Biological H2 from Fuel Gases and from H2o

When photosynthetic bacteria Rhodocyclus gelatinosus and Rhodospirillum rubrum were cultured with CO in the gas phase, a CO-linked pathway was quickly induced. These bacteria perform a water-gas shift reaction with CO and H2O, and produce H2 and CO2 in nearly stoichiometric quantities. The hydrogenase, a terminal enzyme of the linked pathway, is extremely O2 resistant. In order to further investigate its tolerance to O2, a mutant deficient in the uptake hydrogenase was isolated from a blue-green mutant of R. rubrum G9. Upon further purification, we obtained a fraction containing only the CO-linked hydrogenase. This allows for the determination of the inhibitory effect of O2 on this hydrogenase without interference from uptake hydrogenase. Using a mass spectrometer to measure the H2/D2O exchange reaction, we confirmed that the hydrogenase was operative even with the simultaneous presence of up to 7% of O2 for 20 min. More than 70% of the reaction rate was recovered upon the removal of O2, a reversible property not normally observed by hydrogenases from most organisms. The addition of CO to a starved culture also enhanced the hydrogenase reaction rate significantly, although lesser effect was observed with the overall linked pathway. CO, or its oxidized product, may have a stimulatory effect on the longevity of the hydrogenase enzyme. This is not an issue in a scale-up bioreactor system where CO will be fed continuously. This report also proposes a scheme for the isolation of mutants deficient in various components of the CO-linked pathway in order to understand the reaction mechanism and the rate-limiting steps.