ANASTASIA & ALEXANDER SKOROBOGATIY

she/her; he/him | age 16 | Kirkland, QC

Prix Québec Science Distinction Award, Super Expo-Science Hydro-Québec | Prestige Award, Regional Science Fair, l’Institut National de la Recherche Scientifique | Excellence Award, Super Expo-Science

Edited by Manel Zeghal

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INTRODUCTION

Since the 1970s, biologics have been an important advancement in modern medicine (LEEM, 2011). Unlike most chemically synthesized drugs, biologics come from natural sources, i.e., products of living organisms (Center for Biologics Evaluation and Research, 2018), which are large complex molecules that may be composed of hundreds of amino acids (Marrow and Hull Felcone, 2004). These products are used to treat a wide spectrum of diseases and conditions, including diabetes, autoimmune diseases, cancer, etc. (Ogbru, 2019).

Biological drugs are one of the most advanced therapies, however, they are very expensive. For example, a single injection of Remicade, a biologic used to treat autoimmune disorders, costs approximately $1000 (‌Canadian Agency for Drug and Technologies in Health, 2017). That is why there are biosimilar drugs: almost identical replicas of biologics, but produced by other companies, and costing less. For example, a biosimilar of Remicade, Inflectra, costs $525 per injection, which is almost 50% less expensive (‌Canadian Agency for Drug and Technologies in Health, 2017). However, these medications are still very expensive and inaccessible to people with limited income. This is why biohacking was invented.

Broadly defined, biohacking is a new movement aimed at improving health (Craig, 2022; Berning, 2021). One of the branches of biohacking focuses on reproducing and developing treatments for diseases in small laboratories. This intends to reduce the biologics’ high prices and make them accessible to citizens with limited income (Berning, 2021; Vice News, 2019; Berna, 2021). An example of such a movement is the "Open Insulin Foundation" (Berning, 2021; Molnar, 2015).

Insulin is the first true biologic that has been commercialized. This peptide is composed of two chains of amino acids linked by disulfide bonds, and acts as a hormone secreted in the blood system involved in the regulation of blood glucose. The body's inability to produce enough insulin or use it properly leads to diabetes, a chronic metabolic disease. Insulin injections are therefore used to control blood sugar in these patients. Until 1980, porcine insulin was purified and used for treatment (Molnar, 2015). Even though insulin extracted from pork pancreatic glands helped patients manage glucose levels, its use had the potential to lead to allergic reactions and antibody production. Additionally, significant amounts of animal parts are required to produce a low quantity of pure insulin, thus, supply of animal insulin is strongly dependent on animal gland availability (‌FDA History Exhibits, 2022). With the development of recombinant DNA technology, scientists have been able to produce and purify human insulin. Large factories use genetically modified bacteria (E. coli) or yeast cells (S. cerevisiae), which are abundantly cultivated in bioreactors. At the peak of growth, the cells are isolated by filtration, lysed, and maintained at stable temperatures with inhibitors against protein degradation. The two peptide chains are then chemically modified to reproduce disulfide bonds and isolated from other proteins by affinity chromatography, followed by a final purification step (Maloney, 2021).

To identify the challenges in the production chain of biological products, and to understand why they are so expensive, we conducted an experimental study to better understand the steps and the associated difficulties involved in such a process. In addition, we want to know if a small-scale laboratory, such as the one at our school, can produce biologics. After an extensive literature review, we have concluded that with the material at hand, it would be challenging to reproduce biologics like insulin at our school due to the need for genetically modified microorganisms, a tightly regulated sterile growth environment, and an extremely complicated purification process. Therefore, we looked for an alternative to insulin that can still be classified as a biological drug (meaning, a substance that is made from a living organism or its products) while being easier to produce.

As a test system for our study, we chose penicillin production via a biosynthesis route, which shares many similar steps with the production of biologics like insulin. Alexander Fleming's discovery of the production of penicillin by mold revolutionized medicine for treating previously deadly bacterial infections (McFarlane, 2020). After further analysis, we concluded that the production of penicillin, a by-product of mold growth commonly found in our homes, on bread and fruit might be possible in a small, non-specialized laboratory.

Hypothesis:
Using penicillin production via biosynthesis as a model biological system, we can isolate and purify small quantities of penicillin, as detectable by Delvotest SP-NT, in a basic biochemical laboratory.

Figure 1: (left) Growth of mold on bread sprinkled with water after 1 week. The mold with penicillin is in the stage when it goes from white to bluish-green. (right) Preparation of the potato broth agar.

Through a literature search (Vuković, 2022), we developed the following protocol to produce purified penicillin:

1. Isolate Penicillium chrysogenum mold: In this step, we used a slice of bread, lightly sprayed it with water, and placed it in a dark room at room temperature (the optimal conditions for growth). Mold developed in 3-4 days (Fig. 1, left). We kept an eye out for a white mold that turns bluish-green (it’s the one that produces penicillin).

2. Prepare growth medium for recultivation of P. chrysogenum: Further cultivation of the mold is needed to isolate individual colonies and verify morphology. Therefore, we prepared Petri dishes filled with potato broth agar, on which the mold grew (Fig. 2, right). These steps and the one that follows were all performed using aseptic techniques (Bunsen burner) to ensure sterility.

**Figure 2**: (left) *P. chrysogenum* on an agar plate under a microscope after 3 days. (right) The presence of a yellowish colour around the edges of mold growth indicates the presence of penicillin.

3. Transfer the P. chrysogenum mold to the agar. We used an inoculation loop to transfer the mold from the bread to the agar by streaking the mold on the plate, while sterilizing the loop between each streak. Then, the covered petri dishes were set aside at room temperature, away from direct sunlight. We observed the growth under the microscope daily to verify mold morphology and confirm the growth of P. chrysogenum (Fig. 2, left). After 3-4 days, we noticed the presence of a yellowish colour around the edges of our growth, indicating the presence of penicillin (Vuković, 2022) (Fig. 2, right).

Figure 3: (left) Controlled growth after 1 week. (right) Uncontrolled growth after 1 week (Insert: at the beginning).

4. Mold fermentation: Since the mold needs to reproduce in large numbers to produce significant quantities of penicillin, we created a fermentation medium, which we added to a round bottom flask and an Erlenmeyer flask. Next, we fermented the P. chrysogenum spores grown on the plates. Two experiments were carried out in parallel using the same nutrient mixture and isolated colonies. The round bottom flask was aerated using an aquarium air pump at a temperature of 25°C and a pH of 5-6, which are within the optimal growth conditions range for P. chrysogenum (Ayuningtyas, 2021) (Fig. 3, left). At the same time, the Erlenmeyer flask was left in the dark at room temperature 20-21°C (Fig. 3, right).

**Figure 4**: Steps of extraction and purification of penicillin.

5. Extraction and purification of penicillin: After 10-14 days, penicillin should be present in the fermentation medium. We filtered the medium twice (through coffee filters). To separate the penicillin, the pH of the solution was adjusted to 2.0 with HCl, and the filtrate was mixed with ethyl acetate (1:1) in the conical separatory funnel. After shaking, the penicillin dissolved in ethyl acetate. The two liquids were then allowed to separate and the top portion containing penicillin was collected. Finally, potassium acetate was used to precipitate the penicillin, while ethyl acetate was allowed to evaporate for at least one week (Fig. 4).

Materials used in our experiments are summarized in Table 1, while protocols for the preparation of agar growth medium and fermentation media are presented in theAppendix.

**Table 1**: Summary of materials used in our experiments.

RESULTS

**Figure 5**: Tests for the presence of penicillin in various samples.

To test whether penicillin was produced, we used a Delvotest® SP-NT Test Kit (Kukurová & Hozová, 2007), a commercially available product used to detect the presence of penicillin-based antibiotics in dairy products. The test is based on the ability of thermophilic bacteria, Bacillus stearothermophilus, to grow. Each test tube contains Bacillus stearothermophilus in a solid growth medium, purple in colour. When kept at 4°C, the bacterium does not grow, however, once shifted to 62-65°C, it is able to grow. If an antibiotic is present in a sample being tested, the bacteria do not grow, and the growth medium remains purple. If no antibiotics are present, the bacteria grow, and the medium turns yellow.

Distilled water and the fermentation medium were used as negative controls and different concentrations of penicillin V dissolved in distilled water as positive controls. As predicted, penicillin was detected in the positive, but not the negative controls (Fig. 5, right).

As mentioned earlier, we performed two experiments in parallel: controlled (bioreactor) and uncontrolled fermentation cultures. We maintained the optimal conditions in our bioreactor, as described in past literature (Ayuningtyas, 2021). With aeration, we notice that the pH is unstable and that it decreases throughout the fermentation process. To raise the pH, we needed to add 1.25 M sodium hydroxide several times. No presence of penicillin was observed in the controlled medium. It is possible that the addition of sodium hydroxide resulted in the inactivation of P. chrysogenum as 0.5 – 1 M sodium hydroxide solution is used as a sanitizing agent due to its ability to inactivate various viruses, bacteria, and fungi (Cytiva, 2020).

In contrast, the pH of the uncontrolled culture remained stable at around 5.0, and after ~2 weeks of fermentation, we were able to detect penicillin in this culture (Fig. 5, left).

While we were unable to obtain penicillin crystals at the end of the experiment, we obtained dark red-brown precipitates for both uncontrolled and controlled culture media after the ethyl acetate evaporation. The presence of an amorphous precipitate instead of expected crystals suggests that our end product contains impurities. Both precipitates were again tested for the presence of penicillin. As with both culture media, we were unable to detect penicillin in the controlled culture precipitate, whereas it was present in the uncontrolled culture precipitate (red-brown liquid obtained by evaporating the ethyl acetate from the uncontrolled growth medium).

In Table 2 we summarize the results for the mold growth in the controlled and uncontrolled media.

**Table 2**: Observations about controlled and uncontrolled culture growth and penicillin presence.

DISCUSSION

During the experiment, we encountered several challenges that needed to be overcome. When preparing agar plates, we learned that for the agar to be uniform and solid, it had to be completely dissolved in a potato broth before being poured into the petri dishes. Nonetheless, as fungi are known to tolerate a variety of growth conditions, we do not believe this to be a limiting factor for P. chrysogenum growth and ultimately penicillin production. Also, the composition of the fermentation medium had to be modified to ensure an appropriate pH level and to avoid curdling of the milk. For optimal growth conditions, the aeration rate, pH, and temperature should be closely controlled and adjusted during controlled fermentation, which presents a challenging problem during process optimization. In industrial penicillin production, strains of P. chrysogenum expressing additional copies of the penicillin biosynthesis cluster and enzymes involved in its synthesis are used in order to increase the yield of antibiotic (Weber, 2012). While these strains are not available to us for penicillin production in an unspecialized laboratory, we could try manipulating external factors that have been shown to promote penicillin production by P. chrysogenum. It has been reported that penicillin production by fungi is increased under stress conditions such as temperature and salt levels/dehydration (Parsons, n.d). We could try using increased temperature and dehydration in our future experiments to optimize penicillin production. Additionally, sterility should be maintained during all the manipulations for growth parameter adjustment, which is a significant technological problem for a non-specialized laboratory. Finally, the ethyl acetate evaporation took a long time and required a few weeks before it completely evaporated, so care should be taken to avoid contamination of a sample during evaporation.

CONCLUSION

The hypothesis of our experiment is partially confirmed since we succeeded in producing penicillin in liquid culture. However, we could not isolate the purified penicillin in the form of crystals, which can be used for medical applications.

Furthermore, we only detected the presence of antibiotics in the culture with uncontrolled growth conditions, as well as in the red-brown precipitate isolated after the evaporation of ethyl acetate from the uncontrolled growth medium. No presence of penicillin was observed in the controlled culture, which could be explained by fungus inactivation while performing pH adjustments using sodium hydroxide. Additionally, lack of sterility during pH adjustments and culture aeration might have introduced a microorganism that was able to compete with P. chrysogenum, further leading to a decrease in mold growth. This is suggestive from the smell of the controlled culture compared to uncontrolled growth. Sterility is an important factor to maintain in biologics production because only a desired microorganism should continue to grow so as not to compromise the final product.

From our experience, we conclude that it is challenging to synthesize and isolate even the simplest of biological products in a non-specialized laboratory due to sterility issues, the need for research, technology-intensive process optimization, as well as a lack of highly specialized equipment. Therefore, this explains the high prices of biologics and why insulin is not yet accessible via biohacking, since it has a more complex manufacturing process than penicillin and requires even more advanced laboratories and qualified personnel. However, we are confident that with enough determination and commitment from talented scientists, biohacking efforts will soon provide accessible and available biologics.

REFERENCES

‌Ayuningtyas, A. C. (2021). Growth and Penicillin Activities Resulted by Penicillium chrysogenum in Tomato (Solanum Lycopersicum L.) Juice. Malaysian Journal of Medicine and Health Sciences, 17(SUPP2): 16-18. https://medic.upm.edu.my/upload/dokumen/202104291507012020_0876_05.pdf

Berna, M. (2021, September 14). What Is the Monthly Cost of Insulin in Canada? Olympia Benefits Inc. https://www.olympiabenefits.com/blog/what-is-the-monthly-cost-of-insulin-in-canada

Berning, J. (2021, June 26). Biohackers take aim at big pharma’s stranglehold on insulin.

Freethink 2021. https://www.freethink.com/series/just-might-work/how-to-make-insulin

‌Canadian Agency for Drug and Technologies in Health. (2017, November). Cost Comparison – Subsequent Entry Biologic Review Report: Inflectra (infliximab). https://www.ncbi.nlm.nih.gov/books/NBK534733/

Center for Biologics Evaluation and Research. (2018, February 6). What are “biologics” Questions and answers. U.S. Food and Drug Administration. https://www.fda.gov/about-fda/center-biologics-evaluation-and-research-cber/what-are-biologics-questions-and-answers

Craig, P. (2022, September 29). Intriguing and Perhaps Alarming New Approaches to Fitness in 2022. Fitness Business Association. https://member.afsfitness.com/content/intriguing-and-perhaps-alarming-new-approaches-fitness-2022

‌ Cytiva (2020). Use of sodium hydroxide for cleaning and sanitization of chromatography resins and systems. Application note, KA2918110418AN. https://cdn.cytivalifesciences.com/api/public/content/digi-20986-original#:~:text=Sodium%20hydroxide%20has%20shown%20to,yeasts%2C%20fungi%2C%20and%20endotoxins

FDA History Exhibits (2022, June 8). 100 Years of Insulin. U.S. Food and Drug Administration. https://www.fda.gov/about-fda/fda-history-exhibits/100-years-insulin

Kukurová, I., & Hozová, B. (2007). The utilization of disk diffusion method and the Delvotest® for determining synergistic effects of cephalosporin combinations in milk. Journal of Food and Nutrition Research, 46(1), 9–14. https://www.vup.sk/resources/bulletin/jfnr07-1-p009-014-kukurova.pdf

Les entreprises de médicament LEEM. (2011, May 5). Les biomédicaments : nouvelle génération de traitements. https://www.leem.org/les-biomedicaments-une-nouvelle-generation-de-traitements

Maloney, D. (2021, August 23). Open-source insulin: Biohackers aiming for distributed production. Hackaday. https://hackaday.com/2021/08/23/open-source-insulin-biohackers-aiming-for-distributed-production/Marrow, T., & Hull Felcone, L. (2004). Defining the difference: what makes biologics unique. Biotechnology Healthcare 1(4): 24-29. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564302/#:~:text=Some%20would%20apply%20a%20strict,heterogeneous%20structure%20that%20can%20contain

McFarlane, N. (2020, February 4). The History of Penicillin. Allergy and Asthma Center of Boston. https://www.allergyasthmaboston.com/new-blog/2020/2/4/the-history-of-penicillin

Molnar, G. (2015, March 4). Insulin. The Canadian Encyclopedia. https://www.thecanadianencyclopedia.ca/fr/article/insulineOgbru, O. (2019, October 28). Biologics (Biologic Drug Class). MedicineNet. https://www.medicinenet.com/biologics_biologic_drug_class/article.htm

Parsons, A. (n.d). An introduction to penicillin. Future Learn. https://www.futurelearn.com/info/courses/everyday-chemistry/0/steps/22312#:~:text=Interestingly%2C%20penicillin%20is%20only%20produced,increased%20temperature%20or%20salt%20levels

VICE News. (2019, July 22). Inside the factory where most of the world’s insulin is made [Video]. Youtube. https://www.youtube.com/watch?v=nqkggYSVCh4&t=570s

Vuković, D. (2022, August 4). How to make penicillin at home (just in case SHTF). Primal Survivor. https://www.primalsurvivor.net/make-penicillin-home/

Weber,S., Poli, F., Boer, R., Bovenburg, R.A.L., & Driessen, A.J.M. (2012). Increased penicillin production in Penicillium chrysogenum production strains via balanced overexpression of isopenicillin N acyltransferase. Applied Environmental Biology, 78(19): 7107-13. https://pubmed.ncbi.nlm.nih.gov/22865068/

APPENDIx

I. Isolation of Penicillium chrysogenum mold:

Place a piece of bread from a local bakery in a container and spray water on the bread.
Cover with a lid and leave all but one corner closed to keep moisture in.
Place the container out of direct sunlight, at room temperature (20-25°C).
Wait for mold to grow (3-10 days), look for white mold that turns bluish-green.

II. Preparation of agar growth medium for recultivation of P. chrysogenum:

Thinly slice 100g washed potatoes (unpeeled) and transfer them to a 1-liter mason jar.
Fill a mason jar with distilled water and put the lid loosely on without screwing it.
Boil the mason jar in a large saucepan filled with water for 30 minutes, then let cool.
Remove the lid and pour the liquid into the sterilized beaker by straining it through a sterile strainer and cheesecloth.
Add dextrose, agar and mix thoroughly with a sterilized spoon.
Add distilled water to the final volume of 250 ml.
Pour the potato broth onto each of the Petri plates, being careful to respect the sterility of the process by pouring without the containers touching each other.

III. Transfer of the Penicillium mold onto the agar:

Sterilize the inoculating loop by heating it over a flame.
Open a Petri plate and insert the loop into the growth medium to cool it.
Use the loop to touch the bluish-green part of the moldy food. Streak the mold on the surface of the agar in 3 lines. Repeat making 3 more ridges at a 90-degree angle to the previous ridges. Immediately close the lid.
Repeat this operation on all Petri plates, sterilizing the wire between the plates.
Keep the Petrie plates covered and set aside at room temperature, out of direct sunlight.
Wait 3-7 days until growth is apparent. (The mold will have a yellow substance surrounding it on all edges, penicillin).
Check under an optical microscope that it is indeed Penicillium mold that looks like a paintbrush made of smaller round sections. Proceed to the next step only if you are sure the growth is Penicillium mold.

IV. Fermentation:

Measure dextrose, yeast extract, citric acid, milk powder, and sea salt and add them to a sterile graduated cylinder. Top up with distilled water up to 100 ml. Mix until everything is dissolved.
Pour the fermentation medium into the sterilized 1 L Erlenmeyer flask.
Transfer the grown mold from the Petri plates into the Erlenmeyer flask. Practice sterile technique.
Uncontrolled growth: cover the container with sterilized aluminum foil to avoid contaminants and leave it for 7-14 days out of direct sunlight at room temperature.
Controlled growth: close the Erlenmeyer flask or a round bottom flask with a rubber stopper that has three holes. Into the first hole insert a glass tube for airing the growth medium with air from the aquarium pump. In the second hole insert a probe of a temperature/pH meter (sold commercially for testing garden soil). In the third hole insert a glass tube for the excess air to escape the bioreactor. Mount the flask on a hot plate. Adjust pH of a growth medium daily by adding a 1.25M NaOH solution as necessary to maintain the pH of a growth medium around 5.5. Adjust the temperature of a hot plate to keep the temperature of a growth medium between 24-25oC.

V. Extraction and purification:

Chill the bottle of ethyl acetate in a freezer.
Pour the liquid from the fermented vial, through the strainer and coffee filters, into the sterile beaker. Repeat the filtration twice. Use the filtered growth media samples to test for the presence of penicillin.
Use a pH tester to check the pH of the filtered liquid (the pH should be around 5.0 at the end of a growth process). Use a dropper and suck up a few drops of hydrochloric acid. Add a drop to the liquid, stir with a sterile spoon, then check the pH. Continue until the pH reaches 2 or 2.2.
Prepare the conical separatory funnel. Pour the chilled ethyl acetate and the filtered liquid into the funnel (ratio 1:1). Close the top funnel lid and shake vigorously for 30-35 seconds.
Place the funnel on its support. Wait a few minutes until the mixture separates. The top liquid is ethyl acetate where the penicillin is dissolved.
Drain the bottom liquid and transfer the top liquid into the sterile glass beaker.
Add 1 ml of potassium acetate per 100 ml of separated ethyl acetate. Gently mix the solution.
Set the open beaker aside in a well-ventilated area. The ethyl acetate evaporates, leaving the penicillin salt at the bottom.
Store penicillin salt powder in an airtight glass bottle.

VI. Detection of penicillin in liquid culture:

Prepare a 65°C water bath using a hot plate and continue heating it to maintain a stable bath temperature between 62-65°C. Continue checking the water temperature every 20 minutes for the remainder of the experiment.
Use the Delvotest® SP-NT Test Kit. It detects the presence of penicillin-based antibiotics using thermophilic bacteria, Bacillus stearothermophilus. The bacteria do not grow in the presence of penicillin (the growth medium remains purple) and grow when there is no penicillin (the growth medium turns yellow).
Take the Delvotest® SP-NT tubes and carefully lift part of the protective seal (aluminum foil).
Pipette 0.1mL of a test sample into a separate tube. Use a different pipette for each test sample. Negative controls are distilled water and fermentation medium; the positive controls are penicillin V solutions at 1 mg/mL and at 50 mg/mL. Test samples: controlled growth medium, uncontrolled growth medium, and red-brown liquids isolated after evaporation of ethyl acetate from controlled and uncontrolled growth medium.
Cover the protective aluminum sheets of each test tube.
Insert the tubes into the flotation racks and place them in the water bath.
Incubate for 3 hours at 62-65°C.
Observe and record results.

Biohacking of Biologics: A Case Study of Penicillin Biosynthesis