Single-Cell Analysis of Microbial Degradation Mechanisms and Remediation Potential for Emerging Pollutants: A Case Study on Methylnaphthalene

Polycyclic aromatic hydrocarbon (PAH) derivatives, including alkyl-PAHs, are significant emerging environmental contaminants. Traditional culture techniques often fail to isolate functional microorganisms responsible for their <i>in situ</i> degradation, leaving their metabolic interactions and pathways largely uncharacterized. This study employs 2-methylnaphthalene (MP) as a model compound and utilizes Raman-activated cell sorting with stable isotope probing (RACS-SIP) and single-cell genome sequencing to investigate alkyl-PAH degradation in petroleum-contaminated wastewater. RACS-SIP identified two key <i>in situ</i> MP-degrading microbes, <i>Sphingomonas</i> sp. and <i>Pseudomonas</i> sp., showing Raman shifts from 1001 to 968 cm^-1 and 782 to 768 cm^-1. Genome analysis linked these microorganisms to their specific genes and metabolic pathways, revealing distinct degradation mechanisms: <i>Sphingomonas</i> sp. hydroxylates nonmethyl-substituted aromatic rings, while <i>Pseudomonas</i> sp. utilizes both hydroxylation and methyl end oxidation. These findings were substantiated by functional gene quantification, emphasizing the potential of microbial consortia with diverse metabolic pathways to enhance degradation efficiency through cooperative interactions and reduced metabolic load. Moreover, the successful cultivation of RACS-sorted bacteria demonstrated superior pollutant removal compared to traditional methods. This study advances our understanding of microbial biotransformation of emerging contaminants and demonstrates the necessity of precisely identifying and cultivating <i>in situ</i> functional microorganisms to develop robust consortia for sustainable pollutant degradation.