Research statement: Investigating the rate of breakdown of different types of

microplastics by mycelium.

 
Mycelium is the vegetative part of the mushroom that absorbs water and nutrients from the soil
for the fungi to live. Mycelium grows by releasing enzymes from the hyphal tips of the mycelium
to digest the surroundings and then absorb the nutrients. The cells will eventually branch and
continue to branch as it grows to build a vast, filamentous mycelial network.
References about mycelium:
1. Fungi as useful organisms to the ecosystem:
https://decroissons.files.wordpress.com/2014/04/paul-stamets-mycelium-running-how-m
ushrooms-can-help-save-the-world.PDF
2. https://returntonow.net/2018/01/11/7-ways-mushrooms-can-save-world/

References that supports our research:

https://aem.asm.org/content/aem/82/17/5225.full.PDF
Their method: PU(polyurethane)-PE(polyether type of PU) foams were washed and dried,
inoculated with 80mg of 1 week old mycelium growing in static cultures at 30 degrees in petri
dishes. Fungi was grown in mineral medium modified solutions, eg NaH2PO4, K2HPO4, and
potato dextrose agar
They found out that certain fungi found in the soil were able to degrade PU, but PE was unable
to be degraded.

https://teaching.yale-nus.edu.sg/wp-content/uploads/sites/25/2016/02/Plastic-Eating-Fungi-Othe
r-Wonders-Discovered-by-Yale-Undergraduate_Scott-Strobel-.PDF​ (at the bottom)
Plant stems’ inner tissues were isolated and put with agar for the fungal organisms to grow.
Fungi appeared on the agar is then collected and stored. Their conclusion was that some
endophytic fungi was able to degrade polyester polyurethane.

https://www.youtube.com/watch?v=gajEtR--GCg
https://www.fastcompany.com/3039801/this-home-grown-fungi-will-eat-your-leftover-plastic-and-
then-you-can-eat-the-fungi
Video showing the breakdown of plastic by fungi mutation mushroom

Plan for procedure:

1. Collect the macroplastic and identify the number of each category of plastic in each
quadrat
2. Wash the macroplastics and dry them under the sun.
3. Sort all the plastic under the categories
4. iron all the macroplastic so that it is flat
 5. cut the macroplastic into small pieces
6. Grow the mycelium following the link (no1), then we put the plastic in between the
cardboard layers
7. See if the plastic decrease in mass due to the mycelium

1. Collection of macroplastics
1. Ensure that sea is at low tide.

2. Along the coastal trail, use a distance measuring wheel to identify the line of transects A
to H, that are perpendicular to the shoreline and the walking path, at 100 metres
intervals along the trail, with the 0m mark starting after the boardwalk (in front of the mud
experience area)

3. Identify the line of transect A at the first point, 100m from the start point.

4. Along that line of transect, use strings to measure out a 2m by 2m quadrat, laying the
strings on the soil to mark out the area.

5. Use a measuring tape to measure the distance from the pavement to the location of
quadrant, so that the exact quadrant can be easily identified the next time the
experiment is conducted 2 weeks later.

6. Use tongs/hands to collect macro plastics on the surface of the soil within quadrant.

7. Place the macroplastics into a ziplock bag and label it.

8. Repeat steps 4 to 8, at quadrats B to H, which are all 100m apart along the coastal trail.

9. Repeat entire protocol at 2 weeks intervals.

1.5. Classifying macroplastics

1. Classify macroplastics according to their type into 5(?) categories
Categories:
- A: Other Plastic pieces
- B: beverage bottles
- C: Food packages
- D: Plastic bags
- E: Bottle caps
Reference: ​http://www.greenfuture.sg/2014/08/12/insights-on-marine-trash-in-singapore/

2. Growing of mycelium
Materials needed:
- Sealable transparent container A, B, C, D, E, F
 - Corrugated cardboard
- Mushroom with a large base (oyster mushroom)
- Scalpel
- Dark place to store experiment set-up
Procedure:
1. Cut cardboard pieces the size of the container
2. Stack the cardboard inside the container
3. Fill the container with water to the brim for 20minutes
4. Drain out the water
5. Remove the cardboard pieces from the container
6. Use the scalpel to cut the mushroom into sizes 2cm by 2cm, and in thin layers
7. In container A, put a piece of wet cardboard into the container
8. Spread 20 pieces of mushroom onto the cardboard, of equal distance apart
9. Put another layer of cardboard on top of the mushrooms
10. Repeat steps 8 and 9 until the container is fully filled to the brim
11. Repeat steps 7 to 10 for containers B, C, D, E, F

Reference: ​https://www.instructables.com/id/Making-Mycelium/

3. Identifying the types of plastic that mycelium is able to break down

1. Wash the macroplastics which are collected from sites A to F thoroughly

2. Next, categorize the macroplastics into 5 categories. The categories are listed as
followed:

Categories:
- A: Other Plastic pieces
- B: Beverage bottles
- C: Food packages
- D: Plastic bags
- E: Bottle caps

3. Cut macroplastics in categories A, B, C, D, E, F, into small pieces of 5cm by 5cm

Experiment procedure:
1. Measure the mass of 10 pieces of plastics from category A
2. In container A, place one piece of plastic from category A in each layer of cardboard,
until the top layer of the container
3. Every day, open the box to let some air out
 4. Store the containers in a dark room for 2 weeks (we can first experiment use the plastics
from the monday collect one, then our repeat experiment is using the plastics we collect
the nv time)
5. After 2 weeks, open up container A
6. Collect the plastics present in container A
7. Use a weighing balance to measure the total mass of the plastics at the end of
experiment in container A
8. Repeat steps 1 to 6 using containers B to E, and plastics from category B to E
respectively

Dependant variable: rate of breakdown of macroplastics

Independent variable: the enzymes in the oyster mushroom

Control set up:

A negative control is subjected to the same factors as that for the experiment but mycelium are
removed from the experimental set up.
It is expected that the the mass of plastics placed in the container will remain the same before
and after the experiment
This ensures that the rate of breakdown of plastics is due to the mycelium present in the set up.

Backup ideas:
turn plastic into oil
● melt the plastic under ​very high temp (need to find out how to do this in lab) got any link?
● collect the gas
● pass the gas into tap water
● tap water becomes oil
using mealworms to eat plastics
● https://www.treehugger.com/animals/mealworms-can-eat-all-plastic-diet-and-not-die.html
● buy mealworms from fish shop (LOL)
● keep the mealworms and the macroplastics in a container
● we will weight the macroplastics before and after the experiment, thus allowing us to
conclude n verify whether mealworms can reduce plastic
● good points are that the poop can be used as fertilizer for the soil and that mealworms
can survive on a plastic-only diet.
● BUT not recommended bc it takes vvvvv long for them to digest the macroplastics to
give significant results