Lignite samples were obtained from Daliuta Mine in Inner Mongolia (China), crushed in a pulverizer and sieved through a 100 mesh (0.150 mm) stainless steel sieve [13] [14] . Subsequently, the processed samples were sealed in a ziplock bag for anaerobic fermentation gas production experiments. The residual sludge (secondary sedimentation tank) was taken from a wastewater treatment plant in Jiaozuo, China. The percentage of chemical composition in the residual sludge was characterized by 34% carbohydrates, 12% proteins, and 8% fats, which were also stored for experimentation. According to ISO 17246-2010, the lignite’s proximate analysis was performed and the results are shown in Table 1 .

The bacteria were extracted from the residual sludge of a wastewater treatment plant in Jiaozuo, China, and the bacterial enrichment culture was set up in accordance with a method previously described in the literature [15] [16] . The culture medium was used to enrich the microflora and kept in the laboratory for anaerobic fermentation gas production experiments.

M, moisture; A, ash yield; V, volatile matter; C, fixed carbon; ad, air-dry basis; daf, dry ash-free basis; C, carbon; H, hydrogen; O, oxygen; N, nitrogen; S, sulfur.

The residual sludge, diluted to 1000 mg/L and coal fixed at 100 mg/L was utilized in three sets of biochemical methanogenesis experiments with varying VSS mass ratios of S (sludge): L (coal) of 0:1, 1:1, and 3:1. Conical flasks, N₂ was used instead of oxygen in the bottle to achieve an anaerobic environment, were hermetically sealed and placed in a thermostat incubator (35˚C) anaerobic reactions. Until the end of gas production, the composition changes and gas output were notedonce each day. A gas chromatograph (Agilent 7890GC; Agilent Technologies Inc., Santa Clara, CA, USA) was used to examine the composition of the biogas after it had been collected using a gas collecting bag.

In this study, Peak gas production samples were collected from reaction groups and metagenomic libraries (2 × 150 bp) were prepared based on the Illumina MiSeq/NovaSeq/HiSeq high-throughput sequencing platform using the Illumina Nexttera DNA XT kit (Illumina, San Diego, CA, USA). Macrogenomic DNA obtained from fermentation samples was extracted and the V3 - V4 hypervariable region of the bacterial 16S rRNA gene was amplified using primers 338F and 806R. Obtain clean sequences by merging, filtering and quality controlling paired-end reads. The paired-end libraries are constructed using the NEB Next® Ultra™ DNA Library Prep Kit for Illumina®, assembled by splicing with optimized sequences, and then sequenced on the Illumina Genome Analyzer. The resulting genes are annotated for species and function, and categorized. Comparative analysis of samples based on KO data and mapping to KEGG pathway maps was conducted, and MinPat was used to infer the existence and abundance of KEGG pathways. Majorbio BioPharm Technology Co. (Shanghai, China) was the subject of the experiments.

The results of methane yield experiments with different ratios of sludge and lignite in anaerobic fermentation are shown in Figure 1(a)-(b) . The cumulative biogas yield and biomethane yield of the mixed substrate exceeded that of the single lignite. Furthermore, during the anaerobic fermentation methane yield cycle, the gas yield at the daily of incorporating different quantities of sludge exhibited a trend of increasing and then decreasing and essentially halted gas production after 21d. The gas production of the mixed substrate was higher compared with the single lignite. The cumulative methane yield was 87.3 m L/g-VSS for the sludge and lignite mass ratio of 0:1, 123.3 mL/g-VSS for the sludge and lignite ratio of 1:1, and 142.8 mL/g-VSS for the sludge and lignite ratio of 3:1. The methane yield in the 3:1 group was higher compared with the other experimental groups, and the total biogenic gas yield in the 3:1 group reached 1.64 times compared with the 0:1 group. The anaerobic fermentation of the mixed substrate facilitated the biomethane yield. Sludge contains higher quantities of proteins and fatty acids, whereas low-order lignite is characterized by an abundance of side-chain functional groups or free functional groups [17] . These groups are susceptible to chemical bond breaking and reorganization, and concurrently, they serve as readily utilizable substrates for microbial flora transformation, supporting the sustenance of microbial reproduction [5] . Across the three proportioning systems, gas yield efficacy increased with the addition of sludge. Notably, the 3:1 group, featuring a higher percentage of sludge, demonstrated the most favorable

gas yield effect. In summary, sludge addition facilitated the yield of material gas from lignite anaerobic fermentation, enhancing the biomethane potential of lignite, and facilitating the transformation of organic matter in the anaerobic fermentation substrate.

Table 2 presents the results of alpha diversity analysis at the peak of sludge and lignite ratios of 0:1, 1:1, and 3:1 reactions. ACE and Chao1 indices were commonly used to estimate species richness, while the Shannon and Simpson indices were used to measure species diversity. There was a slight difference in species richness and evenness between the three different ratios of both sludge and lignite at the peak of anaerobic fermentation gas production. The community performance was impacted by the increase in community richness with the augmentation of sludge in the reaction system. However, the distribution of strains was non-uniform, with dominant strains holding a prominent position. Given the gas production effect illustrated in Figure 1 , it was evident that greater diversity among dominant flora corresponded to increased participation of microorganisms in the methane conversion process, thereby enhancing the efficiency of biological gas production.

To further elucidate the differences in the Microbial of the three different ratios of sludge and lignite at peak gas production, the Microbial categories of the three sets of reactions were analyzed. Figure 2(a) exhibited the dominant microorganisms at the gate level for the three different ration systems. Bacteroidetes (43%, 15%, and 13%); Proteobacteria (5%, 34%, and 12%); and Euryarchaeota (24%, 10%, and e14%) were the main species. Bacteroidetes contained a wide range of different hydrolytic, acid-producing, and fermentative bacteria. They played an important role in the degradation of complex organic matter, proteins, lipids, and sugars during hydrolysis and acidification, breaking down complex macromolecules into simpler compounds. These were the dominant functional groups of the anaerobic digestion process [18] - [20] The main role of Proteobacteria in wastewater treatment is the removal of organic pollutants, denitrification, and phosphorus removal [21] [22] . Euryarchaeota contained a rich diversity of methanogenic archaea. These archaea can use H₂/CO₂, formic acid, methanol, methylamine and acetic acid to produce CO₂, H₂ and methane. The more abundant Firmicute phylum is also typical of functional phyla that utilize electron acceptors for anaerobic metabolism, are involved in hydrogen and acetic acid production, etc. [23] [24] . As shown in Figure 2(a) , the relative abundance of Euryarchaeota in the sludge–lignite ration ratio of the 3:1 system was 24%. The relative abundance of methanogenic archaea in the 1:1 system was reduced by 4% compared with the 0:1 group, which was reduced by 14%. Furthermore, compared with the 3:1 group, and the abundance of its Bacteroidetes, the phylum of bacteria that hydrolyzed and acidified organic substrate was higher than that in the two groups of the ratio system of 0:1 and 1:1. It was observed that the enrichment of hydrolytic flora, such as Bacteroidetes, facilitated the degradation of organic macromolecules, providing sufficient substrate for methanogenic archaea [25] .

Figure 2(b) and (c) show the dominant bacteria and archaea at genus levels for each proportioning system. The microorganism community composition was dominated by Bacteroidetes, Bacteroidales, Sphaerochaeta, and Aminobacterium. The higher abundance of microorganism at the genus level correlated with the dominant microorganisms exhibited at the phylum level ( Figure 2 ). Among these, Bacteroidetes and Bacteroidales are both anthropomorphic genera, the former only degraded propionic acid, while the latter degraded both a variety of fatty acids ranging from C4 to C8, as well as propionic acid [26] . Members of the genus Sphaerochaeta are able to hydrolyze a wide range of carbohydrates and organic acids [27] [28] . From the data of the top ten relative abundance of bacterial communities in Figure 2(b) , it was observed that among the three different system ratios, the system with 0:1 sludge to lignite ratio contained the lowest Petrimonas and Sphaerochaeta at 0.09%. Additionally, while the system ratio was 3:1, the contents of these types of characteristic microorganisms increased by 1.3% and 1.5%, respectively [29] .

In Figure 2(c) , the composition of the archaea among the three different ration systems at the peak of gas production was dominated by Methanosarcina, Methanothrix, and Methanosarcinales. The most abundant Methanosarcina produced CH₄ by cleaving acetic acid and reducing methyl carbon. Additionally, the hydrogenophilic methane-producing archaea utilized a mixture of CO₂, H₂, and formic acid to produce methane [30] - [32] . Methanothrix was the second most abundant archaeon during the peak gas production period. It is a typical acetic acid-nutrient methanogenic archaeon whose energy metabolism is characterized by the catabolism of acetic acid to CH₄ and CO₂ [33] . Methanothrix content was 9.8% higher than in the 1:1 and 6.30% higher than in the 0:1 in the sludge-to-lignite ratio 3:1 system.

In summary, the relative abundance of hydrolyzing and acidifying bacteria as well as methanogenic archaeal genera at both the phylum and genus levels, was higher compared with the other two groups in the residual sludge-to-lignite ratio of 3:1 system. This resulted in a higher methane production in the 3:1 group compared with the two outer groups.

In anaerobic fermentation, macromolecular organic matter undergoes decomposition into small molecules by extracellular enzymes. The hydrolysis of carbohydrates constitutes a pivotal aspect of this process. Carbohydrate-active enzymes, are functional enzyme systems responsible for the degradation, modification, and synthesis of glycosidic bonds in carbohydrates, serving as the fundamental functional units in the metabolic pathway of saccharides. These enzymes were classified into Glycoside Hydrolases (GHs), GlycosylT-ransferases (GTs), Polysaccharide Lyases (PLs), Carbohydrate Esterases (CEs), and other CAZymes [34] [35] . Complex carbohydrates and glycocomplexes underwent hydrolysis, yielding small molecules. Glycoside Hydrolases are a category of enzymes that catalyze glycosidic bond hydrolysis in diverse sugar-containing compounds to produce monosaccharides, oligosaccharides or glycoconjugates. GH is frequently employed as a key metric for assessing the hydrolytic capacity of carbohydrates [36] [37] . As presented in Table 3 , several GH families with higher abundance contained enzymes, cleaving polysaccharides into monosaccharides, such as GH2 (β-Galactosidase), GH20 (β-Glucosidase), and GH57 (α-galactosidase) [39] . The content of GHs in the 3:1 anaerobic fermentation reactor was higher than that of the corresponding 0:1 and 1:1 GHs. The increase in the abundance of hydrolyzing proteases distinctly characterized the capacity of the colony to hydrolyze macromolecules. The greater the hydrolysis capacity, the smaller molecules were utilized, ultimately enhancing methane production during anaerobic fermentation [39] . Additionally, GH20, GH92, and GH57 contained various GHs such as β-1,6-N-acetylglucosaminidase, α-1,6-mannosidase, β-glucosidase, α-galactosidase, and others, breaking down cell structures and hydrolyzing glycosidic linkages. This played a crucial role in the hydrolysis of sugars and glycoconjugates in organisms [40] . The relative abundance of GH20 and GH92 ( Table 3 ) in the reaction group with a 3:1 ratio system was higher compared with the other two groups, facilitating carbohydrate hydrolysis. GH109 was mainly involved in carbohydrate skeleton degradation (higher content in the 3:1 system compared with the other two groups), facilitating the production of volatile fatty acids [41] . The hydrolases contained in GH13 and GH33 hydrolyzed glucosidic bonds and disrupted cellular structure, which were essential in microbial carbohydrate metabolism. The abundance of these two active enzymes in the 3:1 ratio system ( Table 3 ) was significantly higher compared with that of the other two groups [42] [43] . In summary, the reaction group with a 3:1 ratio of residual sludge to lignite exhibited a higher abundance of hydrolytic extracellular enzymes released by the bacteria during anaerobic fermentation compared with the other two groups. This condition facilitated the hydrolysis of macromolecular organic matter.

Several different metabolic pathways existed within the anaerobic system, and the metabolic pathways present during acid production determined the overall conversion rate of the anaerobic fermentation [44] [45] . As shown in Table 4 , the glycolytic metabolic pathway gene abundance expression was the highest in the three different ration reaction groups, followed by the amino acid pathway, and the fatty acid oxidation pathway was the lowest. This shows that the glycolytic pathway was the most important acid production pathway in anaerobic fermentation. Among the three sets of reactions, the highest abundance of glycolytic genes was found in the sludge-to-lignite ratio of the 3:1 set, followed by the 1:1 set, and the lowest abundance was found in the 0:1 set. This indicated that the higher abundance and activity of acidifying flora in the 3:1 group with higher sludge addition in the three reaction systems facilitated the production of methane in the reaction.

Figure 3 shows the main pathways in glycolysis, as well as the key enzyme genes and their abundances for each pathway. In Figure 3 , the abundance of each key gene in the sludge-to-lignite ratio 3:1 group was generally higher compared with 1:1 and 0:1. The gene abundance of glucokinase (K25026) in the reaction group with a 3:1 ratio was 1.87 and 1.97 times that of 0:1 and 1:1 ratio, respectively. The 6-phosphofructokinase (K00850) abundance was 1.03 and 2.11 times that of 0:1 and 1:1 ratio, respectively. The abundance of expressed genes of phosphoglycerate kinase (K00927) was 1.52 and 1.11 times that of 0:1 and 1:1 ratio, respectively. Thus, the magnitude of activity of certain kinases in glycolysis directly affected the rate and direction of metabolic pathways [46] . The abundance of key enzyme genes associated with acidification in anaerobic fermentation was higher in the reaction group with a 3:1 ratio of residual sludge to lignite. This observation correlated with the distribution pattern of hydrolytic and acidifying flora, as depicted in Figure 2 , where higher abundance at the phylum and genus levels was evident. Consequently, this enhanced abundance facilitated the acidification reaction.

In this study, based on the analysis of the community structure of dominant microorganism at the genus level in Figure 2(c) , the acetate-nutrient methanogenic pathway was the main pathway for the methanogenic process of the three mating systems of residual sludge and lignite.

Volatile fatty acids produced during the acidification stage of hydrolysis were further degraded to acetic acid at this stage, where acetyl coenzyme A served as an important precursor for acetic acid production [47] . It was produced primarily from the following two stages: the reduced acetate coenzyme A pathway and the glycolytic pathway. The two acetic acid synthesis pathways are shown in Figure 4(a) . In the reductive acetyl coenzyme A pathway, microorganisms utilize H as an electron donor and CO2 as an electron acceptor and building block for

biosynthesis, ultimately leading to the generation of acetyl coenzyme A [48] [49] . Furthermore, carbon monoxide dehydrogenase and acetyl-CoA synthetase play an important role in the reaction to produce acetyl-CoA. As shown in Figure 4(b) , the gene abundance of the reaction group with a 3:1 ratio of residual sludge to lignite was higher than that of 1:1 and 0:1 ratios in terms of gene expressions, correlating with the coenzyme A pathway of the reduced acetate.

As shown in Figure 3 , sugars were converted from glucose to pyruvate through the action of hydrolytic acidifying enzymes. In Figure 4(a) , pyruvate is reduced to acetic acid by pyruvate dehydrogenase (EC:1.2.4.1) in the absence of oxygen, and oxidized by dihydrolipoyl lysine-residue acetyltransferase (EC:2.3.1.12) and acetyl-CoA synthetase (EC:6.2.1.1) in the presence of oxygen, generating acetyl coenzyme A [50] . The differences between the three different ratios of residual sludge and lignite in on the glycolytic pathway were more pronounced ( Figure 4(b) .

Overall, there was a positive trend of correlation between sludge addition and gene abundance. Thus, the abundance of gene expression was relatively high in response groups with higher sludge content. Figure 4(c) illustrates several genes with higher abundance. There was a substantial difference in gene abundance within the glycolytic pathway, with genes in the 3:1 ratio being much more prevalent than those in the 1:1 and 0:1 ratios. In the reduced acetyl coenzyme A pathway, although gene abundance was high, the difference was not statistically significant, correlating with the results depicted in Figure 4(b) . As a result, the system featuring a 3:1 ratio of residual sludge to lignite exhibited higher glycolytic metabolic activity. This heightened activity proved conducive, promoting acidification during anaerobic processes.

Biomethane metabolism is associated with organic matter degradation and its transformation during anaerobic fermentation. Furthermore, it was crucial to clarify the metabolic pathways of the archaeal community. To evaluate the process of biomethane metabolism in three groups of the different ration systems, genes encoding key enzymes related to methane production were recorded ( Table 5 ). In order to further elucidate the effect of sludge addition on producer methane, the promotion mechanism was analyzed through the methanogenic metabolic pathway. The results of this analysis are presented in Figure 5 .

Methane metabolism was the last stage of anaerobic fermentation and methanogenic archaea, played a major role in this stage. Furthermore, using the KEGG database to identify the substance metabolic pathways and methanogenic pathways in the anaerobic fermentation process, various types of metabolic pathways in the methanation pathway of three different ratios of residual sludge and lignite systems were analyzed ( Figure 5 and Table 5 ). Figure 5(a) depicts the methanation pathway and the associated biological enzyme species in the methanation pathway. The methanogenic pathways were divided into four: M00356 (methanol methanation), M00357 (decarboxylation of acetic acid), M00563 (methanation of methylamine/d-dimethylamine/trimethylamine), and M00567 (reduction of CO₂).The proportion of acetic acid decarboxylation pathway (9.62% - 15.31%) was higher compared with the CO₂ reduction pathway (7.51% - 14.10%), methylamine/dim-ethylamine/t-rimethylamine methanation pathway (4.93% - 8.12%), and methanol methanation pathway (4.14% - 4.09%) for all three proportioned reaction systems. It was observed that in the process of anaerobic fermentation, the metabolism of acetic acid served as a substrate for heterotrophic microorganisms. The metabolic utilization of acetic acid as a substrate was found to be more competitive compared with other microorganisms during anaerobic fermentation. Meanwhile, archaea with CO₂ as the substrate were expressed in higher abundance in the system with a 3:1 sludge-to-lignite ratio, resulting in a higher proportion of the CO₂ reduction pathway in the methanation pathway in the 3:1 reaction system.

As shown in Table 5 , the key enzymes involved in the conversion of acetic acid during the methanation of acetic acid were acetate kinase (ack, EC: 2.7.2.1), phosphate acetyltransferase (pta, EC: 2.3.1.8), acetyl-CoA synthase (acs, EC:6.2.1.1), and acetyl-CoA synthase [51] . Acetic acid underwent two processes, the conversion of acetic acid to acetyl coenzyme A and the conversion of acetyl coenzyme A to 5-methyl-THMPT, before it is converted to methane. ( Figure 5(a) ). As seen from the data in Table 5 , the gene abundance of the acs system was significantly higher than that of the ack-pta system. This indicated that the pathway involved in the acs system is the main pathway for the conversion of acetic acid to acetyl coenzyme A in the reaction. However, in the hydrogenotrophic (M00567) methanogenesis pathway, as shown in Figure 5 , CO₂ is reduced to a series of intermediates by a variety of enzymes to form Methyl-CoM, which ultimately catalyzes the reduction to CH₄. The differences in gene abundance in the acetic acid decarboxylation pathway among the three reaction systems were small (15% for 3:1, 10% for 0:1, and 9.6% for 1:1). However, in the CO₂ reduction pathway, the gene abundance of the sludge to lignite ration of 3:1 was 186.6% of that of 1:1, and 159.09% of that of 0:1, indicating that the enzyme activity in the CO₂ reduction pathway of the 3:1 reaction system exhibited higher sludge addition. In summary, the biomethanation pathway of anaerobic fermentation of sludge and lignite mainly uses acetic acid and CO₂ as substrates for fermentation. Additionally, combining the typical genes and their abundance data correlated with the methane metabolic pathway in Figure 2(c) and Table 5 . Methanosarcina, Methanothrix and Methanosarcina-les are the main archaea in the methanization pathway. The 3:1 group of the three reaction systems with higher sludge incorporation performed excellently in the methanation pathway.