H<sub>2</sub> Production from Methyl Viologen–Dependent Hydrogenase Activity Monitored by Gas Chromatography

Bio-hydrogen production is an eco-friendly alternative to commercial H2 production, taking advantage of natural systems. Microbial hydrogenases play a main role in biological mechanisms, catalyzing proton reduction to molecular hydrogen (H2) formation under ambient conditions. Direct determination is an important approach to screen bacteria with active hydrogenase and accurately quantify the amount of H2 production. Here, we present a detailed protocol for determining hydrogenase activity based on H2 production using methyl viologen (MV2+) as an artificial reductant, directly monitored by gas chromatography. Recombinant Escherichia coli is used as a hydrogenase-enriched model in this study. Even so, this protocol can be applied to determine hydrogenase activity in all biological samples. Key features • This protocol is optimized for a wide variety of biological samples; both purified hydrogenase (in vitro) and intracellular hydrogenase (in vivo) systems. • Direct, quantitative, and accurate method to detect the amount of H2 by gas chromatography with reproducibility. • Requires only 2 h to complete and allows testing various conditions simultaneously. • Kinetic plot of H2 production allows to analyze kinetic parameters and estimate the efficiency of hydrogenase from different organisms.