Single-Microbe Transcriptomics Reveal Functional Heterogeneity in Sediment Microbiomes

Sediment microbiomes drive global biogeochemical cycling, but their functional heterogeneity and transcriptional activity are hardly resolved at single-cell resolution. We optimized a scalable single-microbe RNA sequencing workflow for environmental muddy sediment samples, achieving high-throughput single cell sequencing across environmental gradients. The method integrates iohexol-based microbial enrichment, optimized enzymatic digestion step, droplet-based single-cell barcoding, and RNA sequencing, then generating a transcriptomic atlas of 55 859 high-quality microbial cells from 8 composite samples covering freshwater, estuarine and nearshore marine habitats. Comparison with metagenomic data showed a high consistency in species composition, supporting the reliability of single-microbe RNA sequencing in community profiling. Taxonomic analysis revealed site-specific microbial communities associated with methane oxidation, sulfur cycling and anaerobic nitrogen metabolism. 15 distinct functional clusters were identified transcriptionally, showing functional heterogeneity within sedimentary microbiome. Co-expression network analysis further resolved coordinated gene modules, with module activities varying across samples. Overall, this work establishes a practical framework for single-microbe transcriptomics in sediment systems and demonstrates the potential of single-cell resolution to uncover functional heterogeneity within complex microbial communities.