For its parental strain Y-50049, cell mass was low and cell growth appeared ceased after 24 h. When cell viability was tested using solid YM of 2% glucose inoculated with the cell cultures at different time point, the parental strain Y-50049 showed a very poor growth response at 24 h and no Savolitinib supplier viable cell growth was observed at any later time points (Figure 2B). On the other hand, the ethanol-tolerant strain Y-50316 displayed a normal growth for samples taken at 24 h till 96 h after the ethanol challenge. Reduced cell
growth and cell lyses were observed for samples taken at 120 to 168 h after ethanol challenge when the fermentation was completed for several days. Figure 2 Cell viability and growth under the ethanol stress. Cell viability of ethanol- and inhibitor-tolerant mutant Saccharomyces cerevisiae NRRL Y-50316 (●) and its parental inhibitor-tolerant strain NRRL Y-50049 (○) in response to 8% (v/v) ethanol challenge as measured by OD600 on a liquid YM of 2% glucose (A) and culture appearance of cell growth on a solid YM of 2% glucose (B). The time point at the addition of ethanol to the medium was designated as 0 h. Cell
growth on YM plate was evaluated 7 days after incubation at 30°C. Glucose consumption and ethanol production With the addition of ethanol at 8% (v/v) 6 h after inoculation, yeast growth of the two strains showed a similar OD VX-689 price reading briefly followed by an obvious separation after 18 h between the ethanol-tolerant strain Y-50316 and its parental strain Y-50049. Strain Y-50316 exhibited a continued growth through a log phase in 48 h to reach an OD600 reading of 1.3 buy AMN-107 when the ethanol concentration was 75.1
g/L (9.5%, v/v) (Figure 3A and 3B). On the other hand, Y-50049 ceased growth since 18 h and apparently went into cell lysis stages mafosfamide and never recovered. Consequently, no glucose consumption and ethanol conversion were observed for Y-50049 under the ethanol challenge (Figure 3B). In contrast, the ethanol-tolerant strain Y-50316 displayed an accelerated glucose consumption and ethanol conversion after 24 h (Figure 3B). At 120 h, glucose was almost exhausted and the total ethanol concentration reached 96 g/L. Production of glycerol and acetic acid under the conditions of this study was insignificant (data not shown). Figure 3 Fermentation profiles under the ethanol stress. Comparison of cell growth and ethanol conversion of Saccharomyces cerevisiae NRRL Y-50316 and NRRL Y-50049 over time in response to 8% (v/v) ethanol challenge on YM medium with 10% glucose. (A) Cell growth as measured by OD600 for Y 50316 (●) and Y-50049 (○). (B) Mean values of glucose consumption (♦) and ethanol concentration (◊) for Y-50316 versus glucose (▲) and ethanol (Δ) for Y-50049. Master equation for qRT-PCR Assays Using CAB as a sole reference to set a manual threshold at 26 Ct for data acquisition (see methods) , raw data were normalized and analyzed for the entire PCR reactions applied in 80 individual 96-well plate runs.