Microanalytical Assessment of Biocompatibility and Microbial Interactions of Nanomaterials with Potential Biological Application

Nanomaterials have attracted significant attention due to their physicochemical properties that improve efficiency and performance across agricultural, pharmaceutical, and food systems. However, they may influence microbial viability, germination, metabolite production, and colonization. Therefore, assessing the biocompatibility of organic nanomaterial-based fertilizers with beneficial microorganisms is crucial to ensure efficacy and prevent unintended impacts on production systems. This study evaluate the biocompatibility and biological interactions of fertilizers based on organic nanomaterials and microorganisms. At the nano–bio interface, nanoparticle (NP) interactions with cellular membranes and organelles are strongly influenced by physicochemical properties such as surface chemistry and charge, which regulate cellular uptake and potential nanotoxic effects [0]. Two nanomaterial systems were engineered: carbon-based nanostructures derived from fullerene C₆₀ and nanocellulose synthesized from sawdust and organic waste using established methodologies [2]. These materials were functionalized and formulated into nano-fertilizers incorporating nutrient sources such as ammonium nitrate, phosphonic acid, zinc oxide, magnesium sulfate, and magnesium chloride [3]. Functionalization and conversion into soluble formulations were achieved through controlled thermal mixing at 70–80 °C under vigorous agitation, promoting surface activation and formation of reactive functional groups [2]. The design of these functional groups was oriented toward addressing practical agricultural challenges, leading to the development of biocompatible nano-fertilizers suitable for both foliar application and ferti-irrigation [4,5]. The formulated materials exhibit enhanced ionic mobility and controlled nutrient-release behavior, supporting sustained nutrient availability while maintaining microbial activity and productivity [6]. Although previous studies have demonstrated their effectiveness in promoting plant growth and improving nutrient use efficiency [5,7], understanding their interactions with microbial systems remains essential to ensure safe and functional agricultural application. The analytical approach integrated multiscale morphological characterization, growth kinetics, and antagonism assays of Trichoderma against Fusarium solani, including cellular and mitochondrial viability analyses. These evaluations assessed the structural, physiological, and functional responses of microbial systems exposed to nano-based fertilizers. To ensure analytical consistency and minimize variability, a standardized sample preparation protocol was implemented, including controlled cell density, synchronized growth phases, and defined physiological conditions. These measures enhanced reproducibility and the reliability of microanalytical results. The experimental design consisted of maintaining Trichoderma under stress conditions for 72 h, corresponding to an active adaptive phase that favored consolidation of interactions with nanomaterials. Each nano-formulation was evaluated at different concentrations (Table 1). Experimental treatments of Trichoderma with nanomaterials *Bh=nanocellulose-based nano formulation; Nf= fullerene-based nano formulation; control treatment consisted of sterile distilled water. Trichoderma was isolated from an aqueous suspension (1 g/L) of a commercial rice flour–based formulation. After filtration, 100 μL of the filtrate was surface-plated onto PDA medium and incubated at 28 °C for 6 days. Compatible colonies were subcultured for purification and macroscopic characterization. For growth kinetics analysis, pre-weighed filters were dried at 50 °C, equilibrated in a desiccator, and weighed prior to use. Twelve inoculated flasks (treatments and control) were prepared, and two were removed every 24 h for biomass determination. Culture contents were filtered through pre-weighed filters and dried at 50 °C to constant weight. Dry biomass was calculated by weight difference, and values were used to construct biomass production curves. Antagonism was assessed using dual culture assays on PDA, confronting Fusarium solani with Trichoderma obtained from the corresponding liquid cultures. Radial growth of F. solani was measured on days 5, 7 and 10, and antagonistic activity was expressed as the percentage of radial growth inhibition (PIRG) relative to the control. Hyphal segregation (obtained from pre-treatment suspension) and cellular clarification were performed to minimize aggregation and improve suspension homogeneity. Subsequently, cell viability was determined using 0.4% trypan blue staining, and mitochondrial activity was evaluated using 0.02% Janus Green B. In the kinetic growth analysis of the nanocellulose-based fertilizer (Fig. 1A), Bh1 exhibited a sustained decline in biomass over time, remaining consistently below the control throughout the experimental period, indicative of a pronounced inhibitory effect. In contrast, Bh2 showed an initial decrease followed by gradual recovery, suggesting partial adaptation. Bh3 displayed a distinct late-phase response, with a marked increase in biomass after 96 h, ultimately surpassing both the control and the other dilutions at 120 h. Fullerene-based treatments (Figure 1B) exhibited a more variable growth pattern. Nf1 showed a transient stimulation around 72 h, followed by a sharp decline at later time points. Nf2 and Nf3 maintained lower biomass levels relative to the control across most time points, consistent with concentration-dependent modulation rather than sustained growth stimulation. Collectively, these patterns support the premise that nanoparticle incorporation may positively or negatively influence microbial systems depending on material characteristics and concentration, with moderate exposure capable of altering metabolic activity without necessarily causing complete growth inhibition [9]. In the same way cellular viability was significantly influenced by treatment (Fig. 2A). The control exhibited the highest viability (~87%). Among nanocellulose-based treatments, Bh1 and Bh3 were classified as “ab,” indicating no significant difference from the control despite moderately reduced values (≈70% and ≈67%, respectively). Bh2 showed a moderate but significant reduction (≈65%). In contrast, fullerene-based treatments resulted in a more pronounced decrease in cellular viability. Nf1 and Nf2 were ≈53% and ≈59%, respectively, while Nf3 showed the lowest viability (~45%), significantly different from the control. Regarding mitochondrial viability (Figure 2B), the control again displayed the highest value (~76). All nanocellulose-based treatments (Bh1, Bh2, Bh3), indicating no significant differences relative to the control, although mean values were slightly reduced (≈61–67%). Among fullerene treatments, Nf1 showed a significant decrease (≈55%), whereas Nf2 and Nf3 reflecting intermediate responses without statistical difference from the control. The divergence observed between cellular membrane integrity and mitochondrial viability suggests that these functional parameters are not strictly coupled under nanomaterial exposure. Such dissociation is consistent with previous reports indicating that nanomaterials can target distinct cellular compartments through differential mechanisms, including membrane perturbation and mitochondria-associated oxidative stress [10]. In nanocellulose-based treatments (Fig. 3A), antagonistic activity showed a clear time-dependent increase. At day 5, inhibitory responses were minimal and, in some cases, negative, particularly in Bh1 (1:10). By day 7, Bh2 (1:100) exhibited a higher antagonistic index compared with Bh1 and Bh3 (1:1000). At day 10, both Bh1 and Bh2 significantly enhanced radial growth inhibition, with Bh2 reaching the highest antagonistic levels, surpassing both the control and Bh3. In contrast, fullerene-based treatments (Fig. 3B) displayed a more gradual and concentration-dependent pattern. Antagonistic activity remained low at day 5 across Nf1 (1:10), Nf2 (1:100), and Nf3 (1:1000). A moderate increase was observed at day 7, particularly in Nf1. By day 10, all fullerene-based treatments improved inhibition, with Nf3 showing the strongest antagonistic effect, exceeding the control as well as Nf1 and Nf2. Overall, nanocellulose-based formulations promoted a more marked early stimulation of antagonistic activity, possibly associated with early modulation of metabolic pathways involved in microbial control, as nanomaterials at sub-toxic concentrations may stimulate microbial metabolic activity through adaptive stress responses that enhance cellular performance during the initial stages of interaction [11]. In contrast, fullerene-based treatments showed progressive improvement, consistent with concentration- and time-dependent physiological responses reported for bioactive nanomaterials. Such effects are likely associated with the interaction of carbon-based nanomaterials with microbial cell membranes, which can disrupt membrane integrity and modulate electron transport chains and redox homeostasis, ultimately influencing cellular metabolism [11]. These findings demonstrate that microscopy and microanalysis constitute powerful tools for addressing the complexity of nanomaterial–microorganism interactions, providing directly observable microstructural and functional criteria for assessing biocompatibility at the nano–bio interface in applied agricultural contexts. Time-dependent biomass production of Trichoderma under Nano formulation. (A) cellulose. (B) fullerene. Evaluation of viability of Trichoderma exposed to nano fertilized. (A) Cellular (B) Mitochondrial. Nanocellulose-based (Bh1–Bh3) and fullerene-based (Nf1–Nf3) nanoformulations. Different letters indicate significant differences among treatments (p < 0.05). Time-dependent modulation of Trichoderma antagonistic activity under nanomaterial-based treatments. (A) Nanocellulose nanomaterials. (B) Fullerene-based nano-fertilizers. The antagonistic index (%) was measured at 5, 7, and 10 days. Different letters indicate statistically significant differences among treatments based on Tukey’s significant difference (HSD) test following two-way ANOVA (p < 0.05).