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OfflineRandoscious_McCray
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Growing in Liquid Medium. Using knowledge from Reactor Des
    #2111589 - 11/16/03 03:27 PM (13 years, 22 days ago)

Dear Advanced Cultivation Group,

I am interested in using the knowledge I have acquired from Biological Reactor Design to help assist and progress the sites current work with growing mycelium in a liquid medium.

Currently in class, we have been using liquid medium to grow microorganisms such as yeast and bacteria, but for a project I would like to try mycellium.

Defining some terminology.

The PF Tek, and most of our methods are based on a 'Batch Reactor'. Substrate and Biomass are put in a vessel, and then no inputs or outputs are connected.

There are however, different types of reactors, and I believe the most beneficial to growing with a liquid medium would be using a 'Continous Stirred Tank Reactor' (CSTR).

A sample CSTR would include a Tank (where biomass is grown), an Influent (in) and Effluent (out) that would supply substrate at a rate determined by a pump.

The benefits of a CSTR, is that if you arrange a set of CSTRs in series, you can control the hydraulic retention time (HRT) and if you set the washout rate greater than the HRT than you can remove contaminants.

Besides grow guides /7943 and /7997 I haven't found too much on liquid growth.

Anyone who knows anything about growth kinetics, who has tried liquid medium, please continue on with this thread. I am away from my school notes, but I will get some diagrams and some of the formulas scanned in.

There is also a program called STELLA, where you can enter values such as the growth and death constants, and you can model the rate of biomass formation and substrate utilization.


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Invisiblemicro
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Randoscious_McCray]
    #2111677 - 11/16/03 03:56 PM (13 years, 22 days ago)

Of course you could grow mycelium in continuous submerged culture, if you had a system set up for it. Unless you need to make mycelium, though, there's not much of a purpose -- the mycelium needs a solid substrate to fruit efficiently. Liquid cultures have only been able to fruit a little bit from the surface culture on top.

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OfflineRandolph_Carter
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: micro]
    #2111764 - 11/16/03 04:25 PM (13 years, 22 days ago)

Now, does the mycellium need the nutrients that the solid substrate provides, or merely the foundation from which to fruit? Just a curiousity.
Stay frosty.
R.C.


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Invisiblemicro
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Randolph_Carter]
    #2111829 - 11/16/03 04:48 PM (13 years, 22 days ago)

That's a tough one. I don't know.

The pinning cycle is controlled by pinning hormone -- it would be interesting to see if anything happened if you added pinning hormone to the liquid media. I can't see any reason why the mycelium should be physically unable to fruit inside of a liquid, but I don't know. The bottom line, I guess, is that it won't really fruit from liquid culture with the exception of a few very tiny fruits on a surface culture in some cases.

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OfflineRandolph_Carter
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: micro]
    #2111936 - 11/16/03 05:24 PM (13 years, 22 days ago)

Well, the thing i would assume to be the problem with fruits on a liquid culture would be the sheer instability of the pin formation..the food's there' the humidity is present, but any attempt to form pins would probably sink, become waterlogged, or fail for any number of reasons... if this is the major problem, then a fine gauge wire mesh that is possibly filled with casing material? would encourage the mycelium to grow into it, and from there they would have a stable base for fruit growth and formation. But what liquid would you use? Is it really required to have more complex nutrients that a solid substrate would provide? Because all the teks i've ever read and seen successful reports from have a very high carbohydrate content...hell, the liquid culture teks (for mycellium innoculation) are based off of simple carbos. I'm unsure, but most of the problem i've anticipated with this would be keeping the liquid Growth medium supplied with enough nutrients to support long term growth and the manufacture of such large structures as a fruiting body, seeing as mycellium has been proven to die off at over 10% sugar concentration in liquids. Assuming that pin formation requires a hormone (probably able to be made invitro via cold shock/temp cycling and light exposure) and that they just need a stable fruiting platform...i wouldn't anticipate a large problem with a full liquid life-cycle. I dont know...done research, but i dont know enough about the actual hardcore specifics of mycological growth and traditional cultivation. Can ya pm me with book suggestions, online sites (preferable) that go into full detail as to the mycellium-thru-fruit production life cycles? I'll try to work out a tek...ya got me thinkin.
Neways,
Stay frosty.
r.c.


--------------------
"..all those molecules thrashing their kinky little tails, hot for destiny and the street."  Gibson


Nuke baby seals for Jesus!

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OfflineRandolph_Carter
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: micro]
    #2111942 - 11/16/03 05:26 PM (13 years, 22 days ago)

Also, can you give references for the experiments that led to the growth of fruiting bodies on liquid, no matter the size? I'd like to see those.
R.C.


--------------------
"..all those molecules thrashing their kinky little tails, hot for destiny and the street."  Gibson


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Invisiblemicro
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Randolph_Carter]
    #2112088 - 11/16/03 06:17 PM (13 years, 22 days ago)

Quote:

Randolph Carter said:
.... but any attempt to form pins would probably sink, become waterlogged, or fail for any number of reasons...



Mycelium has a really good cell wall, so it can withstand a good amount of osmotic stress. Coulds be, though -- who knows? It's also possible that it simply won't pin at all in liquid -- the mycelium only pins under certain conditions and a liquid media might negate this. For example, it would be very unbenificial for a mushroom to fruit inside of a substrate.
Quote:

if this is the major problem, then a fine gauge wire mesh that is possibly filled with casing material? would encourage the mycelium to grow into it, and from there they would have a stable base for fruit growth and formation.



No clue. It might be possible, but with continuous culture you want submerged culture, so you would have to shake it which would mix around the mycelium too much to create a solid network. You'd need a static culture to do this, and in a static culture the mycelial network is really seperated -- the mycelium in the liquid lacks oxygen, and the surface culture lacks nutrients, hence why if fruits do develop they'll be very small. The other problem is everything would have to be 100% sterile or else the media will contaminate, which makes it really inefficient.
Quote:

But what liquid would you use



PDY broth works well, or you could use malt extract. Pretty much any fungal agar recipe without the agar -- there's a long list of them, but PDY and ME seem to be preffered. The simple-sugar method you're talking about works, but it's inefficient for producing quantities of mycelium, and certainly lacks nutrients.

Oh -- the liquid growth thing is in most mycology textbooks -- I know Watkinson+Gooday had a picture of it, somewhere. My recommendation, if you want to learn more, is to simply get a somewhat recent Fungi textbook from the library -- it should have mostly everything you need to know about this.

--
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Invisiblemycofile
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: micro]
    #2116503 - 11/17/03 02:46 PM (13 years, 21 days ago)

Fungus Maximus has a pic of a jar of dextrose and pee pollen fruiting invitro. Don't know if he posted it here, but it can be found in his forum at nansnook.

But, aside from simple scientific masturbation, why bother? I'm not trying to knock you, but I see absolutely no benefit here. I guess an invitro fruiting liquid would be neat to see, but I see no benefits over traditional fruiting methods. Once you add the mesh screen with a casing on it, it's basically what has been theoretically discussed to death as "hydroponic shrooms". You may find some discussions of interest to you if you search the board for that.

IMO, the place of liquid culture is as an innoculate on solid substrates. This requires nothing more than a sealed jar that is periodically shaken. If you want to get fancy, a flask on a magnetic stirrer provides constant agitation. But again, why would you want to fruit it?


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I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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Invisiblemicro
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: mycofile]
    #2116569 - 11/17/03 02:57 PM (13 years, 21 days ago)

You have a link to that pic? Can't find it on Nan's Nook.

Thanks!

--
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OfflineRandoscious_McCray
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Randoscious_McCray]
    #2122234 - 11/19/03 11:19 AM (13 years, 19 days ago)

Clarification(s)

wasn't looking to grow the mushrooms in liquid medium, just the mycelium to create mycelium syringes or harvest the mycelium alone.

using liquid substrate allows for continous aggitation at a constant amount.

using liquid substrate also allows for better modeling of growth kinetics for the mycelium phase.

using liquid medium allows for the effluent (outflow) to be observed with a spectrophotometer to create an absorbance vs. biomass curve, to tell what the % of biomass is.

any mention in the literature of using something besides malt extract? will get to library and see what can be pulled from the academic arena.





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OfflineRandoscious_McCray
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: micro]
    #2122243 - 11/19/03 11:22 AM (13 years, 19 days ago)

Where could I get more information on continuous submerged cultures?


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OfflineRandoscious_McCray
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Randoscious_McCray]
    #2122251 - 11/19/03 11:27 AM (13 years, 19 days ago)


Walked into lab today and a group of students was using diluted cow shit to create methane gas as well as grow biomass.


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OfflineAnnoA
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Randoscious_McCray]
    #2122415 - 11/19/03 12:28 PM (13 years, 19 days ago)

>Where could I get more information on continuous submerged cultures?
I don?t think the process is done continuously, as far I know a batch process is used.
Here:
http://www.fungifun.org/shroomerydocs/


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Invisiblemycofile
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Randoscious_McCray]
    #2123046 - 11/19/03 04:42 PM (13 years, 19 days ago)

McCray, I see your just interested in traditional liquid techniques. Most people in the west simply use large 3000-5000 flasks on magnetic stir plates. The stir plate not only provides agitation, but also oxygenates the liquid. There is a minimum ratio of volume to surface area, Stamets talks about this in GG&MM, I don't remember it off the top of my head.

In the East, they use much larger scale fermenters, airating the liquid with sterile air. This is not popularly done in the east because even a large farms needs can be met by a couple 3 liter flasks. When you consider that only 200cc's are needed to innoculate a gallon of substrate, who needs 500 gallons of liquid innoculate?

Other than malt extract, dextrose is my favorite, many people use honey, many use peptone as an additive. Many people also include small amounts of the spawn media and fruiting substrate so that the enzymatic pathways needed to digest them will be activated prior to innoculation, theoretically allowing for faster colonization.

I assume you are familiar with the liquid teks used by home hobbyists? If not, club 99's honey water tek is hosted here. Nansnook has more info on dextrose solutions (which I prefer because they are crystal clear). GG&MM has lots of info on liquid fermentation, pm me if you want more info on that chapter.
And the invitro fruiting liquid culture/hydro shroom. I actually couldn't find the fruiting pic, but here is what I'm sure is a similar culture. These pics come from Fungus Maximus, they are not mine. I think they consist of 3 tbs finely ground bee pollen and 100cc's water. I don't think they have anything else in them.







So, doesn't show fruits, but one can easily imagine how the top mat could pin.


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PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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Offlineragadinks
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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: mycofile]
    #2123290 - 11/19/03 06:02 PM (13 years, 19 days ago)

Hope this is not a *very* stupid question:

What is the difference between glucose and dextrose and how do they affect the growth of the mycelium ?


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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: ragadinks]
    #2123380 - 11/19/03 06:43 PM (13 years, 19 days ago)

Dextrose is just the d- isomer of glucose. The Difco Manual says that fungi prefer d-glucose, but I'm sure the l- isomer would work, too.

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Offlineragadinks
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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: micro]
    #2123933 - 11/19/03 11:24 PM (13 years, 19 days ago)

And where can I get glucose from? I mean e.g. in honey there is dextrose, right ? So What could I use to provide glucose (white or brown sugar) ? And what would the ratio be ? E.g. how many grams of honey/sugar do I have to use in order to provide X grams of dextrose/glucose ?


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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: ragadinks]
    #2124141 - 11/20/03 01:33 AM (13 years, 19 days ago)

>And where can I get glucose from?

In Germany:


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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: Anno]
    #2124347 - 11/20/03 04:53 AM (13 years, 18 days ago)

Woow, thanks. I did not think of that.  :laugh: 


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Offlineblackout
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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: ragadinks]
    #2126965 - 11/21/03 11:24 AM (13 years, 17 days ago)

glucose/dextrose are often in the baby food section in the supermarket


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