Simplified preparation for the δ15N-analysis in soil [formula omitted] by the denitrifier method

Abstract

We present a simplification of the denitrifier method proposed by Christensen and Tiedje [1988. Sub-parts-per-billion nitrate method—use of an N 2O-producing denitrifier to convert NO 3 - or ( NO 3 - )-N-15 to N 2O. Applied and Environmental Microbiology 54, 1409–1413] and Sigman et al. [2001. A bacterial method for the nitrogen isotopic analysis of nitrate in seawater and freshwater. Analytical Chemistry 73, 4145–4153] for δ 15N analysis of nitrate ( NO 3 - ) and test the method for 15 NO 3 - analysis in soil extracts. A denitrifying strain without N 2O reductase ( Pseudomonas chlororaphis) was used to quantitatively reduce NO 3 - to nitrous oxide (N 2O), which was analysed for δ 15N on an isotope ratio mass spectrometer (IRMS) with a cryo-concentration unit for N 2O. The produced N 2O was collected in dry bottles, thus enabling storage and transportation of N 2O for later isotope analysis. We show both theoretically and by measurement that sampling N 2O from the headspace above the denitrifier culture does not result in a significant loss of precision and accuracy when using bottles with a headspace to culture ratios above 11 (vol/vol). We further tested the effect of P. chlororaphis cell numbers on the conversion and isotopic composition of NO 3 - ; P. chlororaphis from a 6 day culture effectively converted 1–5 μg NO 3 - within a reasonable time window, thus making tedious concentration of cells unnecessary. When applying the denitrifier method to NO 3 - extracted from soil, caution must be exercised to avoid N 2O conversion to N 2 by indigenous denitrifiers. We found this to be a problem in extracts from a soil with high N 2O reductase activity (induced by anaerobic preincubation with N 2O as the sole electron acceptor). However, a brief heating of the soil extracts to 80 °C prior to the addition of P. chlororaphis cells eliminated any indigenous activity from the samples while having no measurable effect on the isotopic composition of the measured NO 3 - . The method has a precision (SD) better than ±0.2‰ for soil extracts with 1–5 μg NO 3 - -N sample −1.