formulation of green nano-fertilizer to enhance the plant...

Journal of Pharmacy Research Vol.5 Issue 11.November 2012

R.Subbaiya et al. / Journal of Pharmacy Research 2012,5(11),5178-5183

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Research ArticleISSN: 0974-6943

Available online throughhttp://jprsolutions.info

*Corresponding author.Mr.R.SubbaiyaAssistant ProfessorDepartment of BiotechnologyK.S.Rangasamy College of TechnologyTiruchengode, Namakkal-637 215Tamil Nadu, India.

INTRODUCTIONNitrogen is naturally present in the soil but it cannot be completely utilizedby the plants. So, it is supplemented to the plants artificially with the helpof fertilizers [1]. Basically the nitrogen applied using conventional fertilizers,with particle size dimensions greater than 100nm, is lost to the soil due toleaching, so nitrogen utilization efficiency (NUE) by plants is low[8]. Thisleaching occurs due to water–soluble nitrates, emission as ammonia andnitrogen oxides and soil microorganisms–mediated incorporation into soilorganic matter [2]. To reduce the leaching and to improve the NUE thesurface area of the fertilizer to nanosize. And this strategy involves theslow–release of fertilizer also [15].

Controlled-release fertilizersControlled-release fertilizers provide an attractive alternative to granularfertilizers. These are fertilizer ‘cocktails’ that slowly release nutrients tothe substrate [4]. The release depends on water availability or soil temperature.Controlled-release fertilizers are more expensive than the more commonwater soluble fertilizers, but they have several advantages:

• The danger of over-fertilizing is reduced as the release of fertilizers occurs gradually.• Fertilizing is necessary only occasionally, sometimes only once in a season• A balanced fertilizer mixture is provided at all times as the plants get what they need at different growth stages.

Formulation of Green Nano-Fertilizer to Enhance the PlantGrowth through Slow and Sustained Release of Nitrogen

R.Subbaiya1, M.Priyanka1, M.Masilamani Selvam2

1Department of Biotechnology, K.S.Rangasamy College of Technology, Tiruchengode,Namakkal-637 215,Tamil Nadu,India.2Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Chennai-600 119,Tamil Nadu,India.

Received on:12-06-2012; Revised on: 17-07-2012; Accepted on:26-08-2012

ABSTRACTFertilizers are chemical compounds applied to promote plant growth. It is applied either through the soil or by foliar feeding. Artificial fertilizers areinorganic fertilizers formulated in appropriate concentration to supply the nutrients. Nitrogen is an important source which is essential for the growth ofplant. Urea is the most widely used water soluble plant nitrogen source. Due to leaching the nitrogen content in the soil gets decreased due to leaching.So NUE (Nitrogen utilization efficiency) is low. Urea modified hydroxyapatite particles have been employed to agriculture, because of their higher NUEand slow release of the nitrogen to the soil. These maximize the NUE utilization by plants and minimize the adverse effects to the environment.Hydroxyapatite was prepared using chemical combination of ortho phosphoric acid and calcium hydroxide. Then prepared colloidal hydroxyapatite waskept in refrigerator for overnight. Powdered hydroxyapatite molecule was prepared in different concentration (10%, 20%, 30% and 40%) and was mixedwith nanourea molecule using ultrasonicator. Urea modified composite molecule was applied to the field green gram (Vigna radiata) as both seed treatedand directly applied to soil. Urea modified HA particle has the highest pH of 7.81 compared to that of normal urea. Seed germination was observed in allthe pot. HA particle treated seed shows the good germination of 100% towards the yield whereas urea alone gives only 70% of seed germination. Heightof the plant clone of HA treated as 11.8cm for 5days time interval in contrast Urea alone treated has only 10cm and density also low compared to HA treat.Nitrogen release was estimated by using nitrate estimation. Nitrate estimation of HA particle was calculated as 100µg/ml whereas urea treated wascalculated as 50µg/ml. In this context, we concluded that Hydroxyapatite molecule shows the good utilization of nitrogen source. So it has the better yieldthan other conventional fertilizer.

Key words: Nitrogen Utilization Efficiency, Hydroxyapatite, Vigna radiata, Fertilizer.

• Nutrients do not leach from the substrate so the plants receive all the nutrients applied[5].

NanoureaNanourea is prepared by reducing its size using some of the reducing agentslike TriSodiumCitrate(TSC) [6]. This could reduce the size of the urea andeffect of urea to the soil, due to this the pH of the soil will be maintained assuch. So the soil does not loss its fertility [7].

HydroxyapatiteHydroxyapatite (Ca10(PO4)6(OH)2) is an important component which pro-vides phosphorus nutrient to the plants. Mostly the application of HAp isfocused on its biomedical applications due to its excellent biocompatibilityand bioactivity[18].While it will not be adequately applied in agriculture. TheHAp can be synthesized by several methods like sol-gel method [3]. HAp ismainly used for bone- replacement because it consist of calcium and phos-phorus. Hydroxyapatite (HAP) microspheres with peculiar spheres-in-sphere morphology were prepared by using oil-in-water emulsions andsolvent evaporation techniques [14].

Composite PreparationHere the surface of the HA is modified with chemically synthesisednanourea. To perform slow release this nanocomposite is prepared [9]. Heretwo types of composites were prepared. They were as follows:1. Hy-droxyapatite nanoparticle + nanourea(chemical synthesis)2. Biologicallysynthesis of nanourea [10]. In biological synthesis two types of concentra-tions were followed: they are, 0.4mM concentration, 200mg.



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MATERIALS AND METHODS

Collection of Rhodococcus CultureRhodococcus spp-2891 was obtained from National Collection of Indus-trial Microorganism, Pune, India. The obtained species was inoculated inMGYP media which acts as growth media for its growth and proliferation.

Preparation of Hydroxyapatite NanoparticlesHA nanoparticles are to be synthesized by using aqueous solution ofCa(OH)2 and H3PO4. First take 19.29gm of Ca(OH)2 are to be dissolved in250ml of distilled water then prepare 0.6M H3PO4(250ml). Add H3PO4 tothe Ca(OH)2 suspension in dropwise, while stirring it vigorously undermechanical agitation (1000 rpm) [11].

6H3PO4+10Ca (OH)2àCa(PO4)6(OH)2+18H2OThe synthesized HA nanoparticles are to be refrigerated overnight to formprecipitate.

Biological SynthesisUrea solution were prepared at different concentrations like 0.2mM, 0.4mM,0.6mM ,0.8mM & 1mM [12].The Rhodococcus species were inoculated andkept at incubation in an incubatory shaker at 370c for 2-3 days. Then theUV-spec reading was taken for that solution to analyze the reduction of theurea particle [13].

Synthesis of Nanourea0.005 mol of urea molecule are to be mixed with 5% of 10ml Trisodiumcitrate.• Mixture is heated to 70ºC for 30minutes• Color gets changed into ash color solution• It indicates the presence of Nanourea molecule• It is further confirmed by using UV spectrophotometer (580nm)

Composite PreparationAbout 0.05gm of nanourea is to be added to the 100ml of HA nanoparticlesand this mixture is subjected to ultrasonic mixing (30 KHz for 1hr). Theresulting mixture is then allowed to settle and the excess liquid is drainedoff. Now the nanourea modified HA particles are subjected to lyophiliza-tion to remove the moisture content present in it. The nanourea modifiedHA nanoparticles are characterized using UV-Spectrophotometer, FTIRand SEM.

Characterization StudiesThe characterization studies were performed through UV-Spectrophotom-eter, FTIR &SEM analysis.EncapsulationThe nanocomposite mixture was then encapsulated into the hydroxyapa-tite using ultrasonicator to enhance the slow release of nitrogen to theplants.

Field StudiesThe soil is collected from K.S.Rangasamy College of Technology and thegreen gram are to be treated with chemically synthesized and biologicallysynthesized nanourea in different concentrations and they are to be pottedin a small pot.

Estimation of nitrogen (AOAC, 1990)Measure one gram of sample in 100 ml conical flask and add 3 ml of salicylicacid (3.2% in concentrated sulphuric acid) and a pinch of sodium thiosulphate.Digestion is done on a hot plate after adding 5 ml of hydrogen peroxide tillthe digest become colourless [16]. Simultaneously, a blank is also run. Thecontents are neutralized with sodium hydroxide and transferred to a 100 mlvolumetric flask, then add one ml of 10% sodium potassium tartarate andone ml of Nessler’s reagent. The solution is made up to the mark withdistilled water and the absorbance is measured at 420 nm using a spectro-

photometer. Ammoniacal nitrogen (ammonium sulphate) is used as thestandard.

Estimation of Urea by DAM method0.2 to 1ml of standard was taken in the test tubes marked as S1-S5.Thegiven unknown solution was made upto 100ml with distilled water and take0.5 and 1ml in test tubes marked as T1 and T2,Simultaneously the blank isalso prepared which is marked as B. All the test tubes were made upto 1mlwith distilled water.3ml of orthophosphoric acid solution. The content ofthe test tubes followed by 3ml of orthophosphoric acid solution. Thecontent of the test tubes was mixed well and kept in boiling water bath for300C for colour development and cooled. Then the colour developed wasread calorimetrically at 540nm. Draw a graph by taking the concentration inX- axis and the optical density on Y-axis. From which calculate the amountof urea present in the whole of the given unknown solution.

Peroxidase AssayPipette out 3ml of already prepared Phosphate buffer (pH 7.0) solution inthe series of test tubes marked as T1, T2, T3 and T4. 0.05ml of Guiacolsolution was added to each test tube. Add 0.03ml of H2O2 solution intoeach test tube. As soon as the solution is transferred into the cuvette, 0.1mlof respective enzyme extract was added, mixed well and the absorbancewas measured at 436nm. The presence of enzyme activity was detected byreading the absorbance for every 10min.

The enzyme activity was calculated using the formula,

Enzyme Activity (U/ml) = ∆436 * 4 * Vt * Dilution factor Min * Molecular Extension Coefficient * Vs

Where,Vt- Volume of total sampleVs- Volume of test sample

RESULTSThe study on chemical and biological synthesis of urea nanoparticles by theRhodococcus spp., and its field studies were carried out in this work usingVigna radiata. For chemical synthesis the appearance of ash colour formationduring heating suggested the formation of urea nanoparticles. The synthesizednanourea particles were encapsulated inside the hydroxyapatite particlesby performing ultra sonication. UV-Spectrophotometer reading was takenfor the conformation of the nanourea particles. The biologically synthesizednanourea have shown the turbidity and for further conformation UV-Spectrophotometer reading was taken, then SEM&FTIR reading was takenand then finally the nanourea was observed in reduced size.The FTIRspectrum of the encapsulated material clearly indicated that the structuralintegrity of nanourea-modified HA nanoparticles was maintained.Thenprepared composite was lyophilized, then the particle size and morphologyof the synthesized samples were studied using a Scanning ElectronMicroscopy(SEM) [17]. The chemical nature and molecular bonding of thesynthesized samples were studied using Fourier Transform Infra RedSpectroscopy(FTIR) in a range from 600 to 4000 cm–1 using attenuatedtotal reflectance (ATR) technique. The field studies were performed usinggreen gram (Vigna radiata) as a plant model.

InoculationThe Rhodococcus spp., was inoculated into the SCN media with differentconcentration of urea solution and kept for incubation (3-4days).The 0.4mMshows the maximum growth and high absorbance at 324nm. So, it was thenmass cultured, filtered using membrane filtration and the sample was lyo-philized. The lyophilized sample was characterized using FTIR, SEM .Thenthe field studies was performed using the lyophilized sample.

The pellet at the concentration of 200 mg shown the maximum growth andhigh absorbance at 206nm. Then the culture was lyophilized and the char-



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acterization studies were performed using FTIR, SEM and the field studieswere carried out using this composite.

FTIR studiesThe chemical nature and molecular bonding of the synthesized sampleswere studied using Fourier Transform Infra Red Spectroscopy(FTIR).

Fig. 1. Supernatant solution.

0.4mM Supernatant SolutionThe supernatant solution does notshows any colour difference in thetubes this indicates the absence ofnanourea synthesis by theRhodococcus spp, extracellurally.

Fig. 2. Pellet Solution.

0.4mM Pellet solutionThe 0.4mM pellet solution showsturbidity in its test tubes thisindicates the reduction of urea size(i.e.,) nanourea synthesis by theRhodococcus spp., intracellurally.

Table.1. Peroxidase assay.Test sample OD at 436nm Enzyme activity (U/ml)

Initial After10min

Sample1 0.084 0.101 0.00848

Calculation for Peroxidase AssayEnzyme Activity (U/ml) = ∆436 * 4 * Vt * Dilution factor Min * Molecular Extension Coefficient * Vs

Where,Vt- Volume of total sampleVs- Volume of test sample

Enzyme Activity (U/ml) = ∆436 * 4 * Vt * Dilution factor Min * Molecular Extension Coefficient * Vs

Characterization Studies

UV-Spec reading for the 0.4mM Rhodococcus solutionThe 0.4mM solution where the Rhodococcus was inoculated has shown themaximum growth and production of nanourea at a 324nm .

UV-Spectrophotometer reading for pellet solutionThe 0.4mM pellet solution has shown the maximum growth and produc-tion of nanourea at a 295nm.

Fig. 3. UV-Spectrophotometerreading for 0.4mM.

Fig. 4. UV-Spec reading for Pelletsolution.

Fig. 5. SEM Image of nanourea particle.

Fig. 6. SEM Image for Hydroxyapatite particles.

Fig. 7. SEM Image of biologically synthesised nanourea particle for 200mg

Fig. 8. SEM Image of biologically synthesised nanourea particle for 0.4mM



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Fig. 9. Fourier transform infrared spectra (FTIR) of lyophilized 0.4mM nanourea sample.

Different forms of composites:The composites was prepared in different forms (i.e.,) chemicallysynthesised and encapsulated into the hydroxyapatite particle,biologicallysynthesised nanourea are given below:

Fig. 10. Fourier transform infrared spectra (FTIR) of lyophilized 200mg nanourea sample.

POT StudiesThe pot studies was carried out using Vigna radiata as a plant model.Chemically and biologically synthesised nanourea was used and the resultsobtained are given below:



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Table 2. Measurement of growth of Vigna radiata by applying differentforms of prepared Nanourea.

S.No Days Biologically Chemically Biologically Normal urea Controlsynthesized synthesized synthesized (Cm) (Cm)nanourea NA-modified nanourea usingusing 0.4mM HAp 200mg of pelletfilterate(Cm) (Cm) solution(Cm)

1. From 4th day 3.6±0.212 3.9±0.188 3.06±0.144 2.9±0.124 2.8±0.1692 5th day 3.80±190 4.1±0.190 3.3±0.169 3.13±0.151 2.9±0.1783. 6th day 4.1±0.222 4.5±0.216 3.56±0.190 3.33±0.151 3.1±0.1784. 7th day 4.4±0.216 4.7±0.241 3.83±0.196 3.6±0.169 3.3±0.2055. 8th day 4.6±0.241 5.1±0.249 4.06±0.222 3.8±0.169 3.4±0.1966. 9th day 4.9±0.242 5.3±0.268 4.33±0.276 4.03±0.196 3.6±0.1967. 10thday 5.2±0.222 5.6±0.294 4.56±0.268 4.36±0.222 3.7±0.1908. 11thday 5.5±0.223 5.8±0.284 4.8±0.294 4.56±0.237 3.9±0.2169. 12thday 5.8±0.241 6.2 ±262 5.0±0.329 4.73±0.259 4.06±0.23710. 13thday 6.1±0.241 6.5±0.241 5.26±0.347 5.03±0.259 4.23±0.25911. 14thday 6.4±0.243 6.8±0.212 5.46±0.349 5.26±0.284 4.36±0.23712. 15thday 6.8±0.243 7.2±0.188 5.66±0.351 5.46±0.284 4.53±0.259

Table 3. Estimation of Urea by DAM method.

Contents Concentration mg/g

Biologically synthesized nanourea(0.4mM) 150Biologically synthesized nanourea(200mg) 100Chemically synthesized Nanourea 258modified –HA particlesTreated with normal urea alone 090Control 050

Contents Concentration (mg/g)

Biologically synthesized nanourea(0.4mM) 29Biologically synthesized nanourea(200mg) 14Nanourea -modified HA nanoparticles 35Treated with urea alone 11Treated with distilled water 8

Table 4. Estimation of nitrogen AOAC,1990).

Fig. 11. Field studies carried out using Vigna radiata

Different types of nanourea was prepared by chemical and biological meth-ods. Rhodococcus spp., was used for biological method. The chemicallysynthesized nanourea was encapsulated into the HAp(Hydroxyapatite par-ticle) to enhance the property of slow and sustained release of nitrogen tothe plants. The prepared nanocomposites were then characterized usingUV-Spectrophotometer, SEM and FTIR studies. Thus of treating theseeds with the nanocomposites, the germination percentage of green gramseeds were all over 99%, which were similar to distilled water. The resultsshowed that using this nanocomposites were safe to the germination ofgreen gram seeds. Applying the 3 kinds of treated slow/controlled releasefertilizer were used. The effects of new type of fertilizer on emergence ofgreen gram seedlings and growth of seedlings was researched under theconditions of pot experiment. Results showed that the emergence rates ofgreen gram seedlings for all treatments were over 95%, which were similarto chemical fertilizer of equal amount of urea fertilizer and no fertilizer.

DISCUSSION• The major advantage of the above slow release fertilizer is to

improve the crop efficiency and higher crop yield by providing thenutrients through slow release to the plants.

• Due to its slow release property it results in reduced environmen-tal damage from leaching of nitrogen, compared to conventionalwater soluble fertilizers.

• This fertilizer composition may maximize the NUE while mini-mizing the adverse effects to the environments due to use of largequantities of fertilizer in agriculture.

ACKNOWLEDGEMENTAuthors are thankful to the management of KSR College of Technology andHead of the Department Dr.P.Ponmurugan for providing the facilities tocarry out the research work.



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Source of support: Nil, Conflict of interest: None Declared

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