Granular Activated Carbon and Organic Loading Interactions in Methane Fermentation: An Inverse Load-Dependent Relationship and Absolute Microbial Abundance Analysis

Abstract

This study addresses volatile fatty acid (VFA) accumulation, a key issue limiting methane fermentation under high organic loading rate (OLR) conditions. Batch experiments were conducted with GAC (0–10%) under various OLRs (1:0.5–1:10) to investigate its effect on biogas yield, methane purity, and microbial interactions. Higher GAC levels (7.5% and 10%) significantly enhanced biogas production (750–800 mL/g VS) and methane concentration (–70%) while shortening stabilization time. A continuous system with 10% GAC showed suppressed VFA accumulation, stable pH (7.0–8.1), and improved organic matter degradation. This work quantitatively evaluates the link between GAC dosage, DIET induction, and microbial community shifts under high OLR. These findings highlight GAC as an operationally simple and potentially cost-beneficial strategy for stabilizing methane fermentation, particularly in decentralized or small-scale applications.

1. Introduction

In response to growing global efforts to reduce greenhouse gas emissions and promote recycling-oriented societies, the utilization of organic waste such as food waste, sewage sludge, and livestock manure has gained momentum [1]. Methane fermentation, a form of anaerobic digestion, has emerged as a promising technology for converting such waste into renewable energy. By decomposing the carbon, hydrogen, and oxygen components of organic matter, methane fermentation not only reduces waste volume and solids but also produces a nutrient-rich digestate suitable for use as fertilizer. This dual benefit of waste minimization and renewable energy production makes methane fermentation a cornerstone of sustainable and circular economy initiatives [2].

Despite this potential, the stable operation of methane fermentation systems remains challenging, especially when processing food waste. Process efficiency is strongly influenced by operational parameters such as temperature, hydraulic retention time (HRT), and organic loading rate (OLR) [3]. Recent advancements have improved methane fermentation efficiency and enabled the development of compact systems. For instance, systems small enough to be introduced at pig farms with approximately 100 pigs have been reported, demonstrating the feasibility of distributed-scale applications [4]. Nevertheless, a major bottleneck persists: the accumulation of volatile fatty acid (VFA) under high OLR conditions, which impairs methanogenesis and destabilizes pH. Process failure commonly occurs when pH falls below 6.0 [5], and inhibition of methanogenic archaea is observed when pH decreases below 6.5 [6]. This instability is particularly pronounced in small-scale systems with limited buffering capacity.

To mitigate these limitations, conductive materials such as granular activated carbon (GAC) have been investigated [7,8,9]. GAC can enhance performance by serving as a microbial support, adsorbing inhibitory compounds, and facilitating direct interspecies electron transfer (DIET) [7,10]. DIET enables more efficient electron exchange between syntrophic partners by bypassing the rate-limiting hydrogen-mediated pathway, thereby promoting methane production [11]. In addition to GAC, other conductive materials such as biochar, magnetite, and iron-based compounds have also been investigated to enhance anaerobic digestion. As summarized in

Table A1. Summary of previous studies on the addition of conductive materials in methane fermentation.

Study, Year	Additive	Substrate	Additive Dosage	Key Quantitative Outcome
Kalantzis et al., 2023 [24]	GAC	Agro-industrial wastewater	5 g/L	Biogas production increased by 32% after the addition of GAC.
Ziganshina et al., 2020 [25]	GAC	Beet pulp and distillers grains with solubles	1–10 g/L	The pH values in reactors remained stable, ranging from 6.91 to 7.57 throughout the experimental period.
Ziganshina et al., 2022 [26]	GAC	Chicken manure	5–10 g/L	Under mesophilic conditions, methane production was highest with 5 g/L GAC, showing an approximately 4.1% increase compared to the control group.
Xu et al., 2018 [27]	GAC	VFA (acetate, propionate, butyrate)	0.5–25 g/L	Methane generation rate increased significantly with GAC supplementation, reducing the lag phase from 4.2 days (0 g/L) to 0.9 days (25 g/L).
Zhang et al., 2024 [28]	GAC+ZVI	Organic fraction of municipal solid waste	1–15% (w/w)	GAC alone (R-GAC) achieved 327.6 mL/gVS, and ZVI alone (R-ZVI) achieved 296.9 mL/gVS, both higher than the control group.
De Sousa e Silva et al., 2025 [29]	GAC	Swine manure	10–30 g/L	GAC enhanced methane generation by promoting Direct Interspecies Electron Transfer (DIET) and adsorbing inhibitory compounds.
Wu et al., 2022 [30]	AC/graphite	Food waste sludge	—	The cumulative biogas production with 100-mesh activated carbon was 468.2 mL/g VSS, which was 13.8% higher than the control group.
Chen et al., 2023 [31]	GAC	Garden waste	50 g/L	The VFA/sCOD ratio in the GAC-amended group reached 70.01%, surpassing the control group’s 49.35%, indicating more efficient hydrolysis and acidogenesis.
Quintana-Najera et al., 2023 [32]	Biochar	Model carbohydrate	0.03–8.0% (w/w)	he addition of biochar showed notable improvements in the digestion of complex substrates rich in lipids and proteins, as well as under stressful conditions.
Hu et al., 2023 [33]	Biochar	Dog food pellets	10–25% (VS basis)	Under high substrate overloading (ISR 0.5), 25% biochar addition enhanced the removal rate of substrate volatile solids (VS).

(Appendix A), these materials have been reported to improve methane yield, methane concentration, and pH stability under various operating conditions by facilitating DIET or mitigating process inhibition. For example, biochar provides a porous surface that supports microbial colonization, while magnetite and iron-based materials can serve as electron mediators to stimulate syntrophic interactions. Although these alternatives are effective, GAC has frequently demonstrated superior performance due to its large surface area, high conductivity, and adsorption capacity. These advantages motivated its selection as the primary focus of the present study. While the potential of GAC has been recognized, many previous studies have been constrained by narrow experimental conditions such as limited OLR ranges or insufficient integration of microbial analysis with performance data [12]. Moreover, microbial communities have typically been analyzed only in relative terms. Comprehensive analyses that systematically map the interaction between OLR and GAC dosage remain scarce [13]. In addition, few studies have quantified microbial populations in absolute abundance to directly link community shifts with process performance [14]. In particular, the load-dependent inversion where increasing GAC dosage reduces methane content at low OLRs but enhances it at high OLRs has not yet been systematically addressed [15].

To address this gap, we investigated the effects of GAC addition (0–10%) on methane fermentation under multiple OLR conditions in both batch and continuous systems. This approach enabled us to determine threshold OLRs at which the effect of GAC inverts, to evaluate both methane content and production yield, and to elucidate microbial mechanisms using absolute quantification. Batch experiments were conducted to assess methane yield, methane content, pH, and microbial community structure, while continuous experiments evaluated biogas yield, methane content, pH, and organic matter degradation.

This study provides novel contributions by systematically mapping GAC dosage against OLR across a wide range of conditions, identifying the inversion point of GAC effects on methane content, and linking process performance with microbial community dynamics quantified in absolute terms. These findings advance the understanding of GAC-mediated stabilization and support its application as a practical strategy for high-load anaerobic digestion, particularly in decentralized or resource-limited settings.

2. Materials and Methods

2.1. Materials and Chemicals

In this study, inoculum sludge was obtained from a methane fermenter operated by a pig farmer in Aichi, Japan, who raises approximately 1000 pigs. This sludge was used as seed inoculum throughout the experiments. Commercial dog food (AIKEN GENKI, Unicharm, Tokyo, Japan), commonly utilized as a model substrate for food waste, was employed as the primary feedstock. The dog food contains 22.0% protein, 10.0% lipids, 4.5% crude fiber, 8.5% crude ash, and 10.0% moisture (w/w). To prepare the substrate solution, the dog food was mixed with an appropriate volume of water and homogenized using a mixer. The concentration of the solution was determined based on volatile solids (VS, g-VS/L) and adjusted to the target level by dilution with water. Low vs. concentrations (2.5 and 5 g-VS/L) represented typical organic loading conditions, while high vs. concentrations (25 and 50 g-VS/L) were used to simulate high organic loading conditions and evaluate system performance under stress. GAC derived from coconut shell (KD-GA-X, UES Corporation, Osaka, Japan) was employed as the conductive material. This GAC corresponds to a CTC65 grade, which indicates that its carbon tetrachloride adsorption capacity reaches approximately 65% a level higher than the standard CTC50–55 grades commonly used for commercial activated carbons. Therefore, the KD-GA-X product can be regarded as a high-purity, high-performance GAC. Its particle size was 4 × 8 mesh, with a BET surface area of 1237 m²/g, a Langmuir surface area of 1423 m²/g, a t-plot micropore area of 1075 m²/g, a micropore volume of 0.41 cm³/g, an average pore diameter of 2.89 nm (BJH method), and a pH of 9.8.

2.2. Anaerobic Digestion Experiments

2.2.1. Effects of Different Addition Rates of GAC on Methanogenesis Under Normal and High Organic Loadings in Batch Fermentation System

Batch fermentation tests were conducted under anaerobic conditions at 37 °C using 75 mL airtight reactors. Each reactor contained 10 mL of inoculum sludge (derived from swine manure), 0.5 mL of reducing agent, and phosphate buffer. The reducing agent was added to rapidly establish anaerobic conditions and lower the redox potential, thereby protecting the activity of anaerobic microorganisms, whereas the phosphate buffer was used to stabilize pH fluctuations during fermentation. Four inoculum-to-substrate ratios were applied (1:0.5, 1:1, 1:5, and 1:10), corresponding to dog food concentrations of 2.5, 5, 25, and 50 g-VS/L, respectively. Each ratio was tested under six GAC dosages: 0% (control), 1%, 2.5%, 5%, 7.5%, and 10% (w/w relative to the liquid phase), all conducted with n = 1. The final liquid volume was adjusted to 20 mL with deionized water. To ensure anaerobic conditions, the reactors were flushed with nitrogen gas for 5 min and sealed. Incubation was carried out at 37 ± 1 °C for 55 days, with manual mixing once daily for 30 s. Biogas samples were collected every 1–5 days, and total biogas volume and methane concentration were measured. Digestate samples were also collected for pH measurement and microbial community analysis.

2.2.2. Effects of GAC Addition on Methanogenesis at High Organic Loading in Continuous Fermentation System

Continuous fermentation was performed in 1 L anaerobic reactors, each seeded with 0.5 L of swine manure digestate. Six GAC dosages were tested (0%, 1%, 2.5%, 5%, 7.5%, and 10% w/w). Dog food substrate was applied at a high OLR of 1:10 (50 g-VS/L) to evaluate the effect of GAC under stress conditions. During fermentation, 25 mL of digestate was withdrawn daily and replaced with fresh substrate, corresponding to a HRT of 20 days. The reactors were maintained at 37 °C and mixed for 30 min per day. Biogas was collected in gas bags, methane concentration was determined by gas chromatography, and total gas volume was measured using a gas flow meter. The pH of the liquid phase was measured periodically using the withdrawn digestate, which was also analyzed for VS degradation.

2.3. Analytical Measurements

2.3.1. Measurement of pH and Methane Concentration

Methane concentration was determined by sampling 0.5 mL of biogas with a stopcock-equipped syringe and analyzing it using gas chromatography (GC-2014, Shimadzu, Kyoto, Japan) with a packed column (Shincarbon ST 50/80, Shinwa Kako, Kyoto, Japan), argon as the carrier gas, and a thermal conductivity detector (TCD). Methane production was calculated from methane concentration and the total biogas volume measured with a gas flow meter (W-NK-0.5 A, Shinagawa Corp., Tokyo, Japan). The pH of the digestate was measured using a glass electrode meter (D-55, Horiba, Kyoto, Japan); in batch experiments, it was determined at the beginning and end of fermentation, and in continuous experiments, it was measured periodically in the withdrawn digestate. Volatile solids (VS) were determined by drying samples at 110 °C for 6 h and combusting at 600 °C for 2 h using a drying oven (DS 410) and muffle furnace (FO 300, Yamato Scientific, Tokyo, Japan). The difference between dry mass and ash mass was used as organic matter. In continuous fermentation, the organic decomposition rate was calculated by measuring the VS of the substrate and the withdrawn digestate, and was expressed as the ratio of digestate VS to substrate VS.

2.3.2. Microbial Analysis

The workflow from DNA extraction to the estimation of absolute microbial abundance is shown in Figure A1 (Appendix A). Microbial DNA was extracted from seed sludge (initial) and digestate (final) using the FastDNA SPIN Kit for Soil (MP Biomedical, California, CA, USA), following the manufacturer’s protocol. Microbial community composition was analyzed via next-generation sequencing (NGS) using the Illumina MiSeq platform (Illumina, California, CA, USA), performed by FASMAC (Kanagawa, Japan). The V4 region of the 16S rRNA gene was amplified using universal primers 515F (GTGYCAGCMGCCGCGGTAA) and 806R (GGACTACNVGGGTWTCTAAT). Quantitative PCR (qPCR) was performed using the same primer set and a Thermal Cycler Dice Real-Time System III (Takara Bio, Shiga, Japan) to quantify total bacterial and archaeal 16S rRNA gene copies. Absolute microbial cell numbers were estimated by combining NGS data with qPCR results and correcting for 16S rRNA gene copy numbers using the rrnDB database.

3. Results

3.1. Batch Fermentation

3.1.1. Effect of Different Levels of GAC on Methanogenesis Under Varying Organic Loading in Batch Fermentation

Figure 1 shows the cumulative biogas production over a 55-day fermentation period in batch systems supplemented with varying concentrations of GAC (1%, 2.5%, 5%, 7.5%, and 10%). It also presents the effects of GAC addition under different OLRs: (A) 1:0.5, (B) 1:1, (C) 1:5, and (D) 1:10. The progression of the curves suggests a dose-dependent effect, with increased GAC concentrations generally resulting in higher biogas yields. Under low to moderate OLR conditions (A–C), biogas production reached saturation within 20 to 30 days across all GAC treatments, with cumulative yields ranging approximately from 600 to 750 mL/g-VS. Under OLR 1:1 and 1:5, GAC concentrations of 5% to 10% showed slightly higher biogas production; however, similar levels were also achieved with 1% and 2.5%, indicating that the dose-dependent effect was limited at these OLR. In contrast, under high OLR conditions (D: 1:10), the presence or absence of GAC significantly affected biogas production. GAC additions of 7.5% and 10% led to a sharp increase in biogas accumulation, reaching approximately 650 mL/g-VS. Meanwhile, lower GAC concentrations (0–5%) resulted in markedly slower production and lower final yields, remaining below 200 mL/g-VS. These results indicate that the effectiveness of GAC addition becomes more pronounced under high organic loading conditions.

Overall, these findings suggest that an OLR of 1:10 imposes greater stress on biogas production compared to lower OLRs. The addition of GAC plays an important role in mitigating this stress and enhancing biogas yield under high OLR conditions, while its effect is limited under low OLRs. This trend implies that higher concentrations of GAC may stimulate microbial activity and improve the efficiency of organic matter degradation under organic overloading stress.

3.1.2. Effect of Different Percentages of GAC on Methanogenesis Under Varying Organic Loading in Batch Fermentation

The variations in methane concentration under different OLR conditions are shown in Figure 2. Under low to moderate OLR conditions (1:0.5, 1:1, and 1:5), the addition of GAC tended to decrease the methane concentration in the produced gas. This suggests that GAC may influence the microbial community structure or metabolic balance in a way that favors non-methanogenic pathways such as acidogenesis or acetogenesis. It is also possible that GAC selectively enriches bacterial groups less involved in methane production, or alters the efficiency of intermediate metabolite (e.g., hydrogen, acetate) utilization. In contrast, under the highest OLR condition (1:10), a clear positive correlation was observed between GAC addition and methane concentration. Notably, GAC additions of 5% or more markedly enhanced methane purity, reaching approximately 70% at 10% GAC. These results suggest that above a certain threshold of GAC concentration (e.g., ≥5–7.5%), methanogenesis via DIET becomes dominant. In addition, GAC’s role in suppressing VFA accumulation and stabilizing pH becomes more pronounced under high loading stress, further supporting stable methanogenic activity. It is also conceivable that, even when GAC facilitates enhanced organic matter degradation, the electrons released may not always be efficiently transferred to methanogens, thereby limiting methane generation. Therefore, it is important to recognize that total biogas production and methane concentration do not always correlate directly.

Moreover, the discrepancy between total methane volume (Figure 1) and methane concentration (Figure 2) highlights the importance of evaluating both the quantity and quality of biogas. While GAC addition may increase total gas production, it may also lead to a higher proportion of non-methane gases such as CO₂ or H₂. Thus, detailed gas composition analysis—including CO₂/H₂ ratio—is essential in future studies to accurately assess the effect of GAC on the anaerobic digestion process.

3.1.3. Change in pH Under High OLR and Different GAC Percentages for Batch Fermentation

Figure 3 presents the changes in pH under different OLR conditions (A) 1:0.5, (B) 1:5, (C) 1:10 and varying GAC supplementation levels. Under low OLR conditions (1:1 and 1:5), the pH values (7.8–8.3) remained relatively constant across all GAC treatments, indicating that GAC addition had little effect on pH regulation in these cases (Figure 3A,B). In contrast, under the highest OLR condition (1:10), the pH values for GAC addition levels of 0%, 2.5%, 5%, and 7.5% were 5.5, 5.7, 7.0 and 7.8, whereas the addition of 10% GAC resulted in a pH of 7.9. Since the optimal pH range for methanogenesis is generally between 6.5 and 8.2, it is suggested that the GAC 0% and 2.5% groups experienced acidification under high OLR conditions, leading to inhibition of biogas production.

The addition of 10% GAC under high OLR (1:10) is therefore considered to have improved biogas production by preventing acidification and influencing the microbial community structure. To further clarify this effect, detailed microbial community analysis is necessary to investigate how 10% GAC supplementation affects microbial populations under high organic loading conditions.

3.1.4. Microflora

The microbial community structure was analyzed in four test sections under high OLR (1:10) conditions: the seed sludge, GAC 0% (no addition), GAC 1%, and GAC 10%, where acidification was effectively suppressed.

Bacterial and Archaeal Abundance and Diversity at the Family and Kingdom

As shown in Figure A2 (Appendix A), the relative abundance and taxonomic composition of microbial communities under high OLR conditions were analyzed at the kingdom and family levels. In all samples, bacteria accounted for most of the community, while archaea comprised only 0.9–3.4% of the total reads. GAC addition influenced community structure, and the 10% GAC group showed a slight increase in archaeal abundance and greater diversity compared to the 0% and 1% GAC groups.

At the bacterial family level, the seed sludge was dominated by Clostridiaceae, Hungateiclostridiaceae, and Bacteroidetes vadinHA17. In contrast, the GAC 0% and 1% groups were enriched in Caloramatoraceae and Ruminococcaceae. The 10% GAC treatment exhibited the greatest bacterial diversity, including higher proportions of Dysgonomonadaceae, Rikenellaceae, and Syntrophomonadaceae—families involved in syntrophic metabolism.

At the archaeal family level, the GAC 0% and 1% groups showed limited diversity, whereas the GAC 10% group displayed a broader range of methanogens, including Methanomicrobiaceae and Methanomassiliicoccaceae. These results suggest that under high-OLR conditions, high GAC concentrations promote both syntrophic bacteria and diverse methanogenic archaea, potentially contributing to enhanced methane production.

Bacterial Abundance and Diversity at the Genus Level

Figure 4 illustrates the major bacterial genera present in digesters operated under high OLR conditions (1:10). The initial microbial community in the seed sludge exhibited high diversity, with a total bacterial abundance of approximately 3.8 × 10¹⁰ cells/mL. Dominant genera included Clostridium sensu stricto 1, Bacteroidetes vadinHA17, DTU014, Fastidiosipila, Smithella, Desulfomicrobium, and Petrimonas. Several of these genera, particularly Bacteroidetes vadinHA17, Fastidiosipila, Smithella, Candidatus Caldatribacterium, and Desulfomicrobium, decreased substantially following GAC addition. In the 0% GAC treatment, overall bacterial diversity declined, with a notable dominance of Caproiciproducens, and reduced abundance of Clostridium sensu stricto 1 compared to the seed sludge and 10% GAC group. In contrast, the 10% GAC treatment supported the highest bacterial abundance (approx. 5.5 × 10¹⁰ cells/mL) and diversity. Several genera, including Clostridium sensu stricto 1, Syntrophomonas, Proteiniphilum, Dysgonomonadaceae, Fermentimonas, and Ruminofilibacter, were enriched. Although some genera such as Bacteroidetes vadinHA17 and DTU014 were still lower than in the seed sludge, their abundance remained higher than in the GAC 0% treatment.

These results indicate that 10% GAC supplementation promotes a more diverse and enriched bacterial community under high organic loading conditions.

Archaeal Abundance and Diversity at the Genus Level

Figure 5 illustrates the archaeal community structure in response to different GAC supplementation levels and seed sludge conditions. The composition of archaeal communities varied considerably among treatments. The seed sludge exhibited moderate diversity, with prominent genera including Methanoculleus, Methanobacterium, Methanosarcina, and Methanosaeta. In contrast, the GAC 0% treatment showed a markedly reduced archaeal abundance and diversity, with a community dominated by Methanobacterium, suggesting that the absence of GAC may shift the community toward hydrogenotrophic methanogens. Notably, the GAC 10% treatment exhibited the highest archaeal cell counts and the greatest diversity, including substantial populations of Methanoculleus, Methanobacterium, Methanosarcina, Methanomassiliicoccus, Methanosaeta, and RumEn M2. The abundance of Methanoculleus increased significantly, from 0.63 × 10⁹ cells/mL in seed sludge to 2.9 × 10⁹ cells/mL under GAC 10% supplementation. Interestingly, Methanomassiliicoccus was detected exclusively under the GAC 10% condition, whereas it remained below the detection limit in seed sludge, GAC 0%, and GAC 1%. In contrast, Methanobacterium and Methanosaeta showed decreased abundance compared to the seed sludge. These findings suggest that high GAC levels promote a more diverse and active methanogenic community under high OLR conditions.

3.2. Continuous Fermentation

3.2.1. Effect of GAC Addition on Biogas and Methanogenesis at High Organic Loadings in Continuous Fermentation

Figure 6 illustrates the temporal changes in biogas production and methane concentration during the continuous methane fermentation system. The addition of 10% GAC clearly outperformed the 0% GAC condition in terms of both biogas production and methane concentration. Biogas production under 0% GAC remained minimal throughout the experiment, consistently yielding low values. In contrast, 10% GAC addition led to a significant increase in biogas production, with peaks observed between days 40 and 60 (Figure 6A). This suggests that GAC enhances microbial activity, stabilizes the fermentation process, and supports higher biogas output. Methane concentration under the 0% GAC condition was unstable, fluctuating between 40 and 60%, and declined markedly after day 50 (Figure 6B). In contrast, the 10% GAC treatment maintained consistently higher methane concentrations, averaging around 60%, indicating improved methanogenesis efficiency. The presence of GAC likely promoted the growth of microorganisms involved in DIET, thereby supporting a more stable methanogenic process. The enhanced microbial activity and DIET facilitation under the 10% GAC condition appear to be key factors driving this improvement, as GAC provides a favorable surface for microbial interaction and electron exchange.

These findings suggest that incorporating GAC at a 10% ratio is an effective strategy to optimize methane fermentation processes, particularly under high organic loading conditions.

3.2.2. pH Change

Figure 7 illustrates the pH variation during the fermentation process. Under GAC 2.5%, GAC 5%, GAC 7.5%, and GAC 10% treatment, pH increased from 7.3 at the beginning to more than pH 8 at the end of 60 days of fermentation. GAC appears to buffer the fermentation environment, preventing acidification, likely due to its role in facilitating DIET, which improves microbial synergy. However, in the GAC 0%, pH decreased significantly over time from 7.8 to 5.8 at the end of the experimental period, indicating acidification due to the accumulation of VFA.

3.2.3. Organic Decomposition Rate

Figure 8 shows the organic matter degradation rates under each GAC supplementation condition. Due to the high OLR setting, all treatments exhibited a gradual decline in degradation efficiency over the fermentation period. Nevertheless, the GAC 10% consistently maintained a degradation rate above 50%, outperforming the other GAC levels. Although the efficiency declined over time in all groups, a proportional relationship was observed between GAC concentration and degradation rate. These results suggest that GAC facilitates the breakdown of organic matter by promoting the growth of syntrophic bacteria and methanogens, thereby enhancing cooperative microbial activity in anaerobic digestion.

4. Discussion

4.1. Enhanced Methane Production by GAC in Batch Fermentation

Under high OLR conditions, biogas production was significantly reduced, and methane generation was completely inhibited in the absence of GAC, likely due to acidification caused by the accumulation of VFA and a drop in pH. This highlights the difficulty of maintaining stable anaerobic digestion under organic overloading and demonstrates the importance of GAC addition in enhancing microbial activity and mitigating process stress. High GAC concentrations (7.5–10%) markedly improved both biogas yield and methane concentration, with methane production increasing up to 7.6-fold compared to the GAC-free condition. These improvements suggest that GAC facilitates microbial electron transfer and promotes efficient methanogenesis under stressed conditions.

The effect of GAC showed a biphasic trend depending on the OLR. At low to moderate OLRs (1:0.5–1:5), GAC addition led to a decrease in methane concentration, possibly due to the preferential activation of acetolactic methanogens, which produce methane and CO₂ in a 1:1 ratio. This trend is consistent with previous studies [16], and since total biogas volume remained stable across GAC concentrations, methane adsorption by GAC is unlikely to be the primary cause. Instead, it is likely that GAC selectively stimulated acetate-utilizing methanogens over hydrogenotrophic methanogens under these conditions. In contrast, under high OLR conditions (1:10), the addition of GAC significantly influenced both the structure and relative abundance of microbial communities. As shown in Figure A2 (Appendix A), bacteria dominated the communities across all treatments, while archaea accounted for less than 3.5%. However, 10% GAC supplementation slightly increased archaeal abundance and greatly expanded archaeal diversity, suggesting improved methanogenic potential.

At the family level, seed sludge was dominated by Clostridiaceae and Hungateiclostridiaceae, both associated with fermentation and syntrophic metabolism. In the GAC 0% and 1% groups, Caloramatoraceae and Ruminococcaceae became dominant, indicating a shift toward stress-tolerant communities under acidic conditions. Conversely, 10% GAC treatment enriched syntrophic families such as Dysgonomonadaceae and Syntrophomonadaceae, supporting a more diverse and functional microbial network. Archaeal community composition (Figure 5) also reflected the effects of GAC. The GAC 0% group exhibited low diversity and was dominated by Methanobacterium, while the 10% GAC treatment promoted a broader range of methanogens, including Methanomicrobiaceae and Methanomassiliicoccaceae. Notably, Methanomassiliicoccus was exclusively detected in the GAC 10% group, and Methanoculleus increased significantly—from 0.62 × 10⁹ cells/mL in seed sludge to 2.9 × 10⁹ cells/mL—indicating a strong enhancement of hydrogenotrophic methanogenesis likely via DIET. Similar trends were observed at the genus level (Figure 4). The 10% GAC condition supported the highest bacterial abundance and diversity, including syntrophic and VFA-degrading genera such as Clostridium sensu stricto 1, Syntrophomonas, and Proteiniphilum, reaching 5.4 × 10¹⁰ cells/mL. In contrast, the GAC 0% group showed limited diversity, with a dominance of Caproiciproducens and reduced Clostridium sensu stricto 1, indicating weakened microbial interactions and possible acidification.

So far, the microbial shifts discussed have mainly reflected how different GAC dosages affected the communities. However, to more fully discuss the role of GAC, it is also important to consider its material properties. In particular, besides the pore structure and surface area, the surface chemistry of GAC may have contributed to the enhanced methane production observed in this study. Previous studies have demonstrated that oxygen-containing functional groups, such as hydroxyl and carboxyl groups, facilitate microbial adhesion, electron transfer, and biofilm development on carbonaceous materials [17]. In addition to surface area and pore structure, the surface charge of GAC is another important property influencing microbial interactions. Li et al. (2002) reported that untreated coconut shell–based activated carbon has a point of zero charge (pHPZC) of approximately 4.5 [18]. Since the operating pH in our reactors (7.0–8.1) was well above the pHPZC reported in previous studies, the GAC surface was likely negatively charged under the fermentation conditions. Although microbial cell surfaces are generally negatively charged due to ionized functional groups such as phosphates and carboxylates, physical entrapment within pores, adsorption via surface functional groups, and the conductive nature of GAC could still promote microbial attachment and facilitate DIET. This mechanism may partially explain the enhanced methane production and microbial enrichment observed with GAC supplementation. Although such groups were not directly characterized in this study, their potential influence represents an important subject for future investigation. Nevertheless, the primary aim of this work was to comprehensively evaluate the optimal GAC dosage under different OLR conditions and to analyze its effects on microbial community dynamics, which we believe has been sufficiently addressed.

Collectively, these findings demonstrate that high GAC concentrations under high OLR conditions enhance the stability and functionality of microbial communities by promoting syntrophic bacteria and diverse methanogenic archaea, thereby improving methane production efficiency. The marked improvements in biogas yield and methane content at GAC ≥ 7.5% are likely due to enhanced DIET, VFA suppression, and pH stabilization. Future studies should further elucidate the electron transfer mechanisms involved and analyze gas composition (e.g., CO₂/H₂ ratios) to deepen understanding of GAC’s role in anaerobic digestion.

4.2. Effect of Different GAC Additions on Methanogenesis Under High Organic Loadings in Continuous Fermentation

To validate the results obtained from batch experiments under high OLR conditions, continuous fermentation tests were conducted using various concentrations of GAC. The batch tests demonstrated that the addition of 10% GAC significantly improved methane production under high OLR conditions. A similar trend was observed in the continuous fermentation experiments with continuous substrate feeding, confirming the sustained effectiveness of 10% GAC in enhancing methane yield. Recent studies have repeatedly reported that supplementing anaerobic digestion with GAC or biochar promotes methane production and enhances process stability [19,20]. This effect is primarily attributed to the stimulation of DIET, which facilitates methane generation. DIET also has advantages over interspecies hydrogen transfer (IHT), such as reduced energy demand and higher electron transfer efficiency [7]. Furthermore, the large specific surface area and porous structure of GAC provide a favorable environment for the immobilization of syntrophic and methanogenic microbes and facilitate substrate utilization [16].

In continuous anaerobic digestion, acidification caused by the continuous withdrawal of digestate and feeding of substrate is a major limiting factor for methane production. Under low GAC concentrations, the pH dropped to 5.8 and 6.6 for the 0% and 1% GAC conditions, respectively, and methane production was inhibited by acidification. In contrast, high GAC concentrations maintained pH values within 7.3 to 8.2, which matches the optimal range for methanogenesis (pH 7–8) [21,22]. This pH stability indicates that GAC supports the consumption of acidic intermediates generated during hydrolysis and acidogenesis, creating an environment suitable for methane production. The buffering capacity of GAC due to its hydroxyl groups is also considered to contribute to this regulatory function.

Although VFA concentrations were not directly quantified in this study due to equipment constraints, the combined evidence from pH trajectories and microbial community shifts provides indirect but consistent insights into VFA dynamics under continuous operation. The pronounced decline in pH observed under low GAC conditions (0–1%) is indicative of acidification typically associated with the transient accumulation of acidic intermediates, whereas the maintenance of pH within 7.3–8.1 under 10% GAC suggests that such accumulation was effectively mitigated. Microbial community data further support this interpretation. The elevated abundance of Fastidiosipila in the 0% GAC condition is consistent with the formation of protein-derived acids such as acetate and butyrate, while the reduced abundance and activity of Methanosaeta and Methanosarcina at 1% GAC indicate a bottleneck in acetoclastic methanogenesis, which may have limited the conversion of acetogenic products to methane. By contrast, the enrichment of hydrolytic and fermentative taxa such as Ruminofilibacter and Fermentimonas under 10% GAC suggests a more balanced upstream metabolism that, when coupled with DIET facilitated on GAC surfaces, enabled more efficient electron transfer to methanogens and alleviated local acidification. Collectively, these findings imply that GAC supplementation promoted the utilization and downstream conversion of acidic intermediates through DIET enabled syntrophy, thereby stabilizing methanogenesis under high organic loading conditions. Although the overall organic decomposition rate decreased across all treatments, GAC 10% achieved a final decomposition rate of 53%, compared to 27% for GAC 0%. This likely resulted from suppressed VFA accumulation, particularly due to increased abundances of Ruminofilibacter and Fermentimonas. Proteiniphilum is known to utilize nitrogen-containing substrates to produce acetate, and its higher abundance under GAC 10% suggests that nitrogen availability supported hydrolysis and acidogenesis. Additionally, butyrate-oxidizing bacteria like Syntrophomonas, which are highly tolerant to ammonia and VFA overload, likely contributed to the stability of anaerobic digestion [23].

Thus, understanding the mechanisms behind GAC’s effectiveness in continuous systems provides important insights for optimizing methane fermentation. Biofilms formed on GAC surfaces may have rapidly decomposed the continuously supplied organic matter and facilitated electron transfer via DIET. However, this DIET effect is especially prominent under high OLR conditions, indicating the need for a gradual increase in OLR to allow microbial acclimation. In this study, continuous experiments were performed only under a single high OLR condition (1:10) to simulate severe overloading stress. Although stepwise increases in OLR were not tested here, separate trials with different substrates showed trends consistent with the batch experiments, namely that higher GAC dosages enhanced methane production and process stability as OLR increased. These observations indicate that GAC is likely to support process stability even when OLR increases. At the same time, it is valuable to position these findings within the broader context of previous literature. As summarized in Table A1 (Appendix A), more than ten studies have reported that the addition of conductive materials, including GAC, enhances methane yield, methane concentration, and pH stability under various operating conditions. The results of this study are consistent with those trends, further confirming the effectiveness of GAC supplementation under high OLR stress. Overall, future systematic studies should examine how GAC performs under fluctuating OLR conditions. In addition, because the fermenter is sealed during anaerobic operation, durability testing and the development of practical installation methods for GAC remain technical challenges.

5. Conclusions

This study demonstrated that the addition of granular activated carbon (GAC) significantly enhanced the stability and efficiency of methane fermentation under high OLR conditions. In batch fermentation, high-dose GAC (7.5–10%) markedly improved performance under high OLR; with 10% GAC, methane concentration increased up to fivefold relative to the GAC-free control and biogas production substantially increased, whereas under low–moderate OLRs (1:0.5–1:5), higher GAC dosages led to a gradual decrease in methane concentration, likely reflecting selective stimulation of acetoclastic methanogens (e.g., Methanosaeta, Methanosarcina) that produce CH₄ and CO₂ at a 1:1 ratio. Microbial community analysis in batch further showed enrichment of hydrogenotrophic methanogens (e.g., Methanoculleus; 0.62 × 10⁹ → 2.94 × 10⁹ cells/mL), broader archaeal diversity, and higher abundances of syntrophic and VFA-degrading bacteria, including Clostridium sensu stricto 1, Syntrophomonas, and Proteiniphilum, which together increased total bacterial counts. These findings are consistent with GAC-mediated DIET and maintenance of pH within the optimal range (7.3–8.1). In the continuous system operated at high OLR (1:10), 10% GAC likewise sustained performance by preventing acidification (pH 5.8–6.6 in low-GAC controls) that would otherwise inhibit methanogenesis, confirming robustness under continuous feeding. Therefore, this study newly demonstrates that the effects of GAC are load-dependent, shifting from inhibitory at low OLRs to beneficial at high OLRs. By quantifying microbial communities in absolute terms, it further links community dynamics directly with process performance, thereby clarifying the mechanistic basis of GAC-mediated stabilization. These insights highlight the potential of optimizing GAC supplementation, particularly at 7.5–10% under high OLR, as a practical strategy for enhancing methane yield and stability in large-scale anaerobic digesters. Nevertheless, a limitation of this study is the absence of a carbon mass balance, and future work should incorporate comprehensive carbon accounting to further validate the robustness of GAC-assisted anaerobic digestion.