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Specification and size were chosen according to the desired amount of production. Next,
the products coming out from the fermenter was purified and treated. Therefore, it has to undergo
a few series of processes before it was ready to be packaged or stored. Firstly, a Rotary Drum
Vacuum Filter was used to separate excess moisture and the broth only contains high concentration
of Penicillin. Next, Penicillin was collected in bulks through adsorption process where activated
carbon in involved. Lastly, freeze drying was done to stabilizes the penicillin. Two steps of drying
were done and results in 1-4% of yield. Material balance were calculated in each tank whereas
energy balance was only calculated at the last tank which is the drying process. The general
equation for penicillin production is as follow:
Number of moles were calculated and atomic balance for each substance were performed
where nitrogen (50.8kmol/hr), sulphur (55.56kmol/hr), carbon(619.6kmol/hr). Full calculation can
be referred at the mass balance part of this paper. Energy balance at the drying tank was calculated
to be at 7.618x10⁸.
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Content Page Number
Executive Summary 1
Table of Contents 2
Introduction 3
Introduction of Product 3
History & Background 4
Industrial Application 5
Process Selection 7
Alternative Production Process 7
Balanced Overexpression of Isopenicillin N 8
Acyltransferase
Final Process Selection 9
Physical and Chemical Properties for each Chemocals 11
Process Equipment 14
Material Balances 19
Energy Balances 26
References 30
2
INTRODUCTION
Introduction of Product
Antibiotics are chemical substances that can inhibit the growth even destroy and harmful
microorganisms. They are derived from special microorganisms or other living systems, and are
produced on an industrial scale using a fermentation process. Although the principles of antibiotic
action were not discovered until the twentieth century, the first known use of antibiotics was by
the Chinese over 2,500 years ago. Today, over 10,000 antibiotic substances have been reported.
Currently, antibiotics represent a multibillion dollar industry that continues to grow each year.
The first discovered natural antibiotic was Penicillin. Penicillins are a class of beta-lactam
antibiotics. Penicillins are generally bactericidal, inhibiting formation of the cell wall. It is used to
treat skin infections, dental infections, ear infections, respiratory tract infections, urinary tract
infections and gonorrhea. There are different types of Penicillin antibiotic products: Amoxicillin
Trihydrate, Ampicillin, Trihydrate Ampicillin, Anhydrous Cloxacillin Sodium, Dicloxacillin
Sodium (Henry, 2007).
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It is less active than benzylpenicillin, however, and is appropriate only in conditions where high
tissue concentrations are not required.
A few years later in 1932, a paper was published which suggested a method for treating
infected wounds using a penicillin preparation. Although these early samples of penicillin were
functional, they were not reliable and further refinements were needed. These improvements came
in the early 1940s when Howard Florey and associates discovered a new strain of Penicillium,
which produced high yields of penicillin.
In December 1945, he and his colleagues Florey and Edward Chain received the Nobel
Prize in medicine for the discovery of penicillin and its therapeutic effect in various infectious
diseases (MedicineNet, n.d). This accidental discovery saved thousands of lives in later years and
had a major impact on pharmaceutical production of various antibioctics. This also allowed large-
scale production of penicillin, which helped launch the modern antibiotics industry. Figure 1 below
shown Alexander Fleming discovered the antibiotic properties of penicillin, produced by the mold
Penicillium chrysogenum.
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Figure 1: Alexander Fleming and his discovery of mold Penicillium chrysogenum
Industrial Application
Other example of penicillin industrial application such as Penicillin G acylases. PGA are
involved in the industrial production of semi-synthetic penicillins, which remain the most widely
used group of antibiotics. Penicillin acylases are useful as biocatalysts in many potentially valuable
reactions such as protection of amino and hydroxyl groups in peptide synthesis, as well as in the
resolution of racemic mixtures of chiral compounds (Foster, Woodruff & Mcdaniel, 2012).
An alternative use of PGA is in peptide synthesis. The acylase can be used for the
protection and de-protection of amino groups of amino acids by direct enzymatic synthesis and
acyl group transfer reactions. For example PGA has been used as a biocatalyst in the synthesis of
the sweetener aspartame, and further use has been in the preparation of D-phenyl dipeptides whose
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esters readily undergo ring closure to the corresponding diketopiperazines (Moyer & Coghill).
Such peptides are used as food additives and as synthons for fungicidal, antiviral and anti-
allergenic compounds. In addition, PGA can hydrolyze phenyl acetyl derivatives of a number of
peptides and re-solve enantiomers of some organic compounds.
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PROCESS SELECTION
Yeast Genetics
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Balanced Overexpression of Isopenicillin N Acyltransferase
Since the discovery of penicillin, classical strain improvement has been the main method
to improve penicillin production. One of the most important phenomena in high-
yielding Penicillium chrysogenum strains is the amplification of the penicillin biosynthetic gene
cluster between tandem repeats (Fierro et al., 1995). These strains contain multiple copies of the
penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)-L-cysteinyl-
D-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase
(IAT). Other changes are the upregulation of genes involved in side chain activation, α-
aminoadipic acid, and valine and cysteine biosynthesis (Van Den Berg et al., 2008). The penicillin
biosynthesis cluster in P. chrysogenum consists of three genes, pcbAB, pcbC, and penDE,
encoding the enzymes that catalyze the key biosynthetic conversions for penicillin production
(Gutiérrez, Fierro, Casqueiro, & Martín, 1999). The phenylacetic acid coenzyme A (CoA) ligase
(PCL) gene encoding the enzyme responsible for the activation of the side chain precursor
phenylacetic acid is localized elsewhere in the genome in a single copy (Koetsier et al., 2010).
Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze
a limiting step in high-yielding strains (Nijland et al., 2010). It is show that penicillin production
in high-yielding strains can be further improved by the overexpression of IAT while at very high
levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL
only marginally stimulates penicillin production.
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Final Process Selection
Various carbon sources have been adopted for this process which include glucose, sucrose
and other crude sugars. Approximately 65% of the carbon is used for cellular maintenance, 25%
for growth and only 10% for penicillin production. Sugar is also used for the regulation of the pH
value during active penicillin production phase (Soltero & Johnson, 1946).
Mini-harvest protocols are usually employed in penicillin fermentation. They involve the
removal of 20-40% of the fermentor contents and its replacement with fresh sterile medium. This
procedure can be repeated several times during this process without yield reduction. Penicillin is
excreted into the medium and recovered at the end of fermentation. Whole broth extraction is best
performed at acidic pH, with a 2-5% improvement in overall extraction efficiency. Solvent
extraction of chilled acidified broth is carried out with amyl, butyl or isobutyl acetate.
Present-day penicillin fermentations are highly automated and computerized. All the
necessary precursors, ammonia, sugar, carbon dioxide, oxygen are controlled, with thorough
monitoring of temperature and pH for optimal antibiotic production. The pH should be between
6.4 and 6.8 during the active production phase.
There are two types of culturing techniques used to grow large amounts of micro-
organism. Batch culturing has all substrates added together are the start and the products harvested
at the end. The cells are in the exponential growth for a smaller period and the amount of product
is limited by the initial amount of substrate. This means batch culturing is less productive but
contamination is less likely. Sometimes it is necessary to use batch culturing: bacteria or fungus
will not produce antibiotic in the exponential phase because there isn't much competition.
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Another process is continuous culture where a new substrate is continually added and
product continually harvested. In this method, the cells are maintained in the exponential growth
phase. However, the fermenter can only be used for one product and contamination is more likely.
The antibiotic Penicillin is obtained from a strain of the mould Penicillium chrysogenum. It is
fermented in a batch culture as if the P. chrysogenum is kept in the exponential phase it uses the
energy to grow rather than make Penicillin.
1) Broth filtration.
2) Filtrate cooling.
3) Additional filtration.
4) Penicillin extraction.
5) Treatment with carbon.
6) Aqueous transfer.
7) Recovery of solvent.
8) Crystallization.
9) Washing of crystals.
10) Drying of crystals.
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Physical and Chemical Properties for each Chemicals.
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Table 4.2: Chemical Properties
12
PROCESS FLOW DIAGRAM
13
PROCESS EQUIPMENT
Penicillin is a group of antibiotic and derived from Penicillium fungi. Generally, industrial
production of penicillin can be classified into two processes ; upstream processing and downstream
processing . Upstream processing is prior input to the fermenter which leads to the synthesis of the
product while the latter one refers to the processes done to purify the output from the fermenter
until desired product is reached.
The first step to produce Penicillin involves fermentation which is a metabolic process of
raw materials being converted into any important products (antibiotic) by microorganism.
Naturally, the particular process is carried out in a fermenter, which is a type of a bioreactor. A
fermenter is defined as a vessel in which sterile nutrient media and pure culture or microorganism
are mixed together where fermentation process is carried out under aseptic and optimum
condition. Most microorganisms grow optimally between a pH of 6 and 8 and a temperature of 20-
40°C
The types of fermenter used for this particular research purposes is an industrial scale
fermenter to fulfil the research requirement which was set up at 50k metric ton. This is because
the particular fermenter sizes ranges from 18927.06litres to 378541 litres (“Fermentation
Equipment And Its Application”, n.d). The materials used for designing of a fermenter must also
be taken into consideration. For example, the fermentation of lactic acid and penicillin must use
fermenter that are made up of wooden material (“Design of A Fermenter”, n.d) .
Based on Figure 2, there are a few major parts of fermenter that plays important roles for
the equipment to work. As such, impellers plays important role to mix the microorganism, media
and oxygen uniformly. The blades however, helps to reduce the size of air bubbles and distributes
them evenly into the fermentation media. However, these bubbles must be kept in check as it can
cause contamination. In the other hand, baffles are important to break the vortex formed during
the agitation process by the impeller. If the vortex is not controlled, the fermentation media may
spilled . Mass production of antibiotic penicillin can be produced via deep-tank batch fermentation
as the first tank. This is because the Penicillium mould are grown in deep-tank batch fermenters
following addition of sugars. The process take place for 6-8 days.
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Figure 1 shows a basic process to produce penicilin
Next, the products coming out from a fermenter are impure and diluted. Therefore, they
need to be purified. The fermentation broth contains intact microorganism, cell fragments and both
soluble and insoluble medium components. As such, the main objectives of this stage is to remove
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large solid particle by either centrifugation or filtration ( . In filtration, microorganisms are
captured in a dense ‘cake’ which consists of sand, sludge or paste. This is why filtration process
is considered to be the most suitable method to remove all the insoluble particle in the broth. The
Rotary Vacuum Filter is the most establish equipment used for penicillin extraction (McGlade &
Lennon, 2015).
As the penicillin extract contain in a broth, the vacuum is needed to remove moisture from
the ‘cake’. Based on Figure 3, The Filter Drum is both cylindrical and hollow. the liquid is drawn
through the filter and a cake of solids builds up on the outer surface. The filter drum, partially
submerged in the trough of broth, rotates slowly. Filtrate and washings are kept separate by the
segments in the drum. Inside the drum, the filtrate is moves from the end of the cylindrical drum
onto a storage tank. Next, the section at the node/knife, which scrapes off the filtrate can get air
pressure to burst out, helping contact with the node. After it passes this particular stage, the broth
collected is rich with penicillin.
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As mentioned earlier, the penicillin is contained in a broth after filtration process take
place. Therefore, as these substance are dissolved in a liquid solution, it need to be removed or
isolation. This is where adsorption process is needed. By definition, adsorption is the adhesion of
molecules or particles from gas and aqueous solution to a surface which creates a film of the
adsorbate where a series of activated carbons was applied for the removal of penicillin. Thus, it
can collect penicillin in bulks. However, this only applies on low concentration aqueous solution
during penicillin removal (Conchi et al., 2000). Adsorption process is widely used due to its
significant advantages. For example, active carbon adsorption consumes low energy and
maintenance costs. It also requires less supervision. Based on Figure 5, the adsorbate being the
peniciliin collected in bulks and the adsorbent being the activated carbon. As such, the penicillin
adheres to the activated carbon and is isolated to undergo the next process.
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The final step for Penicillin production is drying. Being a heat sensitive product, the
particular step is needed to stabilizes the penicillin. Freeze drying method is used to freeze both
organic and inorganic material which includes liquid and fluid substances. The process is done in
a vacuum and evaporation chamber with very low temperature and pressure. This is to create an
optimum environment to start the sublimation process to evaporate the excess solid into vapour .
Therefore, it is the only way to evaporate the aqueous solution of Penicillin to dryness and without
loss of activity (“The Development of Penicillin: Howard Florey’s Surface Culture Method”,
2013). There are 2 types of drying involve in the stage which are primary drying and secondary
drying. As for the primary drying, 95% of frozen water is evaporated while at the secondary drying
the excess water is dried and finally the product yield is at 1-4% (“Penicillin Production Process”,
n.d) .
Among many reason this process was chosen includes giving the product an outstanding
solubility characteristics, viability and longer shelf life. Therefore, this method is the most
common method for manufacturing solid pharmaceutical products. Based on Figure 4, the
essential components includes the chamber (vacuum tight box) and shelves which acts as a heat
exchanger (Patil & Pharm , n.d). Therefore, energy balance must be considered and calculated
thoroughly at this process
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PROCESS & DESIGN IMPROVENT
3. Starch or glucose use in the penicillin production can be substitute with corn syrup.
Allow maximal growth of the culture at the expense of product (antibiotic) formation. This
is because growth and antibiotic production are inversely proportional which more
secondary metabolites during stress phase.
4. Selection of strain
Selection of the best strain depends on the production rate of the secondary metabolite
(penicillin).
Strains are grown on cultures in laboratories and those with best yield is determined.
For example, used of Penicillium notatum (1 mg/dm3) and Penicillium chrysogenum (50
mg/dm3).
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6. Sexual Reproduction
Fungus also has a sexual cycle.
The progenies possess a combination of genes from both mating partners and thus have
new properties, both at the molecular level, as well as in their phenotypes.
Specific environmental conditions; in the dark under oxygen deprivation conditions in a
nutrient medium supplemented with the vitamin biotin.
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Bax R.P. (2009). Clinical trials and the pharmaceutical industry. J Antimicrob Chemother
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Fierro, F., Barredo, J. L., Díez, B., Gutiérrez, S., Fernández, F. J., & Martín, J. F. (1995). The
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