Metataxonomic analysis of a novel anaerobic digester and engineering of isolated bacteria
Mühlbauer, Lukas Frederik
Biomolecular tools such as metataxonomic and metagenomic DNA sequencing approaches have facilitated our detailed understanding of the microbiology of anaerobic digestion (AD) systems in recent years. However, most studies have focussed on single-stage or two-stage AD systems. In this project, the microbiology of a novel multi-stage AD reactor developed by Blue Sky Bio Ltd. (Chester, UK) was characterised for the first time. The results of this analysis suggest that changes in key species abundance follow the longitudinal separation of AD stages (i.e. hydrolysis, acidogenesis, acetogenesis, methanogenesis). Furthermore, the impact of a change in feedstock on the reactor’s microbiology was observed, indicating that microbial communities become less feedstock-specific as biological matter is digested from complex molecules to more simple compounds. Both end products are desirable for their industrially useful properties, mainly as they can be converted or used directly as transport fuels. In particular, butyric acid can be converted into butanol, which is used in the perfume industry but can also be used as a replacement for gasoline, while hydrogen can be a zero-carbon fuel for use in internal combustion engines or to produce electricity in fuel cells, both methods which only result in water as the end product. Still, the work carried out as part of this project has generated insights into multi-stage AD systems and the potential to engineer these newly identified non-model organisms for industrial applications. After characterisation of the community, the isolation and identification of several species involved in the production of volatile fatty acids (VFAs) was carried out. This was finally narrowed down to two species of the genus Clostridium, one of which was shown to be dissimilar enough to other members of the genus to be declared a newly identified species by established delimitation thresholds. The isolates were recognised for their capabilities to produce high amounts of butyric acid as well as hydrogen. Protocols to genetically engineer these bacteria have been established and the GUS reporter from E. coli has been expressed in both bacteria to assess the strength of several promoters. After working native promoters were identified, several hydrogenases and butyrate kinase genes were overexpressed in both isolates using the pMTL80000 shuttle plasmids. Effects on gas production and VFA yield was measured and significant changes were observed, however contrary to the initial hypothesis based on comparative genomics and previous studies in related Clostridium species, observed changes in the isolates’ metabolism were not characterised by a substantial increase in the production of butyric acid and hydrogen.