Exploration of inorganic C and N assimilation by soil microbes with time-of-flight secondary ion mass spectrometry

Abstract

Stable C and N isotopes have long been used to examine properties of various C and N cycling processes in soils. Unfortunately, relatively large sample sizes are needed for accurate gas phase isotope ratio mass spectrometric analysis. This limitation has prevented researchers from addressing C and N cycling issues on microbially meaningful scales. Here we explored the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) to detect 13C and 15N assimilation by individual bacterial cells and to quantify N isotope ratios in bacterial samples and individual fungal hyphae. This was accomplished by measuring the relative abundances of mass 26 (12C14N-) and mass 27 (13C14N- and 12C15N-) ions sputtered with a Ga+ probe from cells adhered to an Si contact slide. TOF-SIMS was successfully used to locate and quantify the relative 15N contents of individual hyphae that grew onto Si contact slides in intimate contact with a model organomineral porous matrix composed of kaolin, straw fragments, and freshly deposited manure that was supplemented with 15NO3-. We observed that the 15N content of fungal hyphae grown on the slides was significantly lower in regions where the hyphae were influenced by N-rich manure than in regions influenced by N-deficient straw. This effect occurred over distances of tens to hundreds of microns. Our data illustrate that TOF-SIMS has the potential to locate N-assimilating microorganisms in soil and to quantify the 15N content of cells that have assimilated 15N-labeled mineral N and shows promise as a tool with which to explore the factors controlling microsite heterogeneities in soil.

FIG. 1.

Schematic representation of a model soil system used to study NH₄⁺ and NO₃⁻ assimilation in fungal hyphae. Pieces of straw and manure were placed on kaolin with 1 mM ¹⁵NO₃⁻ added, covered with an Si contact slide, and incubated for 5 days.

FIG. 2.

Partial high-mass-resolution spectra of smears of P. fluorescens before (A and B) and after (C and D) sputtering, showing the elimination of isobaric signals from adventitious C. Note the scale on the y axis.

FIG. 3.

Comparison of partial negative TOF-SIMS spectra of smears of P. fluorescens grown with natural-abundance NH₄⁺ (A) or 99 atom% ¹⁵NH₄⁺ (B) as the sole N source.

FIG. 4.

Comparison of partial negative TOF-SIMS spectra of smears of N. europaea grown with ¹²CO₂ and ¹⁴NH₄⁺ (A), ¹³CO₂ and ¹⁴NH₄⁺ (B), ¹²CO₂ and ¹⁵NH₄⁺ (C), or ¹³CO₂ and ¹⁵NH₄⁺ (D) as the sole C and N sources.

FIG. 5.

Comparisons of an epifluorescence image (A) and ²⁶CN⁻ (B), ²⁷CN⁻ (C), and ²⁸CN⁻ (D) ion images of DAPI-stained N. europaea cells grown with 99 atom% ¹⁵NH₄⁺ as the sole N and energy source and 98 atom% ¹³CO₂ as the sole C source. Bar, 10 μm.

FIG. 6.

Secondary electron image (A) and light micrographs (B and C) of Si contact slides that were in contact with a model soil system. The system consisted of a straw-manure interface on kaolinite to which ¹⁵NO₃⁻ was added. The area in panel A is bracketed in yellow in panel B. Dark, electron-poor sputtered areas in panel A are represented by white boxes in panel B. The values adjacent to the analysis areas are the ¹⁵N contents (atoms percent) of the region of interest of the fungal hyphae (shaded red). The dashed line represents the straw-manure interface. (C) Light micrograph of an Si contact slide illustrating microsite heterogeneity of differential ¹⁵N assimilation by fungal hyphae growing across a model soil microsite. The arrangement is similar to that of panel B, except that the dashed line represents the straw boundary and the manure boundary is about 10 μm left of and parallel to the panel boundary. Bars, 100 μm.

FIG. 7.

Secondary ion images of an Si contact slide from a clay riparian soil. The soil was labeled with 1 mM ¹⁵NH₄⁺ and 1 mM NO₃⁻. (A) Yellow represents SiO₂⁻, an indicator of the presence of inorganic soil particles; blue represents the sum of the ²⁶CN⁻ and ²⁷CN⁻ ion counts, an indicator of organic matter. Bar, 1.7 μm. (B) The same image as in panel A, except that red represents ²⁶CN⁻ and green represents ²⁷CN⁻, showing the assimilation of ¹⁵N by the putative rod-shaped bacteria. (C) A nearby image showing a fungal hypha and a bacterium; red represents ²⁶CN⁻, and green represents ²⁷CN⁻. Bar, 2.5 μm.