Master of Science / Department of Geology / Matthew Kirk / A growing body of evidence demonstrates that methanogenesis and Fe(III) reduction can occur simultaneously. However, environmental controls on interactions between each are poorly understood. In this study we considered pH as a control on interactions between Fe(III) reduction and methanogenesis in anoxic sediment bioreactors. The reactors consisted of 100mL of synthetic aqueous media, and 1 g of marsh sediment amended with goethite (1mmol). One set of reactors received acidic media (pH 6), and the other alkaline media (pH 7.5). Each set received media containing acetate (0.25 mM) to serve as an electron donor. Control reactors, deficient in acetate, were also included. We maintained a fluid residence time of 35 days by sampling and feeding the reactors every seven days. For pH 6.0 and pH 7.5 reactors, the measured pH of effluent samples averaged 6.33 and 7.37, respectively. The extent of Fe(III) reduction and methanogenesis varied considerably between each set of reactors. More Fe(III) was reduced in the pH 6 reactors (646.39 μmoles on avg.) than the pH 7.5 reactors (31.32 μmoles on avg.). Conversely, more methane formed in pH 7.5 reactors (127.5 μmoles on avg.) than the pH 6 reactors (78.9 μmoles on avg.). Alkalinity concentrations during the middle and end of the experiment averaged 9.6 meq/L and 5.2 meq/L in pH 6 and pH7.5 reactors, respectively Although much less Fe(III) reduction occurred in pH 7.5 reactors, the relative abundance of Fe(III) reducers in them decreased little from levels observed in the pH 6 reactors. Sequences classified within Geobacter, a genus of bacteria known primarily as dissimilatory metal reducers, accounted for 22% and 13.45% of the sequences in the pH 6 and pH 7.5 reactors and only 0.8% of the sequences in the marsh sediment inoculum. In contrast, sequences classified within orders of methanogens were low in abundance, making up only 0.47% and 1.04% of the sequences in the pH 6 and pH 7.5 reactors, respectively. Mass balance calculations demonstrate that the amount of electron donor consumed by each group varied considerably between the sets of reactors. Expressed as a quantity of acetate, the reactions consumed about 160μM of electron donor each in pH 6 reactors. In contrast, methanogenesis consumed over 30 times more electron donor than Fe(III) reduction in the pH 7.5 reactors. Thus, the results of our experiment indicate that the decrease in electron donor consumption by Fe(III) reduction at basic pH was nearly matched by the increase in electron donor consumption by methanogens. Results of geochemical modeling calculations indicate that more energy was available for Fe(III) reduction in the pH 6.0 reactors than the pH 7.5 reactors, matching variation in Fe(III) reduction rates, and that the density of sorbed ferrous iron was higher in pH 6 reactors than pH 7.5 reactors. Thus, the calculation results are consistent with bioenergetics, but not variation in ferrous iron sorption, as a potential mechanism driving variation in the balance between each reaction with pH.

Environmental pollution and health problems created by fossil fuels have led to the development of alternative energies such as solar and wind energies, hydroelectric power, and green hydrogen. The use of biohybrid materials in the development of this type of alternative energies is recent. Biohybrid materials are a unique type of advanced materials that have a biological component that can be a biomolecule or a whole cell and an abiotic or non-biological component that can be a ceramic, a synthetic polymer, or a metal, among others. They have applications in different fields that range from construction (such as bioconcrete) to catalysis (such as artificial enzymes). There are examples in the literature in which bacteria are hybridized with reduced graphene oxide or manganese oxide to catalyze the oxygen evolution of the electrochemical water splitting reaction that produces green hydrogen. The focus of this dissertation is to synthesize efficient biohybrid catalysts following a whole cell approach using electroactive bacteria as the biological component and metallic precursors that form particles ranging from single atoms, nanoclusters, and nanoparticles as the abiotic component. The Fe molecule that is part of the heme group of C-type cytochromes in the outer membrane of Geobacter sulfurreducens acted as the reduction center that allowed the synthesis and hybridization of the metals with the bacteria. Single atom metal catalyst of Ir, Pt, Ru, Cu, and Pd were synthesized and demonstrated a bifunctional catalytic activity towards the hydrogen evolution reaction and the oxygen evolution reaction. Ni single atoms were also synthesized with excellent activity in the water splitting reactions making this biohybrid catalyst very efficient but also green, as Ni is an abundant and cheap metal. Pd nanoclusters with size-control were synthesized by controlling the metal concentration, dosing, and incubation times and were tested in the electrochemical water splitting. Overall, the findings of these studies provide new knowledge on the field of biohybrid materials by contributing with novel methodologies for the synthesis of these materials and the application in the green hydrogen production with high efficiencies.

abstract: The finite supply of current energy production materials has created opportunities for the investigation of alternative energy sources in many fields. One example is the use of microorganisms in bioenergy applications, such as microbial fuel cells. Present in many types of environments, microorganisms with the ability to respire solid electron acceptors have become of increasing relevance to alternative energy and wastewater treatment research. In this dissertation, several aspects of anode respiration are investigated, with the goal of increasing the limited understanding of the mechanisms of electron transport through the use of advanced electrochemical methods. Biofilms of Geobacter sulfurreducens, the model anode respiring organism, as well as its alkaliphilic relative, Geoalkalibacter ferrihydriticus, were investigated using chronoamperometry, electrochemical impedance spectroscopy, and cyclic voltammetry. In G. sulfurreducens, two distinct pathways of electron transport were observed through the application of advanced electrochemical techniques on anode biofilms in microbial electrochemical cells. These pathways were found to be preferentially expressed, based on the poised anode potential (redox potential) of the electrode. In Glk. ferrihydriticus, four pathways for electron transport were found, showing an even greater diversity in electron transport pathway utilization as compared to G. sulfurreducens. These observations provide insights into the diversity of electron transport pathways present in anode-respiring bacteria and introduce the necessity of further characterization for pathway identification. Essential to science today, communication of pressing scientific issues to the lay audience may present certain difficulties. This can be seen especially with the topics that are considered socio-scientific issues, those considered controversial in society but not for scientists. This dissertation explores the presentation of alternative and renewable energy technologies and climate change in undergraduate education. In introductory-level Biology, Chemistry, and Physics textbooks, the content and terminology presented were analyzed for individual textbooks and used to evaluate discipline-based trends. Additional extensions were made between teaching climate change with the active learning technique of citizen science using past research gains from studies of evolution. These observations reveal patterns in textbook content for energy technologies and climate change, as well as exploring new aspects of teaching techniques. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2016

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

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Since the 1900’s it has been known that microorganisms are capable of generating electrical power through extracellular electron transfer by converting the energy found organic compounds (Potter, 1911). Microbial fuel cells (MFCs) has garnered more attention recently, and have shown promise in several applications, including wastewater treatment (Yakar et al., 2018), bioremediation (Rosenbaum & Franks, 2014), biosensors (ElMekawy et al., 2018) desalination (Zhang et al., 2018) and as an alternative renewable energy source in remote areas (Castro et al., 2014). In MFCs catalytic reactions of microorganisms oxidize an electron donor through extracellular electron transfer to the anode, under anaerobic conditions, with the cathode exposed to an electron acceptor, facilitating an electrical current (Zhuwei, Haoran & Tingyue, 2007; Lovley, 2006). For energy production in remote areas a low cost and easily accessible feed stock is required for the MFCs. Sweet sorghum is a drought tolerant feedstock with high biomass and sugar yields, good water-use efficiency, established production systems and the potential for genetic improvements. Because of these advantages sweet sorghum stalks were proposed as an attractive feedstock (Rooney et al., 2010; Matsakas & Christakopoulos, 2013). Dried sweet sorghum stalks were, therefore, tested as a raw material for power generation in a MFC, with anaerobic sludge from a biogas plant as inoculum (Sjöblom et al., 2017a). Using sorghum stalks the maximum voltage obtained was 546±10 mV, the maximum power and current density of 131±8 mW/m2 and 543±29 mA/m2 respectively and the coulombic efficiency was 2.2±0.5%. The Ohmic resistances were dominant, at an internal resistance of 182±17 Ω, calculated from polarization data. Furthermore, hydrolysis of the dried sorghum stalks did not improve the performance of the MFC but slightly increased the total energy per gram of substrate. During the MFC operation, the sugars were quickly fermented to formate, acetate, butyrate, lactate and propionate with acetate and butyrate being the key acids during electricity generation. Efficient electron transfer between the microorganisms and the electrodes is an essential aspect of bio-electrochemical systems such as microbial fuel cells. In order to design more efficient reactors and to modify microorganisms, for enhanced electricity production, understanding the mechanisms and dynamics of the electron transport chain is important. It has been found that outer membrane C-type cytochromes (OMCs) (including omcS and omcZ discussed in this study) play a key role in the electron transport chain of Geobacter sulfurreducens, a well-known, biofilm forming, electro-active microorganism  (Millo et al., 2011; Lovley, 2008). It was found that Raman microscopy is capable of providing biochemical information, i.e., the redox state of c-type cytochromes (cyt-C) without damaging the microbial biofilm, allowing for in-situ observation. Raman microscopy was used to observe the oxidation state of OMCs in a suspended culture, as well as in a biofilm of an MFC. First, the oxidation state of the OMCs of suspended cultures from three G. sulfurreducens strains (PCA, KN400 and ΔpilA) was analyzed. It was found that the oxidation state can also be used as an indicator of the metabolic state of the cells, and it was confirmed that PilA, a structural pilin protein essential for long range electron transfer, is not required for external electron transfer. Furthermore, we designed a continuous, anaerobic MFC enabling in-situ Raman measurements of G. sulfurreducens biofilms during electricity generation, while poised using a potentiostat, in order to monitor and characterize the biofilm. Two strains were used, a wild strain, PCA, and a mutant, ΔOmcS. The cytochrome redox state, observed through the Raman spectra, could be altered by applying different poise voltages to the electrodes. This change was indirectly proportional to the modulation of current transferred from the cytochromes to the electrode. This change in Raman peak area was reproducible and reversible, indicating that the system could be used, in-situ, to analyze the oxidation state of proteins responsible for the electron transfer process and the kinetics thereof.

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