*2.3. Biohydrogen*

The term "biohydrogen" refers to hydrogen created biologically, most typically by algae, bacteria, and archaea, either through cultivating them or from organic waste sources [74]. Hydrogen is considered as ye<sup>t</sup> another sustainable energy source generated by photosynthetic organisms, with a higher energy content of about 122 kJ/g, which is nearly 2.75 times greater than that of hydrocarbon fuels [75]; due to this reason, it has

been considered a viable alternative to fossil fuels and as an carrier of energy. A number of microalgae species, such as *Anabaena* sp. [76], *Chlorella vulgaris* [77], *Nannochoropsis* sp. [78], *Chlamydomonas reinhardtii* [79], *Spirulina maxima* [80], and *Scenedesmus obliquus* [77,81], are capable of generating molecular hydrogen through the photofermentative metabolism. Among the various species, *Chlorella vulgaris* is the most commonly used untreated substrate for hydrogen generation. Hydrogen yield acquired from various species ranged from 0.37 to 19 mL of hydrogen/g VS, and highest hydrogen yield was achieved from *Chlorella vulgaris* [82] and the *Scenedesmus* sp. [83].

Microalgal hydrogen production is facilitated by the solar light, or by fermentation processes and various thermochemical techniques used to convert the biomass. Production of molecular hydrogen by microalgae is carried out in the presence of the enzyme hydrogenase, which utilizes water as the only electron donor. [84].However, as the hydrogenase enzyme that produces biohydrogen is particularly sensitive to oxygen and becomes inactive even at a partial pressure of 2% of oxygen, [85], the ability of microalgae to stimulate hydrogen production shows only after acclimatization to anaerobic environment and is vanished in the presence of even small amount of oxygen [86]. Thus, due to its incompatibility to oxygen, the ability of microalgae lasts only for a short period of time.

In 1939, Gaffron and Rubin conducted the first scientific experiment of generating hydrogen using microalgae. In their study, it was reported that *Scenedesmus obliquus* was able to generate hydrogen at low rates under two conditions, such as in the dark environment and by replacing the culture's atmosphere with nitrogen gas [87]. Thus, it can be stated that the production of hydrogen depends on the mechanism of algae to adapt to the transition phase during which the dark anaerobic condition is switched to oxygenic condition to perform photosynthesis. The electron transport pathway is re-oxidized only through this transition [88].

Due to the low rate of biohydrogen production, the microalgae should be pretreated to convert the complex carbohydrates into simple sugars, thereby increasing the surface area (through the disruption of cell and disintegration of the cell wall) for the action of microbes. To increase the production of biohydrogen, physical, chemical, and biological pretreatments are employed. Physical pretreatment includes mechanical, thermal, and ultrasonication. Chemical pretreatment is carried out by the addition of acid or base or by ozonation.Microbes and enzymes are used in biological pretreatment to disrupt the microalgal biomass and liberate intracellular components, thus increasing the biohydrogen production rate. Enzymes are selected based on the composition of the microalgal cell wall. Another method to increase the hydrogen yield is through metabolic engineering and screening for mutant varieties. The photosynthetic stages within the cell are changed to increase hydrogen production through metabolic engineering.

Hydrogen can be produced inthree different ways such as direct biophotolysis, indirect biophotolysis (as shown in Figure 2), and hydrogen production driven by ATP. In direct photolysis biohydrogen is produced by converting water to hydrogen using solar energy through photosynthesis, and is further used as a substrate for anaerobic bacteria during dark fermentation [76]. Such fermentative reactions are typically faster and produce more hydrogen [89].

In indirect photolysis, the microalga produces hydrogen in two steps. In step 1, carbon dioxide is captured through photosynthesis in the presence of solar light. In other terms, microalgae produce oxygen and build up carbon within the cells. In step 2, production of hydrogen takes place through the degradation of the accumulated carbon through anaerobic fermentation which occurs in the absence of oxygen and involves a series of complex biochemical events involving multi-enzyme systems [36,75]. Hydrogenase enzyme plays an important role in this method. As discussed earlier, it is more sensitive to oxygen, so various research is being carried out to develop hydrogenase enzyme which is not sensitive to oxygen. Closed Photobioreactors can be employed for indirect photolysis (Figure 3). The most widely used photobioreactors have atubular design which consists of numerous transparent tubes. The tubes are usually designed to have a diameter less

than 10 cm to maximize penetration of solar rays. The microalgal broth is pumped through the tubes, where it is exposed to sunlight for photosynthesis, and then recycled back to a reservoir. Using either a mechanical pump or an airlift pump, the algal biomass is kept from settling by maintaining a very turbulent flow within the reactor [46]. A fraction of the algae is usually collected from the solar tubes. Thus, in this way microalgae can be harvested continuously.

**Figure 2.** Biohydrogen Production through photolysis and fermentation.

**Figure 3.** Closed Photobioreactors.

Various literatures have reported that usage of immobilized microalgal cells in the photobioreactors is more advantageous compared to the free cells. Immobilized cells provide increased cell retention time within the bioreactors and enhanced metabolic activity compared to the free cells [90]. Several strategies for improving hydrogen synthesis have been implemented, including varying light intensity, carbon supply, pH, temperature, and sulphur starvation [91,92].

Furthermore, the processs environmental and financial benefits are enhanced by the simultaneous treatment of wastewater and generation of valuable algal biomass [93]. According to Brennan and Owende (2010), in the immediate future, the combination of these processes will be the most reasonable commercial application, and it may be one of the most sustainable methods to generate biofuels [5]. Though the concept of generating

hydrogen through fermentative metabolism was discovered years ago by *Chlamydomonas moewusii* [94]; the challenges faced by construction often hinder the application of algal hydrogen generation in a wastewater environment in terms of renewable energy generation and performance characteristics. Moreover, the action of volatile acids such as acetic acid and butyric acid (released during the anaerobic digestion in the wastewater treatment plant) on depletion of oxygen and consequent generation of biohydrogen and its continuity in producing hydrogen has to be studied [95].
