Surface-Enhanced Raman Spectroscopy Combined with Stable Isotope Probing to Monitor Nitrogen Assimilation at Both Bulk and Single-Cell Level

Microbe-mediated biogeochemical cycle of nitrogen is a critical process in the environment. In this study, surface-enhanced Raman spectroscopy combined with 15N stable isotope probing (SERS–15N SIP) was developed as a new, nondestructive, and robust approach to probe nitrogen assimilation by bacteria at both bulk and single-cell level, and from pure culture to environmental microbial community. Multiple distinguishable SERS band shifts were observed and displayed a linear relationship with 15N content, because of the substitution of "light" nitrogen by "heavier" 15N stable isotope. These shifts, especially in 730 cm–1 band, were highly distinguishable and universal in different bacteria, providing a robust indicator for nitrogen assimilation in bacteria. SERS–15N SIP was also demonstrated in important N2-fixing bacteria via 15N2 incubations. The same prominent shifts as that induced by 15NH4Cl were observed, indicating the applicability of SERS–15N SIP to different nitrogen sources. SERS–15N SIP was further applied to environmental microbial community via 15NH4Cl, 15NO3–, and 15N2 incubation. Bacteria- and nitrogen source-dependent activity in nitrogen assimilation were revealed in environmental microbial community, pointing to the bacterial diversity and necessity of single-cell level investigation. Finally, by mixing optimized ratio of bacteria with Ag NPs, explicit single-cell SERS–15N SIP was obtained. The nondestructive SERS–15N SIP approach will be useful not only to identify active nitrogen-assimilating cells, but also enable Raman activated cell sorting and downstream genomic analysis, which will bring in deep insights into nitrogen metabolism of environmental microorganisms.