[FeFe]-hydrogenase gene quantification and melting curve analysis from hydrogen-fermenting bioreactor samples

In this study, quantitative PCR (qPCR) was used to quantify [FeFe]-hydrogenases and subsequently melting curves were analyzed from hydrogen-fermenting, mixed-culture bioreactor samples. Denaturing gradient gel electrophoresis (DGGE) analysis was also performed to the reactor samples revealing a clostridial dominance in the reactor. Primers targeting [FeFe]-hydrogenases were designed based on known clostridial [FeFe]-hydrogenase gene sequences and tested with several clostridial strains. The results show that amplification efficiencies of four different clostridia are highly similar and melting curves of the clostridial strains were within 1 °C of each other. We compared the melting curves to the hydrogen percentage and observed a correlation between the results. The closer the melting curves were to those of clostridia, the better the hydrogen production. Based on these results, the primers and melting curve analysis of [FeFe]-hydrogenase amplicons can be used for analysing hydrogenase genes from bioreactor samples.