Souichiro Kato - Academia.edu (original) (raw)

Papers by Souichiro Kato

Frontiers in Microbiology, Jan 17, 2020

Conductive materials are known to promote direct interspecies electron transfer (DIET) by electri... more Conductive materials are known to promote direct interspecies electron transfer (DIET) by electrically bridging microbial cells. Previous studies have suggested that supplementation of graphene oxide (GO) based materials, including GO, and reduced GO (rGO), to anaerobic microbial communities, can promote DIET. This promotion mechanism is thought to be involved in electron transfer via rGO or biologically formed rGO. However, concrete evidence that rGO directly promotes DIET is still lacking. Furthermore, the effects of the physicochemical properties of GO-based materials on DIET efficiency have not been elucidated. In the current work, we investigated whether chemically and biologically reduced GO compounds can promote DIET in a defined model coculture system, and also examined the effects of surface properties on DIET-promoting efficiency. Supplementation of GO to a defined DIET coculture composed of an ethanol-oxidizing electron producer Geobacter metallireducens and a methane-producing electron consumer Methanosarcina barkeri promoted methane production from ethanol. X-ray photoelectron spectroscopy revealed that GO was reduced to rGO during cultivation by G. metallireducens activity. The stoichiometry of methane production from ethanol and the isotope labeling experiments clearly showed that biologically reduced GO induced DIET-mediated syntrophic methanogenesis. We also assessed the DIET-promoting efficiency of chemically reduced GO and its derivatives, including hydrophilic amine-functionalized rGO (rGO-NH 2) and hydrophobic octadecylamine-functionalized rGO (rGO-ODA). While all tested rGO derivatives induced DIET, the rGO derivatives with higher hydrophilicity showed higher DIET-promoting efficiency. Optical microscope observation revealed that microbial cells, in particular, G. metallireducens, more quickly adhered to more hydrophilic GO-based materials. The superior ability to recruit microbial cells is a critical feature of the higher DIET-promoting efficiency of the hydrophilic materials. This study demonstrates that biologically and chemically reduced GO can promote DIET-mediated syntrophic methanogenesis. Our results also suggested that the surface hydrophilicity (i.e., affinity toward microbial cells) is one of the important determinants of the DIET-promoting efficiencies. These observations will provide useful guidance for the selection of conductive particles for the improvement of methanogenesis in anaerobic digesters.

Applied and Environmental Microbiology, Oct 1, 2018

Most microorganisms living in the environment have yet to be cultured, owing at least in part to ... more Most microorganisms living in the environment have yet to be cultured, owing at least in part to their slow and poor propagation properties and susceptibility to oxidative stress. Our previous studies demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving the phosphate and agar separately (termed "PS" medium), can greatly improve the culturability of microorganisms by mitigating oxidative stress compared with the use of "PT" medium (autoclaving the phosphate and agar together). Here, we attempted to isolate phylogenetically novel bacteria by combining PS medium with prolonged cultivation. After inoculation with forest soil or pond sediment samples, significantly more colonies appeared on PS medium than on PT medium. A total of 98 and 74 colonies that emerged after more than 7 days of cultivation were isolated as slow growers from PS and PT media, respectively. Sequencing analysis of their 16S rRNA genes revealed that the slow growers recovered from PS medium included more phylogenetically novel bacteria than those from PT medium, including a strain that could be classified into a novel order in the class Alphaproteobacteria. Further physiological analysis of representative strains showed that they were actually slow and poor growers and formed small but visible colonies only on PS medium. This study demonstrates that the culturability of previously uncultured bacteria can be improved by using an isolation strategy that combines a simple modification in medium preparation with an extended incubation time.

Microbes and Environments, 2020

Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 la... more Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca 2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl 2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.

Microbes and Environments, 2013

Some bacteria utilize (semi)conductive iron-oxide minerals as conduits for extracellular electron... more 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 types of EET path; in the absence of iron-oxide minerals, bacterial biofilms rich in extracellular polymeric substances were constructed, while composite networks made of mineral particles and microbial cells (without polymeric substances) were developed in the presence of iron oxides. It was also found that uncharacterized c-type cytochromes were up-regulated in the presence of iron oxides that were different from those found in conductive biofilms. These results suggest the possibility that natural (semi)conductive minerals confer energetic and ecological advantages on Geobacter, facilitating their growth and survival in the natural environment.

Microbes and Environments, 2023

Hydrogen peroxide (H 2 O 2) inhibits microbial growth at a specific concentration. However, we pr... more Hydrogen peroxide (H 2 O 2) inhibits microbial growth at a specific concentration. However, we previously isolated two environmental bacterial strains that exhibited sensitivity to a lower H 2 O 2 concentration in agar plates. Putative catalase genes, which degrade H 2 O 2 , were detected in their genomes. We herein elucidated the characteristics of these putative genes and their products using a self-cloning technique. The products of the cloned genes were identified as functional catalases. The up-regulation of their expression increased the colony-forming ability of host cells under H 2 O 2 pressure. The present results demonstrated high sensitivity to H 2 O 2 even in microbes possessing functional catalase genes.

Frontiers in Microbiology, Feb 1, 2021

Microbes and Environments, 2015

Extracellular electron transfer (EET) is a type of microbial respiration that enables electron tr... more Extracellular electron transfer (EET) is a type of microbial respiration that enables electron transfer between microbial cells and extracellular solid materials, including naturally-occurring metal compounds and artificial electrodes. Microorganisms harboring EET abilities have received considerable attention for their various biotechnological applications, in addition to their contribution to global energy and material cycles. In this review, current knowledge on microbial EET and its application to diverse biotechnologies, including the bioremediation of toxic metals, recovery of useful metals, biocorrosion, and microbial electrochemical systems (microbial fuel cells and microbial electrosynthesis), were introduced. Two potential biotechnologies based on microbial EET, namely the electrochemical control of microbial metabolism and electrochemical stimulation of microbial symbiotic reactions (electric syntrophy), were also discussed.

International Journal of Molecular Sciences, Jan 6, 2017

Geobacter species are capable of utilizing solid-state compounds, including anodic electrodes, as... more Geobacter species are capable of utilizing solid-state compounds, including anodic electrodes, as electron acceptors of respiration via extracellular electron transfer (EET) and have attracted considerable attention for their crucial role as biocatalysts of bioelectrochemical systems (BES's). Recent studies disclosed that anode potentials affect power output and anodic microbial communities, including selection of dominant Geobacter species, in various BES's. However, the details in current-generating properties and responses to anode potentials have been investigated only for a model species, namely Geobacter sulfurreducens. In this study, the effects of anode potentials on the current generation and the EET paths were investigated by cultivating six Geobacter species with different anode potentials, followed by electrochemical analyses. The electrochemical cultivation demonstrated that the G. metallireducens clade species (G. sulfurreducens and G. metallireducens) constantly generate high current densities at a wide range of anode potentials (≥−0.3 or −0.2 V vs. Ag/AgCl), while the subsurface clades species (G. daltonii, G. bemidjensis, G. chapellei, and G. pelophilus) generate a relatively large current only at limited potential regions (−0.1 to −0.3 V vs. Ag/AgCl). The linear sweep voltammetry analyses indicated that the G. metallireducens clade species utilize only one EET path irrespective of the anode potentials, while the subsurface clades species utilize multiple EET paths, which can be optimized depending on the anode potentials. These results clearly demonstrate that the response features to anode potentials are divergent among species (or clades) of Geobacter.

Frontiers in Microbiology, Apr 30, 2020

Screening for bacteria with abilities to accumulate valuable intracellular compounds from an envi... more Screening for bacteria with abilities to accumulate valuable intracellular compounds from an environmental community is difficult and requires strategic methods. Combining the experimental procedure for phenotyping living cells in a microbial community with the cell recovery necessary for further cultivation will allow for an efficient initial screening process. In this study, we developed a strategy for the isolation of polyphosphateaccumulating organisms (PAOs) by combining (i) nontoxic fluorescence staining of polyphosphate granules in viable microbial cells and (ii) fluorescence-activated cell sorting (FACS) for the rapid detection and collection of target cells. To implement this screening approach, cells from wastewater sludge samples were stained with 4'6-diamidino-2-phenylindole (DAPI) to target cells with high polyphosphate (polyP) accumulation. We found a staining procedure (10 µg/ml of DAPI for 30 min) that can visualize polyP granules while maintaining viability for the majority of the cells (>60%). The polyP positive cells were recovered by FACS, purified by colony isolation and phylogenetically identified by 16S rRNA gene sequencing. Follow-up analysis confirmed that these isolates accumulate polyP, indicating that DAPI can be implemented in staining living cells and FACS can effectively and rapidly screen and isolate individual cells from a complex microbial community.

Scientific Reports, Feb 17, 2016

Interest in the interaction of microorganisms with cesium ions (Cs +) has arisen, especially in t... more Interest in the interaction of microorganisms with cesium ions (Cs +) has arisen, especially in terms of their potent ability for radiocesium bioaccumulation and their important roles in biogeochemical cycling. Although high concentrations of Cs + display toxic effects on microorganisms, there have been only limited reports for Cs +-tolerant microorganisms. Here we report enrichment and isolation of Cs +tolerant microorganisms from soil microbiota. Microbial community analysis revealed that bacteria within the phylum Bacteroidetes, especially Flavobacterium spp., dominated in enrichment cultures in the medium supplemented with 50 or 200 mM Cs + , while Gammaproteobacteria was dominant in the control enrichment cultures (in the presence of 50 and 200 mM K + instead of Cs +). The dominant Flavobacterium sp. was successfully isolated from the enrichment culture and was closely related to Flavobacterium chungbukense with 99.5% identity. Growth experiments clearly demonstrated that the isolate has significantly higher tolerance to Cs + compared to its close relatives, suggesting the Cs +-tolerance is a specific trait of this strain, but not a universal trait in the genus Flavobacterium. Measurement of intracellular K + and Cs + concentrations of the Cs +-tolerant isolate and its close relatives suggested that the ability to maintain low intracellular Cs + concentration confers the tolerance against high concentrations of external Cs +. Cesium (Cs) is a Group I alkali metal with atomic number of 55. The physicochemical properties of Cs are highly similar to the other alkali metals, in particular, to potassium (K). Cs generally exists as monovalent cation (Cs +) in solution. Special attention has been paid to the radioisotopes of cesium, especially 134 Cs and 137 Cs, due to the concern of radioactive pollution arising from nuclear weapon testing and from intentional and unintentional discharge from nuclear power plants, relatively long half-lives, high water solubility, and rapid incorporation into biological systems 1,2. Although no essential biological function for Cs + has been identified, Cs + transport and accumulation in living organisms, including bacteria, fungi, and plants, are known to date 1-10. This feature has offered bioaccumulation as a potential alternative to existing methods for decontamination or recovery of radioactive Cs from environments. In microorganisms and plants, Cs + is principally incorporated via K + transport systems because of its chemical similarity to K + (refs 1, 5). For example, Cs + transport in Eschericia coli was shown to occur via a K + uptake system, namely Kup, although the affinity for Cs + is much lower than for K + (ref. 11). Considering the existence of a number of monocation uptake mechanisms, it would not surprising that different microbial species or strains have wide range of ability to transport and accumulate Cs +. High concentration of Cs + , generally above the mM order, is toxic to microorganisms 12,13. Two different mechanisms have been inferred as the inhibitory mechanisms of Cs + , namely, induction of K + starvation and intracellular toxicity of Cs +. K + starvation is induced by an inhibitory effect of Cs + on K +-uptake systems and efflux of K + caused by increasing intracellular Cs + concentration 11,14. Intracellular Cs + is also known to compete with K + for

Microbiology Resource Announcements, 2021

The complete genome of hydrogen peroxide-sensitive alphaproteobacterial strain SO-S41 was sequenc... more The complete genome of hydrogen peroxide-sensitive alphaproteobacterial strain SO-S41 was sequenced. The complete genome contains a single chromosome, is 4,443,179 bp in length, contains a total of 4,632 genes, and has a G+C content of 66.2%.

Frontiers in Microbiology, 2020

Prediction of Neighbor-Dependent Interactions From Limited Data whose growth is dependent on othe... more Prediction of Neighbor-Dependent Interactions From Limited Data whose growth is dependent on other species), we demonstrated that despite data limitation, the proposed new formulation was able to successfully predict interspecies interactions that are consistent with experimentally derived results. Therefore, this technical advancement enhances our ability to predict context-dependent interspecies interactions in a broad range of microbial systems without being limited to specific growth conditions as a prerequisite .

Frontiers in Microbiology, 2021

Microbial reduction of iron contributes to the dissolution and transformation of iron-containing ... more Microbial reduction of iron contributes to the dissolution and transformation of iron-containing minerals in nature. Diverse groups of homoacetogenic bacteria (homoacetogens) have been reported to reduce insoluble Fe(III) oxides, such as hydrous ferric oxide (HFO), an Fe(III) mineral commonly found in soils and sediments. Several members of genus Sporomusa reportedly oxidize Fe(0), indicating the presence of an extracellular electron-uptake mechanism. However, the ability of the genus to reduce insoluble Fe(III) oxides is limited, and the underlying reduction mechanism remains to be elucidated. In this study, the HFO reduction ability of three Sporomusa spp. (Sporomusa sp. strain GT1, Sporomusa sphaeroides, and Sporomusa ovata) and a homoacetogen of a different genus (Acetobacterium woodii) were assayed under organotrophic (ethanol) and lithotrophic (H2 + CO2) conditions without a chelator or reducing reagent. All tested homoacetogens showed acetogenic growth and concomitant reducti...

Microbes and Environments, 2020

Methanogenic microbial communities were enriched from rice paddy soil and anaerobic digester slud... more Methanogenic microbial communities were enriched from rice paddy soil and anaerobic digester sludge using peptidoglycan purified from gram-negative Escherichia coli or gram-positive Micrococcus luteus as the sole substrate. Methane production data suggested the anaerobic degradation of peptidoglycan and also that peptidoglycan from E. coli had lower degradability. The community structures of enrichment cultures fed peptidoglycan from E. coli or M. luteus were similar, but distinctly different. A number of phylogenetically novel and uncultured bacteria, particularly in the phyla Bacteroidetes, WWE1, Armatimonadetes, and Verrucomicrobia, dominated the enrichment cultures, suggesting their involvement in anaerobic peptidoglycan degradation.

Microbes and Environments, 2020

Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 la... more Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca 2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl 2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.

Microbes and Environments, 2020

We previously demonstrated that a simple modification in the preparation of agar media, i.e., aut... more We previously demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving phosphate and agar separately (termed the "PS protocol"), improved the culturability of aerobic microorganisms by reducing the generation of reactive oxygen species. We herein investigated the effects of the PS protocol on the cultivation of anaerobic microorganisms using sludge from a wastewater treatment system as a microbial source. The application of the PS protocol increased colony numbers and the frequency of phylogenetically novel isolates under aerobic, nitrate reduction, and fermentation conditions. The PS protocol is useful for isolating both aerobic and anaerobic microorganisms.

Microbes and Environments, 2019

Supplementation with conductive magnetite particles promoted methanogenic acetate degradation by ... more Supplementation with conductive magnetite particles promoted methanogenic acetate degradation by microbial communities enriched from the production water of a high-temperature petroleum reservoir. A microbial community analysis revealed that Petrothermobacter spp. (phylum Deferribacteres), known as thermophilic Fe(III) reducers, predominated in the magnetitesupplemented enrichment, whereas other types of Fe(III) reducers, such as Thermincola spp. and Thermotoga spp., were dominant under ferrihydrite-reducing conditions. These results suggest that magnetite induced interspecies electron transfer via electric currents through conductive particles between Petrothermobacter spp. and methanogens. This is the first evidence for possible electric syntrophy in high-temperature subsurface environments.

Applied and Environmental Microbiology, 2018

Most microbial species inhabiting natural environments have not yet been isolated. One of the ser... more Most microbial species inhabiting natural environments have not yet been isolated. One of the serious issues preventing their isolation is intrinsically slow and/or poor growth. Moreover, these slow and/or poor growers are likely to be highly sensitive to environmental stresses, especially oxidative stress. We reported previously that interaction between agar and phosphate during autoclave sterilization generates hydrogen peroxide, which adversely affects the culturability of environmental microorganisms, in particular, slow-growing organisms vulnerable to oxidative stress. In this study, we successfully isolated many slow-growing bacterial strains with phylogenetic novelty by simply modifying their cultivation on agar plates, i.e., autoclaving the phosphate and agar separately. The current limited repertoire of culture techniques still has room for improvement in the isolation of microorganisms previously considered unculturable.

International Journal of Molecular Sciences, 2017

Geobacter species are capable of utilizing solid-state compounds, including anodic electrodes, as... more Geobacter species are capable of utilizing solid-state compounds, including anodic electrodes, as electron acceptors of respiration via extracellular electron transfer (EET) and have attracted considerable attention for their crucial role as biocatalysts of bioelectrochemical systems (BES's). Recent studies disclosed that anode potentials affect power output and anodic microbial communities, including selection of dominant Geobacter species, in various BES's. However, the details in current-generating properties and responses to anode potentials have been investigated only for a model species, namely Geobacter sulfurreducens. In this study, the effects of anode potentials on the current generation and the EET paths were investigated by cultivating six Geobacter species with different anode potentials, followed by electrochemical analyses. The electrochemical cultivation demonstrated that the G. metallireducens clade species (G. sulfurreducens and G. metallireducens) constantly generate high current densities at a wide range of anode potentials (≥−0.3 or −0.2 V vs. Ag/AgCl), while the subsurface clades species (G. daltonii, G. bemidjensis, G. chapellei, and G. pelophilus) generate a relatively large current only at limited potential regions (−0.1 to −0.3 V vs. Ag/AgCl). The linear sweep voltammetry analyses indicated that the G. metallireducens clade species utilize only one EET path irrespective of the anode potentials, while the subsurface clades species utilize multiple EET paths, which can be optimized depending on the anode potentials. These results clearly demonstrate that the response features to anode potentials are divergent among species (or clades) of Geobacter.

Scientific reports, Jan 16, 2017

Biological incorporation of cesium ions (Cs(+)) has recently attracted significant attention in t... more Biological incorporation of cesium ions (Cs(+)) has recently attracted significant attention in terms of the possible applications for bioremediation of radiocesium and their significant roles in biogeochemical cycling. Although high concentrations of Cs(+) exhibit cytotoxicity on microorganisms, there are a few reports on the promotive effects of Cs(+) on microbial growth under K(+)-deficient conditions. However, whether this growth-promoting effect is a common phenomenon remains uncertain, and direct correlation between growth promotion and Cs(+) uptake abilities has not been confirmed yet. Here, we validated the growth promotive effects of Cs(+) uptake under K(+)-deficient conditions using an Escherichia coli strain with an inducible expression of the Kup K(+) transporter that has nonspecific Cs(+) transport activities (strain kup-IE). The strain kup-IE exhibited superior growth under the Cs(+)-supplemented and K(+)-deficient conditions compared to the wild type and the kup null ...

Frontiers in Microbiology, Jan 17, 2020

Conductive materials are known to promote direct interspecies electron transfer (DIET) by electri... more Conductive materials are known to promote direct interspecies electron transfer (DIET) by electrically bridging microbial cells. Previous studies have suggested that supplementation of graphene oxide (GO) based materials, including GO, and reduced GO (rGO), to anaerobic microbial communities, can promote DIET. This promotion mechanism is thought to be involved in electron transfer via rGO or biologically formed rGO. However, concrete evidence that rGO directly promotes DIET is still lacking. Furthermore, the effects of the physicochemical properties of GO-based materials on DIET efficiency have not been elucidated. In the current work, we investigated whether chemically and biologically reduced GO compounds can promote DIET in a defined model coculture system, and also examined the effects of surface properties on DIET-promoting efficiency. Supplementation of GO to a defined DIET coculture composed of an ethanol-oxidizing electron producer Geobacter metallireducens and a methane-producing electron consumer Methanosarcina barkeri promoted methane production from ethanol. X-ray photoelectron spectroscopy revealed that GO was reduced to rGO during cultivation by G. metallireducens activity. The stoichiometry of methane production from ethanol and the isotope labeling experiments clearly showed that biologically reduced GO induced DIET-mediated syntrophic methanogenesis. We also assessed the DIET-promoting efficiency of chemically reduced GO and its derivatives, including hydrophilic amine-functionalized rGO (rGO-NH 2) and hydrophobic octadecylamine-functionalized rGO (rGO-ODA). While all tested rGO derivatives induced DIET, the rGO derivatives with higher hydrophilicity showed higher DIET-promoting efficiency. Optical microscope observation revealed that microbial cells, in particular, G. metallireducens, more quickly adhered to more hydrophilic GO-based materials. The superior ability to recruit microbial cells is a critical feature of the higher DIET-promoting efficiency of the hydrophilic materials. This study demonstrates that biologically and chemically reduced GO can promote DIET-mediated syntrophic methanogenesis. Our results also suggested that the surface hydrophilicity (i.e., affinity toward microbial cells) is one of the important determinants of the DIET-promoting efficiencies. These observations will provide useful guidance for the selection of conductive particles for the improvement of methanogenesis in anaerobic digesters.

Applied and Environmental Microbiology, Oct 1, 2018

Most microorganisms living in the environment have yet to be cultured, owing at least in part to ... more Most microorganisms living in the environment have yet to be cultured, owing at least in part to their slow and poor propagation properties and susceptibility to oxidative stress. Our previous studies demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving the phosphate and agar separately (termed "PS" medium), can greatly improve the culturability of microorganisms by mitigating oxidative stress compared with the use of "PT" medium (autoclaving the phosphate and agar together). Here, we attempted to isolate phylogenetically novel bacteria by combining PS medium with prolonged cultivation. After inoculation with forest soil or pond sediment samples, significantly more colonies appeared on PS medium than on PT medium. A total of 98 and 74 colonies that emerged after more than 7 days of cultivation were isolated as slow growers from PS and PT media, respectively. Sequencing analysis of their 16S rRNA genes revealed that the slow growers recovered from PS medium included more phylogenetically novel bacteria than those from PT medium, including a strain that could be classified into a novel order in the class Alphaproteobacteria. Further physiological analysis of representative strains showed that they were actually slow and poor growers and formed small but visible colonies only on PS medium. This study demonstrates that the culturability of previously uncultured bacteria can be improved by using an isolation strategy that combines a simple modification in medium preparation with an extended incubation time.

Microbes and Environments, 2020

Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 la... more Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca 2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl 2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.

Microbes and Environments, 2013

Some bacteria utilize (semi)conductive iron-oxide minerals as conduits for extracellular electron... more 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 types of EET path; in the absence of iron-oxide minerals, bacterial biofilms rich in extracellular polymeric substances were constructed, while composite networks made of mineral particles and microbial cells (without polymeric substances) were developed in the presence of iron oxides. It was also found that uncharacterized c-type cytochromes were up-regulated in the presence of iron oxides that were different from those found in conductive biofilms. These results suggest the possibility that natural (semi)conductive minerals confer energetic and ecological advantages on Geobacter, facilitating their growth and survival in the natural environment.

Microbes and Environments, 2023

Hydrogen peroxide (H 2 O 2) inhibits microbial growth at a specific concentration. However, we pr... more Hydrogen peroxide (H 2 O 2) inhibits microbial growth at a specific concentration. However, we previously isolated two environmental bacterial strains that exhibited sensitivity to a lower H 2 O 2 concentration in agar plates. Putative catalase genes, which degrade H 2 O 2 , were detected in their genomes. We herein elucidated the characteristics of these putative genes and their products using a self-cloning technique. The products of the cloned genes were identified as functional catalases. The up-regulation of their expression increased the colony-forming ability of host cells under H 2 O 2 pressure. The present results demonstrated high sensitivity to H 2 O 2 even in microbes possessing functional catalase genes.

Frontiers in Microbiology, Feb 1, 2021

Microbes and Environments, 2015

Extracellular electron transfer (EET) is a type of microbial respiration that enables electron tr... more Extracellular electron transfer (EET) is a type of microbial respiration that enables electron transfer between microbial cells and extracellular solid materials, including naturally-occurring metal compounds and artificial electrodes. Microorganisms harboring EET abilities have received considerable attention for their various biotechnological applications, in addition to their contribution to global energy and material cycles. In this review, current knowledge on microbial EET and its application to diverse biotechnologies, including the bioremediation of toxic metals, recovery of useful metals, biocorrosion, and microbial electrochemical systems (microbial fuel cells and microbial electrosynthesis), were introduced. Two potential biotechnologies based on microbial EET, namely the electrochemical control of microbial metabolism and electrochemical stimulation of microbial symbiotic reactions (electric syntrophy), were also discussed.

International Journal of Molecular Sciences, Jan 6, 2017

Geobacter species are capable of utilizing solid-state compounds, including anodic electrodes, as... more Geobacter species are capable of utilizing solid-state compounds, including anodic electrodes, as electron acceptors of respiration via extracellular electron transfer (EET) and have attracted considerable attention for their crucial role as biocatalysts of bioelectrochemical systems (BES's). Recent studies disclosed that anode potentials affect power output and anodic microbial communities, including selection of dominant Geobacter species, in various BES's. However, the details in current-generating properties and responses to anode potentials have been investigated only for a model species, namely Geobacter sulfurreducens. In this study, the effects of anode potentials on the current generation and the EET paths were investigated by cultivating six Geobacter species with different anode potentials, followed by electrochemical analyses. The electrochemical cultivation demonstrated that the G. metallireducens clade species (G. sulfurreducens and G. metallireducens) constantly generate high current densities at a wide range of anode potentials (≥−0.3 or −0.2 V vs. Ag/AgCl), while the subsurface clades species (G. daltonii, G. bemidjensis, G. chapellei, and G. pelophilus) generate a relatively large current only at limited potential regions (−0.1 to −0.3 V vs. Ag/AgCl). The linear sweep voltammetry analyses indicated that the G. metallireducens clade species utilize only one EET path irrespective of the anode potentials, while the subsurface clades species utilize multiple EET paths, which can be optimized depending on the anode potentials. These results clearly demonstrate that the response features to anode potentials are divergent among species (or clades) of Geobacter.

Frontiers in Microbiology, Apr 30, 2020

Screening for bacteria with abilities to accumulate valuable intracellular compounds from an envi... more Screening for bacteria with abilities to accumulate valuable intracellular compounds from an environmental community is difficult and requires strategic methods. Combining the experimental procedure for phenotyping living cells in a microbial community with the cell recovery necessary for further cultivation will allow for an efficient initial screening process. In this study, we developed a strategy for the isolation of polyphosphateaccumulating organisms (PAOs) by combining (i) nontoxic fluorescence staining of polyphosphate granules in viable microbial cells and (ii) fluorescence-activated cell sorting (FACS) for the rapid detection and collection of target cells. To implement this screening approach, cells from wastewater sludge samples were stained with 4'6-diamidino-2-phenylindole (DAPI) to target cells with high polyphosphate (polyP) accumulation. We found a staining procedure (10 µg/ml of DAPI for 30 min) that can visualize polyP granules while maintaining viability for the majority of the cells (>60%). The polyP positive cells were recovered by FACS, purified by colony isolation and phylogenetically identified by 16S rRNA gene sequencing. Follow-up analysis confirmed that these isolates accumulate polyP, indicating that DAPI can be implemented in staining living cells and FACS can effectively and rapidly screen and isolate individual cells from a complex microbial community.

Scientific Reports, Feb 17, 2016

Interest in the interaction of microorganisms with cesium ions (Cs +) has arisen, especially in t... more Interest in the interaction of microorganisms with cesium ions (Cs +) has arisen, especially in terms of their potent ability for radiocesium bioaccumulation and their important roles in biogeochemical cycling. Although high concentrations of Cs + display toxic effects on microorganisms, there have been only limited reports for Cs +-tolerant microorganisms. Here we report enrichment and isolation of Cs +tolerant microorganisms from soil microbiota. Microbial community analysis revealed that bacteria within the phylum Bacteroidetes, especially Flavobacterium spp., dominated in enrichment cultures in the medium supplemented with 50 or 200 mM Cs + , while Gammaproteobacteria was dominant in the control enrichment cultures (in the presence of 50 and 200 mM K + instead of Cs +). The dominant Flavobacterium sp. was successfully isolated from the enrichment culture and was closely related to Flavobacterium chungbukense with 99.5% identity. Growth experiments clearly demonstrated that the isolate has significantly higher tolerance to Cs + compared to its close relatives, suggesting the Cs +-tolerance is a specific trait of this strain, but not a universal trait in the genus Flavobacterium. Measurement of intracellular K + and Cs + concentrations of the Cs +-tolerant isolate and its close relatives suggested that the ability to maintain low intracellular Cs + concentration confers the tolerance against high concentrations of external Cs +. Cesium (Cs) is a Group I alkali metal with atomic number of 55. The physicochemical properties of Cs are highly similar to the other alkali metals, in particular, to potassium (K). Cs generally exists as monovalent cation (Cs +) in solution. Special attention has been paid to the radioisotopes of cesium, especially 134 Cs and 137 Cs, due to the concern of radioactive pollution arising from nuclear weapon testing and from intentional and unintentional discharge from nuclear power plants, relatively long half-lives, high water solubility, and rapid incorporation into biological systems 1,2. Although no essential biological function for Cs + has been identified, Cs + transport and accumulation in living organisms, including bacteria, fungi, and plants, are known to date 1-10. This feature has offered bioaccumulation as a potential alternative to existing methods for decontamination or recovery of radioactive Cs from environments. In microorganisms and plants, Cs + is principally incorporated via K + transport systems because of its chemical similarity to K + (refs 1, 5). For example, Cs + transport in Eschericia coli was shown to occur via a K + uptake system, namely Kup, although the affinity for Cs + is much lower than for K + (ref. 11). Considering the existence of a number of monocation uptake mechanisms, it would not surprising that different microbial species or strains have wide range of ability to transport and accumulate Cs +. High concentration of Cs + , generally above the mM order, is toxic to microorganisms 12,13. Two different mechanisms have been inferred as the inhibitory mechanisms of Cs + , namely, induction of K + starvation and intracellular toxicity of Cs +. K + starvation is induced by an inhibitory effect of Cs + on K +-uptake systems and efflux of K + caused by increasing intracellular Cs + concentration 11,14. Intracellular Cs + is also known to compete with K + for

Microbiology Resource Announcements, 2021

The complete genome of hydrogen peroxide-sensitive alphaproteobacterial strain SO-S41 was sequenc... more The complete genome of hydrogen peroxide-sensitive alphaproteobacterial strain SO-S41 was sequenced. The complete genome contains a single chromosome, is 4,443,179 bp in length, contains a total of 4,632 genes, and has a G+C content of 66.2%.

Frontiers in Microbiology, 2020

Prediction of Neighbor-Dependent Interactions From Limited Data whose growth is dependent on othe... more Prediction of Neighbor-Dependent Interactions From Limited Data whose growth is dependent on other species), we demonstrated that despite data limitation, the proposed new formulation was able to successfully predict interspecies interactions that are consistent with experimentally derived results. Therefore, this technical advancement enhances our ability to predict context-dependent interspecies interactions in a broad range of microbial systems without being limited to specific growth conditions as a prerequisite .

Frontiers in Microbiology, 2021

Microbial reduction of iron contributes to the dissolution and transformation of iron-containing ... more Microbial reduction of iron contributes to the dissolution and transformation of iron-containing minerals in nature. Diverse groups of homoacetogenic bacteria (homoacetogens) have been reported to reduce insoluble Fe(III) oxides, such as hydrous ferric oxide (HFO), an Fe(III) mineral commonly found in soils and sediments. Several members of genus Sporomusa reportedly oxidize Fe(0), indicating the presence of an extracellular electron-uptake mechanism. However, the ability of the genus to reduce insoluble Fe(III) oxides is limited, and the underlying reduction mechanism remains to be elucidated. In this study, the HFO reduction ability of three Sporomusa spp. (Sporomusa sp. strain GT1, Sporomusa sphaeroides, and Sporomusa ovata) and a homoacetogen of a different genus (Acetobacterium woodii) were assayed under organotrophic (ethanol) and lithotrophic (H2 + CO2) conditions without a chelator or reducing reagent. All tested homoacetogens showed acetogenic growth and concomitant reducti...

Microbes and Environments, 2020

Methanogenic microbial communities were enriched from rice paddy soil and anaerobic digester slud... more Methanogenic microbial communities were enriched from rice paddy soil and anaerobic digester sludge using peptidoglycan purified from gram-negative Escherichia coli or gram-positive Micrococcus luteus as the sole substrate. Methane production data suggested the anaerobic degradation of peptidoglycan and also that peptidoglycan from E. coli had lower degradability. The community structures of enrichment cultures fed peptidoglycan from E. coli or M. luteus were similar, but distinctly different. A number of phylogenetically novel and uncultured bacteria, particularly in the phyla Bacteroidetes, WWE1, Armatimonadetes, and Verrucomicrobia, dominated the enrichment cultures, suggesting their involvement in anaerobic peptidoglycan degradation.

Microbes and Environments, 2020

Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 la... more Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca 2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl 2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.

Microbes and Environments, 2020

We previously demonstrated that a simple modification in the preparation of agar media, i.e., aut... more We previously demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving phosphate and agar separately (termed the "PS protocol"), improved the culturability of aerobic microorganisms by reducing the generation of reactive oxygen species. We herein investigated the effects of the PS protocol on the cultivation of anaerobic microorganisms using sludge from a wastewater treatment system as a microbial source. The application of the PS protocol increased colony numbers and the frequency of phylogenetically novel isolates under aerobic, nitrate reduction, and fermentation conditions. The PS protocol is useful for isolating both aerobic and anaerobic microorganisms.

Microbes and Environments, 2019

Supplementation with conductive magnetite particles promoted methanogenic acetate degradation by ... more Supplementation with conductive magnetite particles promoted methanogenic acetate degradation by microbial communities enriched from the production water of a high-temperature petroleum reservoir. A microbial community analysis revealed that Petrothermobacter spp. (phylum Deferribacteres), known as thermophilic Fe(III) reducers, predominated in the magnetitesupplemented enrichment, whereas other types of Fe(III) reducers, such as Thermincola spp. and Thermotoga spp., were dominant under ferrihydrite-reducing conditions. These results suggest that magnetite induced interspecies electron transfer via electric currents through conductive particles between Petrothermobacter spp. and methanogens. This is the first evidence for possible electric syntrophy in high-temperature subsurface environments.

Applied and Environmental Microbiology, 2018

Most microbial species inhabiting natural environments have not yet been isolated. One of the ser... more Most microbial species inhabiting natural environments have not yet been isolated. One of the serious issues preventing their isolation is intrinsically slow and/or poor growth. Moreover, these slow and/or poor growers are likely to be highly sensitive to environmental stresses, especially oxidative stress. We reported previously that interaction between agar and phosphate during autoclave sterilization generates hydrogen peroxide, which adversely affects the culturability of environmental microorganisms, in particular, slow-growing organisms vulnerable to oxidative stress. In this study, we successfully isolated many slow-growing bacterial strains with phylogenetic novelty by simply modifying their cultivation on agar plates, i.e., autoclaving the phosphate and agar separately. The current limited repertoire of culture techniques still has room for improvement in the isolation of microorganisms previously considered unculturable.

International Journal of Molecular Sciences, 2017

Geobacter species are capable of utilizing solid-state compounds, including anodic electrodes, as... more Geobacter species are capable of utilizing solid-state compounds, including anodic electrodes, as electron acceptors of respiration via extracellular electron transfer (EET) and have attracted considerable attention for their crucial role as biocatalysts of bioelectrochemical systems (BES's). Recent studies disclosed that anode potentials affect power output and anodic microbial communities, including selection of dominant Geobacter species, in various BES's. However, the details in current-generating properties and responses to anode potentials have been investigated only for a model species, namely Geobacter sulfurreducens. In this study, the effects of anode potentials on the current generation and the EET paths were investigated by cultivating six Geobacter species with different anode potentials, followed by electrochemical analyses. The electrochemical cultivation demonstrated that the G. metallireducens clade species (G. sulfurreducens and G. metallireducens) constantly generate high current densities at a wide range of anode potentials (≥−0.3 or −0.2 V vs. Ag/AgCl), while the subsurface clades species (G. daltonii, G. bemidjensis, G. chapellei, and G. pelophilus) generate a relatively large current only at limited potential regions (−0.1 to −0.3 V vs. Ag/AgCl). The linear sweep voltammetry analyses indicated that the G. metallireducens clade species utilize only one EET path irrespective of the anode potentials, while the subsurface clades species utilize multiple EET paths, which can be optimized depending on the anode potentials. These results clearly demonstrate that the response features to anode potentials are divergent among species (or clades) of Geobacter.

Scientific reports, Jan 16, 2017

Biological incorporation of cesium ions (Cs(+)) has recently attracted significant attention in t... more Biological incorporation of cesium ions (Cs(+)) has recently attracted significant attention in terms of the possible applications for bioremediation of radiocesium and their significant roles in biogeochemical cycling. Although high concentrations of Cs(+) exhibit cytotoxicity on microorganisms, there are a few reports on the promotive effects of Cs(+) on microbial growth under K(+)-deficient conditions. However, whether this growth-promoting effect is a common phenomenon remains uncertain, and direct correlation between growth promotion and Cs(+) uptake abilities has not been confirmed yet. Here, we validated the growth promotive effects of Cs(+) uptake under K(+)-deficient conditions using an Escherichia coli strain with an inducible expression of the Kup K(+) transporter that has nonspecific Cs(+) transport activities (strain kup-IE). The strain kup-IE exhibited superior growth under the Cs(+)-supplemented and K(+)-deficient conditions compared to the wild type and the kup null ...