Effect of Natural Carbon Nanotubes Biotransformed by Trichoderma sp. on the Development of Zea mays

Maize (Zea mays) is a cereal of global economic importance, it is a grass of the Poaceae family, native to Mesoamerica and is currently the cereal with the highest production in the world [1]. At present, for the production of Z. mays, the use of chemical fertilizers has been used indiscriminately, which are harmful to the environment, for which new strategies have been explored as an alternative to reduce or replace them, through the use of nanomaterials and environmentally friendly microorganisms. The use of nanomaterials is being widely studied to improve plant development by retaining water and minerals [3], such as multi-walled carbon nanotubes (MWCNTs) [4]. On the other hand, Juárez et al. [5] evaluated the ability of two fungal species to biotransform MWCNTs, the results indicate structural modifications on the surface of the MWCNTs. This suggests that some fungal enzymes can modify or biotransform the structure and physicochemical properties of graphene-based nanomaterials, and consequently their effects are different in plants than those documented. Therefore, in this work the objective was to characterize the changes in the natural and synthetic MWCNTs in interaction with Trichoderma sp and later to evaluate the effects on the speed and percentage of germination in Z. mays seeds. MWCNTs of natural origin obtained from samples of burned Pinus oocarpa wood from forests in Michoacán, central-western Mexico, were used. The MWCNTs were collected and purified according to Lara-Romero et al., (2017) and presented the following structural characteristics: ∼10 layers of graphene, with inner and outer diameters of 2.52 nm and 12 15 nm, respectively [6]. For the preparation of nanoparticles biotransformed by Trichoderma sp, a liquid medium of potato dextrose was supplemented with natural MWCNTs in concentrations of 10, 20, 30 and 40 µg/ml. Subsequently, they were incubated at 30°C with 150 rpm in a shaker for 14 days. After 14 days of growth of Trichoderma sp. in liquid medium, all H2O was removed in an oven at a temperature of 75°C for 8 hours. Finally, the dry material was pulverized with the fungus-nanomaterials in an orbital mill to homogenize and reduce the particle size. The MWCNTs with and without exposure to the growth of Trichoderma sp. (pulverized) were calcined at 1000°C/3 hrs, later they were characterized to evaluate the effect of Trichoderma sp. on the structural changes of the nanomaterials by means of Fourier Transform Infrared Spectroscopy (FTIR), Raman spectroscopy and Scanning Electron Microscopy (SEM). Bioassays in Zea mays Seeds of Z. mays primed for 24 hours with the inoculum nanoparticles-Trichoderma sp. and nanoparticles at concentrations of 0, 10, 20, 30 and 40 µg/seed, were grown in previously sterile agrolite and kept in the shade. percentage and speed of germination during 3 days after sowing. The result obtained by FTIR in natural MWCNTs, the C-O functional group was identified at 2165 cm-1, when comparing this nanoparticle with the FTIR of the natural MWCNTs exposed to Trichoderma sp., the loss of this C-O functional group was observed, while they were detected. others like C-H at 698, 1366 and C-O at 976 cm-1. Raman spectroscopy analyzes were performed on carbon nanotubes, with and without biotransformation with Trichoderma sp, for their characterization. Peaks ∼1350 and ∼1600 cm-1 are observed, corresponding to the ID and IG bands, respectively. The ID/IG ratio with values <1 indicate that the nanomaterials are more crystalline or have fewer defects in their structure7. The ID/IG values of the MWCNTs showed that the interaction of Trichoderma sp. with these nanoparticles the crystallinity decreased, showing a greater loss of crystallinity where the lowest concentration of MWCNTs was added, as the concentration of these NPs increased the loss of crystallinity was less, however, in all cases an effect of Trichoderma sp. on these NPs (Figure 2). Seed germination results showed that the addition of natural MWCNTs at a concentration of 10 µg/mL without Trichoderma sp. and at 20 µg/ml of biotransformed natural MWCNTs had a positive effect on early seed emergence, compared to the control, and the germination percentage reached was 100% (Figure 3). In general, the treatments caused a promotion in the percentage of germinated seeds compared to the control (Figure 3). Similarly, in recent research they obtained effects in which the seed germination process was improved and accelerated in different plant systems, depending on the aggregate concentrations of the nanoparticles used, when compared with control plants, by graphene oxide in Vicia faba L. (Anjum et al., 2014); Synthetic MWCNTs in Zea mays (Tiwari et al., 2014); Synthetic MWCNTs in Triticum aestivum L. (Joshi, et al., 2018); Natural MWCNTs in Eysenhardtya polystachya (Júarez et al., 2020). The biotransformation of carbon nanotubes by Trichoderma sp. is demonstrated by characterizing by FTIR where the changes in their functional groups are found and by Raman, the loss of crystallinity of the carbon nanotubes is observed. In addition, these biotransformed nanoparticles contributed to the germination percentage of Z. mays and the biotransformed natural MWCNTs at a concentration of 20 µg/ml had a positive effect on early seed emergence. However, studies of these biotransformed nanoparticles in mature stages of different plant systems are still scarce, so it is important to analyze other response variables. FTIR of natural carbon nanotubes with and without biotransformation with Trichoderma sp, in an in vitro experiment with continuous shaking in a shaker at 150 rpm/30°C for 14 days. Raman spectra of carbon nanotubes with and without biotransformation with Trichoderma sp, in an in vitro experiment with continuous shaking in a shaker at 150 rpm/30°C for 14 days. Germination of Zea mayz cultured with 0, 10, 20, 30 and 40 µg/ml of natural carbon nanotubes with and without biotransformation by Trichoderma sp.