Jof 08 01125 v2

Journal of
Fungi
Article
Mechanical, Physical, and Chemical Properties of
Mycelium-Based Composites Produced from Various
Lignocellulosic Residues and Fungal Species
Worawoot Aiduang 1,2,3 , Jaturong Kumla 2,3 , Sirasit Srinuanpan 2,3 , Wandee Thamjaree 4 ,
Saisamorn Lumyong 2,3,5, * and Nakarin Suwannarach 2,3, *
1 Applied Microbiology (International Program), Department of Biology, Faculty of Science,

Chiang Mai University, Chiang Mai 50200, Thailand
2 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
3 Research Center of Microbial Diversity and Sustainable Utilization, Chiang Mai University,
Chiang Mai 50200, Thailand
4 Department of Physics and Materials Science, Faculty of Science, Chiang Mai University,
Chiang Mai 50200, Thailand
5 Academy of Science, The Royal Society of Thailand, Bangkok 10300, Thailand
* Correspondence: [email protected] (S.L.); [email protected] (N.S.); Tel.: +66-865127518 (N.S.)
Abstract: Mycelium-based composites (MBCs) are characterized as biodegradable materials derived

from fungal species. These composites can be employed across a range of industrial applications
that involve the manufacturing of packaging materials as well as the manufacturing of buildings,
furniture, and various other household items. However, different fungal species and substrates can
directly affect the functional properties of MBCs, which ultimately vary their potential to be used in
Citation: Aiduang, W.; Kumla, J.; many applications. In this study, the mechanical, physical, and chemical properties of MBCs made
Srinuanpan, S.; Thamjaree, W.; from four different fungal species (Ganoderma fornicatum, Ganoderma williamsianum, Lentinus sajor-
Lumyong, S.; Suwannarach, N. caju, and Schizophyllum commune) combined with three different types of lignocellulosic residues
Mechanical, Physical, and Chemical (sawdust, corn husk, and rice straw) were investigated. The results indicate that differences in
Properties of Mycelium-Based both the type of lignocellulosic residues and the fungal species could affect the properties of the
Composites Produced from Various obtained MBCs. It was found that the MBCs obtained from sawdust had the highest degree of density.
Lignocellulosic Residues and Fungal
Moreover, MBCs obtained from S. commune with all three types of lignocellulosic residues exhibited
Species. J. Fungi 2022, 8, 1125.
the highest shrinkage value. The greatest degree of water absorption was observed in the MBCs
https://doi.org/10.3390/jof8111125
obtained from rice straw, followed by those obtained from corn husk and sawdust. Additionally,
Academic Editors: Shangxian Xie, the thermal degradation ability of the MBCs was observed to be within a range of 200 to 325 ◦ C,
Susie Dai and Charles M Kenerley which was in accordance with the thermal degradation ability of each type of lignocellulosic residue.
Received: 15 September 2022 The greatest degrees of compressive, flexural, impact, and tensile strength were observed in the
Accepted: 22 October 2022 MBCs of G. williamsianum and L. sajor-caju. The results indicate that the MBCs made from corn
Published: 25 October 2022 husk, combined with each fungal species, exhibited the highest values of flexural, impact, and
tensile strength. Subsequently, an analysis of the chemical properties indicated that the pH value,
Publisher’s Note: MDPI stays neutral
nitrogen content, and organic matter content of the obtained MBCs were within the following ranges:
with regard to jurisdictional claims in
published maps and institutional affil-
4.67–6.12, 1.05–1.37%, and 70.40–86.28%, respectively. The highest degree of electrical conductivity
iations. was observed in MBCs obtained from rice straw. Most of the physical and mechanical properties
of the obtained MBCs were similar to those of polyimide and polystyrene foam. Therefore, these
composites could be used to further develop relevant strategies that may allow manufacturers to
effectively replace polyimide and polystyrene foams in the future.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland. Keywords: agricultural residues; bio-fabrication; biodegradable materials; fungal mycelium;
This article is an open access article residues valorization
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
J. Fungi 2022, 8, 1125. https://doi.org/10.3390/jof8111125 https://www.mdpi.com/journal/jof

J. Fungi 2022, 8, 1125 2 of 21
1. Introduction
Agricultural production has expanded by more than threefold in the last 50 years in
response to global population growth and increases in food demand [1]. The increase in
agricultural production, processing, and consumption has generated a large quantity of
lignocellulosic residue each year [2]. Between 2003 and 2013, Asia has been recognized
as the greatest producer of global agricultural residue (47%), followed by America (29%),
Europe (16%), Africa (6%), and the Oceania region (2%) [3]. Global agricultural residues
are expected to rise to around 2.2 billion tons annually by the year 2025 [4]. Additionally,
the manufacture of forestry wood products can also generate a large amount of residue
(e.g., bark, chips, slabs, and sawdust) [5]. The effective management of agricultural and
wood residues has always been a major concern. Generally, residues are burned and
dumped in landfills in developing countries, particularly in Asia because these are some
of the most convenient and cost-saving methods of residue management. On the other
hand, burning has resulted in a range of air pollution problems that involve increasing
emissions of carbon monoxide (CO), carbon dioxide (CO2 ), and particulate matter (PM10
and PM2.5 ) [6,7]. Air pollution can potentially cause a range of serious health risks, en-
vironmental pollution, and economic problems on both local and regional levels across
the globe [2,8,9]. Accordingly, multiple new research studies have focused on reducing
and recycling agricultural and wood residues into useful and valuable products using a
circular bio-economic approach [10]. Both agricultural and wood residues are defined as
lignocellulosic components that are comprised of cellulose, hemicellulose, lignin, and other
polysaccharides [11]. These residues can be applied in the development of raw materials
that can then be used in the production of other high-value-added products including
animal feed, biofuels, enzymes, and value-added fine chemicals [12–14]. Remarkably,
lignocellulosic residues have also been recognized as a source of nutrients that can facilitate
microbial growth [14].
Turning lignocellulosic residues into biomaterials is a fundamental component in the
process of establishing a circular bio-economic platform [15]. The development of biomate-
rials, which is largely based on lignocellulosic residues, has various potential advantages
that include their diminished environmental impact and the utilization of renewable re-
sources [16]. Mycelium-based composites (MBCs) are a type of biomaterial that holds great
potential for the goal of using agricultural residues in specifically beneficial ways and in the
broader embrace of fungal biotechnology [17]. Saprobic fungi can degrade lignocellulosic
residues into nutrients through certain bio-fabricated processes wherein their mycelia
networks can effectively combine substrate particles together [18]. Ganoderma, Pleurotus,
Pycnoporus, and Trametes are the most commonly used fungal genera in the production of
MBCs [19–21]. Interestingly, MBCs have successfully been employed in the development
of mycelium-based materials that can be applied in the construction of buildings and in the
manufacturing of furniture, packaging, and various other household items [20–23]. These
materials have demonstrated their potential in the mission to replace plastics, synthetic
foams, and some wood composites with eco-friendly and biodegradable materials that
exhibit the appropriate characteristics of ecologically sound sustainable materials [20–24].
Interestingly, MBCs have several major advantages over classical lignocellulosic composites
in that they contain greater amounts of chitin and exhibit higher Young’s modulus and
lower elongation capabilities [21,25]. Presently, the Ecovative Company sells packaging
and board products that are made from MBCs and that are sold on a commercial basis [19].
Foam-like materials made from MBCs are marketed under the name MycoFlexTM [19].
Furthermore, mycelium-based construction materials derived from MBCs have been devel-
oped on a laboratory scale in several cumulative forms, including block materials, particle
board, acoustic materials, thermal insulation, cladding materials, surface materials (thin
sheets and film), and paste materials [19,26–31]. MBCs are generated from various forms of
lignocellulosic residues and fungal species that are available in each country [27,32]. How-
ever, different species of fungi and different substrate types can directly affect the properties
of the final products and the functional aspects of the resulting MBCs [23,24,27,32]. There-
J. Fungi 2022, 8, 1125 3 of 21
fore, this study aimed to produce MBCs from different forms of lignocellulosic residues
(including sawdust, corn husk, and rice straw) and fungal species (Ganoderma fornicatum,
Ganoderma williamsianum, Lentinus sajor-caju, and Schizophyllum commune). Prior to this
study, there have been no reports on MBCs produced from these four selected fungal
species. Therefore, this is the first report of MBCs produced from these fungi. Subsequently,
the mechanical (compression, tensile, flexural, and impact strengths), physical (density,
average shrinkage, thermal degradation, and water absorption), and chemical (final pH
value, electrical conductivity, organic matter content, and nitrogen content) properties
of the obtained MBCs were evaluated. The results of this study can provide valuable
information in the production of MBCs and can be employed to enhance relevant strategies
for the eco-friendly recycling of agricultural residues as well as to fulfill the long-term goal
of replacing plastic and foam in the future.
2. Materials and Methods

2.1. Sources of Fungal Strains and Culture Conditions
Pure cultures of four fungal species, namely, Lentinus sajor-caju CMU-NK0427, Gano-
derma fornicatum CMU-NK0524, Ganoderma williamsianum CMU-NK0540, and Schizophyl-
lum commune CMU-S01, were obtained from the culture collection of the Research Center of
Microbial Diversity and Sustainable Utilization, Faculty of Science, Chiang Mai University,
Thailand. All fungal strains were cultivated on potato dextrose agar (PDA; Conda, Madrid,
Spain) and incubated at 30 ◦ C for 7 days.
2.2. Sources of Lignocellulosic Residues and Preparation

Three different wood and agricultural residues, including the sawdust of rubber tree,
corn husk, and rice straw, were selected and used as substrates in this study. These forms
of residues were obtained from a sawmill and agricultural areas located in Chiang Mai
Province, Thailand. All selected residues were dried in an oven maintained at 60 ◦ C until
they were completely dry. Each substrate was then ground in a wood chipper and sieved.
Particles of about 5–20 mm in size were collected and used in this study.
2.3. Preparation of Mycelium-Based Composites and Mould Design

2.3.1. Inoculum Preparation of Fungal Mycelium
Mycelial inoculum of each fungal species was prepared using sorghum grains. The
sorghum grains were washed and boiled for a period of 20 min. After being allowed to
cool, 100 g of the boiled grains were put into glass bottles that were plugged with cotton
wool and then autoclaved at 121 ◦ C for 20 min. Afterward, the bottles were allowed to cool
down to room temperature over a period of 24 h. Subsequently, mycelial plugs (1 × 1 cm)
of each fungal species obtained from colonies grown on PDA were then transferred into
bottles (5 plugs per bottle). The inoculated bottles were then incubated at 30 ◦ C in darkness.
After two weeks of incubation, the sorghum grains were observed to be completely covered
in fungal mycelia, which were then used as the inoculum [33].
2.3.2. Mycelial Growth on Substrates

Each substrate was supplemented with 5% rice bran, 1% calcium carbonate, 2% cal-
cium sulfate, and 0.2% sodium sulfate on a dry mass basis [34]. The mixtures were then
adjusted to a final relative humidity value of 60% by adding water. Five hundred grams of
mixed substrate was placed in polypropylene bags (3.50 inches wide and 12.5 inches long).
The bags were then sealed with cotton-plugged polyvinyl chloride pipe rings, covered
with pieces of paper, and autoclaved at 121 ◦ C for 60 min. After being allowed to cool
down to room temperature over a period of 24 h, five grams of each mycelial inoculum
was inoculated onto the top of the substrate of each bag. The ratio of fungal inoculum
to substrate mass was 1:100 (w/w). The inoculated bags were then incubated at 30 ◦ C in
darkness [35]. The fungal mycelia were observed to cover the substrate after 14–21 days
of incubation.
J. Fungi 2022, 8, 1125 4 of 21
darkness [35]. The fungal mycelia were observed to cover the substrate after 14–21 days
J. Fungi 2022, 8, 1125 of incubation. 4 of 21
2.3.3. Mold Preparation

2.3.3. For
Mold thePreparation
compression test, molds were made from cylindrical plastic boxes with a ratio
between the diameter and
For the compression height
test, moldsof were
2:1 (86made
mm fromin diameter and plastic
cylindrical 43 mmboxesin height)
with accord-
a ratio
ing to the
between method
the diameter described
and heightby Elsacker
of 2:1 (86etmmal. in
[36]. The mold
diameter andfor
43 the
mmwater absorption
in height) test
according
was
to themade
methodfrom a plasticby
described dish that was
Elsacker 85 mm
et al. [36]. in
Thediameter
mold for and
the13water
mm in height. Moreover,
absorption test was
the molds
made from employed
a plastic dish for that
the tensile,
was 85 flexural, and impact
mm in diameter and tests
13 mmwere designed
in height. from acrylic
Moreover, the
clear sheets
molds that were
employed for thecut into a flexural,
tensile, dumbbell-shaped
and impact segment (165designed
tests were × 19 mm,fromneckacrylic
57 × 13clear
mm,
sheets that were
a rectangular cut into
shape (127 a× dumbbell-shaped
12.7 × 3.2 mm), and segment (165 ×shape
a rectangular 19 mm, neck
(63.5 57 ×× 13
× 12.7 mm,
12.7 mm),a
rectangular shape (127 × 12.7 × 3.2 mm), and a rectangular shape (63.5
respectively, following the standard method of the American Society for Testing and Ma- × 12.7 × 12.7 mm),
respectively,
terials (ASTM) following
[26,37]. the standard method of the American Society for Testing and
Materials (ASTM) [26,37].
2.3.4. Mycelium-Based Composite Fabrication, Preparation for Testing, and Moisture
2.3.4. Mycelium-Based Composite Fabrication, Preparation for Testing, and
Content
Moisture Content
Each substrate that was colonized with each fungal mycelium was put into the mold,
Each substrate that was colonized with each fungal mycelium was put into the mold,
pressed using a unidirectional cold press machine (Shop press ZX0901E-1, New Taipei,
pressed using a unidirectional cold press machine (Shop press ZX0901E-1, New Taipei,
Taiwan) set at 0.5 MPa for 10 min, and incubated at 30 °C. After three days of incubation,
Taiwan) set at 0.5 MPa for 10 min, and incubated at 30 ◦ C. After three days of incubation,
MBCs were removed from the mold and incubated in a plastic box for another three days
MBCs were removed from the mold and incubated in a plastic box for another three
until the mycelia covered the sides that had come into contact with the mold. The obtained
days until the mycelia covered the sides that had come into contact with the mold. The
MBCs were then dried in an oven at 70 °C for 24 to 72 h until their mass was stabilized
obtained MBCs were then dried in an oven at 70 ◦ C for 24 to 72 h until their mass was
[36]. The moisture content of the MBCs was measured by following the standard method
stabilized [36]. The moisture content of the MBCs was measured by following the standard
of ASTM
method ofDASTM
644 [38].
D 644The[38].
percentage moisturemoisture
The percentage content of MBCsofwas
content MBCscalculated from the
was calculated
percentage of mass loss: Moisture content (%) = [(W1 − W2)/W1]
from the percentage of mass loss: Moisture content (%) = [(W1 − W2)/W1] × 100, (where × 100, (where W1 = orig-
inal mass of the sample and W2 = mass of the sample after being
W1 = original mass of the sample and W2 = mass of the sample after being oven dried). oven dried). Ten replica-
tions
Ten were performed
replications on each sample
were performed on eachforsample
each treatment. The driedThe
for each treatment. MBCs were
dried thenwere
MBCs kept
in desiccators for further experimentation (Figure
then kept in desiccators for further experimentation (Figure 1). 1).
Figure1.1.Samples
Figure SamplesofofMBCs
MBCsobtained
obtainedfrom
from Lentinus sajor-caju
Lentinus andand
sajor-caju corn husk
corn in this
husk study:
in this Samples
study: for
Samples
water absorption test (A). Samples for moisture content, density, shrinkage, and compression
for water absorption test (A). Samples for moisture content, density, shrinkage, and compression tests
(B). Samples
tests for impact
(B). Samples strength
for impact test (C).test
strength Samples for tensile
(C). Samples forstrength test (D). test
tensile strength Samples for flexural
(D). Samples for
flexural strength test (E). Scale bars
strength test (E). Scale bars = 2 cm. = 2 cm.
2.4. Scanning Electron Microscope Observations

All dried MBCs were cut into small squares (5 × 5 mm) using a scalpel. The samples
were then attached to a 10 mm2 stub adapter with 2 mm2 double-sided carbon tape
and coated with gold for 2 min under high vacuum mode. Then, the samples were
J. Fungi 2022, 8, 1125 5 of 21
subsequently examined and photographed with a scanning electron microscope (SEM)

JEOL JSM-5910 LV SEM (JEOL, Tokyo, Japan) using an accelerating voltage of 15 kV at the
Science and Technology Service Center, Faculty of Science, Chiang Mai University, Chiang
Mai, Thailand. The surfaces and cross-sectional characteristics of all obtained MBCs were
then examined.
2.5. Determination of Physical Properties

2.5.1. Density
Density was determined using the MBCs for compression testing. After the MBCs
were dried at 70 ◦ C for 24 to 72 h, the density was determined and calculated following
the standard method of the International Organization for Standardization (ISO) 9427 [36]
by the mass and volume of MBCs. Ten replications were performed on each sample for
each treatment.
2.5.2. Water Absorption

The water absorption test was performed according to the standard method of ASTM
C272/C272M-18 [39]. Before they were tested, samples of MBCs were dried at 70 ◦ C until
their mass was stabilized. The samples were then allowed to cool down in a desiccator
for 24 h. The initial mass of the samples was determined. Subsequently, samples were
submerged in deionized water for a total duration of 96 h. Samples were then weighed
after 12, 24, 36, 48, 60, 72, 84, and 96 h. An increase in mass was calculated by applying
the following formula: Mass increasing (%) = [(W − D)/D] × 100, (where W = wet mass,
D = dry mass). Each treatment was applied to each sample over ten replications.
2.5.3. Shrinkage
Shrinkage of the samples was determined and calculated based on wet and dry
volumes according to the method described by Elsacker et al. [36] and expressed as shrink-
age percentage (%) = (V1 − V2/V1) × 100, (where V1 = wet volume of the sample and
V2 = dry volume of the sample). Ten replications were completed for each sample in each
of the treatments.
2.5.4. Thermal Degradation

The degree of thermal degradation of MBCs was determined by thermogravimetric
analysis (TGA) with a thermogravimetric analyzer (Rigaku: Thermo plus EVO2). A mass
of each sample of approximately 10 mg was placed in an alumina crucible and heated from
25 to 600 ◦ C at a heating rate of 10 ◦ C/min in a nitrogen atmosphere. Five replications were
completed for each sample in each of the treatments.
2.6. Determination of Mechanical Properties

2.6.1. Compression Strength
Compression strength was determined following ASTM D 3501 [36] on a Hounsfield-
H10Ks (New York, NY, USA) load bench with a 10 kN capacity and a 1 kN load cell under
ambient conditions (25 ◦ C with 40 to 50% relative humidity). The tests were conducted
with controlled displacement at a rate of 5 mm/min. The load–displacement curve was
converted to a stress–strain curve using the following formulas to calculate the compressive
stress σ and the strain ε: Stress σ = F/A and Stress ε = ∆L/Lo, respectively (where F:
compressive force (N), A: original cross section of the specimen (mm2 ), ∆L: displacement
of the loading surfaces (mm), and Lo: original height of the test piece (mm)). Compres-
sion strength was reported in MPa units. Ten replications were generated for each of
the treatments.
2.6.2. Tensile Strength

Tensile strength was determined following ASTM D 638-14 [26]. The tests were
performed with a Hounsfield-H10Ks universal testing machine (New York, NY, USA) using
J. Fungi 2022, 8, 1125 6 of 21
an elongation rate of 2 mm/min and a maximum force of 1 kN. Data were analyzed in
order to obtain a stress–strain plot and to provide an indication of tensile strength with ten
replications applied to each sample in each treatment.
2.6.3. Flexural Strength

Flexural strength was determined following ASTM D 790-10 [26]. The flexural test
was performed with a Hounsfield-H10Ks universal testing machine (New York, NY, USA)
employing a three-point bending setup that employed the same machine using a cross-head
speed of 2 mm/min and clamp support distance of 40 mm. Ten replications were performed
on each sample in each of the treatments.
2.6.4. Impact Strength

Impact strength was determined by employing the Charpy impact test according to
the standard of ASTM D-256 [37]. Samples were loaded into the machine and exposed
to the pendulum until being fractured. The impact strength values were calculated by
dividing the energy by the cross-sectional area of the sample using the following formula:
Impact strength (kJ/m2 ) = K/A (where K = energy required to fracture the sample (kJ) and
A = cross-sectional area (m2 )). Ten replications were performed on each sample in each of
the treatments.
2.7. Determination of Chemical Properties

Samples of MBCs were ground into small pieces in a blender and sieved to less than
2 mm in particle size. Five grams of each sample was soaked in 50 mL distilled water
for 30 min. Electrical conductivity (EC) and pH value were measured with the use of
conductivity and pH meters, respectively. Moreover, total organic matter and nitrogen
content were determined according to the procedure described by Walkley and Black [40]
and the method employed by Kjeldahl [41], respectively, at the Agricultural Technology
Services Center, Faculty of Agriculture, Chiang Mai University, Ching Mia, Thailand. Five
replications were performed for all samples of each treatment.
2.8. Statistical Analysis

The data of each experiment were analyzed by one-way analysis of variance (ANOVA)
using the SPSS program version 16.0 for Windows. Duncan’s multiple range test was then
used to identify any significant differences (p ≤ 0.05) between the mean values.
3. Results and Discussion

3.1. Scanning Electron Microscope Observations and Moisture Content
The morphological characteristics of MBCs were examined by SEM. It was found that
the surfaces of all obtained MBCs were covered with fungal mycelia (Figure 2A–L). Based on
a visual assessment of photographs in this study, MBCs made from L. sajor-caju and grown
on all substrate types had a high degree of density in terms of mycelia when compared
with other fungal species. Based on an assessment of the cross sections of MBCs, the
fungal mycelia combined substrate particles through a series of mycelial networks and the
air-voids present within the composites (Figure 2M–O). The morphological characteristics
of the MBCs obtained in this study were similar to those described in several previous
studies [26,42]. Remarkably, all uninoculated substrate types were observed to be absent of
both fungal mycelium and air-voids (Figure 2P–R).
The moisture contents of the MBCs obtained in this study are shown in Table 1. The
results indicate that these MBCs were comprised of a moisture content of 61.23 to 74.51%
on a wet-mass basis. The moisture content varied depending on the fungal species and
the type of substrate. The obtained moisture contents were within the ranges reported in
previous studies at 59 to 80% on a wet-mass basis [43,44]. The results of this study agree
J. Fungi 2022, 8, 1125
with the findings of previous studies that reported that the moisture content of MBCs7 was
of 21
dependent upon the fungal species and the type of substrate [43–45].
Figure 2. Scanning
Scanning electron
electron microscopic
microscopic images
images of MBCs obtained in this study: The surfaces of
MBCs
MBCs produced
produced from Ganoderma fornicatum
from Ganoderma fornicatum with
with sawdust
sawdust (A),
(A), corn
corn husk
husk (E),
(E), and
and rice
rice straw
straw (I).
(I). The
The
surface
surface MBCs
MBCs produced
produced from
from Ganoderma
Ganoderma williamsianum
williamsianum with
with sawdust
sawdust (B),
(B), corn
corn husk
husk (F),
(F), and rice
and rice
straw (J). The surface MBCs produced from Lentinus sajor-caju with sawdust (C), corn husk (G), and
straw (J). The surface MBCs produced from Lentinus sajor-caju with sawdust (C), corn husk (G), and
rice straw (K). The surface MBCs produced from Schizophyllum commune with sawdust (D), corn
rice straw (K). The surface MBCs produced from Schizophyllum commune with sawdust (D), corn husk
husk (H), and rice straw (L). The cross sections of MBCs produced from Lentinus sajor-caju with
(H), and rice
sawdust (M),straw (L). The
corn husk (N),cross
and sections of (O).
rice straw MBCs produced
The Lentinus (P),
from sawdust
uncolonized sajor-caju with (Q),
corn husk sawdust
and
(M), corn husk (N), and rice straw (O). The uncolonized sawdust (P), corn husk (Q), and
rice straw (R). Arrows indicated substrate (s), fungal mycelia (m), and air-voids (av). Scale bar; rice straw
(A–
(R).
O) =Arrows
100 μmindicated
and (P–R)substrate
= 50 μm.(s), fungal mycelia (m), and air-voids (av). Scale bar; (A–O) = 100 µm
and (P–R) = 50 µm.
The moisture contents of the MBCs obtained in this study are shown in Table 1. The
results indicate that these MBCs were comprised of a moisture content of 61.23 to 74.51%
on a wet-mass basis. The moisture content varied depending on the fungal species and
the type of substrate. The obtained moisture contents were within the ranges reported in
previous studies at 59 to 80% on a wet-mass basis [43,44]. The results of this study agree
with the findings of previous studies that reported that the moisture content of MBCs was
dependent upon the fungal species and the type of substrate [43–45].
J. Fungi 2022, 8, 1125 8 of 21
Table 1. Moisture content, density, and average shrinkage of MBCs obtained in this study.
Parameters *
Substrates Fungal Species
Moisture Content (%) Density (kg/m3 ) Shrinkage (%)
Ganoderma fornicatum 62.01 ± 0.64 b 337.21 ± 13.36 ab 8.86 ± 1.17 b
Ganoderma williamsianum 61.34 ± 0.73 b 331.44 ± 9.39 a 8.40 ± 1.28 b
Sawdust
Lentinus sajor-caju 61.23 ± 0.53 b 340.31 ± 16.41 a 8.10 ± 1.89 b
Schizophyllum commune 64.23 ± 0.55 a 318.59 ± 8.14 b 10.83 ± 1.39 a
Ganoderma fornicatum 66.98 ± 0.51 b 232.11 ± 11.52 ab 12.64 ± 2.70 b
Ganoderma williamsianum 65.11 ± 0.59 c 239.54 ± 8.65 a 12.32 ± 1.36 b
Corn husk
Lentinus sajor-caju 64.94 ± 0.62 c 240.99 ± 15.61 a 11.91 ± 1.92 b
Ganoderma fornicatum 70.13 ± 0.65 bc 219.46 ± 8.29 a 14.26 ± 2.26 b
Ganoderma williamsianum 69.55 ± 0.48 c 221.05 ± 15.01 a 13.95 ± 0.80 b
Rice straw
Lentinus sajor-caju 70.48 ± 0.56 b 222.76 ± 2.81 a 13.26 ± 1.03 b
* The results are mean ± standard deviation. Different letters in the same column in each substrate type are
considered significantly different according to Duncan’s multiple range test (p ≤ 0.05).
3.2. Determination of Physical Properties

3.2.1. Density
The density values of the obtained MBCs in this study are shown in Table 1. The
obtained density values ranged from 198.84 to 340.31 kg/m3 . The highest degree of density
of MBCs included in this study was found in MBCs produced from sawdust (318.59 to
340.31 kg/m3 ), followed by corn husk (220.74 to 240.99 kg/m3 ) and rice straw (198.84
to 222.76 kg/m3 ). MBCs made from L. sajor-caju and G. williamsianum exhibited higher
degrees of density than the other fungal species. However, the lowest degree of density was
obtained from MBCs made from rice straw and S. commune. It was found that the obtained
density values in this study were within the ranges described in previous reports at 25 to
954 kg/m3 [19,21,26,35,36,39,46–48]. The results of this study are supported by the findings
of several previous studies, which reported that the density of MBCs was significantly influ-
enced by substrate type and fungal species [19,21,26,42]. This outcome is in accordance with
Tacer-Caba et al. [47] who reported that the density of the MBCs produced from A. bisporus,
G. lucidum, and P. ostreatus were grown on rapeseed cakes to produce MBCs with higher
degrees of density than composites grown on oat husk. However, low-density values were
observed in MBCs made from oat husk (25–38 kg/m3 ) and rapeseed cakes (41–58 kg/m3 )
derived from three different fungal species, all of which could potentially replace certain
synthetic foams, namely, polystyrene (11–50 kg/m3 ), polyurethane (30–100 kg/m3 ), and
phenolic formaldehyde resin foam (35–120 kg/m3 ) [19–21]. Moreover, several previous
studies have reported that the pressing process (cold and/or heated pressing) significantly
increased the resulting degree of density of MBCs [19,21,22,26,36,42,46]. Several previous
studies [19–21] have also suggested that the high density (ranging from 440 to 680 kg/m3 )
of MBCs could potentially replace wood-based products (plywood, wood particle board,
and wood insulation board). Moreover, the density of the MBCs was within the range of
lignocellulosic materials (94–1560 kg/m3 ) [49,50]. In this study, the density of the obtained
MBCs was similar to the density of polyimide foam (50 to 400 kg/m3 ) (Table 2).
J. Fungi 2022, 8, 1125 9 of 21
Table 2. Comparison of the properties of MBCs in this study with MBCs from previous studies,
as well as synthetic foams and wood-based composites (modified from Aiduang et al. [19] and
Jones et al. [21]).
Products *
MBCs
Synthetic Foams Wood-Based Composites
Properties
This Previous
PI PS PU PFR PP PW PB IB
Study Studies
198.84–
D (kg/m3 ) 25–954 50–400 11–50 30–100 35–120 895–920 400–800 600–800 170–430
340.31
18.18–
AS (%) 8.1–16.31 6.2–15.0 0.2–1.2 0.2–0.6 - - 1.0–2.5 1–25 0.3–10
30.28
105.07–
WP (%) 24.45–560 1.0–3.0 0.03–9 0.01–72 1–15 0.01–0.03 5–49 30.1–200 55–380
208.82
474.1–
TD (◦ C) 200–325 225–375 318–440 278–379 270–475 360–460 250–380 310–350 150–270
546.8
31.19–
CS (MPa) 0.25–1.87 0.03–4.44 0.6–1.4 0.03–0.69 0.002–48 0.2–0.55 8–25 1.8–3.4 0.1–1.21
48.29
TS (MPa) 0.20–0.87 0.01–1.55 0.44–0.96 0.15–0.7 0.08–103 0.19–0.46 31–41.4 10–44 10–100 0.35–1.38
FS (MPa) 0.06–0.32 0.05–4.40 0.59–1.36 0.07–0.70 0.21–57 0.38–0.78 22–23.2 35–78 1.5–7 2–2.5
IS (kJ/m2 ) 0.21–2.70 - 0.06–0.12 0.01–0.15 1.0–1.2 0.26–1.63 0.02–1 - - -
D = Density, AS = Average shrinkage, WP = Water absorption, TD = Thermal degradation, CS = Compres-
sion strength, TS = Tensile strength, FS = Flexural strength, IS = Impact strength, MBCs = Mycelium-based
composites, PI = Polyimide, PS = polystyrene, PU = polyurethane, PFR = phenolic formaldehyde resin foam,
PP = polypropylene, PW = plywood, PB = particle board, IB = insulation board and “–” = not reported.
* Bruscato et al. [48], Yang [51], Omnexus [52], Du et al. [53], Shi et al. [54], Wang et al. [55], Wei et al. [56],
Li et al. [57], Smirnov et al. [58], Dizon [59], Forest Products Laboratory [60], Stark et al. [61], Ashby [62],
MatWeb LLC. [63], Azahari et al. [64], Filip et al. [65], NPCS Board of Consultants & Engineers [66], Niu and
Wang [67], Jalalian et al. [68], Papadopoulou and Chrissafis [69], Tailor et al. [70], Deng et al. [71], Dou and
Rodrigue [72], Zhu et al. [73], Shen et al. [74], Da Costa Castro et al. [75], Handayani et al. [76], Goulart et al. [77],
Del Menezzi [78], Çolakoğlu and Colak [79], Jivkov et al. [80], Sinha et al. [81], Jamalirad et al. [82], Engineering
Toolbox [83], Fateh [84], Zabihzadeh [85], STRUCTAflor [86], Mawardi et al. [87], Acda and Cabangon [88],
Gößwald et al. [89], Ge et al. [90], Segovia et al. [91], Kallakas et al. [92], Harshavardhan and Muruganandam [93],
and Muthuraj et al. [94].
3.2.2. Water Absorption

The water absorption ability of the obtained MBCs was determined by submerging
the composites in water over a period of 96 h. It was found that the water absorption ability
of the MBCs made from rice straw increased over a 24 h period and slowly stabilized after
36 h, whereas the water absorption ability of the MBCs made from corn husk and sawdust
increased over a period of 60 h and slowly stabilized after 72 h (Figure 3). MBCs obtained
from rice straw displayed the highest degree of water absorption ability, followed by MBCs
obtained from corn husk and sawdust. The results also revealed that MBCs produced from
L. sajor-caju exhibited lower water absorption ability in all the substrate types, while MBCs
produced from S. commune exhibited a significantly high degree of water absorption ability
in all substrate types. This study found that the water absorption ability decreased when
the degree of density was increased. This result is in full agreement with the results of a
number of previous studies, wherein the high degree of density of MBCs was reduced in
accordance with their relevant water absorption ability [19,20,39,47]. After 96 h, the water
absorption ability (105.07–208.82%) was observed to be within the ranges described in
previous reports at 24.45 to 560% when left in contact with water for a period of 24–192 h
(Table 2).
absorption ability of the obtained MBCs and those of synthetic foams and wood-based
composites is shown in Table 2. The water absorption ability of the obtained MBCs was
within the ranges for wood particle and insulation boards, but was higher than that of
synthetic foams and plywood products. Furthermore, the obtained MBCs also exhibited
a water absorption capacity that was similar to those of lignocellulosic composites (53.6–
J. Fungi 2022, 8, 1125 148.8%) [103]. Remarkably, the high-water absorption ability of MBCs remains a major 10 of 21
challenge in terms of the effective applications of these materials.
Waterabsorption
Figure3.3.Water
Figure absorption ability
ability of of
thethe obtained
obtained MBCsMBCs produced
produced fromfrom sawdust
sawdust (A), corn
(A), corn huskhusk
(B),
and
(B),rice
andstraw
rice (C). Data
straw (C).are presented
Data as meansasand
are presented the error
means andbar
theaterror
each bar
point
atindicates the indicates
each point ± stand-
ard
the deviation.
± standard“*” indicates“*”
deviation. a significant
indicates a difference
significant according
difference to Duncan’s
according multiple range
to Duncan’s testrange
multiple (p ≤
0.05) at ≤
test (p each point.
0.05) at each point.
Several research studies have concluded that MBCs can be defined as hydroscopic materials,
while the water absorption ability of MBCs was influenced by the type of substrate and fungal
species. This characteristic is usually associated with a cellulose component (a large number of
accessible hydroxyl groups) and hydrophilic mycelium [19,21,28,29,32,36,42,95–97]. Therefore,
the differences in the water absorption abilities of various MBCs were found to be related
to differences in the chemical components of the composites. Accordingly, the water
absorption ability of MBCs in this study was influenced by the cellulose content in the
substrate, of which rice straw was found to contain higher cellulose content (39–43%
dry mass basis) than corn husk (30–35% dry mass basis) and sawdust (33–38% dry mass
basis) [98–101]. In addition, Robertson et al. [102] found that the absorption ability of
MBCs was reduced when smaller particle-sized substrates were used. An increase in the
hydrophobic mycelia of T. versicolor on the surface of MBCs resulted in a lower degree of
water absorption ability [39]. A comparison of the water absorption ability of the obtained
MBCs and those of synthetic foams and wood-based composites is shown in Table 2. The
water absorption ability of the obtained MBCs was within the ranges for wood particle
and insulation boards, but was higher than that of synthetic foams and plywood products.
Furthermore, the obtained MBCs also exhibited a water absorption capacity that was similar
J. Fungi 2022, 8, 1125 11 of 21
to those of lignocellulosic composites (53.6–148.8%) [103]. Remarkably, the high-water

absorption ability of MBCs remains a major challenge in terms of the effective applications
of these materials.
3.2.3. Shrinkage
The low average shrinkage value of a material can contribute to the shape stability
of the finished product [104]. In this study, MBCs obtained from rice straw exhibited the
highest degree of shrinkage, followed by MBCs obtained from corn husk and sawdust
(Table 1) when all fungal species were compared. MBCs obtained from S. commune in
all three substrate types showed the highest degree of shrinkage. The lowest shrinkage
value was observed in MBCs obtained from L. sajor-caju in all three substrate types, but
these values were not determined to be significantly different from MBCs made from
G. fornicatum and G. williamsianum. These results were supported by the outcomes of
previous studies, which found that the shrinkage value of an MBC can vary depending
on the substrate used [19,36,104]. The shrinkage values recorded in this study were within
the range of some previously studied MBCs and those of wood insulation board (Table 2).
Consequently, they could effectively be used to replace wood insulation boards.
3.2.4. Thermal Degradation

All of the MBCs obtained in this study were found to be similar in terms of the degra-
dation behavior of each lignocellulosic residue throughout all three stages of development
(Figure 4). The first stage (free and chemically linked water evaporation resulting in about
5% mass loss) was observed to occur between 25 and 150 ◦ C, followed by the second stage
(degradation resulting in about 70% mass loss) and the third stage (decomposition), both
of which were observed at temperatures in ranges of 200 and 325 ◦ C and 350 and 375 ◦ C,
respectively. The results of this study were similar to the outcomes of several prior studies,
which reported that the thermal degradation behavior of MBCs was comprised of three
stages identified as the first, second, and third stages, and were recorded at temperatures
within ranges of 25 to 200 ◦ C, 200 to 375 ◦ C, and at temperatures greater than 350 ◦ C,
respectively, all of which were related to the degree of thermal degradation of each used
lignocellulosic residue [26,42,48,95,105–108]. This study found that each uncolonized ligno-
cellulosic residue had a slightly slower mass loss than the MBCs in the same lignocellulosic
residues. This outcome was in accordance with the findings reported by Appels et al. [26]
and could be explained by the fact that fungal colonization can change the composition of
lignocellulosic residues that are susceptible to thermal degradation.
This study found that the thermal degradation values of corn husk (containing 30–35%
cellulose, 31–37% hemicellulose, and 8–14% lignin of dry mass basis) and sawdust (con-
taining 33–38% cellulose, 29–31% hemicellulose, and 28–29% lignin of dry mass basis and
33–38% dry mass basis) were similar [98–101], but they differed from rice straw (containing
39–43% cellulose, 23–25% hemicellulose, and 12–20% lignin of dry mass basis). These
results are supported by the findings of other previous studies, which reported that the
type of lignocellulosic residues had no influence on thermal degradation due to the similar
cellulose content [97,109–111]. The thermal degradation of MBCs was within the range of
most synthetic foams and wood products; however, this was not the case for the thermal
degradation of polypropylene and polyimide foams (Table 2).
peratures within ranges of 25 to 200 °C, 200 to 375 °C, and at temperatures greater than
350 °C, respectively, all of which were related to the degree of thermal degradation of each
used lignocellulosic residue [26,42,48,95,105–108]. This study found that each uncolonized
lignocellulosic residue had a slightly slower mass loss than the MBCs in the same ligno-
cellulosic residues. This outcome was in accordance with the findings reported by Appels
J. Fungi 2022, 8, 1125
et al. [26] and could be explained by the fact that fungal colonization can change the12com-
of 21
position of lignocellulosic residues that are susceptible to thermal degradation.
Thermogravimetric analysis of obtained MBCs produced from sawdust (A), corn husk
Figure 4. Thermogravimetric husk (B),
(B),
and rice straw (C).
3.3. Determination of Mechanical Properties

3.3.1. Compression Strength
The results of this study found that the compression strength of the obtained MBCs
varied depending on the different fungal species and the type of lignocellulosic residue
(Table 3). MBCs produced from sawdust for each fungal species showed higher degrees of
compression strength (1.59 to 1.87 MPa) than MBCs produced from corn husk (0.58 to 0.62)
and rice straw (0.25 to 0.36 MPa). MBCs produced from G. williamsianum and L. sajor-caju
exhibited higher degrees of compression strength when observed in the same substrate,
while MBCs made from S. commune displayed the lowest degree of compression strength.
The results of this study were in accordance with the outcomes of several prior studies,
which found that the compression strength of MBCs varied based on the different fungal
species and the type of lignocellulosic residue [19,21,22,36]. Tacer-Caba et al. [47] found
that the compressive strength of MBC produced from G. lucidum grown on both oat husk
and rapeseed cakes was higher than that of A. bisporus and P. ostreatus when grown on
the same substrate. MBC produced by P. sanguineus grown on pine sawdust had a higher
compression strength than P. albidus [48]. Angelova et al. [112] found that the compressive
strength of MBC of G. resinaceum grown on rose flower residues (1.03 MPa) was significantly
higher than when lavender straw (0.72 MPa) was used. Ghazvinian et al. [113] found that
the MBC of P. ostreatus grown on sawdust (1.02 MPa) had a higher compressive strength
than when straw was used (0.07 MPa). Additionally, Chan et al. [46] and Alemu et al. [18]
found that the act of pressing during the production process effectively increased the
compressive strength of MBCs.
J. Fungi 2022, 8, 1125 13 of 21
Table 3. Compression, tensile, flexural, and impact strengths of MBCs obtained in this study.
Parameters *
Substrates Fungal Species Compression Tensile Strength Flexural Strength Impact Strength
Strength (MPa) (MPa) (MPa) (kJ/m2 )
Ganoderma fornicatum 1.71 ± 0.03 b 0.34 ± 0.02 b 0.07 ± 0.00 bc 0.24 ± 0.00 b
Ganoderma williamsianum 1.85 ± 0.01 a 0.42 ± 0.01 a 0.09 ± 0.02 ab 0.28 ± 0.02 a
Sawdust
Lentinus sajor-caju 1.87 ± 0.03 a 0.44 ± 0.03 a 0.11 ± 0.02 a 0.30 ± 0.02 a
Schizophyllum commune 1.59 ± 0.02 c 0.20 ± 0.01 c 0.06 ± 0.01 c 0.21 ± 0.02 b
Ganoderma fornicatum 0.59 ± 0.01 b 0.67 ± 0.04 bc 0.19 ± 0.01 b 2.05 ± 0.05 c
Ganoderma williamsianum 0.62 ± 0.01 a 0.75 ± 0.06 b 0.28 ± 0.03 a 2.38 ± 0.12 b
Corn husk
Schizophyllum commune 0.58 ± 0.02 b 0.63 ± 0.06 c 0.18 ± 0.04 b 1.49 ± 0.08 d
Ganoderma fornicatum 0.33 ± 0.01 a 0.37 ± 0.04 b 0.10 ± 0.02 b 0.97 ± 0.10 a
Ganoderma williamsianum 0.36 ± 0.02 a 0.46 ± 0.03 a 0.15 ± 0.03 a 0.99 ± 0.07 a
Rice straw
Schizophyllum commune 0.25 ± 0.03 b 0.35 ± 0.01 b 0.07 ± 0.01 b 0.68 ± 0.09 b
* The results are mean ± standard deviation. Different letters in the same column in each substrate type are
considered significantly different according to Duncan’s multiple range test (p ≤ 0.05).
The compression strength value of the obtained MBCs with synthetic foams and wood
products is shown in Table 2. The obtained compressive strength in this study (0.25 to
1.87 MPa) was within the range of compressive strength obtained from previous reports
(0.03 to 4.44 MPa). Moreover, the compression strength values of the obtained MBCs were
within the range of synthetic foams, except for polypropylene, but were lower than the
compression strength values of plywood and particle board products. Thus, the obtained
MBCs might be appropriate for use in packaging and insulating applications that are
typically associated with synthetic foams and wood fiber insulation boards.
3.3.2. Tensile Strength

The tensile strength of MBCs obtained in this study was within the range of 0.20 to
0.87 MPa (Table 3). It was found that tensile strength was affected by the substrate type
and the fungal species. MBCs produced from corn husk showed higher tensile strength
than MBCs produced from rice straw and sawdust for all fungal species. Moreover, it was
found that MBCs produced from G. williamsianum and L. sajor-caju displayed a relatively
high degree of tensile strength. However, MBC made from S. commune had the lowest
tensile strength among all substrates. These results were supported by the outcomes of
several previous studies that found that the tensile strength of MBCs could be influenced
by both the substrate type and the structure of the mycelium binder network [21,22].
Appels et al. [26] found that the tensile strength values of MBCs made from cotton in
P. ostreatus were higher than those of rapeseed straw and beech sawdust. Accordingly,
previous studies have reported that the tensile strength of MBCs can be influenced by
the structure of the mycelium binder network, which varied depending on the type of
mycelium network involved. Generally, monomitic species were associated with lower
tensile strength values than dimitic and trimitic hyphal species [19,21,23,114]. Based on the
three main hyphal types, only the monomitic species displayed generative hyphae, while
the dimitic species formed two hyphal types (generative and skeletal hyphae), and the
trimitic species were composed of all three types of hyphae. Accordingly, binding hyphae
are thick-walled, dense, and hard, all of which contribute to the stiffness of the composite
material [19,21,115,116]. Typically, L. sajor-caju, G. fornicatum, and G. williamsianum have
a trimitic hyphal system, while S. commune has a monomitic hyphal system [117–119].
Therefore, MBCs made from S. commune in this study were associated with a low degree of
tensile strength. Appels et al. [26] found that MBC of T. multicolor (trimitic hyphal system)
exhibited higher tensile strength (0.04 MPa) than P. ostreatus (monomitic hyphal system)
(0.01 MPa) when grown on rapeseed straw. Moreover, Appels et al. [26] found that the
J. Fungi 2022, 8, 1125 14 of 21
heat-pressing process resulted in the highest degree of tensile strength for MBCs, followed
by the cold-pressing and non-pressing methods. The obtained tensile strength values of
MBCs in this study were within the tensile strength values obtained from previous reports
(0.01 to 1.55 MPa). These values were similar to those of polystyrene foam (0.15–0.7 MPa),
phenolic formaldehyde resin foam (0.19–0.46 MPa), and polyimide foam (0.44–0.96 MPa)
(Table 2). Therefore, the MBCs in this study may be used in the future to replace some
synthetic foams and wood fiber insulation boards in packaging and insulation applications.
3.3.3. Flexural Strength

The flexural strength values of MBCs obtained in this study are shown in Table 3.
It was found that the highest degree of flexural strength of MBCs was found in the
MBC produced from corn husk (0.18–0.32 MPa), followed by MBCs produced from rice
straw (0.07–0.15 MPa) and sawdust (0.06–0.11 MPa), respectively. These obtained flexural
strength values were within the ranges described in previous reports from 0.05 to 4.40 MPa
(Table 2). MBCs produced from G. williamsianum and L. sajor-caju exhibited a high degree of
flexural strength on all of the substrates used. The highest flexural strength of the obtained
MBCs in this study was discovered in the MBC made from corn husk and L. sajor-caju.
Appels et al. [26] and Jones et al. [21] suggested that the flexural strength of the MBC was
dependent upon the type of mycelia network and substrate type, as well as the pressing
method used. Lui et al. [120] and Chan et al. [46] found that the MBC made from G. lucidum
grown on cotton stalks resulted in a higher flexural strength value than that of Chinese
albizia sawdust. Moreover, MBCs made from P. ostreatus and rapeseed straw produced
higher flexural strength values than when cotton was used [26].
Subsequently, the type of mycelium binder network used resulted in differing values
of flexural strength [19,23,26]. Generally, MBCs produced from the trimitic fungal species
exhibited higher flexural strength than the monomitic species, which was in accordance
with their tensile strength behavior [20,22,114] and confirmed by the results of this study.
Trametes multicolor (trimitic species) exhibited higher degrees of flexural strength than
P. ostreatus (monomitic species) when rapeseed straw was used as a growing substrate [26].
Accordingly, the MBC exhibited a degree of flexural strength that was similar to that of
polystyrene foam (0.07–0.70 MPa), but this flexural strength value was lower than polyimide
foam, polyurethane foam, phenolic formaldehyde resin foam, polypropylene foam, and
wood products (Table 2). Therefore, in terms of flexural strength, MBCs might not be
appropriate for the structural applications that are generally associated with wood, but
they may serve to replace some of the packaging materials that are currently made from
polystyrene foam.
3.3.4. Impact Strength

The impact strength values of MBCs in this study ranged from 0.21 to 2.70 kJ/m2
(Table 3). MBCs produced from corn husk showed higher impact strength than MBCs pro-
duced from rice straw and sawdust for all fungal species. MBCs produced from G. william-
sianum and L. sajor-caju displayed higher degrees of impact strength than G. fornicatum
and S. commune when all other substrates were used. However, there have been no previ-
ously published reports on the impact strength of MBCs. This study found that both the
substrate type and the type of mycelia network had a considerable influence on impact
strength. MBCs produced from trimitic fungal species were associated with a higher degree
of impact strength than the monomitic fungal species due to the presence of thick-walled,
dense, and hard hyphae. Moreover, previous studies reported that the differences in impact
strength of the differing forms of lignocellulosic residues were dependent upon a number
of other factors, such as fiber and matrix strength, load transfer efficiency, fracture propa-
gation resistance, bonding strength, fiber distribution, and geometry [121,122]. In terms
of impact strength, MBCs produced in this investigation were similar to polyimide foam,
polyurethane foam, and phenolic formaldehyde resin foam (Table 2). Thus, the MBCs
J. Fungi 2022, 8, 1125 15 of 21
created in this study may be used to replace the synthetic foams that are used as insulation
and packaging materials in the future.
3.4. Determination of Chemical Properties

In this study, the chemical properties, including pH value, electrical conductivity (EC),
organic matter content, and nitrogen content of MBCs were investigated, and are presented
in Figure 5. The initial pH values of the three growing substrates used, including sawdust,
corn husk, and rice straw, were averaged at 7.71, 6.96, and 7.49, respectively. The final pH
values of the obtained MBCs in the sawdust, corn husk, and rice straw were within ranges
of 4.78–5.91, 4.67–5.81, and 5.28–6.12, respectively (Figure 5A). This indicates that the final
pH values of MBCs were significantly decreased from the initial pH value of the growing
substrate. This finding was in accordance with the outcomes of several prior studies, which
reported that the finished MBCs exhibited lower pH values than the initial pH values of the
J. Fungi 2022, 8, 1125 growing substrates [18,39,41]. Attias et al. [41] found that the pH levels of MBCs produced
15 of 21
from P. ostreatus and P. salmoneo (grown on woodchips of eucalyptus, pine, and apple trees)
significantly decreased (4.3–4.7) when compared with the initial pH values of all selected
substrates
values wasatgenerally
around 5–5.5.
causedThis decrease
by the in thedigestion
enzymatic pH values was generally
process that tookcaused by the
place during
enzymatic digestion process that took place during the
the growth of the fungal mycelia on the substrate [19,41]. growth of the fungal mycelia on the
substrate [19,41].
The pH
Figure 5. The pH value (A), electrical conductivity
conductivity (B), organic matter content (C), and nitrogen
content (D)
(D) of
ofobtained
obtainedMBCsMBCsand
andthetheinitial substrates
initial in in
substrates this study.
this Data
study. are are
Data presented as means
presented and
as means
and the error
the error bar atbar
eachat point
each point indicates
indicates the ± standard
the ± standard deviation.
deviation. Different
Different letters
letters in in the
the same same ex-
experiment
periment of each substrate
of each substrate type are considered
type are considered significantly
significantly different different
accordingaccording
to Duncan’sto Duncan’s
multiple multi-
range
ple range test
test (p ≤ 0.05).(p ≤ 0.05).
The initial
initial EC
ECvalues
valuesininthe
the sawdust,
sawdust, corn
corn husk,
husk, andandrice rice
straw straw before
before inoculation
inoculation were
were averaged
averaged at1.43,
at 1.08, 1.08,and
1.43,3.12
anddS/m,
3.12 dS/m, respectively.
respectively. After After complete
complete mycelium
mycelium coloni-
colonization,
zation, the ECwere
the EC values values were significantly
significantly increasedincreased inof
in all three allthe
three of the substrates
substrates used (1.14–
used (1.14–1.26 dS/m
in sawdust,
1.26 dS/m in 1.65–2.07
sawdust, dS/m
1.65–2.07in corn
dS/mhusk, and
in corn 3.55–3.94
husk, dS/m indS/m
and 3.55–3.94 rice straw) (Figure(Fig-
in rice straw) 5B).
Accordingly, Hwang Hwang
ure 5B). Accordingly, et al. [123] reported
et al. that thethat
[123] reported EC the
value
EC of the of
value mycelium-colonized
the mycelium-col-
substrate
onized (1.89 dS/m)
substrate (1.89 was
dS/m) higher than that
was higher of that
than the non-colonized substrate
of the non-colonized (1.12 dS/m).
substrate (1.12
dS/m). According to a number of previously published reports, an increase in the EC value
in the substrate that had been colonized by mycelium could be explained by the fact that
the substrate was degraded by an enzyme produced by the fungal mycelium, which also
increased the amounts of inorganic compounds and minerals that were present, indicat-
J. Fungi 2022, 8, 1125 16 of 21
According to a number of previously published reports, an increase in the EC value in

the substrate that had been colonized by mycelium could be explained by the fact that
the substrate was degraded by an enzyme produced by the fungal mycelium, which also
increased the amounts of inorganic compounds and minerals that were present, indicating
a positive correlation with the EC value [98,124,125].
The percentages of the organic matter content of both the uncolonized substrate and
MBCs are shown in Figure 5C. The results indicate that the organic matter content of
the mycelium-colonized substrate was lower than the initial growing substrate. MBCs
produced from L. sajor-caju and G. williamsianum exhibited low organic matter content
in all of the substrates used. These results are supported by the findings of a number of
previous studies, which found that the loss of organic matter in a substrate was caused by
the enzymatic degradation of organic matter and the losses of CO2 and H2 O that occurred
during mycelial metabolism and development [39,41,126]. Moreover, Attias et al. [41]
found that the mycelium colonization and development on a substrate are associated with
the reduced amounts of organic matter in that substrate. Accordingly, a high degree of
mycelium colonization and development resulted in low amounts of organic matter in
that substrate.
The nitrogen content values of the initial growing substrate and the obtained MBCs
are presented in Figure 5D. The results indicate that the nitrogen content of the obtained
MBCs was significantly higher than that of the initial growing substrate among all types.
The nitrogen contents of the obtained MBCs in this study (1.05 to 1.37%) were within the
range of 0.5 to 1.6% as reported in previous studies (Table 2). Similarly, Attias et al. [39,41]
found that the nitrogen content in MBCs was higher than for the control (non-colonized sub-
strate) by a 1.0–1.7-fold increase. The change in nitrogen content that occurred during the
mycelium growth process resulted from mycelium enzymatic digestion. Accordingly, nitro-
gen content is another factor that can be used to assess mycelium growth and development
potential [18,19,21,41].
4. Conclusions
MBC production offers the advantage of using lignocellulosic residues to manufacture
high-value-added products. In this study, three lignocellulosic residues and four different
fungal species, along with their mechanical, physical, and chemical properties, were inves-
tigated for the purpose of developing MBCs. It was found that the properties of MBCs were
directly affected by the type of substrate and fungal species involved. In terms of fungal
species, MBCs of G. williamsianum and L. sajor-caju exhibited higher physical (high density,
low water absorption, and low shrinkage) and mechanical properties (high compression,
tensile, flexural, and impact strengths) than that of G. fornicatum and S. commune. In terms
of substrate type, MBCs obtained from sawdust were associated with the highest degree of
density and compression strength, and the lowest values in terms of both water absorption
and shrinkage. However, MBCs made from corn husk exhibited the highest degrees of
flexural, impact, and tensile strength. Additionally, the thermal degradation ability of
MBCs was observed to be within a range of 200–325 ◦ C, which was similar to the ther-
mal degradation ability of lignocellulosic residues. Changes in the chemical properties of
MBCs were typically caused by the enzymatic digestion processes associated with mycelial
growth and the development of the fungus itself. The obtained MBCs were similar to
those of polyimide and polystyrene foams based mainly on physical and mechanical data.
However, problems of high water absorption and low impact strength were observed when
compared with several synthetic foams. Low mechanical properties were observed when
these composites were compared with wood products. Large-scale production remains a
major challenge that will need to be addressed and improved in the future. Importantly, a
deeper understanding of the biodegradable properties and the period of time required for
the complete degradation of the MBC would result from further investigations. Addition-
ally, further refinement of the standard analytical methods employed should take place
J. Fungi 2022, 8, 1125 17 of 21
in order to better evaluate the suitability of MBCs developed for each specific application,
particularly for those used to produce packaging and insulation materials.
Author Contributions: Conceptualization, N.S. and J.K.; methodology, W.A., N.S., J.K., W.T. and
S.L.; investigation, W.A.; N.S. and J.K., software, W.A. and J.K.; validation, W.A., J.K., W.T., S.S. and
N.S.; formal analysis, W.A., W.T. and J.K.; data curation, W.A., N.S. and J.K.; writing—original draft
preparation, W.A., J.K., W.T. and N.S.; writing—review and editing, W.A., J.K., W.T., S.S., S.L. and N.S.;
supervision, J.K., S.L. and N.S.; project administration, N.S.; funding acquisition, N.S. All authors
have read and agreed to the published version of the manuscript.
Funding: The authors gratefully acknowledge the TA & RA Scholarship, graduate school, and
partially supported by Fundamental Fund 2022 (FF65/067), Chiang Mai University, Thailand.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors are grateful to Russell Kirk Hollis for his kind help with the
English correction.
Conflicts of Interest: The authors declare no conflict of interest.
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1 Applied Microbiology (International Program), Department of Biology, Faculty of Science,

Abstract: Mycelium-based composites (MBCs) are characterized as biodegradable materials derived

J. Fungi 2022, 8, 1125. https://doi.org/10.3390/jof8111125 https://www.mdpi.com/journal/jof

2. Materials and Methods

2.2. Sources of Lignocellulosic Residues and Preparation

2.3. Preparation of Mycelium-Based Composites and Mould Design

2.3.2. Mycelial Growth on Substrates

2.3.3. Mold Preparation

2.4. Scanning Electron Microscope Observations

subsequently examined and photographed with a scanning electron microscope (SEM)

2.5. Determination of Physical Properties

2.5.2. Water Absorption

2.5.4. Thermal Degradation

2.6. Determination of Mechanical Properties

2.6.2. Tensile Strength

2.6.3. Flexural Strength

2.6.4. Impact Strength

2.7. Determination of Chemical Properties

2.8. Statistical Analysis

3. Results and Discussion

3.2. Determination of Physical Properties

3.2.2. Water Absorption

challenge in terms of the effective applications of these materials.

to those of lignocellulosic composites (53.6–148.8%) [103]. Remarkably, the high-water

3.2.4. Thermal Degradation

position of lignocellulosic residues that are susceptible to thermal degradation.

3.3. Determination of Mechanical Properties

3.3.2. Tensile Strength

3.3.3. Flexural Strength

3.3.4. Impact Strength

3.4. Determination of Chemical Properties

According to a number of previously published reports, an increase in the EC value in

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