Biohydrogen Produced via Dark Fermentation: A Review

Abstract

Hydrogen (H₂) is a highly efficient and clean energy source with the potential for renewable energy. The production of H₂ from biological routes such as biophotolysis, photofermentation, dark fermentation, and bioelectrochemical production is characterized as a renewable alternative to current production, which is mainly based on energy-intensive electrochemical and thermochemical processes and responsible for the emission of high amounts of environmentally harmful compounds. Dark fermentation is the most efficient and cost-effective method for producing biohydrogen, making it a key research focus. This article offers a comprehensive overview of the dark fermentation process with the aim of enhancing hydrogen productivity and yields. Aspects related to the main substrates used, the inoculum sources and their pretreatment, and physical-chemical parameters of the process are covered. Furthermore, this manuscript addresses topics such as process integration, genetic and metabolic engineering of fermentative microorganisms, and the main types of bioreactors aimed at greater yields and productivity of biohydrogen to enable its production through dark fermentation on a larger scale.

1. Introduction

The depletion of fossil resources, which represent approximately 80% of the current energy matrix [1], as well as the increase in market prices and the environmental issues related to the release of pollutant gases during their combustion have driven research toward the production of renewable energies and their implementation on an industrial scale, such as solar, wind, geothermal, and biofuels [2,3].

In this context, hydrogen (H₂) is considered the energy vector of the future due to its high energy content (122 kJ g⁻¹, at least 2.75-fold higher than fossil fuels) and the release of H₂O as the only byproduct of its combustion [4,5,6].

Although H₂ can be used as an energy source through its direct application in internal combustion or jet engines or also converted into electrical energy in fuel cells, its current applications are concentrated in the chemical and petrochemical industries aiming at fuel refining, ammonia synthesis, methanol synthesis, and hydrogenation of edible oils [7], generating a global demand of 95 million tons of H₂ in 2022 [8].

H₂ is obtained from energy-intensive processes such as water electrolysis (approximately 4%) and thermochemical methods (approximately 96%) using non-renewable sources such as natural gas, crude oil, and coal (Figure 1) [9], which cause atmospheric emissions of high amounts of carbon, sulfur, nitrogen oxides, ashes, radioactive substances, and heavy metals [10,11,12].

For the H₂ economy to consolidate sustainably, H₂ must be produced from renewable routes, minimizing or avoiding the emission of pollutant compounds [13]. Renewable H₂ production can be carried out from biomass using thermochemical processes such as gasification, pyrolysis, steam reforming, or through biological routes such as dark fermentation and photofermentation (Figure 1) [14]. Biological routes are more attractive than thermochemical processes because they are developed under environmental conditions that demand little energy and cause reduced or no carbon release [15].

Different microorganisms carry out the biological production of hydrogen (bioH₂). They can be categorized into: (1) light-dependent methods such as biophotolysis, performed by photosynthetic microalgae and cyanobacteria, and photofermentation, through the oxidation by photoheterotrophic bacteria of carbon sources such as sugars and organic acids in the presence of light energy, and (2) light-independent methods such as dark fermentation, performed by heterotrophic bacteria from the anaerobic metabolism of pyruvate, and bioelectrochemical production using microbial electrolysis cells. Additionally, these processes can be coupled to improve yields, such as dark fermentation-photofermentation [16] and dark fermentation-microbial electrolysis cells [17].

Regarding energy efficiency and practicality, both light-dependent and independent methods present advantages and disadvantages when compared to one another [18]. However, dark fermentation stands out as the most widely studied biological process for obtaining bioH₂ due to its more advantageous and realistic aspects of the production process [19]. It requires bioreactors with simpler operating conditions than photofermentation, is independent of light, and has higher yield rates than other biological methods [13,20]. It also has lower energy demand, the possibility of integration with other systems like anaerobic digestion for biogas and biomethane production, and the use of a wide variety of organic wastes as substrates. Thus, dark fermentation not only results in the production of bioH₂ but also results in a key biotechnology opportunity in many microbial-based biorefineries to produce valued-added organic compounds (i.e., organic acids, alcohols, biofuels and bioplastics [15,21,22,23].

This work aims to provide a general description of bioH₂ production from dark fermentation and the factors that influence its yield, such as inoculum sources and pretreatment, substrates, pH, temperature, organic loading rate, hydraulic retention time (HRT), partial pressure of H₂, as well as an overview of the main continuous bioreactors used, and technologies employed to achieve higher yields.

2. The Dark Fermentation Metabolic Pathway

Dark fermentation is an anaerobic process of organic compound degradation carried out by strict or facultative anaerobic microorganisms in the absence of light, resulting in bioH₂ production through proton reduction to dissipate excess electrons from organic matter oxidation in the culture medium [24]. As primary byproducts of fermentation, compounds such as alcohols, CO₂, and organic acids are also obtained [25,26,27].

Although various organic substances, including carbohydrates, sugars, proteins, and lipids, can, in principle, serve as substrates for bioH₂ production, the metabolic pathway is commonly demonstrated using glucose as a model substrate (Figure 2). Glucose is converted into pyruvate through the glycolytic pathway, leading to the reduction of NAD to NADH and ADP to ATP [28].

Subsequently, depending on the microorganism’s metabolism, pyruvate can be converted into acetyl-CoA through the mediation of two different enzymes, pyruvate formate lyase (PFL), more commonly used by facultative anaerobic microorganisms but occasionally found in strict anaerobes like Clostridium [29], and pyruvate ferredoxin oxidoreductase (PFOR) used by strict anaerobes [30]. These enzymes are responsible for reducing hydrogenases, directly involved in bioH₂ catalysis. Two main types of hydrogenases are known, [FeFe]-hydrogenases found in both strict and facultative anaerobic pathways, and Ni-Fe hydrogenases, more oxygen tolerant and thus present only in the facultative anaerobic pathway [24,31,32].

Through the PFL enzyme-dependent pathway, facultative anaerobic microorganisms convert pyruvate into acetyl-CoA and formate without immediate production of reducing equivalents [14]. Additionally, pyruvate can be converted into lactate-by-lactate dehydrogenase (LDH) enzyme [33]. Subsequently, formate is oxidized by formate hydrogen lyase (FHL) enzyme, which reduces the hydrogenase enzyme that later catalyzes the reduction of protons H⁺ to H₂, also resulting in CO₂ production [34,35]. Considering that hydrogen is derived from formate and that a maximum of two formates are formed per glucose, maximum bioH₂ yields can be predicted as 2 mol of bioH₂/mol of glucose [31].

The resulting acetyl-CoA from this reaction is oxidized to acetate, through two enzymatic steps. In the first step, the enzyme phosphotransacetylase converts acetyl-CoA into acetyl-phosphate. In the second step, acetyl-phosphate is converted to acetate by the enzyme acetate kinase, a process that also results in the formation of ATP [36,37]. In the PFL enzyme-dependent pathway, the NADH produced during glycolysis cannot be reoxidized for additional bioH₂ production and must be reoxidized by the production of a reduced organic compound, typically ethanol. This conversion initially occurs under the action of the enzyme pyruvate decarboxylase, where acetyl-CoA loses a carboxyl group in the form of CO₂, forming acetaldehyde. Subsequently, acetaldehyde is reduced to ethanol by the enzyme alcohol dehydrogenase [31].

In the PFOR enzyme-dependent pathway, bioH₂ production occurs through the conversion of pyruvate into acetyl-CoA and reduced ferredoxin, a direct electron donor to [FeFe]-hydrogenase, which subsequently reduces protons producing 2 mol of bioH₂ per mol of glucose [31]. However, unlike the PFL-dependent pathway, under ideal bioH₂ partial pressure conditions, acetyl-coA can be converted into acetate by ATP formation, the two NADH molecules generated during glycolysis can be reoxidized to produce two more hydrogen molecules through two other hydrogenases, NADH-dependent hydrogenase (NADH-[FeFe]) and reduced ferredoxin-dependent hydrogenase (NADH-Fd_red-[FeFe]) [27,31,36,38], resulting in a final production of 4 mol of bioH₂ per mol of glucose [39].

BioH₂ production from NADH oxidation only occurs under low bioH₂ partial pressures when the change in free energy is negative [39]. However, during fermentation, the accumulation of bioH₂ in the bioreactor headspace leads to an increase in partial pressure, which increases the concentration of dissolved bioH₂ in the liquid phase, making the oxidation reactions of NADH for bioH₂ production less favorable [40,41].

Thus, under higher bioH₂ partial pressures, the acetyl-CoA formed through pyruvate oxidation is used together with NADH for the formation of reduced organic compounds [42]. The main byproducts in strict anaerobic fermentation are butyric acid, butanol, and acetone, in proportions dependent on the microorganism, substrate, and physicochemical conditions of fermentation [28,35]. It is essential in both pathways that NADH is reoxidized to maintain the cell’s redox balance and allow the continuity of metabolic processes, being crucial to ensure that NAD+ is available to accept electrons during glycolysis.

In addition to limiting bioH₂ yield, the incomplete oxidation of organic matter forming volatile fatty acids can lead to pH drop in the culture medium if not adequately controlled. The acidification under the optimal ranges inhibits microorganisms [43], a disadvantaging dark fermentation. However, to overcome this issue, the effluent from dark fermentation can be used as a substrate for bioH₂ or biogas production in a second stage, using, for example, photofermentation and microbial electrolysis cells [44]. Furthermore, volatile fatty acids can also be purified from fermentation effluent and commercialized [37].

In summary, microorganisms utilize various metabolic pathways for substrate degradation and simultaneous biohydrogen production, including the cellulolytic, butyric, and acetic pathways. The cellulolytic pathway, for instance, involves the degradation of lignocellulosic biomass by microorganisms, converting polysaccharides like cellulose into simple sugars such as glucose. These sugars are then fermented to produce H₂, organic acids (mainly acetic acid and butyric acid), and CO₂. While this method allows for the use of a wide variety of waste materials as substrates, such as abundant and inexpensive lignocellulosic residues, it is important to note that cellulose hydrolysis is a slow process and often requires pretreatment to be efficient due to the presence of lignin, presenting a significant challenge.

The butyric pathway is characterized by the conversion of simple sugars like glucose into 2 moles of H₂, 1 mole of butyric acid, 2 moles of CO₂, and 2 protons (H⁺). The main limitation of this pathway is the accumulation of butyric acid in the cultivation medium, which can lead to a decrease in pH and inhibit the metabolism of hydrogen-producing microorganisms.

The acetic pathway is carried out by a wide variety of microorganisms, such as Clostridium aceticum, and is characterized by the conversion of simple sugars like glucose into 4 moles of H₂, 2 moles of acetate, 2 moles of CO₂, and 2 protons (H⁺). Its main limitation is the accumulation of acetic acid in the cultivation medium, which can affect the metabolism of hydrogen-producing microorganisms.

Although theoretically producing up to 4 moles of H₂ per mole of glucose, acetic fermentation may be less efficient due to CO₂ production and the need to maintain redox balance. Maintaining the redox balance is challenging because acetate formation consumes part of the electrons released during glucose oxidation. The butyric pathway involves the formation of butyric acid and H₂, with less CO₂ production compared to the acetic pathway. The redox balance is more favorable for H₂ production as fewer electrons are used for CO₂ formation and more are directed toward H₂ production.

3. Parameters Affecting Dark Fermentation

3.1. Inoculum and Pretreatments

A diversity of strict anaerobic or facultative microorganisms can conduct dark fermentation, converting organic substrates into energy and biohydrogen as co-product. Among the genera of strict anaerobic bioH₂-producing microorganisms, one can mention microorganisms from the Clostridiaceae Family (Clostridium) [45], as well as facultative anaerobes from the Enterobacteriaceae Family (Escherichia [46]), Enterobacter, Klebsiella [47], Citrobacter [48]), and others.

BioH₂-producing microorganisms can be obtained from different sources such as animal manure [49,50,51,52], anaerobic reactor sludge [53,54,55], organic waste [56], soil [57], and ship wastewater. These biodegradable organic sources are naturally rich in microorganisms capable of performing anaerobic fermentation to produce bioH₂. Dark fermentation systems can be inoculated with a microbial consortium or pure culture [50,58].

The production of bioH₂ using pure bacterial cultures on laboratory-scale facilitates manipulating microbial metabolism through changes in growth conditions and consequent minimization of the production of unwanted byproducts, which can lead to higher yields and process repeatability [27]. Studies with pure strains are essential for understanding metabolic pathways and observing the effects of genetic manipulations. However, this production process is more expensive and needs further investigation for industrial scale-up due to substrate sterilization since pure strains are more sensitive to contamination [14,24,31].

Using mixed cultures is preferable because microorganisms have greater tolerance to metabolic stress and fluctuations in cultivation conditions. Additionally, due to the division of labor, there is more efficient substrate utilization, leading to higher productivity, the possibility of using a wide variety of substrates, and oxygen consumption [18,24]. However, inocula obtained from various locations may also contain H₂-consuming microorganisms such as methanogens, requiring the use of pretreatment to suppress these microorganisms’ activity [13,36].

The inoculum pretreatment can be based on different strategies like Clostridium‘s ability to form spores under adverse conditions, such as high temperatures or extreme pH, unlike methanogens, which are eliminated [27]. However, bioH₂-producing microorganisms that cannot form spores, such as those from the genera Enterobacter and Citrobacter, can also be suppressed or eliminated [14].

A variety of physical and chemical methods are described in the literature for inoculum pretreatment, such as thermal shock, aeration, alternating cycles of light heat-shock and microaeration, microwave irradiation, gamma rays, ultraviolet rays, infrared radiation, ultrasonication, freezing and thawing, or through chemical methods, such as application of acids, bases, or growth inhibitors of bioH₂-consuming microorganisms, such as 2-bromoethanesulfonic acid (BESA), iodopropane, chloroform, higher fatty acids like linoleic acid, or acetylene.

Table 1. Different substrates, inoculums, and physicochemical parameters used in dark fermentation.

Substrate	Inoculum	Inoculum Pretreatment	Predominant Microorganisms	pH	Temperature	bioH2	Reference
Corn stalk	Cow Manure	Microwave for 1.5 min	Clostridium sartagoforme	6.47	35 °C	87.2 mL bioH2/g of corn stalk	[52]
Rice straw	Sludge	Thermal, 95–100 °C for 1 h	Mixed culture	5.5	37 °C	0.77 L bioH2/L culture medium	[59]
Sugarcane bagasse	Anaerobic bioreactor sludge	-	Clostridium bifermentans (62.69% relative abundance), Bacillus coagulans (31.67%) and Enterobacter aerogenes (2.72%)	7.2	37 °C	23.10 mmoL bioH2/L culture medium	[53]
Corn stalk	Cattle manure	Microwave (no description of conditions)	Clostridium butyricum	Without pH adjustment	36° C	92.9 mL bioH2/g corn stalk	[50]
Brewery wastewater and cheese whey	Anaerobic reactor sludge	Thermal, 100 °C for 40 min	Bacillus spp. (25%), Firmicutes Clostridia (20%), Firmicutes bacilli (8%), (<5%) Lactococcus lactis, (<5%) Alcaligenes spp. and (<5%) Paracoccus solventevorans	5.5	35 °C	6.22 mmol bioH2/g DQO	[60]
Glycerol and wastewater from cassava processing	Anaerobic reactor sludge	Thermal, 100 °C for 30 min	Brevundimonas and Bacillus	-	38.5 °C	0.86 L bioH2/L culture medium	[55]
Corn steep liquor and cassava processing water	Vinasse effluent	Thermal, 95 ± 2 °C for 15 min	Porphyromonadaceae 16%, Clostridiaceae 31%, Ruminococcaceae 0.85%, Enterococcaceae 51%, others 1.5%	6	37 °C	107 mL bioH2/g DQO removed	[49,61]
Corn steep liquor and cassava processing water	Chicken manure	Thermal, 95 ± 2 °C for 15 min	Porphyromonadaceae 75%, Clostridiaceae 15%, Ruminococcaceae 6%, Enterococcaceae 3%, others 1%	6	37 °C	83.1 mL bioH2/g DQO removed	[49,61]
Rice mill wastewater	Rice mill wastewater	Thermal, 100 °C for 15 min	Bacillus thuringiensis	5.5	37 °C	1.63 ± 0.14 mol bioH2/mol glucose	[56]
Dairy processing wastewater	Anaerobic reactor sludge	Thermal, 90 °C for 30 min	Mixed culture	5.5	55 °C	254 mL of cumulative bioH2	[54]
Palm oil mill effluent	Anaerobic reactor sludge	Thermal, 85 °C for 60 min	Clostridia, Bacilli, Bacteroidia, Thermoanaerobacteria and Gammaproteobacteria	5.5	60 °C	2.25 mol of bioH2/mol of total soluble carbohydrates	[62]
Food waste	Sludge from a hydrogen-producing reactor	Centrifugation at 5000 rpm for 5 min, freezing for two months, and thermal pretreatment at 90 °C for 30 min	Clostridium, Romboutsia, Sporolactobacillus, Streptococcus, Terrisporobacter and others in smaller fractions	8.2	37 °C	1.12 ± 0.02 mol bioH2/mol glucose	[63]
Food waste	Anaerobic reactor sludge	Alkaline, pH 10 using 5 M NaOH	Clostridium, Paraclostridium, Streptococcus, Lactococcus, Enterococcus and Prevotella	7.5	35 °C	157.25 ± 7.62 mL of bioH2 g/VS	[64]
Food waste	Strain bank	-	Clostridium beijerinckii	5.5	40 °C	128 mL bioH2/g DQO removed	[65]
Food waste	Microorganisms present in the substrate	-	-	5.5	37 °C	118 mL bioH2/g VS	[66]
Algal biomass (Scenedesmus obliquus)	Anaerobic reactor sludge	-	Clostridium butyricum	-	37 °C	116.3 mL bioH2/g VS	[67]
Algal biomass (Chlorella vulgaris)	Anaerobic reactor sludge	Thermal, 90 °C for 60 min	Mixed culture	5.5	35 °C	190.9 mL bioH2/g VS	[68]
Algal biomass (Dunaliella primolecta)	-	-	Thermococcus eurythermalis	-	85 °C	192.35 mL bioH2/g VS	[69]
Algal biomass (Dunaliella tertiolecta)	-	-	Thermococcus eurythermalis	-	85 °C	183.02 mL bioH2/g VS	[69]
Algal biomass (Scenedesmus obliquus)	Strain bank	-	Clostridium butyricum	-	37 °C	113.1 mL bioH2/g VS	[70]
Algal biomass (Scenedesmus obliquus)	Strain bank	-	Enterobacter aerogenes	-	30 °C	57.6 mL bioH2/g VS	[70]
Cattle manure and cheese whey	Digestate	Thermal, 105 °C for 1.5 h	Mixed culture	6–7	35 °C	0.33 L bioH2/L culture medium	[71]
Cattle manure	Cattle manure	Infrared radiation for 2 h	Mixed culture	5.0	36 °C	31.5 mL bioH2/g VS	[51]
Cattle manure	Anaerobic reactor sludge	Acid, pH 2.0 using 6 M HCL	Mixed culture	-	-	44.59 mL bioH2/g VS	[72]
Vinasse and Nejayote	Digestate from mesophilic anaerobic digester treating food waste	Light heat-shock, (30 to 60 °C) for 20–30 min followed by micro aeration	Acetobacter orientalis, 42.94%	5.5	35 °C	115 NmL H2/g VS	[73]

presents various inoculum pretreatments designed to maximize biohydrogen production. BESA and acetylene can inhibit methanogenesis by disrupting the biosynthesis of the cofactor methanofuran, which is essential for the reduction of CO₂ to methane [18]. Chemical inhibitors can increase the efficiency of bioH₂ production; however, issues such as high costs can complicate their use on an industrial scale.

Luo [64] tested the effect of various types of pretreatments on an inoculum from anaerobic reactor sludge for bioH₂ production from food waste. The pretreatments were aeration, acid, base, thermal, thermal + CO₂, free nitrous acid, bromoethanesulfonate, and electric shock. The highest bioH₂ yield was obtained from the alkali-pretreated inoculum (157.25 ± 7.62 mL of bioH₂/g VS). On the other hand, Chen [72] studied the effect of acid, base, and thermal pretreatments on anaerobic reactor sludge for bioH₂ production from rice straw and pig manure, obtaining a maximum cumulative production of 44.59 mL of bioH₂/g VS from the acid-pretreated inoculum. Meanwhile, Elbeshbishy [66] studied the effect of four individual pretreatments (ultrasonication, heat, acid, and base) and three combined pretreatments (ultrasonication with heat, ultrasonication with acid, and ultrasonication with base) for bioH₂ production from food waste. The combined pretreatment of ultrasonication and acid resulted in the highest bioH₂ production of approximately 118 mL/g initial volatile solids, while the control experiment without pretreatment led to a production of 42 mL of bioH₂/g VS.

Although pretreatments result in different effects on the inocula, as observed in many studies listed in Table 1, what can be observed is that different treatments favor the predominance of spore-forming bioH₂-producing microorganisms. In the work of Luo [64], all experiments with inoculum pretreated through physical and chemical methods showed significant enrichment (63–90%) of Firmicutes Phylum microorganisms compared to untreated raw inoculum (29%). Firmicutes are mainly Gram-positive bacteria that include bioH₂-producing microorganisms from the genera Clostridium, Paraclostridium, Streptococcus, and others.

However, despite some methods being more used than others, such as thermal pretreatment (Table 1), there is no superior method over another, as their effects differ based on the inoculum source [13]. Additionally, several parameters must be considered in choosing the pretreatment method, such as operational costs, complexity, time required, scalability, and the formation and disposal of waste.

3.2. Substrates Used in Dark Fermentation

In principle, any organic substrate rich in carbohydrates, fats, and proteins could potentially be considered a viable substrate for bioH₂ production [27]. However, rich-lipid substrates can inhibit dark fermentation, mainly due to the high partial pressures of hydrogen that occur in the process. Soluble and easily accessible sugars are described by Guo [74] to represent the main fraction of biomass that can be converted into bioH₂, as bioH₂-producing microorganisms in dark fermentation show a preference for carbohydrates such as monosaccharides (glucose) and disaccharides (lactose or sucrose) due to their rapid and easy biodegradability [32,71].

Thus, many studies present the use of these pure sugars as model substrates for bioH₂ production [75]. However, these carbohydrates have extremely high prices, making the process unfeasible on an industrial scale [32]. For it to be economically sustainable, dark fermentation must be carried out using abundant, easily accessible, low-cost, and highly biodegradable organic substrates [76].

Widely available residues, such as those from agro-industry, food processing, and urban solid waste like food scraps, are rich in nutrients, and their use in bioH₂ production presents an alternative for energy treatment and recovery [54,77,78].

As observed in Table 1, a wide range of residues has been used as substrates for dark fermentation and can be grouped into: lignocellulosic biomass [50,52,53,59], wastewater from food and beverage processing [49,54,55,56,62], food waste [63,64,65,66], algae biomass [67,68,70,72] and animal husbandry waste [51,71,72]. The most frequently explored types of biomass/waste used as raw materials for bioH₂ production are described below.

3.2.1. Lignocellulosic Biomass

Lignocellulosic biomass is recognized as a promising substrate for dark fermentation due to its wide availability and the presence of sugars in its composition [25,79]. Its polymeric structure is predominantly composed of cellulose (35–55%), hemicellulose (25–40%), and lignin (15–25%) in different proportions, depending on the culture. Cellulose is composed of glucose units and can be broken down by cellulolytic enzymes during dark fermentation. It is an important source of fermentable sugars for bioH₂-producing microorganisms., As previously described simple sugars like glucose are preferred substrates for bioH₂-producing microorganisms. Hemicelluloses is a more complex polysaccharide matrix composed of different sugars such as includes pentoses (xylose, rhamnose, and arabinose), hexoses (glucose, mannose, and galactose), and uronic acids (4-O-methylglucuronic acid, D-glucuronic acid, and D-galacturonic acid) [52,80]. Its degradation during dark fermentation requires the action of various hemicellulolytic enzymes, which vary according to the specific composition of the hemicellulose present in the biomass [52,80]. However, the presence of lignin is a limiting factor for its use due to its difficult biodegradability [76]. Lignin provides rigidity and strength to the plant cell wall structure, making it difficult for hydrolytic enzymes to access the cellulose and hemicellulose molecules encapsulated or protected by its matrix. Thus, pretreatment steps of the biomass are necessary to achieve its hydrolysis and availability of fermentable sugars [20,71].

The pretreatments include physical, chemical, physicochemical, or biological methods. Among the physical methods, milling, microwaves, ultrasound, pyrolysis, and others can be mentioned. Chemical pretreatment is performed using alkalis, acids, ozonolysis [81], organosolvents, and oxidizing agents. Physicochemical pretreatments include techniques such as steam explosion, ammonia fiber expansion (AFEX), supercritical fluids, wet oxidation, hydrothermal pretreatment, among others. Biological pretreatments use fungi, bacteria, or their enzymes [82,83,84].

Although there are several pretreatment options, there is no universal approach due to variations in the composition of the materials. Therefore, it is crucial to adapt pretreatment processes for each type of substrate [40].

Rabelo [53] used hydrothermally pretreated sugarcane bagasse in an autoclave at 121 °C for 15 min to produce bioH₂ using a consortium consisting mainly of Clostridium bifermentans (62.69%), Bacillus coagulans (31.67%), and Enterobacter aerogenes (2.72%). Under optimized conditions of 7.0 g of bagasse/L, pH 7, and 37 °C, a maximum production of 23.10 mmol of bioH₂/L of substrate was achieved.

Additionally, the use of combined pretreatment methods can gather the advantages of different approaches, improving the efficiency of the process [82]. Chemical pretreatment methods, for example, can promote the opening of the cellulose and hemicellulose structure, increasing their susceptibility to subsequent enzymatic digestion [40].

To this end, Sen [59] optimized the pretreatment of rice straw through the combination of acidic and enzymatic hydrolysis. Under the best pretreatment conditions of 100 g/L of rice straw, particle size of 0.15 mm, hydrolysis time of 20 min, and 0.8–1.0 M, approximately 38.6 g/L of total reducing sugar was obtained. Using a microbial consortium, the maximum production of 0.77 L bioH₂/L of culture medium containing 20 g/L of rice straw hydrolysate at pH 5.5 and 37 °C was achieved.

To overcome these difficulties, many studies have focused on the direct use of shredded biomass by enzyme-producing microorganisms that hydrolyze lignocellulosic biomass [52].

Song [50] isolated a strain identified as Clostridium butyricum capable of utilizing lignocellulosic biomass without prior pretreatment. The microorganism was obtained from an inoculum derived from thermally pretreated bovine manure and acclimated through successive cycles in a culture medium containing corn stover as the sole carbon source. Under optimized conditions of 20 g/L of raw corn stover without pH adjustment, a total of 92.9 mL of bioH₂/g of corn stover was obtained.

Similarly, Zhang [52] pretreated an inoculum derived from bovine manure with microwaves and acclimated it in a culture medium containing microcrystalline cellulose as the sole carbon source. A highly bioH₂-producing isolate was identified as Clostridium sartagoforme. Under optimal conditions of 15 g/L substrate concentration, 0.15 M phosphate buffer, 6 g/L urea, and an initial pH of 6.47 at 35 °C, 87.2 mL of bioH₂/g of raw corn straw was obtained after 64 h of fermentation.

Concerning the impact of different proportions of cellulose, hemicellulose, and lignin on the final yield of fermentation, it can be noted that lignocellulosic substrates with a higher cellulose content generally results in a greater availability of fermentable sugars after hydrolysis, potentially increasing fermentation yield. However, higher hemicellulose content, composed of different sugars like xylose, arabinose, and mannose, can be more complex to degrade. Depending on the microorganisms’ ability to ferment these complex sugars, the fermentation yield may vary, with some microorganisms being more efficient in utilizing hemicellulose than others. Biomass with a high proportion of lignin generally results in lower fermentation yields unless effective pretreatment methods are employed to break down the lignocellulosic structure.

Depending on the pretreatments applied to lignocellulosic biomass, like chemical pretreatments using acid or alkali solutions, fermentation inhibitors, such as furfural and 5-hydroxymethylfurfural are formed from the decomposition of sugars and carbohydrates [85]. Furthermore, pretreatments can represent high capital costs, high energy demands, equipment corrosion issues, and additional costs for treating resulting effluents [40,86].

3.2.2. Industrial Processing Residues

Industrial wastewater, such as that from food and beverage processing, is rich in organic matter and can be a potential pollutant if discharged into the environment without proper treatment [87]. For this reason, industrial wastewater has been frequently used as a substrate in bioH₂ production, avoiding the negative effects of improper disposal while simultaneously recovering energy.

Among the industrial wastewater sources explored for bioH₂ production are brewery effluents [60], dairy processing [54], soft drink processing [88] cassava processing [55], palm oil mill effluent [62], sugarcane molasses [89], corn maceration liquor [49], and others.

Ramu [56] used glucose-rich rice mill wastewater for bioH₂ production employing Bacillus thuringiensis as the inoculum. In an optimized medium consisting of 50% rice mill wastewater, pH 5.5, temperature of 37 °C, and agitation at 120 rpm for 72 h, a yield of 1.63 ± 0.14 (mol H₂/mol glucose) and a COD removal of 94.31% were achieved.

Bouchareb [54] employed ultrasonic, thermal, alkaline, acid, and enzymatic pretreatments on dairy industry wastewaters to assess their effects on bioH₂ production. Using thermally pretreated anaerobic sludge at 90 °C for 30 min, pH 5.5, and temperature of 55 °C, the best cumulative production of 254 mL of bioH₂ was achieved through lactase-treated residue.

Arisht [62] studied the effect of various acids and bases in pretreating palm oil mill effluent. It was found that phosphoric acid H₃PO₄ at a concentration of 2.5% resulted in the best pretreatment. Subsequently, pretreated POME was used for bioH₂ production using thermally pretreated anaerobic reactor sludge, under conditions of 60 °C temperature, pH 5.5, 6 h HRT, resulting in a yield of 2.25 mol bioH₂/mol total soluble carbohydrates. The bacterial community responsible for bioH₂ production was genetically analyzed and identified as belonging to the classes Clostridia, Bacilli, Bacteroidia, Thermoanaerobacteria, and Gammaproteobacteria.

Despite being widely available and inexpensive, industrial processing wastewaters often lack nitrogen, phosphate, micronutrients like metallic ions, and even carbon sources essential for biomass and bioH₂ production [13]. To avoid supplementing the culture medium with expensive nutrients like yeast extract, peptone, and pure sugars, the nutritional content of the medium can be enhanced by combining two or more types of residues [90].

Cruz-Lopez [60] conducted co-digestion of brewery wastewaters and cheese whey. The highest percentage of bioH₂ in biogas corresponded to an average yield of 6.22 mmol bioH₂/g COD−1 during reactor operation with a hydraulic retention time (HRT) of 9 h, constant pH of 5.5 ± 0.5, temperature of 35 °C, and a carbon–nitrogen ratio (C/N) of 30.

Martinez-Burgos [49] optimized bioH₂ production with consortia (Vir and Gal) obtained from their previous work [61] through co-digestion of cassava processing wastewater and corn maceration liquor. For the Vir consortium, the ideal cultivation conditions were: initial pH of 6.37 °C, 14% inoculum, 6.5% corn maceration liquor, and 20 s purge time, reaching a maximum yield of 107 mL bioH₂/g COD removed. For the Gal consortium, the ideal cultivation conditions were: initial pH of 6.36 °C, 13% inoculum, 6.5% corn maceration liquor, and 20 s purge time, resulting in a production of 83.1 mL bioH₂/g COD removed. COD removal was approximately 50% and 60% for Vir and Gal, respectively.

Meier [55] conducted co-digestion of glycerol and cassava processing wastewater using anaerobic reactor sludge thermally pretreated (100 °C for 30 min), achieving a maximum yield of 0.86 L bioH₂/L of culture medium.

3.2.3. Food Waste

In 2022, approximately 1.05 billion tons of food were wasted worldwide [91]. These surpluses are usually sent to landfills, where they contribute to the emissions of 8–10% of greenhouse gases, such as methane (CH₄) and carbon dioxide (CO₂), as well as the release of ammonia (NH₃) and leachate [92,93]. Food wastes have a variable composition, being rich in carbohydrates, fats, and proteins [94,95], making them suitable as a substrate in dark fermentation processes. This contributes to energy recovery and mitigates the problems associated with their disposal in landfills [96,97]. To achieve environmental benefits from the production of bioH₂, the process using food waste must not result in new negative ecological impacts, such as soil contamination or the production of harmful effluents. The bioH₂ production per unit of substrate and time must be maximized, and it must also be economically viable, including costs associated with collection, transportation, pretreatment, and processing of food waste.

Jung [63] achieved a yield of 1.12 ± 0.02 mol of bioH₂/mol of glucose using food waste composed of crushed and pretreated rice, potato, onion, orange, pork, cabbage, and radish with H₂SO₄ and an inoculum predominantly composed of Clostridium.

Luo [64] reported a yield of 157.25 ± 7.62 mL of bioH₂/g of volatile solids (VS) using food waste composed of cabbage, rice, and pork, with an inoculum from the sludge of an alkali-treated anaerobic reactor.

Kim [65] investigated the effect of pH, temperature, and high concentrations of organic acids on bioH₂ production using Clostridium beijerincki as an inoculum and food waste collected from a cafeteria as a substrate. The waste was mixed with water at a 1:1 (v/v) ratio and crushed. The experimental results showed that acetic acid (<5000 mg/L) or butyric acid (<3000 mg/L) concentrations significantly inhibit both cell growth and bioH₂ production. Under ideal conditions of 40 °C temperature and controlled pH of 5.5, approximately 128 mL of bioH₂/g COD were produced.

Elbeshbishy [66] studied the effect of various pretreatments on food waste for bioH₂ production. Four individual pretreatments (sonication, heat, acid, and base) and three combined pretreatments (sonication with heat, sonication with acid, and sonication with base) were conducted. After pretreatments, fermentations were carried out at pH 5.5, incubated at 37 °C and 180 rpm, without the addition of inoculum. The combined pretreatment of sonication and acid resulted in the highest increase in soluble carbohydrates of approximately 31% and consequently the highest bioH₂ production, approximately 118 mL bioH₂/g initial VS, while the control experiment without pretreatment led to a production of 42 mL bioH₂/g initial VS.

The large-scale use of food waste as a substrate for dark fermentation poses several practical implications and significant logistical challenges. In terms of collection, the main issue lies in the need for robust infrastructure for efficient separation and transport of food waste from generation points to processing facilities. This often involves coordinating with various waste sources such as supermarkets, restaurants, and households to ensure a continuous and adequate supply. Pretreatment of food waste is crucial to maximize dark fermentation efficiency. The heterogeneity of waste requires effective pretreatment methods to increase the availability of fermentable substrates like cellulose and hemicellulose and reduce the presence of unwanted contaminants that may inhibit the fermentation process. Integration with existing treatment facilities is another critical challenge. Solid waste or liquid effluent treatment units must be adapted to efficiently receive, and process food waste intended for dark fermentation. This may involve investments in infrastructure for biogas treatment, solid–liquid separation, and odor control. Additionally, there are economic implications to consider, such as the cost of implementing and operating these adapted infrastructures, as well as the economic viability of large-scale bioH₂ production compared to other renewable energy sources. Therefore, while food waste holds significant potential as a substrate for dark fermentation, its large-scale utilization requires an integrated approach that considers both technical and logistical aspects, aiming to maximize the energy and environmental efficiency of the process.

3.2.4. Algal Biomass

Microalgae biomass can be used as a substrate for bioH₂ production since they have faster growth rates compared to higher plants, require no arable land or freshwater, avoid the use of fertilizers and pesticides, and have enhanced biodegradability due to the absence of hemicellulose and lignin [67,70]. Moreover, high-value compounds such as pigments, amino acids, or proteins can be extracted from algal biomass before its use in fermentation, thus improving the economic viability of third-generation biofuel production [98,99].

For instance, Ortigueira [67] used biomass from the algae Scenedesmus obliquus as a substrate for bioH₂ production by Clostridium butyricum. The biomass was cultivated in an open pond under nitrogen restriction conditions to promote carbohydrate accumulation. Based on dry weight, the biomass consisted of approximately 307 g/kg of total sugars. The intact cell membranes of microalgae can hinder bioH₂ production; hence, physical milling of the biomass was performed to form a fine powder (<0.5 mm), followed by autoclaving before its addition to the fermentation medium. In BM1 medium containing 50 g/L of algal biomass at 37 °C, a yield of 116.3 mL bioH₂/g of algal biomass was obtained (2.74 mol bioH₂/mol glucose).

As an alternative to biomass milling, Stanislaus [68] performed thermal pretreatment of Chlorella vulgaris biomass under optimized conditions at 100 °C for 60 min. The maximum yield of 190.9 mL bioH₂/g VS was achieved at pH 5.5, temperature 35 °C with constant agitation, and a substrate–inoculum ratio of 8.

Chen [69] used biomass from the species Dunaliella primolecta and Dunaliella tertiolecta with extracted lipids for bioH₂ production via dark fermentation by the microorganism Thermococcus eurythermalis. Since Dunaliella microalgae lack a cell wall, their use as a substrate for dark fermentation would incur lower pretreatment costs. The fermentations were carried out at 85 °C for 18 h. Under optimal conditions of biomass concentration in the culture medium of 2.5 g/L and gas–liquid ratio of 2:1, the yields in hydrogen production from Dunaliella primolecta biomass were 192.35 mL/g VS and from D. tertiolecta 183.02 mL/g VS without sterilization of the culture medium. The yields were only slightly lower than with sterilization, indicating that the high yield was hardly affected by autoclaving, likely due to the high temperature maintained during fermentation.

Batista [70] evaluated bioH₂ production by dark fermentation using Scenedesmus obliquus biomass with two pure cultures: Enterobacter aerogenes and Clostridium butyricum, comparing the use of dry and wet biomass. An ideal biomass concentration of 2.5 g/L was found for maximum bioH₂ production by E. aerogenes, achieving a yield of 56.5 mL bioH₂/g or 57.6 mL/g VS for dry and wet biomass, respectively. For fermentations conducted by C. butyricum, the optimal biomass concentration in the culture medium was 50 g/L, showing significant differences between dry and wet biomass utilization, leading to yields of 113.1 mL/g VS and 80.4 mL/g VS, respectively.

3.2.5. Manure from Animal Farming

Livestock waste includes solid animal manure residues, forage residues (which typically contain a lignocellulosic fraction), and wastewater comprising urine and feces [13]. The large quantities of animal waste currently generated by the livestock sector, as well as the leaching of nutrients and organic matter into the natural environment, pose a constant pollution risk if not managed correctly [100]. Current livestock waste management practices include application to agricultural fields as well as stabilization or biological treatment, such as composting and anaerobic digestion [13].

Hangri [71] employed response surface methodology to optimize the combined pretreatment of H₂O₂ and sonication in bovine manure for subsequent bioH₂ production. The maximum release of carbohydrates (190.4%) was achieved when the H₂O₂ concentration was 0.06 (g/gTS) and the specific ultrasonic energy input was 1419.36 (J/gTS). Subsequently, the pretreated residue was used in bioH₂ production assays using digestate from a cow manure treatment plant. Co-digestion experiments of bovine manure with cheese whey (20% v/v) were also conducted to compare the results. BioH₂ production with untreated and pretreated bovine manure was 22.75 mL/L and 51.25 mL/L, respectively. Adding cheese whey to bovine manure as a co-substrate during dark fermentation improved process performance, reaching a final value of 0.33 L bioH₂/L of culture medium.

Xing [51] applied acid, base, and infrared radiation pretreatments to bovine manure to assess their effects on bioH₂ production, with a solid loading of 10% (w/w) and bovine manure pretreated with infrared radiation for 2 h as the inoculum source. The acid pretreatment was the most efficient, resulting in a yield of 31.5 mL bioH₂/g VS under optimal conditions of substrate concentration of 70 g volatile solids/L, initial pH 7.0, and temperature of 36 °C. The yield without pretreatment was 13.3 ± 0.3 mL bioH₂/g VS.

Due to the high nitrogen content of animal manure, it can be used as a co-digestion substrate to supplement nitrogen from other agricultural residues while maintaining an appropriate carbon–nitrogen ratio for bioH₂ production [13,76].

Chen [72] performed co-fermentation of rice straw and acid-, base-, and heat-pretreated swine manure. The maximum cumulative bioH₂ production of 44.59 mL bioH₂/g volatile solids was obtained from acid-pretreated inoculum under fermentation conditions of pH 7.0 ± 0.1, temperature of 55 ± 1 °C, rice straw–bovine manure ratio of 5:1 (based on TS), and 120 rpm.

3.3. Temperature

The ideal temperature within the reaction system influences the growth rate of microorganisms and the expression and activity of enzymatic systems associated with bioH₂ production processes [40]. Microorganisms can be classified as mesophiles (25–40 °C), thermophiles (40–65 °C), extreme thermophiles (65–80 °C), or even hyperthermophiles (>80 °C) [27,69,101,102]. Several studies have reported good bioH₂ production rates in the mesophilic range (30–40 °C), which is the ideal temperature range for the growth of bioH₂ producers such as many species of the genus Clostridium [50,52]. Easily degradable substrates are more efficiently converted at moderate temperatures than under other temperature conditions [13,40].

Theoretically, bioH₂ production at higher temperatures (equal to or above 60 °C) conducted by thermophilic and hyperthermophilic microorganisms has a more thermodynamically favorable reaction and reduces the activation energy of enzymes, allowing for higher bioH₂ production compared to mesophiles [37,39,69].

The benefits of using higher temperatures include improved substrate solubilization, enhancing yields from complex residues such as lignocellulosic biomass and food waste [103], lower contamination rates, decreased solubility of bioH₂ in the fermentative broth, thus reducing inhibition of bioH₂ production. Additionally, it enables the use of hot industrial effluents directly in fermentative processes [24,103,104].

However, high temperatures can reduce microbial diversity, leading to incomplete substrate degradation. Moreover, the high energy input required to maintain the bioreactor at high temperatures reduces the economic viability of the process [104,105].

Karaday [106] studied the effect of temperature on a mixed culture and found that modifying it led to a change in microbial structure. At temperatures of 37–45 °C, the microbial community was dominated by Clostridium through bioH₂ production via the butyrate pathway. Changing the bioreactor temperature to 60–65 °C resulted in predominance of the thermophilic genus Thermoanaerobacterium, leading to ethanol formation as a fermentation byproduct. Therefore, it can be concluded that besides affecting the thermodynamics of bioH₂ production, temperature also influences changes in the microbial community using mixed cultures, resulting in alterations in the biochemical pathway for bioH₂ production and consequently leading to changes in resulting byproducts [22].

3.4. pH

As observed in Table 1, the pH in bioH₂ production varies depending on the type of substrate and microorganisms used, ranging from 5.5 to 7.5. The pH of the fermentative medium plays an important role in the growth and activity of bioH₂-producing microorganisms as it can cause changes in the cell membrane charge, impacting nutrient absorption, and consequently, the activity of hydrogenase enzymes responsible for catalyzing bioH₂ production [20,24]. The pH also affects the metabolic pathway of bioH₂ production and consequently, the production of byproducts such as organic acids. Fang [107] demonstrated that in bioH₂ production by a mixed culture, the production of byproducts such as ethanol, propionic acid, and lactic acid was favored at higher pH levels, while at lower pH levels, the production of acetic and butyric acids was favored. This may indicate the influence of pH on the microbial community structure, resulting in the prevalence of different microorganisms at different pH levels and consequently leading to the formation of different byproducts.

These organic acids accumulate and lead to acidification of the culture medium and consequent inhibition of the fermentative process if the pH is not controlled [31]. This underscores the crucial importance of pH control and its maintenance at a constant level for bioH₂ production. The kinetic parameters of hydrogen generation by dark fermentation have been described mainly through models such as the modified Gompertz model. The model has been validated using pH curves and the hydrogen measured experimentally [108]. Studies, mainly conducted using batch processes, do not control pH throughout the process but rather focus on the impact of initial pH. Therefore, a distinction should be made between initial pH and fermentation process pH [40]. However, the excessive demand for buffers, acids, or bases to maintain pH can reduce the economy and sustainability of the process, as well as increase the salt concentration of dark fermentation effluents. One sustainable solution could be to explore substrates with higher pH or alkalinity to balance the system [13].

3.5. Hydraulic Retention Time (HRT)

Production of bioH₂ at pilot and industrial scales requires semi-continuous and continuous processes, with the hydraulic retention time (HRT) being a measure of the average time that the substrate remains within the fermentation system and having a significant impact on bioH₂ production [25]. The ideal HRT value depends on the type of substrate used and its biodegradability, needing to be long enough to allow for the solubilization of complex organic matter [25].

Due to differences in the proliferation time of bioH₂-producing microorganisms and consumers, such as methanogens, HRT can be used as a strategy to restrict CH₄ formation within the reactor [104]. The main bioH₂-producing bacteria, such as Clostridium, produce bioH₂ at relatively short HRTs (4–12 h) [109], and it is not recommended to prolong the time to avoid a metabolic shift from acidogenesis to methanogenesis [110].

However, besides being highly variable depending on the substrate, HRT is closely related to other factors such as pH, organic loading rate, and can often vary even for the same substrate [25,110]. For this reason, HRT should be optimized specifically for each bioH₂ production process.

3.6. Partial bioH₂ Pressure

The accumulation of hydrogen in the reactor over its production leads to an increase in its partial pressure causing process inhibition, as discussed in Section 2. To avoid inhibition of bioH₂ production due to pressure, some strategies can be applied, such as agitation of the culture medium, spraying with nitrogen gas (N₂), and carbon dioxide (CO₂) [38,40]. However, despite spraying being a method that improves yield obtained, there is product dilution and additional costs for gas recovery. Thus, a simple method to reduce the reactor’s partial pressure is the continuous removal of the gas phase [13].

3.7. Organic Loading Rate (OLR)

The organic loading rate (OLR) refers to the amount of organic material added to the bioH₂ production system per unit volume over a specific period [111]. It is known that higher substrate concentrations could increase bioH₂ production efficiency, but inhibitions occur in product formation when the organic substrate load exceeds a threshold level [112]. This happens because an excessive amount of substrate increases osmotic pressure, thus inhibiting the growth of bioH₂-producing bacteria. Additionally, excess substrate alters fermentation pathways to produce alcohol and/or organic acids, limiting bioH₂ yields [90].

Studies have demonstrated the effect of OLR on bioH₂ production from various substrates, which varies significantly, from very low rates like 20 g of COD/L/day [113], to high rates like approximately 100 g of COD/L/day [114]. The optimal OLR depends not only on the nature of the substrate but also on pH, temperature, and reactor type [111,115]. The ideal OLR should be determined for each specific raw material [116].

The Organic Loading Rate (OLR) affects fermentation stability and efficiency in different bioreactors. In continuous tank reactors (CSTRs), high OLRs can lead to system overload and inhibit microbial activity. Membrane bioreactors (MBRs) are affected by OLR in terms of biomass formation rate and filtration efficiency. High OLRs can cause rapid biomass growth and membrane fouling. Packed bed reactors (PBRs) are impacted by high OLRs, leading to biomass buildup and reduced process efficiency. Anaerobic fluidized bed reactors (AFBRs) may experience increased biogas production with high OLRs but also risk biomass washout. The upflow anaerobic sludge reactors (UASBRs) can be destabilized with high OLRs, leading to biomass loss and channel formation. Balancing OLR is crucial for reactor stability and efficient organic matter removal.

3.8. Bioreactors Used for bioH₂ Production

BioH₂ can be obtained through batch, semi-continuous, or continuous processes [117]. Batch operations are carried out in a closed system where the substrate and inoculum are added at once, and the product is collected only at the end of fermentation. Batch reactors are easier to operate and are typically used in laboratory-scale studies to assess the bioH₂ production potential of organic substrates and screen for optimal conditions for dark fermentation [118].

Semi-continuous processes are fed continuously, and the product is collected at regular intervals. Compared to batch processes, semi-continuous processes could moderately increase product yield and cycle time to balance productivity and scale, making them more suitable for medium-scale production [40]. Continuous processes are continuously fed and discharged while maintaining stable reaction conditions, high product yields, and short cycle times, providing uninterrupted gas production [118].

Due to higher yields, industrial-scale bioH₂ production requires the use of continuous bioreactors [32], which can be suspended or immobilized [105].

Suspended bioreactors can be classified into continuously stirred tank reactors (CSTRs) (Figure 3A) and membrane bioreactors (MBRs) (Figure 3B). On the other hand, immobilized reactors are classified as packed bed reactors (PBRs) (Figure 3C), anaerobic fluidized bed reactors (AFBRs) (Figure 3D), and upflow anaerobic sludge blanket reactors (UASBs) (Figure 3E) [24,117].

3.8.1. Continuous Stirred Tank Reactors (CSTRs)

Suspended bioreactors are primarily used for raw materials containing a high substrate content, such as urban waste and food waste. Among suspended bioreactors, the Continuous Stirred Tank Reactor (CSTR) is the most commonly used continuous reactor system for bioH₂ production. It features a simple cylindrical construction equipped with a mechanical agitation system, facilitating easy operation and homogeneous mixing to improve bioH₂ removal efficiency from the reaction mixture. This reduces its partial pressure and increases yields [119].

In a CSTR, the substrate and inoculum are suspended in the bioreactor, experiencing the same hydraulic retention time [32]. Various biological or physical immobilization techniques can be employed to enhance biomass retention and hydrogen production capacity in CSTRs [118].

3.8.2. Membrane Bioreactors (MBRs)

Membrane bioreactors (MBRs) differ from CSTRs in that they are equipped with a membrane system that retains microorganisms in the bioreactor, maintaining their concentration high and constant, thus preventing biomass washout, a primary disadvantage of CSTRs [120]. Based on the membrane arrangement, the system can be categorized into two main types: external loop MBRs, where the membrane is located outside and connected to the reactor via a conduit, and submerged MBRs, where the membrane module is placed directly within the bioreactor [121].

In MBRs, the hydraulic retention time (HRT) is not linked to sludge retention time, facilitating HRT regulation [122]. Additionally, the higher biomass concentration in the bioreactor allows for better utilization of organic substrate, reduced reactor volume, decreased sludge production, and absence of microorganisms in the effluent due to their complete retention by the membrane [123].

The main disadvantages of membrane bioreactors include membrane fouling with organic particles or bio-aggregates, resulting in lower mass transfer compared to other types of bioreactors, which is especially detrimental in wastes with high solid content [122]. Integrating nanomaterials into membranes can enhance their surface properties. Nanoparticles can modify membrane properties and improve durability and efficiency. Furthermore, the development of new polymers that offer better chemical and mechanical stability, increased selectivity, and permeability can result in more durable and efficient membranes, thereby reducing the need for frequent replacement. Additionally, combining different materials into composite layers to leverage their individual benefits can lead to the creation of cost-effective, robust membranes with improved fouling retention and resistance.

Furthermore, membrane bioreactors are still an immature technology that needs improvement in terms of cost and bioH₂ productivity. MBRs require significant energy for operations like aeration, circulation, and, in some cases, thermal treatment and membrane cleaning. With decreasing costs of renewable energy sources such as solar and wind, MBR units can be operated at lower operational costs, thereby reducing the overall cost of bioH₂ production. However, with the decreasing cost of renewable electricity and the development of membrane materials, the application of membrane bioreactors is promising [40,123].

3.8.3. Packed Bed Reactors (PBRs)

In packed bed reactors, wastewater is passed over a column that contains a population of microorganisms attached to biological support materials that retain the microorganisms within the bioreactor, preventing biomass washout [120].

Materials used for biofilm formation should be inert and possess a high specific surface area, a rough texture, and significant porosity. Commonly used materials include glass beads, expanded clay, perlite, activated carbon, ceramics, coconut fiber, synthetic polymers, plastic materials, and straw. These materials support the growth of bioH₂-producing microorganisms, which form biofilms arranged in layers within the bioreactor [117,124].

PBRs are simple to operate, and the high cell concentration results in high volumetric bioH₂ production rates with relatively small reactor volumes [125]. Additionally, they can be operated with a high organic loading rate (OLR) and low HRT [120].

Packed bed reactors have lower turbulence, thus higher mass transfer resistance, resulting in incomplete substrate conversion and pH gradients throughout the reactor [126]. To enhance mixing, a recirculation loop is frequently incorporated into PBR systems. Depending on the flow regime within the reactor, PBRs can be categorized as either upflow or downflow [117].

Since the biomass retention time is independent of the substrate retention time, a potential problem with this type of reactor is the loss of bioH₂ through methane formation due to prolonged biomass retention within the reactor, allowing the establishment of slow-growing methanogenic populations [27].

3.8.4. Anaerobic Fluidized Bed Reactors (AFBRs)

AFBRs combine the characteristics of CSTRs and PBRs, being fed with gas from the bottom of the reactor to generate a fluidized bed [25] that improves mass transfer using microorganisms immobilized on inert particles. However, unlike PBRs where biomass is organized in layers, in AFBRs the granules are fluid within the bioreactor, leading to a high precipitation rate due to their weight, which increases the fluid velocity and consequently the sludge activity [120].

AFBRs have positive characteristics such as the ability to work with higher organic loading rates at low hydraulic retention times without biomass washout [118]. The effectiveness of operating at high organic loads and low hydraulic retention times is enabled by carefully maintaining bed fluidization without biomass leaching. This is achieved through precise control techniques of inlet flow and aeration. Proper bed fluidization is crucial to ensure biomass retention within the system, preventing its loss and ensuring active participation in biochemical processes for organic substrate degradation. These strategies are essential to optimize AFBR performance, allowing efficient operation even under high organic loads and reduced hydraulic retention times. However, to maintain the fluidized state of the bed within the bioreactor, the system requires a high level of energy consumption, necessitating the optimization of operating conditions to reduce the required energy input [40]. To reduce these costs and optimize operational conditions, strategies include using efficient aeration and mixing technologies, integrating renewable energy sources, advanced process control, optimizing hydraulic retention time (HRT), and implementing continuous monitoring with preventive maintenance. These measures not only save energy but also enhance sustainability and system performance in bioH₂ production.

3.8.5. Upflow Anaerobic Sludge Blanket Reactors (UASBs)

UASB reactors are commonly used to produce bioH₂ from wastewater of various origins [60,121,127]. They generally have an elongated shape containing a gas/liquid/solid separator at the top, where microbiological granules are formed. These granules settle, creating a thick biomass blanket zone at the bottom [120].

The liquid effluent containing substrates enters the reactor from the bottom, and the liquid exits with minimal substrates from the top. In upflow reactors, biomass is immobilized in granules through the formation of biofilms or trapped in packed support media. The support media include sponge, granular activated carbon, expanded clay, polyethylene-octane elastomer, ceramic balls, alginate gel [25,37]. In a fluidized bed reactor, gas or liquid passes through the accumulated solid matter, generating an upward movement, causing hydraulic turbulence, thereby improving mixing and mass transfer without any mechanical agitation [128].

The granules are formed and grow during the fermentation process through the aggregation of activated sludge. The advantage of the granular form of microorganisms is their higher retention in the reactor and greater resistance to toxic conditions. The advantages of UASB reactors include high efficiency in bioH₂ production, short HRT, and stable operational conditions [14].

Although the UASB has several advantages, its practical application for bioH₂ production requires the development of granules with an ideal size for bioH₂ production, typically requiring a cultivation period of several months [126]. According to Jung [128] an alternative to accelerate granule formation would be the use of CSTR granules as UASB inoculum, resulting in the production of granules with an average size of 1.9 mm after 45 days.

4. Technologies to Increase bioH₂ Production

4.1. Integrated Production Strategies

Although dark fermentation is widely studied, the bioH₂ yield is limited by the production of volatile fatty acids (VFAs) that accumulate in the culture medium, leading to incomplete oxidation of available organic matter and a decrease in pH that inhibits microorganisms [129,130,131].

Therefore, to increase yields in bioH₂ production by recovering energy from the effluent of dark fermentation, many studies have addressed production in two stages, coupling dark fermentation with photofermentation [132] and microbial electrolysis cells [17].

4.1.1. Dark Fermentation—Photofermentation

VFAs present in the liquid residue can be consumed by purple non-sulfur bacteria during an additional photofermentation stage [25], enabling more efficient oxidation of the substrate and consequently higher bioH₂ production [20]. Integrated production can be achieved through co-cultivation of dark fermentative and photofermentative microorganisms [133] or through sequential bioreactors [134].

Integrating dark fermentation and photofermentation can produce the theoretical maximum yield of 12 moles of bioH₂ per mole of glucose, demonstrating that their coupling is more efficient than performing these two types of fermentation separately (Equations (1) and (2)) [135].

Dark fermentation: C₆H₂O₆ + 2H₂O → 2CH₃COOH + CO₂ + 4H₂

(1)

Photofermentation: 2CH₃COOH + 4 H₂O → 4CO₂ + 8H₂

(2)

Rai [136] conducted a two-stage sequential bioH₂ production from hydrolyzed sugarcane bagasse. The dark fermentation was carried out by Enterobacter aerogenes, achieving a yield of 1L of bioH₂ per liter of culture medium. Subsequently, the effluent from the dark fermentation was used in a photofermentation stage by the purple non-sulfur bacterium Rhodopseudomonas BHU 01, resulting in a cumulative maximum production of 0.75 L of bioH₂ per liter of medium in this stage.

Nino-Navarro [134] used fruit and vegetable residues for two-stage bioH₂ production. The dark fermentation was performed by a consortium predominantly composed of Bifidobacterium (85.4%), Klebsiella (9.1%), Lactobacillus (0.97%), Citrobacter (0.21%), Enterobacter (0.27%), and Clostridium (0.18%), achieving a maximum bioH₂ production of 93.4 mL of H₂/g COD. The lactate-rich effluent from the dark fermentation was subsequently diluted (1:2, 1:5, and 1:10) and used for bioH₂ production via photofermentation by the bacterium Rhodopseudomonas palustris, reaching a maximum yield of 1.37 L H₂ per g COD at a 1:10 dilution.

4.1.2. Dark Fermentation—Microbial Electrolysis Cells

Microbial electrolysis cells (MECs) combine the metabolism of microorganisms and bioelectrochemical reactions for bioH₂ production [137]. These cells are composed of one or two chambers, featuring an anode and, in some cases, a membrane separating the electrodes [138,139,140].

Under anaerobic conditions, exoelectrogenic microorganisms capable of catalyzing electron transfer from the substrate to the electrodes, such as those from the genera Geobacter, Shewanella, Acetobacterium, Rhodoferax, Rhodopseudomonas, Ochrobactrum, Enterobacter, Clostridium, among others [141,142], in their pure forms or as part of consortia, form a biofilm on the anode, acting as electrocatalysts [143]. They are responsible for the oxidation of organic matter, generating volatile fatty acids, free electrons, H⁺ protons, and CO₂ [143]. The free electrons are transferred from the anode to a cathode through an electrical circuit under an applied voltage, while the protons diffuse through the separation membrane to the cathode, where they are then reduced to form bioH₂ [144,145].

The expression used to describe bioH₂ production in MECs is commonly based on the utilization of acetate as a carbon source (Equation (3)) [138]. A theoretical maximum yield of approximately 4 mol of bioH₂ per mol of acetate is estimated [146].

The effluent from dark fermentation can also be oxidized through microbial electrolysis cells for additional bioH₂ production [147]. As observed in Equation (3), the theoretical maximum yield of 4 mol bioH₂/mol glucose obtained through dark fermentation (Equation (1)) increases to approximately 8 mol of hydrogen/mol glucose by coupling dark fermentation and microbial electrolysis cells [148].

CH₃COO⁻ + 4H₂O → 2HCO₃ + H⁺ + 4H₂

(3)

Li [149] produced bioH₂ in two stages using corn stover as a substrate. In dark fermentation, they achieved a maximum yield of 129.8 mL of bioH₂/g corn stover. Subsequently, the fermented broth from the first stage was diluted and used as a substrate for bioH₂ production in a microbial electrolysis cell, reaching a maximum yield of 257.3 mL of bioH₂/g corn stover under an applied voltage of 0.8 V and 44% COD removal.

In a similar system, Khongkliang [150] used wastewater from cassava starch processing as a substrate, resulting in a maximum yield of 260 mL bioH₂/g COD removed and COD removal efficiency of 38% during dark fermentation. The second stage using the effluent from dark fermentation in an MEC resulted in a yield of 205 mL bioH₂/g COD removed and COD removal efficiency of 32%.

4.2. Nanoparticles (NPs)

The use of nanoparticles (NPs) composed of metals and metal oxides, such as Ag, Pd, Co, Au, Cu, Ni, Ti, and Fe, has shown potential in enhancing bioH₂ production [151,152]. These trace elements act as cofactors for key enzymes in bioH₂ production metabolism, promote bacterial proliferation [40], act as O₂ scavengers contributing to increased FeFe-hydrogenase enzyme activity, which is more sensitive to oxygen presence, also reduce the redox potential in the system, providing a more favorable environment for bacteria performing dark fermentation [153,154] enrich the microbial community, and promote the growth of bioH₂-producing species such as Clostridium [155].

Among the various elements that can compose nanoparticles, iron (Fe) and nickel (Ni) metal ions are particularly studied due to their involvement in the synthesis of iron-sulfur proteins in ferredoxin and [NiFe] hydrogenases and [FeFe] hydrogenases enzymes [155,156]. Additionally, previous studies found that iron NPs increase the activity of hydrolytic enzymes in anaerobic fermentation, which are essential for biomass hydrolysis and utilization [157].

Do Nascimento Junior [88] synthesized green iron nanoparticles using lignin extracted from empty oil palm fruit bunches. Subsequently, the nanoparticles were applied in a hydrogen fermentation medium composed of wastewater from soft drink production (88%) and corn steep liquor (12%) supplemented with NaHCO₃ (1.0 g/L) and cysteine-HCl (0.5 g/L) and inoculated with Clostridium (64%), Ruminococcus (34.5%), and other genera (1.5%). The yield obtained in fermentation without FeNPs addition resulted in a yield of approximately 180.7 mL of H₂/g COD removed, which increased to 410 mL of H₂/g COD removed with the addition of 200 mg/L of iron nanoparticles.

Sun [158] optimized bioH₂ production from corn straw hydrolysate with the addition of nickel NPs. The bioH₂ yield increased from 0.7 (mol bioH₂/mol glucose) to 1.18 mol bioH₂/mol glucose with the addition of NPs, resulting in a 40% increase in yield.

4.3. Genetic and Metabolic Engineering

The genetic and metabolic engineering of fermentative microorganisms has also been used as a strategy to increase bioH₂ yields, through processes such as the introduction or overexpression of genes related to hydrogenase enzyme production [159,160] and substrate consumption [161], or deletion of genes controlling alternative pathways for organic acid production [162,163].

Jiang [161] introduced the gene encoding exoinulinase into Clostridium tyrobutyricum microorganisms aiming at its use in bioH₂ production from the fermentation of Helianthus tuberosus, a widely available culture rich in a carbohydrate known as inulin, which is unused by wild-type microorganisms. Using the modified strain, a yield of 3.7 mol of bioH₂/mol of inulin was obtained after 96 h in a batch process that combined simultaneous saccharification and fermentation (SSF). Inulinase production was closely linked to the strain’s growth during the exponential phase, achieving peak activity at 28.4 ± 0.26 U/mL.

To increase substrate uptake and yields from glycerol utilization as a substrate for dark fermentation, Sarma [159] produced two recombinant strains of Clostridium pasteurianum. In the first approach, the hydA gene encoding hydrogenase was overexpressed, and in the second approach, a combination of overexpression of dhaD1 and dhaK genes encoding glycerol dehydrogenase and dihydroxyacetone kinase was performed. A yield of 1.1 mol of H₂/mol of glycerol was obtained from the hydA overexpressed strain, and 0.93 mol of H₂/mol of glycerol from the dhaD1K overexpressed strain.

Son [160] performed the recombination of two strains of Clostridium acetobutylicum (CA-zwf and CA-hydA) for the overexpression of zwf and hydA genes responsible for encoding glucose-6-phosphate dehydrogenase and FeFe-hydrogenase enzymes, respectively. The CA-zwf strain achieved a yield of 1.23 (mol of bioH₂/mol of glucose), which is 1.15-fold higher than the wild type. For CA-hydA, the hydrogen yield was 1.49 (mol of bioH₂/mol of glucose), a 39.1% increase compared to the wild type.

Thermophilic anaerobic microorganisms of the species Caldicellulosiruptor bescii can efficiently utilize lignocellulosic biomass without the need for pretreatment, primarily producing lactate, acetate, and bioH₂ as fermentation products. Aiming to increase bioH₂ production by C. bescii and decrease lactate production, [163] deleted the ldh gene responsible for encoding L-lactate dehydrogenase enzyme that catalyzes lactate production. After the modification, the resulting strain did not produce detectable levels of lactate from cellobiose and maltose, instead increasing acetate and bioH₂ production by 21–34%, respectively, compared to the wild type.

Cai [162] blocked the ethanol production pathway by Clostridium butyricum through genetic disruption of the aad gene (encoding aldehyde-alcohol dehydrogenase) using a ClosTron plasmid to increase bioH₂ production yield. The mutant strain showed approximately 20% improved performance in bioH₂ production compared to the wild type.

5. Mathematical Modeling for Biohydrogen Production

Biological hydrogen production is a complex process, since in addition to the energy vector, other intermediate metabolites are also produced, such as acids, alcohols, and other solvents and the biomass itself, which affect the yield or production of the energy vector. However, mathematical models, regressions and artificial neural network systems have been used to evaluate and model the behavior of biological hydrogen production.

The models are based on different criteria, for example, some are based on the stoichiometry of the reactions involved, others on the metabolic pathways of the process, and others on the physical or chemical variables that affect the production of the energy vector [164]. These variables include the types of substrates, quantities and types of inocula, time, temperature, and pH, among others [49].

For example, the Modified Gompertz model (Equation (4)) describes the profile of substrate consumption, microbial biomass growth and the formation of soluble metabolites and hydrogen [164].

$$H=H_{max}exp\left\{-exp\left[{\displaystyle \frac{R_{max}e}{H_{max}}}\left(\mathsf{\lambda }-\mathrm{t}\right)+1\right]\right\}$$

(4)

where H represents the cumulative hydrogen production during fermentation time (t), H_max and R_max represent the maximum amounts and rates of production of the energy vector, respectively. The value 1 represents the latency time and “e” the constant 2.7182818. Nemestóthy [165] evaluated the effect of ionic liquids (1-butyl-3-methylimidazolium acetate and 1-butyl-3-methylimidazolium chloride) on biohydrogen production using the Modified Gompertz model, the results showed changes in hydrogen production parameters such as maximum biohydrogen production rate and lag phase times attributed mainly to ionic compounds.

Another widely used model for evaluating biohydrogen production is the Monod model (Equation (5)). This evaluates the effect of substrate concentration on microbial biomass growth and biohydrogen production.

$$R={\displaystyle \frac{R_{max}S}{K_s+S}}$$

(5)

where R represents the specific rate of hydrogen production at substrate concentration S, R_max and K_s denote the maximum rate of hydrogen production and the Monod half-saturation constant, respectively. These can be used to evaluate biohydrogen production under any condition. While statistical models can only be used under the conditions evaluated and the same type of microorganism employed.

The models mentioned above can be used in any dark fermentation process, regardless of the type of substrates, microorganisms and conditions used. While statistical regression models can only be used in the evaluated ranges. For example, Martinez-Burgos [49] evaluated the effect of the variables, purge time (s) and the percentage of CSL (corn steep liquor), on hydrogen production using two types of microbial consortia (Gal and Vir), obtaining two mathematical models (Equations (6) and (7)) with R² of approximately 95%, indicating an almost perfect fit of the models to the values of the variable, which in this case was the volume of hydrogen produced. ANOVA showed that all linear, quadratic effects and interactions were significant (p ≤ 0.05) in both models.

VH₂ = 4.22 + 0.31X₁ − 1.11X₂ − 1.366X²₁ − 1.32X²₂        (Gal)

(6)

VH₂ = 5.20 + 0.70X₁ − 0.70X₂ − 1.18X²₁ − 2.18X²₂ − 0.6622X₁X₂   (Vir)

(7)

where VH₂: Accumulated volume of biohydrogen; X₁: Purge time (s); X₂: % CSL [88] observed that biohydrogen production was highly affected by fermentation time and substrate concentration. Linear regression analysis of the experimental results generated a second-order polynomial model with a coefficient of determination (R²) of 92%, showing that BioH₂ production is very well represented by the mathematical model (Equation (8)). The linear and quadratic terms were significant (p ≤ 0.05).

BioH₂= 16.47 + 3.35X₁ + 4.94X₂ − 2.83X₁² − 5.0 X₂²

(8)

On the other hand, artificial neural networks have also been used to model hydrogen production via dark fermentation. The networks can be built using the Neural Network Start (nnstart) module of MATLAB software (https://www.mathworks.com/products/matlab.html, accessed on 22 August 2024) using input–output curve fitting models (input–output curve fitting). This tool is based on feed-forward networks with three layers: input, hidden and output.

According to Karthic [166], biohydrogen production is a complex system where the use of artificial networks for process prediction, control and monitoring has great potential. For example, Sydney [167] showed that artificial neural networks can predict the hydrogen production rate with high accuracy R² ≥ 0.98, and Karthic [166] concluded that artificial neural networks are more reliable than classical statistical models, such as the response surface methodology.

6. Advantages and Disadvantages of Dark Fermentation and Other Methods of Renewable Hydrogen Production

Different methods are known to produce renewable hydrogen, among which are those based on the fragmentation of the water molecule and those that use microorganisms to produce the energy vector, among others. Splitting water can be classified into three techniques: electrolysis, thermolysis, and photolysis, while biological methods include dark fermentation, biophotolysis, and photofermentation [168].

In electrolysis, continuous electrical energy is used to fragment the water molecule into hydrogen (H₂) and oxygen (O₂). In addition, the process is catalyzed by electrolytes such as bases, acids and some salts. Renewable electrical energy is generally used in the process, which produces green hydrogen without releasing CO₂ into the atmosphere [169,170].

This is the only process used on an industrial scale to produce renewable hydrogen. In fact, according to Martinez-Burgos [49] 5% of the hydrogen produced in the world is through this process. The main advantages of this technique are that it is a fast process that is carried out under ambient pressure and temperature conditions, is easily scalable and does not emit CO₂ into the atmosphere, since renewable solar or hydraulic energy is generally used. One of its main advantages is the corrosion of the electrodes, so the useful life of the electrodes is quite short. The advantages and disadvantages of the other processes are summarized in

Table 2. Comparison between different methods of renewable hydrogen production.

Process	Methods	Principle	Advantages	Disadvantages	References
Water splitting	Electrolysis	Fragmentation of the water molecule using electric current and some electrolytes such as bases, acids and salts	Easily scale-up Water is used as the main raw material Process carried out at ambient temperatures and pressures Can achieve efficiencies of 60%	Short life of electrodes due to corrosion	[171]
	Thermolysis	Fragmentation of the water molecule using high pressures and temperatures (1800–5000 °C)	Water is used as the main raw material Solar energy and different types of biomass can be used as energy sources	Large amounts of energy are required in the process High pressures and temperatures are required Low efficiency, maximum 40%	[49,172]
	Photolysis	Fragmentation of the water molecule using photons of light	Solar energy can be used	Extremely low process efficiency, between 0.1–1.0% Highly expensive TiO2, IrO2, or RuO2 electrodes must be used	[49]
Biological	Dark Fermentatio	Biological catabolic process carried out by bacteria in which one of the main gaseous bioproducts is hydrogen	Solid or liquid waste is used as substrates The process can be carried out at ambient pressures and temperatures Relatively fast process compared to other biological methods of hydrogen production	Low yield, maximum 4 moles of H2 per mole of glucose Other metabolites are generated during the process that affect the process yield Complex purification processes are required Slow process compared to electrolysis	[173,174]
	Photofermentation	Biological reaction for the production of hydrogen, carried out in two stages. The first stage takes place in the absence of light and the second in the presence of light, the latter being carried out by purple bacteria	Waste can be used as substrate Higher yields can be obtained than dark fermentation Process can be carried out at ambient temperatures and pressures	Light-dependent process Two-step process More time-consuming process Slower process	[175]
	Bio-photolyses	Fractionation of the water molecule by sunlight and catalyzed by photosynthetic microorganisms such as microalgae and cyanobacteria	Waste can be used as substrates The process can be carried out at ambient pressure and temperature conditions	Light-dependent process A process carried out in two stages, in the first stage biomass is produced and hydrogen is obtained in the second stage A slower process than photofermentation	[176]

.

7. Conclusions

Hydrogen is a clean and efficient energy carrier, responsible for emitting only water vapor during combustion and zero greenhouse gas emissions. As a result, extensive research has been conducted to develop a future hydrogen economy. Numerous projects globally focus on renewable hydrogen production, mainly through electrolysis. However, due to the abundance of organic wastes, biological hydrogen production may become attractive.

Among the methods of hydrogen production, dark fermentation has been intensely explored and improved using a wide variety of organic waste as substrates, inoculants, and exploration of operational parameters such as pH, temperature, organic loading rate, partial pressure of bioH₂, hydraulic retention time, and bioreactor design.

However, bioH₂ production through dark fermentation results in limited yields. Therefore, for the bioH₂ economy to consolidate, constant exploration of cheaper raw materials and different inoculants is necessary, along with modeling and optimization of physicochemical parameters that affect the process and the use of approaches that ensure maximum bioH₂ recovery, such as two-stage production, cell immobilization inside bioreactors, and the use of molecular techniques to improve bioH₂-producing strains.