4-28: Application of a multiplex real-time qPCR for detecting degenerated and non-degenerated Clostridium acetobutylicum ATCC 824 in continuous fermentation

Biobutanol, one of the promising biofuels for future, has the potential to meet the needs of sustainable and green energy systems. Solventogenic clostridia lose their solvent formation capabilities during repeated sub-culturing of batch cultures or during a continuous fermentation. This phenomenon is known as ‘degeneration’. Degeneration of solvent producing microorganisms generally occurs during repeated sub-culturing of batch culture or during continuous fermentation. It is one of the limiting factors for stable and high butanol production. The degeneration is caused by the loss of the pSOL1 plasmid that carries essential genes involved in solvent production. Therefore, a method is needed to detect and quantify the pSOL1 plasmid in Clostridium acetobutylicum ATCC 824. In this study, during butanol fermentation, the populations of C. acetobutylicum and the portion of degenerated cells were monitored by using multiplex real-time qPCR with newly designed primers and probe sets, C. aceto set and DGS set on the basis of the 16S rDNA sequence and pSOL1 sequence. To confirm the effectiveness of the technique, the multiplex real-time qPCR results were compared with those obtained by plating. In conclusion, using multiplex real-time qPCR, degenerated Clostridium acetobutylicum was successfully detected and quantified.