Green-fabrication of gold nanomaterials using

0 downloads 0 Views 6MB Size Report
This green technology .... according to the methods described in Bergey's manual of systematic ... N03 and its application in catalytic reduction of nitro aromatic pollutants. Environ Sci ...... cles: technological concepts and future applications.
Environ Sci Pollut Res https://doi.org/10.1007/s11356-017-0617-7

RESEARCH ARTICLE

Green-fabrication of gold nanomaterials using Staphylococcus warneri from Sundarbans estuary: an effective recyclable nanocatalyst for degrading nitro aromatic pollutants Sudip Nag 1 & Arnab Pramanik 1 & Dhrubajyoti Chattopadhyay 2 & Maitree Bhattacharyya 1

Received: 15 July 2017 / Accepted: 24 October 2017 # Springer-Verlag GmbH Germany 2017

Abstract Microbial synthesis of gold nanoparticles (GNPs) has attracted considerable attention in recent times due to their exceptional capability for the bioremediation of industrial wastes and also for the treatment of wastewater. A bacterial strain Staphylococcus warneri, isolated from the estuarine mangroves of Sundarbans region produced highly stable GNPs by reducing hydrogen auric chloride (HAucl4) salt using intracellular protein extract. The nanoparticles were characterized utilizing ultraviolet-visible spectrophotometry, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, X-ray diffraction, and surface enhanced Raman scattering. Highly dispersed, spherically shaped GNPs varied around 15–25 nm in size and were highly crystalline with face-centered cubic structures. Recyclable catalytic activity of as-synthesized GNPs was evidenced by complete degradation of nitro aromatic pollutants like 2-nitroaniline, 4-nitroaniline, 2-nitrophenol and 4-nitrophenol. Our GNPs show excellent and efficient catalytic activity with significantly high rate constant (10−1 order) and high turnover frequency (103 order) in recyclable manner up to three times. To our knowledge, this is the first report of Staphylococcus warneri in the Responsible editor: Santiago V. Luis Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-017-0617-7) contains supplementary material, which is available to authorized users. * Maitree Bhattacharyya [email protected] 1

Department of Biochemistry, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, West Bengal 700019, India

2

Amity University, Major Arterial Road, Action Area II, Rajarhat, Newtown, Kolkata, West Bengal 700156, India

production of gold nanoparticles. This green technology for bioremediation of toxic nitro aromatic pollutants is safe and economically beneficial to challenge the development and sustainability issue. Keywords Bacterial intracellular protein . Gold nanoparticles . Recyclable catalysis . Nitro aromatic pollutants . Wastewater treatment

Introduction In recent years, noble metal nanoparticles such as gold, platinum, and silver have received great attention in research field due to several applications for the sustenance of the societal development (Narayanan and Sakthivel 2010; Sperling et al. 2008). Among these noble metal nanoparticles, especially gold is most important due to its higher biocompatibility, long-term stability, and resistance towards oxidation (Sharma et al. 2012). Gold nanoparticles (GNPs) have found important applications in different areas of therapeutics and industry (Iravani 2014; Fu and Zhang 2015; Biju 2014). Various physical and chemical processes are available for synthesizing GNPs (Sperling et al. 2008; Mohanpuria et al. 2008; Dahl et al. 2007). In chemical process, GNPs are synthesized using toxic compounds including sodium borohydride as reducing agent, toxic molecules containing thiol functional group as stabilizing agents (e.g., dodecanthiolates), and toxic solvents (e.g., ethaline) as a medium (Chirea et al. 2011). These processes are also expensive and energy consuming. Therefore, development of clean, cost-effective, and environmental friendly techniques are needed for synthesizing GNPs. The use of microorganisms might be a prominent substitute to physical and chemical procedures for the synthesis of GNPs in an eco-friendly way (Iravani 2014; Shi et al. 2015; Shankar

Environ Sci Pollut Res

et al. 2004; Hulkoti and Taranath 2014). A number of studies have been reported for the bacterial synthesis of GNPs, such as Geobacillus (Correa-Llantén et al. 2013), Stenotrophomonas maltophilia (Nangia et al. 2009), Escherichia coli (Srivastava et al. 2013), and Rhodopseudomonas (He et al. 2007). Most of the studies were focused on biosynthesis of GNPs using either whole cell (Correa-Llantén et al. 2013) or using cell-free extracellular medium (Manivasagan et al. 2015; Vinay Gopal et al. 2013). But cell-free intracellular extract also contains important reductase enzyme or protein which may also be applied to synthesize GNPs. In recent years, nanoparticles are widely used in catalytic applications especially in case of degrading pollutants in wastewater. Among several water pollutants, nitro aromatic compounds are considered to be the most toxic. These are commonly used in the production of dyes, explosives, and pesticides in industry. Being released into the environment, these compounds show serious carcinogenic and mutagenic threats to human health (Booth 2000). Although, several methods like ion exchange adsorption, filtration, and chemical precipitation are available for eliminating pollutants, but these are not very effective. Therefore, there is always a strong need to eliminate these highly toxic pollutants easily and quickly using efficient catalytic agent. The nanoparticles can work as a better catalyst than bulk metal due to their greater accessibility to surface and low coordination number. Few previous report revealed that nanoparticles could catalyzed the degradation of 4-nitroaniline and 4-nitrophenol (Chirea et al. 2011; Srivastava et al. 2013; Reddy et al. 2013). Generally, the biosynthesized nanoparticles showed better catalytic efficiency than chemically synthesized GNPs. It should be noted that the catalytic behavior of nanoparticles significantly depends on the capping molecules (protein, nucleic acid, polysaccharides, etc.) of biosynthesized nanomaterials (Das et al. 2012; Zhan et al. 2012). In this article, we report the utilization of intracellular protein extract (IPE) of Staphylococcus warneri SuMS_N03 (MCC 3074, NCBI GenBank accession number KP771665) isolated from Sundarbans estuary to produce highly dispersed, stable, and spherical shaped GNPs. The effects of reaction parameters such as initial gold ion concentration, solution pH, and incubation time on the characteristics of synthesized GNPs were assessed. Spectroscopic and microscopic techniques were also used to elucidate the morphology as well as chemical nature of particles. In addition, characterization of the isolate on the basis of 16S rRNA gene sequences as well as determination of their morphological, biochemical, and physiological features have been performed. The catalytic efficiency of as-synthesized GNPs in recyclable mode was investigated during chemical degradation of different nitro aromatic compounds, viz. 2nitroaniline (2-NA), 4-nitroaniline (4-NA), 2-nitrophenol

(2-NP), and 4-nitrophenol (4-NP) in the presence of sodium borohydride. The overall work is depicted in Fig. 1.

Materials and methods Reagents All the reagents purchased and used were of analytical grade. Hydrogen auric chloride (HAucl4), 2-nitroaniline (2-NA), 4-nitroaniline (4-NA), 2-nitrophenol (2-NP), 4nitrophenol (4-NP), sodium borohydride (NaBH4), and Rhodamine 6G (R6G) were purchased from SigmaAldrich; Luria broth and Luria Bertini agar from Himedia Laboratories, India; PCR template preparation kit and Plasmid isolation kit from Roche Diagnostics, India; QIAquick gel extraction kit from Qiagen, Germany; 27F and 1492R primers from IDT, India; and InsTAclone PCR cloning kit from Thermo Scientific, USA. Collection of sample and isolation of bacteria Soil sediments were collected from Sundarbans estuary (21.69 N, 88.565 E), West Bengal, India. The pure colony of strain SuMS_N03 was isolated from this soil sample by pour plate technique on Luria Bertini agar at 37 °C. The strain was maintained in Luria broth media and stored in 20% glycerol at − 80 °C for long-term preservation. The isolated bacterial strain was deposited in the Microbial Culture Collection (MCC), Pune. Characterization of isolated bacteria Phenotypic and biochemical characterization Morphological and physiological characterization of isolated strain was performed using various classical biochemical tests according to the methods described in Bergey’s manual of systematic bacteriology (Boone et al. 2001). Carbon source utilization test was assessed by Biolog GP2 MicroPlate™ (Biolog Inc., Hayward, CA, USA). The detail cell morphology was observed in scanning electron microscope (Zeiss EVO18) with gold coating of 10 nm in coater machine (Quorum Q150T ES). DNA amplification and 16S rRNA gene sequencing Genetic characterization of isolated strain was performed by means of 16S rRNA gene analysis. Genomic DNA was extracted from overnight cultured bacterial cell using high pure PCR template preparation kit (Roche, India). The 16S rRNA gene was amplified by using prokaryotic universal primers 27F and 1492R (Jiang et al. 2006). The

Environ Sci Pollut Res

Fig. 1 Overall schematic representation of biosynthesis of gold nanoparticles using intracellular protein lysate (IPE) of Staphylococcus warneri SuMS_ N03 and its application in catalytic reduction of nitro aromatic pollutants

PCR products were separated electrophoretically on a 1% agarose gel and further purified using QIAquick gel extraction kit (Qiagen). The purified PCR product was subsequently ligated to the pTZ57R/T cloning vector (InsTAclone PCR cloning kit). The vectors with the inserted sequences were then transformed into E. coli DH5α for further sequencing using the ABI Prism 3100 Genetic Analyzer according to previous literature (Chakraborty et al. 2015) with necessary modification.

Phylogenetic analysis The 16S rRNA gene sequences were aligned in the Bioedit 7.2.5 software (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and examined for sequence homology with the archived 16S rDNA sequences from GenBank (http://www.ncbi.nlm.-nih. gov/nucleotide) with the help of the BLAST search program (Altschul et al. 1990) and EzTaxon server (Kim et al. 2007) to identify the nearest taxa. Phylogenetic tree was constructed by neighbor joining (NJ), maximum likelihood (ML), and

minimum evolution (ME) methods using MEGA 6.0 (Tamura et al., 2013). Preparation of intracellular protein extract Overnight grown bacterial cell pellet was collected by centrifugation and washed twice with sterilized phosphate-buffered saline (PBS). Around 1.0 ± 0.02 g of cell pellet was suspended in 10 ml of 0.1 M sodium phosphate buffer, pH 7.0, and disrupted using ultrasonic processor (Hielscher-UP200S) for 10 min with 2-min intervals under 0.7 sonication cycle and 80% amplitude. Finally, supernatant was collected and subsequently filtered using 0.22-μm syringe filters (Merck Millipore, Germany) for the synthesis of gold nanoparticles. Synthesis of GNPs GNPs were synthesized by mixing 1:1 (v/v) of IPE and aqueous solution of HAuCl4 in a conical flask and kept in shaking condition for overnight at 140 rpm. Control was set up as

Environ Sci Pollut Res

above with the exception of the IPE volume being replaced by double-distilled water. Optimization of reaction parameters for synthesis of GNPs IPE was mixed with aqueous solution of HAuCl4 in different sets of pH ranging from 3 to 13 to optimize the pH condition. To estimate the optimum concentration of HAuCl4, IPE was mixed with the HAuCl4 solutions at different concentrations (0.2, 0.4, 0.5, 0.8, 1.0, 1.5, 2.0 mM) and maintained in the above mentioned conditions. To attain the optimized incubation time, IPE and HAuCl4 were mixed at their respective optimal pH and salt concentration. Absorbance spectra were recorded from each sample to find out the optimum reaction parameters. Purification of GNPs After synthesis of GNPs, the purification process was performed by modifying the method described in previous literature (Balasubramanian et al. 2010). To remove excess salt and other byproduct along with maximal recovery of nanoparticles, the solution was centrifuged at 28000g for 30 min at 4 °C (Sigma-3K30). The pellet of the nanoparticles was washed three times by double-distilled water and resuspended in ultrapure water (Invitrogen). The purified nanoparticles solution was stored at 4 °C for long-time storage. Characterization of GNPs UV-Vis spectroscopy The formation of GNPs was observed in the double beam UVVis spectrophotometer (Hitachi-U2900) by recording the spectra in the range 200 to 800 nm with a resolution of 1 nm. Dynamic light scattering (DLS) measurement Hydrodynamic diameter (dh) and zeta potential (ξ) of GNPs was measured by dynamic light scattering using the Zetasizer model NANO ZS90 (Malvern Instruments Ltd., Worcestershire, UK) (5 mW HeNe laser λ = 632 nm). Transmission electron microscopic (TEM), EDAX, and SAED study GNPs were pipetted onto carbon-coated copper grid and allowed to air dry for overnight in dark at room temperature to evaporate the excess solvent. Sample was analyzed in transmission electron microscope (JEOL-JEM 2100, Japan) operated at 200 kV. Selected area electron diffraction (SAED) patterns were taken using the same instrument. Elemental

analyses of synthesized GNPs were conducted by using energy dispersive X-ray analysis which was carried out using transmission electron microscope equipped with an energy dispersive X-ray spectrometer (Oxford-INCA). Field emission scanning electron microscopic (FE-SEM) study GNPs solution was uniformly spread over clean and dry coverslip to make a thin layer and allowed to air dry for overnight at room temperature. The morphology and size were examined on field emission scanning electron microscope (JEOLJSM 7600F, Japan). Atomic force microscopic (AFM) measurement Ultrasonicated GNPs solution was uniformly spread over coverslip and incubated overnight at room temperature to dry properly. Using an atomic force microscope (Bruker AXS Pvt. Ltd., Vecco-Innova), the shape, size, and/or size distribution of individual nanoparticles and group of nanoparticles were determined with visualization and analysis in three dimensions. All these parameters were measured in silicon probes with response frequency 276–318 kHz. X-ray diffraction measurement X-ray diffraction (XRD) is a powerful tools from which crystallographic information about the samples are obtained. For XRD analysis, GNPs solution was lyophilized (LyophilizerEyla-FDU 1200) to get solid, pure powder. The structure and phase purity of GNPs were determined using X-ray diffractometer (D8-Advance, Bruker) using Cu Kα radiation (λ = 1.54 Å). The scanning was done in the region of 2θ from 10° to 90° at 0.02°/min and time constant of 2 s. The size of the nanoparticle was calculated using the Debye-Scherrer’s formula D¼

Kλ β 12 cosθ

where D is the average crystal size, K is shape factor (K = 0.94), λ is the X-ray wavelength (λ = 1.5406A°), θ is Bragg’s angle (2θ), and β1/2 is the full width at half-maximum (FWHM) in radians. Fourier transformed infrared (FTIR) spectroscopic measurement The FTIR measurement was carried out to identify possible functional group involved in intracellular protein extract (IPE) which may be responsible for the synthesis of GNPs. The GNPs solution was lyophilized to get solid powder and was

Environ Sci Pollut Res

mixed with calcium fluoride (CaF) pellet. The spectrums were recorded for the IPE in liquid state and GNPs in solid state at FTIR spectrophotometer (Spectrum 100, Perkin Elmer) in the range of 400–4000 cm−1 at a resolution of 4.0 cm−1.

cooled charge-coupled device (CCD) detector. He-Ne laser with a wavelength of 632.8 nm was used as the excitation light source and a 100× objective with a numerical aperture (NA) of 0.9 was used to get the laser spot diameter of ∼ 0.7 μm.

SDS-PAGE The probable intracellular protein moiety responsible for the synthesis and stabilization of GNPs was identified by SDSPAGE. To separate the capping protein from the surface of nanoparticles, purified GNPs solution was centrifuged at 28000g for 30 min to collect the pellet and treated in three different procedures. First, the pellet was treated with aqueous solution of 2 M guanidine hydrochloride. The supernatant containing capping protein was separated by centrifugation by 28,000g for 15 min. To concentrate protein and removal of guanidine hydrochloride before SDS-PAGE, ethanol precipitation in 1:9 ratio (v/v) was performed. The pellet was collected by centrifugation at 15000g for 10 min and washed with cold ethanol. After aspirating ethanol, it was resuspended in phosphate buffer, pH 7.0. Second, the pellet was mixed with 1% SDS solution and boiled in water bath for 10 min. Third, the pellet was further boiled with loading dye only for 5 min. The supernatant containing capping protein was separated by centrifugation by 28,000g for 15 min. Again, to analyze the whole proteins in intracellular protein extract (IPE), ethanol precipitation in 1:9 ratio (v/v) was performed to concentrate the protein and centrifuged at 15000g for 10 min. The pellet was washed with cold ethanol and resuspended in phosphate buffer, pH 7.0. The four different samples were analyzed in 15% SDS-PAGE. A high molecular weight marker (Thermo Scientific, USA) was used to determine the molecular weight of proteins. To visualize the protein bands in the gel, a standard silver staining method was performed. Surface-enhanced Raman scattering study Surface-enhanced Raman scattering (SERS) is a surfacesensitive powerful technique for the identification of trace amounts of chemical and biological molecules at single molecular level when molecules are adsorbed on the surface of the metal nanoparticles. A wide range of Rhodamine 6G (R6G) solutions having different concentrations (1 × 10−3 to 1 × 10−7 M) were prepared in ultrapure water for the SERS measurements. GNPs solution was mixed with R6G solution of different concentration and shaken well to ensure homogeneous mixing. The mixture was incubated for 2 h and then rinsed with ultrapure water to remove any un-adsorbed R6G molecules. Dried sample was prepared by dropping the solution of R6G and GNPs on a glass substrate and allowing it to evaporate at room temperature. All the measurements were performed using a micro-Raman setup consisting of a spectrometer (Lab RAM HR, Jobin Yvon, Horiba) and a Peltier-

Catalytic degradation of nitro aromatic pollutants using GNPs To test the efficacy of as-synthesized GNPs for their catalytic activity, the degradation of four different nitro aromatic compounds viz. 2-NA, 4-NA, 2-NP, and 4-NP in the presence of NaBH4 was studied as a model reaction accordingly previous literatures (Shi et al. 2015; Gangula et al. 2011; Guria et al. 2016) with necessary modifications. The concentrations of the GNPs were fixed by the intensity of absorbance to 0.3 a.u. In this reaction, ultrapure water (1.59 ml), aqueous solution of 2-NA (0.11 ml, 0.01 M), and freshly prepared ice-cold solution of NaBH4 (1 ml, 0.05 M) were mixed homogeneously. Then 0.3 ml ultrasonically dispersed GNPs solution was added to the reaction mixtures. The catalytic process was investigated by monitoring the alteration of absorbance spectra in a time-dependent manner of 1-min interval in the scanning range of 200 to 800 nm by using a UVVis spectrophotometer (Hitachi-U2900). The same procedure was followed for the degradation of other nitro aromatic compounds with required changes in their concentration depending on their molar absorptivity. The concentrations of each nitro aromatic compounds are given in Supplementary Table S1. We also examined the recyclability of catalytic role of as-synthesized GNPs up to the third cycle in case of all degradation reactions of four nitro aromatic compounds. After every cycle, GNPs are recovered from reaction solution, and the pellet was washed three times with ultrapure water and resuspended in 0.3 ml water to keep the concentration and volume of catalyst constant to be reused in the next cycle of reaction. The same process was repeated up to three cycles. The control reaction was also performed without adding GNPs in the reaction solution. In all these catalysis reactions, the volume and concentration of catalyst as well as NaBH4 solution were also kept constant. To find out the actual concentration of nitro aromatic compound at each point of maximum absorbance, calibration curves of all the compounds were prepared using standard solutions. Statistical analysis All experiments were performed at least three times and the data were reported as mean ± standard deviation. The normality of the GNP size distribution was determined by the Kolmogorov-Smirnov test. All the statistical analysis was performed by the Origin Software 9.0 (Origin Lab Corporation, USA).

Environ Sci Pollut Res

Results Phylogenetic analysis and molecular characterization of isolated bacteria The detail results of basic morphological tests, biochemical tests, and Biolog assay have been summarized in Supplementary Table S2. Scanning electron microscopic image (Fig. 2a) showed that cells were round shaped, distinct, and also attached together forming a cluster. On the basis of nucleotide homology and phylogenetic analysis, the isolated strain SuMS_N03 was found to have the highest level of significant similarity (99.93%) at species level with type strain Staphylococcus warneri ATCC 27836T (see Supplementary Table S3).The phylogenetic position of strain SuMS_N03 (NCBI GenBank accession number KP771665) is shown in dendrogram (Fig. 2b).The phylogenetic position of isolated strain SuMS_N03 was again supported by the maximum evolution (ME) and maximum likelihood (ML) analyses in which analogous result was found having significant branch length and high bootstrap value (94% in ME and 84% in ML).The strain is available in Microbial Culture Collection, Pune, India, with accession no. MCC 3074. Biosynthesis of GNPs and optimization of regulating parameters

(Fig. 3a) indicated the formation of GNPs. The color change is due to excitation of surface plasmon vibration, i.e., collective oscillation of free electrons induced by an interacting electromagnetic field in metallic nanoparticles which is the characteristic feature of metal nanoparticles (Karthick et al. 2014). At the same time, no color change was observed in control experiment. Among different salt concentrations (0.2–2.0 mM), 1.5 mM was found to be the most optimize one to get highest concentration of GNPs (Fig. 3b) with stable and monodisperse particles. During synthesis, no color change was observed at the acidic pH range (3, 5), but faint color change was observed at pH 6 and significant color change was observed at neutral (pH 7) and alkaline pH (9, 11, 13) (see Supplementary Fig. S1). In our study, GNPs were synthesized only at neutral and alkaline condition, but well-dispersed, maximum yield, and most stable particles were formed at pH 7 (Fig. 3c).The GNPs were formed within 15 h incubation time and intensity of SPR peak increased significantly with time up to 75 h without any noticeable peak shift and finally reached a saturation (Fig. 3d).This feature indicates the bio-reduction of Au+3 ion to form gold nanoparticles as Au0 which was increased with time to certain extent. Even after 1 month of synthesis, no significant change was observed in position or intensity of SPR peak (see Supplementary Fig. S2) confirming the high stability of as-synthesized GNPs.

The qualitative analysis GNPs formation using IPE of bacterial strain Staphylococcus warneri SuMS_N03 was carried out based on visual observation of color change from pale yellow to ruby red. The quantitative analysis of GNPs formation was performed using UV-Vis spectroscopy. The intense surface plasmon resonance (SPR) peak at 535 nm

Characterization of GNPs

Fig. 2 a Scanning electron microscopic (SEM) images of Staphylococcus warneri SuMS_N03 (scale bar 200 nm). b Unrooted phylogenetic tree obtained by the neighbor-joining (NJ) method based on 16S rRNA gene sequences representing the position of Staphylococcus warneri SuMS_N03 (KP771665) among its phylogenetic neighbors. Numbers at nodes designate levels of bootstrap support (%)

based on a NJ analysis of 1000 resampled datasets; only values higher than 50% are displayed. Asterisks represent branches that were obtained using the minimum evolution (ME) and maximum likelihood (ML) algorithms. NCBI accession numbers are provided in parentheses. Bar = 0.01 nucleotide substitutions per site. The sequence of Bacillus subtilis DSM10T (AJ276351) was applied as an out-group

HR-TEM, SAED, and EDAX analysis The size, shape, and morphology of the GNPs were represented at different magnification in Fig. 4a. The TEM images

Environ Sci Pollut Res

Fig. 3 UV-Vis absorption spectra of as-synthesized gold nanoparticles under different conditions. a After 24 h, λmax at 535 nm due to surface plasmon resonance. b Effect of initial gold ion (HAucl4) concentration. c Effect of pH of reaction solution. d Effect of incubation time of reaction

indicated that most of the nanoparticles formed at pH 7 were well dispersed and mainly spherical shaped having diameter about 15–25 nm. The morphology and size analysis studies of the GNPs at different pH (9, 11, and 13) were also investigated utilizing HR-TEM (see Supplementary Fig. S3). These TEM images revealed that most of the particles were spherical of diameter 20–30 nm (pH 9), spherical of 20–35 nm (pH 11), and spherical with some triangular shape of 20–40 nm (pH 13). The overall results suggested that the size of synthesized particles may be altered by changing the pH of the reaction solution. The selected area electron diffraction (SAED) pattern (Fig. 4b) of as-synthesized GNPs indicated that the nanoparticles were pure crystalline in nature and the lattice structure is face-centered cubic (FCC) which is again correlated with XRD pattern as discussed later. In energy dispersive X-ray spectroscopic (EDAX) pattern (Fig. 4c), the strong

peaks at 2.1 and 9.9 keV confirmed the presence of gold atom and the peaks for C and Cu were due to background signals from carbon-coated copper grid. The particle size distribution histogram (Fig. 4d) showed that the maximum number of particles was within 15–25 nm. DLS and zeta potential analysis DLS parameter represents the average particle size (Z average) in terms of hydrodynamic diameters. The average size of GNPs was estimated to be 81 nm (see Supplementary Fig. S4A). Zeta potential (ξ) value provides ideas regarding surface charge and colloidal stability of nanoparticles and here zeta potential value of GNPs was observed to be − 36.0 mV (see Supplementary Fig. S4B). This high negative value indicates the higher stability of these nanoparticles in solution because

Environ Sci Pollut Res

Fig. 4 a TEM images of as-synthesized gold nanoparticles (scale bar 20 nm). Inset contains HR-TEM images of spherical shaped assynthesized gold nanoparticle (scale bar 5 nm). b Specific area electron

diffraction (SAED) pattern of as-synthesized gold nanoparticles. c Energy dispersive X-ray (EDX) spectrum of as-synthesized gold nanoparticles. d Particle size distribution histogram determined from the TEM image

the negative–negative repulsive force between nanoparticles in the solution helps to maintain their stability. This high negative charge has been originated from the amino acids present in capping protein of GNPs. DLS analysis also revealed that the polydispersity index of this GNPs was much low being 0.48.

micrographs (Fig. 5a), the as-synthesized particles were well dispersed and spherical shaped having diameter in the range 15–25 nm. The analysis of AFM images (Fig. 5b, c) using the NanoScope Analysis software (Bruker-version 1.40) indicated that the thickness or height of the particles ranged from 26 to 18.8 nm (data not shown).

FE-SEM and AFM analysis

XRD analysis

The morphology, surface pattern, and size distribution of GNPs were estimated using the FE-SEM and AFM. But the difference between this two types is that FE-SEM offers twodimensional visualization or analysis, whereas it is three dimensional in AFM platform. According to FE-SEM

The crystal structures and its phase purity of the assynthesized GNPs were investigated by X-ray diffraction analysis (XRD). In the XRD pattern (Fig. 6), five intense peaks of Bragg’s reflections were observed at 2θ = 38.39°, 44.55°, 64.74°, 77.84°, and 82° corresponding to the (111),

Environ Sci Pollut Res Fig. 5 a FE-SEM image of assynthesized gold nanoparticles (scale bar 100 nm). b, c 2D and 3D view of AFM image of assynthesized gold nanoparticles, respectively

(200), (220), (311), and (222) planes of lattice of metallic gold. According to Bragg’s reflections, the GNPs are distinctly indexed to a face-centered cubic crystal (FCC) structures. This data was correlated with SAED pattern (Fig. 4b) of GNPs. The diffraction peaks was also coincided with the standard data files (International Centre for Diffraction Data) where JCPDS card no. 02-1095, no. 040784, and no. 01-1174 indicated the formation of GNPs.

The absence of any extra peak in Fig. 6 indicated the high purity of the as-synthesized GNPs. The broad bottom area of the peaks in this XRD pattern indirectly indicated the smaller size of the nanoparticles (Binupriya et al. 2010). The size of particles was also estimated to be 19 nm, using Debye-Scherrer’s formula with respect to maximum height peak for (111) plane. Thus, the XRD pattern is a strong evidence in favor of UV-Vis and TEM results for the formation of GNPs.

FTIR spectroscopic analysis

Fig. 6 X-ray diffraction spectrum of as-synthesized gold nanoparticles

The FTIR spectrums of IPE and GNPs are presented in Fig. 7a, b, respectively. It was found that the former higher peak (at 3449 cm−1) has been shifted towards shorter frequency (3429 cm−1) during the formation of GNPs. This result confirmed the role of alcoholic/ carboxylic –OH group for the formation of nanoparticles and also capped around the surface of GNPs (Patra et al. 2015). Again, another peak shift of >C=O stretching vibration of amide linkage from higher frequency (1634 cm−1) to lower frequency (1630 cm−1) indicated the reduction of Au+3 to Au0 (Stalin Dhas et al. 2012). The single peak in dry sample at 1630 cm−1 for >C=O stretching vibration of amide linkage instead of amide I

Environ Sci Pollut Res

extract (lane 4). But in lane 3 (guanidine hydrochloridetreated GNPs), low molecular weight protein bands for capped protein were observed at ~ 70 and ~ 60 kDa. Similar bands were also observed at lane 5 (SDS-treated GNPs) and lane 6 (dye-treated GNPs). Surface plasmon resonance and SERS activity analysis SPR is a unique feature of noble metal nanoparticles. Surface plasmon originates from collective oscillation of free electrons around the surface of nanocore. The assynthesized GNPs showed sharp SPR peak at 535 nm which has been shown in Fig. 3a and the single peak indicated one type of nanostructure. To explore the utility of the as-synthesized GNPs as an efficient SERS substrate, the concentration-dependent measurement has been performed using R6G (a commonly used fluorescent dye) as a Raman probe molecule. The SERS activity of as-synthesized GNPs has been presented as Raman spectrum in Fig. 8. The spontaneous Raman signals for R6G (1 × 10−3 M) was very weak, which was enhanced on the surface of GNPs even at low concentration of R6G (1 × 10−7 M) compared to that of pure R6G. The intensity of Raman signals was enhanced on increasing the concentration of R6G from 1 × 10−7 to 1 × 10−3 M. The peaks were assigned to the in plane xanthene ring deformation and out of plane C–H bending (611 and 773 cm−1), in plane xanthene ring deformation: C–H bending and N–H bending (1180 cm−1); in plane xanthene ring breathing: N–H bending, CH2 wagging (1300.5 cm−1); in plane xanthene ring stretching: C–H Fig. 7 FTIR spectra of a intracellular protein extract (IPE) of Staphylococcus warneri SuMS_N03 and b as-synthesized gold nanoparticles

(1650 cm−1) and amide II (1540 cm−1) indicated the involvement of N–H moieties of intracellular protein with the metal surface. The peak at 1403 cm−1 confirms the contribution of carboxylate ion (−COO−) in the synthesis and present towards outside of metal nanocore (Uma Suganya et al. 2015). Apart from these peaks, some peaks were also observed at different frequencies and the explanation has been represented in Supplementary Table S4.

SDS-PAGE analysis: identification of capped protein After analyzing the gel with silver staining (Supplementary Fig. S5), it was observed that different low molecular size proteins (~ 70 to 15 kDa) presented in intracellular protein

Fig. 8 Raman spectrum of 1 × 10−3 M R6G solution (cyan) and SERS spectra of R6G at concentrations 1 × 10−7 M (red), 1 × 10−6 M (blue), 1 × 10−5 M (green), 1 × 10−4 M (magenta), and 1 × 10−3 M (black) with the excitation wavelength of 632.8 nm

Environ Sci Pollut Res

bending (1351 cm−1); in plane xanthene ring stretching: C–H bending, N–H bending, and C–H stretching (1500 cm−1); in plane xanthene ring stretching: N–H bending (1565 cm−1); in plane xanthene ring stretching: C–H bending (1640 cm−1) (Singha et al. 2015). Catalytic activity of gold nanoparticles: application for the removal of toxic compounds The aqueous solution of 2-NA and 4-NA exhibited vivid yellow color with absorption maxima at about 380 and 412 nm, respectively. Addition of NaBH4 into the aqueous solution of 2-NA and 4-NA did not lead to the reduction reaction. But when GNPs were added to the mixture of 2-NA/4-NA and NaBH4, reduction reaction started immediately. As a result, the solution containing 2-NA turned colorless and the intensity of the characteristic peak at 412 nm decreased progressively with simultaneous appearance of a new peak near 225 nm which is associated with ortho-phenylenediamine (o-PDA) (Anantharaj et al. 2016). The reduction of 2-NA to o-PDA was completed within 5 min in the first cycle, 9 min in the second cycle, and 16 min in the third cycle (Fig. 9a–c). On reduction in the presence of GNPs, the solution of 4-NA turned colorless and the intensity of absorption peak at 380 nm showed progressive decrease with appearance of new absorption peaks at 305 and 240 nm due to the formation of para-phenylenediamine (p-PDA) (Reddy et al. 2013). The reduction reaction was completed within 5 min in the first cycle, 8 min in the second cycle, and 16 min in the third cycle (Fig. 9d–f). Aqueous solution of 2-NP was pale yellow in color and showed two distinct absorption peaks at 278 and 351 nm. When NaBH4 (pH > 12) was added to the aqueous solution of 2-NP, an intense yellow color appeared due to formation of 2-nitrophenolate ion in alkaline medium with a red-shift of the absorption peaks to 281 and 415 nm, respectively (Liu and Yang 2011), but no reduction was observed. Thus, the progress of the reaction was monitored by tracking the changes in the intensity of absorption peak of 2-nitrophenolate ion at 415 nm. Introduction of GNPs into the reaction solution led to the disappearance of the yellow color of the solution progressively with decrease in the intensity of the absorption peak at 415 nm and appearance of a new peak at 229 nm, indicating the formation of the reduction product 2-aminophenol (2-AP). 2-NP was reduced completely within 6 min in the first cycle, 16 min in the second cycle, and 17 min in the third cycle (Fig. 9g–i). On addition of NaBH4 to the aqueous solution of 4-NP, the solution turned into intense yellow color from pale yellow color and the absorption maximum peak at 317 nm was red shifted to 400 nm due to formation of 4-nitrophenolate ion in alkaline medium, although no reduction occurred. Here, the progress of the reduction reaction was monitored by tracking

the changes in the intensity of absorption peak at 400 nm. The reduction process started immediately with addition of GNPs into the solution. The fading of yellow color of the solution and progressive decrease of intensity of absorption peak at 400 nm with simultaneous appearance of new peak at 298 nm indicated the reduction of 4-NP with formation of 4aminophenol (4-AP) (Gangula et al. 2011). This reaction was completed within 10 min in the first cycle, 13 min in the second cycle, and 26 min in the third cycle (Fig. 9j–l). No significant change in color or intensity of absorption peak was observed in control experiments (absence of GNPs) even after several hours indicating no reduction to be possible without addition of GNPs. The rate of reduction should depend on the concentrations of NaBH4 and GNPs. As the concentration of NaBH4 used in reactions was much higher than that of nitro aromatic compounds and GNPs, so it is expected that the concentration of NaBH4 remains constant throughout the reaction. So the rates of these reactions may be considered to be independent of the concentration of NaBH4 and thus the reactions were considered as pseudo first-order reaction with respect to the concentration of corresponding nitro aromatic compound. However, it should be noted that here the rate of the reaction was not influenced by the concentration of nanoparticles due to use of relatively low and fixed concentration of nanoparticles throughout the reaction system. We found a good linear correlation of ln (At/A0) with time (t) where the logarithm of absorption decreased linearly with reaction time (Fig. 10a–l) indicating pseudo first-order kinetics, where At is the absorption of nitro aromatic compound at any time t and A0 is the absorption of nitro aromatic compound at time 0. The rate constant (k) of reaction was calculated from the slope of the linear sections of the plots and has been provided in details in Table 1. Apart from time, order and rate of the reaction, some important quantitative parameters like conversion, selectivity, yield, turnover number (TON), and turnover frequency (TOF) were also derived using calibration curve (see Supplementary Fig. S6A–D) according to the previous literatures (Anantharaj et al. 2016; Kim et al. 2012; Fan and Huang 2014) and the results are shown in Table 1.

Discussion Our study confirms that well dispersed, highly stable GNPs were successfully synthesized using intracellular protein extract of bacterial strain, Staphylococcus warneri SuMS_N03. The strain was isolated from the saline environment of Sundarbans estuary. The tolerance to environmental saline stress forced them to develop specific defense mechanisms to sustain such hostile nature. Sometimes in this condition bacteria produce unique enzyme which may be important for the synthesis of nanoparticles. Marine bacteria like Bacillus

Environ Sci Pollut Res

Fig. 9 Time dependent UV-Vis absorption spectra for the catalytic reduction by NaBH4 in the presence of as-synthesized gold nanoparticles of a–c 2-nitroaniline for the first, second, and third cycles, respectively; d–f

4-nitroaniline for the first, second, and third cycles, respectively; g–i 2-nitrophenol for the first, second, and third cycles, respectively; and j–l 4-nitrophenol for the first, second, and third cycles, respectively

sp. and Actinobacteria from Sundarbans ecosystem are capable to produce unique important enzymes and bioactive compounds (Sana et al. 2007; Sengupta et al. 2015). Previous literature also reported that marine bacteria are also capable

to synthesize nanoparticles (Sharma et al. 2012). A few reports are available regarding the synthesis of nanoparticles using intracellular protein extract of microorganism (Shi et al. 2015; Correa-Llantén et al. 2013). The intracellular

Environ Sci Pollut Res

Fig. 10 First order kinetics plot for the catalytic reduction by NaBH4 in the presence of as-synthesized gold nanoparticles of a–c 2-nitroaniline for the first, second, and third cycles, respectively; d–f 4-nitroaniline for the

first, second, and third cycles, respectively; g–i 2-nitrophenol for the first, second, and third cycles, respectively; and j–l 4-nitrophenol for the first, second, and third cycles, respectively

protein extract can reduce the salt to form nanoparticles and serves as capping agent at the outer surface of nanocore to stabilize them. Attachment of protein with nanoparticles may occur either by interaction through its free amine groups

or amine groups present in the lysine residues or by cysteine residues. Thus, surface-bound protein may be responsible for the stabilization of the nanoparticles (Sharma et al. 2012; Gole et al. 2001). According to previous reports, hydroxyl group

Environ Sci Pollut Res Table 1 Reactant

2-NA

4-NA

2-NP

4-NP

Relevant parameters of catalytic degradation reaction of all nitro aromatic compounds by GNPs Number of cycles

Time (min)

Conversion (%)

Selectivity (%)

Yield (%)

TON(× 103)

TOF (× 103) (h−1)

Rate constant (k) (× 10−1) (min−1)

1

5

80

100

80

1.469

17.604

2.51 ± 0.02

2 3

9 16

76.07 59.6

100 100

76.07 59.6

1.410 1.182

11.29 4.586

1.51 ± 0.005 0.5 ± 0.003

1

5

92.79

100

92.79

1.327

16.31

3.35 ± 0.04

2 3

8 16

92.78 91.75

100 100

92.78 91.75

1.346 1.308

9.812 7.016

1.76 ± 0.013 1.28 ± 0.004

1

6

86.8

100

86.8

1.094

15.449

3.36 ± 0.022

2 3

16 17

84.91 83.92

100 100

84.91 83.92

1.324 1.4

7.463 7.413

1.26 ± 0.006 1.102 ± 0.003

1

10

93.16

100

93.16

0.6

5.182

2.9 ± 0.012

2

13

91.83

100

91.83

0.6

4.235

2.42 ± 0.011

3

26

90.67

100

90.67

0.64

2.543

1.24 ± 0.004

NA nitroaniline, NP nitrophenol, TON turnover number, TOF turnover frequency

(−OH) has a strong affinity towards nanoparticles (Stalin Dhas et al. 2012). FTIR results indicate that intracellular protein having functional groups including hydroxyl, carboxyl, and amine serves as reducing as well capping agent to stabilize the as-synthesized GNPs. Further from SDS-PAGE analysis, it is observed that although different low molecular weight size protein (~ 70 to 15 kDa) present in intracellular protein extract, Fig. 11 Schematic representation of catalytic reduction reaction of 2-nitroaniline (2-NA), 4nitroaniline (4-NA), 2nitrophenol (2-NP), and 4nitrophenol (4-NP) by NaBH4 in the presence of as-synthesized gold nanoparticles. The red color group indicates the participating group during the reaction

but ~ 70 and ~ 60 kDa protein are found to attach with surface of GNPs. Denaturing agent like guanidine hydrochloride, SDS, and β-mercaptoethanol (present in loading dye) removes weak bonding interaction and so detach the capping protein from the surface of GNPs. Thus, for three protein samples from differently treated GNPs, bands appear only for surface capped protein of GNPs. Similar observation from

Environ Sci Pollut Res

three treated samples confirmed that ~ 70 and ~ 60 kDa protein act as capping agent at the outer surface of nanocore and helps in the stability of GNPs. After successful synthesis, the SERS activity of assynthesized GNPs was checked in the present study. Two factors are associated with the enhancement of SERS signals: electromagnetic (EM) effect and the chemical enhancement mechanism. But EM is the dominating factor for signal enhancement when using noble metal nanoparticles as SERS substrate. The EM effect is mainly influenced by the SPR of noble metal nanoparticles. For SERS spectroscopy, the highest EM field enhancement is attained when the irradiation wavelength is resonant with SPR maximum of SERS substrate (Lin et al. 2014; Pu et al. 2013). The as-synthesized GNPs show SPR peak at 535 nm. So 632.8 nm excitation laser has been used as considering the closeness to the SPR wavelength. In this study, R6G has been detected even at lower concentration in the presence of GNPs. Thus, as-synthesized GNPs might be useful as a superior SERS substrate for the ultrasensitive detection of biomolecules and other type of chemical compounds like different pollutants and toxic dye at single molecular level.

The disposal of industrial pollutants like nitro aromatics has emerged to be a great challenge to the industrial development and rapid urbanization. The high toxicity of these nitro aromatic pollutants is of great concern and several technologies are coming up to address this issue. One important process to eliminate nitro aromatics is to convert them into valuable aromatic amines compounds through catalytic reduction by NaBH4. Some products of this chemical reduction are intermediate in the preparation of polymers, hair dyes, and rubber products, developing agent in color photographic film development process, etc. (Singh et al. 2014; Mandlimath and Gopal 2011). Normally, these nitro aromatic compounds are inert to NaBH4 and the reduction occurs only when efficient catalyst is present. Although the reduction reaction of nitro aromatic compounds using aqueous NaBH4 is thermodynamically favorable, but the presence of kinetic barrier due to large redox potential difference between donor and acceptor molecules decreases the possibility of this reaction (Gangula et al. 2011). In reaction, NaBH4 acts both as electron donor and hydrogen supplier. Nanoparticles catalyze this reaction by transferring electron relay from the donor BH4¯ to acceptor

Table 2 Comparison of catalytic activity of as-synthesized GNPs with the reported gold-based nanocatalyst in the reduction of nitro aromatic compounds Reduced compounds

Conversion time (min)

Rate constant (k) (× 10−1) (min−1)

Nanocatalyst

Average size (nm)

Au@SiO2

15

2-NA

21

1.01

Tan et al. 2010

Au@SiO2

102 ± 8

2-NA

20

0.078

Lee et al. 2008

Au-p(NIPAM)

7.18 ± 1.80

2-NA

180

0.127–0.168

Zhu et al. 2012

Au

2.4 ± 0.2

2-NA

23

0.0121

Sharma 2015

Au Au

15–25 9–19

2-NA 4-NA

5, 9, 16 59–108

2.51, 1.51, 0.5 0.45–0.15

This study Reddy et al. 2013

Au

6.07

4-NA

9–40

0.65

Shi et al. 2015

Au

44–61

4-NA

60–240

0.039–0.757

Chiu et al. 2012

Au (nanorods)

22–27

4-NA

56

0.44

Bai et al. 2009

Au NWNs

17.74–23.54

4-NA

51–17

0.3–1.85

Chirea et al. 2011

Au Au

15–25 12–53

4-NA 2-NP

5, 8, 16 8

3.35, 1.76, 1.28 3.2

This study Shen et al. 2016

Au–Pd (bimetallic foams) Au–Ag (bimetallic)

**na 12

2-NP 2-NP

9 10

2.4 1.48

Liu and Yang 2011 Meena Kumari et al. 2015

Fe3O4@SiO2@(PEI–Au)

330

2-NP

**na

1.1

Li et al. 2011

Au Au

15–25 50

2-NP 4-NP

6, 16, 17 10

3.36, 1.26, 1.102 0.124

This study Srivastava et al. 2013

Au Au

23.1–35.8 21–12

4-NP 4-NP

12 60–20

2.2 0.473–1.342

Dash and Bag 2014 Zhu et al. 2012

Au Au

18.9–53.2 17

4-NP 4-NP

**na 17.33

0.325–0.063 1.44

Narayanan and Sakthivel 2011 Wu et al. 2015

Au

15–25

4-NP

10, 13, 26

2.9, 2.42, 1.24

This study

**na not available (indicates that there was no data available regarding particular value in the cited reports)

References

Environ Sci Pollut Res

nitro group of nitro aromatic compounds and the BH4¯ ions also transfer surface hydrogen to the nanoparticles. The nitro aromatic compounds adsorb on the surface of nanoparticles and transfer of hydrogen to that nitro aromatic compounds occur instantly. In the meanwhile, the kinetic barrier of reaction is also overcome and nitro group is reduced to amine group forming corresponding amino aromatic compound. Finally, the product is separated from the surface of nanoparticles (Wunder et al. 2010). In that reaction system, the presence of excess NaBH4 increases the pH of the system, so delaying the degradation of BH4¯, and meanwhile liberated hydrogen removes the oxygen, thereby preventing the aerial oxidation of reduced product, amino aromatic compounds (Esumi et al. 2004). NaBH4 is also toxic but that effect is not significant because the reaction mechanism involves production of sodium metaborate (NaBO2) which has very low toxicity to human health (Weir and Fisher 1972). This is the demand of time to develop suitable catalyst for this chemical reduction and we have successfully achieved this by utilizing as-synthesized GNPs as an efficient catalyst in that reduction process. GNP with (111) facets are known to have more sharp edges and corners and considered to be more active catalytically (Narayanan and El-Sayed 2004). The XRD pattern of as-synthesized GNPs shows that the intensity ratio of (200) and (111) diffraction peaks is 0.35, which is significantly lower than the conventional value (0.52). So there is a possibility to exist the (111) plane in predominant orientation among other planes (Jia et al. 2012; Kannan and Abraham John 2008). Thus, the as-synthesized GNPs are expected to be an efficient catalyst. This work confirms that as-synthesized GNPs catalyze the complete reduction of 2-NA, 4-NA, 2-NP, and 4-NP in the presence of excess NaBH4 without any observable side reactions or by-products. The overall catalytic reactions are depicted in Fig. 11. The rate constant values of reaction are comparable and even higher than the earlier reports (Shi et al. 2015; Ranjan 2014; Wu and Chen 2012) indicating fast reduction of the nitro aromatics. Data analysis shows that conversion of 4-NA, 2-NP, and 4-NP to corresponding products was within the range of 85–93%, and in the case of 2-NA, it was 80–70% which prove that as-synthesized GNPs worked as a very efficient catalyst in this reduction compared to reported works (Reddy et al. 2013; Lin et al. 2013) in terms of percentage of conversion. Till date, few reports are available regarding the use of recyclable gold nanocatalyst in this degradation process. The as-synthesized GNPs catalyzed this reaction efficiently even up to the third cycle. To check the uniformity of the efficiency of catalyst in different cycles, the turnover frequency (TOF) was further analyzed and the values were obtained in the order 103. The slight decrease in TOF value after the first cycle may be due to the number of particles loss during recovery of GNPs from reaction mixtures by washing and centrifugation for several times after each reduction cycle.

The same reason may be involved in the deviation of both rate constant (K) and percentage of conversion value after the first cycle. A brief comparison is shown in Table 2 between the catalytic performance of GNPs of our study and previously reported gold-based nanocatalyst in the reduction of nitro aromatic compounds. Further, a second comparison is also given in Supplementary Table S5 with previously reported nongold-based nanocatalyst used in the reduction of nitro aromatic compounds. It is clearly observed that GNPs of our study are better renewable nanocatalyst in terms of rate constant (K), time of conversion, and sometimes also size of particles than many other reported chemically or biologically synthesized gold-based and non-gold-based nanocatalyst.

Conclusion A simple and effective eco-friendly process alternative to common physical and chemical method for the production of well-dispersed nanoparticles was developed using intracellular protein extract (IPE) of bacterial strain, Staphylococcus warneri SuMS_N03. GNPs were synthesized at room temperature and neutral pH. Most of the nanoparticles were obtained in spherical shape with the size range of 15–25 nm. FTIR and SDS-PAGE analysis inferred that 60 and 70 kDa intracellular protein moieties with functional groups including hydroxyl, carboxyl, and amine were probably responsible both for the reduction of gold ions and stabilization of the particles. Raman spectroscopic analysis revealed that as-synthesized GNPs have superior SERS activity for the detection of compound at single molecular level. Finally, we provided an effective approach to utilize as-synthesized GNPs as recyclable nanocatalyst for the degradation of different toxic nitro aromatic pollutants. The high rate constant values (10−1 order), percentage of conversion (> 80%), and high turnover frequency (103 h−1 order) ensured that as-synthesized GNPs exhibited significant catalytic efficiency even up to the third cycle. The future prospect of nanoscience and nanotechnology primarily depends on the production and use of renewable environment friendly nanoscale-based molecules, which indicated tremendous prospect for our work. Thus, our research unfolds a great possibility of biosynthesized GNPs to be applied as green renewable catalyst in industry to achieve a cleaner environment for tomorrow. Acknowledgements We acknowledge the World Bank, for all the necessary support for the execution of ICZM project, West Bengal. Sudip Nag acknowledges World Bank ICZM project (54-ICZMP/3P) for providing his fellowship and financial support to carry out this work. Dr. Arnab Pramanik is supported by Research Associateship from NCSCM (MoEF, Govt. of India, grant no. 21/RCO/CR/CMR/2013). We would like to acknowledge the continuous encouragement and enthusiasm expressed by Mr. Tapas Paul and Dr. Herbert K. Acquay from the World Bank in our venture to explore the world heritage site,

Environ Sci Pollut Res Sundarbans. We express our sincere gratitude to SPMU, NPMU, and IESWM for their continuous support. We are grateful to Prof. Parimal Karmakar, Jadavpur University, for his support during DLS and zeta potential measurements; Dr. Dipankar Das and Mr. Pallippuram Venkitaraman Rajesh, UGC-DAE, for their help in using XRD facility; Prof. Munna Sarkar, Saha Institute of Nuclear Physics, for her support during FTIR data analysis; and Dr. Subrata Kundu, CSIR-Central Electrochemical Research Institute for the valuable discussion. We are grateful to Dr. Achintya Singha and Mr. Tara Shankar Bhattacharya, Bose Institute, for their active support in performing SERS measurement and analyzing the data. We like to acknowledge UGC-CAS, DST–FIST, DBT-IPLS, UGC-UPE in the Department of Biochemistry, University of Calcutta, and Centre for Research in Nanoscience and Nanotechnology (CRNN), University of Calcutta, for providing the instrumental facility and infrastructural support. It would not be possible to carry out this work without the help and support of the local people of the Sundarbans. We express our inability to acknowledge them individually. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical statement This article does not contain any studies with human participants or animals performed by any of the authors.

References Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S00222836(05)80360-2 Anantharaj S, Jayachandran M, Kundu S (2016) Unprotected and interconnected Ru 0 nano-chain networks: advantages of unprotected surfaces in catalysis and electrocatalysis. Chem Sci 7:3188–3205. https://doi.org/10.1039/C5SC04714E Bai X, Gao Y, Liu H, Zheng L (2009) Synthesis of amphiphilic ionic liquids terminated gold nanorods and their superior catalytic activity for the reduction of nitro compounds. J Phys Chem C 113:17730– 17736. https://doi.org/10.1021/jp906378d Balasubramanian SK, Yang L, Yung L-YL et al (2010) Characterization, purification, and stability of gold nanoparticles. Biomaterials 31: 9023–9030. https://doi.org/10.1016/j.biomaterials.2010.08.012 Biju V (2014) Chemical modifications and bioconjugate reactions of nanomaterials for sensing, imaging, drug delivery and therapy. Chem Soc Rev 43:744–764. https://doi.org/10.1039/C3CS60273G Binupriya AR, Sathishkumar M, Vijayaraghavan K, Yun S-I (2010) Bioreduction of trivalent aurum to nano-crystalline gold particles by active and inactive cells and cell-free extract of Aspergillus oryzae var. viridis. J Hazard Mater 177:539–545. https://doi.org/ 10.1016/j.jhazmat.2009.12.066 Boone DR, Castenholz RW, Garrity GM (eds) (2001) Bergey’s manual of systematic bacteriology, 2nd edn. New York, Springer Booth G (2000) Nitro compounds, aromatic. In: Wiley-VCH Verlag GmbH &Co. KGaA (ed) Ullmann’s encyclopedia of industrial chemistry. Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim, Germany Chakraborty A, Bera A, Mukherjee A et al (2015) Changing bacterial profile of Sundarbans, the world heritage mangrove: impact of anthropogenic interventions. World J Microbiol Biotechnol 31:593– 610. https://doi.org/10.1007/s11274-015-1814-5 Chirea M, Freitas A, Vasile BS et al (2011) Gold nanowire networks: synthesis, characterization, and catalytic activity. Langmuir 27: 3906–3913. https://doi.org/10.1021/la104092b

Chiu C-Y, Chung P-J, Lao K-U et al (2012) Facet-dependent catalytic activity of gold nanocubes, octahedra, and rhombic dodecahedra toward 4-nitroaniline reduction. J Phys Chem C 116:23757– 23763. https://doi.org/10.1021/jp307768h Correa-Llantén DN, Muñoz-Ibacache SA, Castro ME et al (2013) Gold nanoparticles synthesized by Geobacillus sp. strain ID17 a thermophilic bacterium isolated from Deception Island, Antarctica. Microb Cell Fact 12:75. https://doi.org/10.1186/1475–2859–12-75 Dahl JA, Maddux BLS, Hutchison JE (2007) Toward greener nanosynthesis. Chem Rev 107:2228–2269. https://doi.org/10.1021/ cr050943k Das SK, Dickinson C, Lafir F et al (2012) Synthesis, characterization and catalytic activity of gold nanoparticles biosynthesized with Rhizopus oryzae protein extract. Green Chem 14:1322. https://doi. org/10.1039/c2gc16676c Dash SS, Bag BG (2014) Synthesis of gold nanoparticles using renewable Punica granatum juice and study of its catalytic activity. Appl Nanosci 4:55–59. https://doi.org/10.1007/s13204-012-0179-4 Esumi K, Isono R, Yoshimura T (2004) Preparation of PAMAM– and PPI–metal (silver, platinum, and palladium) nanocomposites and their catalytic activities for reduction of 4-nitrophenol. Langmuir 20:237–243. https://doi.org/10.1021/la035440t Fan G-Y, Huang W-J (2014) Synthesis of ruthenium/reduced graphene oxide composites and application for the selective hydrogenation of halonitroaromatics. Chin Chem Lett 25:359–363. https://doi.org/10. 1016/j.cclet.2013.11.044 Fu A, Zhang E (2015) A new strategy for specific imaging of neural cells based on peptide-conjugated gold nanoclusters. Int J Nanomed 2115. doi: https://doi.org/10.2147/IJN.S78554 Gangula A, Podila R, Rao M et al (2011) Catalytic reduction of 4nitrophenol using biogenic gold and silver nanoparticles derived from Breynia rhamnoides. Langmuir 27:15268–15274. https://doi. org/10.1021/la2034559 Gole A, Dash C, Ramakrishnan V et al (2001) Pepsin–gold colloid conjugates: preparation, characterization, and enzymatic activity. Langmuir 17:1674–1679. https://doi.org/10.1021/la001164w Guria MK, Majumdar M, Bhattacharyya M (2016) Green synthesis of protein capped nano-gold particle: an excellent recyclable nanocatalyst for the reduction of nitro-aromatic pollutants at higher concentration. J Mol Liq 222:549–557. https://doi.org/10.1016/j.molliq. 2016.07.087 He S, Guo Z, Zhang Y et al (2007) Biosynthesis of gold nanoparticles using the bacteria Rhodopseudomonas capsulata. Mater Lett 61: 3984–3987. https://doi.org/10.1016/j.matlet.2007.01.018 Hulkoti NI, Taranath TC (2014) Biosynthesis of nanoparticles using microbes—a review. Colloids Surf B Biointerfaces 121:474–483. https://doi.org/10.1016/j.colsurfb.2014.05.027 Iravani S (2014) Bacteria in nanoparticle synthesis: current status and future prospects. Int Sch Res Notices 2014:1–18. https://doi.org/ 10.1155/2014/359316 Jia H, Gao X, Chen Z et al (2012) The high yield synthesis and characterization of gold nanoparticles with superior stability and their catalytic activity. Cryst Eng Comm 14:7600. https://doi.org/10.1039/ c2ce25840d Jiang H, Dong H, Zhang G et al (2006) Microbial diversity in water and sediment of Lake Chaka, an Athalassohaline Lake in northwestern China. Appl Environ Microbiol 72:3832–3845. https://doi.org/10. 1128/AEM.02869-05 Kannan P, Abraham John S (2008) Synthesis of mercaptothiadiazolefunctionalized gold nanoparticles and their self-assembly on Au substrates. Nanotechnology 19:85602. https://doi.org/10.1088/ 0957-4484/19/8/085602 Karthick V, Kumar VG, Dhas TS et al (2014) Effect of biologically synthesized gold nanoparticles on alloxan-induced diabetic rats— an in vivo approach. Colloids Surf B Biointerfaces 122:505–511. https://doi.org/10.1016/j.colsurfb.2014.07.022

Environ Sci Pollut Res Kim BK, Lim Y-W, Kim M et al (2007) EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 57:2259–2261. https://doi.org/ 10.1099/ijs.0.64915-0 Kim JH, Park JH, Chung YK, Park KH (2012) Ruthenium nanoparticlecatalyzed, controlled and Chemoselective hydrogenation of nitroarenes using ethanol as a hydrogen source. Adv Synth Catal 354:2412–2418. https://doi.org/10.1002/adsc.201200356 Lee J, Park JC, Bang JU, Song H (2008) Precise tuning of porosity and surface functionality in Au@SiO 2 nanoreactors for high catalytic efficiency. Chem Mater 20:5839–5844. https://doi.org/10.1021/ cm801149w Li H, Gao S, Zheng Z, Cao R (2011) Bifunctional composite prepared using layer-by-layer assembly of polyelectrolyte–gold nanoparticle films on Fe3O4–silica core–shell microspheres. Catal Sci Technol 1: 1194. https://doi.org/10.1039/c1cy00096a Lin C, Tao K, Hua D et al (2013) Size effect of gold nanoparticles in catalytic reduction of p-nitrophenol with NaBH4. Molecules 18: 12609–12620. https://doi.org/10.3390/molecules181012609 Lin W-H, Lu Y-H, Hsu Y-J (2014) Au nanoplates as robust, recyclable SERS substrates for ultrasensitive chemical sensing. J Colloid Interface Sci 418:87–94. https://doi.org/10.1016/j.jcis.2013.11.082 Liu H, Yang Q (2011) Facile fabrication of nanoporous Au–Pd bimetallic foams with high catalytic activity for 2-nitrophenol reduction and SERS property. J Mater Chem 21:11961. https://doi.org/10.1039/ c1jm10109a Mandlimath TR, Gopal B (2011) Catalytic activity of first row transition metal oxides in the conversion of p-nitrophenol to p-aminophenol. J Mol Catal A Chem 350:9–15. https://doi.org/10.1016/j.molcata. 2011.08.009 Manivasagan P, Alam MS, Kang K-H et al (2015) Extracellular synthesis of gold bionanoparticles by Nocardiopsis sp. and evaluation of its antimicrobial, antioxidant and cytotoxic activities. Bioprocess Biosyst Eng 38:1167–1177. https://doi.org/10. 1007/s00449-015-1358-y Meena Kumari M, Jacob J, Philip D (2015) Green synthesis and applications of Au–Ag bimetallic nanoparticles. Spectrochim Acta A Mol Biomol Spectrosc 137:185–192. https://doi.org/ 10.1016/j.saa.2014.08.079 Mohanpuria P, Rana NK, Yadav SK (2008) Biosynthesis of nanoparticles: technological concepts and future applications. J Nanopart Res 10:507–517. https://doi.org/10.1007/s11051-007-9275-x Nangia Y, Wangoo N, Goyal N et al (2009) A novel bacterial isolate Stenotrophomonas maltophilia as living factory for synthesis of gold nanoparticles. Microb Cell Factories 8:39. https://doi.org/10.1186/ 1475-2859-8-39 Narayanan KB, Sakthivel N (2010) Biological synthesis of metal nanoparticles by microbes. Adv Colloid Interf Sci 156:1–13. https://doi. org/10.1016/j.cis.2010.02.001 Narayanan KB, Sakthivel N (2011) Synthesis and characterization of nano-gold composite using Cylindrocladium floridanum and its heterogeneous catalysis in the degradation of 4-nitrophenol. J Hazard Mater 189:519–525. https://doi.org/10.1016/j.jhazmat.2011.02.069 Narayanan R, El-Sayed MA (2004) Shape-dependent catalytic activity of platinum nanoparticles in colloidal solution. Nano Lett 4:1343– 1348. https://doi.org/10.1021/nl0495256 Patra S, Mukherjee S, Barui AK et al (2015) Green synthesis, characterization of gold and silver nanoparticles and their potential application for cancer therapeutics. Mater Sci Eng C 53:298–309. https:// doi.org/10.1016/j.msec.2015.04.048 Pu Y-C, Wang G, Chang K-D et al (2013) Au nanostructure-decorated TiO2 nanowires exhibiting photoactivity across entire UV-visible region for photoelectrochemical water splitting. Nano Lett 13: 3817–3823. https://doi.org/10.1021/nl4018385 Rajan A, MeenaKumari M, Philip D (2014) Shape tailored green synthesis and catalytic properties of gold nanocrystals. Spectrochim Acta

A Mol Biomol Spectrosc 118:793–799. https://doi.org/10.1016/j. saa.2013.09.086 Reddy V, Torati RS, Oh S, Kim C (2013) Biosynthesis of gold nanoparticles assisted by Sapindus mukorossi Gaertn. Fruit pericarp and their catalytic application for the reduction of p-nitroaniline. Ind Eng Chem Res 52:556–564. https://doi.org/10.1021/ie302037c Sana B, Ghosh D, Saha M, Mukherjee J (2007) Purification and characterization of an extremely dimethylsulfoxide tolerant esterase from a salt-tolerant Bacillus species isolated from the marine environment of the Sundarbans. Process Biochem 42:1571–1578. https://doi.org/ 10.1016/j.procbio.2007.05.026 Sengupta S, Pramanik A, Ghosh A, Bhattacharyya M (2015) Antimicrobial activities of actinomycetes isolated from unexplored regions of Sundarbans mangrove ecosystem. BMC Microbiol 15: 170. https://doi.org/10.1186/s12866-015-0495-4 Shankar SS, Rai A, Ankamwar B et al (2004) Biological synthesis of triangular gold nanoprisms. Nat Mater 3:482–488. https://doi.org/ 10.1038/nmat1152 Sharma N, Pinnaka AK, Raje M et al (2012) Exploitation of marine bacteria for production of gold nanoparticles. Microb Cell Factories 11:86. https://doi.org/10.1186/1475-2859-11-86 Sharma S (2015) Metal dependent catalytic hydrogenation of nitroarenes over water-soluble glutathione capped metal nanoparticles. J Colloid Interface Sci 441:25–29. https://doi.org/10.1016/j.jcis.2014.11.030 Shen W, Qu Y, Pei X et al (2016) Green synthesis of gold nanoparticles by a newly isolated strain Trichosporon montevideense for catalytic hydrogenation of nitroaromatics. Biotechnol Lett 38:1503–1508. https://doi.org/10.1007/s10529-016-2120-5 Shi C, Zhu N, Cao Y, Wu P (2015) Biosynthesis of gold nanoparticles assisted by the intracellular protein extract of Pycnoporus sanguineus and its catalysis in degradation of 4-nitroaniline. Nanoscale Res Lett 10:147. https://doi.org/10.1186/s11671-0150856-9 Singh C, Goyal A, Singhal S (2014) Nickel-doped cobalt ferrite nanoparticles: efficient catalysts for the reduction of nitroaromatic compounds and photo-oxidative degradation of toxic dyes. Nano 6: 7959. https://doi.org/10.1039/c4nr01730g Singha SS, Nandi D, Singha A (2015) Tuning the photoluminescence and ultrasensitive trace detection properties of few-layer MoS2 by decoration with gold nanoparticles. RSC Adv 5:24188–24193. https:// doi.org/10.1039/C5RA01439E Sperling RA, Rivera Gil P, Zhang F et al (2008) Biological applications of gold nanoparticles. Chem Soc Rev 37:1896. https://doi.org/10.1039/ b712170a Srivastava SK, Yamada R, Ogino C, Kondo A (2013) Biogenic synthesis and characterization of gold nanoparticles by Escherichia coli K12 and its heterogeneous catalysis in degradation of 4-nitrophenol. Nanoscale Res Lett 8:70. https://doi.org/10.1186/1556-276X-8-70 Stalin Dhas T, Ganesh Kumar V, Stanley Abraham L et al (2012) Sargassum myriocystum mediated biosynthesis of gold nanoparticles. Spectrochim Acta A Mol Biomol Spectrosc 99:97–101. https:// doi.org/10.1016/j.saa.2012.09.024 Tamura K, Stecher G, Peterson D et al (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30: 2725–2729. https://doi.org/10.1093/molbev/mst197 Tan L, Chen D, Liu H, Tang F (2010) A silica nanorattle with a mesoporous shell: an ideal nanoreactor for the preparation of tunable gold cores. Adv Mater 22:4885–4889. https://doi.org/10.1002/adma. 201002277 Uma Suganya KS, Govindaraju K, Ganesh Kumar V et al (2015) Blue green alga mediated synthesis of gold nanoparticles and its antibacterial efficacy against Gram positive organisms. Mater Sci Eng C 47: 351–356. https://doi.org/10.1016/j.msec.2014.11.043 Vinay Gopal J, Thenmozhi M, Kannabiran K et al (2013) Actinobacteria mediated synthesis of gold nanoparticles using Streptomyces sp.

Environ Sci Pollut Res VITDDK3 and its antifungal activity. Mater Lett 93:360–362. https://doi.org/10.1016/j.matlet.2012.11.125 Weir RJ, Fisher RS (1972) Toxicologic studies on borax and boric acid. Toxicol Appl Pharmacol 23:351–364 Wu C-C, Chen D-H (2012) Spontaneous synthesis of gold nanoparticles on gum arabic-modified iron oxide nanoparticles as a magnetically recoverable nanocatalyst. Nanoscale Res Lett 7:317. https://doi.org/ 10.1186/1556-276X-7-317 Wu X-Q, Wu X-W, Huang Q et al (2015) In situ synthesized gold nanoparticles in hydrogels for catalytic reduction of nitroaromatic compounds. Appl Surf Sci 331:210–218. https://doi.org/10.1016/j. apsusc.2015.01.077

Wunder S, Polzer F, Lu Y et al (2010) Kinetic analysis of catalytic reduction of 4-nitrophenol by metallic nanoparticles immobilized in spherical polyelectrolyte brushes. J Phys Chem C 114:8814–8820. https://doi.org/10.1021/jp101125j Zhan G, Huang J, Du M et al (2012) Liquid phase oxidation of benzyl alcohol to benzaldehyde with novel uncalcined bioreduction Au catalysts: high activity and durability. Chem Eng J 187:232–238. https://doi.org/10.1016/j.cej.2012.01.051 Zhu C-H, Hai Z-B, Cui C-H et al (2012) In situ controlled synthesis of thermosensitive poly(N-isopropylacrylamide)/au nanocomposite hydrogels by gamma radiation for catalytic application. Small 8: 930–936. https://doi.org/10.1002/smll.201102060