08-06-03 09:34
No 452043
Hi!
Swim's paspali is doing great not. They have taken over plenty of agar plates with white fluffy colonies etcetc.
But theres a question regarding the upscaling stage of the fermentation. In the patent: US3038840 and the arcamone article, they make a submerged incubation flask with the right nutrents after the agarplates are nicely overgrown. When the incubation is done, they use some of this to seed a prefermentationflask. They say they use 10% of the destination fluid volume and just "part of the original culture".
As far as i can see, if one wanted say 20 L's of broth in the end, one should start with 200 mL's of incubation liquid -> 2 L prefermentation broth -> 20 L broth, right? Or do they actually mean that only 10% of the incubation liquid should go in the prefermentation liquid, such that really large amounts of incubation broth should be made?
I'm pretty sure it's supposed to go they way i desribed first, but with these babies i want to make sure there are no mistakes(to many anyway ;) )
Regards
Bandil
Size doesn't matter, it's the sound you make when hitting the ground after smoking it, that matters!
(Hive Bee)
08-06-03 21:57
No 452134
Besides Arcamone, almost every other researcher follows this procedure:
1) Isolate Claviceps on agar plate or test tube. After 2 weeks or so, under STERILE conditions, scrape off some mold and chop it up in small amount distilled H2O. (I use a blender, but a sterile scapel and a jar ok)
2) This is then used to innoculate the liquid culture in Shake Flasks. Innoculate 600 - 1000 ml wide mouth Erlenmyer flasks that contain 75 - 200 ml of the "starter" submerged media (See Arcamone Media formula "A"). The flasks are then place on a Rotary Shaker at 24 C., in the DARK, and shaken fairly hard for 2 - 7 days.
The Claviceps mold is VERY particular as to its needs in submerged culture as opposed to non-submerged culture. It needs HUGE amounts of O2 in the media, and unfortunately, if you don't have a Rotary Shaker, I doubt you'll be successful with submerged culture. If you check any Claviceps Patent, you'll see what I mean.
3) Once a good culture is going in the Shaker Flasks, then that can be used to innoculate larger amounts. (Arcamone Media "B") The guidelines are when scaling up is to use 6% to 10% innoculum of the size you want to scale up to; example, for my 2,000 ml bioreactor, I innoculate with 120-200 ml from Shake flasks.
Please also re-read Arcamones article carefully. You'll see that ONLY strains that produced a purple-violet color in liquid media produced any alkaloid. They made many submerged shake flask tests to isolate and re-isolate and re-isolate again purple strains.
Its easy to just grow Claviceps - not so easy to grow AND get alkaloids....
(Hive Bee)
08-07-03 00:56
No 452164
>>As far as i can see, if one wanted say 20 L's of broth in the end, one should start with 200 mL's of incubation liquid -> 2 L prefermentation broth -> 20 L broth, right? <<<
Yes - that's exactly right! You keep scaling up using 10% or so. Using this "10%" method, some patents describe scaling up to 3,000 liter fermenters!! Now I know how they make ergotamine by the kilogram.
