PNS bacteria can use a wide variety of substrates as carbon and nitrogen sources. Different kinds of strains can have different pathways for using substrates to produce hydrogen. Lactate, acetate, butyrate, propionate and succinate are the mostly known simple organic molecules that serve as suitable electron donors.

Glucose can be used as substrate for photofermentative hydrogen production. Photofermentation by Rhodobacter sphaeroides; Rhodobacter capsulatus from glucose achieved rates of 250 and 88 ml/g/h hydrogen, respectively [104, 105]. Using sucrose as the sole carbon source for Rhodobacter capsulatus resulted in 60 ml/g/h hydrogen yield [105]. Lactate and malate are the most studied carbon sources for photofermentative hydrogen production [106]. These two substrates are known as the best carbon sources for PNS bacteria for hydrogen production. By using Rhodobacter sphaeroides as bacterium and lactate as carbon source hydrogen rates varying between 16.7 and 240 ml/l/h have been reported in dif­ferent batch reactor studies [107-110]. Rhodobacter capsulatus can metabolize lactate to hydrogen at rates of 105 and 130 ml/g/h [106, 111]. Rhodopseudomonas palustris and Rhodobacter rubrum can metabolize the lactate with hydrogen production rates of 82.7 and 20 ml/l/h, respectively [112, 113]. Malate is another important carbon source for PNS bacteria. Studies have shown that up to 58 ml/l/h hydrogen rates can be achieved by using different PNS bacteria [107, 112]. Since PNS bacteria can use volatile fatty acids produced in dark fermentation, acetate, butyrate or mixtures of different organic acids were used as carbon sources in different studies. Hydrogen rates changing between 1.6 and 26 ml/l/h were obtained with mainly Rhodobacter capsulatus and Rhodobacter palustris [99, 114].

Non-sulfur photo-heterotrophic bacteria are known to be very effective bio­catalysts for hydrogen production from food, alcohol distilling or sugar industry wastewaters. Environmental benefits could be gained by using wastewaters as substrates. While using the wastewater as a substrate for photofermentative hydrogen production it is necessary to choose a proper pre-treatment that will not damage the main substrate component by sterilization.

Dairy wastewaters can be a good candidate for biohydrogen production with a yield of 3.6 l H2/l-reactor [115]. Since brewery wastewaters contain useful com­pounds like amino acids, proteins, organic acids, sugar as well as vitamins they can be assumed as useful substrates for hydrogen production. By using Rhodobacter sphaeroides 2.24 l H2/l medium hydrogen rate was reported. The best hydrogen production is achieved by R. sphaeroides RV with a yield of 1.23 mol H2/mol glucose from ground wheat [116]. From crude glycerol by Rhodopseudomonas palustris 6 mol of H2/mol glycerol can be produced [117]. In addition, photofer­mentation of glycerol should be used as substrate in a continuous process [117]. Olive mill effluent (OME) with a high content of sugar, volatile fatty acids, polyalcohols and fats and an advantage of low nitrogen content can give a rate of 0.009 l H2/l/h by photofermentation with Rhodobacter sphaeroides [118]. It can be easily seen that PNS bacteria are capable of efficient conversion of organic acids to H2. Using the industrial wastewater with proper modifications of the system can be an ecologically viable solution. Most PNS bacteria cannot metabolize sugar totally but they can metabolize organic acid easily which can be a good solution for dark fermentation end products.