Influence of Metabolism of Firmicutes on the microstructure of anthracite under nitrogen source stimulation

Influence of Metabolism of Firmicutes on the microstructure of anthracite under nitrogen source stimulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of Metabolism of Firmicutes on the microstructure of anthracite under nitrogen source stimulation Chunshan Zheng, Chengcai Zhao, Bingjun Liu, Sheng Xue, Yang Yang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4673807/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Promoting the permeability of deep, low-permeability coal seams through biological means is currently a research hotspot for enhancing the efficiency of coalbed methane extraction. There are few reports in the literature on whether it is possible to promote the development of the microstructure of the coal matrix by the degradation and metabolism of certain groups of functional microorganisms under the stimulation of nitrogen sources. In this study, we selected anthracite coal from Sihe Mine for microbial anaerobic degradation culture experiments. The effects of adding functional microorganisms on the microstructure of anthracite coal under the stimulation of nitrogen source was analyzed by high-throughput sequencing of samples before and after the cultivation and microcharacterization experiments of coal samples. The results showed that the peak amount of residual methane desorption from the coal during the biodegradation process in the experimental group reached 0.640 mL/g coal, and the cumulative amount of methane desorption in the whole period was as high as 1.318 mL/g coal. 16S rRNA high-throughput sequencing results indicated that the bacterial community structure had undergone significant succession after the biodegradation experiments, and that the Firmicutes represented by Bacillus(82.41% of the total) occupied the dominant niche. Metabolic pathway analysis based on KEGG database showed that the degradation of aromatic compounds by microorganisms appeared to be significantly enhanced by the addition of nitrogen sources. Alaso, the relative abundance of a number of key metabolic enzyme genes capable of catalyzing the introduction of oxygen-containing functional groups into the structure of the coal molecule and the de-cyclization reaction were increased. FTIR experiments revealed that biodegradation stimulated by nitrogen source reduced the aromaticity of coal by 59.62% and enhanced the hydroxyl functional group content by 1.822 times.Mercury pressure and low-temperature nitrogen adsorption experiments showed that the micropore pore volume of the treated coal decreased by 34.09%, and the macropore pore volume accounted for an increase of 168.28%, with an average pore size increment of 60.72 nm, and the adsorption level of the gases decreased by 46.1%. Therefore, the nitrogen source can stimulate Firmicutes on the degradation of polycyclic aromatic hydrocarbon and increase the content of oxygen-containing functional groups, which might promote the development of pores in coal and make the difficult-to-desorption methane desorb rapidly. Firmicutes Microbial Degradation Metabolism Coal Microstructure Enhanced Methane Desorption Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction With the gradual depletion of shallow minerals and the gradual shift of mining to the deeper parts of the coal resources under a long period of large-scale development, the gas disaster problems encountered in the coal production process have become increasingly serious. (1)(2) Coalbed methane (mainly composed of methane) has a high calorific value and is cleaner than coal and oil. As an important source of unconventional natural gas, it is enriched in the complex double pore structure of coal seams, (3) especially in Chinese coal seams due to the characteristics of poor pore and fissure development and low permeability, (4),(5) which leads to the difficulty of coalbed methane extraction in mines, and poses a great challenge to the safe production of coal mines. (5) In order to increase the permeability of high gas and low permeability coal seams and strengthen the level of methane transportation, there are mainly physical permeability enhancement technology, (7) chemical permeability enhancement technology (8) and biological permeability enhancement technology. (9) Among them, the physical penetration technology is mainly through water jet, deep hole blasting, mining protective layer technology to make the coal seam decompression rupture coal body, promote the development of pore and fissure structure, improve the transport characteristics of the coal body, so as to increase the permeability of the coal body, and improve the effect of gas extraction, which has been widely used in the coal mine site. (10)(11)(12) For the research of chemical penetration enhancement technology, one part of it cleans the mineral components of the coal body through strong acid solution and transforms the pore and fissure structure of the coal (13) ; the other part of it uses organic solvents to dissolve and remove the small molecule soluble substance in the coal body and then make its molecular structure evolve, thus realizing the penetration of the coal body. (14) Biological permeability enhancement technology has the advantages of green, safe, economic and sustainable, so it has far-reaching and significant theoretical and practical significance in improving the conditions of coalbed methane extraction and developing clean energy. (15) Scott first suggested in 1999 that permeability of coal bodies could be enhanced by microbial degradation. (16) Since then, scholars around the world have carried out laboratory simulations and field experiments to verify the effect and mechanism of microbial anaerobic degradation on coal permeability enhancement. Currently, studies have been conducted to describe the effect of microbial metabolism on the microstructure of coal, some of the studies have proved that the micropore structure of coal will develop into larger pores and improve the pore fracture structure after microbial degradation. Bao et al. found that biodegradation can achieve effects such as pore expansion and seam creation, which are favorable for methane seepage and transport. (17) Wang et al. carried out a simulation of biogenic gas production and found that some micropores and transition pores in the coal samples were transformed into macropores and that side chains and hydroxyls of the coal molecular structure were readily metabolized by methanogenic bacteria, which partially oxidized to form carboxylic acids. (18) Pandey et al. concluded that coal biotransformation resulted in a swelling of the coal matrix, decreasing fractal dimension and contributing to the formation of new pore cleavages. (19) Lu et al.used microorganisms to treat high volatile bituminous coal and sub-bituminous coal, and confirmed that the volume of micropores and mesopores of coal decreased, the volume and porosity of macropores increased, the crystallinity of coal decreased, organic matter degraded, and pore connectivity enhanced. (20) In addition, some scholars have confirmed through microbiology, metabolomics and spectroscopy that the molecular structure and crystal structure of coal will evolve after the action of key products of biometabolism, and its methanophilicity will be reduced, which will help to improve the permeability of coal.Guo et al. found that after the degradation of coal by biometabolism through the FTIR and XRD tests, the oxygen-containing functional groups of coal increased, and the aromatic rings were partially opened and hydroxyl groups introduced at the breaks, and their contents increased accordingly. Hydroxyl groups were introduced at the breaks, and their content increased accordingly, the disorder of macromolecules increased, and the degree of crystallization decreased. (21) Haider et al. found that aromatic and aromatic compounds in coal were degraded after biodegradation experiments using lignite from the Thar Coalfield, and that the degraded organic components may have applications in biomethane synthesis. (22) Yang et al. used a metabolomics approach to characterize the metabolites of the Great South Lake low rank coal addition, P. huatugouensis, and some key metabolites could act on the coal's ester, ether and metal bonds, thus depolymerizing the macromolecular structure into liquid organic molecules such as alcohols, aldehydes, ketones. (23) Xia et al. concluded that microorganisms break down the covalent bonds and functional groups of the macromolecules in coal by secreting extracellular enzymes in the degradation metabolism, which ultimately increase the gas content of coal seams, change the coal's pore structure, and decrease the fractal dimensions of the coal surface to make the coal surface smooth. (24) However, most of the current studies on bio-penetration enhancement are on biodegradation of low-order coal using a single means, which is less effective when applied to low-permeability anthracite. Therefore, it is of great significance to conduct joint research on the whole process of microorganisms driving methane desorption, degradation, and permeability enhancement on high-gas, low-permeability coal mines coals at multiple levels. In this paper, we selected the poorly developed pore space and low permeability anthracite coal from Temple River Mine to add highly efficient degrading bacterial agents for anaerobic degradation indoor cultivation experiments, and monitored the dynamic desorption amount of residual methane in the coal in the process of the experiments. We analyzed the microbial community structure succession law and metabolic function variability by 16s rRNA high-throughput sequencing; jointly characterized the developmental characteristics of the pore structure of the coal samples after the experiments based on pressurized mercury and low-temperature nitrogen adsorption experiments; and investigated the evolution law of the molecular structure of the coal by using FTIR experiments. This paper explores the microstructure evolution of coal by Firmicutes under the stimulation of nitrogen source, reveals the mechanism of microorganisms in the anaerobic degradation of coal, and provides data support for increasing the permeability of high-gas and low-permeability coal seams and strengthening the level of methane transportation. 2 Materials and Methods 2.1 Coal sample collection and coal quality parameters Fresh coal samples were collected from Shanxi Sihe Mine, and immediately after the samples were obtained from underground mining, they were placed in sterile self-sealing bags and wrapped in ice for low-temperature transportation to the laboratory, and were subjected to industrial and elemental analyses respectively, and the results are shown in the following Table 1 . After surface excision of the samples, the samples were crushed, milled, and screened, and the 60–80 mesh of the coal powder was collected and preserved at low temperatures for the subsequent experiments. Table 1 Ultimate/Proximate analysis of raw coal samples. Sample Mad (%) Aad (%) Vad (%) FCad (%) N (%) C (%) H (%) S (%) Coal of Sihe Mine 1.60 20.91 10.08 67.41 1.60 47.61 3.73 1.27 2.2 Experimental design and testing The functional microorganisms added in the experiment were obtained by pumping and filtering the highly efficient degradation functional bacterial agent and using sterile water to mix and then repeat the operation for 3 times. (Bacterial agent composition: Bacillus accounted for 56.80%, Clostridium accounted for 23.28%, Paraclostridium accounted for 19.92%, and the bacterial concentration in the agent was 10 8 ~10 9 cfu/mL). NH 4 Cl 0.81g/L , MgCl 2 ﹒6H 2 O 0.17g/L , CaCl 2 ﹒2H 2 O 0.1g/L ，FeCl 2 ·4H 2 O 0.07g/L，Na 2 HPO 4 ·12H 2 O 1.5g/L，KH 2 PO 4 1.5g/L，sterile water. The control group (CK) was added 50 g of coal sample and 100 mL of sterile water; Treat-1 (T-1) A 50 g coal sample was taken as substrate and placed in a 500 mL culture flask, to which functional microorganisms (120 mL of bacteriophage filtration), 100 mL of basal saline medium, 0.5 g of cysteine hydrochloride, and 2 ml of 10 mmol 2-bromoethanesulfonic acid sodium salt (BES) were added to inhibit the methanogenic potential of the microorganisms and to ensure that the headspace methane was generated from the residual methane in the coal. (25) Treat-2 (T-2) was identical to T-1 except for the addition of tryptone 1g. All three sets of experiments were sealed with nitrile plugs and three parallel experiments were performed. The initial anaerobic environment of the culture bottles was replaced with high-purity nitrogen for three times using a vacuum filtration device and a sterile syringe to replace the headspace air. The culture bottles, centrifuge tubes and culture medium used in the experiment were autoclaved, and the experimental process was carried out on the aseptic operating table. The experiments were carried out at a constant temperature of 35 ℃ for 30 d. In order to obtain pure coal samples, the residual coal was separated from the experimental samples by suction filtration with sterile water for several times, and then the coal samples were dried in an electric blast drying oven at 35 ℃ until constant weight and then sealed and stored. During the incubation experiments, the methane concentration percentage in the headspace gas inside the incubation bottles during the biodegradation process was detected using a capillary analytical gas chromatograph model GC-8900 from Shandong Lunan Xinke. The temperature of the injection chamber of the gas chromatograph was 105 ℃, the temperature of the column chamber was 102 ℃, the temperature of the thermal conductivity cell was 116 ℃, and the bridge current was set at 150 mA. High-purity nitrogen was used to replace the headspace gas in the incubation vials when the amount of methane desorbed was stabilized, i.e., one period of gas desorption. 2.3 High-throughput sequencing of 16S rRNA gene amplicons and gene function analysis experiments MB AquaScreen Fast Extract was used to extract total genomic DNA from CK, T-1 and T-2 on days 0 and 30, which was then stored on dry ice and sent to UW Genetics Ltd. in China, where the microbial communities in the culture system were analyzed by high-throughput sequencing of the 16S rRNA gene amplicons on the HiSeq platform.515F/907R (515F : 5' -GTG CCA GCM GCC GCG G-3'; 907R: 5' -CCG TCA ATT CMT T TR AGT TT-3' ) primer pairs were used to amplify the bacterial 16S rRNA gene. Clustering was performed using UPARSE at 97% similarity, representative sequences of OTUs were obtained and chimeras were identified and deleted using UCHIME (v4.2.40), and classified and annotated according to SILVA reference data (v128). Sequences classified as "mitochondrial" or "unassigned" were removed. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG database), we screened metabolic pathways related to the degradation of methane and molecular structures in anthracite and analyzed the differences in key gene functions. 2.4 Experiments on the determination of coal pore structure parameters Low-temperature liquid nitrogen adsorption experiments were carried out using a high-performance specific surface area and microporous analyzer, BSD-PM, manufactured by Best Instrument Technology (Beijing) Co. About 1.5 g of dried coal samples were taken and placed in a sample tube and degassed under vacuum at 105°C for 6 hrs. The high performance fully automated mercury palyzer AutoPore IV 9500 produced by Shenzhen Huapu General Technology Co.The pressure range was from 1 to 33000 psia, the accuracy of mercury feed was 0.1 µL, and the equilibrium time of each pressure collection point was 10 s. The pressure range was from 1 to 33000 psia, and the accuracy of mercury feed was 0.1 µL. The coal sample used in the experiment was Shanxi Temple River coal, which belongs to anthracite, so the analysis was performed using the method of pore size structure division of high coal rank coal by Qin Yong, i.e., micropore ( 400nm). (26) The results of low-temperature liquid nitrogen adsorption experiments were used for the analysis of micopores and transitional pore for Raw coal, T-1 and T-2 coal samples, and the results of mercury compression experiments were used for the analysis of mesopores and macropores.When dealing with the results of low-temperature liquid nitrogen adsorption experiments, the NLDFT model, which is more accurate for analyzing micropores, was chosen for the pore size distribution at < 15 nm, and the classical BJH desorption method was chosen for the 15–50 nm segment. (27)(28) In addition, due to the existence of voids between the coal samples in the mercury pressure experiment, the pressure point of 0.4 Mpa was selected as the initial mercury absorption reference point in accordance with previous studies . (29) 2.5 Experiments on the determination of coal molecular structure parameters FTIR experiments on residual coal from Raw coal, T-1 & T-2 coal samples were performed using a Bruker INVENIO infrared spectrometer, Germany.The dried coal samples were mixed with KBr powder in the ratio of 1:100, evenly ground and put into a tablet press to make pressed tablets, which were placed in the sample bin of the infrared spectrometer for testing. The infrared spectrograms of the three sets of samples were split-peak fitted using PeakFit v4.12. In order to further quantitatively characterize the law of evolution of the internal micro-molecular structure of coal, it was calculated from the split-peak fitting data. 3 Results and Discussion 3.1 Microbial community structure succession pattern during microbial degradation process The changes in the methane concentration in the headspace gas inside the culture flasks during the 30-day experiments of CK, T-1, and T-2 are shown in Fig. 2 (a).The dynamic trend of methane concentration in CK, T-1 and T-2 was basically similar, showing a rapid increase in methane concentration at the beginning, a leveling off of the growth rate of methane concentration in the middle, and almost no growth or even a decreasing trend at the end of the period of desorption. Among them, the methane concentration in the headspace gas of CK was always less than 1% during the experiment, and there was almost no methane in the headspace gas in the fourth period. In group T-1, the methane concentration in the headspace gas was always higher than that in CK, and the peak methane concentration in the headspace gas was almost the same as that in the first three periods, which was 0.122 ml/g of coal, and there was still methane desorbed continuously in the fourth period. Coal contains a large amount of adsorbed and free methane in different pore structures. (31) In the T-2 group, free methane molecules were rapidly desorbed from the coal at the initial stage of each period, and the methane concentration in the headspace increased rapidly, with a maximum peak value of 0.640 mL/g coal, which was 5.24 times higher than that of T-1. In addition, the methane desorption in the T-2 period showed a stepwise downward trend in sequence, with a decrease of about 50% of that of the previous period, and in the last period, the methane desorption was greatly reduced, and its methane desorption was reduced to 0.132 mL/g, and the growth rate of methane concentration tends to be gentle at 28 d. The dynamic trend of methane concentration in T-1 indicated that the microorganisms were able to enhance the further desorption of residual methane from the coal to a certain extent under the environment of inorganic salt medium, but their metabolic reactions were less active and the effect of enhanced methane desorption was weaker due to the scarcity of metabolic substrates at the initial stage. With the increase of time, the residual methane was gradually desorbed from the coal, and the amount of methane desorption was almost the same in different periods, which indicated that the advantageous flora to enhance the desorption of residual methane evolved in the culture environment during the experimental process, and the metabolism substrate was increased to improve the microbial degradation activity. The dynamic trend of methane concentration in T-2 indicated that after the addition of tryptone to the culture environment, it provided abundant nitrogen and amino acids for the metabolic activity of microorganisms, and its metabolic activity was obviously enhanced, and the effect of methane desorption was significant. However, after a large amount of free methane is gradually desorbed in larger pores, it is necessary to desorption free and adsorbed methane which is difficult to desorption from small and medium-sized pores, so it is difficult to maintain the initial methane desorption rate. (32) The relative abundance of microbial community composition in the three culture environments is shown in Fig. 2 (b).Initial samples were taken at d 0 of the experiment, when the microbial community consisted mainly of the native microorganisms of coal, with Pseudomonas (53.17 ± 10.94%) the dominant genus of in situ microorganisms, followed by Burkholderia-Caballeronia-Paraburkholderia (21.63 ± 9.37%) and Bacillus (2.24 ± 1.81%) were more predominant. The microbial community composition within the culture system changed on the 30th d of the experiment. the original dominant genus Pseudomonas in CK in the aqueous environment was absolutely dominant with a relative abundance of 88.49 ± 5.18%. While Brevibacillus had the highest percentage of microbial community composition at the genus level in T-1, with a relative abundance of 89.26 ± 2.27%, Bacillus (56.53 ± 7.05%) was the dominant genus. T-2 had the most abundant microbial community composition due to the presence of more reaction substrates. Bacillus (56.53 ± 7.05%) had the largest percentage of relative abundance and Tissierella (11.53 ± 8.20%), Clostridium (7.59 ± 7.27%) and Paraclostridium (6.77 ± 6.97%) were the dominant genera. The microbial community in the CK incubation environment did not change significantly due to the presence of water only, and Pseudomonas always dominated. Pseudomonas is thought to have a good solubilizing effect on low rank coal, and has the property of decomposing proteins, glucose, and acid production. (33) In T-1 the microbial community changed and Brevibacillus became the dominant genus. Majid Rasool Kamli et al. concluded that Brevibacillus has a wide range of enzymatic activities and can degrade a wide range of compounds and they are resistant to heavy metals and are effective strains for coal degradation. (34) Thus the increased proportion of Brevibacillus in the microbial community allowed the coal to be degraded to some extent, slightly facilitating the desorption of residual methane within the coal. When a nitrogen source is added to the culture environment, the microbial community undergoes a succession in which the dominant genera Bacillus, Tissierella, Clostridium and Paraclostridium belong to the Firmicutes.Several articles have shown that these four genera are capable of degrading complex biomasses and compounds in anaerobic environments, providing metabolic substrates for other microorganisms through the decomposition of sugars or peptones to produce a mixture of organic acids and alcohols, as well as reducing the nucleation structure of the coal and enhancing the permeability and porosity of the coal. (35)−(38) Among them, Bacillus , as the most dominant genus, has been indicated to be involved in the degradation of organic matter in a variety of environments, e.g., phosphate degradation in contaminated soils, and increasing the degradation efficiency of baijiu lees by increasing cellulase activity. (39),(40) In addition, Bacillus exhibits active carbohydrate and amino acid metabolism, and is characterized not only by strong secretion of enzymes that stimulate the metabolic activity of other microorganisms, but also by the production of bacteriocins that inhibit pathogenic bacteria. The results of microbial community succession in T-2 showed four genera belonging to the Firmicutes, whose mutual stimulatory effects during metabolism enhanced the anaerobic degradation of the coal by microorganisms, which led to a significant increase in desorption of residual methane from coal 3.2 Differential analysis of microbial degradation metabolic functions Figure 3 (a) demonstrates the differential analysis of major metabolic functions by KEGG level 3 for the three groups of experiments, and the effect of microbial degradation of coal may be related to functional genes for degradation of aromatic compounds, etc. (42) The screening of relevant metabolic pathways revealed that the metabolic activities of microorganisms enhanced the degradation of aromatic compounds and fatty acids in coal except nitrotoluene, and this metabolic degradation was more significant under the stimulation of nitrogen sources, especially in the degradation functions of xylene, styrene, and polycyclic aromatic hydrocarbons (PAHs).In addition, the methane metabolism function of the experimental group with the addition of BES showed a decreasing trend indicating that BES inhibited the methane metabolism function of the bacteria, and the methane desorbed in the experiments was derived from the coal residue, and no additional biomethane was synthesized. To further investigate the key degradation metabolites during microbial metabolism, the differences in enzyme genes related to aromatic compound degradation and methane metabolism were analyzed in each experimental group by KEGG database as shown in Fig. 3 (b). In the methane metabolic pathway, acetate and coenzyme M are crucial intermediates for the synthesis of biomethane. (43),(44) Acetate kinase (EC:2.7.2.1, K00925) catalyzes the reversible transfer of phosphate groups from acetylphosphate to ADP, produces acetate and ATP, and plays a central role in methane metabolism. (45) The hyperthermophilic euryarchaeon Methanococcus jannaschi is able to utilize coenzyme M as a terminal methyl carrier in methanogenesis, where phosphosulfolactate synthase (EC: 4.4.1.19, K08097), 2- phosphosulfolactate phosphatase (EC: 3.1.3.71, K05979) can catalyze coenzyme M biosynthesis sequentially. (46)(47) The changes in their relative abundance are consistent with Qiu et al. 2022 who showed that the addition of BES resulted in a decrease in acetate kinase activity and coenzyme M (CoM) content in the methane metabolic pathway.( 48 ) For degradation metabolic pathways of aromatic compounds, toluene monooxygenase system protein A (EC: 1.14.13.236, K15760) can introduce hydroxyl groups on the benzene ring in multiple degradation pathways of toluene; (49) benzoate/toluate 1,2-dioxygenase subunit alpha (EC: 1.14.12.10, K05549) with dihydroxycyclohexadiene carboxylate dehydrogenase (EC: 1.3.1.25. K05783) promote the introduction of hydroxyl groups in the degradation metabolism of benzoate and xylene; (50)(51) While 2,3-dihydroxy-p-cumate/2,3-dihydroxybenzoate 3,4-dioxygenase (EC: 1.13.11.14, K10621), catechol 1,2-dioxygenase (EC: 1.13.11.1, K03381) and catechol 2,3-dioxygenase (EC: 1.13.11.2, K00446) were able to catalyze different ring-opening reactions upon the introduction of oxygen-containing functional groups, such as hydroxyl groups, respectively, and similar degradation of chlorobenzenes and styrenes was achieved by K03381 and K00446; (52)−(54) benzaldehyde dehydrogenase (EC: 1.2.1.28, K00141) catalyzes the synthesis of methyl benzoate from xylene and benzoates from toluene; (55) naphthalene 1,2-dioxygenase ferredoxin reductase component (EC: 1.18.1.7, K14581) enhances the introduction of hydroxyl groups to PAHs and ethylbenzene. (56) 2-pyrone-4,6-dicarboxylate lactonase (EC: 3.1.1.57, K10221) and hydroxyquinol 1,2-dioxygenase (EC: 1.13.11.37, K04098), on the other hand, are able to catalyze aromatic compounds in the metabolic pathway of benzoate degradation cyclization of aromatic compounds in the metabolic pathway of benzoate degradation. (57)(58) The increase in relative abundance of K05979, K05549, K05783, K15760, and K00141 over CK suggests that microbial metabolic functions stimulate the introduction of hydroxyl groups for a variety of aromatic compounds in coal, which enhances the hydrophilicity of coal. The increase in the relative abundance of K00446, K10621, K10221, K04098 and K03381 in T-1 and T-2 indicated that the metabolism of these genes catalyzed the de-cyclization of the coal macromolecule structure after the introduction of benzene rings into the aromatic compounds, which led to the evolution of the coal macromolecules into chains, which was conducive to the microbial pore-expansion and infiltration of the coal. In addition, the abundance of K05979, K05549, K05783, K15760, and K00141 within T-1 and T-2 were similar, while there was a significant difference between K00446, K10621, K10221, K04098, and K03381, confirming that additional nitrogen sources may not have a significant effect on promoting the introduction of oxygen-containing functional groups, such as hydroxyl, into the benzene ring, but the Firmicutes in the stimulated by nitrogen source was able to significantly catalyze the ring-opening reaction of part of the aromatic ring and enhanced biologically driven methane desorption capabilities. As shown in Fig. 4 , the adsorption isotherms of coal samples of groups Raw Coal, T-1 and T-2 are demonstrated, respectively. Among them, the types of adsorption and desorption isotherms of the three groups of coal samples are basically the same, and all of them have hysteresis loops and obvious inflection points. The isothermal adsorption lines were classified into eight types by IUPAC, (59) which can be regarded as different types according to the grading criteria at different relative pressure stages. Firstly, at the stage of low relative pressure, the phenomenon of microporous filling occurs inside the coal body, and the microporous structure has a strong adsorption capacity for nitrogen, so that the adsorption isotherm rises rapidly, and it belongs to the adsorption isotherm onset stage of Class I. After the end of the rapidly rising phase of adsorption isotherm, the curve shows a smooth and slowly rising trend, and in this relative pressure region, the pores of the coal body have successively produced monolayer adsorption and multilayer adsorption effects on nitrogen analysis. With the increasing relative pressure, capillary adsorption cohesion phenomenon, when the relative pressure is about 0.5 when the relative pressure of the desorption branch of the phenomenon of hysteresis, indicating that there are about 4nm pores exist in the coal body, this stage is manifested in the Ⅳ class (a) type. When the relative pressure is close to 1, unlike Type IV (a), the adsorption isotherm rises rapidly and is saturated, showing that there is still a larger pore structure in the coal body, which belongs to the end stage of Type II. In addition to showing the pore size structure of coal, adsorption isotherms can further analyze the geometric properties of coal pores by comparing the adsorption/desorption hysteresis loop type criteria proposed by IUPAC. (59)(60) As shown in Fig. 4 , the hysteresis rings of all three sets of coal samples belong to the H3 type, indicating that a large number of slit-type pore structures are distributed on the coal body. The adsorption/desorption isotherms showed low-pressure hysteresis, and the two curves did not overlap. According to the related research, the reason for the low-pressure hysteresis in this paper may be due to the developed microporous structure of the coal samples, with a large number of tiny "thin-necked bottles" pores, and irreversible adsorption phenomena in the pores led to incomplete adsorption equilibrium. Each desorption branch shows a clear inflection point at a relative pressure of about 0.5 and a wide hysteresis loop, indicating the presence of ink-bottle and semi-open pore structures on the coal body. In addition, at relative pressures greater than 0.45, all of them showed significant hysteresis loops, indicating the presence of a large number of open pore structures on the coal body. Nanoscale micropores are the main factor affecting the methane adsorption capacity of coal. (61) Microbial degradation makes the hysteresis loops of isotherms smaller and flatter, the low-pressure hysteresis phenomenon weaker, and the low-pressure end more skewed towards the X-axis, suggesting that the pore-connecting pores of the coal samples are developing towards mesopores, with the trend of mesopore development being more pronounced in T-1. From the experimental data, the maximum adsorption of nitrogen by Raw Coal was 0.9516 ml/g, and the maximum adsorption of nitrogen by T-1 and T-2 coal samples was 0.4943 ml/g and 0.5127 ml/g, which were reduced by 48.1% and 46.1%, respectively, compared with that of Raw Coal. The metabolic activities of Firmicutes stimulate the activity of microbial communities and the release of more metabolites, and some key metabolites affect the pore distribution, pore size, pore morphology, and surface chemistry of the pore wall in the coal body,etc., (62) so that the volume of micropores inside the coal body will be reduced, and the adsorption space of the methane will become smaller, and the adsorption capacity will be reduced accordingly. 3.4 Coal pore structure development law under microbial action In order to study the developmental characteristics of the full pore size pore structure of coal under microbial degradation, low-temperature liquid nitrogen adsorption test and mercury compression test were carried out on the treated coal samples of groups Raw Coal, T-1 and T-2, respectively. The pore size distributions of the three groups of coal samples are shown in Fig. 5 . In the range of 0–15 nm, the microporous pore size distribution of Raw Coal is more extensive, in which a more uniform regional distribution is presented near 1–2 nm and 7–15 nm, and a bimodal pore distribution is presented out of 2.3-4 nm, and the pores of the Raw Coal are most concentrated at 2.5 nm and 3.1 nm in particular. In the microporous range of T-1 coal samples, the distribution of pores in the range of 1-1.5 nm and 7.5 nm-15 nm is relatively average, while in the range of 1.5–7.5 nm is the distribution of a number of large-scale pore volume peaks, especially in the distribution of pore sizes at about 1.5 nm and 4.7 nm is the largest value. The pore distribution of the T-2 coal samples was similar to that of the residual coals of the T-1 group, with obvious peaks at 2.8 nm and 8.5 nm, where some micropores were concentrated. In the range of 15-50nm, there was no significant difference between the coal samples before and after microbial action. In the range of 50–400 nm, the mesopore pores of coal showed a two-stage development trend. Firstly, in the 50–100 nm region, the pore size distribution pattern of the three groups of coal samples is similar and all of them have a large number of pores distributed near 55–70 nm, in which the pore size distribution values of Raw Coal, T-1 and T-2 decrease in this place in turn. Secondly, in the 100–400 nm region, the Raw Coal pore distribution shows a decreasing trend, and from 180 nm onwards the mesopore pore distribution is extremely small, while the T-1 and T-2 pore size distribution patterns are similar and more evenly distributed. In the macroporous region larger than 400 nm, the pores of Raw Coal coal samples showed a three-stage distribution, mainly in the range around 550 nm, 840–1350 nm, and 2120–3122 nm. The pore size distribution of the T-1 coal sample shows an increasing and then decreasing trend, and the pores are more concentrated at the two ends of the macroporous region.The pore size distribution curve of T-2 coal sample is higher than that of the other two groups, especially after 1000 nm, the trend is significant, and the large pores of the coal sample appear to be greatly developed. Through the analysis of Table 2 , the developmental and evolutionary characteristics of the pore structure of anthracite under the degradation of functional microorganisms can be further explored. After the degradation of coal by microorganisms, the total pore volume of coal samples in both T-1 and T-2 groups increased compared to the total pore volume of Raw Coal, while the total specific surface area decreased and the average pore size increased significantly. Among them, the total pore volume of coal samples in T-1 and T-2 groups increased by 22.70% and 58.11%, respectively, compared with that of Raw Coal; the total specific surface area decreased by 44.11% and 49.61%, respectively, and the average pore diameters were 2.19 and 3.14 times higher than those of Raw Coal, respectively. In addition, the percentage of microporous pore volume and the percentage of specific surface area of T-1 coal sample decreased by 9.03% and 22.82%, respectively, compared with that of Raw Coal, while that of T-2 coal sample decreased by 9.33% and 22.58%, respectively. Since micropores are the main contributor to the specific surface area of coal and have less influence on the pore volume value of coal, (63) microbial degradation has led to the development of a large number of microporous structures in coal, and the proportion of micropores in the total pore structure of coal has decreased dramatically. There was no significant difference in the transitional pore of the three groups of experimental coal samples. T-1 and T-2 showed different developmental trends compared with the Raw Coal samples. the percentage of mesopores in the T-1 samples increased by 5.27%. the percentage of mesopores in the T-2 samples decreased by 15.28%. the percentage of mesopores in the T-2 samples increased by 5.27% and decreased by 15.28%. For the macropore, the pore volume of T-1 and T-2 coal samples is 1.39 and 2.68 times of that of Raw Coal, and the percentage of pore volume increases by 5.08% and 27.31%, respectively. Table 2 Pore structure parameters of three groups of coal samples Sample Total Pore Volume /(cm 3 •g − 1 ) Stage Pore Volume /(cm 3 •g − 1 ) Total Pore Area /(m 2 •g- 1 ) Stage Pore Area /(m2•g-1) Average Pore Diameter Micropore Transition pore Mesopore Macropore Micropore Transition pore Mesopore Macropore Raw Coal 0.00370 0.00044 0.00022 0.00159 0.00145 0.52151 0.42616 0.02402 0.06583 0.00551 28.37913 T-1 0.00454 0.00013 0.00021 0.00219 0.00201 0.29158 0.17174 0.03753 0.07333 0.00898 62.28136 T-2 0.00585 0.00015 0.00019 0.00162 0.00389 0.26263 0.15531 0.03428 0.05924 0.01380 89.09873 Through the joint characterization of the pore structure of the three groups of experimental coal samples, it was confirmed that microbial degradation and metabolism could promote the pore structure of anthracite coal to show a more obvious evolution pattern, and this evolution pattern was more significant after the addition of nitrogen source. Among them, the microbial degradation of anthracite coal in the inorganic salt medium environment after its original micro-small pores to mesopore, macropore evolution and development, the average pore size increased, reducing the adsorption space of methane in coal, showing the effect of pore expansion and pore enlargement. This is consistent with the results of Guo's study. (21) And although the pore structure of micropore and transitional pore had no obvious additional effect after the additional addition of nitrogen source, and the effect of mesopore development was weaker, the macropore presented a particularly significant growth, and the proportion of the coal's whole pore diameter was greatly increased. This suggests that the nitrogen source further stimulated the anaerobic degradation of coal by Firmicutes and changed the evolution law of the pore structure of anthracite coal during microbial degradation. Compared with the developmental trend of micropore favoring mesopore in T-1 coal samples, T-2 micropore showed a large-scale transformation phenomenon to macropore structure, which led to a further decrease in the total specific surface area of the coal, and the effect of expanding and increasing pores was more significant. In addition, tiny pore structure is the main contributor to the adsorption of methane molecules by coal, which can provide more adsorption sites for methane, so the development of tiny pore structure will inevitably reduce the adsorption capacity of coal for methane and improve its transport characteristics, (64)(65) which is important for the enhancement of the level of biologically driven gas desorption. The coal macropore structure is regarded as the "valve that restricts the flow", and the methane flow rate in the macropore controls the desorption rate of the residual methane in the coal. (66) The Firmicutes stimulated by the nitrogen source greatly improves the permeability of the anthracite coal, significantly increases the effective gas diffusion channels of the coal, and promoted the rapid desorption of adsorbed and free methane. 3.5 Characterization of changes in the molecular structure of coal under microbial action In order to study the evolution law of functional groups in low-permeability coals under microbial action, FTIR tests were carried out on three groups of experimental residual coals, respectively. According to a previous study, (67) the infrared spectral map of coal is mainly divided into four parts, in which the range of 900 − 700 corresponds to aromatic hydrocarbons, the range of 1800 − 1000 corresponds to oxygen-containing functional groups, the range of 3000 − 2700 corresponds to aliphatic hydrocarbons as well as the range of 3600 − 3000 corresponds to hydroxyl groups as shown in Fig. 6 (a). In order to visually compare the differences in functional group content under microbial action, quantitative calculations were carried out by peak fitting and the fitted peaks of each group were R 2 > 0.999, and the results obtained after peak fitting are shown in Fig. 6 (b). In the 3600 − 3000 cm − 1 band, there are mainly three kinds of characteristic peaks corresponding to hydroxyl functional groups: hydroxyl-π, self-conjugated hydroxyl, and hydroxyl-N. The relative content of hydroxyl in T-2 coal samples is about 117.231, which is 2.822 times of the hydroxyl content in Raw Coal.The content of hydroxyl in T-1 coal samples is about 2.251 times of that in Raw Coal. Four groups of characteristic peaks corresponding to aliphatic hydrocarbon functional groups mainly existed in the range of 3000 − 2700 cm − 1 : symmetric stretching vibrations of aliphatic methyl and methylene groups and asymmetric stretching vibrations. According to the results of peak fitting, the content of aliphatic hydrocarbon functional groups on the surface of the coal samples showed an increasing trend of different degrees after microbial action. Compared with Raw Coal, the aliphatic hydrocarbon content of T-1 and T-2 increased by 88.46% and 64.01%, respectively. In the 1800 − 1000 cm − 1 band, the contents of oxygen-containing functional groups of T-1 and T-2 coal samples were higher than those of Raw Coal, which were 1.778 and 1.511 times higher than those of Raw Coal, respectively. And at 1600 cm-1 corresponding to aromatic hydrocarbons, the content of Raw Coal coal samples was the highest, and the aromatic hydrocarbons content of T-1 and T-2 decreased sequentially. In addition, carboxyl group is one of the key functional groups to enhance the hydrophilicity of coal and reduce the adsorption capacity of methane. (68) In the region representing the carboxyl functional group around 1700 cm-1, although the carboxyl vibration of anthracite is generally weak, both T-1 and T-2 showed different degrees of growth after microbial metabolism. Within the 700–900 cm-1 region, four groups of characteristic peaks mainly existed in each group of coal samples: benzene ring five-substituted, benzene ring four-substituted, benzene ring three-substituted, and benzene ring two-substituted. Among them, the aromatic hydrocarbon content of Raw Coal was the highest at 9.053, and T-1 and T-2 decreased by 14.01% and 33.78%, respectively, compared with that of Raw Coal, which was also consistent with the changes occurred at the position of 1600 cm-1, and in addition, the percentage of benzene ring disubstitution in the coal samples of the treatment group showed an increasing trend compared with that of the original coal group. In order to further quantitatively characterize the evolutionary law of the internal microscopic molecular structure of coal, it was calculated by fitting the data with split peaks. The calculation results are shown in Table 3 . (30) Table 3 FTIR quantitative parameterization Sample I 1 I 2 I 3 I 4 DOC Raw Coal 21.638 3.415 0.830 0.172 0.445 T-1 14.057 1.557 0.982 0.258 0.413 T-2 11.281 1.379 2.983 0.483 0.336 Among them, I 1 represents the average chain length and degree of branching of aliphatic branched chains of the coal body, which decreased by 35.04% and 47.86% in T-1 and T-2 coal samples, respectively, compared with Raw Coal. The aromaticity of the coal body can be characterized by the ratio of the number of aromatic hydrocarbons to the number of aliphatic hydrocarbons, I 2 , which decreased by 54.41% and 59.62% in the T-1 and T-2 coal samples, respectively, compared with Raw Coal. I 3 is the ratio of the number of hydroxyl and ether groups reflecting the interconversion of oxygen-containing functional groups within the coal body, and the I 3 of T-1 coal was 1.183 times that of Raw Coal, and the conversion of oxygen-containing functional groups prompted by T-2 due to the addition of a nitrogen source to make the microbial metabolism activity more intense was particularly significant, and its I 3 was 3.594 times that of Raw Coal. Carbonyl is the main form of reactive oxygen present in the coal body, (69) I 4 is the ratio of carbonyl to aromatic hydrocarbons represents the degree of reactivity of elemental oxygen in the coal body, T-1 and T-2 coal samples I 4 increased by 50% and 180.814%, respectively. The aromatic ring condensation DOC within the coals was characterized by the ratio of the out-of-plane deformation vibration of -CH in various substituted aromatic hydrocarbons to the stretching vibration of C = C in aromatic hydrocarbons, and Raw Coal had the highest aryl ring condensation, with a decrease in the DOC of T-1 and T-2 coals by 7.19% and 24.49%, respectively. The results of peak fitting and quantitative calculations showed that a large number of ether groups were converted to hydroxyl groups during the microbial metabolism, which introduced various oxygen-containing functional groups, such as hydroxyl groups, into the coal macromolecular structure and thus enhanced the content of oxygens in the coal molecular structure. Because the hydroxyl group can form hydrogen bonds with hydrogen atoms and then form a water film on the coal pore surface, it can strengthen the hydrophilicity of the coal pore structure and weaken the adsorption capacity of methane. (70) Aromatic hydrocarbon functional group is an important component that constitutes the main part of coal, (70) which is transformed from the highly substituted form to the low-substituted form after microbial degradation, the condensation of the aromatic structure decreases, the molecular structure of the coal becomes sparse, and the stabilized benzene ring structure in the body of the coal is gradually transformed to the loose chain structure, and the aroma of the coal decreases. At the same time, the continuous breaking of the coal's fatty chains leads to a decrease in chain length and an increase in branchedness, which can provide more metabolic sites for microorganisms. (21) In addition, the above phenomenon of coal molecular structure evolution was more significant after the addition of nitrogen source, and the transformation effect within the coal oxygen-containing functional groups was significant, especially the transformation efficiency to the key oxygen-containing functional groups, such as hydroxyl group, could be increased by several times. This suggests that the nitrogen source stimulated the metabolic activity of Firmicutes to be more intense, releasing more key metabolites acting on the coal molecular structure, thus enabling the microorganisms to degrade coal more efficiently and promoting the evolution of coal molecular structure. 4 Conclusion The effect of microbial degradation and metabolism on the microstructure of anthracite coal under the stimulation of nitrogen source was investigated by adding functional microorganisms, such as Bacillus sphaericus, to anthracite coal. The results showed that Firmicutes, mainly Bacillus , played a major role in coal degradation under the synergistic effect of coal as a biodegradable carbon source and additional nitrogen source, which reduced the methane adsorption capacity of coal by shedding the alkyl side chains of the coal molecular structure and introducing oxygen-containing functional groups, such as hydroxyl, and increased the diffusion channels of methane within coal by improving the function of the degradation of the cyclic skeleton within the coal, which strengthened microbial-driven methane desorption capacity. During anaerobic culture, Firmicutes, represented by Bacillus , dominated and tended to be stable in the culture group with the best methane desorption. The relative abundance of genes for a variety of key degradation enzymes produced by their metabolism increased, and they were able to promote the catalytic introduction of oxygen-containing functional groups, such as hydroxyls, on the structure of the benzene ring and ring-opening reaction of the coal macromolecule compounds; Secondly, the degradation of microorganisms reduced the aromaticity of coal, shortened the long chain of fat and increased the degree of branching, and increased the content of oxygen-containing functional groups such as hydroxyl groups, which enhanced the hydrophilicity of coal, reduced the adsorption capacity of methane, and loosened the skeleton of coal macromolecules. As a result, the degradation of microorganisms led to the development of the coal's micropore structure towards mesopore and macropore, which led to the improvement of the effective diffusion channels and methane transportation state within the coal, and ultimately led to the desorption of methane in large quantities. Declarations Author Contribution C.Z., C.Z.,B.L., S.X., Y.Y.,T.Z.,X.Z.and J.W. conducted the bulk of the data analysis for the study and co-wrote the manuscript. S.X.,C.Z.and B.L. provided the funding for the study, were involved in the conceptualization of the study, as well as assisted in the writing of the manuscript. All authors read and approved the final manuscript Acknowledgements This work is financially supported by National Key R&D Program of China(NO.2023YFC3009002); Independent Research fund of Joint National-Local Engineering Research Centre for Safe and Precise Coal Mining (Anhui University of Science and Technology)(NO. EC2023006); Graduate Innovation Fund of Anhui University of Science and Technology(No.2022CX2020); National Natural Science Foundation of China (No. 52274171); The Key Research and Development Projects in Anhui Province(NO.2022l07020020). References Niu Y, Wang EY, Li ZH, Gao F, Zhang ZZ, Li BL, Zhang X (2022) Identification of Coal and Gas Outburst-Hazardous Zones by Electric Potential Inversion During Mining Process in Deep Coal Seam. Rock Mech Rock Eng 55:3439–3450 Zhou F, Xia T, Wang X, Zhang Y, Sun Y, Liu J (2016) Recent developments in coal mine methane extraction and utilization in China: A review. J Nat Gas Sci Eng 31:437–458 Moore TA (2012) Coalbed methane: A review. Int J Coal Geol 101:36–81 Wang K, Du F (2020) Coal-gas compound dynamic disasters in China: A review. Process Saf Environ Protect 133:1–17 Wang W, Li H, Liu Y, Liu M, Wang H, Li W (2020) Addressing the gas emission problem of the world’s largest coal producer and consumer: Lessons from the Sihe Coalfield, China. Energy Rep 6:3264–3277 Lu YY, Zhang HD, Zhou Z, Ge ZL, Chen CJ, Hou YD, Ye ML (2021) Current Status and Effective Suggestions for Efficient Exploitation of Coalbed Methane in China: A Review. Energy Fuels 35:9102–9123 Zhang X, Hu J, Xue H, Mao W, Gao Y, Yang J, He M (2020) Innovative approach based on roof cutting by energy-gathering blasting for protecting roadways in coal mines. Tunn Undergr Space Technol 99:103387 Li H, He J, Lu J, Lin B, Lu Y, Shi S, Ye Q (2022) A review of laboratory study on enhancing coal seam permeability via chemical stimulation. Fuel 330:125561 Su X, Zhao W, Xia D, Hou S, Fu H, Zhou Y (2022) Experimental study of advantages of coalbed gas bioengineering. J Nat Gas Sci Eng 102:104585 Wang HF, Cheng YP, Yuan L (2013) Gas outburst disasters and the mining technology of key protective seam in coal seam group in the Huainan coalfield. Nat Hazards 67:763–782 Liu JW, Liu CY, Yao QL, Si GY (2020) The position of hydraulic fracturing to initiate vertical fractures in hard hanging roof for stress relief. Int J Rock Mech Min Sci 132:104328 Duan M, Jiang C, Gan Q, Li M, Peng K, Zhang W (2020) Experimental investigation on the permeability, acoustic emission and energy dissipation of coal under tiered cyclic unloading. J Nat Gas Sci Eng 73:103054 Yi M, Cheng Y, Wang C, Wang Z, Hu B, He X (2021) Effects of composition changes of coal treated with hydrochloric acid on pore structure and fractal characteristics. Fuel 294:120506 Zheng C, Li X, Li H, Jiang B, Chen Z (2023) Experimental Investigation on Pore-Fracture Variations in Coal Affected by Carbon Disulfide. Acs Omega 8:38426–38440 Ritter D, Vinson D, Barnhart E, Akob DM, Fields MW, Cunningham AB, Orem W, McIntosh JC (2015) Enhanced microbial coalbed methane generation: A review of research, commercial activity, and remaining challenges. Int J Coal Geol 146:28–41 Scott AR (1999) In: Mastalerz M, Glikson M, Golding SD (eds) Improving Coal Gas Recovery with Microbially Enhanced Coalbed Methane. Springer Netherlands: Dordrecht, Bao Y, Li Z, An C et al (2023) Multi-method characterization of pore structure evolution characteristics and mechanism of tar-rich coal by anaerobic fermentation. J China Coal Soc 48:891–899 Wang AK, Shao P, Wang QH (2021) Biogenic gas generation effects on anthracite molecular structure and pore structure. Front Earth Sci 15:272–282 Pandey R, Harpalani S (2018) An imaging and fractal approach towards understanding reservoir scale changes in coal due to bioconversion. Fuel 230:282–297 Lu YY, Chai CJ, Zhou Z, Ge ZL, Yang MM (2020) Influence of bioconversion on pore structure of bituminous coal. Asia-Pac J Chem Eng 15e2399 Guo H, Luo Y, Ma J et al Analysis of mechanism and permeability enhancing effect via microbial treatment on different-rank coals. J China Coal Soc 2014,39,1886–1891 Haider R (2017) Coal degradation through fungal isolate RHC2 from Romanian brown coal sample. Energy Sources Part a-Recovery Util . Environ Eff 39:1785–1790 Yang J, Liu X, Yang Z, Zhao S (2023) Biodegradation of Dananhu low-rank coal by Planomicrobium huatugouensis: Target metabolites possessing degradation abilities and their biodegradation pathways. Energy 276:127642 Xia DP, Gu PT, Chen ZH, Chen LY, Wei GQ, Wang ZZ, Cheng S, Zhang YW (2023) Control Mechanism of Microbial Degradation on the Physical Properties of a Coal Reservoir. Processes 11 Webster TM, Smith AL, Reddy RR, Pinto AJ, Hayes KF, Raskin L (2016) Anaerobic microbial community response to methanogenic inhibitors 2-bromoethanesulfonate and propynoic acid. Microbiologyopen 5:537–550 Qin Y, Xu Z, Zhang J (1995) Natural classification of the high-rank coal pore structure and its appliction. J China Coal Soc 03:266–271 Landers J, Gor GY, Neimark AV (2013) Density functional theory methods for characterization of porous materials. Colloids Surf A 437:3–32 Monson PA (2012) Understanding adsorption/desorption hysteresis for fluids in mesoporous materials using simple molecular models and classical density functional theory. Microporous Mesoporous Mat 160:47–66 Suuberg EM, Deevi SC, Yun Y (1995) Elastic behaviour of coals studied by mercury porosimetry. Fuel 74:1522–1530 Li H, Shi S, Lin B, Lu J, Ye Q, Lu Y, Wang Z, Hong Y, Zhu X (2019) Effects of microwave-assisted pyrolysis on the microstructure of bituminous coals. Energy 187:115986 Li Y, Wang Z, Tang S, Elsworth D (2022) Re-evaluating adsorbed and free methane content in coal and its ad- and desorption processes analysis. Chem Eng J 428:131946 Zhao J, Qin Y, Shen J, Zhou B, Li C, Li G (2019) Effects of Pore Structures of Different Maceral Compositions on Methane Adsorption and Diffusion in Anthracite. Appl Sci 9:5130 Kang HL, Liu XR, Zhang YW, Zhao SS, Yang ZW, Du ZP, Zhou AN (2021) Bacteria solubilization of shenmu lignite: influence of surfactants and characterization of the biosolubilization products. Energy Sources Part a-Recovery Util . Environ Eff 43:1162–1180 Rasool Kamli M, Malik A, Sabir SM, Ahmad Rather J, Kim I (2022) Insights into the biodegradation and heavy metal resistance potential of the genus Brevibacillus through comparative genome analyses. Gene 846:146853 Cao Y, He H, Zhan D, Huang HZ, Zhang Y, Fu B, Xu ZM, Ali MI, Liu FJ, Tao XX et al (2023) Changes of Permeability and Porosity of Tiefa Anthracite after Treatment with an Acetogenic Bacterium Clostridium sp. Geomicrobiol J 40:277–284 Du Y, Zou W, Zhang K, Ye G, Yang J (2020) Advances and Applications of Clostridium Co-culture Systems in Biotechnology. Front Microbiol 11 Guo H, Zhang Y, Zhang J, Huang Z, Urynowicz MA, Liang W, Han Z, Liu J (2019) Characterization of Anthracite-Degrading Methanogenic Microflora Enriched from Qinshui Basin in China. Energy Fuels 33:6380–6389 Ikkert OP, Gerasimchuk AL, Bukhtiyarova PA, Tuovinen OH, Karnachuk OV (2013) Characterization of precipitates formed by H2S-producing, Cu-resistant Firmicute isolates of Tissierella from human gut and Desulfosporosinus from mine waste. Antonie Van Leeuwenhoek 103:1221–1234 MENG D, ZHAI L, TIAN Q, GUAN Z, CAI Y, LIAO X (2019) Complete genome sequence of Bacillus amyloliquefaciens YP6, a plant growth rhizobacterium efficiently degrading a wide range of organophosphorus pesticides. J Integr Agric 18:2668–2672 Yang G, Yang D, Wang X, Cao W (2021) A novel thermostable cellulase-producing Bacillus licheniformis A5 acts synergistically with Bacillus subtilis B2 to improve degradation of Chinese distillers’ grains. Bioresour Technol 325:124729 Fira D, Dimkić I, Berić T, Lozo J, Stanković S (2018) Biological control of plant pathogens by Bacillus species. J Biotechnol 285:44–55 Li Y, Qin T, Feng F, Zhang Y, Xue S (2023) Nitrogen amendment enhances the biological methanogenic potential of bituminous coal. Fuel 351:128932 Ferry JG (1997) Enzymology of the fermentation of acetate to methane by Methanosarcina thermophila. BioFactors 6:25–35 Wongnate T, Sliwa D, Ginovska B, Smith D, Wolf MW, Lehnert N, Raugei S, Ragsdale SW (2016) The radical mechanism of biological methane synthesis by methyl-coenzyme M reductase. Science 352:953–958 Chittori S, Savithri HS, Murthy MRN (2011) Preliminary X-ray crystallographic studies on acetate kinase (AckA) fromSalmonella typhimurium in two crystal forms. Acta Crystallogr Sect F Struct Biology Crystallization Commun 67:1658–1661 Graham DE, Xu H, White RH (2002) Identification of Coenzyme M Biosynthetic Phosphosulfolactate Synthase. J Biol Chem 277:13421–13429 Graham DE, Graupner M, Xu H, White RH (2001) Identification of coenzyme M biosynthetic 2-phosphosulfolactate phosphatase. Eur J Biochem 268:5176–5188 Qiu S, Xia W, Xu J, Li Z, Ge S (2023) Impacts of 2-bromoethanesulfonic sodium on methanogenesis: Methanogen metabolism and community structure. Water Res 230:119527 Takami H, Nakasone K, Takaki Y, Maeno G, Sasaki R, Masui N, Fuji F, Hirama C, Nakamura Y, Ogasawara N et al (2000) Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res 28:4317–4331 Veith B, Herzberg C, Steckel S, Feesche J, Maurer KH, Ehrenreich P, Baumer S, Henne A, Liesegang H, Merkl R et al (2004) The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential. J Mol Microbiol Biotechnol 7:204–211 Wrighton KC, Agbo P, Warnecke F, Weber KA, Brodie EL, DeSantis TZ, Hugenholtz P, Andersen GL, Coates J (2008) D. A novel ecological role of the Firmicutes identified in thermophilic microbial fuel cells. Isme J 2:1146–1156 Marín M, Plumeier I, Pieper DH (2012) Degradation of 2,3-Dihydroxybenzoate by a Novel meta-Cleavage Pathway. J Bacteriol 194:3851–3860 Paes F, Liu XK, Mattes TE, Cupples AM (2015) Elucidating carbon uptake from vinyl chloride using stable isotope probing and Illumina sequencing. Appl Microbiol Biotechnol 99:7735–7743 Benjamin RC, Voss JA, Kunz DA (1991) Nucleotide sequence of xylE from the TOL pDK1 plasmid and structural comparison with isofunctional catechol-2,3-dioxygenase genes from TOL, pWW0 and NAH7. J Bacteriol 173:2724–2728 Feng L, Wang W, Cheng JS, Ren Y, Zhao G, Gao CX, Tang Y, Liu XQ, Han WQ, Peng X et al (2007) Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir. Proc. Natl. Acad. Sci. U. S. A . 104, 5602–5607 van Beilen JB, Funhoff EG (2007) Alkane hydroxylases involved in microbial alkane degradation. Appl Microbiol Biotechnol 74:13–21 Zhang SJ, Ma GC, Liu YJ, Ling BP (2015) Theoretical study of the hydrolysis mechanism of 2-pyrone-4,6-dicarboxylate (PDC) catalyzed by LigI. J Mol Graph 61:21–29 Kutsuna R, Miyoshi-Akiyama T, Mori K, Hayashi M, Tomida J, Morita Y, Tanaka K, Kawamura Y et al (2019) Description of Paraclostridium bifermentans subsp. muricolitidis subsp. nov., emended description of Paraclostridium bifermentans (Sasi Jyothsna 2016), and creation of Paraclostridium bifermentans subsp. bifermentans subsp. nov. Microbiol. Immunol. 63, 1–10 Thommes M, Kaneko K, Neimark AV, Olivier JP, Rodriguez-Reinoso F, Rouquerol J, Sing KS (2015) W. Physisorption of gases, with special reference to the evaluation of surface area and pore size distribution (IUPAC Technical Report). Pure Appl. Chem. 87, 1051–1069 Pyun S, Rhee C (2004) An investigation of fractal characteristics of mesoporous carbon electrodes with various pore structures. Electrochim Acta 49:4171–4180 Cai YD, Liu DM, Pan ZJ, Yao YB, Li JQ, Qiu YK (2013) Pore structure and its impact on CH 4 adsorption capacity and flow capability of bituminous and subbituminous coals from Northeast China. Fuel 103:258–268 Strąpoć D, Mastalerz M, Dawson K, Macalady J, Callaghan AV, Wawrik B, Turich C, Ashby M (2011) Biogeochemistry of Microbial Coal-Bed Methane. Annu Rev Earth Planet Sci 39:617–656 Shan CA, Zhang TS, Guo JJ, Zhang Z, Yang Y (2015) Characterization of the micropore systems in high-rank coal reservoirs of the southern Sichuan Basin, China. Aapg Bull 99:2099–2119 Hu B, Cheng YP, He XX, Wang ZY, Jiang ZN, Wang CH, Li W, Wang L (2020) New insights into the CH 4 adsorption capacity of coal based on microscopic pore properties. Fuel 262 Li ZW, Lin BQ, Gao YB, Cao ZD, Cheng YY, Yu JY (2015) Fractal analysis of pore characteristics and their impacts on methane adsorption of coals from Northern China. Int J Oil Gas Coal Technol 10:306–324 Hu B, Cheng YP, Pan ZJ (2023) Classification methods of pore structures in coal: A review and new insight. GAS Sci Eng 110 Ibarra J, Muñoz E, Moliner R (1996) FTIR study of the evolution of coal structure during the coalification process. Org Geochem 24:725–735 Han YN, Bai ZQ, Liao JJ, Bai J, Dai X, Li X, Xu JL, Li W (2016) Effects of phenolic hydroxyl and carboxyl groups on the concentration of different forms of water in brown coal and their dewatering energy. Fuel Process Technol 154:7–18 Xin HH, Wang DM, Dou GL, Qi XY, Xu T, Qi GS (2014) The Infrared Characterization and Mechanism of Oxygen Adsorption in Coal. Spectr Lett 47:664–675 Zhou Y, Albijanic B, Wang YL, Yang JG (2018) Characterizing surface properties of oxidized coal using FTIR and contact angle measurements. Energy Sources Part a-Recovery Util Environ Eff 40:1559–1564 Xia W, Zhang W (2017) Characterization of surface properties of Inner Mongolia coal using FTIR and XPS. Energy Sources Part a-Recovery Util Environ Eff 39:1190–1194 Additional Declarations No competing interests reported. Supplementary Files TOC.png For Table of Contents use only Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 03 Jul, 2024 Submission checks completed at journal 03 Jul, 2024 First submitted to journal 02 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4673807","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322164677,"identity":"dc982ce4-54d8-4cac-a0af-bf9244da7000","order_by":0,"name":"Chunshan Zheng","email":"","orcid":"","institution":"Ministry of Education Key Laboratory of Safe and Effective Coal Mining, Anhui University of Science and Technology, Huainan, Anhui 232001, China","correspondingAuthor":false,"prefix":"","firstName":"Chunshan","middleName":"","lastName":"Zheng","suffix":""},{"id":322164678,"identity":"254ae5b3-3014-492c-9275-8ba9c0ec4a78","order_by":1,"name":"Chengcai Zhao","email":"","orcid":"","institution":"Ministry of Education Key Laboratory of Safe and Effective Coal Mining, Anhui University of Science and Technology, Huainan, Anhui 232001, China","correspondingAuthor":false,"prefix":"","firstName":"Chengcai","middleName":"","lastName":"Zhao","suffix":""},{"id":322164679,"identity":"0a248f12-7318-4ae8-8082-74a66f0bb031","order_by":2,"name":"Bingjun Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACPgmGBAYGNgYZfmbmgw+I0sIG1cIj2c6WbECsFgawFoPzPGYCxGmRbnj4uaCsjsf4MIMZA0ONTTRhLTIHkqVnnGPjMTvMkPaA4VhabgNhhyUkSPO28YC0HDdgbDhMlJbk37xtEjzGzYxtEsRqSQPaYsBjwMzMRqQWmQNp1jznEngkDrMxGyQQ4xd+6Z7k2zxldXL8/ec/PvhQY0NYCwMDTwKCnYBLESpgP0CculEwCkbBKBi5AAAjIzP8gMV9ZgAAAABJRU5ErkJggg==","orcid":"","institution":"State Key Laboratory of Mining Response and Disaster Prevention and Control in Deep Coal Mines, Anhui University of Science and Technology, Huainan, Anhui 232001, China","correspondingAuthor":true,"prefix":"","firstName":"Bingjun","middleName":"","lastName":"Liu","suffix":""},{"id":322164682,"identity":"2253d22a-c9fb-45c3-be9a-73a31fc5b28e","order_by":3,"name":"Sheng Xue","email":"","orcid":"","institution":"State Key Laboratory of Mining Response and Disaster Prevention and Control in Deep Coal Mines, Anhui University of Science and Technology, Huainan, Anhui 232001, China","correspondingAuthor":false,"prefix":"","firstName":"Sheng","middleName":"","lastName":"Xue","suffix":""},{"id":322164684,"identity":"61e2bf59-5062-4283-aaeb-2355e321e100","order_by":4,"name":"Yang Yang","email":"","orcid":"","institution":"Huainan Mining Group Co., Ltd, Huainan, Anhui 232001, China","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Yang","suffix":""},{"id":322164687,"identity":"47bb1505-0827-4fba-8175-13c94fd6a724","order_by":5,"name":"Tianyao Zhou","email":"","orcid":"","institution":"School of Safety Science and Engineering, Anhui University of Science and Technology, Huainan, Anhui 232001, China","correspondingAuthor":false,"prefix":"","firstName":"Tianyao","middleName":"","lastName":"Zhou","suffix":""},{"id":322164690,"identity":"000c0cde-a7bd-4b37-9539-4692c633ad97","order_by":6,"name":"Xun Zhang","email":"","orcid":"","institution":"State Key Laboratory of Mining Response and Disaster Prevention and Control in Deep Coal Mines, Anhui University of Science and Technology, Huainan, Anhui 232001, China","correspondingAuthor":false,"prefix":"","firstName":"Xun","middleName":"","lastName":"Zhang","suffix":""},{"id":322164692,"identity":"2c8fc50e-57bb-41ec-97cd-4bf0fc9713dc","order_by":7,"name":"Junyu Wang","email":"","orcid":"","institution":"State Key Laboratory of Mining Response and Disaster Prevention and Control in Deep Coal Mines, Anhui University of Science and Technology, Huainan, Anhui 232001, China","correspondingAuthor":false,"prefix":"","firstName":"Junyu","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-07-02 11:23:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4673807/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4673807/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61083665,"identity":"fad4ebd8-23f7-469f-8f75-8a275b5ddafa","added_by":"auto","created_at":"2024-07-25 11:20:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93839,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design of microbial anaerobic degradation of anthracite coal\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4673807/v1/97b811432c557b4c70c6fcc9.png"},{"id":61083667,"identity":"b44c558c-d837-4207-a910-3b8174af407e","added_by":"auto","created_at":"2024-07-25 11:20:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83515,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Relative abundance of headspace microbial methane desorption in the three experimental groups; (b) Relative abundance of microbial community composition in the initial samples on d 0 of the experiment versus the three experimental groups on d 30 of the experiment.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4673807/v1/2e3d9d72339478b2dff1ad39.png"},{"id":61084307,"identity":"40cdc7fa-e475-405d-ada6-25c4d7f5973c","added_by":"auto","created_at":"2024-07-25 11:28:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68816,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Differential analysis of major metabolic functions in the three experimental groups; (b) Differential enzyme genes related to aromatic compound degradation and methane metabolism.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4673807/v1/8a09f7eba27cccefc0b809c5.png"},{"id":61083669,"identity":"94e5b175-0933-4d75-9513-0d132924fced","added_by":"auto","created_at":"2024-07-25 11:20:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":26585,"visible":true,"origin":"","legend":"\u003cp\u003eNitrogen adsorption/desorption isotherms of coal samples at the end of three sets of experiments.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4673807/v1/bbd6a5c362bce67f44618980.png"},{"id":61084306,"identity":"0096a1ee-d46f-4ec6-a4a8-e87241f61f42","added_by":"auto","created_at":"2024-07-25 11:28:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":81796,"visible":true,"origin":"","legend":"\u003cp\u003eMulti-scale pore size distribution of Raw Coal, T-1 and T-2 coal samples\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4673807/v1/3c22b84d8bbbcfa575cf0f9a.png"},{"id":61083672,"identity":"96e22d9a-f6b3-464e-9c16-7a257cbe3cd8","added_by":"auto","created_at":"2024-07-25 11:20:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":344969,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Comparison of Infrared Spectra of Raw Coal, T-1 and T-2 Coal Samples; (b) Fitting plots of the main bands in each group of coal samples.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4673807/v1/3e6e8b69a171ae5a8f4db8d6.png"},{"id":61084836,"identity":"afe4109f-400b-48ab-8ed2-77dbaeeee374","added_by":"auto","created_at":"2024-07-25 11:36:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1267058,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4673807/v1/ff58d857-2fa2-43ec-b37d-605ec8816215.pdf"},{"id":61083671,"identity":"855966ab-cb74-4f66-84e3-b86d1dbc2916","added_by":"auto","created_at":"2024-07-25 11:20:44","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":234519,"visible":true,"origin":"","legend":"\u003cp\u003eFor Table of Contents use only\u003c/p\u003e","description":"","filename":"TOC.png","url":"https://assets-eu.researchsquare.com/files/rs-4673807/v1/710b1029859e4c4d0ebf855a.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of Metabolism of Firmicutes on the microstructure of anthracite under nitrogen source stimulation","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eWith the gradual depletion of shallow minerals and the gradual shift of mining to the deeper parts of the coal resources under a long period of large-scale development, the gas disaster problems encountered in the coal production process have become increasingly serious.\u003csup\u003e(1)(2)\u003c/sup\u003e Coalbed methane (mainly composed of methane) has a high calorific value and is cleaner than coal and oil. As an important source of unconventional natural gas, it is enriched in the complex double pore structure of coal seams,\u003csup\u003e(3)\u003c/sup\u003e especially in Chinese coal seams due to the characteristics of poor pore and fissure development and low permeability, \u003csup\u003e(4),(5)\u003c/sup\u003ewhich leads to the difficulty of coalbed methane extraction in mines, and poses a great challenge to the safe production of coal mines.\u003csup\u003e(5)\u003c/sup\u003e In order to increase the permeability of high gas and low permeability coal seams and strengthen the level of methane transportation, there are mainly physical permeability enhancement technology,\u003csup\u003e(7)\u003c/sup\u003e chemical permeability enhancement technology\u003csup\u003e(8)\u003c/sup\u003e and biological permeability enhancement technology.\u003csup\u003e(9)\u003c/sup\u003e Among them, the physical penetration technology is mainly through water jet, deep hole blasting, mining protective layer technology to make the coal seam decompression rupture coal body, promote the development of pore and fissure structure, improve the transport characteristics of the coal body, so as to increase the permeability of the coal body, and improve the effect of gas extraction, which has been widely used in the coal mine site.\u003csup\u003e(10)(11)(12)\u003c/sup\u003e For the research of chemical penetration enhancement technology, one part of it cleans the mineral components of the coal body through strong acid solution and transforms the pore and fissure structure of the coal\u003csup\u003e(13)\u003c/sup\u003e; the other part of it uses organic solvents to dissolve and remove the small molecule soluble substance in the coal body and then make its molecular structure evolve, thus realizing the penetration of the coal body.\u003csup\u003e(14)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBiological permeability enhancement technology has the advantages of green, safe, economic and sustainable, so it has far-reaching and significant theoretical and practical significance in improving the conditions of coalbed methane extraction and developing clean energy.\u003csup\u003e(15)\u003c/sup\u003e Scott first suggested in 1999 that permeability of coal bodies could be enhanced by microbial degradation.\u003csup\u003e(16)\u003c/sup\u003e Since then, scholars around the world have carried out laboratory simulations and field experiments to verify the effect and mechanism of microbial anaerobic degradation on coal permeability enhancement. Currently, studies have been conducted to describe the effect of microbial metabolism on the microstructure of coal, some of the studies have proved that the micropore structure of coal will develop into larger pores and improve the pore fracture structure after microbial degradation. Bao et al. found that biodegradation can achieve effects such as pore expansion and seam creation, which are favorable for methane seepage and transport.\u003csup\u003e(17)\u003c/sup\u003eWang et al. carried out a simulation of biogenic gas production and found that some micropores and transition pores in the coal samples were transformed into macropores and that side chains and hydroxyls of the coal molecular structure were readily metabolized by methanogenic bacteria, which partially oxidized to form carboxylic acids.\u003csup\u003e(18)\u003c/sup\u003e Pandey et al. concluded that coal biotransformation resulted in a swelling of the coal matrix, decreasing fractal dimension and contributing to the formation of new pore cleavages.\u003csup\u003e(19)\u003c/sup\u003e Lu et al.used microorganisms to treat high volatile bituminous coal and sub-bituminous coal, and confirmed that the volume of micropores and mesopores of coal decreased, the volume and porosity of macropores increased, the crystallinity of coal decreased, organic matter degraded, and pore connectivity enhanced.\u003csup\u003e(20)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn addition, some scholars have confirmed through microbiology, metabolomics and spectroscopy that the molecular structure and crystal structure of coal will evolve after the action of key products of biometabolism, and its methanophilicity will be reduced, which will help to improve the permeability of coal.Guo et al. found that after the degradation of coal by biometabolism through the FTIR and XRD tests, the oxygen-containing functional groups of coal increased, and the aromatic rings were partially opened and hydroxyl groups introduced at the breaks, and their contents increased accordingly. Hydroxyl groups were introduced at the breaks, and their content increased accordingly, the disorder of macromolecules increased, and the degree of crystallization decreased.\u003csup\u003e(21)\u003c/sup\u003e Haider et al. found that aromatic and aromatic compounds in coal were degraded after biodegradation experiments using lignite from the Thar Coalfield, and that the degraded organic components may have applications in biomethane synthesis.\u003csup\u003e(22)\u003c/sup\u003eYang et al. used a metabolomics approach to characterize the metabolites of the Great South Lake low rank coal addition, P. huatugouensis, and some key metabolites could act on the coal's ester, ether and metal bonds, thus depolymerizing the macromolecular structure into liquid organic molecules such as alcohols, aldehydes, ketones.\u003csup\u003e(23)\u003c/sup\u003eXia et al. concluded that microorganisms break down the covalent bonds and functional groups of the macromolecules in coal by secreting extracellular enzymes in the degradation metabolism, which ultimately increase the gas content of coal seams, change the coal's pore structure, and decrease the fractal dimensions of the coal surface to make the coal surface smooth.\u003csup\u003e(24)\u003c/sup\u003e However, most of the current studies on bio-penetration enhancement are on biodegradation of low-order coal using a single means, which is less effective when applied to low-permeability anthracite. Therefore, it is of great significance to conduct joint research on the whole process of microorganisms driving methane desorption, degradation, and permeability enhancement on high-gas, low-permeability coal mines coals at multiple levels.\u003c/p\u003e \u003cp\u003eIn this paper, we selected the poorly developed pore space and low permeability anthracite coal from Temple River Mine to add highly efficient degrading bacterial agents for anaerobic degradation indoor cultivation experiments, and monitored the dynamic desorption amount of residual methane in the coal in the process of the experiments. We analyzed the microbial community structure succession law and metabolic function variability by 16s rRNA high-throughput sequencing; jointly characterized the developmental characteristics of the pore structure of the coal samples after the experiments based on pressurized mercury and low-temperature nitrogen adsorption experiments; and investigated the evolution law of the molecular structure of the coal by using FTIR experiments. This paper explores the microstructure evolution of coal by Firmicutes under the stimulation of nitrogen source, reveals the mechanism of microorganisms in the anaerobic degradation of coal, and provides data support for increasing the permeability of high-gas and low-permeability coal seams and strengthening the level of methane transportation.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Coal sample collection and coal quality parameters\u003c/h2\u003e \u003cp\u003eFresh coal samples were collected from Shanxi Sihe Mine, and immediately after the samples were obtained from underground mining, they were placed in sterile self-sealing bags and wrapped in ice for low-temperature transportation to the laboratory, and were subjected to industrial and elemental analyses respectively, and the results are shown in the following Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. After surface excision of the samples, the samples were crushed, milled, and screened, and the 60\u0026ndash;80 mesh of the coal powder was collected and preserved at low temperatures for the subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUltimate/Proximate analysis of raw coal samples.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMad (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAad (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVad (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFCad (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eC (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eH (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoal of Sihe Mine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e47.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental design and testing\u003c/h2\u003e \u003cp\u003eThe functional microorganisms added in the experiment were obtained by pumping and filtering the highly efficient degradation functional bacterial agent and using sterile water to mix and then repeat the operation for 3 times. (Bacterial agent composition: \u003cem\u003eBacillus\u003c/em\u003e accounted for 56.80%, \u003cem\u003eClostridium\u003c/em\u003e accounted for 23.28%, \u003cem\u003eParaclostridium\u003c/em\u003e accounted for 19.92%, and the bacterial concentration in the agent was 10\u003csup\u003e8\u003c/sup\u003e~10\u003csup\u003e9\u003c/sup\u003e cfu/mL).\u003c/p\u003e \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003eCl \u0026nbsp;0.81g/L , MgCl\u003csub\u003e2\u003c/sub\u003e﹒6H\u003csub\u003e2\u003c/sub\u003eO \u0026nbsp;0.17g/L , CaCl\u003csub\u003e2\u003c/sub\u003e﹒2H\u003csub\u003e2\u003c/sub\u003eO \u0026nbsp;0.1g/L ，FeCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO \u0026nbsp;0.07g/L，Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;12H\u003csub\u003e2\u003c/sub\u003eO \u0026nbsp;1.5g/L，KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003e 1.5g/L，sterile water.\u003c/p\u003e\u003cp\u003eThe control group (CK) was added 50 g of coal sample and 100 mL of sterile water; Treat-1 (T-1) A 50 g coal sample was taken as substrate and placed in a 500 mL culture flask, to which functional microorganisms (120 mL of bacteriophage filtration), 100 mL of basal saline medium, 0.5 g of cysteine hydrochloride, and 2 ml of 10 mmol 2-bromoethanesulfonic acid sodium salt (BES) were added to inhibit the methanogenic potential of the microorganisms and to ensure that the headspace methane was generated from the residual methane in the coal.\u003csup\u003e(25)\u003c/sup\u003e Treat-2 (T-2) was identical to T-1 except for the addition of tryptone 1g. All three sets of experiments were sealed with nitrile plugs and three parallel experiments were performed.\u003c/p\u003e \u003cp\u003eThe initial anaerobic environment of the culture bottles was replaced with high-purity nitrogen for three times using a vacuum filtration device and a sterile syringe to replace the headspace air. The culture bottles, centrifuge tubes and culture medium used in the experiment were autoclaved, and the experimental process was carried out on the aseptic operating table. The experiments were carried out at a constant temperature of 35 ℃ for 30 d. In order to obtain pure coal samples, the residual coal was separated from the experimental samples by suction filtration with sterile water for several times, and then the coal samples were dried in an electric blast drying oven at 35 ℃ until constant weight and then sealed and stored.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring the incubation experiments, the methane concentration percentage in the headspace gas inside the incubation bottles during the biodegradation process was detected using a capillary analytical gas chromatograph model GC-8900 from Shandong Lunan Xinke. The temperature of the injection chamber of the gas chromatograph was 105 ℃, the temperature of the column chamber was 102 ℃, the temperature of the thermal conductivity cell was 116 ℃, and the bridge current was set at 150 mA. High-purity nitrogen was used to replace the headspace gas in the incubation vials when the amount of methane desorbed was stabilized, i.e., one period of gas desorption.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 High-throughput sequencing of 16S rRNA gene amplicons and gene function analysis experiments\u003c/h2\u003e \u003cp\u003eMB AquaScreen Fast Extract was used to extract total genomic DNA from CK, T-1 and T-2 on days 0 and 30, which was then stored on dry ice and sent to UW Genetics Ltd. in China, where the microbial communities in the culture system were analyzed by high-throughput sequencing of the 16S rRNA gene amplicons on the HiSeq platform.515F/907R (515F : 5' -GTG CCA GCM GCC GCG G-3'; 907R: 5' -CCG TCA ATT CMT T TR AGT TT-3' ) primer pairs were used to amplify the bacterial 16S rRNA gene. Clustering was performed using UPARSE at 97% similarity, representative sequences of OTUs were obtained and chimeras were identified and deleted using UCHIME (v4.2.40), and classified and annotated according to SILVA reference data (v128). Sequences classified as \"mitochondrial\" or \"unassigned\" were removed.\u003c/p\u003e \u003cp\u003eBased on the Kyoto Encyclopedia of Genes and Genomes (KEGG database), we screened metabolic pathways related to the degradation of methane and molecular structures in anthracite and analyzed the differences in key gene functions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experiments on the determination of coal pore structure parameters\u003c/h2\u003e \u003cp\u003eLow-temperature liquid nitrogen adsorption experiments were carried out using a high-performance specific surface area and microporous analyzer, BSD-PM, manufactured by Best Instrument Technology (Beijing) Co. About 1.5 g of dried coal samples were taken and placed in a sample tube and degassed under vacuum at 105\u0026deg;C for 6 hrs.\u003c/p\u003e \u003cp\u003eThe high performance fully automated mercury palyzer AutoPore IV 9500 produced by Shenzhen Huapu General Technology Co.The pressure range was from 1 to 33000 psia, the accuracy of mercury feed was 0.1 \u0026micro;L, and the equilibrium time of each pressure collection point was 10 s. The pressure range was from 1 to 33000 psia, and the accuracy of mercury feed was 0.1 \u0026micro;L.\u003c/p\u003e \u003cp\u003eThe coal sample used in the experiment was Shanxi Temple River coal, which belongs to anthracite, so the analysis was performed using the method of pore size structure division of high coal rank coal by Qin Yong, i.e., micropore (\u0026lt;\u0026thinsp;15nm), transitional pore (15\u0026thinsp;~\u0026thinsp;50nm), mesopore (50\u0026thinsp;~\u0026thinsp;400nm), macropore (\u0026gt;\u0026thinsp;400nm).\u003csup\u003e(26)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe results of low-temperature liquid nitrogen adsorption experiments were used for the analysis of micopores and transitional pore for Raw coal, T-1 and T-2 coal samples, and the results of mercury compression experiments were used for the analysis of mesopores and macropores.When dealing with the results of low-temperature liquid nitrogen adsorption experiments, the NLDFT model, which is more accurate for analyzing micropores, was chosen for the pore size distribution at \u0026lt;\u0026thinsp;15 nm, and the classical BJH desorption method was chosen for the 15\u0026ndash;50 nm segment.\u003csup\u003e(27)(28)\u003c/sup\u003e In addition, due to the existence of voids between the coal samples in the mercury pressure experiment, the pressure point of 0.4 Mpa was selected as the initial mercury absorption reference point in accordance with previous studies .\u003csup\u003e(29)\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Experiments on the determination of coal molecular structure parameters\u003c/h2\u003e \u003cp\u003eFTIR experiments on residual coal from Raw coal, T-1 \u0026amp; T-2 coal samples were performed using a Bruker INVENIO infrared spectrometer, Germany.The dried coal samples were mixed with KBr powder in the ratio of 1:100, evenly ground and put into a tablet press to make pressed tablets, which were placed in the sample bin of the infrared spectrometer for testing. The infrared spectrograms of the three sets of samples were split-peak fitted using PeakFit v4.12. In order to further quantitatively characterize the law of evolution of the internal micro-molecular structure of coal, it was calculated from the split-peak fitting data.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Microbial community structure succession pattern during microbial degradation process\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe changes in the methane concentration in the headspace gas inside the culture flasks during the 30-day experiments of CK, T-1, and T-2 are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a).The dynamic trend of methane concentration in CK, T-1 and T-2 was basically similar, showing a rapid increase in methane concentration at the beginning, a leveling off of the growth rate of methane concentration in the middle, and almost no growth or even a decreasing trend at the end of the period of desorption. Among them, the methane concentration in the headspace gas of CK was always less than 1% during the experiment, and there was almost no methane in the headspace gas in the fourth period. In group T-1, the methane concentration in the headspace gas was always higher than that in CK, and the peak methane concentration in the headspace gas was almost the same as that in the first three periods, which was 0.122 ml/g of coal, and there was still methane desorbed continuously in the fourth period. Coal contains a large amount of adsorbed and free methane in different pore structures.\u003csup\u003e(31)\u003c/sup\u003e In the T-2 group, free methane molecules were rapidly desorbed from the coal at the initial stage of each period, and the methane concentration in the headspace increased rapidly, with a maximum peak value of 0.640 mL/g coal, which was 5.24 times higher than that of T-1. In addition, the methane desorption in the T-2 period showed a stepwise downward trend in sequence, with a decrease of about 50% of that of the previous period, and in the last period, the methane desorption was greatly reduced, and its methane desorption was reduced to 0.132 mL/g, and the growth rate of methane concentration tends to be gentle at 28 d.\u003c/p\u003e \u003cp\u003eThe dynamic trend of methane concentration in T-1 indicated that the microorganisms were able to enhance the further desorption of residual methane from the coal to a certain extent under the environment of inorganic salt medium, but their metabolic reactions were less active and the effect of enhanced methane desorption was weaker due to the scarcity of metabolic substrates at the initial stage. With the increase of time, the residual methane was gradually desorbed from the coal, and the amount of methane desorption was almost the same in different periods, which indicated that the advantageous flora to enhance the desorption of residual methane evolved in the culture environment during the experimental process, and the metabolism substrate was increased to improve the microbial degradation activity. The dynamic trend of methane concentration in T-2 indicated that after the addition of tryptone to the culture environment, it provided abundant nitrogen and amino acids for the metabolic activity of microorganisms, and its metabolic activity was obviously enhanced, and the effect of methane desorption was significant. However, after a large amount of free methane is gradually desorbed in larger pores, it is necessary to desorption free and adsorbed methane which is difficult to desorption from small and medium-sized pores, so it is difficult to maintain the initial methane desorption rate. \u003csup\u003e(32)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe relative abundance of microbial community composition in the three culture environments is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b).Initial samples were taken at d 0 of the experiment, when the microbial community consisted mainly of the native microorganisms of coal, with \u003cem\u003ePseudomonas\u003c/em\u003e (53.17\u0026thinsp;\u0026plusmn;\u0026thinsp;10.94%) the dominant genus of in situ microorganisms, followed by \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e (21.63\u0026thinsp;\u0026plusmn;\u0026thinsp;9.37%) and \u003cem\u003eBacillus\u003c/em\u003e (2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81%) were more predominant. The microbial community composition within the culture system changed on the 30th d of the experiment. the original dominant genus \u003cem\u003ePseudomonas\u003c/em\u003e in CK in the aqueous environment was absolutely dominant with a relative abundance of 88.49\u0026thinsp;\u0026plusmn;\u0026thinsp;5.18%. While \u003cem\u003eBrevibacillus\u003c/em\u003e had the highest percentage of microbial community composition at the genus level in T-1, with a relative abundance of 89.26\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27%, \u003cem\u003eBacillus\u003c/em\u003e (56.53\u0026thinsp;\u0026plusmn;\u0026thinsp;7.05%) was the dominant genus. T-2 had the most abundant microbial community composition due to the presence of more reaction substrates. \u003cem\u003eBacillus\u003c/em\u003e (56.53\u0026thinsp;\u0026plusmn;\u0026thinsp;7.05%) had the largest percentage of relative abundance and \u003cem\u003eTissierella\u003c/em\u003e (11.53\u0026thinsp;\u0026plusmn;\u0026thinsp;8.20%), \u003cem\u003eClostridium\u003c/em\u003e (7.59\u0026thinsp;\u0026plusmn;\u0026thinsp;7.27%) and \u003cem\u003eParaclostridium\u003c/em\u003e (6.77\u0026thinsp;\u0026plusmn;\u0026thinsp;6.97%) were the dominant genera.\u003c/p\u003e \u003cp\u003eThe microbial community in the CK incubation environment did not change significantly due to the presence of water only, and \u003cem\u003ePseudomonas\u003c/em\u003e always dominated.\u003cem\u003ePseudomonas\u003c/em\u003e is thought to have a good solubilizing effect on low rank coal, and has the property of decomposing proteins, glucose, and acid production.\u003csup\u003e(33)\u003c/sup\u003e In T-1 the microbial community changed and \u003cem\u003eBrevibacillus\u003c/em\u003e became the dominant genus. Majid Rasool Kamli et al. concluded that \u003cem\u003eBrevibacillus\u003c/em\u003e has a wide range of enzymatic activities and can degrade a wide range of compounds and they are resistant to heavy metals and are effective strains for coal degradation.\u003csup\u003e(34)\u003c/sup\u003e Thus the increased proportion of \u003cem\u003eBrevibacillus\u003c/em\u003e in the microbial community allowed the coal to be degraded to some extent, slightly facilitating the desorption of residual methane within the coal. When a nitrogen source is added to the culture environment, the microbial community undergoes a succession in which the dominant genera \u003cem\u003eBacillus, Tissierella, Clostridium\u003c/em\u003e and \u003cem\u003eParaclostridium\u003c/em\u003e belong to the Firmicutes.Several articles have shown that these four genera are capable of degrading complex biomasses and compounds in anaerobic environments, providing metabolic substrates for other microorganisms through the decomposition of sugars or peptones to produce a mixture of organic acids and alcohols, as well as reducing the nucleation structure of the coal and enhancing the permeability and porosity of the coal.\u003csup\u003e(35)\u0026minus;(38)\u003c/sup\u003e Among them, \u003cem\u003eBacillus\u003c/em\u003e, as the most dominant genus, has been indicated to be involved in the degradation of organic matter in a variety of environments, e.g., phosphate degradation in contaminated soils, and increasing the degradation efficiency of baijiu lees by increasing cellulase activity.\u003csup\u003e(39),(40)\u003c/sup\u003e In addition, \u003cem\u003eBacillus\u003c/em\u003e exhibits active carbohydrate and amino acid metabolism, and is characterized not only by strong secretion of enzymes that stimulate the metabolic activity of other microorganisms, but also by the production of bacteriocins that inhibit pathogenic bacteria. The results of microbial community succession in T-2 showed four genera belonging to the Firmicutes, whose mutual stimulatory effects during metabolism enhanced the anaerobic degradation of the coal by microorganisms, which led to a significant increase in desorption of residual methane from coal\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Differential analysis of microbial degradation metabolic functions\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a) demonstrates the differential analysis of major metabolic functions by KEGG level 3 for the three groups of experiments, and the effect of microbial degradation of coal may be related to functional genes for degradation of aromatic compounds, etc.\u003csup\u003e(42)\u003c/sup\u003e The screening of relevant metabolic pathways revealed that the metabolic activities of microorganisms enhanced the degradation of aromatic compounds and fatty acids in coal except nitrotoluene, and this metabolic degradation was more significant under the stimulation of nitrogen sources, especially in the degradation functions of xylene, styrene, and polycyclic aromatic hydrocarbons (PAHs).In addition, the methane metabolism function of the experimental group with the addition of BES showed a decreasing trend indicating that BES inhibited the methane metabolism function of the bacteria, and the methane desorbed in the experiments was derived from the coal residue, and no additional biomethane was synthesized.\u003c/p\u003e \u003cp\u003eTo further investigate the key degradation metabolites during microbial metabolism, the differences in enzyme genes related to aromatic compound degradation and methane metabolism were analyzed in each experimental group by KEGG database as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b). In the methane metabolic pathway, acetate and coenzyme M are crucial intermediates for the synthesis of biomethane.\u003csup\u003e(43),(44)\u003c/sup\u003e Acetate kinase (EC:2.7.2.1, K00925) catalyzes the reversible transfer of phosphate groups from acetylphosphate to ADP, produces acetate and ATP, and plays a central role in methane metabolism.\u003csup\u003e(45)\u003c/sup\u003e The hyperthermophilic euryarchaeon Methanococcus jannaschi is able to utilize coenzyme M as a terminal methyl carrier in methanogenesis, where phosphosulfolactate synthase (EC: 4.4.1.19, K08097), 2- phosphosulfolactate phosphatase (EC: 3.1.3.71, K05979) can catalyze coenzyme M biosynthesis sequentially.\u003csup\u003e(46)(47)\u003c/sup\u003e The changes in their relative abundance are consistent with Qiu et al. 2022 who showed that the addition of BES resulted in a decrease in acetate kinase activity and coenzyme M (CoM) content in the methane metabolic pathway.(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) For degradation metabolic pathways of aromatic compounds, toluene monooxygenase system protein A (EC: 1.14.13.236, K15760) can introduce hydroxyl groups on the benzene ring in multiple degradation pathways of toluene;\u003csup\u003e(49)\u003c/sup\u003e benzoate/toluate 1,2-dioxygenase subunit alpha (EC: 1.14.12.10, K05549) with dihydroxycyclohexadiene carboxylate dehydrogenase (EC: 1.3.1.25. K05783) promote the introduction of hydroxyl groups in the degradation metabolism of benzoate and xylene;\u003csup\u003e(50)(51)\u003c/sup\u003e While 2,3-dihydroxy-p-cumate/2,3-dihydroxybenzoate 3,4-dioxygenase (EC: 1.13.11.14, K10621), catechol 1,2-dioxygenase (EC: 1.13.11.1, K03381) and catechol 2,3-dioxygenase (EC: 1.13.11.2, K00446) were able to catalyze different ring-opening reactions upon the introduction of oxygen-containing functional groups, such as hydroxyl groups, respectively, and similar degradation of chlorobenzenes and styrenes was achieved by K03381 and K00446;\u003csup\u003e(52)\u0026minus;(54)\u003c/sup\u003e benzaldehyde dehydrogenase (EC: 1.2.1.28, K00141) catalyzes the synthesis of methyl benzoate from xylene and benzoates from toluene;\u003csup\u003e(55)\u003c/sup\u003e naphthalene 1,2-dioxygenase ferredoxin reductase component (EC: 1.18.1.7, K14581) enhances the introduction of hydroxyl groups to PAHs and ethylbenzene.\u003csup\u003e(56)\u003c/sup\u003e 2-pyrone-4,6-dicarboxylate lactonase (EC: 3.1.1.57, K10221) and hydroxyquinol 1,2-dioxygenase (EC: 1.13.11.37, K04098), on the other hand, are able to catalyze aromatic compounds in the metabolic pathway of benzoate degradation cyclization of aromatic compounds in the metabolic pathway of benzoate degradation.\u003csup\u003e(57)(58)\u003c/sup\u003e The increase in relative abundance of K05979, K05549, K05783, K15760, and K00141 over CK suggests that microbial metabolic functions stimulate the introduction of hydroxyl groups for a variety of aromatic compounds in coal, which enhances the hydrophilicity of coal. The increase in the relative abundance of K00446, K10621, K10221, K04098 and K03381 in T-1 and T-2 indicated that the metabolism of these genes catalyzed the de-cyclization of the coal macromolecule structure after the introduction of benzene rings into the aromatic compounds, which led to the evolution of the coal macromolecules into chains, which was conducive to the microbial pore-expansion and infiltration of the coal. In addition, the abundance of K05979, K05549, K05783, K15760, and K00141 within T-1 and T-2 were similar, while there was a significant difference between K00446, K10621, K10221, K04098, and K03381, confirming that additional nitrogen sources may not have a significant effect on promoting the introduction of oxygen-containing functional groups, such as hydroxyl, into the benzene ring, but the Firmicutes in the stimulated by nitrogen source was able to significantly catalyze the ring-opening reaction of part of the aromatic ring and enhanced biologically driven methane desorption capabilities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the adsorption isotherms of coal samples of groups Raw Coal, T-1 and T-2 are demonstrated, respectively. Among them, the types of adsorption and desorption isotherms of the three groups of coal samples are basically the same, and all of them have hysteresis loops and obvious inflection points. The isothermal adsorption lines were classified into eight types by IUPAC,\u003csup\u003e(59)\u003c/sup\u003e which can be regarded as different types according to the grading criteria at different relative pressure stages. Firstly, at the stage of low relative pressure, the phenomenon of microporous filling occurs inside the coal body, and the microporous structure has a strong adsorption capacity for nitrogen, so that the adsorption isotherm rises rapidly, and it belongs to the adsorption isotherm onset stage of Class I. After the end of the rapidly rising phase of adsorption isotherm, the curve shows a smooth and slowly rising trend, and in this relative pressure region, the pores of the coal body have successively produced monolayer adsorption and multilayer adsorption effects on nitrogen analysis. With the increasing relative pressure, capillary adsorption cohesion phenomenon, when the relative pressure is about 0.5 when the relative pressure of the desorption branch of the phenomenon of hysteresis, indicating that there are about 4nm pores exist in the coal body, this stage is manifested in the Ⅳ class (a) type. When the relative pressure is close to 1, unlike Type IV (a), the adsorption isotherm rises rapidly and is saturated, showing that there is still a larger pore structure in the coal body, which belongs to the end stage of Type II.\u003c/p\u003e \u003cp\u003eIn addition to showing the pore size structure of coal, adsorption isotherms can further analyze the geometric properties of coal pores by comparing the adsorption/desorption hysteresis loop type criteria proposed by IUPAC.\u003csup\u003e(59)(60)\u003c/sup\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the hysteresis rings of all three sets of coal samples belong to the H3 type, indicating that a large number of slit-type pore structures are distributed on the coal body. The adsorption/desorption isotherms showed low-pressure hysteresis, and the two curves did not overlap. According to the related research, the reason for the low-pressure hysteresis in this paper may be due to the developed microporous structure of the coal samples, with a large number of tiny \"thin-necked bottles\" pores, and irreversible adsorption phenomena in the pores led to incomplete adsorption equilibrium. Each desorption branch shows a clear inflection point at a relative pressure of about 0.5 and a wide hysteresis loop, indicating the presence of ink-bottle and semi-open pore structures on the coal body. In addition, at relative pressures greater than 0.45, all of them showed significant hysteresis loops, indicating the presence of a large number of open pore structures on the coal body. Nanoscale micropores are the main factor affecting the methane adsorption capacity of coal.\u003csup\u003e(61)\u003c/sup\u003e Microbial degradation makes the hysteresis loops of isotherms smaller and flatter, the low-pressure hysteresis phenomenon weaker, and the low-pressure end more skewed towards the X-axis, suggesting that the pore-connecting pores of the coal samples are developing towards mesopores, with the trend of mesopore development being more pronounced in T-1. From the experimental data, the maximum adsorption of nitrogen by Raw Coal was 0.9516 ml/g, and the maximum adsorption of nitrogen by T-1 and T-2 coal samples was 0.4943 ml/g and 0.5127 ml/g, which were reduced by 48.1% and 46.1%, respectively, compared with that of Raw Coal. The metabolic activities of Firmicutes stimulate the activity of microbial communities and the release of more metabolites, and some key metabolites affect the pore distribution, pore size, pore morphology, and surface chemistry of the pore wall in the coal body,etc.,\u003csup\u003e(62)\u003c/sup\u003e so that the volume of micropores inside the coal body will be reduced, and the adsorption space of the methane will become smaller, and the adsorption capacity will be reduced accordingly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Coal pore structure development law under microbial action\u003c/h2\u003e \u003cp\u003eIn order to study the developmental characteristics of the full pore size pore structure of coal under microbial degradation, low-temperature liquid nitrogen adsorption test and mercury compression test were carried out on the treated coal samples of groups Raw Coal, T-1 and T-2, respectively. The pore size distributions of the three groups of coal samples are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. In the range of 0\u0026ndash;15 nm, the microporous pore size distribution of Raw Coal is more extensive, in which a more uniform regional distribution is presented near 1\u0026ndash;2 nm and 7\u0026ndash;15 nm, and a bimodal pore distribution is presented out of 2.3-4 nm, and the pores of the Raw Coal are most concentrated at 2.5 nm and 3.1 nm in particular. In the microporous range of T-1 coal samples, the distribution of pores in the range of 1-1.5 nm and 7.5 nm-15 nm is relatively average, while in the range of 1.5\u0026ndash;7.5 nm is the distribution of a number of large-scale pore volume peaks, especially in the distribution of pore sizes at about 1.5 nm and 4.7 nm is the largest value. The pore distribution of the T-2 coal samples was similar to that of the residual coals of the T-1 group, with obvious peaks at 2.8 nm and 8.5 nm, where some micropores were concentrated. In the range of 15-50nm, there was no significant difference between the coal samples before and after microbial action. In the range of 50\u0026ndash;400 nm, the mesopore pores of coal showed a two-stage development trend. Firstly, in the 50\u0026ndash;100 nm region, the pore size distribution pattern of the three groups of coal samples is similar and all of them have a large number of pores distributed near 55\u0026ndash;70 nm, in which the pore size distribution values of Raw Coal, T-1 and T-2 decrease in this place in turn. Secondly, in the 100\u0026ndash;400 nm region, the Raw Coal pore distribution shows a decreasing trend, and from 180 nm onwards the mesopore pore distribution is extremely small, while the T-1 and T-2 pore size distribution patterns are similar and more evenly distributed. In the macroporous region larger than 400 nm, the pores of Raw Coal coal samples showed a three-stage distribution, mainly in the range around 550 nm, 840\u0026ndash;1350 nm, and 2120\u0026ndash;3122 nm. The pore size distribution of the T-1 coal sample shows an increasing and then decreasing trend, and the pores are more concentrated at the two ends of the macroporous region.The pore size distribution curve of T-2 coal sample is higher than that of the other two groups, especially after 1000 nm, the trend is significant, and the large pores of the coal sample appear to be greatly developed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThrough the analysis of Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the developmental and evolutionary characteristics of the pore structure of anthracite under the degradation of functional microorganisms can be further explored. After the degradation of coal by microorganisms, the total pore volume of coal samples in both T-1 and T-2 groups increased compared to the total pore volume of Raw Coal, while the total specific surface area decreased and the average pore size increased significantly. Among them, the total pore volume of coal samples in T-1 and T-2 groups increased by 22.70% and 58.11%, respectively, compared with that of Raw Coal; the total specific surface area decreased by 44.11% and 49.61%, respectively, and the average pore diameters were 2.19 and 3.14 times higher than those of Raw Coal, respectively. In addition, the percentage of microporous pore volume and the percentage of specific surface area of T-1 coal sample decreased by 9.03% and 22.82%, respectively, compared with that of Raw Coal, while that of T-2 coal sample decreased by 9.33% and 22.58%, respectively. Since micropores are the main contributor to the specific surface area of coal and have less influence on the pore volume value of coal,\u003csup\u003e(63)\u003c/sup\u003e microbial degradation has led to the development of a large number of microporous structures in coal, and the proportion of micropores in the total pore structure of coal has decreased dramatically. There was no significant difference in the transitional pore of the three groups of experimental coal samples. T-1 and T-2 showed different developmental trends compared with the Raw Coal samples. the percentage of mesopores in the T-1 samples increased by 5.27%. the percentage of mesopores in the T-2 samples decreased by 15.28%. the percentage of mesopores in the T-2 samples increased by 5.27% and decreased by 15.28%. For the macropore, the pore volume of T-1 and T-2 coal samples is 1.39 and 2.68 times of that of Raw Coal, and the percentage of pore volume increases by 5.08% and 27.31%, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePore structure parameters of three groups of coal samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTotal Pore Volume /(cm\u003csup\u003e3\u003c/sup\u003e\u0026bull;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eStage Pore Volume /(cm\u003csup\u003e3\u003c/sup\u003e\u0026bull;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTotal Pore Area /(m\u003csup\u003e2\u003c/sup\u003e\u0026bull;g-\u003csup\u003e1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c11\" namest=\"c8\"\u003e \u003cp\u003eStage Pore Area /(m2\u0026bull;g-1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAverage Pore Diameter\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMicropore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTransition pore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMesopore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMacropore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMicropore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTransition pore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMesopore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eMacropore\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRaw Coal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00370\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.00145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.52151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.42616\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.02402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.06583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.00551\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e28.37913\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.00201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.29158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.17174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.03753\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.07333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.00898\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e62.28136\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00585\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.00389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.26263\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.15531\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.03428\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.05924\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.01380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e89.09873\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThrough the joint characterization of the pore structure of the three groups of experimental coal samples, it was confirmed that microbial degradation and metabolism could promote the pore structure of anthracite coal to show a more obvious evolution pattern, and this evolution pattern was more significant after the addition of nitrogen source. Among them, the microbial degradation of anthracite coal in the inorganic salt medium environment after its original micro-small pores to mesopore, macropore evolution and development, the average pore size increased, reducing the adsorption space of methane in coal, showing the effect of pore expansion and pore enlargement. This is consistent with the results of Guo's study.\u003csup\u003e(21)\u003c/sup\u003e And although the pore structure of micropore and transitional pore had no obvious additional effect after the additional addition of nitrogen source, and the effect of mesopore development was weaker, the macropore presented a particularly significant growth, and the proportion of the coal's whole pore diameter was greatly increased. This suggests that the nitrogen source further stimulated the anaerobic degradation of coal by Firmicutes and changed the evolution law of the pore structure of anthracite coal during microbial degradation. Compared with the developmental trend of micropore favoring mesopore in T-1 coal samples, T-2 micropore showed a large-scale transformation phenomenon to macropore structure, which led to a further decrease in the total specific surface area of the coal, and the effect of expanding and increasing pores was more significant. In addition, tiny pore structure is the main contributor to the adsorption of methane molecules by coal, which can provide more adsorption sites for methane, so the development of tiny pore structure will inevitably reduce the adsorption capacity of coal for methane and improve its transport characteristics, \u003csup\u003e(64)(65)\u003c/sup\u003e which is important for the enhancement of the level of biologically driven gas desorption. The coal macropore structure is regarded as the \"valve that restricts the flow\", and the methane flow rate in the macropore controls the desorption rate of the residual methane in the coal. \u003csup\u003e(66)\u003c/sup\u003e The Firmicutes stimulated by the nitrogen source greatly improves the permeability of the anthracite coal, significantly increases the effective gas diffusion channels of the coal, and promoted the rapid desorption of adsorbed and free methane.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Characterization of changes in the molecular structure of coal under microbial action\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to study the evolution law of functional groups in low-permeability coals under microbial action, FTIR tests were carried out on three groups of experimental residual coals, respectively. According to a previous study,\u003csup\u003e(67)\u003c/sup\u003e the infrared spectral map of coal is mainly divided into four parts, in which the range of 900\u0026thinsp;\u0026minus;\u0026thinsp;700 corresponds to aromatic hydrocarbons, the range of 1800\u0026thinsp;\u0026minus;\u0026thinsp;1000 corresponds to oxygen-containing functional groups, the range of 3000\u0026thinsp;\u0026minus;\u0026thinsp;2700 corresponds to aliphatic hydrocarbons as well as the range of 3600\u0026thinsp;\u0026minus;\u0026thinsp;3000 corresponds to hydroxyl groups as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(a). In order to visually compare the differences in functional group content under microbial action, quantitative calculations were carried out by peak fitting and the fitted peaks of each group were R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.999, and the results obtained after peak fitting are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(b).\u003c/p\u003e \u003cp\u003eIn the 3600\u0026thinsp;\u0026minus;\u0026thinsp;3000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e band, there are mainly three kinds of characteristic peaks corresponding to hydroxyl functional groups: hydroxyl-π, self-conjugated hydroxyl, and hydroxyl-N. The relative content of hydroxyl in T-2 coal samples is about 117.231, which is 2.822 times of the hydroxyl content in Raw Coal.The content of hydroxyl in T-1 coal samples is about 2.251 times of that in Raw Coal. Four groups of characteristic peaks corresponding to aliphatic hydrocarbon functional groups mainly existed in the range of 3000\u0026thinsp;\u0026minus;\u0026thinsp;2700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: symmetric stretching vibrations of aliphatic methyl and methylene groups and asymmetric stretching vibrations. According to the results of peak fitting, the content of aliphatic hydrocarbon functional groups on the surface of the coal samples showed an increasing trend of different degrees after microbial action. Compared with Raw Coal, the aliphatic hydrocarbon content of T-1 and T-2 increased by 88.46% and 64.01%, respectively. In the 1800\u0026thinsp;\u0026minus;\u0026thinsp;1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e band, the contents of oxygen-containing functional groups of T-1 and T-2 coal samples were higher than those of Raw Coal, which were 1.778 and 1.511 times higher than those of Raw Coal, respectively. And at 1600 cm-1 corresponding to aromatic hydrocarbons, the content of Raw Coal coal samples was the highest, and the aromatic hydrocarbons content of T-1 and T-2 decreased sequentially. In addition, carboxyl group is one of the key functional groups to enhance the hydrophilicity of coal and reduce the adsorption capacity of methane.\u003csup\u003e(68)\u003c/sup\u003e In the region representing the carboxyl functional group around 1700 cm-1, although the carboxyl vibration of anthracite is generally weak, both T-1 and T-2 showed different degrees of growth after microbial metabolism. Within the 700\u0026ndash;900 cm-1 region, four groups of characteristic peaks mainly existed in each group of coal samples: benzene ring five-substituted, benzene ring four-substituted, benzene ring three-substituted, and benzene ring two-substituted. Among them, the aromatic hydrocarbon content of Raw Coal was the highest at 9.053, and T-1 and T-2 decreased by 14.01% and 33.78%, respectively, compared with that of Raw Coal, which was also consistent with the changes occurred at the position of 1600 cm-1, and in addition, the percentage of benzene ring disubstitution in the coal samples of the treatment group showed an increasing trend compared with that of the original coal group.\u003c/p\u003e \u003cp\u003eIn order to further quantitatively characterize the evolutionary law of the internal microscopic molecular structure of coal, it was calculated by fitting the data with split peaks. The calculation results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003csup\u003e(30)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFTIR quantitative parameterization\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eI\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDOC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRaw Coal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21.638\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.415\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.445\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14.057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.557\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.413\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.983\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.336\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAmong them, I\u003csub\u003e1\u003c/sub\u003e represents the average chain length and degree of branching of aliphatic branched chains of the coal body, which decreased by 35.04% and 47.86% in T-1 and T-2 coal samples, respectively, compared with Raw Coal. The aromaticity of the coal body can be characterized by the ratio of the number of aromatic hydrocarbons to the number of aliphatic hydrocarbons, I\u003csub\u003e2\u003c/sub\u003e, which decreased by 54.41% and 59.62% in the T-1 and T-2 coal samples, respectively, compared with Raw Coal. I\u003csub\u003e3\u003c/sub\u003e is the ratio of the number of hydroxyl and ether groups reflecting the interconversion of oxygen-containing functional groups within the coal body, and the I\u003csub\u003e3\u003c/sub\u003e of T-1 coal was 1.183 times that of Raw Coal, and the conversion of oxygen-containing functional groups prompted by T-2 due to the addition of a nitrogen source to make the microbial metabolism activity more intense was particularly significant, and its I\u003csub\u003e3\u003c/sub\u003e was 3.594 times that of Raw Coal. Carbonyl is the main form of reactive oxygen present in the coal body,\u003csup\u003e(69)\u003c/sup\u003e I\u003csub\u003e4\u003c/sub\u003e is the ratio of carbonyl to aromatic hydrocarbons represents the degree of reactivity of elemental oxygen in the coal body, T-1 and T-2 coal samples I\u003csub\u003e4\u003c/sub\u003e increased by 50% and 180.814%, respectively. The aromatic ring condensation DOC within the coals was characterized by the ratio of the out-of-plane deformation vibration of -CH in various substituted aromatic hydrocarbons to the stretching vibration of C\u0026thinsp;=\u0026thinsp;C in aromatic hydrocarbons, and Raw Coal had the highest aryl ring condensation, with a decrease in the DOC of T-1 and T-2 coals by 7.19% and 24.49%, respectively.\u003c/p\u003e \u003cp\u003eThe results of peak fitting and quantitative calculations showed that a large number of ether groups were converted to hydroxyl groups during the microbial metabolism, which introduced various oxygen-containing functional groups, such as hydroxyl groups, into the coal macromolecular structure and thus enhanced the content of oxygens in the coal molecular structure. Because the hydroxyl group can form hydrogen bonds with hydrogen atoms and then form a water film on the coal pore surface, it can strengthen the hydrophilicity of the coal pore structure and weaken the adsorption capacity of methane.\u003csup\u003e(70)\u003c/sup\u003e Aromatic hydrocarbon functional group is an important component that constitutes the main part of coal,\u003csup\u003e(70)\u003c/sup\u003e which is transformed from the highly substituted form to the low-substituted form after microbial degradation, the condensation of the aromatic structure decreases, the molecular structure of the coal becomes sparse, and the stabilized benzene ring structure in the body of the coal is gradually transformed to the loose chain structure, and the aroma of the coal decreases. At the same time, the continuous breaking of the coal's fatty chains leads to a decrease in chain length and an increase in branchedness, which can provide more metabolic sites for microorganisms.\u003csup\u003e(21)\u003c/sup\u003e In addition, the above phenomenon of coal molecular structure evolution was more significant after the addition of nitrogen source, and the transformation effect within the coal oxygen-containing functional groups was significant, especially the transformation efficiency to the key oxygen-containing functional groups, such as hydroxyl group, could be increased by several times. This suggests that the nitrogen source stimulated the metabolic activity of Firmicutes to be more intense, releasing more key metabolites acting on the coal molecular structure, thus enabling the microorganisms to degrade coal more efficiently and promoting the evolution of coal molecular structure.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eThe effect of microbial degradation and metabolism on the microstructure of anthracite coal under the stimulation of nitrogen source was investigated by adding functional microorganisms, such as \u003cem\u003eBacillus\u003c/em\u003e sphaericus, to anthracite coal. The results showed that Firmicutes, mainly \u003cem\u003eBacillus\u003c/em\u003e, played a major role in coal degradation under the synergistic effect of coal as a biodegradable carbon source and additional nitrogen source, which reduced the methane adsorption capacity of coal by shedding the alkyl side chains of the coal molecular structure and introducing oxygen-containing functional groups, such as hydroxyl, and increased the diffusion channels of methane within coal by improving the function of the degradation of the cyclic skeleton within the coal, which strengthened microbial-driven methane desorption capacity. During anaerobic culture, Firmicutes, represented by \u003cem\u003eBacillus\u003c/em\u003e, dominated and tended to be stable in the culture group with the best methane desorption. The relative abundance of genes for a variety of key degradation enzymes produced by their metabolism increased, and they were able to promote the catalytic introduction of oxygen-containing functional groups, such as hydroxyls, on the structure of the benzene ring and ring-opening reaction of the coal macromolecule compounds; Secondly, the degradation of microorganisms reduced the aromaticity of coal, shortened the long chain of fat and increased the degree of branching, and increased the content of oxygen-containing functional groups such as hydroxyl groups, which enhanced the hydrophilicity of coal, reduced the adsorption capacity of methane, and loosened the skeleton of coal macromolecules. As a result, the degradation of microorganisms led to the development of the coal's micropore structure towards mesopore and macropore, which led to the improvement of the effective diffusion channels and methane transportation state within the coal, and ultimately led to the desorption of methane in large quantities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.Z., C.Z.,B.L., S.X., Y.Y.,T.Z.,X.Z.and J.W. conducted the bulk of the data analysis for the study and co-wrote the manuscript. S.X.,C.Z.and B.L. provided the funding for the study, were involved in the conceptualization of the study, as well as assisted in the writing of the manuscript. All authors read and approved the final manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work is financially supported by National Key R\u0026amp;D Program of China(NO.2023YFC3009002); Independent Research fund of Joint National-Local Engineering Research Centre for Safe and Precise Coal Mining (Anhui University of Science and Technology)(NO. EC2023006); Graduate Innovation Fund of Anhui University of Science and Technology(No.2022CX2020); National Natural Science Foundation of China (No. 52274171); The Key Research and Development Projects in Anhui Province(NO.2022l07020020).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNiu Y, Wang EY, Li ZH, Gao F, Zhang ZZ, Li BL, Zhang X (2022) Identification of Coal and Gas Outburst-Hazardous Zones by Electric Potential Inversion During Mining Process in Deep Coal Seam. Rock Mech Rock Eng 55:3439\u0026ndash;3450\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou F, Xia T, Wang X, Zhang Y, Sun Y, Liu J (2016) Recent developments in coal mine methane extraction and utilization in China: A review. J Nat Gas Sci Eng 31:437\u0026ndash;458\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoore TA (2012) Coalbed methane: A review. Int J Coal Geol 101:36\u0026ndash;81\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K, Du F (2020) Coal-gas compound dynamic disasters in China: A review. Process Saf Environ Protect 133:1\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang W, Li H, Liu Y, Liu M, Wang H, Li W (2020) Addressing the gas emission problem of the world\u0026rsquo;s largest coal producer and consumer: Lessons from the Sihe Coalfield, China. Energy Rep 6:3264\u0026ndash;3277\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu YY, Zhang HD, Zhou Z, Ge ZL, Chen CJ, Hou YD, Ye ML (2021) Current Status and Effective Suggestions for Efficient Exploitation of Coalbed Methane in China: A Review. Energy Fuels 35:9102\u0026ndash;9123\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Hu J, Xue H, Mao W, Gao Y, Yang J, He M (2020) Innovative approach based on roof cutting by energy-gathering blasting for protecting roadways in coal mines. Tunn Undergr Space Technol 99:103387\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, He J, Lu J, Lin B, Lu Y, Shi S, Ye Q (2022) A review of laboratory study on enhancing coal seam permeability via chemical stimulation. Fuel 330:125561\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu X, Zhao W, Xia D, Hou S, Fu H, Zhou Y (2022) Experimental study of advantages of coalbed gas bioengineering. J Nat Gas Sci Eng 102:104585\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang HF, Cheng YP, Yuan L (2013) Gas outburst disasters and the mining technology of key protective seam in coal seam group in the Huainan coalfield. Nat Hazards 67:763\u0026ndash;782\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu JW, Liu CY, Yao QL, Si GY (2020) The position of hydraulic fracturing to initiate vertical fractures in hard hanging roof for stress relief. Int J Rock Mech Min Sci 132:104328\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan M, Jiang C, Gan Q, Li M, Peng K, Zhang W (2020) Experimental investigation on the permeability, acoustic emission and energy dissipation of coal under tiered cyclic unloading. J Nat Gas Sci Eng 73:103054\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi M, Cheng Y, Wang C, Wang Z, Hu B, He X (2021) Effects of composition changes of coal treated with hydrochloric acid on pore structure and fractal characteristics. Fuel 294:120506\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng C, Li X, Li H, Jiang B, Chen Z (2023) Experimental Investigation on Pore-Fracture Variations in Coal Affected by Carbon Disulfide. Acs Omega 8:38426\u0026ndash;38440\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRitter D, Vinson D, Barnhart E, Akob DM, Fields MW, Cunningham AB, Orem W, McIntosh JC (2015) Enhanced microbial coalbed methane generation: A review of research, commercial activity, and remaining challenges. Int J Coal Geol 146:28\u0026ndash;41\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott AR (1999) In: Mastalerz M, Glikson M, Golding SD (eds) Improving Coal Gas Recovery with Microbially Enhanced Coalbed Methane. Springer Netherlands: Dordrecht,\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBao Y, Li Z, An C et al (2023) Multi-method characterization of pore structure evolution characteristics and mechanism of tar-rich coal by anaerobic fermentation. J China Coal Soc 48:891\u0026ndash;899\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang AK, Shao P, Wang QH (2021) Biogenic gas generation effects on anthracite molecular structure and pore structure. Front Earth Sci 15:272\u0026ndash;282\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePandey R, Harpalani S (2018) An imaging and fractal approach towards understanding reservoir scale changes in coal due to bioconversion. Fuel 230:282\u0026ndash;297\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu YY, Chai CJ, Zhou Z, Ge ZL, Yang MM (2020) Influence of bioconversion on pore structure of bituminous coal. Asia-Pac J Chem Eng 15e2399\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo H, Luo Y, Ma J et al Analysis of mechanism and permeability enhancing effect via microbial treatment on different-rank coals. J China Coal Soc 2014,39,1886\u0026ndash;1891\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaider R (2017) Coal degradation through fungal isolate RHC2 from Romanian brown coal sample. \u003cem\u003eEnergy Sources Part a-Recovery Util\u003c/em\u003e. Environ Eff 39:1785\u0026ndash;1790\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Liu X, Yang Z, Zhao S (2023) Biodegradation of Dananhu low-rank coal by Planomicrobium huatugouensis: Target metabolites possessing degradation abilities and their biodegradation pathways. Energy 276:127642\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia DP, Gu PT, Chen ZH, Chen LY, Wei GQ, Wang ZZ, Cheng S, Zhang YW (2023) Control Mechanism of Microbial Degradation on the Physical Properties of a Coal Reservoir. \u003cem\u003eProcesses\u003c/em\u003e 11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWebster TM, Smith AL, Reddy RR, Pinto AJ, Hayes KF, Raskin L (2016) Anaerobic microbial community response to methanogenic inhibitors 2-bromoethanesulfonate and propynoic acid. Microbiologyopen 5:537\u0026ndash;550\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin Y, Xu Z, Zhang J (1995) Natural classification of the high-rank coal pore structure and its appliction. J China Coal Soc 03:266\u0026ndash;271\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanders J, Gor GY, Neimark AV (2013) Density functional theory methods for characterization of porous materials. Colloids Surf A 437:3\u0026ndash;32\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMonson PA (2012) Understanding adsorption/desorption hysteresis for fluids in mesoporous materials using simple molecular models and classical density functional theory. Microporous Mesoporous Mat 160:47\u0026ndash;66\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuuberg EM, Deevi SC, Yun Y (1995) Elastic behaviour of coals studied by mercury porosimetry. Fuel 74:1522\u0026ndash;1530\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Shi S, Lin B, Lu J, Ye Q, Lu Y, Wang Z, Hong Y, Zhu X (2019) Effects of microwave-assisted pyrolysis on the microstructure of bituminous coals. Energy 187:115986\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Wang Z, Tang S, Elsworth D (2022) Re-evaluating adsorbed and free methane content in coal and its ad- and desorption processes analysis. Chem Eng J 428:131946\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao J, Qin Y, Shen J, Zhou B, Li C, Li G (2019) Effects of Pore Structures of Different Maceral Compositions on Methane Adsorption and Diffusion in Anthracite. Appl Sci 9:5130\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang HL, Liu XR, Zhang YW, Zhao SS, Yang ZW, Du ZP, Zhou AN (2021) Bacteria solubilization of shenmu lignite: influence of surfactants and characterization of the biosolubilization products. \u003cem\u003eEnergy Sources Part a-Recovery Util\u003c/em\u003e. Environ Eff 43:1162\u0026ndash;1180\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRasool Kamli M, Malik A, Sabir SM, Ahmad Rather J, Kim I (2022) Insights into the biodegradation and heavy metal resistance potential of the genus Brevibacillus through comparative genome analyses. Gene 846:146853\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao Y, He H, Zhan D, Huang HZ, Zhang Y, Fu B, Xu ZM, Ali MI, Liu FJ, Tao XX et al (2023) Changes of Permeability and Porosity of Tiefa Anthracite after Treatment with an Acetogenic Bacterium Clostridium sp. Geomicrobiol J 40:277\u0026ndash;284\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu Y, Zou W, Zhang K, Ye G, Yang J (2020) Advances and Applications of Clostridium Co-culture Systems in Biotechnology. Front Microbiol 11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo H, Zhang Y, Zhang J, Huang Z, Urynowicz MA, Liang W, Han Z, Liu J (2019) Characterization of Anthracite-Degrading Methanogenic Microflora Enriched from Qinshui Basin in China. Energy Fuels 33:6380\u0026ndash;6389\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIkkert OP, Gerasimchuk AL, Bukhtiyarova PA, Tuovinen OH, Karnachuk OV (2013) Characterization of precipitates formed by H2S-producing, Cu-resistant Firmicute isolates of Tissierella from human gut and Desulfosporosinus from mine waste. Antonie Van Leeuwenhoek 103:1221\u0026ndash;1234\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMENG D, ZHAI L, TIAN Q, GUAN Z, CAI Y, LIAO X (2019) Complete genome sequence of Bacillus amyloliquefaciens YP6, a plant growth rhizobacterium efficiently degrading a wide range of organophosphorus pesticides. J Integr Agric 18:2668\u0026ndash;2672\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang G, Yang D, Wang X, Cao W (2021) A novel thermostable cellulase-producing Bacillus licheniformis A5 acts synergistically with Bacillus subtilis B2 to improve degradation of Chinese distillers\u0026rsquo; grains. Bioresour Technol 325:124729\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFira D, Dimkić I, Berić T, Lozo J, Stanković S (2018) Biological control of plant pathogens by Bacillus species. J Biotechnol 285:44\u0026ndash;55\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Qin T, Feng F, Zhang Y, Xue S (2023) Nitrogen amendment enhances the biological methanogenic potential of bituminous coal. Fuel 351:128932\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerry JG (1997) Enzymology of the fermentation of acetate to methane by Methanosarcina thermophila. BioFactors 6:25\u0026ndash;35\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWongnate T, Sliwa D, Ginovska B, Smith D, Wolf MW, Lehnert N, Raugei S, Ragsdale SW (2016) The radical mechanism of biological methane synthesis by methyl-coenzyme M reductase. Science 352:953\u0026ndash;958\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChittori S, Savithri HS, Murthy MRN (2011) Preliminary X-ray crystallographic studies on acetate kinase (AckA) fromSalmonella typhimurium in two crystal forms. Acta Crystallogr Sect F Struct Biology Crystallization Commun 67:1658\u0026ndash;1661\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGraham DE, Xu H, White RH (2002) Identification of Coenzyme M Biosynthetic Phosphosulfolactate Synthase. J Biol Chem 277:13421\u0026ndash;13429\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGraham DE, Graupner M, Xu H, White RH (2001) Identification of coenzyme M biosynthetic 2-phosphosulfolactate phosphatase. Eur J Biochem 268:5176\u0026ndash;5188\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu S, Xia W, Xu J, Li Z, Ge S (2023) Impacts of 2-bromoethanesulfonic sodium on methanogenesis: Methanogen metabolism and community structure. Water Res 230:119527\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakami H, Nakasone K, Takaki Y, Maeno G, Sasaki R, Masui N, Fuji F, Hirama C, Nakamura Y, Ogasawara N et al (2000) Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res 28:4317\u0026ndash;4331\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVeith B, Herzberg C, Steckel S, Feesche J, Maurer KH, Ehrenreich P, Baumer S, Henne A, Liesegang H, Merkl R et al (2004) The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential. J Mol Microbiol Biotechnol 7:204\u0026ndash;211\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWrighton KC, Agbo P, Warnecke F, Weber KA, Brodie EL, DeSantis TZ, Hugenholtz P, Andersen GL, Coates J (2008) D. A novel ecological role of the Firmicutes identified in thermophilic microbial fuel cells. Isme J 2:1146\u0026ndash;1156\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMar\u0026iacute;n M, Plumeier I, Pieper DH (2012) Degradation of 2,3-Dihydroxybenzoate by a Novel meta-Cleavage Pathway. J Bacteriol 194:3851\u0026ndash;3860\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaes F, Liu XK, Mattes TE, Cupples AM (2015) Elucidating carbon uptake from vinyl chloride using stable isotope probing and Illumina sequencing. Appl Microbiol Biotechnol 99:7735\u0026ndash;7743\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenjamin RC, Voss JA, Kunz DA (1991) Nucleotide sequence of xylE from the TOL pDK1 plasmid and structural comparison with isofunctional catechol-2,3-dioxygenase genes from TOL, pWW0 and NAH7. J Bacteriol 173:2724\u0026ndash;2728\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng L, Wang W, Cheng JS, Ren Y, Zhao G, Gao CX, Tang Y, Liu XQ, Han WQ, Peng X et al (2007) Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir. Proc. Natl. Acad. \u003cem\u003eSci. U. S. A\u003c/em\u003e. 104, 5602\u0026ndash;5607\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Beilen JB, Funhoff EG (2007) Alkane hydroxylases involved in microbial alkane degradation. Appl Microbiol Biotechnol 74:13\u0026ndash;21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang SJ, Ma GC, Liu YJ, Ling BP (2015) Theoretical study of the hydrolysis mechanism of 2-pyrone-4,6-dicarboxylate (PDC) catalyzed by LigI. J Mol Graph 61:21\u0026ndash;29\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKutsuna R, Miyoshi-Akiyama T, Mori K, Hayashi M, Tomida J, Morita Y, Tanaka K, Kawamura Y et al (2019) Description of Paraclostridium bifermentans subsp. muricolitidis subsp. nov., emended description of Paraclostridium bifermentans (Sasi Jyothsna 2016), and creation of Paraclostridium bifermentans subsp. \u003cem\u003ebifermentans subsp. nov. Microbiol. Immunol.\u003c/em\u003e 63, 1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThommes M, Kaneko K, Neimark AV, Olivier JP, Rodriguez-Reinoso F, Rouquerol J, Sing KS (2015) W. Physisorption of gases, with special reference to the evaluation of surface area and pore size distribution (IUPAC Technical Report). \u003cem\u003ePure Appl. Chem.\u003c/em\u003e 87, 1051\u0026ndash;1069\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePyun S, Rhee C (2004) An investigation of fractal characteristics of mesoporous carbon electrodes with various pore structures. Electrochim Acta 49:4171\u0026ndash;4180\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai YD, Liu DM, Pan ZJ, Yao YB, Li JQ, Qiu YK (2013) Pore structure and its impact on CH\u003csub\u003e4\u003c/sub\u003e adsorption capacity and flow capability of bituminous and subbituminous coals from Northeast China. Fuel 103:258\u0026ndash;268\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrąpoć D, Mastalerz M, Dawson K, Macalady J, Callaghan AV, Wawrik B, Turich C, Ashby M (2011) Biogeochemistry of Microbial Coal-Bed Methane. Annu Rev Earth Planet Sci 39:617\u0026ndash;656\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShan CA, Zhang TS, Guo JJ, Zhang Z, Yang Y (2015) Characterization of the micropore systems in high-rank coal reservoirs of the southern Sichuan Basin, China. Aapg Bull 99:2099\u0026ndash;2119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu B, Cheng YP, He XX, Wang ZY, Jiang ZN, Wang CH, Li W, Wang L (2020) New insights into the CH\u003csub\u003e4\u003c/sub\u003e adsorption capacity of coal based on microscopic pore properties. Fuel 262\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi ZW, Lin BQ, Gao YB, Cao ZD, Cheng YY, Yu JY (2015) Fractal analysis of pore characteristics and their impacts on methane adsorption of coals from Northern China. Int J Oil Gas Coal Technol 10:306\u0026ndash;324\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu B, Cheng YP, Pan ZJ (2023) Classification methods of pore structures in coal: A review and new insight. GAS Sci Eng 110\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIbarra J, Mu\u0026ntilde;oz E, Moliner R (1996) FTIR study of the evolution of coal structure during the coalification process. Org Geochem 24:725\u0026ndash;735\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan YN, Bai ZQ, Liao JJ, Bai J, Dai X, Li X, Xu JL, Li W (2016) Effects of phenolic hydroxyl and carboxyl groups on the concentration of different forms of water in brown coal and their dewatering energy. Fuel Process Technol 154:7\u0026ndash;18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXin HH, Wang DM, Dou GL, Qi XY, Xu T, Qi GS (2014) The Infrared Characterization and Mechanism of Oxygen Adsorption in Coal. Spectr Lett 47:664\u0026ndash;675\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Y, Albijanic B, Wang YL, Yang JG (2018) Characterizing surface properties of oxidized coal using FTIR and contact angle measurements. Energy Sources Part a-Recovery Util Environ Eff 40:1559\u0026ndash;1564\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia W, Zhang W (2017) Characterization of surface properties of Inner Mongolia coal using FTIR and XPS. Energy Sources Part a-Recovery Util Environ Eff 39:1190\u0026ndash;1194\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Firmicutes, Microbial Degradation Metabolism, Coal Microstructure, Enhanced Methane Desorption","lastPublishedDoi":"10.21203/rs.3.rs-4673807/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4673807/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePromoting the permeability of deep, low-permeability coal seams through biological means is currently a research hotspot for enhancing the efficiency of coalbed methane extraction. There are few reports in the literature on whether it is possible to promote the development of the microstructure of the coal matrix by the degradation and metabolism of certain groups of functional microorganisms under the stimulation of nitrogen sources. In this study, we selected anthracite coal from Sihe Mine for microbial anaerobic degradation culture experiments. The effects of adding functional microorganisms on the microstructure of anthracite coal under the stimulation of nitrogen source was analyzed by high-throughput sequencing of samples before and after the cultivation and microcharacterization experiments of coal samples. The results showed that the peak amount of residual methane desorption from the coal during the biodegradation process in the experimental group reached 0.640 mL/g coal, and the cumulative amount of methane desorption in the whole period was as high as 1.318 mL/g coal. 16S rRNA high-throughput sequencing results indicated that the bacterial community structure had undergone significant succession after the biodegradation experiments, and that the Firmicutes represented by Bacillus(82.41% of the total) occupied the dominant niche. Metabolic pathway analysis based on KEGG database showed that the degradation of aromatic compounds by microorganisms appeared to be significantly enhanced by the addition of nitrogen sources. Alaso, the relative abundance of a number of key metabolic enzyme genes capable of catalyzing the introduction of oxygen-containing functional groups into the structure of the coal molecule and the de-cyclization reaction were increased. FTIR experiments revealed that biodegradation stimulated by nitrogen source reduced the aromaticity of coal by 59.62% and enhanced the hydroxyl functional group content by 1.822 times.Mercury pressure and low-temperature nitrogen adsorption experiments showed that the micropore pore volume of the treated coal decreased by 34.09%, and the macropore pore volume accounted for an increase of 168.28%, with an average pore size increment of 60.72 nm, and the adsorption level of the gases decreased by 46.1%. Therefore, the nitrogen source can stimulate Firmicutes on the degradation of polycyclic aromatic hydrocarbon and increase the content of oxygen-containing functional groups, which might promote the development of pores in coal and make the difficult-to-desorption methane desorb rapidly.\u003c/p\u003e","manuscriptTitle":"Influence of Metabolism of Firmicutes on the microstructure of anthracite under nitrogen source stimulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-25 11:20:39","doi":"10.21203/rs.3.rs-4673807/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-07-03T10:22:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-03T07:49:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Microbiology","date":"2024-07-02T11:21:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d3395d77-2e20-4988-9c8d-c01a9017c52b","owner":[],"postedDate":"July 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-25T11:20:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-25 11:20:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4673807","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4673807","identity":"rs-4673807","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}