| Abstract: | Biological conversion of solar energy into hydrogen is naturally realized by some microalgae species due to a coupling between the photosynthetic electron transport chain and a plastidial hydrogenase. While promising for the production of clean and sustainable hydrogen, this process requires improvement to be economically viable. Two pathways, called direct and indirect photoproduction, lead to sustained hydrogen production in sulfur-deprived Chlamydomonas reinhardtii cultures. The indirect pathway allows an efficient time-based separation of O2 and H2 production, thus overcoming the O2 sensitivity of the hydrogenase, but its activity is low. With the aim of identifying the limiting step of hydrogen production, we succeeded in overexpressing the plastidial type II NAD(P)H dehydrogenase (NDA2). We report that transplastomic strains overexpressing NDA2 show an increased activity of nonphotochemical reduction of plastoquinones (PQs). While hydrogen production by the direct pathway, involving the linear electron flow from photosystem II to photosystem I, was not affected by NDA2 overexpression, the rate of hydrogen production by the indirect pathway was increased in conditions, such as nutrient limitation, where soluble electron donors are not limiting. An increased intracellular starch was observed in response to nutrient deprivation in strains overexpressing NDA2. It is concluded that activity of the indirect pathway is limited by the nonphotochemical reduction of PQs, either by the pool size of soluble electron donors or by the PQ-reducing activity of NDA2 in nutrient-limited conditions. We discuss these data in relation to limitations and biotechnological improvement of hydrogen photoproduction in microalgae.A number of microalgal and cyanobacterial species are able to convert solar energy into hydrogen by photobiological processes and are therefore considered promising organisms for developing clean and sustainable hydrogen production (Benemann, 2000; Ghirardi et al., 2000; Rupprecht et al., 2006). In microalgae, hydrogen photoproduction results from coupling the photosynthetic electron transport chain and a plastidial FeFe] hydrogenase. Under most conditions, hydrogen photoproduction is a transient phenomenon that lasts from several seconds to a few minutes (Ghirardi et al., 2000; Melis and Happe, 2001). It has been considered a relic of evolution that may now serve, under certain environmental conditions, such as induction of photosynthesis in anoxia (Ghysels et al., 2013), as a safety valve that protects the photosynthetic electron transport chain from photodamage that results from overreduction of electron acceptors (Kessler, 1973; Tolleter et al., 2011). A major limitation to sustained hydrogen photoproduction is due to the oxygen sensitivity of the FeFe] hydrogenase (Happe et al., 2002; Stripp et al., 2009). Melis et al. (2000) proposed an elegant way to overcome this oxygen sensitivity through a time-based separation of hydrogen and oxygen production phases occurring, for instance, in response to sulfur deficiency in a closed environment. Another limitation is related to the electron supply for the hydrogenase coming from the photosynthetic electron transport chain (Cournac et al., 2002). This limitation is partly due to the fact that other metabolic pathways, such as ferredoxin-NADP+ reductase and CO2 fixation, compete with the hydrogenase for the use of reduced ferredoxin (Gaffron and Rubin, 1942; Hemschemeier et al., 2008). This is also due to upstream regulation of the electron transport chain, recently evidenced from the study of a Chlamydomonas reinhardtii mutant affected in proton gradient regulation-like1 (PGRL1)-mediated cyclic electron flow (CEF) around PSI. The strong enhancement of hydrogen production rates observed in the pgrl1 mutant was interpreted as the release of a control exerted by the transthylakoidal pH gradient on electron supply to the hydrogenase (Tolleter et al., 2011).Two pathways, direct or indirect, can supply electrons to the hydrogenase (Benemann, 2000; Melis and Happe, 2001; Chochois et al., 2009). In the direct pathway, the whole electron transport chain is engaged, with PSII supplying electrons to the plastoquinone (PQ) pool, the cytochrome b6/f complex, and, in turn, PSI, ferredoxin, and the FeFe] hydrogenase. Due to the high oxygen sensitivity of the FeFe] hydrogenase and to the fact that O2 is produced during photosynthesis at PSII, the direct pathway only operates when PSII activity is lower than mitochondrial respiration, thereby allowing anaerobiosis to be maintained. Such conditions can be obtained by decreasing PSII activity either by means of sulfur deprivation (Melis et al., 2000) or by decreasing light intensity in the photobioreactor (Degrenne et al., 2010). In the indirect pathway, reducing equivalents, stored as starch during the aerobic phase, are subsequently used to fuel hydrogen production. This implies a nonphotochemical reduction of the PQ pool that is at least in part mediated by NDA2, a type II NADH dehydrogenase discovered in C. reinhardtii chloroplasts (Desplats et al., 2009). RNA interference lines expressing lower levels of NDA2 show lower hydrogen production rates, and it was concluded that NDA2 is involved in hydrogen production by the indirect pathway (Jans et al., 2008; Mignolet et al., 2012). The indirect pathway allows for an efficient time-based separation of O2- and H2-producing phases because it does not involve PSII activity and does not produce O2. However, the indirect pathway has a much lower rate than the direct pathway (Cournac et al., 2002; Antal et al., 2009; Chochois et al., 2009). With the aim to identify limiting steps of hydrogen production in microalgae, we attempted to overexpress NDA2 in C. reinhardtii chloroplasts. We report that algal strains displaying a 2-fold increase in NDA2 show an increased nonphotochemical reduction of PQs and an increased rate of hydrogen production by the indirect pathway, the latter being only observed in conditions where stromal reducing equivalents are available in sufficient amounts. |
