Estimation of growth rate and assessment of absorbed zinc inS. cerevisiae
The effect of Zn 2+ concentration on efficiency of zinc biosorption as well as biomass production in actively growingS. cerevisiae , was evaluated. The ZnSO4 solution, prepared using a 1000 mg/l sterile stock solution of zinc sulfate heptahydrate (Merck, Germany), was diluted to various concentrations of zinc including 0, 10, 25, 50 and 100 mg/l; every concentration was added in a 500 ml Erlenmeyer flask containing 200 ml SDB (sabouraud dextrose broth) culture medium. Then S. cerevisiae pellets (containing ~ 1.5 × 107 cell/ml) were inoculated to these zinc supplemented mediums. The content of all flasks (control and experimental cultures) were incubated in 28 °C in the shaker incubator at agitation speed of 200 rpm/min for 72 h, with the pH adjusted at 5.8. Then, growth rate of S. cerevisiae in present of Zn 2+ as well as the level of Zn 2+absorption by S. cerevisiae were considered as follows:
Growth rate estimation. During cultivation of yeast in the presence of zinc, the yeast growth at 24-hour intervals (0, 24, 48, and 72 h) was estimated; the optical density (OD) of suspension was determined using spectroscopic method at 600 nm wavelength. Also, 10 ml of medium was collected and centrifuged at 4000 rpm in 20°C for 10 min, then immediately supernatant was removed and the weight of sediment was assayed. After that, to determine the content of dry biomass, a two-stage drying procedure including: first temperature at 60°C for 2 h and second stage at 105°C was performed to obtain the constant weight and then, dried biomass was weighed and considered as cell dry weight (CDW).
AAS determination. To determine the total accumulation of zinc in yeast cells at 24-hour intervals, Atomic Absorption Spectrophotometry (AAS) method was performed. In brief, 1 mg dry biomass was digested by adding nitric acid and distilled water (1:1) in 25 ml digestion tubes for 20 min at 160 °C, then the obtained biomass was filtered via filter paper. Finally AAS method was applied to estimate the content of zinc in yeast cell biomass at 213.9 nm wavelength.
Determination of dry content of biomass and AAS for each samples were repeated three times.