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OfflineChem7
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Grain spawn additive experimentation
    #27643465 - 02/03/22 07:13 AM (1 year, 11 months ago)

Hey All! This is my first time posting here, so my apologies if I screw something up. 🙃 I'm a new mushroom cultivator and long time mad scientist, so of course, experimentation is a necessity. I'm posting this here in case it either helps someone or sparks conversation. I'm happy with both!

The objective of the experiment is to test different additives to see what, if anything,
will affect the speed of colonization. I will post my progress as this goes along, and if it garners interest, will post future experiments along this area of exploration. If any of this information is redundant to existing posts, I apologize. The archives are a massive body of information to search through.

Experiment 1:
The grain is wild bird seed mix, washed, sunflower seeds removed, and pressure cooked for 20 minutes. The grain had gypsum added during cooking. After cooking, the excess starch was rinsed from the grain and it was dried in a thin layer until no surface moisture was evident. The grain was separated into 4 equal portions and mixed with additives. Variable group 1 is the control
and only contains gypsum. Variable group 2 has coffee added. Variable group 3 has granular humic acid added. Variable group 4 has both coffee and humic acid added.

The four portions were equally divided into 3 jars each, then pressure cooked for 90 minutes at 15 psi. After cooling, each jar was inoculated with 1 mL of LME liquid culture solution and shaken vigorously. The LC is from a monoculture grown on MEA, and was mixed thoroughly to finely break up the mycelium for equal inoculation of the 12 jars. All jars have been incubated at a consistent 78 - 80 degrees Fahrenheit.  I wanted more than one jar of each variable to be able to have a fair comparison and not be fooled by false results due to random chance, as outliers can occur.

Day 5 post-inoculation:
As expected, the growth is mostly sporadic at this point. I am beginning this post at day 5 because there was not enough growth at day 4 to be noteworthy.

Interestingly, group 3 has a distinct outlier which shows tremendous growth. After only 5 days, this jar is completely colonized. This one is out of the humic acid group. I am fascinated by this result, and wondering what has caused this one jar to grow this way. Did this portion of the grain get a disproportionate amount of the humic acid? Did I somehow overshoot the inoculation amount? I'll definitely have to run some more experiments to quantify this result.

V1: Control. Gypsum only.


V2: Gypsum and fresh ground coffee.


V3: Gypsum and humic acid.


V4: Gypsum, fresh ground coffee, and humic acid.


I welcome any thoughts from the community on the current experiment or future experiments that you would like to see!


Edited by Chem7 (02/04/22 05:42 PM)


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Offlinedyel
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Re: Grain spawn additive experimentation [Re: Chem7]
    #27643639 - 02/03/22 09:40 AM (1 year, 11 months ago)

:takingnotes:


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OfflineChem7
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Re: Grain spawn additive experimentation [Re: Chem7]
    #27644632 - 02/03/22 10:04 PM (1 year, 11 months ago)

Update, day 6:
The grain jars had significant growth in the last 24 hours, and patterns seem to be emerging. As expected following previous growth, the humic acid jars have continued to colonize quickly, and V3a, which had shown the most growth, is now consolidating nicely. Interestingly, the V1 control group has also grown quickly overnight and is now comparable to the V3 humic acid group.

What has surprised me is the two coffee groups, as both of these are growing more slowly than V1 and V3. V4, which also contains humic acid, seems to be growing slightly faster than V2, which has been the slowest overall. Coffee is a common addition to the substrates of many mushroom cultivators to boost nitrogen, so I would have thought that growth would have been improved over the control group.

V1: Control. Gypsum only.



V2: Gypsum and fresh ground coffee.



V3: Gypsum and humic acid.



V4: Gypsum, fresh ground coffee, and humic acid.


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

Hail to the King, Baby


Edited by Chem7 (02/04/22 05:43 PM)


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OfflineChem7
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Re: Grain spawn additive experimentation [Re: Chem7]
    #27646015 - 02/04/22 07:08 PM (1 year, 11 months ago)

Update, day 7:
At this point, the grain in all of the jars is overgrown enough that further observation would be unnecessary. Honestly, these colonized much faster than anticipated. I attribute this to a few factors: I took the extra time to isolate a strong monoculture, the pressure cooked grain has a higher percentage of water, these are smaller jars than I would normally use, and I started using new lids that improve the gas exchange.

V1: Control. Gypsum only.


V2: Gypsum and fresh ground coffee.


V3: Gypsum and humic acid.


V4: Gypsum, fresh ground coffee, and humic acid.


New lids: Leakproof Ball lids, center drilled for inoculation port and drilled 3 times around the edge for gas exchange. These holes are 3/16" and covered with 3M Micropore tape.



Conclusions, Experiment 1:
The growth curve was so short as to make judging results difficult. I can say that humic acid MAY have improved the growth of the mycelium, and that coffee MIGHT have decreased the speed of growth. What I can say definitively is that the results support a need for further testing. For the next iteration, I'll use a larger sample size as well as set stricter parameters. Also, I used fresh coffee in this experiment,  which may have been too acidic, so I'll try spent grounds next time. I may also go to agar and do some comparisons that way.

I know most posts are more of a discussion, whereas this one is becoming more of a journal, but I'll still continue to post the results here as I move forward for the sake of whoever this may benefit. At the end of several experiments, I'll create a new post to summarize the results in an easier to read format, but maybe this post will still entertain those who like to see the journey. 😁


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

Hail to the King, Baby


Edited by Chem7 (02/05/22 11:23 AM)


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InvisibleMysticMycologist
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Re: Grain spawn additive experimentation [Re: Chem7]
    #27648882 - 02/06/22 09:02 PM (1 year, 11 months ago)

You say you are working with a monoculture. How did you isolate it? Serial dilution and microscopy?


--------------------
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OfflineChem7
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Re: Grain spawn additive experimentation [Re: MysticMycologist] * 1
    #27651378 - 02/08/22 07:24 PM (1 year, 11 months ago)

I started with serial dilution of a MSS and inoculated agar plates with a single drop at 1:10,000 dilution. From there, I transferred from individual colonies until I isolated stable monocultures. Out of the 12 monocultures that I was able to transfer out, I chose the one with the fastest and most rhizomorphic growth to transfer to liquid culture.


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

Hail to the King, Baby


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InvisibleScrewdriver
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Re: Grain spawn additive experimentation [Re: Chem7]
    #27656514 - 02/12/22 02:52 PM (1 year, 11 months ago)

This is a rather interesting experiment.
What quantity of additives did each sample get? Did you measure the end pH of the substrates?

I noticed you're using freshly ground coffee whereas most examples of coffee supplementation I've seen utilize brewed/spent coffee grounds. Can't help but wonder if this has any impact on experiment outcome. Hot water extraction would remove a portion of phenolic compounds and tannins, which may have anti fungal properties, with minimal impact on nutrient content of the grounds. Would be interesting to see this include fresh vs. spent coffee grounds.


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OfflineChem7
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Re: Grain spawn additive experimentation [Re: Screwdriver]
    #27666375 - 02/20/22 11:45 AM (1 year, 11 months ago)

Unfortunately, I did not think to test the final pH, and I'm kicking myself for it. I agree that the fresh coffee may have have a negative impact on growth as compared to spent grounds. However, what is interesting is that these grains are now in bulk in fruiting conditions, and the tub using the humic acid grain is beginning to pin before any other tubs. By comparison, I have the other variables from this experiment in their own tubs, along with other tubs that have spent coffee grounds added to the substrate which were spawned to bulk several days earlier (which is actually why this experiment used fresh, as I had just used up all of my spent grounds). Despite being several days younger and being in the same fruiting room, the humic tub has pinned faster than all other tubs that either contained some kind of coffee or the control tub with no coffee or humic.

As for quantities, per quart of grain, I used 1 tablespoon of gypsum, 1 tablespoon of coffee, and 1/4 teaspoon of humic acid. Also of note is that the humic acid that was used for this experiment is a granular product intended for more long-term release in soil, though it did break down significantly during the heating stages of the grain prep. There are other products that are intended to be more water soluble, so the results may have been different with one of those.


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

Hail to the King, Baby


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OfflineRonnyJotton
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Re: Grain spawn additive experimentation [Re: Chem7]
    #27683864 - 03/05/22 01:18 PM (1 year, 10 months ago)

I really like this idea. I also feel like you need a jar with just grain for a control jar.


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OfflineThe Violet Dragon
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Re: Grain spawn additive experimentation [Re: MysticMycologist]
    #27690596 - 03/11/22 06:42 AM (1 year, 10 months ago)

Thank you for sharing this experiment and journaling its progression! How did the different groups end up fruiting? I have to ask, did you put those nifty modified lids on before  you PC'd or after? I found similar lids, but I couldn't find out definitively if they were safe in the PC. Patenting only specified that they were "dishwasher safe."
I'd like to try adding humic acid next time I run grain spawn- I have been researching ways to reach closer to optimal colonization rates. I experienced some issues with my prior inoculated batch... The jars were left to consolidate too long and ended up with more than usual metabolite activity. Also, my yields took a blow as the mycelium reached senescence.
You are greatly appreciated.
Om.


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OfflineChem7
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Re: Grain spawn additive experimentation [Re: RonnyJotton]
    #27695606 - 03/14/22 08:46 PM (1 year, 10 months ago)

That's fair RonnyJotten, but I always add a bit of gypsum to my grain jars to reduce sticking, so for me that is always my baseline control model.


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

Hail to the King, Baby


Edited by Chem7 (03/14/22 09:06 PM)


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OfflineChem7
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Re: Grain spawn additive experimentation [Re: The Violet Dragon]
    #27695628 - 03/14/22 09:05 PM (1 year, 10 months ago)

Violet Dragon, sorry for responding so slowly! Been traveling a lot recently.

I have used these lids in my Instant Pot successfully, but the plastic can deform if the jars are packed at all tightly so they need to have a little extra space.

My goal is to shorten the entire process, so I want to accelerate the grain colonization, the bulk spawn run, and even the agar phase. I do love growing mushrooms, but also love getting things done so I can move on to the next project! That does increase the need for attention to the whole process, because as you said, the mycelium will stress if left too long past full colonization.

As for this experiment, as might have been expected, the tubs mimicked the grain jars. The tub with humic acid grew out first with the control tub close behind. Both of the tubs with coffee grew more slowly and had a more sparse fruiting pattern.

I had another set of grain jars going at the same time (with only gypsum added) that were spawned to bulk with used coffee grounds included in the substrate. Interestingly, even though I spawned these other tubs several days before I completed my experiment, the humic acid tub and control tub both fruited before any of these tubs did. So, while I am sure that the added nitrogen from the coffee has the potential to improve yields, this experiment did not bear that out.

All said and done though, even with the lower producing tubs, the sum total of all my mini tubs has come up to around 250 grams dried, so science has it's rewards. 😋

In future, I want to do some comparisons of growth with different amounts of humic acid, as well as with different amounts of coffee. I have a theory that the coffee's nitrogen doesn't benefit the mycelium if there is a lack of nitrogen-fixing bacteria, so I'll be working with that, as well.

And if anyone has something they would like to see in a future experiment, please let me know! We mad scientist types like to stay busy.


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

Hail to the King, Baby


Edited by Chem7 (03/21/22 12:15 AM)


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Offlineshilohvega
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Re: Grain spawn additive experimentation [Re: Chem7]
    #28099891 - 12/16/22 05:01 AM (1 year, 1 month ago)

Hey mad scientist!!!

“Need input”
“Need input”
“Need input”


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Offlinethegardener
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Re: Grain spawn additive experimentation [Re: Chem7]
    #28117222 - 12/30/22 08:06 AM (1 year, 28 days ago)

Thanks for taking the time to do this, really cool. I was thinking ph as well, would love to know if there was a difference and if it made one. Is ph of high/medium/low importance in regards to healthy growth? Is there a sweet spot for ph?


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