Single cell stable isotope probing with FISH-Raman spectroscopy for deciphering the ecophysiology of uncultured bacteria

Millions of microbial species thrive on Earth, but till today microbiologists have only succeeded in isolating a few thousand bacterial and archaeal species. Uncultured microbes are essential for the health of the planet and provide, due to their enormous genetic diversity, a potentially rich source for new biotechnologically relevant products. However, for most uncultured microbes no information on their physiological capabilities is available. Thus, there is an obvious need for the development of methods which allow to simultaneously identifying and investigating the physiology of microorganisms within their natural habitat. For this purpose, we have coupled fluorescence in situ hybridisation (FISH) with Raman microscopy for simultaneous cultivation-independent identification and determination of 13C incorporation into microbial cells. Cells which were 13C labelled through anabolic incorporation of the isotope exhibited key redshifted spectral peaks in the highly resolved Raman confocal spectra, the calculated ‘red shift ratio’ being highly correlated with the 13C-content of the cells. Subsequently, Raman instrumentation and FISH protocols were optimized to allow combined epifluorescence and Raman imaging of probe-labeled microbial populations at the single cell level. In order to demonstrate the suitability of this technology for structure-function analyses in complex microbial communities, Raman-FISH was deployed to show the importance of Pseudomonas populations during naphthalene degradation in groundwater microcosms.