penicillin prodtn using penicillium chrysogenum

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Industrial Production of Penicillin using Penicillium chrysogenum Industrial Biotechnology 25th October 2013 Presented by: - Nazeer Huda - Seetul Yajeshwar - Cécile Christabelle

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Page 1: Penicillin prodtn using penicillium chrysogenum

Industrial Production of Penicillin using Penicillium

chrysogenum

Industrial Biotechnology25th October 2013

Presented by: - Nazeer Huda- Seetul Yajeshwar- Cécile Christabelle

Page 2: Penicillin prodtn using penicillium chrysogenum

Overview

● Introduction

● What is penicillin?

● History

● General structure of penicillins

● Penicillin derivatives

● How does penicillin work?

● Spectrum of activity

● Fermentors

● Important aspects of fermentors

● Specific conditions for production

● Media consideration & formulation

● Primary & secondary metabolism

● Production method● Stages of production● Production of penicillin● Classification of penicillins● Increase in yield● References

Page 3: Penicillin prodtn using penicillium chrysogenum

Introduction

We have all, at one point or the other, heard about

penicillin. From its accidental discovery and massive use

in World War II to the Nobel Prize for Medicine in 1945

by its rightful owners, penicillin has made its proof as the

“Miracle Drug” that revolutionized the course of the

medical industry. Without further ado, let us take a look

at an important aspect that made penicillin accessible at

large; its industrial production.

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What is Penicillin?

• First true naturally-occurring antibiotic ever discovered:

a great medical breakthrough.

• Group of antibiotics produced by the Penicillium fungi.

It is a group of closely related compounds, not a single

compound.

Examples: Amoxicillin, ampicillin,

phenoxymethylpenicillin.

• Around 50 drugs that are penicillins.

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History: Discovery & Production

• 1928: Scottish biologist, Alexander Fleming discovered

that the Staphylococcus culture he had mistakenly left

growing in open was contaminated with a mould which

had destroyed the bacteria.

• After isolating a sample and testing it, he found that it

belonged to the Penicillium family.

Later the mould was classified as Penicillium notanum.

• At first, it was difficult to convince people about its

potential uses.

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History: Discovery & Production

• But later (1939), using Fleming’s work, two medical

researchers, Howard Florey and Ernst Chain managed

to purify penicillin in a powdered form.

• 1941: They successfully treated a human.

• 1943: They produced penicillin on a large scale.

This helped immensely to treat casualties during the

WWII that had bacterial infections due to their wounds.

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Page 8: Penicillin prodtn using penicillium chrysogenum

General Structure of Penicillins

• Have β-Lactam functional group, thus belong to the β-

Lactam antibiotic group.

• They all have a 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).

The nature of this acyl group produces specific

properties on different types of penicillin.

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Penicillin Derivatives

• Derivatives produced to deal with the problem of

bacterial resistance to penicillin.

• All penicillin or penicillin derivative

has a constant core region which is

the 6-APA.•• The only region that is different from different types of

penicillin derivative is its R group.

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How Does Penicillin Work?

• Inhibits the synthesis of peptidoglycan in cell walls.

o β-Lactam of penicillin binds to the enzyme -

transpeptidase, that is used in the formation of

peptidoglycan cross linking.

o The enzyme is inhibited, thus inability to form cross

linking.

o Cell wall is weakened causing osmotic imbalance in

the cell. This leads to cell death.

• As human cells do not have cell walls, penicillin does

not affect them.

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Page 12: Penicillin prodtn using penicillium chrysogenum

Spectrum of Activity

• Effective against actively growing Gram positive

bacteria - which have thick peptidoglycan.

• Some penicillins like amoxicillin are also effective

against Gram negative bacteria, except Pseudomonas

aeruginosa.

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Fermentors● Purpose of fermentor: provide contained, controlled and

homogeneous environment in which the fermentationcan proceed in a manner that is both safe and practicaland which optimises the particular objectives of thefermentation.

● Other primary factors include cost, reliability and safety.

● For reactor being designed for specific purpose, thereare a number of important parameters that will greatlyaffect performance:

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1. Reactor Size: optimum rates of production?

2. Reactor Configuration: mechanical agitation or will a

bubble column.

3. Mode of operation: Will it be batch fed or continuously

fed?

4. Conditions inside the reactor: how will conditions (pH,

temperature, …) be controlled?

Economic requirements:

● Easy to operate aseptically.

● Reasonably flexible regarding process requirements.

● Low power consumption.

● Stable under fluctuating conditions.

● Cheap, robust, simple and well understood for scale-up.

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Typical Fermentor

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Some Important Aspects● Mass Transfer: good transfer of oxygen across the

liquid interface-the Sparger delivers this oxygenefficiently.

● Heat Transfer: metabolism as a process tends to giveoff heat-achieved through cooling jacket whereby coolwater is passed through.

● Bulk Flow and Mixing: impellers, bubble columns orloop reactors.

● Batch, Fed-Batch and Continuous Culture: hownutrients and substrate will be delivered to a culture in areactor.

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-Batch – a fixed amount of substrate is added at the

beginning whereby the volume of nutrients remains the

same throughout the process.

-Fed Batch - substrate is added in small increments at

various times in the fermentation and consequently volume

increases.

-Continuous - substrate is constantly added to the reactor

while an equal amount of fermented medium is removed.

Volume again remain the same but constantly renewed

with fresh ones.

● Steam: Used to keep the reactor running aseptically.

(temperature/pressure of 121°C/15 psi for 15-30min).

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Specific Conditions for Penicillin Production

● Most penicillins form filamentous broths. This meansthey can be difficult to mix due to their high viscosity.Also the increasing viscosity of the broth can hinderoxygen transfer.

Page 19: Penicillin prodtn using penicillium chrysogenum

- A solution for the viscosity and the filamentous growth of

penicillium species could be bubble columns (air lift

reactors) which would distribute the oxygen equally and

also to agitate the medium.

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● •Penicillin is an aerobic organism; oxygen supply is

critical-reactor must have an efficient oxygen supply

system.

● •The optimum pH for penicillin growth is 6.5-maintain pH

efficiently (pH controller and acid-base reservoir).

● •Strain Stability problems (mutations) - careful strain

maintenance is required.

● •Biomass doubling is about 6h-provisions must be made.

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Media Consideration

● The aim of the media is to:

- provide all the elements required for the synthesis ofcell materials and the formation of the desired product.

- provide favourable environment for the culture inquestion.

- be cost effective.

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Media Formulation● pH 6.5

● Temperature 20-24 °C

● Oxygen

● Nitrogen: corn steep liquor 8.5 %

● Glucose 1%

● 80% ethanol

● phenylacetic acid

● Probenecid

● Lactose 1%

● Calcium Carbonate 1%

● Sodium hydrogen phosphate 0.4%

● Antifoaming agent: vegetable oil

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● Microorganisms require C, H, O, S and N for cell growth

and cell maintenance.

● Also require small amounts of trace elements such as

Cu, Mn and Co (frequently depend on the water source)

or growth factors such as vitamins or amino acids.

● Certain organisms such as Penicillium chrysogenum

that produce antibiotics, enzymes or other secondary

metabolites frequently require precursors like

purine/pyrimidine bases or organic acids to produce

metabolites.

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Primary and Secondary Metabolism

● Primary metabolism is the metabolism of energyproduction for the cell and for its own biosynthesis. Inaerobic organisms (such as Penicillium chrysogenum) itinvolves the conversion of sugars such as glucose topyruvic acid and the production of energy.

● Secondary metabolism regards the production ofmetabolites that are not used in energy production forexample penicillin from Penicillium chrysogenum. Themetabolite is being utilized as a defence mechanismagainst other microorganisms in the environment.Penicillium chrysogenum can kill off the competition toallow itself to propagate efficiently.

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Production Method

● Secondary metabolites are only produced in times ofstress when resources are low and the organism mustproduce these compounds to kill off its competitors toallow it to survive.

● It is these conditions that we wish to duplicate in order to achieve the maximum amount of product from our fermentation.

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Stages of Production1. Primary metabolism will be emphasised. Media for this

stage will be focussed on achieving maximum growthand biomass production.

2. Once the desired biomass has been achieved, starve(Limiting the amount of C and N available to theculture) the culture and induce the kind of stressconditions that trigger the production of the antibiotic.

★ Use the fed-batch method to feed the culture. As statedabove, this allows us to add the substrate to the reactorin small increments and to even change the substrate ifwe so desire.

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Production of Penicillin

● Penicillin was the first important commercial productproduced by an aerobic, submerged fermentation.

● First antibiotic to have been manufactured in bulk.

● At the end of the WWII, penicillin was first made usingthe fungus Penicillium notatum, which produced a yieldof 1 mg/dm3

● Today, using a different species known as Penicilliumchrysogenum, and better extraction procedures, theyield is 50 mg/dm3

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The Industrial Production of Penicillin

This can be broadly classified into two processes namely:

1. Upstream Processing- referring to processes before input to the fermenterand encompases any technology that leads to thesynthesis of a product. It includes the exploration,development and production.

2. Downstream Processing- referring to processes done to purify the output of thefermenter until it reaches to the desired product, suchas extraction and purification of a product fromfermentation.

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Page 30: Penicillin prodtn using penicillium chrysogenum

Simplified Flow Chart

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★Medium for penicillin

1. The Penicillium chrysogenum usually contain its carbonsource which is found in corn steep liquor and glucose.

2. A medium of corn steep liquor and glucose are added tothe fermenter. Medium also consists of salts such asMgSO4, K3PO4 and sodium nitrates. They provide theessential ions required for the fungus metabolic activity.

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★Heat sterilization

3. Medium is sterilized at high heat and high pressure,usually through a holding tube or sterilized together withthe fermenter.

4. The pressurized steam is used and the medium is heatedto 121°C at 30 psi or twice the atm. pressure

Note: High temp. short time conditions are used to minimise degradation of certain components of the media.

Sterilisation machine

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★ Fermentation5. It is done in a fed-batch mode as glucose must not be

added in high amounts at the beginning of growth (whichwill result in low yield of penicillin production asexcessive glucose inhibit penicillin production).

6. The fermentation conditions for the Penicillium mold,usually requires temperatures at 20-24°C while pHconditions are kept at 6.5

7. The pressure in the bioreactor is much higher than theatmospheric pressure (1.02atm). This is to preventcontamination from occurring as it prevents externalcontaminants from entering.

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★ Fermentation

8. It is necessary to mix the culture evenly throughout theculture medium. Fungal cells are able to handle rotationspeed of around 200 rpm.

Fermentors

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★ Seed Culture

9. The seed culture is developed first in the lab by theaddition of Penicillium chrysogenum spores into a liquidmedium. When it has grown to the acceptable amount, itis inoculated into the fermenter.

10. The medium is constantly aerated and agitated.Carbon and nitrogen are added sparingly alongsideprecursor molecules for penicillin fed-batch style.Typical parameters such as pH, temperature, stirrerspeed and dissolved oxygen concentration, are observed.

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★ Seed Culture

11. After about 40 hours, penicillin begins to be secreted bythe fungus.

12. After about 7 days, growth is completed, the pH rises to8.0 or above and penicillin production ceases.

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★Removal of biomass13. Filtration is carried out as bioseparation is required to

remove the biomass from the culture (removing the fungusand other impurities away from the medium, which containsthe penicillin).

14. A Rotary vacuum filter is commonly employed for filtration asit is able to run in continuous mode in any large scaleoperations.

15. After filtration, phosphoric acid,a non-oxidising agent, is introduced,to decrease pH from 8.0 to 6.5 so as to prevent loss of activity of penicillin.

Rotary vacuum filter

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★Addition of solvent

16. Organic solvents such as amyl acetate / butyl acetate areadded to dissolve the penicillin present in the filtrate.

17. At this point, penicillin is present in the solution andany other solids will be considered as waste (can be usedas fertilizers and animal feed).

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★Centrifugal Extraction18. Centrifugation is done to separate the solid waste from the

liquid component which contains the penicillin.

19. Usually a disk centrifuge is used at this point.

20. The supernatant will then be transferred further in the downstream process to continue with extraction.

Disk centrifuge - One of the most common type of centrifuge for large scale production

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21. A series of extraction processes are carried upon thedissolved penicillin, to obtain a better purity of thepenicillin product.

22. The acetate solution is first mixed with a phosphatebuffer, followed by a chloroform solution, and mixedagain with a phosphate buffer and finally in an ethersolution.

23. Penicillin is present in high concentration in the ethersolution and it will be mixed with a solution of sodiumbicarbonate to obtain the penicillin-sodium salt, whichallow penicillin to be stored in a stable powder form atrtp.

★Extraction

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★Extraction24. The penicillin-sodium salt is obtained from the liquid

material by basket centrifugation, in which solids are easilyremoved.

Batch extraction unit Basket Centrifuge-Extremely using in the removal of solids in this case Penicillin salt

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★Fluid bed drying25. Drying is necessary to remove any remaining moisture

present in the powdered penicillin salt.

26. In fluid bed drying, hot gas is pumped from the base of the chamber containing the powdered salt insidea vacuum chamber.

27. Moisture is removed this way, and this result in a much drier form of penicillin.

Fluid bed drying tube

Powdered penicillin being blown by hot air

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★Storage

28. Penicillin is stored in containers and kept in a driedenvironment.

The White Penicillin-Sodium salt

29. The resulting penicillin (called Penicillin G) can bechemically and enzymatically modified to make a variety ofpenicillins with slightly different properties.

30. These can be semi-synthetic penicillins, such as; PenicillinV, Penicillin O, ampicillin and amoxycillin.

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Classification of Penicillins

Four known classes:

1. Natural Penicillins

2. Penicillinase-Resistant Penicillins

3. Aminopenicillins

4. Extended Spectrum Penicillins

Categorized based on their ability to kill types of bacteria and

how effective are they in doing so.

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1. Natural Penicillins:

The natural penicillins are based on the original penicillin-G

structure. They are effective against both gram-positive strains

and gram negative bacteria.

Examples of Natural Penicillins are Penicillin G, Procaine,

Penicillin O, Penicillin V and Benzathine.

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2. Penicillinase-Resistant Penicillins:

The Penicillinase-resistant penicillins have a narrower spectrum

of activity in contrast to the natural penicillins. Their antimicrobial

efficacy is targeted on penicillinase-producing strains of gram-

positive cocci, such as Staphylococcal species and these drugs

are also known as ‘anti-staphylococcal penicillins’.

Examples of Penicillinase-Resistant Penicillins are Dicloxacillin,

Methicillin, Nafcillin, Oxacillin and Cloxacillin.

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3. Aminopenicillins:

Active against gram-negative bacteria (such as E. coli).

Aminopenicillins are acid-resistant-can be administered orally.

Orally administered amoxicillin and ampicillin are used primarily

to treat mild infections such as otitis media, sinusitis, bronchitis,

urinary tract infections and bacterial diarrhoea.

Examples of Aminopenicillins are Amoxicillin, Ampicillin and

Bacampicillin.

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4. Extended Spectrum Penicillins:

These agents have similar spectrums of activity as compared to

the aminopenicillins but it has an additional activity, which is

against several gram negative organisms in the family

Enterobacteriaceae and some strains of Pseudomonas

aeruginosa.

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Increase yield● Adding corn syrup

● Selection of strain

● Mutation and Selection

● Sexual reproduction

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Corn syrup

Allow maximal growth of the culture at the expense of product(antibiotic) formation. This is because growth and antibioticproduction are inversely proportional.

More secondary metabolites during stress phase.

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Selection of strain

● Selection of the best strain depends on the productionrate of the secondary metabolite (penicillin).

● Penicillium notatum (1 mg/dm3) and Penicilliumchrysogenum (50 mg/dm3).

● Strains are grown on cultures in laboratories and thosewith best yield is determined.

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Mutation and Selection

This is based on trial and chance.

● Several strains of Penicillium are cultured.

● Ethyl methanesulphonate (EMS), near-ultraviolet light

in the presence of 8-methoxypsoralen (8MOP) are used

as mutagens.

● Combination of the mutagens leads to a more positiveresult.

● Strains are exposed to the mutagens at different intensitiesand proportions.

● Each time the best strains are selected and are furtherexposed.

● Statistical tests are done to determine the strains withhighest yields.

● Dry conidia is stored in silica gel at 4°C at each stage.

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Sexual Reproduction

● It was assumed that the penicillin-producing mouldfungus Penicillium chrysogenum only reproducedasexually through spores. Fungus also has a sexualcycle(Jan.8 2013)

● The progenies possess a combination of genes from bothmating partners and thus have new properties, both at themolecular level, as well as in their phenotypes.

● Specific environmental conditions; in the dark underoxygen deprivation conditions in a nutrient mediumsupplemented with the vitamin biotin.

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How is Penicillin G enzymatically modified?

● Using PGA (Penicillin G acylase) which is a hydrolytic enzyme.

● It acts on the side chains of penicillin G to produce β-lactam antibiotic. This antibiotic has by-products which has the potential building blocks of semi-synthetic antibiotics, such as ampicillin and amoxicillin.

● The high demand for PGA is being met through a submerged fermentation process that uses genetically manipulated E.coli and Bacillus megateriummicroorganisms.

● Advancements in biotechnology such as screening of microorganisms, site-specific mutagenesis, immobilization techniques, and modifications to the fermentation process could enhance the production of PGA.

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Recombinant DNA Technology

• Increase the gene copy number.

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References● http://penicillin.wikispaces.com/Home (15/09/2013)

● http://penicillin.wikispaces.com/General+bioprocess+flow (15/09/2013)

● http://penicillin.wikispaces.com/Various+types+of+Penicillin(15/09/2013)

● http://microbiologyprocesses.blogspot.com/2011/12/penicillin-production.html (20/09/2013)

● http://inventors.about.com/od/pstartinventions/a/Penicillin.htm(20/09/2013)

● http://www.medicinenet.com/penicillins-oral/article.htm (30/09/2013)

● http://www.eolss.net/Sample-Chapters/C17/E6-58-05-04.pdf (3/10/2013)

● http://mic.sgmjournals.org/content/113/2/209.full.pdf+html (11/10/13)

● http://www.sciencedaily.com/releases/2013/01/130108084138.htm(11/10/13)

● http://archive.bio.ed.ac.uk/jdeacon/microbes/penicill.htm (11/10/13)

● http://www.sciencedirect.com/science/article/pii/S0141022907003560(07/11/13)

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Thank you for your attention!