Q-PCR Methodology for Monitoring the Thermophilic Hydrogen Producers Enriched from Elephant Dung

Abstract

This study aims to create a quantitative polymerase chain reaction (q-PCR) methodology for monitoring the hydrogen-producing mixed cultures enriched from elephant dung using alpha-cellulose as a carbon source through five generations of repetitive sub-culture. The enriched thermophilic mixed cultures from the fifth cultivation cycle gave the highest hydrogen yield of 170.3 mL H2/g cellulose and were used to generate hydrogen from sawdust. Clostridium sp. and Thermoanaerobacterium sp. were the dominant bacteria in thermophilic mixed cultures with high hydrogen yield, according to polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE). q-PCR primers Chis150F and ClostIR, TherF and TherR, and BacdF and BacdR were developed to amplify the 16S rRNA genes of Clostridium sp., Thermoanaerobacterium sp., and Bacillus sp., respectively, for the quantification of hydrogen-producing bacteria in biohydrogen fermentation. Similar q-PCR analysis of Clostridium sp., Thermoanaerobacterium sp., and Bacillus sp. 16S rRNA gene amplification during hydrogen production from cellulose and sawdust revealed increasing gene copy number with time. The molecular approaches developed in this study can be used to monitor microbial communities in hydrogen fermentation processes efficiently.