This work focuses on a novel approach to combine electrical current with cyanobacterial technology, called microbial electrophotosynthesis (MEPS). It involves using genetically modified PSII-less Synechocystis PCC 6803 cells to avoid photoinhibition, a problem that hinders green energy. In the work, a cathodic electron delivery system is employed for growth and synthesis. Photoinhibition leads to the dissipation energy and lower yield, and is a major obstacle to preventing green energy from competing with fossil fuels. However, the urgent need for alternative energy sources is driven by soaring energy consumption and rising atmospheric carbon dioxide levels. When developed, MEPS can contribute to a carbon capture technology while helping with energy demands. It is thought that if PSII electron flux can be replaced with an alternative source photosynthesis could be enhanced for more effective production. MEPS has the potential to address these challenges by serving as a carbon capture technology while meeting energy demands. The idea is to replace PSII electron flux with an alternative source, which can be enhanced for higher yields in light intensities not tolerated with PSII. This research specifically focuses on creating the initiation of electron flux between the cathode and the MEPS cells while controlling and measuring the system in real time. The successful proof-of-concept work shows that MEPS can indeed generate high-light-dependent current at intensities up to 2050 µmol photons m^‒2 s^‒1, delivering 113 µmol electrons h^‒1 mg-chl^‒1. The results were further developed to characterize redox tuning for electron delivery of flux to the photosynthetic electron transport chain and redox-based kinetic analysis to model the limitations of the MEPS system.

Electroactive bacteria connect biology to electricity, acting as livingelectrochemical catalysts. In nature, these bacteria can respire insoluble compounds like iron oxides, and in the laboratory, they are able to respire an electrode and produce an electrical current. This document investigates two of these electroactive bacteria: Geobacter sulfurreducens and Thermincola ferriacetica. G. sulfurreducens is a Gramnegative iron-reducing soil bacterium, and T. ferriacetica is a thermophilic, Grampositive bacterium that can reduce iron minerals and several other electron acceptors. Respiring insoluble electron acceptors like metal oxides presents challenges to a bacterium. The organism must extend its electron transport chain from the inner membrane outside the cell and across a significant distance to the surface of the electron acceptor. G. sulfurreducens is one of the most-studied electroactive bacteria, and despite this there are many gaps in knowledge about its mechanisms for transporting electrons extracellularly. Research in this area is complicated by the presence of multiple pathways that may be concurrently expressed. I used cyclic voltammetry to determine which pathways are present in electroactive biofilms of G. sulfurreducens grown under different conditions and correlated this information with gene expression data from the same conditions. This correlation presented several genes that may be components of specific pathways not just at the inner membrane but along the entire respiratory pathway, and I propose an updated model of the pathways in this organism. I also characterized the composition of G. sulfurreducens and found that it has high iron and lipid content independent of growth condition, and the high iron content is explained by the large abundance of multiheme cytochrome expression that I observed. I used multiple microscopy techniques to examine extracellular respiration in G. sulfurreducens, and in the process discovered a novel organelle: the intracytoplasmic membrane. I show 3D reconstructions of the organelle in G. sulfurreducens and discuss its implications for the cell’s metabolism. Finally, I discuss gene expression in T. ferriacetica in RNA samples collected from an anode-respiring culture and highlight the most abundantly expressed genes related to anode-respiring metabolism.

Trichloroethene (TCE) and hexavalent chromium (Cr (VI)) are ubiquitous subsurface contaminants affecting the water quality and threatening human health. Microorganisms capable of TCE and Cr (VI) reductions can be explored for bioremediation at contaminated sites. The goal of my dissertation research was to address challenges that decrease the efficiency of bioremediation in the subsurface. Specifically, I investigated strategies to (i) promote improve microbial reductive dechlorination extent through the addition of Fe0 and (ii) Cr (VI) bio-reduction through enrichment of specialized microbial consortia. Fe0 can enhance microbial TCE reduction by inducing anoxic conditions and generating H2 (electron donor). I first evaluated the effect of Fe0 on microbial reduction of TCE (with ClO4– as co-contaminant) using semi-batch soil microcosms. Results showed that high concentration of Fe0 expected during in situ remediation inhibited microbial TCE and ClO4– reduction when added together with Dehalococcoides mccartyi-containing cultures. A low concentration of aged Fe0 enhanced microbial TCE dechlorination to ethene and supported complete microbial ClO4– reduction. I then evaluated a decoupled Fe0 and biostimulation/bioaugmentation treatment approach using soil packed columns with continuous flow of groundwater. I demonstrated that microbial TCE reductive dechlorination to ethene can be benefitted by Fe0 abiotic reactions, when biostimulation and bioaugmentation are performed downstream of Fe0 addition. Furthermore, I showed that ethene production can be sustained in the presence of aerobic groundwater (after Fe0 exhaustion) by the addition of organic substrates. I hypothesized that some lessons learned from TCE Bioremediation can be applied also for other pollutants that can benefit from anaerobic reductions, like Cr (VI). Bioremediation of Cr (VI) has historically relied on biostimulation of native microbial communities, partially due to the lack of knowledge of the benefits of adding enriched consortia of specialized microorganisms (bioaugmentation). To determine the merits of a specialized consortium on bio-reduction of Cr (VI), I first enriched a culture on lactate and Cr (VI). The culture had high abundance of putative Morganella species and showed rapid and sustained Cr (VI) bio-reduction compared to a subculture grown with lactate only (without Morganella). Overall, this dissertation work documents possible strategies for synergistic abiotic and biotic chlorinated ethenes reduction, and highlights that specialized consortia may benefit Cr (VI) bio-reduction.

Cyanobacteria contribute to more than a quarter of the primary carbon fixation worldwide. They have evolved a CO2 concentrating mechanism (CCM) to enhance photosynthesis because inorganic carbon species are limited in the aqueous environment. Bicarbonate transporters SbtA and BicA are active components of CCM, and the determination of their structures is important to investigate the bicarbonate transport mechanisms. E. coli was selected as the expression host for these bicarbonate transporters, and optimization of expression and protein purification conditions was performed. Single particle electron cryomicroscopy (cryo-EM) or protein crystallography was carried out for each transporter. In this work, SbtA, BicA and SbtB, a regulator protein of SbtA, were heterologously expressed in E. coli and purified for structural studies. SbtB was highly expressed and two different crystal structures of SbtB were resolved at 2.01 Å and 1.8 Å, showing a trimer and dimer in the asymmetric unit, respectively. The yields of SbtA and BicA after purification reached 0.1 ± 0.04 and 6.5 ± 1.0 mg per liter culture, respectively. Single particle analysis showed a trimeric conformation of purified SbtA and promising interaction between SbtA and SbtB, where the bound SbtB was also possibly trimeric. For some crystallization experiments of these transporters, lipidic cubic phase (LCP) was used. In the case of LCP, often times the crystals grown are generally too tiny to withstand radiation damage from the X-ray beam during an X-ray diffraction experiment. As an alternative approach for this research, the microcrystal electron diffraction (MicroED) method was applied to the LCP-laden crystals because it is a powerful cryo-EM method for high-resolution structure determination from protein microcrystals. The new technique is termed as LCP-MicroED, however, prior to applying LCP-MicroED to the bicarbonate transporters, methods needed to be developed for LCP-MicroED. Therefore the model protein Proteinase K was used and its structure was determined to 2.0 Å by MicroED. Additionally, electron diffraction data from cholesterol and human A2A adenosine receptor crystals were collected at 1.0 Å and 4.5 Å using LCP-MicroED, respectively. Other applications of MicroED to different samples are also discussed.

The increasing concentrations of greenhouse gases into the atmosphere call for urgent measures to use non-fossil feedstock for fuels and chemicals. Synthesis gas (or syngas) is a mixture of three gases: hydrogen (H2), carbon monoxide (CO), and carbon dioxide (CO2). Syngas already is widely used as a non-fossil fuel and a building block for a variety of chemicals using the Fischer-Tropsch process. Recently, syngas fermentation has attracted attention as a more sustainable way for the conversion of syngas to chemicals, since its biocatalysts are self-generating, are resilient, and can utilize a wide range of syngas compositions. However, syngas fermentation has technical and economic limitations. This dissertation, by contributing to the understanding of syngas fermentation, helps to overcome the limitations. A bibliometric analysis showed the topic’s landscape and identified that mass transfer is the biggest challenge for the process. One means to improve syngas mass transfer is to use the membrane biofilm reactor, or MBfR, to deliver syngas to the microorganisms. MBfR experiments delivering pure H2 demonstrated that the H2:IC ratio (IC is inorganic carbon) controlled the overall production rate of organic compounds and their carbon-chain length. Organic chemicals up to eight carbons could be produced with a high H2:IC ratio. A novel asymmetric membrane dramatically improved mass transfer rates for all syngas components, and its low selectivity among them made it ideal for high-rate syngas fermentation. MBfR experiments using syngas and the asymmetric membrane, as well as a conventional symmetric membrane, confirmed that the key parameter for generating long-chain products was a high H2:IC ratio. The fast mass transfer rate of the asymmetric membrane allowed a very high areal production rate of acetate: 253 g.m-2.d-1, the highest reported to date. Since the membrane delivered H2 and C from the syngas feed, the relatively low selectivity of the asymmetric membrane favored acetogenesis over microbial chain elongation. A techno-economic analysis of the MBfR showed that the cost to produce acetate was less than its market price. All results presented in this dissertation support the potential of syngas fermentation using the MBfR as a means to produce commodity chemicals and biofuels from syngas.

Water is a vital resource, and its protection is a priority world-wide. One widespread threat to water quality is contamination by chlorinated solvents. These dry-cleaning and degreasing agents entered the watershed through spills and improper disposal and now are detected in 4% of U.S. aquifers and 4.5-18% of U.S. drinking water sources. The health effects of these contaminants can be severe, as they are associated with damage to the nervous, liver, kidney, and reproductive systems, developmental issues, and possibly cancer. Chlorinated solvents must be removed or transformed to improve water quality and protect human and environmental health. One remedy, bioaugmentation, the subsurface addition of microbial cultures able to transform contaminants, has been implemented successfully at hundreds of sites since the 1990s. Bioaugmentation uses the bacteria Dehalococcoides to transform chlorinated solvents with hydrogen, H2, as the electron donor. At advection limited sites, bioaugmentation can be combined with electrokinetics (EK-Bio) to enhance transport. However, challenges for successful bioremediation remain. In this work I addressed several knowledge gaps surrounding bioaugmentation and EK-Bio. I measured the H2 consuming capacity of soils, detailed the microbial metabolisms driving this demand, and evaluated how these finding relate to reductive dechlorination. I determined which reactions dominated at a contaminated site with mixed geochemistry treated with EK-Bio and compared it to traditional bioaugmentation. Lastly, I assessed the effect of EK-Bio on the microbial community at a field-scale site. Results showed the H2 consuming capacity of soils was greater than that predicted by initial measurements of inorganic electron acceptors and primarily driven by carbon-based microbial metabolisms. Other work demonstrated that, given the benefits of some carbon-based metabolisms to microbial reductive dechlorination, high levels of H2 consumption in soils are not necessarily indicative of hostile conditions for Dehalococcoides. Bench-scale experiments of EK-Bio under mixed geochemical conditions showed EK-Bio out-performed traditional bioaugmentation by facilitating biotic and abiotic transformations. Finally, results of microbial community analysis at a field-scale implementation of EK-Bio showed that while there were significant changes in alpha and beta diversity, the impact of EK-Bio on native microbial communities was minimal.

Eighty-two percent of the United States population reside in urban areas. The centralized treatment of the municipal wastewater produced by this population is a huge energy expenditure, up to three percent of the entire energy budget of the country. A portion of this energy is able to be recovered through the process of anaerobic sludge digestion. Typically, this technology converts the solids separated and generated during the wastewater treatment process into methane, a combustible gas that may be burned to generate electricity. Designing and optimizing anaerobic digestion systems requires the measurement of degradation rates for waste-specific kinetic parameters. In this work, I discuss the ways these kinetic parameters are typically measured. I recommend and demonstrate improvements to these commonly used measuring techniques. I provide experimental results of batch kinetic experiments exploring the effect of sludge pretreatment, a process designed to facilitate rapid breakdown of recalcitrant solids, on energy recovery rates. I explore the use of microbial electrochemical cells, an alternative energy recovery technology able to produce electricity directly from sludge digestion, as precise reporters of degradation kinetics. Finally, I examine a fundamental kinetic limitation of microbial electrochemical cells, acidification of the anode respiring biofilm, to improve their performance as kinetic sensors or energy recovery technologies.

Seeking to address sustainability issues associated with food waste (FW), and fat, oil, and grease (FOG) waste disposal, the City of Mesa commissioned the Biodesign Swette Center for Environmental Biotechnology (BSCEB) at Arizona State University (ASU) to study to the impact of implementing FW/FOG co-digestion at the wastewater treatment plant (WWTP). A key issue for the study was the “souring” of the anaerobic digesters (ADs), which means that the microorganism responsible for organic degradation were deactivated, causing failure of the AD. Several bench-scale reactors soured after the introduction of the FW/FOG feed streams. By comparing measurements from stable with measurements from the souring reactors, I identified two different circumstances responsible for souring events. One set of reactors soured rapidly after the introduction of FW/FOG due to the digester’s hydraulic retention times (HRT) becoming too short for stable operation. A second set of reactors soured after a long period of stability due to steady accumulation of fatty acids (FAs) that depleted bicarbonate alkalinity. FA accumulation was caused by the incomplete hydrolysis/fermentation of feedstock protein, leading to insufficient release of ammonium (NH4+). In contrast, carbohydrates were more rapidly hydrolyzed and fermented to FAs.

The most important contribution of my research is that I identified several leading indicators of souring. In all cases of souring, the accumulation of soluble chemical oxygen demand (SCOD) was an early and easily quantified indicator. A shift in effluent FA concentrations from shorter to longer species also portended souring. A reduction in the yield of methane (CH4) per mass of volatile suspended solids removed (VSSR) also identified souring conditions, but its variability prevented the methane yield from providing advanced warning to allow intervention. For the rapidly soured reactors, reduced bicarbonate alkalinity was the most useful warning sign, and an increasing ratio of SCOD to bicarbonate alkalinity was the clearest sign of souring. Because I buffered the slow-souring reactors with calcium carbonate (CaCO3), I could not rely on bicarbonate alkalinity as an indicator, which put a premium on SCOD as the early warning. I implemented two buffering regimes and demonstrated that early and consistent buffering could lead to reactor recovery.

Electro-Selective Fermentation (ESF) combines Selective Fermentation (SF) and a Microbial Electrolysis Cell (MEC) to selectively degrade carbohydrate and protein in lipid-rich microalgae biomass, enhancing lipid wet-extraction. In addition, saturated long-chain fatty acids (LCFAs) are produced via β-oxidation. This dissertation builds understanding of the biochemical phenomena and microbial interactions occurring among fermenters, lipid biohydrogenaters, and anode respiring bacteria (ARB) in ESF. The work begins by proving that ESF is effective in enhancing lipid wet-extraction from Scenedesmus acutus biomass, while also achieving “biohydrogenation” to produce saturated LCFAs. Increasing anode respiration effectively scavenges short chain fatty acids (SCFAs) generated by fermentation, reducing electron loss. However, the effectiveness of ESF depends on biochemical characteristics of the feeding biomass (FB). Four different FB batches yield different lipid-extraction performances, based on the composition of FB’s cellular structure. Finally, starting an ESF reactor with a long solid retention time (SRT), but then switching it to a short SRT provides high lipid extractability and volumetric production with low lipid los. Lipid fermenters can be flushed out with short a SRT, but starting with a short SRT fails achieve good results because fermenters needed to degrading algal protective layers also are flushed out and fail to recover when a long SRT is imposed. These results point to a potentially useful technology to harvest lipid from microalgae, as well as insight about how this technology can be best managed.

Microbial electrochemical cells (MxCs) are a novel technology that use anode-respiring bacteria (ARB) to bioremediate wastewaters and respire an electrical current, which can then be used directly to produce value-added products like hydrogen peroxide (H2O2). Ninety-five percent of the world’s H2O2 is currently produced using the anthraquinone process, whose production requires expensive and potentially carcinogenic catalysts and high amounts of electricity. However, the amount of H2O2 that can be produced from these microbial peroxide-producing cells (MPPCs) has not been thoroughly investigated. Predicting potential H2O2 production in MxCs is further complicated by a lack of mathematical models to predict performance utilizing complex waste streams like primary sludge (PS).

A reactor design methodology was developed for MPPCs to systematically optimize H2O2 production with minimal energy consumption. H2O2 stability was evaluated with different catholytes, membranes, and catalysts materials, and the findings used to design and operate long-term a dual-chamber, flat-plate MPPC using different catholytes, ferrochelating stabilizers, and hydraulic retention times (HRT). Up to 3.1 ± 0.37 g H2O2 L-1 was produced at a 4-h HRT in an MPPC with as little as 1.13 W-h g-1 H2O2 power input using NaCl catholytes. Attempts to improve H2O2 production by using weak acid buffers as catholytes or ferrochelating stabilizers failed for different reasons.

A non-steady-state mathematical model, MYAnode, was developed combinging existing wastewater treatment, anode biofilm, and chemical speciation models to predict MxC performance utilizing complex substrates. The model simulated the large-scale trends observed when operating an MPPC with PS substrate. At HRTs ≥ 12-d, the model demonstrated up to 20% Coulombic recovery. At these conditions, ARB required additional alkalinity production by ≥ 100 mgVSS/L of acetoclastic methanogens to prevent pH inhibition when little influent alkalinity is available. At lower HRTs, methanogens are unable to produce the alkalinity required to prevent ARB inhibition due to washout and rapid acidification of the system during fermentation. At ≥ 100 mgVSS/L of methanogens, increasing the diffusion layer thickness from 500 to 1000 μm improved Coulombic efficiency by 13.9%, while increasing particulate COD hydrolysis rates to 0.25/d only improved Coulombic efficiency by 3.9%.