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

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 C-13 and N-15 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 ((CN-)-C-12-N-14) and mass 27 ((CN-)-C-13-N-14 and (CN-)-C-12-N-15) 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 (NO3-)-N-15. We observed that the N-15 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 N-15 content of cells that have assimilated N-15-labeled mineral N and shows promise as a tool with which to explore the factors controlling microsite heterogeneities in soil.