Subtleties of Production of Penicillin: Fermentation Technology

Introduction

Penicillins are antibacterial drugs that are used against a wide range of bacteria. It is obtained from Penicillium fungi, which is taken orally or as an injection. It was discovered by Alexander Fleming from Penicillium notatum, which is a blue-green mold. They act directly on peptidoglycans and in turn burst the bacterial cell wall. Penicillin is produced during the stationary phase of the growth of bacteria since it is a secondary metabolite. The production of Penicillin is grouped into three processes: Upstream process, bioreactor, and downstream process.

Upstream processing refers to the technology that leads to the synthesis of the product and includes the development and production. The extraction, purification of the product, and subsequent processing is referred to as Downstream processing. Penicillin is produced by Fermentation by the Fed-batch process. it is carried out aseptically in stainless steel tank reactor with 30 to 100 thousand gallons capacity. The time cycle ranges from 120 to 200 hours which involves two to three initial seed growth phases.

Various carbon sources have been used such as glucose, sucrose, and other crude sugars which is used by the cell for cell maintenance, growth, regulation of pH, and also for penicillin production. Mini-harvest protocols are usually employed in penicillin fermentation. They involve the removal of 20-40% of the fermentor contents and its replacement with a fresh sterile medium. This procedure can be repeated several times during this process without yield reduction; quite the opposite, it can enhance the total penicillin yield per fermentor.

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. Nowadays penicillin production are highly monitored and they are usually computerized. The necessary precursors such as ammonia, sugar, carbon dioxide, and oxygen are controlled, with thorough monitoring of temperature and pH for optimal antibiotic production using the computerized system. The pH should be between 6.4 and 6.8 during the active production phase.

Initially, penicillin was made from Penicillium notatum but the process yielded one milligram per cubic decimeter. Today, by using a different species (Penicillium chrysogenum) and improved extraction procedures the yield is 50 grams per cubic decimeter. The yield of penicillin was further increased by improving the composition of the medium, isolating  Penicillium chrysogenum which grows better in the huge deep fermentation tank, but also via the development of submerged culture technique for cultivation of mold in a large volume of the liquid medium through which sterile air is forced. Still, classical strain improvement has been the mainstay of penicillin production. The amplification of the penicillin biosynthetic gene cluster represents one of the most important phenomena in high-yielding Penicillium chrysogenum strains.

Although the therapeutic need for penicillin was great, laboratories in the United Kingdom were unable to dedicate sufficient resources to improve the production methods. They soon discovered that a strain of Penicillium chrysogenum obtained from a moldy cantaloupe in a Peoria farmer’s market generated higher levels of penicillin than those previously studied.  Farmer’s market strain was used as a base, to which scientists applied x-rays and ultraviolet light to produce even higher penicillin-producing mutants. Subsequent experiments revealed that growing efficiency could be improved by growing Penicillium in submerged culture media instead of on a plate surface and that changing the nutrient base from sucrose to lactose or corn-steep liquor also increased yield.

Modern Production Methods

Significant improvements in modern production methods have increased production and decreased cost. Today, commercial producing strains of Penicillium chrysogenum are grown using submerged culture in constant agitation and aeration in 50,000-gallon stainless steel tanks.  These industrial strains can now produce 40-50 grams of penicillin per liter of culture with a 90% recovery yield. In order to achieve these production rates, modern Penicillium strains display a host of genetic and cellular modifications that result in increased production, including amplification of the penicillin biosynthesis gene cluster, an increased number of peroxisomes, and elevated levels of transporter proteins that secrete newly produced penicillin out of the peroxisomes and the cell.

Penicillin now costs $10 per kilogram versus $300 per kilogram in 1953. Although Europe is the major producer of beta-lactam antibiotics, newer manufacturing facilities are relocating to China and other regions of Asia where labor and energy costs are lower.

Upstream Processing

The Fermentation media for the production comprises of sources of carbon, nitrogen, amino acids, salts, and other precursors. It should also provide growth of mycelium, accumulation, extraction, and purification of penicillin.

• Carbon source – Glucose, Lactose, and Sucrose.
• Nitrogen source – Ammonium sulfate, Ammonium acetate, Ammonium lactate.
• Amino acids – Corn steep liquor.
• Precursors for penicillin G – phenylacetic acid.
• Precursors for penicillin X – Hydroxyphenylacetic acid.
• Precursors for penicillin V – Phenoxyacetic acid

Optimization of Media

Inoculum preparation:

The strain  Penicillium chrysogenum gave a yield of 200 units/ ml. After the strain improvement, the strain gave a yield of 761 units /ml by using the submerged cultured method. This strain improvement is carried out by genetic mutation so this strain is highly unstable so this strain should be maintained properly. The strain is maintained in a dormant state by using the lyophilization technique, stored in liquid nitrogen in a frozen state or in spore form.

In the process of inoculum preparation, the pure culture of Penicillium chrysogenum is prepared inadequate amount for the production of penicillin. The primary stock is added in special agar the special agar should provide sporulation of spores the sporulating medium is used to prepare this working stock. The sporulated spores are suspended in an SLS solution that is sodium lauryl sulphonate in a proportion of 1:10,000 further this spores are added in a nutrient medium of wheat bran plus nutrient and the flask are incubated for 5 to 7 days at 24 ° C. This medium is used for heavy sporulation. Now, these spores are used as an inoculum in the fermentation tank.

Fermentation process

Fermentation of penicillin is carried out in trays or by the submerged culture method. The 10 % of inoculum is added in the fermentor aseptically. The temperature of about 25 ° C to 26 ° C is maintained. The sterile air supply is provided continuously as the fermentation is aerobic fermentation. The fermentation is carried out for about 3 to 5 days. During this fermentation process, the samples are withdrawn aseptically and checked for a yield of penicillin, pH, and contamination. Checking for contamination is very important if the fermentation media gets contaminated by organisms producing penicillinase enzyme then it can result in a great economic loss to the industry. The fermentation process is monitored for foam formation also if the foam is produced it is controlled by an antifoaming agent.

Initially, the pH of fermentation media remains constant as the inoculum initially utilizes carbon as a source of energy but further when the concentration of carbon is reduced the micro-organism starts utilization of nitrogen as a source of energy so at this point the pH of the fermentation media increases to 7.0 to 7.5 due to deamination and release of ammonia. Now at this point, the micro-organism starts product synthesis by utilization of lactose and production of penicillin. After product formation, the concentration of lactose is decreased and pH rises to 8 or even more which results in autolysis of mycelium. Here at this point, the fermentation is stopped and recovery and harvesting of product is started. Initially, during 20 to 30 hours the fungal spores utilize carbohydrates and corn steep liquor and fungal spores develop as mycelium, and further in the duration of 48 to 96 hours, the mycelium starts production of penicillin product. The yield obtained is 3 % to 5 % and 1500 units per milliliter of fermentation medium.

Different Aspects

Procedure Volume 300-500m3, made from stainless steel.

Agitation is provided at a rate of 100 to 300 rpm.

Temperature is controlled by using cooling coils.

Parameters Monitoring

Dissolved oxygen and other parameters should be continuously controlled, this is achieved by the withdrawal of small volumes of broth in the fermenter.

Aeration

Vigorous supplied from the bottom of fermenters by ring or tube sparges.

Common Problems

Due to the high viscosity of the broth, oxygen transfer is a major problem in penicillin fermentation.

Downstream Processing

The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing. Downstream processing is relatively easy since penicillin is secreted into the medium so there is no need to break open the fungal cells. However, the product needs to be very pure, since it is being used as a therapeutic medical drug, so it is dissolved and then precipitated as a potassium salt to separate it from other substances in the medium.

Harvest and Recovery

Initially, the mycelium and other solid suspended particles are removed by the filtration process. Further, the filtrate is treated with a solvent extraction procedure for the separation of penicillin. Penicillin is converted to anionic form by using phosphoric acid and sulphuric acid. Further, the broth is extracted by using an organic solvent like methyl isobutyl ketone or amyl acetate. The extracted penicillin insolvent is back-extracted in water by use of potassium hydroxide and sodium hydroxide in form of salt. The aqueous penicillin is acidified and re-extracted by methyl isobutyl ketone. This process of extraction of penicillin in water and organic solvents separates and purifies penicillin. Further, the aqueous penicillin is evaporated and crystallized in the form of sodium penicillin. Then finally penicillin is standardized. To remove the moisture present in the penicillin salt. The hot gas is pumped from the base of the chamber and the powdered salt is contained in a vacuum chamber.It results in the powdered form of penicillin.

Purification

1. Adsorption.

Carbon adsorption is used to remove impurities and pigments from the penicillin-rich solvent after extraction. Several activated carbon columns in series can be used for this purpose.

2.  Crystallization.

Na and penicillin concentrations, pH, and temperature need to be adjusted for crystallization.

Excess amounts of Na are added to the penicillin-rich solvent before crystallization in an agitated vessel.  The crystals are separated by a rotary vacuum filter. The crystals are washed and predried with anhydrous butyl alcohol to remove some impurities.  Large horizontal belt filters are used for the collection and drying of the crystals.

Storage

Penicillin is stored in containers and kept in a dried environment. Then packaged into :

• Liquid penicillin
• Penicillin in pills

Types of Fermentation

There are mainly 3 types of fermentation used in the production of penicillin

1. Solid-state fermentation
2. Fed-Batch fermentation
3. Deep Tank fermentation

Solid-State Fermentation

Solid-state fermentation (SSF) is an old method which is revaluated and modernized lately for the production of protein and enzymes. Penicillin was produced by a non-sterile solid-state fermentation (SSF) on a bagasse impregnated with a culture medium. Using concentrated media, greatly enhanced the antibiotic production in this system. It was observed that adequate initial moisture content (70%) of the impregnated solid medium results in higher production.

Solid-state fermentation (SSF) is defined as the microbial cultivation process in the absence or near absence of free water in the substrate. However, there must be enough moisture present to support cell growth. Bacteria and filamentous fungi grow typically in nature on solid substrates, such as wood, seeds, stems, roots, and leaves of plants in symbiotic associations.

Advantages of Solid-State Fermentation:

• SSF is more economical mainly due to the cheap and abundant availability of agricultural wastes which can be used as substrates.
• The economy of space needed for fermentation.
• The fermentation media is simple.
• Has higher fermentation productivity.
• Has higher end-concentration of products.
• Has higher product stability.
• Lower catabolic repression.
• Cultivation of microorganisms specialized for water-insoluble substrates.
• Lower demand of sterility due to the low water activity used in SSF and no requirement for complex machinery.
• Greater compactness of the fermentation vessel provides a lower water volume; greater product yield; reduced energy demand; lower capital and low recurring expenditures in industrial operation.
• Provides easier scale up processes.
• A lesser volume of solvent is required for product recovery.
• Absence of foam build up and easier control of contaminants due to the low moisture level in the system.

Deep Tank Fermentation

Deep tank fermentation was developed by Pfizer for the production of citric acid. It was later used to produce other substances such as gluconic acid and itaconic acid. The production of gluconic acid using deep tank fermentation led to the mass production of penicillin for commercial use.

Penicillin Production by Deep Tank Fermentation

The British government sought help from Pfizer to solve the need for huge amounts of Penicillin. The company plunged into the mass production of the wonder drug. Initially, Jasper Kane used flasks and pans for producing penicillin similar to the setup of citric acid, later it was suggested and changed to deep tank fermentation.

The company took the gamble and went onto purchase an old plant which had the equipment required, rebuilding it to the first large-scale factory for penicillin. The company started producing five times more penicillin than expected. The production was started with a sterile culture of penicillin mold, which was then moved to huge fermenter tanks, called seed tanks where the mold was allowed to grow for two to four days. The trickiest part out of the whole process was extracting penicillin from the broth. The extracted penicillin was purified and stored. The scientists later discovered that the yellow color of penicillin indicated the presence of impurities which was corrected to produce white penicillin which was stable at room temperature and also retained its potency for years.

Discussion

The production of Penicillin has increased significantly from the time when it was discovered first by using various methods. The fermentation processes used initially were developed radically by scientists and engineers to give rise to the techniques that are used at present. Genetically modified versions of the fungi have also been used today. The yield per liter has also been increased. Different fermentation processes have been used and modified for the production process. Online tools and computer-aided techniques are used for controlling and optimizing the standard conditions used during the fermentation process. Further, techniques which increase the yield per liter may be developed and could be incorporated. Problems related to the fermentation techniques have to be avoided to get better quality of the drug. Commercial strains of Penicillium chrysogenum are grown using submerged culture in constant agitation and aerated in 50,000-gallon stainless steel tanks. Thesel strains can produce 40-50 grams of penicillin per liter of culture with a 90% yield.  This is an overwhelming improvement from the earliest Peoria farmer’s market strain that only produced 0.15 grams per liter with very low recovery rates. In order to achieve these production rates, modern Penicillium strains display a host of genetic and cellular modifications that result in increased production, including amplification of the penicillin biosynthesis gene cluster, an increased number of peroxisomes, and elevated levels of transporter proteins that secrete newly produced penicillin out of the peroxisomes and the cell.