Cultivation and characterization of functional-yet-uncultivable phenanthrene degraders by stable-isotope-probing and metagenomic-binning directed cultivation (SIP-MDC)

• Published in: Environment international 2024-03, Vol.185, p.108555-108555, Article 108555
• Main Authors: Li, Jibing, Luo, Chunling, Cai, Xixi, Dai, Yeliang, Zhang, Dayi, Zhang, Gan
• Format: Article
• Language: English
• Subjects: Achromobacter; antibiotic resistance; biodegradation; Cultivation; environment; genes; Metagenome binning; metagenomics; microbiology; Phenanthrene degrader; phenanthrenes; Stable isotope probing
• ISSN: 0160-4120; 1873-6750
• DOI: 10.1016/j.envint.2024.108555

[Display omitted] •Development of SIP and metagenomic-binning directed cultivation (SIP-MDC) approach.•First use of SIP-MDC in identifying and isolating active-yet-uncultivable microbes.•Cultivation medium modification with the aid of metagenomic binning.•Successful cultivation of uncultivable Achromobacter-like phenanthrene degrader. High-throughput identification and cultivation of functional-yet-uncultivable microorganisms is a fundamental goal in environmental microbiology. It remains as a critical challenge due to the lack of routine and effective approaches. Here, we firstly proposed an approach of stable-isotope-probing and metagenomic-binning directed cultivation (SIP-MDC) to isolate and characterize the active phenanthrene degraders from petroleum-contaminated soils. From SIP and metagenome, we assembled 13 high-quality metagenomic bins from 13C-DNA, and successfully obtained the genome of an active PHE degrader Achromobacter (genome-MB) from 13C-DNA metagenomes, which was confirmed by gyrB gene comparison and average nucleotide/amino identity (ANI/AAI), as well as the quantification of PAH dioxygenase and antibiotic resistance genes. Thereinto, we modified the traditional cultivation medium with antibiotics and specific growth factors (e.g., vitamins and metals), and separated an active phenanthrene degrader Achromobacter sp. LJB-25 via directed isolation. Strain LJB-25 could degrade phenanthrene and its identity was confirmed by ANI/AAI values between its genome and genome-MB (>99 %). Our results hinted at the feasibility of SIP-MDC to identify, isolate and cultivate functional-yet-uncultivable microorganisms (active phenanthrene degraders) from their natural habitats. Our findings developed a state-of-the-art SIP-MDC approach, expanded our knowledge on phenanthrene biodegradation mechanisms, and proposed a strategy to mine functional-yet-uncultivable microorganisms.