Introduction Ethanol Is Also Called Ethyl Alcohol, 625c13

INTRODUCTION Ethanol is also called ethyl alcohol, pure alcohol, grain alcohol or dri nking alcohol is a volatile, flammable, colorless, liquid .It is a psychoactive drug, best known as the type of alcohol found in alcoholic beverages and in mode rn thermometers. It is produce by fermentation of sugar. Also called as ‘Sprit E thanol’ is straight chain alcohol. STRUCTURE:-

CHEMICAL PROPERTY:PROPERTIES IUPAC Name Ethanol Other name Ethyl alcohol; Grain alcohol; pure alcohol; hydroxyl ethane; Dri nking alcohol; Ethyl hydrate; Absolute alcohol Molecular Formula C2H6O or C2H5OH. Molecular Mass 46.07 g/ml. Density 0.789 g cm-3 . Melting point −114.3 °C, 159 K, 174 °F. Boiling point 78.4 °C, 352 K, 173 °F. Solubility in water Miscible Odor pH 15.9 Related Compound

Methanol, Propanol.

Amount of waste fruits are generated every year because they are more prone to s poilage than cereals due to their nature and composition and this spoilage occu rs at the time of harvesting handling , transportation ,storage ,marketing and p rocessing results in waste .Recycling of agricultural waste is one of the most i mportant reactions of utilizing number of innovative ways yielding a new produc ts of meeting the requirements of essential products in humans ,animals and pla nt nutrition as well as in the pharmaceutical industry. Microbial technique is a vailable for recycling and processing of agricultural waste and following the pr oducts can be made out the different process. Means agricultural waste can be us ed for production of economically important products like ethanol. It is a clean , burning renewable resource that can be produce from fermentation of glucose ri ch substrate. After ethanol production, the by product is used as cattle feed .B ioethanol is an alternative petroleum. It’s wide spread used of substances inclu ding scents, flavoring, coloring, medicines and alcoholic beverages. C6H12O6 → 2 CH3CH2OH + 2 CO2 . (Glucose) (Ethanol) (Carbon dioxide) List of Agricultural waste: 1) Sugar cane 2)

Orange pulp

3)

Mango juice

4)

Banana stem powder

5)

Pomegranate

6)

Tomato

7)

Maize

8)

Wheat

9)

Potato

10)

Rice

11)

Jawar

12)

Bazara. etc

REVIEW OF LITERATURE On of limited supply of oil, ethanol has emerged as an alternative for p etroleum based liquid fuels .Now a day’s its use in automobiles as an alternativ e fuel has attracted worldwide attention for its production on large scale while maintaining the economics status of a country. In present state of energy crise s efforts are being made to reduce the dependence upon nonrenewable energy sourc es, one of which is fuel alcohol produced by fermentation .An efficient ethanol production requires four components: Fermentable carbohydrates, an efficient yea st strain, a few nutrient and simple culture conditions. Approximately 80% of wo rld supply of alcohol is produced by fermentation of sugar. India present there are ( 285 Distilleries ) many distilleries producing ethanol by traditional batc h fermentation process. However use of this system followed by distillation to r ecover the ethanol is rather uneconomical. In order to produce ethanol in large quantities and reasonable costs, the parameter is important .The important para meter that could affect ethyl alcohol fermentation may be mentioned ,availabilit y and ferment ability of substrate ,possible isolation of new potent strain and improvement of the available strain towards the higher productivity ,improvemen t of fermentation technology and reduction in by product formed during the ferme ntation process .As India one of the largest sugarcane and fruit producing count ry. Lots of waste are generated every year that is available in plenty at cheap rate is mostly used as raw material per fermentation. Hence, literature available on ethanol production from agricultural waste about all these aspects is reviewed under following heads. 1) History 2) Organisms 3) Production procedure 4) Biochemistry of ethanol fermentation 5) Recovery 6) A pplication History of Ethanol Ethanol has been used by humans since prehistory as the in toxicating ingredient s of alcoholic beverages, dried residues on 1,000 years old pottery found in chi na imply that alcoholic beverages were used even among Neolithic people. Its isolation as relatively pure compound was first achieved by the persion alch emist, Muhmmad ibn zakariya Razi ( Phazes 865 925). Two other chemists who contributed to the development of distillation to techniq ue were Gerber and Al kind. In 19796, Johann Tobia Lowitz obtained pure ethanol by filtering distilled etha nol through activated charcoal. Antoine Lavoisier described ethanol as compound carbon hydrogen and oxygen and i n 1908 Nicolas Theodore de Saussure determined ethanol’s chemical formula. Fifty years later, Archibald Scott couper published the structural formula of et hanol. In 1828, Michael Faraday prepared ethanol by acid catalyzed hydration of ethylene a process similar to that which is used today for industrial ethanol sy nthesis.

Ethanol was used as lamp fuel in the united state at early as 1840. From 1908 on wards Ford Model T automobiles could be adopted to run on ethanol.

Alcohol Production (in India) (in million liters) Alcohol Year Production of Alcohol es Surplus Availability

Industrial Use Potable Use

1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

238.2 455.8 462.7 527.7 597.5 627.5 665.8 742.3 822.8 813.4

99 00 01 02 03 04 05 06 07 08

1411.8 1654.0 1685.9 1775.2 1869.7 1969.2 2074.5 2187.0 2300.4 2500.9

534.4 518.9 529.3 539.8 550.5 578.0 606.9 619.0 631.4 731.8

584.0 622.7 635.1 647.8 660.7 693.7 728.3 746.5 765.2 869.8

55.2 57.6 58.8 59.9 61.0 70.0 73.5 77.2 81.0 85.0

Other Us

(Source: http://www.ethanolindia.net/) BIOLOGICAL SYSTEM USED: In fermentation, of the various ethanol producing microorganism (Bhatt et al 19 87, Leplace et. al. 1992 and 1998) yeast belonging to Saccharomyces cerevisiae have been used most commonly. Ok and Hashinanga (1987) isolated yeast from spoil ed high sugar foods. Skotnicki et. Al. compared the rates of growth and ethanol production by 11 different strain of Zymomonas , Zymomonas mobilis is one of the most promising ethanol producing organism .This bacterium can produce up to 1.9 mol of ethanol from each mole of glucose fermented . Renu Bensal and R.S.Singh (2003) did comparative study on ethanol production using Saccharomyces cerevisia e and Zymomonas mobilis .Yeast was found to be more ethanol tolerant and produce d more ethanol at sugar concentration 15 %(v/v). Uma and Polasa (1990) isolated Saccharomyces cerevisiae from palm wine, which produced increased amount of etha nol in yeast extract peptone dextrose medium(PDA). Bertolini et. al. (1991) isolated new strains of Saccharomyces cerevisiae an bas al medium containing 48% Sucrose from fermentating sample. Collected from fermen ting sample collected from Brazilian alcohol factories.

Different types of ethanol producing strain Strain % of Ethanol production Saccharomyces cerevisiae 5.8 11.16 ZygoSaccharomyces spp. 4.2 Saccharomyces ellipsoids 9.7 Schizo.pombe 8.7 Schizo.mallaeri 7.8 Zymomonas mobilis 6.2 (Source: Recycling, residues of agriculture and industry, M.S. kalra, P.p.202,)

MEDIA COMPONENTS Carbon Source: Several raw materials as waste sugar cane , Banana stem, Pomegranate, Orange p ulp, Mango, have been employed as substrates for ethanol production. Means anaer obic fermentation of these agricultural wastes to produce ethanol. Generally ethanol is produce by fermentation using inexpensive raw materials. Raw material Strain %Yield(Ethanol) Sugar cane Saccharomyces cerevisiae Saccharomyces spp. 0.20 Cassava roots Saccharomyces spp. 0.18 Maize grain Saccharomyces cerevisiae Saccharomyces ovarum 0.36 0.30 0.35 Sugar cane stalks Saccharomyces spp. 0.07 Sweet potato roots Saccharomyces pp. 0.12 Wood

Saccharomyces cerevisiae 0.16 (Source: http://www.engineeringtoolbox.com/ethanol bio fuel d_1357.html

) PRODUCTION PROCESS Ethanol production by fermentation of agricultural waste is the most eco nomical and widely used way of obtaining product. More than its advantages as op erations are simple, stable, energy consumption is lower. Needs for production A) Microorganisms: a. Inoculums Preparation. b. Growth form. c. Strain Improvement. Production Technique Anaerobic Fermentation: The production ethanol by anaerobic condition is called as “Alcoholic fermentati on”. The fermentation is characterized by development of microorganism which con verted the sugar present in agricultural waste in to ethanol that organism was S accharomyces cerevisiae . They isolated from Brewers yeast then grow into Sabour aud Medium. These Yeast inoculums were inoculated in Yeast extract Peptone Dextr ose (YPD) fermentative media at pH 5.6 and at room temperature in anaerobic cond ition. Inoculate the agricultural waste in fermentative media in place of the de xtrose. Before added these agricultural waste in to fermentative media, Di nitro Salicylic Acid (DNSA) was carried for determination of sugar concentration whic h present in agricultural waste. The fermentation process was completed with in 48 hours under optimal conditions.

Biochemical Pathway: Glucose

Fructose bis phosphate 2ATP

2ADP

2 Phosphoglyceric acids. 2ADP 2ATP

2 Pyruvic acid.

2 trios’ phosphate

2 NADH2

2NAD+

2 Acetaldehyde + Co2 2 Ethanol. Fig .1:Biochemical pathway of ethanol. Fermentation is an ATP regenerating metabolic process in which degradation prod ucts of organic substrates serves as hydrogen acceptor. The reaction that leads to phosphorylation of ADP is oxidations. The oxidized carbon compound is finally released from the cell as carbon dioxide. The oxidizing steps are dehydrogenati ons in which the hydrogen is transferred to a cofactor like NAD. Intermediately a product of substrate degradation then serves as acceptor for the hydrogen from NADH. The reduced products formed by the regeneration of NAD are exerted by the cell. Organic substrate (Energy and Carbon Source)

Catabolism via Fructose 6 phosphate Pathway

Carbon dioxide. (Co2)

Reduced product.

Fig. 2: Biochemical pathway of reduced product formation. The conversion of glucose to ethanol was formulated in1815 by Gay lussacs law in its present form – C6H12O6 2Co2 + 2C2H5OH. Glucose (1kg) (0.489kg) (0.511kg) Normal Fermentation: Fermentation of glucose to ethanol and carbon dioxide by yeast proceeds via the fructose bis phosphate pathway. The conversion of pyrvate to ethanol then compro mises two steps In first, Pyrvate is decarboxylated by pyrvate decarboxylase with the participatio n of thiamine pyrophosphate to acetaldehyde this is then reduced to ethanol by a lcohol dehydrogenase with NADH2 as the hydrogen donor. Glucose

2 Pyravate

2 Ethanol 2 Acetaldehyde Fig 3: In this hydrogen transfer, the hydrogen obtained during the dehydrogenation of t rose phosphate is utilized ,the oxidation reduction balance is there for maintai ned. RECOVERY Ethanol is separated from the fermented broth in continuous stills resembling th

ose utilized for recovery of acetone, butanol and ethanol of 95 % concentration is obtained by successive distillation in these stills. To obtained alcohol conc entration greater than 95 % however requires special distillation technique beca use of the ability of alcohol to form and azeotropic mixture containing 5% water . Thus to prepare absolute ethanol, the 5% water is remove by forming and azeotr opic mixture of benzene , water and ethanol which then is distilled with increas ing temperature increment. This procedure removes first the azeotrophic benzene Water Ethanol mixture, then an ethanol Benzene azeotrophic mixture so that absol ute ethanol remains. The absolute ethanol as well as 95 % ethanol is marketed wi th and without denaturation. Other products of fermentation except for the carbon dioxide which is often coll ected for commercial reuse are present in only small amounts. Thus trace amount of succinic acid and slightly larger amount of glycerol occur. Some acrolein a lso may be present in distillate, having been formed by microbiological contamin ants attacking the glycerol.

APPLICATION 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13)

Bio fuels Alternative source for petroleum. Agricultural wastes are reused. Pharmaceuticals. Detergent. Ink, Paints and Adhesive. Vinegar and chemicals. Gasoline production. Flavors and Chemical Industry. Disinfectant. Alcoholic Beverages. Good Solvent. Clean source of energy. Antidote use.

OBJECTIVES Isolation of ethanol producing organism from brewer’s yeast. Characteristic and identification of ethanol producing organism. Media optimization –pH, Temperature Sugar concentration. Ethanol production using various agricultural waste such as, sugar cane, Banana stem, Orange pulp, Pomegranate, Mango Juice, Maize, etc. To conformed desired product (Ethanol) CAN test was used.

MATERIAL AND METHOD Raw Material: 1)

Brewer’s yeast. 2) Agricultural waste.

Primary Screening: 1) Isolation of Saccharomyces cerevisiae : Media: Sabouraud dextrose agar medium( For isolation of Saccharomyces cerevisiae. 2) Slide culture technique: Slide Culture assembly (U Shaped rod,Filter paper, Slide, Cover Slip). Glycerol. Sterile Nutrient broth. Lacto phenol cotton blue.

3) Residual Sugar Estimation: Di nitro Salicylic acid (DNSA Reagent). Working Stock (Maltose). Distilled Water. Test Sample. 4) Fermentation: Yeast Peptone Dextrose Media (YPD) Fermentative media. 5) Distillation: 6) Cerric Ammonium Nitrate Test (CAN): Cerric Ammonium Nitrate. Distillate. Distilled Water.

Screening of Saccharomyces cerevisiae for ethanol production: 1) Isolation of Saccharomyces cerevisiae from brewer’s yeast by microbial technique. Spread plate technique. Pour plate technique. 2) Using microbial and biochemical method for characterization of Saccharomyces cerevisiae. Slide culture technique Eosin Methylene blue test (EMB) 3) Materials used agricultural waste like sugar cane, Banana stem, Orange pulp,

Pomegranate, Mango Juice, Maize, etc. 4) Di nitro salicylic acid (DNSA). 5) Fermentation. 6) Distillation. 7) Cerric Ammonium nitrate Test. (CAN) Methodology 1) Isolation of Organism Saccharomyces cerevisiae. Isolated from brewer’s yeas t: Place Location NASHIK N. G. Wines Khaedgaon. Tal: Dindori, Dist: Nashik The yeast granules was diluted in sterile saline so as to get 1 / 100 ,1/1000, d ilution of each sample .this dilutions (0.1ml) of Each sample were spread separa tely on sterile sabouraud dextrose agar medium. Plates incubated at 280 for 48 h ours. Isolated culture were observe then purified by sub culturing on sabouraud dextrose agar slant . 2) Slide culture technique: For culturing microscopic examination of yeast , slide culture technique is used. 3) Eosin Methyelene Blue agar Medium: This is the confirmatory test for isolation of Saccharomyces cerevisiae , The colony of S. cerevisiae shows bulky white and sticky colony on eosin methyle ne blue agar medium. Bacteria and Fungi shows the Blackish colony on the same me dium. Isolated colony showed bulky white colure on EMB agar, that means isolated colon y was the Saccharomyces cerevisiae was conform. 4) Di nitro Salicylic acid (DNSA): Before adding agricultural waste in to fermentative media, to determine the suga r concentration present in agricultural waste by DNSA . Principle: DNSA reagent has been employed which assay sugar by its reducing property under acidic condition 3,5,dinitro salicylic acid is converted to 3 amino 5 nitro sali cylic acid .After heating , the color changes from color Orange red which can be determined by colorimetric at 540 nm. Calculation: Unknown concentration =

Absorbance of Test. Slope.

Procedure: I. Pipette out 0.1, 0.2 ….1 ml, of working standard into series of tubes. II. Pipette out 0.9, 0.8 ….0.1 ml of distilled water into each tube. III. Make up the volume to 1 ml in the all test tube .A tube with 1 ml of wat

er serves as blank. IV. Add 1ml of DNSA reagent in each of the tube V. Transfer the tubes to boiling water bath for 10 min. VI. Take the readings at 540 nm. VII. Draw standard graph and calculate the amount of residual sugar in the sa mple. 5) Fermentation: The agricultural waste is added into fermentative media (YPD) in place of Dextro se. Agricultural waste act as sugar source added isolated Saccharomyces cerevisi ae and then anaerobic fermentation was carried out for 48 hours at Room temperat ure. In this fermentation Saccharomyces cerevisiae converts the sugar of agricultural waste into ethanol and carbon dioxide. C6H12O6 Glucose (1kg)

→

2 CH3CH2OH + 2 CO2. Ethanol (0.489 kg)

Carbon dioxide (0.511kg

) 7) Distillation: After 48 hours fermentation, the distillation process was carried out at 80 0 temperature. Due to distillation ethanol is separated from other material. Ethanol at 96 % (w/w) is produced in the distillation process. Normally two dis tillation columns were used and some byproduct such as aldehyde and heavy alcoho l are recovered. Stillage that is a mixture of the by product was separated and then about 70% of this liquid mixture sent again to the fermentation process for increasing process efficiency. Finally the still age is carried to the stillage treatment plant; the solids are separated and sent to the composting plant, whe re they are mixed with ashes and residual biomass to obtain an organic fertilize r. At the end of process the product is dehydrated using molecular sieves to pro duce anhydrous ethanol at 99.08 % (w/w). 7) Cerric Ammonium Nitrate Test : ( CAN) This alcohol detection test. This test is use for detection of distill ate contains alcohol or not. Principle: Alcoholic compounds on reaction with cerric ammonium nitrate give a red colorati on due to the formation of a complex. (NH4)2 [Ce (NO3)6] + [3ROH] → [Ce (NO3)4(ROH) 3] + 2NH4NO3 Cerric ammonium Red complex. Nitrate.

Protocol: Distilled Water Distillate Blank 2.5 ml _ 2.5 ml Test _ 2.5 ml 2.5 ml

Cerric Ammonium Nitrate Optical Density

When cerric ammonium nitrate added in to distillate then colored changed change from yellow to orange. It was the conformation for the distillate contains ethan ol. Calculation: Practical Yield: 1) Amount of alcohol =

O.D of Test. O.D. of Standard.

Practical yield = …………….g m% 2)

Percent yield =

Practical yield.

Theoretical yield. 3) Theoretical yield was calculated by Gay Lussac law equation. 1 Mole Glucose = 2 Moles of alcohol. i.e. 180 gm of Sugar = 92 gm of alcohol. Media Optimization Fermentation process carried out by yeast is known to vary with respect to subst rate concentration, temperature pH, It therefore imperative to optimize the ferm entation condition for yeast cells so that production efficiency increases. Sr.No Yeast extract (gm/lit.) Peptone (gm/lit.) Dextrose (gm/lit.) pH Temp. 0C % of Alcohol yield 1 10.0 20.0 15 6.5 30 5.2 2 10.0 20.0 20 6.5 30 9.0 3 10.0 20.0 25 6.5 30 2.50 4 10.0 20.0 30 6.5 30 1.78 1) Effect of the Sugar concentration on ethanol production: Sugar concentration is one of the major constraints than determines the ethanol production. To know optimum sugar concentration for ethanol production, the solu tion contains 15 %, 20 %, 25% 30% Sugar concentration and studied the ethanol p roduction at different concentration. A low ethanol production 1.78 % at 30% sug ar concentration in 48 hours. As shown in table at 20 % of sugar concentration, the ethanol yield was maximum and turned out to be 2.89 %. However increasing th e sugar concentration belong 20 % the ethanol production was decreased. So 20 % was selected as optimum sugar concentration for ethanol production. 2) Effect of pH on Ethanol production: Sr.No Yeast extract (gm/lit.) Peptone (gm/lit.) Dextrose (gm/lit.) pH Temp. 0C % of Alcohol yield 1 10.0 20.0 10 5 30 7.9 2 10.0 20.0 15 6 30 11.6 3 10.0 20.0 20 7 30 8.6 4 10.0 20.0 25 8 30 9.7 Initial Sugar concentration of 20 % and optimum temperature of 300C was selected for further studies and subjected to pH treatment 5,6,7,8.The result are shown in table . At pH 5 took place but it gave low ethanol content. Best results were obtained a t pH 6 where Maximum ethanol production was noticed. Yadav et al (1997) found an increase in alcohol concentration, productivity as w ell as efficiency with increase pH from4.0 to 5.0 and found that optimum pH rang e for Saccharomyces cerevisiae to be between pH 4.5 to 5.0 .Based on fermentatio n of efficiency. The pH 6.0 was selected for further experiments. 3) Effect of temperature on ethanol production: Sr.No Yeast extract (gm/lit.) Peptone (gm/lit.) Dextrose (gm/lit.) pH Temp.

0C % of Alcohol yield 1 10.0 20.0 10 5 25 6.8 2 10.0 20.0 15 6 30 11.6 3 10.0 20.0 20 7 35 8.2 4 10.0 20.0 25 8 40 3.0 The temperature is one of the major constraints that determine the ethanol produ ction. To know the optimum temperature for ethanol fermentation the solutions we re kept at 250C, 300C, 350C and 400C with 20% initial sugar concentration. Then studied the effect of various temperature on ethanol production fermentation was carried out at 48 hours. A low ethanol yield of 6.8 % was observed at 250C in 4 8 hours. As shown in table at 300C, the ethanol yield was maximum and turned out to be 11 %. How ever increasing the temperature beyond the 300C the growth as well as concen tration of alcohol decreased. This decrease was pronounced at 400C, so 300C was selected as optimum temperature for ethanol production. PROTOCOL FOR RECOVERY: Fermented broth. Distillation at 700C Temperature. Collect the distillate. Detection of ethanol. Orange red Or Red color observed. Ethanol was present in distillate. Store in small bottles. RESULT AND DISCUSSION Saccharomyces cerevisiae: Cultural characteristics Colonies were isolated on Sabouraud dextrose agar medium, growing rapidly about 1 mm diameter in to two days incubation having creamy color. Size Shape Color Margin Elevation Character Motility 1mm Circular Creamy Entire Flate cci Motile

Opacity Consistency

Gram

Opaque Smooth Gram positive Co

Four agricultural waste were used for ethanol production that were Suga rcane, Banana Stem powder, Orange pulp ,Mango Juice ,Pomegranate among these sug ar cane give higher amount of ethanol (14.7% ) and Orange pulp give small amount of ethanol (5.53%) Banana stem powder gave 7.83% ethanol, Mango gave 8.00% of e thanol and pomegranate gave 9.23% of ethanol.

Media optimization: 1) Yeast extract –Peptone Dextrose (Lab fermentation Media): Dextrose (gm/lit.) % of Alcohol yield pH %of Alcohol yield Temp. %of Alcohol yield 15 2.49 5 7.9 25 6.8 20 2.65 6 11.6 30 11.6 25 2.89 7 8.6 35 8.2 30 1.78 8 9.7 40 3.2 Fig: Media optimization of YPD Media. Comment: When the culture were tested for variation in sugar concentration , pH, and Temp erature it was reported that at 20 % of sugar concentration ,6 pH, and at 30o C temperature gave higher amount of ethanol optimum condition for ethanol produc tion. Sugar concentration =20gm/lit. Temperature = 300 C pH = 6 2) Sugar cane juice : Dextrose (gm/lit.) % of yield pH %of yield Temp. %of yiel d 15 10.3 5 7.9 25 6.8 20 14.7 6 11.6 30 11.6 25 11.2 7 8.6 35 8.2 30 7.2 8 9.7 40 3.2 35 5.0 9 6.2 45 0 Fig: Ethanol production by using agricultural waste Sugar cane. Comment: When the culture were tested for variation in sugar cane juice concentra tion , pH, and Temperature it was reported that at 20 % of sugar concentration ,6 pH, and at 30o C temperature gave higher amount of ethanol optimum condition for ethanol production by using sugar cane juice was . Sugar concentration =20gm/lit. Temperature = 300 C pH = 6 3) Banana stem powder : Dextrose (gm/lit.) % of yield d 15 5.8 5 20 7.8 6 25 6.4 7 30 4.8 8 35 4.0 9

pH

%of yield

5.4 7.4 6.0 6.5 3.9

25 30 35 40 45

Temp.

%of yiel

4.2 7.4 5.0 4.2 0

Fig: Ethanol production by using agricultural waste Banana stems powder. Comment: When the culture were tested for variation in Banana stem (powder) sugar concentration , pH, and Temperature it was reported that at 20 % of sugar con centration ,6 pH, and at 30o C temperature gave higher amount of ethanol optimum condition for ethanol production by using Banana stem (powder) was . Sugar concentration =20gm/lit. Temperature = 300 C pH = 6

4) Orange pulp Dextrose (gm/lit.) d 15 20 25 30 35

% of yield 3.3 5.5 4.3 4.4 2.5

5 6 7 8 9

pH

%of yield

3.7 5.2 4.9 5.0 1.6

25 30 35 40 45

Temp.

%of yiel

3.6 5.1 4.8 2.3 0

Fig: Ethanol production by using agricultural waste Orange pulp. Comment: When the culture were tested for variation in Orange pulp sugar concentr ation , pH, and Temperature it was reported that at 20 % of sugar concentratio n ,6 pH, and at 30o C temperature gave higher amount of ethanol optimum conditio n for ethanol production by using Orange pulp was . Sugar concentration =20gm/lit. Temperature = 300 C pH = 6 5) Mango Dextrose (gm/lit.) % of yield pH %of yield Temp. %of yiel d 15 6.2 5 4.8 25 5.1 20 8.0 6 7.2 30 7.4 25 7.4 7 5.6 35 6.3 30 4.0 8 6.8 40 4.2 35 3.2 9 3.2 45 0 Fig: Ethanol production by using agricultural waste Mango. Comment: When the culture were tested for variation in Mango. Sugar concentration , pH, and Temperature it was reported that at 20 % of sugar concentration, 6 pH, and at 30o C temperature gave higher amount of ethanol optimum condition for et hanol production by using Orange pulp was. Sugar concentration =20gm/lit. Temperature = 300 C. pH = 6 6) Pomegranate Dextrose (gm/lit.) d 15 20 25 30 35

% of yield 6.2 8.0 7.4 4.0 3.2

5 6 7 8 9

pH

%of yield

4.8 7.2 5.6 6.8 3.2

25 30 35 40 45

Temp.

%of yiel

5.1 7.4 6.3 4.2 0

Fig: Ethanol production by using agricultural waste Pomegranate. Comment: When the culture were tested for variation in Pomegranate. Sugar concent ration, pH, and Temperature it was reported that at 20 % of sugar concentration, 6 pH, and at 30o C temperature gave higher amount of ethanol optimum condition for ethanol production by using Pomegranate was. Sugar concentration =20gm/lit. Temperature = 300 C. pH = 6

7) Reducing Sugar estimation of agricultural waste by DNSA method: Sr.No. Stock Solution (in ml) D/w (ml) Concentration (in ml) DNSA Reagent Incubation Optical density (540 nm) 1 0.1 0.9 100 1ml Keeping boiling 0.06 2 0.2 3 0.3 4 0.4 5 0.5 6 0.6 7 0.7 8 0.8 9 0.9 10 1 11

water bath for 10 min. 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 1ml

200 300 400 500 600 700 800 900 1000

1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml Blank

0.18 0.24 0.30 0.43 0.50 0.63 0.75 0.86 0.94 1ml

Conclusion: • The investigation made under this project can be summarized as follows: • The isolate found was to be potential procedure of ethanol at a sugar co ncentration of 20gm /lit with productivity range 2.89 % • Ethanol yield was maximum (11.6 %) at pH 6. • Isolate found at temperature 30 0C to give yield maximum i.e. 11.6%. • Isolate organisms Saccharomyces cerevisiae was successfully produced et hanol from agricultural waste like Sugar cane, Banana stem (powder ),Orange pulp , Pomegranate, Mango by anaerobic fermentation sugar cane was giving maximum eth anol that was 14.7%, and Orange pulp gave small amount of ethanol that was 5.53% .

Path Forward: • Fermentation was used to evaluate the potential of ethanol production. T he enhancement of ethanol production from various fermentation systems and the a pplication of fungal ethanol to biorediation can be achieved from the following future work. • To isolate the organism which having the capacity of rapid fermentation with in short period. • To study the effect of various mutagenic strain on ethanol production.

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