3-05: High-rate hydrogen production from biomass sugars via cell-free metabolic engineering

Hydrogen, an important commodity chemical and potential energy carrier, is currently produced from natural gas using a capital- and energy-intensive process.  A renewable alternative, biohydrogen, offers reduced greenhouse gas emissions and great potential for reducing the cost of delivered hydrogen.  In the system described here, biohydrogen is produced from biomass sugars, catalyzed by a cell-free cascade consisting of 15 thermostable enzymes in one reactor.  Substrates used include glucose, xylose, sucrose, cellulose, and pretreated biomass—the latter two enabled by combining enzymatic cellulose hydrolysis with the hydrogen production cascade.  Rate and yield were then increased using a metabolic model informed by enzyme kinetics and metabolomics assays of the reaction mixture.  As a result, we have achieved a reaction rate of 157 mmol H2/L/h, which is on the order of the highest biological hydrogen production rate achieved and is very close to the rate required for industrial application.  These important results highlight the excellent potential of cell-free conversions for the production of biocommodities such as hydrogen and demonstrate the power of metabolomics and modeling to greatly improve the performance of cell-free systems.