Methane production from the biodegradation of lignite with different sizes by mixed fungi-methanogen microflora.

Biogenic coalbed methane (CBM) is a developing clean energy source. However, it is unclear how the mechanisms of bio-methane production with different sizes of coal. In this work, pulverized coal (PC) and lump coal (LC) were used for methane production by mixed fungi-methanogen microflora. The lower methane production from LC was observed. The aromatic carbon of coal was degraded slightly by 2.17% in LC, while 11.28% in PC. It is attributed to the proportion of lignin-degrading fungi, especially Penicillium, which was reached 67.57% in PC on the 7th day, higher than that of 11.38% in LC. The results suggested that the limited interaction area in LC led to microorganisms hardly utilize aromatics. It also led the accumulation of aromatic organics in the fermentation broth in PC. Increasing the reaction area of coal and facilitating the conversion of aromatic carbon are suggested means to increase methane production in situ.

Krumholzibacteriota and Deltaproteobacteria contain rare genetic potential to liberate carbon from monoaromatic compounds in subsurface coal seams.

Biogenic methane in subsurface coal seam environments is produced by diverse consortia of microbes. Although this methane is useful for global energy security, it remains unclear which microbes can liberate carbon from the coal. Most of this carbon is relatively resistant to biodegradation, as it is contained within aromatic rings. Thus, to explore for coal-degrading taxa in the subsurface, this study reconstructed relevant metagenome-assembled genomes (MAGs) from coal seams by using a key genomic marker for the anaerobic degradation of monoaromatic compounds as a guide: the benzoyl-CoA reductase gene (bcrABCD). Three MAGs were identified with this genetic potential. The first represented a novel taxon from the Krumholzibacteriota phylum, which this study is the first to describe. This Krumholzibacteriota MAG contained a full set of genes for benzoyl-CoA dearomatization, in addition to other genes for anaerobic catabolism of monoaromatics. Analysis of Krumholzibacteriota MAGs from other environments revealed that this genetic potential may be common, and thus, Krumholzibacteriota may be important organisms for the liberation of recalcitrant carbon in a broad range of environments. Moreover, the assembly and characterization of two Syntrophorhabdus aromaticivorans MAGs from different continents and a Syntrophaceae sp. MAG implicate the Deltaproteobacteria class in coal seam monoaromatic degradation. Each of these taxa are potential rate-limiting organisms for subsurface coal-to-methane biodegradation. Their description here provides some understanding of their function within the coal seam microbiome and will help inform future efforts in coal bed methane stimulation, anoxic bioremediation of organic pollutants, and assessments of anoxic, subsurface carbon cycling and emissions.IMPORTANCESubsurface coal seams are highly anoxic, oligotrophic environments, where the main source of carbon is "locked away" within aromatic rings. Despite these challenges, many coal seams accumulate biogenic methane, implying that the coal seam microbiome is "unlocking" this carbon source in situ. For over two decades, researchers have endeavored to understand which organisms perform these processes. This study provides the first descriptions of organisms with this genetic potential from the coal seam environment. Here, we report metagenomic insights into carbon liberation from aromatic molecules and the degradation pathways involved and describe a Krumholzibacteriota, two Syntrophorhabdus aromaticivorans, and a Syntrophaceae MAG that contain this genetic potential. This is also the first time that the Krumholzibacteriota phylum has been implicated in anaerobic dearomatization of aromatic hydrocarbons. This potential is identified here in numerous MAGs from other terrestrial and marine subsurface habitats, implicating the Krumholzibacteriota in carbon-cycling processes across a broad range of environments.

Selective trace elements significantly enhanced methane production in coal bed methane systems by stimulating microbial activity.

Trace elements are associated with the microbial degradation of organic matter and methanogenesis, as enzymes in metabolic pathways often employ trace elements as essential cofactors. However, only a few studies investigated the effects of trace elements on the metabolic activity of microbial communities associated with biogenic coalbed methane production. We aimed to determine the effects of strategically selected trace elements on structure and function of active bacterial and methanogenic communities to stimulate methane production in subsurface coalbeds. Microcosms were established with produced water and coal from coalbed methane wells located in the Powder River Basin, Wyoming, USA. In initial pilot experiments with eight different trace elements, individual amendments of Co, Cu, and Mo lead to significantly higher methane production. Transcript levels of mcrA, the key marker gene for methanogenesis, positively correlated with increased methane production. Phylogenetic analysis of the mcrA cDNA library demonstrated compositional shifts of the active methanogenic community and increase of their diversity, particularly of hydrogenotrophic methanogens. High-throughput sequencing of cDNA obtained from 16S rRNA demonstrated active and abundant bacterial groups in response to trace element amendments. Active Acetobacterium members increased in response to Co, Cu, and Mo additions. The findings of this study yield new insights into the importance of essential trace elements on the metabolic activity of microbial communities involved in subsurface coalbed methane and provide a better understanding of how microbial community composition is shaped by trace elements.IMPORTANCEMicrobial life in the deep subsurface of coal beds is limited by nutrient replenishment. While coal bed microbial communities are surrounded by carbon sources, we hypothesized that other nutrients such as trace elements needed as cofactors for enzymes are missing. Amendment of selected trace elements resulted in compositional shifts of the active methanogenic and bacterial communities and correlated with higher transcript levels of mcrA. The findings of this study yield new insights to not only identify possible limitations of microbes by replenishment of trace elements within their specific hydrological placement but also into the importance of essential trace elements for the metabolic activity of microbial communities involved in subsurface coalbed methane production and provides a better understanding of how microbial community composition is shaped by trace elements. Furthermore, this finding might help to revive already spent coal bed methane well systems with the ultimate goal to stimulate methane production.

Coexistence of Psychrophilic, Mesophilic, and Thermophilic Sulfate-Reducing Bacteria in a Deep Subsurface Aquifer Associated with Coal-Bed Methane Production.

The microbial community of subsurface environments remains understudied due to limited access to deep strata and aquifers. Coal-bed methane (CBM) production is associated with a large number of wells pumping water out of coal seams. CBM wells provide access to deep biotopes associated with coal-bed water. Temperature is one of the key constraints for the distribution and activity of subsurface microorganisms, including sulfate-reducing prokaryotes (SRP). The 16S rRNA gene amplicon sequencing coupled with in situ sulfate reduction rate (SRR) measurements with a radioactive tracer and cultivation at various temperatures revealed that the SRP community of the coal bed water of the Kuzbass coal basin is characterized by an overlapping mesophilic-psychrophilic boundary. The genus Desulfovibrio comprised a significant share of the SRP community. The D. psychrotolerans strain 1203, which has a growth optimum below 20 °C, dominated the cultivated SRP. SRR in coal bed water varied from 0.154 ± 0.07 to 2.04 ± 0.048 nmol S cm-3 day-1. Despite the ambient water temperature of ~ 10-20 °C, an active thermophilic SRP community occurred in the fracture water, which reduced sulfate with the rate of 0.159 ± 0.023 to 0.198 ± 0.007 nmol S cm-3 day-1 at 55 °C. A novel moderately thermophilic "Desulforudis audaxviator"-clade SRP has been isolated in pure culture from the coal-bed water.

The variation of microorganisms and organics during methane production from lignite under an electric field.

OBJECTIVES: The succession of microbial communities and intermediates during methane production was determined by pyrosequencing and GC-MS to investigate the mechanism of biomethanation enhancement from coal. RESULTS: The maximum methane production at 1.2 V was significantly higher than that at 0 V. Bacterial flora have been changed as a result of the addition of an electric field, e.g., the abundance of Pseudomonas significantly increased to enhance the coal degradation which improved the methane yield by facilitating the electron transfer. The fungal structure was also found stabilized by the electric field when compared to the control after 7 days of cultivation. The predominance of Methanosarcina could also stimulate interspecies electron transfer. The GC-MS analysis revealed that the electric field can selectively promote the metabolism of refractory intermediates such as esters and aromatics during coal biodegradation. CONCLUSION: The application of an electric field could enhance methane production from coal by changing the structure and succession of microbial communities, improving electron transfer, and enhancing the fermentation of intermediates during coal biodegradation.

Physical, chemical, and bio-pretreatments on microbial gas production in Baode Block coal.

Currently, the exploitation of Baode Block as a biogenic coal-bed gas field has been in the later stage of stable production; hence, exploration and activation of microbial gas production are of great practical significance for the enhancement and stabilization of block production. Pretreatment is the key process to improve anaerobic biodegradation performance and increase yield and production rate of gas. In this study, we examine physical, chemical, and biological pretreatment methods and compare their effectiveness toward microbial gas production in the coal seam. The obtained results indicate that: (1) grinding can enhance contact between the coal sample and bacteria liquid, and coal powder has greater gas-producing performance than the coal lump. (2) Chemical pretreatment of coal samples using acid and base can enhance gas production capacity. NaOH treatment has better gas-producing performance than HCl treatment, and the activity of microbial flora is higher after treatment. (3) Biological pretreatment can greatly enhance the microbial degradation of coal bed. The highest gas yield after white rot fungus pretreatment is 11.65 m3/t, and gas production cycle is shorter than before. This may be due to the white rot fungus effectively degrading macromolecules and, therefore, shortening the duration of methanogenic hydrolysis, which provides more organic matter for methanogens to decompose. During production, in addition to selecting a proper pretreatment method, the treatment cost and balance between energy input of pretreatment and gas energy output must also be considered. The joint pretreatment between different reagents and treatment methods is a possible solution to the problem and a current research trend to realize the large-scale degradation of coal. The simulated microbial methane production of coal seam is feasible for Baode Block in Ordos, where coal samples in this block have great gas-producing potential after treatment, and provides good references for further in-field tests.

Desulfuromonas sp. 'CSMB_57', isolation and genomic insights from the most abundant bacterial taxon in eastern Australian coals.

One of the most abundant and ubiquitous taxa observed in eastern Australian coal seams is an uncultured Desulfuromonas species and part of the Coal Seam Microbiome dataset assigned as 'CSMB_57'. Despite this abundance and ubiquity, knowledge about this taxon is limited. The present study aimed to generate an enrichment culture of Desulfuromonas sp. 'CSMB_57' using culturing strategies that exploit its sulphur-reducing capabilities by utilizing a polysulfide solution in a liquid medium. Using dilution to extinction methods, a highly enriched culture was successfully generated. The full-length 16S rRNA sequence revealed that all closely related taxa were observed in subsurface environments suggesting that D. sp. 'CSMB_57' may be a subsurface specialist. Subsequently, the DNA from the enrichment culture was sequenced and the genome of D. sp. 'CSMB_57' was assembled. Genomic annotation revealed a high number of CRISPR arrays for viral defence, a large array of ABC transporters for amino acid and peptide uptake, as well as genes likely associated with syntrophy such as genes associated with type-IVa pilus, often used for direct interspecies electron transfer, and multiple hydrogenases capable of producing hydrogen. From the various genomic observations, a conceptual ecological model was developed that explores its possible syntrophic roles with hydrogenotrophic methanogens and acetogenic bacteria within coal-seam environments.

The ecological roles of assembling genomes for Bacillales and Clostridiales in coal seams.

Biogenic coalbed methane is produced by biological processes mediated by synergistic interactions of microbial complexes in coal seams. However, the ecological role of functional bacteria in biogenic coalbed methane remains poorly understood. Here, we studied the metagenome assembled genomes (MAGs) of Bacillales and Clostridiales from coal seams, revealing further expansion of hydrogen and acetogen producers involved in organic matter decomposition. In this study, Bacillales and Clostridiales were dominant orders (91.85 ± 0.94%) in cultured coal seams, and a total of 16 MAGs from six families, including Bacillus, Paenibacillus, Staphylococcus, Anaerosalibacter, Hungatella and Paeniclostridium, were reconstructed. These microbial groups possessed multiple metabolic pathways (glycolysis/gluconeogenesis, pentose phosphate, ß-oxidation, TCA cycle, assimilatory sulfate reduction, nitrogen metabolism and encoding hydrogenase) that provided metabolic substrates (acetate and/or H2) for the methanogenic processes. Therein, the hydrogenase-encoding gene and hydrogenase maturation factors were merely found in all the Clostridiales MAGs. ß-oxidation was the main metabolic pathway involved in short-chain fatty acid degradation and acetate production, and most of these pathways were detected and exhibited different operon structures in Bacillales MAGs. In addition, assimilatory sulfate reduction and nitrogen metabolism processes were also detected in some MAGs, and these processes were also closely related to acetate production and/or organic matter degradation according to their operon structures and metabolic pathways. In summary, this study enabled a better understanding of the ecological roles of Bacillales and Clostridiales in biogenic methane in coal seams based on a combination of bioinformatic techniques.

Methanogenic archaea in subsurface coal seams are biogeographically distinct: an analysis of metagenomically-derived mcrA sequences.

The production of methane as an end-product of organic matter degradation in the absence of other terminal electron acceptors is common, and has often been studied in environments such as animal guts, soils and wetlands due to its potency as a greenhouse gas. To date, however, the study of the biogeographic distribution of methanogens across coal seam environments has been minimal. Here, we show that coal seams are host to a diverse range of methanogens, which are distinctive to each geological basin. Based on comparisons to close relatives from other methanogenic environments, the dominant methanogenic pathway in these basins is hydrogenotrophic, with acetoclastic being a second major pathway in the Surat Basin. Finally, mcrA and 16S rRNA gene primer biases were predominantly seen to affect the detection of Methanocellales, Methanomicrobiales and Methanosarcinales taxa in this study. Subsurface coal methanogenic community distributions and pathways presented here provide insights into important metabolites and bacterial partners for in situ coal biodegradation.

Two new dihydroisocoumarins with antimicrobial activities from the fungus Penicillium sp. XR046 collected from Xinren coal area.

Two new dihydroisocoumarins (1 and 2), together with six known compounds (3-8), were isolated from the fungus Penicillium sp. XR046 collected from the Xinren coal area of Guizhou province in China. Their structures were elucidated on the basis of spectroscopic analysis. The absolute configurations of C-3 in 1 and 2 were established by comparison of their CD data with those of known compounds. Compounds 1-6 showed anti-microbial activities with MIC values in the range of 50∼100 µg/mL against Candida albicans, Staphylococcus epidermidis, Bacillus subtilis, and Escherichia coli.

Effects of Microbes from Coal-Related Commercial Humic Substances on Hydroponic Crop Cultivation: A Microbiological View for Agronomical Use of Humic Substances.

Here, coal-related humic substances (HSs) were examined to confirm whether sterilization treatments induce their inferior ability to stimulate lettuce in hydroponic cultivations. Interestingly, a drastic reduction in both lettuce biomass and microbial colony-forming units of the crop culture solutions was observed when the autoclaved HSs were treated. Some microbial genera (i.e., Bacillus and Aspergillus) identifiable in the bare HS-treated hydroponic systems were able to be isolated by direct inoculation of bare HS powders on conventional microbial nutrients, supporting that flourishing microbes in the hydroponic cultivations derive from bare HSs-treated. Moreover, coincubation of some isolated bacterial and fungal strains (i.e., Bacillus and Aspergillus genera) from HSs with lettuce resulted in a significant increase in plant biomass and enhanced resistance to NaCl-related abiotic stresses. Microbial volatile organic compounds renowned for plant stimulation were detected by using solid-phase microextraction coupled with gas chromatography-mass spectrometry. It was finally confirmed that the isolates are capable of utilizing carbon substrates such as pectin and tween 20 or 40, which are relevant to those of microbes isolated from peat and leonardite (i.e., HS extraction sources). Overall, our results suggest that microbiological factors could be considered when commercial coal-related HSs are applied in hydroponic crop cultivations.

Early reprecipitation of sulfate salts in coal biodesulfurization processes using acidophilic chemolithotrophic bacteria.

This study evaluated the effect of three sulfate salt-based culture media on the reprecipitation of sulfur under the action of two types of bacterial inoculum, a pure strain of Acidithiobacillus ferrooxidans (ATCC 23270) and a consortium of this strain and Acidithiobacillus thiooxidans (ATCC 15494), in a biodesulfurization process for coal (particle size < 0.25 mm) from the 'La Guacamaya' mine (Puerto Libertador, Córdoba, Colombia). All of the experiments were periodically monitored, with measurements taken of pH, cell concentration, iron concentration, and pyrite oxidation. Additionally, mineralogical analyses were conducted on the initial and final coal samples, through scanning electron microscopy with an energy-dispersive X-ray spectrometer. The results showed that sulfate reprecipitation occurred primarily, and nearly entirely, during the first 3 days of the process. While all the treatments obtained high levels of mineral oxidation, the reprecipitation processes decreased in media with low concentrations of sulfate, leading to the higher final removal of inorganic sulfur. The bioassays revealed that after 15 days, the maximum pyrite oxidation (86%) and inorganic sulfur removal (53%) was obtained with the treatments using the Kos and McCready culture media. The bacteria evaluated were found to have a great ability to adapt to very simple culture media with minimal nutrient concentrations, and even with some nutrients absent (as in the case of magnesium).

The effect of NaOH pretreatment on coal structure and biomethane production.

Biogenic CBM is an important component of detected CBM, which is formed by coal biodegradation and can be regenerated by anaerobic microorganisms. One of the rate-limiting factors for microbial degradation is the bioavailability of coal molecules, especially for anthracite which is more condense and has higher aromaticity compared with low-rank coal. In this paper, NaOH solution with different concentrations and treating time was employed to pretreat anthracite from Qinshui Basin to alter the coal structure and facilitate the biodegradation. The results showed that the optimal pretreatment conditions were 1.5 M NaOH treating for 12 h, under which the biomethane production was increased by 17.65% compared with untreated coal. The results of FTIR and XRD showed that NaOH pretreatment mainly reduced the multi-substituted aromatics, increased the C-O in alcohols and aromatic ethers and the branching degree of aliphatic chain, and decreased the aromatic ring structure, resulting in the improvement of coal bioavailability and enhancement of biomethane yield. And some organics with potential to generate methane were released to filtrate as revealed by GC-MS. Our results suggested that NaOH was an effective solution for pretreating coal to enhance biogenic methane production, and anthracite after treating with NaOH could be the better substrate for methanogenesis.

Genome Mining Revealed a High Biosynthetic Potential for Antifungal Streptomyces sp. S-2 Isolated from Black Soot.

The increasing resistance of fungal pathogens has heightened the necessity of searching for new organisms and compounds to combat their spread. Streptomyces are bacteria that are well-known for the production of many antibiotics. To find novel antibiotic agents, researchers have turned to previously neglected and extreme environments. Here, we isolated a new strain, Streptomyces sp. S-2, for the first time, from black soot after hard coal combustion (collected from an in-use household chimney). We examined its antifungal properties against plant pathogens and against fungi that potentially pose threat to human health (Fusarium avenaceum, Aspergillus niger and the environmental isolates Trichoderma citrinoviridae Cin-9, Nigrospora oryzae sp. roseF7, and Curvularia coatesieae sp. junF9). Furthermore, we obtained the genome sequence of S-2 and examined its potential for secondary metabolites production using anti-SMASH software. The S-2 strain shows activity against all of the tested fungi. Genome mining elucidated a vast number of biosynthetic gene clusters (55), which distinguish this strain from closely related strains. The majority of the predicted clusters were assigned to non-ribosomal peptide synthetases or type 1 polyketide synthetases, groups known to produce compounds with antimicrobial activity. A high number of the gene clusters showed no, or low similarity to those in the database, raising the possibility that S-2 could be a producer of novel antibiotics. Future studies on Streptomyces sp. S-2 will elucidate its full biotechnological potential.

Gas seepage pockmark microbiomes suggest the presence of sedimentary coal seams in the Öxarfjörður graben of northeastern Iceland.

Natural gas seepage pockmarks are found off- and onshore in the Öxarfjörður graben, Iceland. The bacterial communities of two onshore seepage sites were analysed by 16S rRNA gene amplicon sequencing; the geochemical characteristics, hydrocarbon content, and the carbon isotope composition of the sites were also determined. While one site was found to be characterised by biogenic origin of methane gas, with a carbon isotope ratio (δ13C (‰)) of -63.2, high contents of organic matter and complex hydrocarbons, the other site showed a mixed origin of the methane gas (δ13C (‰) = -26.6) with geothermal characteristics and lower organic matter content. While both sites harboured Proteobacteria as the most abundant bacterial phyla, the Deltaproteobacteria were more abundant at the geothermal site and the Alphaproteobacteria at the biogenic site. The Dehalococcoidia class of phylum Chloroflexi was abundant at the geothermal site while the Anaerolineae class was more abundant at the biogenic site. Bacterial strains from the seepage pockmarks were isolated on a variety of selective media targeting bacteria with bioremediation potential. A total of 106 strains were isolated and characterised, including representatives from the phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. This article describes the first microbial study on gas seepage pockmarks in Iceland.

Genomic and phenotypic insights point to diverse ecological strategies by facultative anaerobes obtained from subsurface coal seams.

Microbes in subsurface coal seams are responsible for the conversion of the organic matter in coal to methane, resulting in vast reserves of coal seam gas. This process is important from both environmental and economic perspectives as coal seam gas is rapidly becoming a popular fuel source worldwide and is a less carbon intensive fuel than coal. Despite the importance of this process, little is known about the roles of individual bacterial taxa in the microbial communities carrying out this process. Of particular interest is the role of members of the genus Pseudomonas, a typically aerobic taxa which is ubiquitous in coal seam microbial communities worldwide and which has been shown to be abundant at early time points in studies of ecological succession on coal. The current study performed aerobic isolations of coal seam microbial taxa generating ten facultative anaerobic isolates from three coal seam formation waters across eastern Australia. Subsequent genomic sequencing and phenotypic analysis revealed a range of ecological strategies and roles for these facultative anaerobes in biomass recycling, suggesting that this group of organisms is involved in the degradation of accumulated biomass in coal seams, funnelling nutrients back into the microbial communities degrading coal to methane.

Who eats what? Unravelling microbial conversion of coal to methane.

Microbial communities in subsurface coal seams are responsible for the conversion of coal organic matter to methane. This process has important implications for both energy production and our understanding of global carbon cycling. Despite the environmental and economic importance of this process, little is known about which components of the heterogeneous coal organic matter are biodegradable under methanogenic conditions. Similarly, little is known about which taxa in coal seams carry out the initial stages of coal organics degradation. To identify the biodegradable components of coal and the microorganisms responsible for their breakdown, a subbituminous coal was fractionated into a number of chemical compound classes which were used as the sole carbon source for growth by a coal seam microbial community. This study identifies 65 microbial taxa able to proliferate on specific coal fractions and demonstrates a surprising level of substrate specificity among members of this coal-degrading microbial consortia. Additionally, coal kerogen, the solvent-insoluble organic component of coal often considered recalcitrant to microbial degradation, appeared to be readily converted to methane by microbial degradation. These findings challenge our understanding of coal organic matter catabolism and provide insights into the catabolic roles of individual coal seam bacteria.

Analysis of methanogens adsorption and biogas production characteristics from different coal surfaces.

The aim of this study was to examine the biogas production and the adsorption aspect of microorganism from different coals. Coal samples were obtained from Qianqiu mine and Guandi mine. Microbial populations were cultured from the coal mine drainage. After an anaerobic reaction period at about 35 °C, adsorption rate was determined by the spectrophotometer, while a scanning electron microscopy was used to observe the microorganisms on the coal and the headspace methane was analyzed using gas chromatography. Results show that the coal rank and particle size serve as important factors influencing the adsorption of microorganism and biogenic methane production. With decreasing particle size, the Qianqiu coal produced a considerable adsorption rate between 75 and 79%, while the adsorption rate of Guandi coal was between 52 and 74%. Meanwhile, the density of microorganisms from the Qianqiu coal surface demonstrated a higher level of adsorption than that of Guandi coal following the scanning electron microscopy images. Additionally, Qianqiu coal produced a higher level of biogas production (391.766-629.199 µmol/g) than that of Guandi coal (292.835-393.744 µmol/g) and the Qianqiu coal also generated a higher concentration of methane during the incubation. When the adsorption rate decreasing, the biogas production from various pulverized coals appeared to be decreased and demonstrated a positive correlation to the adsorption rate. The results of this study suggest that the adsorption behavior of microorganisms is closely related to the effect of coal biodegradation and contributes to the biogenic methane production potential.

Long-term succession in a coal seam microbiome during in situ biostimulation of coalbed-methane generation.

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.

Ecological selection for small microbial genomes along a temperate-to-thermal soil gradient.

Small bacterial and archaeal genomes provide insights into the minimal requirements for life1 and are phylogenetically widespread2. However, the precise environmental pressures that constrain genome size in free-living microorganisms are unknown. A study including isolates has shown that thermophiles and other bacteria with high optimum growth temperatures often have small genomes3. It is unclear whether this relationship extends generally to microorganisms in nature4,5 and more specifically to microorganisms that inhabit complex and highly variable environments, such as soils3,6,7. To understand the genomic traits of thermally adapted microorganisms, here we investigated metagenomes from a 45 °C gradient of temperate-to-thermal soils that lie over the ongoing Centralia, Pennsylvania (USA) coal-seam fire. We found that hot soils harboured distinct communities with small genomes and small cell sizes relative to those in ambient soils. Hot soils notably lacked genes that encode known two-component regulatory systems, and antimicrobial production and resistance genes. Our results provide field evidence for the inverse relationship between microbial genome size and temperature in a diverse, free-living community over a wide range of temperatures that support microbial life.