeted metaproteomes, and is extensible to other microbial communities where rare but active microorganisms underpin essential ecosystem functions or services.

Text

2024-09-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/85890

Genome Science and Technology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/