Along a spore’s journey to developing into a mushroom, there are several integral phases. A spore will bloom into highly branched structures reminiscent of treetops called hyphae. A forest of hyphae is called mycelium (Figure 1), which acts like roots reaching deeper into organic matter to obtain water and nutrients, breaking down large molecules (like cellulose) into simpler ones (like glucose) that the fungi can utilize.
A hardened mass of mycelium, called sclerotia, may form. The sclerotia acts as an emergency food bank, helping fungi survive environmental extremes. Some sclerotia are edible like the growths on birch trees known as chaga mushrooms (Figure 2). And the sclerotia of mushrooms like Psilocybe cubensis or Psilocybe mexicana contain the psychedelic molecules psilocybin and psilocin.
Psilocybin Culture, Subculture
When Albert Hoffman discovered the hallucinogenic properties of P. mexicana , there was interest amongst other researchers to unlock more of the mushroom’s secrets. To do so requires more of the compounds, and traditional ways of cultivating mushrooms can be very time-consuming, potentially requiring many months. The question was how to quicken the pace to obtaining larger stockpiles of psilocybin and psilocin? A mushroom culture provides a proliferative way to get more mushrooms by reproducing them in a growth medium under controlled conditions. A subculture involves removing some or all cells to put them into new batches of growth media. This method can lengthen the life of and augment the number of cells in the culture.
Submerged culture fermentation, where the fungi is placed within a closed flask containing a rich nutrient broth, has become one of the most popular methods for mycelium proliferation. A 1964 paper, in fact, explored whether this now common technique would prove viable for psilocybin and psilocin production.  Mycelial cultures were added to flasks containing 30 mL of nutrient media and were put on a shaker for 4 to 7 days at 25°C. The mycelium that grew were homogenized and added to new flasks to serve as inocula for subsequent growth over a period of 5 to 15 days.
Replacement cultures were created much the same but used a longer homogenization step. This homogenized product was added to 300 mL of medium and put on the shaker. Mycelium pellets were washed with distilled water and aliquots were removed and added to the replacement medium. The researchers also evaluated the addition of the amino acid tryptophan since it was speculated as being a biosynthetic precursor to psilocybin.
Old School Analytics
The mycelium pellets were ground, extracted with methanol on the shaker for eight hours, and centrifuged to remove solids. The cleaned-up extracts were spotted on filter paper and “formed” with a mixture of n-butanol-acetic acid-water (4:1:5). Old school paper chromatography! Indole derivatives were identified by spraying the paper with a 2% solution of p-dimethylaminobenzaldehyde in hydrochloric acid and drying the sprayed sheet with a heat gun. A minimum quantity of 1 µg psilocybin could be detected. Ultraviolet spectrophotometry was used to confirm the presence of psilocybin.
The P. cubensis mycelium pellets grown via submerged culture produced psilocybin, but two other Psilocybe species (P. cyanescens Wakefield and P. pelliculosa (Smith) Singer & Smith) did not. Psilocin was not detected.
The researchers determined the optimum growth period and how it related to maximum psilocybin production over 5 to 15 days. Peak psilocybin (~ 0.54%) was obtained on the 7th day when the pH of the medium was between 4 and 5. The maximum growth of mycelium was achieved after nine days, but mycelium and psilocybin production decreased thereafter as the pH rose from acidic conditions to a slightly basic pH 8.
Once the optimum conditions were demonstrated, the researchers purposefully left out media constituents like glucose, glycine, thiamine, yeast extract, and ammonium succinate. The omission of glucose, a vital source of carbon, dramatically inhibited growth of mycelium and no psilocybin was produced. Excluding yeast extract initially stunted growth, but interestingly, the researchers report that “the organism adapted to this deficiency and subsequently underwent vigorous growth and comparable psilocybin production.” This produced a peak psilocybin concentration of 0.86%. When glycine was left out, psilocybin production slightly increased to approximately 0.75%.
Conversely, the researchers evaluated using different concentrations of media ingredients. When the glucose concentration was raised from 0.25 to 1.0%, an acidic medium pH was sustained, thereby providing a boost in psilocybin production (1.06%) on the 7th day. This growth didn’t last over time, however, as psilocybin concentration plummeted to 0.16% by day 11.
The addition of tryptophan to the growth medium did not boost psilocybin production. The authors scaled their submerged culture, increasing the volume from 30 to 300 mL without measuring much difference in psilocybin concentration after seven days.
There once was a time where psilocybin mushrooms were studied for their prospective medicinal benefits. Fortunately, many within the scientific community have resurrected their interest. Although humanity was strongarmed into lives without these mushrooms, it was a short separation in what has otherwise been an ancient relationship between the mushrooms and the human beings who ate them.
References Hofmann A, Heim R, Brack A. et al. Psilocybin, ein psychotroper Wirkstoff aus dem mexikanischen Rauschpilz Psilocybe mexicana Heim. Experientia. 1958;14:107–109. [journal impact factor = 6.496; times cited = 84 (Semantic Scholar)]
 Catalfomo P, Tyler V. The production of psilocybin in submerged culture by Psilocybe cubensis. Lloydia. 1964; 27:53-63. [journal impact factor = 3.779; times cited = 24 (Semantic Scholar)]