2.2 Classification and identification of yeast.........................................................14
2.3 Yeast killer toxin..............................................................................................21
2.4 Ethanol fermentation.......................................................................................25
2.4.1 Thermotolerant yeasts and their ethanol production.............................26
2.4.1.1 Kluyveromyces.........................................................................27
2.4.1.2 Saccharomyces.........................................................................28
2.4.1.3 Candida....................................................................................30
2.4.2 Effect of ethanol on yeast fermentation................................................32
2.5 Effects of nutrients and pH on ethanol production by thermotolerant yeasts.33
2.5.1 Nutrients................................................................................................33
2.5.2 pH..........................................................................................................34
III MATERIALS AND METHODS.......................................................................36
3.1 Isolation of thermolerant yeasts.....................................................................36
3.2 Selection of thermotolerant yeast with ethanol production activity...............36
3.3 Optimization of temperature for yeast growth...............................................37
3.4 Detection of yeast killer toxin........................................................................37
3.5 Detection of ethanol tolerance of thermotolerant yeast.................................38
3.6 Identification of the selected thermotolerant yeast........................................39
3.6.1 Morphological characterization...........................................................39
3.6.1.1 Growth in solid medium..........................................................39
3.6.1.1.1 Ascospore formation...............................................39
3.6.1.1.2 Pseudomycelium formation....................................40
VIII
CONTENTS (Continued)
Page
3.6.1.2 Growth in liquid medium.........................................................40
3.6.2 Biochemical characterization................................................................41
3.6.3 Molecular characterization....................................................................41
3.6.3.1 Genomic DNA extraction........................................................41
3.6.1.2 PCR amplification....................................................................42
3.6.1.3 Sequence analysis.....................................................................43
3.7 Optimization of some ethanol production condition.......................................43
3.7.1 Nitrogen source.....................................................................................43
3.7.2 Glucose concentration...........................................................................44
3.7.3 Initial pH................................................................................................45
3.8 Ethanol production in 2-L fermenter...............................................................45
IV RESULTS AND DISCUSSIONS........................................................................47
4.1 Isolation of thermotolerant yeasts...................................................................47
4.2 Selection of thermotolerant yeasts with ethanol production activity..............47
4.2.1 Screening of yeast morphology under microscope...............................47
4.2.2 Study of optimum growth temperature of selected thermotolerant
yeast......................................................................................................48
4.3 Identification of the thermotolerant yeast strain S1........................................49
4.3.1 Morphological and physiological characteristics..................................45
4.3.2 Genetic analysis.....................................................................................53
4.3.3 18S rDNA sequence analysis................................................................54
4.4 Some characteristics of the selected thermotolerant yeast strain....................56