Visible light induced bactericidal and photocatalytic activity of hydrothermally synthesized BiVO4 nano-octahedrals

    Visible light induced bactericidal and photocatalytic activity of hydrothermally synthesized BiVO4 nano-octahedrals Rishabh Sharma, Uma, Sonal Singh, Ajit Verma, Manika Khanuja PII: DOI: Reference:

S1011-1344(16)30246-9 doi: 10.1016/j.jphotobiol.2016.06.035 JPB 10441

To appear in: Received date: Accepted date:

7 April 2016 10 June 2016

Please cite this article as: Rishabh Sharma, Uma, Sonal Singh, Ajit Verma, Manika Khanuja, Visible light induced bactericidal and photocatalytic activity of hydrothermally synthesized BiVO4 nano-octahedrals, (2016), doi: 10.1016/j.jphotobiol.2016.06.035

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Visible light induced bactericidal and photocatalytic activity of hydrothermally synthesized BiVO4 nano-octahedrals

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Rishabh Sharmaa; Umab; Sonal Singhc; Ajit Vermab; Manika Khanujad* [email protected] a

Amity Institute of Nanotechnology, Amity University, Uttar Pradesh, Sector-125, Noida-201303, India.

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b

Amity Institute of Microbial Technology, Amity University, Uttar Pradesh, Sector-125, Noida-201303, India.

c

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University of Petroleum and Energy Studies (UPES), VPO Bidholi, PO Prem Nagar, Dehradun 248007, India

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Centre for Nanoscience and Nanotechnology,Jamia Millia Islamia (A Central University), New Delhi – 110025, India.

Corresponding author.

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*

Abstract

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In the present work, monoclinic bismuth vanadate (m-BiVO4) nanostructures have been synthesized via simple

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hydrothermal method and employed for visible light driven antimicrobial and photocatalytic activity. Morphology (octahedral) and size (200-300 nm) of the m-BiVO4 are studied using transmission electron microscopy (TEM). The crystal structure of m-BiVO4 (monoclinic scheelite structure) is confirmed by high resolution-TEM (HRTEM) and

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X-ray diffraction (XRD) studies. The band gap of m-BiVO4 was estimated to be ca. 2.42 eV through Kubelka-Munk function F(R∞) using diffuse reflectance spectroscopy (DRS). Antimicrobial action of m-BiVO4 is anticipated by (i) shake flask method, (ii) MTT [3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-Diphenyltetrazolium Bromide] assay for cytotoxicity. SEM analysis has been carried onEscherichia coli (E.coli) before and after treatment with nanostructure materials to reveal the mechanism underlying the antimicrobial action. Antimicrobial activity is studied as a function of m-BiVO4 concentration viz. 20, 40, 60 and 80 ppm.The bacterial growth is decreased 80% to 96%, with the increase in m-BiVO4 concentration from 20 ppm to 80 ppm, respectively, in 2 hours. Photocatalytic activity and rate kinetics of m-BiVO4 nanostructures have been studied as a function of time on methylene blue (MB) dye degradation which is one of the waste products of textile industries and responsible for water pollution. Keywords: m-BiVO4, Antimicrobial, Photocatalytic, Octahedral, E coli

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1. INTRODUCTION Antimicrobial agents are of utmost importance when there is a need to fight against infectious diseases.

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However, the major problem with the use of these antimicrobial agents is the emergence of resistance which bacteria’s are gaining against these agents [1]. Resistance is most often an evolutionary processes taking place

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during, for example, antibiotic therapy, and leads to inheritable resistance. Thus, infectious diseases continue to be one of the greatest health challenges worldwide as bacteria have developed resistance against many

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common antibacterial agents. In addition, there are various adverse side effectsof these conventional antimicrobial agents [2]. Drug resistances often generate intolerable toxicity by enforcement of high-dose administration of antibiotics. This has driven the development of alternative strategies for the treatment of these bacterial

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diseases .

Taking into account several recent advancements of nanotechnology and its potential application in the

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field of medical and health sciences, nano-structured materials have emerged as one of the most promising and novel therapeutic and antimicrobial agents [3-6]. Since the synthesis and morphology[7,8](size, surface properties,

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crystal arrangement) of nano-structures plays an crucial role in deciding the efficiency of photo-assisted antimicrobial and catalytic activity of materials, several wet techniques such as sol-gel, polymeric precursor method,

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co-precipitation, hydrothermal methods have been used so far for synthesis[9-15]. Microwave-hydrothermal method is an another important route of particle synthesis[16, 17].Thus, the preparation and surface modification of nanoparticles open up possibilities for many new nanomaterials that exhibit excellent biocidal action against bacteria and other harmful organisms. Photo-induced antimicrobial activity of various metal oxide (MO) nanoparticles has been extensively investigated. Titanium dioxide(TiO2), Zinc Oxide (ZnO) and Iron(III) Oxide (alpha-Fe2O3) [18-20] are examples of a few majorly known MO nanoparticles. Organic materials are also used for antimicrobial activities in different forms such as inorganic/organic nanocompositecoating[21].They are potential candidates and show high antimicrobial as well as photocatalytic activity but owing to their large band gap can be fully utilized only in UV region (4%) of solar spectrum[22,23] and thus poses a drawback. Therefore, there is an urgent need for new materials having visible light driven (VLD) photocatalytic and photo-antimicrobial properties with high efficiency under sunlight[24]. In this direction, bismuth vanadate is emerging as a promising candidate for photocatalytic activity in visible region for various applications including organic contaminants decomposition[25], CO2 reduction [26-30], and solar to hydrogen efficiency [31]. Bismuth vanadate appears in three main crystalline phases: monoclinic, tetragonal zircon, and tetragonal scheelite [32]. Their photocatalytic performances are closely related with the phase, morphology, structure, size, band gap, surface area, crystallinity, rate of the charge transfer, efficiency of charge separation and so on [33]. Among the three crystalline phases, monoclinic BiVO 4 (m-BiVO4) is the best VLD semiconductor photocatalyst because of its efficient photocatalytic activity, high stability, low cost and non-toxicity [24].

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So far, only a few studies reports about the bactericidal action of bismuth vanadate [34-36]. In the present work, we report photo induced high antimicrobial activity of bare monoclinic-BiVO4 nanoparticles with octahedral

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morphology synthesized by hydrothermal method at 200ºC temperature without the use of any co catalyst in contrary to previous studies[35,36]. The material has also been tested for environmental remediation application

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based on its photocatalytic activity targeting organic dyes that are primarily accountable for water pollution.

2. MATERIALS AND METHODS

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2.1. Experimental

All chemicals were analytical grade and used in experimentation without any further purification. In the

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synthesis process, 6 mM (2 g) of Bi (NO3)3.5H2O (Bismuth Nitrate Pentahydrate) was dissolved in 50 ml of 2.4 M HNO3 (Nitric Acid) solution and 6 mM NH4VO3 (Ammonium Metavanadate) was dissolved in another 50 ml of 1.9 M NaOH(Sodium Hydroxide) solution. Then, 1 g of Sodium Benzene Dodecyl Benzene Sulphonate(SDBS) was

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added to both the solutions. After stirring both solutions for 30 min, they were mixed to get yellow precursor

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solution. The precursor solution was then poured to the 100 ml teflon lined stainless steel autoclave so that it occupies 80% of autoclave volume. The autoclave was heated at 200ºC for 4 hours and after that it was cooled to

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room temperature to get brilliant yellow color precipitate [37]. Precipitate was centrifuged and washed with DI water several times and was dried at 60ºC for 24 hours. 2.2. Characterization

Morphology of the samples was studied by Transmission electron microscopy (TEM, FEI Tecnai G2, 200 kV). TEM sample was prepared by placing a drop of colloidal solution of BiVO 4 on carbon coated copper grid followed by drying in air and transferred it to the microscope operated at an accelerated voltage of 200kV. The HRTEM studies provide the interplanar spacing confirming monoclinic phase of BiVO 4. The Phase structure of hydrothermally synthesized Bismuth Vanadate nanopowder was studied by X-Ray Diffraction taken using Philips X’Pert PRO-PW 3040. Samples were scanned from 20° to 80º with a glancing angle of 1º. Elemental composition of sample was carried by energy dispersive spectroscopy (EDS). UV-Vis spectra were recorded by using Shimadzu UV 2600 in the diffuse reflectance mode (R) and transformed to a magnitude proportional to the extinction coefficient (K) through the Kubelka-Munk function, F (R∞). Samples were mixed with BaSO4 that does not absorb in the UVVis radiation range (white standard). Scanning range was 250 - 800 nm with step size of 0.5 nm.

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2.3. ANTIBACTERIAL ASSAYS TO DETERMINE MICROBIAL TOXICITY

Shake Flask method

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2.3.1.

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The antibacterial activity of m-BiVO4 nanoparticles, were assessed using following analytical techniques:

Escherichia coli ATCC 25922 was obtained from Department of Microbiology, Rajiv Gandhi Cancer Research

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Institute, Delhi, India. The test strain were maintained on nutrient agar slants at 4° and subcultured on to nutrient broth for 24 h prior to testing. This bacteria served as test pathogen for antibacterial activity assay. The effect of bismuth vanadate nanoparticles on Gram-negative Escherichia coli was investigated by examining the bacterial cells

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in liquid medium treated with bismuth vanadate nanoparticles. Nutrient Broth (NB, 100 mL) was inoculated with fresh colonies of the Escherichia coli growing on the liquid medium. Culture broth was incubated in aerobic

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conditions at 37ºC for 24 h under constant rotary shaking in a shaking incubator. Subsequently, an aliquot from above was added to 100 mL NB broth embedded with 20, 40, 60, 80 ppm of nanoparticles under visible light irradiation. NB broth without bismuth vanadate nanoparticles was used as a control. Bacterial growth was

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2.3.2 MTT Assay

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determined by measuring the optical density at 600 nm after fixed time interval of 2 h.

Cytotoxic effect of m-BiVO4nanoparticles was determined by 3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-

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Diphenyltetrazolium Bromide (MTT) assay. Nutrient broth was freshly prepared and inoculated with fresh Escherichia coli culture and allowed to grow for 24 h in shaking incubator at 37 ºC.

BiVO4 of different

concentrations of 20, 40 and 60 ppm was dissolved in freshly inoculated E.coli culture. 0.5 ml of the MTT was added in 5 ml of BiVO4 treated E.coli culture and control (E.coli culture without any treatment). Samples were kept in the incubator at 37°C for 4 h followed by gentle shaking. A tetrazolium salt was enzymatically reduced by NADPH- dependent cellular oxidoreductase present in living cells. The reduction product was water insoluble formazan that was prepared by addition of isopropanol in 0.1 N HCl to make up the volume to 125 ml and then, added 10% triton in it. After 4 h of incubation, 5 ml of reduction product was added in BiVO4 treated E.coli culture to dissolve crystals of formazan. The absorbance of BiVO4 treated E.coli culture was measured at 600 nm with respect to control. Cell viability was determined by (1) (2) Where, At is the absorbance of treated sample and Ac is the absorbance of control

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2.4. SCANNING ELECTRON MICROSCOPY

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SEM (EVO 18 Special edition, ZEISS) analysis had been carried on Gram negative E.coli before and after treated with BiVO4 nanoparticles. The culture of E.coli was grown overnight in NB medium at 37 ± 2°C. BiVO 4 sample (60 ppm) was added to the culture, and the mixture was incubated at 37°C ± 2°C for 2 h with continuous

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shaking. The bacteria cell suspension was given washing with 0.1 M PBS buffer solution (PH 7.4) to remove

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residues of glutaraldehyde and afterwards with distilled water which was further isolated by centrifugation. The samples were then fixed in 2.5% glutaraldehyde at 4ºC for 2 h, and finally added drop-wise on the cover slip and kept for drying. The E.coli culture without BiVO4 treatment was served as control. The bacterial cells coated with

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a thin gold film (<10 nm).

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2.5. PHOTOCATALYTIC STUDIES

Photocatalytic efficiency of m-BiVO4nanoparticles were measured by the degradation of methylene blue (MB) in water as a function of time. 5 mg of BiVO 4 nanoparticles as a photocatalyst were added in 100 ml of 10 µM

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aqueous solution of MB. Solution was ultra-sonicated for 30 mins followed by 2 hours stirring in dark to remove agglomeration, resulting in uniform distribution of nanoparticles in MB solution. Concentration of MB in water was

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estimated after every 15 minutes by recording absorption spectra using UV-Vis Spectroscopy. Photocatalytic

Where

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efficiency of BiVO4 was measured using following relation: (3)

initial absorbance of MB before visible light irradiation and C is absorbance of MB at time t simulated

under AM 1.5 solar illuminations 100 mW/cm2 from xenon arc lamp in ambient atmosphere

3.

RESULTS & DISCUSSION

Fig. 1 (a) shows the TEM micrograph of BiVO4 nanostructures. As revealed from the micrograph, BiVO4nanoparticles are of octahedral shape of size 200-300 nm. Fig. 1 (b) shows the HRTEM micrograph and inset shows the interplanar spacing of 0.296 nm corresponding to (004) plane as confirmed by JCPDS File number (140688). Fig. 2 shows the XRD diffractogram of the pure BiVO 4. The observed diffraction peaks are in good match with the monoclinic scheelite BiVO4 (m- BiVO4) phase, as also confirmed by JCPDS file number (14-0688). Fig.3 shows the EDS studies and elemental composition analysis as shown in the Fig.3 inset.EDS spectrum shows the presence of Bi, V, and O elements in the sample. As synthesized BiVO4 is in good stoichiometric ratio as can be observed from atomic percent of Bismuth, Vanadium and Oxygen (1:1:~4) in EDS elemental composition Table.

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The diffuse reflectance of the samples is shown in the Fig. 4. Barium sulphate was used as a standard. In the sample, strong absorption in the visible range (500-800 nm) is observed. The acquired diffuse reflectance is converted to

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Kubelka-Munk function F(R∞) for optical band gap calculations. Kubelka-Munk function is defined as

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, where, R is the reflectance of the material.

Using the Kubelka-Munk function, the [hυF(R∞)] 2 was plotted against hυ.A line tangent was drawn to the

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plotted curve inflection point with the horizontal (hυ) axis. The point of intersection of the tangent and the horizontal axis gives the band gap (Eg) value. The band gap was found to be 2.42 eV for sample. The observed band gap values of m-BiVO4 sample are in good match with the band gap of m-BiVO4 phase i.e 2.4 eV [38]. Thus, synthesized m-

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BiVO4 sample can effectively show VLD photocatalytic activity. 3.

Changes in the morphological structure of E.coli cells after treatment

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with m-BiVO4

the bacterial strain. SEM micrograph of E.coli in control, seemed to retain their rod shaped morphology with intact and well preserved cell walls and membranes as shown in Fig. 5 (a) whereas m-BiVO4 nanoparticles

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(b)

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SEM analysis of the prepared bacterial samples was carried out showing the changes in morphological structure of

treated E.coli cells as shown in Fig. 5 (b) shows the dead aggregated colonies. In addition, aberrant morphology of the treated cells was accompanied by complete cellular degradation. The treated E.coli cells undergo lysis and finally get disrupted, resulting in the release of their cytoplasmic contents into their surrounding environment. 3.1. Growth of Bacterial cells exposed to different concentrations of m-BiVO4 nanoparticles Antimicrobial action of m-BiVO4 nanoparticles against E.coli was studied as a function of m-BiVO4 concentration using shake flask method as shown in Fig. 6. Antimicrobial efficacy (X) is determined by: (4) Where Y is the absorbance of E.coli culture without nanoparticle and Z is the absorbance of the E.coli culture with nanoparticle. Time versus absorbance graph was plotted to determine the growth reduction in microbial activity with different concentrations of nanoparticle. Table I summarizes the antibacterial activity of m-BiVO4 nanoparticle. It can be inferred from the Fig. 6 and Table I that the growth of bacterial cells in presence of m-BiVO4 nanoparticles was lower than that of cells in the control, indicating that m-BiVO4 nanoparticles could inhibit the growth of bacterial cell.In 2 h, the bacterial growth is

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attributed due to an increase in the bacterial membrane permeability for the entry of m-BiVO4nanoparticles of octahedral shape with abrasive texture, which causes disorganization of the membrane and changes occurs at the

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protein level. This eventually leads to inhibition of cellular metabolism leading to bacterial cell death. The toxicity of m-BiVO4 nanoparticles against the bacterial species is heightened due to the persistent contact of the

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nanoparticles with the bacterial cellular membrane[39].The toxic effect of m-BiVO4 nanoparticles on bacterial cell death is due to the photo generated reactive oxidative species such as •OHads, H2O2 and •HO2/•O2–[40]. Also, the

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hydrothermal synthesis at 200°C resulted in high crystalline octahedral shaped nanoparticles which offer high surface areaand better stabilitywhich further contributes to photo-inactivation of bacteria [41, 42].

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3.2. MTT

E.coli cells were exposed to Bismuth vanadate nanoparticles at the concentration of 20, 40, and 60 ppm for 24 h and cytotoxicity were determined by MTT assay. As the concentration of nanoparticles increased to 20 to 60 ppm, in

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MTT assay, cell viability dropped drastically to 28.4% to 12.2% respectively ((Fig. 7 and Table II). Thus, the

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cytotoxicity assays had shown that the m-BiVO4 nanoparticles could produce significant cytotoxicity to cells.Furthermore, Bismuth vanadate nanoparticles at the concentration of 60 ppm showed 88% cytotoxicity to E.colicells. The cytotoxicity induced by Bismuth vanadate nanoparticles is attributed to the direct interaction of

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nanoparticle with the E.coli cells [39].

3.3. Photocatalysis

In Photocatalytic process, when photocatalytic material was illuminated by photons with energy more than the band gap energy of that material than electrons and hole pairs are generated in conduction and valence bands, respectively due to the migration of electrons from valence band to conduction band after absorbing energy from the incident photon. If these electrons and holes pair have suitable energy they ionizes water to OH and O free radicals which act as a powerful agents to oxidize dyes [23]. Photocatalytic degradation of methylene blue without and with m-BiVO4photocatalyst for different visible light irradiation times (15, 30, 45, 60, 75, 90, 105, 120 and 135 minutes) is as shown in the Fig. 8 (a) and Fig. 8 (b), respectively. The intensity of absorbance peak of MB at 664 nm wavelength in UV-Vis spectrum was monitored at above given time interval to calculate degradation percentage with and without m-BiVO4. Degradation efficiency (η) of MB without photocatalyst is: 6%, 11%, 16%, 20%, 23%, 27%, 31%, and 33% and with photocatalyst is: 17%, 27%, 40%, 45%, 51%, 56%, and 62%. After 135 minutes it was observed that addition of m-BiVO4 photocatalyst resulted in approximately twice increase in degradation percent of MB. Study of the photo-degradation kinetics of

ACCEPTED MANUSCRIPT methylene blue (MB) catalyzed by m-BiVO4 nanostructures are shown in Fig.9. The photocatalytic activity follows the pseudo first order kinetics and is given by:

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(5)

(6)

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Alternatively,

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Where, C0 is the initial concentration; Ct is the concentration after time t and k is the rate constant of pseudo first order reaction[23]. A linear relationship between ln (C o/Ct) and time (t) was observed. The first order degradation rate constants of MB without photocatalyst and with m-BiVO4photocatalyst are found to be k 0.00281 min and

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MB and

k(MB +BiVO4) =

0.0073 min , respectively. Thus, degradation rate of MB by m-BiVO4 photocatalyst is

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-1

approximately three times faster as compared to without photocatalyst.

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3.5. Mechanism of action`

Bismuth vanadate nanoparticle was used as an effective VLD photocatalyst for inactivation of Escherichia coli as

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shown in the schematic diagram Fig. 10. The mechanism of the VLD photocatalytic bacterial inactivation by mBiVO4 did not allow any bacterial regrowth. The photo generated H+ and reactive oxidative species derived from

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H+, such as •OHads, H2O2 and •HO2/•O2, were the major reactive species for bacterial inactivation [23, 43]. The inactivation by H+ and •OHads required close contact between the m-BiVO4 nanoparticles and bacterial cells, and

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only a limited amount of H2O2 could diffuse into the solution to inactivate bacterial cells exposed to m-BiVO4.

Electron hole pairs are generated in photocatalyst when illuminated with light radiation as shown in equation 7.

Electrons in conduction band are absorbed by free oxygen molecules adsorbed on photocatalyst surface producing super oxide radicals

. These super oxide radicals and hydroxyl ions

produced by water redox reaction lose

their electrons to the holes present in valence band of photocatalyst thus producing reactive species

Super oxide ions react with free H+ ionsandH2O to produce

and H2O2 reactive species.

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Reactive oxygen species formed above results in degradation of dye

The inactivation and decomposition of bacterial cells might be due to attack from photogenerated H 2O2. The

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destruction of the cell envelope (i.e., cell wall and cell membrane) and the leakage of intracellular macromolecules

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may be caused by extracellular reactive oxygen species (ROSs), such as H 2O2, which penetrates into the bacterial cell and cause dramatically elevated intracellular ROSs levels [23,43,44]. The leaked nucleic acids and proteins from bacterial cells are believed to be segmented or oxidatively damaged and further attacked by intracellular and

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extracellular produced ROSs, leading to degradation and complete mineralization. Also m-BiVO4 nanoparticles penetrates into the bacterial membrane leading to inactivation of enzymes related to respiratory system and

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enhances the outflow of cytoplasmic contents, which results in damages to its membrane and ultimately kills the bacteria [40-44].

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4. CONCLUSION

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In the present work, monoclinic BiVO4 octahedral nanostructures have been successfully synthesized via hydrothermal method. In particular, the m-BiVO4 nanostructures for the first time demonstrate the multifunctional property of bacteria inactivation and photocatalysis.They display outstanding photocatalytic performance under

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visible light irradiation for degradation of methylene blue (MB) in aqueous solution, as well as enhanced photo induced antimicrobial activity towards E. coli. Shake flask method had shown that the growth of bacterial cells decreased 80% to 100% with the increase in m-BiVO4 nanoparticle concentration from 20 ppm to 80 ppm in the duration of 2 hours. The photodegradation rate of MB was enhanced upto 3 times in the presence of m-BiVO4 nanoparticles as catalysts in comparison to pure MB. Therefore, it is anticipated that the m-BiVO4 nanostructures will lead to many possibilities for creating more novel nano-dimensional bismuth containing compounds with multiple functionalities.

ACKNOWLEDGEMENT The authors are thankful to Prof. B. R. Mehta from Indian Institute of Technology Delhi, New Delhi India, for providing all necessary research facilities during this research work.

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BiVO4 Concentration 80 ppm 96.5 99.9 99.9 100

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Antibacterial Activity (%) Time control (h) 20 ppm 40 ppm 0 0 2 0 80.9 4 0 84.9 6 0 92.5 8 0 94.2

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Table II. Cell viability (%) and cell cytotoxicity (%) of BiVO4 nanostructures against E.coli

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Synthesis and characterization (structural confirmation by XRD and HRTEM, band gap by K-M function using diffuse reflectance spectroscopy) of monoclinic BiVO4 nanostructures. First time monoclinic BiVO4 nanostructures are used for antimicrobial activity using (a) Shake flask method, (b) MTT Assay, (c) SEM analysis showing antimicrobial action in E.coli before and after treatment with BiVO4. Photocatalytic activity and rate kinetics of m- BiVO4 as a function of time is carried on methylene blue (MB) dye. Mechanisms that underlie the antimicrobial and photocatalytic activity are elucidated in the present work.

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ACCEPTED MANUSCRIPT Highlights of Work: Synthesis of m-BiVO4 by hydrothermal method

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Characterization by TEM, HRTEM, XRD and DRS

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Antimicrobial Activity: Shake Flask, MTT assay and SEM analysis

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Photocatalytic activity and rate kinetics study

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Mechanism elucidating antimicrobial and photocatalytic activity

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