Fungi in Mycelium-Based Composites: Usage and Recommendations

Abstract

Mycelium-Based Composites (MBCs) are innovative engineering materials made from lignocellulosic by-products bonded with fungal mycelium. While some performance characteristics of MBCs are inferior to those of currently used engineering materials, these composites nevertheless prove to be superior in ecological aspects. Improving the properties of MBCs may be achieved using an adequate substrate type, fungus species, and manufacturing technology. This article presents scientifically verified guiding principles for choosing a fungus species to obtain the desired effect. This aim was realized based on analyses of scientific articles concerning MBCs, mycological literature, and patent documents. Based on these analyses, over 70 fungi species used to manufacture MBC have been identified and the most commonly used combinations of fungi species-substrate-manufacturing technology are presented. The main result of this review was to demonstrate the characteristics of the fungi considered optimal in terms of the resulting engineering material properties. Thus, a list of the 11 main fungus characteristics that increase the effectiveness in the engineering material formation include: rapid hyphae growth, high virulence, dimitic or trimitic hyphal system, white rot decay type, high versatility in nutrition, high tolerance to a substrate, environmental parameters, susceptibility to readily controlled factors, easy to deactivate, saprophytic, non-mycotoxic, and capability to biosynthesize natural active substances. An additional analysis result is a list of the names of fungus species, the types of substrates used, the applications of the material produced, and the main findings reported in the scientific literature.

1. Introduction

Mycelium-Based Composites (MBC) consist of defragmented lignocellulosic particles bonded with dense chitinous mycelium. These innovative biomaterials show eco-friendly characteristics: waste materials usage, low energy demand during production, the production does not generate waste, and the products are readily recycled [1]. The performance properties of MBC are usually inferior to those of the materials used so far. However, their advantages are revealed in some areas, such as high acoustic attenuation, fire resistance, the absence of harmful synthetic chemical components [2,3,4], and advantages connected with aesthetics. In turn, the drawbacks of MBC, which need to be eliminated, include excessive hygroscopicity, low tensile strength, susceptibility to biological corrosion, and the need to deactivate the fungus. Improving the properties of this innovative material is the goal of many scientific and commercial endeavors [5,6]. Thus, the potential applications of MBC may be found in architecture [7,8], packaging [9], the automotive industry [10], as a furniture material, in art [8], and in manufacturing various chitin- and β-glucan-based flexible materials, such as foams or paper, as well as textile substitutes [11]. The scientific literature describes the biocomposites as pure mycelium bio-materials, consisting only of mycelial biomass, e.g., myco-leather, as a substitute for petrochemically produced and animal-based leather [12]. There are also concepts for the use of mycelium to grow monolithic buildings from the functionalized fungal substrate [13] and as self-repairing wearable electronics, using various fungus properties (memristors, oscillators, pressure, and optical and chemical sensors) [14]. The results of our own feasibility studies on different surface structures of MBC required in art and architecture uses are shown in Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5. In all cases, the Ganoderma lucidum was the binding agent, the substrates contained admixtures.

The practical difficulty in the production of MBC is connected with an appropriate selection of the fungi species, substrate, and production technology. The produced MBC should be technologically feasible, profitable, provide expected physical properties in the entire volume, and be acceptable for humans. Difficulties in the appropriate selection of the fungi species result from the large variety of fungus species and available substrates, problems associated with combining a specific fungi species with a specific substrate in terms of mycelium growth, and inactivation parameters and different requirements for biocomposites [15]. Many fungi form mycotoxins, attract insects, or become invasive species [16]. The factors that may cause the biocomposite properties to differ from expectations are shown in Figure 6.

Current mycelium-based engineering materials are innovative with many advantages but have some disadvantages. The selection of an appropriate species of fungus for the substrate or the use of species that have not been used so far could eliminate these disadvantages. This choice could be adequately supported by the quantitative analysis of the fungi species described in the scientific documents to create a biocomposite with expected properties. There are no review articles comparing the intensity of studies of individual species of fungi and analyzing the most common combinations of fungus species–substrate. As is known, there are millions of fungi species, but only a few dozen are used to produce biomaterials. Furthermore, no general guidelines have been formulated in the literature to find new species to create mycelium-based materials. This review fills the research gaps in this regard. The present review is expected to contribute to discovering optimal combinations of species of fungus–substrate based on current research. The review also aims to propose scientifically justified criteria to be met by a newly used fungus specie to make available the optimal production of Mycelium-Based Composites.

2. Fungus Species in the Scientific Literature

Fungi are a group of organisms classified into separate kingdom. Defining characteristics include the presence of chitin in their cell walls, heterotrophism, and cosmopolitism [17]. The total number of fungi species is not known. To date, as few as approx. 150,000 species [18] have been described from the estimated number of 1.5 million up to 5.1 million species [19]. Commonly used databases containing updated information on fungi are Species Fungorum (www.speciesfungorum.org, Centre for Agriculture and Bioscience International (CABI), Wallingford, Oxfordshire, UK, accessed on 8 August 2022), and MycoBank (www.mycobank.org, Westerdijk Fungal Biodiversity Institute, Utrecht, Belgium, accessed on 8 August 2022). Fungi are classified using a phylogenetic tree [20], which orders these organisms into hierarchic groups. In nature, fungi are associated with other organisms through symbiosis and commensalism as parasites or reducers. Considering these dependencies, fungi are classified as harmful (causing disease or depreciation) or beneficial organisms (mycorrhizas).

From 2012 to 2022, almost 100 original articles were published [21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113], presenting almost 70 species of fungi used to produce Mycelium-Based Composites; these species are listed in

Table 1. Fungus species in scientific publications related to Mycelium-Based Composites.

Decay Type	Fungus Species and Literature References
Brown rot	Fomitopsispinicola [63]; Gloeophyllum sepiarium [63]; Laetiporus sulphureus [63]; Phaeolus schweinitzii [63];
Soft rot	Acremonium sp. [96]; Fusarium oxysporum [94]; Oudemansiella radicata [96]; Trichoderma asperellum [77], T. asperellum [77];
White rot	Agaricus bisporus [59,77,87]; Auricularia polytricha [81]; Ceriporia lacerata [30]; Colorius sp. [61]; Cyclocybe aegerita (specified as Aaegerita agrocibe) [36]; Coprinopsis cinerea [62]; Daedaleopsis confragosa [44]; Flammulina velutipes [77]; Fomes fomentarius [38,83,85,87,108]; Fomitopsis pinicola [63]; “Ganoderma sp.” [21,41,44,61,68,77,110], G. applanatum [87], G. boninense [75], G. lucidum [22,25,31,32,41,69,70,72,73,74,77,79,80,81,82,83,89,100,102,106,109,112], G. resinaceum [44,49,86,93,101], G. sessile [61,110]; Inonotus obliquus [67]; Irpex lacteus [42]; Kuehneromyces mutabilis [77]; Laetiporus sulphureus [63]; Lentinula edodes [32,64,77]; Lentinus velutinus [67]; Megasporaporia minor [49]; Oxyporus latermarginatus [49]; Phaeolus schweinitzii [63]; Piptoporus betulinus [63]; “Pleurotus sp.” [33], P. albidus [67], P. citrinopileatus [74], P. djamor [62], P. eryngii [74], P. ostreatus [26,29,32,35,36,37,38,41,46,56,57,63,74,77,81,82,84,88,90,91,94,96,99,103,105,106], P. ostraceus florida [77], P. ostraceus sajorcaju caju [77], P. salmoneo-stramineus [36]; Polyporus arcularius [63], P. brumalis [59], P. pulmonarius [36]; Pycnoporus sanguineus [67,83,92]; Trametes sp. [53,61]; Trametes hirsuta [83,99,104], T. multicolor [46,57,110], T. pubescens [63], T. suaveolens [63], T. versicolor [29,36,44,50,59,65,66,78,86,87,101,110], Trichaptum abietinu [63]; Schizophyllum commune [46,48,53,57]; “white-rot saprotrophic fungi, endemic to Alaska” [42]
Probably white rot	Specified as “phylum Basidiomycetes” [24,40,51]

. The growth conditions used in the cited studies, inactivation methods, and the results achieved are listed in Appendix A.

As can be seen from Table 1, most studies on Mycelium-Based Composites concern white rot fungi. Some scientific publications describe the results of comparative analyses for various fungus species. The visualization of the frequency of research and the frequency of scientific comparisons of different species of fungi is given in Figure 7. The size of the circle shows the popularity of the fungus species in the scientific literature and the lines indicate the most frequently used comparisons of the fungus species in scientific publications.

It results from Figure 7 that two fungus, Pleurotus ostreatus (mentioned in 22 documents) and Ganoderma lucidum (mentioned in 20 documents), are most frequently mentioned in scientific publications. Another commonly used species is Trametes versicolor (10 times). P. ostreatus and G. lucidum are the most frequently compared species. All these species cause white rot. A detailed list of fungus species, substrates, technological parameters, research aims, and main findings based on almost 100 original articles is given in Appendix A.

3. Fungus Species in Patent Documents

There are several hundred patent documents concerning Mycelium-Based Materials [8]. The oldest document was filed at the United States Patent and Trademark Office on 12 December 2007 [114]. Patent documents mention several dozen fungus species. They are listed in

Table 2. Fungus species in patent documents.

Division	Order	Fungus Species	No. of Patent Documents	Ref. to the Oldest Patent Document
Basidiomycota	Agaricales	Agaricus sp.	19	[114]
		Agrocybe sp./Agrocybe aegerita/A. brasiliensis	6/13/10	[115]/[116]/[114]
		Coprinus comatus	24	[114]
		Flammulina velutipes	13	[114]
		Hypsizygus sp. (as “Hypsizygous sp.”)	2	[115]
		Hypholoma capnoides/H. sublaterium	11/10	[114]/[114]
		Lentinula edodes	17	[116]
		Macrolepiota procera	11	[114]
		Omphalotus sp.	2	[115]
		Pleurotus djamor/P. eryngii/P. ostreatus var. columbines/P. ostreatus	16/15/13/45	[116]/[116]/[116]/[114]
		Schizophyllum sp.	14	[117]
	Hymenochaetales	Inonotus obliquus	24	[114]
	Polyporales	Ceriporiopsis sp.	2	[115]
		Fomes fomentarius	13	[118]
		Ganoderma appalantum/G. lucidum (also as “lucidem”)/G. oregonense/G. resinaceum, G. tsugae	3/42/23/11/27	[118]/[118]/[116]/[119]/[114]
		Grifola frondosa	15	[116]
		Laetiporus sp.	2	[115]
		Phanerochaete sp.	7	[117]
		Piptoporous betulina (as “betulinus”)	8	[120]
		Polyporellus sp.	2	[115]
		Polyporus avleolaris/P. mylittae/P. squamosus	3/3/8	[118]/[118]/[118]
		Pycnoporus cinnabarinus	4	[121]
		Trametes versicolor	19	[120]
	Russulales	Hericium erinaceus	4	[122]
Ascomycota	Pezizales	Morchella angusticeps	11	[114]
	Xylariales	Xylaria polymorpha/X. hypoxylon/X. filiformis/X. longipes	4/4/3/1	[117]/[117]/[117]/[123]
Zygomycota	n.d.	n.d.	1	[124]

, giving the specie names, the number of patent documents specifying a given species or family, and references to the first patent document in which this species was mentioned.

It is worth highlighting that patent documents do not provide detailed knowledge concerning the effectiveness of the mentioned fungus species, as is typically seen in scientific documents. Admittedly, all patent documents disclose the essence of the invention, but conversely, providing too much information is clearly against the interests of the owner of the invention. For this reason, patent documents contain a minimum of knowledge and simultaneously make producing a similar solution as complicated as possible [125].

4. Substrate Type Analysis

Substrates for the production of Mycelium-Based Composites originate from three sources: agricultural by-products, industrial waste, and post-consumer waste. In terms of their composition, these substrates can be divided into annual plants, softwood, and hardwood. Common bulk substrates include several components: wood chips or sawdust, mulched straws (wheat, rice, and others), chopped corncobs, recycled paper, nut and seed hulls or meal, coffee pulp or grounds, and brewer’s grain. An ideal substrate contains enough nitrogen and carbohydrates for rapid fungal mycelium growth. Various substrates are compared in scientific analyses or combined as mixtures in different proportions. Combinations of various substrates in scientific experiments, described in 85 scientific publications [21,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,40,41,42,45,46,49,50,51,52,53,57,58,59,60,61,62,63,64,65,66,67,68,70,72,73,74,75,76,77,78,79,80,81,82,83,84,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112], are presented in Figure 8. The size of the circle shows the popularity of the substrate and the lines indicate the most frequently used comparisons of substrates in scientific publications.

It can be seen from Figure 8 that pine wood is the wood material most commonly used as a substrate. In turn, fibrous plants with high cellulose contents, i.e., hemp, cotton, and wheat straw, were the most frequently used among annual plants. In terms of the expected strength, the substrate materials should be long (strand type), thus wood chips and straw are preferred. Regarding technological requirements, an abundant and uniform supply of substrate materials is needed, while low acquisition cost is essential in the production economy.

All these raw materials are lignocellulose materials. They are composed of 30–50% cellulose, 15–30% lignin, and 25–35% hemicelluloses as well as non-structural substances (e.g., pectins, waxes, pigments, tannins, lipids, and minerals). Their composition is dependent on their origin and species [126,127,128]. Cellulose is the primary structural component of all plant fibers [129]. It is a natural polymer. Cellulose molecules consist of glucose units linked together in long chains (β-1,4 glycoside linkages join the repeating units of D-anhydro glucose C₆H₁₁O₅), which in turn are linked together in bundles called microfibrils. This principal component provides them with strength, stiffness, and stability. Hemicelluloses are polysaccharides bonded together in relatively short, branching chains. They are closely associated with cellulose microfibrils, embedding cellulose in a matrix. Hemicelluloses are highly hydrophilic. The molecular weights of hemicelluloses are lower than that of cellulose. Lignin is a complex aromatic hydrocarbon polymer that imparts rigidity to plants. Without lignin, plants could not attain great heights. Lignin is a three-dimensional polymer with an amorphous structure and a high molecular weight, and it is less polar than cellulose. It serves as a chemical adhesive within and between fibers. Lignin acts primarily as a structural component by adding strength and rigidity to the cell walls. However, it also allows the transport of water and solutes through the vascular system of plants and provides physical barriers against invasions of phytopathogens and other environmental stresses. It consists of three basic phenylpropanoic monomers known as monolignols: p-coumaryl, coniferyl, and sinapyl alcohols [130].

When incorporated into the lignin polymer, the units that originated from the monolignols are called p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units, respectively. The amount of lignin varies according to the origin of the lignocellulosic starting material. At the same time, the proportion of different monolignols and chemical bonds in the lignin structure also depends on the lignocellulosic biomass, because these vary between hardwood, softwood, or grass. In softwoods, lignin is mainly composed of guaiacyl units linked by ether and carbon-carbon bonds, whereas in hardwoods, lignin has equal amounts of guaiacyl and syringyl units. The grass lignin is characterized by guaiacyl, syringyl, and hydroxyphenyl units.

Several methods are employed to sterilize the substrate, thereby rendering the substrate inert. This can be provided (1) by temperature, i.e., heat treatment, such as autoclaving and pasteurization, or (2) treatment with chemical or microbial agents (Appendix A). Sterilization in an autoclave is typically run at temperatures ranging from 115 to 121 °C for 15 to 120 min. In turn, the pasteurization is run in water at a temperature of 100 °C for approx. 100 min. Substrates may also be subjected to the action of a hydrogen peroxide solution at a concentration ranging from 0.3% to 10%.

The substrate has to contain the nutrients required for fungus growth to improve the growth rate and modify mechanical strength properties, which the mycelium matrix attains after growth. Simple sugars, such as glucose, are used as additives. The addition of glucose to the lignocellulose material results in the lesser degradation of holocellulose at the preliminary stage of degradation caused by fungi. Figure 9 illustrates lignocellulose substrates linked with various fungus species in original articles related to Mycelium-based Composites [21,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,40,41,42,45,46,49,50,51,52,53,57,58,59,60,61,62,63,64,65,66,67,68,70,72,73,74,75,76,77,78,79,80,81,82,83,84,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112]. As with Figure 7 and Figure 8, the size of the circle shows the popularity of the mushroom species or substrate, and the lines indicate the most common combinations of fungal species and substrates in scientific publications.

Substrates derived from both hardwood and softwood materials were typically combined with white rot fungi, i.e., T. versicolor and P. ostreatus. Additionally, composites based on fibrous plants were obtained mainly using white rot fungi T. versicolor, P. ostreatus, and G. lucidum. The critical observation is that the white rot fungi can degrade lignin in the plant cellwall by skipping cellulose, unlike the other wood degrading fungi. The following patterns have been found when studying lignin bioconversion by basidiomycetes: (1) the first stages include lignin demethoxylation and subsequent hydroxylation, which is accompanied by a decrease in the number of methoxy groups and an increase in hydroxyl groups; (2) then the αC–βC bond is broken with oxidation of the first hydroxyl to carboxyl group; and (3) the aromatic ring in lignin is broken [131].

Following mycelium growth, the resulting composite materials may be removed from molds and hot pressed, dried in an oven or air-dried to dehydrate the obtained material and neutralize the fungus. Consequently, fungi may no longer grow or spread while the composite material is rigidified. Hot pressing and oven drying are preferred treatment methods in industrial practice because they are the fastest dehydration methods. As a result of hot pressing, the material is consolidated and condensed, which results in higher values of mechanical strength properties.

A systematic review of applied MBC growth parameters is given in Appendix A.

5. Discussion: Fungus Species Recommendations

The main purpose of the literature review described in this article is to indicate attributes of an ideal fungus species to create mycelium-based materials. The 11 such attributes have been identified: (1) rapid growth of hyphae, (2) high virulence, (3) dimitic or trimitic hyphal structures, (4) white rot fungi, (5) high versatility in nutrition, (6) tolerance to a wide range of substrate parameters and environmental conditions, (7) susceptible to readily controlled ecological factors, (8) easy to deactivate, (9) saprophytic, (10) non-mycotoxic, and (11) having the ability to biosynthesis natural active substances.

(1–2) A Mycelium-based Composite (MBC) for engineering usage needs to exhibit isotropic physical properties, thus an optimal organism should bind the organic matrix into a composite with such properties. In this case, it is best to select an appropriate organism assuming the division proposed by Harper [132] into modular and autonomous organisms. Modular organisms develop through repeated iterations of modules, and such a repeatable structure facilitates the exploitation of a static resource (substrate) by an immobile organism (a fungus). Mycelium hyphae absorb nutrients serving the role of building components in the area where they are growing. A solution to the problem of nutrient depletion around hyphae is offered by the regrowth of hyphae from the depleted substrate zone at the simultaneous production of successive modules, i.e., hyphae located so that the zones are devoid of nutrients do not overlap. Harper showed a lack of mutual overgrowth of young hyphae. This mechanism ensures the rapid colonization of large substrate areas acting as a matrix in the biocomposite. Modular organisms may find food using two strategies: guerrilla and phalanx. Fungi degrading lignocellulose materials find food using the phalanx method. This phalanx growth type involves extensively branched hyphae facilitating colonization of such a substrate. This type of growth is observed in white rot fungi. Hyphae produce high local concentrations of extracellular enzymes and other chemical substances, preventing the colonization of the substrate by other organisms. This mechanism supports the axenic culture during biocomposite production. The hyphae’s anatomy and the modular structure ensures fungal survival in the case of mechanical damage to the mycelium. Internal organelles, such as Woronin’s bodies, can plug the septal pores to prevent cytoplasm loss from hyphae. Every single hypha may reproduce, forming another organism. This property makes it possible to obtain large amounts of the material used as an inoculum within a short time.

Hyphae can regrow from the substrate, facilitating their transition through the gas phase to penetrate new sites abundant in nutrients. This is because most currently known fungi are organisms living in the terrestrial environment with the predominance of the gas phase over the liquid phase. This property considerably facilitates the colonization of a loose lignocellulose material. As it results from the above, the fungal mycelium seems to be the most adequate for biocomposite production among all the organisms colonizing our planet. More details concerning the modular structure of mycelium may be found in a publication by Calile [133].

The rapid growth of hyphae, combined with the possibility to initiate the development of new mycelium by its fragment, makes it possible to obtain large amounts of inoculum within a short time. Increased inoculum density in the substrate results in a reduction in lag phase time, increased specific growth rate, improved maximum efficiency, and lowered substrate degradation. Jones et al. [134] were of the opinion that the optimum inoculation density is 10–32% inoculum to substrate ratio (by volume) depending on the used inoculum, whether in the liquid or solid form. In terms of the efficient formation of the biocomposite, it is desirable to minimize the lag phase and provide optimal environmental conditions and abundance of nutrients to maximize growth rate and efficiency and prevent the premature transition to the stationary growth phase.

An isotropic composite has to be manufactured under sterile conditions, which is required for the rapid and uniform colonization of the substrate in the axenic culture (monoculture). This increases the chance of obtaining a material exhibiting comparable properties over the entire material volume. In the case of incomplete substrate sterilization, the produced biocomposite may exhibit various physical properties differing from those assumed [135]. The colonization of dead wood by fungi under natural conditions takes the form of microbial succession. Wood is first colonized by rapidly growing more primitive microorganisms (e.g., mitosporic fungi), which are next replaced by higher fungi (white, brown, and grey rot fungi). This is not an absolute requirement, but it depends rather on the local conditions and present fungal strains. In the case of axenic cultures in mycelium-based composite formation, we need to consider the phenomenon of the succession of microorganisms colonizing the substrate. The division into three groups based on the colonization rate of all substances also needs to be remembered. Primary colonizers appear as the first microorganisms. A rapid growth rate characterizes them; they spread fast and degrade simple compounds. Secondary colonizers rely on primary colonizers, which partially degrade the organic matter before digestion of more complex compounds. Tertiary colonizers appear towards the end of the degradation process, taking advantage of the conditions created by primary and secondary colonizers. When the dependencies mentioned earlier are not considered, the substrate colonization rate by the fungus used to produce the biocomposite may be slower than initially assumed. This harms the economic aspect of biocomposite manufacture.

(3) When considering fungus species for producing biomaterials, the species producing leathery or woody fruiting bodies should be considered. They have a complex system of dimitic and trimitic hyphae. The function of fungal hyphae is to bind the biocomposite matrix. This is achieved most effectively by dimitic or trimitic hyphae, providing mycelium with better physical properties than the mycelium containing only generative hyphae (monomitic fungi). Apart from the generation of a branched network structure, the increased contact area with the composite matrix hyphae should contain adhesive substances, such as hydrophobins.

(4) Fungi used to produce biocomposites need to cause a simultaneous white rot of the substrate. Regarding MBC strength, the cellulose in the substrate must remain undegraded, therefore selective white rot is the preferred type of degradation during mycelial growth. The selection of a fungus causing this type of degradation prevents the defibration of wood during degradation even in a highly advanced process [136]. White rot fungi causes xylem defibration, which will provide a composite with poorer physical parameters. Fungi have to degrade lignin more effectively than holocellulose, thanks to which better physical properties of the substrate are maintained, compared to fungi causing brown or grey rot [137]. White rot fungi cause a uniform volumetric shrinkage of the isotropic substrate, observable only when the loss of substrate mass exceeds 40–50% [138]. It minimizes volumetric changes in the composite matrix during the production process. This is also reflected in the compressive strength of MBC, which is dependent on the substrate structure (matrix). In the case of fungi causing brown or grey rot, the volumetric changes of wood are anisotropic and found at a much earlier stage of degradation. Brown and grey rot fungi cause an adverse loss of holocellulose, so the composite matrix has much poorer physical parameters than the original parameters of wood.

(5) Optimal fungi for composite production must colonize and degrade many different lignocellulose materials and other waste generated by the agricultural, forestry, and food industries. Moreover, these fungus species should biodegrade various synthetic chemical substances providing a wider range of potential substrate types to manufacture composites. This makes it possible to use lignocellulose matrices contaminated with other substances.

(6) The properties of Mycelium-Based Composites are significantly affected by their production parameters, such as growth time and conditions, incubation temperature, the pH and moisture content of the substrate, access to light, and the material drying methods. These parameters vary for different fungal strains and used substrates. Manufacturing parameters may be modified to influence the properties of produced biocomposites. Incubation time depends on substrate volume and ranges from 5 to 42 days depending on the fungus species. The optimal incubation temperature ranges from 21 to 30 °C for different fungus species.

(7) Similarly, the substrate pH level for optimal growth in the case of various fungi ranges from 5 to 8, while humidity from 80 up to 100%. Because of biocomposite production, the used fungal species have to be readily maintained in the anamorphic stage, not producing fruiting bodies. This process may be controlled using CO₂ concentration, elevated temperature (30–35 °C), and lack of access to light. It results from the above that the fungus species should be thermophilic and tolerate the CO₂ content in the culture chamber atmosphere.

Preferential conditions for the production of a biomaterial with high mycelium density include a lack of light radiation, increased carbon dioxide concentration at a simultaneous reduced oxygen concentration, and elevated temperature (18–35 °C). Figure 10 presents parameters causing changes in the fungus development stage.

(8) Mycelium deactivation in MBCs may consist in heat denaturation or otherwise. The heat denaturation requires the element made from an MBC to be placed in a drier. To improve an economic efficiency of biocomposite production, the mycelium should be deactivated at the lowest possible temperature, e.g., 60 °C, or applying other safe and, at the same time economically viable methods, e.g., microwave radiation. This can facilitate the deactivation process and provide the deactivation of large-sized elements.

(9) Fungi used in biocomposite production have to be saprophytic, not parasitic, since the latter are frequently pathogenic. Using saprophytes to produce biocomposites will reduce health hazards for humans and other organisms, particularly homeothermic animals, in the case of an uncontrolled release of biomaterials to the natural environment. Such a situation may occur when no effective deactivation is performed following the culture process.

(10) An important feature of pathogenic fungi is connected with the synthesis of mycotoxins and microbial volatile organic compounds (MVOC). These substances of natural origin very often cause various diseases in humans.

(11) For this reason, it seems advisable to consider either medicinal effects or the neutral effect on the homeothermic organisms in the course of production and the use of biocomposites. This will reduce the risk during composite production and use in environmental protection aspects. It may even reduce manufacturing costs thanks to the production of biologically active substances, such as medications. Secondary fungal metabolites, which exhibit antimicrobial action, may be applied in materials used in the food industry. The biocomposite obtained using mycelium synthesizing active substances may have contact with food if the used organism is edible and free from toxic substances. Such properties may be found in edible mushrooms and fungi used in natural medicine to provide medicinal substances.

6. Summary and Conclusions

The most important reasons for using Mycelium-Based Biocomposites (MBC) include the management of by-products, the storage of carbon dioxide from the atmosphere, reduced need for petrochemicals in produced materials, and recyclability as well as interesting aesthetic features. Substrates for the manufacture of MBCs come from three primary sources: agricultural by-products, industrial waste, and post-consumer waste. In the case of substrates for industrial MBC production, it is vital to ensure their constant, abundant supply and availability. The functional properties of MBC are usually inferior to those of the materials used currently; however, in some areas, advantages of this innovative material need to be stressed, such as high acoustic attenuation, fire resistance, absence of chemicals, and, finally, aesthetic features, even though the latter is difficult to parameterize. The appropriate selection of the fungus species for the substrate is key to achieving the expected MBC properties. Millions of fungus species are still unknown to science, thus providing an excellent opportunity to identify fungi capable of producing MBCs with even better characteristics. Based on the analysis of many literature sources, 11 features were formulated to increase the effectiveness of fungi in the manufacture of MBC:

• Rapid linear growth of hyphae will facilitate the production of large amounts of inoculum in a short time and will contribute to the minimization of the biocomposite production time. Moreover, the substrate will not be excessively degraded by mycelium.
• High virulence. Fungi must be able to rapidly colonize the substrate before other microorganisms do. The aim is to obtain an axenic, uniform, dense fungal culture in the substrate. Thus, a biocomposite with isotropic physical properties is obtained.
• Hyphal structures. The hyphae of the fungus, which provide a lattice for biocomposites, should be dimitic or trimitic, thus producing mycelium with better strength properties than the mycelium containing only generative hyphae (monomitic fungi). For this reason, the mycelia of mushrooms with hard leathery or woody fruiting bodies need to be used because they form mainly dimitic and trimitic hyphae.
• White rot fungi. Fungi that cause white rot are preferred. These fungi degrade lignin in the cell walls of woody plants to a greater degree than they do with cellulose—thus, the composite matric has better physical parameters compared to the application of brown rot or grey rot fungi.
• High versatility in nutrition. The fungus used in MBC needs to grow on a wide range of lignocellulose materials and on many other materials, e.g., plastics. The availability of various substrates will reduce the manufacturing costs of materials.
• High tolerance to a wide range of substrate parameters and environmental conditions. Selected fungal specie should exhibit high tolerance to various environmental conditions, i.e., temperature and humidity, as well as the analogous parameters of the substrate, including non-uniform substrate moisture content and pH. This can simplify an MBC manufacturing technology.
• Fungi susceptible to readily controlled ecological factors, such as temperature, light intensity, carbon dioxide concentration, oxygen concentration, or other technological factors. These parameters may promote the rapid linear growth of hyphae while preventing the formation of fruiting bodies.
• Mycelium easy to deactivate. Mycelium in an MBC should be susceptible to deactivation using various methods. This will enable the production of large MBC elements and increase the human acceptance level of manufactured MBC products.
• Saprophytic fungi. Fungi for the production of MBC may not be facultative parasites, since otherwise, the produced biocomposite may be hazardous for humans.
• Non-mycotoxic fungi. The fungus should not synthesize harmful metabolites, e.g., mycotoxins or microbial volatile organic compounds (mVOC). Mycotoxins and mVOC may cause disease or even death in humans and other animals.
• The biosynthesis of natural active substances. Fungi preferred in the production of biocomposites might synthesize natural active substances. This will reduce production costs and provide biocomposites with unique properties.