*4.4. Emerging Synergies between Bioremediation, and the Production of Food, Materials, and Medicine*

This project is a first step towards a model in which fungi, especially their mycelium, can serve to decontaminate substrates, while producing fruiting bodies, medicinal drugs, fertilizers, and materials (e.g., for buildings or packaging). It stems from a circular model described by Paul Stamets, in which fungi are used for food, medicine, bioremediation, and fertilizer [51]. The updated model can be implemented in the two following examples.

The first example aims at reducing the environmental dispersal of chemicals that negatively impact the environment. The run-off of nitrates and phosphates, coming from agricultural fertilizers, is the main cause of harmful algae blooms in the Great Lakes [61,62]. The bloom of harmful algae and cyanobacteria lowers water oxygen levels, kills marine life, and negatively affects human health and agriculture [63]. Furthermore, the dispersal in the environment of these limited resources contributes to their increasing scarcity. As a response, hyper-accumulators of nitrates and phosphates, in the form of prairie grasses, could be planted along the edge of over-fertilized agricultural fields to limit environmental dispersal of these chemicals. The prairie grasses could then be harvested according to the growing seasons. They would then serve as a substrate for fungal growth to filter and uptake the substances, thus limiting their return in the environment. A variety of fungal species could be grown on this substrate to provide various functions, such as harvesting scarce substances in the fruiting bodies, producing mycelium-based materials, supplying food through edible fruiting bodies, and manufacturing of medicinal drugs. Chemical testing would be conducted throughout the process to track the substances and ensure safe levels depending on the targeted application. At the end of their life cycle, mycelium-based materials are biodegradable and can be reused as compost for fertilizers or as substrate for new generations of mycelium growth. Furthermore, a wider variety of organic substrates could be used to increase the impact of this upcycling process. For instance, algae can be harvested using algal turf scrubbers to produce living materials and various substances [4,64–68].

Similarly, organic waste from building and demolition sites (e.g., wood) can serve as substrate for mycelium growth to produce food and materials while decontaminating it. A large quantity of waste placed in landfills comes from construction and demolition sites, and its toxicity can contaminate the environment, including ground water [2,3]. Various studies have shown that children from various countries, such as USA, Nigeria, and France, are being poisoned with lead from buildings, especially through paint degrading into dust particles [69–74]. As a response, various fungal species can serve to substantially reduce lead concentration in aqueous substrates [75]. Fungi secrete enzymes known to break down aromatic compounds, including Polycyclic Aromatic Hydrocarbons (PAHs), present in waste from construction materials [76]. *Coprinus comatus*, a bio-accumulator of heavy metals, is a species recommended for decontamination of substrates with nitrates and phosphorusbound toxins [51]. Research shows that using biochar and mycoremediation can lock away contaminants, such as lead, from the water cycle and bioavailability [75,77,78]. Chemical analysis should be performed based on the substrate and fungal species used to quantify the accumulation and breakdown of the various chemicals contaminating the substrate. Such analysis, called Toxicity Characteristic Leaching Procedures (TLCPs), would evaluate the effect of mycelium growth on bioremediation while ensuring the edibility of the fruiting bodies.

### **5. Conclusions**

The diversity of fungal species and their functions has the capability to serve multiple functions in our industrialized society, from decontamination to production of food,

materials, and medicine. This study shows the potential of two fungal species commonly used in bioremediation practices to produce building materials. These mycelium-based materials showed Young's and elastic moduli comparable to or slightly higher than those from other mycelium-based materials, depending on the variables studied. The effect of the selected variables (e.g., fungal species, substrate particle size, and post-growth treatments) tested in this study enhances knowledge about which parameters serve to tune mechanical properties of the final material. Density of composite materials was positively correlated with mechanical performance. The presence of micro-particles and compaction of grown material (as a post-growth treatment) increased density and the resulting Young's modulus, in addition to the elastic modulus. Differences between baking or drying grown material were less pronounced, with baking leading to slightly higher compressive moduli in most cases. The standard deviations observed within samples exemplify the need to better understand variables affecting material properties and to develop more accurate manufacturing procedures. Heterogeneity of produced materials was particularly apparent when analyzing the fracture location's eccentricity of materials under bending. The study of shrinkage during growth and drying periods provides valuable insight into predicting the materials' final dimensions. Both fungal species led to similar mechanical behavior. In addition to *Pleurotus ostreatus*, *Coprinus comatus*, a species known for bioremediation and food production, can also serve for material production and, potentially, a combination of all three. Integration of material production is suggested as an enhancement of the circular model described by Paul Stamets [51]. Further studies still need to be conducted to validate the combined use of these species' mycelium as a binder for building materials while decontaminating substrate and producing edible fruiting bodies.

**Author Contributions:** Conceptualization, C.M., P.G. and T.H.; methodology, C.M., P.G. and T.H.; software, T.H.; validation, T.H. and P.G.; formal analysis, T.H.; investigation, T.H. and R.N.; resources, C.M. and P.G.; data curation, T.H., P.G. and R.N.; writing—original draft preparation, T.H.; writing review and editing, T.H., P.G., C.M. and R.N..; visualization, T.H.; supervision, P.G. and C.M.; project administration, P.G.; funding acquisition, P.G. and C.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by USDA—Rural Business Development Grant (RBDG) under the Project Title: "Develop prototype panels for customer evaluation" processed by the University of Akron Research Foundation (UARF) in 2019 and 2020.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is available upon request to the authors.

**Acknowledgments:** The authors would like to thank: John Burmeister from Valley City Fungi for providing knowledge and help with pasteurizing substrates; Henry Astley for helping with writing python code to extract particle size dimensions; Hunter King for his suggestions to measure particle dimensions of sieved materials; Jiansheng Feng for his support with mechanical testing; David McVaney for his ideas on mechanical testing procedures; Nicholas Mazzocca, Derek Jurestovsky and Jessica Tingle for their help with basics of making plots in R; Randy Mitchell and Richard Einsporn for their advice with data analysis; and Elena Stachew for her comments and suggestions throughout the project.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **Appendix A**

The percentages of each ingredient used in inoculation recipes from the literature are presented here. The GrAB project team used the following averaged percentages: 10 wt% of *Pleurotus ostreatus* spawn, 14 wt% of substrate, and 76 wt% of water [79]. Jones et al., 2018, soaked the substrate in Type 1 Milli-Q® water and sterilized the mixture before adding 25 wt% of *Trametes versicolor* spawn, while explaining that lower percentages of spawn slowed mycelial growth and increased risks of contamination [18]. Elsacker et al., 2019,

used the following recipe in weight percentages: 10 wt% of *Trametes versicolor* spawn, 20 wt% of substrate (chipped, sieved and sterilize), and 70 wt% of sterile demineralized water [9]. The LIWAS project used the following recipe: 6 wt% of *Hypsizygus ulmarius* mycelium spawn, 34.5 wt% of substrate, and 59.5 wt% of water [5]. In comparison with other projects, these last percentages produced mixed mycelium growth results, likely due to the low amounts of mycelium spawn provided [18].
