Recovery of Clostridium from soil using Heat Shock enrichment technique and Agar Deeps

Some members of the genus Clostridium can produce butanol and hydrogen from renewable substrates contributing to biofuel production. In recent years, there has been a growing social demand for its utilization to realize a sustainable society. Clostridium is also attracting attention in the medical field, and research is being conducted to use it as a prodrug treatment and an intestinal bacterium to reduce cancer. Clostridium is a very attractive research subject. However, because Clostridium is an obligatory anaerobe, it requires expensive equipment not used to culture aerobic bacteria, making it difficult to start research on it. In this study, we developed an inexpensive and highly selective method for isolating Clostridium that does not require special equipment. This method combines two methods to increase the selectivity of Clostridium: the heat shock enrichment method, which selects non-spore-forming bacteria, and the agar deep method, which selects anaerobic bacteria based on their oxygen requirement. Using the isolation method developed in this study, we succeeded in isolating 11 new species of Clostridium. In total, 17 species of 3 genera were obtained from soil samples (0.2 g each) from only eight locations. Surprisingly, this represents 7.5 % of Clostridium reported to date.