A wide range of different organic substrates can be used for biohydrogen pro­duction by dark fermentation. As noted above, carbohydrates are the most suitable, and thus the most studied carbon sources, since they have a hydrogen production potential 20 times higher than with fat and proteins [62]. Biodegradability, availability, cost and carbohydrate content are the most important factors for selection of substrates for hydrogen production. Glucose, sucrose and lactose are the most widely studied simple sugars. Carbohydrate-rich substrates undergoing dark fermentation by mixed anaerobic bacteria produce H2, CO2 and organic acids (Fig. 10.2).

Pure mono and disaccharides are mostly used for dark fermentation with pure cultures. Dark fermentative hydrogen production from glucose with different pure cultures has given different hydrogen yields; Enterobacter cloacae 2.2 mol H2/mol [63]; Clostridium beijerinckii 2.4 mol H2/mol [64]; Thermoanaerobacterium
thermosaccharolyticum 2.42 mol H2/mol [65]; Pantoea agglomerans, 1.6 mol H2/ mol [66]; Escherichia coli 0.23 mol H2/mol [67]; Caldicellulosiruptor saccharo — lyticus 3.6 mol H2/mol [68]. Dark fermentative hydrogen production from glucose with mixed cultures resulted in yields between 1.70 and 2.75 mol H2/mol glucose [69-72]. Sucrose is another widely used pure substrate. Hydrogen yields from sucrose have been examined with pure cultures: Clostridium butyricum 0.5 mol H2/mol [73]; Thermoanaerobacterium thermosaccharolyticum 1.89 mol H2/mol [74]. Using mixed cultures to produce hydrogen from sucrose resulted in yields between 0.87 and 1.72 mol H2/mol [69, 75].

Pure substrates are not an effective approach for large-scale applications. Instead, using a waste or wastewater which has a high organic content will create a win-win solution, reducing wastewater disposal costs at the same time as creating an energy source, hydrogen, in a cost effective manner. In addition to various residues and wastes, energy crops can be a very good substrate option for dark fermentative hydrogen production. Among the different types of crops; sugar, starch, lignocellulosic based, the first two groups have been more widely used. The use of different kinds of energy crops resulted in different hydrogen yields; wheat starch 1.9 mol H2/mol glucose [76]; sweet sorghum plant 0.86 mol H2/mol glu­cose [77]; starch from paper mill 1.5 mol H2/mol glucose [46]; molasses 3.47 mol H2/mol glucose [78]. Some types of energy crops, especially lignocellulosic based, need pre-treatment before use for hydrogen production, increasing the process costs.

As well, various types of wastes and wastewaters have been used as substrates for dark fermentative hydrogen production with different hydrogen yields such as: sugar factory wastewater; 2.6 mol H2/mol hexose [79]; olive mill wastewater, 0.15 ml H2/ml OMW [4]; rice winery wastewater, 2.14 mol H2/mol hexose [80]; food waste, 1.8 mol H2/mol hexose [81]; cheese whey, 5.9 mol H2/mol lactose, 0.9 mol H2/mol hexose, 22 mmol H2/g COD, 10 mM/gCOD [82-86]. In these cases, high yields of hydrogen production by microbial processes can be achieved without any pretreatment. However, in most cases dilution is necessary to lower organic loading, and to prevent possible toxic effects of the substrate on the bacteria. In general, wastes can include a variety of organic and inorganic chemicals, some of which can be inhibitory for hydrogen production. Therefore, it is important to know the composition of the wastewater before using it as a substrate.