Raman Stable Isotope Probing of Bacteria in Visible and Deep UV-Ranges

Raman stable isotope probing (Raman-SIP) is an excellent technique that can be used to access the overall metabolism of microorganisms. Recent studies have mainly used an excitation wavelength in the visible range to characterize isotopically labeled bacteria. In this work, we used UV resonance Raman spectroscopy (UVRR) to evaluate the spectral red-shifts caused by the uptake of isotopes (¹³C, ¹⁵N, ²H(D) and ¹⁸O) in E. coli cells. Moreover, we present a new approach based on the extraction of labeled DNA in combination with UVRR to identify metabolically active cells. The proof-of-principle study on E. coli revealed heterogeneities in the Raman features of both the bacterial cells and the extracted DNA after labeling with ¹³C, ¹⁵N, and D. The wavelength of choice for studying ¹⁸O- and deuterium-labeled cells is 532 nm is, while ¹³C-labeled cells can be investigated with visible and deep UV wavelengths. However, ¹⁵N-labeled cells are best studied at the excitation wavelength of 244 nm since nucleic acids are in resonance at this wavelength. These results highlight the potential of the presented approach to identify active bacterial cells. This work can serve as a basis for the development of new techniques for the rapid and efficient detection of active bacteria cells without the need for a cultivation step.