Large scale production of antibiotics

1. PENICILLIN AND
STREPTOMYCIN
PRESENTED BY,
REVATHY K.S
2ND MSC BOTANY

2. INTRODUCTION
o Industrially produced antibiotics are produced by fermentation,
where the source of microorganism is grown in large liquid
growth medium.
o Oxygen concentration, temperature, PH and nutrient levels
must be optimal and are closely monitored and adjusted if
necessary.
o As the antibiotics are the secondary metabolites, the
population size must be controlled very carefully to ensure that
maximum yield is obtained before the cell die.
o Once the process is complete, the antibiotic must be extracted
and purified to a crystalline product.

3. WHAT IS ANTIBIOTICS?
o Compound that kill or inhibit the growth of other
organisms.
o Most antibiotics are produced by filamentous fungi or
actinomycetes.
o They are derived from special microorganisms or other
living systems , and are produced on an industrial scale
using a fermentation process.
o Today, over 10,000 antibiotics substances have been
reported.
o The process may take a few days to obtain an extractable
amount of product.
o Antibiotic production is done by the Batch process.

4.  LARGE SCALE PRODUCTION OF PENICILLIN
o Penicillin is an antibiotic. It is used to treat infections
caused by bacteria.
o It is a secondary metabolite , thus it is only produced in the
stationary phase.
o The industrial production of penicillin was generally
classified into two processes:
1. UPSTREAM PROCESSING
2. DOWNSTREAM PROCESSING

5. o Upstream processing encompasses any technology that
leads to the synthesis of a product.
o Upstream includes the exploration, development and
production.
o The extraction and purification of a biotechnological
product from fermentation is referred to as
downstream processing.
Any large scale microbial process occurring with or
without air is called Fermentation

6. STEPS IN THE PRODUCTION OF PENICILLIN
SELECTION OF THE MICROORGANISM: A suitable
strain of Penicillium is selected for the large scale
production of penicillin.
Early Penicillium notatum was used, currently high
yielding strains of Penicillium chrysogenum are preferred.
PREPARATION OF INOCULUM: A small amount of the
selected strain is transferred to a sterile nutrient rich
medium in a small fermenter. This stage is called the seed
stage.

7. GROWTH OF INOCULUM: The seed culture is allowed to
grow under controlled conditions to obtain a large number
of cells.
TRANSFER OF THE INOCULUM: The cells from the seed
culture are transferred to a larger fermenter in the
production stage.
FERMENTATION: The cells are grown in the production
fermenter under controlled conditions that optimize the
production of penicillin.
It is a Fed Batch process that is carried out aseptically in
stainless steel tank reactors with a capacity of 30 to 100
thousand gallons.

8. Various carbon sources have been adopted for this process-
including Glucose, Sucrose, and other crude sugars.
Approximately, 65% of the carbon is used for cellular
maintenance, 25% for growth and only 10% for penicillin
production.
Sugar is also used for the regulation of the pH value during
active penicillin production stage.
HARVESTING: When the fermentation process is complete,
the penicillin is harvested from the fermentation broth.
SEPERATION AND PURIFICATION: The penicillin is
separated from the fermentation broth using various
separation technique such as filtration, centrifugation and
chromatography.

9. CONCENTRATION: The penicillin obtained from the
separation and purification stage is concentrated to obtain the
final product.
QUALITY CONTROL: The final product undergoes rigorous
quality control measures to ensure that it meets the required
purity and potency standards.
PACKAGING AND DISTRIBUTION: The final product is
packaged and distributed to hospitals, clinics and pharmacies
for use in treating bacterial infections.
Overall, the large scale production of penicillin is a complex and multi-
step process that requires careful monitoring and optimization at each
stage to ensure maximum yield and purity

11. LARGE SCALE PRODUCTION OF STREPTOMYCIN
o Streptomycin is an antibiotic, that shows a broad spectrum
against the gram –ve bacteria and it is the 1st antibiotic to
cure TB.
o Streptomyces griseus is used in the large scale production
of streptomycin, which utilizes carbon, nitrogen, mineral
source etc. incorporated in the fermentation medium.
o S. griseus is commonly present in the soil, and it is gram
+ve bacteria.
o Its production process is complex, which goes through a
series of operations under the sterile conditions.

12. STEPS OF PRODUCTION
1. GROWTH OF STREPTOMYCES GRISEUS
o Maintain the hymns of Streptomyces griseus spores in the
stock soil culture.
o Then, inoculate the inoculum of S. griseus into the agar media
at 28℃.
o After that, the spores of S. griseus sporulate and build up the
mycelial network in the flask or seed tanks.
o The sporulation occurs well in a liquid culture medium, and it
forms endospores in low nutrient availability.
o The growth requires a pH range of 5- 11.

13. 2. PREPARATION OF MEDIUM
o The production of streptomycin requires Hockenhull
nutrient medium with the following media:
o For the production of the medium, there must be need
energy sources like carbon and nitrogen etc.
• Glucose : 2.5%
• Soy meal : 4%
• Sodium chloride : 0.25%
• Distillers dried soluble : 0.5%
• pH : 7.6 - 8

14. A. CARBON SOURCE : Monosaccharide like glucose
provides the best carbon source that helps in building of
streptomycin. Sugars like fructose, maltose , starch etc.
can also be used as the carbon source.
B. NITROGEN SOURCE : Contents like soy meal ,
meat extract, ammonium salts etc. serve as the nitrogen
source.
3. FERMENTATION
o Sterilize all the above contents and fed into the stirred
tank fermenter.
o Then transfer the bacterial inoculum of about 4-5%to the
medium.

15. o Maintain a temperature between the range of 25-30℃ ,pH
between 7.6-8 inside the fermenter.
o The production requires continuous fermentation, and the
growth of S . griseus requires continuous aeration and
agitation.
o The fermentation process takes 4-10 days.
o This process involves 3 consecutive phases:
FIRST PHASE : During 1st phase, the S . Griseus grows by
utilizing soya meal and releases ammonia through proteolytic
activity. S . Griseus slowly utilizes the carbon source present in
the soy meal and the glucose and thereby producing
streptomycin at a slower rate.

16. SECOND PHASE : After the utilization of soy meal and
glucose , the ammonia is secreted , and the streptomycin is
synthesized rapidly. In this phase , the incubation period lasts
for 24 hours to 6-7 days. There is little production of mycelium.
THIRD PHASE : It is the final phase of fermentation , where
concentration of glucose depletes from the medium that results
in cessation of streptomycin production and the bacterial cell
begin to lyse. Before the lysis , harvest the fermentative
medium for recovery and purification.

17. 4. RECOVERY OF STREPTOMYCIN
The recovery of streptomycin includes the following stages:
a. FILTERATION : In the recovery of streptomycin separate the
bacterial biomass from the fermentative broth through
Oliver’s pre- coated pressure filter. After filtration , the
remaining liquid is 1st acidified and then neutralized.
b. ADSORPTION: Mix the activated carbon with the clear
broth
obtained after filtration in a series of three adsorption tanks.
Separate streptomycin from the broth to remove impurities.

18. c. ELUTION: Elute the streptomycin via ion exchange method
by adding dilute HCl . In this process, the impurities like
metallic salts are removed from the solution , which can
contaminate the streptomycin.
d. CONCENTRATION: Concentrate the remaining solution at
60℃ under vacuum by the series of 3 single phase
evaporators.
e. CRYSTALLIZATION : Firstly, dissolve the streptomycin in
methanol and then filter it again . Then treat the filtrate with
acetone to obtain a precipitate. Rewash the precipitate with
the acetone and later dry it under vacuum.

19. FINAL OPERATIONS
• Then, dissolve the crystalline streptomycin in the solution
containing pyrogen free water.
• Pass the solution through the special biological filter like
Seitz filter to eliminate the microorganisms that contaminate
streptomycin.
• Then, dry the filtrate under vacuum to form a dried cake.
• Crush the dried cake into a powdered form by employing
high vacuum sublimation using sterile steel balls.
• After that, weigh the powdered streptomycin into sterile
vials. The whole process needs to be carried out in the air-
conditioned room.

20. • Finally package of powdered streptomycin in the rooms
having 10% relative humidity.
• To avoid the chances of contamination, we can use
special microbial filters and UV lamps . All the factors
maintain sterile condition throughout the production
process.

21. CONCLUSION
 Development of antibiotics is difficult, whereas many drug
discoveries have been a result of concerted effort and
intensive research and development, antibiotics have
seemingly been discovered by chance.
 Since 1987 there have been no discoveries or development
of a new class of antibiotics.
 As most are produced biosynthetically they require an
organism to produce.