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Woodlover-Regeneration-TEK: Densification of Rhizomorphs by Layering


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#1 Rhizoo

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Posted 14 September 2007 - 06:32 PM

Introduction

In this thread I will first show you the properties of mycelial growth if fresh substrate is aplied in layers instead of the conventional mixing, and then how those properties can be used to regenerate weak mycelium or handle hard-to-replace mycelium. This TEK is mainly useful for woodlovers, which are cultivated unsterile on low-in-nutrient substrates like wood.

This is a summary of my experiments posted in a german-speaking forum; the complete thread is here:
TEK: Rhizomorphen-Verdichtung durch Schichtung

In december, i got a spawn bag of ps. cyanescens but had problems with growth when i used the conventional mixing method. I tried different substrates, but found little differences. After I read a thread by an user named "Blauer Kahlkopf" who reported that he propagated his mycelium just by putting fresh substrate on top of his outdoor patches, I tried this not for propagation but for regeneration.

The technique of propagation by layering has been mentioned elsewhere too, for instance:
Delivery systems for mycotechnologies, mycofiltration and mycoremediation - Patent 20050176583 patent by Stamets, scroll down to 0058 which mentions "layer spawning"
http://mycotopia.net...dlover-tek.html Waylit's WoodLover Tek
http://mycotopia.net...tml#post410712 Island Effect by Workman


Over the next days I will post several more layering experiments I had done this spring.

#2 Rhizoo

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Posted 14 September 2007 - 06:45 PM

Experiment 1 - Comparision mixed vs. layered


Day 0.

This is the weak Ps. cyanescens growing on beech woodchips with additional hemp fibre and straw. The layer is approx 1 cm thick:
http://mycotopia.net...=1&d=1189813377

As you can see there are very few rhizomorphs. This detail is the best looking rhizomorph:

http://mycotopia.net...=1&d=1189813377

I seperated this container into three areas. The little area remained unchanged all the time. On both other areas I apllied a 1cm layer of fresh substrate, but only in the middle area this was mixed in - the right area remained in the layered fashion.

http://mycotopia.net...=1&d=1189813377

This was stored at 18-20°C and air was exchanged daily.

...

Day 16:
http://mycotopia.net...=1&d=1189813377

The small left area remained unchanged. On both other areas fresh substrate was applied for a second time, but only in the middle area it was mixed in again

....

Now observe how the the mycelium grows (like when milk gets poured into a bowl of cornflakes):

After 25 days:
http://mycotopia.net...=1&d=1189813377

After 27 days:
http://mycotopia.net...=1&d=1189813377

After 29 days:
http://mycotopia.net...=1&d=1189813377

Attached Thumbnails

  • rhizotek_29d_fullview.jpg
  • rhizotek_25d_fullview.jpg
  • rhizotek_27d_fullview.jpg
  • rhizotek_0d_original.jpg
  • rhizotek_0d_original_m.jpg
  • rhizotek_16d_fullview.jpg
  • rhizotek_0d_diagview.jpg


#3 Rhizoo

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Posted 14 September 2007 - 07:02 PM

Lets take a closer look at the individual areas:

the original, unchanged area:
http://mycotopia.net...=1&d=1189814088



the "mixed" area:
http://mycotopia.net...=1&d=1189814088

http://mycotopia.net...=1&d=1189814088

http://mycotopia.net...=1&d=1189814088



the "layered" area:
http://mycotopia.net...=1&d=1189814088

http://mycotopia.net...=1&d=1189814088

http://mycotopia.net...=1&d=1189814088


Observations:

In the unchanged area there was very little growth. The woodchips were already reached by the mycelium, no densification occured.

In the mixed area, the mycelium had to grow starting from seperate, individual woodchips. The growth was successful only on some parts of the area, some bits of substrate are only sparsely overgrown or not at all. Because of the mixing, there are probably more such areas inside the whole mixed area.

In the layered area, uniform dense growth can be seen. Of course, no growth can be seen on the surface after an fresh layer of substrate is applied, but later, when the mycelium "rises like milk", the mycelium reaches all of the surface at the same time. This uniform densified growth appears only in the newly overgrown substrate, the initial layer at the bottom did not densify (this is consistent with the unchanged area).



Now, only on the layered area another layer of fresh substrate was applied, the mixed area remained untouched.

Attached Thumbnails

  • rhizotek_29d_original.jpg
  • rhizotek_29d_mixed_m2.jpg
  • rhizotek_29d_mixed_m1.jpg
  • rhizotek_29d_mixed.jpg
  • rhizotek_29d_layer_m1.jpg
  • rhizotek_29d_layer_m2.jpg
  • rhizotek_29d_layer.jpg


#4 Rhizoo

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Posted 14 September 2007 - 07:11 PM

After 45 days:

mixed area
http://mycotopia.net...=1&d=1189815163

As you can see, the mixed area, which remained untouched this time, did only change in the parts which before were not overgrown at all (top border) - and in this parts the growth is now densified. This is a sort of local layer effect.


layered area
http://mycotopia.net...=1&d=1189815163


In the new (3th) layer, an even more densified mycelium appeared.

...

btw, this is an other container made frome the same ps. cynescens mycelium after +/- 5 layers:
http://mycotopia.net...=1&d=1189815163

Attached Thumbnails

  • rhizotek_xxd.jpg
  • rhizotek_45d_mixed.jpg
  • rhizotek_45d_layer.jpg


#5 Rhizoo

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Posted 14 September 2007 - 07:23 PM

.

Conclusions


The most astonishing result of this experiment is that the method of applying fresh subtrate has such an extreme impact on the growth structure of mycelium. Remember that substrate as well as the climate was the same on all areas.

In my opinion, there are several explanatory approaches for the layer effect:

Starting from the horizontal mycelium layer, the hyphae can only grow vertical to reach fresh substrate. By this way of growing, the the hyphae do not need to (and can not) spread out - in the mixed method the hyphae have to spread out spherically, which gives a weaker growth because the available energy has to dispersed in many directions. (Note again that densification only occurs in the fresh growth, not the initial mycelium.)

Faster hyphae will reach fresh subrate first and cut off the slower hyphae from nutrients. This leads to selection of fast hyphae as more layers are apllied, especially in multi-substrain mycelium (from multispore). The selected fast substrains then concentrate in the top layer, which can be used for a new container. With the mixed method, all substrains remain forever in the container.

The horizontal mycelium layer (and all newly overgrown layers) are allready well connected and do not have to waste energy for repairing broken hyphae. This increases the robustness especially in otherwise weak mycelium (which often dies if it is mixed).

Note also that in the natural woodlover habitat mixing rarely occurs...



This leads to several consequences for regeneration of weak or handling of hard-to-replace mycelium:

A layer of substrate just on top of the the existing mycelium is the best choice. Substrate at the sides will be colonized slower and weaker because the mycelium has to spread out. Chose the size of the container accordingly.

If the mycelium is very weak, do not destroy the existing hyphal connections. For instance, if you got an ziplock with spawn lay it flat into a container, cut away the top foil and apply a fresh layer directly. Do not crumble the spawn. If the mycelium is allready densified crumbling is much less a problem.

The fresh layer has to have a thickness which can be grown through completly without contaminating. The weaker the mycelium is, the more nutrients the fresh subtrate has, or the higher the sporecount in your air is, the thinner the fresh layer hast to be. Start with 5mm or so, and afterwards use thicker layers if this worked well. Note that if you start with dried-out mycelium it takes severeal weeks at high humidity before the mycelium will start growing again so you better exchange the layer severeal times until you notice growth (because if not overgrown, even wood will contaminate after while).

#6 waylitjim

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Posted 14 September 2007 - 07:32 PM

Cool experiment, nice writeup. :thumbup:

#7 Jake420

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Posted 14 September 2007 - 10:44 PM

This sort of thing has been well documented in certain books concerning mushroom cultivation (at least those that have sections on outdoor woodlovers.) The concept is that if the mycelium is restricted on growing outward it can put more energy into growing upward, which happens to be the natural course of things for most fruiting fungi. Bricks have been used in outdoor beds to much the same success as you have encountered with the tubs.

#8 Rhizoo

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Posted 15 September 2007 - 07:25 PM

.

Experiment 3: Mixing with densified mycelium


This is an small extension to the first experiment. I took off colonized woodchips from both the "mixed" and the "layered" area, two portions of each area. All four portions were put in glasses and filled with fresh substrate in a ratio of 1:3.

http://mycotopia.net...=1&d=1189901790


The chips in all four glasses now were mixed.

...

After 28 days, the glasses looked like this:

http://mycotopia.net...=1&d=1189901790
from left to right: top layer of "layered", second layer of "layered", top layer of "mixed", second layer of "mixed".

glasses viewed from the the reverse side:
http://mycotopia.net...=1&d=1189901811

glasses viewed from top:
http://mycotopia.net...=1&d=1189901811
left "layered", right "mixed". Oberste Lage jeweils im unteren Glas.


Results:

The best glass was the one with the densified mycelium from the top layer of the "layered" area. Almost the complete substrate was colonized, and the growth was the most dense of all four. After that followed second layer "layered", first layer "mixed", second layer "mixed. Note that in the "mixed" glasses, the growth was less rhizomorphic.

In contrast to layering, with mixing there was no improvement of the style of growth, the style remained more ore less the same.

Some areas between the initial mycelium did get contaminated after the second week, which slowed down the colonization (although after several weeks more even the "mixed" glasses had overgrown those contamns). In my opinion, this is the main problem with mixing: contamns can grow on uncolonized substrate independent of the method, but with growing from a layer the mycelium has a much better starting base combatting a contamn than when growing from seperate pieces of mycelium.

Although because of the contamns this experiment was not really representative, it showed that the densified mycelium does not only look nice but has the better growth properties.

Attached Thumbnails

  • rhizotek3_28d_top.jpg
  • rhizotek3_28d_back.jpg
  • rhizotek3_28d_front.jpg
  • rhizotek3_spawn_3.jpg


#9 mycobri

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Posted 15 September 2007 - 07:32 PM

nice experiment rhizoo :thumbup:

i am about to spawn some woodlovers and
'wood' love to try this out ;)

looks like open air colonization
what are you covering the glasses and trays with during recovery between added layers?

#10 Rhizoo

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Posted 15 September 2007 - 07:33 PM

.

Experiment 2: Elimination of bacterial contamination


I have an small outdoor patch of Ps. azurescens, and last spring I took frome it some colonized woodchips/soil to cultivate the mycelium indoors. However, quickly a bacterial contamn showed up. In the garbage dump smell the azure did not want to grow. This seems to be a common distinction between indoor and outdoor cultivation: many contamns which pose no problem outside get really nasty indoors.

I put the well colonized woodchips in a layer, and apllied a layer of fresh substrate. The first layer was too thick and got contaminated again, so I removed it. Now apllied only a thin layer where every piece of fresh substrate was in contact with mycelium directly. Only for a short time the garbage smell appeared again, but then it vanished. I did more thin layers, and the contamn never reappeared again.

The fact that woodlovers can overgrow contamns is well known. However, the point here is to give the mycelium an advantage over the contamn, and this can be done by layering. Another advantage of this method is that mycelium which was never in contact with the contamn can be taken from the top layer.

This is how it looked after several layers:
[IMG]http://http://mycotopia.net/forums/attachment.php?attachmentid=61727&stc=1&d=1189902457[/IMG][IMG]http://mycotopia.net/forums/attachment.php?attachmentid=61727&stc=1&d=1189902457[/IMG]


PS: If you work with contamns, you do this at your own risk.

Attached Thumbnails

  • rhizotek2_azur_seite.jpg


#11 cheshire

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Posted 15 September 2007 - 07:45 PM

is this only seen to be had in woodlovers or could the same results be seen to extend to cubes also?

#12 Rhizoo

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Posted 15 September 2007 - 08:05 PM

@waylitjim & mycobri: thanks ;)

In indoor cultivation I usually use plastic flower boxes enwrapped with saran wrap. Very cheap setup :D

The nice thing about the flower boxes is that holes can be drilled in the boxes easily if needed. The glasses in experiment 2&3 were a compromise I only used because of the transparent sides needed for photos.

I do not poke holes in the saran wrap because I do not want insects to be able to get inside, and the high humidity is also good for the growth - if you provide humidity by another way then you can probably do it open-air. If I put flower boxes outside for fruiting I do not use the wrap also, just inside.



BTW, I here is some nice Mycoporn showing mycelium growing over the sides of a flower box on the search for food :D

[IMG]http://http://mycotopia.net/forums/attachment.php?attachmentid=61728&stc=1&d=1189904302[/IMG][IMG]http://mycotopia.net/forums/attachment.php?attachmentid=61728&stc=1&d=1189904302[/IMG]

[IMG]http://mycotopia.net/forums/attachment.php?attachmentid=61729&stc=1&d=1189904302[/IMG]

Even the water drops get overgrown:

[IMG]http://mycotopia.net/forums/attachment.php?attachmentid=61730&stc=1&d=1189904302[/IMG]

[IMG]http://mycotopia.net/forums/attachment.php?attachmentid=61731&stc=1&d=1189904302[/IMG]

Attached Thumbnails

  • rhizotek5_drops_on_the_wall_1.jpg
  • rhizotek5_drops_on_the_wall_2.jpg
  • rhizotek5_rhizos_on_the_wall_2.jpg
  • rhizotek5_rhizos_on_the_wall.jpg


#13 Rhizoo

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Posted 15 September 2007 - 08:18 PM

@cheshire:

the difference is the sterility respective the nutritional value of the substrate. Wood is low in nutrients and does take a lot more time to contaminate than the high nutritional substrates like grain used for cubes.

On the other hand, you probably do not need this TEK with cubes because mycelium growing from high-nutrient substrates is usually strong and dense already.

#14 mycobri

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Posted 15 September 2007 - 08:25 PM

these are some great pics.
:cool:
'the water droplets getting overgrown'
nominated for Picture Of the Month

#15 Rhizoo

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Posted 19 September 2007 - 03:46 PM

Thanks mycobri :kiss:

The next posts will be not so much about regeneration but propagation by layering. I tried to determine how layers should be made for an balanced relation between secure growth and propagation rate.

Note that this is all indoors - outdoors usually regeneration as propagation is faster (if it's neither hot and dry nor ground frost), but you can't keep a culture pure outdoors - and the quantitative results are only valid for this cyan.

#16 Rhizoo

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Posted 19 September 2007 - 04:01 PM

.

Experiment 4: stirred vs. connected

This is an rather simple experiment. In one flower box with 4cm densified colonized wood I destroyed the mycelial connections between the individual woodchips:

http://mycotopia.net...=1&d=1190235602

Then I applied a 1cm layer of fresh substrate.

After 10 days:
http://mycotopia.net...=1&d=1190235602

after 14 days:
http://mycotopia.net...=1&d=1190235602


With the densified mycelium, the cyan was able to reconnect itself rather fast. The delay at the left side was approx. 1-2 days, and the density is almost the same at both sides. However, this is only valid for rather thick initial layers, the next both experiments therefore use thinner layers.

Attached Thumbnails

  • rhizotek4_14d.jpg
  • rhizotek4_10d.jpg
  • rhizotek4_0d_mixed.jpg


#17 Rhizoo

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Posted 19 September 2007 - 05:04 PM

.

Experiment 5: single vs. double layered


In this experiment, I compared two ways of layering: one thick (2cm) layer colonized wood between thick layers of fresh substrate, and the same done with thin (1cm) layers but two of this stacked above each other. Thereby the colonized wood to fresh substrate ratio was the same at both sides. I better show you pictures so this becomes clear ;)

http://mycotopia.net...=1&d=1190236879
http://mycotopia.net...=1&d=1190236879
http://mycotopia.net...=1&d=1190236879
http://mycotopia.net...=1&d=1190236879
http://mycotopia.net...=1&d=1190236879
http://mycotopia.net...=1&d=1190236879

When the mycelium reached the surface at a side (not full colonization!), I apllied on the respective side an additional 1cm layer. The idea here is to compare the speed and the densification (or loss thereof).


After 12 days, the side with the double thin layers was ready for this next layer:
http://mycotopia.net...=1&d=1190237305

With the thin layers there is some loss of densification. The mycelium had to grow in both directions, so there was only 0.5cm per direction.


At 17 days, the
other side with the single thick layers was ready for the new layer too:
http://mycotopia.net...=1&d=1190237305

There was less loss in densifcation because there was more initial mycelium per direction and the distance was higher so it could re-densify better. However, because of this it took more time (but not much more).

So, now on both sides there was one additional layer.


After 29 days:
http://mycotopia.net...=1&d=1190237305

after 32 days:
http://mycotopia.net...=1&d=1190237305

In the end, the left side reached the same densification as the right side, and was a little faster.

I guess both seperate layers at the left side grew together, and this added additional strenght to to growth there. This stacked layering is rather useful if one wants a great height right from the beginning, for example for an outdoor patch.

If one wants a high thorough densification (to use the colonized wood again for propagation), thicker initial layer(s) may be better. But the thickness of the fresh substrate can't be extended indefinitely in turn because of the risk of contamination the longer the colonization takes.

Attached Thumbnails

  • rhizotek5_29d_fullview_.jpg
  • rhizotek5_32d_fullview_.jpg
  • rhizotek5_17d_1x.jpg
  • rhizotek5_layer6.jpg
  • rhizotek5_12d_2x.jpg
  • rhizotek5_layer5.jpg
  • rhizotek5_layer3.jpg
  • rhizotek5_layer4.jpg
  • rhizotek5_layer2.jpg
  • rhizotek5_layer1.jpg


#18 Rhizoo

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Posted 19 September 2007 - 05:41 PM

.

Experiment 6: thickness gradient


Now, on the left side there was 1cm layer of fresh substrate, and nothing on the right side. Then colonized wood was apllied on both sides in the same way: the thickness of the colonized layer rises from 1.5cm to 6cm.

http://mycotopia.net...=1&d=1190240917

On top, 1cm fresh substrate was apllied on both sides.

After 13 days:
right (no bottom layer)
http://mycotopia.net...=1&d=1190240917
and left (with bottom layer)
http://mycotopia.net...=1&d=1190240917

After 17 days:
http://mycotopia.net...=1&d=1190240917
http://mycotopia.net...=1&d=1190240917

After 21 days:
http://mycotopia.net...=1&d=1190240917
http://mycotopia.net...=1&d=1190240917
(on the right side of the last picture mycelium grew around the separating plate)


Conclusions: thickness of the colonized layer is important. And on the left side with the bottom layer, the mycelium is less strong because it has to grow into both directions.

For this cyan i come to the following results: Best densification and speed above 3cm initial thickness, if the growth is in both directions then multiply by 1.5. Therefore, when working with weak mycelium, only growth in one direction is recommended (see first post).

btw, this flower box was later layered several times on the left side. Now it has a height of 3-12 cm. It is now standing outside to test which influence the height has on fruiting :D




[/CENTER]

Attached Thumbnails

  • rhizotek6_21d_2l.jpg
  • rhizotek6_21d_1l.jpg
  • rhizotek6_17d_1l.jpg
  • rhizotek6_17d_2l.jpg
  • rhizotek6_13d_2l.jpg
  • rhizotek6_13d_1l.jpg
  • rhizotek6_0d_c.jpg


#19 waylitjim

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Posted 19 September 2007 - 05:57 PM

All these wood chips are gettin' me excited! :loveeyes:

#20 python

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Posted 19 September 2007 - 06:45 PM

Me too ................ nice experiments and photo-log of them

can't wait to see the results :bow:




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