Spinach chloroplasts and Clostridium butyricum cells were immobilized in 2% agar gel. Crude ferredoxin isolated from spinach and benzyl viologen were used as electron carriers. The optimum pH for both NADP reduction by immobilized chloroplasts and for hydrogen evolution by immobilized Cl. butyricum was 8.0. The optimum temperature was between 25 and 30°C for NADP reduction by immobilized chloroplasts, and 37°C for hydrogen evolution by immobilized cells. The total amount of hydrogen evolved in 6 h was 41 μmol/mg Chl for the immobilized chloroplast-benzyl viologen-immobilized Cl. butyricum system, and 11 μmol/mg Chl for the immobilized chloroplast-ferredoxin-Cl. butyricum system. The systems evolved only a trace amount of hydrogen when dichlorophenyldimethylurea was added. The immobilized chloroplast-benzyl viologen-immobilized Cl. butyricum system evolved hydrogen continuously for 6 h, and immobilized Cl. butyricum retained the initial hydrogenase activity. However, the photoreduction activity of chloroplasts decreased to 30% of the initial activity after 6 h of reaction.
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