Recovery of urea nitrogen from seawater for measurement of 15N abundance in urea regeneration studies using the isotope-dilution approach

Abstract

Two procedures for extracting urea nitrogen from seawater for determination of its 15N abundance in urea production measurements are described. They involve hydrolysis of urea in the presence of urease and subsequent concentration of the liberated ammonium by either vacuum distillation with diffusion (distillation-diffusion method) or solely by diffusion (diffusion method). Recovery tests, both for total nitrogen and 15N content, showed that these methods are suitable for application to isotope-dilution experiments. Sufficient nitrogen for mass spectrometric analysis is recovered from 300-ml filtrate samples with low initial urea concentrations by adding ∼ 6 μmol of 14N-urea carrier. Comparisons between theoretically expected and measured isotopic compositions of urea-nitrogen standards gave a near-1: 1 correspondence for abundances ranging from 0.32 to 86.0 at.% excess 15N. The best precision obtained for isotopic determination enables the detection of differences in enrichment of 0.28 at.% excess 15N for samples with an initial enrichment (before 15N-urea carrier addition) of ∼ 4.0 at.% excess 15N. Results from an application of one of the two procedures (diffusion method) to a field study (time-course experiment) indicated that (1) urea uptake and production are strongly time dependent, (2) ignoring 15N-isotope dilution resulting from urea production leads to underestimates in calculated uptake rates of up to 25%, and (3) during the early stage of incubation (< 1 hh) up to 88% of 15N leaving the dissolved phase is not recovered in the particulate phase. A possible mechanism responsible for the apparent missing 15N suggests efflux of urea as 15N-labelled ammonia/ammonium from algal cells.