Bioreactors for H2 production by purple nonsulfur bacteria.

Two types of laboratory-scale bioreactors were designed for H(2) production by purple nonsulfur bacteria. The bioreactors employed a unique type of hydrogenase activity found in some photosynthetic bacteria that functions in darkness to shift CO (and H2O) into H(2) (and CO2). The mass transport of gaseous CO into an aqueous bacterial suspension was the rate-limiting step and the main challenge for bioreactor design. Hollow-fiber and bubble-train bioreactors employing immobilized and free-living bacteria have proven effective for enhancing the mass transfer of CO. The hollow-fiber bioreactor was designed so that both a growth medium and CO (10% in N(2)) passed from the inside of the fibers to the outside within the bioreactor. Bacteria were immobilized on the outer surface of the hollow fibers. Hydrogen production from CO at an average rate of 125 ml g cdw(-1) h(-1) (maximum rate of 700 ml g cdw(-1) h(-1)) was observed for more than 8 months. The bubble-train bioreactor was built using polyvinyl chloride (PVC) tubing, wound helically on a vertical cylindrical supporting structure. Small bubbles containing CO were injected continuously through a needle/septum connection from the gas reservoir (20% CO). Up to 140 ml g cdw(-1) h(-1) of H(2) production activity was observed using this bioreactor for more than 10 days.

Characterization of the oxygen tolerance of a hydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivax gelatinosus.

A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O2. When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O2, 79% N2), its H2 evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O2 for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O2 inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 microM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O2, potential applications of this hydrogenase are discussed.