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1 INT J CURR SCI 2012, RESEARCH ARTICLE ISSN Comparative study of continuous ethanol fermentation from molasses by using Saccharomyces cervisiae and Schizosaccharomyces pombe Hemamalini V*, S.G.E. Saraswathy, C. Hema and S. Geetha Department of Plant Biology and Biotechnology, Arignar Anna Govt Arts College for Women, Walajapet, Tamil Nadu, India Abstract *Corresponding author: hemaraju97@yahoo.com; Phone: Ethanol production has been carried out from molasses by yeast Saccharomyces cervisiae and Schizosaccharomyces pombe. The level of alcohol production from molasses by the use of both these organisms in continuous ethanol fermentation. The highest percentage of alcohol production by Saccharomyces cervisiae with specific gravity was 7.20% whereas Schizosaccharomyces pombe showed 5.25% of alcohol production with specific gravity Batch fermentation was conducted by these organism for the role of bio protease enzyme in ethanol production. Higher alcohol production was notiied in flask which contained enzymes with Saccharomyces cervisiae and Schizosaccharomyces pombe. It was found that Saccharomyces cervisiae with enzyme added batch fermentation showed 8% of alcohol production is higher compared with free cells as only 6% of alcohol production. Amount of total reducing sugar and fermentation efficiency of the substrate molasses were calculated and tabulated. Molasses is a complex substrate that has wide range of nutrients that are not often completely metabolized by the microbial inoculums. Analysis of COD reduction rate of Saccharomyces cervisiae used waste water was 41.50%. Analysis of COD reduction rate of Schizosaccharomyces pombe used waste water was 50.78%. From this study, we conclude that Saccharomyces cervisiae yielded better results in alcohol production compared to Schizosaccharomyces pombe. Keywords: Saccharomyces cervisiae, Schizosaccharomyces pombe, ethanol fermentation, molasses Received: 19 th December; Revised: 29 th December; Accepted: 30 th January; IJCS New Liberty Group 2012 Introduction Microorganisms play an important role in biotransformation of waste products into human, animal and plant consumables. Yeast cells are used in household fermentation, food production, industrial fermentation and biotransformation process. Fermentation is a process in which complex molecules are converted into simple sugar by the role of microorganisms (Patel, 1991). Ethanol is an important industrial solvent and chemical feed stock for the synthesis of pharmaceuticals, detergents, adhesives, plastics, plasticizers and host for other chemicals (Prescott and Dunn, 1959). It is also an energy feed stock and serve as a fuel in internal combustion and possible future decline in oil supplies are stimulating more use of ethanol as an octane enhancer in unblended gasoline and as a gasoline substitute (Kiem and Venkatasubramanian, 1989). Today alcohol technology is again reviving the production of alcohol as a fuel in being given attention with the world energy crisis (Jogdand, 1993). It has enhanced interest in renewable sources gives no net contribution to Green gases vehicles that turn straight alcohol are cleaner with respect to emission of hydrocarbon, carbon monoxide and one better to drive in many aspects (Wyman and Good man, 1993). Ethanol had been trusted as an alternate fuel for the further utilization of a world-wide interest in the utilization of bio-ethanol as an energy source which in turn stimulate studies on the cost and efficiency of fermentative organisms. Cheapest fermentation substrates and optimum environmental conditions are needed for fermentation. (Gunasekaran and Chandraraj, 1999). The main raw material used in India for the ethanol production is sugarcane molasses. The demand for ethanol is also increasing day by day. The prerequisite for achieving process improvement in alcohol production depends on the selection of suitable yeast strains for high ethanol yield to maximize substrate utilization, minimize fermentation capital costs and reduce ethanol recovery costs. Molasses is

2 dark coloured syrup left after extraction of sugar from sugarcane. It contains about 62% of carbohydrates in the form of 30% uncrystalized sucrose and about 32% of invert sugar which is a mixture of glucose and fructose (Patel, 1999). The sugar, which is converted into molasses, is adjusted to 14-16%, which permits an alcohol content of 8-10 volume percentage in the fermented wort. (Krotzschmar, 1995). Ethanol production of fuel has been industrialized in a number of developing countries. Saccharomyces spp. has been used to ferment molasses for ethanol production, tolerance of these strains to high alcohol levels and sugar concentration limits the concentration of alcohol that can be obtained from such process. (Ezeogu and Okolo, 1994). Saccharomyces cervisiae is a high ethanol yielding yeast which was used for ethanol production from the ultra filtrates of whey that was hydrolyzed first to give glucose and glactose (Roland and Alm, 1975). Saccharomyces cervisiae were examined for their production ability in molasses than tapioca and sorghum. It gave high efficiency of ethanol production by using substates like sorghum, tapioca (Brown and Oliver, 1982). Schizosaccharomyces pombe is fission yeast, which has high growth rate increased fermentation ratio, high ethanol tolerance temperature tolerance, and osmotolerent. Schizosaccharomyces pombe produced maximum bimass. Traditionally ethanol is produced from cane molasses by fermentation with yeasts. Due to product inhibition ethanol concentration is usually limited to 8-9% by volume (Heraldson and Bjorling, 1981). Ethanol fermentation is a continuous process, the molasses flow in and fermented wash flows out of the fermentor. The concentration of yeast cell cycle can be segregated in different fermentors for the yeast cell growth and carbon dioxide evolved. The process is continued and yeast cells remain in suspension. Finally the yeast cells are removed and clear wash is taken for distillation. The yeast strains normally employed in industrial process show a limited tolerance to ethanol, temperature and high osmotic pressure of the medium (Casey and lngledew, 1986; Amore et al., 1990; Bertolini et al., 1991). An important aspect in ethanol production, in search of new strains succeeding in industry depends upon the characters like thermo tolerance, capacity to grow in gravity works and tolerance to high concentration of ethanol. Protease is one of the most important industrial enzymes accounting for 60% of total worldwide enzyme scale. The major market for enzyme is Western Europe and is found to be the emerging market. In the next fifteen years, Asia is expected to account for nearly 20% of the global enzyme market. The leading enzyme marketers in India are Biocon Pvt. Ltd., Advanced Bio Chemicals Ltd., Maps industries; Textan chemicals; Anil starch products Ltd., Maize products and SPIC Ltd. Ethanol production has been carried out from molasses by yeast Saccharomyces cervisiae and Schizosaccharomyces pombe. Continuous ethanol fermentation from molasses by the use of various yeast culture such as Saccharomyces cervisiae and Schizosaccharomyces pombe Batch fermentation was conducted by these various yeast with Bio-protease enzyme Analysis of molasses medium Find out and calculate the COD reduction rate in spent wash and recycle sample. Collection of scum sample from fermentor. Effect of Bio-protease enzyme Materials and methods Collection of sample and source of microbial strains Molasses sample was collected from the distillery unit of Dharani Sugars and Chemicals Pvt. Ltd., Vasudevanallur, Thirunelveli district. The ethanol producing strains Saccharomyces cervisiae and Schizosaccharomyces pombe were obtained from microbial type culture collection (MTCC), Chandigarh (India). Maintanance of culture Saccharomyces cervisiae was routinely sub cultured and maintained in MGYP agar slant at every 15 days intervals and stored at 4C. Schizosaccharomyces pombe was subcultured and maintained in molasses agar slants. Inoculum preparations for ethanol production The yeast inoculum was prepared (Nellaiah and Gunasekaran, 1991). To a 50 ml of sterile molasses medium and glucose medium, a loop full of yeast Schizosaccharomyces pombe and Saccharomyces cervisiae

3 from fresh slant culture were inoculated separately. The inoculum was incubated at room temperature for 24 hrs. Continuous ethanol fermentation Continuous ethanol fermentation was done in 500 ml molasses medium, which was prepared at brix specific gravity, autoclaved and cooled. Yeast inoculums Schizosaccharomyces pombe and Saccharomyces cervisiae was inoculated into separate flasks. Incubate the flasks for 8 hrs at 37C and thus yeast was grown. The fermentation of foam indicates the growth of the yeast cells. Take another 1500 ml molasses medium, which was prepared at brix specific gravity and sterilized. Add this molasses medium into the yeast inoculums and the total volume of 2000 ml culture medium was incubated for 8 hrs at 37 C. After 8 hrs, the total 2000 ml of culture medium was added to 2500 ml of freshly prepared molasses medium with1.032 brix specific gravity and incubated for 8 hrs at 37C. The total amount of culture medium is 4500 ml. After 8 hrs 250 ml fermentated wash was taken from the flask and check the specific gravity of the sample i.e. called initial gravity. Gravity was highly decreased and then 100 ml was taken and discarded from the flask. Then freshly prepared 100 ml molasses medium with high specific gravity (1.070 brix specific gravity) was added to the flask culture. After every one hour the specific gravity of the culture medium was analyzed and the same procedure was followed every one hour. After every four hours residual sugar level, alcohol percentage and total volatile acid were noted. The same procedure was continued for 5 days. Alcohol percentage depends upon the reducing sugar level, if the sugar level decreases, the alcohol percentage increases. Sugar was converted into ethanol by the action of yeast cultures such as (Schizosaccharomyces pombe and Saccharomyces cervisiae). When sugar was present in high level the organism utilizes the sugar slowly resulting in production of ethanol. According to this procedure, alcohol percentage and residual sugar levels were noted. At the end of the experiment, as the sugar level has decreased the alcohol percentage had increased to a higher level. The specific gravity of the solution was set up as 1.032, and etc. Batch fermentation Bio protease enzyme was added with molasses medium for ethanol fermentation. Molasses as the substrate was prepared by diluting with distilled water, until brix gravity of sugar concentration had reached. The ph was adjusted as 4.5, 5.0 and 5.5. The sugar concentration (15 to 18%) and 10 ppm of our enzyme solution (0.5 g) were added. To this fermentation broth 10% of yeast inoculums Saccharomyces cervisiae was inoculated. The same procedure was followed in another flask and inoculated with 10% of yeast inoculums Schizosaccharomyces pombe. It was incubated after room temperature 37C for 48 hrs. After incubation period the RS, alcohol percentage and TVA was analyzed. In the same manner two control flask were also run without adding bio protease enzyme. Estimation of residual sugar (RS) Incubate 20% of fermented wash taken for titration. 5 ml of Fehling s solution A and B with 100 ml of distilled water were taken in a 250 ml conical flask and heated. Few drops of methylene Blue indicator were added while heating. The solution turned blue in colour and the titration continued till brick red colour. Reducing sugar = 27.2 / titre value i.e Fehling s factor value. Estimation of alcohol content The fermented wash (250 ml) was taken and made up to 500 ml with distilled water. The sample was transferred into the round bottom flask and allowed for condensation. Then 250 ml of condensate was collected and temperature of the condensate was noted. The condensate was poured into the measuring jar. The alcohol content was estimated by a hydrometer which is designed for measuring only alcohol percentage. 100-alcohol percentage / 1.66 (alcohol standard value) Estimation of total volatile acids (TVA) Fermented wash 100 ml and 100 ml distilled water was mixed with 5 ml of sulphuric acid (50%). This

4 mixture was taken in a round bottom flask and distillated using distillation unit. Exactly collected 150 ml distillate and transferred the distillate into the conical flask. To this added 5 drops phenolphthalein as an indicator. The burette was filled with Sodium Hydroxide solution (0.5N). Sodium hydroxide was titrated against distillate with indicator until the pale pink colour appears. The end point (pale pink colour) was noted. Total Volatile Acid = Titer value X Analysis of fermentation medium Biological conversion of carbohydrate residue mainly involves traditional fermentation processes. Estimation of total reducing sugar 5 gm of molasses was dissolved in 100 ml of distilled water and 5 ml of concentrated hydrochloric acid was added. Then it was boiled at 70C and cooled. The solution was neutralized by the addition of 6 N Sodium hydroxide until the red litmus paper turned to blue. The neutralized solution was made up to 1000 ml taken in the burette solution and titrated against 5 ml of both Fehling s A&B solution till wine red developed. 4 drops of methylene blue were added as an indicator. The titration was continued till the blue colour disappeared and there was an appearance of permanent brick red, which was noted as the end point. Total Reducing Sugar = / TV x FF x DF FF Fehling s Factor, TV Titre Value, DF Dilution Factor (0.005) / 26.5 = Estimation of unfermentable sugar 25 gm of raw molasses was weighed and mixed with 25 gm of dry yeast. This mixture was made up to 150 ml with ordinary water. Add a few grams of urea and diammonium phosphate. Allow it to ferment for 24 hrs. After fermentation, the 150 ml was made up to 250 ml, 100 ml of tabletop centrifuge. Yeast sludge was precipitated. The 50ml of supernentant was taken and make up to 100 ml. It was taken in the burette solution and titrated against 5 ml of both Fehling s A&B solution till wine red colour developed. A few drops of methylene blue was added. The titration was continued till the blue colour disappeared and there was an appearance of permanent brick red, which was noted as the end point / FF x TV x DF / 26 = Estimation fermentation efficacy 50 gm of molasses was taken and mixed with water. Added two grams of di-ammonium Phosphate and adjusted the ph of the flask to 4.5 with dilute H 2 SO 4. Made up the volume up to 300 ml. Sterilized the flask at 15 lbs pressure for about 10 min. After sterilization cool the flask at room temperature. Added 2 gm of yeast to the flask and kept in the incubator at 37C for 48 hrs. Find out the specific gravity by specific gravity method and determined the initial total reducing sugar. Fermented wash 250 ml was taken and added with 250 ml of distilled water. This mixture was taken in a round bottom flask and distilled. 250 ml of distillate corrections are applied, and the alcohol percentage was calculated from the standard table. Fermentable sugar (%) = Total Reducing sugar Unfermentable sugar. Theoretical yield: C12H22O C6H12O6 C6H12O C2H5OH + 2CO2 From this molecular formula, 100 gm of sugar gives 64.4 ml of ethanol. 100 gm of sugar = 64.4 ml of alcohol. Theoritical yield Fermentable Sugars x 64.4 Fermentation Efficacy (%) Actual Percentage of alcohol / Theoritical yield x 100 Bioenergy potential of distillery effluent There are 285 distilleries in India producing 2.7 billion litres of alcohol generating 40 billion litres of waste water annually. The enormous distillery waste water has potential to produce 1100 milion cubic meters of biogas. Distillery waste water known as spent wash. According to a recent estimate, the alcohol production in India has reached the 27 million litre mark. The proportions of waste water, generally known as spend wash, in nearly 15 times the total alcohol production. This massive quantity,

5 approximately 40 billion litres of effluent, if disposed untreated can cause considerable stress on the water causes leading to widespread damage to aquatic life. Bio-gas normally 60% methane gases which is a well recognized fuel gas with minimum air pollution potential. This is more significant in Indian context and many distilleries are adopting this technology. Composition of spent wash Spent wash is characterized by its colour, high temperature, low ph, high ash content and contains high percentage of dissolved organic and inorganic matter of which 50% may be present as reducing sugars. It contains about 90 93% water and 7 10% solids; sugar being 2-20% and protein 10 11% in the dry spent wash. Indian spent wash contain very high amounts of potassium, calcium chloride, sulphate and BOD (around 50,000 mg/lt) when compared to spent wash in other countries. Biomethanation plant Spent wash is used as a feed for Bio-gas plant. In Dharani Sugar anaerobic digester contain plastic media in the centre of the digester tank. This is used for microbes to stick on media in the centre of the digester tank. This is used for microbes to stick on media surface and active for long time to produce bio-gas. Methanogenic and acetogenic bacteria are used for bio gas production. The top of the plant space called Gas zone. Recirculation pump was present at the bottom of the tank which is used for even mixing of feed and bacteria. The capacity of the digester is 4000 m 3 (40,00,000). Microbiological processes involved in anaerobic digestion centre around Methanogenic bacteria differ significantly from acid forming bacteria in terms of physiology; nutritional requirements and sensitivity to formers are separated. The efficacy of the system is enhanced. The methane forming bacteria could be effectively and eliminating the potential problems before the methane forming bacteria are subjected to stress. Post methanation wastewater is called Recycle. If used carefully for irrigation of agricultural crops can produce more than 30,000 tonnes of bio mass annually. Estimation of chemical oxygen demand 100 ml of sample (spent wash/recycle) was taken and centrifuged. From that 1 ml of solvent wash sample was taken and made up to 500 ml (Dilution factor for spent wash = 50). (Dilution factor for recycle = 25). From that 1 ml of recycle sample was taken and made up to 250 ml. Diluted solutions were taken in COD flask. An ounce of mercuric sulphate was added. Then 5ml of potassium dichromate is added and 15 ml of concentrated H 2 SO 4 was added in the flask. It was cooled in water and it was refluxed for two hours in heating mantle. Then it was left to cool down and totrated against 0.1N FAS solution. Ferroin was used as an indicator. End point was the appearance of brick red colour. Ins blank flask was also kept along this. Blank sample x Normality of FAS x DF x 8000 / 20 = mg / 1 Dilution factor For spent wash For recycle COD reduction rate Spent wash recycle / spent wash x 100 Estimation of total volatile acid inspent wash and recycle samples A mixture of 100 ml sample and 100 ml distilled water were taken and mixed with 5 ml of sulphuric acid (50%). This mixture was distilled in distillation unit. Exactly, 150 ml of distillate was collected and five drops of phenolphthalein as an indicator was added. 0.5 N sodium hydroxide was taken in burrete and titrated against distillate with indicator till pale pink colour appeared. The end point was noted. Titre value x 0.5 x 1000 / 70s Results and discussion Saccharomyces cerivisiae and Schizosaccharomyces pombe are promising strains for the ethanol production that are actively researched worldwide. Saccharomyces cervisiae is used all over the world as the major ethanol producing organism. Industrial usefulness of Schizosaccharomyces pombe was also recorded by Gomes et al. (2002), where this strain was recorded to have osmotolerant, alcohol tolerance and sustained alcohol

6 production. The continuous ethanol fermentation by both these organisms was reported in table 1 and 2. Table 1. Continuous ethanol production by Sacharomyce cervisiae Substrate Added Setup Gravity Initial Gravity 100ml ml ml ml Alcohol % Reducing Sugar 250ml % ml ml ml ml ml % ml ml ml % ml ml ml ml % ml ml ml ml % ml ml ml ml % ml ml ml ml % ml ml ml ml ml % ml ml ml % ml ml ml % ml ml ml % ml ml % ml ml ml % ml ml ml ml ml % ml ml ml ml % ml ml ml % ml ml ml

7 250ml % ml ml % ml ml ml ml ml % ml ml ml ml ml % 1.21 Table 2. Continuous ethanol production by Schizosaccharomyces pombe Substrate Added Setup Gravity Initial Gravity Alcohol % Reducing Sugar 100ml ml ml ml ml % ml ml ml ml ml % ml ml ml ml % ml ml % ml ml ml ml ml % ml ml ml ml ml % ml ml ml ml ml % ml ml ml ml ml % ml ml ml ml ml % ml ml ml ml % ml ml % ml % ml % % ml ml ml ml ml % ml % ml % ml ml % 1.36

8 5.20% ml ml ml % 1.37 On this basis Saccharomyces cervisiae and Schizosaccharomyces pombe were employed individually to understand their efficacy in ethanol fermentation. The highest percentage of alcohol production by Saccharomyces cervisiae with specific gravity was 7.20%, whereas Schizosaccharomyces pombe showed 5.25% of alcohol production with specific gravity (Mandeep Kawr and Kocher, 2002). Batch fermentation results were noted and tabulated in table 3 and 3A. Table 3. Batch fermentation by Saccharomyces cervisiae with enzyme and without enzyme Factor Initial gravity Control Enzyme Added 1, Final gravity Alcohol RS TVA Table 3 A. Batch fermentation by Schizosaccharomyces pombe with enzyme and without enzyme Factor Control Enzyme added Initial gravity 1, Final gravity Alcohol RS TVA For the study of role of protease enzyme in ethanol production, the batch fermentation of ethanol was carried out with protease enzyme. The control flask was also maintained without protease enzyme by using molasses as the fermentation substract. The RS, TVA and alcohol percentage were noted for the both, enzyme added flask as well as the free cells contained flask. Table 4. Analysis of fermentation medium molasses Factors Percentage TRS % UFS 4.60% FS 45.31% AIC 4.96% FE 51.01% TRS Total Reducing Sugar UFS Unfermentable Sugar FS Fermentable Sugar AIC - Alcohol FE Fermentation efficiency Concentration of bio protease enzyme 5 mg was added with 100 ml molasses medium with specific gravity. From our result, alcohol production was enhanced only in the enzyme added flask. The control flasks produced alcohol in lesser amount, when compared to the experimental flask. Higher alcohol production was notified between various type of yeast strains such as Saccharomyces cervisiae with enzyme containing flask and Schizosaccharomyces pombe with enzyme containing flask. Table 5. Analysis of COD reduction rate in Saccharomyces cervisiae used waste water Factor Control Enzyme added Blank Spent Wash 19 1,06,000 Recycle ,000 COD reduction % The yeast Saccharomyces cervisiae was highly fermentative when it was added with enzyme. It produces better result for ethanol production than Schizosaccharomyces pombe. It was concluded that Saccharomyces cervisiae with enzyme added batch fermentation showed 80% of alcohol production, as higher

9 when compared with free cells as only 6% of alcohol production (Heraldson and Bjorling, 1981). Table 5a. Analysis of COD reduction rate in Schizosaccharomyces pombe used waste water Enzyme Factor Control added Blank Spent ,25,000 Wash Recycle 18 63,000 COD reduction % Amount of total reducing sugar and fermentation efficiency of the substrate molasses were calculated and tabulated in table 4. Molasses is a complex substrate that has wide range of nutrients that are not often completely metabolized by the microbial inoculums. Attempts are being made throughout the world to increase ethanol production by making the substrates more metabolizable. In this idea protease enzyme was added to simplify these complex molecules. Industrial exploitation of renewable raw materials has become greater interest. This work was especially focused on substrate problems concerned with industrial requirement for alcoholic fermentation. The COD reduction rate was analyzed from the industrial effluent like spent wash and recycle samples. The result were noted and tabulated in table 5 and 5A. Spent wash and recycle sample was analyzed and find out the chemical oxygen demand level present in these effluents. Saccharomyces cervisiae used type of industrial effluent contains lesser rate of COD level. But the yeast strain Schizosaccharomyces pombe used type of effluent contain higher rate of COD level. Conclusions The present study was undertaken with the aim to compare the activity of two yeast strains in ethanol production.the sugarcane industry waste (molasses) was subjected for ethanol production by using an osmotolerant strain Saccharomyces cervisiae and Schizosaccharomyces pombe. The yeasts Saccharomyces cervisiae and Schizosaccharomyces pombe were used as free cells in continuous ethanol fermentation and the ethanol yield was noted as 5.25% and 7.20% respectively. The Bio-protease enzyme, obtained from Biocon enhanced the ethanol yield. In batch fermentation, Saccharomyces cervisiae and Schizosaccharomyces pombe was added with enzyme, yielded higher ethanol than they were used as free cells (6.74% and 8.0%). When yeast inoculums Saccharomyces cervisiae and Schizosaccharomyces pombe were used as free cells, the ethanol yield was 5.0% and 6.24% respectively. The above indications emphasize that the use of Schizosaccharomyces pombe was not efficient when compared to Saccharomyces cervisiae. These two strains were highly efficient when added with enzymes. However, Saccharomyces cervisiae is a highly efficient fermentative organism. From this study, we concluded that Saccharomyces cervisiae yielded better result in ethanol production than Schizosaccharomyces pombe. References Brown RL, K. Oliver (1982). Efficiency of ethanol production by using yeast culture, J. Bio. Tech., 5: Casey GP, WM Ingledew (1986). Ethanol tolerance in yeast. Crit. Rev. Microbio. 13: Chorles pascal C (1987). Ethanol fermentation efficiency of yeast (S. cerevisiae), J. Bio. Tech., 8: Gunasekaran P, Chandra Raj K (1994). Ethanol fermentation technology. Zymomonas mobilis, 77: Kiem CR, Venkatasubramanian K (1989). Trends Biotechnology, 7: 22. Merrit NR (1996). Byproducts formation during ethanol fermentation by distiller s yeasts. J. Inst. Browing, 72: 374. Mandeep K, Kocher K (2002). Ethanol production from molasses and sugarcane juice by an adapted strain of S. cerevisiae. J. Microbiology, 60:

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