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International Journal of Biological Macromolecules 65 (2014) 362–368

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac

nO nanoparticles induced exopolysaccharide productiony B. subtilis strain JCT1 for arid soil applications

amesh Raliyaa,∗, J.C. Tarafdarb, H. Mahawarb, Rajesh Kumarb, Priya Guptab,anu Mathurb, R.K. Kaulb, Praveen-Kumarb, A. Kaliac, R. Gautamb,.K. Singhb, H.S. Gehlotd

Washington University, St. Louis, MO 63130, USACentral Arid Zone Research Institute, Jodhpur, Rajasthan 342003, IndiaPunjab Agricultural University, Ludhiana 141004, IndiaJ. N. Vyas University, Jodhpur, Rajasthan 342005, India

r t i c l e i n f o

rticle history:eceived 7 January 2014eceived in revised form 24 January 2014ccepted 27 January 2014vailable online 3 February 2014

eywords:rid soils

a b s t r a c t

ZnO nanoparticle induced exopolysaccharide (EPS) production from Bacillus subtilis strain JCT1 (NCBIGenBank Accession No. JN194187) is a novel approach for arid soil applications. In the series of inves-tigations, environmentally benign protocol was followed for the synthesis of ZnO nanoparticles usingextracellular enzymes obtained from Aspergillus fumigatus TFR8. Putative characterization techniqueswere employed for confirmation of size, shape, surface structure, crystalline nature and elemental pro-portion of ZnO nanoparticles. Results established an average size of ZnO nanoparticles to be 2.9 nm atleast at one dimension and oblate spherical in structure. The qualitative composition of the nanoparticles

xopolysaccharidenO nanoparticles

exhibited 97.5% Zn element atom percentage. Biosynthesized ZnO nanoparticles enhanced exopolysac-charide production by 596.1% as compared to control and further EPS amelioration led to enhancedsoil aggregation (up to 82%), moisture retention (10.7–14.2%) and soil organic carbon. Soil aggre-gation stability was further confirmed by Fourier transform infra-red spectroscopy. A possible ZnOnanoparticle mediated biological mechanism for enhancing exopolysaccharide production has beendiscussed.

. Introduction

Polysaccharides are macromolecules synthesized and secretedy several microorganisms including fungi, bacteria and yeasturing their growth. Exopolysaccharides (EPS) play importantiological roles in all living beings [1]. These are the integralonstituents of membrane and cell wall, provide physical andhemical protection from the environment, ensure nutrition stor-ge, compose antigens that increase or repress defense strategyuring infection, affect protein folding, help in molecular recogni-ion (bio-film formation/Quorum sensing) and cell adhesion [2,3].

n nature these EPS are produced as a result of microbial exudates,lays pivotal role in defense mechanism for environmental influ-nces, bacteriophage attack and attachment to surfaces, nutrient

∗ Corresponding author at: Department of Energy, Environmental & Chemicalngineering, Washington University in St. Louis, Brauer Hall, Room 3029, One Brook-ngs Drive, Campus Box 1180, St. Louis, MO 63130, USA. Tel.: +1 (314) 748 0038.

E-mail addresses: rameshraliya@gmail.com, rameshraliya@seas.wustl.eduR. Raliya).

141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2014.01.060

© 2014 Elsevier B.V. All rights reserved.

gathering and antigenicity [4,5]. Microbial polysaccharide hasindustrial application as food supplements, cosmetics, pharma-ceutical and oil, used as thickening, stabilizing and emulsifyingagents [6]. About 20 different types of microbial polysaccharidesof commercial importance have been reported but still constitutesmall fraction of the total polysaccharides used [7].

Besides industrial application, recently microbial EPS have alsobeen used for the stabilization of deserts by trapping and retain-ing humidity in sandy soils, that helps in the increasing moistureretention capability in the upper layers of sandy soils and reduc-ing water infiltration, protecting the soil from erosion [8] becauseof its high viscosity, pseudo plasticity and the rheological proper-ties over wide range of temperatures and pH [9]. Many metals andmetalloids (e.g., Zn, Cu, Mn) are essentially required for the func-tioning of living organisms as micronutrients serve as structuralcomponents of proteins and pigment, used in the redox processes,regulation of the osmotic pressure, maintaining the ionic balance

and enzyme component of the cells [10]. Although some metalssuch as Zn are essential trace elements for bacterial growth, but atits high concentrations most bacterial growth is inhibited. In somecases, proteins possess functional groups that have strong affinity

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R. Raliya et al. / International Journal of B

or metals (Fe2+, Cu2+ and Zn2+). Additionally, metals are needed fornzymes at catalytic or functional sites, and as co-factors in catalyticrocess. Nevertheless, the effect of zinc nanomaterial at nontoxiconcentrations focusing on bacterial extracellular polysaccharideroduction is not fully addressed. Keeping this in view, the present

nvestigation was undertaken to study the role of zinc oxide (ZnO)anoparticles in EPS production by bacteria Bacillus subtilis strain

CT1 for its application in arid soils. A possible ZnO nanoparticleediated biological mechanism for enhancing exopolysaccharide

roduction has been discussed.

. Materials and methods

.1. Isolation of B. subtilis strain JCT1

The bacteria, B. subtilis strain JCT1 (NCBI GenBank Accessiono. JN194187) was isolated from plant rhizospheric soil of cen-

ral agricultural research farm (26◦18′ N 73◦ 01′ E) of Central Aridone Research Institute (CAZRI), Jodhpur, India. To isolate bacteria,oil samples were collected in sterilized polyethylene bags. Oneram of soil was mixed thoroughly with 10 mL of autoclaved dou-le distil water and 1 mL aliquot was dispensed and incubated inutrient broth (HiMedia, India) for 24 h at 37 ◦C. After incubation,0 mL of the culture was inoculated in 90 mL of fresh Minimal Saltsedium containing 0.5 g K2HPO4, 0.04 g H2PO4, 0.1 g NaCl, 0.002 g

aCl2·2H2O, 0.2 g (NH4)2SO4, 0.02 g MgSO4·7H2O, 0.001 g FeSO4 periter. pH of the medium was adjusted to 7.0 and incubated at 37 ◦Cn BOD incubator shaker (make Surana Scientific) at 125 rpm. Bac-erial culture was further spread by streaking on nutrient mediaHiMedia) followed by incubation at 37 ◦C in BOD incubator. Indi-idual colony picked up and sub-cultured in broth media for furthertudy.

.2. Morphological and biochemical characterization of bacteria

The bacterial strain was identified through colony morphol-gy, microscopic examination, and biochemical characteristics.ram staining, morphology and motility were observed by Opticalesearch microscope using phase contrast condenser at 100× oil

mmersion objective (Leica DM 5000B, Germany). Physiological andiochemical properties examined following methods suggested bymibert and Krieg [11].

.3. Molecular characterization of B. subtilis strain JCT1

The molecular identification of isolated bacterial strain was car-ied out on the basis of 16S rRNA gene sequencing using universalrimers viz., EUB1 and EUB 2. Each amplification was performedsing PCR in a total volume of 50 �L containing 0.2 �L Taq DNAolymerase (5 U/�L of Promega), 5 �L of 10× PCR buffer (10 mMris HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2, Sigma Chem.), 0.4 �LNTP mix 2.0 mM each A, T, C, G (MBI Fermentas), 1 �L of eachUB 1 and EUB 2 primers, 2 �L of 5% (v/v) glycerol, 4 �L of genomicNA and 36.4 �L dH2O. The reactions were performed in a ther-al cycler (Corbett Research, USA) with following conditions i.e.

min denaturation at 95 ◦C, 30 s annealing at 50 ◦C, 1 min 20 slongation at 72 ◦C, repetition 34 times with a final elongationtep of 10 min at 72 ◦C. The PCR products were visualized on 1%w/v) Agarose gel in Tris–acetic acid EDTA (1× TAE) buffer at 60 Vor 100 min. Agarose gel was stained with ethidium bromide and

hotographed under UV light using gel documentation systemSyngene, USA).

Each amplified PCR product was subjected to direct sequenc-ng using big dye terminator method on ABI prism DNA sequence.

cal Macromolecules 65 (2014) 362–368 363

DNA amplified fragments were about 993 bps. Nucleotide sequencecomparisons were performed using Basic Local Alignment SearchTool (BLAST) network services of the National Center for Biotech-nology Information (NCBI) database. The bacterial strain designatedto the sequenced analyses based on similarity with the best alignedsequence of BLAST search. The complete sequence of B. subtilisstrain JCT1 was submitted to NCBI database and Gen Accessionnumbers obtained.

2.4. Synthesis and characterization of ZnO nanoparticles

Synthesis of ZnO nanoparticle was carried by the method pre-viously reported from our laboratory [12]. Details of nanoparticlebiosynthesis are described under supplementary materials of thisarticle. Biosynthesized ZnO nanoparticles were characterized byDynamic Light Scattering analysis (DLS; Beckman DelsaNano C,USA) to determine the hydrodynamic particle size which is use-ful to predict behavior and transport of ZnO nanoparticles incomplex soil and biological/cellular systems. Transmission Elec-tron Microscopy (TEM JEM-2100F) measurements were carriedout using drop method at 200 kV accelerating voltage to con-firm the size and shape of the synthesized ZnO NPs. ScanningElectron Microscope (SEM, model Hitachi S-3400-N) was per-formed to determine the surface topography. Instrument wasoperated at 30 kV accelerating voltage. Elemental analysis on sin-gle particles was carried out using Thermo-Noran EDS attachmentequipped with TEM (JEM-2100F). It was performed for determi-nation of the elemental composition and purity of the sample byatom%.

2.5. Production and quantification of EPS

B. subtilis strain JCT1 (NCBI GenBank Accession No. JN194187)was grown in 100 mL of synthetic medium for EPS productioncontaining 20 g sucrose, 5 g yeast extract, 3 g malt extract, 5 gpeptone, 1.5 g MgSO4·7H20, 0.3 g KH2PO4, and 8 mg biosynthe-sized ZnO nanoparticle (2.9 ± 0.2 nm size and oblate spherical inshape) per liter of distilled water in 500 mL Erlenmeyer flask, pHof the medium was adjusted to 6.8 and kept on a rotary shaker(150 rpm) at 37 ◦C for 48 h. After incubation bacterial cells wereseparated from the culture media by centrifugation (6000 rpm,10 min at 4 ◦C) by addition of 0.5 mL of Tri chloro acetic acid(TCA) to precipitate extracellular proteins that is and separatedby centrifugation (8000 rpm, 10 min at 4 ◦C). Subsequently, 1 mLof protein free supernatant was used for EPS quantitative anal-ysis by Anthrone – sulphuric acid method suggested by Yemmand Willims [13] and remaining solution was added with doublevolume of chilled ethanol to precipitate. Polysaccharide precip-itate was dried under hot air oven at 55 ◦C till constant weightof EPS powder was obtained for further study of EPS potencytest.

2.6. Physicochemical characteristics of soil used for the study ofEPS potency test

The experimental soil was collected from Central Research Farmof Central Arid Zone Research Institute (26◦18′ N 73◦01′ E), Jodhpur.The climate of the region was arid with a mean annual precipitationof 360 mm and loamy sand soil (hypothermic typic haplocambids).EC (soil/water ratio 1:2.5) and pH of the soil samples were deter-mined by Conductivity Bridge and a glass electrode, respectively.Organic carbon (OC) was estimated following the method of Walk-

ley and Black [14] using 1 N potassium dichromate and back titratedwith 0.5 N ferrous ammonium sulphate solution. N, P and K in soilsamples were estimated as described by Jackson [15]. Data charac-terizing the soils are given in Table 1.

364 R. Raliya et al. / International Journal of Biological Macromolecules 65 (2014) 362–368

Table 1Characteristics of the experimental soil used.

Parameter Quantity

Sand (%) 85.1 ± 2.2Slit (%) 6.9 ± 1.5Clay (%) 7.5 ± 1.0pH 8.1 ± 0.1EC (dS m−1) 0.3 ± 0.05Organic matter (%) 0.35 ± 0.03Total N (mg kg−1) 462 ± 18.5Total P (mg kg−1) 733 ± 23.5

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Table 2Morphological and biochemical characterization of isolated bacterial strain JCT 1.

Characteristics Results

Colony morphological featureShape RoundSize LargeColor WhiteSurface DullMargin EntireCell morphology BacillusGram staining +

Biochemical testsCasein hydrolysis +Starch hydrolysis +Lipid hydrolysis +

Total K (mg kg−1) 695 ± 21.9

.7. Potency test of nanoparticle induced EPS for soil aggregationtability, moisture retention and organic carbon buildup

ZnO nanoparticle induced EPS was used to study the potency foroil aggregation, moisture retention and organic carbon buildup inrid soil. Soil aggregation was measured according to method sug-ested by Rennie et al. [16]. Aggregate analysis was conducted onpray-wetted soil samples. The percentage of water stable aggre-ates >0.01, >0.5 and 0.18 mm in diameter were used as an indexf sol aggregation.

To study effect of EPS on moisture retention, 1–6% of the EPSbtained from B. subtilis strain JCT1 (NCBI GenBank Accession No.N194187) were mixed with 50 g of soil taken in to 100 mL capacitycrew cap polypropyline containers. The contents were moistenedt 60% water holding capacity and EPS mixed for 2 h on spin-ing mixer. The moisture contents of treated and untreated soilamples were determined at 110 ◦C by an closed chamber thermoalance moisture analyzer (i-Thermo 163M, Columbia, MD) at dif-erent time intervals until a complete release of the water appliedo thesoils was achieved.

Changes in soil organic carbon (SOC) were estimated by follow-ng the method of Walkley and Black [14]. Changes in the bondtrength and vibration among soil particles owing to addition ofPS were further studied by FTIR analyses.

.8. Fourier transform infrared (FTIR) study

To study the aggregation of soil particles owing to addition ofanoinduced EPS was further confirmed by FTIR analyses. For FTIRach sample was prepared in IR grade potassium bromide (KBr;igma Aldrich, St. Louis, MO, USA) as a pellet under 1:99 ratio ofample and KBr, and it was recorded by ABB FTLA 2000-100 (Que-ec, Canada) at a resolution limit of 16 cm−1 [17].

. Results and discussions

.1. Isolation of EPS producing bacterial strain

Serially diluted soil suspension on nutrient agar plates werellowed to grow for 24 h at 37 ◦C produced nine different bacte-ial colonies. After enrichment for 25 days with minimal media,nly one bacterial strain was found to have the ability to producehe maximum amount of EPS, designated as strain JCT 1.

.2. Morphological and biochemical characterization of bacteria

Morphological and biochemical characteristic features of iso-ated bacterial strain JCT 1 are given in Table 2. Studied bacterialtrain JCT 1 exhibited white and round shape colony on nutrientgar plate. Microscopic examination revealed cell morphology with

Oxidase +Catalase +Nitrate reduction +

positive gram staining, and showed positive response for casein,starch and lipid hydrolysis as well as for nitrate reduction, oxidaseand catalase.

3.3. Molecular characterization of strain JCT1

Molecular characterization of B. subtilis strain JCT 1 was per-formed on the basis of 16S rRNA gene sequencing. Forward primerEUB1 (5′-AGA GTT TGA TCC TGG CTC A-3′) and reverse primerEUB 2 (5′-GCT CGT TGC GGG ACT TAT CC-3′) were used forselective gene amplification. The sequence was compared usingBLAST services of NCBI and the submitted sequence (NCBI Gen-Bank Accession No. JN194187) is now available in public domainhttp://www.ncbi.nlm.nih.gov.

3.4. Synthesis and characterization of ZnO nanoparticles

The extracellular synthesis of ZnO nanoparticles was carriedout by exposure of a precursor salt zinc nitrate (ZnNO3) solutionof 0.1 mM concentration to fungal cell-free filtrate obtained byincubating the fungus Aspergillus fumigatus TFR-8 in an aqueoussolution. The reaction was carried out for 72 h.

Particle size of biotransformed ZnO nanoparticles was analyzedby DLS using particle size analyzer (Fig. 1a–c). Histogram of inten-sity (Fig. 1a), number (Fig. 1b) and volume distribution (Fig. 1c)shows particle size ranges from 1.4 to 5.3 nm and possess anaverage size of 2.9 nm. The poly-dispersity index (PDI) was 0.128shows high mono-dispersity of the particle in the solution. Inten-sity distribution histogram shows noise peak of agglomeration dueintermolecular multiple weak interactions, that was confirmed bynumber and volume distribution histograms. The size was furtherconfirmed by TEM and SEM analysis.

TEM measurements were used to determine the morphologi-cal i.e. size, shape etc. study of biosynthesized ZnO nanoparticles.A TEM micrograph (Fig. 2) showed homogenous distribution ofZnO nanoparticle at the measurement scale bar of 50 nm. Insetof Fig. 2 shows magnified micrograph of ZnO NPs confirms shapeand size. The nanoparticles were oblate spherical with clear edgeof crystal and lattice structure observed in HR-TEM micrograph(sub inset of Fig. 2), which supports the crystalline nature of ZnOnanoparticle.

SEM micrograph revealed the structural and topology of ZnOnanoparticles (Fig. 3). The EDS spectrum (full scan mode) of dropcoated ZnO nanoparticle shown in Fig. 4, confirms the purity of

ZnO nanoparticles. The spectrum shows strong peak intensity ofZn (97.5 ± 0.02 atom%) at 1.1 keV. Oxygen peaks were also foundat 0.6 keV confirms the presence of oxygen molecule because of itsoxide form of Zinc.

R. Raliya et al. / International Journal of Biological Macromolecules 65 (2014) 362–368 365

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Fig. 1. Histogram of particle size distribution obtained from DLS (a) i

.5. EPS production, quantification and potency test for soilggregation, moisture retention and soil organic carbon contents

n arid soils

The effect of ZnO nanoparticle induction for enhancing the pro-uction of EPS in B. subtilis strain JCT 1 was studied (Fig. 5a). It

ity distribution, (b) number distribution and (c) volume distribution.

was observed that addition ZnO NPs (2.3 nm with oblate sphericalshape) in minimal media tremendously enhances quantity (596.1%

over control) of EPS production from bacterial strain JCT 1 (Table 3).Produced EPS (Fig. 5b) was dried under hot air oven to get EPSin powder (Fig. 5c) form for further application in soil study. Itwas observed that when 1% of EPS powder was mixed with arid

366 R. Raliya et al. / International Journal of Biological Macromolecules 65 (2014) 362–368

Fig. 2. TEM micrograph of biosynthesized ZnO nanoparticles used for enhancing theproduction of bacterial EPS; inset of picture showing magnified image of ZnO NPs;sub-inset picture showing HR-TEM micrograph, supports crystalline nature of NPs.

Fig. 3. SEM micrograph of ZnO nanoparticles.

Fig. 4. EDX spectrum showing elemental proportion of biotransformed product.

Table 3Effect of ZnO NPs on production of EPS by Bacillus subtilis strain JCT1.

Treatments EPS contents (�g mL−1)

Control 402.7ZnO NPs induced 2803 (596.1)a

a Figure in parenthesis indicate % increase over control.

Table 4Effect of nano-induced EPS powder on soil aggregation stability.

% EPS applied % improvement of aggregate size over control after30 days (1%, w/v)

1.0 mm 0.5 mm 0.18 mm

1 33.4 82.9 56.4

Table 5Effect of ZnO NPs induced EPS powder on improvement in soil moisture retentiona

in arid soils.

EPS percentage Improvement inmoisture retention (%)

1 10.72 12.23 12.54 12.85 13.66 14.2

LSD (P = 0.05) 2.3

a After 4 weeks of application.

soil, significant improvement was observed in soil aggregation sta-bility (Table 4). The maximum aggregation stability was found at>0.5 mm after 30 days of EPS application. A clear visual differencein soil aggregated owing to EPS addition can also be seen in Fig. 6.In arid soil, moisture is a major factor that determines interactionof various biotic communities in plant rhizosphere. Upon additionof EPS (1–6%) in arid soils, moisture retention was also found to beincreased from 10.7 to 14.2% (Table 5). Further addition of EPS doesnot show any significant effect on moisture retention. EPS play veryimportant role in enhancement of SOC in arid soil probably dueto carbon skeleton of the polymeric chain in its constituents. AsEPS concentration increased SOC was found to be increase simulta-neously (Table 6). The enhanced SOC content decreased after 7 daysand thereafter continuously either due to degradation of polymericsubstances or fixation of carbon contents.

3.6. FTIR study of aggregated soil in response of EPS

FTIR analysis was performed to confirm the interaction of soilparticles by shifting and formation of bond among soil particlesowing to ZnO NPs induced EPS addition. FTIR analyses were carriedout with different time intervals i.e. 7, 14, 21, 28 days after additionof EPS (Fig. 7). It was observed that as number of day’s increases,new strong % transmission peak formation was fond at 802 cm−1,

1256 cm−1, 1504 cm−1, 1813 cm−1 and 2992 cm−1 which corre-sponds to C H bending, C N stretch, C C stretch, C O stretchand O H stretch respectively.

Table 6Effect of nano-induced polysaccharide powder on soil organic carbon build up.

% EPS applied % Improvement of organic C in soil (days after application)

7 14 21 28

1 41.0 30.6 27.1 21.04 61.0 52.0 47.3 36.85 63.0 54.0 48.5 44.6

R. Raliya et al. / International Journal of Biological Macromolecules 65 (2014) 362–368 367

Fig. 5. Steps of EPS powder production, (a) bacteria strain JCT 1 spread over agar media, (b) flocculent of EPS in ethanol and (c) dehydrated EPS powder.

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including temperature, pressure and light intensity [22–24]. TheseEPSs affect the way in which microorganisms interact with theexternal environment, whether the environment is liquid orsolid.

Fig. 6. Arid soil after 30 days of treatment, (A) soil m

.7. Probable biological mechanism to explain the enhancedroduction of EPS by bacteria

To understand the mechanism for enhanced EPS production byacteria, we draw a probable biological mechanism (Fig. 8) in whichnO nanoparticles escalate the activities of various trans-peptidasenzymes involved in cell wall formation of bacteria, and may alsonhance expression level of genes involved in cell wall polysac-haride formation. At molecular level, gene expression of wzz andzy gene leads to polysaccharide formation in bacterial cell [18,19].ole of zinc as cofactor for DNA and RNA polymerase enzyme isell known [20]. Nano ZnO may enhance the activity of these

nzymes. Below toxic concentration of zinc essential for bacterialrowth [21] as it play pivotal role in physiological and to accom-

lish nutritional demands. The physiological role of EPSs dependsn the biotope of the microorganisms producing them. EPS pro-uction is a direct response to selective environmental pressures,

ig. 7. FTIR analysis of soil sample mixed with EPS, (A) 7 days, (B) 14 days, (C) 21ays and (D) 28 days after treatment.

ith 1% EPS and (B) soil mixed with water (control).

Fig. 8. Hypothetical mechanism to explain duel role of ZnO NPs for enhancing EPSproduction by bacterial strain JCT 1, 1: monomers of sugar, 2: polymeric sugarresidues linked by glycosidic bonds, 3: enzymes responsible for polymerizationof sugar by forming glycodic bonds that catalyzed by ZnO NPs, 4: techoic acid, 5:repeated subunits of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM)joined by peptide chain, 6: protein embedded in lipid bilayer 7: lipid bilayer of bac-terial plasma membrane, 8: polymerase enzyme involved in synthesis of RNA, 9:ZnO NPs inside bacterial cell, play catalytic role, 10: m RNA for further synthesis ofenzyme involved in carbohydrate subunit polymerization.

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[25] A.L. Godinho, S. Bhosle, Curr. Microbiol. 58 (2009) 616–621.

68 R. Raliya et al. / International Journal of B

Because of inherent charge capacities and the nature of theharge present at surfaces and edges, polymeric flocculation of thelay fraction of the soil at certain specific pH of the solution or itsnteraction with organic colloids results in a polymeric stability ofhe soil aggregates. Similarly ZnO NPs induced EPS also enhancesrid soil aggregation stability which helps greatly in curbing soilrosion particularly from the desert area. According to Godinhond Bhosle [25], a polymeric weak stability phenomenon occurshen the addition of EPS in a soil colloid system causes the partiale flocculation of the clay dispersion. Further FTIR study revealednd supports the obtained results by correspondence shifting of IReaks owing to stretching and bending of various bonds i.e. C H,

N, C C, C O, O H etc.EPS can provide a microenvironment that holds water and dries

ore slowly than its surroundings [26]. Soil is an extremely het-rogeneous environment, and wetting and drying do not proceedniformly throughout it and rhizospheric communities depend onhis heterogeneity. The present results of increased moisture reten-ion up to 14.2% in arid soils may help in microbial communitytructure build up that often play role in nutrient mobilization forlants and other biotic communities.

Soil organic carbon, the major component of soil organic mat-er, is extremely important in all soil processes. Organic material inhe soil is essentially derived from residual plant and animal mate-ial, synthesized by microbes and decomposed under the influencef temperature, moisture and ambient soil conditions [27,28]. EPSound as an alternative option to enhance SOC in arid soils whichre often low in SOC. Increase in C may leads to higher productionnd microbial activity in the rhizosphere for nutrient recycling andobilization.

. Conclusion

In the present study nanoparticles of ZnO were biosynthesizedy fungal enzymes and used for enhancing EPS production by B.ubtilis strain JCT1 (NCBI GenBank Accession No. JN194187) in aridoils. Biosynthesized ZnO nanoparticles enhanced EPS productiony 596.1% and improved soil aggregation, moisture retention andoil organic carbon. Soil aggregation stability was validated byTIR spectroscopy. A possible biological mechanism for enhancingPS production in response to ZnO nanoparticles is also conferred.indings of present study may be of significance in containingesertification and fortification of arid soils.

onflicts of interest

The authors declare that they have no conflicts of interest.

cknowledgements

Authors are appreciative of World Bank – Indian Council of Agri-ultural Research (ICAR), National Agricultural Innovation Project

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cal Macromolecules 65 (2014) 362–368

(NAIP/C4/C-2032) for financial assistance. RR is thankful to CAZRIJodhpur for providing lab space. Authors are thankful to AIRF, JNU,Delhi; EMN Lab, PAU, Ludhiana for TEM characterization and SEM-EDS characterization, respectively.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2014.01.060.

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