Applications and impacts of stable isotope probing for analysis of microbial interactions.

Probing the interactions between microbes and their environment with stable isotopes became a powerful technique over the last years. While quadruple mass spectrometry or isotope ratio mass spectrometry (IRMS) require at least 300,000 bacterial cells, analysis at the single-cell level is possible with secondary ion mass spectrometry (SIMS) or Raman microspectrometry. While SIMS needs enrichments of more than 0.1 and Raman microscopy of more than 25 at.-%, IRMS can deal with 0.0001 at.-%. To find out who eats what, one has to discern between the different species in a community. Several methods have been introduced to discern between the different taxa in microbial communities, e.g., by using fatty acids as biomarkers, density centrifugation of DNA/RNA, or fluorescent in situ hybridization (FISH) with phylogenetic probes. While the biomarker approach can be coupled with the high sensitivity of the IRMS, the DNA approach gives in general a better phylogenetic resolution of the metabolic active microbes. A combination of both is the separation via coupling of FISH-probes to magnetic beads or fluorescent assisted cell sorting (FACS) of stained cells leading to fractions which can be analyzed by IRMS. Applying these techniques over a time course can reveal the metabolic kinetics and food webs. In this review, the different methods are presented with examples and their advantages and disadvantages are discussed. An outlook on the combination of the various techniques and their applications in microbial ecology is given.