LEARNING OUTCOME

To be able to explain the development of

bioprocess for commercial production of
Penicillin.
MICROBIAL CELL CULTIVATIONS

• Used since prehistoric times in the preparation of Food,

Alcoholic beverages, Milk products, Textiles, etc.

• Use of microbes has been extended to

pharmaceuticals, industrial chemicals, enzymes,
agricultural chemicals, wastewater treatment, etc.
BACTERIA

SHAPE:
• Unicellular microscopic organisms
• ~1500 species in environment
• Cell diameter ranges between 0.5-1.0 m
• Variety of shapes: cocci, bacilli, spirilla

GROWTH PATTERN:
• Reproduce predominantly by binary fission: asexual
process
BACTERIA: 90% WATER

ELEMENTAL COMPOSITION: C50%, O20%, N14%, H8%, P3%, S1%,

Mg4%

NUTRITIONAL REQUIREMENTS:
Source of energy: radiant energy (phototrophs) or chemical
reactions in dark (chemotrophs)
Source of carbon: CO2 or inorganic carbon (autotrophs); one
or more organic source of carbon (heterotrophs)
Source of nitrogen: atm N2 , inorganic nitrogen or other
sources
Source of sulphur: inorganic, organic or elementary S
Metallic elements: sodium, potassium, calcium, magnesium,
manganese, iron, zinc, copper and cobalt
Vitamins
 BACTERIA
PHYSICAL CONDITIONS
• Temperature: psychrophiles (-7 to 35 °C), mesophiles (7-45
°C), thermophiles (40-75 °C)
• Gaseous environment (aerobic O2, anaerobic O2, facultative
anaerobe O2 & O2, microaerophilic O2 )
• pH (acidophile <2; alkaliphile >9; neutralophile 5.5-8.5)

SPORULATION
• Some form spores
• Form under starvation and unfavorable conditions
• Increased resistance than normal cells toward heat,
radiation, chemicals & drying
• Can convert to normal cells under favorable conditions
CULTURE MEDIA

GROWTH OF MICROBIAL POLUTAION IN ARTIFICIAL

ENVIRONMENT IS CULTIVATION
PURE CUTURE: only one kind of microorganism
MIXED CULTURE: more than one kind of microorganism

Steps involved:
1. Preparation of optimum culture medium
2. Culture vessel and media sterilization
3. Inoculation in the medium to facilitate rapid growth
CULTURE MEDIUM

Natural (Chemically undefined composition):

• Based on experience, no exact information on
composition and mode of action
• Contain peptones, beef extracts, yeast extract, etc.
• Solid medium requires addition of agents like agar and
gelatin
• Readymade media: nutrient agar/broth

Synthetic (Chemically defined and reproducible

composition)
• Known and reproducible concentrations of pure
inorganic and organic compounds
 Sterilization
• Commonly used method:
• steam sterilization (moist heat) under pressure in autoclave.
• Conditions: 15 psi at 121 °C
• Time of sterilization depends on nature of material, type of
vessel, volume.

Inoculation
• Seeding of culture vessel with microorganisms
• Use of metal wire or loop
• Liquid culture in sterile pipette
• Done in LAF to minimize contamination risk
 Penicillin story
• The first antibiotic discovered in 1896 by Ernest Duchesne
and "rediscovered" by Alexander Flemming in 1928 from the
filamentous fungus Penicilium notatum.

• In 1928, Sir Alexander Fleming, a Scottish biologist, observed

that Penicillium notatum, a common mold, had destroyed
staphylococcus bacteria in culture.
• He took a sample of the mold from the
contaminated plate.

• He found that it was from the Penicillium family,

later specified as Penicillium notatum.

• Fleming presented his findings in 1929,

published a report on penicillin and its
potential uses in the British Journal of
Experimental Pathology.

• Penicillin was first produced on a large scale for

human use in 1943.

• Many lives were saved during World War II

 How Penicillin Works?

https://www.slideshare.net/seetaram443/penicillins-53561419
 Production of Penicillin
• Like most
antibiotics,
penicillin is a
secondary
metabolite, so is
only produced in
non-growth
associated phase.
 Penicillin production
• Successful scale-up of a laboratory scale cultivation of a
microorganism into a profitable industrial-scale process
• Despite the demand rising to 650 billion per month, the
cost to 20 $ to 60 cents, per 10,000 units.
• The yield of penicillin was increased by 1000 times

Factors responsible:
• Improvement in medium composition: use of corn steep
liquor, replacing glucose with lactose, phenylacetic acid
addition
• Development of submerged fermentation technique
• Strain improvement using X-ray and UV radiation
 INDUSTRIAL PRODUCTION OF PENICILLIN

Broadly classified in to two processes:

• Upstream processing
• Downstream processing
 UPSTREAM PROCESSING

• Upstream processing encompasses any

technology that leads to the synthesis of a
product. Upstream includes the exploration,
development and production.
 DOWNSTREAM PROCESSING

• The extraction and purification of a

biotechnological product from fermentation is
referred to as downstream processing.
 UPSTREAM PROCESSING

• The medium is designed to provide the

organism with all the nutrients that it requires.

• Inoculation method- submerged technique

• Spores -major source of inoculum

 UPSTREAM -
MEDIUM FORMULATION
• CARBON SOURCES:
Lactose acts as a very satisfactory carbon compound, provided that is used
in a concentration of 6%. Others such as glucose & sucrose may be used.
NITROGEN SOURCES:
• Corn steep liquor (CSL)
• Ammonium sulphate and ammonium acetate can be used as nitrogenous
sources.
MINERAL SOURCES:
Elements namely potassium, phosphorus, magnesium, sulphur, zinc and
copper are essential for penicillin production. Some of these are applied
by corn steep liquor.

• Calcium can be added in the form of chalk to counter the natural acidity of
CSL

• Phenylacetic acid (PAA)- precursor

 UPSTREAM-FERMENTATION PROCESS

• The medium is inoculated with a suspension

of conidia of Penicillium chrysogenum.
• The medium is constantly aerated and
agitated, and the mould grows throughout as
pellets.
• After about seven days, growth is complete,
the pH rises to 8.0 or above, and penicillin
production ceases
 DOWNSTREAM PROCESSING
• Products in a fermenter are impure and dilute,
so need to be purified by downstream
processing

• This usually involves filtration to separate the

microbial cells from the liquid medium, followed
by chemical purification and concentration of the
product

• Downstream processing can account for 50% of

the cost of a process.
 STAGES IN DOWNSTREAM PROCESSING

• Downstream processing is relatively easy since penicillin is

secreted into the medium (to kill other cells), so there is no
need to break open the fungal cells.

• However, the product needs to be very pure, since it being

used as a therapeutic medical drug, so it is dissolved and then
precipitated as a potassium salt to separate it from other
substances in the medium.
 ISOLATION OF BENZYL PENICILLIN

• The pH is adjusted to 2-2.5 with the help of phosphoric or

sulphuric acids.
• In aqueous solution at low pH values there is a partition
coefficient in favor of certain organic solvents such as butyl
acetate.
• This step has to be carried out quickly for penicillin is very
unstable at low pH values.
• Antibiotic is then extracted back into an aqueous buffer at a
pH of 7.5, the partition coefficient now being strongly in favor
of the aqueous phase. The resulting aqueous solution is again
acidified & re-extracted with an organic solvent.
• These shifts between the water and solvent help in the
purification of penicillin.
• The treatment of the crude penicillin extract varies
according to the objective, but involves the formation of
an appropriate penicillin salt.

• The solvent extract recovered in the previous stage is

carefully extracted back with aqueous sodium hydroxide.

• This is followed by charcoal treatment to eliminate

pyrogens and by sterilization.

• Pure metal salts of penicillin can be safely sterilized by dry

heat, if desired. Thereafter, the aqueous solution of
penicillin is subjected to crystallization.
 OTHER PRODUCTS:

• The resulting penicillin (called penicillin G) can be chemically

and enzymatically modified to make a variety of penicillins
with slightly different properties.

• These semi-synthetic penicillins include penicillin V, penicillin

O, ampicillin and amoxycillin.
 PRODUCTION OF PENICILLIN V
• Phenoxy methyl penicillin
• Addition of different Acyl groups
• Phenoxyacetic acid as precursor instead of
phenyl acetic acid.
 FURTHER PROCESSING

• For use, the antibiotic is packed in sterile vials as a

powder or suspension.
• For oral use, it is tabletted usually now with a film
coating.
• Searching tests (ex: for purity, potency) are performed
on the appreciable number of random samples of the
finished product.
• It must satisfy fully all the strict government standards
before being marketed
 SUMMARY
Main stages of Penicillin production