Catalytic Innovations for High-Yield Biohydrogen Production in Integrated Dark Fermentation and Microbial Electrolysis Systems

Abstract

Biohydrogen, a low-carbon footprint technology, can play a significant role in decarbonizing the energy system. It uses existing infrastructure, is easily transportable, and produces no greenhouse gas emissions. Four technologies can be used to produce biohydrogen: photosynthetic biohydrogen, dark fermentation (DF), photo-fermentation, and microbial electrolysis cells (MECs). DF produces more biohydrogen and is flexible with organic substrates, making it a sustainable method of waste repurposing. However, low achievable biohydrogen yields are a common issue. To overcome this, catalytic mechanisms, including enzymatic systems such as [Fe-Fe]- and [Ni-Fe]-hydrogenases in DF and electroactive microbial consortia in MECs, alongside advanced electrode catalysts which collectively surmount thermodynamic and kinetic constraints, and the two stage system, such as DF connection to photo-fermentation and anaerobic digestion (AD) to microbial electrolysis cells (MECs), have been investigated. MECs can generate biohydrogen at better yields by using sugars or organic acids, and combining DF and MEC technologies could improve biohydrogen production. As such, this review highlights the challenges and possible solutions for coupling DF–MEC while also offering knowledge regarding the technical and microbiological aspects.

1. Introduction

Life on Earth has evolved according to natural rules throughout recorded history. Human action, however, is responsible for most of the resulting changes. The current generation needs to be more cautious in order to secure the future of the world. Fossil fuel depletion, pollution, climate change, and biodiversity loss are the main problems of the twenty-first century [1]. All these issues are a result of the world’s population growing faster than ever. Demand and supply for energy are critical to the advancement of human civilization [2]. Fossil fuels like natural gas, petroleum, and crude oil provide the majority of energy today, but they are rapidly running out [3]. Finding a more sustainable and alternative energy source is currently crucial because of the negative environmental effects of current energy sources, their depletion, and the instability of energy markets caused by political and economic issues. Several energy forms have been produced using renewable resources, such as hydropower, wind, and solar. Even though these clean energy sources have many enticing benefits, their sporadic nature and reliance on environmental factors create difficult circumstances due to unpredictable energy supply and price fluctuations [4]. One renewable energy source that is seen to have the ability to displace the economy’s reliance on fossil fuels is hydrogen (H₂). It is widely acknowledged that the decarbonization of the energy system would depend heavily on bio-hydrogen, or hydrogen produced via low-carbon footprint technologies. Hydrogen is an energy vector, a fuel, and a chemical feedstock. Since burning biohydrogen emits no greenhouse gases, it offers a practical means of decarbonizing enterprises, transportation, buildings, and power systems. Furthermore, biohydrogen has garnered a lot of interest as a clean fuel and energy carrier due to its higher energy content (142 kJ/g) compared to competing fuels (52 kJ/g for natural gas, 24 kJ/g for coal, or 23 kJ/g for methanol) [5,6].

Biohydrogen production is possible via four distinct technologies: dark fermentation (DF), photofermentation, photosynthetic biohydrogen, and microbial electrolysis cells (MECs) [7]. DF is more flexible with organic substrate and produces more H₂, both of which reduce operating costs and make it a sustainable waste repurposing method. The benefits of DF include (i) quick gas generation rates, (ii) easy reactors design requirement, and (iii) the ability to valorize a variety of organics matter, such as wastewaters and solid resources obtained from biomass [8]. There are several current problems with this technology that need to be resolved. One of the most frequently mentioned of them is low achievable H₂ yields, which indicates the amount of gas produced for a given quantity of substrate are used [9,10,11].

Several solutions, including stage system such DF connection to photo fermentation and anaerobic digestion (AD) to microbials electrolysis cells (MECs), have been investigated in an effort to overcome low H₂ yields. It’s interesting to note that MEC can generate H₂ at better yields by using sugars or organic acids. When 100% of the electron equivalent is used for H₂ production, the maximum theoretical yields are 12 mol H₂/mol glucose and 4 mol H₂/mol acetates. Acetate has shown the highest productivities in Microbial Electrochemical Cells (MECs) compared to other substrates, achieving 50 m³.H₂/m₃. It is thought that combining DF and MEC technologies will improve H₂ production because acetate is a common acid in DF effluents. In this way, dead-end fermentation products like VFAs would be used as substrates for MECs, increasing H₂ yields. The Microbial Electrolysis Cell (MEC) and Microbial Fuel Cell (MFC) operate essentially on the same concept. The products that are produced as a consequence of the biological conversion are, in theory, one significant distinction between the two processes: MFCs produce usable bioelectricity directly, whereas MECs produce H₂ gas at the cathode [12,13,14].

The general operating principle of MECs, which are based on two-chambered cells, is organic oxidation at anodes and reduction processes at cathodes. Another name for this procedure is electro fermentation. This kind of biocatalyzed electrolysis cell produces electrons, protons, and carbon dioxide when microorganisms at the anode catalyze the oxidation of an organic substrate. Subsequently, a proton exchange membrane (PEM) and an external circuit, often an electric wire, are employed to transport protons and electrons to the cathode, respectively. When the protons and electrons combine, reduction reactions produce biohydrogen. Furthermore, MECs and DF work well together because DF breaks down complex organic chemicals into simpler ones that may be used by MECs’ anodes-respiring bacteria for current generation. Many studies have looked into coupled DF-MEC in an effort to produce high H₂ production [14,15].

In recent years, several reviews have examined specific facets of producing biohydrogen using microbial electrolysis cells (MECs) or dark fermentation (DF), with an emphasis on issues like system configurations for increased hydrogen yield, DF microbial consortia optimization, and MEC electrode materials. The integration of DF and MEC into a single, synergistic DF–MEC system has received little attention in recent evaluations, which have mostly viewed them as distinct processes. The linked DF–MEC approach is the focus of this review, which is unique in that it summarizes and synthesizes the most recent studies on its operational concepts, system designs, and performance improvements. This analysis highlights the technological, financial, and sustainability-related issues that need to be resolved for widespread adoption by combining findings from both domains. Additionally, it offers fresh perspectives on how DF–MEC integration may enhance process economics, reduce environmental effects, and maximize hydrogen generation efficiency a thorough viewpoint that has been absent from earlier research.

2. Strategies for Producing Biohydrogen

Hydrogen is basically produced by a sustainable and ecologically safe biological process. Hydrogen can be produced from a variety of feedstocks, including water, urban solid waste, agricultural residue, industrial effluents, and household garbage. Direct and indirect bio-photolysis, microbial electrolysis, dark-fermentation (DF), and photo-fermentation are the primary methods for producing biohydrogen. The two main groups of these processes are light dependent and light independent. While photo-fermentation and indirect and direct bio-photolysis are light-dependent processes, dark-fermentation and electro-hydrogenesis are not; Figure 1 indicates the several methods and microorganisms utilized in the creation of hydrogen.

2.1. Direct Photolysis

This mechanism uses the same pathways as photosynthesis in algae and plants, but it modifies them to produce hydrogen gas rather than carbon-based biomass. Water molecules are converted into H₂ and O₂ during photosynthesis with the aid of sunlight’s photons. Solar photons in the reducing sites of photosystem I (PSI) generate hydrogen ions in anaerobic conditions or when too much energy is collected. The decreased enzyme Fe-Fe hydrogenase in the algal cell then provides electrons to turn it into H₂ gas in a medium. Molecular oxygen is simultaneously produced by the oxidizing sides of photosystem-II (PSII). The following is the reaction that took place during the process:

2H₂O + Light Energy → 2H₂ + O₂

(1)

Hydrogenase activity has been found in a variety of green algae, such as Scenedesmus obliquus, Chlamydomonas reinhardtii, Platymonas subcordiformis, Chlorella fusca, and Chlorococcum littorale. Direct photolysis has the potential to produce hydrogen in theory. However, there are some problems with the procedure. First off, because the hydrogenase enzyme is so sensitive to O₂, direct photolysis generates a lot less hydrogen. Second, the yield of hydrogen is reduced due to the limitations of light. However, the issues need to be resolved in order to make the process more feasible [16].

2.2. Indirect Photolysis

A variation on direct bio-photolysis, indirect bio-photolysis separates the synthesis of hydrogen and oxygen either temporally by carrying out the reactions one after the other in the same system or spatially by employing different reactors for each step. Because of this separation, oxygen’s inhibitory influence on enzymes that evolve hydrogen is reduced. Cyanobacteria are frequently chosen for this procedure because of their low nutritional needs and metabolic flexibility.

The mechanism involves two main stages. First, CO₂ is fixed via photosynthesis to form storage carbohydrates such as starch and glycogen, shown in Equation (2):

6H₂O + 6CO₂ + Light Energy → C₆H₁₂O₆ + 6O₂

(2)

In the second stage, these carbohydrates are anaerobically degraded to produce hydrogen, either through dark fermentation alone or in combination with photo-fermentation, as shown in Equation (3). During dark fermentation, the carbohydrates are converted into hydrogen, acetic acid, and CO₂, with the acetic acid further transformed into hydrogen and CO₂ under illuminated anaerobic conditions:

C₆H₁₂O₆ + 6H₂O + Light Energy → 12H₂ + 6CO₂

(3)

An extensive group of cyanobacteria species, some of which may fix nitrogen or not, are the primary drivers of this process. non-marine Anabaena sp., Calothrix sp., and Oscillatoria sp. are N₂ fixing cyanobacteria’s; Synechococcus sp., Gloebacter sp., and marine Anabaena sp. are the non-N₂ fixing cyanobacteria. It has a number of real-world restrictions, much like direct photolysis, which make it difficult to scale up and commercialize the process [16].

2.3. Photo-Fermentation

Different photosynthetic bacteria work in anaerobic conditions to convert organic chemicals like acetic acid into hydrogen through a biological process called photofermentation (PF). Purple nonsulfur (PNS) bacteria are the main subject of study since they can eat a wide range of feedstocks. PNS bacteria employ nitrogenase to make hydrogen when nitrogen is scarce, and absorption hydrogenase consumes hydrogen. Hydrogen is produced by PNS bacteria such as Rhodopseudomonas palustris, Rhodobacter capsulatus, Rhodobacter sphaeroides, and Rhodospirillum rubrum. Despite producing a significant amount of hydrogen, the method has a number of disadvantages, including a high energy requirement, limited photosynthetic conversion, and a low volumetric production rate [17]. Process of photofermentation can be seen in Figure 2.

2.4. Dark Fermentation

Anaerobic dark fermentation is the process by which certain acidogenic bacteria convert organic materials, primarily carbohydrates, to H₂ gas. Microorganisms produce ATP, or anaerobic energy, for cells by obstructing the Tricarboxylic Acid (TCA) cycle. As a result, metabolic end products like ethanol and volatile fatty acids are formed using the additional electron that was created. Because of its high rate of production and yield, compared to alternative methods of producing biohydrogen, anaerobic dark fermentation has several advantages. It makes it possible to produce hydrogen with little energy input from a variety of carbohydrate-rich substrates, such as different organic wastes and renewable biomass. The procedure lowers operating complexity and cost by using moderate conditions and very simple reactor setups. With short hydraulic retention durations and high hydrogen production rates and yields, it is appropriate for large-scale or continuous applications. To improve overall energy recovery and waste valorization, dark fermentation can also be combined with other procedures like photofermentation or anaerobic digestion [18]. Further details regarding dark fermentation are provided in the following section [17]. Process of dark fermentation can be seen in Figure 3.

2.5. Microbial Electrolysis Cell (MEC)

An external electric current and exoelectrogenic bacteria are used by the microbial electrolysis cell (MEC), a modified microbial fuel cell (MFC), to transform organic molecules into molecular hydrogen. The proton exchange membrane (PEM), cathode, and anode are the three primary components of the system. Exoelectrogens that are most frequently found are Escherichia coli, Rhodoferax ferrireducens, Citrobacter sp., Burkholderia sp., Geobacter sp., Pseudomonas sp., and Shewanella sp. Further details regarding MEC are provided in the following section [17].

Table 1. Advantages and Disadvantages of various hydrogen production technologies [18].

Techniques	Merits of the Technique	Demerits of the Technique
Photo-fermentation	1. A broad spectrum of light energy can be used by the bacteria in use. 2. Fit for a range of organic waste kinds.	1. The produced O2 inhibits the nitrogenase. 2. Slow hydrogen production 3. High implementation cost.
Thermochemical gasification	Maximize the substrate’s conversion to biohydrogen.	The specifications for the unique circumstances of gas maintenance
Supercritical conversion.	1. An ongoing procedure that is nighttime operational. 2. Elevated hydrogen concentration 3. The substrate does not need to be dried 4. High H2 selectivity and gasification efficiency.	Conditions for choosing a supercritical media.
Indirect bio-photolysis	1. The blue–green algae use water to create hydrogen. 2. The atmospheric fixation of nitrogen gas.	1. Certain hydrogenase enzymes must be eliminated in order to halt the H2 breakdown process. 2. Oxygen makes up 30% of the gas produced.
Dark fermentation	1. The procedure is easy to use and economical. 2. Attained a high rate of H2 production 3. Ability to produce H2 in the absence of light	1. O2 has the ability to inhibit hydrogenase 2. Reduce the output of biohydrogen. 3. An increase in H2 pressure makes the process less favorable thermodynamically.
Direct bio-photolysis	1. Uses only water and sunlight to generate H2 2. Cost-effective process 3. Energy discussion significantly increased when compared to other biomass (crops or forests).	1. The requirement for strong lighting. 2. The simultaneous production of H2 and O2, with the latter having a detrimental effect on the entire system. 3. Reduced efficiency of photochemistry
Microbial electrolysis	1. Direct production of biohydrogen from waste streams 2. A viable and efficient method for producing hydrogen from wastewater in the future	1. We still have a good understanding of the metabolic pathways involved. 2. Reduced H2 generation at low electrode power densities

compares the benefits and drawbacks of multiple hydrogen generation strategies with microbial electrolysis cells.

3. Catalysts in Dark Fermentation: Enzymatic and Biocatalytic Systems

Microbial consortia and enzymatic systems are essential for the breakdown of organic substrates in dark fermentation, a crucial anaerobic process for the creation of biohydrogen. Catalysts improve hydrogen generation, substrate conversion, and reaction efficiency, especially enzymatic and biocatalytic systems. These systems play a key role in breaking down kinetic and thermodynamic barriers, which makes dark fermentation a viable option for producing energy sustainably. In dark fermentation, hydrogenases and other hydrolytic enzymes are the main enzymatic catalysts. A crucial stage in the fermentation process, proton reduction to molecular hydrogen is facilitated by hydrogenases such [Fe-Fe]- and [Ni-Fe]-hydrogenases. These enzymes’ active sites are tailored for electron transfer, making them extremely selective. Multiple enzymatic pathways are integrated by biocatalytic systems, such as microbial consortia and tailored strains, to enhance the process. For example, the pyruvate fermentation process, which uses enzymes like pyruvate, is how Clostridium and Enterobacter species, which are frequently employed in dark fermentation, produce hydrogen: A key role for ferredoxin oxidoreductase [19].

Temperature, pH, and substrate type are some of the variables that affect these systems’ efficiency. Enzyme stability and activity are improved under ideal circumstances, which are often 35–55 °C and pH 5.5–7.0. Recent bioreactor investigations have shown that immobilized enzymes or whole-cell biocatalysts further increase process stability, lowering costs and permitting continuous operation [20].

3.1. Types of Enzymatic Catalysts in Dark Fermentation

Anaerobic bacteria are used in dark fermentation, a key process in the production of biohydrogen, to transform organic substrates into metabolites such as carbon dioxide and hydrogen. Hydrogenases, hydrolases, and oxidoreductases are essential enzymatic catalysts that drive these biological events as shown in

Table 2. Types of enzymatic catalysts in dark fermentation.

Enzyme	Microorganism	Role in Dark Fermentation	Active Site	Turnover Frequency (k_cat)	Key Characteristics	Reference
[Fe-Fe] Hydrogenase	Clostridium spp. (e.g., C. acetobutylicum, C. pasteurianum)	Catalyzes proton reduction to H2 using electrons from reduced ferredoxin	Di-iron H-cluster with CN− and CO ligands	~104 s−1	High efficiency, low redox potential, sensitive to O2 and high H2 partial pressure	[22]
[Ni-Fe] Hydrogenase	Enterobacter spp., Desulfovibrio spp.	Facilitates H2 production via proton reduction with electrons from ferredoxin or NADH	Nickel-iron with cysteine ligands	102–103 s−1	Moderate efficiency, higher redox potential, less sensitive to O2 than [Fe-Fe]	[23]
Pyruvate-Ferredoxin Oxidoreductase (PFOR)	Clostridium spp., Thermotoga maritima	Converts pyruvate to acetyl-CoA, reducing ferredoxin for hydrogenase activity	Iron-sulfur clusters	~102 s−1	Essential for electron transfer to hydrogenases, operates in acetate pathway	[24]
NADH-Ferredoxin Oxidoreductase (NFOR)	Clostridium thermocellum, C. butyricum	Regenerates reduced ferredoxin from NADH, supporting [Fe-Fe] hydrogenase activity	Iron-sulfur clusters	~101–102 s−1	Enhances electron flow, critical for sustained H2 production	[25]
Pyruvate-Formate Lyase (PFL)	Enterobacter aerogenes, Escherichia coli	Converts pyruvate to formate, providing an alternative electron donor for H2 production	Glycyl radical	~102 s−1	Active in mixed acid fermentation, less common in strict anaerobes	[26]

, with high efficiency and selectivity and with an emphasis on improving the production of biohydrogen, this article offers a thorough examination of the various kinds of enzyme catalysts used in dark fermentation, as well as their mechanisms, structural complexities, uses, and difficulties [21].

3.2. Hydrogenases: The Core of Hydrogen Production

Table 3. Michaelis–Menten kinetic parameters for selected catalysts used in biohydrogen production.

Catalyst/Enzyme System	Substrate	VmaxV (mmol H2·h−1·cm−2 or μmol·min−1·mg−1)	KmK_m (mM)	Temperature (°C)	pH	Notes on Performance
Ni–Mo alloy cathode	Acetate	0.145	2.5	30	7.0	High activity under neutral conditions
Pt/C catalyst	Glucose	0.160	3.0	25	7.0	Low overpotential, high hydrogen yield
MoS2 nanosheets	Lactate	0.125	1.8	35	7.0	Good stability, suitable for long-term operation
[FeFe]-hydrogenase immobilized on CNTs	Reduced ferredoxin	0.210	0.05	25	8.0	Very low KmK_m, high specificity for electron donor

illustrates how hydrogenases, which are metalloenzymes, catalyze the reversible reduction of protons to molecular hydrogen (H₂), a crucial stage in dark fermentation. According to the metal composition of their active sites, they are divided into [Fe-Fe]-hydrogenases, [Ni-Fe]-hydrogenases, and [Fe]-hydrogenases (Hmd). The structural and functional characteristics of each type vary, which affects their catalytic effectiveness and industrial applicability [27].

3.3. [Fe-Fe]-Hydrogenases

The high catalytic efficiency of [Fe-Fe]-hydrogenases, which are found in anaerobic bacteria like Clostridium pasteurianum and Desulfovibrio vulgaris, is well known. Cyanide (CN⁻), carbon monoxide (CO), and dithiolate ligands coordinate the bimetallic [Fe-Fe] active site in the H-cluster of these enzymes. Rapid electron transport is made possible by the H-cluster in conjunction with iron-sulfur clusters ([4Fe-4S]), which can achieve turnover frequencies of 1000 to 10,000 s⁻¹. For high-yield hydrogen synthesis in dark fermentation, [Fe-Fe]-hydrogenases are hence perfect [22].

[Fe-Fe]-hydrogenases and pyruvate: ferredoxin oxidoreductase combine in Clostridium species to convert reduced ferredoxin produced during pyruvate metabolism into H₂. Because the active site of the enzyme is extremely sensitive to oxygen, it can oxidize the H-cluster irreversibly, stopping catalysis [22]. To improve oxygen tolerance, recent developments in protein engineering have concentrated on altering the gas diffusion channels. Hydrogenases in Clostridium, for instance, decreased oxygen sensitivity without sacrificing activity. Furthermore, immobilization methods such encapsulation in alginate beads have increased the stability of enzymes in bioreactors, allowing for continuous operation [28].

3.4. [Ni-Fe]-Hydrogenases

The bimetallic [Ni-Fe] active site of [Ni-Fe]-hydrogenases, which are present in bacteria such as Escherichia coli and Enterobacter aerogenes, is synchronized by CN⁻/CO ligands and cysteine residues. With turnover frequencies of 100–1000 s⁻¹, these enzymes are less effective than [Fe-Fe]-hydrogenases; nevertheless, because of their higher oxygen tolerance, they can be used in mixed microbial consortia. Bidirectional [Ni-Fe]-hydrogenases help to maintain cellular redox balance during fermentation by catalyzing both H₂ generation and absorption based on redox circumstances [29].

[Ni-Fe]-hydrogenases utilize electrons from formate metabolism or NADH to increase hydrogen output during dark fermentation. Clostridium and Desulfovibrio [Ni-Fe]-hydrogenases have been incorporated into microbial fuel cells, resulting in stable H₂ production over long periods of time. Their stability has led to applications in bioelectrochemical systems, where enzymes are immobilized on carbon electrodes to facilitate direct electron transfer. But because of their lower catalytic rates, contemporary synthetic biology initiatives must co-express them with other enzymes in order to maximize yields [30].

3.5. [Fe]-Hydrogenases

Dark fermentation is only little influenced by [Fe]-hydrogenases, or Hmd, which are mostly present in methanogenic archaea. These enzymes lack the iron-sulfur clusters found in other hydrogenases, instead having a single iron atom coordinated by a guanylylpyridinol cofactor. Hmd’s applicability to biohydrogen systems is limited because it catalyzes hydrogen transfer in methanogenesis rather than direct H₂ generation. Although [Fe]-hydrogenases’ low catalytic efficiency limits their wider use, research on them focuses on their evolutionary importance and possible niche applications in archaeal-based bioreactors [30].

By breaking down complex organic substrates into fermentable monomers, hydrolases start the dark fermentation process. For the processing of lignocellulosic biomass, starchy wastes, and protein-rich residues, respectively, these enzymes—which include cellulases, amylases, and proteases—are essential. The availability of fermentable substrates, a crucial bottleneck in the creation of hydrogen, is determined by their efficiency [30].

3.6. Cellulases

A significant part of lignocellulosic biomass, cellulose, is broken down into glucose and cellobiose by cellulases, which are produced by bacteria such as Clostridium thermocellum and Trichoderma reesei. Endoglucanases (which break internal β-1,4-glycosidic linkages), exoglucanases (which liberate cellobiose from chain ends), and β-glucosidases (which hydrolyze cellobiose to glucose) make up the cellulase complex. Although their combined action is necessary for effective biomass breakdown, accessibility is frequently hampered by the cellulose’s crystallinity and lignin content [31].

Cellulases make it possible to employ energy crops and agricultural waste as substrates for dark fermentation. Pretreatment techniques that provide enzyme access to cellulose, including steam explosion or acid hydrolysis, increase the rates of hydrolysis. Recent advances in enzyme cocktails that combine hemicellulases and cellulases have resulted in a 20–30% improvement in sugar yields from lignocellulosic feedstocks. Furthermore, Caldicellulosiruptor species’ thermophilic cellulases have benefits in high-temperature bioreactors, matching ideal fermentation conditions (35–55 °C) [32].

3.7. Amylases

Amylases help ferment starchy wastes, such as leftovers from food processing, by hydrolyzing starch into glucose. While glucoamylase releases glucose units from non-reducing ends, α-amylase randomly breaks down α-1,4-glycosidic bonds in starch to produce dextrins. For dark fermentation, bacteria such as Bacillus subtilis and Thermoanaerobacterium species generate amylases that are highly active at mesophilic temperatures (30–40 °C) and pH 5.5–7.0 [33].

When digesting inexpensive substrates, such potato or corn processing waste, amylase efficiency is essential. Amylase stability has been improved by genetic engineering; Bacillus strains expressing thermostable variants exhibit 50% greater activity at higher temperatures, and co-culture systems that combine bacteria that produce hydrogen and amylase have increased overall hydrogen yields by expediting substrate conversion [33].

3.8. Proteases

Proteases hydrolyze proteins into amino acids and peptides. acting as fermentation substrates or sources of nitrogen. Utilizing protein-rich wastes, including slaughterhouse leftovers or dairy byproducts, is made possible by the proteases found in Clostridium and Enterobacter species during dark fermentation. Serine, cysteine, and metalloproteases are among the enzymes that are active at pH 5.5–6.5, which is in line with fermentation conditions. However, the protease activity increases substrate diversity, enabling dark fermentation to treat a variety of waste streams. Nevertheless, excessive protein breakdown can result in ammonia buildup, which stops microorganisms from growing. Protease expression has been adjusted in mixed consortia in recent studies to balance the production of hydrogen and the supply of nitrogen [34].

3.9. Oxidorductases: Redox Balance and Electron Transfer

In dark fermentation, oxidoreductases such as dehydrogenase, pyruvate: ferredoxin oxidoreductase (PFOR), and NADH: ferredoxin oxidoreductase—are metabolic centers that coordinate electron transfer to preserve redox balance. By directing electrons to cofactors such as ferredoxin, NAD⁺, or flavins, these enzymes connect substrate oxidation to hydrogen generation, which in turn fuels hydrogenase activity. Because they control the flow of reducing equivalents in intricate fermentation routes, their effectiveness is essential for optimizing hydrogen yields. Temperature (35–55 °C) and pH (5.5–7.0) have a major impact on their activity, and thermophilic strains frequently perform better. Improvements in synthetic biology and metabolic engineering have increased their expression and stability, increasing the efficiency of hydrogen production. For example, in certain bioreactor configurations, co-expression of oxidoreductases and hydrogenases in engineered bacteria has raised hydrogen production by as much as 25%. Furthermore, oxidoreductases have become more stable and reusable when immobilized on nanostructured supports like carbon nanotubes, which lowers operating costs in continuous fermentation systems [35].

3.10. Pyruvate:Ferredoxin Oxidoreductase

A key enzyme in dark fermentation, pyruvate: ferredoxin oxidoreductase (PFOR) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO₂ while reducing ferredoxin, which then transfers electrons to hydrogenases for the generation of H₂. A tetrameric enzyme with iron-sulfur clusters that promote electron transport, PFOR is found in Clostridium and Thermotoga species. Because acetyl-CoA inhibits the product and competes with pyruvate at the active site, its activity is rate-limiting. Site-directed mutagenesis to lower acetyl-CoA binding affinity has further improved catalytic efficiency, and overexpression of PFOR in modified Clostridium strains has raised hydrogen yields by 15% to 20% [24].

Thermophilic bacteria such as Thermotoga maritima can function at 60 to 80 °C thanks to PFOR’s thermostability, which complements high-temperature bioreactors that increase substrate solubility and lower the danger of contamination. The incorporation of PFOR into synthetic metabolic pathways has been investigated recently; in optimal systems, it co-expresses with hydrogenases and electron carriers to facilitate electron flow and produce up to 30% greater H₂ yields. Furthermore, by mixing Clostridium and Enterobacter in co-culture systems, PFOR increases total hydrogen generation through a variety of metabolic outputs. Nevertheless, there are still issues, such as oxygen sensitivity and the high energy expenditures of enzyme synthesis, which has led to studies on stabilization using nanoparticles and enzyme immobilization [36].

3.11. NADH Ferredoxin Oxidoreductase

Hydrogenase activity is indirectly supported by NADH ferredoxin oxidoreductase (NFOR), which transfers electrons from NADH to ferredoxin to replenish NAD⁺. In routes like those seen in Enterobacter and Klebsiella species, where NADH buildup prevents glycolysis, this enzyme is essential. FAD or FMN cofactors help NFOR function as a flavoprotein by promoting electron transport. Its activity depends on pH, and it works best at 5.5 to 6.5, which corresponds to dark fermentation conditions [37].

Optimizing electron flow through metabolic engineering to increase NFOR expression in Enterobacter aerogenes, batch fermentations have seen a 10–15% increase in hydrogen yields. NFOR has been included into synthetic gene circuits in recent research, where it works in tandem with hydrogenases to balance NADH/NAD⁺ ratios and enhance metabolic efficiency. NFOR’s half-life has been increased by 50% through immobilization on graphene-based supports, allowing for continuous bioreactor operation. Furthermore, NFOR’s function in mixed fermentation systems, where it supplements PFOR, has a variety of electron sinks that decrease the formation of byproducts (such as ethanol) and increase H₂ output. Nevertheless, the challenges include low catalytic rates in comparison to PFOR and sensitivity to high NADH concentrations, which are being addressed by ongoing research through cofactor engineering and directed evolution [38].

3.12. Synthesis and Preparation of Enzymatic Catalysts

Enzymatic catalysts are essential for promoting biological reactions that transform organic substrates into useful products like hydrogen gas (H₂), especially when using dark fermentation (DF) for the synthesis of biohydrogen [22] Microorganisms physiologically manufacture these catalysts, which are mainly enzymes like formate dehydrogenases and hydrogenases. A variety of strategies are used in the creation of these enzymatic catalysts, such as pretreatment procedures to increase enzyme synthesis and activity, genetic engineering approaches, and microbial growth condition optimization [39].

3.12.1. Microbial Synthesis of Enzymatic Catalysts

Microorganisms that have metabolic pathways that support the production of enzymes, including as bacteria (such as Clostridium, Enterobacter, and Thermotoga) and some archaea, create enzymatic catalysts in DF. Proton reduction to molecular hydrogen is catalyzed by [FeFe]-hydrogenases, [NiFe]-hydrogenases, and nitrogenases, which are the main enzymes involved in H₂ synthesis [40]. Under the influence of genetic instructions included in the organism’s DNA, these enzymes are produced inside the microbial cell during active development and metabolism [22].

The transcription and translation of genes encoding enzymes, like hydA in Clostridium species, which codes for [FeFe]-hydrogenase, initiates the synthesis process. Environmental variables and cellular cues, such as substrate availability, pH, and redox state, strongly control the expression of these genes. Hydrogenase genes, for instance, are upregulated when fermentable carbohydrates such as glucose or sucrose are present because these substrates are broken down by glycolysis and subsequent fermentation pathways, producing reducing equivalents (such as NADH and Fd_red) that hydrogenases need [41]. The growing conditions of the host microorganisms have a significant impact on the efficiency of enzymatic catalyst production.

The pH of the fermentation medium significantly affects microbial metabolism and enzyme stability. Most H₂-producing microorganisms thrive in a slightly acidic pH range of 5.0–6.5, as this range supports the activity of hydrogenases while inhibiting competing metabolic pathways, such as methanogenesis. For instance, Clostridium species exhibit optimal hydrogenase activity at pH 5.5–6.0. Maintaining pH within this range requires buffering agents (e.g., phosphate or bicarbonate buffers) or continuous pH monitoring with automated acid/base addition systems. Deviations from the optimal pH can lead to reduced enzyme synthesis, as extreme acidity or alkalinity disrupts cellular homeostasis and denatures enzymes [42].

3.12.2. Temperature

A crucial element is temperature, as various microbial strains have varying thermal preferences. Thermotoga neapolitana and other thermophilic strains of bacteria thrive at 50–60 °C, whereas mesophilic species, including Clostridium acetobutylicum, function best around 30–40 °C. Enzyme synthesis is frequently improved by thermophilic settings because they raise metabolic rates and lower the possibility of contamination from mesophilic rivals. The greater energy inputs needed by thermophilic systems must be weighed against the yield gains. Using bioreactors with water baths or heating/cooling jackets, temperature control is accomplished, guaranteeing steady temperatures during the fermentation process [43].

3.12.3. Substrate Composition

The substrate is a source of carbon and energy for microbial development; it has a direct effect on enzyme synthesis. Simple sugars (glucose, sucrose), starch, and lignocellulosic biomass (such as wood chips and agricultural waste) are common substrates in DF. Although glucose and sucrose are easily fermented and produce significant levels of hydrogenase expression, their price may be too high for large-scale uses. Despite being inexpensive and plentiful, lignocellulosic biomass needs to be pretreated (such as by acid hydrolysis or enzymatic saccharification) in order to liberate fermentable sugars since its complex structure (cellulose, hemicellulose, and lignin) resists direct microbial consumption. The fermentation pathway is also influenced by the makeup of the substrate; in Clostridium, glucose promotes the acetate-butyrate pathway, which increases H₂ generation and hydrogenase activity [44].

3.12.4. Nutrient Availability

Microbes need carbon sources, but they also need nitrogen, phosphorus, and trace elements (such iron and nickel) to synthesize enzymes. The catalytic centers of [FeFe]- and [NiFe]-hydrogenases, respectively, are formed by iron and nickel, making them especially important for hydrogenase synthesis. Microbial growth and enzyme expression are improved by nutrient supplementation, such as the addition of ammonium sulfate for nitrogen or FeSO₄ for iron. But high nutrient levels might cause osmotic stress or the buildup of byproducts (like ammonia), therefore it’s important to carefully optimize nutritional amounts [45].

3.12.5. Genetic Engineering for Enhanced Enzyme Production

In enzyme production and activity, genetic engineering techniques have completely changed the manufacture of enzymatic catalysts. Overexpression of hydrogenase genes, like hox in Enterobacter or hydA in Clostridium, is a typical tactic to increase enzyme levels. To do this, recombinant plasmids with the target gene under the control of potent promoters that guarantee high transcription rates are introduced. Escherichia coli, for instance, has been modified to overexpress Clostridium hydrogenases, which results in noticeably greater H₂ production [46].

To reroute metabolic flow toward the synthesis of H₂, another strategy is to eliminate competing metabolic pathways. For example, Clostridium’s lactate dehydrogenase genes can be deleted to decrease lactate production and increase the amount of reducing equivalents that hydrogenases can access. These changes have been simplified by CRISPR-Cas9 and other gene-editing technologies, enabling accurate changes to microbial genomes [46].

Designing new enzyme variations with enhanced catalytic qualities, including increased substrate affinity or thermal stability, is also made possible by synthetic biology. Under DF circumstances, hydrogenases with higher activity have been produced through directed evolution approaches, which involve randomly mutating enzyme genes and screening them for improved performance. By reintroducing these modified enzymes into microbial hosts, customized biocatalysts for particular uses are produced [46].

3.12.6. Pretreatment of Inocula

A crucial stage in the synthesis of enzymatic catalysts, especially in mixed-culture DF systems, is the pretreatment of microbial inocula. The goal of pretreatments is to decrease unwanted species, including methanogens, which use H₂ as a source of methane, while favoring bacteria that create H₂. and the inoculum (such as anaerobic sludge) is heated to 80–100 °C for 15–30 min in order to induce heat shock. Spore-forming H₂ generators, such as Clostridium, are preserved whereas non-spore-forming methanogens are eliminated. Although heat shock is easy to use and efficient, it may decrease microbial diversity, which could restrict the variety of substrates that can be used [47].

Acid treatment selectively inhibits methanogens and other acid-sensitive microorganisms by exposing the inoculum to a low pH (for example, pH 3.0–4.0) for a number of hours. This technique can be used in conjunction with pH modification throughout fermentation to maintain constant selective pressure and is especially useful for enriching acid-tolerant H₂ producers [48].

Methanogens are specifically targeted by chemical inhibitors like 2-bromoethanesulfonate (BES), which interfere with their metabolic processes. The pretreatment increases the overall enzymatic activity by establishing a microbial community dominated by H₂-producing species, which indirectly increases hydrogenase expression and H₂ yields. However, chemical treatments, although effective, can be expensive and may cause environmental issues, necessitating careful consideration in large-scale applications. The source of the inoculum, the intended microbial makeup, and operational limitations all influence the pretreatment selection [49].

3.12.7. Catalytic Activity and Kinetics

In dark fermentation (DF) for the synthesis of biohydrogen, catalytic activity in hydrogenases is essential, and [Fe-Fe] and [Ni-Fe] hydrogenases operate differently. In DF, glucose is converted to hydrogen by hydrogenases, which produce 2–4 mol H₂ for every mol of glucose. The efficiency of metabolic pathways, particularly the acetate and butyrate pathways that control hydrogen emission, is reflected in this yield [50]. [Fe-Fe] hydrogenases exhibit superior catalytic efficiency, with turnover frequencies (k_cat) reaching approximately 10⁴ s⁻¹, compared to [Ni-Fe] hydrogenases, which range from 10² to 10³ s⁻¹. The higher k_cat of [Fe-Fe] hydrogenases stems from their structural adaptability, enabling faster proton reduction and hydrogen release and the Kinetic studies reveal that hydrogen production is constrained by substrate availability and product inhibition. Glucose, the primary substrate, is metabolized by microorganisms like Clostridium spp., with Michaelis-Menten kinetics indicating K_m values of 1–10 mM. The K_m reflects the enzyme’s affinity for glucose, where lower values suggest efficient substrate binding. Nevertheless, environmental variables like pH and enzyme concentration affect V_max, the maximal reaction rate. While pH variations can lower V_max and limit hydrogen output, optimal pH increases enzyme stability and activity. Kinetics are further complicated by product inhibition by volatile fatty acids (VFAs), such as butyrate and acetate. During fermentation, VFAs build up and compete with substrates for active sites, decreasing catalytic efficiency. However, because of its larger hydrogen output, the acetate pathway (Equation (4)) is kinetically preferred over the butyrate pathway (Equation (5)). Due to the full oxidation of glucose to acetate, which releases additional electrons for proton reduction, the acetate route generates 4 mol H₂ per mol glucose. The butyrate route, on the other hand, reduces available electrons by diverting carbon to butyrate, producing only 2 mol H₂ per mol glucose [51] and under ideal circumstances, metabolic flow favors acetate, demonstrating the preference for the acetate route in Clostridium species. High VFA levels, however, have the potential to change metabolism to butyrate, which would reduce hydrogen yields [51].

Kinetics is strongly influenced by environmental conditions, such as pH and VFA buildup. Acidic environments destabilize hydrogenases, lowering k_cat and εvώ VFAs inhibit enzymes by changing the conformations of their active sites [52] and continuous VFA elimination and pH buffering are two methods to lessen inhibition while maintaining catalytic activity. Furthermore, in low-glucose conditions, substrate availability limits response rates, requiring improved feeding strategies to maintain steady-state kinetics [53].

3.12.8. Catalytic Mechanisms in Dark Fermentation

Hydrogenases and accompanying enzymes catalyze the microbial process known as “dark fermentation” (DF), which produces biohydrogen. Through a sequence of enzymatic processes, this process which is mostly controlled by anaerobic bacteria like Clostridium spp.—transforms organic substrates like glucose into hydrogen gas (H₂). Optimizing DF efficiency requires an understanding of the catalytic mechanisms and their logical sequence [54].

The first step in the process is glycolysis, which produces ATP and NADH by oxidizing glucose to pyruvate. Glycolytic enzymes, not hydrogenases, catalyze this phase, which prepares the way for electron transfer to pathways that produce hydrogen. After that, pyruvate is further metabolized through two important pathways: the butyrate pathway, which produces 2 mol H₂ per mol glucose, or the acetate pathway, which produces 4 mol H₂ per mol glucose. Because there are more electrons available in the acetate pathway, it is preferable for increased H₂ output. However, pyruvate-ferredoxin oxidoreductase (PFOR) reduces ferredoxin by converting pyruvate to acetyl-CoA. Hydrogenases use reduced ferredoxin as an electron source. The H-cluster of [Fe-Fe] hydrogenases, which are predominant in Clostridium, has a di-iron active site that is synchronized by cyanide and carbon monoxide ligands. The distal iron is reduced when ferredoxin electrons enter the H-cluster. When protons (H⁺) attach to this location, a hydride intermediate is created. H₂ is released when a second proton joins the hydride. With turnover frequencies (k_cat) of about 10⁴ s⁻¹, this process is quite effective and is fueled by the quick electron transfer and low redox potential of the H-cluster [29,54].

On the other hand, the nickel-iron active site of [Ni-Fe] hydrogenases, which are present in Enterobacter species, has cysteine ligands. Ferredoxin or NADH electrons cycle between the Ni²⁺ and Ni³⁺ states via reducing the Ni center. Iron is bound by protons, creating a hydride that combines with another proton to generate H₂. Higher redox potentials and slower electron transfer result in a lower k_cat (10²–10³ s⁻¹), which reduces the efficiency of [Ni-Fe] hydrogenases [29].

4. DF–MEC Combination

4.1. Stage I: Dark Fermentation Description

First, complex organic compounds like proteins, lipids, and carbohydrates are broken down into their monomers, which are sugars, amino acids, and long chain fatty acids, by hydrolytic bacteria during dark fermentation (Figure 4). Later, acidogenic bacteria convert these monomers into volatile fatty acids (VFAs), hydrogen dioxide (H₂), and other organic molecules including lactic acid or ethanol. Acetogenic bacteria can then create CO₂, H₂, and acetic acid [55]. The primary process that produces H₂ is the breakdown of carbohydrates into VFAs [56,57,58]. With acetic acid production, the maximum H₂ yield is 4 mol H₂/mol glucose (Equation (4))) while, with butyric acid production in AF, this is 2 mol H₂/mol glucose (Equation (5)).

C₆H₁₂O₆ + 2H₂O → 2CH₃COOH + 2CO₂ + 4H₂

(4)

C₆H₁₂O₆ → 2CH₃ (CH₂)₂ COOH + 2CO₂ + 2H₂

(5)

However, the ultimate result of DF is typically a mixture of volatile organic compounds (VOCs), which is dependent upon the characteristics and content of the substrate as well as the operational parameters that are put in place in the reactors. The best substrates to get a high H₂ yield include dimers like lactose and sucrose, as well as monomers of glucose and xylose [59,60]. However, DF requires cheap substrates to be profitable on a large scale, since sugar-based feedstocks can be employed for other applications that yield greater economic profits. Therefore, other organic leftovers have been investigated as feedstocks in an effort to find alternative appropriate substrates. Because they are high in carbohydrates, food, industrial, and agricultural wastes have received the greatest attention among these alternate substrates. Both substrate in DF and wastewater from agro-sectors, such as the dairy or paper and pulp industries, have been investigated. One of the main elements of plant walls is lignin, which is resistant to microbial deterioration. In fact, pretreatments are frequently used to achieve fermentable sugars (mono- and disaccharides) by disrupting the connections between polysaccharides and lignin complexes. Another material that has been investigated as a possible substrate for DF is activated sludge, which is produced by aerobic wastewater treatment. Since the bulk of the activated sludge is made up of microbial cells, their cell walls must be broken down in order to liberate the intracellular organic material [56,61,62,63,64,65]. Microorganisms can more readily access organic matter through this method. Because VFAs are synthesized concurrently and act as hydrogen scavengers, DF has low hydrogen production yields. Examples of volatile fatty acids (VFAs) are propionic, caproic, (iso)butyric, (iso)valeric, and acetic. Volatile fatty acids (VFAs) find use across various industries such as textile, food preservation, cosmetics, and medicines. Moreover, they can be utilized as a carbon source to remove phosphate and nitrogen from wastewater. They can also be the precursor to the synthesis of medium chain fatty acids (MCFA) and the building block of biodegradable polymers like polyhydroxyalkanoates (PHAs). Because the method is so expensive, recovering and isolating individual volatile fatty acids (VFA) is still difficult. Consequently, it would be advantageous to have a method that could make use of these VFAs without requiring their extraction or purification. Two bioreactors are linked together in a two-stage anaerobic fermentation process. The first bioreactor is designed to produce H₂ and has specific operating conditions that encourage hydrolysis and acidogenesis, while preventing methanogenesis by using a low hydraulic retention time (HRT) or maintaining an acidic pH. The remaining organic matter, which consists mostly of the volatile fuels produced in the initial phase, is then transformed into biogas by operating a second reactor with a longer hydraulic retention time (HRT) and the optimal pH for methanogenic activity. This method causes the biogas to contain more methane and increases the rate at which methane is produced in the second reactor. Utilizing hybrid reactor systems to separate the microbial retention time from the hydraulic retention time (HRT) or adding carriers that can aid in the attachment of methanogens are two more creative methods for producing both hydrogen (H₂) and methane in a single reaction [66,67,68,69,70,71,72].

Despite the feasibility of the two-stage DF-AD method in improving substrate energy recovery, it is important to note that hydrogen (H₂) has a higher energy content than methane, with 141.6 kJ/g compared to 55.5 kJ/g. Consequently, further strategies to enhance the overall generation of H₂ are now being investigated. One example of DF is photofermentation. The limited potential of DF-photo-fermentation systems is constrained by the poor solar conversion rate of the microorganisms involved in this stage, which results in modest energy recoveries. The MECs systems have emerged as an intriguing option in this scenario [73,74,75]. Few previous works on DF given in

Table 4. Biohydrogen production using dark fermentation.

S.No	Inoculum Used	Substrate Used	H2 Production Rate	Reference
1	Cow Dung Compost	Corn Stalk	1.71 L H2/L−d	[78]
2	Digested sludge from wastewater treatment	Cheese Whey	1.54 L H2/L−d	[79]
3	Anaerobic digester sludge	Glucose	1.1 L H2/L−d	[80]
4	Cow dung compost	Pretreated corn stalk	1.7 L H2/L−d	[81]
5	Anaerobic digested sludge	Sugar beet juice	1.91 L H2/L−d	[82]

. Anaerobes that can produce H₂ through DF can be either facultative or obligatory. The most prevalent facultative anaerobes under mesophilic conditions are Enterobacteriaceae, while the obligatory anaerobes are Clostridiaceae. It has been determined that the majority of H₂ generating microbes belong to the genus Clostridium. This genus can manufacture H₂ via a number of metabolic pathways, the most notable of which is the acetate-butyrate pathway, which yields the largest amounts of H₂ from glucose. Inocula mixes are frequently employed to reduce sterility expenses and, more importantly, to guarantee the activity of the inoculum against alterations in substrate composition or environmental conditions by encouraging more biodiversity, even if they produce a substantial amount of hydrogen (H₂) [4,76,77].

4.2. Stage II: Microbial Electrolysis Cell (MEC)

From a conceptual standpoint, MECs are identified as modified MFCs that need to expend a specific amount of energy in order for the anaerobic cathode side to generate hydrogen. An external voltage is frequently supplied thereafter to induce this non-spontaneous response and create a drop in protons at the cathode surface [40,83]. Over the last decade, MECs have been identified for resource recovery and wastewater treatment. The production of hydrogen gas as a renewable energy source has recently gained prominence. The three main components of the system are the anode, cathode, and proton exchange membrane (PEM) (Figure 5). Microorganisms in MECs produce CO₂, protons, and electrons as a consequence of substrate oxidation, which was previously discussed. While electrons traverse a membrane, protons approach the cathode through an electric wire. Protons at the cathode are reduced and converted to H₂ when an electric voltage is applied. In order to overcome the thermodynamic barriers between the anode and cathode, voltage is introduced into MEC. The voltage (0.2–0.8 V) that is applied to the cathode in order to produce hydrogen gas is notably lower in comparison to the voltage (1.8–3.5 V) required for water electrolysis. The ability of carbon substrates (e.g., wastewater, glycerol, acetate, and glucose) to generate H₂ in MECs has been demonstrated. For its favorable MEC performance and susceptibility to degradation by electroactive or electrogenous bacteria, acetate has been employed as the model substrate in MEC [84,85,86,87,88]. The chemical reactions occurring in the MECs are given below:

At anode:

CH₃COOH + 2H₂O → 2CO₂ + 8e⁻ + 8H⁺

(6)

At cathode:

8e⁻ + 8H⁺ → 4H₂

(7)

Overall reaction:

CH₃COOH + 2H₂O → 2CO₂ + 4H₂

(8)

Protons are produced and consumed during redox reactions, as the equations above illustrate. Therefore, a major factor influencing the electrode potential values is the solution’s pH. The equilibrium voltage of the cell can be significantly impacted by phosphorus changes near the anode and cathode. This is because the electrode potential values for both processes protons being reduced at the cathode and acetate serving as an electron donor at the anode are influenced by pH. These pH gradients can be significantly increased by using membranes as separators. As a result of their high diffusivity, membranes are intended to facilitate the movement of H⁺ ions from the anode chamber to the cathode chamber. Moreover, by preventing the dispersion of reagents between the anode and cathode, the membrane enables the production of purified H₂ devoid of impurities such as CO₂, which is generated when organic molecules undergo oxidation at the anode. In contrast to single chamber systems, the substantial internal resistance attenuated by membranes in two chamber microbial electrolysis cell (MEC) systems is a substantial drawback. These membranes obstruct the transit of particular ions, causing the voltage required for hydrogen (H₂) production to increase significantly [4,83]. Few previous works on MECs are given in Table 5. The electroactive bacteria that make up MECs are their primary constituents, and one of the key elements in determining how well the bacteria colonize at the anode is the inoculum. Electroactive bacteria have been cultivated using a range of inoculum sources, including soil, cow excrement, industrial effluent, and municipal wastewater. Bradyrhizobium, Geobacter, Pseudomonas, and Shewanella are the most promising electroactive bacteria because they can respire through a variety of terminal electron acceptors. In addition to rivers, compost, soil, and anaerobic wastewater treatment sludge, soil also contains electroactive microorganisms. Numerous phyla include electroactive bacteria, including Firmicutes (Clostridium), Acidobacteria (Geothrix), and Proteobacteria (Pseudomonas, Shewanella, Geobacter, and Desulfovibrio) [4,89].

Figure 5. Factors affecting performance of DF–MEC system.

Figure 5. Factors affecting performance of DF–MEC system.

Table 5. Biohydrogen production using microbial electrochemical cell (MEC).

S.No	Inoculum Used	Anode	Cathode	H2 Production Rate	Reference
1	Waste water MEC effluent	Graphite felt	Ni-containing electrode	0.54 L H2/L−d	[79]
2	Wastewater	Graphite brush	Carbon cloth with Pt content	1.7 L H2/L−d	[90]
3	Anaerobic Sludge	Graphite felt	Ni-containing electrode	1.46 L H2/L−d	[91]
4	Municipal Wastewater	Graphite cloth	Carbon paper with Pt content	0.07 L H2/L−d	[92]
5	Anaerobic Sludge	Graphite felt	SS Mesh	0.05 L H2/L−d	[93]

Table 5. Biohydrogen production using microbial electrochemical cell (MEC).

S.No	Inoculum Used	Anode	Cathode	H2 Production Rate	Reference
1	Waste water MEC effluent	Graphite felt	Ni-containing electrode	0.54 L H2/L−d	[79]
2	Wastewater	Graphite brush	Carbon cloth with Pt content	1.7 L H2/L−d	[90]
3	Anaerobic Sludge	Graphite felt	Ni-containing electrode	1.46 L H2/L−d	[91]
4	Municipal Wastewater	Graphite cloth	Carbon paper with Pt content	0.07 L H2/L−d	[92]
5	Anaerobic Sludge	Graphite felt	SS Mesh	0.05 L H2/L−d	[93]

4.3. DF–MEC System Coupled for H₂ Production

Due to their high energy requirements for hydrogen generation, current technologies like pyrolysis and hydrocarbon reforming are costly and non-sustainable. Therefore, the most popular way to produce H₂ is by biological means. While DF may degrade wastes and produce significant concentrations of volatile organic compounds (VOCs), its production of hydrogen is not up to par. However, MEC is thought to be a promising technology for producing H₂ because it can consume more substrate than other techniques. Given the complementary properties of DF and MEC, it is anticipated that their further improved linkage will generate significant H₂ production yields [4]. Figure 4 depicts the two-stage procedure for producing hydrogen.

Metal-encapsulated catalysts (MECs) are an innovative technology that can enhance low hydrogen (H₂) production rates and address the treatment of wastewater containing dissolved organic compounds (DF effluents). The relationship between DF and MEC can be stated as follows: DF is utilized to treat wastewater and produce short-chain volatile organic compounds (VOCs), which are then employed by MEC to produce additional H₂. By integrating these two technologies, MECs can obtain cost-effective substrates for microbial oxidation. Indeed, acetate is a prominent component of DF effluents and is well studied as a substrate in MECs. Anaerobic fermentation effluents contain a variety of acids referred to as volatile fatty acids (VFAs), even though there are elevated acetate levels in these effluents. As a result, both the production yields and the processes that give rise to H₂ may differ from Equation (8). Equation (8) illustrates that MECs can potentially release 4 moles of H₂ for every mole of acetate. It’s interesting to note that lactic, propionic, and butyric acids can all serve as electron donors in MEC. Additionally, using volatile fatty acids (VFAs) from fermentation effluents rather than artificial acids has a benefit for electrolyte conductivity, which is a common problem in wastewater-supplied microbial electrolysis cell (MEC) systems. The DF effluent’s natural augmentation of ionic conductivity by the inclusion of extra acids, minerals, and salts removes the need for additional materials [94].

Later section displays previous research demonstrating a higher H₂ yield in a DF-MEC coupled system. Therefore, it can be concluded that there is ample evidence to support the claim that coupled DF-MEC systems generate a greater amount of H₂ compared to single-stage DF or MEC systems. Electroactive bacteria have higher effectiveness in oxidising organic material when volatile organic compounds (VOCs) are derived from complex organic molecules, compared to when complex organic effluents are directly supplied to the MEC. The higher crop production is most likely due to this reason. DF effluent has more organic acids than synthetic or glucose acids, which makes them a more economical and environmentally friendly choice. There are a number of factors that can affect the relationship between DF and MEC, most of them have to do with how differently DF and MEC operate. As a result, the difficulties related to achieving greater H₂ yields with DF-MEC are well covered here [4,94].

5. Factors Affecting Coupled DF–MEC System Performance

A two-stage treatment technique that combines DF and MEC can reduce the thermodynamic barrier to H₂ generation. To achieve an increased H₂ yield from DF-MEC, there are a number of operational and design considerations to consider. The following section discusses the main elements, which include substrates, overpotential, pH, electrode difficulties, and unwanted electron sinks. Factors affecting performance of DF-MEC system can be seen in Figure 4.

5.1. Substrates

The effectiveness and performance of MECs can vary depending on the type of waste. Understanding how DF effluent interacts with MECs is crucial to appreciating this technology’s potential for producing H₂. The efficiency and performance of H₂ production with various wastes have been demonstrated to differ, even though numerous research have combined DF with MEC and employed various waste sources as a secondary treatment. VFAs make up most DF effluents, with acetic and butyric acid being the most prevalent. Because it gives higher results, acetic acid is the preferred organic substrate for H₂ generation when it comes to MECs, rather than butyric. It’s also important to consider how different substrates suit different microorganisms. Li et al. (2014) found that the consumption of butyric acid was only 4–5%, while the consumption of acetic acid was reported to be 80–90% [78]. This suggests the necessity of developing a productive method to oxidize butyric acid to increase the production of hydrogen in a microbial electrolysis cell (MEC). In response to this goal, several tactics have been developed to aid in the adaptation of biofilms. One suggestion is to feed a biofilm with various materials that encourage the growth of the target species [95].

Acetate, butyrate, and fermentation effluent were supplied to an MFC in order to create a microbial anode biofilm. Examining the effects of anode acclimation on the microbial electrolysis cell (MEC) of the maize stalk fermentation effluent was the aim. After the alteration, the biofilm was moved to the MEC. It is imperative to comprehend the characteristics of the waste liquid that is produced when DF is combined with MEC. While DF’s effluent generally contains electroactive bacteria such as VFAs, there are instances when these substances vanish. The performance of MEC may drastically decline under such circumstances. To obtain H₂, Rivera et al. (2015) used wastewater from both natural and artificial sources [92]. The wastewater was treated using a dark fermentation (DF) technique, and the resultant effluent was then added to a microbial electrolysis cell (MEC). Comparing the study’s synthetic wastewater to residential wastewater, the former produced more H₂ (81 mL /H₂ L^−d) than the latter (30 mL H₂ /L^−d). The study found that when VFAs were present in the DF effluent, MEC performed similarly to synthetic wastewater. But it was not as good as that; when the wastewater contained carbs, methane was produced instead of H₂ [82,92].

5.2. Increased Potential at the Electrodes

Anode and cathode potentials are crucial components of MEC for the generation of H₂. However, the current flow in MEC creates an overpotential, which allows potential deviations from equilibrium to occur. Independent adjustments are made to the anode and cathode potentials. Their potential is only influenced by the kind of reactions taking place at that specific electrode and the current density. Therefore, independent research should be done on electrode overpotential. MEC electrodes are another important component that might contribute to possible losses during operation. Thermodynamic analysis of the anode and cathode shows that a minimum of 0.14 V of external energy is required to reduce the thermodynamic potential and start the processes that produce H₂. To start producing H₂, an additional energy input of approximately 0.25 V is necessary to account for any losses that could occur at the electrodes, membranes, and electrolytes during MEC operation [96]. Three different kinds of overpotential can occur at the electrode: activation losses, ohmic losses, and concentration losses [97,98,99].

The rates of mass transport in the substrate significantly influence the decrease in concentration. The electrode surface modulates the substrate concentration by influencing the current flow in the system. Concentration loss is seen when the substrate is restricted. The main causes of concentration loss include imbalanced ratios of oxidized and reduced species, inappropriate substrate mixing, and substrate flow to the biofilm. The deviation of the electrode from its equilibrium position is caused by all these causes. Concentration loss may be an issue in the case of DF-MEC because an imbalance between oxidized and reduced products in the MEC system may result from improperly defined and managed DF effluent.

Bacterial metabolic losses are the primary determinant of ohmic losses. This metric is based on how many electrons are recovered as current compared to how many electrons are accessible in the substrate to produce H₂. The potential differential between the electrode and substrate determines the development of the bacteria. The energy needed to create H₂ likewise drops when the potential difference diminishes, but the bacteria’s energy gain is reduced. Consequently, potential differences ought to be adjusted to facilitate bacterial energy gain while also achieving increased H₂ generation.

The quantity of energy cell needed to overcome the difficulties posed by the redox process is determined by the activation energy. The relationship between anode electrodes and microbes, as well as the constraints on electron transport from the bacteria to the electrode, are mostly explained by activation overpotential. Enhancing H₂ generation requires the use of an electrode with a large surface area for reaction and a high catalytic capacity. Overpotential issues can be avoided in part by employing a low-cost anode, such as a carbon-based electrode, that can efficiently transfer electrons from bacteria to the electrodes. Larger catalytic cathode electrodes can lower the system’s internal resistance and overpotential, but the cathode is always a crucial part of MECs.

5.3. pH

While electroactive microorganisms can only thrive in MEC systems when the pH is between neutral and 6.5, methanogen growth can be prevented by running DF at a pH of between 5.0 and 6.5. The anode gets more acidic, and the cathode becomes more alkaline during H₂ production in MECs with restricted electrolyte conductivities. Consequently, at high pH levels, certain cation-related deposits, such calcium and magnesium, may accumulate at the cathode. Geobacter or Shewanella are the most common exoelectrogen bacteria that inoculate the biofilm at the anode. Because their metabolisms function best in neutral pH environments, many bacteria are sensitive to low pH media. Furthermore, low pH can affect the efficiency of H₂ generation by deactivating the hydrogenase enzyme. Instead of using non-adapted microbiota as the inoculum in MEC, exoelectrogen microorganisms produced from DF could be employed to get around the low pH-related problems of the bioanodes. Combining DF and MEC may result in the best possible microbiome because DF is where most acidogenic microbial systems grow. This indicates that the microbes have evolved to function at a pH that is somewhat lower. It appears plausible that the fermentation effluent’s high level of organic materials can block the electroactive bacteria. Consequently, it has been determined that the dilution of the DF effluent is necessary to increase the production of H₂ by preventing excessive organic content. Hence, if the appropriate concentration and composition are not thoroughly examined, DF effluents may pose challenges when used as a substrate in MEC [100,101,102].

5.4. Electrode Difficulties

Hydrogen evolution process (HER) at the electrode surface is triggered by the reduction of protons or water. Either electroactive microorganisms (biotic cathode) or inorganic catalysts (abiotic cathode) can induce HER at the cathode. At an approximate equilibrium potential of -0.41 V or pH level close to neutral, the process in MECs takes place. Both carbon-based materials and stainless steel are considered to have low reactivity for hydrogen evolution processes (HER) at the cathode. Consequently, cathode electrodes made of platinum are commonly used because they have a low demand for overpotential in the synthesis of hydrogen. Under neutral pH conditions, the platinum electrode experiences a decline in its catalytic activity. Furthermore, platinum is an expensive catalyst that, when removed, pollutes the environment severely and is quickly rendered worthless by sulphide poisoning. Consequently, a variety of electrode materials, including nickel, molybdenum, and stainless steel with a high concentration of nickel, have been used as the cathode electrode [103,104].

Metals and materials based on carbon are the conventional materials utilized as microbial anodes. Due to their inexpensiveness, chemical stability, and biocompatibility, carbon-based materials have been utilized extensively. Carbon and graphite felt as well as fabric are examples of such substances. Carbon and graphite felts are renowned within the carbon-based anode community for possessing a denser and more permeable composition in comparison to carbon cloth anodes. When compared to metal anodes, carbon-based electrodes exhibit a reduced conductivity. Despite their increased susceptibility to corrosion, metal anodes have been employed in MECs. As the anode’s surface area and porosity rise, so does the surface area that is open to microbial adhesion. Better contact with electroactive bacteria is now possible due to enhanced metal anode surface volume ratios resulting from the creation of mesh structures. Logan et al. (2007) created brush anodes, a unique design [105]. A metal conductor is interconnected with carbon or graphite fibers in the central region of the structure. These anodes, which were first used in MFCs, have become more and more well-liked in laboratory and pilot-scale applications because they combine the benefits of metal- and carbon-based electrodes with a high surface-to-volume ratio, superior conductivity, and low resistance [106,107,108].

The potential application of microorganisms as cathode catalysts has also been studied recently, much like bioanodes. The self-generation potential and affordability of microbial cathodes, also known as bio-cathodes, are their primary benefits. Specific electroactive microorganisms can extract electrons from the cathode to initiate hydrogen evolution processes, catalyzing the reduction reaction. Biocathode-associated microbiological communities may include bacteria with hydrogenases or c-type cytochromes on their outer membranes, which promote HER and electron transport. These traits have been seen in Desulfovibrio sp., a sulphate-reducing bacterium. In general, there is less research on the biocathode than there is on the bioanode. To fully capitalise on the manifold opportunities presented by these microbial systems, considerable effort should be devoted to the development of comprehensive biological MECs. The technology for accelerating the HER by ensuring permanent biofilm adhesion to the electrode surface is still in its infancy [105,109,110,111,112].

5.5. Unwanted Electron Sinks

Most people agree that the current density of MECs accurately represents their capacity to produce hydrogen. High H₂ generation yield is generally connected with high current density. Because there are other electron acceptors besides the electrodes, this isn’t always the case. The created H₂ can also be converted to methane by archaea, just like in traditional anaerobic bioprocesses. The presence of methanogenic archaea in DF effluent can lead to a decrease in H₂ generation over time in DF-MFC due to the prevalence of methanogenesis. Instead of producing H₂, that results in the generation of methane. When a single chamber MEC has no separator in place, methanogenesis typically occurs. According to reports, in low acetate environments like MECs, acetoclastic methanogens outcompete electroactive bacteria that oxidize acetate [113].

Methanogenic competition occurs when methanogens that are hydrogenotrophic consume the H₂, whereas acetoclastic methanogens consume the acetate. The presence of hydrogenotrophic methanogens could be problematic due to the fact that DF-MEC requires 4 moles of H₂ to produce 1 mole of methane. Methanogenesis is less of an issue in double chamber MEC since separators are used to limit the dispersion of H₂. To circumvent these competing microorganisms in single chamber MECs, a variety of chemical and physical techniques for inoculum pretreatment have been suggested. Chemical procedures involve the utilization of alkaline and acidic solutions, in addition to the incorporation of inhibitory compounds such as acetylene, chloroform, iodopropane, and 2-bromo ethanesulfonic acid (BESA). O₂ exposure is an additional method for reducing methanogenesis; however, prolonged O₂ exposure can also diminish the activity of electroactive bacteria [114].

In stirred tank reactors, low retention times (RTs) can be employed to lower the methanogenic archaea population, provided that the solids retention time (SRT) and hydraulic retention time (HRT) are equal. Methanogenic archaea have been induced to wash away by using the fact that they develop more slowly than acidogenic bacteria. Heat shock treatment, chemical pretreatment, or aeration of the inoculum are all viable pretreatments that can be applied to DF in order to eliminate methanogens responsible for H₂ production [115]. The best course of action for reducing the difficulties posed by electroactive bacteria is to extract H₂ from MEC more quickly rather than storing it in the cells for a longer period of time [116,117,118].

6. Current Major Research in the Field of Coupled DF–MEC

DF effluent molded by process pH has an impact on MEC performance. In this regard, the MEC favored consuming acetate since it was more abundant at pH 7, but because this component was absent and butyrate was significantly more available at pH 5.5, the MEC produced lower hydrogen yields. The assessment of the DF effluents in MECs showed that the pH 7 effluents eliminated more volatile fatty acids (VFAs) (84–95%) and produced more hydrogen (568–723 mL H₂/g COD_in) than the pH 5.5 effluents (175–188 mL H₂/g COD_in and 29–59%). Varanasi and Das achieved a hydrogen yield of 67.7 L of hydrogen per kilogram of chemical oxygen demand consumed in 2020 by employing a two-stage DF-MEC approach. The yields from the single-stage DF and MEC procedures were 41.4 and 42.9 L H₂/kg COD_consumed, respectively. When DF and MEC were utilized individually, the net energy recovery rates were 8.2% and 7.4% respectively. However, when they were combined, the rate increased significantly to 67.7%. In 2017, Marone et al. 2017 investigated the use of coupled DF-MEC to boost production of biohydrogen from agro-industrial waste and wastewater [119]. Two phases of assessment were conducted to determine the viability of manufacturing hydrogen from six different wastewaters and industrial by-products from plants that produce cheese, fruit juice, paper, sugar, processed fruit, and spirits. The total amount of hydrogen produced was increased up to 13 times greater when dark fermentation and microbial electrolysis were coupled than when fermentation was done alone. An overall hydrogen generation of 1609.5 ± 266.5 mLH₂/gCOD_consumed and a maximum COD elimination of 78.3 ± 5.6% were attained.

7. Challenges of MEC–DF Integration

Despite the benefits of MEC-DF, it still encounters certain problems that must be addressed to maintain its long-term viability in industrial settings. Initially, the initiation phase of the MEC-DF start-up typically requires a duration ranging from several weeks to several months, hence imposing a constraint on the feasibility of implementing industrial-scale applications. Hence, it is imperative to validate a viable approach to expedite the initiation of MEC-DF for ensuring the long-term viability of this application. The selection of cathodic catalysts and electrode materials that meet the requirements of the MEC-DF integrated system is an additional significant barrier to its widespread adoption. Electrodes comprised of carbon-based materials are widely employed in numerous applications. When the cathodic potential becomes excessively negative, however, these electrodes are susceptible to corrosion, significant voltage loss, and high overpotentials. Moreover, the long-term build-up of surface impurities and the quick onset of corrosion brought on by the intricate makeup of the material used in the MEC-DF worsen the degradation of electrode materials. Therefore, it is essential to create a new electrode with exceptional catalytic activity, conductivity, stability, affordability, long-lasting durability, and corrosion resistance in order to ensure its dependable deployment. To overcome the thermodynamic barrier and lower overpotentials to the optimal level and achieve significant electrosynthesis efficiency, platinum is frequently used as a catalyst in MEC. Nevertheless, this economic application is not suitable for industrialization due to its exorbitant cost and lack of cost-effectiveness. Additionally, its usage is limited to a specific timeframe. Alternatively, it is crucial to focus on exploring the viability of utilizing a biological cathode that solely relies on microbial cells as a biocatalyst. The utilization of electroactive microorganisms in the microbial biocathode has several advantages compared to metal-based cathodes, including cost-effectiveness, regular rejuvenation, and immunity to corrosion. Nevertheless, the process of developing and maturing the microbial biofilm remains intricate and time-consuming. Consequently, there is a pressing need for straightforward and efficient methods to promote long-term biofilm formation in the MEC-DF system. Another challenge is that a number of factors, including the origin of the original microorganisms, the make-up of the feedstock, the materials utilized to make the electrodes, and the operating environment, might influence how effective the biofilm’s catalytic activity is. These issues hinder the practical application of MEC-DF in industrial settings. The investigators must exercise meticulousness in order to ensure the efficacy and uninterrupted biocatalytic activity on a wide scale [14].

8. Techno-Economic Analysis of DF–MEC

8.1. TEA–LCA

As a sustainable and cutting-edge energy source, hydrogen is gradually replacing fossil fuels, which are major contributors to greenhouse gas emissions. Empirical studies have demonstrated that the intricate interaction of thermochemical and biological processes is necessary for the synthesis of biohydrogen from biomass feedstocks. A reliable technique for assessing the viability of biohydrogen generation systems from an economic standpoint is techno-economic analysis (TEA). Pyrolysis and gasification are two essential thermochemical conversion processes that turn carbon-rich feedstocks into useful products including biochar, bio-oil, syngas, and biohydrogen. Lignocellulosic biomass, food waste, animal waste, municipal solid waste, agricultural residues, and plastic waste are just a few of the varied types of feedstocks that fall under this category. By calculating the overall costs of turning waste feedstocks into biohydrogen, TEA makes it easier to conduct a thorough economic analysis of biohydrogen production technology. Critical economic metrics, including operating costs and capital expenditure, are employed to determine profitability and the minimum selling price of biohydrogen. Capital expenditure encompasses costs related to the establishment, procurement, and overall development of the production facility [120,121].

In accordance with international environmental standards and energy laws, economic and environmental indicators are measured by allocating monetary values to different units. These units take into consideration the management of waste disposal and the replacement of fossil fuels. Historically, around 98% of the hydrogen produced worldwide has come from the gasification of coal and other fossil fuels, and this process is well known for being economically feasible. However, due to worries about climate change and global warming, biomass has become the preferred feedstock for gasification in recent years, replacing fossil fuels [120,121]. Gasification, pyrolysis, solid-state fermentation, dark fermentation (DF), photo-fermentation (PF), and microbial electrolysis cells (MEC) are the main processes used to produce biohydrogen. Applying TEA to biomass feedstocks is a novel way to evaluate these technologies’ potential for both immediate and long-term commercialization. Figure 6 shows a life cycle assessment (LCA) block diagram for the production of biohydrogen.

High H₂ generation can be achieved with the coupling of MEC into DF because to the significant cost reductions. Less energy is required for MEC because of the biocatalysis that is brought forth by the anode’s biofilm development. Since DF can manufacture the carbon source (acetic acid), which is required in MEC as a substrate, the operational cost of DF with MEC decreases even further. By lowering overpotentials, the DF substrate also improves MEC’s conductivity, which eventually lowers the price of high-performing electrodes. Electrodes utilized in MEC come with a hefty price tag, to start. A total of 94% of the MEC building expenses are related to the anode and cathode. It is, on the other hand, less energy-intensive to create H₂ when bacteria are present near the anode. Anode substrate oxidation accounts for about 35 percent of the total energy, This technical breakthrough has a favorable impact on the environment in addition to the profitability of the process [122,123,124,125,126].

The cost per kilogram of COD utilized for wastewater treatment in MEC is theoretically estimated to be 0.21 USD for H₂ production. According to Li et al. (2012), producing hydrogen (H₂) using dark fermentation (DF) with agricultural waste and beverage effluent has considerable financial benefits [125]. The research indicates that there is potential for commercializing biohydrogen production, with estimated annual revenues of up to $2,001,000 for agricultural waste and $2,658,000 for beverage effluent. When DF and MEC are combined, H₂ production can be produced at the lowest possible cost as opposed to when DF and MEC. According to their estimates, producing 1 L H₂/g of organic waste would cost 2.8 USD/g. The MEC’s design has a huge impact on the cost of manufacturing H₂ on a large scale. According to a thorough MEC study, each MEC unit will cost 1186 USD per m³ to treat wastewater and produce H₂, assuming a COD input of 17.5 kg m⁻³ d⁻¹. Long-term application of the technology is also essential for preserving economic competitiveness and a stable energy balance. Most of the time, the efficiency of microbial electrolysis cells in generating hydrogen decreases gradually because of reasons like reduced electroactive bacterial activity, clogging, membrane fouling, and cathode failure or corrosion. To ensure the system operates for a long period, it is crucial to use biocompatible materials and monitor the growth and behavior of electroactive bacteria in the system [122,123,124,125,126].

8.2. Cost Analysis

The reactor architecture and microalgae growing system have a major role in determining the cost of producing biohydrogen. For instance, growing biohydrogen in a 14-hectare photobioreactor or a 140-hectare open pond is required to produce it using a biophotolysis system. The open pond costs $6 per square meter, while the photobioreactor costs $100. In addition, there are $10 million in yearly operating expenditures and a $43 million capital cost. As a result, $10 per gigajoule is the manufacturing cost. About 90% of the total cost was allocated to the capital investment, with a 25% yearly capital charge. With a 90% operating capacity, the system aims to produce 1200 TJ of biohydrogen per year. However, the costs of managing and storing hydrogen were not included in the full estimate, even though they are significant cost components that can amount to around $0.58 million [127]. James et al. (2009) attempted to calculate the cost of producing biohydrogen using oxygen-tolerant microalgae and found that it was $1.38 kg⁻¹, without including storage expenses [128]. The photobioreactor’s operation accounted for 42% of the total expenses, while biohydrogen collecting resources accounted for 34% and recycling and circulation systems for 13%. Additionally, the microalgae feed assembly accounted for 4% of the overall costs as shown in Figure 7, with consumables and other control systems accounting for the remainder sums. Storage serves as a major cost limitation, making it an important consideration. It is recommended to raise the conversion efficiency to 13.4% and the biomass generation to 9.4% in order to guarantee cost-effectiveness in direct bio-photolysis.

When examining the economic viability of producing biohydrogen using the newest technology, there is ambiguity because most cost studies were finished more than ten years ago. Moreover, many of the recent research concerning the economic aspects of biohydrogen present a very optimistic picture. Show et al. (2019) calculated that the cost of manufacturing one GJ of biohydrogen is approximately $10–$20 [129]. Nevertheless, biohydrogen’s production costs are too expensive to make it a competitive and feasible replacement for gasoline, which costs $0.33 GJ−1. Subsequent investigations ought to concentrate on doing comprehensive evaluations of the feasibility and functionality of the facilities, in addition to examining the financial ramifications of the materials and operations, the influence of weather and light, the need for land, the mixing of biomass, and stability. Recycling biomass and residual metabolites can improve viability at a lower cost.

9. Circular Economy

Population expansion and urbanization have driven global energy consumption exponentially. Conventional fossil fuels cannot meet this demand, causing price rises and significant environmental damage from greenhouse gas emissions. Hydrogen has high energy density and purity. Biological approaches have gained appeal because of the high cost, high energy consumption, and negative environmental impact of present hydrogen generating systems. Utilizing dark fermentation and photosynthesis are the primary biological methods for producing hydrogen from organic materials. Anaerobic fermentation of carbohydrates-rich substances in the absence of light or oxygen results in the production of acids. The economic analysis of hydrogen generating shows that current H₂ synthesis methods outperform conventional fuel production. H₂ generation may be made cheaper by reducing photobioreactor costs and improving storage. Biological H₂ technology research and development must be constructive. Biohydrogen generation must be economically viable for a circular economy and sustainable development.

Utilizing a variety of waste sources, including sewage, dairy, poultry farm waste, industrial waste, and agricultural waste (especially lignocellulosic biomass), biological processes including dark and photofermentation generate hydrogen gas (H₂). Compared to chemical methods, fermentation processes provide specific advantages. However, these methods are beset by low H₂ output and slow production rates. The combination of dark and photofermentation yields a controllable 8 mol H₂ per mole of glucose, which can assist alleviate this problem. The waste product from dark fermentation is the first substrate for photofermentation; it is a two-step sequential process. The necessary procedures for preparing the dark fermentation effluent include altering the concentration, optimising the nutritional composition, adjusting pH, and modifying the amounts of volatile fatty acids and NH₄⁺. These measures are crucial before proceeding with photofermentation. Large amounts of separator/pretreatment equipment and fermenter units are required for the successive dark and photo fermentation processes. To enhance the economic viability of H₂ production, potential options include reducing the expenses associated with photobioreactor technology and implementing an efficient storage system. To guarantee the financial viability of H₂ production methods, a focus on biological technique research and development is necessary. To improve quality of life, society ultimately requires a favorable and nurturing atmosphere.

10. Integrating Dark Fermentation with Other Processes

10.1. Anaerobic Digestion

The process of decomposing organic waste in the absence of oxygen is called anaerobic digestion, or AD. A collection of microorganisms known as digestate, which is rich in nutrients, and biogas, which is mostly composed of CH₄ and CO₂, are responsible for this. Biogas is a very energy-dense material that can be used to produce heat and electricity. Digestate from the byproduct of digestion is a helpful biofertilizer. The four steps of anaerobic digestion are acetogenesis, methanogenesis, hydrolysis, and acidogenesis. Both facultative and obligatory anaerobes break down complex chemicals in the first stage into soluble molecules like glucose and amino acids. The subsequent breakdown of these substances by acidogens results in the production of alcohols, acetic acids, short-chain fatty acids, H₂, and CO₂ in the second step. Further breakdown of the VFAs occurs during the acetogenesis stage of digestion, producing acetic acids, H₂, and CO₂. The fourth step, called methanogenesis, is when acetotrophic or acetoclastic methanogens transform the acetic acids into methane. At the same time, H₂ is turned into methane by hydrogenotrophic methanogens as shown in Figure 8 [130].

It is possible to use the waste products from the DF process as substrates for the AD process. While the DF process can produce acetic acid, which can proceed through the methanogenic phase of the AD process to produce biomethane, butyric acid or ethanol produced from the DF process can proceed through acetogenesis prior to methanogenesis. Biogas and bioenergy are produced in significantly greater quantities when the DF process is coupled with the AD process than when the DF method is utilized alone. Thus, the integration of the DF with the AD process increases the DF’s potential for widespread adoption. The two-stage method yields biohydrogen and biomethane, which can be combined to create bio-hythane, which is a better fuel than either H₂ or CH₄. The anaerobic digestion (AD) process not only improves energy recovery but also contributes to the stabilization of wastewater generated by the dry fermentation (DF) process. Organic matter is converted into biogas more quickly, hence the effluent from the two-stage process contains much less organic matter. Consequently, a more robust end product is produced that can be applied as an organic fertilizer [131].

When combining DF with AD or any other system, the primary difficulty lies in effectively utilizing two or three bioreactors to manage the multiple ongoing activities. Consequently, this causes the costs related to large-scale investments to increase significantly. Furthermore, compared to a single-stage process, a two-stage process is typically more sophisticated and requires higher operating and maintenance expenses. Furthermore, a neutralization phase needs to be completed before the DF effluent is exposed to the AD process due to the high quantities of volatile fatty acids and its acidic pH. Furthermore, a significant amount of alkali might be required, which would result in a digestate that is extremely concentrated in salts and has less potential to be used as a biofertilizer in the future. Another challenge is that substantial concentrations of specific substances, including metal ions and ammonia, in the DF effluent may prevent methanogen activity in the second stage [130,131].

10.2. Photo-Fermentation

Phototrophic fermentation, or PF for short, is the biochemical process through which the metabolic activity of photosynthetic bacteria converts organic materials into hydrogen gas (H₂) and carbon dioxide (CO₂). This phenomenon occurs under specific conditions: light is present, but the atmosphere is devoid of nitrogen and oxygen. As a kind of photosynthetic bacteria, purple non-sulfur bacteria have the ability to create ATP by utilizing light energy and organic acids as electron sources. During this process, the electrons are guided to nitrogenase by Fd. The nitrogenase enzyme uses ATP to catalyze the reduction of protons in an environment lacking in nitrogen, producing H₂. The effluent from the dark fermentation (DF) process, which contains a variety of volatile fatty acids (VFA), is a suitable substrate for the photosynthesis (PF) process because photosynthetic bacteria may use organic acids as electron donors to create H₂. By employing the interconnected DF/PF processes, bioenergy production is increased, which raises the efficiency of energy conversion or energy recovery. The primary disadvantage of photobioreactors (PF) is their need for light and their ability to penetrate light, which often leads to more complex reactor designs. This problem is made even more difficult to deal with on a pilot to large scale because of the increased reactor capacities, which in turn makes light penetration more challenging. It appears that attempts to integrate the DF and PF processes in order to remedy this issue have not been successful. Alternatively, in order to improve light penetration, the discharge from the DF must be diluted before being utilized as substrates for the PF. Therefore, the widespread application of DF/PF as a two-step process will necessitate an additional intermediate step (for dilution), which will raise investment costs once more. The fact that the DF effluent contains significant levels of VFAs and NH4+, which may interfere with the PF process, is another drawback of the combined DF/PF process [120,130].

10.3. Microbial Fuel Cell

At the anode of a microbial fuel cell (MFC), organic substrates can be converted by microorganisms into CO₂, protons, and electrons in anaerobic conditions—an environment devoid of oxygen. A metabolite found in DF effluent termed acetic acid undergoes oxidation, leading to the production of protons, electrons, and carbon dioxide. Electricity is produced as a result of the electrons traveling to the cathode via an external circuit. Simultaneously, protons pass through a semi-permeable membrane to the cathode, where they mix with oxygen to create water. The waste product generated by the DF process can assist the MFC in producing bio-energy, which can then be utilized to generate bio-electricity [119].

MFCs function in a neutral environment and, like the AD process, require a neutralization phase before treating the DF effluent in the MFC. Consequently, a substantial volume of alkali is needed on a big scale, and the MFC waste is heavily polluted with salts, necessitating additional treatment before disposal. Reduced cathode and cathode catalyst prices, along with excellent efficiency levels, are prerequisites for the widespread adoption of MFC. Moreover, prior to the extensive application of MFCs (Microbial Fuel Cells), it is critical to minimize the ohmic voltage losses as well as the pH gradients that arise from the use of semi-permeable membranes. As was already noted, there are currently significant challenges in carrying out two-stage processes on a wide scale. Nevertheless, these problems are made worse when three or more phases are taken into account, largely because of higher capital costs and other related variables [4].

11. Future Perspective

The combination of DF-MEC has enormous potential for producing H₂ while consuming the least amount of energy and reducing pollutants in the environment. To improve the performance of DF- MEC, the currently available research indicates that a few crucial factors must be considered. One of those elements has to do with designing the DF so that the ideal distribution profile and inoculum and VFA concentrations are obtained for the right MEC performance. The key elements derived from DF in the linked DF-MEC system that contribute to enhanced H₂ production are the inoculum, substrate, and conductivity. High conductivity allows for the reduction of resistance and charge transfer rate in MEC, which promotes effective charge transfer.

The contamination of H₂ with other gases is the other main problem with DF-MEC. The arrangement of MECs and the selection of microorganisms are also critical in minimizing this issue. Acidogens and methanogens, two types of bacteria that are not electroactive, can negatively impact the production of H₂. Apart from the microbiological components, the future development of this technology could also be influenced by the configuration of the microbial electrolyzes’ cell architecture. These systems are right now the focus of basic research on a laboratory scale. However, these developments must proceed to a scale where they can be used, e.g., flow reactors, where the primary reactions, particularly those involving bacteria, can occur effectively. In order to deliver an energy efficient cell performance, MEC’s study area will need to increase with the ongoing emergence of different cell shapes combined with either double chamber or single chamber. These cell structures include, for example, adaptive flow manifolds, flow-by designs, and flow-through designs [132].

12. Conclusions

Combining dark fermentation (DF) and microbial electrolysis cells (MECs) provides a revolutionary method of producing biohydrogen by utilizing DF’s capacity to decompose complex organic substrates into volatile fatty acids (VFAs), which MECs then transform into high-yield hydrogen, outperforming DF or MEC systems alone. By overcoming thermodynamic and kinetic limitations, enzymatic catalysts such as [Fe-Fe]- and [Ni-Fe]-hydrogenases, in conjunction with electroactive microbial consortia, can achieve up to 13 times higher hydrogen yields and significantly reduce COD from waste streams and the Scalability is hampered by issues such methanogenic archaea competition, expensive and corrosive electrodes, and the lengthy microbial biofilm formation. With future developments in biofilm optimization and affordable electrodes, DF-MEC systems will be positioned as a sustainable solution for decarbonized energy production. This is made possible by advancements in electrode materials, microbial pretreatment, and reactor design, as well as techno-economic benefits from waste utilization.