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 (13)C and (15)N 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 ((12)C(14)N(−)) and mass 27 ((13)C(14)N(−) and (12)C(15)N(−)) 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 (15)N 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 (15)NO(3)(−). We observed that the (15)N 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 (15)N content of cells that have assimilated (15)N-labeled mineral N and shows promise as a tool with which to explore the factors controlling microsite heterogeneities in soil.