isolation and selection of thermo tolerant yeasts from alcoholic fermentation starter

ABSTRACT In this research, 40 yeast strains that were isolated from alcoholic fermentation starters were examined for morphology, physiology, biochemistry, thermo-tolerant ability at 30,

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY

ISOLATION AND SELECTION OF THERMO-TOLERANT YEASTS FROM ALCOHOLIC FERMENTATION STARTER

Assoc.Prof NGO THI PHUONG DUNG HUYNH QUOC KHANH

Studentcode: 3102818 Session: 36 (2010-2014)

Can Tho, 2014

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS

THE ADVANCED PROGRAM IN BIOTECHNOLOGY

ISOLATION AND SELECTION OF THERMO-TOLERANT YEASTS FROM ALCOHOLIC FERMENTATION STARTER

Assoc.Prof NGO THI PHUONG DUNG HUYNH QUOC KHANH

Studentcode: 3102818 Session: 36 (2010-2014)

Can Tho, 2014

APPROVAL

Can Tho, December , 2014

PRESIDENT OF EXAMINATION COMMITTEE

ABSTRACT

In this research, 40 yeast strains that were isolated from alcoholic fermentation starters were examined for morphology, physiology, biochemistry, thermo-tolerant ability at 30, 35, 37, 40, 43, and 45ºC, and for their ethanol tolerant ability at 3, 6, 9, and 12% v/v ethanol The yeast isolates with high thermo-tolerant and ethanol tolerant ability were further tested for ethanol fermentation ability in 2% w/v glucose liquid and ethanol fermentation ability in molasses at high temperatures (30, 35, 40 and 42ºC) The results showed that most of the yeast’s cells are spherel, oval and ellipse, size range from (5-15) µm x (2-10) µm Based on the characteristics of morphology, physiology and biochemistry, 40 yeast isolates were classified as follows: Saccharomyces, Kluyveromyces and Hansenspora 27 yeast isolates were found to be able for growth at temperature 43 o C 29 yeast isolates were able for growth in the medium containing 12% v/v ethanol Among them, three yeast isolates including CT, HG2 and TO1 had the high capacity of ethanol fermentation in 2% w/v glucose medium than others CT, HG2 and TO1 were able to produce ethanol at temperature

of 42 o C and with ethanol concentrations in the medium folowed 3.03% v/v, 2.69% v/v and 2.77% v/v respectively The result of molecular analysis of ITS1, 5.8S rDNA, and ITS2 sequence showed that the TO1 yeast isolate belonged to Saccharomyces cerevisiae with 93% homogeneous level

Key words: ethanol fermentation, ethanol tolerant ability, Saccharomyces cerevisiae,

thermo-tolerant ability, thermo-tolerant yeast

CONTENTS

APPROVAL i

ABSTRACT ii

CONTENTS iii

CHAPTER 1: INTRODUCTION 1

1.1 Introduction 1

1.2 Objectives 2

CHAPTER 2: MATERIALS AND METHODS 3

2.1 Materials 3

2.2 Method 3

2.2.1 Isolation 3

2.2.2 Morphological, physiological and biochemical Characteristics 3

2.2.3 Testing thermo-tolerant ability of yeast isolates 4

2.2.4 Testing ethanol tolerant ability of yeast isolates 4

2.2.5 Study on ethanol fermentation ability of selected yeast isolates in glucose liquid 4

2.2.6 Study on ethanol fermentation ability of selected yeast isolates at high temperature 5

2.2.7 Identification of the selected thermo-tolerant yeast isolates 5

2.2.8 Statistical data analysis 5

CHAPTER 3 RESULTS AND DISCUSSION 6

3.1 Isolation 6

3.2 Morphological, physiological and biochemical tests 6

3.3 Testing thermo-tolerant ability of yeast isolates 14

3.4 Testing ethanol tolerant ability of yeast isolates 16

3.5 Ethanol fermentation ability of selected yeast isolates in glucose liquid 19

3.6 Ethanol fermentation ability of CT1, HG2 and TO1 yeast isolates at high

temperature 21

3.7 Identification of the TO1 yeast isolate 22

CHAPTER 4 CONCLUSIONS AND SUGGESTIONS 24

4.1 Conclusions 24

4.2 Suggestions 24

REFERENCES 25

CHAPTER 1: INTRODUCTION

1.1 Introduction

For a long time, ethanol has many applications in life and industry Ethanol is commonly known as beverages, or it can be used as an antiseptic, inhibitor or material for synthesisother organic compound, depending on the concentration and uses Furthermore, ethanol was also assessed as a bio-fuel that has the potential to replace fossil fuels (Alfenore et al., 2002)

The use of yeast to produce ethanol from sugar has a great advantage because sugar may be obtained from agricultural residues such as straw, corn stalks, molasses These are cheap and diversematerials in agriculture based countries However, there are many factors affecting the activity of the yeast as carbon sources, nitrogen sources,

pH, concentration of ethanol, temperature

Temperature is an important factor affecting the efficiency of ethanol fermentation process, especially in the tropics, where the average temperature is high throughout the year The advantage of the high-temperature fermentation is that fermentation will occur more quickly, reducing the risk of microbial contamination, saving cooling costs (Roehr, 2001)

However, in case the temperature is too high or ethanol concentrations exceed the tolerant of yeast, fermentation will be inhibited, leading to low efficiency of fermentation Therefore, the selection of yeast strains that is capable of growing, fermentating at high temperatures and withstanding high ethanol concentration is very promising for the production of ethanol

The climate in the Mekong Delta region is hot and humid all year-round, it is very suitable to growth andenhance yeast and yeast populations are The probability to obtain yeast strains possessing the desirable characteristics for developing original starter for industrial production and research on ethanol fermentation in high temperature conditions is entirely feasible

Currently, the country had few researchs on thermo-tolerant yeast in alcoholic fermentation starter Consequently, researchs on isolating yeast strains to apply in the

ethanol production are in need That was the reasons why the topic "Isolation and selectionthermo-tolerant yeast in alcoholic fermentation starter" is done

CHAPTER 2: MATERIALS AND METHODS

Devices: oven, microscope, electric stove, flasks, pH meter, water-lock, refractometer…

Media: + YPD agar (2% D-glucose, 2% agar, 0.5% yeast extract, 0.5% peptone w/v, adding tetracyclin 50 mg/500 mL media)

+ YPD broth (2% D-glucose, 0.5% yeast extract, 0.5% peptone w/v) Chemicals: C2H5OH, NaCl, NaHSO3, NaOH, HCl

2.2 Method

2.2.1 Isolation

Purpose: selecting pure yeast strains from alcoholic fermentation starter

Method: weighing 1 g alcoholic fermentation starter and increasing biomass in

YPD mediumfor 48 hours on a shaker Diluting media into lower concentraions, the suspension of each concentration was transplanted into a petri dish containing YPD agar medium, incubate at 30oC for 48 hours, observedcolonies, continued to transplant each different single colony to a petri dish until having got pure strains

2.2.2 Morphological, physiological and biochemical Characteristics

a MorphologicalCharacteristics

Purpose: identifying the characteristics including shape and size of yeast

colonies and cells

Method: yeastisolates were transplanted into a petri dish containing YPD agar

medium incubate at 30oC for 48 hours, measured the shape and size of colonies

Observing the morphology of yeast under a X100 objective optical microscope andmeasuring the size and shape and photograph yeast cells

b Testing physiology and biochemistry characteristics

Determination ethanol fermentation ability of yeast isolates in glucose, saccharose and maltose liquid: ethanol fermentation ability of yeast isolates in

glucose liquid was determined by measuring the CO2 height in Durham test tubes produced by yeasts Yeast isolates were inoculated into YPD medium for 24 hours Then, suspensions of yeast isolates were inoculated into Durham tubes containing 2% w/v glucose liquid, and incubated at room temperature Measure the accumulation of

CO2 gas in the inner Durham tubes at 4, 8, 12, 16, 20, and 24 hours Do same as above with saccharose and maltose liquid

Determination urease enzyme activity: christensen urea broth media was

prepared and sterilized at 115oC for 1 hour Then, yeast isolates were transplanted and incubatedat 30oC for 48 hours A positive result could be observed in case the medium color turns into pink

2.2.3 Testing thermo-tolerant ability of yeast isolates

Yeasts were inoculated into YPD medium for 48 hours Thermo-tolerant ability

of yeast isolates was determined by culturing the yeasts on YM agar medium and incubating at 30, 35, 37, 40, 43 and 45ºC in 48 hours The isolates forming colonies at high temperatures were recorded

2.2.4 Testing ethanol tolerant ability of yeast isolates

Yeast isolates were inoculated into YPD medium for 48 hours Ethanol tolerant ability of yeast isolates was determined by culturing the yeasts on YM agar medium supplemented with pure ethanol at levels of 3, 6, 9, and 12% v/v and incubating at 30ºC in 48 hours The isolates forming colonies on high ethanol concentration supplemented medium were recorded

2.2.5 Study on ethanol fermentation ability of selected yeast isolates in glucose

liquid

Ethanol fermentation ability of yeasts was determined by measuring the CO2 height in Durham test tubes produced by yeasts Yeast isolates were inoculated into YPD medium for 24 hours Then, suspension of yeast cells was inoculated into

Durham tubes containing 2% w/v glucose liquid of, and incubated at room temperature Measure the accumulation of CO2 gas in the inner Durham tubes each 2 hours in 24 hours

2.2.6 Study on ethanol fermentation ability of selected yeast isolates at high

2.2.7 Identification of the selected thermo-tolerant yeast isolates

Yeast strain are sent to the Microgene company for DNA extraction and amplification regions ITS1, ITS2 and 5.8S rDNA by PCR Nucleotide sequence was aligned and compared with the data obtained from Gene Bank (http://www.ncbi.nlm.nih.gov/) and determine the species of yeast strains

2.2.8 Statistical data analysis

The statistical data were analyzed by Microsoft Office Excel 2010 and Statgraphics centurion XVI (USA) software

CHAPTER 3 RESULTS AND DISCUSSION

3.1 Isolation

Forty isolates were obtained from alcoholic fermentation starter sources are denoted: AA1, TV1, AG*, AG1, AG1.1, AG2, AG3.3, BD1, BD2, BL1, BL2, BL3, BL4, CM2, CM2.2, CM3, OM1, CM4.4, OM2, OM3, CT1, CT2, TV2, TV3, OM4, DT1, HG1, HG2, TV4, NK2, NK4, ST2, TB1, TG1, DT2, TNN3, TNN4, TO1, TO2, ST1

3.2 Morphological, physiological and biochemical tests

According to Nguyen Lan Dung et al (1998) and Nguyen Duc Luong et al (2004) yeast strains can be identified preliminarily base on morphological and physiological characteristics Morphological characteristics include: description of the yeast cell morphology when grown on the PGA medium for 2 days, the formation of budding cells and budding style when cultured on peptone - yeast extract – glucose medium after a period of 2 or 3 days incubation, the sporulation of yeast cultured on agar – water medium after a period of 14 days Physiological characteristics include: the ability to ferment sugars and urea assimilation ability of yeast

a Morphological characteristics of yeast

General morphology of yeast strains includes the following types: 40 strains isolates were variety of shapes and sizes Yeast colonies had a convex shape, about 1-3

mm in diameter and 0.1 mm thickness Some colonies surfaced smooth or rough Most

of colonieshad white color The shapes of typical yeast colonies are shown in Figure 1

µm Based on morphological characteristics of the yeast cell, 40 yeast strainsisolates could be divided into five main groups These group names were shown in Table 1

Table 1 Shape of yeast colonies

Group Yeast cell

Group 1 Large Sphere AA1, BD1, CM2.2, DT1, ST2, TB1,

Group 3 Small Oval AG1, BD2, BL4, CM3, TO2, OM1 6

Group 4 Large Ellipse AG*, AG1.1, AG3.3, OM3, CT2, HG1,

Group 5 Small Ellipse BL1, BL2,BL3, CM2, CM4.4, OM2,

OM4, TV2, TV3, NK2, NK4, TNN4 12

Main yeast cells shapes are showed in figure 2

Figure 2 Main shapes of five yeast trains group at objective lens X100

Specific detailed characteristics of each yeast strain forms are listed in in Table 2

Group 3: Small Oval

Group 4: Large Ellipse Group 5: Small Ellipse Goup1: Large Sphere Group 2: Large Oval

Table 2 Morphological Characteristics of yeast strains isolates

Convex/

crateriform Surface,Margin Color

Size (µm) Shape

1 AA1 2-2.5 0.1 Convex Smooth, entire White 10x10 Large

8 BD2 2-2.3 0.1 Convex Rough,

filiform White 4x3

Small Oval

9 BL1 1.5-2 0.1 Convex Smooth, entire White 5x3 Small

12 BL4 1.5-2 0.1 Convex Rough,

undulate White 4x2

Small Oval

13 CM2 1.5-2 0.1 Convex Rough,

undulate White 6x3

Small Ellipse

14 CM2.2 1.5-2 0.1 Convex Rough,

Filiform White 8x8

Large Sphere

15 CM3 2-2.5 0.1 Convex Smooth,

undulate White 4x3

Small Oval

16 CM4.4 1-1.5 0.1 Convex Smooth, entire White 6x2 Small

Ellipse

17 CT1 1-1.5 0.1 Convex Rough, entire White 12x12 Large

21 HG1 1.5-2 0.1 Convex Smooth, entire White 10x5 Ellipse

26 OM2 2-2.5 0.1 Convex Smooth, entire White 5x3 Small

36 TO2 2-2.5 0.1 Convex Smooth, entire White 6x5 Small

b Testing physiology and biochemistry characteristics

Determination ethanol fermentation ability of yeast isolates in glucose, saccharose and maltose liquid:

The results of test showed that 40 yeast strains isolates could be classified into 3 groups: group I (capable of fermenting), group II (weak fermentation) and group III (incapable of fermentaion) shown in Table 3

Table 3 Fermentation ablility of yeast strains isolates Types of

Glucose

AA1, AG*, AG1, AG1.1, AG2, BD2, BL1, BL2, BL3, BL4, CM2, CM2.2, CM3, CM4.4, CT1, DT1, DT2, HG1, HG2, NK2, NK4, OM1, OM2, OM4, TB1, TG1, TNN3, TNN4, TO1, TO2, TS2,TV1, TV2

AG3.3, BD1, CT2, OM3, ST2, TV3, TV4

Saccharose

AA1, BL1, BL2, CM2.2, CM4.4, CT1, DT1, DT2, HG2, ST2, TB1, TG1, TNN3, TNN4, TO1

AG*, AG1.1, AG2, AG3.3, BD1, BL3, BL4, CM2, CT2, HG1, NK2, NK4, OM2, OM3, OM4, TV1, TV2, TV3, TV4

AG1, BD2, BL4, CM3, OM1, TO2

Maltose

AA1, AG1, AG1.1, AG3.3, BD1, BD2, BL1, BL2, BL3, BL4, CM2, CM2.2, CM3, CM4.4, CT1, CT2, DT1, DT2, HG2, NK2, NK4, OM1, OM2, OM3, OM4, TB1, TG1, TNN3, TNN4, TV2, TV3, TO1, TO2, TS2

AG*, AG2, HG1, ST2, TV1, TV4

The results showed that all yeast isolateshad the ability to use maltose and glucose Meanwhile, there were 34 yeast strains capable to ferment saccharose

The ability to ferment sugars is an important feature in ethanol producing process Therefore, the testof ethanol fermentation ability of yeast isolates in glucose, saccharose and maltose liquidwill be one of the criteria for classification of yeast, while it is the initial selection of the appropriate yeast strains for fermentation of various types of substrates in order to take full advantage of sugar source in substrate, because composition and proportion of sugar in each type of substrate is very different