Molecular detection of the clostridia in an anaerobic biohydrogen fermentation system by hydrogenase mRNA-targeted reverse transcription-PCR

Abstract

Molecular biological approaches were developed to monitor the potential biohydrogen-producing clostridia in an anaerobic semisolid fermentation system that used brewery yeast waste as the fermentation substrate. The denaturing gradient gel electrophoresis with 16S rDNA gene-targeted polymerase chain reaction (PCR) analysis was employed to confirm the existence of clostridia in the system. Remarkably, reproducible nucleotide sequences of clostridia were obtained from different hydrogen production stages by using hydrogenase gene-targeted reverse transcription (RT)-PCR. These RNA-based information suggested that the predominant hydrogen-producing strains possess either a specific Clostridium pasteurianum-like or a specific Clostridium saccharobutylicum-like hydrogenase sequence. Comparison of the hydrogenase gene-targeted sequence profiles between PCR and RT-PCR revealed that the specific C. pasteurianum-like hydrogenase harboring bacterial strains were dominant in both mRNA and bacterial population level. On the other hand, the specific C. saccharobutylicum-like hydrogenase harboring strains expressed high level of hydrogenase mRNA but may not be dominant in population. Furthermore, quantitative real-time RT-PCR analysis showed the expression pattern of the clostridial hydrogenase mRNA and may serve as an activity index for the system.

Novel Clostridium populations involved in the anaerobic degradation of Microcystis blooms

“…During the anaerobic incubation of the Microcystis slurry, Clostridium species appear to have the collaborative ability to hydrolyze initial substrates and produce low molecular weight intermediates, such as acetate and ethanol, and even directly generate biohydrogen gas (Chang et al, 2006;Yokoyama et al, 2007;Seedorf et al, 2008). The occurrence of these processes in the fermentation systems was confirmed by the presence of methanogenic Archaea (our unpublished data), which anaerobically utilize low molecular weight intermediates (for example, acetate or H 2 /CO 2 ) to produce CH 4 (Supplementary Figure S6).…”

Succession of the Bacterial Community and Dynamics of Hydrogen Producers in a Hydrogen-Producing Bioreactor

“…The Fe-hydrogenase gene (hydA), which is usually involved in proton reduction (H 2 production) to dispose of excess reducing equivalents (1,2,22) in Clostridium spp. and sulfate reducers, has recently been used as a molecular marker to distinguish potential hydrogen-producing bacteria in mixed cultures (3,8,23). Therefore, in this study, the V3 regions of the 16S rRNA gene and the hydA gene were used as biomarkers to investigate the succession of the bacterial community during hydrogen production in a batch culture.…”

“…Anaerobic mixed cultures are frequently used as inocula in hydrogen production (3,4,10). Compared with pure cultures, mixed cultures have the advantage of more technical feasibility and the potential for using complex carbohydrates as substrates (14), possibly because different members of the bacterial community play complementary or mutually beneficial roles in utilizing substrates, providing growth factors, eliminating feedback inhibition, etc.…”

Genetic Diversity of Hydrogen-Producing Bacteria in an Acidophilic Ethanol-H2-Coproducing System, Analyzed Using the [Fe]-Hydrogenase Gene

“…They have been divided into hydA (large-subunit) and hydB (small-subunit) classes. Chang et al specifically detected clostridia in an anaerobic H 2 biofermentation system by use of [Fe]-hydA (9). However, specific detection of all fermentative HPB in microbial communities based on universal…”