PENICILLIN PRODUCTION

BY DAKSHITA NAITHANI

Penicillin, which is now an essential element of our antibacterial arsenal had a huge influence on the twentieth century’s second half. Deep-fermentation techniques, which were established largely for the synthesis of Penicillin during WWII, paved the way for the creation of medications and aided the emergence of the young biotechnology sector in the 1970s. In the presence of blood, pus, and bodily fluids, it is effective against some gram-positive bacteria. It is soluble in water, acetone, ethyl alcohol, and ether, but less so in benzene, chloroform, and other organic solvents.

 It is a secondary metabolite, like other antimicrobials, and is only generated in the stationary phase. The industrial manufacturing may be divided into two processes: upstream and downstream.

When the development of the fungus is hindered by stress, it produces certain species of Penicillium. Penicillin production is likewise restricted by feedback in the synthesis process.

α-ketoglutarate + AcCoA → homocitrate → L-α-aminoadipic acid → L-lysine + β-lactam

Because the by-product L-lysine hinders homocitrate synthesis, exogenous lysine should be avoided in its manufacture.

The cells are cultivated using a process known as fed-batch culture, in which the cells are continually exposed to strain, which is necessary for inducing its synthesis. It’s also crucial to consider the carbon sources available: glucose inhibits its synthesis, but lactose does not. The pH of the phases, as well as the amounts of nitrogen, lysine, phosphate, and oxygen, must all be monitored closely.

THE FERMENTATION PROCESS

INOCULUM: The source of inoculum is master stock (spores). They are cultivated working samples are immersed in water and mixed with wheat bran and nutrition solution in a flask. A four-day-old shaking flask culture is inoculated into a seed tank for three days.

THE MEDIUM: In 1958, Jackson created a medium for the manufacture of penicillin. Fermentable carbohydrate (corn steep liquor (3.5%), organic nitrogen source, lactose (3.5%), glucose (1%), potassium di-hydrogen phosphate (0.4%), calcium carbonate (as a buffer) (1%),  phenyl acetic acid precursor, edible oil (0.25%), pH near 6.

As temperature is very important aspect during its production it should be around 280 degree Celsius and the supply of oxygen in the bioreactor acts as a limiting factor in its production the aeration speed should be between 3.0-1.5.

Fermentation is the method through which Penicillin is commercially produced. It’s a fed-batch technique performed in aseptic conditions in stainless steel tank reactors with capacities ranging from 30 to 100 thousand gallons. The fermentation process consists of two to three seed development phases, followed by a fermentation production phase that lasts 120 to 200 hours. For this procedure, a variety of carbon sources have been used. During the active Penicillin synthesis phase, sugar is also utilised to regulate the pH value.

During fermentation, mini-harvest techniques are commonly used. They entail removing 20-40 percent of the overall of the fermenter’s contents and replacing it with new sterile medium. This method can be done multiple times during the process without lowering the overall Penicillin yield per fermenter; in fact, it can increase it.

Penicillin is discharged into the fermentation medium and collected at the end. With a 2-5 percent increase in total extraction efficiency, whole broth extraction is best conducted at an acidic pH. Amyl, butyl, or isobutyl acetate is used to extract cooled acidified broth from a solvent.

These fermentations are extremely mechanised and computerised in today’s world. For optimum antibiotic synthesis, all essential precursors, such as ammonia, sugar, carbon dioxide, and oxygen, are carefully monitored, along with temperature and pH. During the active manufacturing phase, the pH should be between 6.4-6.8.

PURSUIT FOR A BETTER YIELD

Penicillin was first produced using the fungus Penicillium notatum toward the conclusion of World War II, yielding one milligramme per cubic decimeter. Today’s yield is 50 grammes per cubic decimeter, thanks to the use of a new species (Penicillium chrysogenum) and better extraction techniques.

These yields can be increased further by improving the medium’s composition, isolating the above- mentioned Penicillium chrysogenum, which grows better in large deep fermentation tanks, and developing a submerged culture technique for mould cultivation in large volumes of liquid medium through which sterile air is forced.

Its manufacturing has remained mostly reliant on traditional strain improvement. The most important occurrences in high-yielding Penicillium chrysogenum strains are the expansion of the Penicillin biosynthetic gene cluster between tandem repeats. There have also been created molecular methods that are not based on increasing biosynthetic gene dosages.

 THE EARLY PRODUCTION OF PENICILLIN

The consortium of British and American experts came together to enhance manufacturing processes and their initial objective was to find the strains of Penicillium chrysogenum that generated the most penicillin. They quickly discovered that a Penicillium chrysogenum strain acquired from a mouldy cantaloupe at a Peoria local farmers market produced greater amounts of Penicillin than those recently tested. Scientists utilised x-rays and ultraviolet light to produce even more mutants from the farmer’s market strain.

Following those experiments, it was discovered that growing Penicillium in immersed culture media rather than on a plate surface enhanced growing efficiency, and that changing the nutrient base from sucrose to lactose or corn-steep liquor (a nutrient-rich by-product of corn processing) also increased yield. 

MODERN PRODUCTION METHODS

Major advances in contemporary manufacturing processes have improved output while lowering costs. Nowadays, commercial generating strains of Penicillium chrysogenum are produced utilising submerged culture in 50,000-gallon stainless steel tanks that are continually agitated and aerated. With a 90 percent recovery rate, these commercial strains can now produce 40-50 gram of Penicillin per litre of culture. This is a huge leap forward over the first Peoria farmer’s market strain, which only produced 0.15 grams per litre and had extremely low recovery rates.

Amplification of the biosynthesis gene cluster, an increasing amount of peroxisomes, and increased levels of transporter proteins that secrete newly production out of the peroxisomes and the cell are among the genetic and cellular modifications that result in increased production in modern Penicillium strains.

Penicillin related antibiotics now generate more than $15 billion in annual sales worldwide. Despite the fact that costs are at an all-time minimum, these sales figures exist. Penicillin currently costs $10 per kilogramme, compared to $300 in 1953. Though Europe is the world’s largest manufacturer of beta-lactam antibiotics, newer production facilities are moving to China and other Asian countries with reduced labour and energy prices.

FORGOTTEN VICTIMS……..COMFORT WOMEN

Around 150,000 to 200,000 women during the Second World War were coerced, abducted and forced into being sex slaves for the Imperial Japanese Army. These women were lured in with promises of nurse jobs, waitresses, to work in an office or to work as an entertainer but there taken to military bases and kept in comfort stations. The reason for this brutal and inhumane legally sanctioned rape was to mitigate or to reduce the rape crime by the Japanese Imperial Army due to rising anti-Japanese sentiment. The rising anti-Japanese sentiment was due to incidents like the rape of Nanking. By forming comfort stations where women were ready to service the Japanese army men then they could confine rapes and abuse to the military stations and such atrocities would not reach international news.
The first Comfort station was formed in Shanghai in 1932. The women working there were voluntary Japanese prostitutes or kidnapped Japanese women but later when the resources dried up in Japan, the army started to look beyond its country. Women from Japanese concessions and Japanese colonies were recruited. Women were falsely misled into thinking that they were about to get a job as a nurse or waitress or a simple office worker but were taken to Japanese military stations where they were made to learn a routine and were coerced into having sex multiple times with different people. Because of such inhumane conditions, most of the comfort women would contract sexually transmitted diseases. But the Japanese military doctors would carefully and regularly monitor comfort women to detect STDs and to stop it from reaching the troops.


The women were 80 per cent Koreans and the rest were Chinese / Filipina /Dutch/women who were abducted. Many such women were taken in and were raped 30 -40 times every day. Those who refused were beaten severely. Girls who were virgins while entering the stations were broken in or raped. So Tetsuo who was a medical physician in the Imperial Japanese Army wrote that comfort was referred to as female ammunition and was seen as things to be used rather than human beings. Comfort women were also forced to donate blood to save wounded soldiers. When a soldier would enter a comfort station, where photographs of comfort women were displayed and soldiers could choose from there. Women who got pregnant were forced to get abortions. Any human being with a little shred of humanity would know that this is wrong but this issue went unnoticed. Most comfort women were told and the theory that white men were cannibals were indoctrinated into them. When the Allied forces came close to capturing Japanese military stations, most of these women were forced to commit suicide or were killed by the soldiers. Many comfort women killed themselves as they didn’t want to be eaten by white men as this fact that white men were cannibals were drilled into them.


The Japanese military used the drug called salvarsan which was used to sterilize women; many condoms were given out for free to prevent the spread of sexually transmitted diseases. Japan has issued apologies to the countries where it had forcefully taken women from. Prime Minister Shinto Abe stated in a newspaper interview that there was zero evidence that the Japanese army had used sex slaves but the fact that the Japanese government had admitted that they used brothels in a statement issued in 1993. But the country had issued an apology for the inhumane treatment of women and the deep psychological trauma that it had inflicted on the countries citizens.