This document discusses the production of vitamin B12 and riboflavin through microbial fermentation. It outlines the key microorganisms used, including Streptomyces griseus and Streptomyces olivaceus for vitamin B12 production, and Ashbya gossypii for riboflavin production. The document also provides an overview of the fermentation process, noting that it is carried out through submerged fermentation over 3-5 days, requiring aeration and agitation of the growth medium. Details are given on inoculum preparation and scaling up for vitamin B12 production using S. olivaceus.

1. FERMENTATION
PRESENTING BY:
Mr. Purushotham K N
Assistant Professor
Department of Pharma.Chemistry
SACCP
2022-2023

2. FERMENTATION
• The word Fermentation is derived from Latin word fervere which means to boil.
• But the conventional definition of Fermentation is to break down of larger molecules
into smaller and simple molecules using microorganisms.
• In Biotechnology, Fermentation means any process by which microorganisms are
grown in large quantities to produce any type of useful materials.
• In other words fermentation may be define as the process of growing a culture of
organisms in a nutrient media and thereby converting feed into its desired end product.
Its is sometimes described as biochemical reaction in which microorganisms serve
(bacteria or fungi) as biocatalyst.
Any metabolic process that releases energy from a sugar or other organic molecules,
does not require oxygen or an electron transport system, and uses an organic molecule as
the final electron acceptor.

3. • This process is carried out in an equipment called as fermentor.
• Fermentor can be defined as a vessel in which sterile nutrient media and pure
culture of microorganism are mixed and fermentation process is carried out under
aseptic and optimum condition.
• The fermentor provides sterile environment, an optimum condition that are
important for microorganism to grow.

4. TYPES OF INDUSTRIAL FERMENTATION
1. Batch Fermentations
2. Continuous Fermentations
3. Aerobic Fermentations
4. Anaerobic Fermentations

5. BATCH FERMENTATION:
• A tank of fermenter is filled with the prepared mash of raw materials to be
fermented. 'The temperature and pH for microbial fermentation is properly
adjusted, and occasionally nutritive supplements are added to the prepared mash.
The mash is steam-sterilized in a pure culture process.
• The inoculums ofa pure culture is added to the fennenter, are taken out for further
processing.
• The fermenter is cleaned and the process is repeated. Thus each fermentation is
discontinuous process divided into batches.

6. CONTINUOUS FERMENTATION
• Growth of microorganisms during batch fermentation confirms to the
characteristic growth curve, with a lag phase followed by a logarithmic phase. is
because of limitation of one or more of The essential nutrients. 'This , in turn, is
terminated by progressive decrements in the essential nutrients.
• In continuous fermentation, the substrate is added to the fermenter continuously at
a fixed rate. 'This maintains the organisms in the logarithmic growth phase .

7. AEROBIC FERMENTATION:
• A number of industrial processes, although called "fermentations", are carried out
by microorganisms under aerobic conditions.
• In older aerobic processes it was necessary to furnish a large surface area by
exposing fermentation media to air.
• In modern fermentation processes aerobic conditions are maintained in a closed
fermenter with submerged cultures.
• The contents of the fermenter are agitated with auimpeller and aerated by forcing
sterilized air.

8. ANAEROBIC FERMENTATION:
• Basically a fermenter designed to operate under micro aerophilic conditions will
be the same as that designed to operate under aerobic conditions, except that
arrangements for intense agitation and aeration are unnecessary.
• Many anaerobic fermentations do, however, require mild aeration for the initial
growth phase, and sufficient 'N agitation for mixing and maintenance of
temperature.

9. Penicillium chrysogenum
• Also known as Penicillium notatum.
• It is common in temperate and subtropical regions and can be found on salted food
products, but it is mostly found in indoor environments, especially in damp or
water-damaged buildings.
• It is the source of several ß-lactam antibiotics, most significantly penicillin which
inhibits the biosynthesis of bacterial cell walls affecting lysis of the cell.

10. Classification
• Kingdom: Fungi
• Division: Ascomycota
• Class: Eurotiomycetes
• Order: Eurotiales
• Family: Trichocomaceae
• Genus: Penicillium
• Species: Chrysogenum
• Cell structure
• Penicillium chrysogenum exhibits typical eukaryotic cell structure; it has a tubulin
cytoskeleton which is used for motility.

11. In P. chrysogenum, the conidia are blue to blue-green.
These conidia are the cause of pathogenicity in humans as in the cases of allergy and
endophthalmitis.
The conidia originate from complexes known as conidiophores.
The growth of conidiophores begins when a stalk sprouts out of a foot cell.
The stalk swells at the end and forms a vesicle. Sterigmata form from the vesicle
which give way to long chains of conidia.

12. Application to Biotechnology
• It produces the hydrophobic ß-lactam compound penicillin.
• Penicillium chrysogenum remains the primary producer of Penicilian G and
Penicilian V
• P. chrysogenum has been used industrially to produce Penicilian G and Penicilian
V and Xanthocillin X, and to produce the enzymes polyamine oxidase,
phosphogluconate dehydrogenase, and glucose oxidase.
• Penicillium chrysogenum can be used to assist crops to fight off other pathogenic
species.

13. Production of penicillin:
P. chrysogenum is high yielding strain and therefore most widely used as production
strain.
Inoculum Preparation:
Purpose is to develop a pure inoculum in an adequate amount.
To do so various sequential steps are necessary like:
1 A starter culture is needed for inoculation.
2 After getting growth on solid media, one or two growth stages should allowed in
shaken flask cultures to create a suspension, which can be transferred to seed tanks
for further growth.
3 After about 24-28 hours, the content of the seed tanks is transferred to the primary
fermentation tanks.

14. • All the bio parameters like temperature, PH, aeration,agitation etc. should be
properly maintained.
Bio parameters
• PH: near 6.5
• Temperature: 260C to 280C
• Aeration: a continuous stream of sterilized air is pumped into it.
• Agitation: have baffles which allow constant agitation (200rpm).

15. Nutrient media
Fermentation broth contains all the necessary elements required for the proliferation
of the microorganisms.
Generally, it contains a carbon source, nitrogen source,mineral source, precrsors and
antifoam agents.
Carbon Source Lactose in concentration of 6%.
Other carbohydrates like glucose & sucrose.
Complex as well as cheap sources like molasses, orsoya meal can also be used
which are made up of lactose and glucose sugars.

16. • Nitrogen Source
• Ammonium salts such as ammonium sulphate,
• ammonium acetate, ammonium lactate or ammonia gas
• are used for this reason.
• Sometime corn steep liquor may be used.
• Mineral Source
• These elements include phosphorus, sulphur,
• magnesium, zinc, iron, and copper which generally
• added in the fom of water soluble salts.
• Precursors
• Various types of precursors are added into production
• medium to produce specific type of penicillin.

17. For example, if phenyl acetic acid is provided then only penicillin-G will be produced
but if hydroxy phenyl acetic acid is provided then penicillin-X will be produced.
Phenoxy acetic acid is provided as precursor for penicillin-V production.
corn steep liquor is provided as nitrogen source, When it also provides phenyl acetic
acid derivatives; therefore it is widely used in the production of penicillin-G.
Anti-foam agents
Anti-foaming agents such as lard oil, octadecanol and silicones are used to prevent
foaming during fermentation.
Recovery
The recovery of penicillin is carried out in three
successive stages:
1. Removal of mycelium
2. Counter current solvent extraction of penicillin
3. Treatment of crude extracts

18. At harvest the fermentation broth is filtered on a rotatory vacuum filter to remove the
mycelium and other solids.
Phosphoric or sulfuric acids are added to lower the pH (2 to2.5) in order to transform
the penicillin to the aniomc form.
Then the broth is directly extracted in a Podbielniak Counter Current Solvent Extractor
with an organic solvent.
such as methyl isobutyl ketone, amyl acetate or butyl acetate.
Penicillin is then again extracted into water from the organic solvent by adding an
adequate amount of potassium or sodium hydroxide to form a salt of the penicillin.
The resulting aqueous solution is again acidified & re-extracted with methyl isobutyl
ketone.

19. This shifts between water and solvent help in purification of the penicillin.
The solvent extract is carefully back extracted with NaOH and from this aqueous
solution; various
procedures are utilized to cause the penicillin to crystalize as sodium or potassium
penicillinate.
The resulting crystalline penicillin salts are then washed and dried.
Sometimes the crude extract of penicillin is passed out from charcoal treatment to
eliminate pyrogens; even sterilization can also be done.

20. Flow diagram for Penicillin G Production
Master stock of P. chrysogenum
Sporulation (flask culture)
Spore suspension
Germination /seed tank
Production fermenter
Filtration
Filtrate

21. Streptomycin
Streptomycin is an antibiotic
It was discovered by Schatz, Bugie and Waksman (1944) in one of the soil isolates
Streptomyces griseus.
Most of the industry used this parent strain for streptomycin production today also.
Mutation and selection employed to increase yields to the present day levels.
But,nowadays also Streptomycin is produced by Streptomyces griseus and only a
few strains of this organism have the ability to produce reasonable yields of the
antibiotic.
It is active against gram Positive and Negative bacteria and against the tuberculosis
organism Mycobacterium tuberculi.

22. Uses
• It has been used therapeutically in the treatment of infections caused by organisms
resistant to Penicillin
• It is also used in the treatment of plant diseases caused by bacteria
• It is used in the treatment of tuberculosis caused by Mycobacterium tuberculi.

23. Chemical structure:
• Streptomycin and dihydrostreptomycin are basic compounds.
• They are usually prepared as salts.
• Streptomycin is available as the hydrochloride C21H39N202.3HCL as a
crystalline hydrochloride double salt with calcium chloride or as the
• phosphate or sulphate Dihydrostreptomycin as the hydrochloride or Sulfate.
• Depending on the strain of this organism being used or on the production of
medium,small amounts of mannosidostreptomycin or hydroxy streptomycin are
accumulated in addition to streptomycin
• Some mannosido streptomycin is produced early in the fermentation, but this
antibioticis largely enzymatically degraded by Streptomyces griseus to
sterptyomycin at the time of harvest.

24. • the mannosidostreptomycin is not desired because of its low antibiotic activity.
• The use of precursors does not increase yields of streptomycin.
• Most of the carbon of the streptomycin molecule has shown to originate from
glucose and not from the more complex carbon compounds of the medium ,
although some of the carbon molecule originate from carbon di oxide.
• The carbonyl function on the streptose moiety is involved in the antibiotic activity
of streptomycin.
• Most chemical additions to the carbonyl group destroy the antibiotic activity.

25. Production —medium
Two types of medium were used
1. Woodruff and Mc. Daniel (1954)
1% soyabean meal
1% glucose
0.5% sodium chloride
2. Hockenhull (1963)
2.5% glucose
4% soyabean meal
0.5% distillers dried soluble
0.25% sodium chloride
pH -7-3 to 7-5 before sterilization

26. Inoculum preparation
High yielding mutated strains of Streptomyces griseus are genetically unstable , a
fact to be considered in maintenance of stock cultures.
Because of this consideration, spores of the organism usually are maintain soil
stocks or are lyophilized in a carrier such as sterile skim milk .
Spores from these stock cultures are then transferred to a sporulation medium to
sporulated to initiate liquid culture buildup of growth
mycelial inoculum in flasks or inoculum tanks.

27. STREPTOMYCIN PRODUCTION
Commercial streptomycin fermentation passes through three phases:
I phase: lasts approximately 24hours.
With rapid growth of the mycelium.
Proteolytic activity of streptomyces griseus releases ammonia to the medium from
the soyabean meal.
Carbon nutrients of the medium utilized for the growth
Glucose of the medium is utilized slowly during this period
Only slight streptomycin production occurs
During this period, the pH of the medium rises from approximately 6.7 or 6.8 to 7-5
or slightly higher

28. II phase
Streptomycin is produced at high rate
Lasts approximately 24 hours to 6 or7 days of incubation
Almost, no mycelium growth, weight of the mycelium remains constant
The ammonia is utilized and the pH remains fairly constant in a range of
approximately about 7.6-8
Glucose and oxygen are required in a large quantity.

29. III phase
• Sugar has been depleted from the medium
• Streptomycin production ceases
• Mycelium undergoes autolysis,releasing ammonia and the pH value rises
• The fermentation , however usually is harvested before cell lysis.

30. Harvest and Recovery of streptomycin
After completion of fermentation the mycelium is separated from the broth by
filtration. Streptomycin is recovered by several methods. The choice of procedure
depending on the industrial concern.
In one procedure,
The streptomycin is adsorbed from the broth onto the activated carbon and then
eluted from the carbon with dilute acid. The eluted streptomycin is precipitated by
acetone, filtered and dried before further purification.

31. In an alternative procedure,
The fermentation broth is acidified, filtered and neutralized. It is then passed
through a column containing a cation exchange resin to adsorb the streptomycin
from the broth.
The column is then washed with water and theantibiotic is eluted with hydrochloric
acid or cyclohexanol or phosphoric acid. It is then concentrated at about 600C under
vacuum almost to dryness.
The streptomycin is then dissolved in methanol and filtered and acetone is added to
the filtrate to precipitate the antibiotic. The precipitate is again washed with acetone
and vacuum dried.
It is purified further by dissolving in methanol. The streptomycin in
pure form is extracted as calcium chloride complex.

32. vitamins
 Vitamins are essential micronutrients required in trace quantities that cannot be
synthesized by mammals.
 They are essential for metabolism for all living organism
 presently few of the vitamins are chemically synthesized or via extraction
processes.

33. Vitamin B 12 also known as cyanocobalamin, is a water-soluble vitamin
with a key role in normal functioning of the brain, nervous system and
for the formation of blood.

34. Structure of Vitamin B12
• The chemical structure of the molecule was determined by Dorothy
crowfoot Hodgkin and her team in 1956 based on crystallographic
data.

38. MICRO-ORGANISM IN INDUSTRIAL
PRODUCTION OF VITAMIN B12
Streptomyces griseus,
S. olivaceus,
Bacillus megaterium,
Pseudomonas denitrificans,
Propionibacterium freudenreichii,
P. Shremanii & mixed fermentation of a proteus spp and a pseudomonas sp.

39. Manufactured by submerged fermentation
Aeration and agitation of medium essential
Fermentation process completed in 3 to 5 days

40. Vitamin B 12 production using
Streptomyces olivaceus NRRL B-1125
Pure slant culture of streptomyces olivaceus NRRL B 1125 is inoculated
and grown in 100 to 250 ml of inoculum medium.
Seeded flask are kept on shaker for incubation
Flask cultures are used to inoculate large amount of inoculum media
arranged in series of tank
Two or three successive transfers are made to obtain required amount of
inoculum cultures.

44. Vitamin B2/Riboflavin

45. Microorganism in industrial production of Vitamin B 2

46. Production process of riboflavin
• Industrial production of riboflavin is mostly carried out with the organism,
Ashbya gossypii by using simple sugars such as glucose and corn steep liquor.
• Glucose can be replaced by sucrose or maltose for the supply of carbon source.
• In recent years, lipids such as corn oil, when added to the medium for energy
purpose, have a profound influence on riboflavin production.
• Further, supplementation of the medium with yeast extract, peptones, glycine,
inositol, purines also increase the yield of riboflavin.

47. • It is essential to carefully sterilize the medium for good yield of riboflavin.
• The initial pH of the culture medium is adjusted to around 6- 7.5.
• The fermentation is conducted at temperature 26-28°C with an aeration rate
0.3 vvm.
• The process is carried out for about 5-7 days by submerged aerated
fermentation.

48. • Fermentation through phases:
• Phase I: This phase is characterized by rapid growth of the organism utilizing
glucose.
• As pyruvic acid accumulates, pH becomes acidic.
• The growth of the organism stops as glucose gets exhausted. In phase I, there is no
production of riboflavin.
• Phase II: Sporulation occurs in this phase, and pyruvate concentration decreases.
Simultaneously, there is an accumulation of ammonia (due to enhanced deaminase
activity) which makes the medium alkaline.
• Phase II is characterized by a maximal production of riboflavin. But this is mostly
in the form of FAD and a small portion of it as FMN.

49. Phase III: In this last phase, cells get disrupted by a process of
autolysis. This allows release of FAD, FMN and free riboflavin into the
medium.
• Recovery: Riboflavin is found in fermentation broth and in a bound
form to the cells. The latter can be released by heat treatment i.e. 120°C
for about 1 hour.
• The cells can be discarded after filtration or centrifugation. The filtrate
can be further purified and dried, as per the requirements.

50. STATINS
 Hyper-lipoproteinemias (HPL) are conditions in which the concentration of cholesterol or
triglyceride carrying lipoproteins in the plasma is elevated above normal.
 Increase in lipoprotein can hasten the development of atherosclerosis and is a risk factors
for myocardial infarction (MI)
 Lipids and proteins form complexes called lipoproteins & circulate in blood vessels.

51. Hypolipidaemias
• These are the drugs which lower the levels of lipids and lipoproteins in blood used to
prevent cardiovascular disease by retarding the atherosclerosis in hyper-lipidaemia
individuals
• Drugs used to treat hyperlipidaemia are
 Lovastatin, Simvastatin, Atorvastatin
 Cholestyramine, Colestipol
 Clofibrate, Gemfibrozil
 Nicotinic Acid

53. Mechanism of Action of Statins (HMG CoA
reductase Inhibitors)
• Hydroxymethyl glutaryl CoA is the rate controlling enzyme in the biosynthesis of cholesterol
• Lovastatin and its congeners are structurally similar to HMG CoA and therefore competitive
inhibitors of the enzyme HMG-CoA reductase. The synthesis of cholesterol in the liver is reduced.
There is an increase in the expression of hepatic LDL receptor. So that more of LDL is taken up
from the circulation. As a result plasma levels of LDL cholesterol and triglycerides fall.
• Concentration of HDL- cholesterol increases by 10%

54. Statins
Inhibit the HMG CoA reductase
Decrease synthesis of Cholesterol
Decrease LDL
Statins to be useful lowering morbidity and mortality in patients with coronary heart
disease. Hence they are used in patients with MI, angina etc

55.  Lovastatin was the first statin drug which was approved by USFDA In the
year 1987
 It is a potent drug that is used to control increased serum cholesterol level,
there by preventing hypercholesterolemia and associated health issue.
 It is a competitive inhibitor of the enzyme HMG CoA reductase which
catalyses rate limiting step in cholesterol biosynthesis

56. Fungal organism reported for lovastatin
production
• Aspergillus
• Penicillium citrinum
• Monascus ruber
• Trichoderma
• Pleurotus widely reported soil fungi capable of lovastatin production
• Commercial production of lovastatin employs a Aspergillus terreus soil fungus
• Several production media have been evaluated for lovastatin production by SmF ( submerged fermentation)
• Increased yield by supplementation with carbon, nitrogen, aminoacids, vitamins, etc.

57. Fermentation Techniques
• Different fermentation techniques including solid state fermentation (SSF)
and submerged fermentation (SMF) can be used for statin production.
• Large scale commercial production utilises submerged batch fermentation
• There is a controlled aeration & agitation in a bioreactor during SMF, which
increases the oxygen mass transfer and constant distribution of nutrients to
fungal mycelia, resulting in increased production of statins.

60. Wheat bran (40g) as substrate
Inoculated with spore suspension (107/8ml spores) of A. terreus
(KM017963)
Incubated at 280C for 7 days
Materials and Methods
Culturing of Aspergillus terreus by Solid State Fermentation (SSF)

61. EXTRACTION
Inoculated substrate was dried at 40◦C for 24h
Crushed to powder
Ethyl acetate (150 ml) was added
Filtrate was dried using rotary vacuum evaporator

62. Purification of lovastatin
One gram of dried crude lovastatin extract was loaded on to pre-
packed silica gel column
Elution with benzene (100%), and combination of
Benzene: Acetonitrile in the following ratio 95:5, 90:10, 85:15, 80:20
Acetonitrile (100%)
Thin Layer Chromatography (TLC)

63. Detection of Lovastatin by Thin Layer
Chromatography (TLC)
Organic phase (20 µl) was spotted on TLC plate
Dichloromethane: Ethyl acetate (70:30)
Rf comparison with standard Lovastatin

64. THANK YOU