Protoplast formation, L-colony growth, and regeneration of Clostridium beijerinckii NRRL B-592 and B-593 and Clostridium acetobutylicum ATCC 10132

Abstract

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. © 1989 Society for Industrial Microbiology.