Image Cytometry and Kinetic Modelling Reveal How Aged Leaf Biomass Dose Regulates Microbial Physiology and PAH Degradation

Purpose: Organic amendments are widely applied to enhance bioremediation of PAH-contaminated soils; however, remediation performance varies with amendment dosage, and the mechanisms linking dose, microbial physiological state, and degradation kinetics remain unclear. This study integrated kinetic modelling with imaging-enabled cytometry and machine learning to determine how aged leaf-biomass dose regulates microbial physiology and PAH degradation efficiency. Methods: PAH-contaminated soil containing phenanthrene and pyrene (249.15 mg kg⁻¹ combined) was amended with pine–poplar leaf litter at 10%, 30%, and 70% (w/w), alongside unamended (SS) and leaf-only (AL) treatments, followed by 30-day aerobic aging. Enriched consortia and tolerant isolates were evaluated in liquid minimal salt medium (40 mg L⁻¹ PAH mixture). Degradation kinetics were modelled using first-order decay and validated by GC–FID. Imaging flow cytometry coupled with supervised machine learning classified viable and debris-associated cells using morphology- and fluorescence-derived features. Multivariate analyses quantified associations between cellular phenotypes and degradation rate constants. Results: Amended systems achieved 60–80% PAH removal within 10 days, whereas unamended soil retained approximately 70% residual PAHs. The 10% amendment exhibited the highest degradation rate (k ≈ 0.153 day⁻¹; t½ ≈ 4.5 days), compared with slower kinetics in unamended soil (k ≈ 0.052 day⁻¹). Imaging cytometry revealed sustained viable-cell fractions in amended treatments and elevated mortality under PAH-only conditions. Machine learning classifiers achieved near-perfect discrimination (ROC–AUC ≈ 1.00). Morphology-derived cellular metrics showed the strongest correlation with degradation rates (ρ ≈ 0.89), exceeding viability-based associations (ρ ≈ 0.83). Conclusion: Moderate aged-leaf biomass doses optimized PAH degradation by preserving microbial physiological integrity rather than merely increasing biomass. Imaging-derived cellular phenotypes quantitatively predicted degradation kinetics, demonstrating that amendment dose regulates remediation efficiency through microbial physiological control. Integrating amendment optimization with cytometry-based physiological monitoring provides a scalable framework for predictive and cost-efficient PAH bioremediation.