Preparation of Refractile Spores of Clostridium thermosaccharolyticum Involves a Solventogenic Phase

Conversion of vegetative cells of Clostridium thermosaccharolyticum to refractile endospores was achieved by sequential transfer and dilution at each generation, with a final dilution into a sporulation medium that contained xylan supplemented with excess calcium. The subsequent growth was synchronous and resulted in elongated, solventogenic cells that were then shifted to 35 degrees C to permit further differentiation without cell division. The synchronized cells grown in xylan medium supplemented with Ca gluconate produced total solvents that reached 9.63% (vol/vol). One hundred percent of these elongated solventogenic cells (4.84 x 10 cells per ml) entered the sporangial stage and continued to differentiate into refractile spores. Only cells sequentially transferred and diluted at a critical time of the growth cycle are synchronized, induced to elongate (>/=fourfold), become highly solventogenic in the presence of excess calcium, and are converted to a homogeneous population of refractile spores.