Effect of Biotic-Origin Carbon Nanotubes Biotransformed by Trichoderma sp. on the Development of Avena sativa

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The use of nanotechnology in agriculture has gained significant relevance in recent years due to its potential to improve crop yields and reduce dependence on conventional agrochemicals. In this context, multi-walled carbon nanotubes (MWCNTs) have been explored for their unique physicochemical properties, which can enhance nutrient retention and stimulate plant growth. However, their biocompatibility and behavior in agricultural environments still require further understanding [1]. The biotransformation of nanomaterials by microorganisms represents an innovative strategy to enhance their functionality and mitigate potential adverse effects on ecosystems [2]. Trichoderma sp., a fungus widely studied for its ability to promote plant growth and act as a biocontrol agent [3], has been shown to modify the structure of carbon nanotubes, altering their physicochemical properties and enhancing their interaction with crops [4]. In this study, the biotransformation of naturally sourced MWCNTs was evaluated through their interaction with Trichoderma sp., aiming to characterize the structural changes induced by the fungus and analyze its effect on the growth stimulation of Avena sativa. This research seeks to contribute to the development of sustainable strategies that integrate nanotechnology and beneficial microorganisms in agriculture. In this work, naturally sourced MWCNTs were collected and purified following the methodology of Lara-Romero et al. (2017), presenting the following structural characteristics: ∼10 graphene layers, with inner and outer diameters of 2.52 nm and 12–15 nm, respectively [5]. For the preparation of Trichoderma sp.-biotransformed nanoparticles (NPs), a potato dextrose liquid medium was supplemented with natural MWCNTs at concentrations of 10 and 20 µg/ml. The cultures were then incubated at 30°C with shaking at 150 rpm for 14 days. After Trichoderma sp. had grown for 14 days in the liquid medium, all water was removed by drying at 75°C for 8 hours. The samples were then calcined at 1000°C for 3 hours, followed by nanomaterial characterization to assess the effect of Trichoderma sp. on the structural changes of the nanomaterials. Root architecture and biomass of A. sativa were measured 100 days after sowing in seeds previously impregnated for 24 hours with the Trichoderma sp.-nanoparticle inoculum and the corresponding nanoparticles for each treatment. The FTIR results for natural MWCNTs revealed the presence of functional groups, including C-H at 2975 cm-1, C-O at 2165 and 1441 cm-1, and C=C at 1597 cm-1. When comparing these nanoparticles to the FTIR spectra of natural MWCNTs exposed to Trichoderma sp., the loss of functional groups such as C=C and OH was observed, while new groups such as C-H at 617, 1397, and 304 cm-1 were detected (Fig. 1-A). Figure 1-B presents the Raman analysis spectra of the carbon-based nanoparticles, showing peaks around ∼1350 and 1600 cm-1, corresponding to the ID and IG bands, respectively. Based on these data, the ID/IG ratio was calculated, where values of ID/IG < 1 indicate higher crystallinity or fewer structural defects in the nanomaterials [6]. The ID/IG values of the MWCNTs suggested that their interaction with Trichoderma sp. reduced crystallinity in areas with lower NP concentrations. In contrast, non-biotransformed natural MWCNTs exhibited a higher index, attributed to the presence of organic matter during analysis. SEM images revealed a clear biotransformation of the nanoparticles (Fig. 2), displaying changes in both morphology and size. Additionally, concentration-dependent variations were observed. Notably, in MWCNTs, Trichoderma sp. caused partial degradation (Fig. 2). The effects of natural MWCNTs on A. sativa root architecture were evaluated 100 days after sowing (Fig. 3). The addition of NPs without Trichoderma sp., at a concentration of 10 µg/ml, stimulated root length compared to control plants. Similarly, exposure of Trichoderma sp. to 20 µg/ml of natural MWCNTs during its growth also promoted root development compared to the control without NPs (Fig. 3-A). Regarding root volume, the treatment with 10 µg/ml of biotransformed MWCNTs showed the most significant increase compared to the control. Furthermore, other treatments also exhibited significant differences (Fig. 3-B). The addition of NPs to A. sativa stimulated fresh weight. Likewise, the exposure of these NPs during Trichoderma sp. growth also enhanced fresh weight, showing significant differences compared to control plants (Fig. 4-A). Moreover, dry weight was significantly increased with the addition of NPs and with the exposure of NPs to Trichoderma sp. during its growth, favoring the dry biomass of this grass species (Fig. 4-B). The results of this study demonstrate the ability of Trichoderma sp. to biotransform NPs under the evaluated experimental conditions. Furthermore, these biotransformed nanoparticles contributed to promoting plant growth. The findings indicate that both natural MWCNTs and the fungus positively influenced root architecture and biomass accumulation in Avena sativa. FTIR and Raman analysis of carbon nanotubes with and without biotransformation by Trichoderma sp. in an in vitro experiment under continuous shaking at 150 rpm/30°C for 14 days. SEM micrographs of natural MWCNTs with and without biotransformation by Trichoderma sp., obtained at 10 kV X10,000 and X20,000 (08 30 SEI). The line represents a scale of 10 µm and 1 µm. Effect of natural MWCNTs biotransformed by Trichoderma sp. on the root architecture of Avena sativa. The root architecture was analyzed 100 days after sowing using WinRhizo software, and the data were analyzed with ANOVA-Tukey at p ≤ 0.05 (n=5). Different letters indicate significant differences. Effect of natural MWCNTs biotransformed by Trichoderma sp. on the biomass of Avena sativa. Data were analyzed 100 days after sowing using ANOVA-Tukey at p ≤ 0.05 (n=5). Different letters indicate significant differences.

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