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6th International Conference on Biotechnology Engineering (ICBioE 2021) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 1192 (2021) 012016 doi:10.1088/1757-899X/1192/1/012016

Chitin Fiber from Mushroom as Reinforcement for Biobased

Polymer

M I M Zin1, N S Shamsudin1, F Ali1, W M F W Nawawi1,2

1
Department of Biotechnology Engineering, Kulliyyah of Engineering, International
Islamic University Malaysia (IIUM), P.O. Box 10, 50728, Gombak, Kuala Lumpur,
Malaysia
2
Corresponding Author’s Email: [email protected]

Abstract. This project aimed to study the reinforcement effect of fungal chitin at different
loading on chitin/PLA composite. The chitin nanofibers were extracted from three mushroom
species (oyster mushroom Pleurotus ostreatus, shiitake mushroom Lentinula edodes, enoki
mushroom Flammulina velutipes) and used as a reinforcement element in PLA. The chitin/PLA
composite was fabricated using a solvent-casting method followed by the hot-compress molding
method. In the solvent-casting method, the chitin nanofibers were dispersed in PLA/chloroform
mixture and the mixture was left for solvent evaporation. The solvent-free chitin/PLA thin film
was then filled in dog bone mold before proceeded with hot-compress molding at 190°C and 70
bar. The samples with different chitin loading were tested with tensile test to study the
mechanical performance of nanocomposite. The chitin/PLA composite from oyster mushroom
shows the optimum result (σ= 43 MPa, E= 12 MPa) at 5% chitin loading. The increment of the
chitin loading leads to a decrease in both strength and strain. However, for the samples from
enoki and shitake mushrooms, the optimum chitin loading is 10% with 55 MPa and 56 MPa
tensile strength, respectively. This study suggests the potential of fungal chitin as reinforcement
in PLA.

1. Introduction
Chitin is a natural fiber that has been recently studied for its beneficial properties as reinforcing materials
in biopolymers [1-3]. Its is abundantly found in crustacean shell, insect cuticle and cell wall of fungi.
Fungal chitin has gained attention as the alternative to chitin source since it can provide continuous and
consistent supply [4, 5]. Since fungal chitin has excellent properties such as biodegradability and
biocompatibility, it can be used as a reinforcing element for biobased materials such as polylactic acid
(PLA) [6].
PLA is the most promising biodegradable bioplastic which is considered as eco-friendly material
to replace conventional plastics, especially in the packaging industry. However, its brittleness and poor
stability limit its industrial applications. Therefore, modification of PLA with the addition of plasticizers,
coupling and antistatic agents was done as a strategy to improve its performance so that it can compete
with the conventional polymers used in the packaging industry [3]. Coltelli, et al. [7] discovered that
incorporating PLA with chitin nanofibrils increased the elongation of the polymer by 180%. The chitin
loading in PLA affects the mechanical properties of PLA composite [1, 8, 9] Another study by Maskur,
et al. [10] reported that the incorporation of 5 wt% crustacean chitin resulted in higher tensile strength
and modulus. However, no study has been done yet on the effect of fungal chitin loading in PLA.

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6th International Conference on Biotechnology Engineering (ICBioE 2021) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 1192 (2021) 012016 doi:10.1088/1757-899X/1192/1/012016

Therefore, this study demonstrates the effect of fungal chitin loading on the mechanical properties
of chitin/PLA composite. The fungal chitin in this study was extracted from oyster (Pleurotus ostreatus),
Shiitake (Lentinula edodes) and Enoki mushroom (Flammulina velutipes) before the chitin powder was
mixed with PLA using chloroform. The mechanical properties of chitin/PLA composite were studied
using a universal tensile machine.

2. Materials and Methods

2.1. Materials
Three different mushrooms which are oyster (Pleurotus ostreatus), Shiitake (Lentinula edodes) and
Enoki mushroom (Flammulina velutipes) were used as fungal chitin sources in this study. The
mushrooms were frozen at least overnight at -20°C. Sodium hydroxide (Merck, pellets) was used for
chitin extraction process. The extracted chitin and PLA (Nature Works, Ingeo 3251D and Ingeo 3052D)
were mixed using chloroform (Merck).

2.2. Chitin Nanofiber Extraction

The extraction was carried out according to a procedure performed by Nawawi, et al. [11]. The frozen
mushrooms were thawed and washed with distilled water to remove any impurities before blending in a
kitchen blender (Vita Mixer Innofood, SX766) for 5 minutes to break the cell wall. Distilled water was
added to the blended mushrooms and the suspension was heated at 85°C for 1 hour to remove any water-
soluble components. The water-soluble components were removed by filtration process using a cotton
cloth. A vacuum pump (VALUE, VE 115N) was used to facilitate the filtration process. Then, the filter
cake was soaked in 1 M NaOH (65°C, 3 hours) to remove protein, lipid and alkaline-soluble
polysaccharides before neutralizing with distilled water until pH 7 was indicated by universal test paper.
The neutralized chitin was freeze-dried to remove the excess moisture and ground into powder (150 µm)
for further use.

2.3. Chitin/PLA Composite Fabrication

Prior to chitin/PLA composite fabrication, the PLA was dried overnight at 70°C to remove moisture
content. Then, the fabrication process started by first dispersing the chitin powder (5%, 10%, 15% and
20%) in chloroform before dispersing in PLA/chloroform mixture. The mixture was then stirred at high
speed using the magnetic hot plate (IKA®, G-MAG HS 7) until the chitin was homogenously dispersed
within the mixture. The mixture was poured into glass petri dish covered with aluminium foil and left
in the fume hood until the chloroform was fully evaporated, leaving behind chitin/PLA film composite.
The film composite was ground and filled into the dog bone mold cavities (Type IV, ASTM D638) that
were coated with a releasing agent (Loctite 770-NC Frekote) to ease the removal process. The step was
proceeded with compression molding by hot press (SUN RUN, SPH-900) at 190°C and 70 bar for 15
minutes. As a control, neat PLA sample was prepared by adding the PLA granule until it fully occupied
the dog bone mold cavities.

3. Results and Discussion

3.1. Chitin/PLA Composite Fabrication

The agglomeration of chitin during composite fabrication was minimized by grinding freeze-dried chitin
into powder form. The smaller size of chitin powder increases the contact area with PLA/chloroform
mixture which contributes to a homogenous dispersion within PLA polymeric matric. Still, stirring was
required to promote a homogenous dispersion of chitin since the chitin is not easily solubilized in a
common solvent, such as chloroform [12]. During the solvent casting method, the chloroform was
slowly evaporated by covering the glass petri dish with aluminium foil. This step was important to
prevent bubble formation in the film which would affect the preparation of tensile test specimens via
compression molding. Figure 1 shows the chitin/PLA film with different chitin loading. The
accumulation spots of chitin become more visible as higher chitin loading is introduced. This formation

2
6th International Conference on Biotechnology Engineering (ICBioE 2021) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 1192 (2021) 012016 doi:10.1088/1757-899X/1192/1/012016

describes the properties of chitin that attracted and crystallized when less solvent was present. The higher
chitin loading in PLA induces more interaction between chitin nanocrystals and the solvent.

Figure 1. Chitin/PLA composite with (a) 5 wt%, (b) 10 wt%, (c) 15 wt% and (d) 20 wt%.

Figure 2 shows the tensile specimen of neat PLA. No bubble formation was observed within the
tensile specimen. This was achieved by pre-dried PLA and grinding the film before tensile specimen
preparation. The formation of bubbles in the samples acts as a weak point for crack propagation to
initiate. Thus, will affect the mechanical properties.

Figure 2. Tensile specimen of neat PLA.

3.2. Mechanical Properties of Chitin/PLA composite

The mechanical properties of oyster chitin/PLA composite were compared to neat PLA (Ingeo 3052D).
While for shitake and enoki chitin/PLA composite, PLA (Ingeo 3251D) was used since we are unable
to purchase the same product due to technical limitations. However, the effect of fungal chitin loading

3
6th International Conference on Biotechnology Engineering (ICBioE 2021) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 1192 (2021) 012016 doi:10.1088/1757-899X/1192/1/012016

in PLA still could be discussed except that we could not compare the result for oyster with shitake and
enoki chitin/PLA composite. Table 1 shows the mechanical properties of oyster chitin/PLA at different
chitin loading.

Table 1. Mechanical properties of oyster chitin/PLA at different chitin loading.

Sample Tensile strength Young modulus Tensile strain Tensile toughness
(MPa) (MPa) (%) (MJ/m2)
NP1 17.76 ± 7.83 12.86 ± 0.5 1.56 ± 0.73 0.42 ± 0.36

CP1O5 43.04 ± 2.45 12.15 ± 0.14 4.35 ± 0.45 0.91 ± 0.08

CP1O10 41.52 ± 0.54 19.15 ± 9.24 3.16 ± 1.13 0.73 ± 0.26

CP1O15 30.37 ± 0.86 12.07 ± 0.51 2.77 ± 0.05 0.44 ± 0.01

CP1O20 34.75 ± 4.10 12.54 ± 0.3 3.10 ± 0.40 0.59 ± 0.15

*The numbers represent the chitin loading percentage in PLA.

The strength and toughness of PLA were improved when oyster chitin was added. The composite
containing 5 wt % of oyster chitin recorded the highest tensile strength and toughness with the value of
43 MPa and 0.91 MJ/m2, respectively. The result was in agreement with a study by Maskur, et al. [10]
when they investigate the effect of crustacean chitin loading in PLA. A lower chitin loading promotes a
better homogenous dispersion within PLA matrix. Since chitin is less hydrophilic, lower chitin loading
makes it more compatible with the hydrophobic PLA matrix [8]. As the result, the affinity of chitin and
PLA matrix contributes to the properties of composite [2]. Figure 3 shows the trends of tensile strength
when more chitin loading was introduced. When the chitin loading was increased to 10, 15 and 20 wt%,
the tensile strength and strain were decreased. At higher chitin loading, the compatibility of chitin with
PLA is weaker and the chitin tends to agglomerate. Therefore, the bigger chitin particle gives a negative
impact on the strength of the composite. However, there is no reduction of strain to failure observed,
proposing that no damage on fibers occurred during the extraction process.

50 5
45 4.5
40 4
Tensile Stength (MPa)

Tensile Strain (%)

35 3.5
30 3
25 2.5
20 2
15 1.5
10 1
5 0.5
0 0
PLA 5 10 15 20
Chitin wt% for oyster mushroom
Tensile strength Tensile strain

Figure 3. Tensile strength and strain of chitin/PLA nanocomposite for oyster mushroom.

Table 2 presents the mechanical properties of enoki and shitake chitin/PLA composite. For both
enoki and shitake chitin/PLA composite samples, the highest tensile strength and toughness were

4
6th International Conference on Biotechnology Engineering (ICBioE 2021) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 1192 (2021) 012016 doi:10.1088/1757-899X/1192/1/012016

exhibited when 10 wt% chitin was added into PLA. The result showed that PLA (3251D) promotes
better chitin compatibility up to 10 wt% chitin loading compared to PLA (3052D) which only up to 5
wt% chitin loading. An increase in fiber content in a composite system theoretically would increase the
tensile strength [9, 13, 14]. However, the compatibility between the fiber with PLA matrix also
contributes to the tensile strength. Therefore, if better compatibility at higher fiber content could be
maintained, the tensile strength probably would be improved further.
Table 2. The mechanical properties on chitin/PLA nanocomposite of Enokii and Shiitake mushroom.
Sample Tensile strength Young modulus Tensile strain Tensile toughness
(MPa) (MPa) (%) (MJ/m2)
NP2 48.63 ± 11.83 12.22 ± 0.42 5.97 ± 2.22 1.90 ± 1.08

CP2E5 53.18 ± 4.98 12.37 ± 0.20 6.06 ± 0.87 1.91 ± 0.45

CP2E10 55.87 ± 0.56 10.27 ± 4.16 7.21 ± 0.34 2.33 ± 0.10

CP2E15 53.97 ± 0.71 9.78 ± 0.48 7.27 ± 0.59 2.22 ±0.24

CP2E20 48.77 ± 0.22 13.68 ± 0.04 4.68 ± 0.07 0.68 ± 0.67

CP2S5 52.05 ± 1.19 12.13 ± 0.21 5.35 ± 0.19 1.58 ± 0.10

CP2S10 56.85 ± 2.01 11.85 ± 0.45 6.46 ± 0.50 2.17 ± 0.25

CP2S15 51.33 ± 4.47 13.02 ± 0.20 5.15 ±0.52 1.53 ± 0.29

CP2S20 47.27 ± 4.78 13.86 ± 0.36 4.37 ± 0.66 1.20 ± 0.33

*CP2E and CP2S indicate the samples from chitin enoki and shiitake respectively. The numbers represent the
chitin loading percentage in PLA.

The highest modulus was recorded at 20 wt% chitin loading for both enoki and shitake chitin/PLA
samples. According to Rizvi, et al. [9], the modulus is expected to increase when the nanofiller content
increases up to some limit. Differ from tensile strength, the modulus is probably not affected by the
compatibility of chitin and PLA (3251D) since the result showed a different pattern. Nevertheless, the
tensile strain of enoki chitin/PLA composite showed a slight increase up to 15 wt% of chitin before it
plummeted at 20 wt% (Figure 4). The tensile strain for enoki chitin/PLA at 15 wt% showed no
significant difference with the one at 10 wt% chitin loading. Therefore, we can claim that the tensile
strain pattern for both enoki and shitake chitin/PLA composite was quite similar.
8
7
Tensile Strain (%)

6
5
4
3
2
1
0
PLA 5 10 15 20
Chitin wt%

Enokii Shiitake

Figure 4. Tensile strain of enoki and shitake chitin/PLA nanocomposite at different chitin loading.

5
6th International Conference on Biotechnology Engineering (ICBioE 2021) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 1192 (2021) 012016 doi:10.1088/1757-899X/1192/1/012016

4. Conclusions
The use of fungal chitin nanofiber as a reinforcement element in PLA has a positive effect to enhance
its mechanical properties. The chitin/PLA (3052D) composite has optimum tensile strength and
toughness when 5 wt% oyster chitin was incorporated into the PLA. However, for enoki and shitake
chitin/PLA (3251D) composite, the optimum tensile strength and toughness were recorded at 10 wt%
chitin loading. The overall result suggested the potential of fungal chitin nanofiber as a reinforcement
element in the development of sustainable material.

Acknowledgements
The authors acknowledge the Ministry of Education Malaysia and International Islamic University
Malaysia (FRGS/1/2018/TK05/UIAM/03/3) for providing financial support to this project.

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