Production of Penicillin Through Fermentation

Abstract: Antibiotics are among the most frequently prescribed medications in modern medicine. Antibiotics cure disease by killing bacteria and keeping them from reproducing. Penicillin was the first antibiotic, discovered accidentally from a mold culture. Presently, over 100 different antibiotics are available in the market to cure minor discomforts as well as lifethreatening infections. Antibiotics are very useful in a wide variety of infections, but they only treat bacterial infections. Antibiotics are useless against viral infections (for example, the common cold) and fungal infections (such as ringworm).

Introduction: Penicillin is a group of antibiotics derived from Penicillium fungi. Penicillin is a historically significant drug being the first antibiotic discovered. Penicillin is a Betalactam antibiotic and is used in the treatment of bacterial infections caused by susceptible, usually Gram-positive, organisms. Figure1: Core structure of Penicillin: R-C9H11N2O4S History: In September 1928, Alexander Fleming accidentally discovered penicillin when he was engaged in studying different kinds of bacteria and molds in his laboratory.

He once opened one of his Petri dishes for a few seconds to smear it with a strain of staphylococcus which is a bacterium that typically occurs in clusters resembling grapes. Fleming noticed a halo of inhibition of bacterial growth around a contaminant blue-green mold staphylococcus plate culture. He concluded that the mold was releasing a substance that was inhibiting bacterial growth and lysing the bacteria. He grew a pure culture of the mold and discovered that it was a penisillium mold, now known to be penicillium notatum.

He then demionstrated that this material had powerful antimicrobial properties and named the product penicillin. Fleming carefully preserved the culture, but the discovery lay essentially dormant for over a decade. World War 2 provided the impetus to resurrect the discovery. The development of penicillin for the medical use is attributed to an Australian nobleman called Howard Walter Florey who worked with a team at Oxford University. They wanted to investigate the biochemical and biological properties of antibacterial substances that microorganisms might possess.

The team eventually discovered that penicillin was a molecule and not an enzyme as physicians used to consider it. The team also discovered that penicillin was very unstable unlike other simple molecules. Florey’s team members were able to produce stable penicillin by reducing the temperature of a water solution of penicillin by freeze drying it. Then, Florey experimented 1 penicillin on animals before he applied it on human beings. Penicillin was produced by a surface culture method early in World War II. Sub-merged culture methods were introduced by 1943 and are now almost exclusively employed.

Parameters that affect fermenetation: Penicillin is a secondary metabolite of fungus Penicillium that is produced when growth of the fungus is inhibited by stress. It is not produced during active growth. Production is also limited by feedback in the synthesis pathway of penicillin. ?-ketoglutarate + AcCoA > homocitrate > L-? -aminoadipic acid > L-Lysine + ? -lactam The by-product L-Lysine inhibits the production of homocitrate, so the presence of exogenous lysine should be avoided in penicillin production.

The Penicillium cells are grown using a technique called fed-batch culture, in which the cells are constantly subject to stress and will produce plenty of penicillin. The carbon sources that are available are also important: glucose inhibits penicillin, whereas lactose does not. Penicillin production needs strict asceptic conditions. Contamination by other microorganisms reduces the yield of penicillin. The pH and the levels of nitrogen, lysine, phosphate, and oxygen of the batches must be controlled. Temperature is an important factor affecting the performance of the cells.

As the temperature is increased toward optimal growth temperature, the growth rate increases. Above the optimal temperature range, the growth rate decreases and thermal death occurs. There are two temperature optima connected with penicillin production. One, about 30oC, best for mycelium-producing phase; the other, about 200C, best for penicillin producing phase. 2 The effect of cultivation conditions on the penicillin production can be summarized as: 1) The maximum production amount of penicillin was achieved when cultivation temperature and pH were kept at 22±2°C and 6.

5, respectively. 2) The maximum production yield of penicillin for substrate was obtainable when the concentration of lactose and that of cornsteep liquor in the basal medium were adjusted at 60 kg/m3 and 30 kg/m3, respectively. 3) The rate of oxygen supply is critical for the fermentation. Thus, the reactor must have an efficient oxygen supply system for optimum yield. Transport Phenomena in Fermenter: Mass Transfer is seen when there is mixing between two components of varying concentrations. This is very important when we are attempting to grow aerobic microbes in media.

When the oxygen is bubbled through the fermenter tank, there is high concentrations of oxygen near the bubbles but low concentration everywhere else in the tank. Since oxygen concentration is such a vital component of aerobic metabolism, it is essential that there is good transfer of oxygen across the gas/liquid interface. The Sparger delivers this oxygen. At low stirrer speeds, gas dispersion is inadequate and the insufficient oxygen transfer rate is a limiting factor. At higher stirrer speeds, the oxygen supply to indivisual cells is improved and more cell mass is formed, but foaming increases.

Heat Transfer is important because metabolism as a process tends to give off heat, as does the mechanical mixing by the impellers (less significant in terms of contribution to the heat generation). If the reactor needs to be operated at a constant temperature of range of temperatures, then it is critical to have an efficient system to cool the reactor. This is normally achieved through a combination of a cooling jacket and cool water being passed around the tank. Reactor Design and Downstream Processing: The Penicillium is produced using a fed-batch culture.

A Semi-batch fermentation Reactor is such that the reaction mixture can be charged into and product can be drawn periodically. The final reaction product mixture is further sent to downstream processing. The fermented broth from the reactor is sent to a surge tank. Further it is sent to series of centrifugal extractors. The extraction of penicillin is based on the fact that penicillin is a weak acid which, in aqueous solutions of increasing acidity (pH less than 4) it become less and less dissociated and thus more soluble in organic solvents.

From the solutions of pH not less than 6, it is easy to extract the penicillin from organic solvents in to the aqueous phase. 3 This procedure of extracting into the solvents and re-extracting from the solvents is usually carried out twice. Generally, amylacetate is used in the first extraction and chloroform in second extraction. The final result of extraction processes is a dilute solution of sodium or calcium salt of penicillin, depending whether NaOH or CaOH was used in the final neutralizing extraction.

The salt solution is dried using the method of freeze drying and results into a product yield of about 50-80% of the penicillin initially present in the broth. Figure 2: A typical batch fermenter. Issues in Scale Up: Scale up is the process by which a vegetative culture in a conical flask is transfered to a fermenter having a size of about 10,000 litres. The major issues in scale up are difficulty in maintaining: ?? homogeneity in large systems, ?? changes in surface to column ratios, ?? changes in the cultures themselves due to the increased length of culture time. 4

The reactor must be capable of running aseptically and the design must reflect safety and contamination requirements. Also, the presence of penicillin in the reactor is itself inhibitory to the production of penicillin. Therefore, efficient method for the removal of this product and maintenance of constant volume in the reactor is required. The mixing of the microbes with the broth is a big challenge in large fermenter, the reaction mixture may not have uniform concentration through out the reactor and as a result, the productivity may go down. The reactor must be designed to ensure homogeneity inside the reactor.

Efficient heat removal is also a big issue. Large reactors use internal coils or jacketed vessel for heat removal. Foaming is another major problem. If it escapes from the fermenter, it can wet filters, increasing pressure drop and decreasing gas flow. Moreover it provides a pathway for contaminating cells to enter the fermenter. Head space must be provided inside the fermenter for the gas to disengage from the liquid. Conclusion: Alexander Fleming predicted that the use of penicillin would, in time, be of limited value because bacteria would eventually recombine genetically to resist the effects of penicillin.

By as early as 1952, as much as three-fifths of all staph infections were penicillin resistant. Various steps were taken so as to continue the use of penicillin. However through time, their value has been diminished by the widespread development of resistance among target microorganisms and also by some people’s allergic reaction to penicillin (1 in 10 people are allergic to penicillin). The use of antibiotics must be strictly controlled if they are to remain useful as a defense against infection, and technology must struggle to keep up with the advances of nature.

References: Bioprocess Engineering Basic Concepts; Michael L. Shuler and Fikret Kargi, Second Edition http://en. wikipedia. org/wiki/Penicillin http://www. sciencedirect. com http://www. ncbi. nlm. nih. gov/pmc/articles/PMC1057142/? page=1 http://www. freepatentsonline. com/2482938. pdf http://itech. dickinson. edu/chemistry/? p=105 http://www. springerlink. com/content/m1g0336k56wwjp08/ http://www. gbanalysts. com/Reading%20Room/Situation%20Analysis/EnergyTechAnalysis/r xtordesignbfr. html 5.

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