The microbial ecology of anaerobic digestion: characterising novel biogas configurations through molecular and statistical methods

The microbiological generation of methane from organic substrates is a process with incredible flexibility, which has allowed continual innovations in its application as a source of renewable energy. Ireland’s renewable energy sector is gradually expanding, and although biogas is as-yet a minor component, it currently presents an opportunity to capitalise on prevalent feedstocks and existing gaps in the country’s energy infrastructure. In order for anaerobic digestion to succeed in this context, several criteria need to be optimised, particularly improving process stability and yields in order to make the technology competitive and deliver on the promise of biogas. Despite the efforts to harness anaerobic digestion as a sustainable energy technology, it remains a microbial phenomenon which is incompletely characterised and difficult to scale to industrial requirements. Indeed, many applications of anaerobic digestion for biogas require microbial communities to endure sustained stresses (high organic loading rates, high concentrations of inhibitive breakdown products, high temperatures) which can further destabilise the process and make certain configurations appear unfeasible. An alternative application of anaerobic digestion focuses on a subset of the biogas community (the methanogenic archaea) to convert supplies of CO2 and H2 to biomethane at efficiencies approaching natural gas production (i.e. near-pure methane). This technology is highly attractive as it could allow conversion of both exogenous/industrially-produced H2 and CO2 to biomethane, as well as upgrading the methane content of ‘raw’ biogas. However, addition of hydrogen to anaerobic communities can paradoxically prevent biogas formation, by disrupting the finely-balanced thermodynamics of fermentation. It has been suggested that to avoid the inhibition caused by adding hydrogen to the microbial community in situ, methanogens could be cultured and fed CO2 and H2 in a specialised ex situ reactor, independent of feedstock hydrolysis and fermentation. Although hydrogenotrophic methanogens are autotrophs and can fix CO2 to cellular material, it has not been clear what sort of x microbial communities are encouraged by upgrading setups, nor is it clear how the supply of H2 disrupts the biogas community. This thesis explores the microbial ecology underlying several anaerobic digestion configurations relevant to renewable energy production in both Irish and international contexts. Microbial community structures were considered in relation to feedstock composition and biogas output, and shifts in the abundance of functional microbial populations were related to changes in reactor environment and biomethane output, thereby identifying factors which appear to inhibit or support biogas production in these novel setups. Chapters 2 and 3 consider anaerobic digestion of feedstocks which represent promising biogas resources in Ireland and beyond (seaweed/dairy slurry and grass silage/dairy slurry respectively), but can be recalcitrant or problematic for digestion due to their compositions. In both chapters, next-generation sequencing of 16S microbial community profiles clearly indicates disruption between the metabolism of end-fermentation products and subsequent methanogenesis when operated at relatively high loading rates of substrate (2-3kgVSm-3d -1 ). In the case of seaweed/dairy slurry digestion (chapter 2), large quantities of seaweed release excess ammonia, correlating with collapse of the methanogen populations (acetoclastic Methanosarcina) necessary to metabolise accumulating acetate, ultimately leading to reactor failure. In the case of grass silage/dairy slurry digestion, high organic loading of grass silage appears to inhibit or exhaust fermenting bacteria (Clostridia), as shown through a supplementation regime which restores process function and increases abundance of fermenting bacteria. Surprisingly, this treatment does not encourage archaeal populations (Methanobacterium), and may even reduce their abundance. In both chapters, conditions which inhibit biogas production lead to microbial communities which are distinct from those seen when inhibition was resolved. xi Chapters 4 and 5 explore biogas upgrading communities, charting how these communities relate to variables in the upgrading process. Chapter 4 characterises the microbial community in a minimal example of ex situ biogas upgrading at two thermophilic temperatures (55°C, 65°C), demonstrating the persistence and stability of methanogen populations (in particular, the family Methanobacteriaceae) alongside a surprisingly complex bacterial community. Chapter 5 expands on this finding, comparing the upgrading communities between in situ and ex situ operation during increasing flow rates of H2, showing that the presence (in situ) or absence (ex situ) of feedstock delineates these communities. This in turn governs community response to rates of H2 supply, with in situ communities showing a far greater susceptibility to inhibition, population flux, and displacement of methanogens (Methanothermobacter). In comparison, ex situ upgrading communities saw little change at much higher H2 flow rates, and instead encouraged larger populations of closely related methanogens (Methanobacterium). These large ex situ hydrogenotrophic populations were significantly associated with smaller, ‘satellite’ populations of hydrogen-producing bacteria, indicating a thermophilic upgrading community centred on biogas metabolism