Reassessment of hydrogen tolerance in Caldicellulosiruptor saccharolyticus

Caldicellulosiruptor saccharolyticus has the ability to produce hydrogen (H2) at high yields from a wide spectrum of carbon sources, and has therefore gained industrial interest. For a cost-effective biohydrogen process, the ability of an organism to tolerate high partial pressures of H2 (PH2) is a critical aspect to eliminate the need for continuous stripping of the produced H2 from the bioreactor. Herein, we demonstrate that, under given conditions, growth and H2 production in C. saccharolyticus can be sustained at PH2 up to 67 kPa in a chemostat. At this PH2, 38% and 16% of the pyruvate flux was redirected to lactate and ethanol, respectively, to maintain a relatively low cytosolic NADH/NAD ratio (0.12 mol/mol). To investigate the effect of the redox ratio on the glycolytic flux, a kinetic model describing the activity of the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was developed. Indeed, at NADH/NAD ratios of 0.12 mol/mol (Ki of NADH = 0.03 ± 0.01 mM) GAPDH activity was inhibited by only 50% allowing still a high glycolytic flux (3.2 ± 0.4 mM/h). Even at high NADH/NAD ratios up to 1 mol/mol the enzyme was not completely inhibited. During batch cultivations, hydrogen tolerance of C. saccharolyticus was dependent on the growth phase of the organism as well as the carbon and energy source used. The obtained results were analyzed, based on thermodynamic and enzyme kinetic considerations, to gain insight in the mechanism underlying the unique ability of C. saccharolyticus to grow and produce H2 under relatively high PH2. C. saccharolyticus is able to grow and produce hydrogen at high PH2, hence eliminating the need of gas sparging in its cultures. Under this condition, it has a unique ability to fine tune its metabolism by maintaining the glycolytic flux through regulating GAPDH activity and redistribution of pyruvate flux. Concerning the later, xylose-rich feedstock should be preferred over the sucrose-rich one for better H2 yield.