Determination of yeast count in Bioethanol production

Biofuel development and production have dramatically increased over the last years. For production of biofuel utilizes the baker’s yeasts, Saccharomyces cerevisiae with high ethanol tolerance. To perform fermentation usually are used sugar cane, corn meal, polysaccharides, and Waste Water. To accomplish high production of bioethanol it is necessary to made the continuous control of the total and vital cell. Determining the cell count and their viability in bioethanol production from corn and wheat bran has major problems because of the inhomogeneity of the samples. The analysis in made in heterogeneous environment, due to the presence of solid particles of bran. These solid particles cause problems, because the used fluorescent DNA dye has background fluorescence. These particles absorb the dye and begin emitting fluorescence signal.

Good example for this is the propidium iodide dye. To avoid this problem there are two solutions:

First solution is the removal of solid particles by filtration or centrifugation. In this case, however, the cells are subtracted from their natural environment and this may result in changes of their morphology and vitality.

Second solution is to use fluorescence staining, which has insignificant back ground fluorescence and high fluorescence intensity, when dye binds with the cells DNA. To solve this complicated question some authors (Chan, US 2014/0024074 A1), offer a direct determination of the number of cells in the sample without removing the solid particles, but by bringing the sample to pH 10. In this analysis, the well-known fluorescent dye for dead cells - propidium iodide is used. The purpose of the alkaline pH is hydrolyzing of the solid particles of corn and wheat bran, thereby avoiding the staining of the side impurities. This option is also not very good because it is possible to break the integrity of the cells.

Our company offers a better approach to addressing this problem - the use of a new DNA fluorescent dye – SOFIA GREEN. It is synthesized by our company, has a strong fluorescence intensity when binding to cell DNA and no background noise. A comparison of the fluorescence spectra of the dyes SOFIA GREEN and propidium iodide by spectrophotometer was carried (fig. 1). The figure shows that the fluorescence intensity of SOFIA GREEN upon its binding to DNA is greater than the intensity of Propidium iodide-DNA. The efficiency of the intensity (If/I0) of conjugate Sofia Green-DNA is 6 times greater than that of the propidium iodide-DNA. In addition to that the background signal of Sofia green is insignificant in comparison with propidium iodide.

Fluorescence spectra of SOFIA GREEN
Fluorescence spectra of propidium iodide
Fig. 1. Fluorescence spectra of SOFIA GREEN (left) and propidium iodide (right) without DNA (red) and DNA (syn) Ratio SOFIA GREEN (50 μg / ml): DNA (50 μg / ml) = 1: 12.5. Ratio Propidium iodide (50 μg / ml): DNA (500 μg / ml) = 1: 1. The range is 650 V for both spectra.

Dye λеx (nm) dye + dsDNA λеm (nm) dye + dsDNA Fluorescence of free dye (I0) Fluorescence in presence of dsDNA ( If) If/I0
Propidium Iodidе 535 617 6.5 85 13
SOFIA GREEN 494 514 5.7 455 80
Table 1. Fluorescence intensities of SOFIA GREEN and propidium iodide without DNA and with DNA

SOFIA GREEN dye is used to stained cells of samples taken from fermenter of bioethanol production.

The fermentation was made with cornstarch and Saccharomyces cerevisiae yeast with high ethanol tolerance. The stained cells were observed on the Olympus fluorescence microscope (Fig. 2 and 3). In parallel, bright field images of the same samples were also made. From the bright field pictures in Fig. 2 it can be seen that the observed corn starch impurities do not stain with SOFIA GREEN.

The Fig 2 shows only the stained yeast cells that corresponded perfectly to the cells into the bright microscopic photo.

Fluorescence spectra of SOFIA GREEN
Fluorescence spectra of propidium iodide
Fig. 2. bright field and fluorescence photos of alcoholic yeast using the SOFIA GREEN and fluorescent microscope Olympus.

For comparison, we did the same experiments with the fluorescent propidium iodide dye.

On the contrary, on Fig.3 can be seen that the cornstarch impurities are stained with propidium iodide (see squares). Obviously, this disadvantage is avoided when using the new SOFIA GREEN dye.

Fluorescence spectra of SOFIA GREEN
Fluorescence spectra of propidium iodide
Fig. 3. bright field and fluorescence photos of alcoholic yeast using Propidium Iodide dye and Fluorescent Microscope Olympus

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