Microbiological approaches for the improvement of industrial biogas production

Biogas is an alternative fuel source, composed of methane and carbon dioxide, formed during the microbial process of anaerobic digestion (AD). Biogas production has the potential to be used as a substitute for natural gas and ease problems associated with global warming, energy security and waste management. However, AD can often be unreliable due to the instabilities that can arise during the process. The work described in this thesis, focuses on the discovery and development of micro-organisms for use in synthetic bio-methane producing microbial communities. Two main sources of instability within AD were focused on: oxygen-sensitivity of methane-producing microbes (methanogens) and volatile fatty acid (VFA) accumulation. The estuarine tidal mudflats of the River Exe Estuary, Exmouth, Devon, UK, were chosen for the search of potentially oxygen-tolerant methanogens, due to its intermittent oxygen exposure. Initially, 16S rRNA screening indicated the presence of methanogens in the very surface layer of the sediment, which was later confirmed by enrichment experiments. Whole genome shot-gun sequencing suggested Methanosarcina mazei, a methanogen known to have oxygen-tolerant capabilities, or a closely related organism, had been successfully enriched. The second part of this investigation focused on the use of E. coli as a model organism, for the engineering of a negative feedback loop against acetate production, a common VFA. The glutamine synthetase promoter, found to be sensitive to external pH and acetate concentration, was used to control the production of antisense RNA targeting the pta gene, involved in acetate production. Results indicated that expression of pta was successfully reduced, however overall acetate production was not found to decrease