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Development of formaldehyde-free bio-board produced from mushroom

mycelium and substrate waste

Article  in  Journal of Hazardous Materials · June 2020

DOI: 10.1016/j.jhazmat.2020.123296

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 Journal of Hazardous Materials 400 (2020) 123296

Contents lists available at ScienceDirect

Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Development of formaldehyde-free bio-board produced from mushroom T

mycelium and substrate waste
Shing Ching Khooa,b,1, Wan Xi Penga,1, Yan Yanga, Sheng Bo Gea,c, Chin Fhong Soond,
Nyuk Ling Mab,a,e,*, Christian Sonnef,a,*
a
Henan Province Engineering Research Centre for Biomass Value-Added Products, Henan Agricultural University, Zhengzhou, 450002, China
b
Faculty of Science and Marine Environment, University Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia
c
School of Materials Science and Engineering, Central South University of Forestry and Technology, Changsha 410004, China
d
Microelectronics & Nanotechnology - Shamsudin Research Centre (MiNT-SRC), Institute for Integrated Engineering, Universiti Tun Hussein Onn Malaysia, 86400 Parit
Raja, Malaysia
e
Anhui Juke Graphene Technology Co., Ltd. Bozhou, 233600, China
f
Department of Bioscience, Aarhus University, Arctic Research Center (ARC), Frederiksborgvej 399, PO box 358, DK-4000 Roskilde, Denmark

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Editor: Rinklebe Jörg Synthetic adhesives in the plywood industry are usually volatile compounds such as formaldehyde-based che-
Keywords: mical which are costly and hazardous to health and the environment. This phenomenon promotes an interest in
FTIR developing bio-boards without synthetic adhesives. This study proposed a novel application of natural mycelium
Green technology produced during mushroom cultivation as natural bio-adhesive material that convert spent mushroom substrate
RAMAN spectroscopy (SMS) into high-performance bio-board material. Different types of spent mushroom substrates were compressed
Spent mushroom substrate with specific designed mould with optimal temperature at 160 °C and 10 mPa for 20 min. The bio-board made
from Ganoderma lucidum SMS had the highest internal bonding strength up to 2.51 mPa. This is far above the
0.4−0.8 range of China and US national standards. In addition, the material had high water and fire resistance,
high bonding and densified structures despite free of any adhesive chemicals. These properties and the low cost
one step procedure show the potential as a zero-waste economy chain for sustainable agricultural practice for
waste and remediation.

⁎
Corresponding authors.
E-mail addresses: nyukling@umt.edu.my (N.L. Ma), cs@bios.au.dk (C. Sonne).
1
First authors: These authors contributed equally in preparation of this manuscript.

https://doi.org/10.1016/j.jhazmat.2020.123296
Received 30 April 2020; Received in revised form 13 June 2020; Accepted 22 June 2020
Available online 24 June 2020
0304-3894/ © 2020 Elsevier B.V. All rights reserved.
S.C. Khoo, et al. Journal of Hazardous Materials 400 (2020) 123296

1. Introduction
Mushroom represent a food product with high protein and nutrient
value (Deepalakshmi and Sankaran, 2014). With the increasing demand
from global mushroom consumption, the volume of spent mushroom
substrate (SMS) generated also increases and bring many challenges in
term of waste management and disposal of SMS. The global consump-
tion of mushroom market increases annually and is projected to reach
20.84 billion tons by 2026 (Fortune Business Insight, 2019). Approxi-
mately 5 kg of spent mushroom wastes is generated per kilogram
mushroom production (Lau et al., 2003), and that will be 104.2 billion
tons of wastes generated annually by 2026. Apparently, the massive
accumulation of SMS could be a breeding bed for insect and flies, re-
lease of awful smell from SMS dumping site, and causing ground water
contamination (Najafi et al., 2019). The application of SMS in agri-
culture such as animal feedstock, fertilizer and compost were reported
but is not popular within farmer (Ma et al., 2014). Farmers are favored
to dispose SMS through conventional methods such as discarded into
landfill, burning, and burying on land that required low technical
knowledge. Hence, a new green technology should be developed to
manage this large amount of SMS generated and recycled into useful
materials for sustainable circular economic in the mushroom industry.
Plywood including medium-density fiberboard (MDF) and particle
board has been one of the most widely used construction material in
recent decades (Chapman, 2006). It is an engineered wood produced
using chemical synthetic adhesive to bind layers of wood veneers
during high heat and pressure (Cai and Ross, 2010). The widely used
chemical synthesis adhesives are urea-formaldehyde (UF) and phenol-
formaldehyde (PF) to improve the internal bonding strength for wider
application (Lei et al., 2010). Generally, the plywood for indoor usage
are applied with cheaper UF glue and plywood for outdoor usage is
manufactured using high water resistant PF glue (Mamza et al., 2014).
The use of different adhesive types determines the quality of plywood
including high strength and stability, fire and water resistant, preven-
tion of microbial degradation in high humidity condition, which enable
the vast application such as timber joinery products, flooring, external
cladding, stairs, doors, internal rails, and balustrades (Berglund and
Rowell, 2005).
Most of the synthetic adhesive are volatile organic compounds.
Therefore, all the plywood, MDF and particle board have the potential
to release formaldehyde slowly to the environment (Mantanis et al.,
2017). Long term exposure to formaldehyde gas, known as a portable
human carcinogen, may induce irritation to eye and respiratory system
(Deepalakshmi and Sankaran, 2014). According to the Environment
Protection Agency (EPA) reports, new furniture release formaldehyde
levels up to more than 0.3 ppm and this particularly constitutes a health
issue and call for stricter legislations of volatile organic compounds
(VOCs) emissions from the plywood production (Mathias et al., 2016;
Nina, 2019). Therefore, a new trend towards replacing the current
chemical adhesives with bio-based adhesives for commercial interest is
taking place accelerated by international legislation mainly in the US
and EU that reduces the use of toxic materials in the automotive and
building industries (Mathias et al., 2016). These demands for green
adhesive materials and sealant market is projected to reach USD 73.8
billion in 2024 compared to the global market of a few billions in 2019
(Rearch and Market, 2019).
The mycelium coated on SMS is the vegetative part of mushroom to
support the growth and development of mushroom. Mycelium has a
great potential to be used as green adhesive material to replace syn-
thetic adhensive in wood production (Kittel, 2014). The solid white
mycelium will melt during hot-compression process and then penetrate
to form tight nets of cohesion and incoherent material within the SMS
matrix which enhance the adhesive and bonding resulting in a strong
bio-composite (Elsacker et al., 2019). In addition, these thick mycelium
layer also showed good water resistance properties (Appels et al.,
2019). The large SMS waste generated annually and the motivation to
replace chemical adhesive in the production of wood board had driven
motivation to discover the potential to produce formaldehyde-free bio-
board from SMS. In this study, the utilization of renewable bio-com-
posite using mycelium skin of different mushroom species: Ganoderma
lucidum, Pleutorus ostreatus and Auricularia polytricha as natural bio-
adhesives for the bio-board production were examined. Besides ex-
amined on the different mushroom species of SMS, a comparison of SMS
from different raw material such as sawdust, combination of food waste
and diaper waste substrate had been performed in this study as well.
This is the first study that examined the potential of recycle food waste
such as coffee ground, banana skin, sugarcane bagasse, eggshell and
used diaper core as SMS and its effect on the characters of produced bio-
board. The heat compression technique used together with specific
modified mold contributed to high mechanical strength in the absence
of chemical adhesive could replace the used of current synthetic ad-
hesive materials and that definitely contribute to greener production in
wood panel and bio-composite industries. In addition, the potential of
utilizing mycelium from different mushroom cultivar for bio-boards
production were also explored.
2. Materials and method
2.1. Synthesis of spent mushroom substrate (SMS)
The SMS consist of growth substrates formulated from food waste,
diaper waste and sawdust were used to grow different types of mush-
room (Table 1). All SMS were collected from mushroom farm developed
by Plant Biotechnology Research Group, Universiti Malaysia Ter-
engganu, except SMS of B2 and B3 were collected from Wonder Farm,
Taiping Perak (Table 1). The plastic that covered the SMS were cut
open, and the SMS were heat dried under the sun to remove moisture
content. The dried samples, including its outer layer of mycelium skins,
were then blended into powder form using Universal Blending Machine
(Nippon NBL-C501SS Stainless Steel Blender). The SMS powder were
then used as feedstock for bio-board production.
2.2. Bio-board formation process
About 25 g of SMS was put into the mold and pressed using a
thermo-compressor for 20 min under a pressure of 10 mPa at 160 °C to
produce bio-board with a dimension of 50 × 50 × 7 mm (Henan Ming
Sai Machinery Equipment Co. Ltd). The densified fibre was allowed to

Table 1
Types of spent mushroom substrate (SMS) used for bio-board production. Details on preparation and cultivation of SMS is found in (Lam et al., 2019a).
SMS Types of SMS

C (Control) Sawdust without mycelium spreading (Rubber tree Hevea brasliensis sawdust: rice bran: lime powder at ratio of 100:10:1)
B1 Sawdust from Reishi mushroom species Ganoderma lucidum
B2 Sawdust from white oyster mushroom species Pleurotus ostreatus
B3 Sawdust from cloud ear fungus mushroom species Auricularia polytricha
B4 Sawdust with 20 g/l diaper cores from Ganoderma lucidum mushroom
B5 50 % commercial sawdust and 50 % food waste (diaper core: coffee: banana skin: eggshell: sugarcane in 5:2:1:1:1)
B6 Food waste composition (diaper core: coffee: banana skin: eggshell: sugarcane in 5:2:1:1:1)

2
S.C. Khoo, et al. Journal of Hazardous Materials 400 (2020) 123296

Fig. 1. The schematic overview of the Bio-board production from recycle of SMS.

cool at room temperature for 3 h and the compressed bio-board was strength of the bio-board among treatments. The data are compared
then removed (Fig. 1). with the higher IBS showed higher bonding strength between fiber
matrix and higher quality against cracking and temperature changes
whereas lower IBS showed higher degree of stratification with lower
2.3. Characterization of bio-board
bonding strength.
2.3.1. Internal bonding strength (IBS)
IBS was analyzed using computer controlled electronic universal
2.3.2. Water immersion test
tensile machine (Model HD-B604B-S, Haida Testing Equipment
Water immersion test was performed to examine the water re-
Manufacturer Co., Ltd., China) and measured according to the China
sistance properties of bio-board and to detect the presence of bubbles or
National Standard (2013). IBS is a measurement of tensile strength of
delamination on the surface of bio-board. The bio-board was subjected
bio-board when uniform tension is applied to it. The front and back
to water immersion for cumulative time of 3, 6, 9, 12, 24 and 48 h. The
surface of bio-board was evenly applied with epoxy resin and then
surface of the bio-board was wiped with tissue paper once the bio-board
clamped on two loading medal hooks of tensile machine (Fig. 2b). The
were removed from the water immersion container (China National
two loading medal hooks were then loaded in the tensile machine and
Standard, 2013). The thickness of bio-board after immersion were
ready for IBS testing. The IBS data were generated from the machine
measured with digital caliper gauze (Model RTPEL_06IG, OEM, China).
linked software TM2010 (Haida). IBS testing was examined on the ratio
The thickness of swelling rate (TSR) is calculated as:
maximum failure tension on the vertical surface of bio-board to its
surface area and qualified the bonding strength and mechanical

Fig. 2. a: The internal bonding strength of bio-boards (C-B6), standard of particleboard (Pb), standard of medium densified fiberboard (MDF). The small letter a and c
represented to US and China respectively. b: The structural diagram of universal tensile machine with a magnification view of the bio-board loading between two
loading metal.

3
S.C. Khoo, et al.
Tf Ti
TSR = X100%
Ti (1)
where Tf is final value of thickness and Ti is initial thickness.
2.3.3. Contact angle analysis
Contact angle analysis was performed to study the wettability
properties of the bio-board specimens by using VCA Optima Contact
Angle System (AST Product Inc., 2019). VCA Optima System was
equipped with PC technology and high-resolution video camera to
capture the angle of sessile droplet on solid material. A drop of distilled
water (approximately 20 μL) was deposited on the surface of bio-board.
The contact angles between the water droplet and the surface of the bio-
board was determined using the VCA Optima XF software. Water con-
tact angle measurement was used to estimate the hydrophobicity or
hydrophilicity of a solid surface according to the ASTMD7334 standard.
2.3.4. Thermogravimetric analysis (TGA)
TGA analysis was performed using Thermal Gravimetric Analyzer
(Mettler Toledo, Polaris Parkway Columbus, USA) to study on the
thermal behaviour, thermal stability, stage of decomposition, kinetics
reaction of bio-board under inert condition. Nitrogen gas was used as
carrier gas. About 10 mg of bio-board meshed powder for each treat-
ment was heated in the TGA analyser at a heating rate of 10 °C/min and
gas flow rate of 25 mL/min. The applied temperature for TGA analysis
for the samples was set from 45 to 900 °C (Nam et al., 2018).
2.3.5. Density and weight reduction after compression
For each bio-board, 25 g of SMS was used to load into the mold. The
weight of the bio-board after heat compression were recorded using
weighing balance. The weight reduction and were written as:
25 FW
Weight reduction = X100%
25 (2)
FW
Density =
V (3)
where the FW was final weight of bio-board after compression, and V is
the multiplication of bio-board thickness with the surface area of bio-
board at 5 × 5 cm.
2.3.6. Microscopic observation
The morphological observation of bio-board was obtained using
scanning electron microscope (SEM) (JEOL Ltd., Tokyo, Japan). SEM
analysis had been performed in Institute of Oceanography and
Environment, University Malaysia Terengganu. The separated cross
section views of bio-board specimens were used to observe the internal
surface structure of the bio-board. The specimens were then rinsed with
acetone to remove unwanted residue and fingerprints on the surface of
the bio-board. The bio-board specimens were cut at the cross section of
bio-board and into 2 × 2 cm square and then transferred to the spe-
cimen stub. The bio-board specimen was then subjected to a thin layer
of gold coating using auto-fine coater (JEOL Ltd., Tokyo, Japan). The
gold coated specimen was then loaded into the SEM to observe the
surface morphology at high magnification.
2.3.7. Fourier transform infrared spectroscopy (FTIR) analysis
FTIR analysis was conducted to examine the changes in molecular
structure and distinguish the internal functional groups involved in the
conversion of spent mushroom substrate into bio-board. Little portion
of bio-board samples were further blended into powder prior to FTIR
analysis. The powder was mixed with potassium bromide at a ratio of
1:70 using mortar and pestle. The acquisition of FTIR spectrum were
obtained by using a FTIR spectrophotometer (FT/IR6700, Jasco, US)
equipped with potassium bromide disk at the wavelength from 4000
cm−1 to 400 cm−1 and 64 scans.
4
Journal of Hazardous Materials 400 (2020) 123296
2.3.8. Raman spectroscopy analysis
Raman analysis was performed using Raman Spectrometers (X-
ploRA TM Plus, Horiba, US). The Raman spectra were obtained with
532 nm wavelength of Argon laser and the spectra were recorded over
the range of 3500−50 cm−1. Spectra processing including baseline
correction, background correction and spectra analysis were processed
using Origin Pro software.
2.3.9. X-ray diffraction analysis
X-ray diffraction analysis was performed to examine the crystal-
linity presented within the bio-board (AST Product Inc., 2019). X-ray
diffractograms obtained from a XD-2 diffractometer with Cu-Ka radia-
tion (λ =1.5406 Å). The X-ray tubes used are Cu tube and 36 kV was
set for pipe potential and 20 mA for pipe current with 2θ/θ continuous
scanning. A graphite crystal monochromator was used with a slit device
set at DS = 1°, SS = 1°, RS = 0.3 mm. The rotary half-cone angle 2θ
was set from 5 to 42° with 2°/min of scanning velocity and 0.01 of scan
step angle. The relative crystallinity of bio-board was calculated based
on the formula Cr = (Ac/Aa x 100 %), where Ac = total area of
crystallinate peaks, Aa = area of all peaks including both crystallinate
and amorphous region, Cr is the relative percentage of crystallinity (Ji
et al., 2018a). The area of the Ac and Aa was analysed by the Origin Pro
Statistical Analyser.
2.4. Statistical analysis
All of the bio-board characterization were analysed in triplicate. The
experimental data were analysed using Statistical Package for Social
Science (SPSS) software. In SPSS, one-way analysis of variance
(ANOVA), homogenous subsets test and Post Hoc Test using equal
variances assumed Turkey’s HSD for p ≤ 5% were conducted to de-
termine possible differences among means. The data were recorded in
the mean with n = 3 and different letters expressed on the top of the
bars showed significantly different at P < 0.05.
3. Result and discussion
3.1. Internal bonding strength (IBS)
The IBS of bio-boards C-B6 were compared with the IBS standard in
America (US) and China as in Fig. 2a. All the bio-boards produced from
SMS (B1-B6) in this study surpassed the IBS standard requirement for
wood panels in US and China standards (Fig. 2a) (China National
Standard, 2013; American National Standard, 2009). IBS of B1-B6
ranged from 1.337 to 2.509 mPa showed higher IBS in comparison to
chitosan adhesive wood fiber composite and industrial particleboard
with internal bonding strength (0.14–1.86 mPa) and (0.31–1.17 mPa)
respectively.
3.2. Water resistance
After 48 h of immersion, the B1 and B2 bio-board from SMS of
Ganoderma lucidum and Pleurotus ostreatus, showed the least thickness
swelling rate with only 4.1 % and 4.4 % respectively (Fig. 3a). The
symbol - denotes for no sight of delamination, + represents small de-
lamination areas occur without cracking on the surface, and + - is large
delamination areas with cracking on the surface (Fig. 3a). B1 and B2
were found also still intact without any delamination after 48 h
(Fig. 3a). This is due to the mycelium formation by Ganoderma lucidum
and Pleurotus ostreatus being thick and forming rigid impermeable layer
at the outer spent mushroom substrate (Jafari et al., 2007). In addition,
water resistance is enhanced by the strong adhesion between mycelium
colonized sawdust fibers which tightly adhere all fiber components
together (Pickering et al., 2007). The bio-boards produced from SMS of
recycle food waste (B4-B6) had a very poor water stability with the
highest thickness swelling rate of B4 being 42.28 % and showed
S.C. Khoo, et al.
Fig. 3. Water resistance properties. a: TSR analysis with the degree of delamination. b: Bar graph of the contact angle analysis. c-e: the contact angle diagrams
obtained from treatment C, B1 and B6, respectively.
delamination after 48 h immersion in water. This might probably due to
the content of super absorbent polymers (SAP) in diaper core that has
good water absorption ability (Khoo et al., 2019). The reason of adding
small amount of diaper core into the mushroom growth substrate (B4-
B6) was because the biochar produced from pyrolysis of baby diaper
shown excellence results in our previous study, but the SMS produced
are less resistance to water damage (Lam et al., 2019a, b). The contact
angle analysis added new understanding of the water affinity of bio-
boards towards water (Fig. 3 c–e). The angle of surface that is favour-
able to liquid droplet is calculated by the degree of spread over at < 90°
(Lafuma and Quere, 2003). The angle created between the water dro-
plet and the bio-board was measured, the contact angle < 90° indicates
hydrophilic properties or poor water resistance, whereas the contact
angle > 90° indicates hydrophobic properties or high water resistance
(Yuan and Lee, 2013). B1 bio-board has the largest degree of non-
contact area > 120.33° (Fig. 3d) and it is significantly different from
the control and B4-B6 SMS (Fig. 3 b). The hyrophilic properties of B4-
B6 is proven by < 90° in contact angle analysis (Fig. 3b).
The materials for plywood or timber-based construction contain
high lignocellulosic components (fibers) that are usually hydrophilic in
contact with water (Kabir et al., 2012). The fiber cell walls contain
many OeH bonds within the macromolecules which in contact with
water forms new hydrogen bonds between H group of water molecules
and OeH group of the fiber matrix (Zakaria and Kok Poh, 2002). These
interactions caused the swelling within the fiber matrix and that
weaken the interface bonding strengths which literally result in the
matrix cracking, dimensional instability and poor mechanical proper-
ties (Zakaria and Kok Poh, 2002). In conventional plywood production,
coating chemicals such as polyurethane, varnish or lacquer are used to
remove or block the OeH groups in the fiber structures (Sun et al.,
2010). Interestingly, this ability can be replaced by the mycelium
5
Journal of Hazardous Materials 400 (2020) 123296
coating. Mycelium can act as impermeable coating layer to reduce the
water absorption. This explanation further supported the bio-board
with thick mycelium in B1-B3 has better water resistance compared to
control (Fig. 3).
3.3. Thermal stability test
The thermal stability of bio-boards was analyzed using the ther-
mogravimetric analysis (TGA). The loss of mass was obtained in inlet
environment enclosed with nitrogen gas supplied and single decom-
position stage was observed in all treatment except treatment B5 and B6
with multiple decomposition stages (Fig. 4a). B5 and B6 seem to be
more thermal stable as the rate of degradation is less than 40 % at high
temperature (600 °C) while other bioboards began to degrade at 255 °C
and more than half of mass loss was observed when the temperature
reached 350 °C. The degradation of conventional wood materials oc-
cured at a much lower temperature from 120 °C, hence the bio-board
showed improved thermal stability and resistance to thermal degrada-
tion in the event of burning during fire. For high fire risk application,
additional processing to enhance the fire resistance are needed because
it may reach tempartures of 1650 °C (Guimarães et al., 2009). Nor-
mally, the thermal decomposition of cellulose and hemicellulose within
the fibres in SMS take place at 200–300 °C while the thermal decom-
position of lignin is around 400 °C (Helmenstine, 2020). Therefore, the
sharp decrease in thermal degradation of all bio-boards around
255–340 °C are probably due to the thermal degradation of the cellu-
lose and hemicellulose fibres that occur within the SMS (Fig. 4a).
Weight reduction of the SMS in all treatments demonstrate the
thermal decomposition of the bio-board after the heat compression
process (Fig. 4b). Based on the thermal stability observed in TGA and
weight reduction, B1 and B3 are more thermal stable after B5 and B6
S.C. Khoo, et al. Journal of Hazardous Materials 400 (2020) 123296

Fig. 4. Thermal stability. a: TGA analysis from 40-900 °C; b: weight reduction of SMS after high heat and pressure compression; c: density of bio-board after high heat
and pressure compression.

but also very light in weight (Fig. 4c). Interestingly, our bio-boards compared to the control (Fig. 6a). The spectrum from all ten bio-boards
produced in this study have higher density ranged from 1.2 to 1.4 were compared and peaks were identified (Table 2). The IR peak of bio-
gcm−1 compared to 0.45 – 0.6 gcm−1 of some hardwood species result board B1-B5 at 3450−3350 cm−1 was corresponding to OeH sym-
from the high compression pressure (Phillips et al., 2019). metric stretching (Li et al., 2009) and showed higher intensity absor-
bance counts compared to C and B6. These might attribute to amide
linkages formed between amine groups and the carbonyl group of the
3.4. Scanning electron microscope (SEM) and chemometric analysis by
SMS (Lord and Howard, 2004) and hydrogen bonds formed between the
FTIR
fibers of SMS (Agustina et al., 2019; Sun et al., 2019). In the IR shift
within the range of 3500−3100 cm−1, possibly N-H bonding were
The C bio-board (raw mushroom subtract without mushroom
observed. The presence of amide group of mycelium-based adhesive
growth) shows mostly well-arranged microfilaments and microtubules,
was further supported by the higher intensity of C]O bonds detected at
only partially rupture microtubes within the cells were observed
the IR peak around 1760−1750 cm−1 in mycelium-based bio-board
(Fig. 5a & b). This reflect that the enzymes produced during mushroom
compared to control (Fig. 6a). In Fig. 6a, it is reflected that the NeH
degradation is important for the degradation and digest the raw parti-
bonding in the mycelium-based adhesive is around 3500–3100 cm−1
cles into smaller molecules. Thus, in the absence of mycelium de-
which might overlap with the OeH bonding peak in the IR spectra. The
gradation, the internal surface after compaction in control remained as
result was supported by C]O bonds in the amide group as reflected in
big particles; the microfilaments were found still in contact, creating
the IR peak around 1760−1750 cm−1. Three major peaks with huge
big holes within molecules (Fig. 5 a&b), this is probably the reason for
variation were located at 3350−3450 cm−1, 2900−2950 cm−1 and
poor internal bonding strength (Fig. 2). In bio-boards B1, the 200X
2850 cm−1 in the IR spectra which corresponds to OeH symmetric
magnification image show fine size molecules were compacted and
stretching, C-H asymmetric stretching and CeH stretching, respectively
densified into nicely packed structure (Fig. 5c). The B1 bio-board has
(Li et al., 2009) (Fig. 6a and Table 2). There are multiples peaks ob-
the strongest internal bonding with microtubules and microfilaments
served within the range of 400−1800 cm−1, all these bands can be
being degraded into tiny fragments with aid of mycelium forming an
explained by the characteristic peak of carbohydrates and lignin (Bisht
intertwined fiber network after heat compression (Fig. 5c&d). Lig-
et al., 2017). IR frequency of 1756, 1637−1620, 1437, 1370–1380,
nocellulosic degradation of cell wall materials by mycelium enzymes
1250−1260 cm−1 were attributed to C]O stretching vibration, C]C
probably accelerated the rupture and collapse of SMS fibers in addition
bending stretch (aromatic ring), C-C stretch (aromatic ring), C-H
to the high pressure and heat compression process (Ji et al., 2018b).
bending stretch, (O)eCeH stretch respectively (Aboulkas et al., 2017).
The combination of mycelium digestion and heat press contributed to
The FT-IR spectra of bio-board C, B1 and B6 before and after heating
the formation of hydrogen bonds between polymers within the bio-
compression is shown in Fig. 6b. Overall, the peak intensity of the bio-
board (Song et al., 2018).
board before heat pressure showed a lower peak intensity. These peaks
The FT-IR spectrum was in agreement with the SEM observation of
are located at 1654, 1647 and 1639 cm−1 in bio-board C, B1 and B6,
the spectra profile of bio-board with mycelium being highly different

6
S.C. Khoo, et al.
Fig. 5. Scanning electron microscopy (SEM) images of internal surface of bio-board C in magnification of (a) 75X and (b) 450X; internal surface of bio-board B1 in
magnification of (c) 200X and (d) 500X.
Fig. 6. a: FTIR spectra of all bio-boards; b: FTIR spectra of bio-board C, B1 and B6 before compression (black line) and after compression (red line).
respectively, might be due to in changes of ester linkage after heat
compression. According to Ji et al. (2018b), the decline showed that
hemicelluloses within the bio-board was degraded after heat compres-
sion process. Weak and less intense peaks at 1657, 1234, 1047 was
observed in bio-board B1 compared with C and B6. These three peaks
had been reported to be associated with C]C (aromatic ring), C-C
(aromatic ring) and CeH (out of plane) stretching vibration respec-
tively (Essabir et al., 2016) and the peaks also belongs to the compo-
nents of lignin (Dai and Fan, 2015). Hence, the result suggested de-
gradation of lignin components in bio-board B1.
3.5. Detection of Raman spectroscopy analysis
Formaldehyde is a volatile toxic pollutant with a chemical structure
7
Journal of Hazardous Materials 400 (2020) 123296
of H2-C = O that is found in particle boards, medium density fibre-
boards and most wood veneer products. Salem et al. (Salem and Böhm,
2013) reported that some wood sources may leak low volumes of for-
maldehyde in the absence of adhesive aldehyde resin base products.
Formaldehyde is known as a human carcinogen compound at exposure
limit as low as 0.08 ppm for 30 min according to National Toxicology
Program of Department of Health and Human Services (Suresh and
Bandosz, 2018). In this study, the bio-board production utilized green
technology by replacing mycelium as nature adhesive instead of urea
formaldehyde and phenol formaldehyde which are commercially used
in the wood panel production. Zhang et al. (2014) reported the Raman
spectra for formaldehyde peaks were occurred at 873, 1275, 1401,
1511 cm−1 at high intensity (ranged within 5000–25000) counts.
Compared to the published literature, bio-boards produced from SMS-
S.C. Khoo, et al. Journal of Hazardous Materials 400 (2020) 123296

Table 2 2824 to 2798 (Chazallon et al., 2008) are not detected in this study.
FT-IR spectra and the assignment of peaks from C and B1-B6 bio-boards in Hence, we conclude that the bio-board produced in this study are green
Fig. 5e. material free of formaldehyde.
IR shift (cm−1) Peak Detected Primary Possible The Raman spectra in bio-board C show an overall higher intensity
(cm−1) Assignment compound compared to other bio-board (Fig. 7b). The peak at 1534 in bio-board C,
1550 in bio-board B1 and 1582 cm−1 in bio-board B6 represent the
3600−3300 3417, 3409 O-H s Various, alkaloids
C]C aromatic rings stretching bond of lignin structure (Fig. 7b-d)
3050−2900 2929, 2924 C–H as Various
2900−2800 28,602,857 C–H s Various (Prieur et al., 2017). These spectra further supported the lignin de-
1750−1690 1732, 1756 C=O ar Esters gradation during mushroom cultivation by mycelium via the lig-
1680−1570 1637, 1624, 1620 C=C s, ar Ester, Aromatic nocellulosic enzymatic reaction as higher intensity of C]C bind present
1525−1400 1437 C-C b, ar Aromatic
in the control bio-board compared to the mycelium-based bio-board
1380−1370 1374 C–H s, b Various
1280−1200 1254 C–H s, ar Saccharides
(Agarwal, 2019). The use of mycelium for the production of bio-board
1200−1000 1120, 1057 C-O s Saccharides contributed to the overall lignin degradation and enhance the forma-
Below 1000 872,779 C–H (deformation) Saccharides tion of bonds either covalent, hydrogen bond, dipole or van der Waal
forces within the fiber and matrix. These bonds are crucial for adequate
The letters s, as, ar, and b denoted for the symmetric, asymmetric, aromatic ring adhesion to achieve optimal mechanical properties of wood-fiber
and bending stretching vibration.
composites (Almgren, 2010). In sum, the result in Raman spectra
proven that the bio-boards produced by heat press of SMS are for-
grown mushrooms were free from formaldehyde. Even though 2 similar
maldehyde free and the high strength of internal bonding are due to
peaks at 873 and 1275 were detected in bio-board C and B1, the in-
various bonding formations that form strong linkages within SMS par-
tensities detected were extremely low below 800 intensity count in bio-
ticles.
board C and only 50 intensity count in B1 (Fig. 7b &c). There is an
assumption for the low intensity peak observed at 873 cm−1 could
more possibly due to HeC]O bending stretching of cellulose fiber 3.6. XRD analysis
which also associate with the peaks at 859 cm−1 and 913 cm-1 (Fig. 7b)
(Pedersen, 2015). The peak at 1275 cm-1 is associated with aromatic The mass fraction of the crystalline region in cellulose material play
ether (ReOeR) in G unit of lignin which had been found in the peak of as key role on the chemical, mechanical, and physical properties of
1240 cm-1 in C and 1289 cm−1 in B1 (Pedersen, 2015; Lourenço and cellulose (Agarwal, 2019). X-ray diffraction and the relative crystal-
Pereira, 2018). Therefore, the two peaks 873 and 1275 cm−1 observed linity of bio-board C, B1, B6 were compared in (Fig. 8). The sharp peak
in this study can’t represent the formaldehyde. Moreover, frequent region and the mass multiple short peak region were classified as the
peaks for formaldehyde compound (H2-C = O) within the range of crystalline region and amorphous region respectively. The XRD patterns

Fig. 7. Raman spectroscopy analysis. a: stack plot of the Raman spectra of all bio-boards; b: Raman spectra of bio-board C; c: Raman spectra of bio-board B1; and d:
Raman spectra of bio-board B6, respectively.

8
S.C. Khoo, et al.
Fig. 8. XRD result. a-c: The comparison of the chemical changes after heat compression of the bio-board for C, B1 and B6 respectively where the black line refers to
the before heat compression and red line refer to after heat compression of the bio-board. d: The relative crystallinity of bio-board using Origin Pro software.
showed different ordered structures of the crystalline regions for the
bio-boards C, B1 and B6. The major peaks only observed at 2θ angles in
bio-board B1 (22.35, 26.43) and B6 (22.35, 23.15) representing Type I
cellulose which can contributed to high crystal stability (Pickering
et al., 2007).The result in Fig. 6b is consistent with the X-rays diffrac-
tion patterns obtained in Guimarães et al. (2009) that reported for
lignocellulosic fibres exhibiting mainly the Cellulose Type I structure.
Type I cellulose formed crystal in XRD analysis either by triclinic cel-
lulose Iα or monoclinic cellulose Iβ through the intra molecular hy-
drogen bonding within the cellulose matrix (Goldberg et al., 2015).
Goldberg et al. (2015) showed that the weak hydrogen bond in C6-H→
O2′ can contributed to the crystal instability. The higher crystallinity
found within the bio-board after the heat compression revealed that the
lignin fibres had been reduced after the heat compression (Pickering
et al., 2007). Other reports also show that the high crystallinity re-
presenting the neat packaging of cellulose chains, therefore is the key
for higher mechanical strength, higher density and higher dimensional
stability (Agarwal et al., 2010). Interestingly, we found higher crys-
tallinity formed in the bio-board B1 and B6 with both showing the
crystallinity values of 50 % (Fig. 8d) but not in control sample, this
prove that the thermally stable and higher hardness properties observed
in B1 and B6 are due to lignin degradation of mycelium in addition to
heat compression (Carraher, 2003; Menges et al., 2011). Theoretically,
the higher the temperature, the higher the kinetic energy to stretch the
polymer within the lignocellulose matrix resulting in higher degree
formation of crystal (Gillespie, 2018). However, there is a decrease in
crystallinity after heat compression in bio-board C. The possible reason
might be due to the larger size of complex structure in bio-board C
compared to B1 and B6. The degree of crystallinity not only affected by
temperature, it can be influenced by other factors such as chemical
structure and size of the polymers (Bhuiyan et al., 2001). Strong in-
termolecular bonding result to densified molecules (Polymerdatabase,
9
Journal of Hazardous Materials 400 (2020) 123296
2015). The result also come into agreement to the SEM observation
presented in Fig. 5c-d where the mycelium-based bio-board showed
more densified and compact structure.
3.7. Considerations
The combination findings of SEM, FTIR and Raman analysis sup-
ported the mycelium-based bio-board showed more densified structure
with the presence of the chemical bonding which provide high me-
chanical properties. Most importantly, the bio-board produced in this
study are free of formaldehyde through the Raman analysis. The com-
bination of the major factors such as hydrogen bonding, amide linkages
between fiber matrix, lignocellulosic enzymatic degradation by myce-
lium fungi, structure conversion to low molecular weight lead to highly
inter-connectivity between fiber matrix and formed dense interface
surface structure, which literally contributed to excellent performance
for bio-board composite.
4. Conclusion
This study demonstrated the green production of high-quality bio-
board without the application of chemical synthetic adhesive. All the
produced bio-boards had higher mechanical strength compared to
control and also the industrial standard requirement of US and China.
In particular, SMS derived from mushroom Ganoderma lucidum present
an outstanding feature such as IBS of 2.51 mPa, high water resistance
and hydrophobic properties, and displayed strong densified structure.
Thus, the mycelium of Ganoderma lucidum could be an ideal bio-ad-
hesive to replace formaldehyde-based adhesive in wood composite in-
dustry. As such, the new bio-boards produced could be used in many
applications especially for kid toys and furniture to reduce environ-
mental health hazard. Moreover, using SMS for wood panels production
S.C. Khoo, et al.
is feasible and could address the problem of waste spent mushroom
block since it has been massively produced due to the high demand for
mushroom.
CRediT authorship contribution statement
Shing Ching Khoo: Project administration, Writing - original draft,
Data curation. Wan Xi Peng: Visualization, Validation, Resources. Yan
Yang: Project administration. Sheng Bo Ge: Formal analysis. Chin
Fhong Soon: Formal analysis, Validation. Nyuk Ling Ma: Writing -
review & editing, Supervision, Conceptualization. Christian Sonne:
Supervision, Writing - review & editing, Conceptualization.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The authors gratefully acknowledge the Ministry of Education
Malaysia for the financial support under FRGS research grant (Vot
59482). The authors also acknowledge Henan Agricultural University
for the financial, facility, and technical support throughout this re-
search under a Research Collaboration Agreement (RCA) and Golden
Goose Research Grant (Vot 55191) with Universiti Malaysia
Terengganu, and Siong Hoong Mushroom farm and Wonder Farm Sdn
Bhd, Taiping in providing the research platform to conduct the re-
search.
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