Development and application of a functional CE-SSCP fingerprinting method based on [Fe–Fe]-hydrogenase genes for monitoring hydrogen-producing Clostridium in mixed cultures
Abstract
A Capillary Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP) method based on functional [Fe Fe]-hydrogenase genes was developed for monitoring the hydrogen (H-2)-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 H-2-producing mixed cultures incubated at temperatures ranging from 25 C to 45 degrees C. The hydA CE-SSCP profiles clearly differed between temperatures tested. Hence, they varied according to variations of the H-2 production performances. The HydA sequences amplified with the new primer set indicated that diverse Clostridium strains impacted the H-2 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 H-2-producing clostridial population dynamics in H-2 dark fermentation bioreactors.