Study of the Metabolic Activity of Saccharomyces Yeasts in Brewing and Bioethanol Industry
The metabolic activity of brewing yeasts and yeasts used in bioethanol production was investigated using carboxyfluorescein diacetate (cFDA). cFDA penetrates the cell membrane, then active esterase enzymes inside the cell cleaved acetate residues, and the released fluorescein stains the cell in green. The optimal pH of two type of cell staining was determined – pH 4. The optimal temperature for esterase hydrolization of cFDA of brewing cells was 37 0C, and for bioethanol cells – 40 0C. The cFDA optimal concentration for metabolic activity determination of the brewing yeast was found to be 50 μg/ml, and for yeasts in bioethanol production was found to be 200 μg/ml. The incubation time for cell staining was varied with both the strains. It is noteworthy that at 5 minutes the number of stained cells of the brewing yeast Saccharomyces carlsbergensis is 2 times greater than that of the bioethanol producing species. At 10 minutes for both strains the number of stained cells reaches the maximum. At 20 minutes, the released fluorescein from esterase hydrolysis of cFDA begins to leak from the brewing yeast cells and their count decreases. For bioethanol cells the leakage begins at 40 minutes. The metabolic activity of cells during the cultivation of two investigated strains were studied. The same samples were simultaneously tested for determination of total cell count, dead cell count and viable cell count. The results convincingly show that for different cell types the penetration of cFDA into the cells and the leakage of the released fluorescein is different and the conditions of the assay need to be specified.
Keywords - Brewing Yeast, Ethanologenic Yeast, Fluorescence Cytometry, Metabolic Activity, Vitality