The Spanish Government provided funds through the Ministerio de Educaci&#243;n as a predoctoral fellowship to C.P-L. (FPU12-00644) and research grants of the Ministerio de Economia y Competitividad: NitroPir (CGL2010-19737), Lacus (CGL2013-45348-P), Transfer (CGL2016-80124-C2-1-P). The REPLIM project (INRE - INTERREG Programme. EUUN - European Union. EFA056/15) supported the final writing of the protocol.

1. Preparation
NOTE: Begin this on the day before the measurements are taken.
<ol>
	<li>Assemble the measurement setup (Figure 1a, see the Table of Materials). 
	NOTE: To ensure a constant and high-quality power supply, the measurement device is connected to the grip via an uninterruptible power supply (UPS) that can also act as a backup. In the case of a long-duration power failure, a car battery serve as an extra...

<ol>
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The authors have nothing to disclose.

The main advantages of the described method are the use of minimally disturbed sediment core samples and the continuous recording of the N2O accumulation. These allow estimation of relatively low denitrification rates that are likely similar to those occurring in situ. Nonetheless, some aspects concerning the coring, sensor performance, and potential improvements are discussed.
An apparently simple but critical step of the method is good core recovery. The sediment/water in...

Anthropogenic alteration of the nitrogen cycle is one of the most challenging problems for the Earth system1. Human activity has at least doubled the levels of reactive nitrogen available to the biosphere2. However, there remain large uncertainties regarding how the global N cycle is evaluated. A few flux estimates have been quantified with less than &#177;20% error, and many have uncertainties of &#177;50% and larger3. These uncertainties indicate the need for accurate estimations of denitrification rates across ecosystems and an understanding of the underlying mechanisms of variation. Denitrification is a microbial activity through which nitrogenous oxides, mainly nitrate and nitrite, are reduced to dinitrogen gasses, N2O and N24. The pathway is highly relevant to the biosphere availability of reactive nitrogen because it is the primary process of removal5. N2O is a greenhouse gas with a warming potential nearly 300 times that of CO2 over 100 years, and it is the current major cause of stratospheric ozone depletion due to the large quantities being emitted6,7.
In the following, we present a protocol for estimating sediment denitrification rates using cores and N2O microsensors experimentally (Figure 1). Denitrification rates are estimated using the acetylene inhibition method8,9 and measurements of the accumulation of N2O during a defined period (Figure 2 and Figure&#160;3). We demonstrate the method by applying it to mountain lake sediments. This case study highlights the performance of the method for detecting relatively low rates with minimal disturbance to the physical structure of the sediments.
Denitrification is particularly difficult to measure10. There are several alternative approaches and methods, each with advantages and disadvantages. Drawbacks to available methods include their use of expensive resources, insufficient sensitivity, and the need to modify the substrate levels or alter the physical configuration of the process using disturbed samples10. An even more fundamental challenge to measuring N2 is its elevated background levels in the environment10. The reduction of N2O to N2 is inhibited by acetylene (C2H2)8,9. Thus, denitrification can be quantified by measuring the accumulated N2O in the presence of C2H2, which is feasible due to low environmental N2O levels.
The use of C2H2 to measure denitrification rates in sediments was developed about 40 years ago11, and the incorporation of N2O sensors occurred about 10 years later12. The most widely applied acetylene-based approach is the &#34;static core&#34;. The accumulated N2O is measured during an incubation period of up to 24 h after the C2H2 is added to the headspace of the sealed sediment core10. The method described here follows this procedure with some innovations. We add the C2H2 by bubbling the gas in the water phase of the core for some minutes, and we fill all the headspace with sample water before measuring the accumulation of N2O with a microsensor. We also include a stirring system that prevents the stratification of the water without resuspending the sediment. The procedure quantifies the denitrification rate per sediment surface area (e.g., &#181;mol N2O m-2 h-1).
The high spatial and temporal variation of denitrification presents another difficulty in its accurate quantification10. Usually, N2O accumulation is measured sequentially by gas chromatography of headspace samples that are collected during the incubation. The method described provides improved monitoring of the temporal variation of the N2O accumulation, because the microsensor provides a continuous signal. The microsensor multimeter is a digital microsensor amplifier (picoammeter) that interfaces with the sensor(s) and the computer (Figure 1a). The multimeter allows several N2O microsensors to be used at the same time. For instance, up to four sediment cores from the same study site can be measured simultaneously to account for the spatial variability.
The core approach barely disturbs the sediment structure compared to some other methods (e.g., slurries). If the integrity of the sediments is altered, this leads to unrealistic denitrification rates13 that are only adequate for relative comparisons. Higher rates are always obtained with slurry methods compared to core methods14, because the latter preserves the limitation of denitrification by substrate diffusion15. Slurry measures cannot be considered representative of in situ rates16; they provide relative measures for comparisons made with the exact same procedure.
The method described is appropriate for estimating denitrification rates in any sediment type that can be cored. We particularly recommend the method for performing experimental manipulations of some of the driving factors. Examples are experiments that modify nitrate availability and temperature as needed for estimating the energy activation (Ea) of denitrification17 (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58553/58553fig1v2.jpg" /> 
Figure 1: Experimental setup. (a) General experimental setup to estimate sediment denitrification rates using cores and N2O microsensors. The incubation chamber ensures darkness and controlled-temperature (&#177;0.3 &#176;C) conditions. Five intact sediment cores can be processed simultaneously using their respective N2O sensors. (b) N2O sensor calibration chamber. We adapted it with rubber stoppers and syringes to mix the N2O water (see protocol step 3.4.3). There is a thermometer to control the water temperature. (c) Close-up of a sediment core sample with the sensor inserted into the central hole of the PVC cover and the joints sealed with adhesive tape. The stirrer is hanging in the water, and the electromagnet is close to it and fixed to the external part of the acrylic tube. (d) Close-up of the N2O microsensor tip protected by a metal piece. (e) A sediment core that has just been recovered. It was sampled from a boat in a deep lake; the acrylic tube with the core is still fixed to the messenger-adapted gravity corer19. See the Table of Materials for all the items needed to perform this method. <a href="https://www.jove.com/files/ftp_upload/58553/58553fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" style="width:595px;" width="594">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Messenger-adapted gravity corer</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">Reference in the manuscript. Made by Glew, J.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">Sampling tube</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">Acrylic. Dimensions: 100 cm (h) &#215; 6.35 cm (d) &#215; 6.50 cm (D). Sharpen the edge of the sampling tube that penetrates into the sediment to minimize the disturbance in the recovered sediment core sample.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Handheld sounder</td>
			<td style="width:65px;">Plastimo</td>
			<td style="width:100px;">38074</td>
			<td style="width:203px;">Echotest II Depth Sounder.</td>
		</tr>
		<tr height="128">
			<td height="128" style="height:128px;width:227px;">Rubber stopper</td>
			<td style="width:65px;">VWR</td>
			<td style="width:100px;">DENE1012114</td>
			<td style="width:203px;">With two holes, used to mix the N2O-water in the calibration chamber. Dimensions: 20 mm (h) &#215; 14 mm (d) &#215; 18 mm (D) (3 mm hole (D)).</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:227px;">Rubber stopper</td>
			<td style="width:65px;">VWR</td>
			<td style="width:100px;">217-0125</td>
			<td style="width:203px;">To seal the bottom part of the methacrylate tube and to sample in shallow water bodies. Dimensions: 45 mm (h) &#215; 56 mm (d) &#215; 65 mm (D).</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:227px;">PVC cover</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">To seal the top side part of the acrylic tube. Dimensions: 45 mm (h) &#215; 56 mm (d) &#215; 65 mm (D). Dimensions: 65 mm (D).</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:227px;">Adhesive tape</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">Waterproof. To ensure all joints (PVC cover sampling tube and PVC cover sensor) and to avoid water leaks.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:227px;">Thermometer</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">Portable and waterproof, to measure the temperature in the water overlying the sediment just after sampling the cores.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">GPS</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">To save the location of a new sampling site or to arrive at a previous site.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Wader</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">For littoral or shallow site samplings.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:227px;">Boat</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">An inflatable boat is the best option for its lightness if the sampling site is not accessible by car.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:227px;">Rope</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">Rope with marks showing its length (e.g., marked with a color code to distinguish each meter).</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:227px;">N2O gas bottle and pressure reducer</td>
			<td style="width:65px;">Abell&#243; Linde</td>
			<td style="width:100px;">32768-100</td>
			<td style="width:203px;">Gas bottle reference.</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:227px;">C2H2 gas bottle and pressure reducer</td>
			<td style="width:65px;">Abell&#243; Linde</td>
			<td style="width:100px;">32468-100</td>
			<td style="width:203px;">Gas bottle reference.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:227px;">Tube used to evacuate the excess of water</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">Consists of a solid part (e.g., a 5 ml pipette tip without its narrowest end) inserted in a silicone tube.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Nitrous Oxide Minisensor w/ Cap</td>
			<td style="width:65px;">Unisense</td>
			<td style="width:100px;">N2O-R</td>
			<td style="width:203px;">We use 4 sensors at a time.</td>
		</tr>
		<tr height="235">
			<td height="235" style="height:235px;width:227px;">Microsensor multimeter 4 Ch. 4 pA channels</td>
			<td style="width:65px;">Unisense</td>
			<td style="width:100px;">Multimeter</td>
			<td style="width:203px;">Picoammeter logged to a laptop. The standard device allows for 2 sensor picoammeter connections (e.g., N2O sensor), one pH/mV and a thermometer. We ordered a device with four picoammeter connections, allowing the use of 4 N2O sensors simultaneously.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">SensorTrace Basic 3.0 Windows software</td>
			<td style="width:65px;">Unisense</td>
			<td style="width:100px;"></td>
			<td style="width:203px;">Sensor data acquisition software.</td>
		</tr>
		<tr height="107">
			<td height="107" style="height:107px;width:227px;">Calibration Chamber incl. pump</td>
			<td style="width:65px;">Unisense</td>
			<td style="width:100px;">CAL300</td>
			<td style="width:203px;">Calibration chamber. We tuned it with rubber stoppers and syringes to mix the N2O-water without making bubbles.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:227px;">Incubation chamber</td>
			<td style="width:65px;">Ibercex</td>
			<td style="width:100px;">E-600-BV</td>
			<td style="width:203px;">Indispensable equipment for working at a constant temperature (&#177;0.3 &#176;C). It also allows control of the photoperiod.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:227px;">Electric stirrer</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">Part of the stirring system. It hangs in the water, overlying the sediment subject, by a fishing line that is hooked to the PVC cover.</td>
		</tr>
		<tr height="252">
			<td height="252" style="height:252px;width:227px;">Electromagnet</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">Part of the stirring system. It is fixed to the outside of the acrylic tube, approximately at the same level as the stirrer. It is activated episodically (ca. 1 on-off per s) by a circuit, attracting the stirrer when it is on and releasing it when it is off, thereby generating the movement that agitates the water.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:227px;">Electromagnetic pulse circuit</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">Part of the stirring system. It is connected by wires to the electromagnet and sends pulses of current that turn the electromagnet on and off.</td>
		</tr>
		<tr height="170">
			<td height="170" style="height:170px;width:227px;">Uninterruptible power supply (UPS)</td>
			<td style="width:65px;">-</td>
			<td style="width:100px;">-</td>
			<td style="width:203px;">It improves the quality of the electrical energy that reaches the measurement device, filtering the highs and low of the voltage, thereby ensuring a more constant and stable N2O sensor signal.</td>
		</tr>
	</tbody>
</table>

estimating sediment denitrification rates using cores and n2o microsensors

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has become particularly relevant for assessing the anthropogenic-altered global nitrogen cycle and the emission of greenhouse gases (i.e., N2O). Several methods are available for measuring denitrification, but none of them are completely satisfactory. Problems with existing methods include their insufficient sensitivity, and the need to modify the substrate levels or alter the physical configuration of the process using disturbed samples.This work describes a method for estimating sediment denitrification rates that combines coring, acetylene inhibition, and microsensor measurements of the accumulated N2O. The main advantages of this method are a low disturbance of the sediment structure and the collection of a continuous record of N2O accumulation; these enable estimates of reliable denitrification rates with minimum values up to 0.4-1 &#181;mol N2O m-2 h-1. The ability to manipulate key factors is an additional advantage for obtaining experimental insights. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate. The method is appropriate for estimating denitrification rates in any aquatic system with retrievable sediment cores. If the N2O concentration is above the detection limit of the sensor, the acetylene inhibition step can be omitted to estimate the N2O emission instead of denitrification. We show how to estimate both actual and potential denitrification rates by increasing nitrate availability as well as the temperature dependence of the process. We illustrate the procedure using mountain lake sediments and discuss the advantages and weaknesses of the technique compared to other methods. This method can be modified for particular purposes; for instance, it can be combined with 15N tracers to assess nitrification and denitrification or field in situ measurements of denitrification rates.

A total of 468 denitrification rates were estimated using the protocol above in sediments from Pyrenean mountain lakes over the period 2013-2014. We show some of these results to illustrate the procedure (Figure 2 and Figure&#160;3). In general, the linear model between the N2O concentration and time has good correlation (R2 &#8805; 0.9). The slope of the relationship provides an estimate of the denitrificat...

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Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

This method estimates sediment denitrification rates in sediment cores using the acetylene inhibition technique and microsensor measurements of the accumulated N2O. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate.

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has ...

This method can help answer key questions in biogeochemistry related to denitrification in sediments which are relevant for understanding the nitrogen cycle and the greenhouse gas emissions. The main advantages of this technique are the low disturbance of the sediment structure and the container recording of nitrogen oxide accumulation for reliable denitrification rate estimation. For deep water bodies, select the sampling point according to the investigation aims.Take note of the position using GPS coordinates and take the measurement depth using a handheld sounder. Deploy a messenger-adapted gravity coring system until the sampling tube is approximately one meter from the sediment. Stabilize the sampling equipment for 60 seconds to ensure the correct sediment penetration and recovery of a scarcely disturbed sediment core.Release approximately one meter more rope so that the sampling tube penetrates the sediment. Be aware that if the sampling tube penetrates too much, it can disturb the water-sediment interface. Release the messenger while trying to keep tension in the rope so that the corer remains fixed and in a vertical position.Recover the corer by pulling the rope constantly and gently. Once the corer is close to the surface but still entirely submerged, place a rubber stopper at the bottom of the sampling tube. Uplift the entire coring system from the water.Inspect the water-sediment interface. It should be clear and not visibly disturbed. Release the sample tube from the corer and place a PVC cover on the top.Seal the tube with adhesive tape avoiding the formation of air space. When sampling from literal habitats and shallow water bodies, dress in a wader for sampling in very shallow waters. Manually insert the sampling tube into the sediment.Place a rubber stopper in the top side of the sampling tube to obtain a vacuum. Remove the corer from the sediment and quickly introduce another rubber stopper at the tube bottom. For the calibration value with zero nitrous oxide, first read the sensor signal keeping the sensor tip submersed in deionized water.To calibrate with nitrous oxide water at the desired concentration, obtain nitrous oxide saturated water by bubbling nitrous oxide in deionized water for a few minutes. Dilute the nitrous oxide saturated water by adding a specific volume of saturated nitrous oxide water to a volume of deionized water. Gently mix the nitrous oxide saturated water with deionized water in the calibration vessel to dilute it to the desired concentration.When mixing the solution, be careful not to generate bubbles as this would eliminate nitrous oxide from the calibration solution. Now, read the sensor signal when it is constant. This reading is the calibration value with X micromolar nitrous oxide water.Change the PVC cover located at the top of each sediment core to another cover with a hole in the center and a hanging magnetic stirrer. Reseal the junction with adhesive tape. Reduce the water phase of each sample to an approximate height of 12 centimeters.For this, first insert a silicon tube in the central hole. Then put the sediment core in a cylinder and push the bottom stopper to create pressure. The stopper and sediment sample go up and the excess water passes through the tube.Collect the water in a recipient vessel. Perform the acetylene inhibition by bubbling with acetylene gas in the water phase of the core for approximately 10 minutes. Avoid resuspending the sediment.Fill all the air space with the previous leftover water before sealing the junction sensor PVC cover. Place the sensor in the sediment core through the central hole of the top side PVC cover. The tip of the sensor should be located in the water phase above the stirrer.Switch on the electromagnetic pulse circuit that is part of the stirring system. Move the electromagnet around the external part of the acrylic tube until the stirrer moves continuously and then fix it in place using adhesive tape. Close the incubation chamber to ensure a constant temperature.Press the record button on the sensor software to start recording the sensor signal. Then press the stop button at the end of the measurement period. Wait at least 10 minutes with the sensor's tip submerged in free nitrous oxide water before reading the signal of the zero nitrous oxide calibration measure.After performing a final sensor calibration, save the file using the sensor software. To perform denitrification rate calculations, start with a tabulated output file generated by the sensor's software that contains the record of the sensor's signal in millivolts and micromolar nitrous oxide and the calibration data. Block the sensor's signal against time to visualize the nitrous oxide accumulation trend.Use only the time range with a linear accumulation excluding the initial acclimation period of the sample and a possible final saturation due to substrate limitation. Denitrification rates were estimated using this protocol in sediments from Pyrenean mountain lakes over the period of 2013 to 2014. Here, the rates are measured from Lake Plan without nitrate addition.Measurements are noisy and only in some cases can the rates be properly estimated. In this figure, the same samples shown with nitrate addition exhibit more stable readings and good estimation of the potential rates. While this procedure approximates denitrification and see the rates, it also provides a way to experimentally change key factors controlling this activity.To test temperature and substrates, don't forget that a good control of temperature is fundamental for a good and stable measurement. Furthermore, an undisturbed sediment-water interface during the core collection is the first and critical requirement for a reliable estimation. Following this procedure, other methods like N15 ratios can be combined to investigate nitrification, denitrification coupling, and other nitrogen cycle processes.

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8.1K Views.  CREAF. This method can help answer key questions in biogeochemistry related to denitrification in sediments which are relevant for understanding the nitrogen cycle and the greenhouse gas emissions. The main advantages of this technique are the low disturbance of the sediment structure and the container recording of nitrogen oxide accumulation for reliable denitrification rate estimation. For deep water bodies, select the sampling point according to the investigation aims.Take note of the posi...