Souichiro Kato

In general, metabolism in living cells is closely related to the intracellular redox state. Focusing on the fact, a lot of studies examined the electrochemical regulation of cellular metabolism using lipophilic electron mediators with transmembrane ability. In these systems, electron mediators transport electrons between the intracellular redox active species and the extracellular redox active species or electrodes, across cell membranes. This process is called extracellular electron transfer (EET). Although conventional lipophilic mediators show cytotoxicity and are not suitable for long-term cultivation of living cells, we have recently developed a redox-active polymer as a biocompatible electron mediator (hereafter, the mediator is called PMF)[1]. PMF is an amphiphilic, transmembrane random copolymer polymerized by a free radical polymerization of hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC) and hydrophobic vinyl ferrocene (VFc). The MPC and the VFc unit plays a role as a biocompatible and a redox active unit, respectively. Using thus synthesized PMF, we have successfully achieved electrochemical regulations of microbial metabolism without any genetic manipulations[2]. In the present work, with the aim of expanding the potential of this method, we attempted to regulate EET efficiency by tuning the molecular structure of the PMF polymers. It is known that the transmembrane ability is one of the rate determining factors in EET with conventional lipophilic electron mediators. Therefore, we assumed that cell membrane permeability could also influence the efficiency of the PMF-mediated EET. To verify this hypothesis, we added ethylene diamine tetra acetic acid (EDTA), which is known as a reagent to increase the cell-membrane permeability of gram-negative bacteria, during the measurement of the EET current from E. coli. Consequently, the EET current was increased by the addition of EDTA, and thus, the importance of the cell-membrane permeability was confirmed also for the case of the PMF-mediated EET. On the basis of the results, we decided to control the transmembrane ability of PMF for improving the EET efficiency. Theoretical calculations on amphiphilic copolymers have indicated that there is an suitable ratio between hydrophilic and hydrophobic units (R) for efficient cell-membrane permeability[3]. In addition, in the case of random copolymers including PMFs, cell-membrane permeability is expected to increase with decreasing molecular weight (Mw ). Based on these assumption, three kinds of PMF polymers with different Mw and R values (in this case, the ratio of VFc in the PMF polymer) were synthesized in this study. To evaluate the effects of three PMF polymers on EET, EET current from E. coli and S. cerevisiae was measured. Here, E. coli and S. cerevisiae were used as model living cells as prokaryotic cells and eukaryotic cells, respectively. From both experiments, the tendency of the relationship between EET efficiency and properties of PMF was revealed to be nearly the same for these two species. Experimental results suggested that R has a more significant influence on EET efficiency. Furthermore, it was also suggested that the EET efficiency could be increased with decreasing Mw at similar R values, as expected. Next, we tried to investigate the relationship between EET efficiency and the metabolisms by evaluating the activity of anaerobic glycolysis of S. cerevisiae as a model system. It is reported that lipophilic electron mediators with the redox potential lower than redox active species in electron transport chain (ETC) change the metabolic pathway of S. cerevisiae from the anaerobic glycolysis to anaerobic respiration by intercepting intracellular electrons from the ETC[4]. To evaluate the change of the metabolic pathway, we measured ethanol concentrations and the biomass in cell culture cultivated in the presence or the absence of the oxidized form of PMF. It was anticipated that the ethanol production becomes lower and the biomass becomes higher. In fact, the metabolic pathway was revealed to change in the presence of the oxidized form of PMF. Reasonably, the degree of the metabolism change was higher when the PMF with higher EET efficiency was used. These results indicated that the properties of the PMFs affects the efficiency of electrochemical regulation of cellular metabolism. Reference [1]Nishio et al, Chemphyschem, 2013, 14, 2159–2163. [2]Nishio et al, Environ. Sci. Technol. Lett., 2014, 1, 40–43. [3]Werner et al, Biomacromolecules, 2015, 16, 125–135. [4]Zhao et al, Anal. Chim. Acta, 2007, 597, 67–74. Figure 1

ABSTRACT Go with the flow: Shewanella is capable of transferring respiratory electrons to solid-state metal-oxides through physical contact (direct pathway) or by using electron shuttles (indirect pathway). We reveal, by using an electrochemical approach, that these pathways are switched depending on the redox state of the cytochromes.

Biological assimilation of CO2 to produce sugars occurs in metabolic cycles with an autocatalytic nature, such as the Calvin cycle and reverse citric acid cycle. The formose reaction, in which sugars are non-enzymatically synthesized from formaldehyde in alkaline solutions, involves such an autocatalytic cycle and has attracted much interest from the viewpoint of the abiotic chemical synthesis of sugars. However, many side reactions are indiscriminately accelerated by hydroxide ions in an alkaline aqueous solution, which results in a very low selectivity of sugar formation. Here we report non-enzymatic sugar synthesis in a neutral aqueous solution using mono-oxometalate as a catalyst to form an autocatalytic cycle. The construction of an autocatalytic reaction system in a neutral aqueous solution significantly improved the selectivity of sugar formation. It was also demonstrated that abiotically synthesized sugars could sustain the growth of microbial cells.

H2 is an important fermentation intermediate in anaerobic environments. Although H2 occurs at very low partial pressures in the environments, the culture and isolation of H2-utilizing microorganisms is usually carried out under very high H2 pressures, which might have hampered the discovery and understanding of microorganisms adapting to low H2 environments. Here we constructed a culture system designated the “iron corrosion-assisted H2-supplying (iCH) system” by connecting the gas phases of two vials (one for the iron corrosion reaction and the other for culturing microorganisms) to achieve cultures of microorganisms under low H2 pressures. We conducted enrichment cultures for methanogens and acetogens using rice paddy field soil as the microbial source. In the enrichment culture of methanogens under canonical high H2 pressures, only Methanobacterium spp. were enriched. By contrast, Methanocella spp. and Methanoculleus spp., methanogens adapting to low H2 pressures, were specifical...

Anaerobic digester is one of the attractive technologies for treatment of organic wastes and wastewater, while continuous development and improvements on their stable operation with efficient organic removal are required. Particles of conductive iron oxides (e.g., magnetite) are known to facilitate microbial interspecies electron transfer (termed as electric syntrophy). Electric syntrophy has been reported to enhance methanogenic degradation of organic acids by mesophilic communities in soil and anaerobic digester. Here we investigated the effects of supplementation of conductive iron oxides (magnetite) on thermophilic methanogenic microbial communities derived from a thermophilic anaerobic digester. Supplementation of magnetite accelerated methanogenesis from acetate and propionate under thermophilic conditions, while supplementation of ferrihydrite also accelerated methanogenesis from propionate. Microbial community analysis revealed that supplementation of magnetite drastically c...

Microbial extracellular electron transfer (EET) to solid-state electron acceptors such as anodes and metal oxides, which was originally identified in dissimilatory metal-reducing bacteria, is a key process in microbial electricity generation and the biogeochemical cycling of metals. Although it is now known that photosynthetic microorganisms can also generate (photo)currents via EET, which has attracted much interest in the field of biophotovoltaics, little is known about the reduction of metal (hydr)oxides via photosynthetic microbial EET. The present work quantitatively assessed the reduction of ferrihydrite in conjunction with the EET of the photosynthetic microbe Synechocystis sp. PCC 6803. Microbial reduction of ferrihydrite was found to be initiated in response to light but proceeded at higher rates when exogenous glucose was added, even under dark conditions. These results indicate that current generation from Synechocystis cells does not always need light irradiation. The qu...

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 occur...

Investigation of microbial interspecies interactions is essential for elucidating the function and stability of microbial ecosystems. However, community-based analyses including molecular-fingerprinting methods have limitations for precise understanding of interspecies interactions. Construction of model microbial consortia consisting of defined mixed cultures of isolated microorganisms is an excellent method for research on interspecies interactions. In this study, a model microbial consortium consisting of microorganisms that convert acetate into methane directly (Methanosaeta thermophila) and syntrophically (Thermacetogenium phaeum and Methanothermobacter thermautotrophicus) was constructed and the effects of elevated CO2 concentrations on intermicrobial competition were investigated. Analyses on the community dynamics by quantitative RT-PCR and fluorescent in situ hybridization targeting their 16S rRNAs revealed that high concentrations of CO2 have suppressive effects on the syn...

Corrosion of iron occurring under anoxic conditions, which is termed microbiologically influenced corrosion (MIC) or biocorrosion, is mostly caused by microbial activities. Microbial activity that enhances corrosion via uptake of electrons from metallic iron [Fe(0)] has been regarded as one of the major causative factors. In addition to sulfate-reducing bacteria and methanogenic archaea in marine environments, acetogenic bacteria in freshwater environments have recently been suggested to cause MIC under anoxic conditions. However, no microorganisms that perform acetogenesis-dependent MIC have been isolated or had their MIC-inducing mechanisms characterized. Here, we enriched and isolated acetogenic bacteria that induce iron corrosion by utilizing Fe(0) as the sole electron donor under freshwater, sulfate-free, and anoxic conditions. The enriched communities produced significantly larger amounts of Fe(II) than the abiotic controls and produced acetate coupled with Fe(0) oxidation pri...

The inhibitory effects of ammonia on two different degradation pathways of methanogenic acetate were evaluated using a pure culture (Methanosaeta thermophila strain PT) and defined co-culture (Methanothermobacter thermautotrophicus strain TM and Thermacetogenium phaeum strain PB), which represented aceticlastic and syntrophic methanogenesis, respectively. Growth experiments with high concentrations of ammonia clearly demonstrated that sensitivity to ammonia stress was markedly higher in M. thermophila PT than in the syntrophic co-culture. M. thermophila PT also exhibited higher sensitivity to high pH stress, which indicated that an inability to maintain pH homeostasis is an underlying cause of ammonia inhibition. Methanogenesis was inhibited in the resting cells of M. thermophila PT with moderate concentrations of ammonia, suggesting that the inhibition of enzymes involved in methanogenesis may be one of the major factors responsible for ammonia toxicity. Transcriptomic analysis rev...

Three thermophilic methanogens (Methanothermobacter thermautotrophicus, Methanosaeta thermophila, and Methanosarcina thermophila) were investigated for their ability to reduce poorly crystalline Fe(III) oxides (ferrihydrite) and the inhibitory effects of ferrihydrite on their methanogenesis. This study demonstrated that Fe(II) generation from ferrihydrite occurs in the cultures of the three thermophilic methanogens only when H2 was supplied as the source of reducing equivalents, even in the cultures of Mst. thermophila that do not grow on and produce CH4 from H2/CO2. While supplementation of ferrihydrite resulted in complete inhibition or suppression of methanogenesis by the thermophilic methanogens, ferrihydrite reduction by the methanogens at least partially alleviates the inhibitory effects. Microscopic and crystallographic analyses on the ferrihydrite-reducing Msr. thermophila cultures exhibited generation of magnetite on its cell surfaces through partial reduction of ferrihydri...

The addition of ferrihydrite to methanogenic microbial communities obtained from a thermophilic anaerobic digester suppressed methanogenesis in a dose-dependent manner. The amount of reducing equivalents consumed by the reduction of iron was significantly smaller than that expected from the decrease in the production of CH4, which suggested that competition between iron-reducing microorganisms and methanogens was not the most significant cause for the suppression of methanogenesis. Microbial community analyses revealed that the presence of ferrihydrite markedly affected the bacterial composition, but not the archaeal composition. These results indicate that the presence of ferrihydrite directly and indirectly suppresses thermophilic methanogenesis.

Some bacteria utilize (semi)conductive iron-oxide minerals as conduits for extracellular electron transfer (EET) to distant, insoluble electron acceptors. A previous study demonstrated that microbe/mineral conductive networks are constructed in soil ecosystems, in which Geobacter spp. share dominant populations. In order to examine how (semi)conductive iron-oxide minerals affect EET paths of Geobacter spp., the present study grew five representative Geobacter strains on electrodes as the sole electron acceptors in the absence or presence of (semi)conductive iron oxides. It was found that iron-oxide minerals enhanced current generation by three Geobacter strains, while no effect was observed in another strain. Geobacter sulfurreducens was the only strain that generated substantial amounts of currents both in the presence and absence of the iron oxides. Microscopic, electrochemical and transcriptomic analyses of G. sulfurreducens disclosed that this strain constructed two distinct typ...

In anaerobic biota, reducing equivalents (electrons) are transferred between different species of microbes [interspecies electron transfer (IET)], establishing the basis of cooperative behaviors and community functions. IET mechanisms described so far are based on diffusion of redox chemical species and/or direct contact in cell aggregates. Here, we show another possibility that IET also occurs via electric currents through natural conductive minerals. Our investigation revealed that electrically conductive magnetite nanoparticles facilitated IET from Geobacter sulfurreducens to Thiobacillus denitrificans, accomplishing acetate oxidation coupled to nitrate reduction. This two-species cooperative catabolism also occurred, albeit one order of magnitude slower, in the presence of Fe ions that worked as diffusive redox species. Semiconductive and insulating iron-oxide nanoparticles did not accelerate the cooperative catabolism. Our results suggest that microbes use conductive mineral pa...

We focused on bacterial interspecies relationships at the air-liquid interface where the formation of pellicles by aerobes was observed. Although an obligate aerobe (Brevibacillus sp. M1-5) was initially dominant in the pellicle population, a facultative aerobe (Pseudoxanthomonas sp. M1-3) emerged and the viability of M1-5 rapidly decreased due to severe competition for oxygen. Supplementation of the medium with carbohydrates allowed the two species to coexist at the air-liquid interface. These results indicate that the population dynamics within pellicles are primarily governed by oxygen utilization which was affected by a combination of carbon sources.

In natural and engineered ecosystems, diverse species of microbes coexist and interact, resulting in the emergence of community functions. Since microbes have evolved under such circumstances, it is reasonable to deduce that they have acquired strategies for specific interspecies interactions in complex microbial communities. In this review, we discuss the ecological and evolutionary interactions in syntrophic methanogenic consortia comprised of organic acid-oxidizing bacteria and methanogenic archaea. These microbes are known to exhibit mutual interactions (syntrophy), although the molecular mechanisms underlying these sophisticated partnerships have only just been discovered. In addition, recent genomic studies have provided insights into evolutionary interactions among members of methanogenic consortia, from which a novel concept termed "niche-associated evolution" has been proposed for interpreting how specialists evolve in a biological community. We suggest that micro...

A novel anaerobic, thermophilic and cellulolytic bacterium (strain CSK1(T)) was isolated from a cellulose-degrading bacterial community. On the basis of 16S rRNA gene sequence similarity, strain CSK1(T) was mapped to cluster III of the genus Clostridium. Strain CSK1(T) is closely related to Clostridium thermocellum (96.2 %) and Clostridium aldrichii (95.1 %). Strain CSK1(T) is a non-motile, spore-forming, straight or slightly curved rod. The optimum temperature and initial pH for its growth and cellulose degradation are 50-55 degrees C and pH 7.5. Strain CSK1(T) grew under a gas phase containing up to 4 % O(2). Phylogenetic and phenotypic analyses support the differentiation of strain CSK1(T) from its closest relatives. Strain CSK1(T) therefore represents a novel species, for which the name Clostridium straminisolvens sp. nov. is proposed, with CSK1(T) (=DSM 16021(T)=IAM 15070(T)) as the type strain.

We report here molecular mechanisms underlying a bacteria-archaeon symbiosis. We found that a fermentative bacterium used its flagellum for interaction with a specific methanogenic archaeon. The archaeon perceived a bacterial flagellum protein and activated its metabolism (methanogenesis). Transcriptome analyses showed that a substantial number of genes in the archaeon, including those involved in the methanogenesis pathway, were up-regulated after the contact with the flagellum protein. These findings suggest that the bacterium communicates with the archaeon by using its flagellum.