Protoplast Formation, L-Colony Growth and Regeneration of Clostridium beijerinckii NRRL B-592 and B-593 and Clostridium acetobutylicum ATCC 10132.

摘要

Clostridium beijerinckii (butylicum) and Clostridium acetobutylicum have industrial potential because of their ability to produce solvents from various carbohydrates via a two-stage fermentation. In the acidogenic stage acetate and butyrate are the primary products produced, while small amounts or no ethanol may also be produced. In the solventogenic stage industrially significant amounts of acetone and butanol are produced by C. acetobutylicum. Similar amounts of butanol and ethanol are produced by solventogenic C. beijerinckii along with either acetone or isopropanol, or both. Propanol and 1,2-propanediol are also produced in this stage by C. beijerinckii when grown on certain pentoses. Protocols for protoplast formation, L-colony cultivation, and regeneration of Clostridium beijerinckii NRRL B-592, B-593 and C. acetobutylicum ATCC 10132 were developed. Two osmotically reinforced media were formulated. Protoplasts of B-592, B-593, and ATCC 10132 grew as cell wall-deficient forms (L-colonies) when plated on the first medium (BLM) and continued to do so through at least 3 passages on this medium. The second (BRM) permitted the L-colonies to regenerate cell walls after transfer to this medium. Transferred C. beijerinckii B-592 L-colonies reverted to bacillary colonies at a frequency of 25%. Likewise, L-colonies of B-593 and C. acetobutylicum ATCC 10132 could be regenerated at frequencies of 7.0 and 8.6%, respectively. Thus, these procedures are suitable for genetic engineering of these industrial microorganisms using protoplast manipulation techniques. Reprints. (KT)