Document [original]

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

https://doi.org/10.1186/s40694-022-00133-y

RESEARCH

Establishment ofthebasidiomycete Fomes

fomentarius fortheproduction ofcomposite

materials

Carsten Pohl1† , Bertram Schmidt1† , Tamara Nunez Guitar1, Sophie Klemm2 , Hans‑Jörg Gusovius3 ,

Stefan Platzk4 , Harald Kruggel‑Emden4, Andre Klunker5 , Christina Völlmecke5 , Claudia Fleck2* and

Vera Meyer1*

Abstract

Background: Filamentous fungi of the phylum Basidiomycota are considered as an attractive source for the biotech‑

nological production of composite materials. The ability of many basidiomycetes to accept residual lignocellulosic

plant biomass from agriculture and forestry such as straw, shives and sawdust as substrates and to bind and glue

together these otherwise loose but reinforcing substrate particles into their mycelial network, makes them ideal can‑

didates to produce biological composites to replace petroleum‑based synthetic plastics and foams in the near future.

Results: Here, we describe for the first time the application potential of the tinder fungus Fomes fomentarius for lab‑

scale production of mycelium composites. We used fine, medium and coarse particle fractions of hemp shives and

rapeseed straw to produce a set of diverse composite materials and show that the mechanical materials properties

are dependent on the nature and particle size of the substrates. Compression tests and scanning electron microscopy

were used to characterize composite material properties and to model their compression behaviour by numerical

simulations. Their properties were compared amongst each other and with the benchmark expanded polystyrene

(EPS), a petroleum‑based foam used for thermal isolation in the construction industry. Our analyses uncovered that

EPS shows an elastic modulus of 2.37 ± 0.17 MPa which is 4‑times higher compared to the F. fomentarius composite

materials whereas the compressive strength of 0.09 ± 0.003 MPa is in the range of the fungal composite material.

However, when comparing the ability to take up compressive forces at higher strain values, the fungal composites

performed better than EPS. Hemp‑shive based composites were able to resist a compressive force of 0.2 MPa at 50%

compression, rapeseed composites 0.3 MPa but EPS only 0.15 MPa.

Conclusion: The data obtained in this study suggest that F. fomentarius constitutes a promising cell factory for the

future production of fungal composite materials with similar mechanical behaviour as synthetic foams such as EPS.

Future work will focus on designing materials characteristics through optimizing substrate properties, cultivation

conditions and by modulating growth and cell wall composition of F. fomentarius, i.e. factors that contribute on the

meso‑ and microscale level to the composite behaviour.

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Open Access

Fungal Biology and

Biotechnology

*Correspondence: claudia.fleck@tu‑berlin.de; vera.meyer@tu‑berlin.de

†Carsten Pohl and Bertram Schmidt shared first co‑authorship

1 Chair of Applied and Molecular Microbiology, Technische Universität

Berlin, Str. des 17. Juni 135, 10623 Berlin, Germany

2 Chair of Materials Science and Engineering, Technische Universität

Berlin, Str. des 17. Juni 135, 10623 Berlin, Germany

Full list of author information is available at the end of the article

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Pohletal. Fungal Biology and Biotechnology (2022) 9:4

Introduction

Fungal biotechnology is an innovation driver for the bio-

economy with its principles of circular economy and sus-

tainability [1, 2]. Especially filamentous fungi have a rich

and very versatile metabolism that forms the basis for a

diverse palette of products, which become harnessed by

the food, beverage, pharmaceutical, biofuel, textile, feed,

automotive, packaging and chemical industries. How-

ever, filamentous fungi are not only masters of biosynthe-

sis, they are also masters of decomposition. Their ability

to degrade and transform lignocellulosic substrates into

composite materials is unique in nature and attracted a

lot of interest recently [1, 2]. In several interdisciplinary

endeavours, fungal bio(techno)logists, designers, process

engineers and material scientists have collaborated to

turn by-products from agriculture and forestry with the

help of basidiomycetes into composite materials as high-

lighted in recent reviews [3–5]. The vision is surprising

and fascinating, yet plausible and thus hopefully achiev-

able in the near future: Plastics, foams, textiles and other

materials derived from petroleum-based resources could

soon be functionally replaced by a new class of bioma-

terials produced by fungal biotechnology [2]. Given the

urgent need to reduce global carbon dioxide emission

and plastic pollution, the pressure to innovate is indeed

high. Within the last 5years, the ability of fungal myce-

lium not only to digest but also to bind and connect loose

plant-based particles into firmer composite materials

has thus led to a substantial increase in publications that

pioneered the manufacturing process and that described

some characteristics of mycelium-based materials [6–12].

Potential applications for fungal composite materials that

have been discussed so far are as diverse as disruptive—

soon packaging material, thermal insulation, acoustic

insulation, construction material as well as leather could

be produced by filamentous fungi of the phylum Basidi-

omycota [2–4, 13–15].

To contribute to these research efforts, we ran a bio-

prospecting program in 2018 in our Berlin-Brandenburg

area to explore the local biodiversity of mushroom-form-

ing fungi and to build up a strain collection of basidiomy-

cetes that reflects the predominant regional biodiversity

and that feeds well on regional renewable plant resources.

As recently described [16], we could isolate and identify

about 75 basidiomycetes, most of which were assigned

to the order Polyporales, including the tinder fungus

Fomes fomentarius, the fire sponge Phellinus robustus,

Ganoderma adspersum, the artist´s bracket Ganoderma

applanatum and the turkey tail Trametes versicolor. Also,

representatives of the order Agaricales became members

of the strain collection including the oyster mushroom

Pleurotus ostreatus, the stump mushroom Armillaria

ostoyae and the similar looking Pholiota limonella [16].

In growth experiments on different substrates from

regional agricultural residual streams, the white-rot fungi

F. fomentarius, P. ostreatus and T. versicolor excelled with

the best performance [16].

Various considerations let us to focus our further

research on the tinder fungus F. fomentarius. This basidi-

omycete, which is prevalent throughout the temperate

climate zone of the northern hemisphere, is well-known

to traditional medicine and thus has a rich ethnomyco-

logical tradition [17, 18]. Furthermore, the trama of its

fruiting bodies has been safely used by mankind for hun-

dreds of years as wound dressing and leather alternative

[19]. Remarkably, the fruiting bodies are water-repellent,

very stable and light-weighted. Interestingly, the hyme-

mium follows a hierarchically honeycomb structure and

was previously already subjected to mechanical test-

ing [20], showing compressive stress–strain curves of

foams, where an initially linear course is followed by an

extended plateau region [20]. Given that such charac-

teristics could be adjusted in the future for laboratory

cultivated F. fomentarius mycelia that were fed on renew-

able plant biomass, new materials for lightweight appli-

cations, specifically for anisotropic loading conditions

could be developed. Finally, the genome sequence of

one F. fomentarius isolate identified in France has been

recently published (strain CIRM-BRFM 1821) [21] and

uncovered many genes in its genome predicted to encode

lignin-active peroxidases and manganese peroxidases

which are key for the breakdown of lignin. As its genome

sequence contains less genes predicted to encode cellu-

lases, it grows less well on cellulose, which is typical for

white-rot fungi. F. fomentarius was thus recently ranked

with a moderate hyphal expansion rate on lignocellulosic

substrates but a high rate of decomposition of its sub-

strate when compared to another 20 basidiomycetes [22].

Another important premise for our decision was that F.

fomentarius grows well on local agricultural residues such

as hemp shives or rapeseed straw [16]. Hemp was once an

important source for fibres for the textile industry, but its

cultivation and use declined in the last century because

cotton and synthetic fibres became more popular. The

Keywords: Filamentous fungi, Fomes fomentarius, Circular economy, Bioeconomy, Hemp, Rapeseed, Composite

material, Mycelium, Neo‑Hooke model, Finite element method FEM, Mechanical properties, Compressive strength,

Stiffness

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Pohletal. Fungal Biology and Biotechnology (2022) 9:4

worldwide annual area of hemp harvested mainly for

seeds and fibres is reported with about 150,000ha for

2018 [23]. In Europe alone, the area under cultivation

has increased to over 40,000ha in recent years, which

represents a potential use for at least 60,000 t of shives

[23]. Hemp fibres currently experience a resurgence of

interest by the textile industry as an environmentally

friendly alternative to cotton, the cultivation of which is

high in water demand, pesticide use and soil salinization

[23]. In contrast, hemp is a frugal but high-yielding plant

that has no pesticide and low fertilizer demand but uses

water about six times more efficiently for biomass forma-

tion than cotton [24, 25]. Thus, hemp can grow well even

under hot and dry conditions and on poor-soil sites such

as prevalent in the Berlin-Brandenburg area and beyond

[26]. The second main product after hemp fibre separa-

tion, the shives, are currently very often under-valued in

applications like animal bedding. But with its content of

about 48% w/w cellulose, 21 to 25% w/w hemicellulose

and 17–19% w/w lignin [27], hemp shives are ideal sub-

strates for both white-rot and brown-rot basidiomycetes.

Rapeseed will remain an important source of oil pro-

duced for food and feed as well as technical use, although

it has a high water and fertilizer demand and the land use

efficiency can be regarded as critical in terms of biodiesel

production due to the low energy efficiency [28]. The

Food and Agriculture Organization of the United Nations

lists the harvested area of rapeseed as 36.96 Mio ha. In

Brandenburg, winter rapeseed is the most important oil-

seed crop with an acreage of about 77,000 ha [29], which

takes up about 10% of the arable land [30]. The composi-

tion of rapeseed straw is very similar to hemp shives with

about 37% w/w cellulose, 24% w/w hemicellulose and

about 17% w/w lignin [31] and thus well suited as a sub-

strate for both white-rot and brown-rot basidiomycetes.

In the current study, we describe the cultivation of F.

fomentarius on both hemp shives and rapeseed straw

for the production of composite materials. We applied

compression tests to determine the compressive Young’s

Modulus as recently described for composite materials

obtained with Schizophyllum, Ganoderma and Trametes

species, respectively [7, 32, 33] and used scanning elec-

tron microscopy to characterize the composite structure

and mechanical properties. We used the experimental

data for numerical simulations of the compression behav-

iour. We furthermore studied the impact of the substrate’s

particle sizes on the composite material properties and

used fine, medium and coarse fractions of hemp shives

and rapeseed straw to produce a set of diverse composite

materials. Their properties were compared amongst each

other and with the benchmark expanded polystyrene

(EPS), a petroleum-based foam used for thermal isolation

in the construction industry.

Results anddiscussion

Substrate preparation andclassification

The particle size of both hemp shives and rapeseed straw

substrates were reduced by means of a laboratory cutting

mill. To estimate the mass percentages of the subsequent

classification products, sieve analyses of the milling prod-

ucts were carried out using analytical sieves and shakers.

Rapeseed straw showed significantly larger amounts of

screening residue of mesh sizes above 8mm (Additional

file1). Furthermore, fine fractions below 0.63mm mesh

size were found at about 5% weight fraction. To achieve

a mass distribution of approximately one third each for

small, medium, and large fraction, the results suggested

classification cut sizes of 2mm and 3.15mm, given the

available screens. Consequently, these mesh sizes were

utilized during the following classification processes via

a Mogensen Sizer. Simultaneously, a 0.65mm screen was

used with the intention to reduce the finest particles such

as dust. Hence, three particle fractions were prepared for

each substrate: small (0.63–2mm), medium (2–3.15mm)

and large (> 3.15mm–6.3mm). The resulting mass per-

centages are shown in Fig.1.

Fig. 1 Mass distribution of plant substrate fractions after classification

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Pohletal. Fungal Biology and Biotechnology (2022) 9:4

Finally, bulk density was determined for all fractions

by measuring the total bulk volumes of the fractions

(Additional file2). Each fraction was subjected to further

analysis with respect to particle size and shape by means

of digital image analysis. A significantly larger number

of finest particles were found within all rapeseed straw

samples compared to hemp shives, possibly due to differ-

ences in abrasion resistance (note that results from image

analysis are based on the number of particles rather than

mass percentages). In Fig.2, histograms of the ratios of

minimum to maximum Feret diameters of hemp shives

and rapeseed straw middle fractions are depicted. While

the rapeseed straw’s modal value is smaller than the cor-

responding value for hemp shives, its distribution is

broader and leans towards larger Feret ratios. Within the

examined fraction, hemp shives display thinner and more

elongated shapes.

Cultivation ofF. fomentarius andmanufacturing

ofcomposite materials

F. fomentarius grows well on malt extract agar (MEA),

glucose-based complete medium (CM) and on lignocel-

lulosic substrates such as hemp shives and forms hyphae

with a mean diameter of 2.8µm (n = 300, SD = 0.7, Fig.3).

A three-stage laboratory manufacturing process was

established for F. fomentarius (for details see “Methods”

section). In the first stage, mycelium harvested from malt

agar plates (Fig.4A) was used to inoculate millet grains

Fig. 2 Histograms of minimum to maximum Feret diameter ratios for hemp shives (left) and rapeseed straw (right)

Fig. 3 A F. fomentarius colonies after incubation at 25 °C in the dark for 96 h and 186 h. Doubling time of colony surface area on MEA and CM are

6 h (n = 8, SD = 1) and 14 h (n = 8, SD = 2) respectively. As the fungus also grows into the shives and towards the bottom of the agar plate, it is

impossible to estimate a doubling time based on radial growth measurement when cultivated on hemp shives inoculated with pure F. fomentarius

mycelium or with millet spawn. B Light microscopic images of F. fomentarius hyphae when cultivated in liquid CM for 96 h and 186 h, respectively at

400× magnification

Page 5 of 13

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

to obtain precultures of F. fomentarius during a 2-week

cultivation (Fig.4B). This ‘millet spawn’ then served as

inoculum to inoculate 3-L bag cultures of hemp shives

and rapeseed straw for the second stage cultivation. For

future industrial upscaling efforts, however, we propose

that the millet preculture should be substituted by non-

food plant substrates that become mixed with non-inoc-

ulated plant substrates to avoid extensive use of cereal

grains. After the 2-week cultivation in substrate bags, the

overgrown substrates were shred and transferred into

sterile cylindrical moulds (Fig.4C and D), to allow for a

final cultivation with the duration of 2weeks, whereby

the moulds were removed after one week (Fig.4E). For

each condition tested (substrate, particle fraction), at

least six biological replicates were produced. The final

composite materials obtained with this manufacturing

process were optically inspected after cutting, revealing

a gradient of fungal growth within the test specimens

Fig. 4 Laboratory manufacturing process for F. fomentarius composite materials. A Inoculation of sterile millet with for F. fomentarius mycelium

followed by an incubation for 2 weeks at 25 °C in the dark. B Inoculation of hemp shives (or rapeseed straw) cultivation bags with the millet spawn

followed by an incubation for 1 week at 25 °C in the dark. Note that the use of millet spawn for inoculation has the advantage of good mixing

properties in the 3‑L cultivation bags used and thus generation of more homogeneous growth throughout the plant substrates. C Shredding of

preliminary hemp shives (or rapeseed straw) composites and transfer of the material into moulds. D Filled moulds before cultivation for 1 week at

25 °C in the dark. E Sample appearance after 1 week of cultivation. Moulds are removed to allow thorough overgrowth of the samples for another

week. F Drying in an oven at 60 °C for 2 days and final appearance of composites used for compression tests

Page 6 of 13

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

(Additional file3). The outer shell of the material is cov-

ered by a dense pure mycelium of F. fomentarius and

the inner part is less overgrown but still consists of suf-

ficient hyphal material that covers and embeds all plant

particles to keep them in place after cutting. Scanning

electron microscopy (SEM) revealed that the composite

material is formed by hyphae that form an isotropic net-

work andinteract with the hemp shives particles (Fig.5).

As for example described for Trametes versicolor and dif-

ferent types of agricultural feedstocks [33], these interac-

tions eventually define the mechanical properties of the

composite material at the microscale, something, which

remains to be shown for F. fomentarius in future stud-

ies. On the macroscale, it appears that the outer layer is

clearly beneficial to achieve a certain resistance against

abrasion (Additional file3).

Compression tests ofcomposites

Cylindrical composite specimens containing fine,

medium and large particles of rapeseed straw or hemp

shives where subjected to compression tests. At least six

biological replicates per condition were used to deter-

mine their deformation behaviour. The results were

compared with EPS. Hereby, the strain (the change in

shape, that is the decrease in height divided by the origi-

nal height of the specimen) depending on the stress (the

force acting on a cylinder divided by the original cross-

sectional area) was measured and expressed in stress–

strain curves. Note that during elastic deformation,

the relationship between stress and strain is linear and

reversible and described by the elastic modulus. With

further loading, above a yield point, a specimen becomes

plastically deformed or exhibits cracks, resulting in

permanent deformation even after unloading from the

previously applied force.

During compression loading up to 1.8 kN (i.e. a force

exerted by a weight of 180kg), all mycelium compos-

ites showed elastic–plastic deformation behaviour. The

stress–strain curves rise only slightly at the very begin-

ning due to deformation of the surface mycelium on

top of the composites. Following this region, the slope

increases continuously until the end of the experiment.

With increasing load, the specimens became clearly

deformed (Fig.6A, B). The elastic deformation recovered

immediately after unloading from the compression tests,

while a certain fraction of plastic deformation remained,

which is depicted in Fig.6B. While the specimens showed

nearly no cracks and visible damage on the surface, we

cannot exclude that cracks within the mycelium and/or

delamination at the mycelium-reinforcement interfaces

added to the remaining deformation.

Figures6C–D compare the mean stress–strain curves

with a confidence interval of 95% (shaded region) of com-

posite material with different substrate material in the

three different particle sizes large, medium and small.

Notably, a significant difference can be seen between the

individual particle sizes. Composites with large particle

size performed less well in terms of compression stability

compared to materials based on medium-sized particles.

The composite materials with small particle performed

the best, i.e. displayed the lowest elastic–plastic defor-

mation for the same stress. Compared to EPS (Fig.6E),

however, the stress–strain relationships for the fungal

composites scatter to a much greater extent, presumably

due to inhomogeneous growth of F. fomentarius around

and into the substrate particles and inhomogeneous sub-

strate characteristics (see Fig.5). However, when scaling

Fig. 5 SEM images of F. fomentarius grown on hemp shives. A Overview of mycelium embedding a central cluster of hemp shives (centre); B Close

up of hemp shives overgrown with mycelium, demonstrating that a dense mesh of mycelium connects the substrate particles. Sample specimens

were taken from the outer zone of a composite body

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Pohletal. Fungal Biology and Biotechnology (2022) 9:4

fungal composite production from laboratory to indus-

trial conditions, it will become possible to standardize

substrate density, e.g. by applying precompression and

weighing filled moulds, thus reducing biological varia-

tion. However, scatter as low as observed for purely syn-

thetic material such as EPS will likely not be possible due

to within- and between-subject variations which is an

inherent property of biological systems.

For calculation of the elastic modulus m, we decided to

evaluate the range of the curve, where the cross-section

of the samples was equally loaded, namely from 10%

strain (unequal loading is due to the inclination of some

samples) up to a strain value estimated by the elastic

recovery. The slope of this part of the curve corresponds

to the elastic modulus. Furthermore, the compres-

sive strength σst was evaluated at 20% strain. Figure7

and Additional file4 compare the elastic modulus and

the compression strength for the composites based on

different substrate particle sizes. Remarkably, as com-

pared to composites with hemp shives, composites with

Fig. 6 A Stress–strain curve of mycelium composite with medium sized substrate and optical micrographs highlighting the deformation at defined

strain levels. B Compression test sample with medium size substrate particles before (left) and after (right) compression. C–E Compression stress–

strain curves of (C) mycelium composite with rapeseed straw and (D) hemp shives of different particle sizes (RL, RM, RS—large, medium, small for

rapeseed straw; HL, HM, HS—large, medium and small for hemp shives) and E EPS

Fig. 7 The elastic modulus m (A) and the compressive strength σst (B) dependent on the particle sizes large (L), medium (M) and small (S) of

rapeseed straw and hemp shives, respectively. For additional data, see Additional file 5

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Pohletal. Fungal Biology and Biotechnology (2022) 9:4

rapeseed straw particles performed slightly better in the

medium and small size range (compressive strength of

0.103 ± 0.014MPa for medium and 0.134 ± 0.029 MPa

for small size compared to 0.072 ± 0.010 MPa and

0.104 ± 0.040MPa for medium and small hemp shives).

In contrast, composites based on large hemp shive par-

ticles performed better compared to those with large

rapeseed straw particles (compressive strength of

0.056 ± 0.005MPa for rapeseed straw, 0.072 ± 0.005MPa

for hemp shives). Interestingly, the influence of particle

size on the elastic moduli is the same as for the strength:

large hemp shive particles lead to a higher modulus than

large rapeseed straw particles, whereas medium and

small rapeseed straw particles lead to higher modulus

values as compared to hemp shive particles in that size

range.

The stress–strain behaviour of EPS is significantly dif-

ferent from the mycelium composites. It shows an elas-

tic modulus of 2.37 ± 0.17MPa, which is 4-times higher

compared to the F. fomentarius composite materials, but

it has a compressive strength of 0.09 ± 0.003MPa, which

is in the range of the fungal composite material. However,

if the load bearing capability at higher strains, e.g. 50% is

compared, the composites exhibit stress values compara-

ble to EPS.

Numerical simulations ofthecomposite material

behaviour

Numerical simulations of the compression tests on the

composite materials were performed using the finite ele-

ment method (FEM) [34]. One practical approach is to

consider the specimens as homogenous isotropic sol-

ids. However, given the large range of strains exhibited

by the specimens in the compression tests before plastic

deformation, the usage of a linear strain tensor in a model

is erroneous. Thus, fully non-linear kinematics must be

applied and a compressible variant of the hyperelastic

Neo-Hookean model [35] was chosen as the constitutive

model (see “Methods” section).

An example 3D visualization before a simulated com-

pression test is shown in Fig.8 and the force–displace-

ment simulation results are shown in Fig.9. Although the

model reflects the qualitative nonlinear behaviour well,

quantitative discrepancies can be identified. These are

expected and can be traced back to various reasons. Most

importantly, some important mechanical effects occur-

ring in the composite material are not reflected in the

homogenous Neo-Hookean model. This includes damage

as well as substrate debonding, but also surface effects

(see Additional file6) and differences between real speci-

men geometry and idealized cylindrical mesh geom-

etries. Furthermore, although slanted geometries have

been taken into account, the initial phase of the com-

pression test where the specimen settles and full contact

between stamp and specimen surface is established is not

sufficiently well reflected in the simulations. This can be

seen best in the data obtained for large rapeseed straw

particles (RL, Fig.9). A better representation of the speci-

men geometry could thus improve the simulation.

Remedy to the mechanical flaws of the model can

either be provided by explicitly simulating the compos-

ite including damage and debonding or using a dedicated

homogenous model specially developed for the novel

material combination at hand. Nevertheless, based on the

data the model used provides a good qualitative reflec-

tion of the composite’s behaviour in compression.

Fig. 8. 3D FEM simulation of compression tests. A Slanted mesh used in simulations to reflect the initial. B Deformed configuration due to contact

pressure, colouring represents equivalent stress in the specimen. For details regarding numerical simulations see “Methods”

Page 9 of 13

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

Conclusions

In this study, we investigated the impact of rapeseed

straw and hemp shives particle sizes on the characteris-

tics and compression behaviour of mycelium composite

materials produced by the basidiomycete F. fomentarius.

In general, the characteristics of mycelium compos-

ites are defined on multiscale levels. On the microscale

level (µm), the smallest mechanically effective compo-

nent of the material is the fungal network of hyphae that

form the matrix, and whose elastic properties are mainly

dependent on the cell wall chitin content [8]. On the

mesoscale level (mm) are reinforcing substrate particles

such as hemp shives and rapeseed straw that become

bonded randomly by the mycelial network. Bonding in

this heterogeneous material is discontinuous at the par-

ticle–matrix interface. The macroscale level is defined

by the size of the final mycelium composites and can be

measured by stress–strain curves. Our first simulation

analyses, based on experimentally obtained compres-

sion data, cannot reflect yet the mechanisms occurring at

Fig. 9 Force–displacement data as computed using FEM with the Neo‑Hookean model (dashed line) and experimental data (straight line). Particle

sizes are RL, RM, RS—large, medium, small for rapeseed straw; HL, HM, HS—large, medium and small for hemp shives

Page 10 of 13

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

the microscale and mesoscale and further experimental

investigations are thus necessary. However, the chosen

two-parameter model can qualitatively reflect the global

force displacement behaviour and thus serve as a base

model which can be augmented when additional experi-

mental data is available to achieve a better quantitative

agreement. To this end, a micromechanically informed

damage model may be included to reflect the decreasing

stiffness observed in some of the specimens. The differ-

ent particle sizes are represented by significantly differing

values for the constitutive parameters of the hyperelastic

model chosen for the FEM simulations.

Notably, the impact of particle size on compression

behaviour was more profound for large rapeseed straw

particles, whereas stress–strain curves of other combina-

tions of particle sizes and substrates appeared more clus-

tered. In addition, the stress–strain curves of mycelium

composites differed from stress–strain curves obtained

for EPS. On the one hand, less force is required for initial

deformation of the mycelium surface of the composites,

showing that surface damage of the material occurs more

easily than with EPS. However, when comparing the

ability to take up compressive forces, both hemp shive-

based and rapeseed-based composites take up more load

compared to EPS before being deformed. At 50% com-

pression, hemp-shive-based and rapeseed-based com-

posites were able to resist compressive forces of 0.2MPa

and 0.3MPa, respectively, whereas EPS only sustained

0.15MPa. Thus, the composite materials obtained with

F. fomentarius are in the same range or slightly above of

the values obtained for EPS. The data obtained in this

study thus suggest that F. fomentarius potentially con-

stitutes a promising cell factory for the future develop-

ment of fungal composite materials that could replace

synthetic foams such as EPS. However, other important

material properties such as thermal insulation, water

resistance, long-term stability, aging, biodegradability to

name but a few need to be studied and likely optimized

in future experiments. The advantage of composite mate-

rials based on F. fomentarius over synthetic ones is that

their characteristics can likely be easily modulated at the

nano- and micro-scale through variations of the cultiva-

tion conditions of F. fomentarius and thus its cell wall

composition [36].

Methods

Substrate preparation andclassification

Two types of lignocellulosic substrate were tested: hemp

shives (Hemparade) and rapeseed straw (Optistraw) from

European agriculture, both purchased from Futtermittel

Louven e.K. For each substrate, three particle fractions

were prepared: small (0.63–2mm), medium (2–3.15mm)

and large (> 3.15mm–6.3mm). Processing was tailored

to the specific requirements of the substrate type. Prelim-

inary size reduction was optionally carried out by means

of a laboratory cutting mill with an 8 mm discharge

screen, constraining the particle size distribution if nec-

essary. To achieve fractionation, each substrate was clas-

sified by using a three-deck Mogensen sizer with mesh

sizes of 0.63mm, 2mm, and 3.15mm. The densities of

the resulting fractions were determined by measuring

their total masses and bulk volumes. The fractions were

finally subjected to detailed analysis of particle size and

shape. The images were taken via a flatbed scanner and

digital microscopy; Zeiss Zen software was employed for

image analysis.

Isolation andcultivation ofF. fomentarius

The F. fomentarius isolate GaG41 is particularly suit-

able to produce composite materials from agricultural

raw materials, as described previously [16]. As growth

performance of fungi is often strain-dependent, we iso-

lated additional F. fomentarius strains form the Ber-

lin-Brandenburg area (Germany). One of them, strain

PaPF11, showed better growth rate on malt extract agar,

glucose agar medium, millet culture (used for millet

spawn production) and solid lignocellulosic substrates

such as hemp shives and rapeseed straw compared to

GaG41 (data not shown) and was thus used in the cur-

rent study.

In brief, strain PaPF11 was isolated using a fruiting

body collected from a birch tree trunk. Isolation was

performed by cutting slants (3 × 3 mm) from differ-

ent internal zones of the fruiting body, followed by dip-

ping into 4% H2O2 solution for 30s to reduce bacterial

burden. Slants were placed on malt extract agar plates

(Roth, Germany) supplemented with 50µg/ml ampicillin

sodium salt (Sigma-Aldrich) and 50µg/ml streptomycin

sulfate (Applichem) to suppress bacterial growth. Myce-

lium outgrown from the slants was transferred twice to

new plates using sterile toothpicks to obtain axenic cul-

tures. Strain identity was confirmed by Sanger sequenc-

ing of the internally transcribed spacer (ITS) region using

primer ITS1 (TCC GTA GGT GAA CCT GCG G) and ITS4

(TCC TCC GCT TAT TGA TAT GC) as described earlier

[37]. For strain maintenance, cultures were grown for

about 2weeks in the dark at 25–27°C followed by cold

storage at 2–8°C and subsequent transfer of mycelium

pieces to new medium plates. The same cultivation con-

ditions were also used for preparation of mycelium plates

for millet spawn inoculation.

Manufacturing ofcomposite materials

Strain PaPF11 was harvested from an agar plate after

5–7days of cultivation and used to inoculate a brown

millet culture (purchased from Mühle Schlingemann,

Page 11 of 13

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

Germany), which served as preculture to inoculate the

bulk solid substrate. Brown millet was supplemented with

1wt.% calcium sulfate dihydrate (Roth) and 150wt.% dis-

tilled water. The mixture was sterilized by autoclaving

(VX-150 autoclave, Systec GmbH, Germany) and incu-

bated for 14days at 25°C in the dark after inoculation.

Particle fractions from hemp shives and rapeseeds straw

were hydrated with 150wt% of water in separate cultiva-

tion bags (SacO2, Belgium) and autoclaved. 5wt% over-

grown millet spawn was added to the wet substrate and

mixed by kneading. The bags were then heat sealed and

incubated at 25°C in the dark. After 7days of incubation,

the bags were mixed to promote homogeneous growth

and incubation was continued for another 7 days. The

overgrown solid substrate was then crushed using a disin-

fected shredder (Rapid AXT 2000, Bosch, Germany) and

manually transferred into a plastic tube of 7cm diameter

and 6–7cm height which served as a cylindrical mould.

Note that it was impossible to control exactly the amount

of pre-compression of the crushed mycelium-substrate

mix to the tube. Therefore, for each condition tested

(substrate, particle fraction), six biological replicates

were produced. The samples were incubated for 7days

in the mould followed by another 7days after removing

the mould to allow surface growth of F. fomentarius. To

reduce the risk of contamination and ensure a high rela-

tive humidity of 80–100%, incubation was done in a disin-

fected closed plastic box (IKEA, Sweden) with two sterile

sponges soaked in sterile distilled water. Finally, growth of

F. fomentarius was stopped by drying the samples in an

oven (B5090E, Heraeus, Germany) at 60°C for 2days. The

weight and geometry parameters of the produced samples

were recorded, and the density of the specimen calculated

to access the reproducibility of this manufacturing process

(data not shown). As a reference for expanded polystyrene

(EPS), commercially available EPS plates (FIW, Germany)

with a thermal conductivity coefficient of 0.035W/mK

according to DIN 4102-1: B1 where cut to the same geom-

etry as fungal samples using a scalpel.

Microstructural characterisation

Scanning electron microscopy (SEM, CamScan Series

2, Obducat, Sweden) was performed to analyse hyphal

growth of F. fomentarius on lignocellulosic substrates.

In brief, SEM was used in the high vacuum, second-

ary electron mode with an accelerating voltage of 14kV.

The specimen was gold sputtered (Cressington Sputter

Coater, 108 Auto, Tescan GmbH, Dortmund, Germany)

for 40s at 30mA.

Compression testing

Cylindrical specimens (at least six biological replicates for

each substrate/particle fraction combination) underwent

compression testing in a universal testing machine type

0008.00 (UTS Testsysteme GmbH, Ulm, Germany) with a

crosshead speed of 10mm/min and a pre-load of 1N. Load

and displacement were measured by a 2kN load cell (res-

olution 0.01%) and the in-built displacement transducer

(resolution 0.001%). The dimensions of each specimen

were measured with callipers to calculate the stress σ and

the strain ε (note that stretch in material science is called

‘strain’ which is different from the meaning of the term

‘strain’ in microbiology). The tests ended automatically at a

load of 1.8 kN or earlier when a certain displacement (RS

32mm, RM 42mm, RL 44mm, HS 30mm, HM 35, HL 40)

was reached. Immediately after the test, the height of the

samples was determined.

The stress–strain curves were evaluated according to the

German standard for compression testing of foams DIN

50134:2008-10 [38]. The load–displacement curves were

converted to stress–strain curves, using the following for-

mulas to calculate the stress σ and the strain ε:

[MPa

]

and

�L/L0[

−

]

where F equals the compressive force

[N], A is the original cross-sectional area of the specimen

[mm2], ΔL is the obtained displacement [mm] and Lo cor-

responds to the original height of the specimen [mm].

Numerical simulations

A compressible variant of the hyperelastic Neo-Hookean

model [35] was chosen as the constitutive model to

describe the characteristics of the composite materi-

als. The associated hyperelastic potential, i.e. the strain

energy density, reads

Here,

i

are the principal stretches, and

and

are

material parameters which need to be identified using

experimental data. For the parameter identification pro-

cess based on the 1D experimental data at hand a uni-

axial simplification of the model is needed. The second

Piola–Kirchhoff stress tensor

is then given as deriva-

tive of the potential which in the present case of uniaxial

compression can be simplified to

The requirement that the specimen be stress free in

directions perpendicular to the loading direction (

)

leads to a closed form expression for the uniaxial stress

S33

as a function of the stretch



3

in loading direction:

(i)=c



2

1+2

2+2



−3−2 ln (123)+d(123−1)2

=2∂

∂C=2



∂

(



∂i

∂



∂C=



i

∂

∂i

ei⊗ei

33()=2c



1−1

2



+dψ()

3



2ψ()−1

,

Page 12 of 13

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

The above expression for the uniaxial stress is used to

fit the model to the average experimental stress strain

curve across specimens of identical substrate material

and particle sizes. Using this approach, the influence of

substrate type and particle size is entirely reflected in the

values of the two model parameters.

With the constitutive parameters

and

at hand

(Additional files 5 and 6) the compression tests are sim-

ulated using the open source finite element computing

platform FeniCS [39]. A penalty contact algorithm was

implemented together with slightly slanted top surfaces

of the specimen meshes according to measured geometry

data to model the compression boundary conditions.

Supplementary Information

The online version contains supplementary material available at https:// doi.

org/ 10. 1186/ s40694‑ 022‑ 00133‑y.

Additional file1: Particle size distribution.

Additional file2: Bulk densities of substrate fractions.

Additional file3: Cut section of a composite.

Additional file4: Elastic modulus and compression strengths of

composites.

Additional file5: Neo‑Hookean material parameters.

Additional file6: Comparison of the Neo‑Hookean model data with

experimental data.

Acknowledgements

We would like to thank Paul Zaslansky (Charité—Universitätsmedizin Berlin)

for the use of the SEM and acknowledge the German DEAL consortium for

open access funding.

Authors’ contributions

VM initiated this study which was jointly designed by all authors who also

jointly interpreted the data. SP fractionated hemp shives and rapeseed straw

substrates, BS and CP established all protocols for F. fomentarius cultivation

and composite material manufacturing, TNA produced cylindrical specimen

for composite material tests, TNA and SK performed compression tests, and

ASK the numerical simulations. CP and VM co‑wrote the manuscript and

were supported by all co‑authors with text contributions and discussions. All

authors read and approved the final manuscript.

Funding

Open Access funding enabled and organized by Projekt DEAL. This work was

partially funded by the Citizen science project ‘Mind the Fungi’ granted by the

Technische Universität Berlin to V.M.

Availability of data and materials

The raw datasets generated in this study are available from the corresponding

authors upon request.

Declarations

Ethics approval and consent to participate

Vera Meyer is an Editor‑in‑Chief of Fungal Biology and Biotechnology.

()=−



2+



+

d

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

1 Chair of Applied and Molecular Microbiology, Technische Universität

Berlin, Str. des 17. Juni 135, 10623 Berlin, Germany. 2 Chair of Materials Sci‑

ence and Engineering, Technische Universität Berlin, Str. des 17. Juni 135,

10623 Berlin, Germany. 3 Department of Post Harvest Technology, Leibniz‑

Institute for Agricultural Engineering and Bioeconomy (ATB), Max‑Eyth‑Allee

100, 14469 Potsdam, Germany. 4 Chair of Mechanical Process Engineering

and Solids Processing (MVTA), Technische Universität Berlin, Str. des 17. Juni

135, 10623 Berlin, Germany. 5 Stability and Failure of Functionally Opti‑

mized Structures Group, Technische Universität Berlin, Str. des 17. Juni 135,

10623 Berlin, Germany.

Received: 20 October 2021 Accepted: 27 January 2022

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