期刊
FEMS MICROBIOLOGY ECOLOGY
卷 91, 期 10, 页码 -出版社
OXFORD UNIV PRESS
DOI: 10.1093/femsec/fiv106
关键词
soil microorganisms; single-cell methods; NanoSIMS; Raman microspectroscopy; Nycodenz; stable isotopes
类别
资金
- Marie Curie International Incoming Fellowship [300807]
- Marie Curie Career Integration Grant [321742]
- Austrian Science Fund FWF [P25700-B20]
- ERC [294343]
- DOE Office of Science User Facility [DE-AC02-05CH11231]
- JGI Emerging Technologies Opportunity Program
- Austrian Science Fund (FWF) [P25700] Funding Source: Austrian Science Fund (FWF)
- European Research Council (ERC) [294343] Funding Source: European Research Council (ERC)
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 N-2-fixing and cellulose-responsive microorganisms via N-15(2) and C-13-UL-cellulose incubations, respectively. The same procedure was also applicable for Raman microspectroscopic analyses of soil microorganisms, assessed via microcosm incubations with a C-13-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.
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