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Groundwater sampling is a critical step in subsurface microbial ecology. Here, we compared two different sampling methods: commonly used disposable bailers (unimproved sampler) and an impro...

While meteoric water recharge is known to stimulate biogenic methane formation in shale and coal seams, the underlying mechanisms are currently unresolved. To this end, we conducted fieldwork in the Tempoku Coal Field, Japan. Pore water in core samples and well water in boreholes were analyzed for dissolved components and isotopic compositions. The hydraulic gradient was determined using values of borehole hydraulic head. Cl− concentrations (38 mg L−1 to 16,600 mg L−1), δ18O(H2O) values (−10.5‰ to −2.4‰), and δD(H2O) values (−71.6‰ to −17.8‰) increased with depth (

A novel mesophilic sulfate-reducing bacterium, strain HN2T, was isolated from groundwater sampled from the subsurface siliceous mudstone of the Wakkanai Formation located in Horonobe, Hokkaido, Japan. The bacterium was Gram-negative and vibrio-shaped, and its motility was conferred by a single polar flagellum. Cells had desulfoviridin. Catalase and oxidase activities were not detected. It grew in the temperature range of 25–40 °C (optimum, 35 °C) and pH range of 6.3–8.1 (optimum, pH 7.2–7.6). It used sulfate, thiosulfate, dimethyl sulfoxide, anthraquinone-2,6-disulfonate, Fe3+, and manganese oxide, but not elemental sulfur, nitrite, nitrate, or fumarate as electron acceptors. The strain showed weak growth with sulfite as the electron acceptor. Fermentative growth with pyruvate, lactate and cysteine was observed in the absence of sulfate, but not with malate or fumarate. NaCl was not required, but the strain tolerated up to 40 g l–1. Strain HN2T did not require vitamins. The major cellular fatty acids were iso-C15 : 0 (23.8 %), C18 : 1 ω9t (18.4 %), C18 : 0 (15.0 %), C16 : 0 (14.5 %), and anteiso-C17 :0 (10.1 %). The major respiratory quinone was menaquinone MK-6(H2). The G+C content of the genomic DNA was 56.7 mol%. Based on 16S rRNA gene sequence analysis, the closest phylogenetic relative of strain HN2T is Desulfovibrio psychrotolerans JS1T (97.0 %). Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values of the strains HN2T and D. psychrotolerans JS1T were 22.2 and 79.8 %, respectively. Based on the phenotypic and molecular genetic evidence, we propose a novel species, D. subterraneus sp. nov. with the type strain HN2T (=DSM 101010T=NBRC 112213T).

A microbial consortium associated with coal from the coal-bearing Soya Formation in the Tempoku Coalfield (northern Hokkaido, Japan) was cultivated with reaction solutions of lignite and hydrogen peroxide (H2O2) (i.e., chemically solubilized lignite) to evaluate in situ biogenic methane generation. Column experiments with H2O2 flowing through pulverized lignite achieved maximum concentrations of dissolved organic carbon, acetic acid, and formic acid of 6330, 612, and 1810 mg/L, respectively. Cultivation experiments using the above reaction solution as a substrate for methanogens produced nearly 6 cm3 CH4 per g lignite with a maximum rate of 0.14 cm3 per g per day. Pyrosequencing analysis of the microbial consortium after cultivation confirmed the presence of methanogens, such as Methanoregulaceae and Methanosarcinaceae, as the dominant archaea in the culture. The observed production of biogenic methane from a reaction solution of lignite and H2O2 under the simulated conditions indicates the potential for successful in situ microbially enhanced coalbed methane generation using H2O2.

Future world energy consumption is projected to increase along with the high economic growth of emerging countries. This will make it necessary to expand the development of unconventional resources such as shale oil and gas and coalbed methane. The production of secondary methane gas using microorganisms at oil fields and coal mines is another area of great interest in the development of unconventional resources. This paper describes a feasibility study involving a new method to convert unused organic matter contained in sedimentary rock in northern Hokkaido into biogenic methane. The new method, subsurface cultivation and gasification (SCG), is proposed for the production of biogenic methane gas in the subsurface environment. Our approach uses hydrogen peroxide (H2O2) to decompose organic matter rapidly into usable substrates for methanogens. Two wells were drilled through several lignite seams in the Soya coal-bearing formations in the Tempoku coalfield in northern Hokkaido, Japan, and lignite cores were recovered. Batch tests using a 1 wt% H2O2 solution were performed on the lignite to estimate the amount of low-molecular-weight organic acids as substrates for methanogens. Oxidative decomposition of the lignite produced a high yield of low-molecular-weight organic acids. Moreover, it was confirmed that methanogens converted the H2O2 reaction solution containing organic acids into methane. H2O2 would be useful for effective SCG at lignite seams, and conversion of organic matter from lignite into biogenic methane with the help of microorganisms is expected to be highly profitable. The results reveal that using SCG to produce biogenic methane in this coalfield shows great commercial promise.

Geobacter spp., regarded as strict anaerobes, have been reported to grow under aerobic conditions. To elucidate the role of fatty acids in aerobiosis of Geobacter spp., we studied the effect of aerobiosis on fatty acid composition and turnover in G. bemidjiensis BemT. G. bemidjiensis BemT was grown under the following different culture conditions: anaerobic culture for 4 days (type 1) and type 1 culture followed by 2-day anaerobic (type 2) or aerobic culture (anaerobic-to-aerobic shift; type 3). The mean cell weight of the type 3 culture was approximately 2.5-fold greater than that of type 1 and 2 cultures. The fatty acid methyl ester and hydrocarbon fraction contained hexadecanoic (16:0), 9-cis-hexadecenoic [16:1(9c)], tetradecanoic (14:0), tetradecenoic [14:1(7c)] acids, hentriacontanonaene, and hopanoids, but not long-chain polyunsaturated fatty acids. The type 3 culture contained higher levels of 14:0 and 14:1(7c) and lower levels of 16:0 and 16:1(9c) compared with type 1 and 2 cultures. The weight ratio of extracted lipid per dry cell was lower in the type 3 culture than in the type 1 and 2 cultures. We concluded that anaerobically-grown G. bemidjiensis BemT followed by aerobiosis were enhanced in growth, fatty acid turnover, and de novo fatty acid synthesis.

Since the beginning of the 21st century, various works have sought to enhance coalbed methane (CBM) production by stimulating indigenous microbes in coal seams. This concept of microbially enhanced coalbed methane (MECBM) generation relies greatly on the solubilization of the coal, which determines the rate of microbial conversion of the coal into biogenic methane. This study examined the potential of using dilute hydrogen peroxide (H2O2) (0.3%) to solubilize lignite at common subsurface temperatures (10–50 °C) via batch experiments at a solid/liquid ratio of 1 g/150 mL. Dissolved organic carbon (maximum 450 mg C/L) and organic acid (formic, acetic, oxalic, malonic, and succinic acid, maximum total of 145 mg C/L) concentrations reached their maximum levels within periods of 1.2 days (at 50 °C) to 45 days (at 10 °C). H2O2 consumption was a first-order reaction with respect to its concentration, and the linear (r = 0.998) Arrhenius plot for the rate constants yielded its activation energy to be 73 kJ/mol. If the methanogens convert acetic and formic acids to methane, under the current experimental conditions they can produce about 6.0 m3 of methane per ton of lignite. Dilute H2O2 would thus be useful for effective MECBM production at lignite seams. The aromatic-C content of the lignite markedly decreased by about 30% during the reaction, implying high reactivity of catechol structures in the lignite with H2O2.

We have characterized a member of the profilin (PROF) family protein as a common antigen in three pathogens-Babesia bovis (B. bovis), Babesia bigemina (B. bigemina), and Babesia microti (B. microti)-and evaluated its immunogenic and cross-protective properties against a challenge infection with B. microti in BALB/c mice. The recombinant PROF proteins of B. bovis, B. bigemina, and B. microti were successfully expressed in Escherichia coli (E. coli) as soluble GST fusion proteins (rBboPROF, rBbigPROF, and rBmPROF, respectively), and they were found to be antigenic. On probing with mouse anti-rPROF serum, green fluorescence was observed on the parasites' cytosols by confocal laser microscopy. Immunization regimes in BALB/c mice using rPROFs induced cross-protective immunity against B. microti infection based on high levels of cytokines and immunoglobulin (IgG) titers, a reduction in peak parasitemia levels, and earlier clearance of the parasite as compared with control mice. The findings of the present study indicate that PROF is a common antigen among bovine and murine Babesia parasites, and it might be used as a common vaccine candidate against babesiosis.

Kushiro Coalfield is one of the largest coalfields in Japan, and Kushiro Coal Mine (KCM) is currently the only underground coal mine in the country. At KCM, coalbed methane (CBM) has been extracted from the Eocene Harutori Formation and water-dissolved gas from the Paleocene Shiomi Formation. This study investigates the origin of these gases and their formation environments. A positive correlation between δD(CH4) of the sampled gas and δD(H2O) of the groundwater, following δD(CH4) = δD(H2O) − 180 ± 20, indicates that CBM and water-dissolved gas in KCM are classified as secondary biogenic methane and were formed within in situ groundwater. The lower δ13C(CO2) values (less than −6.9‰) from KCM compared with representative CBM of biogenic origin from other coalfields imply the contribution of oxidants (e.g., SO42−) in the biodegradation of sedimentary organic matter (SOM) to CO2 in KCM. Conversely, high δ13C(CaCO3) values (>12.5‰) of calcite deposited in the coal seams of KCM imply negligible contributions of oxidants for SOM biodegradation when the calcite was formed. The methanogenic conditions at the time of calcite formation may have arisen when the rate of groundwater flow in the coal seams was higher than at present.

The effect of oxidative weathering on the composition of sedimentary organic matter (SOM) of lignite, subbituminous coal, quartzose arenite, siliceous mudstone, siliceous shale and diatomaceous shale was investigated in Hokkaido, Japan, in a subarctic humid climate. For 15 samples in total, a humic substance extraction method was applied to divide SOM into lipid-C, humin-C and alkali soluble-C. The alkali soluble-C was further divided into fulvic acid (FA)-C, humic acid (HA)-C and a smaller molecular size fraction (SMSF)-C, which comprised organic molecules able to pass through a dialysis membrane (

application/pdf, Oocyst stage of Toxoplasma gondii is characterized by a durable wall that confers a strong protection to this protozoan parasite in face of harsh environmental conditions. Thus, it is considered the key for transmission of T. gondii. Analysis of oocyst wall composition is mandatory therefore; the aim of this study was to identify novel T. gondii oocyst wall proteins and test their use in detection of these oocysts in environmental samples. Five candidates of novel T. gondii oocyst wall proteins (TgOWPs) were identified and named TgOWP8 through TgOWP12. Recombinant protein of TgOWP8 was expressed in E. coli using glutathione S-transferase as fusion protein. Polyclonal antibody was produced and validated by indirect immunofluorescence antibody assay (IFA). For detection by IFA, we used different methods for fixation and permeabilization of oocysts to improve the antigen-antibody detection. Specificity to wall of T. gondii oocyst was confirmed and revealed absence of cross reactivity with bradyzoite cyst wall and tachyzoites. Although some TgOWPs were identified previously, our study represents a continuation of molecular investigations of oocyst wall proteins as an essential structure for the longevity and infectivity of this stage and also provided new trial to improve T. gondii oocysts detection. c 2017 The Authors

Geobacter spp., regarded as strict anaerobes, have been reported to grow under aerobic conditions. To elucidate the role of fatty acids in aerobiosis of Geobacter spp., we studied the effect of aerobiosis on fatty acid composition and turnover in G. bemidjiensis Bem

Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of 14C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth’s subsurface.

A methanogenic archaeon, strain HC-2T, was isolated from a deep diatomaceous shale formation. The strain grew on methanol, monomethylamine, dimethylamine, trimethylamine and dimethylsulphide, but not on acetate, H2/CO2, formate, 2-propanol, 2-butanol or cyclopentanol. Cells were Gram-stain-negative, non-motile, and coccus-like, 0.9–1.4 µm in diameter, and occurred singly, in pairs, or as aggregates. The strain grew at 10–40 °C (optimum 35 °C), pH 5.9–7.4 (optimum pH 6.6–6.8) and in 0–0.6 M NaCl (optimum 0.1–0.2 M). The genomic DNA G+C content was 41.5 mol% and the 16S rRNA gene sequence was closely related to those of Methanosarcina lacustris DSM 13486T (99.1 %) and Methanosarcina siciliae DSM 3028T (98.3 %). Values for DNA–DNA hybridization with these strains were less than 30 %. The phenotypic and phylogenetic features of HC-2T indicate that it represents a novel species of the genus Methanosarcina , for which the name Methanosarcina subterranea sp. nov. is proposed. The type strain is HC-2T ( = DSM 22503T = JCM 15540T = NBRC 102578T).

application/pdf, The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.