Phase variation of manganese oxide in the MnO@ZnO nanocomposite with calcination temperature and its effect on structural and biological activities

Having powerful antibacterial and antioxidant effects, zinc oxide and manganese oxide nanomaterials are of great interest. Here we have synthesized manganese oxide decorated zinc oxide (MZO) nanocomposites by co-precipitation method, calcined at different temperatures (300–750 °C) and studied various properties. Here the crystalline structure of the nanocomposite and phase change of the manganese oxide are observed with calcination temperature. The average crystalline size increases and the dislocation density and microstrain decrease with the increase in calcined temperature for the same structural features. The formation of composites was confirmed by XRD pattern and SEM images. EDAX spectra proved the high purity of the composites. Here, different biological properties change with the calcination temperature for different shapes, sizes and structures of the nanocomposite. Nanomaterial calcined at 750 °C provides the best anti-microbial activity against Escherichia coli, Salmonella typhimurium, Shigella flexneri (gram-negative), Bacillus subtilis and Bacillus megaterium (gram-positive) bacterial strain at 300 µg/mL concentration. The nanomaterial with calcination temperatures of 300 °C and 450 °C provided better antioxidant properties.

6H 2 O was added to 200 mL distilled water and stirred at 545 rpm for 40 min.50 mL homogeneous solution of manganese sulfate (3.0003 g MnSO 4 ⋅H 2 O in 50 mL distilled water and stirred for 20 min) was added dropwise to the solution and stirred for 20 min.Then 10 mL ethylene glycol is added and stirred for another 20 min.The pH of the solution was adjusted to ~ 9 using NaOH solution.Then the total solution was stirred for 2 h and dried to get nanopowder.After that, it was calcined at 300 °C, 450 °C, 600 °C, and 750 °C to get MZ 1, MZ 2, MZ 3 and MZ 4 respectively.

Characterization
At room temperature, the structures of the synthesized nanomaterials were examined using an X-ray diffractometer (XRD) with a Bruker D8 Advance diffractometer and Cu Kα (λ = 1.5418Å).A Perkin-Elmer Paragon 1000 FT-IR spectrometer was used to record FTIR spectra across a specific range of 450-4000 cm −1 at ambient temperature.To examine the surface morphologies of nanocomposites and average grain sizes, SEM pictures were acquired using a JEOL JSM-5800 working at an accelerating voltage of 10 kV.Energy dispersive X-ray spectroscopy (EDAX) (Hitachi SU8010 Series) was employed for the elemental analysis.

Antibacterial activity
The antibacterial activity of differently calcinated MZO nanocomposites was assessed by adopting the disc diffusion method 32,33 .In this study, the antibacterial activity of test compounds was evaluated against five bacterial strains including Escherichia coli, Salmonella typhimurium, Shigella flexneri (gram-negative), Bacillus subtilis and Bacillus megaterium (gram-positive).A loopful of each bacterial strain was inoculated in sterile nutrient broth and incubated for 24 h at 37 °C to get fresh and viable bacterial inoculum.After evenly mixing the test organism (200 µL of bacterial suspense) in a nutrient agar plate, it was allowed to harden.The test compounds dissolved in DMSO to obtain a concentration of 100, 200 and 300 µg/mL.Sterile paper disks impregnated with the test compounds were placed on the inoculated plates using sterile forceps and the plates were incubated at 37 °C for 24 h.The bactericidal effect of different grades of MZO nanocomposites was initially assessed using the highest concentration of the tested sample (300 µg/mL) and only the effective grade of nanocomposites was further tested in different concentrations.The diameter of the inhibition zone was measured in millimeters using a ruler.

Antioxidant activity
The antioxidant propensity of MZO nanocomposites (synthesized using different calcination temperatures) was investigated following the colorimetric decolorization technique against ABTS + , DPPH, superoxide and nitric oxide radical according to the protocol mentioned previously 34,35 .The antioxidant efficacy of the tested samples was determined by calculating the inhibition percentage and the IC 50 values, which represent the theoretical concentration of the tested sample at which 50% of the free radicals are scavenged, were used to express the antioxidant activity of the sample.

Data analysis and program used
Growth parameters and plant biomass were calculated by taking readings from 20 replicas.For plant biochemical, antioxidant, and antimicrobial study three replications were taken into consideration and results were presented as average with standard deviation.Statistical differences were carried out by Tukey's HSD test at p ≤ 0.05 (for the germination test) and a two-tailed t-test at 95% confidence level (plant growth and biochemical parameters).

Result and discussions
Characterization of the nanocomposites X-ray diffraction (XRD) is a powerful tool that is frequently used and widely recognized for both qualitative and quantitative studies of crystalline phases present in materials 40 .A careful examination of the diffraction patterns can reveal much more information, including the characterization of solid substances, crystallite size and shape, crystal orientation, and internal elastic strains/stresses at various levels 41 .From Fig. 1a 202) and (104) planes respectively predicting the hexagonal structured of zinc oxide (ZnO) with P63mc space group and lattice parameters were found to be a = b = 3.3420 Å and c = 5.176 obtained from literature studied with PDF code no.00-001-1136 43 .In MZ 1 and MZ 2 there is the existence of two different phases in both and the peak intensity slightly increases from MZ 1 to MZ 2, i.e. with an increase in calcination temperature.
The average crystalline size can be calculated by using Scherer's equation where, D is the average crystalline size, λ is the wavelength of the radiation (0.154 nm), θ is the angle of diffraction, β is the full width at half maximum intensity 45 .Also dislocation density (δ), crystallinity and microstrain (ɛ) can be calculated using the following equations 46 : (2) D = 0.9 βcosθ  S1 and S2).Again, the calculated average crystalline sizes of MZ 3 and MZ 4 are 20.98 nm and 26.12 nm respectively.Here, size and crystallinity increase (crystallinity 66.78% for MZ 3 and 78.91% for MZ 4) as well as dislocation density and microstrain decrease with an increase in calcination temperature from 600 to 750 °C (Tables S3 and S4).
Figure 2, represents the FTIR spectra of the nanocomposites, the broad peak for all of the nanocomposites at wavenumber 3300-3600 cm −1 is caused by the presence of O-H stretching arising from the H 2 O molecule 45 .Two small, sharp peaks at 2923 cm −1 and 2854 cm −1 are the asymmetric stretching and vibrations of -CH 2 -present in aliphatic chains 47 .At high calcination temperatures, such peaks get less intense.The peak arises in the range of 1626-1630 cm −1 due to the O-H bending vibrational mode for moisture 45 .As a result, the intensity of the peak is decreased as enhanced the calcination temperatures of the nanocomposites.The peak is visible at 1384 cm −1 due to the C-H bending vibrational bands 48 .Peaks for the C-O stretching vibration are found to be in the frequency range of 1110-1115 cm −149 .The stretching frequency in the range of 500 to 1000 cm −1 corresponds to the metal-oxygen bond present in the prepared nanocomposites.It is normal to see the unique stretching vibration frequency of Zn-O at 915 cm −1 and 750 cm −1 for the presence of Zn-O bonds 50 .The distinct Zn-O peaks in composites appeared at 987 cm −1 and 793 cm −1 , respectively, due to a shift to a higher frequency 51 .The SEM is a technique that can be used to evaluate the surface texture 54 .In Fig. 3 the SEM images of the composites, the presence of two different instinct shapes of particles confirms the formation of composites.The porous nature and morphology of the samples at various synthesized temperatures from 300 to 750 °C are shown by the SEM pictures in Fig. 3.The morphology of the samples exhibits a noticeable shift in the SEM pictures of MZ 2 to MZ 3, i.e. increasing calcination temperatures of the synthesized nanocomposites.These may be due to the formation of different phase of manganese oxides.Quite large low-density agglomerates are generated, according to SEM examination.Continuous agglomeration of small particles occurs at higher calcining temperatures.
Using the EDAX spectrum, the elemental analysis of MZ 1, MZ 2, MZ 3 and MZ 4 were examined as depicted in Fig. 4. The existence of peaks attributable to manganese, zinc and oxygen was visible in the EDAX spectrum, which demonstrated that carbon and other organic residue had been removed from the template in the calcined samples.Zn, Mn and O are the sample's main constituents, according to the strong signal at 8.6 and 1.0 keV for Zn; 0.6 and 5.9 keV for Mn and 0.

Antibacterial effect of MZO nanocomposites
The results of the present study demonstrate the antibacterial activity of MZO nanocomposites against five different bacterial strains (Fig. 5).From the initial screening with the highest concentration of sample, it was found that MZ 4 was the best (Fig. 5).The synergistic effect of different phases of manganese oxide and ZnO nanoparticles in MZ 4 may contribute to its enhanced antibacterial activity.Thus, MZ 4 was further tested against the bacterial strains at three different concentrations (100, 200 and 300 µg/mL).It highlighted the importance of optimizing the calcination temperature of nanocomposites to obtain the best antibacterial activity 55 .In the present study, the superior activity of MZ 4 could be attributed to the changes in the morphology, crystalline structure, and composition of the nanocomposites that occurred during calcination at 750 °C.The attributed antibacterial activity may be due to the generation of reactive oxygen species (ROS) by the nanocomposites, which caused damage to the bacterial cell membrane and cytoplasmic structures.There are such reported facts in a manganese oxide and zinc oxide nanocomposites study 56 .
The antibacterial activity of the nanocomposites was tested at different concentrations, and it was observed that the zone of inhibition increased with an increase in concentration.This trend was observed for all the bacterial strains tested.Several previous reports support this concentration-dependent antibacterial activity against different bacterial strains 57 .The highest zone of inhibition was observed for Escherichia coli (28 ± 1.00) at a concentration of 300 µg/mL (Figs. 6, 7).Shigella flexneri was also found to be susceptible to the nanocomposites, whereas Salmonella typhimurium among gram negatives and B. megaterium among gram positives were the least susceptible, with no zone of inhibition observed at a concentration of 100 µg/mL (Table S5).In the current study, it was observed that the gram-negative strains showed higher susceptibility than the positive ones.The observed variation in antibacterial activity could be attributed to the differences in the cell wall structures and physiological characteristics of the bacterial strains.The findings of the present study are consistent with previous studies 58 .

Antioxidant activity of MZO nanocomposites
Several types of chemical entities with one or more unpaired electrons are known as free radicals.These free radicals destroy other molecules by extracting their electrons to become stable.More importantly, these free radicals are generated all the time inside the body system as a by-product of several important reactions.In the current investigation, antioxidant activity was expressed as an IC 50 value, and presented in Table S6.The high IC 50 value of a sample indicates comparatively low antioxidant activity.From the presented table and Fig. 8, it was observed that in maximum cases against all the studied free radicals no such strong differences in antioxidant activity were observed among MZ 1 (IC 50 values are 36.845± 0.126 µg/mL for ABTS, 46.127 ± 1.622 µg/mL for DPPH, 62.194 ± 0.180 µg/mL for superoxide and 91.487 ± 5.754 µg/mL for NO) and MZ 2 (36.974 ± 0.481 µg/mL for ABTS, 47.145 ± 0.681 µg/mL for DPPH, 66.456 ± 0.110 µg/mL for superoxide and 95.347 ± 6.459 µg/mL for  NO).Furthermore, with increasing calcination temperature, the antioxidant efficacy of all the studied samples gradually decreases.These may be due to the increase in particle size and decrease in active surface to capture the radicals 59 .It is reported that the best antioxidant activity of iron oxide nanoparticles calcined for 2-3 h, maintaining the temperature in the range of 200-300 °C, which coincides with our present study 60 .They further reported a strong reduction in antioxidant activity above 500 °C, which also supported our findings.Overall results depicted that synthesized MZO nanocomposites demonstrated the highest activity against ABTS + radical and the least against nitric oxide radical.

Effect of MZO nanocomposites on wheat seed germination and seedling growth
The process of seed germination is fundamental to the emergence of new plants, but it is of great significance as it ultimately determines factors such as crop quality and yields.A seed germination study was carried out by taking 200 seeds and it was found that MZ 1 treatment was the most effective that can germinate almost 89% of seeds.Though among other grades of MZO nanocomposites (i.e.MZ 2, MZ 3 and MZ 4), no such strong statistical variability (at p ≤ 0.05) in seed germination was observed (Fig. 9).From the germination assay, among different grades of nanocomposites MZ 1 was chosen as it demonstrates 25.87% more seed germination capability than the control, and for that reason, further seedling growth study was carried out with this particular treatment (MZ 1) in comparison to control.On evaluating phenotypic appearance, the same trends of results were found, where almost every growth parameter such as shoot height, root length, and plant biomass identified to be strongly influenced by MZ 1 treatment (Fig. 10).This particular treatment increased seedling shoot height and root length by 21.63% and 20% respectively in comparison to control seedling.Root length improvement as observed through seeds priming with MZ 1 nanocomposites may be attributed to the activation of cell cycling and respiration during the priming process.This activation, along with the translocation of assimilated nutrients and withering of the seed coat, leads to faster root emergence, as described in previous studies 61 .Root parameters improvement through MZO nanocomposites might be due to the influence of outside factors like the presence of mineral nutrition, which regulates numerous morpho-physiological processes.Plant biomass was also enhanced by 23% from control by application of MZ 1 through seed priming and subsequent irrigation.

Effect of MZO nanocomposites on wheat seedling biochemical attributes
The most important factors influencing plant growth and development include the rate of photosynthesis, the assimilation of sugars, and the amount of protein present.The application of MZO nanocomposites was found to have a moderate to strong effect on biochemical parameters such as chlorophyll, sugar, protein, and phenol content.In contrast to control seedlings, those treated with MZ 1 exhibited a 20.83% increase in chlorophyll content.Several studies that have been published claimed that the use of nanoparticles significantly increased the amount of chlorophyll by creating more light-harvesting complexes and absorbing the most amount of light energy, which in turn increased of the rate photosynthesis 62,63 .Though the excessive application of zinc was reported to inhibit chlorophyll synthesis by interfering with the iron metabolism of plants 64 , in the present study no such effect of chlorophyll retardation was observed.It might be due to the co-application of Zn and Mn working in a balanced system as Mn has an immense role in chlorophyll biosynthesis, and oxygen evolution during photosynthesis and is found to be involved in the chloroplast 65,66 .Optimal grade MZO nanocomposites (MZ 1) treated wheat seedlings had the highest total carbohydrate content, surpassing the control by 31.63%.Indeed, sugars are essential as regulatory molecules in the central signaling system that control the expression of genes involved in metabolism, growth, and development 67 .The application of MZ 1 promisingly enhances (39.47% over control) the protein content of the treated wheat seedling.The amount of protein in leaves is extremely important and plays a significant function in plant growth, reproduction, and ultimately grain yield 68 .A lot of published works reported the role of micronutrients like Fe, Mn, Zn and Cu (applied in their nano form) in the improvement of plant protein content and subsequent growth and development 37,69,70 .On analysis of total phenol content, it was observed that there was no such strong statistical difference between control and treated seedlings (Table 1).

Conclusion
In our work, we study the effects of calcination temperature on structural, anti-bacterial, anti-oxidant and seed germination processes.The prepared nanocomposites were characterized using different techniques like XRD, FTIR, SEM and EDX.The characterization results showed that with the variation of calcination temperatures, the synthesized nanocomposite MZO transformed into different structures and sizes.At low calcination temperatures the composite exists as Zn 1.41 Mn 1.59 O 4 /ZnO and is converted into ZnMnO 3 /ZnO at higher calcination temperatures.The bioactivities are greatly influenced by the varied temperature.For this reason, the bioactivities of the composite changes with the different calcination temperatures.Composite shows the best antibacterial at 750 0 C and best antioxidant and seed germination properties at 300 0 C indicating the temperature controlling activities are pronouncedly found in the nanocomposites giving a new way for future research.
Table 1.Various biochemical attributes of treated and control wheat seedlings.Results were presented as mean ± standard deviation, whereas '*' symbol indicated that they are statistically significant from their respective control at 95% confidence level as observed through two-tailed t-test.

Figure 6 .
Figure 6.Antibacterial activity of MZ 4 against the tested bacterial strain.

Figure 7 .Figure 8 .
Figure 7. Zone of inhibition of MZ 4 against different bacterial strain at different concentration.

Figure 9 .
Figure 9. Germination percentage of wheat seed as influenced by different types of MZO nanocomposites.The vertical bar above column represents the standard deviation (n = 3).Different letters (a, b, c etc.) indicated that they are statistically different at p ≤ 0.05 following Tukey's HSD test.

Figure 10 .
Figure 10.Phenotypic appearance (A) and growth attributes (B) of treated and control and treated wheat seedlings.The vertical bar above the column represents the standard deviation (n = 20).Asterisk: symbol indicated that they are statistically significant from their respective control at 95% confidence level as observed through two-tailed t-test.