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Ubaid Tariq Bhat
M.Pharm Biotechnology
Delhi Pharmaceutical Science Research University
E. Mail: bhatubaid55@gmail.com
1
2
 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 β-lactam antibiotics, most
significantly penicillin which inhibits the biosynthesis
of bacterial cell walls affecting lysis of the cell.
3
 Kingdom: Fungi
 Division: Ascomycota
 Class: Eurotiomycetes
 Order: Eurotiales
 Family: Trichocomaceae
 Genus: Penicillium
 Species: Chrysogenum
4
 Penicillium chrysogenum exhibits typical eukaryotic
cell structure; it has a tubulin cytoskeleton which is
used for motility.
5
TAM structure of P.
Chrysogenum
Structure of P. Chrysogenum
 This image displays the typical filamentous hyphae that
contain many conidia.
 The oblong structures in the image are conidia, the asexual
spores of the fungus.
 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.
6
 It produces the hydrophobic β-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.
7
 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.
8
4) All the bio parameters like temperature, pH, aeration,
agitation etc. should be properly maintained.
Bio parameters
 pH: near 6.5
 Temperature: 26°C to 28°C
 Aeration: a continuous stream of sterilized air is
pumped into it.
 Agitation: have baffles which allow constant agitation
(200rpm).
9
 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 a concentration of 6%.
 Other carbohydrates like glucose & sucrose.
 Complex as well as cheap sources like molasses, or
soya meal can also be used which are made up of
lactose and glucose sugars.
10
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 form of water soluble salts.
Precursors
 Various types of precursors are added into production
medium to produce specific type of penicillin.
11
 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.
 When corn steep liquor is provided as nitrogen source,
it also provides phenyl acetic acid derivatives; therefore
it is widely used in the production of penicillin-G.
12
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
13
 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 to
2.5) in order to transform the penicillin to the anionic 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.
14
 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.
15
16

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Production of Penicillin by Fermentation

  • 1. Ubaid Tariq Bhat M.Pharm Biotechnology Delhi Pharmaceutical Science Research University E. Mail: bhatubaid55@gmail.com 1
  • 2. 2
  • 3.  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 β-lactam antibiotics, most significantly penicillin which inhibits the biosynthesis of bacterial cell walls affecting lysis of the cell. 3
  • 4.  Kingdom: Fungi  Division: Ascomycota  Class: Eurotiomycetes  Order: Eurotiales  Family: Trichocomaceae  Genus: Penicillium  Species: Chrysogenum 4
  • 5.  Penicillium chrysogenum exhibits typical eukaryotic cell structure; it has a tubulin cytoskeleton which is used for motility. 5 TAM structure of P. Chrysogenum Structure of P. Chrysogenum
  • 6.  This image displays the typical filamentous hyphae that contain many conidia.  The oblong structures in the image are conidia, the asexual spores of the fungus.  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. 6
  • 7.  It produces the hydrophobic β-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. 7
  • 8.  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. 8
  • 9. 4) All the bio parameters like temperature, pH, aeration, agitation etc. should be properly maintained. Bio parameters  pH: near 6.5  Temperature: 26°C to 28°C  Aeration: a continuous stream of sterilized air is pumped into it.  Agitation: have baffles which allow constant agitation (200rpm). 9
  • 10.  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 a concentration of 6%.  Other carbohydrates like glucose & sucrose.  Complex as well as cheap sources like molasses, or soya meal can also be used which are made up of lactose and glucose sugars. 10
  • 11. 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 form of water soluble salts. Precursors  Various types of precursors are added into production medium to produce specific type of penicillin. 11
  • 12.  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.  When corn steep liquor is provided as nitrogen source, it also provides phenyl acetic acid derivatives; therefore it is widely used in the production of penicillin-G. 12
  • 13. 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 13
  • 14.  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 to 2.5) in order to transform the penicillin to the anionic 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. 14
  • 15.  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. 15
  • 16. 16