pubs.acs.

org/acsagscitech Article

Urea−Lignin/Chitosan Nanocomposite as Slow-Release

Nanofertilizer
Published as part of the ACS Agricultural Science & Technology virtual special issue “The Future of Agriculture
and Food: Sustainable Approaches to Achieve Zero Hunger”.
M. Latha, K. S. Subramanian,* D. Jeya Sundara Sharmila, K. Raja, S. K. Rajkishore, and T. Chitdeshwari

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ABSTRACT: Urea is a universal fertilizer, but its use efficiency hardly exceeds 30−35%, and more than 70% is lost to the
environment, causing eutrophication in aquatic systems. This research focuses on encapsulating urea molecules using a lignin-
chitosan composite. The nanocomposite was developed as a suitable carrier for entrapping nitrogenous fertilizer. Coconut coir
contains plenty of lignin, which was extracted using the organosolv process by using ethanol as a solvent. Organosolv lignin (OSL)
yielded spherical lignin nanoparticles (LNPs) via the solvent displacement method. Finally, a nitrogen source was loaded into the
chitosan/lignin nanocomposite fertilizer (lignorea), which consists of LNPs, chitosan, and a cross-linker. The nanocomposite was
characterized using PSA, SEM, TEM, UV−vis, FTIR, and XRD. The morphology of the OSL and LNPs was examined under SEM
(626 ± 40 nm) and TEM (26 ± 9 nm). The noncrystallinity of both lignin and crystallinity after entrapment in nanocomposite was
validated by XRD. The specific functional group pertaining to the lignin backbone had aromatic vibrations at 1511 cm−1 (OSL) and
1507 cm−1 (LNPs), and the presence of an amide peak after loading N was observed in FTIR. The lignin samples have their
corresponding spectral absorbance at 281 and 288 nm wavelengths obtained in UV−vis. The size and stability of the extracted OSL
and LNPs were verified by a particle size analyzer and zeta potential, respectively. The compatibility and interactions of the
developed nanofertilizer were confirmed with molecular modeling and simulation. The developed nanofertilizer has total nitrogen of
30−35% which slowly releases up to 15 days in the soil. The study clearly suggests that lignin with chitosan served as a perfect
template to hold and release N in a regulated pattern that collectively contributes to improved N use efficiency.
KEYWORDS: Organosolv lignin, lignin nanoparticles, nanocomposite, urea entrapment, slow release

1. INTRODUCTION nanofibers 12,13 were attempted and found effective in

The food requirement of the globe will continue to increase by
35−56% from 2010 to 2050, closely commensurating with
population growth. Such an increase in food production can be
achieved only through higher productivity using fertilizers.1
Among the fertilizers, nitrogen is a vital element for plant
growth and is primarily applied in the form of urea.2 Urea is a
widely used water-soluble nitrogenous fertilizer with 46%N
ratio that aids in photosynthesis and promotes plants with
green leafy growth. After the field application of urea, most of
N is lost in various forms due to its mobile nature. The N must
be controlled to make its use efficient.3 The nitrogen nutrient
is lost in processes such as ammonia volatilization, leaching,
and runoff, As much as 60% can be lost leading to poor
nutrient use efficiency and ultimately reducing crop yield.
Therefore, it is necessary to develop methodologies that could
aid in the smart release of nitrogen from urea with crop
demand.4 Nanotechnology holds a promise to enable atom-by-
atom manipulation that facilitates slow and regulated release of
nutrients; thereby nutrient use efficiencies can be improved
while ensuring environmental safety. Several approaches such
as the use of nanozeolites,5−7 intercalation of urea with
biochar,8−10 polymer coatings,4,11 and encapsulation using
improving the nutrient use efficiencies and productivity of
crops. The world’s first nanourea developed by IFFCO (Indian
Farmers and Fertilizers Cooperative) was released in 2021 and
widely adopted across India since then.14 The literature serves
as the base data to indicate nanotechnologies that have a
potential to improve the use efficiencies and reduce the
quantities of fertilizer used. The polymer coatings can prevent
N losses by controlling the release.
Polymers are widely used for the encapsulation of nutrients
to regulate the release and improve the nutrient use
efficiencies. In the encapsulation process, nutrients are loaded
in the core surrounded by polymers as sheaths. Recently,
multinutrients have been successfully loaded, and balanced
crop nutrition has been achieved through seed encapsulation
with nanofibers fortified with nutrients.15 A coated slow-release
Received: December 21, 2022
Revised: April 14, 2023
Accepted: April 17, 2023
Published: May 15, 2023

© 2023 American Chemical Society https://doi.org/10.1021/acsagscitech.2c00334

463 ACS Agric. Sci. Technol. 2023, 3, 463−476
ACS Agricultural Science & Technology
urea was developed with S/CO-based polyurethane inter-
penetrating polymer networks and was found to be an effective
coating material.16
The world is moving toward sustainable and renewable
sources to cut down the effects caused by environmental
hazardous substances. There is a growing demand for using
biodegradable resources in various fields that can reduce future
thrust to increase the economy of the nation. Many industries
use strong chemicals to process products and, in the case of
extraction, design, and value addition, may result in awful
conditions for the environment and wellbeing. By adopting the
12 green chemistry principles, these situations can be
avoided.17 The agro-residue wastes are important source of
lignocellulose that can be used to develop nanofertilizers.
Lignocellulose consists of cellulose, hemicelluloses, and lignin
in different proportions.18 Lignin is the second most abundant
biopolymer next to cellulose which imparts rigidity and
strength to the plant cell wall.19
Lignin has a crucial role in the construction of cell walls in
wood and bark, as they provide stiffness and resistance against
rot. Lignin fills the void present in the cell wall and is especially
found in xylem and vascular bundles, which helps in the
transport of water and nutrients through plant stem20 and
inhibits enzymatic degradation of other components.21
Structurally, lignin is a complex heterogenic aromatic polymer
made of three phenylpropane monomeric precursors, p-
hydroxyphenyl units (H), guaicyl units (G), and syringyl
units (S), with their corresponding monolignols, p-coumaryl
alcohol, coniferyl alcohol, and sinapyl alcohol, that undergo
polymerization to produce lignin.22 The lignin present in
hardwood mainly consists of G and S units with some traces of
H units. This lignin composition variability is much higher in
hardwood than in softwoods.23 Lignin derived from plant
sources was widely used in textile materials, polymer
composites, fabrics, binders, adhesives, additives, emulsifiers,
stabilizers, adhesives, fuel, energy, and agriculture due to its
less toxicity and versatility.
The major portions of agricultural lignocellulosic wastes are
bound to unexploited use of renewable sources. One of the
nonpreferred byproducts from paper and pulp industries after
the cellulose extraction is lignin, considered as a waste of over
50 million tons every year, only after 2% is utilized for making
products and the remaining is left as waste and burnt for
energy generation.24−26 The growing interest in waste
recycling probes the use of lignin which was generated from
the cellulose industry. Other than the paper industry, lignin is
found unused in many ways after product processing from
plants. Among them, the major agricultural output of coconut
is coconut oil, and its byproducts, like fiber, shell, pith, and
husk, are largely discarded as waste.27 Coconut (Cocos nucifera
L.) is one of the major plantation crops in India (Kerela,
Karnataka, and Tamil Nadu) accounting for 84.26% of the area
under cultivation.28 Apart from their food and feed sources,
coconut wastes are reutilized in one or another manner.29
Among the various lignocelluloses, coconut fiber has a higher
amount of lignin, on average 46% next to cellulose.30 The
lignin-based biodegradable polymers could mitigate environ-
mental issues and could eliminate resource shortfalls when
compared to mineral oil-based polymers.31 The extraction of
lignin from plants depends on the extraction process and the
chemical structure present in the sources. The delignification
process was classified as sulfur-bearing, and sulfur-free
extraction is based on the future goals. The sulfur-bearing
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lignin is toxic to the ecosystem and causes adverse effects. The
sulfur-free delignification process comprises organosolv and
soda lignin and results in higher purity. The use of organic
solvents (ethanol, methanol, acetone) or organic acids (acetic
acid/formic acid) for extracting the pulp gives the term
organosolv extraction. In this study, organosolv lignin was
extracted from coconut coir and characterized under PSA,
SEM, TEM, UV−vis, FTIR, and XRD. In the organosolv
extraction process, ethanol and hydrochloric acid were used as
solvent and acid catalysts. The adopted procedure for lignin
extraction was advantageous and generates more homogeneous
organosolv lignin from the coir.
Large-scale uses and industrial processing of lignin are
severely constrained by its insolubility in water. However,
lignin nanoparticle preparation has been demonstrated to be a
viable option.32 Currently, there is a ton of study being done
on the use of nanolignin. By manipulating the reaction
conditions of solvent/antisolvent, the concentration of lignin,
the temperature, and pH of the solution, etc., lignin
nanoparticles with various morphologies have been success-
fully synthesized.33 The lignin micro- and nanoparticles are
used in a wide range of material science applications, such as
drug delivery, the delivery of hydrophobic molecules, and as a
UV barrier, to enhance the mechanical, thermal stability,
barrier, antibacterial, and antioxidant properties of polymer
nanocomposites due to their less toxicity and biocompati-
bility.33,34 In recent years, many lignin-based nanoparticles
have been synthesized using a variety of techniques, including
solvent exchange, acid precipitation, cross-linking, self-
assembly, polymerization, ultrasonication, and CO2 antisol-
vent.35 In this study, using the solvent exchange method, lignin
nanoparticles were prepared, characterized, and used as
nanofiller in the experiment. Recently, de Azevedo et al.
(2021) have shown that the lignin derived from various parts
of coconuts has varying surface area (leaf 52; other parts 252
m2/g biomass). The surface area of lignin is important to use
as a template for the adsorption of nutrients and the regulation
of nutrients using another polymer, chitosan. It is hypothesized
that lignin derived from agro-wastes combined with chitosan
serves a nanocomposite to adsorb and desorb N in urea, and
such a process regulates the release of N from the composite
leading to the development of slow-release nanofertilizers. This
research encompasses extraction of lignin from coconut wastes,
design and fabrication of the nanocomposite, loading of urea,
molecular modeling to confirm the N loading, and release
kinetics of N.
2. MATERIALS AND METHODS
2.1. Materials. Coconut coir was collected from the local market
in Coimbatore, Tamil Nadu. Coir was cut and ground into powder
using liquid nitrogen. The chemical used were ethanol (99.9% purity),
and distilled water was used for the extraction and nanoparticle
synthesis process. Laboratory-grade hydrochloric acid (35%) was used
as a catalyst supplied by Molychem (Mumbai). For the nano-
composite preparation, the synthesized LNPs, commercial grade
chitosan was used with urea (46%N) and citric acid supplied by
Himedia.
2.2. Synthesis of OSL and LNPs. The organosolv procedure
utilized to extract lignin from coconut coir was a slightly modified
version of that in the literature.36−38 In the extraction process, the
solvents of 50% (v/v) ethanol and 1 mL of 35% hydrochloric acid
were added to the coconut coir, and taken in a round-bottom flask.
The mixture was boiled in a heating mantle with a refluxed condenser
setup for 5 h at 60 ± 10 °C. The resultant mixture was brought down
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ACS Agricultural Science & Technology
Figure 1. Graphical representation of (a) electrostatic cross-linking mechanism among lignorea components and (b) preparation of lignorea
nanofertilizer.
to room temperature and then filtered. The liquor was separated from
the spent extract through manually squeezing using a muslin cloth.
The spent was washed with 50% (v/v) aqueous ethanol (∼30 mL).
The filtrate along with washing was collected, and the liquor
containing lignin was precipitated using four times distilled water.
After the sedimentation of colloidal lignin for 24 h, it was centrifuged
and the pellet was collected. The pellet was oven-dried at 60 °C and
stored for further characterization.
The lignin nanoparticles were synthesized by self-assembly using
the solvent displacement method.39,40 The OSL was dissolved in
ethanol at the rate of 1%, and undissolved portions were removed by
filtering with Whatman filter paper grade no. 40. The buret containing
distilled water was added dropwise at the rate of 2 mL/min to the
filtrate under stirring. The synthesized nanoparticles were allowed to
settle down for 32 h and then centrifuged to collect the pellet. The
pellet was oven-dried at 50 °C for 1 h and stored for further
characterization.
2.3. Lignorea Preparation. The entrapment of urea in
nanocomposite was achieved by a cross-linking mechanism due to
the electrostatic attraction among the compounds. The binding takes
place as chitosan acts as binder and LNPs act as polymeric
nanoparticle. These two, made into a complex polymer, act as the
matrix material cross-linked with citric acid to hold urea (Figure 1).
The positive charged chitosan strand clutches all the negatively
charged ingredients to form nanocomposites. The chitosan being a
cationic polymer possesses positively charged linear polysaccharide
due to the presence of an amino group (N-acetyl-D-glucosamine).
This nature of chitosan enables the electrostatic behavior between
oppositely charged lignin molecules, thereby forming a matrix to hold
urea and providing a cross-linker for enhancing the ionic cross-
linkage.
Chitosan 1%, prepared by dissolving in 1% of glacial acetic acid by
constant stirring at 200 rpm, and citric acid were dissolved in distilled
water. Lignorea stock solution was prepared by mixing the 0.3% (w/
v) chitosan solution, LNPs 1.2−1.3% (w/v), and citric acid 0.025−
0.033% (w/v), and finally 25% (w/w) of urea was added. This
solution mix was stirred continuously for 2 h to obtain a
homogeneous mixture. The solution was poured in a plate and air-
dried to scraping consistency and pelletized using a mold with 0.5 cm
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diameter. The pellets were properly dried and stored for further
characterizations.
2.4. Characterization. 2.4.1. Particle Size Analyzer and
Stability. The lignin samples were evenly dispersed in water, and
the size of the colloidal particle was measured by the dynamic light
scattering method in a Horiba Scientific Nanoparticle SZ-100
(nanoparticle analyzer), Japan. The stability of the samples was
measured by zeta potential (Nanopartical SZ-100, Horiba Scientific,
Japan), giving the surface charge of the nanoparticles in the range of
−200 mV to 200 mV.
2.4.2. UV−Vis Spectroscopy. UV−vis spectra were recorded on
UV−visible spectroscope (model SPECORD plus 210 BU, Analytik
Jena AG, Germany). The extracted organosolv lignin and LNPs were
dissolved in the solvent at the ratio of 1:20 and were taken with
ethanol/water as a reference. Absorbance scan mode range from 200
to 800 nm at the rate of 5 nm per second was measured.
2.4.3. Fourier Transform Infrared Spectroscopy (FTIR). The FTIR
spectra of both lignins were recorded on FT/IR-6800 typeA equipped
with attenuated total reflectant unit (ATR Pro One) sensor. The
background spectrum was run initially to subtract it from the resulting
spectrum. The spectra were obtained by accumulating 64 scans at a
resolution of 4 cm−1 in an absorbance mode between 400 and 4000
cm−1.
2.4.4. X-ray Diffraction (XRD). Using a Cu Kα radiation source, the
sample XRD patterns were recorded between 10 and 90°, 2θ at 10°/
min, 40 kV/30 mA by powder diffraction method in X-ray diffraction
system (model Ultima IV, Rigaku, Japan). The organosolv lignin and
lignin nanoparticles and lignorea, each 1 g, were placed in the sample
holder and analyzed under the same experimental conditions. The
crystallite size was calculated using the Scherrer equation,
K
crystallite size (L) =
cos
2.4.5. Scanning Electron Microscope (SEM). The surface
morphology of samples was studied using SEM (Quanta 250, FEI,
and Netherlands). The samples were evenly dispersed in water and
sputter-coated with gold. The images were collected under E-SEM
mode at different magnifications.
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2.4.6. Field Emission Scanning Electron Microscope (FESEM).
The lignorea sample was analyzed in FESEM (model: Sigma with
Gemini Column, Carl Zeiss (USA)) with a resolution of 1.5 nm. The
lignorea pellet was vacuum-dried completely for 8 h to get rid of
moisture, and imaging was done with different magnifications.
2.4.7. Transmission Electron Microscope (TEM). Transmission
electron microscopy was performed on TEM FEI Technai Spirit
operated at 20−120 kV. The samples were evenly dispersed in water
dropped on a copper grid and incubated with enough time for drying
at room temperature.
2.5. Lignin Determination. The extraction of organosolv lignin
from coir was qualitatively determined by the Wiesner method
(phloroglucinol-HCl). The staining solution (5%) was prepared by
dissolving phloroglucinol-HCl in 2.5% ethanol. The raw coconut coir
and after-lignin-extracted coir (spent) were taken separately in a 15
mL glass vial, to which phloroglucinol stain was added (Figure 2).
Figure 2. Qualitative determination of lignin by Wiesner method
(phloroglucinol-HCl).
The above assay was incubated for 12 h in dark conditions to avoid
photodegradation of phloroglucinol. After incubation, both coirs were
examined using a light microscope and a scanning electron
microscope.
2.6. Lignin Yield. The percentage of lignin recovery through the
adopted organosolv extraction was calculated. The amount of lignin
recovered from 10 g coir using 50% ethanol concentration was
calculated with the formula,
wt of lignin in fraction (g)
lignin yield(%) = 100
wt of the initial biomass (g)
2.7. Nitrogen Content (%) Estimation. The amount of nitrogen
present in lignorea was estimated using the Kjeldahl method on a
KELPLUS-ELITE EX instrument (Pelican equipment). The lignorea
sample was taken and analyzed by the Kjeldahl method.41 The sample
undergoes digestion, distillation, and estimation by titrating with
sulfuric acid, giving the percentage amount of nitrogen present in the
sample. For this, 1 g of lignorea sample was powdered and dissolved
in 50% (v/v) ethanol and water. Then 50 mL of the solution was
taken in a Kjeldahl digestion tube, to which, a mixture of copper
sulfate and potassium sulfate was added as catalyst. The known
amount of concn H2SO4 was added, and the mixture was digested in
the sand bath until the development of an apple green color. Then,
the final solution was cooled, diluted with water, and filtered. Later, 10
mL of NaOH (40%) was added to the solution, and the mixture was
distilled up to 6 min. The ammonia vapors were collected into boric
acid that was kept at the delivery end. Finally, the solution was titrated
using N/10 sulfuric acid until an end point detection. The volume of
0.1N H2SO4 consumed for titration was noted and the amount of N
was calculated using the formula
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Total N(%) = [(normality of H2SO4 used for titration)
× (vol. of H2SO4 consumed for titration)
× (makeup volume)]
/[(quantity of solution taken for analysis)
× (g of sample taken for digestion)] × 100
2.8. Encapsulation Efficiency. The encapsulation efficiency of
urea entrapped in lignorea was estimated using standard graphs. The
p-dimethylamino-benzaldehyde (p-DMAB) colorimetric method42
was used to measure spectrophotometrically the amount of urea
loaded in the nanocomposite. In 100 mL of 95% ethyl alcohol and 10
mL of concentrated hydrochloric acid, the coloring ingredient (2 g)
was dissolved. A known concentration of urea solution (1 ppm to
1000 ppm) was made, and 1.5 mL of it was mixed to 1.5 mL of p-
DMAB coloring reagent. A UV−visible spectrophotometer was used
to measure the yellow-green color complex’s absorbance at 420 nm.
From the absorbance values, a straight line of standard graph was
plotted against urea concentration.
The lignorea of 100 mg was powdered and dispersed in 100 mL of
water, and the mixture was homogenized constantly with a magnetic
stirrer for 30 min @ 200 rpm under room temperature. Then 1.5 mL
of this solution was taken and mixed with 1.5 mL of p-DMAB
coloring reagent. Optical density of the samples was measured at 420
nm for urea. Water and DMAB mix were used as the reference. The
OD values were compared with the standard graph to arrive at the
respective concentrations. The encapsulation efficiency was calculated
using the given formula,
encapsulation efficiency(%)
amount of urea present in lignorea
= × 100
amount of urea initially added
2.9. Release Kinetics of Lignorea in Water. The p-
dimethylamino-benzaldehyde (p-DMAB) colorimetric method was
detailed in section 2.8. The lignorea of 100 mg was immersed in 100
mL of water and kept undisturbed for 75 days. For analysis, 1.5 mL of
this solution was mixed with 1.5 mL of p-DMAB coloring reagent.
Then, the solution was subjected to a UV−visible spectroscopy
method, and absorbance values were measured for 75 days at 24 h
intervals. By entering the absorbance of the sample against the
standard graph, the unknown urea concentration was calculated.
2.10. Nutrient Release Pattern of Lignorea in Soil. The
percolation reactor system was used to study the nitrogen release
pattern. It was made up of glass and was cylindrical in shape having 4
pore holes, with 2.5 and 15 cm in internal diameter and height,
respectively. Ten grams of soil and one gram of fertilizer (urea and
lignorea) were placed inside the reactor, and 50 mL of distilled water
was added from 1 to 20 days. The leachate solution was collected to
estimate the ammonium and nitrate ions as per the water quality
standard protocol.43 By conducting this experiment, the slow-release
pattern of nitrogen from lignorea was estimated.
2.11. Molecular Simulation. The molecular dynamics compu-
tations were performed with the NAMD version-2.11 for Linux-
x86_64-multicore package that includes the CHARMM36 force field.
The trajectory investigations and graphical representations were
realized by means of VMD tool. The potential energy graph illustrates
the energetically stable nature, and the root mean square deviation
(RMSD) profile explains the equilibration nature of the compound
throughout the 100 ns MD simulation. The generation of hydrogen
bonds is one of the major steps in analyzing the energy of a system
which forms bonds between acceptor and donor.
3. RESULTS AND DISCUSSION
3.1. Lignin Determination. One of the most widely used
qualitative methods for determining lignin through color stain
is the Wiesner test. It elucidates the presence or absence of
lignin in the development of stains.44 Lignin’s cinnamaldehyde
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Figure 3. SEM image of before (A) and after extracting lignin (B).
end groups react with phloroglucinol-HCl to produce a
reddish-violet hue.45 reported the procedure for this experi-
ment in a beginner’s guide to the study of plant structure, and
insisted that the red color develops due to the presence of
lignin. In this study, the raw coir developed strong dark brown-
red color compared to spent where the color intensity was
reduced due to the removal of lignin. This result was further
supported by SEM. The SEM images displayed the integrated
structure of the coir without any damage (Figure 3A) whereas
in Figure 3B, after the removal of lignin, the structure
disintegrates, collapsed exposing the pits indicating the lignin
was removed effectively. The results were similar to that in ref
46 where the tyloses present on the parenchyma surface and
residual lignins were removed, and thereby the fibrils lose the
compactness in the coir.
3.2. Lignin Yield. Coir has been widely found as
unexploited waste material in South India. In this research,
the green solvent was used for the lignin extraction and
characterized. The percentage of lignin recovery through
adopted organosolv extraction was calculated. The percentage
of lignin recovery from 50% ethanol concentration yields about
29% of total lignin.
The organic solvent used here was ethanol which extracts
both water-soluble and oil-soluble compounds as it has both
polar and nonpolar groups which make it able to extract both
groups. It is a good solvent for polyphenol extraction, and it is
less toxic and cheaper than other organic solvents.47 A study
on the effects of different ethanol concentrations on organo-
Figure 4. Particle size of OSL and LNPs (left) and zeta potential of OSL and LNPs (right).
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solv lignin precipitation performed by the authors of ref 37
reported that when the ethanol content declined, lignin
recovery enhanced. The authors of reference 48 reported
that lignin with good yield, purity, and solubility can be
extracted using a relatively safe and fast method with ethanol−
water mixtures. The cleavage of lignin was favored by
nucleophilic solvents, water and ethanol can dissolve the
aryl−aryl and ether fragments. On increasing water in
nucleophilic agents, the capability of solvent for delignification
decreases due to the hydrophobicity of the lignin polymer.49
Three factors primarily influence lignin solubility in solvents
and lignin aggregation: (i) hydrogen-bonding interaction; (ii)
π−π interactions between the aromatic structure of solvents
and the aromatic compounds of lignin; (iii) Brønsted acidic
protons; (iv) van der Waals forces, and chain entangle-
ment.50,51
3.3. Characterization Results. 3.3.1. Particle Size
Analysis and Zeta Potential. The size and polydispersity
index of extracted organosolv lignin were measured by
dynamic light scattering and recorded at 1229.7 nm size
(83.7% of particles) with 0.75 distributions due to agglomer-
ation of particles. The lignin nanoparticles recorded 330 nm
size (49.9% of particles) with 0.5 PDI. The surface charge of
lignin was −44 mV (OSL) and −45.6 mV (LNPs), showing
the negative charge and good stability (Figure 4). Due to the
surface charge caused by the hydroxyl groups and carboxyl
groups, electrical double layers may have formed a negative
potential with good stability.52
3.3.2. Scanning electron microscope (SEM) and trans-
mission electron microscope (TEM). The SEM pictures show
that the surface morphology of the extracted organosolv lignin
and synthesized lignin nanoparticles have changed in their size
as a result of the conversion process. Figure 5A and 5B were
taken at 20,000× magnification showing that the particles were
spherical in shape. The size of the OSL was 626.7 nm to 1.078
μm (Figure 5A), whereas the lignin nanoparticle showed sizes
in the range of 189.4 nm to 335.1 nm (Figure 5B). A
comparison between the structure of organosolv lignin and
synthesized lignin nanoparticles showed that the surface of
nanoparticles has a hollow cavity on it, whereas it was not
observed in the macroscale lignin. The form of the hole in each
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Figure 5. Scanning electron microscope image of organosolv lignin (A) and lignin nanoparticles (B). SEM image of lignorea (C and D) and
FESEM of lignorea (E and F).

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Figure 6. Transmission electron microscope image of organosolv lignin (A) and lignin nanoparticles (B).
Figure 7. UV−vis absorbance spectra of organosolv lignin and lignin
nanoparticles.
hollow nanosphere is produced by a pressure gradient between
the layer of the membrane at a two-phase interface between
water and ethanol.53 This occurs due to lignin molecules being
redistributed in an ethanol/water mixture as per their polarity
and hydrophobicity during a phase separation process that
occurs in the creation of lignin nanoparticles. Due to their poor
capacity to dissolve in water, the most hydrophobic
components of the lignin molecules are probably formed
first. The most polar molecules persist solvated in the lignin/
ethanol solvent when too much water is added, and as the
solvent evaporates, they form a low-density layer (Figure 6A)
that is adsorbed onto the surface of the nanoparticle.40
The pellets having entrapped urea was viewed under SEM,
and images clearly showed the rod structure of urea formed by
crystallization. The rods on the surface and in cross section
confirmed the presence of urea entrapped in composite
(Figure 5C and 5D). The lignorea pellets were viewed under
FESEM for in depth analysis, and images clearly showed the
presence of spherical LNPs (marked in circle) in the
nanocomposite along with entrapment of urea (Figure 5E
and 5F).
The middle and shell of the lignin are clearly contrasted in a
transmission electron microscopy image of OSL showing the
size in the range of 356 nm to 187 nm (Figure 6A), providing
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additional confirmation that they are hollow (doughnut-like)
with a nanoparticle size in the range of 26 nm to 36 nm
(Figure 6B). The morphological structure of developed LNPs
was in line with that in refs 40 and 54 where the synthesis was
controlled by initial lignin concentration, water dropping
speed, and stirring rate.
3.3.3. UV−Vis Spectroscopy. The UV−vis spectroscopy
results showed the minima and maxima position of absorbance
spectra for the organosolv lignin and its nanoparticles based on
separation from coconut coir, purity, and molecular weight.
UV−vis spectral characterization (Figure 7) showed the least
specific absorbance spectra at 281 nm for oragnosolv lignin due
to its heterogeneous molecular structure and trace fractions of
coir compounds. The lignin nanoparticles synthesized from
organosolv lignin have a maxima absorbance peak at 288 nm
due to the high organo-solvent solubility and high purity.
These two specific absorbance spectra were prompted from the
nonconjugated phenolic groups present in the organosolv
lignin and lignin nanoparticles synthesized from the acid-
catalyzed organosolv treatment of coir. A similar kind of results
was updated in ref 55 where the authors found that organosolv
lignin extracted from wheat straw has an absorbance peak at
274 nm due to corresponding nonconjugated phenolic groups
present in lignin. The synthesized lignin nanoparticles have a
higher absorbance coefficient than organosolv lignin because
the nanoparticles were devoid of nonlignin traces like ash,
polysaccharides, and proteins by complete hydrolyzation
during the synthesis process. The resulting wavelength is in
coordination with that in ref 56 where the author reported that
unconjugated guaiacyl and 3,4-dimethoxy-phenyl model
compounds have narrow absorption minima that occur in
the 277−282 nm wavelength region.
3.3.4. Fourier Transform Infrared Spectroscopy (FTIR).
The main functional groups and structural changes resulting
from the extraction of organosolv lignin and synthesis of lignin
nanoparticles were studied by FTIR (Figure 8A). The peak
analysis was navigated based on a previous report.30 The peak
difference in the fingerprint region of lignin was compared and
presented in (Table 1). The OSL and LNPs samples have the
stretching of O−H groups at 3326 and 3356 cm−1; stretching
of methyl C−H groups at 2978 and 2977 cm−1; and methylene
C−H groups stretch at 2857 and 2859 cm−1, respectively. The
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Figure 8. FTIR spectra of (A) coir, organosolv lignin, and lignin nanoparticles; (B) raw compounds and nanocomposite fertilizer.
significant region for detecting the core structure of lignin was
between 2000 and 500 cm−1. The stretching vibrations of ester
carbonyl C�O groups were found in organosolv lignin at
(1734 and 1148 cm−1) and nanoparticles at (1737 and 1162
cm−1). Both lignin samples have peaks related to lignin
backbones like an aromatic skeleton ring C�C stretching
between 1500 cm−1 and 1600 cm−1 and aromatic C−H
deformation at around 1424 and 1413 cm−1. The intensities of
the lignin fingerprint were observed to be higher in the lignin
nanoparticles spectra than in the organosolv lignin due to the
absence of nonlignin contents. The presence of carbohydrate
residuals in the organosolv lignin samples shows less intensity
of characteristic lignin peaks.57 The transmittance bands of
syringyl and guaiacyl C−O stretch were raised in the region of
1210 cm−1 to 1300 cm−1. The bands found at 1124 and 1112
cm−1 were from the C−H group deformation of aromatic
guaiacyl units. The signals at 881 cm−1 and 873 cm−1 came
from the C−H deformation of syringyl units. The O−H
deformation of OSL and LNPs existed at 1027, 1034, 1354,
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and 1329 cm−1. The coir sample has all the spectra of lignin
and cellulose components. The FT-IR spectra of different
lignin samples were compared and found with high similarity
in peaks, less transmittance difference, and structure.58
The functional groups present in lignorea and its
components were analyzed by scanning the samples under
FTIR. The lignin nanoparticle has the intense peaks at 1267
and 1211 cm−1 due to the vibrational stretching of syringyl and
guaiacyl C−O, deformation of aromatic syringyl and guaiacyl
C−H at 873 and 1112 cm−1, stretching of aromatic methoxyl
and methyl C−H at 2977 and 2859 cm−1, and phenolic and
aliphatic alcohol O−H stretching at 3356 and 1034 cm−1, and
aromatic C−H and phenolic OH deformation were observed
at 1413 and 1329 cm−1, respectively. The characteristic peaks
of LNPs were discussed above. The spectral scanning of
chitosan confirmed its characteristic peaks at 1645, 1590, and
1525 cm−1 due to the stretching of primary amide C�O and
primary amine NH bends and also showed the presence of
functional group sharp peaks at 988 cm−1 (free amino group
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Table 1. FTIR Peak Wavenumbers of Coir, Organosolv Table 2. 2θ Intensity Peaks of OSL, LNPs, and Lignorea
Lignin, and Lignin Nanoparticles from X-ray Diffraction
organosolv lignin OSL LNPs Lignorea
vibrations coir lignin nanoparticles
2θ mean size 2θ mean size 2θ mean size
Phenolic and aliphatic OH 3346 3326 3356 (deg) (nm) (deg) (nm) (deg) (nm)
stretch
22.24 352.48 21.24 120.65 22.54 31.14
Methyl CH group stretch 2972 2978 2977
21.40 211.19 21.14 179.66 29.60 28.00
Methylene CH group stretch 2866 2857 2859
21.36 422.35 21.04 179.63 24.90 28.48
Unconjugated C�O stretch 1736 1734 1737
21.32 211.16 21.04 118.99 35.78 23.56
Aromatic C�C stretch 1597 1596 1587
Aromatic C�C stretch 1520 1520 1507
Aromatic C−H deformation 1428 1424 1413
were confirmed by FTIR spectral scanning. The spectral
Phenolic OH group deformation 1361 1354 1329
scanning of lignorea confirmed the entrapment of urea the in
Syringyl C−O stretch 1278 1284 1267 chitosan/lignin nanocomposite by showing the corresponding
Guaiacyl C−O stretch 1224 1224 1211 functional group peaks of urea at 3442 cm−1 (N−H stretching)
Ester C�O stretch 1159 1148 1162 and 1684 cm−1 (stretching of C�O). The presence of the urea
Aromatic guaiacyl C−H 1113 1124 1112 amide peak was confirmed by refs 62 and 63, where it was
deformation reported that N−H stretching and C�O regions were
Aliphatic O−H stretch 1030 1027 1034 observed after loading urea in hydroxyapatite nanoparticles.
Aromatic syringyl C−H 883 881 873 A similar study also reported the presence of C�O stretching
deformation vibrations (1667 cm−1) corresponding to urea in layered
double hydroxides and montmorillonite clay nanohybrid
−NH2), 1154 cm−1 (C−O−C stretching), 1373 cm−1 (C−O structures, confirmed by FTIR spectra. The presence of lignin
stretching of starch), 1422 cm−1 (C−H bending), 2850 cm−1 groups in lignorea was confirmed by its characteristic peaks
(CH2 stretching), and 3304 cm−1 (OH stretching). A similar with a slight shift at 1508 cm−1 (aromatic C�C stretching),
kind of functional groups for standard chitosan was reported in 1410 cm−1 (aromatic C−H deformation), 1289 cm−1 (guaiacyl
ref 59 in which the authors observed the peaks for NH bending C−O stretching), and 1176 cm−1 (ester C�O stretching) and
(711 cm−1), C−O−C stretching (1174 cm-1), CH2 stretching 2979 cm−1 (aromatic C−H stretch). Lignorea have the
(2865 cm−1), OH stretching (3564 cm−1), and amide C�O chitosan peaks at 2861 and 998 cm−1 due to the stretching
group stretching (1604, 1598, and 1592 cm−1). The peaks at of aromatic C−H and free amino group −NH2. The peak at
3286 cm−1 (O−H stretching), 1728 cm−1 (C�O stretching), 783 cm−1 was due to the rocking of CH2 of citric acid, and
1108 cm−1 (C−OH stretching), and 781 cm−1 (CH2 rocking) 3394 cm−1 (O−H stretching) was the common functional
were attributed to the vibration of citric acid molecules.60 The group present in the components of lignorea. The spectral
functional groups of citric acid were confirmed by analyzing scanning of lignorea confirmed the presence of urea in the
under FITR which showed its respective peaks at 3290.12 composite nanocarrier by showing its respective component
cm−1 (−OH stretch), 1721.69 cm−1 (C�O stretch), 1105.85 peaks along with slight shifting in the functional molecule
cm−1 (C−OH stretch), and 778.78 cm−1 (CH2 rocking). FTIR vibration (Figure 8B).
spectra of urea showed the peaks at 3434 cm−1 (N−H 3.3.5. X-ray Diffraction (XRD). OSL presents the mean size
stretching), 1677 cm−1 (stretching of C�O), 1594 cm−1 (N− domains in the range of 211 nm to 422 nm with the maximum
H deformation), and 1464 cm−1 (C−N stretching).61 2θ intensity peaks, whereas in LNPs the mean size is further
Reported peaks like N−H stretching, C�O stretching, and reduced to 118 nm. Even though the mean differences were
C−N stretching corresponding to the urea functional groups observed, two forms of lignin exhibit amorphous structures, as

Figure 9. (A) XRD spectra of organosolv lignin and lignin nanoparticles. (B) XRD spectra of lignorea.

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Figure 10. Release kinetics of urea from lignorea in water.
Figure 11. Desorption of ammonium and nitrate nitrogen on lignorea.
per XRD (Figure 9) as was also confirmed in refs 64 and 65. As
lignin is a biopolymer made of amorphous phenyl propylene
units, crystallinity decreases with increasing lignin content.
Their corresponding maximum 2θ intensity peaks are
tabulated with their mean size (Table 2) calculated by
Scherrer’s equation. The curves were analyzed using the
software Origin 8.5.
Nanolignin was amorphous in nature due to its phenyl
propane units, whereas urea was crystalline in nature. The
crystalline peaks were observed later in the entrapment of urea
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in the nanocomposite (Figure 9B). The amorphous nature
disappears after the addition of urea and peculiar diffracted
crystalline peaks of urea appeared in nanocomposite after
loading nitrogen at 22.54°, 29.60°, 24.90°, 35.78°. The specific
intensity peaks of lignorea were not clear due to the masking of
urea crystalline peaks.
3.4. Nitrogen Content (%) Estimation. The total N
present in the developed lignorea was 30−35%, estimated
through KELPLUS instrument. Similarly, ref 66 estimated total
N in chars before and after loading N using the Kjeldahl
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Figure 12. Multiplot potential energy profile.
Figure 13. RMSD profile.
method. Ref 7 reported the nitrogen content was higher in
nanozeourea (28%N) compared to zeourea.
3.5. Encapsulation Efficiency. Encapsulation efficiency of
urea in nanocomposite was analyzed in UV−vis spectroscopy
to quantify the amount of urea loaded. The optical density
value recorded for each concentration was compared with the
standard graph, and the loading efficiency was computed. The
urea loading in lignorea had the entrapping efficiency value of
76%.
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Figure 14. Hydrogen bond analysis.
3.6. Release Kinetics of Lignorea in Water. The amount
of urea released from lignorea in water was determined
spectrophotometrically using the (p-DMAB) colorimetric
method. For this, the mix of urea released solution and
DMAB solution was subjected to a UV−visible spectroscopy
method, and absorbance values were measured. On the first
day of the experiment, 31.71% (277.8 ppm) of urea was burst
released for lignorea. Similarly, 90.10% (789.48 ppm) of urea
was released gradually up to the 75th day from lignorea. The
urea release from lignorea in water was extended up to 72 days
and gets saturated from the 75th day (Figure 10). Similar study
was reported by the authors in ref 67 in which the entrapped
urea in kaolinite nanocomposite released about 87% up to 150
h.
3.7. Percolation Reactor Study. The release of NH4+-N
and NO3−-N from urea and lignorea was represented in Figure
11. On the first day of the experiment, 64.9 and 39.7% of
NH4+-N was released from urea, and lignorea, respectively.
The leachate solution collected from the setup had 2719 and
1665.4 mg L−1 of NH4+-N and 489 and 256 mg L−1 of NO3−-
N in urea and lignorea, respectively. A similar kind of nitrogen
release pattern in nanoporous zeolite-based N fertilizer was
earlier reported by ref 68. Irrespective of NH4+-N and NO3−-
N, the N release from urea was ended within 5 days, but
lignorea released N slowly up to 15 days and ceased. Thus, the
data collected from the percolation reactor experiment strongly
recommended that the lignorea is a potential nitrogen fertilizer
for steady and prolonged release of N in soil. A parallel kind of
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Figure 15. Typical 2D and 3D view of lignorea and its components after MD simulation (red, chitosan; pink, nanolignin; green, citric acid; navy
blue, urea; turquoise, water).
nutrients slow-release pattern from nanocarriers was reported
by refs 6, 9, and 69.
3.8. Molecular Simulation. The stability of lignorea was
reflected in the potential energy profile, where a decrease in the
potential energy indicates the firmness of the nanocomposite
(Figure 12). The RMSD profile (Figure 13) gives the deviation
of compounds from its initial frame. In the water environment,
the composite component citric acid readily dissolves and
deviates in a plateau. The chitosan and nanolignin are
hydrophobic and less deviated, wherein nanolignin deviates
step by step which helps in holding the active molecule. The
RMSD of urea clearly shows the highest deviation from its
initial frame in the range of 30−58.8 Å which indicates that the
urea is released from the rigid nanocomposite. The primary
wetting of nanolignin (marked in box) is responsible for the
burst release in lignorea. The later sustained release was
observed due to chitosan, nanolignin, and citric acid cross-
linking with urea. The urea interaction was sustained in the
range of 108−132 numbers (Figure 14). The MD simulation
trajectory of the designed nanofertilizer system resulted in the
remarkable development of a nanocomposite in which urea
was not only firmly enclosed but also compatible with all other
components (Figure 15). The similar molecular docking
results were reported by70 for nanoemulsion in which medium
chain triglyceride and sorbitan monolaurate at definite
quantities would increase the encapsulation of the keto-
Curcumin in protein through its specific molecular inter-
actions.
■ CONCLUSION
The focus of this study is to develop nanofertilizer, for which
lignin was extracted and converted to nanolignin from biomass
waste using an eco-friendly safe procedure. In this study, the
results showed that an organosolv process optimized with 50%
(v/v) aqueous ethanol can extract lignin from coir yielding
about 29% recovery. Later, the extracted organosolv lignin was
converted to lignin nanoparticles by a solvent shifting method
where water was used as an antisolvent to improve the
functional properties. The characterization results confirmed
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the lignin and their structural morphologies. Despite these
interesting and promising results, this basic methodology of
extraction can be improved to scale-up the yield with a reactor-
oriented extraction process to cope with wide applications.
Second, the developed nanocomposite successfully entrapped
nitrogen at 30−33.5% and was characterized. Molecular
simulation results confirmed the compatibility among the
components along with the sustained release of the nutrient.
The results from the MD model confirm the simple solution
behavior of chitosan, citric acid, and urea with confirmed
number of hydrogen bonds of approximately #250nos, #50nos,
#100nos, respectively, when they are not involved in the
encapsulation. However, it is interesting to note the initial
bulging of nanolignin due to the absorption of H2O molecules
and a greater number of hydrogen bonds, approximately
#320nos, i.e., sustained at #280nos throughout the trajectory.
Besides the bulging of nanolignin, it releases the urea not only
with the initial burst (#460nos) but also for the control and
sustained release (#418nos). Hence the encapsulation of urea
in lignorea enhances the presence of the nutrients (urea) in the
aqueous medium by 98%. The developed urea-chitosan/
nanolignin based slow-release fertilizer would solidly improve
nutrient use efficiency and crop growth. It also overcomes the
demerits of conventional fertilizer by minimizing nutrient loss
through encapsulation and increased use efficiency which
enhances the crop productivity and yield. The urea loading
efficiency of 76% percent was observed in lignorea. Urea
released rapidly in water at about 277.8 ppm (31.71%) at the
first day of experiment and released 90.10% (789.48 ppm) at
the end of experiment, and the remaining urea was bound
inside. The urea release from lignorea in water was extended
up to 72 days and was saturated from the 75th day. Lignorea is
capable of releasing N up to 15 days, whereas conventional
urea takes over a period of 3 days to release N in soil. In order
to validate nutrient absorption, it is necessary to undertake
trials on various crops under controlled environments, as well
as on- and off-farm investigations.
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■ AUTHOR INFORMATION
Corresponding Author
K. S. Subramanian − Centre for Agricultural Nanotechnology,
Tamil Nadu Agricultural University, Coimbatore 641003,
India; Email: kss@tnau.ac.in
Authors
M. Latha − Centre for Agricultural Nanotechnology, Tamil
Nadu Agricultural University, Coimbatore 641003, India;
orcid.org/0000-0002-5254-091X
D. Jeya Sundara Sharmila − Centre for Agricultural
Nanotechnology, Tamil Nadu Agricultural University,
Coimbatore 641003, India
K. Raja − Anbil Dharmalingam Agricultural College, Tamil
Nadu Agricultural University, Trichy 620027, India
S. K. Rajkishore − Agricultural research station, Tamil Nadu
Agricultural University, Bhavanisagar 638451, India
T. Chitdeshwari − Department of Soil Science & Agricultural
Chemistry, Tamil Nadu Agricultural University, Coimbatore
641003, India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsagscitech.2c00334
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We gratefully acknowledge the support from IFFCO, India, for
funding the project functioning at the Centre for Agricultural
Nanotechnology, Tamil Nadu Agricultural University, Coim-
batore. We also acknowledge PSG iTech for FESEM analysis.
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