**4. Discussion**

## *4.1. Mycelial Growth*

4.1.1. Reducing Contamination and Promote Mycelial Growth

The mycelium-based materials recipe and processes impact the growth of contamination and mycelium, which impacts the performance of the materials. For the two batches grown in this study, the substrate was either sterilized (batch 1) or pasteurized (batch 2). Pasteurization is known to kill pests and competitors while minimizing the loss of beneficial micro-organisms [59]. In this regard, pasteurization resulted in lower contamination levels during mycelium growth over the substrate than sterilization [5]. In our study, medium levels of contamination were observed throughout all specimens during mycelium growth over sterilized substrate (i.e., both mixtures of batch 1). By comparison, specimens grown with pasteurized substrate (i.e., batch 2) were either fully contaminated (i.e., those grown with mixture L) or only displayed nil to very low amounts of contamination (i.e., those grown with mixture S containing fine particles). Since no significant difference in contamination was observed between specimens grown with both mixtures in batch 1, and mixtures with fine particles are harder to sterilize or pasteurize than those without, it seems unlikely that the mixture was the cause of the contamination levels. The variation in contamination levels between mixtures of batch 2 was most probably due to the success of the pasteurization process. Even if both sterilization/pasteurization processes were conducted on two different fungal species, pasteurization seems more effective as it can inhibit all contamination if successful [5]. The absence of contamination increases the process' potential for replication. In conclusion, future research should prioritize pasteurization of the substrate.

Furthermore, the growth of contaminants competes with the growth of the inoculated fungal mycelium species. Existing research projects have shown variations in growth time ranging from 6 days up to months [7,13,34,35]. In our case, access to the growth lab was limited due to COVID-19 regulations. Twenty-one days after inoculation, a thin mycelium mat was observed over most of the specimens' visible surfaces. The growth time was set to 46 days for both batches to ensure complete growth over all specimens. The addition of more-nutritious substrates or higher concentrations of mycelium spawn enhances mycelial growth and reduces contamination, resulting in higher mechanical properties [13]. However, this addition (not used in this project) decreases the sustainability of these materials as the substrate is composed of products other than waste or byproducts.

### 4.1.2. Food Production

During the growth and drying phases, fruiting bodies were collected on the myceliumbased materials grown with *Pleurotus ostreatus*, but not on those grown with *Coprinus comatus*. Yet, materials were grown in the same dark environment with the same temperature and relative humidity. However, they were not grown during the same season (i.e., March–May for *Pleurotus ostreatus* and June–August for *Coprinus comatus*). The formation of *Pleurotus ostreatus* fruiting bodies is most abundant in spring in temperatures ranging from 4 to 24 ◦C [51], which is the season in which our materials were grown. *Coprinus comatus* fruiting bodies are most prolific under spring and fall temperatures ranging from 4 to 16 ◦C [51], whereas our materials were grown in the summer. Therefore, the variation in mushroom yield may result from the difference in the fungal species and/or growth season.

The weight of *Pleurotus ostreatus* fruiting bodies harvested from compression and bending specimens, respectively, equaled 9.45 ± 10.70% and 9.85 ± 9.10% of the initial specimens' weight. Therefore, the biological efficiency (i.e., the effectiveness of the process for mushroom production) equaled 9.45% for the compression specimens and 9.85% for the bending specimens. A wide variation of yield was observed among our specimens. The biological efficiency ranged from 0.00 to 35.94% for the compression specimens and from 0.00 to 20.51% for the bending specimens. As a reference, the biological efficiency of various substrates grown with the same fungal species, *Pleurotus ostreatus*, ranged from none for elephant grass to 61.04% for composted sawdust [60]. However, our process mainly focused on producing mycelium-based materials and was not optimized for mushroom yield. Materials were grown inside a PVC tube or a flat rectangular mold enclosed with housewrap in a dark environment. Fruiting bodies were able to grow through the housewrap material that sealed the PVC tubes by enlarging the small holes already present in this material. Therefore, they could be harvested without removing the housewrap and exposing the mycelium-based material to potential contaminants. Mushrooms were not harvested in multiple flushes and their growth was stopped by drying the materials. Therefore, the biological efficiency is presented as a reference and only shows the potential of combined material and food production.
