Strain development of yeasts for efficient alcoholic fermentation utilizing xylose
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Date
2010
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Punjab Agricultural University Ludhiana
Abstract
Two strains of yeasts, Saccharomyces cerevisiae G and Pachysolen tannophilus MTCC1077 were used
to develop improved strains of yeasts for efficient alcoholic fermentation utilizing xylose as carbon
source by means of mutagenesis and protoplast fusion. Mutagenesis of xylose fermenting yeast, P.
tannophilus MTCC1077 using physical mutagen, UV rays gave rise to 103 ethanol negative variants
with the inability to use ethanol as carbon source for growth. The parent P tannophilus MTCC1077
was found to give a maximum ethanol yield of 0.339g/g with a corresponding fermentation efficiency
of 66.47% from 20g/l xylose when fermentation was carried out at 30ºC at a pH of 5.0 with inoculum
of 6% and an aeration of 100rpm. Two ethanol negative variants UVmut 62 and UVmut 41 gave
ethanol yields significantly higher than the parent strain. UVmut 41 showed highest yield of ethanol of
0.350g/g with a fermentation efficiency of 68.53% while UVmut 62 gave a yield of 0.347 g/g with a
fermentation efficiency of 67.92%. Mutagenesis of xylose fermenting yeast, P. tannophilus
MTCC1077 using chemical mutagen, ethylmethanesulfonate (EMS) gave rise to 102 ethanol negative
variants with the inability to use ethanol as carbon source for growth. Three ethanol negative variants
EMSmut14, EMSmut39 and EMSmut26 showed significantly higher ethanol producing capacity than
the parent strain. EMSmut26 gave the highest ethanol yield of 0.364g/g with a fermentation efficiency
of 71.32% followed by EMSmut39 and EMSmut14 giving a yield of 0.350g/g and 0.346g/g
respectively. Protoplast isolation rates of 99% for S. cerevisiae and 97% for P. tannophilus achieved
with action of lyticase enzyme (0.08mg/ml) on cells for 60min at 30°C with 0.8M sorbitol, 1mM
EDTA and 10mM Tris buffer at pH 7.5. Protoplast fusion was obtained when cells were treated for
30min with chemical fusogen, polyethyleneglycol (PEG mol wt. 6000) and highest protoplast
regeneration frequency of 21% was obtained with 35% PEG and 0.8M sorbitol at 30ºC. Two fusants
were able to produce ethanol from a mixture of glucose and xylose. Parent S. cerevisiae gave an
ethanol yield of 0.430g/g with a fermentation efficiency of 84.21%. FUS1 giving a yield of 0.244g/g
with a fermentation efficiency of 47.82% while FUS2 gave a yield of 0.273g/g with fermentation
efficiency of ethanol at 72hr of fermentation. Scanning electron microscopy (SEM) study of parent
strains and fusants revealed differences in the cell surface and also in budding pattern as compared to
that seen with either of the individual parent strains. DNA contents of the two fusants were larger than
that of any single parental strains, suggesting that these fusants have inherited genetic material from
both parental strains.