Sustainable Materials and Technologies 28 (2021) e00256

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Sustainable Materials and Technologies

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

Fungal textiles: Wet spinning of fungal microfibers to produce

monofilament yarns
Sofie E. Svensson a,⁎, Jorge A. Ferreira a, Minna Hakkarainen b, Karin H. Adolfsson b, Akram Zamani a,⁎
a
Swedish Centre for Resource Recovery, University of Borås, 501 90 Borås, Sweden
b
Department of Fibre and Polymer Technology, KTH Royal Institute of Technology, 100 44 Stockholm, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: The cell wall of a zygomycetes fungus was successfully wet spun into monofilament yarns and demonstrated as a
Received 18 December 2020 novel resource for production of sustainable textiles. Furthermore, the fungus could be cultivated on bread waste,
Received in revised form 19 January 2021 an abundant food waste with large negative environmental impact if not further utilized. Rhizopus delemar was
Accepted 25 January 2021
first cultivated in bread waste in a bubble column bioreactor. The fungal cell wall collected through alkali treat-
ment of fungal biomass contained 36 and 23% glucosamine and N-acetyl glucosamine representing chitosan and
Keywords:
chitin in the cell wall, respectively. The amino groups of chitosan were protonated by utilizing acetic or lactic acid.
Chitin This resulted in the formation of a uniform hydrogel of fungal microfibers. The obtained hydrogel was wet spun
Chitosan into an ethanol coagulation bath to form an aggregated monofilament, which was finally dried. SEM images con-
Filamentous fungi firmed the alignment of fungal microfibers along the monofilament axis. The wet spun monofilaments had ten-
Zygomycetes sile strengths up to 69.5 MPa and Young's modulus of 4.97 GPa. This work demonstrates an environmentally
Wet spinning benign procedure to fabricate renewable fibers from fungal cell wall cultivated on abundant food waste, which
Monofilaments opens a window to creation of sustainable fungal textiles.
© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).

1. Introduction
Textile fibers with sustainable profile would significantly advance
the transition to circular bioeconomy. Currently both natural and syn-
thetic fibers pose environmental problems along the production chain
[1]. Cultivation of cotton, and other natural fibers obtained from agricul-
ture, requires irrigation and usage of land, along with fertilizers and pes-
ticides. Synthetic fibers, produced from a non-renewable resource, have
high energy consumption during production and a large amount of
chemicals are needed [2]. Furthermore, synthetic fibers cause concerns
for the impact on the marine environment due to the microplastic con-
tamination [3]. In addition to the problems related to raw materials and
production of textiles, recycling of textiles is often challenging [4].
Chitin is the major component of exoskeleton of crustacean and chi-
tosan is the deacetylated derivative of chitin. Chitosan is regarded as
non-toxic, biodegradable, and biocompatible, and therefore chitosan-
based materials are possibly suitable for medical applications [5] in
the form of, among others, sutures [6], bandages [7] and scaffolds [8].
Chitosan is also a suitable candidate for pharmaceutical purposes, for
example in drug delivery systems [9] in the form of a biodegradable re-
lease system carrying e.g. drug molecules [10]. Furthermore, chitosan
⁎ Corresponding authors.
E-mail addresses: sofie.svensson@hb.se (S.E. Svensson), akram.zamani@hb.se
(A. Zamani).
has been used for food applications, such as antimicrobial agent and
emulsifier [11]. Biopolymer based textiles can be a potential solution
to the environmental issues related to the textile industry. Since many
biopolymers of natural origin, such as chitin and chitosan, are not possi-
ble to melt [5], they are spun into filaments by using alternative
methods, for example wet spinning.
Chitosan can be dissolved in aqueous acid solution and wet-spun
into coagulation baths [12,13]. After alignment of the fibers and coagu-
lation, filaments are obtained. More recently, chitosan was combined or
reinforced with chitin nanofibrils [14] and nanocrystals [15] or polyvi-
nyl alcohol [16] to improve the properties of the wet spun fibers. Chito-
san textile fibers have been commercially implemented, however in
comparison with synthetic fibers, the mechanical properties of chitosan
fibers need improvement and a more economically viable production
process is needed [9].
Chitin has been dissolved in alkaline aqueous solvent and wet-spun
to produce fibers with high strength and biocompatibility [17].
Nanochitin [18,19] which forms a homogeneous hydrogel in water has
also been used for wet-spinning. Instant dehydration of the hydrogels
occurs upon injection of the nanofiber hydrogels which leads to aggre-
gation of the material to a fibrous structure. Another biopolymer that
has been subjected to a like-wise mechanism of fiber formation by
wet-spinning of a hydrogel, is nanocellulose [20,21].
The cell wall of the zygomycetes fungi is the only source in which
chitosan naturally exists together with chitin. The cell wall also contains

https://doi.org/10.1016/j.susmat.2021.e00256
2214-9937/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
S.E. Svensson, J.A. Ferreira, M. Hakkarainen et al.
some other biopolymers such as polyphosphates and polyglucuronic
acid [22,23]. Although fibers have earlier been produced from the
crustacean's chitin and chitosan, wet-spun monofilaments have not
yet been produced from fungal chitin and chitosan. To separate the
cell wall skeleton from zygomycetes fungal biomass, an alkali treatment
is applied, which removes proteins and other alkali soluble materials.
The alkali insoluble material (AIM) is therefore mainly composed of
the cell wall material. Our preliminary experiments indicated that, the
AIM exhibits high water binding capacity and thus results in a highly
viscous hydrogel in water.
Filamentous fungi such as zygomycetes can utilize many different
sources of nutrients [24], and therefore it is possible to use for example
food waste as a substrate in the production of fungal biomass. Studying
the streams of food waste from a Swedish supermarket [25] revealed
that bread waste was one of the fractions that contributed most to the
environmental footprint. Therefore, a possible utilization of the bread
waste fraction from supermarkets could be in the cultivation of fungal
biomass.
Growing fungi on abundant food waste resources could enable pro-
duction of materials rich in biopolymers, at the same time as the high
negative environmental impact of food waste is reduced. This could fur-
ther enable the creation of sustainable textile materials. This hypothesis
was demonstrated by development of the first wet-spun monofilament
yarns from microfibers of the zygomycete fungus Rhizopus delemar. The
fungal cell wall material was extracted from the obtained fungal bio-
mass after cultivation of the filamentous fungus on bread waste. The
cell wall material was processed to a spinnable hydrogel and spun
into a monofilament yarn. The cell wall material was analyzed for its
contents, and with automated time lapse microscopy the approximate
size of the fungal microfibers was investigated. The produced wet-
spun fungal monofilament yarns were studied for their morphological
and mechanical properties.
2. Material and methods
2.1. Chemicals and substrate
L-(+)-Lactic acid solution (88–92%) was purchased from Sigma Al-
drich. Acetic acid (glacial, 99.8%) was purchased from Sharlau and eth-
anol (Absolute ethanol) was purchased from Scharlau and VWR.
Chitin (from shrimp shells, practical grade) and chitosan were pur-
chased from Sigma Aldrich. Bread waste was supplied by ICA City
Borås (Sweden) in the form of different unsold bake-off bread, baked
in the store from pre-made dough. The bread waste was dried at room
temperature and then milled in a rotor beater mill (SM 100, Retsch
Technology GmbH, Germany). The milled bread was sieved before cul-
tivation to obtain particles of <1 mm. The sieving was performed to im-
prove the composition of the harvested fungal biomass, as visible
residual bread particles entangled in the fungal biomass were antici-
pated to mainly originate from larger bread particles of >1 mm.
2.2. Cultivation
Rhizopus delemar CBS 145940 (Centraalbureau voor Schimmel-
cultures, Utrecht, The Netherlands) was cultivated for 48 h in a 26 L bub-
ble column bioreactor (Bioengineering, Switzerland) containing 7.5%
milled bread suspension in ultrapure water. The bioreactor was empty
in-situ sterilized by injection of steam at 130 °C for 20 min, while milled
bread and ultrapure water were sterilized at 121 °C for 20 min in an au-
toclave (Systec, Germany). The aeration rate was 1 vvm (volume of air
per volume of liquid per minute) and the cultivation was performed
at 35 °C and without pH control. The initial pH at start was 5.5 and at
harvesting around pH 3.4.
To produce the pre-inoculum, agar plates (17 g/L agar, 20 g/L glu-
cose, 4 g/L peptone, pH 5.5) containing R. delemar, which had been incu-
bated for 4 days at 35 °C, were prepared. Spore suspension was made by
2
Sustainable Materials and Technologies 28 (2021) e00256
addition of 20 mL sterile distilled water to each agar plate with
R. delemar. The spore suspension was added to flasks with 7.5% bread
and cultivated for 24 h to obtain a pre-inoculum, which was thereafter
fed to the bioreactor together with bread and ultrapure water.
After 48 h of cultivation, the biomass was extracted by pumping out
the suspension of biomass and medium from the bioreactor. The sus-
pension was sieved with a kitchen sieve to remove the liquid fraction,
and the biomass was washed two times with distilled water, pressed
and stored in freezer until use.
2.3. Preparation of alkali insoluble material (AIM)
AIM was extracted from the fungal biomass by passing it through a
meat grinder followed by treatment for 1 min in a kitchen blender to
disperse the biomass in the alkali solution (100 mL 0.1 M NaOH/g dry
biomass). The blended solution was then autoclaved at 121 °C for
20 min. After reaching room temperature, the solution was centrifuged
in 50 ml tubes (10 min, 5000 g), and the AIM fraction was collected and
either washed with distilled water until neutral pH, or immediately
used for the preparation of a spinnable hydrogel. The washing was con-
ducted by filling 50 mL centrifuge tubes with 20–30 mL of AIM and the
remaining volume with distilled water. The suspension was mixed
manually and thereafter centrifuged again. This was repeated until,
after 5 repetitions, neutral pH was reached in the supernatant as tested
by pH paper indicator. The obtained AIM is the cell wall skeleton of the
fungus, which was kept at 4 °C until further use. The yield of the AIM (g
AIM/g dry biomass) was measured after freeze-drying (Labconco, USA).
2.4. Cell wall analysis
The composition of the cell wall was investigated by determining the
glucosamine (GlcN) and N-Acetyl glucosamine (GlcNAc) content in the
freeze-dried AIM according to the method developed by Mohammadi
et al. [26]. The phosphate content was determined with the Ammonium
Molybdate Spectrometric Method [27]. To determine the ash content,
0.1 g AIM was ashed in a muffle furnace at 550 °C until constant weight.
2.5. Preparation of spinnable hydrogels
Hydrogels were prepared from three different AIM modification:
washed AIM without any further mechanical treatment, as well as
washed and non-washed AIM that were subjected to further processing
(Fig. 1). The mechanical treatment consisted of dispersing the AIM in
distilled water (100 mL/g dry AIM), and processing with a kitchen
blender for 1 min followed by centrifugation (5 min, 5000 g), further
treatment with an IKA T25 Ultra-Turrax homogenizer in distilled
water (200 mL/g dry AIM) for 5 min and finally centrifugation (5 min,
5000 g).
Acetic acid or lactic acid was added to the three forms of AIM. Two
different concentrations of acetic acid, (3.5 M or 17.5 M) or lactic acid
(3.5 M or 11.7 M) were added to the AIM on the basis of 1 mL acid/
0.1 g AIM dry weight. After 1 h of gelation time, the gels were concen-
trated for 10 min in spin column concentrators (3 kDa MWCO, GE
Healthcare), where liquid was collected at the bottom and discarded.
The gels were stored at 8 °C until further use.
2.6. Wet spinning
The prepared gels were transferred to 10 mL syringes and injected at
room temperature into a coagulation bath with ethanol using a syringe
pump (Cole-Parmer, United States). The syringe needle (50 mm length,
1.2 mm diameter) was submerged directly into the bath through a hole
(Fig. 2) and the injection speed was set to 20 mL/h. After 2 min of coag-
ulation time, the filaments were collected from the bath and dried at
ambient temperature while fixed between two points.
S.E. Svensson, J.A. Ferreira, M. Hakkarainen et al. Sustainable Materials and Technologies 28 (2021) e00256

Table 1
Dry weight of hydrogels and diameter of wet-spun monofilaments.

Sample name Acid concentration Dry weight (%) hydrogel Diameter (mm)

W-AIM-AA-L 3.5 M 2.8 ± 0.1 0.14 ± 0.01

W-AIM-AA-H 17.5 M 2.9 ± 0.1 0.17 ± 0.02
W-AIM-LA-L 3.5 M 5.0 ± 0.4 0.16 ± 0.01
W-AIM-LA-H 11.7 M 10.9 ± 1.4 0.18 ± 0.01
P-AIM-AA-L 3.5 M 3.6 ± 0.1 0.19 ± 0.02
P-AIM-AA-H 17.5 M 3.0 ± 0.1 0.22 ± 0.04
P-AIM-LA-L 3.5 M 5.5 ± 0.5 0.21 ± 0.03
P-AIM-LA-H 11.7 M 11.3 ± 1.8 0.27 ± 0.03
WP-AIM-AA-L 3.5 M 3.1 ± 0.1 0.17 ± 0.02
WP-AIM-AA-H 17.5 M 3.1 ± 0.1 0.16 ± 0.02
WP-AIM-LA-L 3.5 M 4.8 ± 0.2 0.24 ± 0.05
WP-AIM-LA-H 11.7 M 8.9 ± 1.3 0.24 ± 0.03

Note: The samples were labelled according to type of treatment, where W is washed, P is
processed and WP is washed and processed. Type of acid is indicated with AA as acetic acid
or LA as lactic acid, and finally the acid concentration by L as low concentration (3.5 M) and
H as high concentration (17.5 M or 11.7 M).

rapidly frozen with the help of liquid nitrogen to maintain their mor-
phological structure and thereafter freeze-dried. The images were ac-
quired by ultrahigh-resolution FE-SEM (Hitachi S4800). Samples were
coated with 2 nm Pd/Pt layer and 3 kV acceleration voltage was used.

2.8. FluidScope™ scanning technology (oCelloscope)

The automated time lapse microscopy pictures recorded with the

Fig. 1. Process scheme from biomass to wet-spinning where three different treatment
routes are marked: W - washed AIM, WP - washed and processed AIM and P -
processed AIM.
FluidScope™ scanning technology with the oCelloScope (BioSense Solu-
tions ApS, Farum, Denmark) were utilized to evaluate the size of the
fungal fibers in the AIM and in the hydrogels. The hydrogel samples
were diluted with aqueous solution of lactic acid (pH ~3.2) or acetic
acid (pH ~3.2). The AIM samples were diluted with distilled water to at-
tain suitable fiber dispersions for the observation of individual fibers in
the pictures. For the image acquisition, default settings for a 96-wells
plate were used (2 ms illumination time, 4.9 μm imagine distance and
0 μm focus above bottom) and the number of images for each sample
was set to 20. The size distribution of the fiber diameters was estimated
with 50 measurements per sample.

2.9. Tensile testing

Mechanical properties of the monofilaments were studied by tensile

testing conducted on an INSTRON 5944 module equipped with pneu-
matic grips. The fibers were glued onto paper to secure the attachment
of the fiber to the grips. A 50 N load cell was used for the measurements,
with a gauge length of 20 mm, a crosshead speed of 5%/min and a 0.01 N
pre-load. The fibers had average diameters of 0.143–0.266 mm as deter-
mined by SEM. Before analysis, the fiber samples were preconditioned
overnight at 50% ± 5% relative humidity and 23 °C ± 1 °C. Young's mod-
ulus was calculated from the obtained stress-strain curves between 0.1
and 0.5% strain.
Fig. 2. Wet spinning set up showing the injection of the spinning gel into a coagulation
bath with ethanol.
2.10. Fourier transform infrared spectroscopy (FT-IR)

FT-IR spectra of the washed AIM, the hydrogels made by addition of

In Table 1, sample descriptions and diameters are given for the pro-
duced monofilaments as well as the dry weight of the hydrogels.
2.7. Scanning Electron Microscopy (SEM)
The morphology of the spinning gels and the produced monofila-
ments were analyzed by scanning electron microscopy. The gels were
acetic and lactic acid and their corresponding spun fibers were captured.
The hydrogel samples were freeze-dried and then prepared and ana-
lyzed as finely ground and dried powders. As references, pure chitosan
and chitin were analyzed. The spectra were obtained on a Nicolet iS10
FT-IR spectrometer (Thermo Scientific, USA) in absorbance mode,
with 64 scans and a resolution of 4 cm−1. The intensity of obtained spec-
tra was normalized at the C\\O stretching band around 1023 cm−1.

3
S.E. Svensson, J.A. Ferreira, M. Hakkarainen et al.
3. Results and discussion
There is increasing request for textile fibers with more environ-
mentally benign profile. Here, an abundant food waste source with
high negative environmental impact as discarded waste [25], was
utilized for creating a value-added sustainable material for textile
applications. A filamentous fungus was successfully grown on the
bread waste, and the produced fibrous cell walls were wet-spun
to monofilament yarns. The impacts of mechanical processing and
addition of acid to the cell wall material was evaluated based on
morphology and mechanical properties of the produced wet-spun
monofilament yarns.
3.1. Fungal biomass and alkali insoluble material (AIM)
Zygomycetes fungi can grow on a great variety of substrates and
are known to be enzyme producers [24]. In a previous work [28] Rhi-
zopus delemar displayed an excellent growth on bread waste and was
able to consume nearly all 7.5% bread within 48 h without the need
for addition of enzymes or other nutrients. Rhizopus delemar (previ-
ously Rhizomucor CCUG 61147) was originally isolated from leaves
used for tempe preparation [29]. The 48 h cultivation of Rhizopus
delemar yielded 10.7 g/L ± 0.73 g/L biomass. The obtained biomass
contained residues of bread particles, which were to some extent re-
moved in the washing step with distilled water. The obtained bio-
mass was subjected to alkali treatment and the fibrous cell wall
material was isolated with yield of 0.23 g AIM/g biomass (Table 2).
The AIM had a gel-like appearance due to the high water-holding ca-
pacity of the microfibers. However it lacked a complete homogeneous
consistency.
The cell wall represents a vegetative phase of the fungi, having the
purpose of protecting the inner cell components from the exposure to
the external environment [22]. Depending on the strain, the composi-
tion of the cell wall biopolymers differs, however, chitin and chitosan
are among the main components of cell wall of zygomycetes fungi. Glu-
cosamine and N-acetyl glucosamine are the dominant monomers of chi-
tosan and chitin, respectively [30]. Here, the analysis of glucosamine
and N-acetyl glucosamine content, showed that they made up 36.4
and 22.8% of the cell wall of the fungus (AIM) (Table 2), which can be
directly translated to the chitosan and chitin content in the produced
fungal cell wall.
Phosphates are reported to be another major component of cell wall
of zygomycetes fungi [23]. The AIM from the fungus cultivated on bread
waste, exhibited low phosphate content (Table 2). The bread substrate
used in this study had a very low phosphate content (<0.1%) and there-
fore, the obtained fungus had not accumulated phosphate in the cell
wall. Previous studies on Zygomycetes cultivated on phosphate-
containing medium, revealed that around 4–20% of the polyphosphates
were stored in the cell wall material [31].
The total amount of GlcN and GlcNAc constitute approximately 60%
of the cell wall content, this together with the absence of
polyphosphates, give an indication of other components in the cell
wall material. Previous studies have shown up to around 12 wt-% of
polyglucuronic acid in the cell wall of the zygomycetes strains Mucor
mucedo [32] and Mucor rouxii [33]. Therefore, the missing fraction in
the cell wall of the fungus produced here might be polyglucuronic
acid. However, further investigations are needed to confirm this
hypothesis.
Table 2
Yield of AIM, glucosamine (GlcN), N-Acetyl glucosamine (GlcNAc), phosphates and ash content.
AIM (g/g biomass) GlcN (g/g AIM)
0.227 ± 0.011 0.364 ± 0.012
Sustainable Materials and Technologies 28 (2021) e00256
3.2. Preparation of spinnable hydrogel from cell wall material
Different treatment procedures were investigated to develop a ma-
terial that would be suitable for spinning of monofilaments. Firstly, a
comparison of different alkali treatment procedures in autoclave, at
boiling temperature or at room temperature was performed. The most
homogenous and gel-like AIM was obtained from the treatment in an
autoclave (20 min, 121 °C). Therefore, the autoclave treatment was cho-
sen as the procedure for extracting the cell wall material and for further
development for processing into a spinnable hydrogel.
The AIM obtained after autoclave treatment was injected using a sy-
ringe and a needle placed immediately into a coagulation bath filled
with ethanol. This revealed that the outcoming material from the needle
was scattered in pieces and no fiber formation occurred. The non-
washed AIM contained larger remaining substrate particles after the al-
kali treatment of the biomass, which gave rise to clogging in the needle.
Therefore, only washed AIM could be spun as described. To mitigate co-
agulation and formation of monofilaments, acetic or lactic acid were
added to the washed AIM. This modification resulted in the formation
of a homogeneous hydrogel from the AIM. When this hydrogel was ap-
plied to the wet spinning procedure monofilaments were formed, and it
was possible to extract these manually. In order to improve the stability
of the as spun monofilaments, the gels were concentrated after addition
of acid, using spin column concentrators in which only liquid could pass
through to avoid any loss of dissolved chitosan. The fungus tested in this
work belongs to the family of zygomycetes fungi, which contain chitin-
chitosan in their cell wall. Further investigations are needed to deter-
mine whether other types of fungi can be subjected to a similar method
for production of monofilament yarns.
3.3. AIM and hydrogels pictured by automated lapse microscopy
The structure of many natural biopolymers is severely altered upon
drying, making for example size measurements or morphological anal-
yses a difficult task. Therefore, to be able to approximate the size of the
fungal fibers in the AIM and the acid treated derivatives of AIM, the ma-
terials were diluted in distilled water or acid solution and directly, at
room temperature, subjected to automated time lapse microscopy.
This made it possible to study the materials in their wet-state.
The results revealed the mycelium in a typical branch like state,
which represents the cell wall material of the fungus (Fig. 3). From
this, it was possible to conduct both diameter and length approximation
of the fibers. The length of the fibers often exceeded the size of the pic-
tures, and therefore the fiber length was concluded to, a great extent, be
in the range of ≥500 μm. The length approximation measurements of in-
dividual fibers are presented in the Supplementary material (Fig. A.1
and A.2). The diameters of the branched fibers were mainly in the
range of 6–10 μm, which agrees with the previous descriptions for typ-
ical mycelia of zygomycetes [34]. The diameter of the fibers was some-
what smaller in the washed AIM compared to the gels after addition of
lactic or acetic acid. This indicates that the gels were subjected to swell-
ing due to the addition of acids.
3.4. Wet spinning of the fungal hydrogels and the production of
monofilament yarn
The fungal hydrogels, which were prepared according to three differ-
ent routes and with modification by two different acids with higher and
GlcNAc (g/g AIM) Phosphates (g/g AIM) Ash (g/g AIM)
0.228 ± 0.015 <0.001 0.008 ± 0.001
4
S.E. Svensson, J.A. Ferreira, M. Hakkarainen et al.
Fig. 3. Microscopy pictures obtained with oCelloScope showing the fungal fibers and their corresponding range of diameters in a) washed AIM, b) gel with acetic acid and c) gel with lactic
acid. The average value of diameter is presented in the up-right corner.
lower acid concentration, were wet-spun and characterised. The wet-
spun filaments produced from the mechanically processed hydrogels, il-
lustrated an uneven distribution of strength. However, the most influenc-
ing factor seemed to be the choice of the acid and its concentration. Here,
the properties of the monofilaments are discussed with regards to mor-
phology, chemical changes and mechanical properties.
Addition of acidic aqueous solution to pure chitosan protonates the
amino groups, which increases the interactions between chitosan and
water, leading to dissolution. Since the AIM is a network structure of
fungal fibers which consist of a complex of chitosan with other mole-
cules, the fungal chitosan is not easily extractable without breakage of
this complex [35]. Therefore, upon addition of acid the amino groups
of chitosan were pronated, and the AIM exhibited the form of a gel.
5
Sustainable Materials and Technologies 28 (2021) e00256
This was confirmed by the gel- and sponge like appearance for wet-
state AIM, while AIM with added acid had the character of a homoge-
neous hydrogel. The induced improved properties upon addition of
acid resulted in a spinnable gel, which formed monofilament yarns
with a fibrous structure upon coagulation.
SEM analysis indicated an entangled fibrous structure for the fungal
biomass (Fig. 4e). A more porous entangled fibrous structure was ob-
served for non-washed AIM (Fig. 4b) which might be because of disper-
sion of the fungal fibers during the course of alkali treatment. This alkali
treated material was swollen compared to the original biomass. This in-
dicates that sodium hydroxide solution was entangled inside the struc-
ture of AIM and therefore leaves a more porous structure upon freeze-
drying. A more swollen hydrogel was also obtained by repeated
S.E. Svensson, J.A. Ferreira, M. Hakkarainen et al. Sustainable Materials and Technologies 28 (2021) e00256

Fig. 4. SEM pictures of a) biomass b) NW-AIM (non-washed AIM) c) W-AIM d) NW-AIM-AA-L e) W-AIM-AA-L f) P-AIM-AA-H and g) WP-AIM-AA-H. All samples were prepared for SEM by
freezing in liquid nitrogen and thereafter freeze-drying under vacuum.

6
S.E. Svensson, J.A. Ferreira, M. Hakkarainen et al.
washing of AIM. Washing of AIM results in diffusion of more water to
the fibrous structure of AIM and removal of the sodium ions. Higher
swelling capacity resulted in larger space between the fungal
microfibers in washed AIM (Fig. 4c). Wang et al. [36] and Tang et al.
[37] also reported higher swelling capacity of hydrogels after reduction
of the sodium ion concentration. Additionally, the repeated washing
procedure removed traces of proteins, lipids, and alkali soluble carbohy-
drates to a higher extent for the washed AIM (Fig. 4b and c), which re-
sulted in thinner fungal fibers and some separation and spacing
between the fibers.
After addition of acetic or lactic acid (Fig. 4d and e), a clear gelling be-
havior could be noted instantly. This was confirmed by the SEM images,
which showed that the randomly structured fibers were swollen and
had formed a flaked interconnected structure. This can be explained
Fig. 5. SEM pictures of a) W-AIM, b) W-AIM-AA-L, c) W-AIM-AA-H, d) W-AIM-LA-L and e) W-AIM-LA-H.
7
Sustainable Materials and Technologies 28 (2021) e00256
by the protonation of the free amino groups that occurs when chitosan
molecules are dissolved in dilute organic acids [5]. After addition of
acetic or lactic acid, the non-washed and washed AIM still differed in
the clear spacing of fungal fibers as observed for the AIM washed with
acetic acid (Fig. 4e). The images of the materials after further treatment
with blender and homogenizer (Fig. 4f and g) revealed that the fibers
were slightly reduced in size. The difference in washed or non-washed
AIM was not significant, probably due to the repeated addition and re-
moval (by centrifugation) of water during the treatments, which
washed off dissolved parts from the AIM material. The pH of both
washed AIM and unwashed AIM was neutral after the homogenizer
treatment as indicated by pH paper. The mechanical treatment lead to
a better dispersion of the AIM as seen in Fig. 4g, which illustrates
more swollen fibers compared with washed AIM (Fig. 4c). This might
S.E. Svensson, J.A. Ferreira, M. Hakkarainen et al.
indicate that the mechanically treated material was better dispersed,
which could facilitate the protonation of the amino group of the fungal
chitosan by the acids.
A comparison between the acetic acid and lactic acid treated AIM
(Fig. 5) indicated that the fungal fibers were in general more swollen
in the case of lactic acid treated materials. An even clearer difference
was visible when AIM was treated with concentrated acids. The use of
concentrated acid resulted in the formation of an interconnected struc-
ture where lactic acid treated material was significantly more swollen
with less and smaller voids compared to the acetic acid treated material
(Fig. 5c and e). This difference was also confirmed by the water content
of the gels, where lactic acid treated materials contained considerably
less water compared to the acetic acid treated materials (Table 1).
To identify if any chemical changes occurred during the process, FT-
IR spectra of the materials were recorded. The absorption bands in the
spectra correspond to different functional groups present in the AIM.
By comparing the peaks with reference materials of chitin and chitosan,
the functional groups could be identified, thus confirming the structure
and any chemical changes caused by the different processing methods.
Fig. 6 shows the FT-IR spectrum of the washed AIM, and its gels and
spun monofilaments. The spectra were compared to the spectra of com-
mercially available pure chitin and chitosan powders. For the character-
istic bands of chitin and chitosan, O\\H stretching and N\\H stretching
appear around 3260–3430 cm−1 (Fig. 6a). The amide band I, II and III are
located at 1654 cm−1 , 1560 cm−1 (1551 cm−1 for chitin), and around
−1
1315–1306 cm respectively. The AIM sample has a similar profile to
Fig. 6. FT-IR spectra of a) commercial chitin (commercial, obtained from crab shells), chitosan, washed AIM, gel and monofilaments with acetic acid (W-AIM-AA-L) and gel and spun
monofilaments with lactic acid (W-AIM-LA-L) and b) peak appearance at 1741 cm−1. The asterisk (*) marks the monofilament samples.
8
Sustainable Materials and Technologies 28 (2021) e00256
that of chitosan, confirming the domination of glucosamine in accor-
dance with the analysis of the cell wall. At 1741 cm−1, a peak appeared
with low intensity for the AIM, and with increased intensity for the W-
AIM-AA-L sample of gel and fiber (Fig. 6b). For the W-AIM-LA-L sample
of gel and fiber, the peak is shifted to 1735 cm−1, which could be due to
creation of an ionic complex between the protonated NH+ 3 of chitosan
and the lactate [38] after addition of lactic acid, which could result in a
more intense C_O peak in the spectra. Similarly, an ionic complex in-
duced by acetic acid could be the reason for the increased intensity for
C_O absorption band for the AIM-AA-L gel and fiber, compared to the
AIM. The peak with low intensity at 1741 cm−1 in the AIM spectra can
possibly be assigned to the presence of the C_O group of glucuronic
acid [39], which is generally present in the cell wall of zygomycetes
fungi.
Table 1 shows the type of acid, acid concentration, and diameter for
all the wet-spun fibers. For the fibers prepared with lactic acid, the di-
ameter clearly increased. The monofilaments prepared with acetic
acid had a smaller diameter, resulting from a more complete drying,
while the lactic acid monofilaments possibly retained more moisture,
which can also be seen in the lower mechanical properties and higher
flexibility. The processed AIM materials in general exhibited a larger di-
ameter, while the washed AIM resulted in quite similar diameters for
the fibers. This indicates that when the AIM has been mechanically
processed, the aqueous acid can easier infiltrate the structure and
thus increase the volume of the wet spun fibers. The addition of lactic
acid instead of acetic acid also increased the fiber diameter in all cases,
S.E. Svensson, J.A. Ferreira, M. Hakkarainen et al. Sustainable Materials and Technologies 28 (2021) e00256

Fig. 7. SEM pictures of the surface of wet spun monofilaments.

which was especially evident for the monofilaments obtained from
processed AIM.
The SEM pictures of the monofilaments are presented in Fig. 7,
showing the surface of the wet spun filaments. The parallel pattern,
which is visible on the surface of the filaments, shows that the wet-
spinning procedure has aligned the fungal fibers along the monofila-
ment axis. Therefore, in comparison with the non-oriented structure
of the freeze-dried gels in Fig. 5, the wet-spun monofilaments have an
oriented structure. The effect of adding acetic or lactic acid seems to
be that the acetic acid addition, and the following wet spinning, resulted
in a filament with more ordered and aligned fibers and consequently
smaller diameter.
3.5. Mechanical properties of monofilaments
High flexibility and strength of the textile fibers are of great impor-
tance to enable the processing into final woven fabrics. The results
from tensile testing showed that the filaments after lactic acid modifica-
tion had lower tensile strength compared to the filaments prepared
with acetic acid modification (Fig. 8a). The determination of tensile

Fig. 8. Mechanical properties of the produced monofilaments spun from hydrogels with 3.5 M acetic or lactic acid modification are shown as a) tensile stress b) elongation at break and
c) Young's Modulus.

9
S.E. Svensson, J.A. Ferreira, M. Hakkarainen et al.
properties confirmed that the lactic acid filaments were more flexible
and exhibited lower strength, which correlates with the morphological
differences (less fiber alignment during the spinning procedure). How-
ever, the filaments prepared from washed AIM without any further
treatment did not exhibit large differences in the tensile strength. For
the monofilament yarns W-AIM-AA-L and W-AIM-LA-L, the tensile
strength reached 69.5 MPa and 55 MPa respectively. From the filaments
prepared from further processed AIM (samples marked with P and WP)
(Fig. 8a), the differences in tensile strength and strain became more ev-
ident, and the monofilaments prepared with lactic acid had substan-
tially lower strength and higher elongation (Fig. 8b). The modulus
followed same trend as the tensile strength, where the highest value ob-
tained was 4.97 GPa (Fig. 8c). The results show the possibility of tuning
the mechanical properties, by either reaching a high strength with
acetic acid modification or high flexibility via lactic acid modification.
This is further illustrated in typical stress-strain curves for monofila-
ment with acetic acid or lactic acid modification in Fig. A.3.
The dry weight for the spinning gels prepared with acetic acid,
which ranged around 3 wt-%, seemed to be independent of which con-
centration of acid was added. However, the dry weight for hydrogels
with lactic acid had an uneven distribution of dry weights, depending
on both the concentration of added acid and to which material
(Table 1). The gels with concentrated lactic acid had a high dry weight
(around 9–11 wt-%) after the centrifugation with concentrator filters,
compared to the gels with less concentrated lactic acid added (around
5 wt-%). Considering tensile strength, the highly concentrated gel with
lactic acid resulted in lower values (Table A.1). This might have been
caused by the lesser free volume, which prohibited the alignment of
the fibers during the injection to the coagulation bath. However, the
final strength is not only a function of the gel concentration but may
also depend on degree of fibrillation of fungal microfibers, therefore fur-
ther investigations are needed.
In this work, the maximum tensile strength achieved was 72.3 MPa
(acetic acid hydrogel) and 55 MPa (lactic acid hydrogel) which is lower
than the strength reported earlier for wet-spun chitin nanofibers
(91 MPa [19] and 245.8 MPa [18]), where the improved strength was
due to the reinforcement effect of the nanosized fibrils. This is a differ-
ence to the fungal materials, which contain micro-sized fungal fibers
comprising of different polysaccharides. For wet-spun nanocellulose,
the obtained strength can be considerably higher, around 321 MPa
was achieved by Iwamoto et al. [21]. However, the nanocellulose fila-
ments were quite brittle with lower elongation at break (around
1.5–3.1%). The previously reported wet-spun chitin nanofibers demon-
strated somewhat higher elongation at break, around 2–14% [19] and
4.2–10.6% [18]. The elongation at break of the wet-spun fungal filaments
produced in this work was in the same range or higher than those re-
ported for chitin nanofibers. Elongation at break of 2.7–27.4% and
2.8–6.7% were recorded for filaments spun after lactic acid and acetic
acid modification, respectively. Fibers have previously been wet-spun
from another polysaccharide material, namely alginate. Alginate fibers
have high absorption capacity, porous structure, and they are suitable
for medical applications because of their non-toxicity, biodegradability
and biocompatibility [5]. However, alginate fibers do not exhibit excel-
lent mechanical strength. When chitosan whiskers were incorporated
with alginate, the wet-spun fibers had improved stiffness [40]. This
shows the reinforcement potential of the chitosan fibrils, since chitosan
has a semi-crystalline structure. The strength of the yarns produced
from the fungal cell wall filaments, obtained through mild and resource
efficient process, is promising and it is probable it can be further im-
proved. In the wet-spinning procedure, the hydrogel stability and
strength are crucial for a steadily outcoming fiber with a regular diam-
eter and smooth surface in the coagulation bath. The coagulation
could probably be further improved by tuning the coagulation solvent
according to the material's components tendency to aggregate into a
stable fibrous structure.
10
Sustainable Materials and Technologies 28 (2021) e00256
4. Conclusions
First demonstration of wet spinning of monofilament yarn directly
from the cell wall of a zygomycete fungus was presented. Throughout
the process, environmentally benign solvents and acids were used e.g.
acetic acid and lactic acid for preparation of the materials and ethanol
for the regeneration of monofilaments. The cultivation of Rhizopus
delemar on bread waste yielded a cell wall composition comprising of
chitosan and chitin. This enabled the production of hydrogels that
could be protonated at low pH followed by successful wet spinning to
monofilament yarns. Modification of the cell wall material with either
acetic acid or lactic acid showed that monofilaments with high strength
respectively high flexibility could be obtained. Altogether this work
demonstrated a promising route for decreasing negative impacts of
food waste through utilization as resource for materials production at
the same time as a door is opened to sustainable fungal textiles.
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgements
Funding: This work was supported by Vinnova, Sweden, through the
project Sustainable Fungal Textiles: A novel approach for reuse of food
waste [Reference number: 2018-04093].
Appendix A. Supplementary data
In the supplementary material, complete data for mechanical prop-
erties is given, typical stress-strain curves and oCelloScope pictures
with length approximations of fungal microfibers. Supplementary data
to this article can be found online at [doi: https://doi.org/10.1016/j.
susmat.2021.e00256].
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