Effect of Citric Acid Surface Modification on Solubility of

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Effect of Citric Acid Surface Modification on Solubility of Hydroxyapatite Nanoparticles Ranuri Samavini, Chanaka Sandaruwan, Madhavi de Silva, Gayan Priyadarshana, Nilwala Kottegoda, and Veranja Karunaratne J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05544 • Publication Date (Web): 22 Mar 2018 Downloaded from http://pubs.acs.org on March 22, 2018

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Effect of Citric Acid Surface Modification on Solubility of Hydroxyapatite Nanoparticles

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Ranuri Samavini,1 Chanaka Sandaruwan,2 Madhavi De Silva,1, 2, 3 Gayan Priyadarshana,2 Nilwala

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Kottegoda,*1,2, 3 Veranja Karunaratne2

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1 Department of Chemistry, University of Sri Jayewardenepura, Nugegoda, Sri Lanka.

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2 Sri Lanka Institute of Nanotechnology (SLINTEC), Nanotechnology and Science Park,

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Center for Excellence in Nanotechnology, Mahenwatte, Pitipana, Homagama, Sri Lanka. 3 Center for Advanced Materials Research (CAMR), Faculty of Applied Sciences,

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University of Sri Jayewardenepura, Nugegoda, Sri Lanka.

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*[email protected], [email protected], Tel: + 94 11 4650509, Fax: + 94 11 4741995

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Abstract

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Worldwide, there is an amplified interest in nanotechnology based approaches to develop

12

efficient nitrogen, phosphorous and potassium fertilizers to address major challenges pertaining

13

to food security. However, there are significant challenges associated with fertilizer manufacture

14

and supply as well as cost in both economic and environmental terms. The main issues relating

15

to nitrogen fertilizer surround the use of fossil fuels in its production and the emission of

16

greenhouse gases resulting from its use in agriculture; phosphorus being a mineral source, make

17

it non-renewable and casts a shadow on its sustainable use in agriculture. This study focuses on

18

development of an efficient P nutrient system that could overcome the inherent problems arising

19

from current P fertilizers. Here, attempts are made to synthesize citric acid surface modified

20

hydroxyapatite nanoparticles using wet chemical precipitation. The resulting nanohybrids were

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characterized using powder X-Ray diffraction to extract the crystallographic data while

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functional group analysis was done by Fourier transform infrared spectroscopy. Morphology and

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particle size were studied using scanning electron microscopy along with elemental analysis

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using energy dispersive X-Ray diffraction spectroscopy. Its effectiveness as a source of P were

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investigated using water release studies and bio availability studies using Zea mays as the model

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crop. Both tests demonstrated the increased availability of P from nanohybrids in the presence of

27

an organic acid compared to pure hydroxyapatite nanoparticles and rock phosphate.

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Keywords: P fertilizers, hydroxyapatite nanoparticles, organic acids, citric acid modified

29

hydroxyapatite nanoparticle, Nanohybrids, bioavailability

30

Introduction

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Due to advances in agriculture during the ‘green revolution’, food production has increased

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globally, in order to meet the requirements of a growing population.1 Agricultural food

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production basically relies on the external supply of major nutrients such as N, P, and K to the

34

plants for their proper growth as soil itself does not have enough nutrient quantities.2 However,

35

due to low nitrogen utilizing efficiency (NUE) by plants, high cost of N fertilizers together with

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environmental issues have spurred a research towards developing slow release N fertilizers that

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focused mainly on various polymer based strategies3-16 and considering the sustainability

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challenges of P due to its non-renewable nature, effective use of P resources have also drawn a

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greater attention worldwide.1, 17-19

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There are different sources of P used in agriculture such as ground rock phosphate and

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commercial fertilizers for instance mono-ammonium phosphates (MAP), di-ammonium

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phosphates (DAP), triple superphosphates (TSP).20-21 Importantly, the plant availability and

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water solubility of these sources are different from each other. Rock phosphates are not water-

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soluble whereas commercial P fertilizers are highly water-soluble. As a result the availability of

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P from rock phosphates is less for plants whereas major portion of water soluble commercial

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fertilizers are wasted without being available for plants making major environmental issues, such

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as eutrophication.2, 20-21

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The major P source, rock phosphate, being a mineral source makes it a non- renewable resource

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and the absence of a gaseous phase in its biogeochemical cycle makes its depletion harder to

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replenish.22 Since P plays a critical role in living organisms including respiration, photosynthesis,

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protein and nucleic acid synthesis6, its gradual scarcity does not bode well for advances in the

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agriculture and food production in the future. Especially, plants will not gain their maximum

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yield without adequate levels of P. As a result, external fertilizer application is needed for crop

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plants where soil has no adequate amounts of P.2 As agriculture uses ~ 80-90% of mined P, its

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sustainable use at a global level is an urgent priority.1 Therefore, effective use of P through

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smart fertilizers with minimum leaching and optimized solubility6,

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research proposition.

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In this connection, reports on the use of synthetic hydroxyapatite nanoparticles (HANP) for

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controlled P release in soybean6, use of HANP as a P fertilizer in wheat24, the work carried out

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by Giroto et al. (2015) where HANP was incorporated into biodegradable, soluble host

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matrixes25 and our work where urea coated HANP was used for slow release of N in rice, there is

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a paucity of research on controlled release of P in agriculture. In this paper, we report citric acid

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(CA) modified hydroxyapatite nanoparticles (CMHANPs) to achieve optimum release of P in

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corn.

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Materials and Methods

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All reagents and chemicals used in this study were purchased from the Sigma Aldrich Company,

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USA and were of analytical grade and used without further purification. Eppawala Rock

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phosphate (ERP), triple superphosphates (TSP) were used as the commercial phosphate sources

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becomes an attractive

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during experiments, purchased from Lanka Phosphate Ltd. All solutions were prepared using

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distilled water.

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Experimental methods

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Synthesis of pure hydroxyapatite nanoparticles (HANPs)

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Pure HANPs were synthesized according to the wet chemical precipitation method discussed by

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Kottegoda et al.11, 15 Calcium hydroxide (8.12 g) was dissolved in distilled water (100 cm3) by

75

stirring.

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Into that slurry, 0.6 mol dm-3 orthophosphoric acid (100.0 cm3) was added drop wise under 1000

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rpm of continuous agitation. The stoichiometry of the reaction was maintained such that Ca:P is

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1.67 which is in agreement with that in hydroxyapatite. The pH of the reaction mixture was

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checked during the reaction in order to assure basic pH medium at 10.

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Synthesis of citric acid surface modified hydroxyapatite nanoparticles (CMHANPs)

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In-situ modification method

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Here, CA modification was performed while synthesizing HANP. CA solution of 50 mM was

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prepared in 100.00 cm3 volumetric flask using CA monohydrate. Calcium hydroxide (8.12 g)

84

was dispersed in distilled water (200.00 cm3) by mechanical agitation at 1000 rpm. Phosphoric

85

acid (0.6 mol dm-3, 100.00 cm3) was prepared to meet the molar ratio of Ca:P, 1.67 in

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hydroxyapatite. Into the slurry of Ca(OH)2, two acid solutions were added in a controlled

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manner. Each drop of phosphoric acid added was followed by the addition of a drop of CA under

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continuous agitation (~0.4 mL/min flow rate of each solution was maintained resulting ~ 4 ½ h

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of reaction time). Reaction mixture pH was monitored and maintained at 10 by adding

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ammonium hydroxide (25% w/w). Same procedure was followed as above for the CA solutions

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of 150 mM and 250 mM to prepare different compositions of CMHANPs (In50mM CMHANPs,

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In150mM CMHANPs and In250mM CMHANPs).

93 94

Ex-situ modification method

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Here, CA modification was performed after the synthesis of HANP. Firstly, phosphoric acid (0.6

96

mol dm-3,100.00 cm3) was added to a slurry of calcium hydroxide (8.12 g, 200.00 cm3) under

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continuous stirring to prepare HANP suspension. Then, the CA solution (50 mM, 100.00 cm3)

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was added drop wise to HANP suspension maintaining pH at 10 by adding ammonium

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hydroxide (25% w/w). Same procedure was repeated with 150 mM and 250 mM CA

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concentrations to prepare Ex50mM CMHANPs, Ex150mM CMHANPs, Ex250mM CMHANPs

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nanohybrids.

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Characterization of nanohybrids

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Scanning Electron Microscopy (SEM) characterization

104

The SEM images of the synthesized nanohybrids were obtained using the Hitachi SU6600

105

scanning electron microscope. The secondary electron mode was used for imaging. Samples

106

were coated with a thin layer of gold in order to make it conductive.

107

Energy Dispersive X-ray Spectroscopy (EDXS) analysis

108

EDXS analysis was conducted using Hitachi SU6600 SEM operating in secondary electron mode

109

with an accelerating voltage of 20 kV and working distance of 10 mm, equipped with EDXS

110

detector to determine elemental composition.

111

Fourier Transform Infra-Red (FTIR) spectroscopy

112

Nicolet IS 10 in diffuse reflection mode with the frequency range 4000 cm-1 to 500 cm-1 was

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used to determine the chemical structure and the bonding nature of resulted nanohybrids.

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Powder X-Ray Diffraction (PXRD) studies

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PXRD patterns of powdered samples of HANPs and CMHANP composites were obtained using

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Rigaku X-ray, Ultima IV diffractometer using Cu Kα radiation of wavelength 1.540 Å in 2θ scan

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axis over scan range of 5 to 80 degree with a scanning speed of 2.0 degrees/min.

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UV-Visible spectroscopy

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Genesys 10s UV-Vis spectrometer using Vision Lite software was used to determine total P

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content of resulted hybrids.

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Total P content determination

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The standard procedure given by SLS 645 for total P determination in organic fertilizers was

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followed.26

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Then, the absorbance was measured at 420 nm in 1 cm cell using 5.00 mg solution as the blank.

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HANP (2.50 g) was mixed with calcium oxide (0.50 g) and calcined at 500°C to ash the sample

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and allowed to cool. Then, 50.00 cm3 of distilled water was added and stirred thoroughly. This

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was then heated to the boiling point. Conc. HCl (5 cm3) followed by conc. HNO3 (5 cm3) were

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added to the boiling solution. Solution was boiled for 10 min until the solution became clear. The

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prepared sample was allowed to cool, filtered and transferred into a 250.00 cm3 (Vo) volumetric

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flask, and diluted to the mark. V1 volume from this solution was used for the test using 12.50

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cm3 of vanadomolybdate reagent and top up to mark with distilled water. Relevant P2O5 content

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was calculated as follows.

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𝑃2 𝑂5 % 𝑏𝑦 𝑚𝑎𝑠𝑠 =

𝑦 × 𝐷𝐹 × 𝑉𝑜 × 100 1000 × 𝑉1 × 𝑀

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y - Concentration relevant to absorbance from standard curve

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DF- Dilution Factor

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M - Mass of the sample

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Above procedure was followed for CMHANP composites. Same procedure was followed for

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pure HANP, ERP, and TSP samples without calcination due to the absence of organic

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components.

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P release study

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P release studies were carried out according to the standard dialysis membrane method described

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in the literature.27 Each prepared sample in solid form (1.00 g) was weighed and put into a semi-

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permeable membrane bag. Then, 50.00 cm3 of distilled water was added into 100 cm3 beakers

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and initial pH was measured. Then, the membrane bags having each sample was dipped in the

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beaker and kept closed. From each setup 1.00 cm3 was sampled out periodically after proper

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stirring to make sure that the sample resembles the whole solution. This sample was then

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treated with the standard vanadomolybdate method developed for the examination of water and

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waste water.28 Then, P content was quantified using UV- Vis spectrometer. Final pH of the

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solution is measured and the solution was topped up with distilled water after each withdrawal of

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the sample. This was repeated for CMHANP composites, HANP and ERP.

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Release kinetics

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P release kinetics for HANP and CMHANP nanohybrids were studied using existing release

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models. Here, release data from each experiment were fitted with the zero order model, first

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order model, Higuchi and Korsmeyer-Pappas model and the R2 values of the linear graphs were

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compared to derive the mechanism of the release of phosphate.29

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Bio-availability studies

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Corn (Zea mays) plants were used for bio-availability studies.30 Fertilizer application was

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conducted as recommended by the Department of Agriculture (DOA), Sri Lanka as given in the

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Table 1. To minimize the complexity arisen from the natural soil, coco peat which can be used as

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an inert growing medium was used in the experiment.31

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Table 1: DOA fertilizer recommendation for Zea mays. DOA recommendation for Time of application Before

Amounts per pot

irrigated cultivation for corn Urea

TSP

(kg/ha) (kg/ha) 75

100

MOP

Urea

TSP

MOP

(kg/ha)

(g/pot)

(g/pot)

(g/pot)

50

2

3

1.4

3

1.4

planting 4 weeks after

250

7

planting Total

325

100

50

9

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To minimize the interferences from the complex soil structure, coco peat was used as the inert

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growing medium. Three sets of ten fertilizing schemes were conducted as (T1) Pure HANP

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(T2) In50mM CMHANP (T3) In150mM CMHANP (T4) In250mM CMHANP (T5) Ex50mM

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CMHANP (T6) Ex150mM CMHANP (T7) Ex250mM CMHANP (T8) ERP (T9) TSP, (T10)

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Fertilizer without P, each pot having two plants. All the pots were treated in a similar manner

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including N, P, K fertilizer application and watering, except for different sources of P. Urea

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and muriate of potash (MOP) were used as N and K sources, respectively. External

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micronutrient solution (50 cm3) was supplied for all experimental pots in order to minimize

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other nutrient deficiencies (See supplementary-Table S1).32 Total P of each sample was

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determined as described in methods and an equal amount of P was supplied (See

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supplementary-Table S2) to each pot at the same time period based on TSP amount, before

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planting. The growth of the plants was monitored by measuring the height of plants and

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number of unrolled leaves weekly. Once the lifecycle of the plants were completed, the

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yield of cobs was monitored. Dry mass yield of the plants were also measured compared to

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the regular P treatments.

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Results and Discussion

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The SEM image of pure HA (Figure 1(a)) exhibits the nanorod like morphology with a diameter

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less than 50 nm confirming the synthesis of HA in nanoscale. As shown in Figure 1 (b) the

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morphology of CMHANP in ex-situ modification is retained more or less even after the

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modification while the diameter of the particle has increased (100 nm) due to surface coating of

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the CA.

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Figure 1: SEM images of (a) pure HANP (b) ex-situ modified CMHANP nanohybrids (c) in-situ

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modified CMHANP nanohybrids.

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Interestingly, in in-situ modified nanoparticles, the nanorod like morphology has changed into

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blunt ended spherical particles with a diameter of ~500 nm (Figure 1 (C)). This observation

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reveals that the two synthetic approaches have an effect on the morphology of the CA surface

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modified HANP. According to the EDXS analysis, an increment of the C% in the samples were

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observed (Table 2). The maximum CA loading is observed for In250mM CMHANP.

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Table 2: Elemental composition of resulting nanohybrids Sample

Elemental Composition (Average %) C

O

P

Ca

Pure HANP In50mM CMHANP

0.00 23.18

58.45 36.02

14.17 11.03

27.38 35.33

In150mM CMHANP

31.19

19.98

13.51

35.33

In250mM CMHANP

49.23

41.19

2.28

7.30

Ex50mM CMHANP

42.48

37.24

7.41

12.87

Ex50mM CMHANP

45.81

42.62

3.56

8.05

Ex50mM CMHANP

47.20

37.75

2.58

12.26

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In order to understand the nature of interactions between the nanoparticles and CA, FTIR spectra

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were compared with that of pure HANP. Peak shifts, peak broadenings, new peaks appearing or

201

disappearing due to phosphate, hydroxyl, and carbonyl regions were compared to obtain

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conclusive evidence. A significant shift in the peak due to stretching vibrations of the phosphate

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group, from 1041 cm-1 to ~1020 cm-1 was observed for all in-situ modified samples (Figure 2

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(i)).

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Figure 2: FTIR spectra for the (i) PO43- stretching, (ii) C=O stretching and OH bending, and (iii)

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OH stretching regions of (a) Pure HANP, (b) In50mM CMHANP, (c) In150mM CMHANP, (d)

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In250mM CMHANP, and (e) Pure CA.

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Other than that, the peak intensity of phosphate stretching mode decreased with increasing the

210

loading of CA. According to the literature, inorganic phosphate group stretching appears around

211

~1050

212

compounds like aliphatic groups which explains the observed peak shift. This can be further

213

explained by observing the lowering of intensity of the phosphate peak with increasing CA

214

concentrations.

cm-1 and shifts to ~1020 cm-1

(1050-990 cm-1)33 upon interaction with organic

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The peak at 1395 cm-1 (1310-1410 cm-1) is assigned to the bending mode of tertiary OH group in

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CA while the peak at 1475 cm-1 is corresponding to the bending modes of CH in methylene

217

group of CA (Figure 2 (ii)). Peak around 1725 cm-1 represents the carbonyl group of carboxylic

218

acid group in pure CA and C-O stretching mode of carboxylic acid appears as a shoulder peak at

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~1425 cm-1 while the bending mode of OH in the carboxylic acid group is given around ~1600

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cm-1 which are in agreement with those reported by Coates et al.33 The weak carbonyl stretching

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peak has disappeared after modification with CA suggesting the appearance of strong

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interactions between HA and CA molecules. In CMHANP composites, hydroxyl group of HANP

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is bonded with the carbonyl groups and hydroxyl groups arising from the CA molecule. Bending

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mode of OH in CA has appeared with a reduced intensity with considerable peak shifts from

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1600 cm-1 to around ~1570 cm-1 (Figure 2 (ii)) in CMHANP composites further confirming the

226

presence of strong H bonding between HANP and CMHANP.

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According to literature, the carbonyl group of carboxylate anion can be assigned to the peak

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around 1610–1550 cm-1.33 Therefore, the peak centered on ~1570 cm-1 in in- situ modified

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samples was assigned to the carbonyl group in carboxylate anion form which suggests the

230

presence of carboxylate anions in the nanocomposite. The appearance of this peak in the

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nanocomposite with the disappearance of the peak relevant to carbonyl group in pure CA (~1725

232

cm-1) suggests that favorable interactions exist between the two species (Figure 2 (ii)). Further

233

elaboration of this behavior interpret that some CA molecules can substitute as an anion with

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phosphates in the HANP crystal structure. The peak centered around ~3450 cm-1 in pure HANP

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spectrum was broaden in CMHANP composites with the center of the peak being shifted towards

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lower frequencies around 3300 cm-1 (Figure 2 (iii)) which concluded that H-bonding has taken

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place during the binding of CA onto HA.33

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Importantly, similar behavior of shifts was also observed for ex-situ modified nanocomposites.

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Table 3 summarizes the major FTIR peak position values for both in-situ modified and ex-situ

240

modified nanocomposites. Here, in all nanocomposites, the region of tertiary OH bending of

241

citric acid is overlapped with C-O stretching of carboxylic group to give a single peak which has

242

been denoted using * mark. Notably, higher extent of interactions can be seen for in-situ

243

modified samples.

244

Table 3: Major FTIR peak value comparison of in-situ and ex-situ modified nanocomposites Sample

FTIR peaks positions (cm-1) P-O OH stretching stretching

OH bending of CA

Pure HANP

1041

3450

-

In50mM CMHANP

1020

3350

1573

OH bending of tertiary - alcohol group of CA 1416*

C-O stretching of CA

In150mM CMHANP

1019

3346

1569

1413*

-

In250mM CMHANP

1019

3349

1570

1412*

-

Ex50mM CMHANP

1019

3344

1577

1416*

-

Ex150mM CMHANP

1022

3265

1566

1428*

-

Ex250mM CMHANP

1025

3225

1560

1408*

-

Pure CA

-

32003500

1600

1395

-

C=O stretching of CA -

1425

1725

245 246

PXRD characterization was done in order to extract crystallographic data of synthesized HANP

247

and CMHANP samples. Except for the In250mM CMHANP sample, all other samples were

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verified as hydroxyapatite (HA) crystalline structure according to the reported powder diffraction

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data (PDF No: 01-089-4405). However, most of the peaks corresponding to the HA were

250

identified together with the peaks arising from CA. Peaks denoted by an * represents the

251

corresponding peaks for the CA. It is clear that the major mineral phase of the modified samples

252

were HA and with the increment of citric acid loading, the extra peaks relevant for citric acid

253

also appeared. Interestingly, both in-situ and ex-situ modified samples have broader diffraction

254

peaks than those of the pure HANP and the three major peaks (e), (f), and (g) have been merged.

255

These observations suggest that some extent of modification has occurred in HA crystalline

256

structure without changing its major mineral phase. Merging of nearby peaks due to broadening

257

is characteristic for monocrystalline materials.34 This confirms the successful synthesis of

258

monocrystalline CMHANP. As shown in Figures 3 (i) and (ii), the peaks denoted by a-l

259

represents the major peaks relevant to pure HA in comparison with the modified samples.

260 261

Figure 3: Comparison of PXRD pattern of (a) Pure HANP, and (i) in-situ modified

262

nanocomposites of (b) In50mM

263

CMHANP and (ii) ex-situ modified nanocomposites of (b) Ex50mM CMHANP, (c) Ex150mM

264

CMHANP, and (d) Ex250mM CMHANP.

CMHANP, (c) In150mM

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Table S3 and table S4 summarizes the major crystallography planes for both in-situ modified and

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ex-situ modified nanocomposites (see supplementary).

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Total P content of all nanocomposites, pure HANP, TSP and ERP was calculated and given in

268

Table 4.

269

Table 4: Total P content as % P2O5 by mass Sample

Total P content as % P2O5 by mass

Pure HANP

35.59

In50mM CMHANP

34.62

In150mM CMHANP

32.24

In250mM CMHANP

27.43

Ex50mM CMHANP

35.02

Ex150mM CMHANP

33.78

Ex250mM CMHANP

28.10

TSP

50.31

ERP

43.26

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The total P content of ex-situ modified samples was higher compared to the corresponding in-situ

271

modified samples. This observation suggests that in the in-situ modification there is greater

272

interactions of CA with HANP prior to agglomeration due to the presence of CA in the reaction

273

system whereas in the ex-situ method, there is more time for the agglomeration of HANP that

274

lower the surface area, which leads to lower interactions between CA and HANP.

275

Release behavior of phosphate ions from the nanohybrid was studied in distilled water (pH=6.8).

276

Final pH of the released medium was recorded as 5.9 for pure HANP sample, 6.0 for ERP and

277

5.8-6.0 for CMHANP nanohybrids. Interestingly, lowering of the pH can be observed due to the

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release of phosphate ions from nanohybrids accompanied by the leaching of citric acid into the

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released medium. CMHANP nanohybrids showed an increased release of phosphorus compared

280

to that of pure HANP and ERP (Figure 4). As shown in Figure 4, the ex-situ modified samples

281

exhibited an initial burst release of phosphorus followed by a slow release whereas in-situ

282

modified samples showed a gradual increment of phosphate release. Release profiles of ex-situ

283

and in-situ modified samples can be correlated with the synthesis pathway used. For an example,

284

CA modification was done after the synthesis of HANP in ex-situ modification, while HANP

285

synthesis was carried out in the presence of CA in solution in the in-situ modification. As a

286

result, in ex-situ method there is a chance of agglomeration of HANP where the binding of CA

287

on to HANP is poorly expected. The initial rapid phosphate release of ex-situ modified samples

288

is owing to rapid pH increment due to weakly bound CA on HANP being released into the

289

medium. Once the nanohybrid is exposed to the water medium, the hydrogen bonded surface

290

interactions with citric acid is diminished resulting in an equilibrium with water to give H+ to the

291

medium which leads to greater HA dissolution.35 Compared to in-situ modified samples, ex-situ

292

modified samples reached this equilibrium faster resulting in increased dissolution of HANP in

293

the initial stages. The slow release rate of phosphate in the later stage is due higher particle size

294

compared to in-situ modified samples. This means that due to agglomeration, the surface area of

295

the solid that H+ can attack is lower in the ex-situ modified sample.

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Figure 4: Overall comparison of release profiles, (a) Ex250mM CMHANP, (b) In250mM

298

CMHANP, (c) Ex150mM CMHANP, (d) In150mM CMHANP, (e) Ex50mM CMHANP (f)

299

In50mM CMHANP, (g) Pure HANP, and (h) ERP.

300

Gradual increment of phosphorus release throughout 60 days was observed (Figure 4). Since in-

301

situ modification was done simultaneously with the synthesis of HANP, citrate ions could be

302

replaced with phosphate ions in the HA crystal structure which was also evident from reduced

303

crystallinity as shown by PXRD data compared to pure HANP. The dissolution in in-situ

304

modified samples thus aided by two factors in the aspect of release of H+ from surface bonded

305

CA and citrate ions that have been replaced in HA lattice. Based on the release kinetics and the

306

chemical model used to explain the dissolution, two types of interactions (a) surface addition,

307

and (b) Intercalation, can be deduced between CA and HANP. The release data was further

308

analyzed by fitting with different kinetic models relevant to controlled release formulations.

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Using this data the mechanism behind the release of P from the nanohybrid was derived. As

310

shown in the Table 5, four different models were manipulated to derive the release kinetics of

311

modified HANP.

312

Table 5: Comparison of release kinetics for CMHANP nanohybrids Sample

R2 value for each kinetic model Zero-order First-order Higuchi Korsmeyer

Best fitted model

(ZOM)

(FOM)

model

-Peppas

(HM)

(KPM)

Pure HANP

0.937

0.925

0.690

0.972

KPM

In50mM CMHANP

0.958

0.814

0.773

0.962

KPM

In150mM CMHANP

0.988

0.806

0.843

0.984

ZOM

In250mM CMHANP

0.950

0.887

0.937

0.992

KPM

Ex50mM CMHANP

0.972

0.770

0.931

0.934

ZOM

Ex150mM CMHANP

0.115

0.620

0.818

0.883

KPM

Ex250mM CMHANP

0.237

0.462

0.647

0.736

KPM

ERP

0.905

0.954

0.671

0.992

KPM

313 314

Based on the results, samples had the best fit with the zero order model and the Korsmeyer-

315

Pappas model as shown in the Table 5. Even though In150mM CMHANP and Ex50mM

316

CMHANP release profiles best fitted the zero order model with R2 values of 0.988 and 0.972,

317

respectively; the R2 values for the Korsmeyer-Pappas model were 0.984 and 0.934 respectively,

318

showing close agreement. Except these two samples, all other systems behave according to the

319

Korsmeyer-Pappas model. Korsmeyer–Peppas model is applied for controlled release systems

320

where the matrix is cylindrical which release active species by diffusion. Further investigation of

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the release profiles that were fitted with the Korsmeyer–Peppas model reveals the release

322

mechanism in relation to its exponential factor as given in the table S5 (See supplementary).

323

According to the results, the kinetic model of the nanohybrid can be explained as the

324

Korsmeyer–Peppas model which follows a non Fickian transport mechanism.

325

Maximum average height of the plants after 9th week was identified for In250mM CMHANP

326

treatment. Average height of the plants are significantly higher compared to No P, pure HANP,

327

and ERP treatments while in-situ modified samples depicted higher growth than the

328

corresponding ex-situ modified treatments Figure 5.

329 330

Figure 5: Comparison of maximum heights of the plants at 9th week (a) No P, (b) Pure HANP,

331

(c) TSP, (d) ERP, (e) In50mM CMHANP, (f) In150mM CMHANP, (g) In250mM CMHANP,

332

(h) Ex50mM CMHANP, (i) Ex150mM CMHANP, and (j) Ex250mM CMHANP.

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Interestingly, maximum average dry weight of the cobs resulted for In250mM CMHANP and

334

In150mM CMHANP treatments (Figure 6).

335 336

Figure 6: Average dry weight of the cobs from plants under different treatments.

337

This observation can be justified by referring to the extent of growth of the root system. As can

338

be seen by figure 7, the roots are efficiently developed for the treatments f and g which received

339

highest amount of P while all the treatments received same amount of nitrogen. Therefore, the

340

similar plant height was observed for in-situ and ex-situ while cob development was retarded by

341

the unavailability of sufficient P. This observation can also be further explained by referring to

342

the water release profiles given in figure 4 where efficient and gradual release of P was observed

343

for in-situ composites.

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Figure 7: The root system of plants under different treatments

346

The results from the pot experiments, the optimum growth parameters were obtained with the in-

347

situ modified samples compared to the conventional P fertilizers. In250mM CMHANP treatment

348

displayed the best crop productivity over ERP and TSP treatment used as conventional

349

fertilizers. A significant increment of the growth and crop productivity of all nanohybrid

350

treatments were observed compared to ERP or TSP. According to the results from the release

351

study and plant uptake studies, it can be concluded that the developed nanohybrids have opened

352

up a new pathways for development of an efficient P nutrient system with increased solubility

353

and plant availability.

354 355 356 357 358 359 360 361 362 363

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Supporting information

365

Table S1: General micronutrient formulation as recommended. Nutrient

Concentration (ppm)

Ca

180

Mg

48

S

336

Fe

3

Cu

0.02

Zn

0.11

Mn

0.62

B

0.44

366 367

Table S2: Amount of sample used as the source of P in pot experiments Source of P

Total P content as % P2O5

Mass

of

each

sample

applied to the experimental by mass (pot / g) Pure HANP

35.59

4.24

In50mM CMHANP

34.62

4.36

In150mM CMHANP

32.24

4.68

In250mM CMHANP

27.43

5.50

Ex50mM CMHANP

35.02

4.30

Ex150mM CMHANP

33.78

4.47

Ex250mM CMHANP

28.10

5.37

TSP

50.31

3.00

ERP

43.26

3.49

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Table S3: Major peaks assigned for PXRD pattern of in-situ modified samples. Peak Peak

Pure HANP

In50mM CMHANP 2θ d (nm)

Index



d (nm)

a

100

10.78

0.820

b

200

25.76

0.346

25.79

0.345

d

210

29.10

0.307

28.43

0.314

e

211

31.84

0.281

31.97

f

112

32.90

0.272

g

300

33.94

0.264

h

310

39.88

0.226

39.60

i

222

46.69

0.194

j

312

48.07

0.189

k

213

49.38

0.184

49.68

0.183

l

400

52.98

0.173

53.19

0.172

In150mM CMHANP 2θ d (nm)

In250mM CMHANP 2θ d (nm) 11.51

0.768

25.86

0.344

26.01

0.342

0.280

32.13

0.278

31.94

0.280

0.227

39.46

0.228

40.10

0.225

46.36

0.196

49.72

0.183

53.13

0.172

49.38

369 370 371 372 373 374 375 376 377

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Table S4: Major peaks assigned for PXRD pattern of ex-situ modified samples. Peak Peak

Pure HANP

Ex50mM CMHANP 2θ d (nm)

Ex150mM CMHANP 2θ d (nm)

Ex250mM CMHANP 2θ d (nm)

10.76

0.822

11.51

0.768

25.85

0.344

25.83

0.345

28.90

0.309

31.85

0.281

32.85

0.272

Index



d (nm)

a

100

10.78

0.820

b

200

25.76

0.346

25.81

0.345

d

210

29.10

0.307

28.39

0.314

e

211

31.84

0.281

31.85

0.281

f

112

32.90

0.272

g

300

33.94

0.264

33.91

0.264

33.98

0.264

33.97

0.264

h

310

39.88

0.226

39.78

0.226

39.81

0.226

39.79

0.226

i

222

46.69

0.194

46.77

0.194

46.65

0.195

j

312

48.07

0.189

k

213

49.38

0.184

49.41

0.184

49.31

0.185

l

400

52.98

0.173

53.17

0.172

53.06

0.172

49.71

0.183

31.86

379 380 381 382 383 384 385 386 387

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Table S5: Exponential factor and relevant mechanism of the samples behaving KPM Sample

Exponential factor (n)

Transport mechanism

Pure HANP

1.461

Super case II transport

In50mM CMHANP

2.188

Super case II transport

In250mM CMHANP

0.618

Non -Fickian transport

Ex150mM CMHANP

0.770

Non -Fickian transport

Ex250mM CMHANP

0.919

Super case II transport

ERP

1.774

Super case II transport

389 390

Acknowledgements

391

Authors wish to acknowledge University of Sri Jayewardenepura for offering facilities for the

392

research.

393

Author Contributions

394

NK developed the idea, designed the project, supervised, provided the leadership and finalized

395

the manuscript. VK mentored the project and finalized the manuscript. RS designed and

396

performed the experiments, analyzed the data and participated in manuscript preparation. CS

397

designed the pot experiments, analyzed the data and prepared the manuscript. MS and GP

398

analyzed the data and participated in manuscript preparation.

399 400

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TOC Graphic

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SEM images of (a) pure HANP (b) ex-situ modified CMHANP nanohybrids (c) in-situ modified CMHANP nanohybrids 752x564mm (96 x 96 DPI)

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FTIR spectra for the (i) PO43- stretching, (ii) C=O stretching and OH bending, and (iii) OH stretching regions of (a) Pure HANP, (b) In50mM CMHANP, (c) In150mM CMHANP, (d) In250mM CMHANP, and (e) Pure CA 1695x1442mm (96 x 96 DPI)

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Comparison of PXRD pattern of (a) Pure HANP, and (i) in-situ modified nanocomposites of (b) In50mM CMHANP, (c) In150mM CMHANP, and (d) In250mM CMHANP and (ii) ex-situ modified nanocomposites of (b) Ex50mM CMHANP, (c) Ex150mM CMHANP, and (d) Ex250mM CMHANP 1699x717mm (96 x 96 DPI)

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Overall comparison of release profiles, (a) Ex250mM CMHANP, (b) In250mM CMHANP, (c) Ex150mM CMHANP, (d) In150mM CMHANP, (e) Ex50mM CMHANP (f) In50mM CMHANP, (g) Pure HANP, and (h) ERP 271x229mm (300 x 300 DPI)

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Comparison of maximum heights of the plants at 9th week (a) No P, (b) Pure HANP, (c) TSP, (d) ERP, (e) In50mM CMHANP, (f) In150mM CMHANP, (g) In250mM CMHANP, (h) Ex50mM CMHANP, (i) Ex150mM CMHANP, and (j) Ex250mM CMHANP 265x228mm (300 x 300 DPI)

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Average dry weight of the cobs from plants under different treatments 260x229mm (300 x 300 DPI)

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The root system of plants under different treatments 1449x345mm (96 x 96 DPI)

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2376x1040mm (96 x 96 DPI)

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