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.

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.

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.

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.

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.

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.

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.

Development of a CRISPR interference system for selective gene knockdown in Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is an iron-oxidizing chemolithotroph used for bioleaching of precious metals and is also regarded as a potential host for bioelectrochemical production of value-added chemicals. Despite its industrial utility, however, it is difficult to genetically engineer A. ferrooxidans due to low transformation and recombination efficiencies. Here, we developed a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system that can selectively repress the expression of a target gene in A. ferrooxidans. The mutated gene encoding a nuclease-deactivated Cas9 protein was cloned into the broad-host-range plasmid pBBR1-MCS2, and the applicability of the CRISPRi system was examined using the nitrogenase nifH gene as a knockdown target. Introduction of the CRISPRi plasmid into A. ferrooxidans resulted in decreased nifH transcription and retarded cell growth in the absence of nitrogen sources, demonstrating that the CRISPRi system altered the phenotype of this bacterium via selective gene knockdown. We suggest that the CRISPRi system developed in this study provides an efficient technique for constructing A. ferrooxidans knockdown mutants that are useful for the genetic dissection of this bacterium.

Codh/Acs-Deficient Methanogens Are Prevalent in Anaerobic Digesters.

Methanogens are archaea that grow by producing methane as a catabolic end product and thrive in diverse anaerobic habitats, including soil, sediments, oil reservoirs, digestive tracts, and anaerobic digesters. Methanogens have typically been classified into three types-namely, hydrogenotrophic, acetoclastic, and methylotrophic methanogens. In addition, studies have found methanogens that require both hydrogen/CO2 and organics, such as acetate, for growth. Genomic analyses have shown that these methanogens lack genes for carbon monoxide dehydrogenase/acetyl-CoA synthase (Codh/Acs), one of the oldest enzymes that catalyzes the central step in the Wood-Ljungdahl pathway. Since these methanogens have been found dominant in such habitats as digestive tracts and anaerobic digesters, it is suggested that the loss of Codh/Acs confers ecological advantages on methanogens in these habitats. Comparisons in genomes of methanogens suggest the possibility that these methanogens have emerged recently in anaerobic digesters and are currently under the process of prevalence. We propose that an understanding of the genetic and ecological processes associated with the emergence and prevalence of these methanogens in anaerobic digesters would offer novel evolutionary insights into microbial ecology.

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.

Shewanella oneidensis MR-1 as a bacterial platform for electro-biotechnology.

The genus Shewanella comprises over 70 species of heterotrophic bacteria with versatile respiratory capacities. Some of these bacteria are known to be pathogens of fishes and animals, while many are non-pathogens considered to play important roles in the global carbon cycle. A representative strain is Shewanella oneidensis MR-1 that has been intensively studied for its ability to respire diverse electron acceptors, such as oxygen, nitrate, sulfur compounds, metals, and organics. In addition, studies have been focused on its ability as an electrochemically active bacterium that is capable of discharging electrons to and receiving electrons from electrodes in bioelectrochemical systems (BESs) for balancing intracellular redox states. This ability is expected to be applied to electro-fermentation (EF) for producing value-added chemicals that conventional fermentation technologies are difficult to produce efficiently. Researchers are also attempting to utilize its electrochemical ability for controlling gene expression, for which electro-genetics (EG) has been coined. Here we review fundamental knowledge on this bacterium and discuss future directions of studies on its applications to electro-biotechnology (EB).

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.

Hydrogen-dependent current generation and energy conservation by Shewanella oneidensis MR-1 in bioelectrochemical systems.

Bioelectrochemical systems (BESs) are engineered systems that utilize electrochemical interactions between electrochemically active bacteria (EAB) and electrodes. BESs have attracted considerable attention for their utility in biotechnological processes. In a BES, hydrogen is generated by the reduction of water on low-potential cathode electrodes. However, limited information is available on the effect of hydrogen on the metabolism and growth of EAB and current generation in BESs. Here, we investigated the effect of hydrogen on current generation by a model EAB, Shewanella oneidensis MR-1. We found that this strain utilizes hydrogen as an electron donor for electrode respiration, thereby facilitating current generation and cell growth in the presence of organic substrates. Inner membrane (IM) quinones (i.e., ubiquinone and menaquinone), IM quinone-reactive hydrogenase Hya, and IM-bound quinone reductase CymA are involved in hydrogen-dependent current generation, suggesting that the redox cycling of IM quinones catalyzed by Hya and CymA contributes to the generation of the proton motive force and the synthesis of ATP via F0F1-ATPase. These findings indicate that the evolution of hydrogen on the cathode facilitates energy metabolism and growth of hydrogen-utilizing EAB associated with anodes. The results also suggest that hydrogen cycling between cathodes and anodes can hinder quantitative evaluation of organic substrate-dependent current generation in BESs.

Towards Application of Electro-Fermentation for the Production of Value-Added Chemicals From Biomass Feedstocks.

According to recent social demands for sustainable developments, the value of biomass as feedstocks for chemical industry is increasing. With the aid of metabolic engineering and genome editing, microbial fermentation has been developed for producing value-added chemicals from biomass feedstocks, while further improvements are desired for producing more diverse chemicals and increasing the production efficiency. The major intrinsic limitation in conventional fermentation technologies is associated with the need for balancing the net redox equivalents between substrates and products, resulting in limited repertories of fermentation products. One solution for this limitation would be "electro-fermentation (EF)" that utilizes bioelectrochemical systems for modifying the intracellular redox state of electrochemically active bacteria, thereby overcoming the redox constraint of fermentation. Recent studies have attempted the production of chemicals based on the concept of EF, while its utility has not been sufficiently demonstrated in terms of low production efficiencies. Here we discuss EF in terms of its concept, current status and future directions, which help us develop its practical applications to sustainable chemical industries.

Enhanced electricity generation in rice paddy-field microbial fuel cells supplemented with iron powders.

Microbial fuel cells installed in rice paddy fields (RP-MFCs) are able to serve as on-site batteries for operating low-power environmental sensors. In order to increase the utility and reliability of RP-MFCs, however, further research is necessary for boosting the power output. Here we examined several powdered iron species, including zero valent iron (ZVI), goethite, and magnetite, for their application to increasing power outputs from RP-MFCs. Soil around anodes was supplemented with either of these iron species, and RP-MFCs were operated for several months during the transplanting and harvesting. It was found that power outputs from RP-MFCs supplemented with ZVI were more than double the outputs from control (not supplemented with iron species) and other RP-MFCs, even after iron corrosion was ceased, and the maximum power density reached 130 mW/m2 (per projected area of the anode). Metabarcoding of 16S rRNA gene amplicons suggested that several taxa represented by fermentative and exoelectrogenic bacteria were substantially increased in MFCs supplemented with ZVI. Results suggest that ZVI lowers oxidation/reduction potential around anodes, activates anaerobic microbes involved in the conversion of organic matter into electricity and increases power output from RP-MFCs.

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.

Shewanella algae Relatives Capable of Generating Electricity from Acetate Contribute to Coastal-Sediment Microbial Fuel Cells Treating Complex Organic Matter.

To identify exoelectrogens involved in the generation of electricity from complex organic matter in coastal sediment (CS) microbial fuel cells (MFCs), MFCs were inoculated with CS obtained from tidal flats and estuaries in the Tokyo bay and supplemented with starch, peptone, and fish extract as substrates. Power output was dependent on the CS used as inocula and ranged between 100 and 600 mW m-2 (based on the projected area of the anode). Analyses of anode microbiomes using 16S rRNA gene amplicons revealed that the read abundance of some bacteria, including those related to Shewanella algae, positively correlated with power outputs from MFCs. Some fermentative bacteria were also detected as major populations in anode microbiomes. A bacterial strain related to S. algae was isolated from MFC using an electrode plate-culture device, and pure-culture experiments demonstrated that this strain exhibited the ability to generate electricity from organic acids, including acetate. These results suggest that acetate-oxidizing S. algae relatives generate electricity from fermentation products in CS-MFCs that decompose complex organic matter.

Isolation of an Obligate Mixotrophic Methanogen That Represents the Major Population in Thermophilic Fixed-Bed Anaerobic Digesters.

Methanothermobacter Met2 is a metagenome-assembled genome (MAG) that encodes a putative mixotrophic methanogen constituting the major populations in thermophilic fixed-bed anaerobic digesters. In order to characterize its physiology, the present work isolated an archaeon (strain Met2-1) that represents Met2-type methanogens by using a combination of enrichments under a nitrogen atmosphere, colony formation on solid media and limiting dilution under high partial pressures of hydrogen. Strain Met2-1 utilizes hydrogen and carbon dioxide for methanogenesis, while the growth is observed only when culture media are additionally supplemented with acetate. It does not grow on acetate in the absence of hydrogen. The results demonstrate that Methanothermobacter sp. strain Met2-1 is a novel methanogen that exhibits obligate mixotrophy.

CRISPR/Cas9-mediated genome editing of Shewanella oneidensis MR-1 using a broad host-range pBBR1-based plasmid.

Here, we developed an all-in-one, broad host-range CRISPR/Cas9 vector system widely applicable to genome editing of proteobacteria. Plasmid pBBR1-Cas9 was constructed by cloning the cas9 gene from Streptococcus pyogenes into the broad host-range plasmid pBBR1MCS-2. We evaluated its applicability for frameshift mutagenesis of Shewanella oneidensis MR-1. Significant cell death was observed when MR-1 cells were transformed with a pBBR1-Cas9 derivative that expressed a single-guide RNA targeting the crp gene. However, cell death was partially prevented when a donor DNA fragment containing a modified crp sequence with a frameshift mutation was introduced using the same vector. All transformants (9 colonies) contained the expected frameshift mutation in their chromosomal crp genes. These results indicate that this vector system efficiently introduced CRISPR/Cas9-mediated double-strand DNA breaks and subsequent homology-directed repair. This work provides a simple and powerful genome-editing tool for proteobacteria that can harbor pBBR1-based plasmids.

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.