Construction of a Synthetic Microbial Community for Enzymatic Pretreatment of Wheat Straw for Biogas Production via Anaerobic Digestion.

Biological pretreatment is a viable method for enhancing biogas production from straw crops, with the improvement in lignocellulose degradation efficiency being a crucial factor in this process. Herein, a metagenomic approach was used to screen core microorganisms (Bacillus subtilis, Acinetobacter johnsonii, Trichoderma viride, and Aspergillus niger) possessing lignocellulose-degrading abilities among samples from three environments: pile retting wheat straw (WS), WS returned to soil, and forest soil. Subsequently, synthetic microbial communities were constructed for fermentation-enzyme production. The crude enzyme solution obtained was used to pretreat WS and was compared with two commercial enzymes. The synthetic microbial community enzyme-producing pretreatment (SMCEP) yielded the highest enzymatic digestion efficacy for WS, yielding cellulose, hemicellulose, and lignin degradation rates of 39.85, 36.99, and 19.21%, respectively. Furthermore, pretreatment of WS with an enzyme solution, followed by anaerobic digestion achieved satisfactory results. SMCEP displayed the highest cumulative biogas production at 801.16 mL/g TS, which was 38.79% higher than that observed for WS, 22.15% higher than that of solid-state commercial enzyme pretreatment and 25.41% higher than that of liquid commercial enzyme pretreatment. These results indicate that enzyme-pretreated WS can significantly enhance biogas production. This study represents a solution to the environmental burden and energy use of crop residues.

Solid-state fermentation of corn straw using synthetic microbiome to produce fermented feed: The feed quality and conversion mechanism.

Straw is a typical biomass resource which can be converted into high nutritional value feed via microbial fermentation. The degradation and conversion of straw using a synthetic microbial community (SMC-8) was functionally investigated to characterise its nitrogen conversion and carbon metabolism. Four species of bacteria were found to utilise >20 % of the inorganic nitrogen within 15 h, and the ratio of the diameter of fungal transparent circles (D) to the diameter of the colony (d) of the four fungal species was >1. Solid-state fermentation of corn straw increased the total amino acid (AA) content by 41.69 %. The absolute digestibility of fermented corn straw dry weight (DW) and true protein was 34.34 % and 45.29 %, respectively. Comprehensive analysis of functional proteins revealed that Aspergillus niger, Trichoderma viride, Cladosporium cladosporioides, Bacillus subtilis and Acinetobacter johnsonii produce a complex enzyme system during corn straw fermentation, which plays a key role in the degradation of lignocellulose. This study provided a new insight in utilizing corn straw.

Pretreatment of Luzhou distiller's grains for feed protein production using crude enzymes produced by a synthetic microbial consortium.

Chinese distillers' grains (CDGs) have low fermentation efficiency due to the presence of lignocellulosic components, such as rice husk. In this study, a microbial consortium synthesized was used based on the "functional complementarity" principle to produce lignocellulolytic crude enzyme. The crude enzyme was used to hydrolyze CDGs. After enzymatic hydrolysis, lignocellulose was damaged to varying degrees and the crystallinity decreased. Subsequently, the feed protein was produced using yeast through two pathways. The results showed that the crude enzyme produced by the microbial consortium (comprising Trichoderma reesei, Aspergillus niger, and Penicillium) exhibited excellent enzymatic efficiency, yielding 27.88%, 19.64%, and 10.88% of reducing sugar, cellulose, and hemicellulose. The true protein content of CDGs increased by 53.49% and 48.35% through the first and second pathways, respectively. Notably, the second pathway demonstrated higher economic benefits to produce feed protein. This study provides a pathway for high-quality utilization of CDGs.

Construction of a synthetic microbial community based on multiomics linkage technology and analysis of the mechanism of lignocellulose degradation.

The efficient degradation of lignocellulose is a bottleneck for its integrated utilization. This research performed species analysis and made functional predictions in various ecosystems using multiomics coupling to construct a core synthetic microbial community with efficient lignocellulose degradation function. The synthetic microbial community was employed to degrade corn straw via solid-state fermentation. The degradation mechanisms were resolved using proteomics. The optimum culture conditions included 10% inoculum level (w/v), 4% nitrogen source ratio and a fermentation time of 23 d. Under these conditions, the degradation rates of cellulose, hemicellulose, and lignin were 34.91%, 45.94%, and 23.34%, respectively. Proteomic analysis revealed that lignin 1,4-ß-xylanase, ß-xylosidase and endo-1,4-ß-xylanase were closely related to lignocellulose degradation. The metabolic pathways involved in lignocellulose degradation and the functional roles of eight strains were obtained. The synthesis of a microbial community via multiomics linkage technology can effectively decompose lignocellulose, which is useful for their further utilization.

Effect of aeration/micro-aeration on lignocellulosic decomposition, maturity and seedling phytotoxicity during full-scale biogas residues composting.

With the accelerated construction of biogas plants, the amount of biogas residues are expanding. Composting has been widely implemented to deal with biogas residues. Aeration regulation is the main factor affecting the post-composting treatment of biogas residues as high-quality fertilizer or soil amendment. Therefore, this study aimed to investigate the impact of different aeration regulations on full-scale biogas residues compost maturity by controlling oxygen concentration under micro-aeration and aeration conditions. Results showed that micro-aerobic extended the thermophilic stage of 17 days at above 55 ℃ and facilitated the mineralization process of organic nitrogen into nitrate nitrogen to retain higher N nutrition levels compared to aerobic treatment. For biogas residues with high moisture, aeration should be regulated at different full-scale composting stages. Total organic carbon (TOC), NH4+-N, NO3--N, total potassium (TK), total phosphorus (TP) and the germination index (GI) could be used to evaluate stabilization, fertilizer efficiency and phytotoxicity of compost with frequent monitoring times. However, seedling growth trials were still necessary in full-scale composting plants when changing of composting process or biogas residues feedstock.

Microbial insights towards understanding the role of hydrochar in enhancing phenol degradation in anaerobic digestion.

Anaerobic digestion (AD) of wastewater is the most promising bioprocess for organic conversion, however, phenol is toxic and resistant to anaerobic degradation. The current study compared the effect of hydrochar and granular activated carbon (GAC) on AD of phenol at four concentrations (100 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L). Results demonstrated that hydrochar significantly improved the methane production rate and reduced the lag phase at all concentrations of phenol. The methane production rate was improved by about 50% at both 100 mg/L and 250 mg/L phenol, while it was raised by >160% at 500 mg/L and 1000 mg/L phenol by hydrochar. The GAC only increased the methane production rate at 500 mg/L and 1000 mg/L due to high adsorption capacity. Further, the adsorption of phenol by hydrochar had no apparent impact on the methane production rate, even though certain amounts of phenol were adsorbed. At 500 mg/L, the amount of methane produced significantly increased, so 16S rRNA transcripts sequencing and metabolomic analysis were conducted. 16S rRNA transcripts sequencing analysis indicated that hydrochar resulted in the enrichment of syntrophic bacteria (e.g., Syntrophorhabdus & Syntrophobacter) and Methanosaeta, which might be related with direct interspecies electron transfer. Further, it was noticed that the growth of Methanobacterium was repressed at 500 mg/L phenol, while hydrochar promoted its growth. Phenol was degraded into L-tyrosine and then followed the benzoate degradation pathway for methane production as revealed by metabolomic analysis. In addition, metabolomic analysis also revealed that hydrochar promoted the degradation of all metabolites and enhanced the phenol degradation into methane.

An innovative way to treat cash crop wastes: The fermentation characteristics and functional microbial community using different substrates to produce Agricultural Jiaosu.

With the increase of global demand for cash crops, a large of cash crop waste was produced and caused severe environmental issues. To produce Agricultural Jiaosu (AJ) using these wastes is a sustainable waste disposal method. However, the fermentation mechanism, metabolites, and microbial characteristics of AJ fermented with different substrates remain unclear. In this study, the effects of different substrates (fruit and vegetable waste and Chinese herbal medicine waste) on the fermentation characteristics of AJ, including metabolites and microbial community properties, were investigated. The results revealed that AJ fermentation was a process of converting organic matter into organic acids and other metabolites, mainly including hydrolysis, acidogenesis, and maturation stages. At the genus level, Lactobacillus, Acetobacter, Hydrogenibacillus, Halomonas, and Prevotella_1 were the dominant bacteria in the fermentation system. The bacterial diversity of composite substrate AJ was higher than that of single substrate AJ. The organic acids and secondary metabolites concentration and the composition of key microorganisms depended on the substrate type. Furthermore, AJ's potential functional genes were mainly concentrated in cofactors and vitamin, carbohydrate, and amino acid metabolism. The findings of this study indicated that AJ is an innovative eco-friendly technology that can convert cash crop wastes into sustainable eco-products, and that its characteristics depend on the substrate type. Therefore, the substrate used to produce AJ should be carefully selected according to the application field.

Biogas Residues Improved Microbial Diversity and Disease Suppression Function under Extent Indigenous Soil Microbial Biomass.

Indigenous soil microbial biomass (ISMB) plays a key role in maintaining essential functions and biodiversity of soil health. One of the critical unknowns is how the indigenous microorganisms respond to different fertilizers which is directly related to agricultural production. Therefore, we used Mi-Seq sequencing and network analyses to compare the response of ISMB to biogas residue and chemical fertilizers. The results showed that crop production was profoundly influenced by levels of ISMB present and is further dependent on the strategy of fertilizer application. Higher ISMB primarily manifests through retention of richer microbial abundance, a balanced community structure, and tightened co-occurrence within a certain proportion of Nitrospirae, Rhizophlyctidaceae, and Gemmatimonadetes. Compared to chemical fertilizer, biogas residue resulted in higher production with more strongly linked nodes such as Actinobacteria, Chloroflexi and Gemmatimonadetes. Under the same level of ISMB, the microbial diversity was richer and co-occurrence was tighter when biogas residues were applied compared with chemical fertilizer. In addition, the higher level of ISMB with biogas residue applied had a lower abundance of potential fungal pathogens in both bulk and rhizosphere soil compared with chemical fertilizer. This study provides critical data to understand the influence of ISMB and biogas residue on soil ecological system.

Trace elements' deficiency in energy production through methanogenesis process: Focus on the characteristics of organic solid wastes.

Excessive or insufficient supplementation of trace elements (TEs) limits the progression of anaerobic digestion. The main reason for this is the lack of sufficient understanding of digestion substrate characteristics, which significantly affects the demand for TEs. In this review, the relationship between TEs requirements and substrate characteristics is discussed. We mainly focus on three aspects. 1) The basis for TE optimization and existing problems: The optimization of TEs often based on the total solids (TS) or volatile solids (VS) of substrates, does not fully consider substrate characteristics. 2) TE deficiency mechanisms for different types of substrates: nitrogen-rich, sulfur-rich, TE-poor, and easily hydrolyzed substrates are the four main types of substrates. The mechanisms underlying TEs deficiency in the different substrates are investigated. 3) Regulation of TE bioavailability: characteristics of substrates affect digestion parameters, which disturb the bioavailability TE. Therefore, methods for regulating bioavailability of TEs are discussed.

Syntrophic consortium with the aid of coconut shell-derived biochar enhances methane recovery from ammonia-inhibited anaerobic digestion.

Anaerobic digestion (AD) of nitrogen-rich substrates often suffers from the issue of ammonia inhibition. Although bioaugmentation has been used to assist AD with high ammonia concentration, the combined effect of domesticated syntrophic consortium (MC) together with biochar on ammonia inhibited AD are still unknown. In the present study, MC was adapted and enriched by purposive domestication. As a novel strategy, coconut shell-derived biochar was used as a carrier to aid the MC. The results showed that the digestion system deteriorated completely without the assistance of MC and biochar when the TAN concentration exceeded 8.0 g L-1. The combination of biochar and MC (B-MC treatment) could restore ammonia inhibition in 10 days and achieved a high methane yield of 357.5 mL g-1 volatile solid, which was 7.5 % higher than that of MC treatment. Syntrophomonas, Syntrophobacter, and Methanoculleus in MC played a critical role in reducing propionic acid and butyric acid content and efficiently producing methane. Their abundances increased 12-fold, 10-fold, and 2-fold, respectively. With the assistance of biochar, MC had a better performance in relieving ammonia inhibition. This could be attributed to two aspects. First, biochar encouraged the growth or colonization of key microorganisms such as propionate and butyrate oxidizing bacteria and ammonia-tolerant archaea. Second, biochar induced the growth of conductive microorganisms such as Geobacter. From the perspective of enzyme genes, biochar increased the abundance of related enzyme genes in butyrate and propionate degradation, acetoclastic and hydrogenotrophic pathways. In conclusion, MC combined with biochar is a potential approach to alleviate ammonia nitrogen inhibition.

Two microbial consortia obtained through purposive acclimatization as biological additives to relieve ammonia inhibition in anaerobic digestion.

Ammonia inhibition is a challenging issue in the anaerobic digestion (AD) of nitrogen-rich substrates and hinders the energy recovery from organic wastes. Bioaugmentation is promising strategy to stabilize AD systems with high ammonia concentration. The composition of microbial consortia often determines their effectiveness in bioaugmentation. Up to now, the effect of various microbial consortia as biological additives on the AD systems is not fully understood. In this study, two microbial consortia (syntrophic microbial consortium, MC, and hydrogenotrophic methanogen consortium, SS) were obtained through two domestication methods, and were applied in a nitrogen-rich AD system. The results showed that the MC and SS treatments could restore AD performance within 21 days and 83 days, respectively. The recovery of digestion performance depended on the methanogenic archaea Methanospirillum, Methanothermobacter, and Methanoculleus in the early and later stages. Analysis of the 13C isotope indicated that both MC and SS enhanced the hydrogenotrophic pathway. The KEGG analysis showed that the MC not only promoted the key enzyme genes in the hydrogenotrophic pathway but also had a positive effect on the related enzyme genes of propionate and butyrate degradation, which was affected by the abundant short-chain fatty acids degrading bacteria, such as Syntrophomonas, Syntrophobacter, and Tissierella in the MC. After recovery of digestion performance, there was no significant difference (p > 0.05) in methane yield between the MS and SS treatments. Therefore, the best intervention period for bioaugmentation is when the digestion performance of the AD system is unstable.

The greenhouse gas emission potential and phytotoxicity of biogas slurry in static storage under different temperatures.

With the booming development of biogas industry to treat organic waste in China, the by-product of biogas slurry was accompanied with a huge amount. Static storage process of biogas slurry was normally operated under different seasons before application to land which would cause nutrition decomposition and greenhouse gas emission. Thus, the aim of this study was to investigate the nutrition decomposition, greenhouse gas emission (CH4 and N2O), and phytotoxicity of biogas slurry under different static temperatures, furthermore to illuminate the network among them and functional microorganism. According to the results, higher temperature at 30 °C contributed to fast and complete degradation of COD. In addition, more quantity of NH4+ conversion and NO3- formation appeared at 30 °C. These factors resulted in relatively less crop toxicity together. CH4 was the dominant greenhouse gas emission than N2O and was highest in 30 °C treatment with total emission of 273.7 L/(m3·d) and greenhouse gas emission of 20.01 kg CO2e (carbon dioxide equivalent). Lower temperature was conductive to N reservation and reduction of greenhouse gas emission, but making against with stabilization of organic matter and crop safety. At the same dilution times (≤3) of biogas slurry with deionized water, higher temperature at 30 °C could reduce 30 days of storage time, but 10 °C was still unsafe for crop. Structural equation model was further illustrated the positive effect of temperature on NO3-, CH4, GI, and N2O and negative on COD and NH4+. These results could help to monitor the environmental risk, evaluate the maturity, guide the irrigation scheme, and regulate the static storage condition of biogas slurry under different seasons.

An innovative strategy to enhance the ensiling quality and methane production of excessively wilted wheat straw: Using acetic acid or hetero-fermentative lactic acid bacterial community as additives.

Ensiling is an effective storage strategy for agricultural biomass, especially for energy crops (mainly energy grasses and maize). However, the ensiling of excessively wilted crop straw is limited due to material characteristics, such as a high lignocellulosic content and low water-soluble carbohydrate and moisture contents. In this study, acetic acid or hetero-fermentative lactic acid bacterial community (hetero-fermentative LAB) were employed as silage additives to improve the ensiling process of excessively wilted wheat straw (EWS). The results showed that the additives inhibited the growth of Enterobacteriaceae and Clostridium_sensu_stricto_12, whose abundances decreased from 55.8% to 0.03-0.2%, respectively. The growth of Lactobacillus was accelerated, and the abundances increased from 1.3% to 80.1-98.4% during the ensiling process. Lactic acid fermentation was the dominant metabolic pathway in the no additive treatment. The additives increased acetic acid fermentation and preserved the hemicellulose and cellulose contents, increasing the methane yield by 17.7-23.9%. This study shows that ensiling with acetic acid or hetero-fermentative LAB is an effective preservation and storage strategy for efficient methane production from EWS.

Bioaugmentation with methanogens cultured in a micro-aerobic microbial community for overloaded anaerobic digestion recovery.

Anaerobic digestion (AD) is widely used for conversion of waste materials into biogas, but inhibition of methane production caused by overloading can be a major problem. The micro-aerobic microbial community MC1 was used to successfully culture methanogens, Methanosarcina acetivorans C2A and Methanosaeta thermophila NBRC 101360. The maximum 16S rRNA gene concentrations of Methanosarcina acetivorans C2A and Methanosaeta thermophila NBRC101360 were 1.06 × 106 and 1.35 × 103 copies/mL, respectively. The five key bacteria in MC1 were quantified to assess the effect of inoculation on the abundances of the bacteria in the mixed culture. The original MC1 total 16S rRNA gene concentration was 1.93 × 108 copies/mL, and the total 16S rRNA gene concentration had increased to 4.79 × 109 copies/mL on day 9 (p < 0.05). The proportions of the key strains in MC1+MST had changed by day 9. Cells were harvested and used to bioaugment and increase the pH values of the high- and medium-temperature anaerobic systems. After bioaugmentation, thermophilic AD recovered well. The cumulative amounts of gas produced were 44.78% and 28.28% higher in the MC1+MST and MC1 groups, respectively, than the sterilized control. The MC1+MST group gave better results than the chemical addition control group (CaCO3). There was no clear effect of bioaugmentation in mesophilic AD. When compared with traditional pure culture of methanogens as inoculants, methanogen cultivation in MC1 was simple and there was no need to separate and purify the target strains. This simplified methanogenic bioaugmentation agent was useful to study the mechanism of bioaugmentation for the recovery from low pH inhibition, showing the potential for practical application.

The role of biochar on alleviating ammonia toxicity in anaerobic digestion of nitrogen-rich wastes: A review.

This paper reviewed the mechanisms of biochar in relieving ammonia inhibition. Biochar affects nitrogen-rich waste's anaerobic digestion (AD) performance through four ways: promotion of direct interspecies electron transfer (DIET) and microbial growth, adsorption, pH buffering, and provision of nutrients. Biochar enhances the DIET pathway by acting as an electron carrier. The role of DIET in relieving ammonia nitrogen may be exaggerated because many related studies don't provide definite evidence. Therefore, some bioinformatics technology should be used to assist in investigating DIET. Biochar absorbs ammonia nitrogen by chemical adsorption (electrostatic attraction, ion exchange, and complexation) and physical adsorption. The absorption efficiency, mainly affected by the properties of biochar, pH and temperature of AD, can reach 50 mg g-1 on average. The biochar addition can buffer pH by reducing the concentrations of VFAs, alleviating ammonia inhibition. In addition, biochar can release trace elements and increase the bioavailability of trace elements.

The absolute concentration and bioavailability of trace elements: Two vital parameters affecting anaerobic digestion performance of chicken manure leachate.

It is currently unclear whether trace elements (TEs) deficiency is due to low bioavailability or low absolute TEs concentrations, especially in high-pH anaerobic digestion (AD) systems. A mixed solution of TEs and EDTA-Na2 were used separately in mono-AD of chicken manure (CM) leachate to investigate this research gap. The results showed relatively low bioavailability of Fe, Mn, and Zn. The bioavailability of all TEs remained stable along with a gradual increase in total ammonia nitrogen concentration. Both TE and EDTA-Na2 supplementation improved the bioavailability of TEs, but TEs supplementation also gave a high proportion of soluble TEs. Adding TEs improved methane production efficiency (+38.3%) and decreased the H2S content. The exchangeable fraction of specific TE (Mo) in H2/CO2 pathway was higher in the TEs treatment. TEs bioavailability and absolute concentrations of available TEs are critical aspects that need to be scrutinized to assess the risk of TEs deficiency.

The characteristics of multi-substrates (low and high C/N) anaerobic digestion: focus on energy recovery and the succession of methanogenic pathway.

Anaerobic digestion of common rural wastes (human feces (HF), food waste (FW) and lawn grass (LG)) were studied considering the specific methane yield, process parameters and microbial characteristics (mainly microbial community and pathogenic bacteria). The results showed that co-digestion of multiple substrates obtained high digestion performance when the total solid (TS) was 4%. The optimal co-digestion ratio of HF, FW, and LG was 33-56%, 21-38% and 20-40%, respectively. The digestion system containing HF underwent ammonia inhibition, which leads to the succession of the methanogenesis pathway from the acetoclastic pathway to the hydrogenotrophic pathway. Simultaneously, the dominant methanogenic archaea changed from Methanosaeta to Methanobacterium and Methanosarcina. Co-digestion reduced Salmonella's absolute concentration. The recovered energy and nitrogen could meet 52-109 % energy demand of rural community and all nitrogen demand in lawn fertilization, respectively. The main rural organic wastes could be recycled by anaerobic digestion, considering the flexibility of substrate ratio.

Exploring the combined effect of total ammonia nitrogen, pH and temperature on anaerobic digestion of chicken manure using response surface methodology and two kinetic models.

Ammonia inhibition is the most challenging issue in the anaerobic digestion (AD) of nitrogen-rich substrates. Total ammonia nitrogen (TAN) concentration, temperature and pH are the main operational parameters affecting the chemical equilibrium between free ammonium nitrogen (NH3; FAN) and ammonium ions (NH4+). However, it is still unclear how these parameters together affect digestion performance by influencing this equilibrium. To determine the effect and linkages of these parameters, a Box-Behnken design-response surface methodology (RSM), correlation analysis and two kinetic models were carried out. The results revealed that the linear effect of TAN, the interaction effect between TAN and temperature, and temperature and pH were significant, however, the quadratic effect of TAN, temperature and pH were also significant. Furthermore, TAN and temperature were positively correlated with Mmax, k and Rmax. Findings from this study could provide a theoretical basis to develop the way of relieving ammonia inhibition.

Obstacles faced by methanogenic archaea originating from substrate-driven toxicants in anaerobic digestion.

Anaerobic digestion (AD) is used to treat waste and produce bioenergy. However, toxicants, which originate from the substrate, can inhibit or damage the digestion process. Methanogenic archaea (MA), which are the executor in the methanogenesis stage, are more sensitive than bacteria to these toxicants. This review discusses the effects of substrate-driven toxicants, namely, antibiotics, H2S and sulfate, heavy metals (HMs), long-chain fatty acids (LCFAs), and ammonia nitrogen, on the activity of MAs, methanogenic pathways, and the inter-genus succession of MAs. The adverse effects of these five toxicants on MA include effects on pH, damages to cell membranes, the prevention of protein synthesis, changes in hydrogen partial pressure, a reduction in the bioavailability of trace elements, and hindrance of mass transfer. These effects cause a reduction in MA activity and the succession of MAs and methanogenic pathways, which affect AD performance. Under the stress of these toxicants, succession occurs among HA (hydrogenotrophic methanogen), AA (acetoclastic methanogen), and MM (methylotrophic methanogen), especially HA gradually replaces AA as the dominant MA. Simultaneously, the dominant methanogenic pathway also changes from the aceticlastic pathway to other methanogenic pathways. A comprehensive understanding of the impact of toxicants on MA permits more specific targeting when developing strategies to mitigate or eliminate the effects of these toxicants.

The effects of C/N (10-25) on the relationship of substrates, metabolites, and microorganisms in "inhibited steady-state" of anaerobic digestion.

The carbon/nitrogen ratio (C/N) is a key parameter that affects the performance of anaerobic digestion (AD). Recent AD research has focused on optimizing the C/N of feedstock. The so-called "inhibited steady-state" refers to a special state of ammonia inhibition of AD that often occurs at low-C/N (below 25) when degradable nitrogen-rich substrates, such as livestock manure, are used as feedstock. However, the mechanism behind the "inhibited steady-state" is still unknown. In the current study, co-digestion and recirculation were used to create a C/N gradient in the influent to explore the relationship between substrates, metabolites, and microorganisms in the "inhibited steady-state." Data were collected at the macro, microbial, and genetic levels. Three CSTRs were successfully made run into the "inhibited steady-state" using influent C/Ns of 10-12. Digestion performance levels of R10-R12 were low and stable, transitioning from an aceticlastic methane-producing pathway to a hydrogenotrophic pathway as the C/N gradually decreased. As the abundance of the hydrogenophilic methanogens increased, the abundance of syntrophic acetate-oxidizing bacteria (SAOB) also increased. The succession between populations of Methanosaeta and Methanosarcina may be used as a microbiological indicator of ammonia inhibition. Under high-C/Ns, cooperation among bacteria was high, while under low-C/Ns, competition among bacteria was high. These results clarify the processes underlying the "inhibited steady-state," which is a condition often faced in actual large-scale biogas facilities that use degradable nitrogen-rich substrates. Moreover, practical guidelines for evaluating ammonia inhibition are provided, and strategies to alleviate ammonia suppression are developed.