Biological systems have long been known to utilize two processes for energy conservation: substrate-level phosphorylation and electron transport phosphorylation. Recently, a new bioenergetic process was discovered that increases ATP yields: flavin-based electron bifurcation (FBEB). This process couples an energetically favorable reaction with an energetically unfavorable one to conserve energy in the organism. Currently, the mechanisms of enzymes that perform FBEB are unknown. In this work, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn), a FBEB enzyme, is used as a model system to study this phenomenon. Nfn is a heterodimeric enzyme that reversibly couples the exergonic reduction of NADP+ by reduced ferredoxin with the endergonic reduction of NADP+ by NADH. Protein film electrochemistry (PFE) has been utilized to characterize the catalytic properties of three ferredoxins, possible substrates for Nfn enzymes, from organisms that perform FBEB: Pyrococcus furiosus (PfFd), Thermotoga maritima (TmFd), and Caldicellulosiruptor bescii (CbFd). Additionally, PFE is utilized to characterize three Nfn enzymes from two different archaea in the family Thermococcaceae: two from P. furiosus (PfNfnI and PfXfn), and one from Thermococcus sibiricus (TsNfnABC). Key results are as follows. The reduction potentials of the [4Fe4S]2+/1+ couple for all three ferredoxins are pH independent and modestly temperature dependent, and the Marcus reorganization energies of PfFd and TmFd are relatively small, suggesting optimized electron transfer. Electrocatalytic experiments show that PfNfnI is tuned for NADP+ reduction by both fast rates and a low binding constant for NADP+. A PfNfnI variant engineered to have only cysteines as coordinating ligands for its [FeS] clusters has significantly altered rates of electrocatalysis, substrate binding, and FBEB activity. This suggests that the heteroligands in the primary coordination sphere of the [FeS] clusters play a role in controlling catalysis by Nfn. Furthermore, a variant of PfNfnI lacking its small subunit, designed to probe allosteric effects at the bifurcating site, has altered substrate binding at the NADP(H) binding site, i.e. the bifurcation site. PfXfn and TsNfnABC, representing different types of Nfn enzymes, have different electrocatalytic properties than PfNfnI, including slower rates of FBEB. This suggests that Nfn enzymes vary significantly over phylogenetically similar organisms despite relatively high primary sequence homology.

This research explores microbial chain elongation as a pathway for production of complex organic compounds in soils with implication for the carbon cycle. In chain elongation, simple substrates such as ethanol and short chain carboxylates such as acetate can be converted to longer carbon chain carboxylates under anaerobic conditions through cyclic, reverse β oxidation. This pathway elongates the carboxylate by two carbons. The chain elongation process is overall thermodynamically feasible, and microorganisms gain energy through this process. There have been limited insights into the versatility of chain elongating substrates, understanding the chain elongating microbial community, and its importance in sequestering carbon in the soils.

We used ethanol, methanol, butanol, and hydrogen as electron donors and acetate and propionate as electron acceptors to test the occurrence of microbial chain elongation in four soils with different physicochemical properties and microbial communities. Common chain elongation products were the even numbered chains butyrate, caproate, and butanol, the odd numbered carboxylates valerate and heptanoate, along with molecular hydrogen. At a near neutral pH and mesophilic temperature, we observed a stable and sustained production of longer fatty acids along with hydrogen. Microbial community analysis show phylotypes from families such as Clostridiaceae, Bacillaceae, and Ruminococcaceae in all tested conditions. Through chain elongation, the products formed are less biodegradable. They may undergo transformations and end up as organic carbon, decreasing the greenhouse gas emissions, thus, making this process important to study.

The aims of this project are to demonstrate the design and implementation of separations modalities for 1) in situ product recovery and 2) upstream pretreatment of toxic feedstocks. Many value-added bioproducts such as alcohols (ethanol and butanol) developed for the transportation sector are known to be integral to a sustainable future. Likewise, bioproduced aromatic building blocks for sustainable manufacturing such as phenol will be equally important. The production of these compounds is often limited by product toxicity at 2- 20 g/L, whereas it may desirable to produce 20-200 g/L for economically feasible scale up. While low-cost feedstocks are desirable for economical production, they contain highly cytotoxic value-added byproducts such as furfural. It is therefore desirable to design facile detoxification methods for lignocellulose-derived feedstocks to isolate and recover furfural preceding ethanol fermentation by Escherichia coli. Correspondingly it is desirable to design efficient facile in situ recovery modalities for bioalcohols and phenolic bioproducts. Accordingly, in-situ removal modalities were designed for simultaneous acetone, butanol, and ethanol recovery. Additionally, a furfural removal modality from lignocellulosic hydrolysates was designed for upstream pretreatment. Solid-liquid adsorption was found to serve well each of the recovery modalities characterized here. More hydrophobic compounds such as butanol and furfural are readily recovered from aqueous solutions via adsorption. The primary operational drawback to adsorption is adsorbent recovery and subsequent desorption of the product. Novel magnetically separable mesoporous carbon powders (MMCPs) were characterized and found to be rapidly separable from solutions at 91% recovery by mass. Thermal desorption of value added products was found efficient for recovery of butanol and furfural. Fufural was desorbed from the MMCPs up to 57% by mass with repeated adsorption/thermal desorption cycles. Butanol was recovered from MMCPs up to an average 93% by mass via thermal desorption. As another valuable renewable fermentation product, phenol was also collected via in-situ adsorption onto Dowex Optipore L-493 resin. Phenol recovery from the resins was efficiently accomplished with tert-butyl methyl ether up to 77% after 3 washes.

One solution to mitigating global climate change is using cyanobacteria or single-celled algae (collectively microalgae) to replace petroleum-based fuels and products, thereby reducing the net release of carbon dioxide. This work develops and evaluates a mechanistic kinetic model for light-dependent microalgal growth. Light interacts with microalgae in a variety of positive and negative ways that are captured by the model: light intensity (LI) attenuates through a microalgal culture, light absorption provides the energy and electron flows that drive photosynthesis, microalgae pool absorbed light energy, microalgae acclimate to different LI conditions, too-high LI causes damage to the cells’ photosystems, and sharp increases in light cause severe photoinhibition that inhibits growth. The model accounts for all these phenomena by using a set of state variables that represent the pooled light energy, photoacclimation, PSII photo-damage, PSII repair inhibition and PSI photodamage. Sets of experiments were conducted with the cyanobacterium Synechocystis sp. PCC 6803 during step-changes in light intensity and flashing light. The model was able to represent and explain all phenomena observed in the experiments. This included the spike and depression in growth rate following an increasing light step, the temporary depression in growth rate following a decreasing light step, the shape of the steady-state growth-irradiance curve, and the “blending” of light and dark periods under rapid flashes of light. The LI model is a marked improvement over previous light-dependent growth models, and can be used to design and interpret future experiments and practical systems for generating renewable feedstock to replace petroleum.

The application of microalgal biofilms in wastewater treatment has great advantages such as abolishing the need for energy intensive aerators and recovering nutrients as energy, thus reducing the energy requirement of wastewater treatment several-fold. A 162 cm2 algal biofilm reactor with good wastewater treatment performance and a regular harvesting procedure was studied at lab scale to gain an understanding of effectual parameters such as hydraulic retention time (HRT; 2.6 and 1.3 hrs), liquid level (LL; 0.5 and 1.0 cm), and solids retention time (SRT; 3 and 1.5 wks). A revised synthetic wastewater “Syntho 3.7” was used as a surrogate of domestic primary effluent for nutrient concentration consistency in the feed lines. In the base case (2.6 hr HRT, 0.5 cm LL, and 3 wk SRT), percent removals of 69 ± 2 for total nitrogen (TN), 54 ± 21 for total phosphorous (TP), and 60 ± 7 for chemical oxygen demand (COD) were achieved and 4.0 ± 1.6 g/m2/d dry biomass was produced. A diffusion limitation was encountered when increasing the liquid level, while the potential to further decrease the HRT remains. Nonlinear growth kinetics was observed in comparing SRT variations, and promoting autotrophic growth seems possible. Future work will look towards producing a mathematical model and further testing the aptness of this system for large-scale implementation.

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.

Microbial electrochemical cells (MXCs) serve as an alternative anaerobic technology to anaerobic digestion for efficient energy recovery from high-strength organic wastes such as primary sludge (PS). The overarching goal of my research was to address energy conversion from PS to useful resources (e.g. hydrogen or hydrogen peroxide) through bio- and electro-chemical anaerobic conversion processes in MXCs.

First, a new flat-pate microbial electrolysis cell (MEC) was designed with high surface area anodes using carbon fibers, but without creating a large distance between the anode and the cathode (<0.5 cm) to reduce Ohmic overpotential. Through the improved design, operation, and electrochemical characterization, the applied voltages were reduced from 1.1 to ~0.85 V, at 10 A m-2. Second, PS conversion was examined through hydrolysis, fermentation, methanogenesis, and/or anode respiration. Since pretreatment often is required to accelerate hydrolysis of organic solids, I evaluated pulsed electric field technology on PS showing a modest improvement of energy conversion through methanogenesis and fermentation, as compared to the conversion from waste activated sludge (WAS) or WAS+PS. Then, a two-stage system (prefermented PS-fed MEC) yielded successful performance in terms of Coulombic efficiency (95%), Coulombic recovery (CR, 80%), and COD-removal efficiency (85%). However, overall PS conversion to electrical current (or CR) through pre-fermentation and MEC, was just ~16%. Next, a single-stage system (direct PS-fed MEC) with semi-continuous operation showed 34% CR at a 9-day hydraulic retention time. The PS-fed MEC also showed an important pH dependency, in which high pH (> 8) in the anode chamber improved anode respiration along with methanogen inhibition. Finally, H2O2 was produced in a PS-fed microbial electrochemical cell with a low energy requirement (~0.87 kWh per kg H2O2). These research developments will provide groundbreaking knowledge for MXC design, commercial application, and anaerobic energy conversion from other high-strength organic wastes to resources.

Biomass synthesis is a competing factor in biological systems geared towards generation of commodity and specialty chemicals, ultimately limiting maximum titer and yield; in this thesis, a widely generalizable, modular approach focused on decoupling biomass synthesis from the production of the phenylalanine in a genetically modified strain of E. coli BW25113 was explored with the use of synthetic trans-encoded small RNA (sRNA) to achieve greater efficiency. The naturally occurring sRNA MicC was used as a scaffold, and combined on a plasmid with a promoter for anhydrous tetracycline (aTc) and a T1/TE terminator. The coding sequence corresponding to the target binding site for fourteen potentially growth-essential gene targets as well as non-essential lacZ was placed in the seed region of the of the sRNA scaffold and transformed into BW25113, effectively generating a unique strain for each gene target. The BW25113 strain corresponding to each gene target was screened in M9 minimal media; decreased optical density and elongated cell morphology changes were observed and quantified in all induced sRNA cases where growth-essential genes were targeted. Six of the strains targeting different aspects of cell division that effectively suppressed growth and resulted in increased cell size were then screened for viability and metabolic activity in a scaled-up shaker flask experiment; all six strains were shown to be viable during stationary phase, and a metabolite analysis showed increased specific glucose consumption rates in induced strains, with unaffected specific glucose consumption rates in uninduced strains. The growth suppression, morphology and metabolic activity of the induced strains in BW25113 was compared to the bacteriostatic additives chloramphenicol, tetracycline, and streptomycin. At this same scale, the sRNA plasmid targeting the gene murA was transformed into BW25113 pINT-GA, a phenylalanine overproducer with the feedback resistant genes aroG and pheA overexpressed. Two induction times were explored during exponential phase, and while the optimal induction time was found to increase titer and yield amongst the BW25113 pINT-GA murA sRNA variant, overall this did not have as great a titer or yield as the BW25113 pINT-GA strain without the sRNA plasmid; this may be a result of the cell filamentation.

Microbial Electrochemical Cell (MXC) technology harnesses the power stored in wastewater by using anode respiring bacteria (ARB) as a biofilm catalyst to convert the energy stored in waste into hydrogen or electricity. ARB, or exoelectrogens, are able to convert the chemical energy stored in wastes into electrical energy by transporting electrons extracellularly and then transferring them to an electrode. If MXC technology is to be feasible for ‘real world’ applications, it is essential that diverse ARB are discovered and their unique physiologies elucidated- ones which are capable of consuming a broad spectrum of wastes from different contaminated water sources.

This dissertation examines the use of Gram-positive thermophilic (60 ◦C) ARB in MXCs since very little is known regarding the behavior of these microorganisms in this setting. Here, we begin with the draft sequence of the Thermincola ferriacetica genome and reveal the presence of 35 multiheme c-type cytochromes. In addition, we employ electrochemical techniques including cyclic voltammetry (CV) and chronoamperometry (CA) to gain insight into the presence of multiple pathways for extracellular electron transport (EET) and current production (j) limitations in T. ferriacetica biofilms.

Next, Thermoanaerobacter pseudethanolicus, a fermentative ARB, is investigated for its ability to ferment pentose and hexose sugars prior to using its fermentation products, including acetate and lactate, for current production in an MXC. Using CA, current production is tracked over time with the generation and consumption of fermentation products. Using CV, the midpoint potential (EKA) of the T. pseudethanolicus EET pathway is revealed.



Lastly, a cellulolytic microbial consortium was employed for the purpose ofassessing the feasibility of using thermophilic MXCs for the conversion of solid waste into current production. Here, a highly enriched consortium of bacteria, predominately from the Firmicutes phylum, is capable of generating current from solid cellulosic materials.

Bioremediation of trichloroethene (TCE) using Dehalococcoides mccartyi-containing microbial cultures is a recognized and successful remediation technology. Our work with an upflow anaerobic sludge blanket (UASB) reactor has shown that high-performance, fast-rate dechlorination of TCE can be achieved by promoting bioflocculation of Dehalococcoides mccartyi-containing cultures. The bioreactor achieved high maximum conversion rates of 1.63 ± 0.012 mmol Cl- Lculture-1 h-1 at an HRT of 3.6 hours and >97% dechlorination of TCE to ethene while continuously fed 2 mM TCE. The UASB generated bioflocs from a microbially heterogeneous dechlorinating culture and produced Dehalococcoides mccartyi densities of 1.73x10-13 cells Lculture-1 indicating that bioflocculation of Dehalococcoides mccartyi-containing cultures can lead to high density inocula and high-performance, fast-rate bioaugmentation culture for in situ treatment. The successful operation of our pilot scale bioreactor led to the assessment of the technology as an onsite ex-situ treatment system. The bioreactor was then fed TCE-contaminated groundwater from the Motorola Inc. 52nd Street Plant Superfund site in Phoenix, AZ augmented with the lactate and methanol. The bioreactor maintained >99% dechlorination of TCE to ethene during continuous operation at an HRT of 3.2 hours. Microbial community analysis under both experimental conditions reveals shifts in the community structure although maintaining high rate dechlorination. High density dechlorinating cultures containing bioflocs can provide new ways to 1) produce dense bioaugmentation cultures, 2) perform ex-situ bioremediation of TCE, and 3) increase our understanding of Dehalococcoides mccartyi critical microbial interactions that can be exploited at contaminated sites in order to improve long-term bioremediation schemes.