Characterization of microbial community dynamics during anaerobic digestion of wheat distillery waste

Anaerobic digestion of agricultural wastes provides an opportunity for renewable energy production while reducing emissions of greenhouse gasses such as carbon dioxide and methane from crop and livestock production. While anaerobic digestion is possible under a wide range of temperatures and reactor configurations, it does require a stable methanogenic community composed of hydrolytic and fermentative bacteria and methanogenic archaea in order to maintain robust methane production. Research focused on characterizing and optimizing the microbial community during anaerobic digestion is increasingly exploiting DNA-based methods. In addition to providing an in-depth phylogenetic survey, these techniques permit examination of dynamic changes in α- and β-diversity during the digestion process and in response to perturbations in the system. This study used universal target amplification, next generation sequencing, and quantitative PCR to characterize the Bacteria and Archaea in digestate from thermophilic batch anaerobic digesters processing different combinations wheat ethanol stillage waste and cattle manure. The results indicated that the bacterial community was composed primarily of Firmicutes, with Proteobacteria and Bacteroidetes also numerically abundant. While less phylogenetically diverse, the archaeal community showed robust populations of both hydrogenotrophic and acetoclastic methanogens. A core microbiome present across all reactors was identified and differences in the relative abundances of the bacteria within the core community suggested significant niche overlap and metabolic redundancy in the reactors. A time-course study correlating the abundances of individual Bacteria and Archaea to methane production and volatile fatty acid catabolization identified several microorganisms hypothesized to be critical to both hydrogenotrophic and acetoclastic methanogenesis. Individual Bacteria most closely related to Clostridium spp. and Acetivibrio spp. were 10-1000-fold less abundant in reactors suffering from volatile fatty acid accumulation and inhibition of methanogenesis. Additionally, failing reactors were devoid of robust populations of acetoclastic methanogens. Microorganisms identified as critical during the time-course study were targeted for isolation in vitro and a robust methanogenic consortium consisting of at least 9 bacteria and both a hydrogenotrophic and an acetoclastic methanogen was stably propagated. Addition of this bioaugmentation consortium to digesters experiencing classic symptoms of acid crisis resulted in reduced acetate accumulation and initiation of methanogenesis. One acetoclastic methanogen, most likely a novel species from the genus Methanosarcina, showed particularly robust growth in the recovered bioaugmented reactors, increasing 100-fold in the first 7 days post-treatment. A combination of Illumina shotgun and Roche 454 paired-end sequencing chemistry was used to generate a high quality draft genome for this organism. Analysis of the annotated genome revealed diverse metabolic potential with a full complement of genes for acetoclastic, hydrogenotrophic and methylotrophic methanogenesis pathways represented. Taken as a whole, this thesis provides the foundation for using microbial community characterization to inform anaerobic digester design and operation. By identifying organisms of interest, correlating their abundance to specific biochemical functions and confirming their hypothesized functions in situ, microorganisms critical for robust methane production were acquired. The logical extension of this work is to establish monitoring tools for microorganisms identified as critical to specific performance parameters, to enumerate them in real-time, and to use that data to improve reactor operation