Unveiling soil health recovery after phytoremediation: Insights from a metaproteomic approach

The recovery of soil health in multi-contaminated sites remains a critical environmental challenge due to the simultaneous presence of organic and inorganic pollutants. While laboratory-scale experiments provide promising insights, in-field long-term validation is essential to assess soil health recovery under real conditions. In this study, a previously optimized phytoremediation approach was applied in a multi-contaminated site to evaluate its effectiveness in reducing pollutant loads and restoring microbial communities. The design included three treated pilot areas and an untreated control. Metaproteomics analyzed microbial functional activity and taxonomic shifts, supported by a protein extraction protocol for complex matrix. Results showed a marked reduction of contaminants: the sum of polychlorinated biphenyls (PCBs) decreased from 8.35 to 7.68 mg/kg in the control after 5 years, while in treated pilots areas it dropped to 0.68 mg/kg. Among heavy metals, significant declines were observed when comparing the untreated bulk control (B1) with the pilot areas treated through the biotechnological phytoremediation approach, with average reductions of about 92 % for mercury, 70 % for cadmium, 56 % for zinc, and 61 % for lead. Metaproteomic analysis revealed a restored microbial metabolic profile in treated soils, with increased abundance of metabolic enzymes (e.g., GAPDH, isocitrate dehydrogenase), stress proteins (GroEL, HSP70), and biosynthetic enzymes for amino acid and nucleotide production. Microbial taxa enriched in treated areas included Pseudomonas knackmussii and Microbacterium hydrocarbonoxydans. Conversely, control soils showed stress-dominated proteomes with limited metabolic capacity. These findings support the efficacy of phytoremediation and demonstrate the power of metaproteomics in monitoring ecological recovery.