The methylotrophic yeast Hansenula pozymorpha was grown in a chemostat with a medium containing a mixture of glucose (c6) and methanol (C,) (87.8% c6: 12.2% C1, w/w) as sole carbon source and NH,+ as sole nitrogen source. At a constant growth rate (D 0.10 h-l) the influence of the carbon :nitrogen ratio (C : N) of the inflowing medium on the cellular and enzymic composition of the cells was studied. Three distinct growth regimes were recognized. A medium with a C : N ratio c 12 resulted in carbon-limited growth (high cellular protein content, low carbohydrate content) and under these conditions glucose and methanol were utilized simultaneously. A medium with a C : N ratio >31 resulted in nitrogen-limited growth (low protein but high carbohydrate content of the cells) and the cells metabolized only glucose. A transition growth regime was observed during growth on media with intermediate C : N ratios (12 < C:N > 31). When assessed from both substrate consumption and cellular composition, growth was double-substrate (carbon and nitrogen)-limited. In this transition growth regime, changes in carbon metabolism and the cellular and enzymic composition of the cells were found. With increasing C: N ratios in the growth medium a gradual repression of the synthesis of methanol-assimilating and dissimilating enzymes was found. This effect was most pronounced for alcohol oxidase, and as a consequence the cells switched from the utilization of the carbon substrate mixture to growth on glucose alone. The data presented suggest that the range within which double-substrate-limited growth can be expected is predictable from the composition of cells grown under single substrate limitation.
