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 15 N stable isotope probing (SERS- 15 N 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 15 N content, because of the substitution of "light" nitrogen by "heavier" 15 N 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- 15 N SIP was also demonstrated in important N 2 -fixing bacteria via 15 N 2 incubations. The same prominent shifts as that induced by 15 NH 4 Cl were observed, indicating the applicability of SERS- 15 N SIP to different nitrogen sources. SERS- 15 N SIP was further applied to environmental microbial community via 15 NH 4 Cl, 15 NO 3 - , and 15 N 2 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- 15 N SIP was obtained. The nondestructive SERS- 15 N 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.