Atsushi Kouzuma

Electron shuttles in biotechnology.

Electron-shuttling compounds (electron shuttles [ESs], or redox mediators) are essential components in intracellular electron transfer, while microbes also utilize self-produced and naturally present ESs for extracellular electron transfer. These compounds assist in microbial energy metabolism by facilitating electron transfer between microbes, from electron-donating substances to microbes, and/or from microbes to electron-accepting substances. Artificially supplemented ESs can create new routes of electron flow in the microbial energy metabolism, thereby opening up new possibilities for the application of microbes to biotechnology processes. Typical examples of such processes include halogenated-organics bioremediation, azo-dye decolorization, and microbial fuel cells. Herein we suggest that ESs can be applied widely to create new microbial biotechnology processes.

Disruption of the Putative Cell Surface Polysaccharide Biosynthesis Gene SO3177 in Shewanella oneidensis MR-1 Enhances Adhesion to Electrodes and Current Generation in Microbial Fuel Cells

ABSTRACT A microbial fuel cell (MFC) was inoculated with a random transposon insertion mutant library of Shewanella oneidensis MR-1 and operated with lactate as the sole fuel to select for mutants that preferentially grew in it. Agar plate cultivation of the resultant MFC enrichment culture detected an increased number of colonies exhibiting rough morphology. One such isolate, strain 4A, generated 50% more current in an MFC than wild-type MR-1. Determination of the transposon insertion site in strain 4A followed by deletion and complementation experiments revealed that the SO3177 gene, encoding a putative formyltransferase and situated in a cell surface polysaccharide biosynthesis gene cluster, was responsible for the increased current. Transmission electron microscopy showed that a layered structure at the cell surface, stainable with ruthenium red, was impaired in the SO3177 mutant (ΔSO3177), confirming that SO3177 is involved in the biosynthesis of cell surface polysaccharides. Compared to the wild type, ΔSO3177 cells preferentially attached to graphite felt anodes in MFCs, while physicochemical analyses revealed that the cell surface of ΔSO3177 was more hydrophobic. These results demonstrate that cell surface polysaccharides affect not only the cell adhesion to graphite anodes but also the current generation in MFCs.

Microbial interspecies interactions: recent findings in syntrophic consortia

Microbes are ubiquitous in our biosphere, and inevitably live in communities. They excrete a variety of metabolites and support the growth of other microbes in a community. According to the law of chemical equilibrium, the consumption of excreted metabolites by recipient microbes can accelerate the metabolism of donor microbes. This is the concept of syntrophy, which is a type of mutualism and governs the metabolism and growth of diverse microbes in natural and engineered ecosystems. A representative example of syntrophy is found in methanogenic communities, where reducing equivalents, e.g., hydrogen and formate, transfer between syntrophic partners. Studies have revealed that microbes involved in syntrophy have evolved molecular mechanisms to establish specific partnerships and interspecies communication, resulting in efficient metabolic cooperation. In addition, recent studies have provided evidence suggesting that microbial interspecies transfer of reducing equivalents also occurs as electric current via biotic (e.g., pili) and abiotic (e.g., conductive mineral and carbon particles) electric conduits. In this review, we describe these findings as examples of sophisticated cooperative behavior between different microbial species. We suggest that these interactions have fundamental roles in shaping the structure and activity of microbial communities.

Exploring the potential of algae/bacteria interactions.

Algae are primary producers in aquatic ecosystems, where heterotrophic bacteria grow on organics produced by algae and recycle nutrients. Ecological studies have identified the co-occurrence of particular species of algae and bacteria, suggesting the presence of their specific interactions. Algae/bacteria interactions are categorized into nutrient exchange, signal transduction and gene transfer. Studies have examined how these interactions shape aquatic communities and influence geochemical cycles in the natural environment. In parallel, efforts have been made to exploit algae for biotechnology processes, such as water treatment and bioenergy production, where bacteria influence algal activities in various ways. We suggest that better understanding of mechanisms underlying algae/bacteria interactions will facilitate the development of more efficient and/or as-yet-unexploited biotechnology processes.

Comparative metagenomics of anode-associated microbiomes developed in rice paddy-field microbial fuel cells.

In sediment-type microbial fuel cells (sMFCs) operating in rice paddy fields, rice-root exudates are converted to electricity by anode-associated rhizosphere microbes. Previous studies have shown that members of the family Geobacteraceae are enriched on the anodes of rhizosphere sMFCs. To deepen our understanding of rhizosphere microbes involved in electricity generation in sMFCs, here, we conducted comparative analyses of anode-associated microbiomes in three MFC systems: a rice paddy-field sMFC, and acetate- and glucose-fed MFCs in which pieces of graphite felt that had functioned as anodes in rice paddy-field sMFC were used as rhizosphere microbe-bearing anodes. After electric outputs became stable, microbiomes associated with the anodes of these MFC systems were analyzed by pyrotag sequencing of 16S rRNA gene amplicons and Illumina shotgun metagenomics. Pyrotag sequencing showed that Geobacteraceae bacteria were associated with the anodes of all three systems, but the dominant Geobacter species in each MFC were different. Specifically, species closely related to G. metallireducens comprised 90% of the anode Geobacteraceae in the acetate-fed MFC, but were only relatively minor components of the rhizosphere sMFC and glucose-fed MFC, whereas species closely related to G. psychrophilus were abundantly detected. This trend was confirmed by the phylogenetic assignments of predicted genes in shotgun metagenome sequences of the anode microbiomes. Our findings suggest that G. psychrophilus and its related species preferentially grow on the anodes of rhizosphere sMFCs and generate electricity through syntrophic interactions with organisms that excrete electron donors.

Roles of siderophore in manganese-oxide reduction by Shewanella oneidensis MR-1

Dissimilatory metal-reducing bacteria (DMRB), such as Shewanella oneidensis MR-1, are of great interest for their importance in the biogeochemical cycling of metals and utility in biotechnological processes, such as bioremediation and microbial fuel cells. To identify genes necessary for metal reduction, this study constructed a random transposon-insertion mutant library of MR-1 and screened it for isolating mutants that were deficient in metal reduction. Examination of approximately 5000 mutants on lactate minimal-medium plates containing MnO(2) resulted in the isolation of one mutant, strain N22-7, that showed a decreased MnO(2)-reduction activity. Determination of a transposon-insertion site in N22-7 followed by deletion and complementation experiments revealed that the disruption of SO3030, a siderophore biosynthesis gene, was responsible for the decreased MnO(2)-reduction activity. In ΔSO3030 cells, iron and cytochrome contents were decreased to approximately 50% of those in the wild-type cells, when they were incubated under MnO(2)-reduction conditions. In addition, the transcription of genes encoding outer-membrane cytochromes necessary for metal reduction was repressed in ΔSO3030 under MnO(2)-reduction conditions, while their transcription was upregulated after supplementation of culture media with ferrous iron. These results suggest that siderophore is important for S. oneidensis MR-1 to respire MnO(2), because iron availability influences the expression of cytochromes necessary for metal reduction.

Catabolic and regulatory systems in Shewanella oneidensis MR-1 involved in electricity generation in microbial fuel cells

Shewanella oneidensis MR-1 is a facultative anaerobe that respires using a variety of inorganic and organic compounds. MR-1 is also capable of utilizing extracellular solid materials, including anodes in microbial fuel cells (MFCs), as electron acceptors, thereby enabling electricity generation. As MFCs have the potential to generate electricity from biomass waste and wastewater, MR-1 has been extensively studied to identify the molecular systems that are involved in electricity generation in MFCs. These studies have demonstrated the importance of extracellular electron-transfer (EET) pathways that electrically connect the quinone pool in the cytoplasmic membrane to extracellular electron acceptors. Electricity generation is also dependent on intracellular catabolic pathways that oxidize electron donors, such as lactate, and regulatory systems that control the expression of genes encoding the components of catabolic and electron-transfer pathways. In addition, recent findings suggest that cell-surface polymers, e.g., exopolysaccharides, and secreted chemicals, which function as electron shuttles, are also involved in electricity generation. Despite these advances in our knowledge on the EET processes in MR-1, further efforts are necessary to fully understand the underlying intra- and extracellular molecular systems for electricity generation in MFCs. We suggest that investigating how MR-1 coordinates these systems to efficiently transfer electrons to electrodes and conserve electrochemical energy for cell proliferation is important for establishing the biological basis for MFCs.

Electrochemical Gating of Tricarboxylic Acid Cycle in Electricity-Producing Bacterial Cells of Shewanella

Energy-conversion systems mediated by bacterial metabolism have recently attracted much attention, and therefore, demands for tuning of bacterial metabolism are increasing. It is widely recognized that intracellular redox atmosphere which is generally tuned by dissolved oxygen concentration or by appropriate selection of an electron acceptor for respiration is one of the important factors determining the bacterial metabolism. In general, electrochemical approaches are valuable for regulation of redox-active objects. However, the intracellular redox conditions are extremely difficult to control electrochemically because of the presence of insulative phospholipid bilayer membranes. In the present work, the limitation can be overcome by use of the bacterial genus Shewanella , which consists of species that are able to respire via cytochromes abundantly expressed in their outer-membrane with solid-state electron acceptors, including anodes. The electrochemical characterization and the gene expression analysis revealed that the activity of tricarboxylic acid (TCA) cycle in Shewanella cells can be reversibly gated simply by changing the anode potential. Importantly, our present results for Shewanella cells cultured in an electrochemical system under poised potential conditions showed the opposite relationship between the current and electron acceptor energy level, and indicate that this unique behavior originates from deactivation of the TCA cycle in the (over-)oxidative region. Our result obtained in this study is the first demonstration of the electrochemical gating of TCA cycle of living cells. And we believe that our findings will contribute to a deeper understanding of redox-dependent regulation systems in living cells, in which the intracellular redox atmosphere is a critical factor determining the regulation of various metabolic and genetic processes.

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

BackgroundShewanella 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.ResultsHere, we investigated the transcriptional mechanisms for the mtr genes and demonstrated that omcA and mtrC are transcribed from two upstream promoters, PomcA and PmtrC, 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 PomcA and PmtrC. However, the expression of omcA and mtrC was differentially regulated in response to culture conditions; specifically, the expression from PmtrC was higher under aerobic conditions than that under anaerobic conditions with fumarate as an electron acceptor, whereas expression from PomcA exhibited the opposite trend. Deletion of the region upstream of the CRP-binding site of PomcA resulted in a significant increase in promoter activity under aerobic conditions, demonstrating that the deleted region is involved in the negative regulation of PomcA.ConclusionsTaken 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.

Metagenomic Analyses Reveal the Involvement of Syntrophic Consortia in Methanol/Electricity Conversion in Microbial Fuel Cells

Methanol is widely used in industrial processes, and as such, is discharged in large quantities in wastewater. Microbial fuel cells (MFCs) have the potential to recover electric energy from organic pollutants in wastewater; however, the use of MFCs to generate electricity from methanol has not been reported. In the present study, we developed single-chamber MFCs that generated electricity from methanol at the maximum power density of 220 mW m−2 (based on the projected area of the anode). In order to reveal how microbes generate electricity from methanol, pyrosequencing of 16S rRNA-gene amplicons and Illumina shotgun sequencing of metagenome were conducted. The pyrosequencing detected in abundance Dysgonomonas, Sporomusa, and Desulfovibrio in the electrolyte and anode and cathode biofilms, while Geobacter was detected only in the anode biofilm. Based on known physiological properties of these bacteria, it is considered that Sporomusa converts methanol into acetate, which is then utilized by Geobacter to generate electricity. This speculation is supported by results of shotgun metagenomics of the anode-biofilm microbes, which reconstructed relevant catabolic pathways in these bacteria. These results suggest that methanol is anaerobically catabolized by syntrophic bacterial consortia with electrodes as electron acceptors.