Author Topic: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali) (Read 1616 times)

Tsathoggua · « **Reply #100 on:** April 13, 2011, 08:30:39 PM »

Whilst I am willing to lay plates spiked with nitrosamines, EMS, or bombard them with radiation...I draw the line at urushiol. 2-50micrograms being sufficient to cause blistering....shit, thats quite a lot worse than lewisite or sulfur mustard.

Vesp · « **Reply #101 on:** April 13, 2011, 09:01:36 PM »

The amount needed is probably very insignificant, and it is not exactly urushiol. Probably a crude extract of whatever plant is found to contain the useful compound would be good enough - if this stuff actually plans out like the literature has lead me to believe.

Bluebottle · « **Reply #102 on:** April 13, 2011, 09:25:27 PM »

Umm, I was thinking something more along the lines of tea. But if you want to play around with urushiol [or wheat extracts what have you], haha okay.

[Also, the obvious, whatever it is, it should be added after the medium has been colonised.]

Vesp · « **Reply #103 on:** April 13, 2011, 09:40:59 PM »

Yeah, i was thinking of a "tea" made from rye material - to give the exact chemical that induces alkaloids in nature, just purified a bit better than a normal tea - though, maybe not needed. Like i said, its more so alkylresorcinols than urushiol - but if urushiol worked, or its analogue found in Gingko Biloba -- it might be worth extracting to a crude amount.

Bluebottle · « **Reply #104 on:** April 16, 2011, 12:39:54 AM »

What is the best amount of potato per liter? It depends on the potato I guess. Russet, for example?

It seems to me that maximum growth (corn steep solids, for protein right? but potatoes also have that) and then add the "trigger": an HAT inhibitor... and tryptophan, extra sugar/citrate maybe, salts, arsenic, throw what one has at it. For this, I'm thinking some kind of concentrated green tea extract, curcumin or EGCG, garcinol if it's available because that's been proven, and/or a cocktail of the other stuff.

The question, is how much can you streamline it, and my bet is that every little thing helps. This trick, if it works, doesn't really change much, it could potentially make things much much easier. But it does not remove nutritional requirements! And those could still be sorted out a little better, direct me if otherwise.

The two-phase dual purpose extraction oxygenation though, what kind of organic solvent would be ideal for selectively dissolving alkaloids? And to what extent would they be soluble in it? I can't imagine anyone has investigated the solubility of ergotamine in olive oil, but if you have that info, please tell me.

The optimum pH of the fermentation is 5.2 if I recall, so, equilibrium with the salts, but something's gotta seep through, no? Anyway I'm almost confident that with the proper secondary extraction (mildly acidic H2O) and then replacing it, it would not really disrupt the ergot's growth. If a more or less continuous production/extraction could be maintained, then efficiency would go through the roof. A shaken culture would be necessary for that setup, as an emulsifier would make settling it, inconvenient.

Alginate microspheres, undoubtedly helpful but something of a hassle, if they can be done without so much the better.

Tsathoggua · « **Reply #105 on:** April 16, 2011, 08:11:59 PM »

I think much better done with. Generating them doesn't look that hard based on what I have read. Encapsulation stabilizes the culture in a state that makes the fungus think it has formed sclerotia, and its much, much more stable. Many more cycles can be run in a fermenter with an immobilized culture than a floating blob of mycelium goo, before it degrades and stops producing.

When we are talking grams of lysergic acid..one run is worth quite a lot of effort.

Urushiol is just out for me, no bloody way, like I said, on a dose basis, its far, far more effective as a vesicant than mustard gas, probably not close to being as carcinogenic due to its being immunotoxic rather than an alkylating agent, but in some people, 2ug is enough to cause a reaction. Makes the arsenical, ergotamine-laden nutrient soup one might use for culture medium look like something you might drink.

aniracetam · « **Reply #106 on:** April 16, 2011, 11:16:27 PM »

so with the alginate encapsulation...
culture aliquots can just be transferred via peristaltic pumps
from spent jug of fermentation broth to a fresh jug of ferm broth?

overunity33 · « **Reply #107 on:** April 17, 2011, 12:00:34 AM »

Quote from: aniracetam on April 16, 2011, 11:16:27 PM

so with the alginate encapsulation...
culture aliquots can just be transferred via peristaltic pumps
from spent jug of fermentation broth to a fresh jug of ferm broth?

Yeah its about that easy theoretically, but you have to remember in those references where they got such high yields they often filter out the mycelium that has seeped out of its encapsulation and re encapsulate it. Also yields are stated in grams per liter, but what they dont mention is that, yeah, you can get 6g/L but its over the course of a few months with multiple culture changes. Also the phosphate that is necessarily present in our culture solutions will interfere with the encapsulation.

It looks good on paper but why even bother when you can change out your nutrients and mycelium every run and not have to bother with contamination, re-encapsulation and other problems inherent to this method? Not to mention sterile technique is hard enough without having to deal with the algination protocol...

The fact that algination comes up so often (not talking about you specifically aniracetam) while the finer details of traditional fermentations are being ignored shows that people are looking at this more in a theoretical sense then a realistic, practical sense...

You are better off buying a peristaltic pump and ph electrode to constantly adjust fermentation ph, this alone will get you a huge increase in yield, add temperature control and you will be doing good enough to not worry about immobilisation.

Now when you are designing a 10,000 gallon reactor I guess that additional yield with less switchouts will mean thousands in saved materials, etc, but for us its simply not worth it. Its like that guy everyone knows that decides to grow weed and buys a badass aeroponics system with co2, but has no understanding of the finer points and does much worst then the guy that knows what hes doing with a few plants in soil.

Vesp · « **Reply #108 on:** April 18, 2011, 10:32:02 PM »

You know... that beer wort, -- which is kind of like malt extract has been known to produce alkaloids, and a sexual sporulation of claviceps on agar while... it also makes perfect sense that it would contain alkylresorcinols!

Makes a bit of sense

aniracetam · « **Reply #109 on:** April 18, 2011, 10:35:26 PM »

so PMA plating of the s&h strain?

Vesp · « **Reply #110 on:** April 18, 2011, 10:39:15 PM »

What does PMA mean?

aniracetam · « **Reply #111 on:** April 18, 2011, 10:40:33 PM »

potato-malt-agar, as opposed to potato-dextrose-agar.
that is, substitute malt extract in place of d-glucose

Vesp · « **Reply #112 on:** April 18, 2011, 10:41:46 PM »

Oh, I don't know. I was just saying that the possibly presecents of those phenolic compounds in the malt might explain the function of beer wort than just "having the sugars and other compounds in the right ratio"

Tsathoggua · « **Reply #113 on:** April 22, 2011, 04:53:44 AM »

Just noticed that new avatar of yours vesp. That really seriously kicks arse! Thats one mean looking little fungal fuck. Looks half gorilla, half sclerotia

Vesp · « **Reply #114 on:** April 22, 2011, 08:48:25 AM »

bigger picture of it: http://olegti.design.ru/diaryi/008.jpg

@aniracetam -- what concentration of sugar are you using? sucrose is supposed to be best also.... right?

Tsathoggua · « **Reply #115 on:** April 22, 2011, 09:02:43 AM »

It has been used. However sugar alcohols appear to be better, although I have a distinct feeling that a mixture of sucrose and a polyol would be better, the sucrose being present to allow for a limited quantity of rapidly utilizeable sugar during the initial seeding/colonization part of the growth phase.

Mannitol, sorbitol and IIRC xylitol have all been used to some success.

Specifically though, avoid glucose. It leads to the fungus producing a whole load of polymeric polysaccharide gloop, which buggers up O2 transport by increasing the viscosity of the medium.

Vesp · « **Reply #116 on:** April 22, 2011, 09:00:40 PM »

And malt extract is primarily glucose from my understanding.

aniracetam · « **Reply #117 on:** April 22, 2011, 09:50:10 PM »

it's a dimer of two glucose molecules.

and i'm using 1% sucrose in solution, 5% mannitol. I don't see any of this gloopy stuff people speak of. the viscosity is rather low, and aeration is high. cells are roughly spherical like they're supposed to be

overunity33 · « **Reply #118 on:** April 24, 2011, 12:35:44 AM »

Looks like the FDA finally did something right:
http://www.fda.gov/food/scienceresearch/researchareas/safepracticesforfoodprocesses/ucm101662.htm

This process is perfect for sterilising large growth mediums, appears to not affect the medium composition unlike UV sterilisation..

Vesp · « **Reply #119 on:** April 30, 2011, 08:41:51 AM »

Is this under a blacklight as well?
The yellow circle is ergot alkaloids fluorescing?

Author Topic: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali) (Read 1616 times)

Tsathoggua

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Vesp

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Bluebottle

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Vesp

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Bluebottle

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Tsathoggua

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aniracetam

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overunity33

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Vesp

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aniracetam

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Vesp

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aniracetam

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Vesp

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Tsathoggua

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Vesp

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Tsathoggua

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Vesp

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aniracetam

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overunity33

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Vesp

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