Industrial Biotechnology

UNIT 4

Unit 4. Production of Microbial Products-Brief account of the

following products obtained by industrial microbiological
fermentation, Alcohol- Ethanol, Alcoholic Beverage – Beer,
Organic acid – Citric acid, Antibiotic – Penicillin, Amino acids –
Glutamic acid, Vitamin – B12. Brief account of Steroid
biotransformation
 Alcoholic fermentation
✓ Conversion of sugar into alcohol by microbial enzymes is called alcoholic
fermentation.
✓ Alcohol fermentation process using the unicellular fungus yeast
(Saccharomyces cerevisiae) has become a very sophisticated in industry.
✓ Alcoholic drink known today are beer, wine, whisky, brandy, rum and gin.
 Ethanol
❖Ethanol (ethyl alcohol, EtOH) is a clear, colorless liquid with a characteristic, pleasant odour.

❖Ethanol (also called ethyl alcohol, grain alcohol) is a chemical compound.

❖The chemical formula is C2H5OH.

❖ Ethanol production from agricultural products has been in practice for more than 100 years.
Ethanol can be produced from many kinds of raw materials that contain starch, sugar or
cellulose etc.

❖ In general there are three groups of raw materials from which ethanol can be produced:
1) beet, sugar cane, sweet sorghum and fruits
2) starchy material such as corn, milo, wheat, rice, potatoes, cassava, sweet potatoes etc.
3) cellulose materials like wood, used paper, crop residues etc.
 Ethanol
MICROORGANISMS
The choice of fermentation organism for industrial alcohol production depends to some
extent, on the type of carbohydrate present in the medium.
Bacteria: clostridium, Zymomonas mobilis.
Yeast: candida spp. Saccharomyces cerevisiae, Saccharomyces uvarum, Kluyveromyces fragilis
Filamentous fungi: Fusarium, Mucor sp.
Starch and sugar raw material: Specially selected strains of Saccharomyces cerevisiae is used.
Strain selection
• Must grow rapidly and tolerant to high concentration of sugar.
• Must produce large amount of alcohol.
• Relatively resistant to alcohol.
Nitrogen source:
Ethanol
• Urea is the most suitable source.
• Gaseous ammonium increases the pH of the medium.
• Ammonium sulfate can lead to incrustation.
Phosphorus source:
• Diammonium phosphate used as a source.
Hydrolytic enzymes:
• They can also be added to convert biopolymers and non-fermentable substances in the
molasses to monosaccharides or amino acids.
Process
Classical fermentation can be achieved in three steps:
❑During the first phase (22-24 h), yeast cells multiply aerobically by consuming oxygen
present in the mash.
❑In the middle phase (24-48 h), alcohol production occurs with post saccharification of sugars
and multiplication of yeast falls off.
❑The decrease in alcohol formation along with insignificant yeast growth at the final stage
(48-72 h).
 Ethanol
• Made since ancient times by fermentation of sugars (beverage ethanol is still made by this
process: simple sugars are the raw material, and Zymase an enzyme from yeast, changes the
simple sugars into ethanol and carbon dioxide by this rxn:)
• C6H12O6 ---> 2 CH3CH2OH + 2 CO2
Biosynthesis of ethanol
 Ethanol
Uses of Ethanol:
(i) Use as a chemical feed stock : In the chemical industry, ethanol is an intermediate in many
chemical processes because of its great reactivity. It is thus a very important chemical feed
stock. it tis used for sterilization process in industries and laboratory. Sterilizing agent
(ii) Solvent use : Ethanol is widely used in industry as a solvent for dyes, oils, waxes,
explosives, cosmetics etc.
(iii) General utility : Alcohol is used as a disinfectant in hospitals, for cleaning and lighting in
the home, and in the laboratory as a solvent.
(iv) Fuel : Ethanol is mixed with petrol or gasoline upto 10% and known as gasohol and used in
automobiles.
 Beer
•Brewing is the process of production of malt beverages. Beers, ale and lagers are the main
malt beverages produced by a method called brewing.
•Brewing is a complex fermentation process.
•It differs from other industrial fermentation because flavor, aroma, clarity, color, foam
production, foam stability and percentage of alcohol are the factors associated with finished
product.
•The starting substrate is barley malt which is germinated barley grains that are dried and
powdered.
•Barley malt contains a mixture of amylases and proteinases.
•Production of beer from entirely malted barley is done in some European countries .
•In most of the places beer is made from malted barley supplemented with corn, rice, or
wheat as adjuncts. These are contains carbohydrate for ethanol productions
 Beer
Types of beer
•Top fermented beer (yeast at the top)
•Bottom fermented beer (yeast at the bottom)
Process of beer production
•Malting
•Cleaning and milling
•Mashing
•Mash operation
•Wort boiling
•Fermentation
•Laggering
•Packaging
 Beer
Malting
❖ The process of preparing malt is called malting.
❖ Malting is prepared from barley, wheat and oats.
❖ The grains are cleaned free from dust and foreign materials.
❖ Malting process is carried out in malt house ( special are earmarked for the purpose)
❖ Cleaned barley grains are soaked in water for 12 hours water is drained and sprouting is
allowed to continue for 5-7 days until desired growth of embryo is attained.
❖ During this time the grains have to be frequently turned for aeration and for uniform
germinations.
❖ At the end of the process malt is killed to prevent the further growth.
❖ Proper humidity and temperature are maintained in malt house to produce good quality
malt
 Beer
Malting
❖ Malt acquires during germination process, enzyme which degrades starch (amylases)
❖ Purpose of malting is to develop amylases and proteases in grain
❖ These enzymes are produced in germinating barley to enable it to break carbs and proteins
present in grain
❖ This germination is halted by warm air and at this step α-amylase and β-amylase are
produced.
Cleaning and milling
•Germinating barley is transferred to the brewing tower.
•At the top of this malt added where its cleaning is done.
•Fermentation will occur at the bottom
•Any dust, metal or impurity will be separated
 Beer
Mashing
•Central part of beer production
•It determines the type of wort present also determine the type of beer
•2 parts of grains (starch 55%, proteins 10-12%) are break during the mashing process
•During the mashing process malt is mixed with water to allow the amylases present in malt
to hydrolizes starch and other polysaccharides to simple sugars (maltose and dextrin)
•The adjuncts are corn and rice starch which are boiled in water for gelatinization.
•Maltose gets fermented into alcohol and carbon dioxide during fermentation with yeast
•Dextrins and malt proteins remain to give the body of beer and impart the typical foam and
flavor to beer.
•The starch breakdown into α & β-amylase
 Beer
Mashing
•Protein breakdown done with the help of protease enzyme, peptones and polypeptides are
converted into amino acids
•After completion conversion of starch mash is heated to a temperature of 70 ℃ to arrest all
enzymatic activity.
•Mash is allowed to stand for a few hours. The insoluble materials in mash settle down
leaving a clear liquid above called wort
•Alternatively insoluble materials may be removed by filtration.

Boiling of wort
The filtrate is then boiled with stirring for 2-3 hours and hop flowers are added at various
interval during boiling.
 Beer
•Reasons for boiling of wort:
• For extraction of hop flavor from hop flower
• Boiling coagulate remaining protein and partially hydrolyze protein and help in removal
of protein
• Boiling inactivates enzymes that were active during mashing, otherwise causes
caramelization of sugar
• Boiling also sterilize and concentrate the wort
Hops:
•Hops are dried female flower of hop plant Humulus lupulus. Approximately one quarter
pound of hop flower is added per barrel of beer
Advantages of hop addition in beer are;
• Provide beer with its pungent and aromatic character
• Provide tannin which helps in coagulation of remaining protein
• Contains α-resin and β-resin which gives bitter flavor as well as preservative action
against gram Positive bacteria
• Contains pectin which is responsible for foam characteristic of beer
 Beer
Brewing process
•Beer production utilize strain of Saccharomyces carlsbergens and S. varum which are bottom
yeast and S. cerevisiae which is a top yeast.
•Yeast cells for inoculation are usually recover from previous fermentation tank by treatment
with phosphoric acid, tartaric acid or ammonium persulphate to reduce the pH and removed
considerable bacterial contamination.
•Fermentation is usually carried out at 3-4 °C but it may range from 3- 14° Fermentation
usually completes in 14 days.
•During fermentation yeast converts sugar mainly into ethanol and CO2 plus some amount of
glycerol and acetic acid.
 Beer
•For fermentation open tank fermenter can be used however closed fermenter tank is
preferred, so that CO2 liberated during fermentation can be collected for later carbonation
step.
•CO2 evolution is maximum by fifth day of fermentation, there is no evolution of CO2 by 7-9
days because yeast cells become inactive and flocculate.
•Most beer contains 3.5-5% alcohol.
Flow chart in beer processing
Step1: Malting ( soaked barley is allowed to sprout for 5-7 days and then killed
Step2: mashing ( malt is ground and supplemented with adjuncts, rice and corn starch)
Step 3: Preparation of Wort (after enzymatic degradation of starch by amylases in malt, the
liquid containing maltose and dextrin's is wort . Wort is separated by sedimentation or
filtration
 Beer
•Step 4 – brewing ( wort is subjected to fermentation by selected strains of yeast; hops added
for unique flavor of beer fermentation temperature is 6- 12 ℃
•Step 5- carbonization (Final beer is filtered, bottled and injected with Carbon dioxide
•Step 6 – pasteurization (pasteurization at 60 – 61 ℃ is necessary to prevent souring of beer
•Step 7 –Aging ( Keeping the beer for 2 weeks to several months is necessary to get improved
flavor
Ale
Ale is somewhat similar to beer but the yeast used is Saccharomyces cerevisiae.
The fermentation is carried out at temperature ranging from 12 to 25℃ for 5 – 7 days.
The yeast cells do not settle to the bottom but are carried upward.
Higher amount of Hops is added to ale than beer
The concentration of alcohol is higher compared to beer.
 CITRIC ACID
❖ Citric acid is the most important organic acid produced in tonnage and is extensively used
in food and pharmaceutical industries.
❖ It is produced mainly by submerged fermentation using Aspergillus niger or Candida sp.
from different sources of carbohydrates, such as molasses and starch based media.
❖ However, other fermentation techniques, e.g. solid state fermentation and surface
fermentation, and alternative sources of carbon such as agro-industrial residues have been
intensively studied showing great perspective to its production
❖ Citric acid is formed as an intermediate in the Kreb’s cycle, but it is accumulated in greater
quantities in the fungus Aspergillus niger, may be due to metabolic abnormality.
❖ Citric acid is the most important commercial product which is found in almost all plant and
animal tissues.
 CITRIC ACID
❖ The molecular formula of citric acid is C6H8O7, 2-hydroxy-1,2,3-propane tricarboxylic acid.
❖ It is widely used organic acid in the field of food (60%) and pharmaceuticals (10%).
Microorganism used for Citric Acid Production
❖ A large number of micro-organisms have been employed to produce citric acid. These
include bacteria, fungi, and yeasts. But A.Niger and saccharomycopsis sp. are employed
for commercial production because it has several advantages.
Advantages of using this micro-organism are:
•Its ease of handling.
•Its ability to ferment a variety of cheap raw materials.
•It provides high yields.
 CITRIC ACID
Yeasts
•Candida tropicalis
•C.oleophila
•C.guilliermondii
•C.Citroformans
•Hansenula anamola
•Yarrowia lipolytica
Bacteria:
•Bacillus licheniformis
•Arthrobacter paraffinens
•Corynebacterium species
Fungi:
•Aspergillus nagger
•A.aculeatus
•A.awamori
•A. carbonarius
 CITRIC ACID
Steps Surface culture method
Selection of fungal strain
✓ High yield strain of Aspergillus niger is selected.
✓ The selected strain should not produce other organic acid such as oxalic acid.
✓ It should be fast growing and should have uniform biochemical characteristics.
Preparation of inoculum
✓ Spores from the stock cultures are transferred to a solid sporulation medium and
incubates at 25℃ for 10 -14 days
✓ From the cultures, spore suspension is obtained by suspending the harvested spores in a
proper diluent such as water with a wetting agent.
✓ The spores suspension is transferred to a fermenting medium so that the spores are kept
floating on the surface.
 CITRIC ACID
Fermenting medium and fermentation process
❖ A typical medium for citric acid fermentation contains sugar or molasses, ammonium
nitrate, magnesium sulphate and potassium phosphate
❖ A low pH is maintained (around 2.2-3.5)
Recovery of citric acid
❖ After the fermentation process, the product has to be recovered.
❖ The fermentation broth containing citric acid is filtered to remove mycelia and other
debris.
❖ The mother liquor is treated with small amounts of hydrated lime (calcium hydroxide) with
heating to 80-90 ℃ to allow precipitation of oxalic acid.
❖ Citric acid is then precipitated as calcium citrate.
❖ The precipitate is washed several times with water and it is treated with sulphuric acid.
 CITRIC ACID
❖ The solution is filtered to remove CaSO4.
❖ The mother liquor containing citric acid is then decolorized by charcoal and passed
through ion exchange resin columns.
❖ The liquid is concentrated in vaccum and finally crystallized.
The fermentation can also be carried out by submerged cultures method with continuous
aeration. The citric acid is finally recovered as calcium citrate crystals.
B)Submerged fermentation process
❖ In this process, fermentation is carried out in large stirred tank fermenter.
❖ The fungus Aspergillus niger is grown the same way for inoculum production and spore
suspension is transferred to the fermenter with sterilized liquid medium for the same
composition as mentioned for surface cultivation.
 CITRIC ACID
❖ Aeration is the key factor with increase in aeration growth rate and product formation can
be enhanced up to optimum level
C) Solid state fermentation
❖ In this process, the fungus is grown in a medium coated on to a solid carrier. Here, increase
in surface area results in increased aeration.
❖ However the growth of the fungus will be on the surface of the solid carrier only, and
product formation will be comparatively poor.
Industrially, citric acid is used in the following ways:
1. It is used in the production of carbonated beverages
 CITRIC ACID
2. As a chelating and sequestering agent in the tanning and textile industry.
3. Citrate esters are used as plasticizer.
4. It is abundantly used in food industry as an acidulent in the preparation of food items like
jams, preserved fruits and fruit juices etc.
5. It is used in frozen foods to prevent its change in colour and flavour.
6. Metal painting industry
7. In pharmaceutical industry
8. In the manufacture of astringent, hair rinser and hair setting fluids.
9. In beverage industry as a preservative to prevent oxidation of alcohol, emulsifier of dairy
products like cheese and ice creams.
10. It is used as preservative and to prevent change in colour, flavour and in the oxidation of
alcohol.
 STEPS IN CITRIC ACID
❖ Stock culture of Aspergillus niger
❖ Transfer to sporulation medium
❖ Spore suspension
❖ Transfer to fermentation medium
❖ Fermentation on surface for 7-10days at 25-30
❖ Settling tank-Separation of mycelia
❖ Broth transferred to precipitator and separator
❖ Filter and transfer to Acidulator (Add sulphuric acid)
❖ Filter CaSO4
❖ Concentrator
❖ Purifier (Add charcoal /Ion exchanger)
❖ Crystallizer
❖ Drier
❖ Pure citric acid
 Penicillin
❖ Penicillin belongs to a group of antibiotics generally called as penicillin's, derived
directly from Penicillium or through chemical modifications
❖ These are all effective against gram positive bacteria.
❖ All penicillin have the basic structure called beta lactam ring, and therefore they are
called beta lactam antibiotics.
a) Selection of the fungus and strain development
✓ The species of penicillin selected for commercial production should be able to
produce high yield of penicillin.
✓ By the process of mutation selection high yielding strains of Penicillium
chrysogenum have been produced for commercial production.
 Penicillin
b) Culture maintenance
As the mutant high yielding strains are not very stable they have to be preserved carefully.
Otherwise they will lose their characteristics
1) Spore suspension is mixed with sterile finely divided soil or sand and desiccated when
there is no moisture, growth and metabolism will be arrested the cells will remain as they
are without change.
2) Spores are lyophilized or freeze dried
3) Spore suspension is frozen and stored under liquid nitrogen.
C) Inoculum preparation
❖ For inoculum preparation, the primary inoculum is spores stored in soil or in freeze dried
condition.
❖ The spores are placed in solid medium because this medium supports good sporulation
 Penicillin
d) Production medium
❖ From this spores are harvested and transferred
Component Quality (%)
to shake cultures and then to inoculum
production tanks Glucose or Molasses 10
Corn steep liquor 4
❖ Inoculum is raised in wheat bran nutrient
Phenyl acetic acid 0.50
solution by allowing the cultures to incubate for Potassium hydrogen 0.40
1 week at 24 ℃ phosphate
❖The cultures raised in flasks are transferred to Edible oil (vegetable 0.25
inoculum tanks and aerated for 1-2 days to oil)
produce more inoculum with heavy mycelia Calcium carbonate 1
growth.
pH:5.5-6.0
❖After desirable growth is achieved this can be
used as a inoculum for penicillin production
 Penicillin
✓ Phenylacetic acid is the percussor used for the synthesizing the benzene ring side chain of
penicillin-G
✓ The addition of the percussor stimulates the fungus to produce more Penicillin G
✓ The pH of the medium is adjusted to 5.5-6.0 and the temperature is maintained at 25-26 ℃
e)Fermentation Process
✓ The production is carried out in huge production tank which are well aerated and the fungus
grows as a submerged cultures mostly as hyphal balls
✓ The fermentation is allowed to go on for 7 days.
✓ During the first day of fermentation mycelia growth takes place with utilization of
carbohydrates.
✓ Reduction in the carbohydrate concentration leads to a condition favouring penicillin
 Penicillin
✓ production from the second day, and the penicillin production continuous from second day
at this seventh day.
✓ The pH rised to 8.0 as the fermentation goes on and penicillin production at this stage.
F)Antibiotic recovery
❖ When the maximal concentration of penicillin that can be produced is reached, the
medium containing penicillin is separated from mycelia using rotating vacuum filter.
❖ The fungal biomass is separated and used as an animal feed supplement
❖ Penicillin is extracted from the broth through an organic solvent (Amyl acetate or Butyl
acetate).
❖ From this antibiotic is extracted back into water (from the organic solvent) by adding
enough potassium hydroxide.
 Penicillin
❖ Penicillin G is finally obtained as a potassium salt.
❖ Final product is dried and packaged.
Flow chart of penicillin production
Step 1: Culture of Penicillin chrysogenum
Step 2: transfer to culture flask
Step 3: Transfer to inoculum production tank (seed tank)
Step 4: Filter through rotatory filter
Step 5: First extractor Step 6: Second extractor
Step 7: Third extractor
Step 8: Third extractor
Step 9: ceramic filter
Step 10: Vacuum drier
 Penicillin
Step 11 : Packaging into Vials

Successful production of
penicillin depends on the
following criteria
1) Selection of the fungus and
strain development
2) Culture maintenance
3) Incolumn preparation
4) Production medium
5) Fermentation process
6) Antibiotic recovery
 L-Glutamic acid or (L-Glutmate)
❖ L-Glutamic acid was the first amino acid to be mass produced through microbial fermentation

❖ In this form of its salt L-glutamate.

❖ Even though Corynebacterium glutamicum was the first bacterial species to be used for this

purpose, later several other bacterial species have been harnessed for commercial production

❖ Species of Micrococcus, Arthrobacter and Brevibacterium are also used for industrial

production of L glutamic acid and monosodium glutamate (MSG)

❖ Corynebacterium glutamicum and Brevibacterium flavam are widely used in the large scale

production of MSG

❖ Production of glutamic acid in large quantities is now carried out using mutant of
 L-Glutamic acid or (L-Glutmate)
❖ Corynebacterium glutamicum that have limited ability to process the citric acid cycle
(Krebs cycle) intermediate alpha ketoglutarate to succinyl coenzyme A.
Production strains
❖ For efficient production any products has to be released from the cell and should not
remain intracellular.
❖ The amino acid is generally retained in the cell or on the cell membrane
❖ In the discovery production of L glutamate from C. glutamicum, it was shown that L-
glutamate was released into the medium when the cells were limited by biotin (biotin
starvation)
 L-Glutamic acid or (L-Glutmate)
Medium and production process
❖ The relevant factors influencing L-glutamate formation are ammonium concentration,
dissolved oxygen concentration and pH.
❖ Ammonium is added in low concentration in the beginning of fermentation and then
added continuously during the course of fermentation
❖ Th medium consists of carbohydrates generally glucose, peptone, inorganic salts eg:
ammonium chloride and biotin.
❖ The concentration of biotin has a significant influence on the yield of glutamic acid
❖ Alpha ketoglutaric acid produced via the tricarboxylic acid (Krebs cycle) is the precussor of
L-glutamic acid.
❖ The pH of the medium has to be well aerated, but well controlled as excessive oxygen may
result in the accumulation of alpha ketoglutarate
 L-Glutamic acid or (L-Glutmate)
❖ L glutamic acid produced inside cells may be released to the medium by addition of
penicillin or surfactant (Tween)mor by other methods which makes the cells leaky.
❖ At the end of fermentation process, L –glutamate is in the form of ammonium salt
Purification process
❖ The cells are separated from the broth and the clear broth containing L-glutamate is passes
through basic anion exchange resin.
❖ L glutamate will be bound to the resin and ammonia is released.
❖ Ammonia can be recovered through distillation.
❖ Elution of basic anion exchange resin with sodium hydroxide will result in MSG.
❖ From the eluate, MSG can be crystallized at a specific pH
 Vitamin B12
❖ Among vitamins of B complex only two vitamins are produced thorough microbial
fermentation they are vitamin B12 and riboflavin (vitamin B2)
❖ Vitamin B12 (cyanocobalamin) is one of the most essential vitamins produced through
microbial sources.
Chemical structures
✓ Vitamin B12 is not a single compound but is a group of closely related cobamide which
shows varying effect on animal growth.
✓ It is also known as cyanocobalamin and consists of a cobamide linked to a nucleotide.
✓ The nucleotide is a typical in having 5,6,-dimethyl benzimidazole as its base instead of
purine or pyrimidine base.
✓ The cobamide molecules has a central atom of cobalt linked to a cyanide group.
✓ This is surrounded by four reduced pyrrole rings to form a macro ring.
 Vitamin B12
✓ A number of carbon atoms carry methyl or other substituent groups.
Production of vitamin B12
The organism
• Vitamin B12 is produced by bacteria and actinomycetes. Streptomyces olivaceus,
Pseudomonas denitrificans, Propionibacterium shermanii and Propionibacterium
freudenreichii are mainly used for commercial production of vitamin B12.
• Generally, vitamin B12 is prepared by the submerged culture process.
• Vitamin B12 can be produced by a large number of bacteria such as Bacillus megaterium,
B.coagulans, Psedomonas dentirificans, Propionibacterium shermanii. It is a by product of
streptomycin fermentation and also Aureomycin fermentation by streptomyces species.
• However Streptomyces olivaceus is normally used for large scale production of vitamin B12
Vitamin B12
 Vitamin B12
Inoculum preparation ❖ The composition of
❖ From a pure culture of Streptomyces olivaceus a small fermentation medium is as
portion is transferred to inoculum medium is a 250 ml flask. follows:
The medium is generally Bennett’s medium.
Ingredients Percentage (%)
❖ The flask are kept in mechanical shaker and incubated at a Distiller’s solubles 4
(Soybean meal;
temperature of 27℃ until thick growth is visible. meal)
Dextrose 5-10
❖ The culture thus raised is used for inoculating the
CaCO3 0.5
fermenting medium in the fermenter. CoCl3.6H2o 1.5-10

Fermenting medium
❖ A cobalt salt in the form of cobalt chloride (CoCl3) is added
to the fermentation medium as a percussor to increase the
yield of vitamin B12
Fermentation Process Vitamin B12
❖ After proper sterilization of the fermentation medium in the fermenter, the inoculum
suspension is added to initiate fermentation.
❖ The pH of the medium has to be maintained at 7.
❖ The pH rises after 2 days of fermentation due to lysis of the cells of the organism.
❖ The pH can be reduced by the addition of required amount of dilute sulphuric acid
❖ The temperature of the fermenting broth is maintained at 27℃ for optimum production
of vitamin B12. Duration of the fermentation is about 4 days
❖ Aeration and agitation of the fermenting broth is very important
❖ However aeration rated higher than optimum will result in foaming.
❖ Optimum aeration is 0.5 volume of air/1 volume of medium/minute.
❖ Addition of antifoam agents such as soybean oil, corn oil, lard oil or silicones.
Product recovery Vitamin B12
❖ After removing of cyanocobalamin from the adsorbents it is treated with water acetone
to forma an organic solvent of vitamin B12. From the organic solvent it can be
crystallized.
 STEROID BIOTRANSFORMATION
❖ Steroids are biologically active compounds similar to hormones
(often called steroid hormones) produced by testes, ovary
adrenal cortex, placenta.
❖ They are derived form cholesterol.
❖ Cortisone has been found to relieve pain caused by theumatoid
arthritis.
❖ Various other derivatives of cortisone are useful in alleviating
allergic and inflammatory responses of human body (anti-
inflammatory drugs)
❖ Steroids are organic compounds characterized by the presence of
steroid nucleus or steroid carbon Skelton composed of four rings
 STEROID BIOTRANSFORMATION
❖ The most common locations of functional group are C-3,C-
4,C-7, C-11, C-12 and C-17.
❖ The structures has thus six asymmetric carbons, which
provides for many possible stero isomers, and this provides
for the varied functions of steroid hormones.
❖ Additional C atom (18 to 27 )may be present depending on
the side chains.
❖ For estane is a C-18 steroid (where C-19 CH3 group is absent)
❖ Cholestane is C-27 steroid where R is CH3 CH3
❖ CH CH2 CH2 CH2 CH
❖ CH3
STEROID BIOTRANSFORMATION
 STEROID BIOTRANSFORMATION
MECHANISM OF MICROBIAL STERIOD TRANSOFRMATION
❖ The major difficult in chemically synthesizing cortisone is the need to insert an oxygen
atom at the 11th carbon of the steroid ring.
❖ This can be easily accomplished by a microorganism such as the fungus Rhizopus nigricans
hydroxylates progesterone forming a steroid with the introduction of the OH group at the
11th position.
❖ This remarkable ability of the fungus is to introducer oxygen at C 11 has enabled
microbiologist and chemist to produce cortisone and hydrocortisone economically .
❖ The fungus Cunninghamella blakesleeana can hydroxylate cortexolone to form 11-
hydrocortisone
❖ The use of microorganism in steroid hormone production has reduced the cost of these
hormones by about 400 times.
STEROID BIOTRANSFORMATION
 STEROID BIOTRANSFORMATION
❖ Scientist have screened large number of fungal cultures for their ability to
monohydroxylate steroid (hydroxylate carbon at a single point in the molecule) at
unusual sites, have identified several fungal strain capable of dehydrogenation ring B of
progesterone and androstenedione at position C6 and C7.
❖ Microbiological dehydrogenation at this site has only been recently reported.
❖ The structure of the metabolite isolated from progesterone and the producing fungi are
Compound Producing fungi
6-dehydroprogesterone Botryodiplodia theobromae
11 α hydroxy-6-dehydroprogesterone Botryosphaerica obtusa, Mucor
racemosus and Nigrospora sphaerica
12 α, 15β and 16 α hydroxy -6- Botryosphaerica obtusa
dehydroprogesterones
4 α hydroxy-6-6-dehydroprogesterones Apiocrea chrysosperma
 STEROID BIOTRANSFORMATION
❖ Microbial biotransformation of steroids takes
place by the following mechanism
1) Hydroxylation
Microbial hydroxylation of steroid molecule involves
the replacement of the hydrogen atom in the given
carbon with hydroxyl group. For example species of
Rhizopus nigricans and Aspergillus ochraceus covert
progesterone into 11 α hydroxyprogesterone
 STEROID BIOTRANSFORMATION
2)Dehydrogenation
Bacteria and fungi are capable of dehydrogenizing the secondary alcohol groups of
steroids with the help of enzyme dehydrogenase
C1 dehydrogenation results in a double bond between C1 and C2.
Example is the conversion of cortisone into prednisone by the bacteria Corynebacterium
simplex and Streptomyxa affinis . The enzyme involved in C1 dehydrogenase .
STEROID BIOTRANSFORMATION
 STEROID BIOTRANSFORMATION
3) Hydrogenation
Hydrogenation includes addition of hydrogen to specific side chains eg: conversion of =O
to –OH. Example is the conversion of prednisone to cortisone by the bacterium , Bacillus
megaterium.
 STEROID BIOTRANSFORMATION
4) Epoxidation
Epoxidation takes place during the conversion of 11-deoxycorticol into corticol by fungi
Cunninghamella blakesleeana and Curvularia lumata
Production process and product recovery
❖ In the production process employed in industry steroid to be transformed into another
is added to culture of the fungus which has been allowed to grow for a day and has
achieved a good biomass.
❖ The usual substrate necessary for the fungal growth are provided in the fermentation
tank for achieving the initial biomass.
❖ The fermenter has to be aerated properly.
STEROID BIOTRANSFORMATION
 STEROID BIOTRANSFORMATION
❖ After the growth of the fungus for a day or two the steroid eg: progesterone is
added dissolved in a water miscible solvent such as acetone, alcohol or propylene
glycol. The solvent should be in small quantities to avoid toxicity to the fungus.
❖ The transformation may require several hours to day depending on the type of
transformation.
❖ The product is extracted with a suitable solvent such as methylene chloride or
chloroform
❖ It is purified by column chromatography and crystallized.