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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.

Anaerobic digestion (AD) is a widespread technology for treating waste streams such as livestock manure. During AD, biogas is produced and subsequently used as renewable energy for certain purposes, such as injection into the natural gas grid or as fuel for transportation. Despite its many advantages, AD can be limited by various factors, including process instability against volatile fatty acids (VFA), nitrogen overloads, or the presence of inhibitors, as well as the need for biogas post-processing to increase its methane content. Therefore, strategies are needed to monitor the AD process, control the effluent quality and upgrade the biogas recovered. Microbial electrochemical technologies (MET) have the potential to optimize AD. MET are systems in which oxidation and/or reduction reactions are catalyzed by electroactive microorganisms (EAM) on the surface of an electrode. Typically, EAM used in AD-MET combinations are dominated by Geobacter spp., that form multilayer biofilms on electrodes (e.g., anodes) used as solid terminal electron acceptors. However, using Geobacter spp. dominated biofilm anodes in AD-MET combinations has so far encountered several hurdles, ranging from biofilm dispersal to inhibition of biofilm performance. Thus, the intention of the present thesis was to identify and to address the different inhibition processes of Geobacter spp. dominated biofilm anodes in AD-MET combinations. Particular attention was devoted to the impact that planktonic methanogens, particles and dissolved components present in AD effluents may have on the activity, stability and microbial community of Geobacter spp. dominated biofilm anodes. This was achieved by investigating the effect of biofilm age, applied anode potentials as well as the role played by specific methanogens with different metabolisms on the activity, stability and microbial community of Geobacter spp. dominated biofilms. The results indicated that older Geobacter spp. dominated biofilm anodes (≥ 5-week-old) are far more active and stable than younger biofilms (≤ 3-week-old) in AD environments. Compared to high applied anode potential (0.4 V vs. Ag/AgCl sat. KCl), low applied anode potentials (-0.2 V to 0.2 V vs. Ag/AgCl sat. KCl) resulted in higher activities of Geobacter spp. dominated biofilm anodes in AD environments. Other results indicated that AD effluents dominated by strict acetoclastic methanogens (e.g., Methanothrix spp.) cause deterioration in biofilm stability and activity, in contrast to AD effluents dominated by hydrogenotrophic methanogens (e.g., Methanobacterium spp.). In conclusion, the thesis provides useful information for understanding and improving the performance of AD-MET combinations and ways to overcome the multiple hurdles encountered so far.:1 Introduction 12 1.1 Basics of microbiology of anaerobic digestion 12 1.1.1 Methanogens and their functionality 12 1.1.2 Anaerobic digestion - methanogens and bacteria 13 1.1.3 Microbial diversity in anaerobic digestion: focus on methanogens 14 1.1.4 Metabolism and syntrophy in anaerobic digestion 16 1.1.5 Parameters influencing anaerobic digestion 18 1.2 Microbial electrochemical technologies 19 1.2.1 Primary microbial electrochemical technologies - possible applications 19 1.2.2 Basic examples of primary microbial electrochemical technologies 21 1.2.3 Geobacter spp. as model electroactive microorganism in microbial electrochemical technologies 23 1.2.4 Modes of electron transfer between electroactive microorganisms and electrodes 24 1.2.5 Electrochemical characterization of biofilm anodes 27 1.3 Combination of anaerobic digestion and microbial electrochemical technologies 28 1.3.1 Benefits: biotechnological applications 28 1.3.2 Challenges: factors limiting the combination anaerobic digestion - microbial electrochemical technologies 29 1.4 Aims and thesis outline 30 2 Publication 1: Benefits of Age – Improved Resistance of Mature Electroactive Biofilm Anodes in Anaerobic Digestion 33 2.1 Supplementary information for “Benefits of Age – Improved Resistance of Mature Electroactive Biofilm Anodes in Anaerobic Digestion” 43 3 Publication 2: Combining Geobacter spp. dominated biofilms and anaerobic digestion effluents - the effect of effluent composition and electrode potential on biofilm activity and stability 55 3.1 Supplementary information for “Combining Geobacter spp. dominated biofilms and anaerobic digestion effluents - the effect of effluent composition and electrode potential on biofilm activity and stability” 67 4 Publication 3: A unified and simple medium for growing model methanogens 91 4.1 Supplementary information for “A unified and simple medium for growing model methanogens” 105 5 Publication 4: Effect of model methanogens on the activity, stability, and microbial community structure of Geobacter spp. dominated biofilm anodes 117 5.1 Supplementary information for “Effect of model methanogens on the activity, stability, and microbial community structure of Geobacter spp. dominated biofilm anodes” 153 6 Discussion 167 6.1 The older the biofilm, the higher its activity and resistance when combined with anaerobic digestion effluents 167 6.2 Low applied anode potential leads to high activity of Geobacter spp. dominated biofilm in anaerobic digestion environments. 168 6.3 The role of methanogens, abiotic particles, dissolved components in the combination anaerobic digestion – microbial electrochemical technologies 170 6.3.1 Abiotic particles, dissolved components present in anaerobic digestion effluents do not always interfere with the activity, stability and community of Geobacter spp. dominated biofilm anodes 170 6.3.2 The activity and community of Geobacter spp. dominated biofilm anodes in anaerobic digestion environments vary with the predominant group of methanogens 171 7 Conclusions and future prospects 173 8 References 176 9 Appendix 187 9.1 Author contribution statements of published articles 187 9.2 Curriculum vitae 193 9.3 List of publications and conference contributions 196 9.4 Acknowledgment 199 9.5 Declaration of authorship 201