Supplementation with Amino Acid Sources Facilitates Fermentative Growth of Shewanella oneidensis MR-1 in Defined Media.

Shewanella oneidensis MR-1 is a facultative anaerobe that grows by respiration using a variety of electron acceptors. This organism serves as a model to study how bacteria thrive in redox-stratified environments. A glucose-utilizing engineered derivative of MR-1 has been reported to be unable to grow in glucose minimal medium (GMM) in the absence of electron acceptors, despite this strain having a complete set of genes for reconstructing glucose to lactate fermentative pathways. To gain insights into why MR-1 is incapable of fermentative growth, this study examined a hypothesis that this strain is programmed to repress the expression of some carbon metabolic genes in the absence of electron acceptors. Comparative transcriptomic analyses of the MR-1 derivative were conducted in the presence and absence of fumarate as an electron acceptor, and these found that the expression of many genes involved in carbon metabolism required for cell growth, including several tricarboxylic acid (TCA) cycle genes, was significantly downregulated in the absence of fumarate. This finding suggests a possibility that MR-1 is unable to grow fermentatively on glucose in minimal media owing to the shortage of nutrients essential for cell growth, such as amino acids. This idea was demonstrated in subsequent experiments that showed that the MR-1 derivative fermentatively grows in GMM containing tryptone or a defined mixture of amino acids. We suggest that gene regulatory circuits in MR-1 are tuned to minimize energy consumption under electron acceptor-depleted conditions, and that this results in defective fermentative growth in minimal media. IMPORTANCE It is an enigma why S. oneidensis MR-1 is incapable of fermentative growth despite having complete sets of genes for reconstructing fermentative pathways. Understanding the molecular mechanisms behind this defect will facilitate the development of novel fermentation technologies for the production of value-added chemicals from biomass feedstocks, such as electro-fermentation. The information provided in this study will also improve our understanding of the ecological strategies of bacteria living in redox-stratified environments.

Improved electrochemical properties of graphite electrodes incubated with iron powders in rice-paddy fields boost power outputs from microbial fuel cells.

Studies have shown that the supplementation of anode-surrounding soil with zero-valent iron (ZVI) boosts power outputs from rice paddy-field microbial fuel cells (RP-MFCs). In order to understand mechanisms by which ZVI boosts outputs from RP-MFCs, the present study operated RP-MFCs with and without ZVI, and compositions of anode-associated bacteria and electrochemical properties of graphite anodes were analyzed after 3-month operation. Metabarcoding using 16S rRNA gene fragments showed that bacterial compositions did not largely differ among these RP-MFCs. Cyclic voltammetry showed improved electrochemical properties of anodes recovered from ZVI-supplemented RP-MFCs, and this was attributed to the adhesion of iron-oxide films onto graphite surfaces. Bioelectrochemical devices equipped with graphite anodes recovered from ZVI-supplemented RP-MFCs generated higher currents than those with fresh graphite anodes. These results suggest that ZVI is oxidized to iron oxides in paddy-field soil and adheres onto graphite anodes, resulting in the boost of power outputs from RP-MFCs.

Identification of a Diguanylate Cyclase That Facilitates Biofilm Formation on Electrodes by Shewanella oneidensis MR-1.

In many bacteria, cyclic diguanosine monophosphate (c-di-GMP), synthesized by diguanylate cyclase (DGC), serves as a second messenger involved in the regulation of biofilm formation. Although studies have suggested that c-di-GMP also regulates the formation of electrochemically active biofilms (EABFs) by Shewanella oneidensis MR-1, DGCs involved in this process remained to be identified. Here, we report that the SO_1646 gene, hereafter named dgcS, is upregulated under medium flow conditions in electrochemical flow cells (EFCs), and its product (DgcS) functions as a major DGC in MR-1. In vitro assays demonstrated that purified DgcS catalyzed the synthesis of c-di-GMP from GTP. Comparisons of intracellular c-di-GMP levels in the wild-type strain and a dgcS deletion mutant (ΔdgcS mutant) showed that production of c-di-GMP was markedly reduced in the ΔdgcS mutant when cells were grown in batch cultures and on electrodes in EFCs. Cultivation of the ΔdgcS mutant in EFCs also revealed that the loss of DgcS resulted in impaired biofilm formation and decreased current generation. These findings demonstrate that MR-1 uses DgcS to synthesize c-di-GMP under medium flow conditions, thereby activating biofilm formation on electrodes.IMPORTANCE Bioelectrochemical systems (BESs) have attracted wide attention owing to their utility in sustainable biotechnology processes, such as microbial fuel cells and electrofermentation systems. In BESs, electrochemically active bacteria (EAB) form biofilms on electrode surfaces, thereby serving as effective catalysts for the interconversion between chemical and electric energy. It is therefore important to understand mechanisms for the formation of biofilm by EAB grown on electrodes. Here, we show that a model EAB, S. oneidensis MR-1, expresses DgcS as a major DGC, thereby activating the formation of biofilms on electrodes via c-di-GMP-dependent signal transduction cascades. The findings presented herein provide the molecular basis for improving electrochemical interactions between EAB and electrodes in BESs. The results also offer molecular insights into how Shewanella regulates biofilm formation on solid surfaces in the natural environment.

Molecular mechanisms regulating the catabolic and electrochemical activities of Shewanella oneidensis MR-1.

Electrochemically active bacteria (EAB) interact electrochemically with electrodes via extracellular electron transfer (EET) pathways. These bacteria have attracted significant attention due to their utility in environmental-friendly bioelectrochemical systems (BESs), including microbial fuel cells and electrofermentation systems. The electrochemical activity of EAB is dependent on their carbon catabolism and respiration; thus, understanding how these processes are regulated will provide insights into the development of a more efficient BES. The process of biofilm formation by EAB on BES electrodes is also important for electric current generation because it facilitates physical and electrochemical interactions between EAB cells and electrodes. This article summarizes the current knowledge on EET-related metabolic and cellular functions of a model EAB, Shewanella oneidensis MR-1, focusing specifically on regulatory systems for carbon catabolism, EET pathways, and biofilm formation. Based on recent developments, the author also discusses potential uses of engineered S. oneidensis strains for various biotechnological applications.

Identification of an extracytoplasmic function sigma factor that facilitates c-type cytochrome maturation and current generation under electrolyte-flow conditions in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 was cultured on electrodes in electrochemical flow cells (EFCs), and transcriptome profiles of electrode-attached cells grown under electrolyte-flow conditions were compared with those under static (nonflow) conditions. Results revealed that, along with genes related to c-type cytochrome maturation (e.g., dsbD), the SO_3096 gene encoding a putative extracytoplasmic function (ECF) sigma factor was significantly upregulated under electrolyte-flow conditions. Compared to wild-type MR-1 (WT), an SO_3096-deletion mutant (∆SO_3096) showed impaired biofilm formation and decreased current generation in EFCs, suggesting that SO_3096 plays critical roles in the interaction of MR-1 cells with electrodes under electrolyte-flow conditions. We also compared transcriptome profiles of WT and ∆SO_3096 grown in EFCs, confirming that many genes upregulated under the electrolyte-flow conditions, including dsbD, are regulated by SO_3096. LacZ reporter assays showed that transcription from a promoter upstream of dsbD is activated by SO_3096. Measurement of current generated by a dsbD-deletion mutant revealed that this gene is essential for the transfer of electrons to electrodes. These results indicate that the SO_3096 gene product facilitates c-type cytochrome maturation and current generation under electrolyte-flow conditions. The results also offer ecophysiological insights into how Shewanella regulates extracellular electron transfer to solid surfaces in the natural environment.

Metatranscriptomic Evidence for Magnetite Nanoparticle-Stimulated Acetoclastic Methanogenesis under Continuous Agitation.

Conductive nanomaterials have been reported to accelerate methanogenesis by promoting direct interspecies electron transfer (DIET), while their effects seem to vary depending on operational conditions. The present study examined the effects of magnetite nanoparticles (MNPs) on methanogenesis from acetate by soil-derived anaerobic cultures under continuous agitation. We found that MNPs accelerated methanogenesis in agitated cultures, as has been observed previously for static cultures. Metabarcoding of 16S rRNA gene amplicons showed that Methanosarcina substantially increased in the presence of MNPs, while DIET-related Geobacter did not occur. Metagenomic and metatranscriptomic analyses confirmed the predominance of Methanosarcina in MNP-supplemented agitated cultures. In addition, genes coding for acetoclastic methanogenesis, but not those for hydrogenotrophic methanogenesis, were abundantly expressed in the dominant Methanosarcina in the presence of MNPs. These results suggest that MNPs stimulate acetoclastic methanogenesis under continuous agitation.IMPORTANCE Previous studies have shown that conductive nanoparticles, such as MNPs, accelerate methanogenesis and suggested that MNPs facilitate DIET between exoelectrogenic bacteria and methanogenic archaea. In these methanogens, electrons thus obtained are considered to be used for hydrogenotrophic methanogenesis. However, the present work provides evidence that shows that MNPs accelerate DIET-independent acetoclastic methanogenesis under continuous agitation. Since most of previous studies have examined effects of MNPs in static or weakly agitated methanogenic cultures, results obtained in the present work suggest that hydraulic conditions definitively determine how MNPs accelerate methanogenesis. In addition, the knowledge obtained in this study is useful for engineers operating stirred-tank anaerobic digesters, since we show that MNPs accelerate methanogenesis under continuous agitation.

Roles of d-Lactate Dehydrogenases in the Anaerobic Growth of Shewanella oneidensis MR-1 on Sugars.

Shewanella oneidensis MR-1 is a facultative anaerobe that respires using a variety of electron acceptors. Although this organism is incapable of fermentative growth in the absence of electron acceptors, its genome encodes LdhA (a putative fermentative NADH-dependent d-lactate dehydrogenase [d-LDH]) and Dld (a respiratory quinone-dependent d-LDH). However, the physiological roles of LdhA in MR-1 are unclear. Here, we examined the activity, transcriptional regulation, and traits of deletion mutants to gain insight into the roles of LdhA in the anaerobic growth of MR-1. Analyses of d-LDH activity in MR-1 and the ldhA deletion mutant confirmed that LdhA functions as an NADH-dependent d-LDH that catalyzes the reduction of pyruvate to d-lactate. In vivo and in vitro assays revealed that ldhA expression was positively regulated by the cyclic-AMP receptor protein, a global transcription factor that regulates anaerobic respiratory pathways in MR-1, suggesting that LdhA functions in coordination with anaerobic respiration. Notably, we found that a deletion mutant of all four NADH dehydrogenases (NDHs) in MR-1 (ΔNDH mutant) retained the ability to grow on N-acetylglucosamine under fumarate-respiring conditions, while an additional deletion of ldhA or dld deprived the ΔNDH mutant of this growth ability. These results indicate that LdhA-Dld serves as a bypass of NDH in electron transfer from NADH to quinones. Our findings suggest that the LdhA-Dld system manages intracellular redox balance by utilizing d-lactate as a temporal electron sink under electron acceptor-limited conditions.IMPORTANCE NADH-dependent LDHs are conserved among diverse organisms and contribute to NAD+ regeneration in lactic acid fermentation. However, this type of LDH is also present in nonfermentative bacteria, including members of the genus Shewanella, while their physiological roles in these bacteria remain unknown. Here, we show that LdhA (an NADH-dependent d-LDH) works in concert with Dld (a quinone-dependent d-LDH) to transfer electrons from NADH to quinones during sugar catabolism in S. oneidensis MR-1. Our results indicate that d-lactate acts as an intracellular electron mediator to transfer electrons from NADH to membrane quinones. In addition, d-lactate serves as a temporal electron sink when respiratory electron acceptors are not available. Our study suggests novel physiological roles for d-LDHs in providing nonfermentative bacteria with catabolic flexibility under electron acceptor-limited conditions.

Conductive carbon nanoparticles inhibit methanogens and stabilize hydrogen production in microbial electrolysis cells.

Nanosized conductive carbon materials have been reported to stimulate methanogenesis by anaerobic microbiomes, while other studies have shown their antimicrobial activities. The present study examined effects of conductive carbon nanoparticles (carbon black Vulcan, CB) on methanogenesis from glucose by anaerobic sludge. We found that a relatively high concentration (e.g., 2% w/v) of CB entirely inhibited the methanogenesis, where a substantial amount of acetate was accumulated after degradation of glucose. Quantitative real-time PCR assays and metabarcoding of 16S rRNA amplicons revealed that, while bacteria were stably present irrespective of the presence and absence of CB, archaea, in particular methanogens, were largely decreased in the presence of CB. Pure-culture experiments showed that methanogenic archaea were more seriously damaged by CB than fermentative bacteria. These results demonstrate that CB specifically inhibits methanogens in anaerobic sludge. We attempted to supplement cathode chambers of microbial electrolysis cells with CB for inhibiting methanogenesis from hydrogen, demonstrating that hydrogen is stably produced in the presence of CB.

Methylomusa anaerophila gen. nov., sp. nov., an anaerobic methanol-utilizing bacterium isolated from a microbial fuel cell.

Abacterial strain, designated MMFC1T, was isolated from a methanol-fed microbial fuel cell that had been inoculated with sludge obtained from a wastewater-treatmentfacility in a chemical plant. The strain grows by fermenting methanol to produce acetate under anaerobic conditions, while homoacetogenic growth is not observed. MMFC1T also grows on pyruvate and lactate but not on sugars and other organic acids. Cells are curved rods and motile, have peritrichous flagella, and form endospores. The genome sequence of strain MMFC1T supports the physiological data. Phylogenetic analysis based on the 16S rRNA gene sequence shows that strain MMFC1T is affiliated with the family Sporomusaceae, while the closest relative is Sporomusa ovata with nucleotide-sequencesimilarity of 93.5 %. Major fatty acids are iso-C13 : 0 3-OH, C16 : 1ω9 and iso-C17 : 0. On the basis of its physiological, genomic and phylogenetic features, a novel genus and species are proposed to accommodate strain MMFC1T, with the name Methylomusa anaerophila gen. nov., sp. nov. The type strain of Methylomusa anaerophila is MMFC1T (=JCM 31821T = KCTC 15592T).

CpdA is involved in amino acid metabolism in Shewanella oneidensis MR-1.

Cyclic 3',5'-adenosine monophosphate (cAMP) phosphodiesterase (CPD) is an enzyme that catalyzes the hydrolysis of cAMP, a signaling molecule affecting diverse cellular and metabolic processes in bacteria. Some CPDs are also known to function in cAMP-independent manners, while their physiological roles remain largely unknown. Here, we investigated physiological roles of CPD in Shewanella oneidensis MR-1, a model environmental bacterium, and report that CPD is involved in amino-acid metabolism. We found that a CPD-deficient mutant of MR-1 (ΔcpdA) showed decreased expression of genes for the synthesis of methionine, S-adenosylmethionine, and histidine and required these three compounds to grow in minimal media. Interestingly, deletion of adenylate cyclases in ΔcpdA did not restore the ability to grow in minimal media, indicating that the amino acid requirements were not due to the accumulation of cAMP. These results suggest that CPD is involved in the regulation of amino acid metabolism in MR-1 in a cAMP-independent manner.

Structures, Compositions, and Activities of Live Shewanella Biofilms Formed on Graphite Electrodes in Electrochemical Flow Cells.

An electrochemical flow cell equipped with a graphite working electrode (WE) at the bottom was inoculated with Shewanella oneidensis MR-1 expressing an anaerobic fluorescent protein, and biofilm formation on the WE was observed over time during current generation at WE potentials of +0.4 and 0 V (versus standard hydrogen electrodes), under electrolyte-flow conditions. Electrochemical analyses suggested the presence of unique electron-transfer mechanisms in the +0.4-V biofilm. Microscopic analyses revealed that, in contrast to aerobic biofilms, current-generating biofilm (at +0.4 V) was thin and flat (∼10 µm in thickness), and cells were evenly and densely distributed in the biofilm. In contrast, cells were unevenly distributed in biofilm formed at 0 V. In situ fluorescence staining and biofilm recovery experiments showed that the amounts of extracellular polysaccharides (EPSs) in the +0.4-V biofilm were much smaller than those in the aerobic and 0-V biofilms, suggesting that Shewanella cells suppress the production of EPSs at +0.4 V under flow conditions. We suggest that Shewanella cells perceive electrode potentials and modulate the structure and composition of biofilms to efficiently transfer electrons to electrodes.IMPORTANCE A promising application of microbial fuel cells (MFCs) is to save energy in wastewater treatment. Since current is generated in these MFCs by biofilm microbes under horizontal flows of wastewater, it is important to understand the mechanisms for biofilm formation and current generation under water-flow conditions. Although massive work has been done to analyze the molecular mechanisms for current generation by model exoelectrogenic bacteria, such as Shewanella oneidensis, limited information is available regarding the formation of current-generating biofilms over time under water-flow conditions. The present study developed electrochemical flow cells and used them to examine the electrochemical and structural features of current-generating biofilms under water-flow conditions. We show unique features of mature biofilms actively generating current, creating opportunities to search for as-yet-undiscovered current-generating mechanisms in Shewanella biofilms. Furthermore, information provided in the present study is useful for researchers attempting to develop anode architectures suitable for wastewater treatment MFCs.

Transcriptional mechanisms for differential expression of outer membrane cytochrome genes omcA and mtrC in Shewanella oneidensis MR-1.

BACKGROUND: Shewanella oneidensis MR-1 is capable of reducing extracellular electron acceptors, such as metals and electrodes, through the Mtr respiratory pathway, which consists of the outer membrane cytochromes OmcA and MtrC and associated proteins MtrA and MtrB. These proteins are encoded in the mtr gene cluster (omcA-mtrCAB) in the MR-1 chromosome. RESULTS: Here, we investigated the transcriptional mechanisms for the mtr genes and demonstrated that omcA and mtrC are transcribed from two upstream promoters, P omcA and P mtrC, respectively. In vivo transcription and in vitro electrophoretic mobility shift assays revealed that a cAMP receptor protein (CRP) positively regulates the expression of the mtr genes by binding to the upstream regions of P omcA and P mtrC. However, the expression of omcA and mtrC was differentially regulated in response to culture conditions; specifically, the expression from P mtrC was higher under aerobic conditions than that under anaerobic conditions with fumarate as an electron acceptor, whereas expression from P omcA exhibited the opposite trend. Deletion of the region upstream of the CRP-binding site of P omcA resulted in a significant increase in promoter activity under aerobic conditions, demonstrating that the deleted region is involved in the negative regulation of P omcA. CONCLUSIONS: Taken together, the present results indicate that transcription of the mtr genes is regulated by multiple promoters and regulatory systems, including the CRP/cAMP-dependent regulatory system and yet-unidentified negative regulators.

Electrochemical selection and characterization of a high current-generating Shewanella oneidensis mutant with altered cell-surface morphology and biofilm-related gene expression.

BACKGROUND: Shewanella oneidensis MR-1 exhibits extracellular electron transfer (EET) activity that is influenced by various cellular components, including outer-membrane cytochromes, cell-surface polysaccharides (CPS), and regulatory proteins. Here, a random transposon-insertion mutant library of S. oneidensis MR-1 was screened after extended cultivation in electrochemical cells (ECs) with a working electrode poised at +0.2 V (vs. Ag/AgCl) to isolate mutants that adapted to electrode-respiring conditions and identify as-yet-unknown EET-related factors. RESULTS: Several mutants isolated from the enrichment culture exhibited rough morphology and extraordinarily large colonies on agar plates compared to wild-type MR-1. One of the isolated mutants, designated strain EC-2, produced 90% higher electric current than wild-type MR-1 in ECs and was found to have a transposon inserted in the SO_1860 (uvrY) gene, which encodes a DNA-binding response regulator of the BarA/UvrY two-component regulatory system. However, an in-frame deletion mutant of SO_1860 (∆SO_1860) did not exhibit a similar level of current generation as that of EC-2, suggesting that the enhanced current-generating capability of EC-2 was not simply due to the disruption of SO_1860. In both EC-2 and ∆SO_1860, the transcription of genes related to CPS synthesis was decreased compared to wild-type MR-1, suggesting that CPS negatively affects current generation. In addition, transcriptome analyses revealed that a number of genes, including those involved in biofilm formation, were differentially expressed in EC-2 compared to those in ∆SO_1860. CONCLUSIONS: The present results indicate that the altered expression of the genes related to CPS biosynthesis and biofilm formation is associated with the distinct morphotype and high current-generating capability of strain EC-2, suggesting an important role of these genes in determining the EET activity of S. oneidensis.

Microbial electricity generation in rice paddy fields: recent advances and perspectives in rhizosphere microbial fuel cells.

Microbial fuel cells (MFCs) are devices that use living microbes for the conversion of organic matter into electricity. MFC systems can be applied to the generation of electricity at water/sediment interfaces in the environment, such as bay areas, wetlands, and rice paddy fields. Using these systems, electricity generation in paddy fields as high as ∼80 mW m(-2) (based on the projected anode area) has been demonstrated, and evidence suggests that rhizosphere microbes preferentially utilize organic exudates from rice roots for generating electricity. Phylogenetic and metagenomic analyses have been conducted to identify the microbial species and catabolic pathways that are involved in the conversion of root exudates into electricity, suggesting the importance of syntrophic interactions. In parallel, pot cultures of rice and other aquatic plants have been used for rhizosphere MFC experiments under controlled laboratory conditions. The findings from these studies have demonstrated the potential of electricity generation for mitigating methane emission from the rhizosphere. Notably, however, the presence of large amounts of organics in the rhizosphere drastically reduces the effect of electricity generation on methane production. Further studies are necessary to evaluate the potential of these systems for mitigating methane emission from rice paddy fields. We suggest that paddy-field MFCs represent a promising approach for harvesting latent energy of the natural world.

Complete genome sequence of Geobacter sp. strain 60473, an electrochemically active bacterium isolated from lakeshore mud.

Geobacter sp. strain 60473 is an electrochemically active bacterium (EAB) isolated from mud taken from the shore of lake Suwa in Japan. Here, we report the complete genome sequence of strain 60473, which helps deepen our understanding of common and strain-specific genomic features of EAB affiliated with the genus Geobacter.

A non-methanogenic archaeon within the order Methanocellales.

Serpentinization, a geochemical process found on modern and ancient Earth, provides an ultra-reducing environment that can support microbial methanogenesis and acetogenesis. Several groups of archaea, such as the order Methanocellales, are characterized by their ability to produce methane. Here, we generate metagenomic sequences from serpentinized springs in The Cedars, California, and construct a circularized metagenome-assembled genome of a Methanocellales archaeon, termed Met12, that lacks essential methanogenesis genes. The genome includes genes for an acetyl-CoA pathway, but lacks genes encoding methanogenesis enzymes such as methyl-coenzyme M reductase, heterodisulfide reductases and hydrogenases. In situ transcriptomic analyses reveal high expression of a multi-heme c-type cytochrome, and heterologous expression of this protein in a model bacterium demonstrates that it is capable of accepting electrons. Our results suggest that Met12, within the order Methanocellales, is not a methanogen but a CO2-reducing, electron-fueled acetogen without electron bifurcation.

Use of Microbial Fuel Cells for the Treatment of Residue Effluents Discharged from an Anaerobic Digester Treating Food Wastes.

One of practical challenges in anaerobic-digestion (AD) technology is the cost-effective treatment of residue effluents containing high concentrations of organics, nitrogen and phosphorus (CNP). In order to evaluate the utility of microbial fuel cells (MFCs) for treating anaerobic-digester effluents (ADEs) and generating power from them, laboratory-scale single-chamber MFCs were filled with ADE obtained from a commercial AD plant treating food wastes and thereafter operated by routinely supplying ADE at different hydraulic residence times (HRTs, 5 to 20 days). It is shown that MFCs were able to reduce not only organics in ADE but also nitrogen and phosphorus. For instance, data demonstrated that over 50% of CNP was removed in MFCs operated at an HRT of 10 days, at which the maximum power density reached over 200 mW m-2 (based on the projected area of anode). Metabarcoding of 16S rRNA genes showed that some bacteria were specifically enriched in anode biofilms, suggesting their involvement in power generation. Our study suggests that MFCs are applicable to reducing CNP in ADEs at reasonable rates, and provides subsequent work with fundamental data useful for setting targets for further developments.

Electrogenetic control of gene expression in Shewanella oneidensis MR-1 using Arc-dependent transcriptional promoters.

Electrochemically active bacteria (EAB) are capable of electrically interacting with electrodes, enabling their application in bioelectrochemical systems (BESs). As the performance of BES is related to the metabolic activities of EAB, the development of methods to control their metabolic activities is important to facilitate BES applications. A recent study found that the EAB Shewanella oneidensis MR-1 uses the Arc system to regulate the expression of catabolic genes in response to electrode potentials, suggesting that a methodology for electrical control of gene expression in EAB, referred to as electrogenetics, can be developed by using electrode potential-responsive, Arc-dependent transcriptional promoters. Here, we explored Arc-dependent promoters in the genomes of S. oneidensis MR-1 and Escherichia coli to identify electrode potential-responsive promoters that are differentially activated in MR-1 cells exposed to high- and low-potential electrodes. LacZ reporter assays using electrode-associated cells of MR-1 derivatives revealed that the activities of promoters located upstream of the E. coli feo gene (Pfeo) and the MR-1 nqrA2 (SO_0902) gene (Pnqr2) were significantly increased when S. oneidensis cells were exposed to electrodes poised at +0.7 V and -0.4 V (versus the standard hydrogen electrode), respectively. Additionally, we developed a microscopic system for in situ monitoring of promoter activity in electrode-associated cells and found that Pnqr2 activity was persistently induced in MR-1 cells associated with an electrode poised at -0.4 V. Our results indicate that these electrode potential-responsive promoters enable efficient regulation of gene expression in EAB, providing a molecular basis for the development of electrogenetics.

Bioaugmentation of microbial electrolysis cells with Geobacter sulfurreducens YM18 for enhanced hydrogen production from starch.

Double-chamber microbial electrolysis cells (MECs) were operated using starch-based medium as the anolyte and rice paddy-field soil as the anode inoculum, and hydrogen production from the cathode chamber was examined. In order to enhance current generation and hydrogen production, the anode chamber was bioaugmented with Geobacter sulfurreducens strain YM18, and its effects were evaluated based on the performances of non-bioaugmented controls. Results show that the bioaugmented MEC generated threefold greater current during one-month operation and produced sixfold greater amounts of hydrogen than those of the non-bioaugmented control. Quantitative PCR and metabarcoding analyses confirmed successful colonization of anode surfaces with YM18, suggesting the utility of bioaugmentation with YM18 for enhancing the performance of bioelectrochemical systems, including MECs treating biomass wastes.

Influences of aerobic respiration on current generation by Shewanella oneidensis MR-1 in single-chamber microbial fuel cells.

In single-chamber microbial fuel cells (SC-MFCs), oxygen molecules diffuse through air cathodes into electrolytes and compete against anodes in accepting electrons. In this study, we constructed multiple gene-knockout mutants for terminal oxidases (SO4607, SO2364, and SO3286) in Shewanella oneidensis MR-1 and examined their abilities to generate electric currents in SC-MFCs. Although single-knockout mutants generated levels of current similar to that of the wild type (WT), an SO4607/SO2364 double-knockout mutant (DO) generated 50% higher current than WT. A triple-knockout mutant did not grow in SC-MFC. The Coulombic efficiencies in SC-MFC were, however, not substantially different between WT and DO. In aerobically grown DO cells, the transcription levels of the genes involved in extracellular electron transfer (mtrC and crp) were increased compared to those in WT cells. These results suggest that suppression of aerobic respiration activates the expression of genes related to the extracellular electron transfer and increases the electric output from SC-MFCs.