Complete degradation of 1 mol of glucose can yield a 12 mol of hydrogen by combining fermentation and photofermentation in a two stage system. According to the Gibbs free energy of this reaction, complete oxidation of glucose into hydrogen and carbon dioxide is not feasible thermodynamically (Eq. 10.15).

C6H12O6 + 6H2O! 12H2 + 6CO2 AG0 = +3.2 kJ/mol (10.15)

Photon energy in photofermentation can be a useful external energy supply to reach the theoretical values of conversion. To achieve this, external light source is needed. In the case that an external light source cannot be applied and with only dark fermentation process by glucose consumption maximum 4 mol of hydrogen with acetate as a by-product will be produced (Eq. 10.1)

The by-product of dark fermentation stage acetate can be oxidized by photo­synthetic bacteria to produce hydrogen energy and to complete the oxidation of glucose totally into H2 and CO2 (Eq. 10.16).

CH3COOH + 2H2O + ‘elight energy’ ! 4H2 + 2CO2 AG0 = +104 kJ/mol

(10.16)

Integrating the photofermentation with dark fermentation process (Fig. 10.4) can result as the maximum yield of hydrogen production [67].

There are some studies reported about two-stage systems that are a combination of dark fermentation step with pure cultures and photofermentation step. The combination of dark fermentation and photofermentation steps with pure cultures like Caldicellulusiruptor saccharolyticus and Rhodobacter capsulatus, Rhodob — acter capsulatus hup-mutant and Rhodopseudomonas palustris [132] to produce hydrogen from beet molasses; Caldicellulusiruptor saccharolyticus and Rhodob — acter capsulatus to produce hydrogen from glucose, potato steam peels and molasses [119]; Clostridium butyricum and Rhodopseudomonas palustris to pro­duce hydrogen from glucose [133], Clostiridium pasterianum and Rhodopseudo­monas palustris from sucrose [134], Clostridium saccharoperbutylacetonicum and Rhodobacter sphaeroides from glucose [135] resulted in higher hydrogen pro­duction values in comparison with single systems.

Glucose and the sucrose are the most studied organic substrates for hydrogen production by two-stage systems. Using the glucose in a dark fermentation process with mixed anaerobic bacteria 1.36 mol H2/mol hexose yield achieved. By using the effluents of this system which includes mainly acetate, propionate and butyrate in a photobioreactor inoculated by Rhodopseudomonas capsulatus the overall yield increased to 4.46 mol H2/mol hexose [81]. Cattle dung batch at 38°C was

used as inoculum in the dark fermentation stage to produce hydrogen from sucrose and 1.29 mol H2/mol hexose, hydrogen yields are achieved. Using the effluents of first stage in photofermentation stage by Rhodobacter sphaeroides increased the overall yield to 3.32 mol H2/mol hexose [136].

One of the main advantages of two-stage systems is the usability of organic wastes and wastewaters. Carbohydrate-rich raw materials, especially starch and cellulose containing renewable biomass resources, are used in many studies of two-stage systems. After hydrolyzing by acidic or enzymatic pre-treatment methods wheat starch becomes a suitable substrate for two-stage systems [137]. After dark fermentation with anaerobic sludge a concentration of 1950 mg/l vol­atile fatty acids was produced. By using the produced volatile fatty acids 27 ml H2/ l/day hydrogen production rate was achieved at 72 h HRT with a PC controlled fermenter by R. sphaeroides (NRRL — B1727) [138]. For using wheat powder as a carbon source it is important to keep the concentrations at low levels to prevent the system from substrate inhibition [137]. A two-stage system is used with mixed bacterial cultures in dark fermentation and Rhodopseudomonas palustris in photofermentation to produce hydrogen from cassava. Cassava has a high content of 15-20% starch and 4-6% free sugar and it is a low cost biomass. It is a good source for biohydrogen production of 6.07 mol H2/mol hexose totally which was 2.53 mol H2/mol hexose after dark fermentation step [1]. Pre-treating the cassava starch by hydrolyzing with amylase and glucoamylase could increase the hydrogen production rates from 84.4 to 172 and 262 mlH2/h, respectively. The overall hydrogen yields were improved from 240 ml H2/g starch by dark fermentation to 402 ml H2/g starch by adding the photofermentation to the system [8] which shows that hydrogen production from cassava starch using a combination of dark fermentation and photofermentation is feasible. While using agricultural wastes as a carbon source it is important to choose the best pre-treatment option from the view of both system efficiencies and overall costs of the system. Acid pretreatment is decided to be the best option for corncob which is a cellulose-rich waste used to obtain biohydrogen. It is found that by dark fermentation of corncob with anaer­obic mixed culture 120 mL H2/g corncob and using the effluents of dark

fermentation in photofermentation resulted in 713 ml H2/g COD [139]. With a high content of carbohydrates sugar beet molasses is another good candidate for biohydrogen production by two-stage systems. With a low nitrogen content olive mill effluent (OME) is a good source for photofermentation but the dark color of color affects the light penetration negatively. Because of the high organic content and dark color of OME combining the system with dark fermentation could improve the overall yields. Treating the OME with active sludge cultures in dark fermentation step and using the effluents of this process in photofermentation step by R. sphaeroides O. U.001 resulted in 29 l H2/l OME hydrogen production [140]. The main wastewater of the cheese processing industry, cheese whey wastewater is used as carbon source in a two-stage system which is a combination of dark fermentation with anaerobic mixed sludge and photofermentation with Rhodo — pseudomonas palustris. Diluting the wastewater by 1/5 ratio with malic acid gave the highest yield of 349 ml H2/g COD [85]. Potato homogenate (PH) is utilized in an integrated study by combining dark and photofermentation sequentially. Dark fermentation was conducted by anaerobic mixed bacteria obtained from silo pit liquid and resulted as 0.7 mol H2/mol glucose and 350 mM VFA production with a concentration of 400 g/l medium. High fermentation effluents concentration negatively affected the performance of photofermentation therefore diluted efflu­ents were used with supplementation of Fe/Mg/phosphate. By using Rhodobacter capsulatus 4.9 mol H2/mol glucose hydrogen yields were achieved by using 5% fermentation effluent [141].

10.5 Conclusion

Producing hydrogen by biological methods have some advantages compared to chemical and physical methods such as; possibility to use sunlight and organic wastes as substrates which help environmental conversions and use of moderate conditions like room temperature which is very economical compared with sys­tems that need high energy. By combining the systems the individual problems of the systems can be solved and the overall performance can be improved. Com­bining dark and photofermentation is one of the most promising technologies for biological hydrogen production. This type of combinations can be operated in continuous mode for several days. It can be easily seen that the performance of the integrated systems is more than the individual systems. Moreover integration of dark and photofermentation could be an economical solution in terms of waste reduction. Taking into account all the system requirements and deciding the best reactor configurations hydrogen production yields can be improved effectively. While using the dark fermenter effluents for photofermentative hydrogen com­position it is important to adjust the composition for best biomass growth therefore biohydrogen production. Treating the highly concentrated wastes within the dark fermentation step by mixed anaerobic cultures which are already modified from wastewater treatment systems without sterilization then using the organic acids produced in photofermentation process is a very effective way of biohydrogen production

Acknowledgments Biofuels research in the laboratory of PCH is supported by FQRNT (Le Fonds quebecois de la recherche sur la nature et les technologies), Tugba Keskin thanks the TUBITAK DB-2214 (Turkey) for support.