Reduced extracellular electron shuttles increase hydrogen production and substrate utilization in pure culture Clostridium beijerinckii fermentation

Biological hydrogen production is considered one reasonable alternative for generating H2 as a fuel that can be linked to electricity production in standard fuel cells. However, the molar yields are currently too low to be economically feasible and the inefficient production rates lead to the oversizing of bioreactors for possible industrial application. Lignocellulosic materials, the most abundant biomaterial on the earth, have been considered promising renewable substrates for biofuel production; however, better strategies need to be developed to increase sugar utilization in the fermentation process. This study aims to bridge the gap between the current status of biological hydrogen production and future industrial applications by developing an innovative physiological strategy (adding reduced electron shuttles) to increase the hydrogen production yield, kinetics and substrate utilization in a model hydrogen producing microorganism-Clostridium beijerinckii fermentation system.;Results show that adding extracellular hydroquinones (anthrahydroquinone disulfonate; AH2QDS) increased the hydrogen yield by 24-37% and the extent of xylose utilization by up to 56% when xylose was the sole substrate. Electron mass balance suggested that the increase of hydrogen yield was correlated to the pathway shift from butyrate production pathway to the acetate production pathway. Electron flows at the pathway level were examined to show that reduced electron shuttles increased the utilization of reduced ferredoxin to generate hydrogen, which contributed to increased hydrogen molar yield. Adding reduced electron shuttles also stimulated the kinetics of hydrogen production from different substrates including xylose, glucose and cellobiose. The increases of hydrogen production kinetics were consistent with the increases of substrate utilization rates and apparent growth rates. Further tests using mixed sugar at different glucose:xylose ratios demonstrated that adding reduced electron shuttles increased the total substrate utilization and hydrogen production by improving the xylose utilization, while concomitantly increasing the kinetics of hydrogen production and substrate utilization. Increasing concentrations of AH2QDS increased hydrogen production and xylose utilization at fixed glucose:xylose ratio of 1:1.;The results from this study show that adding reduced electron shuttles stimulated the hydrogen production rate at all tested sugar systems, increased hydrogen molar yield in xylose fermentation, and increased the xylose utilization in mixed sugar fermentation. Possible mechanisms have been proposed to address the influence of reduced electron shuttles on the fermentative physiology. Results suggest that it might be promising to use reduced electron shuttles to develop better strategies to extract biohydrogen from the lignocellulosic materials by allowing us to manipulate the fermentative metabolism at the physiological level.