PRODUCTION OF ETHANOL FROM CHEAP SUBSTRATESBY T.KRUPA RANI(Y7BT844) U.HARIKA(Y7BT845)

INTRODUCTIONy 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.

properties Ethanol, also known as ethyl alcohol or grain alcohol, is a volatile, flammable,colorless chemical compound. It is a monohydric primary alcohol and it boils at78.5C. It is miscible (i.e., 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. Ethanol forms a constant-boiling mixture, orazeotrope, with water that contains 95% ethanol and 5% water and that boils at78.15C. Ethanol is a psychoactive agent and it produces a variety ofphysiological and behavioral effects.

Production rutes1 Ethylene hydration y Ethanol for use as industrial feedstock is most often made from petrochemical feed stocks, typically by the acid-catalyzed hydration of ethylene, represented by the chemical equation C2H4 + H2O _ CH3CH2OHy The catalyst is most commonly phosphoric acid,

adsorbed onto a porous support such as diatomaceous earth or charcoal; this catalyst was first used for large scale.

2 Fermentationy Ethanol for use in alcoholic beverages, and the vast majority of ethanol

for useas fuel, is produced by fermentation: when certain species of yeast (mostimportantly, Saccharomyces cerevisiae) metabolize sugar in the absence ofoxygen, they produce ethanol and carbon dioxide. 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. Brewing can only produce relatively dilute concentrations of ethanolin water; concentrated ethanol solutions are toxic to yeast. The most ethanoltolerantstrains of yeast can survive in up to about 25% ethanol (by volume).In order to produce ethanol from starchy materials such as cereal grains, thestarch must first be broken down into sugars

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

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

Different types of ethanol producing strainsy Strain % Ethanol produced y S.cerevisiae 5.8-11.16 y Zygosaccharomyces sp. 4.2 y S.ellipsoids 9.7 y Schizo.pombe 8.7 y Schizo.mallaeri 7.8

Factors affecting fermentation1 Effect of sugar concentrationy Use of concentrated sugar substrate is one of the ways

to obtain high ethanol yield during fermentation. However high substrate concentrations are inhibitory to fermentation (Jones et al 1981) due to osmotic stress

y batch fermentation system was used to produce

ethanol using an osmotolerant S.cerevisiae immobilized on calcium alginate. Fermentation was carried out with initial concentration of 150, 200, 250 glucose per liter at 30C

y The maximum amount of ethanol produced by

immobilization cells using 150, 200 and 250 g/l glucose was 72.5, 93 and 83g ethanol per liter at 30C after 48h. Maximum yield was obtained at initial sugar of 20% with fermentation efficiency of 90%.

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

2 Effect of temperaturey 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, metabolism and survival o the fermenting organism.

y Fermentation in industries is usually carried out at

ambient temperature of 25-35C but temperature exceeds 40C during fermentation especially in northern regions which decreases the cell viability and productivity.y Maintenance of high cell viability is a major

characteristic of fermentation to get high ethanol yield.

y Fermentation at 35-40C or above has advantages such

as ethanol recovery and significant savings on operational costs of refrigeration control in distilleries for alcohol production.y Therefore many studies have been carried out for

development of yeast to ferment at high temperature of up to 40-45C.

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

from various substrates.

Composition YEPDy y y y y y y

Yeast extract 3.0g Peptone 10.0g Dextrose 20.0g Distilled water 1.0L Agar 1.5g Ph 5.5 The medium was sterilized in an autoclave at 15psi for 15minutes.

Strain maintenance conditionsy y y y y y

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

Inoculum and inoculationy The yeast inoculum was prepared in YEPD broth. A

loopful of twenty four hour old culture was inoculated at 28C on a rotary shaker (200rpm) for twenty-four hours. y This inoculum was used at 10 percent or as specified to inoculate sterilized production medium.

DNS METHOD:y Different aliquots of glucose 0.1-1ml are taken in test y y y y

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

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water bath 10-15 mins

ALCOHOL METHOD:y Different aliquots of alcohol 0.1-1ml are taken in test y y y y y y

tubes and mark them. Volume made up to 1ml with distilled water. Add 1ml 10% k2cr2o7 and mix it. Transfer the tubes to ice bath. Add 5ml of concentric H2SO4 to each tube. Add 3ml of distilled water each tube and mix well. Read the absorbance at 600nm.

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Selection of suitable cheap sugary substrate

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. y Apple pomace is the processing waste generated after apple juice manufacturing and represents up to 30% of the original fruit. This solid residue consists of a complex mixture of peel, core, seed, calyx, stem, and soft tissue.

y Bagasse obtained as a by-product of sugar cane

processing, is composed of fiber, pith, non-soluble solids and water; fiber represents about half of all components, and includes cellulose, hemicelluloses and lignin of low molecular weight.

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.

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 6.0

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

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

Optimization of fermentation processy Fermentation process carried out by yeast is known to

vary with respect to substrate concentration, temperature,ph. y It is therefore imperative to optimize the fermentation conditions for yeast cells so that the production efficiency increases. y Various factors were investigated affecting ethanol production from sugarcane bagasse.

Effect of sugar concentrationy To study the effect of sugar concentration on ethanol

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

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. 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.ie 16%.

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Effect of pH

y pH of 4.0, 5.0, 6.0, 7.0 and 8.0 were tested for

fermentation using sugarcane bagasse extract with 16% sugar concentration (best of previous experiment) and temperature of 29 1C. Low pH inhibits the yeast multiplication.ie 6.

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Effect of temperature:y To optimize the fermentation temperature,

fermentation was carried out at 35,37 and 45C. 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.

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RESULT:y The agricultural and agro-industrial wastes, such as

wheat bran, rice husk, corn straw, corn cob, fruit peels, paper industry wastes and orange and sugarcane bagasse have high organic matter content and their disposal arise both economic and environmental problems.y Finally conclusion is that we can produce high

amount of ethanol by using cheap substrates i.e. sugar cane bagasse .