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.
