Ethanol, also known as alcohol, is produced through the fermentation of sugars by yeast. It has been produced for thousands of years and is used in alcoholic beverages as well as fuels. Ethanol is commonly fermented from sugars in grains like corn or from sugarcane. New technologies allow ethanol to be produced from cellulosic biomass as well through enzymatic or thermal processes. The fermentation process involves inoculating sugar-containing substrates with yeast, which converts the sugars to ethanol and carbon dioxide through a series of biochemical reactions. Proper fermentation conditions like temperature, pH, and nutrients maximize ethanol yield.

1. ETHANOL
• commonly called alcohol, ethyl alcohol, and drinking alcohol
• the principal type of alcohol found in alcoholic beverages,
produced by the fermentation of sugars by yeasts.
• It is a neurotoxinc, psychoactive drug, and one of the
oldest recreational drugs. It can cause alcohol
intoxication when consumed in sufficient quantity.
• Ethanol is a volatile, flammable, colorless liquid with a slight
chemical odor.
• Empirical formula is C2H5OH

2. CHEMICAL STRUCTURE

3. HISTORY OF ETHANOL
• The fermentation of sugar into ethanol is one of the earliest organic
reactions that man learned to carry out and the history of man-made
ethanol is very long.
• Dried ethanol residue have been found on 9 000 year old pottery in
China which indicates that Neolithic people in this part of the world
may have consumed alcoholic beverages.
• Distillation was well known by the early Greeks and Arabs. Greek
alchemists working in Alexandria during the first century A.D.
carried out distillation.
• Fractional distillation was invented by Tadeo Alderotti in the 13th
century.
• The year 1796 is significant for ethanol history because this is when
Johann Tobias Lowitz obtained pure ethanol by filtering distilled
ethanol through activated charcoal.
• In mid 1800s, ethanol became one of the first structural formulas to
be determined The scientist behind the description was Scottish
chemist Archibald Scott

4. HOW TO MAKE ETHANOL ?
Ethanol can be produced in different ways:
• through fermentation - biological process
• through ethylene hydration - a petrochemical
process
• Wet milling process
• Dry milling processes

5. PRODUCTION OF ETHANOL BY FERMENTATION
• Fermentation is the oldest way for humans to produce
ethanol, and this is the traditional way of making
alcoholic beverages. It is also the process used for the
vast majority of ethanol fuels on the market.
• When certain species of yeast metabolize sugar, the
end result is ethanol and carbon dioxide. One
example of such a species is Saccharomyces
cerevisiae, which has been used by brewers since
ancient times.
• In Greek Saccharo means sugar and myces means
fungus

6. • This is the chemical formula for turning sugar into
ethanol and carbon dioxide:
C6 H12 O6 2 CH3CH2OH + 2 CO2
C6 H12 O6 is simple sugar, also known as
dextrose or D-glucose.
CH3CH2OH is ethanol
CO2 is carbon dioxide
• Most types of yeast will stop reproducing when the
alcohol content reaches 15% ethanol by volume, or
even earlier, putting a natural limit on the alcohol
concentration achieved through fermentation

7. BIOCHEMICAL PROCESS

8. MICROORGANISMS USED
 Bacteria
• Zymomonas mobilis
• Clostridium acetobutylicum
• Klebseilla pnemonia
• Candida brassica
 Fungi
• Saccharomyces cerevisiae
• Schizosaccharomyces

9. BIOETHANOL PRODUCTION
Bioethanol can be produced in three ways:
 First generation bioethanol
• SUGAR ETHANOL
• STARCH SUGAR ETHANOL
 Second generation bioethanol
• CELLULOSE OR HEMICELLULOSE ETHANOL
 Third generation bioethanol
• ALGAE SUGAR ETHANOL

10. CROPS USED IN FIRST GENERATION
BIOETHANOL PRODUCTION
• INDIA : SUGARCANE
• BRAZIL : SUGARCANE
• USA : CORN
• EUROPE : WHEAT AND BARLEY

11. CHOICE OF RAW MATERIAL DEPENDS
UPON
• Ease of processing of various plant materials
• Prevailing conditions of climate
• Landscape and soil composition
• Sugar content

12. SECOND GENERATION BIOETHANOL
PRODUCTION
• Second generation biofuels are made from lignocellulosic biomass or
woody crops, agricultural residues or waste, which makes it harder to
extract the required fuel.
• Second generation biofuel technologies have been developed
because first generation biofuels manufacture has important limitations.
• The goal of second generation biofuel processes is to extend the amount of
biofuel that can be produced sustainably by using biomass consisting of the
residual non-food parts of current crops, such
as stems, leaves and husks that are left behind once the food crop has been
extracted, as well as other crops that are not used for food purposes (non-
food crops) which contains lignin, cellulose and hemicellulose.
• Lignocellulosic biofuels reduces greenhouse gas emissions with 60-90%
when compared with fossil petroleum (Börjesson.P. et al. 2013)

14. Bioethanol from cellulose
• 2nd generation of bioethanol
• Researchers are exploring ways to make ethanol from other feedstocks or
plant materials.
• Cellulosic ethanol production starts with the biomass or plant materials and
breaks down the cell wall to release the starch or sugars in the plants leaves
and stems. These simpler compounds are then fermented into ethanol.
• The combination of lignin, hemicellulose and cellulose in plant material is
very resistant to breakdown into the molecular sugar components. Two
main pathways to converting lignocellulose to biofuels are:
• Biological – specialized enzymes or microbes break pre-treated biomass-
based cellulose into sugars, which are then fermented into alcohols.
• Thermochemical – biomass is converted by pyrolysis, gasification, or
liquefaction into gaseous and liquid chemical "building blocks. It is
recombined through catalytic processes into a variety of fuels and
chemicals.

15. THIRD GENERATION BIOETHANOL
PRODUCTION
• Major feedstock is – ALGAE
• Advantages
Don’t occupy agricultural lands
Do not need any fertilizer for cultivation
Significant carbohydrate content, higher ethanol yields are obtained
Algae can grow in every season and everywhere such as salty waters,
fresh waters, lakes, deserts and marginal fields etc
 Two types of algae
• Microalgae - high amount of lipid, protein and carbohydrate mainly starch
• Macroalgae - carrageenan, laminaran, mannitol, alginate
 Chlamydomonas reinhardtii has highest amount of acrbohydrate content

17. CONDITIONS FOR FERMENTATION
• Carbon source- Pure sugar or molasses(10-18% , a
concentration higher than this is detrimental to the yeast
strain)
• Nitrogen Source- Mostly available in form of
Ammonium Sulphate
• Growth Factor- can be provided in form of molasses.
• pH- 4.8 – 5
• Temperature- 32˚C-45˚C
• Time- depend on strain, usually between 30 to 70 hrs(2-
3 days)

18. PRODUCTION PROCESS
PRETREATMENT
• Most of the raw material require some degree of
pre-treatment .
• In general sugary material require mild or no pre-
treatment while cellulosic material require
extensive pretreatment.
• This is because cellulosic material have to be
subjected to acidic or enzyme hydrolysis to
release monosaccharide units that are required for
alcohol fermentation.

19. • Production process of alcohol is carried out in
3 stages
1. Inoculum
2. Proper fermentation
3. Recovery

20. PREPARATION OF INOCULAM
• After selection of desired organism and its in
pure form the Inoculam is prepared under
aseptic condition.
• For this condition organism is fist culture in
flask to increase the size of the inoculam
which increases the size of the inoculam which
can then be used for inoculation.

21. PROPER FERMENTATION
• Previously Batch fermentation was used but now
Continuous fermentation is used as it increases
the alcohol production by 10-12 fold as compare
to batch fermentation.
• Large fermentor of size 1,25000 gallon are used .
• Initially aeration is required for good growth of
micro organism .Later anaerobic conditions are
created by withdrawal of oxygen .
• It take 2-3 days for the fermentation to complete.

22. A STUDY
• Fermentation of sugar by Saccaharomyces cervisiae for
production of ethanol in a batch experiment .
• When the temperature was increased to 45°C, the system
still showed high cell growth and ethanol production rates,
while it was inhibited at 50°C. The maximum specific
growth rate and the maximum specific ethanol production
rate were observed between 30-45°C.
• pH 4.0-5.0 was the optimal range for the ethanol production
process. The highest specific ethanol production rate for all
the batch experiments was achieved at pH5.0. Formation of
acetic acid was increased when the pH was below 4.0, while
butyric acid was produced when the pH was higher than 5.0

23. BATCH FERMENTATION
• In Batch fermentation bacteria are inoculated
into the bioreactor .
• Then under optimal condition(temp, pH etc)
the bacteria go through all the growth
phases(LAG, exponential and stationary).
• It may be necessary to add acid or alkali to
maintain pH and antifoaming agents to
minimize foam under optimal condition for
growth.

24. PHASES
• Lag phase
• Acceleration phase
• Logarithmic (log) phase
• Deceleration phase
• Stationary phase
• Death phase

25. LAG PHASE
• initial period just after the inoculation
• During lag phase micro organisms adapt to the
new environment (available nutrient ,pH etc)
• No increase in cell number, although cellular
weight may slightly increase.
ACCELERATION PHASE
• Brief transient period during which cell start
growing slowly .
• Connect lag phase to log phase.

26. LOG PHASE
• Most active growth of micro- organism and multiplication
occur during log phase.
• Number of cells and rate of population increases doubles
with each consecutive time period.
• Growth rate of microbes in log phase is dependent on
substrate(nutrient supply)
STATTIONARY PHASE
• Depletion of nutrients and accumulation of metabolic end
product .
• Microbial growth may either completely slow down or
completely stop
• Biomass may remain constant .
• The number of cells produced is limited by growth factor
and as a result the rate of cell growth matches the rate of
cell detah.

27. DEATH PHASE
• Cells die at an exponential rate

28. • At last fermentation is stopped and product is
collected.
• Then after cleaning and sterilization of
fermentor is ready for another batch

29. ADVANTAGES
• ADVANTAGES
• VERSATILE- can be used for different
reaction everyday
• SAFE- can be properly sterilized. Little risk of
infection or strain mutation. Complete
conversion of substrate is possible.

30. DISADVANTAGS
• High labor cost: Skilled labor is required.
• High proportion of Down time between
batches
• Product Variability – The quality and quantity
(to some extent ) may vary from one batch to
other batch.
• Safety Problem- when cleaning, filling and
emptying.

31. CONTINIOUS FERMENTATION
• In, continuous fermentation fresh medium flows
into the fermentor continuously, and a part of the
medium is withdrawn from the fermentor at the
same flow rate of the inlet flow.(so that working
volume remain constant)
• Chemostat bioreactor are frequently used in
industrial manufacturing of ethanol.
• BY changing the rate at which the medium is
added to the bioreactor the specific growth rate of
the micro-organism can be easily controlled

33. ADVANTAGES
• Low labor cost
• Growth rate is higher as nutrients are
continuously added to the reactor.
• More efficient as the fermentor operates
continuously.(No down time )
DISADVANTAGE
• If contamination occur huge volume of product
may be lost.

34. Power Alcohol
• Ethyl alcohol which is used to generate power; for example as an
additive to motor fuels to act as fuel for internal combustion engine
is called power alcohol. It has generally 80% petrol and 20%
alcohol.
• There have been decades of motor fuel application experience in the
United States and other countries with ethanol. Ethanol has been
blended with fuels to produce more efficiency.
• The practice of blending ethanol started in India in 2001.
Government of India mandated blending of 5% ethanol with petrol.
• Rather than using chemically produced alcohol, nowadays ethanol
prepared by biomass is being used for this blending. This is
considered more environment friendly and hence are called biofuels.
These biofuels have overtaken the traditional fossil fuels over the
past few years all over the world.

35. BIOFUELS
‘Biofuels’ are those liquid or gaseous fuels produced from biomass
resources and used in place of, or in addition to, diesel, petrol or
other fossil fuels for transport, stationary, portable and other
applications.
Three broad categories of bio-fuels are identified in India:
- ‘bio-ethanol’: ethanol produced from biomass such as sugar
containing materials, like sugar cane, sugar beet, sweet sorghum,
etc.; starch containing materials such as corn, cassava, algae etc.;
and, cellulosic materials such as bagasse, wood waste, agricultural
and forestry residues etc.;
-‘biodiesel’: a methyl or ethyl ester of fatty acids produced from
vegetable oils, both edible and non-edible, or animal fat of diesel
quality; and
-other biofuels: biomethanol, bio CNG, biosynthetic fuels etc.

36. PRODUCTION OF BIOETHANOL
Wet Milling:
Wet milling is used to produce many products besides fuel ethanol. Wet
milling is called “wet” because the first step in the process involves soaking
the grain in water (steeping) to soften the grain and make it easier to
separate (fractionate) the various components
Dry Grind:
In the dry-grind ethanol process, the whole grain is processed, and the
residual components are separated at the end of the process. There are five
major steps in the dry-grind method of ethanol production:
1. Milling
2. Liquefaction
3. Saccharification
4. Fermentation
5. Distillation and recovery

37. PRODUCTION OF BIOETHANOL

38. BIOETHANOL PRODUCTION USING CORN
•First generation ethanol production
1. Milling: involves processing the corn through a hammer mill .This
whole corn flour is slurried with water, and heat-stable enzyme (a-
amylase) is added.
2. Liquefaction:
- accomplished using jet-cookers that inject steam into the corn flour
slurry to cook it at temperatures above 100°C (212°F).
- The heat and mechanical shear of the cooking process break apart the
starch, and the enzymes break down the starch polymer into small
fragments.
- The cooked corn mash is then allowed to cool to 80-90°C (175-
195°F), additional enzyme (a-amylase) is added, and the slurry is
allowed to continue liquefying for at least 30 minutes.
3. Saccharification :
-The slurry is cooled to approximately 30°C (86°F), and a second
enzyme glucoamylase is added. Glucoamylase completes the breakdown
of the starch into simple sugar.

39. 4. Fermentation:
Yeast grown in seed tanks are added to the corn mash to begin the
process of converting the simple sugars to ethanol.
5. Distillation and Recovery :
• After fermentation, the liquid portion of the slurry has 8-12%
ethanol by weight. Conventional distillation/rectification systems
can produce ethanol at 92-95% purity.
• The residual water and corn solids that remain after the distillation
called “stillage.” is then centrifuged to separate the liquid (thin
stillage) from the solid fragments (wet cake or distillers’ grains).
• The thin stillage is passed through evaporators to remove a
significant portion of the water to produce thickened syrup. Usually,
the syrup is blended with the distillers’ grains and dried to produce
an animal feed called “distillers’ dried grains with solubles”
(DDGS).

41. BIOETHANOL PRODUCTION USING SUGARCANE
•First generation ethanol production
• Cutting , washing and drying of sugarcane.
• Buffer was used to dilute the enzyme alpha-amylase ( phosphate buffer) and
glucoamylase (sodium acetate buffer)
• Liquefaction of sugarcane: Sugarcane was weighed and NaOH was added to
maintain pH at 4.5. Alpha amylase enzyme was added to it and heated until
500C. Alpha- amylases will breakdown the cellulose into smaller size called
dextrin.
• Saccharifaction of sugarcane bagasse- The mixture was cooled down to
400C. Glucoamylase was added to the mixture which hydrolyzed the dextrin
to fermentable glucose. The mixture was cooled to 320C and Saccharomyces
cerevisiea (baker yeast) was added.
• Fermentation of sugarcane - Saccharomyces cerevisiea (baker yeast) was
used to ferment the simple sugar to ethanol and carbon dioxide.
• Distillation: The bioethanol was distilled using rotary evaporator. The
sample was heated at 800C to get the bioethanol.

43. WHY IS BIOETHANOL GOOD
• Availability - It is renewable. Unlike fossil fuels, biofuels can be
easily produced from raw agricultural materials.
• Cleaner fuels - which means they produce fewer emissions on
burning.
• Reduce Greenhouse Gases: Fossil fuels, when burnt, produce large
amount of greenhouse gases i.e. carbon dioxide in the atmosphere
leading to global warming. Studies suggests that biofuels reduces
greenhouse gases up to 65 percent.
• They release lower levels of carbon dioxide and other emissions
when burnt. Although the production of biofuels creates carbon
dioxide as a byproduct, it is frequently used to grow the plants that
will be converted into the fuel. This allows it to become something
close to a self sustaining system.
• If more people start shifting towards biofuels, a country can reduce
its dependence on fossil fuels and also imported fuels from other
country can be reduced leading to economic development of our
country.

44. IS BIOFUEL HARMFUL?
• High Cost of Production: Even with all the benefits associated
with biofuels, they are quite expensive to produce in the current
market.
• Monoculture: Growing same crop every year may deprive the
soil of nutrients that are put back into the soil through crop
rotation.
• Industrial Pollution: The process with which they are produced
is largely dependent on lots of water and oil. Large scale
industries meant for churning out biofuel are known to emit
large amounts of emissions and cause small scale water
pollution as well.

45. Distillation and Ethanol
Recovery