Biological Routes for Biohydrogen Production: A Clean and Carbon‐Free Fuel

ABSTRACT

Hydrogen (H₂) is a clean, renewable, and sustainable energy source that holds great promise as an alternative fuel and is expected to play a central role in the future transportation energy economy. However, the hydrogen yield from microorganisms remains insufficient, presenting a significant challenge. Biohydrogen (bio‐H₂) production pathways are well established and can be categorized into four main processes: (1) direct biological photolysis of water by green algae; (2) indirect biological photolysis by cyanobacteria, a combination of green algae and photosynthetic microorganisms, or a separate two‐stage photolysis using only green algae; (3) photo‐fermentation by purple bacteria, photosynthetic bacteria, or fermentative bacteria; and (4) dark anaerobic fermentation by fermentative bacteria. Among these processes, dark fermentation shows great potential for practical applications, such as organic waste treatment. This review summarizes recent advances in bio‐H₂ production, including both fundamental research and applied studies.

Graphical Abstract and Lay Summary

Biological routes for hydrogen (H₂) production are mainly classified into four pathways: 1) direct bio‐photolysis, 2) indirect bio‐photolysis, 3) photo‐fermentation, and 4) dark fermentation. Of these pathways, dark fermentation produces the highest levels of H₂ production. For this reason, research for improve H₂ production has been primarily focused on manipulating the dark fermentation pathway using enzyme engineering, metabolic engineering, and process engineering.

1. Introduction

Fossil fuels pose a continuous and serious threat to the environment, contributing to the greenhouse effect, global climate change, and fine dust pollution. Additionally, fossil fuel reserves are rapidly depleting and will soon be exhausted [1, 2]. Biofuels, such as bioethanol, biodiesel, and biohydrogen (bio‐H₂), offer promising alternatives that can help mitigate these environmental issues. Biofuels have evolved through four generations (Table 1). First‐generation biofuels are derived from food crops. Second‐generation biofuels are produced from non‐food crops or waste materials. Third‐generation biofuels are generated using microorganisms. The most recent, fourth‐generation biofuels utilize genetic engineering to enhance biological systems for improved biofuel production [3]. More details on each generation are provided in Table 1. Biofuel technology, which enables hydrogen (H₂) production from water, organic waste, and biomass using solar energy and microorganisms, is gaining attention as an environmentally friendly energy solution. Bio‐H₂, particularly, is a clean and renewable alternative to fossil fuels, especially for transportation, and can be produced from various sources of plant‐based biomass [4, 5].

TABLE 1.

Description of biofuel generations, their substrates, biofuels, advantages, and disadvantages.

Generation	Substrates	Biofuels	Advantages	Disadvantages
1st Generation	Edible food sources (grains, seeds, maize, sugarcane, soybean, etc.)	Bioethanol or biobutanol (microbial fermentation)	‐Simple production process with minimal pretreatment requirements ‐Cost‐effective production	‐Utilization of food crops (e.g., sugarcane, edible oils) for energy production may reduce their availability for food consumption
2nd Generation	Lignocellulosic biomass, including industrial/agricultural wastes (wood, organic residues, etc.)	Bioethanol or biobutanol (enzymatic hydrolysis) Biomethanol (anaerobic digestion)	‐Reduction of greenhouse gas emissions ‐Renewable energy source ‐Highly compatible with internal combustion engines ‐Primarily used in the transportation sector	‐Release of nitrates, which contribute to acid rain ‐Requires extensive pretreatment
3rd Generation	Algae, Seaweeds, CO2, light energy	Biodiesel (from algae) Biohydrogen (from green algae/microorganisms)	‐Direct CO2 capture ‐Reduced freshwater consumption ‐High efficiency ‐Can grow in diverse environmental conditions	‐High downstream processing costs ‐Still in the developmental stage
4th Generation	Genetically modified carbon‐negative crops and genetically engineered microorganisms (including microalgae, yeast, fungi, and cyanobacteria) that photosynthesize CO2 into fuel		‐Potential to significantly reduce greenhouse gas emissions beyond 2nd generation biofuels	‐Still under development

In South Korea, the government has set ambitious targets to expand hydrogen usage from the current 220,000 tons to 3.9 million tons by 2030 and 27.9 million tons by 2050. The share of clean hydrogen is also expected to rise to 50% by 2030 and 100% by 2050 [6]. Globally, the H₂ market is valued at approximately USD 242.7 billion in 2023 and is projected to reach USD 410.6 billion by 2030, with a compound annual growth rate (CAGR) of 7.8% from 2023 to 2030 [7].

Interest in and utilization of H₂ are rapidly increasing due to the following advantages of bio‐H₂: (1) Bio‐H₂ is readily available, as hydrogen is a fundamental element on Earth and an abundant, clean energy source [8]. (2) When used as fuel, it does not emit CO₂, a greenhouse gas [9]. (3) Bio‐H₂ is non‐toxic and safe, unlike coal, nuclear power, and gasoline, which are associated with toxic or hazardous environments [10, 11]. (4) H₂ is more efficient at generating electricity than other energy sources. For instance, traditional combustion‐based power plants typically achieve an efficiency of 33%–35%, whereas hydrogen fuel cells can reach up to 65% efficiency [12]. Due to these advantages, H₂ has the potential to become a major chemical energy carrier and should be utilized as a high‐energy storage medium in transportation vehicles.

Microorganisms that produce hydrogen are broadly classified into fermentative bacteria, photosynthetic bacteria, cyanobacteria, and green microalgae [13]. Photosynthetic bacteria are further categorized into three families: Rhodospirillaceae, Chromatiaceae, and Chlorobiaceae, commonly referred to as purple non‐sulfur bacteria, purple sulfur bacteria, and green sulfur bacteria, respectively [14]. Algae are divided into two groups: green algae and blue‐green algae (cyanobacteria), with Chlamydomonas reinhardtii being the most extensively studied green algal strain for hydrogen production [15]. Anaerobic bacteria are also classified into two types: strict anaerobes and facultative anaerobes.

Bio‐H₂ production utilizes water, organic substances, and gases as substrates, with various technologies available based on different microbial mechanisms. Research on new technologies and hydrogen‐producing microorganisms remains active. As illustrated in Figure 1, there are four representative metabolic pathways for hydrogen production: (1) Direct bio‐photolysis, a hydrogen production process in which green algae generate hydrogen by supplying protons and electrons from water through photosynthesis [16]. (2) Indirect bio‐photolysis, a process that decomposes water to generate oxygen via a synthetic reaction while simultaneously fixing atmospheric carbon dioxide into polymer storage materials within the cell. Hydrogen is then produced through anaerobic or photosynthetic fermentation. (3) Photo‐fermentation, a process in which red bacteria, under anaerobic culture conditions and in the presence of light, use organic matter as a substrate to produce hydrogen [17]. (4) Dark fermentation (anaerobic fermentation), a process in which anaerobic microorganisms ferment organic substrates in the absence of light, producing hydrogen and organic acids [18, 19].

FIGURE 1.

Four major biohydrogen‐producing processes.

In this review, we discuss various bio‐H₂ production pathways. Chapter 1 introduces hydrogen production and its potential pathways, Chapter 2 details direct and indirect photolysis, Chapter 3 focuses on photo‐fermentation, and Chapter 4 provides an in‐depth discussion of dark fermentation. Finally, Chapter 5 summarizes the different H₂ production pathways and explores future directions for achieving satisfactory H₂ yields.

2. Bio‐Photolysis

Some microorganisms, particularly green algae and cyanobacteria, undergo two biological processes: bio‐photosynthesis and bio‐photolysis. These processes are essential for microbial energy production and growth. Photosynthesis converts solar energy into chemical energy in the form of carbohydrates while releasing oxygen as a byproduct [20]. Microorganisms utilize glucose (a carbohydrate) as an energy source for their metabolic activities. Under specific conditions, such as anaerobic environments, these microorganisms can also harness solar energy to split water molecules into hydrogen ions (protons) and oxygen through photosystem II (PSII). PSII absorbs light in the form of photons and generates strong oxidants, enabling the direct cleavage of water molecules [21]. This biological process, known as bio‐photolysis, supplies electrons to the photosynthetic electron transport chain and occurs via two distinct mechanisms: direct and indirect bio‐photolysis (Figure 2A,B) [22].

FIGURE 2.

Overview of hydrogen production through microorganism‐mediated photolysis. (A) Direct photolysis process for hydrogen production. (B) Indirect photolysis process for hydrogen production.

2.1. Direct Bio‐Photolysis

Research on microbial H₂ production began in the late 19th century, with early studies focusing on algae and bacteria. In 1886, Jackson and Ellmes isolated Anabaena cylindrica from lake algae and conducted hydrogen production experiments, discovering that photosynthetic microorganisms could generate H₂ from water. Later, in 1942, Gaffron and Rubin were the first to report hydrogen production via photosynthesis in the green alga Scenedesmus obliquus. Various green algae, including C. reinhardtii, Chlorella fusca, S. obliquus, and Ulva lactuca, as well as blue‐green algae such as A. cylindrica, Synechococcus elongatus, Synechocystis spp., and Nostoc muscorum, have been reported to produce H₂ [23].

Algae generate oxygen and reducing agents through photosynthesis [24, 25]. During this process, water is split, releasing oxygen, while carbon dioxide is fixed and protons are reduced to H₂. In direct bio‐photolysis, electrons are sequentially transferred from water through PSII and photosystem I (PSI), ultimately reaching hydrogenase, a hydrogen‐producing enzyme, via ferredoxin (Fd), an electron carrier [26]. The key enzymes involved in hydrogen production in algae and cyanobacteria are hydrogenase and nitrogenase. Depending on the microbial strain, either or both enzymes contribute to H₂ production. These enzymes are metal ion‐containing polymers that exist in different forms depending on their Ni, Fe, Mo, or V content, which significantly influences their activity [27, 28]. Among them, nitrogenase, which is unique to blue‐green algae, has been extensively studied for its role in H₂ production. Typically, nitrogenase reduces nitrogen to ammonia, but under nitrogen‐limited conditions, it reduces protons to generate H₂ gas [29]. However, this process is highly energy‐intensive, requiring four ATP molecules per H₂ molecule [28]. Furthermore, nitrogenase is less efficient than hydrogenase, consuming nearly twice the metabolic energy, and is also large, slow‐reacting, and highly sensitive to external conditions [30]. Therefore, to maximize H₂ yields, minimizing or eliminating nitrogenase activity is essential.

Most algae grow under photosynthetic conditions and later, after a period of adaptation to dark and anaerobic environments, synthesize hydrogenases. This enzyme is then activated for a limited time, enabling H₂ production. This phenomenon occurs due to the breakdown of organic compounds accumulated during photosynthesis. However, when normal photosynthesis resumes, H₂ production often decreases and becomes unstable because carbon dioxide is fixed, and oxygen is generated from water in the process [31, 32]. Direct water splitting and H₂ production through photosynthetic decomposition are inhibited by the oxygen produced during photosynthesis. In other words, both the hydrogenase enzyme and the reaction itself are highly sensitive to oxygen, which is generated simultaneously, thereby preventing sustained H₂ production [33]. To address this, laboratory experiments have been conducted to remove oxygen as it is generated within the reactor. However, this approach is not practical for large‐scale production.

If a technology capable of simultaneously producing oxygen and hydrogen through the photolysis of water by photosynthetic microorganisms were successfully developed, it would provide an ideal, sustainable method for hydrogen production using only water and solar energy. However, significant technical challenges remain. Future advancements must focus on developing microbial technologies that can efficiently harness the reducing power of microorganisms to maximize sunlight conversion and hydrogenase activity. Additionally, further innovation is needed to establish cost‐effective bioreactor systems that facilitate efficient bio‐H₂ production from water and sunlight while optimizing H₂ collection. The overall reaction for direct photolysis is summarized in Equation (1) and illustrated in Figure 2A [34, 35, 36].

$$2{\mathrm{H}}_2\mathrm{O}+\text{energy from sunlight}\to 2{\mathrm{H}}_2+{\mathrm{O}}_2$$ (1)

2.2. Indirect Bio‐Photolysis

In photosynthesis, autotrophic organisms produce carbohydrate biomass by absorbing carbon dioxide from the atmosphere in the presence of solar radiation and water. This process, known as indirect bio‐photolysis (Figure 2B), occurs in two distinct steps [35, 37]. Unlike direct bio‐photolysis, where hydrogen is directly generated, indirect bio‐photolysis separates photosynthesis from hydrogen production by using carbon dioxide as an intermediate electron carrier in the first step [35]. This process is carried out by nitrogen‐fixing cyanobacteria that possess specialized cells called heterocysts, which play a crucial role in microbial taxonomy [35]. Cyanobacteria undergoing indirect bio‐photolysis consist of two types of cells: vegetative cells, which generate oxygen and fix carbon dioxide, and heterocysts, which fix nitrogen or produce hydrogen in the absence of nitrogen due to their thick cell walls. Since hydrogen production is compartmentalized within heterocysts and separated from nitrogenase activity, this method avoids the oxygen sensitivity issues associated with hydrogenase in direct bio‐photolysis [35]. During photosynthesis, nitrogenase receives electrons from organic compounds formed via carbon dioxide fixation, while Fd, generated by PSI in cyanobacteria, functions as an electron transporter [38]. The efficiency of hydrogen production depends on the availability of organic substrates generated through carbon fixation. However, nitrogenase is an energy‐intensive enzyme, requiring four ATP per H₂ molecule, making it less efficient than other hydrogen‐producing enzymes [39].

The second step of indirect bio‐photolysis can be further categorized into two approaches. The first approach employs a single type of photosynthetic microorganism, such as algae or cyanobacteria, cultured under two different conditions. The second approach utilizes two or more types of microorganisms, such as photosynthetic bacteria, to enhance hydrogen production. In the first step, algae are cultivated in an open pond, where they accumulate carbohydrates by fixing atmospheric carbon dioxide [40]. In the second step, hydrogenase is induced under dark and anaerobic conditions within a fermenter, leading to H₂ production [41]. After hydrogen production, the algae return to the first step, where they resume photosynthesis to accumulate carbohydrates for the next cycle. In summary, algae first produce polymer substrates for hydrogen production through photosynthesis (first step), after which photosynthetic bacteria generate hydrogen in the second step. The overall reactions for indirect bio‐photolysis are summarized in Equations (2) and (3) and illustrated in Figure 2B [35].

First‐step:

$$12{\mathrm{H}}_2\mathrm{O}+6\mathrm{C}{\mathrm{O}}_2+\mathrm{sunlight}\mathrm{energy}\to {\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6+6{\mathrm{O}}_2+6{\mathrm{H}}_2\mathrm{O}$$ (2)

Second‐step:

$${\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6+6{\mathrm{H}}_2\mathrm{O}+\mathrm{sunlight}\mathrm{energy}\to 12{\mathrm{H}}_2+6\mathrm{C}{\mathrm{O}}_2+6{\mathrm{H}}_2\mathrm{O}$$ (3)

3. Photo‐Fermentation

Microorganisms capable of generating or absorbing molecular hydrogen do not commonly occur in nature, with only certain photosynthetic microorganisms being able to do so. This phenomenon was first observed in the red sulfur bacterium Allochromatium vinosum, which fixes carbon dioxide in the presence of light and uses hydrogen as an electron donor [42]. Similarly, Rhodospirillum rubrum, a representative nitrogen‐fixing, purple non‐sulfur bacterium, produces hydrogen by decomposing formate into hydrogen and carbon dioxide under dark fermentation conditions. It also generates hydrogen in the presence of light [43]. In the absence of nitrogen gas and ammonia ions, nitrogenase reduces protons to molecular hydrogen, facilitating hydrogen production. Within photosynthetic microorganisms, the mechanisms of hydrogen production under light and dark conditions differ. The production of hydrogen also varies depending on the type of substrate used and the specific enzyme systems present in the microorganisms.

Photosynthetic bacteria capable of hydrogen production include R. rubrum [43], Rhodobacter sphaeroides [44], Chromatium okenii [45], Thiocystis violacea, Thiosarcina rosea, Thiospirillum sanguineum, Thiocapsa roseopersicina, Lamprocystis roseopersicina, Thiodictyon elegans, Thiopedia rosea, Amobobacter pendens, Ectothiorhodospira mobilis, Chlorobium limicola, Prosthecochloris aestuarii, Pelodictyon clathratiforme, and Chlorochloris sulfateca. These bacteria belong to three families: Rhodospirillaceae, Chromatiaceae, and Chlorobiaceae. Among them, the Rhodospirillaceae family is commonly referred to as purple non‐sulfur bacteria. These bacteria utilize bacteriochlorophyll (BChl), a photosynthetic pigment found in various phototrophic bacteria, to fix nitrogen through an electron transfer mechanism when exposed to long‐wavelength light. The final enzyme involved in nitrogen fixation is nitrogenase, which reduces protons (H⁺) to H₂ in the absence of nitrogen sources such as nitrogen gas, ammonia, or ammonium ions (NH₄ ⁺). The protons and electrons required for this process originate from organic substances, reduced sulfur compounds, or molecular hydrogen. H₂ production by this process reaches its maximum rate under anaerobic conditions, optimal light intensity, a limited nitrogen supply, and an efficient substrate supply, although the exact conditions vary depending on the microorganism. Among organic substrates, organic acids serve as efficient carbon sources and electron donors for hydrogen production, while glutamate is considered an ideal nitrogen source. For example, Rhodopseudomonas palustris P4 converted acetate to hydrogen with a yield of approximately 70% of the theoretical maximum, while Rhodopseudomonas palustris WP 3–5 achieved a 75% hydrogen yield from glucose [46]. Similarly, Rhodopseudomonas capsulata, one of the most extensively studied red non‐sulfur bacteria, converted sucrose to hydrogen with a 53% yield [47]. Red bacteria, named for their natural pigmentation, carry out photosynthesis in their cell membranes, where BChl a or b absorbs light and drives photosynthetic activity. Red bacteria are generally classified into red sulfur bacteria and red non‐sulfur bacteria, which can be distinguished by their physiological and ecological characteristics. Red sulfur bacteria are photosynthetic autotrophs that use H₂S as an electron donor and are obligate anaerobic, meaning they rely on hydrogen sulfide instead of water. In contrast, red non‐sulfur bacteria are photosynthetic heterotrophs. Since red non‐sulfur bacteria are sensitive to H₂S, only some species can oxidize and utilize trace amounts of H₂S as a reducing agent under anaerobic conditions. However, for most species, even relatively low H₂S concentrations can inhibit growth.

Unlike algae, which use both PSI and PSII for photosynthesis, photosynthetic bacteria rely solely on PSI for photosynthesis and hydrogen production from organic compounds [48]. Photosynthetic microorganisms possess a chlorophyll (Chl) pigment‐protein complex, known as the reaction center, which converts light energy into chemical energy [49]. During this process, electrons supplied from the substrate reduce the Fd electron transporter in the nitrogenase enzyme system. Utilizing this reducing power along with ATP, nitrogenase catalyzes the production of molecular hydrogen (H₂) in the absence of a nitrogen source.

Photosynthetic bacteria exhibit metabolic diversity, allowing them to grow under both aerobic and anaerobic dark conditions. They are known for their ability to perform photosynthesis and can also be cultured via fermentation. Due to this metabolic flexibility, they can utilize various organic substrates, including monosaccharides, disaccharides, and organic acids, though substrate utilization efficiency varies by species. The theoretical H₂ production yields are 12 molecules of hydrogen gas per molecule of glucose and 4, 6, and 8 molecules of hydrogen gas per molecule of acetic acid, lactic acid, and butyric acid, respectively [50].

Several strategies have been explored to maximize photosynthetic hydrogen production from organic substances in the genus Rhodobacter. Genetic engineering research has focused on modifying genes related to hydrogen production, including alterations to the nitrogenase gene, which plays a key role in hydrogen synthesis, and deletion of the uptake hydrogenase (Hup) gene, which otherwise recycles hydrogen back into metabolism. Additionally, modifications to light‐harvesting system genes have been investigated to improve light utilization efficiency [51]. Research is also being conducted on optimizing and scaling up bioreactors to enhance hydrogen production, with an emphasis on efficient light supply and distribution. Various types of light‐utilizing bioreactors have been developed, including stirred‐tank bioreactors, open‐pond systems, vertical and horizontal tubular reactors, helical (or coil) reactors, internal gas‐exchange tubular reactors (horizontal or inclined), and vertical modular reactors. Cultivation technology is equally important, as securing high‐performing microbial strains and optimizing reactor design directly impact bio‐H₂ production. Key research areas include cell immobilization techniques to maximize bacterial density in culture media, pretreatment methods to remove inhibitory salts and ammonia when using organic wastewater or waste resources and optimizing temperature and light intensity to enhance hydrogen production efficiency. In summary, photo‐fermentation is a highly efficient route for hydrogen production, as organic compounds are broken down into smaller molecules in the presence of light [37]. During this process, organic substrates are converted to H₂ and carbon dioxide via Fd and nitrogenase. A schematic overview of photo‐fermentation is provided in Figure 3, and the reaction is as follows [52]:

$$\begin{array}{c}\mathrm{Organic}\mathrm{compounds}\left(\mathrm{C}{\mathrm{H}}_2\mathrm{O}\right)+\mathrm{light}+\mathrm{water}\to \\ \to \mathrm{ferredoxin}\to \mathrm{nitrogenase}\to {\mathrm{H}}_2\end{array}$$ (4)

FIGURE 3.

Biohydrogen production via photo‐fermentation.

4. Dark Fermentation

Microorganisms that biologically produce H₂ from organic substances are classified into photosynthetic microorganisms, which perform photo‐fermentation and require light (Figure 3), and non‐photosynthetic microorganisms, which carry out dark fermentation and do not require light (Figure 4) [53]. Dark fermentation is an anaerobic process in which microorganisms convert organic and inorganic substrates into bio‐H₂ without the need for light [53]. Unlike anaerobic digestion, which primarily aims at biogas production, dark fermentation is specifically directed toward hydrogen generation [53]. Microorganisms capable of hydrogen production through dark fermentation include Escherichia coli [54], Porphyridium cruentum [55], Klebsiella pneumoniae [56], Alcaligenes eutrophus [57], Desulfovibrio vulgaris [58], Clostridium butyricum [59], C. pasteurianum [60], Methanobacterium spp. [61], Rhizobium leguminosarum [62], Azotobacter vinelandii [63], and Enterobacter aerogenes [64]. These strains produce hydrogen along with various organic acids during anaerobic fermentation (Figure 4) and exhibit broad substrate utilization, breaking down complex carbohydrates such as starch, xylan, pectin, mannitol, sorbitol, glycerol, cellobiose, and sucrose. Among these, C. butyricum [65], C. pasteurianum [66], C. aceticum [67], C. kluyveri [68], E. aerogenes [69], and Caldicellulosiruptor bescii [4, 5, 70] are the most well‐known hydrogen‐producing anaerobic bacteria, and extensive research is being conducted to optimize hydrogen production using these strains.

FIGURE 4.

Hydrogen production by Clostridium spp. through dark fermentation.

The hydrogen production pathway in Clostridium species follows the Embden–Meyerhof–Parnas (EMP) pathway, where carbohydrates are converted into organic acids, including acetate, lactate, butyrate, propionate, and succinate. Under nitrogen‐rich conditions, these bacteria can also produce alcohols (butanol, isopropanol) and solvents (acetone) as fermentation byproducts (Figure 4). The mechanism of hydrogen production differs between obligate anaerobes, such as Clostridium spp., and facultative anaerobes, such as Enterobacter spp., E. coli, and Bacillus spp. [71]. Obligate anaerobes generate H₂ through the oxidation of NAD(P)H mediated by Fd and hydrogenase, where pyruvate acts as an intermediate metabolite. In contrast, facultative anaerobes produce H₂ via the formate pathway, utilizing cytochrome c and hydrogenase. The biochemical reactions involved in the production of acetate (Equation 5) and butyrate (Equation 6) from glucose, along with hydrogen generation, are presented below [72]:

$${\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6+2{\mathrm{H}}_2\mathrm{O}\to 2\mathrm{C}{\mathrm{H}}_3\mathrm{COO}-+2{\mathrm{H}}^{+}+2\mathrm{C}{\mathrm{O}}_2+4{\mathrm{H}}_2$$ (5)

$${\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6\to \mathrm{C}{\mathrm{H}}_3{\left(\mathrm{C}{\mathrm{H}}_2\right)}_2\mathrm{COO}-+{\mathrm{H}}^{+}+2\mathrm{C}{\mathrm{O}}_2+2{\mathrm{H}}_2$$ (6)

Hydrogen production through anaerobic fermentation yields up to 4 mol of hydrogen along with 2 mol of acetic acid (Figure 4) [73], which accounts for only about 33% of the theoretical maximum of 12 mol of hydrogen that can be derived from 1 mol of glucose. In practice, well‐known wild‐type genera such as Clostridium and Enterobacter typically produce around 1–2 mol of hydrogen (Table 2). However, due to the rapid hydrogen production rate of dark fermentation, this method is considered the most commercially viable. As previously mentioned, anaerobic microorganisms capable of producing hydrogen utilize organic substances in the absence of oxygen while also generating organic acids. The yield of hydrogen and organic acids from one molecule of glucose varies depending on the microorganism. However, approximately 2–4 molecules of hydrogen are theoretically produced alongside acetic, butyric, and succinic acids (Table 2). This hydrogen production is a distinct biochemical feature of microorganisms, and external factors such as temperature, dissolved oxygen, and culture medium acidity can be regulated to optimize the process. Recent research efforts have focused on genetically engineering microorganisms to enhance enzyme activity and increase hydrogen accumulation. Unlike photosynthetic bacteria, anaerobic bacteria do not require light for fermentation, allowing hydrogen production to occur continuously, regardless of the time of day, using organic matter as a substrate. Additionally, their rapid cell growth rate makes them well‐suited for continuous cultivation and the operation of large‐scale facilities.

TABLE 2.

Hydrogen production by dark fermentation of microorganisms.

Microorganisms	H2 yield (mol H2/mL glucose)	References
Clostridium acetobutylicum 1CICC 8012	1.2–2.5	[85]
Clostridium acetobutylicum 2ATCC 824	0.41–0.63	[86]
Clostridium beijerinckii	2.81	[87]
Clostridium butyricum	3.57	[88]
Clostridium butyricum 3KCCM 35433	1.23–1.42	[89]
Clostridium butyricum SC‐E1	1.3–2.2	[90]
Clostridium butyricum 4DSM 10702	3.2	[91]
Clostridium thermocellum 4DSM 1313	0.43–0.68	[92]
Escherichia coli	1.4	[93]
Enterobacter aerogenes	1.92	[94]
Enterobacter aerogenes E.82005	1.1	[95]
Enterobacter aerogenes HO‐39	0.9	[96]
Klebsiella pneumoniae	2.7	[97]
Thermoanaerobacter tengcongensis	4.0	[98]
Thermotoga maritima 4DSM 3109	4.0	[99]
Thermococcus kodakarensis KOD1	3.3	[100]

Abbreviations: ATCC, The American Type Culture Collection, KCCM: Korean Culture Center of Microorganisms; DSM, German Collection of Microorganisms, Deutsche Sammlung von Mikroorganismen; CICC, China Center of Industrial Culture Collection.

Hydrogen plays a crucial role in the metabolism of many anaerobic microorganisms, as they can utilize energy‐rich hydrogen molecules from their surroundings and oxidize them to generate electrons for energy production. In the absence of external electron acceptors, some microorganisms manage excess electrons produced during metabolism by reducing protons to form hydrogen. In both cases, hydrogenase is the key enzyme involved. There are two types of hydrogenases, [Ni‐Fe] hydrogenase and [Fe─Fe] hydrogenase [37], classified based on the metal component in their active center. Generally, [Ni─Fe] hydrogenase functions as a hydrogen oxidation catalyst, whereas [Fe─Fe] hydrogenase exhibits high activity in proton reduction [74].

5. Hydrogenases and Nitrogenases

Hydrogenase and nitrogenase are essential catalysts involved in bio‐H₂ production and can be applied to various biological conditions. They are key enzymes for energy metabolism and nutrient cycling, but their functions are different. Hydrogenase catalyzes the reversible reaction: (1) the oxidation of H₂ to protons and electrons, or (2) the reduction of protons to produce H₂. Unlike this hydrogenase, nitrogenase takes the irreversible reaction and reduces nitrogen (N₂) to ammonia (NH₃) and produces H₂ as a by‐product, the main step for nitrogen fixation, under anaerobic and nitrogen‐deficient conditions [28].

5.1. Hydrogenases

In the dark fermentation pathway, carbon (carbohydrate) goes to lactate or acetyl‐CoA, and electrons go to lactate and H₂ from pyruvate, making it a central metabolic branch point. In theory, many microorganisms via the pathway produce relatively low yields of H₂ (4 moles of H₂ per mole of glucose) [75]. H₂ production is coupled with acetate production, which simultaneously combines with the electron carriers NADH (a two‐electron carrier) and ferredoxin (a one‐electron carrier). Here, protons are reduced by the hydrogenase to form H₂. The hydrogenases are all metalloenzymes classified by the metal content in the center of the active site. There are two representative types of hydrogens, [Ni─Fe] hydrogenase and [Fe─Fe] hydrogenase, that provide a redox mechanism to store and utilize energy. [Ni─Fe] hydrogenase is the most widely distributed membrane‐bound heteroenzyme in nature, and its electron donor should be reduced ferredoxin alone. The hydrogen‐consuming reaction is catalyzed by [Ni─Fe] hydrogenase. The consumed H₂ is oxidized to H⁺, recovering the energy lost in the N₂ fixation process. [Fe─Fe] hydrogenase, which requires NADH/NADPH and ferredoxin, is oxidized simultaneously in a 1:1 stoichiometric ratio, catalyzing the production of H₂ [28, 76].

In most microorganisms, [Fe─Fe] hydrogenase appears to be a key enzyme in the H₂ production catalytic process. [Fe─Fe] hydrogenase is very sensitive to oxygen and is inhibited under aerobic conditions. Oxygen produced through bio‐photolysis inhibits the activity of hydrogenase and reduces hydrogen production efficiency. In contrast, [Ni─Fe] hydrogenase is an enzyme that is more aerobic and more tolerant to O₂ availability than [Fe─Fe] hydrogenase. [Ni─Fe] hydrogenase is a minor enzyme in H₂ production but plays a vital role in pumping protons across the cell membrane and generating the proton motive force (MPF), ultimately catalyzing ATP synthesis via chemiosmosis [28, 76].

5.2. Nitrogenases

Nitrogenase is an essential enzyme in the nitrogen cycle, involved in biological nitrogen fixation, and produces H₂ gas as a by‐product. This process occurs when nitrogenase reduces dinitrogen (N₂) to ammonia (NH₃), which releases H₂. The nitrogenases are classified into three groups by a unique metal center: Mo‐nitrogenase, V‐nitrogenase, and Fe‐nitrogenase, containing molybdenum, vanadium, and iron, respectively. As mentioned previously, during nitrogen fixation, nitrogenase transfers electrons and reduces protons (H⁺) to produce H₂ gas. This H₂ production is a result of the enzyme's electron transfer mechanism, in which some of the electrons intended for N₂ reduction are instead used to reduce protons to H₂. Although this enzyme can produce H₂ gas, the problem with this enzyme is that it is also very sensitive to oxygen and can be irreversibly damaged. This limits the conditions available for hydrogen production. Another issue with nitrogenases is that H₂ production dependent on nitrogenase is an energy‐intensive process, requiring lots of ATP to catalyze nitrogen reduction [28].

Hydrogenase and nitrogenase are involved in various metabolic pathways, such as the methanogenesis pathway, nitrogen fixation through nitrogenase‐hydrogenase co‐regulation, treatment of toxic heavy metals, and virulence of pathogenic bacteria and parasites. Therefore, understanding the overall mechanism of both enzymes is very important to improve H₂ production.

6. Strategies for Improving Microbial H₂ Production

There are several strategies based on enzyme engineering, metabolic engineering, and process engineering to improve process efficiency and scalability of H₂ production. Enzyme engineering focuses on modifying hydrogenases and nitrogenases (the enzymes responsible for producing H₂) to improve their activity and specificity [77]. Metabolic engineering refers to manipulating microbial metabolism to optimize H₂ production by modifying pathways, increasing substrate utilization, and reducing by‐product formation. Process engineering covers the practical aspects of H₂ production, including bioreactor design, fermentation conditions, and downstream processes to increase process efficiency and scale‐up.

6.1. Enzyme Engineering

This field focuses on improving the performance of hydrogenases, which are essential for hydrogen production. Techniques such as protein engineering and directed evolution are used to improve the activity, stability, and specificity of enzymes, thereby increasing hydrogen yields. For example, most studies have focused on coupling hydrogenases to electron carriers or PSI itself. The high catalytic rate of [Fe─Fe] hydrogenases shows significant potential in these applications, but as mentioned earlier, oxygen sensitivity and irreversible inactivation are major obstacles. This is why it is necessary to explore ways to develop oxygen‐insensitive [Fe─Fe] hydrogenases through enzyme engineering. Caserta et al. reported [FeFe]‐hydrogenases (HydAs) from Megasphaera elsdenii (MeHydA) that show significant resistance to O₂. They constructed a shorter version of this enzyme (MeH‐HydA) without the N‐terminal domain harboring the accessory FeS clusters. This study demonstrated that it is possible to engineer HydA to produce an active hydrogenase that combines the resistance properties of the most resistant HydA [78].

6.2. Metabolic Engineering

Metabolic engineering offers a very promising strategy to improve hydrogen yields by re‐directing biochemical pathways. There are several biological techniques to alter metabolic fluxes to increase hydrogen production. This involves modifying microbial metabolism to optimize H₂ production pathways. Strategies include: (1) elimination of competing pathways, such as eliminating or modifying the lactate pathway to store electrons for H₂ formation by combining protons and electrons; (2) homologous and heterologous expression of genes involved in H₂ production, such as hydrogenase and nitrogenase; and (3) modifying metabolic pathways to utilize different carbon sources to improve substrate utilization [4, 5, 79].

6.3. Process Engineering

This field focuses on optimizing the practical aspects of H₂ production processes. This includes: (1) designing efficient bioreactors to accommodate microbial culture and H₂ production, (2) optimizing fermentation conditions such as temperature, pH, and nutrient availability to promote H₂ production, and (3) developing hydrogen recovery and purification strategies [80].

As mentioned here, the efficiency of biohydrogen production is determined not only by the efficiency of the key catalyst but is also influenced by microbial metabolism, which is further complicated by substrate type, fermentation process, and reactor design and condition [81]. To ensure the economic feasibility of biohydrogen production, it is necessary to comprehensively consider all aspects from enzymes to processes. Several studies based on different strategies have reported manipulating microbial strains and enzymes to increase H₂ production. To summarize these, there are possible techniques to increase H₂ production, such as (1) overexpression of evolving H₂ases, (2) elimination/downregulation of uptake H₂ases, (3) elimination of pathways competing for electrons, (4) improvement of substrate catabolism, and (5) engineering electron and redox metabolism [82]. Therefore, an improvement in H₂ yield over 4 mol/mol glucose would significantly improve the economic sustainability of the overall H₂ production process, especially the dark fermentation.

7. Conclusion

In this review, we discussed different biological routes for hydrogen production, including direct water‐splitting hydrogen production (direct bio‐photolysis), indirect water‐splitting hydrogen production (indirect bio‐photolysis), photosynthetic fermentation (photo‐fermentation) by red bacteria under anaerobic culture conditions in the presence of light, and anaerobic fermentation (dark fermentation) in the absence of light. As summarized in Table 3 [34, 83, 84], dark fermentation has been the most extensively studied and has produced greater amounts of hydrogen (Table 3). To enhance hydrogen production, it is essential to optimize bioreactor conditions on a large scale and manipulate genes related to hydrogen production, such as hydrogenase‐ and nitrogenase‐encoding genes, using genetic engineering tools. Additionally, genetic modifications targeting the electron transport chain could further improve bio‐H₂ production efficiency.

TABLE 3.

Evaluation of the efficiency, scalability, and economic viability of hydrogen production.

The type of biohydrogen production	Efficiency	Issues/ Benefits	Economic viability
Direct biophotolysis	‐ Sunlight to hydrogen (<1.5%) ‐ 3%–10% by removing the oxygen produced	‐ Low efficiency of hydrogen production ‐ High sensitivity of hydrogenase enzymes to oxygen	‐ High cost ‐ Requirement of technological advancements to improve efficiency and reduce costs
Indirect biophotolysis	‐ Sunlight to hydrogen (10%–15%)
Photo‐fermentation	‐  A maximum light conversion efficiency: 9.3% ‐ A maximum hydrogen production: 80%	‐ Optimization of processes to keep product quality and efficiency ‐ Scalability ‐ Cost‐effectiveness	‐ Economic viability to be ensured due to efficiency and waste utilization potential ‐ Requirement to optimize substrate costs, improve hydrogen yields, and explore byproduct value creation strategies for economic sustainability
Dark fermentation	‐  Conversion efficiency: 60%–80%

Author Contributions

Minseok Cha: writing – original draft, writing – review and editing, conceptualization, investigation. Min‐Seo Park: visualization, investigation. Soo‐Jung Kim: conceptualization, funding acquisition, writing – review and editing, supervision.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.