FERMENTATION
PRODUCTION OF ETHANOL FROM
CHEAP SUBSTRATES
BY
T.KRUPA RANI(Y7BT844)
U.HARIKA(Y7BT845)
INTRODUCTION
Ethanol has been used by humans since prehistory as
the intoxicating ingredientin alcoholic beverages.
Ethanol was first prepared synthetically in 1826,
through theindependent efforts of Henry Hennel in
Britain and S.G. Serullas in France.
Michael Faraday prepared ethanol by the acid-
catalyzed hydration of ethylene in1828, in a process
similar to that used for industrial ethanol synthesis
today.
Fermentation (formerly called zymosis) is the
anaerobicmetabolic breakdown of a nutrient
molecule, such as glucose, without net oxidation.
Depending on which organism it is taking place in,
fermentation mayyield lactate, acetic acid, ethanol or
other reduced metabolites.
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 at78.5°C. 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 at78.15°C. Ethanol is a psychoactive agent and it
produces a variety ofphysiological and behavioral effects.
Production rutes
1 Ethylene hydration
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 _ CH3CH2OH
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 Fermentation
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
The largest single use of ethanol is as a motor fuel and fuel
additive.
Most new cars sold in Brazil are flexible-fuel vehicles that can
run on ethanol, gasoline, or any blend of the two.
The United States fuel ethanol industry is based largely on
corn. Thailand,
India, China and Japan have now launched their national
gasohol policies.
Ethanol with water content of 2% or less can be used as the
alcohol in the
production of biodiesel, replacing methanol, which is quite
dangerous to work with.
Alcoholic beverages
Alcoholic beverages vary considerably in their ethanol
content and in the foodstuffs from which they are produced.
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.
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.
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
strains
Strain % Ethanol produced
S.cerevisiae 5.8-11.16
Zygosaccharomyces sp. 4.2
S.ellipsoids 9.7
Schizo.pombe 8.7
Schizo.mallaeri 7.8
Factors affecting fermentation
1 Effect of sugar concentration
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
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 30ºC
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 30ºC after 48h.
Maximum yield was obtained at initial sugar of 20%
with fermentation efficiency of 90%.
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.
Maximum productivity was calculated from the
experimental data of ethanol Concentration.
2 Effect of temperature
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.
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.
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.
Maintenance of high cell viability is a major
characteristic of fermentation to get high ethanol
yield.
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.
Therefore many studies have been carried out for
development of yeast to ferment at high temperature
of up to 40-45°C.
MATERIALS AND METHODS
MATERIALS
The yeast used was Saccharomyces cerevisiae.
The strain was found efficient in producing alcohol
from various substrates.
Composition YEPD
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 conditions
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 inoculation
The yeast inoculum was prepared in YEPD broth. A
loopful of twenty four hour old culture was inoculated
at 28ºC on a rotary shaker (200rpm) for twenty-four
hours.
This inoculum was used at 10 percent or as specified to
inoculate sterilized production medium.
DNS METHOD:
Different aliquots of glucose 0.1-1ml are taken in test
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.
TABLE
water
bath
10-15
mins
ALCOHOL METHOD:
Different aliquots of alcohol 0.1-1ml are taken in test
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.
TABLE
Selection of suitable cheap sugary substrate
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.
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.
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.
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:
Take sugar cane and apple pulp in the separate
flasks and add 50ml water to both the flasks and
put into the shaker.
Finally a extract taken by using the filter paper.
Composition Production medium (g/L)
Malt extract 5
Yeast extract 4
MgSO4 5
KH2PO4 1
(NH4)2PO4 5
Distilled water 1L
pH 6.0
The production medium supplemented with diluted sugarcane
bagasse and apple pomace was inoculated with overnight grown
yeast culture.
Sugar utilized was estimated by DNS method after every 24 hrs.
Alcohol produced was estimated by K2Cr2O7 method after every
24 hrs.
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.
The primary inoculum prepared in YEPD broth was transferred
to sterilized production media taken in 250ml flask and
incubated at 30ºC under shaking conditions.
TABLE:
Sugarcane bagasse was showing high
production after 48 hrs, so further studies
were done with sugarcane bagasse.
Optimization of fermentation process
Fermentation process carried out by yeast is known to
vary with respect to substrate concentration,
temperature,ph.
It is therefore imperative to optimize the
fermentation conditions for yeast cells so that the
production efficiency increases.
Various factors were investigated affecting ethanol
production from sugarcane bagasse.
Effect of sugar concentration
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.
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.
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.
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%.
TABLE:
Effect of pH
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 ± 1ºC. Low pH inhibits the yeast
multiplication.ie 6.
TABLE:
Effect of temperature:
To optimize the fermentation temperature,
fermentation was carried out at 35,37 and 45ºC.
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.
TABLE:
RESULT:
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.
Finally conclusion is that we can produce high
amount of ethanol by using cheap substrates i.e. sugar
cane bagasse….
