Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on ¹⁴NH₄⁺/¹⁵NH₄⁺ Analyses via Sequential Conversion to N₂O

Coastal and the marine ecosystems are important as reservoirs, for removing nitrate from terrestrial ecosystems. Nitrate in aquatic environment can be consumed by several processes simultaneously. Such as denitrification, anammox and DNRA.

While previous studies have shown that DNRA is potentially culpable to denitrification, studies measuring DNRA activity as still very limited compared to those measuring denitrification. In our protocol, we provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. We believe that potential DNRA rate can be calculated from N15 labeled Ammonium accumulation with N15 labeled Nitrate addition.

The advantage of our method compared to other methods, is that Ammonium is ultimately converted to Nitrous oxide, which has a low atmospheric background. Also it is not wise that we measured Nitrous oxide via quadrupole gas chromatography mass spectrometer. Which is less expensive and easy to manage than an isotope-ratio mass spectrometer.

First, place a 60 millimeter piece of PTFE tape on a small sheet of aluminum foil. Ash a glass fiber filter at 450 degrees Celsius for four hours in a muffle furnace. Then place the glass fiber filter a little above the mid point of the longer axis of the tape.

Next, spot 20 microliters of 0.9 moles per litre of sulfuric acid on the center of the glass fiber filter, and immediately fold the PTFE tape using flat ended stamp and straight ended tweezers. Flip the PTFE tape over the glass fiber filter, then seal both sides of the PTFE tape by folding and then tightly pressing the edge with the tweezers. Using the tweezers, fold the open end of the PTFE tape, and press the edge.

Seal the open end of the PTFE tape by tightly pressing the edge with the tweezers, taking care not to press the glass fiber filter. Transfer 30 milligrams of ashed Magnesium oxide, to a 20 milliliter glass vial, and place the PTFE envelope in the vial. Transfer five milliliters of a previously prepared sample or standard into the vial containing the Magnesium oxide and the PTFE envelope.

And immediately close the vial with a gray butyl rubber stopper. Then, seal the vial with an aluminum cap. Shake the vials at 150 RPM for three hours at four degrees Celsius under dark conditions.

Following this, remove the aluminum cap and the butyl rubber stopper from each vial. Remove the PTFE envelope from each vial using point ended tweezers, and thoroughly rinse the envelope and the tweezers with ion exchanged water. Then, wipe the envelope and the tweezers with wiping paper, and place the envelope on fresh wiping paper.

Open the PTFE envelope with flat ended and pointed ended tweezers in reverse order of the folding steps. Using flat ended tweezers, hold the peripheral area of the glass fiber filter, where the sulfuric acid is supposed to be unabsorbed. And transfer it into an 11 millimeter screw cap test tube.

Rinse the tweezers with ion exchanged water and wipe them with wiping paper. Add one milliliter of ion exchanged water to each test tube. Close the test tubes with PTFE lined screw caps, and allow them to stand for at least 30 minutes at room temperature, to completely elude the ammonium cation from the glass fiber filter.

Following this, open the screw cap, add two milliliters of a previously prepared persulfate oxidizing solution reagent to the test tube, and close the tube tightly with a screw cap, to prevent any loss or contamination during the following steps. Stand the test tubes on a rack, wrap them in double layered aluminum foil, and autoclave them in an upright position for one hour at 121 degrees Celsius. Mix 100 milliliters of sterile 40 millimoles per liter phosphate buffer, and 100 milliliters of sterile 30 millimoles per liter glucose aseptically.

Add a glycerol stock of P.chlororaphis to 200 milliliters of the phosphate buffered glucose solution, in a 300 milliliter Erlenmeyer flask. And purge with an ultra pure helium stream for one hour. Next, dispense two milliliters of a previously prepared denitrifier suspension into five milliliter vials.

Cap each vial with a gray butyl rubber stopper, and an aluminum closure. Replace the Headspace air with ultra pure helium, by vacuuming for three minutes and charging the helium for one minute. Set the Headspace gas positive pressure to 1.3 atmospheres, to avoid unintentional air contamination.

Inject one milliliter of a sample or standard through the butyl rubber stopper, using a one milliliter disposable syringe. Then, incubate the vials overnight at 25 degrees Celsius, under dark conditions. On the following day, inject 0.3 milliliters of six moles per liter sodium hydroxide to stop the denitrification and absorb the Headspace carbon dioxide, which will otherwise disturb the nitrous oxide analysis.

Because carbon dioxide and nitrous oxide have the same molecular weight. Determine the amounts of nitrous oxide with a molecular weight of 44, 45 and 46, in the Headspace gas using quadrupole GC/MS with a modified injection port. The representative results were derived from 15 nitrogen tracing experiments of salt marsh sediments created from the 2011 Great East Japan Earthquake in the moon area of Kesennuma city and Miyagi prefecture, Japan.

An increase in the labeled ammonium concentration throughout the incubation period was observed for all sediments collected in the subtidal and intertidal zones. The DNRA rates were within the range of 24.8 to 177. And are comparable to values reported in previous studies, but higher than values derived from similar environments.

The high DNRA rate, may be explained by the salt marsh being used as a cultivation field before the earthquake. Consistent with the speculation, the DNRA rate in the intertidal zone, which is rich and organic compounds compared to the subtidal zone, was higher. Our protocol is widely applicable to analysis of metabolic pathways, which involves ammonium formation and N15 trace additions.