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.
Highlights
Plastid Overexpression of NDA2 in Chlamydomonas reinhardtii such as ferredoxin-NADP1 reductase and CO2 fixation, compete with the hydrogenase for the use of reduced ferredoxin (Gaffron and Rubin, 1942; Hemschemeier et al, 2008)
Immunodetection, realized using an antibody produced against recombinant Chlamydomonas reinhardtii NDA2 (CrNDA2) (Desplats et al, 2009), showed that CrNDA2 was overexpressed by about 1.7-fold in the transplastomic CrNDA2+ strain compared with the control strain (Fig. 1C)
We first checked that growth performances and photosynthetic electron transport activity are similar in CrNDA2+ and control cells (Supplemental Fig. S2)
Summary
Plastid Overexpression of NDA2 in Chlamydomonas reinhardtii such as ferredoxin-NADP1 reductase and CO2 fixation, compete with the hydrogenase for the use of reduced ferredoxin (Gaffron and Rubin, 1942; Hemschemeier et al, 2008). 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). 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
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