Microfluidic Droplet-Assisted Single-Cell Raman Sorting for High-Throughput Screening of Functional Lignin-Degrading Microbes

Lignin, a major component of plant biomass, poses significant challenges for sustainable valorization due to its structural recalcitrance. Traditional microbial screening methods are hindered by culturability limitations and time-consuming workflows. This study introduces a novel Microfluidic Droplet-assisted Single-Cell Raman sorting system (MD-SCR sorting) to address these bottlenecks. By integrating droplet microfluidics with single-cell Raman spectroscopy (SCRS), the platform enables non-destructive, high-throughput screening of lignin-degrading microbes directly from environmental samples. A deuterium tracing strategy was developed, where microbial strains were cultured in lignin media containing 30% D ₂ O. Raman detection of C-D bond vibrations (2040–2300 cm ⁻¹ ) allowed in situ identification of active degraders within 12 hours. The MD-SCR system achieved precise single-cell encapsulation, cultivation, and sorting using a custom-designed three-layered microfluidic chip. Validation experiments demonstrated strong correlation (R ² =0.92) between %C-D values (quantifying deuterium assimilation) and traditional shake-flask degradation efficiency. Two lignin-degrading fungi (Pleurotus ostreatus G5 and P. eryngii PB) exhibited distinct C-D peaks, while non-degrading controls showed no deuterium incorporation. Laser intensity optimization (5% power) ensured spectral fidelity with >95% cell viability. The platform’s label-free, culture-independent workflow facilitates rapid functional screening of unculturable microbes, overcoming limitations of fluorescence-based methods. This technology not only advances lignin bioconversion by enabling targeted isolation of high-efficiency degraders but also establishes a scalable framework for exploring complex microbiomes. By bridging metabolic phenotyping with single-cell resolution sorting, the MD-SCR system offers transformative potential for sustainable resource utilization and environmental biotechnology.