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Previously, we found that marine Chlorella pyrenoidosa strain IOAC707S could generate hydrogen (H 2 ) under nitrogen deprivation in seawater medium, without dark incubation, N 2 flushing or addition of protonophore. In this work, the H 2 yield of C. pyrenoidosa was enhanced, and the mechanism of H 2 photoproduction was investigated. It was found that C. pyrenoidosa could produce H 2 under hypoxic conditions, which indicated the hydrogenase of C.pyrenoidosa could tolerate a low concentration of O 2 . Nitrogen deprivation could lower the efficiency of photosyntem (PS) II photochemical activity and PSII oxygenic activities, which were favorable for rapid establishment of anoxia and efficient H 2 photoproduction, and thus significantly enhance H 2 photoproduction of C. pyrenoidosa . Pre-culture in nitrogen-deprived medium could further increase the H 2 yield of C. pyrenoidosa , for its more severe inhibition of the oxygen-evolving complex, electron transport and photochemical efficiency of PSII, and PSII oxygenic activities, which facilitated the establishment of anaerobiosis and activation of hydrogenase. [ABSTRACT FROM AUTHOR]

Abstract: The green alga Chlamydomonas reinhardtii is capable of photoproducing molecular hydrogen following sulphur deprivation, which results in anaerobiosis and a suppression of oxygen evolution and thus an alleviation of the inhibitory effect of oxygen on the hydrogenase. At the same time it transiently maintains a limited supply of electrons arising from photosystem II (PSII) to the hydrogenase (Melis and Happe Plant Physiol 2001; 127:740–748). In this work, using fast chl a fluorescence and P700 measurements, we show that ascorbate (Asc), a naturally occurring PSII alternative electron donor, is capable of donating electrons to PSII in heat-treated and sulphur-deprived cells and this can be significantly accelerated by supplementing the culture with 10 mM Asc. It also enhances, about three-fold, the photoproduction of hydrogen in cells subjected to sulphur deprivation as shown by gas chromatography. Similar stimulation was obtained in the presence of diphenylcarbazide (DPC), an artificial PSII electron donor. Asc and DPC also facilitated the anaerobiosis of cells, probably via super reducing the oxygen evolving complex while feeding electrons to PSII reaction centres and the linear electron transport chain, and ultimately to the hydrogenase – as shown by the significant DCMU-sensitivity of the light-induced Asc- and DPC-dependent re-reduction of P700+ and hydrogen evolution. [Copyright &y& Elsevier]