Metagenomic insights into nitrate- and sulfate-enhanced anoxic biodegradation of PAHs in subsurface soil

Anoxic biodegradation is pivotal for remediating PAH-contaminated subsurface soils, yet its mechanisms remain poorly understood. In this study, nitrate and sulfate were used as electron acceptors to stimulate the anoxic biodegradation of PAHs in soil by indigenous bacteria. A 180-day anoxic incubation experiment was conducted, coupled with high-throughput sequencing for bacterial community composition, quantitative PCR for microbial abundance, metagenomic sequencing for functional gene profiling, and gas chromatography-mass spectrometry for PAH quantification, to characterize microbial community properties, key functional genes, and their contributions to PAH degradation. After 180 days of incubation, the addition of electron acceptors significantly increased the abundances of total and potential PAH-degrading bacteria (which increased by 0.11-0.24 and 0.09-0.46 orders of magnitude per gram of soil, respectively) and promoted the removal of 3- and 4-ring PAHs (59-64% and 26-33%, respectively). Notably, the degradation efficiency followed the order of NO₃⁻ > mixed electron acceptors > SO₄²⁻, revealing a clear preference for nitrate. Nitrate amendment selectively enriched key PAH-degrading taxa like Bacillus. Metagenomic analysis revealed the underlying microbial mechanisms: the functional pathway ko00624 (PAH degradation) was enriched, and the abundances of 15 key genes (e.g., pcaH, ligB, and pht5) involved in upstream and downstream metabolic steps were positively correlated with degradation efficiency. Comparative analysis showed that differences across treatments stemmed primarily from ‌elevated expression of shared core genes (e.g., pht4, phdG, nidB), with nitrate (SN) treatment showing the greatest enrichment. These findings elucidate electron acceptor-driven anoxic PAH transformation, highlighting nitrate's dual role as a nutrient and favorable electron acceptor, and provide a basis for targeted subsurface bioremediation.