SOIL PLATE LUNCH: DIY Myco-remediation soil toxin Experiment Design

While myco-remediation is being explored as an important component of bioremediation efforts, there are few resources for in-classroom experiments which middle and high school students can replicate without expensive tools and reagents. In Hawaii, there is no EPA-certified water testing facility and water samples need to be sent to the continental US. With the growing number of disasters such as the fires on Maui and the Navy Red Hill fuel leaks on Oahu, there is a need to drive innovation in tool design and solutions design locally from a place of understanding of the unique criteria & constraints of the Hawaiian ecosystems. A uniquely Hawaiian-focused bioremediation educational program that is low cost, collaborates with locally sourced invasive and endemic species, and which uses accessible lab protocols will expand the base of research in younger generations rooted here.

Question

Can mycelium (propagated from locally grown oyster mushrooms in a range of invasive species fire fuel grasses) be used in accessible in-classroom soil toxin bioremediation experiment designs? In lab notes 1, we demonstrated that we can successfully grow mycelium by collaborating with chopping up mushrooms and growing them out in pasteurized invasive guinea grass and in haole koa seed pods.

Hypothesis

By placing mycelium myco-socks and myco-sheets into plastic containers filled with soil, we can test the bioremediation efficacy of oyster mushroom mycelium in remediation of a mock soil toxin. The soil group receiving myco-remediation will show a reduced presence of chemicals than the soil that did not receive the myco-remediation. 

 

Initial experiment test concept sketches

 

Inital test experiment concept sketches

Materials

Plastic bin (maybe not the plate lunch container I used, but individual separate ones instead), soil sourced from garden or school grounds. Disposable gloves, cookie sheets or wax paper, spray bottles of 70% isopropyl alcohol, Pine-sol cleaner, quaternary test strips, chopsticks, towels or paper towels, permanent marker, pre grown myco-sheets and myco-sock (see lab notes 1)

Procedure Options

There are two directions we were initial considering for this experiment:

1. Using gold flakes as a heavy metal soil toxin stand in. Testing would use a DIY Microbit nail soil hygrometer to assess change in conductivity of the soil and mycelium before and after oyster mushroom myco-sock or myco-sheet grown in it. Small amounts of gold foil would be sourced in small amounts from an online culinary source or at a baking shop.

2. Using Pine-sol as a mock soil toxin, Testing would include adding easily accessible household cleaner in soil. The soil and mycelium growth would be tested before and after by using quarternary test strips.

Testint with Pine-sol as mock toxin procedure

Set up work area as BSL-1 on a cookie sheet. Basic safety protocols followed (gloves, goggles, hair back and no dangling jewelry, as well as no food or drink in area)

1. collect soil from school garden or grounds

2.  

   

   collect soil in container to be carefully measured back in classroom.

   evenly distribute soil in two container compartments. One will be control and other will be the test soil. Students can measure amounts using measuring cups or a gram scale 3/4 cup soil for control and 3/4 for experiment soil.

    

   

   Plate lunch container with 3/4 cup soil in top two sections and 1.25 cups soil in lower section

3. Students measure 3 TBS Pine-sol or Lysol ( or another cleaner with high ammonium compound levels) and add it to one container section of 3/4 cup soil. Label "experiment 1"

    

   

   Adding 3 TBS Pine-sol

4. Students measure 3 TBS water and add it to the other container section of 3/4 cup soil. Label "control"

5. 2 inch strips of quaternary strips are taped to two chopsticks and stuck into the soil for 2 minutes. Remove and wipe off soil seperately with different clean towels or paper towels.

    

   

   chopsticks with quaternary strips attached to bottom.

6.  

   

   compare the two, label and note from which container the quarternary colorimetric test strip comes from and photograph. The green blue color indicates high presence of our mock toxin ammonium compounds in the test soil.

7. A growing mycelium sheet growing in guinea grass or haole koa is added to each of the soil sections. These are flipped about and nestled in between a top and bottom layer of soil. In this first experiment I added wedges that were about 3/4 sized of the container.

    

   

   container growing oyster mushroom mycelium in Guinea grass for 3 weeks.

    

   

   mycelium sheet cut into smaller pieces for use in experiment.

8. a quatarnary strip is tested against the damp mycelium in the control container.

    

   

   Left is control soil, middle is mycelium, right is soil with pinesol. Since mycelium creates chemicals similar to Lysol and Pine-sol, its test strip is showing some of this type of chemical present.

9. An additional experiment is to add a myco-sock to a soil container. In this test, since the mycelium had not grown densley throughout the substrate, I added it to 1.25 cups of soil and am seeing if it continues to grow. If it does, I will add pine-sol in a week to the soil for testing.

    

   

   myco-sock added into bin of 1.25 cups soil. The soil is pressed to the side to accomodate the sock. No chemicals have been added to this yet as I want to just see if it can continue to grow when set in soil with this miminal amount of mycelium growth. The top rght section has Pine-sol, the bottom right is control, and the left larger section has a myco-sock.

10. Lightly cover bin. After a month, all the soil container sections will be tested again with the quartenary strips attached to chopsticks to see if the test strips indicate a visible change in chemical concentration.

     

    

    Day 1 of test. Plate lunch container with mycelium and soil in each section.

Next steps:

1. Exploration of a new test system with a free downloadable peristaltic pump and a stepper motor driven by a microbit or arduino could be set up to have a continuous flow of liquid in the test system and it would enable easier testing of chemicals via the liquid in the system. I have just started exploring these systems on Thiniverse 

2. Trial of the gold foil in soil experiment mentioned earlier to see if mycelium uptakes gold in anyway that is detectable by a DIY nail hygrometer system (which is actually a system testing for conductivity). It has been highlighted to me that this probably will not work without both sophisticated tools and nanoparticle gold. So I might table that.

3. Sharing test systems developed so far with Kumu Rose Bailey Ho’omanawanui who is the Maui Hawaiian educator/practicioner on our team.

4. Add in a succesional bioremediation step (Vermicomposting) that was suggested by both Dr. Cruz Perez and field mycologist Taylor Bright.

 

sketch of a test system with nested containers draining into eachother and a peristaltic pump and stepper motor system.

 

Peristaltic pump from Thingiverse: https://www.thingiverse.com/thing:454702 and here is another one: https://www.thingiverse.com/thing:8914