Worldwide, abandoned coal mines release substantial amounts of methane, which is largely of biogenic origin. The aim of this study was to understand the microbial processes involved in mine-gas formation. Therefore, coal and timber samples and anaerobic enrichments from two abandoned coal mines in Germany were subjected to DGGE analyses and quantitative PCR. The primers used were specific for Bacteria, Archaea, Fungi, and the key functional genes for sulfate reduction (dsrA) and methanogenesis (mcrA). A broad spectrum of facultative anaerobic bacteria and acetogens belonging to all five groups (α-ϵ) of the Proteobacteria, as well as the Bacteroidetes, Tenericutes, Actinobacteria, Chlorobi and Chloroflexi were detected. Archaea were represented by acetoclastic Methanosarcinales and Crenarchaeota with an unknown metabolism. Fungi formed thick biofilms particularly on timber, and were identified as typical wood degraders belonging to the Ascomycetes and Basidiomycetes. The community analysis as well as the environmental conditions and the metabolites detected in a previous study are consistent with the following scenario of methane release: Weathering of coal and timber is initiated by wood-degrading Fungi and Bacteria under a suboxic atmosphere. In the lower, oxygen-depleted layers Fungi and Bacteria perform incomplete oxidation and release reduced substrates which can be channeled into methanogenesis. Acetate appeared to be the main precursor of the biogenic methane in the investigated coal mines.
Abstract Despite the significance of biogenic methane generation in coal beds, there has never been a systematic long-term evaluation of the ecological response to biostimulation for enhanced methanogenesis in situ. Biostimulation tests in a gas-free coal seam were analysed over 1.5 years encompassing methane production, cell abundance, planktonic and surface associated community composition and chemical parameters of the coal formation water. Evidence is presented that sulfate reducing bacteria are energy limited whilst methanogenic archaea are nutrient limited. Methane production was highest in a nutrient amended well after an oxic preincubation phase to enhance coal biofragmentation (calcium peroxide amendment). Compound-specific isotope analyses indicated the predominance of acetoclastic methanogenesis. Acetoclastic methanogenic archaea of the Methanosaeta and Methanosarcina genera increased with methane concentration. Acetate was the main precursor for methanogenesis, however more acetate was consumed than methane produced in an acetate amended well. DNA stable isotope probing showed incorporation of 13C-labelled acetate into methanogenic archaea, Geobacter species and sulfate reducing bacteria. Community characterisation of coal surfaces confirmed that methanogenic archaea make up a substantial proportion of coal associated biofilm communities. Ultimately, methane production from a gas-free subbituminous coal seam was stimulated despite high concentrations of sulfate and sulfate-reducing bacteria in the coal formation water. These findings provide a new conceptual framework for understanding the coal reservoir biosphere.
Pelagic redoxclines represent chemical gradients of elevated microbial activities. While chemolithoautotrophic microorganisms in these systems are well known as catalysts of major biogeochemical cycles, comparable knowledge on heterotrophic organisms is scarce. Thus, in this study, identity and biogeochemical involvement of active heterotrophs were investigated in stimulation experiments and activity measurements based on samples collected from pelagic redoxclines of the central Baltic Sea in 2005 and 2009. In the 2009 samples, (13)C-acetate 16S rRNA stable isotope probing (16S rRNA-SIP) identified gammaproteobacteria affiliated with Colwellia sp. and Neptunomonas sp. in addition to epsilonproteobacteria related to Arcobacter spp. as active heterotrophs at the oxic-anoxic interface layer. Incubations from sulfidic waters were dominated by two phylogenetic subgroups of Arcobacter. In the 2005 samples, organics, manganese(IV), and iron(III) were added to the sulfidic waters, followed by the determination of metal reduction and identification of the stimulated organisms. Here, the same Arcobacter and Colwellia subgroups were stimulated as in 2009, with Arcobacter predominating in samples, in which manganese(IV) reduction was highest. Our results offer new insights into the heterotrophic bacterial assemblage of Baltic Sea pelagic redoxclines and suggest Arcobacter spp. as a heterotroph with presumed relevance also for manganese cycling.
Quantification of microbes in water systems is essential to industrial practices ranging from drinking water and wastewater treatment to groundwater remediation. While quantification using DNA-based molecular methods is precise, the accuracy is dependent on DNA extraction efficiencies. We show that the DNA yield is strongly impacted by the cell concentration in groundwater samples (r = -0.92, P < 0.0001). This has major implications for industrial applications using quantitative polymerase chain reaction (qPCR) to determine cell concentrations in water, including bioremediation. We propose a simple normalization method using a DNA recovery ratio, calculated with the total cell count and DNA yield. Application of this method to enumeration of bacteria and archaea in groundwater samples targeting phylogenetic markers (16S rRNA) demonstrated an increased goodness of fit after normalization (7.04 vs 0.94 difference in Akaike's information criteria). Furthermore, normalization was applied to qPCR quantification of functional genes and combined with DNA sequencing of archaeal and bacterial 16S rRNA genes to monitor changes in abundance of methanogenic archaea and sulphate-reducing bacteria in groundwater. The integration of qPCR and DNA sequencing with appropriate normalization enables high-throughput quantification of microbial groups using increasingly affordable and accessible techniques. This research has implications for microbial ecology and engineering research as well as industrial practice.
Pristine hydrocarbon-rich river sediments in the Greater Blue Mountains World Heritage Area (Australia) release substantial amounts of methane. The present study aimed to unravel for the first time the active methanogens mediating methane formation and exploiting the bacterial diversity potentially involved in the trophic network. Quantitative PCR of 16S rRNA gene and functional genes as well as 454 pyrosequencing were used to address the unknown microbial diversity and abundance. Methane-releasing sediment cores derived from three different river sites of the Tootie River. Highest methane production rates of 10.8 ± 0.5 μg g(-1)(wet weight) day(-1) were detected in 40 cm sediment depth being in congruence with the detection of the highest abundances of the archaeal 16S rRNA gene and the methyl-coenzyme M reductase (mcrA) genes. Stable carbon and hydrogen isotopic signatures of the produced methane indicated an acetoclastic origin. Long-term enrichment cultures amended with either acetate or H2/CO2 revealed acetoclastic methanogenesis as key methane-formation process mediated by members of the order Methanosarcinales. Conditions prevailing in the river sediments might be suitable for hydrocarbon-degrading bacteria observed in the river sediments that were previously unclassified or closely related to the Bacteroidetes/Chlorobi group, the Firmicutes and the Chloroflexi group fuelling acetoclastic methanogensis in pristine river sediments.
Anaerobic oxidation of methane (AOM) and the environmental conditions supporting AOM on continental margins is an essential component to global methane budgets. Diagnostic lipid biomarkers and their compound specific isotope analysis preserved in authigenic carbonates at cold seeps can serve as “fingerprints” to archaeal−bacterial consortia involved in AOM. However, despite the discovery of several hundreds of seeps along the United States Atlantic Margin (USAM), there are relatively few biomarker investigations of cold seep carbonates along this passive margin. A lipid biomarker, carbon isotope, and DNA marker gene study was therefore undertaken to determine the microbial origins of authigenic carbonates from two USAM seeps, Norfolk and the Baltimore Canyon seep fields. Results from this study capture a distinct archaeal lipid signature from putative methanotrophic archaea, including archaeol (I), sn-2-hydroxyarchaeol, 2,6,10,15,19-pentamethylicosane (PMI), and crocetane. The 13C-depleted AOM-related archaeal lipid samples (i.e., archaeol: −91.6‰, sn-2-hydroxyarchaeol: −129.2‰, PMI −92.8‰, and crocetane: −70.9‰) confirm the dominance of methane assimilation and isotope fractionation during AOM. These results are consistent with the detection of archaeal anaerobic methanotrophs (ANMEs) based on 16S rRNA gene sequencing. The Norfolk authigenic carbonate contained ANME-1a, -1b, 2a-2b, and 2c whereas only the ANME-2 clade was detected at Baltimore and present as the subclusters 2a-2b and -2b. The ANME-2d clade may also be present, particularly at the Baltimore seep site, given the high abundance of Candidatus Methanoperedens nitroreducens detected in the mcrA gene sequencing. The presence of terminally branched fatty acids, antesio- and iso-C15:0 components, as well as C16:1ω7 with δ13C values as low as −107.6‰, are indicative of sulfate-reducing bacteria (SRB) at the Norfolk seep site and supports syntrophy of SRB with methane-oxidizing archaea. In contrast, nitrate-driven AOM in syntrophy M. nitroreducens at the Baltimore seep site is consistent with elevated fatty acid δ13C values and lack of Deltaproteobacteria at the Baltimore seep site. Taken together, the range in lipid composition, distribution, and carbon isotopic composition observed at the Norfolk and Baltimore seep sites suggests AOM is performed by multiple archaea instead of a single species and may be paired with either or both nitrate- and sulfate-reduction. Given the heterogeneous nature of cold seep ecosystems, this study fills a critical spatial gap in our knowledge of AOM activity at two seep sites along a passive margin.
Identifying the source of methane (CH4) in groundwater is often complicated due to various production, degradation and migration pathways, particularly in settings where there are multiple groundwater recharge pathways. This study demonstrates the ability to constrain the origin of CH4 within an alluvial aquifer that could be sourced from in situ microbiological production or underlying formations at depth. To characterise the hydrochemical and microbiological processes active within the alluvium, previously reported hydrochemical data (major ion chemistry and isotopic tracers (3H, 14C, 36Cl)) were interpreted in the context of CH4 and carbon dioxide (CO2) isotopic chemistry, and the microbial community composition in the groundwater. The rate of observed oxidation of CH4 within the aquifer was then characterised using a Rayleigh fractionation model. The stratification of the hydrochemical facies and microbiological community populations is interpreted to be a result of the gradational mixing of water from river leakage and floodwater recharge with water from basal artesian inflow. Within the aquifer there is a low abundance of methanogenic archaea indicating that there is limited biological potential for microbial CH4 production. Our results show that the resulting interconnection between hydrochemistry and microbial community composition affects the occurrence and oxidation of CH4 within the alluvial aquifer, constraining the source of CH4 in the groundwater to the geological formations beneath the alluvium.
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