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A comprehensive framework for the production of mycelium-based

lignocellulosic composites

Article  in  Science of The Total Environment · April 2020

DOI: 10.1016/[Link].2020.138431

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A comprehensive framework for the production of mycelium-based lignocellulosic
composites
Elise Elsacker a ,b ,⁎ , Simon Vandelook b , Aurélie Van Wylick a ,b , Joske Ruytinx b , Lars De Laet a ,

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Eveline Peeters b ,⁎⁎
a
Architectural Engineering Research Group, Department of Architectural Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
b
Research Group of Microbiology, Department of Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

ARTICLE INFO ABSTRACT

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Article history: Environmental pollution and scarcity of natural resources lead to an increased interest in developing more sus-
Received 30 November 2019 tainable materials. For example, the traditional construction industry, which is largely based on the extraction of
Received in revised form 1 April 2020 fossil fuels and raw materials, is called into question. A solution can be found in biologically augmented materials
Accepted 2 April 2020
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that are made by growing mycelium-forming fungal microorganisms on natural fibres rich in cellulose, hemicel-
Available online xxx
lulose and lignin. In this way, organic waste streams, such as agricultural waste, are valorised while creating a
Editor: Konstantinos G Moustakas
material that is biodegradable at the end of its life cycle – a process that fits in the spirit of circular economy.
Mycelium-based materials have properties that are promising for a wide range of applications, including the use
Keywords as construction materials. Despite this promise, the applicability and the practicality of these materials are largely
Mycelium-based composites unexplored and moreover, individual studies use a wide range of different experimental approaches and non-stan-
fungi dardized procedures. In this review, we critically evaluate existing data on the composition of mycelium-based
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Lignocellulosic fibres materials and process variables with the aim of providing a comprehensive framework of the production process.
Natural fibre reinforcement
The framework illustrates the many input factors during the production that have an impact on the final char-
Mechanical characteristics
acteristics of the material, and the unique potential to deploy more tuneable levels in the fabrications process
Manufacturing variables
that can serve to prototype a diversity of new unprecedented applications. Furthermore, we determine the ap-
plicability of existing data and identify knowledge gaps. This framework is valuable in identifying standardized
approaches for future studies and in informing the design and process of new applications of mycelium-based
materials.
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© 2020

1. Introduction Regarding medium-density fibreboards (MDF) and particleboards, pe-

troleum-derived adhesives or other formaldehyde-containing glues and
At the dawn of the third millennium, our societies are experienc- resins are needed for their manufacturing. Due to the scarcity and pol-
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ing major upheavals linked to perils on the living conditions of hu- lution of those resources, increased attention is devoted to the transition
manity. The extraction of fossil fuels and raw materials cause climatic towards circular building methods with biological production of materi-
disturbances and pollution generated by anthropogenic activities als from renewable resources.
(Moldalieva, n.d.; IPCC, 2018; United Nations, Environment As- Mycelium-based composite materials, making up a promising class
sembly of the United Nations Environment Programme, 2017; of bio-based materials, are produced by growing mycelium, the vege-
United Nations Environment Programme, International Energy tative part of filamentous fungi, on solid organic substrates (Jones et
Agency, 2018). In particular, the construction sector in Europe ac- al., 2017a). Fungi are eukaryotic microorganisms that are ubiquitous.
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counts for about half of all extracted materials and energy consump- They display an enormous diversity in morphology and lifestyle. Many
tion and for about a third of overall water consumption and waste fungal species play a central role in terrestrial ecosystems by degrad-
generation (European Commission, 2010). ing (ligno-)cellulosic biomass and recycling carbon (Hawksworth and
Lücking, 2017). Given their metabolic and physiological characteris-
tics, they have a large biotechnological potential and are likely to pro-
⁎
Correspondence to: E. Elsacker, Architectural Engineering Research Group, Department vide resilient, efficient and cost-effective solutions to current environ-
of Architectural Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, mental challenges (Chambergo and Valencia, 2016). When grow-
Belgium. ing mycelium on solid organic substrates, a lightweight composite is
⁎⁎
Corresponding author. obtained consisting of a three-dimensional interwoven network of nat-
E-mail addresses: [Link]@[Link] (E. Elsacker); [Link]@[Link]
(E. Peeters)
ural reinforcement fibres pre

[Link]
0048-9697/© 2020.
2 E. Elsacker et al. / Science of the Total Environment xxx (xxxx) 138431

sent in the feedstock (e.g. jute, flax or straw) on one hand and fila- A wealth of biological information is available on fungi, but these
mentous mycelial cells on the other hand (Elsacker et al., 2019). The are typically placed in relation to microbial production of chemicals or
organism itself is an essential constituent of these composite materials enzymes for applications in biofuels, medicine and food. Similarly, an
not only because of its cellular components harbouring unique proper- abundance of knowledge exists about lignocellulosic and polymer com-
ties, but also because of its behaviour during the growth process thereby posites. Yet, there is a lack of understanding how these scientific aspects
mediating self-assembly by acting as a “natural glue” and avoiding the relate to each other and to the production methods of the emerging ap-

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requirement for fossil-derived thermoplastic polymer binders (Jones plications of these promising natural composites. The available litera-
et al., 2017b). After substrate colonization, the material is heated ture and knowledge on mycelium materials is very fragmented. In ad-
to kill the organism and eliminate the moisture (Bayer and McIn- dition, methodologies used in different published studies are typically

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tyre, 2016). The entire production process is considered to be environ- different, and no standardized and comparative overview of the produc-
mentally friendly due to the valorisation of waste streams ([Link], tion parameters and mechanical properties of mycelium composites ex-
2019a), thereby preventing the destruction of ecosystems through the ists. Moreover, with the view of using mycelium composites as construc-
mining of resources. Furthermore, a broad diversity of vegetal substrates tion material, there are no studies that work towards the assessment of
can be used as feedstock (Elsacker et al., 2019). Importantly, the ob- norms, not even an overview is available that can serve as a basis. These
tained mycelium-based composite materials, which only contain bio- are essential for the implementation of the material for large-scale appli-
compatible components, can be naturally decomposed after use, en- cations. In this review paper, an integrated study is presented across dis-
abling an entirely circular production model. ciplines, thereby aiming at contributing to the growing field of bio-com-

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Mycelium materials find their way in many (potential) applications, posite manufacturing processes and new applications.
for example as insulating or packaging materials ([Link], 2019a; We provide an integrative and comprehensive framework of the as-
Holt et al., 2012a; Yang et al., 2017; Cerimi et al., 2019). The liv- pects that should be considered during the production of fungi-based
ing organism is capable of adaptive growth in response to environmental lignocellulosic composites, including i) the matrix selection, namely the
conditions. In combination with the use of diverse feedstocks, this bio- fungal species or community, ii) the morphology of the mycelial net-
logical adaptiveness leads to an enormous palette of materials with dif- work, including cell wall composition, tip extension and branching, hy-
ferent characteristics that are envisaged to be produced. Although this phal fusion and hyphal density, and how this is affected by environ-

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variability offers many potential benefits for product design and archi- mental conditions, iii) the interaction between fungi and their feedstock,
tecture (Appels et al., 2018a), it also brings engineering challenges iv) the feedstock selection, namely the type of lignocellulosic biomass,
(United Nations Environment Programme, International Energy v) the process variables during manufacturing – including the protocol,
Agency, 2018; European Commission, 2010). Indeed, it has been sterilization, inoculation, packing, incubation, growing period, and dry-
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demonstrated that the properties of the resulting composite material de- ing method and finally, vi) the resulting applications. In addition to pre-
pend on a plethora of biological, (physico-)chemical and process engi- vious descriptive overviews of the final characteristics of mycelium ma-
neering factors (Appels et al., 2018a; Haneef et al., 2017). terials (Jones et al., 2017b; Girometta et al., 2019), this work aims
A wealth of biological information is available about fungal species, at evaluating the impact of every variable during the production process
but these are always put in relation with the production of chemicals on mechanical and physical properties. A better understanding of the
or enzymes for applications in biofuels, medicine and food. Similarly, relative contribution of each variable can ultimately lead to the opti-
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an abundance of knowledge exists about lignocellulosic and polymer mization of the production process in order to enable and control diver-
composites. Yet, a lack of scientific insights and research that place the sification depending on the desired material outcome.
factors in relation with each other, with the production methods and
with the emerging applications of these promising natural composites, 2. Mycelium-based composites: The contribution of all its
prevails. The available literature and knowledge are very fragmented. components
The methodologies of the published studies are typically different, and
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no standardized and comparative overview of the production parame- 2.1. Why does the choice of the fungal species matter?
ters and mechanical properties of mycelium composites exists. More-
over, with the view of using mycelium composites as a construction ma- 2.1.1. Phylogenetic diversity
terial, there are no studies that work towards the assessment of norms, Given that fungal hyphae are an integral part of a mycelium com-
not even an overview can serve as a basis. These are essential for the posite material (Fig. 1), their biological characteristics influence both
implementation of the material for large-scale applications. The hybrid the production process and material characteristics. To date, 36 fun-
investigation across disciplines developed within this review paper aims gal species have been used or are mentioned in patents (Cerimi et
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at contributing to the growing field of bio-composite manufacturing al., 2019) for mycelium-material applications. This group of species
processes and new applications. are restricted to the Basidiomycota phylum and includes Trametes ver-
sicolor (Jones et al., 2017a; Lelivelt et al., 2015a), Fomes fomen-
tarius (Moser et al., 2017), Gandoderma lucidum (Haneef et al.,
2017; Holt et al., 2012b; TRAVAGLINI et al., 2016;
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Fig. 1. Schematic representation of the mycelium materials at different scales.

 E. Elsacker et al. / Science of the Total Environment xxx (xxxx) 138431 3

Travaglini et al., n.d.), Irpex lacteus (Yang et al., 2017), Pleurotus the selection of the most suited species in function of the desired mater-
djamor (Ahmadi, 2016), Pleurotus ostreatus (Haneef et al., 2017; Le- ial outcome.
livelt et al., 2015a; Moser et al., 2017; He et al., 2014) and Schizo-
phyllum commune (Appels et al., 2018b) (Fig. 2). These examples all 2.1.2. Hyphal growth, branching and fusion
have a saprotrophic lifestyle and Basidiomycetes categorized as primary Hyphal growth is a highly polarized process called tip extension (Vi-
decomposers are able to grow on a large variety of lignocellulosic bio- dal-Diez de Ulzurrun et al., 2017). As hyphae continue to grow,

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mass, even low-quality organic waste streams like sawdust and straw, branches are repeatedly formed at an angle of between 42 and 47 de-
therefor making them ideal candidates for growing mycelium materi- grees (Papagianni, 2004; Gooday, 1977) to the long axis of the initial
als (Martin et al., 2011). Another remarkable trait is their ability to hypha. Multiple branching events lead to an interwoven network of hy-

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grow almost infinite large networks of hyphae, which is only limited by phal filaments referred to as mycelium (Howard, 1981; Nordberg et
the availability of their growth substrate. Nevertheless, fungal diversity al., 2014; Heaton et al., 2010). Mycelium grows in and around dis-
is estimated on 2.2–3.8 millions of species (Hawksworth and Lück- crete particles thereby binding those particles together into a solid com-
ing, 2017) with contrasting lifestyles ranging from wood decay over posite. This mechanism results in efficient substrate colonization and
mycorrhizal mutualism to plant and animal (Stajich, 2017). Given the surface to volume maximalisation allowing a very efficient way for nu-
phylogenetic diversity of fungi (Jones et al., 2018), it could be en- trient uptake, due to the large surface area, similar to gut microvilli in
visaged that there is an unexplored wealth of species with potential to animals.
build mycelium materials of superior quality. Besides investigating di- When the colony continues to stretch out, hyphal fusions (anasto-

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versity of natural strains, genetic engineering can be another approach mose) may occur, producing the lattice-like structure of mycelium tis-
to improve the application. As an example, a genetically modified strain sues (Rayner et al., 1999). The hyphae subsequently adapt during
of Schizophyllum commune resulted in a sterile strain by targeting mole- their development and in response to their environment. Moreover,
cular mechanisms involved in fruiting body formation. This resulted in mycelium can respond to local damage by reinforcing, regrowing and
deleting the unwanted ability of fruiting body and spore formation and reconnecting neighbouring branches, which is of interest in the produc-
on top of that, this mutant strain displayed a 3-fold higher growth speed tion process of mycelium-materials. The strengthening of the branches
than wildtype strains (Heath, 1995). and the alternation of pathways improves not only the transport ca-

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Morphological, biochemical and physicochemical characteristics of pacity of the channels, but also their robustness to damage. Indeed, if
filamentous hyphae, the network they build and the way they inter- the finest hyphae are continuously trimmed, more local branching is
act with the provided lignocellulosic substrate (feedstock) vary sig- stimulated, resulting in an accentuated growth of connections (Gross,
nificantly among species depending on their phylogeny, ecology and 2009). Consequently, damaging or cracking the mycelial network dur-
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lifestyle (Vidal Diez de Ulzurrun, 2017). In the next sections, we ing the growth stimulates the formation of a more robust and denser net-
delineate how fungal characteristics such as growth, cell wall com- work, and the regression of other regions to recycle redundant material
position and potential to colonize and degrade lignocellulosic feed- (Heaton et al., 2010; Falconer et al., 2005; Fricker et al., 2007).
stock differ among species and we discuss current insights in their Overall, the processes of tip extension and radial branching into
relationship with material properties. A better understanding of this an interconnected lattice-like network, are regulated by a range of en-
relationship is needed to identify promising species and guide vironmental conditions which are specific to the concerned species,
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its ecology and lifestyle. Indeed, different species show a dif

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Fig. 2. Phylogenetic representation at the order level of the diversity of filamentous fungi species used in the application of growing mycelium materials. The box on the right side display
the number of species belonging to the respective order that have been mentioned in scientific articles or [Link] adapted from Jennings (2008).
4 E. Elsacker et al. / Science of the Total Environment xxx (xxxx) 138431

ferent hyphal and mycelial morphology when grown on the same als compared to P. ostreatus (Lelivelt et al., 2015a) (Fig. 3). P. ostrea-
medium. For example, P. ostreatus hyphae are characterized by larger di- tus-based materials are stiffer (2-fold, E: 28 MPa) than G. lucidum-based
ameters and more compressed filaments than those of G. lucidum when (E: 12 MPa) ones. This can be explained by a higher polysaccharides'
cultured in medium containing cellulose and potato dextrose (Haneef content in the P. ostreatus-based material (Haneef et al., 2017).
et al., 1292). Jones et al. (2018) assessed the mycelial growth rate G. lucidum, on the other hand, presents a larger elongation at frac-
and density of 18 randomly selected species with contrasting ecology ture (3-fold, ε: 33%) and higher values in strength (σ: 0.8 MPa) com-

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and lifestyle within the Basidiomycota. The white rot species Polyporus pared to P. ostreatus (ε: 9% and σ: 0.7 MPa) because it consists of a larger
brumalis and P. djamor showed highest mycelial growth rate and density, amount of proteic and lipid constituents associated with the infrared
respectively. However, overall lifestyle or phylogenetic position did not absorption spectra of the mycelia, which may act as plasticisers. G. lu-

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correlate with growth performance which seemed to vary rather arbi- cidum-based materials also display a broader Young's modulus distrib-
trarily (Jones et al., 2018). ution (Haneef et al., 2017). The tensile properties of non-pressed T.
versicolor (σ: 0.04 MPa, E: 4 MPa) compared to P. ostreatus (σ: 0.01 MPa,
2.1.3. Cell wall composition E: 2 MPa) grown on rapeseed straw are contradicting the properties of
Fungal cells have tube-like structures with a complex chemical com- heat-pressed T. versicolor (σ: 0.15 MPa, E: 59 MPa) compared to P. os-
position with the cell walls being characterized by layers consisting of treatus (σ: 0.24 MPa, E: 97 MPa) (Appels et al., 2018a). The forma-
extensively cross-linked glucan and chitin components in addition to
tion of materials with the highest tensile strength can be attributed to
proteins such as hydrophobins and mannoproteins (Bowman and Free,

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S. commune (Appels et al., 2018b) (Fig. 4). The Young's modulus (E:
2006; NAR et al., 2017). Chitin is a homopolymer of β-(1,4)-linked
913 MPa) and tensile strength (σ: 9.5 MPa) of S. commune wild-type are
N-acetylglucosamine, and although it only constitutes a minor part of
up to 76 and 9-fold stronger compared to G. lucidum and respectively
the entire cell envelope, it greatly contributes to its structural integrity
33 and 9-fold for P. ostreatus. However, elongation at rupture was up to
and to that of the organism as a whole (NAR et al., 2017). The Young's
23-fold higher for G. lucidum and 6-fold higher for P. ostreatus (Haneef
modulus and the tensile strength of pure chitin films vary between 1.2
et al., 2017; Appels et al., 2018b).
and 3.7 GPa and 38.3 and 77.2 MPa, respectively, depending on the
Although most studies have been performed with wild-type strains,
method of preparation (Ofem et al., 2017). Consequently, the presence
strains that are genetically modified in a targeted manner could also

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of chitin-containing fungal cell walls in mycelial composite materials –
hold much promise for use in mycelium-based materials. For example,
even at minor fractions – is crucial for the material's structural and me- the genetic modification of S. commune by deletion of the hydrophobin
chanical properties. As fungal cell walls exhibit a large degree of pheno- gene sc3 impacts the water absorbance, the Young's modulus (3-fold, E:
typical diversity and plasticity (NAR et al., 2017), this contributes to
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2727 MPa), and the tensile strength (4-fold, σ: 40.4 MPa) as compared
the material's engineerability. to the wild-type (Appels et al., 2018b) (Fig. 4).
Although limited studies have been performed on how material
characteristics alter with the used fungal species or strain (Figs. 3 2.1.4. Lifestyle and capacity to degrade lignocellulose
and 4), they point to an important correlation between (phylo-)ge- Feedstock or substrate to be colonized by filamentous fungi during
netic nature and mechanical properties which can, among other rea- production of mycelial materials exists of lignocellulosic waste streams
sons, be attributed to cell wall composition. For instance, a higher
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(Elsacker et al., 2019). Ideally, fungal mycelium grows

compressive strength and stiffness is found with T. versicolor materi
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Fig. 3. Compressive strength (MPa) of mycelium-materials (data not normalised to density). Ganoderma sp. on processed cotton plant (Holt et al., 2012a), Pleurotus sp. on crop residues
of Triticum with edible coating (López Nava et al., 2016), Unknown species on fibre cotton down woven mat, on hemp pith woven mat, on hemp pith & and non-mat, Trametes versicolor
on hemp hurds, chopped hemp, pre-compressed hemp hurds, flax hurds, chopped flax, flax treated tow, flax untreated tow, pre-compressed flax hurds, flax waste hurds, pre-compressed
flax waste hurds, pine soft wood shavings (Elsacker et al., 2019).
 E. Elsacker et al. / Science of the Total Environment xxx (xxxx) 138431 5

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Fig. 4. Tensile strength (MPa) of mycelium-materials (data not normalised to density). a. P. ostreatus on cellulose (Haneef et al., 2017), b. P. ostreatus on cellulose-PDB (Haneef et al.,
2017), c. P. ostreatus on rapeseed straw non-pressed (Appels et al., 2018a), d. P. ostreatus on rapeseed straw cold-pressed (Appels et al., 2018a), e. P. ostreatus on rapeseed straw
heat-pressed (Appels et al., 2018a), f. P. ostreatus on cotton cold-pressed (Appels et al., 2018a), g. P. ostreatus on cotton heat-pressed (Appels et al., 2018a), h. wild type S. commune
(dark 400 ppm CO2) (Appels et al., 2018b), i. wild type S. commune (dark 70,000 ppm low CO2) (Appels et al., 2018b), j. wild type S. commune (light 400 ppm CO2) (Appels et

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al., 2018b), k. wild type S. commune (light 70,000 ppm CO2) (Appels et al., 2018b), l. ∆sc3 S. commune (dark 400 ppm CO2) (Appels et al., 2018b), m. ∆sc3 S. commune (dark
70,000 ppm CO2) (Appels et al., 2018b), n. ∆sc3 S. commune (light 400 ppm CO2) (Appels et al., 2018b), o. ∆sc3 S. commune (light 70,000 ppm CO2) (Appels et al., 2018b), p.
G. lucidum on cellulose (Haneef et al., 2017), q. G. lucidum on cellulose-PDB (Haneef et al., 2017), r. G. lucidum on red oak (TRAVAGLINI et al., 2016), s. T. versicolor on rapeseed
straw non-pressed (Appels et al., 2018a), t. T. versicolor on beech sawdust non-pressed (Appels et al., 2018a), u. T. versicolor on rapeseed straw heat-pressed (Appels et al., 2018a).
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(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

into these fibres and on wood blocks by degrading lignocellulose in or- Another characteristic of white rot wood decay is, as the name already
der to effectively glue the substrate. By secreting redox-active enzymes suggests, a visual indication of white colonizing fungal biomass. Con-
such as laccases and peroxidases, the ability to degrade lignocellulose cerning brown rot species, it has been observed that they lack functional
allows the fungus to scavenge highly sequestered nutrient sources pre- peroxidases that play a central role in ligninolytic wood decay, although
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sent in lignified plant biomass. The chemical structure of lignin changes they evolved from white rots, and thus lost this ability by shifting to-
during the enzymatic oxidation. Lignin-based radicals are cross-linked wards a non-ligninolytic type of wood decomposition where lignin poly-
after the oxidation process and can subsequently form an adhesive by mers are being modified to give access to cellulose and hemicellulose
the increased bonding between the fibres (Widsten and Kandelbauer, (Floudas et al., 2012). Soft rot fungi are less efficient lignin degraders
2008a). Results from Unbehaun et al. (2000) show that the bending compared to white rot, but they tend to possess better cellulolytic, hemi-
strength of enzymatically pre-treated fibreboards increased by approx- cellulolytic and pectinolytic abilities (AKS and Qin, 2017). Their pres-
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imately 50%, while the perpendicular tensile strength of the materials ence on decomposing wood can often be assessed as a greyish decoloura-
doubled and the thickness swell decreased to half (Unbehaun et al., tion.
2000). Wood delignification using fungal ligninolytic enzymes, aiming Most filamentous species used for the application of growing
to improve the mechanical properties of the fibreboards, was already de- mycelium composites belong to the group of white rot fungi (Fig. 2,
veloped in 1949 by Luthardt W. at the Technical University of Dresden Supplementary Table S1). This can probably be attributed to their ability
(Unbehaun et al., 2000). The properties of fibreboards prepared from of displaying a relatively good substrate colonization rate and of feeding
enzymatically activated wood fibres were shown to depend strongly on on a large variety of lignified plant biomass as well as detoxifying sub-
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the fabrication method, including the pre-heating temperature, the incu- strates containing toxic aromatic compounds such as terpenes. Never-
bation conditions and pressing conditions (Widsten and Kandelbauer, theless, this does not exclude the potential of brown- and soft-rot species
2008a). as suitable candidates for the application of mycelium-based materials.
Many fungal species, including those with a mycorrhizal, endo- Indeed, given that only a small fraction of fungal biodiversity has been
phytic or pathogenic lifestyle show the potential to degrade plant cell tested thus far, many more species and strains might have suitable char-
wall material (lignocellulose) in a search for essential but limited nu- acteristics for their use in the application. Based on the observation that
trients such as nitrogen (Mycorrhizal Genomics Initiative Consor- growth properties, hyphal extension, cell wall composition and meta-
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tium et al., 2015; Lindahl and Tunlid, 2015; Martino et al., bolic characteristics vary on a species- but also on a strain-level, it is
2018). However, the most effective degraders of lignin are those us- difficult to discern phylogenetic trends and it is advised to assess fungal
ing wood as a carbon source, i.e. wood-decaying saprotrophs. There are species and strains on an individual basis to establish their suitability for
three main groups of wood-decaying fungi: white, brown and soft rot composite manufacturing applications.
fungi, which reflect the effectiveness and pattern specificity of wood
decay that is directly related to the array of secreted ligninolytic en-
zymes (Kameshwar and Qin, 2016). The group of the white rots
is characterized by the singular ability of complete lignin degrada-
tion together with excellent decay of wood polysaccharides.
6 E. Elsacker et al. / Science of the Total Environment xxx (xxxx) 138431

2.2. How does the type of feedstock matter? agarose can be added to the fibres to retain the moisture or enhance the
viscosity of the substrate (Ross, 2016).
2.2.1. Nature of lignocellulosic feedstock The type of fibres also influences the flammability of the mycelium
Most of the feedstocks used for the production of mycelium-based composite (Jones et al., 2017a). The char formation is favourable
materials contain lignocellulose. Lignocellulose is the primary struc- when supplemented with high amounts of silica (for example present
tural component of many plants, crops and trees and consists of cellu- in rice husk). Lignin contains stable cyclic rings which decompose in

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lose, hemicellulose and lignin, with small amounts of ash, proteins and aromatic fragments, the principal constituents of char (Jones et al.,
pectin. The composition and proportions of these three compounds are 2017a), while cellulose increases combustion (Mouritz and Gibson,
variable and depend on the type of vegetation (Malherbe and Cloete, 2006).

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2002; Prasad et al., 2007; Sánchez, 2009), and also influence the
mechanical performances (Pickering et al., 2016). Cellulose is one of 2.2.3. Impact of feedstock on fungal biology
the most abundant organic molecules on Earth (Sánchez, 2009), fol- Not only the genetic nature, but also the feedstock characteristics in-
lowed by hemicellulose, with both macromolecules being constructed fluence morphological properties of the fungal organism: for example,
from different sugars in different polymeric structures (Sánchez, 2009). the hyphal structure is affected when changing the feedstock (Haneef
Lignin, linked to both hemicellulose and cellulose, is an aromatic poly- et al., 2017). Upon growth on potato dextrose broth (PDB), P. ostrea-
mer synthesised from phenylpropanoid precursors. It is known to give tus hyphae collapse along their central part and show a lower width of
the cellular wall of plants structural support and resistance against mi- the filaments as compared to growth on cellulose substrate, an obser-

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crobial attack and oxidative stress (Sánchez, 2009). vation linked to a lower abundance of chitin synthesis (Haneef et al.,
2017). As a result, the material with a low chitin/polysaccharide ratio
2.2.2. Direct impact of the feedstock type on the material properties displays more water uptake, a lower Young's modulus and higher elon-
The mechanical properties of the natural fibres used when growing gation (Haneef et al., 2017). Nutritional preferences depend on the
mycelial materials can be influenced by the processing of the fibres, used species or strain: for example, when fed on a wood-based substrate,
their chemical composition, and the environmental conditions during P. ostreatus leaves cellulose almost intact and utilises hemicellulose as
growth, which all influence in turn the properties of the mycelium com- the primary energy and carbon source (Chi et al., 2007). Although

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posites themselves. Important factors to take into account when select- not always well-described, similar principles regarding feedstock prefer-
ing the highest-potential fibres, are their dimensions, defects, strength, ences and effects can be expected to govern other fungal species.
and structure (Faruk et al., 2012). A considerable amount of literature
about lignocellulosic fibres and composites already exists, and is beyond 3. The impact of processing variables on the material properties
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the scope of this review, however, here we will focus on the role of the
fibres for mycelium-based materials in particular. 3.1. A general protocol for the production of mycelium-based materials
Substrates containing natural fibres that are not degraded during
the production of mycelial materials exhibit a strain-hardening behav- Besides parameters such as the genetic nature of the used strain and
iour because the natural fibres serve to reinforce and prevent shear fail- the characteristics of the feedstock type, there are many variable para-
ure (Yang et al., 2017). The natural fibres minimise surface crack- meters underling the production process (Fig. 5) that are dependent on
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ing during shear measurements and also improve the Young's modu- specific laboratory conditions and which are not always well-described
lus and compressive strength (Yang et al., 2017). Research on dif- because of pending IP or due to incompleteness. Examples are temper-
ferent types of fibres suggests that the fibre condition influences the ature during growth, drying methods or post-processing procedures. Al-
compressive Young's modulus, as chopped hemp fibres (<5 mm) result though the available data on these parameters are currently limited and
in a Young's modulus which is 1.6 times higher than loose hurds (El- fragmented, we hereby aim to associate results from different studies,
sacker et al., 2019) (Fig. 3). Another study indicates that the com- enabling to further build upon the body of existing knowledge.
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pressive strength of materials containing natural fibres (wood chips) in- The general protocol, compiled from open-source manuals (BioFab-
creases up to 300% when sand or gravel aggregates are mixed, com- Forum, n.d.; Fungal Materials and Biofabrication, n.d.), research
pared to pure materials (Moser et al., 2017). Additives such as sil- papers (Elsacker et al., 2019; Yang et al., 2017; Appels et al.,
ica, clay, perlite, methylcellulose, glycerine, 2018a; Haneef et al., 2017; Moser et al., 2017; Holt et al.,
2012b; TRAVAGLINI et al., 2016; Travaglini et al., n.d.; Ah-
madi, 2016; He et al., 2014; Lelivelt et al., 2015b; Jiang et
al., 2017; Jiang et al., 2013; Arifin and Yusuf, 2013; Islam et
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Fig. 5. Process flow chart showing the applied fabrication method of mycelium-based [Link] from Elsacker et al. (2019).
 E. Elsacker et al. / Science of the Total Environment xxx (xxxx) 138431 7

al., 2017) and patents (Bayer and McIntyre, 2016; Ross, 2016; (Streptomyces alboniger, Streptomyces natalensis and Bacillus subtilis) and/
Kalisz and Rocco, 2012) (Fig. 5), is as follows: or fungal species (Ganoderma sp.) (Lindahl and Tunlid, 2015). The
excretion of antimicrobial compounds will reduce the competition be-
(1) The mycelium is initially grown on agar plates, in grain-substrate, tween unwanted microbes and the selected fungal species (Schaak and
in a liquid nutrient solution, or in the pre-grown homogenised sub- Lucht, 2016). Alternatively, fungal strains could be genetically modi-
strate; fied to overexpress antimicrobials in situ (Schaak and Lucht, 2016).

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(2) The substrate is autoclaved or pasteurised to eliminate any type of In several studies fungal growth was analysed after using different
already present microorganisms on the substrate and thereby pre- substrate sterilization methods such as hot water treatment for 30 min,
venting contamination during the growth and incubation process; autoclaving at 15 lbs. pressure for 20 min, treatment with formaldehyde

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(3) A specific amount of the mycelium tissue is added to the substrate. solution (50 mL/L water), treatment with bavistin (2 g/L water). It was
If the substrate was not humidified before autoclaving (step 2) an found that substrates sterilized by autoclaving took less time for spawn
amount of sterile water is added. To improve growth, a sterile solu- run (Kalita, 2015; Atila, 2016).
tion of nutrients can also be added;
(4) The inoculated substrate is hand-packed in a sterilized mould which 3.3. Inoculation method
has the desired shape. The mould is sealed with a filtered air-perme-
able cover to maintain a micro-climate; Different approaches can be used to inoculate the substrate with
(5) The mycelium grows trough the substrate in a controlled environ- mycelium. When grown on malt extract peptone agar plates, one or

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ment. The material can be grown in two phases, first in a mould to more agar plugs (10 mm × 10 mm) from a fully colonized plate are cut
bind the fibres, and secondly outside the mould to solidify the outer and inoculated directly in the substrate (Haneef et al., 2017; Moser
skin of the material during a period; et al., 2017; Islam et al., 2017). The mycelium can also be grown on
(6) The grown material is heat-treated at a specific temperature for sev- grain spawn (Elsacker et al., 2019; Lelivelt et al., 2015a; Holt et
eral hours to end the growth process and dehydrate the material; al., 2012b; Ahmadi, 2016) before inoculation and this is subsequently
(7) A coating or post-processing can be applied to the material to im- used to inoculate the substrate with mycelium (10% to 20% of the sub-
prove its properties. strate's weight) (Lelivelt et al., 2015a) (Fig. 6a). For a homogenous

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distribution of the fungal spores and easy use during the inoculation
Many procedural variations on this general protocol exist for which process, a < 20% volume/volume (Schaak and Lucht, 2016) liquid
only fragmented information is available (Table 1). inoculum can be applied to the blends (Holt et al., 2012b; Appels et
al., 2018b). Alternatively, a pre-grown homogenised substrate consti-
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3.2. Substrate sterilization method tuted of the fungal biomass can be distributed evenly within the sub-
strate. During the inoculation process, the pre-grown substrate is mixed,
Several methods are employed for the sterilization of the substrate, and the mycelial biomass is pulled apart into smaller pieces (He et al.,
thereby rendering the substrate inert. This can happen (1) by tempera- 2014; Jiang et al., 2017; Jiang et al., 2013). This process stimulates
ture such as autoclaving and pasteurization or (2) treatment with chem- the hyphae to grow and to become more robust.
ical or microbial agents. Previous research findings have highlighted the difference between
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Firstly, autoclaving the substrate can be performed at different tem- grain-based inoculation and liquid-based inoculation and thereby identi-
peratures, between 115 °C and 121 °C (Elsacker et al., 2019; Ap- fied an influence on the dimensional stability after drying. A grain-based
pels et al., 2018a; Haneef et al., 2017; Holt et al., 2012b; Ar- inoculum, combined with a substrate with relatively small particle sizes
ifin and Yusuf, 2013) for varying durations between 15 and 28 min. (0.1–12 mm), has a small surface area contraction (0.64%), whereas a
Pasteurization is not always described (Yang et al., 2017), but comes liquid-based inoculum with larger substrate particle sizes (28–51 mm)
down to incubating the substrate in boiling water at ~ 100 °C dur- exhibited more substantial surface contraction (2.4%) (Holt et al.,
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ing approximately 100 min (Lelivelt et al., 2015a). By applying this 2012b). The more variable an inoculum/substrate combination is, the
method, harmless or possibly helpful organisms to mycelium growth are more unpredictable the dimensional tolerances are. In addition, the flex-
preserved. Little information exists about the effect of the sterilization ural Young's modulus is remarkably higher (0.67 MPa) for a liquid in-
method on the material properties. Schirp et al. (2006) found that oculum compared to a grain-based inoculation (0.25 MPa) (Holt et al.,
sterilized, inoculated straw was less thermally stable than unsterilized, 2012b).
inoculated straw (Schirp et al., 2006). The heat-treatment procedure Remarkably, liquid inoculation also results in a higher compressive
may also play a role in the defibrillation of the natural fibres, possi-
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strength (0.07 MPa) as compared to grain inoculation (0.002 MPa) (Fig.

bly resulting in an increased elasticity of the fibres (Fan et al., 2011). 3). This difference might be the result of the easier distribution of the
Heat may also facilitate the breakdown of plant cell walls and soften the fungal spores within the centre of the substrate, knowing that fungal fu-
chemical components, like lignin and hemicellulose, due to their differ- sion of filaments and enzyme expression occur in the centre of colonies
ent melting points (Fan et al., 2011). By applying high temperatures, rather than at the peripheries (Yang et al., 2017; Glass, 2004).
the lignin is plasticized and fibre separation occurs (Widsten and Kan-
delbauer, 2008b). Accordingly, the surface of the separated fibres is 3.4. Packing method
rich in lignin (Widsten et al., 2004).
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Secondly, the substrate can also be sterilized by addition of a 0.3% The air content influences the materials because high air content
(Lelivelt et al., 2015a) or 10% (Jiang et al., 2017) hydrogen per- causes a low thermal conductivity (Elsacker et al., 2019; Lelivelt
oxide solution. The mould and the equipment can be sterilized by et al., 2015a; Jahangiri et al., 2014). In addition, void spaces be-
rubbing an alcohol-based solution (70% ethanol) (Elsacker et al., tween the fibres also lead to a high porosity (Ahmadi, 2016). It is
2019; Jiang et al., 2017). This procedure might decrease the growth interesting to observe that the dry density of loosely packed materi-
rate of mycelium-forming fungi. Finally, unwanted microbial devel- als (Fig. 6d) with non-degraded natural fibres is considerably lower
opment can be avoided by co-cultivation of bacterial as compared to those without natural fibres (Yang et al., 2017). As
Table 1
Overview of the variety in protocols developed by different researchers, ordered alphabetically by fungal species.

Fungal Sterilization Inoculation Growth

species Feedstock method method Packing method Growth conditions time Drying method Application Ref

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Coriolus Hemp hurd, wood chips, Boiling water 10 or 20% pre- Not specified Dark conditions. 90 to 100% 30 days Oven at 125 °C and dried for 2 h. Foam (Lelivelt et
(Trametes) hemp mat, hemp fibres, for 100 min or grown spawn RH. Fresh air and CO2 content al., 2015a)
versicolor non-woven mats, 0,3% cultivated on should be kept high, Room

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and hydrogen- rye temperature.
Pleurotus peroxide
ostreatus

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Ganoderma Sawdust wood Not specified Not specified In polymer bag 25–35 C̊ , 5.0 pH, and low light 14 days Heat processing above 70 °C, 5% (TRAVAGLINI
lucidum levels moisture content et al., 2016)

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Ganoderma Quercus kelloggii (Red Oak) Not specified Inoculated into Not specified Pending IP prevents full Not 220 °C for 120 min. 10–20% moisture Foams / The (Travaglini et
lucidum wood, macerated into moulds disclosure of the nutrient specified content, via convective heating using core of al., n.d.)

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5.0–15.0 mm chips solution and growth conditions. solar dryers. sandwich
structures
Ganoderma cellulose and cellulose-PDB Autoclaved at Agar plug N/A 25–30 °C and 70–80% RH 20 days Oven for 2 h at 60 °C Fibrous film (Haneef et al.,
lucidum and 120 °C for 2017)

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Pleurotus 15 min
ostreatus

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Ganoderma Cotton carpel sized to be Sterilized at Liquid Plastic mould and hand- 21 °C 5 days 60 °C convection oven for 8 h Moulded (Holt et al.,
sp. within the range of 0.1 to 115 °C for inoculum and packed packaging 2012b)

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51 mm. 28 min grain inoculum material
Irpex lacteus Macerated sawdust pulp of Pasteurization Not specified Loose packing, naturally Not specified 14–42 days Dryer at 60 °C for 24 h. Foam (Yang et al.,
Alaska birch of 5 mm or deposited without 2017)

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smaller in size, millet grain, compaction and dense
wheat bran, a natural fibre, packing with approximately

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and calcium sulfate. twice the original volume of
materials packed

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Not Biotex Jute, Biotex Flax, Sterilized by Pre-grown Thermoforming in plastic 24 °C 5 days Dried completely in a convection The core of (Jiang et al.,
specified. BioMid cellulous plain 10% hydrogen inoculum – a mould. oven at 82 °C for 12 h and 93 °C for sandwich 2017)
Developed weave peroxide kenaf/hemp 8 h. Thermally pressed and dried for structures

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by solution mix and a corn 20 min at 250 °C to the specified
Ecovative stover/hemp thicknesses.
Design, LLC

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mix (both 50/
(Green 50% by weight)
Island, NY)

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Not Woven jute textile + Not specified Filled with A wooden male mould and Not specified 5–7 days Microwave heating, IR (infrared) The core of (Jiang et al.,
specified. natural fibres as inoculated core thermoformed plastic oven heating, heated tooling sandwich 2013)
Developed reinforcement, agricultural material. female mould (conduction), IR lamp heating, a structures

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by waste material (e.g., Kenaf combination of a heated tool & IR
Ecovative pith) as core lamp heating, and, finally, air drying.

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Design, LLC
(Green
Island, NY)
Not Rice husk 50% + wheat 121 °C for Not specified Polypropylene container Not specified Not Drying machine at 50 °C for 46 h. Foam (Arifin and
specified. grain 50%, Rice husk 15–20 min specified Yusuf, 2013)
Developed 70% + wheat grain 30%,
by Rice husk 30% + wheat
Ecovative grain 70%
Design, LLC
(Green
Island, NY)
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Table 1 (Continued)

Fungal Sterilization Inoculation Growth

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species Feedstock method method Packing method Growth conditions time Drying method Application Ref

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Not Not specified. Developed by Not specified Agar plug Tiles Not specified Not Dried at “elevated temperature”, for Tiles (Islam et al.,
specified. Ecovative Design, LLC specified “several hours” 2017)
Developed (Green Island, NY)

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by
Ecovative

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Design, LLC
(Green
Island, NY)
Oyster sp. Cotton seed hulls Not specified Crushed Pressed by hand into a self- Dark and humid room. Then the 5–7 days Dried in the oven Building (He et al.,

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mycelium made glass mould plastic wrap was removed for boards 2014)
ventilation to supply oxygen.

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Pleurotus Beech, European oak pear Autoclaving Malt extract Not specified 25 °C– 28 °C 14–28 days Baked at 95 °C until it weighs ≤50% Construction (Moser et al.,
ostreatus and spruce processed into peptone agar of its original weight material 2017)

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and Fomes wood chips of 0.2–5.0 mm plugs
fomentarius and 0.75–3.5 mm + ratios

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of sand or gravel aggregates
Pleurotus A standard Northern Not specified Organic rye Not specified 20–25 °C, pH 5–8, 80% RH, 5–25 days Oven at 55 °C for 2 h Foam (Ahmadi,

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djamor Bleached Softwood Kraft grain spawn Darkness 2016)
(NBSK) pulp sheets
Trametes Beech sawdust and Not specified Pre-grown Plastic thermo-formed 25 °C in the dark for 14 days. 24 days Heat (150 °C) or cold (20 °C) pressing Particleboards (Appels et al.,

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ochracea rapeseed straw, substrate moulds (34 × 34 × 4 cm, Plates were demoulded and kept was performed with a mechanical / foam 2018a)
and supplemented with bran. PET-G). Hand-pressed to at the same conditions for 10 multi-plate press for 20 min at

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Pleurotus Non-woven low-quality distribute the substrate as more days. Humidity of 55–70% F < 30 kN. Materials exposed to heat
ostreatus cotton fibre. uniform as possible and pressing were cooled at room
covered with perforated temperature, whereas non-pressed or

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cellophane foil cold-pressed materials were dried at
environmental conditions for 24–48 h

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Trametes Flax, flax dust, flax long Autoclaved at 10% grain The 20% wt of fibres, 70% In a micro box with a depth- 16 days 70 ºC for 5 to 10 h Thermal (Elsacker et
versicolor treated fibres, flax long 121 C
̊ for spawn wt of sterile demineralised filtration system at 28 ºC for insulation al., 2019)
untreated fibres, flax waste, 20 min H2O and 10% wt of 8 days. After 8 days, the

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wheat straw dust, wheat mycelium spawn were samples were demoulded in the
straw, hemp fibres and pine mixed together and put in laminar flow and incubated in a

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softwood shavings the PVC moulds micro box for another minimum
of 8 days without mould
10 E. Elsacker et al. / Science of the Total Environment xxx (xxxx) 138431

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Fig. 6. Laboratory conditions for growing mycelium-materials. a. Inoculation method of sterilized fibres with mycelium on grain spawn in a laminar flow, b. Drying method by convection
heating, c. Growth chamber with specific environmental conditions. d. Loosely packed material with a mix of natural fibres and mycelium spawn. e. Fully grown sample after a second
growth phase out of the mould which allowed a homogenous colonization on the sides that were previously in contact with the mould.(a-c: ©Elise Elsacker, d-e:Reprint with permission
©Søren Jensen Consulting Engineers)

the hyphae grow, the fractional volume of the mycelium increases, first loading, resulting in a more compact material. The stress-strain
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which leads to a densely packed material (Ahmadi, 2016). curve exhibits strain-softening behaviour for densely packed samples
Pre-compressing the materials during the manufacturing influences and strain-hardening behaviour when loosely packed (Yang et al.,
their mechanical response in terms of compressive stiffness, resulting 2017). Dense samples exhibit higher stiffness than loose samples and in
in a Young's modulus that is 2.4 times higher for pre-compressed loose general Young's moduli are higher than shear moduli.
hemp, 4.3 times higher for pre-compressed loose flax, and 4.6 times Hyphae grow aerial and when encountering a PVC mould verti-
higher for pre-compressed loose flax waste, compared with the non-com- cally along the surface to reach for oxygen, forming an engulfing hy-
pressed equivalent (Elsacker et al., 2019) (Fig. 3). The porosity phal protective layer in contact with air. Therefore, a second growth
and high content of air also explain the increase in stress and stiff- phase out of the mould allows a homogenous colonization on the sides
ness after reloading the materials in compression tests (Lelivelt et that were previously in contact with the mould (Fig. 6e) (Elsacker
al., 2015a) because the air is pushed out during the et al., 2019). This results in a barrier where the produced
 E. Elsacker et al. / Science of the Total Environment xxx (xxxx) 138431 11

heat during the growth is unable to dissipate in the central parts of the temperatures such as 360 °C (Ahmadi, 2016). More extensive incuba-
material (Lelivelt et al., 2015a). It is unclear whether the accumu- tion times such as 42 days result in a negative impact on the elastic and
lation of heat in the centre of the materials slows down the formation shear moduli (Yang et al., 2017). This can be explained by the fur-
of hyphae. On the other hand, the mycelial outer layer results in low ther degradation of the substrate by the fungi, which is otherwise essen-
water absorption (24–30%) (Elsacker et al., 2019) compared to heat- tial to contribute to the elastic stiffness. In contrast, such a long incu-
and cold-pressed mycelium -composites (180% and 350%) (Girometta bation time has a positive impact on the compressive strength with the

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et al., 2019; Ziegler et al., 2016). largest absolute value increase from 350 to 570 kPa, or over 60%, for
The fibres' orientation and the surface direction of the sandwich pan- the pre-incubated in filtered bags and densely packed samples (Yang et
els influence the dimensional reduction. The length and width have a al., 2017). As the mycelium continues to grow, the spaces between the

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higher reduction (4–5%) compared to the thickness reduction (0.06%) fibres are filled, the substrate is bonded more strongly together, and the
because of the chosen packing method (Jiang et al., 2016). Young's density is enlarged. The prolonged incubation time positively influences
and shear moduli in the horizontal direction are much higher than the Poisson's ratio, varying between 0.25 and 0.5, with less scattering.
those in the vertical direction (20 to 60%), so a strong elastic modulus The Poisson's ratio in the horizontal direction is larger than in the verti-
anisotropy is induced by the horizontal layers or fabric formed during cal direction (Yang et al., 2017).
packing and a tough layer is formed on the circumferential surface of
the materials (Yang et al., 2017). 3.7. Drying method

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3.5. Growth conditions There are numerous ways, durations and temperatures to dehy-
drate and denature the materials (Table 1), such as microwave heating
The optimal growth conditions are reached under specific environ- (Jiang et al., 2013), infrared (IR) oven heating (Jiang et al., 2013),
mental conditions which differ for every fungal species/strain on a dif- IR lamp heating (Jiang et al., 2013), a combination of a heated tool
ferent substrate (Fig. 6c). The incubation temperatures vary between & IR lamp heating (Jiang et al., 2013), oven backing at 60 °C–125 °C
21 and 30 °C (Lelivelt et al., 2015a; Holt et al., 2012b) (Haneef et for 2 h (Haneef et al., 2017; Ahmadi, 2016) (TRAVAGLINI et al.,
al., 2017; Moser et al., 2017; TRAVAGLINI et al., 2016; Ahmadi, 2016) (Moser et al., 2017) (Lelivelt et al., 2015a), convection

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2016; Jiang et al., 2017), depending on the species. An overview of heating (Fig. 6b) (using solar dryers) at 60 °C for 8 h (Holt et al.,
the different temperatures, related to the species and feedstock is given 2012b), at 82 °C for 12 h (Jiang et al., 2017) or at 220 °C for 2 h
in Table 1. Few studies specify the exact moisture level of the substrate, (TRAVAGLINI et al., 2016), thermal pressing at 250 °C for 20 min
yet proprietary knowledge indicates a hydration level of approximately (Jiang et al., 2017), the use of a drying machine at 50 °C for 46 h (Ar-
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66% (Ross, 2016). The total weight after hydration is composed of 2/3 ifin and Yusuf, 2013) or at 60 °C for 24 h (Jiang et al., 2013), and
water and 1/3 substrate. The relative humidity content typically ranges finally, air drying (Jiang et al., 2013).
between 70 and 100% (Haneef et al., 2017; Lelivelt et al., 2015a; The dried samples are not 100% dry due to the labs' conditions
Ahmadi, 2016; Yadav and Tripathi, 1991). The pH level ranges be- which contain humid air. The moisture content of the dried materials is
tween 5 and 8 (TRAVAGLINI et al., 2016; Ahmadi, 2016). The in- between 0.6% and 20%. In order to extract most of the moisture, the
cubation is mostly performed in the dark, in a controlled environment samples should be dried in an oven with air circulation. As expected,
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where gas exchange and air circulation are possible. the drying process influences the thermal conductivity of the materials.
High material densities are obtained under a low CO2 concentration As living materials consist of a high moisture content, the thermal con-
and in the absence of light or at a high CO2 concentration in the pres- ductivity is higher with more varying amounts of moisture compared to
ence of light (Appels et al., 2018b). Mycelium requires O2 to grow and thoroughly dry materials where moisture is replaced by air (Yang et
produces CO2. A low CO2-content is an indication for a basidiomycete al., 2017). Finally, when the mycelium growth stops by thermal treat-
that an outside surface is reached, triggering the formation of fruiting ment, its filaments are not anymore supported by the internal hydrosta-
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bodies. Therefore, the CO2 content should be kept high (Lelivelt et al., tic pressure, and for this reason they appear to be flatten (Haneef et al.,
2015a). The Young's modulus doubles as a result of increased CO2 lev- 2017). Therefore, elastic moduli of live samples are smaller compared
els in the light, while the opposite effect is observed in the dark. Similar to the dried samples (Yang et al., 2017).
outcomes are noted with the tensile strength (Appels et al., 2018b).
3.8. Post-processing
3.6. Growth time
Several post-processing methods exist for the mycelium materials, in-
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The incubation time depends on the size for the material, and is usu- cluding re-growth, the applications of natural resins and coatings. De-
ally determined in-situ due to the poor predictability. Growth periods pending on the function of the material, different types of coatings can
can range from 5 to 7 (Holt et al., 2012b; He et al., 2014; Jiang et be used. To render the bio-based composite weather-proof, natural oils
al., 2017; Jiang et al., 2013), 14 (Yang et al., 2017; TRAVAGLINI and shellac (a natural polymer) represent two options to seal the pores
et al., 2016), 20 (Haneef et al., 2017), 25 (Ahmadi, 2016), 30 (Le- of the material (Insights Into Mycelium||Critical Concrete, 2018).
livelt et al., 2015a) to 42 days (Yang et al., 2017; Moser et al., For example, the bricks of the Hy-Fi tower by David Benjamin were
2017). An overview of the growth period, related to the species and subjected to a natural oil penetration (Mycotecture? More-mycelium
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feedstock, is given in Table 1. in Construction. In: Muck, Waste, Trash, Emissions......................

Materials which have grown during an increased period are less to Money?, 2019) and Philip Ross uses both linseed oil and shel-
porous and therefore result in a decrease of the hydraulic permeabil- lac to preserve and protect his fungi-based furniture. He sanitizes the
ity (e.g. 0.40 m2 after 5 days as compared to 0.20 m2 after 25 days) material by means of essential oils prior to the addition of the coat-
(Ahmadi, 2016). Similarly, the increased period of mycelium growth ing (Scott, 2012). In another study, edible films were applied made
improves the thermal stability. The best result in the thermal decom- of carrageenan, chitosan and xanthan gum to create a physical bar-
position was obtained after 25 days of mycelial growth, which de- rier to water with antimicrobial properties
layed the onset of thermal decomposition upon exposure to higher
12 E. Elsacker et al. / Science of the Total Environment xxx (xxxx) 138431

(López Nava et al., 2016). No significant positive impact was wit- Block Research Group (Insights Into Mycelium||Critical Concrete,
nessed. If however the aim is to maintain the porous identity of the 2018), a spatial construction made from load-bearing mycelium compo-
material to, for example, allow a wall to breath, other solutions are nents with a mix of sugarcane and waste of cassava roots (average com-
proposed by Maurizio Montalti. Nanoparticles, such as nanocellulose, pressive stress of 0.61 MPa at 5% deformation). Compared with other
would be able to penetrate the pores and simultaneously keep the struc- load-bearing materials such as brick masonry (average compressive
ture open (Insights Into Mycelium||Critical Concrete, 2018). Besides strength 5.7 MPa) or concrete (average compressive strength 22.5 MPa),

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from the constructional point of view, other industries provide differ- mycelium-based materials cannot compete. MOGU's mycelium-based
ent possibilities. A moisture barrier strategy based on water-insoluble floor tiles made from a bio-based coating comply to the CE standard EN
proteins is developed by the leather industry. These proteins include 14041 for Resilient Floor coverings ([Link], 2019b), complying to

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corn zein, wheat gluten, and fish myofibrillar protein, which are ex- the requirements of slip resistance, thermal resistance, emission of dan-
tracted from agricultural by-products (Deeg et al., n.d.). Apart from gerous substances into indoor climate and electrical behaviour.
weather-proof applications, natural resins can be used as well to in- The current mushroom industry shows high potential to transition-
crease the material's mechanical properties. Both strength and stiffness ing towards the production of mycelium-based materials. Recently, the
are improved by infusing a bioresin into the reinforcement skins of the company CNC Exotic Mushrooms announced a partnership with Ecova-
mycelium composite when used as a sandwich structure (Jiang et al., tive to implement mycelium materials further in the European market
2017; Jiang et al., 2014). Vacuum infusing processing is therefore an (Deeg et al., n.d.). The spawn producer Mycelia also hosts research
adequate and widely used method (Jiang et al., 2014). However, a and development of mycelium-materials (Mycelia BVBA, n.d.). Grimm

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wax should be added to the material's core's edges to prevent excessive and Wösten describe that mycelium materials can be part of the produc-
resin absorption; a significant impregnation will result in unfavourable tion process of edible mushrooms (Grimm and Wösten, 2018). Indeed,
characteristics for a sandwich structure (Jiang et al., 2014). the mushroom industry offers the infrastructure and knowledge on the
material, whereas the design companies active in the field of mycelium
4. How do the material properties impact the applications? materials can provide future perspectives to the industry while taking
advantage of its facilities.
The versatility and high potential of the material leads to a broad

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spectrum of innovative applications and services, which can be classified 4.2. Design sector
in different sectors, with the construction and design sector being the
most prominent. In addition, the development of mycelium-based mate- Not only “classical” materials with application as construction ma-
rials is also expected to be an important proof-of-concept application in terials can be envisaged. The American companies Mycoworks (My-
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creating societal awareness for the transition to a circular economy. coworks Homepage, n.d.) and Bolt Threads (Bolt Threads, n.d.) are
currently developing mycelium-based leather. Mushroom leather grown
4.1. Construction sector with the fruiting body of the fungi Phellinus ellipsoideus are sold as
Muskin by Life Materials (Scott, 2012). Apart from leather, various
Currently, the composites are commercially developed as packag- artists and designers created all types of objects, such as mycelium-based
ing materials, insulating panels and acoustic tiles by Ecovative De- textiles used in fashion by Aniela Hoitink (Neffa, n.d.), 3D-printed fur-
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sign (Atila, 2016), Krown Design (Krown – Beautiful Products With niture by Eric Klarenbeek (Klarenbeek, n.d.), grown lamps by Jonas
Fungus and Biomass, n.d.) and MOGU (Home/MOGU • MOGU, Edvard (Edvard, n.d.), and even shoes, backpacks and book covers by
n.d.). The Italian company MOGU also offers a range of commercial Maurizio Montalti (Officina Corpuscoli, n.d.) (Fig. 7).
mycelium-based products on the European market, suitable for inte-
rior design applications (Home/MOGU • MOGU, n.d.). Acoustic pan- 4.3. Educational sector
els are sold with a Noise-Reduction-Coefficient of 0.4–0.53 ([Link],
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2019a) as a unique alternative for commercial acoustical ceiling tiles Mycelium materials are a gratifying subject to stimulate transdis-
(NRC 0.644) (Pelletier et al., 2017). Moreover, the mycelium-based ciplinary collaborations between scientists and citizens about sustain-
acoustic products also perform well as thermal insulation panels able and future-oriented innovative solutions because the components
(0.05 W/mK), being a possible substitute for polystyrene (0.03–0.04 W/ are easily and locally accessible. Worldwide, organisations are devel-
mK) and polyurethane (0.006–0.18 W/mK) foam. The engineering office oping workshops and educational materials for children, disadvantaged
Arup is developing sound-absorbing surfaces for interior fittings in col- groups, teachers, citizens and designers with do-it-yourself (DIY)
mycelium materials (Mycelium Material Workshops – Daniel Par-
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laboration with the start-up company MOGU (Bio-composite Systems

for Interior Fit-outs - Arup, 2019). Usage of the material as a build- nitzke, n.d.; Grow-it-yourself Mushroom Material Workshop – Part
ing block has shown encouraging results: this was demonstrated by us- 2, 2018; Mycelium Materialen Workshop, 2018; Training, n.d.).
ing mycelium bricks in construction for the annual pavilion Hy-Fi of Several worldwide active communities of citizen experts discuss and
The Living for MoMA Ps1 (Schaak and Lucht, 2016; Glass, 2004). share their experiments, for example, BioFabForum (BioFabForum,
Designer Pascal Leboucq created The Growing Pavilion as an ode to n.d.), and Fungal Materials and Biofabrication (Fungal Materials and
the beauty and power of biobased materials (Leboucq, 2019) (Fig. Biofabrication, n.d.). Such digital platforms are open source data-
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7). The experiments by Philip Ross, one of the first artists to explore bases enabling experts and hobbyists to share inspiration, best-prac-
the potential of mycelium as a material for design, show the ability tices and manuals. The domestication of this technology empowers
of a mycelium material to grow components together or to incorpo- many transdisciplinary collaborations in finding sustainable, ethical,
rate other elements, such as wooden beams (Ross, n.d.). The Amer- and affordable material solutions for many wasted resources. It nour-
ican architects' office Redhouse (Jahangiri et al., 2014) goes one ishes a decentralised manufacturing approach, as a substitute for the
step further by using construction waste, such as wooden panels or complex and moral issues on patents and IP control. Moreover, the
window frames that are crushed into flakes. In Indonesia the com- richness of an open and distributed approach can be of economic,
pany Mycotech (Yadav and Tripathi, 1991) manufactures panels social and environmental
and has also produced a prototype called “MycoTree” together with
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Fig. 7. a. The Growing Pavilion during DDW at the Ketelhuisplein in Eindhoven by Pascal Leboucq in collaboration with Erik Klarenbeek's studio Krown Design (Reprinted with permission
of ©Oscar Vinck and Company New Heroes). b. Mycelium Plates. c. Mycelium Vases (b-c: Reprint with permission ©Officina Corpuscoli / Maurizio Montalti).

benefit for localities and industry through the rapid diffusion and adop- 5.1. Future perspectives
tion of the technology (Raworth, 2017). The regenerative potential of
circular production here is the creation of various unique local solutions Crucial knowledge gaps and many questions still remain in spite of
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with global open knowledge commons. the rapid growth of our understanding during the last decade. Future
studies in various fields are therefore recommended.
5. Discussion In the field of biology further research is needed to understand which
factors and diffusible signals regulate the formation and differentiation
Altogether, the discussed studies provide important understandings of hyphae during the growth. How can the robustness of hyphae be im-
about the impact of every process parameter on the final characteristics proved by influencing the growth process? Also, material characteris-
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of the materials. In the past, very little attention was given to asses all tics could be further diversified by exploring fungal (phylo-)genetic di-
the variables individually, which led to many gaps in the manufacturing versity, taking into account the varying growth properties and cell wall
description. The main contribution of this paper is the disentanglement composition.
of the different process parameters, ranging from the fungal morphol- Progressions in the field of synthetic biology involve the investiga-
ogy, feedstock type to processing conditions and mechanical materials, tion of genetically engineered fungal strains by, for example, removing
and the demonstration of the strong interrelationships between this mul- specific natural functions with the purpose to better predict the fabrica-
titude of parameters. The framework (Fig. 8) for mycelium-based mate- tion process and improve the characteristics of the mycelium materials.
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rial production was derived from three underlying categories: I) Compo- If living matter is accomplished through engineering, it would be inter-
nents II) Processing variables III) Applications. In contrast to existing re- esting to understand how those new materials can contain the unique
views, this framework accounts for the complexity and interdependence qualities of the organisms, such as self-organisation, adaptation, respon-
of many factors. Overall, Fig. 8 illustrates the unique potential of the siveness, entropy and decay. Indeed, attempting to engineer living mat-
framework to deploy more tuneable levels in the fabrication process of ter brings about a whole new set of challenges, like the understanding
mycelium-based materials, specifically, to work towards a standardized of the evolutionary selective pressure, and how this information can be
production at the building scale. The featured physical, mechanical and used to develop new tools and protocols with stable final material prop-
chemical characteristics serve to prototype a diversity of new unprece- erties.
dented applications of bio-hybrid matter. Looking at the feedstock, many lignified plant biomass sources can
be used for the production of mycelium-materials. How can nat
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Fig. 8. Illustrative framework of the main factors affecting the production methods of mycelium-based materials and the resulting impact on the characterisation. The framework encom-
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passes the factors from I) Components II) Processing variables III) Applications. a) Influence of the fungal species. b) Impact of the lignocellulosic feedstock on the properties. c) Substrate
sterilization method. d) Inoculation method. e) Packing method. f) Growth conditions. g) Growth time. h) Drying method. i) Post-processing. j) Impact of the properties on the applications.

ural fibres be engineered to improve the mechanical and physical prop- Considerably more work is needed to optimise the structural prop-
erties of the material, for example by layering fibre mats or adding erties of mycelium-materials, by for example heat-pressing (densifica-
supplements such as inorganic components, nutrients or nanoparticles? tion), sandwich structures containing wooden beams, nanoparticles or
Since some fungi are known to detoxifying substrates containing aro- improvement of the inner growth.
matic compounds it is feasible to use waste-stream feedstocks containing Future research in design and architectural practices can concen-
pollutants and microplastics. trate on how the unique temporality of these materials can be de-
ployed as a quality, not as definitive decay, but as a process of re
 E. Elsacker et al. / Science of the Total Environment xxx (xxxx) 138431 15

generation, reintegration in the cycle. How can the properties of living SB PhD fellowships to Elise Vanden Elsacker (grant number 1S36417N)
organisms serve as design rules? Furthermore, from the environmental and Simon Vandelook (grant number 1SC9220N)].
point of view, the material's sustainability is currently praised but re-
quires however a scientific substantiation. References

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