Development and use of NanoSIMS-ISH to decipher functional networks of uncultured microbes in complex ecosystems

International audienceConsiderable attention has been drawn on methods allowing simultaneous observation of microbial identity and function such as FISH-MAR or RAMAN-FISH. We have developed a new method called NanoSIMS-ISH (Nano Secondary Ion Mass Spectrometry-In Situ Hybridization) that allows the visualization of microbial identity together with the exclusive possibility to measure isotopic ratios at the single-cell level. Proof of concept was obtained by hybridization of 16S rRNA targeted iodized oligonucleotide probes on cell mixtures of E. coli, B. subtilis and P. abyssi exhibiting various 13C and 15N contents. Isotopic ratios obtained with IRMS and NanoSIMS at the single cell level exhibit some differences that might be due to fixation and hybridization procedure (Li et al., 2008). To illustrate the application of this new methodology to complex environmental ecosystems, 13C-methanol injection experiments were carried out in anaerobic digester samples. Physico-chemical analysis revealed that degradation was processing through a complex scheme. A rDNA SIP-survey was conducted revealing the implication of multiple groups related to phyla Firmicutes, Proteobacteria and to class Methanomicrobia. Specific probes were designed and used for NanoSIMS-ISH. Different patterns of cellular 13C-enrichment evolution during time could be distinguished. A mathematical model was designed and allowed us to propose a network of functional relationships between microbial community members. This study shows that evolution during time of the different phylotypes' isotopic composition can be specifically monitored at the single-cell level using NanoSIMS-ISH. This new data can be interpreted using mathematical models to shed light into functional networks of uncultured microorganisms