Microbial nutrient stimulation enhances in situ methanogenesis ~ 900 m depth in coalbeds: evidence from long-term field trial in Hancheng Block

Abstract

To enhance in-situ bioconversion of coalbed methane by microbial nutrient solution, two long-term operating coalbed methane wells in the Hancheng block of Shaanxi Province, China (up to 2010) were employed as the research objects. The thickness of the main coal seam is approximately 26–115 m, the salinity is appropriate (3,000–12,000 mg/L), and the permeability is low (0.001–3.503 md), which is suitable for large-scale coalbed methane exploration. After nutrient solution (660 m³) was injected, the pH value gradually increased from 6.5 to 7.5 (100th day) and then slowly decreased to 7.0 (300th day) as the accumulated VFAs were rapidly utilized by the inoculated bacteria. The bottom hole pressure recovery rate after injection was 0.37–0.95 MPa (15th day), which was better than 0.7–0.8 MPa (40th day) before injection. The microbial community structure indicated that the bacteria in both wells were more dominant, while the archaea in the test well increased to 6.3%. The relative abundance of Methanobacterium formicicum increased from 0.25% to 4.14%. At the species level, the dominant microorganisms in the control well were Ralstonia pickettii and Variovorax sp., while those in the test well were Stutzerimonas stutzeri and Variovorax paradoxus. The typical denitrifying microorganisms Stutzerimonas stutzeri and Comamonas sp. were significantly increased by 14.29% and 5.53%. The abundance of Ralstonia pickettii decreased by 18.41%, and the methanooxidizing bacteria genus accounted for 15.89%. Gene function enrichment analysis indicated that compared with the phenylpropionate degradation mode of the control group, the metabolic mode after microbial intervention shifted to pyruvate metabolism.

Keywords

CBM / Microbial nutrient / In-situ bioconversion / Community structure

References

[1] Aitken CM, Jones DM, Larter SR. Anaerobic hydrocarbon biodegradation in deep subsurface oil reservoirs. 431 (2004)

[2] Barker JF, Fritz P. Carbon isotope fractionation during microbial methane oxidation. Nature, 1981, 293: 289-291.

[3] Barnhart EP, Ruppert LF, Hiebert R, Smith HJ, Schweitzer HD, Clark AC, Weeks EP, Orem WH, Varonka MS, Platt G, Shelton JL, Davis KJ, Hyatt RJ, McIntosh JC, Ashley K, Ono S, Martini AM, Hackley KC, Gerlach R, Spangler L, Phillips AJ, Barry M, Cunningham AB, Fields MW. In situ enhancement and isotopic labeling of biogenic coalbed methane. Environ Sci Technol, 2022, 56: 3225-3233.

[4] Boone DR, Johnson RL, Liu Y. Diffusion of the interspecies electron carriers h2 and formate in methanogenic ecosystems and its implications in the measurement of k m for h2 or formate uptake. Appl Environ Microbiol, 1989, 55: 1735-1741.

[5] Bowers RM, Kyrpides NC, Stepanauskas R, Harmon-Smith M, Doud D, Reddy TBK, Schulz F, Woyke T. Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea (2017)

[6] Campbell BC, Gong S, Greenfield P, Midgley DJ, Paulsen IT, George SC. Aromatic compound-degrading taxa in an anoxic coal seam microbiome from the Surat Basin, Australia. FEMS Microbiol Ecol, 2021, 97. ArticleID: fiab053

[7] Campbell BC, Greenfield P, Barnhart EP, Gong S, Midgley DJ, Paulsen IT, George SC. Krumholzibacteriota and Deltaproteobacteria contain rare genetic potential to liberate carbon from monoaromatic compounds in subsurface coal seams. Mbio, 2024, 15. ArticleID: e01735-23

[8] Feng G, Zeng Y, Wang H-Z, Chen Y-T, Tang Y-Q. Proteiniphilum and Methanothrix harundinacea became dominant acetate utilizers in a methanogenic reactor operated under strong ammonia stress. Front Microbiol, 2023, 13. ArticleID: 1098814

[9] Gieg LM, Fowler SJ, Berdugo-Clavijo C. Syntrophic biodegradation of hydrocarbon contaminants. Curr Opin Biotechnol, 2014, 27: 21-29.

[10] Grossman EL, Coffman BK, Fritz SJ, Wada H. Bacterial production of methane and its influence on ground-water chemistry in east-central Texas aquifers. Geol, 1989, 17: 495.

[11] Guo H, Zhang M, Chen Z, Shen Y, Lv J, Xu X, Yu H. The metabolic process of methane production by combined fermentation of coal and corn straw. Biores Technol, 2021, 337. ArticleID: 125437

[12] Hanson CA, Fuhrman JA, Horner-Devine MC, Martiny JBH. Beyond biogeographic patterns: processes shaping the microbial landscape. Nat Rev Microbiol, 2012, 10: 497-506.

[13] Hou Z, Zhou Q, Mo F, Kang W, Ouyang S. Enhanced carbon emission driven by the interaction between functional microbial community and hydrocarbons: an enlightenment for carbon cycle. Sci Total Environ, 2023, 867. ArticleID: 161402

[14] Hu L, Yang Y, Yan X, Zhang T, Xiang J, Gao Z, Chen Y, Yang S, Fei Q. Molecular mechanism associated with the impact of methane/oxygen gas supply ratios on cell growth of Methylomicrobium buryatense 5GB1 through RNA-Seq. Front Bioeng Biotechnol, 2020, 8: 263.

[15] In ’T Zandt MH, Beckmann S, Rijkers R, Jetten MSM, Manefield M, Welte CU. Nutrient and acetate amendment leads to acetoclastic methane production and microbial community change in a non‐producing Australian coal well. Microb Biotechnol, 2018, 11: 626-638.

[16] Izadi P, Izadi P, Eldyasti A, Cheng C, Beckley M. Influence of vitamin coupled with micronutrient supplement on the biomethane production, process stability, and performance of mesophilic anaerobic digestion. Biomass Bioenerg, 2020, 141. ArticleID: 105706

[17] Jung W, Cho S, Choi Y, Lee H, Na J-G, Lee J. Methane capture, utilization, and sequestration technology based on biological ectoine production using Methylotuvimicrobium alcaliphilum 20Z. Chem Eng J, 2024, 498. ArticleID: 155339

[18] Kim NY, Kim SN, Kim OB. Long-term adaptation of Escherichia coli to methanogenic co-culture enhanced succinate production from crude glycerol. J Ind Microbiol Biotechnol, 2018, 45: 71-76.

[19] Kohtz AJ, Petrosian N, Krukenberg V, Jay ZJ, Pilhofer M, Hatzenpichler R. Cultivation and visualization of a methanogen of the phylum Thermoproteota. Nature, 2024, 632: 1118-1123.

[20] Lalucat J, Gomila M, Mulet M, Zaruma A, García-Valdés E. Past, present and future of the boundaries of the Pseudomonas genus: proposal of Stutzerimonas gen. Nov.. Syst Appl Microbiol, 2022, 45. ArticleID: 126289

[21] Li Y, Zhang J, Wen X, Mazarji M, Chen S, Liu Q, Zhao S, Feng L, Li G, Zhou H, Pan J. Advancing anaerobic digestion with MnO2-modified biochar: insights into performance and mechanisms. Sci Total Environ, 2024, 954. ArticleID: 176303

[22] Lovley DR. Syntrophy goes electric: direct interspecies electron transfer. Annu Rev Microbiol, 2017, 71: 643-664.

[23] Mayumi D, Tamaki H, Kato S, Igarashi K, Lalk E, Nishikawa Y, Minagawa H, Sato T, Ono S, Kamagata Y, Sakata S. Hydrogenotrophic methanogens overwrite isotope signals of subsurface methane. Science, 2024, 386: 1372-1376.

[24] Midgley DJ, Hendry P, Pinetown KL, Fuentes D, Gong S, Mitchell DL, Faiz M. Characterisation of a microbial community associated with a deep, coal seam methane reservoir in the Gippsland Basin, Australia. Int J Coal Geol, 2010, 82: 232-239.

[25] Mlinar S, Weig AR, Freitag R. Influence of NH₃ and NH₄⁺ on anaerobic digestion and microbial population structure at increasing total ammonia nitrogen concentrations. Bioresour Technol, 2022, 361. ArticleID: 127638

[26] Papendick SL, Downs KR, Vo KD, Hamilton SK, Dawson GKW, Golding SD, Gilcrease PC. Biogenic methane potential for Surat Basin, Queensland coal seams. Int J Coal Geol, 2011, 88: 123-134.

[27] Penner TJ, Foght JM, Budwill K. Microbial diversity of Western Canadian subsurface coal beds and methanogenic coal enrichment cultures. Int J Coal Geol, 2010, 82: 81-93.

[28] Qi M, Su X, Zhao W, Wang Q, Zhou Y. Metabolic mechanisms of carbon and nitrogen interactions during anaerobic digestion of coal. Renewable Energy, 2024, 237. ArticleID: 121653

[29] Raghoebarsing AA, Pol A, Van De Pas-Schoonen KT, Smolders AJP, Ettwig KF, Rijpstra WIC, Schouten S, Damsté JSS, Op Den Camp HJM, Jetten MSM, Strous M. A microbial consortium couples anaerobic methane oxidation to denitrification. Nature, 2006, 440: 918-921.

[30] Ruan Z, Chen K, Cao W, Meng L, Yang B, Xu M, Xing Y, Li P, Freilich S, Chen C, Gao Y, Jiang J, Xu X. Engineering natural microbiomes toward enhanced bioremediation by microbiome modeling. Nat Commun, 2024, 15. ArticleID: 4694

[31] Schmidt MP, Mamet SD, Senger C, Schebel A, Ota M, Tian TW, Aziz U, Stein LY, Regier T, Stanley K, Peak D, Siciliano SD. Positron‐emitting radiotracers spatially resolve unexpected biogeochemical relationships linked with methane oxidation in Arctic soils. Glob Change Biol, 2022, 28: 4211-4224.

[32] Scott AR. Mastalerz M, Glikson M, Golding SD. Improving coal gas recovery with microbially enhanced coalbed methane. Coalbed methane: scientific, environmental and economic evaluation, 1999. Dordrecht, Springer: 89-110.

[33] Singh A, Schnürer A, Dolfing J, Westerholm M. Syntrophic entanglements for propionate and acetate oxidation under thermophilic and high-ammonia conditions. ISME J, 2023, 17: 1966-1978.

[34] Stone K, Hilliard M, Badr K, Bradford A, He QP, Wang J. Comparative study of oxygen-limited and methane-limited growth phenotypes of Methylomicrobium buryatense 5GB1. Biochem Eng J, 2020, 161. ArticleID: 107707

[35] Tang D, Chen M, Huang X, Zhang G, Zeng L, Zhang G, Wu S, Wang Y. SRplot: a free online platform for data visualization and graphing. PLoS ONE, 2023, 18. ArticleID: e0294236

[36] Tian H, Du Y, Deng Y, Sun X, Xu J, Gan Y, Wang Y. Identification of methane cycling pathways in Quaternary alluvial-lacustrine aquifers using multiple isotope and microbial indicators. Water Res, 2024, 250. ArticleID: 121027

[37] Wang B, Wang Y, Cui X, Zhang Y, Yu Z. Bioconversion of coal to methane by microbial communities from soil and from an opencast mine in the Xilingol grassland of northeast China. Biotechnol Biofuels, 2019, 12. ArticleID: 236

[38] Wang J, Gao P, Pan X, Fan K, Li Y, Gao Y, Gao Y. An aerobic denitrifier Pseudomonas stutzeri Y23 from an oil reservoir and its heterotrophic denitrification performance in laboratory-scale sequencing batch reactors. Int Biodeterior Biodegrad, 2022, 174. ArticleID: 105471

[39] Wang J, Zhao Y, Zhou M, Hu J, Hu B. Aerobic and denitrifying methanotrophs: dual wheels driving soil methane emission reduction. Sci Total Environ, 2023, 867. ArticleID: 161437

[40] Wang C, Xiao Y, Wang Y, Liu Y, Yao Q, Zhu H. Comparative genomics and transcriptomics insight into myxobacterial metabolism potentials and multiple predatory strategies. Front Microbiol, 2023, 14. ArticleID: 1146523

[41] Wang L, Cheng X, Guo Y, Cao J, Sun M, Hwang J-S, Liu R, Fang J. Novel isolates of hydrogen-oxidizing chemolithoautotrophic Sulfurospirillum provide insight to the functions and adaptation mechanisms of Campylobacteria in shallow-water hydrothermal vents. mSystems, 2024, 9. ArticleID: e00148-24

[42] Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, Feng T, Zhou L, Tang W, Zhan L, Fu X, Liu S, Bo X, Yu G. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation Camb Mass, 2021, 2: 100141

[43] Wu Z, Liu G, Ji Y, Li P, Yu X, Qiao W, Wang B, Shi K, Liu W, Liang B, Wang D, Yanuka-Golub K, Freilich S, Jiang J. Electron acceptors determine the BTEX degradation capacity of anaerobic microbiota via regulating the microbial community. Environ Res, 2022, 215. ArticleID: 114420

[44] Wu K, Zhou L, Tahon G, Liu L, Li J, Zhang J, Zheng F, Deng C, Han W, Bai L, Fu L, Dong X, Zhang C, Ettema TJG, Sousa DZ, Cheng L. Isolation of a methyl-reducing methanogen outside the Euryarchaeota. Nature, 2024, 632: 1124-1130.

[45] Xia DP, Zhang HW, Huang S, Dong ZW. Change and mechanism of liquid phase products in coal fermentation cogeneration of hydrogen and methane. J China Coal Soc, 2019, 10: 3098-3106

[46] Xia Q, Liu F, Sun S, Huang W, Zhao Z, Yang F, Lei Z, Huang W, Yi X. Coupling iron sludge addition and intermittent aeration for achieving simultaneous methanogenesis, feammox, and denitrification in a single reactor treating fish sludge. Environ Sci Technol, 2023, 57: 15065-15075.

[47] Xie J, Chen Y, Cai G, Cai R, Hu Z, Wang H. Tree visualization by one table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic Acids Res, 2023, 51: W587-W592.

[48] Xu F, Hou W, Xiong X, Xu B, Wu P, Wang H, Feng K, Yun J, Li S, Zhang L, Yan X, Fang H, Lu Q, Mao D. The status and development strategy of coalbed methane industry in China. Pet Explor Dev, 2023, 50: 765-783.

[49] Xu Z, Wang Y, Qian Y, Zhang J, Tu Y, Gu S, Sun M. Distribution and leaching characteristics of metal elements in coal gasification fine slag: insights into inorganic and organic acid systems. Sep Purif Technol, 2025, 361. ArticleID: 131517

[50] Yadav S, Koenen M, Bale NJ, Reitsma W, Engelmann JC, Stefanova K, Damsté JSS, Villanueva L. Organic matter degradation in the deep, sulfidic waters of the Black Sea: insights into the ecophysiology of novel anaerobic bacteria. Microbiome, 2024, 12: 98.

[51] Yu J, Zhao L, Yao Z, Feng J, Yuan X, Wang H, Liang Y, Chen J, Du Y, Shen R. Deeper insights into the synergy of material transformation, microbial network, and energy balance during pilot thermophilic and mesophilic dry anaerobic digestion systems. Sci Total Environ, 2023, 891. ArticleID: 164410

[52] Yue J, Xu J, Zhang J, Shi B, Zhang M, Li Y, Wang C. Gas displacement characteristics during the water wetting process of gas-bearing coal and microscopic influence mechanism. Sci Total Environ, 2024, 949. ArticleID: 175034

[53] Zhang Y, Li C, Yuan ZW, Wang R, Angelidaki I, Zhu G. Syntrophy mechanism, microbial population, and process optimization for volatile fatty acids metabolism in anaerobic digestion. Chem Eng J, 2023, 452. ArticleID: 139137

[54] Zhang A, Xu L, Wu L, Zhou Y, Zhong J, Li W, Guo J, Chen Z, Cheng H, Zhou H, Wang Y. Environmental heterogeneity and geographic isolation drive prokaryotic community succession in acid mine drainage from abandoned coal mines. J Environ Manage, 2025, 395. ArticleID: 127780

[55] Zhao C, Khan A, Wei Z, Jinghong W, Fangzheng Z, Guinan S, Yanhua H, Dan W, Zongjun C, Weidong W. Metabolic pathway analysis of methane from methanol as substrate in microbial consortium. Bioresour Technol, 2024, 413. ArticleID: 131517

[56] Zhao Y, Yuan X, Du Z, Niu J, Song J, Zhai S, Liu Y, Nuramkhaan M. New insights into N₂O emission and electron competition under different chemical oxygen demand to nitrogen ratios in a biofilm system. Sci Total Environ, 2024, 949. ArticleID: 175265

[57] Zhou Z, Zhang C, Liu P, Fu L, Laso-Pérez R, Yang L, Bai L, Li J, Yang M, Lin J, Wang W, Wegener G, Li M, Cheng L. Non-syntrophic methanogenic hydrocarbon degradation by an archaeal species. Nature, 2022, 601: 257-262.

[58] Zhou Y, Su X, Zhao W, Wang L, Fu H. Enhanced coal biomethanation by microbial electrolysis and graphene in the anaerobic digestion. Renew Energy, 2023, 219. ArticleID: 119527

Funding

National Natural Science Foundation of China(32471540, 32371540, 21878128)

National Key Research and Development Program of China(2022YFC3401300; 2024YFC3015001)

Basic Research Program of Jiangsu and supported by the Jiangsu Basic Research Center for Synthetic Biology(Grant No. BK20233003)

RIGHTS & PERMISSIONS

Jiangnan University