Name: measurement of the potential rates of dissimilatory nitrate reduction to ammonium based on 14nh4+/15nh4+ analyses via sequential conversion to n2o
Uploaded: 2020-10-07T12:30:00
Description: Watch this Scientific Journal Video about Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O at JoVE.com

We thank Naoto Tanaka for helping data collection and developing the protocol. The collection of samples was supported by JSPS KAKENHI Grant Number 17K15286.

1. Preparation of a PTFE envelope for quantitatively capturing gaseous NH3
<ol>
	<li>Place a 60-mm piece of polytetrafluoroethylene (PTFE) tape (25 mm in width) on a small sheet of aluminum foil (approximately 300 mm x 450 mm in size, wiped with ethanol).</li>
	<li>Ash a glass fiber filter (10 mm in diameter with a pore size of 2.7 &#956;m) at 450 &#176;C for 4 h in a muffle furnace. Place the glass fiber filter a little above the midpoint of the longer axis of the tape (Figure 1a</stron...

The concentration and isotope ratio of NH4+ for the DNRA analysis was quantified using several methods. The concentrations and isotope ratios of NH4+ are generally measured separately. The NH4+ concentration is typically measured using colorimetric methods including an autoanalyzer4,10,15,16,17....

The artificial synthesis of nitrogen fertilizer and its widespread application have greatly perturbed the global nitrogen cycle. It is estimated that the transfer of reactive nitrogen from terrestrial to coastal systems has doubled since pre-industrial times1. A significant portion of fertilizers applied to a given field is washed away from the soil to rivers or groundwater, primarily as NO3&#8722; 2. This may cause environmental problems such as drinking water pollution, eutrophication, and the formation of hypoxia. NO3&#8722; in water environments is removed from or retained in the ecosystem via biological assimilation and various microbial dissimilatory processes. Denitrification and anammox are known to be major microbial removal processes for NO3&#8722;. Denitrification is the microbial reduction of NO3&#8722; to gaseous N products (NO, N2O, and N2) coupled with the oxidation of an electron donor, such as organic substances, thereby reducing the risk of the above-mentioned problems. Anammox also produces N2 from NO2&#8722; and NH4+; therefore, it removes inorganic N from an ecosystem. Conversely, DNRA works to retain N in an ecosystem; it is generally accepted that DNRA is performed primarily by fermentative bacteria or chemolithoautotrophic bacteria and that they reduce dissimilatory NO3&#8722; to bioavailable and less mobile NH4+.
Studies on DNRA have primarily been performed in marine or estuarine ecosystems, such as oceanic or estuarine sediments and water, salt or brackish marsh soil, and mangrove soil. Coastal or marine ecosystems are important as reservoirs for removing NO3&#8722; from terrestrial ecosystems, and in previous studies DNRA has been shown to contribute over a very wide range of NO3&#8722; removal (0&#8211;99%)3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18. Further, the existence of DNRA has been demonstrated in a wide range of environments including freshwater environments19, rice paddy soils20, and forest soils21. While these studies have shown that DNRA is potentially comparable to denitrification for NO3&#8722; removal, studies measuring the DNRA activity are still very limited compared to those measuring denitrification.
The DNRA rate has been evaluated using 15N-labeling techniques in conjunction with data analysis via analytical or numerical models. One analytical solution to calculate the DNRA rate is based on the increase in the 15N enrichment of the NH4+ pool after the addition of 15NO3&#8722; as a tracer. 15N-labeled NO3&#8722; is added to a sample and incubated, and the DNRA rate can then be calculated from the concentration and isotope ratio changes in NH4+ before and after a certain period of time. In this paper, a method to quantify the NH4+ concentration and the isotope ratio, which are required to calculate the DNRA rate, is described in detail. Basically, the method reported here is a combination of several previously reported techniques22,23,24,25,26 with modifications added to some procedures. The method is comprised of a series of five component procedures: (1) incubation of an environmental sample with the amendment of a stable isotope tracer, 15NO3&#8722;, (2) extraction and recovery of NH4+ using a &#8220;diffusion procedure&#8221; with modifications, (3) persulfate oxidation of NH4+ in the sample, consisting of indigenous NH4+ and 15NH4+ derived from 15NO3&#8722; via DNRA activity, into NO3&#8722; and 15NO3&#8722;, (4) subsequent microbial transformation of NO3&#8722; and 15NO3&#8722; to N2O isotopomers via the modified denitrifier method, and (5) quantification of the N2O isotopomers using gas chromatography&#8211;mass spectrometry (GC/MS). In the following section, first, the preparation for procedures (2) and (4) is described and then, subsequently, all five component procedures are described in detail.

<table>
	<tbody>
		<tr>
			<td>15N-KNO3</td>
			<td>SHOKO SCIENCE</td>
			<td>N15-0197</td>
			<td></td>
		</tr>
		<tr>
			<td>15N-NH4Cl</td>
			<td>SHOKO SCIENCE</td>
			<td>N15-0034</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL PP bottle</td>
			<td>SANPLATEC</td>
			<td>61-3210-18</td>
			<td>Wide-mouth</td>
		</tr>
		<tr>
			<td>Aluminum cap</td>
			<td>Maruemu</td>
			<td>1307-13</td>
			<td>No. 20, with hole</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Wako</td>
			<td>021-02195</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>HITACHI</td>
			<td>Himac CR21G II</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxygenized Gas Pressure &#38; Replace Injector</td>
			<td>SANSIN INDUSTRIAL</td>
			<td>IP-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable cellulose acetate membrane filter</td>
			<td>ADVANTEC</td>
			<td>25CS020AS</td>
			<td>Pore size 0.22 &#181;m, 25 mm in diameter</td>
		</tr>
		<tr>
			<td>Disposable syringe</td>
			<td>Termo</td>
			<td>SS-10SZ</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Disposable syringe</td>
			<td>Termo</td>
			<td>SS-01T</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (-)</td>
			<td>NISSUI PHARMACEUTICAL</td>
			<td>5913</td>
			<td></td>
		</tr>
		<tr>
			<td>Gastight syringe</td>
			<td>VICI Valco Instruments</td>
			<td>4075-15010</td>
			<td>Series A-2, 100 &#181;L</td>
		</tr>
		<tr>
			<td>GC/MS</td>
			<td>shimadzu</td>
			<td>GCMS-QP2010ultra</td>
			<td></td>
		</tr>
		<tr>
			<td>GF/D</td>
			<td>Whatman</td>
			<td>1823-010</td>
			<td>10 mm in diameter</td>
		</tr>
		<tr>
			<td>Glass vial</td>
			<td>Maruemu</td>
			<td>0501-06</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>Gray butyl rubber stopper</td>
			<td>Maruemu</td>
			<td>1306-03</td>
			<td>No.20-S</td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Wako</td>
			<td>192-04696</td>
			<td>Guaranteed Reagent</td>
		</tr>
		<tr>
			<td>K2S2O8</td>
			<td>Wako</td>
			<td>169-11891</td>
			<td>Nitrogen and Phosphorus analysis grade</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Wako</td>
			<td>163-03545</td>
			<td>Guaranteed Reagent</td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>Wako</td>
			<td>160-04035</td>
			<td>Guaranteed Reagent</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Wako</td>
			<td>191-08625</td>
			<td>Nitrogen compounds analysis grade</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Wako</td>
			<td>017-02995</td>
			<td>Guaranteed Reagent</td>
		</tr>
		<tr>
			<td>Plastic centrifuge tube</td>
			<td>ASONE</td>
			<td>1-3500-22</td>
			<td>50 mL, VIO-50BN</td>
		</tr>
		<tr>
			<td>Pseudomonas chlororaphis subsp. aureofaciens</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td>ATCC 13985</td>
			<td>Freeze-dried, the type strain of Pseudomonas aureofaciens</td>
		</tr>
		<tr>
			<td>PTFE sealing tape</td>
			<td>Sigma-Aldrich</td>
			<td>Z221880</td>
			<td>25 mm in width</td>
		</tr>
		<tr>
			<td>Reciprocating shaker</td>
			<td>TAITEC</td>
			<td>0000207-000</td>
			<td>NR-10</td>
		</tr>
		<tr>
			<td>Screw-cap test tube</td>
			<td>IWAKI</td>
			<td>84-0252</td>
			<td>11 mL</td>
		</tr>
		<tr>
			<td>PTFE-lined cap for test tube</td>
			<td>IWAKI</td>
			<td>84-0262</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>Difco Laboratories</td>
			<td>211825</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of the potential rates of dissimilatory nitrate reduction to ammonium based on 14nh4+/15nh4+ analyses via sequential conversion to n2o

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has been increasing because global nitrogen loads have dramatically increased following industrialization. Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are both microbial processes that use NO3&#8722; for respiration. Compared to denitrification, quantitative determinations of the DNRA activity have been carried out only to a limited extent. This has led to an insufficient understanding of the importance of DNRA in NO3&#8722; transformations and the regulating factors of this process. The objective of this paper is to provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. In brief, the potential DNRA rate can be calculated from the 15N-labeled ammonium (15NH4+) accumulation rate in 15NO3&#8722; added incubation. The determination of the 14NH4+ and 15NH4+ concentrations described in this paper is comprised of the following steps. First, the NH4+ in the sample is extracted and trapped on an acidified glass filter as ammonium salt. Second, the trapped ammonium is eluted and oxidized to NO3&#8722; via persulfate oxidation. Third, the NO3&#8722; is converted to N2O via an N2O reductase deficient denitrifier. Finally, the converted N2O is analyzed using a previously developed quadrupole gas chromatography&#8211;mass spectrometry system. We applied this method to salt marsh sediments and calculated their potential DNRA rates, demonstrating that the proposed procedures allow a simple and more rapid determination compared to previously described methods.

The representative results presented in this paper were derived from 15N-tracing experiments of salt marsh sediments. The sampled salt marsh was newly created in the aftermath of the 2011 Great East Japan Earthquake in the Moune area of Kesen-numa city in Miyagi Prefecture, Japan. In September 2017, surface sediments (0&#8211;3 cm) were collected at two sites in the subtidal and intertidal zones. First, immediately after collection, the sediment was sieved through a 4-mm mesh t...

Watch this Scientific Journal Video about Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O at JoVE.com

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

A series of methods to determine the potential DNRA rate based on 14NH4+/15NH4+ analyses is provided in detail. NH4+ is converted into N2O via several steps and analyzed using quadrupole gas chromatography&#8211;mass spectrometry.

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

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has ...

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