*Article* **Mycelium-Based Composite Graded Materials: Assessing the Effects of Time and Substrate Mixture on Mechanical Properties**

**Ali Ghazvinian \* and Benay Gürsoy**

Department of Architecture, Penn State University, University Park, PA 16802, USA; bug61@psu.edu **\*** Correspondence: axg1370@psu.edu; Tel.: +1-(618)-425-5170

**Abstract:** Mycelium-based composites (MBC) are biodegradable, lightweight, and regenerative materials. Mycelium is the vegetative root of fungi through which they decompose organic matter. The proper treatment of the decomposition process results in MBC. MBC have been used in different industries to substitute common materials to address several challenges such as limited resources and large landfill waste after the lifecycle. One of the industries which started using this material is the architecture, engineering, and construction (AEC) industry. Therefore, scholars have made several efforts to introduce this material to the building industry. The cultivation process of MBC includes multiple parameters that affect the material properties of the outcome. In this paper, as a part of a larger research on defining a framework to use MBC as a structural material in the building industry, we defined different grades of MBC to address various functions. Furthermore, we tested the role of substrate mixture and the cultivation time on the mechanical behavior of the material. Our tests show a direct relationship between the density of the substrate and the mechanical strength. At the same time, there is a reverse relation between the cultivation time and the material mechanical performance.

**Keywords:** mycelium-based composites; compressive structures; compressive strength; digital image correlation; masonry

### **1. Introduction**

The architecture, engineering, and construction (AEC) industry consumes half of the mineral resources and contributes the most to landfill waste [1]. Therefore, there is a need for alternative construction materials and greener energy resources to reduce the AEC industry's global greenhouse gas emissions and landfill waste. A circular approach must replace the current linear approach of extract-produce-use-dump. This approach emphasizes reuse, remanufacturing, refurbishment, repair, and upgrading of materials and utilization of solar, wind, biomass, and waste-derived energy during the product's life cycle. [2]. One possible path to conform with circular economies is through the use of bio-based materials as alternatives to conventional materials in construction [3,4]. These materials can be produced using waste as one of their initial ingredients and can become reusable, recyclable, or compostable at the end of their lifecycles.

Due to the rapid population growth worldwide, the global demand for food and agricultural wastes and byproducts has increased [5]. Besides, this growing population needs affordable habitat. Since the traditional ways of dumping agricultural waste into landfills or burning them impact global warming [6], converting agricultural waste into building components seems an optimal solution for these problems. Various bio-based materials are studied in this context [7]. The development of these materials can be costly, time-consuming, and inefficient due to the problematic methods of processing and functionalization [8], although they have a multitude of advantages. One of these materials is mycelium-based composites (MBC). MBC is manufactured using a low-energy and natural process that sequesters carbon and uses waste as the input. There are several applications of mycelium-based matter and fungal biotechnologies [9]. This research focuses on using MBC as load-bearing masonry components in construction.

**Citation:** Ghazvinian, A.; Gürsoy, B. Mycelium-Based Composite Graded Materials: Assessing the Effects of Time and Substrate Mixture on Mechanical Properties. *Biomimetics* **2022**, *7*, 48. https://doi.org/10.3390/ biomimetics7020048

Academic Editors: Andrew Adamatzky, Phil Ayres and Han A.B. Wösten

Received: 15 March 2022 Accepted: 16 April 2022 Published: 19 April 2022

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**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/).

### **2. Background 2. Background**

Mycelium is the vegetative root of fungi. Mycelium has a long, branching, and filamentous structure called hyphae that secrete enzymes to break down the biopolymers into simpler bodies of digestible carbon-based nutrients. The outcome of this process is an organic colony of hyphae. The organic matter bounds with this hyphal structure and forms fungal skin during this process. When this process is ceased through drying or heating, the incomplete process results in MBC. This composite material is made of the substrate as the filler and the hyphal mycelium as the binder. Without the hyphal binder, the substrate works as an inconsistent mass of particles and shows negligible mechanical performance. This bio-based composite can be shaped to produce panels, bricks, or various objects [10]. The properties of MBC depend on various cultivation parameters, such as the fungal species, substrates, growth conditions, processing of material, and additives [5,11]. This dependence on the controllable parameters enables MBC to meet specific application requirements [5]. Among these applications are acoustic insulation [12,13], thermal insulation [14–17], packaging [18–21], fire retardants [22–24], and structural building components [11,25–35]. Mycelium is the vegetative root of fungi. Mycelium has a long, branching, and filamentous structure called hyphae that secrete enzymes to break down the biopolymers into simpler bodies of digestible carbon-based nutrients. The outcome of this process is an organic colony of hyphae. The organic matter bounds with this hyphal structure and forms fungal skin during this process. When this process is ceased through drying or heating, the incomplete process results in MBC. This composite material is made of the substrate as the filler and the hyphal mycelium as the binder. Without the hyphal binder, the substrate works as an inconsistent mass of particles and shows negligible mechanical performance. This bio-based composite can be shaped to produce panels, bricks, or various objects [10]. The properties of MBC depend on various cultivation parameters, such as the fungal species, substrates, growth conditions, processing of material, and additives [5,11]. This dependence on the controllable parameters enables MBC to meet specific application requirements [5]. Among these applications are acoustic insulation [12,13], thermal insulation [14–17], packaging [18–21], fire retardants [22–24], and structural building components [11,25–35].

time-consuming, and inefficient due to the problematic methods of processing and functionalization [8], although they have a multitude of advantages. One of these materials is mycelium-based composites (MBC). MBC is manufactured using a low-energy and natural process that sequesters carbon and uses waste as the input. There are several applications of mycelium-based matter and fungal biotechnologies [9]. This research focuses on

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 2 of 15

using MBC as load-bearing masonry components in construction.

Scholars are making various efforts to make MBC meet the performance requirements of the AEC industry. One approach is to enhance the material properties of MBC by investigating the cultivation parameters. Another approach is to develop novel design and fabrication techniques around the specific material properties of MBC via geometry and form optimizations [32]. This paper focuses on the former approach. Scholars are making various efforts to make MBC meet the performance requirements of the AEC industry. One approach is to enhance the material properties of MBC by investigating the cultivation parameters. Another approach is to develop novel design and fabrication techniques around the specific material properties of MBC via geometry and form optimizations [32]. This paper focuses on the former approach.

### *2.1. The Cultivation Process of MBC 2.1. The Cultivation Process of MBC*

The cultivation process of MBC has three major phases: inoculation, growth, and ceasing. Figure 1 illustrates the cultivation process of MBC. The cultivation process of MBC has three major phases: inoculation, growth, and ceasing. Figure 1 illustrates the cultivation process of MBC.

**Figure 1.** MBC cultivation process. **Figure 1.** MBC cultivation process.

The substrate mixture is first prepared with organic matter as the filler, optional supplements that provide additional carbon and nitrogen, and water in the inoculation phase. This mixture is then pasteurized or sterilized to eliminate any other living organism that can pose a threat to mycelium growth. The substrate mixture is typically placed within autoclavable bags and heated in autoclave machines for less than an hour at 121 °C. Alternative methods such as the use of herbal remedies [15] and heating in lower tempera-The substrate mixture is first prepared with organic matter as the filler, optional supplements that provide additional carbon and nitrogen, and water in the inoculation phase. This mixture is then pasteurized or sterilized to eliminate any other living organism that can pose a threat to mycelium growth. The substrate mixture is typically placed within autoclavable bags and heated in autoclave machines for less than an hour at 121 ◦C. Alternative methods such as the use of herbal remedies [15] and heating in lower temperatures for longer durations are also available. Once the sterilization is complete, the substrate mixture is cooled down to room temperature, and the fungal spawns are added. The ingredients of the substrate mixture and the fungal species used in inoculation affect the material properties of MBC [36,37]. The second phase of the MBC cultivation process, the growth, starts in the autoclavable bags. Depending on the application, growth can continue within formworks [38,39]. Some studies explored the extrusion of the substrate mixtures through additive manufacturing [40–48]. Some of the parameters that affect the resulting material properties in this phase are the duration of growth, environmental conditions

such as temperature and relative humidity, and CO<sup>2</sup> concentration [49,50]. The third phase, ceasing, also influences the resulting MBC. In this phase, mycelial growth is stopped by either drying or heating the colonized substrate mixture. Researchers have also explored experimental processes such as rubbing herbal oils and hot or cold pressing the composite. The method of ceasing and parameters associated with the chosen method (for example, pressing temperature) [51] can alter the material properties of MBC.

### *2.2. Role of Cultivation Parameters on the Mechanical Behavior of MBC*

Various scholars have studied the mechanical behavior of MBC, specifically compressive strength. The consensus is that the mechanical behavior of MBC is comparable to that of synthetic foams, with room for enhancement [10]. MBC made of low-weight substrate mixtures have similar compressive strength to polystyrene foams and are weaker than polyurethane and phenolic formaldehyde resins [5].

Among the various research that explore the role of cultivation parameters on the outcome, Holt et al. (2012) [20] studied the substrate mixtures of six different cotton plant biomass. Yang et al. (2017) [7] experimented with the degree of compaction of substrates within formworks. They also tested the role of the duration of cultivation (two and six weeks) on the outcome. Their results show that the densely packed samples have higher compressive strength and elastic moduli. In comparison, the longer duration of cultivation results in better compressive strength and lower elastic moduli. Islam et al. (2018) [52] defined three sizes: small (from 0.4 to 0.9 mm), medium (from 0.9 to 1.7 mm), and large (from 1.7 to 6.7 mm) fillers (such as sawdust), and a mixture of these three to study the effects of filler size on compressive strength. They reported that the mechanical behavior of MBC is not affected by the filler size. On the contrary, the experiments by Elsacker et al. (2019) [53] show that fiber size is more influential on the mechanical strength than the type of fibrous substrate used. Except for the dust material that yielded poor growth and mechanical properties, the more chopped material resulted in better strength. In conformation with Yang et al. (2017) [7], their experiments showed that densely packed substrates had better mechanical properties than loose fibers.

Attias et al. (2019) [54] experimented with three different spawns and two growing protocols. They used *Colorius*, *Trametes*, and *Ganoderma* species and cultivated them with a 7-day difference in incubation time to establish their final experiments on the suitability of these conditions. They continued their study in Attias et al. (2020) [25] and reported better mechanical behavior for the samples cultivated with *Ganoderma* species. They also reported a reverse relation between the mycelium colonization and compressive strength, suggesting that shorter incubation periods restrict the organic matter digestion and preserve the mechanical characteristics of the substrate mixture. On the other hand, longer incubation times change the material content of the digestible substrate more and weaken the produced MBC. Bruscato et al. (2019) [55] utilized three different species of *Pycnoporus sanguineus*, *Pleurotus albidus*, and *Lentinus velutinus* for cultivation with sawdust and wheat bran. They found *L. velutinus* to be resulting in weaker composites because of the way mycelium colonizes during the growth. They suggest that the colonization of this species is more accentuated around the interstices of the mixture fillers than the overall agglomerate, which is different from the other two species, and that this is the reason for lower mechanical strength.

Appels et al. (2019) [50] Studied the role of different species, substrates, and pressing conditions on material behavior. They tested the bending capacity of MBC in their studies, with *T. multicolor* and *P. ostreatus* growing on rapeseed straw and beech sawdust. They also used three different conditions for ceasing: non-pressed, cold-pressed, and heat-pressed. Their most important result is the direct relation of mechanical strength and elastic moduli with the pressing, mainly through hot-pressing. They reported that heat-pressing shifts MBC performance from foam-like to wood-like. They also explained that colonization of mycelium occurs better at the outer parts of the substrates than the cores, emphasizing the importance of forcing air through the center of the substrate. Additional research on the

optimal temperatures for hot-pressing the substrates reveals that lower temperatures result in weaker materials, and higher temperatures may burn the materials [51].

Ghazvinian et al. (2019) [28] studied the role of supplements on two different substrates with *P. ostreatus*. Their results show slightly stronger materials with 7% wheat bran in the inoculation phase. There is also a considerable difference between MBC cultivated with oak sawdust and wheat straw. Ongpeng et al. (2020) [33] utilized clay, rice bran, and sawdust mixed with different waste materials to make MBC bricks and tested them to compare with masonry minimum limits. They also used the compressed substrates without mycelium to study the role of mycelium as the binding agent in these bricks. For the clay samples, the mycelium content was not modifying the characteristics, while for the other samples, mycelium bound the substrates, which resulted in stronger materials. Besides, all the mycelium-based bricks passed the minimum compressive strength for masonry bricks. One other important aspect studied by Zimele et al. (2020) [27] is the biodegradability of this material after use. Compared to hemp magnesium oxychloride concrete and cemented wood wool panel, two other bio-based materials, MBC showed quadruple biodegradability. This biodegradability is a testimony of the circularity of MBC when used in the AEC industry. An LCA analysis of MBC bricks on the lab and industrial scale shows reductions in most impact categories. Biodegradability might reduce the AEC industry's environmental footprint if conventional building materials can be substituted with MBC [56].

In a more recent study, Elsacker et al. (2021) [57] investigated the addition of other organisms, such as bacterial cellulose to *T. versicolor* inoculated on hemp-based substrates to make particleboards. They found the enhancing role of bacterial cellulose in improving internal bonding.

### **3. Materials and Methods**

This paper studied the effects of three different MBC cultivation parameters on compressive strength. The first parameter studied is the substrate mixture type. We created various mixtures by combining particle-based (i.e., sawdust) and fibrous (i.e., straw) materials. The other two parameters we studied are related to the duration of cultivation. The entire and partial cultivation time in bags and formworks has been investigated.

As mentioned before, there are three primary phases in the cultivation process of MBC. Figure 2 illustrates the materials and methods employed in these phases as part of this research. *Biomimetics* **2022**, *7*, x FOR PEER REVIEW 5 of 15

### **Figure 2.** Details of MBC cultivation. **Figure 2.** Details of MBC cultivation.

### *3.1. Substrate Mixtures Preparation 3.1. Substrate Mixtures Preparation*

Five different substrate mixtures are created for the experiment based on the sawdust to straw ratios. Oakwood pellets (Atlanta, GA, USA) and wheat straws (chopped, 3 cm long) are the base materials used for the mixtures. The various mixtures have straw and sawdust ratios of 1 to 1, 2, 3, 7, and one with sawdust only, as shown in Table 1. In addition, unbleached whole wheat flour (Bentonville, AR, USA) has been added to the mix-Five different substrate mixtures are created for the experiment based on the sawdust to straw ratios. Oakwood pellets (Atlanta, GA, USA) and wheat straws (chopped, 3 cm long) are the base materials used for the mixtures. The various mixtures have straw and sawdust ratios of 1 to 1, 2, 3, 7, and one with sawdust only, as shown in Table 1. In addition, unbleached whole wheat flour (Bentonville, AR, USA) has been added to the mixtures by

tures by 7% of the dry weight to enhance the growth process. The water content of the mixtures has been controlled to stay between 65% to 70%, following the best practices for

A 1 0 65–70% 7% DW 7% DW B 1 7 65–70% 7% DW 7% DW C 1 3 65–70% 7% DW 7% DW D 1 2 65–70% 7% DW 7% DW E 1 1 65–70% 7% DW 7% DW

The five substrate mixtures have been hand-mixed for 120 s to distribute the ingredients and water evenly. They were then moved to autoclavable bags (Impresa Mushroom Growing Bags) for sterilization and test paper bags for humidity check. Bags were sterilized in the autoclave machine at 121 °C temperature for 40 min and then removed from

The fungal spawns of *Pleurotus ostreatus* were purchased from local suppliers (Lambert Spawn, Coatesville, PA, USA). Oyster mushroom spawn has been used because 1) it is widely available locally, and 2) satisfactory results have been obtained with similar genera according to the literature [8]. Sterilized substrates were inoculated with the spawns by 7% of the dry weight in a sterilized environment. The bags were then placed in a growth room with environmental control. The temperature was set to 21 °C, and the rel-

To study the effects of different durations of growth on the mechanical behavior of MBC, three different durations of growth for 5, 6, and 7 weeks were selected, regarding the best results from the literature [7]. The growth process of MBC has been divided into two phases: within bags and formworks. To compare the partial growth of MBC in bags and formworks, the substrate mixtures have been placed in formworks at different times.

the autoclave machine and left to cool down (overnight, at room temperature).

ative humidity to 95%. The room was kept dark to help with the growth process.

**Water Content**  **Wheat Flour Content** 

*Fungal* **sp. Content** 

**Straw Ratio** 

**Table 1.** Characteristics of different mixtures.

**Ratio** 

**Mixture Sawdust** 

DW = dry weight of the mixture.

*3.2. Cultivation of Materials* 

7% of the dry weight to enhance the growth process. The water content of the mixtures has been controlled to stay between 65% to 70%, following the best practices for *Pleurotus ostreatus* mushroom cultivation.

**Table 1.** Characteristics of different mixtures.


DW = dry weight of the mixture.

The five substrate mixtures have been hand-mixed for 120 s to distribute the ingredients and water evenly. They were then moved to autoclavable bags (Impresa Mushroom Growing Bags) for sterilization and test paper bags for humidity check. Bags were sterilized in the autoclave machine at 121 ◦C temperature for 40 min and then removed from the autoclave machine and left to cool down (overnight, at room temperature).
