Biochar pre-conditioning reduces nanoplastic toxicity to plant growth-promoting bacteria

Abstract

Nanoplastics are emerging environmental pollutants that threaten soil microbial communities, especially plant growth-promoting bacteria. Here, we investigate whether sugar maple biochar-widely recognized for its soil amendment benefits-can reduce nanoplastic toxicity. Using confocal microscopy, scanning electron microscopy (SEM), and fluorescence spectroscopy, we characterized the interactions between biochar and nanoplastics and observed extensive nanoplastic aggregation on biochar surfaces. Pre-conditioning nanoplastics with biochar (i.e., allowing nanoplastics to interact with biochar before bacterial exposure) lowered their effective concentration in solution and reduced surface coverage on bacterial cells. Growth assays confirmed that biochar pre-conditioning improved both planktonic and biofilm growth of Pseudomonas defensor, a plant growth-promoting bacteria, at nanoplastic concentrations up to 100 μg mL^-1. Our results highlight biochar's potential to sequester nanoplastics and mitigate their toxicity, offering a sustainable strategy for protecting microbial communities in plastic-contaminated soils.

Fig. 1. Interaction of PS–NH₂ with sugar maple biochar. (A) Confocal images of biochar without nanoplastic (top row); 12.5 μg per mL PS–NH₂ without biochar (middle row); 12.5 μg per mL PS–NH₂ with 100 μg per mL biochar (bottom row). (B) SEM images of 0, 12.5 and 50 μg per mL PS–NH₂ with 100 μg per mL biochar. Scale top row: 5 μm, bottom row: 1 μm. (C) Fluorescence intensity of 0–200 μg per mL PS–NH₂ after 0, 100 or 200 μg per mL biochar interaction, diluted 30×. Error bars represent standard deviation of at least six replicates from two trials. One-way ANOVA was used followed by Tukey test where ** represents p < 0.01.

Fig. 2. Biochar reduces nanoplastic on bacterial surface. (A) Confocal microscopy images of bacteria with 0 or 200 μg per mL biochar under simultaneous or prior exposure to 100 μg per mL PS–NH₂. Scale: 5 μm. (B) Nanoplastic fluorescence mean intensity on bacteria with 0 or 200 μg per mL biochar during simultaneous or prior exposure to PS–NH₂. Error bars represent standard deviation of three replicates, where *, **, and *** represent p < 0.05, 0.01 and 0.001, respectively, from one-way ANOVA followed by Tukey test. (C) SEM images showing PS–NH₂ on bacteria after prior exposure condition from 0–50 μg per mL PS–NH₂. Scale: 500 nm.

Fig. 3. Biochar pre-conditioning to nanoplastic improves bacterial growth. (A) Bacterial growth for 20 h recorded every hour in nutrient-rich media with 0–200 μg per mL PS–NH₂ with 0 or 200 μg per mL biochar. (B) Maximum growth of bacteria obtained from growth curves in (A). Bars represent standard deviation of at least three replicates from three trials, where n.s. is non-significant, * and ** from t-test represent p < 0.05 and p < 0.01, respectively.

Fig. 4. Biofilms on agar and surface coverage after nanoplastic conditioning with biochar. (A) 1 d biofilms on agar after exposure to PS–NH₂ without or with prior exposure of 200 μg per mL biochar. Scale: 1 cm. (B) Average biofilm surface coverage after 1 d growth on agar. Bars represent standard deviation of at least three biofilms and experiments were repeated at least three times, where ** represents p < 0.01 from t-test. (C) 3 d biofilms on agar after exposure to 0, 50 and 100 μg per mL PS–NH₂ with or without biochar. Biofilm images were taken from the top of the agar plate.