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PRODUCTION OF ETHANOL FROM CHEAP SUBSTRATES

BY T.KRUPA RANI(Y7BT844) U.HARIKA(Y7BT845)

INTRODUCTION
y Ethanol has been used by humans since prehistory as

the intoxicating ingredientin alcoholic beverages.
y Ethanol was first prepared synthetically in 1826,

through theindependent efforts of Henry Hennel in Britain and S.G. Serullas in France.

y Michael Faraday prepared ethanol by the acid-

catalyzed hydration of ethylene in1828, in a process similar to that used for industrial ethanol synthesis today.

y Fermentation (formerly called zymosis) is the

anaerobicmetabolic breakdown of a nutrient molecule, such as glucose, without net oxidation.

y Depending on which organism it is taking place in,

fermentation mayyield lactate, acetic acid, ethanol or other reduced metabolites.

y Distillation is the process by which ethanol isboiled

from the fermented mixture and captured, producing a liquid with a muchhigher concentration of alcohol.

e. also known as ethyl alcohol or grain alcohol. orazeotrope. ‡ Ethanol forms a constant-boiling mixture..colorless chemical compound. with water that contains 95% ethanol and 5% water and that boils at78. It is miscible (i.properties ‡ Ethanol. .5°C. It is a monohydric primary alcohol and it boils at78.15°C. Ethanol is a psychoactive agent and it produces a variety ofphysiological and behavioral effects. flammable. is a volatile. mixes without separation) with water in all proportionsand is separated from water only with difficulty. ethanol that is completely free ofwater is called absolute ethanol.

typically by the acid-catalyzed hydration of ethylene. adsorbed onto a porous support such as diatomaceous earth or charcoal. represented by the chemical equation C2H4 + H2O _ CH3CH2OH y The catalyst is most commonly phosphoric acid.Production rutes 1 Ethylene hydration y Ethanol for use as industrial feedstock is most often made from petrochemical feed stocks. this catalyst was first used for large scale. .

they produce ethanol and carbon dioxide.In order to produce ethanol from starchy materials such as cereal grains. Saccharomyces cerevisiae) metabolize sugar in the absence ofoxygen. and the vast majority of ethanol for useas fuel. thestarch must first be broken down into sugars . The overall chemical reactionconducted by the yeast may be represented by the chemical equation C6H12O6 _ 2 CH3CH2OH + 2 CO2  The process of culturing yeast under conditions to produce alcohol is referred toas brewing. is produced by fermentation: when certain species of yeast (mostimportantly. Brewing can only produce relatively dilute concentrations of ethanolin water.2 Fermentation y Ethanol for use in alcoholic beverages. concentrated ethanol solutions are toxic to yeast. The most ethanoltolerantstrains of yeast can survive in up to about 25% ethanol (by volume).

y Most new cars sold in Brazil are flexible-fuel vehicles that can run on ethanol. y Ethanol with water content of 2% or less can be used as the alcohol in the y production of biodiesel. replacing methanol. y The United States fuel ethanol industry is based largely on corn. or any blend of the two. gasoline. . y India. Thailand. China and Japan have now launched their national gasohol policies.Uses: As a fuel y The largest single use of ethanol is as a motor fuel and fuel additive. which is quite dangerous to work with.

and meads from honey. the upper limit being set by the yeast's tolerance for ethanol. or as distilled beverages. beverages whose preparation involves concentrating the ethanol in fermented beverages by distillation.Alcoholic beverages y Alcoholic beverages vary considerably in their ethanol content and in the foodstuffs from which they are produced. . Beers are made from cereal grains or other starchy materials. or by the amount of sugar in the starting material. wines and ciders from fruit juices. beverages made by the action of yeast on sugary foodstuffs. y Fermented beverages may contain up to 15 20% ethanol by volume. y Most alcoholic beverages can be broadly classified as fermented beverages. y Fermented beverages can be broadly classified by the foodstuff from which they are fermented.

8 .mallaeri 7.Different types of ethanol producing strains y Strain % Ethanol produced y S.16 y Zygosaccharomyces sp.7 y Schizo.8-11.cerevisiae 5.pombe 8.7 y Schizo.ellipsoids 9.2 y S. 4.

However high substrate concentrations are inhibitory to fermentation (Jones et al 1981) due to osmotic stress .Factors affecting fermentation 1 Effect of sugar concentration y Use of concentrated sugar substrate is one of the ways to obtain high ethanol yield during fermentation.

Fermentation was carried out with initial concentration of 150. . 200. 250 glucose per liter at 30ºC y The maximum amount of ethanol produced by immobilization cells using 150. 93 and 83g ethanol per liter at 30ºC after 48h. Maximum yield was obtained at initial sugar of 20% with fermentation efficiency of 90%.cerevisiae immobilized on calcium alginate. 200 and 250 g/l glucose was 72.5.y batch fermentation system was used to produce ethanol using an osmotolerant S.

y The inhibition of the fermentation of oakhemicellulose acid hydrolysates by minor sugars. y Maximum productivity was calculated from the experimental data of ethanol Concentration. . Synthetic xylose media and detoxified oak hemicellulose acid hydrolysates were fermented batch wise.

metabolism and survival o the fermenting organism.2 Effect of temperature y The fermentation process is always accompa nied with evolution of heat thatraises the temperature of the fermenter. As a result it becomes necessary to cool the large fermenters in the distilleries. . y This necessity often becomes a major operation and a cost factor in the production of ethanol. Temperature exerts aprofound effect on growth.

. y Maintenance of high cell viability is a major characteristic of fermentation to get high ethanol yield.y Fermentation in industries is usually carried out at ambient temperature of 25-35ºC but temperature exceeds 40ºC during fermentation especially in northern regions which decreases the cell viability and productivity.

y Therefore many studies have been carried out for development of yeast to ferment at high temperature of up to 40-45°C. .y Fermentation at 35-40°C or above has advantages such as ethanol recovery and significant savings on operational costs of refrigeration control in distilleries for alcohol production.

.MATERIALS AND METHODS MATERIALS y The yeast used was Saccharomyces cerevisiae. y The strain was found efficient in producing alcohol from various substrates.

5g Ph 5.0g Dextrose 20. .Composition YEPD y y y y y y y Yeast extract 3.0g Peptone 10.5 The medium was sterilized in an autoclave at 15psi for 15minutes.0g Distilled water 1.0L Agar 1.

cylindrical plate assay for any strain. no vitamin requirement.Strain maintenance conditions y y y y y y Growth medium YEPD Growth condition Aerobic Temperature 30C Incubation time 24 hrs Subculture 60 days Special feature: distillery strain. . and production of ethanol.

Inoculum and inoculation y The yeast inoculum was prepared in YEPD broth. . y This inoculum was used at 10 percent or as specified to inoculate sterilized production medium. A loopful of twenty four hour old culture was inoculated at 28ºC on a rotary shaker (200rpm) for twenty-four hours.

.1-1ml are taken in test y y y y tubes and mark them. Transfer the tubes into water bath for 10-15min.DNS METHOD: y Different aliquots of glucose 0. Volume made up to 1ml with distilled water. Add the 1ml of DNS reagent and mix it. Read the absorbance in each test tube at 540nm.

TABLE water bath 10-15 mins .

ALCOHOL METHOD: y Different aliquots of alcohol 0. Volume made up to 1ml with distilled water. Transfer the tubes to ice bath. Add 5ml of concentric H2SO4 to each tube. .1-1ml are taken in test y y y y y y tubes and mark them. Add 3ml of distilled water each tube and mix well. Read the absorbance at 600nm. Add 1ml 10% k2cr2o7 and mix it.

TABLE .

Selection of suitable cheap sugary substrate .

This solid residue consists of a complex mixture of peel. core. and soft tissue. . seed. calyx. y Apple pomace is the processing waste generated after apple juice manufacturing and represents up to 30% of the original fruit.y In the present study two sugary substrates apple pomace and sugarcane bagasse obtained from fruit and sugar processing industries were evaluated to select the efficient alcohol producer among them. stem.

hemicelluloses and lignin of low molecular weight. fiber represents about half of all components. non-soluble solids and water. and includes cellulose.y Bagasse obtained as a by-product of sugar cane processing. pith. . is composed of fiber. y A extract of waste material was prepared and obtained extract was 10 fold diluted and added to production medium @ 2%.

Sugar cane and apple extract preparation: y Take sugar cane and apple pulp in the separate flasks and add 50ml water to both the flasks and put into the shaker. . y Finally a extract taken by using the filter paper.

0 .Composition Production medium (g/L) y y y y y y y Malt extract Yeast extract MgSO4 KH2PO4 (NH4)2PO4 Distilled water pH 5 4 5 1 5 1L 6.

y The production medium supplemented with diluted sugarcane bagasse and apple pomace was inoculated with overnight grown yeast culture. y The primary inoculum prepared in YEPD broth was transferred to sterilized production media taken in 250ml flask and incubated at 30ºC under shaking conditions. temperature and total reducing sugars was studied. y For fermentation studies 24 h old inoculum was used to inoculate the production media and the effect of variable parameters like pH. y Alcohol produced was estimated by K2Cr2O7 method after every 24 hrs. . y Sugar utilized was estimated by DNS method after every 24 hrs.

. so further studies were done with sugarcane bagasse.TABLE: Sugarcane bagasse was showing high production after 48 hrs.

. y Various factors were investigated affecting ethanol production from sugarcane bagasse. y It is therefore imperative to optimize the fermentation conditions for yeast cells so that the production efficiency increases.Optimization of fermentation process y Fermentation process carried out by yeast is known to vary with respect to substrate concentration. temperature.ph.

8. .16 &20 percent with distilled water and filtered through ordinary filter paper to remove suspended particles.12. the production media was prepared by diluting sugarcane bagasse to sugar Concentration of 4. y Fermentation was carried out in 250 ml conical flasks.cerevisiae. A twenty four hour old inoculum of yeast was added at the rate of 10 percent to the medium.Effect of sugar concentration y To study the effect of sugar concentration on ethanol production by S.

y Samples were withdrawn after every 12-hour interval and estimated for residual sugars as well as ethanol content in the media by K2Cr2O7 colorimetric method for estimating the percentage of ethanol was employed.ie 16%. . y The initial sugar concentration that was efficiently utilized by the yeast for ethanol production was selected and maintained in fermentation media for further use.

TABLE: .

6.0.0 and 8.0 were tested for fermentation using sugarcane bagasse extract with 16% sugar concentration (best of previous experiment) and temperature of 29 ± 1ºC.0.0. . Low pH inhibits the yeast multiplication.Effect of pH y pH of 4. 5.ie 6. 7.

TABLE: .

fermentation was carried out at 35.Effect of temperature: y To optimize the fermentation temperature. y Sugarcane bagasse extract diluted to 16% sugars in production medium was used as production media and fermentation was carried out at different temperatures. The periodic samples were analyzed for reducing sugars and ethanol content. .37 and 45ºC.

TABLE: .

. y Finally conclusion is that we can produce high amount of ethanol by using cheap substrates i.RESULT: y The agricultural and agro-industrial wastes. rice husk. fruit peels.e. paper industry wastes and orange and sugarcane bagasse have high organic matter content and their disposal arise both economic and environmental problems. corn cob. sugar cane bagasse . such as wheat bran. corn straw.