isolation and selection of thermo tolerant yeasts from alcoholic fermentation starter

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 SUPERVISOR STUDENT 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 SUPERVISOR STUDENT Assoc.Prof. NGO THI PHUONG DUNG HUYNH QUOC KHANH Studentcode: 3102818 Session: 36 (2010-2014) Can Tho, 2014 APPROVAL SUPERVISOR STUDENT NGO THI PHUONG DUNG HUYNH QUOC KHANH Can Tho, December , 2014 PRESIDENT OF EXAMINATION COMMITTEE i 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 43oC. 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 42oC 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. ii 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 iii 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 iv 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 1 ethanol production are in need. That was the reasons why the topic "Isolation and selectionthermo-tolerant yeast in alcoholic fermentation starter" is done. 1.2. Objectives The aim of this study is isolation and selection of thermo-tolerant and ethanol- tolerant yeast from alcoholic fermentation starter and capable of fermentation ethanol at high temperature in molasses. 2 CHAPTER 2: MATERIALS AND METHODS 2.1. Materials Molasses (collected from Phung Hiep Sugar Factory, Nga Bay Town, Hau Giang Province). Alcoholic fermentation starter collected from markets in the location: Chau Phu District and Long Xuyen City, An Giang Province; Bac Lieu City, Bac Lieu Province; Ca Mau City, Ca Mau Province; Ninh Kieu District, O Mon District and Thot Not District, Can Tho City; Lap Vo District, Dong Thap Province; Phung Hiep District, Hau Giang Province; Vinh Chau Town, Soc Trang Province; Tra Cu District, Tra Vinh Province; Tra On District, Vinh Long Province. 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. 3 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 4 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 temperature Yeast isolates were inoculated into YPD medium for 24 hours. The 1 mL of pre-culture yeasts determined the density of 106 cells/mL was inoculated into 99 mL of 22oBrix molasses medium sterilized at 121ºC for 15 minutes. Incubate the molasses medium anaerobically at 30, 35, 37, 40, and 42ºC for five days. Ethanol content after fermentation was determined by distillation method. 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. 5 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. 6 1a 1b 1c 1d Figure 1.The shape of typical colonies of yeast strains *Note: (1a): HG2, (1b): CT2, (1c): BL4, (1d): TNN4 The result of yeast cells morphological features observed under a microscope showed that the shape of yeast cells were very diverse, consisting mainly sphere, oval and ellipse. The size of the yeast cells ranged 5-15 µm and a width in the range of 2-10 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. 7 Table 1. Shape of yeast colonies Group Yeast cell Yeast strains shapes Quantity AA1, BD1, CM2.2, DT1, ST2, TB1, Group 1 Large Sphere Group 2 Large Oval DT2, CT1, HG2, ST1, TO1 5 Group 3 Small Oval AG1, BD2, BL4, CM3, TO2, OM1 6 Group 4 Large Ellipse Group 5 Small Ellipse TG1, TNN3 AG*, AG1.1, AG3.3, OM3, CT2, HG1, AG2, TV1, TV4 BL1, BL2,BL3, CM2, CM4.4, OM2, OM4, TV2, TV3, NK2, NK4, TNN4 Total 8 9 12 40 Main yeast cells shapes are showed in figure 2 Goup1: Large Sphere Group 2: Large Oval Group 3: Small Oval Group 4: Large Ellipse Group 5: Small Ellipse 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 8 Table 2. Morphological Characteristics of yeast strains isolates Number Yeast strains Colonies description Diameter Thickness Convex/ (mm) (mm) crateriform Cells Features Surface,Margin Color Size (µm) 1 AA1 2-2.5 0.1 Convex Smooth, entire White 10x10 2 AG* 2-2.5 0.1 Convex Smooth, entire White 10x5 3 AG1 2-2.3 0.1 Convex Smooth, entire White 6x5 4 AG1.1 2-2.4 0.1 Convex Smooth, entire White 10x5 5 AG2 1-1.5 0.1 Convex Rough, entire White 10x5 6 AG3.3 2-2.2 0.1 Convex Rough, entire White 10x5 7 BD1 1.5-2 0.1 Convex 8 BD2 2-2.3 0.1 Convex 9 BL1 1.5-2 0.1 Convex 10 BL2 1.5-2 0.1 Convex 11 BL3 2-2.5 0.1 Convex 12 BL4 1.5-2 0.1 Convex 13 CM2 1.5-2 0.1 Convex 14 CM2.2 1.5-2 0.1 Convex 15 CM3 2-2.5 0.1 Convex 16 CM4.4 1-1.5 0.1 Convex 9 Rough, filiform Rough, White 10x10 White 4x3 Smooth, entire White 5x3 Rough, entire White 4x3 White 7x3 White 4x2 White 6x3 White 8x8 White 4x3 White 6x2 filiform Smooth, Filiform Rough, undulate Rough, undulate Rough, Filiform Smooth, undulate Smooth, entire Shape Large Sphere Large Ellipse Small Oval Large Ellipse Large Ellipse Large Ellipse Large Sphere Small Oval Small Ellipse Small Ellipse Small Ellipse Small Oval Small Ellipse Large Sphere Small Oval Small Ellipse 17 CT1 1-1.5 0.1 Convex Rough, entire White 12x12 18 CT2 2-2.5 0.1 Convex Smooth, entire White 12x5 19 DT1 2-2.6 0.1 Convex Rough, entire White 9x9 20 DT2 2-2.3 0.1 Convex White 8x7 21 HG1 1.5-2 0.1 Convex Smooth, entire White 10x5 22 HG2 1-2 0.1 Convex Rough, entire White 10x8 23 NK2 2-2.5 0.1 Convex Smooth, entire White 8x4 24 NK4 1.5-2 0.1 Convex Smooth, entire White 6x4 25 OM1 2-2.5 0.1 Convex White 3x2 26 OM2 2-2.5 0.1 Convex Smooth, entire White 5x3 27 OM3 1-2 0.1 Convex Smooth, entire White 10x5 28 OM4 2-2.5 0.1 Convex Rough, entire White 6x4 29 ST1 1.5-2 0.1 Convex Smooth, entire White 8x7 30 ST2 2-2.5 0.1 Convex Smooth, entire White 10x10 31 TB1 2-2.5 0.1 Convex Smooth, entire White 10x10 32 TG1 1- 2 0.1 Convex Rough, entire White 13x13 33 TNN3 1.5-2 0.1 Convex Rough, entire White 12x12 34 TNN4 2-2.5 0.1 Convex Smooth, entire White 7x4 35 TO1 1.5-2 0.1 Convex Smooth, entire White 8x7 10 Rough, undulate Smooth, Filiform Large Sphere Large Ellipse Large Sphere Large Oval Ellipse lớn Large Oval Small Ellipse Small Ellipse Small Oval Small Ellipse Large Ellipse Small Ellipse Large Oval Large Sphere Large Sphere Large Sphere Large Sphere Small Ellipse Small Oval 36 TO2 2-2.5 0.1 Convex Smooth, entire White 6x5 37 TV1 2-2.5 0.1 Convex Smooth, entire White 13x6 38 TV2 1-1.5 0.1 Convex Smooth, entire White 8x4 39 TV3 1-2 0.1 Convex Smooth, entire White 7x4 40 TV4 2-2.5 0.1 Convex Smooth, entire White 10x6 Small Oval Large Ellipse Small Ellipse Small Ellipse Large Ellipse 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. 11 Table 3. Fermentation ablility of yeast strains isolates Types of sugar Group I Group II Group III AA1, AG*, AG1, AG1.1, AG2, BD2, BL1, BL2, BL3, BL4, CM2, CM2.2, CM3, Glucose CM4.4, CT1, DT1, DT2, AG3.3, BD1, CT2, HG1, HG2, NK2, NK4, OM3, ST2, TV3, TV4. OM1, OM2, OM4, TB1, TG1, TNN3, TNN4, TO1, TO2, TS2,TV1, TV2. Saccharose AA1, BL1, BL2, CM2.2, AG*, AG1.1, AG2, CM4.4, CT1, DT1, DT2, AG3.3, BD1, BL3, BL4, HG2, ST2, TB1, TG1, CM2, CT2, HG1, NK2, TNN3, TNN4, NK4, OM2, OM3, OM4, TO1. TV1, TV2, TV3, TV4. AG1, BD2, BL4, CM3, OM1, TO2. . AA1, AG1, AG1.1, AG3.3, BD1, BD2, BL1, BL2, BL3, BL4, CM2, CM2.2, CM3, Maltose CM4.4, CT1, CT2, DT1, AG*, AG2, HG1, ST2, DT2, HG2, NK2, NK4, TV1, TV4. OM1, OM2, OM3, OM4, TB1, TG1, TNN3, TNN4, TV2, TV3, TO1, TO2, TS2 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. 12 Determination urease enzyme activity: a b c + d - e Figure 3: Urea media vitroes before and after 48 hours incubation *Note: (3a): Urea media before yeast transplanting (3b), (3c), (3d): Urea media after yeast transplanting and 48 hours incubation (3e): positive (left) and negative (right) control samples Table 4 shows the results of determination urease enzyme activity of yeast strains isolates. Table 4. Urease enzyme activity of yeast strains isolates Result Positive (+) Nagative (-) Yeast strains BL2, BL1, HG1. AA1, AG*, AG1, AG1.1, AG2, AG3.3, BD1, BD2, BL3, BL4, CM2, CM2.2, CM3, CM4.4, CT1, CT2, DT1, DT2, HG2, NK2, NK4, OM1, OM2, OM3, OM4, ST1, ST2, TB1, TG1, TNN3, TNN4, TO1, TO2, TV1, TV2, TV3, TV4. quantity 3/40 37/40 *Note: "+" medium turned into pink, "-"medium remained previous color. Values in the table are the average of three repetitions. The results showed that, after 48 hours incubation, 3 tubes turned into pink color: BL2, BL1, HG1 and 37 tubes all of yeast strains belong to groups 1, 2 and 3 remained yellow color, it means 37 yeast trains are incapable to analyze urea. 13 According to Booth and Vishniac (1987), the presence of urease enzyme plays a key role in the urea hydrolysis in yeast. In Urea Broth medium with color indicator phenol red, with a pH of 6.8 ± 0.2, medium color is orange. However, if the yeast strains are able to create urease enzyme to hydrolyze urea, CO2 and NH3 will be formed. The formation of NH3 will make pH increase and environment change to pink.  Preliminary Identification of yeast strains Yeast strains can be identified preliminarily based on morphological, physiological andbiochemical characteristics. 40 yeast strains isolates were identified preliminarily in Table 5 Table 5. The result of preliminary identification of 40 yeast strains islolates Morphological Physiological, biochemical characteristics characteristics Preliminary Group Cells shape 1 2 3 4 5 Large Budding Saccharose Maltose fermentation multiple Urea identification fermentation hydrolysis + + - Saccharomyces multiple directions +/- +/- - Saccharomyces Oval multiple directions - +/- -/+ Hanseniaspora Large bipolarity +/- +/- -/+ Kluyveromyces +/- +/- -/+ Kluyveromyces Sphere directions Large Oval Small Ellipse Small multiple Ellipse directions *Note: "+": capability, "-": incapability The result showed that yeast strains in Group 1 and 2 belong to Saccharosemyces spp., yeast strains in Group 3 belong to Hanseniaspora spp., yeast strains in Group 4 and 5 belong to Kluyveromyces spp. 3.3. Testing thermo-tolerant ability of yeast isolates The thermo-tolerant ability of yeasts was determined based on the growth of colonies at high temperature after 48 hours. 14 Thermal-tolenrant ability of 40 yeast strains isolates are shown in Table 6: Table 6. Thermal-tolenrantablitiyof 40 yeast strains isolates Name 30oC 35oC 37oC 40oC 43oC 45oC 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 + + + + - - 15 TB1 + + + + + - TG1 + + + + + - DT2 + + + + - - TNN3 + + + + + - TNN4 + + + + + - TO1 + + + + - - TO2 + + + + + - TS2 + + + + + - Total 40 40 40 39 27 0 *Note: “+”: forming colonies, “–”: no forming colonies 2 1 3 6 4 5 Figure 4. Some thermo-tolerant yeast at 43oC *Note: (1): TO1, (2): CT1, (3): HG1, (4): TNN4, (5) : TB1, (6) :HG2 After 48 hours incubation, all 44 yeast isolates formed colonies at 30 - 37ºC, 39 and 27 yeast isolates formed colonies at 40ºC and 43ºC. At 45ºC, there were no yeast isolates formed colonies. It showed that temperature is one of the major factors effecting on the development of yeasts. The higher the temperature is, the smallernumbers of yeast colony forming are. 27 yeast strains grown at high temperature (43oC) were used in further experiments. 3.4. Testing ethanol tolerant ability of yeast isolates The ethanol tolerant ability of yeasts was determined based on the growth of forming colonies at high ethanol concentration after 48 hours. The growth of yeast strains isolated in medium with addition ethanol concentration 3, 6, 9 and 12% are shown in Table 7. 16 Table 7. Ethanol tolerant ability of yeast isolates Yeast strains 3% 6% 9% 12% 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 + + + + 17 HG2 + + + - TV4 + + + + NK2 + + + + NK4 + + + + ST2 + + + - TB1 + + + + TG1 + + + - DT2 + + + + TNN3 + + + + TNN4 + + + - TO1 + + + + TO2 + + + - ST1 + + + - Sum 40 40 39 29 *Note: “+”: forming colonies, “–”: no forming colonies 2 1 6 3 5 4 Figure 5. Some ethanol tolerant yeast trainsat ethanol concentration 12% *Note: (1): TO1, (2) : CT1, (3): HG2 (4): HG2, (5) : TNN4 (6) : 18 After 48 hours incubation, all yeast isolates formed colonies in the medium supplemented with 3% and 6% of ethanol. In 9% ethanol contained medium, 39 isolates formed colonies. The number of yeast isolates forming colonies decreased when the supplemented ethanol concentration increased. As the results, 29 yeast isolates formed colonies in the medium containing 12% ethanol. This proved that ethanol was one of the major factors affecting on the growth of yeast strains. Ethanol inhibited the growth of yeast. By the way, higher concentrations of ethanol could cause poison for yeast. From the results of 4.1 and 4.2, 27 thermol-tolerant and ethanol tolerant yeast were selected: TV1, AG*, AG1, AG1.1, AG2, BD2, BL3, BL4, CM2.2, CM3, CM4.4, OM2, OM3, CT1, TV2, TV3, OM4, HG2, TV4, NK2, NK4, TB1, TG1, TNN3, TNN4, TO1, ST1. Twenty-seven lines of yeast were used in further experiments. 3.5. Ethanol fermentation ability of selected yeast isolates in glucose liquid Ethanol fermentation ability of seven yeast isolates was determined based on their CO2 production in Durham test tubes after 24 hours of fermentation. The results were presented in Table 8. 19 Table 8. Height of CO2 column produced in Durham test tubes Height of CO2 (mm) at fermentation moments (hours) Yeast Strains 2 4 AG* 1 3 fghij 6 8 16 18 a 7.67 30a AG1 0f 3fghij 9ghi 13.33efg 17.67d 22.67b 25bc 30a 30a AG1.1 0f 2hijk 8.67hij 12efhg 18d 19c 21d 24.67b 30a AG2 1de 4.33efgh 8.33hij 10.33hij 15def 17cd 22cd 30a 30a BD2 0f 3.67fghi 15.67d 20.33bc 30a 30a 30a 30a 30a BL3 0f 0k 3kl 5.33kl 13.67efg 19.33c 30a 30a 30a BL4 0f 4.67efg 6.67j 11.67fgh 16de 17.67cd 26.33b 30a 30a CM2.2 2cd 6.33de 14.67de 20.33bc 25bc 30a 30a 30a 30a CM3 2cd 4.33efgh 13ef 22.33bc 30a 30a 30a 30a 30a CM4.4 0f 4.67efg 10.33gh 21bc 25bc 30a 30a 30a 30a CT1 8.67b 17.67b 30a 30a 30a 30a 30a 30a 30a HG2 11.3a 20.67a 30a 30a 30a 30a 30a 30a 30a NK2 0f 2.33ghijk 11fg 15.67def 25bc 30a 30a 30a 30a NK4 1de 3.67fghi 11fg 16de 22c 30a 30a 30a 30a OM2 0f 0k 3.33k 7.33jk 15def 17.67cd 25bc 30a 30a OM3 0f 0k 3.33k 5.67kl 10.33g 13e 20d 25.33b 30a OM4 2.33cd 5def 12.67ef 18.67bcd 23.67bc 30a 30a 30a 30a ST1 0f 1.33ijk 7.33ij 18.33cd 30a 30a 30a 30a 30a TB1 0f 4efgh 18.67c 30a 30a 30a 30a 30a 30a TG1 2.67c 11.67c 22b 22.67b 30a 30a 30a 30a 30a TNN3 0f 2.67fghij 15.33d 22bc 30a 30a 30a 30a 30a TNN4 0f 7d 22b 30a 30a 30a 30a 30a 30a TO1 0f 10.33c 30a 30a 30a 30a 30a 30a 30a TV1 2cd 5def 13ef 19.33bcd 26.67ab 30a 30a 30a 30a TV2 0f 3.67fghi 9ghi 13.33efg 16de 19.67bc 24.67bc 30a 30a TV3 0f 1.0jk 2kl 8ijk 12fg 15.67de 20d 24.67b 30a TV4 0f 0k 1l 2l 4h 5.67f 10.67e 15.33c 21b ij 10.33 10 hij de 17 12 18.67 14 cd 23.33 bcd 30 *Note: The maximum height of CO2 trapped in Durham tube is 30 mm. Value in the table was average value of triplication; the average values with the same letter were not significantly different at the 95% confidence level. Glucose fermentation in the Durham tube assessed the preliminary fermentation ability of selected yeast strains. After 2 hours, CO2 appeared in tubes containing TV1, AG2, CM2.2, CM3, CT1, OM4, HG2, NK4, TG1, TO1 isolates then in others tubes (4 and 6 hours). At 4, 20 and 6 hours, height of CO2 column in tubes containing TV1, AG2, CM2.2, CM3, CT1, OM4, HG2, NK4, TG1, TO1 isolates was always higher than others. After 6 hours, CT1, HG2 and TO1 isolates reached the maximum height of Durham tubes (30 mm). The ethanol fermentation ability of CT1, HG2 and TO1 isolates in 2% w/v glucose medium was faster and stronger than other yeast isolates. Thus, these yeast isolates was selected for further experiments to find favorable conditions for yeast producing ethanol from molasses at high temperature 3.6. Ethanol fermentation ability of CT1, HG2 and TO1 yeast isolates at high temperature Ethanol fermentation ability at high temperature of CT1, HG2 and TO1 yeast isolates was determined by measuring the ethanol concentration after five fermentation days at room temperature, 35, 40, and 45ºC. The results were presented in Figure 6 Ethanol concentration (%v/v) 9 8 a ab 8.05ab 8.18 8.12 7.89abc 7.97ab7.85bc cd 7.47d 7.49d 7.6 30°C 7 35°C 6 37°C 5 40°C 4 42°C 3.31e 3.03ef 3 2.77fg 2.84fg 2.69g 2.77fg 2 1 0 CT1 HG2 Yeast strains TO1 Figure 6. Graph showing the concentration of ethanol produced by CT1, HG2 and TO1 at temperatures of 30, 35, 37, 40 and 42oC *Note: Value in the figure was average value of triplication; the average values with the same letter were not significantly different at the 95% confidence level The results showed that, in all three yeast strains, ethanol concentration produced decreased when the temperature increased from 30oC to 42oC. In CT1 strain, highest ethanol concentration generated at 35oC (8.18% v/v) and reduced significantly when temperature increased from 37oC to 40oC (reduced from 8.12% v/v to 3.31% v/v). 21 In HG2 strain, highest ethanol concentration generated at 35oC (7.97% v/v) and reduced significantly when temperature increased from 37oC to 40oC (reduced from 7.85% v/v to 2.77% v/v). In TO1 strain, highest ethanol concentration generated at 37oC (7.6% v/v) and reduced significantly when temperature increased from 37oC to 40oC (reduced from 7.6% v/v to 2.84% v/v). In 3 yeast trains, the ethanol concentration among threetemperatures 30, 35 and 37oC were not significantly different at the 95% confidence level. The content of ethanol among two experimental temperatures 40 and 42oC were not significantly different at the 95% confidence level, besides, between 3 experimental temperatures 30oC, 35oC, 37oC and 2 experimental temperatures 40oC, 42oC were significantly different at the 95% confidence level. All three yeast strains selected fermented well at temperature from 30oC to 37oC and began to reduce when threshold 40oC. From the above results, it can be said that temperature is an important factor affecting the ethanol fermentation ability of yeast. According to research by Nanba and Nagai (1987) showed that, when the temperature was high, the ability of the activity of intracellular enzyme of yeast would be diminished, thereby ethanolproduced concentration was lower. Besides, according to Navarro and Durand (1978), when the temperature was high, the amount of ethanol intracellular accumulation within the yeast cells increase and disrupt the growth of yeast, and then fermentation activity of yeast is inhibited, cause a lower amount of ethanol produced. It was found that the fermentation activity of the yeast greatly affected by temperature, the higher the temperature, the lower ethanol produced concentration. 3.7. Identification of the TO1 yeast isolate On YPD agar medium, TO1 isolate could form white smooth entire colonies. The colonies were about 1 – 1.5 mm of size and 0.1 mm of height (Figure 7). Microscope images showed that the diameter of the TO1 cells ranged from 7 to 8 μm, and the cells’ shape is oval (Figure 8). 22 Figure 7. TO1 colonies Figure 8. TO1 cells under microscope X100 The selected sequences were homologous with ITS1, 5.8 rDNA, and ITS2 sequences of Saccharomyces cerevisiae species with 93% homogeneous level. Thus, TO1 belonged to the Saccharomyces cerevisiae. According to Araque Edgardo et al. (2008), 11 Saccharomyces cerevisiae strains are able to grow and ferment glucose in a temperature range of 35–45oC. 23 CHAPTER 4. CONCLUSIONS AND SUGGESTIONS 4.1. Conclusions 40 yeast trains were isolated from 12 sources of alcoholic fermentation starter. In particular, there were 27 yeast strains able to grow at temperature 42oC, 29 yeast strains able to grow at 12% ethanol concentration. The selected 27 yeast strains tolerated heat (43oC) and tolerated ethanol (12%): TV1, AG*, AG1, AG1.1, AG2, BD2, BL3, BL4, CM2.2, CM3, CM4. 4, OM2, OM3, CT1, TV2, TV3, OM4, HG2, TV4, NK2, NK4, TB1, TG1, TNN3, TNN4, TO1, ST1. Three yeast strains CT1, HG2 and TO1 had fast and strong fermentation ability in 2% w/v glucose medium. (CO2 column height reached 30 mm in 6 hours). CT1 produced 3.03% v/v of ethanol in molasses medium at 42ºC. It was significantly different from 37oC (8.12% v/v), 35oC (8.18% v/v) and 30oC (8.05% v/v) at the 95% confidence level. HG2 produced 2.69% v/v of ethanol in molasses medium at 42ºC. It was significantly different from 37oC (7.85% v/v), 35oC (7.97% v/v) and 30oC (7.89% v/v) at the 95% confidence level. TO1 produced 2.77% v/v of ethanol in molasses medium at 42ºC. It was significantly different from 37oC (7.67% v/v), 35oC (7.49% v/v) and 30oC (7.47% v/v) at the 95% confidence level. Molecular analysis showed that TO1 belonged to the Saccharomyces cerevisiae. 4.2. Suggestions Testing factors affecting the fermentation ability of 3 yeast 3 strains CT1, HG2 and TO1 as isolated yeast cell number, pH, Brix, fermentation time... in high temperatures condition. Testing ethanol ability of 3 yeast strains isolates in other kinds of medium: fruit juices, sugar cane juice… 24 REFERENCES Vietnamese Nguyễn Lân Dũng. 1998. Vi sinh vật học. Nhà xuất bản Giáo dục. Nguyễn Đức Lượng. 2004. Công nghệ sinh học tập 1 – Cơ sở vi sinh vật công nghiệp. NXB Nông nghiệp. English Alfenore, S., C. Molina, S.E. Guilouet, J. L. Uribelarrea, G. Goma and L. Bendadis. 2002. Improving ethanol producing and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process. Applied Microbiology and Biotehnology. 60:67-72. Araque Edgardo, Parra Carolina, Rodríguez Manuel, Freer Juanita and Jaime Baeza. 2008. Selection of thermotolerant yeast strains Saccharomyces cerevisiae for bioethanol production. Enzyme and Microbial Technology Volume 43, Issue 2, Pages 120–123. Booth, J.L., and H.S. Vishniac.1987. Urease testing and yeast taxonomy. Can. J. Microbiol. 33. 396-404. Nanba, A. and Nagai S. 1987. Kinetic analysis of batch ethanol fermentation of S. cerevisiae.Journal of Fermentation Technology, 65:277-283. Navarro, J. M. and G. Durand. 1978. Alcohol fermentation: effect of temperature on ethanol accumulation within within yeast cells. Annals of Microbiology. Roehr, M. 2001. The Biotechnology of Ethanol: Classical and Future Applications. Chichester: Wiley-VCH, pp. 232. 25 [...]... J.L., and H.S Vishniac.1987 Urease testing and yeast taxonomy Can J Microbiol 33 396-404 Nanba, A and Nagai S 1987 Kinetic analysis of batch ethanol fermentation of S cerevisiae.Journal of Fermentation Technology, 65:277-283 Navarro, J M and G Durand 1978 Alcohol fermentation: effect of temperature on ethanol accumulation within within yeast cells Annals of Microbiology Roehr, M 2001 The Biotechnology of. .. 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 5 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,... yeast strains in Group 1 and 2 belong to Saccharosemyces spp., yeast strains in Group 3 belong to Hanseniaspora spp., yeast strains in Group 4 and 5 belong to Kluyveromyces spp 3.3 Testing thermo- tolerant ability of yeast isolates The thermo- tolerant ability of yeasts was determined based on the growth of colonies at high temperature after 48 hours 14 Thermal-tolenrant ability of 40 yeast strains isolates... formed colonies in the medium containing 12% ethanol This proved that ethanol was one of the major factors affecting on the growth of yeast strains Ethanol inhibited the growth of yeast By the way, higher concentrations of ethanol could cause poison for yeast From the results of 4.1 and 4.2, 27 thermol -tolerant and ethanol tolerant yeast were selected: TV1, AG*, AG1, AG1.1, AG2, BD2, BL3, BL4, CM2.2, CM3,... concentration was lower Besides, according to Navarro and Durand (1978), when the temperature was high, the amount of ethanol intracellular accumulation within the yeast cells increase and disrupt the growth of yeast, and then fermentation activity of yeast is inhibited, cause a lower amount of ethanol produced It was found that the fermentation activity of the yeast greatly affected by temperature, the... 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 11 Table 3 Fermentation ablility of yeast... Uribelarrea, G Goma and L Bendadis 2002 Improving ethanol producing and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process Applied Microbiology and Biotehnology 60:67-72 Araque Edgardo, Parra Carolina, Rodríguez Manuel, Freer Juanita and Jaime Baeza 2008 Selection of thermotolerant yeast strains Saccharomyces cerevisiae for bioethanol production Enzyme and Microbial... lines of yeast were used in further experiments 3.5 Ethanol fermentation ability of selected yeast isolates in glucose liquid Ethanol fermentation ability of seven yeast isolates was determined based on their CO2 production in Durham test tubes after 24 hours of fermentation The results were presented in Table 8 19 Table 8 Height of CO2 column produced in Durham test tubes Height of CO2 (mm) at fermentation. .. 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... anaerobically at 30, 35, 37, 40, and 42ºC for five days Ethanol content after fermentation was determined by distillation method 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/) ... topic "Isolation and selectionthermo -tolerant yeast in alcoholic fermentation starter" is done 1.2 Objectives The aim of this study is isolation and selection of thermo- tolerant and ethanol- tolerant. .. analyzed by Microsoft Office Excel 2010 and Statgraphics centurion XVI (USA) software CHAPTER RESULTS AND DISCUSSION 3.1 Isolation Forty isolates were obtained from alcoholic fermentation starter sources... tolerant yeast from alcoholic fermentation starter and capable of fermentation ethanol at high temperature in molasses CHAPTER 2: MATERIALS AND METHODS 2.1 Materials Molasses (collected from Phung