A Simple, High-Precision, High-Sensitivity Tracer Assay for N(inf2) Fixation

Abstract

We describe a simple, precise, and sensitive experimental protocol for direct measurement of N 2 fixation using the conversion of 15 N 2 to organic N. Our protocol greatly reduces the limit of detection for N 2 fixation by taking advantage of the high sensitivity of a modern, multiple-collector isotope ratio mass spectrometer. This instrument allowed measurement of N 2 fixation by natural assemblages of plankton in incubations lasting several hours in the presence of relatively low-level (ca. 10 atom%) tracer additions of 15 N 2 to the ambient pool of N 2. The sensitivity and precision of this tracer method are comparable to or better than those associated with the C 2 H 2 reduction assay. Data obtained in a series of experiments in the Gotland Basin of the Baltic Sea showed excellent agreement between 15 N 2 tracer and C 2 H 2 reduction measurements, with the largest discrepancies between the methods occurring at very low fixation rates. The ratio of C 2 H 2 reduced to N 2 fixed was 4.68 ؎ 0.11 (mean ؎ standard error, n ‫؍‬ 39). In these experiments, the rate of C 2 H 2 reduction was relatively insensitive to assay volume. Our results, the first for planktonic diazotroph populations of the Baltic, confirm the validity of the C 2 H 2 reduction method as a quantitative measure of N 2 fixation in this system. Our 15 N 2 protocols are comparable to standard C 2 H 2 reduction procedures, which should promote use of direct 15 N 2 fixation measurements in other systems.

Nitrogenase activity in cyanobacteria measured by the acetylene reduction assay: a comparison between batch incubation and on‐line monitoring

“…Sampling of the gas phase of a batch incubation can be done with a timing accuracy of 10±30 s. In order to obtain an error by time registration of less than 1%, incubation times of at least 15±40 min are necessary. The same is true for 15 N 2 incubations but, in this case, filtration of the samples may lead to an additional time uncertainty of up to 10 min (Montaya et al, 1996). This makes it impractical to carry out incubations shorter than 1 h, unless filtration has been done before incubation.…”

“…Some researchers have considered a conversion factor of 3 to be more appropriate; they assume that H 2 produced during nitrogen fixation is taken up by a unidirectional hydrogenase (Houchins, 1984). On the other hand, calibration of acetylene reduction in natural environments by 15 N 2 fixation has yielded conversion factors varying between 1 and 50 (Montaya et al, 1996). These differences in conversion factors were mostly unexplained, but one should note that ARA and 15 N 2 incorporation do not necessarily measure the same parameters.…”

Diel regulation of photosynthetic activity in the oceanic unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501

“…The samples were incubated at 288C in the dark for 1 h. Subsamples of the headspace were taken immediately after acetylene addition and then at the end of the incubation to measure their ethylene content with a flame ionization gas chromatograph (HRGC 5300, Carlo Erba Instruments). Ethylene production during the incubation was analysed, and produced ethylene was calculated according to Breitbarth (Breitbarth et al, 2004) and converted to fixed N 2 using a theoretical molar ratio of acetylene reduction to cellular N 2 reduction of 4:1 (Montoya et al, 1996).…”

High single‐cell diversity in carbon and nitrogen assimilations by a chain‐forming diatom across a century

“…The stable isotope incubations were terminated by filtering samples onto precombusted (450 C) GF/F filters (Montoya et al, 1996). The filters were first dried (60 C, overnight) and then decalcified by exposure to HCl fumes overnight in a glass desiccator, before being packed into tin cups.…”