Ethanol

Production

Introduction
Ethanol produced as a component of
alcoholic
fermentation
Used as chemical feed stock and fuel
supplement
World wide- 4 million tons of ethanol
produced annually
[ 80% is by fermentation]
Used as solvent

Biosynthesis of Ethanol

Produced by fermentation f sugar rich produ

of yeast- Saccharomyces cerevisiae or som
Kluyveromyces fragilis

Bacteria also used ( widely used one is Zymo

S.cerevisiae suitable for fermentation of h

K.fragilis- suitable source is lactose
Candida sps. suitable source is pentose

Most microbes requires readily available sug

for fermentation ( eg. Sugar cane ,sugar be
Molasses)

Ethanol Biosynthesis Pathway

Carbohydrate
Hydrolysis
Glucose
Glycolysis
Pyruvate
Pyruvate
decarboxylase, Mg2+
Acetaldehyde + CO2
Alcohol
dehydrogenase
Ethanol

Schematic representation of Ethanol pr

Raw Materials
(Starch,Cellulose,
Molasse))

Separat
ion

Distillat
ion

Pretreatme
nt of
substrates

Fermenta
tion

Ethanol
(94.5% w/v)

Sterilizatio
n

Ethanol Production

Ethanol is produced in 3 steps1. Preparation of substrate

2. Fermentation
3. Distillation

1.Preparation of substrate
3 types of substrate are used
a) Starch containing roots , tubers, or
grains
b) Molasses or juice from sugar cane or
sugar beet
c) Wood or agricultural waste or wood
waste
Root starch is derived from Manihot esculenta
(Root contains 20-30% of starch and 1 to 2 %
protein)
S.cerevisiae donot produce amylase; so

Glucose production glucoamylase and alpha

amylase are added after boiling and cooling
process
1 tone of starch app. 1 liter of alpha amylase
and 3.5 L of glucoamylase are used

Molasses byproduct of sugar ,commonly

used
Wood- Is not yet used in commercial
production
Sulfite waste liquor- source of sugar
formed during production of paper from

2. Fermentation process

Both Batch and continuous fermentation was emp

ethanol production.

When compared to batch , continuous fermentatio

useful for ethanol production

Industrial production of alcohol is carried out in lar

upto a size 5 lakh liters
Inoculum volume is around 4 % of the fermentor
pH around 4-4.5

Initial temperature is kept between 21-26 C, Durin

temp. rise to around 30 C

Cooling devices are necessary to maintain temp.

Duration is 2 -3days, but actual time period depe

materials used and incubation temp.

After fermentation , spent medium contains etha

range of 6-9% by volume. This represents 90-95 %

converted in to ethanol

3. Distillation
Ethanol is recovered from fermented medium by
distillation
Cell mass is separated initially by centrifugation
or sedimentation
1st Distillation 80 % of pure ethanol is obtained
Repeated Distillation- 99.4% or more ethanol is
achieved
Final dessication is by chemical dessicants and
by absorbtion
Ethanol 92.4% - used as solvent

Methods to increase Productivity

Process should be carried out in large scale

fermentation

Continuous operation with cell recycle- res

conversion of substrates to ethanol

Maximum productivity with glucose as carb

82 g/L/h by yeast
120g/L/h by Z. mobilis

Use of immobilized cells ( high cell concent

fluidized bed bioreactor)

If Molasses is used as substrate

Batch system yield 2g/L/h of ethanol

Continuous system yield 3.35g/L/h of et

Immobilized cells with continuos system

yeild 28.6g/l/h of ethanol

Solvents - Acetone , Butanol &

Acetone Butanol Production

Acetone used as solvent for fats ,oils, resins,
waxes
- used as gelatinizing agent for
nitrocellulose
- used in dyeing industry
Butanol- used in production of detergent and
brake fluids
- used as solvents for fats, waxes and
resins
Butanol is better fuel additive than ethanol
due to its low vapour pressure, low solubility
with water, complete solubility with diesel fuel
Acetone, Butanol, Butyric acid & isopropanol

Products will vary and it depends o strain and ferm

Acetone-butanol fermentation C. acetobu

Butanol-Isopropanol fermentation- C. buty

Butyric acid-Acetic acid fementation-C. bu

Clostridium acetobutylicum strict anaero

starch in to glucose & maltose

Biosynthesis
Glucose
Glycolysis
Pyruvate
Acetyl CoA
AcetylCoA

Acetyl Phosphate

Acetic Acid

AcetylCoA
AcetoacetylCoA

-HydroxybutyrylCoA

Crotonyl CoA

Hydroxy -methyl g

Acetoacetic acid

Acetone

Butyric Acid
Isopropanol

Butanol

Production process
Cane Molasses ( Raw Material)
C. acetobutylicum
Low inoculum is inoculated into
production fermenters
(1:3000)
Fermentation
(pH 5.8-6.0),Temp.34 C, Fermenter

1.After sterilization of fermentor, it is gassed

with CO2
2.Before and after inoculation, the medium
contents are also
stirred with CO2
This is to ensure that it will support the
growth of the organism
To remove any residual O2 present in the
fermenter
To maintain anaerobic condition

Fermentation takes 36 hours to complete

First Phase (18hrs) pH decreases to 5.2 {format

Second phase (18hrs) - pH increases { formation

Third phase pH value remain constant 5.8

{growth and solvent production s

Continuous distillation & frac

Product recovered

* Contamination by phages & Lactobacilli is a com

Several ways to improve solvent yield

Addition of Acetic & Butyric acids

High agitation (300rpm) during acid phase followe

agitation (25 rpm) during solvent phase

Simultaneous removal of inhibitory products (A/B

and adsorption

Continuous or fed batch operations improve solve

Use of immobilized cultures
Gene manipulations of C.acetobutylicum

Glycerol Production

Glycerol is formed by yeast along with ethanol

during alcoholic fermentation
Usually glycerol is formed in low amount . By
modifying the fermentation balance high
amount of glycerol is produced
Acetaldehyde is the intermediary product in
ethanol fermentation
When sodium sulfite is added, it blocks alcohol
production and diverts the pathway for large
scale production of glycerol.
Sodium sulfite reacts with CO2 in the medium
and gets converted to Sodium bisulfite

NADH2 formed during glycolysis usually reduces

acetaldehyde to ethanol
Due to unavailability of acetaldehyde ,NADH2
reduces
Dihydroxyacetone phosphate to form glycerol 3phosphate then
glycerol
Glycerol fermentation carried out in 1000m3 size
of fermenters
Fermentation duration within 2 to 3 days
Osmotolerant yeast are used
Commonly used strains S. rouxii & Torulopsis

Glycerol Production Using Algae

Dunaliella salina is halophilic algae commonly
found in salty lakes.
While growing in hypersaline conditions ,this
algae synthesizes glycerol within the cells to
balance the high osmotic pressure in the
surroundings (due to salts)
So Higher the salt concentration in the
surroundings results in high intracellular glycerol
production.
Extraction of glycerol from algae
Collect the algal cells and place it in low salt
environmental condition.
Sudden shift in high salt to low salt condition will