Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils

FEMS Microbiology Ecology
Stephanie A EichorstDagmar Woebken

Abstract

The combined approach of incubating environmental samples with stable isotope-labeled substrates followed by single-cell analyses through high-resolution secondary ion mass spectrometry (NanoSIMS) or Raman microspectroscopy provides insights into the in situ function of microorganisms. This approach has found limited application in soils presumably due to the dispersal of microbial cells in a large background of particles. We developed a pipeline for the efficient preparation of cell extracts from soils for subsequent single-cell methods by combining cell detachment with separation of cells and soil particles followed by cell concentration. The procedure was evaluated by examining its influence on cell recoveries and microbial community composition across two soils. This approach generated a cell fraction with considerably reduced soil particle load and of sufficient small size to allow single-cell analysis by NanoSIMS, as shown when detecting active N2-fixing and cellulose-responsive microorganisms via (15)N2 and (13)C-UL-cellulose incubations, respectively. The same procedure was also applicable for Raman microspectroscopic analyses of soil microorganisms, assessed via microcosm incubations with a (13)C-labeled carbon source ...Continue Reading

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Methods Mentioned

BETA
Raman
amplicon sequencing
amplicon
PCR
scanning electron microscopy
density gradient centrifugation
nucleic acid extraction
Fluorescence Activated Cell Sorting
single-cell sequencing

Software Mentioned

UCHIME
NanoSIMS
Chao and Coverage Estimator
FLASH
WinImage
Horiba
R

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