Hydrogen production by cyanobacteria and microalge through photosynthesis can be represented by the following reactions:

In indirect biophotolysis, the electrons are derived from water by photoautotrophic cells. As presented in reactions [13.3] and [13.4], the process consists of two stages in series: the first one is photosynthesis for carbohydrate accumulation and the second one, is dark fermentation of the endogenous carbohydrates for hydrogen production. In this way, the oxygen and hydrogen evolutions may be temporally and/or spatially separated (Benemann, 1996). This separation not only avoids the incompatibility of oxygen and hydrogen evolution (e. g. enzyme deactivation and the explosive property of the gas mixture), which are key barriers to direct biophotolysis, but also makes hydrogen purification relatively easy, because CO2 can be conveniently removed from the generated H2/CO2 mixture (Belafi-Bako et al., 2006).

Cyanobacteria have attracted more research interest for hydrogen production via indirect biophotolysis than microalgae. Such cyanobacteria species include Anabaena sp., Spirulina sp., marine cyanobacteria such as Calothrix sp., Synechococcus sp. and Geobacter sp. Anabaena cylindrical is a well-known hydrogen producing cyanobacterium, but Anabaena variabilis has received more attention in the recent years, because of higher hydrogen yields compared to the other species (Masukawa et al., 2001). Emphasis has been given to increase the activity of hydrogen producing enzymes and to develop mutants of Anabaena sp. to increase the rate of hydrogen production. However, at the present time, the hydrogen production rate by Anabaena sp. is considerably lower than that obtained by dark or photo­fermentations which are described below (Pinto et al., 2002; Liu et al., 2006a).

Nowadays, indirect biophotolysis, just like direct biophotolysis, is an immature technology, applied only at laboratory scale. It should be noted that indirect water photolysis, is under active research and development, since several factors are still crucial for further improvement in technology. Environmental conditions, such as light, temperature, salinity, nutrient availability and gas atmosphere (the presence of oxygen, nitrogen or methane) play an important role in the hydrogen production efficiency (Dutta et al., 2005). In addition, in order to improve hydrogen production rates and yields using cyanobacteria, methods such as screening of wild-type strains possessing highly active hydrogen evolving enzymes (nitogenases and/or hydrogenases) (Pinto et al., 2002), or genetic modification of strains to increase the hydrogenase activity, are under investigation. Finally, optimization of cultivation conditions such as light intensity, pH, temperature, and nutrient content, will contribute to increased H2 production.