Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase

The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGA-CThydA) using a log-phase expression promoter P( pckA ) to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum FeFe]-hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38?°C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40?°C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight.