*1.1. Problem Statement of Current Building Materials*

Today's architectural practices negatively impact the planet's ecosystems. The building industry and its energy-intensive material manufacturing processes were responsible for a total of 37% of global carbon emissions in 2020, and these processes contribute significantly to anthropogenic climate change and resulting extreme weather events [1]. The push for sustainability in architecture needs to encompass the entire life cycle of materials and buildings, from resource extraction to repurposing or disposal. Even simple materials used in the building industry are becoming scarce and limited. At the other end of the building process, 600 million tons of construction and demolition waste were generated in 2018, with 145 million tons sent to landfills [2]. As a result, landfilled hazardous materials, such as lead, can contaminate ground water [3]. Even after waste removal, sites contaminated with heavy metals and toxic chemicals from industry still need to be cleaned to limit run-off and spread to the environment. Therefore, the building industry urgently needs to focus on cleaner materials that can be repurposed, to reduce both material scarcity and waste generation.

**Citation:** Houette, T.; Maurer, C.; Niewiarowski, R.; Gruber, P. Growth and Mechanical Characterization of Mycelium-Based Composites towards Future Bioremediation and Food Production in the Material Manufacturing Cycle. *Biomimetics* **2022**, *7*, 103. https://doi.org/ 10.3390/biomimetics7030103

Academic Editor: Stanislav N. Gorb

Received: 7 May 2022 Accepted: 23 July 2022 Published: 28 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

## *1.2. Towards Living Materials*

In response to the current limitations of traditional architectural materials and their manufacturing practices, living materials (i.e., materials integrating biological organisms) have emerged in recent decades. Specifically, Engineered Living Materials (ELMs) are defined as genetically or mechanically "engineered materials composed of living cells that form or assemble the material itself, or modulate the functional performance of the material in some manner" [4]. The implementation of living organisms in technological materials allows engineers to benefit from the qualities of biological growth [5]. The overall advantages of ELMs are self-production, clean chemistry, sustainability, adaptability, self-healing, and the potential for added functionality through genetic and mechanical engineering. The wide scope, potential, and limitations of ELMs have been discussed in multiple reviews [4,6]. Examples of ELMs include microbially manufactured polymer matrices, soft living robots, smart living surfaces, living carbon composites, bacteria-based self-healing concrete, bacterial cellulose, biologically fabricated bricks, and myceliumbased materials. The long-term goal of ELMs is to build large-scale hierarchical material systems from simple autonomous micro-entities in situ. However, more research needs to be conducted to keep organisms alive in the applied setting, scale up production of a laboratory environment, and predict organisms' behavior.

### *1.3. Mycelium-Based Materials*

Fungi are used for ELMs because of their mycelium, which forms a 3D binding network, secretion of enzymes, diversity of properties between species, and wide range of material applications. Mycelium-based materials are among the most successful largescale living materials [4,7]. Mycelium-based materials are produced by growing fungal mycelium on an organic substrate (e.g., often agricultural byproducts) in a mold. A variety of post-growth treatments are applied based on the desired application. The material properties are highly tunable based on the selected substrate type (i.e., chemically, and related to size), fungal species, growth environment, and post-growth treatments [5,7–13]. The ability to fine tune the properties of mycelium-based materials increases their range of application, including packaging, electronics, acoustic absorbers, footwear, insulators, fire protection, and self-healing materials [8,14–23]. At the end of their life cycle these composite materials are biodegradable and can even be used as a substrate for growing new iterations of materials. Therefore, mycelium-based materials promote organic waste upcycling, lowenergy material manufacturing, and biodegradable materials, making them an alternative to current architectural materials. To showcase their architectural potential, temporary installations have been built with mycelium-based materials [5,24–29]. To address the limitations of ELMs, more studies need to be conducted before their implementation in permanent buildings by exploring new substrate/fungus combinations, evaluating the effect of various parameters (e.g., fungal species, substrate type and size, growth environment, and post-growth treatments) on material properties, predicting material behavior, ensuring homogeneous material properties, and characterizing the accumulation and decomposition of toxic chemicals [5].

### *1.4. State of the Art of the Production and Mechanical Properties of Mycelium-Based Materials*

Throughout the production process of myceliated materials, various factors influence their acoustic, thermal, mechanical, and physical characteristics [12,13]. Different fungal species will feed on different substrates and grow at different rates [30]. The mycelium anatomy and structure differ between species, with three main categories found in basidiomycetes: monomitic (i.e., generative), dimitic (i.e., generative, and skeletal), and trimitic (i.e., generative, binding, and skeletal) [13]. For example, myceliated materials grown from trimitic species (e.g., *Trametes versicolor*) display a higher compressive, tensile, and flexural strength than those grown from monomitic species (e.g., *Pleurotus ostreatus*) [31,32]. The substrate forms the base of the composite materials as it is not completely decomposed by the fungus during the mycelial growth process. Therefore, the substrate composition

and particle size affect the material properties of the end material as it serves as its backbone structure [13,33]. To successfully introduce the desired fungus, the substrate must be cleaned of other species. By removing competing species, this fungus can grow over the entire substrate and produce a coherent material. Different techniques are used to kill competing species: sterilization (e.g., autoclaving, bathing in hydrogen peroxide, bathing in a basic solution, baking) or pasteurization (e.g., steaming) [5,13]. These techniques affect the mycelial growth speed and removal of competing species differently.

During inoculation, the proportions of the ingredients (e.g., dry substrate, water, mycelium spawn, nutrients) for optimal mycelial growth vary between fungal species. As mycelial growth highly depends on the nutrient profile of the substrate, food with high nutritious content is often added to the agricultural byproducts. Since mechanical failure always occurs in the mycelium binder, mycelial growth and, therefore, the nutrient profile of the substrate, are especially important for mechanical performance [13]. However, the use of more-nutritious substrate makes the material less impactful in terms of sustainability, waste upcycling, and in general, resource efficiency. Percentages of ingredients used in previous research projects are shown in Appendix A.

The growth environment (i.e., temperature, relative humidity, access to oxygen, clean/ventilated air exchange, and lighting conditions) also impacts mycelium growth. The temperature should be kept at around 25–30 ◦C [7,13,34] and the relative humidity around 70–80% [7]. Based on the fungal species, substrate, growth environment, and level of growth desired, the growth time may vary from 6 days [35] to 20 days [7], and up to months [13,34]. In the literature, the growth of fruiting bodies in the production of mycelium-based materials is avoided as it can consume resources otherwise used for mycelium growth, modify material shape, increase composite heterogeneity, require maintenance (i.e., harvest required in an environment that should remain as clean and undisturbed as possible to avoid contamination), and release spores that could cause allergic reactions or infections [32,36–41]. Fruiting body formation can be inhibited by controlling both the lighting conditions and carbon dioxide concentration (i.e., dark with low CO2) or by introducing GSK-3 inhibitors [36,37]. During the growth process and the drying period, myceliated materials shrink. Knowing the shrinkage of the material is important to estimate and target specific product dimensions. For cylindrical specimens, the shrinkage has been estimated to be around 17% in height (vertical shrinkage) and around 10% in diameter (horizontal shrinkage) [9]. For rectangular specimens, shrinkages of 5.56% horizontally and 2.78% vertically have also been observed [42].

After the growth process, various treatments (e.g., drying, baking, compacting, coating) can be applied to the myceliated materials to tune their properties [13]. Existing studies do not usually differentiate drying and baking of the specimens. Furthermore, there is no standard practice for drying or baking, as their success depends on specimens' dimensions. For example, various research teams used the following drying/baking techniques: 40 ◦C for 72 h plus 2 h at 100 ◦C [33], 60 ◦C for 24 h [8], 60 ◦C for 2 h [7], 70 ◦C for 5 to 10 h until the weight is stabilized [9], 80 ◦C up to a constant weight [21], 80 ◦C for 24 h [32], 100 ◦C for 4 h [35,43], and 100 ◦C for several hours [44]. The mycelial structure is believed to degrade with temperatures of approximatively 225 to 300 ◦C [7].

Various research articles studied the mechanical properties of mycelium-based materials, which have been described by a two-phase particulate model with the mycelium as the matrix and substrate as the dispersed phase [7–11,42]. The differences in the production, dimensions, description, and testing procedures of such bio-composite materials make data comparison between studies difficult, as they severely impact their mechanical behavior [42]. The mechanical behavior of mycelium-based materials highly depends on the anisotropic substrate matrix. A study recently looked at the effect of fiber orientation within the substrate on the compressive behavior of mycelium-based materials [42]. The study found that adding fibers oriented in the direction of loading increased Young's modulus. Conversely, fibers oriented perpendicular to the loading direction produced a decrease in Young's modulus and ultimate strength. A study found that myceliated materials made

from loose substrate had lower compressive Young's moduli than those from chopped substrates [9]. The same study also found variability in compressive Young's moduli based on substrate used, despite their similar density (around 100 kg/m<sup>3</sup> ): from 0.14 MPa for loose pine shavings samples up to around 1.25 MPa for pre-compacted hemp and flax samples. Myceliated samples grown with *Pleurotus ostreatus* on hemp mat had a compressive strength of 0.19 MPa compared to 0.26 MPa for *Trametes versicolor* [31]. Another study found a compressive modulus of 1.3 MPa for *Ganoderma lucidum* grown on macerated red oak wood chips (5–15 mm) and a nutrient solution with a final density of 318 kg/m<sup>3</sup> [45]. Vidholdová et al., 2019, grew low-density mycelial boards having a density of 103 kg/m<sup>3</sup> , which resulted in a compressive resistance at 20% strain of 23.95 kPa [11]. Compressive performance of porous materials increases with increasing density, which may result from a variety of parameters including the substrate used, its particle size, the degree of compaction, and the amount of substrate digested by the fungus [13,46,47]. Islam et al., 2017, studied the correlation between density and uniaxial elastic modulus on mycelium boards from Ecovative Design LLC (Green Island, NY, USA) [48]. They found that densities in the range of around 150 to 160 kg/m<sup>3</sup> lead to elastic moduli in the range of 0.5 MPa to 1.1 MPa, respectively. Yang et al., 2017 found various Young's moduli ranging from around 5 to 50 MPa for densities ranging from around 160 to 280 kg/m<sup>3</sup> made with *Irpex lacteus* mycelium grown on a variety of substrates including wood pulp, millet grain, wheat bran, natural fiber, and calcium sulfate [8]. In terms of bending properties, a study looked at the mechanical properties of bioresin-infused mycelium-based sandwich composite materials under 3 pt bending, which led to an elastic modulus of 1.13 MPa for a density of 121.7 kg/m<sup>3</sup> [49]. Appels et al., 2019, found an increase in flexural moduli from nonpressed (ranging from 1 to 9 MPa for densities from 100 to 170 kg/m<sup>3</sup> ) to cold-pressed (ranging from 12 to 15 MPa for a mean density of 240 kg/m<sup>3</sup> ) and hot-pressed (ranging from 34 to 80 MPa for densities from 350 to 390 kg/m<sup>3</sup> ) in materials grown from *Trametes multicolor* or *Pleurotus ostreatus* on rapeseed straw, beech sawdust, or cotton [32]. Further, Appels et al., 2018, found that controlling the lighting conditions and carbon dioxide levels of the growth environment (i.e., light with CO<sup>2</sup> content), in addition to deleting the hydrophobin gene *sc3*, increased composite density and resulting Young's modulus [38].

Due to the novelty of the research field of mycelium-based composites, many research questions still need to be addressed before their implementation in permanent architectural projects [5]. For instance, more studies should explore particle shapes, composition, and distributions due to their significant influence on mechanical properties [42]. The substrate particle size for an optimal balance between mycelial growth and mechanical performances is still subject to research. Higher substrate density lowers air transmission, resulting in limited mycelial growth inside the substrate if the substrate is not artificially aerated [13]. However, Islam et al., 2018, found that the generic trends of the stress–strain curves from compressing mycelial composite made of different particle sizes were not sensitive to the particle size, suggesting that the myceliated materials' compressive response is independent of substrate particle size [43]. In their study, they compared five different substrate particle sizes with varying aspect ratios (2, 5, or 8), sizes (2.5, 5, or 10 mm), and diameters (0.5, 1, or 2 mm). In another study, materials made from medium particles (0.75–3.0 mm) led to higher density, Young's modulus, and ultimate strength than others made from smaller (0.5–1.0 mm), larger (4.0–12.0 mm), or more diverse (0.5–12.0 mm) particles [42]. This study also showed that higher density did not increase compressive performance in all cases. To the author's knowledge, no study has questioned the effect of the very fine micro-particles on mycelial growth speed and mechanical performance. Moreover, the substrate particle size will likely influence the effects of different post-growth treatments. To further extend the analysis of substrate size, more studies should evaluate the subsequent effects of postgrowth treatments on composites grown with these various substrate sizes. As a result, the combined effects of mutually responsive variables need to be addressed in future research.

### *1.5. Underutilized Benefits of Fungal Mycelium*

The variety of fungal species possess many qualities unexploited in current myceliumbased materials. Fungi are known for their nutritional, medicinal, and bioremediation benefits [50,51]. Fungi release enzymes to break down substances that they can feed upon. This behavior makes them very interesting for mycofiltration (i.e., use of fungi to filter water) and mycoremediation (i.e., use of fungi to decontaminate/depollute the environment). Whereas mycofiltration relates to filtering water, mycoremediation aims at decontaminating a substrate. Mycoremediation is a subset of bioremediation practices, in which fungi serve to uptake and break down pollutants. It has been referenced as the cheapest remediation technique for polycyclic aromatic hydrocarbons, at approximately 50 USD/ton [51]. Paul Stamets described a circular model in which fungi serve to produce food and medicine, remediate soil, and facilitate plant growth [51]. Thanks to recent mycelium-based materials research, the production of building materials can now be integrated in this model by growing mycelium materials on contaminated substrate and/or harvesting fruiting bodies during the growth process. Depending on the type of contamination and fungal species employed, toxins present in the substrate are decomposed by the fungus, stored in the mycelium, or accumulated in the fruiting bodies, which are harvested for treatment. In all cases, the myceliated substrate can serve for producing composite materials once their toxicity is evaluated. As stated in Section 1.4, the growth of fruiting bodies is currently avoided in the production of composite materials for various reasons. In terms of mechanical performance, the authors did not find any study comparing the mechanical behavior of mycelium-based materials grown with and without fruiting bodies. Therefore, a study should be conducted to validate the hypothesis that fruiting body formation lowers mechanical performance of these materials. Depending on the targeted application, the authors believe that the benefits of producing fruiting bodies for food or medicinal applications outweigh the potential reduction in mechanical performance. For instance, fruiting bodies could be harvested during the mycelium growth of composite materials. Specific frames should be used to allow harvest without disturbing the growth environment and introducing contaminants. Another solution is to upcycle the fruiting block serving for mushroom production after it is spent, by using it as myceliated substrate for material production [52]. In conclusion, more studies should be performed to evaluate the potential integration of material production in the circular model of current utilizations of fungi.

## *1.6. Overall Goal of the Project*

A potential for this updated model is to grow bioremediating species on contaminated organic substrate from sources including the agriculture and building industries. In addition to decontaminating the substrate, food (i.e., fruiting bodies) and materials (i.e., mycelium-based composites) can be produced after toxicity evaluation. Depending on the chemicals used to accumulate or decompose and the desired application, various bioremediating fungal species can be employed. For instance, *Pleurotus ostreatus* (commonly named oyster mushroom) is a recommended species to decontaminate petroleum products [51,53]. Another species often seen in polluted soils, *Coprinus comatus* (commonly named shaggy mane), is a bio-accumulator of heavy metals and a species recommended for decontamination of substrates with nitrates and phosphorus-bound toxins [51]. Therefore, specific fungal species can be used depending on the chemicals present in the substrate. During mycelial growth over the enriched substrate, fruiting bodies are harvested for chemical treatment or food based on their toxicity. Upon full mycelial coverage, the composite material receives various treatments to tune its properties. Finally, the mycelium-based material and fruiting bodies produced would be chemically tested to ensure that they are safe for the desired application.

This article presents a first step towards the implementation of this model in a casestudy. Two bioremediating species (*Coprinus comatus* and *Pleurotus ostreatus*) were grown on an agricultural byproduct (straw) to produce composite materials. The mycelium-based materials produced were mechanically characterized through compression and bending

testing to assess their potential for architectural purposes. These tests sought to evaluate the effects of the following variables on composite mechanical behavior: substrate size (with or without micro-particles), fungal species (*Coprinus comatus* or *Pleurotus ostreatus*), and post-growth treatment (dried, baked, compacted then dried, compacted then baked). sought to evaluate the effects of the following variables on composite mechanical behavior: substrate size (with or without micro-particles), fungal species (*Coprinus comatus* or *Pleurotus ostreatus*), and post-growth treatment (dried, baked, compacted then dried, compacted then baked).

sent in the substrate. During mycelial growth over the enriched substrate, fruiting bodies are harvested for chemical treatment or food based on their toxicity. Upon full mycelial coverage, the composite material receives various treatments to tune its properties. Finally, the mycelium-based material and fruiting bodies produced would be chemically

This article presents a first step towards the implementation of this model in a case-study. Two bioremediating species (*Coprinus comatus* and *Pleurotus ostreatus*) were grown on an agricultural byproduct (straw) to produce composite materials. The mycelium-based materials produced were mechanically characterized through compression and bending testing to assess their potential for architectural purposes. These tests

To complete the model validation, chemical tests would need to be performed in a further study. These tests were not generated in this part of the project due to a cut in resources (expertise and money) and delays emerging from COVID-19 regulations. However, all mycelium-based materials and samples of both substrate mixtures were stored to be used in future chemical analysis. To complete the model validation, chemical tests would need to be performed in a further study. These tests were not generated in this part of the project due to a cut in resources (expertise and money) and delays emerging from COVID-19 regulations. However, all mycelium-based materials and samples of both substrate mixtures were stored to be used in future chemical analysis.

### **2. Materials and Methods 2. Materials and Methods**

The main steps of the project and variables studied are illustrated in Figure 1. The main steps of the project and variables studied are illustrated in Figure 1.

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 6 of 36

tested to ensure that they are safe for the desired application.

**Figure 1.** This figure shows the overall process of the project, including the variables evaluated (i.e., fungal species, substrate particle size, and post-growth treatment) for their effect on the mechanical properties (i.e., under compression and bending) of the composite material. **Figure 1.** This figure shows the overall process of the project, including the variables evaluated (i.e., fungal species, substrate particle size, and post-growth treatment) for their effect on the mechanical properties (i.e., under compression and bending) of the composite material.

### *2.1. Substrate and Fungal Species 2.1. Substrate and Fungal Species*

The substrate used for mycelium growth was straw, collected in bales from Dussel Farm, Ohio, USA. Different batches of mycelium-based materials were produced with two different fungal species (*Pleurotus ostreatus* and *Coprinus comatus*). Due to limited availability of resources and the scale of the project, the batches were not conducted at the same time. Mycelium-based materials grown with *Pleurotus ostreatus* were manufactured from January to June 2020, while those with *Coprinus comatus* were produced from June to September 2020. The same process was used for both batches: preparation of the The substrate used for mycelium growth was straw, collected in bales from Dussel Farm, Ohio, USA. Different batches of mycelium-based materials were produced with two different fungal species (*Pleurotus ostreatus* and *Coprinus comatus*). Due to limited availability of resources and the scale of the project, the batches were not conducted at the same time. Mycelium-based materials grown with *Pleurotus ostreatus* were manufactured from January to June 2020, while those with *Coprinus comatus* were produced from June to September 2020. The same process was used for both batches: preparation of the straw substrate, inoculation, mycelium growth, application of post-growth treatments, and mechanical testing.

### *2.2. Substrate and Mold Preparation*

## 2.2.1. Chipping

To reduce the size of the substrate particles, the straw was chipped (Done Right Chipper Shredder Premier 300, Generac Power Systems, Inc., Waukesha, WI, USA). Chipping straw increases substrate density, as a reduction in particle dimension increases packing density. The density of the straw increased from 121.69 kg/m<sup>3</sup> before chipping to 177.28 kg/m<sup>3</sup> after. The chipper produced particles with varying lengths, including microparticles. Due to the dispersal of particles into the environment, 17.93% and 21.62% of the straw weight was lost during the chipping process for the first and second batch, respectively. Both batches were chipped at different times of the year (January and June). The

difference in straw weight loss likely results from the divergent environmental conditions (i.e., mostly humidity and wind) during chipping.

### 2.2.2. Sieving

Since the chipper produced particles of variable dimensions, the chipped particles were sieved to control the particle size. Two different particle size sets were produced for this research project to compare their effect on mycelial growth speed and mechanical performance. The first set of particles (S for small) was chipped and sieved through a 5.7 mm sieve to exclude large particles. The second set of particles (L for large) was similarly chipped and sieved through a 5.7 mm sieve. In addition, the particles were then sieved through a 1.5 mm sieve; only the material stuck on the sieve was kept and fine particles were removed. In conclusion, fine particles (passing through a 1.5 mm sieve) were kept in mixture S, while they were removed from mixture L. The two mixtures of particles (L and S) served as substrate to assess the effect of the fine particles on the mycelial growth and mechanical performance. The meshes of the 5.7 mm sieve (0.22-inch opening) and 1.5 mm sieve (0.06-inch opening) are respectively closest to standard US meshes No. 30 and No. 14. Additional details on sieving non-spherical objects and the sieving procedure are given in Appendix B.

For the first batch, the sieving process only resulted in 1.05% weight loss for the 5.7 mm sieve, and 0.51% for the 1.5 mm sieve. After the chipping and sieving processes of the first batch, 80.57% of the unchipped straw weight (100%) remained as the small mixture. Due to the removal of fine particles, only 42.73% of the unchipped straw weight remained as the large mixture. For the second batch, 66.13% and 38.52% of the unchipped straw weight resulted in the small and large mixtures, respectively. Both batches were sieved by a different individual, which may be the cause of the different percentages observed despite following the same procedure.

### 2.2.3. Quantifying Particle Sizes

As stated in Section 2.2.2., the dimensions of the particles passing through a sieve may be larger than the opening size of the sieve. Therefore, a technique was developed to quantify the mean size of the particles of mixtures S and L. The same chipping and sieving processes were performed on a known quantity of straw to produce both mixtures. Particles from each sieving step were then measured by placing them on a light table; a picture of them was taken and their 2D dimensions were extracted using an image analysis algorithm. This process is described in detail in Appendix C. For each set of particle sizes, this entire process was repeated five times with new particles from the same mixture.

### 2.2.4. Sterilization and Pasteurization

Due to inaccessibility of specific resources (e.g., pasteurization and inoculation equipment and workforce) under COVID-19 regulations, the first batch of substrate was sterilized and the second was pasteurized. For the first batch, the dry chipped and sieved substrate was sterilized in 10 autoclaving bags weighing around 2422 ± 109 g for mixture L and 1918 ± 152 g for mixture S. Each bag was sterilized for 30 min at 121 ◦C and 16 psi inside an autoclave at the Biology Department of The University of Akron. Autoclaved bags remained sealed until inoculation to reduce the entry of contaminants. For the second batch, both mixtures were pasteurized one after the other at Valley City Fungi, OH, USA. Mixture L was pasteurized first to avoid the transfer of fine particles from mixture S to L. The pasteurization process is described in Appendix D. Both mixtures were finally placed into sealed bags to avoid contamination in transport and following manipulations.

### 2.2.5. Mold Preparation

The molds into which the inoculated substrate was placed for mycelium growth were built based on the ASTM testing requirements. Due to the absence of ASTM standards specific to mycelium-based materials, various related standards had to be analyzed. Based

on their similarity with mycelium-based physical properties and previous research studies, ASTM D2166/D2166M-13 was used for compression testing and ASTM D1037-12 for bending testing [8,9,54,55]. According to these standards, cylindrical molds (i.e., PVC pipes) were built for specimens to be tested in compression and rectangular molds (i.e., wooden frames) for those to be tested in bending.

For compression testing, the targeted dimensions were 7 cm in diameter and 14 cm in height (i.e., height-to-diameter ratio of 2:1) as described in ASTM D2166/D2166M-13 [55]. However, it was anticipated that specimens would shrink during the drying process. To anticipate shrinkage and test specimens following the dimensions specified by the ASTM standard, specimens were made 8% larger in diameter and 20% larger in height based on estimations from the literature and preliminary experiments. Furthermore, it was expected that half of the specimens would be compacted to half of their height after the growth period (post-growth treatment detailed in Section 2.4). In this regard, specimens were grown inside 3 in. × 10 ft. PVC tubes (Charlotte PVC 40 Plain-End DWV Pipes, Charlotte Pipe and Foundry, Charlotte, NC, USA), with a diameter of 7.7 cm and a height of 17 or 34 cm.

For the bending test in ASTM D1037-12, materials thicker than 0.6 cm should have a width of 7.6 ± 0.1 cm and a length of (5.1 cm + 24 × the nominal thickness) [54]. Bending tests were performed with an Instron 5567 electrochemical testing system (Instron, Norwood, MA, USA), which can fit specimens up to a length of around 35.56 cm. Therefore, the specimens' dimensions were selected to be as follows: a length of 32.66 cm, a width of 7.60 cm, and a thickness of 1.15 cm. The same shrinkage percentages as per compression testing specimens were used to produce specimens 8% larger in width and length and 20% thicker. To optimize space and material, each mold was built to contain six specimens side by side. Half of the specimens were also grown twice as thick to prepare for compaction to half of their thickness. These molds were made of <sup>1</sup> 2 in. medium-density fiberboard (MDF) boards (Home Depot, Atlanta, GA, USA) to grow a large mycelium-based specimen measuring 35.5 cm in width, 50 cm in length, and 1.4 or 2.8 cm in thickness. A layer of plastic made from polyethylene resin (Home Depot, USA) was laid inside the mold to keep the mycelium from growing on and sticking to the wooden mold. Additional rectangular molds measuring 49.2 cm in width, 55 cm in length, and 1.4 or 2.8 cm in thickness were produced to grow leftover material.
