Microbiome studies on anaerobic digestion using genome–resolved multi–omics

Anaerobic digestion (AD) is a mainstay of modern wastewater treatment. In this process, biodegradable compounds are converted into biogas containing methane, which can be a source of renewable energy. Despite its wide application, the ecology of the AD ecosystem is only partially understood. The overarching objective of this thesis is to explore the underlying biological processes from an ecological perspective to better understand how specific members of the microbial community and their activities affect the performance of the AD process. To this end, three studies are conducted on full- and laboratory- scale anaerobic digesters. In the first study, the AD microbial community is examined through a genome-resolved metagenomics and metatranscriptomics approach. The sequencing data generated in a previous project are used to construct the metagenome assembled genomes (MAGs) in three replicate full-scale anaerobic digesters. Next the recovered MAGs are analysed to understand the phylogeny, community structure, potential and active functions of the AD communities. This approach reveals several novel lineages of microorganisms present in the full-scale anaerobic digesters in Singapore. In addition, the functional analysis of the recovered MAGs leads to the annotation of unexpected functional modules in the genus Methanothrix. These functional modules are then incorporated into a novel complex methanogenesis pathway, RuBisCo - Wood Ljungdahl mediated methanogenesis. The second study explores the effect of the frequency of a disturbance on the AD ecosystem in laboratory-scale anaerobic digesters. Twelve laboratory-scale mesophilic anaerobic digesters are operated for 90 days after an 8-month acclimation period. These digesters are arranged in four experimental groups, each receiving a constant number of disturbances at varied frequencies to test the intermediate stochasticity hypothesis (ISH) in the AD ecosystem. Both process performance and the community structure and dynamics are followed throughout the experiment. Results show that the species richness (zero-order Hill number) peaks at intermediate frequencies of disturbance, in accordance with the ISH. On the contrary, a U-shaped pattern emerges for higher order Hill numbers. In addition, the assembly mechanisms shift toward more stochasticity at intermediate levels of disturbance. Finally, trade-offs in ecosystem functions are observed in different experimental groups. In the third study, the biodiversity – ecosystem functioning relationships in laboratory-scale anaerobic digesters are investigated to probe the response of the AD ecosystem to a disturbance occurring at varied frequencies. The Grime trait-based life-history strategy framework reveals that competitor, ruderal, and stress tolerant life-history strategies are adopted by undisturbed, intermediately disturbed, and press disturbed communities, respectively. Overall, the findings of this thesis deepen our understanding of the ecology of anaerobic digestion. By employing advanced bioinformatics techniques, the inter-relationships of complex microbial communities are studied and novel lineages identified. In addition, ecological theories are employed to study microbial ecosystems. Finally, the outcomes may have implications for microbial resource management using anaerobic digestion to preserve or enhance ecosystem services.Doctor of Philosoph