Effects of fermentation conditions on ethanol production from corn by Saccharomyces cerevisiae MS42

Abstract: Fermentation is a complicated process which depends on a variety of conditions. In order to gain high fermentation efficiency with a low level of toxins and impurities to obtain good products, it is essential to determine suitable conditions for the process. Within the scope of this study, the effects of some key conditions was estimated (i.e. pH, temperature, SO2 level in fermentation broth, the inoculation rates of yeast cells, sugar level) for the fermentation of corn with Saccharomyces cerevisiae MS42. The analyses of fermentation efficiency, remaining amount of sugar, alcohol content, toxic components, impurities, and sensory evaluation were conducted to determine optimal conditions. The results show that the fermentation broth from corn (100%) with pH: 4.5 - 5.0; temperature from 25 °C to 30 °C; K2S2O5: 0.2 - 0.3 (g/L); the inoculation rate of yeast cells: 7 - 10%; sugar concentration: 140- 160 g/L during 144 - 156 hours are optimal for the fermentation process by Saccharomyces cerevisiae MS42.

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JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2016-0070 Natural Sci. 2016, Vol. 61, No. 9, pp. 169-176 This paper is available online at 169 EFFECTS OF FERMENTATION CONDITIONS ON ETHANOL PRODUCTION FROM CORN BY Saccharomyces cerevisiae MS42 Nguyen Quang Thao 1 , Nguyen Van Quyen 2 , Nguyen Thao Anh 3 and Nguyen Thanh Dat 4 1 Department for Science & Technology, Ministry of Industry and Trade 2 Faculty of Natural Science, Hai Duong Teacher Training College 3 School of Bio-technology and Food Technology, Hanoi University of Science and Technology 4 Faculty of Biology, Hanoi National University of Education Abstract: Fermentation is a complicated process which depends on a variety of conditions. In order to gain high fermentation efficiency with a low level of toxins and impurities to obtain good products, it is essential to determine suitable conditions for the process. Within the scope of this study, the effects of some key conditions was estimated (i.e. pH, temperature, SO2 level in fermentation broth, the inoculation rates of yeast cells, sugar level) for the fermentation of corn with Saccharomyces cerevisiae MS42. The analyses of fermentation efficiency, remaining amount of sugar, alcohol content, toxic components, impurities, and sensory evaluation were conducted to determine optimal conditions. The results show that the fermentation broth from corn (100%) with pH: 4.5 - 5.0; temperature from 25 °C to 30 °C; K2S2O5: 0.2 - 0.3 (g/L); the inoculation rate of yeast cells: 7 - 10%; sugar concentration: 140- 160 g/L during 144 - 156 hours are optimal for the fermentation process by Saccharomyces cerevisiae MS42. Keywords: Fermentation, corn, Saccharomyces cerevisiae MS42, optimal conditions. 1. Introduction Ethanol fermentation is a complicated process to biotransform sugar to ethanol by EMP (Embden-Meyerhof-Parnas pathway), release CO2 an energy (117.6 KJ) [1]. Acording to Ivanov, A. Lebedep, Neiberg and Mayerhoff [2], the fermentation by Saccharomyces cerevisae is divided into five phases and generalized by chemical equation below: C6H12O6 + 2ADP + 2H3PO4 → 2C2H5OH + 2CO2 + 2ATP + H2O Several studies showed that the growth of yeast and ethanol fermentation depends on finding conditions such as: pH, sugar concentration, temperature, SO2. Thus, study on determind optimal conditions for ethanol fermentation is necessary to create a good quality product with high efficiency, less toxins and impurities. Received September 23, 2016. Accepted December 10, 2016. Contact Nguyen Van Quyen, e-mail address: vanquyengv@gmail.com Nguyen Quang Thao, Nguyen Van Quyen, Nguyen Thao Anh and Nguyen Thanh Dat 170 The objectives of study is identifying a number of basic conditions affecting the process of fermentation of Saccharomyces cerevisiae MS42 in from corn liquor and identifying kinetic process of alcoholic fermentation of Saccharomyces cerevisiae MS42 in from corn liquor. 2. Content 2.1. Materials and methods 2.1.1. Materials, chemicals and equipments Fermentation medium (N) from 100% of corn (cultivar DK9955 from Vietnam), corn liquid was made by crushing (d ≤ 0.5mm) with water (ratio 1:2.5), adding enzyme Termamyl (0.35%) activated at 45 o C in 20 minutes then rose to 85 o C in 45 minutes and heating up to 100 o C in 30 minutes starch hydrolysis. We continued adding Termamyl 0.05% to avoid clot then change temperature to 64 o C in 90 minutes plus enzyme Dextrozyme 1.0% for saccharification. The sugar concentration of corn liquid was 250 g/L, protein: 3.82 g/L, FAN 60.88 mg/L. - Yeast strain: Saccharomyces cerevisae MS42 was isolated from traditional baker’s starter in Mau Son (Lang Son). - Corn cultivar DK9955 from Vietnam. - Chemicals: purified chemicals, glucose, pepton, meat extract, agar (Merk, USA); NaCl, KH2PO4, (NH4)2SO4, MgSO4.7H2O (PA). Enzyme Termamyl®; Dextrozyme ® GA (Novozymes, Denmark). - Equipments: light microscope Olympus BX - 41, refrigerated incubator FOC-225E, Agilent 6890 GC Gas Chromatograph Series, SMICO alcohol distillation equipment, sugar measurement device, alcohol measurement device. 2.1.2. Methods - Sensory analysis by 20-point scale (TCVN 3215 - 79) and analytical chromatography was used to determine the existence, possible analytes concentration in the samples. - The cell concentration was determined by Thoma cell counting chamber [2]. - Maltose and glucose sugars in the fluid were measured by DNS method, comparing with Bectran method [2, 3]. y = 10.335x + 2.2915 R2 = 0.9982 0.00 2.00 4.00 6.00 8.00 10.00 12.00 0 0.2 0.4 0.6 0.8 1 Opitical density C o n te n t g lu c o s e ( m ic ro m o l/ m l) y = 10.878x + 2.4679 R2 = 0.9976 0.00 2.00 4.00 6.00 8.00 10.00 12.00 0 0.2 0.4 0.6 0.8 1 Opitical density M a n to s e c o n te n t (m ic ro m o l/ m l) Fugue 1. Standard graph for Glucose Fugue 2. Standard graph for Maltose - Determining the level of acidity using acid neutralization method [2]. - Determining the pH level of the fermentation broth using a pH meter [2]. - Optimize conditions by using Design – Expert software. Effects of fermentation conditions on ethanol production from corn by Saccharomyces cerevisiae MS42 171 2.2. Results and discussions 2.2.1. Effect of sugar concentration on ethanol fermentation The fermentation and the quality of products are affected by different amount of sugar. In this research, the corn liquid was used and diluted to get the sugar concentration: 120, 140, 160, 180, 200, 220 (g/L). The medium pH value was 5.0, inoculation rate was 7% (equivalent to 15 × 10 6 cells/mL) at 25 o C. The analytical results are shown in the Table 1. Table 1. Effects of sugar concentration on the fermentation process Sugar concentration (g/L) Analytical index 120 140 160 180 200 220 Fermentation time (hours) 104 ± 2 108 ± 2 144 ± 3 154 ± 3 168 ± 3 192 ± 3 Alcohol content (%V) 6.70 7.84 8.93 9.95 10.81 11.64 Efficiency (%) 90.60 90.50 90.23 89.25 88.18 86.71 Remaining amount of sugar (g/L) 5.1 ± 0.1 5.4 ± 0.1 6.3 ± 0.2 6.7 ± 0.2 9.6 ± 0.2 11.5 ± 0.2 The results showed that when the amount of sugar increased from 120 g/L to 180 g/L, the fermentation efficiency decreased by contrast; this can be explain that the osmotic pressure and the amount of alcohol created inhibited the growth of yeast during process. The highest efficiency was 90.6% at 120 g/L however the alcohol accumulation was low (6.7%V), this might lead to infection by unwanted microorganisms. From the content of 140 g/L to 160 g/L, the efficiency decreased slightly compared to 120 g sugar, however, it can be seen that the efficiency went up significantly which can prevent liquor from infection. The amount of sugar from 180 g/L to 220 g/L indicated that the efficiency declined, remaining amount of sugar was higher than other samples’s. However, this is a disadvantage for the growth of yeast because of effects on yeast cell membrane by osmotic pressure. In author’s opinion, 140 - 160 g/L of sugar concentration in corn liquid was optimal for whisky fermentation. Although the time of fermentation was short, the process efficiency still high, the amount of alcohol was 7.84 - 8.93%V which can prevent unwanted microorganisms effectively. For following experiments, we chose using 160 g/L of sugar in corn liquid. 2.2.2. Effect of pH on ethanol fermentation The medium was set with 160 g/L sugar concentration, inoculation rate was 7%, temperature was 25 o C, using acid citric and NaHCO3 to adjust pH level to 4.0, 4.5, 5.0, 5.5, 6.0. The results are shown in Table 2. Nguyen Quang Thao, Nguyen Van Quyen, Nguyen Thao Anh and Nguyen Thanh Dat 172 Table 2. Effect of pH level on the fermentation pH level Analytical index 4.0 4.5 5.0 5.5 6.0 Alcohol content (%V) 8.7 8.8 8.9 8.9 8.7 Fermentation efficiency (%) 87.67 88.56 89.57 89.57 87.55 Remaining amount of sugar (g/L) 6.2 ± 0.1 6.1 ± 0.1 5.9 ± 0.1 5.9 ± 0.1 5.8 ± 0.1 Acetaldehyde (mg/L) 25.13 ± 1.0 33.26 ± 1.0 35.82 ± 1.0 40.29 ± 1.0 56.21 ± 1.0 Methanol (mg/L) 5.32 ± 0.1 4.26 ± 0.1 4.19 ± 0.1 4.21 ± 0.1 4.28 ± 0.1 Higher alcohols (mg/L) 237.8 ± 2.0 294.5 ± 2.0 317.2 ± 2.0 375.2 ± 2.0 378.1 ± 2.0 Furfurol (mg/L) 1.26 ± 0.05 1.56 ± 0.05 1.68 ± 0.05 1.75 ± 0.05 1.81 ± 0.05 Sensory evaluation 15.3 15.3 15.6 15.9 15.5 At pH level of 4.5 - 5.5, the alcohol content, efficiency both were very high while the amount of methanol was low, however, at 5.0, the results were optimal. In contrast, the efficiency fell considerably, aldehyde and higher alcohols increased. According to [4-6], pH = 5.0 is appropriate for enzymes activation to bio-transform glucose to alcohol, which is also similar to our results. Based on that, we decided to use medium with pH level at 5.0 for following experiments. 2.2.3. Effect of temperature on ethanol fermentation Table 3. Effect of temperature on the fermentation Temperature ( o C) Analytical index 16 20 25 30 35 Fermentation time (hours) 168 ± 6 160 ± 6 150 ± 6 146 ± 6 143 ± 6 Alcohol content (%V) 9.02 8.95 8.90 8.86 8.61 Fermentation efficiency (%) 90.87 90.34 89.82 89.30 86.81 Remaining amount of sugar (g/L) 5.8 ± 0.1 6.0 ± 0.1 6.1 ± 0.1 5.9 ± 0.1 5.8 ± 0.1 Acetaldehyde (mg/L) 21.53 ± 0.5 30.26 ± 0.5 36.85 ± 0.5 46.85 ± 0.5 61.59 ± 0.5 Methanol (mg/L) 3.45 ± 0.1 3.96 ± 0.1 4.37 ± 0.1 6.13 ± 0.1 8.21 ± 0.1 Higher alcohols (mg/L) 225.61 ± 2.0 241.53 ± 2.0 319.28 ± 2.0 382.25 ± 2.0 396.11 ± 2.0 Furfural (mg/L) 1.2 ± 0.05 1.41 ± 0.05 1.72 ± 0.05 1.85 ± 0.05 2.01 ± 0.05 Sensory evaluation 16 15.8 15.5 15.1 14.8 The corn liquid has compositions: sugar concentration: 160g/L, inoculation rate 7%, pH = 5.0 with different temperature: 16 o C, 20 o C, 25 o C, 30 o C, 35 o C. Effects of fermentation conditions on ethanol production from corn by Saccharomyces cerevisiae MS42 173 The result has been shown clearly that at low temperatures, it took the time longer but the quality of products was better with less methanol, acetaldehyde, impurities. When temperature reached higher, the speed of process was faster but the amount of methanol, acetaldehyde, higher alcohols and impurities also higher. Although the temperature from 16 o C to 20 o C shows the best result but it could not be apply to industrial scale because of economic effective (low temperature is required). For further research, we chose temperature from 25 o C to 30 o C for industrial scale to create quality products [7]. 2.2.4. Effect of SO2 content on ethanol fermentation SO2 was used to inhibit activities of bacteria on fermentation broth. However, using SO2 uncontrollably will reduce efficiency and quality of products. The experiment was set: 160 g/L sugar, pH: 5.0, temperature was 28 o C, inoculation rate: 7%, SO2 from K2S2O5 with different amounts (g/L): 0.15, 0.20, 0.25, 0.30, 0.40. Table 4. Effect of SO2 content on the fermentation K2S2O5(g/L) Analytical index 0.15 0.20 0.25 0.30 0.4 Alcohol content (%V) 8.78 8.96 8.95 8.95 8.87 Fermentation efficiency (%) 88.54 90.50 90.50 90.50 89.82 Remaining amount of sugar (g/L) 5.93 ± 0.1 6.12 ± 0.1 6.26 ± 0.1 6.31 ± 0.1 6.52 ± 0.1 Acetaldehyde (mg/L) 23.55 ± 1.0 32.26 ± 1.0 39.89 ± 1.0 56.75 ± 1.0 75.52 ± 1.0 Methanol (mg/L) 3.92 ± 0.1 4.75 ± 0.1 5.31 ± 0.1 7.12 ± 0.1 9.15 ± 0.1 Higher alcohols (mg/L) 236.66 ± 2.0 245.58 ± 2.0 329.26 ± 2.0 405.25 ± 2.0 426.18 ± 2.0 Furfurol (mg/L) 1.28 ± 0.05 1.65 ± 0.05 1.95 ± 0.05 1.98 ± 0.05 2.32 ± 0.05 Sensory evaluation 15.3 15.5 15.2 15.2 14.5 The results above showed that using less than 0.2 g/L K2S2O5 could lower the methanol content but on the other side, the efficiency slightly decreased, this could be explain that samples might be infected. With 0.4 g/L K2S2O5 in the fluid, both fermentation efficiency and the quality of products are lower than others because the growth of yeast was inhibited by high amount of K2S2O5. Based on these, 0.2 - 0.3 g/L K2S2O5 is optimal for the growth of yeast strain MS42 which inhibited unwanted microorganisms as well. This conclusion also consistent with published papers researching on effect of SO2 content (maximum amount is 490 ppm). For further experiments, we decided using 0.3 g/L K2S2O5 (the concentration of SO2 is 110 - 150 approximately) to get higher efficiency on large scale [8, 9]. Nguyen Quang Thao, Nguyen Van Quyen, Nguyen Thao Anh and Nguyen Thanh Dat 174 2.2.5. Effect of yeast inoculation rate on ethanol fermentation The inoculation rate of yeast has a directly influence to the fermentation. To study on the effect of inoculation rate, the experiments was conducted similar to above parts (pH = 5 .0, 160 g/L sugar concentration, t o = 28 o C, K2S2O5 = 0.3 g/L [8, 9]) only changing the inoculation rate of yeast cells: 5%, 7%, 12% (appropiated (10, 14, 20, 24) ×10 6 cells/mL). The results are shown in Table 5. Table 5. Effect of yeast inoculation rate on ethanol fermentation Inoculation rates Analytical index 5% 7% 10% 15% Fermentation time (h) 160 ± 3 152 ± 3 146 ± 3 143 ± 3 Alcohol content ( 0 V) 8.83 9.00 9.06 8.96 Fermentation efficiency (%) 89.71 90.87 91.40 90.34 Remaining amount of sugar (g/L) 7.05 ± 0.1 6.08 ± 0.1 5.92 ± 0.1 5.85 ± 0.1 The contrast between time and inoculation rate is shown in Table 5, the changing of efficiency, alcohol content, remaining amount of sugar is showing well but not considerable. This is logical because the yeast strain still were on log phase when inoculation, so the higher inoculation yeast rate is, the faster fermentation is. However, at inoculation rate of 15%, the fermentation time and efficiency seem slightly decreased. Thus, we chose the yeast inoculation rate was 7 - 10% to control process and shorten the time. 2.2.6. Research on ethanol fermentation by Saccharomyces cerevisiae MS42 In this study, the corn broth (made from 100% corn) was used as a medium and conditions were adjusted based on previous results: sugar 160 g/L, pH = 5.0, K2S2O5: 0.3 (g/L), yeast inoculation rate: 8% at 28 0 C in fermentor capacity 15L. Samples were collected and analysed every 12 hours. Results are shown in Table 6. It can be observed that: At the period of first 24 - 72 hours, the number of yeast cells is stable with an average of 204 × 10 6 cell/mL then declined slowly; the highest number was marked at 36 hours (216 × 10 6 cell/ml). After 108 hours, the number of cells dropped quickly and then stayed steadily after 144 hours. The changing of cell numbers can be explain that: at the first 24 hours, yeast were on log phase in a nutrition medium with low alcohol content which is an advantage for the growth of yeast; after 60 hours, the nutrition content decreased while the alcohol content went up in anaerobic condition which inhibited the yeast growth; besides, a deposit of cells at the bottom also repressed yeast growth through a long fermentation time. The line graph of sugar and alcohol content provide some information: in spite of the fact that sugar concentration declined substantially, the alcohol content showed a slowly upward trend. After 96 hours of fermentation, sugar concentration went down slightly and remained stable after 144 hours. The maximum content of alcohol was 9.1% at the period of 144 - 156 hours. Effects of fermentation conditions on ethanol production from corn by Saccharomyces cerevisiae MS42 175 Table 6. Changes of sugar concentration, alcohol, acid levels and number of yeast cells during fermentation Analytical index Time Sugar concentration (g/L) Alcohol content (%V) Number of yeast cell (×10 6 cell/mL) Total acid (based on acetic acid) mg/L 0 h 160.0 ± 0.5 0.2 17 ± 1 418.5 ± 1 12 h 144.5 ± 0.5 1.2 135 ± 3 426.8 ± 1 24 h 125.5 ± 0.5 2.8 208 ± 3 426.5 ± 2 36 h 110.1 ± 0.5 4.5 216 ± 3 455.9 ± 2 48 h 80.4 ± 0.3 6.1 215 ± 3 461.8 ± 2 60 h 65.8 ± 0.3 6.8 200 ± 3 461.8 ± 2 72 h 45.2 ± 0.3 7.2 165 ± 2 482.3 ± 2 84 h 30.6 ± 0.3 7.5 115 ± 2 482.3 ± 2 96 h 26.8 ± 0.3 7.7 105 ± 2 486.9 ± 2 108 h 18.3 ± 0.3 7.9 86 ± 2 490.5 ± 1 120 h 13.6 ± 0.2 8.3 70 ± 2 506.8 ± 1 132 h 11.2 ± 0.2 8.6 61 ± 2 506.8 ± 1 144 h 8.1 ± 0.2 9.0 49 ± 2 511.7 ± 1 156 h 6.2 ± 0.1 9.1 46 ± 1 514.3 ± 0.5 168 h 6.1 ± 0.1 9.0 45 ± 1 514.8 ± 0.5 Compared with the fermentation from malt (100%), the fermentation from corn took 10 – 12 hours longer because the amount of free amino acid in malt wort (FAN = 192.5 mg/L) is higher than in corn liquor (FAN = 60.88 mg/L); however, there are more sugar concentrations such as maltose, maltotriose, in corn liquor than in malt wort. Total acid content increased gradually and lower pH because not only the sugar biotransforms into alcohol but also into organic acids such as: acetic acid, malic acid, this process still continued by the activation of enzymes even after the ethanol fermentation. 0 50 100 150 200 250 300 350 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 Fermentation time (hours) c o n te n t Sugar content (g/l) Alcohol by volume (%V) Number of yeast cell (millon cell/ml) Fugue 3. Changes in content during fermentation by S. cerevision MS42 Nguyen Quang Thao, Nguyen Van Quyen, Nguyen Thao Anh and Nguyen Thanh Dat 176 The results in this research are corresponding with a number of public studies [4, 7, 8, 10]. In author’s suggestion, the optimal time is 144 - 156 hours; it should not take longer because of the influence to the economic efficiency and the quality of products. 3. Conclusion Optimal conditions for whisky fermentation from 100% corn with yeast strain Saccharomyces cerevisiae MS42 was found: sugar concentration: 140 - 160 g/L, pH: 4.5 - 5.0, temperature from 25 to 30 0 C, the amount of K2S2O5: 0.2 - 0.3 (g/L), inoculation rate: 7 - 10% during 144 - 156 hours. REFERENCES [1] D'Amato, D., Corbo, M.R., Del Nobile, M.A., Sinigaglia, M., 2006. Effects of temperature, ammonium and glucose concentrations on yeast growth in a model wine system. International Journal of Food Science & Technology 41, pp. 1152-1157. [2] Le Thanh Mai, Nguyen Thi Hien, Pham Thu Thuy and Nguyen Thanh Huong, Le Thi Lan Chi. 2006. The analysis methods of fermentation industry. Publishers of Technical Sciences, p. 28, 42-51 (in Vietnamese). [3] AOAC, 2012. Official Methods of Analysis. 19th Ediction, p. 210-235. [4] Nguyen Thanh Dat. 2012, Basic biology: Microorganisms. Volume 1. Publisher University of Education, p. 28-42, 116, 197-205, 233-240 (in Vietnamese). 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