Biogeochemical stratification governs microbial hydrocarbon degradation potential in a petrochemical contaminated...

The success of in situ bioremediation at petrochemical-contaminated sites (defined as those impacted by petrochemical production or accidental release) depends on the structure and functional capacities of indigenous microbial communities, as well as the physicochemical gradients that shape their metabolic potential. While microbial degradation mechanisms are well-documented in homogeneous environments, systematic evaluations of microbial dynamics across environmental compartments (e.g., surface/middle/deep soil vs. groundwater) remain scarce. In this study, we combined chemical profiling with shotgun metagenomic sequencing to characterize layer-specific microbial assemblages and hydrocarbon degradation pathways at a historically contaminated petrochemical site. Total petroleum hydrocarbons (TPHs) were markedly higher in the middle and deep soil layers compared to the surface. In subsurface layers, elevated organic carbon and moisture limited oxygen diffusion, and high concentrations of hydrophobic hydrocarbons imposed toxic stress, together creating a reducing environment that favored anaerobic taxa and pathways such as benzoyl-CoA reductase. In contrast, surface soils, with greater aeration and lower pollutant levels, harbored approximately 50 % greater microbial α-diversity (P < 0.05) and supported more diverse and complex metabolic capabilities. Groundwater showed an 83 % detection frequency of naphthalene, and its high solubility and toxicity selected for specialized degraders. Principal Coordinates Analysis (PCoA) revealed distinct depth-dependent community clustering of microbial communities (P < 0.001), with middle/deep soil microbiomes showing significantly reduced metabolic versatility for xenobiotics (BTEX, PAHs, and derivatives) compared to surface soils and groundwater at the community level. Functional gene annotation identified rate-limiting enzymes in aerobic/anaerobic degradation pathways (dmpK [benzene], badA [ethylbenzene], nahA [naphthalene], and fadA [fatty acid β-oxidation]), and Metagenome-Assembled Genomes (MAGs)-based reconstructions revealed a systemic bias towards anaerobic degradation. These mechanistic insights guide layer-specific in situ bioremediation, integrate environmental gradients with microbial functional potential for targeted treatments, and provide a framework for predicting community succession and functional resilience at petrochemical-contaminated sites.