13C-Labeled Single-Cell Raman Sorting Reveals Sulfur-Driven Dark Carbon Fixation in Coastal Sediments

ABSTRACT Chemoautotrophs drive carbon fixation in coastal sediments, but most of them remain uncultured with poorly characterized in situ activities. In this study, a cultivation-independent single-cell approach combining Raman spectroscopy with 13C-stable isotope probing was developed to enable direct identification of active chemoautotrophs in coastal sediments using function-specific spectral biomarkers, targeted metagenomic sequencing and pure culture verification. 13C-induced shifts in cytochrome c (749, 1129, 1312, 1589 cm-1) and phenylalanine (1002 cm-1) Raman bands were systematically evaluated and applied as functional biomarkers through investigations of both representative chemoautotrophic strains and environmental samples. The combined analysis of targeted sorting of active chemoautotrophic cells and metagenomic sequencing revealed dominant species and a complete Calvin-Benson-Bassham cycle pathway in sulfur-oxidizing guilds. Remarkably, a novel sulfur-oxidizing chemoautotroph, Guyparkeria sp. TX1, which showed ≥99% gene sequence similarity to contigs recovered from sorted-cell metagenomes, was isolated from enrichment cultures. Its significant carbon fixation capacity provided experimental validation for the effectiveness of Raman-based in situ functional screening. This study establishes Raman-based functional biomarkers applicable to chemoautotrophic carbon fixation, enabling in situ mapping of microbial carbon fluxes. By integrating single-cell phenotypic activity with genomic potential, this work advances the mechanistic understanding of sulfur-driven dark carbon fixation, which sustains coastal blue carbon ecosystems as a keystone process.