Identifying the Active Phenanthrene Degraders and Characterizing Their Metabolic Activities at the Single-Cell Level by the Combination of Magnetic-Nanoparticle-Mediated Isolation, Stable-Isotope Probing, and Raman-Activated Cell Sorting (MMI-SIP-RACS).

Magnetic-nanoparticle-mediated isolation coupled with stable-isotope probing (MMI-SIP) is a cultivation-independent higher-resolution approach for isolating active degraders in their natural habitats. However, it addresses the community level and cannot directly link the microbial identities, phenotypes, and in situ functions of the active degraders at the single-cell level within complex microbial communities. Here, we used 13C-labeled phenanthrene as the target and developed a new method coupling MMI-SIP and Raman-activated cell sorting (RACS), namely, MMI-SIP-RACS, to identify the active phenanthrene-degrading bacterial cells from polycyclic aromatic hydrocarbon (PAH)-contaminated wastewater. MMI-SIP-RACS significantly enriched the active phenanthrene degraders and successfully isolated the representative single cells. Amplicon sequencing analysis by SIP, 13C shift of the single cell in Raman spectra, and the 16S rRNA gene from single cell sequencing via RACS confirmed that Novosphingobium was the active phenanthrene degrader. Additionally, MMI-SIP-RACS reconstructed the phenanthrene metabolic pathway and genes of Novosphingobium, including two novel genes encoding phenanthrene dioxygenase and naphthalene dioxygenase. Our findings suggested that MMI-SIP-RACS is a powerful method to efficiently and precisely isolate active PAH degraders from complex microbial communities and directly link their identities to functions at the single-cell level.