4a). For the analysis of photohydrogen production in C. reinhardtii, O2 (PSII activity, respiration), CO2 (CO2 assimilation,

respiration, and fermentation), and H2 are the relevant gases. Moreover, mass spectrometric analyses allow differentiating between different isotopes of one element, so that O2 and CO2 production can be separated from O2 and CO2 consumption. Lindberg et al. (2004) described gas-exchange analyses in the filamentous cyanobacterium Nostoc punctiforme, in which isotopic tracing was applied. The addition of 18O2 allowed the calculation of respiratory activity, since photosynthetic activity mainly produces 16O2. In a similar manner, GSK923295 nmr the uptake of 13CO2 has been used as criterion for CO2 assimilation during photosynthesis, since 12CO2 production originates mostly from the oxidation of stored carbohydrates. For the analysis of the H2 metabolism of whole cells or the activity of hydrogenase enzymes, the exchange of heavy hydrogen (D2) (HD-exchange) has been described as being a valuable tool to monitor enzyme activities within the cells C646 in vivo (Cournac et al. 2004; Lindberg et al. 2004) or to study gas diffusion in isolated hydrogenases (Leroux et al.

2008) (in references Cournac et al. 2004 and Leroux et al. 2008; the HD-exchange technology and calculations are described in some detail). The analysis of photohydrogen production in C. reinhardtii has also benefited from this system. For instance, the direct (real-time) effect of the PSII inhibtor DCMU on H2 evolution could be analyzed utilizing the mass-spectrometric setup (Fig. 4b),

thereby allowing to show that the residual PSII activity of S-deprived algal cells only partially contributes to the ongoing in vivo H2-production rates (Hemschemeier et al. 2008). Combined with other experiments involving DCMU treatment, this observation allowed to affirm the model stated by Melis et al. (2000). This model already postulated that PSII activity in the first few hours of S deprivation is essential for H2 production since it is essential for starch Nutlin3a accumulation, but that water-splitting becomes dispensable during the H2-production phase, since the latter occurs mainly at the expense of accumulated organic reserves (Melis et al. 2000; Fouchard et al. 2005; Hemschemeier et al. 2008). Furthermore, the application 5-Fluoracil nmr of 13CO2 permitted to verify the strong decrease of in vivo CO2 uptake activity (Hemschemeier et al. 2008), which had been concluded from the degradation of the Rubisco before (Zhang et al. 2002). In vivo hydrogen production in microalgal cultures If neither a MS system nor a photobioreactor equipped with several electrodes is available, key parameters of S-deprived C. reinhardtii cells have to be analyzed in independent samples. If this is the case, the measuring conditions of the utilized devices should as much as possible be adapted to the conditions of the incubation flasks.