Biodegradation tests and other experiments: a wrap up

Hi backers!

I know I have not been too active in publishing my work lately but here is a lab note to summarize all I could achieve thanks to your help! And I have been doing a lot of things!

Plastic biodegradation tests

One of the purposes of the project was to assess how well different fungal strains were suited to biodegrade textile that is cellulose but also plastic based. All the strains I had are suitable for cellulose biodegradation but in order to decipher what strain could be potentially used for plastic based waste, I first carried some tests by growing all my strains on some in-house made Polyurethane agar (PUR-agar) and Polyethylene terephthalate agar (PET-agar).

During this stage of the research, I had to develop and optimize a way to test the use PUR or PET as a sole carbon source. Making an agar composed of only mineral medium and a plastic in such a DIY lab comes with challenges related to hydrophobicity of the plastic and aggregates formation, but it was made possible by following (with a twist) protocols described in Russel et. al. 2011 for PUR and in Charnock 2021 for PET.

 

Image 1: PET agar plates before (left) and after (right) protocol optimization.

All the strains ( Pleurotus ostreatus, Omphalotus nidiformis, Pestalotiopsis microspora, Trametes versicolor and Ganoderma lucidum) were tested for both PET and PUR biodegradation, and biodegradation was assessed by the measuring the apparition of a clear halo (decoloration indicating the mycelium is metabolizing the plastic) on the petri dish. To do that, images were modified using ImageJ's lookup tables and a difference in pixel values was measured.

 

Image 2: P. microspora (left) and P. ostreatus (right) grown on PUR-agar. On the left, we can clearly see the apparition of a clear halo indicating biodegradation. Image treated with ImageJ.

From those tests, some strains were identified as potential biodegrading candidates, even if the biodegradation was very light in most cases. Considering that the next tests to carry were on fibers and that for those, 3 biological replicates x 3 technical replicates are done for each condition (9 experiments), the purpose of these tests was to limit the amount of experiments to carry when possible. In this sense, all the strais were tested for cotton biodegradation and only the strains that displayed signs of PUR or PET biodegradation were tested for different types of fibers.

Textile biodegradation tests

To test out fabric biodegradation, I had to develop a protocol that was limiting the amount of nutrient the mycelium would recieve, restricting its feedstock to the fibers, but would still provide it with the necessary minerals.

The protocol I developped included a first phase of growth on some minimum liquid medium, followed by inoculation of fibers supplemented with mineral medium.

 

Image 3: P. microspora textile biodegradation test on polyester, cotton and spandex fibers + controls.

Those tests led to very weak composites and most of the strains were not strong enough for plastic biodegradation. This difference with the first results can be explained by the fact that in agar, the plastic used is in amorphous phase while in fibers, it is cristalline which makes it much more complicated to biodegrade. Some strains had very poor results even for cotton. However, some of them, such as Omphalotus nidiformis, Pleurotus ostreatus or Pestalotiopsis microspora showed growth and the hyphae entanglement with the textile fibers could be observed on the microscope.

 

Image 4: P. microspora grown on cotton textile waste. On the right, we can see the hyphae fibers growing with the cotton fibers.

 

Image 5: G. lucidum mycelium growing on cotton waste fibers, formation of a mycelium skin.

The strongest samples were heat pressed to see their potential as a composite material and some results were very similar to cardboard. From those experiments, the best results were obtained from Omphalotus nidiformis, Pleurotus ostreatus and Ganoderma lucidum.

Strains screening for pure mycelium leather making

In parallel from the previously described experiments, I wanted to test out all the strains I had in-house to see their suitability for pure mycelium leather growth. I had already experimented with liquid state fermentation in 2022 and made a tutorial video about how to make mycelium leather, but wanted to optimize the procotocol for upscaling to larger sheets.

 

Image 6: "The mycelium battle" of all the strains we have at the lab.

Considering the final purpose of the project is to make a textile and food waste composite material, those results would also be useful for the choice of the strain and optimal conditions. This additional research allowed me to master liquid state surface fermentation but also submerged fermentation and thus diversify my practice and be able to explore materials a bit further. And some of the steps are really beautiful, like those myceliums clouds, stars, caterpillars !

 

Image 7: different morphologies mycelium can adopt in submerged liquid fermentation setups.

By following the same principle as described in the video but using mycelium grown on a liquid state submerged fermentation as inoculum, I was able to test all the strains for pure mycelium leather production. Pestalotiopsis microspora gave interesting results in terms of growth time and texture of the final material was very latex/plastic-like, while Omphalotus nidiformis led to results ressembling leather.

 

Image 8: Omphalotus nidiformis liquid culture and mycelium leather sample (left) and Pestalotiopsis microspora (right).

Conclusions

The results from those 3 rounds of experiments led me to various conclusions:

• plastic biodegradation and textile waste biodegradation tests will lead to different outcomes as biodegrading a cristalline product is a more complex process than an amorphous polymer.

• cotton waste biodegradation should continue being explored paired with a more nutritious substrate using the three Omphalotus nidiformis, Pleurotus ostreatus and Pestalotiopsis microspora strains.

• If some results do not align with results found in litterature, it might be due to differences between strains. Every strain is different and results might differ, even within one species. For instance, I would have expected Trametes versicolor to have better biodegradation and material properties according to information found in litterature, but the strain we have in the lab might not be the best.

As this post comes as a wrap up, I would also like to put the emphasis on all what this crowdfunding campaign has allowed me to achieve. This past year has been a very intense journey, during which I built a lab in partnership with my colleague Jessica Kate Dias. I grew and learnt a lot of scientific and non-scientific things, ranging from finding lab suppliers, mastering logistics to buy lab equippement, managing a budget, optimizing a lab workflow, networking, applying for grants, making plastic agar, strategically designing experiments, making scanned images of my results, doing bibliographic research, optimizing the amount of experiments, operating new machines, shifting from liquid to solid fermentation, developping post-processes...

So I would like to truly thank you for allowing me to achieve all this. And thanks to that, I have also been able to push the project further: I will now be continuing this research in the best environment possible at the VUB university of Brussels by doing a PhD on textile biodegradation using mycelium, and intenting to develop a new textile recyling technique !