You might like this foxy2 unless you've seen it before.. it's ancient, but it's got to be one of my favorite.
-Zen
Ref: Catalfomo, P. and V.E. Tyler, Jr. "The Production of psilocybin in submerged culture of Psilocybe cubensis" LLoydia 27:53-63, 1964
Pellets of Psilocybe cubensis produced in medium no. 1 [ammonium succinate (1 g), Glycine (9 g), Glucose (5 g), yeast extract (.5 g), KH2PO4 (.1 g), thiamine hydrochloride (.003 g), (NH4)6Mo7O24-4H2O (.05 mg), ZnSO4-7H2O (.3 mg), MnCl2-4H2O (.35 mg), FeSO4-7H2O (2.5 mg), CuSO4-5H2O (0.5 mg), MgSO4-7H2O (0.5 g), distilled water, to make (1.0 L), adjust to pH 5.5 with hydrochloric acid] accumulated psilocybin but not psilocin. Maximum production of the former compound occurred on the seventh day (0.52 per cent, dry weight of mycelium), whereas growth attained its maximum (average 112.6 mg, dry weight of mycelium per 30 mL of medium in a 125 mL flask) on the ninth day.
Maximum yields of both psilocybin and mycelium occurred in the acid pH range (4.0-4.6). However, the acidid nature of the mycelium does not preclude the possibility that thte internal pH of the organism is maintained at a different level by an efficient buffering system. Failure to find psilocybin in the medium may be attributed to its instability in the vigorously agitated acid medium, although permability factors are probably also involved.
In the absence of a readily assimilable carbon source (glucose), detectable amounts of psilocybin did not accumulate. Omission of ammonium succinate did not significantly alter the pH of the medium but it did lower psilocybin yields. Without yeast extract, mycelial production was retarded at five days, and culture liquors remained acidic through the eleventh day. Adaption and/or synthesis of necessary precursors nevertheless permitted a continued increase in growth which was accompanied by a rapid increase inthe yield of psilocybin by the seventh day. Similar results were obtained for media from which both yeast extract and thiamine had been omitted, except psilocybin levels remained lower.
According to Cochrane (5) most fungi have an absolute requirement for thiamine; however, where low levels are needed, synthesis takes place after initiation of growth. Under conditions of thiamine deficiency, glucose utilization is impaired, which may account for initial inhibition of growth when yeast extract or that ingredient and thiamine were omitted from the medium. Although glycine constituted more than one-half of the total weight of the dissolved solids in medium no. 1, the organism grew and metabolized efficiently in its absence.
The influence of different concentrations of glucose, ammonium succinate, and potassium acid phosphate in medium no. 1 was noted. A low level of glucose was associated with a rapid rise in exracellular pH, and lower levels of product accumulated. Doubling the normal amount of carbohydrate promoted psilocybin accumulation which reached a level of 1.02 percent (dry weight) by the seventh day. After eleven days, this level dropped to 0.15 percent; a similar sudden decline in psilocybin content was also noted in those flasks containing a reduced phosphate concentration. The significance of the low pH levels after eleven days is unknown. In contrast, levels of ammonium succinate which were one-half or twice that of the normal medium had no significant effect upon pH or psilocybin production.
Extracellular tryptophan added to replacement flasks did not enhance psilocybin production, but, instead, underwent degradative reactions of the type reported to occur in Neurospora and other fungi (5). This conclusion is based on the observation that a progressive increase in kynurenine, a catabolic product of tryptophan metabolism, paralleled the disappearance of tryptophan. These results are not necessarily contradictory to those obtained by Brack, et al. (2) since they used a different organism and different experimental design which preclude a direct comparison. However, our results do establish the existance of a direct relationship between psilocybin production and mycelial growth. Utilization of th replacement culture technique served to separate growth and psilocybin production, whereas the method of Brack, et al., did not afford this distinction. It was concluded that psilocybin production is so intimately related to mycelial proliferation that the nutrient-deficient replacement medium was of little value inthe study of psilocybin biosynthesis. However, the technique had previously proven satisfactory for biosynthetic studies of other indole derivatives in fungi (3).
Increasing the scale of fermentation from 30 mL of medium in 125-mL flasks to 300 mL in 2800-mL flasks markedly affected psilocybin production. Significatn differences in yields did not appear until after the seventh day when accumulation of product ceased, and a more rapid decline occurred in the large flasks as the pellets became physiologically older. Although the cause of this phenomenon was not established, it way be attributed, at least in part, to differences in the efficiency of aeration of the cultures.