Making Penicillin, Part 4: Fermentation

Introduction
Most bioproducts (products produced biologically, often by microorganisms) are produced by fermentation. Microbes are grown in conditions that make them divide rapidly until the culture is dense. When the easily "digestible" nutrients are spent, the culture reaches maximum density and the microbes "switch metabolic gears." This is usually when they start producing secondary metabolites - the product you want. This is fermentation.

Glucose is a simple sugar that acts as a quick carbon source, jumpstarting the rapid growth of your culture. The idea is to increase the density of your Penicillium so that you have a lot of cells producing penicillin. Yeast extract is a bunch of yeast cells that have broken themselves down (a process called “autolysis”) into component carbohydrates and amino acids. This provides a lot of nutrients and longer-term energy once the glucose is depleted (which happens pretty quickly). Citric acid acts as a buffer, to prevent the pH of your medium from fluctuating and stressing your cells. Lactose is a simple sugar (glucose bonded to galactose) that is often used because unlike glucose, lactose doesn’t inhibit penicillin production. It’s important to use sea salt rather than regular table salt for two reasons: 1) table salt uses iodine as an anti-caking agent, which is great for people (we have thyroid problems if we don’t get enough iodine), but can inhibit microbial growth; and 2) aside from the iodine, table salt is pure sodium chloride, and your cultures need the other minerals and micronutrients found in sea salt.

Materials
Erlenmeyer flask/glass jar
graduated cylinder
100mL distilled water
2g glucose (aka dextrose)
1g yeast extract
2g citric acid
10g powdered nonfat milk

• While this concentration provides enough lactose (a simple sugar) to promote a high concentration of penicillin, this will make the medium thick, and some may not even dissolve. At this concentration, you'll see the growth, but you can dial back the powdered milk if you'd like a clearer view

1g sea salt
forceps/inoculation

Procedure
1. Prepare the medium. Take a clean, dry Erlenmeyer flask or glass jar. Lab scale fermentation is typically performed in shake flasks. As a rule of thumb, whatever the volume of your Erlenmeyer flask is, you never want it more than 20% full with medium. Thus, if for example you have a 500mL flask, only put 100mL of medium in it. Keeping the liquid volume low increases the surface area of the liquid, allowing for faster diffusion of oxygen from the air to the medium, and Penicillium definitely needs oxygen to make penicillin.¹

If you’re shooting for 100mL of inoculated medium, put 2g glucose (aka dextrose), 1g yeast extract, 2g citric acid, 10g powdered nonfat milk and 1g sea salt to your graduated cylinder. Fill the graduated cylinder with distilled water until it reaches the 100mL level. Pour the whole thing into your flask and mix until everything’s dissolved. Cover the top of the flask with a folded pieced of aluminum (it keeps errant microbes out but lets oxygen in), and autoclave.

2. Inoculate with your isolate. Once the medium has cooled to room temperature, aseptically add spores of your Penicillium culture with sterile forceps or inoculation loop. Once you have replaced your sterile aluminum foil cover (after flaming both), you can let the culture sit at room temperature (give it an occasional swirl) for at least a week (but not longer than two weeks).

References
1. Rolison, G. N. 1952. "Respiration of Penicillium chrysogenum in penicillin fermentations." J. gen. Microbiol. 6: 336-343