The discovery of photosynthetic H2 production is based on the classic work of Gaffron and Rubin in 1942 (12). However, only since 1973, at the time of the energy crisis, has photosynthetic H2 production been investigated as a potential source of energy (13-41). Sustained photoevolution of H2 and 02 by microalgae was first demonstrated by Greenbaum in 1980 (15). Under anaerobic conditions, sustained photoevolution of H2 and 02 in microalgae can be readily demonstrated using a reactor-flow-detection system (Figure 2). Figure 3 presents a typical measurement of H2 and 02 production in Chlamydomonas in a helium atmosphere using the reactor-flow-detection system. The data clearly demonstrate that photoevolution of H2 and 02 can occur stably with a stoichiometric ratio of H2 to 02 of nearly 2:1 as expected for water splitting. Photoevolution of H2 and 02 can be sustained for weeks.

The advantage of the simultaneous H2 and 02 photoevolution is that it can potentially have a high energy conversion efficiency since electrons energized by the light reactions are used directly in the reduction of protons to produce H2 by the Fd/hydrogenase pathway (Figure 1). However, H2 production by this mechanism requires gas product separation since both H2 and 02 are produced simultaneously in the same volume. Furthermore, until an 02-insensitive hydrogenase is developed (42, 43), the 02 concentration in the algal suspension has to be kept low to maintain H2 production since the hydrogenase is sensitive to 02. Therefore, an efficient and inexpensive technique to separate and remove gas products is needed.

Another important aspect is that the photoevolution of H2 and 02 in microalgae is often saturated at a relatively low actinic intensity. This is probably due to three factors: (1) accumulation of a back-proton gradient because of the limited permeability of the thylakoid membrane to protons and the loss of ATP utilization due to the inactivation of the Calvin cycle, (2) partial inactivation of PSII activity owing to loss of C02 binding at a regulatory site on the PSII reaction center in the absence of C02, and (3) the normal nonlinear response of the light saturation curve of photosynthesis. Therefore, the potential still exists to improve the efficiency of photosynthetic H2 production. Further research is needed to eliminate these limiting factors. The first limitation may be solved on a short-term basis by using an appropriate proton uncoupler that dissipates the proton gradient across the thylakoid membrane (44), whereas the second could be overcome by eliminating the requirement of C02 binding through molecular engineering. The third limitation can, in principle, be overcome by reducing the antenna size of the photosynthetic reaction centers.

To avoid gas product separation and to increase efficiency, we have previously proposed a PSI and PSII reactor system that can potentially produce H2 and 02 in two separate compartments (32). This reactor system is based on the structure and function of isolated PSI and PSII reaction centers and on the catalytic activity of metallic platinum and osmium for H2 production (45-49). As illustrated in Figure 4, 02 and

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Figure 2. Schematic illustration of a dual-reactor-flow system for simulta­neous detection of H2, 02, and C02. The dual-reactor system permits two independent experiments to be performed simultaneously.

Figure 3. Sustained photoassimilation of H2 and 02 in Chlamydomonas 137c under anaerobic conditions and in the absence of C02. (Reproduced with permission from Ref. 35 Copyright 1996 Macmillan Magazines Limited)

Figure 4. A photosynthetic reactor system made of PSI and PSII electro­optical cells for production of H2 and 02 in separate compartments. (Reproduced with permission from Ref. 32 Copyright 1996 Human Press Inc.)

protons are produced by water splitting by an array of PSII reaction centers in the PSII compartment. The electrons acquired from water splitting are wired to the reducing side of a PSI array where the electrons are energized again by PSI photochemistry. The PSI-energized electrons are then used to evolve H2 by platinum — (or osmium-) catalyzed reduction of protons that come from the PSII compartment through a proton-conducting channel. As described previously, this system should be able to operate continuously since the number of protons and electrons generated can be balanced with the number consumed. We believe this is an important direction for future research. In this laboratory, research progress has been made in this direction (50-52). Recently, we have constructed a two-dimensional spatial array of PSI reaction centers on a gold surface at nanometer scale by a platinization anchoring technique (52).