[Bio] pH discovery that explains why my Stipticus experiments are probably failing

Frantisek Apfelbeck algoldor at yahoo.com
Wed Nov 9 03:48:47 PST 2011


Message: 2
Date: Tue, 8 Nov 2011 20:36:14 -0800 (PST)
From: Roger H <domitron at yahoo.com>
Subject: [Bio] pH discovery that explains why my Stipticus experiments
    are    probably failing
To: Rikke Rasmussen <rikke.c.rasmussen at gmail.com>,    Matthew Downs
    <downs.matt at gmail.com>
Cc: "tastebridge at lists.noisebridge.net"
    <tastebridge at lists.noisebridge.net>,    "bio at lists.noisebridge.net"
    <bio at lists.noisebridge.net>
Message-ID:
    <1320813374.28901.YahooMailNeo at web83008.mail.mud.yahoo.com>
Content-Type: text/plain; charset="iso-8859-1"

I
 believe I have discovered my mistake in growing the Stipticus. The 
starting pH of a wood-decomposing substrate should be higher than the 
growth pH.? White-rot wood-rotting fungi such as Stipticus decrease pH 
through the release of oxalic acid.? The starting pH in the research 
paper I found online* suggests that the fungi is well accustomed to a 
starting wood pH of around 5.1, which a white-rot fungus takes to 3.9 
(ideal is 3.5-3.8 in this species).? Thus by me starting the pH of the 
liquid culture at 3.8, the fungus probably cannot grow to release oxalic
 acid to lower the pH as it does in nature.? And my Burning Man 2007 
bags DID start at a pH of about 5, actually by mistake according to my 
notes!? So, I think I have solved the problem of why nothing is working 
when I lower the pH to the optimal growth pH.? I will start a new liquid
 culture at a pH of 5.0 and bags accordingly and allow the fungus to 
lower the pH to the ideal growth level as it
does in nature.

Roger

* See: http://les.bf.uni-lj.si/fileadmin/datoteke_asistentov/mhumar/clanki/2001_pHafterdecay_holz_als_roh.pdf

PSS
 - A 4% dextrose/light malt liquid culture as I was using is around 5.3 
pH without anything added.? Under these conditions the mycelium was 
growing very well, which correlates with my hunch that I should not be 
dropping the pH to the optimal growth-stage pH but rather let the 
mycelium handle the drop.


>> Hello,
>> In my experiments with lignocellulose degrading fungus Talaromyces emersonii the growing pH is 5.5 and I do not think that we were adding buffer (materials and methods are copied below). Many fungal enzymes work fine to pH below 3, lots of times the activities would be really good around pH 4-5. I'm not sure about growing conditions of this particular strand but I would assume lower pH than 7 for start of the cultivation, and to be honest I would expect growing pH to be around 4.5 - 6 with the natural course getting more acidic.

>> Anyway I'm curious about the next experiment.
>> Sincerely,
>> Frantisek

 
2.2.2 Fungal
Cultivation and Enzyme production (Talaromyces emersonii)

Inocula
for these studies were taken from laboratory stocks of the
microorganism and were routinely subcultured, at 45oC,
on Sabouraud Dextrose Agar (SDA)  in the form of plate culture. Longer
term preservation was achieved by storing 1 cm2pieces of mycelial mat, in a sterile solution of 20% (v/v) glycerol,
at -70oC
(typically 3-4 pieces per 10 mLaliquot of 20% (v/v) glycerol; Tuohy et
al.,
1990). Liquid cultures of Talaromyces
emersoniiIMI 393751, were grown at 45oC,
in the mineral/salts inducing medium described by Moloney et
al.,
(1983), with inclusion of the appropriate carbon source. This medium
contained corn steep liquor (0.5% w/v), yeast extract (0.1% w/v),
KH2PO4(0.5% w/v), and (NH4)2SO4 (1.5%
w/v) as a source of inorganic nitrogen. Trace mineral salts
FeSO4.7H2O
(62.5 mg/l),
ZnSO4.7H2O,
H3BO3,
MnSO4.4H2O,
Na2MoO4,
CoCl3.6H2O
and KI (all at a concentration of 12.5 mg/mL)
were included in the medium. MgSO4.7H2O
(0.05% w/v), CaCl2.2H2O
(0.05% w/v), and anhydrous Na2SO4(0.10% w/v), were added to the medium, as described earlier (Moloney et
al.,
1983). The medium was sterilized by autoclaving at 121oC,
15 p.s.i. for 20 min..  

Glucose
‘starter’ cultures were prepared by aseptically inoculating
sterile medium containing 2.0% (w/v) glucose (generally 100 mLnutrient medium with 2 g glucose per 250 mLErlenmeyer flask), with 2-3 1 cm2pieces of mycelial mat taken from the outer edges of actively growing
agar-plate cultures. Samples, 10% (v/v) from 24-36 h ‘starter’
cultures served as inocula for primary induction media, which was
prepared as described above, with the inclusion of the appropriate
carbon source. Primary induction cultures were grown under the same
conditions for 36 h. A further induction cycle was repeated
(secondary induction, using a 10% mL(v/v) inoculum (taken from the primary induction culture)), for the
purpose of screening or fermentation scale-up. In the various
induction studies, the carbon source substrate was added at
concentration 2.0% (w/v). After induction with various carbon
sources,the culture filtrates (which would contain secreted enzymes) were
recovered by filtration of the culture broth  through several layers
of fine grade muslin to remove the fungal biomass or mycelia. The
crude filtrate was centrifuged at 4000 gin Du Pont RC-58
Refrigerated Superspeed Centrifuge fitted with GSA rotor. The
supernatant fraction, hereinafter referred to as thermozymes or
thermozyme cocktail, was stored at 4oC,
if not for immediate use.


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________________________________
From: "bio-request at lists.noisebridge.net" <bio-request at lists.noisebridge.net>
To: bio at lists.noisebridge.net
Sent: Wednesday, November 9, 2011 9:04 AM
Subject: Bio Digest, Vol 8, Issue 3

Send Bio mailing list submissions to
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Today's Topics:

   1. Re: Bio Digest, Vol 8, Issue 2 (Ethan Chew)
   2. pH discovery that explains why my Stipticus experiments are
      probably failing (Roger H)
   3. Re: pH discovery that explains why my Stipticus experiments
      are probably failing (miloh)
   4. Re: pH discovery that explains why my Stipticus experiments
      are probably failing (Roger H)


----------------------------------------------------------------------

Message: 1
Date: Tue, 8 Nov 2011 14:56:41 -0600
From: Ethan Chew <spacefelix at gmail.com>
Subject: Re: [Bio] Bio Digest, Vol 8, Issue 2
To: bio at lists.noisebridge.net
Message-ID:
    <CACr872wUt5S3oG=dfjwrOytvwrC2Duy7bDpN_8EmPj=zQ=vscA at mail.gmail.com>
Content-Type: text/plain; charset="iso-8859-1"

Hello,

      Is there a means by which I could telecon into the meeting?  I am
based in Huntsville, AL and would like to learn more about mycoremediation.
Please let me know.

        - Ethan

On Tue, Nov 8, 2011 at 2:00 PM, <bio-request at lists.noisebridge.net> wrote:

> Send Bio mailing list submissions to
>        bio at lists.noisebridge.net
>
> To subscribe or unsubscribe via the World Wide Web, visit
>        https://www.noisebridge.net/mailman/listinfo/bio
> or, via email, send a message with subject or body 'help' to
>        bio-request at lists.noisebridge.net
>
> You can reach the person managing the list at
>        bio-owner at lists.noisebridge.net
>
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Bio digest..."
>
>
> Today's Topics:
>
>   1. mycoremediation (Nevada M.)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 7 Nov 2011 23:34:49 -0800
> From: "Nevada M." <bramble.greenbrier at gmail.com>
> Subject: [Bio] mycoremediation
> To: Matthew Downs <downs.matt at gmail.com>
> Cc: "tastebridge at lists.noisebridge.net"
>        <tastebridge at lists.noisebridge.net>,    bio at lists.noisebridge.net
> Message-ID:
>        <CAJz_BgYAs=jJw_eK6a=if5RWFeTyR5tHs9VTMZJkfXAQOuZtGA at mail.gmail.com
> >
> Content-Type: text/plain; charset="iso-8859-1"
>
> Hey folks,
>
> So I haven't had the time to follow up on getting resources on
> mycoremediation from the folks who put together the radical mycology
> convergence up in Washington state earlier this year (though the
> convergence do have a webpage with links and stuff on it).  However, I just
> came across a mention that there is currently a radical mycology group
> meeting on a weekly basis in the East Bay, which is "studying the role of
> mushrooms in decomposition, bioremediation, and human culture."  That might
> be a good place for folks to go to get connected with others who have
> interests along those lines.  The group is meeting every Saturdays at 2pm
> (Nov 5th, Nov 19th, Dec 3rd and Dec 17th are the meetings currently
> submitted to the East Bay Free Skool calendar).  The meetings happen at the
> Long Haul, 3124 Shattuck @ Woolsey which is I believe one block into the
> city of Berkeley.  The meetings are being hosted by Joey, 805 813 2099.
>
> Cheers,
>
>
>
> Nevada
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Message: 2
Date: Tue, 8 Nov 2011 20:36:14 -0800 (PST)
From: Roger H <domitron at yahoo.com>
Subject: [Bio] pH discovery that explains why my Stipticus experiments
    are    probably failing
To: Rikke Rasmussen <rikke.c.rasmussen at gmail.com>,    Matthew Downs
    <downs.matt at gmail.com>
Cc: "tastebridge at lists.noisebridge.net"
    <tastebridge at lists.noisebridge.net>,    "bio at lists.noisebridge.net"
    <bio at lists.noisebridge.net>
Message-ID:
    <1320813374.28901.YahooMailNeo at web83008.mail.mud.yahoo.com>
Content-Type: text/plain; charset="iso-8859-1"

I believe I have discovered my mistake in growing the Stipticus. The starting pH of a wood-decomposing substrate should be higher than the growth pH.? White-rot wood-rotting fungi such as Stipticus decrease pH through the release of oxalic acid.? The starting pH in the research paper I found online* suggests that the fungi is well accustomed to a starting wood pH of around 5.1, which a white-rot fungus takes to 3.9 (ideal is 3.5-3.8 in this species).? Thus by me starting the pH of the liquid culture at 3.8, the fungus probably cannot grow to release oxalic acid to lower the pH as it does in nature.? And my Burning Man 2007 bags DID start at a pH of about 5, actually by mistake according to my notes!? So, I think I have solved the problem of why nothing is working when I lower the pH to the optimal growth pH.? I will start a new liquid culture at a pH of 5.0 and bags accordingly and allow the fungus to lower the pH to the ideal growth level as it
does in nature.


Roger

* See: http://les.bf.uni-lj.si/fileadmin/datoteke_asistentov/mhumar/clanki/2001_pHafterdecay_holz_als_roh.pdf

PSS - A 4% dextrose/light malt liquid culture as I was using is around 5.3 pH without anything added.? Under these conditions the mycelium was growing very well, which correlates with my hunch that I should not be dropping the pH to the optimal growth-stage pH but rather let the mycelium handle the drop.
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Message: 3
Date: Tue, 8 Nov 2011 22:36:50 -0800
From: miloh <froggytoad at gmail.com>
Subject: Re: [Bio] pH discovery that explains why my Stipticus
    experiments are probably failing
To: Roger H <domitron at yahoo.com>
Cc: "bio at lists.noisebridge.net" <bio at lists.noisebridge.net>,
    "tastebridge at lists.noisebridge.net"
    <tastebridge at lists.noisebridge.net>,    Matthew Downs
    <downs.matt at gmail.com>
Message-ID:
    <CAE4k+fsW0C_zOfeOy3jo6UiK8AZH58LGfw5OXTmzqGS0EuVvtA at mail.gmail.com>
Content-Type: text/plain; charset="iso-8859-1"

Is it me, or is 3 a hella low ph?

Good notekeeping!
On Nov 8, 2011 8:36 PM, "Roger H" <domitron at yahoo.com> wrote:

> I believe I have discovered my mistake in growing the Stipticus. The
> starting pH of a wood-decomposing substrate should be higher than the
> growth pH.  White-rot wood-rotting fungi such as Stipticus decrease pH
> through the release of oxalic acid.  The starting pH in the research paper
> I found online* suggests that the fungi is well accustomed to a starting
> wood pH of around 5.1, which a white-rot fungus takes to 3.9 (ideal is
> 3.5-3.8 in this species).  Thus by me starting the pH of the liquid culture
> at 3.8, the fungus probably cannot grow to release oxalic acid to lower the
> pH as it does in nature.  And my Burning Man 2007 bags DID start at a pH of
> about 5, actually by mistake according to my notes!  So, I think I have
> solved the problem of why nothing is working when I lower the pH to the
> optimal growth pH.  I will start a new liquid culture at a pH of 5.0 and
> bags accordingly and allow the fungus to lower the pH to the ideal growth
> level as it does in nature.
>
> Roger
>
> * See:
> http://les.bf.uni-lj.si/fileadmin/datoteke_asistentov/mhumar/clanki/2001_pHafterdecay_holz_als_roh.pdf
>
> PSS - A 4% dextrose/light malt liquid culture as I was using is around 5.3
> pH without anything added.  Under these conditions the mycelium was growing
> very well, which correlates with my hunch that I should not be dropping the
> pH to the optimal growth-stage pH but rather let the mycelium handle the
> drop.
>
>
> _______________________________________________
> Bio mailing list
> Bio at lists.noisebridge.net
> https://www.noisebridge.net/mailman/listinfo/bio
>
>
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Message: 4
Date: Wed, 9 Nov 2011 01:04:12 -0800 (PST)
From: Roger H <domitron at yahoo.com>
Subject: Re: [Bio] pH discovery that explains why my Stipticus
    experiments    are probably failing
To: miloh <froggytoad at gmail.com>
Cc: "bio at lists.noisebridge.net" <bio at lists.noisebridge.net>,
    "tastebridge at lists.noisebridge.net"
    <tastebridge at lists.noisebridge.net>,    Matthew Downs
    <downs.matt at gmail.com>
Message-ID:
    <1320829452.31402.YahooMailNeo at web83006.mail.mud.yahoo.com>
Content-Type: text/plain; charset="iso-8859-1"

3.8 is more acidic than even orange juice and would kill many bacteria and fungi.? It also is the ideal pH for this species but apparently the substrate is turned that low by the mycelium in time, not that the original substrate should be that low.? That was the part I did not get originally.? The pH of the substrate should not be that low initially.? 


Roger



________________________________
From: miloh <froggytoad at gmail.com>
To: Roger H <domitron at yahoo.com>
Cc: Rikke Rasmussen <rikke.c.rasmussen at gmail.com>; "bio at lists.noisebridge.net" <bio at lists.noisebridge.net>; "tastebridge at lists.noisebridge.net" <tastebridge at lists.noisebridge.net>; Matthew Downs <downs.matt at gmail.com>
Sent: Tuesday, November 8, 2011 10:36 PM
Subject: Re: [Bio] pH discovery that explains why my Stipticus experiments are probably failing


Is it me, or is 3 a hella low ph?
Good notekeeping!
On Nov 8, 2011 8:36 PM, "Roger H" <domitron at yahoo.com> wrote:

I believe I have discovered my mistake in growing the Stipticus. The starting pH of a wood-decomposing substrate should be higher than the growth pH.? White-rot wood-rotting fungi such as Stipticus decrease pH through the release of oxalic acid.? The starting pH in the research paper I found online* suggests that the fungi is well accustomed to a starting wood pH of around 5.1, which a white-rot fungus takes to 3.9 (ideal is 3.5-3.8 in this species).? Thus by me starting the pH of the liquid culture at 3.8, the fungus probably cannot grow to release oxalic acid to lower the pH as it does in nature.? And my Burning Man 2007 bags DID start at a pH of about 5, actually by mistake according to my notes!? So, I think I have solved the problem of why nothing is working when I lower the pH to the optimal growth pH.? I will start a new liquid culture at a pH of 5.0 and bags accordingly and allow the fungus to lower the pH to the ideal growth level as it
does in nature.
>
>
>
>Roger
>
>
>* See: http://les.bf.uni-lj.si/fileadmin/datoteke_asistentov/mhumar/clanki/2001_pHafterdecay_holz_als_roh.pdf
>
>
>PSS - A 4% dextrose/light malt liquid culture as I was using is around 5.3 pH without anything added.? Under these conditions the mycelium was growing very well, which correlates with my hunch that I should not be dropping the pH to the optimal growth-stage pH but rather let the mycelium handle the drop.
>
>
>_______________________________________________
>Bio mailing list
>Bio at lists.noisebridge.net
>https://www.noisebridge.net/mailman/listinfo/bio
>
>
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