Fig. 6

Time course analysis of biomass levels, carbon sources concentrations and number of healthy P. pastoris cells at different stages of the optimal point estimated by the ANN model [30°C, 2.9 mL/L/H methanol constant flow-rate and 6% (v/v) DMSO]. a Biomass levels measured by spectrophotometric determination at 600 nm and methanol and glycerol levels measurements by HPLC with RID; (each value represents the mean of three independent samples). b Dot plots of green fluorescence of cells (BOX, x-axis) plotted against red fluorescence (PI, y-axis) obtained with cell samples taken at different stages of the optimum point retrieved from the ANN modelling. Three main subpopulations of cells can be distinguished corresponding to: healthy cells, no staining; cells with depolarized membranes, stained with BOX; and cells with permeabilized membranes, stained with PI. A total of 10,000 events were collected for these analysis. The variation on the percentage of healthy cells at different stages of the bioprocess is depicted in the graph. Each experiment was conducted in duplicate.