Various pretreatment strategies have been studied to use waste materials for bio-hydrogen production. Pretreatment of the substrate is essential for efficient bio-hydrogen production. Pretreatment facilitates the substrate breakdown into simple sugars, a prerequisite for microbial growth. Fermentation at high operating temperatures (65–70 °C) improves the substrate degradation rate and thus increases H
2 production rate
[9][17]. Various wastes or wastewaters, such as olive mill effluent
[10][4], are ideal for photo-biological H
2 production as they contain ethanol, acetate, butyrate, and propionate. Sewage sludge and tofu wastewater are regarded as the most promising substrates for the production of H
2. Before being utilized as a substrate, sewage sludge is pretreated for 1 h at 150 °C temperature under 10 atm after alkali treatment. During photo-fermentation, 0.17–0.28 L H
2/L broth/day is constantly produced
[11][18]. Cassava pulp is a by-product of the starch industry that contains up to 60% starch as dry matter and contains carbohydrates, including cellulose and hemicelluloses. Cassava pulp’s cellulose and hemicellulose may be acid hydrolyzed and utilized as a substrate for fermentative hydrogen production by anaerobic mixed cultures, under optimum hydrolysis conditions; 0.5% H
2SO
4 at a ratio of 1:15 (dry
w/v) for 30 min, resulting in 27.4 g/L of total sugar yield
[5][14]. Using various organic-rich domestic, agricultural, and industrial waste products for biohydrogen production decreases the cost of scaling up while also effectively removing the organic load
[12][19]. Cheese whey is an excellent substrate for fermentative H
2 generation since it comprises 4.6%lactose, 1.2% crude protein, 0.6% ash, 0.3% fat, 5–8% total solids, and 92.7% water
[13][7][9,10]. Dairy wastewaters contain polysaccharides, proteins, and lipids, which undergo hydrolysis to sugars, amino acids, and fatty acids
[11][18]. During fermentation, they are transformed into volatile fatty acids (VFAs), which are then degraded by acetogens to produce acetate, CO
2, and H
2 [14][20]. Pretreatment of complex biomass containing lignocelluloses is prevalent, as most complex substrates are unsuitable and must be broken down into simple components for easier access during fermentation
[15][21] (
Figure 12).
After selecting a microbial consortium enriched in hydrogen-producing ability, the accessibility of easily decomposable substrate is essential for the growth of microbes and hydrogen production. Algal biomass is enriched in large sugar residues and is considered an ideal substrate for H
2 production
[16][22]. Algal biomass (such as other organic waste) can be used for H
2 generation due to its high protein, fat, and carbohydrate content
[17][5][5,14]. Nguyen et al. (2010) used accumulated starch present within the green algae
C. reinhardtii as a substrate. Later, the hyperthermophilic eubacterium
T. neapolitana was used to convert that accumulated starch into H
2 gas
[17][5]. The algal starch could be directly fermented into H
2 by a bacterium with amylase activity (1.8–2.2 mol H
2/mol)
[16][22]. These wastes’ cellulose and hemicellulose percentages must be hydrolyzed to degrade carbohydrates to achieve high H
2 production. The resultant treated waste may be regarded as the potential substrate for fermentative hydrogen production. The main purpose of substrate pretreatment technology is to break down the complex structure of less degradable organic compounds and improve their solubility, thereby upgrading H
2 yield. The type of substrate and pretreatment method used may affect the yield of hydrogen and the characteristics of the effluents. Ideally, the best substrate pretreatment method should be selected, with high hydrogen yield, cost efficiency, process sustainability, and low energy requirements.