Various studies focused on the advantages and limitations of mixed microbial consortia compared with pure cultures for maximum hydrogen yield via dark fermentation. Some studies state that pure cultures provide high metabolic specificity and reproducibility, allowing precise methodological analysis and high hydrogen yields under controlled, sterile conditions (Chaganti et al., 2012) . Pure cultures are highly sensitive to contamination and operational fluctuations. Compared to pure cultures, mixed microbial consortia exhibit greater functional diversity and process stability, particularly when treating complex and heterogeneous substrates such as wastewater and agricultural wastes (Ghosh et al., 2012). This increased reliability occurs with metabolic competition, as hydrogen-producing microorganisms coexist with hydrogen-consuming and solvent-producing species. In spite of this challenge, many studies have shown that suitable inoculum pre-treatment and careful control of operating conditions can preferentially enrich hydrogen-producing groups within mixed cultures, enabling stable and sustained hydrogen production without the need for precise sterility.

The attributes of the inoculum have a strong impact on the initial development and proliferation of the microbial community in dark fermentation systems. Cultures obtained from anaerobic sludge, compost, manure, and soil consists of different microbial assemblies with varying hydrogen producing capacity. The most preferred source of inoculum is anaerobic sludge and compost, as they contain a large number of fermentative and spore-forming bacteria such as Clostridium species, which have a well-adapted hydrogenogenic metabolism (S.-C. Zhang et al., 2016). For suppression of existing methanogens, various treatments such as heat shock, aeration, and acid exposures are applied to these cultures which inhibit the growth of methanogens and other hydrogen consuming microorganism, enabling rapid enrichment of hydrogen producing colonies. The inoculates obtained from untreated soil and manure often contain competing populations that enables the formation of ethanol and lactate diverting the process to non-hydrogen pathways, resulting in delayed hydrogen production and lower process stability (Pineda-Muñoz et al., 2020). All the microbial communities are continuously evolved as per the operational conditions. The microbial population is strongly influenced by various parameters such as pH, temperature, hydraulic retention time (HRT), and organic loading rate in all the metabolic pathways (Akutsu et al., 2009; Chaganti et al., 2013; Chandra and Venkata Mohan, 2014). Different molecular tools such as terminal restriction fragment length polymorphism (TRFLP), denaturing gradient gel electrophoresis (DGGE), and 16S rRNA gene sequencing have demonstrated that microbial communities in dark fermentation change over time (Balachandar et al., 2020). At the start of fermentation, facultative fermenting bacteria usually dominate as they quickly adapt to fluctuating conditions and residual oxygen. After the environment inside a dark fermentation reactor becomes strictly anaerobic and selective parameters such as acidic pH and short retention time are established, these early growing populations are replaced by obligate anaerobes, specially Clostridium  species, which are most suitable for sustained hydrogen production (Ren et al., 2011). Amidst various operating factors, pH plays an important role because even a small change in the production medium pH can highly affect the enzyme activity, hydrogenase function, and interactions between the microbial population (Z.-K. Lee et al., 2008; T. Zhang et al., 2022). Even the slight variations in pH shift the microbial metabolism from hydrogen producing pathway to ethanol or lactate formation routes, resulting in reduced hydrogen yields (Aruwajoye et al., 2020; Hay et al., 2013). Maintaining mesophilic conditions is also an important aspect, as temperature ranging between 20-45℃ supports the rapid growth of microbial population whereas thermophilic conditions (>45℃) selectively enrich the hydrogen producing bacteria, which helps to achieve higher hydrogen production rates. Sustained production of hydrogen via extended operation requires continuous maintenance of operational conditions that favours the acetate and butyrate pathway for fermentation while limiting the proliferation of hydrogen consuming organisms such as homoacetogens and methanogens (O-Thong et al., 2016). These studies emphasize that effective hydrogen depends on coordinated control of the interaction between inoculum properties and operation conditions.

6.       Operational Parameters for Effective Dark Fermentation Performance

The stability and efficiency of dark fermentation using agricultural residues are highly impacted by the operational parameters that stimulate microbial metabolism and process kinetics. These parameters as in interconnected manner, determine the direction of metabolic flux towards the hydrogen producing pathway or whether the process is being shifted towards reduced end product formation. pH is one of the most crucial parameters which directly affects the enzyme activity, structure of microbial community, and selection of specific metabolic pathway. Various studies report an optimal pH range of 5.5-6.5 for efficient acetate and butyrate type fermentation increasing the net hydrogen yield (Sarangi and Nanda, 2020; Sivaramakrishnan et al., 2021). At a lower pH (<5.0), the activity of hydrogenase enzyme is inhibited due to proton stress, and the microbial metabolism shift towards lactate production pathway, reducing the hydrogen yield. Inappropriately high acidic conditions also suppress the microbial activity, which results in reduced substrate utilization hence low nor no hydrogen production. High acidic conditions support the growth of hydrogen consuming methanogens specially in continuous operation systems having longer retention time (Balachandar et al., 2020). High organic loading also reduces the pH and promotes the accumulation of VFAs, which destabilizes the production medium and thus reduced hydrogen yields. Therefore, active control over pH is important to maintain metabolic balance.

                Dark fermentation of agricultural residues is generally operated under mesophilic conditions (30-40℃). Maintaining optimal mesophilic conditions efficiently determines enzymatic reaction rates and microbial growth kinetics, and are stable and energy-efficient which supports the existing microbial consortia that can tolerate fluctuations in feedstock composition (Yukesh Kannah et al., 2019). However, the hydrogen yields under mesophilic conditions are moderate. On the other hand, thermophilic conditions (50-60℃) enhance the hydrolysis rates and accelerate the metabolic reactions, while suppressing the growth of hydrogen consuming methanogens, resulting in higher specific rates of production (De Vrije et al., 2007). Hydraulic retention time (HRT) is also an important parameter in continuous flow reactors, as it controls the amount of time of residence for microbial population and their washout (Chaganti et al., 2013; H.-G. Lee and Dulany, 2025). Various studies demonstrate that HRTs generally in the range of few hours, depending upon the configuration of reactor and characteristics of the inoculum gives suitable results. However, short HRTs can result in biomass washout, incomplete substrate conversion, and unstable hydrogen production (Krupp and Widmann, 2009).

                Another factor influencing the hydrogen yields is Organic Loading Rate (OLRs), along with initial substrate concentration often represented as chemical oxygen demand (COD) (H.-G. Lee and Dulany, 2025). Slight increase in OLR generally gives enhanced hydrogen yield by providing sufficient fermentable substrates whereas, excessive increase in initial COD concentrations frequently result in inhibition in substrate utilization, leading to rapid accumulation of VFAs, decline in pH and metabolic stress (Lin et al., 2008). High COD levels can also increase hydrogen partial pressure and osmotic stress, suppressing the overall hydrogenase activity and resulting in lower hydrogen yields. After efficient pretreatment of agricultural residues, large amount of soluble COD is released into the medium, causing interruption in the stable operation. Therefore, controlled organic loading rates and staged feeding strategies are important for efficient hydrogen production. Supplementation of nutrients, especially the carbon-to-nitrogen ratio (C/N), plays an important role in directing the metabolic flux towards hydrogen production instead of biomass synthesis (Anzola-Rojas et al., 2015). Agricultural residues are generally rich in carbon sources but lack nitrogen, which requires the addition of external nutrients to the medium for supporting microbial growth and effective enzyme activity. Low C/N ratios promote biomass formation, diverting the carbon atoms away from hydrogen evolution whereas high C/N ratio inflicts nitrogen limitation, restricting synthesis of enzyme and reducing hydrogen yields. Several studies also highlight the importance of phosphorus and trace elements, indicating that effective nutrient management requires a balanced approach rather than optimization of a single parameter. Inhibitory compounds in dark fermentation arise from both substrate characteristics and process operation. Process-derived inhibitors include VFAs and ammonia. Accumulation of VFAs lowers pH and disrupts intracellular redox balance, while elevated ammonia concentrations, often associated with protein-rich substrates or excessive nitrogen supplementation, exert toxic effects on hydrogen-producing bacteria (Lin et al., 2008; Raj et al., 2015; Sarangi and Nanda, 2020). Substrate-derived inhibitors are particularly relevant for lignocellulosic agricultural residues. Pretreatment processes can generate furans and phenolic compounds, which inhibit microbial activity and hydrogenase function even at low concentrations (Garcia-Maraver et al., 2013; Yukesh Kannah et al., 2019). The severity of inhibition depends on pretreatment conditions and feedstock composition, often necessitating detoxification steps or mild pretreatment strategies. Importantly, pH control and appropriate OLR management can partially mitigate the effects of inhibitory compounds, reinforcing the interconnected nature of operational parameters.

7.       Metabolic By-products, Carbon Flow Control, and Biorefinery Integration

The metabolite profile generated during dark fermentation offers direct insight into the system’s metabolic state and its efficiency in hydrogen production. Across multiple studies, volatile fatty acids (VFAs) consistently predominate the liquid phase, with acetate and butyrate identified as the principal end products under hydrogenogenic conditions (Sarangi and Nanda, 2020). This trend appears independent of reactor configuration or inoculum source, indicating that the acetate–butyrate pathways of pyruvate metabolism constitute the fundamental biochemical routes driving hydrogen evolution. Agricultural residues rich in readily fermentable carbohydrates—such as starch-based wastes, molasses, and well-pretreated lignocellulosic hydrolysates—tend to favor butyrate-dominant metabolite profiles, whereas substrates with lower sugar content or incomplete hydrolysis exhibit higher acetate proportions and more diverse metabolite distributions (Lin et al., 2008; Raj et al., 2015). These variations reflect substrate-induced shifts in redox balance and carbon flux rather than simple changes in microbial community composition.

Hydrogen yield correlates closely with the relative distribution of acetate and butyrate, commonly represented by the acetate-to-butyrate (A/B) ratio. Although acetate formation theoretically yields the highest hydrogen, the literature indicates that stable and high hydrogen production rarely coincides with exclusive acetate fermentation (H.-G. Lee and Dulany, 2025; Lin et al., 2008). Optimal hydrogenogenic performance is generally observed at intermediate A/B ratios where butyrate predominates but acetate remains significant. Excessive acetate fractions often precede system instability, while pronounced butyrate accumulation is associated with elevated hydrogen partial pressure and diminished net hydrogen recovery. Thus, the A/B ratio serves as a practical metabolic indicator of hydrogenogenic stability rather than a strict predictor of theoretical yield, enabling real-time process monitoring.

A persistent challenge identified across the dataset is the diversion of carbon and electrons from hydrogen production toward competing metabolic pathways (Ren et al., 2011). Solventogenesis, marked by ethanol and butanol formation, is frequently linked to environmental stressors such as rapid pH decline and accumulation of undissociated acids. Under these conditions, reducing equivalents are redirected toward alcohol synthesis to maintain intracellular redox balance, causing sharp declines in hydrogen yield despite ongoing substrate consumption. Homoacetogenesis represents another, often less apparent, loss mechanism whereby hydrogen is consumed to convert carbon dioxide into acetate. Unlike methanogens, homoacetogenic bacteria often survive common inoculum pretreatments and become active under increased hydrogen partial pressure or prolonged retention times (Cao et al., 2009). Several continuous-flow studies report stable acetate production concurrent with declining hydrogen output, indicative of internal hydrogen recycling rather than net generation. The consensus from reviewed studies is that suppressing these competing pathways requires coordinated operational control rather than reliance on a single parameter. Maintaining pH within a narrow acidic range favorable to hydrogenase activity, ensuring short hydraulic retention times, and promoting efficient gas removal consistently limit both solventogenesis and homoacetogenesis. Nutrient balance further influences carbon diversion; inappropriate carbon-to-nitrogen (C/N) or carbon-to-phosphorus (C/P) ratios stimulate excessive biomass growth and secondary metabolism, indirectly fostering hydrogen-consuming pathways. Collectively, these findings underscore that effective hydrogen production depends on deliberate management of carbon flux and redox pressure within the bioreactor.

Importantly, the liquid effluent from dark fermentation is increasingly recognized not as waste but as valuable intermediate rich in short-chain organic acids. Even under optimized hydrogenogenic conditions, a substantial portion of the initial chemical oxygen demand remains in the effluent, underscoring both the limitation of single-stage hydrogen production and the opportunity for downstream valorization. Biomethanation is the most established integration strategy; multiple studies demonstrate that coupling dark fermentation with a secondary anaerobic digestion stage enables near-complete recovery of residual energy as methane while enhancing overall process stability. Two-stage systems consistently outperform single-stage digestion by decoupling acidogenesis and methanogenesis, allowing each phase to operate under optimal conditions. Photo-fermentation has also been shown to convert acetate- and butyrate-rich effluents into additional hydrogen, thereby increasing total hydrogen recovery from the same carbon pool, although light requirements and reactor complexity currently constrain scalability (Hay et al., 2013b). Emerging research further reveals the feasibility of utilizing dark fermentation effluents as substrates for biopolymer synthesis, particularly polyhydroxyalkanoates, leveraging the predictable VFA composition generated under hydrogenogenic conditions. While still nascent, these approaches illustrate how byproduct management can transform dark fermentation from a single-product process into a multifunctional platform. Taken together, the literature positions dark fermentation most convincingly as a central unit operation within integrated biorefineries rather than as a standalone hydrogen production technology (Balachandar et al., 2020; Lin et al., 2008; Raj et al., 2015; Sarangi and Nanda, 2020). As a solitary process, dark fermentation is constrained by stoichiometric limits and incomplete carbon conversion. However, when embedded within cascading systems that couple hydrogen production with methane generation, secondary hydrogen recovery, or bioproduct synthesis, agricultural residues can be sequentially exploited to maximize energy recovery and economic value. From a circular bioeconomy perspective, such integration enhances the overall energy balance, reduces effluent treatment demands, and improves process robustness—highlighting that the true potential of dark fermentation lies not in maximizing isolated hydrogen yield but in enabling flexible, integrated pathways for resource utilization

8.       Scale Up Challenges

Translating dark fermentation of agricultural residues from laboratory-scale demonstrations to commercially viable systems remains a significant challenge in the field of biological hydrogen production. Although numerous bench-scale studies report promising hydrogen yields, only a limited number have advanced to pilot-scale operations, with even fewer achieving sustained performance under industrial conditions. The primary obstacles are not related to biological feasibility but rather to maintaining process stability, optimizing energy efficiency, and ensuring economic competitiveness when scaling beyond controlled laboratory settings.

8.1 Scale-up and Pilot Scale Performance

Pilot-scale investigations consistently show a decline in hydrogen yields upon scaling, despite using identical substrates and inocula. This reduction largely stems from hydrodynamic inconsistencies, uneven biomass distribution, and gradients in pH and substrate concentration, which are negligible at small scales but become significant at larger volumes. Transitioning from reactors ranging from 2 to 10 liters to systems of 50 liters, 1 cubic meter, or even 10 cubic meters highlights the challenge of maintaining short hydraulic retention times (HRTs) without biomass washout (Balachandar et al., 2020)Advanced reactor designs such as fixed-bed, fluidized-bed, and granular sludge reactors improve biomass retention compared to conventional continuous stirred-tank reactors (CSTRs), though they increase design complexity and capital costs. Importantly, pilot-scale studies using agro-industrial wastes (e.g., molasses, distillery effluents) show that co-substrate supplementation with nitrogen-rich residues like oilseed cakes can partially mitigate yield losses by stabilizing microbial metabolism and alleviating nutrient limitations (Yukesh Kannah et al., 2019) Another critical scale-up issue is hydrogen gas–liquid mass transfer; accumulation of dissolved hydrogen raises its partial pressure, thermodynamically inhibiting hydrogenase activity and shifting metabolic pathways toward reduced end products. Inefficient gas disengagement at scale exacerbates this inhibition. Reactor designs that incorporate continuous gas stripping or headspace flushing reduce hydrogen inhibition but add operational energy demand and complexity.

While laboratory scale systems have demonstrated the potential of biohydrogen from agricultural waste, the upscaling process to industrial applications is still constrained by critical technical and economic bottle necks (Balachandar et al., 2020). These limitations are intrinsically linked throughout the production pathway, where inefficiency in pre-treatment, reactor stability, and downstream processing collectively compromising the potential of sustainable industrial deployment, the advancement of Technology readiness level (TRL) and economic competitiveness of large-scale biohydrogen production(Abawalo et al., 2025). Although the agricultural feedstocks are abundant and form the substrate foundation for dark fermentation, but their dependence introduces significant supply chain complexity due to pronounced compositional and temporal variability (Kabeyi & Olanrewaju, 2022). Variations in lignocellulosic profiles such as the carbohydrate fractions, lignin recalcitrance, and C/N ratios, moisture, ash content, and organic load rate create inter batch inconsistency, which destabilizes steady state reactor performance. To mitigate these discontinuities, industrial facilities must adopt rigorous feedstock management protocols which include long term storage and homogenization which inherently escalates the operational expenditures (OPEX) and logistical footprint of the plant (Balachandar et al., 2020; Krupp & Widmann, 2009). Apart from consistent feedstock supply, Pre-treatment of the substrate is the most important yet an intricate step in scaling dark fermentation of agricultural residues. At laboratory scale pre-treatment methods like thermal, chemical, and physicochemical pre-treatments significantly enhance the biodegradability of the substrate and hydrogen yields at laboratory scale yet their translation to pilot scale requires high energy and chemical consumption, corrosion risks, and generates secondary waste streams (Yukesh Kannah et al., 2019). Particularly, acid and alkaline pre-treatment require corrosion resistant construction materials and effluent neutralization which increases both capital and operational costs. Other methods such as heat shock or acidification of inoculum are potent in supressing the growth of hydrogen consuming microorganisms, but are economically and operationally challenging at larger volumes in terms of material requirement for reactor fabrication and effluent handling (Xue et al., 2024). A cost-effective alternative is following the substrate driven acidification strategy, but it would not be much beneficial in terms of operational challenges as it requires precise control of organic loading rates to avoid incomplete acidification at low substrate concentration or high loadings that cause inhibitory conditions. Therefore, maintaining this balance is a bottleneck in large reactors (Xue et al., 2024).

Beyond the limitations in engineering of the reactors, the process of dark fermentation is also limited by metabolic thermodynamics. The fluctuations in hydrogen yields are inherently governed by distribution of electrons towards the reduced fermentation products like acetate, butyrate, lactate, and ethanol, with practical yields remaining critically low in comparison to theoretical yields (Aruwajoye et al., 2020). At large scale production process, accumulation of volatile fatty acids (VFAs) inhibits the hydrogenase activity, causes pH instability, and escalates the overall cost of downstream effluent management. These existing limitations restrict the high hydrogen yields and reduce the economic viability of dark fermentation pathway. Therefore, to address these limitations present efforts focus on coupling dark fermentation with downstream technologies like microbial electrolysis cells, anaerobic digestion, or VFA recovery pathways (García-Depraect et al., 2025; Marone et al., 2017).  While these hybrid systems successfully boost overall energy yields, they also complicate the engineering landscape, requiring higher initial investments and more rigorous control protocols to manage the increased system intricacy.

8.2 Energy Balance and Techno-Economic Considerations

Dark fermentation benefits from mild operating conditions, such as ambient pressure and mesophilic temperatures, minimizing energy inputs. However, pre-treatment of agricultural residues often dominates the overall energy demand. While high-severity chemical or thermal pre-treatments enhance substrate fermentability, they can offset net energy gains from hydrogen production. Life-cycle and energy analyses suggest that low-severity or hybrid pre-treatments combined with co-fermentation strategies provide the most favorable net energy balance (Hay et al., 2013; Kumar et al., 2018). Economic assessments identify feedstock handling, pre-treatment, and downstream gas purification as the primary cost drivers. Although biologically produced hydrogen is valued as a clean fuel, its economic competitiveness is limited compared to hydrogen from steam methane reforming or water electrolysis using low-cost electricity. Consequently, dark fermentation is unlikely to be economically viable as a standalone hydrogen production method in the near future. Its value proposition improves significantly when integrated with waste management, wastewater treatment, or biorefinery systems, where waste disposal costs are offset and co-products such as volatile fatty acids (VFAs) or methane are valorized (Hay et al., 2013; Jain et al., 2022).

8.3 Microbial Community Stability and Control

Findings across recent studies demonstrate that the stability of microbial community is a key challenge in scaling up the process of dark fermentation for hydrogen production. It is comparatively easier to enrich and maintain the hydrogen producing microbes at laboratory scale. On the other hand, as the volume of reactor increases, maintaining the dominance of hydrogenogenic pathway becomes challenging due to competition from methanogens, homoacetogens, and lactic acid bacteria (Dzulkarnain et al., 2022; Hay et al., 2013; Marone et al., 2017). These competing microbial colonies divert electrons away from the hydrogen producing pathway, thus resulting in lower hydrogen yields and reduced process stability over long term operation (Jain et al., 2024b). The instability in microbial communities directly affects the operational factors that intensify at large scale. Various factors that create microenvironments that favour non-hydrogen producing pathways such as variability in  feedstocks, uneven substrate hydrolysis, accumulation of VFAs, and localised pH gradients (Adjalle et al., 2017; García-Depraect et al., 2025; Kumar et al., 2018). While inoculum pre-treatment and substrate-induced acidification can suppress hydrogen consuming microbes at bench scale, their effectiveness reduces in large scale reactors where maintaining uniform conditions is challenging and operational disturbances are more frequent (Xue et al., 2024). As these microbial limitations are persistent, dark fermentation is rarely feasible as a standalone process at large scale. As a result, most scale-up approaches combine dark fermentation process with complementary technologies such as anaerobic digestion, microbial electrolysis cell, or the valorisation of volatile fatty acids (Domińska et al., 2025; García-Depraect et al., 2025; Marone et al., 2017). These integrated systems enable additional energy recovery from fermentation by products formed due to microbial limitations. Therefore, it can be concluded that the stable operation and good yields depends on the coordinated performance of both biological and engineering units (García-Depraect et al., 2025).

8.4 Reactor Operations & Data limitations

The stable operations of reactors for efficient biohydrogen production highly depends upon the microbial community dynamics (Tiegam Tagne et al., 2024). Major challenge persists in maintaining the microbial stability in large reactor volumes. The laboratory scale systems generally succeed in maintaining homogenous conditions favourable for hydrogen producing bacteria, but replicating this uniformity in large reactors presents significant engineering challenges.  Hindrance in mixing, mass transfer, heat transfer leads to quantifiable variations in pH of the medium, concentration of substrate consumption and hydrogen partial pressure, these changes create a favourable environment for hydrogen consuming microorganisms to grow rapidly, thus decreasing the overall hydrogen yield (Gałązka et al., 2025; Sivaramakrishnan et al., 2021). The configuration of conventional continuous stirred-tank reactors are liable to biomass washout at lesser HRTs (Hydraulic retention time), eventually lowering the preservation of hydrogen producing micro-organisms(Kongjan et al., 2010; Mizuno et al., 2000; Pawar et al., 2013). Whereas, fixed bed reactors along with attached growth reactors improve biomass retention but usually come across complications like clogging, channelling, and uneven substrate distribution when processing fibrous agricultural residues (Anzola-Rojas et al., 2015; Jayachandran et al., 2022; O-Thong et al., 2016). These operational issues hinder microbial structure and performance, limiting sustained hydrogen production under continuous conditions.

                Progress in scaling up dark fermentation is further limited by a significant gap in long-term, and appropriate experimental data for upscaling. Number of researches focus on short-term hydrogen production, often overlooking the changes in microbial structure and its recovery after disturbances occurred from operational problems. In consequence, these short-term studies limit the understanding of how microbial populations evolve under realistic operating conditions or how reactor respond to feedstock variability and process upsets (Vidal et al., 2025; Zagrodnik & Laniecki, 2015). This gap in available data restricts the development of reliable control strategies required to maintain stable hydrogen-producing communities at larger scales. Although data-driven and machine learning approaches are increasingly suggested for improving process monitoring and control, their practical application remains limited. This is mainly due to the scarcity of long-term datasets and the lack of  consistency across reactor designs and feedstocks (Ganguly et al., 2025). Without comprehensive data that clearly corelates with the reactor operation and microbial behaviour over extended periods, the transition from laboratory experiments to stable pilot-scale systems continues to be slow, highlighting the importance of both reactor design and data availability for achieving microbial stability during scale-up.

8.5 Economic Implications

One of the major concerns governing the scalability of dark fermentation is high operational costs (Ben Said et al., 2026). Although the process offers various advantages like the use of different types of raw materials and can be operated under mild conditions, the process does not proves to be cost effective as a single stage system due to often yielding low amounts of hydrogen and incomplete substrate breakdown and inefficient energy recovery thus limiting its commercial potential (Ahmadi et al., 2025). The economic burden is further increased by the costs related to feedstock preparation, addition of various chemicals and nutrients to regulate pH, and the purification of gas. The process of pre-treatment particularly for lignocellulosic residues such as plant waste, alone can represent a large share of the total production expenses. Moreover, the downstream processing of the produced hydrogen gas is essential to meet the quality standards which requires further investments (Kundu et al., 2025). Precise process control systems are also required to maintain the stability of the microbial population, which is an energy intensive process and thus leads to higher operational complexity in large scale reactors. The economic viability of dark fermentation process at large scale is directly impacted by technical and biological uncertainties. As there is a lack of robust technical data referring to the long-term operation and performance of the large-scale reactors which makes it difficult to evaluate the costs related to maintenance of the system and its durability, also calculating hydrogen yields etc. Therefore, scaling up the process beyond pilot scale still remains stalled, with only a few studies proving to be cost effective in practice (Ben Said et al., 2026). Recent studies also highlight the importance of integrated process configurations to make dark fermentation process economically viable. The process of dark fermentation accompanied by secondary steps like anaerobic digestion or microbial cell electrolysis would allow the operators to recover additional energy from fermentation by-products, thus improving the overall use of resources (Ahmadi et al., 2025; Md. S. Islam et al., 2018). Therefore, it is presumed that the potential of dark fermentation to become cost-effective depends on efficient production system designing, long-term data of consistent large-scale operations, and the capability to extract value from all by-products. The entire production process must be optimised to reduce the generation of waste and maximize energy recovery via integrated approaches.

9.       Comparison with Other Hydrogen Production Technologies

Other than Dark Fermentation, Hydrogen can be produced via thermochemical, electrochemical and biological routes comprising of steam methane reforming (SMR), water electrolysis, and photo-fermentation. Each pathway varies in terms of efficiency, environmental impact, technological readiness, and complexity of the process. To objectively assess the viability of the Dark fermentation process against these established and developing alternatives, a critical comparison is required. Steam Methane Reforming (SMR) is currently an economic benchmark as an industrial technology for hydrogen production (Yukesh Kannah et al., 2019). Although this process is economically efficient and gives higher yields, it possesses a negative environment impact by substantial greenhouse gas emissions caused by using fossil fuels. Even with its integration with carbon capture technology, SMR is an energy intensive process and proves to be harmful for the climate (Krupp & Widmann, 2009). Therefore, SMR can be considered as a transitional pathway rather than a sustainable solution.

                Production of hydrogen following the electrolysis of waste water is considered as a cleaner alternative as it can achieve near zero emissions, when utilising renewable energy sources (Jalil et al., 2025; Marone et al., 2017). Various studies state that theoretically this route of hydrogen production possesses no direct emissions and shall be a key part of future hydrogen goals, yet using this technology for large scale hydrogen production possess various challenges as it requires a large amount of electricity and big start-up investment. Therefore, the high cost of implementation restricts its use to specific regions that do not have access to abundant water and low-cost energy sources.

                Among biological pathways for hydrogen production, dark fermentation when compared with thermochemical routes, offers significant environmental advantages, such as lower greenhouse gas emissions and use of waste-based feedstocks providing a sustainable alternative. The process of dark fermentation can be efficiently operated under mild conditions, in ambient temperature and pressure, does not requires light energy and external renewable resources (Chaganti et al., 2012; Fan et al., 2006; Vidal et al., 2025). Despite these potential benefits, this technology possesses limitations in its metabolic stoichiometry. A number of studies demonstrate a maximum theoretical yield of 4 mol H₂ per mol of glucose, whereas practical yields obtained using microbial consortium is found to be below 2.5 mol H₂ per mol of hexose because of competing metabolic pathways of microorganisms(Ghimire et al., 2015; Jayachandran et al., 2022). Other biological pathways such as photo-fermentation and microbial cell electrolysis can efficiently convert fermentation by-products into hydrogen, improving the overall hydrogen recovery but require these secondary steps require additional inputs such as constant light resources and electricity. Due to these extensive energy  requirements, various studies suggest an effective approach such as hybrid systems that combines the process of dark fermentation to secondary downstream processes to enhance the overall efficiency of the process (Ben Said et al., 2026; Dębowski et al., 2025). Within this framework of integrated biorefinery, dark fermentation plays a crucial role as primary conversion step. It facilitates the simultaneous treatment of wastes and efficient production of hydrogen, while generating by products that can further be utilised by combined pathways, increasing the overall hydrogen yield. Therefore, instead of serving dark fermentation as a standalone solution, it would be proved as a valuable component of sustainable, multi-product biorefinery systems.

10.     Emerging Advances

Recent researches increasingly acknowledge that, along with the optimization of operational parameters for the process of dark fermentation, and to acquire the desired results, its integration with advanced technologies such as genetic engineering, microbial electrolysis cells (MECs) and AI-driven data analysis would serve as a breakthrough to overcome yield limits and improved energy recovery. These enhancing technologies would be complementary enablers to enhance the efficiency of conventional DF process rather than replacing it.

10.1 Genetic and Metabolic Engineering

Various advances in systems biology have enabled the researchers to better understand the complex metabolic networks that regulate production of hydrogen in fermentative organisms. In dark fermentation, due to generation of reducing equivalents formed during glycolysis cause diversion of microbial communities towards multiple competing pathways, resulting in formation of ethanol lactate and other solvents. This diversion of microbes eventually results in low hydrogen yields (Jalil et al., 2025; Kongjan et al., 2010). Therefore, to redirect this electron flux towards hydrogenase-mediated proton reduction by suppressing the competitive pathways and enhance hydrogenase activity, metabolic engineering strategies are being used. Various laboratory studies on model organisms such as Clostridium and Enterobacter have demonstrated that modifying the genes by deleting the target gene or over expressing the key redox enzymes prove to increase the hydrogen yields under controlled operational conditions (Domińska et al., 2025; Ganguly et al., 2025). In recent studies, it is stated that the genetic modification has proven to be much more effective in pure cultures rather than mixed cultures. As pure cultures allow the precise control of metabolic fluxes and are often sensitive to even slight fluctuations in pH, substrate composition, and by the presence of different inhibitory compounds in waste-derived feedstocks. In contrast, mixed cultures are difficult to engineer at gene level and present challenges related to genetic stability, risks of contamination and regulatory acceptance, specifically in large scale operation set ups(Kongjan et al., 2010; Lin et al., 2008; K. Zhang et al., 2011). Therefore, metabolic engineering is currently more suitable for targeted or integrated process systems, instead of considering it as an independent solution for large scale industrial dark fermentation.

10.2 Coupling Dark-Fermentation with Microbial Electrolysis Cell Technology

Microbial electrolysis cells (MECs) have been demonstrated as a promising technique to recover additional hydrogen from fermentation by-products that would contrarily remain as unrecovered energy. In this system, electroactive microbes breakdown the residual VFAs, such as acetate and butyrate at the positive electrode (anode) and a small amount of external current leads to the generation of hydrogen at the negative electrode (cathode)(Ganguly et al., 2025; Jalil et al., 2025). MECs when integrated with dark fermentation, creates a hybrid configuration which enables the effective conversion of both primary substrates and fermentation by products into hydrogen, resulting in higher yields and increased energy recovery. Various comparative studies have demonstrated that integrating MECs with dark fermentation outperforms independent systems in terms of progressive hydrogen yields. Since dark fermentation as a singular system is theoretically limited to 2-4 mol H₂ per mol of hexose, the addition of an MEC to the system enables the partial recovery of the chemical energy present in accumulated VFAs, bridging the gap between theoretical and practical yields (Ahmadi et al., 2025; Carosia et al., 2017; Hay et al., 2013). Studies conducted at pilot-scale suggest that these integrated systems are the cost-effective alternates, specifically when low-cost renewable electricity is available to cover the additional power inputs required by MECs. Despite the potential, the efficient performance of MEC depends on quality of electrode material, internal resistance, and the stability of microbial film (biofilm). All these factors contribute to the investment and operational costs. Furthermore, as the technology requires additional power input, its actual cost depends upon the electricity prices and carbon intensity, which complicates the life cycle assessments, making it difficult to validate MEC as a feasible technology. As a result, the deployment of MEC at large scale is still at developing stage.

10.3 AI-Assisted Monitoring and Optimisation of Biohydrogen Production

The process of dark fermentation is strenuous to optimise using conventional mechanistic models due to its complexity and unpredictable behaviour. As the process involves constant change of dynamic interactions between microbial communities and operating conditions, nature of feedstock and internal environment of the reactor, it is difficult to map the optimization process mathematically. Important factors for optimisation of the fermentation process include pH of the medium, hydraulic retention time (HRT), balanced addition of chemicals and nutrients, substrate loading rate and temperature interact in interdependent way that is tough to predict and represent them accurately using standard theoretical equations (C. Liu et al., 2025; C.-M. Liu et al., 2014). Recent studies demonstrate the use of AI and machine learning tools as an alternate to better understand the system behaviour and generate accurate experimental and operational data. Researches demonstrate that data-driven models can efficiently predict exact hydrogen yields and continuous production rates by comparing multiple process variables as the same time. These techniques are specially valuable for real time data generation and process control(C. Liu et al., 2025). Machine learning algorithms are also capable of identifying process imbalances at the very initial phase which enables the operator to perform corrective actions before the complete process setup gets damaged. (Aghajani Delavar & Wang, 2021).

Collectively, genetic engineering, integrating dark fermentation with microbial electrolysis cells (MECs), and AI-driven process optimization provide with a corelative approach to address the interconnected challenges such as low hydrogen yields, pH and microbial stability that occur during dark fermentation, thus enhancing the overall process efficiency. Primarily, these technologies work more efficiently within an integrated biorefinery approach, producing hydrogen alongside utilisation of other valuable chemicals. Such frameworks prove to be economically viable and helps allocate financial and operational risks across multiple value streams (Chatterjee & Venkata Mohan, 2021; Marone et al., 2017; Rambo et al., 2015). While this convergence of technologies presents a promising future, certain barriers limit their industrial scale transitions due to lack of long-term operational studies that clearly demonstrate the process viability and limitations. Additionally, techno-economic and life-cycle assessments are also required to evaluate the feasibility of these technologies under real-time operating conditions and to identify the most suitable integration for commercial deployment.

11.    Conclusion

Dark fermentation is advancing from an experimental stage to a viable technology for sustainable hydrogen production. Recent progresses in genetic and metabolic engineering have demonstrated improved hydrogen yields. On the other hand, integration of MECs (microbial electrolysis cells) have shown the potential to enhance hydrogen yields via additional energy recovery from fermentation by-products. Simultaneously, data-driven and AI assisted process optimisation technologies have proven to efficiently manage complex process variables, enabling pH stability, nutrient balance and hydraulic conditions. Altogether, the advancements in these technologies have demonstrated innovations that are no longer limited to improving an independent system but a coordinated system optimization approach. Moreover, dark fermentation within an integrated biorefinery framework efficiently utilises the residual organic acids and carbon streams through downstream processes such as methanogenesis, MEC assisted conversion, thereby enhancing overall carbon utilisation and energy recovery. When combined with waste-based substrates, dark fermentation efficiently conforms resource recovery and waste management objectives, strengthening its environmental and economic relevance. In conclusion, dark fermentation of agricultural residues represents a promising yet emerging pathway for sustainable hydrogen production. The successful transition of this method from research to real-time application will require integrated biorefinery thinking, robust strategies for efficient deployment to commercial scale. Equally important are standardized evaluation frameworks and supportive policy environments that recognize the broader environmental and societal benefits of waste-based biohydrogen. Rather than competing directly with established hydrogen technologies, dark fermentation is most likely to succeed as a component of integrated biorefineries and circular bio economy systems, where hydrogen production is coupled with waste treatment and co-product recovery.

Conflict of interest Author declares that there is no conflict of interest. 

Funding information not applicable.

Ethical approval not applicable.

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