Abstract: Background and Objective: Nanoparticles with a little size to an enormous surface (1-100 nm) have expected clinical, mechanical and agricultural applications. This study aimed to produce nano Zinc Oxide (ZnO) and nano Copper Oxide (CuO) particles by green synthesis. Materials and Methods: Two strains of Pseudomonas fluorescens i.e., PSI and PSII, both cell culture supernatants and cell pellets from the two strains were examined separately in CuSO4 or ZnSO4 solutions. The supernatants from both strains produced color changes in both solutions referring to the formation of nano CuO or ZnO particles. The solutions were examined for nano-particle characteristics using UV-spectroscopy, particle size and morphology were tested using a scanning electron microscope and transmission electron microscopy. Results: UV-Vis absorption spectrum of solutions at a wavelength range 200-800 nm exhibits a distinct absorption peak in the region of 238-331 and at 303-366 nm for CuO or ZnO NPs, respectively. Absorption bands and the characteristic Surface Plasmon Resonance (SPR) spectra confirm the existence of CuO and ZnO NPs. SEM analysis micrographs indicated that CuO NPs were formed as spherical particles, while the exact shape of ZnO NPs could be identified as oval aggregates. Conclusion: Changes of color occurred in both solutions of two strains referring to the formation of nano CuO or ZnO particles.
INTRODUCTION
Bio-nanotechnology is a combination of biological science and nanotechnology dependent on the standards and substance pathways of living organisms1,2. The biological method with many benefits is preferred to other methods for the synthesis of non-toxic, cheap nanoparticles (NPs) and eco-friendly (green chemistry concept) products3-5. Green nanotechnology has been adopted to enhance the environmental sustainability of nanomaterial products6,7 was one of the earliest studies on the production of nanoparticles by microorganisms (bacteria). Therefore, nowadays production of metallic nanoparticles has been more deeply investigated, due to its competitiveness, effectiveness and low operational cost8.
The production of metallic nanoparticles by microorganisms is relatively a novel approach. There is a wide variation in the production of metallic nanoparticles by living cells (e.g., organelles and compounds responsible for production, shape and size of nanoparticles), which depends on the mechanisms of metal ions bioreduction9,10. Metallic nanoparticle producers (living cells) have unique characteristics, these metallic nanoparticles are produced by both intra and extracellular bio-compounds9. Usually, the production of metallic nanoparticles by bacteria occurs during the stationary phase. In theory, when compared to the logarithmic phase, greater metabolic stress is observed during the stationary phase10. Thus, these metabolites can reduce metal ions, which lead to the production of metallic nanoparticles3,11,12.
In this concern, different bacteria have different mechanisms for the synthesis of NPs. Despite major differences in a mechanism, NPs are usually formed by detoxification/reduction mechanism of the metal ions to less toxic metal salts, metal-ion efflux system and decreases in the membrane permeability (soluble metals (insoluble nanosized structures)), since ions lead to change in the helical structure by cross-linking and, consequently, to many biochemical pathways reported an easy, fast and cost-effective production of copper NPs by the non-pathogenic bacteria Pseudomonas stutzeri13-16. The delivered copper nanoparticles indicated extraordinary strength. Along these lines, the metabolites from P. stutzeri created copper nanoparticles other than settling them. Generally, the metabolic process may be responsible for the bio-reduction of NPs in the living bacteria. However, for dead entities, metal and metalloid ions are bound to the bacteria cells that provide nucleation sites for NPs17. The primary requirements of green synthesis of nanoparticles are metal ion solution and a reducing biological agent. In most cases, reducing agents or other constituents present in the cells act as stabilizing and capping agents, so there is no need to add capping and stabilizing agents from outside5.
Bacteria possess a remarkable ability to reduce heavy metal ions and one of the best candidates for nanoparticle synthesis. For instance, some bacterial species have developed the ability to resort to specific defense mechanisms to quell stresses like the toxicity of heavy metal ions or metals. It was observed that some of Pseudomonas sp. could survive and grow even at high metal ion concentrations18,19.
The present study was an attempt to produce nano CuO or ZnO particles in a green approach by two strains of Pseudomonas fluorescens and to study the physical and chemical characteristics of the produced nanomaterials.
MATERIALS AND METHODS
Study area: The study was carried out at the Department of Microbiology and Complex Research Laboratories from the University of Cairo, Faculty of Agriculture (Cairo University Research Park, CURP), Egypt from June, 2019-February, 2020.
Bacterial strains collection: Two bacterial strains of P. fluorescens PSI and PSII namely, kindly supplied by the Department of Microbiology, Soil, Water and Environmental Research Institute, Agriculture Research Center (ARC), Egypt were used in this study for synthesizing nano-Zinc and Copper. The bacterial strains were grown in the specific medium F-Base (Kings B medium) for pseudomonas containing (g L–1): Peptone, 20, dipotassium hydrogen phosphate, 1.5, magnesium sulphate, 1.5, Agar, 15, glycerol, 10 mL and pH was adjusted at 7.2. using pH meter (pH 211 Microprocessor, Hanna Instruments, Rhode Island, USA). An inoculum of 1×10–3 dm3 was transferred to 50×10–3 dm3, F- Base media and maintained at 30°C on a shaker incubator (Sartorius Stedim Biotech, Aubagne, France) at 150 rpm, according to Shantkriti and Rani20. All chemicals were purchased from Algomhoria Company, Cairo, Egypt.
Biosynthesis of CuO and ZnO NPs by P. fluorescens strains: A 48-old culture of either strain in an F-base medium was centrifuged at 5000 rpm for 30 min at 30°C (High Speed laboratory centrifuge, YINGTAI INSTRUMENT, Shenzhen, China) where the cell pellet as well as cell-free culture supernatant, was recovered and independently suspended in 40 mL of 1000 ppm CuSO4 or ZnSO4 solutions then incubated for 24-48 hrs at 30°C on a shaker incubator (Sartorius Stedim Biotech, Aubagne, France) at 150 rpm and examined for production of CuO or ZnO NPs. The suspensions were subjected to the following tests for characterization of the produced Nps.
Characterization of CuO and ZnO Nps: Formation of bio CuO and ZnO NPs was firstly observed by visual color change. Further UV-Vis spectroscopy, SEM and TEM techniques were applied in the Research Park of the Faculty of Agriculture, Cairo University, Egypt. The reduction of metal in the prepared mixtures was monitored by Ultraviolet-Visible (UV-Vis) spectral analysis from 200-800 nm using UV-Vis spectrophotometer (Thermo Scientific HERYIOSγ, UV-Vis, USA) ("Thermo Scientific HERYIOSγ"). The suspensions were dried and subjected to Scanning Electron Microscopy (SEM) using a JEOL MODEL a JEOL MODEL JSM 6360 SEM, Tokyo,Japan, JSM 6360 SEM, samples surface was observed at a voltage of 25 kV with different magnifications. Morphology and size of NPs were examined by Transmission Electron Microscopy (TEM) onto an amorphous carbon-coated copper grid, dried and analyzed using a Jeol JEM 2100 TEM operated at a voltage of 80 kV (Jeol JEM 2100 TEM, Tokyo,Japan).
Determination of Cu or Zn NPs concentration: The concentrations of Cu or Zn NPs in samples were determined by using Inductively Coupled Plasma (ICP-AES), (Thermo Sci, model: iCAP6000 series, USA), Thermo Sci, model: iCAP6000 series, in the Research park of Faculty of Agriculture, Cairo University, Egypt. Advanced microwave digestion system was used for digestion of samples by Milestone (Milestone ETHOS lab station, Seoul, Korea) ETHOS lab station with easy wave or easy control software HPR1000/10S high pressure-segment rotor. Argon gas was used for the excitation of the element atom. The blank values for each element were deduced from the sample’s values.
RESULTS AND DISCUSSION
Visual identification: The cell-free supernatants of both examined culture strains P. fluorescens were added separately to CuSO4 or ZnSO4 solutions and incubated for 48 hrs, the reaction mixtures color changed to dark green or brownish yellow (Fig. 1a) indicating the formation and oxidation of CuO or ZnO NPs, respectively. The visual color change of the culture suspension is a tentative evidence for nanoparticles synthesis21-23. Previous works considered color change of cell free supernatant after addition of 5 mM CuSO4 or ZnNO3 solution, respectively as evidence on nanoparticle production by Morganella sp. and Aspergillus niger24,25. Addition of various concentrations of CuSO4 or ZnSO4 solutions to cell pellets did not reveal any color change (Fig. 1b). Colour change of media reflects physical indication of nanoparticle formation and it was further verified using UV visible spectrum15.
| Fig. 1(a-b): | Visual Identification of CuO and ZnO NPs Color changes in the supernatant (a) and No color change for pellet (b) |
| Fig. 2(a-b): | Distinct absorption peak of (a) CuO and (b) ZnO NPs for 2 strains of P. fluorescens |
The color changes of the culture because of Localized Surface Plasmon Resonance (LSPR). LSPR is a typical phenomenon that is considered the feedback of NPs formation26. The coherent oscillation of electron gas at the surface of NPs is the origin of LSPR, which was generated by a coupling between the conduction electrons oscillation modes and the incident electromagnetic radiation and leading to different colors and the intensity at a specific absorption wavelength in the UV-Vis spectrum is enhanced27-30.
UV-Vis Spectroscopy: The biosynthesis of NPs was monitored by optical measurements. The UV-Vis absorption spectrum of these solutions was screened at wavelength range 200-800 nm and exhibits a distinct absorption peak in the region of 238-331 nm (Fig. 2a) and at 303-366 nm (Fig. 2b) for CuO or ZnO NPs, respectively. There are 2 strong peaks for strain PSII at 326 and 331 nm and 321 and 324 nm for Cu and Zn oxide nanoparticles, respectively. Moreover, in the visible range at 400-800 nm did not find any peaks. Absorption bands and the characteristic Surface Plasmon Resonance (SPR) spectra as shown confirms the existence of CuO and ZnO NPs in cultures11,20,31. The exact position of the SPR band may shift depending on individual particle properties including size, shape and capping agents32. Another study of Gao et al.33 showed the color change of A. paniculata extract treated with Zinc nitrate might be because of vibrations in surface plasmon resonance. This result correlates with the already reported results, in which Zinc oxide absorption peak was found at 350 and 360 nm, also specified the UV spectrum range of ZnO was measured at 380 nm34-36. Like this, the study examined the UV spectrum range of ZnO is 320-390 nm37,25.
| Fig. 3(a-b): | SEM of (a) CuO and (b) ZnO NPs for 2 strains of P. fluorescens |
Biplab et al.38 showed the UV-Visible absorption spectrum of the as-synthesized copper oxide nanoparticles was a strong absorption peak positioned at a wavelength of 258 nm and was features of the surface plasmon resonance of nano-sized particles39. Berra et al.29 revealed that spectra of copper oxide nanoparticles synthesized at a strong peak at about 275 nm. The presence of this assimilation pinnacle can be ascribed to the presence of the Cuprous Oxide (Cu2O) period of the CONPs. Essentially, the presence of an assimilation top for cuprous oxide has just been accounted by Abboud et al.14. Another broad absorption peak centered at 553-620 nm40-44. The presence of the Copper Oxide (CuO) stage in the incorporated CONPs is answerable for this expansive retention top. Subsequently, the UV-noticeable retention range of the copper oxide nanoparticles additionally affirms the conjunction of both the CuO and Cu2O stages in the CONPs14,38. The cell-free filtrate of B. cereus SWSD1 was incubated with CuSO4 solution at optimum conditions and SPR of synthesized NPs were observed in two peaks in different regions of the UV-Vis spectrum: one in the visible range at 570-630 nm and the other intensity peak in the ultraviolet range at 300-350 nm than that in the visible range22.
SEM and TEM: SEM analysis micrographs at (Fig. 3a) indicated that CuO NPs were formed as spherical particles, while (Fig. 3b) the exact shape of ZnO NPs could be identified as oval aggregates. The appearance of elements was may be due to media components or other biomolecules secreted by the bacteria. TEM analysis provided further insight into the shape, size and distribution of CuO and ZnO NPs. TEM images of copper nanoparticles (Fig. 4a) obtained after 48 hrs of the reaction of cell-free supernatant with CuSO4, the particle size ranged between 7.92-18.5 and 2.67-10.0 nm for strain 1 and 2, respectively, with the presence of spherical nanoparticles. While ZnO NPs (Fig. 4b) appears as scattered particles with tiny shape ranged from 19.3-33.0 and 2.29-4.14 nm for strain 1 and 2, respectively. The results referring that these particles are polydispersity with variable diameter ranged from 2.29-33.0 nm of biosynthesis by strains of P. fluorescens and range at 2.67-18.5 and 2.29-33.0 nm for CuO and ZnO NPs, respectively and these particles uniformly distributed without any agglomeration. According to these results, strain 2 was most efficient for biosynthesis very tiny nanoparticles ranged from 2.29-10.0 nm.
| Fig. 4(a-b): | TEM and SAED ring pattern of (a) CuO and (b) ZnO NPs for 2 strains of P. fluorescens |
To the best of our knowledge, the first report of biosynthesis of ZnO NPs by bacteria was using Aeromonas hydrophila as an eco-friendly reducing and capping agent, Jayaseelan et al.45 showed the morphology of the NPs to be oval with an average size of 57.72 nm. Mirhendi et al.46 reported the production of magnetite-ZnO NPs, along with Zn and ZnS, with 16.35 and 22 nm in size on the surface of Pseudomonas stutzeri and Brevundimonas diminuta cells, respectively. While Singh et al.47 examine ZnO nanoparticles biosynthesis by Pseudomonas aeruginosa was Spherical shape with 35-80 nm in size. Emad et al.48 also examined the practices and morphology of ZnO NPs and found that the size is spherical in shape and the size range is 41-75 nm. Radhika and Suman49 and Ghashghaei and Emtiazi50 showed that P. Fluorescens after 48 hrs, NPs was occasional aggregation and spherical with 50-70 nm).
Moreover, Varshney et al.16 also characterized copper nanoparticles synthesized by Pseudomonas stutzeri with CuSO4 source as spherical shape and 8-15 nm in diameter. Gargi et al.40 confirms the spherical shape of Cu NPs and the average size was 5.7±1.8 nm. Ashutosh, K. S. and Siavash5 and Siavash18 revealed the size ranges from 8-140 nm and spherical shape of CuNPs. Ismail44 synthesized CuNPs were spherical and the sizes were in the range of 7-24 nm. This confirms the hypothesis that the extract work as a capping agent to acquire the dominant fashioning of spherical CuNPs. TEM analysis revealed the presence of widely dispersed copper nanoparticles of a size range of 14-50 nm (Rajesh et al.51) and 40-80 nm (Nazar et al.43). CuNPs were found to be spherical shaped according to findings by Prabhu et al.52 and Gu et al.53. Bacillus cereus mediated CuNPs synthesis was synthesized to be 50 nm and the presence of spherical-shaped particles42. The size and shape of the synthesized nanoparticles revealed that they are spherical with a size ranging from 28-45 nm.
Nanoparticles are uniform in shape and are polycrystalline when examined under SAED pattern54. Selected Area Electron Diffraction (SAED) pattern (Fig. 4) of formed nanoparticles confirmed that Cu and Zn NPs were formed and SAED spot confirms the presence of planes. SAED analysis of nanoparticles clearly shows the crystalline nature of the nanoparticles. Due to the presence of proteins surrounding the particles, the SAED showed characteristics like that found in amorphous materials, which suggests the possibility of these agents acting as capping agents55. It is revealed that the average particle size of the produced Cu and Zn were in the nano-size range. As seen in TEM-EDS confirmed that copper and zinc are present in a sample compared to the grid. The diffraction rings correlate to Copper and Zinc.
Determine the elemental concentration: Elements concentration was determined by ICP-AES for further future prospective biostudies, which Cu concentration was 1092 and 1649 ppm, while Zn was 773 and 1158 ppm for strain 1and 2, respectively. These results confirmed that, strain 2 was most efficient for biosynthesis Cu and Zn NPs.
CONCLUSION
Summing up, results indicated that copper oxide and zinc oxide could be produced as nanoparticles using bacteria. Data of microbiological studies proved that two strains of Pseudomonas fluorescens can convert copper and zinc from inorganic form to nano form by high concentration and that effect appeared in changes colors in media of bacteria.
SIGNIFICANCE STATEMENT
This study discovers the possibility to produce copper oxide and zinc oxide as nanoparticles using bacteria. This way considers eco-friendly and more efficient because bacteria produce elements in an effective form. Thus, the elements produced by bacteria will be used as an alternative source in animal feeding to replace the inorganic source of zinc and copper in feed premixes.