Details about Penicillin and its Industrial Productions in various modes

1. SHRI SHIVAJI EDUCATION SOCIETY AMRAVATI
SHRI SHIVAJI COLLEGE OF ARTS COMMERCE
AND SCIENCE AKOLA(MS)
NAAC REACCREDITED WITH A GRADE(CGPA OF( 3.24)COLLEGE WITH POTENTIAL FOR
EXCELLENCE(STATUS BY UGC)LEAD COLLEGE (STATUS BY SGBAU, AMRAVATI)
DST-FIST SUPPORT
DEPARTMENT OF BOTANY
BY
DR. RASIKA N. PATIL
ASSISTANT PROFESSOR
SHRI SHIVAJI COLLEGE OF ARTS COMMERCE AND SCIENCE
AKOLA

2. INTRODUCTION
 We have all, at one point or the other, heard about
penicillin. From its accidental discovery and massive
use in World War II to the Nobel Prize for Medicine in
1945 by its rightful owners, penicillin has made its
proof as the “Miracle Drug” that revolutionized the
course of the medical industry.
 Without further ado, let us take a look at an important
aspect that made penicillin accessible at large; its
industrial production.

3. WHAT IS PENICILLIN?
● First true naturally-occurring antibiotic ever discovered: a
great medical breakthrough.
● Group of antibiotics produced by the Penicillium fungi.
It is a group of closely related compounds, not a single
compound.
Examples: Amoxicillin, ampicillin, phenoxymethylpenicillin.
● Around 50 drugs that are penicillins.

4. HISTORY
● In year 1928: Scottish biologist, Alexander
Fleming discovered that the Staphylococcus
culture he had mistakenly left growing in open
was contaminated with a mould which had
destroyed the bacteria.
● After isolating a sample and testing it, he found
that it belonged to the Penicillium family.
Later the mould was classified as Penicillium
notanum.
● At first, it was difficult to convince people about
its potential uses.

5. HISTORY:DISCOVERY&PRODUCTION
● But later (1939), using Fleming’s work, two
medical researchers, Howard Florey and
Ernst Chain managed to purify penicillin in a
powdered form.
● In Year 1941: They successfully treated a human.
● In year 1943: They produced penicillin on a large
scale.
This helped immensely to treat casualties during
the WWII that had bacterial infections due to
their wounds.

6. GENERAL STRUCTUREOF
PENICILLIN

7. HOWDOES PENICILLIN WORK?
● Inhibits the synthesis of peptidoglycan in cell walls.
○ β-Lactam of penicillin binds to the enzyme - transpeptidase,
that is used in the formation of peptidoglycan cross linking.
○ The enzyme is inhibited, thus inability to form cross linking.
○ Cell wall is weakened causing osmotic imbalance in the cell.
This leads to cell death.
● As human cells do not have cell walls, penicillin does not affect
them.

8. WHATIS FERMENTER
● Purpose of fermenter:
provide contained, controlled and homogeneous environment in
which the fermentation can proceed in a manner that is both
safe and practical and which optimises the particular objectives
of the fermentation.
● Other primary factors include cost, reliability and safety.
● For reactor being designed for specific purpose, there are a
number of important parameters that will greatly affect
performance:

9. TYPICAL FERMENTOR

10. SOME IMPORTANT ASPECTS
● Mass Transfer: good transfer of oxygen across the liquid
interface-the Sparger delivers this oxygen efficiently.
● Heat Transfer: metabolism as a process tends to give off heat-
achieved through cooling jacket whereby cool water is passed
through.
● Bulk Flow and Mixing: impellers, bubble columns or loop
reactors.
● Batch, Fed-Batch and Continuous Culture: how nutrients
and substrate will be delivered to a culture in a reactor.
● Steam: Used to keep the reactor running aseptically.
(temperature/pressure of 121°C/15 psi for 15-30min).

11. ● Batch: fixed amount of substrate is added at the beginning
whereby the volume of nutrients remains the same throughout
the process.
● Fed Batch: substrate is added in small increments at various times
in the fermentation and consequently volume increases.
● Continuous: substrate is constantly added to the reactor while an
equal amount of fermented medium is removed. Volume again
remain the same but constantly renewed with fresh ones.
BATCH,FED-BATCH ANDCONTINUOUS
CULTURE

12. ● Penicillin is an aerobic organism; oxygen supply is critical: reactor
must have an efficient oxygen supply system.
● The optimum pH for penicillin
efficiently (pH controller
growth is 6.5: maintain pH
and acid-base reservoir).
● Strain Stability problems (mutations): careful strain maintenance is
required.
● Biomass doubling is about 6h: provisions must be made.
SPECIFIC CONDITIONS FOR PENICILLIN
PRODUCTION

13. MEDIA CONSIDERATION
● The aim of the media is to:
○ Provide all the elements required for the
synthesis of cell materials and the formation of the
desired product.
○ provide favourable environment for the culture in
question.
○ be cost effective.

14. ● Microorganisms require C, H, O, S and N for cell growth and cell
maintenance.
● Also require small amounts of trace elements such as Cu, Mn and
Co (frequently depend on the water source) or growth factors such
as vitamins or amino acids.
● Certain organisms such as Penicillium chrysogenum that produce
antibiotics, enzymes or other secondary metabolites frequently
require precursors like purine/pyrimidine bases or organic acids to
produce metabolites.
MEDIA FORMULATION

15. PRIMARY ANDSECONDARY
MET
ABOLISM
● Primary metabolism is the metabolism of energy production for
the cell and for its own biosynthesis. In aerobic organisms (such as
Penicillium chrysogenum) it involves the conversion of sugars such
as glucose to pyruvic acid and the production of energy.
● Secondary metabolism regards the production of metabolites
that are not used in energy production for example penicillin from
Penicillium chrysogenum. The metabolite is being utilized as a
defence mechanism against other microorganisms in the
environment.
● Penicillium chrysogenum can kill off the competition to allow
itself to propagate efficiently.

16. STAGES OF PRODUCTION
1. Primary metabolism will be emphasised. Media for this stage will
be focussed on achieving maximum growth and biomass
production.
2. Once the desired biomass has been achieved, starve (Limiting the
amount of C and N available to the culture) the culture and induce
the kind of stress conditions that trigger the production of the
antibiotic.
★ Use the fed-batch method to feed the culture. As stated
above, this allows us to add the substrate to the reactor
in small increments and to even change the substrate if
we so desire.

17. PRODUCTIONOF PENICILLIN
● Penicillin was the first important commercial product produced by
an aerobic, submerged fermentation.
● First antibiotic to have been manufactured in bulk.
● At the end of the WWII, penicillin was first made using the fungus
Penicillium notatum, which produced a yield of 1 mg/dm3
● Today, using a different species known as Penicillium
chrysogenum, and better extraction procedures, the yield is 50
mg/dm3

18. THEINDUSTRIALPRODUCTION
OF PENICILLIN
This can be broadly classified into two processes namely:
1. Upstream Processing
-referring to processes before input to the fermenter and
encompases any technology that leads to the synthesis of a product.
It includes the exploration, development and production.
2. Downstream Processing
-referring to processes done to purify the output of the fermenter
until it reaches to the desired product, such as extraction and
purification of a product from fermentation.

19. SIMPLIFIEDFLOWCHART

20. MEDIUM FORPENICILLIN
 The Penicillium chrysogenum usually contain its carbon source
which is found in corn steep liquor and glucose.
 A medium of corn steep liquor and glucose are added to the
fermenter. Medium also consists of salts such as MgSO4, K3PO4 and
sodium nitrates. They provide the essential ions required for the
fungus metabolic activity.

21. HEAT STERILIZATION
 Medium is sterilized at high heat and high pressure, usually through a
holding tube or sterilized together with the fermenter.
 The pressurized steam is used and the medium is heated to 121°C at
30 psi or twice the atm. pressure
Sterilisation machine

22. FERMENTATION PROCESS
 The fermentation conditions for the Penicillium mold, usually
requires temperatures at 20-24°C while pH conditions are kept at
6.5
 The pressure in the bioreactor is much higher than the atmospheric
pressure (1.02atm). This is to prevent contamination from occurring
as it prevents external contaminants from entering.

23. FERMENTATION
 It is necessary to mix the culture evenly throughout the culture
medium. Fungal cells are able to handle rotation speed of around
200 rpm.
Fermentors

24. SEED CULTURE
 After about 40 hours, penicillin begins to be secreted by the
fungus.
 After about 7 days, growth is completed, the pH rises to 8.0 or
above and penicillin production ceases.
The Penicillium fungus

25. REMOVALOF BIOMASS
 Filtration is carried out as bioseparation is required to remove the
biomass from the culture (removing the fungus and other impurities
away from the medium, which contains the penicillin).
 A Rotary vacuum filter is commonly employed for filtration as it is
able to run in continuous mode in any large scale operations.
 After filtration, phosphoric acid,
a non-oxidising agent, is
introduced, to decrease pH
from 8.0 to 6.5 so as
 to prevent loss of activity of penicillin.
Rotary vacuum filter

26. ADDITIONOF SOLVENT
 Organic solvents such as amyl acetate /
butyl acetate are added to dissolve the
penicillin present in the filtrate.
 At this point, penicillin is present in the
solution and any other solids will be
considered as waste (can be used as
fertilizers and animal feed).

27. CENTRIFUGAL EXTRACTION
 Centrifugation is done to separate the solid
waste from the liquid component which
contains the penicillin.
 Usually a disk centrifuge is used at this point.
 The supernatant will then be transferred
further in the downstream process to
continue with extraction.
Disk centrifuge - One of the most
common type of centrifuge for large
scale production

28.  A series of extraction processes are carried upon the dissolved
penicillin, to obtain a better purity of the penicillin product.
 The acetate solution is first mixed with a phosphate buffer, followed
by a chloroform solution, and mixed again with a phosphate buffer
and finally in an ether solution.
 Penicillin is present in high concentration in the ether solution and
it will be mixed with a solution of sodium bicarbonate to obtain the
penicillin-sodium salt, which allow penicillin to be stored in a
stable powder form.
EXTRACTION

29. EXTRACTION
 The penicillin-sodium salt is obtained from the liquid material by basket
centrifugation, in which solids are easily removed.
Batch extraction unit Basket Centrifuge: Extremely useful in the
removal of solids in this case Penicillin salt

30. FLUIDBEDDRYING
 Drying is necessary to remove any
remaining moisture present in the
powdered penicillin salt.
 In fluid bed drying, hot gas is
pumped from the base of the
chamber containing the powdered
salt inside a vacuum chamber.
 Moisture is removed this way, and
this result in a much drier form of
penicillin. Powdered penicillinbeing
blown by hot air
Fluid bed drying
tube

31. STORAGE
Penicillin is stored in containers and kept in a dried
environment.
The White Penicillin-Sodium salt
 The resulting penicillin (called Penicillin G) can be chemically and
enzymatically modified to make a variety of penicillins with slightly
different properties.
 These can be semi-synthetic penicillins, such as; Penicillin V,
Penicillin O, ampicillin and amoxycillin.

32. Thank you for your attention !