Carbohydrate Polymers 245 (2020) 116513

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Effect of different conditions of synthesis on properties of silver T

nanoparticles stabilized by nanocellulose from carrot pomace
Jolanta Cieśla, Monika Chylińska, Artur Zdunek, Monika Szymańska-Chargot*
Institute of Agrophysics, Polish Academy of Sciences, Doświadczalna 4, 20-290, Lublin, Poland

A R T I C LE I N FO A B S T R A C T

Keywords: The silver nanoparticles (AgNPs) can exhibit different optical properties depending on their size and shape as a
Nanocellulose result of synthesis method and the stabilizer used. In this research the synthesis of AgNPs in the presence of
Silver nanoparticles nanocellulose obtained from carrot pomace was investigated. The influence of silver nitrate concentration,
Optical properties temperature and mechanical agitation on size and shape of AgNPs was studied. The mixing of reagents during
Stabilizing agent
synthesis, regardless temperature, led to obtain AgNPs of various sizes and shapes. It was confirmed by different
colors of samples with absorbance maximum from 334 to 779 nm, the transmission electron microscopy images
and dynamic light scattering results. In unmixed samples only spherical nanoparticles with absorbance max-
imum at 408 nm were observed. Obtained results have demonstrated that mechanical agitation and an appro-
priate silver nitrate concentration combined with stabilizing effect of nanocellulose allow to obtain AgNPs in
different shapes and sizes.

1. Introduction
Colloidal metal nanoparticles have received great attention due to
their unique optical, electronic, magnetic and antimicrobial properties.
Their small size, large specific surface area and tunable physicochem-
ical properties that differ significantly from the bulk analogues led to
intense research on their use in composite materials (Lee & Jun, 2019)
and are reason for exploiting them in large variety of fields from
chemistry to medicin (Burdușel et al., 2018). Due to the low cost of
obtaining and desirable physicochemical properties silver nanoparticles
(AgNPs) are most commonly used among metal nanoparticles
(Chouhan, 2018). Therefore, it is crucial to determine the conditions of
their synthesis such as dissolution rate (i.e. reactivity of the silver salt in
the solution and the release of silver ions) and the type of reducing and
stabilizing agents used to obtain structures with given surface proper-
ties, size distribution, morphology and composition of particles. So far
AgNPs have been produced in various shapes such as spheres (Jose &
Sakthivel, 2014), plates (hexagonal or triangular) (Sarkar & Das, 2016;
Thiele et al., 2015), rods (Rekha, Nayar, & Gopchandran, 2018) and
cubics (Sun & Xia, 2002). Methods of AgNPs production can be divided
into top-down, which are usually physical methods and, more popular,
bottom-up method which involves chemical or photoinduced synthesis
(Lee & Jun, 2019; Liu, Wang, Song, & Shang, 2011; Szymańska-Chargot
et al., 2009; Wang, Jiang, Hsieh, & Nitin, 2014). The chemical synthesis
requires the use of reducing and stabilizing agents and its advantage is
that by choosing conditions and substrate the AgNPs with various
properties can be obtained. The most commonly used reductants are:
dimethylaminoborane, sodium borohydride, glucose, hydrazine, so-
dium ascorbate, ethylene glycol, N-dimethylformamide and sodium
citrate. Due to the fact that metal nanoparticles are generally thermo-
dynamically unstable, stabilizing agents are used to prevent aggrega-
tion. The polyacrylic acid, sodium salt, polyvinyl alcohol, poly-
vinylpyrrolidone, chitosan, cellulose, polyethylene glycol,
polymethacrylic acid or polymethacrylate are the most commonly used
stabilizers (Lee & Jun, 2019). However, general problem of all methods
of AgNPs synthesis is to control the formation and growth of AgNPs
with narrowly defined optical properties (Jiang, Chen, Chen, Xiong, &
Yu, 2011). That’s why still are searching optimal conditions of synthesis
involving different light lengths (photo assisted synthesis), different
temperatures, time of synthesis, or mentioned above different com-
pounds used as reductants and stabilizing agents.
Generally, polysaccharides, thanks to their ability to coordinate
metal ions, can act as both reducing and stabilizing agents. The metal
ion complex with the polysaccharide can then be reduced under mild
conditions, resulting in the formation of smaller size particles and
narrower size distribution than those that are obtained in the absence of
stabilizer (Emam & Ahmed, 2016). In recent years, nanocellulose due to
its large surface area, reducing functional groups on the surface and the

⁎
Corresponding author.
E-mail address: m.szymanska@ipan.lublin.pl (M. Szymańska-Chargot).

https://doi.org/10.1016/j.carbpol.2020.116513
Received 27 March 2020; Received in revised form 20 May 2020; Accepted 23 May 2020
Available online 02 June 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.
J. Cieśla, et al.
ability to form stable suspensions in water has found its use as stabi-
lizing and/or reducing agent in AgNPs synthesis process (Emam &
Ahmed, 2016; Gopiraman et al., 2015; Liu et al., 2011; Wang et al.,
2014). An important feature of cellulose nanostructures is the nu-
merous hydroxyl groups on the surface introduced during preparation
via for example TEMPO-oxidation, which results in their high reactivity
(Kaushik & Moores, 2016; Lokanathan, Uddin, Rojas, & Laine, 2014).
The possibility of using the fruit and vegetable pomaces as the
source of cellulose has recently been presented (Szymańska-Chargot,
Chylińska, Gdula, Kozioł, & Zdunek, 2017; Szymańska-Chargot,
Chylińska, Pieczywek, & Zdunek, 2019), since the fruit and vegetable
pomaces constitute residues after food processing and therefore popular
waste. Under the mild acid-alkali conditions, cellulose from pomaces
was extracted and nanocellulose was produced using a high-intensity
ultrasonication process. It has also been shown that the choice of cel-
lulose source can lead to the acquisition of nanocellulose with different
structures, i.e. high-intensity ultrasonication of apple cellulose resulted
in the formation of nanofibrils, while carrot nanocellulose was obtained
in the form of large whiskers (Szymańska-Chargot et al., 2019). In the
present study, the carrot nanocellulose with an average diameter of
3.31 nm and crystallinity degree of 80.09 % was used (Szymańska-
Chargot et al., 2019).
In this paper the hypothesis that silver nitrate concentration, tem-
perature and mechanical agitation (mixing) has influence on formation
of AgNPs stabilized by the carrot nanocellulose was tested. As a re-
ductant the sodium borohydride was used and nanocellulose from
carrot pomace was a stabilizing agent. The distribution of AgNPs shapes
and sizes was characterized with transmission electron microscopy
(TEM), UV–vis spectroscopy and Dynamic Light Scattering (DLS).
Stability of AgNPs/CNF systems were checked using Laser Doppler
Electrophoresis (LDE). To the best of our knowledge, it is first time
when nanoparticles in diverse shapes, sizes and therefore optical
properties were obtained using the carrot nanocellulose as a stabilizing
agent.
2. Materials and methods
2.1. Chemicals
Silver nitrate (AgNO3), sodium borohydride (NaBH4), hydrochloric
acid (HCl), sodium hydroxide (NaOH), sodium hypochlorite (NaClO)
were purchased in POCH Poland (Avantor Performance Materials
Poland S.A., Gliwice, Poland) and were of analytical grade. Deionized
water with conductivity of 0.11 μS/cm was used along all processes.
2.2. Nanocellulose isolation
The nanocellulose (CNF) was obtained from carrot pomace as de-
scribed by Szymańska-Chargot et al. (2017). Briefly, the thermo-
chemical process was based on the extraction of pectic and hemi-
cellulosic polysaccharides in first acidic and then alkaline environments
followed by lignin removal in sodium hypochlorite solution. After the
treatment, carrot cellulose was obtained as water suspension with
concentration around 4 % wt. The nanocellulose from cellulose was
prepared via an ultrasonication treatment with a Sonics Vibracell ul-
trasonic homogenizer (VCX-130FSJ; Sonics & Materials, Inc., Newtown,
CT, USA). The sonication system contained a temperature probe, and to
avoid heating the samples, an ice bath was used. The operation am-
plitude of ultrasonic homogenizer was maintained at 90 % of the
nominal amplitude (Szymańska-Chargot et al., 2019). Finally, carrot
cellulose nanofibers (CNF) in concentration of 0.1 % wt. were obtained.
2.3. Silver nanoparticles synthesis
Sodium borohydride was added to the suspension of CNF in water
(0.1 %) (so that the final concentration of NaBH4 was 2 mM). The initial
2
Carbohydrate Polymers 245 (2020) 116513
temperature of CNF was 5 °C or 20 °C depending on the temperature of
further synthesis. The whole was mixed using a magnetic stirrer for
around 10 min until complete dissolution of NaBH4. Thereafter, 3 ml of
an AgNO3 solution of appropriate concentration (from 0.01 mM to 0.3
mM) was added dropwise to the 12 mL of CNF/NaBH4 suspensions (Fig.
S1). Immediately, the containers with suspensions were put onto la-
boratory rotator operating with constant velocity of 10 rpm. The re-
action was continued for 48 h at either 5 or 20 °C with simultaneous
mixing and no light. Visible during the first hours of synthesis, gas
bubbles originated from the hydrogen formed as a result of the reaction
between NaBH4 and water. Simultaneously, the analogous set of solu-
tions were prepared in the same route with difference that no me-
chanical agitation was used.
Additionally, to evaluate whether the nanocellulose plays role as
only a stabilizer or also as reducing agent during AgNPs synthesis the
system containing the 0.08 mM of AgNO3 was investigated deeply.
Following dispersions were prepared: CNF in H2O, CNF/AgNO3 (0.08
mM), CNF/NaBH4 (2 mM) and CNF/NaBH4 (2 mM)/AgNO3 (0.08 mM).
Moreover, CNF dispersions with NaCl (0.08 mM): CNF/NaCl and CNF/
NaBH4 (2 mM)/NaCl (0.08 mM) were obtained as the systems similar to
those containing AgNO3. Samples were prepared with (mixed) or
without (unmixed) mixing in dark for 48 h at 20 °C. The UV–vis ab-
sorbance, pH, electrolytic conductivity, zeta potential and the particles
size were investigated.
2.4. Spectroscopy UV–vis
Spectrophotometric studies on the formed AgNPs were carried out.
Absorbance of AgNPs/CNF colloids was characterized by Genesys 10 s
UV–vis (Thermo Scientific). Quartz cuvettes with optical path of 10 mm
were used. The absorbance spectra were recorded in the range of
190–1000 nm. As a reference sample the 0.1 % CNF suspension was
used. The measurements were taken after 48 h from instilling a solution
of silver nitrate.
2.5. Transmission electron microscopy (TEM)
Liquid samples were placed on copper grids covered with Formvar
(Ted Pella) and carbon and left to dry. Then they were examined using
high performance transmission electron microscope JEM 1400 (JEOL
Co.) equipped with 11 Mpx TEM camera MORADA G2 (EMSIS GmbH).
Several dozens of images were obtained for each sample and re-
presentative ones are shown.
2.6. Nanoparticles size and zeta potential of AgNPs/CNF dispersion
Dynamic Light Scattering (DLS) method (ISO 22412 2017) was used
to determine the mean hydrodynamic diameter, polydispersity index
and particles size distributions of the systems studied. Measurements
were done using Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern,
UK) equipped with the He–Ne laser (633 nm) as a source of light. The
173° (back-scattering) and 12.3° (forward-scattering) detection angles
were applied.
Electrophoretic mobility of dispersed particles was determined
using Zetasizer Nano ZS apparatus and Laser Doppler Electrophoresis
(LDE) method (Mayinger, 1994). Zeta potential was calculated using
the Henry’s equation (Delgado, Gonzales-Caballero, Hunter, Koopal, &
Lyklema, 2007). Electrolytic conductivity of samples was determined
simultaneously with electrophoretic mobility using a general purpose
model by the apparatus software. All measurements were carried out in
six repetitions at 20 °C.
An influence of the procedure of samples preparation (a mixing,
different temperatures, AgNO3 concentration) on the obtained results
was checked using the multi- and one-way ANOVA (Statistica v. 12,
StatSoft, Cracow, Poland).
For better determination of the changes in particles size and zeta
J. Cieśla, et al.
potential connected with the AgNPs presence the differential distribu-
tions of the above parameters were obtained. It was done by subtraction
of the appropriate curve (relationship between the particles size or zeta
potential and percentage of the scattered light intensity) characterizing
the pure nanocellulose/NaBH4 from this determined for the sample
containing AgNPs. Only the positive effects were considered further and
the area normalization was used to obtain the new curves.
3. Results
3.1. Optical properties of AgNPs stabilized by CNF
The color change occurred in all CNF suspensions after the injection
of AgNO3 solution. It was preliminary evidence that AgNPs were
formed. Initially, it was a yellow color with varying intensity dependent
on AgNO3 starting concentration. However, after 48 h differences in the
color of suspensions with the same initial concentrations of AgNO3
between mixed and unmixed suspensions were noticeable (Fig. S2).
Unmixed suspensions remained yellow, and only the intensity of this
color depended on the initial concentration of AgNO3. While mixed
suspensions changed their color which was influenced by the initial
concentration of AgNO3. Only for samples with initial AgNO3 con-
centrations above 0.15 mM the color was the same regardless of whe-
ther mechanical agitation was applied or not. Temperature had little
effect on the color differences of mixed suspensions with the same in-
itial concentration of AgNO3 and had no effect on the unmixed sus-
pensions (Fig. S2).
Fig.1 presents UV–vis spectra of AgNPs as function of AgNO3 con-
centration from 0.01 mM to 0.3 mM at constant concentration of CNF
(stabilizing agent) and NaBH4 (reducing agent). The high intensity band
with maximum at 190 nm is connected with presence of NaBH4, while
broad band with maximum around 220 nm is proportional to Ag con-
centration in solution (Van Hyning & Zukoski, 1998). Apart that two
absorbance regions can be distinguished in UV–vis spectra: (1)
300−450 nm and (2) 450−800 nm. The first one with maximum ab-
sorbance around 403 nm is characteristic for all samples, but its in-
tensity decreased gradually with decreasing concentration of AgNO3.
For samples whose initial concentration of AgNO3 was below 0.15 mM
and were mixed the absorbance bands in the second region could be
observed. The maximum of absorbance was shifted towards longer
wavelengths (red shift) with decreasing initial concentration of AgNO3
and also small band with maximum around 338 nm occurred for these
samples. The absorbance region with maximum around 400 nm is
usually obtained for spherical AgNPs (Lee & Jun, 2019). Evolution of
another absorbance region can be proof that AgNPs were obtained in
sizes and shapes different from spherical (Huang & Nancy Xu, 2010;
Rivero, Goicoechea, Urrutia, & Arregui, 2013). Also shift to longer
wavelengths (red-shift) could be connected with either reduction of
particles numbers or increase of their size (Huang & Nancy Xu, 2010).
According to Kawabata-Kubo effect the increase in size of nanoparticles
cause the red-shift of the absorption bands (Van Hyning & Zukoski,
1998). (Thiele et al., 2015) demonstrated that the shift of absorbance
maximum to the longer wavelength and change in silver colloid color
from orange through pink and purple to blue is due to growth of silver
nanotriangles. The temperature influence on nanoparticles formation
was also observed. For samples mixed in 20 °C absorbance maxima
were shifted towards longer wavelengths (difference was even 200 nm)
comparing with samples mixed in 5 °C. Absorbance maxima of samples
which weren’t mixed also were slightly shifted but they were within the
range of 403−424 nm.
The absorbance maxima obtained for each AgNO3 concentration are
presented in details in Table 1. Beginning from silver salt concentration
of 0.10 mM its further decrease resulted in the split of absorbance
maxima in AgNPs UV-vis spectra to two or three for samples obtained
under mixing no matter in which temperature the synthesis was con-
ducted. For all this samples the maximum between 410–430 nm is
3
Carbohydrate Polymers 245 (2020) 116513
present which could be connected with the presence of spherical
AgNPs. The peak around 330–338 nm could be connected with in-plane
and out-plane quadrupole plasmon resonances and sharper edges of
formed nanoparticles in shapes different than spherical (Rivero et al.,
2013). But also it could be connected with polydispersity of nano-
particles size (Van Hyning & Zukoski, 1998). The presence of more than
one maximum influences the shade and intensity of colloid color.
3.2. Shape of AgNPs stabilized by CNF
In addition, TEM images were obtained on which nanoparticles of
various shapes are visible. In the case of unmixed samples (Fig. 2), the
spherical nanoparticles with averaged relatively small diameter were
obtained regardless of the initial concentration of AgNO3. However,
very large nanoparticles were also present.
In the case of AgNPs/CNF mixed systems (Fig. 3), the shape and size
of the nanoparticles related to the initial concentration of AgNO3. Tri-
angular, hexagonal, nanorods, spherical nanoparticles conglomerates
were obtained, however variability of different shapes increased with
decreasing initial concentration of AgNO3. In particular, for the initial
concentration of 0.15 mM or above only spheres with small diameter
were observed in TEM images. In the case of 0.1 mM triangles appeared
apart of spherical nanoparticles (Fig. 3 downward panel). From con-
centration of 0.09 mM the proportion of triangular to spherical nano-
particles increased, but also other shapes of AgNPs appeared such as
hexagons (from concentration 0.07 mM) or rods (0.06 mM). Also size of
nanoparticles evidently increased with decreasing initial concentration
of AgNO3. The increase of AgNPs size is accompanied by decrease of
spherical nanoparticles number. This is the most visible for con-
centration of 0.02 – 0.04 mM where triangles or quasi-triangles are
dominant (Fig. 3 upward panel).
3.3. Physical and physicochemical properties of AgNPs/CNF colloids
The colloids samples shown in Fig. S2 were characterized in respect
to the size of dispersed particles using DLS method. It was found, that
the AgNO3 concentration, temperature and mechanical agitation had a
significant (multi–way ANOVA, p < 0.05) influence on the mean hy-
drodynamic diameter and polydispersity index. Generally, the particles
size in mixed samples was lower and the polydispersity was higher than
in the unmixed ones. It is consistent with the results of spectroscopic
and microscopic analyses, discussed above. Furthermore, the samples
synthesized at 20 °C contained smaller and more inhomogeneous in size
particles than the colloids prepared at 5 °C. It can be related to an in-
crease of effectiveness of AgNPs synthesis due to the mechanical and
thermal energy application. An initial concentration of AgNO3 differ-
entially modified the samples properties, dependently on the tem-
perature and the mixing applied.
The mean hydrodynamic diameter of particles synthesized at 5 °C
without the mixing (88.0 ± 1.1 nm) (Fig. 4a) was not significantly
different from the CNF control sample (87.8 ± 1.6 nm) and was not
affected by the initial concentration of AgNO3 (p > 0.05). However, the
polydispersity index increased linearly with the increasing content of
silver salt (r = 0.61), confirming the presence of various-sized particles.
In the case of synthesis without the mixing at 20 °C (Fig. 4c), both the
mean hydrodynamic diameter (p > 0.05) and polydispersity index
(p > 0.05) were independent of the AgNO3 concentration. The CNF
particles size was 86.1 ± 4.0 nm and the mean size of particles in
systems containing AgNPs was 81.9 ± 2.1 nm.
In the systems obtained with the use of mixing both the hydro-
dynamic diameter and polydispersity index were variable with the
silver salt concentration. For synthesis at 5 °C (Fig. 4b) the pure CNF
particles size was 87.2 ± 0.6 nm at 0.31 ± 0.05 polydispersity. The
mean hydrodynamic diameter decreased from 83.0 ± 3.0 nm to
62.5 ± 2.3 nm, whereas polydispersity index increased from
0.39 ± 0.02 to 0.52 ± 0.02 with the AgNO3 initial concentration
J. Cieśla, et al. Carbohydrate Polymers 245 (2020) 116513

Fig. 1. Absorbance spectra obtained for various initial concentrations of AgNO3 after 48 h from instillation to a suspension of nanocellulose from carrot pomace
(CNF, 0.1 % wt.). Synthesis was carried out in 5 °C (upward panel) or 20 °C (downward panel). The decreasing intensity of peaks is connected with decreasing
concentration of AgNPs. Capital B in photo denotes blank (reference sample 0.1 % CNF).

increasing from 0.01 to 0.05 mM. Further increase of silver salt con-
centration to 0.1 mM led to increase of mean hydrodynamic diameter to
83.6 ± 1.6 nm and decrease of polydispersity index to 0.37 ± 0.01. At
the AgNO3 concentration ranging from 0.1 to 0.25 mM the particles size
increased to 86.5 ± 0.9 nm but polydispersity slightly decreased to
0.34 ± 0.04. At the highest concentration of silver salt (0.3 mM) the
mean hydrodynamic diameter increased to 106 ± 4 nm with the
polydispersity index increasing to 0.48 ± 0.09. Such increase could be
an effect of the agglomerates formation. At the synthesis temperature of
20 °C (Fig. 4d) the dependence of mean hydrodynamic diameter and
polydispersity index on the AgNO3 content was more complicated.
Firstly, the size of particles decreased from 87.8 ± 3.7 nm to 49.5 ± 3.3
nm and polydispersity increased from 0.37 ± 0.02 to 0.54 ± 0.02 at the
concentration increasing from 0.01 to 0.02 mM. In the AgNO3 con-
centrations ranged from 0.02 to 0.04 mM the increase of mean hy-
drodynamic diameter to 85.0 ± 3.4 nm with decrease of polydispersity
index to 0.42 ± 0.04 was observed. Further increase of silver salt
concentration to 0.08 mM resulted in decrease of mean hydrodynamic
diameter of particles to 55.2 ± 2.1 nm and an increase of polydispersity
index to 0.52 ± 0.03. Next increase of AgNO3 concentration led to in-
crease of the particles size (94.4 ± 0.2 nm at 0.3 mM) and decrease of
polydispersity (0.40 ± 0.03 at 0.3 mM). The variability of mean hy-
drodynamic diameter and polydispersity index values with the silver
salt concentration in the systems obtained by using the mechanical
agitation corresponds to that connected with the differential shapes and
optical properties of dispersed AgNPs, determined by TEM and UV–vis
methods.
Considering that the formation of AgNPs can have an influence also
on three-dimensional structure of colloids studied, the ratio of mean
hydrodynamic diameters obtained from the back- and forward- light
scattering was additionally investigated (Fig. S3). In all cases, the di-
mension based on the forward scatter (dforward) was longer than that
obtained from back-scattering (dback). It was probably due to the pre-
sence of CNF macromolecules which are rod-like particles dominating
in the studied dispersions. Assuming that the dback/dforward ratio reflects
the minimum diameter to maximum diameter value, it could be treated
as an estimation of aspect ratio. In this case, the value close to 0 points
out the elongated shape, whereas the value of 1 indicates a spherical
one. The dback/dforward ratio ranged from 0.15 to 0.35 for systems ob-
tained at 5 °C and was not affected by the silver salt concentration. At
20 °C the AgNPs synthesis led to increase of this ratio value in respect to
that of pure nanocellulose. It was visible especially for the unmixed
system, where the dback/dforward increased from 0.3 to about 0.5 at
AgNO3 concentration increasing from 0.01 to 0.09 mM but the further
increase of silver salt content did not change this value. Systems which
were synthesized with the mixing applied were more differentiated in

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J. Cieśla, et al. Carbohydrate Polymers 245 (2020) 116513

Table 1 S4. For all samples the addition of AgNO3 resulted in an increase of the
Absorbance maxima obtained for various initial concentrations of AgNO3 after peaks’ height at the particles diameter of ∼100 and 20 nm. For samples
48 h from injection to a suspension of CNF from carrot pomace (0.1 % wt.). prepared with the mechanical agitation at the silver salt concentration
Sample 5 °C 20 °C lower than 0.1 mM the peaks from several-nanometer (especially at 20
°C) and about 10-nm particles were visible. At the mixing applied and
UNMIXED MIXED UNMIXED MIXED AgNO3 concentration ranging from 0.01 to 0.1 mM over 50 % of total
λmax [nm] λmax [nm] λmax [nm] λmax [nm]
area of peaks referred to the size lower than 100 nm (Fig. S5). This
0.01 mM 413 441 424 412 percentage decreased with an increasing concentration of the silver salt,
0.02 mM 412 335 419 341 which could be a consequence of the agglomerates formation or
733 589 stronger immobilization of small nanoparticles in nanocellulose net-
0.03 mM 412 334 420 338
work (see Fig. 3). For the samples prepared without mixing the area of
625 567
779
peaks of particles lower than 100 nm was about 20–50 %.
0.04 mM 409 337 417 An initial amount of AgNO3 as well as the method of sample pre-
418 340 paration had a significant (p < 0.05) influence on the electrolytic
544 470 conductivity and zeta potential values. Generally, the electrolytic con-
0.05 mM 408 336 420 336
ductivity and zeta potential (absolute value) in mixed samples were
412 429
577 602 higher than those obtained for unmixed ones. The samples synthesized
0.06 mM 408 336 412 336 at 20 °C were characterized by lower electrolytic conductivity and more
422 418 negative values of zeta potential than the dispersions prepared at 5 °C.
532 589
Relationship between the initial concentration of silver salt and both
0.07 mM 408 337 411 336
414 414
the electrolytic conductivity and zeta potential is shown in Fig. 5.
517 573 The negative sign of zeta potential for both the CNF and AgNPs/CNF
0.08 mM 408 337 410 336 pointed out that the synthesized AgNPs were electrostatically stabilized
414 412 by nanocellulose. The value of electrokinetic potential ranged from
518 563
−60 to −40 mV that corresponds to the highly stable systems (de
0.09 mM 407 338 410 336
412 410 Barros, Cruz, Mayrink, & Tasic, 2018). The range of zeta potential va-
488 546 lues of the new-formed particles was very wide (Fig. S6). Even the
0.10 mM 407 338 409 338 positive or close to 0 values were detected, however, in most cases, over
410 407
50 % of the total area of peaks represented the absolute value of zeta
486 512
0.15 mM 405 405 409 406
potential higher than 30 mV (Fig. S7).
0.20 mM 405 403 409 404
0.25 mM 405 403 408 403 3.4. Evaluation of the role of CNF in AgNPs formation
0.30 mM 404 403 407 403

The system containing the 0.08 mM of AgNO3 was chosen to eval-

respect to value of this ratio in all range of AgNO3 concentration used.
As it can be seen in Fig. 4b and d, the synthesis of AgNPs resulted in
a mean hydrodynamic diameter lower and the particles size hetero-
geneity higher than those of pure nanocellulose. However, the obtained
results did not show directly the size of AgNPs due to the presence of
nanocellulose particles significantly larger than them. To more pre-
cisely describe the investigated systems the particles size distributions
and the peaks’ location were analyzed. The ‘differential’ particles size
distributions, reflecting the size of particles resulting in an increase of
the peaks height in respect to those of pure cellulose, were obtained.
The location of peaks’ maxima and the peaks’ width are shown in Fig.
uate whether the nanocellulose plays just a role of stabilizer or also a
role of reducing agent of silver ions. Investigations were performed for
the following dispersions: CNF in H2O, CNF/AgNO3 (0.08 mM), CNF/
NaBH4 (2 mM) and CNF/NaBH4 (2 mM)/AgNO3 (0.08 mM).
Additionally, CNF dispersions containing NaCl (0.08 mM) instead of
AgNO3 were prepared. Samples were obtained using the mechanical
agitation (mixed) or not (unmixed) in dark for 48 h at 20 °C. The UV–vis
absorbance, pH, electrolytic conductivity, zeta potential and the parti-
cles size were investigated (please see Supplementary Material for de-
tailed description of results).
Absorbance spectra of CNF dispersions with NaBH4, NaCl and
AgNO3 which were mixed or unmixed are presented in Fig. S8a,

Fig. 2. TEM micrographs of obtained AgNPs stabilised by CNF from carrot pomace. Synthesis condtions: temperature of 5 °C and without mixing.

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J. Cieśla, et al. Carbohydrate Polymers 245 (2020) 116513

Fig. 3. TEM micrographs of obtained AgNPs stabilised by CNF from carrot pomace. Synthesis condtions: temperature of 5 °C and with mixing.

Fig. 4. Mean hydrodynamic diameter and polydispersity index determined for the suspension of nanocellulose from carrot pomace (CNF, 0.1 % wt.) with various
initial concentrations of AgNO3 after 48 h from instillation. The AgNPs synthesis was carried out in 5 °C (upward panel) or 20 °C (downward panel).

6
J. Cieśla, et al.
Fig. 5. Zeta potential and electrolytic conductivity determined for the suspension of nanocellulose from carrot pomace (CNF, 0.1 % wt.) with various initial
concentrations of AgNO3 after 48 h from instillation. The AgNPs synthesis was carried out in 5 °C (upward panel) or 20 °C (downward panel).
showing only maxima characteristic for ions. The spectra show that
only in the case of systems where NaBH4 was present the absorbance
maxima occur at 410 nm (unmixed) and 340 and 510 nm (mixed) (Fig.
S8b). This shows that the NaBH4 served as reducing agent, while CNF
had no such properties. Moreover, only factor having influence on
position of absorption maximum was mechanical agitation (mixing).
The samples preparation (unmixed and mixed) had a slight effect on pH
(Fig. S9). For unmixed samples, where the diffusion process and the
cations mobility affected the H+ exchange on CNF surface, pH obtained
in NaCl was lower than in AgNO3 solution. The mixing eliminated these
differences. An effect of mixing/unmixing on electrolytic conductivity,
zeta potential (Fig. S10) and polydispersity index (Fig. S11) was in-
significant. For the mechanically agitated systems the CNF/NaBH4 and
CNF/NaBH4/NaCl were less polydisperse than CNF. In the case of
samples containing NaBH4 and AgNO3 or NaCl with mixing applied, the
particles diameter in CNF/NaBH4/AgNO3 was significantly lower than
in CNF/NaBH4 and CNF/NaBH4/NaCl systems (Fig. S11). High number
of small AgNPs synthesized led to decrease the mean hydrodynamic
diameter (Fig. 4). This effect was not visible in the unmixed CNF/
NaBH4/AgNO3 (the mean value of diameter was comparable with the
size of CNF) due to the bigger size of AgNPs and their low concentration
and in the systems without NaBH4, where the presence of AgNO3 led to
increase of particles size. It can be an indirect evidence, that only
NaBH4 was a reducing agent in systems studied.
4. Discussion
Previously, several research showed influence of changing propor-
tions of reducing and stabilizing agents’ concentrations on shape and
size of AgNPs (Rivero et al., 2013; Huang & Nancy Xu, 2010). Here, the
higher concentration of stabilizing agent (as CNF from carrot pomace)
7
Carbohydrate Polymers 245 (2020) 116513
and reducing agent (NaBH4) than the concentration of AgNO3 was, the
formed AgNPs were in diverse shapes. Similar results were presented by
Huang and Nancy Xu (2010) who also used NaBH4 as reductant al-
though with PVP as stabilizing agent. Also, Rivero et al. (2013) showed
that the higher concentration of stabilizing agent (PAA) in relation to
AgNO3 concentration was, the more diverse shapes of AgNPs were
formed. However, the same effect on AgNPs formation had decreasing
concentration of reducing agent (DMAB) (Rivero et al., 2013). Kuo et al.
(2015) stated that temperature is key factor influencing the morphology
of AgNPs obtained by photo-assisted synthesis. For temperatures below
40 °C the nanodecahedral large nanoparticles (low ratio of specific
surface to volume) were formed while at temperatures 50−60 °C
mostly nanoplates and at 80 °C - nanorods were visible. Piñero, Camero,
and Blanco (2017) noticed that only temperature of synthesis exceeding
30−40 °C influences the intensity of absorbance bands of AgNPs col-
loids and the non-uniform, non-spherical AgNPs are formed then.
Spherical and quite homogeneous AgNPs were obtained by Das,
Yeasmin, Khatua, Acharya, and Bandyopadhyay (2015) during synth-
esis in presence of guar gum and PVA (stabilizing agents) with the
stirring applied at temperature range of 25–100 °C. However, to long
time of heating caused the large particles formation (Jiang et al., 2011).
Results presented by various authors showed that the size and zeta
potential of AgNPs synthesized in presence of polysaccharides are dif-
ferentiated. de Moura, Mattoso, and Zucolotto (2012) observed that the
higher speed of mixing was applied (500 rpm and 1000 rpm), the
smaller (100 nm and 41 nm) and more stable (zeta potential of −17
and −38 mV) polyvinyl alcohol- coated AgNPs were obtained in na-
nocellulose dispersion (de Moura et al., 2012). The size of carbox-
ymethyl tamarind polysaccharide -capped AgNPs, synthesized by
Sanyasi et al. (2013), was several hundred of nm at zeta potential value
of −36.7 mV. Chook et al. (2015) by in situ synthesis obtained the
J. Cieśla, et al.
AgNPs with average size from about 22 nm to more than 100 nm
(clusters), whereas Bhui and Misra (2012) synthesized AgNPs (0 °C) in
methyl cellulose and determined spherical (1 mM AgNO3) and worm-
like (10 mM AgNO3) particles with the size of 6–22 nm. Patel et al.
(2017) obtained 159-nm AgNPs in sodium carboxylmethylcellulose.
Other authors determined the particles size of 48 nm and zeta potential
−19 mV for AgNPs synthesized using orange peel extract (de Barros
et al., 2018). For the AgNPs obtained using the hesperidin and nano-
cellulose from orange the particles size was about 25 nm and zeta po-
tential −28 mV. In that research, both the steric and electrostatic sta-
bilization of AgNPs by nanocellulose was stressed.
In this study the NaBH4 reduced Ag+ and led to the formation of
metallic silver (Ag°), which was followed by agglomeration into col-
loidal silver particles. Nanocellulose (CNF) played the role of stabilizer
in the process of AgNPs synthesis, what was confirmed by the results of
performed experiments. Usually stabilizers in form of polymers can
absorb or bind nanoparticles to their surfaces, avoiding their agglom-
eration (Iravani, Korbekandi, Mirmohammadi, & Zolfaghari, 2014). In
case of the carrot CNF studied here, the interactions between AgNPs
and carboxylic groups on the surface of cellulose nanofibers (CNF)
could occur. Additionally, at the high pH conditions the H+ is released
from nanocellulose hydroxyl groups at C(3) position leading to forma-
tion of alkoxylate radical R-O−. This radical interacts with silver ions
chemically. One silver ion can interact with hydroxyl groups between
two adjacent nanofibrils forming chelate compound, which also is the
reason of lack of AgNPs migration (Kim, Kwon, & Ostler, 2009).
Moreover, nanocellulose form spatial structure which probably lead to
separate the formed AgNPs (Fig. 6). In the unmixed systems the coa-
lescence of Ag particles is limited to the space provided by CNF. Process
leads to saturation and spherical nanoparticles are formed. While in
systems where mechanical agitation is introduced the interaction be-
tween forming nanoparticles is forced by mixing and, therefore, more
probable. In this case, nanoparticles with other than spherical shape can
be formed. According the LaMer theory, when the number of particles
in solution is fixed, the nanoparticles’ growth takes place by addition of
monomers to the surface of already formed nanoparticles (Van Hyning
& Zukoski, 1998). Also stirring was found to have effect on nucleation
and aggregation of AgNPs. Li and Kaner (2006) showed that stirring
triggers heterogeneous nucleation and induces aggregation. This was
also proved by Raza et al. (2016) who in the case of continuous stirring
during wet chemical synthesis obtained silver nanotriangles (Raza
et al., 2016).
Fig. 6. Probable mechanism of the studied formation of AgNPs in the carrot CNF dispersion.
8
Carbohydrate Polymers 245 (2020) 116513
5. Conclusions
It was shown in this studies using different analytical methods (UV
Vis, TEM, DLS and LDE), that AgNPs with size lower than 100 nm can
be effectively synthesized in reaction of AgNO3 and NaBH4 in the
presence of CNF obtained from the carrot pomace. Independently from
the conditions of AgNPs synthesis, the carrot nanocellulose was very
good stabilizing agent being the source of high absolute value of zeta
potential (40−60 mV) of AgNPs/CNF systems.
The dynamic conditions (mixing) of reaction and the AgNO3 con-
centration lower than 0.15 mM were crucial for the obtaining the dif-
ferent-shape AgNPs at a stable concentration of the reducing (2 mM of
NaBH4) and stabilizing agents (0.1 % CNF). For these systems synthe-
sized both at 5 °C and 20 °C, an increase of initial concentration of
AgNO3 resulted in the shifting the absorbance maxima towards longer
wavelengths. TEM micrographs revealed that the triangular shape was
the most common, but hexagons, rods or spherical conglomerates were
also visible. Differently sized and shaped AgNPs, present next to CNF,
affected the light scattering, that caused the high variability of DLS
results (hydrodynamic diameter and polydispersity index) with in-
creasing initial concentration of AgNO3. Beginning from the AgNO3
concentration of 0.15 mM the absorbance maxima were at 403−406
nm, the spherical particles were visible using TEM and the agglomerate
formation was determined by DLS.
For the AgNPs/CNF systems obtained in static conditions (no-
mixing) in all range of AgNO3 concentration studied the maximum of
absorbance was shifted from 403 nm to 411 nm and the spherical na-
noparticles were formed. An initial concentration of AgNO3 did not
have an influence on the mean hydrodynamic diameter of particles.
Changing the temperature of AgNPs synthesis from 5 to 20 °C re-
sulted in red-shift of absorption maximum in relation to the same
concentrations of AgNO3, decrease of particles size and increase of
polydispersity (determined by DLS).
Contribution of authors
MS-C conceived and designed the experiment, wrote the manu-
script, and obtained and analyzed the UV–vis spectra and TEM images.
MS-C and MC designed and performed the experiments involving cel-
lulose isolation and nanocellulose preparation and silver nanoparticles
synthesis. MC participated in the results analysis and discussions. AZ
revised and discussed manuscript. JC designed, performed and dis-
cussed the results of nanoparticles size, polydispersity and electro-
kinetic measurements.
J. Cieśla, et al.
CRediT authorship contribution statement
Jolanta Cieśla: Conceptualization, Methodology, Investigation,
Visualization, Writing - original draft, Writing - review & editing.
Monika Chylińska: Methodology, Investigation. Artur Zdunek:
Writing - review & editing. Monika Szymańska-Chargot:
Conceptualization, Methodology, Investigation, Visualization, Writing -
original draft, Writing - review & editing, Project administration,
Funding acquisition, Supervision.
Declaration of Competing Interest
The authors declare no competing interests.
Acknowledgments
This study was partially supported by research grant: LIDER/300/L-
6/14/NCBR/2015 from the Polish National Center for Research and
Development. TEM studies were performed using JEM 1400 microscope
(Jeol Co. Japan) installed in the Laboratory of Electron Microscopy, the
Nencki Institute of Experimental Biology, Polish Academy of Sciences.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116513.
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