Metabolic Characterization and Geochemical Drivers of Active Hydrocarbon‐Degrading Microorganisms

Abstract Understanding the metabolic characteristics and controlled geochemical factors of functional microorganisms in petroleum‐contaminated areas at different locations is pivotal for enhancing pollutant removal strategies. To address the existing research gap in this domain, we employed stable‐isotope‐probing (SIP) with multi‐isotope labeling substrates, combined with 16S amplicon sequencing, metagenomic sequencing, and geochemical factor analysis. Utilizing n ‐hexadecane and phenanthrene as model compounds, our study revealed location‐specific differences in the composition of functional microorganisms. Despite these variances, key players such as Pseudomonas , Marinobacter , Alcanivorax , Ochrobactrum , and Sphingomonas consistently emerged as active degraders of n ‐hexadecane and/or phenanthrene. Several genera, including Pseudomonas , Ochrobactrum , Alcanivorax , Nitriliruptoraceae , and Sphingobacterium , demonstrated versatility by effectively degrading both contaminants. SIP‐metagenomic binning facilitated the acquisition of genomes from key active degraders, such as Pseudomonas sp., Ochrobactrum sp., Sphingomonas sp., and Shinella sp. This enabled a comprehensive analysis of petroleum hydrocarbon degradation pathways and genes, encompassing PAH dioxygenase genes, alkB genes, phthalate, and salicylate‐related pathways. Environmental factor and variation partitioning analysis revealed that oil pollution significantly influences the functional microbial community (12%), followed by available potassium and available nitrogen. Geochemical parameters and geographic location independently explained 14% and 21% of total variations, respectively. Intriguingly, more than half (51%) of the variation in functional microbial community structure remains unexplained, possibly due to unmeasured environmental variables. Our study contributes valuable insights into the in situ bioremediation mechanism for petroleum‐contaminated soil, elucidating factors influencing functional microbial structures across locations. These findings provide a vital theoretical reference for in situ regulation and bioremediation of petroleum hydrocarbon pollution in diverse environmental contexts.