Abstract
Dark fermentation is the conversion of various biomass wastes into biohydrogen. It occurs in the absence of light. The purpose of this review is to provide the latest advances in biohydrogen production via dark fermentation, focusing on feedstock characteristics, microbial community dynamics, and approaches to system integration. Carbohydrate-based substrates are ideal for the production of biohydrogen due to their high fermentative potential. In contrast, lignocellulosic biomass requires pretreatment to enhance its susceptibility to microbial degradation. Hydrogen-producing microorganisms such as Clostridium spp were considered, focusing on metabolic processes, changes in the microbial communities involved, and process stability at different process parameters. The hydrogen partial pressure, byproducts, and inhibition due to competition between methanogens, lactic acid bacteria, and hydrogen-utilizing organisms were reviewed along with control mechanisms. The use of fermentation by-products, volatile fatty acids, and acidified slurry has been considered within biorefinery concepts. This approach addresses the economic challenges associated with the biohydrogen production technology. Although some success has been achieved, several issues remain. These include scalability and feedstock variation. These factors present important bottlenecks for future commercial applications of this technology.
Highlights
Carbohydrate-rich wastes yield higher biohydrogen in DF.
Mixed consortia outperform pure cultures in stability.
Bio-H2 yield is limited by thermodynamics and by-product formation.
Co-digestion improves yield, stability, and nutrient balance.
Integration boosts recovery but increases system complexity.
1 Introduction
1.1 Background
Energy is one of the key requirements for the existence, development, and wellbeing of human beings on planet Earth. For instance, energy is required for cooking, lighting, industries, farming, transport systems, medical facilities, and communication systems (). The global energy demand is increasing due to population growth, urbanization, and industrialization. In 2024, global energy consumption increased by 2.2%, alongside a global GDP growth of 3.2%. The energy mix consisted of natural gas at 28%, coal at 15%, oil at 11%, nuclear energy at 8%, and renewable energy at 38% (). In addition, the International Energy Outlook projects that global energy consumption will increase by 56% between 2010 and 2040. Approximately 78% of this energy demand is derived from fossil fuels (). This high percentage indicates that most of the world’s energy demand still relies on fossil fuels (Yaashikaa et al., 2022). The heavy dependence on fossil fuels such as petroleum, coal, and natural gas has contributed to carbon dioxide emissions, thus causing climate and environmental change. Another challenge of heavy dependence on fossil fuels is their limited sustainability, as fossil fuel reserves are non-renewable in nature. Their long-term availability is therefore increasingly uncertain due to continued extraction and growing global energy demand (Zhang, 2024). In general, concerns about climate change, rising oil prices, and the depletion of fossil fuels have driven efforts to replace them with more environmentally sustainable energy sources (). The shift from fossil-fuel-based energy systems to renewable energy systems is crucial to ensuring global energy sustainability ().
Investment in renewable energy plays a major role in reducing dependency on fossil fuels. Clean energy investments have increased sharply since 2020. World investment in energy reached US$3 trillion for the first time in 2024. From these global energy investment budgets, two trillion dollars were invested in clean energy expansion and dissemination (World Energy Investment, 2024).
Hydrogen is a better alternative fuel because of its non-toxicity and high heating value (; Sivaramakrishnan et al., 2021; Yusuf et al., 2021). Hydrogen has emerged as one of the most promising clean energy sources. This is due to its high-energy content per unit mass. It also produces eco-friendly by-products during combustion. Globally, hydrogen demand is predicted to rise from 70 million tonnes in 2019 to 120 million tonnes in 2024 (Osman et al., 2024). Australia currently hosts the largest number of biohydrogen production facilities (96 plants), followed by India (18 plants). The Sub-Saharan Africans, the Middle East, and North Africa are emerging as potential hubs for biohydrogen production by 2050 due to the availability of biomass resources and increasing investment in renewable energy technologies. Currently, the majority of the estimated 50 million metric tons of hydrogen produced each year is derived from fossil fuels, primarily through natural gas reforming and coal gasification, and is mainly utilized in oil refining and ammonia production. In contrast, biohydrogen production remains at its early stage of development ().
Among the different hydrogen production routes, biohydrogen represents a promising alternative, especially from an environmental viability and green energy production. Nevertheless, no single pathway universally satisfies all criteria of economic viability, scalability, and sustainability, as these depend on technological maturity, feedstock availability, and system boundaries. However, the scalability challenge in biohydrogen still needs to be addressed for large-scale implementation (Vidal et al., 2025). In addition, biohydrogen production has attracted attention due to its ability to utilize organic waste substrates under mild operating conditions. However, its energy and economic advantages relative to electrochemical (water electrolysis) and thermochemical (e.g., gasification and pyrolysis) hydrogen production processes remain dependent on process efficiency and scale (). Biohydrogen production is produced via biophotolysis, dark fermentation, microbial electrolysis cells, and photo-fermentation processes (; ). Among these biohydrogen production pathways, dark fermentation is promising due to its high production rate and substrate flexibility, despite its low hydrogen yield (; Khesareh and Ataei, 2024; Rani et al., 2024).
The main difficulties in biohydrogen production using dark fermentation are low hydrogen yield, process instability, and inhibitory compounds produced in the process (Taggar et al., 2024). Low hydrogen yield caused by substrate inhibition, byproduct formation, and microbial community composition. Elevated substrate concentrations can inhibit microbial growth used for hydrogen production and diminish hydrogen generation (). Dark fermentation often produces other chemicals, such as acetic acid, butyric acid, and lactic acid, that reduce the biohydrogen yield (). The exact types and amounts of microorganisms in the fermentation process have a considerable impact on hydrogen production. Some bacteria may produce more hydrogen, whereas others may prefer to make other metabolites or even consume hydrogen, influencing overall yield (; ).
Other major issues associated with the production of biohydrogen via dark fermentation are changes in operational parameters, including temperature, pH, substrate concentrations, and the presence of inhibitive compounds (). Hydrogen-producing microorganisms have an ideal temperature ranging from mesophiles to thermophiles. Failure to maintain this ideal range leads to cracking, disrupting the enzyme, microbial growth rates, and low hydrogen yield. On the other hand, temperature changes will also cause instability and inhibit microbial population (Zisha et al., 2025). In addition to temperature, pH also plays an important role in biohydrogen production. The microbes require an optimal pH environment to produce biohydrogen ().
In addition to these process-related constraints, oxygen sensitivity, cost-effectiveness, and industrial-level production pose challenges for dark fermentation-based biohydrogen production. For instance, hydrogenase enzymes, which are involved in the final step of hydrogen gas synthesis, are very oxygen-sensitive. This has contributed to enzyme damage and inefficient hydrogen gas production (Woon et al., 2023). In addition, the viability of producing biohydrogen from dark fermentation at an industrial scale is influenced by a range of factors, including capital and operating costs and requirements for setting up bioreactors, preparing substrates for fermentation, and managing process conditions (e.g., pH, temperature, and anaerobic conditions) (). However, there are still some technological challenges that need to be addressed during the dark fermentation process on a larger scale. Such difficulties are associated with microbial stability, heat management, and mass transfer limitations. ().
Co-digestion is another method for increasing biohydrogen production. Using mixed substrates to complement one another and increase overall hydrogen output (Teke et al., 2024). Developing strategies to control hydrogen-consuming microorganisms, such as methanogens, enhances biohydrogen production (). Additionally, researchers found that mixed cultures of different microbes are more efficient in producing biohydrogen than pure microbes. Mixed cultures can effectively address operational challenges, such as metabolic shifts and the demand for sterility ().
Numerous studies have explored key aspects of dark fermentation, including feedstock characteristics, microbial community behavior, and reactor configurations. Despite extensive research on biohydrogen production via dark fermentation, existing studies largely examine feedstock characteristics, microbial community dynamics, and reactor configurations in isolation, limiting a comprehensive understanding of their interdependent effects on process performance. Moreover, critical aspects such as techno-economic feasibility and life-cycle sustainability are sufficiently integrated in the current reviews. Therefore, a holistic, system-level synthesis is needed. This synthesis should integrate these dimensions. It will help identify key bottlenecks. It will also guide the scalable and sustainable development of dark fermentation technologies. This review covers: (i) the relationship between feedstock compositions and microbial population structure, (ii) the effects of microbial population dynamics on the performance of the reactor and biohydrogen production, and (iii) the importance of system integration approaches in overcoming process bottlenecks. Moreover, a new section on techno-economic feasibility and life-cycle analysis has been included to provide a more holistic analysis of scalability and sustainability, aspects that were often neglected in earlier reviews.
2 Metabolic routes of dark fermentation
Dark fermentation is an important anaerobic bioconversion process. It involves a series of sequential biochemical reactions. In this process, biomass is converted into hydrogen and carbon dioxide. It also produces reduced end-products such as butyrate, acetate, and ethanol. This occurs without the use of light (Ren et al., 2022). In this process, sugar-rich organic substrates are metabolized by anaerobic microorganisms under suitable environmental conditions, including optimal temperature, pH, pressure, and nutrient availability, leading to the production of H2, CO2, and various metabolic by-products (). Theoretically, a wide variety of organic compounds, such as carbohydrates, sugars, proteins, and lipids, can be utilized as substrates for biohydrogen production. However, glucose is most frequently employed as a model substrate to demonstrate and analyze the metabolic processes involved ().
In this process, complex biomass is first hydrolyzed into fermentable sugars such as glucose. Hydrogen-producing microorganisms convert glucose into pyruvate through glycolysis. This process produces adenosine triphosphate (ATP) from adenosine diphosphate (ADP). It also generates reduced nicotinamide adenine dinucleotide (NADH). NADH plays a key role in subsequent hydrogen production pathways (). Pyruvate is further acidified by enzymes such as pyruvate decarboxylase and formate hydrogenase, resulting in the release of H+ ions. The electrons required for hydrogen production are released during the oxidation of NADH, which is produced as a reducing equivalent during glycolysis. Finally, hydrogenase enzymes catalyze the reduction of protons using these electrons, resulting in the formation and release of molecular hydrogen (). The metabolic pathways of biohydrogen production via dark fermentation are presented in Figure 1.
FIGURE 1
Pyruvate conversion varies among microorganisms and is a key branching point in the metabolic pathway (). Pyruvate is converted to acetyl-CoA by two major enzymes. The first enzyme is pyruvate formate lyase (PFL). PFL is mainly found in facultative anaerobes but can also occur in strict anaerobes such as Clostridium species. The second key enzyme is pyruvate ferredoxin oxidoreductase (PFOR). PFOR is characteristic of obligate anaerobic microorganisms (). Facultative anaerobes employ the PFL enzyme to oxidize pyruvate to acetyl-CoA and formate without generating reducing equivalents immediately (Shi et al., 2023). Similarly, pyruvate is oxidized to lactate by the enzyme lactate dehydrogenase. Formate is oxidized by the formate hydrogen lyase enzyme to produce carbon dioxide. During this process, hydrogenase reduces protons (H+) to form H2 (). In obligate anaerobes such as Clostridia, glycolysis is followed by the oxidation of pyruvate to acetyl-CoA. This reaction is catalyzed by the pyruvate-ferredoxin oxidoreductase enzyme. It contributes to the formation of hydrogen gas. Acetyl-CoA yields acetate and ATP molecules. In addition, pyruvate is oxidized to acetyl-CoA with the involvement of reduced ferredoxin (Fdred). The reduced ferredoxin then donates electrons to the ferredoxin-dependent [FeFe]-hydrogenase enzyme. [FeFe] Hydrogenase oxidizes Fd to produce H2 gas. Four moles of H2 gas are yielded per glucose molecule when pyruvate is oxidized to Acetate. When pyruvate is ultimately converted to butyrate, 2 mol of H2 are produced per mole of glucose. Therefore, in microbes that use mixed-acid fermentation pathways, the acetate-to-butyrate ratio strongly influences hydrogen production (Xuan et al., 2023).
Formate hydrogen lyase (FHL) and [FeFe]-hydrogenase are the common enzymes involved in the dark fermentation process during biohydrogen production. The organisms involved in the biohydrogen production process use one of these two enzymes (). Hydrogenases are classified as either Ni–Fe or Fe-only enzymes, the latter also referred to as [Fe–Fe] hydrogenases. The two types of enzymes differ in molecular weight, location, subunit composition, electron carrier selectivity, sensitivity to O2 inactivation (with [Fe-Fe] being more sensitive), and physiological significance (Ren et al., 2022). Ni–Fe hydrogenases are mainly involved in hydrogen (H2) uptake, although they can also participate in H2 evolution. In contrast, [Fe–Fe] hydrogenases are primarily responsible for H2 evolution and can exhibit rates that are over a hundred times faster than those of [Ni–Fe] enzymes. They are therefore a promising option for biohydrogen production. Hydrogenases are classified as Ni–Fe or Fe-only enzymes, the latter also known as [Fe–Fe] hydrogenases. These enzymes differ in several properties, including molecular weight, cellular location, and subunit composition. They also vary in electron carrier specificity, oxygen sensitivity (with [Fe–Fe] hydrogenases being more sensitive), and physiological roles. Ni-Fe hydrogenases are mainly involved in hydrogen (H2) uptake, although they can also participate in H2 evolution. In contrast, [Fe–Fe] hydrogenases are primarily responsible for H2 evolution. Ni–Fe hydrogenases are primarily involved in H2 uptake activities, although they can also contribute to H2 evolution. [Fe–Fe]-hydrogenases are widely found in anaerobic bacteria. However, they are also present in aerobic cyanobacteria and green algae. They contain iron–sulfur clusters that bind cyanide and carbon monoxide. This forms an unusual structure. This structure is suitable for the active sites of biological enzymes ().
Nitrogenases are found solely in prokaryotes and are irreversible catalysts that can only evolve, but not take up, H2. Their primary role is to fix N2, and while some H2 evolution occurs during N2 fixation, it occurs significantly more in the absence of N2. Nitrogenases exhibit significantly lower specific activity (H2 production per unit enzyme mass per unit time) compared to [Fe–Fe] hydrogenases (Radan et al., 2025). Nitrogenases require approximately twice as much metabolic energy (ATP) to produce H2 compared to hydrogenases. Thus, nitrogenases are impractical for producing biohydrogen. Nitrogen-fixing bacteria are ideal models for high-rate H2 synthesis because they possess efficient pathways for generating reducing equivalents. Nitrogen-fixing bacteria can produce H2 if the inefficient nitrogenase is substituted with [Fe-Fe] hydrogenase (Xuan et al., 2023).
Hydrogenases adsorb and desorb H2 with a free-energy change comparable to platinum, one of the most efficient catalysts for electrochemical hydrogen production. The genetics, regulation, and molecular characterization of hydrogenases and nitrogenases have advanced significantly in recent years (Xuan et al., 2023). These developments include the high-resolution 3-dimensional structures of these enzymes that display the locations of iron-sulfur clusters, the site where the reductant (such as ferredoxin or flavodoxin) docks, and, in the case of hydrogenases, the putative gas channels that are thought to allow H2 molecules to exit the active site, among other details. The development of entirely novel hydrogenases, including oxygen-insensitive enzymes, is increasingly being anticipated ().
3 Factors affecting biohydrogen production via dark fermentation
Biohydrogen generation output is influenced by numerous factors, several of which are controllable within the bioreactors (). A graphical summary of the factors that affect the production of biohydrogen via dark fermentation is presented in Figure 2.
FIGURE 2
3.1 Inoculum sources
The initiation of hydrogen production requires a seed culture (inoculum) that is capable of producing hydrogen. Mixed cultures of hydrogen-producing microorganisms are usually preferred over pure cultures due to their cost-effectiveness, feasibility, and easier operational control (Omer et al., 2025). The nature of the microbial culture used to kick off dark fermentation (DF) bioprocess is an important factor influencing hydrogen production (). Fermentative microorganisms, including Bacillus, Clostridium, Enterobacter, Citrobacter, and Rhodopseudomonas, are commonly used as inoculum during biohydrogen production (). These microbes can be obtained from animal dung, anaerobic sludge in reactors, organic waste, soil, and effluent in a different treatment plant (). Biodegradable organic substances are naturally rich in anaerobic fermenting bacteria, which can contribute to biohydrogen production. Dark fermentation bioreactors can be operated using either pure microbial cultures or mixed microbial consortia (). The microbial culture used throughout the dark fermentation start-up process significantly affects hydrogen productivity. Biohydrogen production was determined by screening suitable inocula and optimizing pretreatment time (Tiegam Tagne et al., 2024).
Pretreatment of inoculum is essential in dark fermentation to suppress hydrogen-consuming microorganisms (e.g., methanogens) and enrich hydrogen-producing microflora (). The effectiveness of pretreatment depends on inoculum characteristics, substrate type, and operating conditions. Common methods include physical techniques such as heat shock, aeration, and freezing, as well as chemical treatments such as acid, alkali, and 2-bromoethanesulfonate. These approaches can enhance process stability and hydrogen production; however, their performance remains system-specific. Pretreatment also influences the start-up (lag) phase of hydrogen production. Recent studies indicate that heat-shock pretreatment typically results in shorter lag times (approximately 12–48 h) due to rapid enrichment of spore-forming hydrogen producers, whereas chemical pretreatments often exhibit longer lag phases (1–3 days) because of microbial stress and adaptation requirements. Therefore, the selection of an appropriate pretreatment strategy should be tailored to the inoculum source and process conditions (; Sun et al., 2025).
3.2 Substrate composition
The macromolecules present in biomass—carbohydrates, proteins, and lipids (including structural fractions such as cellulose and hemicellulose)—can be converted into biohydrogen via dark fermentation. The type of feedstock significantly influences both the rate and yield of hydrogen production. Carbohydrates generally exhibit the highest hydrogen yields due to their relatively simple structure and favorable metabolic pathways, with theoretical yields reaching up to 4 mol H2/mol glucose and typical practical yields ranging from 1.5 to 3.0 mol H2/mol hexose hexose (; ; Pawar et al., 2026). In contrast, proteins and lipids produce lower hydrogen yields due to their more complex molecular structures and metabolic pathways. Protein-rich substrates typically yield around 0.5–1.5 mol H2/mol substrate, while lipid-based substrates often result in even lower or more variable yields due to long-chain fatty acid inhibition (). Lignocellulosic components such as cellulose and hemicellulose can also contribute to hydrogen production after hydrolysis, with yields generally ranging from 1.0 to 2.5 mol H2/mol sugar equivalent, depending on pretreatment efficiency and microbial activity ().
The cost and feedstock used directly impact the efficiency and yield of biohydrogen production (Tsegaye et al., 2023). The substrates used for hydrogen production contain carbon, nitrogen, minerals, and vitamins; each plays a specific role in fermentation. Carbon is essential for cell growth, product formation, and ATP generation. Nitrogen provides a component of the proteins used for cell growth and cellular division. Minerals and vitamins provide the necessary components that act as cofactors for metabolic processes. Moreover, the combination of carbon and nitrogen sources must be carefully considered when producing H2. Their balance affects microbial metabolism. It also influences microbial growth. Ultimately, it determines the overall hydrogen yield. The combination of trace metals such as Fe, Ca, Na, Cu, Ni, Mg, and K, along with vitamins in hydrogen-producing media, plays an important role in biohydrogen production. The presence of these substances enhances the biohydrogen production (). Dark fermentation uses microorganisms that can produce biohydrogen. These microorganisms can also rapidly break down simple sugars such as glucose, lactose, and sucrose. However, the use of carbohydrate-rich substrates creates competition with other sectors. These sectors also depend on the same feedstocks. This is especially true for the animal feed industry and food supply chains. As a result, their availability for biohydrogen production is significantly limited (; Pengadeth et al., 2024; Rani et al., 2024). In addition, there is growing interest in using food waste for bioenergy. This highlights the need to prioritize waste-based substrates. Edible carbohydrate sources should be avoided. This helps ensure sustainability. It also reduces conflicts between food, energy, and animal feed uses (). This occurs because these sugars are easy to use, highly biodegradable, and are readily available ().
Dark fermentation can be inhibited by lipid-rich substrates. These substrates can lead to high hydrogen partial pressure during the process. This, in turn, reduces hydrogen production efficiency. Co-digestion of mixed substrates can produce biohydrogen while improving nutrient balance, decreasing hazardous substances, and controlling pH levels without the need for additional alkali (). Studies have shown that using co-fermented complementary substrates can improve hydrogen production rates. It can also modify metabolic pathways. In addition, it helps resolve problems caused by specific microbial strains developed on a single substrate (). Pretreatment can increase the rate-limiting stage of dark fermentation, specifically hydrolysis. Pretreatment increases H2 production by breaking down the substrate into smaller molecules and making it easier for microorganisms to metabolize ().
3.3 PH value
The pH of the fermentation medium is a key operational parameter in dark fermentation. It strongly influences the overall process performance (). It affects microbial activity, enzyme function (particularly hydrogenase), metabolic pathway distribution, and lag phase duration (; ). Optimal hydrogen production occurs within a narrow pH range. This range supports hydrogenase activity. When the medium becomes too acidic, hydrogen production decreases. Acidification occurs due to the accumulation of organic acids. This condition can inhibit microbial growth. It can also shift metabolism toward solvent and organic acid production instead of hydrogen production (). Overall, hydrogen ion concentration regulates enzymatic and microbial processes, thereby determining hydrogen yield and the composition of fermentation by-products such as acetic, lactic, butyric, and propionic acids. Controlling VFA generation during acidogenic fermentation requires careful attention to pH. Extreme pH levels affect microbial activity. Most enzymes are also affected by extreme pH conditions. The pH during fermentation affects both hydrolysis and acidogenesis; the ideal pH for VFA synthesis should support both processes. Methanogens thrive at pH 7.0, while acidogens grow well at pH 5.5–6.5. Maintaining an acidic pH can limit methanogen activity and cause VFA buildup (Pandey et al., 2022). Higher pH levels in the fermenter generally lead to lower H2 production. However, very low pH conditions (typically below 4.5) also negatively affect hydrogen-producing cultures. (Wadjeam et al., 2019). Therefore, the optimal pH of the fermentation broth is critical to the effective and consistent operation of the fermentation process. Failure to maintain optimal pH creates operational instability. It can also lead to suboptimal microbial activity. As a result, the microbes may function inefficiently or behave unpredictably ().
Different microorganisms exhibit distinct optimal pH ranges that influence their growth, survival, and metabolic performance (Zheng et al., 2022). For instance, Clostridium species can produce higher biohydrogen at optimal pH levels ranging from 5.0 to 6.5. Clostridium butyricum at pH 6.0 promotes the acetate-butyrate fermentation pathway, which produces the highest biohydrogen production. On the other hand, a high pH range up to 8.0 facilitates the growth of Propionibacterium species. This promotes propionate production and limits hydrogen production. Overall, several studies have reported that lowering the pH below 5.0 inhibits hydrogenase enzyme activity. In contrast, pH values above 6.5 can increase hydrogen consumption by competing microorganisms ().
3.4 Temperature
The operational temperature affects hydrogen yield in the dark fermentation process (). Temperature changes significantly affect H2 generation rates, substrate consumption, biohydrogen yield, volatile fatty acid synthesis, and microbial presence in the system (). Depending on their conditions of optimal growth, fermentative bacteria can be divided into groups based on the temperatures at which they grow best: Psychrophiles at 10 °C–20 °C; Mesophiles at 30 °C–40 °C; Thermophiles at 50 °C–60 °C; Extreme thermophiles (>65 °C) (Rani et al., 2024). Psychrophiles and low-temperature-adapted bacteria include genera such as Clostridium and Enterobacter. These microorganisms can produce hydrogen under sub-mesophilic conditions. The mesophiles Clostridium butyricum, Clostridium acetobutylicum, and Enterobacter aerogenes are most commonly used because of their wider versatility and constant production of hydrogen. Bacteria involved in biohydrogen production under thermophilic conditions include species such as Thermoanaerobacterium thermosaccharolyticum and Clostridium thermocellum. These microorganisms have higher substrate conversion capabilities. They also have a lower risk of contamination. On the other hand, extremely thermophilic bacteria such as Thermotoga neapolitana and Caldicellulosiruptor saccharolyticus are highly efficient hydrogen producers at temperatures above 70 °C (; ).
Thermophilic conditions enhance thermodynamic efficiency and enzymatic activity. This leads to higher biohydrogen yields. It also increases VFA production compared to mesophilic conditions (). The temperature of dark fermentation will depend on the type of bacterial culture and the carbon source used. Mesophilic conditions (35 °C–40 °C) are most commonly used for cultivating bacteria for hydrogen production (). High temperatures (over 60 °C) have been shown to dramatically reduce H2 production activity. Pyrococcus furious and Thermotoga maritima are the only bacteria capable of producing hydrogen at severe temperatures. These bacteria can produce hydrogen at temperatures above 80 °C (Sim et al., 2023; Yang et al., 2025).
3.5 Organic loading rates (OLR)
The organic loading rate is the quantity of degradable organic material fed to the biological system per unit reactor volume and time, which ultimately influences hydrogen generation (). At low OLR, there is limited substrate available for microbial activity, resulting in low hydrogen production rates. As the OLR increases, hydrogen production improves due to greater substrate availability and increased microbial activity. However, beyond the optimal OLR, hydrogen production declines (). In dark fermentation, biohydrogen production is governed by the interaction between HRT, OLR, and cell retention time (CRT/SRT), which together control substrate availability and microbial stability. A higher CRT than HRT helps retain hydrogen-producing microorganisms and improves process performance, while an imbalance among these parameters may lead to washout or inhibition due to organic overloading (; ).
According to recent research, the use of a moderate OLR is vital in achieving a balance between hydrogen production and volumetric productivity in dark fermentation processes using continuous and semi-continuous reactors, especially when food waste and agricultural residues are treated. In continuous systems, OLR is closely linked with HRT and microbial retention. High OLR (often associated with shorter HRT) can result in biomass washout, especially for slow-growing hydrogen-producing bacteria. This destabilizes the microbial community and reduces hydrogen production performance ().
Another important effect of OLR is increased production of VFAs, such as acetate, butyrate, and propionate. Moderate production of VFAs indicates active hydrogen-producing reactions. However, excessive accumulation of VFAs can lower the pH. It can also inhibit enzyme activity. In addition, it can suppress hydrogenase activity. Consequently, hydrogen production is decreased, and the system becomes acidic. A moderate OLR prevents inhibitory effects induced by VFA accumulation. It therefore promotes hydrogen production (Regueira-Marcos et al., 2024). The OLR has a significant impact on the distribution of metabolic pathways during dark fermentation. The acetate/butyrate pathway predominates under an optimum OLR, resulting in maximum hydrogen yield. But when the OLR is increased, the lactate/ethanol/propionate pathway becomes predominant, leading to low hydrogen yield because the electrons flow to form reduced compounds (). Higher OLR values tend to increase hydrogen production. This leads to higher levels of partial hydrogen pressure (Ph2) in the reactor. An increase in hydrogen partial pressure will thermodynamically inhibit the formation of hydrogen, hence favoring product reduction. Efficient gas stripping is thus vital at elevated OLR levels (). In general, the OLR determines the amount of substrate available, microbial activity, and overall reactor stability during dark fermentation.
3.6 Hydraulic retention time (HRT)
HRT is the time that a substrate remains in contact with microorganisms in a bioreactor to achieve the desired bioconversion. The ideal retention time for each substrate varies depending on several factors, including reaction rates, substrate type and concentration, microbial concentration, substrate composition, and temperature (). HRT is a very important factor in the operation of both continuous and semi-continuous dark fermentative processes. Recent research has shown that optimizing HRT has an important role in the biohydrogen production process ().
The use of shorter HRTs tends to increase hydrogen production in volume terms owing to improved substrate flow through the reactor and enhanced organic loadings. However, overly short HRTs may lead to inadequate substrate utilization and microbial washout, especially when using CSTRs. On the other hand, higher HRT promotes feedstock degradation and microorganism adaptation. It also promotes the accumulation of VFAs and the development of methanogens in the processes. Higher HRT thus leads to a metabolic switch from hydrogen generation through acidogenesis to methanogenesis, reducing hydrogen production efficiency ().
HRT selection depends on several factors, including feed composition, feed handling, reactor size, and digestion temperature. Carbohydrate-rich substrates such as glucose-, starch-, and sugar-containing wastes are rapidly metabolized during dark fermentation. Therefore, they typically require short HRTs of 2–12 h. In contrast, lignocellulosic substrates such as agricultural residues generally require longer HRTs (24–72 h) due to the slower hydrolysis of cellulose and hemicellulose fractions (). Importantly, lignin is largely resistant to biodegradation under dark fermentation conditions and may hinder substrate accessibility and hydrogen production efficiency (Sun et al., 2025). Recently, there has been an increasing focus on reactor designs in which HRT can be completely decoupled from solids retention time (SRT). Such systems include membrane bioreactors, packed-bed reactors, granular sludge reactors, and immobilized cell systems. These systems improve biomass retention and allow stable operation at shorter HRT without significant microbial washout. Advanced fixed-film and structured-bed reactors have also demonstrated enhanced substrate conversion efficiency and hydrogen evolution under high-rate continuous operation ().
3.7 Partial pressure of hydrogen
The accumulation of H2 gas in the headspace results in an increase in the total H2 in the liquid phase (dissolved H2). It has been reported that the partial pressure of H2 in the bioreactor headspace can approach inhibitory levels (≈10–3 atm), negatively affect hydrogenase activity, and metabolic pathways away from hydrogen production. To mitigate this effect, several strategies are employed to continuously remove hydrogen from the system, including gas sparging with inert gases (e.g., N2 or CO2), increasing mixing intensity, applying vacuum conditions, or using gas-permeable membranes to enhance hydrogen stripping. These approaches reduce hydrogen partial pressure, thereby promoting favorable thermodynamic conditions for hydrogen production (; Sun et al., 2025). Furthermore, temperature plays an important role in hydrogen dissolution: increasing temperature decreases gas solubility in the liquid phase, facilitating hydrogen release from the broth and reducing inhibition. Consequently, thermophilic conditions are often advantageous for maintaining lower dissolved hydrogen concentrations and improving overall hydrogen yield (; Prado and Rial, 2025).
3.8 By-broduct accumulation
In dark fermentations, substrates are converted to biohydrogen in different pathways. Acetate and butyrate pathways produce more hydrogen yield than other production pathways. In the acetate pathway, the theoretical maximum hydrogen production can be up to 4 mol of hydrogen per mole of glucose. On the other hand, other fermentation processes, such as lactate, ethanol, and propionate fermentation, yield lesser amounts of hydrogen. This is because these pathways utilize reducing power in the form of NADH, which limits hydrogen generation because of a lack of electrons (; ).
An essential inhibitory mechanism in this context is feedback inhibition concerning thermodynamic and metabolic aspects. Increased amounts of by-products in the reactor shift the redox state of the microbial cells. Thus, hydrogenase activity gets inhibited. Furthermore, the development of volatile fatty acids changes the pH level. Thus, the resulting acidic condition inhibits microbial metabolic reactions. Low pH levels allow undissociated acids to enter the cell membrane, thus reducing hydrogen formation ().
Currently, new approaches have been developed to minimize by-product formation through reactor engineering and process optimization. Continuous or semi-continuous reactors, including CSTRs, anaerobic baffled reactors (ABRs), and membrane bioreactors (MBRs), have demonstrated higher hydrogen production due to reduced metabolite buildup and increased mass flow rates. In membrane systems, metabolites that inhibit the process, along with other dissolved gases, can be selectively removed, thus increasing process stability (; ; Sahil et al., 2024). Another approach involves a two-stage fermentation process followed by photofermentation. By doing so, the produced VFAs in stage one are upgraded to hydrogen in stage two, resulting in increased efficiency of hydrogen production and substrate conversion (). The use of conductive and carbon materials, specifically biochar, has proven to be an effective method of controlling by-product formation. Biochar facilitates extracellular electron transport, improves microbial immobilization and stabilization, favors acetate-butryate pathways, and suppresses lactate and propionate production. Biochar also acts as a buffer, neutralizing any pH decrease caused by VFA production (; Roychowdhury and Ghosh, 2024).
Another emerging approach to enhance biohydrogen production is microbial community engineering and omics methods. These include metagenomics, metabolomics, and transcriptomics techniques. Such techniques contribute to improving the knowledge of metabolic regulation. They also provide insight into microbial interactions in the bioreactor during fermentation processes. Selective enrichment of hydrogen-generating bacteria (Clostridium spp.) and inhibition of competitive bacteria such as homoacetogens and methanogens are some ways to control the formation of unwanted products (). Moreover, artificial intelligence and machine learning are becoming prevalent in predicting patterns of by-product formation and optimizing operational parameters, including pH, temperature, HRT, and OLR. These technologies allow for process optimization and help achieve scalability (Shi et al., 2024).
Despite the progress made, a number of issues remain. The complex nature of microorganism communities (), the variability in the quality of actual waste material (), and their interdependence, such as the influence of various factors including pH value, hydrogen pressure, and accumulation of metabolites, complicate the predictability of the process ().
3.9 Influence of bioreactor types
Bioreactors are essential components of every biological process. They can be designed and constructed to meet the specific requirements of the microorganisms used, ensuring optimal growth and performance (Regonesi, 2023). The technological foundation of biohydrogen production relies on the effectiveness of bioreactors, which are designed to maximize biohydrogen production. Conditions in the bioreactor influence the success of biological processes such as anaerobic digestion, dark fermentation, and biohydrogen generation. Bioreactors are critical components in biohydrogen production processes. The common configurations are anaerobic sequencing batch reactors (ASBR), MBR, up-flow anaerobic sludge blanket reactors (UASBR), CSTR, and anaerobic fluidized bed reactors (AFBR) (Raut et al., 2025). Dark fermentation bioreactors can be classified into two main categories: batch and continuous bioreactors (Yahaya et al., 2022). Different studies indicate that batch processes yield higher amounts of biohydrogen than continuous processes. This is attributed to the more favorable conditions created by high initial substrate concentrations in batch systems, compared to the lower substrate concentrations typically maintained in continuous digesters (Sanghvi et al., 2024). One major issue using batch reactors for biohydrogen production via dark fermentation is the downtime associated with filling and emptying. Thus, a reduction in total efficiency results (). The effects of bioreactor type on the dark fermentation process are summarized in Table 1.
TABLE 1
| Reactors types | Substrates used | Temp (0C) | pH | H2 yield | References |
|---|---|---|---|---|---|
| Batch | Rice mill wastewater | 37 | 5.5 | 1.2 L H2/L | Ramu et al. (2021) |
| Batch | Food waste water | 37 | 7.5 | 0.566 ± 0.004 LH2/L | |
| Batch | Sugar beet molasses | 37 | 4.4 | 208.3 LH2/L | Sanghvi et al. (2024) |
| Batch | Rice straw | 55 | 7.0 | 0.069 L H2/L | |
| Batch | Rice straw | 37 | 6.8 | 2.25 L H2/L | Tondro et al. (2020) |
| CSTR | Poplar leaves | 30–40 | 5–8 | 18 L H2/L | |
| CSTR | Date waste | 80 | 7.0 | 1.08–9.14 L H2/L | Saidi et al. (2023) |
| CSTR | Peanut shell | 60 | 4.85–5 | 0.58 LH2/L | Qi et al. (2023) |
| CSTR | Acidic cheese whey | 55 | 5 | 3.2 LH2/L | Ordoñez-Frías et al. (2024) |
| CSTR | Pig manure | 27 | 6.5 | 3.84 LH2/L | |
| CSTR | Xylose | 30 | 7.5 | 30.26 ± 1.19 LH2/L | |
| CSTR | Rice straw | 55 | 6.5 | 24.8 LH2/L | |
| Membrane bioreactor (MBR) | Diluted grape deposits | 37 | 5.5 | 4.5 LH2/L | Renaudie et al. (2024) |
| MBR | Mixture of glucose, xylose and arabinose | _ | _ | 28.52 ± 0.85 L H2/L | |
| Liquid/gas MBR | Glucose | 37 | 5.1 ± 0.3 | 2.56 ± 0.25 L H2/L | Renaudie et al. (2021) |
| DMBR | Xylose | 37 | 7.5 | 30.26 ± 1.19 L H2/L | |
| DMBR | Pretreated red algal | - | - | 21.6 L H2/L | Sim et al. (2021) |
| DMBR | Rice straw | - | - | 33.8 L H2/L | |
| DMBR | Mixture of glucose, xylose and arabinose | - | - | 28.5 L H2/L |
Effects of the type of bioreactor on biohydrogen production.
According to Table 1, the configuration of the bioreactors affects substrate conversion, microbial population, and mass transport in the reactor. These factors inhibit hydrogen yield. Continuous stirred tank reactors have become very popular because of their simple design and effective mixing, which allows for maximising substrate contact and pressure removal of hydrogen from the liquid phase by reducing the inhibitory partial pressures on the substrate. However, the drawback of CSTRs is the washout effects at high organic loading rates unless additional measures are used. MBR is an innovative platforms that enhance cell retention and electron recovery. It provides enhanced retention of microbial biomass. It is independent of hydraulic retention time. It provides enhanced retention of microbial biomass regardless of its dependence on hydraulic retention time. However, MBRs present significant membrane fouling challenges that must be addressed during high-solid waste fermentation (). Other types of advanced reactor systems include the packed-bed bioreactor, fluidized-bed reactor, and two-stage bioreactor configurations, all of which can greatly improve bulk biomass retention and volumetric productivity, but they do result in more complexity and cost compared to typical MBR and CSTR systems (Magdalena et al., 2024). Figure 3 shows a schematic representation of the CSTR and MBR systems.
FIGURE 3
The availability, low cost, and high carbohydrate content of lignocellulosic biomass make it a prospective renewable feedstock for biohydrogen generation. Cellulose and hemicellulose present in agricultural by-products, including corn stover, wheat straw, rice straw, sugarcane bagasse, and coffee husk, can be hydrolyzed to form fermentable sugars through dark fermentation. But lignin, which forms the recalcitrant lignin matrix, needs to be pretreated using techniques such as dilute acid, alkaline, steam explosion, and hydrothermal pretreatment to enhance hydrolysis and increase the amount of sugars obtained. Although pretreatment improves fermentability, it can also lead to the formation of inhibitory compounds, including furfural, 5-hydroxymethylfurfural (HMF), phenolics, and weak acids. These compounds negatively affect microbial growth, hydrogenase activity, and biohydrogen yield. Studies have shown that several approaches can improve biohydrogen fermentation performance. These include detoxification, microbial acclimatization, membrane-assisted fermentation, co-fermentation, and the use of conductive additives such as biochar (; ; ; Zhao et al., 2024).
Agricultural waste feed sources that contain large quantities of simple sugars are better suited for producing biohydrogen than other types of organic feed sources when run under identical bioreactor conditions. Examples of fruit and vegetable peel wastes (FVPWs), such as watermelon and melon peels, have shown high potential for biohydrogen production. When fermented using Clostridium butyricum NE95, they achieved high maximum hydrogen yields. The large volume of easily hydrolysable carbohydrates present in these substrates was rapidly metabolized by the microbes present (). Simple carbohydrate sugars such as glucose are preferentially metabolized by hydrogen-producing microbes. This occurs regardless of prior metabolism of other sugars. This preference is mainly due to their simpler structure and the lower enzymatic effort required for their breakdown (). Based on typical dark fermentation performance, approximately 50–100 kg of lignocellulosic biomass is required to produce 1 kg of biohydrogen. In contrast, water electrolysis requires only about 10–15 L of water to produce the same amount of biohydrogen. This difference reflects the lower conversion efficiency and higher material demand of biological hydrogen production systems (; Zhao et al., 2024).
Tests on various substrates for hydrogen production found that wheat starch produced the most hydrogen yield (∼140.29 mL of H2 per g COD), while corn waste and brewers’ spent grain were next highest. Glucose-derived substrates produced a greater hydrogen yield than lignocellulosic hydrolysates, as hydrogen-producing bacteria can more readily utilize monosaccharide-containing substrates compared to those containing mixed sugar fractions that result from hydrolyzing hemicellulose (Sarkar et al., 2025). Pretreated sugarcane bagasse has been shown to produce high volumetric hydrogen yields (approximately 6,980 mL H2/L). This is higher than materials containing significant amounts of lignin (Rani et al., 2024). The presence of lignin and other complex polysaccharides limits the rapid hydrolysis of lignocellulosic biomass. Therefore, pretreatment methods such as enzymatic treatment or sugar extraction are required to obtain fermentable sugars. It is necessary to enable efficient conversion of substrate to hydrogen by microorganisms. If this is not done, the accessibility of fermentable sugars to microorganisms will be greatly diminished ().
Protein- and lipid-rich organic materials, such as dairy whey and slaughterhouse waste, generally produce lower hydrogen yields per unit of substrate. This is lower than the yields obtained from carbohydrate-rich organic waste streams in the same reactors. Ammonia produced from protein degradation can inhibit the activity of hydrogen-producing microorganisms. Likewise, long-chain fatty acids produced from lipid degradation are often toxic. They can also redirect hydrogen-producing microorganisms away from hydrogen gas production (). Several studies have shown that co-substrate systems combining carbohydrates with proteins or lipids can increase hydrogen yields. These yields are higher than those obtained from using protein or lipid substrates alone. This improvement is attributable to the limitations of single-substrate utilization (). Many metabolites produced from the breakdown of proteins and lipids, including ammonia and long-chain fatty acids, inhibit hydrogen production. They also divert electron flow away from hydrogen generation ().
Recent studies have shown that combining different feedstocks can increase hydrogen production. For example, whey protein and pre-treated sugarcane can produce more hydrogen than single-feedstock fermentation under identical reactor conditions. This is due to a more balanced nutrient ratio and the availability of multiple metabolic pathways. In co-fermentation, the proper balance between carbon and nitrogen increases biohydrogen production. This is because the conditions formed enable the growth of microorganisms on the substrate ().
4 Challenges and mitigations in biohydrogen production processes
Dark fermentation for the production of biohydrogen is constrained and challenged in three areas of development: technology, biology, and economics (). Thermodynamic constraints, including partial pressures, transport difficulties, reactor designs, biomass retention, hydrodynamic issues, feed handling problems, and the need for instrumentation/control to maintain continuous operation, are all technical limitations (). From thermodynamics, the maximum yield of hydrogen gas would be 4 mol for every mole of glucose. But this is not usually achieved because some electrons are used in forming VFAs, alcohols, and biomass (). Because of the high concentration of H2 in dark fermentation atmospheres and because CO2 (and CH4 intermittently) are also present, separation of H2 from these gases will be necessary, as well as the capital/equipment and operational (CapEx/OpEx) costs associated with their separation. According to several publications, the biohydrogen generated by the dark fermentation contains incombustible gases, including CO2, trace amounts of hydrogen sulfide, and moisture. Enriching and upgrading hydrogen is equally important as producing it sustainably. It is important to remember that H2 can only be used as an energy fuel in specified applications if its purity is at least 99.99% (). Effectively purifying and storing biohydrogen produced via dark fermentation is challenging because of its high diffusivity and low density (). Precautions can be taken by utilizing gas-stripping techniques such as vacuuming, membrane contactors, alkaline scrubbing, and coupling with PSA/membrane units, which inhibit hydrogen-producing bacteria, often leading to crashes or shifts to unanticipated pathways. Mitigating the environment through buffer methods like bicarbonate, CaCO3, biochar, pH-controlled feeding, continuous or partial elimination of VFAs, and careful OLR ramping during startup (; ).
Accumulated H2 increases partial pressure, hindering H2-producing reactions and leading to solventogenesis or other reduced products. Poorly soluble H2 levels necessitate efficient removal; inadequate gas-liquid transfer and poor mixing reduce volumetric productivity and create concentration gradients (gas pockets, dead zones). The use of reactor geometries that increase kLa (such as fluidized/hybrid beds and membrane bioreactors), optimized agitation/gas sparging techniques, and CFD-guided scale design are among the mitigation measures (). Short HRTs, used to suppress methanogens in bacteria, can reduce CSTR productivity due to the slow growth of hydrogen-producing microbes. Immobilization can be achieved using biochar, granules, packed-bed or expanded-bed designs, utilizing the nutrient-rich media of bacterial cells as a dietary supplement (Wodołażski and Smoliński, 2022). Other operational headaches caused by particles, lipid-rich substances, carrier scouring, and persistent foaming add to downtime. The mitigation methods include pre-screening and homogenization, grease/lipid removal steps, antifoamer and mechanical defoamers, and modular cartridge/carrier designs for easy cleaning (; ).
Lignocellulosic feedstock hydrolysis needs technical pretreatment. Enzymatic, thermal, or chemical pretreatment is necessary to release fermentable sugars from lignocellulosic skeletal wastes. This adds cost and process complexity. Two-stage systems (hydrolysis DF), low-energy pretreatments, and co-digestion with easily fermentable wastes are effective methods for reducing process times (; Roychowdhury and Ghosh, 2024). Stable DF requires precise control of pH, temperature, gas composition, and flow, as well as instrumentation/monitoring and process control. System complexity and operating expenses can be heightened by high-resolution monitoring and automated control (Teke et al., 2024). Some of the challenges of scaling up include mixing issues, heat transfer, process reproducibility, hydrogen management, mass flow rate, and contamination. When these parameters are scaled up, many of them exhibit nonlinear behavior. Consequently, it is hard to achieve the high biohydrogen production that is possible in laboratory settings at larger scales. The optimal mitigation measures are modular scale-up, CFD (Computational Fluid Dynamics)-based design in pilots, standard testing protocols, and phased risk de-risking (). Due to its lower flammability limit (LFL) of 4%, H2 poses significant fire and explosion risks at both smaller and larger scales. The mitigation measures include intrinsic safety design, continuous H2 detectors (for example, when working on gas), ventilation, flame arrestors, and strict SOPs (Standard Operating Procedures) for gas handling (Teke et al., 2024).
While different hydrogen-producing strategies have advantages, they also pose substantial obstacles that require additional exploration (Osman et al., 2023). Barriers to biohydrogen production include low energy conversion efficiency due to metabolic restrictions and byproduct losses. In addition to yield limitations due to the electron transfer, hydrogenase enzymes are vulnerable to environmental stresses. Inhibitors in renewable feedstocks and unpredictable substrates reduce process efficiency. Scaled-up systems face issues such as contamination, inconsistent yields, and difficulty maintaining optimal conditions. Biohydrogen generation remains limited due to regulatory hurdles, high operational costs, and delayed reaction kinetics (Omer et al., 2025). In experiments, dark fermentation was found to be more cost-effective than photo fermentation, which was more efficient but more expensive ().
The industrial sector requires hydrogen for its operations. However, biohydrogen production in the laboratory setting has limited practical value for large-scale applications. Moreover, biohydrogen cannot be calculated with the same precision as many other forms of hydrogen production. This is because it is not easy to determine how much biohydrogen can be obtained from particular organic waste or even from biomass altogether. For instance, producing 1 kg of hydrogen via water electrolysis would require much more than a single litre of water per day ().
Generally, microbial abnormalities during biohydrogen production pose significant challenges. Microbes may struggle to break down chemical components in feedstock, or the feedstock itself may impede them. Another concern may be the microorganisms’ lifespan. Microbes have a limited lifespan, lasting from hours to a few days under optimum conditions. Hydrogen-producing microorganisms, such as Synechocystis, Anabaena, Clostridium, Escherichia coli, and Methanogens, can live from a day to weeks depending on cultivating procedures. Lifetime is affected by pH, temperature, by-product toxicity, light intensity, nutrition availability (particularly nitrogen), and substrate availability (). Microbial populations can be maintained over an extended period under both efficient and inefficient conditions by producing more than 100 cm3 of hydrogen from 100 cm3/g COD molasses. Furthermore, lactate co-fermentation was also found to improve microbial population stability and increase hydrogen generation ().
Biohydrogen can be produced efficiently from carbohydrate-based feedstocks due to their high fermentability and favorable metabolic pathways. Industrial-scale biomass utilization should rely on cheap, non-food, and underutilized waste materials, including wastes from fruit and vegetable processing. Non-food agro-industrial residues such as molasses and cassava waste are also appropriate. In addition, lignocellulosic agricultural residues, including crop straws, coffee husks, and bagasse, are viable options. Using these materials helps avoid competition with food and animal feed value chains (; Vidal et al., 2025). In contrast, diverting substrates already widely used in animal feeding (e.g., grains, oilseed cakes) may increase feed costs and disrupt livestock supply chains, raising economic and ethical concerns. Similarly, many organic wastes (e.g., manure, food waste) are already valorized in anaerobic digestion (biogas) systems, where they contribute to stable methane production and nutrient recycling; redirecting these substrates toward biohydrogen production could reduce overall energy recovery unless integrated approaches are adopted (). From a systems perspective, replacing biogas pathways with standalone dark fermentation may be suboptimal, whereas two-stage processes (biohydrogen followed by biomethane production) can maximize total energy yield and ensure compatibility with existing infrastructure.
Instability in the process can be attributed to several factors, including variation in substrate, microbial adaptation, poor mass transfer, and economic considerations. Thus, stable microbial consortia and operational strategies should be considered to enhance system efficiency. The integrated system is difficult to run, but it can recover energy more effectively. Despite its benefits, the system suffers from more problems. This is mainly due to the use of lignocellulosic materials. One key point to consider in large-scale deployment is the management of the acidified slurry (effluent) produced through dark fermentation. These substrates have high VFAs and nutrients. Some viable ways to exploit this resource could be: (i) secondary anaerobic digestion for hydrogen-methane production, (ii) recovery and upgrading of VFAs for production of valuable chemicals, (iii) recovery of nutrients for use in biofertilizers after stabilization, and (iv) using it in biorefinery or microbial electrochemical systems (; Zhao et al., 2024). Overall, utilizing these waste as substrates, avoiding conflict with existing food and energy resources, and utilizing by-products are crucial for sustainable large-scale biohydrogen deployment.
5 Technological integrations to enhance biohydrogen production
Recent advances in biohydrogen production through dark fermentation have focused on integrating dark fermentation with other complementary processes. This strategy enables one to address problems associated with low yield and inhibition of the fermentation process. Pre-treatment and co-digestion strategies for enhancing the bioavailability of complex organic wastes include physical, chemical, or enzymatic pre-treatments or the mixing of different feedstocks (; Singh et al., 2022). These methods increase the content of fermentable sugars, optimize nutrient balance, and improve hydrogen production. Integration with bioelectrochemical and photofermentation has emerged as a promising route to further increase productivity (). Microbial electrolysis cells recover electrons from the intermediate metabolites produced during dark fermentation to create additional hydrogen, whereas photofermentation uses light-driven microbes to change residual volatile fatty acids into hydrogen. Such combined systems extend substrate utilization and significantly improve overall hydrogen yield from organic wastes ().
Designs of different bioreactors and process control play an important role in dark fermentation during biohydrogen production from biomass. Some forms of CSTR and UASB reactors (including the hybrid variant) utilize fermentation to achieve balanced mass transfer, hydraulic retention, and microbial retention (i.e., the combination of these forms) during the scale-up and stable operation of the bioconversion process (Pandit et al., 2025). Coupled with automated control over pH, temperature, substrate feeding, and hydrogen partial pressure, these strategies maintain optimal microbial activity and metabolic flux. Further, engineering robust microbial consortia through adaptive evolution or co-culturing enhances resilience to inhibitory conditions and maximizes substrate-to-hydrogen conversion. Collectively, these integrated approaches put together a comprehensive framework for scalable, efficient, and sustainable biohydrogen production from organic wastes (; Santhosh and Mohan, 2025). Some of the biohydrogen production integrated systems are summarized in Table 2.
TABLE 2
| Integrated technology | Quantitative yield/Performance improvement | Key benefit | Advantage of integration | References |
|---|---|---|---|---|
| DIET and microbial consortia | 10%–30% increase in H2 yield | Improved microbial stability | Enhanced electron transfer between microbes | |
| Dark fermentation + photo-fermentation | 2–3× increase in total H2 yield | Sequential substrate utilization | Maximizes VFA conversion to H2 | , Putatunda et al. (2023) |
| DF + MEC | 30%–40% additional H2 recovery | Utilization of residual organics | Improves total energy recovery | |
| Thermochemical integration | Improved overall energy recovery | Full biomass utilization | Combines biochemical and thermal conversion | |
| High-solids fermentation | 20%–50% higher productivity | Reduced water usage | Higher reactor efficiency | Silva-Martínez et al. (2025), Zhang et al. (2023) |
| Heat-shock pretreatment | 20%–45% increase in H2 yield | Suppresses methanogens | Enhances hydrogen-producing bacteria | |
| Biochar-assisted DF | 15%–40% increase in H2 production | Improved kinetics | Reduces inhibition and enhances microbes | |
| Microbiome engineering | 0.47–1.88 mol H2/mol glucose | Improved metabolic control | Targets hydrogen-producing pathways | |
| Pilot-scale modeling | Improved economic performance | Scale-up feasibility | Bridges lab to industry | Sivaranjani et al. (2023) |
Integrated technology for biohydrogen production.
Table 2 presents recent strategies for improving the performance of biohydrogen production processes. The integration of technologies leads to higher hydrogen production efficiency. It also improves system stability. In addition, it enhances overall energy recovery compared to dark fermentation alone. Research is shifting from modifying individual microbial strains to multi-microbial consortia. These consortia can directly transfer electrons between microbes. This improves metabolic coupling. It also enhances hydrogen production. DIET reduces the accumulation of VFAs. It does this by bypassing the diffusion limitations of hydrogen and formate intermediates. This improves system stability. It also increases productivity (Parra et al., 2025). Two-stage fermentation and photofermentation can improve overall hydrogen yield. They use dark fermentation (DF) effluent as an input. This effluent is then used for light-driven hydrogen recovery. The process is passed through a two-step fermentation system. Thermochemical coupling can be achieved by combining dark fermentation (DF) with hydrothermal gasification or other high-temperature processes. These processes convert fermentation residues into hydrogen-rich syngas. They also provide an additional pretreatment stage with higher recovery efficiency (). High-solids DF can be used to reduce pretreatment requirements and improve energy balance by operating at higher solids concentrations, such as municipal solid materials or co-digested feed (Silva-Martínez et al., 2025). Heat-shock pretreatment can be applied to both the inoculum and the feedstock. This can increase the overall hydrogen yield. This suggests that integrated preprocessing could enhance both microbial activity and substrate availability (). Complex waste types are undergoing further investigation to better understand DF microbial communities. By comprehending the specific roles and interactions of niches, targeted stabilization and performance improvement can be achieved (). Optimization of performance and scale-up conditions can enhance biohydrogen production. The use of fed-batch/semi-continuous processes is on the rise due to their ability to decrease substrate inhibition and enhance metabolic management, making them more effective than batch or continuous modes. Up to 3,000 years of pilot tests and models. On the large scale, there is actual potential, but further development is required to achieve a robust design and financial stability (Teke et al., 2024). In general, advanced technologies such as photoperiodic electrolysis, microbial fermentation techniques, and co-digestion can improve biohydrogen production from residual waste (Ram et al., 2024).
6 Techno-economic feasibility and life-cycle assessment for biohydrogen production
6.1 Techno-economic feasibility analysis (TEA)
TEA is an essential technique for analyzing the economic feasibility of biohydrogen production through DF. Although DF offers various benefits, such as being a renewable and environmentally friendly energy resource, the economic viability of this process is challenging due to its high cost compared to hydrogen produced using fossil fuels. According to recent studies, the cost of biohydrogen production via DF typically ranges between 3.2 and 48.96 USD/kg H2, depending on the feedstock, reactor type, and integration techniques. The cost of feedstock can contribute up to 80% of the total production cost. The pretreatment step alone can account for more than 50% of the operational costs in some processes (Osman et al., 2024; Sanghvi et al., 2024).
However, the potential of such integration techniques has proven to be highly more efficient. The use of DF technology, along with AD or membranes, enhances hydrogen yield by decreasing shock load and stabilizes the process. The results of techno-economic assessment conducted on a pilot scale revealed hydrogen production costs of around 3.84–3.86 USD/kg H2 for DF integration processes, with payback periods being about 1.3 years ().
6.2 Life-cycle assessment (LCA) of dark fermentation systems
Life cycle assessment serves as a holistic approach to assess the sustainability of DF-based hydrogen energy, including all stages ranging from feedstock extraction through to the end use of hydrogen. Recent studies indicate that DF has a better environmental suitability than fossil fuel-based hydrogen energy because of its capability to use organic waste streams. The global warming potential (GWP) of DF is lower than that of steam methane reforming (SMR) hydrogen production processes, with approximately 0.79 kg CO2-eq/kg H2 difference for an integrated DF-AD system (). Moreover, comparative LCA studies show that hydrogen production from biomass can result in significantly lower CO2 emissions. This is especially true when waste-based feedstocks are used instead of fossil fuels for biohydrogen (Osman et al., 2024; Sanghvi et al., 2024).
However, environmental performance is heavily dependent on energy use for heating, mixing, and pre-treatment, accounting for more than 70% of emissions from DF technologies. Thus, electricity consumption and heat energy production have been recognized as significant environmental hotspots in the life cycle inventories. Furthermore, differences in system boundaries, functional units, and inventory data reported in many studies make direct comparisons difficult, suggesting the need for standardization in LCA approaches in future studies (Sivaranjani et al., 2023). Despite these shortcomings, DF is a promising technology for incorporation into the circular bioeconomic system ().
7 Conclusion and future directions
Dark fermentation is a promising pathway for converting organic wastes into biohydrogen within a circular bioeconomy framework. However, its large-scale deployment remains constrained by thermodynamic limits, process instability, and economic and environmental trade-offs. Current studies indicate that carbohydrate-rich feedstocks offer the most reliable performance. In contrast, lignocellulosic substrates are constrained by high pretreatment energy demand and associated environmental impacts, as highlighted in the TEA/LCA analysis. Mixed anaerobic consortia provide the most robust and scalable inoculum strategy, and highly enriched cultures require careful control to maintain stability. Improvements in reactor designs and process configurations, including CSTRs, hybrid reactors, and membrane bioreactors, enhance volumetric productivities and hydrogen yields. However, there are still challenges due to thermodynamic constraints, hydrogen partial pressure, and process instability arising from the accumulation of VFAs. In addition, energy-intensive steps and gas purification significantly affect the techno-economic and environmental aspects of the processes. The approach of system integration has gained importance in solving these problems. The combination of DF with subsequent processes, including anaerobic fermentation, microbial electrolysis cells, and photo-fermentation, has led to improved biohydrogen production. But this makes the whole process complex and calls for economic optimization. In general, dark fermentation holds strong potential as a waste-to-biohydrogen technology. However, its successful commercialization depends on moving beyond isolated laboratory optimizations. It requires integrated, system-level solutions. These solutions must address biological, engineering, economic, and environmental constraints simultaneously.
To advance DF toward industrial application, the following priority areas are recommended.
Future studies should focus on low-energy and environmentally sustainable pretreatment methods, as well as optimized co-digestion strategies to enhance substrate utilization while minimizing costs and environmental burdens.
Greater emphasis is needed on developing robust, self-regulating microbial communities through microbial ecology, adaptive evolution, and metabolic engineering approaches to improve stability and hydrogen yield under real operating conditions.
Research should prioritize advanced reactor configurations that enhance mass transfer, biomass retention, and hydrogen removal, including membrane-based and hybrid systems, supported by CFD-based design and scale-up studies.
c. Integrated systems combining DF with anaerobic digestion, microbial electrolysis, or photo-fermentation should be further explored to maximize energy recovery and improve overall system efficiency.
Future developments must be guided by integrated TEA and LCA frameworks. This will ensure that improvements in biohydrogen production translate into real-world economic viability and environmental sustainability
More pilot- and demonstration-scale studies are required to address scale-dependent challenges such as process control, contamination risks, and operational stability.
Statements
Author contributions
TB: Writing – review and editing, Visualization, Validation, Software, Methodology, Investigation, Formal Analysis, Conceptualization. SN: Writing – review and editing, Software, Formal Analysis, Conceptualization. VA: Writing – review and editing, Visualization, Validation, Software, Methodology, Investigation, Formal Analysis, Conceptualization. SA: Writing – review and editing, Visualization, Validation, Software, Methodology, Investigation, Formal Analysis, Conceptualization. TM: Writing – review and editing, Software, Formal Analysis, Conceptualization. EJ: Writing – review and editing, Software, Formal Analysis, Conceptualization. M-MN: Writing – review and editing, Software, Formal Analysis, Conceptualization.
Funding
The author(s) declared that financial support was received for this work and/or its publication. The publication fees were supported by University of Rostock.
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Summary
Keywords
biohydrogen, dark fermentation, microbial dynamics, substrate utilization, system integration
Citation
Binchebo TL, Narra S, Ancha VR, Asefa SK, Mengesha TT, Jiru EB and Narra M-M (2026) Recent developments in biohydrogen production through dark fermentation of biomass waste: feedstocks, microbial population dynamics, and system integration. Front. Energy Res. 14:1819152. doi: 10.3389/fenrg.2026.1819152
Received
27 February 2026
Revised
04 June 2026
Accepted
08 July 2026
Published
20 August 2026
Volume
14 - 2026
Edited by
Ioannis A. Panagiotopoulos, Intergeo, Greece
Reviewed by
Xiomar Arleth Gomez, University of León, Spain
Young Eun Song, Berkeley Lab (DOE), United States
Updates
Copyright
© 2026 Binchebo, Narra, Ancha, Asefa, Mengesha, Jiru and Narra.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Tarekegn Limore Binchebo, tarekegn.limore@ju.edu.et
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