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bioRxiv · 10.1101/2025.04.25.650636

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

Jia, T.·Zhang, M.·Chen, H.·Lu, L.·Wang, L.·Wang, Y.·Zheng, G.
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Abstract

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% D2O. 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 (R2=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 platforms 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.

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Jia, T., Zhang, M., Chen, H., Lu, L., Wang, L., Wang, Y., Zheng, G.. 2025-04-25. Microfluidic Droplet-Assisted Single-Cell Raman Sorting for High-Throughput Screening of Functional Lignin-Degrading Microbes. https://doi.org/10.1101/2025.04.25.650636

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