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Biosynthesis of Novel Zinc Oxide Nanoparticles (ZnO NPs) Using Endophytic Bacteria Sphingobacterium thalpophilum Neethipathi Rajabairavi, Chellappan Soundar Raju, Chandrasekaran Karthikeyan, Kandhan Varutharaju, Shanmugam Nethaji, Abdulrahman Syedahamed Haja Hameed and Appakan Shajahan

Abstract In the present work, we describe the synthesis of zinc oxide nanoparticles (ZnO NPs) using culture supernatant of endophytic bacterial isolate Sphingobacterium thalpophilum and their antibacterial efficiency against bacterial pathogens. In the process of reduction aqueous zinc nitrate being extra-cellular which lead to the development of an easy bioprocess for synthesis of ZnO NPs. The ZnO NPs were characterized by X-ray Powder Diffraction (XRD), Field emission scanning electron microscopy (FESEM) and Energy dispersive X-ray analysis (EDAX). Ultraviolet-Visible (UV-Vis) Spectrometer and Fourier transfer infrared rays (FTIR) analysis. Antibacterial efficiency of ZnO NPs are tested with two bacterial pathogens Pseudomonas aeruginosa and Enterobacter aerogens using the disc diffusion method to determine their ability as potential antimicrobial agents. This ZnO NPs showed improved antibacterial activity on both tested strains. This was confirmed by zone of inhibition measurements.

N. Rajabairavi (&) Department of Biotechnology, Selvam Arts and Science College, Namakkal 637003, Tamil Nadu, India e-mail: [email protected] C.S. Raju Department of Botany, Vivekananda College, Tiruvedakam West, 625234 Madurai, Tamil Nadu, India C. Karthikeyan A.S.H. Hameed Department of Physics, Jamal Mohamed College, Tiruchirappalli 620 020, Tamil Nadu, India C.S. Raju K. Varutharaju A. Shajahan Department of Botany, Jamal Mohamed College, Tiruchirappalli 620020, Tamil Nadu, India S. Nethaji Department of Biochemistry, Marudupandiyar College, Thanjavur 613 403, Tamil Nadu, India © Springer International Publishing Switzerland 2017 J. Ebenezar (ed.), Recent Trends in Materials Science and Applications, Springer Proceedings in Physics 189, DOI 10.1007/978-3-319-44890-9_23

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1 Introduction Zinc oxide (ZnO) could have many applications; such as optical, piezoelectric, magnetic, and gas sensing, ceramics, rubber processing, wastewater treatment, food package and antimicrobial agent [1–3]. Zinc oxide nanoparticles (ZnO NPs) are as a generally recognized as safe (GRAS) material, nontoxic and biocompatible [4]. Several reports stated that metal oxides induce cell death and cytotoxicity [5, 6]. The size, shape, surface area, surface reactivity, its charge, chemical composition and media interactions are the unique physicochemical properties of nanoparticles. Inorganic nanoparticles structures exhibit significantly novel and improved physical, chemical, and biological properties [1]. These nanoparticles have a vast anti-potential against pathogenic microorganisms, increase with decreasing particle size [7]. Conventional methods of nanotechnology are the organic compounds as a reducing agent, lower time consumption and rapid production of nanoparticles [8]. The presence of toxic chemicals on the surface led to adverse effects [1]. The development of reliable processes for the synthesis of metal nanomaterial has its great importance in the field of nanotechnology. Biological synthesis of nanoparticles is a cost effective and eco-friendly technology [9, 10]. There are several reports in the literature on the cell-associated biosynthesis of nanoparticles using several microorganisms [8, 11]. Cell mass or their leached cell components of microorganisms reduce the size of the metal ions. Our attempt is to isolate endophytic bacteria Sphingobacterium thalpophilum from sterilized plant parts of Withania somnifera. This is subjected to extra-cellular biosynthesis of ZnO NPs. This study is the first report describing the culture supernatant of S. thalpophilum for biosynthesis of ZnO NPs and their antibacterial efficiency against pathogenic bacteria.

2 Materials and Methods 2.1

Bacterial Strains and Culture Conditions

The fresh bacterial strain S. thalpophilum (from our collection; Genbank accession: KM019199.1) was maintained on nutrient agar medium at 37 °C for 24 h. Further, the culture was subcultured into nutrient broth medium at continuous orbital shaking in on 150 rpm (LM-570RD, Yihder, Thiwan), 37 °C for 24 h. The supernatant was collected after centrifugation at 5000 rpm for 5 min in overnight bacterial culture and it is used for synthesis of ZnO NPs.

2.2

Biosynthesis of Zinc Oxide Nanoparticles

The 100 ml of cell free supernatant and 100 ml of Zinc nitrate solution (1 mM) was taken in 500 ml beaker. The beaker containing mixture solution was placed on

Biosynthesis of Novel Zinc Oxide Nanoparticles (ZnO NPs) …

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magnetic stirrer at room temperature for 24 h and it was dried at 120 °C. This dried sample was annealed at 700 °C for 5 h because the energy from the heat can enhance the vibration and diffusion of the lattice atoms for crystallization.

2.3

Characterization Techniques

The ZnO NPs were characterized by X-ray diffractometer (model: X’Pert Pro Pan alytical). The diffraction patterns were recorded in the range of 20o to 80o for the ZnO samples where the monochromatic wavelength of 1.54 Å was used. The synthesized nanoparticles retained the wurtzite hexagonal structure, which was confirmed by X-ray diffraction studies (XRD). The size and morphology of ZnO NPs were analyzed by Field emission scanning electron microscope (FESEM; model: Supra 55) with EDAX (model: Ultra 55). The excitation spectra of these samples were measured by Ultraviolet visible spectrophotometer (UV-Vis; model: Lamda 35) operated at a resolution of 1 nm. A Fourier transfer infrared rays (Nicolet FT-IR Avatar 360, America) was employed to monitor the isocyanate concentration.

2.4

Antibacterial Studies

An agar disc diffusion method for antibacterial tests was carried out using nutrient agar plate. The inoculums were prepared with nutrient broth cultures of test bacteria Pseudomonas aeruginosa and Enterobacter aerogens. The bacterial suspension was loaded on a sterile cotton swab that was rotated several times and pressed firmly against the inside wall of the tube to remove excess inoculum from the swab. The dried surface of a nutrient agar plate was inoculated by streaking the swab over the entire sterile agar surface. The discs with the synthesized of ZnO NPs (0.3 g, 11 mm diameter) were placed on the top of the inoculated plates and incubated at 37 °C for 24 h. The antibacterial activity was evaluated by measuring the zone of growth inhibition surrounding the discs. The larger diameter zone of inhibition was the greater antimicrobial activity [7].

3 Results and Discussion 3.1

Biosynthesis of Zinc Oxide Nanoparticles

The formation of ZnO NPs by the culture supernatants of S. thalpophilum was investigated. The appearance of a brown color in the reaction vessels suggested the

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Fig. 1 Solutions of ZnO before (left) and after (right) exposure to the culture supernatant of S. thalpophilum

formation of nanoparticles. In the present study before reaction, ZnO solution was colorless but its color was changed into brown after reacted with S. thalpophilum culture supernatant (Fig. 1). This result indicates the synthesis of nanoparticles.

3.2

X-ray Diffraction Analysis

The X-ray diffraction peaks of ZnO NPs synthesized using S. thalpophilum supernatant (Fig. 2). The XRD peaks are located at angles (2h) of 31.761, 34.424 and 36.26242 corresponding to (100), (002) and (101) planes of the ZnO NPs. Similarly, other peaks found at angles (2h) of 47.541, 56.61, 62.85 and 67.97 are corresponding to (102), (110), (103) and (112) planes of ZnO NPs. The standard diffraction peaks show the hexagonal wurtzite structure of ZnO NPs with the p63mc space group. This is also confirmed by the JCPDS data (Card no: 36-1451). The lattice constants ‘a’ and ‘c’ of wurtzite structure can be calculated by using the relation [12] and the formula as follows, 1 4 h2 þ hk þ k2 l2 ¼ þ d2 3 a2 c2 With the first order approximation (n = 1) for the (100) plane, the lattice constant ‘a’ is obtained through the relation a ¼ pffiffi3ksinh and lattice constant ‘c’ is derived k for the plane (002) by the relation c ¼ sinh . For ZnO NPs, values of the lattice parameters ‘a’ and ‘c’ are estimated 3.2506 and 5.2063 Å respectively. The average crystal size of the samples is calculated after appropriate background correction from X-ray line broadening of the diffraction peaks of (101) plane using Debye Scherrer’s formula [13],


(101)

Fig. 2 X-ray powder diffraction patterns of ZnO NPs

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Intensity (a.u)

(103) (200) (112) (201)

(102)

(110)

(100) (002)

ZnO

S. thalpophilum with ZnO NPs asprepared

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2 Theta (degree)

Average crystal size D ¼

kk bD cosh

Where D-is the size in nanometers, k is the wavelength of the radiation (1.5406 Å for CuKa), k is a constant (0.94), bD-is the peak width at half-maximum in radian along (101) plane and is Bragg’s diffraction angle. The ZnO NPs average particle size is 37 nm respectively.

3.3

Field Emission Scanning Electron Microscope

The FESEM images of the ZnO NPs synthesized using S. thalpophilum supernatant. From the figures, we can find that the ZnO NPs form a triangle chips-like structure with uniform grain boundary formed (Fig. 3a, b). The chips average sizes of 112 nm are found respectively.

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Fig. 3 FESEM images of ZnO NPs. a 10 KX image shows triangle chips-like structure with uniform grains of ZnO NPs X. b 50 KX image

3.4

Energy Dispersive X-ray Analysis

The compositional analysis of the ZnO synthesized using S. thalpophilum supernatant is carried out using EDAX. From the EDAX analysis, ZnO NPs atomic percentage of Zn and O are found to be 48.76 and 51.24 % respectively (Fig. 4a).

3.5

Ultraviolet Visible Spectrophotometer

The adsorption of ZnO NPs was determinate 379 nm by UV-Vis spectrophotometer (Fig. 4b). It has been found that the key factors which play important role in protein


(a)

Absorbance (a.u)

(b) 379 nm

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400

500

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700

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Wavelength (nm)

(c)

Transmittance (%)

Fig. 4 Characterization of ZnO NPs. a EDAX spectrum, b UV-spectrum and c FT-IR spectrum of synthesized ZnO NPs using S. thalpophilum culture supernatant

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4000

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Wavenumber

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(cm-1)

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adsorption on various materials are the electrostatic interaction, hydrophobic interaction and specific chemical interaction between protein and nanoparticles surface.

3.6

Fourier Transform Infrared Spectroscopy

The standard peaks of ZnO NPs around 488.34 cm−1 (Fig. 4c). This peak was attributed to the ZnO stretching frequency of Zn–O bonds. It further confirms the formation of ZnO NPs synthesized by using S. thalpophilum culture supernatant.

3.7

Antimicrobial Studies

Antibacterial activity of ZnO NPs was compared with water control and ZnO NPs with S. thalpophilum culture (Fig. 5a, b). ZnO NPs synthesized by endophytic bacteria S. thalpophilum tested against pathogenic bacteria is showed in the zone of inhibition of 14.3 mm with P. aeruginosa and 11.1 mm with E. aerogens with minimum concentration of ZnO NPs (Fig. 6). None of the effects were observed from ZnO NPs with S. thalpophilum culture and water control. The inhibition of bacterial growth by ZnO NPs could be attributed to damage of the bacterial cell membrane and extrusion of the cytoplasmic contents thereby resulting in the death of the bacterium. This depends on the nature of surface of different microbes. Sharma et al. [14] was also reported that the antimicrobial activity dependent on the concentration of the ZnO NPs and impact of the types of surfactant used. The result was concordant with Zhang et al. [15]. Similarly, reports of Sunkar and Valli Nachiyar

Fig. 5 Antibacterial efficiency of ZnO NPs against a P. aeruginosa and b E. aerogens (Note 1— ZnO NPs; 2—ZnO NPs with S. thalpophilum culture; 3—water control)


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Fig. 6 Zone inhibition of ZnO NPs on bacterial pathogens

[16] regarding antibacterial activity of nanoparticles, produced by endophytic bacterium, Bacillus cereus isolated from Garcinia xanthochymus showed zone of inhibition at higher level.

4 Conclusion In this study we demonstrated the extra-cellular synthesis of ZnO NPs by endophytic bacterial isolate S. thalpophilum for antimicrobial agent. The ZnO NPs were characterized by XRD studies, UV-Vis spectrometer, FTIR analysis, FESEM and EDAX. The antimicrobial activity of the zinc oxide nanoparticles has been examined on two common bacterial pathogens P. aeruginosa and E. aerogens. The ZnO NPs exhibits the better bactericidal activity which eradicated both bacterial species. Inorganic metal oxides may serve as effective disinfectants, due to their relatively non-toxic profile, chemical stability and efficient antibacterial activity. Zinc oxide is one of the metal oxide which has the significant bacterial growth inhibition efficiency, by catalysing the development of reactive oxygen species (ROS) from water and oxygen, which disrupt the integrity of the bacterial membrane [17]. ROS contain the most reactive hydroxyl radical (OH), the less toxic superoxide anion radical (O2−) and hydrogen peroxide with a weaker oxidizer (H2O2). This can damage DNA, cell membranes etc., leading to cell death. Generally, nanoparticles with better photocatalytic activity have a larger specific surface area and a smaller crystal size which increase oxygen vacancies, resulting in more ROS [18, 19].

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