2003), the use of synchronized versus non-synchronized cultures (Kosourov et al. 2002), certain amounts of sulphate in the medium (Zhang et al. 2002; Kosourov et al. 2002), as well as temperature

and the growth YM155 phase of the pre-culture (the authors’ own unpublished results) have significant effects on the time it takes for the algal culture to start producing H2 and on the amounts of H2 that are accumulated. Light intensity has a particular impact on the development of S-depleted C. reinhardtii cultures (Laurinavichene et al. 2004) similar to that the culture density has (Kosourov et al. 2002), since the latter determines the amount of light that can penetrate the cell suspension. Furthermore, Saracatinib clinical trial the availability of carbon (C) sources strongly influences the H2 metabolism of S-deprived C. reinhardtii cultures. Standard TAP medium contains acetate, which can be used by this species as a C source both for growth and respiration. Chlamydomonas can be grown in TAP without supplemental CO2, whereas some researchers use TAP as growth medium

but furthermore provide extra CO2 (up to 5%), and in some laboratories, C. reinhardtii is grown photoautotrophically in HSM medium or other minimal media (Harris 1989, 2009). For H2 production upon S deprivation, acetate is essential for the establishment of anaerobic conditions (Fouchard et al. 2005), unless PSII find more activity is rapidly diminished by applying light stress to the cells grown in dimmed light (Tsygankov et al. 2006; Kosourov et al. 2007). On the other hand, the attempts of several researchers to rapidly induce H2 production in illuminated algae by applying the PSII inhibitor DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) did not result in any H2 accumulation because of the dependence on electrons provided by organic reserves which were

built up using electrons provided by PSII (Fouchard et al. 2005; Hemschemeier et al. 2008). Not in the least, the activity of the Calvin Benson cycle plays a significant role in H2 production by C. reinhardtii, since it acts as a competing electron sink. For instance, it has been shown that a Ribulosebisphosphate carboxylase/oxygenase (Rubisco)-deficient strain produces H2 in full TAP medium (Hemschemeier below et al. 2008). On the other hand, C. reinhardtii transformants having a reduced ratio of photosynthetic O2 evolution and respiratory O2 uptake establish anaerobiosis and develop in vitro hydrogenase activity in full medium upon illumination, but they do not produce significant amounts of H2 unless the Calvin Benson cycle is inhibited (Rühle et al. 2008). As a consequence of all these affecting parameters, we recommend the following to stably establish photohydrogen production in S-deprived C. reinhardtii cells: The pre-culture should have a chlorophyll content of 20–25 μg ml−1. Too thin cultures will not establish anaerobic conditions; too dense cultures will have a less efficient photosynthetic activity.