Bioremediation of naphthenic acid by Bacillus subtilis: Degradation kinetics and pathway elucidation

Naphthenic acids, toxic and persistent carboxylic acids found in petroleum contaminated water, pose a significant environmental challenge, but bioremediation offers a promising and cost-effective solution for their treatment. The present study illustrates the ability of Bacillus subtilis to degrade commercial naphthenic acid (100 mg/L) in aerobic and microaerobic settings under optimized conditions (temperature 36 °C, pH 6.0, and salinity 0.5 %). The degradation was confirmed by 47.61 ± 3.609 % reduction in total organic carbon levels within 144 h, indicating the microbial potential to mineralize organic naphthenic acid in aqueous medium as a sole carbon source. Naphthenic acids, being structurally complex and comprising a diverse array of carboxylic acids, were further studied using two representative models, hexanoic acid (linear) and benzoic acid (aromatic). These representative acids were selected to investigate the degradation kinetics and to elucidate the underlying degradation mechanism. The growth kinetics of B. subtilis on hexanoic acid and benzoic acid followed the Monod growth model, with maximum specific growth rates (μ_max) of 0.17344 ± 0.004 and 0.15088 ± 0.006 day^-1 respectively. The biodegradation kinetics followed a non-linear first-order rate model, with rate constants (k) of 0.43 ± 0.084 h^-1 for hexanoic acid and 0.12 ± 0.02 h^-1 for benzoic acid. Corresponding half-lives (t₁/₂) were determined as 13.37 h for hexanoic acid and 29.52 h for benzoic acid, demonstrating a faster degradation rate for hexanoic acid compared to benzoic acid. GC-MS analysis elucidated the degradation pathway, catechol and muconic acid were identified as the key intermediates, which suggest a potential metabolic breakdown. Consequently, it demonstrates the potential of Bacillus subtilis for the effective removal of naphthenic acids from polluted wastewater.