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)

Abstract

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 13 C-labeled phenanthrene as the target and developed a new method coupling MMI−SIP and Ramanactivated 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, 13 C 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.

Electron Transfer in the Biogeochemical Sulfur Cycle

“…Additionally, electrochemical measurement methods are crucial for characterizing the electrochemical activity of redox proteins or nanowires [137]. By combining recent molecular microbiological approaches, such as genome editing or sequencing [100,138], isotope probing coupled with Ramanactivated cell sorting [139,140], and microbiomics integrated with data mining [141,142], we can develop a powerful tool for identifying key proteins or extracellular components expressed by microorganisms involved in electron transfer. Additionally, this combined approach can facilitate the faster and more accurate discovery of novel electroactive microorganisms.…”

Advances and perspectives of using stable isotope probing (SIP)-based technologies in contaminant biodegradation

“…This challenge was partially addressed by a study using magnetic nanoparticles (MNP) to assist isolation of active phenol degraders and compared to those identified through DNA-SIP application of 13 C-labeleld phenol ( Zhang et al., 2015 ). Subsequent applications of MNP with SIP and cell-sorting techniques have been reported to identify active acetonitrile degraders ( Sun et al., 2021 ) and phenanthrene degraders ( Li et al., 2018 , 2022 ). Yet, as magnetic Fe 3 O 4 nanoparticles have been shown toxicity to microbes ( Manke et al., 2013 ), this aspect needs to be thoroughly investigated to determine whether MNP can truly capture the targeted metabolically active microbes under various environmental conditions.…”

Targeted Cell Labeling and Sorting of Prokaryotes for Cultivation and Omics Approaches

“…RACS has been shown to be capable of in situ single-cell detection, identification, and sorting of active cells in complex environments and was successfully used to sort carotenoid-containing cells (Pantoea spp., Legionella spp., Massilia spp., Pseudomonas spp., and Pedobacter spp.) from mock bacterial communities [Jing et al, 2021;Li et al, 2022]. Although Raman-based characterization performs well in the absence of markers, combining it with SIP by the preceding addition of, e.g., D 2 O substantially expands the technique's potential.…”