Penicillin

Mass production of penicillin in flasks.

Penicillins are a group of beta-lactam antibiotics produced by the mold species Penicillium chrysogenum and P. rubens, which were first discovered in 1928 by Sir Alexander Fleming. Penicillin has a long history of use, ever since 1939 when it was first isolated by a team lead by Howard Florey and Ernst Chain, and was the second antibacterial drug to be used in humans, after the compound sulfanilamide. Penicillin is much safer, much more potent, and more broad spectrum than sulfanilamide, allowing it to be used in many more infections.

However it should be noted that some infections will not respond to penicillin. This may be due to innate immunity of the bacterium or from developed resistance to the effects of the drug. Most forms of bacterial GI tract infections do not respond to treatment with penicillin (although, fortunately, many will respond to sulfonamide treatment). Most forms of pneumonia, meningitis, and gangrene respond well to treatment with penicillin. There is no universal way of knowing how effective penicillin will be without possessing a culture of the bacterium. If a culture has been produced, however, it is simple to form dilutions of the antibiotic and assay its efficacy by that manner. This also helps in the process of calculating doses and allows for as much of the precious drug to be conserved as possible.

Resistance

Seeing as penicillin is such an old and widely used antibiotic, resistance to it is relatively widespread among infectious bacteria. The most notable penicillin-resistant bacterium is Staphylococcus aureus, a very common cause of wound infections. In fact, most Staphylococcus spp. now possess some degree of resistance to penicillin. Streptococci have been much slower in developing resistance, and penicillin may still be used to treat streptococcal pneumonia, the most common form of pneumonia, and scarlet fever. However, some strains of S. pneumoniae have managed to develop resistance.

Developed resistance is not the only source of problems, however. Certain bacteria have an innate resistance to it. The most clinically significant class of these are the enterobacteria, pathogenic and symbiotic members of the family Enterobacteriaceae. These bacteria are common causes of bacterial diarrhea, both opportunistic and otherwise. Pseudomonas aeruginosa, a soil bacterium commonly implicated in wound infections characterized by a greenish-blue exudate, have long had penicillin resistance and many strains are in fact multi-drug resistant.

Perhaps the most consequential bacterium with innate resistance, however, is Mycobacterium tuberculosis the causative agent of tuberculosis. Tuberculosis is infamously difficult to treat, and even today, the only way to cure the infection is with a harsh cocktail of broad-spectrum antibiotics and antituberculars. Penicillin is wholly ineffective at treating tuberculosis.

Production

Penicillins are a relatively simple class of antibiotics to produce. Penicillium spp. are cosmopolitan in their distribution, are easily cultured, and they are very harmless. In addition, Penicillium spp. are unique in their morphology relative to other genus, making it much easier to identify the mold. Bear in mind that the following procedure will produce penicillin G (see Use section below).

Mold Identification

The first step to the production of penicillin is to identify the mold. Penicillium spp. generally like to grow in acidic environments, and thus the classic story of an ultra-high producing strain being discovered on an Illinois cantaloupe is not without merit, as cantaloupes are mildly acidic fruits. Although morphology could easily be used to differentiate geni, Penicillium chrysogenum would be difficult to discern from other species of Penicillium from morphology. Therefore, it makes sense to make use of its antibiotic production to identify it. Simply cultivate the mold to be tested along with some bacterium known to be susceptible to penicillin in Petri dishes. Lactococci and Streptcocci (from fermented milk products and saliva, respectively) work well for this purpose, however care must be taken when handling the latter, which can be pathogenic. If the mold is penicillin-producing, the characteristic "lytic ring" will form around the mold colony (a ring with no bacterial growth) and the agar will turn a golden yellow. Once this activity is identified, simply subculture the mold which will be used in the next step.

Materials and Equipment

Bioreactor Selection

Now it is necessarily to select the container to be used in the cultivation of the mold. This is of greater importance than one might imagine, as using an improperly chosen container will massively impair yield. Most forms of Penicillium chrysogenum are top fermenting, meaning that they will not grow in media even if it is aerated. Seeing, in addition, that it is unlikely that complex aeration equipment would be available to anyone seeking to rebuild society, it is necessary to rely on the mold's natural inclination to grow at the top of the culture medium. However, this poses new problems, as it means that only the topmost section of the broth will be colonized by the mold. Therefore, the containers in which the mold is cultivated must be relatively flat. Florey and Chain used modified bedpans placed on bookshelves to produce the massive amounts of culture necessary for their experiments.

Culture Medium

Next, one must select the culture medium to be used for the production. The culture which Florey and Chain used was Czapek-Dox medium, a medium composed primarily of sucrose and potassium nitrate with some substances to provide ions and buffer the solution, however, this is a very inefficient way of growing the mold. For reasons still not entirely known, surface cultures using a by-product of corn processing called corn steep liquor allows for a truly monumental increase in yield, from 5 to 20 times more penicillin than Czapek-Dox medium. It is very important to note that distilled water must be used for all preparations made for the cultivation of Penicillium chrysogenum. While the mold is scarcely affected by metal ions in water, the penicillin it produces very much is, to the point that even a bit of metallic contamination can completely spoil an entire batch. This medium is then inoculated with the mold previously isolated.

Example Medium Ingredients

In 1 liter of sterile distilled water, dissolve:

• 15 g yeast extract
• 10 g malt extract
• 10 g peptone
• 10 g glucose
• 35 g lactose
• 3 g ammonium nitrate
• 4 g monopotassium phosphate

Mix thoroughly and add a magnetic stir bar.

Culturing

Simple Culturing

The mold is then grown at room temperature (the mold is very hardy and does not require temperature controls). After approximately 10 days, the mycelium will have completely colonized the broth.

Bioreactor Culturing

Reactor Setup and Sterilization

1. Transfer the media into a 1.5 L autoclavable bioreactor.
2. Cap with sterile silicone covers for all hose barbs.
3. Autoclave the entire reactor assembly.
4. Allow to cool completely.

Inoculation

1. Inside a sterile environment, inject overnight mold culture via the inoculation port.
2. Replace silicone caps on inlet/outlet ports with sterile tubing and inline 0.2 µm filters.

Fermentation

1. Set bioreactor conditions to:

• Temperature: 25°C
• Stirring: Continuous (magnetic stir)
• Duration: 7–10 days (longer may improve yield)

1. Observe formation of a thick mold mass (pelle).

Harvesting Culture

1. Open the bioreactor. Use glass rods to remove the thick mold mat (resembles scrambled eggs).
2. Transfer mass to a secondary beaker.
3. Filter liquid phase through a fritted funnel.
4. Rinse mold mass with fresh water, stir, and filter again.
5. Combine filtrates.

Acid-Base Extraction

Pure penicillin G is a water-insoluble, acidic compound, so it can be quite easily separated from solution by first acidifying the broth and extracting with an immiscible solvent. Traditionally diethyl ether, chloroform and DCM would also work, and even ethyl acetate could be substituted, although with a significant loss of yield. Next, the immiscible solvent would be separated from the broth and the penicillin rendered soluble in water with a base (typically sodium or potassium carbonate, as hydroxides are too harsh and will destroy the unstable penicillin). This process would continue multiple times, transferring the penicillin between diethyl ether and water. At the end, the diethyl ether could be evaporated and a crude form of penicillin would be left. An example process is below:

Acidify and Salt

1. Add:

• 40 g ammonium sulfate
• 100 g sodium chloride

1. Slowly add hydrochloric acid until pH reaches 2–3.

Organic Extraction

1. Add 150 mL ethyl acetate.
2. Mix thoroughly to extract penicillin into organic layer.
3. Transfer to separatory funnel.
4. Allow layers to separate. Collect organic (upper) layer.

Optional: Pigment Removal

1. Add activated charcoal.
2. Stir and filter through cotton or filter paper.

Note: Regular charcoal is ineffective.

Back-Extraction

1. Prepare 30 mL water adjusted to pH 8 using sodium carbonate.
2. Add to organic layer. Mix well.
3. Use separatory funnel to recover aqueous layer.
4. This is your crude penicillin solution.

Final Purification

1. Freeze-dry the aqueous extract.
2. Recover powder.
3. Typical yield: ~125 mg/L (low on first attempt; optimize growth for more)

Testing

This penicillin, although no longer toxic, would still require further purification to be used with absolute safety, as in this form it has a much higher chance of causing anaphylactic shock. This purification would typically be in the form of a chromatography column. However, it is nonetheless pure enough to have the chance of helping more than it injures when injected, which the broth obviously could never do. Below are instructions for bioactivity confirmation:

Plate Setup

1. Prepare sterile LB agar plate.
2. Spread E. coli lawn evenly.

Disk Diffusion

1. Sterile filter paper disc: Add 20 µL of penicillin solution.
2. Place onto bacterial lawn.
3. Incubate overnight.

Result Interpretation

• Look for a clear "dead zone" around the disc.
• This indicates antibiotic activity.

Use

Not all penicillins are created equal and some are more useful than others for certain applications. The majority of penicillin produced by the mold is penicillin G. Penicillin G is not bioavailable orally and is almost entirely destroyed by the stomach acid. As a consequence of this, penicillin G must always be injected, typically intravenously, as it is extremely painful to inject the soluble salt into a muscle (it is, nonetheless, not typically dangerous to do it this way).

The form of penicillin most people are familiar with is penicillin V, which is a chemical modification of penicillin G which allows it to be bioavailable. It would be almost entirely impossible to produce penicillin V on a small scale, and so any attempted treatment with penicillin necessitates the use of IV injection. This is one major reason why penicillin should only be used when absolutely necessary. Not only is it incredibly difficult to produce, it also requires the use of IV injection, often in situations where modern sanitation and sterilization standards are impractical or impossible.

Verifications

I Grew Real Penicillin from MOLD, The Thought Emporium, YouTube, May 5, 2025. Duration: 18:50.