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The formate pathway and NADH pathway as two common hydrogen-producing metabolic pathways have been well characterized to understand and improve biohydrogen production. These two pathways have been thought to be separate and have been independently investigated. However, in this study, perturbation of genes (hycA, fdhF, fhlA, ldhA, nuoB, hybO, fdh1, narP, and ppk) in Enterobacter aerogenes related to the formate pathway or NADH pathway revealed that these two pathways affected each other. Further metabolic analysis suggested that a linear relationship existed between the relative change of hydrogen yield in the formate pathway or NADH pathway and the relative change of NADH yield or ATP yield. Thus, this finding provides new insight into the role of cellular reducing power and energy level in the hydrogen metabolism. It also establishes a rationale for improving hydrogen production from …