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- All Subjects: Biochemistry
- All Subjects: Fermentation
Description
Microbial electrochemical cells (MXCs) offer an alternative to methane production in anaerobic water treatment and the recapture of energy in waste waters. MXCs use anode respiring bacteria (ARB) to oxidize organic compounds and generate electrical current. In both anaerobic digestion and MXCs, an anaerobic food web connects the metabolisms of different microorganisms, using hydrolysis, fermentation and either methanogenesis or anode respiration to break down organic compounds, convert them to acetate and hydrogen, and then convert those intermediates into either methane or current. In this dissertation, understanding and managing the interactions among fermenters, methanogens, and ARB were critical to making developments in MXCs. Deep sequencing technologies were used in order to identify key community members, understand their role in the community, and identify selective pressures that drove the structure of microbial communities. This work goes from developing ARB communities by finding and using the best partners to managing ARB communities with undesirable partners. First, the foundation of MXCs, namely the ARB they rely on, was expanded by identifying novel ARB, the genus Geoalkalibacter, and demonstrating the presence of ARB in 7 out of 13 different environmental samples. Second, a new microbial community which converted butyrate to electricity at ~70% Coulombic efficiency was assembled and demonstrated that mixed communities can be used to assemble efficient ARB communities. Third, varying the concentrations of sugars and ethanol fed to methanogenic communities showed how increasing ED concentration drove decreases in methane production and increases in both fatty acids and the propionate producing genera Bacteroides and Clostridium. Finally, methanogenic batch cultures, fed glucose and sucrose, and exposed to 0.15 – 6 g N-NH4+ L-1 showed that increased NH4+ inhibited methane production, drove fatty acid and lactate production, and enriched Lactobacillales (up to 40% abundance) above 4 g N-NH4+ L-1. Further, 4 g N-NH4+ L-1 improved Coulombic efficiencies in MXCs fed with glucose and sucrose, and showed that MXC communities, especially the biofilm, are more resilient to high NH4+ than comparable methanogenic communities. These developments offer new opportunities for MXC applications, guidance for efficient operation of MXCs, and insights into fermentative microbial communities.
ContributorsMiceli, Joseph (Author) / Torres, César I (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Rittmann, Bruce (Committee member) / Arizona State University (Publisher)
Created2015
Description
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.
ContributorsJennings, David Peter (Author) / Jones, Anne K (Thesis advisor) / Redding, Kevin E (Committee member) / Torres, César I (Committee member) / Arizona State University (Publisher)
Created2018
Description
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.
ContributorsHerschel, Daniel Jordan (Author) / Nielsen, David R (Thesis advisor) / Torres, César I (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016
Description
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.
ContributorsLiu, Yuanzhen (Author) / Rittmann, Bruce E. (Thesis advisor) / Torres, César I (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Arizona State University (Publisher)
Created2019