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Title: 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 15N2 and 13C-UL-cellulose incubations, respectively. The same procedure was also applicable for Raman microspectroscopic analyses of soil microorganisms, assessed via microcosm incubations with a 13C-labeled carbon source and deuterium oxide (D2O, a general activity marker). Lastly, 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.
Authors:
 [1] ;  [1] ;  [2] ;  [1] ;  [1] ;  [1] ;  [3]
  1. Univ. of Vienna, Vienna (Austria)
  2. USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
  3. Rutgers Univ., New Brunswick, NJ (United States)
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
FEMS Microbiology Ecology (Online)
Additional Journal Information:
Journal Name: FEMS Microbiology Ecology (Online); Journal Volume: 91; Journal Issue: 10; Journal ID: ISSN 1574-6941
Publisher:
Federation of European Microbiological Societies
Research Org:
USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; soil microorganisms; single-cell methods; NanoSIMS; Raman microspectroscopy; Nycodenz; stable isotopes
OSTI Identifier:
1342958