Accepted Manuscript Extracellular polymeric substances of a marine bacterium mediated synthesis of CdS nanoparticles for removal of cadmium from aqueous solution Ritu Raj, Kalpana Dalei, Jaya Chakraborty, Surajit Das PII: DOI: Reference:

S0021-9797(15)30249-6 http://dx.doi.org/10.1016/j.jcis.2015.10.004 YJCIS 20790

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Journal of Colloid and Interface Science

Received Date: Accepted Date:

27 August 2015 3 October 2015

Please cite this article as: R. Raj, K. Dalei, J. Chakraborty, S. Das, Extracellular polymeric substances of a marine bacterium mediated synthesis of CdS nanoparticles for removal of cadmium from aqueous solution, Journal of Colloid and Interface Science (2015), doi: http://dx.doi.org/10.1016/j.jcis.2015.10.004

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Extracellular polymeric substances of a marine bacterium mediated synthesis of CdS nanoparticles for removal of cadmium from aqueous solution Ritu Raj#, Kalpana Dalei#, Jaya Chakraborty and Surajit Das* Laboratory of Environmental Microbiology and Ecology (LEnME), Department of Life Science, National Institute of Technology, Rourkela- 769 008, Odisha, India *Corresponding author. Tel. +91661 2462684; Fax. +91661 2462022 E-mail address: [email protected] or [email protected] (S. Das). #contributed equally Abstract Hypothesis: Microbial extracellular polymeric substances (EPS) are natural metal adsorbent and effective bio-reductant. In the biosynthesis of CdS NPs, functional groups of EPS act as capping and stabilizing agents. The NPs enriched EPS have enhanced adsorption capacity of cadmium ions, which may be due to increased adsorptive sites. Experiment: The current study demonstrates, an efficient biosynthesis method to prepare CdS NPs using EPS extracted from a marine bacterium Pseudomonas aeruginosa JP-11 and its comparison with chemical method using D-glucose. The synthesized NPs were characterized by Ultraviolet–visible spectroscopy, ATR-FTIR spectrometry, X-ray diffraction, Field emission scanning electron microscopy and Transmission electron microscopy. Atomic absorption spectroscope (AAS) was used to study adsorption capacity of pristine EPS, functionalized EPS and NPs incorporated functionalized EPS. Findings: Spherical CdS NPs of 20-40 nm diameter were synthesized with high crystallinity confirmed by XRD, FESEM and TEM analysis. The ATR-FTIR peaks within 2300 to 2600 cm-1 range showed a prominent shift in sulphydryl group (-SH). The cadmium removal efficiency by CdS NPs incorporated functionalized EPS (88.66 %) was higher than functionalized EPS (80.81%) and pristine EPS (61.88%) after 48 h of incubation. The experimental data of adsorption thermodynamics and kinetics of Cd by NPs incorporated functionalized EPS was fitted in Langmuir isotherm model. Keywords: Nanoparticles, Marine bacteria, Extracellular polymeric substances, Cadmium sulphide, functionalization. 1 Introduction Cadmium sulphide (CdS) nanoparticles (NPs) belong to II-VI group semiconductor that has discrete energy levels, tuneable band gap with good chemical stability can be used as

potential biosensors [1]. Synthesis of CdS NPs by physical top-down or chemical bottom-up approaches are usually not favourable for biological uses due to lack of homogeneity and residual toxic elements respectively [2]. However, the biological process of CdS NPs synthesis involves plants, microorganisms and their products which are usually non-toxic, less expensive and more environment-friendly. Biosynthesis of CdS NPs has been achieved by using microorganisms that grab the Cd+2 ions from their metal solution and accumulate them in a reduced state by various intracellular or extracellular enzymes and metabolites [3]. Intracellular synthesis of CdS NPs by several microorganisms has been reported, for example Schizosaccharomyces pombe and Candida glabrata (yeasts) and Escherichia coli [4, 5]. Extracellular synthesis of CdS NPs have also been shown in Clostridium thermoaceticum, Klebsiella pneumonia, Rhodobacter sphairoides, bacterial cellulose nanofibres matrix and Aspergillus versicolor fungal matrix [6-10]. Besides several reports on microbial assisted synthesis of CdS NPs, none has explored the extracellular synthesis process of CdS NPs by bacterial extracellular polymeric substances (EPS). EPS are high molecular weight polymers of long chain sugar residues with proteins and nucleic acids secreted into the surroundings by bacteria [11]. Apart from being a protective measure for bacteria, it has many other utilities as a surface adherent and stabilizer that make it useful for several industrial applications like emulsification, gel formation, film formation, absorption and bioremediation [12]. Bacterial EPS due to charged functional groups, have a specific adsorptive and adhesive property that helps in binding of other charged moieties including metal ions [13]. This metal binding and high adsorption capacity of EPS has paved the way for its applications in the bioremediation of different toxic metals like nickel, mercury and cadmium [14]. Biologically active multitasking bacteria apart from being a preferred choice for nanoparticle synthesis also have a very important role in the remediation of contaminants from environment [15]. Among which, cadmium (Cd) is one of the most toxic metal found as an industrial effluent [16]. Cadmium ions are non-biodegradable in nature and accumulated in living organisms at a higher concentration causing various health hazards [17, 18]. Microbial bioremediation assisted with EPS is a new promising eco-friendly approach for removing these toxic metals from the environment. However, large scale site-specific remediation of toxic metals for aquatic system needs high affinity towards metal and a short time to reach equilibrium. To enhance the adsorption capacity of EPS, its surface functionalization with suitable metal binding groups is a recommended measure [19]. Generally, amines, polyethylamine, crown ethers, sulphur bearing groups like thiols, dithiophosphate and xanthate 2

have been used for functionalization of a substrate. In the previous reports, mycelia of Aspergillus versicolor fungus has been surface functionalized to increase cadmium ion adsorption along with the synthesis of CdS NPs [10]. There has been no such report on bacterial EPS based CdS NPs synthesis and cadmium ion adsorption from aqueous solution. The current study presents a sustainable eco-friendly biological mode of synthesis of CdS NPs and simultaneous removal of metal ions from the aqueous solutions. The batch adsorption study of cadmium ions from aqueous solution and its removal efficiency by different adsorbents (pristine EPS, functionalized EPS and CdS NPs incorporated functionalized EPS) showed significant practical application of EPS in terms of biosynthesis of NPs and bioremediation of heavy metals. 2 Experimental methods Chemicals Cd (NO3)2.4H2O and Na2S were purchased from Merck (Germany). Microbiological media were procured from Himedia (India). All the reagents were of analytical reagent (AR) grade and used without further purification. Production and extraction of EPS from Marine Bacteria EPS producing and biofilm forming marine bacterium Pseudomonas aeruginosa JP-11 (NCBI GenBank accession number KC771235) was cultured aerobically for 24 h in Luria Bertani (LB) broth at 37°C with continuous shaking at 120 rpm [35]. The bacterial culture was then centrifuged at 6900 rpm for 15 min to collect cell pellet. The cell pellet was again inoculated in Minimal Broth Davis (MBD) medium supplemented with filter sterile 1% glucose and 20 mM CaCl2 solution and incubated at 37°C with shaking at 120 rpm. The cells were grown up to 48 h and after that the culture was centrifuged at 6900 rpm for 30 min at 4°C to separate the cell pellet. In the obtained supernatant, an equal volume of chilled ethanol (99.9%) was added slowly and incubated overnight at 4°C for precipitation of EPS. After incubation, EPS pellet was collected by centrifugation at 6900 rpm at 4°C for 20 min and further dried in a desiccator. Xanthate Functionalization of Pristine Bacterial EPS Following the method reported by Panda et al. [14], 750 µL of 4M NaOH solution was added in extracted dried EPS (50 mg) followed by 50 µL carbon disulphide and incubated for 5 h at 120 rpm and 30°C. After incubation, functionalized EPS was collected as yellow pellet by centrifugation. The residues were washed thoroughly with double distilled water and finally dried by washing with acetone. The dried functionalized EPS was then stored in the refrigerator for further use. 3

Synthesis of Cadmium sulphide (CdS) NPs Cadmium nitrate [Cd (NO3)2] as a source of Cd +2 ion and sodium sulphide (Na2S) as a chalcogen ion S -2 source were used as precursors for the synthesis of CdS NPs. D-glucose (C6H12O6) or EPS was used as a capping agent for the reaction mixture. In the freshly prepared Cd (NO3)2 solution, an equal volume and concentration of Na2S solution was added drop wise with vigorous continuous stirring at 50°C for 20h. This was followed for three concentrations (0.1M, 0.01M, and 0.001M) of both precursors in six set with 1 mg and 2 mg D-glucose and EPS. The colour of reaction mixture is changed from colourless to pale yellow, then glucose or EPS (1 mg and 2 mg) was added to the solution that turned it into orange yellow colour. After 20 h of stirring, the solution was centrifuged at 12000 rpm at 25°C for 30 min to collect the pellet. The obtained pellet was washed three times with ethanol (99.9%) and lyophilised for further use. Estimation of EPS constituents The carbohydrate, protein and nucleic acid (DNA) quantification of extracted pristine EPS were estimated by phenol sulphuric acid method, Bradford method and diphenylamine method respectively [36-38]. Functionalized EPS as a precursor and capping agent 20 mg of functionalized EPS was added to 10 mL of 0.01M Cd (NO3)2 solution and vigorously stirred at 50°C for 20 h. After 20 h of stirring, the pale yellow solution was centrifuged at 12000 rpm at 25°C for 30 min. The pellet was collected and washed three times with ethanol (99.9%) and then lyophilised for further use. Batch adsorption experiment for cadmium removal from aqueous solutions Cadmium adsorption capacity of pristine EPS, functionalized EPS and NPs incorporated EPS as substrate were conducted through batch adsorption experiments. The optimum pH for adsorption was determined by suspending 4 mg/mL pristine EPS and functionalized EPS in 15 mL falcon tubes containing 50 ppm cadmium ions of different pH values (2.0-7.0) where, 0.1M citric acid and 0.2M dibasic sodium phosphate were used to obtain the desired pH. The adsorption experiments were performed by continuous mechanical shaking of 50 mg of desired adsorbent (pristine EPS, functionalized EPS and NPs incorporated EPS) in aqueous solution of desired concentration (25, 50, 75 and 100 ppm) of cadmium salt at optimum pH at required temperature (298 K, 308 K and 318 K) for 24 h and 48 h in triplicates [39]. The adsorption set up temperature was maintained by the use of a constant temperature water bath. After incubation, the solution was centrifuged to separate the adsorbent from the supernatant to measure the metal-ion concentration (Cd2+) by atomic absorption spectroscopy 4

(AAS) using flame ionization method. The amount of Cd bound with NPs incorporated EPS at equilibrium, qe (mg g-1) was calculated according to following equation. qe = (Co –Ce) V/m…………

(1)

Where, qe is the equilibrium adsorption capacity of Cd adsorbed on unit mass of adsorbate (mg g-1) here, NPs incorporated EPS; Co and Ce are the initial concentration of Cd in the solution and Cd concentration at equilibrium (mg L-1) respectively; V is the total volume of the solution (L), m the mass of the adsorbate (g). From the above studies, the effect of temperature on the equilibrium concentration of the sorbed Cd was studied to investigate the thermodynamics parameters by fitting experimental data into Langmuir isotherm model. The percentage removal capacity of different adsorbents was also calculated using the following equation. Re (%) = [(Co-Ce)/ Co] × 100………… (2) Where, Re is removal capacity at equilibrium.

3 Instruments Optical absorption spectra of the suspended samples were recorded using (UV/Vis spectrometer, Perkin Elmer, USA) double beam spectrophotometer in the range 700–200 nm. X-ray powder diffractograms (XRD) of the lyophilized samples were taken on a Rigaku Miniflex, X-ray diffractometer (Japan). The radiation source used was CuKα (λ =1.542 Å), operating at 40 kV-30mA. FTIR analysis of suspended samples were taken by (ALPHA‘s Platinum ATR, Bruker, Germany) single reflection diamond module in the range of 4000-500 cm-1. FESEM observations of the samples were performed using Nova Nano SEM Field Emission Scanning Electron Microscope (USA). The atomic composition of the samples were analysed by Energy Dispersive X-ray Spectroscopy (EDX, Hitachi, Japan) attached with FESEM. The transmission electron microscopy (TEM, JEOL JEM 2100 HR, USA) images of the CdS NPs were obtained by dispersing samples in Milli-Q water, and a drop of the solution was casted on a carbon-coated copper grid. A completely dried sample was examined in TEM sample chamber at 200 KV. Cadmium concentration in aqueous solutions was estimated by AAS (Atomic Absorption Spectrometer, iCE 3000, Thermo Scientific, India) using cadmium lamp at 228.8 nm with 0.2 nm band pass. Surface charge (Zeta potential) of pristine and functionalized EPS (dissolved in citrate phosphate buffer) at different pH were measured by using Nano Zetasizer (Nano series ZS90, Malvern Instruments Ltd., UK) at room temperature.

5

4 Results and discussion The addition of equal volume and concentration of Na2S solution into Cd (NO3)2 solution with continuous stirring changed the colour of Cd (NO3)2 solution from colourless to pale yellow. Further addition of D-glucose changed the colour to orange yellow that indicated the formation of CdS NPs. The addition of pristine bacterial EPS, instead of D-glucose has also yielded the same colour change pattern, confirming the synthesis of CdS NPs. The pale orange coloured NPs synthesized by these methods were collected by centrifugation and washed three times with ethanol (99.9%). Obtained precipitate was dried by lyophilisation and dispersed in Milli-Q water for characterization. 4.1 UV-Vis spectroscopy The optical absorption of materials in UV-vis region is a useful preliminary method to investigate the change in the size of materials based on their optical transition and electronic band structure. UV-Vis spectra of dispersed solutions were recorded by UV-Vis spectrophotometer in the range of 700–200 nm. The absorption maxima between 400-500 nm for analysed solutions had confirmed the formation of smaller particles of CdS (Fig. 1) which was much below than 512-515 nm absorption peaks for the bulk CdS [20, 21]. The blue shift observed in the UV-Vis spectrum was due to surface plasmon and quantum confinement effect of CdS NPs, which was in the accordance with the work carried out by Khan et al. (2012) [22]. Here, this blue shift could be attributed to the transition from CdS bulk material to CdS NPs in the presence of capping agent D-glucose or EPS which prevented the aggregation of NPs into bulk material [2]. This shift is consistent with the quantum confinement effect due to decreasing particle size as the higher concentration of capping agent is unfavourable to the nucleation and growth of CdS NPs. The synthesis of EPS capped CdS NPS involves the reaction between sulphur and cadmium ions, where electron-deficient atoms of cadmium serve as binding sites to anchor organic capping agent (EPS). The EPS hinders further growth of nanocrystal grains of CdS NPs by stabilizing its surface energy and these combined effects of sulphur and EPS resulted in the formation of nanosized crystals of CdS [23]. 4.2 X-Ray analysis The X-Ray diffraction (XRD) analysis was performed for all the NPs obtained with EPS and D- glucose after lyophilisation. The observed patterns of synthesized NPs also confirmed the formation of CdS in both the methods followed using D-glucose and pristine EPS. The XRD pattern exhibited diffraction peaks at the range of 26.33˚ to 26.70˚, 43.65˚ to 44.16˚ and 51.6˚ to 52.11˚ corresponding to (111), (220), (311) planes of cubic phase CdS NPs (Joint Committee for Powder Diffraction Standards (JCPDS) 10-454). The crystalline nature 6

was significantly influenced by the presence of capping agents. This result is in concert to the previously reported XRD patterns of CdS NPs synthesised by bacterial cellulose nanofibers [9]. It was also observed that the diffraction peaks in the presence of pristine EPS (1 mg and 2 mg) in 1mM solution of reactants [Cd (NO3)2 and Na2S] were broader compared to other samples (Fig. 2). This signifies the reduction in crystal size of the synthesized NPs and can be attributed by atomic structure studies of CdS NPs by calculating the crystallites size from diffraction peaks [24]. Based on Scherrer equation, the crystal size of synthesized CdS NPs were in the range of 21.54 - 38.28 nm and 24.14 - 38.81 nm for D-glucose and EPS synthesized CdS NPs respectively (Table 1). Based on calculation using the width of the (111), (220) and (311) peaks respectively, these results showed that pristine EPS yielded in similar size crystals for respective planes compared to D-glucose [25]. However, the presence of pristine EPS did not change the crystal structure of the prepared CdS NPs, which could be co-related to CdS NPs synthesis by organic ligand 3-mercaptopropionic acid (MPA) [23]. 4.3 ATR-FTIR analysis The capping effects of D-glucose and EPS molecules have been investigated by Attenuated Total Reflectance Fourier Transform Infra-Red Spectrometer (ATR-FTIR). The FTIR spectra of the CdS NPs synthesized by using D-glucose and pristine EPS were compared with pristine EPS, functionalized EPS and cadmium treated functionalized EPS (Fig. 3). The prominent peaks at the range of 3846.05 to 3617.66 cm-1 may be due to overlapping of O-H and N-H stretching vibrations, which were consistent at a range of 1011.47 to 1064.49 cm

-1

in

all the samples. In D-glucose, synthesized CdS NPs consecutive peaks at 1712.98 and 1680.35 cm-1 correspond to aldehyde group present in D-glucose γC=O. According to FTIR spectroscopic data table, the downfield shift peak between 2300 cm-1 to 2600 cm-1 showed the presence of -SH functional group. The CdS NPs synthesized by using D-glucose and EPS showed peak at 2345.15 cm-1, which also confirmed the presence of –SH group. Even pristine EPS showed minor peak at 2345.15 cm-1, an evidence of sulphydryl group. Surface functionalization of pristine EPS has shown –SH peak shifted upward and became more prominent at 2353.31 cm-1. The presence of sulphur groups in the functionalized EPS had been identified by the appearance of new peaks at 2304.37 and 664.80 cm-1 corresponding to γS-H and γC–S. The ATR-FTIR spectra thus confirmed the presence of sulphur group that can bind with cadmium and form CdS NPs. The N–H and C=O stretching vibrations shift from 1529.45 to 1525.37 and 1712.98, 1680.35 to 1708.90 and 1676.27 cm−1 was also observed. The absorption band of CdS stretch was not observed in the current scale of the spectrum as it appears at around 250 cm-1. The results showed that there exists no adsorption band for CdS 7

NPs in the current scale but a downfield shifts of peak at 2330-2360 cm-1 was seen, which corresponds to sulphydryl (-SH) functional group [26]. A previous study also deduced that this group acts as an important attachment group in the EPS [27]. The observed results confirm the capping effect of D-glucose and EPS molecules on CdS NPs. The sulphur group acts as the binding site for cadmium ion and upon binding synthesizes CdS NPs [10]. The proteins present in pristine EPS either contain the sulphur group or any other charged group which after binding with cadmium synthesizes the CdS NPs. 4.4 Electron microscopy The surface morphology study of CdS NPs synthesized by using D-glucose and pristine EPS was performed under Field Emission Scanning Electron Microscope (FESEM) (Fig. 4). The average size of CdS NPs synthesized using D-glucose was found to be 17 nm and 20 nm when D-glucose concentration was 1 mg and 2 mg respectively. However, the average size ranged between 22 nm and 24 nm were found when pristine EPS was used instead of Dglucose. The energy dispersive X-ray (EDX) analysis of synthesized CdS NPs from both the capping agents aforementioned had shown the presence of cadmium and sulphur in the analysed samples. FESEM images of pristine EPS, functionalized EPS and cadmium treated functionalized EPS showed morphological changes in the surface of EPS (Fig. 5). This assures the binding of sulphur groups to the surface of EPS after functionalization and synthesis of CdS NPs after treatment with Cd solution. The average size of CdS NPs synthesized by using D-glucose was comparable to those synthesized using pristine EPS. It was clearly noticed that microbial EPS can effectively synthesize CdS NPs with an average diameter of 20 nm comparatively smaller than synthesis by bacterial cellulose [9]. The FESEM images showed that the synthesized NPs were homogenous with regular shape and uniform morphology. EDX analysis confirmed the presence of Cd and S in the synthesized CdS NPs, however, there were prominent peaks of Ca, Na, Si and O, which might be due to glass surface (source of Si, and Ca) used for sample mounting, PBS buffer (source of Na) used in synthesis process as solvent. However, the presence of oxygen in samples might be due to surface bound water molecules [28]. Transmission electron microscope (TEM) analysis of CdS NPs at different magnifications was also performed to observe the fine details of morphology and crystal structure of synthesized CdS NPs. The obtained TEM images showed CdS NPS were embedded in EPS matrix and formed homogenous uniform sized NPs in the range of 20-40 nm that was in accordance with XRD and FESEM analysis (Fig. 6). The lattice parameters observed also suggested a high level of crystallinity as observed in XRD patterns [8]. The TEM 8

studies also confirmed the capping effect of EPS matrix which stabilized the size of CdS NPs. This observation coincides with FESEM and ATR-FTIR analyses of CdS NPs and EPS molecules. These results assured facile synthesis of homogenous CdS NPs of uniform size by microbial EPS compared to fungal biomass as reported by Das et al. [10]. 4.5 Batch adsorption studies To investigate adsorption capability of EPS of Pseudomonas aeruginosa JP-11, its carbohydrate, protein and nucleic acid (DNA) constituents were analysed. The total carbohydrate, protein and nucleic acid (DNA) contents were found to be 4919.18 μg/mL, 2160.92 μg/mL and 24.08 μg/mL respectively. The composition of extracted EPS has similarity to previously cited work, where carbohydrates (glucose and galactose) found as major EPS constituents [29]. This suggests carbohydrate and protein are having an important role in capping action and adsorption of metal. AAS analysis of samples after 24 and 48 h of incubation, showed a decrease in Cd concentration in solution with an increase in incubation time (Table 2). The percentage of cadmium removal by the pristine EPS, functionalized EPS and CdS NPs incorporated functionalized EPS at 24 h and 48 h were found to be 57.41%, 61.88%; 77.07%, 80.81% and 86.46%, 88.66% respectively. In the batch studies, pH was found to be an important factor in the adsorption of metal ions by changing the surface charge density of both adsorbent and adsorbate. Moreover, the metal speciation, sequestration, and mobility were strongly influenced by pH of the solution; with an optimum pH range of 4.0-7.0. It was observed that pH 4.6 was optimum pH for adsorption of metal ions because at this pH, the net charge on cadmium is positive which corresponds to strong affinity for the thio functionalized EPS as compared to non-functionalized EPS [10]. Zeta potential values of pristine and functionalized EPS at pH 2.6, 4.6 and 6.6 respectively have shown huge shift in surface charge at pH 4.6 compared to others, which also validate role of pH in adsorption (Fig. 7). Adsorption isotherms are used to predict favourable and feasible parameters for sorption. Therefore, in this study, Langmuir isotherm models are used to verify experimental adsorption data of cadmium metal ion concentration from 25 to 100 mg L-1 (Table 3). The experimental results obtained from adsorption kinetics were fitted in Langmuir model indicating that Cd adsorption was irreversible (Fig. 8). The obtained isotherm parameters very well supported the feasibility of adsorption capacity of NPs incorporated functionalized EPS (Table 4). Further study of Cd adsorption by NPs incorporated functionalized EPS at different temperatures suggested that an increase in temperature would eventually enhance adsorption capacity of the adsorbent. Van't

9

Hoff graph (between lnKL and 1/T) also supported exothermic adsorption of Cd by selected adsorbent (Fig. 9). Usually, bacterial EPS has an affinity towards metal ions and helps in their adsorption. But less number of functional groups on surface limit adsorption efficiency of EPS to achieve desired results [30-31]. Therefore, surface functionalization of EPS with thio group was studied to increase the uptake capacity of bacterial pristine EPS. The adsorption studies suggest that presence of various functional groups in the EPS could effectively be harnessed by surface functionalization for better removal metal ions from aqueous solutions. Simultaneously, these functional groups probably also help in the synthesis of NPs [32]. Adsorption results suggested that NPs incorporated functionalized EPS was having highest adsorption ability followed by functionalized EPS then pristine EPS. However, CdS NPs alone did not show significant adsorption capacity. This may be because the NPs incorporated functionalized EPS provide enhanced surface area with increased number of functional groups with higher affinity for Cd+2 ions [33]. The sulphur groups (S2-) of functional moieties, act as a soft base against soft acid Cd2+ to form a stable complex that also suggests increased adsorption capacity of pristine EPS after xanthate surface functionalization [34]. 4.6 Adsorption characteristics of cadmium on functionalized EPS The UV-Vis spectra of cadmium treated functionalized EPS showed the absorption maxima at 451.78 nm wavelength (Fig. 10 A) which is the characteristic peak for CdS NPs [21, 22]. However, the XRD pattern of pristine EPS and functionalized EPS did not show peaks of CdS but when the functionalized EPS was treated with cadmium, the peak was observed at 26.42° which corresponds to (111) Miller plane of cubic phase of CdS [8]. Moreover, the CdS NPs incorporated functionalized EPS when treated with Cd also showed a peak at 26.6° (Fig. 10 B). The CdS NPs incorporated functionalized EPS showed the highest affinity for metal ion adsorption. Moreover, the functionalized EPS contains sulphur groups that bind with the cadmium when added in aqueous solution and eventually form CdS NPs similar to synthesis of the CdS NPs by sulphur functionalized fungal biomass [10]. To further confirm the binding of sulphur groups to the surface of functionalized EPS synthesized CdS NPs after treatment with Cd solution, FESEM analysis was performed. The distinguished morphological changes in functionalized EPS suggested an interaction between cadmium ions and surface functional groups of EPS. This obtained result are well supported by the previous study which illustrated that functionalized mycelia of Aspergillus versicolor could remove cadmium from its aqueous solution and simultaneously synthesize CdS NPs on its surface [10]. However, the cadmium ion removal efficiency of functionalized fungal mycelia 10

has about 50% increase compared to pristine mycelia [10]. In perspective to fungal mycelia, this work has used surface functionalized bacterial EPS for the synthesis of CdS NPs and removal of cadmium from its aqueous solution with 88.66 % efficiency compared to pristine EPS. Thus, the illustrated work could be a novel and most efficient technique for bioremediation of toxic metals along with the synthesis of economically important NPs. 5 Conclusion The synthesis of NPs using physical and chemical methods involves various chemical and environmental hazards, so biosynthesis of nanoparticles using microorganisms is an emerging approach in the field of nanotechnology. Extracellular polymeric substances (EPS) can serve as binding sites for various metal ions and also act as a capping agent in the synthesis of NPs. Surface functionalization of EPS with suitable functional groups can enhance the adsorption of metal. Among the various functional groups, sulphur is mostly preferred due to its high affinity toward metals with high stability constant, low solubility products and easy preparation procedure. The sulphur group can easily bind with the cadmium ions in the aqueous medium and synthesize CdS NPs. The CdS NPs incorporated into the functionalized EPS has a greater adsorption potential for metal ions than the functionalized and pristine EPS. The present work showcased a novel method for the synthesis of CdS NPs using the bacterial EPS that also helps in removal of toxic metal pollutant cadmium from aqueous solution.

Acknowledgements Authors would like to acknowledge the authorities of NIT, Rourkela for providing facilities. XRD and FESEM of the Department of Physics and Ceramic Engineering of National Institute of Technology, Rourkela respectively were accessed during this study. Thanks are due to the Centre for Research in Nanoscience and Nanotechnology, University of Calcutta, Kolkata and Bose Institute, Kolkata for providing the TEM and AAS facility respectively.

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References 1

P. Bansal, N. Jaggi, and S. K. Rohilla , Res. J. Chem. Sci.,2012, ISSN, 2231, 606X.

2

R. Bhattacharya and S. Saha, Pramana, 2008, 71,187.

3

R. A. Mousavi, A. A. Sepahy and M. R. Fazeli, Proc Int Conf Nanomat., 2012, 1, 1862.

4

C. T. Dameron, R. N. Reese, R. K. Mehra, A. R. Kortan, P. J. Carrollm. L. Steigerwaldl. E. Brus and D. R. Winge, Nature,1989, 338, 596.

5

R. Y. Sweeney, C. Mao, X. Gao, J. L. Burt, A. M. Belcher, G. Georgiou and B. L. Iverson, Chem. Biol., 2004, 11, 155.

6

D. P. Cunningham, and L. L. Lundie, Appl. Environ. Microb., 1993. 59, 7.

7

J. D. Holmes, D. J. Richardson, S. Saed, R. Evans-Gowing, D. A. Russell and J. R. Sodeau, Microbiology+,1997, 143, 2521.

8

H.J. Bai, Z.M. Zhang, Y. Guo, G.E.Yang, Colloid. Surfaces. B., 2009,70, 142.

9

X. Li, S. Chen, W. Hu, S. Shi, W. Shen, X. Zhang and H. Wang, Carbohyd. Polym, 2009, 76, 509.

10 S. K. Das, I. Shome, and A. K. Guha, RSC Advances, 2012, 2, 3000. 11 R. M. Weiner, Mar. Biotech., 1997, 15, 390. 12 Y. K. Lee, H. W. Kim, C. L. Liu and H.K. Lee, J. Microbiol. Meth., 2003, 52, 245. 13 A.W. Decho, and G. R. Lopez, Limnol. Oceanogr.,1993, 38, 1633. 14 G. C. Panda, S. K. Das, A. K. Guha, Colloid. Surfaces B.,2008, 62, 173. 15 S. Chen, Y. Zou, Z. Yan, W. Shen, S. Shi, X. Zhang, and H. Wang, J. Hazard. Mater., 2009, 161, 1355. 16 R. S. Lokhande, P. U. Singare and D. S. Pimple, Resour. Environ., 2011, 1, 13. 17 J. O. Duruibe, M. O. C. Ogwuegbu, J.N. Egwurugwu, Int. J. Phys. Sci., 2007, 2, 112. 18 R. Singh, N. Gautam, A. Mishra and R. Gupta, Indian J. Pharm 2011, 43, 246. 19 G. C. Panda, S. K. Das and A. K. Guha, Colloid. Surf. B., 2008, 62(2), 173-179. 20 X. Li, H. Xu, Z. S. Chen and G. Chen, J. Nanomater., 2011, 2011. 21 J. Barman, J. P. Borah, K. C. Sarma, Optoelectron. Adv. Mat. Rapid commun., 2008, 2,770. 22 Z. R Khan, M. Zulfequar and M. S. Khan, J. Mater. Sci., 2011, 46, 5412. 23 A. Khan, J. Nanomater., 2012, 2012, 66. 24 A. S. Vorokh, and A. A. Rempel, Phys Solid State +., 2007, 49, 148. 25 V. Drits, J. Srodon and D. D. Eberl, Clay. Clay. Miner., 1997, 45, 461

12

26 D. Pavia, G. Lampman, G. Kriz and J. Vyvyan, in Introduction to Spectroscopy, ed. D. Pavia, G. Lampman, G. Kriz and J. Vyvyan, Cengage Learning, USA, 4th edn., 2008, ch. 2, pp. 15-86. 27 S. R. Rao, Rev. Inorg. Chem., 1992,12, 183. 28 E. S. Windsor, C. J. Zeissler, S. A. Wight, E. B. Steel and D. H. Blackburn, Microsc. Microanal., 2002, 8, 1494 29 I. Dogsa, M. Kriechbaum, D. Stopar and P. Laggner, Biophys. J., 2005, 89, 2711. 30 G. Bayramoglu, A. Deizli, S. Sektas, and M.Y. Arica, Microchem. J., 2002, 72, 63. 31 L. Hall-Stoodley, J. W. Costerton and P. Stoodley, Nat. Rev. Microbiol., 2004,2, 95. 32 M.J. Winter, Complexes, d-block Chemistry, Oxford University Press, New York. 1994. 33 Y. Feng, J. L. Gong, G. M. Zeng, Q. Y. Niu, H. Y. Zhang, C. G. Niu and M. Yan, Chem. Eng. J., 2010, 162, 487. 34 R. G. Pearson, J. AM. Chem. Soc., 1963, 85, 3533. 35 J. Chakraborty, S. Das, Environ. Sci. Pollut. Res., 2014, 21, 14188. 36 M. Dubois, K.A. Gilles, J. K. Hamilton, P. A. Rebers and F. Smith, Anal. Chem., 1956, 28, 350. 37 M.M. Bradford, Anal. Biochem., 1976,72, 248. 38 K. Burton, Biochem. J.1956, 62, 315. 39 C. A. Eligwe, N. B. Okolue, C. O. Nwambu and C. I. Nwoko., 1999, Chem. Eng. Technol, 22, 45.

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Table 1. Crystal size of synthesized CdS NPs by D-glucose and pristine EPS at 0.1 M conc. of Cd (NO3)2/Na2S based on XRD analysis Miller indices

D-Glucose 1mg

110 220 311

21.54 36.07 38.28

Pristine EPS

2mg Crystal size (nm) 24.04 36.67 37.50

1mg

2mg

24.14 38.12 38.81

25.10 34.15 38.61

Table 2. Percentage removal of cadmium from aqueous solution Adsorbents

% removal of Cd (24 h) % removal of Cd (48 h)

Pristine EPS

57.41±0.48

61.88±0.45

Functionalized EPS

77.07±0.99

80.81±0.89

CdS NPs incorporated

86.46±0.71

88.66±0.61

functionalized EPS

Table 3. Calculated values of Cd at equilibrium (Ce), equilibrium adsorption capacity of Cd (qe) and Ce/qe as obtained from Langmuir plot Temperature (K) Co (mg L-1)

298

308

318

298

Ce (mg L-1) 2.74

308

318

298

qe (mg g-1)

308

318

Ce/qe

25

2.98

2.58 220.2 222.6 224.2

50

6.13 5.732

75

11.78 10.91

9.87 632.2 640.9 651.3

0.01863

0.017022 0.015154

100

17.23 15.36 13.66 827.7 846.4 863.4

0.02081

0.018147 0.015821

0.01353

0.012309 0.011507

5.52 438.7 444.8 430.7 0.013973 0.012886 0.012816

Table 4. Langmuir isotherm parameters for the adsorption of Cd onto NPs incorporated functionalized EPS Temperature (K)

q max (mg g-1)

b (l mg-1)

R2

298

5.5072e-4

11.48

0.93797

308

4.7782e-4

11.20

0.96025

318

-4

10.84

0.92909

3.9233e

14

Figure legends Fig. 1. UV-Visible spectroscopy of CdS NPs synthesised by using pristine EPS and D-glucose at 0.1 M, 0.01 M and 0.001 M concentrations of Cd (NO3)2/Na2S (a) Cd (NO3)2 /Na2S with 1 mg pristine EPS and only pristine EPS as control (b) Cd (NO3)2 /Na2S with 2 mg pristine EPS (c) Cd (NO3)2 /Na2S with 1 mg D-glucose and (d) Cd (NO3)2 /Na2S with 2 mg D-glucose. Fig. 2. X-ray diffraction patterns of CdS NPs synthesised by using D-glucose and pristine EPS at 0.1 M, 0.01 M and 0.001 M concentrations of Cd (NO3)2/Na2S (A) Cd (NO3)2 /Na2S with 1 mg D-glucose and (B) Cd (NO3)2 /Na2S with 2 mg D-glucose (C) Cd (NO3)2 /Na2S with 1 mg pristine EPS (D) Cd (NO3)2 /Na2S with 2 mg pristine EPS. Fig. 3. ATR-FTIR spectra of CdS NPs synthesised by using (a) D-glucose and (b) pristine EPS. Followed by spectrum of (c) pristine EPS, (d) functionalized EPS and (e) functionalized EPS with adsorbed Cd. Fig. 4. Field Emission Scanning Electron micrographs (200 000X magnification) of CdS NPs synthesized by (a) 1 mg pristine EPS and (c) by 1 mg D-glucose in 0.1 M concentrations of Cd (NO3)2/Na2S respectively. The electron diffraction X-ray analysis (EDXA) showing presence of cadmium and sulphur in CdS NPs synthesized by (b) 1 mg pristine EPS and (d) by 1 mg D-glucose in 0.1 M concentrations of Cd (NO3)2/Na2S respectively. Fig. 5. Field Emission Scanning Electron micrographs (at 10000X magnification) of (a) Pristine EPS (b) functionalized EPS and (c) CdS NPs adsorbed on functionalized EPS. Fig. 6. Transmission electron micrographs of CdS NPs at different magnifications showing (a) CdS NPs embedded in EPS matrix, (b) exhibits the particle size in the range 20-40 nm in distribution histogram, (c) at higher magnification show large number of dispersed NPs and (d) lattice parameter of CdS NPs with high level of crystallinity at 5 nm resolution. Fig. 7. Zeta potential of pristine and functionalized EPS at 2.6, 4.6 and 6.6 pH respectively. Fig. 8. Langmuir plots for interaction of CdS NPs incorporated functionalized EPS and Cd at different temperatures. Fig. 9. Van't Hoff plot for Cd adsorption on NPs incorporated functionalized EPS. Fig. 10. (A) UV-visible spectra of pristine EPS and functionalized EPS after treatment with cadmium solution. (B) XRD patterns of (a) pristine EPS (b) functionalized EPS (c) pristine EPS after treatment with cadmium solution and (d) functionalized EPS after treatment with cadmium solution. 15

Fig. 1

16

Fig. 2

Fig. 3 17

Fig. 4

Fig. 5

18

Fig. 6

Fig. 7

19

Fig. 8

20

Fig. 9

Fig. 10 21

Graphical Abstract