Check our references thread, not only is citric acid cheaper and more available but it increases yields.

At least for C.purpurea, I believe a mixture of mostly mannitol, with the remainder being sorbitol, and a LITTLE sucrose, to support initial growth, both very slowly absorbed, thus growth will be slower, but who cares about a week-ish more waiting maybe, when the potential is a for a shitload more alkaloids?



Avoid glucose AT ALL COSTS, it leads to rapid formation of a load of viscous, gloopy polysaccharide crap that severely interferes with oxygen transport.

Mechanical stress pisses them all off, big time, such as aeration, stirring, although some is needed of course.

My project is crawling ahead, achingly, infuriatingly slowly thanks to a disability benefit income for one having to support two, it ends up with the likes of a bag of phosphate in two weeks, a chromatography column in another fortnight, ferric chloride scrounged from somebody who makes circuits, and hey in another month, I will probably be able to afford some agar.

So fucking frustrating, I hate having no work.

For the culture medium, I think using a 40-50% (but no higher) perfluorooctyl bromide or a fully perfluorinated alkane in water emulsion, whilst it may interfere with morphology, it does significantly increase yield.

Also, using those long ass flexible ultrafine air diffusion stones available from pet shops would be sweet, to divide air intake equally throughout the bioreactor, as well as supplemental oxygenation of the airsupply, give it a richer mix than normal air should do wonders, and lessen the need for damaging, but vital stirring speed.

Alginate immobilization lowers the rate of degredation of the cultured fungal cells HUGELY and increases productive life, also it does decrease O2 diffusion, so the pellets MUST be, in my opinion and reading, produced by electrostatic droplet formation (think syringe driver pumping alginate/fungus suspension through electrically charged needles, at about 14 kV with the other positive end connected both to ground and to the reciever full of CaCl2)

Thinner the needle bore the smaller the microspheres, higher the electric potential the smaller, lower the alginate flow rate, the smaller, less the flow rate through each needle (using a massive multiple needle array, with a low flow rate per needle is fine)

Lower alginate/fungus suspension flow rate per needle increases yield, as does adding a perfluorocarbon to the bead mixture before polymerization in CaCl2.

Some citrate/succinate is needed, but it MUST be kept in a low concentration relative to CaCl2 in the mix if it not to chelate out the counter-ion that sits in the alginate guluronic acid block binding sites to crosslink it to form the hardened form of the polymeric alginate.


Hm vesp, that simple sorbitol/succinate/phosphate/MgSO4/ammonia medium looks like utter shite for growth and yield.

For purpurea at least it NEEDS trace transition metal salts, copper, iron, some sulfate is needed but over a certain level it impacts yield, MgSO4 at normal levels, but I would add a few of the trace elements where feasible in the form of sulfates, and salts that are insoluable as sulfates, as long as they are trace elements only, as nitrates, as almost ALL transition metal nitrates are soluable, with one or two exceptions.

Much nitrate fucks it up though, but adding some trace elements otherwise insoluable as the sulfate as a nitrate isn't going to hurt at a few tens to hundreds of milligrams per liter of medium.

Looks like in 2.5-3% alginate solution (more alginate=less O2 diffusability but greater mechanical stability, plus less stress on the fungus, however having a shitload of perfluorocarbons everydamnwhere should offset the O2 uptake issue, if coupled with sub-100 micrometer beads. (150 micrometers reaches, without perfluorocarbon in beads themselves, fuck knows about it in medium as regards to anoxic zones, the aforementioned anoxic zone, so keep em tiny, keep well oxygenated)

What I REALLY want to know, is what happens when one forces polyploidy on ergots.

What I do know about that, from the SINGLE study I found, is that it MASSIVELY increases growth vigour, but since it was a short paper, dealing solely with uptake of radiolabelled sugars and acetate, there was fuck all discussion whatsoever of ergopeptide/lysergide/ergoamide yield, I mean, not a sentence, fuck all whatsoever, none.

But, it didn't harm the fungus, it beefed up growth like a shot in the arse of a high potency anabolic steroid, I mean, big time, and since other research indicates conidial strains are usually haploid, and at least diploidy is necessary for alkaloid production to a meaningful degree, one would reasonably, tentatively but educatedly hazard a guess that getting one's lil babies to be freakishly polyploid as hell with colchicine or other mitotic poisons would further beef up the production of an already decent strain post selection/mutation.

Other things I wish to know, and I do not believe it has ever been done, or if it has, it has not been published, are what happens when one exposes it to a mitogen such as pokeweed mitogen, or that phytohaemagglutinin lectin-type toxin found in uncooked red kidney beans, which could easily be pulled out on a multiple gram scale.

And, for that matter, when one tries to first induce mitosis, and get those cells dividing, and as such, doubling chromosomes, THEN, whilst in the middle of it, after chromosome multiplication but before cell division occurs, arresting the process during anaphase, using colchicine again, which would simultaneously both double chromosome count, PLUS induce polyploidy.

Any ideas as to what might happen? I am guessing it either seriously fucks up and ends up with some aneuploid, the fungal equivalent of down's syndrome, or hurler's , turner's or triple X, etc. (NOT to disrespect those with downs, or anything else, I have as much respect for these people as for my fellow auties and aspies, but merely to illustrate an analogy)

Of course, aneuploidy in plants is much different to the effects in animal life forms, and often increases growth and yield outputs, whilst in humans, it can result in anything from higher intelligence, to lowered intelligence, to embryonic lethal genetic conditions that lead to eyeless fetuses with brains hanging out of the back of their heads.

What happens in fungi, I truly do not know, but I sure as hell wish to find out.

Grrr, fucking jesus, so much to experiment on, so little funding, too bad nobody in hell is going to give a research grant to a semi-clandestine biochemist operating from a back-garden shed with a bioreactor hacked up from an autistic sensory toy and some oxygen cylinders :/

Tsathoggua, this thread is only focusing on culture mediums to increase alkaloid yield. Not strain, and is also ignoring methods to increase oxygen and alginate use as stated in the first post.

I am looking for a mg/L type of mixture that, from all of the research seems to be the best.
Lets stay on topic.
We can worry about oxygen and alginate later.

This medium is specifically for C. paspali:

The seed-stage medium:
5ml/l tween 40,60, or 80
10ml/l propylene glycol
40 g/l mannitol
10 g/l glucose
10 g/l succinic acid,
3g/l KH2PO4,
.3g/l MgSO4
2 g/l chicken peameal in tap water


The production-stage medium:
5ml/l tween 40,60, or 80
10ml/l propylene glycol
50 g/l mannitol,
30 g/1 succinic acid
2g g/l KH2PO4
0.3 g/l MgSO4
20g/l NaCl in tap water
1g dl-tryptophane (~+800ug/ml)

pH of 5.2 with ammonia before sterilisation, maintain pH with NaOH for massive yields...

It should be noted that many of these extras came from studies where they used a standard medium and changed one factor, each of these mods alone increases yields 30-70%.  It really is a crapshoot to estimate how they will work together.  Also, since alkaloid product will be so high you may be able to tweak certain things up (bringing kh2po4 up to a max of 10g/l, increasing tryptophane) and max out yields.

This medium came from studying many papers and patents over the course of the last 6 months or so, some methods are omitted for practical simplicity such as the arsenate method.  Things like adjusting the pH on the fly and temperature control matter a lot more then things you can add to the medium.

One thing I haven't found a good reference for but know works is adding DSMO to the brew... You could end up very spun very fast if you added DMSO and spilled an alkaloid dense solution on yourself...

you could add biotin, I haven't found a reference for this but it has been alluded to... The aresenate trick requires you to increase kh2po4 to 10g/l and use 1/20th-1/50th that molar amount in aresenate.  I left this out because I don't know how it will effect a culture that is already near 3g/l.. you may have to tweak the amounts...

You are right the 20g nacl is for the osmotic pressure, you use 50g mannitol + 20g nacl instead of 100g mannitol.  For these high yielding conditions it may actually be smart to use 100g of mannitol instead...

This isnt about pumping a culture full of the right ingredients, its about having the proper conditions.  You can have all the fancy tricks you want including cells immobilized in alginate but until you get your temp, aeration and ph adjustment perfect it wont matter much...

The biotin paper you attached links success with spore production and increased cell lipid content, two things which will be drastically different between their strain and c. paspali...

One book I read does mention it works with paspali, it referenced the article: (Gupta A, Tiwari KP (1989) J Microb Biotechnol 4 : 54 88).  You would have to pull that one to find out concentrations but the book said it works.

Using tweens AND glycols at the same time was really something I assumed to be a good idea.  In the reference they never used both at the same time, each increased yields separately.  The reason I think they should be used together is because even though propylene glycol increased yields just like tweens did, while tweens cut the surface tension in half, propylene glycol barely effected the surface tension, meaning it probably acts directly on the cells..

pH and temperature are both examined in the R2 reference in the LSA bioreactor thread... the optimal numbers are pH 5.2 and 21-23C

well span-80 seems to be the way to go for immobilised cells.. tween 80 did better for free cells... The alkaloids in g/liters from immobilised cells is not really an accurate representation of alkaloid producing ability.  They did get 8.35g/l but that was over 60 days and changing out nutes 6 times...

you can make a really simple still using a pressure cooker and some piping (I use copper).  Easy way to get distilled water, and or delicious liquor spirits without tying up your glassware (assuming you have any).

Almost certainly. I have a most extensive collection. Only reason its taking me so damnably long to actually get culturing and mutating, is the cost, and the fact that I also have other projects that are higher priority, including an AMPAkine, for my cognitive and memory issues which I need to finish, as I have a lot of functional difficulty at the moment.

I have some sort of nasty virus (as if there is any other sort)on my own computer at the moment and I need to fix the fucker, so it might take me some time to hunt down a full list. Currently stuck with somebody else's computer.

So is buying a 15k$ fermentor/bioreactor suspicious? Just curious if anyone is on to this yet.


also I keep trying to download the patents for ergot cultivation,  3224945 and 3219545  along with a few others, and they all come out corrupted, like some conspiracy or something. can someone else check the freepatent  website and see if they are having the same trouble?

or better yet, can someone post them here(I  know, wrong forum)

thanks in advance

peace

TM