µ-Raman and GC-MS to interrogate the heterogeneity of deuterium-label incorporation on the single cell vs. fatty acid isotopologue level

Abstract: Understanding microbial metabolic functions at both bulk biomolecular and single-cell biomass levels requires precise tracing of isotope incorporation. Despite the established nature of compound-specific and single-cell stable isotope probing (SIP) approaches, a systematic comparison remains lacking. This study presents a comparative analysis of two SIP methods, utilizing deuterium from labelled glucose and heavy water to track incorporation into microbial fatty acids through gas chromatography-mass spectrometry (FAME-GC-MS) and into single cells via stable isotope Raman microspectroscopy (SIRM). Both methods exhibited consistent trends in deuterium incorporation across bacterial samples of Sphingomonas koreensis grown on various (deuterated) substrates in the presence of differing concentrations of D2O. The deuteration levels of single cells and fatty acids supported one another quantitatively. Besides demonstrating good agreement between both datasets, they were also shown to synergistically complement each other. Information on different cell generations was derived from clusters of varying degrees of cell deuteration, and heterogeneities between different fatty acids and their isotopologues were revealed. Additionally, variations in carbon and deuterium sources influenced fatty acid profiles and biomass deuterations. This proof-of-principle study underscores the consistent and complementary nature of lipid-SIP and single-cell-SIP approaches, providing a benchmark for future research on microbial community dynamics and organic substrate biodegradation.