◦Industrial ProductionofPenicillin
Dr. Muhammad Nawaz

● First true naturally-occurring antibiotic ever discovered
● Penicillin is a secondary metabolite of certain species of Penicillium
Examples: Amoxicillin, ampicillin, phenoxymethylpenicillin.
What is Penicillin

History: Discovery &
Production
● 1928: Scottish biologist, Alexander Fleming
accidently discovered Penicillin. He found
that the fungus Penicillium notatum
prevented growth of bacteria Staphylococcus
spp.
● 1932: Clutterbuck and his colleagues studied
the nature of Penicillin and found it as an
organic acid

History: Discovery &
Production
● 1940; Howard Florey and Ernst Chain managed to
purify penicillin in a powdered form.
● 1942:Purified protein was used to treat streptococcal
meningitis
● 1943: Penicillin on a large scale.
This helped immensely to treat casualties during the
WWII that had bacterial infections due to their
wounds.

Classes of Penicillins
◦ Natural Penicillins:
◦ Penicillin G (same as Benzylpenicillin)
◦ Penicillin V (same as Phenoxymethylpenicillin)
◦ Aminopenicillins
◦ Ampicillin
◦ Amoxicillin
◦ Hetacillin
◦ Penicillinase-resistant Penicillins (Antistaphylococcal Penicillins)
◦ Methicillin (prototype)
◦ Cloxacillin
◦ Dicloxacillin
◦ Nafcillin
◦ Oxacillin

Classes of Penicillins
◦ Extended Spectrum Penicillins (Antipseudomonal Penicilllins)
◦ Azlocillin
◦ Carbenicillin
◦ Mezlocillin
◦ Piperacillin
◦ Ticarcillin
◦ Beta-lactamase Inhibitors
◦ Clavulanic acid

The basic chemical structure of all penicillin group consists of a beta-lactam ring,
a thiazolidine ring, and a side chain (6-aminopenicillanic acid)
Structure ofPenicillin:

Penicillin Derivative
●Derivatives produced to deal with the
problemof bacterial resistance to penicillin.
●All penicillin or penicillin derivative have a
constant core region which is the 6-APA.
●The only region that is different from different
types of penicillin derivative is its R group.

Penicillin derivatives:

Biosynthesis of Penicillin:
◦ Three main steps
Catalytic step.
Oxidative step.
Exchange of different chains.

Biosynthesis of Penicillin:
◦ The catalytic step involves an ACV synthetase enzyme that condenses
the lateral chain of cysteine, valine, and alpha aminoadipate into
tripeptide ACV.
◦ In the second step, tripeptide ACV forms a bicyclic ring by oxidative ring
closure. Isopenicillin N synthase is involved resulting in isopenicillin N
which is a bioactive intermediate in the pathway.
◦ The third step involves the exchange of L-aminoadipate. Acyl-CoA
synthetase and Acyl-CoA racemase, a two enzyme system is involved
that helps in converting isopenicillin N into Penicillin G.

Biosynthesis of Penicillin:

Stagesof
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.

Penicillin production process:

Penicillin production steps:
1. Seed Culture development in laboratory.
2. After three days of incubation, the content is used for inoculation and
kept in a fermentor that is well equipped with optimum conditions.
3. The content is filtered after six days of incubation which contains
penicillin.
4. The penicillin is extracted into amyl or butyl acetate and is transferred
into an aqueous solution with phosphate buffer.

Penicillin production steps:
5. Acidify the extract and again re-extract penicillin into butyl acetate
6. In the solvent extract potassium acetated is added to a crystallization
tank to crystallize as a potassium salt.
7. Crystals were recovered and further sterilization of salt is done.

Strains for Penicillin Production
◦ Penicillium notatum
◦ Penicillium chrysogenem

Strain Improvement
• At the end of the WWII, penicillin was first made using the fungus
Penicillium notatum, which produced a yield of 1mg/dm3
● Today, using a different species known as Penicillium
chrysogenum, and better extraction procedures, the yield is 50
mg/dm3

Media
Formulation
● pH 6.5
● Temperature 20-24 °C
● Oxygen
● Nitrogen: corn steep liquor 8.5 %
● Glucose 1%
● 80% ethanol
● phenylacetic acid
● Probenecid
● Lactose 1%
● Calcium Carbonate 1%
● Sodium hydrogen phosphate 0.4%
● Antifoaming agent: vegetable oil

★ Medium for
penicillin
1. The Penicillium chrysogenum usually contain its carbon source
which is found in corn steep liquor and glucose.
2. 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.

Production Method
● Secondary metabolites are only produced in times of stress when
resources are low and the organism must produce these compounds
to kill off its competitors to allow it to survive.
● It is these conditions that we wish to duplicate in order to achieve
the maximum amount of product from our fermentation.

Heat sterilization
3.Medium is sterilized at high heat and high pressure, usually through a
holding tube or sterilized together with the fermenter.
4.The pressurized steam is used and the medium is heated to 121°C at 30
psi or twice the atm. pressure

Seed Culture
9. The seed culture is developed first in the lab by the addition of
Penicillium chrysogenum spores into a liquid medium. When it has
grown to the acceptable amount, it is inoculated into the fermenter.
10. The medium is constantly aerated and agitated. Carbon and
nitrogen are added sparingly alongside precursor molecules for
penicillin fed-batch style. Typical parameters such as pH, temperature,
stirrer speed and dissolved oxygen concentration, are observed.

Seed Culture
11. After about 40 hours, penicillin begins to be secreted by the fungus.
12.After about 7 days, growth is completed, the pH rises to 8.0 or above
and penicillin production ceases.
The Penicillium fungus

Fermentation:
5.It is done in a fed-batch mode as glucose must not be added in high
amounts at the beginning of growth (which will result in low yield of
penicillin production as excessive glucose inhibit penicillin production).
6.The fermentation conditions for the Penicillium mold, usually
requires temperatures at 20-24°C while pH conditions are kept at 6.5
7.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.

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

Product Recovery
• Harvest broth from fermented tank by filtration (rotary vaccum
filtration) chill to 5-10 C (because penicillin is highly reactive and
destroyed by alkali and enzyme)
• Acidify filtrate to pH 2.0-2.5 with H2SO4 ( to convert penicillin to its
anionic form)
• Extract penicillin from aqueous filtrate into butyl acetate or amyl
acetate (at this very low pH as soon as possible in centrifugal
counter current extractor)
• Discard aqueous fraction
Rotary vacuum filter

◦ Allow the organic solvent to pass through charcoal
to remove impurities and extract penicillin from
butylacetate to 2% aqueous phosphate buffer at pH
7.5
◦ Acidify the aq. Fraction to pH 2-2.5 and re-extract
penicillin into fresh butylacetate ( it concentrated up
to 80-100 times)
◦ Add potassium acetate to the solvent extract in a
crystallization tank to crystalize as potassium salt
◦ Recover crystal in filter centrifuge
◦ Sterilization
◦ Further processing
◦ Packaging

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.

SpecificConditions forPenicillin
Production
● Most penicillins form filamentous broths. This means they can be
difficult to mix due to their high viscosity. Also the increasing
viscosity of the broth can hinder oxygen transfer.

A solution for the viscosity and the
filamentous growth of penicillium
species could be bubble columns (air lift
reactors) which would distribute the
oxygen equally and also to agitate the
medium.
SpecificConditions forPenicillin
Production

● 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.
SpecificConditions forPenicillin
Production

Thankyouforyourattention!