Background: Sustainable biohydrogen production can be achieved by dark fermentation of organic wastes, and studies were carried out using a range of substrates and inocula. The bacterial populations involved were mainly identified as facultative anaerobes (Enterobacteriaceae) and strictly anaerobes (Clostridiales), and their dynamics in relation to H2 and metabolite production depends on the physical and environmental conditions of the bioreactor. This study has evaluated the use of length heterogeneity (LH)-PCR fingerprinting to detect changes in the microbial community during continuous hydrogen production under dark fermentation. Methods: A mesophilic continuous dark fermentation was established using coastal lake sediment as an inoculum in a synthetic medium with glucose as a substrate. The LH-PCR profiling associated to the sequencing of 16S rRNA genes was used for the characterization of the bacterial community and identification of species during a continuous production of H2. Results: The resulting dominant units of the communities present at both 24 h (batch) and 210 h (continuous culture) were affiliated to species of the genus Clostridium. Fluctuations based on their relative abundance over time were observed. At 24 h, a higher ratio was detected for the group Clostridium butyricum - Clostridium tertium followed by Clostridium bifermentans and Clostridium perfringens, while at 210 h, the group Clostridium aurantibutyricum - Clostridium acetobutylicum was the most abundant. Conclusions: The LH-PCR profiling has proven to be a sensitive and rapid method for the evaluation of the dynamics of a functional consortium formed by species of the genus Clostridium and has a potential for studies aimed at the optimization of biohydrogen production. © 2013 Di Bonito et al.; licensee Springer.

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