Carbon-Nitrogen-Sulfur-Related Microbial Taxa and Genes Maintained the Stability of Microbial Communities in Coals.

Coal microbes are the predominant form of life in the subsurface ecosystem, which play a vital role in biogeochemical cycles. However, the systematic information about carbon-nitrogen-sulfur (C-N-S)-related microbial communities in coal seams is limited. In this study, 16S rRNA gene data from a total of 93 microbial communities in coals were collected for meta-analysis. The results showed that 718 functional genera were related to the C-N-S cycle, wherein N2 fixation, denitrification, and C degradation groups dominated in relative abundance, Chao1 richness, Shannon diversity, and niche width. Genus Pseudomonas having the most C-N-S-related functions showed the highest relative abundance, and genus Herbaspirillum with a higher abundance participated in C degradation, CH4 oxidation, N2 fixation, ammoxidation, and denitrification. Such Herbaspirillum was a core genus in the co-occurrence network of microbial prokaryotes and showed higher levels in weight degree, betweenness centrality, and eigenvector centrality. In addition, most of the methanogens could fix N2 and dominated in the N2 fixation groups. Among them, genera Methanoculleus and Methanosaeta showed higher levels in the betweenness centrality index. In addition, the genus Clostridium was linked to the methanogenesis co-occurrence network module. In parallel, the S reduction gene was present in the highest total relative abundance of genes, followed by the C degradation and the denitrification genes, and S genes (especially cys genes) were the main genes linked to the co-occurrence network of the C-N-S-related genes. In summary, this study strengthened our knowledge regarding the C-N-S-related coal microbial communities, which is of great significance in understanding the microbial ecology and geochemical cycle of coals.

Introduction

Coal is the most vital fossil fuel on the Earth.[1,2] The formation of coals is driven by geological events,[3] geologic settings,[4] and microorganisms.[5] Among them, microbes are the predominant form of life in the subsurface ecosystem including coals and play a vital role in biogeochemical cycles,[1] which have accompanied the evolution of coals over tens to hundreds of millions of years.[6]

Microbial activities run throughout the whole process from humus deposition to anthracite formation.[6] During the humus deposition period, anaerobic or facultative anaerobic bacteria participated in the decomposition of peat or low-rank coals in the anaerobic environment; and in the coalification metamorphic stage, microbes in surface water and groundwater infiltrated into coal seams by surface uplifting, which could act on n-alkanes and other organic matters in coals. Previous studies observed that some organic substances in coals could be degraded by a variety of microorganisms following a quasi-step-by-step biodegradation process.[1,6] The macromolecular substances of coals and/or peats were degraded into single molecules and oligomers by hydrolysis and fermentation bacteria at first, and then some intermediate products were generated by different acidifying bacteria, acetic acid-producing bacteria, and hydrogen-producing bacteria. The resulting products could further generate methane under the action of methanogens. However, these studies mainly monitored the biomarkers in coals, but the important factors influencing the coal biodegradation[7] and the interaction between microorganisms have received extensive attention[8,9] merely in recent years.

Except for these above C metabolic processes, the biogeochemical processes of N and S also have an impact on the coal biodegradation. Guo et al.[10] detected the microbial taxa related to N metabolism in coal seams, including N2 fixing taxa and denitrifying taxa. The participation of these microorganisms in N metabolism can increase the N availability of coal seam ecosystems because bioavailable N is a major limiting factor in the extreme oligotrophic environments[11] including coal seams. Shi et al.[12] found that microbial N metabolism had an effect on organic matter decomposition in coals such as the decomposition of cellulose and carbohydrate.

In contrast, S in coals is the most notorious environmental pollutant, and its geochemical processes are closely related to the deposition and formation of coals.[13] For example, most of the S in coals was derived from the seawater submerged in the peat swamp during the peat accumulation process. A large amount of seawater sulfate diffused into the bottom peat and was reduced to H2S, S, and polysulfides by microorganisms.[14] In addition, the release of H2S due to sulfate reduction would be detrimental to the methanogenesis process during the coal biodegradation.[15] The process of anaerobic fermentation of coals might also be affected by degraded intermediates and final products (such as sulfides), whose high concentrations affected pH, disrupted cell membranes, prevented protein synthesis, altered hydrogen partial pressure, reduced bioavailability of trace elements, and hindered mass transfer, and thereby disrupted the anaerobic degradation chain.[16] Among these inhibitory compounds, sulfide is formed by the microbial reduction of sulfate and the degradation of S-containing organic matter under anaerobic conditions, and microorganisms involved in the sulfate reduction could compete with other anaerobic bacteria in an environment with low redox potential.[16,17] On the other hand, the microbial S metabolism process is not necessarily completely detrimental to the biogeochemical processes of coals. Among the bacterial groups, there are a large number of microbial groups closely related to Desulfomonas. These sulfate-reducing bacteria can use kerogen as an end-point autoacceptor or shuttle to oxidize acetic acid or other simple fatty acids, which is the key to the degradation of organic matter in coal seams.[18] Meslé et al.[19] pointed that the depletion of bitumen by solvent extraction resulted in an increase in methane volume in some shales, indicating the methanogenic potential of the shale matrix. These shale-associated microbial communities were able to produce more acetate when grown on the fulvic acid fraction than on ether extracts of the same shale, wherein the microbes were grown under sulfate-reducing conditions rather than under methane-producing conditions.[20]

In summary, C–N–S-related microbial communities play an important role in the decomposition of coal organic matter and coal evolution. Therefore, it is of great significance to systematically describe the C–N–S-related microbial communities in coal seams. In this study, 16S rRNA data of microbial composition in coal samples from the NCBI database were extracted and reanalyzed. The study aims to (1) describe the levels of the C–N–S-related microbial communities and functional genes in coal seams, (2) explore the important role of C–N–S-related groups in the microbial community, and (3) clarify the correlation among C–N–S-related groups in coal seams.

Materials and Methods

Data Sets

Until February 2022, literature retrieval was conducted through the Web of Science database, and the published papers[5,8,21−30] of “coal” and “microbial communities” were retrieved. The fastQ files according to the accession numbers of the 16S rRNA gene data from coal samples were downloaded. 16S rRNA gene data from 93 microbial communities in coals were collected for meta-analysis. The detailed sample information is shown in Table S1.

Bioinformatics Analysis

For microbial community (bacteria and archaea) analysis, the reads from 16S genes were merged and the raw sequences were quality filtered using the QIIME pipeline. The chimeric sequences were identified by the “identify_chimeric_seqs.py” command and removed with the “filter_fasta.py” command according to the UCHIME algorithm. The selection and taxonomic assignment of operational taxonomic units (OTUs) were performed based on the SILVA reference data (version 128) at 97% similarity. Reads that did not align to the anticipated region of reference alignment were removed as chimeras by the UCHIME algorithm. Also, reads that were classified as “chloroplast”, “mitochondria”, or “unassigned” were removed.

The predictive functional abundance was predicted by PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) with “picrust2_pipeline.py” (https://github.com/picrust/picrust2),[31] which performed four key steps including sequence placement, hidden-state prediction of genomes, metagenome prediction, and pathway-level predictions. In addition, the additional output file Predicted Enzyme Commission (EC) number copy numbers was used to screen the C–N–S-related microbial genera.

Data Analysis

To avoid differences in amplified fragments among different samples, microbiological analysis was performed at the genus level according to the classification. The Shannon diversity and Chao1 richness were determined according to the relative abundance of genera. In addition, Bray–Curtis dissimilarity was calculated based on the relative abundance of genera matrix in the Vegan package of R v 4.1.2. Also, nonmetric multidimensional scaling (NMDS) was applied based on the Bray–Curtis dissimilarity by Vegan’s metaMDS function. Spearman’s correlations between the Shannon diversity, NMDS1, and relative abundance of C–N–S-related groups were performed by PerformanceAnalytics package in R. Random forest machine learning was performed with the caret and random forest package in R. These C–N–S-related groups with nonzero abundance values in at least 10% of the samples were preselected and z-score standardized prior to model training. Network analysis was used to explore co-occurrence patterns of microbial groups by ggClusterNet package in R. Spearman’s correlations between the relative abundance of the C–N–S-related genera and genes were considered. Also, Gephi (v0.9.1) and Cytoscape (v3.9.1) were used to visualize the co-occurrence networks for the C–N–S-related microbial communities and the C–N–S-related genes, respectively.

Results

Relative Abundance and Diversity of C–N–S-Related Microbial Communities

Based on the predicted EC number for each OTUs in the coal microbial communities, a total of 718 functional genera related to C (C degradation, methanogenesis, and CH4 oxidation), N (N2 fixation, ammoxidation, denitrification, and dissimilatory nitrate reduction to ammonium (DNRA)) and S (S reduction) cycles were detected (Table S1). Among the relative abundance of eight microbial groups (Figure a), the relative abundance of N2 fixation taxa was the highest (43.85 ± 2.35%, ranging from 2.40 to 97.08%), followed by denitrification taxa (41.49 ± 2.36%, ranging from 0.68 to 96.04%), and C degradation taxa (32.58 ± 2.25%, ranging from 1.34 to 94.24%). The relative abundance of methanogenesis taxa was the lowest (5.77 ± 1.54%, ranging from 0.00 to 63.98%). In addition, the regularity of the α diversity indexes and niche width was slightly different from that of relative abundance (Figure ). The Chao1 richness, Shannon diversity, and niche width of denitrification taxa were the highest (71.91 ± 6.74, 2.34 ± 0.12, and 3.84 ± 0.40, respectively).

Figure 1

Relative abundance (a), niche width (b), Chao1 index (c), and Shannon index (d) for the C–N–S-related microbial communities. Difference letters indicated a significant difference at p < 0.05.

Main C–N–S-Related Microbial Genera and Genes

Among 718 functional genera, many microbial communities participated in multiple element cycles (Table S2). For example, the vast majority of methanogens can fix N2. The top 20 C–N–S-related functional genera are shown in Figure . Among them, the genus Pseudomonas has the most C–N–S functions except methanogenesis and showed the highest relative abundance (12.02 ± 1.97%, ranging from 0.00 to 89.15%). In addition, the genus Herbaspirillum participated in C degradation, CH4 oxidation, N2 fixation, ammoxidation, and denitrification, which accounted for 8.84 ± 1.79% (ranging from 0.00 to 86.34%). The methanogen genera such as Methanobacterium, Methanosaeta, Methanolobus, Methanosarcina, Methanobrevibacter, and Candidatus Methanoperedens were also the main N2 fixation groups in coals. In addition, some common anaerobic taxa such as Clostridium sensu stricto 1 were also widely involved in the C–N–S cycles (C degradation, N2 fixation, and S reduction) of the coal seam environment.

Figure 2

Relative abundance of the top 20 genera for the C–N–S-related microbial communities: (a) C degradation; (b) methanogenesis; (c) CH4 oxidation; (d) N2 fixation; (e) ammoxidation; (f) denitrification; (g) DNRA; and (h) S reduction. Different letters indicated a significant difference at p < 0.05.

Based on the relative abundance of predictive functional genes (Figure ), the total relative abundance of S reduction genes was the highest (0.21 ± 0.01%), followed by the total relative abundance of C degradation genes (2.46 × 10–4 ± 0.42 × 10–4) and denitrification genes (0.82 × 10–4 ± 0.12 × 10–4). Among the C-related genes, the celF gene (6-phospho-β-glucosidase) and the pmoA-amoA gene (methane/ammonia monooxygenase subunit A) had the highest relative abundance of C degradation (celF, 1.36 × 10–4 ± 0.37 × 10–4) and CH4 oxidation (pmoA-amoA, 5.03 × 10–6 ± 1.13 × 10–6), respectively, and the methanogenesis genes showed no difference in the relative abundance. Among the N-related genes, the nif genes (nifH, nifD, and nifK), the nar/nxr genes (narG, narZ, nxrA, narH, narY, nxrB, narI, and narV), and the nirB gene had the highest relative abundance of N2 fixation, denitrification, and DNRA, respectively. Among the S reduction genes, cysJ, cysH, and cysD genes were the highest in the relative abundance (3.41 × 10–4 ± 0.27 × 10–4, 3.61 × 10–4 ± 0.17 × 10–4, and 3.44 × 10–4 ± 0.21 × 10–4, respectively).

Figure 3

Relative abundance for the C–N–S-related genes. (a) Total gene relative abundance (%) for the C–N–S-related genes. (b) Relative abundance of the C-related genes. (c) Relative abundance of the N-related genes. (d) Relative abundance of the S-related genes. Difference letters indicated a significant difference at p < 0.05.

Effect of the C–N–S-Related Group on the Total Microbial Communities

The diagnostic value of microbiome (n = 93) in coals was further evaluated by applying a random forest machine learning classification and regression analysis with the diversity index, relative abundance, and gene abundance of functional genera. The effectiveness of functional genera in reducing uncertainty and variance within the machine learning algorithm was measured by the decrease in mean accuracy for classification and mean-squared error (% Inc. MSE) for regression (Figure ). The most important diversity indexes of functional taxa for microbial diversity (Shannon diversity and NMDS1) mainly included the diversity (abundance, Shannon diversity, and NMDS1) of denitrification, DNRA, ammoxidation, N2 fixation, and C degradation. The most important genes and genera for microbial Shannon diversity mainly included DNRA genes (nrfA and nrfH), S reduction genes (dsrB, cysD, cysH, dsrA, and sir), N2 fixation genes (nifH, nifD, and nifK), and Enhydrobacter. The most important genes and genera for microbial NMDS1 mainly included denitrification genes (norB, norC, nosZ, and nirS), S reduction genes (cysNC and nirS), and C degradation genes (MAN2C1, celF, and chitinase). In addition, the Shannon diversity and NMDS1 of microbial communities were significantly related to the Shannon diversity of denitrification communities and NMDS1 of DNRA communities, respectively (Figure b,d).

Figure 4

Random forest machine learning. (a) Thirty most important C–N–S-related microbial indexes that reduce the uncertainty in the prediction of microbial Shannon index based on their mean decrease in accuracy. (b) Relationship of the Shannon index between denitrification groups and microbial communities. (c) Thirty most important C–N–S-related microbial indexes that reduce the uncertainty in the prediction of microbial β-diversity (NMDS1) based on their mean decrease in accuracy. (d) Relationship of the β-diversity index (NMDS1) between dissimilatory nitrate reduction to ammonium (DNRA) groups and microbial communities.

In ecosystem studies, a co-occurrence network has become an essential tool for understanding the symbiotic patterns of microbial communities in ecosystem studies.[32] The co-occurrence network of microbial prokaryotes constructed from correlations with r ≥ |0.60| and p < 0.05 had 161 nodes (including 96 C–N–S-related genera) and 679 edges (Figure a). The correlations identified were predominantly positive. The top 20 genera with the highest values of weight degree, betweenness centrality, and eigenvector centrality were listed. Among them, C–N–S-related taxa had 11, 7, and 10 in the top 20 genera of such three node centrality indices (Figure b–d). Herbaspirillum ranked in the top 20 in all three node centrality indices, and such genus participated in C degradation, CH4 oxidation, N2 fixation, ammoxidation, and denitrification. Methanogenesis genera Methanoculleus and Methanosaeta were the main hub microbes with a higher betweenness centrality index. In addition, unclassified Comamonadaceae, unclassified Alphaproteobacteria, and unclassified Clostridiales had the highest values of weight degree, betweenness centrality, and eigenvector centrality, respectively.

Figure 5

Co-occurrence networks of microbial groups in coals based on a correlation analysis (a). Top 20 genera with the highest values of weight degree (b), betweenness centrality (c), and eigenvector centrality (d).

Coupling Relationship between C–N–S-Related Groups

Pearson correlation showed that the Shannon diversities, NMDS1, and relative abundances of the majority of the C–N–S-related genera (expect methanogenesis ones) were significantly related to each other (Figure ). The methanogenesis group merely had a significantly positive correlation with the N2 fixation group in Shannon diversity and relative abundance, and methanogenesis genes were also significantly related to N2 fixation genes at the level of relative abundance.

Figure 6

Pearson correlation for the Shannon diversities (a), NMDS1 (b), genera abundance (c), and gene abundance (d) among the C–N–S-related groups.

The network of correlations (r ≥ |0.60| and p < 0.05) between the relative abundance of the C–N–S-related genera included 88 nodes and 154 edges (Figure a). All these correlations identified were positive. Multiple modules were shown in the co-occurrence network of the C–N–S-related genera. Two genera Clostridium sensu stricto 3 and Clostridium sensu stricto 5 linked to the module enriched with various methanogenesis genera. In addition, the genera with higher degree indexes mainly possessed the functions of N2 fixation, denitrification, DNRA, C degradation, and S reduction except for methanogenesis (Figure b).

Figure 7

Co-occurrence networks of the C–N–S-related genera groups in coals based on a correlation analysis (a). Top 20 genera with the highest values of degree (b). Co-occurrence networks of the C–N–S-related genes (c).

The network of correlations (r ≥ |0.60| and p < 0.05) between the relative abundance of the C–N–S-related genes included 63 nodes and 207 edges (Figure c). These correlations identified were predominantly positive. Multiple modules were shown in the co-occurrence network of the C–N–S-related genes and S-related genes (including sir, cysNC, cysH, cysD, cysI, cysN, and cysJ) and celF gene were the main genes linking the co-occurrence network of the C–N–S-related genes.

Discussion

This study comprehensively demonstrated the C–N–S-related microbial taxa and functional genes in coals. It was mainly based on the coal microbial data released in NCBI. To avoid OTU sequence differences caused by various amplified primers, the analyzed taxonomic unit was used at the genus level. In the field of coal seam microbial researches including these referenced researches, most attention has been paid to these groups related to the formation of biogenic coal bed methane,[33,34] and these studies are the key hubs for applying microbial knowledge to practical production. However, coal seams were important habitats for the coexistence of underground microbial communities, and the stable microecology in coal seams was inseparable from the synergy of multiple functional microorganisms.

Characterizing the functional properties and diversity is critical for understanding the community assembly and function relationships of biodiversity and ecosystem.[35] In particular, the methanogenesis taxa in coal seams, which have attracted wide attention, had the lowest abundance, biodiversity, and niche width among C–N–S-related microbial taxa (Figure ). It showed that the number and relative abundance of phylotypes[36] and the range of environmental conditions that a species may tolerate[37] were lower than those of other functional groups. The C degradation and N2 fixation groups that provided available C and N for coals and the denitrification groups with nitrate as electron acceptor had higher abundance, biodiversity, and niche width (Figure ). Microbial growth is influenced by many factors, the most important of which is the availability of nutrients. Therefore, the availability of nutrients determined microbial community assembly.[38] Although the coal is mainly composed of the C element, the lack of available nutrients (especially available C and N) limits the microbial activity in coal seams.[39] Therefore, the addition of nutrients[40,41] was considered to improve the coal bed microbial activities and further stimulate the production potential of biogenic methane. In the coal seam environments where the available nutrients were extremely deficient, the transfer of nutrients and energy along the trophic level during the assimilation and dissimilatory biomass utilization was the basis of the ecosystem.[42] The C degradation, N2 fixation, and denitrification could provide available C, N, and energy for the microecology in coal seams and ensure the exchange of metabolites in the microbial communities.[42]

For functional genes in this study, the total abundance of genes related to the S reduction process was the highest among the different functional genes investigated. The S reduction genes here were mainly dominated by the cys genes but not the dsr genes encoding sulfate respiration (Figure ). These cys genes were also the core linking the co-occurrence network of the C–N–S-related genes (Figure ). There are a large number of microorganisms related to S reduction in coal seams. For example, Midgley et al.[43] found that some fermentative desulfurizing bacteria could produce H2S, which might be related to the symphoretic relationship of other bacteria and might also favor coal degradation. Beckmann et al.[44] considered that a high sulfate concentration and sulfate-reducing bacteria did not prevent the growth of methanogenic archaea, but sulfate-reducing bacteria had limited energy and competed with methanogenic archaea for acetate. This study found that sulfate in coal seams was mainly utilized by bacteria through the assimilatory sulfate reduction pathway. Several cys enzymes were used to synthesize sulfites and convert sulfates into sulfides, and the existence of sulfate utilization enhanced the bacterial ability to produce amino acids, such as cysteine and methionine.[45] These processes provided biosulfur for the microbial communities in coals. Gene celF was another core gene linking the co-occurrence network of the C–N–S-related genes (Figure ), which was dominant in C degradation genes (Figure ). Such a gene was the key gene that encodes glycosidases hydrolyzing O- and S-glycosyl compounds[46] and played a vital role in the degradation of oligo- and polysaccharides[47] in coal degradation.

The study found that there were generally diverse functional microbial groups in many coal seams, among which some microorganisms with a variety of special functions had a high abundance, such as genera Pseudomonas and Herbaspirillum (Figure ). The C–N–S functional communities dominated the diversity and composition of microbial communities (Figure ) and the co-occurrence network of microbial prokaryotes (Figure ), and the genus Herbaspirillum also ranked in the top 20 in all three node centrality indexes. Pseudomonas is a bacterial genus that has been reported to be ubiquitous in coal seams.[48] This is precisely because that such genus has different metabolic potentials, allowing it to persist and grow in a wide range of coal seam environments and to utilize a variety of C compounds under special environmental conditions. Their lifestyle may be opportunotrophic, which was described by Singer et al.[49] Vick et al.[48] observed two Pseudomonas species with markedly different metabolic and ecological lifestyles, reflecting the broad metabolic and lifestyle diversity within such taxa, from parasitic to mutually beneficial[50] and free-living lifestyles. Genus Herbaspirillum has raised wide attention due to its ability to fix N2 under microaerobic or anaerobic conditions;[51] in addition, it is widely involved in the C–N metabolic process including aromatic compounds metabolization[52] and nitrate reduction.[53] In addition, methanogenesis genera Methanoculleus and Methanosaeta were the main hub microbes with a higher betweenness centrality index. Methanoculleus has been reported in a coal seam in Hokkaido as the dominant methanogen.[54] Zhang et al.[55] found that the existence of Methanosaeta with Pseudomonas could enhance direct interspecies electron transfer and further promote the anaerobic degradation. These methanogens were the terminal carriers for the transformation of coal organic matter into methane and were an important driving force for the geochemical cycle of C, N, S, and other elements between the lithosphere and the atmosphere.

In contrast, the methanogenesis group was merely related to the N2 fixation group at the genus and gene levels (Figure ). It fully showed the deficiency of nitrogen source in the coal seams. The N2 fixation taxa were widely found in many coal seams, such as Jharia coal bed[56] and Alberta coal beds.[57] Here, genera Clostridium linked the module that was enriched in various methanogenesis-related genera (Figure ). These Clostridium taxa might exist in a wide pH and temperature range and metabolize a wide range of substrates including cellobiose, glucose, xylose, vanillate, ferulate, lactate, propanol, and formate,[58] and were considered important substrate suppliers for methanogens.[59,60] In addition, such taxa contained a variety of regulatory genes responsible for regulating and absorbing N and urea.[61]

In conclusion, this study comprehensively demonstrated C–N–S-related microbial taxa and functional genes in coals. There are a large number of C–N–S-related groups in coal seams. The inter-relationship of these taxa ultimately affects the microhabitat and has important implications for the decomposition of organic matter and the geochemical cycles in coal seams. Together, this study strengthens our knowledge regarding the microbial diversity and community composition of coals.