biomimetics

Article
Isolation and Characterization of Nanocellulose from
Polypodiophyta Fern Using Chemo-Mechanical Method
Katja Vasić 1 , Monika Dokl 1 , Željko Knez 1,2 and Maja Leitgeb 1,2, *

1 Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova ulica 17,
2000 Maribor, Slovenia; [email protected] (K.V.); [email protected] (M.D.); [email protected] (Ž.K.)
2 Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia
* Correspondence: [email protected]; Tel.: +386-2-2297-462

Abstract: Nanocellulose is considered a promising and sustainable biomaterial, with excellent

properties of biorenewability with improved mechanical properties. As a unique natural biopolymer,
it has been applied to many different industries, where efficient and environmentally friendly
productions are in demand. For the first time, ferns from the class Polypodiopsida were used for the
isolation of cellulose fibers, which was performed using a chemo-mechanical method. As chemical
treatment plays a crucial role in the isolation of nanocellulose, it affects the efficiency of the extraction
process, as well as the properties of the resulting nanocellulose. Therefore, mechanical fibrillation
was performed via grinding, while the chemical process consisted of three different treatments:
alkali treatment, bleaching, and acid hydrolysis. In three different experiments, each treatment was
separately prolonged to investigate the differing properties of isolated nanocellulose. Structural
analysis and morphological analysis were investigated by SEM, EDS, FT-IR, and DLS. The thermal
stability of cellulose fibers was investigated by TGA/DSC. The morphology of obtained nanocellulose
was confirmed via SEM analysis for all samples, with particles ranging from 20 nm up to 600 nm, while
the most consistent sizes were observed for NC3, ranging from 20 to 60 nm. FT-IR spectra showed
prominent absorption peaks corresponding to cellulose, as well as the absence of absorption peaks,
corresponding to lignin and hemicellulose. The EDS confirmed the elemental purity of nanocellulose,
while TGA/DSC indicated higher thermal stability of nanocellulose, compared to untreated fern,
Citation: Vasić, K.; Dokl, M.; Knez, Ž.; which started to degrade earlier than nanocellulose. Such characteristics with unique properties
Leitgeb, M. Isolation and make nanocellulose a versatile biomaterial for the industrial production of cellulosic materials.
Characterization of Nanocellulose
from Polypodiophyta Fern Using Keywords: nanocellulose; isolation; Polypodiophyta fern; chemo-mechanical method; biomaterial;
Chemo-Mechanical Method.
biopolymer
Biomimetics 2024, 9, 624. https://
doi.org/10.3390/biomimetics9100624

Academic Editor: Florian Ion

Tiberiu Petrescu 1. Introduction
Received: 2 September 2024 Biomaterials from renewable sources are gaining immense attention as environmental
Revised: 10 October 2024 sustainability dictates today’s world of science and everyday life. Environmentally friendly
Accepted: 11 October 2024 approaches are used to try to avoid environmental issues caused by the use of synthetic
Published: 14 October 2024 materials and biopolymers. Therefore, such materials are being replaced by innovative
and renewable biomaterials, namely biopolymers. In that manner, agricultural feedstocks
are the most abundant alternative, as well as the biggest source for such biomaterials, as
they can be used to obtain various high-value-added products with minimum impact on
Copyright: © 2024 by the authors.
the environment and its ecosystem. Cellulose, being the most abundant biopolymer and
Licensee MDPI, Basel, Switzerland.
the oldest on Earth, plays a significant role in the polymer industry for the synthesis of
This article is an open access article
green-oriented products and was used in the forms of cotton, fiber, or hemp as its first
distributed under the terms and
sources in ancient times. Cellulose is a natural biopolymer with many advantages since it
conditions of the Creative Commons
Attribution (CC BY) license (https://
displays non-toxicity, inherent biocompatibility, biodegradability, renewability, low cost,
creativecommons.org/licenses/by/
and colloidal stability. It can also be derived from many different biomass feedstocks [1–3].
4.0/). However, when starting the process, pretreatment of cellulose is often required, in order to

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Biomimetics 2024, 9, 624 2 of 16

efficiently fractionate cellulose from other lignocellulosic fractions [4]. With the purpose
of recovering cellulosic nanosized biomaterials, it is necessary to perform the purification
of cellulosic fractions by solubilizing the existing non-cellulosic compounds. As different
physiochemical methods can be used for the pretreatment of cellulose, a wide range of
structurally diverse nanocellulosic biomaterials can be produced [5,6]. The high-demand
interest in nanocellulose (NC) is not only in its distinctive nanoscaled structure but also in its
intrinsic properties, which include nanoscaled sizes, large specific surface areas, high aspect
ratios, and ease of surface chemistry modifications. As it is derived from sustainable sources,
it also possesses many desirable functions, which widen its use in different sustainability-
oriented industries, such as biomedical applications, sensors, paper, packaging, coatings,
and cosmetics. Based on its biocompatible nature, such NC-oriented biomaterials have
widened the range of use in environmental remediation as adsorbents and photocatalysts,
as well as flocculants, and show high binding affinities and adsorption capacities. Cellulose
is a linear polysaccharide with the formula (C6 H11 O5 )n consisting of many repeating β-
D-glucopyranose molecules, where the so-called cellobiose units are a dimer of glucose,
which are covalently linked through glycosidic bonds between the hydroxyl groups in
the carbon C1 and carbon C4 atoms of glucose units [7]. Elementary fibrils are the basic
building block units of cellulose fibers, which are the smallest morphological units that
form microfibrils [8].
Naturally occurring cellulose is known as cellulose I, which exists in parallel strands
without intersheet hydrogen bonding. Cellulose II is thermodynamically more stable and
exists in antiparallel strains with intersheet hydrogen bonding. Both cellulose I and II are
the main polymorphs of cellulose, as they exist as a variety of different conformations
and are a result of different cellulosic chain orientations of hydrogen bonds within the
network [9]. The difference in properties of cellulose I and II arises due to changes in
crystal structure. The non-cellulosic components, such as lignin, and hemicellulose, as
well as other compounds, are removed by pretreatment. Afterwards, NC is isolated from
cellulose fibrils using different isolation methods. For biomass pretreatment, alkaline
treatment and acid hydrolysis are used [10,11], also known as the delignification process.
After the bleaching process, the white-colored fibers of holocellulose (mainly consisting
of hemicellulose and cellulose) indicate that lignin and other impurities were successfully
removed. The main isolation methods mainly include acid and enzymatic hydrolysis, as
well as mechanical processes. Various types of acids, mostly sulfuric acid, formic acid,
hydrochloric acid, stearic acid, and maleic acid, were used for acid hydrolysis of cellu-
losic biomass [12–14]. Sulfuric acid causes strong isolation of nanocrystalline cellulose
and esterification of hydroxyl groups by sulfate ions. It removes amorphous regions and
increases the crystallinity of the fibers. Treatment success is affected by acid concentration,
reaction time, and temperature [15]. Different reaction times (5, 10, 15, 30, and 60 min) with
a sulfuric acid concentration of 64% (w/w) were studied by Prado et al., and after 30 min
of reaction, the cellulose-based nanoparticles were isolated [16]. Also, TEMPO-mediated
oxidation was investigated for the isolation of cellulose nanofibers, which involves the
2,2,6,6-tetramethylpiperidine-1-oxyl radical [17,18]. Enzymatic hydrolysis may be con-
ducted by a variety of enzymes (commonly named cellulases), which are used to break
down the β-(1,4)-glycosidic linkages [19]. Due to an impact on the defibrillation of cellulose
fibers, several intensive mechanical treatments may be performed. These processes consist
of grinding, high-pressure homogenization, steam explosion, cryocrushing, and high-
intensity treatments with ultrasound. A combination of chemical and mechanical treatment
was reported in many research studies. For example, deep eutectic solvent pretreatment
for lignin-containing nanocellulose production was investigated, which displayed good
thermal stability and high crystallinity [20]. A report by Almeida et al. describes the produc-
tion of functionalized nanocellulose using deep eutectic solvents, which resulted in highly
fibrillated and functionalized lignocellulose nanofibers [21]. Additionally, Wang et al. report
on a new nanocellulose from waste coconut shell fibers, which was based on an ultrasonic
method [22], while Zhang et al. investigated the extraction of nanocellulose from banana
Biomimetics 2024, 9, 624 3 of 16

pseudo-stem, which was conducted using a chemical–mechanical method [23]. NC was also
isolated from palm oil mesocarp fiber with hydrothermal treatment, which was coupled with
weak acid hydrolysis, such as oxalic acid dihydrate. Two different concentrations of oxalic
acid dihydrate were selected for the acid hydrolysis process, where average particle sizes of
NC were measured using TEM and were approx. 25 nm [24]. Isolation of nanocellulose from
hardwood pulp was performed via phytic acid pretreatment, which yielded around 90%
of nanocellulose with a ribbon-like structure [25]. Another study reports on the formation
of NC through the two-step approach of delignification, which was microwave-assisted
followed by ultrasonic extraction, which produced cellulose of purity 93% and hemicel-
lulose of purity 99% [26]. Also, NC crystals were prepared via acid hydrolysis from tea
stalks in China, which reports improved physical and chemical properties, as well as better
stability [27]. NC, being a result of the isolation process from lignocellulosic biomaterials,
has many advantages; besides biodegradability and renewability, it also has high specific
strength and modulus. NC can be divided into two types, namely cellulose nanofibrils
(CNFs), which are traditionally extracted via acid treatment, where the amorphous regions
are cleaved, obtaining highly crystalline and rigid nanostructures. Since, during acid hydrol-
ysis, the incorporation of acid results in the surface charge of CNCs, colloidal dispersion
is quickly formed [28]. Cellulose nanocrystals (CNCs) are most commonly prepared by a
mechanical disintegration process. Additionally, various other methods, such as enzymatic
treatment, chemical treatment, high-pressure homogenization, ball milling, and ultrasonic
fiber delamination, are other approaches used to obtain CNFs [28,29]. Both CNCs and CNFs
are obtained via mechanical treatments, such as homogenization, ultrasonication, steam
explosion, nanofibrillation, and biologically or chemically assisted milling [30]. It can also
be produced with enzymatic treatment, acid hydrolysis, ultrasonication, or hydrothermal
treatment [31]. Fern, from the class Polypodiopsida, which is a class of nonflowering vascular
plants, is a carbohydrate-based source with essential characteristics of plant-fiber-based
nanocellulose, since it possesses stems, true roots, and complex leaves and is produced by
spores. Such characteristics of this class are molecular, mechanical, and tensile properties, as
well as its biodegradability potential. Since ferns are extremely diverse in form, habitat, and
reproductive methods, they are abundant in damp and warm places [32,33]. Consequently,
they are a cheap and, most importantly, carbohydrate- and nanocellulose-rich source with
outstanding structures and features. With ever-growing interest and a combination of
multidisciplinary fields, such as life sciences, chemistry, biology, physics, and engineering,
they are an important part in the evolution of natural and nano-based biomaterials [34],
which are used for constructing tissue replacements or in tissue regeneration and repair, as
implants, as well as in drug delivery and biosensing [35,36].
There are a lot of possible applications for such biomaterials. As far as we know, there
is no information on isolating NC from ferns; therefore, in the present study, Polypodiopsida
ferns were used as a raw plant biomaterial for the first time and NC was successfully
isolated via a combination of mechanical and chemical treatment. Each chemical treatment
(alkali, bleaching, and acid hydrolysis) was prolonged in three different experiments, to
investigate the differing properties of resulting nanocellulose. After chemo-mechanical
treatment, isolated NC was further characterized, where morphology, chemical structure,
and composition were investigated. This work introduces a potential material that may
be used in dye extraction, the removal of ion pollution, and the monitoring of emerging
pollutants, as well as in drug delivery systems and neuroendovascular treatments.

2. Materials and Methods

2.1. Raw Materials
Ferns (class Polypodiopsida) were collected from a forest in Maribor, Slovenia. Sodium
hydroxide (NaOH, microgranular, pure) was obtained from Poch, Poland. Hydrogen
peroxide (H2 O2 , 30%, p.a.) was obtained from Merck, Germany. Hydrochloric acid (HCl,
37%) and sulfuric acid (H2 SO4 , 95–97%, p.a.) were obtained from Honeywell, Fluka,
Charlotte, NC, USA.
 Biomimetics 2024, 9, x FOR PEER REVIEW 4 of 17

Biomimetics 2024, 9, 624 4 of 16

Ferns (class Polypodiopsida) were collected from a forest in Maribor, Slovenia. Sodium
hydroxide (NaOH, microgranular, pure) was obtained from Poch, Poland. Hydrogen per-
oxide (H2O2, 30%, p.a.) was obtained from Merck, Germany. Hydrochloric acid (HCl, 37%)
and sulfuric acid (H2SO4, 95–97%, p.a.) were obtained from Honeywell, Fluka, Charlotte,
2.2.
NC, Isolation
USA. of Nanocellulose
The isolation of nanocellulose is schematically presented in Figure 1. Cellulose was
2.2. Isolation of Nanocellulose
isolated in three different ways. The experimental setup for each sample is presented in
The isolation of nanocellulose is schematically presented in Figure 1. Cellulose was
Table
isolated1.in For
three all experiments,
different a small amount
ways. The experimental of sample
setup for each untreated raw inmaterial (150 g) was first
is presented
Table 1. For
ground all experiments,
into small piecesa small amount
using of untreated rawmiller
a commercial materialat(150 g) was
room first
temperature and 20,000 rpm.
ground into small pieces using a commercial miller at room temperature and 20,000 rpm.
Later, 100 g of grounded material was treated with a 2% aqueous
Later, 100 g of grounded material was treated with a 2% aqueous NaOH solution heated
NaOH solution heated to
80 ◦ for 4 or 24 h under continuous stirring.
to 80C °C for 4 or 24 h under continuous stirring.

Figure 1. Isolation process of nanocellulose from Polypodiophyta fern.

Figure 1. Isolation process of nanocellulose from Polypodiophyta fern.
The alkali-treated material was then bleached in a 5% H2O2 aqueous solution at 80 °C
Table 1.24Experimental
for 4 or conditions
h under continuous stirring. for
Theeach sample.
bleached material then went through acid
hydrolysis with a 2% aqueous HCl solution at 80 °C for 4 or 24 h under continuous stirring.
The distilled water/material
Alkali suspension
Treatmentwas put in Bleaching
an ice bath and treated
Acidwith 60% con-
Hydrolysis Sulfuric Acid
Experiment
centrated H2SO4, which was(NaOH)
added dropwise. The suspension
(H2 O2 ) was heated to 50(HCl)
°C for 1 h, Treatment (H2 SO4 )
while continuously stirred.
NC1 24 h 4h 4h 1h
Table 1. Experimental conditions for each sample.
NC2 4h 4h 24 h 1h
NC3 Alkali Treat-4 h Bleaching Acid 24 h
Hydrolysis 4 hTreat-
Sulfuric Acid 1h
Experiment
ment (NaOH) (H2O2) (HCl) ment (H2SO4)
NC1 24 h 4h 4h 1h
The alkali-treated
NC2 4h material
4 hwas then bleached
24 h 1 hH2 O2 aqueous solution at 80 ◦ C
in a 5%
for NC3 4 h 24 h 4 h 1h
4 or 24 h under continuous stirring. The bleached material then went through acid
hydrolysis
After each with a 2% suspensions
treatment, aqueous HCl solution
were filtered and at 80 ◦ Cwith
washed fordistilled
4 or 24water.
h under continuous stirring.
The
Since distilled
the solutionwater/material suspension
was highly acidic after the treatment, was put in
the material wasan ice bath
washed with and treated with 60%
distilled water until
concentrated H2the
SOsolution reached neutral pH 7. Obtained fibers were eventually ◦
4 , which was added dropwise. The suspension was heated to 50 C for
dried for 24 h at 60 °C. The fiber-to-solution ratio used in all treatments was 1:10 (in g/mL).
1The
h, reaction
while timecontinuously
during H2SO4 stirred.
treatment was the same in each experiment. The reaction
time ofAfter
alkali each treatment,
treatment, bleaching, suspensions were
and acid hydrolysis filtered
varied. and washed
All experiments with distilled water. Since
were per-
formed in triplicate with a standard deviation of less than ±3%.
the solution was highly acidic after the treatment, the material was washed with distilled
water until the solution reached neutral pH 7. Obtained fibers were eventually dried for
24 h at 60 ◦ C. The fiber-to-solution ratio used in all treatments was 1:10 (in g/mL). The
reaction time during H2 SO4 treatment was the same in each experiment. The reaction time
of alkali treatment, bleaching, and acid hydrolysis varied. All experiments were performed
in triplicate with a standard deviation of less than ±3%.

2.3. Isolation of Nanocellulose

2.3.1. Scanning Electron Microscope (SEM)
The morphology and surface of the isolated samples was analyzed by scanning elec-
tron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). The samples were
covered with gold coating and performed on a Scanning Electron Microscope (FE, SEM
SIRIN, 400 NC, FEI).

2.3.2. Energy-Dispersive X-ray Spectroscopy (EDS)

The elemental composition of the cellulose samples was measured by energy-dispersive
X-ray spectroscopy (EDS). Measurements of samples covered with gold coating were per-
formed on a Scanning Electron Microscope (FE, SEM SIRIN, 400 NC, FEI).
Biomimetics 2024, 9, 624 5 of 16

2.3.3. Fourier-Transform Infrared (FTIR) Spectroscopy

The functional groups in fibers were analyzed by FTIR analysis and determined in a
Perkin Elmer 1600 spectrometer by taking the spectrum in the range of 500 to 4000 cm−1 .
The samples were ground into powder and blended with KBr followed by pressing the
mixture into thin pellets.

2.3.4. Thermogravimetric Analysis (TGA)

The thermal stability of the cellulose samples was analyzed with a TGA/DSC 1
analyzer, STARe System, Mettler Toledo, Greifensee, Switzerland at a heating rate of
10 ◦ C/min, ranging from 30 ◦ C to 600 ◦ C. All measurements were performed under
nitrogen flow to prevent thermo-oxidative degradation.

2.3.5. Differential Scanning Calorimetry (DSC)

Using a TGA/DSC 1 analyzer, STARe System, Mettler Toledo, the thermal behavior of
the cellulose sample was analyzed at a heating rate of 10 ◦ C/min, ranging from 30 ◦ C to
600 ◦ C under a nitrogen atmosphere.

2.3.6. Dynamic Light Scattering (DLS)

The particle size of the isolated samples was determined by Dynamic Light Scattering
using a particle size analyzer from the Malvern Zetasizer Nano series. Analyses of aqueous
suspensions were carried out for 50 s in the following conditions: water refractive index
1.330, viscosity 0.8872 mPas, and temperature 25 ◦ C.

3. Results
Isolation of NC derived from various sources underwent different treatments as per the
literature. However, the isolated materials did not possess the same crystal characteristics,
as there is a wide range of cellulosic feedstocks, such as plants, animals, bacteria, fungi, or
marine algae being used for NC isolation. There are also different (pre)treatments used to
obtain such NC. In our study, isolation of NC was performed using Polypodiopsida fern for the
first time, where it was successfully achieved by three modified treatments, based on different
experimental conditions. Different parts of the treatment were varied. As prolonging the time
of each chemical treatment could result in different characteristics of isolated nanocellulose,
each treatment was carried out for 24 h in three different experiments. In the first experiment
(NC1), the alkali treatment was performed for 24 h, which was followed by 4 h of bleaching
and 4 h of acid hydrolysis. In the second experiment (NC2), the acid hydrolysis treatment was
performed for 24 h, to which the sample was modified beforehand with 4 h of alkali treatment
and 4 h of bleaching. In the third experiment (NC3), the alkali treatment was performed
for 4 h, which was followed by 24 h of bleaching treatment, and treatment continued for
4 h with acid treatment. All three kinds of treatments were followed by 1 h of sulfuric acid
treatment, while alkali bleaching and acid treatment were concluded. Details about each
experimental condition and for how long each treatment lasted are presented in Table 1. After
the treatments were concluded, the characterization of all obtained samples was examined
to investigate how each treatment affected the morphological and structural properties of
the samples, as well as the size of the obtained nanoparticles and their distribution. Thus,
characterization was examined by performing SEM, EDS, FT-IR, TGA, DSC, and DLS analysis.

3.1. Microscopic Morphology Analysis by SEM

The surface of the isolated fibers was studied by SEM. Depending on the treatment
conditions, fibers and particles of different shapes attached to them were found. The particle
size was determined at higher magnification levels. The sample obtained under prolonged
alkali treatment contained straight and unbroken fibers measuring approximately 30 µm
wide (Figure 2a). According to Figure 2c,e, two clustered particles measuring approximately
5 µm in length are shown. The particles were also arranged in layers (Figure 2b) or dispersed
over fibers (Figure 2e). The diameters of the individual particles and the particles grouped
Biomimetics 2024, 9, 624 6 of 16

together were between 100 and 400 nm. The straight, torn, twisted, and porous fibers were
found in SEM images of sample NC2 (Figure 2f–j). The straight fibers were 10 to 20 µm
wide. The porosity of fiber as the holes with diameter within the range of 50–100 nm was
observed (Figure 2h), indicating the absence of particles that size. The 60 to 600 nm large
particles were located on the fiber surface (Figure 2j) with a lightly brushed surface. The
prolonged bleaching treatment resulted in straight and twisted fibers (Figure 2k,o). At
Biomimetics 2024, 9, x FOR PEER REVIEW 7 of 17
higher magnification levels, large layers and clusters of small particles ranging from 27 to
60 nm were determined (Figure 2k–o).

Figure2.2.SEM
Figure SEM of
of samples
samplesNC1
NC1(a–e), NC2
(a–e), (f–j),
NC2 andand
(f–j), NC3NC3
(k–o)(k–o)
at various magnification
at various levels. levels.
magnification

3.2. It
EDS Elemental
was alreadyAnalysis
investigated and reported in the literature that nanocellulose and other
The insight
nanocellulosic into the chemical
structures, such ascomposition of nanocellulose
cellulose nanocrystals samples was
or cellulose provided by
nanofibrils, consist of
EDS analysis.
chains On theorder
with different SEM degrees,
images, two measurement
which vary from locations of each
arrangements sample
that were de-
are highly crystalline
totermined, one on the chain
slightly unsettled particle, and the other
distributions on the
[37]. Forremaining
sample NC1fiber.(Figure
In the EDS profiles
2a–e), in varies
the size
Figure 3, well-defined peaks characteristic of carbon, oxygen, and nitrogen due
around 200 and 300 nm, while sample NC2 (Figure 2f–j) displays crystal sizes from 65 to to gold
coatings were found. For all samples, a high carbon content and a lower oxygen content
86 nm, as well as around 600 nm. For sample NC3, the crystal sizes decreased and ranged
were revealed. The difference in peak expressivity was large because of dehydration [38].
from 27 to 43 nm (Figure 2k–o). With each treatment, the nanocellulose is distinct and clear.
The difference in nitrogen content of particle and fiber was observed in all samples and
Notably,
might bethe
dueNC3 sample
to the exhibits
different shapessmaller and being
of the parts more measured.
even sizes,Because
while NC1 and NC2 exhibit
measurements
were performed at a low voltage (5 kV), some elements such as sulfur could not be de-
tected. They may be present in the amorphous regions in the samples by reason of higher
exposure of hydroxyl groups to esterification [39].
Biomimetics 2024, 9, 624 7 of 16

more uneven sizes, which is attributed to the irregular distribution of lignin. NC2 and NC3
demonstrate more uneven sizes, indicating a high aspect ratio. This characteristic can be
valuable in composite engineering applications, where such ratios are desirable.

3.2. EDS Elemental Analysis

The insight into the chemical composition of nanocellulose samples was provided
by EDS analysis. On the SEM images, two measurement locations of each sample were
determined, one on the particle, and the other on the remaining fiber. In the EDS profiles
in Figure 3, well-defined peaks characteristic of carbon, oxygen, and nitrogen due to gold
coatings were found. For all samples, a high carbon content and a lower oxygen content
were revealed. The difference in peak expressivity was large because of dehydration [38].
The difference in nitrogen content of particle and fiber was observed in all samples and
might be due to the different shapes of the parts being measured. Because measurements
were performed at a low voltage (5 kV), some elements such as sulfur could not be detected.
Biomimetics 2024, 9, x FOR PEER REVIEW 8 of exposure
They may be present in the amorphous regions in the samples by reason of higher 17
of hydroxyl groups to esterification [39].

Figure 3.
Figure 3. Scanning
Scanning electron
electronmicrographs
micrographsand
andline
linescanning EDS
scanning profiles
EDS of isolated
profiles cellulose
of isolated sam-samples:
cellulose
ples:
(a) (a) NC1,
NC1, (b) NC2,
(b) NC2, and and (c) NC3.
(c) NC3.

3.3. FT-IR Analysis

FT-IR spectroscopy has been extensively used in research regarding cellulose and NC
research, to study the functional groups and to obtain information on chemical changes
occurring during various chemical and mechanical treatments. The three main compo-
nents found in samples are cellulose, hemicellulose, and lignin, which are mainly com-
Biomimetics 2024, 9, 624 8 of 16

3.3. FT-IR Analysis

FT-IR spectroscopy has been extensively used in research regarding cellulose and NC
research, to study the functional groups and to obtain information on chemical changes
occurring during various chemical and mechanical treatments. The three main components
found in samples are cellulose, hemicellulose, and lignin, which are mainly composed of
alkanes, aromatic, esters, ketones, and alcohols [40]. Determined functional groups in the
structure of the samples NC1, NC2, NC3, and untreated raw material by infrared transmit-
tance spectra are shown in Figure 4. A broad peak at the range from 3400 to 3440 cm−1 was
observed in all samples (Figure 4a–c), which can be associated with free O-H stretching
vibrations of -OH groups bending due to the presence of inter- and intramolecular hydro-
gen bonds, which allow chain linkage and structure stability [41]. Consequently, due to
the formation of hydrogen bonds via hydroxyl groups during drying, small particles were
united into crystalline structures, which increased the surface tension of the fibers. The
vibration and elongation of C-H bonds in cellulose and hemicellulose was observed in all
three samples, which was pronounced at 2922 cm−1 , while in Figure 4c, the peak was less
pronounced, but still indicates the removal of hemicellulose, as in the other two samples
(Figure 4a,b). All presented samples’ characteristic peaks around 1510 cm−1 corresponded
to the vibration of aromatic C=C in-plane symmetrical stretching vibration of the aromatic
ring in lignin [42]. However, all bands assigned to lignin were not observed in the NC1–
NC3 samples, which may be successfully removed through the chemo-mechanical process.
The absorption bands at approximately 1300–1500 cm−1 correspond to the aromatic ring in
lignin; hence, the presence of lignin was confirmed for raw material, which was successfully
removed in all NC1–NC3 samples. In Figure 4c, a smaller peak was observed, indicating
a successful removal of the lignin. Also, the observation of the less pronounced peak at
1647 cm−1 correlated to the lower content of absorbed water in cellulose compared to
the other two samples [43]. A group of three peaks in the range from 1010 to 1165 cm−1
was observed in all samples, suggesting that the groups -CH, -CH2 , and -OH formed a
C-O-C glycosidic ether band and caused vibrations of the skeletal pyranose ring [44]. In
Figure 4c, the pronounced peak at 668 cm−1 occurred due to the presence of a β-glycosidic
bond, and thereby, Iβ cellulose was determined [45]. FT-IR analysis successfully identified
functional groups that were present in each sample and indicated typical band patterns for
cellulose, hemicellulose, and lignin, and that were present in all three samples. However,
all of the above characteristics indicated that each sample maintained its original structural
characteristics. Nevertheless, it was evident that there was a difference in the bandwidth
of each spectrum around 2900 cm−1 , 1600 cm−1 , and 660 cm−1 . It can be observed that
with an increase in the intensity of bleaching treatment using alkaline hydrogen perox-
ide, sample NC3 had a wider adsorption peak at the mentioned wavenumber positions,
as compared to the other two samples, NC1 and NC2. All these characteristics proved
that each treatment that occurred had an impact on treated cellulose, where it attacked
corresponding functional groups.

3.4. Thermogravimetric Analysis

As one of the most valuable and important characteristics of NC, the thermal stability
of the obtained NC was investigated by thermogravimetric analysis (TGA). Thermal stabil-
ity presents an important feature of NC, as it is intended as a reinforcing agent in various
biomimetic composites. The thermogravimetric curves of the samples (Figure 5) represent
weight losses at certain temperatures, and the contents of the samples were determined.
 tained its original structural characteristics. Nevertheless, it was evident that there was a
difference in the bandwidth of each spectrum around 2900 cm−1, 1600 cm−1, and 660 cm−1.
It can be observed that with an increase in the intensity of bleaching treatment using al-
kaline hydrogen peroxide, sample NC3 had a wider adsorption peak at the mentioned
wavenumber positions, as compared to the other two samples, NC1 and NC2. All these
Biomimetics 2024, 9, 624
characteristics proved that each treatment that occurred had an impact on9treated
of 16 cellu-
lose, where it attacked corresponding functional groups.

Figure
Figure 4. FTIR spectra of 4.the
FTIR spectrananocellulose
isolated of the isolated samples:
nanocellulose samples:
(a) NC1, (b) (a) NC1,
NC2, (c)(b) NC2,
NC3, and(c) NC3, and (d) raw
(d) raw
untreated material.
untreated material.
3.4. Thermogravimetric Analysis
All TGA curves showed thermal degradation of untreated material (Figure 5d) and
treated nanocelluloseAs one of the most valuable and important characteristics of NC, the thermal stability
(NC1–NC3) in the N2 atmosphere. Generally, the thermostability of
of the obtained NC was investigated by thermogravimetric analysis (TGA). Thermal
the isolated nanocellulose decreases with an increase in pretreatment intensity. For example,
compared with untreated cellulose, the maximum decomposition temperature of the NC1,
NC2, and NC3 gradually decreased from 340 ◦ C to 285 ◦ C, 305 ◦ C, and 280 ◦ C, respectively.
Thermal decomposition of cellulose is divided into three main stages. The initial weight
loss was observed in the first stage in the temperature range of 35 ◦ C to 120 ◦ C, which
was due to the physically adsorbed water and light volatiles, which were observed in
many reports [46,47]. Water evaporates when it is chemically absorbed or loosely bound
by intermolecular hydrogen bonds on the surfaces of cellulose. Water can bind to the
cellulose structure where sulfate amorphous sites are located and where hemicellulose
and lignin have been removed. Since prolonged alkaline treatment was performed, a
greater loss of water could be observed in the first sample (Figure 5a), as has also been
confirmed in the literature, since this treatment leaves the cellulose structure more open [44].
The second degradation step began at about 230 ◦ C when the sample mass was lost
through dehydration, decarboxylation, and decarbonylation. Thermal depolymerization
of hemicellulose occurred [48], lasting up to about 320 ◦ C for all samples. Hemicellulose
consists of various saccharide groups, such as mannose, xylose, galactose, and glucose in
an amorphous structure, which is full of branches that can easily be removed from the
main stem at low temperatures [49]. Cellulose pyrolysis begins at higher temperatures
because it consists of a long polymer of glucose, which is without branches but has a
stronger structure and high thermal stability. In the 230 ◦ C to 280 ◦ C temperature range,
crystalline areas began to decompose. With flatter curves in this area for the second sample
(Figure 5b), a smaller proportion of crystalline areas were determined. The prolonged acid
treatment confirms that this kind of treatment increased the amount of amorphous sites.
Hydrocarbon residues and monomeric glucopyranose units break up into free radicals
up to about 420 ◦ C [45]. Lignin is decomposed at high temperatures and is considered to
be the most thermally stable of the organic components. Because of its complex structure
with numerous ether linkages and hydroxyl and methoxy groups, the decomposition of
lignin was carried out over a wide temperature range. Decomposition began with the
evaporation, melting, and degradation of polysaccharide residues, resulting in loosely
bound functional groups. The lignin degradation was in accordance with previously
published works [46,47,50]. In the second part, the bigger mass loss occurred, due to the
Biomimetics 2024, 9, 624 10 of 16

cleavage of the glycosidic bonds and depolymerization of xylan units [40,49]. At 600 ◦ C,
33.45%, 38.64%, and 31.45% of the initial mass of NC1 (Figure 5a), NC2 (Figure 5b), and NC3
(Figure 5c) remained in the samples, respectively. The residue was represented by inorganic
substances [51] and non-combustible sulfate esters [45]. Also, a reduction in polymerization
degree and particle size was observed in all samples, particularly in sample NC3 (Figure 5c),
which was confirmed with TGA. It can be observed from the TGA graphs that the onset
of degradation temperature was seen to shift to a higher temperature, indicating that the
thermal stability has improved. For NC1, the first step occurs before reaching 300 ◦ C, while
for NC2, the first step is visible after 300 ◦ C. Two-stage degradation can be observed in
sample NC3, which contributes to the evaporation of water and polysaccharide residues.
As an alternative to other alkaline treatments, oxidative alkaline treatments, which use
hydrogen peroxide, have reported synergy with many pretreatment methods, such as acetic
acid organosolv treatment [52], steam explosion [53], and alkaline extraction [54]. Because
Biomimetics 2024, 9, x FOR PEER REVIEW 11 of 17
of its mild treatment conditions, as well as its low impact on the environment, hydrogen
peroxide has become a popular treatment method for biomass fractionation [55].

Figure5.5.TGA
Figure TGAprofiles
profilesof
ofthe
theisolated
isolatedcellulose
cellulosesamples:
samples:(a)
(a) NC1,
NC1, (b)
(b) NC2,
NC2, (c) NC3, and (d) untreated
rawmaterial.
raw material.

3.5. Differential Scanning Calorimetry (DSC) Analysis

With DSC analysis, additional insights into the thermal stability of the samples were
provided. The thermal degradation of the samples as shown with TGA and in Figure 6
occurred in three steps. Firstly, decomposition occurred with the removal of unbound and
bound water molecules. The curves have, in addition to the main one, several smaller
Biomimetics 2024, 9, 624 11 of 16

3.5. Differential Scanning Calorimetry (DSC) Analysis

With DSC analysis, additional insights into the thermal stability of the samples were
provided. The thermal degradation of the samples as shown with TGA and in Figure 6
occurred in three steps. Firstly, decomposition occurred with the removal of unbound and
bound water molecules. The curves have, in addition to the main one, several smaller
peaks due to other components that differentially bound and retained water in the struc-
ture [44]. The weight loss was distributed over the entire temperature range throughout the
diagram because the sample consisted of cellulose, lignin, hemicellulose, pectin, and other
components having different melting temperatures. All three DSC thermograms of the
samples exhibited distinct endothermic changes occurring within the temperature range
since the nature of endotherms is quite characteristic of the composition of the material. The
initial endotherm could be observed in all three samples, which occurred at much lower
temperatures than 100 ◦ C and happened due to evaporation, causing moisture loss. Such
moisture loss occurred also due to sulfuric acid treatment since the sulfuric acid acted as a
dehydrating catalyst. Moreover, cellulose crystals were sulfonated on the active surface,
which reduced the affinity for moisture absorption. Therefore, some moisture could be
adsorbed on the surface; however, it was loosely bound and could evaporate at a much
lower temperature. The main degradation step included dehydration reactions and the
formation of volatile products. Most cellulose and hemicellulose decomposed in the range
of 200–290 ◦ C, which is evident from Figure 6d, and most of the decomposition for raw ma-
terial occurred at 100 ◦ C. The third degradation step for samples NC1–NC3 occurred above
400 ◦ C with a long swing of the curve corresponding to the decay of lignin. Degradation
of the first sample took place at a higher temperature than the other two samples, which
proved higher thermal stability. As a result of the alkaline treatment, its structure was more
orderly. After the removal of hemicellulose and lignin, the hydrolysate could diffuse into
the fibers more easily. Treatment with sulfuric acid involved the esterification of hydroxyl
groups, and sulfates acted as catalysts for the hydrolysis of pyranose bonds, which linked
cellulose units to longer chains. Owing to the size and abundance of the sulfate groups, the
cellulose structure was rearranged and the amount of amorphous regions increased, which
led to an extensive reduction in the polymerization degree and, consequently, particle
size [44].

3.6. Particle Size Distribution Studies

The DLS technique is commonly used to obtain and verify particle sizes and their
stability, but also to report on the statistical distribution of the particles present in NC.
Obtained measurements considered spherical particles, and the values depended on the
orientation of the fibers in suspension; in most cases, they were higher than those measured
by microscopic analysis. According to Figure 7a, suspension NC1 showed two peaks with
mean values around 200 nm and 83 nm, with intensities of 64.7% and 33.8%, respectively.
For suspension NC2 (Figure 7b), a distribution in three different ranges was observed,
where mean values were near 76 nm, with an intensity of 32.2%, and a more significant
population (59.3%) with average sizes of 227 nm and 877 nm and an intensity of 8.5%. For
suspension NC3 (Figure 7c), the measured particles were within the 50–100 nm range.
The most consistent particle sizes were observed in sample NC3, where bleaching
treatment was prolonged. Alkaline bleaching using hydrogen peroxide is essentially a
delignification process, which can result in the solubilization of a considerable amount of
hemicellulose since it induces depolymerization and cleavage of ester bonds cross-linking
lignin and hemicellulose [56]. The DLS analysis is in accordance with results obtained
from SEM and TGA, which confirms that the most consistent sizes are obtained when the
cellulose is treated with prolonged bleaching treatment using alkaline hydrogen peroxide.
 could diffuse into the fibers more easily. Treatment with sulfuric acid involved the esteri-
fication of hydroxyl groups, and sulfates acted as catalysts for the hydrolysis of pyranose
bonds, which linked cellulose units to longer chains. Owing to the size and abundance of
the sulfate groups, the cellulose structure was rearranged and the amount of amorphous
Biomimetics 2024, 9, 624 regions increased, which led to an extensive reduction in the polymerization degree
12 of 16 and,
consequently, particle size [44].

Biomimetics 2024, 9, x FOR PEER REVIEW 13 of 17

The DLS technique is commonly used to obtain and verify particle sizes and their
stability, but also to report on the statistical distribution of the particles present in NC.
Obtained measurements considered spherical particles, and the values depended on the
orientation of the fibers in suspension; in most cases, they were higher than those meas-
ured by microscopic analysis. According to Figure 7a, suspension NC1 showed two peaks
with mean values around 200 nm and 83 nm, with intensities of 64.7% and 33.8%, respec-
tively. For suspension NC2 (Figure 7b), a distribution in three different ranges was ob-
served, where mean values were near 76 nm, with an intensity of 32.2%, and a more sig-
nificant population (59.3%) with average sizes of 227 nm and 877 nm and an intensity of
Figure
8.5%.
Figure 6.6.DSC
For DSCprofile
profilecurves
suspension curves
NC3 of ofthe
theisolated
(Figure isolated cellulose
7c), the measured
cellulose samples:
(a)(a)NC1,
particles
samples: NC1,(b)(b)
were NC2,
(c)(c)
within
NC2, NC3,
the
NC3, and (d)
50–100
and nm
untreated raw material.
(d) untreated raw material.
range.
3.6. Particle Size Distribution Studies

Figure 7. Cont.
Biomimetics 2024, 9, 624 13 of 16

7. Particle
Figure 7.
Figure Particlesize
sizedistribution
distributionprofiles of isolated
profiles cellulose
of isolated samples.
cellulose samples: (a) NC1, (b) NC2, and (c)
NC3.
4. Conclusions
In the present study, nanocellulose was isolated from Polypodiopsida fern under three
The most consistent particle sizes were observed in sample NC3, where bleaching
different prolonged treatment conditions. Each chemical process consisted of alkaline
treatment was prolonged. Alkaline bleaching using hydrogen peroxide is essentially a del-
and acid treatment, as well as bleaching and sulfuric acid treatment. In three different
ignification
experiments,process,
alkaline, which canand
bleaching, result
acidinhydrolysis
the solubilization
treatmentsof a considerable
were prolonged to amount
24 h. of
hemicellulose
The prolonged since it induces
alkaline treatment depolymerization
resulted in a moreand cleavage
orderly of ester
structure bonds cross-linking
of nanocellulose in
lignin
sampleand NC1,hemicellulose [56]. The
while the prolonged acidDLS analysis
treatment is in accordance
confirmed with sites
more amorphous results obtained
on the
from
surface SEM and TGA, which
of nanocellulose confirms
in sample NC3.that the analysis
FT-IR most consistent sizes
confirmed thatare obtained
each when the
prolonged
treatmentis
cellulose had an impact
treated withon the isolated
prolonged nanocellulose
bleaching in all samples
treatment of NC1, hydrogen
using alkaline NC2, and NC3.
peroxide.
It is evident from our study that varying the time of each chemical process by prolonging
its time of treatment can affect the end characteristics of isolated nanocellulose and in doing
so, enhance its quality and potential for use in various industrial applications. Such new
NC biomaterials may be used for industrial coatings and suspensions, as well as stabilizing
agents for water-based paints. Such advanced product development and its optimization
are crucial in adding value to derived nanocellulose, which is necessary in the development
of advanced and feasible nanocellulose-based biopolymer materials. Additionally, to
overcome increasing environmental and economic concerns, milder reaction conditions
and easier recyclability are needed.

Author Contributions: K.V.: conceptualization, methodology, formal analysis, investigation, writing—

original draft preparation, writing—review and editing, funding acquisition; M.D.: formal analysis
and investigation, writing—original draft preparation; Ž.K.: funding acquisition, resources; M.L.:
conceptualization, writing—review and editing, funding acquisition, resources, supervision. All
authors have read and agreed to the published version of the manuscript.
Funding: Funding was provided by the Slovenian Research and Innovation Agency (ARIS), Program
P2-0046 “Separation Processes and Product Design” and Research Projects J2-3037 “Bionanotechnol-
ogy as a tool for stabilization and applications of bioactive substances from natural sources”; Z2-4431
“Functional biocomposites for biomedical and sustainable applications”; and L2-4430 “Production,
isolation and formulation of health beneficial substances from Helichrysum italicum for applications
Biomimetics 2024, 9, 624 14 of 16

in cosmetic industry”. The authors acknowledge the use of research equipment for the production
of biological substances and their detection, procured within the project “Upgrading national re-
search infrastructures—RIUM”, which was co-financed by the Republic of Slovenia, the Ministry of
Higher Education, Science and Innovation, and the European Union from the European Regional
Development Fund, and the system of downstream processes for performance of obtaining biological
substances was used (Package 21, ARIS).
Institutional Review Board Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this
published article.
Acknowledgments: The authors acknowledge the Slovenian Research and Innovation Agency (ARIS).
Conflicts of Interest: The authors declare that they have no conflicts of interest.

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