Study on the production of ethanol from mango peel

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MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY STUDY ON THE PRODUCTION OF ETHANOL FROM MANGO PEEL SUPERVISOR STUDENT Dr. NGO THI PHUONG DUNG DUONG THI THANH XUAN Student code: 5085861 Session: 34 (2008 – 2013) Can Tho, 2013 APPROVAL SUPERVISOR Dr. NGO THI PHUONG DUNG STUDENT DUONG THI THANH XUAN Can Tho, 2013 PRESIDENT OF EXAMINATION COMMITTEE i Abstract The increasing interest in biotechnological processes employing lignocellulosic residues is justifiable because these materials are cheap, renewable and widespread sugar sources. Biotechnological conversion of biomass into fuels and chemicals requires hydrolysis of the polysaccharide fraction into monomeric sugars that are converted into ethanol. The objective of this study was to examine the feasibility for the production of ethanol from mango peel. The experimental activities included: studying the effects of H2SO4 concentration, time and temperature on the hydrolysis of mango peel, assessing the effects of yeast inoculum levels, pH levels to the fermentation capacity, determining the favourable time and temperature for ethanol fermentation. The results showed that the hydrolysis of mango peel with H2SO4 of 3% at 121oC for 1 hour released 8.49% reducing sugars. The fermentation conditions including yeast inoculum levels at 10 5cells/mL and pH 5.5 were found to be favourable for ethanol fermentation. In the treatment of 7 incubation days at 30°, the ethanol concentration of 3.08% (v/v) was obtained. Keyword: ethanol, fermentation, hydrolysis, mango peel, Saccharomyces cerevisiae. i CONTENTS Abstract i Content ii 1. Introduction 1 2. Materials and methods 4 3. Results and discussion 8 3.1. Hydrolysis of mango peel 3.1.1. The effect of the concentration of H2SO4 on 8 8 hydrolysis 3.1.2. The effect of time and temperature on 10 hydrolysis 3.2 Fermentation with Saccharomyces cerevisiae 13 3.2.1. The effect of yeast density on fermentation 13 3.2.2. The effect of pH on fermentation 14 3.2.3. The effects of temperature and time on 16 fermentation 4. Conclusion and Suggestions 20 4.1 Conclusion 20 4.2 Suggestions 20 References 21 ii 1. INTRODUCTION Nowadays, ethanol is an important industrial chemical with emerging potential as a bio-fuel to replace non-renewable fossil fuels (Alfenore et al., 2002). Ethanol may be produced commercially by chemical synthesis or biosynthesis. To produce ethanol using substrates such as molasses, fruit pulps etc. are well known. The economics of ethanol production by fermentation is significantly influenced by the cost of the raw materials. It accounts for more than half of the production costs. To achieve a lower production cost, the supply of cheap raw material is thus a necessity. Production of value added products from agro-industrial and food processing wastes is now an area of focus because it reduces pollution in the environment in addition to the energy generation. The major part of this kind of waste is mostly discarded and it is the main source for increasing the pollution in environment on occasions and also, the discarding process become a very expensive step due to high transportation costs. Majority of fruit and vegetable wastes available from their processing industries are seasonal and do not decompose rapidly. Moreover, carbohydrate is the main source for ethanol production which is easily found in various plantparts. It was reported that natural resources along with S. cerevisiae are the highest bidders for the commercial production of ethanol. Mango is native to the Indian subcontinent, belonging to family Anacardiaceae, is the most cultivated and favourite fruit of the tropics (Purseglove, 1972). The king of fruits is nutritionally very rich, unique in flavor and smell thus account for 1 approximately half of all tropical fruits produced globally. Ripe mango flesh contains carbohydrate and fiber. Carotene, thiamine, riboflavin, niacin, ascorbic acid, tryptophan, lysine and minerals are also present in the fruit (Goldsmith, 1976). Mango peel fibre is a good source of dietary fibre and its chemical composition may be compared to that of citrus fibre. The peel fibre also shows higher values of antioxidant activity, glucose retardation and its aroma and flavour are pleasant (Reyes and Vega, 1988) Mango is processed to a maximum extent, thereby producing high quantity of solid and liquid wastes. Solid wastes, stones, stalks, trimmings and fibrous materials are obtained during the preparation of raw material. Liquid waste is the waste material that comes out of a factory after washing of fruits, packaging, blanching, cooling and plant and machinery clean up and so on. Utilization of this mango waste is both a necessity and a challenge. In mango peel, there is a high amount of lignocellulose and reducing sugars. Lignocellulosic materials, are constituted primarily of lignin, hemicellulose and cellulose. The carbohydrate fraction (hemicellulose and cellulose) can be depolymerised into sugars which act as a primary carbon source for the microbial biocatalysts for the production of xylitol, ethanol, organic acids, industrial enzymes, etc. (Carvalheiro et al. 2008; Mussatto and Teixeira 2010; Chandel et al. 2011a). The present study was carried out to produce ethanol using mango peel, an agro-industry waste. 2 Objectives: Production of ethanol from mango peel using Saccharomyces cervisiae Activities in the research: - Study on the effect of the concentration of H2SO4 on hydrolysis - Study on the effect of time and temperature on hydrolysis - Study on the effect of yeast density on fermentation - Study on the effect of pH on fermentation - Study on the effects of temperature and time on fermentation 3 2. MATERIALS AND METHODS 2.1 Materials - Mango peel collected from Tien Giang Vegetables and Fruits Joint Stock Company. - Saccharomyces cerivisiae strain from Biotechnology Research & Develop Institute of Can Tho University (notated as 2.1). - Bien Hoa sugar from Co. op Mart - Medium: YPG (Yeast extract 0.3%, potato 20%, and Glucose 20%) - Chemicals, equipments in Food Biotechnology laboratory. 2.2 Methods 2.2.1. Preparation of mango peel After collected, mango peel was washed and then dried in the sun for 2-3 days, followed by being chopped into pieces of 2-3 cm. The material was transferred into an oven to dry at 65oC in 24 hours until the weight was unchanged and ground it in mixer into a fine powder. 2.2.2 Hydrolysis of mango peel 2.2.2.1. Study on the effect of the concentration of acid H2SO4 on hydrolysis - The activity was designed with one factor – the concentration of H2SO4 with five levels: 0.5, 1, 2, 3 and 4%. The total number of experimental units was 5 treatments x 3 replicates = 15 units. - 10 g mango peel powder was mixed with 100 mL dilute acid of different concentrations and autoclaved at 121ºC for 15 minutes (Arumugam và Manikandan, 2011). 4 - The acid pretreated samples were cooled and filtered. - pH of the hydrolysate was adjusted to 6.0 with NaOH (20N). - The reducing sugar content in the hydrolysates was determined by DNS method (Walia et al, 2013) and the amount of soluble solids was measured by Brix meter. 2.2.2.2. Study on the effect of time and temperature on hydrolysis - The experiment was carried out with two factors: time with five levels (15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours) and temperature at 30, 60, 90, 100 and 121oC. Total number of treatments was 5 x 5 = 25 treatments. The total number of experimental units was 25 treatments x 3 replicates = 75 units. - 10 g mango peel powder was mixed with 100 mL dilute acid with the concentration chosen from the previous activity. The hydrolysis was carried out in the conditions of time and temperature as in the experimental arrangement. - The acid pretreated samples were cooled and filtered - pH of the hydrolysate was adjusted to 6 with NaOH (20N). - The reducing sugar content in the hydrolysates was determined by DNS method (Walia et al, 2013) and the amount of soluble solids was measured by Brix meter. 5 2.2.3. Fermentation with Saccharomyces cerevisiae 2.2.3.1. Study on the effect of yeast density on fermentation - The activity was designed with one factor: yeast density 4 at 10 , 105 and 106 cells/mL. The total number of experimental units was 3 treatments x 3 replicates = 9 units. - Transfered 100 mL hydrolyzed mango peel solution (pH 6) into flasks. - Sterilized the solution by NaHSO3 140 mg/L in 30 minutes. - Inoculated yeast suspension with experimental levels. Cover flasks by water-locks and ferment for 7 days at room temperature. - Monitored Brix level, pH and ethanol content after fermentation. Analyze the data with statistics program STATGRAPHICS Plus v3.0 2.2.3.2. Study on the effect of pH on fermentation - The effect of pH was assessed with five levels: 4, 4.5, 5, 5.5 and 6. The total number of experimental units was 5 treatments x 3 replicates = 15 units. - Adjusted pH of the hydrolysate to mentioned levels and transfer 100 mL into flasks. - Sterilized the solution with NaHSO3 140mg/l in 30 minutes. - Inoculated yeast suspension as the result of previous activity. Cover flasks by water-locks and ferment in the conditions of time and temperature mentioned. 6 - Monitored Brix level, pH and ethanol content after fermentation. Analyzed the data with statistics program STATGRAPHICS Plus v3.0. 2.2.3.3. Study on the effects of temperature and time on fermentation The activity was designed with two factors: fermentation temperatures at: 25°C, 30°C and 35°C; fermentation time at: 3, 5, 7 and 9 days. Total number of treatments was 3 x 4 = 12 treatments. The total number of experimental units was 12 treatments x 3 replicates = 36 units. - Transferred 100 mL hydrolyzed mango peel solution into flasks with pH as the result of activity before. - Sterilized the solution with NaHSO3 140mg/l in 30 minutes. Inoculate yeast suspension as the result of previous activity. Cover flasks by water-locks and ferment in the conditions of time and temperature mentioned. - Monitored Brix level, pH and ethanol content after fermentation. Analyze the data STATGRAPHICS Plus v3.0. 7 with statistics program 3. RESULTS AND DISCUSSION 3.1. Hydrolysis of mango peel 3.1.1. Effect of the concentration of acid H2SO4 on hydrolysis Among factors affecting the efficiency of acid hydrolysis, acid concentration is considered one of the most important factors regarding the release of sugars. High concentrations of acid may decompose the hemicellulosic structure, producing inhibitors and also causing damage to the equipment used. Therefore, an appropriate acid concentration is essential for acid hydrolysis of lignocellulose (Taherzadeh and Karimi, 2007). The results in Table 3 showed that the concentration of the acid had a great impact on hydrolysis, especially the amount of reducing sugars produced which increased with the growth of the concentration of acid. The catalyst activity was proportional to H+ concentration. The more hydrogen ions were formed in the solution, the more rapid the hydrolysis process occurred (Mosier et al, 2002). Therefore, the breaking of glucosidic bounds would increase, leading to the high conversion of hemicellulose fraction into xylose. Under harsh conditions if high acid concentration was applied, the cellulose fraction would be disrupted and glucose would be generated. After 15 minutes hydrolyzing, the maximum reducing sugars was observed in the treatment of H2SO4 4% (7.74%) and the minimum was observed in the case of H2SO4 0.5% (6.14%). Soluble solids were also greater when mango peel was hydrolyzed with higher concentration of acid. Specifically, with 4% acid, hydrolysate had the highest Brix level (11.7 oBrix) 8 while the lowest (6.5oBrix) was seen in the treatment of 0.5% acid. Table 3: Effect of the concentration of acid H2SO4 on hydrolysis Concentration of H2SO4 CV 0,5% 1% 2% 3% 4% (%) 6.14c 6.21bc 6.47b 7.61a 7.74a 1.75 6.5 7.5 8.7 11.2 11.7 Amount of reducing sugars (%) Brix level Note: The difference was statistically significant mean only in rows. The same characters show no difference statistically significant at 95%. The determination of acid’s suitable concentration was very important to the efficiency of ethanol production from mango peel. The low level of acid (0.5%) led to low performance of hydrolysis whereas the hydrolysis occurred faster with high concentration of acid (4%) in spite of its high cost for the amount of acid used and acid neutralization later. According to the statistical result, there was no significant difference between the percentages of reducing sugars produced in the treatment of H2SO4 3% and that of H2SO4 4% (7.61% and 7.74%, respectively). Therefore, the concentration of 3% H2SO4 was suitable for the hydrolysis of mango peel. 9 3.1.2. Effect of time and temperature on hydrolysis The results were presented in Table 4. It was indicated that the hydrolysis rate gradually increased with respect to temperature. Kim (1999) showed that temperature plays an important role in the reaction rate of acid hydrolysis. Based on Arrhenius equation, at high temperatures, reaction should be faster. According to Xiang et al., breakage of hydrogen bonds into cellulose and hemicellulose fractions takes place steeply in response to temperature. At low temperature (30 – 90oC) the hydrolysis occurred more slowly than at high temperature (100 – 121oC). Particularly, the amount of reducing sugars doubled (about 8%) at high temperature (100 – 121oC) in comparison with lower levels of temperature. According to Gírio et al. (2010), temperature for hemicellulose hydrolysis was between 121 and 160oC. The extension of hydrolysis time also contributed to the increase of reducing sugars. As can be seen in Table 4, in all treatments, after 1 hours hydrolyzing, the amount of reducing sugars grew to the levels that were significantly different from that in 15 and 30 minutes. Remarkably, the rate of reaction was high at 121oC in 1 hour, producing reducing sugars with 8.49%. After that, it increased to insignificantly different levels (8.39% after 2 hours and 8.58% after 3 hours). Soluble solids also went up according to temperature and time. The highest Brix level (12.5%) was seen at 121oC in 3 hours while at the temperature lower than 100oC it ranged only between 5 – 9.17oBrix. Therefore, the hydrolysis of lignocellulosic compounds in mango peel occurred fast at 121oC in 1 hour. In order to get higher yield, 10 time can be extended. However, it will be time-consuming, reducing the efficiency of production. Meanwhile, according to Lenihan et al, (2011), hydrolysis at too high temperature and in too long time, monosaccharides can be degraded into undesirable chemicals inhibiting the fermentation later such as furfural, hydroxylmethylfurfural-HMF, acetic acid, levulinic acid, formic acid, uronic acid, vanillic acid, phenol, cinnamaldehyde, formaldehyde… Thereby, 1 hour was adequate time for efficient hydrolysis. The proportion of crude fiber in mango peel is 9.33% (Ashoush, 2011), so with 8.49% reducing sugars from the treatment of 121oC and 1 hour the efficiency of hydrolysis was 79.21%. This figure was high and logical since in crude fiber there is lignin, a component that cannot be hydrolyzed (Palmquist and Hahn-Hagerdal, 2000; Taherzadeh, 1999). In comparison with 38,6% in hydrolysis on the sugar cane bagasse at 100oC, 2% phosphoric acid concentration for 300 minutes (Gamez et al., 2004). The amount of reducing sugars released from the hydrolysis of durian peel is 5.6% (w/v) at 121oC in 45 minutes with 2% sulphuric acid (Matura et al., 2012). The hydrolysis of red macroalgae (Dwi, 2012) gave the highest level of reducing sugars (3.28%, w/v) in the conditions of 121oC and 1 hour. Therefore, it could be concluded that the hydrolysis of mango peel at 121oC in 1 hour was feasible and efficient because high reducing sugars were produced and the degradation of these sugars could be avoided. In addition, this choice is particularly important in making the process more economic and less timeconsuming. 11 Table 4: Effect of time and temperature on hydrolysis Temperature (oC) 30 60 90 100oC 121oC Amount of reducing sugars(% 1.84l 3.54l 4.07k 4.10k 4.33k 3.29l 3.33l 4.02k 4.18k 4.42ik 4.83hi 5.07gh 6.17de 5.85def 5.50fg 5.76ef 6.22d 7.25c 7.67bc 8.03b 7.53c 7.58c 8.49a 8.53a 8.58a 3.69 Time 15 mins 30 mins 1 hr 2 hrs 3 hrs 15 mins 30 mins 1 hr 2 hrs 3 hrs 15 mins 30 mins 1 hr 2 hrs 3 hrs 15 mins 30 mins 1 hr 2 hrs 3 hrs 15 mins 30 mins 1 hr 2 hrs 3 hrs CV (%) Brix level 5 6.17 6.33 6.33 7.17 6.17 6.83 7 7.67 8.17 7.83 8.17 8.17 9 9.17 9.33 9.83 9.83 10.17 10.33 10.83 11.33 12.17 12.33 12.5 Note: The difference was statistically significant mean only in columns. The same characters show no difference statistically significant at 95%. 12 3.2 Fermentation with Saccharomyces cerevisiae 3.2.1. Effect of yeast density on fermentation The initial density of yeast inoculated effects considerably the fermentation. With the sufficient level, the fermentation takes place effectively, creating good-quality product. The process will be slow if there are not enough yeast cells added, whereas in the case of abundant yeast cells, it will be wasteful and the product will have strange smell. The result of the study on the effect of yeast density on fermentation was shown in the Table 5. Table 5: Effect of yeast density on fermentation Yeast pH after Brix level after density fermentation fermentation Log 6 tb/mL Log 5 tb/mL Log 4 tb/mL Ethanol content (% v/v ở 20°C) 5.25 9 3.47a 5.37 9.5 3.21a 5.91 11.5 0.37b CV (%) 14.56 Note: The difference was statistically significant mean only in columns. The same characters show no difference statistically significant at 95%. After fermentation, pH of all fermented liquid was lower than that before fermentation (initial pH was 6.0). pH of hydrolysates decreased gradually through stages of fermentation, 13 ranging between 5.25 and 5.91. This decrease caused by sugars oxidation into organic acids. During fermentation process, yeasts converted sugars into alcohol and other intermediate products organic acids, reducing the pH of the fermenting liquid. Besides, the results proved that the fermentation occurred in the good way, with yeast overwhelming acidic bacteria that could cause pH to decrease to the level 3.0 – 3.5. Similarly, there was a difference in Brix level before and after fermentation. The reason was the conversion of sugars into ethanol by yeast. With the initial point of 12%, Brix level of the treatment of log 6 cells/mL decreased to 9oBrix and 9.5oBrix in log 5 cell/mL. There was little decrease in Brix level in the treatment of log 4 cell/mL (11% after fermentation). Statistical analysis with Statgraphics plus v3.0 releaved that average ethanol of triplcates in the density log 5 cell/mL and log 6 cells/mL (3.21% and 3.47%, respectively) was higher and significantly different from that of the density log 4 cells/mL (0.37%). Therefore, the density log 5 cells/mL (105 cells/mL) was selected for fermentation in order to produce high level of ethanol as well as save a great amount of yeast in comparison with the treatment of log 6 cells/mL. 3.2.2. Effect of pH on fermentation pH has a big influence on the growth of yeast which depends on different fermentation medium. Therefore, determining a suitable pH for the solution of hydrolyzed mango peel is very important. 14
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