Investigation of microbial community structure and function underpinning grass and food waste anaerobic digestion
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Anaerobic digestion (AD) of feedstocks, such as grass and food waste, presents a sustainable and cost efficient technology for the generation of bioenergy and value added products. However, to fully optimize the process, the microbial communities involved and their functional roles must be understood. Mixed microbial AD sample analysis requires a robust methodological framework for investigation of community structure and function. The objective of this thesis was, firstly, to develop a successful co-extraction methodology to recover nucleic acids and proteins from AD bioreactors treating grass and food waste for 16S rRNA analysis and metaproteomics analyses. Methods for sample preparation, cell lysis, protein extraction and separation were investigated. Once a metaproteomic workflow was established, a co-extraction methodology was developed for both grass AD and food waste AD samples. The established co-extraction workflow using the Norgen Biotek kit was employed to analyse DNA, RNA and protein from grass biofilm and leachate fractions of triplicate leach-bed grass anaerobic bioreactors. The combined 16S rRNA analysis and metaproteomics revealed the microbial structure of the triplicate bioreactors and their microbial functions. Clostridiales, Bacteroidales and Prevotella were involved in grass degradation, the production of methane was mainly attributed to Methanosarcinales, while Prevotella, Megasphaera, Clostridiales, Bacteroides, Azotobacter and Dysogonomas were responsible for VFA production. A metaproteomic approach was also employed in the context of food AD, where digestate fractions of triplicate bioreactors were sampled as a function of time. Previous 16S rRNA analysis had revealed community shifts during the reactor trial, and a functional-based approach was employed to investigate the metabolic activities taking place throughout the reactor run (3 time-points). The majority of proteins identified were assigned to Lactobacillales, while Enterobacteriales were mainly responsible for food waste hydrolysis. There was no evidence of methane production, however proteins were identified for the production of acetate, butyrate and propionate, which was in agreement with process observations. Overall, the workflow established and integrated analysis employed in this research uncovered the community structure and function of grass AD and food waste AD. The metabolic activities of the microbial communities supported the observations at the process level. Microorganisms responsible for the degradation of substrates were uncovered, while those involved in the production of end-products were also identified. This work provides a platform for process optimization, whereby enrichment strategies could be employed to favour the growth of key players in the process, encompassing bioaugmentation approaches and tailoring of environmental conditions.