Research on Chemical Intermediates

https://doi.org/10.1007/s11164-018-3630-7

Preparation of NPK nanofertilizer based on chitosan

nanoparticles and its effect on biophysical characteristics
and growth of coffee in green house

Ngoc Minh Chau Ha1 · Thi Huyen Nguyen1 · San‑Lang Wang2 ·

Anh Dzung Nguyen1

Received: 10 July 2016 / Accepted: 23 August 2016

© Springer Nature B.V. 2018

Abstract
NPK nanofertilizer was prepared by loading nitrogen (N), phosphorous (P) and
potassium (K) into chitosan nanoparticles. The chitosan nanoparticles were prepared
via ionic gelation of tripolyphosphate and chitosan solution. The chitosan nanopar‑
ticles were characterized by SEM, TEM, zeta potential and size distribution. The
results showed that size distribution was from 300 to 750 nm and zeta potential of
around 50 mV. The released kinetics of nitrogen, phosphorous and potassium in
nanofertilizer were also investigated for 240 h. The nanofertilizer was applied to
coffee seedlings in a greenhouse condition. The results showed that the nanofer‑
tilizer enhanced uptake of nutrients, photosynthesis and growth of coffee plants.
Application of the nanofertilizer improved 17.04% nitrogen, 16.31% phosphorous
and 67.50% potassium content in the leaves of treated plots compared to the con‑
trol; total chlorophyll content increased up to 30.68% and 71.7% of photosynthesis
net rate. Application of nanofertilizer also enhanced leaf number, plant height and
leaf area of the coffee seedlings. Using the nanofertilizer may be a potential way to
enhance use efficiency of fertilizers for coffee.

Keywords Nanofertilizers · Chitosan nanoparticles · Coffee · Photosynthesis ·

Chlorophylls

* San‑Lang Wang
[email protected]
* Anh Dzung Nguyen
[email protected]
1
Institute of Biotechnology and Environment, Tay Nguyen University, Buon Ma Thuot 567,
Vietnam
2
Life Science Development Center, Tamkang University, New Taipei City 25137, Taiwan

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 N. M. C. Ha et al.

Introduction

Fertilizers are indispensible to crops, provide necessary nutrients for crop growth,
and increase crop yield and crop quality. The most important nutrients for almost
all crops are macronutrients such as nitrogen, phosphorous and potassium. Nowa‑
days, farmers have used much more chemical fertilizers to harvest higher yield
and enhance economical effeciency. However, macronutrients use efficiency for
crops is very low, such as use efficiency of nitrogen, potassium and phosphorous
is about 30–60%; 30–50% and 10–20%, respectively. It means that there are about
50–70% nitrogen and potassium, and 80–90% phosphorous fertilizer that could
not be taken up by crops and released to the environment. The result in this case
is that it does not only increase input of production, but also reduces economical
efficiency and enhances environmental pollution [1, 2].
Recently, there has been a new orientation to prepare and apply the nanoferti‑
lizers to enhance use efficiency of fertilizer, increase yield and quality of crops,
reduce harmful effects of chemical fertilizers on environment and develop a green
and sustainable agriculture [3–12]. Urea nanofertilizer prepared by coating urea
into nanofilm has been successful using it for Canola [13]. Nano nitrogen fer‑
tilizer carried out by coating urea with sulfur and nano nitrogen chelate were
effectively applied for potato to enhance yield and reduce nitrate leaching [12]. In
recent years, Complex NPK nanofertilizer has been studied by many researchers.
Wu and Liu [11] investigated loading NPK fertilizer into chitosan and coating
the outer by poly (acrylic acid-co-acrylamide). Complex NPK nanofertilizer was
prepared by trapping the fertilzer in polyacrylic hydrogel and invertigating the
NPK slow release control of the nanofertilizer [14]. Chitosan and chitosan nano‑
particles were applied as a useful matrix for loading nutrients for crops. Complex
NPK fertilizer was loaded into chitosan nanoparticles, and chitosan nanoparticles
with polymethacrylic acid (MMA) [15, 16].
The materials being used for preparation of nanofertilizer could be from
inorganic, organic, synthesis polymer, natural polymer, and grafted copolymers
between carbohydrate and synthetic polymer materials [4, 5, 9]. However, using
synthetic polymer materials to prepare nanofertilizers seems to have some dis‑
advantages such as their nonbiodegradable property that can be harmful to the
environment and public health. In addition, synthetic polymers are much more
expensive in producing nanofertilizer on an industrial scale.
Chitosan is a biopolymer of glucosamine and N-acetyl glucosamine residues,
processed from seafood wastes. Chitosans are non-toxic, biocompatible, biode‑
gradable, and friendly to the environment and have a great potential for agricul‑
tural application. Chitosan enhances growth and crop yield due to their bioactivi‑
ties to plants such as stimulating growth of plants, seed germination, enhancing
nutrient uptake, and antibacterial and antifungal activity [17–33]. Moreover, chi‑
tosan nanoparticles have also a positive effect on biophysical aspects and growth
of crops [34]. In our previous investigation, chitosan nanoparticles have effects on
photosynthesis, nutrient uptake and growth of coffee seedlings [34]. Therefore, in
this work chitosan nanoparticles were selected to use as nanomaterial for loading

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Preparation of NPK nanofertilizer based on chitosan…

NPK with the idea of applying both bioactive effects of chitosan nanoparticles
and NPK macronutrients to crops.
This work is to evaluate loading efficiency, slow release control of NPK from the
nanoparticles and their effects on biophysical characteristics and the growth of cof‑
fee seedlings in greenhouse conditions.

Experimental

Materials and Plants

Chitosan was purchased from Biosics, Canada. The degree of chitosan deacetyla‑
tion was approximately 80–90%, as determined by IR spectroscopy. The molecu‑
lar weight of chitosan was 130 cPs, determined via its viscosity (Brookfield
viscometer).
TPP (tripolyphosphate), acetic acid and ­KNO3 were high grade chemicals and
purchased from Merck (Germany).
In this work, the TR4 clones of coffee seedlings were purchased from Western
Highland Agricultural Scientific Technological Institute (WASI), Buon Ma Thuot,
Vietnam to make sure that all the plants are not different genetically.

Preparation of nanofertilizer

Chitosan nanoparticles were prepared according to a modified ionic gelation method

described by Gan et al. [35]. In brief, 0.1% chitosan solution was prepared by dis‑
solving 0.1 g chitosan powder in 100 mL of 0.35% (w/v) acetic acid solution and
kept overnight at room temperature. The chitosan solution was adjusted to pH 5.5
by 0.5 M NaOH solutions. 0.25% (w/v) TPP solution was dissolved in deionized
water. And then, chitosan nanoparticles were prepared by dropping 0.25% TPP solu‑
tion into the chitosan solution at a chitosan: TPP ratio of 6: 1 (w/w) while stirring at
900 rpm with a magnetic stirrer for 60 min at room temperature.
The nanofertilizer was prepared by adding 0.3% ­KNO3 into chitosan nanoparti‑
cles emulsion.

Particles size, zeta potential value and morphology of the nanoparticles

The morphology of the nanoparticles was investigated by field emission scanning

electron microscopy (FE-SEM; model JSM 7401F, JEOL, Ltd., Japan) at an accel‑
eration voltage of 15 kV. Zeta potential value and size distribution were determined
by a Zetasizer Nano ZS (Malvern, UK).

Slow released control of NPK from nanofertilizer

Chitosan nanoparticles were loaded with 0.3% K ­ NO3 (w/v) and studied for slow
release for 240 h. After every 24 h, 1 mL of the nanofertilizer emulsion was collected

13
 N. M. C. Ha et al.

and centrifuged at 15,000 rpm. The supernatant was measured for content of N, P
and K to determine kinetics of slow release of N, P and K elements. Total phospho‑
rous was measured by the colorimetric molybdate blue method by Spectrophotome‑
ter, Jasco, V630, Japan. Total potassium was determined by AAS (A7000, Shimazu,
Japan). Total nitrogen was determined by the micro Kjeldahl method (Velp, Italia).

Experiment for coffee seedlings in greenhouse

The experiment was conducted in the greenhouse of the Faculty of Agriculture, Tay
Nguyen University. The experiment was performed with six plots sprayed with water
for control, 10, 20, 30, 40 and 50 ppm of NPK nanofertilizer emulsion on two sides
of the leaves in triplicate and volume of 50 mL for 20 coffee seedlings of each plot.
Coffee seedlings were planted in black polyethylene bags (size 15 × 25 cm), consist‑
ing of Ferrasols mixed with 5% degraded manure. During the experiment, applica‑
tion of NPK nanofertilizer was used three times with period of 15 days. The irriga‑
tion control assured that water content in the soil of all plots were similar. Fertilizing
regime and the microclimate conditions were identical (average air temperature was
25 °C, humidity was 85% and light intensity was around 5000 lx). The growth of the
coffee seedlings was determined by height of the plants, and diameter of the stem.
The height of the coffee seedlings was measured by a ruler from ground to pinnacle
of the plants. Diameter of the stem of coffee seedlings was measured by Palmer.
Area of the leaf was determined by measuring length and diameter of the leaf by the
following equation:
S = K ⋅ a ⋅ b,
where S is leaf area; a and b are length and diameter of the leaf. K is 0.66 (specific
index of Robusta coffee leaf). All parameters of the growth of coffee seedlings were
measured for ten plants for each plot.
Parameters of photosynthesis such as CO2 concentration in cells, stomatal con‑
ductance, transpiration and photosynthesis intensity were determined by PPsystem,
PTS 2 (USA).

Plant analysis

After 8 weeks spraying the NPK nanofertilizer with three applications, the matured
leaves of the coffee seedlings were collected to analyze the nutrient content. The
leaves were dried at 105 °C in the convention oven until their weight was unchanged
and ground up for analysis. Total phosphorous was measured by the colorimetric
molybdate blue method following Kjeldahl digestion (UV Vis spectrophotometer,
Jasco V630, Japan). Total potassium, calcium and magnesium were measured by
AAS (A7000, Shimazu, Japan).
Total nitrogen of the leaves was determined by the micro Kjeldahl method
(Velp, Italia). Content of chlorophyll and carotenoid was determined from Yoshida
and Forno [36] as follows: The leaves were cut in small pieces and immersed in
80% acetone solvent to extract pigments. The extraction solvent was diluted and

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Preparation of NPK nanofertilizer based on chitosan…

measured with a spectrophotometer (UV Vis, Jasco V 630, Japan) at 663, 645 and
440.5 nm. The content of pigments was calculated by the equation:

Ca mg g−1 fresh leaf = 0.0127. D663 − 0.00269. D645;

( )

Cb mg g−1 fresh leaf = 0.0299. D645 − 0.00468. D663;

( )

Ccar mg g−1 fresh leaf = 0.004695. D440.5 − 0.000268 (Ca + Cb).

( )

where Ca, Cb (mg g−1 fresh leaf) are content of chlorophyll a and b; and Ccar
(mg g−1 fresh leaf) is content of carotenoid. D663, D645 and D440.5 are optical
density of the extract solution at 663, 645 and 440.5 nm.

Statistical analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA)

and followed by Duncan’s multiple range tests with triplicate by MSTATC software.
α ≤ 0.05 considered as significant.

Results and discussion

Characteristics of nanofertilizer prepared by loading NPK on chitosan

nanoparticles

Preparation of Chitosan nanoparticles by ionic gelation with TPP is a relatively

simple method, and cheap in expense. Therefore, it is a suitable method for agri‑
culture application. The morphology of chitosan nanoparticles, characterized by
SEM, TEM, size distribution and zeta potential value, affected the loading capacity
of NPK nutrients. The results shown in Fig. 1 indicated that chitosan nanoparticles
were a spherical surface with homogenous size distribution, stable and of fine shape.
The spherical shape of chitosan nanoparticles possesses the highest surface area that
is suitable for loading NPK nutrients on the surface of the nanoparticles. Sizes of
the chitosan nanoparticles were distributed from 300 nm to 750 nm and mean size
was around 500 nm (Fig. 2) and the zeta potential value of 50 mV. The higher zeta
potential value nanoparticles have more adsorption capacity of negative charge ions
such as ­NO3− and ­PO43− on their surface.
Chitosan nanoparticles, prepared by different methods such as ionic gelation
with TPP, spray drying, nanocomposite of chitosan with polyacrylic acid, poly‑
methacrylic acid…have different characteristics [4, 5, 7]. Moreover, the size dis‑
tribution of the nanoparticles also depended on molecular weight, concentration
and ratio of chitosan with other polymer and cross-linking agents. La et al. [25]
that reported that the size distribution of chitosan nanoparticles prepared by spray
drying is in a range of 95.5–395 nm and by spray drying combined with ionic
gelation was in the size distribution of 166–1230 nm with the zeta potential value
ranging from 34.9 to 59 mV [29]. In our previous work it was indicated that the

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 N. M. C. Ha et al.

Fig. 1  SEM of chitosan nanoparticles prepared by ionic gelation with TPP under condition: 0.1% chi‑
tosan solution, 0.25% TPP; Chitosan: TPP ratio 6:1 (w/w). The chitosan nanoparticle images were taken
by field emission scanning electron microscopy FE-SEM (left) model JSM 7401F, JEOL, Japan) at an
acceleration voltage of 15 kV. TEM (right) was taken by JEM-1400, JEOL, Japan

Fig. 2  Size distribution (left) and zeta potential (right) of the chitosan nanoparticles under condition:
0.1% chitosan solution; 0.25% TPP; chitosan: TPP ratio 6:1 (w/w). Zeta potential value and size distribu‑
tion were determined by a Zetasizer Nano ZS (Malvern, UK)

the medium size distribution of chitosan nanoparticles (average size of 750 nm)
had better effect on nutrient uptake, and growth of the coffee than bigger and
smaller sizes [34]. Recent works that reported that a smaller size of nanofertilzer
prepared by chitosan and polymethacrylic acid was distributed from 80 to 100 nm
[15, 37]. According to the review of Ghormade et al. [4], the size of nanopar‑
ticles applied for nanofertilizers, nanopesticides and nanoherbicides in agricul‑
ture fields was normally from 100 to 500 nm. Therefore, the mean size of nano‑
particles in this work was around 500 nm to be suitable for preparation of NPK
nanofertilizer.

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Preparation of NPK nanofertilizer based on chitosan…

NPK nanoparticles were prepared by loading a suitable amount of ­KNO3 solution

at pH 5.5 into chitosan nanoparticles. After loading, the total contents of N, P and
K nutrients in nanofertilzer were analyzed for the loading capacity. The results indi‑
cated that loading capacity of NPK in nanofertilizer was in the amount of 0.21% N,
0.0004% ­P2O5 and 0.116% ­K2O which was higher than that of other NPK nanofer‑
tilizers. Hasaneen [38] and Corradini [15] prepared NPK nanofertilizer by a combi‑
nation of chitosan nanoparticles with polymethacrylic acid and then loading urea,
calcium phosphate and potassium chloride into the chitosan nanoparticles. The
results indicated that the maximum N, P, K loaded into chitosan nanoparticles was
500 ppm N (0.05%), 400 ppm K (0.04%) and 60 ppm P (0.006%) which was lower
than current study works. It could be because the chitosan nanoparticles will pre‑
cipate at higher concentration [15, 16]. Wu and Liu [11] reported that a new NPK
fertilizer granular prepared by coating three layers of chitosan with poly (acrylic
-co- polyacrylmide) contained 7.98% ­K2O, 8.14% P ­ 2O5 and 8.06% N which was rel‑
atively high compared to current work and Hassaneen and Corradini’s works. How‑
ever, this NPK fertilizer in a granular shape was of a bigger size than nanoparticles.
Moreover, NPK fertilizers prepared by Wu & Liu and Hassaneen were synthesized
in nonbiodegradable polymers as polymethacrylic acid, polyacrylic acid and poly‑
acrylamide. Therefore, these NPK fertilizers can be harmful to the environment and
public health [11, 16].

Slow release kinetics of the NPK nanofertilizer

The slow release kinetics of NPK nanofertilizer were analyzed by immersing it in

pH 5.5 solution. The result of N, P and K slow release from the nanofertilizer is
shown in Table 1. It indicated that nitrogen nutrient ­(NO3−) was slowly released

Table 1  Slow release kinetic of the NPK nanofertilizer

Release time Slow release kinetic of N, P and K
(h)
Content of Relative Content of Relative Content of Relative
released nitro‑ release released phos‑ release released potas‑ release
gen (%) (%) phorous (%) (%) sium (%) (%)

0 0 0 0 0 0 0
12 0.014 6.6 0.00013 3.1 0.076 45.5
24 0.021 10.0 0.00012 3.0 0.064 50.1
48 0.028 13.3 0.00012 3.0 0.064 52.1
72 0.126 60.0 0.00011 2.6 0.062 55.4
96 0.126 61.0 0.00011 2.6 0.067 57.7
120 0.133 63.3 0.00011 2.6 0.065 56.0
144 0.133 63.3 0.00011 2.6 0.068 58.6
168 0.126 60.0 0.00011 2.6 0.067 57.7
192 0.133 63.2 0.00011 2.6 0.068 58.6
216 0.147 70.0 0.00010 2.6 0.067 57.7
240 0.140 66.7 0.00013 3.1 0.067 57.7

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 N. M. C. Ha et al.

from nanoparticles for the first 48 h with the relative release of 13.3%, and then
quickly increased up to 60.0% from 48 h to 72 h later. Afterward, the relative release
of nitrogen seemed to be unchanged with the release percentage from 60.0 to 63.2%
from 72 to 192 h and got the release of about 66.7% after 240 h. There was, accord‑
ing to the results of Chao et al., [21] indication that the release of nitrogen of NPK
fertilizer prepared by loading on the membrane of chitosan cross linked with suber‑
oyl chloride was 60% nitrogen after the third h. The results in this work were accord‑
ance with Wu and L ­ iu’s work (2008) that indicated that NPK released from chi‑
tosan nanoparticles was 15% by the third day and 75% by the 30th day, respectively
[11]. The slow release of nitrogen in NPK fertilizer could be explained due to the
ionic bond force between chitosan nanoparticles in positive charge (around 50 mV
as shown in Fig. 2) and nitrogen in negative charge (­ NO3−). The slow release kinet‑
ics of potassium ions (Table 1) indicated that the fast release of K from chitosan
nanoparticles was shown in the first 72 h, with the percentage of about 55.4% and
stable from 55 to 58% for 240 h later. These results were in accordance with Santos
[39]. In this work, chitosan nanospheres were prepared with montmorillonite and
their mean size was of 200 nm and then, loading with ­KNO3. The result showed that
release of K was also fast for first 3 days.
The slowest release kinetic amongst the NPK in naofertilizer was found in
phosphorous nutrient with the relative release of about 3% and unchanged for the
first 240 h. This result could be explained by content of phosphorous in the NPK
nanofertilizer coming from TPP which was cross-linked with N ­ H3+ residues in the
chitosan chain.

Effect of NPK nanofertilizer on biophysical characteristics and growth of coffee

seedlings in a greenhouse

NPK nanofertilizer was sprayed with different doses on the leaves of the coffee
seedlings in a greenhouse to evaluate its effect on nutrient uptake, photosynthesis
and growth of the coffee which are shown in Table 2. Results represented that NPK
nanofertilizer significantly enhanced nitrogen and potassium content in the leaves
of investigated coffee plants compared to that of control. Nitrogen content in the
leaves increased from 3.15 to 3.57% compared to 3.05% in the control and increase

Table 2  Effect of application Experiments Nutrients in the coffee leaf after 60 days of
dose of NPK nanofertilizer on experiment (%)
nutrient uptake of the coffee
N P K Ca Mg

NF 0 3.05 0.130 1.169 1.13 0.86

NF 10 3.29 0.139 1.809 0.94 0.76
NF 20 3.15 0.143 1.892 1.02 0.80
NF 30 3.57 0.147 1.959 1.15 0.74
NF 40 3.57 0.147 1.668 0.98 0.76
NF 50 3.43 0.143 1.386 0.87 0.75

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Preparation of NPK nanofertilizer based on chitosan…

in the doses trended to improving the uptake of nitrogen in the coffee leaves. Potas‑
sium content in the leaves increased from 1.169% in the control (NF0) to 1.959%
in the NF30 experiment. In contrast, uptake of phosphorous, calcium and magne‑
sium nutrients was not affected by spraying NPK nanofertilizer. The results shown
in Table 2 also indicated that suitable doses of NPK nanofertilizer for improvement
of nutrients uptake of coffee plant seedings in a green house was of 30 to 40 ppm.
The results in the current study are in accordance with some other works on using
chitosan nanoparticles and nanofertilizer [34, 37]. Using only chitosan nanoparticles
increased nutrient uptake as 9.8–27.4% N and 30–45% K [34]. Some works have
reported that chitosan may directly affect gene expression, metabolism and induce
many biological responses in the plants [3, 4]. These affects may directly or indi‑
rectly make an increase in uptake of nutrients in the leaves of plants. Ledezma-Del‑
gadillo [37] reported that using N nanofertilizer based on chitosan nanoparticles and
methacrylic acid possessed a higher N use efficiency compared to using free urea in
the control. Nitrogen content in the leaves of lettuce in the use of urea nanofertilzer
was the same as free urea in the control, but the amount of urea used in nanoferti‑
lizer accounted for only 16% of the control. It indicated that increasing uptake of
nutrients may be effected by both chitosan nanoparticles and N, P and K nutrients
loaded into chitosan nanoparticles.
Effects of the NPK nanofertilizer on photosynthesis of the coffee, the content
of pigments and data of photosynthesis process were investigated and shown in
Tables 3 and 4.
The results shown in Table 3 indicated that the nanofertilizer enhanced contents
of chlorophyll and carotenoids in the coffee leaves with an incease in chlorophyll a,
chlorophyll b and carotenoid of the percentage of 14.8–27.7% of, 14.7–35.2% of and
10.7–25.0%, respectively, compared to the control. These results may be due to the
improvement of the nutrients uptake of the nanofertilizer as shown in Table 2, and
in accordance with some previous works [28, 34]. Limpanavech [40] proposed that
chitosan can induce expression of chloroplast gene in plants that lead to the increase
in chlorophyll content of leaves [26].
Photosynthesis is the most important physiological process of plants; it affects
directly the growth, biomass, yield and quality of crops. Effect of NPK nanofertilizer
on data of photosynthesis is shown in Table 4 which indicates that the nanofertilizer

Table 3  Effect of NPK nanofertilizer on pigment content in the coffee leaves

Experiments The content of pigments in the coffee leaf (mg g−1 fresh leaf)
Chlorophyll a Chlorophyll b Total chlorophyll Carotenoids

NF 0 0.54 0.34 0.88 0.28

NF 10 0.66 0.43 1.09 0.33
NF 20 0.69 0.45 1.14 0.35
NF 30 0.69 0.46 1.15 0.34
NF 40 0.63 0.40 1.03 0.32
NF 50 0.62 0.39 1.01 0.31

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 N. M. C. Ha et al.

Table 4  Effect of NPK nanofertilizer on photosynthesis of coffee leaves in a greenhouse

Experiments Transpiration rate Stomatal Photosynthesis net CO2 concentration inte‑
(mmol m−2 s−1) conductance rate (µmol m−2 s−1) grated cells (µmol mol−1)
(mmol m−2 s−1)

NF 0 0.22 24 4.6 230

NF 10 0.28 27 4.8 221
NF 20 0.31 31 6.2 237
NF 30 0.38 37 7.9 340
NF 40 0.39 35 7.7 328
NF 50 0.37 28 7.0 302

had a remarkable effect on photosynthesis of coffee as photosynthesis net rate, ­CO2

concentration integrated cells, stomatal conductance and transpiration rate. All pho‑
tosynthesis data in Table 4 were increased when treating with NPK nanofertilizer.
Stomatal conductance improved from 12.5 to 54.1% of treated plots compared to
the control. Increase in stomatal conductance lead to the improvement of ­CO2 con‑
centration in integrated cells up to 31.3–47.8% of NF30, NF40 and NF50 plots.
These results can be explained that positive charge of chitosan nanoparticles and
potassium may increase osmosis pressure of stomatal cells that lead to improving
the opening of stomatal cells and stomatal conductance. As C ­ O2 gas is a very impor‑
tant component for photosynthesis reaction of plants, these results contributed to the
enhancement of the photosynthesis net rate of the coffee. Photosynthesis net rate
was of 4.6 µmol m−2 ­s−1 in the control and increased from 6.2 to 7.9 µmol m−2 ­s−1
(34.7–71.7%) in the NF20 to NF50 plots. In comparison to our previous work, the
results also showed that NPK nanofertilizer had a greater effect on the photosynthe‑
sis process of the coffee than only chitosan nanoparticles [34].
The affects of NPK nanofertilizer on the enhancement of nutrient uptake, chlo‑
rophyll content and photosynthesis of the coffee contributed to the improvement of
growth data as shown in Table 5.
The results shown in Table 5 indicate that before experiments, all growth data
such as leaf number, leaf area, and plant height and stem diameter were not signifi‑
cantly different between all plots but after 60 days of the application of the nanofer‑
tilizer, all growth data of the coffee were higher than the control. Leaf number was
of 3.07 in the control compared to 3.77 in the NF30 plot and leaf area dramatically
increased from 540.13 cm2 in the control to 793.36 cm2 in the NF30 plot. The dif‑
ference of leaf area and leaf number between plots was of significant difference
(α < 0.05) (see Table 5). Moreover, the plant height in the treated plots was from
36.13 to 40.27 cm which was higher than the 35.72 cm of the control. The applica‑
tion of nanofertilizer also increased stem diameter of the coffee up to 0.59 cm of
NF30 compared to 0.46 cm of the not treated coffee seedlings. The results shown in
Table 5 also indicate that the suitable dose of the nanofertilizer for the growth of the
coffee seedlings was of 30 ppm and with each two weeks for application.
In recent years, the effects of chitosan, chitosan nanoparticles and nanofertilizer
based on chitosan nanoparticles on growth and yield crops have been reported [4,

13
 Table 5  Effect of NPK nanofertilizer on the growth of coffee seedlings in a greenhouse
Experiments Growth data of coffee seedlings in a greenhouse

Before experiment After 60 days of application

2
Leaf number Leaf area ­(cm ) Plant height (cm) Sterm diam‑ Leaf number Leaf area ­(cm2) Plant height (cm) Sterm
eter (cm) diameter
(cm)

NF 0 2.27a 129.11a 31.97a 0.44a 3.07c 540.13d 35.72a 0.46b

Preparation of NPK nanofertilizer based on chitosan…

a a a a bc cd a
NF 10 2.73 132.13 31.73 0.47 3.13 565.29 36.13 0.51ab
a a a a bc bc a
NF 20 2.53 144.78 31.03 0.41 3.33 624.41 37.83 0.52ab
a a a a a a a
NF 30 2.33 151.83 30.70 0.41 3.77 793.36 40.27 0.59a
a a a a ab b a
NF 40 2.60 147.51 31.10 0.43 3.50 663.43 37.93 0.55a
a a a a abc bc a
NF 50 2.53 136.42 30.69 0.47 3.43 644.12 37.40 0.53ab
CV% 13.33 2.77 4.38 10.91 6.59 7.02 8.50 7.44

Superscripts a, b and c, d are ranged when compared with LSD 0.05 (Least Significant Difference at alpha 0.05). Different superscripts in the same columm are signifi‑
cantly different between the treatments at 5% level according to Duncan’s Multiples Range Test
CV% is coefficiency of variation

13
 N. M. C. Ha et al.

5, 12, 34, 37, 41, 42]. To enhance N use efficiency of nano nitrogen chelate, sul‑
fur coated nano nitrogen chelate and sulfur coated urea were applied for potatoes
in the field. The results indicated that potato yield increased by 56.10, 59.61 and
49.76%, respectively. Application of nano nitrogen fertilizer also reduced nitrate
leaching in potato to improve the quality [12]. Ledezma-Delgadillo [37] applied
nitrogen nanofertilizer based on chitosan nanoparticles and polymethacrylic acid.
Amount of nitrogen (urea) use was only 16% compared to free urea for lettuce. It
means that nanofertilizer can be slow release and this property leads to using ferti‑
lizer with higher efficiency [37]. Foliar application of chitosan oligomer with molec‑
ular weight of 2 kDa and concentration of 40 ppm increased the growth and yield of
coffee in the field [28]. Application of chitosan oligomer also improved the growth
and yield of peanut, soybean and other crops [7, 17].

Conclusions

Chitosan nanoparticles prepared by ionic gelation with TPP can be a potential

controlled slow release carrier to produce NPK nanofertilizer. Moreover, the NPK
nanofertlizer had a strong effect on biophysical characteristics such as nutrient
uptake, chlorophyll content, photosynthesis process and growth of the coffee seed‑
lings in a greenhouse. These results concluded that NPK nanofertilizer can be a
novel fertilizer for developing a green and sustainable agriculture in the near future.

Acknowledgements The authors express to thank Ministry of Education and Training, Vietnam sup‑
ported a grant for this work (Code: B2014-15-71) and a part of the grant by the Ministry of Science and
Technology, Taiwan (NSC 102-2313-B-032-001-MY3).

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