Effect of Acetic Acid on Saccharomyces Carlsbergensis ATCC 6269 Batch Ethanol Production Monitored by Flow Cytometry

Abstract

Bioethanol produced from lignocellulosic materials has been considered a sustainable alternative fuel. Such type of raw materials have a huge potential, but their hydrolysis into mono-sugars releases toxic compounds such as weak acids, which affect the microorganisms' physiology, inhibiting the growth and ethanol production. Acetic acid (HAc) is the most abundant weak acid in the lignocellulosic materials hydrolysates. In order to understand the physiological changes of Saccharomyces carlsbergensis when fermenting in the presence of different acetic acid (HAc) concentrations, the yeast growth was monitored by multi-parameter flow cytometry at same time that the ethanol production was assessed. The membrane potential stain DiOC(6)(3) fluorescence intensity decreased as the HAc concentration increased, which was attributed to the plasmic membrane potential reduction as a result of the toxic effect of the HAc undissociated form. Nevertheless, the proportion of cells with permeabilized membrane did not increase with the HAc concentration increase. Fermentations ending at lower external pH and higher ethanol concentrations depicted the highest proportions of permeabilized cells and cells with increased reactive oxygen species levels. Flow cytometry allowed monitoring, near real time (at-line), the physiological states of the yeast during the fermentations. The information obtained can be used to optimize culture conditions to improve bioethanol production.

Metabolic Engineering Strategies for Improved Lipid Production and Cellular Physiological Responses in Yeast Saccharomyces cerevisiae

“…In addition, sphingolipids and sterols in the yeast cell membrane coordinately regulate cell membrane integrity [ 134 ]. The membranes of yeast may also be affected by metabolites secreted into the cell culture medium, such as acetic acid and ethanol [ 135 ]. Furthermore, the fatty acid unsaturation degree and chain length affect the cell membrane fluidity of S. cerevisiae and hence the organism’s resistance to metabolites in the medium [ 136 , 137 ].…”

Updates on the applications of flow cytometry in microbiology

“…The DNA content of yeast cells provides information about the cell cycle and is thus a powerful tool to assess the effects of propagation parameters on the yeast cell cycle and yeast physiology. FCM assays were developed for assessing the physiological status of the yeast cells during fermentation process [24,25]), for determination of yeast viability and cell number in a brewery [26][27][28][29][30][31][32], predictions of flocculation ability of brewer yeasts, separation of the prototroph yeast fusants [33], assessment of Saccharomyces cerevisiae vitality [34,35,36], detection of spoilage microorganisms [24,37], the influence of beer process conditions on beer stability [38], identification of haploid strains of industrial brewer's yeast, fermentation process control [20,39,40]. The use of FCM for age assessment of a yeast population and its application in beer fermentation was reviewed in the publication [41].…”

Action mechanisms involved in the bioprotective effect of Lactobacillus harbinensis K.V9.3.1.Np against Yarrowia lipolytica in fermented milk

“…To assess the cell membrane potential of Y. lipolytica, a mixture of 3,3'dihexyloxacarbocyanine iodide (DIOC 6 (3); λ ex /λ em : 484/501 nm) (Molecular probes, France) and PI dyes was used (Freitas et al, 2012). DIOC 6 (3) is a carbocyanine derivative with short alkyl tails that accumulates in polarized and hyperpolarized cytoplasmic and mitochondrial membranes, due to its positive charge.…”

“…To perform this analysis, dihydrorodhamine 123 was used as previously described (Freitas et al 2012). Dihydrorodhamine 123 is a non-fluorescent ROS indicator than can passively diffuse across membranes and can be oxidized to cationic rodhamine 123, which exhibits a green fluorescence informing on the degree of ROS accumulation in cells.…”