Mio Takeuchi

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.

Rate determination and distribution of anammox activity in activated sludge treating swine wastewater.

This paper presents a quantitative investigation and analysis of anammox activity in sludge taken from biological swine wastewater treatment plants. An incubation experiment using a (15)N tracer technique showed anammox activity in sludge taken from 6 out of 13 plants with the rate ranging from 0.0036 micromol-N(2)/g-VSS/h to 3.1 micromol-N(2)/g-VSS/h, and in a biofilm with the highest activity at 25.8 micromol-N(2)/g-VSS/h. It is notable that 9 out of 11 sludges in which the pH was maintained between 6.6 and 8.1 retained anammox activity, while those with either a lower or higher pH did not. Moreover, anammox-positive sludge had a significantly higher concentration of NO(2)(-)-N plus NO(3)(-)-N than did anammox-negative sludge. A significant difference was not observed between anammox-positive and -negative sludge regarding BOD/NH(4)(+)-N in the influent, DO concentration in aeration tanks, and the concentrations of NH(4)(+)-N, free nitric acid, and free ammonia.

Methylocaldum marinum sp. nov., a thermotolerant, methane-oxidizing bacterium isolated from marine sediments, and emended description of the genus Methylocaldum.

An aerobic, methane-oxidizing bacterium (strain S8(T)) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain is closely related to members of the genus Methylocaldum (97.6-97.9 % similarity) within the class Gammaproteobacteria. Strain S8(T) was a Gram-staining-negative, non-motile, coccoid or short rod-shaped organism. The temperature range for growth of strain S8(T) was 20-47 °C (optimum growth at 36 °C). It required NaCl (>0.5 %), tolerated up to 5 % NaCl and utilized methane and methanol. The major cellular fatty acid and major respiratory quinone were C16 : 0 and 18-methylene ubiquinone 8, respectively. The DNA G+C content was 59.7 mol%. Strain S8(T) possessed mmoX, which encodes soluble methane monooxygenase, as well as pmoA, which encodes the particulate methane monooxygenase. On the basis of this morphological, physiological, biochemical and genetic information, the first marine species in the genus Methylocaldum is proposed, with the name Methylocaldum marinum sp. nov. The type strain is S8(T) ( = NBRC 109686(T) = DSM 27392(T)). An emended description of the genus Methylocaldum is also provided.

Bacterial and Archaeal 16S rRNA Genes in Late Pleistocene to Holocene Muddy Sediments from the Kanto Plain of Japan

Microbial communities in ancient marine sediments composed of clay and silt obtained from the terrestrial subsurface were phylogenetically analyzed based on their 16S rRNA gene sequences. Chloroflexi and Miscellaneous Crenarchaeotic Group were predominant in bacterial and archaeal clone libraries, respectively. Of 44 operational taxonomic units (OTUs) that had close relatives in the database, 30 were close to sequences obtained from marine environments. Some sequences belonged to the candidate groups JS1, ANME-I, and Marine Benthic Group-C, which are typically found in marine sediments. Low chloride concentrations in the sediments suggest that these marine-affiliated sequences may not reflect currently active microbial communities. Our results indicate the existence of long-term preserved DNA or descendants of ancient oceanic microbial components in subsurface muddy sediments in a temperate region, which may reflect indigenous population of paleoenvironments.

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.

Comparative study of microbial dechlorination of chlorinated ethenes in an aquifer and a clayey aquitard.

In order to determine whether natural attenuation of chlorinated ethenes by microbial activity occurs in aquitards, sediments at a site contaminated with tetrachloroethene were vertically studied by drilling. The distribution of microbes (Dehalococcoides group and anaerobic hydrogen producers) and the ability of the sediments to sustain microbial dechlorination were determined in an aquitard as well as in an aquifer. Close-spaced sampling revealed the existence of large populations of Dehalococcoides and H(2)-producing bacteria, especially in the organic-rich clayey aquitard rather than in the aquifer. The vinyl chloride reductase gene was also detected in the clay layer. Furthermore, incubation experiments indicated that the clay sediment could sustain transformations of tetrachloroethene at least to vinyl chloride. In contrast, no significant transformation was observed in the aquifer sand. Our results indicate that dechlorination of tetrachloroethene by bacteria can take place in an organic-rich clayey aquitard, and that organic-rich clay may also be important in the natural attenuation in an adjacent aquifer, possibly supplying a carbon source or an electron donor.

Chemistry of fly ash and cyclone ash leachate from waste materials and effects of ash leachates on bacterial growth, nitrogen-transformation activity, and metal accumulation.

The effects of waste ash leachates on soil microorganism were evaluated along with a chemical characterization of ash leachates. Thirty fly ash samples and cyclone ash samples obtained from the incineration of municipal solid waste, plastic waste, and construction waste were used. Twenty-one and 22 samples inhibited N transformation activity of soil microorganism and growth of Bacillus subtilis, respectively. On the other hand, 11 and 18 samples stimulated bacterial activity and growth, respectively, at low concentrations. Generally, cyclone ash contained a smaller amount of toxic metals than fly ash. Our results suggest that cyclone ash can be further studied for reuse, perhaps as a soil amendment. Pb was found to be highly accumulated in B. subtilis cells, and should be carefully monitored when waste ash is reused in the environment.

Tepidicaulis marinus gen. nov., sp. nov., a marine bacterium that reduces nitrate to nitrous oxide under strictly microaerobic conditions

A moderately thermophilic, aerobic, stalked bacterium (strain MA2T) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain MA2T was most closely related to the genera Rhodobium,Parvibaculum, and Rhodoligotrophos (92-93 % similarity) within the class Alphaproteobacteria. Strain MA2T was a Gram-stain-negative and stalked dimorphic bacteria. The temperature range for growth was 16-48 °C (optimum growth at 42 °C). This strain required yeast extract and NaCl (>1 %, w/v) for growth, tolerated up to 11 % (w/v) NaCl, and was capable of utilizing various carbon sources. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The DNA G+C content was 60.7 mol%. Strain MA2T performed denitrification and produced N2O from nitrate under strictly microaerobic conditions. Strain MA2T possessed periplasmic nitrate reductase (Nap) genes but not membrane-bound nitrate reductase (Nar) genes. On the basis of this morphological, physiological, biochemical and genetic information a novel genus and species, Tepidicaulis marinus gen. nov., sp. nov., are proposed, with MA2T ( = NBRC 109643T = DSM 27167T) as the type strain of the species.

Microbial community structure in deep natural gas-bearing aquifers subjected to sulfate-containing fluid injection

In the natural gas field located in central Japan, high concentrations of natural gases and iodide ions are dissolved in formation water and commercially produced in deep aquifers. In the iodine recovery process, the produced formation water is amended with sulfate, and this fluid is injected into gas-bearing aquifers, which may lead to infrastructure corrosion by hydrogen sulfide. In this study, we examined the microbial community in aquifers subjected to sulfate-containing fluid injection. Formation water samples were collected from production wells located at different distances from the injection wells. The chemical analysis showed that the injection fluid contained oxygen, nitrate, nitrite and sulfate, in contrast to the formation water, which had previously been shown to be depleted in these components. Sulfur isotopic analysis indicated that sulfate derived from the injection fluid was present in the sample collected from near the injection wells. Quantitative and sequencing analysis of dissimilatory sulfite reductase and 16S rRNA genes revealed that sulfate-reducing bacteria (SRB), sulfur-oxidizing bacteria, and anaerobic methanotrophic archaea (ANME) in the wells located near injection wells were more abundant than those in wells located far from the injection wells, suggesting that fluid injection stimulated these microorganisms through the addition of oxygen, nitrate, nitrite and sulfate to the methane-rich aquifers. The predominant taxa were assigned to the ANME-2 group, its sulfate-reducing partner SEEP-SRB1 cluster and sulfur-oxidizing Epsilonproteobacteria. These results provide important insights for future studies to support the development of natural gas and iodine resources in Japan.