The use of hydrogen (H 2 ) as a fuel offers enhanced energy conversion efficiency and tremendous potential to decrease greenhouse gas emissions, but producing it in a distributed, carbon-neutral, low-cost manner requires new technologies. Herein we demonstrate the complete conversion of glucose and xylose from plant biomass to H 2 and CO 2 based on an in vitro synthetic enzymatic pathway. Glucose and xylose were simultaneously converted to H 2 with a yield of two H 2 per carbon, the maximum possible yield. Parameters of a nonlinear kinetic model were fitted with experimental data using a genetic algorithm, and a global sensitivity analysis was used to identify the enzymes that have the greatest impact on reaction rate and yield. After optimizing enzyme loadings using this model, volumetric H 2 productivity was increased 3-fold to 32 mmol H 2 ·L. The productivity was further enhanced to 54 mmol H 2 ·L −1 ·h −1 by increasing reaction temperature, substrate, and enzyme concentrations-an increase of 67-fold compared with the initial studies using this method. The production of hydrogen from locally produced biomass is a promising means to achieve global green energy production.hydrogen | biomass | in vitro metabolic engineering | metabolic network modeling | global sensitivity analysis

In vitro hydrogen generation represents a clear opportunity for novel bioreactor and system design. Hydrogen, already a globally important commodity chemical, has the potential to become the dominant transportation fuel of the future. Technologies such as in vitro synthetic pathway biotransformation (SyPaB)-the use of more than 10 purified enzymes to catalyze unnatural catabolic pathways-enable the storage of hydrogen in the form of carbohydrates. Biohydrogen production from local carbohydrate resources offers a solution to the most pressing challenges to vehicular and bioenergy uses: small-size distributed production, minimization of CO 2 emissions, and potential low cost, driven by high yield and volumetric productivity. In this study, we introduce a novel bioreactor that provides the oxygen-free gas phase necessary for enzymatic hydrogen generation while regulating temperature and reactor volume. A variety of techniques are currently used for laboratory detection of biohydrogen, but the most information is provided by a continuous low-cost hydrogen sensor. Most such systems currently use electrolysis for calibration; here an alternative method, flow calibration, is introduced. This system is further demonstrated here with the conversion of glucose to hydrogen at a high rate, and the production of hydrogen from glucose 6-phosphate at a greatly increased reaction rate, 157 mmol/L/h at 60°C.