ReportMicrobial Physiology

Methane production from coal by a single methanogen

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Science  14 Oct 2016:
Vol. 354, Issue 6309, pp. 222-225
DOI: 10.1126/science.aaf8821

Microbes make methane from coal

Methane associated with coal beds is an important global resource of natural gas. Much of the methane in coal comes from microbial methanogenesis. Mayumi et al. characterized a strain of Methermicoccus shengliensis that, unexpectedly, is capable of making methane from the dozens of methoxylated aromatic compounds found in a variety of coal types (see the Perspective by Welte). Isotope tracer experiments showed that this organism could also incorporate carbon dioxide into methane.

Science, this issue p. 222; see also p. 184

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.

Coal-bed methane (CBM), a form of natural gas distributed in coal seams or adjacent sandstones, is a relatively untapped energy source with a large potential: The global reserves in 2014 were estimated at 50 trillion m3, equivalent to 11% of conventional natural gas resources (1). Large-scale CBM production has been implemented in the United States, Canada, Australia, and other countries worldwide. The contribution of biogenic methane to CBM is quite large (2, 3); geochemical studies have estimated that 40% of CBM produced in the United States is of microbial origin (4). Live microbial communities are present in coal seams and are associated with methanogenesis from coal in subsurface environments (510). Geomicrobiological studies have shown that enhanced CBM production in coal seams might be achieved by the stimulation of methanogenic activity (11). Although extensive efforts have been made to develop this technology, very little is known about what components of coal can be used for methanogenesis and which microorganisms possess the metabolic capabilities to do so.

Coal is an extremely complex and heterogeneous material whose structure consists of single and condensed aromatic rings (12, 13). Aromatic compounds in coal are derived from lignin monolignols and are often substituted with hydroxyl, methoxy, and carboxyl groups (14, 15). Methoxy groups are especially abundant and common in immature coal (14, 16). Because methanogenesis from coal tends to occur in immature coal rather than in mature coal (4, 17), coal-bed microorganisms may produce methane from methoxy groups. Methanogenic microorganisms in CBM fields are commonly dominated by methylotrophic methanogens belonging to the archaeal order Methanosarcinales (5, 18, 19). The methylotrophic methanogens are capable of using methyl compounds such as methanol, methylamines, and/or dimethylsulfide (20), but it is unclear whether they can directly use methoxylated aromatic compounds (MACs) as substrates.

To investigate the possibility of MACs as substrates for methylotrophic methanogens, we tested the methane production ability of one archaeal isolate (Methermicoccus shengliensis strain AmaM) obtained from a high-temperature deep subsurface oil reservoir in this study (fig. S1), and of 10 type strains belonging to the genera Methanosarcina, Methanolobus, Methanohalophilus, Methanosaeta, Methanomicrococcus, Methanococcoides, Methanohalobium, Methanosalsum, Methanomethylovorans, and Methermicoccus (i.e., M. shengliensis strain ZC-1) (21) in the order Methanosarcinales using seven types of MACs (2-methoxy-benzoate, 3-methoxy-benzoate, 4-methoxy-benzoate, 3,4,5-trimethoxy-benzoate, 3,4,5-trimethoxy-cinnamate, 1,2,3-trimethoxy-benzene, and 3,4,5-trimethoxy-benzylalcohol) as substrates. We only observed substantial methane production in the incubation of Methermicoccus shengliensis strains AmaM and ZC-1 with seven and six types of MACs, respectively (Fig. 1A). To investigate their substrate ranges, we incubated the strains AmaM and ZC-1 with 40 commercially available MACs. The results showed that the strains AmaM and ZC-1 used 35 and 34 types of MACs, respectively (Fig. 1B and table S1).

Fig. 1 Methoxydotrophic methanogenesis from various MACs.

(A) Methanogenesis from seven types of MACs by 10 type strains and one isolate belonging to the order Methanosarcinales. Each MAC was supplied with methoxy groups to a final concentration of 30 mM. Methane produced was measured after incubation for 9 months. Data are means of three individual incubations; error bars represent SD of these triplicates. (B) Substrate ranges of Methermicoccus shengliensis strains AmaM and ZC-1 for 40 types of MACs; an asterisk designates MACs analyzed for the media with coal samples. For each substrate, the average amount of methane produced (n = 3) is expressed by one of four ranges. Detailed methane production data are shown in table S1.

To confirm whether these “methoxydotrophic methanogens” could produce methane from coal, we incubated M. shengliensis AmaM with coals of different maturity levels: lignite (most immature) and subbituminous and bituminous (most mature) coals (fig. S2). A small but substantial amount of methane (7.5 to 10.8 μmol/g-coal) was produced in all of the three lignites (L-A, L-B, and L-C) and even in the subbituminous coal S-A and the bituminous coal B-A (Fig. 2A). M. shengliensis AmaM used MACs, methanol, and methylamines as substrates for methanogenesis (Fig. 1 and fig. S1), which suggests that coal may have provided some of these substrates. We analyzed growth media with coal before incubation for the 26 types of MACs usable for M. shengliensis AmaM (marked with asterisks in Fig. 1B) by gas chromatography–mass spectrometry (GC-MS). We detected either two or three types of methoxylated benzoates in each medium from which methane production was observed (Fig. 2B). The total concentrations of methanol and methylamines as well as of MACs detected in the media were too low to account for the concentrations of methane produced in the coal cultures alone (Fig. 2); for example, in bituminous coal culture B-A, 0.09 μmol/g-coal of MACs was detected but 9.39 μmol/g-coal of methane was produced. This result suggests that M. shengliensis AmaM produced methane from undetectable MACs dissolved in the media as well as those chemically or physically bound to the coal surface. This was supported by the ability of M. shengliensis AmaM to use a wide variety of MACs (>30 types of MACs) for methanogenesis (Fig. 1B).

Fig. 2 Methanogenesis from coal samples by Methermicoccus shengliensis AmaM.

(A) Methane production in a medium with lignite, subbituminous coal, or bituminous coal. Information for each coal sample is shown in fig. S2. A small amount of methane in the media without substrate was detected as a result of carryover from methanol-grown preculture. Data are means of three individual incubations; error bars represent SD of these triplicates. +, inoculated; –, not inoculated. (B) MACs, methanol, and methylamines detected in the media with coal samples before inoculation of M. shengliensis AmaM. The GC-MS analyses were performed in duplicate; average concentrations are shown. ND, not detected; TR, detected but below the range of concentration for which the calibration curve could be applied.

If methoxydotrophic methanogenesis proceeds in analogy to methylotrophic methanogenesis, it is expected from stoichiometry that ¾ mol of methane is produced from 1 mol of the methoxy group (4Ar-OCH3 + 2H2O → 4Ar-OH + 3CH4 + CO2, where Ar denotes any aromatic group). During incubation with 2-methoxy-benzoate, M. shengliensis AmaM produced methane and 2-hydroxy-benzoate with a decrease in 2-methoxy-benzoate (Fig. 3). The 2-hydroxy-benzoate produced was nearly equivalent to the 2-methoxy-benzoate consumed, which suggests that M. shengliensis AmaM produced methane via O-demethylation of the methoxy group.

Fig. 3 Methanogenesis from 2-methoxy-benzoate by Methermicoccus shengliensis AmaM.

All symbols represent means of three individual incubations; error bars represent SD of these triplicates.

We conducted stable isotope tracer experiments to elucidate the mode of metabolism in the methoxydotrophic methanogenesis. In the incubation of M. shengliensis AmaM with 2-[13C]methoxy-benzoate, the 13C contents of methane increased with increasing contents of 13C at the methoxy group (Fig. 4A), indicating that the methoxy carbon was incorporated into methane. However, the incorporation efficiency from the methoxy group to methane, estimated as the slope of the regression line between the 13C contents of methane and the methoxy group, was 63.6% (Fig. 4A). By contrast, in the incubation with [13C]methanol, we estimated that nearly all (96.4%) of the methane carbon came from the substrate methanol. This implies that additional carbon (other than methoxy carbon) is incorporated into methane during MACs-driven methanogenesis.

Fig. 4 Stable isotope tracer experiments to elucidate the mode of metabolism in methoxydotrophic methanogenesis.

(A) Carbon isotopic relationship between methane produced by M. shengliensis AmaM and either methanol or the methoxy group of 2-methoxy-benzoate added to the media. The slopes of the regression lines show the efficiency of carbon incorporation from each substrate into methane. (B) Carbon isotopic relationship between methane produced from either methanol or 2-methoxy-benzoate and carbon dioxide. The slopes of the regression lines show the efficiency of carbon incorporation from carbon dioxide into methane. Each symbol represents one of three individual incubations.

To identify this additional carbon, we evaluated whether CO2 was incorporated into methane via CO2 reduction. We incubated M. shengliensis AmaM with 2-methoxy-benzoate or methanol in the medium amended with [13C]bicarbonate. Although the 13C contents of methane increased only slightly in the presence of methanol, those of methane in the presence of 2-methoxy-benzoate increased far more substantially (Fig. 4B), indicating that CO2 is also incorporated into methane via CO2 reduction in methoxydotrophic methanogenesis. The incorporation efficiency from CO2 into methane, estimated as the slope of the regression line (Fig. 4B), was 29.6%. Considering that the incorporation efficiency from the methoxy group to methane is 63.6% (Fig. 4A), approximately one-third of methane carbon is derived from CO2 and two-thirds of methane carbon from the methoxy group. In the incubation of M. shengliensis AmaM with a variety of coals in the presence of [13C]bicarbonate, we observed the production of highly 13C-enriched methane, which indicates that methane produced from coal was mostly derived from MACs (fig. S3).

Further tracer experiments incubating M. shengliensis AmaM with 2-methoxy-benzoate in the medium amended with [2-13C]acetate revealed a small but substantial incorporation of the acetate methyl carbon into methane (fig. S4). The acetate concentration in the medium did not change during growth, implying no intentional uptake of extracellular acetate through acetoclastic methanogenesis. We therefore infer that acetyl–coenzyme A (CoA) could be a catabolic intermediate in the methoxydotrophic methanogenesis. M. shengliensis AmaM genes encode acetyl-CoA synthesis, acetyl-CoA oxidation, and CO2-reducing methanogenesis, but its genome lacks known acetogen-associated genes for O-demethylation of the methoxy group (e.g., Mtv system) (22, 23) and electron transport systems (e.g., Rnf, complete Fpo complex, etc.) (20) necessary for conventional methanogenesis (table S2). Although details of the metabolic pathway in the methoxydotrophic methanogenesis remain elusive, all the results indicate that the mode of metabolism is clearly different from the conventional methylotrophic methanogenesis.

Our finding that MACs serve as a direct substrate for methanogens may not be limited to coal-bed environments. In the deep subsurface, MACs are contained in sedimentary organic matter derived from lignin in higher plants, namely kerogen, with quantitative variation depending on the maturity (24). In fact, alkyl-methoxy-phenols with a short C1-C3 chain have been detected in the pyrolysates of immature kerogen extracted from a Cretaceous (Cenomanian) black shale (25). Kerogen is ubiquitous in sediments and accounts for most of the organic matter in subsurface environments (26). Microorganisms from the genus Methermicoccus and related clones have often been detected in deep subsurface environments worldwide (fig. S5). Methoxydotrophic methanogenesis may therefore play an important role in the biogeochemical carbon cycle in Earth as well as in the formation of biogenic gas, which accounts for more than 20% of natural gas resources, including CBM (27).

Supplementary Materials

www.sciencemag.org/content/354/6309/222/suppl/DC1

Materials and Methods

Figs. S1 to S5

Tables S1 and S2

References (2832)

References and Notes

  1. Acknowledgments: We thank Japan Petroleum Exploration Co., Ltd. (JAPEX) for providing samples from an oil reservoir; F. Nozawa, K. Shuin, Y. Shinotsuka, T. Ujiie, and X. Meng for technical support; and M. Nobu for valuable comments. Supported by JSPS KAKENHI grants JP26709070, JP25289333, JP26710012, and JP26106004. M. shengliensis strain AmaM has been deposited as accession number NBRC 112467 in the Biological Resource Center, National Institute of Technology and Evaluation (NBRC). Genomic data of M. shengliensis strain AmaM are available in the Integrated Microbial Genomes system of the U.S. Department of Energy Joint Genome Institute with ID no. 2516653088 (Gold Project ID: Gp0021722).
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