One of the most remarkable events in investigating H2 metabolism in green algae was the discovery of sus­tained H2 photoproduction in C. reinhardtii cultures un­der sulfur-deprived conditions (Melis et al., 2000; Ghirardi et al., 2000). In this approach, the long-term H2 photoproduction is possible due to a metabolic switch occurring in sulfur-deprived algal cells, which separate temporarily the O2-evolving, aerobic (Eqn (21.5)) and H2-producing, anaerobic (Eqn (21.6)) stages in the same culture.

Sulfur deprivation causes the partial and reversible inhibition of PSII-dependent water-splitting activity in algae. As demonstrated by Wykoff et al. (1998), C. rein — hardtii cells lose gradually up to 75% of the initial PSII ac­tivity within the first 24 h of sulfur starvation. The reduction of H2O-splitting activity was also shown under deprivation of other nutrients, such as nitrogen, phosphorus, Fe and Mn (Wykoff et al., 1998; Ghirardi et al., 2000; Philipps et al., 2012) but usually with a significant delay, as compared to sulfur starvation. The repression of the linear electron flow from the PSII centers under nutrient starvation is a common phenom­enon not only for green algae but also for cyanobacteria (Sauer et al., 2001) and high plants (Dietz and Heilos, 1990; Ferreira and Teixeira, 1992), and is a good example of how photosynthetic organisms adjust the rate of photosynthesis to the stress conditions. Continuous nutrient starvation reduces the capacity for de novo pro­tein biosynthesis and CO2 fixation, and, as a result, decreases the demand of the cells in the photosynthetic reductants (Grossman, 2000). Under these conditions, the repression of the O2-evolving activity and linear electron flow protects the photosynthetic apparatus from overreduction, generation of reactive oxygen spe­cies and photoinhibition. Numerous experiments showed that the inhibition of O2-evolving activity in nutrient-deprived cells is mostly caused by the loss of PSII centers (Kolber et al., 1988; Wykoff et al., 1998). In the absence of basic nutrients such as nitrogen, phos­phorus or sulfur, the cells cannot efficiently resynthesize D1 protein, the key component of the PSII complex, and the PSII repair cycle is blocked (Melis and Chen, 2005).

Nutrient deprivation, however, has little effect on cellular respiration, especially in the first few days (Melis et al., 2000). As a result, the rate of photosynthetic

O2 evolution falls below the rate of respiratory O2 up­take and algal cultures, if sealed in photobioreactors with a little headspace volume, become anaerobic in the light (Melis et al., 2000). In sulfur-deprived cultures, this usually happens within the first 24 h. The establish­ment of anaerobiosis in the sealed photobioreactor induces the expression of [Fe—Fe]-hydrogenase

enzymes in algal cells (Happe and Kaminski, 2002; Forestier et al., 2003). [Fe-Fe]-hydrogenase accepts elec­trons from the photosynthetic electron-transport chain and algae start producing H2 in the light. If not opti­mized, H2 photoproduction lasts for several days (Melis et al., 2000; Ghirardi et al., 2000). Under continuous flow of the medium containing sulfur in a micromolar range, algae produce H2 gas for several months, although at substantially low rates (Fedorov et al., 2005; Laurinavi — chene et al., 2006). The most interesting results obtained from sulfur-deprivation experiments are summarized in Table 21.1. As shown in the table, the rates and the yields of H2 photoproduction in algal cultures vary depending on the experimental conditions. In C. reinhardtii wild — type strains the rate usually does not exceed 13 mmol mg/Chlh, while some genetically modified strains are able to produce H2 with rates up to 27 mmol mg/Chl h.

In green algae, sulfur deprivation demonstrates the strongest inhibitory effect on PSII (Wykoff et al., 1998; Ghirardi et al., 2000) most probably due to the lowest intracellular sulfur reserves. The later studies showed that the same principle works for phosphorus — deprived (Batyrova et al., 2012) and nitrogen-deprived (Philipps et al., 2012) microalgae. Phosphorus-depleted cultures start producing H2 gas only after the initial growth period on the phosphorus-free medium. Growing algae utilize an intracellular pool of reserved phosphorus. When they reach the point of phosphorus starvation, PSII in algal cells is inactivated in a manner similar to sulfur-starved algae. Despite a considerable delay in the establishment of anaerobic conditions, phosphorus-deprived algae produce only slightly less H2 gas than sulfur-deprived cultures under the same experimental conditions, but they also accumulate less starch reserves during the growth stage (Batyrova et al., 2012). Nitrogen-deprived algae behave in a similar way. They produce H2, but with a significant delay (Philipps et al., 2012). In contrast to phosphorus — deprived cells, the delay in nitrogen-deprived cultures seems to be caused by slower inactivation of PSII cen­ters. These algae also accumulate significantly more starch reserves than sulfur-deprived algae, but degrade them slower. As a result, they produce considerably less H2 overall. Inability to efficiently channel electrons from carbohydrate oxidation toward the hydrogenase enzyme likely causes the degradation of the Cyt bg/ complex upon nitrogen starvation and lowers amounts of PetF. Nevertheless, nitrogen-deprived cultures may have a higher potential for the light-independent H2 production pathway (Philipps et al., 2012).

The vast majority of experiments completed on H2 production by nutrient-deprived microalgae have been undertaken so far with C. reinhardtii cultures. However, other species of green algae also produce H2 gas under this condition (Winkler et al., 2002; Skjanes et al., 2008; Meuser et al., 2009). Successful H2 production has been demonstrated by sulfur-depriving C. noctigama and Chlamydomonas euryale (Skjanes et al., 2008). Sulfur — deprived cultures of S. obliquus, Platymonas subcordi/or — mis, Scenedesmus vacuolatus, Chlamydomonas vectensis, Chlamydomonas pyrenoidosa, Desmodesmus subspicatus, Pseudokirchneriella subcapitata, Chlamydomonas moewusii and Lobochlamys culleus generate only minor amounts of H2 gas (Winkler et al., 2002; Guan et al., 2004; Skjanes et al., 2008; Meuser et al., 2009). Some other tested spe­cies, such as Dunaliella salina and C. vulgaris demonstrate no detectible hydrogenase activities and do not produce H2 under sulfur-deprived conditions (Cao et al., 2001; Winkler et al., 2002).

H2 photoproduction in nutrient-deprived algae de­pends both on the residual PSII activity remaining in cells after inactivation (Antal et al., 2003; Kosourov et al., 2003) and on the catabolism of starch accumulated during the first 18—24 h of sulfur deprivation (Fouchard et al., 2005; Ghirardi et al., 2000; Kosourov et al., 2003; Tsygankov et al., 2002; Zhang et al., 2002). The contribu­tion of these two pathways in H2 photoproduction varies depending on the stage of sulfur deprivation (Laurinavichene et al., 2004) and, most probably, on the strain used in the experiment (Chochois et al., 2009). In the wild-type C. reinhardtii CC-124 strain, starch degradation may donate up to 20% electrons to hydrog- enase enzymes in the middle of the H2 production stage (Kosourov et al., 2003). Besides contribution to H2 photoproduction, the degradation of starch and other stored organic substrates fuels the respiratory consump­tion of O2 produced by the residual PSII activity and therefore is responsible for maintaining culture anaero — biosis and for protecting hydrogenase enzymes from O2 inactivation (Fouchard et al., 2005; Kosourov et al., 2007). The importance of efficient respiration for H2 photoproduction was further proved by inhibitory analysis (Antal et al., 2009) and in the respiratory — deficient mutants (Table 21.1).

Under photoheterotrophic conditions (when acetate is the only substrate), accumulation of starch in algae in the beginning of sulfur deprivation is tightly linked to consumption of acetate from the medium. The respi­ration of acetate provides the cells with a substrate for CO2 fixation. It also helps with the establishment of anaerobiosis in the photobioreactor (Kosourov et al., 2007). The use of acetate in the growth medium, however, increases the expense associated with maintenance of

TABLE 21.1 The Rates and Yields of H2 Photoproduction by the Sulfur-Deprived, Wild-Type C. reinhardtii Strains and Some Mutants under Different Experimental Conditions

the system and should therefore be avoided. Recently, Tsygankov et al. (2006) showed that H2 photoproduc­tion in green algae is also possible under autotrophic conditions, when cultures are supplied with CO2 gas instead of acetate. In this experiment, authors used the microprocessor-controlled bioreactor system for a controllable addition of CO2 gas. The unique aspect of this system is that cells are provided with appropriate amounts of CO2, in accordance with the demands of the culture. Under these conditions, algae accumulate enough starch that can later be used for the establish­ment of anaerobiosis in the culture and for the removal of O2 during the H2 production stage (Kosourov et al., 2007). Using the special light regime, the authors gener­ated almost the same amounts of H2 gas as in photoheter­otrophic cultures (Tsygankov et al., 2006; Tolstygina et al., 2009).