Enzyme-driven bioremediation: hydrocarbon degradation and phytotoxicity alleviation in petroleum-contaminated soils using Bacillus subtilis α-amylase

Petroleum hydrocarbon contamination severely degrades soil quality and limits agricultural productivity, creating the need for sustainable, biologically driven remediation strategies. This study evaluated the catalytic properties, environmental persistence, and soil plant interaction effects of Bacillus subtilis α-amylase in hydrocarbon-contaminated systems. The enzyme was characterized using starch hydrolysis assays, DNS analysis, TLC, and activity profiling across temperature and pH ranges. Enzyme-amended hydrocarbon-spiked soils were monitored using FTIR and TLC to assess hydrocarbon transformation and enzyme stability, followed by pot experiments with tomato (Solanum lycopersicum) to evaluate plant growth, oxidative stress markers, and antioxidant enzyme responses over 30 days. α-Amylase showed optimal activity at 37 °C and pH 7.0 (73.2 ± 0.8 U/mg protein) with moderate thermostability (activation energy 25.6 kJ/mol). FTIR spectra revealed marked attenuation of aliphatic hydrocarbon bands and formation of oxidative degradation products by Day 30, while soil TLC and DNS assays confirmed enzyme persistence post-application. Enzyme treatment enhanced tomato shoot and root growth by 23.7% and 13.1%, respectively, and mitigated hydrocarbon-induced growth inhibition by 40–70%. Oxidative stress was reduced, as indicated by decreased H2O2 levels (from 5.0 ± 0.3 to 3.0 ± 0.2 µmol/g FW), moderated proline accumulation, and elevated antioxidant enzyme activities. Overall, the findings demonstrate that B. subtilis α-amylase is catalytically efficient, environmentally stable, and effective in promoting hydrocarbon degradation while promoting plant growth and enhancing tolerance to oxidative stress, highlighting its potential for sustainable remediation of petroleum-contaminated soils.