Article
Lignin-Mediated Biosynthesis of ZnO and TiO2
Nanocomposites for Enhanced Antimicrobial Activity
Kanchan M. Samb-Joshi 1 , Yogesh A. Sethi 2 , Anuradha A. Ambalkar 2 , Hiralal B. Sonawane 3 ,
Suresh P. Rasale 1 , Rajendra P. Panmand 2 , Rajendra Patil 4 , Bharat B. Kale 2, * and
Manohar G. Chaskar 1, *
1
2
3
4
*
Department of Chemistry, Prof. Ramkrishna More Arts, Commerce and Science College, Pune,
Maharashtra 411044, India
Nanocrystalline Laboratory, Centre for Material for Electronic Technology (CMET), Pune,
Maharashtra 411008, India
Department of Botany, Prof. Ramkrishna More Arts, Commerce and Science College, Pune,
Maharashtra 411044, India
Department of Biotechnology, Savitribai Phule Pune University, Pune, Maharashtra 411007, India
Correspondence: bbkale1@gmail.com (B.B.K.); manohar_c@hotmail.com (M.G.C.)
Received: 30 June 2019; Accepted: 9 September 2019; Published: 13 September 2019
Abstract: In this work, we report the synthesis of fragmented lignin (FL) assisted zinc oxide (ZnO) and
titanium oxide (TiO2 ) nanocomposites. The fragmented lignin synthesized from biomass (sugarcane
bagasse) was used as a template to generate the morphology and crystallite structure of metal oxide
nanomaterial. The nanocomposites were synthesized by a simple precipitation method, wherein
fragmented lignin is used in alkaline medium as a template. X-ray diffraction (XRD) analysis shows
the phase formation of hexagonal wurtzite ZnO and mixed phase formation of TiO2 as rutile and
anatase. The morphology was studied by using field emission scanning electron microscopy (FE-SEM)
and high-resolution transmission electron microscopy (HRTEM). The FE-SEM of pristine ZnO
nanocomposites showed a cluster of particles whereas FL–ZnO NPs showed self-aligned nanoparticles
in the form of rod shaped having average size 30–70 nm. Pristine TiO2 nanoparticles showed clusters
of particles and FL–TiO2 nanocomposites showed well crystalline 41nm size nanocomposites. The FL
acts as a surfactant which restrict the cluster formations. The band gap determined by diffuse
reflectance spectra is 3.10 eV and 3.20 eV for FL–ZnO and FL–TiO2 nanocomposites, respectively.
Photoluminescence spectra of both nanocomposites showed structural defects in the visible region.
Further, the antimicrobial activity of pristine ZnO and TiO2 nanoparticles, and FL–ZnO and FL–TiO2
nanocomposites against Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923) were studied
under UV-A (315-400 nm) (8W) for 30min.
Keywords: ZnO; TiO2 ; fragmented lignin; E. coli; S. aureus. Nanorods
1. Introduction
One of the greatest challenges of the twenty-first century is the spread of multidrug resistance in
microorganisms [1]. Microbial infections are common in humans, but the development and spread of
drug resistance in microorganisms has made the present antimicrobial therapy ineffective [2]. Infections
that were once easily treatable have become increasingly more difficult to treat and result in higher
morbidity and mortality. Therefore, in order to overcome the inability of antibiotics to deal with the
rising issue of resistance in microorganisms, the need for the development of novel, broad spectrum
antimicrobials have arisen [3,4]. Various contemporary novel approaches including combination
drug therapy, bacteriophage therapy, fecal microbial transplantation, antimicrobial adjuvants, and
J. Compos. Sci. 2019, 3, 90; doi:10.3390/jcs3030090
www.mdpi.com/journal/jcs
J. Compos. Sci. 2019, 3, 90
2 of 13
antimicrobial peptides have been tried to address the issues of resistance in microbes, however, either
these have met with limited success or have failed to achieve the goal [5,6]. Recently, the advancement
in field of nanotechnology has contributed towards the synthesis various antimicrobial nanomaterials
with the aim to develop meta and metal oxide-based antimicrobials [7]. The nanomaterials are generally
considered to be particles with at least one dimension measuring around 1–100 nm and show unique
properties which change significantly with size and differ from bulk material properties, due to
the great increase in surface area to volume ratio, leading to an increased number of surface atom
interactions with their surroundings. Amongst all nanomaterials, semiconductor-based nanomaterials
have showed wide applications including antimicrobial agents [8]. Among them, one dimensional
(1D) nanostructures such as nanowires, nanorods and nanobelts have shown potentials antimicrobial
activities due to their distinctive geometrical morphologies, novel physical and chemical properties [9].
One dimensional ZnO nanomaterials with a characteristic direct wide band gap of about 3.37 eV
and large excitation binding energy of 60 meV at room temperature have been shown to be nontoxic
and good chemical stabilities were widely explored for antimicrobial applications due to its photo
catalytically phenomenon associated with it [10]. To date, several approaches have been developed for
the synthesis of 1D ZnO nanostructures, such as sputtering method, physical, chemical, and pulsed
laser vapor deposition [11]. However, most of these methods often faced with problems such as
complex procedures, high temperature, and cost. The other problem being the aggregations behaviors
and difficult dispersibility which reduces the interface compatibility of nanomaterials with other
surfaces [12]. Thus, the template-based chemical-based method which has an advantage of simplicity
and low cost, has become the most promising methods for synthesis of ZnO nanomaterials [13].
In chemical-based method, ZnO are synthesized on the template of organic polymer to achieve
polydispersity and increase the interface compatibility of nanomaterials with other surfaces. Several
natural polymers such as DNA, silk, albumen, orange juice, pea starch, peptide structures, etc., have
been used as templates for the synthesis of ZnO [14]. However, one of the natural polymers, called
lignin, which is available on earth in a huge quantity is relatively unexplored for the synthesis of ZnO
nanomaterials [15]. The lignin is a “renewable chemical resource” formed from the assembling of
functionalized aromatic entities with phenolic hydroxyl, alcoholic hydroxyl, carboxyl, or methoxy
groups [16,17]. Lignin has many potential value-added applications with significant impact on industry.
For example, derivation of lignin leads to functional polymers with a role of dispersant for pesticide,
surfactants, additive in oil drilling, stabilizers in colloidal suspensions, antioxidants, antiviral, antibiotic,
and/or anticarcinogenic agents, etc. [18–20]. Importantly, over 50 million tons of industrial lignin
is produced from the paper-making industry as a by-product every year [21]. However, only 2%
of lignin is effectively unitized and more than 98% of lignin is combusted as fuel, which not only
causes environmental problems but also causes a huge waste of the resources [22]. Therefore, in the
present work, a simple and environmentally friendly in-situ template fragmented lignin (FL)-assisted
synthesis method is reported for controllable preparation of FL–ZnO and FL–TiO2 nanocomposites.
The FL–ZnO and FL–TiO2 nanocomposites were characterized by UV-Visible, high resolution mass
spectrometry (HRMS), X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM),
TEM and photoluminescence (PL) spectroscopy. Finally, the antimicrobial potential of the FL–ZnO and
FL–TiO2 nanocomposites were explored by using model organisms, Escherichia coli (ATCC25922) and
Staphylococcus aureus (ATCC25923). The main objective of this research is to determine the functional
significance of fragmented lignin (template) assisted synthesis of ZnO and TiO2 nanocomposites for
improving the antimicrobial property of ZnO and TiO2 nanoparticles.
2. Materials and Methods
2.1. Material
The materials in this work are Bagasse, Zinc acetate, Titanium isoproxide, Sodium hydroxide, and
Escherichia coli (ATCC25922), and Staphylococcus aureus (ATCC25923).
J. Compos. Sci. 2019, 3, 90
3 of 13
2.1.1. Fragmentation of Lignin
Commercial lignin (Sigma-Aldrich, St. Louis, MO, USA) was used to prepare fragments of
lignin. 5 g of purified and air-dried lignin was dissolved in 50 mL 0.1M sodium hydroxide (NaOH)
solution (pH 12) then stirred for 30 min at temperature 40–50 ◦ C. After this, 100 mL 1% hydrogen
peroxide (H2 O2 ) solution was added to above solution in a drop wise manner over the period of 1 h.
The resultant solution was filtered, cooled and acidified with sulphuric acid (H2 SO4 ) up to pH 4.5–5
and the ivory colour precipitation used as FL.
2.1.2. Synthesis of FL–ZnO Nanocomposites and Pristine ZnO Nanoparticles.
All reagents are of analytical grade without further purification, supplied by Loba Chemie Pvt.
Ltd. (Mumbai, India). 0.1 g FL and 0.1 M 100 mL NaOH were sonicate for 1 h to get the homogenous
solution. After sonication the solution was heated as well as stirred and maintaining temperature
between 80 to 90 ◦ C. Then 2 g of zinc acetate was added to the solution over a period of 30 min. again
stirred for 1 h. A white color precipitate was obtained. It was wash with water followed by absolute
ethanol to remove the residual fragmented lignin and sodium hydroxide. The precipitate was dried in
an oven at 90 ◦ C for 1 h, followed by calcination at 450 ◦ C in muffle furnace for 3h. Similar procedure
was followed to prepare pristine ZnO nanoparticles except the addition of FL.
2.1.3. Synthesis of FL–TiO2 Nanocomposites and Pristine TiO2 Nanoparticles
4 mL Titanium isopropoxide and 25 mL isopropanol mixture was added slowly in the solution of
1 N 100 mL NaOH and 0.1 g of FL at 0 ◦ C up to 3 h. A precipitation formed was washed several times
with 0.01 N hydrochloric acid, water and finally with ethanol. Then it was dried in an oven at 110 ◦ C
for 1 h followed calcination at 450 ◦ C for 3 h. Similar procedure was followed to prepare pristine TiO2
nanoparticles except the addition of FL.
3. Characterization
The phase purity and crystallinity of the nanocomposites and nanoparticles were performed
by X-ray diffraction studies by using Cu Kα1 (1.5406 angstrom) radiation scanned in the 2θ range
from 10◦ to 80◦ , and photoluminescence (PL) spectroscopy optical properties by using UV-visible
spectrophotometer in the range from 300-800 nm, morphology by using field emission scanning
electron microscopy (FE-SEM) and transmission electron microscopy (TEM).
Antimicrobial Property of Biosynthesis FL–ZnO and FL–TiO2 Nanomaterial with Pristine ZnO and
TiO2 Material
The standard strain of Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923)
obtain from Microbiology Laboratory Fergusson College Pune were used for antimicrobial studies.
The antimicrobial activity was performed as per the guideline of Clinical and Laboratory Standards
Institute [23]. It was performed by measuring the minimum bactericidal concentration (MBC) values of
composites and pristine nanoparticles against standard strains of microorganisms. In short, 0.1 mL of
1 × 104 cells/mL was inoculated into 1 mL volume of nutrient broth containing different concentrations of
nanocomposites and pristine nanoparticles (0.5–1.5 mg/mL). After inoculation all sets were irradiated in
UV-A for 30 min. Then each sample was spread on nutrient agar plate and incubated at 35 ± 2 ◦ C for 24 h.
Colonies appeared were counted to calculate the MBC value. Nutrient agar plate with microorganism
and without nanoparticles served as control. Similar procedure with standard antibiotics, amoxicillin,
served as positive control. The experiments were performed in triplicate and the average measurement
were reported.
J. Compos. Sci. 2019, 3, 90
4 of 13
4. Results and Discussions
4.1. Structural Study
Crystalline phase and lattice parameters of FL–ZnO and FL–TiO2 nanocomposites, and pristine
ZnO and TiO2 nanoparticle were obtained from recorded XRD spectra (Figure 1). The crystal size was
calculated by using the Debye–Scherrer equation:
β(2θ) =
Kλ
,
LCosθ′
where K—Scherrer constant. K varies from 0.68 to 2.08. K = 0.94 for spherical crystallites with cubic
symmetry, L—volume average of the crystal thickness in the directional normal to the reflecting planes,
λ—X-ray wavelength. For Mini XRD, Cu Kα average = 1.54178 Å, θ—XRD peak position, one half
of 2θ.
The major peaks in XRD of FL–ZnO nanocomposites and pristine ZnO do not show any difference
in peak positions but an appreciable difference in peak intensities were observed. The observed peak
at 2θ = 31.610, 34.320, 36.140, 47.370,56. 480, 62.710 and 68.280 correspond to (100), (002), (101), (110),
(103) and (201) planes these are characteristic peaks of hexagonal crystalline structure of ZnO (JCPDS
file 36-1451 of ZnO). Sharp and intense peaks in the XRD analyses of both F–ZnO nanocomposites and
pristine ZnO indicate high crystallinity and the polycrystalline nature of ZnO nanoparticles (curves a
and b Figure 1). Moreover, no extra peaks other than hexagonal crystalline phase of ZnO were found in
XRD. Pristine ZnO has average crystallite size 43.76. High intensity of peak at (101) plane shows a weak
preferential growth in FL–ZnO nanoparticles along c-axis [24] with an average crystallite size 25.34 nm.
The XRD of FL–TiO2 nanocomposites showed a mix phase of anatase and rutile (JCPDS anatase 33-1381,
and Rutile 34-0180) which shows the major peaks at 2θ = 25.30,27.380, 36.08 37.730, 41.24 48.020, 54.320,
55.068, 62.840, 68.780, and 70.250 (curve c in Figure 1). The observed peaks (27.23, 36.08 41.24, and
54.32) at (110) (101), (111) and (211) can be indexed is a typical characteristic of rutile phase TiO2 .
The peaks 25.30, 37.730, 48.020, 53.068, 62.11, 62.840, and 68.780, and 70.250 at (101), (004), (200), (105),
(211), (213), (116) and (220) is typical of anatase phase TiO2 (curve d in Figure 1). The pristine TiO2
showed pure anatase phase (curve d in Figure 1) with major peaks 25.380, 37.730, 48.910, 53.980, 55.980,
62.840, 68.830, 70.160 and 75.030 indexed at (101), (004), (200), (105), (211), (213), (116), (220), and (215)
planes. The FTIR and HRMS spectra of FL gives the information of functional groups present in the
FL such as 679, 850, 1029–1150, 1629, 2829–2926, and 3354 cm1 for aromatic ring, aromatic ring (Para
Substituted), CNO stretching, C–O–C stretching, aromatic compound, CH2 stretching, hydroxyl group
broad band (Intramolecular Hydrogen Bonding), HRMS showed five different fragments as (1) m/z 276
gives 4-(4-(hydroxymethyl)-3-methoxyphenoxy)-2-methoxyphenol, (2) m/z 290 suggest the β-etheral
linkage of phenolic structure, (3) m/z 322 is for Sinapic acid (3-(4-hydroxy 3, 5-dimethyl phenyl), (4)
m/z 405 is for dimeric fragments, (5)534m/z for β-O-4 dimer with attachment of different carbon atom
or –CH2 group present in the molecule, 1 H NMR of FL. 1 H NMR (400MHz, MeOD): δ0.84, δ1.2, δ1.52,
δ2, δ2.3, δ3.8, and δ6.5–7.8 showed presence of methylene proton, ethylene proton, acetylene proton
and aromatic proton (SEI-1 FTIR of Fragmented Lignin, SEI-2 1 H-NMR of Fragmented Lignin and
SEI-3 HRMS of Fragmented Lignin in Supplementary Materials).
J. Compos. Sci. 2019, 3, 90
5 of 13
Figure 1. X-ray diffraction (XRD) of (a) Pristine ZnO, (b) FL–ZnO, (c) FL–TiO2 and (d) Pristine TiO2 .
4.2. Surface Morphological Studies
The surface morphology was studied by FE-SEM. The FE-SEM image of FL–ZnO exhibits rod
shape morphology given in Figure 2c,d, it is possible due to the inhibition of the growth of nanoparticle
in third dimension by fragmented lignin.
The FE-SEM images of pristine ZnO (Figure 2a,b) showed an agglomeration. FL contain many
polar functional groups mainly –OH, –OCH3˃, >C=O and C–O–C which might play an important
role in the formation of ZnO rod nanoparticle.˃Due to the presence of these functional groups lignin
fragments may have acted as a complexing, capping or stabilizing agent for Zn+2 ions. The polymeric
nature of lignin fragments can create protective and functionalized surrounding for metal ions which
might be playing a structure directing role in the formation of rod shaped ZnO nanoparticles [25].
Difference in particle morphology of of pristine TiO2 and FL–TiO2 was observed in Figure 2e,f and
Figure 2g,h. FL–TiO2 showed uniformity in grain size and shape. The grains were roughly spherical
to spherically elongate in shapes. The average grain size for this FL–TiO2 was 41 nm. Pristine TiO2
showed grains of large and varied size with roughly spherical in shape with an average grain size of
277 nm. Here the anisotropic effect was observed in both samples. The peak of FL–ZnO and FL–TiO2
show broad peak width due to the decrease in crystal size as compare to the pristine ZnO and TiO2 .
Figure 2. Field emission scanning electron microscopy (FE-SEM): (a,b) Pristine ZnO, (c,d) FL–ZnO,
(e,f) Pristine TiO2 and (g,h) FL–TiO2 .
J. Compos. Sci. 2019, 3, 90
6 of 13
4.3. TEM
Under TEM, FL–TiO2 showed spherical morphology with average particle size is 14 nm (Figure 3a).
FL–ZnO has rod shaped structure composed of spherical particles with self-aligned nature having
average size 20 nm diameter. This is one directional growth along (101) plane [26]. XRD showed the
highest intensity peak (101) plane of the FL–ZnO, which may be favored by biosynthesis method using
fragments lignin as a template given in Figure 3b.
Figure 3. TEM of (a) Spherical morphology of FL–TiO2 , (b) self-aligned in one direction Spherical
particle of FL–ZnO.
High-Resolution Transmission Electron Microscopy (HRTEM)
The interplanar distance of FL–ZnO is 0.247 nm for (101) plane. Both the technique XRD and
HRTEM shows the mixed phase of FL–TiO2 nanoparticles. For anatase plane (110) with an inter planer
distance of 0.266 nm and for the rutile phase (211) the inter planer distance is 0.324 nm. The mixed
phase formation of FL–TiO2 and one directional growth of nanoparticle were due to the use of FL
which act as a surfactant as shown in Figure 4a,b.
The fast Fourier transformation (FFT) gives the information of the lattice fringes on HRTEM image
and it can use to index the observed spots. In given Figure 4c,d corresponding to the fast Fourier
transmission (FFT) pattern of the nanoparticle is closed to single crystalline. From these observations,
it is reasonable that the as-prepared ZnO and TiO2 nanoparticle is of nanocrystalline structures, which
means that they are constituted by well-crystallized ZnO and TiO2 nanoparticles.
Figure 4. High-resolution transmission electron microscopy (HRTEM): (a) FL–ZnO, (b) FL–TiO2 , (c) FFT
FL–ZnO and (d) FFT FL–TiO2 .
J. Compos. Sci. 2019, 3, 90
7 of 13
4.4. Optical Study
The Ultra-Violet diffuse reflectance spectra (UVDRS) of pristine ZnO, TiO2 , FL–ZnO and FL–TiO2
were shown in set of Figure 5. It was calculated from a UV-Visible spectrum and Tauc’s plot.
All these materials showed absorbance in a range of 350–400 nm. The calculated band gap of FL–ZnO
nanoparticles in presence of FL at 3.10 eV and pristine ZnO is 3.26 eV. More is the oxygen defects lower
is the band gap [27]. Deviation in the band gaps of FL–TiO2 , and pristine TiO2 was observed in curves
b and d in Figure 5. FL–TiO2 showed the band gap of 3.20 eV while pristine TiO2 has band gap of
3.37 eV.
Figure 5. UV-Visible spectrum and UV-diffuse reflectance spectrum of ZnO and TiO2 as (a) FL–ZnO
3.10 eV, (b) FL–TiO2, 3.20 eV. (c)Pristine ZnO 3.20 eV and (d) Pristine TiO2 3.37 eV.
4.5. Photoluminescence Study of Pristine ZnO, FL–ZnO, Pristine TiO2 and FL–TiO2
Photoluminescence study gives us the information of the structural defects in nanoparticles.
The Figures 6 and 7 showed photoluminescence property of nanoparticles of pristine ZnO, FL–ZnO,
pristine TiO2 , and FL–TiO2 synthesized. ZnO samples were excited at 390 nm excitation wavelength
and TiO2 samples were excited at 395 nm excitation wavelength. The emission spectrum of pristine
ZnO, FL–ZnO, Pristine TiO2 and FL–TiO2 samples were showed in Figures 6 and 7. The different
types of peaks were observed in visible region from 400 to 600 nm, such as 400–436 nm (violet region),
436–497 nm (blue region), 497–568 nm (green region) and 568–592 nm (yellow region).
Figure 6. Photoluminescence spectra of ZnO materials excited at 390 nm excitation wavelength (a)
FL–ZnO and (b) Pristine ZnO.
J. Compos. Sci. 2019, 3, 90
8 of 13
Figure 7. Photoluminescence spectra of TiO2 material excited at 395 nm excitation wavelength
(a) FL–TiO2 and (b) Pristine TiO2 .
FL–ZnO has highest peak intensity than pristine ZnO where pristine ZnO has no specific peak
shoulder in spectrum (Figure 6). For FL–ZnO the peak intensity area was 400–436 nm (violet region),
436–497 nm (blue region) which gives more oxygen defect in visible region. The blue green region are
due to the lattice defects which can produce compressive strain at intrinsic crystal lattice [28]. It causes
the transition of energy from conduction band or zinc interstitials, which results the recombination
of holes and electrons in the valences band and conduction band [29]. Pristine TiO2 showed broad
emission band because agglomerated lattice sites and peak intensity shift towards 568–592 nm
(yellow–red region) which is significant for increase in particle size (Figure 7) caused by oxygen
interstitials zinc vacancies and oxygen interstitials. It may conclude that O-/O2 - ion concentration
increases intrinsic strain in the lattice of the materials, which gives transition from conduction band to
the oxygen interstitial position yellow emission [30,31]. In FL–TiO2 only two peaks were observed,
one at 400–436 nm (violet region), and the other at 497–568 nm (green region) due to structural defects
related to the deep level emission [32].
5. Antimicrobial Property
Nanoparticles have emerged as an effective antimicrobial-agent alternative to traditional organic
based drugs, primarily due to actions that specifically target and minimize toxicity [33–35]. Most studies
reported bacteriostatic or bactericidal effect of nanoparticles due to disruption of their cell membrane.
The nanoparticle can accommodate a large number of ligands present on microbial cells due to its
large surface area to volume ratio. Several types of metal and metal oxide nanoparticles have been
already reported to possess anti-microbial property like titanium oxide, gold, silver, copper, iron, zinc
oxide, copper oxide and iron oxide nanoparticles [36–42]. However, of all nanoparticles, TiO2 and
ZnO nanoparticles have gained considerable attention because of their unique electronic, optical and
medicinal properties [43,44]. These nanoparticles are highly biocompatible and therefore have found
various applications in biological field [45]. Recently, a green chemistry approach has been reported to
synthesis various nanoparticles including ZnO and TiO2 wherein natural products such as silk fibroin,
cellulose, starch, humic acid, carbohydrates, lignin, etc. have been used as reducing and stabilizing
agents. Among them, lignin is interesting natural products because: (i) it comes from the wood pulp
industry; (ii) it is the second most abundant on earth, just second to cellulose; (iii) it is a nanocrystalline
and heterogeneous polymer with a network structure; (iv) it is the only kind of biomass constituent that
belong to the aromatic compounds; (v) structurally, it contains many aliphatic and aromatic hydroxyls,
several aromatic methoxy, carboxyl, carbonyl and ethereal moieties and (vi) it has many aliphatic
hydroxyl groups, therefore, it can be used in reduction of metal salt to metal nanoparticles [46–48], for
J. Compos. Sci. 2019, 3, 90
9 of 13
changing surface morphology and nanoroughness of metal oxide, it can affect on for cell adhesion
and proliferation [49]. Antibacterial activity of pristine ZnO and TiO2 nanoparticles and FL–ZnO and
FL–TiO2 nanocomposites on E. coli and S. aureus were as shown in Figures 8 and 9, respectively.
FL-ZnO
Pristine ZnO
FL-TiO2
FL-ZnO
Pristine
PristineZnO
TiO2
FL-TiO2
E.coli, Antimicrobial Activity
2.5
E.coli, Antimicrobial Activity
2.5
2.0
Pristine TiO2
Log (CFU/mL)
Log (CFU/mL)
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
0.0
0.0
0.5
1.0
Conc (mg/mL)
0.5
1.0
Conc (mg/mL)
1.5
1.5
Figure 8. Antimicrobial activity of E. coli against pristine ZnO (48%), FL–ZnO (5.2%), pristine TiO2
(52%) and FL–TiO2 (6%).
2.5
2.5
S.aureus, Antimicrobial acivity
S.aureus, Antimicrobial acivity
FL-ZnO
Pristine ZnO
FL-ZnO
FL-TiO
Pristine2ZnO
Pristine TiO2
FL-TiO
2
2.0
Pristine TiO2
Log Log
(CFU/mL)
(CFU/mL)
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
0.0
0.0
0.5
1.0
Conc (mg/mL)
0.5
1.0
1.5
1.5
(mg/mL)
Figure 9. Antimicrobial activity of S. aureusConc
against;
growth shown for Pristine ZnO (32%), FL–ZnO
(3.2%), Pristine TiO2 (36%) and FL–TiO2 (5%).
As can be observed, FL–ZnO and FL–TiO2 nanocomposites showed a better antimicrobial activity
than pristine ZnO and TiO2 nanoparticles. The nanocomposites were more effective on cells of S. aureus
than on cells of E. coli. The antimicrobial action of FL–ZnO and FL–TiO2 nanocomposites were
concentration dependent. When the cells of E. coli and S. aureus were independently subjected to
various concentrations of ZnO and TiO2 nanoparticles and FL–ZnO and FL–TiO2 nanocomposites,
there was a decrease in the number of viable cells. An initial 2.4 log CFU/mL was reduced to less than
0.5 log CFU/mL in presence of FL–ZnO and 0.7 log CFU/mL in presence of FL–TiO2 nanocomposites.
However, the log CFU/mL in presence of pristine ZnO and TiO2 nanoparticles were respectively, 2.2
and 2.1. The reduction was more prominent in presence of FL–ZnO and FL–TiO2 nanocomposites.
The colony count of E. coli and S. aureus, in presence of FL–ZnO and FL–TiO2 nanocomposites also,
was less (Figure 10). Small particles size of material showed enhancement in the bioactivity because
increased in surface area to the volume ratio. The smaller particles can easily bind higher number
of bacterial colonies which result in the large number of active oxygen species to burst the cell wall
J. Compos. Sci. 2019, 3, 90
10 of 13
of the bacteria also the structural defects, oxygen defect and oxygen interstitials help to increase the
antimicrobial activity of nanomaterial of FL–ZnO and FL–TiO2 . Whereas, pristine ZnO and TiO2
nanoparticles have agglomeration, bulky size and less surface to the volume ration can decreases
contribution to the antimicrobial activity [50].
Figure 10. Antimicrobial activity of E. coli and S. aureus against TiO2 and ZnO nanomaterials. (a) Control
of S. aureus; (b) FL–ZnO against S. aureus; (c) FL–ZnO against E. coli; (d). Control of E. coli; (e) TiO2
against S. aureus; (f) TiO2 against E. coli.
The antimicrobial activities of FL–ZnO and FL–TiO2 were in agreement with previous reports.
The powder extract of dry ginger rhizome was used as a reducing material as well as surface stabilizing
agent to synthesize ZnO nanoparticles and were shown to possess antimicrobial activity against
bacteria and fungi [51]. Recently Coptidis rhizoma-mediated ZnO nanoparticles were tested for its
antimicrobial activity against four disease-causing pathogens Bacillus megatherium, B. pumilus, B. cereus
and E. coli [52]. ZnO nanoparticles adopt a series of mechanisms to act as an anti-bacterial agent.
Loss of phospholipid bilayer cell membrane integrity is considered as one of the most important
mechanisms of ZnO and TiO2 NPs due to the oxidative stress induced by reactive oxygen species
(ROS). This ROS molecule further causes cell death by inhibiting or altering DNA replication, protein
synthesis, and membrane potentials [53]. In the present study, FL–TiO2 and FL–ZnO nanocomposites
have shown a prominent action on cells of S. aureus because of the difference in the mechanism of
attachment of Gram- positive and Gram negative bacteria to nanoparticles, their transport inside
the cell, and differences in their different membrane structure. Gram positive bacteria have a thick
layer of peptidoglycan and have teichoic acid and lipoteichoic acid, the later serves as binding sites to
nanocomposites. They also chelate metal ions from nanoparticles and transport inside the cell. Gram
negative bacteria have a triple layer of peptidoglycan in their cell wall which imparts additional barrier
for nanoparticles to enter inside cells [54]. Upon illumination, ZnO and TiO2 nanoparticles generate
the electron hole pairs, and produce reactive oxygen species (ROS), which oxidizes organic matter
and, thus, imparts biocidal property to nanoparticles [55]. However, ZnO and TiO2 nanoparticles are
having a very low efficiency for the separation of electron hole pairs due to fast recombination of
charge carriers, therefore, it is very essential to suppression of the recombination of photogenerated
electron-hole pairs in ZnO and nanoparticles. FL which contains different organic function groups,
such as aromatic, phenolic, hydroxyl, etheral, alkene and methoxy groups, are hypothesized to prevent
this recombination of electron hole pair. In the present study, the biosynthesized FL–ZnO and FL–TiO2
nanocomposites have porous structure and low band gap with several defect as was observed in
photoluminescence spectrum. FL–TiO2 nanocomposites have mixed phase morphology of anatase
and rutile geometry. Therefore, such an environment is favorable for ejection and stabilization of
electrons to create hole and electron pairs and imparts FL–ZnO and FL–TiO2 nanocomposites as an
antimicrobial property.
J. Compos. Sci. 2019, 3, 90
11 of 13
6. Conclusions
The FL results in five different fragments which contain aldehydic, etheral, alcoholic, aromatic,
hydroxyl and phenolic groups, which may act as surfactant and restrict the cluster formations. Hence,
these groups play an important role in synthesis of FL–ZnO and FL–TiO2 nanocomposites by a
biosynthesis method. These organic materials were coated on the nanomaterial during fabrication
which creates defects to generate holes and electrons, which easily react with the cell membrane and
DNA to result in cell death. Furthermore, the large surface area and special morphology of ZnO gives
a more pronounced effect. In this way, both of these biosynthesized materials show better results in
bactericidal activity compared to the pristine material.
Supplementary Materials: The following are available online at http://www.mdpi.com/2504-477X/3/3/90/s1.
Figure S1. FTIR of Fragmented Lignin, Figure S2. 1H-NMR of Fragmented Lignin, Figure S3. HRMS of
Fragmented Lignin.
Author Contributions: Conceptualization, K.M.S.-J., M.G.C.; Methodology, K.M.S.-J., H.B.S., R.P.; Software,
K.M.S.-J., Y.A.S., A.A.A.; Validation, K.M.S.-J., R.P.P.; Formal Analysis, K.M.S.-J., R.P.P., S.P.R.; Investigation, B.B.K.,
M.G.C.; Data Curation, K.M.S.-J., R.P.; Writing, K.M.S.-J., R.P., S.P.R.; Original Draft Preparation, K.M.S.-J., R.P.;
Writing–Review & Editing, K.M.S.-J., R.P.; Supervision, R.P., B.B.K., M.G.C.
Funding: This research received no external funding
Acknowledgments: Manohar G. Chaskar would like to thank Prof. Ramakrishna More A.S.C College and
B.G. College Sangavi pune for providing research facilities. Also, we thank for C-MET Pune. Rajendra Patil
acknowledges Departmental Research and Development Grant 2018-19.
Conflicts of Interest: The authors declare no conflict of interest
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Kaul, G.; Kapoor, E.; Dasgupta, A.; Chopra, S. Management of multidrug-resistant tuberculosis in the 21st
century. Drugs Today 2019, 55, 215–224. [CrossRef] [PubMed]
Richardson, L.A. Understanding and overcoming antibiotic resistance. PLoS Biol. 2017, 15, e2003775.
[CrossRef] [PubMed]
Paule, A.; Frezza, D.; Edeas, M. Microbiota and Phage Therapy: Future Challenges in Medicine. Med. Sci.
2018, 6, 86. [CrossRef] [PubMed]
Spaulding, C.N.; Klein, R.D.; Schreiber, H.L.; Janetka, J.W.; Hultgren, S.J. Precision antimicrobial therapeutics
the path of least resistance? NPJ Biofilms Microbiomes 2018, 4, 4. [CrossRef] [PubMed]
Baker, S.J.; Payne, D.J.; Rappuoli, R.; De Gregorio, E. Technologies to Address Antimicrobial Resistance; National
Acadamey of Science of United State of America: New Havan, CT, USA„ 2018; Volume 115, pp. 12887–12895.
Gupta, A.; Mumtaz, S.; Li, C.H.; Hussain, I.; Rotello, V.M. Combatting antibiotic-resistant bacteria using
nanomaterials. Chem. Soc. Rev. 2019, 48, 415–427. [CrossRef]
Raghunath, A.; Perumal, E. Metal oxide nanoparticles as antimicrobial agents: a promise for the future. Int. J.
Antimicrob. Agents 2017, 49, 137–152. [CrossRef]
Baranwal, A.; Srivastava, A.; Kumar, P.; Bajpai, V.K.; Maurya, P.K.; Chandra, P. Prospects of Nanostructure
Materials and Their Composites as Antimicrobial Agents. Front. Microbiol. 2018, 9, 422. [CrossRef]
Mantravadi, P.K.; Kalesh, K.A.; Dobson, R.C.J.; Hudson, A.O.; Parthasarathy, A. The Quest for Novel
Antimicrobial Compounds: Emerging Trends in Research, Development, and Technologies. Antibiotics 2019,
8, E8. [CrossRef]
Mahamuni, P.P.; Patil, M.; Dhanavade, M.J.; Badiger, M.V.; Shadija, P.G.; Lokhande, A.C.; Bohara, R.A.
Synthesis and characterization of zinc oxide nanoparticles by using polyol chemistry for their antimicrobial
and antibiofilm activity. Biochem. Biophys. Rep. 2018, 17, 71–80. [CrossRef]
Siddiqi, K.S.; Rahman, A.; Tajuddin, U.R.; Husen, A. Properties of Zinc Oxide Nanoparticles and Their
Activity Against Microbes. Nanoscale Res. Lett. 2018, 13, 141. [CrossRef]
Wilhelm, S.; Kaiser, M.; Würth, C.; Heiland, J.; Carrillo-Carrion, C.; Muhr, V.; Wolfbeis, O.S.; Parak, W.J.;
Resch-Genger, U.; Hirsch, T. Water dispersible. Upconverting nanoparticles: effects of surface modification
on their luminescence and colloidal stability. Nanoscale 2015, 7, 1403–1410. [CrossRef] [PubMed]
J. Compos. Sci. 2019, 3, 90
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
12 of 13
Xie, Y.; Kocaefe, D.; Chen, C.; Kocaefe, Y. Review of Research on Template Methods in Preparation of
Nanomaterials. J. Nanomater. 2016, 2016, 10. [CrossRef]
Bhandari, S.; Mondal, D.; Nataraj, S.K.; Balakrishna, R.G. Biomolecule-derived quantum dots for sustainable
optoelectronics. Nanoscale Adv. 2019, 1, 913–936. [CrossRef]
Wang, J.; Vermerris, W. Antimicrobial nanomaterials derived from natural products—A review. Materials
2016, 9, 255. [CrossRef] [PubMed]
Huang, J.; Fu, S.; Gan, L. Chapter 2—Structure and Characteristics of Lignin 2019. In Lignin Chemistry and
Applications; Elsevier: Atlanta, GA, USA, 2019; pp. 25–50.
Katahira, R.; Elder, T.J.; Beckham, G.T. Chapter 1 A Brief Introduction to Lignin Structure. In Lignin
Valorization: Emerging Approaches; The Royal Society of Chemistry: Philadelphia, PA, USA, 2018; Volume 1,
p. 20.
Acosta, J.E.; Torres-Chávez, L.P.I.; Wong, B.R.; Saiz, C.M.L.; Leyva, B.M. Antioxidant, Antimicrobial, and
Antimutagenic Properties of Technical Lignins and Their Applications. J. BioResour. 2016, 2, 5452–5481.
Vinardell, M.P.; Mitjans, M. Lignins and Their Derivatives with Beneficial Effects on Human Health. Int. J.
Mol. Sci. 2017, 18, 1219. [CrossRef] [PubMed]
Gordobil, O.; Herrera, R.; Yahyaoui, M.; Ilk, S.; Kaya, M.; Labidi, J. Potential use of kraft and organosolv
lignins as a natural additive for healthcare products. RSC Adv. 2018, 8, 24525–24533. [CrossRef]
Ge, Y.; Li, Z. Application of Lignin and Its Derivatives in Adsorption of Heavy Metal Ions in Water: A Review.
ACS Sustain. Chem. Eng. 2018, 6, 7181–7192. [CrossRef]
Lora, J.H.; Glasser, W.G. Recent Industrial Applications of Lignin: A Sustainable Alternative to Nonrenewable
Materials. J. Polym. Environ. 2002, 10, 39–48. [CrossRef]
Anand, N. Chapter 2.2 Biological Methods; The Indian Pharmacopeia Commission: Ghaziabad, India, 2010;
Volume 1, pp. 32–54, ISBN 81-903436-6-1 (VoU).
Joshi, K.M.; Shinde, D.R.; Nikam, L.K.; Panmad, R.; Kale, B.B.; Chaskar, M.G. Fragmented lignin-assisted
synthesis of a hierachical ZnO nanostructure for ammonia gas sensing. RSC Adv. 2019, 9, 2487. [CrossRef]
.Rubin, J.E.; Ball, K.R.; Trejo, M.C. Antimicrobial susceptibility of Staphylococcus aureus and Staphylococcus
pseudintermedius isolated from various animals. Can. Vet. J. 2011, 52, 153. [PubMed]
Fu, Y.Q.; Luo, J.K.; Nguyen, N.T.; Walton, A.J.; Flewitt, A.J.; Zu, X.T.; Du, H. Advances in piezoelectric thin
films for acoustic biosensors, acoustofluidics and lab-on-chip applications. Prog. Mater. Sci. 2017, 89, 31–91.
[CrossRef]
Miao, T.T.; Sun, D.X.; Guo, Y.R.; Li, C.; Li, Y.; Ma, G.Z. Low temperature precipitation synthesis of flower like
ZnO with lignin amine and its optical properties. Nanoscale Res. Lett. 2013, 8, 431. [CrossRef] [PubMed]
Khan, M.F.; Ansari, A.H.; Hameedullah, M.; Ahmad, E.; Husain, F.M.; Zia, Q.; Zaheer, B.M.R.; Alams, M.M.;
Khan, A.M.; Alothman, Z.A.; et al. Sole-gel synthesis of thorn like ZnO nanoparticles endorosing mechanical
stirring effect and their antimicrobial activity: Potential role as nano-antibiotics. Sci. Rep. 2016, 6, 27689.
[CrossRef] [PubMed]
Roy, J.S.; Majumder, T.P.; Dabrowski, R. Photoluminescence behavior of TiO2 nanoparticles doped with
liquid crustal. J. Mol. Struct. 2015, 1098, 351–354. [CrossRef]
Faixl, O.; Grunwald, C.; Beinhoff, O. Determination of phenolic Hydroxylic group content of Milled Wood
Lignins (MWUs) Different Botanical Origins Using Selective Aminolysis, FTIR, and UVSpectoscopy. Int. J.
Biol. Chem. Phys. Technol. Wood 1992, 46, 428.
Haque, F.Z.; Nandanwar, R.; Singh, P. Evaluating photodegradation properties of anatase and rutile TiO2
nanoparticles for organic compounds. Optik 2017, 128, 191–200. [CrossRef]
Shi, L.; Shen, H.; Jiang, L.; Li, X. Co-emission of UV, violet and green photoluminescence of ZnO/TiO2 thin
film. Mater. Lett. 2007, 61, 4735–4737. [CrossRef]
Chithra, M.J.; Sathya, M.; Pushpanathan, K. Effect of pH on Crystal Size and Photoluminescence Property by
Chemical Precipitation Method; Springer: New Delhi, India, 2015; Volume 28, p. 3.
Kim, B.Y.S.; Rutka, J.T.; Chan, W.C.W. Effect of Coumarate 3-zhydroxylase Down regulation on lignin
structure. Nanomed. N. Engl. J. Med. 2010, 363, 2434–2443. [CrossRef]
Akhtar, M.S.; Swamy, M.K.; Umar, A.; Abdullah, A.; Sahli, A. Biosynthesis and characterization of silver
nanoparticles from methanol leaf extract of Cassia didymobotyra and assessment of their antioxidant and
antibacterial activities. Nanosci. Nanotechnol. 2015, 15, 1–6. [CrossRef]
J. Compos. Sci. 2019, 3, 90
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
13 of 13
Rudramurthy, G.R.; Swamy, M.K.; Sinniah, U.R.; Ghasemzadeh, A. Nanoparticles: Alternatives against
drug-resistant. Molecules 2016, 21, 1–30. [CrossRef] [PubMed]
Lee, H.; Ryu, D.; Choi, S.; Lee, D. Antibacterial activity of silver- nanoparticles against Staphylococcus aureus
and Escherichia coli. Korea J. Microbiol. Biotechnol. 2011, 39, 77–85.
Mohamed, M.M.; Fouad, S.A.; Elshoky, H.A.; Mohammed, G.M.; Alaheldin, T.A. Antibacterial effect of gold
nanoparticles against Corynebacterium pseudotuberculosis. Int. J. Vet. Sci. Med. 2017, 5, 23–29. [CrossRef]
[PubMed]
Pacheco, G.J.; sánchez, M.E.; martínez, A.R.; Ruiz, F.; Jasso, M.E.C. Antimicrobial properties of
copper nanoparticles and amino acid chelated copper nanoparticles produced by using a soya extract.
Bioinorgan. Chem. Appl. 2017, 15, 17.
Naseem, T.; Farrukh, M.A. Antibacterial activity of Green synthesis of iron nanoparticles using lawsonia
inermis and gardenia jasminoides leaves extract. J. Chem. 2015, 2015, 7. [CrossRef]
Jesline, A.; John, P.N.; Narayanan, P.M.; Vani, C.; Murugan, S. Antimicrobial activity of zinc and titanium
dioxide nanoparticles against biofilm-producing methicillinresistant Staphylococcus aureus. Appl. Nanosci.
2015, 5, 157–162. [CrossRef]
Ren, G.; Hu, D.; Cheng, E.W.C.; Vargas-reus, M.A.; Reip, P.; Allaker, R.P. Characterisation of copper oxide
nanoparticles for antimicrobial applications. Int. J. Antimicrob. Agents 2009, 33, 587–590. [CrossRef] [PubMed]
Ismail, R.A.; Sulaiman, G.M.; Abdulrahman, S.A.; Marzoog, T.R. Antibacterial activity of magnetic iron
oxide nanoparticles synthesised by laser ablation in liquid. Mater. Sci. Eng. C 2015, 53, 286–297. [CrossRef]
[PubMed]
Agarwal, H.; Menon, S.; Kumar, S.V.; Rajeshkumar, S. Mechanistic study on antibacterial action of zinc oxide
nanoparticles synthesized using green route. Chemico-Biol. Interact. 2018, 286, 60–70. [CrossRef] [PubMed]
Singh, A.V.; Mehta, K.K.; Worley, K.; Dordick, J.S.; Kane, R.S.; Wan, L.Q. Carbon nanotube-induced loss
of multicellular chirality on micropatternedsubstrate is mediated by oxidative stress. ACS Nano 2014, 8,
2196–2205. [CrossRef]
Reddy, L.S.; Nisha, M.M.; Joice, M.; Shilpa, P.N. Antimicrobial activity of zinc oxide(ZnO) nanoparticle
against Klebsiella pneumoniae. Pharm. Biol. 2014, 52, 1388–1397. [CrossRef] [PubMed]
Huang, Y.; Wu, C.; Aronstam, R.S. Toxicity of transition metal oxide nanoparticles: recent insights from
in vitro studies. Materials 2010, 3, 4842–4859. [CrossRef] [PubMed]
Boeriu, C.G.; Bravo, D.; Gosselink, R.J.; van Dam, J.E. Characterisation of structure-dependent functional
properties of lignin with infrared spectroscopy. Ind. Crops Prod. 2004, 20, 205–218. [CrossRef]
Singh, A.V.; Ferri, M.; Tamplenizza, M.; Borghi, F.; Divitini, G.; Ducati, C.; Lenardi, C.; Piazzoni, C.; Merlini, M.;
Podest, A.; et al. Bottom- up engineering of the suface roughness of nanostructured cubic zirconia to control
cell adhesion. Nanotechnology 2012, 23, 475101. [CrossRef] [PubMed]
Padmavathy, N.; Vijayaraghavan, R. Enhanced bioactivity of ZnO nanoparticles—An antimicrobial study.
Sci. Technol. Adv. Mater. 2008, 9, 035004. [CrossRef] [PubMed]
Gosselink, R.; Snijder, M.; Kranenbarg, A.; Keijsers, E.; de Jong, E.; Stigsson, L.L. Characterisation and
application of NovaFiber lignin. Ind. Crops Prod. 2004, 20, 191–203. [CrossRef]
Janaki, A.C.; Sailatha, E.; Gunasekaran, S. Synthesis, characteristics and antimicrobial activity of ZnO
nanoparticles. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 2015, 144, 17–22. [CrossRef] [PubMed]
Nagajyothi, P.C.; Sreekanth, T.V.M.; Tettey, C.O.; Jun, Y.I.; Mook, S.H. Characterization, antibacterial,
antioxidant, and cytotoxic activities of ZnO nanoparticles using Coptidis rhizoma. Bioorg. Med. Chem. Lett.
2014, 24, 4298–4303. [CrossRef]
Tiwari, V.; Mishra, N.; Gadani, K.; Solanki, P.S.; Shah, N.A.; Tiwari, M. Mechanism of Anti-bacterial Activity
of Zinc Oxide Nanoparticle Against Carbapenem-Resistant Acinetobacter baumannii. Front. Microbiol. 2018,
9, 1218. [CrossRef] [PubMed]
Pesci, F.M.; Wang, G.; Klug, D.R.; Li, Y.; Cowan, A.J. Efficient Suppression of Electron–Hole Recombination
in Oxygen-Deficient Hydrogen-Treated TiO2 Nanowires for Photoelectrochemical Water Splitting. J. Phys.
Chem. C 2013, 117, 25837–25844. [CrossRef]
© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).