<i>In situ</i> Raman spectroscopic relative quantitative analysis of sulfur metabolic dynamics in deep-sea microorganisms

Deciphering dynamic regulation of microbial sulfur metabolism in deep-sea environments is critical for understanding global biogeochemical cycles and climate feedback mechanisms. Current analytical approaches face limitations in achieving quantitative, <i>in situ</i> visualization of microbial metabolic processes, including susceptibility to environmental interference during sampling and analysis, leading to impaired data accuracy. This study developed an innovative method based on confocal Raman spectroscopy utilizing nitrogen as an internal standard for metabolite quantification. Taking sulfate, which is a major component of seawater and essential for the sulfur cycle, as a model, we quantified it in solid medium and monitored the <i>in situ</i> metabolic processes of deep-sea <i>Erythrobacter flavus</i> 21-3. The non-invasive technique revealed previously unrecognized light-dependent differences in microbial metabolic patterns between deep-sea and laboratory conditions through spectral visualization and relative quantification. We found that natural light exposure promoted sulfate production and enhanced zero-valent sulfur (cyclooctasulfur S₈) accumulation near the surface, accompanied by co-enrichment of carotenoids, suggesting the presence of light-driven sulfur metabolic processes. In contrast, dark conditions favored S₈ storage in the subsurface layers, potentially supported by abundant internal organic carbon sources as energy reserves. These findings may provide new insights into photo-regulated sulfur transformation mechanisms. Our approach establishes an analytical framework for <i>in situ</i> quantitative investigation of microbially mediated elemental cycling processes.IMPORTANCEMicrobial sulfur metabolism in the deep ocean is critical to global biogeochemical cycles, yet its regulatory mechanisms remain poorly understood, largely due to methodological limitations. In this study, we introduce an innovative non-invasive, quantitative approach using confocal Raman spectroscopy with molecular nitrogen (N₂) as an internal standard, overcoming major obstacles in real-time metabolic monitoring. Our results demonstrate light-dependent adaptations in sulfur metabolism among deep-sea bacteria, unveiling previously unrecognized photo-regulated sulfur transformations that refine our understanding of microbial ecological strategies in these environments. The established analytical framework provides a versatile platform for <i>in situ</i> investigation of microbial-driven elemental cycling across diverse extreme ecosystems.