Enhancing Methane Production from Lignite via Pyrite-Electrochemical Coupling and Microbial Community Restructuring

Abstract

Coalbed methane enhancement technologies are critical for clean and efficient utilization of low-rank coal resources. However, current biological conversion efficiencies remain limited because of low electron transfer rates and insufficient activity of methanogenic bacteria. In this study, a synergistic enhancement strategy combining pyrite addition and electrical stimulation was proposed to improve methane production from brown coal during anaerobic fermentation by promoting microbial electron transport and metabolic activity. Gradient experiments were conducted under varying pyrite dosages (0–32%) and voltage conditions (0–6 V), coupled with 16S rRNA high-throughput sequencing to systematically analyze gas production, key physicochemical parameters, and microbial community shifts. The results indicated that the combined treatment (16% pyrite + 3 V) increased the cumulative methane yield to 4220 mL over a 30 day fermentation period, representing a 767% increase compared with the untreated control. Concurrently, the relative abundance of Methanobacterium increased from 1.42% to 9.13%. Pyrite functioned as an Fe²⁺/S^2– electron shuttle to facilitate direct electron transfer, whereas electrical stimulation enhanced H₂ production and jointly optimized the hydrogenotrophic methanogenesis pathway. A threshold effect was also identified, whereby excessive pyrite or high voltage induced Fe³⁺/S^2– toxicity and water electrolysis side reactions, compromising system stability. This synergistic strategy achieved a 7-fold increase in methane yield without the addition of conventional chemical additives such as nutrients, enzymes, or synthetic mediators, highlighting its potential for application in the biological intensification of low-rank coal exploitation.

1. Introduction

With the continuous rise in global energy demand, traditional energy sources are facing the dual challenges of resource depletion and environmental pollution. Although coal remains a major fossil fuel with abundant reserves, its utilization often causes considerable environmental harm. Therefore, the development of clean and efficient energy conversion technologies has emerged as a global research focus. As a methane gas generated during the coalification process, coalbed methane (CBM) represents a key unconventional natural gas resource characterized by high energy conversion efficiency and low environmental impact. As the demand for clean energy intensifies, the extraction and utilization of CBM has become a critical area of investigation.

Despite the abundance of CBM resources, current extraction technologies remain constrained by diverse technical bottlenecks, particularly high costs and low recovery rates. , Traditional CBM extraction methods primarily rely on physical and chemical processes. Although partially effective, these methods are associated with high energy consumption and limited efficiency. , Thus, enhancing CBM production, particularly from high-rank coal, has emerged as a critical challenge. In this context, biological coalbed methane (B-CBM) technology has attracted increasing attention as a cost-effective and environmentally friendly energy conversion approach. − This technology relies on microbial degradation of coal-derived organic matter to generate methane. , Research has focused on optimizing microbial consortia and regulating environmental conditions. Low-rank coal (e.g., lignite) has been reported to be more readily degraded by microbes than high-rank coal (such as bituminous coal), rendering B-CBM more effective in low-rank coal applications. However, high-rank coal’s complex organic structure and low biodegradability result in limited microbial activity and methane yields. In contrast, lignite, as a low-rank coal with a simpler macromolecular structure and higher volatile matter content, offers favorable substrate properties for microbial conversion. Therefore, this study focuses on lignite to explore strategies for enhancing biomethane production. , Recent studies have demonstrated that codigestion with biodegradable plastics can enhance methane production while facilitating the degradation of recalcitrant substrates, offering a promising strategy for waste resource utilization. Therefore, improving the biodegradability of high-rank coal, particularly through external factor optimization, has become a key research focus in biogasification technologies.

In recent years, increasing efforts to enhance coal biodegradation and methane production efficiency have highlighted pyrite (FeS₂) and electrical stimulation as promising external stimulants for improving coal bioconversion. , Pyrite contributes significantly to anaerobic coal fermentation by releasing Fe²⁺ and S^2– ions, optimizing microbial metabolic conditions, and facilitating interspecies electron transfer. , Its role as an electron donor has been shown to enhance microbial activity and substantially increase methane production rates. − Appropriate pyrite addition improves the coal microenvironment, reduces the accumulation of volatile fatty acids (VFAs) and acidic compounds, and stabilizes the system pH and oxidation–reduction potential (ORP), thereby promoting methanogenesis. However, a threshold effect has been observed, where excessive pyrite leads to elevated Fe³⁺ and sulfide concentrations, inhibiting microbial metabolism and reducing methane yield. , In contrast to pyrite, electrical stimulation enhances electron transfer efficiency via an external electric field, which can increase hydrogen production and promote the activity of hydrogenotrophic methanogens. It accelerates the hydrogen generation, providing additional substrate for methanogenesis and significantly improving the methane production efficiency. The electrical stimulation also optimizes the microbial community structure, suppresses excessive VFA accumulation, improves the anaerobic fermentation environment, and enhances the system stability and sustainability. , Experimental results have demonstrated that the voltage modulation can significantly promote the microbial metabolic activity. However, excessively high voltages can induce water electrolysis, altering gas composition and consequently reducing methane yield and purity. − Although both pyrite and electrical stimulation have demonstrated individual potential for improving coal biodegradation and methane production, certain challenges remain in their practical application. Pyrite dosage should be optimized based on coal quality to avoid potential toxicity, whereas electrical stimulation requires precise voltage regulation to mitigate side effects. Furthermore, the synergistic potential of combining these two approaches remains largely unexplored. Specifically, whether their combined application can further enhance methane production efficiency across various coal types and environmental conditions remains an unresolved scientific question. Thus, this study aimed to investigate the synergistic effects of pyrite and electrical stimulation on low-rank coal bioconversion. The main objectives were to evaluate their combined effects on (1) lignite biodegradation efficiency, (2) methane production, and (3) microbial community structure and metabolic pathways. These findings are expected to elucidate the mechanisms underlying CBM generation and support efficient utilization of CBM resources.

2. Materials and Methods

2.1. Sample Collection and Preparation

Brown coal (L) used in this study was obtained from a fresh coal seam at a depth of 200 m in the Shanxin Village coal mine, Milu City, Honghe Prefecture, Yunnan Province, China. To prevent oxidation during transport, the samples were sealed in plastic wrap, dried in a forced-air oven at 105 °C for 72 h, ground using a planetary ball mill (QM-3SP2), and sieved to a particle size of less than 100 mesh. The resulting coal powder was stored in brown reagent bottles under low temperature conditions. The inoculum sludge was sourced from the anaerobic digestion system of the wastewater treatment plant operated by Shandong Qixin Environmental Engineering Co., Ltd. and stored at 4 °C. Collected from the Xiangshan Sulfur Iron Mine in Maanshan City, pyrite (FeS₂) was ground, sieved to below 80 mesh, dried, and sealed for storage. The components of the brown coal and anaerobic sludge are listed in Tables and . The high volatile matter content, low sulfur content, and moderate ash content of brown coal indicate its suitability as a substrate for anaerobic fermentation. The anaerobic sludge with 71.88% moisture, an initial pH of 6.96, and 35.06% organic matter demonstrated a high abundance of methanogens and its appropriateness for cofermentation with brown coal.

1. Industrial Analysis of Lignite.

	Aad (%)	Mar (%)	Mad (%)	Vdaf (%)	Vda (%)	St d (%)
lignite	11.03	43.64	0.23	55.29	49.08	0.739

2. Basic Indicators of Anaerobic Activated Sludge.

	MC (%)	pH	ORP (mV)	OM (%)	SV (%)	MLSS (mg/L)
sludge	71.88	6.96	0	35.06	20	3000

2.2. Experimental Scheme

The anaerobic fermentation system consisted of a digital temperature-controlled water bath (HH-4), multiple 1 L wide-mouth bottles, 1 L graduated cylinders, and collection buckets. All components were connected using PVC tubing, with sealing points reinforced using petroleum jelly and sealing film. Prior to fermentation, nitrogen was purged through the sampling port for 3 min to remove oxygen and establish an anaerobic environment. A pH meter and a pressure gauge were inserted into each fermentation bottle to monitor real-time fermentation conditions. Gas production was measured using the water displacement method, with a 1 L graduated cylinder serving as the gas collection device. Two system configurations with and without a CO₂ absorption bottle were evaluated. To enable observation of CO₂ fluctuations, a configuration without a CO₂ absorption bottle was adopted (Figure a). For the power supply setup, a variable-voltage adapter (KSJ-1509, 0–10 V range) was integrated into the system and connected to copper wires and graphite electrodes (Figure b).

1.

Drawing of the overall experimental setup (a) and electrically stimulated fermenting flask (b).

Each fermentation bottle was loaded with 40 g of brown coal and 100 g of anaerobic sludge as substrates and then incubated in a water bath at 55 °C. This ratio was chosen based on our preliminary experiments, which showed that it produced the highest methane yield under comparable conditions. Previous studies have identified 55 °C as the optimal temperature for high-temperature anaerobic coal digestion to produce methane. Initially, the effect of pyrite addition on gas production was investigated, with pyrite added at 0%, 4%, 8%, 16%, and 32% of the brown coal mass. Pure water was added to reach a total volume of 800 mL. This phase lasted for 30 d. Each group was repeated three times, totaling 15 samples. Upon completion, the optimal pyrite dosage was identified. Subsequently, an electro-stimulated fermentation experiment was conducted using an optimal pyrite concentration (16%) under varying voltage conditions (0, 1.5, 3, 4.5, and 6 V). In each group, 16% pyrite was added, and the total volume was adjusted to 800 mL with pure water. This experiment lasted for 30 d, with three replicates per group, totaling 15 samples. Methane production was recorded every 2 d, and the fermentation broth was analyzed regularly for physicochemical indicators. The gas collected by the measuring cylinder was transferred to a PTFE (polytetrafluoroethylene) gas sampling bag for subsequent analysis to ensure minimal gas loss and avoid chemical interference. The experimental factor level values are listed in Table .

3. Experimental Factor Levels.

coal step	coal sample (g)	sludge sample (g)	coal particle size (mesh)	Temp (°C)	pyrite quantity (g)	applied voltage (V)	repetition
lignite	40	100	100	55	0	–	3
lignite	40	100	100	55	1.6	–	3
lignite	40	100	100	55	3.2	–	3
lignite	40	100	100	55	6.4	–	3
lignite	40	100	100	55	12.8	–	3
lignite	40	100	100	55	6.4	0	3
lignite	40	100	100	55	6.4	1.5	3
lignite	40	100	100	55	6.4	3	3
lignite	40	100	100	55	6.4	4.5	3
lignite	40	100	100	55	6.4	6	3

2.3. Instrumentation and Methods

The primary instruments used in this experiment included a digital constant-temperature water bath (HH-4, Changzhou Jintan Honghua Instrument Factory), a portable pH/ORP meter (PHB-4, Shanghai Leizhi Instrument Factory), a high-speed centrifuge (KL04A, Hunan Kaida Scientific Instrument Co., Ltd.), an electronic balance (T series, Changshu Shuangjie Instrument Factory), and a portable biogas analyzer (BIOGAS 5000, Geotech, UK; detection accuracy: ± 0.5% for CH₄ and CO₂). Anaerobic fermentation was conducted in a 1-L wide-mouth tank used as a reactor, and the gas produced was directed through a PVC pipe into a gas collection system. Daily and cumulative methane production was measured using the water displacement method. Gas composition was analyzed using a portable biogas analyzer, focusing on methane (CH₄) and carbon dioxide (CO₂) concentrations. An inverted fluorescence microscope (IX-83, Olympus) was used later in the experiment to observe the abundance of methanogenic bacteria under different conditions.

During fermentation, pH was measured using a pH meter, and the ORP was determined using an ORP meter (PHB-4) to provide environmental data for analyzing anaerobic fermentation conditions. Both pH and ORP were measured on the same day as gas sampling to ensure data consistency. Ammonia nitrogen concentrations were determined using the Nessler reagent spectrophotometric method based on the HJ535-2009 standard. VFAs were quantified by colorimetry using a UV-1800APC spectrophotometer. These parameters were monitored every 2 d to track organic acid production. Changes in the abundance of methanogens were regularly observed in fermentation broth samples using an inverted fluorescence microscope. The microbial community structure was assessed at peak gas production (day 10), with 50 mL of broth collected from both the control group (0 V: CK1, CK2, and CK3) and the treatment group (3 V: T1, T2, and T3). The samples were subjected to 16S rRNA high-throughput sequencing using the Illumina MiSeq platform to characterize the bacterial and methanogenic archaeal communities. The bacterial primers were 338F (5′-ACT­CCT­ACG­GGA­GGCA­GCAG-3′) and 806R (5′-GGA­CTA­CHV­GGG­TWTC­TAAT-3′), and the methanogenic archaeal primers were MLfF (5′-GGT­GGT­GTM­GGA­TTC­ACA­CAR­TAY­GCWA­CAGC-3′) and MLrR (5′-TTC­ATT­GCR­TAG­TTW­GGRT­AGTT-3′).

2.4. Gompertz Model Analysis

To evaluate the kinetic behavior of methane production under various conditions, the Gompertz model was applied for data fitting. This model has been widely used to characterize biological processes exhibiting S-shaped growth patterns, and its mathematical expression is as follows:

$$y=A\exp \{-\exp [\frac{{\mu }_{\mathrm{m}}\exp (1)}{A}(\lambda -\mathit{t})+1]\}$$ 1

where y is the cumulative specific methane production (mL/g), A is the maximum specific methane production potential (mL/g), μ_m is the maximum specific methane productivity (mL/day), and λ is the lag time (days) and t is the incubation time (days), This model was used to fit the methane production profiles of the 0 and 3 V experimental groups and to evaluate the influence of voltage on the methane production rate and lag phase. The experimental data were fitted using the least-squares method to estimate the model parameters and characterize the dynamic behavior of methane generation. The model can provide a theoretical foundation for further analysis of the roles of electrical stimulation and pyrite in enhancing methane production.

3. Results and Discussion

3.1. Methane Production Dynamics

3.1.1. Impact of Pyrite Addition on Methane Yield

Figure a illustrates that the methane yield from brown coal fermentation exhibited a significant upward trend with increasing pyrite addition. A one-way ANOVA on the cumulative gas production yielded p < 0.01. Based on the average of three parallel replicates, the group with 16% pyrite addition produced 1906.33 mL of methane, approximately 24% higher than that of the control group (1533.33 mL). Compared with the untreated sample, 16% pyrite addition increased the cumulative gas production by 24% and methane yield by 29%.

2.

Analytical curves of cumulative gas production (a) and daily gas production (b) with pyrite added.

Further analysis revealed that the pyrite addition significantly enhanced the gas production rate during fermentation. The group with 16% pyrite addition achieved the highest daily gas yield (166 mL/d) on day 7 compared with 143 mL/d in the control group, which represented an increase of 15.6% (Figure b). This result indicated that as a trace element source, pyrite promoted microbial metabolic activity and accelerated lignite fermentation. However, at higher pyrite dosages, the rate of increase in gas production diminished, and at 32% addition, the methane yield declined. The 32% pyrite group produced 1653.33 mL of methane, 15.3% lower than that of the 16% group. This trend suggested that the excessive pyrite inhibited the fermentation process, reducing the methane production efficiency. The inhibitory effect could be due to the release of excessive metal ions (e.g., Fe²⁺ and Fe³⁺) or sulfides, which adversely affected the microbial community structure and suppress methanogen activity. ,,

3.1.2. Gas Composition Variations with Pyrite Addition

Gas composition serves as a key indicator for evaluating anaerobic fermentation efficiency. As shown in Table , the methane concentration in the 16% pyrite group reached 36.7%, exceeding that of the control group (35.4%). This indicated that pyrite addition not only enhanced methane production but also altered the gas composition. However, at a 32% pyrite dosage, the methane concentration declined to 35.3%, suggesting a reduced methane conversion rate at higher concentrations. This outcome may be attributed to the adverse effects of excessive pyrite on microbial communities. While the appropriate levels of pyrite can supply the essential trace elements, promote the growth and metabolism of methanogens, and enhance the methane production, the excessive pyrite may increase the generation of hydrogen (H₂) and other intermediates, intensifying the competition for substrates and thus reducing the methane production efficiency. Additionally, sulfides released from pyrite may inhibit the methanogenic activity, leading to a decline in methane concentration. The anaerobic digestion flowchart is shown in Figure .

4. Accumulated Gas Composition.

pyrite usage (%)	CH4 (vol %)	CO2 (vol %)	O2 (vol %)	H2S (ppm)
0	35.4	0.3	4.8	1
4	35.2	0.3	4.2	4
8	35.8	0.3	4.8	2
16	36.7	0.4	6.9	11
32	35.3	1.6	4.5	2

3.

Anaerobic digestion flowchart.

3.1.3. pH and ORP Dynamics

The pH of the fermentation liquid serves as a critical indicator of the anaerobic environment, reflecting the accumulation of acidic compounds during the fermentation process. Figure a demonstrates that pyrite addition initially lowered the pH of the fermentation broth but did not significantly affect the overall system pH, indicating a minimal impact on the system acidity or alkalinity. Throughout fermentation, pH remained within a fluctuating range. During the initial stage, hydrolysis dominated, leading to a pH decrease. Hydrolysis dominated the first 4 d of fermentation, during which pH declined. Thereafter, pH gradually increased, corresponding to the peak gas production phase, as acidic compounds were consumed. By day 20, the system entered a stagnation phase, and pH stabilized. Throughout the process, pH ranged from 6.8 to 7.5. These values did not inhibit fermentation, indicating that methane production from brown coal with pyrite addition proceeded under favorable conditions. Compared with the control group without pyrite, the experimental group exhibited greater overall pH fluctuations, indicating higher levels of acid production and consumption and a more active fermentation process. Additionally, elevated pyrite concentrations may further lower the pH of the fermentation broth, potentially reducing methane production efficiency.

4.

Analytical curves of pH (a), ORP (b), and ammonia nitrogen (c) of fermentation broths with extra pyrite.

Figure b illustrates the ORP trends in each group during lignite anaerobic fermentation with pyrite addition. Overall, the ORP variation in the pyrite group followed a pattern similar to that of the control group: a slight initial increase, a sharp decline, and a gradual subsequent increase. The ORP values generally decreased from approximately −20 mV to a minimum of approximately −45 mV and then recovered to −20 mV. A pronounced decline occurred between days 13 and 19. This could be attributed to the depletion of dissolved oxygen (DO) and subsequent microbial reduction of oxidized nitrogen species to nitrogen gas, which led to a 75% ORP drop in the reactor. Days 15–19 corresponded to the peak fermentation phase, during which ORP remained unstable. Under microbial activity, pyrite can undergo a reduction-dissolution reaction, releasing electrons and key ions:

$${\mathrm{FeS}}_2+7{\mathrm{H}}_2\mathrm{O}\to {\mathrm{Fe}}^{2+}+2{{\mathrm{SO}}_4}^{2-}+14{\mathrm{H}}^{+}+13{\mathrm{e}}^{-}$$ 2

These electrons can be transferred to methanogens via a direct interspecies electron transfer (DIET) mechanism, thereby enhancing their syntrophic metabolic activity and promoting methane production.

3.1.4. Ammonia Nitrogen Concentration Fluctuations

The ammonia nitrogen concentration served as a key indicator for evaluating nitrogen conversion during anaerobic fermentation. In the pyrite addition group, the ammonia nitrogen levels increased significantly with increasing pyrite dosage. Figure c suggests that the ammonia nitrogen concentrations remained relatively low during coal fermentation, fluctuating between 50 and 350 mg/L. Within the first 7 d, the levels rose sharply from an initial 82–101 to 294–334 mg/L. A brief decline followed, reaching approximately 250 mg/L, before increasing again to 340 mg/L by day 10. Subsequently, the concentration dropped to approximately 200 mg/L by day 19 and then stabilized, coinciding with minimal gas production as fermentation ceased. Compared with the pyrite-treated group, the control group (0% pyrite) exhibited lower ammonia nitrogen production and consumption. In this group, the highest and lowest recorded values were 290 and 250 mg/L, respectively, with less overall variation than in the pyrite addition group.

During the initial stage of fermentation, microbial activity in the anaerobic system was relatively high. The nitrogen-containing organic compounds, such as proteins and amino acids, were gradually degraded, resulting in incomplete degradation and utilization of organic nitrogen, thereby increasing the ammonia nitrogen levels. As fermentation progressed to the midstage, ammonia nitrogen concentrations decreased as microorganisms assimilated the available nitrogen. This alternating pattern of production and consumption caused significant fluctuations in ammonia nitrogen levels during the midfermentation phase. The gas production peaked on days 9 and 15, which corresponded with extensive nitrogen utilization, leading to a significant decline in ammonia nitrogen concentrations. After day 20, the ammonia nitrogen levels stabilized due to the reduced microbial activity or complete substrate depletion, and the system gradually entered a stagnation phase.

3.2. Effects of Electric Stimulation on Fermentation

3.2.1. Influence of Electric Voltage on Methane Yield and Gas Composition

As shown in Figure a, the applied voltage significantly influenced the gas production during fermentation, with the electron release promoting the biological conversion of coal to methane. The SPSS analysis yielded a p value <0.01, indicating the significant differences among groups. The fermentation ceased after approximately 20 d in the 0 and 1.5 V groups, while the groups subjected to the voltages above 1.5 V continued fermenting beyond 30 d, which demonstrated the extended reaction durations. The daily gas production varied markedly under different voltage conditions. The maximum daily gas production in the control group (0 V) was 163 mL, while the 1.5 V group peaked at 193 mL (Figure b). Both groups showed similar trends of gradual increase and stabilization before cessation on day 20. In contrast, the 3 V group reached a peak daily production of 626 mL. Further voltage increases to 4.5 and 6 V yielded peak values of 1826 and 2843 mL, respectively. After 30 d, the cumulative gas production reached 1933 and 1860 mL in the 0 and 1.5 V groups, respectively, compared to 12,523, 26,500, and 28,860 mL in the 3, 4.5, and 6 V groups. These results indicated that voltages of 3 V or higher substantially enhanced both daily and cumulative gas production compared to the control.

5.

Analytical curves of cumulative gas production (a) and daily gas production (b) for the synergistic effect of pyrite and electrical stimulation.

Although the gas production rates were high in the 4.5 and 6 V groups, the biogas analyzer results (Table ) revealed that the methane concentrations were extremely low, accounting for only ∼3% of the total gas volume. In contrast, concentrations of CO₂, O₂, and H₂S were significantly elevated. At voltages of 0 and 1.5 V, methane comprised approximately 36% of the gas, whereas at 3 V, it accounted for ∼34%. Overall, methane production was the highest at 3 V. The control group produced 700 mL of CH₄ over 30 d, whereas the 3 V group generated 4258 mL, denoting an increase of approximately 608%. These findings indicated that electrical stimulation significantly enhanced hydrogen production through the external voltage application. The applied electric field accelerated electron transfer and promoted the reduction of hydrogen ions (H⁺), thereby increasing the hydrogen gas output. This supplied more substrates to hydrogenotrophic methanogens such as Methanobacterium. As hydrogen served as the primary substrate for methane biosynthesis, the increased hydrogen availability stimulated the methanogen activity, resulting in greater methane yields.

5. Accumulated Gas Composition.

applied voltage (V)	CH4 (vol %)	CO2 (vol %)	O2 (vol %)	H2S (ppm)
0	36.2	0.5	2..6	2
1.5	36.4	1.6	2.9	2
3	33.7	1.5	9	19
4.5	3.3	12.1	5.5	1004
6	3.1	11.9	6.1	985

During the experiment, increasing the voltage to 4.5 and 6 V elevated the current density and reduced the cycle time, causing the gas generated from hydrolysis to be expelled from the fermentation system before utilization. Additionally, water electrolysis contributed to elevated concentrations of H₂, CO₂, O₂, and H₂S, while the methane content declined. This could explain why methane accounted for only ∼3% of the total gas volume under high-voltage conditions. At 3 V, the lower voltage promoted H₂ generation at a level suitable for uptake by hydrogenotrophic methanogens, thereby facilitating methane production. The extended gas retention time under this condition could enhance methane accumulation. , However, hydrogen generated through hydrolysis and the initial decomposition of organic matter may escape into the gas collection system, reducing the methane concentration during the early fermentation stage compared with the nonelectrified group. When the brown coal and sludge were cofermented, the voltages below 1.5 V did not significantly improve the gas production. This may be due to the insufficient voltage to maintain the electron transfer and associated biochemical processes required in such a complex fermentation system.

3.2.2. pH and ORP Dynamics under Electric Stimulation

As shown in Figure a, the pH of the fermentation broth exhibited distinct stage-dependent characteristics. In all groups, pH declined sharply during the first 4 d and began to diverge after day 7, where the 0 and 1.5 V groups recovered to 7.3 ± 0.2 and stabilized, the 3 V group remained steady at 6.5 ± 0.2, and the groups with the voltages above 3 V continued decreasing to 5.0–5.5 by day 20 before partially recovering to below 6.0. In the early stage, the pH drop was primarily driven by the production of propionic and valeric acids. In the midstage, the enhanced system conductivity accelerated H⁺ consumption at the cathode, and organic acid adsorption by biochar contributed to pH recovery. At 3 V, a dynamic equilibrium between acid production and consumption was maintained. However, voltages above 3 V inhibited microbial activity, leading to the suppression of methanogenesis and accumulation of organic acids and protons, thereby inducing sustained acidification.

6.

Analytical curves of pH (a) and ORP (b) of fermentation broth with synergistic effects of pyrite and electrical stimulation.

As shown in Figure b, the ORP of the fermentation broth increased with the rising applied voltage. When the voltage exceeded 3 V, the ORP continued to increase until day 7, briefly stabilized, and then increased further before exhibiting slight fluctuations after day 16. By day 30, the 4.5 and 6 V groups reached ORP values of 87 and 121 mV, respectively, with a pronounced increase observed in the 6 V group. In contrast, the 3 V group exhibited an initial ORP peak, followed by a decline and subsequent stable fluctuations. The groups subjected to voltages below 3 V maintained ORP values primarily below 0 mV. After peaking at 9 mV within the first 7 d, ORP declined and stabilized within a narrow range of −41 to 9 mV. Although all groups exhibited an initial rise followed by fluctuations, the high-voltage groups (≥3 V) consistently exhibited significantly higher overall ORP levels than the low-voltage groups.

During the early stage of fermentation, the oxidation level of the system was relatively high. As the load increased, a large amount of reduced coenzyme I (NADH) in the substrate was oxidized to nicotinamide adenine dinucleotide (NAD⁺), leading to a gradual increase in the ORP. In the late stage, when the applied voltage exceeded 3 V, the elevated voltage maintained a high ORP level, creating conditions unfavorable for anaerobic fermentation. In contrast, the 0, 1.5, and 3 V groups exhibited declining ORP values during the later stages owing to continued substrate degradation.

3.2.3. Ammonia Nitrogen and VFA Profiles in Response to Electric Stimulation

The ammonia nitrogen concentration during fermentation exhibited a stage-specific pattern (Figure a). The content increased rapidly during the first 4 d, followed by a slower increase. In the midto-late stages, the trends varied with the applied voltage. After day 16, the ammonia nitrogen decreased in the 0 and 1.5 V groups but continued to rise and fluctuate in groups with voltages over 1.5 V. The peak concentrations and corresponding time points were as follows: 0 V on day 7 (210 mg/L), 1.5 V on day 28 (214 mg/L), 3 V on day 28 (291 mg/L), 4.5 V on day 22 (371 mg/L), and 6 V on day 22 (284 mg/L). These results indicated that the voltages exceeding 1.5 V markedly increased the cumulative ammonia nitrogen production.

7.

Analytical profiles of ammoniacal nitrogen (a) and volatile fatty acids (b) in fermentation broths under the synergistic effects of pyrite and electrical stimulation.

Ammonia nitrogen is generated by microbial degradation. During the initial fermentation stage (days 1–4), high microbial activity led to a rapid increase in ammonia nitrogen concentration. As fermentation progressed (days 4–20), a balance between production and consumption was gradually established, resulting in minimal fluctuations and a relatively stable state. In the later stage, the microbial abundance and activity declined in the 0 and 1.5 V groups, leading to the reduced ammonia nitrogen generation and a downward trend in concentration. However, when the applied voltage exceeded 1.5 V, the dominant microbial populations continued to proliferate, maintaining the strong anaerobic digestion activity and causing the continued fluctuations in ammonia nitrogen levels.

As shown in Figure b, electrical stimulation markedly increased VFA concentrations, with distinct differences observed across voltage levels. The experimental groups subjected to voltage, particularly ≥3 V, exhibited higher total VFA levels that persisted in the late fermentation stage. In the 0 V group, the VFA concentration peaked at 21,200 mg/L on day 10, then declined steadily to 1294 mg/L by day 30, demonstrating a trend similar to that of the 1.5 V group. In contrast, the ≥3 V groups reached the highest peaks of 44,542–46,575 mg/L on day 10, followed by a decline during days 13–16, a subsequent rise, a second drop around day 25, and a final rebound. This indicated sustained fluctuations during the mid to late fermentation period, suggesting that electrical stimulation not only enhanced VFA production but also altered its dynamic temporal profile.

VFAs in anaerobic fermentation primarily include acetic, propionic, and butyric acids. During the initial fermentation stage, microorganisms degrade substrates into VFAs, ethanol, and other intermediates. Simultaneously, acetogenic bacteria convert H₂, CO, and CO₂ into acetic acid, further contributing to an increase in VFA concentrations. , In particular, acetic acid can serve as a key precursor for methane formation.

$${\mathrm{CH}}_3\mathrm{COOH}\to {\mathrm{CH}}_4+{\mathrm{CO}}_2$$ 3

Subsequently, microbial metabolic activity can convert acid into methane, leading to a reduction in VFA concentrations. The application of external voltage accelerated VFA production, with concentrations exceeding twice those of the control group when the voltage exceeded 3 V. However, under high organic loading, prolonged VFA accumulation inhibits methanogenesis, resulting in decreased methane output and reduced VFA utilization. The fatty acid accumulation could also be observed in the later stages of fermentation.

3.3. Synergistic Effects of Pyrite and Electric Stimulation

Under the combined influence of pyrite and electrical stimulation, the key parameters of lignite anaerobic fermentation, including gas production, pH, ORP, ammonia nitrogen, and VFAs, underwent significant changes. Specifically, at a voltage of 3 V, the effects were most pronounced. The methane yield of the 3 V group reached 4220 mL, representing a 648.2% increase compared to the 0 V group (651 mL) (Figure a). This result indicated that electrical stimulation substantially enhanced gas production by promoting microbial metabolic activity, primarily through accelerated electron transfer and increased hydrogen generation, thereby stimulating methanogen activity. These findings underscore the critical role of electrical stimulation in improving methane production efficiency.

In this study, the Gompertz model was applied to fit the methane production profiles from brown coal anaerobic fermentation under 0 and 3 V conditions. As shown in Figure a (0 V) and Figure b (3 V), electrical stimulation significantly enhanced methane production, particularly at 3 V. Under the 0 V conditions, the fitted curve exhibited a typical S-shaped growth pattern, with the maximum gas production of 1933 mL, a reaction rate constant of 156.22, and a lag phase of 3.78 d. These results indicated that in the absence of electrical stimulation, pyrite functioned as an electron donor and moderately promoted methane generation. However, the effect was limited, the production rate was slow, and system stabilization occurred early.

8.

Fitted methane production curves at 0 V (a) and 3 V (b). Pm: maximum gas production; Rm: reaction rate constant; λ: delay time.

At 3 V, gas production increased dramatically, with a maximum yield of 12,523 mL, a reaction rate constant of 560.28, and a significantly reduced lag time of 0.49 d. Gas production at 3 V was significantly higher than that at 0 V. Electrical stimulation enhanced hydrogen production and optimized electron transfer, thereby accelerating methane generation. The applied electric field provided hydrogenotrophic methanogens (e.g., Methanobacterium) with additional substrates, promoting their metabolic activity and increasing methane yield. Further analysis indicated that the 3 V voltage not only improved methane production but also markedly reduced the lag phase, demonstrating that the electric field accelerated microbial activation. In contrast, the 0 V group, relying solely on natural microbial metabolism, exhibited a longer lag phase and lower methane production efficiency.

In summary, electrical stimulation significantly enhanced methane production, particularly at 3 V. By promoting hydrogen generation and optimizing electron transfer, it markedly improved methane yield, demonstrating its strong potential for application in lignite anaerobic fermentation. These findings provide a theoretical foundation for optimizing electrical stimulation for the biological conversion of low-rank coal.

As shown in Figure a, the pH in the 3 V group demonstrated a substantial decrease during the initial fermentation period (0–7 d) and then stabilized. Compared to the 0 V group, the 3 V group exhibited a more pronounced pH decline, with stabilization occurring after day 7. This suggests that electrical stimulation accelerates organic matter decomposition and acidification in the early stage, leading to a rapid pH drop. With increasing voltage, the enhanced degradation of organic substrates generated more acidic compounds, such as VFAs, directly contributing to pH reduction. In the 0 V group, pH remained relatively stable but exhibited a gradual downward trend, reaching a low point by day 7. This indicated that acidification occurred even in the absence of electrical stimulation during anaerobic fermentation:

$${\mathrm{CH}}_3{\mathrm{CHNH}}_2\mathrm{COOH}+2{\mathrm{H}}_2\mathrm{O}\to {\mathrm{CH}}_3\mathrm{COOH}+{\mathrm{NH}}_3+{\mathrm{CO}}_2+4{\mathrm{H}}^{+}$$ 4

$$2{\mathrm{C}}_3{\mathrm{H}}_6{\mathrm{O}}_3+2{\mathrm{H}}_2\mathrm{O}\to {\mathrm{CH}}_3{\mathrm{CH}}_2{\mathrm{CH}}_2\mathrm{COOH}+2{\mathrm{H}}_2+2{\mathrm{H}}^{+}+2{{\mathrm{HCO}}_3}^{-}$$ 5

In contrast, the 3 V group presented greater pH fluctuations following the initial rapid decline, eventually stabilizing at a relatively consistent level.

Electrical stimulation optimized microbial metabolic processes, promoted the conversion and consumption of organic acids, and mitigated the risk of excessive acidification. Although pH declined sharply in the early stage of the 3 V group, it stabilized in the later stage, providing a favorable environment for microbial growth, particularly for methanogens. Thus, electrical stimulation not only accelerated organic matter degradation but also maintained a suitable pH by improving the acid–base balance, thereby promoting methane production. As shown in Figure b, the ORP values in the 3 V group gradually increased during the initial fermentation phase (0–7 d), reaching a peak of ∼50 mV on day 7 before stabilizing. In contrast, the ORP values in the 0 V group remained near 50 mV initially but gradually increased, reaching ∼100 mV by day 28. This trend indicated that the electrical stimulation, particularly at 3 V, significantly improved the reducing environment of the system by accelerating the electron transfer and enhancing the microbial metabolic activity, thus creating favorable conditions for methanogens and enhancing methane yield.

As shown in Figure a, the 3 V group exhibited a marked increase in ammonia nitrogen concentration during the initial fermentation stage, reaching 294 mg/L by day 10, followed by fluctuations. Compared to the 0 V group, the ammonia nitrogen levels were consistently higher in the 3 V group, indicating that electrical stimulation promoted both the decomposition of organic nitrogen and the accelerated release of ammonia nitrogen. In the later stages, the continued electrical stimulation facilitated the microbial uptake of ammonia nitrogen, providing additional nitrogen sources to support methane production.

As shown in Figure b, the combined effect of electrical stimulation and pyrite markedly increased VFA concentrations, particularly in the voltage groups ≥3 V. In the 3 V group, the VFA concentration peaked at 44,542 mg/L on day 10, markedly higher than the 21,200 mg/L observed in the 0 V group. This indicated that electrical stimulation enhanced VFA production by promoting hydrolysis and acidification of organic matter, thereby providing more substrates for methanogenesis. The VFA concentrations continued to fluctuate upward over 23 d, reflecting the sustained stimulation of anaerobic fermentation by the applied electric field.

Overall, the combined application of pyrite and electrical stimulation significantly enhanced the gas production efficiency of brown coal anaerobic fermentation. Electrical stimulation improved the anaerobic conditions, such as pH and ORP, thereby promoting microbial metabolism and markedly increasing the production of ammonia nitrogen and VFAs, which served as the essential substrates for methanogenesis. Pyrite (FeS₂) underwent a reduction-dissolution reaction in anaerobic environments, releasing electrons as donors for methane production and stimulating the activity of hydrogenotrophic methanogens. Furthermore, electrical stimulation induced hydrogen generation via water electrolysis, providing further substrate for hydrogenotrophic methanogens. Simultaneously, hydrogen reacted with carbon dioxide, further accelerating methane formation. The corresponding reaction is as follows:

$${\mathrm{FeS}}_2+3.5{\mathrm{O}}_2+3.5{\mathrm{H}}_2\mathrm{O}\to 2{\mathrm{Fe}}^{3+}+4{{\mathrm{SO}}_4}^{2-}+4{\mathrm{H}}^{+}$$ 6

$$2{\mathrm{H}}_2\mathrm{O}\to {\mathrm{O}}_2+4{\mathrm{H}}^{+}+4{\mathrm{e}}^{-}$$ 7

$$4{\mathrm{H}}^{+}+4{\mathrm{e}}^{-}\to 2{\mathrm{H}}_2$$ 8

$${\mathrm{CO}}_2+4{\mathrm{H}}_2\to {\mathrm{CH}}_4+2{\mathrm{H}}_2\mathrm{O}$$ 9

Pyrite functions as an electron donor, releasing electrons via the reduction-dissolution reaction to support microbial metabolic processes. Notably, the 3 V group achieved a substantial increase in methane yield by promoting electron transfer, enhancing hydrogen production, and optimizing microbial metabolic pathways. The electrical stimulation played a vital role in the lignite anaerobic fermentation, significantly improving the methane production efficiency by enhancing the physicochemical environment and facilitating the formation of key metabolic substrates.

3.4. Microbial Community Structural Dynamics

3.4.1. Relative Abundance of Methanogenic Bacteria

Microbial community structure was assessed on day 10 (corresponding to peak gas production). From the control (0 V; CK1, CK2, and CK3) and experimental (3 V; T1, T2, and T3) groups, 50 mL of fermentation broth was taken for each sample. The samples were subjected to 16S rRNA high-throughput sequencing on the Illumina MiSeq platform to analyze the bacterial and methanogenic community composition. The results revealed that the combined application of electrical stimulation and pyrite significantly altered the abundance of methanogens, particularly hydrogenotrophic methanogens (e.g., Methanobacterium) and acetoclastic methanogens (e.g., Methanosarcina). , Figure (A) and (B) demonstrated that under the 3 V treatment, the relative abundance of Methanobacterium increased from 1.42% in the control group to 9.13%, whereas Methanosarcina decreased from 39.7% to 21.36%. These findings indicated that the electrical stimulation enhanced the hydrogenotrophic methanogenesis by promoting the hydrogen availability, while concurrently inhibiting acetoclastic pathways. The significant enrichment of Methanobacterium in the cotreatment group confirmed that the electrical stimulation optimized the hydrogen-dependent methane production pathway.

9.

Community structure of methanogenic archaea at the genus level in control group 0 V (A) and experimental group 3 V (B) at the peak of fermentation.

Meanwhile, the relative abundance of Methanobrevibacter producing methanogens through utilizing CO₂ and H₂ as substrates significantly decreased in the combined treatment group, from 3.86% in the control group to 0.33%. This suggested that the electrical stimulation inhibited the growth of hydrogenotrophic methanogens sensitive to H₂/CO₂ metabolism, potentially by increasing H₂ concentrations or modifying the electron transfer pathway.

3.4.2. Microbial Diversity and Community Evenness Alterations

Microbial community diversity is a key indicator for evaluating the ecosystem stability and functionality. In this study, community diversity across treatment groups was assessed using the Shannon and Simpson indices. The combined application of electrical stimulation and pyrite significantly influenced the community diversity (Table ), particularly through the variations in the relative abundance of two dominant methanogenic genera: Methanobacterium and Methanosarcina. In the experimental groups, both indices reflected the high microbial diversity, with the T1 group (3 V and pyrite) exhibiting the highest Shannon index of 5.71. This indicated that the microbial community under combined treatment was more even and diverse, suggesting that the electrical stimulation and pyrite jointly contributed to the optimization of community structure.

6. Alpha Diversity Indices of Bacterial and Archaeal Communities.

sample		Sobs	Shannon	Simpson	Ace	Chao	coverage
bacteria	CK1	1262	5.488382	0.015542	1280.917152	1267.390244	0.998229
	CK2	1326	5.645157	0.012203	1347.40108	1331.422535	0.998092
	CK3	1392	5.769624	0.010316	1392	1392	1
	T1	1262	5.488382	0.015542	1280.917152	1267.390244	0.998229
	T2	1421	5.717474	0.012163	1431.97715	1422.343931	0.998944
	T3	1291	5.558944	0.014968	1303.244893	1292.927835	0.998842
methanogens	CK1	3	0.017909	0.995358	0	3	1
	CK2	3	0.008096	0.998051	3	3	1
	CK3	3	0.015703	0.99571	3	3	1
	T1	13	0.853946	0.546529	13	13	1
	T2	13	0.765152	0.599712	13	13	1
	T3	15	0.781783	0.573085	15	15	1

3.4.3. Phylum-Level Shifts in Microbial Communities

At the phylum level, the combined application of electrical stimulation and pyrite significantly altered microbial community composition. As shown in Figures and , the relative abundances of key anaerobic fermentative phyla, such as Firmicutes, Proteobacteria, and Actinobacteriota, increased under the combined treatment. Specifically, Figure presents that under the 3 V voltage and 16% pyrite conditions, Firmicutes increased from 35.6% to 41.8%, and Proteobacteria from 24.3% to 29.4%. These shifts suggests that the combined treatment not only promotes the proliferation of methanogens but also improves the community’s overall metabolic capacity and optimized organic matter degradation. A simplified mechanistic model summarizing these findings is shown in Figure .

10.

Bacterial community structure at the phylum level in the control and experimental groups at the peak of fermentation.

11.

Bacterial community structure at the genus level in control and experimental groups during peak fermentation.

12.

Mechanistic sketch of the experimental results.

3.4.4. Dynamic Shifts in Microbial Populations

With increasing fermentation time, dynamic changes in the microbial community structure further highlighted the impact of the combined application of electrical stimulation and pyrite on the anaerobic fermentation process. By day 10, the relative abundances of Methanobacterium and Methanosarcina continued to rise, whereas Methanobrevibacter declined (Figure (A) and (B)). These trends indicated that the electrical stimulation promoted the dominance of hydrogenotrophic methanogens by enhancing the hydrogen availability and optimizing the electron transfer, while concurrently inhibiting the activity of acetoclastic and CO-utilizing methanogens.

13.

Comparison of the abundance of control (A) and experimental (B) strains under an IX83 microscope.

3.4.5. Synergistic Impact on Microbial Community Structure

The combined application of pyrite and electrical stimulation significantly optimized the microbial community structure, particularly with respect to methanogen abundance, community diversity, and the relative abundance of the dominant microbial phyla. Electrical stimulation enhanced hydrogen production, thereby promoting the activity of hydrogenotrophic methanogens (e.g., Methanobacterium) while inhibiting acetoclastic methanogens (e.g., Methanosarcina). Pyrite supplementation supplied essential trace elements, further enhancing the microbial metabolic capacity.

The combined treatment optimized microbial community diversity, balanced the community structure, and significantly enhanced methane production efficiency. Notably, the synergistic effects of electrical stimulation and pyrite were reflected in the increased abundance of key phyla, such as Firmicutes and Proteobacteria, highlighting their roles in community optimization and methane enhancement.

4. Conclusions

This study systematically investigated the synergistic effects of pyrite (0–32%) and external voltage (0–6 V) on anaerobic methane production from lignite. By analyzing gas production, physicochemical parameters (pH, ORP, VFAs, and ammonia nitrogen), and microbial community structure, the underlying mechanisms were elucidated. This study provides a novel pyrite-electrochemical strategy for enhancing methane yield through multifactor coupling. The main conclusions are

• (1)A 16% pyrite dosage combined with a 3 V electric field significantly enhanced methane production, achieving 4220 mL (a 7.67-fold increase vs the control), primarily via Fe²⁺/S^2–-mediated DIET. However, excessive pyrite (>16%) caused Fe³⁺/S^2– toxicity, reducing yield by 15.3%.
• (2)Combined treatment shifted methanogenic pathways. The abundance of hydrogenotrophic Methanobacterium increased (from 1.42% to 9.13%), whereas that of acetoclastic Methanosarcina decreased (from 39.7% to 21.36%). Pyrite provided trace nutrients, whereas electricity promoted H₂-driven methanogenesis. At the phylum level, Firmicutes (41.8%) and Proteobacteria (29.4%) enrichment confirmed improved degradation capacity.
• (3)Methane yield declined when pyrite exceeded 16% or voltage surpassed 3 V. At 32% pyrite, gas yield dropped by 19.9% (584 vs 700 mL), and a 6 V treatment yielded only 3% methane. Excessive metal/sulfide release and acidification (pH 5.0–5.5; ORP 121 mV) impaired archaeal activity.
• (4)This pyrite-electrochemical strategy achieved 4220 mL of methane over 30 d without external additives, demonstrating strong potential for practical applications. Key thresholds of pH, ORP, and VFAs identified in this study may aid in the process control and scale-up of lignite bioconversion systems. Nonetheless, challenges such as electric field uniformity and sustainable pyrite sourcing warrant further investigation.

The authors declare no competing financial interest.