THE WONDER Student name: Yousra

Mohamed

DRUG Student No.:

21806757
Antibiotics
◦ The term antibiotic has been defined by Selman Waksman as being an organic compound
produced by one microorganism that inhibits the growth of or kills a group of harmful
microorganisms (Bacteria).
◦ These enter and stick to important parts (think of targets) of the bacterial cell, and interfere
with its ability to survive and multiply [1].
If the bacteria are susceptible to the antibiotic, then they will stop growing or simply die.
◦ These important parts include:
◦ Proteins/sugars in the bacterial wall.
◦ Important enzymes that make new bacterial DNA or proteins[2].
Bacteria, and Only Bacteria!
 First Naturally-occuring Antibiotic
◦ Fleming was already well known upon
his return from a long holiday in September
1928, he noted that one petri dish contained
colonies of staphylococcus bacteria except
for clear area contaminated with a fungus
that appeared to inhibit bacterial growth.
The mold was found to be penicillium
notatum[3].
Figure 1:shows penicillin mold on bacterial petri dish.
 Staphylococcus Aureus
◦ Staphylococcus is a genus of Gram-positive bacteria. Under the microscope, they
appear spherical, and form in grape-like clusters. It can cause scarlet fever,
pneumonia or sepsis [3].

Figure 2: illustrations of staphylococci bacteria and its arrangement.

 General Structure
of Penicillin
 Penicillin is not a single compound but a
group of closely related compounds, all with
the same basic ring-like structure (a β-
lactam) derived from two amino acids
(valine and cysteine) via a tripeptide
intermediate. 
The third amino acid of this tripeptide is
replaced by an acyl group (R) and the nature
Figure 3: Shows the chemical Structure of Penicillins
of this acyl group produces specific
group.
properties on different types of penicillin.[4].
Figure 4: Pathway of penicillin G by Penicillium Chrysogenum[4].
Types of β-lactam antibiotics:
◦ Penicillins are either :
1. Natural penicillin.
2. Semisynthetic penicillin.
Penicillin Production
Aim:
To provide sterile, controlled and homogeneous environment in which the fermentation can
carryout in a manner that is safe and practical and which optimizes the Penicillin production for
ultimate use.
UPSTREAM PROCESSING :
It the steps taken that lead to the synthesis of a product.
Upstream includes the development and production. 
DOWNSTREAM PROCESSING :
The extraction and purification of a  product from fermentation.
Step 1: Inoculum Preparation.
◦ Inoculum: A small amount of material containing bacteria, viruses,
or other microorganisms that is used to start a culture[5].

◦ Out of various species of the fungus Penicillium, mainly two species are used
in the fermentation:
◦ These are P. notatum & P. chrysogenum but P. chrysogenum is high yielding
strain and therefore most widely used as production strain[6].
Penicillium Chrysogenum
◦ It is able to synthesize penicillins with specific hydrophobic side chains when
the appropriate precursor is fed to the production medium.
◦ PA appears to be the best precursor, but the phenyl group can be substituted or
replaced by other ring systems.
◦ penicillin G, penicillin V, or penicillin O. In the absence of an exogenous side
chain precursor, P. chrysogenum produces mainly 6-aminopenicillanic acid.
 Myceilium
◦ The mycelium is well developed and
copiously branched.
◦  It is composed of colourless,
slender, tubular, branched and septate
hyphae.
◦  The hyphae run in all directions and
become intertwined with one another
to form a loose network of hyphae
Figure 5: Shows the Mycelium structure and how constituting the mycelium.
spores germinate.
◦ Myceilium is the vegitative part of
the mold [7].
 Secondary
Metabolite
◦ penicillin is a secondary
metabolite, so is only produced in
the stationary phase. 
◦ Note that: it is only produced in
times of stress when resources are
low and the organism must produce
these compounds to kill off its
Figure 6: Variation in number of cells of competitors to allow it to survive
Microorganism during time in days.
Figure 7: Difference between Primary metabolite (Alcohol Fermentation and Secondry Metabolite
(Penicillin fermentation).
Maintenance of Strain.
◦ The production strain should be carefully maintained  because
appropriate maintenance and production of reliable pure cultures with
desirable quality is a key operation.
◦ The Microorganism was maintained as lyophilized spore
suspension until usage.
Step 2: Nutrients Sterilization.
◦ Throughout the fermentation industry, heating using steam is widely used as a
sterilization method, since it is reliable and easy to control.
◦ Sterilization of culture is necessary to prevent contamination.
Step 3: Inoculum Build-up.
◦ Here the chief purpose is to develop a pure inoculum in an adequate amount
and in the fast growing phase for the production stage fermenter.
1. Starter culture is transferred into agar-plate to allow growth.
When spores are placed on a vegetative media , the spores start to germinate and
form myceilia.
2. after growth on agar-plate, Cultures were incubated for approximately 70 h in
a rotary shaker at 200 rpm and 25C, to improve oxygen diffusion.
3.  two growth stages (2 days incubation)are allowed upon transfer into seed
fermenter.
Step 4: Seed Fermenters.
◦ Initial stages of fermentation are designed for considerable microbial growth and
the can be distiguished from main Bioreactors by several factors; these are:
1. Smaller in size than the main fermenters approx. 2 Liter capacity
2. Made of stainless steel.
3. Equipped with agitators, which allow continuous mixing of growth medium.
4. Provided with pump to deliver sterilized, filtered air.

◦ Must contain all the nutrients including Growth factors.

◦ After about 24-28 hours, the content of the seed tanks is transferred to fermentation
tank.
Step 5: Bioreactor Set-up.
Fed-Batch culture: to feed a batch by controlled addition of a carbon source
and/or other nutrients, resulting in a fed-batch culture.
Air-lift Bioreactor:
◦ Advantages: 
considerably higher volumetric and specific productivities since the
phenotype of the cell is under better control[8].
Bioreactor Specifications
Table 1: The different parameters in a bioreactor.

Different Procedure
Aspects
Volume 300-500m3, made from stainless steel. 
agitation provided at a rate of 100 to 300 rpm. 

Temperature controlled by using cooling coils. 

Parameters Dissolved oxygen and other parameters should be continuously controlled,
Monitoring this is achieved  by the withdrawal of small volumes of broth in the
fermenter.
Aeration Vigorous supplied from the bottom of fermenters by ring or tube sparges. 
Common Due to the high viscosity of the broth, oxygen transfer is a major
Problems problem in penicillin fermentations.
Broth preparation:
Raw Materials
 Raw materials are primary requirement to design the fermentation broth for
antibiotic production.
◦  Fermentation broth contains all the necessary elements required for the
proliferation of the microorganisms.
Carbon Source:
Lactose acts as a very satisfactory carbon compound when it is used as a food
source for the microorganism. with slow feeding rate.
◦ Corn oil supplemented with lactose results in fast production of highly
concentrated penicillin. 
◦ Glucose, fructose, galactose and etc. Are easily utilized carbon sources reduce
penicillin titer while the titer in lactose is high.

◦Note: In inoculum medium lactose is generally absent because it induces

penicillin production and retarding the growth of production strain.
Nitrogen Source:
◦ Another essential compound for metabolism of organisms is nitrogen.
◦ We use Corn steep liquor (CSL), since it results in higher penicillin yields as
compared to the other nitrogen sources. Some compounds in CSL are
converted to phenylacetic acid or other side-chain precursors.

◦ Cottonseed flour or soybean meal may also be used as nitrogen sources;

however, they are more expensive than CSL [9].
Ammonium Source.
◦ Continuous addition of ammonium sulfate to keep the ammonium
concentration around 250–300 mg/L is required for continued synthesis of
penicillin and to avoid lysis of the mycelium.
◦ The omission of ammonium nitrate decreased the penicillin activity in the
original fermentation medium (Table I).
◦ This result agrees with the findings of [10] they reported that ammonium
nitrogen is a limiting factor in penicillin production. 
Mineral Source:
◦Additionally, some minerals are necessary for the proper growth of these
organisms. are included.
◦ These elements include phosphorus, sulfur, magnesium, zinc, iron, and copper
which generally added in the form of water soluble salts.

◦For example:
◦However, the pH at the end of the fermentation period is slightly lower, when
low concentrations of calcium carbonate were used[10]
AntiFoam Agents:
Anti-foaming agents such as lard oil to reduce foam formation.
Disadvantages of Foaming:
1. Reduce process productivity since bursting bubbles can damage proteins[11],
2. Can result in loss of sterility if the foam escapes the bioreactor[13].
3. leads to over-pressure if a foam-out blocks an exit filter.
 Precursors.
◦ Certain
precursors of the penicillin side chain need to be added into the
fermentation medium. 
◦ This constitutes a major cost item. Penicillin G requires 0.47g sodium phenylacetate.
◦ the precursor must be added repeatedly in small amounts during the fermentation:
why?
In order to avoid toxic effects.
◦ The Phenylacetic acid appears to be the best precursor, but it can be substituted or
replaced by other ring systems[14]. it is transported across the plasma membrane by
free diffusion into myceilium.
Table 2: Constitutes of penicillin G fermentation broth.

Media Percentage%
Lactose 3-4

Glucose 10

Corn steep liquor 4

CaCO3 1

Antifoam 0.25-0.5
Phenyl acetic acid 0.5
KH2PO4 0.4
Step 6: Production.
Table 3: Parameters monitored in penicillin fermentation.

Parameter Status

PH Around 6.5-7

Temperature 26°C to 28°C

Aeration continuous stream of sterilized air is

pumped into it

Agitation baffles which allow constant agitation

Inside the Bioreactor.
First phase-
growth of the mycelium occurs, yield of antibiotic is quite low.Lactic acid present in corn steep liquor is
utilized at the maximum rate by the microorganisms. Lactose is used slowly. Ammonia is liberated into
the medium resulting into the rise in pH.
Second phase-
There was intense synthesis of penicillin in this phase, due to rapidconsumption of lactose and ammonia
nitrogen. The mycelia mass increases, the pH remainunchanged.
Third phase-
The concentration of antibiotic decreases in the medium. The autolysis of mycelium starts, liberation of
ammonia and slight rise in pH.
Final product.
◦ Benzylpenicillin (Penicillin G)[15].

Figure 8: Biosynthesis of 6(APA) to Penicillin g.

 Kinetic Models
◦ The original model has been extended by including additional input variables such as agitation power
and aeration rate [12].
Initial conditions and kinetic parameters.
Table 4:Original fermentation Kinetic model and its initial conditions [12].
Figure 9: Glucose and penicillin concentrations at initial glucose concentrations of 15, 25, 30 g/l
[12].
Step 7: Recovery
1. Filtration.
The penicillin-rich filtrate is cooled to 2–4ºC to avoid chemical or
enzymatic degradation of the penicillin.Filtration is usually achieved by using
high-capacity, rotary vacuum drum filters for separation of the mycelia. The
mycelia are washed on the filter and disposed.
2.   Extraction. 
◦ solvent extraction is accomplished at low pH such as 2.5–3, using
amyl acetate as solvent. 
◦ Continuous, countercurrent, multistage centrifugal extractors are used for this purpose. To avoid
degradation of penicillin during solvent extraction at low pH, temperature is kept around 2–4°C and
filtration time is kept very short (1–2 min). 
◦ Two extractors used in series result in nearly 99% penicillin recovery.
Step 8:Purification
1. Adsorption.
◦ Carbon adsorption is used to remove impurities and pigments from penicillin-
rich solvent after extraction. Several activated carbon columns in series can
be used for this purpose.
2.   Crystallization.
◦ Na and penicillin concentrations, pH, and temperature need to be adjusted for
crystallization. 
◦ Excess amounts of Na are added to the penicillin-rich solvent before
crystallization in an agitated vessel.  The crystals are separated by a rotary
vacuum filter.  
◦ The crystals are washed and predried with anhydrous butyl alcohol to remove some
impurities.  Large horizontal belt filters are used for collection and drying of the crystals. 
Figure 9: Main Bioreactor Configuration
[16].
Figure 10: Simplification of penicillin G industrial production
 References
[1] Waksman, S.A. and Woodruff, H.B., 1940. The soil as a source of microorganisms antagonistic to disease-
producing bacteria. Journal of bacteriology, 40(4), p.581.
[2] Levy, S.B., 1998. The challenge of antibiotic resistance. Scientific American, 278(3), pp.46-53.
[3]Bennett, J.W. and Chung, K.T., 2001. Alexander Fleming and the discovery of penicillin.
[4]Elander, R.P., 2003. Industrial production of β-lactam antibiotics. Applied microbiology and
biotechnology, 61(5-6), pp.385-392.
[5]"inoculum." A Dictionary of Biology. . Encyclopedia.com. 7 Dec. 2018 <https://www.encyclopedia.com>.
[6] Patnaik, P.R., 2001. Penicillin fermentation: mechanisms and models for industrial-scale bioreactors. Critical
reviews in microbiology, 27(1), pp.25-39.
[7] http://www.biologydiscussion.com/fungi/life-cycle-of-penicillium-with-diagram-fungi/63497, accessed
09/12/2018.
[8] Meyer, H.P., Minas, W. and Schmidhalter, D., 2017. Industrial-scale fermentation. Industrial Biotechnology: Products
and Processes, pp.1-53.
[9]Foster, J.W., Woodruff, H.B., Perlman, D., McDaniel, L.E., Wilker, B.L. and Hendlin, D., 1946. Microbiological Aspects
of Penicillin: IX. Cottonseed Meal as a Substitute for Corn Steep Liquor in Penicillin Production. Journal of
bacteriology, 51(6), p.695.
[10]Soltero, F.V. and Johnson, M.J., 1953. The effect of the carbohydrate nutrition on penicillin production by Penicillium
chrysogenum Q-176. Applied microbiology, 1(1), p.52.
[11]Holmes, W., Smith, R. and Bill, R., 2006. Evaluation of antifoams in the expression of a recombinant FC fusion protein
in shake flask cultures of Saccharomyces cerevisiae & Pichia pastoris. Microbial Cell Factories, 5(1), p.P30
[12] Bajpai, R.K. and Reuss, M., 1980. A mechanistic model for penicillin production. Journal of Chemical Technology and
Biotechnology, 30(1), pp.332-344.
[13]Varley, J., Brown, A.K., Boyd, J.W.R., Dodd, P.W. and Gallagher, S., 2004. Dynamic multi-point measurement of foam
behaviour for a continuous fermentation over a range of key process variables. Biochemical engineering journal, 20(1),
pp.61-72.
[14]Hillenga, D.J., Versantvoort, H., van der Molen, S., Driessen, A. and Konings, W.N., 1995. Penicillium chrysogenum
takes up the penicillin G precursor phenylacetic acid by passive diffusion. Applied and environmental microbiology, 61(7),
pp.2589-2595.
[15]Deindoerfer, F.H., 1957. Calculation of heat sterilization times for fermentation media. Applied microbiology, 5(4),
p.221.
[16] Moore, D., Robson, G.D. and Trinci, A.P., 2011. 21st century guidebook to fungi with CD. Cambridge University Press.