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Applied and Environmental Microbiology, August 2002, p. 4067-4073, Vol. 68, No. 8
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.8.4067-4073.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Exploration of Inorganic C and N Assimilation by Soil Microbes with Time-of-Flight Secondary Ion Mass Spectrometry

Department of Crop and Soil Science, Oregon State University, Corvallis, Oregon 97331-7306,¹ William R. Wiley Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352²

Received 28 December 2001/ Accepted 25 April 2002

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.

Oregon Agricultural Experiment Station technical paper 11812.

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