Rapid resonance Raman microspectroscopy to probe carbon dioxide fixation by single cells in microbial communities

Photosynthetic microorganisms play crucial roles in aquatic ecosystems and are the major primary producers in global marine ecosystems. The discovery of new bacteria and microalgae that play key roles in CO₂ fixation is hampered by the lack of methods to identify hitherto-unculturable microorganisms. To overcome this problem we studied single microbial cells using stable-isotope probing (SIP) together with resonance Raman (RR) microspectroscopy of carotenoids, the light-absorbing pigments present in most photosynthetic microorganisms. We show that fixation of ¹³CO₂ into carotenoids produces a red shift in single-cell RR (SCRR) spectra and that this SCRR–SIP technique is sufficiently sensitive to detect as little as 10% of ¹³C incorporation. Mass spectrometry (MS) analysis of labelled cellular proteins verifies that the red shift in carotenoid SCRR spectra acts as a reporter of the ¹³C content of single cells. Millisecond Raman imaging of cells in mixed cultures and natural seawater samples was used to identify cells actively fixing CO₂, demonstrating that the SCRR–SIP is a noninvasive method for the rapid and quantitative detection of CO₂ fixation at the single cell level in a microbial community. The SCRR–SIP technique may provide a direct method for screening environmental samples, and could help to reveal the ecophysiology of hitherto-unculturable microorganisms, linking microbial species to their ecological function in the natural environment.