Risk assessment of fungal materials

Sustainable fungal materials have a high potential to replace non-sustainable materials such as those used for packaging or as an alternative for leather and textile. The properties of fungal materials depend on the type of fungus and substrate, the growth conditions and post-treatment of the material. So far, fungal materials are mainly made with species from the phylum Basidiomycota, selected for the mechanical and physical properties they provide. However, for mycelium materials to be implemented in society on a large scale, selection of fungal species should also be based on a risk assessment of the potential to be pathogenic, form mycotoxins, attract insects, or become an invasive species. Moreover, production processes should be standardized to ensure reproducibility and safety of the product.


The need for sustainable materials
The world population is predicted to increase to 9.7 billion people in 2050 [1], thereby putting even more pressure on natural resources than is happening today. The materials used in industries are often non-sustainable and dominated by the linear economic model to make, use and dispose [2]. This linear approach is not sustainable and is already taken its toll on global ecosystems. Fungal materials have high potential to replace nonsustainable and/or polluting products and production processes. For instance, they could replace, at least in part, non-sustainable plastics, textiles, leather and construction materials. Petrochemical based plastics are widely used as packaging material. The amount of plastics produced globally amounts over 360 million metric tons per year [3] and has been growing annually by 8.4% in the period 1950-2015 [4]. Only 35% of the consumer plastics was recycled in Europe in 2020, 42% was incinerated for energy production, while 23% ended up in landfills [3]. Spreading of (micro-)plastics as litter or from landfills into the environment poses significant hazard to both terrestrial and marine ecosystems. Cotton production also impacts the environment by its high water demand and intense use of pesticides [5], while the leather industry uses chemicals such as chrome for tanning [6]. Finally, 4.2 giga tonnes of cement were produced worldwide in 2019, which coincided with a concerning amount of CO 2 emissions [7]. Clearly, we need to shift towards a sustainable economy. The use of fungal materials may offer a fundamental change in our current way of manufacturing because these materials are produced from waste streams. Moreover, the fungal materials can be recycled at their end of life to make new materials, to use as feed or fertilizer, or to improve soil structure [8,9]. In this review, we will discuss the risks of mycelium materials for workers, researchers, consumers and the natural environment due to the fungal life style and the production process of these materials. Based on this, we will make recommendations for their safe introduction on the market.

Fungal materials
The growing interest in fungal materials during the past decade is illustrated by the increase in the number of publications, patent applications and start-ups in this field. Moreover, established companies like Adidas and Hermès have opened their doors for fungal materials [10]. Mycelium composites and pure fungal materials are distinguished. In the former case, a network of hyphae binds substrate particles together, whereas pure mycelium materials consists solely of fungal biomass. Wood degrading basidiomycetes are mainly used to produce these mycelium materials but species from other phyla have also been explored [11] (Table 1).
Composite mycelium materials are usually made by growing the fungus in a substrate, often a low-cost organic waste stream. During colonization, the mycelium acts as a glue that binds the substrate particles together. The first step in the process of making a composite material is the selection of the species and substrate. Commonly used substrates are for instance hemp shives, different types of straw, and sawdust. The pasteurized or sterilized substrate is inoculated with colonized substrate from a previous batch or with spawn (i.e. a highly nutritious substrate like grains that is colonized by the fungus). Another approach is the use of mycelium that has been blended in water or medium. The inoculated substrate is grown in a mould for several days up to a month depending on species, substrate, sample dimension and growth conditions [12,13]. Alternatively, the substrate is pre-grown in for instance bags and then transferred to a mould. At a certain moment, the colonized substrate is removed from the mould and dried or growth is prolonged to mature the material followed by drying. The drying process is essential to metabolically inactivate or to kill the fungus. Drying temperature can range from room temperature to 100 °C, while drying time varies between a few hours to several days [12,14]. Drying at room temperature will normally inactivate but not kill the fungus. For instance, dried material of Ganoderma sp. could regrow a year after it had been dried at ambient temperature [15]. In contrast, a temperature of ≥ 60 °C will normally kill the fungus. The resulting mycelium composite has foam-like properties with a density of 60-300 kg/m 3 . It can be used for insulation because of its inherent low thermal conductivity and high acoustic absorption [13]. Mycelium composite materials absorbs 70-75% of the sound at frequencies < 1500 Hz [16] and have a thermal conductivity of 0.04-0.08 W/(m K) [13], both of which are similar to traditional insulation materials. Dried mycelium composite can be (heat) pressed to obtain materials with cork-and wood-like mechanical properties [12]. These pressed composites can be coated with resin and used as flooring [13], while the use of pressed mycelium composite as building materials is also being explored.
Besides inactivating (e.g. by drying at ambient temperature) or killing (e.g. by heat-drying) the fungus can also be maintained metabolically active to create a biocomputational material. For instance, a living composite made with Ganoderma resinaceum responds to pressure by changing its electrical activity [17]. Moreover, mycelium can be maintained active to enable production of large size mycelium composites, for instance to make mycelium connections between mycelium panels [15,18,19].
Pure mycelium materials are made by using liquid-or solid-state fermentation. Growing pure mycelium in a solid-state fermentation is similar to the way mycelium composites are produced. In the case of pure mycelium, however, the fungal skin that develops at the substrateair interface is harvested. A CO 2 concentration of 5-7% by volume and a temperature of 30-35 °C is used to inhibit mushroom development of Ganoderma sp. and to stimulate aerial hyphae formation, resulting in a thick felty skin [20]. Pressing the skin during and after growth is common practice to obtain a desired density [20]. Mushrooms resulting from solid-state fermentation can also be used to make pure mycelium materials. Mycelium films can be obtained by casting and drying blended mushrooms. Mycelium can also be processed before casting. For instance, white button mushrooms (Agaricus bisporus) has been used to make chitin-glucan based nanopaper [21][22][23][24].
Static and dynamic conditions can be used for liquid-state fermentation. Growth conditions such as agitation, pH, oxygen, temperature, light, medium composition and amount of inoculum are being optimized for each species. After the growth phase the fungal biomass is harvested. In the case of liquid static cultures, a sheet of mycelium is harvested that has formed at the water-air interface [25,26]. In contrast, total mycelium is separated from the spent medium by filtration or centrifugation from liquid shaken or bioreactor cultures resulting in a "pulp" of biomass. The biomass can be directly casted and dried or first homogenized before casting and drying. The mycelium films resulting from static or liquid fermentations can be processed to modify properties. For instance, treatment with the plasticizer glycerol (≥ 8%) results in elastomer-like materials that are more hydrophilic than untreated material [27].
Pure mycelium materials can be used as a foam, a cellular scaffold or as a meat alternative [20]. Moreover, the material can be physically and/or chemically processed to manufacture leather-like materials. The use as textiles is also being explored. Like mycelium composites, one may wish to keep the pure mycelium metabolically active in the final product to use it for instance as smart wearables [28]. By measuring electrical activity it was shown that pure Ganoderma resinaceum mycelium responds to mechanical and optical stimulation [29]. This opens up a completely new range of fungal material applications, such as sensors and biocomputers. The fungal life style The Kingdom Fungi consists of nine phyla, of which the Ascomycota and Basidiomycota represent most species [30]. According to the Catalogue of Life the current number of identified fungal species exceeds 146.000 [31] but the total number of species is predicted to range between 1.5 and 12 million [32,33]. Fungi play a vital role in most ecosystems by interacting with other living organisms such as plants, animals, and algae [32]. A fraction of the fungi can establish mutual beneficial interactions with for instance plants (mycorrhizae) and algae and/or cyanobacteria (lichens). On the other hand, fungi can be pathogens of for instance animals, plants and other fungi. A distinction is made between opportunistic and classical pathogens that infect weakened or healthy individuals, respectively. Fungal pathogens may prevent species to become too dominant in ecosystems but can have devastating effects as well. For instance, members of the genus Armillaria (Basidiomycota) are aggressive pathogens causing root disease that affect trees and shrubs worldwide [32,34]. Even more impactful, around 600 fungal species can infect humans. The far majority of these fungi are opportunistic fungi that infect individuals with a compromised immune system [35]. For instance, Pleurotus ostreatus (oyster mushroom) (Basidiomycota) and Saccharomyces cerevisiae (Ascomycota) that are commonly used as food or to produce food, respectively, are opportunistic pathogens that can cause allergies and serious infections, albeit at low frequency [36,37]. Apart from infections, fungi can also give rise to disease by causing allergies and by production of mycotoxins. In addition, fungi play an important role in nature in nutrient cycling by degrading organic waste streams such as plant material. In fact, fungi are the main degraders of lignin in wood [30]. Saprotrophic fungi that degrade wood are classified as white-, brown-and soft-rot fungi. White-rot fungi (mainly Basidiomycota) degrade cellulose, hemicellulose and lignin, whereas brown-rot (Basidiomycota) degrade cellulose and hemicellulose, but do not depolymerize lignin [30,32,38,39]. Soft-rot is dominated by soil-inhabiting Ascomycota that break down cellulose and hemicellulose and lignin as well, albeit at a much lower rate [32]. Fungi are typically opportunistic by adopting their life style when (a)biotic conditions change. Endophytes adopt the various fungal life styles and illustrate fungal opportunism. Endophytes reside in plants, either actively colonizing the host or simply being present in a dormant state [40,41]. Endophytes can be harmless or beneficial, for instance by producing alkaloids that protect against grazing [32,40] or by promoting plant growth and fruit production [40,42]. However, endophytes can also be pathogens or switch from a mutualistic to a parasitic mode of growth due to changes in the (a)biotic environment of the fungus [40,41].

Dispersion of fungi in nature
Fungi reproduce by producing (a)sexual spores [43]. For instance, asexual conidia are formed by specialized structures in ascomycetes, while asexual chlamydospores are formed by vegetative hyphae of Ascomycota and Basidiomycota [32,44,45]. Moreover, Ascomycota and Basidiomycota form sexual asco-and basidio-spores. Part of the spores (mainly sexual spores) are considered hardly motile by being immobilized in the fruiting body. Other spores (both sexual and asexual) will be dispersed by water, air or by other vectors such as animals. Typically, every cubic meter of air contains 1000 to 10,000 spores [43]. Spores can be very stress resistant. For instance, Paecilomyces variotii (Ascomycota) forms the most heat resistant conidia reported to date with a decimal reduction time of more than 20 min at 60 °C [46]. Ascospores can be even more heat resistant. For instance, those of Talaromyces flavus (Ascomycota) have a decimal reduction time exceeding 5 min at 91 °C [47]. Spores can also be highly resistant to drought, salt, radiation and oxidative stress conditions. For example, conidia of Aspergillus niger (Ascomycota) are extremely resistant against X-ray, cosmic and UV-C radiation thereby likely to survive space travel [48]. Spores germinate when conditions are favourable. Notably, only part of the conidia will germinate when exposed to such conditions [49]. Only 20% of the conidia of A. niger germinate in the presence of 50 mM glucose. The majority of these spores thus remain in their stress resistant resting state. This provides a bet hedging strategy to prevent for instance that all germlings die when temperature exceeds the cardinal temperature of 47 °C during daytime [49].
Less common is the formation of a specialized structure known as sclerotium [32,50] that is extremely stressresistant and able to survive for years [32,51]. Sclerotia are aggregates of hyphae with a tough outer layer of thick pigmented hyphae [32,50] and their formation is triggered by stressful conditions [51]. Sclerotia of L. mylittae can even germinate and form a basidiocarp without external water [32].
The dispersion of fungi can have a huge impact on food production, human health and biodiversity. For instance, they cause extinction of amphibians [52]. A single basidiocarp is able to release 1 billion spores a day [53] but human activities such as trade, transport and travel are also important ways of spreading spores [32,52]. For instance, transport of plant material and presence of the fungus on shoes, clothes and equipment are important factors of spreading Fusarium TR4 (Ascomycota),

Pathogenic fungi
Some of the fungal species that are used or have been proposed to use for mycelium materials have been reported to be pathogens of humans, animals and/ or plants (Table 2). However, none of these animal or human pathogens are considered classical pathogens. The majority of the species that are used to make mycelium materials are white-rot basidiomycetes belonging to the subphylum Agaricomycotina [30]. Some of these wood degrading fungi can incidentally cause disease in human as opportunistic human pathogens. Exposure to high numbers of basidiospores can cause respiratory problems as observed in growers of the oyster mushroom P. ostreatus [36,55]. Moreover, agaricomycetes can infect humans with a compromised immune system. For instance, a total of 71 S. commune infections (mainly broncho-pulmonary mycosis and sinusitis) have been reported worldwide until 2013 [56]. This number of infections should be related to the 57 reported cases of fungemia (until 2003) caused by S. cerevisae that is widely used in baking and brewing and as a probiotic [37] and the 150 million severe cases of fungal infections each year, of which 1.7 million patients die [57]. The opportunistic nature of agaricomycetes makes that one can work with these fungi at the lowest biosafety level during their production, although regulations may differ between countries. Plants are more prone to fungal infections than animals. Quite some fungi listed in Table 1 have been reported to be pathogenic for plants (Table 2). In fact, Botrytis cinerea, Phytophthora cinnamomi and Fusarium spp. are listed as regulated non-quarantine organisms in Europe, while Fusarium oxysporum f. sp. albedinis is even listed as a quarantine organism. Moreover, B. cinerea, F. oxysporum and Fusarium graminearum that are listed in Table 2 are considered to be in the top 5 of fungal pathogens based on scientific or economic importance [58]. In all other cases, fungi listed in Table 2 are not mentioned by the European Food Safety Authority. This implies that one can work with these fungi at the lowest biosafety level during material production. Yet, some of them are considered serious pathogens. Most of the 43 polypores listed in Table 1 are strictly saprotrophic, some grow and on dead wood in living trees, and a small number can invade and kill living wood [59,60]. The polypores of Ganoderma that are often used to make mycelium material (Table 1) are considered serious pathogens for plantations and natural forests especially in Southeast Asia, causing tremendous economic loss [45,61,62]. For instance, Ganoderma boninense causes a destructive disease in palm plantations known as basal stem rot [45,61]. In the past this disease was mainly found on older plants, but nowadays even young plants are affected [61]. Stem rot caused by Ganoderma species is also a disease for coconut palms [63]. White-rots (both polypore and gilled fungi) have been described as pathogens of grapevines [64,65]. The main cause of white rot in grapevine is Fomitiporia mediterranea, however, other opportunistic species have also been described as a causative agent [64]. For instance, Flammulina velutipes, P. ostreatus, S. commune and Trametes hirsuta have been observed on grapevine in Europe, generally on weakened plants that have other diseases or wounds [65]. Another study identified white-rot fungi, also used for mycelium materials, on living fruit and nut trees at the West Coast of the United States [66] (Table 2). Occurrence was in most cases associated with wounded trees. Among others, Oxyporus, Ganoderma and Trametes species were often found on cherry trees, whereas species belonging to Pleurotus and Laetiporus were more common on walnut trees. In addition, white-and brown-rots can cause decay of wooden structures [67]. In particular, Gloeophyllum sepiarium causes decay in houses [59] and wooden objects such as railroad and utility poles [68].
A fraction of the fungi listed in Table 1 have been reported as endophytic fungi (Table 3). Endophytes should be used with caution, especially when introduced from a different continent since co-evolution between host and the fungus did not take place. Hence, resistance has not evolved which can make hosts highly susceptible [40]. It has been described that a change in lifestyle from endophytic to pathogenic can be caused by a mutation in a single locus [41]. The fact that disease is often only detected when sporocarps are formed complicates risk management. Furthermore, little is known how the intra-species genetic variation affects the life style of the fungus [32,33]. This genetic variation can be high. For instance, a diversity of 0.2 has been found within synonymous sites of S. commune [69].

Mycotoxin production
Some fungi listed in Table 1 produce mycotoxins, some of which can even be used as a biological weapon. Isolates of F. oxysporum [70], F. graminearum [58] and Aspergillus [71] are known to produce a variety of mycotoxins. Basidiomycetes also have the ability to produce toxins, leading to hundreds of deaths every year [72]. For instance, the white-rot fungus Galerina marginata is considered highly poisonous due to its ability to produce amatoxin [73,74]. Mycotoxin levels are strictly monitored in food, but it may be relevant for fungal materials as well depending on their application. For instance, mycelium materials may be in direct contact with human  Table 2 Pathogenic species used or proposed to make mycelium materials *Also part of Table 3 Species

Impact on the biotic environment
Fungi and insects are abundant in nature and they have evolved different interactions [76]. Insects benefit from fungi as food source, mechanical protection and antimicrobial defence. Fungi benefit from insects in a similar way, while insects also serve as a vector for fungal spore dispersal [76]. Fungi are known to produce complex mixtures of volatiles. The composition of volatile compounds can vary depending on growth conditions [77,78] and developmental stage [79]. Hundreds of volatiles have been identified, including alcohols, aldehydes, esters, phenols and ketones [80,81]. Volatiles are synthesized as by-products of metabolism and can have a protective or attractive role in interaction with animals [75,76]. A well-known compound is the alcohol 1-octen-3-ol [80,81], which can act both as attractant and repellent depending on the fungus-insect interaction [82]. Female flies are attracted by volatiles to lay eggs on the fruiting body to provide larvae with fungal tissue as a food source [32,83]. In some cases, this is mutually beneficial when dispersal of fungal propagules by the insect takes place [76,83]. These interactions can also result in the attraction of generalist predator insects to prey on fungusinsects [84]. Ants can also be attracted to mushrooms [85][86][87]. For instance, the ant species Euprenolepis procera is a specialist in harvesting of and living on fruiting bodies [85,86]. A range of volatile compounds have been identified in species listed in Table 1. Most publications are about volatile compounds from (fresh) basidiocarps [80,84], while few discuss volatile compounds of vegetative mycelium. The mycelial volatile compounds of the commonly used species Trametes versicolor and P. ostreatus are listed in Tables 4 and 5, respectively. An important aspect for insect herbivores to recognize host plants is the perception of the whole blend of volatiles. Therefore, testing individual compounds may not always be representative for insect behaviour [88]. Studying the natural effects of fungal volatile Table 3 Endophytes used or proposed to make mycelium materials *Also part of Table 2 Species

Recommendations
Fungal materials have attracted a lot of interest during the last decade, showing analogy with the interest in collected and cultivated edible mushrooms. The latter prompted the Nordic countries to re-assess safety of mushrooms that are being traded and / or collected and consumed by individuals [90]. Clearly, risk assessment of fungi for materials is different from that of consumption but we can learn from such assessments. The list of species used or proposed to make mycelium materials (Table 1) is a fraction of the (wood degrading) fungi found in nature. Effective screens will be needed to unravel the potential of fungal species to produce mycelium materials. So far, only three publications [11,91,92] reported screening of ≥ 10 species for their applicability to make composite materials. This should be scaled up selecting not only for mechanical properties but also for instance for rate of colonization, robustness of performance, the amount of CO 2 emission, visual appearance and haptic properties, genetic stability of the strain, and the ability to easily maintain stocks of the strains. Several methods have still to be developed, while others have been established such as those to quantify mechanical properties [92], to store fungi [93], or to monitor homogeneity and rate of colonization in a 3D substrate [94].
On top of the mechanical and other performative properties, fungi should be screened based on a risk assessment. Such a risk assessment could be partly based on a history of safe use, for instance as a food. Risk assessment relies on a correct identification of the fungal species that is used. Therefore, standards of identification should be used such as proposed recently [95]. This identification should be linked to biosafety. In particular, fungi should be selected that can be used at Bio Safety Level 1 during production of the mycelium materials (see [96,97]). Pathogenic fungi and fungi producing mycotoxins should not be used when the final product contains living mycelium. In addition, species should be avoided that attract insects when grown in certain substrates. Still, little is known about release of volatiles by fungi and their impact on insect communities. Species that are commonly used to produce fungal materials have been selected based on their speed of colonizing substrates, thereby being potentially highly competitive in nature. Especially when exotic fungi are used they may become invasive, replacing part of the local fungal communities [98].
Fruiting should be repressed during formation and later use of mycelium materials to prevent spreading in the natural environment. Mushroom production in Coprinopsis cinerea and Pleurotus djamor is repressed by glycogen synthase kinase-3 (GSK-3) inhibitors like lithium chloride and CHIR99021 trihydrochloride. These inhibitors have been proposed to be included in the substrates to inhibit fruiting body formation on fungal materials and regulating mycelium growth [99]. Alternatively, one could use non-sporulating strains. Such strains are already being used in the cultivation of P. ostreatus to protect workers from exposure to spores [36,55]. Clearly, the most practical solution to prevent spreading in the natural environment is to kill the fungus before it leaves the production facility. Use of local strains not only prevents introduction of invasive Table 5 Volatile compounds of P. ostreatus when grown on sugarcane bagasse [78], wheat straw [164] or Raper medium [78] in the absence of mushroom formation  species, it also is the easiest way to comply with the Nagoya protocol. This protocol was put in place in 2014 to enforce fair and equitable sharing of benefits arising from the use of genetic resources from countries [100]. When working with fungi standardisation is key [9]. This is not only essential to compare screens in different laboratories but also to ensure reproducible manufacturing and material properties. For instance, drying of mycelium materials should be standardized. So far, drying is done at room temperature, in an oven, or a drier. An important aspect is the insulation property of mycelium composites, with surrounding material potentially keeping the inner section viable and/ or moist. Therefore, studies should unveil viability of mycelium composites after drying and heat treatment by plating and counting colony-forming units. Possibly, substrates, species and materials dimensions as well as methods of drying should be optimized. In addition, studies have to be performed to demonstrate stability of the material in time. Only recently an article assessed the impact of tropical weathering conditions (75 ± 15% relative humidity and 27.5 ± 2.5 °C) on the mechanical properties of composite material [101]. Mechanical properties of uncoated samples substantially dropped over 35 days, whereas applying an oil-based coating reduced the weathering effect, albeit only significant for tensile strength. This was explained by the high porosity of the composite material that prevented the coating from forming a perfect sealed surface, thus enabling moisture from entering. Finally, biodegradability of the mycelium material after use should be assessed as well as the bioavailability of the nutrients contained in the material. So far, these topics have not been addressed in the literature.
Together, the following recommendations are made.
• Fungi used for fungal materials should be identified via standardized procedures; • Use standardized methods to select fungal species and strains for specific applications; • Fungi should be selected that are not pathogenic to humans, animals and plants; • Fungi should be selected that do not produce mycotoxins, even when the fungal materials have non-food applications; • Use spore-less strains; • Preferably use local fungi to produce mycelium materials; • Work at biological safety levels as dictated by the local authorities; • Preferably kill the fungus in the mycelium material before it is leaves the production facility; • Regularly confirm efficacy of the killing procedure.

Conclusions
Fungal materials have a very high potential to replace non-sustainable products on the market. In fact, fungal materials may even have properties that are not yet provided by other materials. Given their potential, fungal materials may be used at a very large scale. In the future, people may be surrounded by these materials in their houses, at work, and may even wear it. This requires a critical assessment of the risks associated with fungal materials. This includes the selection of species used for making the materials, the conditions used during the production process and when they leave the production facility, as well as measures to prevent impact on the environment when the products are used in society (Fig. 1). Our assessment of pathogenicity and mycotoxin data indicates that fungal species that have been described in scientific publications to produce fungal materials show low risk, if at all, for workers, consumers and the environment. Genetic modification could be used to improve properties of mycelium materials, and to reduce pathogenicity, invasiveness, mycotoxin production, spreading in the environment, and/or attraction by insects. In this case too, introduction on the market should be accompanied by a risk assessment. This would be particularly needed when the mycelium product contains a living fungus.