Development and Application of Fluxomics Tools for Analyzing Metabolisms in Non-Model Microorganisms

Decoding microbial metabolism is of great importance in revealing the mechanisms governing the physiology of microbes and rewiring the cellular functions in metabolic engineering. Complementing the genomics, transcriptomics, proteinomics and metabolomics analysis of microbial metabolism, fluxomics tools can measure and simulate the in vivo enzymatic reactions as direct readouts of microbial metabolism. This dissertation develops and applies broad-scope tools in metabolic flux analysis to investigate metabolic insights of non-model environmental microorganisms. 13C-based pathway analysis has been applied to analyze specific carbon metabolic routes by tracing and analyzing isotopomer labeling patterns of different metabolites after growing cells with 13C-labeled substrates. Novel pathways, including Re-type citrate synthase in tricarboxylic acid cycle and citramalate pathways as an alternate route for isoleucine biosynthesis, have been identified in Thermoanaerobacter X514 and other environmental microorganisms. Via the same approach, the utilizations of diverse carbon/nitrogen substrates and productions of hydrogen during mixotrophic metabolism in Cyanothece 51142 have been characterized, and the medium for a slow-growing bacterium, Dehalococcoides ethenogenes 195, has been optimized. In addition, 13C-based metabolic flux analysis has been developed to quantitatively profile flux distributions in central metabolisms in a green sulfur bacterium, Chlorobaculum tepidum, and thermophilic ethanol-producing Thermoanaerobacter X514. The impact of isotope discrimination on 13C-based metabolic flux analysis has also been estimated. A constraint-based flux analysis approach was newly developed to integrate the bioprocess model into genome-scale flux balance analysis to decipher the dynamic metabolisms of Shewanella oneidensis MR-1. The sub-optimal metabolism and the time-dependent metabolic fluxes were profiled in a genome-scale metabolic network. A web-based platform was constructed for high-throughput metabolic model drafting to bridge the gap between fast-paced genome-sequencing and slow-paced metabolic model reconstruction. The platform provides over 1,000 sequenced genomes for model drafting and diverse customized tools for model reconstruction. The in silico simulation of flux distributions in both metabolic steady state and dynamic state can be achieved via flux balance analysis and dynamic flux balance analysis embedded in this platform. Cutting-edge fluxomics tools for functional characterization and metabolic prediction continue to be developed in the future. Broad-scope systems biology tools with integration of transcriptomics, proteinomics and fluxomics can reveal cell-wide regulations and speed up the metabolic engineering of non-model microorganisms for diverse bioenergy and environmental applications

Engineering Microbial Consortia for High-Performance Cellulosic Hydrolyzates-Fed Microbial Fuel Cells

Microbial fuel cells (MFCs) are eco-friendly bio-electrochemical reactors that use exoelectrogens as biocatalyst for electricity harvest from organic biomass, which could also be used as biosensors for long-term environmental monitoring. Glucose and xylose, as the primary ingredients from cellulose hydrolyzates, is an appealing substrate for MFC. Nevertheless, neither xylose nor glucose can be utilized as carbon source by well-studied exoelectrogens such as Shewanella oneidensis. In this study, to harvest the electricity by rapidly harnessing xylose and glucose from corn stalk hydrolysate, we herein firstly designed glucose and xylose co-fed engineered Klebsiella pneumoniae-S. oneidensis microbial consortium, in which K. pneumoniae as the fermenter converted glucose and xylose into lactate to feed the exoelectrogens (S. oneidensis). To produce more lactate in K. pneumoniae, we eliminated the ethanol and acetate pathway via deleting pta (phosphotransacetylase gene) and adhE (alcohol dehydrogenase gene) and further constructed a synthesis and delivery system through expressing ldhD (lactate dehydrogenase gene) and lldP (lactate transporter gene). To facilitate extracellular electron transfer (EET) of S. oneidensis, a biosynthetic flavins pathway from Bacillus subtilis was expressed in a highly hydrophobic S. oneidensis CP-S1, which not only improved direct-contacted EET via enhancing S. oneidensis adhesion to the carbon electrode but also accelerated the flavins-mediated EET via increasing flavins synthesis. Furthermore, we optimized the ratio of glucose and xylose concentration to provide a stable carbon source supply in MFCs for higher power density. The glucose and xylose co-fed MFC inoculated with the recombinant consortium generated a maximum power density of 104.7 ± 10.0 mW/m2, which was 7.2-folds higher than that of the wild-type consortium (12.7 ± 8.0 mW/m2). Lastly, we used this synthetic microbial consortium in the corn straw hydrolyzates-fed MFC, obtaining a power density 23.5 ± 6.0 mW/m2

Anaerobic Shewanella physiology: An unusual respiratory substrate and an unusual respiratory partner

University of Minnesota M.S. thesis.August 2017. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Jeffrey Gralnick. 1 computer file (PDF); vi, 39 pages.With an elegant and flexible electron transport chain, Shewanella oneidensis strain MR-1, is the most versatile respiratory organism known to date. MR-1 is a diverse respiratory heterotroph that lives in complex aquatic communities, often in association with other microorganism or eukaryotes, such as fish and algae. Fish produce TMAO as an osmoprotector, which also serve as a respiratory substrate for Shewanella isolates that are able to respire it. Although, TMAO is readily found in the aquatic environments where MR-1 is known to be found, MR-1 metabolism under TMAO respiring conditions is not fully understood. In addition, bacteria are usually studied as monocultures in laboratory conditions, however, microorganisms exist in nature as members of communities that interact with each other. Therefore, the factors that shape microbial behavior and interactions in communities remain largely undefined. The work presented in this thesis aims to further elucidate the metabolic strategy of MR-1 under TMAO respiring conditions, as well as, in a commensal interaction with Geobacter sulfurreducens. Coupled to the reduction of terminal electron acceptors, MR-1 has an aerobic branch, as well as, an anaerobic branch for the oxidation of carbon sources. However, in conditions where TMAO is the sole electron acceptor, the oxidation pathway for carbon sources is not fully understood. Furthermore, at the electron transport chain level, TMAO is reduced differently from other anaerobic compounds. Therefore, we aim to understand electron and carbon flux under growth conditions with this important electron acceptor. We have made gene deletions of key enzymes in both aerobic and anaerobic metabolic pathways in MR-1, and assayed for growth under conditions where TMAO is the sole terminal electron acceptor. We aim to begin to explore MR-1 metabolism in a more complex system with two organisms instead of one. For this purpose, we have engineered a close physical associationbetween S. oneidensis and Geobacter sulfurreducens. G. sulfurreducens is an anaerobic subsurface bacterium and another well-characterized organism capable of metal reduction and extracellular electron transfer. By performing laboratory evolution of this synthetic co-culture we aim to identify genes implicated in community interaction and understand how these genes influence their metabolism

Economic and empirical investigation of bioelectrochemical systems for CO₂ utilization

PhD ThesisThis thesis investigates economically and empirically the feasibility of CO2 reduction into high grade chemicals using microbial electrosynthesis (MES). An economic evaluation was initially performed for MES and anaerobic fermentation (AF) for 100 tonnes per year (t/y) acetic acid production. MES and AF incurred high investment and production costs; however, integrating MES and AF decreased investment costs, doubled production rates, and set production cost at 0.24 £/Kg which is market competitive (0.48 £/Kg). Although integrating MES and AF processes showed to be cost effective, it generated no positive return across 15 years of operation. Similar analyses were used to evaluate MES as stand-alone process for the production of acetic, formic and propionic acids, methanol, and ethanol at higher production rates (1000 t/y). High returns were evaluated for formic acid (21%) and ethanol (14%) compared to the minimum requirements of the industry (11.60%) making these products economically attractive. Experimentally, volatile fatty acid bioproduction was investigated in H-shape bioelectrochemical systems using Shewanella Oneidensis MR-1 as biocatalyst and CO2 as a substrate on polarised carbon cloth electrodes. Biofilm and mediated driven systems were used to evaluate the influence of electron transfer on bioproduction. It was found that mediated systems (0.66 mmol/L) produced more volatile fatty acids than biofilm systems (0.53 mmol/L), suggesting that the use of mediators enhances electron transfer. Different polarizations (-0.2, -0.4, -0.6 and -0.8 V) were also evaluated on biofilm driven systems, revealing that volatile fatty acid production was not affected by polarization (p=0.192) and incurred low cathode capture (13-77%) and energy (0.0009-0.6%) efficiencies which suggests a biochemical process rather than respiration. This was later confirmed using extracted proteins from Shewanella Oneidensis MR-1 cells. The effects of operating conditions (i.e. temperature and agitation) and biofilm development technique: open circuit (OCP) and closed circuit (CCP) potential, were further assessed for energy production. It was found that energy production increased with high temperature (30 oC) and slow agitation (90 rpm), as reflected by higher current generation (median = 12.05 μA), more live cells number (median=2.3×106 cells), and better electrode bacterial coverage (median=35.29%). In addition, using OCP biofilms offered further advantages by reducing the lag phase (1-2 days). The effect of OCP and CCP biofilms operating at the best operating conditions found were then examined for chemical production. OCP and CCP biofilms resulted in the synthesis of different chemicals suggesting that the bacterial metabolism is dependent on the biofilm development conditions. These findings offer insights on MR-1 performance and reveal a bright opportunity towards the use and scale-up of MES for a technically and economically viable bioprocess

Quorum Sensing durch das bakterielle Signal Autoinducer-2: Phylogenetische Verbreitung des Synthese-Genes LuxS und dessen Rolle in Shewanella oneidensis

Pseudomonas aeruginosa, an important human pathogen, does not produce AI-2. However, it is possible that a few of its quorum sensing controlled virulence genes are stimulated in the presence of AI-2. This stimulation could not be confirmed here. AI-2 is the side product of the Bacteria-specific LuxS enzyme and has an important role for detoxification of S-adenosyl-homocysteine (SAH) in the activated methyl cycle. Some bacteria, however, use the SAH hydrolase for the same purpose. In this study, 164 marine isolates were tested for the presence of the luxS and sahH genes. Within one phylogenetic group, phylum or genus, these isolates used one pathway consistently and in congruence with fully-sequenced organisms. Therefore the presence of these genes and also AI-2 production are determined by the phylogenetic position and biased only slightly by the life mode of a species. To elucidate the role of the luxS gene in Shewanella oneidensis, insertion and deletion luxS mutants as well as an insertion control were constructed. The insertion luxS mutant and its control (luxS-ins and WTKm) were compared in their growth, AI-2 production, secretome, biofilm growth and siderophore production. Both strains grew at identical growth rates. The WTKm strain produced AI-2 at wildtype levels, while the luxS-ins mutant did not. In the secretome study, marginal differences were observed in a complex LB medium. In a defined minimal medium, the biofilm of the luxS-ins mutant developed faster and exhibited a less- structured, less-compact biofilm structure than that of the WTKm strain. Also in a defined minimal medium, the luxS-ins mutant produced more siderophores than WTKm strains after two days’ incubation. If these phenotype changes of the luxS mutant were caused by the absence of AI-2, this can be determined first after complementation with AI-2.P. aeruginosa, ein wichtiges Humanpathogen, produziert kein AI-2. Dennoch können einige seiner Quorum Sensing kontrollierten Virulenzgene in Gegenwart von AI-2 leicht stimuliert werden. Diese Stimulation konnte nicht bestätigt werden. AI-2 ist ein Nebenprodukt des für Bakterien spezifischen LuxS Enzyms, das eine wichtige Rolle für die Entgiftung des Moleküls S-Adenosyl-Homocystein (SAH) im aktivierten Methylzyklus spielt. Manche Bakterien nutzen jedoch SAH Hydrolase für die Entgiftung. In dieser Studie, wurden 164 marine Isolate auf die Präsenz der luxS und sahH Gene getestet. Die Isolate sowie die vollsequenzierten Organismen nutzen ausschließlich einen dieser Wege, darüber hinaus denselben Weg innerhalb einer phylogenetischen Gruppe, Phylum oder Genus. Somit wird die Präsenz dieser Gene und daher die AI-2 Produktion durch die phylogenetische Position und nur geringfügig durch die Lebensweise einer Spezies bestimmt. Um die Rolle des luxS Genes in Shewanella oneidensis zu klären, wurden Insertions- und Deletionsmutanten des luxS Genes, sowie eine Insertionskontrolle konstruiert. Die Insertionsmutante und deren Kontrolle (luxS-ins and WTKm) wurden in den Phänotypen Wachstum, AI-2 Produktion, Sekretom, Biofilmwachstum und Siderophore-Produktion verglichen. Beide Stämme wuchsen identisch schnell. Der WTKm Stamm produzierte AI-2 auf dem Niveau des Wildtypes, die luxS-ins Mutante produzierte jedoch kein AI-2. In der Sekretom-Analyse konnten marginale Unterschiede im komplexen Medium festgestellt werden. Im definierten Minimalmedium, entwickelte sich der Biofilm der luxS-ins Mutante schneller und zeigte eine weniger differenzierte, weniger feste Biofilmstruktur als der WTKm Stamm. Ebenso im definierten Minimalmedium, produzierte die luxS-ins Mutante mehr Siderophore als der WTKm Stamm nach zwei Tagen Inkubation. Ob die Phänotypen der luxS Mutante durch den Mangel an AI-2 verursacht wurden, kann erst durch die Komplementierung mit AI-2 bestätigt werden

Valorisation of CO 2 into Value-Added Products via Microbial Electrosynthesis (MES) and Electro-Fermentation Technology

Microbial electrocatalysis reckons on microbes as catalysts for reactions occurring at electrodes. Microbial fuel cells and microbial electrolysis cells are well-known in this context; both prefer the oxidation of organic and inorganic matter for producing electricity. Notably, the synthesis of high energy-density chemicals (fuels) or their precursors by microorganisms using bio-cathode to yield electrical energy is called Microbial Electrosynthesis (MES), giving an exceptionally appealing novel way for producing beneficial products from electricity and wastewater. This review accentuates the concept, importance and opportunities of MES, as an emerging discipline at the nexus of microbiology and electrochemistry. Production of organic compounds from MES is considered as an effective technique for the generation of various beneficial reduced end-products (like acetate and butyrate) as well as in reducing the load of CO2 from the atmosphere to mitigate the harmful effect of greenhouse gases in global warming. Although MES is still an emerging technology, this method is not thoroughly known. The authors have focused on MES, as it is the next transformative, viable alternative technology to decrease the repercussions of surplus carbon dioxide in the environment along with conserving energy

The genetics of geochemistry

Bacteria are remarkable in their metabolic diversity due to their ability to harvest energy from myriad oxidation and reduction reactions. In some cases, their metabolisms involve redox transformations of metal(loid)s, which lead to the precipitation, transformation, or dissolution of minerals. Microorganism/mineral interactions not only affect the geochemistry of modern environments, but may also have contributed to shaping the near-surface environment of the early Earth. For example, bacterial anaerobic respiration of ferric iron or the toxic metalloid arsenic is well known to affect water quality in many parts of the world today, whereas the utilization of ferrous iron as an electron donor in anoxygenic photosynthesis may help explain the origin of Banded Iron Formations, a class of ancient sedimentary deposits. Bacterial genetics holds the key to understanding how these metabolisms work. Once the genes and gene products that catalyze geochemically relevant reactions are understood, as well as the conditions that trigger their expression, we may begin to predict when and to what extent these metabolisms influence modern geochemical cycles, as well as develop a basis for deciphering their origins and how organisms that utilized them may have altered the chemical and physical features of our planet

Periplasmatische Elektronentransfer-Reaktionen in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 ist einer der Modellorganismen zur Erforschung der dissimilatorischen Eisenreduktion (DIR). Zur Etablierung von extrazellulärem Elektronentransfer ist die Übertragung der Elektronen durch das Periplasma Gram-negativer Bakterien obligatorisch. Diese Arbeit untersucht die Vorgänge, die periplasmatischen Elektronentransport in S. oneidensis vermitteln