Penicillin Production

Tom O’Hare & Lynne White Abstract The discovery of penicillin and its medicinal uses was arguably the most important scientific discovery of the 20th century. From the observation of an accidental inoculation to the application of knowledge and technology, the production of penicillin is a process that has changed dramatically since its beginnings in the late 1930’s and early 1940’s. As new ways were found to ensure that more penicillin was being produced and that the purification process was as effective as possible, the production could increase, allowing the use of penicillin in times of war to save the lives of soldiers who otherwise would have died due to infection of their wounds. Since then, the development of large scale production has allowed penicillin to be used whenever needed to kill off bacteria and prevent serious infection, however, this has also been its downfall. The ability of some bacteria to now produce penicillinase to break down and render penicillin completely useless has come about due to the wide scale use of the drug and has therefore limited the effectiveness of penicillin as a clinical treatment. In order to combat this, scientists have turned to semi-synthetic derivatives of penicillin in the hope that these will have the properties necessary to beat the resistance problem.

Penicillin and its history
Penicillin was the first naturally occurring antibiotic discovered (Prontosil, the first chemical used to cure certain infectious diseases, had been discovered in 1933 but had serious side effects). There are now more than 60 antibiotics, which are substances that are produced by microbes and that fight bacteria, fungi and other microbes harmful to humans - the word means against (anti) life (bio). Penicillin is obtained in a number of forms from Penicillium moulds.

Penicillin: C16H18N2O5S 1

all with the same basic ring-like structure (a β-lactam) derived from two amino acids (valine and cysteine) via a tripeptide intermediate. which cross connects long polymers of sugars that form the bacterial cell wall. but it stops new ones forming. There are all kinds of what are called semisynthetic derivatives of penicillin . As a result. These compounds consist of the basic Penicillin structure. so the disease can't spread. etc. They enlarge to about twice their size before the DNA chromosome is copied. newly-formed cell walls will be structurally weak in some areas. a degree of resistance to penicillinase (or β-lactamase) (a penicillin-destroying enzyme produced by some bacteria) and an extended range of activity against some Gram-negative bacteria. 2 . Penicillin G (or benzylpenicillin) is the most widely used form and the same one we get in a shot (hypodermic) form.As shown in the above diagram. Biosynthetic penicillin is natural penicillin that is harvested from the mould itself through fermentation. the new cell wall won't be able to form. Penicillin acts by blocking the activity of the enzyme transpeptidase. Bacteria reproduce by dividing to produce two new cells. This means the bacteria can't reproduce. These modern semi-synthetic penicillins have various specific properties such as resistance to stomach acids so that they can be taken orally. The third amino acid of this tripeptide is replaced by an acyl group (R in the diagram below) and the nature of this acyl group produces specific properties on different types of penicillin. The two new chromosomes move apart and a cell wall forms between Ampicillin. Penicillin V. The β-lactam ring on penicillin (see structure) irreversibly blocks the activity of the enzyme by covalently bonding with the functional end of the enzyme. There are two different categories of penicillin. penicillin is not a single compound but a group of closely related compounds. but have been purposefully modified chemically by removing the acyl group to leave 6-aminopenicillanic acid and then adding acyl groups that produce new properties. Methicillin. Carbenicillin. The other form of penicillin is known as semi-synthetic. It doesn't harm old bacterial cell walls. But if penicillin is present. causing water to rush in and rupture the cell. Oxacillin.

This history clearly displays that discovery in itself is not enough. Mary’s Hospital in London. a bit of blue-green mould had fallen into a discarded culture plate containing Staphylococcus aureus. while working in St. bacteriologist Alexander Fleming was conducting research on the flu.Penicillin G Penicillin G is not stable in the presence of acid (it is therefore said to be acid-labile). influenced by his wartime experience. there must also be a drive (in this case the huge numbers of soldiers dying from secondary infections in the second world war) to utilise this new discovery to its fullest. The history of penicillin is a fascinating reminder of how the most freak and seemingly random of occurrences can lead to very significant scientific discoveries. Also seeing as how the discovery of penicillin is arguably man’s most important medical (if scientific) advance to date. after the Penicillium notatum mould that produced it and 1929. He had witnessed the deaths of many soldiers that died.0). he published the results of his investigations. the compound would be destroyed in our stomach before it could be absorbed into the bloodstream. He had been searching for antibacterial agents. Many of the semi-synthetic penicillins can be taken orally. It is for this reason that penicillin G must be taken by intramuscular injection . forming a clear patch in the surrounding area. some discussion of its origins are necessary. and would therefore not be any good to us as a treatment for infection somewhere in our body. In 1928. Since our stomach has a lot of hydrochloric acid in it (the pH can be around 2. While he was on holidays. but from secondary infections of those wounds. if we were to ingest penicillin get the compound in our bloodstream. which is not acidic at all. He named the antibiotic penicillin. not from the wounds they received during combat. From this he could conclude that the mould was producing an antibiotic substance. noting that his discovery might have therapeutic value if it could be 3 .

a 48-year-old London policeman had developed septicaemia as a result of a small cut on his face. Because the United States intended to enter into World War II in another few months the penicillin project. This underwent a slow purification process to produce the large amounts of clinically usable penicillin that became available for military use in early 1940's. It was Chain who began extracting penicillin into a purified and powerful antibiotic. Florey and his team were able to use beer-brewing technology to produce the huge amounts of the mouldy liquor needed for penicillin production. which became declared a war project. In Peoria. Scientists began to try to increase the amount of penicillin produced by P. While Fleming’s lab was poorly equipped with no staff support. Chain.produced in quantity. Albert Alexander. When treated with penicillin Alexander began to recover within the day. This mould was identified as Penicillium chrysogenum and produced approximately 200 times as much penicillin than what Florey’s team were working with (Penicillium notatum). All of the untreated mice died the next day while the treated mice all recovered. At first penicillin was made using old dairy equipment and after a great deal of effort. by irradiating it with X-rays and UV rays in order to induce mutations of this species. enough was extracted for experimentation to begin. followed by injection of penicillin in half the mice. Eight white mice were inoculated with deadly Streptococcus germs. and it would be 10 years before another significant leap forward for penicillin would occur. Now it was time for the first human test. In the same year Florey travelled to the United States (which at the time was still neutral) to continue his work with penicillin. At the same time scientists began to grow the mould in the first deep tank fermenters. Ernst B. Florey came across Fleming's paper on penicillin (which oddly had languished in obscurity). Howard W. However by 1941. was given top priority (and funding). Unfortunately it couldn’t. In 1938 Dr. They succeeded and developed a mutant that produced 1000 times the amount of penicillin than Fleming's original culture. it was acknowledged that penicillin was indeed a worthwhile drug and could save thousands of lives. Illinois a blue-green mould was found growing on a mouldy cantaloupe in a market. chrysogenum. However Florey's team didn't have enough of the drug to see the patient through to a full recovery and he later re-lapsed and died. Florey's lab was well equipped and staffed with a team of scientists at Oxford University that included Dr. 4 .

Reactor Size: How large does the reactor need to be in order to achieve optimum rates of production? 2. Flexibility is frequently a major objective in a lab. it all began with a bit of blue-green mould falling onto a discarded culture plate. Conditions inside the reactor: What temperature and pH should the reactor be maintained at? How will contamination be avoided? And how will these conditions be controlled? Regardless of how the above parameters are approached. including the Merck. In 1945 Fleming was awarded the Nobel Prize in Physiology and Medicine along with Florey and Chain. the purpose of a fermenter is to provide a contained. When a reactor is being designed for a specific purpose there are a number of important parameters that will greatly affect the reactors process performance. Generally.By late 1943. Some of the most common are summarized below: Functional: • High gas/liquid mass transfer. 5 . controlled. reliability and safety. 1. there is a huge range of variability and flexibility. strains and techniques were improved upon and Penicillin saved tens of thousands of lives. mass production of the drug had commenced and by the end of the war. As reactors become larger and larger there is less and less room for error. Reactor Configuration: How will the reactor be configured? For example should impellers be used to exert mechanical agitation (stirred tank) or will an air-jet system be used for mixing (bubble column)? 3. In the coming years. Fermenters An important part of any fermentation process (of which penicillin is an example) is the type and configuration of fermenter being used for the fermentation. It should be noted however that this flexibility tends to decrease with scale. However. Other primary factors include cost. Squibb and Pfizer. many companies were manufacturing the drug. Mode of operation: How will substrate be added? Will it be batch fed or continuously fed? 4. Within these parameters. there are still are number of common desired features that are requirements (both functional and economic) for a good working reactor. homogenous environment in which the fermentation can proceed in a manner that is both safe and practical and which optimises the particular objectives of the fermentation.

robust. Economic: • • • • • Easy to operate aseptically. Good bulk-flow and mixing to prevent the creation of dead zones in the reactor. Stable under fluctuating conditions. A good example is a small amount of dye being dropped into a bucket of water. Mass Transfer is seen when there is mixing between two components of varying concentrations. we need to take a step backwards to define some of the above terms. When the oxygen is bubbled through the fermenter tank. Low power consumption. Reasonably flexible regarding process requirements. Doran. Mass Transfer processes are responsible for the movement of the dye through the water until equilibrium is reached.• • • • Reasonable heat-transfer. Pauline M. before we examine the above design. simple and well understood for scale-up. Prevention of aggregation without damaging microorganisms. Below is an example of a typical reactor configuration: However. Cheap. 6 . Since oxygen concentration is such a 1 Bioprocess Engineering Principles. Good nutrient transfer.1 This is very important when we are attempting to grow aerobic microbes in media. there will be high concentrations of oxygen near the bubbles but low concentration everywhere else in the tank.

the reactor must have an efficient oxygen supply system. The impeller that serves to break up the foam generally relieves this. Most reactors use the Rushton Flat-bladed disc turbine seen in the diagram. Batch simply means that a fixed amount of substrate is added at the beginning of the process. This means they can be difficult to mix due to their high (and not constant) viscosity. Thus. • Penicillin is an aerobic organism. there are a few specific characteristics of penicillin that must be considered when attempting fermentation: • Most penicillins form filamentous broths that are pseudoplastic (nonNewtonian) in nature. then it is critical that there is some kind of system to cool the reactor. Also the increasing viscosity of the broth can hinder oxygen transfer. Continuous means that substrate is constantly added to the reactor while an equal amount of fermented medium is removed. What follows is a brief description of the specific fermentation conditions necessary for the production of penicillin. it is essential that there is good transfer of oxygen across the gas/liquid interface. However many fluids are non-Newtonian and are described as Bingham or Pseudoplastic. 7 . One problem with gas/liquid interfaces is the formation of foam. Fed-Batch and Continuous Culture are terms used to describe how nutrients and substrate will be delivered to a culture in a reactor. Fed Batch means that substrate is added in small increments at various times in the fermentation. Batch. So much for our discussion of basic reactor design. Heat Transfer is important because metabolism as a process tends to give off heat. Steam is used to keep the reactor running aseptically. This is normally achieved through a combination of a cooling jacket and cool water being passed around the tank. Which leads to the next point. as does the mechanical mixing by the impellers. Bulk Flow and Mixing are achieved by the impellers. Newtonian fluids show constant viscosity and linear shear rate against shear stress. therefore the rate of oxygen supply is critical to the fermentation. Special fermenter Conditions for the production of Penicillin Aside from what is described above. The Sparger delivers this oxygen. This is achieved because the reactor is designed as a pressure vessel and steam is sent through at a minimum temperature/pressure of 1210C/15 psi for 15-30min. If the reactor needs to be operated at a constant temperature of range of temperatures. Fluid flow in a reactor can be described as Newtonian or non-Newtonian.vital component of aerobic metabolism.

Also.• • • The optimum pH for penicillin growth is 6. Strain Stability problems do exist and careful strain maintenance is required. Primary metabolism is the metabolism of energy production for the cell and for its own biosynthesis. all microorganisms require Carbon. the selection of media is of critical importance to the overall performance of the fermentation. Mn and Co (this will frequently depend on the water source as most water sources contain small amounts of the elements) or growth factors such as vitamins or amino acids as well. it is necessary to give a brief discussion of the differences between primary and secondary metabolism. 8 . Media Considerations When performing any kind of fermentation.5. At this stage. enzymes or other secondary metabolites frequently require precursors like purine/pyrimidine bases or organic acids to produce said metabolites. Typically. Biomass doubling is about 6h. Hydrogen. in aerobic organisms (such as Penicillium chrysogenum) it involves the conversion of sugars such as glucose to pyruvic acid2 and the production of energy via the TCA cycle. Thus the reactor must maintain pH efficiently (this is frequently done by addition of NaOH). Sulphur and Nitrogen for cell growth and cell maintenance. The last sentence is particularly important. In many cases. Here it should be noted that these secondary metabolites are only produced in times of stress when resources are low and the organism much produce these compounds to kill off its competitors to allow it 2 As an aside in anaerobic organisms this pyruvic acid can be converted into other commercially important compounds such as alcohol and lactic acid. The aim of the media is to provide all the elements required for the synthesis of cell materials and the formation of the desired product. microorganisms require small amounts of trace elements such as Cu. certain organisms such as Penicillium chrysogenum that produce antibiotics. In essence Penicillium chrysogenum can “kill off the competition” to allow itself to propagate efficiently. At the same time. In this case the metabolite is being utilized as a defence mechanism against other microorganisms in the environment. the media must provide a favourable environment for the culture in question (an example of this would be control of pH by addition of calcium carbonate or inorganic phosphates). As well as this it must remain cost effective. Secondary metabolism regards the production of metabolites that are not used in energy production for example penicillin from Penicillium chrysogenum. Typically. Oxygen.

It is also very important that the nitrogen source is carefully controlled. In the first stage. This is because this sugar is the most easily used for energy metabolism and thus gives the best yields in terms of growth. The solution to this is to use the fed-batch method to feed the culture. First of all. Readily available carbon and nitrogen sources tend to inhibit antibiotic production (this is known as Catabolite Repression. at this stage there is frequently a need for the addition of precursor molecules (e. In the next stage. the amount of carbon source that is added must be carefully controlled. the production survive. This leads us onto the next point. As stated above. It is these conditions that we wish to duplicate in order to achieve the maximum amount of product from our fermentation. this kind of fermentation is done in stages. 9 . where a secondary metabolite is inhibited by the presence of a more readily usable substrate).g. In order to do this. we wish to do almost the exact opposite as the first stage. These also must be considered with the media. as excess nitrogen will greatly inhibit antibiotic production. At this stage. There are also several other considerations regarding this stage of production. Media for this stage will typically be focussed on achieving maximum growth and biomass production. this allows us to add the substrate to the reactor in small increments and to even change the substrate if we so desire. Glucose (Starch) will usually be the primary source of carbohydrate for the culture. This is because growth and antibiotic production are mutually interrelated and inversely proportionate. primary metabolism will be emphasised. Both these substrates allow maximal growth of the culture at the expense of product (antibiotic) formation. Once the desired biomass has been achieved. Other additives can include the addition of inorganic phosphates to alter pH and chemical anti-foaming agents to prevent the development of foam. Hence. At this stage a substrate is frequently added that will give a readily usable source of Nitrogen as well such as corn steep liquor. Limiting the amount of carbon and nitrogen available to the culture typically does this. Generally. purine/pyrimidine) into the medium. lactose is frequently used as carbohydrate source in this part of the fermentation. This is the best way to change from one production stage to another. we wish to “starve” the culture and induce the kind of stress conditions that trigger the production of the antibiotic. as mentioned above.

a number of these spores will be introduced into a small (normally 250-500ml) conical flask where it will be incubated for several days. from the initial activation of a preserved culture right up to the full-scale industrial production of the antibiotic of interest. To begin the fermentation process. At this stage. Typical parameters observed include pH. there are numerous factors that must considered. This reactor will be fitted with a number of instruments to allow the culture to be better observed than it was in the shake flask. explosive growth is the most desired parameter and as such the medium in the flask will contain high amounts of easily utilisable carbon and nitrogen sources. This is a kind of stock of established culture from which other cultures are grown. modifications will be made to the process and/or the reactor. 10 . Once the overall conditions for growth have been established and there is a viable vegetative culture active inside the flask. At this point. the spores will begin to revive and form vegetative cells. stirrer speed and dissolved oxygen concentration. there may not be enough oxygen getting to the culture and hence it will be oxygen starved. although there may be some changes made to facilitate optimum growth. this will be a stock that has been modified over time (genetically or not) to give the best possible yield of product and as such is generally very valuable. Often the best way to preserve these cultures is in the form of inactive spores. The majority of optimisation of the process occurs here in the bench-top reactor as well as gathering of important information about the activity of the cells. Almost all fermentations begin with the preserved culture. it is often desirable to use as little of this stock as possible to initiate the fermentation process. In this way the cells can be preserved for many years. Microbead system) and frozen. temperature. This is because spores are very resistant structures that can survive heat. To increase their longevity.Process considerations and Scale-up This section deals with the various steps in a fermentation process. low pH and mechanical forces that would typically kill a normal vegetative cell. Another example would be high shear-rate leading to cell damage. This allows “tweaking” of the process to occur and difficulties to be examined. To resolve these problems. For example. As can be imagined.g. Temperature is normally maintained at 23-280C and pH at ~6. The flask will often have baffles in it and be on a shaking apparatus to improve oxygen diffusion in the flask. For this reason. these spores are often kept in a cyropreservative fluid (e. each playing a different role at different stages in the fermentation. desiccation. At this stage. Frequently. such as starch and corn-steep liquor.5. it will be transferred to a 1 or 2 litre benchtop reactor.

hopefully growth will continue as before.000 bacterial cells capable of causing an infection. At this stage. we must have an efficient method for the removal of this product and to maintain constant volume in the reactor. Carbon and nitrogen will be added sparingly alongside precursor molecules for penicillin fed-batch style. Therefore. (If there are 100. Various steps were taken so as to continue the use of antibiotics. and only one cell in this population can make penicillinase. Once this has been successful the process is scaled-up again to a pilot-scale bioreactor.000 penicillin-resistant disease-causing bacteria!) By as early as 1952. Other systems. as much as three-fifths of all staph infections were penicillin resistant.and to therefore reproduce .now we can have 100. This reactor will be similar in design to the bench-top reactor except it will have a size of about 100-1000 litres. If all this goes to plan. be of limited value because bacteria would eventually recombine genetically to resist the effects of penicillin. The reactor must be capable of running aseptically and the design must reflect safety and contamination requirements. As before. At this stage the medium being added to the reactor will change.the cells should be showing filamentous morphology. as this is preferred for penicillin production. This can be due to changes in the morphology of the culture (remember Penicillium chrysogenum is a filamentous fungi and hence pseudoplastic) that may or may not be correctable. If the pilot-plant stage is successful then work can begin on an industrial scale operation. their value has been diminished by the widespread development 3 It should be noted that a full discussion of downstream processing is beyond the scope of this document. we should have fully functioning reactor that is production penicillin ready for downstream processing3. The aim here is to examine the effect of scale-up on the culture. there are often sudden changes or loss in performance. Although the penicillins are still used clinically. 11 . Now we wish to emphasis penicillin production over growth while maintaining a constant volume. then treating the infection with penicillin will allow only this cell to survive . This is now very much an engineering problem. This reactor is often viewed as a type of prototype for the larger fermentations that follow. From here it can be refined and packaged for marketing and distribution to a global market. must also be considered. Resistance Alexander Fleming predicted that the use of penicillin would. cell growth is priority at this stage. however. in time. Another note is that the presence of penicillin in the reactor is itself inhibitory to the production of penicillin. such as cooling water supply.

htm http://nobelprize. including MRSA. Bu'Lock Bioprocess engineering principles / Pauline M.J. Staphylococcus aureus is found on many individuals skin and causes no problems. and technology must struggle to keep up with the advances of References Biochemistry of Antimicrobial Action. J. Franklin and G. The discovery of the naturallyoccuring penicillinase inhibitor clavulanic acid has dramatically opened the way for chemically modifying β-lactamase-susceptible agents such as ampicillin and amoxicillin.wikipedia.html Ward 12 . Staphylococcus principles.ed. This compound irreversibly binds to penicillinase and prevents the enzyme from working. The discovery of clavulanic acid is an important one in the fight against these organisms but bacteria are very adaptable. if worried about a particular bacterium being resistant to penicillin. Meyrath. Snow. clavulanic acid can be given along with one of the semi-synthetic penicillins.A.htm Biotechnology and fungal differentiation / edited by J. The use of antibiotics must be strictly controlled if they are to remain useful as a defence against infection. processes. Methicillin-resistant Staphylococcus Aureus (MRSA) is a type of bacteria that is resistant to certain antibiotics. inclluding methicillin.ed. Doran Fermentation Biotechnology. However if it gets inside the body. occur most frequently among persons in hospitals and healthcare facilities who have weakened immune systems. penicillin and amoxicillin.of resistance among target microorganisms and also by some people's allergic reaction to penicillin (1 in 10 people are allergic to penicillin). products / Owen P. oxacillin. T. There is no tried and tested method for defeating organisms like So. it can cause infections such as boils or pneumonia. new ways of treating such resistant bacteria are being developed and will hopefully be effective in years to come. but as science http://www.htm Fourth Edition http://inventors.about.

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