1. Introduction
The growing global emphasis on renewable energy production and sustainable waste management has established anaerobic digestion (AD) as a critical technology for addressing energy insecurity stemming from inadequate solid waste management, a challenge that remains one of the most persistent global challenges. Annually, more than two billion tons of solid waste are generated, causing significant environmental harm [1]. AD is a biological process that decomposes organic material in the absence of oxygen, producing biogas, a methane–rich mixture (CH4) suitable as an energy source, and a nutrient-rich digestate that can serve as fertilizer [2]. The AD process comprises four main stages: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. During hydrolysis, carbohydrates, lipids, and proteins are broken down into simpler molecules such as glucose and amino acids. Acidogenesis then converts these molecules into acetic acid, carbon dioxide (CO2), and hydrogen (H2). Acetogenesis further transforms these products into methanogenic substrates, including volatile fatty acids (VFAs). In the final stage, methanogenesis, methanogenic bacteria convert VFAs into CH4 [2]. The resulting biogas is rich in CH4 and CO2, with trace amounts of other gases such as hydrogen sulfide (H2S) and carbon monoxide (CO). Transforming biomass into biogas through AD is considered one of the most energy, and environmentally, efficient approaches [3]. The versatility of AD allows for the treatment of diverse substrates, including food waste, animal manure, sewage sludge, agro-industrial waste, and other organic materials [1,4–9].
Temperature is a critical parameter that significantly influences the efficiency and stability of the AD process [10–12] . It directly affects microbial activity, metabolic pathways, and substrate solubilization, which, in turn, impact the rate and yield of biogas production, as well as the composition of the microbial community within the digester [13–16]. Reported temperature regimes for AD include the psychrophilic range (below 30°C) [17], mesophilic range (approximately 30 to 40°C) [18–20], and thermophilic range (approximately 50 to 60°C) [12,13,21], as well as strategies involving temperature shifts [5,22,23]. Elucidating microbial dynamics during the digester recovery period is essential for understanding the relationship between microbial communities and digester performance. Microbial communities respond to parameters such as substrate, temperature, and hydrogen potential (pH) during periods of stability, disturbance, and recovery. Identifying microbial populations that optimally respond to AD conditions can enhance methanogenesis by generating more CH4 content in biogas. This knowledge supports the development of effective strategies to prevent inhibitors in biogas production [24,25].
Correspondingly, pretreatments have been identified as effective strategies to optimize this process, aiming to improve the biodegradability of the substrate by disintegrating complex structures, such as lignocellulose in agricultural waste, or by solubilizing organic matter in sludge [7,26]. The most common methods are physical, chemical and biological pretreatments, as well as a combination of these [27,28]. Among them, physical pretreatment has a simple operation model, thus less environmental pollution, but its cost is high [27]. Biological pretreatments are highly effective, but the type of substrate is a determinant factor in their application [10,29]. Also, chemical methods are more cost-effective, easier to operate, and less polluting [27]. Physical pretreatments, such as electric field pulses to disintegrate biomass, maceration, ultrasonic disintegration, have been widely applied in recent years [30,31]. The results of such studies indicate that hydrolysis and methanogenesis can be improved by up to 30% in physical pretreatment. Chemical pretreatment methods, e.g., addition of acids and alkalis, thermochemical pretreatment, and thermobaric treatments, have also been shown to increase hydrolysis and CH4 yield by up to 60% [31–33]. However, such effects are attributed to the type of substrate; consequently, some of these differ in results [34]. Nevertheless, there is a need to synthesize current knowledge on the factors that enhance methanogenesis and biogas production, including temperature as primary methanogenesis accelerator, regarding yielding more CH4, and as a conditional modulator that depends on different organic wastes and parameters. While numerous studies have explored this factor, the variety of substrates and operating strategies may lead to seemingly contradictory results and incomplete understanding of optimal temperature conditions across different scenarios [3,17,35].
Among techniques for renewable energy generation, various studies have reported that microbial electrochemical systems (MESs) comprise a diverse group of bioelectrochemical technologies, including microbial fuel cells (MFCs) [36], sediment microbial fuel cells (SMFCs) [37], microbial electrolysis cells (MEC) [38–40], microbial desalination cells (MDCs) [41], microbial reverse-electrodialysis cells (MRCs) [42], and microbial electrosynthesis cells (MESCs) [43], which have been explored for a wide range of applications such as electricity generation, hydrogen production, desalination, and the synthesis of value-added chemicals. Although these systems share common electrochemical principles, their targeted biological processes and energy carriers differ substantially.
In this context, the present review places particular emphasis on MES configurations that are directly associated with CH4 production, namely MECs operated under electromethanogenic conditions. This focus does not aim to encompass the full spectrum of MES applications, but rather to highlight those systems in which methanogenic pathways are explicitly involved, allowing temperature-related effects on CH4 formation, microbial adaptation, and process performance to be examined in a more consistent and comparable manner [38,44,45].
Against this background, a Systematic Literature Review (SLR) is conducted to examine how temperature is addressed within studies focused on biogas production, particularly in relation to the methanogenic step. Rather than treating temperature as an isolated or universal intensification factor, this review adopts temperature as a selection criterion to organize and analyze the available evidence on methane production under different operational regimes. In doing so, the review aims to identify patterns, limitations, and emerging approaches that have not yet been sufficiently consolidated in the literature, thereby contributing to the broader understanding of renewable energy systems and environmental sustainability. Accordingly, the main objective guiding this work is to assess whether temperature functions as a primary accelerator or a conditional modulator of methanogenesis in biogas production, based on a cross-study comparative synthesis of anaerobic digestion strategies. Additional figures and tables are provided in the supplementary materials (Table S1 and Fig. S1).
2. Methodology
Given the uneasiness in investigating scientific approaches to accelerating methanogenesis by increasing CH4 yield in biogas production, this study used a systematic review methodology. The present one was feasible according to the updated PRISMA2020 (Preferred Reporting Items for Systematic Review and Meta-Analyses) guidelines [46], which provide a framework for selecting, evaluating, and synthesizing studies for research of this type. Along these lines, to select reliable and objective sources, the search for studies was conducted using research publication, such as original articles. For this purpose, recognized journals indexed in specialized databases were consulted: Scopus, a database of multidisciplinary character due to the availability of huge publications in various academic fields [47]; and Web of Science (WoS), a database with a considerable scientific content, of high impact worldwide, in the fields of engineering, science, social sciences, art and humanities [48].
2.1. Search Strategy
In short, the PICO (Population/Problem, Intervention, Comparison, Outcomes) methodology was used, which allowed us to formulate the research question, an essential action for a systematic review. For this assumption, only three components of this method were selected, which were: “Population (P)”, “Intervention (I)” and “Outcomes (O)”. Thus, in the first instance, the search terms were selected for each of the “PIO” components resulted from PICO method, which are presented in English as the default language.
For the selection of information sources, keywords related to the specific research questions previously presented in the supplementary materials (Fig. S1) were employed, which made it possible to construct the following search string in Scopus: (TITLE-ABS-KEY(“biogas production” OR “biogas generation”) AND TITLE-ABS-KEY(temperature OR t) AND TITLE-ABS-KEY (methanogenesis OR “methane production”)), which was taken to the Scopus database, and were applied to WoS database, yielding 897 and 1148 scientific articles, respectively.
The initial search was conducted on January 30th, 2025, without applying filters, using advanced search string that combined Boolean operators and wildcards to identify studies on biogas production regarding temperature influence on methanogenesis enhancing through CH4 content increase. In this search string, the “OR” operator was used to broaden coverage by including articles that contained any of the terms within the same thematic conceptual domain, while the “AND” operator was used to combine different terms, ensuring that the results simultaneously included concepts related to biogas production, methane production increase through methanogenesis acceleration, and the influence of temperature in these keyword cluster.
In the first section of the string, TITLE-ABS-KEY(“biogas production” OR “biogas generation”) terms related to the renewable energy “biogas” were grouped. In the second section, TITLE-ABS-KEY (temperature or T), the intervention variable was induced to prove its influence on methanogenesis acceleration through an enhancement in CH4 content. In the third section, TITLE-ABS-KEY (methanogenesis OR “methane production”), the main interest in this regard was on capturing studies related to the last stage of biogas production as the result foreseen within conventional AD processes.
After the initial unrestricted search, predefined filters were subsequently applied to refine the retrieved records and enhance the relevance of the evidence base. The first filter applied was the publication year (PUBYEAR > 2018 AND PUBYEAR < 2025), specifically covering the period from 2018 to 2024, to ensure that the information is recent. The second filter was applied for document type (LIMIT-TO(DOCTYPE, “ar”)) to include only articles, thereby ensuring that the sources provide information aligned to the objectives of this research. Also, the language filter (LIMIT-TO (LANGUAGE, “English”)), restricted the results to English only, as it is the predominant language in these publications. Finally, the open access filter (LIMIT-TO (OA, “all”)) was used to select all publications that are publicly available. Overall, these filters were implemented to align the search output with the objectives of this review, ensuring the inclusion of studies that met specific methodological, thematic, and temporal criteria.
After applying the filters described above, the following search string was obtained and applied to the Scopus and WoS databases, identifying a total of 390 scientific publications, 124 articles in Scopus, and 266 articles in WoS. (TITLE-ABS-KEY (“biogas production” OR “biogas generation”) AND TITLE-ABS-KEY (temperature OR t) AND TITLE-ABS-KEY (methanogenesis OR “methane production”)) AND PUBYEAR > 2018 AND PUBYEAR < 2025 AND (LIMIT-TO (DOCTYPE, “ar”)) AND (LIMIT-TO (LANGUAGE, “English”)) AND (LIMIT-TO (OA, “all”))
2.2. Eligibility Criteria
Following this same line, the information obtained from both databases in question was downloaded and transferred to Comma Separated Values (CSV) format for Scopus, and Excel format for WoS, with the aim of submitting it to a quantitative and qualitative analysis that will provide answers to the questions posed in this research. Subsequently, Fig. 1 visually presents the PRISMA2020 methodological flow applied throughout the entire process of screening and filtering the scientific literature [46].
In the first stage, referred to as Identification, the results were refined to remove duplicate articles from both databases consulted. Through this strategy, 95 duplicate documents were excluded, reducing the initial sample to 295 unique publications and ensuring a more accurate analytical basis.
In the second stage, referred to as Screening, the titles and abstracts of each article were reviewed to verify their relevance to the object of study. This process led to the exclusion of 144 articles after determining that their content was not related to the biogas production and did not provide relevant information for the influence of temperature on methanogenesis acceleration through CH4 content increase. As a result, 151 articles were retained as the filtered sample in the second stage.
In the third stage, referred to as Inclusion, a full-text analysis of the presented articles was carried out. During this final phase, the inclusion (IC) and exclusion (EX) criteria were rigorously applied. For inclusion criteria, it was considered articles with open access availability to ensure information accessibility (IC1), studies addressing the use of biogas as a renewable energy source (IC2), studies investigating empirical and theoretical methods for accelerating methanogenesis (IC3), and research articles analyzing the influence of temperature on biogas production (IC4). For exclusion criteria, the main topic of this article restricted studies whose relationship between parameters in biogas production is not relevant or objective (EX1), studies that do not address biogas production without explicitly or systematically assessing temperature-driven effects on the optimization of the methanogenesis process, including CH4 yield (EX2), articles of purely theoretical projections, with nothing original in terms of the effectiveness of the proposed methods for biogas production (EX3), and finally, studies that were written in another language than English (EX4).
Of the 151 articles, it was not feasible to retrieve 12 of them because these articles were restricted to subscription-based or paid access only (EX1), resulting in 139 articles evaluated for eligibility. Furthermore, 27 articles were excluded for deviation from the main research objective (EX2), 40 of these for lack of relevance in terms of approaches to biogas production, without a specific relationship to the optimization of the methanogenesis process (EX3), 20 for studies that only show theoretical projections, and 1 for being in a language other than English (EX4). Finally, 51 scientific articles were selected, as described in the PRISMA2020 flowchart model [49], in Fig. 2. Finally, the ZOTERO tool [50] allowed the storage of the final studies included for this review, providing an optimization in the analysis of the data corresponding to the questions of this research.
3. Results
From the analysis of the 51 articles selected according to the PRISMA2020 methodology, it was found that 36% were able to respond completely to the three questions of the PICO strategy. Specifically, 100%, 60.7% and 74.5% of the articles responded to component P, I, and O, respectively. This report is shown in Table S1, highlighting the capacity of the articles reviewed, using the PRISMA2020 statement, to contribute significantly to the research.
3.1. Critical Factors in Biogas Production
For component P, as shown in Fig. 2, biogas production is enabled by two main factors: substrate type and process parameters. Substrate biochemical composition (carbohydrates, lipids, proteins, lignin, and microbial content) determines yield and quality [4,51], with substrates like olive oil residues, manure, sludge, and agro-wastes commonly studied. Key operational parameters, such as temperature, hydraulic retention time (HRT), organic loading rate (OLR), pH, and carbon-to-nitrogen ratio (C/N) further control production efficiency [2].
3.1.1. Main substrates
In this context, the importance of the variety of substrates is highlighted, specifically, the efficiency of each one for specific processes, thus obtaining a greater trend in animal manure as the main substrate, followed by a similar trend in sludge from wastewater treatment plants and, finally, food waste.
3.1.1.1. Manure
In the first instance, 31.4% of the articles in this study address the type of substrate "Animal manure" as the biomass of choice for biogas production. Mutungwazi et al. [52] reported technological advances in metagenomics and next-generation sequencing (NGS) using four types of animal manure: bovine, equine, swine, and poultry. The substrates’ elemental composition, linked to animal diet and metabolism, significantly affects biogas production by shaping the bacterial community through carbon, nitrogen, hydrogen and sulfur levels. These factors promote specific rumen bacteria, enhancing biogas yield by reducing start-up time, and stabilizing the process [7].
On the one hand, cattle manure supports microbial growth, while equine, swine, and poultry manure require co-digestion with high-nitrogen substrates (e.g., food waste) to yield significant biogas production. G. Ren et al. [53] evaluated a co-digestion of rumen and food waste, finding that cattle manure acted as an effective pH stabilizer, thereby enhancing substrate degradation and promoting the growth of methanogenic bacteria. Additionally, it played a key role in lignocellulosic biomass pretreatment, enabling efficient VFAs degradation exceeding 75% [54].
On the other hand, Rahman et al. [55] reported a 16% increase in biogas yield when poultry manure was used as a co-substrate, attributed to its buffering capacity from alkaline compounds such as ammonia and ammonium ions, which stabilize digester pH and support microbial proliferation. However, Alkarimiah [20] highlighted several challenges associated with using chicken manure alone in AD, including high total ammonia nitrogen (TAN) and the presence of inorganic particles. Co-digestion with nitrogen-rich substrates or pretreatment is recommended to address these limitations [52]. In contrast, cattle manure’s higher C/N reduces inhibitory effects and improves biogas production [7]. Using swine manure as an inoculum with food waste and cattle manure [56] further increased biogas yields and process stability, due to its buffering capacity, alkalinity, and nitrogen content [21].
3.1.1.2. Sludge from wastewater treatment plants
Secondly, 29.4% of the articles presented discuss the use of sludge from wastewater treatment plants (SWTP). Öztep et al [57] stated that a small amount of SWTP is a crucial factor, given that this parameter allows a better purification of the organic content of the digester, favoring its dissolution for a better degradation of the organic matter by the methanogenic bacteria of the process, and a stabilization in the anaerobic digestion; consequently, a greater stability in the production of biogas, if it is combined, in turn, with another type of substrate.
This assumption is ratified by [22], who noted that SWTP must be co-digested to boost biogas production. Its high moisture, low dewaterability, and strict regulations hinder treatment. Alone, its limited biodegradability cannot mitigate the effects of inhibitors [33]. Along the same lines, Ramírez et al. [18] also found that co-digestion with biochar improves methane yields.
3.1.1.3. Food Waste
Ultimately, 17.6% of the articles correspond to food waste as the substrate of choice; thus, Adesiyan et al. [2] reported that most food waste contains high levels of dry matter, proteins, carbohydrates and lipids, which confer necessary micro and macronutrients for the purpose of being a potential substrate for biogas generation. However, this substrate is not sustainable on its own, due to its highly perishable characteristics by producing large amounts of volatile fatty acids and ammonium compounds that often inhibit the anaerobic digestion process, as a consequence of a high carbon to nitrogen ratio, which leads to acidification in the digester, requiring co-digestion with another substrate that can optimize nutrient levels, alkalinity and stability in the digester [1,56,58].
3.1.2. Essential process parameters
The relevance of certain parameters in biogas production is indicated, with temperature standing out above all others, which is an essential indicator for the intervention elucidated in this review. In addition, the pH and chemical oxygen demand factors stood out among the others.
3.1.2.1. Temperature
60.8% of the articles under consideration demonstrate a direct relationship in the influence of temperature on biogas production. Temperature has a significant impact on anaerobic digestion, affecting microbial activity, metabolic processes, and biogas yield [54]. Mesophilic (optimal range: 30 to 40°C), thermophilic (50 to 60°C) and psychrophilic (low temperatures; below 30°C) conditions influence microbial communities according to substrate type and pretreatment, which, in turn, affects methane production [17,21,59]. Temperature should be evaluated alongside factors such as pH, OLR, COD, toxins and nutrients [60,61]. Its influence also depends on substrate availability, underscoring the importance of controlling hydraulic retention time [62].
3.1.2.2. Chemical oxygen demand
About 29.4% of the studies identify chemical oxygen demand (COD) as a key factor in biogas production, with a clear link with temperature. Higher COD indicates greater organic availability, as seen in the digestion of food waste and other materials, and co-digestion studies with food waste and poultry manure that achieved high biogas yields [4,51,58]. COD is also used to assess methanogenic potential and process efficiency through its reduction during digestion [54,63]. Generally, higher COD levels enhance biogas production when the material is biodegradable and conditions are optimal. Nonetheless, certain substrates and parameters can inhibit different AD stages, thereby lowering productivity; identifying these inhibitors is therefore essential [6,64].
3.1.2.3. Hydrogen potential
27.5% of the articles implicitly suggest a relationship between hydrogen potential (pH), as an indicator, and biogas production. Although they do not focus in detail on a direct analysis of its influence on methanogenesis, its effect on the first stages of biogas production is evident, indicating that a variability in pH is directly related to CH4 production. Reinforcing the previous argument, Mohamed et al. [65] assumed pH to be a critical parameter for biogas production, requiring continuous monitoring. On day 8, pH dropped to 4.2 due to VFAs accumulation during hydrolysis, creating an acidic medium. By day 21, it rose to 5.77, favoring methanogen growth. Alkaline conditions improve buffering and recovery [53], while neutral pH (6.5–7.7) yields optimal performance [59]. A pH of 7 or higher supports methanogenic activity [17,66], whereas highly acidic environments inhibit it [5].
3.2. Methanogenesis Enhancement Techniques Regarding CH4 Production
To enable a structured comparison across heterogeneous AD studies, the results were organized according to three major technical approaches: co-digestion, pretreatment, and reactor design. For each approach, operating temperature, CH4 production metrics, and reported inhibitory effects were extracted using a unified framework. This organization allows temperature to be examined not only as an operational condition, but also as an intervening variable shaping process performance across techniques.
As summarized in Table 1, methane yields and inhibition profiles exhibit consistent temperature-dependent patterns across techniques, although the role of temperature differs markedly between co-digestion, pretreatment, and reactor configuration studies. In co-digestion systems, temperature primarily modulates process stability under mixed substrates; in pretreatment-based approaches, it governs treatment severity and inhibitory risk, and in reactor design studies, temperature is increasingly embedded within the process architecture itself [1,5,27]. These cross-study contrasts provide the empirical basis for identifying technique-specific trade-offs and unresolved mechanistic gaps.
3.2.1. Co-digestion
The co-digestion studies compiled in this section indicate that temperature primarily acts as a modulator of synergy, stability, and inhibition intensity rather than as an isolated driver for CH4 maximization. Under mesophilic conditions close to 32–37°C, co-digestion of food waste and manure with functional additives consistently enhanced biogas and CH4 production, effects that were associated with ammonia buffering and increased volatile solids removal, without reports of inhibitory phenomena [4,56]. Collectively, these findings suggest that within this thermal range, temperature promotes a robust operational balance when substrate composition is appropriately managed.
Conversely, when the thermal range is expanded, temperature exerts an intensifying effect on methanogenesis, leading to higher CH4 production but at the expense of increased instability. Mrosso et al. [1] showed that raising temperature from 20 to 40°C increased cumulative biogas yield by more than 70%; however, VFAs accumulation associated with banana peel-rich fractions triggered acidification and process collapse. Similarly, under thermophilic conditions (55°C), co-digestion resulted in significant gains in methanogenic potential but with clear trade-offs between yield and stability, particularly when high proportions of readily fermentable substrates were applied [21,67].
In this context, co-digestion emerges as a strategy in which temperature amplifies both system benefits and limitations. While elevated temperatures accelerate hydrolysis and CH4 production, they also intensify the accumulation of inhibitory intermediates, such as VFAs, propionate, and phenolic compounds, thereby narrowing the stable operational window [1,21,73].
The incorporation of functional materials, such as biochar, has proven to be an effective approach to decouple this thermal trade-off by significantly reducing VFAs and NH4+ accumulation [18]. Overall, these studies indicate that in co-digestion, temperature does not act independently but rather modulates the interaction among substrate composition, intermediate generation, and buffering mechanisms.
3.2.2. Pretreatments
Chemical pretreatments consistently show that temperature acts as a severity amplifier, enhancing substrate solubilization and methanogenic potential when applied within moderate ranges. Ambient-temperature conditions combined with alkaline or oxidative agents improved digestibility and CH4 yields by shortening pretreatment times; however, increased thermal severity promoted early VFAs accumulation and organic overloading, ultimately constraining subsequent methanogenesis [27,32,33,68]. In aggregate, these results indicate that temperature enhances pretreatment efficacy while simultaneously reducing operational stability.
In physical and biological pretreatments, temperature plays a more constrained role, acting primarily as a supportive operating condition under mesophilic regimes (approximately 30–37°C). Within this range, technologies such as AHP and PEF increased CH4 production and shortened lag phase without inducing persistent inhibition [30,69]. Comparably, biological pretreatments achieved peak performance at moderate temperatures when treatment duration was limited, whereas prolonged exposure promoted nutrient depletion, release of phenolic compounds, or transient acidification, offsetting initial benefits [29,72]. More thermally severe approaches, such as organosolv and torrefaction, reinforce this pattern by enhancing solubilization at the expense of releasing inhibitory intermediates unless downstream digestion is carefully controlled [70,71].
3.2.3. Reactor design
The reviewed studies on reactor design and configuration indicate that temperature shifts from being merely an operating condition to a structural component of the process, capable of redefining system stability boundaries. Fixed-bed configurations with high biomass retention sustained CH4 production under extreme psychrophilic conditions, maintaining methanogenic activity down to 4°C, albeit with a clear thermal threshold marked by severe VFAs accumulation and acidification at 3°C [17]. In mesophilic fixed-bed systems operated at high organic loading rates, thermal stability was governed by the reactor’s capacity to buffer VFAs, LCFA, and ammonia accumulation, highlighting that temperature interacts more strongly with organic loading and biomass retention than with the intrinsic substrate yield [51].
By the same token, digestion under thermally differentiated phases enabled the decoupling of accelerated hydrolysis at thermophilic temperatures from more stable methanogenesis under mesophilic conditions, reinforcing the role of temperature as a process control tool. This configuration resulted in higher yields and improved pH recovery through the more efficient conversion of VFAs [5,11].
Nevertheless, direct comparisons between mesophilic and thermophilic operation reveal clear trade-offs: while thermophilic operation can suppress competing processes such as sulfidogenesis, it may also drastically reduce CH4 production under high loading conditions or sulfur-rich substrates; in contrast, mesophilic operation sustains higher CH4 yields but promotes H2S accumulation [13]. These results indicate that reactor design can redistribute temperature-induced inhibition but cannot fully eliminate it.
Taken together, the comparative analysis of co-digestion, pretreatments, and reactor design shows that temperature consistently governs stability, intermediate accumulation, and methanogenic performance, yet its role remains fragmented across techniques. In co-digestion, temperature modulates system resilience and amplifies substrate-dependent synergies or inhibitions, without transferable thermal thresholds. In pretreatments, it is applied as a severity parameter to maximize solubilization, with limited integration of its downstream effects on metabolic reorganization during digestion. In reactor design, temperature is incorporated as an engineering tool to spatially and temporally redistribute inhibition, although its effects are interpreted largely at a macroscopic level. This crosscutting disconnect explains the coexistence of performance gains with stability losses within similar thermal ranges and highlights a central gap in the literature: the absence of a unified framework linking temperature to dominant metabolic pathways and the observed trade-offs across techniques. Addressing this gap is essential to move beyond empirical comparisons toward a mechanistic understanding that enables the rational design of temperature-dependent AD systems.
3.3. Intervention of Temperature on Methanogenic Metabolic Pathways
Regarding this assumption, the intervention of this review is shown to be the question of component I, shown in Fig. 3, in which the various articles were identified that conceived the temperature factor as an influential parameter, explicitly and implicitly, in biogas production, due to its crucial role in the anaerobic digestion process, significantly affecting microbial activity and metabolic pathways [22,54].
3.3.1. Psychrophilic regime
On the one hand of the 31 studies addressing component I (Intervention), 16.1% focus on the psychrophilic range. Psychrophilic digestion is advantageous in cold regions where heating is unfeasible, as adapted microbes function well under low temperatures due to specialized membrane proteins, lipids, and thermal response mechanisms, though with slower reaction rates [74]. Under psychrophilic conditions (≤ 20°C), the reviewed studies consistently indicate that methanogenic performance is governed primarily by kinetic constraints rather than thermodynamic limitations. Low temperature suppresses hydrolysis and acetogenesis rates, leading to transient accumulation of intermediates, particularly acetate (CH3OOH), whose direct conversion becomes unfavorable. Consequently, methanogenesis is predominantly sustained via hydrogenotrophic pathways coupled to syntrophic acetate oxidation (SAO), forming highly interdependent consortia. This is evidenced by the predominance of Methanomicrobiales, accounting for 67.1% of the archaeal community in a fixed-bed reactor operated between 10 and 4°C [17], markedly exceeding Methanosarcinaceae and Methanosaetaceae, especially in the attached sludge fraction, which exhibits higher metabolic activity [75,76]. Given that Methanomicrobiales are strictly hydrogenotrophic, their dominance confirms CO2/H2 conversion to CH4 as the functionally prevailing methanogenic route at low temperatures, with genera such as Methanoculleus playing a central role due to their high H2 affinity [17,66].
In contrast, acetoclastic methanogenesis plays a limited role under this thermal regime. Although Methanosaeta and Methanosarcina are detected, the accumulation of acetate and propionate at ≤ 4°C indicates that this pathway is kinetically constrained and unable to sustain CH4 production, reflecting a loss of acetoclastic efficiency and a concomitant shift toward hydrogenotrophic communities [66,77].
3.3.2. Mesophilic Regime
On the other hand, 83.8% of the studies focus on the mesophilic range, which favors microbial adaptation at moderate temperatures and supports stable organic matter degradation and biogas production [26,78]. Diverse “thermotolerant consortia” thrive between 30–45°C, enhancing process stability and tolerance to fluctuations while requiring less temperature control [22,78]. However, factors such as substrate type, OLR, inoculum, and pH can trigger abrupt temperature shifts, potentially harming microbial communities, particularly methanogens. For that matter, lots of works investigated a crucial temperature on mesophilic conditions (37°C) for ensuring process stability through initial acclimatization of methanogens [10,14,55,63,65]. For instance, [2] reported that mesophilic digestion at 37°C produced 50% more biogas that thermophilic digestion at 55°C, mainly due to ammonia inhibitions at higher temperatures and the lack of thermophilic microbes in food waste. In contrast, mesophilic conditions favor greater microbial stability and gas yield.
Along the same lines, mesophilic stability at this temperature range is reinforced by higher microbial diversity and functional redundancy, as well as by the presence of archaea with high metabolic plasticity capable of tolerating moderate variations in organic loading and inhibitors [6,14]. Accordingly, mesophilic conditions are commonly used as a baseline reference across the reviewed literature, both in terms of energy performance and process controllability, even then they do not represent absolute productivity maxima [5,57].
This is attributed to the dominance of direct acetoclastic methanogenesis, which reduces the need for complex syntrophic couplings and confers high resilience to operational fluctuations [26,79,80]. The analyzed studies consistently indicate that acetoclastic methanogenesis is the prevailing metabolic pathway under mesophilic regimes, supported by a clear dominance of archaea specialized in direct acetate consumption [57,81]. For instance, Yan et al. [66] showed that at 25 and 35°C, more than 50% of VFAs corresponded to acetate, which was directly associated with a high relative abundance of Methanosaeta (65–67%) within the archaeal community, confirming acetate-to-CH4 conversion as the primary CH4 production mechanism under mesophilic conditions. This pattern was consistent with process stability, as evidenced by the absence of significant propionate accumulation and CH4 contents exceeding 78% during the active digestion phase.
Complementarily, [6] reported that in reactors operated within the mesophilic range (38–42°C), the methanogenic community was dominated by members of the families Methanosarcinaceae and Methanosaetacea, whose functional coexistence enabled sustained high conversion rates without system destabilization. The study demonstrated that under mesophilic conditions, the acetoclastic pathway was not only active but also operationally resilient [80], remaining stable under variations in organic loading without requiring dominant coupling to hydrogenotrophic routes [82,83].
Across the reviewed studies, shifts toward Methanosarcina or hydrogenotrophic methanogens were generally observed when generally observed when systems were exposed to temperature increases beyond the mesophilic range [14,22,23,84] . However, [84] demonstrated that Methanosaeta exhibits marked thermotolerance, persisting across both mesophilic and thermophilic regimes, and can therefore be regarded as a highly resilient acetoclastic archaeon in terms of CH4 production. Consistent with this mesophilic behavior, Methanosaeta dominance may nevertheless be displaced by Methanosarcina even in the absence of thermal shifts, when environmental perturbations compromise direct acetoclastic kinetics.
Factors such as increased organic loading, pH reduction, high acetate availability, or the presence of salts and ammoniacal nitrogen do not alter the dominant metabolic pathway but do favor organisms with higher tolerance and specific growth rates, allowing Methanosarcina to function as a stabilizing mechanism within the mesophilic regime [85–87]. In this sense, mesophilia should not be interpreted as a fixed microbial state, but rather as a stable metabolic space within which community structure can reorganize without losing the dominant methanogenic pathway.
3.3.3. Thermophilic regime
Lastly, from the 31 articles reviewed to address the second question, 70.9% focused on the thermophilic temperature range. Higher temperatures in this range can accelerate reaction rates and, in some cases, increase biogas and methane production [5,54,65,88]. However, maintaining these temperatures requires more energy and increases sensitivity to fluctuations in methane output, often due to fermentation failures caused by power outages, mechanical issues, or human error, which can reduce biogas yields [22,55,62].
Under this framework, the reviewed studies converge on a central finding: hydrogenotrophic methanogenesis emerges as the dominant metabolic route, accompanied by a profound reconfiguration of the archaeal community and a loss of functional relevance of direct acetoclastic pathways. Yan et al. [66] showed that increasing temperature from 35 to 45 and 55°C induced a sharp decline in Methanosaeta abundance, from 66% to <3%, concomitant with a marked rise of Methanosarcina at 45°C and, subsequently, strictly hydrogenotrophic methanogens such as Methanoculleus and Methanothermobacter at 55°C. This taxonomic shift correlated with an explicit transition in methane formation mechanisms from acetoclastic to hydrogenotrophic pathways, as evidenced by VFA profiles, relative propionate accumulation at advanced stages, and the intensification of hydrogen metabolism-related functions, indicating a greater relative contribution of H2/CO2 -associated routes [82].
Likewise, these shifts from Methanosaeta to Methanosarcina, and subsequently toward hydrogenotrophic methanogens during thermal transitions between mesophilic and thermophilic ranges, highlight that stabilization of thermophilic performance occurs only after a period of microbial adaptation. This confirms that increased CH4 production is not a direct effect of temperature elevation, but rather the outcome of metabolic reorganization within the microbial consortium [14,22,84,89].
Nevertheless, from an energy performance perspective, [6] and [66] reported that once a thermophilic community is established, cumulative CH4 production can substantially exceed mesophilic reference values, with increases of up to 81% at 55°C. These studies also agree that such overperformance is coupled with heightened sensitivity to VFAs accumulation and narrower operational margins, clearly distinguishing thermophilia as a high-productivity yet intrinsically less robust regime [26]. Overall, the evidence suggests that hydrogenotrophic methanogenic routes are more promising than acetoclastic ones [90], while indicating that thermophilic energy recovery relies on highly specialized communities whose functionality is tightly linked to biological adaptation and fine control of operating conditions, including temperature [21,91].
4. Discussions
4.1. Temperature-induced Transitions between Acetoclastic and Hydrogenotrophic Methanogenesis: Microbial and Thermodynamic Performance Integration in AD
The evidence compiled in Table 2, summarizing the analyzed systems, operating temperature regimes, reported microbial taxa, identified inhibitors and enhancers, functional evidence, and implications for future research, indicates that temperature does not exert a linear effect on AD performance. Instead, temperature acts as a selective ecological pressure that reshapes microbial community structure and redirects dominant metabolic pathways.
Across the reviewed studies, thermal perturbations, particularly within the mesophilic-thermophilic transition window (approximately 41–50°C), consistently triggered syntrophic disruption, accumulation of VFAs, and transient loss of key methanogenic populations, ultimately resulting in abrupt declines in methane productivity [51,57,84]. This recurrent “critical temperature zone” was documented by multiple authors through pronounced reductions in CH4 yield and volumetric productivity, confirming that inhibition at intermediate temperatures is not system-specific but rather a reproducible ecological phenomenon.
A second convergent pattern emerging from the reviewed studies is the biphasic response of microbial consortia to thermal stress. Initial inhibitory phases, characterized by community destabilization, proliferation of competing guilds (e.g., sulfate-reducing bacteria), accumulation of inhibitory intermediates, were systematically followed by adaptive reconfiguration once thermal exposure was sustained over time [14,15].
This adaptive phase was marked by the enrichment of hydrogenotrophic methanogens (e.g., Methanothermobacter, Methanobacterium), together with the persistent presence of archaeal taxa such as Methanosaeta across psychrophilic, mesophilic and thermophilic regimes, and the emergence of syntrophic acetate-oxidizing bacteria (SAOB). Collectively, these shifts enabled a functional transition from acetoclastic methanogenesis toward Syntrophic acetate oxidation (SAO) + H2/CO2 – driven pathways [14,82]. The recovery, and in several cases, enhancement of CH4 production following adaptation demonstrates that energy performance is governed less by absolute temperature and more by the capacity of microbial networks to reorganize under thermal pressure [6,66,70,92].
The correlations among studies, together with the compiled microbial datasets, indicate that temperature regulates AD through a coupled ecological-thermodynamic selection mechanism rather than through a simple increase in metabolic activity. Across the reviewed systems, sub-mesophilic and mesophilic conditions (25–37°C) consistently support relatively stable microbial communities over moderate residence times (approximately 20 to 30 days), where Methanosaeta/Methanothrix dominates the archaeal assemblage (approximately 65–67%) alongside smaller proportions of Methanobacterium (14–16%), [57,66,84], as shown in Fig. 5(c). This community structure favors acetoclastic methanogenesis, in which acetate is produced by fermentative bacteria such as Firmicutes, Coprothermobacter, Clostridium, and Tepidanaerobacter, and whose relatively stable abundances under mesophilic conditions are illustrated in Fig. 5(d), converted to methane through the Eq. (1), which has a standard Gibbs free energy change (ΔG°) of approximately −31.6 kJ mol−1.
Correspondingly, CH4 yields across these mesophilic systems remain stable (132 mL CH4 g−1 VS and 298 mL CH4 g−1 COD) [14,57], as observed in Fig. 4(e) and Fig. 4(f), indicating that acetate turnover remains thermodynamically balanced. From a cross-study perspective, mesophilic digestion therefore represents a kinetically stable but metabolically specialized regime, where microbial interactions are optimized for acetate-based methanogenesis but remain sensitive to environmental perturbations such as temperature shifts. This pattern indicates that temperature acts primarily as a selective ecological filter rather than merely as a kinetic accelerator of metabolic reactions.
When temperature increases into the 40–45°C range, the reviewed studies consistently identify a transitional ecological and kinetic phase, in which the incubation time required for microbial adaptation becomes critical. Within this thermal window, as illustrated in Fig. 4(a–d), CH4 yields decrease under temperature shifts despite constant residence times. At a microbial level, Fig. 5(c) shows that the abundance of Methanosaeta can decline sharply from 65% to below 3%, while Methanosarcina expands to values approaching 67%, and hydrogenotrophic methanogens such as Methanoculleus (approx. 20%) begin to emerge [66,84], while in Fig. 5(d) and Fig. 5(e), thermotolerant fermentative bacteria including Anaerobaculum, Coprothermobacter, and Clostridium, increase in relative abundance (30 to 45%), intensifying the production of H2 and CO2 [22,66]. However, this microbial restructuring does not occur immediately. Several studies report that during the initial incubation periods following temperature increases, CH4 yields decline, for instance, from 132 to 82.8–79.5 mL CH4 g−1 TVS at 40–45°C, while fermentation intermediates accumulate [57]. Mechanistically, this destabilization reflects the temporary disruption of syntrophic hydrogen turnover: when methanogenic consumption of hydrogen decreases, reactions such as propionate oxidation reaction depicted in Eq. (2), with ΔG° around + 76 kJ mol−1 become thermodynamically unfavourable. Consequently, VFAs accumulate, reaching concentrations up to 905 mg L−1, indicating that the 40–45°C interval represents a microbial adaptation window in which digestion performance becomes strongly dependent on residence time and community restructuring [57].
Once systems reach the thermophilic regime (48–55°C) and the microbial consortium completes its adaptation period, a consistent ecological reorganization emerges across the studies. As illustrated in Fig. 5(c), the methanogenic community shifts toward sustained hydrogenotrophic dominance, characterized by increases in Methanothermobacter (31%), Methanoculleus (15%), and Methanobacterium (12%), while Methanosaeta declines to below 2% [66,84]. Simultaneously, Fig. 5(e–g), thermophilic fermentative bacteria such as Coprothermobacter, Petrotoga, Kosmotoga, and Thermodesulfovibrio become ecologically prominent, producing hydrogen and acetate that are rapidly consumed by hydrogenotrophic methanogens [22,84]. Under these conditions, CH4 formation proceeds primarily through the Eq. (3), which is substantially more exergonic than acetoclastic methanogenesis.
When microbial communities successfully reorganize toward this thermophilic syntrophic network, as observed in Fig. 4(e), CH4 productivity can increase markedly, with reported yields of 905 ± 50 mL CH4 g−1 VS at 55°C, approximately 81% higher than those observed at 25°C [66]. These results indicate that thermophilic digestion can achieve higher CH4 productivity once microbial adaptation stabilizes hydrogen-mediated electron transfer within the reactor. Overall, the comparative synthesis suggests that AD under thermal gradients follows a time-dependent metabolic adaptation model governed jointly by temperature and incubation time. Initially, mesophilic systems operate through stable acetoclastic networks sustained by moderate residence times; as temperature increases, the system undergoes a microbial reorganization phase in which performance depends on the time required for syntrophic interactions to reestablish metabolic equilibrium; finally, after thermophilic adaptation, a community dominated by hydrogenotrophic methanogens and thermotolerant fermentative bacteria becomes established.
Importantly, the temperature-driven “acceleration of methanogenesis” reported across studies reflects two distinct process mechanisms. In mesophilic regimes, improvements in CH4 production primarily represent kinetic enhancement, where increased temperature accelerates enzymatic reaction rates while the dominant metabolic pathway remains acetoclastic [14,66]. In contrast, the higher CH4 yields frequently observed under thermophilic conditions reflect thermodynamic capacity, arising from ecological selection of hydrogenotrophic pathways that are energetically more favorable for CH4 formation [66]. Collectively, these observations support an emergent metabolic temperature model of AD, in which temperature acts as an ecological selection pressure that restructures microbial communities, reorganizes syntrophic metabolic networks, and ultimately determines the thermodynamic efficiency of CH4 production. Thus, temperature-driven changes in AD should be interpreted as ecosystem-level reorganizations of metabolic networks rather than simple thermally accelerated reactions.
4.3. Electromethanogenesis-based Systems: MES/MEC as Strategies to Overcome Temperature Constraints
Despite the general trends identified, the cross-study analysis reveals significant inconsistencies in the effect of temperature on AD performance. While some systems show substantial increases in CH4 under thermophilic conditions (e.g., 905 ± 50 mL CH4 g−1 VS in [66]), others exhibits pronounced declines when temperature is increased (e.g., from 132.2 to 76.5 mL CH4 g−1 TVS in [57]), as reflected in Fig. 4. This contradiction indicates that temperature alone is not a deterministic factor, but rather interacts with microbial adaptability, substrate characteristics, and syntrophic balance. Although the transition from acetoclastic to hydrogenotrophic methanogenesis is consistently observed (Fig. 5(a–c)), the rate, extent, and stability of this shift remain poorly understood, especially within the transitional 40–45°C range where process instability and VFA accumulation frequently occur. Moreover, the dual role of fermentative bacteria (Fig. 5(d–g)) introduces further uncertainty, as increased hydrogen production can either enhance CH4 formation or destabilize the system depending on methanogenic consumption capacity.
These limitations also have direct implications for the integration of AD with microbial electrochemical systems (MES/MECs). In particular, the recurrent instability observed in the 40–45°C transitional range and the frequent uncoupling between fermentative hydrogen production and methanogenic consumption suggest that electron flow, rather than substrate availability alone, becomes the limiting factor under temperature stress [38,93]. MES/MEC technologies offer a potential solution by facilitating extracellular electron transfer, thereby stabilizing redox balance and enhancing syntrophic interactions when conventional hydrogen-mediated pathways are kinetically or thermodynamically constrained. This exposes a conceptual and technological gap between adaptive AD and ecologically directed process control.
In this context, Bhatt et al. [92] provide direct evidence that thermal inhibition is not an absolute constraint on the energy performance of AD, but rather a condition amenable to modulation through MEC substantially reversed the kinetic limitation imposed by low temperature: at 18°C, conventional operation yielded nearly negligible biogas production (approx.. 0.2 cm3), whereas electro-assisted operation increased production to 13 cm3, representing an order of magnitude enhancement, along with a marked improvement in energy conversion, as reflected by the increase in CH4 content from 30% to 80%.
The authors attribute this behavior to the reinforcement of interspecies electron transfer, facilitated by an external electron supply, and to the sustained activity of hydrogenotrophic methanogens (primarily Methanobacterium), even under unfavorable thermal conditions [94,95]. Notably, these findings are particularly relevant considering the widespread inhibition observed at low temperatures in conventional AD systems, as they demonstrate that thermal constraints are not fixed barriers but rather controllable parameters from a redox and process-engineering perspective. Accordingly, this low-temperature electromethanogenesis evidence, characterized by increased biogas production and substantial improvements in CH4 fraction, confirms that energy performance can be restored through the reinforcement of hydrogenotrophic pathways and interspecies electron transfer [35].
By comparison, the evidence synthesized in Table 3 allows temperature-induced inhibition and enhancement patterns observed in conventional AD to be reinterpreted as design-contingent limitations rather than intrinsic constraints of methanogenic metabolisms. Specifically, studies in Table 3 lacking MES/MEC consistently report transient collapses in energy efficiency during thermal upshifts and downshifts, which are associated with kinetic imbalances in CH4 production, VFAs accumulation, free NH3 stress, or loss of acetoclastic archaea. In contrast, the studies summarized in Table 3, which incorporate electrochemical systems, demonstrate that methanogenic activity can be sustained, or even intensified, without the need for strict thermal optimization [35,96].
The inclusion of MES, particularly MECs oriented to electromethanogenesis, should be interpreted within the broader framework of temperature-regulated AD rather than as an independent technological pathway. Across the studies reviewed, temperature was consistently identified as a key factor governing methanogenic community structure, metabolic pathway dominance, and system stability. In particular, the recurrent dominance of electrostimulated hydrogenotrophic archaea, such as Methanobacterium, Methanoculleus, Methanosarcina and Methanobrevibacter, in methanogenic biocathodes, together with efficient operation at low applied voltages and ambient temperatures within the mesophilic range [44,96], can offset the kinetic penalties associated with suboptimal thermal conditions. This indicates that the applied potential functions as a selective ecological filter, enforcing metabolically robust pathways that are more resilient to thermal and operational fluctuations.
This is because hydrogenotrophic microorganisms play a pivotal role in methanogenesis by consuming H2 and CO2 to produce CH4, thereby maintaining low H2 partial pressures that allow fermentative bacteria to remain thermodynamically favorable [99–101]. Accordingly, their enhancement in AD-MEC systems can be synergistically coupled with other electroactive microorganisms present at the cathode, improving interspecies electron transfer. This metabolic integration promotes pathway complementarity and, consequently, accelerates methanogenesis, resulting in increased CH4 production [102–104].
Similarly, several studies report that hydrogenotrophic methanogenesis contributes approximately 30–86% of total CH4 production in biogas systems [105,106], positioning hydrogenotrophic methanogens as highly productive agents. Their persistence and proliferation are attributed to their tolerance to environmental stressors, including cathodic redox imbalances arising from limited H2 evolution catalysis, as well as ammonia toxicity, organic acid accumulation, and temperature fluctuations [45,96].
4.3.1. Temperature regulation and electrochemical modulation of methanogenesis
Temperature is widely recognized as a key factor regulating methanogenic activity in AD, as it directly influences microbial metabolism, mass transfer rates of electroactive bacteria (EAB), enzymatic kinetic, and community structure. Variations in temperature determine the predominance of specific metabolic pathways and microbial guilds, often inducing shifts between acetoclastic and hydrogenotrophic methanogenesis. In combination with factors such as inoculum characteristics, substrate type, electrode material, and reactor configuration, temperature interacts with operational parameters including pH, HRT, OLR, and COD, ultimately influencing CH4 production and biogas yield [107–110].
Within this temperature-regulated framework, microbial electrochemical systems, particularly methane-oriented MECs, provide additional insight into how thermal constraints can be modulated. The key distinction lies in the applied voltage or controlled electrode potential, which modifies how temperature affects microbial metabolism [38]. By introducing an electrochemical driving force that supplies reducing equivalents directly to methanogenic pathways, these systems shift process dependence from purely temperature-driven enzyme kinetics toward electron availability. As a result, several studies report stable or enhanced CH4 production without requiring thermophilic operation [44,96].
According to the studies analyzed, MEC/MES systems are frequently operated at ambient or sub-mesophilic temperatures (approximately 25–32°C). Compared with conventional AD systems [6,22,64], AD-MEC configurations can maintain methanogenic activity under these conditions without additional heating, thereby reducing operational costs and minimizing inhibition risks while improving overall energy efficiency [105,109]. The dominance of hydrogenotrophic methanogens under these conditions further supports this interpretation, as voltage-mediated H2 generation or direct electron transfer compensates for reduced metabolic rates at lower temperatures.
Experimental evidence highlights this interaction between temperature and electrochemical control. For example, [111] demonstrated that bioelectrochemical systems enhanced CH4 yield, and acetate removal even at 10°C. In a combined BES-AD configuration operated at a cathodic potential of −0.9 V (Ag/AgCl), CH4 yield reached 31 mg CH4-COD/g VSS, representing a 5.3–6.6-fold increase compared with conventional AD at the same temperature. Similarly, studies conducted at constant ambient temperature [35,97] show that adjusting electrode potential alters CH4 productivity and microbial community composition without modifying thermal conditions, confirming voltage as an independent control variable capable of mitigating temperature-related limitations [112,113].
Nevertheless, the applied electrical potential significantly influences microbial diversity and CH4 production [114,115]. Accordingly, several studies report optimal voltage ranges for external application, typically between 0.2–0.8 V and 0.1–1.8 V [38,109,116]. For instance, Guo et al. [117] showed that low voltages (≤ 0.5 V), even with increased cathode surface-to-anode volume ratios (1–4 cm2 cm−3), had negligible effects on CH4 yield, whereas higher applied voltages (0.7–0.9 V) markedly enhanced CH4 production kinetics. Similarly, Park et al. [118] reported a 51% decrease in CH4 production when interelectrode spacing increased from 1 to 5 cm at an applied voltage of 0.3 V. Collectively, these findings confirm that retention of a methanogenic biofilm at the cathode is critical for enhancing CH4 production rates, as increased electrode spacing likely raises internal resistance, reduces effective current, and promotes parasitic losses, thereby limiting electron flux available for stimulating productive metabolic pathways.
Overall, these findings indicate that electrochemical control does not replace the role of temperature but introduces an additional regulatory layer capable of modulating its influence. While conventional AD relies primarily on thermal optimization to enhance microbial kinetics, electromethanogenic systems demonstrate that redox control can partially alleviate temperature constraints while sustaining CH4 production. Consequently, MES/MEC studies complement the understanding of temperature-driven AD by showing that thermal effects on methanogenesis can be modulated through electrochemical intervention.
5. Future Directions for Temperature-resilient Methanogenesis Systems
Therefore, it is imperative to consider voltage-associated factors, such as interelectrode spacing, when optimizing this independent control variable and assessing its coupled effects on process temperature and pH. Taken together, the evidence supports a conceptual reinterpretation of MES/MEC-assisted systems, in which temperature becomes a secondary and bounded variable, while voltage emerges as the primary lever governing energy performance and metabolic pathway selection. Rather than optimizing operation within a narrow thermal window, future systems may operate across broader temperature ranges by tuning electrochemical parameters, coupling applied electrical potential, pH regulation, and microbial adaptation [35,98]. Under this framework, temperature is transformed from a rigid operational constraint into a flexible background condition actively shaped by electrochemical intervention. From a future research perspective, this opens a promising avenue for temperature-electroactivity coupling strategies, where MES/MEC systems are used to regulate electron flux during thermal transitions. Key priorities include evaluating how electrode-assisted electron transfer influences microbial adaptation timescales, determining whether MES integration can mitigate VFA accumulation in the 40–45°C range, and assessing the role of electroactive fermentative consortia in enhancing process stability. Ultimately, combining temperature control with bioelectrochemical regulation may enable a shift from passive thermal optimization toward active control of microbial metabolism, offering a more robust framework for high-efficiency CH4 production.
A first critical research gap lies in the decoupling of temperature from metabolic control. The reviewed literature shows that most AD studies interpret temperature as an external stressor, rather than as a variable whose effects are mediated by electron availability and syntrophic coordination. Future research should explicitly test whether thermal sensitivity is a consequence of insufficient interspecies electron transfer rather than an intrinsic temperature limit. This requires experimental designs where temperature transitions are systematically combined with controlled redox manipulation, rather than in isolation.
A major prospect concerns the engineering of functional redundancy and resilience. Conventional thermophilic systems often exhibit reduced redundancy and higher vulnerability to perturbations, whereas MES-assisted systems show that selective enrichment of hydrogenotrophic methanogens can be imposed independently of inoculum composition. Future work should focus on whether electrochemical stimulation can deliberately enhance functional redundancy, particularly during temperature transitions, by stabilizing syntrophic networks and preventing community collapse.
From a microbiological standpoint, future studies should move beyond taxonomic descriptions and focus on electron-transfer functionality as a core selection pressure. The recurring dominance of Methanobacterium under electrochemical control suggests that electroactivity and hydrogen-scavenging capacity may outweigh temperature adaptation alone. Linking metagenomics, electrochemical performance, and metabolic flux analysis will be critical to establish causal relationships between electron supply, pathway dominance, and temperature tolerance.
Finally, from a process-engineering perspective, temperature gradients are inevitable at scale, yet none of the MEC studies explicitly examine how coaxial geometries respond to axial or radial thermal heterogeneity. This represents a third gap. Future investigations should assess whether coaxial electrodes, by maintaining uniform interelectrode distance and minimizing ohmic losses, can buffer localized temperature drops and sustain methanogenesis more homogeneously than planar designs. Such studies are especially relevant for low-energy or decentralized applications where active heating is undesirable.
6. Conclusions
This systematic review, conducted under the PRISMA2020 guidelines and based on a PIO-driven screening of 51 articles scientific studies, highlights the multifactorial nature of methanogenesis acceleration for optimized biogas production. The evidence confirms that both substrate characteristics, particularly animal manure, sewage treatment plant sludge, food waste, and process parameters, with emphasis on temperature, pH, and COD, jointly determine CH4 production performance. Temperature emerges not as universal accelerator, but as a conditional regulator of microbial activity and metabolic routing. Among the selected studies, 31 articles (60.7%) explicitly examined temperature effects, with reported distributions of 16.1% (psychrophilic), 83.6% (mesophilic), and 70.9% (thermophilic) regimes. Overall, mesophilic conditions (approximately, 30–40°C) consistently provided the most balanced performance, combining stable operation and high CH4 yields. In contrast, psychrophilic regimes were commonly associated with kinetic limitations, while thermophilic operation, despite enhanced reaction rates, frequently entailed higher energy demand, reduced process stability, and increased susceptibility to inhibition.
From an operational perspective, these findings indicate that temperature optimization must be weighed against heating energy requirements and inhibition risks, rather than CH4 yield alone. Importantly, evidence from microbial electrochemical systems (MES), particularly methane-oriented MEC configurations, suggests that electrochemical assistance can partially decouple methanogenesis from strict thermal optimization, sustaining hydrogenotrophic pathways and CH4 production under ambient or sub-mesophilic conditions. Future research should move toward integrated MES/MEC-assisted anaerobic systems in which electrochemical control, rather than temperature alone, governs metabolic routing and process performance. Priority should be given to experimentally decoupling temperature from metabolic regulation through controlled redox manipulation, assessing the role of electron availability and syntrophic coordination during temperature transitions. From a microbiological and engineering standpoint, advancing functional redundancy, resilience under thermal heterogeneity, and scalable electrode configurations will be critical, alongside standardized reporting of thermal conditions in electrochemical systems. Collectively, these directions reposition temperature as a bounded background variable, enabling the design of anaerobic processes that are energetically efficient, robust and adaptable to fluctuating operational conditions.
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