In acetate-limited chemostat cultures started with single-colony cultures of Thiobacilus versutus, a mutant appeared after approximately 85 volume changes. The inhomogeneity of the culture was detected by the development of two different types of colonies on agar plates. When a pure culture of the mutant was grown in a chemostat, parent colonies appeared after almost the same period of time. Electron micrographs of the mutant grown on butyrate showed the presence of fibrils surrounding the cells. The cells of the parent strain were bald when grown under the same conditions. The growth kinetics of the parent and the mutant were investigated in batch cultures with a variety of substrates and were found to be identical. Major differences between the two strains were observed during growth on mannitol; the mutant attained a lower yield and excreted large amounts of extracellular polysaccharides.The loss of homogeneity of a bacterial culture during cultivation in a rigidly controlled environment is not an unknown phenomenon. Calcott (3) recently summarized a number of studies in which parent-mutant transitions in chemostat cultures were observed. In continuous cultures, at nutrient concentrations much lower than the Ks, the outcome of the competition between parent and mutant depends on the pmaxIKs ratio. At higher substrate concentrations, the competition is always won by the organism with the lowest Ks, except for the situation in which the Monod curves cross over. In this situation, at substrate concentrations below the crossover point, K, is decisive, but at higher concentrations the organism with the highest growth rate is selected (16). Physiological causes of parent-mutant systems have been investigated in a number of cases (3). Although some of these studies have led to an understanding of the observed phenomenon, in some transitions take-over by a mutant without obvious reasons has been noticed.There are two possible ways to cope with the problem of the appearance of mutants in a pure culture. The first is to maintain a chemostat culture only for a short period of time, as suggested by Tempest (14); the second one is to wait until the most adapted organism has established itself (8).Here we report the appearance of a mutant with a different colonial morphology in a pure chemostat culture of Thiobacillus versutus. To identify the nature of this parentmutant transition, some growth kinetic and physiological parameters of both strains have been investigated in chemostat and batch cultures.
MATERIALS AND METHODSOrganism, media, and cultivation. A T. versutus strain formerly called A2 (ATCC 25364) (5) was a gift from J. G. Kuenen (Delft, The Netherlands); in this paper this strain is called WS. The organism was maintained on thiosulfate agar slants stored at 4°C and subcultured every 2 months. The basal medium for growth in batch culture was that of Taylor and Hoare (13), containing 2 ml instead of 5 ml of trace element solution as described by Gottschal and Kuenen (4).For growth on methanol, formate, methylamine...