ISOLATION AND CHARACTERIZATION OF CLOSTRIDIUM-BUTYRICUM DSM-5431 MUTANTS WITH INCREASED RESISTANCE TO 1,3-PROPANEDIOL AND ALTERED PRODUCTION OF ACIDS

Clostridium butyricum mutants were isolated from the parent strain DSM 5431 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and two selection procedures: osmotic pressure and the proton suicide meth od, Isolated mutants were more resistant to glycerol and to 1,3-propan ediol (1,3-PD) than was the wild type, and they produced more biomass. In batch culture on 62 g of glycerol per liter, the wild type produce d more acetic acid than butyrate, with an acetate/butyrate ratio of 5. 0, whereas the mutants produced almost the same quantities of both aci ds or more butyrate than acetate with acetate/butyrate ratios from 0.6 to 1.1. The total acid formation was higher in the wild-type strain. Results of analysis of key metabolic enzymatic activities were in acco rdance with the pattern of fermentation product formation: either the butyrate kinase activity increased or the acetate kinase activity decr eased in cell extracts of the mutants. A decreased level of the hydrog enase and NADH-ferredoxin activities concomitant with an increase in f erredoxin-NAD(+) reductase activities supports the conclusion that the maximum percentage of NADH available and used for the formation of 1, 3-PD was higher for the mutants (97 to 100%) than for the wild type (7 0%). In fed-batch culture, at the end of the fermentation (72 h for th e wild-type strain and 80 to 85 h for the mutants), 44% more glycerol was consumed and 50% more 1,3-PD was produced by the mutants than by t he wild-type strain.