Seaweed-Derived Nontoxic Functionalized Graphene Sheets as

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Seaweed derived non-toxic functionalized graphene sheets as sustainable materials for the efficient removal of fluoride from high fluoride containing drinking water Mukesh Sharma, Dibyendu Mondal, Nripat Singh, Kapil Upadhyay, Akhilesh Rawat, Ranjitsinh V. Devkar, Rosy Alphons Sequeira, and Kamalesh Prasad ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b00198 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 9, 2017

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ACS Sustainable Chemistry & Engineering

Seaweed derived non-toxic functionalized graphene sheets as sustainable materials for the efficient removal of fluoride from high fluoride containing drinking water Mukesh Sharma,abc† Dibyendu Mondal,c† Nripat Singh,ab Kapil Upadhyay,d Akhilesh Rawat,e Ranjitsinh V. Devkar d, Rosy Alphons Sequeira ab and Kamalesh Prasad ab* a

Natural Products & Green Chemistry Division, CSIR-Central Salt & Marine Chemicals Research Institute, G. B Marg, Bhavnagar-364002 (Gujarat), India. b

AcSIR- Central Salt & Marine Chemicals Research Institute, G. B Marg, Bhavnagar364002 (Gujarat), India. c

Departamento de Química, Universidade de Aveiro, Campus Universitário de Santiago 3810–193 Aveiro, Portugal d

Department of Zoology, Faculty of Science, The M.S. University of Baroda, Vadodara 390 002, Gujarat, India. e

Department of Chemistry, University of Rajasthan, J. L. N. Marg, Jaipur – 302004, Rajasthan, India. *Correspondence: Dr. Kamalesh Prasad [[email protected]; [email protected]] Phone No.: +91-278 2567760. Fax No. +91-278-256756. KEY WORDS: Biomass, functionalized graphene, non-toxic, fluoride removal ABSTRACT:

Herein we present a sustainable and cost effective approach for the

preparation of functionalized graphene nanosheets (GNs) directly from seaweed and deep eutectic solvents (DES). The seaweed granules remained after the recovery of juice from fresh brown seaweed, Sargassum tenerrimum was utilized as a raw material and DES generated by the complexation of choline chloride and metal salts were employed as solvent and catalyst for the large scale and facile production of metal oxide functionalized GNs. 1 ACS Paragon Plus Environment


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Moreover considering the biological application of such GNs, where nontoxic nature of substrates are desirable, we have also evaluated the cytotoxicity of the functionalized GNs (Fe3O4/Fe or SnO2/SnO/Sn or ZnO/Zn-functionalized GNs) and most of them were found to be non-toxic against human lung carcinoma cells (A549). Thereafter, efficiency of these GNs was assessed for the removal of F– from fluoride contaminated ground water (2.72 to 6.71 mg L-1) used for drinking purposes. After treatment with GNs, the concentration of fluoride was found to reduce to 0.36 to 1.69 mg L-1 (75-87% removal efficiency). Moreover, after recovery of GNs from the water, no significant contamination of metal ions was found in the remaining water. Thus, seaweed derived non-toxic GNs can be utilized to produce safe drinking water with permissible fluoride content as per WHO norms.

INTRODUCTION The honeycomb decorated 2D planner carbon allotrope is termed as “Graphene” which is made up of conjugated sp2 carbon.1 The unique properties such as high thermal and electrical conductivity, superior mechanical strength, high specific surface area etc., make it a potential material for applications in energy storage devices, as membranes in separations and as sensors.

2-5

There are several methods available for the

commercial production of graphene from graphite including Hummer’s method, scotch tape exfoliation of graphite and chemical vapour deposition (CVD) of volatile organics on metallic surfaces etc.1,6 However, these methods poses some limitations, for instance uses of toxic chemicals, need of metallic surfaces, high temperature, irregular deposition of carbon and high cost of production. 7 Thus, development of cost effective technology for large scale production of graphene is important and ever growing research arena. Use of biomass as resources for the production of graphene has gained considerable attention in last few years. 8-14 The production of single layer

2 ACS Paragon Plus Environment

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N-doped graphene film through the pyrolysis of chitosan under an inert atmosphere and porous graphene-like nanosheets from coconut shells has been reported.

8,9

Chen

et al., (2016) reported the production of high-quality graphene from wheat straw without using any catalyst. 10 Shams et al. (2015) also developed a scalable method for the production of graphene from dead camphor leaves (Cinnamomum Camphora).11 Rice husk biomass has been utilized as a biomass resource for the controllable synthesis of graphene quantum dots.12 The carbon sources such as food, insects and waste has also been used for the production of single layer graphene on the surface of copper through pyrolysis.13 In a recent review article, different methods for the preparation of sustainable carbon materials from biomass have been disclosed.14 All the literature reported methods for the production of graphene nanosheets have some drawbacks/limitations such as need of metal surfaces, requirement of further treatment of as obtained graphene nanosheets to obtain functionalized graphene nanosheets etc.,. Besides the use of terrestrial biomass which have ‘food-fuel’ dilemma,15 seaweed biomass/seaweed polysaccharides are considered to be a substitute for nonrenewable fossil resources16 and various synthetic materials used in a number of high end applications.13

A number of seaweeds can be successfully cultivated, which

ensures the availability of seaweed biomass for large scale industrial utilization.17 Other than several uses, seaweed biomass can be applicable for the production of graphene from economic, sustainability and scalability point of view. In this direction, N- and S-doped porous carbon structure was obtained by the pyrolysis of Undaria pinnatifida, a brown seaweed, without using any template.18 Recently, Fe3O4/Fe functionalized graphene nanocomposites (GNs) derived from brown seaweed has been reported as potential electro-catalyst for oxygen reduction reaction (ORR) in an alkaline fuel cell.19,20



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The non-toxic graphene nanosheets and graphene nanosheets-based materials at the explored concentration could be applicable for the safer biotechnological, electronics

and

biomedical

applications.

Wang

et

al.,

demonstrated

the

biocompatibility of the graphene oxide (GO) on human fibroblast cells and the cells were found dead when the dose was higher than 50 μg mL-1.21 Liu et al., have reported the highly biocompatible graphene nanosheets functionalized with gelatin and this gelatin functionalized graphene nanosheets can be used for the drug delivery and cellular imaging.

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Here also a facile method for the production of biocompatible and

nontoxic nitrogen doped graphene nanosheets useful as biomimetic electrochemical sensor for the detection of nitric oxide was reported. 23 Considering the harmful effects of fluoride due its excessive presence in drinking water it is mandatory to eliminate the excessive amount to provide safe drinking water. In view of above, the present work report a facile method for the scalable production of metal oxide functionalized GNs (Fe 3O4/Fe or SnO2/SnO/Sn or ZnO/Znfunctionalized GNs) directly from the naturally abundant seaweed biomass, Sargassum tenerrimum (S. tenerrimum), using deep eutectic solvents (DESs) obtained by the combination of

choline chloride (ChCl) as hydrogen bond acceptors and

different metal salts (FeCl3, SnCl2 and ZnCl2) as hydrogen bond donors. DESs were utilized because it can simultaneously act as solvent, soft template and catalyst for the growth of GNs.19,20 Among the GNs synthesized and used, Fe3O4/Fe functionalized GNs was previously reported by us.19,20 The cytotoxicity and biocompatibility of the functionalized GNs were evaluated against human carcinoma lung cell (A549). The GNs thus produced were also explored for the efficient removal of fluoride from fluoride rich drinking water.


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Scheme 1: Schematic representation of mechanical expulsion of sap from fresh seaweed Sargassum tenerrimum to obtain seaweed granules and deep eutectic solvent promoted preparation of functionalized graphene nanosheets from seaweed granules.

EXPERIMENTAL SECTION

Materials. The chemicals used for the synthesis of DESs such as choline chloride, ferric chloride (FeCl3), zinc chloride (ZnCl2), stannous chloride (SnCl2) and sodium fluoride (NaF) was purchased from S.D. Fine Chemicals, Mumbai, India and used without any further purification. The brown seaweed S. tenerrimum was collected from Okha (Geographical location: 22° 28ʹ N, 69° 4ʹ E), Gujarat (India). Fluoride contaminated drinking water was collected from natural ground water sources from the village Govind garh, Ajmer, Rajasthan, India (Geographical location: 26° 45ʹ N, 74° 38ʹ E) known to have high fluoride content in drinking water. The water samples were collected from three different locations in the village which were free from excessive dust. The collected samples were packed in airtight pre-autoclaved polypropylene bottles and transported to the laboratory for further analyses.



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Synthesis of deep eutectic solvents. DES [ChCl-FeCl3 (1: 2), ChCl-ZnCl2 (1: 2) and ChCl-SnCl2 (1: 2)] were prepared following the method described by Abbott et al.,.

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(supporting information). Method for extraction of seaweed sap and production of granules: The freshly harvested S. tenerrimum seaweed was mechanically crushed followed by centrifugation to obtain liquid sap as supernatant which was stored under refrigeration (1 Kg fresh S. tenerrimum yielded 600 mL sap). The remaining solid residue (granules) was dried in oven to obtaindried seaweed granules (75 g of dry granules with 14.2 % moisture was obtained from 1 Kg of fresh seaweed). Preparation of graphitic carbon nanosheets : Process for the preparation of GNs from indigenous seaweeds was prepared as per the patented process (Scheme 1)19,20 In a typical experiment, the solid Sargassum tenerrimum granules and DESs were mixed with each other followed by heating at 80 °C for 30 min to obtain a semisolid composite mass. The semisolid composite mass thus obtained was pyrolysed at three different temperatures in the range of 700-900 °C under inert atmosphere (95% N2 and 5% H2) followed by washing with water for several times to obtain functionalized GNs. The functionalized GNs thus prepared were characterized for its elemental compositions and data are provided in Supporting Information, Table S1 (for GNs such as SAR-Fe-700, SAR-Fe-800, SAR-Fe-900, SAR-Sn-700, SAR-Sn-800, SARSn-900, SAR-Zn-700, SAR-Zn-800 and SAR-Zn-900). Method for the removal of fluoride from fluoride rich drinking water: Stock solution (500 ppm) for fluoride was prepared by dissolving sodium fluoride (NaF) in Milli-Q water. This was further diluted to prepare 0.5, 1, 2, 3, 4, 5, 10, 25, 50 and 100 ppm standard solutions. In a typical procedure, 0.025 g of GNs was added in 50 mL fluoride solution in a 150 mL conical flask. The solution was then homogenized by ultra-sonication for 5 min. The homogenized solution thus obtained was shaken at room temperature for 30-120 min


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(for optimization). After that the suspension was filtered through a Whatman filter paper of 0.45 µm. The fluoride concentration in the filtrate was measured on an Orion Versa Star ISE meter fitted with an ionplus sure-flow fluoride electrode (Thermo Scientific, USA).. The similar experiment was carried out for real fluoride contaminated drinking water samples which was collected from real water sources (pH 7.7-7.8). The amount of fluoride removed by GNs at equilibrium was calculated using the following equation (1):

……………………….. (1) Where C0 and Ce is the initial and equilibrium concentrations of fluoride (ppm) respectively, m is the mass of GNs (g) and v is the volume of the solution (L). Determination of adsorption isotherms : a) Freundlich adsorption isotherm equation ………………………….. (2) Where, Ce is the concentration of fluoride at equilibrium (mg L−1), KF is the binding constant and 1/n is the empirical parameter in Freundlich adsorption isotherms equation. b) Langmuir adsorption isotherm equation ………..…………………….. (3) Where, qm is the measurements of the sorption capacity (mg g−1) and KL is the binding constant of Langmuir adsorption isotherms.

Characterization : Characterization of GNs was

carried out using number of

analytical techniques as described in the previous work

19

supporting information.

RESULTS AND DISCUSSION


and mentioned briefly in the


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Preparation of functionalized graphene nanosheets : The freshly harvested seaweed biomass was utilized to prepare functionalized GNs. The seaweed biomass was crushed to make a semisolid blend and then centrifuged to separate the juice (6070% v/w of fresh seaweed biomass) and solid granules as shown in Scheme 1. The solid granules were allowed to dry in oven to obtain dry seaweed granules (75 g dry granules with ~14% moisture were obtained from 1 Kg of fresh seaweed biomass). The seaweed granules thus obtained was characterized for its elemental composition in which concentrations of Na, K, Ca, Mg, C, H, N and S was found to be significant and characteristic of the seaweed as shown in Table S1. Scheme 1 shows the procedure for the synthesis of functionalized GNs by using solid seaweed granules and DESs. The functionalized GNs s prepared were characterized for its elemental compositions and data are provided in Supporting Information, Table S1 (for GNs such as SAR-Fe-700, SAR-Fe-800, SAR-Fe-900, SAR-Sn-700, SAR-Sn-800, SAR-Sn-900, SAR-Zn-700, SAR-Zn-800 and SAR-Zn-900). As can be seen from Table S1, concentration (in %) of N and S was reduced with the increase in pyrolysis temperature, whereas amount of Fe was found to increase, however the amount of Sn and Zn was found to decrease with the increase in pyrolysis temperature. Among the GNs reported herein SAR-Fe700, SAR-Fe-800 and SAR-Fe-900 was previously reported by us as elctrocatalyst for oxygen reduction reaction,19,20 however, neither the cytotoxicity of these GNs nor their F– removal efficiency was evaluated. Characterization of functionalized graphene nanosheets :

All the products

obtained after pyrolysis of Sargassum granules and DESs [ChCl-FeCl3 (1: 2), ChClSnCl2 (1:2) and ChoCl-ZnCl2 (1:2)] mixture were characterized by various spectroscopic and microscopic techniques for the confirmation of synthesis of GNs. Fig. 1a & 1b shows the FT-IR spectra of Sn- and Zn-functionalized GNs obtained after


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pyrolysis of semisolid composite of seaweed granules and DESs at different pyrolysis temperature (700-900 oC). As can be seen from the spectra, the peaks appear at ~1636 cm‒1 and ~1576 cm‒1 can be assigned to stretching of C=C bond, stretching of C‒N bond

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, respectively. The band at ~1576 cm‒1 appears in the SAR-Sn-700 and SAR-

Sn-800 GNs which indicates the presence of C‒N bond in both GNs. For SAR-Sn900, there was no band found for C‒N bond from FT-IR spectra and the absence of nitrogen in these GNs is also supported by elemental analysis (Table S1). The band appeared at ~600 cm‒1 is ascribed to stretching vibrations of Sn‒O or Zn‒O bond.

26,27

This indicates the presence of tin oxide (SnO and SnO2) in SAR-Sn-700, SAR-Sn-800 & SAR-Sn-900 and zinc oxide (ZnO) in SAR-Zn-700 & SAR-Zn-800 whereas no band appeared at ~600 cm‒1 in the case of SAR-Zn-900 indicates the absence of ZnO. The FT-IR spectra of Fe-functionalized GNs (SAR-Fe-700, SAR-Fe-800 & SAR-Fe900 GNs) which is already reported in a previous article. 19 The presence and absence of metal oxide (SnO, SnO2, and ZnO) in GNs was further confirmed by powder XRD as discussed below in Fig. 1e & 1f. Raman spectroscopy is frequently used for the confirmation of graphene like structure present in pyrolysed samples.28 The peaks at 1350 cm‒1 (D-band), 1570 cm‒1 (G-band) and 2700 cm‒1 (2D-band) in Raman spectrum are of characteristic signature of graphene. The existence of sp2-hybridized carbon atoms demonstrated by G-band, whereas D-band reflects the defects present in graphene and the 2D-band gives indication of number of layers in graphene sample. 29 The properties of the graphene materials are also studied by the relative intensities ratio of the G-band (IG) to D-band (ID), lesser the ratio of ID/IG superior is quality of graphene.28 As can be seen from Fig. 1c & 1d for Sn and Zn-functionalized GNs, D-band and G-band was found to be present in all these samples, however the 2D-bands were not found in all cases.



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Besides, the ID/IG ratio was found to be 1.13, 1.25, 1.11, 1.13, 1.27 and 1.12 for SARSn-700, SAR-Sn-800, SAR-Sn-900, SAR-Zn-700, SAR-Zn-800 and SAR-Zn-900 GNs respectively. The Raman spectra for Fe-functionalized GNs is already reported in a previous article, here also the D-band and G-band was found to be present in all samples, however the 2D band was found only in SAR-Fe-700 and SAR-Fe-800 GNs.19 Thus, the overall results indicates that the quality of graphene was better in case of SAR-Sn-900, SAR-Zn-900, SAR-Fe-700 and SAR-Fe-800.

Figure 1: FT-IR spectra (a & b), FT-Raman spectra (c & d) and Powder X-Ray diffraction pattern (d & f) of Sn- and Zn-functionalized graphene nanosheets obtained at varying pyrolysis temperature


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Fig. 1e & 1f shows the powder x-ray diffraction pattern of Sn and Znfuctionalized GNs. As can be seen from Fig. 1e & 1f, SAR-Sn-700 and SAR-Zn-700 GNs shows the presence of SnO/SnO2 and ZnO phase when the semisolid composite of seaweed granules and DES pyrolysed at 700 oC (Ref. JCPDS file no.: 04-007-0731, 04-012-0775 and 04-014-0069) respectively. These phases remain similar in SAR-Sn800 and SAR-Zn-800 GNs when the pyrolysis temperature increased to 800 oC. Upon further increase in the pyrolysis temperature up to 900 oC, all phases were disappeared indicates the absence of metal oxide in SAR-Sn-900 and SAR-Zn-900 GNs. Whereas some other peaks appear in SAR-Sn-900 and SAR-Zn-900 GNs with weak intensity shows the presence of metal particles indicating the GNs are functionalized with Sn and Zn metal particles respectively. The PXRD pattern of Fe-functionalised GNs such as SAR-Fe-700, SAR-Fe-800 and SAR-Fe-900 is already reported in a previous article.19 The x-ray photoelectron spectroscopy (XPS) is a potential technique to investigate the chemical environment of graphene lattices modified by foreign doping.30 The XPS survey of both the Sn- and Zn-functionalized GNs as shown in Fig. 2a & 2b shows the bands of C, O, N and Sn/Zn with a trace of sulphur. The appearance of oxygen and Sn/Zn band in XPS survey spectrum of Sn- and Zn-functionalized GNs indicates the presence of SnO/SnO2 and ZnO in the GNs respectively and the results are consistent with the powder XRD spectra (Fig. 1e & 1f) and elemental analyses data (Table S1). The high resolution C 1s spectrum of Sn-functionalized GNs presented in the Fig. S1a, shows a high concentration of sp2 carbon with a broad asymmetric tail towards a higher binding energies, a typical signature of graphene samples. The Znfunctionalized GNs also showed a broad asymmetric tail of sp2 carbon towards a higher binding energies (Fig. S2a). The existense of sp2 carbon in Sn- and Zn-



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functionalized GNs can be seen at ~283.1 eV and ~294 eV for C 1s which is from the interaction of graphitic carbon in C-OH with O 1s at ~531 eV and ~541 eV, as shown in the XPS survey spectra, Fig. 2a & 2b. A small band of nitrogen (N 1s peak) also appeared at ~399 eV and ~409 eV for Sn- and Zn-functionalized GNs respectively, confirms that nitrogen interacts with carbon by attaching to the edge of two graphitic carbon nanosheets (Fig. S1b & S2b). From XPS survey spectra, it is also cleared that the presence of SnO/SnO2 and ZnO peak edges in Sn- and Zn-functionalized GNs respectively (Fig. S1c & S2c). In Sn-functionalized GNs, two peaks are appreared for tin at ~486 eV for Sn 3d5/2 and ~495 eV for Sn 3d3/2 (Fig. S1c). Zn-functionalised GNs also showed two peaks of zinc at ~1031 eV for Zn 2p 3/2 and ~1055 eV for Zn 2p1/2 (Fig. S2c). Thus, the XPS spectra of Sn- and Zn-functionalized GNs indicates the presence of metal oxide in all the GNs except SAR-Zn-900, because there are no band appeared in the range of 1025-1060 eV indicates the absence or present in very less amount of zinc particle on the GNs surface (Fig. S2c), this is also supported by PXRD (Fig. 1f) and elemental analyses data as shown in Table S1. The XPS survey spectrum and their high resolution N 1s and Fe 2p spectrum for Fe-functionalized GNs such as SAR-Fe-700, SAR-Fe-800 and SAR-Fe-900 are reported in a previous article.19

Figure 2: The XPS survey spectrum of (a) Sn- and (b) Zn-functionalized graphene nanosheets showing inherent doping of highlighted elements.


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After confirming the structural properties of functionalized GNs by FT-IR, PXRD, Raman and XPS spectroscopic techniques, the sheet like morphology of the GNs were confirmed by SEM and TEM microscopic images. Fig. 3a-c shows the SEM images of GNs such as SAR-Fe-700, SAR-Sn-700 and SAR-Zn-700 respectively. The sheet like morphology of graphitic carbon was visible in SEM images, which was consistence with those observed in TEM images. SEM-EDX element mapping was performed for the investigation of the distribution of metal oxide (Fe3O4, SnO/SnO2 and ZnO)/metal on the surface of GNs (Fig. 3d-f). The SEM-EDX element mapping images of GNs showed the homogenous distribution of metal oxide/metal particles on the surface of GNs, (red and green colour indicated the presence of oxygen and metal elements, Fe/Sn/Zn, respectively). The EDX profile diagram showed the presence of elements such as C, N, O and metal particles in appropriate amount in these GNs (Fig. 3g-i). Therefore, the GNs thus prepared directly from the pyrolysis of seaweed granules and DES in inert atmosphere is functionalized with metal oxide/metal particles and the presence of metal oxide/metal particles is also confirmed by FT-IR, PXRD, XPS and elemental analysis of GNs as discussed above. The SEM images of others GNs such as SAR-Fe-800, SAR-Fe-900, SAR-Sn-800, SAR-Sn-900, SAR-Zn-800 and SAR-Zn900 showed the sheets like morphology in all these GNs and the SEM images of these GNs are provided in supporting information as Fig. S3.



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Figure 3: (a-c) SEM images of SAR-Fe-700, SAR-Sn-700 and SAR-Zn-700 graphene nanocomposites respectively, (d-f) SEM-EDX element mapping on above graphene nanocomposites (Red and green colour shows oxygen and metal element such as Fe, Sn and Zn, respectively) and (g-i) SEM-EDX profile diagram for above functionalized graphene nanocomposites. Fig. 4a-c shows the TEM images of the GNs such as SAR-Fe-700, SAR-Sn-700 and SAR-Zn-700 and it can be observed from the TEM images that the GNs has sheet like morphology with nano scale structural distribution. Fig. 4d-f provides the high resolution TEM (HR-TEM) images of these GNs. TEM images (Fig. 4g-i) also showed homogenous distribution of metal particles on the surface of GNs indicated the GNs are doped with metal particles. The TEM images of others GNs such as SARFe-800, SAR-Fe-900, SAR-Sn-800, SAR-Sn-900, SAR-Zn-800 and SAR-Zn-900


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showed the sheets like structure in all these GNs and the TEM images of these GNs are provided in supporting information as Fig. S4. After characterization of structural and functional properties of the GNs, their surface properties were also evaluated. Surface area of the GNs was measured by N2 adsorption-desorption isotherm and was calculated using Brunauer-Emmett-Teller (BET) equation. The N2 adsorption-desorption isotherm and pore volume distributions of functionalized GNs showed in Figs. 6 & S6 and the surface area, pore volume and pore size data are provided in Table 1. In the pore size analysis of the GNs, pore volume was found to be in the range of 0.042 cm3 g−1 to 0.233 cm3 g−1 (Table 1). In the same order, average pore diameter were noted to be in the range of 2.68 nm to 4.81 nm (Table 1), which is a signature of mesoporous materials. The pore structures of functionalized GNs are complex in nature and tend to be made up of interconnected networks with different size and shape. Thus, the mesoporous nature of functionalized GNs dominates micropore volume of the materials.



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Figure 4: (a-c) TEM images of SAR-Fe-700, SAR-Sn-700 and SAR-Zn-700 respectively, (d-f) HR-TEM images of above graphene nanocomposites and (g-i) HRTEM images of above graphene nanocomposites showed homogenous distribution of metal particles on the surface of graphene nanocomposites.

Table 1: BET Surface area, pore volume and pore size of functionalized graphene nanosheets obtained at different pyrolysis temperature range. Sample code

SAR-Fe-700 SAR-Fe-800 SAR-Fe-900 SAR-Sn-700 SAR-Sn-800 SAR-Sn-900 SAR-Zn-700 SAR-Zn-800 SAR-Zn-900

BET surface area (m2 g‒1) 220.73 167.82 132.02 270.72 298.38 331.76 250.65 223.15 216.14

Pore volume (cm3 g‒1)

Pore diameter (nm)

0.063 0.107 0.193 0.145 0.142 0.132 0.077 0.089 0.233

2.80 2.79 2.18 3.07 3.69 3.71 2.68 2.94 2.81

The Surface area of the GNs were measured by BET method and found to be high (Table 1). As can be seen from Table 1, the surface area of Fe and Zn-doped GNs decreased with increasing pyrolysis temperature. This may be due to the collapse of pores with the increase of temperature. Whereas, in case of Sn-doped GNs, the surface area increased with increasing pyrolysis temperature. The highest BET surface area 331.76 m2 g−1, was observed for SAR-Sn-900 (Sn-doped GN). It is because of the flaky properties of GN and highly exfoliation during pyrolysis of semi-solid composite of seaweed granules and DES as shown in Scheme 1. The lowest surface area 132.02 m2 g−1, was observed for SAR-Fe-900 (Fe-doped GN) and it is because of its lower exfoliation during pyrolysis of semi-solid composite.19 According to IUPAC technical report, the N2 adsorption-desorption isotherm plots (Hysteresis loop) are classified in various types.

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Thus, the physisorption isotherm of functionalized GNs is type-4 and 16 ACS Paragon Plus Environment

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it comes under hysteresis loop type H2 of all functionalized GNs (Figures S5a, S5c and S6a). So the GNs with type-4 physisorption isotherm and H2 type loop is indicated the mesoporous adsorbent type materials and it may be effective for the adsorption of fluoride ion from fluoride rich drinking water as discussed lateron. Cytotoxicity assay of functionalized graphene nanosheets tested on human lung carcinoma cells. Human lung carcinoma cells (A549) were used for the cytotoxicity MTT assay of functionalized GNs (details are described in supporting information). In the present study, GNs did not record cytotoxicity at the highest dose of 200 µg and hence their effective dose (ED50) could not be calculated. These results were in agreement with cytological observations wherein, cells were adherent and spindle shaped at the end of treatment (Supporting Fig. S7) and were comparable to that of the control cells (Fig. S8). But SAR-Sn-700 GN recorded 20% decrement in the cell viability at 10 µg without major alterations in cell morphology (Fig. S7d), while the highest dose of 200 µg also elicited similar level of toxicity response (Fig. 5d ). Hence, it can be inferred that SAR-Sn-700 shows moderate toxicity even at highest dose whereas other GNs such as SAR-Fe-700, SAR-Fe-800, SAR-Fe-900, SAR-Sn-800, SAR-Sn-900, SAR-Zn-700, SAR-Zn-800 and SAR-Zn-900 are non-toxic (Fig. 5). These observations are vital for safety assessment of functionalized GNs and opens new avenues for their use in biomedical applications and removal of fluoride from fluoride rich drinking water to obtain safe-drinking water. Efficient removal of excess fluoride from fluoride rich drinking water and their kinetic study : The non-toxic GNs (mesoporous adsorbent) with high specific surface area has opened up a new green, sustainable and cost-effective way to remove excess fluoride from fluoride rich drinking water. In a typical experimental procedure, 0.025 g of the GNs was added to the 50 mL of fluoride rich drinking water and has shaken at



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room temperature for 30-150 min followed by filtration through Whatman filter paper to optimize the time of adsorption. The effect of time on fluoride adsorption by Fedoped GNs obtained at different pyrolysis temperature was studied and the time dependent study shown in Fig. 6a . It can be seen that the adsorption rate of fluoride was increased with time and become constant after 120 min, which was considered as a optimized time duration for maximum fluoride adsorption (Fig. 6a ).

Figure 5: Cytotoxicity potential of graphene nanosheets such as (a) SAR-Fe-700, (b) SAR-Fe-800, (c) SAR-Fe-900, (d) SAR-Sn-700, (e) SAR-Sn-800, (f) SAR-Sn-900, (g) SAR-Zn-700, (h) SAR-Zn-800 and (i) SAR-Zn-900 on human lung carcinoma cells (A549). Results are expressed as mean ± SD for triplicate samples wherein ns = nonsignificant, * p

Seaweed-Derived Nontoxic Functionalized Graphene Sheets as

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