This document discusses the production of two industrial solvents - ethyl alcohol and butanediol. It describes the microbial production of ethyl alcohol from molasses using Saccharomyces cerevisiae yeast. Key conditions for fermentation are listed. Commercial production involves diluting molasses, adding nutrients and yeast, then distilling the fermented product. Butanediol is produced similarly using bacteria like Bacillus and Enterobacter. Its uses include fuels, chemicals and polymers. Production methods aim to optimize yields under aerobic conditions with controlled pH and sugar concentrations.

1. SEMESTER – IV
FERMENTATION AND INDUSTRIAL
MICROBIOLOGY
SEMINAR TOPIC: SOLVENTS (ETHYL ALCOHOL
AND BUTANEDIOL)
SUBBMITED TO,
Dr. S. VISWANATHAN
HEAD DEPARTMENT OF
MICROBIOLOGY
SUBMITTED BY,
M.RESHMA
II M.SC MICROBIOLOGY
REG NO : 20201232516117

3. Solvent – definition:

4. Characteristics of solvent:
◦ Available in bulk quantity
◦ Biocompatibility
◦ Chemical and thermal stability.
◦ Favourable distribution coefficient for product and substrate.
◦ Favourable properties for product recovery.
◦ High selectivity.
◦ Inexpensive.
◦ Low emulsion forming tendency
◦ Low aqueous solubility.
◦ Non biodegradability.
◦ Non hazardous.

5. Types of solvents:

6. Industrial solvents:
◦ Industrial solvents are widely used in chemical as well as fermentation industries.
◦ For example,Ethanol is used as a solvents and precursor of many synthetic
chemicals.
◦ On the other hand, extraction procedures of certain antibiotics from fermented broth
(eg, Penicillin) need suitable solvents. These chemical compounds have been
produced by fermentation process.
◦ Today fermentation have virtually disappeared as a result of the development of
chemical ones. this has happened for the following reasons;
1. . High prices of the fermentable substrates.
2. . Increasing economic competition from the petrochemical industry.
◦ These chemical compounds are produced as by products during the refining of
petroleum.

7. Ethyl alcohol production
◦ Ethyl alcohol is the most common solvent and raw material.It is next to water
and it is used in the laboratory and chemical industries.
◦ Ethyl alcohol is also called as Ethanol.
◦ Ethanol is produced by biological process in which sugars such as glucose,
fructose, sucrose are converted into Alcohol and carbon dioxide.
◦ The fermentation is carried out under anaerobic conditions
◦ Much of this alcohol is obtained synthetically from ethylene.
◦ It is a clear, colourless liquid with a characteristic, pleasant odour.
◦ Ethyl alcohol is the intoxicating component in beer ,wine and other alcoholic
beverages.

8. ◦ In aqueous solution,it has some sweet flavours, but in more concentrated
solutions it has a burning taste.
◦ Alcohol fermentation was the first fermentation known to mankind. Distilleries
began to appear in Europe in the middle of the17th century.At first alcohol was
used only for human consumption.Later , the demand for alcohol as a
universal solvent and chemical raw material developed.
◦ The distilling industries grew very rapidly.The annual production in the United
States alone has now reached one billion gallons.
◦ In India the research claims that ethanol production was around 335 crore
litres in 2021.

9. Uses of ethyl alcohol (Ethanol):
◦ Use as a chemical feed stock: In the chemical industry, ethanol is an
intermediate in many chemical processes of its great reactivity,it is thus
a very important chemical feed stock.
◦ Solvent use: Ethanol is widely used used in industry as a solvent for
dyes ,oils,waxes, Explosives, cosmetics etc
◦ General utility: Alcohol is used as a disinfectant in hospitals. For
cleaning and lighting in the home and in the laboratory it is used as a
solvent next to water.
◦ Fuel : Ethanol is mixed with petrol or gasoline upto 10 percentage and
known as gasohol and used in automobiles.

10. Microbial production of ethyl alcohol:
ETHANOL PRODUCING MICRO-ORGANISMS:
◦ The MICRO-ORGANISMS responsible for alcoholic
fermentation of molasses are yeast species of Saccharomyces
, Torulopsis, kloekera, Candida (C.tropicalis) ,kluyeromyces
fragilis and certain species of mucor and bacteria such as
Zymomonas mobilis, Clostridium thermocellum.
◦ The most important among them are the yeast species
belonging to genus Saccharomyces.
◦ There are two types of Saccharomyces species,
. Saccharomyces cerevisiae ( Top fermenter yeast) are active
at the top surface of the fermenting liquid.
Saccharomyces carlsbergensis (bottom fermenter yeast)
develop only at the bottom of the fermentation vats. They are
whirled up in the liquid owing to carbondioxide evolution.
◦ The strain of yeast to be used for alcohol fermentation should
possess the following selective features:

11. 1. It should be an efficient strain.
2. It should be a fast growing strain.
3. It should have a tolerance to alcohol,as well as to osmotic pressure.
4. It should possess uniform and stable biochemical properties.
◦ The choice of microorganisms depends upon the type of raw materials to be used for alcohol fermentation.
◦ Generally, strains of S. cerevisiae are selected when starchy material, saccharide material like molasses are used as
raw materials.
◦ Strains of Candida are preferred when whey and waste sulphate liquor are employed as raw material in alcohol
fermentation.
PREPARATION OF INOCULUM:
◦ A large amount of yeast culture is required in the industrial production of ethyl alcohol
◦ The size of inoculum ranges from 8-10 percent with an average of 4percent v/v.
◦ The medium employed in the preparation of inoculum is the sane as that of the actual fermentation process.
◦ A high degree of agitation and aeration is necessary,since this favours rapid growth of yeast and producing a large
amount of cell mass.
◦ Optimum pH is 4.8 – 5.
◦ Optimum temperature is 28°C -30°C

12. Steps involved in inoculum preparation:
1. 10 ml of sterile medium in a test tube is inoculated with pure culture
of yeast and tube incubated at 28 to 30 °C for a suitable period.
2. The tube culture is transferred to a flask containing 200ml of sterile
medium. Flask is incubated at 28 to 30 °C to obtain sufficient cell
mass
3. The flask culture is transferred to a suitable glass container
containing about 4 litres of sterile medium and incubated at 28 – 30
°C .
4. Finally this culture is used to inoculate a small tank containing about
10- 40 gallons of sterile medium .
5. After incubation, the inoculum is used to inoculate the fermentation
medium in fermenter.

14. RAW MATERIALS:
◦ A variety of carbohydrates available as waste products of agri cultural industries are
used for the preparation of fermentation medium.On the basis of their chemical
nature,they may be grouped as:
◦ Saccharide materials:
Molasses,hydrol,whey,technical glucose, fruit juices.
◦ Starchy materials:
Potato starch, corn starch, wheat flour, cereals (eg oats)
◦ Cellulosic materials:
Sulphite waste liquor and others.
PREPARATION OF FERMENTATION MEDIUM:
Generally, molasses is used as the main raw materials in the preparation of a
fermentation medium. However, Starchy and cellulosic materials may be also for the
preparation of fermentation medium. These materials require treatment before their use.

15. ◦ For example, starchy material is subjected to saccharification,where starch gets
hydrolysed, thus forming fermentable sugars, maltose and glucose.
FERMENTATION CONDITIONS:
◦ CARBON SOURCES : sugar concentration in the range of 10 to 18 % sugar
concentration of 12% is often used. For example, cane molasses contains
sucrose (cane sugar)as a carbon source.The sucrose of this raw material is
about 10 percentage sugars. High concentration affects yeast and low
concentration makes the process uneconomical.
◦ NITROGEN SOURCES : Many organic,as well as inorganic nitrogenous
compounds may be used in this fermentation.Mostly nitrogen source is in the
form of ammonium sulphate. It promotes the growth of yeast cells.but inhibiting
the fermentation process.
◦ GROWTH FACTORS : It is not necessary to add the growth factors separately in
the preparation of the fermentation medium,since the raw materials eg ., Cane
molasses used to satisfy the requirements of the culture.

16. ◦ pH : pH of the fermentation medium is in the range of 4.8 to 5.The highest value
of pH increase the chances of contamination, whereas lowers the pH values
inhibit the yoeld of ethyl alcohol.
◦ TEMPERATURE : Usually the temperature range is 32° C – 45°C (70°F – 80° F).
As there is heat evolution during fermentation, the temperature in the
fermentation tank rises gradually and is controlled by means of cooling coils.
Intermittent agitation is required for the uniform cooling of the medium.Higher
temperature favours the growth of bacteria and also cause loss of ethyl alcohol
due to evaporation.
◦ TIME : Fermentation starts within a few hours after the addition of yeast. Duration
of ethyl alcohol fermentation is usually 30 to 72hours when the specific gravity of
the fermented liquid becomes constant.At this stage it contains 6 to 8 percent of
ethyl alcohol.
◦ YIELD : On an average 0-4 gallons of ethyl alcohol is obtained from one gallon of
molasses. About 90% of carbohydrates is converted into Alcohol.

17. Commercial production of Alcohol using
molasses as a raw material:
◦ Molasses contains about 50 percent fermentable carbohydrates (sugars).Big
deep tanks of steel or stainless Steel are used as a containers in the
industrial production method.
◦ Molasses is diluted to suitable sugar concentration (15- 16%) a small
quantity of nitrogen source and sulfuric acid is added in it.
◦ pH : 5.0 and yeast (S. cerevisiae) Culture is added in it. The fermentation
starts and is allowed for 24 -40 hrs at 25 – 30° C. The fermented medium is
then distilled and passes through rectifying columns to obtain ethyl alcohol
◦ The yield of ethyl alcohol ranges about 50 percent of fermentable sugar
concentration present in the medium. The large amount of CO2 which is
produced during the fermentation process as a result of decarboxylation,is
recovered and compressed to its solid state.
◦ The yeast recovered is usually used as a animal feed.

19. Commercial production of ethyl alcohol
using molasses as raw material:

20. Commercial production of ethyl alcohol
using starch as raw material :
◦ When starch such as corn are used
as the raw materials they have first
to be hydrolysed to release simple
fermentable sugars.
◦ The hydrolysis can be accomplished
with enzymes from barley Malt or
molds.eg : Aspergillus oryzae or
heat treatment of acidified materials.

21. Product recovery in laboratory:

22. Product recovery in large scale:

23. Byproducts:

24. Advantages and Disadvantages:
◦ Allows for domestically
dependent energy sources.
◦ Renewable in nature.
◦ Readily metabolizes in cells
and the environment .
◦ Produces profitable by
products.
◦ Reduces carbon relase by
incorporating the carbon
cycle.

25. Butanediol production:

26. 2,3-butanediol and its uses:
◦ 2,3 – butanediol is an odourless ,colorless and transparent liquid at
normal temperature, which is widely used in
Chemicals, food, fuels, aeronautical and other fields.
◦ 2,3 – Butanediol (also called as diol or 2,3- butylene glycol) is a possible
source for 1,3- butadiene (used in the synthesis of rubber).
◦ It is also a source for a permanent –type antifreeze for its low freezing
point.
◦ Moreover, other compounds, readily derived from this mother compound
2,3- butanediol includes methyl ethyl ketone, methyl vinyl carbinol and
methyl vinyl ketone.
◦ Methyl vinyl carbinol and methyl vinyl ketone are used in the plastic
industry.

27. ◦ The dehydration esterification forms of 2,3-butanediol is the synthesis
of polyimide precursors,which can be applied to
. Drugs, cosmetics, lotion etc;
◦ In China, 2,3 – butanediol is have been added to the spirits to improve
the wine flavour.
◦ 2,3- butanediol is a highly valuable fuel with the burning value of 27198
J/g,which is comparable to methyl ethyl ketone by sulfuric acid
catalyzed dehydration.
◦ After combination with methyl ethyl ketone and hydrogenation reaction,
2,3-butanediol can be converted into octane,which is used to produce
high quality aviation fuel.

28. Microbial production of butanediol :
◦ MICROORGANISMS :
◦ 2,3-Butanediol is known to be produced by a range of sugar
or citrate fermenting microbes.
◦ The organisms are –
Bacillus amyloliquefaciens
Bacillus subtilis
Enterobacter aerogenes
Klebsiella pneumoniae
Bacillus polymyxa
Serratia marcescens

29. Raw materials:
Raw materials for the manufacture of 2,3-butanediol are
grouped in three major classes:
1. Starchy substrates (eg., Corn, wheat, barley, sweet
potatoes and potatoes),
2. Sugars and sugar residues ( e.g. beet and cane molasses)
3. Wood hydrolysates and pulp mill residues (eg. Waste
sulphite liquor)

30. Comparison of yields of various fermentation
products:

32. ◦ All the 2,3-butanediol fermentations are sensitive to changes in fermentation
conditions (ie., Air supply, pHor sugar concentration).
◦ For example, dissimilation of glucose under aerobic conditions by these
bacterial species takes place as under :

33. ◦ Under anaerobic conditions,free hydrogen production is lowered for the three
bacterial species.
◦ Serratia marcescens gives little formic acid, and Bacillus subtilis (Ford’s type)
gives rarely any glycerol.
◦ Generally,the yields of 2,3 –butanediol or its oxidation product,acetoin,are increased
as compared to the other products under aerobic conditions.
◦ Oxygen retards ethyl alcohol formation by both Bacillus polymyxa and Aerobacter
aerogenes.
◦ Automatic pH control is necessary to obtain the highest yield of 2, 3-butanediol in
the shortest possible Fermentation time.
◦ Optimum Fermentation conditions for an aerobic dissimilation of sucrose by
Bacillus polymyxa are in a growth medium of 8percent sugar concentration at 33°C.
◦ pH at 6.2 by automatic addition of ammonium hydroxide.
◦ Fermentation time – 30hours, giving130 moles of 2, 3-butanediol and 122moles of
ethyl alcohol/100 moles of sucrose used.

34. ◦ Bacillus polymyxa is a unique bacterial species in that it produces its
own amylolytic enzymes, hydrolyzing whole grains mashes during
fermentation.

35. Commercial production of 2,3-butanediol:

36. A new route for the industrial production of
2,3 butanediol:

37. ◦ As a bio-polyol project with the annual output of 200,000 tons has been put into
produced by Changchun Dacheng Group has been successfully developed the
catalyst technology .
◦ This catalyst has a high selectivity to the unique hydroxyl structure of biomass
molecules and can turn the biomass materials like
1. Corn Starch into glucose by hydrolysis.
2. The glucose is converted into sorbitol after catalytic hydrogenation, it cracks
into the mixture of C2-4 dihydroxy alcohol and polyol.
3. After the heavy ends(including organic salts)has been removed, distilled, and
refined, propylene glycol, ethylene glycol and butanediol with purity more than
95 Percent can be obtained, in which the yield rate of 2,3-butanediol is
5percent.

38. Product recovery:
◦ Recovery of 2,3 butanediol from fermented mashes is rather difficult
because of its high boiling point (179-180°C).
◦ The product was recovered by the various procedures:
◦ They are – spray drying, drum drying, chemical conversion, dialysis,
solvent extraction and steam stripping.
◦ In most of these procedures, the 1st step consists of elimination of ethyl
alcohol.
◦ Therefore, liquor is subjected to filtration or screening, followed by
concentration by evaporation.
◦ With grain fermentations the most feasible procedure of recovery was
found to be vapour phase steam extraction.

39. References:
◦ Industrial Microbiology – A.H.Patel.
◦ M.J.Syu,”Biological production of 2, 3-butanediol” Applied Microbiology
and biotechnology, vol.55,2001.
◦ S.K.Garg and A.Jain “Fermentative production of 2, 3 –butanediol.A
review,Bioresource Technology,1995,vol.51.
◦ R.J.Magee and N.Kosaric,”The Microbial production of 2, 3 –butanediol
“Advance in Applied Microbiology,vol.32,1987.