Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils.
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 and deuterium oxide (D2O, a general activity marker). The described sample preparation procedure enables single-cell analysis of soil microorganisms using NanoSIMS and Raman microspectroscopy, but should also facilitate single-cell sorting and sequencing.
Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, Research network 'Chemistry meets Microbiology', University of Vienna, Vienna 1090 Austria.
Stephanie A
SA
Eichorst
Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, Research network 'Chemistry meets Microbiology', University of Vienna, Vienna 1090 Austria.
Florian
F
Strasser
DOE Joint Genome Institute, Walnut Creek, CA 94598, USA.
Tanja
T
Woyke
Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, Research network 'Chemistry meets Microbiology', University of Vienna, Vienna 1090 Austria Large-Instrument Facility for Advanced Isotope Research, University of Vienna, Vienna 1090 Austria.
Arno
A
Schintlmeister
Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, Research network 'Chemistry meets Microbiology', University of Vienna, Vienna 1090 Austria Large-Instrument Facility for Advanced Isotope Research, University of Vienna, Vienna 1090 Austria.
Michael
M
Wagner
Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, Research network 'Chemistry meets Microbiology', University of Vienna, Vienna 1090 Austria woebken@microbial-ecology.net.
Dagmar
D
Woebken
D001105:Archaea / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D001419:Bacteria / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism; D017666:Deuterium Oxide / Q000378:metabolism; D005658:Fungi / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods*; D012988:Soil Microbiology; D018629:Spectrometry, Mass, Secondary Ion; D013059:Spectrum Analysis, Raman / Q000379:methods*
D012336:RNA, Ribosomal, 16S; D002244:Carbon; D017666:Deuterium Oxide
NanoSIMS; Nycodenz; Raman microspectroscopy; single-cell methods; soil microorganisms; stable isotopes