Role of Carbonyl Cyanide m-Chlorophenylhydrazone in Enhancing Photobiological Hydrogen Production by Marine Green Alga Platymonas subcordiformis Chunqiu Ran, ²,‡ Xingju Yu, ² Meifang Jin, ² and Wei Zhang* ,²,§ Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China, Graduate School of Chinese Academy of Sciences, Beijing 100039, China, and Department of Medical Biotechnology, School of Medicine, Flinders University, Bedford Park SA 5042, Adelaide, Australia We demonstrated that a significant volume of H 2 gas could be photobiologically produced by a marine green alga Platymonas subcordiformis when an uncoupler of photophosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), was added after 32 h of anaerobic dark incubation, whereas a negligible volume of H 2 gas was produced without CCCP. The role of CCCP in enhancing photobiological H 2 production was delineated. CCCP as an ADRY agent (agent accelerating the deactivation reactions of water-splitting enzyme system Y) rapidly inhibited the photosystem II (PSII) activity of P. subcordiformis cells, resulting in a markedly decline in the coupled oxygen evolution. The mitochondrial oxidative respiration was only slightly inactivated by CCCP, which depleted O 2 in the light. As a result, anaerobiosis during the stage of photobiological H 2 evolution was established, preventing severe O 2 inactivation of the reversible hydrogenase in P. subcordiformis. The uncoupling effect of CCCP accelerates electron transfer from water due to a disruption of the proton motive force and release of ΔpH across the thylakoid membrane and thus enhances the accessibility of electron and H + to hydrogenase. The electrons for hydrogen photoevolution are mainly from the photolysis of water (90%). Upon the addition of CCCP, Chl a/b ratio increased, which implies a decrease in the light-harvesting PSII antennae or an increase in PSII/PSI ratio, possibly resulting in higher efficiency of utilization of light energy. The enhancement of H 2 evolution by the addition of CCCP is mostly due to the combination of the above three mechanisms. However, the disruption of the proton gradient across the thylakoid membrane may prevent a sustained photobiological H 2 evolution due to a shortfall of ATP generation essential for the maintenance and repair functions of the cells. Introduction Severe environmental pollution resulting from the use of fossil fuels and their limited supply prompts increasing research on clean and renewable energy. Hydrogen, the third most abundant element on the earth’s surface, is a clean and renewable energy that has attracted great attention during the past several decades. The first report on H 2 production in both dark and light by the green alga Scenedesmus obliquus under anaerobic conditions dated back to about 60 years ago (1, 2). However, H 2 was produced transiently at a very low yield, which was rapidly stopped in the light due to rapid deactivation of hydrogenase by photoevolved O 2 (3, 4). The fundamental studies on H 2 evolution in green algae have elucidated the basic pathways of hydrogen metabolism (5, 6). Under normal physiological conditions, PSII (photosystem II) utilizes solar energy in photosynthesis for the oxidation of water molecules, to release electrons, protons, and oxygen. Under the effect of electrical potential, these electrons are transported sequentially from Q A (the primary quinone acceptor of PSII) to Q B (the secondary quinone acceptor of PSII), PQ (plasto- quinone), Cyt/b 6 -f (cytochrome b 6 -f complexes), PC (plasto- cyanin), and PSI (photosynthesis system I). Thereafter, electrons are driven by light absorbed by PSI to NADP reductase. During the process of photosynthetic electron transfer, ATP and NADPH are generated to provide energy essential to the survival of algal cells. However, under anaerobic condition, electrons are eventually transferred to the Fe-S protein ferredoxin on the reducing side of PSI, which is an efficient electron donor to the hydrogenase. Protons released by the water oxidation enter into the lumen and transport across the membrane of the thylakoid via photosynthetic phosphorylation (7). The reversible hydrogenase in the stroma of the algal chloroplast accepts electrons from reduced ferredoxin and donates to 2H + to produce one H 2 molecule (5, 6, 8-10). One of the critical and necessary conditions for H 2 photo- production by green algae is to maintain an anaerobic environ- ment. It has been shown that oxygen severely inhibits the hydrogenase activity with a complete inactivation at partial pressures above 2% (11). Therefore, hydrogenase cannot be induced in algae cells under natural physiological condition, due to the powerful production of oxygen. To sustain H 2 production in green algae, photosynthetic O 2 evolution must be separated or uncoupled spatially and/or temporally from H 2 photoevolution (3). The only one successful example is the sustained H 2 photoproduction using a freshwater green alga, Chlamydomonas reinhardtii, by incubating the cells in a sulfur- * To whom correspondence should be addressed. Fax: +86-411- 84379069. E-mail: weizhang@dicp.ac.cn. ² Chinese Academy of Sciences. ‡ Graduate School of Chinese Academy of Sciences. § Flinders University. 438 Biotechnol. Prog. 2006, 22, 438-443 10.1021/bp050289u CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical Engineers Published on Web 01/28/2006