Tracking heavy water (D₂O) incorporation for identifying and sorting active microbial cells

Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D ₂O) combined with Raman microspectroscopy. Incorporation of D ₂O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D ₂O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D ₂O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics. Significance Measuring activity patterns of microbes in their natural environment is essential for understanding ecosystems and the multifaceted interactions of microorganisms with eukaryotes. In this study, we developed a technique that allows fast and nondestructive activity measurements of microbial communities on a single-cell level. Microbial communities were amended with heavy water (D ₂O), a treatment that does not change the available substrate pool. After incubation, physiologically active cells are rapidly identified with Raman microspectroscopy by measuring cellular D incorporation. Using this approach, we characterized the activity patterns of two dominant microbes in mouse cecum samples amended with different carbohydrates and discovered previously unidentified bacteria stimulated by mucin and/or glucosamine by co