Microalgae contain pigment molecules capable of absorbing solar energy and converting it into chemical energy by simultaneously splitting water into oxygen (O₂) and protons (H⁺). The photosynthetic electron transfer constitutes light and dark reactions. The light reaction helps to obtain electrons by splitting water in photosystem II (PSII) and transfer the electrons through an electron transport chain from PSII to Photosystem I (PSI). This results in the generation of Adenosine triphosphate (ATP) and strong reductants (NAD(P)H). Photobiological bioH₂ production is associated with photosynthesis, where the final electron acceptor, ferredoxin (Fd), donates electrons to enzymes involved in H₂ metabolism ^[1][28]. Biophotolysis is the initial step of microalgal bioH₂ production. In direct biophotolysis, microalgae convert solar energy into chemical energy, and H₂ is derived from the electrons and protons generated by the water splitting at PSII. Nevertheless, some of the restrictions of biophotolysis include O₂ generation by the activity of PSII, the requirement for a customized photobioreactor, the sensitivity of H₂ase to O₂, and low yield ^[2][3][29,30]. For indirect biophotolysis, electrons and protons are mainly supplied by the degradation of intracellular carbon compounds. Indirect photolysis has two stages: first, the carbohydrate biomass is generated from photosynthesis, and in the next stage, H₂ and CO₂ are produced due to the fermentation of carbohydrate-rich biomass. In these two steps, oxygen and H₂ will be separated. This prevents enzyme deactivation and removes CO₂ from the H₂ and CO₂ mixture, making H₂ purification easier. Some drawbacks include high H₂ selectivity, the restricted effect of O₂ on the H₂ase, and low yield ^[4][31]. Figure 1 shows a schematic diagram of hydrogen production by direct and indirect biophotolysis.

Dark fermentation has gained attention because of its relatively high bioH₂ production rates. The complex organic substances (lipids, carbohydrates, and proteins) are subjected to four stages: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. Firstly, lipids, carbohydrates, and proteins are hydrolyzed into sugars, fatty acids, and amino acids. In the acidogenesis stage, the hydrolyzed products are acidified to form H₂, CO_2, fatty acids, and other intermediates. In the next step, acetogenesis, the fatty acids produced are again fermented to generate H₂ and acetate. The final products, methane (CH₄) and CO₂, are formed from the decarboxylation of acetate by the acetoclastic methanogens. H₂-utilizing methanogens consume H₂ gas as an electron donor while reducing the carbon dioxide to methane. In dark fermentation, H₂ is produced as an intermediate metabolite in the acidogenesis and acetogenesis transformations. Two mechanisms which involve specific coenzymes are responsible for the evolution of H₂ gas, either by formic acid catabolic transformation or by re-oxidation of nicotinamide adenine dinucleotide (NADH) catalyzed by H₂ase pathway; H₂ase is the major enzyme in the process ^[5][6][7][32,33,34]. Dark fermentation has many advantages: it is capable of continually producing H₂ without depending on sunlight, has high energy efficiency, is an eco-friendly and economical process, is easy to commercialize, has a less complicated bioreactor design, and has a wide range of organic acid as byproducts. The major disadvantages are low bioH₂ production due to the accumulation of O₂, methanogenic bacteria utilizing H₂ as an electron donor, more tedious and expensive H₂ recovery due to the generation of CO₂ and other gaseous products, and low substrate conversion efficiency ^[5][8][9][24,25,32].

In photo fermentation, H₂ is produced by the photosynthetic bacteria by breaking down the organic compounds with the help of nitrogenase (N₂ase) enzymes under nitrogen-deprived conditions. Atmospheric nitrogen is converted into ammonium ions used by microorganisms as a nitrogen source through nitrogen (N₂) fixation by nitrogenase. They are only found in cyanobacteria and non-sulfur purple and green sulfur bacteria. These bacteria consume acetic acid and use ATP as an energy source. This leads to the transfer of the electrons by ferredoxin to the enzyme nitrogenase and results in N₂ fixation. Moreover, nitrogenase protons are converted to H₂ in the absence of nitrogen ^[6][9][25,33]. The major advantages are that photosynthetic bacteria use various spectral energy and substrates, can treat effluents from dark fermentation, have higher substrate transformation efficiency, and hence have a high H₂ yield. One disadvantage is the requirement of a light source; the photosynthesis efficiency directly depends on the availability of light. Moreover, it requires a large area and an anaerobic bioreactor, which increases costs ^[5][8][24,32].

Thermochemical processes include liquefaction, gasification, and pyrolysis. The wet microalgae biomass is converted into gaseous bioH₂ during hydrothermal gasification. The process includes heating biomass at higher temperatures in a compressed water medium. The reaction is quick due to the higher temperatures; the main products obtained are H₂, CO_2, and methane (CH₄). The compressed water medium usually has a low percentage of effluents and can be reused ^[10][11][35,36]. The microalgae should be dried up to low moisture content in conventional thermal gasification for higher efficiency. Nevertheless, the high moisture content of microalgae often results in high energy consumption during thermal gasification. Thus, the main advantage of applying supercritical water gasification (SCWG) is that it can be conducted without the drying process, but rather in an aqueous state. Lastly, pyrolysis occurs in the absence of oxygen at higher temperatures. Dry microalgae are needed to be fed to the reactor, which demands a large amount of energy. The conversion efficiency of microalgae depends on parameters such as reaction temperature, retention time, and the composition of feedstock ^[12][13][9,37].

Bioelectrochemical systems are an alternate method for bioH₂ production. Microalgae catalyze the oxidation–reduction at the anode and cathode, respectively, and act as electrochemical catalysts. Microbial fuel cells and microbial electrolysis cells are the two categories of bioelectrochemical systems. Microbial Fuel Cells (MFCs) are eco-friendly bioelectrochemical devices that produce electrical energy from chemical energy obtained from biomass. The H₂ forms at the cathodes as a result of the reduction reaction and is collected by an external system. The primary restriction of MFC is membrane fouling, which occurs due to the long-term growth of biofilm in separators. It can also occur due to the accumulation of microbes, which leads to the formation of thick biofilm on the surface of the membrane. This prevents the transfer of H₂ ions from the anode to the cathode. Another restriction is the removal of heavy metals, which causes lower efficiency performance of microbes and pH imbalance. The higher cost of the membrane prevents the large-scale expansion of MFC. Microbial Electrolysis Cells (MECs) are a different form of MFC; the bioH₂ is produced by the oxidation of organic matter, which is catalyzed by electroactive biofilms. It is yet to be developed in an efficient and scalable design. One major advantage of MEC is the higher efficiency in metal ions removal. The drawbacks are the higher cost as well as the H₂ loss and contamination ^[9][14][15][25,38,39].

Efficient production of bioH₂ yield from microalgae biomass depends on factors such as nutrients, pH, temperature, light intensity, photoreactor configuration, substrate concentration, and cell density. Compared to a near-neutral pH, higher H₂ productivity was observed when the pH was around 6. At a highly acidic pH, H₂ yield declines because of the inactivation of the acetate-producing bacteria. Temperature is another parameter that influences the metabolic pathways of H₂ase. A temperature range of 15–35 °C is good for microalgal growth. Moreover, the proper configuration of the photoreactor is important as it is critical for the effective use of light and the provision of sufficient surface area for the growth of microalgae. Additive subtracts such as biotin, cyanocobalamin, and thiamine are required to add to the culture to support the maximum cell growth and bioH₂ production. For optimal bioH₂ production, the culture needs a balance of carbohydrate-based substrate. Moreover, a carbon source is also required for the microalgae to flourish in all conditions except photoautotrophic conditions ^[16][17][15,40].

Furthermore, high bioH₂ production and good microalgal growth can be achieved by introducing the proper fraction of nutrients such as nitrogen, phosphorus, and trace elements. The nitrogen element mainly regulates the protein synthesis and growth metabolites of microalgae, while the phosphorus element regulates most of the cell’s activities and metabolism. The trace elements like magnesium, sodium, and zinc are important supplements that play a role in improving microalgal cultivation. The problem associated with the inhibition of H₂ase is caused by the presence of oxygen and can be resolved by sulfur deprivation. BioH₂ production is relatively low at lower light intensities. Exposure to high light intensity can increase bioH₂ production rates by inhibiting photosynthetic O₂ ^[9][18][25,41]. In addition, optimal light conditions can reduce the lag period of microalgae and increase H₂ yield ^[9][18][25,41]. Cell density controls the amount of light that passes through the microalgal cell, and it depends on the nature of the cultivation process. The low cell concentration will not allow the uptake of the dissolved O₂ into the microalgae culture. In contrast, a high cell density may cause the cumulation of starch and hinder the productivity rate. Therefore, to have significant bioH₂ production, an active growth phase and cell density for the culture should be maintained ^[16][15]. The application of bioH₂ production using microalgae is still limited due to the lack of proper distribution, capture, storage, and transformation technologies.

Various strategies can be adopted to improve bioH₂ production using microalgae, such as immobilization of microalgae, pretreatment techniques, nanoparticles, and genetic engineering. The pretreatment immediately disrupts the microalgal cell walls and enhances the accessibility of carbohydrates present in the cells. Different pretreatment methods include chemical, thermal, mechanical, enzyme, and combined methods. The best pretreatment method and its optimal conditions are yet to be determined ^[9][16][15,25].

Microalgal immobilization is the mechanism of the entrapment of microalgal cells on or into solid support. It has many advantages, such as high cell density, alleviating manipulation of cultures, and easy microalgae cell harvesting. Further, this approach protects the cells from unwanted contaminations and sudden changes in other culture parameters. This also results in high bioH₂ production due to the enhanced permeability of cell walls. In addition, the microalgal cells wash out, get reduced, and cause an overall increase in H₂ yields. The major drawbacks are the slow infusion of nutrients from the medium into microalgae and the high sunlight gradient within the cells because of high cell density ^[19][20][12,13]. Nanotechnology is capable of bioH₂ production due to its role in intracellular electron transfer, microalgal growths, and enzymes involved in bioH₂ generation. Genetic engineering and metabolic engineering can be used to modify specific pathways to increase bioH₂ production. The photosynthetic barriers and inhibition factors can be suppressed ^[8][24].