Why is the ratio of H2/acetate over 2 in glucose fermentation by Caldicellulosiruptor saccharolyticus?

The ratio of H2/acetate in glucose fermentation shall be equal or less than 2. However, the ratio over 2 is found in the literature. Two possible reasons were proposed in this study: acetate oxidation or biomass growth via acetyl-CoA. In order to find out the right reason, glucose fermentation by Caldicellulosiruptor saccharolyticus was investigated. Under different glucose concentrations (1.0 g/L, 2.0 g/L, 3.5 g/L and 7.5 g/L), the Optical Density (OD) reached a maximum value of 0.35, 0.48, 0.53 and 0.55, respectively. It was found that the ratios of H2/acetate under different glucose concentrations were all greater than 2. When CH3-13COOH was added to the system, 60% of CH3-13COOH was converted to isotope ethanol. About the same amount of 13CO2 (0.01 mmol) was detected in both the control and isotopic experiments, illustrating acetate oxidation didnʹt occur in this study. The corrected ratio of H2/acetate after the compensation from biomass growth was around 2, demonstrating the biomass growth from acetyl-CoA was the right reason for the abnormal high ratio.