Enhanced thermophilic high-solids anaerobic digestion of organic fraction of municipal solid waste with spatial separation from conductive materials in a single reactor volume.

Despite benefits such as lower water and working volume requirements, thermophilic high solids anaerobic digestion (THSAD) often fails due to the rapid build-up of volatile fatty acids (VFAs) and the associated drop in pH. Use of conductive materials (CM) can promote THSAD through stimulation of direct interspecies electron transfer (DIET), while the need for their constant dosing due to poor separation from effluent impairs economic feasibility. This study used an approach of spatially separating magnetite and granular activated carbon (GAC) from the organic fraction of municipal solid waste (OFMSW) in a single reactor for THSAD. GAC and magnetite addition could both mitigate the severe inhibition of methanogenesis after VFAs build-up to ∼28-30 g/L, while negligible methane production was observed in the control group. The highest methane yield (286 mL CH4/g volatile solids (VS)) was achieved in magnetite-added reactors, while the highest maximum CH4 production rates (26.38 mL CH4/g VS/d) and lowest lag-phase (2.83 days) were obtained in GAC-added reactors. The enrichment of GAC and magnetite biofilms with various syntrophic and potentially electroactive microbial groups (Ruminiclostridium 1, Clostridia MBA03, Defluviitoga, Lentimicrobiaceae) in different relative abundances indicates the existence of specific preferences of these groups for the nature of CM. According to predicted basic metabolic functions, CM can enhance cellular processes and signals, lipid transport and metabolism, and methane metabolism, resulting in improved methane production. Rearrangement of metabolic pathways, formation of pili-like structures, enrichment of biofilms with electroactive groups and a significant improvement in THSAD performance was attributed to the enhancement of the DIET pathway. Promising results obtained in this work due to the spatial separation of the bulk OFMSW and CM can be useful for modeling larger-scale THSAD systems with better recovery of CM and cost-effectiveness.

Comparison of metabolic kinetics during high and low solids anaerobic digestion of fecal sludge.

High solids anaerobic digestion (HS-AD) is an attractive energy-producing technology; however, high total solids (TS) content may inhibit methanogens due to high volatile fatty acid (VFA) and total ammonia nitrogen concentrations. The objective of this paper is to quantify rate-limiting metabolic kinetic parameters to determine the influence of TS content during anaerobic digestion of fecal sludge. Two TS content: 11% and 17% microcosms were analyzed. Good performance was observed in both systems, with volatile solid (VS) removal greater than 50%, CH4 yield between 0.44 and 0.56 m3 CH4 /g VS added and cumulative CH4 production between 1.78 and 2.03 m3 CH4 /m3 digester-day. At 11% TS VFA consumption and VS removal had a positive correlation to CH4 production while the 17% TS microcosm had a negative correlation with both. This is the first study to determine the kinetic parameters for hydrolysis, VFA consumption, and methanogenesis during digestion of fecal sludge. These kinetic parameters are necessary in the design and operation of anaerobic digestion systems treating fecal sludge.

Semi-continuous mono-digestion of OFMSW and Co-digestion of OFMSW with beech sawdust: Assessment of the maximum operational total solid content.

In this study, mono-digestion of the organic fraction of municipal solid waste (OFMSW) and co-digestion of OFMSW with beech sawdust, simulating green waste, were used to investigate the maximum operational total solid (TS) content in semi-continuous high-solids anaerobic digestion (HS-AD). To alleviate substrate overloading in HS-AD, the effluent mass was relatively reduced compared to the influent mass, extending the mass retention time. To this aim, the reactor mass was daily evaluated, permitting to assess the reactor content removal by biogas production. During mono-digestion of OFMSW, the NH3 inhibition and the rapid TS removal prevented to maintain HS-AD conditions (i.e. TS ≥ 10%), without exacerbating the risk of reactor acidification. In contrast, the inclusion of sawdust in OFMSW permitted to operate HS-AD up to 30% TS, before acidification occurred. Therefore, including a lignocellulosic substrate in OFMSW can prevent acidification and stabilize HS-AD at very high TS contents (i.e. 20-30%).

Modelling non-ideal bio-physical-chemical effects on high-solids anaerobic digestion of the organic fraction of municipal solid waste.

This study evaluates the main effects of including 'non-ideal' bio-physical-chemical corrections in high-solids anaerobic digestion (HS-AD) of the organic fraction of municipal solid waste (OFMSW), at total solid (TS) between 10 and 40%. As a novel approach, a simple 'non-ideal' module, accounting for the effects of ionic strength (I) on the main acid-base equilibriums, was coupled to a HS-AD model, to jointly evaluate the effects of 'non-ideality' and the TS content dynamics on the HS-AD bio-physical-chemistry. 'Non-ideality' influenced the pH, concentration of inhibitors (i.e. NH3), and liquid-gas transfer (i.e. CO2), particularly at higher TS (i.e. ≥ 20%). Meanwhile, fitting the experimental data for batch assays at 15% TS showed that HS-AD of OFMSW might be operated at I ≥ 0.5 M. Therefore, all HS-AD simulations should account for 'non-ideal' corrections, when assessing the main inhibitory mechanisms (i.e. NH3 buildup and acidification) potentially occurring in HS-AD of OFMSW.

High-solids anaerobic mono-digestion of riverbank grass under thermophilic conditions.

The purpose of this study was to investigate the potential of high-solids anaerobic mono-digestion of riverbank grass under thermophilic conditions, focusing on the effects of the strength and the amount of inoculum. Ensiled grass was inoculated with three different inocula; inoculum from liquid anaerobic digester (LI), inoculum from dry anaerobic digester (DI), and mixture of LI and DI (MI), at feedstock-to-inoculum ratio (FIR) of 1, 2 and 4. The ensiling process of riverbank grass reduced moisture content (p>0.05), while the hemicellulose content was significantly increased from 30.88% to 35.15% (p<0.05), on dry matter basis. The highest methane production was at an FIR of 2 with MI (167L/kg VSadded), which was significantly higher (p<0.05) than with DI, but not significant compared to LI (p>0.05). At an FIR of 4, digesters inoculated with LI and DI failed to produce methane, whereas 135LCH4/kg VSadded was obtained with MI. The kinetic studies showed that at an FIR of 1 with LI and MI, the inoculum had less of effects on the hydrolysis rate constant (0.269day-1 and 0.245day-1) and methane production (135 versus 149L/kg VSadded); rather, it affected the lag phase. In a thermophilic HS-AD of riverbank grass, the mixture of inoculum with low and high total solids content (TS) helps increase the TS of inoculum and digestion process. An FIR of 2 was deducted to be the limit for a better startup time and higher volumetric productivity of methane.

From grass to gas and beyond: Anaerobic digestion as a key enabling technology for a residual grass biorefinery.

Roadside grass clippings hold potential as a sustainable source of bioenergy as they do not compete with crops for land use, and are only partially utilized for low-value applications. In this study, we proposed using roadside grass as a sole feedstock for anaerobic digestion (AD) in three different settings, and assessed the potential of producing biomaterials and fertilizers from grass-based digestate. Wet continuous digestion at pilot scale and dry batch digestion at pilot and large scales resulted in biogas yields up to 700 Nm3.t-1 DOM with a methane content of 49-55 %. Despite promising results, wet AD had operational problems such as clogging and poor mixing; once upscaled, the dry digestion initially also presented an operational problem with acidification, which was overcome by the second trial. Digested grass fibers from the pilot dry AD were processed into biomaterials and performed similarly or better than the undigested fibers, while around 20 % performance reduction was observed when compared to reference wood fibers. A mass balance indicated reduced fiber recovery when higher biogas production was obtained. The liquid fraction from the pilot dry AD was characterized for its nutrient content and used as a biofertilizer in another study. In contrast, the leachate collected from the large-scale dry AD had a low nitrogen content and high chloride content that could hinder its further use. Finally, a regional market analysis was conducted showing that the biocomposites produced with the available grass fibers could substitute at least half of the current European market based on our results.

Use of nanobubble water bioaugmented anaerobically digested sludge for high-efficacy energy production from high-solids anaerobic digestion of corn straw.

An increasing attention has been paid to the secure and sustainable management of agricultural wastes, especially lignocellulosic biomass. Nanobubble water (NBW) contains 106-108 bubbles/mL with diameter <1000 nm. Although previous studies have examined the enhancement effects of NBW on methane production from organic solid wastes, the NBW-based anaerobic digestion (AD) system is still restrained from practical application due to the large increase in AD reactor volume, generation of wastewater, and increase in energy consumption as well. In this study, NBW bioaugmentation of anaerobically digested sludge for the first time was performed for high-solids AD of corn straw. Results show that cellulase, xylanases and lignin peroxidase activities were increased by 2-55% during the NBW bioaugmentation process. Significant enrichment of hydrolytic/acidogenic bacteria and methanogenic archaea were noticed in the NBW bioaugmented sludge. This study clearly demonstrated 47% increase in methane production from high-solids AD of corn straw when O2-NBW bioaugmented sludge was applied, achieving a net energy gain of 5138 MJ/t-volatile solids of corn straw with an energy recovery of 34%. The NBW-based high-solids AD system can provide a novel and sustainable management solution for renewable energy production from agricultural wastes, targeting the reduction of environmental pollution and energy crisis.

Mass Transfer Enhancement in High-Solids Anaerobic Digestion of Organic Fraction of Municipal Solid Wastes: A Review.

The increasing global population and urbanization have led to a pressing need for effective solutions to manage the organic fraction of municipal solid waste (OFMSW). High-solids anaerobic digestion (HS-AD) has garnered attention as a sustainable technology that offers reduced water demand and energy consumption, and an increased biogas production rate. However, challenges such as rheology complexities and slow mass transfer hinder its widespread application. To address these limitations, this review emphasizes the importance of process optimization and the mass transfer enhancement of HS-AD, and summarizes various strategies for enhancing mass transfer in the field of HS-AD for the OFMSW, including substrate pretreatments, mixing strategies, and the addition of biochar. Additionally, the incorporation of innovative reactor designs, substrate pretreatment, the use of advanced modeling and simulation techniques, and the novel conductive materials need to be investigated in future studies to promote a better coupling between mass transfer and methane production. This review provides support and guidance to promote HS-AD technology as a more viable solution for sustainable waste management and resource recovery.

Carbon cloth amendment for boosting high-solids anaerobic digestion with percolate recirculation: Spatial patterns of microbial communities.

The addition of conductive materials in anaerobic digestion (AD) is a promising method for boosting biomethane recovery from organic waste. However, conductive additives have rarely been investigated for the high-solids anaerobic digestion (HSAD). Here, the impact of adding carbon cloth in the solid phase of an HSAD system with percolate recirculation was investigated. Furthermore, spatial patterns of microbial communities in suspended biomass, percolate, and carbon cloth attached biofilm were assessed. Carbon cloth increased biomethane yield from source-separated organics (SSO) by 20% more than the unamended control by shortening the lag phase (by 15%) and marginally improving the methanogenesis rate constant (by ∼8%) under a batch operation for 50 days. Microbial community analysis demonstrated higher relative abundances of the archaeal population in the carbon cloth amended reactor than in unamended control (12%-21% vs. 5%-15%). Compared to percolate and suspension, carbon cloth attached microbial community showed higher enrichment of known electroactive Pseudomonas species along with Methanosarcina and Methanobacterium species, indicating the possibility of DIET-based syntrophy among these species.

Effects of organic loading rates on high-solids anaerobic digestion of food waste in horizontal flow reactor: Methane production, stability and mechanism.

To maximize the methane production efficiency of high-solids anaerobic digestion (HSAD) of food waste (FW), a horizontal flow reactor was operated under mesophilic, semi-continuous condition at organic loading rates (OLRs) ranging from 1.00 to 13.80 kg-VS/(m3 d). The gas production, substrate transformation, and microbial community characteristics of the horizontal flow HSAD reactor were evaluated. The results indicated that the methane yield (0.173-0.516 L/(g d)) fluctuated with the increasing OLR, volumetric methane production rate (0.25-5.69 L/(L d)) increased with increasing OLR, and the volatile solids (VS) reduction rate ranged between 83.30% and 93.05%. The relationship of biogas or methane production with OLR and HRT in the horizontal flow HSAD reactor were characterized with an empirical equation. The concentrations of soluble COD and volatile fatty acid exhibited significant fluctuations, and free ammonia-nitrogen peaked at the OLR of 13.80 kg-VS/(m3 d). Microbial community analysis revealed that the methanogenic metabolic pathway changes along the propelling direction of the horizontal flow HSAD reactor from CH3COOH and H2/CO2 pathways to CH3COOH, H2/CO2, and H2/methyl co-dominant pathways. These results provide theoretical support for stable methane production from FW and deeper insight into horizontal flow HSAD for FW treatment.

Insights into high-solids anaerobic digestion of food waste enhanced by activated carbon via promoting direct interspecies electron transfer.

High-solids anaerobic digestion (HS-AD) of food waste frequently confronted the acidification and failure under high organic loading rates (OLRs). Results indicated powdered activated carbon (PAC) addition significantly enhanced methane production and process stability than granular activated carbon, and columnar activated carbon at higher OLRs via accelerating the propionate consumption. Potential direct interspecies electron transfer (DIET) partners, including various syntrophic oxidation bacteria and methanogens, were enriched with the activated carbon (AC) addition. Furthermore, DIET contribution to methane production was 35% by PAC, predicated by the modified Anaerobic Digestion Model No.1 (ADM1). This study deeply elucidated the DIET mechanism and offered the potential foundations for the selection and applications of AC-based materials in HS-AD of food waste.

High-solids anaerobic digestion of organic fraction of municipal solid waste: Effects of feedstock to inoculum ratio and percolate recirculation time.

The effects of feedstock to inoculum (F:I) ratio and percolate recirculation time (PRT) were studied for the high-solids anaerobic digestion (HSAD) of the organic fraction of municipal solid waste (OFMSW). Six mesophilic HSAD systems were operated at different F:I ratios (1 to 3 kg VS/kg VS; PRT = 2.5 h/d) and PRTs (1.5 to 3.5 h/d; F:I = 2 kg VS/kg VS). The F:I ratio of 1 provided up to 86% of the theoretical methane potential of OFMSW. In contrast, F:I ratio of 3 provided only 34% methane recovery due to volatile fatty acids (VFAs) accumulation and pH drop. Despite F:I ratio of 2 could provide 70% methane recovery, it could enable almost 45% higher organics processing capacity (VS basis) and lower solids washout during percolate recirculation, as compared to the F:I ratio of 1. However, different examined PRTs showed marginal impacts on methane yields with comparable changes in profiles of percolate characteristics.

The implications of facility design and enabling policies on the economics of dry anaerobic digestion.

Diverting organic waste from landfills provides significant emissions benefits in addition to preserving landfill capacity and creating value-added energy and compost products. Dry anaerobic digestion (AD) is particularly attractive for managing the organic fraction of municipal solid waste because of its high-solids composition and minimal water requirements. This study utilizes empirical data from operational facilities in California in order to explore the key drivers of dry AD facility profitability, impacts of market forces, and the efficacy of policy incentives. The study finds that dry AD facilities can achieve meaningful economies of scale with organic waste intake amounts larger than 75,000 tonnes per year. Materials handling costs, including the disposal of inorganic residuals from contaminated waste streams and post-digester mass (digestate) management, are both the largest and the most uncertain facility costs. Facilities that utilize the biogas for vehicle fueling and earn associated fuel credits collect revenues that are 4-6x greater than those of facilities generating and selling electricity and 10-12x greater than facilities selling natural gas at market prices. The results suggest important facility design elements and enabling policies to support an increased scale of organic waste handling infrastructure.

Fate of disposable face masks in high-solids anaerobic digestion: Experimental observations and review of potential environmental implications.

Face masks became a part of our daily life amid the global COVID-19 (SARS-CoV-2) pandemic. Most of the face masks are made for single-use and primarily disposed of in garbage bins with other non-recyclable wastes. To date, little is known about how disposable face masks in municipal solid waste (MSW) would interfere with high-solids anaerobic digestion (HSAD) in waste management facilities. Here, we first report preliminary results from a lab experiment conducted with the organic fraction of municipal solid waste (OFMSW) amended with used disposable face masks. The lab-scale HSAD systems were operated with percolate recirculation comparable to commercial HSAD systems typically used for full-scale processing of OFMSW. The results suggested that the presence of face masks in OFMSW could negatively affect methane productivity and kinetics. In the digesters amended with face masks, total cumulative methane production decreased by up to 18%, along with a 12-29% decrease in maximum methane production rates than the control digester (without face masks). Moreover, lag phases increased by 7-14%. The results also suggested that the type of polymeric materials used in face masks would be more critical than their total number/loading in the digester, which warrants further investigation. The visual inspection of digestate showed that the face masks were mostly undegraded after 40 days of operation. Much remains unknown about how the undegraded face masks will affect the digestate management practices, such as composting, land application, and landfilling. However, the review of existing literature suggested that they can be a potential source of plastic and microplastic pollution and amplify transmission of antibiotic resistance genes to the ecosystem. In summary, this study underscores the importance of developing safe and reliable disposal guidelines and management plans for single-use face masks.

Transitions of microbial communities in the solid and liquid phases during high-solids anaerobic digestion of organic fraction of municipal solid waste.

This study presents a microbiological diagnosis of a mesophilic high-solids anaerobic digestion (HSAD) system with percolate recirculation. The results demonstrated a significant decrease in microbial diversity in both the solid digestate and the liquid percolate. Also, the digestate from the top and middle sections of the digester had similar diversity, whereas the digestate from the bottom of the tank had a slightly lower diversity. These results suggest that despite percolate recirculation, substrate gradients might have developed across the system. Archaeal communities showed shifts towards known hydrogenotrophic and ammonia-tolerant methanogens (genera Methanocelleus, Methanolinea, Methanosarcina, vadin CA11, etc.), which was a consequence of decreased volatile fatty acids and increased ammonia-nitrogen levels over time. Compared to initial solid and liquid inoculum, the relative abundances of some bacteria (phyla Proteobacteria and Firmicutes) and archaea of the genus Methanosarcina changed between two phases in the opposite direction, indicating a shift of microbes between two phases.

Processing High-Solid and High-Ammonia Rich Manures in a Two-Stage (Liquid-Solid) Low-Temperature Anaerobic Digestion Process: Start-Up and Operating Strategies.

Globally, livestock and poultry production leads to total emissions of 7.1 Gigatonnes of CO2-equiv per year, representing 14.5% of all anthropogenic greenhouse gas emissions. Anaerobic digestion (AD) is one of the sustainable approaches to generate methane (CH4) from manure, but the risk of ammonia inhibition in high-solids AD can limit the process. Our objective was to develop a two-stage (liquid-solid) AD biotechnology, treating chicken (CM) + dairy cow (DM) manure mixtures at 20 °C using adapted liquid inoculum that could make livestock farming more sustainable. The effect of organic loading rates (OLR), cycle length, and the mode of operation (particularly liquid inoculum recirculation-percolation mode) was evaluated in a two-stage closed-loop system. After the inoculum adaptation phase, aforementioned two-stage batch-mode AD operation was conducted for the co-digestion of CM + DM (Total Solids (TS): 48-51% and Total Kjeldahl Nitrogen (TKN): 13.5 g/L) at an OLR of 3.7-4.7 gVS/L.d. Two cycles of different cycle lengths (112-d and 78-d for cycles 1 and 2, respectively) were operated with a CM:DM mix ratio of 1:1 (w/w) based on a fresh weight basis. Specific methane yield (SMY) of 0.35 ± 0.11 L CH4g/VSfed was obtained with a CH4 concentration of above 60% for both the cycles and Soluble Chemical Oxygen Demand (CODs) and volatile solid (VS) reductions up to 85% and 60%, respectively. For a comparison purpose, a similar batch-mode operation was conducted for mono-digestion of CM (TS: 65-73% and TKN: 21-23 g/L), which resulted in a SMY of 0.52 ± 0.13 L CH4g/VSfed. In terms of efficiency towards methane-rich biogas production and ammonia inhibitions, CM + DM co-digestion showed comparatively better quality methane and generated lower free ammonia than CM mono-digestion. Further study is underway to optimize the operating parameters for the co-digestion process and to overcome inhibitions and high energy demand, especially for cold countries.

Influence of cationic polyacrilamide flocculant on high-solids' anaerobic digestion of sewage sludge under thermophilic conditions.

Treatment of sewage sludge (SS) by biodegradable polyacrylamide-based flocculants (PAM) is considered to be an effective way to increase total solids' (TS) content prior to anaerobic digestion (AD). However, data on how PAM addition influences the efficiency of AD process are quite contradictory; moreover, no data are available for thermophilic AD (TAD). This study showed that at an optimal inoculum-to-substrate ratio (ISR, 55/45), PAM addition resulted in some decrease in initial methane production during the TAD of SS due to the formation of large flocs (up to 2-3 mm in diameter), which deteriorated the mass transfer. However, at non-optimal ISR (40/60), which led to the destabilization of TAD, PAM addition (40 mg/g TS) could restore the methanogenesis despite the inhibiting accumulation of volatile fatty acids (14-15 g/l) and pH drop (5.5). The observed positive effect of PAM-forced flocculation proposes a new interesting alternative for recovery of 'soured' reactors.

Biogas production from high solids anaerobic co-digestion of food waste, yard waste and waste activated sludge.

High-solids anaerobic co-digestion (HS-AcD) has the potential to recover energy and reduce environmental impacts of the organic fraction of municipal solid waste and waste activated sludge. We investigated the impact of substrate to inoculum (S/I) ratios, alkalinity sources (sodium bicarbonate and oyster shells), co-substrate mixing ratios and inoculum acclimation on HS-AcD of food waste, yard waste, and waste activated sludge using batch studies. Long-term HS-AcD performance was evaluated under the optimal conditions through a semi-continuous biodigester study with leachate recirculation. The digester with S/I = 1 using a mixture of crushed oyster shells and sodium bicarbonate as alkalinity sources had the highest methane yields (183 mL CH4/g VS). Addition of waste activated sludge to food waste and yard waste alleviated acidification (pH 6.86 ±â€¯0.12) during the start-up period, which and improved digester stability. Mixtures with FW/YW/WAS = 0.8:1.7:0.5 had higher methane yields (134 ±â€¯15 mL CH4/gVS) than mixtures with FW/YW/WAS = 1:1:1, but required a longer time (10 days) for self-recovery from volatile fatty acid inhibition. The use of an acclimated inoculum eliminated the lag time during start-up and produced 38% higher methane yield. In the semi-continuous biodigester, an average volatile solids reduction of 38% and methane yield of 186 mL/gVS was achieved. Improved performance in the semi-continuous biodigester compared with batch reactors was likely due to leachate recirculation, which can improve mass transfer of substrates to the microbial community. Digestate produced from HS-AcD of waste organics had a 1.7-2.3 fold higher nitrogen, similar phosphorous and 0.2-0.3 fold lower potassium content than commercially available bioorganic fertilizer.

Assessing practical identifiability during calibration and cross-validation of a structured model for high-solids anaerobic digestion.

High-solids anaerobic digestion (HS-AD) of the organic fraction of municipal solid waste (OFMSW) is operated at a total solid (TS) content ≥ 10% to enhance the waste treatment economy, though it might be associated to free ammonia (NH3) inhibition. This study aimed to calibrate and cross-validate a HS-AD model for homogenized reactors in order to assess the effects of high NH3 levels in HS-AD of OFMSW, but also to evaluate the suitability of the reversible non-competitive inhibition function to reproduce the effect of NH3 on the main acetogenic and methanogenic populations. The practical identifiability of structural/biochemical parameters (i.e. 35) and initial conditions (i.e. 32) was evaluated using batch experiments at different TS and/or inoculum-to-substrate ratios. Variance-based global sensitivity analysis and approximate Bayesian computation were used for parameter optimization. The experimental data in this study permitted to estimate up to 8 biochemical parameters, whereas the rest of parameters and biomass contents were poorly identifiable. The study also showed the relatively high levels of NH3 (i.e. up to 2.3 g N/L) and ionic strength (i.e. up to 0.9 M) when increasing TS in HS-AD of OFMSW. However, the NH3 non-competitive function was unable to capture the acetogenic/methanogenic inhibition. Therefore, the calibration emphasized the need for target-oriented experimental data to enhance the practical identifiability and the predictive capabilities of structured HS-AD models, but also the need for further testing the NH3 inhibition function used in these simulations.

Long-term high-solids anaerobic digestion of food waste: Effects of ammonia on process performance and microbial community.

A long-term high solids anaerobic digestion of food waste was conducted to identify microbial mechanisms of ammonia inhibition during digestion and to clarify correlations between ammonia accumulation, microbial community dynamics (diversity, composition, and interactions), and process stability. Results show that the effects of ammonia on process performance and microbial community were indirectly caused by volatile fatty acid accumulation. Excess free ammonia blocked acetate metabolism, leading to process instability. Accumulated acetate caused feedback inhibition at the acetogenesis stage, which resulted in considerable accumulation of propionate, valerate, and other long-chain fatty acids. This high concentration of volatile fatty acids reduced the abundance of syntrophic acetogenic bacteria and allowed hydrolytic fermentative bacteria to dominate. The normally interactive and orderly metabolic network was broken, which further exacerbated the process instability. These results improve the understanding of microbial mechanisms which contribute to process instability and provide guidance for the microbial management of anaerobic digesters.