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

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 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.

Keywords: NanoSIMS; Nycodenz; Raman microspectroscopy; single-cell methods; soil microorganisms; stable isotopes.

Graphical Abstract Figure.

A procedure is presented that allows one to investigate the activity of soil microorganisms at the single-cell level by combining stable isotope-labeled substrate incubations with NanoSIMS or Raman microspectroscopy.

Figure 1.

Box plot diagram of recovered cells (g dry wt soil) ⁻¹ based on DAPI staining across the tested cell detachment treatments (n = 3 per treatment) for Klausen-Leopoldsdorf (beech forest soil) (panel a) and Neustift (alpine meadow soil) (panel b). Gray box plots represent initially formaldehyde-fixed samples, whereas white box plots represent unfixed cells. Significant differences (p < 0.05) in cell recovery between formaldehyde-fixed and unfixed samples are depicted with an asterisk.

Figure 2.

Relative abundance of dominant phyla based on 16S rRNA gene amplicon sequencing for Klausen-Leopoldsdorf (beech forest soil) (panel a) and Neustift (alpine meadow soil) (panel b). Biological replicates are depicted for each treatment (T1 = PVP, T2 = combination, T3 = sonication and T4 = Tween20).

Figure 3.

Agglomerative hierarchical cluster dendrogram of triplicate microbial communities of ‘native soil’, ‘homogenized soil’, ‘cell detached soil’ and ‘cell fraction’ samples from Klausen-Leopoldsdorf soil (beech forest soil) (panel a) and Neustift soil (alpine meadow soil) (panel b) based on the Bray–Curtis distance. Scale bar indicates the similarity of the communities.

Figure 4.

SEM images of polycarbonate filters carrying an untreated soil sample exhibiting copious amounts of soil particles (panel a), whereas a Nycodenz-treated soil sample exhibited low soil particle load (panel b). Inlet in panel b represents a higher magnification image depicting cells (arrows) on the filter surface. The untreated soil had to be diluted 1:100 compared to the amount of soil in the ‘cell fraction’ so it was possible to be imaged by SEM due to the high load of particles.

Figure 5.

Single-cell isotope measurements of cells extracted from soil microcosms amended with stable isotope-labeled substrates by NanoSIMS. Panels a and b: secondary electron (e⁻) and carbon isotope composition (¹³C/(¹²C + ¹³C), given in at%) images of cells from soil microcosms amended with ¹³C-UL-cellulose. Microcosms were incubated for a 15-day period. Images depict single cells enriched in ¹³C stemming from the utilization of ¹³C-UL-cellulose. Panels c and d: secondary electron (e⁻) and nitrogen isotope composition (¹⁵N/(¹⁴N + ¹⁵N), given in at%) images of cells from soil microcosms incubated with ¹⁵N₂ gas for 21 days. Images depict single cells enriched in ¹⁵N stemming from nitrogen fixation of ¹⁵N₂ gas.

Figure 6.

Raman spectra from soil microorganisms as a function of different added amino acid concentrations, ranging from 0 to 1.0 g L⁻¹. Representative Raman spectra from soil microorganisms incubated in the presence of varying ¹³C-labeled amino acid concentrations and ¹²C glucose (panel a) and varying ¹²C-labeled amino acid concentration and ¹³C glucose (panel c). Red regions in panels a and c depict the ¹³C-phenylalanine peak region around 964 cm⁻¹. Panels b and d depict the ratios of phenylalanine peak area associated with ¹²C carbon [peak calculated between wavenumbers 1000–1005 (cm⁻¹)] and ¹³C carbon [peak calculated between wavenumbers 960–970 (cm⁻¹)] from these respective incubations. Each triangle represents a Raman spectrum of one cell. Graphical inlets depict the relationship when plotted on the log scale.

Figure 7.

D₂O incubations to monitor activity of soil microorganisms in the presence and absence of glucose as an additional energy and carbon source in soil microcosm experiments. Panel a: representative Raman spectra of soil microorganisms incubated with D₂O and glucose (blue spectrum), D₂O without carbon source (purple spectrum) and the water (H₂O) control (gray spectrum). Panel b: intensity of deuterium incorporation in randomly selected soil cells incubated in the presence of D₂O, with (blue) and without (purple) glucose, measured by %CD. The control values for the H₂O incubated cells are depicted in gray. The red horizontal line depicts the threshold for a D₂O-labeled soil cell calculated based on the H₂O control values (2.75 %CD, mean + 3 SD of % CD).