Droplet-Shielded Micropore Raman-Activated Cell Sorting Improves Recovery of Emerging-Contaminant-Degrading Microorganisms

Abstract Identifying, isolating, and cultivating functional microorganisms from complex environmental microbiomes remains a central challenge in linking microbial identity with contaminant transformation. Conventional Raman-activated cell sorting (RACS) enables label-free single-cell phenotyping but often compromises cell viability during laser interrogation and ejection, limiting the cultivation of functional indigenous microorganisms. Tire-wear-derived 6PPD-quinone (6PPDQ) is increasingly recognized as a highly toxic emerging contaminant in roadside soils, yet the indigenous microorganisms associated with its biological attenuation in roadside soils remain poorly characterized. Using 6PPDQ as a model emerging contaminant, we developed a droplet-shielded RACS platform based on a monolayer micropore-array chip that confines individual cells within picoliter-scale aqueous droplets during Raman phenotyping and laser ejection. Under the tested workflow-specific conditions, droplet-shielded RACS increased postsorting cultivation success of Escherichia coli from 13.9% to 95.6% and improved the cultivation success of 13C-6PPDQ-assimilating soil cells from 6.67% to 36.7%. Isotopic red shifts at 1001 and 1668 cm–1 enabled identification of cells assimilating 13C-6PPDQ-derived carbon. Guided by this strategy, we recovered indigenous 6PPDQ degraders affiliated with Bordetella and Brucella and confirmed their degradation capacity in pure culture. DNA-SIP provided complementary evidence for assimilation of 13C-labeled ring-derived carbon by the corresponding taxa. Single-cell genome amplification and sequencing identified cytochrome P450-, protocatechuate-, and catechol-metabolism-associated genes, indicating predicted metabolic potential in 6PPDQ transformation. Overall, droplet-shielded RACS provides a high-viability workflow for recovering contaminant-associated microorganisms from complex environmental microbiomes and enables their subsequent cultivation and functional characterization.