Removal of Cr(VI) by magnetic iron oxide nanoparticles synthesized from extracellular polymeric substances of chromium resistant acid-tolerant bacterium Lysinibacillus sphaericus RTA-01

Abstract

Background

Extracellular polymeric substances (EPS) from Cr(VI) resistant acid-tolerant biofilm forming bacterium (CrRAtBb) Lysinibacillus sphaericus RTA-01 was used for synthesis of magnetic iron oxide nanoparticles (MIONPs) in removal of Cr(VI).

Methods

MIONPs synthesized in EPS matrix were characterized by UV-Vis, DLS, ATR-FTIR, XRD, FESEM, HRTEM and VSM. Primarily, the synthesis of MIONPs was established by the formation of black-colored precipitate through surface plasmon resonance (SPR) peak in between 330 and 450 nm.

Results

The size of the spherical MIONPs with diameter range 13.75–106 nm was confirmed by DLS, XRD and FESEM analysis. HRTEM study confirmed the size of the MIONPs in the range of 10–65 nm. Moreover, the EDX and SAED confirmed the purity and polycrystalline nature of MIONPs. The ATR-FTIR peaks below 1000 cm⁻¹ designated the synthesis of MIONPs. Also, the magnetic property of MIONPs was confirmed for separation from the aqueous solution. MIONPs were further checked for the adsorption of Cr(VI) with initial concentration range of 50–200 mg L⁻¹. An adsorption isotherm and thermodynamic study were also carried out and the experimental data was best fitted in Langmuir isotherm model with maximum adsorption percent of 1052.63 mg g⁻¹ of Cr(VI). Post interaction with Cr(VI), the surface characteristic of MIONPs in EPS matrix was evaluated by zeta potential, EDX, ATR-FTIR and XRD.

Conclusion

This study ascertained the adsorption of Cr(VI) over EPS stabilized MIONPs whereas the zeta potential and XRD analysis confirmed the presence of reduced Cr(IV) on the adsorbent surface.

Electronic supplementary material

The online version of this article (10.1007/s40201-019-00415-5) contains supplementary material, which is available to authorized users.

Introduction

Chromium (Cr) in form of hexavalent Cr [Cr(VI)] is one among the several toxic metals, generated from variety of industrial operations like electroplating, tanneries, acid mines, textile plants, paper mills and rubber industries [1–4]. In aquatic system, Cr(VI) exists in many different toxic forms such as chromate (CrO₄²⁻), dichromate (H₂CrO₄²⁻), and Cr2O₇⁻ which act as powerful oxidizing agents. Cr(VI) exhibits high water solubility, skin irritating and corrosive behavior leading to severe damage to human health [5]. US Environmental Protection Agency (US-EPA) has classified Cr(VI) as highly toxic pollutant and categorized under Class A human carcinogen [6]. Considering the potential toxicity of Cr(VI), several physical and chemical methods have been widely used for selective detoxification of Cr(VI). However, such conventional techniques involve synthetic chemicals and high energy for heavy metal remediation which further leads to secondary pollution [7]. However, biological method for reduction or adsorption of Cr(VI) is considered as one of the most adopted strategies utilizing microbial biomass as cost effective, sustainable and eco-friendly approach for environmental remediation. The efficiency of biological remediation depends on many factors such as physicochemical parameters of environment and existence of microbial population having potential to degrade or reduce the pollutants. Further, the removal rate and effect of reduction method of Cr(VI) are greatly influenced by the pH of industrial effluents which plays very crucial role in chromium speciation and solubility [8]. Metabolic activity of microorganisms also decides the efficiency of bioremediation process. The complex composition of acidic industrial effluents seizes the growth of microorganisms and poses a great challenge for bioremediation process [9]. This manifests a potential problem in the current biological remediation technologies to remediate the acidic effluents containing toxic metals such as Cr(VI).

Nano-bioremediation approach has been developed as a completely new dimension to achieve improved bio based detoxification of various inorganic pollutants mitigating potential limits of bioremediation. Among the nanoscale materials, magnetic iron oxide nanoparticles (MIONPs) have been widely studied due to large surface area, high reactivity, tunable properties, specific magnetic properties, strong reducing power and ability to adsorb a wide range of toxic metals and metalloids [10, 11]. Synthesis of MIONPs by conventional methods i.e. physical and chemical is not encouraging due to the use of harsh chemicals and high energy demand [12]. Therefore, the biological process involving plants and microorganisms holds an immense potential in green synthesis of MIONPs [13]. Biosynthesis of MIONPs has been attained by reduction of bulk iron to the iron nanoparticles with small size distribution [14, 15]. Although many microorganism like bacteria, fungi, yeast, actinomycetes and algae are capable of synthesizing nanoparticles both intra- and extracellularly, bacteria possess several advantages over other microbial community for nanoparticle synthesis due to smaller size, shorter generation time and wide distribution even in extreme environments [16–20]. Bacterial community thrives in low pH and toxic metal containing environment develops certain defence mechanisms like biotransformation, biomineralization and bioreduction to combat metal toxicity which in turn leads to nanoparticle synthesis [12]. Additionally, they build up a protective layer of extracellular polymeric substances (EPS) to survive under adverse conditions.

EPS are functional organic polymers of high molecular weight that usually bound with bacterial cell surface either as capsular or secreted forms [21, 22]. The components of EPS are elongated chain sugar residues with proteins and nucleic acids [21]. Apart from protective role, bacterial EPS plays a major role in surface adherence, biofilm formation, adsorption and detoxification of organic and inorganic pollutants [23]. Typically, bacterial EPS possess several functional groups like phosphate, sulphate, hydroxyl, carboxyl and amide etc. which play an important role in chelation, reduction, transformation of metal ions to nanoscale particles on EPS matrix and stabilize the colloidal state of nanoparticles [24]. Although, microbial mediated synthesis of MIONPs has been studied extensively, use of extracellular polymeric substances (EPS) of bacteria for extracellular synthesis of nanoparticles has not been yet explored. Therefore, the present study describes the role of bacterial EPS, extracted from Cr(VI) resistant acid tolerant biofilm forming bacterium Lysinibacillus sphaericus RTA-01, in synthesis and stabilization of MIONPs and explains the mechanism of Cr(VI) sequestration from the aqueous solution using EPS stabilized MIONPs.

Materials and experimental methods

Media and chemicals

Microbiological media were obtained from HiMedia (India). All the chemicals, reagents and microbiological media used in present study were of analytical grade. Nutrient broth contains (g L⁻¹) 1 g beef extract, 2 g yeast extract, 5 g peptone and 5 g NaCl and 15 g agar. Stock solution (1000 mg L⁻¹) of Cr(VI) was prepared by dissolving K₂Cr₂O₇ (Rankem, India) in microbiological grade water (Millipore, USA). A required volume of Cr(VI) from the stock solution was taken, sterilized and added to the media before inoculation to achieve desired Cr(VI) concentration. Diphenylcarbazide (2%, w/v) was freshly prepared by dissolving in 95% acetone. Sulphuric acid (98%, sp. Gr. 1.85, Merck, India) was diluted to the required volume to make 6 N.

Isolation and identification of EPS synthesizing CrRAtBb

Water and sediment sample were collected from rubber latex processing units of Tripura, India which generates acidic effluents. The EPS synthesizing chromium resistant and acid-tolerant biofilm forming bacterium (CrRAtBb) was isolated on nutrient broth medium, supplemented with 50 μg ml⁻¹ Cr(VI) of K₂Cr₂O₇ at pH 5.5 [25]. Under 37 °C incubation temperature for 24 h, the bacterium Lysinibacillus sphaericus RTA-01 was obtained. Post isolation, EPS synthesizing CrRAtBb Lysinibacillus sphaericus RTA-01 was used for further study. The isolated strain has been identified by 16S rRNA gene amplification and sequencing technology.

Microscopic examination and characterization of EPS synthesizing CrRAtBb

The overnight grown culture of isolate Lysinibacillus sphaericus RTA-01 was diluted to 1:100 in Nutrient broth (Himedia, India). 3 ml of the culture was transferred to 6-well plate with a glass slide (2 × 2 cm) and incubated at 37 °C under static condition. pH 5.5 was maintained during the synthesis of EPS. At 48 h, slide was washed with sterilized distilled water twice and stained with 0.02% aqueous solution of acridine orange for 5 min. After 5 min, slide was washed again with sterilized distilled water and observed under fluorescence microscope (Olympus 1 × 71, Tokyo, Japan) under 20X objective lens. The images obtained through fluorescence microscope were evaluated using IMAGE J ver 1.46. Biofilm-EPS production has also been investigated through Field emission scanning electron microscope (FESEM, Nova NanoSEM, USA). To perform FESEM analysis, biofilm was grown on glass slides of dimension 1 × 1 cm for 48 h followed by fixation with 2.5% glutaraldehyde at 4 °C for 24 h. Slide was dehydrated through 30, 70 and 100% alcohol followed by 1% aqueous solution of tannic acid. Fixed slide was then coated with platinum and observed under FESEM at different magnifications.

Production and extraction of EPS

Lysinibacillus sphaericus RTA-01 was aerobically cultured at 37 °C for 24 h in Nutrient broth (Himedia, India) with continuous shaking at 120 rpm. 0.1% of log phase culture was inoculated in fresh and sterilized NB broth followed by maintaining the culture up to 48 h. After 48 h of incubation, the bacterial culture was centrifuged at 5600 g for 30 min at 4 °C to separate the cell pellet. The obtained cell free supernatant was added with chilled absolute ethanol in ratio of 1:1 (v/v) and kept at 4 °C overnight to precipitate the EPS. Post centrifugation at 5600 g for 20 min at 4 °C, the precipitate (EPS pellet) was collected. EPS pellet was further dried in a desiccator and stored at 4 °C. As per the requirement, EPS was re-suspended in milli-Q water and further used as template for manufacture of nanoparticles. The derived EPS of Lysinibacillus sphaericus RTA-01 was further analyzed for estimation of major organic biomolecules. Carbohydrate and protein were estimated through Phenol sulfuric acid method, Bradford method measuring absorbance (λ_max) at 490 nm and 595 nm respectively [26, 27] while quantification of nucleic acid (eDNA) was carried out using NanoDrop (Eppendorf, Germany) at 260 nm.

Synthesis of MIONPs

Anhydrous FeCl₃/FeSO₄ and EPS were used as initial salt precursors and capping agent respectively for synthesis of MIONPs. Freshly prepared aqueous solution of EPS and iron salts (prepared in 1:1 ratio of 0.1 M FeCl₃ and 0.1 M FeSO₄) were kept ready in two separate containers [28]. An equal volume of EPS was added in drop wise manner to the freshly prepared aqueous iron solutions at 120 rpm shaking for 48 h at 37 °C. The flask was protected with aluminum foil to avoid photo-reduction. The MIONPs production of the reaction mixture was visually monitored after 48 h. The MIONPs was collected using centrifugation at 11,000 g for 25 min and washed with milli-Q water to obtain maximum purity of the nanoparticles.

Characterization of MIONPs

The preliminary confirmation for synthesis of MIONPs was monitored through UV-visible spectroscopy. A small aliquot of MIONPs sample was scanned using UV-visible spectrophotometer (Perkin-Elmer, USA) in the wavelength range of 700–200 nm. The Z-average size (Hydrodynamic diameter), size distribution (Polydispersity index) and zeta potential (Surface charge) of MIONPs were determined through Zeta sizer (ZS 90, Malvern Instruments Ltd., Malvern, UK). X-ray powder diffractogram (XRD) of the lyophilized MIONPs were studied to identify the solid state nature of the particles using X-ray diffractometer (Rigaku Miniflex, Japan). The XRD instrument was equipped with radiation source Cu Kα (k = 1.54056 Å), operating at 40 kV. The interaction of capping agent (EPS) with MIONPs was explored through Attenuated Total Reflectance Fourier Transform Infra-Red Spectrometer (ATR-FTIR, Bruker, Germany). The FTIR spectrum was recorded in the wavelength range of 4000–400 cm⁻¹. FESEM analysis of the MIONPs was performed using Field Emission Scanning Electron Microscope (Nova, USA) to study the surface topology and size of the MIONPs nanoparticles. The atomic level composition of the MIONPs was investigated by Energy Dispersive X-ray Spectroscopy (EDX) (Hitachi, Japan) attached with FESEM (Nova NanoSEM, USA). HRTEM (High resolution transmission electron microscopy) (FEI, Tecnai G2 F30 Twin TEM, USA) micrographs at different magnification for MIONPs were acquired by dispersing samples in milli-Q water. Whereas, the texture of MIONPs was obtained through selective area electron diffraction (SAED) pattern. The magnetic property of MIONPs was studied under an applied magnetic field of ±1.67 Tesla at 300 K using a Vibrating Sample Magnetometer (VSM, Lake Shore-7407, Japan).

Batch adsorption experiment for Cr(VI) removal from aqueous solution

To evaluate the efficacy of biosynthesized MIONPs for the removal of Cr(VI), batch mode experiments were designed and performed. The impact of pH for adsorption study was determined using initial Cr(VI) concentration 50 mg L⁻¹ and an adsorbent dose of 5 mg ml⁻¹ for 12 h. The adsorption study was conducted by suspending 50 mg of desired EPS stabilized MIONPs in 15 ml falcon tube containing 50–200 mg L⁻¹ of Cr(VI) salt at optimum pH at 300 K, 310 K and 318 K temperature for 24 h under continuous shaking (120 rpm). Under different initial Cr(VI) concentration range (50 to 200 mg L⁻¹), the changes in the adsorption capability of adsorbents were determined for 24 h. The effect of change in contact time (24–72 h) for adsorption study was determined using initial Cr(VI) concentration 50 mg L⁻¹ and an adsorbent dose of 5 mg ml⁻¹. After incubation at specified condition, the aqueous solution of reaction mixture was centrifuged at 2500 g for 20 min to separate the suspended MIONPs. Furthermore, the obtained supernatant was filtered using 0.22 μm syringe driven micropore filter. Finally, the residual Cr(VI) concentration was determined following diphenyl carbazide complexation procedure 7196A of USEPA, 1992.

Adsorption isotherms models and thermodynamic study

The adsorption isotherm for Cr(VI) removal through MIONPs were evaluated by Langmuir, Freundlich and Dubunin-Radushkevich (D-R) isotherm models [29]. Langmuir model is based on monolayer adsorption over the surface of adsorbent. A plot of 1/q_e versus 1/C_e yielded a straight line and the values of q_max and K_L were computed from slope and intercept. Freundlich model graph was plotted between logq_e versus logC_e which yielded a straight line with a slope and intercept that correspond to 1/n and logK_F. The D-R isotherm model was plotted as a function of logarithmic of amount adsorbed lnq_e versus ε². Then, the regression co-efficient (R²) was calculated and compared to evaluate the best fitted model for adsorption of Cr(VI) through MIONPs. Vant’t hoff graph was plotted between lnK_L versus 1/T to determine the nature of enthalpy during adsorption reaction. The obtained value for K_L at different temperatures was further used to calculate the Gibbs energy (∆G⁰) and corresponding standard enthalpy (∆H⁰) and the standard entropy (∆S⁰) for Cr(VI) interaction with MIONPs.

Deciphering Cr (VI) removal mechanism by MIONPs

Surface charge of MIONPs after interaction with Cr(VI) was investigated by adding 10 mg L⁻¹ of MIONPs to the distilled water at different pH ranging from 3.0 to 5.8. Solution with MIONPs was equilibrated for 1 h. Zeta potentials were measured by particle size analyzer (ZS 90, Malvern Instruments Ltd., Malvern, UK). The surface elemental analysis of Cr(VI) interacted MIONPs was performed using EDX (JEOL-JSM-6480, USA). The surface chemical analysis of Cr(VI) interacted MIONPs was characterized by ATR-FTIR spectroscopy (Bruker, Germany). The different phases of Cr(VI) interacted EPS and MIONPs were determined using powder X-ray diffraction operating in the reflection mode Cu Kα radiation. The X-ray diffractogram was recorded over an angular range of 10°–80° to observe the Cr(VI) adsorption and reduction. Peaks were identified using the Joint Committee on Powder Diffraction Standards (JCPDS) diffraction database. In order to differentiate the adsorption and reduction of Cr(VI) by MIONPs, 50 mg L⁻¹ of Cr(VI) was treated 5 mg ml⁻¹ of adsorbent dose at pH 5.2 and 318 K for 72 h. Removal of total Cr was determined by Atomic adsorption spectroscopy (AAS, Perkin Elmer, USA). Residual concentration of Cr(VI) was evaluated spectroscopically to quantify the reduced species of Cr(VI) [30].

Statistical analysis

The obtained results were checked for statistical significance by one way analysis of variance (ANOVA) coupled with Tukey’s and Dunnett’s multiple comparison tests using GraphPad Prism 7 software. For entire study, P < 0.05 was taken as statistical significance.

Results and discussion

Isolation, identification and microscopy of biofilm-EPS

Several CrRAtBb were isolated in nutrient broth medium supplemented with Cr(VI) at pH 5.5. The potent bacterium was selected based on the EPS producing characteristic under slightly acidic condition. Strain was identified through 16S rRNA gene amplification and sequencing. The strain was identified as Lysinibacillus sphaericus RTA-01. Partial sequence of 16S rRNA gene of Lysinibacillus sphaericus RTA-01 was submitted to NCBI GenBank database with accession number KP986590. The biofilm-EPS architecture of Lysinibacillus sphaericus RTA-01 was obtained after staining with acridine orange (Fig. 1a and b). The bacterium exhibited the biofilm-EPS growth over the surface of glass slide. Fluorescence image displayed the small developing micro-colonies connecting to each other followed by dispersion of the biofilm-EPS architecture. The raw integrated density (31,805,228 ± 4,656,460) was calculated by taking ten images from different points. Previous finding also suggested that the smaller micro-colonies are bound together and grows further with the help of transient binding agents of EPS counterpart of biofilm [31]. Moreover, the EPS synthesizing characteristic of the isolate Lysinibacillus sphaericus RTA-01 was confirmed by field emission scanning electron microscopy (Fig. 1c and d). Fluorescence and electron microscopy investigations of biofilm-EPS study of Lysinibacillus sphaericus RTA-01 showed clusters of cells within the polymeric matrix. Similar observation was reported in SEM analysis of EPS in Bacillus pseudomycoides U10 [32]. Additionally, the EPS has great relevance in microbial biofilm system by providing varieties of functions. The components of EPS for instance polysaccharide, proteins, DNA whose functions confers redox activity, sorption of nutrients and toxic metal ions, enzymatic activity, stabilization and many more [31]. The insight view of (Fig. 1d), depicts the secreted EPS around the surface of cells. The polymers in EPS mechanically stabilize the microbial aggregates via many interactions, such as dispersion forces, electrostatic interaction and hydrogen bonds. The formation of a jelly like tridimensional structure around the cells allows the microorganisms to be retained near each other to establish stable biofilm [31, 33].

Fig. 1.

Microscopic image for EPS synthesizing bacterium Lysinibacillus sphaericus RTA-01 a and b represent the 2D and 3D plot of fluorescence images of EPS respectively; c and d show the FESEM of biofilm-EPS associated bacterial cells. The insight depicts the close view of bacterial cells surrounded by EPS matrix at higher magnification 30,000×

EPS mediated synthesis and characterization of MIONPs

MIONPs has gained more attention and exporation in multiple dimensions such as environmental and biomedical field for eco-friendly means of synthesis. However, the controlled synthesis of monodispersed nanoparticles with high stability has been a major challenge in the field of nanoscience. In this context, the use of EPS as capping agent in synthesis of several nanoparticles has shown a newer and better prospect in controlled synthesis of nanoparticles. Recently, iron oxide nanoparticles using bacterial EPS was developed [34]. Conversely, this study did not report the utility of EPS-stabilized iron oxide nanoparticles for removal of environmental contaminants. The stabilized nanoparticles have less surface energy and least possibility of aggregation can enhance the rate for removal of toxic metals. Previously, Raj et al. [35] established the role of EPS in capping and stabilizing characteristic for CdS nanoparticles synthesis. The study hypothesized that the growth of nanocrystal occurred inside the EPS milieu due to presence of reducing precursors which reduced iron to iron nanocrystals. As the growth of iron nanocrystals takes place, they start getting trapped in to the network of EPS and provide stability to the MIONPs.

The addition of equal volume of EPS in Fe^3+/2+ aqueous solutions with continuous shaking changed the color of reaction mixture. The presence of dark black color of reaction mixture after incubation substantiated the synthesis of MIONPs. Subsequently, aqueous solution of suspended nanoparticles was obtained using centrifugation followed by washing with ethanol (99.9%) to eliminate the traces of water molecules. Obtained precipitate of MIONPs was further dried in desiccator and dispersed in milli-Q water for further characterization. The synthesis of MIONPs has been characterized by UV-Visible spectroscopy, DLS, zeta potential, X-ray diffractometer, FESEM-EDX and VSM techniques.

UV-visible spectroscopy

UV-Visible spectra were recorded in the range of 700–200 nm to evaluate the optical properties due to change in the size and shape of the materials. The change in color of the reaction mixture showed a suitable spectroscopic monogram to indicate the synthesis of MIONPs. Additionally, the optical absorption maxima of MIONPs produced a SPR peak in between 330 and 450 nm which ascertained the synthesis of MIONPs. The UV-Visible spectra of MIONPs are depicted in Fig. 2a1. A peak at around 390 nm confirmed the presence of MIONPs which was somewhat shifted as compared to only aqueous solution of precursor iron (Fe^3+/2+) salts. The blue shift was detected in the UV-Visible spectrum due to excitation of SPR and the confinement in the dimension of MIONPs. Due to the SPR, MIONPs exhibited a distinctive optical absorption peak at 390 nm which is in agreement with the previous report of Rahman et al. [36] showing an absorption band in the region of 330–450 nm. The blue shift may possibly be attributed to the shift from Fe^3+/2+ bulk material to MIONPs in the presence of capping agent (EPS).

Fig. 2.

a Characterization of MIONPs (a1) EPS stabilized MIONPs (a2) iron precursor salts b Dynamic light scattering (DLS) c Surface zeta potential d X-ray diffraction pattern, and e FTIR spectra of EPS and biosynthesized MIONPs showed variations in the transmittance

Dynamic light scattering, surface zeta potential and poly dispersity index

DLS analysis was accompanied to determine the hydrodynamic size (Z-average diameter) of the nanoparticles when they are dispersed in a liquid medium. In this study, obtained MIONPs are allowed for DLS study to determine the size and dispersity in aqueous solution. Figure 2b shows the average hydrodynamic size with poly dispersive index of MIONPs. Hydrodynamic size was determined as 106.2 nm and the dispersive index was calculated as 0.164. Furthermore, based on the surface charge of synthesized MIONPs, the stability of particles was calculated in aqueous solution. Figure 2c shows the surface zeta potential of synthesized MIONPs was −18.9 mV. A polydispersity index (PDI) from DLS study for MIONPs was found to be lesser than 0.5 which clearly designated their monodisperse phase with least possibilities of aggregation. The negative zeta potential value (−18.9 mV) of MIONPs manifests incipient stability (from ±10 to ±30 mV). Surface charge analysis of EPS was measured to be −53.4 mV. Due to negative surface charge, the electrostatic attraction between EPS and the iron ions in solution influenced the driving force for the iron reduction and stabilization [37]. It has also been observed that the growth of MIONPs took place inside the EPS matrix which corroborates with the previous result [35].

X-ray diffraction

To further characterize the phase, orientation and grain size of synthesized MIONPs, XRD pattern was evaluated by X-ray diffractometer. Figure 2d depicts the XRD pattern and a major characteristic peak for magnetite at 2θ is 36.03° with some additional peaks at 31.5°, 43.02° and 56.5° corresponding to the plane (311), (220), (400) and (511) respectively. The obtained XRD pattern was subsequently matched and compared with the JCPDS using X’ pert high score software. This reveals that synthesized NPs have Fe₃O₄ crystals with JCPDS reference code–75-0449. The particle size of MIONPs was calculated according to the Scherrer’s equation.

Where λ is the wavelength of X-ray (1.54056 Å), K = 0.9, θ is the Braggs angle and β is the full width at half maximum in radians. This equation was applied for determining average size of the MIONPs and found to be 40.65 ± 0.77 nm. XRD of MIONPs suggested the prominent sharp peak at (311) planes unveiling the presence of dominant population of magnetite nanoparticles. The lattice parameter for magnetite nanoparticles is proposed as 8.31 at 2θ = 35.62°. In present study, the lattice parameter for MIONPs was determined to be 8.31, confirming that the synthesized nanoparticles contain magnetite in higher population. For further corroboration, the 2θ value of plane (311) was considered. In accordance with preceding report, the standard 2θ values for this plane (311) for magnetite nanoparticles (NPs) are in range of 35.48° to 35.62° [38]. The diffraction angle was determined as 36.03° for the synthesized MIONPs, which corroborates with previously specified value by Nuryono et al. [39].

ATR-FTIR analysis

The interaction and capping effects of EPS molecules in synthesis of MIONPs have been examined by ATR-FTIR. Figure 2e represents the FTIR spectra for MIONPs synthesized from EPS the pristine EPS. A band at 681 cm⁻¹ (in between 400 and 850 cm⁻¹) below 1000 cm⁻¹, implies the Fe-O bond vibration which designated the presence of MIONPs. The prominent peaks in the range of 3837–3621 cm⁻¹ could possibly be caused by overlapping of O-H and N-H stretching vibrations. The peak between 2300 cm⁻¹ and 2600 cm⁻¹ showed the presence of -SH functional group as per FTIR spectroscopic data table. The appearance of sharp peak at 2345.15 cm⁻¹, suggested the presence of –SH group which indicated the binding of MIONPs with the capping agents (EPS) whereas the peak for pristine EPS showed the minor peak at 2337 cm⁻¹, an evidence of sulfhydryl group. Thus, ATR-FTIR spectra confirmed the presence of sulfur group that can bind with iron and form MIONPs. Furthermore, the prominent vibration of C=O and N-H at 1708 cm⁻¹ and 1529 cm⁻¹ were also observed in iron interacted EPS spectrum, which denoted the O=C=O stretching and N-H bending vibration respectively. FTIR analysis of MIONPs demonstrated a band at 681 cm⁻¹ corroborating the Fe-O-Fe stretching vibration in the region of 850–400 cm⁻¹ revealing metal oxide bond vibrations present in the magnetite nanoparticles [40]. The binding and slightly shift in peak at around 2337–2345 cm⁻¹ of pristine EPS spectra revealing the presence of attachment group in EPS. A prominent sharp band at 2345 cm⁻¹ depicted the presence of –SH group, which might possibly be interacted with Fe and form MIONPs. The band at 1708 cm⁻¹ is attributed to the presence of carbonyl groups (1630–1780 cm⁻¹) and organic functional groups such as –NH or –OH (3200–3600 cm⁻¹). In addition, the band at 1031 cm⁻¹ represents the presence of C-O stretching in the region of 1300–1000 cm⁻¹ which could be possibly due to existence of covalent bonding of ether or ester groups of the nanoparticles [34]. Thus, FTIR spectrum of MIONPs depicted that carboxyl and hydroxyl groups are involved in the reduction and stabilization of MIONPs. These results clearly assured that the functional group of microbial EPS can efficiently help in synthesis and stabilization of MIONPs with an average diameter of 37.5 nm comparatively smaller than other green synthesis [40, 41]. The coating of MIONPs with the functional group of EPS showed an excellent dispersity of MIONPs by reducing surface energies. EPS mediated synthesis of MIONPs has advantage over chemical co-precipitation method where iron oxide nanoparticles tend to aggregate to each other due to Van der Waals force [42].

Electron microscopy

The surface morphology as well as topology of MIONPs was obtained by Field Emission Scanning Electron Microscope (FESEM). Figure 3a illustrate the FESEM image for MIONPs that are spherical in shape and the size ranges from 13.75 ± 30.05 nm. Furthermore, the energy dispersive X-ray (EDX) investigation of synthesized MIONPs from the capping agents designated the presence of iron (Fig. 3b). The presence of sharp peak of Si and O might possibly be appeared due to glass surface used for sample mounting and presence of oxygen in samples due to surface bound water molecules. High resolution transmission electron microscopy (HRTEM) analysis of MIONPs performed to detect the details of morphology and crystal structure for synthesized MIONPs (Fig. 3c). The obtained HRTEM micrograph showed MIONPs in the range of 10–65 nm (Fig. 3d). The lattice parameters determined through SAED analysis also suggested the production of crystalline MIONPs as observed in XRD analysis. SAED showed ring pattern made up of small spots which revealed the polycrystalline property of synthesized MIONPs (Fig. 3e). The HRTEM studies also established the capping effect of EPS matrix which stabilized the size of the MIONPs. The MIONPs embedding with in the EPS milieu is shown in Fig. S1a (Supplementary data) which indicated the interaction of EPS with nanoparticles and increased the potential of EPS to adsorb enhanced amount of Cr(VI) in their vicinity. This observation also coincided with FESEM and ATR-FTIR results in which the interaction of iron with EPS molecule formed the MIONPs and functional groups of EPS acted as reducing agents. The d spacing values calculated through diffraction analysis inside HRTEM was matched with obtained XRD data showing the presence of (220) and (311) planes in Fig. S1b. Electron microscopy results also exhibited that the synthesized MIONPs were homogenous with regular shape and uniform morphology. In this study, MIONPs produced through EPS based template, displayed the size range determined by XRD, FESEM and HRTEM are 13.75–65 nm whereas 75–120 nm as per the previous report [43]. HRTEM study revealed no contacts between MIONPs inside the EPS matrix in the solution, indicating the role of EPS as capping and stabilizing agent for production of MIONPs even at the lower size range. As per the SAED, the synthesized MIONPs showed the diffraction pattern of polycrystalline particles. This result is in agreement with the previously reported SAED pattern of iron oxide nanoparticles through green synthesis route [40].

Fig. 3.

Microscopy of the MIONPs a Field Emission Scanning Electron microscopy images of MIONPs at 100,000× magnifications. The insight shows the morphology of particle size at 200,000× magnification b Electron diffraction X-ray (EDX) analysis for elemental composition c High resolution transmission electron microscopy images of MIONPs d particle size distribution histogram, and e Selective area electron diffraction pattern (SAED) analysis

Vibrating sample magnetometry (VSM)

To study the magnetic feature of MIONPs, magnetic hysteresis measurement was carried out at 300 K in the specified magnetic field of ±1.67 Tesla. The super paramagnetic behavior is observed based on the magnetic hysteresis loop obtained from MIONPs which has been shown in Fig. 4. MIONPs exhibited a magnetic property in existence of magnetic field (insight of Fig. 4). The dispersed MIONPs in aqueous solution of falcon tube were kept in between the electromagnet showed a good response under applied magnetic field. MIONPs were displayed the attraction towards the magnet and were dissolved when the magnetic field around particles was removed. Therefore, MIONPs can be detached or recycled from the medium with the simple magnetic device. The MIONPs exhibited a magnetic property at 300 K determined by VSM in the given specified magnetic field. It is evident that at 300 K the magnetic interactions of the sample are super paramagnetic with saturation magnetization (Ms) of 40.7 emu g⁻¹. The MIONPs presented a good response under the additional permanent magnetization, which made the quick separation of MIONPs from liquid phase. This super paramagnetic behavior of MIONPs is well corroborated with previous report for green sysnthesis of Fe₃O₄ nanoparticles using aqueous extract of seaweed Sargassum muticum [44].

Fig. 4.

M–H curve measured at 300 K for MIONPs. The insight shows the attraction of MIONPs to the magnet

EPS characterization, batch adsorption and thermodynamic study

EPS contain a remarkable potential to serve as reducer, stabilizer, and capping agent. To the best of our knowledge, very little study has been conducted for the synthesis of MIONPs by using the microbial EPS. MIONPs have a tremendous potential in the remediation of several organic and inorganic contaminants. Several studies have shown the potential of MIONPs in the remediation of toxic metals for example Cd, As, Cr, Pb, and Ni and found to be highly effective in removal of toxic metals from the contaminated sites [28, 45–47]. Furthermore, the ease of its in-situ application broaden its application and demand for production of inexpensive, eco-friendly route of MIONPs synthesis along with capability to eliminate toxic metals [40, 48].

The carbohydrate, protein and nucleic acid (DNA) constituents of EPS of L. sphaericus RTA-01 were analyzed. The carbohydrate, protein and nucleic acid (DNA) contents were found to be 1570 μg ml⁻¹, 567 μg ml⁻¹and 28.84 μg ml⁻¹ respectively. The composition of carbohydrate in extracted EPS is well in agreement as per previous reports where carbohydrate found as major EPS constituents followed by protein [35]. Thus, the present study also suggests that the carbohydrate and protein play major role in the capping functionality and adsorption of metals.

The adsorption of EPS stabilized MIONPs with Cr(VI) has been determined at different operational condition to obtain the optimum condition. The effect of temperature on Cr(VI) adsorption by MIONPs was studied. The change in adsorption percent with respect to temperature range was significantly detected (P < 0.05) by One-way ANOVA coupled with Dunnett’s multiple comparison test (Fig. 5a). The effect of temperature on Cr(VI) adsorption over magnetic multi-wall carbon nanotubes showed higher adsorption percent of Cr(VI), which ascribed to the stability of binding sites of adsorbents even at higher temperature [49]. With increase in the temperature from 300 K to 318 K, the adsorption percent of MIONP for Cr(VI) was determined to be significantly increased by one-way ANOVA coupled with Tukey’s multiple comparison test (P < 0.05). Adsorption experiments were conducted for the MIONPs at pH ranging from 3 to 8.2 (Fig. 5b). Maximum percentage of adsorption was measured at pH 5.2. With increase in further pH beyond 5.2, the adsorption percent decreased. The change in adsorption behaviour of MIONPs with increasing pH from 3 to 7, 4.2 to 8.2 and 5.2 to 8.2 were found to be statistically significant (P < 0.05, One-way ANOVA Tukey’s multiple comparison test). The change in pH (from acidic to basic) exhibited the existence of different forms of chromate ions. Below the pH 4, Cr(VI) primarily exists in the form of monovalent HCrO₄⁻ whereas above the pH 4, Cr(VI) exists in the form of Cr₂O₇²⁻ [50]. The adsorption feature of negatively charged oxyanionic chromate with adsorbents synthesized in this study was occurred due to the electrostatic interaction. This was happened due to protonation of –NH₂ group originated from EPS stabilized MIONPs, which drive the electrostatic interaction with Cr(VI) ions. pH 5.2 was decided as optimal pH for the adsorption of chromate ions by EPS stabilized MIONPs.

Fig. 5.

Effect of various parameters on adsorption of Cr(VI) by MIONPs a Temperature b pH c Initial Cr(VI) concentration, and d contact time

The surface charge of MIONPs was studied with variation in pH. The relative positive zeta potential value obtained for EPS stabilized MIONPs was found to be decreased with increase in the pH from 3 to 5.8. A net decrease in the positive charge from EPS stabilized MIONPs after pH 5.2 might have contributed in decreasing the adsorption capacities, as observed at pH 5.8, 7.0 and 8.2. This study was well in agreement with the previous Cr(VI) adsorption studies, which used the magnetite multi-wall carbon nanotubes as an adsorbent [49]. Martínez et al. [51] demonstrated the adsorption of Cr(VI) dependent on pH and temperature. Similarly, the impact of initial Cr(VI) concentration on adsorption was analyzed using increasing dosages (50–200 mg L⁻¹) in Fig. 5c. The adsorption percent increased as the Cr(VI) concentration reached up to 100 mg L⁻¹, after which it started decreasing. The decrease in adsorption percent with increase in initial Cr(VI) concentration was found to be statistically significant (P < 0.05, One-way ANOVA Tukey’s multiple comparison test). The effect of initial Cr(VI) concentration on adsorption was determined using increasing doses of Cr(VI) from 50 to 200 mg L⁻¹. Adsorption percent was increased due to greater availability of the metal ions. Therefore, 50 mg L⁻¹ of initial Cr(VI) concentration was considered to be equilibrium metal ion concentration and imparted a driving force to overcome the mass transfer resistance between the aqueous solutions and solid adsorbents. The decrease in adsorption capacities with further increase in the initial Cr(VI) concentration shows the saturation of binding sites [50]. The effect of contact time on to the Cr(VI) adsorption was found to be increased with the increase of contact time from 24 to 72 h (Fig. 5d). The effect of contact time for adsorption initially yielded high adsorption percent within 24 h and sustained till 72 h. They were found to be statistically significant (P < 0.05, One-way ANOVA Tukey’s multiple comparison test).

Moreover, adsorption isotherm models namely Langmuir, Freundlich and D-R were used to study adsorption mechanism involved in case of EPS mediated synthesis of MIONPs. After adsorption of Cr(VI) with MIONPs, the adsorption parameters were computed in these models. The Langmuir adsorption model is represented as linear form.

Where q_max signifies the maximum amount of Cr(VI) ion per unit weight of adsorbent for formation of complete monolayer at high equilibrium concentration C_e, while K_L implies the affinity of Cr(VI) to binding sites of adsorbent. q_e denotes a particle limiting adsorption percent when the surface is entirely covered with solute. Langmuir parameters, such as q_max and K_L were calculated from the slope and intercept of the linear plots of 1/q_e vs 1/C_e is given in Table 1.

Table 1.

Adsorption isotherm parameters for Cr(VI) adsorption of MIONPs

Isotherm parameters	Temperatures
	300 K	310 K	318 K
Langmuir isotherm
qmax (mg g−1g)	877.19	990.09	1052.63
KL (L g−1)	11.40	14.65	15.78
R2	0.98	0.97	0.96
Freundlich isotherm
Kf (mg g−1)	1559.55	1840.77	2032.35
n	1.376	1.440	1.492
R2	0.96	0.94	0.92
D-R isotherm
Kads	−1.18688E-6	−9.91519E-7	−8.98815E-7
Qm (mg g−1)	14.72287	14.02544	13.72218
R2	0.98	0.98	0.98
E (kJ mol−1)	0.653	0.714	0.769

The Freundlich model designates the heterogeneity of the adsorbent surface and considers multilayer adsorption. Where K_F and 1/n represent the constant for adsorption percent and adsorption intensity (heterogeneity factor) respectively. The values of K_F and 1/n were obtained from the slope and intercept of the linear Freundlich plot of log q_e vs log C_e is given in Table 1.

The concept of D-R isotherm model is not based on homogenous monolayer adsorption like Langmuir but considers more common features of adsorption. Where Q_m is the theoretical adsorption percent (mg g⁻¹), K_ads is the constant related to adsorption energy, Ɛ is the polyani potential, R is the gas constant (kJ mol⁻¹ K⁻¹), T is the temperature (K). The plot of ln q_e vs Ɛ² gives the value Q_m. The value of K_ads and Q_m is given in Table 1.

Based on the coefficient of correlation (R²) value, the Langmuir model was found to be best fitted to the experimental data than Freundlich and D-R isotherm in Fig. 6a. R_L is an added significant characteristic of the Langmuir isotherm which depicts the nature and feasibility of adsorption process. It designates the shape of the isotherms, unfavourable if R_L > 1, linear if R_L = 1, favourable if 0 < R_L < 1 or irreversible if R_L = 0. Based on the Langmuir constant, the value of R_L for Cr(VI) adsorption with MIONPs was found between 0 and 1, which indicated the favorable reaction. The plot for R_L Vs C_e in Langmuir adsorption model was calculated at three different temperatures (Fig. S2). Additionally, a constant ‘n’ value of the Freundlich model was found to be greater than 1, which indicated that EPS stabilized MIONPs was an appropriate adsorbent and beneficial for the adsorption of Cr(VI) from aqueous solution (Table 1). The Freundlich plot for the Cr(VI) adsorption at different temperatures with MIONPs incorporated with EPS is shown in Fig. S3. Based on D-R isotherm in Table 1, the calculated value of E as free energy of adsorption were 0.653, 0.714 and 0.769 at temperatures 300 K, 310 K and 318 K respectively. The D-R isotherm plot for the Cr(VI) adsorption at different temperatures with EPS incorporated MIONPs is shown in Fig. S4. The magnitude of E provides the type of adsorption process. When value of E is between 8 and 16 kJ mol⁻¹, the adsorption type can be an ion exchange process. When E < 8 kJ mol⁻¹, it could represent a physical adsorption process. The value of E, in the present study was found to be <8 kJ mol⁻¹, suggesting that the adsorption was led by a physical process. The adsorption characteristic of MIONPs for Cr(VI) has been determined by evaluating three different adsorption isotherm models.

Fig. 6.

Plots for adsorption of Cr(VI) with MIONPs at different temperatures a Langmuir plots at different temperatures, and b Van’t Hoff plot

In the present study, the experimental results obtained from adsorption kinetics were best fitted in Langmuir isotherm model indicating that Cr(VI) adsorption was a favourable process. The obtained isotherm parameters supported the feasibility and adsorption percent of MIONPs. The Cr(VI) removal efficiency determined by starch functionalized iron oxide nanoparticles showed highest adsorption potential than only iron oxide nanoparticles [52]. The Langmuir isotherm model provided the adsorption percent of EPS-incorporated MIONPs for Cr(VI) in the range of 877.19 to 1052.63 mg g⁻¹of EPS-MIONPs. This attained value is greater than other studies, i.e. 2–51.7 mg g⁻¹ [53–55]. Furthermore, the study of Cr(VI) adsorption by MIONPs at different temperatures suggested that an increase in the temperature eventually enhanced the adsorption percent of the adsorbent. The Cr(VI) removal potential of Synechocystis spp. (Synechocystis sp. BASO672 and Synechocystis sp. BASO670) varied in terms of EPS production and showed only 33% removal of Cr(VI) in EPS producing strain within 7 days at 10 mg L⁻¹ Cr(VI) load [56]. This study showed 82.8% removal of Cr(VI) within 72 h at 50 mg L⁻¹ Cr(VI) load under acidic condition. The present study used EPS stabilized MIONPs for removal of Cr(VI) from aqueous solution with 82.8% efficiency due to highest adsorption ability caused by enhanced surface area and functional groups compared to pristine EPS [35]. Thus, this study illustrates the novel and potentially most effective technique for removal of Cr(VI) from acidic contaminated sites and synthesis of economically feasible NPs.

Moreover, the thermodynamic parameters for adsorption, namely standard free energy change (ΔG⁰), standard enthalpy change (ΔH⁰), and standard entropy change (ΔS⁰) were calculated by using standard methods. ΔG⁰ was obtained by putting the lnK_L value in standard equation at specified temperature. ΔH⁰ was obtained from slope of the plot of ∆G/T against 1/T. The ΔS⁰ was calculated by putting the obtained ΔH⁰ and ΔG⁰ value in standard equation and their values are given in Table 2. As shown in the table, the values of ΔG⁰ for adsorption of Cr(VI) are −6.068, −6.905 and − 7.279 kJ mol⁻¹ at 300 K, 310 K and 318 K respectively. The ΔG⁰ value is negative at all studied temperature, inferring that adsorption of Cr(VI) onto EPS stabilized MIONPs follow the spontaneous process and favourable trend. The obtained value of ∆H⁰ (13.09 kJ mol⁻¹) and ∆S⁰ (63.86–64.50 J mol⁻¹ K⁻¹) are in positive magnitude, indicating endothermic reaction. Vant’t hoff plot also supported the endorthermic reaction of Cr(VI) adsorption over EPS stabilized MIONPs (Fig. 6b).

Table 2.

Thermodynamics parameters of Cr(VI) adsorption of MIONPs

Temperature (K)	ln KL	1/T	ΔG	ΔH	ΔS
		(K−1)	(kJ mol−1)	(kJ mol−1)	(J mol−1 K−1)
300	2.433	0.003333	−6.068		63.86
310	2.684	0.003226	−6.905	13.09	64.50
318	2.758	0.003145	−7.279		64.05

Thermodynamic parameters were also evaluated in this study which provides the additional information regarding the inherent energetic change associated with the adsorption process [57]. With increase in temperature from 300 K to 318 K, the ΔG⁰ value was decreased which exhibits an increase in adsorption of Cr(VI) [49]. This result could be ascribed due to increasing in the mobility of the Cr(VI) molecules and number of active sites for the adsorption with rising temperature. The positive value of ΔH⁰ (13.09 kJ mol⁻¹) confirmed the endothermic reaction. Additionally, the Van’t Hoff plot also indicated the endothermic reaction of Cr(VI) over MIONPs. The positive value of ΔS⁰ (63.86–64.11 J mol⁻¹ K⁻¹) showed the increased in the randomness at the boundary of adsorbate and adsorbent with some structural alterations and an affinity of the adsorbent towards the Cr(VI) aqueous solution [58]. The results indicated that the number of adsorption sites is higher in EPS stabilized MIONPs in comparison to other materials studied (Table 3). Hence, the prepared EPS stabilized MIONPs is found to be proficient adsorbent for elimination of Cr(VI) element from acidic industrial effluents.

Table 3.

Comparison for adsorption capacity of MIONPs with other reported adsorbents

Adsorbents	qmax (mg g−1)	Initial Cr(VI) concentration (mg L−1)	pH	Reference
3-Mercaptopropionic acid coated superparamagnetic iron oxide nanoparticles	55	10–50	1–6	[47]
Maghemite nanoparticles	19.2	50/100	2.5	[48]
Iron nanoparticles in orange peel pith biocomposite	5.37	10–50	1–7	[54]
Fe-MBC	35.7	50	5	[55]
Diatomite-supported/ unsupported magnetite nanoparticles	12.31–20.16	50	2–3	[59]
Starch functionalized iron oxide nanoparticles	6.87–9.02	40	2–8	[60]
EDA-Fe3O4 nanoparticles	81.5	60–100	2	[61]
Magnetite nanoparticles	15.4	5–100	2.0	[62]
Sandwitched Nanocomposite	374.53	300	2	[63]
FeS/CMC/biochar	138.7	100	2.3	[64]
Brown Seaweed Dictyopteris polypodioides	21.78	50	1	[65]
Sargassum seaweed biomass	333.39	350	2	[66]
Peanut shell	4.42	40	2	[67]
Leucaena leucocephala seed shell activated carbon	27.53	71.49	4.22	[68]
EPS stabilized MIONPs	1052.63	50	5.2	This study

Mechanism of Cr(VI) removal by MIONPs

The zeta potential study measured the surface charge of adsorbents with variation in pH (3.0–5.8). The positive zeta potential value obtained for EPS stabilized MIONPs were decreased with increase in pH, whereas it was increased after Cr(VI) interaction as shown in Fig. 7a. The elemental composition analysis of surface of Cr(VI) interacted MIONPs through EDX spectroscopy confirmed the presence of Cr ions over the surface of nano-adsorbents (Fig. S5). The amount of chromium build up (in weight percent) over the adsorbent was found to be 0.52%. The role of functional group of EPS stabilized MIONPs in interaction with Cr(VI) were determined by ATR-FTIR spectroscopy (Fig. 7b). The band intensity of both –OH and –NH functional group ranging from 3833 to 3173 cm⁻¹ corresponds to polysaccharides and proteins as constituents of EPS stabilized MIONPs were decreased and shifted. These two major groups have participated in reaction whereas the band at 2796 cm⁻¹ which confirms the formation of –C=O containing functional group i.e. H–C=O (2830–2695 cm⁻¹) from –OH, and designates the reduction process of Cr(VI) [69]. At the same time, the anionic functional groups as –OH stretching vibration from sugar (3700–3100 cm⁻¹) at 3173 cm⁻¹ was decreased, suggesting interaction with reduced byproduct of Cr(VI) ions [70]. Post interaction, the peak intensity of amide I band (1700–1600 cm-1) at 1708 cm⁻¹ and amide II band (1580–1510 cm⁻¹) at 1529.45 cm⁻¹ which corresponds to protein as constituents of EPS stabilized MIONPs were disappeared. This disappearance of these two sharp band supported their involvement in process of interaction with Cr(VI). This study under acidic condition exhibits proton environment around amine group which facilitates the formation of ammonium (-NH₃⁺) on the surface of EPS stabilized MIONPs, subsequently drives the adsorption of Cr(VI) ions through electrostatic interaction [71]. pH plays an important role in modifying the surface charge of the adsorbents. Recently, Ferreira et al. [72] reported the similar trend where magnetite nanoparticles were modified with various kinds of polyionic liquid. Polymer phase incorporates functional groups over the surface of MIONPs which can be protonated at low pH values and impart positive charges on the surfaces of the adsorbents, thereby improves the interaction with Cr(VI) due to electrostatic interaction [70, 72]. Moreover, the peak at 1031.86 cm⁻¹ corresponding to sugar region (1200–900 cm⁻¹) disappeared, suggesting that the glycosidic bond was also involved in the reaction [73, 74].

Fig. 7.

Characterization of EPS stabilized MIONPs before and after interaction with Cr(VI) a Zeta potential, and b ATR-FTIR spectra

Upon interaction with Cr(VI), a new peak conforming to Cr-O vibrations at 833.98 cm⁻¹ formed in fingerprint region, which also confirmed the adsorption of Cr on the adsorbent surface [75]. The variation of the absorption peaks at 2345.15 cm⁻¹ indicated the participation of thiol group in the reaction. Thus, the decrease in the intensity of the peaks for –SH, –NH, -OH and –C=O groups depicted the interaction of EPS stabilized MIONPs with Cr element. The contribution of these functional groups in interaction with metal was in agreement with previous reports [76]. On the other hand, previous study also stated that Cr(VI) can be reduced to Cr(III) in acidic solution in presence of organic matter [77]. The decrease in band intensity at 681.11 cm⁻¹ indicated the involvement of the Fe-O bond in the interaction and probable complexation of Cr ions with Fe of EPS stabilized MIONPs. XRD patterns of the pristine EPS, EPS stabilized MIONPs and EPS stabilized MIONPs after Cr(VI) interaction is shown in Fig. 8. The XRD profile of pristine EPS showed a broad diffractogram revealing the amorphous nature of EPS (Fig. 8a). XRD for EPS stabilized MIONPs indicates the peak at 36.03° and 31.5° which corresponds to the (311) and (220) plane of iron (JCPDS no. 75–0449), and confirmed the presence of Fe₃O₄ nanoparticles (Fig. 8b). The XRD analysis of EPS stabilized MIONPs after interaction with Cr(VI) indicate the presence of peak at 2θ = 30°, 35.5°, 43°, 57° and 62.6° which corresponds to (220), (311), (400), (511) and (440) Miller plane of cubic phase of Cr₂FeO₄ (JCPDS no. 24–0512) in Fig. 8c. The effect of Cr(VI) interaction with EPS stabilized MIONPs were further studied. The formation of positively charged Cr(III) hydroxide complexes such as Cr(OH)²⁺ and Cr(OH)₂⁺ at pH 3.0, 4.2 and 5.8 was attributed to the significant increase in the magnitude of zeta potential after interaction of MIONPs with Cr(VI). The zeta potential data suggested the redox reaction during the interaction. The subsequent products of redox reaction such as Cr(OH)²⁺ and Cr(OH)₂⁺ were adsorbed by EPS stabilized MIONPs, which might be the reason for change in the zeta potential measurements. Similar observation was reported while Cr(III) was interacted with magnetite and functionalized magnetic mesoporous silica materials [78]. Moreover, the XRD analysis of EPS stabilized MIONPs after interaction with Cr(VI) depicted the significant change in the XRD spectra. The XRD profile of EPS stabilized MIONPs before and after Cr(VI) interaction were found to be significantly different suggesting that involvement of iron core of EPS stabilized MIONPs for interaction with Cr(VI). The interaction of EPS stabilized MIONPs after Cr(VI) interaction showed peak at 2θ value of 35.5° corresponded to the (311) Miller plane of cubic phase of Cr₂FeO₄. Thus, the appearance of Cr(III) in EPS stabilized MIONPs after reduction demonstrated the occurrence of redox reaction [78]. Further, AAS analysis revealed that MIONPs were able to remove 80.43% of total Cr while 1.08 mg L⁻¹ Cr(III) was left in the aqueous solution. This finding suggested that the majority of Cr(VI) was initially reduced to Cr(III) by EPS stabilized MIONPs followed by adsorption of Cr(III) on to the surface of the adsorbent [79].

Fig. 8.

XRD patterns of a pristine EPS b EPS stabilized MIONPs, and c EPS stabilized MIONPs interacted with Cr(VI)

Conclusion

Green synthesis of nanomaterials would be an alternative and emerging approach in the field of nanotechnology to lessen the environmental pollution emerging from industrial effluents containing Cr(VI). EPS matrix of biofilm has been used in the present study as a safe and green approach. It consists of several binding site for various metal ions and also act as a capping agent in the synthesis of MIONPs. Furthermore, from this study it may be concluded that EPS produced by CrRAtBb Lysinibacillus sphaericus RTA-01 might serve as better reducing, stabilising and capping potential for the synthesis of nanoparticles. The application of these MIONPs functionalised by EPS showed enhanced capability to adsorb Cr(VI). The present work elaborated an approach for green synthesis of MIONPs using biofilm-EPS and its application in the removal of Cr(VI) element from the aqueous solution.

Compliance with ethical standards

Conflict of interest

Authors declare no conflict of interest.