Enhancing acetone production from H2 and CO2 using supplemental electron acceptors in an engineered Moorella thermoacetica.

Acetogens grow autotrophically and use hydrogen (H2) as the energy source to fix carbon dioxide (CO2). This feature can be applied to gas fermentation, contributing to a circular economy. A challenge is the gain of cellular energy from H2 oxidation, which is substantially low, especially when acetate formation coupled with ATP production is diverted to other chemicals in engineered strains. Indeed, an engineered strain of the thermophilic acetogen Moorella thermoacetica that produces acetone lost autotrophic growth on H2 and CO2. We aimed to recover autotrophic growth and enhance acetone production, in which ATP production was assumed to be a limiting factor, by supplementing with electron acceptors. Among the four selected electron acceptors, thiosulfate and dimethyl sulfoxide (DMSO) enhanced both bacterial growth and acetone titers. DMSO was the most effective and was further analyzed. We showed that DMSO supplementation enhanced intracellular ATP levels, leading to increased acetone production. Although DMSO is an organic compound, it functions as an electron acceptor, not a carbon source. Thus, supplying electron acceptors is a potential strategy to complement the low ATP production caused by metabolic engineering and to improve chemical production from H2 and CO2.

The effect of not-anaerobicization and discolored bacteria on uranium reduction by Shewanella sp. RCRI7.

Shewanella sp. RCRI7 is a native strain capable of reducing uranium in anaerobic conditions. In order to employ this bacterium for the bioremediation, the mutual effects of uranium and the bacteria are studied in two different approaches. The optimal settings for the bacterial proliferation capacity and uranium reduction without anaerobicization of the environment, as well as the related effects of bioremediation and bacterial color under uranium-reducing conditions, have been investigated in this study. Uranium reduction procedure was analyzed using XRD, spectrophotometry and ICP-AES. In addition, the uranium's effect on the population of the first-generation of the bacteria as well as the color and growth of the second-generation were investigated using neobar lam and CFU (Colony Forming Unit), respectively. Uranium toxicity reduced the population of non-anaerobicized bacteria more than the anaerobicized bacteria after one day of incubation, while the amount of uranium extracted by the bacteria was almost the same. In both situations, the bacteria were able to reduce uranium after two weeks of incubation. In addition to the cell counts, uranium toxicity disrupts the growth and development of healthy second-generation anaerobicized bacteria, as created creamy-colored colonies grow slower than red-colored colonies. Furthermore, due to malfunctioning cytochromes, unlike red bacteria, creamy-colored bacteria were unable to extract the optimum amount of uranium. This study reveals that reduced uranium can be produced in a deprived environment without anaerobicization. Creamy-colored Shewanella can remove soluble uranium, however the most effective bacteria have red cytochromes. These findings represent a big step forward in the industrialization of uranium bioremediation.

Iodate Reduction by Shewanella oneidensis Requires Genes Encoding an Extracellular Dimethylsulfoxide Reductase.

Microbial iodate (IO3 -) reduction is a major component of the iodine biogeochemical reaction network in anaerobic marine basins and radioactive iodine-contaminated subsurface environments. Alternative iodine remediation technologies include microbial reduction of IO3 - to iodide (I-) and microbial methylation of I- to volatile gases. The metal reduction pathway is required for anaerobic IO3 - respiration by the gammaproteobacterium Shewanella oneidensis. However, the terminal IO3 - reductase and additional enzymes involved in the S. oneidensis IO3 - electron transport chain have not yet been identified. In this study, gene deletion mutants deficient in four extracellular electron conduits (EECs; ΔmtrA, ΔmtrA-ΔmtrDEF, ΔmtrA-ΔdmsEF, ΔmtrA-ΔSO4360) and DMSO reductase (ΔdmsB) of S. oneidensis were constructed and examined for anaerobic IO3 - reduction activity with either 20 mM lactate or formate as an electron donor. IO3 - reduction rate experiments were conducted under anaerobic conditions in defined minimal medium amended with 250 µM IO3 - as anaerobic electron acceptor. Only the ΔmtrA mutant displayed a severe deficiency in IO3 - reduction activity with lactate as the electron donor, which suggested that the EEC-associated decaheme cytochrome was required for lactate-dependent IO3 - reduction. The ΔmtrA-ΔdmsEF triple mutant displayed a severe deficiency in IO3 - reduction activity with formate as the electron donor, whereas ΔmtrA-ΔmtrDEF and ΔmtrA-ΔSO4360 retained moderate IO3 - reduction activity, which suggested that the EEC-associated dimethylsulfoxide (DMSO) reductase membrane-spanning protein DmsE, but not MtrA, was required for formate-dependent IO3 - reduction. Furthermore, gene deletion mutant ΔdmsB (deficient in the extracellular terminal DMSO reductase protein DmsB) and wild-type cells grown with tungsten replacing molybdenum (a required co-factor for DmsA catalytic activity) in defined growth medium were unable to reduce IO3 - with either lactate or formate as the electron donor, which indicated that the DmsAB complex functions as an extracellular IO3 - terminal reductase for both electron donors. Results of this study provide complementary genetic and phenotypic evidence that the extracellular DMSO reductase complex DmsAB of S. oneidensis displays broad substrate specificity and reduces IO3 - as an alternate terminal electron acceptor.

U (VI) tolerance affects Shewanella sp. RCRI7 biological responses: growth, morphology and bioreduction ability.

Native Shewanella sp. RCRI7 is recently counted as an operative bacterium in the uranium bio-reduction. The aim of this study was to investigate the effects of uranium tolerance on the morphology and population of RCRI7, following its potential removal capacity in different time intervals. In this research, the bacterial growth and uranium removal kinetic were evaluated in aerobic TSB medium, uranium-reducing condition (URC), aerobic uranium-containing (AUC) and anaerobic uranium-free (AUF) solution, following evaluations of omcAB gene expressions. In addition, spectrophotometry analyses were performed in URC confirming the bio-reduction mechanism. It was found that the bacteria can grow efficiently in the presence of 0.5 mM uranium anaerobically, unlike AUC and AUF solutions. Since the bacterium's adsorption capacity is quickly saturated, it can be deduced that uranium reduction should be dominant as incubation times proceed up to 84 h in URC. In 92 h incubation, the adsorbed uranium containing unreduced and reduced (U (IV) monomeric), was released to the solution due to either increased pH or bacterial death. In AUC and AUF, improper conditions lead to the reduced bacterial size (coccus-shape formation) and increased bacterial aggregations; however, membrane vesicles produced by the bacteria avoid the uranium incrustation in AUC. In overall, this study implies that Shewanella sp. RCRI7 are well tolerated by uranium under anaerobic conditions and the amount of regenerated uranium increases over time in the reduced form.

Selenium respiration in anaerobic bacteria: Does energy generation pay off?

Selenium (Se) respiration in bacteria was revealed for the first time at the end of 1980s. Although thermodynamically-favorable, energy-dense and documented in phylogenetically-diverse bacteria, this metabolic process appears to be accompanied by a number of challenges and numerous unanswered questions. Selenium oxyanions, SeO42- and SeO32-, are reduced to elemental Se (Se0) through anaerobic respiration, the end product being solid and displaying a considerable size (up to 500 nm) at the bacterial scale. Compared to other electron acceptors used in anaerobic respiration (e.g. N, S, Fe, Mn, and As), Se is one of the few elements whose end product is solid. Furthermore, unlike other known bacterial intracellular accumulations such as volutin (inorganic polyphosphate), S0, glycogen or magnetite, Se0 has not been shown to play a nutritional or ecological role for its host. In the context of anaerobic respiration of Se oxyanions, biogenic Se0 appears to be a by-product, a waste that needs proper handling, and this raises the question of the evolutionary implications of this process. Why would bacteria use a respiratory substrate that is useful, in the first place, and then highly detrimental? Interestingly, in certain artificial ecosystems (e.g. upflow bioreactors) Se0 might help bacterial cells to increase their density and buoyancy and thus avoid biomass wash-out, ensuring survival. This review article provides an in-depth analysis of selenium respiration (model selenium respiring bacteria, thermodynamics, respiratory enzymes, and genetic determinants), complemented by an extensive discussion about the evolutionary implications and the properties of biogenic Se0 using published and original/unpublished results.

Secreted Flavin Cofactors for Anaerobic Respiration of Fumarate and Urocanate by Shewanella oneidensis: Cost and Role.

Shewanella oneidensis strain MR-1, a facultative anaerobe and model organism for dissimilatory metal reduction, uses a periplasmic flavocytochrome, FccA, both as a terminal fumarate reductase and as a periplasmic electron transfer hub for extracellular respiration of a variety of substrates. It is currently unclear how maturation of FccA and other periplasmic flavoproteins is achieved, specifically in the context of flavin cofactor loading, and the fitness cost of flavin secretion has not been quantified. We demonstrate that deletion of the inner membrane flavin adenine dinucleotide (FAD) exporter Bfe results in a 23% slower growth rate than that of the wild type during fumarate respiration and an 80 to 90% loss in fumarate reductase activity. Exogenous flavin supplementation does not restore FccA activity in a Δbfe mutant unless the gene encoding the periplasmic FAD hydrolase UshA is also deleted. We demonstrate that the small Bfe-independent pool of FccA is sufficient for anaerobic growth with fumarate. Strains lacking Bfe were unable to grow using urocanate as the sole electron acceptor, which relies on the periplasmic flavoprotein UrdA. We show that periplasmic flavoprotein maturation occurs in careful balance with periplasmic FAD hydrolysis, and that the current model for periplasmic flavin cofactor loading must account for a Bfe-independent mechanism for flavin transport. Finally, we determine that the metabolic burden of flavin secretion is not significant during growth with flavin-independent anaerobic electron acceptors. Our work helps frame the physiological motivations that drove evolution of flavin secretion by ShewanellaIMPORTANCEShewanella species are prevalent in marine and aquatic environments, throughout stratified water columns, in mineral-rich sediments, and in association with multicellular marine and aquatic organisms. The diversity of niches shewanellae can occupy are due largely to their respiratory versatility. Shewanella oneidensis is a model organism for dissimilatory metal reduction and can respire a diverse array of organic and inorganic compounds, including dissolved and solid metal oxides. The fumarate reductase FccA is a highly abundant multifunctional periplasmic protein that acts to bridge the periplasm and temporarily store electrons in a variety of respiratory nodes, including metal, nitrate, and dimethyl sulfoxide respiration. However, maturation of this central protein, particularly flavin cofactor acquisition, is poorly understood. Here, we quantify the fitness cost of flavin secretion and describe how free flavins are acquired by FccA and a homologous periplasmic flavoprotein, UrdA.

Parameter Selection for a Microvolume Electrochemical Escherichia coli Detector for Pairing with a Concentration Device.

Waterborne infections are responsible for health problems worldwide and their prompt and sensitive detection in recreational and potable water is of great importance. Bacterial identification and enumeration in water samples ensures water is safe for its intended use. Culture-based methods can be time consuming and are usually performed offsite. There is a need to for automated and distributed at-source detectors for water quality monitoring. Herein we demonstrate a microvolume Escherichia coli (E. coli) detector based on a screen printed electrode (SPE) bioelectroanalytical system and explore to what extent performance can be improved by coupling it with a filtration device. To confidently benchmark detector performance, we applied a statistical assessment method to target optimal detection of a simulated concentrated sample. Our aim was to arrive at a holistic understanding of device performance and to demonstrate system improvements based on these insights. The best achievable detection time for a simulated 1 CFU mL-1 sample was 4.3 (±0.6) h assuming no loss of performance in the filtration step. The real filtered samples fell short of this, extending detection time to 16-18 h. The loss in performance is likely to arise from stress imposed by the filtration step which inhibited microbial growth rates.

Enhancing hydrogen-dependent growth of and carbon dioxide fixation by Clostridium ljungdahlii through nitrate supplementation.

Synthesis gas (syngas) fermentation via the Wood-Ljungdahl pathway is receiving growing attention as a possible platform for the fixation of CO2 and renewable production of fuels and chemicals. However, the pathway operates near the thermodynamic limit of life, resulting in minimal adenosine triphosphate (ATP) production and long doubling times. This calls into question the feasibility of producing high-energy compounds at industrially relevant levels. In this study, we investigated the possibility of co-utilizing nitrate as an inexpensive additional electron acceptor to enhance ATP production during H2 -dependent growth of Clostridium ljungdahlii, Moorella thermoacetica, and Acetobacterium woodii. In contrast to other acetogens tested, growth rate and final biomass titer were improved for C. ljungdahlii growing on a mixture of H2 and CO2 when supplemented with nitrate. Transcriptomic analysis, 13CO2 labeling, and an electron balance were used to understand how electron flux was partitioned between CO2 and nitrate. We further show that, with nitrate supplementation, the ATP/adenosine diphosphate (ADP) ratio and acetyl-CoA pools were increased by fivefold and threefold, respectively, suggesting that this strategy could be useful for the production of ATP-intensive heterologous products from acetyl-CoA. Finally, we propose a pathway for enhanced ATP production from nitrate and use this as a basis to calculate theoretical yields for a variety of products. This study demonstrates a viable strategy for the decoupling of ATP production from carbon dioxide fixation, which will serve to significantly improve the CO2 fixation rate and the production metrics of other chemicals from CO2 and H2 in this host.

Transcriptional regulation of dimethyl sulfoxide respiration in a haloarchaeon, Haloferax volcanii.

The halophilic euryarchaeon Haloferax volcanii can grow anaerobically by DMSO respiration. DMSO reductase was induced by DMSO respiration not only under anaerobic growth conditions but also in denitrifying cells of H. volcanii. Deletion of the dmsR gene, encoding a putative regulator for the DMSO reductase, resulted in the loss of anaerobic growth by DMSO respiration. Reporter experiments revealed that only the anaerobic condition was essential for transcription of the dmsEABCD genes encoding DMSO reductase and that transcription was enhanced threefold by supplementation of DMSO. In the ∆dmsR mutant, transcription of the dmsEABCD genes induced by the anaerobic condition was not enhanced by DMSO, suggesting that DmsR is a DMSO-responsive regulator. Transcriptions of the dmsR and mgd genes for Mo-bisMGD biosynthesis were regulated in the same manner as the dmsEABCD genes. These results suggest that the genetic regulation of DMSO respiration in H. volcanii is controlled by at least two systems: one is the DMSO-responsive DmsR, and the other is an unknown anaerobic regulator.

Aggregatibacter actinomycetemcomitans QseBC is activated by catecholamines and iron and regulates genes encoding proteins associated with anaerobic respiration and metabolism.

Aggregatibacter actinomycetemcomitans QseBC regulates its own expression and is essential for biofilm growth and virulence. However, the signal that activates the QseC sensor has not been identified and the qseBC regulon has not been defined. In this study, we show that QseC is activated by catecholamine hormones and iron but not by either component alone. Activation of QseC requires an EYRDD motif in the periplasmic domain of the sensor and site-specific mutations in EYRDD or the deletion of the periplasmic domain inhibits catecholamine/iron-dependent induction of the ygiW-qseBC operon. Catecholamine/iron-dependent induction of transcription also requires interaction of the QseB response regulator with its binding site in the ygiW-qseBC promoter. Whole genome microarrays were used to compare gene expression profiles of A. actinomycetemcomitans grown in a chemically defined medium with and without catecholamine and iron supplementation. Approximately 11.5% of the A. actinomycetemcomitans genome was differentially expressed by at least two-fold upon exposure to catecholamines and iron. The expression of ferritin was strongly induced, suggesting that intracellular iron storage capacity is increased upon QseBC activation. Consistent with this, genes encoding iron binding and transport proteins were down-regulated by QseBC. Strikingly, 57% of the QseBC up-regulated genes (56/99) encode proteins associated with anaerobic metabolism and respiration. Most of these up-regulated genes were recently reported to be induced during in vivo growth of A. actinomycetemcomitans. These results suggest that detection of catecholamines and iron by QseBC may alter the cellular metabolism of A. actinomycetemcomitans for increased fitness and growth in an anaerobic host environment.