Takefumi Shimoyama

Characterization of a filamentous biofilm community established in a cellulose-fed microbial fuel cell

BackgroundMicrobial fuel cells (MFCs) are devices that exploit microorganisms to generate electric power from organic matter. Despite the development of efficient MFC reactors, the microbiology of electricity generation remains to be sufficiently understood.ResultsA laboratory-scale two-chamber microbial fuel cell (MFC) was inoculated with rice paddy field soil and fed cellulose as the carbon and energy source. Electricity-generating microorganisms were enriched by subculturing biofilms that attached onto anode electrodes. An electric current of 0.2 mA was generated from the first enrichment culture, and ratios of the major metabolites (e.g., electric current, methane and acetate) became stable after the forth enrichment. In order to investigate the electrogenic microbial community in the anode biofilm, it was morphologically analyzed by electron microscopy, and community members were phylogenetically identified by 16S rRNA gene clone-library analyses. Electron microscopy revealed that filamentous cells and rod-shaped cells with prosthecae-like filamentous appendages were abundantly present in the biofilm. Filamentous cells and appendages were interconnected via thin filaments. The clone library analyses frequently detected phylotypes affiliated with Clostridiales, Chloroflexi, Rhizobiales and Methanobacterium. Fluorescence in-situ hybridization revealed that the Rhizobiales population represented rod-shaped cells with filamentous appendages and constituted over 30% of the total population.ConclusionBacteria affiliated with the Rhizobiales constituted the major population in the cellulose-fed MFC and exhibited unique morphology with filamentous appendages. They are considered to play important roles in the cellulose-degrading electrogenic community.

Flagellum Mediates Symbiosis

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.

The genome of Pelotomaculum thermopropionicum reveals niche-associated evolution in anaerobic microbiota

The anaerobic biodegradation of organic matter is accomplished by sequential syntrophic catabolism by microbes in different niches. Pelotomaculum thermopropionicum is a representative syntrophic bacterium that catalyzes the intermediate bottleneck step in the anaerobic-biodegradation process, whereby volatile fatty acids (VFAs) and alcohols produced by upstream fermenting bacteria are converted to acetate, hydrogen, and carbon dioxide (substrates for downstream methanogenic archaea). To reveal genomic features that contribute to our understanding of the ecological niche and evolution of P. thermopropionicum, we sequenced its 3,025,375-bp genome and performed comparative analyses with genomes of other community members available in the databases. In the genome, 2920 coding sequences (CDSs) were identified. These CDSs showed a distinct distribution pattern in the functional categories of the Clusters of Orthologous Groups database, which is considered to reflect the niche of this organism. P. thermopropionicum has simple catabolic pathways, in which the propionate-oxidizing methylmalonyl-CoA pathway constitutes the backbone and is linked to several peripheral pathways. Genes for most of the important catabolic enzymes are physically linked to those for PAS-domain-containing regulators, suggesting that the catabolic pathways are regulated in response to environmental conditions and/or global cellular situations rather than specific substrates. Comparative analyses of codon usages revealed close evolutionary relationships between P. thermopropionicum and other niche members, while it was distant from phylogenetically related sugar-fermenting bacteria. These analyses suggest that P. thermopropionicum has evolved as a syntrophy specialist by interacting with niche-associated microbes.

Cloning, expression, and characterization of cis-polyprenyl diphosphate synthase from the thermoacidophilic archaeon Sulfolobus acidocaldarius.

cis-polyprenyl diphosphate synthases are involved in the biosynthesis of the glycosyl carrier lipid in most organisms. However, only little is known about this enzyme of archaea. In this report, we isolated the gene of cis-polyprenyl diphosphate synthase from a thermoacidophilic archaeon, Sulfolobus acidocaldarius, and characterized the recombinant enzyme.

Molecular Biological Analysis of Microflora in a Garbage Treatment Process under Thermoacidophilic Conditions

In our efforts to solve problems associated with the treatment of garbage wastes, a novel, efficient process utilizing a small bioreactor equipped with a heating and an agitating apparatus was developed. The use of this process, which reduces and stabilizes garbage wastes, can be distinguished from other similar treatment processes that utilize similar equipment by its highly stable operation. This advantage led us to consider a characteristic microflora that would play an important role in the process. Thus, we analyzed the structure of the microflora in the process using molecular biological methods. The major microorganisms inhabiting the treatment environment were usually maintained for several weeks although garbage waste was added to the system each weekday. Moreover, surprisingly, lactic acid bacteria constituted a large majority in the microflorae in spite of the thermoacidophilic conditions in the reactor. These analyses permitted a better understanding of the mechanism of the process, especially of its stability.

Dysgonomonas oryzarvi sp. nov., isolated from a microbial fuel cell

A Gram-stain-negative, non-motile and coccoid- to short-rod-shaped bacterium, designated strain Dy73(T), was isolated from a microbial fuel cell that had been inoculated with rice paddy field soil and fed starch, peptone and fish extract as fuels. On the basis of 16S rRNA gene sequence phylogeny, strain Dy73(T) was affiliated with the genus Dysgonomonas in the phylum Bacteroidetes, and most closely related to Dysgonomonas mossii CCUG 43457(T) with a 16S rRNA gene sequence similarity value of 99.7 %. However, the DNA-DNA relatedness value between strain Dy73(T) and Dysgonomonas mossii CCUG 43457(T) was 34.8%. In addition, strain Dy73(T) was found to be different from other recognized species of the genus Dysgonomonas in taxonomically important traits, including habitat, DNA G+C content, bile resistance and fatty-acid composition. Based on these characteristics, strain Dy73(T) represents a novel species of the genus Dysgonomonas for which the name Dysgonomonas oryzarvi sp. nov. is proposed. The type strain is Dy73(T) ( = JCM 16859(T) = KCTC 5936(T)).

Ecophysiological consequences of alcoholism on human gut microbiota: Implications for ethanol-related pathogenesis of colon cancer

Chronic consumption of excess ethanol increases the risk of colorectal cancer. The pathogenesis of ethanol-related colorectal cancer (ER-CRC) is thought to be partly mediated by gut microbes. Specifically, bacteria in the colon and rectum convert ethanol to acetaldehyde (AcH), which is carcinogenic. However, the effects of chronic ethanol consumption on the human gut microbiome are poorly understood, and the role of gut microbes in the proposed AcH-mediated pathogenesis of ER-CRC remains to be elaborated. Here we analyse and compare the gut microbiota structures of non-alcoholics and alcoholics. The gut microbiotas of alcoholics were diminished in dominant obligate anaerobes (e.g., Bacteroides and Ruminococcus) and enriched in Streptococcus and other minor species. This alteration might be exacerbated by habitual smoking. These observations could at least partly be explained by the susceptibility of obligate anaerobes to reactive oxygen species, which are increased by chronic exposure of the gut mucosa to ethanol. The AcH productivity from ethanol was much lower in the faeces of alcoholic patients than in faeces of non-alcoholic subjects. The faecal phenotype of the alcoholics could be rationalised based on their gut microbiota structures and the ability of gut bacteria to accumulate AcH from ethanol.

Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of microbial community structure in landfill leachate.

The structures of microbial communities in water samples obtained from a landfill site that had been a source of environmental pollution by emitting hydrogen sulfide were elucidated using polymerase chain reaction-denaturing gradient gel electrophoresis. The microbial communities, which consisted of a limited number of major microorganisms, were stable for several months. Microorganisms capable of degrading such chemical compounds as 2-hydroxybenzothiazole and bisphenol A were observed in landfill leachate. Microorganisms responsible for the production of hydrogen sulfide were not the primary microbes detected, even in water samples obtained from the site of gas emission.

Paenibacillus relictisesami sp. nov., isolated from sesame oil cake.

A facultatively anaerobic, Gram-stain-positive, rod-shaped bacterium, designated strain KB0549T, was isolated from sesame oil cake. Cells were motile, round-ended rods, and produced central or terminal spores. The cell wall peptidoglycan contained meso-diaminopimelic acid as the diamino acid. The major fatty acids were anteiso-C15:0 and anteiso-C17:0. The DNA G+C content of strain KB0549T was 51.9 mol%. On the basis of 16S rRNA gene sequence phylogeny, strain KB0549T was affiliated with the genus Paenibacillus in the phylum Firmicutes and was most closely related to Paenibacillus cookii with 97.4% sequence similarity. Strain KB0549T was physiologically differentiated from P. cookii by the high content of anteiso-C17:0, inability to grow at 50 °C, spore position, and negative Voges-Proskauer reaction. Based on these unique physiological and phylogenetic characteristics, it is proposed that the isolate represents a novel species, Paenibacillus relictisesami sp. nov.; the type strain is KB0549T (=JCM 18068T=DSM 25385T).

Purification, Gene Cloning, and Biochemical Characterization of a β-Glucosidase Capable of Hydrolyzing Sesaminol Triglucoside from Paenibacillus sp. KB0549

The triglucoside of sesaminol, i.e., 2,6-O-di(β-D-glucopyranosyl)-β-D- glucopyranosylsesaminol (STG), occurs abundantly in sesame seeds and sesame oil cake and serves as an inexpensive source for the industrial production of sesaminol, an anti-oxidant that displays a number of bioactivities beneficial to human health. However, STG has been shown to be highly resistant to the action of β-glucosidases, in part due to its branched-chain glycon structure, and these circumstances hampered the efficient utilization of STG. We found that a strain (KB0549) of the genus Paenibacillus produced a novel enzyme capable of efficiently hydrolyzing STG. This enzyme, termed PSTG, was a tetrameric protein consisting of identical subunits with an approximate molecular mass of 80 kDa. The PSTG gene was cloned on the basis of the partial amino acid sequences of the purified enzyme. Sequence comparison showed that the enzyme belonged to the glycoside hydrolase family 3, with significant similarities to the Paenibacillus glucocerebrosidase (63% identity) and to Bgl3B of Thermotoga neapolitana (37% identity). The recombinant enzyme (rPSTG) was highly specific for β-glucosidic linkage, and k cat and k cat/K m values for the rPSTG-catalyzed hydrolysis of p-nitrophenyl-β-glucopyraniside at 37°C and pH 6.5 were 44 s−1 and 426 s−1 mM−1, respectively. The specificity analyses also revealed that the enzyme acted more efficiently on sophorose than on cellobiose and gentiobiose. Thus, rPSTG is the first example of a β-glucosidase with higher reactivity for β-1,2-glucosidic linkage than for β-1,4- and β-1,6-glucosidic linkages, as far as could be ascertained. This unique specificity is, at least in part, responsible for the enzyme’s ability to efficiently decompose STG.