Biomaterial Functionalized Graphene-Magnetite Nanocomposite: A

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Biomaterial Functionalized Graphene-Magnetite Nanocomposite: A Novel Approach for Simultaneous Removal of Anionic Dyes and Heavy-Metal Ions Baskaran Ramalingam, Thanusu Parandhaman, Priyadarshani Choudhary, and Sujoy K. Das ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00139 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018

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ACS Sustainable Chemistry & Engineering

Biomaterial Functionalized Graphene-Magnetite Nanocomposite: A Novel Approach for Simultaneous Removal of Anionic Dyes and Heavy-Metal Ions

Baskaran Ramalingam,a,b Thanusu Parandhaman,a,c Priyadarshani Choudhary,a,c Sujoy K Das,a,c,* a

Biological Materials Laboratory, Council of Scientific and Industrial Research (CSIR)Central Leather Research institute (CLRI), Chennai600020, India, bAnna University, Chennai600020, India, and cAcademy of Scientific and Innovative Research (AcSIR), New Delhi110001, India.

Abstract. Despite of immense application potential of graphene in wastewater treatment, the colloidal stability, aggregation and recyclability remains a major challenge. To address this issue, we report biomaterial functionalized graphene-magnetite (Bio-GM) nanocomposite as a novel recyclable material for treatment of wastewater containing dyes and heavy metal. The integration of biomaterial including living cells of Shewanella oneidensis with graphene-magnetite nanocomposite was characterized through UV-vis, FTIR, FESEM and fluorescent microscopic studies. The contact angle measurement depicted the hydrophilic property (water contact-angle 27.93), while VSM result demonstrated super paramagnetic behavior of the nanocomposite with saturation magnetization value of 30.2 emu/g. The Bio-GM nanocomposite exhibited excellent adsorption capacity towards dyes and Cr6+ in both single and multicomponent system with removal capacity of 189.63 ± 7.11, and 222.2 ± 8.64 mg/g of dyes and Cr+6, respectively, suggesting selective binding capacity and high adsorption efficiency of Bio-GM nanocomposite. In the adsorption coupled redox reaction, the Cr+6 was reduced to Cr+3 through biocatalytic activity of Bio-GM nanocomposite. The nanocomposite could be easily regenerated and reused for multiple cycles of adsorption-desorption studies without release of graphene and magnetite, and thus eliminating the potential hazardous risk of nanomaterial to the environment. The proposed biomaterial functionalized graphene-magnetite nanocomposite thus offers a novel way for sustainable, affordable and efficient removal of coexisting toxic pollutants of dyes and heavy metal.

Keywords: Graphene, Magnetite, Nanocomposite, Adsorption, Biocatalytic activity * Email: [email protected]; [email protected] 1 ACS Paragon Plus Environment

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Introduction. Environmental issues are becoming a major concern with intensification of industrial and agricultural activities along with rapid population growth. Discharging of various organic and inorganic hazardous chemicals recklessly pollutes the water bodies.1,2 Owing to the high toxicity and environmental persistency the accumulation of heavy metals and dyes imparts severe ecotoxicological impact and also possess serious health concern.3,4 As a consequence, considerable attention has been devoted to develop robust technologies to mitigate the water pollution. Various technologies

such

as

physicochemical,

biological,

electrochemical

and

multistep

biophysicochemical process have been emerged for wastewater treatment either separately or in integrated process.5-7 Among these, adsorption process has received increasing attention because of low energy inputs, operational simplicity, cost-effectiveness, and less amount of sludge production.8,9 Recent advancement of nanotechnology provides an opportunity to further improve the efficacy of current wastewater treatment technology using different types of nanostructured adsorbents including metal, metal oxide and carbon based nanomaterials.10-17 The discovery of graphene and graphene oxide has aroused significant interest in the development of cost-effective and ecofriendly water purification because of their unique and fascinating physicochemical properties.16,17 Especially graphene, a thin nanosheet of single layer carbon material, is of particular interest due to its largest surface area, high thermal and mechanical strength, good electrical conductivity, excellent stability under acidic and alkali conditions, and biocompatibility compared to the other nanomaterials.18,19 In addition, graphene represents an excellent support for chemical functionalizations, making it an ideal material for efficient removal of contaminants involving adsorption or surface reactions.20 Several literature reports demonstrated excellent adsorption efficacy of graphene to tackle organic, inorganic, and biological contaminants.17,21-24 For instance, Vilela et al.21 prepared graphene based microbots to remove heavy metals from water, while Yusuf et al.24 reported application of graphene and its derivatives as an adsorbent for removal of heavy metal and dye. Despite the great potentiality in water remediation applications, there is a significant concern about dispersion of graphene in aqueous solution. Unlike graphene oxide, graphene exhibits less water dispersibility due to the low availability of intrinsic functional groups and thus easily aggregated into aquatic system, which reduces the effective surface area and ultimately limits their potential application.25,26 At the same time the accumulation of graphene into the environment raises serious eco-toxicological concern.27,28 Poor adsorption performance towards various anionic species like 2 ACS Paragon Plus Environment

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chromate, phosphates, arsenates and anionic dyes is also another major problem.29 Functionalization of graphene could offer a potential solution to enhance the adsorption performance, colloidal stability, easy recovery, and decrease the rate of aggregation.30-32 Of particular interest, the hybridization of graphene with magnetic nanoparticles (e.g., iron or iron oxide) is the most common approach to prepare reusable graphene-magnetite nanocomposite, which eases the separation and recovery of the nanocomposite and also minimize the potential environmental hazard.33-35 Several strategies have been explored to prepare hybrid graphene-magnetite nanocomposite as reusable adsorbents for removal of heavy metals and dyes.36-38 Nevertheless, their application is restricted due to susceptibility of magnetic nanoparticles to oxidation and/or dissolution in aquatic environment, resulting in short life-span.39 Furthermore, it fails to improve the adsorption efficiency towards anionic species. Therefore, designing and fabrication of multifunctional adsorbent is need of the hour to address the pressing environmental challenges. In

this

study,

we

fabricated

biomaterial

functionalized

graphene-magnetite

(Bio-GM)

nanocomposite as a novel reusable adsorbent for simultaneous removal of anionic dyes and heavy metal from multi-component system. The integration of biomaterial including living cells of S. oneidensis with graphene-magnetite (GM) nanocomposite may enhance the dyes and heavy metal adsorption efficiency due to the synergistic biocatalytic activity of S. oneidensis cells with the functionality of graphene-magnetite nanocomposite. To achieve a robust and stable cellular network structure on GM nanocomposite, S. oneidensis cells was initially immobilized into the GM nanocomposite through covalent and  interactions, which were grown further to form magnetically separable biomaterial integrated graphene-magnetite nanocomposite through in situ growth process. This tailored made Bio-GM nanocomposite exhibits excellent thermal and physicochemical stability, magnetic separation, removal efficiency and recyclability. We therefore, believe that the unique combination of graphene, magnetic nanoparticles and together with network structure of S. oneidensis cells can offer fabrication of a novel nanocomposite for sustainable, efficient, and affordable wastewater purification.

Experimental Section Materials. Acid blue 113 (AB 113), Acid black 52 (AB 52), Acid yellow 110 (AY 110), Congo red (CR), potassium dichromate (Cr+6), ferric chloride, ferrous sulfate, graphite powder, hydrogen peroxide, magnesium chloride, sodium bicarbonate, sodium citrate, sodium lactate, and all other chemicals were purchased from Merck (Bangalore, India). Luria-Bertani media and other 3 ACS Paragon Plus Environment


microbiological ingredients were purchased from Hi-Media (Mumbai, India). All chemicals were used as such received without any further purification. Microorganism. The Shewanella oneidensis MR-1 (ATCC700550) was purchased from American Tissue Culture Collection (Manassas, VA 20110 USA) and sub-cultured at regular intervals of 15 days in Luria-Bertani (LB) agar (1% peptone, 0.5% yeast extract, 0.5% sodium chloride and 1.5% agar, pH 7.0) slant to maintain the cell viability. Preparation and Estimation of Dye and Heavy Metal Solution. Stock solution (1500 mg/L) of AB 113, AB 52, AY 110, and CR were prepared separately in deionized and double distilled water and further diluted to get required concentration. The calibration curves of dyes were prepared with appropriate dilution at maximum absorbance (λmax) values and the concentration of dye was measured by UV–vis spectrophotometer (V650, JASCO, Japan). The multicomponent dye-metal solution was prepared by mixing all dyes and Cr+6 solution and maximum absorbance (λmax) of the dye was determined by UV–vis spectrophotometer. The concentration of mixed dye was then measured spectrophotometrically as described above. Similarly stock solution (1500 mg/L) of Cr6+ was prepared using potassium dichromate in deionized and double distilled water. Further it was diluted to get desired concentration. The total chromium concentration was measured by flame atomic absorption spectrometer (Nova 350, Analytic Jena, Germany) using standard chromium solution with appropriate dilution, while Cr+6 concentration was determined spectrophotometrically at 540 nm after complexation with 1,5-diphenylcarbazide.40 Synthesis of Graphene-Magnetite Nanocomposite. Water dispersible graphene oxide (GO) was synthesized initially from graphite powder by modified Hummer‟s method.41 The synthesis of grapheneFe3O4 nanocomposite was carried out by co-precipitation method. In a typical experiment, a pellucid solution containing 1.5 mM sodium citrate, 6 mM sodium hydroxide, and 0.2 M sodium nitrate was prepared in 15 mL of double distilled water and kept at 100 °C for 1 h. Simultaneously, 1 mL of 2 M ferrous sulfate solution was added to the GO dispersion under shaking and incubated at room temperature (30 ± 2 °C) for 5 h. This reaction mixture was added dropwise into the pellucid solution under vigorous stirring and maintained the final concentration of ferrous sulfate and GO at 0.1 M and 3.5 mg/mL, respectively. The mixture was incubated initially at 100 °C for 1 h and allowed to cool down to room temperature (30 ± 2 °C) naturally. The pH of the reaction mixture was then increased to 12.0 using 1 M sodium hydroxide and further incubated at 100 °C for 1 h. A 4 ACS Paragon Plus Environment

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black precipitation was obtained, which was separated using an external magnet, washed with deionized water several times and finally dried at 60 °C. The as-prepared graphene-magnetite (GM) nanocomposite was re-dispersed in deionized and double distilled water by sonication for 20 min and used for further application. Fabrication of Biomaterial Functionalized Graphene-Magnetite Nanocomposite. S. oneidensis was grown aerobically in LB broth (1% peptone, 0.5% yeast extract, and 0.5% sodium chloride) at 30 °C under shaking (120 rpm) for 24 h. The cells (109 CFU/mL) were harvested by centrifugation at 6600g for 20 min (Velocity 14R, Dynamica, UK) and dispersed in 50 mL of modified M1 medium containing 30 mM HEPES, 7.5 mM sodium hydroxide, 28 mM ammonium chloride, 1.34 mM potassium chloride, 4.35 mM sodium dihydrogen phosphate, 0.7 mM calcium chloride and 5% of LB broth. A sterile dispersion of GM nanocomposite (0.2%) was prepared in modified M1 media containing S. oneidensis and incubated under anaerobic condition with shaking (120 rpm) for 2 days at 30 °C in presence of 10 mM sodium lactate and 10 mM ferric chloride as the electron donor and acceptor, respectively. Following incubation, GM nanocomposite containing cells was separated from the culture media and washed with sterile deionized water to remove loosely attached cells. Freshly prepared modified M1 media was then added and further incubated at 30 °C under aerobic and static condition to allow the formation of cellular structure network on the nanocomposite. The media was changed every day and at the end of the sixth day the biomaterial functionalized GM nanocomposite was formed, designated as Bio-GM nanocomposite. Estimation of Polymeric Substances on Bio-GM Nanocomposite. The extra polymeric substances was extracted from Bio-GM nanocomposite by ethanol extraction method as described by Gong et al.42 and protein and carbohydrate concentration were estimated by Bradford43 and phenol-sulfuric acid methods,44 respectively. The detailed experimental procedures are described in supporting information. Characterization of GM and Bio-GM Nanocomposite. The morphology and chemical composition of GO, Fe3O4, GM, and Bio-GM were characterized by various spectroscopic and microscopic techniques. Prior to that, GO, Fe3O4, and GM nanocomposite were dispersed in deionized water by ultra-sonication for 10 min, while Bio-GM nanocomposite was vortexed for 15 min. The UV–vis spectra of the samples were recorded on UV–vis spectrophotometer from 200 to 800 nm. The morphology and structural characteristics of the drop casted samples on glass cover 5 ACS Paragon Plus Environment


slip were investigated by field emission scanning electron microscopy (FESEM, JEOL JSM 6700F, Japan) operated at an accelerating voltage of 20 kV equipped with an energy dispersive X–ray analysis (EDXA) system. Fourier transform infrared spectra of dried lyophilized samples were recorded on FTIR spectrometer (Nicolet Nexus 470, ThermoFisher Scientific, USA) with a spectral resolution of 2 cm-1. Crystallographic pattern and thermal stability of the dried samples were measured at 080  by X-ray diffractometer (MiniFlex-II, Rigaku, Japan) with a Cu Kα radiation (λ = 0.154 nm) and TGA (SDT Q600 TA Instrument, USA) at 20800 °C with a heating flow rate of 10 °C/min, respectively. The magnetic hysteresis loop of the samples was obtained using vibrating sample magnetometer (VSM, Lakeshore 7410, Lake Shore Cryotronics, Inc. USA) at room temperature (30 °C) using Teflon as a blank and high purity nickel sphere as a standard reference. The cell viability in Bio-GM nanocomposite was evaluated by fluorescence microscopic image following staining with Live/Dead viability kit (Invitrogen, USA). The image was recorded on a Fluorescence microscope (DMi8, Leica, Germany) and analyzed using Leica Application Suit X (LAS X) software. The contact angle of the samples was measured on a contact angle meter (HOIAD-CAM-01B, Holmarc Opto-Mechatronics Pvt. Ltd. India). The total chromium concentration was measured by flame atomic absorption spectrometer. Removal of Azo Dye and Heavy Metal. The adsorptive removal of azo dyes and heavy metals was carried out through incubation of dye and metal ion solution with as-prepared Bio-GM nanocomposite in 100 mL Erlenmeyer flask. Initially, 50 mg Bio-GM nanocomposite was incubated in 50 mM acetate (pH 4.0-5.0) or phosphate buffers (pH 6.0-8.0) for 20 min. The pre-conditioned Bio-GM nanocomposite was separated using an external magnet, and washed with deionized and double distilled water. Further, it was treated with 20 mL of 100 mg/L of AB 113, AB 52, AY 110, CR, CD, and Cr6+ solution containing 1% LB broth separately under shaking (120 rpm) at 30 C. After treatment, the nanocomposite was recovered using an external magnet and the residual concentrations of dye and metal ions were measured spectrophotometrically as described above. Similarly, the effect of pH and temperature was investigated at different pH value (4.0-8.0) and temperature (20-50 °C), respectively without changing other parameters. The kinetic studies were performed at pH 4.0 and room temperature (30 ± 2 °C). The equilibrium adsorption isotherm was carried out by varying the initial dye and metal ions concentrations at optimum pH and temperature. The adsorptive removal of dyes and Cr+6 was also studied in multi-component system by mixing all dyes and Cr+6. The effect pH, temperature, contact time and adsorption isotherm were carried out in 6 ACS Paragon Plus Environment

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a similar fashion as described above. The control experiment was performed under identical condition in absence of Bio-GM nanocomposite. All the experiments were repeated five times and data represent an average of five independent experiments ± SD (standard deviation) shown by error bar. Two-tailed Student‟s „t‟ test was performed for calculating the statistical significance at p value < 0.05. The removal efficiency (R %) and amount of dye and metal ions adsorbed (qe) were calculated using the following equations.45 Eq. 1 Eq. 2 Eq. 3

where, qt and qe are the amount of dye or metal ions adsorbed (mg/g) on Bio-GM at time „t‟ and equilibrium, respectively, Co, and Ce are initial and equilibrium concentration of dye or metal ions (mg/L), respectively, V is the volume of the dye or metal ions solution (L), and M is the mass of the Bio-GM nanocomposite (g). Recycling and Reusability of Bio-GM Nanocomposite. The Bio-GM nanocomposite was separated following adsorptive removal process using an external magnet and washed with deionized and double distilled water several times. The adsorbed dyes and Cr+6 were eluted using different elution buffers such as buffer I (50 mM phosphate buffer, pH 8.0), buffer II (50 mM phosphate buffer, pH 8.0 and 20% ethanol) and buffer III (50 mM acetate buffer, pH 4.0). The dye and Cr+6 adsorbed Bio-GM were incubated with above mentioned elution buffers separately for 1 h at 30 °C and concentration of dyes and Cr+6 were measured spectrophotometrically as described above. The regenerated Bio-GM was further incubated in modified M1 medium for 24 h at 30 °C and subsequently processed for next cycle of dye and Cr+6 removal as described above. The concentration of leachable graphene and magnetic nanoparticles in aqueous solution was also measured by UV-vis and atomic absorption spectroscopic analysis, respectively. Stability of Bio-GM Nanocomposite. The amount of cells mass on Bio-GM nanocomposite before and after adsorptive removal was measured by crystal violet staining46 in order to understand the stability of Bio-GM nanocomposite in aqueous environment. Further, the crystal structure and

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chemical composition of Bio-GM nanocomposite after desorption was studied by AAS, XRD, FTIR, and VSM analyses as described above. RESULTS AND DISCUSSION Synthesis and Characterization of GM Nanocomposite. In the process of GM nanocomposite preparation, GO (Figure 1A, left panel) was initially prepared by modified Hummer‟s method and treated with Fe+2 ions to electrostatically bind them through hydroxyl, carboxyl or epoxy groups of GO sheets (ζ values = 34.3). In the next step, GO–Fe+2 was converted into graphene–Fe3O4 (GM) nanocomposite by co-precipitation process in presence of excess citrate and nitrate ions. The asprepared GM nanocomposite (Figure 1A, second panel) carried negative surface charge (ζ values =18.6) and therefore, homogeneously dispersed in water due to columbic repulsion. The UV-vis spectroscopic analysis showed (Figure 1B) that GO exhibits a peak at 232 nm with a shoulder at ~300 nm due to π−π* transitions of the aromatic C−C bonds and n−π* transitions of the C=O bonds, respectively.47 On the other hand, Fe3O4 nanoparticle synthesized by co-precipitation did not show any such peak in the range of 200–800 nm (data not shown). The UV-vis spectra of GM demonstrated an absorption peak at 260 nm correspond to aromatic CC bonds indicating the reduction of GO to graphene and restoration of the ππ conjugate structure of few layer graphene sheets. During the co-precipitation process, the reduction of GO to reduce graphene oxide might be accompanied by reducing action of sodium hydroxide. At the same time, Fe+2 is converted into Fe3O4 resulting in formation of magnetically separable GM nanocomposite (Figure 1A, right panel). The X-ray diffraction pattern (Figure 1C) of GO showed sharp peaks at 2θ value of 11.2, and 43° corresponds to [001], and [100] planes respectively, indicating a two-dimensional structure of exfoliated GO. Notably, these peaks of GO disappeared and a new broad peak appeared at around 23° in GM, which is likely due to the reduction of exfoliated layered structures of GO and partial restacking of exfoliated graphene layers. In addition, it exhibited strong diffraction peaks at 2θ values of 30.3, 35.7, 43.3, 54, 57.3, and 63° corresponds to [220], [311], [400], [422], [511], and [440] planes of Fe3O4 nanoparticle,48 demonstrating the coexistence of Fe3O4 and reduced GO in GM nanocomposite. The result further demonstrates that crystalline nature of magnetic nanoparticle did not get change following hybridization with graphene.

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Fabrication of Biomaterial Functionalized GM Nanocomposite. The fabrication of magnetically separable Bio-GM nanocomposite is schematically represented in Figure 2A, which involves initial attachment of S. oneidensis cells on GM nanocomposite followed by growth of the attached cells to form cellular network structure on GM nanocomposite. The initial attachment of the cells and subsequent cellular growth was monitored through UV-vis spectroscopic measurement (Supporting Information Figure S1). A red shifting of absorption peak of graphene to 250 nm indicates attachment of the cells with graphene surface. Over the course of time a new peak at 600 nm appeared (Figure 1A), which confirmed that the attached cells grew further leading to the formation of cellular network structure on GM nanocomposite surface. During the integration process, the attached cells were grown with excretion of extracellular polymeric substance, which played an important role in adhesion of S. oneidensis cells and formation of cellular network structure on the GM nanocomposite surface. The protein and carbohydrate concentration measurement revealed that the Bio-GM nanocomposite contains high amount of protein (27.06 ± 1.95 mg/g) and carbohydrate (13.17 ± 1.11 mg/g), and therefore contribute significantly in Bio-GM nanocomposite. The scanning electron and fluorescence microscopic images were recorded to study the surface morphology of Bio-GM nanocomposite. The FESEM micrograph of GM nanocomposite (Figure 2B) exhibited sheet-like structure of graphene with irregular shape and many pores between these graphene sheets. The lateral dimensions of the graphene sheet varied from 100 nm to few micrometers with average thickness of 2.93 ± 0.53 nm. The higher magnified image (Figure 2C) further showed folded structure of the graphene sheet with electron dense bright dots of Fe3O4 nanoparticles distributed throughout the surface of the graphene sheets. The EDXA spectrum (Supporting Information Figure S2) further depicted the presence of C, O and Fe peaks, confirming the formation and hybridization of Fe3O4 nanoparticle with graphene. Conspicuous change of surface morphology of GM was noticed following interaction with S. oneidensis. Thick layer of S. oneidensis cells on Bio-GM nanocomposite surface is observed in Figure 2D. The FESEM image also showed strongly adhered, well distributed, and rod-shaped S. oneidensis cells attached over the entire surface of Bio-GM nanocomposite. The high magnified image further demonstrated that bacterial cells are interconnected through well-known bacterial nanowires, as indicated by circle in Figure 2E, which effectively attached the cells with the surface of the graphene sheets to form cellular network structure. It is reported that Shewanella produces electrically conductive nanowires under oxygen limiting condition, which facilitates the electron transfer to the external surfaces.49 The EDXA spectrum (Figure 2F) of Bio-GM nanocomposite 9 ACS Paragon Plus Environment


depicted the peaks of C, O, Fe including N, P and S, supporting the functionalization of biomaterial with GM nanocomposite. The metabolic activity of S. oneidensis cells in Bio-GM nanocomposite was studied using fluorescence microscopy following staining with Live/Dead cell viability kit containing differential membrane permeable dyes such as Syto 9 and Propidium iodide (PI). Syto 9 stains the metabolically active live cells with green fluorescent color, whereas PI stains dead cells emitting red fluorescence. The fluorescent microscopic image (Figure 2G) showed strong green fluorescent color, implying the formation of thick layers of metabolically active cells on Bio-GM nanocomposite. The large surface area (2630 m2/g)50 and pores between layers of graphene sheets thus favors the attachment of S. oneidensis cells with GM nanocomposite and the diffusion of nutrients through these pores provides favorable environment for cellular growth, keeping the cells in metabolically active state. In addition, the strong chemotactic behavior of this dissimilatory metal-reducing bacteria towards Fe3O4, due to high affinity of outer membrane c-type cytochromes (MtrC and OmcA) and Fe+3 oxide51,52 also stimulates the attachment of S. oneidensis cells with GM nanocomposite. The integration of biomaterials with GM nanocomposite and chemical composition of Bio-GM nanocomposite was further investigated through FTIR and XRD spectroscopic analysis. The FTIR spectrum of GO showed (Figure 3A) strong and intense signal for –OH and CH stretching vibrations at 3356 and 2920 cm-1, respectively. The other IR signatures appeared at 1718, 1598, 1395, 1247 and 1057 cm-1 attributes to –C=O carbonyl stretching vibrations and –COOH carboxyl stretching, –OH deformation, C–OH and C–O stretching in C–O–C (epoxide group), respectively. The IR spectrum of Fe3O4 nanoparticle exhibited peaks at 1620 and 1381 cm-1, respectively due to stretching vibrations of asymmetric and symmetric –COO- group of surface citrate molecules. The strong peak at 573 cm-1 corresponds to the stretching vibration of Fe–O.53 The IR spectrum of GM nanocomposite displayed significant alteration in comparison to GO. Decreased in peak intensities and shifting of few peaks are noticed in the spectrum. The peaks appeared at 1718 and 1247 cm-1 in GO is disappeared in GM, confirming the removal of most oxygen containing functional groups from the GO precursor during co-precipitation process. In addition, the peaks at 1620, and 1381 cm1

related to Fe3O4 nanoparticle is also appeared and Fe-O peak shifted to 566 cm-1 indicating the

binding of Fe3O4 nanoparticle with graphene.53 The FTIR spectrum of S. oneidensis showed characteristic peaks at 1648, 1543, 1465, 1403, 1234, and 1065 cm-1 corresponds to amide I, amide II, carboxyl and phosphate groups of proteins, phospholipids, and lipopolysaccharides,54,55 which provides binding sites for interaction with GM nanocomposite. Similarly, the Bio-GM 10 ACS Paragon Plus Environment

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nanocomposite exhibited IR signature at 1648, 1536, 1465, 1409, 1241, 1058, 988 and 573 cm-1, demonstrating coexistence of S. oneidensis and GM nanocomposite. However, the shifting of amide, carboxyl and phosphate bands of S. oneidensis cells and Fe-O of Fe3O4 suggests chemical bond formation between S. oneidensis cells and GM nanocomposite. The XRD pattern (Figure 1C) of Bio-GM nanocomposite exhibited diffraction peaks at 2θ value of 24, 30.3, 35.7, 43.3, 54, 57.3, and 63°, similar to that of GM excepting the peak intensities reduced significantly due to the surface coverage of GM nanocomposite by biomaterials like S. oneidensis cells, proteins and carbohydrate. The thermal stability of Bio-GM nanocomposite was also studied by TGA and thermogram of GO, Fe3O4, GM, S. oneidensis and Bio-GM nanocomposite are shown in Figure 3B. The TGA curve of GO showed the initial weight loss of 17.8% at below 150 °C is due to the evaporation of absorbed or bound water. The second stage contributed of about 21.5% weight loss in the range of 150650 °C. The third and final stage, which started at 650 °C and ended at 800 °C demonstrated 12.2% weight loss due to the release CO, and CO2 from the most labile functional groups.56 Therefore, a total weight loss of about 51.5 % was recorded in GO precursor. On the other hand, the Fe3O4 nanoparticle exhibited only 5.5% weight loss due to evaporation of bound and crystalline water in the entire temperature range, while S. oneidensis biomass recorded ~75% weight loss due to degradation of the cells. Likewise, GM nanocomposite exhibited initial weight loss of 4.5% till 200 °C and the subsequent gradual weight loss of 11.6% between 250800 °C due to oxidation of carbonaceous material. This clearly demonstrates that incorporation of Fe3O4 nanoparticle with graphene increases the thermal stability of GM nanocomposite. The thermogram of Bio-GM nanocomposite showed an interesting three stage weight loss. The initial weight loss of 4% up to 150 °C is due to evaporation of bound water content. A drastic reduction in weight loss of about 36.3% in between 180 to 450 °C and gradual weight loss of 20.6% till 800 °C was observed due to decomposition of bacterial cells, proteins and carbohydrate along with oxidation of carbonaceous material graphene. The TGA analysis further revealed that biomaterial corresponds to nearly 20% in Bio-GM nanocomposite. The magnetic property of the samples was further studied by VSM analysis at room temperature to evaluate the magnetic separation of the Bio-GM nanocomposite. Figure 3C shows an “S”-shaped magnetic hysteresis loop of Fe3O4 nanoparticles, GM and Bio-GM samples. The saturation magnetization value of GM was recorded at 49.6 emu/g, whereas the coercivity and retentivity values were 0.08 kOe and 4.3 emu/g, respectively (inset Figure 3C). On the other hand, the 11 ACS Paragon Plus Environment


saturation magnetization, coercivity and retentivity values of Fe3O4 nanoparticle are found at 59.1 emu/g, 0.07 kOe and 5.1 emu/g, respectively. In comparison to pristine Fe3O4 nanoparticles and GM, the Bio-GM nanocomposite exhibited less saturation magnetization value of 30.2 emu/g. The reduction of saturation magnetization value after S. oneidensis growth is attributed to the formation of cellular network structure and biomaterial functionalization over GM nanocomposite. The coercivity and retentivity values of Bio-GM are 0.11 kOe, and 3.8 emu/g, respectively. The hysteresis loop analysis of pure Teflon was also recoded as a blank, which showed (Supporting Information Figure S3) the coercivity values varied in between 0.06-0.12 kOe. Since, the coercivity values of all the three samples are within the error limit of the VSM instrument; this confirms that Mag, GM, and Bio-GM have superparamagnetic behaviour. Most importantly, the saturation magnetization value of Bio-GM nanocomposite is high enough for its facile magnetic separation using an external magnetic field (Figure 1B, right panel). Hydrophobic-hydrophilic properties of the material have great effluence in the adsorption process, while a good hydrophilic property is required to facilitate efficient adsorption of dyes and heavy metal ions from aqueous solution. To understand the hydrophilic property of the Bio-GM nanocomposite, contact angle of Bio-GM surface was recorded. The result showed (Figure 3D) that GM nanocomposite retain considerable hydrophobicity exhibiting contact angle of 74.47 ± 7.7. In comparison, the Bio-GM surface demonstrated very good hydrophilicity with contact angle of 27.93 ± 3.2, which can be attributed to high bacterial cells density along with protein and carbohydrate content in Bio-GM nanocomposite. Therefore, the magnetic separation behavior, high bacterial cell density, protein and carbohydrate content along with good hydrophilicity could favor the adsorptive removal of dyes and heavy metal ions from aqueous solution by Bio-GM nanocomposite. Adsorptive Removal of Dyes and Heavy Metal Ions in Single Component System. The anionic dyes i.e. AB 113, AB 52, AY 110, CR and Cr+6 (Figure 4A) were chosen as model dyes and heavy metal, respectively owing to their wide use in leather dying and tanning processes. The effect of different environmental factors such as solution pH, contact time, temperature and initial concentration of dyes and Cr+6 were investigated to optimize the adsorption efficiency of Bio-GM nanocomposite. Considering the cell viability of S. oneidensis, the effect of pH was studied using preconditioned Bio-GM nanocomposite at different pH value of 4.08.0. At low pH value the nanocomposite surface is protonated favoring binding of negatively charged dyes and chromate ions54 through electrostatic interaction. The Figure 4B shows color photograph of all dyes and Cr+6 12 ACS Paragon Plus Environment

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solutions before and after treatment with preconditioned Bio-GM nanocomposite, which clearly demonstrated complete removal of dyes as well as Cr+6 at pH value of 4.0. The adsorption data at various pH values further revealed that removal efficiency gradually increased with decrease in pH values (Figure 4B) and almost 99.5% of these anionic dyes and Cr+6 were adsorbed by Bio-GM nanocomposite conditioned at low pH. The FTIR spectrum of Bio-GM nanocomposite (Figure 3A) demonstrated that cell wall component of S. oneidensis and extracellular protein and carbohydrate contains amine, hydroxyl, carboxyl, and phosphate groups. At low pH value these functional groups got protonated (ζ value = 5.35 ± 1.73), and thus electrostatically bound negatively charged anionic dyes and Cr+6 ions. Likewise, deprotonation of these functional groups at high pH value (8.0) hindered the adsorption process due to coulombic repulsion. As the pH value decreases, the number of positively charged sites increases and vice versa. Apart from surface charge, the metal speciation and mobility of the metal species also influenced the adsorption of Cr+6 ions. At low pH value Cr+6 exists in various oxyanionic species40,57 such as HCrO4−, CrO4−2, HCr2O7−, and Cr2O7−2 having strong affinity towards positively charged functional groups and therefore enhanced the adsorption efficiency. The adsorption experiments was restricted at pH 4.0 due to low metabolic activity and reduced cell viability of S. oneidensis at pH value 40 ºC. In addition, beyond survival temperature of the cells, the biochemical composition of Bio-GM nanocomposite gets damage resulting in low adsorption. Since, adsorption efficiency of Bio-GM nanocomposite did not exhibit significant difference (p

Biomaterial Functionalized Graphene-Magnetite Nanocomposite: A

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