By Dr Tomislav Meštrović, MD, PhD and Susan Chow, PhD, ELS
Penicillin, which remains an important part of our antimicrobial armamentarium, had a significant impact on the second half of the twentieth century. Deep-fermentation methods, which were primarily developed for the production of penicillin during the war, gave rise to the development of antibiotics and contributed to the nascent biotechnology industry which appeared in the 1970s.
Akin to other antimicrobials, penicillin is a secondary metabolite, thus it is only produced in the stationary phase. The industrial production of penicillin was generally classified into two processes – upstream processing and downstream processing.
Upstream processing encompasses any technology that leads to the synthesis of a product and includes the exploration, development and production. The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing.
The Fermentation Process
Fermentation is the technique used for the commercial production of penicillin. It is a fed-batch process that is carried out aseptically in stainless steel tank reactors with a capacity of 30 to 100 thousand gallons. The fermentation involves two to three initial seed growth phases, followed by a fermentation production phase with a time cycle ranging from 120 to 200 hours.
Various carbon sources have been adopted for this process – including glucose, sucrose and other crude sugars. Approximately 65% of the carbon is used for cellular maintenance, 25% for growth and only 10% for penicillin production. Sugar is also used for the regulation of the pH value during active penicillin production phase.
Mini-harvest protocols are usually employed in penicillin fermentation. They involve the removal of 20-40% of the fermentor contents and its replacement with 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.
Present-day penicillin fermentations are highly automated and computerized. All the necessary precursors, ammonia, sugar, carbon dioxide, oxygen are controlled, with thorough monitoring of temperature and pH for optimal antibiotic production. The pH should be between 6.4 and 6.8 during the active production phase.
Pursuit for a Better Yield
When penicillin was initially made at the end of the Second World War using the fungus Penicillium notatum, the process yielded one milligram per cubic decimeter. Today, with a use of a different species (Penicillium chrysogenum) and improved extraction procedures the yield is 50 grams per cubic decimeter.
The yield of penicillin can be further increased by improving the composition of the medium, isolating aforementioned Penicillium chrysogenum which grows better in huge deep fermentation tank, but also via the development of submerged culture technique for cultivation of mold in large volume of 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 between tandem repeats represents one of the most important phenomena in high-yielding Penicillium chrysogenum strains. Molecular strategies that differ from those involving an increase in biosynthesis gene doses have also been developed.
Early Penicillin Production
Although the therapeutic need for penicillin was great, laboratories in the United Kingdom were unable to dedicate sufficient resources to improving production methods because industrial manufacturing efforts were focused on generating supplies for World War II. Therefore, in 1941, Howard Florey and his colleague Norman Heatley travelled to the United States to work with scientists at the U.S. Department of Agriculture’s Northern Regional Research Laboratory (NRRL) in Peoria, Illinois. Together, this group of British and American scientists set out to improve production methods. Their first task was to identify Penicillium chrysogenum strains that produced the highest levels of penicillin. 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. Using the farmer’s market strain as a base, scientists used x-rays and ultraviolet light to create 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 (a nutrient-rich by-product of corn processing) also increased yield. Even though production methods were crude, this earliest form of penicillin was in high demand during World War II and is thought to have saved thousands of lives.
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 constantly agitating and aerated 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. 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.
Worldwide sales of penicillin and other beta-lactam antibiotics is now greater than $15 billion (U.S. dollars) per year. These sales numbers exist despite the fact that cost is now at an all-time low. 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.