INTRODUCTION:

Alcohol plays a major role in various production units of chemicals in perfumery manufacture in pharmaceutical industry production of drugs and it even used at a fuel. The rising use of motor vehicle is causing serious air pollution mainly in urban areas. Ethanol is a clean, renewable, domestically produced fuel that can reduce dependence on imported petroleum, while benefitting the Nation‘s farmers. Cost is the key impediment to the widespread use of ethanol in highway vehicles. The use of ethanol as a fuel is seeing tremendous growth driven by changes in legislation. In North America, demand for fuel ethanol is already growing rapidly, mainly driven by the replacement of methyl tertiary butyl ether (MTBE) which has been eliminated from gasoline as an additive.

In Europe, the use of bioethanol (ethanol from fermentation) in fuel has had a slow start but is still expected to grow rapidly. Because of it, they are facing an urgent need to develop about 20 years ago, the domestic oil production had been approximately demand, and thus they had been depending on imports for only about 20% of their consumption. However, because of recent increasing oil demand, they have to depend on imports for 70% of circumstances, the ethanol production acts like a triple-purpose solution for curing air pollution, agriculture promotion, and decreasing energy dependence on foreign suppliers and it calls attention as use of renewable for countering global warming¹. Nowadays people are looking for an alternate renewable resource for fuel energy. Since researchers informed that the motor fuel is going to get exhausted within few years. Also today’s trend is to produce useful and economical produces from wastes. India is one of the largest sugar producers in the world. Sugarcane is one of the major crops in India. The sugar factories after extraction of crystalline sugar to the distilleries leave molasses as a waste, which it is converted into alcohol by yeast fermentation. In recent years, attention has been focused on effective utilization of agro-byproducts to produce fuel using Zymononas mobilis in this microbes investigation of molecular biology and biochemistry of ethanol production by of molecular biology and biochemistry of ethanol production by Z. mobilis has been accomplished. Transformation of E.coli with genes (pet) from Z. mobilis for alcohol production has been successfully carried out however, Z. mobilis only utilizes glucose or fructose or sucrose for ethanol production². The alcohol produced is extracted and purified by distillation column setup. The foam formed is controlled by antifoaming agents like soap oil (or) turkey red oil many heat exchangers are employed for cooling and condensing purposes of both in fermentation and distillation. A maximum yield of 96% ethyl alcohol is obtained. To judge the yield and economical profit, laboratory tests are carried out earlier³.

Ethanol Fermentation with Bacteria:

A great number of bacteria are capable of ethanol formation. Many of these microorganisms, however, generate multiple end products in addition to ethyl alcohol. These include other alcohols (butanol, isopropylalcohol, 2,3-butanediol), organic acid (acetic acid, formic acid, and lactic acids), polyols (arabitol, glycerol and xylitol), ketones (acetone) or various gases (methane, carbon dioxide, hydrogen). Z. mobilis degrades sugars to pyruvate using the Entner-Doudoroff pathway. The pyruvate is then fermentated to produce ethanol and carbon dioxide as the only products (analogous to yeast). An interesting characteristic of Z. mobilis is that its plasma membrane contains hopanoids, pentacyclic compounds similar to eukaryotic sterols. This allows it to have an extraordinary tolerance to ethanol in its environment, around 13% glucose medium.

Ethanol Fermentation with Yeast:

The organisms of primary interest to industrial operations in fermentation of ethanol include Saccharomyces cerevisiae, S. uvarum, saccharomyces pombe, and Kluyueromyces species. Yeast, under anaerobic conditions; metabolize glucose to ethanol primarily by way of the Embden-Meyerhof pathway. Yeast are carefully selected for its high growth and fermentation rate, high ethanol yield, ethanol and glucose tolerance, osmo tolerance, Low pH fermentation optimum, High temperature fermentation optimum, General hardness under physical and chemical stress.

The yeast Saccharomyces cerevisiae and facultative bacterium Z. mobilis are better candidates for industrial alcohol production. Z. mobilis possesses advantages over S. cerevisiae with respect to ethanol productivity and tolerance⁴.

MATERIALS AND METHODS:

Microorganism and Culture Conditions:

Saccharomyces cerevisae were obtained from National Chemical Laboratory (NCL, Pune, India). Pure culture was maintained on Sabouraud’s Dextrose Agar slants and stored in test tubes at 4⁰C and sub-cultured monthly. Sabouraud’s Dextrose Agar containing (g L^-1) dextrose, 40; peptone, 10; Agar, 15; pH 5.6 at 30⁰C for 5 days. Zymomonas mobilis and Erwinia carotovora were obtained from National Chemical Laboratory (NCL, Pune, India). Pure culture was maintained on Nutrient Agar slants and stored in test tubes at 4⁰C and sub-cultured monthly. Nutrient Agar containing (g L^-1) Peptic digest of animal tissue, 5; Sodium chloride, 5; Beef extract, 1.5; Yeast extract, 1.5; Agar, 15; pH 7.4 at 37⁰C for 3 days.

Analytical Methods:

Analysis of Brix:

The total unfermentable solid of molasses was determined. 50g of molasses was weighed and dissolved in 530ml of water. The brix hydrometer reading and temperature was noted. Using the brix table, the table reading corresponding to the hydrometer value and the temperature value was noted.

Determination of total reducing sugar:

The amount of total reducing sugar present in molasses was determined. 12.5 g of molasses was weighed and dissolved in 100ml of water. 25ml of lead acetate solution was added. It was making up to 250ml in a standard flask with water. The solution was allowed to stand and settle. 50ml of the top layer was collected and filtered. To this10ml of deleading solution was added. It was making up to 250ml in a standard flask with water. The solution was allowed to stand and settle. 50ml of the top layer was collected and filtered. 5 ml of concentrated HCl was added. The solution was boiled to 69^oC and then cooled to room temperature. Then 5 drops of phenolphthalein indicator was added. To the solution 6N NaOH was added till the colour changes from yellow to purple. It was making up to 100ml with water and was poured in burette. 5ml each of Fehlings A and Fehlings B was taken in a conical flask. It was titrated against the burette solution along with boiling. To this 5-6 drops of methylene blue indicator was added. Titration was carried out. The end point is the colour changes from blue to brick red colour.

Wash solution:

Wash is the dilution of molasses with water.150g of molasses was dissolved in distilled water150G OF

MOLASSES

till the gravity was set to 1.082. This is now called as wash.

The following analyses are carried out for wash:

Determination of specific gravity:

The specific gravity was determined by hydrometer. Molasses was diluted with water so that the specific gravity of wash was 1.08.

Determination of Unfermentable solids:

The amount of unfermentable sugars present in wash was determined. 10ml of wash was diluted with 100ml of water and was filled in burette. 5ml each of Fehlings A and Fehlings B solution along with 20ml of water was taken in a conical flask. It was titrated against the burette solution up to 10-15ml of burette. The conical flask was heated. After boiling, the methylene blue indicator was added and then it was heated until the blue colour disappeared. It was titrated further. The end point was the appearance of brick red colour.

Ethanol production using Saccharomyces cerevisae:

Determination of Unfermentable matter:

The amount of unfermentable matter present in molasses was determined. 20g of molasses was weighed and dissolved in 150ml of water. To this 2ml of H₂SO₄ was dropped in order to maintain the pH. 25 g of yeast was added. The medium was kept for 24 hrs incubation at room temperature. After that the solution was taken and 20ml of lead acetate solution was added. It was making up to 250ml in a standard flask with water and was allowed to stand and settle. 125ml of the top layer was collected and filtered. To this10ml of deleading solution was added. It was making up to 200ml in a standard flask with water. The solution was taken in burette. 5ml each of Fehlings A and Fehlings B solution along with 5ml of water was taken in a conical flask. It was titrated against the burette solution upto 10ml of burette solution. The conical flask was heated. After boiling, the methylene blue indicator was added and then it was heated until the blue colour disappeared. It was titrated further. The end point was the appearance of brick red colour.

Preparation of wash:

150g of molasses was dissolved in distilled water150G OF MOLASSES till the gravity was set to 1.082.

Fermentation process:

The culture was mixed with wash solution. To this antibiotics and nutrients (penicillin, urea, DAP) were added. It was kept for 24 hours incubation at 32^oC. The pH was maintained around 4-6. After the incubation period, the pH was calculated and the hydrometer reading was noted. The alcohol produced was analyzed.

Analysis of alcohol:

In this test the percentage of alcohol present in the wash was determined. 150ml of wash was dissolved in 150ml of water. It was boiled at 100^oC. The vapours were condensed. 150ml of condensed solution was collected. The hydrometer reading and the temperature in (^oF) were noted. The table reading corresponding to the temperature and hydrometer reading was noted. Alcohol is measured in terms of proof litres. Proof spirit is the ethanol containing 49.28% of alcohol by weight (or) 57.10% of alcohol by volume.

Yield:

The yield of alcohol can be calculated from the alcohol percentage mathematically.

Ethanol production using Zymomonas mobilis

Determination of Unfermentable matter:

The amount of unfermentable matter present in molasses was determined. 20g of molasses was weighed and dissolved in 150ml of water. To this 2ml of H₂SO₄ was dropped in order to maintain the pH. Zymomonas mobilis was added. The medium was kept for 24 hrs incubation at 37^oC temperature. After that the solution was taken and 20ml of lead acetate solution was added. It was making up to 250ml in a standard flask with water and was allowed to stand and settle. 125ml of the top layer was collected and filtered. To this10ml of deleading solution was added. It was making up to 200ml in a standard flask with water. The solution was taken in burette. 5ml each of Fehlings A and Fehlings B solution along with 5ml of water was taken in a conical flask. It was titrated against the burette solution upto 10ml of burette solution. The conical flask was heated. After boiling, the methylene blue indicator was added and then it was heated until the blue colour disappeared. It was titrated further. The end point was the appearance of brick red colour.

Preparation of wash:

150g of molasses was dissolved in distilled water150G OF MOLASSES till the gravity was set to 1.082.

Fermentation process:

The culture was mixed with wash solution. To this nutrients (urea, DAP) were added. It was kept for 24 hours incubation at 37^oC. The pH was maintained around 7. After the incubation period, the pH was calculated and the hydrometer reading was noted. The alcohol produced was analyzed and yield is calculated.

Analysis of alcohol:

In this test the percentage of alcohol present in the wash was determined. 150ml of wash was dissolved in 150ml of water. It was boiled at 100⁰C. The vapours were condensed. 150ml of condensed solution was collected. The hydrometer reading and the temperature in degree Fahrenheit (⁰F) were noted. The table reading corresponding to the temperature and hydrometer reading was noted. Alcohol is measured in terms of proof litres. Proof spirit is the ethanol containing 49.28% of alcohol by weight (or) 57.10% of alcohol by volume.

Yield:

The yield of alcohol can be calculated from the alcohol percentage mathematically.

Ethanol production using Erwinia carotovora

Determination of Unfermentable matter:

The amount of unfermentable matter present in molasses was determined. 20g of molasses was weighed and dissolved in 150ml of water. To this 2ml of H₂SO₄ was dropped in order to maintain the pH. Erwinia carotovora was added. The medium was kept for 24 hrs incubation at 37⁰C temperature. After that the solution was taken and 20ml of lead acetate solution was added. It was making up to 250ml in a standard flask with water and was allowed to stand and settle. 125ml of the top layer was collected and filtered. To this10ml of deleading solution was added. It was making up to 200ml in a standard flask with water. The solution was taken in burette. 5ml each of Fehlings A and Fehlings B solution along with 5ml of water was taken in a conical flask. It was titrated against the burette solution upto 10ml of burette solution. The conical flask was heated. After boiling, the methylene blue indicator was added and then it was heated until the blue colour disappeared. It was titrated further. The end point was the appearance of brick red colour.

Preparation of wash:

150g of molasses was dissolved in distilled water150G OF MOLASSES till the gravity was set to 1.082.

Fermentation process:

The culture was mixed with wash solution. To this nutrients (urea, DAP) were added. It was kept for 72 hours incubation at 37⁰C. The pH was maintained around 7. After the incubation period, the pH was calculated and the hydrometer reading was noted. The alcohol produced was analyzed and yield is calculated.

Analysis of alcohol:

The percentage of alcohol present in the wash was determined. 150ml of wash was dissolved in 150ml of water. It was boiled at 100⁰C. The vapours were condensed. 150ml of condensed solution was collected. The hydrometer reading and the temperature in (⁰F) were noted. The table reading corresponding to the temperature and hydrometer reading was noted. Alcohol is measured in terms of proof litres. Proof spirit is the ethanol containing 49.28% of alcohol by weight (or) 57.10% of alcohol by volume.

Yield:

The yield of alcohol can be calculated from the alcohol percentage mathematically.

ANALYSIS OF MOLASSES:

Analysis of Brix:

Brix = Hydrometer reading + [Table reading × Dilution factor]

Determination of total reducing sugar:

TRS = 5.128 / [Titre Value × Fehlings Factor × Dilution factor]

Determination of total reducing sugar:

UFM = 5.128 / [Titre value × Fehlings factor × Dilution factor]

ANALYSIS OF WASH:

Determination of Unfermentable solids:

Analysis of alcohol:

Alcohol Percentage = (100-Table reading) × 0.57

Yield:

RESULTS AND DISCUSSION:

Alcohol is widely used for many different purposes. In this application of fermentation, energy is obtained when sugar is changed to ethanol and carbon dioxide. All beverage ethanol and more than half of industrial ethanol is made by this process. Ethanol production is highly economical from molasses. It is very possible that the ethanol project, using molasses as a raw material will be profitable as it contains a substantial amount of unextracted sugar, along with various inorganic salts, organic salts, and organic polymers. No further sugar may be recovered from final molasses by standard production methods. Any substrate other than molasses, containing hexose sugars or materials that can be converted to hexose sugars can be used as raw material. However most distilleries in India are using cane molasses-c as a feedstock for alcohol production. Molasses-c differs from other feed stocks for alcohol production such as corn, sorghum, and potatoes etc. which have their carbohydrate content stored as starch which is usually precooked and hydrolyzed into fermentable sugars. Molasses doesn’t require pre-treatment as the carbohydrates are already in the form of sugars⁵. During the sugar process, one of the important by-products is molasses-c. Molasses is used to produce ethanol, citric acid, yeast and cattle feed. Over 20 billion litres of ethanol is produced annually from molasses⁶.

It contains sucrose which can be degraded easily using microorganisms. The sample was taken in the company and were analysed in all the parameters. The alcohol produced is extracted and purified by distillation column setup. Many yeast and bacterial species are capable of producing alcohol. Yeast feeds on the sugar and causes the fermentation.  As the fungi feeds on the sugar, it produces alcohol (ethanol) and carbon dioxide.  In fermentation, the ethanol retains much of the energy that was originally in the sugar, and explains why ethanol is an excellent fuel.

Table 1: Analysis of molasses and wash

Analyses of molasses
Analysis / Value	Normal Value	Obtained Value
Analysis of Brix	85-88	89
Determination of total reducing sugar	45-50%	47%
Analysis of wash
Determination of specific gravity	1.40-1.45	1.41
Determination of unfermentable solids	3-4%	4.5%

Table 2: Ethanol production using S. cerevisae, Z. mobilis and E. carotovora:

Analysis / Strain	*S.cerevisae*	*Z.mobilis*	*E.carotovora*
Unfermentable matter	1.5%	4.2%	4.3%
Analysis of alcohol	6.5%	8.1%	4.54 %
Yield	220.40%	291.61%	169.64%

The efficiency of Z. mobilis in alcohol production is high compared to that of yeast. Z. mobilis degrades sugars to pyruvate using the Entner-Doudoroff pathway. The pyruvate is then fermentated to produce ethanol and carbon dioxide as the only products beside that the Entner-Doudoroff pathway is an additional means of glucose consumption in many bacteria. Even up to 14% of alcohol can be obtained using Z. mobilis. The optimum temperature and the pH for ethanol production are 37⁰C and 7 respectively. In order to maintain this condition, high energy is utilized. Heating systems are employed for the process. This will improve the cost of production. For these reasons, yeast is the choice of industries.8-9% of alcohol can be obtained using yeast.

The alcohol production is 96% pure at the maximum. The distillation process can be improved so that the alcohol yield could be increased. The optimum temperature is room temperature. Production cost is lesser compared to that of Z. mobilis or other species. When seeing Erwinia carotovora, the production of alcohol is not economical. Incubation period is high compared to that of other species. This even reduces the production of alcohol per day. For yeast, the incubation period is one day. Erwinia carotovora will improve the cost of production of ethanol. The development of various recombinant microorganisms including yeasts (e.g., Saccharomyces species, Pichia species) and bacteria (Zymomonas mobilis, Escherichia coli, Klebsiella species.) capable of converting both C₅ sugars (mainly xylose and arabinose) and C₆ sugars (mainly glucose) to ethanol at relatively high rates and efficiencies.

The advantages of Z. mobilis over S. cerevisiae with respect to producing bio ethanol are higher sugar uptake and ethanol yield, lower biomass production, higher ethanol tolerance, does not require controlled addition of oxygen during the fermentation, amenability to genetic manipulations. However, it has a severe limitation compared to yeast: its utilizable substrate range is restricted to glucose, fructose, and sucrose. Using biotechnological methods, scientists are currently trying to overcome this. A variant of Z. mobilis that is able to use certain pentose as a carbon source has been developed. A great number of bacteria are capable of ethanol formation. Many of these microorganisms, however, generate multiple end products in addition to ethyl alcohol. These include other alcohols (butanol, isopropylalcohol, 2, 3-butanediol), organic acid (acetic acid, formic acid, and lactic acids), polyols (arabitol, glycerol and xylitol), ketones (acetone) or various gases (methane, carbon dioxide, hydrogen).

S. cerevisiae can change its morphology due to an inadequate nutrient supply. In this case, the proper supply of nitrogen should be used to overcome the morphological changes in culture yeast⁷.

Advances in technology are being made to further reduce the large amounts of energy needed for distillation. Technologies expected to be adopted include: steeping with gas injection of sulphur dioxide, membrane saccharification, high-tolerance yeast, yeast immobilization and bacterial fermentation. These advances help to reduce the costs and make producing ethanol more economical.

CONCLUSIONS:

The yield and efficiency of ethanol were determined (Table 2). Z. mobilis gave the better yield. The percentage of alcohol is 8.1 for Z. mobilis. However, industrially it is not better regarding the parameters like temperature and other maintenance factors. Erwinia is not good for industrial alcohol production. The percentage of alcohol production is less than 1 for Erwinia species. Hence, it is concluded that Saccharomyces cerevisiae is better for industrial alcohol production. The alcohol production is around 6.5%. The fermentation process can be done efficiently at room temperature using yeast.

REFERENCES:

1. Fiechter, A. and W. Seghezzi –. Regulation of glucose metabolism in growing yeast cells. Journal of Biotechnology. 27; 1992: 27-45.

2. Graeme M. Walker. Yeast Physiology and Biotechnology. John Wiley & Sons England. 1998.

3. Lewis, S.M Fermentation alcohol, Industrial Enzymology (2^nd Edition). Edited by: T. Godfrey and S, west Macmillan press Ltd., London UK. 1996.

4. Gunasekaran.P, T., Karunakaran and M. Kasthuribai, 1986 Fermentation pattern of Zymomonas mobilis strains on different substrates— a comparative study, Journal of Biosciences. 10 (2); 1986: 181-186.

5. Marcia S., Tan João B. Buzato and Maria Antonia P. C. Celligoio., “Sugar Cane Juice fermentation by Zymomonas mobilis CP4 Subjected to Inhibition by Ethanol and High Initial Concentration of Substrate”, Brazilian Archives of Biology and Technology. 43 (4); 2000.

6. Thomas, K.C., S.J. Hynes and W.M. Ingledow, 1996. Practical and theoretical considerations of alcohol by fermentation. Process Biochemistry, 31: 321-331.

7. Kalmokoff, M.L. and W.M. Ingledew. Evaluation of ethanol tolerance in selected saccharomyces strains. Journal of the American Society of Brewing Chemists. 43; 1985:75-83.

Received on 26.08.2013 Accepted on 01.09.2013

Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 174-178