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EXECUTIVE SUMMARY

This research was conducted to produce a batch of Penicilin antibiotic. A certain


requirement was established and Penicilin was produced at 50,000 metric tonnes per year. Penicilin
is the first discovered natural antibiotic which obtained from ‘Penicillium molds’. Nowadays, the
properties of the particular substance are studied further and it is the most versatile antibiotic ever
recorded. It is also used in the industry as an alternative use in peptide synthesis. The chemicals
involved in the production process includes glucose, sulfuric acid, water and ammonium. The first
process involve is a deep-tank fermentation which converts the raw material into Penicilin under
an optimum condition.

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:

C6H12 + SO4 + NH3 C16H1804N2S + H20 + C4H703N

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 Flow Diagram 13

Process Equipment 14

Material Balances 19

Energy Balances 26

Process & Design Improvement 28

References 30

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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).

Penicillin was obtained from multicellular fungi, “Penicillium molds”. Penicillin is a


group of compounds having common basic nucleus, 6-amino penicillinic acid (6-APA). 6-APA
contains ring like structure termed as a β-lactam ring. Penicillin consist of two different types
which are Natural Penicillin and Semi-synthetic Penicillin. Natural Penicillin is formed during the
process of mold fermentation and the semisynthetic penicillins (those in which the structure of a
chemical substance—6-aminopenicillanic acid—found in all penicillins is altered in various
ways). There are two common types of natural penicillin used in medical industrial namely,
Penicillin G and Penicillin V.

Benzylpenicillin, commonly known as Penicillin G, is the gold standard penicillin.


Penicillin G is typically given by a parenteral route of administration (not orally) because it is
unstable in the hydrochloric acid of the stomach. Because the drug is given parenterally, higher
tissue concentrations of penicillin G can be achieved than is possible with phenoxymethylpenicillin
(Bajpai & Reuss, 2009). These higher concentrations translate to increased antibacterial activity.
Phenoxymethylpenicillin, commonly known as Penicillin V, is the orally active form of penicillin.

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It is less active than benzylpenicillin, however, and is appropriate only in conditions where high
tissue concentrations are not required.

History & Background

In September 1928, Alexander Fleming, a professor of bacteriology at St. Mary’s Medical


School in London, made one of the most important contributions to the field of antibiotics. He first
observed the antibiotic properties & therapeutic value of penicillin. He found that a strain of green
Penicillium mould inhibited the growth of bacteria on an agar plate in his experimental study. He
observed that mould had developed accidentally on a Staphylococcus aureus culture plate that was
left on the laboratory bench and that the mould had created a bacteria-free circle around itself (ACS
Chemistry for Life, 2012). He was inspired to further experiment and he found that a mould culture
prevented growth of Staphylococcus even when diluted 800 times. He named the active substance
penicillin. This led to the development of the first modern era antibiotic which is penicillin.

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

In recent years, penicillin antibiotics mainly created large contribution in pharmaceutical


industry as medication purposes. Penicillin production significantly higher rate in the market for
antibiotics. There are several different kinds of penicillins. Each is used to treat different kinds of
infections. One kind of penicillin usually may not be used in place of another. In addition,
penicillins are used to treat bacterial infections in many different parts of the body. They are
sometimes given with other antibacterial medicines (antibiotics). For example, Penicillin V
potassium is used to treat many different types of infections including strep and staph infections,
pneumonia, rheumatic fever, and infections affecting the mouth or throat (Bax, 2009).
Furthermore, Penicillin V potassium is also used to prevent infections of the heart valves in people
with certain heart conditions who need to have dental work or surgery.

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

Alternative Production Process

Yeast Genetics

The industrial production of this β-lactam antibiotic is confined to the filamentous


fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized
producer of pharmaceuticals, represents an attractive alternative (Stöckmann et al., 2009).
Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS)
δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the
production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene
encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example
of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with
the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the
two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase
(PCL) resulted in production of biologically active penicillin, which was efficiently secreted (Van
De Kamp, Driessen, & Konings, 1999). The amount of secreted penicillin was similar to that
produced by the original P. chrysogenum NRRL1951 strain. Penicillin production was decreased
over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of
IAT and PCL is important for efficient penicillin production (Gidijala, Van Der Klei, Veenhuis, &
Kiel, 2007). The breakthroughs of this work enable exploration of new yeast-based cell factories
for the production of β-lactam antibiotics as well as other natural and semi-synthetic peptides,
whose production involves NRPS's.

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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

After finalizing a few ways to produce Penicillin, production of Penicillin by fermentation


was chosen. Fermentation is the technique used for the commercial production of penicillin. It is
a fed-batch process that is carried out aseptically in stainless steel tank reactors with a capacity of
30 to 100 thousand gallons. The fermentation involves two to three initial seed growth phases,
followed by a fermentation production phase with a time cycle ranging from 120 to 200 hours
(Elander, 2003).

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.

Because penicillin is produced naturally by fungi after fermentation penicillin must be


flushed of any impurities. Downstream processing purifies the end product and ensures the
penicillin produced is as pure and healthy as possible. There are ten steps to the downstream
processing of 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.

Table 4.1: Physical Properties

Physical Glucose Sulfuric Water Ammonium Penicillin Carbon Dioxide


Properties Acid

Synonym Monosaccharide - Hydrogen Ammonium Penicillin V -


sugar Oxide Ion

Molecular C6H12O6 H2SO4 H2O NH4 C16H18N2O5S CO2


Formula
Molecular 180.156 98.072 18.01 18.039 350.39 44.01
Weight
(g/mol)
CAS 50-99-7 7664- 7732-18- 14798-03-9 87-08-1 124-38-9
Number 93-9 5
Density 1.02 1.840 0.9998 0.91 g/cm3 - 1.43 0.001977
(g/mL) 0.88 g/cm3
Boiling >146°C 337 °C 99.98 37.7 °C - −78.5 °C
Point (°C)
Melting 146 °C 10 °C 0 −57.5 °C - 120-128 ºC −56.6 °C
Point (°C) −91.5 °C

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Table 4.2: Chemical Properties

Chemical Glucose Sulfuric Acid Water Carbon Dioxide


Properties

Std molar 209.2 157 69.95 214


entropy (J/mol.K)
Std enthalpy of −1271 −814 -285.83 −393.5
formation
(kJ/mol)
Enthalpy of −2805 - - -
combustion
(kJ/mol)
Heat capacity 218.6 - 75.375 37.135
(J/mol.K)

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PROCESS FLOW DIAGRAM

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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

Figure 2 shows the design of a fermenter

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.

Figure 3 shows a Rotary Drum Vacuum Filter

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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.

Figure 5 shows Adsorption 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

Figure 4 shows the components of freeze drying

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PROCESS & DESIGN IMPROVENT

1. Improvement at the downstream process


 The castellation of penicillin can be increase with use of proper equipment.
 For example by using rotary drum filtration, the yield of the product can be increase as the
rotary drum keep oscillate, thus remove the impurities on the penicillin as well as increase
the product.

2. Improvement at the upstream process


 Yield of the penicillin can be increase by increase the feed of the production.
 For example by using large volume of fed-batch fermenter, the amount of the feed also
can be increase. Thus increase the production of the penicillin.

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).

5. Mutation and Selection


 Several strains of Penicillium are cultured.
 Combination of the mutagens leads to a more positive result.
 Strains are exposed to the mutagens at different intensities and proportions.
 Each time the best strains are selected and are further exposed.
 Statistical tests are done to determine the strains with highest yields.

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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.

7. Select the best temperature


 Temperature of the fermenter will affect the yield of the penicillin.
 Penicillin are make from microorganism and microorganism has specific temperature for
them to be culture and growth.
 For example, the used of temperature at 25°C is the best temperature as its increase the
yield of the penicillin.

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