Iron oxide nano-material: physicochemical traits and in vitro antibacterial propensity against multidrug resistant bacteria

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Contents lists available at ScienceDirect

Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec 1 2 3

Iron oxide nano-material: physicochemical traits and in vitro antibacterial propensity against multidrug resistant bacteria

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Q1 M.

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a

Harshiny a, C. Nivedhini Iswarya a, N. Samsudeen a, P. Saravanan b,c, M. Matheswaran a,*

Department of Chemical Engineering, National Institute of Technology, Tiruchirappalli 620015, India Nanotechnology and Catalysis Research Center (NANOCAT), University of Malaya, 50603 Kuala Lumpur, Malaysia c Department of environmental science and engineering, Indian School of Mines, Dhanbad 826004, Jharkhand, India b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 May 2016 Received in revised form 22 August 2016 Accepted 2 September 2016 Available online xxx

The synthesis of Azadirachta indica leaf extract mediated Iron oxide nanoparticles (FeO-NPs) from ferric chloride were investigated. The characterization results confirmed that FeO-NPs were having good physicochemical traits. The FeO-NPs hold the resistance in ohmic range and coercivity value reached 126emu/g. The FeO-Nps antioxidant efficiency was observed to be 95% of inhibition against 1, 1diphenyl-2-picrylhydrazyl. The indigo-carmine dye decolourization of 79 and 85% under sunlight and UV-light irradiance were attained for FeO-NPs. The antibacterial and antibiofilm activities of FeO-NPs against bacterial pathogen were tested. The results confirmed that FeO-NPs have a virtuous propensity against gram negative than positive bacteria. ß 2016 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.

Keywords: Azadirachta indica Iron oxide Physicochemical Antioxidant Antimicrobial Photocatalytic

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Introduction

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The emergence of multidrug-resistant microbes (bacteria) has become an imperative universal health concern in the current era. Bacterial strains like P. aeruginosa, S. aureus, K. pneumonia, L.sphaericus and Bacillus sp have the potential to create slime that assists the adhesion and the formation of biofilms in any adverse surface [1]. The bioflim of P.aeruginosa, S. aureus, K. pneumonia causing severe issues in the biomedical fields and industrial water systems by contamination of surfaces, increase spoilage and cause safety issues [2]. Biofilms are high resistant to antibiotics than freefloating bacteria also very complex to treat medically [3]. Lateef et al., reported that B.safensis was salt-tolerating, plant growth promoting rhizobacteria as well as an extreme environment relies on their efficient physiological and genotypic characteristics [4]. Berry et al., concluded that L. sphaericus bacteria were used as an insect pathogen owe to the presence of parasporal crystal endotoxins [5]. Moreover, L. sphaericus and B. safensis show resistant against the multidrug. Hence, it is essential to develop an alternate and effective therapeutic strategy for eradicating harmful biofilms in clinical and food industry [6].

* Corresponding author. Tel.: +91 431 2503120; fax: +91 431 2500133. E-mail address: [email protected] (M. Matheswaran).

In the last few decades, numerous new antibiotics were developed, although; which was holding adverse toxic effects. Therefore, the discovery of new drugs necessitates to eradicating the fore-mentioned drawback [7,8]. Metallic nanoparticles such as silver, gold, copper, zinc, titanium and selenium oxide were used as an agent in food safety and wastewater technology. However, these nanoparticles showing an adverse noxious effect [9– 11]. Harshiny et al reported that among different types of nanoparticles, iron oxide nanoparticles hold few ideal properties such as chemical stability, conductivity and magnetic behaviour. FeO NPs were widely used in a variety of scientific and technological applications from environmental to biomedical field [12]. Various chemical and physical methods have been reported for the synthesis of nanoparticles, but in those methods toxic solvents were used for preperation. It could produce hazardous byproducts and utilize high energy [13,14]. The plant extract mediated Nps were more stable and the rate of synthesis is faster than conventional methods [15,16]. The biosynthesized metal Nps can also used as an anticoagulant, antimicrobial, antioxidant agent in the various fields [17– 19]. Arokiyara et al., synthesized FeO Nps using A. Mexicana.L extract as a reducing agent and proved to have outstanding antimicrobial efficacy against the pathogenic bacteria’s [20]. Naseem and Farrukh synthesized leaf extract mediated iron nanoparticles and showed good antibacterial activity against the

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human pathogens [21]. The biosynthesis of iron nanoparticles using leaf extracts are an economic and eco-friendly benefit over chemical and physical synthesis [22]. The leaf extract of A. indica contained multifunctional organic compounds such as phenolics, tannins, flavonoids, alkaloids, saponins; which act as non-toxic capping stabilizing agents and it reduces metal ions into respective metal or metal oxide NPs [23]. A.indica. L extract also having the remedial properties such as antioxidant antibacterial, antifungal, and antitumor activity, due to the presence of biomolecules [24]. Zhejiang et al., synthesized iron–polyphenols nanoparticles using plant extracts and investigated the catalytic performance by decolorization of azo dye [25]. Hu et al., reported that nanoparticles with photocatalytic property exhibits an enhanced inhibition of microbial growth [26]. These initiates, the green synthesis of FeO Nps using A. indica leaf extract for enhancing its physicochemical properties. The aim of the present study was to synthesis, iron oxide nanoparticles from ferric chloride using hydro-alcoholic A. indica leaf extract as a reducing agent. The shape, size, surface area, surface potential, optical, magnetic, electrical properties and stability of FeO Nps were characterized using analytical techniques. The antioxidant potency estimation of FeO Nps was carried out using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Since, DPPH is one of the rapid, facile, economical and extensively used method to evaluate the capacity of molecules to act as free radical scavengers or hydrogen donors [27]. The photocatalytic performance of NPs was tested by decolourization of Indigo carmine dye under sunlight and UV irradiation. The FeO NPs antibacterial activity against human pathogenic bacteria such as P.aeruginosa, S.aureus, K. pneumoniae, L. sphaericus and B. safensis were investigated. The standard growth kinetics, disc diffusion method and minimum inhibitory concentration (MIC) were performed to assess the antibacterial qualitative and quantitative propensity of FeO NPs [28]. The antibiofilm activity of FeO Nps was studied using test tube crystal violet (CV) assay; which is an ease and efficient tool for observing the reduction of biofilm [29].

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Experimental

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Microbial cultures and chemicals

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The test organism such as P. aeruginosa (MTCC-2488), S. aureus (MTCC-9542) and K. pneumoniae (MTCC-4032) was obtained from the microbial type culture collection (MTCC). L. sphaericus (KR996141.1) and B.safensis (KT003213.1) bacteria were isolated from distillery waste water. The chemicals such as Ferric chloride (FeCl3), Sodium borohydride (NaBH4), sodium hydroxide (NaOH), hydrochloric acid (HCl), DPPH, ethanol, crystal violet, Dimethyl sulfoxide, nutrient broth and agar were obtained from Merck and Himedia, India. All chemicals were used without any further purification in this study.

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Synthesis of nanoparticles using A. indica leaf extract

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The A.indica leaf extraction process as well as antioxidant capacity was investigated and conferred S1.1 (see supplementary). The FeO Nps was synthesized using FeCl3 and hydro-alcoholic A.indica leaf extracts as a precursor and reducing agent respectively. The solution pH was adjusted using 0.1 N HCl and 0.1 N NaOH. 25 mL of 1000 ppm leaf extract (pH 6.3) was added drop by drop using a burette to 50 mL of 0.5 M FeCl3 in 150 mL Erlenmeyer flask. The solution was continuously stirred with a magnetic stirrer (250 rpm) and maintained at 37  1 8C for 60 mins. The solution color changes from brown into a blackish green colour; which confirmed the formation of nanoparticles. The precipitated of of FeO NPs was acquired by vacuum filtration. The collected filtrate was

washed with absolute ethanol and then dried in a hot air oven at 70 8C for 180 min. The obtained samples were stored in sealed bottles under dry conditions prior to use.

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Characterization

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The X-ray diffraction (XRD) pattern was obtained by Rigaku Ultima III by step scan technique with Cu-Ka radiation (1.500 A, 40 kV, 30 mA). The IR spectra of the leaf extract and nanoparticles were recorded by fourier transforms infrared spectroscopy (FTIRThermo Scientific99 TM Inc. NicoletTM iSTM5) over a spectral range of 400–4000 cm1. The morphologies and composition were analyzed using Transmission Electron Microscope with Energy Dispersive X-ray Analysis (TEM-EDAX- Technal Spitit G2) operating at accelerating voltages of 120 KV. The Particle Size Distribution (PSD) and zeta potential were measured by Hiroba SZ-100 nanopartica. The surface area analysis was done by N2 adsorption isotherm using a Micromertics ASAP 2020 V3.04 H. Prior to analysis, the samples were degassed with nitrogen at 300 8C for an hour. The thermal properties were determined using the thermogravimetric analysis (TGA 4000 – PerkinElmer). XPS technique was carried out on a K-Alpha instrument supplied by Thermoscientific, USA. The electrochemical measurements were carried out using Metrohm B.V. Multi Autolab-ATUM101X work station for determination of sample conductivity. Electrochemical studies were performed in a conventional three electrode system using glassy carbon as a working electrode, Ag/AgCl (saturated KCl) as a reference electrode and Pt wire as a counter electrode in a single compartment cell. The cyclic voltammograms were recorded at scan rates of 10 mVs1. Optima 8000- Perkin Elmer Inductively Coupled Plasma Atomic Emission Spectroscopy ICPOES was used to determine the stability of nanoparticles. The Nikon Eclipse, fluorescence microscope was employed for imaging bacteria cells (control) and NPs treated bacterial cells was observed.

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Photocatalytic activity

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The photocatalytic decolourization of IC dye using Nps was investigated. 1000 mL of 100 ppm IC (pH-11) solution was taken and added 250 mg/L of synthesized Nps in the photocatalytic reactor. The mixture was continuously stirred using a magnetic stirrer (100 rpm) under the average natural sunlight irradiation of 1000 W/m2 and maintained a temperature at 38  2 8C. The decolourization experiments were carried in the daytime between 10 am and 6 pm, during April–May, 2016. Solar radiation intensities were measured with a regular interval of time using pyranometer. The photocatalytic activity of Nps under UV irradiation were carried in a cylindrical double jacket vessel equipped with 16 W UV lamps under same experiments out condition. Samples were collected at regular intervals of time during the experiments, then percentage of colour removal were assessed using UV–visible spectrophotometer with a wavelength range of 200–800 nm [30]. The photocatalytic decolourization efficiency for IC was calculated using the following equation:

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Percentage decolourization ¼

A0 At 100 A0

(1)

Where Ao and At is the absorbance of the initial concentration and after time ‘t’ of IC solution.

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Antioxidant activity

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The antioxidant activity of NPs was examined in terms of hydrogen donating potential using DPPH assay. Different concen-

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tration of NPs from 50 to 250 mg/mL were mixed with DPPH (25 mg/L) and incubated in the dark condition for 5 mins. The concentration of DPPH was measured at lmax 515 nm [31]. The percentage of inhibition was calculated using the following equation, Percentage Inhibition ¼

AB AS 100 AB

(2)

3

violet adsorbed by bacterial biofilm. The absorbance of CV color was measured by observing the absorbance at 630 nm [33]. The percentage reduction of biofilm was calculated using equation. Biofilm reductionð%Þ ¼

ðCBÞðTBÞ 100 CB

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(3)

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where, AB and As is the absorbance of the blank and samples.

where B, C and T denotes, the average absorbance of blank, control and treated biofilm tubes. The experiment was performed in triplicates.

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Growth curve studies

Result and discussion

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The bactericidal activity of the FeO NPs was tested by growth inhibition studies. Sterile Erlenmeyer flasks containing 500 mL nutrient broth and 1 mL of the freshly prepared bacterial suspension were inoculated. The desired amount of FeO Nps was added in the inoculm for testing the growth inhibition. The devoid of nanoparticles was also monitored as a control. The flasks were incubated in a rotary shaker for 150 rpm at 37 8C. The growth kinetic studies were performed by measuring optical density (O.D.) at 600 nm using a spectrophotometer at regular time interval. The growth rate was determined by plotting the optical density versus time [32].

Characterization of nanoparticles using analytical techniques

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Evaluation of FeO NPs MIC

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The MIC of FeO NPs was studied for each of the gram positive (+ve) and gram negative (ve) bacteria. To test bacterial inhibition various concentrations (50 mg to 250 mg/mL) of NPs were added to sterile test tube each containing 5 mL nutrient broth and freshly prepared bacterial cultures were inoculated. The prepared culture media were incubated for 24 h (37 8C) in an orbital shaker at 150 rpm. Optical density was measured for each test organism to determine the inhibitory efficacy of FeO NPs [33].

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Disc diffusion method

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Antibacterial activity of FeO NPs was conducted in-vitro by disc diffusion assay method against P. aeruginosa, S. aureus, K. pneumoniae L. sphaericus and B. safensis. The result was compared with leaf extract and Streptomycin 25 mg as a standard. The sterile Muller Hinton agar was poured on sterile petri dishes and inoculated with the fresh overnight test cultures using cotton swabs. Sterile discs of 6 mm width had been impregnated with (10, 15 and 20 mL) of test solution from (5 mg/mL). The upper layer of the prepared disc was introduced onto the seeded agar plate. The plates were incubated overnight at 37 8C. Antibacterial activity was evaluated and compared by measuring zones of inhibition that formed around the disc [34]. The mean values were calculated by repeating thrice the experiment.

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Antibiofilm activities

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The biofilm development of test organism was examined using the execution of the crystal violet assay. The test cultute was separately inoculated in equal volume in different sterile test tubes. To each tube (1, 2 and 5 mg/mL) of nanoparticles were added and incubated at 37 8C for 24 h. The devoid of nanoparticles was used as a control. After the incubation, culture broths from test tube were removed. Tubes were washed twice with sterile Milli Q water and 150 mL of 0.4% crystal violet was added. Then incubated for 15 min at room temperature. Subsequently, a crystal violet solution was discarded from each tube and washed with sterile Milli Q water twice for removal unabsorbed crystal violet. The 200 mL of 30% acetic acid was added to each tube to dissolve crystal

The XRD patterns of FeO NPs was shown in Fig. 1a. Peaks appearing at 2u value of 35.68 and 56.178 were matched to JCPDS:01-073-0603 FeO crystal structure. It also confirmed (1 1 0), (2 1 1) planes of rhombohedral phase structures in iron oxide. The broadening of the XRD lines, as seen in the pattern may owe to the bio-molecules are merged with nanoparticles. The identified diffraction peaks belonged to FeO Nps and iron-oxohydroxide Nps (FeOOH) [35]. The leaf extract on the surface of FeO NPs functional group was confirmed by FTIR as shown in Fig. 1b. A band at 3346 cm1 was attributed to the stretching vibration of OH group. The IR results of two bands at 1497 cm1 attributed to the COO– molecules immobilized on the FeO NPs surface. While, the band at 880 cm1, assigned to the CH strong vibration of A.indca was shifted to 828 cm1. The presence of two absorption peaks at 508 and 457 cm1 corresponding to the Fe–O group; These IR results revealed that organic groups from leaf were bonded to the FeO Nps surface which clearly confirmed A.indca leaf extract which acts as a reducing agent and stabilizer for the formation of FeO NPs [30]. The feasible formation mechanism of FeO nanoparticles using A.indica; also IR spectrum of a leaf extract were conferred (see supplementary S.1 and 2). The thermal behaviour of A.indca leaf extract mediated FeO NPs shown in Fig. 1c. There were four mass losses in the TGA curve. The first mass loss of 4.8% in the temperature region from 37 to 90.5 8C is most probably due to the loss of water molecules. The second, percentage of mass loss is about 8.84% in the temperature region from 167 to 206.5 8C are due to the removal of bio-molecules on the surface of FeO NPs. At 315 8C, the third percentage mass loss is about 14.689%, which confirms strong binding between the biomolecules and nanoparticles. The last percentage of mass loss is about 7.5% at 630 8C which was attributed to the phase transition nanoparticles, because FeO is thermodynamically stable only above 570 8C. For comparison, plant extract and sodium bhorohydride mediated iron oxide nanoparticles thermal stability were also given in Fig. 1c. The sodium bhorohydride mediated iron oxide thermograms shows weight loss of about 2 and 18% due to loss of residual water and the phase Fe–O transition [35,36]. Degradation of the plant samples indicates three weight loss. The initial loss occurred at 48–130 8C owing to the evaporation of water from the extract. The weight loss in the temperature from 180 to 350 8C was due to the degradation of simple organic components in extract, The above 350 8C losses may due to degraded of volatile products like phenolics, alcohols and aldehyde acids [37]. The NPs functional group, thermographs and binding energy results (see supplementary S2.2) confirmed the implying of higher organic molecules and FeO in the prepared samples, it was believed that bio mediated FeO Nps are going to play an active role in stability and applications. The particle size analysis has shown the average particle size of 80 nm for FeO NPs and was shown in Fig. 1d. The zeta potential range was observed from 60 to 81 mV. The potential range

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Fig. 1. XRD pattern (a), FTIR spectrum (b), TGA analysis (c) and Particle size distribution of FeO Nps (d).

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demonstrates less agglomeration and apparently depicts that FeO NPs have less size and high potential as shown in Fig. 2a. This was due to bio-molecules binding to the surface of FeO NPs and significantly increasing their surface charge that enhances their stability by inhibiting aggregation[2]. The nitrogen adsorption–desorption isotherms were carried out to investigate the surface area of FeO NPs was shown in Fig. 2b. The specific surface area of nanoparticles was calculated using the BET surface area plot and found to be 55 m2/g for FeO NPs. The surface morphology of the synthesized nanoparticles was investigated using TEM; Fig. 2c. shows the typical TEM micrograph of FeO NPs appears to possess a characteristic of spherical like morphology and less aggregation. The EDAX analysis of synthesized nanoparticles confirms the composition of FeO as shown in Fig. 2d. The elements appearing in the EDAX spectrum are also observed using XPS spectra, as shown in Figure S4 (see supplementary).

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Stability of nanoparticles

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The absorbance and size distributions of NPs were varied depending on size (diameter), shape, band gap, surface structure and agglomeration state; which helps to evaluate stability of nanoparticles [2]. Therefore, the nanoparticles stability was studied using Uv-Vis spectrum and particle size analysis for different time intervals of 1, 15, 30 and 60 days after the synthesis. The Uv-Vis spectrum confirmed the FeO NPs absorbance at 274 nm and bandgap was calculated from absorbance spectrum using tauc plot as shown in figures S5a,b (see supplementary). The band gap of Nps was slightly decreased after 30 and 60 days respectively.

The particle size analysis clearly depicts that Nps size does not show any significant change up to 15 days, but average sizes of NPs increased from 80 to 99 and 106 nm for over 1 and 2 months respectively were shown in S5 c, d, e (see supplementary). The plot (S f see supplementary) clearly depicts that optical bandgap energy decreases with increase in particle size. This slight changes may due to physicochemical effects. The stability of nanoparticles was further confirmed by ICP-OES analysis, the result does not show any Fe content for samples prepared in Milli Q water up to 100 h. The leaf extract mediated nanoparticles are less soluble in water and having better stability. It was attributed to Phenolic compounds, acting as capping agents, improve stabilization of the colloidal suspension and also avoid nanoparticle aggregation[38]. The results conform that leaf extract mediated nanoparticles showed good yield, smaller size and better surface with less aggregation compared to sodium borohydrate mediated FeO Nps as reported in earlier studies [19].

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Electrical and magnetic properties of nanoparticles

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The electrical conductivity of FeO NPs was measured using impedance spectrum at room temperature in the 1 k Hz to 10 MHz. Fig. 3a represents the Nyquist plot of FeO NPs. It was apparently depicted that the impedance spectra exhibit a semi-circle loop. The corresponding circuit of the impedence spectra was confirmed using Z-view software. Fig. 3b reveals that circuit contains a resistor (R) in series with the RC parallel circuit. The circuit element was estimated manually by reading the real axis intercept at both high frequency (initial solution resistance) and low frequency

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Fig. 2. Zeta potential (a), nitrogen adsorption desorption isotherm (b), TEM micrograph (c) and EDAX spectrum of FeO Nps (d).

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(sum of initial solution resistance and polarization resistance) side. The relaxation time t was calculated as 3.98 ms. The estimated values of the components of the proposed equivalent circuit were illustrated in Fig. 3b. This clearly shows that the FeO-Nps is a promising candidate for electrochemical application. The capacitance was calculated using the following equation 2pymax C dl Rp ¼ 1

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where ymax (251188.6) is the frequency corresponding to the imaginary maximum value, Rp is the polarization resistance and Cdl is double layer capacitance value. The calculated DC (Direct Current) electrical conductivity (sdc) using the following equation

s dc ¼ 354 353 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369

0

(2 )

l AR

was 7:77107 S cm1

0

(3 )

where R be the total resistance, A be the area of analyzed sample and l be the thickness of the sample. Fig. 3c shows the conductance spectra of FeO NPs. The predominant of DC at low frequency, was due to independent of frequency and this conductivity was owing to band conduction which occurs at grain boundaries [39]. Conversely, at high frequency region AC conductivity is high. The hopping frequency was 0.441 kHz. Likewise, the carrier concentration (N) was calculated as 1.761  109. It was believed that synthesized nanoparticles can be used effectively for electrochemical applications. The magnetization measurements were recorded using VSM at room temperature. Fig. 3d shows a hysteresis loop recorded for FeO NPs. It reveals that co-ercivity values reached 126 emu/g [35]. Baskaran et al reported that saturation magnetizations of uncoated FeO NPs were 70.3 emu g1 and a Ms value of 59.8 emu g1 was obtained for glycol chitosan coated

FeO NPs, which is smaller than for the uncoated FeO NPs and proposed that of implying the polymer or coating influenced the magnetization[40]. Although, it confirmed that the leaf extract does not affect the magnetic properties of synthesized Nps

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Photocatalytic activity

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The decolorization potential of 250 mg/L FeO NPs were investigated using 100 ppm IC at pH 11 under sunlight and UVlight irradiance for 5 h was shown in Figure S5 (see supplementary). The decolorization efficiencies of IC dye 79 and 85% were observed using NPs under sunlight and UV-light irradiance. Due to biomoleclues encapsulated on a larger number of FeO NPs holds better surface area and lesser band gap. Hence, the photoelectron passed through a large number of NPs boundaries that can improve the formation of hydroxyl radical and conferred the decolorization [30]. Wang et al synthesized FeO Nps from Eucalyptus tereticornis, Melaleuca nesophila and Rosmarinus officinalis, moreover used as photocatalyst for decolourization of Acid black 194 dye. FeO NPs showed complete decolourization of dye in 200 min without any pH adjustment and at an initial concentration of 50 ppm [25]. Wen et al demonstrated nanoparticles having a relatively high surface area, improved electrical conductivity, excellent stability and so on, offer a great potential for both fundamental study and industrial applications in catalysis, energy storage and conversion systems [41].

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Effect of FeONPs on bacterial growth kinetics

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The growth kinetic studies of P.aeruginosa, S.aureus, K. pneumoniae, L. sphaericus and B. safensis in the presence and absence of FeO NPs was shown in Fig. 4. The growth curve of

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Fig. 3. Nyquist plot (a) and equivalent circuit (b), plots of ac conductivity with the frequency (d) and Saturation magnetization of FeO NPs (d).

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control experiments attained lag, log, stationary and death phases are nearly from 0th to 4th, 5th to 11th, 12th to 24th and 27th hours was observed for P.aeruginosa, S.aureus, K. pneumoniae. However L. sphaericus and B. safensis attained lag at 4th hour, then maintained log phase from 4th to 23rd hour and express constant pattern of growth in the subsequent stationary phase from 24th to 37th hour; finally it starts death phases at 38th hour. The growth inhibition of bacteria in the presence of nanoparticles compared to devoid of nanoparticles. The kinetics depicted that, P.aeruginosa, K. Pneumonia growth was inhibited in the initial stages; there was no obvious

Fig. 4. Growth kinetics of gram +ve and–ve bacteria in the presence and absence of FeO NPs.

growth after the fifth hour. On the other hand S.aureus, L. sphaericus and B. safensis showed their evident growth up to 10th hour. At the stationary phase control culture and effect of interaction pattern on NPs antimicrobial propensity was visualized under microscope as shown in Fig. 5. For microscopic image, the samples were prepared using the procedure reported by Arakha et al., with a few changes. Micrographic images of green fluorescence shown the presence of viable bacterial cells and a mixture of red and green fluorescence images confirming a mixture of viable and non-viable cells. Also, due to FeO NPs bacterial membrane physical rupture, reduced size and count were observed for all cultures. The kinetic data indicate that nanoparticles holds antimicrobial propensity against gram ve than gram +ve. Taylor et al reported that FeO Nps have inherent antibacterial properties due to superparamagnetic properties that could allow such particles to be directed inside the cell with a magnetic field; gram +ve bacteria are found to be less sensitive to FeO NPs than gram ve bacteria owing to the presence of a thicker peptidoglycan layer [42]. Considering, the presence of a thick peptidoglycan layer as an outer membrane in gram +ve species that functions as a permeability barrier also vulnerable to the antibacterial effect of nanoparticles [43].

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MIC of FeO Nps

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At which lowest concentration NPs exhibits antimicrobial activity was determined using the standard MIC method, as shown in Fig. 6. It is clearly seen that nanoparticles have evident bactericidal efficacy on bacteria; when compared to +ve controls. The viability of P.aeruginosa and K. Pneumonia reduced 85 and 90% in the presence of 250 mg/mL of nanoparticles. The data indicate a strong antimicrobial propensity of against gram–ve bacterial strains. It also apparently depicts at higher concentrations the growth inhibition of S. aureus, L. sphaericus and B. safensis gram +ve

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Fig. 5. Control K. Pneumonia (a, a1), P.aeruginosa (c, c1), S.aureus (e, e1), B. Safensis (g, g1) and L. Sphaericus (i, i1) microscopic small and higher magnification images and FeO Nps treated K. Pneumonia (b, b1) P.aeruginosa (d, d1), S.aureus (f, f1), B. Safensis (h, h1) and L. Sphaericus (j, j1) small and higher magnification microscopic images.

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bacteria were occurred. Nhiem et al reported that at the highest dose of iron oxide (3 mg/mL), was inhibited the growth of S. aureus significantly [44]. The higher concentrations of nanoparticles in the test solutions are enhancing ROS production. A principal reason for the antimicrobial propensity of nanoparticles; increasing net interactive interaction between nano-bacteria interface [42]. Above a certain concentration of nanoparticles, the net

Fig. 6. MIC of test bacteria exhibiting varied growth and the inhibition against FeO Nps concentrations.

interactive interaction enhances ROS production at the interface [43].

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Disc diffusion assay

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The effect of different concentration, such as 10,15 and 20 mL of FeO Nps was also tested by a qualitative disc diffusion assay for both gram +ve and gram -ve bacteria. Table 1 shows the inhibition zone diameter measurements of leaf extract, FeO NPs and streptomycin. The results indicate that nanoparticles were more effective against gram -ve bacterial strains compared to gram +ve bacterial strains as shown in Fig. 7. Due to the thick peptidoglycan layer as an outer membrane in gram +ve species FeO NPs showed less antibacterial. In case of gram -ve bacteria outer cellular membranes were thin and pores in the nanometre range [45]. Therefore nanoparticles will cross the cell membrane easily compared to other biomedicine [46]. Also, it was speculated that the positive zeta potential of synthesized FeO NPs promotes its interactions through cell membranes as well as damages proteins, inner membranes and DNA it leads to reduction in viability [47]. Likewise, control streptomycin and leaf extract also show the effect against gram -ve bacterial strains compared to gram +ve bacterial strains. Sumathi et al tested anethole coated FeO NPs antibacterial and antibiofilm activity were assessed using the organisms such as E. coli, S. aureus, B. subtilus, P. aeroginosa and K. Pneumonia. The zone of inhibition of anethole coated FeO NPs were found to be highest of 13 mm [48]. It apparently depicted that, A. indica hydro-alcoholic leaf extract mediated FeO-Nps have enhanced antibacterial activity.

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Table 1 ZOI (mm) against test bacteria and at different concentrations of Nps, 25 mg of Streptomycin and 200 mg/mL of prepared leaf extract. Organism

Streptomycin 25 mg ZOI (mm)

Leaf extract

FeO NPs

200 mg/mL

10 mL

15 mL

20 mL

11 10 7 Nil Nil

16 14 10 Nil Nil

18 16 11 Nil 7

ZOI (mm) K. Pneumonia P.aeruginosa S.aureus L. sphaericus B. safensis

26 25 24 22 23

10 9 7 Nil 7

473

Inhibition of biofilms and DPHH

474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489

The antibiofilm effect of FeO NPs were observed against gram +ve and gram -ve. FeO NPs. The maximum biofilm inhibition of 81.9, 89 and 59.5% against P.aeruginosa, K. Pneumonia and S.aureus were reduced by Nps. While, very less inhibition of biofilm formation was observed for L. sphaericus and B. safensis as shown in Fig. 8a. The figure shows, as increasing the concentration Nps conferred high inhibition of biofilm formation. Madhubala et al also studied the influence of gold and iron-oxide nanoparticles on biofilm-forming P. aeruginosa and S. aureus; also found that at higher concentrations (0.05, 0.10 and 0.15 mg/mL) of FeO Nps shows a significant reduction in biofilm growth was observed compared to low concentration Nps [49]. The result further confirmed greatest antimicrobial activity against gram -ve than +ve bacteria. Hajipour et al illustrated that structure of the cell wall played a major role in susceptibility of biofilm in the presence of nanoparticles. The diffusion inside biofilm matrixes by altering

surface from hydrophilic to an awfully hydrophobic towards nanoparticles and transform the expression of cell wall proteinase [50]. Furthermore, analysis are necessary to assess the influence of direct physical effect and release of metal ions from nanoparticles to a bacterial cell. On other hand, the biofilms of L. sphaericus and B. safensis are integral part of the purification process in wastewater treatment such as bioremediation of heavy metals [51,52] and bioelectricity production by oxidizing a variety of organic compounds [53]. Peng et al suggested among nanoparticles, FeO Nps holds advantages of good biocompatibility, environmental stability, low cost and unique electric property, making it as a good material for electrochemical capacitors[54]. Hence biofilms are not only an issue to abolish, but are also a promising sustainable solution to environmental issues. Thus, the isolated bacteria such as L. sphaericus and B. safensis are going to use in environmental applications and synthesized FeO NPs may apply as an effective material from clinical to environmental application. The antioxidant potentials of FeO NPs were also investigated using the DPPH assay. The results clearly depict that 50–250 mg/ mL of FeO NPs confirmed the inhibition of DPPH radical 30.46– 95.02% with an increase in concentration from 50 to 250 mg/mL. On the other hand, increases the concentration higher than 250 mg/mL inhibition of DPPH radical showing saturated activity as shown in Fig. 8b. Due to phyto-constituents such as, Phenolic compounds from plants are known to be good natural antioxidants; this group bound to FeO Nps, which can react further with stable biomolecule attachment to the metals and its consequent impact on the activity of the antioxidant agent[30]. In DPPH assay shifting of the electron to be found in oxygen to the odd electron located at surface orbits of oxygen in OH and O2 radicals

Fig. 7. Inhibition zone measurements of Streptomycin and FeO-NPs against B. Safensis (a, b) and L. Sphaericus (c, d).

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Fig. 8. Percentage reduction of biofilms (a) and Antioxidant activites of FeO Nps (b).

522 523 524 525 526 527 528

[12]. Jyoti Prasad et al reported ferric oxide nanoparticles DPPH scavenging capacity enhanced up to 13.94% with the help of sonication [55]. However, A.indica leaf extract mediated 250 mg/ mL of FeO NPs conferred complete decolourization in 5 min incubation and shows fine evidence toward antioxidant capacity. The significant FeO NPs antioxidant activity might be a potential materials for diverse biomedical uses.

529

Conclusion

530 531 532 533 534 535 536 537 538 539 540 541 542 543 544

The hydroalcoholic A.indica leaf extract mediated FeO Nps was successfully prepared. The analytical techniques confirmed physicochemical properties of FeO NPs such as stability, size, surface area, conductivity and magnetic properties making it as a good material for bioelectrochemical application. The decolourization of Indigo carmine dye study confirmed the photocatalytic property of synthesized FeO NPs. The antimicrobial results indicated that of FeO NPs more effective against gram ve strains than Gram +ve bacterial strains. The antioxidant efficiency was also observed to be high against DPPH. The antimicrobial and antioxidant properties are enabling FeO NPs application in the clinical sector. In conclusion, A.indica leaf extract mediated FeO Nps will significant practical application from biological to biomedical applications. Simultaneously influence of Nps toxicity is also currently underway.

9

Acknowledgments

545

The authors M.M are grateful to Department of Science and Technology (NO.SB/FT/CS-047/2012) and M.M and M.H Department of Biotechnology (BT/PR6080/GBD/27/503/2013) Government of India for the financial assistance. Also authors sincerely thank the. Dr. Jaffar Ali B.M, Centre for Green Energy Technology, Pondicherry University for VSM interpretation. A.V.Karthikeyani, Indian Oil Corporation, R&D Centre, for BET interpretation. Dr. J. Sarat Chandra Babu and G.Bhuvaneswari Department of Chemical Engineering, NIT Trichy for Particle Size analysis and ICPOES interpretation. Dr.T. Selvalakshmi, The Standard Fireworks RajaRatnam College for women, Sivakasi, for spectral interpretation.

546 547 548 549 550 551 552 553 554 555 556

Appendix A. Supplementary data

557

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jiec.2016.09.014.

558 559

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