Author Topic: Zen and the Art of Hydroponic Mycology  (Read 2873 times)

0 Members and 1 Guest are viewing this topic.

Zen

  • Guest
Zen and the Art of Hydroponic Mycology
« on: March 09, 2002, 05:45:00 AM »

-Zen

Zen

  • Guest
Re: Zen and the Art of Hydroponic Mycology
« Reply #1 on: March 09, 2002, 05:48:00 AM »
A synthetic nutrient solution that provides Psilocybe Cubensis with all essential nutrients is as follows:

Product:  60 g of concentrated synthetic nutrient solution
Concentration:  100 g will produce 8 L of aqueous nutrient solution.
Acidity:  7.5 g of concentrate should produce 1 L of solution with a pH of 5.5 
Quality:  Does not need to meet reagent purity, food grade is acceptable.
Make Up:  Relevance of definition in the order of Formula, Name, then CAS RN:

1.  Glucose  (C6H12O6) [40 g] CAS: [50-99-7]
2.  Ammonium Succinate (C4H12N2O4) [8 g] CAS: [2226-88-2]
3.  Yeast Extract (Organic Extract) [4 g] CAS: [8013-01-2]
4.  Magnesium Sulfate (MgSO4-7H2O) [4 g] CAS: [7487-88-9]
5.  Glycine (C2H5NO2) [3 g] CAS: [CAS 56-40-6]
6.  Potassium Phosphate (KH2PO4) [800 mg] CAS: [7778-77-0]
7.  Thiamine Hydrochloride (C12H17ClN4OS HCl) [24 mg] CAS: [67-03-8]
8.  Ferrous Sulfate,Heptahydrate (FeSO4-7H2O) [20 mg] CAS: [7720-78-7]
9.  Cupric Sulfate,5-Hydrate (CuSO4-5H2O) [4 mg] CAS: [7758-98-7]
10. Manganese Chloride, 4-Hydrate (MnCl2-4H2O) [2.8 mg] CAS: [7773-01-5]
11. Zinc Sulfate, Heptahydrate (ZnSO4-7H2O) [2.4 mg] CAS: [7733-02-0]
12. Ammonium Molybdate, 4-Hydrate ((NH4)6Mo7O24-4H2O) [0.4 mg] CAS: [12027-67-7]
13. DiHydrogen Oxide (H2O) [146.4 mg - X] CAS: [7732-18-5]
14. Hydrochloric Acid (HCl) [X mg] adjusted to balance solution pH when properly diluted.

   This solution is typically used for the production of psilocybin in submerged mycelium cultures of Ps.C. When well aerated this solution comes to complete submerged growth in about 11 days. It is typically ready for harvest and at highest alkaloid content in 7 days. Solution fermented for up to 50 days did not display any primordia or fruiting responses. Nutrient solution saturated agar surface cultures display rapid growth, primordia formation, and raised alkaloid content. Omission of essential nutrients suppresses growth and lowers alkaloid content, in some cases  to undetectable amounts, in both submerged and surface cultures.

   I believe the idea of nutrient depletion and deficiency acting as a triggering response to be false. Perhaps this is a misinterpretation of data, whereby a nutrient deficient mycelia culture was used to inoculate a nutrient rich substrate. The resulting mycelium is unable to begin fruiting until significant alkaloid creation has taken place. Nutrient rich mycelium cultures are capable of primordia formation with in as little as 3 days of exposure to an open-air interface.

   The traditional triggers of the fruiting response are a drop in temperature, a drop in humidity, and an exposure to light, these stimuli correspond to mycelia surfacing in nature. I further purpose a  set of internal triggers that directly relate to alkaloid levels within the mycelium, whereby primordia formation is seldom initiated in the absence of significant psilocybin levels. Further evidence to support psilocybin as a growth regulator comes from the evaluation of the organism's biomechanics.

   Of most interest is this organism's management of phosphors, it is essential in the production of DNA, and this organisms spore reproduction method requires the production of allot of DNA. There is also an isolatable enzyme in the biomass that actively removes the phosphorus from psilocybin. We have a theory at "the lab" that assumes this enzyme is responsible for the acquisition of phosphorus to be used in the production DNA. The resulting levels of psilocin production and lowered psilocybin content are thereby directly related to the production of spores. The production of psilocybin seems to be a mechanism by which the organism can "sense" its environments chemical makeup for, as well as store, the nutrients necessary in the process of  reproduction.

   Since submerged cultures are constantly at +100% humidity the fruiting response is usually suppressed, even with lowered temperatures and exposure to light. It is possible a mutant strain could "adapt" and attempt to fruit in an aquatic environment, but it is more likely the response was initiated at the open-air interface and then produced abnormal submerged growth from that point.

   These solutions can be aerated using an aquarium air pump, an inline HEPA filter, and some sterilized tubing. Sandstones can be quite useful but difficult to sterilize ( submerge in alcohol then rinse in distilled water and H2O2 ). The substrate in a hydroponics setup is typically inert since all the nutrients are provided directly from the nutrient solution.

   In a supportive nutrient solution, a slightly acidic environment, and raised glucose levels the submerged mycelium exhibits rapid and aggressive growth. By doubling glucose levels from 5 g/l to 10 g/l the resulting mycelium had slightly higher mass and the psilocybin content was doubled to 1.01% by day 7, unfortunately the glucose induced hyper activity resulted in a rapid degradation and psilocybin content diminished to .1% by day 11.

  Aside from the addition of the essential nutrients needed for efficient growth and raised glucose levels the addition of glucuronic acid and homobrassinolide have shown to stimulate mycelia and fruit growth. The growth promoting effect of glucuronic acid at 2% may be that of an additional carbon source in regards to Ps.C. The strong growth promoting effect of pollen at 2% and 10% is possibly the effect of homobrassinolide, a brassinosteroid hormone, which was detected at concentrations of 100 ng/g in pollen. 10% pollen in a medium would correspond to a hormone concentration of 10 µg/l, a level that has been shown to enhance the growth of Psilocybe cubensis. Glucuronic acid at 2% had a weak growth promoting effect and pollen at 2% and 10% had a strong growth promoting effect.

References available upon request.

-Zen

paranoid

  • Guest
Re: Zen and the Art of Hydroponic Mycology
« Reply #2 on: March 09, 2002, 12:22:00 PM »
This is positively brilliant Zen!!  Man I love your style.  Now this is a setup worth investing in.  I mean, it's fun to fruit the shrooms but personally I feel that it's not very efficient or convenient.



"   Of most interest is this organism's management of phosphors, it is essential in the production of DNA, and this organisms spore reproduction method requires the production of allot of DNA. There is also an isolatable enzyme in the biomass that actively removes the phosphorus from psilocybin. We have a theory at "the lab" that assumes this enzyme is responsible for the acquisition of phosphorus to be used in the production DNA. The resulting levels of psilocin production and lowered psilocybin content are thereby directly related to the production of spores. The production of psilocybin seems to be a mechanism by which the organism can "sense" its environments chemical makeup for, as well as store, the nutrients necessary in the process of  reproduction."

This portion raises some interesting points.  Now i am by no means well versed in biochemistry, although I am somewhat versed in cell biology.  I've never really understood the rationale behind the production of psilocybin/psilocin in the shrooms - a defense mechanism perhaps to ward off potenial consumers?  This above though seems to desginate the psilocybin as phosphorus storage for enhanced DNA production during spore production.  Would this be a sole purpose for the psilocybin production or is it a two-fold effect of deterrance and storage?  Or am I just missing a large chunk of info, whereby due to particular cellular metabolism the psilocybin evolved as a convenient end-product in which it also serves the above purposes (evolution is a funny thing)?

I don't think it was stated directly but I am I to understand the the expected alkaloid content from this method is approximately 0.5%?  What are the psilocyn/psilocybin ratios?  For long term storage considerations, what is the best way to prevent degradation of the psilocin (other than rapid drying, addition of anti-oxidants such as ascorbic acid, and freezing storage temps). 

Sorry about all the questions but you've renewed SWIMs interest in psilo production for personal consumption.

bujinkan

  • Guest
Re: Zen and the Art of Hydroponic Mycology
« Reply #3 on: March 10, 2002, 03:10:00 PM »
we should talk about the possibility of modifying this setup for ergot cultures.

meme

  • Guest
Re: Zen and the Art of Hydroponic Mycology
« Reply #4 on: March 12, 2002, 10:00:00 AM »
It has been my experiance that the only way to grow indoor psillocybin fungus in large enough (to really be profitible) yields is using liquid culture, because the space required by fruiting is too high.  A liquid culture fill volume, and can truly meet demand.

Look forward to a detailed reply to this post, and subject, by the years end.

superman

  • Guest
worth doing?
« Reply #5 on: January 18, 2004, 12:23:00 PM »
can anyone confirm whether or not this nutrient solution works?   i'd like to try a large scale batch, a hundred litres maybe.  if my calculations are correct a 100L batch should produce at least 250 doses w/ cubensis.

Bubbleplate

  • Guest
ZEN = Liquid Culture of Mycelia?
« Reply #6 on: January 18, 2004, 03:58:00 PM »
Perhaps not. I don't mean to discourage you, but here's some food for thought:
Your media is obviously taken from the scientific paper on the subject, P. Catalfomo & V.E. Tyler's " The Production of Psilocybin in Submerged Culture by Psilocybe Cubensis" circa the heady days of the Pyschedelic era, as appeared in Lloydia Vol. 27, 1-4, 1964.
First, any media containing Yeast extract and organic compounds like Glycine are very susceptible to Bacterial contamination. Even when they are thoroughly sterilized and HEPA filtered air is pumped through. All it takes is for a few bacteria to multiply into BILLIONS.
Also the actual over yield of mycelia they got was extremely low - from 300 ml portions of media, they obtained, at best, 114 milligrams dry weight which contained .5 % (that’s POINT 5 % ) i.e. .57 milligrams of psilocybin or about 1/20 the amount need for a minimum dose. A modifed media did get the % of alkaloid up to .86%, but then the mycelia weight went down to 81 milligrams. Not too good...
You said: “I believe the idea of nutrient depletion and deficiency acting as a triggering response to be false.”
I disagree. The mushroom mycelium, given suitable substrate (food) will grow and grow until it’s used up. The mycelium, sensing that the food is all gone, knows that it needs to produce spores, and that means mushrooms, if there is to be a next generation.
It’s the reason that the brilliant “PF Tek” method of shroom growing works so well. Fill a jar with damp sterile brown rice powder and Vermiculite, and cover with a non-nutritive substrate, in this case Vermiculite. Innoculate the rice powder with spores, and the mycelium will grow and consume all the rice powder. Once it starts growing into the non-nutritive Vermiculite, that is enough to trigger the formation of the Primordia or baby mushrooms.  No other triggering mechanisms are required – I’ve seen jars put in a dark closet for a month and they always make primordia.
Morel mushrooms are another example: growers tried for decades to grow them indoors, without success, and it wasn’t until one stumbled upon the need for the mycelium to grow into a non-nutrative substrate that indoor growth was possible.
Bottom line is, it is MUCH easier to grow mushrooms, or even the mycelia for that matter, on an organic substrate like cow dung or straw or rice powder, then it is to try and grow it on a liquid artificial media in submerged culture. You may want to consider growing out the mycelia on large amounts of grain and then extracting the alkaloids.
But good luck and success with your experiments!

paranoid

  • Guest
"First, any media containing Yeast ...
« Reply #7 on: January 19, 2004, 09:25:00 PM »
"First, any media containing Yeast extract and organic compounds like Glycine are very susceptible to Bacterial contamination. Even when they are thoroughly sterilized and HEPA filtered air is pumped through. All it takes is for a few bacteria to multiply into BILLIONS."

This is of course always a possibility.  However these nutrient compounds are rather standard laboratory fare.  I understand the concern regarding a large scale setup, where contamination is more of an issue.  In respose, I know that there has been use with the various shrooming TECs with introduction of H2O2 as a prophylactic measure.  However, given the sensitivity of a developing mycelial culture versus a fruiting one, it may be a useless idea.  Worth considering though.


Mr_Bronson

  • Guest
H2O2 mycelium sensitivity
« Reply #8 on: January 20, 2004, 04:03:00 PM »
Is it not the case that the mycelium can colonise media with a fairly high H2O2 concentration due to the production of neutralising enzymes? Many bacterial and fungal spores are killed by the same concentration of H2O2; some are not: contamination of the growth medium is still possible.

Bubbleplate

  • Guest
You Are Correct Mr. Bronson
« Reply #9 on: January 21, 2004, 03:22:00 PM »
There is more going on at many levels than meets the eye...
Some plants actually generate peroxides in response to attack by pathogenic organisms; and some pathogenic organisms, including fungi like ergot, generate peroxides to increase their virulence against plants!

http://www.auf.uni-rostock.de/iup/phyto/version_d/EU-Projekt.htm


http://www.blackwell-synergy.com/links/doi/10.1111/j.1364-3703.2004.00201.x/abs/


http://www.biochemj.org/bj/371/0783/bj3710783.htm


http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=32009


Mr_Bronson

  • Guest
Interesting links
« Reply #10 on: January 21, 2004, 05:07:00 PM »
I suffered a denied access message for the 2nd link (blackwell).

The more you know, the more you realise you don't know. It is striking how complicated so-called 'simple organisms' are (and people then profess to understand the human body).

I enjoy reading your posts; biosynthesis and other left-field processes such as electrochemistry are very interesting to me. Claviseps culture is rather beyond me at the moment but maybe one day. I hope one day. A couple of kilos of HBWR seeds might be a more practical proposition for someone less skilled in this area.

There is something strangely satisfying about cultivating fungus and even more strangely, it is not yet illegal in the UK.

Charles Bronson

adroit_synth

  • Guest
Hello again Mr_Bronson!
« Reply #11 on: January 22, 2004, 10:10:00 PM »
Not illegal in the UK huh? Well fuck I am thinking about moving now.
So bubbleplate, this technique is practically useless in your opinion? OR merely requires some updating and optimization?


Mr_Bronson

  • Guest
Triggering Fruiting
« Reply #12 on: January 23, 2004, 04:59:00 PM »
Bubbleplate: Is it possible for fruiting to occur in liquid media or does it require an open-air environment? If it is, could fruiting be triggered somehow by a low enough nutrient concentration? I was also wondering whether mycelial colonisation of the liquid media might inhibit growth of competing organisms in a similar way to solid media? Maybe a combination of good sterile techniques, H2O2 and rapid colonisation might be sufficient to prevent contamination.

adroit_synth: Mushrooms aside, you probably wouldn't like the food and the weather, well it has been raining for the past 2 months. On the plus side, fresh mushrooms are not restricted and the classification of wacky-backy is soon to be reduced to class C.

Bubbleplate

  • Guest
Fruiting Mr. Bronson
« Reply #13 on: January 24, 2004, 03:18:00 PM »
I would tend to doubt that fruiting would occur in liquid media, especially if one is pumping air through it. Also, the amount of nutrients required for good mycelia growth, i.e. high concentration, vs. that required to trigger fruiting, i.e. very low concentration, is the limiting factor.
In my own experiments with growing P. cubensis mycelium, even without pumping air through the media, I've never had fruiting. BTW, bacterial infection was a problem.
However, one most likely COULD get fruiting on a "semi-liquid" media - that is a media that has a small amount of agar added to make a "semi-gel". Here's some info I found about mycelium (Psilocybe mexicana) growing experiments by R. Heim, who worked with both Gordon Wasson and Albert Hofmann. Quoted from the book "Biochemistry of Industrial Microorganisms", 1963, pub. by Academic Press. page 371:
"In the second method, a complex organic nutrient such as beer wort or malt extract, supplemented with minerals and PARTIALLY SOLIDIFIED WITH AGAR was used. The concentration of the nutrient was found to control the type of growth obtained. At the highest concentrations only mycelia formed. At intermediate ranges, sclerotia accompanied by mycelium, and in very dilute or in old cultures where the nutrient was almost exhausted, normal carpophores (mushrooms) were produced. Light was essential to carpophore, but not sclerotia formation."
Personally, I've had better luck growing the mycelium on an organic substrate (brown rice powder or grain like bird seed, rye, etc) and then extracting that substrate/mycelium. Organic material is more "forgiving", that is, doesn't encourage rampant bacterial growth like a rich liquid medium.
And you are correct in assuming that good sterile techniques and rapid colonisation are sufficient to prevent contamination.

7is

  • Guest
Excised blocks of fungal mycelia were ...
« Reply #14 on: January 26, 2004, 10:40:00 AM »
Excised blocks of fungal mycelia were transferred from cultures growing on slants into sterile Petri dishes, cut into small pieces (less than 0.5 mm) using a sterile scalpel and inoculated into liquid CYM (0.46 g KH2PO4, 1.0 g K2HPO4·3H20, 0.5 g MgSO4·7H20, 20.9 g D-glucose, 2.0 g of Difco Yeast Extract and 2.0 g of Difco Bacto Peptone per liter). The cultures were then grown at room temperature for 15 days or until mycelia covered the plate. Mycelia were harvested by filtration through cheesecloth and rinsed with double distilled water.

Kimberly G. Nugent and Barry J. Saville.
Forensic analysis of hallucinogenic fungi: a DNA-based approach. Forensic Science International. In Press.

ning

  • Guest
Wonder how much psilocybin was in that water
« Reply #15 on: April 27, 2004, 10:22:00 PM »
I do hope they save the water they filtered...perhaps a little A/B would turn up the other half of the alkaloids...