Structural characterization of MWCNTs
Dynamic light scattering and zeta potential of MWCNTs
DLS scanning size distribution Fig. 1a of MWCNTs was observed to be of diameter 37.84 nm which can be taken up efficiently by the plant cells [7], and the surface charge Fig. 1b was observed to be −55.5 mV which specified that nanotubes were highly stable in double-distilled water after sonication with noteworthy diameter for uptake by plants.
X-ray diffraction (X-RD) analysis of MWCNTs
Usually, the XRD examination of MWCNTs reflects a few extra peaks. “This peak family is generated because of atomic planes declining where 3D structure with regular stacking nanotube layers has to be established” [44]. Figure 2 depicts the crystallinity of MWCNTs. The most substantial diffraction peak was witnessed at the angle (2θ) of 25.903°, which can be attributed to the C (002) reflection of the hexagonal graphite structure of MWCNTs. The other characteristic diffraction peaks of graphite were at 2θ of about 43.23, 53.13, 77.92 in accordance with C (002), C (004), and C (110), and all the peaks were in reference to JCPDS file number 74–1602. This X-RD graph was plotted with Xpert high score.
SEM and FE-SEM characterization analysis of MWCNTs
To understand the sample surface morphology, SEM and FE-SEM analyses were performed. MWCNTs sample was dispersed in double-distilled water to visualize under the FE-SEM and SEM to observe carbon nanotubes’ morphological structures and size distribution. Since most of the biological cell structures lie in the range of nanometres, the size and structure of CNTs must comply with them. Figure 3a is a SEM image analysis after magnification up to 15000X. The SEM image presented the micromorphology of MWCNTs as long tubes and aggregated clusters. The same water-dispersed MWCNTs sample was visualized under FE-SEM, presenting better image resolution and focus than the SEM. The photograph of FE-SEM Fig. 3b shows the micromorphology of individual MWCNTs with high resolution and focus at 100000X magnification.
UV–visible spectroscopy
The absorption range of pure MWCNTs falls between 250–260 nm [32]. From the UV–Vis spectroscopy characterization, it has been shown that the characteristic existence of absorption peak (Fig. 4) at 258 nm which aligns with the characteristic absorption peak range of pure MWCNTs.
Phenotypical analysis of Basella alba after MWCNT treatment
The plant pots (Fig. 5b C (Control), S1, S2, S3, and S4 were treated with different concentrations 0 µg ml−1 (control), 50 µg ml−1, 100 µg ml−1, 150 µg ml−1, and 200 µg ml−1 of MWCNTs treatment sample, respectively.
Germination percentage
It was observed that out of 30 Basella alba plant seeds, all seeds that were soaked in double-distilled water were germinated successfully, and the germination percentage was calculated to be 100%.
Vigour index calculation
Comparative analysis of root and stem was performed after harvesting from pots. Figure 5a graph depicts the highest mean length of the root system, which was found to be 8.9 cm in the S3 sample treated with 150 µg ml−1, and the highest mean of the shoot length was found to be 48 cm in the S4 sample which was treated with 200 µg ml−1 concentration of MWCNTs. Statistically significant difference was found between the control group and S4 sample group; however, no such significant difference was found between S1, S2 and S3 with a p ≥ 0.05. When the S1 sample was treated with a concentration of 50 g ml−1 MWCNTs, the root and shoot lengths were seen to be shorter. Basella alba is an annual crop with a short harvesting period and without a long taproot system. In comparison to the control plant sample, plant shoot length increased and root length decreased as the MWCNTs treatment concentration increased.
Vigour index
The highest vigour index was observed to be at 4390.2 in the S4 sample (treated with 200 µg ml−1 concentration of MWCNTs) (Fig. 6), which was statistically significant, giving p = 0.001 while being compared to the control, S1 and S2 sample, while no significant difference was found between S3 and S4 as declared by p ≥ 0.05. Treated plant samples showed substantial vigour index. From this study, it can be inferred that the plant samples supplemented with a higher concentration ranging from 150 µg ml−1 to 200 µg ml−1 of MWCNTs can grow in sub-optimum conditions very well, in comparison to the control.
Phenotypical analysis—leaf count
The phenotypical analysis offers statistical data regarding morphological changes that occurred in the sample plants. This study was done to determine the impact of MWCNTs on the growth of Basella alba plant leaf samples after the treatment. Before harvesting, the number of leaves was counted in all the sample plants, and their average was considered for the study.
From the above data (Fig. 7), it was evident that the plants treated with a higher concentration of MWCNTs have shown good and healthy growth compared to the control sample. The plants that were treated with the 50 µg ml−1 showed low leaf count, and the plants treated with 100 µg ml−1 showed a similar number of leaves as the control Basella alba plant sample. As compared to the control, these two samples (S1 and S2) have not shown statistically significant differences, hence raising the p ≥ 0.05. While the plants treated with a higher concentration of MWCNTs of 150 µg ml−1 and 200 µg ml−1 were shown to have the highest mean number of leaves in comparison with the control Basella alba plant sample, **p = 0.00078. From the phenotypical analysis, a promising increment in the growth of the average number of plant leaves has been seen in the treated samples with high MWCNTs concentration. Both 150 µg ml−1 and 200 µg ml−1 concentrations of MWCNTs were found to be effective for obtaining better output in leaf production.
Phenotypical analysis—average height of the plants
Available statistical data on the height of the plant sample treated with a low concentration, i.e. 50 µg ml−1 and 100 µg ml−1 of MWCNTs sample, have shown a lower mean height (Fig. 8). While the plant samples treated with 150 µg ml−1 and 200 µg ml−1 have shown increased plant height as compared to the control plant sample. Hence, maximum plant growth has been observed in the samples which were treated with a higher concentration of 150 µg ml−1 and 200 µg ml−1 MWCNTs.
Upon comparison between S4, S3, and the control, the p-value obtained was 0.013724, while the comparison between the control group and S1 and S2 sample groups did not show any statistically significant increment in plant height. From the above study, it was affirmed that Basella alba plant height observation is significant and true to conclude that MWCNTs is showing its impact on the height of S3 and S4 treated Basella alba plant sample.
SDS-PAGE analysis
Positive results have been achieved regarding protein content in MWCNTs-treated Basella alba plant leaves. In the SDS gel, each well was filled with different plant protein samples (C, S1, S2, S3, and S4) of Basella alba plants. The loaded protein samples confirmed that dark and thick bands at molecular weights 42 kDa and 18 kDa of plant protein samples were obtained in plants treated with a high concentration of MWCNTs. From the SDS-PAGE gel image (Fig. 9) it is evident that the sample loaded in the S3 well showed bright and thick bands compared to the control plant protein sample. Here in the S1 sample well, all protein bands are very light and thin, indicating less protein content. This concludes that plants treated with a lower concentration of MWCNTs had lesser protein content than the treated plant sample. 150 µg ml−1 concentration of MWCNTs has been proven to increase the protein concentration in Basella alba plants.
Chlorophyll content estimation analysis by UV–visible spectroscopy
The chlorophyll extraction from Basella alba plant samples of C, S1, S2, S3, and S4 has been done using the acetone method. Leaf extractions samples were taken for UV–Vis spectroscopy analysis to identify chlorophyll concentration present in each plant sample. The absorption spectroscopic scan was performed between 600 and 800 nm, expecting sharp peaks in the chlorophyll absorption wavelength range. The absorption spectroscopy results disclosed that all sharp peaks were observed between 650 and 675 nm wavelength, which is the absorption spectrum of chlorophyll pigment, and it has been demonstrated in the above graph (Fig. 10) obtained from UV–visible spectroscopy. Comparing the Basella alba plants treated with 200 g ml−1 MWCNTs to the control, it was found that they exhibited the maximum absorption peaks. Hence it can be established that treatment with a 200 µg ml−1 concentration of MWCNTs resulted in an elevated chlorophyll pigment concentration. The biochemical pathway and the underlying mechanism involved in increased chlorophyll content are yet to be ventured.
SEM–EDX (scanning electron microscopy–energy dispersive X-ray diffraction)
Basella alba leaf was investigated for the presence of MWCNTs, and certain other elements in the control leaf sample and MWCNTs-treated leaf sample. EDX has been used to examine the elemental composition present in the samples. To ensure that MWCNTs were uptaken by the plants, EDX spectra are considered. In SEM analysis, the sample’s surface morphology has been considered for further analysis. Basella alba plant leaf samples were investigated with EDX analysis to obtain the carbon weight percentage in control, and MWCNTs-treated plant leaf samples. The elemental scan using EDX declared clearly that the control carbon weight percentage is much less than treated Basella alba plant leaves (Fig. 11). The observed carbon weight percentage in control Basella alba plant leaves is 49.85, and the treated carbon weight percentage in Basella alba plant leaves is 65.45. The SEM imaging disclosed that MWCNTs are absorbed and accumulated in the form of clumps in Basella alba leaves. The SEM–EDX spectra from control and treated Basella alba plant leaves have shown the expected results for the absorption of MWCNTs.
Study of soil microbiota after MWCNT supplementation
Impact of MWCNTs on soil microbiota
For plants to absorb nutrients, they must first be processed into their elemental form, predominantly by soil bacteria. This symbiotic interaction between microorganisms and plants must be strengthened for enhanced growth parameter results. To determine the effect of MWCNTs on the microorganisms population in the rhizosphere, we undertook a study through the plating method.
The microbial growth was at a slow pace after incubation of 24 h. However, after 48 h of incubation, a considerable boost in microbial growth was noticed as the treatment concentration was increased. The highest number of microbial colonies were observed in Basella alba plant-soil S4, which was treated with 200 µg ml−1 of MWCNTs, while the lowest number of colonies were observed in the control soil sample (Fig. 12). This response of the soil microbial culture plating revealed that MWCNTs are boosting the microbial community development in the soil microbiota surrounding the rhizosphere. The additional carbon source provided by MWCNTs for these microbial strains is responsible for the enhanced soil microbiota population.