Materials Today Communications 25 (2020) 101671

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Materials Today Communications

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

Dual electrospinning of a nanocomposites biofilm: Potential use as an

antimicrobial barrier
Judith Vergara-Figueroa a, c, *, Serguei Alejandro-Martin b, c, Fabiola Cerda-Leal d,
William Gacitúa a, b, c
a
Centro de Biomateriales y Nanotecnología, Universidad del Bío-Bío, 4030000 Concepción, Chile
b
Departamento de Ingeniería en Maderas, Facultad de Ingeniería, Universidad del Bío-Bío, Concepción 4030000, Chile
c
Nanomaterials and Catalysts for Sustainable Processes Group (NanoCatpPS), Universidad del Bío-Bío, 4030000 Concepción, Chile
d
Departamento de Ingeniería en Alimentos, Universidad del Bío-Bío, Chillán 3780000, Chile

A R T I C L E I N F O A B S T R A C T

Keywords: The accumulation of plastic waste and infectious diseases has led the scientific community to search for new
Polylactic acid materials to make nanocomposites biofilms. With the help of nanotechnology, the materials that make up the
Nanocellulose new nanocomposites biofilm can be manipulated, achieving an improvement in its mechanical properties and
Copper ions
granting antimicrobial powers. Biofilms based on electrospun fibers as a barrier material can protect against
Nanozeolite
Electrospinning
infectious microorganisms. For these applications, the electrospinning method allows the encapsulation of active
Biofilm agents in the polymeric fibers. This article presents the development of a nanocomposites biofilm with potential
Antibacterial activity use as an antimicrobial barrier, which incorporates polylactic acid microfibers with active agents. Among them,
copper ions supported on Chilean natural zeolite nanoparticles and reinforced with acetylated cellulose nano­
fibers. The study begins with obtaining cellulose nanofibers through mechanical methods. Nanofibers were
acetylated to ensure adequate dispersion in the polymer matrix and to improve the mechanical properties of
biofilms. Polymers and nanoparticles were incorporated into biofilms through a dual configuration (simulta­
neous injection) electrospinning stage.
Results indicated that acetylated cellulose nanofibers with an average diameter of 83 nm were obtained. The
FT-IR characterization confirmed the appearance of bands of adhered acetyl groups in primary hydroxyl groups
of the nanocellulose. Morphological analysis of biofilms indicated the presence of continuous, randomly oriented
microfibrillar structures and average fiber diameter from 1.7 μm to 2.4 μm. According to copper distribution
maps, it was confirmed that the electrospinning technique was able to encapsulate nanoparticles of Chilean
natural zeolite with copper ions. For all biofilms to which acetylated nanofibers were added, there was an in­
crease in mechanical properties. The thermal degradation profile revealed that the addition of nanoparticles
favored the thermal degradation resistance of films. The water vapor permeability test showed that the increased
contraction of nanoparticles in the biofilms does not cause an increase in this parameter. The microbiological
tests confirmed the biofilms antibacterial activity towards the Gram (-) Salmonella Typhimurium bacteria
(responsible for foodborne diseases in poultry products) and Gram (+) Staphylococcus aureus (whose presence
accounts for of sanitary and hygienic quality in food processing).

1. Introduction droplets produced by the cough, exhalation of infected people, by

The increase of pathogenic microorganisms worldwide is a constant
concern for human health. The presence of pathogenic microorganisms
(viruses, fungi, parasites, and bacteria) poses a severe risk to humanity
due to the infectious and contagious diseases generated by them. Path­
ogenic microorganisms are usually spread through small contaminated
ingestion of contaminated food, and by contact with contaminated el­
ements or surfaces [1–3].
The use of nanotechnology to develop antimicrobial biofilms, as a
cutting-edge research area, allowed the combination of materials to
obtain new improved biofilms. In addition to the increase in its barrier,
mechanical, heat resistance, and biodegradability properties, biofilms

* Corresponding author at: Centro de Biomateriales y Nanotecnología, Universidad del Bío-Bío, 4030000 Concepción, Chile.
E-mail address: judith.vergara1601@alumnos.ubiobio.cl (J. Vergara-Figueroa).

https://doi.org/10.1016/j.mtcomm.2020.101671
Received 1 June 2020; Received in revised form 10 September 2020; Accepted 11 September 2020
Available online 19 September 2020
2352-4928/© 2020 Elsevier Ltd. All rights reserved.
J. Vergara-Figueroa et al.
can be endowed with antimicrobial properties. Incorporating antimi­
crobial compounds into the biofilm matrix could allow a functional ef­
fect on a given surface that is in contact with the biofilm [4]. Polylactic
acid (PLA) is a biodegradable, biocompatible, and non-toxic natural
polymer, with the ability to produce fibers by electrospinning. These
fibers form films with possible applications as a barrier material, with
attributes according to the required needs. Much research highlights its
versatility in forming hydrophobic biocomposites, mechanical strength,
and the ability to encapsulate controlled release nanocomposites
[5–14].
Previous studies showed a suitable method to incorporate antimi­
crobials in a film, mainly based on biodegradable polymers and natural
components [4,6,8,15,16]. The reported method is a potential alterna­
tive to fight pathogenic microorganisms. The reduction and inhibition of
microorganisms’ growth over antimicrobial film have been demon­
strated before [17–19]. Copper oxide nanoparticles in a polymeric
structure have a more substantial antimicrobial effect than microparti­
cles or metallic surfaces, by facilitating the adsorption of microorgan­
isms on the polymer surface. Copper ions and associated hydroxyl
radicals cause denaturation of DNA and RNA, damaging their helical
structures and biomolecules, causing lysis in microorganisms [20,21].
Although the use of biopolymers is an alternative for the manufac­
ture of antimicrobial films, its application is limited by its mechanical
properties and high permeability to water and oxygen. These charac­
teristics are mainly due to the number of hydroxyl groups present in its
structure [22]. To improve the physical-mechanical properties of
biopolymer-based composite materials, the route of reinforcing them
with other elements, including fibers and nanoparticles, has been stud­
ied. This procedure will allow satisfying some of the technical disad­
vantages of biopolymeric materials, incorporating properties that are
related to the size and characteristics of the particles that reinforce it
[23]. The use of nanofibers allowed the elaboration of an
environment-friendly biocomposite (matrix and reinforcement),
enhancing the physical-mechanical properties of the resulting hybrid
material. Plant fibers are currently being used for this purpose. Chemical
or mechanical processes can be used to isolate cellulose nanofibers or
nanoparticles. Through these processes, two types of nanocellulose can
be obtained: cellulose nanofibers (CNF) or nanocrystalline cellulose
(CNC). CNF is achieved through mechanical procedures based mainly on
high-pressure homogenization and grinding. The CNC is achieved
through chemical treatments, generally based on cellulose hydrolysis
[24].
Electrospinning technology has attracted great interest in recent
decades, thanks to its advantage to process, quickly and effectively, a
wide range of polymeric materials in the form of nano and microfibres.
Compared to other nanofiber manufacturing processes, such as self-
assembly and phase separation, electrospinning has the advantages of
being versatile and flexible in terms of material selection and control
over fiber morphology and diameter. These characteristics, together
with a too high surface to volume ratio, make electrospun fibers the
nanomaterials of choice in applications such as sensors, energy,
biomedicine, filtration, and antimicrobial film [17–19,25,26]. During
the process, nano and microfibres are generated by applying an electric
field between the electrodes of the equipment, causing rapid movement
of the solution, creating a jet of charged and stretched polymer, evap­
orating the solvent, and solidifying the jet to form the fiber. The
morphology and diameter of the fibers manufactured by electrospinning
depend on several parameters, including voltage, the distance between
the needle and the collector, concentration of the solution, the surface
tension of the diluted polymer, viscosity, environmental conditions,
among others. Electrospinning of natural resources composite fibers can
be used in different fields, especially in composite films and films. Its
application on antimicrobial films was reported elsewhere [26–30].
Nanocellulose is an abundant, biodegradable, and biocompatible
polymer. For these attributes, it came to the consideration of researchers
in nanotechnology to be used as a mechanical reinforcement element in
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Materials Today Communications 25 (2020) 101671
films developed by electrospinning [22,31–33]. For its part, the elec­
trospinning of zeolite-loaded fibers allows fibers to be endowed with
catalytic, antibacterial, filtration, and membranes for medical use
[34–39]. Natural nanozeolite particles exchanged with copper salt ions
(Cu2+) can be directly incorporated into a film. The purpose is to allow
the retention of ions in the zeolite framework, thus functioning as a
carrier. The ions must be kept within the zeolite structure to perform an
antimicrobial activity. Natural zeolite by itself is not an effective anti­
bacterial but works as the ion support that can be used as an antimi­
crobial agent. The ion exchange is carried out by replacing the sodium
that is found as a compensation cation in the zeolite structure by other
ions, such as copper [40]. Nanoparticles made of copper oxides are
inexpensive, non-toxic to humans, abundant, and have stable physical
and chemical properties. These properties give it advantages over other
metal oxides and make them used worldwide.
Furthermore, they can be easily mixed with polymers and other
matrices, which can be prepared with various surface morphologies
[41]. The antimicrobial activity exhibited by the nanostructures of metal
oxides could have an application to potential use as an antimicrobial
barrier. Its nanometric dimensions and its large specific surface allows it
to interact with bacteria [20,21].
This research aims to show the development of a nanocomposites
biofilm with potential use as an antimicrobial barrier, based on PLA
microfibers and manufactured using the electrospinning technique in
dual configuration. The biofilm was loaded with copper ions supported
on zeolite nanoparticles (nZH-Cu) and reinforced with cellulose nano­
fibers (CNF). The biofilms developed underwent morphological anal­
ysis, evaluation of their physical-mechanical properties, thermal
degradation profile, and permeability to water vapor. Finally, the anti­
bacterial activity of biofilms that showed better characteristics was
evaluated against Gram (-) Salmonella Typhimurium and Gram (+)
Staphylococcus aureus bacteria.
2. Materials and methods
2.1. Materials
PLA was used in pellets from the company Natureworks polymer
2002 D, with a molecular weight of 200,000 g / mol. The reagents used
were: chloroform, acetone, acetic acid, acetic anhydride, tartaric acid,
hydrochloric acid, and sodium hydroxide, all of them purchased from
Merck (p.a). Deionized water was used to prepare solutions. The copper
salt ion-exchange zeolite nanoparticles (nZH-Cu) were obtained
following a reported procedure of a previous study [42]. Zeolite nano­
particles showed antimicrobial properties under laboratory conditions
[42]. The cellulose nanofibers were obtained from bleached reject kraft
pulp purchased from the CMPC Laja Cellulose Plant. To prepare the
biofilms, ascending vertical Dual Electrospinning equipment was used
(Inovenso NE300). For the microbiological tests, the Gram (-) Salmonella
Typhimurium strain (S. Typhi, ATCC 14028) and the Gram (+) Staph­
ylococcus aureus strain (S. aureus, ATCC 25923), American Type Culture
Collection (ATCC), from the collection unit of the Departamento de
Ingeniería en Alimentos of the Universidad del Bío-Bío, Chillán.
2.2. Methods
2.2.1. Obtaining cellulose nanofibers from rejected bleached kraft pulp
The protocols described by Junka et al. (2012) and Kalia et al. (2014)
with some modifications. The pulp was initially subjected to a dry
milling process. It was then immersed for 12 h in a 0.5 % v/v NaOH
solution, from a 5% w/v NaOH stock solution. Then, in the refining
process, the sample was taken to the SUPER MASSCOLLOIDER equip­
ment (MASUKO, Kawaguchi-city, JAPAN) with a disc opening of 0.5 μm,
for two continuous hours, at a pulp consistency of 10 % w/v. ext, the
sample was subjected to a high shear mechanical process in the Micro­
fluidics LM10 equipment (MicrofluidicsTM, Massachusetts, MA, USA)
J. Vergara-Figueroa et al.
for nine continuous cycles, 1000 bar, at a pulp consistency of 1% w/v.
The sample was ultra-centrifuged YINGTAI INSTRUMENT (12,000
min− 1, 30 min, 18 ◦ C) [43,44]. This sample was named as CNF.
2.2.2. Acetylation of cellulose nanofibers
This treatment improves the dispersion and interfacial adhesion of
the CNF with apolar polymers. The acetylation method developed by
Foresti et al. (2014) and Ávila et al. (2014) was used. 0.5 g of CNF (dry
base) was placed with magnetic stirring with 30 mL of acetic acid
(15 min, room temperature). Subsequently, the sample was ultra-
centrifuged (8000 min-1, 10 min, 15 ◦ C). Next, the sample was
magnetically stirred with 50 mL of acetic anhydride and 0.47 g of tar­
taric acid (3 h, 120 ◦ C). The sample was ultra-centrifuged (8000 min-1,
10 min, 15 ◦ C). Then, the sample was washed with 30 mL of acetone
(magnetic stirring, 15 min). Finally, the sample was ultra-centrifuged at
8000 min-1 for 10 min at 15 ◦ C [32,45]. This sample was named as
acetylated CNF.
2.2.3. Manufacture of biofilms from PLA
Two polymer blends were used to make the biofilms. One of the
mixtures carried nZH-Cu as an antimicrobial agent, and the other
mixture carried acetylated CNFs as reinforcement. Both mixtures were
injected simultaneously using the electrospinning method in dual
configuration, and a rotary drum type collector was used to receive the
sample [11,17,35]. The concentration of these elements was added ac­
cording to the biopolymer amount (dry weight) in mixtures. For the
elaboration of the biofilms, a General Factorial Design was used, with
three replicas. Design-Expert 10.0.3 software was used [46]. The factors,
levels, and response variables are observed in Table 1.
For each polymer mixture, 10 ml of solution was prepared, 10 % of
this solution corresponds to PLA and the remaining 90 % to solvent
(chloroform: acetone 2:1 v/v) [47,48]. Table 2 describes the mixtures
used and their preparation.
The best experimental conditions of electrospinning equipment re­
ported by the Gaitán and Gacitúa (2018), was conducted in this study
due to the similarity of the equipment and type of PLA used. Distance
from the tip of the injector to the collector of 20 cm, manufacturing
voltage of 24 kV, the flow of the solution was estimated at 0.1 mL/hr
[48].
2.3. Characterizations
2.3.1. Atomic force microscopy, AFM
The CNF gel samples were observed using atomic force microscopy
(AFM), using the Nanosurf Naio AFM apparatus. The images obtained
were processed and measured using Image J software. From this data, a
histogram of particle size distribution was constructed.
2.3.2. Fourier transform infrared spectrometry, FTIR
Fourier transform infrared spectrometry (FTIR) was used to check
the binding of acetyl groups on the surface of the CNF. A Perkin Elmer
Table 1
General Factorial Experimental Design: elaboration of a biofilm employing the
electrospinning method in dual configuration.
Factors Levels Variable response
1 placed on each square. The Petri dishes were then incubated at 37 ◦ C for
nZH-Cu (%) 2 Morphology.
•
3 • Average fiber diameter.
1 • Elastic module.
Acetylated CNF (%) 1.5 • Permeability to the transfer of water vapor.
2 incubated for 48 h at 37 ◦ C. At the end of the time, the bacterial colonies
nZH-Cu: concentration of zeolite nanoparticles exchanged with copper ions, in
polymer mixture.
Acetylated CNF: concentration of acetylated fibrillated nanocellulose, in poly­
mer mixture.
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Materials Today Communications 25 (2020) 101671
Table 2
Mixtures used for the manufacture of biofilms employing electrospinning.
Mix Type Reagents Stirring
Control PLA in chloroform, 12 h + acetone Magnetic stir at
1000 min− 1, 1 h
Reinforcement PLA in chloroform, 12 h + acetylated Magnetic stir at
CNF in acetone 1000 min− 1, 1 h
Active Agents PLA in chloroform, 12 h + nZH-Cu in Magnetic stir at
acetone 1000 min− 1, 1 h
Spectrum equipment was used in the Attenuated Total Reflection tech­
nique (ATR), registered spectra were collected for CNF and acetylated
CNF samples. Data were processed using OriginPro 8 software.
2.3.3. Scanning electron microscopy with an elemental detector, SEM-EDX
The morphological analysis consisted of characterizing the fibers
that make up the biofilms. Homogeneity, continuity, sizes, and porosity
were analyzed. The characterization was carried out using scanning
electron microscopy and elemental surface analysis (SEM-EDX), in a
BRUKER QUANTAX EDS XFlash® 6 apparatus (BRUKER, Billerica, MA,
USA). The images obtained were processed using the Image J software.
The elemental analysis confirmed the presence of copper in the devel­
oped biofilms, as the spatial map distribution showed.
2.3.4. Mechanical properties
Tensile tests were carried out on the biofilms developed following
the protocol of the norm "Standard test method for tensile properties of
thin plastic sheeting, D882-12" [49]. Samples of 2 cm wide by 10 cm
long were used in the tests. A universal machine, Zwick Roell, with a
20 kN cell load, was used. The test speed was calibrated at 500 mm/min.
Values of Modulus of elasticity (E, MPa), Tensile Strength, (σ, MPa) y
Elongation at failure (ε, %) were obtained.
2.3.5. Thermogravimetric analysis, TGA
For the analysis of thermal degradation to the developed biofilms,
the TGA Q50 equipment, TA Instruments, was used. The heating rate
was 10 ◦ C/min, from 25 ◦ C to 600 ◦ C. Nitrogen gas was used as a purge
at 100 mL/min. The maximum degradation temperature was obtained
using the first derivative of TGA as a function of time (DTG, %/min).
2.3.6. Water vapor transmission, WVT
The procedure was adapted to the provisions of the E96/E96M-14
standard and following the protocols used by the authors Graciano
et al. (2006) and Valderrama et al. (2016) with slight modifications. The
test was carried out in a glass chamber (25 ◦ C, 75 % relative humidity).
The biofilms were placed on hermetically sealed permeability cups,
containing granular anhydrous calcium chloride inside. Biofilm weights
were recorded every 1 h for eight continuous hours [50–52].
2.3.7. Determination of antibacterial activity
Biofilms with 2% acetylated CNF were tested since they presented
better characteristics in mechanical, thermal, and WVT resistance. In
these biofilms, the different concentrations of nZH-Cu (1, 2 and 3%)
were evaluated. The test was performed according to the methodology
dictated by ASTM E2180–07. Square samples (3 cm2) of biofilms were
used and placed in Petri dishes. A melted agar solution was inoculated
with standardized cultures of the S. Typhi and S. aureus strains were
24 h. Then, the inoculated agar solution was recovered from each Petri
dish, and they were eluted in neutralizing broth. Serial dilutions were
made and poured into Petri dishes with agar. They were subsequently
of each dilution series were counted and recorded, using the official
method of the Association of Official Agricultural Chemists (AOAC)
[53]. The Arithmetic Mean of the number of organisms recovered after the
J. Vergara-Figueroa et al.
incubation period and the calculation of the Reduction Percentage of
bacteria were applied [54]. The trials were performed in triplicate with a
randomized experimental design, at a confidence level of 95 %. ANOVA
statistical analysis and Multiple Comparison Test were performed in the
Statgraphics Centurion XVI program.
3. Results and discussions
3.1. Morphological characterization of cellulose nanofibers after the size
reduction process
The morphology of the fibers was characterized by observation in
AFM, after the size reduction processes. The AFM micrograph Fig. 1. (a)
show the CNF sample as a fibrillated structure of variable diameter. The
diameter had a distribution from 55 to 173 nm, with an average of
91 nm (Fig. 1. (b)). The microfluidization process has been widely used
for the production of CNF [44]. In the microfluidizer, the suspension is
forced to pass through a narrow channel of defined fixed geometry at
high pressure (1500–2100 bar). There the suspension accelerates at high
speed and creates a cutting speed in the sample that exceeds 106 s− 1. As
a result, intense particle collisions occur, and cavitation causes disinte­
gration of cellulose fibers in elemental building blocks [44].
From the AFM micrograph Fig. 1. (c), it can be seen that the acety­
lation process did not affect the morphology of the fibers, nor did it
degrade them. Acetylated CNFs had diameters ranging from 47 to
138 nm, with an average of 83 nm (Fig. 1. (d)). The acetylation process is
a chemical treatment that includes a thermal step [32], possibly causing
the diameter of the CNF to decrease slightly.
Fig. 1. AFM images. (a) CNF; (b) CNF histogram; (c) acetylated CNF; (d) acetylated CNF histogram.
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Materials Today Communications 25 (2020) 101671
3.2. Characterization of cellulose nanofibers using FTIR
Fig. 2 presents the spectra obtained from the CNF and acetylated CNF
samples. The characteristic peaks for CNF are 3309 cm− 1 (OH stretch),
2892 cm− 1 (CH stretch), 1338 cm− 1 (flexural vibration of aromatic rings
C–H and C–O in polysaccharides), and 1014 cm− 1 (the stretch of C–O,
and O–H) and 556 cm− 1. When compared, these peaks show similar­
ities in the absorbances of the works reported by Jonoobi et al. (2010)
and Tingaut et al. (2010) [55,56].
It can be seen in the spectra that the acetylated CNF incorporates
signals at 1732 cm− 1 (stretch C– – O), 1370 cm− 1 (deformation C–H of
CH3), and 1237 cm− 1 (stretch C–O). The appearance of these bands
indicates the presence of acetyl groups, a consequence of the acetylation
of primary hydroxyl groups. These results are similar to those reported
by Tingaut et al. (2010) and Foresti et al. (2014) [32,56]. It is relevant to
indicate that the absence of absorption in the region of
1850− 1760 cm− 1 indicates that the acetylated samples were recovered
free of unreacted acetic anhydride [57].
3.3. Scanning electron microscopy with an elemental detector, SEM-EDX
3.3.1. Morphological analysis and detection of copper in the developed
biofilms
Fig. 3. (a) - (j) shows SEM micrographs, histograms of the average
fiber diameters, and EDX map for the distribution of copper in the
developed biofilms. When analyzing SEM (a1) - (j1) micrographs,
microfibrillar structures can generally be observed. They show porosity
and variable average diameters ranging from 1.7 μm to 2.4 μm, with a R2
value of 0.8 (ANOVA, α 5%). This value finds explanation in that the
microfibers of the films are not all the same; they are deposited
J. Vergara-Figueroa et al.
Fig. 2. FTIR spectra of CNF and acetylated CNF samples.
randomly on the collector. Furthermore, the contraction of nano­
particles and the dual process may have influenced the value of R2 [58].
A transition from heterogeneous to homogeneous fibers is observed,
related to the insertion of nanoparticles. The average diameter of the
fibers of the control biofilm (a1) is wider than those with nanoparticles.
This phenomenon can be explained by the fact that the fibers become
more uniform and thinner with a higher particle content due to the
improved electrical conductivity of the electrospinning solutions [34,37,
38,59,60]. It was observed in some cases that the insertion of nano­
particles in the biofilms showed helical type imperfections on the fibers
(2% CNF / 1% nZH-Cu). This type of imperfection can be caused by the
deformation of the jet due to the impact with the collecting plate or
drum. The concentration of the polymer solution promotes this
behavior. The angle of incidence of the jet influences the obtaining of
this type of fibers [61]. The presentation of pores on the surface of the
fibers for all the treatments could possibly be caused by the relative
humidity of the environment and the vapor pressure of the solvents used
(chloroform: acetone, 2: 1) in the elaboration of the polymeric mixtures.
Their appearance can be taken as an advantage, since they can promote
transfer and migration of molecules between their interstices [37].
From Fig. 3 (d) onwards, it is observed that the incorporation of
nanoparticles induces a decrease in the diameter of the fibers. Further­
more, it is evident that the diameter distribution narrows, which is
consistent with that reported by other authors [37,38,59,60,62]. When
comparing the results of the morphological analysis with those pre­
sented by Dai et al. (2018), it can be indicated that they show similar
characteristics in the sense that in both works, it is demonstrated that the
addition of nanoparticles can adjust the diameter of the PLA fibers [62].
The decrease in diameter can be attributed to the fact that the addition
of the nanoparticles with copper ions reduces the surface tension.
nZH-Cu particles could be responsible for causing an improvement in
electrical conductivity. Barakat et al. (2009) indicate that the addition of
salts to the solution increases the conductivity and, therefore, the elec­
tric force for the jet stretching [63]. Under these conditions, thinner jets
are generated, causing a reduction in the fiber diameter distribution [34,
37,38,60,64,65]. The surface tension coefficient in the mixture depends
on the polymer and solvent [65]. In this case, increasing the concen­
tration of acetylated CNF in the mixture could also contribute to the
reduction of surface tension. The CNF sample is in a colloidal state, that
is, a gel loaded with nanofibers with superficial apolarity dispersed in
acetone. Acetone is a solvent typically used in polymer blends used for
electrospinning processes [66].
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Materials Today Communications 25 (2020) 101671
According to the copper distribution maps (a3) - (j3), it can be
confirmed that the electrospinning technique is capable of encapsulating
nanoparticles [34,35,37–39,67]. Nanozeolite charged with copper ions
can play a fundamental role by providing biofilms with antibacterial
characteristics [8,15,68]. It can be inferred that the electrospinning
process does not produce a copper loss.
3.3.2. Mechanical properties
Table 3 shows the results of the mechanical properties carried out on
the biofilms developed to demonstrate the mechanical reinforcement.
Results confirmed that for all treatments in which acetylated CNF was
added to the mixture, an increase in the elastic modulus (E) was re­
ported, in comparison with the pure PLA biofilm. The R2 value was
0.912 (ANOVA, α 5%). Those results can be explained by the fact that
the combination of PLA with natural polymers such as cellulose induces
an improvement in the mechanical properties of the new composite
material [5]. The incorporation of acetyl groups in the CNF causes a
change in the polarity of the CNF [32,45], which induces an improve­
ment in dispersion and interfacial adhesion with nonpolar polymers
such as PLA [5].
It is observed that keeping constant the percentage of acetylated CNF
and increasing the amount of nZH-Cu, the E value decreases slightly.
This can be explained by the fact that low nZH-Cu contents have a
uniform distribution in the polymer matrix. However, they tend to
agglomerate when added in large quantities in the mixtures [62]. The
pure PLA biofilm obtained an E of 274 MPa. The highest E was 808 MPa
corresponding to 2% acetylated CNF/ 1% nZH-Cu, followed by 755 MPa,
corresponding to 2% acetylated CNF/ 2% nZH-Cu. In other words, the
insertion of nanoparticles gave a maximum increase of 195 % for 2%
acetylated CNF/ 1% nZH-Cu, followed by 175 % for 2% acetylated CNF/
2% nZH-Cu, concerning pure PLA. The incorporation of clays or zeolites
gives rigidity to the environment in which they are dispersed. However,
the increase in them causes the mechanical properties of the composite
material to be diminished because they tend to agglomerate and occupy
the spaces that correspond to the continuous matrix [62,69].
The tensile strength (σ) tends to increase as the addition of nano­
particles in the mixtures increases. In other words, the biofilm shows
greater support for rupture when acetylated CNF and nZH-Cu particles
are found in its dispersed phase. Although the contribution of these
nanoparticles indeed provides an improvement in the phys­
icomechanical properties of the new composite material, not all its pa­
rameters were favored [62,69]. In fact, in our results (Table 3) it is
J. Vergara-Figueroa et al. Materials Today Communications 25 (2020) 101671

Fig. 3. SEM micrographs, average fiber histograms, and EDX map for copper distribution in biofilms developed by electrospinning. Small light dots represent the
presence of copper. (a): 0% Acetylated CNF/0% nZH-Cu; (b): 1% Acetylated CNF/1% nZH-Cu; (c): 1% Acetylated CNF/2% nZH-Cu; (d): 1% Acetylated CNF/3%
nZH-Cu; (e): 1.5 % Acetylated CNF/1% nZH-Cu; (f): 1.5 % Acetylated CNF/2% nZH-Cu; (g): 1.5 % Acetylated CNF/3% nZH-Cu; (h): 2% Acetylated CNF/1% nZH-Cu;
(i): 2% Acetylated CNF/2% nZH-Cu; (j): 2% Acetylated CNF/3% nZH-Cu.

6
J. Vergara-Figueroa et al. Materials Today Communications 25 (2020) 101671

Fig. 3. (continued).

observed that the elongation at fault (ε) decreases as the concentration
of nZH-Cu increases. It is evidenced that only the incorporation of
acetylated CNF in the mixture favors ε, while the increase in the con­
centration of nZH-Cu causes a decrease in ε. This result may be because
the nZH-Cu nanoparticles were encapsulated between the PLA fibers, no
entanglement between the molecules make up the matrix and nZH-Cu.
On the contrary, the hydrophobicity of CNF (produced by the
incorporation of acetyl groups in the nanofibers) caused adequate
interfacial adhesion with the PLA [32,45]; this is reflected in the in­
crease in E, σ and ε at a higher concentration of acetylated CNF in the
mixture. The mechanical parameters can be impaired with the addition
of excess nanoparticles since it removes the rigidity of the new

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J. Vergara-Figueroa et al. Materials Today Communications 25 (2020) 101671

Table 3
Mechanical properties of biofilms after the electrospinning process.
Nanoparticles (%) Mechanical properties Increase (%) of mechanical properties

Acetylated CNF nZH-Cu E (MPa) σ (MPa) ε (%) E σ ε

0 0 274.1 ± 29g 2.2 ± 0.3e 4.2 ± 1.2d – – –
1 1 665.4 ± 110d 3.1 ± 0.1b 5.7 ± 1.6e 142.8d 41.4c 36.8f
1 2 602.2 ± 144e 2.3 ± 0.3e 4.1 ± 1.0d 119.7e 5.0f ≤ 0.00h
1 3 579.1 ± 16f 2.7 ± 0.3d 4.7 ± 1.0d 111.3e 23.0e 13.1g
1.5 1 654.9 ± 33d 3.1 ± 0.4b 10.1 ± 1.4c 138.9d 37.4c 140.1c
1.5 2 704.2 ± 29c 3.3 ± 0.1a 8.3 ± 2.9d 156.9c 46.8b 96.9d
1.5 3 640.2 ± 102d 3.3 ± 0.3a 6.7 ± 1.4e 133.5d 50.5a 57.5e
2 1 807.9 ± 89a 3.0 ± 0.3c 14.8 ± 3.0a 194.8a 33.3d 252.7a
2 2 754.7 ± 73b 3.2 ± 0.2a 13.1 ± 2.6b 175.3b 45.5b 211.7b
2 3 660.7 ± 60d 3.1 ± 0.3b 10.3 ± 2.6c 139.5d 40.1c 146.1c

E (MPa): Modulus of elasticity; σ (MPa): Tensile Strength; ε (%): Elongation at failure.

Values are presented as means ± SD. N = 5. Values followed by different superscript letters in the same column indicate significant differences (p > 0.05).

composite nanomaterial [5,48,62,69]. The work done by Shankar et al.
(2014) showed that the incorporation of copper nanoparticles in agar
biofilms showed a slight decrease in E and σ [68]. In turn, Romero et al.
(2011) indicated that the incorporation of antibacterial agents such as
salts and oils could cause a reduction in the stress of biofilms [15].
Gómez et al. (2013) reported that the orientation of the fibers and
their diameter are essential parameters to consider when analyzing the
mechanical properties of a film composed of PLA fibers. The importance
of these parameters is that nanofibers and nanoparticles can improve the
physical-mechanical properties of a biocomposite material. Thus, as the
polymer chains are oriented along the central axis of nanofibers, the
elastic modulus increases in this direction. As known, the covalent bonds
between the carbon atoms in the polymer chain are stronger than the
intermolecular bonds between the polymer chains (Van der Waals,

Fig. 4. Thermogravimetric curves. TGA (a) and DTG (b) of the biofilms after the electrospinning process.

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J. Vergara-Figueroa et al. Materials Today Communications 25 (2020) 101671

hydrogen bonds, and others). Their results indicated that the fibers
oriented in the same direction had an E value of 881 MPa and σ of
25 MPa [70]. The fibers that make up the biofilms obtained in the pre­
sent study are randomly oriented (Fig. 3), which could induce a lower
capacity for uniaxial mechanical resistance. The best values obtained in
the present study are lower (Table 3) than those obtained by
Gómez-Pachón et al. (2013).
The orientation of the PLA fibers reported by Zhang et al. (2016) was
random and used poly trimethylene carbonate (PTMC) as their rein­
forcement. The best results presented by them were: E 310 MPa and σ
7.5 MPa [71]. When comparing the results of the work carried out by
Zhang et al. (2016) with those obtained here (Table 3), it can be indi­
cated that Zhang et al. (2016) obtained a lower E and a higher σ. Dai
et al. (2018) developed a PLA fiber membranes with a zeolitic imida­
zolate framework-8 (ZIF-8) insert, using the electrospinning technique.
Their results in terms of mechanical characterization showed a similar
trend to the present investigation, since the values of E, σ and ε
demonstrate that they reach a maximum when the content of ZIF-8 is 2%
by weight [62].
3.3.3. Thermogravimetric analysis, TGA
The effect of the content of acetylated CNF and nZH-Cu on the
thermal stability of all developed PLA biofilms was evaluated by TGA, as
shown in Fig. 4. Table 4 summarizes the results of the initial degradation
temperature (Td) and maximum degradation temperature (Tmax) ana­
lyzes for all the developed biofilms. From Fig. 4. (a), it is observed that in
general, the samples present decomposition in a simple process in a
single step. There was no weight loss below 200 ◦ C. When analyzing the
results shown in Table 4. it can be mentioned that the control sample
showed a Td of 212 ◦ C and a Tmax of 324 ◦ C. PLA films with nanoparticle
insertion withstood the test temperature rise. They presented a higher Td
and Tmax compared to the control sample. The sample with the lowest
concentration of nanoparticles began to degrade at 220 ◦ C, and its Tmax
was 335 ◦ C. The sample that presented the best mechanical character­
istics (2% acetylated CNF/ 2% nZH-Cu) began to degrade at 226 ◦ C, and
its Tmax was 358 ◦ C. The sample with the highest concentration of 2%
acetylated CNF/ 3% nZH-Cu nanoparticles began to degrade at 222 ◦ C,
and its Tmax was 330 ◦ C.
The results demonstrate that the addition of nanoparticles favors the
resistance to thermal degradation of films based on PLA. They present a
similar trend and coincide with the range of values reported by Yin et al.
(2018). They developed films based on PLA, manufactured using the
electrospinning technique, and as reinforcement, they used microcrys­
talline cellulose. Their results indicated that the Td for the pure PLA films
was 220 ◦ C, and for the PLA films with 5%, 10 %, and 15 % crystalline
cellulose, the Td was 278 ◦ C. As Tmax, the pure PLA film obtained 329 ◦ C,
Table 4
Results of the TGA and DTG analysis for all biofilms after the electrospinning
process.
Nanoparticles (%)
Initial Td Tmax Remaining mass (%) in
Acetylated nZH- (◦ C) (◦ C) Tmax
CNF Cu
0 0 212d 324g 77b
1 1 220c 335e 84a
1 2 219c 341d 73c
and between 350 ◦ C to 355 ◦ C for the PLA films with microcrystalline
cellulose [72]. It should be noted that the samples 1% acetylated CNF/
3% nZH-Cu and 1.5 % acetylated CNF/ 1% nZH-Cu present double peaks
of Tmax and remaining mass (Fig. 4. (b)), which indicates a multistage
decomposition with a stable intermediate product.
Regarding the mass loss at Tmax, it can be mentioned that there is an
important remnant of the sample (41%–84%). It is observed from
Table 4. that in most cases, there is practically 50 % of the sample even
without degrading (remaining mass). Possibly zeolite nanoparticles with
copper ions manage to protect the biopolymer matrix from the action of
temperature. Han (2014) indicates that the incorporation of clay in the
biopolymeric matrix can improve thermal stability. This phenomenon is
because the dispersed clay acts as an insulator for heat transfer and a
barrier for mass transfer to the volatile products generated during
thermal decomposition [73]. However, it must be taken into consider­
ation that excessive incorporation of nanoparticles in composite mate­
rials does not ensure their thermal stability, as a result of the
agglomeration of the nanoparticles, followed by a lower dispersion.
3.3.4. Water vapor transmission, WVT
The effect of the concentration of acetylated CNF and nZH-Cu on the
water vapor transmission (WVT) of the developed biofilms was evalu­
ated. The results are presented in Table 5. The analysis indicates that the
biofilms did not show statistically significant differences (ANOVA) in
WVT by an increased concentration of acetylated CNF or nZH-Cu in the
mixtures. The control sample shows lower WVT because one of the main
characteristics of PLA is its hydrophobicity [14,70,71,74]. The incor­
poration of particles as the dispersed phase causes the adhesion "matrix"
- "dispersed phase" to depend mainly on the physical, mechanical or
chemical compatibility between them [75]. According to the results of
this research, it is possible to demonstrate that the interface (the limit
zone where a discontinuity occurs) manifested strong adhesion between
nanofibers, nanozeolites, and the biopolymer matrix. Possibly the
acetylation of the CNFs helped that the moisture absorbed in the cell
wall of the fibers was reduced, caused by acetylation of the hydroxyl
groups present in the vegetable fiber [76].
The trend of the results is comparable with that reported by Espino-
Pérez et al. (2013). They made PLA films with elongated cellulose
nanocrystalline grafts. Regarding the insertion of nanocrystals, the WVT
did not increase significantly. This was due to the inclusion of a hy­
drophobic graft in the mixture (chemical modification of elongated
cellulose nanocrystals with n-octadecyl isocyanate), which could coun­
teract the effect of water vapor permeability [74]. Regarding the
incorporation of nZH-Cu, it can be pointed out that the zeolite nature
can hydrate and dehydrate reversibly [77]. As reported by García et al.
2019 there is a trend to increase WVT with the addition of zeolite and
bentonite, on chitosan films [78]. Furthermore, as reported by Rhim
(2013), who prepared multilayer films composed of PLA and nanoclay
Table 5
WVT for all biofilms after the electrospinning process.
Nanoparticles (%) WVT
(g/Pa*s*m)
15 16
Acetylated CNF nZH-Cu (x 10− ) (x 10− )
0 0 4.98 ± 3.00 b

1 3 223b 347c 42e 1 1 5.75 ± 2.50a

361a 66d 1 2 5.79 ± 2.81a
1.5 1 224b 346c 41e 1 3 5.69 ± 4.59a
360a 65d 1.5 1 5.73 ± 3.72a
1.5 2 226a 355b 66d 1.5 2 5.87 ± 1.39a
1.5 3 221c 335e 69d 1.5 3 5.81 ± 8.05a
2 1 224b 347c 44e 2 1 5.88 ± 3.15a
2 2 226a 358a 72c 2 2 5.69 ± 1.40a
2 3 222c 330f 76b 2 3 5.73 ± 4.65a

Values followed by different superscript letters in the same column indicate Values are presented as means ± SD. N = 3. Values followed by different su­
significant differences (p > 0.05). perscript letters in the same column indicate significant differences (p > 0.05).

9
J. Vergara-Figueroa et al. Materials Today Communications 25 (2020) 101671

(Cloisite® Na+), it was shown that both the mixture and its nano­ Table 6
composite films exhibited a considerably lower water resistance prop­ Determination of the antibacterial activity of biofilms against S. aureus and S.
erty due to their hydrophilic nature. Only the formation of multilayers of Typhi.
PLA film with nanoparticles allowed to decrease the permeability to Recovered microorganisms Microorganism reduction
water vapor [79]. Treatment (CFU/mL Log10) (%)
In the present study, WVT was not affected by the increased con­ S. aureus S. Typhi S. aureus S. Typhi
centration of nanoparticles in biofilms. Possibly, both the incorporation a a e
Control 9.2 ± 0.0 9.2 ± 0.0 0.0 ± 0.0 0.0 ± 0.0d
of acetylated CNF and that of nZH-Cu in the biopolymer matrix could PLA- 0% nZH-Cu 4.5 ± 0.4b 3.3 ± 0.2b 51.0 ± 3.8c 64.5 ± 2.6b
have prevented the permeability effect in the biofilms. According to our PLA- 3% nZH 4.7 ± 0.4b 3.3 ± 0.2b 48.8 ± 3.8d 64.5 ± 2.6b
results, the nanoparticles dispersed in the film prevented the transfer of PLA- 1% nZH-Cu 4.1 ± 0.2c 3.4 ± 0.1b 55.3 ± 2.2b 62.7 ± 1.1c
water vapor. The explanation can be found in that possibly, the incor­ PLA- 2% nZH-Cu 0.0 ± 0.0d 0.0 ± 0.0c 100.0 ± 0.0a 100.0 ± 0.0a
PLA- 3% nZH-Cu 0.0 ± 0.0d 0.0 ± 0.0c 100.0 ± 0.0a 100.0 ± 0.0a
poration of acetylated CNF formed bonds with the biopolymer matrix.
Also, the inclusion of copper in the porous interstices of the nanozeolite Values are presented as means ± SD. N = 3. Values followed by different su­
occupied the active sites of the zeolite, causing the inability to react with perscript letters in the same column indicate significant differences (p> 0.05).
the environment (water vapor). Similar WVT trends were obtained when
copper and silver nanoparticles were mixed in an agar film [68,80]. processes of chromosomal DNA and plasmid, so signal transduction in
bacteria is affected [20].
3.3.5. Determination of the antibacterial activity of biofilms
The ASTM E2180–07 in vitro assay was used to evaluate the anti­ 4. Conclusions
bacterial capacity of biofilms with different concentrations of nZH-Cu.
Table 6 presents the count and reduction results for the S. aureus and The production of cellulose nanofibers was achieved by two me­
S. Typhi strains. It is observed that the different treatments affect the chanical methods, defibration and high shear microfluidization. It was
recovery of microorganisms since as the concentration of nZH-Cu in the found that the acetylation process catalyzed with tartaric acid modified
biofilms increases, a significant reduction in the bacterial count is the surface polarity of the NFC, which was evidenced in the FTIR
generated, compared to the control. The PLA-1% nZH-Cu biofilm analysis with the appearance of bands of acetyl groups. Biofilms were
reduced 55.3 % of S. aureus and 62.7 % of S. Typhi. It is observed that developed based on the PLA polymer with the insertion of acetylated
with the biofilm PLA- 2% nZH-Cu and PLA- 3% nZH-Cu, there was no CNF and nZH-Cu, using the electrospinning technique. These nano­
bacterial growth (100 % reduction) against both strains. These results particles were deposited on and between the fibers that make up the
indicate that the biofilms show antibacterial activity, potentially broad- biofilms. SEM and morphological analyzes revealed a successful biofilm
spectrum, as it is effective on the Gram (-) and Gram (+) strain and that manufacturing process made up of uniform, continuous, and randomly
the total reduction of microorganisms is achieved from the PLA-2% nZH- arranged microfibers. Furthermore, a homogeneous distribution of
Cu biofilm. copper in the biofilms was verified by SEM-EDX. The addition of acet­
The in vitro results indicate that biofilms formed by PLA microfibers ylated CNF to the biofilm mixture causes an increase in its mechanical
with the insertion of copper ions in zeolite nanoparticles are useful properties, determined by mechanical tests. The addition of nano­
materials to slow the growth of bacteria [29,41,42,81]. They present particles was found to enhance the resistance to thermal degradation of
antibacterial activity against S. Typhi bacteria, the primary bacteria biofilms, as revealed by the TGA degradation profile. The WVT assay
responsible for foodborne diseases in poultry products, and against demonstrated that the insertion of nanoparticles does not affect the
S. aureus, whose presence accounts for the sanitary and hygienic quality permeability of biofilms based on PLA-nanoparticles. Through in vitro
in the preparation of food [82–85]. The results obtained here are com­ tests, it was verified that the biofilms show antibacterial activity against
parable to those reported by other authors since it is corroborated that the S. Typhi bacteria and S. aureus. The trials and tests applied in this
the antibacterial activity present in hydrophobic composite materials research reveal that the biofilms developed have potential use as an
depends on the concentration of agents incorporated in the materials antimicrobial barrier. They show adequate characteristics to withstand
[26,86]. handling in the hands of the consumer, as well as prevent bacterial
There are several mechanisms and theories on the bactericidal effect growth. Additionally, its biodegradability characteristics would help
of supported metal ions on nanostructures. Antimicrobial activity can be reduce contamination problems caused by the accumulation of plastic
associated with nanometric dimensions and a large specific surface, waste.
which allows it to interact with bacteria [41]. It has been suggested that
the formation of reactive oxygen species can produce harmful effects on
cells such as damage to DNA damage due to oxidation of poly­ Declaration of Competing Interest
unsaturated fatty acids and amino acids, causing oxidative stress in the
cell. On the other hand, the polysaccharides of bacterial membranes The authors report no declarations of interest.
have electronegative groups, sites of attraction of metal cations. The
difference in charge between them causes electrostatic attraction. They Acknowledgments
accumulate on the surface of the bacteria, altering the structure and
permeability of the cell membrane. Gram (-) bacteria, like the S. Typhi The authors wish to thanks the financial support of the Doctoral
strain, have a higher negative charge than Gram (+). Therefore the Scholarship and Research Grant from the University of Bio-Bio and
electrostatic interaction will be stronger in Gram (-) strains. Bacterial Project Grant EQM 170077.
membranes have pores of nanometric sizes, so the smaller the bacteri­
cidal particle size, the larger its surface area will be, leading to greater References
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