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4.2.1Chemicals and media

The chemical mutagens namely EMS and MNNG and fluorescent dye Flourescein di-acetate (FDA) were purchased from Sigma-Aldrich. Rest of the chemicals were purchased from Merck. The fermentation media and buffers used in the study are presented in Table 2 and 3.

4.2.2 Sequential mutagenesis

Mutagenesis of the fusant strain was conducted by eight different combinations of mutagenic treatments using EMS, MNNG, near UV and far UV radiations as mutagens. The fusant yeast cells grown overnight in YPD medium were harvested and washed with 0.1M sodium phosphate buffer (pH 6.8). Cells were sonicated for 5 sec and re-suspended in the buffer.

Chemical mutagenesis was carried out by adding mutagenic agent (50 µL EMS or 50 µg MNNG) to 2 mL of cell suspension. The suspension was then incubated at 30^oC with shaking at 120 rpm. After 45 min, 0.5 mL cell suspension was taken out, washed with 3.0 mL of sodium thiosulfate (5% w/v) and resuspended in buffer.

Mutagenesis of fusant was also induced by far (254 nm) and near (290-350 nm) ultraviolet radiations using UV lamps (30W, Philips, India). About 2 mL of cell suspension in buffer was transferred to sterile petriplate and exposed to UV radiation for 90 sec. After serial dilution in buffer, cells were plated on YPD plates and incubated at 30^oC. Colonies with better morphology and size were selected from the plate and analysed for stress tolerance studies and ethanol production.

4.2.3 Cell Viability assay

After each stage of mutagenesis, the viability of cells was checked by FDA-PI dual staining to assay the lethality of mutagen. The cells were washed and suspended in phosphate buffer. 50 µL of FDA solution (5 mg/mL in acetone) was added to 1mL of cells suspension, the resulting suspension was centrifuged at 500 rpm for 3 min and the cells were incubated in dark for 5 min. The cells were then stained with propidium iodide (PI) by adding 20 µL of PI per mL of cell suspension. The viability of cells was assayed using flow cytometer (LSR

Fortessa, BD Biosciences) and the LD 50 (Lethal dose 50) was estimated after each step of mutagenesis.

4.2.4 Stress tolerance study

The stress tolerance in mutants was evaluated by conducting fermentation experiment using glucose-xylose mixture as model lignocellulosic substrates (3:1 ratio). The fermentation temperature and agitation were kept at 30^oC and 150 rpm throughout the fermentation experiment. The ethanol tolerance and thermotolerance were examined by growing mutants in YPD medium containing different concentrations of ethanol (6-10% v/v) and temperature range (38-42^oC) respectively. The tolerance of the mutants to toxic inhibitors was studied by adding known amount of inhibitors (vanillin, furfural and acetic acid) in the fermentation medium. Mutant grown at 30^oC in YPD medium containing 175 gL^-1 glucose-xylose mixture without ethanol or inhibitors was used as control. The study of combined effect of thermal and inhibitor stress was carried out by fermentation of 150 gL^-1 glucose–xylose mixture at 39^oC and 150 rpm in presence of inhibitors. The combined effect of ethanol and inhibitor stress was investigated by carrying out fermentation experiment at 30^oC using fermentation medium supplemented with 5% ethanol and the three fermentation inhibitors. Furthermore, the combined effect of thermal, inhibitors, ethanol and substrate stress was investigated with the fermentation medium containing 250 gL^-1 glucose–xylose mixture, 5% ethanol and fermentation inhibitors at 39^oC. The amount of inhibitors used were 0.25-1.0 gL^-1 vanillin, 0.25-2.0 gL^-1 furfural and 2-8 gL^-1 acetic acid in each fermentation experiment. The range of the inhibitors was selected on the basis of published literature [82-84]. The range of temperature and ethanol concentrations has also been selected based on the published literature and the knowledge of the most optimum conditions for yeast growth [36, 85].

4.2.5 Evaluation of ethanol production of mutants under normal conditions

The performance of the mutants towards ethanol production was studied by fermentation of glucose-xylose mixture in a 250 mL Erlenmeyer flask at 30^oC and 150 rpm. The fermentation medium with 175 gL^-1 glucose-xylose mixture (3:1 ratio) was used for fermentation unless

otherwise specified. Initial pH and inoculum size were maintained at 4.5 and 10%

respectively.

4.2.6 Trehalose assay

Trehalose content of yeast cells was estimated following the methods previously described by Swan and Watson [86]. In brief, mutant and fusant yeast cells were centrifuged and washed with distilled water and with 0.1M sodium phosphate buffer (pH 6.8). The cells were then suspended in 2 mL buffer in test tubes and the tubes were placed on ice and 4 ml of 0.5M cold tri-chloro acetic acid (TCA) was added to each of them. The cell suspension was shaken gently at intervals of 10 min for 30 min, suspension was centrifuged at 4000 rpm and the supernatant were collected. This step of suspending cells in TCA and collection of supernatant was repeated till about 20 mL of supernatant was obtained. The volume of supernatant was made to 50 mL by addition of distilled water. 1mL of diluted supernatant was added to 5 mL of anthrone reagent and tubes were placed in boiling water bath for 10 min.

The optical density at 620 nm was determined for each sample. The trehalose content of each sample was compared with the standard curve and recorded.

4.2.7 Ergosterol content

The ergosterol content of the cells was determined by the spectrophotometric method of Breivik and Owades [87]. The cells grown in YPD media for 18 h were digested in 25% w/v alcoholic KOH at 90^oC for 3h. The saponified samples were then extracted into n-heptane and ergosterol content was determined spectrophotometrically at 281.5 nm.

4.2.8 Stability study

The stability of mutants was examined after every 15 days for a period of 9 months by assessing their substrate utilization and ethanol fermentation efficiencies using glucose-xylose mixture as explained in section 4.1.9.

4.2.9 Genetic characterization of mutant

RAPD and partial DNA sequencing of the mutants was done to investigate the changes brought in their genetic constitution after mutagenesis. The detailed procedure of steps of genetic characterization has been mentioned in section 4.1.10.