Understanding the Symbiosis in Anaerobic Oxidation of Methane Through Metabolic, Biosynthetic and Transcriptomic Activities

Abstract

Microorganisms provide essential ecological services to our planet. Their combined activities control and shape our environment as we know today. In the deep sea, a microbial mediated process known as anaerobic oxidation of methane (AOM) consumes large amounts of methane, a potent greenhouse gas and a valuable energy resource. How this symbiosis works is poorly understood. In this thesis, I tested current hypotheses on the symbiotic mechanisms in AOM microbial consortia, consisting of a partnership between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Sediments collected from methane seeps offshore Oregon and California and dominated by AOM consortia were used in these investigations. A range of compounds were amended to sediment microcosms, and their effects on the metabolic activities of ANME or SRB were monitored by tracking the rates of methane oxidation or sulfate reduction on timescales varying from hours to months. A lack of stimulation or inhibition on the AOM consortia, combined with long-term community profiles, suggest that diffusible compounds are unlikely to be involved in the symbiosis in AOM. I further examine ANME genomes, focusing the role of sulfur in methane seep ecosystems. Phylogenetic analyses revealed multiple poorly characterized genes in the sulfur pathway, and comparisons with methanogenic archaea related to ANME provided a better understanding of their roles in the cell. Transcriptional responses combined with protein modeling were used to predict the potential substrate of a sulfite reductase related enzyme. These predictions were validated using genetics, and together point to an assimilatory rather than dissimilatory sulfur pathway in methane-utilizing archaea in general. Then, the AOM symbiosis was decoupled for the first time using soluble electron acceptors. ANME remained metabolically and biosynthetically active without their SRB partner, suggesting that the electrons are transferred directly in this partnership. This observation was investigated to a greater depth with transcriptomics. Membrane proteins and multiheme cytochromes critical in extracellular electron transfer in ANME and SRB were expressed. These results together illuminate the path electrons may take to exit or enter the AOM consortia. Overall, multiple activity analyses used here piece together a clearer view on how the symbiosis in AOM works, with potential applications in future energy generation from methane.