Development and application of a functional CE-SSCP fingerprinting method based on [Fe–Fe]-hydrogenase genes for monitoring hydrogen-producing Clostridium in mixed cultures

A Capillary Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP) method based on functional [Fe–Fe]-hydrogenase genes was developed for monitoring the hydrogen (H2)-producing clostridial population in mixed-culture bioprocesses. New non-degenerated primers were designed and then validated on their specific PCR detection of a broad range of clostridial hydA genes. The hydA-based CE-SSCP method gave a specific and discriminating profile for each of the Clostridium strains tested. This method was validated using H2-producing mixed cultures incubated at temperatures ranging from 25 °C to 45 °C. The hydA CE-SSCP profiles clearly differed between temperatures tested. Hence, they varied according to variations of the H2 production performances. The HydA sequences amplified with the new primer set indicated that diverse Clostridium strains impacted the H2 production yields. The highest performances were related to the dominance of Clostridium sporogenes-like hydA sequences. This CE-SSCP tool offers highly reliable and throughput analysis of the functional diversity and structure of the hydA genes for better understanding of the H2-producing clostridial population dynamics in H2 dark fermentation bioreactors.