**1. Introduction**

In recent years, the exciting characteristics of filamentous fungi did not go unnoticed in the context of biodegradable materials, providing a low-cost and environmentally sustainable solution compared to the production and life cycle of petroleum-based materials [1–7]. These composite materials are realized by growing the fungi into lignocellulosic fibers, thereby valorizing organic waste streams, and generating dense materials with a construction material application [8]. Typically, composites are composed of a matrix and a reinforcement. For mycelium materials, the matrix is mycelium, and the reinforcement is the natural fiber. Mycelium surrounds the fibers and maintains their relative position inside the material. The fibers, in turn, largely influence the mechanical and physical properties of the composite [1]. A key aspect of improving the mechanical and physical properties of mycelium materials is the implementation of organic or inorganic particles [9–11].

**Citation:** Elsacker, E.; De Laet, L.; Peeters, E. Functional Grading of Mycelium Materials with Inorganic Particles: The Effect of Nanoclay on the Biological, Chemical and Mechanical Properties. *Biomimetics* **2022**, *7*, 57. https://doi.org/10.3390/ biomimetics7020057

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

Received: 23 March 2022 Accepted: 3 May 2022 Published: 5 May 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/).

Due to the wide variety of reinforcement particles, mycelium nanocomposites can potentially be designed for specific functions and applications, such as fire resistance and mechanical improvement.

Recent studies of such hybridization using glass improved the fire performance of mycelium materials as a result of significantly higher silica (inflammable) concentrations and low combustible material content [11]. It takes almost six times as much time for mycelium materials incorporating 50 wt.% glass fines to flash over as it takes synthetic materials, such as extruded polystyrene insulation foam, and two times as much as particleboard [11]. This study suggests that mycelium materials are very economical and exhibit far better fire safety parameters than the traditional construction materials tested (extruded polystyrene foam and particleboard made from wood flakes and bonded with moisture-resistant synthetic resin) [11]. However, a major problem with incorporating high contents of inorganic matter in the substrate might be the biocompatibility with white-rot fungi. Minerals limit the growth of the hyphae over poor nutritional surfaces and can therefore influence the bond between mycelium and the lignocellulosic fibers that must hold the material together.

To maintain sufficient mycelial growth, glass fines that comprise primarily silica (SiO2) and up to 30 wt.% organic surface matter were used in the particular research by Jones et al. [11]. Very limited research has further investigated the integration of additives in mycelium materials, with the exception of a study that indicates that the compressive strength of mycelium materials containing sand or gravel aggregates and wood chips increases up to 300% [12]. A patent makes note of the addition of components such as silica, clay and perlite to the fibers to retain moisture or enhance the viscosity of the substrate [13]. Thus far, no other study has investigated the fabrication, growth methods and mechanical properties of mycelium materials that incorporate inorganic (nano)particles.

Yet, nanotechnology and nanomaterials have great potential to improve the properties of different materials. Nanoparticles, like nanoclay, are widely used in various industries and areas of research, such as computing, adhesives, textiles, pharmaceutical and automotive [14–16]. For example, the reinforcement of particleboard and plywood panels with nanoSiO2, nanoAl2O3, and nanoZnO was reported to significantly decrease formaldehyde emission [17,18]. During the past decades, rapid developments have occurred in the area of polymer/clay nanocomposites. Most early studies focused on synthetic polymer, such as polyamides [18], polyimides [19], methacrylates [20,21] or polystyrene [22]. Nanoclay also offers an improved dimensional stability in wood–plastic composites [23]. Moreover, for wood–plastic composites, it is reported that flexural strength, tensile strength and elongation and water absorption are improved by the addition of nanoclay; the most interesting properties are observed in specimens with 5% of nanoclay content [24]. This improvement is due to the formation of bonds between the hydroxyl groups of nanoclay and the wood flour components. The addition of clay nanoparticles to cotton-based polymer composites resulted in increased char yield, therefore rendering them flame retardant [25].

One of the most common nanoclay forms is montmorillonite with a particle thickness of 1 nm, crosswise 70 to 100 nm [26,27]. Montmorillonite clays have a layered structure, and each layer is constructed from tetrahedrally coordinated Si atoms fused onto an edge-shared octahedral plane of either Al(OH)<sup>3</sup> or Mg(OH)<sup>2</sup> [26]. The layers exhibit excellent mechanical properties parallel to the layer direction due to the nature of the bonding between these atoms [26]. The principle for using nanoclay is to separate not only clay aggregates, but also individual silicate layers in a polymer [26]. The choice for montmorillonite nanoparticles is mainly motivated by their wide availability and inexpensiveness [15,28]. The main advantage is that a minimal content (1–5 wt.%) of such additives can improve the reinforcement of the polymer matrix [15,29,30]. Moreover, several studies have shown that the resulting organic–inorganic hybrids possess tremendous improvement in tensile strength and modulus, gas permeability, heat distortion temperature, and flammability [31].

This paper focuses on the incorporation of nanoparticles and the development of intricate gradients in the material's arrangement. The goal of this work is to investigate the production of organic–inorganic hybrids and their material properties. The influence of nanoclay on the physical and mechanical properties of mycelium materials is studied. The hypothesis is that the hybridization of mycelium composites with nanoparticles improves mechanical properties as the nanoclay can reinforces the internal vessels of the lignocellulosic fibers. This is the first study undertaking a longitudinal analysis of nanoclay– mycelium hybrid materials by using different characterization methods, such as SEM, FTIR and mechanical testing. production of organic–inorganic hybrids and their material properties. The influence of nanoclay on the physical and mechanical properties of mycelium materials is studied. The hypothesis is that the hybridization of mycelium composites with nanoparticles improves mechanical properties as the nanoclay can reinforces the internal vessels of the lignocellulosic fibers. This is the first study undertaking a longitudinal analysis of nanoclay–mycelium hybrid materials by using different characterization methods, such as SEM, FTIR and mechanical testing.

This paper focuses on the incorporation of nanoparticles and the development of intricate gradients in the material's arrangement. The goal of this work is to investigate the

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 3 of 23

The methodological approach focusses on the fabrication of nanoclay-coated fibers, inoculated with mycelium as binder (Figure 1). The experimental design was developed gradually and then combined in this work. The effect of nanoclay particles on the fabrication process and properties was mapped by analyzing the surface colonization rate, microscopic structure, chemical changes and mechanical properties. The methodological approach focusses on the fabrication of nanoclay-coated fibers, inoculated with mycelium as binder (Figure 1). The experimental design was developed gradually and then combined in this work. The effect of nanoclay particles on the fabrication process and properties was mapped by analyzing the surface colonization rate, microscopic structure, chemical changes and mechanical properties.

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sites.

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

### *2.1. Fungal Species*

*Trametes versicolor* (M9912) spawn was purchased from Mycelia bvba (Veldeken 38A, 9850 Nevele, Belgium). The species were conserved on a grain mixture at 4 ◦C in a breathing Microsac 5 L bag (Sac O2 nv, Nevele, Belgium).

### *2.2. Materials*

Studies were performed on the following fiber types: 5–25 mm hemp fibers (Aniserco S.A, Groot-Bijgaarden, Belgium). The superfine powder Ventoux montmorillonite clay was obtained from EMSPAC (Mons, France) in an untreated state.
