Synthesis and Characterization of Ca, Mg, Fe and Zn Nanoparticles and its Impact on Bacillus thuringiensis 

The insecticidal bacterium Bt has been proven to be one of the most effective tool for controlling lepidopteran insect pests  including S. litura (Vimala devi  et al., 2005). It is a naturally occurring, soil-dwelling bacterium that is widely used as a biological pesticide. It produces proteins that are toxic to certain insect pests. Bt toxins have been used as topical insecticides against lepidopteran pests and many others (Vimala devi and Vineela, 2014).
       
According to Dominguez (2004), calcium (Ca) ions support the maintenance of cell motility, transport, structure and differentiation processes such as sporulation, heterocyst formation and fruiting body growth. The divalent cation Mg2+ is necessary for a number of enzymatic processes, including the regulation of membranes and ribosomes, neutralisation of nucleic acids and as a cofactor (Groisman et al., 2013). The most crucial cellular processes in which iron is involved are oxygen transport, Adenosine triphosphate (ATP) production, cell development and proliferation and detoxification (Duan et al., 2023). It is an enzyme coenzyme that catalyses the conversion of ribonucleotides to deoxyribonucleotidides, specifically the conversion of deoxyuridine to thymidine. Ribonucleotide reductase is a crucial enzyme for Deoxyribonucleic acid (DNA) production (Thelander et al., 1983). The survival of all living things depends on the metal ion Zn, which is engaged in numerous essential biological processes. They are present in every creature and are only found as parts of proteins, such as transcription factors, storage proteins and enzymes (Hood and Skaar, 2012).
       
The NPs exhibit distinctive chemical, physical and biological properties at a size of 10-9 m in diameter, as well as increased strength, strong electrical conductivity and additional chemical reactivity (Nykypanchuk et al., 2008, Sabbour, 2013, Nikita Sehgal et al., 2023, Sharma Kumar Rajesh et al., 2022, Pabitra Barik, et al., 2023). Additionally, it is predicted that the creation of nanostructured catalysts in the upcoming years will enable pesticides to be more effective at lower doses. The present investigation aimed synthesis of metal nanoparticles and its impact of nanoparticles on multiplication and growth of Bacillus thuringiensis.

The present investigation on synthesis and characterization of Ca, Mg, Fe and Zn nanoparticles (NPs) was carried out at the Department of Entomology, Sri Venkateswara Agricultural College (SVAC) and Nanotechnology Laboratory, Department of Soil Science, Institute of Frontier Technology (IFT), Tirupati. Standardization of doses of  nanomaterials with Bt (375 strain) was carried out in IFT, Regional Agricultural Research Station (RARS), Tirupati during Kharif, 2016-19.
 
Synthesis and characterization of ZnNP, FeNP, CaNP and MgNP
 
The oxides of Zn, Fe, Ca and Mg were synthesized by using sol-gel method (Wahab et al., 2007) where oxide forms of nanoparticles were used. The homogeneous sol  was produced from the precursors and converted into a gel. The solvent in the gel is then removed from the gel structure and the remaining gel is dried.
       
The properties of the dried gel depend significantly on the drying method and characterized using following methods.
 
UV–Visible spectrum       
 
Electronic transitions within the sample cause absorption in the near ultraviolet range. The apparent colour of the chemicals involved is directly impacted by the visible spectrum absorption. Understanding the electronic nature of the material’s optical band gap aided by studying the spectral patterns of UV-Vis absorption.
 
Fourier transform infrared spectroscopy (FTIR) analysis
 
The interferometer’s intensity-time output is transformed into the intensity frequency of the groups of infra-red spectrum using a Fourier Transform. FTIR produces percentage transmission and wave number as its output, the atomic arrangement and concentrations of chemical bonds found in the materials have been identified.
 
X-ray diffraction (XRD) analysis
 
XRD is used to find the structural determination of materials. The wave length of X-rays is on the atomic scale, so XRD is a primary tool for probing structure of materials.
 
Particle size and zeta potential analysis
 
Nanopartical SZ-100 was used to quantify particle size and zeta potential (HORIBA). Dynamic light scattering is a method to record the hydrodynamic diameter (HDD) of the suspended particulates, while zeta potential helps in determining the bonding of the charged particles in dispersion.
 
Transmission electron microscopy (TEM)
 
An electron beam is used in the TEM technique to image the sample’s surface, giving it a much higher resolution than is feasible with light-based imaging methods. To directly measure the size, shape, size distribution and morphology of NPs, TEM is the technique of choice. We used a JEOL 1010 transmission electron microscope with a 100 keV accelerating voltage and an AMT XR41-B 4-megapixel (2048) bottom mount CCD camera for the current study. For magnifications up to 150,000x, the camera’s finite-conjugate optical coupler offers excellent resolution, flat focus and less than 0.1% distortion (Surender and Sunithdevi (2010) and Robina et al., (2013).
 
Effect of NPs on  multiplication and growth of Bt
 
Preparation of nanosolutions
 
In order to investigate the catalytic activity of nanomaterials on the Bt, Zn, Fe, Ca and Mg at 10 ppm, 20 ppm, 50 ppm, 100 ppm and 500 ppm were added to the Luria Bertani Agar media in a 1:9 ratio (1 ml of nanoparticulate solution to 9 ml of Luria Bertani Agar (LBA) media). This was done before sterilization.
 
Inoculation of Bt
 
A single loop of bacteria (Bt strain 375) was placed into 1 ml of Luria Bertani (LB) broth and kept as such for 24 hours at 25°C. Then, the  culture was diluted 10-9 times in sterile water by serial dilution method. A total of 0.1 ml from the sample was inoculated into LBA media impregnated with metal nanoparticles and spread with an L-shaped rod and covered in glass lid nder a laminar air flow chamber. These dishes of 21 treatments with 3 replications were incubated for 24 hours at 28°C (Plate 1).


 
Colony assessment
 
The colony forming units (Cfu) of Bt were counted with the aid of a colony metre  after 24-hours of incubation as per the formular proposed by  formula (Vimala devi and Vineela 2014).
 

 

 
Statistical analysis
 
Completely Randomized Design (CRD) statistical method used to standardize the effective concentration of different NPs on Bt  cfu/g in present studies.

Characterization of CaO, MgO, FeO and ZnO nanoparticles
 
UV-Vis Spectroscopy
 
UV-Vis spectroscopy recorded the absorbance peak  of CaONPs at 340 nm, while MgONPs  at 310 nm, FeONPs at 370 nm and ZnONPs at 300 nm suggesting successful formation of nanoparticles (Fig 1 to 4).



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Fourier transform infrared (FTIR) spectroscopy
 
CaO
 
The spectrum showed the bands for the functional groups located at 3419, 2191, 1420, 680, 825 cm^-1. The peaks at 3419 and 2191represent the presence of -OH group and medial alkyne di substitution respectively. Skeletal C-C vibrations were recorded consecutively from 985 cm-1 to 877 cm^-1. The bands near 680 cm^-1 and 693 cm^-1 indicates the presence of aryl thioesters. (Fig 5).

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MgO
 
The spectrum showed the bands for the functional groups located at 2064, 713 and 1388 cm^-1. The peaks at 2064 and 1388 cm^-1. represent the presence of isothiocyanate group and trimethyl/3º butyl groups respectively. Skeletal C-C vibrations were recorded at 713 cm^-1. (Fig 6).

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FeO
 
The spectrum displays the peaks for the functional groups located at 2119, 1908, 1459, 1654 and 1131 cm^-1 while the peak at 2119 represents the cyanide / thiocyanate or related ions and 1908 denotes aromatic combination bands. Aromatic ring stretch was recorded at 1459 cm^-1, open chain imino groups and C-C vibrations were evident from the bands near 1654 cm^-1 and 1131 cm^-1 respectively (Fig 7).

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ZnO
 
The peaks in the spectrum refer to the functional groups located at 1460, 1886, 1457 and 1371 cm^-1. The peaks at 1460 and 1371 represent the presence of methyl C-H asymmetric / symmetric band whereas the peak at 1886 cm^-1. refers to the presence of transition metal carbonyls. Carbonate ion was recorded at 1457 cm^-1. band and to conclude, the change in wave number of the functional groups  might due to the synthetic protocol (Fig 8).

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X-ray diffraction (XRD) analysis
 
The CaO’s XRD pattern demonstrates that the cubic CaO can be used to identify the NPs. Their lattice parameters match the standard numbers provided in JCPDS PDF 82-1690 quite well (CaO). The diffraction intensities were recorded from 10°-80° at 2θ. Seven different and important characteristic peaks recorded at 32.2°, 35.7°, 37.3°, 48.5°, 54.5°, 64.5° and 67.3° correspond to the (111), (110), (200), (024), (202), (311) and (222) crystal planes, respectively (Fig 4.3.1). Using Scherrer’s equation, the average crystallite size (3.15 nm) was calculated from the broadenings of matching peaks:
 

                               

Where:
D= Average crystallite domain size perpendicular to the reflecting planes.
l (1.5429x10-10 )= X-ray wave length of the incident beam in nm.
θ= Diffraction angle.
b= Full width at half the maximum intensity in radians (Fig 9).

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The hexagonal phase of MgO in the synthesised substance was demonstrated by the XRD pattern. The absence of impurities in this pattern suggests that hexagonal phase MgO could be produced using the current synthetic method and all of the crystal structures complied with the JCPDS data that had been published. The Scherrer formula was used to determine the size of the crystallites and was applied to the main peaks (Fig 10).

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The examples are polycrystalline and primarily contain (100), (101) and (110) MgO, according to the XRD pattern. Within the XRD measurement range, no additional peaks could be located. It is discovered that the respective peak intensities for (100), (101) and (110) are greater than 2. (4.3.3). It has been demonstrated that polycrystalline MgO barriers with a higher percentage of the (100) textured phase produce very high values (Fig 11).

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The crystalline of the samples was analyzed by powder XRD. The diffraction profiles of  FeO NPs samples shown some disturbance peak there is only one major peak seen ( theta angle at 35.5°) showing (220) indicating the presence of FeO, 28.7° showing (111), 38.9° showing (311), 41.2° showing (400), 48.9° showing (422), 55.4° showing (511) and 66.4° showing (220).
       
All peaks were found to correspond to an FeO spinel structure and this observation is significant as it mainly consist of magnetite partially oxidized at their surface (Fig 11).
       
The prepared material contains particles in the nanoscale region, as indicated by a clear line broadening of the XRD peaks. Our study of the XRD patterns allowed us to identify the peak’s intensity, position and width as well as its full-width at half-maximum (FWHM) data. The hexagonal wurtzite phase of ZnO has been sharply enumerated as the diffraction peaks at 31.84°, 34.52°, 36.33°, 47.63°, 56.71°, 62.96°, 68.13° and 69.18° (Fig 12).

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The fact that the XRD lines have widened indicates that the material contains NPs. Some notable peaks in the XRD were taken into consideration and the associated d-values were compared to the norm [JCPDS file No. 80-0075]. The hexagonal structure of pure ZnO in the metal oxide was verified by X-ray diffraction.
 
Particle size and zeta potential analysis
 
The measured hydrodynamic diameter (HDD) of ZnO NPs was 42.7 nm and that of CaO, MgO NPs and FeO NPs were 98.5 nm, 36.8 nm and 112.2 nm respectively Greater stability of the prepared NPs is indicated by a larger zeta potential value. The measured HDD of the prepared nanoscale oxide particles (Zn, Ca, Mg and Fe) were well comparable with the measured sizes (25 nm, 50 nm, 30 nm and 80 nm) from the TEM micrographs.
       
Zeta potential of the substance is an important indicator of the stability of the respective substance. The measured zeta potentials of nanoscale ZnO, CaO, MgO and FeO were -20.1 mV, -28.9 mV, -44.7 and  -20.1mV,  respectively clearly indicated that the prepared nanoscale oxide materials were highly stable (Fig 13 to 16).

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TEM analysis
 
The micrographs of transmission electron microscope showed well defined, monodispersed nanoscale oxide particles of Ca, Fe, Mg and Zn. From the micrographs relatively spherical shaped CaO NPs were observed with the mean particle size of 60nm. Slight agglomeration of the particles was observed and is due to the absence of protective ligands on the surface of the NPs (Plate 2 to 5).



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Rod shaped and occasional spherical shaped FeO NPs monodispersity with the slight agglomeration of FeO NPs also been observed with uniform surface morphological features and with mean size of 80 nm.
       
Relatively spherical shaped MgO NPs with mean size of 50 nm. The agglomeration of the particles is due to the absence of the protective ligands on the surface.
Polydispersed spherical shaped ZnO NPs with mean size of 25 nm. The absence of the protective ligands on the surface caused the agglomeration of the NPs (Table 1).


       
The results pertaining to characterisation of nanoCaO using UV-absorbance spectrum, FTIR and XRD, DLS and TEM were closely related with Ashwini et al., (2016) and Mirghiasi et al., (2014). The synthesis and characterisation of nanoMgO was closely related to Wahab et al., (2007), Suresh et al., (2014) and Agarwal et al., (2015). The nanoFeO synthesis and characterisation results were in close conformity with Surender and Sunithadevi (2010). Similarly the nanoZnO characterisation results were in agreement with Vafaeea and Sasani (2007), Robina et al., (2013) and Jurablu et al., (2015).
 
Effect oof nanoparticles on Bt
 
The maximum cfu count (17.3x10⁹ Cfu/g) was recorded with MgO at 50 ppm concentration followed by 9.8x10⁹ with 100 ppm, 8.4x10⁹ with 500 ppm and 20 ppm and 7.8x10⁹ with 10 ppm. Similarly, 16.9x10⁹ with CaO 20 ppm, 14.3x10⁹ with 50 ppm, 8.4x10⁹ with 100 ppm, 8.1x10⁹ with 10 ppm and 7.4x10⁹ with 500 ppm. Simultaneously with ZnO the maximum number of 16.5x10⁹ Cfu/g recorded at 10 ppm, followed by 10.7x10⁹, 8.4x10⁹, 7.8x10⁹ and 7.4x10⁹ with 20 ppm, 50 ppm, 100 ppm and 500 ppm respectively.  With FeO the highest number of 15.0x10⁹ Cfu/g recorded with 50 ppm, followed by 9.1x10⁹ with 100 ppm, 8.9x10⁹ with 10 ppm, with 500 ppm, 8.3 x 10⁹ with 20 ppm, as against 6.7x10⁹ Cfu/g in control (Table 2). During 2017, the maximum number of 17.3x10⁹ Cfu/g were recorded with MgO at 50 ppm concentration followed by 16.9x10⁹ with CaO at 20 ppm, 16.5x10⁹ with ZnO at 10 ppm, 15.0x10⁹ Cfu/g  with  FeO at 50 ppm  as against 6.7x10⁹ Cfu/g in control.


       
UV absorption spectrums of the CaO nano-particles band positions at 300 and 365 nm correspond to CaO NPs. The UV spectrum of Ca NPs shows the absorbent peak rose at 340 nm. The formation of MgO NPs metal oxide in the material is indicated by the absorbance at 310 nm from UV-visible spectroscopy.
       
Prepared and annealed Fe₂O₃ NPs were achieved at wavelength near 370 nm whereas for ZnO NPs the peak oBtained at 300 nm clearly demonstrates the presence of the reaction mixture.
       
The nanoCa spectrum showed the bands for the functional groups located at 3419, 2191, 1420, 680, 825 cm^-1. The peaks at 3419 and 2191 represent the presence of –OH group and medial alkyne disubstitution respectively. Skeletal C-C vibrations were recorded consecutively from 985 cm-1 to 877 cm^-1. The bands near 680 cm-1 and 693 cm^-1 indicates the presence of aryl thioesters.
       
The nanoMgO spectrum showed the bands for the functional groups located at 2064, 713, 1388 cm^-1. The peaks at 2064 and 1388 cm-1.represent the presence of isothiocyanate group and trimethyl/3º butyl groups respectively. Skeletal C-C vibrations were recorded at 713 cm^-1.
       
The nanoFeO spectrum displays the peaks for the functional groups located at 2119, 1908, 1459, 1654, 1131 cm-1. 2119 represents the cyanide/thiocyanate or related ions at 1908 cm^-1 denotes aromatic combination bands. Aromatic ring stretch was recorded at 1459 cm^-1, open chain imino groups and C-C vibrations were evident from the bands near 1654 cm^-1 and 1131 cm^-1 respectively.
       
The nanoZnO peaks in the spectrum refer to the functional groups located at 1460, 1886, 1457, 1371 cm^-1. The peaks at 1460 and 1371 represent the presence of methyl C-H asymmetric/symmetric bend whereas the peak at 1886 cm-1. refers to the presence of transition metal carbonyls.
       
The CaO’s XRD pattern demonstrates that the cubic CaO can be used to identify the NPs. Their lattice parameters match the standard numbers listed in JCPDS PDF# 82-1690 quite well (CaO).The diffraction intensities were recorded from 10°-80° at 2è. Seven different and important characteristic peaks recorded at 32.2°, 35.7°, 37.3°, 48.5°, 54.5°, 64.5° and 67.3° correspond to the (111), (110), (200), (024), (202), (311) and (222) crystal planes, respectively.
       
XRD pattern proved that the synthesized material is MgO of hexagonal phase. The crystalline of the samples was analyzed by powder XRD. The diffraction profiles of FeO NPs samples showed some disturbance peak with only one major peak observed (220) ( theta angle at 35.5°) indicating the presence of FeO  111 at  28.7°, 311 at 38.9°, 400 at 41.2°, 422 at 48.9°, 511 at 55.4° and 220 at 66.4°.
       
All peaks were found to correspond to an FeO spinel structure and this observation is significant as it mainly consisted of magnetite partially oxidized at their surface.
       
ZnO NPs’ XRD results revealed a distinct line broadening of the XRD peaks, indicating that the prepared substance contained nanoscale-sized particles. The peak intensity, position and width from this study of the XRD patterns using full width at half-maximum (FWHM) data. It has been determined with great precision that the diffraction maxima at 31.84°, 34.52°, 36.33°, 47.63°, 56.71°, 62.96°, 68.13° and 69.18° belong to the hexagonal wurtzite phase of ZnO.
       
The measured hydrodynamic diameter (HDD) of ZnO NPs was 42.7 nm and that of CaO, MgO NPs and FeO NPs were 98.5 nm, 36.8 nm and 112.2 nm, respectively. The measured hydrodynamic diameters of the prepared nanoscale oxide particles (Zn, Ca, Mg and Fe) were well comparable with the measured sizes (25 nm, 50 nm, 30 nm and 80 nm) from the TEM micrographs.
       
Zeta potential of the substance is an important indicator of the stability of the respective substance. The measured zeta potentials of nanoscale ZnO, CaO, MgO and FeO were -20.1 mV, -28.9 mV, -44.7 and -20.1 mV respectively which clearly indicated that the prepared nanoscale oxide materials were highly stable.
       
The micrographs of transmission electron microscope showed well defined, monodispersed nanoscale oxide particles of Ca, Fe, Mg and Zn.
       
The NPs enriched media recorded maximum cfu/g was with B.thuringiensis with MgO at 50ppm, CaO at 20 ppm, ZnO at 10 ppm and FeO 50 ppm. The results are on par with Dominguez, 2004, Groisman et al., 2013, Duan et al., 2023, Hood and Skaar, 2012 who identified the Calcium (Ca), Mg2+, Zn and iron ions importance in sporulation, enzymatic processes including the regulation of membranes and ribosomes, cell development and proliferation and for the synthesis of proteins and enzymes respectively.

The synthesis of NPs viz., Mg, Ca, Fe and Zn were carried out by using sol-gel method. Characterisation of synthesized NPs was done by using UV-visible spectrum, Zeta potential analyser, FTIR, XRD and TEM.
        Characterization of NPs revealed that the particles were of 25 to 80 nm size, zeta potential of 25 to 60 mV with spherical (Cao) and rod shaped (FeO) structures, with good electronic transitions that demonstrated the presence of the reaction mixture, presence of atomic arrangement and the concentrations of the chemical bonds. Maximum cfu/g was recorded with B.thuringiensis with MgO at 50ppm, CaO at 20 ppm,  ZnO at 10 ppm and FeO 50 ppm.
 

I extend my immense thanks to S.V.AGRICULTURE COLLEGE AND IFT TIRUPATHI, ANGRAU for providing financial assistance and all other facilities for my research work.
 
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The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

There are no conflicts of interest regarding the publication of this article.