Daisuke Mayumi

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.

Methane production from coal by a single methanogen.

Microbes make methane from coal Methane associated with coal beds is an important global resource of natural gas. Much of the methane in coal comes from microbial methanogenesis. Mayumi et al. characterized a strain of Methermicoccus shengliensis that, unexpectedly, is capable of making methane from the dozens of methoxylated aromatic compounds found in a variety of coal types (see the Perspective by Welte). Isotope tracer experiments showed that this organism could also incorporate carbon dioxide into methane. Science, this issue p. 222; see also p. 184 Microbes can produce methane directly from the complex aromatic compounds found in a variety of coal substrates. Coal-bed methane is one of the largest unconventional natural gas resources. Although microbial activity may greatly contribute to coal-bed methane formation, it is unclear whether the complex aromatic organic compounds present in coal can be used for methanogenesis. We show that deep subsurface–derived Methermicoccus methanogens can produce methane from more than 30 types of methoxylated aromatic compounds (MACs) as well as from coals containing MACs. In contrast to known methanogenesis pathways involving one- and two-carbon compounds, this “methoxydotrophic” mode of methanogenesis couples O-demethylation, CO2 reduction, and possibly acetyl–coenzyme A metabolism. Because MACs derived from lignin may occur widely in subsurface sediments, methoxydotrophic methanogenesis would play an important role in the formation of natural gas not limited to coal-bed methane and in the global carbon cycle.

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.

A distinct freshwater‐adapted subgroup of ANME‐1 dominates active archaeal communities in terrestrial subsurfaces in Japan

Anaerobic methane-oxidizing archaea (ANME) are known to play an important role in methane flux, especially in marine sediments. The 16S rRNA genes of ANME have been detected in terrestrial freshwater subsurfaces. However, it is unclear whether ANME are actively involved in methane oxidation in these environments. To address this issue, Holocene sediments in the subsurface of the Kanto Plain in Japan were collected for biogeochemical and molecular analysis. The potential activity of the anaerobic oxidation of methane (AOM) (0.38-3.54 nmol cm⁻³ day⁻¹) was detected in sediment slurry incubation experiments with a (13) CH(4) tracer. Higher AOM activity was observed in low-salinity treatment compared with high-salinity condition (20‰), which supports the adaptation of ANME in freshwater habitats. The 16S rRNA sequence analysis clearly revealed the presence of a distinct subgroup of ANME-1, designated ANME-1a-FW. Phylogenetic analysis of the mcrA genes also implied the presence of the distinct subgroup in ANME-1. ANME-1a-FW was found to be the most dominant active group in the archaeal communities on the basis of 16S rRNA analysis (75.0-93.8% of total archaeal 16S rRNA clones). Sulfate-reducing bacteria previously known as the syntrophic bacterial partners of ANME-1 was not detected. Our results showed that ANME-1a-FW is adapted to freshwater habitats and is responsible for AOM in terrestrial freshwater subsurface environments.

Identification of active and taxonomically diverse 1,4-dioxane degraders in a full-scale activated sludge system by high-sensitivity stable isotope probing

Abstract1,4-Dioxane is one of the most common and persistent artificial pollutants in petrochemical industrial wastewaters and chlorinated solvent groundwater plumes. Despite its possible biological treatment in natural environments, the identity and dynamics of the microorganisms involved are largely unknown. Here, we identified active and diverse 1,4-dioxane-degrading microorganisms from activated sludge by high-sensitivity stable isotope probing of rRNA. By rigorously analyzing 16S rRNA molecules in RNA density fractions of 13C-labeled and unlabeled 1,4-dioxane treatments, we discovered 10 significantly 13C-incorporating microbial species from the complex microbial community. 16S rRNA expression assays revealed that 9 of the 10 species, including the well-known degrader Pseudonocardia dioxanivorans, an ammonia-oxidizing bacterium and phylogenetically novel bacteria, increased their metabolic activities shortly after exposure to 1,4-dioxane. Moreover, high-resolution monitoring showed that, during a single year of operation of the full-scale activated sludge system, the nine identified species exhibited yearly averaged relative abundances of 0.001–1.523%, and yet showed different responses to changes in the 1,4-dioxane removal efficiency. Hence, the co-existence and individually distinct dynamics of various 1,4-dioxane-degrading microorganisms, including hitherto unidentified species, played pivotal roles in the maintenance of the biological system removing the recalcitrant pollutant.

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).