Making Penicillin, Part 5: Extraction

Introduction

Penicillin-producing fungus will excrete penicillin into the fermentation medium. This means that you don't have to lyse (bust open) the cells in order to recover your product. If you wish, you can siphon off some of the supernatant (the liquid portion of the fermentation) to extract penicillin, and add fresh media to the remainder of the fermentation to continue the culture.

If you're very lucky, you'll recover 1g/L of supernatant. According to Rxlist.com, it takes up to 24 million units (about 15g) per day (divided over 4-6 doses) to treat septicemia, pneumonia, endocarditis, meningitis and syphilis. For treatments lasting 14 days (as is often the case with syphilis), that's 210g of penicillin phosphate - 200 liters' worth of fermentation for the treatment of one person. It is difficult enough to produce that volume of antibiotic, but it is even more difficult given penicillin's finite shelf-life. Pharmaceutical-grade penicillin has an expiry of a year; even in a post-apocalypse scenario, I wouldn't hold onto homemade penicillin any longer than that.

Materials

Penicillium fermentation
coffee filter
hydrogen chloride (aka HCl or hydrochloric acid)
ethyl acetate
pH strips
separatory funnel
ring stand
potassium acetate

• The link above is to potassium acetate salt, which you can use to make a 1% solution. To do so, add 0.5g potassium acetate salt to 50mL of distilled water and mix until the salt completely dissolves.

Procedure

1. Separate solids from liquid. You'll notice growth forming solid structures within the fermentation medium. The first step is to separate the liquid (in which the penicillin is secreted) from the solids. One way to do this is to place the separatory funnel in the ring stand, remove the stopper, curl a coffee filter into a cone shape and fit the apex of the filter-cone into the neck of the separatory funnel. Make sure the stop cock at the bottom of the separatory funnel is in the closed position, and pour the fermentation through the coffee filter.

2. Refrigerate. Once the fermentation has been filtered, remove the solids, stopper the separatory funnel and place it in the fridge. While the liquid is cooling, put the ethyl acetate in the freezer (it won't freeze), and put the hydrochloric acid in a bucket of ice to chill. Leave the fermentation liquid in the fridge for a couple of hours.

3. Adjust the pH. Add the hydrochloric acid solution in a few drops at a time, mix and test the pH. The fermentation should start at a pH of around 5; the more acid you add, the lower the pH will go. You'll probably have to repeat adding acid and testing several times to get a solution to a pH of 2.2, especially since the citric acid in your solution acts as a buffer. This means that as you at acid (protons), citric acid will soak them up (to a point), and your solution's pH won't appear to change much. Try not to get impatient and pour acid into your mix; there comes a point where the citric acid has soaks up all the protons it can, and suddenly the pH will change dramatically with a small addition of acid.

4. Take collection beaker. Take a clean glass vessel (such as a mason jar), measure the mass with your analytic balance. Write the mass in permanent marker on the outside of the jar (you may even want to put a piece of clear tape over your writing to make certain it's legible later). When you've collected your penicillin, you can subtract the weight of the glass vessel from the total weight to calculate the amount of penicillin you produced.

5. Add cold ethyl acetate. On the molecular level, ethyl acetate looks a lot more like penicillin than acidic water - i. e., penicillin is more soluble in ethyl acetate. Add cold ethyl acetate, put the stopper back in the neck of the separatory funnel and shake the whole thing vigorously for at least 30 seconds. This will allow the penicillin to move from the acid water to the ethyl acetate. Ethyl acetate is denser than water, so it will settle on the bottom of the separatory funnel. Put a beaker or container under the separatory funnel, open the stop cock slowly and pour off the ethyl acetate (ideally in a glass container that has been chilled in the freezer). To make sure you've extracted as much penicillin as you can add more ethyl acetate to the remaining acid water solution, shake, and pour off the ethyl acetate layer into the collection beaker.

6. Add potassium acetate to your ethyl acetate solution. A good way to keep your ethyl acetate solution cold is to seat the glass vessel (e. g., mason jar) within a dish of crushed ice. With a 1% solution of potassium acetate, you will have 1g of potassium acetate per 100mL of the solution. At best, you will only get about 1g of penicillin per liter (0.1g in your entire 100mL fermentation), so you won't need a lot of potassium acetate; 100mL should be plenty.¹

7. Evaporate the ethyl acetate. Put your solution in a a well-ventilated area (ideally away from living areas, such as in a garage with a fan - you don't want to breathe the fumes). This will allow the water and ethyl acetate to evaporate, leaving only potassium acetate salt (which will act as a penicillin preservative) and potassium penicillin G salt as the precipitates. It's important that all the liquid has evaporated before you weigh the vessel; any remaining liquid will add weight to the vessel and falsely inflate the calculated amount of penicillin you produced.

8. Weigh and calculate. Weigh the vessel with the penicillin salt in it, then subtract the tared mass of the empty vessel (from step 4). You will also need to subtract the amount of potassium acetate from the total mass to get the final weight of penicillin:

Total mass - empty vessel mass - (1g potassium acetate/100mL solution X mL of solution used) = mass of penicillin

9. Refrigerate. Even with the potassium acetate acting as a preservative, penicillin G has a limited shelf-life (you probably shouldn't use it after a year).

References

1. Sajjad-Ur-Rahman; Rasool, M.; Rafi, M. 2012. "Penicillin production by wild isolates of Penicillium chrysogenum in Pakistan." Braz J Microbiol. 43(2): 476-481