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1.
ISME J ; 15(12): 3549-3565, 2021 12.
Article in English | MEDLINE | ID: mdl-34145392

ABSTRACT

Methane-generating archaea drive the final step in anaerobic organic compound mineralization and dictate the carbon flow of Earth's diverse anoxic ecosystems in the absence of inorganic electron acceptors. Although such Archaea were presumed to be restricted to life on simple compounds like hydrogen (H2), acetate or methanol, an archaeon, Methermicoccus shengliensis, was recently found to convert methoxylated aromatic compounds to methane. Methoxylated aromatic compounds are important components of lignin and coal, and are present in most subsurface sediments. Despite the novelty of such a methoxydotrophic archaeon its metabolism has not yet been explored. In this study, transcriptomics and proteomics reveal that under methoxydotrophic growth M. shengliensis expresses an O-demethylation/methyltransferase system related to the one used by acetogenic bacteria. Enzymatic assays provide evidence for a two step-mechanisms in which the methyl-group from the methoxy compound is (1) transferred on cobalamin and (2) further transferred on the C1-carrier tetrahydromethanopterin, a mechanism distinct from conventional methanogenic methyl-transfer systems which use coenzyme M as final acceptor. We further hypothesize that this likely leads to an atypical use of the methanogenesis pathway that derives cellular energy from methyl transfer (Mtr) rather than electron transfer (F420H2 re-oxidation) as found for methylotrophic methanogenesis.


Subject(s)
Euryarchaeota , Methane/metabolism , Methyltransferases , Euryarchaeota/enzymology , Euryarchaeota/genetics , Methyltransferases/genetics
2.
Syst Appl Microbiol ; 44(1): 126154, 2021 Jan.
Article in English | MEDLINE | ID: mdl-33227632

ABSTRACT

An anaerobic thermophilic, rod-shaped bacterium possessing a unique non-lipid sheathed-like structure enveloping a single-membraned cell, designated strain NRmbB1T was isolated from at the deep subsurface oil field located in Yamagata Prefecture, Japan. Growth occurred with 40-60°C (optimum, 55°C), 0-2% (2%), NaCl and pH 6.0-8.5 (8.0). Fermentative growth with various sugars was observed. Glucose-grown cells generated acetate, hydrogen, pyruvate and lactate as the main end products. Syntrophic growth occurred with glucose, pyruvate and 3,4,5-trimethoxybenzoate in the presence of an H2-scavenging partner, and growth on 3,4,5-trimethoxybenzoate was only observed under syntrophic condition. The predominant cellular fatty acids were C16:0, iso-C16:0, anteiso-C15:0, and iso-C14:0. Respiratory quinone was not detected. The genomic G+C content was 40.8mol%. Based on 16S rRNA gene phylogeny, strain NRmbB1T belongs to a distinct order-level clade in the class Clostridia of the phylum Firmicutes, sharing low similarity with other isolated organisms (i.e., 87.5% for top hit Moorella thermoacetica DSM 2955T). In total, chemotaxonomic, phylogenetic and genomic characterization revealed that strain NRmbB1T (=KCTC 25035T, =JCM 39120T) represents a novel species of a new genus. In addition, we also propose the associated family and order as Koleobacteraceae fam. nov and Koleobacterales ord. nov., respectively.


Subject(s)
Clostridiales/classification , Oil and Gas Fields/microbiology , Phylogeny , Bacterial Typing Techniques , Base Composition , Clostridiales/isolation & purification , DNA, Bacterial/genetics , Fatty Acids/chemistry , Japan , Sequence Analysis, DNA
3.
Nat Commun ; 11(1): 5825, 2020 11 17.
Article in English | MEDLINE | ID: mdl-33203858

ABSTRACT

At marine cold seeps, gaseous and liquid hydrocarbons migrate from deep subsurface origins to the sediment-water interface. Cold seep sediments are known to host taxonomically diverse microorganisms, but little is known about their metabolic potential and depth distribution in relation to hydrocarbon and electron acceptor availability. Here we combined geophysical, geochemical, metagenomic and metabolomic measurements to profile microbial activities at a newly discovered cold seep in the deep sea. Metagenomic profiling revealed compositional and functional differentiation between near-surface sediments and deeper subsurface layers. In both sulfate-rich and sulfate-depleted depths, various archaeal and bacterial community members are actively oxidizing thermogenic hydrocarbons anaerobically. Depth distributions of hydrocarbon-oxidizing archaea revealed that they are not necessarily associated with sulfate reduction, which is especially surprising for anaerobic ethane and butane oxidizers. Overall, these findings link subseafloor microbiomes to various biochemical mechanisms for the anaerobic degradation of deeply-sourced thermogenic hydrocarbons.


Subject(s)
Geologic Sediments/microbiology , Hydrocarbons/metabolism , Metagenome/physiology , Adaptation, Biological , Alkanes/chemistry , Alkanes/metabolism , Anaerobiosis , Biodegradation, Environmental , Biodiversity , Chloroflexi/genetics , Chloroflexi/metabolism , Deltaproteobacteria/genetics , Deltaproteobacteria/metabolism , Genome, Microbial , Marine Biology , Metagenome/genetics , Methane/metabolism , Nova Scotia , Oceans and Seas , Phylogeny , RNA, Ribosomal, 16S
4.
Sci Rep ; 10(1): 19124, 2020 11 05.
Article in English | MEDLINE | ID: mdl-33154519

ABSTRACT

H2 is an important fermentation intermediate in anaerobic environments. Although H2 occurs at very low partial pressures in the environments, the culture and isolation of H2-utilizing microorganisms is usually carried out under very high H2 pressures, which might have hampered the discovery and understanding of microorganisms adapting to low H2 environments. Here we constructed a culture system designated the "iron corrosion-assisted H2-supplying (iCH) system" by connecting the gas phases of two vials (one for the iron corrosion reaction and the other for culturing microorganisms) to achieve cultures of microorganisms under low H2 pressures. We conducted enrichment cultures for methanogens and acetogens using rice paddy field soil as the microbial source. In the enrichment culture of methanogens under canonical high H2 pressures, only Methanobacterium spp. were enriched. By contrast, Methanocella spp. and Methanoculleus spp., methanogens adapting to low H2 pressures, were specifically enriched in the iCH cultures. We also observed selective enrichment of acetogen species by the iCH system (Acetobacterium spp. and Sporomusa spp.), whereas Clostridium spp. predominated in the high H2 cultures. These results demonstrate that the iCH system facilitates culture of anaerobic microorganisms under low H2 pressures, which will enable the selective culture of microorganisms adapting to low H2 environments.

5.
Water Res ; 176: 115750, 2020 Jun 01.
Article in English | MEDLINE | ID: mdl-32272322

ABSTRACT

In anaerobic membrane bioreactor (AnMBR) treating organic solid waste, acetate is one of the most important precursors to CH4. However, the identity and diversity of anaerobic acetate degraders are largely unknown, possibly due to their slow growth rates and low abundances. Here, we identified acetate-degrading microorganisms in the AnMBR sludges by high-sensitivity stable isotope probing. Degradation of the amended 13C-acetate coincided with production of 13CH4 and 13CO2 during the sludge incubation. High-throughput sequencing of RNA density fractions indicated that the aceticlastic and hydrogenotrophic methanogens, i.e., Methanosaeta sp. (acetate dissimilator) and Methanolinea sp. (acetate assimilator), incorporated 13C-acetate significantly. Remarkably, 22 bacterial species incorporating 13C-acetate were identified, whereas their majority was distantly related to the cultured representatives. Only two of them were the class Deltaproteobacteria-affiliated lineages with syntrophic volatile fatty acid oxidation activities. Phylogenetic tree analysis and population dynamics tracing revealed that novel species of the hydrolyzing and/or fermenting taxa, such as the phyla Bacteroidetes, Chloroflexi and Lentisphaerae, exhibited low relative abundances comparable to that of Methanolinea sp. (0.00011%) during the AnMBR operation, suggesting that these bacteria were involved in anaerobic acetate assimilation. Meanwhile, novel species of the phyla Firmicutes, Synergistetes and Caldiserica, the candidate phyla Aminicenantes and Atribacteria and the candidate division GOUTA4-related clade, as well as the known Deltaproteobacteria members, existed at relatively high abundances (0.00031%-0.31121%) in the reactor, suggesting that these bacterial species participated in anaerobic dissimilation of acetate, e.g., syntrophic acetate oxidation. The results of this study demonstrated the unexpected diversity and ecophysiological features of the anaerobic acetate degraders in the AnMBR treating organic solid waste.


Subject(s)
Methane , Solid Waste , Acetates , Anaerobiosis , Bioreactors , Isotopes , Phylogeny
6.
Nat Commun ; 10(1): 1816, 2019 04 18.
Article in English | MEDLINE | ID: mdl-31000700

ABSTRACT

The lack of microbial genomes and isolates from the deep seabed means that very little is known about the ecology of this vast habitat. Here, we investigate energy and carbon acquisition strategies of microbial communities from three deep seabed petroleum seeps (3 km water depth) in the Eastern Gulf of Mexico. Shotgun metagenomic analysis reveals that each sediment harbors diverse communities of chemoheterotrophs and chemolithotrophs. We recovered 82 metagenome-assembled genomes affiliated with 21 different archaeal and bacterial phyla. Multiple genomes encode enzymes for anaerobic oxidation of aliphatic and aromatic compounds, including those of candidate phyla Aerophobetes, Aminicenantes, TA06 and Bathyarchaeota. Microbial interactions are predicted to be driven by acetate and molecular hydrogen. These findings are supported by sediment geochemistry, metabolomics, and thermodynamic modelling. Overall, we infer that deep-sea sediments experiencing thermogenic hydrocarbon inputs harbor phylogenetically and functionally diverse communities potentially sustained through anaerobic hydrocarbon, acetate and hydrogen metabolism.


Subject(s)
Archaea/metabolism , Bacteria/metabolism , Geologic Sediments/microbiology , Microbiota/physiology , Petroleum/metabolism , Acetates/metabolism , Archaea/genetics , Archaea/isolation & purification , Bacteria/genetics , Bacteria/isolation & purification , Geologic Sediments/chemistry , Hydrocarbons/metabolism , Hydrogen/metabolism , Metagenome , Metagenomics/methods , Mexico , Microbial Interactions/physiology
7.
Microbes Environ ; 34(1): 95-98, 2019 Mar 30.
Article in English | MEDLINE | ID: mdl-30773516

ABSTRACT

Supplementation with conductive magnetite particles promoted methanogenic acetate degradation by microbial communities enriched from the production water of a high-temperature petroleum reservoir. A microbial community analysis revealed that Petrothermobacter spp. (phylum Deferribacteres), known as thermophilic Fe(III) reducers, predominated in the magnetite-supplemented enrichment, whereas other types of Fe(III) reducers, such as Thermincola spp. and Thermotoga spp., were dominant under ferrihydrite-reducing conditions. These results suggest that magnetite induced interspecies electron transfer via electric currents through conductive particles between Petrothermobacter spp. and methanogens. This is the first evidence for possible electric syntrophy in high-temperature subsurface environments.


Subject(s)
Acetates/metabolism , Ferrosoferric Oxide/chemistry , Methane/biosynthesis , Microbiota , Petroleum/microbiology , Bacteria/classification , Bacteria/genetics , Bacteria/isolation & purification , Bacteria/metabolism , Electron Transport , Euryarchaeota/metabolism , Ferric Compounds/chemistry , Ferrosoferric Oxide/antagonists & inhibitors , Hot Temperature , Oxidation-Reduction , Petroleum/metabolism , RNA, Ribosomal, 16S/genetics
8.
Chemosphere ; 219: 202-208, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30543954

ABSTRACT

In recent years, bioremediation has been used as an effective technique for the cleaning of polluted sites. However, bioremediation treatment efficacy varies considerably; thus, characterization of indigenous pollutant-degrading soil microorganisms and assessment of the changes in microbial composition by pollutants are essential for designing efficient bioremediation methods. In this study, an ecological impact evaluation method that is cost-efficient and has low contamination risk was developed to assess the indigenous microbial composition. An "in situ microcosm" was constructed using a porous ceramic arrowhead. Phenol, a common environmental pollutant, was used to assess the evaluation efficacy of this method. Our data showed that phenol gradually percolated into the soil adjacent to the arrowhead and stimulated unique indigenous microorganisms (Bacillus sp., Streptomyces sp., and Cupriavidus sp.). Furthermore, the arrowhead approach enabled efficient evaluation of the ecological impact of phenol on soil microorganisms. Thus, the arrowhead method will contribute to the development of bioremediation methods.


Subject(s)
Biodegradation, Environmental , Soil Pollutants/chemistry , Soil/chemistry , Soil Microbiology
9.
ISME J ; 12(10): 2376-2388, 2018 10.
Article in English | MEDLINE | ID: mdl-29899516

ABSTRACT

1,4-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.


Subject(s)
Bacteria/metabolism , Dioxanes/metabolism , Sewage/microbiology , Bacteria/classification , Dioxanes/chemistry , Isotopes , Microbial Consortia/genetics , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Wastewater/microbiology
10.
Int J Syst Evol Microbiol ; 67(10): 3982-3986, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28893364

ABSTRACT

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).


Subject(s)
Bacteria, Anaerobic/classification , Oil and Gas Fields/microbiology , Phylogeny , Bacteria, Anaerobic/genetics , Bacteria, Anaerobic/isolation & purification , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Fatty Acids/chemistry , Ferric Compounds/metabolism , Japan , Manganese/metabolism , Nitrates/metabolism , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sulfates/metabolism , Vitamin K 2/analogs & derivatives , Vitamin K 2/chemistry
11.
Science ; 354(6309): 222-225, 2016 10 14.
Article in English | MEDLINE | ID: mdl-27738170

ABSTRACT

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.


Subject(s)
Coal/microbiology , Methane/metabolism , Methanosarcinales/metabolism , Acetyl Coenzyme A/metabolism , Carbon Dioxide/metabolism , Carbon Isotopes/metabolism , Hydroxybenzoate Ethers/metabolism , Methanol/metabolism , Methanosarcinales/enzymology , Methylation , Oxidation-Reduction , Radioactive Tracers
12.
Nat Commun ; 4: 1998, 2013.
Article in English | MEDLINE | ID: mdl-23759740

ABSTRACT

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.


Subject(s)
Carbon Dioxide/pharmacology , Methane/biosynthesis , Microbiota/drug effects , Oil and Gas Fields/microbiology , Acetates/metabolism , Archaea/drug effects , Archaea/metabolism , Carbon Isotopes , Molecular Sequence Data , Oxidation-Reduction/drug effects , Partial Pressure , Thermodynamics
13.
J Biosci Bioeng ; 113(1): 84-7, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22014785

ABSTRACT

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.


Subject(s)
Bacteria/metabolism , Industrial Microbiology/methods , Methane/biosynthesis , Oil and Gas Fields/microbiology , Bacteria/genetics , Colony Count, Microbial , Culture Media/chemistry , DNA, Bacterial/genetics , Ecosystem , Petroleum/microbiology , Phylogeny , Water Microbiology , Yeasts/chemistry
14.
J Biosci Bioeng ; 113(2): 204-10, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22019404

ABSTRACT

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.


Subject(s)
Archaea/classification , Bacteria/classification , Biodiversity , Petroleum/microbiology , Archaea/genetics , Bacteria/genetics , Base Sequence , Gene Library , Genes, rRNA , Hot Temperature , Molecular Sequence Data , Oil and Gas Fields , Phylogeny , RNA, Ribosomal, 16S/genetics , Water/chemistry , Water Microbiology
15.
Environ Microbiol ; 13(12): 3206-18, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21651687

ABSTRACT

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.


Subject(s)
Archaea/classification , Fresh Water/microbiology , Geologic Sediments/microbiology , Methane/metabolism , Phylogeny , Adaptation, Biological , Anaerobiosis , Archaea/genetics , Archaea/metabolism , Bacteria/classification , Bacteria/genetics , DNA, Archaeal/genetics , Ecosystem , Fresh Water/chemistry , Gene Library , Geologic Sediments/chemistry , Japan , Molecular Sequence Data , Oxidation-Reduction , RNA, Ribosomal, 16S/genetics , Salinity , Water Microbiology
16.
Environ Microbiol ; 13(8): 1995-2006, 2011 Aug.
Article in English | MEDLINE | ID: mdl-20860731

ABSTRACT

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.


Subject(s)
Acetates/metabolism , Archaea/metabolism , Bacteria/metabolism , Hot Temperature , Methane/biosynthesis , Oil and Gas Fields/microbiology , Archaea/classification , Archaea/genetics , Bacteria/classification , Bacteria/genetics , Biodiversity , Environmental Microbiology , Japan , Molecular Sequence Data , Petroleum/metabolism , Phylogeny , Pressure , RNA, Ribosomal, 16S
17.
Microbes Environ ; 25(3): 156-63, 2010.
Article in English | MEDLINE | ID: mdl-21576868

ABSTRACT

The community structure of methane-oxidizing bacteria (methanotrophs) is affected by concentrations of methane and oxygen. In rice fields, concentrations of both gases differ significantly between the flooded and drained seasons. We investigated the active methanotrophic community structures in flooded and drained soils by DNA-based stable isotope probing. Active methanotrophic diversity was assessed with clone library-based analyses of the 16S rRNA gene and the particulate methane monooxygenase gene (pmoA). The active methanotrophic populations were also estimated by group-specific quantitative real-time PCR assays targeting the 16S rRNA gene and the pmoA gene in (13)C-labeled DNA. These molecular biological analyses showed that the flooded rice field soil was dominated by Type II methanotrophs closely related to the genera Methylocystis and Methylosinus, whereas the drained rice field soil was dominated by Type I methanotrophs closely related to the genera Methylomonas, Methylosarcina, and Methylomicrobium. The alternating conditions in a rice field select for methanotrophs adapted to each environment, resulting in a dramatic change in methanotrophic community structure from one season to another.


Subject(s)
Bacteria/growth & development , Bacteria/metabolism , Biodiversity , Isotope Labeling/methods , Methane/metabolism , Polymerase Chain Reaction/methods , Soil Microbiology , Bacteria/classification , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Molecular Sequence Data , Oryza/growth & development , Oxygenases/genetics , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA
18.
Appl Microbiol Biotechnol ; 79(5): 743-50, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18461319

ABSTRACT

Many poly(lactic acid) (PLA)-degrading microorganisms have been isolated from the natural environment by culture-based methods, but there is no study about unculturable PLA-degrading microorganisms. In this study, we constructed a metagenomic library consisting of the DNA extracted from PLA disks buried in compost. We identified three PLA-degrading genes encoding lipase or hydrolase. The purified enzymes degraded not only PLA, but also various aliphatic polyesters, tributyrin, and p-nitrophenyl esters. From their substrate specificities, the PLA depolymerases were classified into an esterase rather than a lipase. Among the PLA depolymerases, PlaM4 exhibited thermophilic properties; that is, it showed the highest activity at 70 degrees C and was stable even after incubation for 1 h at 50 degrees C. PlaM4 had absorption and degradation activities for solid PLA at 60 degrees C, which indicates that the enzyme can effectively degrade PLA in a high-temperature environment. On the other hand, the enzyme classification based on amino acid sequences showed that the other PLA depolymerases, PlaM7 and PlaM9, were not classified into known lipases or esterases. This is the first report on the identification and characterization of PLA depolymerase from a metagenome.


Subject(s)
Bacteria/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Esterases/chemistry , Esterases/genetics , Genome, Bacterial , Lactic Acid/metabolism , Polymers/metabolism , Bacteria/chemistry , Bacteria/classification , Bacteria/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Biodiversity , Cloning, Molecular , Enzyme Stability , Esterases/isolation & purification , Esterases/metabolism , Gene Library , Lipase/chemistry , Lipase/genetics , Lipase/isolation & purification , Lipase/metabolism , Molecular Sequence Data , Phylogeny , Polyesters , Sequence Analysis, DNA , Soil Microbiology , Substrate Specificity
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