Rates of N2 fixation were measured using the 15N2-enriched seawater method42 . 15N2 enriched seawater was prepared by injecting 1:100 (v/v) 15N2 gas (99%, Cambridge Isotopes) into filtered (0.2 μm, PALL) and degassed (MiniModule G543) seawater collected at the study site. The enriched seawater stock was then vigorously shaken to completely dissolve the 15N2 gas, and aliquots (225 mL) were added to the experimental bottles (5% of total sample volume). After two days of incubation under either ambient light or complete dark conditions, the samples were filtered through pre-combusted (450 °C, 4.5 h) 25 mm Whatman GF/F and dried overnight in an oven at 60 °C. The samples were analyzed on a CE Instruments NC2500 elemental analyzer interfaced to a Thermo-Finningan Delta Plus XP isotope ratio mass spectrometer (IRMS). For isotope ratio mass spectrometry, a standard curve to determine N mass was generated for each sample run. Based on natural abundance, N mass on the filters, incubation times, and precision of the mass spectrometer, our calculated detection limit for 15N uptake was 0.02 nmol N L−1 d−1.
15n2 gas
Manufactured by Cambridge Isotopes
Sourced in United States
15N2 gas is a stable isotope of nitrogen that is used in various scientific and industrial applications. It has a molecular formula of 15N2, where the nitrogen atoms have an atomic mass of 15 atomic mass units. The product is a colorless, odorless gas.
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4 protocols using 15n2 gas
1
Measuring N2 Fixation Rates in Seawater
2
Nitrogen Fixation Quantification in Cyanobacteria
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All strains were grown under light/dark conditions as mentioned above, and 50-ml cultures were transferred into a 125-ml airtight glass vial. DCMU (10 μM) was added to the culture, vials were flushed with pure N2, and cultures were incubated under light/dark conditions. Cells in the sealed vials were cultured overnight, and, at the D1 time point, 8 ml of headspace gas was removed followed by injection of 8 ml of 15N2 gas (Cambridge Isotope Laboratories, Inc.) (98%+). After 8 h of incubation at 30°C in light (50 µmol photons ⋅ m−2 ⋅ s−1), the cultures were collected and dried in a laboratory oven at 50°C to 60°C for 24 h. The dried pellets were ground, weighed, and sealed into tin capsules. Isotope ratios were measured by elemental analyzer-isotope ratio mass spectrometry (EA-IRMS; Thermo Fisher Scientific), and values are indicated as δ15N (‰), where the number represents a linear transform of the 15N/14N isotope ratios, representing the per-mille difference between the isotope ratios in a sample and in atmospheric N2 (49 (link)). Data presented are mean values determined on the basis of results from at least two biological replicate cultures.
3
Measuring C and N2 Fixation in Trichodesmium
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To measure rates of C and N2 fixation, single Trichodesmium colonies were incubated with 15N2 gas (Cambridge Isotope Laboratories, Tewksbury, MA, USA) and NaH13CO3 (Sigma Aldrich) in 5.9 ml vials (Exetainer, Labco, Lampeter, UK) at the respective pCO2 level. Solutions of 15N2 gas and NaH13CO3 in filtered seawater were prepared according to Klawonn et al. (2015b) . The atom percent excess (AT% excess) for 13C at the beginning of incubations was 4.2±0.2 (ambient pCO2) and 3.8±0.1 (high pCO2; quantified by gas chromatography isotope ratio mass spectrometry; n=6). The AT% excess for 15N was 4.2±0.4 (ambient pCO2) and 3.5±0.3 (high pCO2; quantified by membrane inlet mass spectrometry; n=7). Day and night incubations were conducted for 11.5 h, respectively, in an on-deck incubator shaded to 50% surface irradiance (blue acrylic shielding #2069, Delvie's Plastic Inc., Salt Lake City, UT, USA) at sea-surface temperature, allowing for gentle movement of the vials to minimize diffusion limitation to the colonies. Subsequent to incubations, colonies were fixed with paraformaldehyde (2% final concentration; Electron Microscopy Sciences, Hatfield, PA, USA) for 24 h at 4 °C in darkness, filtered onto polycarbonate filters (type GTTP, 0.2 μm, Millipore, Merck, Darmstadt, Germany), washed with milliQ water and stored at room temperature.
4
Measuring Nitrogen Fixation in Aquatic Environments
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Measurements were done as described by Mohr et al.64 . Briefly, an enriched 15N2 medium was prepared by injecting 15N2 gas (99%, Cambridge Isotopes) into pre-filtered (0.2 µm) artificial medium at a 1:100 (vol:vol) ratio. The enriched stock was vigorously shaken to completely dissolve the 15N2 gas bubble, and then added to the experimental bottles (0.5% and 5% of monoculture media and Qishon sample volume, respectively18 (link)). This procedure assured adequate equilibration of the 15N2 with the media/estuary water. Following two days of dark incubation, the samples were filtered through pre-combusted 25-mm GF/F (450 °C, 4.5 h) and dried overnight at 60 °C. The samples were analyzed on a Thermo-Finningan Delta Plus XP isotope ratio mass spectrometer (IRMS) interfaced to a CE Instruments NC2500 elemental analyzer. A standard curve to determine N mass was done with each sample run. The detection limit was 0.02 nmol N L−1 d−1.
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