Microbial Community and Metabolic Pathways in Anaerobic Digestion of Organic Solid Wastes: Progress, Challenges and Prospects

Abstract

Anaerobic digestion (AD) is a sustainable and widely adopted technology for the treatment of organic solid wastes (OSWs). However, AD efficiency varies significantly across different substrates, primarily due to differences in the microbial community and metabolic pathways. This review provides a comprehensive summary of the AD processes for four types of typical OSWs (i.e., sewage sludge, food waste, livestock manure, and straw), with an emphasis on their universal characteristics across global contexts, focusing mainly on the electron transfer mechanisms, essential microbial communities, and key metabolic pathways. Special attention was given to the mechanisms by which substrate-specific structural differences influence anaerobic digestion efficiency, with a focused analysis and discussion on how different components affect microbial communities and metabolic pathways. This study concluded that the hydrogenotrophic methanogenesis pathway, TCA cycle, and the Wood–Ljungdahl pathway serve as critical breakthrough points for enhancing methane production potential. This research not only provides a theoretical foundation for optimizing AD efficiency, but also offers crucial scientific insights for resource recovery and energy utilization of OSWs, making significant contributions to advancing sustainable waste management practices.

1. Introduction

In recent years, the increased generation of organic solid wastes (OSWs) has emerged as a pressing global concern [1]. Statistical data indicate that approximately 1.3 billion tonnes of municipal solid waste (MSW) are produced annually worldwide, with projections suggesting an increase to 2.2 billion tonnes by 2025 [2]. These OSW streams contain valuable resources, including proteins, carbohydrates, and minerals, which can be metabolized by microorganisms and used as biosynthetic precursors or transformed into a wide range of bio-based products [3]. Anaerobic digestion (AD) is a sequential, multi-stage biological process that facilitates the decomposition and stabilization of OSWs under anaerobic conditions. With the participation of diverse anaerobic microbial communities, various OSWs can be converted into methane, a renewable energy source, which can serve as a sustainable alternative to fossil fuels for heat and electricity generation [4]. Life cycle assessments have demonstrated that AD offers significant environmental advantages over conventional waste management practices, including reduced atmospheric emissions, lower global warming potential, and minimized fossil fuel consumption [5].

AD process comprises four sequential stages: hydrolysis, acidogenesis, acetogenesis, and methanogenesis [6]. During these processes, OSWs are biologically converted to biogas (primarily a mixture of carbon dioxide (CO₂) and methane (CH₄)) through the metabolic activities of diverse anaerobic microbial communities, as illustrated in

Table 1. The reactions that occur in the biological treatment unit under anaerobic conditions [8].

Participants	Reactions
Denitrifying bacteria	${0.4\mathrm{N}\mathrm{O}}_3^{-}+{\mathrm{H}}_2+0.4{\mathrm{H}}^{+}\to 0.2{\mathrm{N}}_2+{1.2\mathrm{H}}_2\mathrm{O}$
	${1.6\mathrm{N}\mathrm{O}}_3^{-}+{\mathrm{C}\mathrm{H}}_3{\mathrm{C}\mathrm{O}\mathrm{O}}^{-}+0.6{\mathrm{H}}^{+}\to 0.8{\mathrm{N}}_2+{0.8\mathrm{H}}_2\mathrm{O}+{2\mathrm{H}\mathrm{C}\mathrm{O}}_3^{-}$
PAOs	$\mathrm{A}\mathrm{T}\mathrm{P}+{\mathrm{H}}_2\mathrm{O}\to \mathrm{A}\mathrm{D}\mathrm{P}+{\mathrm{H}}_3\mathrm{P}{\mathrm{O}}_4+\mathrm{e}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}$
Sulfate-reducing bacteria	${\mathrm{H}}_2+0.25{\mathrm{S}\mathrm{O}}_4^{2-}+0.25{\mathrm{H}}^{+}\to {0.25\mathrm{H}\mathrm{S}}^{-}+{\mathrm{H}}_2\mathrm{O}$
	${\mathrm{C}\mathrm{H}}_3\mathrm{C}{\mathrm{O}\mathrm{O}}^{-}+{\mathrm{S}\mathrm{O}}_4^{2-}\to {\mathrm{H}\mathrm{S}}^{-}+{2\mathrm{H}\mathrm{C}\mathrm{O}}_3^{-}$
Methanogens	${\mathrm{H}}_2+0.25{\mathrm{H}\mathrm{C}\mathrm{O}}_3^{-}+0.25{\mathrm{H}}^{+}\to {0.25\mathrm{C}\mathrm{H}}_4+{0.75\mathrm{H}}_2\mathrm{O}$
	${\mathrm{C}\mathrm{H}}_3\mathrm{C}{\mathrm{O}\mathrm{O}}^{-}+{\mathrm{H}}_2\mathrm{O}\to {\mathrm{C}\mathrm{H}}_4+{\mathrm{H}\mathrm{C}\mathrm{O}}_3^{-}$
Fe3+-reducing bacteria	${\mathrm{H}}_2+{2\mathrm{F}\mathrm{e}}^{3+}\to {2\mathrm{F}\mathrm{e}}^{2+}+{2\mathrm{H}}^{+}$
	${\mathrm{C}\mathrm{H}}_3\mathrm{C}{\mathrm{O}\mathrm{O}}^{-}+{4\mathrm{F}\mathrm{e}}^{3+}+{4\mathrm{H}}_2\mathrm{O}\to {4\mathrm{F}\mathrm{e}}^{2+}+{5\mathrm{H}}^{+}{2\mathrm{H}\mathrm{C}\mathrm{O}}_3^{-}$

. The transformations of OSWs are driven by intricate microbial interactions and metabolic pathways. Recent advancements in microbial metabolomics have shifted research focus from the macroscopic chemical properties of AD systems to the underlying structure and metabolic mechanisms of microbial communities. For example, high-throughput sequencing of 16S rRNA gene amplicons has significantly enhanced resolution for analyzing the structure of microbial communities. Metagenomics offers insights into the physiological potential of the communities, while metatranscriptomics enables the measurement of in situ gene expression, highlighting the active members within the community. Metaproteomics further extends this understanding by identifying catalytic enzymes, entire metabolic pathways, and novel functional proteins, as well as elucidates the distribution of metabolic activities and cooperation or competition among microbial populations. Additionally, metabolomics provides qualitative and quantitative analyses of low-molecular-weight molecules involved in metabolic reactions, facilitating deeper inferences into metabolic mechanisms [7]. Collectively, these advanced techniques provide powerful tools to investigate how varying substrates and environmental conditions influence microbial community structure and metabolic pathways.

Given the current knowledge gaps in understanding the metabolic pathways associated with distinct microbial community structures under varying substrate conditions in AD, this study aims to elucidate the microbial community composition, mutualistic interactions, and metabolic processes associated with different types of OSWs. Specifically, we examine the characteristics of substrate components in AD systems treating OSWs, detailing their impact on microbial community structure and metabolic pathways. Furthermore, we analyze how systems with distinct characteristics influence underlying mechanisms of AD. Finally, we summarize the similarities and differences in the AD metabolic pathways of four OSWs, providing theoretical support at the metabolic pathway level for efficiency optimization strategies. This work provides a theoretical foundation for enhancing the metabolic capacity and overall efficiency of AD systems.

2. Microbial Community Structure in AD

In AD systems, the microbial taxa performing specialized functional roles remain relatively stable; however, the dominant microbial populations vary significantly depending on substrate composition and environmental conditions. Changes in gene expression in microbial communities during AD are relatively stable at the order level and highly variable at family level [9]. For instance, metagenomic analyses of biogas plant feedstocks have consistently identified Firmicutes, Bacteroidetes, and Proteobacteria as the dominant phyla in AD systems [10]. Among them, Firmicutes are particularly abundant due to their resilience to environmental fluctuations. At the order level, Clostridiales and Bacteroidales dominate the immediate response (IR) microbial community of AD, serving as keystone taxa that drive critical metabolic processes. The processes include the metabolism of amino acids (AAs), carbohydrates, nucleotides and cofactors/vitamins, genetic translation, and membrane transport systems [9].

The hydrolysis stage involves the breakdown of complex macromolecular organic compounds, such as carbohydrates, lipids, and proteins, into simpler and soluble organic compounds including monosaccharides, fatty acids, AAs, and alcohols. This stage is primarily mediated by bacterial phyla such as Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Chloroflexi, which are the most common bacterial phyla in this phase. During the acidogenesis stage, the hydrolysis products are further metabolized by acidogens into volatile fatty acids (VFAs, e.g., acetate, propionate, butyrate, and valerate) and alcohols. The dominant bacteria phyla in this stage mainly include Bacteroidetes, Firmicutes, and Clostridium. In the subsequent acetogenesis stage, VFAs are catalytically converted into acetate, hydrogen (H₂), and CO₂. Additionally, the syntrophic acetate-oxidizing bacteria (SAOB) can oxidize acetate into H₂ and CO₂ through the Wood–Ljungdahl pathway [11]. The final stage, methanogenesis, involves the conversion of CO₂, H₂, and acetate into methane by methanogens. Methanogens are broadly categorized into three main types: acetotrophic methanogens, hydrogenotrophic methanogens, and methylotrophic methanogens. Acetotrophic methanogens, primarily represented by genera Methanosaeta and Methanosarcina, convert acetate to CH₄ and CO₂. Notably, most Methanosarcina can also utilize H₂/CO₂ and C-1 compounds. Hydrogenotrophic methanogens, such as Methanobacterium, Methanobrevibacter, Methanoculleus, Methanospirillum, and Methanothermobacter, utilize H₂ or formate to reduce CO₂ to CH₄, playing a critical role in scavenging H₂ and maintaining low H₂ partial pressures essential for AD. Methylotrophic methanogens, including Methanobacterium, Methanococcus, Methanosclerotinia, Methanosalsus, and Methanosalts, produce CH₄ from methyl compounds such as methanol, methylamine, and methyl sulphides [12].

In AD systems, microbial species do not function in isolation but engage in syntrophic interactions, where one species lives off the metabolic by-products of another [13]. Syntrophy in AD is characterized by a mutually dependent thermodynamic relationship between bacteria and archaea (methanogens). Specifically, bacteria degrade organic substrates into intermediates such as acetate, H₂, CO₂, or formate, which are subsequently utilized by methanogens as energy sources. This syntrophic partnership is illustrated in Figure 1 [14].

Syntrophy is a fundamental mechanism that sustains microbial communities involved in AD, enabling electron transfer primarily through interspecies electron transfer (IET). IET occurs via three distinct modes: interspecies hydrogen transfer (IHT), interspecies formate transfer (IFT), and direct interspecies electron transfer (DIET) [18]. Among these, IET and IFT are categorized as indirect interspecies electron transfer (IIET) due to their reliance on diffusible electron carriers, as illustrated in Figure 2. In contrast, DIET exhibits higher electron transfer efficiency than IIET [19]. Based on this, a more efficient electron transfer mechanism, termed proton-coupled electron transfer (PCET), has been identified in AD systems [20,21]. PCET involves transmembrane proton and electron exchange via microbial respiration, driven by enzymatic reactions that couple proton pumping to electron transfer across cell membranes [18]. PCET is energetically favorable due to its lower activation barrier, making it a more thermodynamically efficient process [20]. In summary, microbial communities play a pivotal role in AD systems. Systems characterized by high functional redundancy and community diversity tend to exhibit greater stability, as the loss of or reduction in one species due to environmental perturbations can be compensated by other functionally similar but more resilient species [12].

The AD process relies on a diverse consortium of microorganisms that engage in symbiotic relationships, utilizing their unique enzymatic capabilities to drive the degradation of organic matter. During the hydrolysis stage, fermentative microorganisms secrete extracellular enzymes to break down large, complex polymers into smaller organic molecules, which can then be transported across membranes for further degradation [24]. The acidogenesis stage is critical for determining the metabolic pathways, as the VFAs formed during this phase dictate subsequent reactions [11]. Pyruvate, generated via the Embden–Meyerhof-Parnas (EMP) pathway, serves as a key intermediate and is further converted into acetate, propionate, butyrate, and their corresponding alcohols through various enzymatic pathways. The specific products and their quantities depend on the substrate composition, environmental conditions, and the dominant microbial strains [11,25]. Notably, acetate is produced not only through the acetogenesis pathway but also via the Wood–Ljungdahl (W-L) pathway, where SAOB convert H₂/CO₂ into acetate [26]. In addition to this, homoacetogenic bacteria, specialized anaerobes, utilize the Wood–Ljungdahl pathway to synthesize acetyl–CoA, fix CO₂, and conserve energy for cellular carbon production. Among the products of the acetogenesis stage, only formate, acetate, and H₂/CO₂ can be directly utilized by methanogens as substrates for methane production, while other products must first be oxidized into them for further utilization [27]. The last stage of AD is methanogenesis, as illustrated in Figure 3. CH₄ can be derived through three primary pathways: acetotrophic methanogenesis (utilizing acetate), hydrogenotrophic methanogenesis (utilizing H₂/CO₂), and methylotrophic methanogenesis (utilizing methylamines or methanol). Hydrogenotrophic methanogens play a crucial role in regulating H₂ levels within the system, maintaining the H₂ partial pressure at 10⁻⁶ to 10⁻⁴ atm, which is essential for the syntrophic reactions between hydrogenogenic bacteria and methanogens [8]. H₂ is primarily generated during the breakdown of propionate and butyrate into acetate by specialized hydrogenogenic bacteria. However, these reactions are thermodynamically unfavorable (∆G > 0) and are inhibited by the accumulation of H₂. To facilitate these reactions, hydrogenotrophic methanogens rapidly consume H₂, ensuring that the actual free energy change (∆G) becomes negative, thereby enabling the reactions to proceed spontaneously [28]. Approximately 70% of methane produced during methanogenesis is derived from acetate [12].

With the continuous advancement of metabolomics technologies, our observations of microbial reaction processes have become increasingly refined. Consequently, it is particularly crucial to systematically summarize key metabolic pathways. Although both universal patterns and substrate-specific traits coexist, identifying these commonalities can provide a fundamental scientific basis for enhancing methanogenic efficiency in AD systems. In this section, we summarize the functional microorganisms commonly present in each stage of AD. These microbes produce methane through similar energy conversion mechanisms and metabolic pathways. However, due to differences in the relative abundance of key functional microorganisms and critical genes involved in these metabolic processes, their methane production potential varies. Since different OSWs exhibit variations in methane production potential and key components, we will further investigate how their specific inhibitory factors influence microbial communities and associated metabolic pathways.

3. Anaerobic Digestion of Organic Solid Wastes

Based on the summary of microbes producing methane through similar energy conversion mechanisms and metabolic pathways, we further review how the specific inhibitory factors of waste-activated sludge (WAS), food waste (FW), manure, and lignocellulosic biomass (LB) influence microbial communities and associated metabolic pathways; these wastes are all characterized by their abundant availability and have been extensively studied. As shown in Figure 4, combined with the metabolic process and the structure of microbial communities in AD systems, we observed that the methanogenic efficiency varies significantly across different types of OSWs, despite the involvement of similar microbial consortia. The specific performance is as follows: the methane potentials of WAS, FW, manure, and LB are 126.1 ± 47.6, 627.8 ± 242.4, 230.2 ± 53.4, and 134.3 ± 71.9 (mL CH₄/g VS), respectively, with FW being significantly higher than the other three OSWs (p < 0.001 for WAS and LB; p < 0.01 for manure), followed by manure, which is significantly higher than WAS and LB (p < 0.01 for WAS and p < 0.05 for LB). This variation is likely attributed to the distinct compositional characteristics of the substrates. The different components of substrates can regulate anaerobic digestion efficiency by altering the relative abundance of microbial populations and some of their associated metabolic pathways.

3.1. AD of Sludge

The organic matters in sludge mainly include proteins, polysaccharides, nucleic acids, humic substances, and lipids, which collectively form the extracellular polymeric substances (EPS) and constitute a significant component of sludge [48,49]. Among these, proteins, humic substances, and polysaccharides occupy 40–60%, 10–30%, and 10–20% of organic components in sludge, respectively, making them the top three organic components in sludge. Due to the relatively high protein content, the sludge exhibits a low C/N ratio of 6–10. A low C/N ratio can cause insufficient utilization of the carbon source and a high amount of ammonia, which inhibits microbial growth [50]. In substrates with high nitrogen content, excessive ammonia production during AD can elevate pH levels, causing inhibitory effects and eventual process deterioration. Microbial communities in AD systems adapt to high ammonia–ammonium–pH conditions through domestication, shifting from acetotrophic to hydrogenotrophic methanogens, which exhibit greater tolerance to ammonia stress [51,52]. The prominent issue of ammonia inhibition will be discussed in detail in Section 3.4.

Humic substances, specific components of sludge, contain functional groups such as quinone, nitrogen, and sulfur. These groups can act as electron acceptors, accelerating key metabolic reactions, including the conversion of glyceradehyde-3P → D-glycerate 1,3-diphosphate, and pyruvate → acetyl-CoA, thus promoting the acidogenesis stage. However, during the hydrolysis stage, the predominantly negative charge of humic substances creates a repulsive effect that overrides the hydrophobic interaction between humic substances and hydrolytic enzymes, potentially hindering enzymatic activity. Moreover, increasing concentrations of humic substances have been shown to inhibit the activity of coenzyme F420, a key enzyme in the methanogenic stage, leading to a decreased methanogenic efficiency [53]. Furthermore, studies have demonstrated that humic substances exert inhibitory effects on Firmicutes and methanogens, particularly hydrogenotrophic methanogens. This inhibition is hypothesized to result from the suppression of hydrogenase activity by humic substances [54].

Wastewater treatment plants (WWTPs) receive both municipal sewage and industrial wastewater, leading to the accumulation of undesirable pollutants in the primary and secondary sedimentation tanks, such as heavy metals, exogenous organic matter, microplastics and so on, which have different influences on the microbial metabolism and community structure in the AD [53]. These pollutants originate from the breakdown of household products and waste plastics produced by equipment and fillers in WWTPs. Microplastics, in particular, pose significant challenges by adhering to the surface of sludge, thereby inhibiting direct contact between microorganisms and sludge particles. This interaction increases the van der Waals free energy of the sludge surface, further hindering microbial utilization [55]. Owing to their large specific surface area and hydrophobic nature, microplastics readily adsorb organic pollutants and heavy metals from the environment, exacerbating their adverse effects on AD systems [56]. The presence of microplastics has been shown to reduce the abundance of key microbial genera essential for hydrolysis and acidification, e.g., Firmicutes, Bacterioides sp., Clostridium_sensu_stricto_12, Proteobacyeria, Chloroflex, as well as archaea (methanogens), e.g., Euryarchaeota [36,57]. Microplastics can adhere to and damage cell membranes, altering membrane permeability and disrupting redox cycling. This damage compromises metabolic functions and protein activity, as evidenced by the decreased relative abundance of key functional genes such as mcrA (methyl-coenzyme M reductase) and ACS (acetyl-CoA synthetase) [58]. Additionally, microplastics induce the production of intracellular reactive oxygen species (ROS), leading to toxic oxidative stress, disruption of redox signaling pathways, and impaired intramembrane electron transfer [59]. Antibiotics can adsorb onto microplastics through mechanisms such as hydrogen bonding, electrostatic interactions and hydrophobicity. Interestingly, this adsorption can reduce the direct interaction between antibiotics and microorganisms, thereby mitigating the biotoxicity of antibiotics [37].

Metals in sludge may originate from the adsorption of microplastics or the addition of flocculants during wastewater treatment processes [60]. The predominant metals in sludge mainly include calcium (Ca), magnesium (Mg), aluminum (Al), and iron (Fe). High-valent metal ions can bridge chemical complexes with macromolecular organic matter (e.g., EPS containing carboxyl, hydroxyl, and sulfonic groups). These complexes occupy binding sites on organic matter, bacteria, and enzymes, thereby interfering with their functions [61]. The removal of metals has been shown to enhance the relative abundance of metabolically relevant genes and microbial metabolic activities. For instance, fabG, which encodes acetyl reductase, utilizes NAD⁺ or NADP⁺ as electron acceptors to facilitate lipid metabolism and fatty acid synthesis. Studies demonstrated that reducing metal contents can also improve the relative abundance of key genes involved in carbohydrate and lipid metabolism, such as fas, fabHY, accABCD, bccA, and four methanogenic modules (M00357, M00567, M00356, M00563) along with their associated genes [60].

Some exogenous organic pollutants, such as antibiotics and surfactants, can significantly influence the AD of sludge. Antibiotics, due to their antimicrobial properties, directly affected the metabolic processes of anaerobic methane synthesis by inhibiting ATP and protein synthesis as well as damaging cells through interference with DNA/RNA replication and cell wall formation [62,63]. Bai et al. [64] investigated the impact of antibiotics on sludge AD by comparing microbial communities before and after antibiotic addition. Their findings revealed that high antibiotic concentrations decreased the microbial community abundance in the AD system, as indicated by declines in the Chao1 and ACE indices. Antibiotics can decrease the relative abundance of Firmicutes (from 74.7% to 65.5%), which are essential for the bioconversion of glutamate to ammonia, CO₂, acetate, butyrate, and H₂ in the hydrolysis and acidogenic stages [53,65,66]. They can decrease the relative abundance of genes associated with the glycolytic pathway, inhibit the conversion of serine to pyruvate, and suppress the hydrotrophic methanogenic pathway [37]. For the methanogenesis stage, antibiotics can decrease the relative abundance of methyl-coenzyme M reductase (mcrA) and increase the relative abundance of Methanosarcina, which is more suitable for acetate-scarce environments [67].

Surfactants, commonly used as detergents in domestic and industrial settings, enter WWTPs through sewage networks and become enriched in secondary sludge. Surfactants exhibit dual regulatory effects on the metabolic processes of sludge AD, which depend on the concentration and the length of the alkyl chain [68]. On the one hand, several studies have proved that surfactants can enhance the solubility of organic matter in sludge, thereby improving the efficiency of hydrolysis and acidogenesis stages. The addition of surfactants has been shown to enrich some of the tolerant hydrolytic and acidogenic bacteria, such as Firmicutes, Acetoanaerobium, and Fususibacter, as well as functional genes associated with hydrolysis and acidification of proteoglycan (e.g., glsA, MAN2C1, ALDO, and fabK) [69,70]. On the other hand, their unique amphiphilic structure allows them to interact with proteins via hydrophilic portions and with membrane lipids via hydrophobic portions [71]. When the surfactant concentration reaches a certain threshold (e.g., 400 ppm for Sodium Dodecyl Benzene Sulfonate), it can induce curvature stress in cell membranes, leading to disordering and cell lysis [72]. Surfactants can inhibit enzymes (including the activity of coenzyme F420), decelerate microbial growth, and promote VFA accumulation, adversely affecting methanogenesis and reducing methane production efficiency [73,74].

3.2. AD of Straw

Straw is an ideal substrate for biogas production through AD due to its abundance, low cost, widespread availability, and lack of direct competition with food and feed production [75]. Globally, the annual production of lignocellulosic biomass, including agricultural residues and greenhouse biomass, is estimated at approximately 181.5 billion tonnes [76]. Structurally, straw is composed of a complex matrix of cross-linked cellulose, hemicellulose, glycosylated proteins, and lignin. Within this matrix, cellulose microfilaments are interconnected by hemicellulose and pectin while being covered by lignin. This intricate structure creates a barrier that limits the accessibility of microbial cellulose-degrading enzymes, hindering efficient degradation [77,78]. To overcome this challenge, various pretreatment methods have been proposed to disrupt the recalcitrant fibrous structure of straw. These methods aim to alter key properties such as chemical composition, cellulose crystallinity, polymerization, accessible surface area, enzyme adsorption and desorption, hemicellulose acetylation, and hydrolysis capacity. By modifying these factors, pretreatments enhance the accessibility of hydrolytic enzymes and microorganisms to cellulose, thereby improving the overall efficiency of biomass hydrolysis and subsequent biogas production. This metabolic process is also specific to lignin-containing biomass and is mainly carried out by some anaerobic bacteria and fungi. Among the bacterial phyla, Firmicutes, Bacteroidetes, and Proteobacteria dominate the microbiota during straw AD. Several genera belonging to these phyla, such as Prevotella, Eubacteria, Clostridium, Lachnoclostridium, Cellvibrio, Luteimonas, Fibrobacter, and Proteiniphilum, appear to play a significant role in lignocellulose degradation by providing an array of carbohydrate-active enzymes [79]. The liquid fraction of cellulosic hydrolysates is rich in hemicellulosic components, mainly acetate and xylose [80]. Proteiniphilum and Fermentimonas exhibit significant syntrophic interactions with methanogens to produce acetate [79]. Therefore, the conversion of acetate in the methanogenesis process plays a dominant role in AD metabolism compared to other OSWs.

Straw typically exhibits a high C/N ratio; for instance, wheat straw has a C/N ratio of approximately 100. This high ratio is characterized by abundant carbohydrates and elevated carbon content but low nitrogen levels, which poses a major limitation for biogas production. Studies indicate that an optimal C/N ratio for AD ranges between 20 and 30.1 [6], as this balance supports optimal microbial activity and process efficiency. An imbalanced C/N ratio negatively affects microbial functions, often leading to process instability and a deterioration phase. Specifically, a high C/N ratio can trigger irreversible acidification due to rapid degradation of carbohydrates, destabilizing the AD process. In addition, nitrogen is one of the important nutrients for methanogens; a high C/N ratio means lower availability of nitrogen, further hindering methanogenic activity [81,82]. To address this issue, co-digestion with nitrogen-rich substrates, such as livestock manure, which has a lower C/N ratio, has been proposed as an effective solution [83]. For example, Li et al. [84] demonstrated that co-fermentation of cow dung with straw increased the methane content of biogas by 4.9–7.4% compared to that of corn stover alone, while also improving process stability. Cheng et al. [42] found that the addition of swine manure to cotton straw in a batch reactor increased methane yield (mL CH₄/g VS_added) by a factor of 2.2 times. Additionally, adjusting the C/N ratio to 20–30 during AD shifts bacterial communities from halophilic to non-halophilic types [85]. Halophilic communities, which are anaerobic bacterium capable of producing H₂ from glucose, facilitate hydrogenotrophic methanogenesis through IET. Sun et al. [78] found that Firmicutes and Bacteroidetes, as the dominant bacteria, were responsible for cellulose degradation, as revealed by targeting glycoside hydrolase genes through T-RFLP analysis.

Straw is often deficient in essential micronutrients such as Fe and nickel (Ni), which are critical components of hydrogenase enzymes during AD [86]. Hydrogenase is mainly classified into Ni-Fe hydrogenase and Fe-Fe hydrogenase, and it plays a pivotal role in the metabolism of the AD system [87]. Consequently, the deficiency of Fe and Ni in straw can compromise hydrogenase activity, thereby hindering electron transfer efficiency and overall AD performance. Hydrogenase is the core of hydrogen-producing bioprocesses and hydrogenotrophic methanogenic pathway, as it facilitates electron transfer using H₂ as an electron carrier; this supports the evidence for the dominance of acetotrophic methanogenesis in the AD of straw.

3.3. AD of Food Waste

Food waste (FW) has high biodegradability and rapid hydrolysis, making it a highly promising substrate for AD [88,89]. Life cycle comparing production, transportation, and compost preparation have demonstrated that AD can serve as a sustainable alternative to incineration and landfill. However, the application of FW in AD presents unique challenges that distinguish it from other types of OSWs.

First and foremost, FW is characterized by its high salt content, primarily sodium chloride (NaCl), which significantly inhibits the activity of microorganisms and enzymes involved in AD, thereby disrupting the overall metabolic process. In China, the NaCl concentration in FW typically ranges from 7 to 12 g/L, exerting adverse effects across various stages of AD [90]. Sodium ions (Na⁺) play a dual role in AD: At low concentrations, they are essential for cell synthesis, growth, and metabolism, thereby promoting enzyme activity, maintaining biofilm homeostasis, and regulating osmotic pressure. However, at high concentrations, Na⁺ increases osmotic pressure, leading to intracellular water loss in methanogens and reducing the activity of key enzymes [38]. He et al. [91] further investigated that at NaCl concentrations exceeding 10 g/L, AA metabolism shifts toward decarboxylation, impairing the conversion of AAs to VFAs and inhibiting acidification. Additionally, the diversity of microbial community structure indicated a transition from butyrate to propionate fermentation under high salt conditions, with Actinobacteria exhibiting greater salt tolerance compared to Bacteroidetes. Zhang et al. [92] highlighted that methanogens are particularly sensitive to elevated salt concentrations, as dehydration induced by high osmotic pressure inhibits their growth and can lead to cell death, ultimately destabilizing the AD system. In summary, the high NaCl content in FW exerts inhibitory effects on AD at multiple stages. These effects can be mitigated through co-digestion with sludge or other OSWs, which helps dilute salt concentrations and stabilize the AD process.

The high C/N ratio and high biochemistry of FW often lead to rapid acidification in the AD process. This rapid acidification promotes the proliferation of acidogenic bacteria, which in turn inhibits methanogenic activity, leading to the accumulation of VFAs [52]. A shift in the dominant methanogenic pathway from hydrogenotrophic to acetotrophic methanogenesis can be observed with increasing C/N ratio, concomitant with a declining relative abundance of Methanosaeta [93]. When the rate of VFA consumption is less than the rate of production, the resulting pH drop further exacerbates the inhibition of methanogenesis. The phyla Bacteroidetes, Firmicutes and Chloroflexi are always dominant in the AD system of FW [94]. Under high C/N ratio conditions, the metabolic advantage of Lactobacillus species becomes more pronounced, as their growth requires relatively less nitrogen and their ability to produce amylase [95]. Lactate can serve as an electron donor and can be fermented to propionate and acetate by Megasphaera elsdenii and Clostridium propionicum, while the reducing equivalents are eliminated via the reduction of lactate to propionate through a linear pathway involving HS-CoA derivatives [96].

The impact of high lipid content and capsaicin in AD of FW has recently garnered attention. Lipids, which are abundant in FW, make the degradation process slower and tend to encapsulate microorganisms, thereby hindering the utilization of macromolecular carbohydrates and proteins [97]. In addition, in high-lipid environments, specific functional bacterial taxa, such as Clostridium and Longilinea, thrive due to their ability to degrade long-chain fatty acids (LCFAs) into smaller organic molecules [98]. Capsaicin, a lipid-soluble organic compound, typically present in FW at concentrations ranging from 0.043 to 1.254 mg/g, has been shown to adversely affect AD systems [99]. Yue et al. [39] examined AD of lipids (i.e., glycerol trioleate) and FW in the presence of an N-vanillin-based nonanamide (a synthetic capsaicin). Their findings revealed that capsaicin compromises cellular integrity and disrupts metabolic functions, leading to a significant reduction in the abundance and diversity of microbial communities. Additionally, the activity of key enzymes associated with methanogenic metabolic processes (e.g., CoA, AK, F420, CoM, etc.) was markedly inhibited. Cyclic voltammetry analysis further demonstrated a 99% reduction in electron transfer rates, highlighting the profound inhibitory effects of capsaicin on AD performance.

3.4. AD of Livestock Manure

Livestock manure, a biodegradable byproduct, is unsuitable for landfill disposal due to its high nutrient content and potential pathogen load. Improper management of livestock manure can lead to significant environmental pollution, adversely affecting soil, air, and water quality, while also promoting the accumulation of harmful microorganisms in ecosystems. The high nutrient concentration in livestock manure exacerbates issues such as water eutrophication and heavy-metal toxicity [100]. AD offers a sustainable solution by converting livestock manure into bioenergy and nutrient-rich soil conditioner, while simultaneously reducing greenhouse gas emissions and mitigating odor-related atmospheric impacts. Additionally, the presence of antibiotics in livestock manure, a growing concern due to their classification as emerging pollutants, has been shown to negatively impact AD performance [101]. Antibiotics can induce bacterial resistance over time, facilitate the spread of resistance genes within ecosystems, and disrupt microbial communities. For example, they can inhibit the growth of hydrolyzers and acidogens, thereby suppressing the hydrolysis stage; however, their negative impact on archaea (i.e., methanogens) is comparatively less pronounced [102]. Turker et al. [101] found that acetotrophic methanogens are more severely inhibited by antibiotics compared to hydrogenotrophic methanogens, leading to the dominance of hydrogenotrophic methanogens, particularly those within the order Methanobacteria, which exhibit the highest cellular activity.

Studies have demonstrated that livestock manure possesses excellent buffering capacity, preventing pH drops and reactor acidification [103]. However, its low organic load, high nitrogen concentration, and minimal lipid content can inhibit methanogen activity [81,100]. Microorganisms require ammonia, a byproduct of the digestion of nucleic acids, proteins, and urea, to synthesize cellular protoplasm for growth and reproduction. Ammonia is essential for microbial activity, while excessive levels can inhibit the performance of microorganisms involved in AD [6]. During the biodegradation of nitrogenous compounds, nitrogen is primarily converted into ammonia, which exists in two forms: ammonium nitrogen (NH₄⁺) and free ammonia (NH₃). Among these, NH₃ exerts a stronger inhibitory role in the AD process [104]. The diffusion of NH₃ into the cell disrupts proton homeostasis, increases energy demands for cellular maintenance, depletes potassium, and interacts antagonistically with calcium (Ca²⁺) and sodium (Na⁺) ions, ultimately inhibiting enzymatic reactions [6,103]. Methanogens are particularly sensitive to high ammonia concentrations, with hydrogenotrophic methanogens exhibiting greater tolerance compared to acetotrophic methanogens. At elevated ammonia levels, some acetotrophic methanogens may even disappear entirely [41]. In high-ammonia environments, the syntrophic interactions between hydrogenotrophic methanogens and SAOB are enhanced. Additionally, genes associated with hydrotrophic methanogenesis (eha, ehc, ehb) and those involved in energy conservation and osmoprotectant synthesis (ablB, kch, BCCT) are upregulated to facilitate adaptation to the increased ammonia concentrations [105]. Jiménez et al. [41] found that in the AD of swine manure, the phylum Firmicutes and the genus Methanosarcina dominate the microbial community. While Methanosarcina can use H₂, CO₂, and acetate for methane production, it preferentially synthesizes methane from H₂ and CO₂ in manure-based systems [41,101]. Fotids et al. [40] achieved a methane recovery rate exceeding 30% by bioaugmenting manure-based digesters with hydrogenotrophic methanogens. Similarly, Xu et al. [81] demonstrated that adding manure to the co-digestion process of MSW and FW increased the abundance of Clostridium and further enhanced the AD process.

In addition to the challenges associated with the low AD yield of manure due to its inherent composition, the management of the residual digestate poses a significant concern. Consequently, livestock manure is more appropriately utilized as a substrate for cultivating hydrotrophic methanogens and as an inoculum to promote the AD of other wastes. However, further research is required to optimize the specific dosage and purification methods for these applications.

3.5. Summary on Main Metabolisms in AD of Four OSWs

In summary, we have further summarized how the specific inhibitory factors of OSWs influence microbial communities and associated metabolic pathways, as shown in Table 2. Among AD systems of OSWs, Firmicutes and Bacteroidetes serve as the dominant phyla. At the order level, Clostridiales and Bacteroidales specifically play a pivotal role in the hydrolysis and acidification steps for various OSWs. Additionally, lignin degradation occurs mostly among anaerobic fungi. Some bacteria also have the capacity for degradation but usually do not play a dominant role. In the methanogenesis process, acetotrophic and hydrogenotrophic types mainly dominate, but their contributions vary depending on substrate properties. Combining Figure 4 with Figure 5, manure demonstrates significant advantages in the hydrogenotrophic methanogenesis pathway and H⁺/H₂ conversion efficiency compared to other OSWs, suggesting its considerable potential for hydrogen production. Notably, hydrogenotrophic methanogens exhibit three key advantages that significantly enhance methanogenic efficiency: (1) The hydrogenotrophic methanogenic pathway is more thermodynamically advantageous, as shown by the Gibbs free energy in Table 3. (2) The hydrogenotrophic pathway plays a crucial role in maintaining hydrogen partial pressure, while the acetotrophic methanogenic pathway is subject to more stringent hydrogen partial pressure constraints when dominant, compared to the hydrogenotrophic pathway, which has been proven in our previous research [21]. (3) The enhanced tolerance to most of the environmental stressors, such as high concentrations of NH₄⁺, antibiotics, surfactants, and high temperature, may explain the improved methane production efficiency correlated with the increased dominance of the hydrogenotrophic methanogenic pathway.

It can be concluded that the TCA cycle and the W-L pathway show strong stabilization, as shown in Figure 5, and are less affected by the types of substrates. This may provide a new direction for the enhancement of AD efficiency. For instance, the TCA cycle is linked to oxygen consumption, as the oxidized electron carriers required to continue the cycle are regenerated by the electron transfer chain (ETC), and the concerted activity of these two pathways allows for the generation of significant amounts of ATP. In anaerobic environment, TCA cycle is thus inhibited; at the same time, the reaction system will be more focused on the glycolysis and AA metabolism to produce energy to maintain the necessary functions [106]. Likewise, the W-L pathway is used in the reductive direction for energy conservation and autotrophic carbon assimilation in acetogens. When hydrogenotrophic methanogens dominate the system, acetogens use the W-L pathway in the reductive direction for CO₂ fixation, which could serve as a critical breakthrough point for enhancing anaerobic hydrogen production efficiency. At the same time, hydrogenotrophic methanogens conserve energy by converting H₂ + CO₂ to methane [107]. It has also been proven that the W-L pathway is the most energy-efficient way to fix carbon (less than 1 mol ATP) [108].

Table 2. Effects of inhibitory factors of each type of OSWs on microbial communities and metabolic pathways.

Type of OSWs	Key Components	Inhibitory Factors	Effects on Microbial Communities	Effects on Metabolic Pathways	References
Sludge	Proteins, humic substances, and polysaccharides occupy 40–60%, 10–30% and 10–20% of organic components in sludge, respectively.	Low C/N ratio (6–10)	Methanogens shifting from acetotrophic to hydrogenotrophic.	Consistent with the NH4+ inhibition mechanisms outlined in livestock manure.	[51,52,109]
		Presence of humic substances	Inhibitory effects on Firmicutes and methanogens, particularly hydrogenotrophic methanogens.	(1) Acceleration of the conversion of glyceradehyde-3P → D-glycerate 1,3-diphosphate, and pyruvate → acetyl-CoA; (2) Inhibition of the activity of coenzyme F420.	[53]
		Presence of microplastics	Reduction the abundance of Firmicutes, Bacterioides sp., Clostridium_sensu_stricto_12, Proteobacyeria, and Chloroflex, as well as archaea (methanogens), e.g., Euryarchaeota.	(1) Reduction of the relative abundance of mcrA (methyl-coenzyme M reductase) and ACS (acetyl-CoA synthetase); (2) Toxic oxidative stress, disruption of redox signaling pathways, and impaired intramembrane electron transfer.	[36,57,58,59]
		Presence of metals	Reduction of the relative abundance of fabG, fas, fabHY, accABCD, and bccA in the hydrolysis and acidogenic stages and four methanogenic modules (M00357, M00567, M00356, M00563).		[60]
		Antibiotics	(1) Reduction of the relative abundance of Firmicutes. (2) Improvement of the relative abundance of Methanosarcina.	(1) Inhibition of ATP and protein synthesis as well as damaging the cells through interference with DNA/RNA replication and cell wall formation; (2) Reduction of the relative abundance of genes associated with the glycolytic pathway, inhibiting the conversion of serine to pyruvate and decreasing the relative abundance of methyl-coenzyme M reductase (mcrA) from methanogenesis.	[37,62,63,64,67]
		Surfactants	Enrichment of Firmicutes, Acetoanaerobium and Fususibacter	(1) Disruption of the function and integrity of biological membranes, affecting cellular viability; (2) Inhibiting enzymes (including the activity of coenzyme F420), decelerating microbial growth, and promoting VFA accumulation adversely affect methanogenesis, thereby reducing methane production efficiency.	[69,70,71]
Straw	A complex matrix of cross-linked cellulose, hemicellulose, glycosylated proteins, and lignin	A-rich ligin	(1) Firmicutes, Bacteroidetes, and Proteobacteria dominate the straw AD; (2) Prevotella, Eubacteria, Clostridium, Lachnoclostridium, Cellvibrio, Luteimonas, Fibrobacter, and Proteiniphilum play a significant role in lignocellulose degradation by providing an array of carbohydrate-active enzymes; (3) Proteiniphilum and Fermentimonas exhibit significant syntrophic interactions with methanogens to produce acetate.		[77,78,79]
		Lack of essential micronutrients such as Fe and Ni	Acetotrophic methanogens are dominant in the system, due to the lack of hydrogenase.	Reduction of electron transfer efficiency.	[86,87]
Food waste	High biodegradability and rapid hydrolysis	High NaCl content	Actinobacteria exhibit greater salt tolerance compared to Bacteroidetes.	(1) Intracellular water loss in methanogens and reduction of key enzyme activity; (2) Inhibition of acidification; (3) Transition from butyrate to propionate fermentation.	[38,91]
		High C/N ratio	(1) Rapid acidification promotes the proliferation of acidogens, which in turn inhibits methanogenic activity, leading to the accumulation of VFAs; (2) A shift occurs in the dominant methanogenic pathway from hydrogenotrophic to acetotrophic methanogenesis, concomitant with a declining relative abundance of Methanosaeta; (3) The metabolic advantage of Lactobacillus species becomes more pronounced.	Lactate can serve as an electron donor and be fermented to propionate and acetate by Megasphaera elsdenii and Clostridium propionicum, while the reducing equivalents are eliminated via the reduction of lactate to propionate through a linear pathway involving HS-CoA derivatives.	[52,93,96]
		High-lipid	Clostridium and Longilinea thrive due to their ability to degrade long-chain fatty acids (LCFAs) into smaller organic molecules.		[98]
		Capsaicin	Capsaicin compromises cellular integrity and disrupts metabolic functions, leading to a significant reduction in the abundance and diversity of microbial communities.	(1) The activity of key enzymes associated with methanogenic metabolic processes (e.g., CoA, AK, F420, CoM, etc.) was markedly inhibited; (2) There was a 99% reduction in electron transfer rates.	[39]
Livestock manure	High nutrient content and potential pathogen load.	Antibiotics	(1) Suppression of the hydrolysis stage through the inhibition of hydrolytic/acidogenic bacterial growth. (2) The dominance of hydrogenotrophic methanogens, particularly those within the order Methanobacteria, which exhibit the highest cellular activity.		[102]
		Low C/N ratio	(1) The diffusion of NH3 into the cell disrupts proton homeostasis, increases energy demands for cellular maintenance, depletes potassium, and interacts antagonistically with Ca2+ and Na+, ultimately inhibiting enzymatic reactions; (2) Hydrogenotrophic methanogens have greater tolerance; (3) The syntrophic interactions between hydrogenotrophic methanogens and SAOB are enhanced; (4) The phylum Firmicutes and the genus Methanosarcina dominate the microbial community.	Genes associated with hydrotrophic methanogenesis (eha, ehc, ehb) and those involved in energy conservation and osmoprotectant synthesis (ablB, kch, BCCT) are upregulated to facilitate adaptation to the increased ammonia concentrations.	[6,41,101,103,105]

Table 2. Effects of inhibitory factors of each type of OSWs on microbial communities and metabolic pathways.

Type of OSWs	Key Components	Inhibitory Factors	Effects on Microbial Communities	Effects on Metabolic Pathways	References
Sludge	Proteins, humic substances, and polysaccharides occupy 40–60%, 10–30% and 10–20% of organic components in sludge, respectively.	Low C/N ratio (6–10)	Methanogens shifting from acetotrophic to hydrogenotrophic.	Consistent with the NH4+ inhibition mechanisms outlined in livestock manure.	[51,52,109]
		Presence of humic substances	Inhibitory effects on Firmicutes and methanogens, particularly hydrogenotrophic methanogens.	(1) Acceleration of the conversion of glyceradehyde-3P → D-glycerate 1,3-diphosphate, and pyruvate → acetyl-CoA; (2) Inhibition of the activity of coenzyme F420.	[53]
		Presence of microplastics	Reduction the abundance of Firmicutes, Bacterioides sp., Clostridium_sensu_stricto_12, Proteobacyeria, and Chloroflex, as well as archaea (methanogens), e.g., Euryarchaeota.	(1) Reduction of the relative abundance of mcrA (methyl-coenzyme M reductase) and ACS (acetyl-CoA synthetase); (2) Toxic oxidative stress, disruption of redox signaling pathways, and impaired intramembrane electron transfer.	[36,57,58,59]
		Presence of metals	Reduction of the relative abundance of fabG, fas, fabHY, accABCD, and bccA in the hydrolysis and acidogenic stages and four methanogenic modules (M00357, M00567, M00356, M00563).		[60]
		Antibiotics	(1) Reduction of the relative abundance of Firmicutes. (2) Improvement of the relative abundance of Methanosarcina.	(1) Inhibition of ATP and protein synthesis as well as damaging the cells through interference with DNA/RNA replication and cell wall formation; (2) Reduction of the relative abundance of genes associated with the glycolytic pathway, inhibiting the conversion of serine to pyruvate and decreasing the relative abundance of methyl-coenzyme M reductase (mcrA) from methanogenesis.	[37,62,63,64,67]
		Surfactants	Enrichment of Firmicutes, Acetoanaerobium and Fususibacter	(1) Disruption of the function and integrity of biological membranes, affecting cellular viability; (2) Inhibiting enzymes (including the activity of coenzyme F420), decelerating microbial growth, and promoting VFA accumulation adversely affect methanogenesis, thereby reducing methane production efficiency.	[69,70,71]
Straw	A complex matrix of cross-linked cellulose, hemicellulose, glycosylated proteins, and lignin	A-rich ligin	(1) Firmicutes, Bacteroidetes, and Proteobacteria dominate the straw AD; (2) Prevotella, Eubacteria, Clostridium, Lachnoclostridium, Cellvibrio, Luteimonas, Fibrobacter, and Proteiniphilum play a significant role in lignocellulose degradation by providing an array of carbohydrate-active enzymes; (3) Proteiniphilum and Fermentimonas exhibit significant syntrophic interactions with methanogens to produce acetate.		[77,78,79]
		Lack of essential micronutrients such as Fe and Ni	Acetotrophic methanogens are dominant in the system, due to the lack of hydrogenase.	Reduction of electron transfer efficiency.	[86,87]
Food waste	High biodegradability and rapid hydrolysis	High NaCl content	Actinobacteria exhibit greater salt tolerance compared to Bacteroidetes.	(1) Intracellular water loss in methanogens and reduction of key enzyme activity; (2) Inhibition of acidification; (3) Transition from butyrate to propionate fermentation.	[38,91]
		High C/N ratio	(1) Rapid acidification promotes the proliferation of acidogens, which in turn inhibits methanogenic activity, leading to the accumulation of VFAs; (2) A shift occurs in the dominant methanogenic pathway from hydrogenotrophic to acetotrophic methanogenesis, concomitant with a declining relative abundance of Methanosaeta; (3) The metabolic advantage of Lactobacillus species becomes more pronounced.	Lactate can serve as an electron donor and be fermented to propionate and acetate by Megasphaera elsdenii and Clostridium propionicum, while the reducing equivalents are eliminated via the reduction of lactate to propionate through a linear pathway involving HS-CoA derivatives.	[52,93,96]
		High-lipid	Clostridium and Longilinea thrive due to their ability to degrade long-chain fatty acids (LCFAs) into smaller organic molecules.		[98]
		Capsaicin	Capsaicin compromises cellular integrity and disrupts metabolic functions, leading to a significant reduction in the abundance and diversity of microbial communities.	(1) The activity of key enzymes associated with methanogenic metabolic processes (e.g., CoA, AK, F420, CoM, etc.) was markedly inhibited; (2) There was a 99% reduction in electron transfer rates.	[39]
Livestock manure	High nutrient content and potential pathogen load.	Antibiotics	(1) Suppression of the hydrolysis stage through the inhibition of hydrolytic/acidogenic bacterial growth. (2) The dominance of hydrogenotrophic methanogens, particularly those within the order Methanobacteria, which exhibit the highest cellular activity.		[102]
		Low C/N ratio	(1) The diffusion of NH3 into the cell disrupts proton homeostasis, increases energy demands for cellular maintenance, depletes potassium, and interacts antagonistically with Ca2+ and Na+, ultimately inhibiting enzymatic reactions; (2) Hydrogenotrophic methanogens have greater tolerance; (3) The syntrophic interactions between hydrogenotrophic methanogens and SAOB are enhanced; (4) The phylum Firmicutes and the genus Methanosarcina dominate the microbial community.	Genes associated with hydrotrophic methanogenesis (eha, ehc, ehb) and those involved in energy conservation and osmoprotectant synthesis (ablB, kch, BCCT) are upregulated to facilitate adaptation to the increased ammonia concentrations.	[6,41,101,103,105]

Table 3. The corresponding free energy of ( $∆{G°}^{\prime }$) important methanogenic reactions [8].

Functional Step	Reaction	$∆{\mathit{G}°}^{\mathit{\prime }}$ (kJ/mol)
Acetotrophic methanogenesis	${\mathrm{C}\mathrm{H}}_3^{-}{\mathrm{C}\mathrm{O}\mathrm{O}}^{-}+{\mathrm{H}}_2\mathrm{O}\to {\mathrm{C}\mathrm{H}}_4+{\mathrm{H}\mathrm{C}\mathrm{O}}_3^{-}$	−31
Hydrogenotrophic methanogenesis	${\mathrm{C}\mathrm{O}}_2+4{\mathrm{H}}_2\to {\mathrm{C}\mathrm{H}}_4{+2\mathrm{H}}_2\mathrm{O}$	−131

Table 3. The corresponding free energy of ( $∆{G°}^{\prime }$) important methanogenic reactions [8].

Functional Step	Reaction	$∆{\mathit{G}°}^{\mathit{\prime }}$ (kJ/mol)
Acetotrophic methanogenesis	${\mathrm{C}\mathrm{H}}_3^{-}{\mathrm{C}\mathrm{O}\mathrm{O}}^{-}+{\mathrm{H}}_2\mathrm{O}\to {\mathrm{C}\mathrm{H}}_4+{\mathrm{H}\mathrm{C}\mathrm{O}}_3^{-}$	−31
Hydrogenotrophic methanogenesis	${\mathrm{C}\mathrm{O}}_2+4{\mathrm{H}}_2\to {\mathrm{C}\mathrm{H}}_4{+2\mathrm{H}}_2\mathrm{O}$	−131

4. Challenges and Prospects

In the context of the ‘dual carbon’ goals, minimizing greenhouse gases during the formation and transport of ‘waste streams’ is also a topic of concern. Anaerobic digestion (AD), a sustainable and widely adopted technology for the treatment of organic solid wastes (OSWs), plays a pivotal role in this endeavor. While previous research has predominantly focused on material changes at the macro-level, microbial dynamics at the microscopic level has received less attention. Advances in microbial metabolomics now enable a more detailed exploration of anaerobic treatment mechanisms at the microbial scale. This review addresses the varying methanogenic efficiencies observed during AD of different OSWs. We systematically summarize the shared electron transfer mechanisms, functional microorganisms, and metabolic processes across various OSWs, as well as the distinct microbial community structures and metabolic pathways arising from OSW-specific compositions.

Due to the thermodynamic advantages and stability under certain adverse conditions of the hydrogenotrophic methanogenic pathway, it is feasible to consider transformation of the dominant methanogens from acetotrophic methanogenesis towards hydrogenotrophic methanogenesis through external interventions, thereby improving system stability and methane production efficiency. Furthermore, systems where the hydrogenotrophic methanogenic pathway predominates could be utilized for hydrogen production by inhibiting the methanogenesis stage, particularly when using substrates with low C/N ratios, such as WAS or livestock manure.

Additionally, the TCA cycle and W-L pathway demonstrate remarkable stability, showing minimal susceptibility to inhibitory factors across different OSWs. Under anaerobic environments, the TCA cycle becomes suppressed; thus, the system enhances glycolysis and amino acid metabolism pathways to maintain essential energy supply. Similarly, acetogens utilize the W-L pathway for both energy conservation and autotrophic carbon assimilation. Therefore, destabilizing the TCA cycle could serve as a critical breakthrough for significantly enhancing AD efficiency. This approach would reduce anaerobic bacteria’s reliance on the TCA cycle for energy production, thereby promoting a more thorough utilization of organic substrates such as glucose and amino acids to maintain energy supply.

5. Conclusions

This study investigated the impact mechanisms of different inhibitory factors on the microbial community structure and metabolic pathways during the AD of four types of widely available and abundant OSWs, including waste-activated sludge, food waste, livestock manure, and straw. This research revealed several similarities and differences in the functional microbial community structure and metabolic pathways under varying substrate compositions, with particular emphasis on the synergistic interactions among key functional microorganisms (such as hydrolytic bacteria, hydrogen-producing bacteria, acetogens, and methanogens) and their response characteristics to environmental conditions. The main conclusions of the review are as follows: (1) The hydrogenotrophic methanogenesis pathway exhibits significant advantages in enhancing the methane production potential of AD. (2) The systems dominated by the hydrogenotrophic methanogenesis pathway also demonstrate advantages in hydrogen production. (3) Furthermore, the TCA cycle and the Wood–Ljungdahl pathway serve as critical regulatory points that affect methanogenesis and hydrogen production efficiency. This study not only provides crucial theoretical support for optimizing AD processes but also offers key scientific insights and technology pathways for improving energy (CH₄) recovery efficiency from OSWs. It holds significant importance for advancing sustainable waste management practices.