*2.4.1 Biophotolysis*

Biological photolysis occurs when light or a microbiological species is present and leads to the dissociation of H2O into molecular H2 and O2. Biophotolysis is a metabolic mechanism that is reliant on light and can be classified into two types: direct photolysis and indirect photolysis [44, 45].

#### **Figure 4.** *Four-step anaerobic digestion process adapted from [43] with modifications.*

Direct Biophotolysis is a light-dependent route for hydrogen formation which occurs in two stages: first, the breakdown of H2O molecules in photosynthesis (Eq. 1), accompanied by the synthesis of hydrogen facilitated by hydrogenases (Eq. 2), which occurs in green algae and cyanobacteria and depends on light energy [46].

$$\text{2H}\_2\text{O} \rightarrow \text{4H}^+ + \text{4e}^- + \text{O}\_2 \tag{1}$$

$$\text{4H}^+ + \text{4e}^- \rightarrow \text{2H}\_2\tag{2}$$

Direct bio-photolysis comprises H2O oxidation as well as a light-dependent electron exchange to the [Fe] hydrogenase, which leads to H2 generation through photosynthesis [47]. Direct bio-photolysis was based on the photosynthetic ability of microalgae and cyanobacteria to quickly breakdown H2O into oxygen and hydrogen. Microalgae can employ solar energy via proton and electron obtained from the H2O—splitting process, but cyanobacteria receive their energy from photosynthetic activity to enhance H2 generation, which takes place by direct adsorption of light and electron transfer to two enzyme cateories—hydrogenase and nitrogenase [48]—responsible for the enhancement of the transformation of hydrogen ions to hydrogen gas [49]. These techniques showed tremendous potential, but they also had major limitations, such as the discrepancies of direct bio-photolysis to simultaneously generate H2 and O2, as well as the fact that the O2 produced by bacteria throughout the procedure prevents considerable H2 production from being achieved.

During indirect biophototlysis, photosynthetic H2 can be formed by green algae amid sulfur deprivation conditions, as opposed to direct bio photolysis [48]. The restriction of sulfur—nutrients in the growth media of green algae prompted a reversible impediment in the O2 photosynthetic operation of the green algae. Sulfur deprivation triggers a decrease in the activity of the photosystem II (PSII), which is responsible for enhancing electron extraction from water through photochemical oxidation, and the photosynthetic process decreases below the respiration activity, resulting in a decrease in oxygen discharge below the amount of oxygen expended by respiration [50]. The synergistic effect between photosynthesis and respiration attributed to sulfur deprivation leads to a net utilization of oxygen by cells, which enables the growth environment to become anoxic [51]. The potential to develop ways to reuse constituents of the photobioreactor and optimize the cost of chemical nutrients that aids algae development which account for around 80% of the overall operational costs are two of the setbacks of efficient commercial application of indirect bio-photolysis for biogas synthesis [52, 53].

#### *2.4.2 Photo fermentation*

Under anoxic environments with light, photosynthetic microbes are capable of converting the majority of organic acids or volatile fatty acids (VFA) into biohydrogen and carbon dioxide [54]. Nitrogenase is the enzyme responsible for the majority of the biohydrogen produced by photosynthetic bacteria. Luminous light has a significant effect on the synthesis of nitrogenase [55]. It is essential for biohydrogen synthesis that the feedstock have an appropriate ratio of carbon and nitrogen sources (C/N ratio). Nitrogen constraints have been shown to modify the metabolic activities of photosynthetic bacteria, directing it more towards the discharge of extra energy and reducing power in the form of biohydrogen. The process of photo fermentation is

influenced by some variables, such as light intensity, inoculum age, nutrient type, and temperature. Temperature has a significant impact on the metabolic routes' ability to shift to greater biohydrogen synthesis [56]. The biohydrogen metabolism of purple non-sulfur bacteria is primarily controlled by the activity of the enzymes; nitrogenase and hydrogenase [56]. As part of the process, the nitrogenase enzyme generates biohydrogen under nitrogen-deficient environments (Eq. 3), where the hydrogenase enzyme oxidizes the biohydrogen in order to reuse electrons, protons, and ATP for employ in energy metabolism [57, 58]. Because hydrogenase enzyme can operate in any direction, according to Eq. 4, some of them are physiologically dedicated to utilizing biohydrogen (in the presence of appropriate electron acceptors) while others are responsible for the synthesis of biohydrogen under stringent anaerobiosis [59].

Light

$$\text{2H}^+ + \text{2e}^- + \text{4ATP} \rightarrow \text{H2v} + \text{4ADP} + \text{4Pi} \tag{3}$$

Nitrogenase

$$\text{H}\_2 \rightarrow 2\text{H}^+ + 2\text{e}^- \tag{4}$$

Nitrogenase

The overall metabolic route for the photo fermentation system is given as:

Substrate ! TCA cycle ! NAD*=*NADH ! Ferredoxin ! Nitrogenase ! H2*:*

#### *2.4.3 Dark fermentation*

Anoxic and certain microalgae (green algae) perform heterotrophic fermentation on carbohydrate-based substrates in the absence of light energy, resulting in the synthesis of hydrogen [60]. When it comes to dark fermentation, the practicality of producing hydrogen is dependent on the fact that hydrogen can be generated by heterotopic bacteria satellites that are situated in the algae biomass slurries. The impediment of H2-consuming microorganisms in a multi-microbial consortium that disintegrates algal biomass for the generation of H2 is a vital issue that presents a barrier to the effective use of dark fermentation technology. Dark fermentation is a mechanism in which organic feedstock are transformed by fermentative bacteria into biohydrogen, volatile fatty acids (VFA), and carbon dioxide in the absence of light. Carbohydrates (mostly glucose) are the primary energy sources for this mechanism, which results in the production of biohydrogen as well as volatile fatty acids (VFAs) such as acetic acid and butyric acid. Eqs. (5) and (6) demonstrate variation in product yield when acetic acid or butyric acid is the sole VFA product, the highest output of 4 and 2 mol H2/mol glucose respectively can be obtained. A lesser output is frequently attained in reality, because glucose is not only utilized for biohydrogen generation, but also to nourish and sustain the development of the microbes [60]. Biohydrogen generation via this approach can be influenced by substrate, inoculum, bacteria growth conditions, and other operating parameters.

$$\text{C}\_6\text{H}\_{12}\text{O}\_6 + 2\text{H}\_2\text{O} \to 4\text{H}\_2 + 2\text{CH}\_3\text{COOH} + 2\text{CO}\_2\tag{5}$$

$$\text{C}\_6\text{H}\_{12}\text{O}\_6 + 2\text{H}\_2\text{O} \rightarrow 2\text{H}\_2 + \text{CH}\_3\text{CH}\_2\text{CH}\_2\text{COOH} + 2\text{CO}\_2\tag{6}$$

#### *2.4.4 Proposed multi-stage bioreactor for biogas production*

A multi-stage bioreactor can be employed for the production of biohydrogen or hythane. A four-stage bioreactor produces significant amounts of hydrogen and recovers energy. In the first step which involves direct biophotolysis, blue-green algae employ visible light whereas photosynthetic microorganisms utilize unfiltered infrared rays in the second stage photo-fermentative reactor. The second phase photosynthetic reactor discharge is passed to a third stage dark fermentation for microbial transformation of substrates into H2 and organic acids. The fourth stage involves converting organic acid (from dark fermentation) into biohydrogen via microbial cell electrolysis in the dark (ideally at night or in low light) [47]. The growing interest in hythane has led to substantial study into dark fermentation of biomass for hythane generation in two-stage processes. Hythane is a gaseous combination of H2 (10–30%) and CH4 (70–90%) used as a substitute to methane in the automotive sector. Hythane is now produced mostly from fossil fuels, however using sustainable sources will significantly minimize greenhouse gas emissions. The efficient biotechnology process of two-stage anaerobic digestion (AD) can generate biohythane in two-stages, dark fermentation, and methanogenic phases, for H2 and CH4 synthesis respectively. Because H2 is a sustainable energy source, its existence in hythane facilitates the reduction of CO2 and NOx emissions. This product (hythane) is a clean-burning green energy that could be used as industrial biogas [61]. However, various issues need to be addressed before the multi-stage bioreactor technology may be efficiently utilized [62].
