Masayuki Ikarashi

Evidence for syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis in the high-temperature petroleum reservoir of Yabase oil field (Japan).

The methanogenic communities and pathways in a high-temperature petroleum reservoir were investigated through incubations of the production water and crude oil, combined with radiotracer experiments and molecular biological analyses. The incubations were conducted without any substrate amendment and under high-temperature and pressurized conditions that mimicked the in situ environment (55°C, 5 MPa). Changes in methane and acetate concentrations during the incubations indicated stoichiometric production of methane from acetate. Rates of hydrogenotrophic methanogenesis measured using [(14)C]-bicarbonate were 42-68 times those of acetoclastic methanogenesis measured using [2-(14) C]-acetate, implying the dominance of methane production by syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis in the environment. 16S rRNA gene sequence analyses of the incubated production water showed bacterial communities dominated by the genus Thermacetogenium, known as a thermophilic syntrophic acetate-oxidizing bacterium, and archaeal communities dominated by thermophilic hydrogenotrophic methanogens belonging to the genus Methanothermobacter. Furthermore, group-specific real-time PCR assays revealed that 16S rRNA gene copy numbers of the hydrogenotrophic methanogens affiliated with the order Methanobacteriales were almost identical to those of archaeal 16S rRNA genes. This study demonstrates that syntrophic acetate oxidation is the main methanogenic pathway in a high-temperature petroleum reservoir.

Carbon dioxide concentration dictates alternative methanogenic pathways in oil reservoirs

Deep subsurface formations (for example, high-temperature oil reservoirs) are candidate sites for carbon capture and storage technology. However, very little is known about how the subsurface microbial community would respond to an increase in CO2 pressure resulting from carbon capture and storage. Here we construct microcosms mimicking reservoir conditions (55 °C, 5 MPa) using high-temperature oil reservoir samples. Methanogenesis occurs under both high and low CO2 conditions in the microcosms. However, the increase in CO2 pressure accelerates the rate of methanogenesis to more than twice than that under low CO2 conditions. Isotope tracer and molecular analyses show that high CO2 conditions invoke acetoclastic methanogenesis in place of syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis that typically occurs in this environment (low CO2 conditions). Our results present a possibility of carbon capture and storage for enhanced microbial energy production in deep subsurface environments that can mitigate global warming and energy depletion.

Phylogenetic diversity of microbial communities associated with the crude-oil, large-insoluble-particle and formation-water components of the reservoir fluid from a non-flooded high-temperature petroleum reservoir

The diversity of microbial communities associated with non-water-flooded high-temperature reservoir of the Niibori oilfield was characterized. Analysis of saturated hydrocarbons revealed that n-alkanes in crude oil from the reservoir were selectively depleted, suggesting that crude oil might be mildly biodegraded in the reservoir. To examine if any specific microorganism(s) preferentially attached to the crude oil or the other components (large insoluble particles and formation water) of the reservoir fluid, 16S rRNA gene clone libraries were constructed from each component of the reservoir fluid. The clones in the archaeal libraries (414 clones in total) represented 16 phylotypes, many of which were closely related to methanogens. The bacterial libraries (700 clones in total) were composed of 49 phylotypes belonging to one of 16 phylum-level groupings, with Firmicutes containing the greatest diversity of the phylotypes. In the crude-oil- and large-insoluble-particle-associated communities, a Methanosaeta-related phylotype dominated the archaeal sequences, whereas hydrogenotrophic methanogens occupied a major portion of sequences in the library of the formation-water-associated community. The crude-oil associated bacterial community showed the largest diversity, containing 35 phylotypes, 16 of which were not detected in the other bacterial communities. Thus, although the populations associated with the reservoir-fluid components largely shared common phylogenetic context, a specific fraction of microbial species preferentially attached to the crude oil and insoluble particles.

Analysis of methane production by microorganisms indigenous to a depleted oil reservoir for application in Microbial Enhanced Oil Recovery

We examined methane production by microorganisms collected from a depleted oilfield. Our results indicated that microorganisms indigenous to the petroleum reservoir could effectively utilize yeast extract, suggesting that the indigenous microorganisms and proteinaceous nutrients could be recruitable for Microbially Enhanced Oil Recovery.

Petrothermobacter organivorans gen. nov., sp. nov., a thermophilic, strictly anaerobic bacterium of the phylum Deferribacteres isolated from a deep subsurface oil reservoir

A novel thermophilic, anaerobic, chemoheterotrophic, acetate-oxidizing and iron(III)-, manganese(IV)-, nitrate- and sulfate-reducing bacterium, designated strain ANAT, was isolated from a deep subsurface oil field in Japan (Yabase oil field, Akita Pref.). Cells of strain ANAT were Gram-stain-negative, non-motile, non-spore forming and slightly curved or twisted rods (1.5-5.0 µm long and 0.6-0.7 µm wide). The isolate grew at 25-60 °C (optimum 55 °C) and pH 6.0-8.0 (optimum pH 7.0). The isolate was capable of reducing iron(III), manganese(IV), nitrate and sulfate as an electron acceptor. The isolate utilized a limited range of electron donors such as acetate, lactate, pyruvate and yeast extract for iron reduction. Strain ANAT also used pyruvate, fumarate, succinate, malate, yeast extract and peptone for fermentative growth. The major respiratory quinones were menaquinone-7(H8) and menaquinone-8. The strain contained C18 : 0, iso-C18 : 0 and C16 : 0 as the major cellular fatty acids. The G+C content of the genomic DNA was 34.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ANAT was closely related to Calditerrivibrio nitroreducens in the phylum Deferribacteres with low sequence similarities (89.5 %), and formed a distinct clade within the family Deferribacteraceae. In addition, the isolate is the first sulfate-reducing member of the phylum Deferribacteres. Based on phenotypic, chemotaxonomic and phylogenetic properties, a novel genus and species, Petrothermobacter organivorans gen. nov., sp. nov., is proposed for the isolate (type strain=ANAT= NBRC 112621T=DSM 105015T).