Evaluation of the ethanol tolerance for wild and mutant Synechocystis strains by flow cytometry.

Flow cytometry was used to evaluate the effect of initial ethanol concentrations on cyanobacterial strains of Synechocystis PCC 6803 [wild-type (WT), and ethanol producing recombinants (UL 004 and UL 030)] in batch cultures. Ethanol recombinants, containing one or two metabolically engineered cassettes, were designed towards the development of an economically competitive process for the direct production of bioethanol from microalgae through an exclusive autotrophic route. It can be concluded that the recombinant Synechocystis UL 030 containing two copies of the genes per genome was the most tolerant to ethanol. Nevertheless, to implement a production process using recombinant strains, the bioethanol produced will be required to be continuously extracted from the culture media via a membrane-based technological process for example to prevent detrimental effects on the biomass. The results presented here are of significance in defining the maximum threshold for bulk ethanol concentration in production media.

Effect of Furfural on Saccharomyces carlsbergensis Growth, Physiology and Ethanol Production.

This work described the effect of furfural, a product resulting from the lignocellulosic material pretreatment, on Saccharomyces carlsbergensis growth and ethanol production. Flow cytometry was used to evaluate the yeast membrane potential, membrane integrity, reactive oxygen species production and lipid content. Above 0.3 g/L of furfural, a progressive decrease in the maximal specific growth rate was observed, reaching 53% of the value obtained in the absence of toxic when the cells were grown in the presence of 4 g/L of furfural. In general, the yeast biomass concentration and yield were less affected by the furfural presence than the specific growth rate, and a maximum reduction of 25% was observed for the assay at 4 g/L. The ethanol production was even less affected by the furfural presence than the yeast growth. At 4 g/L of furfural, the maximum ethanol concentration was reduced by only 10% relatively to the maximum ethanol concentration observed in the absence of toxic. At 5 g/L of furfural, the yeast cells were barely able to keep metabolic functions and produced a final ethanol concentration of 0.87 g/L although growth was undetectable. S. carlsbergensis membrane potential was affected by the furfural presence, concomitantly with the ethanol production. However, at 4 g/L, most of the yeast cells (90%) displayed the cytoplasmic membrane depolarized. The proportion of cells with increasing reactive oxygen species (ROS) production levels increased for the experiments at 0-4 g/L. For the experiment at 4.5 g/L of furfural, ROS production was observed for only 11% of the yeast cells. The yeast lipid content was also severely affected by the furfural presence. Both polar and neutral lipids decreased in the presence of furfural, and this reduction was more notorious during the stationary phase.

Using Flow Cytometry to Evaluate the Stress Physiological Response of the Yeast Saccharomyces carlsbergensis ATCC 6269 to the Presence of 5-Hydroxymethylfurfural During Ethanol Fermentations.

Lignocellulosic materials have been considered low-cost effective substrates for bioethanol production. However, lignocellulosic pretreatment releases toxic compounds such as 5-hydroxymethylfurfural (HMF) that is known to inhibit the yeast growth and ethanol production. In this work, flow cytometry was used to monitor the physiological response of the yeast Saccharomyces carlsbergensis ATCC 6269 in the presence of different initial HMF concentrations within the range of 0-15 g/L, in terms of cell membrane integrity, potential, and intracellular lipids. It was observed that the HMF presence affected more significantly the yeast growth than the ethanol production. At 15 g/L HMF, the yeast growth and fermentation ability were completely inhibited. The cell membrane integrity and potential decreased as the initial HMF concentration increased. At the end of the fermentation process with 10 g/L HMF, the yeast culture contained 45 % of cells with depolarized plasma membrane, 52 % of cells with permeabilized plasma membrane, and 53 % of cells with increasing reactive oxygen species (ROS) levels. Using the Nile Red stain, it was observed that intracellular polar lipids were more affected by the initial HMF concentration than the neutral lipids, probably due to the extensive membrane damage.

Use of multi-parameter flow cytometry as tool to monitor the impact of formic acid on Saccharomyces carlsbergensis batch ethanol fermentations.

The use of lignocellulosic materials as substrate for bioethanol production is considered a cost-effective approach to make the biofuel production process economically sustainable. However, lignocellulosic hydrolysis releases toxic compounds such as weak acids which inhibit microorganism growth and ethanol production. In order to understand the physiological response of Saccharomyces carlsbergensis when fermenting glucose in the presence of formic acid (HF), the yeast growth was monitored by multi-parameter flow cytometry. Cytoplasmic membrane potential decreased as the HF concentration increased and as the yeast culture reached the stationary phase. However, the proportion of cells with permeabilized membrane did not increase with the HF concentration increase. The accumulation of reactive oxygen species was also monitored. Control and fermentations at low HF concentrations (<1 g/L) resulted in a high proportion of highly oxidized cells at the stationary phase. The multi-parameter flow cytometry approach proved to be a useful tool to monitor the physiological stress response of S. carlsbergensis growth and ethanol production in the presence of HF, an inhibitor present in lignocellulosic hydrolysates. The information here obtained at near real time can be used to enhance second-generation bioethanol production process efficiency.

Effect of acetic acid on Saccharomyces carlsbergensis ATCC 6269 batch ethanol production monitored by flow cytometry.

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.