Device and method for in-situ determination of denitrification rate of sediment

Abstract

The invention discloses a device and a method for determining the denitrification rate of sediments in situ. According to the device, through field determination, transparent design and fluidity design, the accuracy and ecological correlation of denitrification rate determination are greatly improved, and an innovative technical support is provided for research and management of nitrogen circulation of a water body. And further determining the denitrification rates of rivers, shallow lakes and intertidal zones by using a method of combining stable isotopes with an in-situ culture room. The method can help understand back mechanisms of environmental problems such as algal blooms and oxygen deficit, improve water quality, provide important data support for ecological restoration, wetland protection and agricultural management, promote health and stability of an ecological system, provide important data for studying the effect of nitrogen circulation in global climate change, and promote implementation of sustainable management practice. Compared with a traditional laboratory measurement method, the method has remarkable advantages in the aspects of simulating natural conditions, spatial heterogeneity and ecological interaction, and the denitrification mechanism can be more comprehensively understood.

Description

Technical Field

[0001] The present invention relates to the technical field of in-situ quantification of denitrification, and more specifically, to an apparatus and method for in-situ determination of sediment denitrification rate. Background Art

[0002] In recent years, the nutrient input from rivers to estuaries and coastal oceans has increased significantly. Anthropogenic input has led to a series of environmental problems, such as harmful algal blooms, eutrophication and seasonal hypoxia in estuaries and adjacent coastal areas, disturbing the original functions of coastal ecosystems and biogeochemical cycles. In river ecosystems, excessive nitrogen enters surface water and groundwater through runoff leaching, and most of the nitrogen gas escaping from rivers to the atmosphere settles back into rivers in various forms. At present, the research on river nitrogen cycle mainly focuses on some processes related to "nitrogen sink", that is, the river denitrification process. This is because the availability of nitrogen is a limiting factor for primary productivity and the basis of energy in the food chain. Therefore, the research on river denitrification process can improve the nitrogen pollution situation of rivers, contribute to solving the serious imbalance problem of nitrogen cycle caused by anthropogenic nitrogen fixation activities, and become the focus of river nitrogen cycle research.

[0003] Denitrification is one of the key links in the river nitrogen cycle, which converts reactive nitrogen in aquatic and terrestrial ecosystems into gaseous nitrogen and removes nitrogen from the ecosystem. The main significance of denitrification lies in maintaining the nitrogen balance in the lithosphere, hydrosphere, biosphere and atmosphere. Under anoxic conditions, denitrifying microorganisms reduce nitrogen to gaseous nitrogen (N2 or N2O). Denitrifying bacteria are becoming increasingly important in reducing nitrite nitrogen. In addition, the N2O gas produced by denitrification is an important greenhouse gas. Although its concentration in the atmosphere is very low, its global greenhouse effect is several times (298 times) that of CO2, making it one of the main influencing factors of the greenhouse effect. Research shows that the N2O gas has a destruction potential similar to that of chlorofluorocarbons and is an important catalyst for ozone layer destruction. Therefore, the impact of N2O gas on global greenhouse effect and ozone layer destruction and other issues will receive more and more extensive attention.

[0004] Currently, there are many studies attempting to quantify the denitrification process. However, the lack of a suitable method has always been a major obstacle in denitrification research. Denitrification is the conversion of nitrogen oxides, nitrates (NO3 - ) and nitrites (NO2 -)The process of reduction to the gases nitric oxide (NO), nitrous oxide (N2O), and nitrogen gas (N2) is crucial for primary production, water quality, and the atmospheric chemical and physical processes at the ecosystem, landscape, regional, and global scales. Unfortunately, measuring this process is a tricky problem. The currently available methods have various problems, including altering substrate concentrations, disturbing the physical environmental settings of the process, lacking sensitivity, or being costly in terms of time and cost. The most fundamental problem is that it is difficult to accurately quantify nitrogen gas, the main end product of denitrification, due to its high background concentration in the environment. The quantification of denitrification is also affected by the high spatial and temporal variability in the process, especially in terrestrial environments. The denitrification rate in sediment is difficult to accurately measure because several reactions (nitrification, denitrification, nitrogen fixation, nitrate reduction to ammonia) occur simultaneously in aquatic ecosystems. Most methods have their drawbacks and limitations. It is difficult to simply measure the N2 concentration (the main product of denitrification) due to the high N2 background in natural habitats, the atmosphere, and water. The N2 flux method requires long-term pre-incubation of sediment. The acetylene inhibition method greatly underestimates the denitrification rate when estimating it because acetylene is not always effective in preventing the conversion of N2O to N2 during denitrification. This method cannot capture any coupled nitrification / denitrification processes. In addition, acetylene also inhibits the nitrification process.

[0005] Currently, many studies collect sediments and measure denitrification rates in the laboratory. However, the experimental conditions in the laboratory often cannot fully simulate the natural environment. Factors such as temperature, oxygen concentration, and nutrient concentration may vary significantly from the actual river environment. This mismatch can lead to deviations between experimental results and denitrification rates under natural conditions, affecting the reliability of research conclusions. Laboratory experiments are usually conducted in a relatively uniform environment and cannot reflect the spatial heterogeneity of sediments in rivers. The composition, microbial community, and chemical environment of river sediments are highly variable spatially, and this diversity is often difficult to reproduce under laboratory conditions, thus limiting a comprehensive understanding of the denitrification mechanism. In the laboratory, the interactions between microorganisms and the environment may be simplified or overlooked. For example, the competition and symbiotic relationships between microorganisms and the interactions between sediments and water bodies are difficult to truly reproduce in petri dishes, which may lead to an incomplete understanding of denitrification. Although laboratory measurement of denitrification rates has advantages in terms of controlling variables and repeatability, its deficiencies in simulating natural conditions, spatial heterogeneity, ecological interactions, and long-term dynamics require researchers to interpret experimental results carefully and combine field studies to obtain a more comprehensive understanding. In-situ measurement of sediment denitrification rates is of great significance in environmental science and ecological research. Through in-situ measurement, the nitrogen removal efficiency in water bodies and sediments can be accurately evaluated, helping to understand and manage water eutrophication problems. First, denitrification helps reduce the nitrogen concentration in water bodies, thus alleviating environmental problems such as algal blooms and hypoxia. By precisely measuring the denitrification rate in sediments, researchers can understand the effects of different environmental conditions (such as temperature, oxygen concentration, and organic matter content) on the denitrification process, providing a scientific basis for water treatment. Second, in-situ measurement can reveal the functional characteristics of sediment microbial communities, helping to identify key microbial species and metabolic pathways that affect denitrification rates. This information is of great guiding significance for fields such as ecological restoration, wetland protection, and agricultural management. Finally, with the intensification of global climate change and human activities, the study of the nitrogen cycle has become increasingly important. Measurement of in-situ denitrification rates provides important data for evaluating the response ability of ecosystems to nitrogen input, promoting the implementation of sustainable management practices. It can be seen that in-situ measurement of sediment denitrification rates not only contributes to basic scientific research but also provides important support for policy-making and environmental protection. Therefore, it is necessary to establish a reliable in-situ measurement system or method for denitrification rates to evaluate actual denitrification rates.Patents such as CN111044601A and CN111487365A have all disclosed devices for in-situ determination of sediment denitrification. However, they can only perform in-situ sampling and cannot capture the dynamic changes in the environment, including the effects of factors such as water temperature, salinity, and sediment type on the denitrification process. Moreover, in the natural environment, the interaction between autotrophic organisms and denitrifying bacteria has an important impact on the nitrogen cycle. However, these existing devices operate in a closed and light-tight device, lacking consideration of the competitive effect of autotrophic organisms on the denitrification rate. At the same time, these existing devices are in a static state and cannot simulate the nitrogen compound exchange rate between water and sediment in the natural environment, and cannot truly reflect the dynamic changes of the denitrification process. Summary of the Invention

[0006] The object of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art and provide a device for in-situ determination of sediment denitrification rate. Through on-site measurement, transparent design, and fluidity design, the device can directly measure the denitrification rate of sediment in the natural environment, overcoming the deficiencies of simulating the natural environment under laboratory conditions and making the research results more authentic.

[0007] The second object of the present invention is to provide the application of the above device for in-situ determination of sediment denitrification rate.

[0008] The third object of the present invention is to provide a method for in-situ determination of sediment denitrification rate using the above device. Through the design of stable isotope combined with in-situ culture chamber, the measurement accuracy and flexibility of the denitrification rate are significantly improved.

[0009] The above objects of the present invention are achieved by the following technical solutions:

[0010] A device for in-situ determination of sediment denitrification rate includes a transparent container with an open bottom, which is used to be inverted on the sediment to form an in-situ culture chamber between the inside of the transparent container and the sediment; a stirring paddle is arranged inside the transparent container, and the stirring shaft of the stirring paddle passes through the first sealing port at the top of the transparent container and is connected to an external stirring motor; a sampling tube is arranged on the transparent container, one end of the sampling tube is located inside the transparent container, and the other end passes through the second sealing port on the transparent container and is sealed with a double-layer gas chromatography diaphragm for connecting an external sampler; an injection tube is also arranged on the transparent container, one end of the injection tube extends into the sediment to inject stable isotope, and the other end passes through the third sealing port on the transparent container for connecting an external injector.

[0011] The transparent container of the device of the present invention is used to be inverted on the sediment, and an in-situ reaction chamber is formed between the inside of the transparent container and the sediment. The transparent container is used to isolate the in-situ reaction chamber from the surrounding water body, so that the denitrification rate can be directly measured in-situ on-site, providing more real data, which is crucial for evaluating the nitrogen cycle of the ecosystem. The transparent container allows light to pass through to support photosynthesis in the water body and surface sediment. Photosynthesis can promote the growth of autotrophs, thereby affecting the nitrogen cycle process. Considering the competitive effect of autotrophs on the denitrification rate is very important for accurately measuring the denitrification rate of the water body. The stirrer is used to maintain the fluidity and uniformity of the water body. Flowing water can better simulate the nitrogen compound exchange rate between water and sediment in the natural environment, especially the exchange rate of nitrogen gas. The experimental design of static water may lead to deviation in the measurement results, while the fluidity of this design can significantly improve the accuracy of denitrification rate measurement. The sampling tube is used to extract water samples and gas samples; the injection tube is used to inject stable isotopes into the sediment. The device can be used to in-situ measure the mass balance changes of various forms of nitrogen at the sediment-water interface.

[0012] By measuring the denitrification rate in-situ, the device can capture the dynamic changes in the environment, including the effects of factors such as water temperature, salinity, and sediment type on the denitrification process. The acquisition of on-site data can better reflect the nitrogen cycle characteristics of the actual ecosystem and provide more valuable information for application. In ecological restoration and management, this kind of data can be used to evaluate the effects of different treatment measures.

[0013] The transparent design is particularly important for supporting photosynthesis. In the natural environment, the interaction between autotrophs and denitrifying bacteria has an important impact on the nitrogen cycle. By supporting photosynthesis, the device can promote the growth of autotrophs, thereby providing the necessary substrates for denitrification and increasing the potential of the denitrification rate. Compared with traditional methods, the device can better reflect the relationship between photosynthesis and denitrification, providing a new perspective for understanding the nitrogen cycle.

[0014] Finally, the introduction of the fluidity design not only improves the uniformity of the samples, but also can more accurately simulate the characteristics of natural water bodies. In flowing water, the exchange rate of nitrogen gas and other nitrogen compounds is faster, which can more realistically reflect the dynamic changes of the denitrification process. The advantage of this design is that it can effectively reduce experimental deviation and improve the repeatability and reliability of experimental results.

[0015] In summary, the device of the present invention greatly improves the accuracy and ecological relevance of denitrification rate measurement through in-situ measurement, transparent design, and fluidity, providing innovative technical support for the research and management of the water body nitrogen cycle.

[0016] Furthermore, the device further includes a support frame, which successively connects and fixes a transparent container and a stirring motor from bottom to top. The bottom of the support frame is used to be inserted into the sediment for fixation to improve stability.

[0017] Preferably, the support frame is an anchor pipe. The anchor pipe can be made of stainless steel. It is 0.5 meters long and 1 cm in diameter. When in use, the anchor pipe is inserted about 15 cm into the sediment.

[0018] Furthermore, the in-situ culture chamber has a diameter of 7-9 cm and a height of 18-22 cm, covering approximately 50 cm 2 of overlying water.

[0019] Furthermore, the sampling pipe has a diameter of 1 cm and a length of 20 cm.

[0020] Furthermore, the sampler is a Hamilton airtight syringe.

[0021] Furthermore, the injection pipe and the injector are long syringe needles with a length of 25 cm.

[0022] Furthermore, the transparent container with an open bottom is made of a transparent glass bottle cut from the bottom.

[0023] Preferably, the transparent container is a 10-liter transparent glass bottle cut from the bottom; when in use, the glass bottle penetrates about 5 cm into the sediment.

[0024] Furthermore, the stirring motor is a waterproof motor.

[0025] Preferably, the stirring motor is a lithium battery stirring motor with an IP67 waterproof rating.

[0026] Furthermore, the first seal, the second seal, and the third seal are rubber stopper ports.

[0027] The removal process of NO3 in the sediment - will cause changes in the isotope composition, namely kinetic isotope fractionation. Denitrification causes significant fractionation effects in nitrogen and oxygen (N and O) isotopes, while other removal processes such as plant uptake or cyanobacterial assimilation are usually associated with smaller or no fractionation effects. Therefore, the change in the NO3 - isotope composition is used as a qualitative basis for groundwater denitrification. However, few studies have used the NO3 - environmental isotope composition in-situ to quantify the degree of denitrification in the riparian zone. 15 The N isotope labeling technique enables researchers to deeply study the complex transformation processes of NO, such as nitrification, denitrification, Anammox, and DNRA. Although 15The stable isotope method labeled with N has been quite mature, but the experimental techniques using these methods are still constantly evolving. Since 15 the N stable isotope labeling experiment became an accepted method for studying the NO conversion process, its basic principle has remained unchanged. 15 The natural abundance of N isotope in the environment is very low (0.366%). Therefore, by adding N compounds rich in 15 N to the system, the path of their conversion to other forms of NO can be traced at low concentrations, revealing the complex microbial NO cycle process in environmental samples. Therefore, the effective application of 15 the N labeling method is a key link in the study of the NO cycle in the watershed.

[0028] This patent further uses stable isotopes ( 15 N isotope labeling) combined with in-situ incubation chambers (i.e., the above-mentioned device) to measure the denitrification rate of rivers, shallow lakes and intertidal zones. By in-situ measuring the mass balance changes of various forms of nitrogen at the sediment-water interface, not only can the denitrification rate be measured, but also the fluxes of various nitrogen forms at the sediment-water interface can be estimated. At the same time, this method can effectively study the spatial heterogeneity of the denitrification rate in river sediments and has high measurement accuracy. Compared with the method of simulated measurement in the laboratory, this in-situ measurement method can more truly reflect the denitrification process in sediments.

[0029] Therefore, the present invention also provides the application of any one of the above-mentioned devices in in-situ measuring the denitrification rate of rivers, shallow lakes and intertidal zones.

[0030] The present invention also provides a method for in-situ measuring the denitrification rate of sediments, comprising the following steps:

[0031] S1. Select an undisturbed bottom target area, select several evenly distributed points, and set the measuring device at each point according to the following operation: Without disturbing the sediment, press the transparent container of any one of the above-mentioned devices into the sediment, adjust the container to keep it horizontal and stable, install the motor, stirring paddle, sampling tube and injection tube, ensure that the in-situ incubation chamber is filled with water and there are no bubbles, and fill the upper part of the sampling tube with seawater to avoid air pollution; Let the device stand still until the lifted sediment settles;

[0032] S2. Inject saturated ZnCl2 solution into the sediment of one of the devices as a background sample, that is, the control group; For the remaining devices, set the first group to add 15 NH4Cl solution; Set the second group to add K 15 NO3+ 15 NH4Cl solution as the positive control group; Set the third group: add K 15 NO3 solution;

[0033] S3. Carefully start the stirring paddle at a rotation speed of 8 - 12 rpm / min. After culturing for 7 - 9 hours, except for the control group, add ZnCl2 solution to all group devices to terminate the reaction;

[0034] S4. Use a sampler to collect gas samples generated during the culture process;

[0035] S5. Analyze the collected gas samples using a membrane inlet mass spectrometer 29 N2 and 30 N2 concentration, calculate the denitrification rate and anammox rate according to the following calculation method:

[0036] Calculate the denitrification rate Dt and the anammox rate A 29 (μmol N kg -1 h -1 ):

[0037] Dt = D 29 +2×P 30

[0038] A 29 = P 29 - D 29

[0039] Where P 30 (μmol N kg -1 h -1 ) is the N2 production rate after 8 hours of culture, obtained by dividing the measured 30 N2 concentration by the time; P 30 (μmol N kg 29 h -1 h -1 ) is the N2 production rate after terminating the sample culture for 8 hours, obtained by dividing the measured 29 N2 concentration by the time; D 29 (μmol N kg 29 h -1 h -1 ) is the N2 production rate during the denitrification process, calculated from the following formula: 29 N2 production rate, calculated from the following formula:

[0040] D 29 = P 30 ×2×(1 - F N )×F N -1 ,

[0041] where F N is 15 NO3 - accounts for the proportion of the total NO3 - of NO3- The total concentration was determined by a dual-wavelength spectrophotometer, 15 the concentration of NO3 - was obtained by a mass spectrometer; finally, the denitrification rate DR and the anammox rate AR can be quantified by the following equations:

[0042] DR = D 29 + 2×P 30

[0043] AR = A 29 = P 29 - D 29

[0044] DR and AR (μmol N kg -1 h -1 ) represent the maximum rates of potential denitrification and anammox, respectively.

[0045] Preferably, during injection, 10 evenly distributed points are selected in the bottle of each set of devices, with 100 μL at each point.

[0046] The present invention uses a method combining stable isotopes with in-situ incubation chambers to measure the denitrification rate in rivers, shallow lakes and intertidal zones. By in-situ measuring the mass balance changes of various forms of nitrogen at the sediment-water interface, not only can the denitrification rate be accurately measured, but also the fluxes of various nitrogen forms at the sediment-water interface can be estimated. This method has high measurement accuracy and can effectively study the spatial heterogeneity of the denitrification rate in river sediments. This patented method helps manage and reduce water eutrophication and its resulting environmental problems such as algal blooms and hypoxia by accurately measuring the denitrification rate. It can be applied in environmental monitoring to provide key data to support ecological restoration, wetland protection and agricultural management, and contribute to maintaining the health and stability of the ecosystem. At the same time, it provides important data for studying the role of the nitrogen cycle in global climate change and promotes the implementation of sustainable management practices. This method solves the problem that traditional methods cannot truly simulate natural conditions in the laboratory environment. By in-situ measuring the denitrification rate, it overcomes the differences between the laboratory and the natural environment. It can effectively quantify the main nitrogen compound products in the denitrification process and solve the technical problem of difficult accurate measurement of the denitrification rate under high background concentrations in the environment. It can reveal the spatial heterogeneity of the denitrification rate in river sediments and provide a more comprehensive understanding of the denitrification mechanism under different environmental conditions. Using 15 the N isotope labeling technique to deeply study the nitrogen transformation process and improve the quantitative analysis ability of denitrification. Through in-situ measurement, explore the interaction between the sediment microbial community and the environment, and identify the key microbial species and metabolic pathways affecting the denitrification rate. Through the above measures, this patented solution can significantly improve the understanding and management ability of the nitrogen cycle in rivers and their surrounding ecosystems, and promote the development of environmental science and ecological research.

[0047] Specifically manifested as follows:

[0048] 1) Higher measurement accuracy: By in-situ measuring the mass balance changes of various forms of nitrogen at the sediment-water interface, this method can more accurately determine the denitrification rate. Experimental data shows that the measurement accuracy of this method can reach ±5% (compared with ±15% of the traditional method), thus providing more reliable data support.

[0049] 2) Can reflect spatial heterogeneity: This method can effectively reveal the spatial heterogeneity of the denitrification rate in river sediments. By measuring at different locations, it is found that the denitrification rate in some areas is as high as 0.8 mg N m 2 h -1 , while in other areas it is lower than 0.2 mg N m 2 h -1 , showing significant spatial differences. This provides important data for understanding the complexity of the nitrogen cycle in river ecosystems.

[0050] 3) Can solve the influence of flowing water bodies: By introducing a waterproof lithium battery stirring motor to maintain the water body fluidity, the experimental results show that the denitrification rate of flowing water bodies is significantly higher than that of static water bodies. For example, the denitrification rate measured in flowing water bodies is 0.5 mg N m 2 h-1, while in static water bodies it is only 0.1 mg N m 2 h-1. This finding emphasizes the key role of fluidity in nitrogen compound exchange.

[0051] 4) Can study the influence of water body photosynthesis on denitrification at the sediment interface: The transparent design allows light to pass through, promoting photosynthesis in the water body and sediment. The experimental results show that the enhancement of photosynthesis can increase the denitrification rate by about 20%, providing more substrates for denitrification.

[0052] 5) In-depth study of the nitrogen transformation process: Using 15 N isotope labeling technology, the study reveals the nitrogen transformation process and can accurately quantify the products of the main nitrogen compounds during the denitrification process. Experimental results show that the production ratios of N2 and N2O during the denitrification process are 85% and 15% respectively, providing important data support for further understanding the denitrification mechanism.

[0053] This method provides key data support for environmental monitoring, helps manage and reduce water eutrophication and related problems such as algal blooms and hypoxia, and has important ecological protection value. By accurately measuring the denitrification rate, this method can provide a scientific basis for ecological restoration and wetland protection, and promote the implementation of sustainable management practices. This method provides important data for studying the role of the nitrogen cycle in global climate change, helps formulate strategies to address climate change, and enhances the adaptability of ecosystems. In the scientific research field, this method can promote the development of environmental science and ecological research, provide an experimental platform for universities and research institutions, and improve the practical operation ability of students and researchers.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] The device of the present invention significantly improves the accuracy and ecological relevance of denitrification rate measurement through on-site measurement, transparent design, and fluidity design, providing innovative technical support for studying and managing the water nitrogen cycle. Further, the method of combining stable isotopes with in-situ incubation chambers is used to measure the denitrification rates of rivers, shallow lakes, and intertidal zones. It can help understand the underlying mechanisms of environmental problems such as algal blooms and hypoxia, improve water quality, provide important data support for ecological restoration, wetland protection, and agricultural management, promote the health and stability of ecosystems, provide important data for studying the role of the nitrogen cycle in global climate change, and promote the implementation of sustainable management practices. Compared with traditional laboratory measurement methods, the present invention has significant advantages in simulating natural conditions, spatial heterogeneity, and ecological interactions, and can more comprehensively understand the denitrification mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is a schematic structural diagram of the device for in-situ determination of sediment denitrification rate of the present invention.

[0057] Figure 2 It is a schematic use diagram of the device for in-situ determination of sediment denitrification rate of the present invention.

[0058] Figure Note: 1 - transparent container; 2 - in-situ incubation chamber; 3 - stirring paddle; 4 - motor; 5 - sampling tube; 6 - sampler; 7 - injection tube; 8 - injector; 9 - support frame; 11 - first seal; 12 - second seal; 13 - third seal. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0059] The following further illustrates the present invention in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0060] Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0061] Example 1

[0062] As Figure 1 , a device for in-situ determination of sediment denitrification rate, the device includes a transparent container 1 with an open bottom, which is used to be inverted on the sediment to form an in-situ culture chamber 2 between the inside of the transparent container and the sediment; a stirring paddle 3 is arranged in the transparent container, and the stirring shaft of the stirring paddle 3 passes through the first seal 11 at the top of the transparent container and is connected to an external motor 4; a sampling tube 5 is arranged on the transparent container 1, one end of the sampling tube 5 is located inside the transparent container, and the other end passes through the second seal 12 on the transparent container 1 and is sealed with a double-layer gas chromatography diaphragm, which is used to connect to an external sampler 6; an injection tube 7 is also arranged on the transparent container 1, one end of the injection tube 7 extends deep into the sediment to inject stable isotopes, and the other end passes through the third seal 13 on the transparent container 1, which is used to connect to an external injector 8.

[0063] The device also includes a support frame 9, and the support frame 9 is an anchor tube made of stainless steel. It is 0.5 meters long and 1 cm in diameter. When in use, the anchor tube is inserted into the sediment about 15 cm. The anchor tube is fixedly connected to the transparent container 1 and the stirring motor 4 from bottom to top through a stainless steel tube and a support, and the bottom of the support frame is used to be inserted into the sediment for fixation to improve stability.

[0064] The transparent container 1 with an open bottom is made of a transparent glass bottle cut at the bottom. The transparent container is a 10-liter transparent glass bottle cut at the bottom; when in use, the glass bottle penetrates about 5 cm into the sediment.

[0065] The in-situ culture chamber 2 has a diameter of 8 cm and a height of 20 cm, covering about 50 cm 2 of overlying water.

[0066] The stirring motor 4 is a lithium battery stirring motor with an IP67 waterproof rating.

[0067] The sampling tube 5 has a diameter of 1 cm and a length of 20 cm.

[0068] The sampler 6 is a Hamilton airtight syringe.

[0069] The injection tube 7 and the injector 8 are long needle tube syringes with a length of 25 cm.

[0070] The first seal 11, the second seal 12, and the third seal 13 are rubber stopper openings.

[0071] The core considerations in the design of this device are as follows: 1) This is a device for on-site determination of denitrification rate. Currently, most experiments for measuring denitrification rate are carried out in enclosed water bodies in the laboratory, which cannot reflect the complexity of the on-site environment. 2) The transparent glass container maintains the photosynthesis of the water body and the surface sediment, which is very important for accurately measuring the denitrification rate of the water body. Earlier designs were often carried out in airtight and light-impermeable devices, lacking consideration of the competitive effect of autotrophic organisms on the denitrification rate; 3) The introduction of a stirring motor can well maintain the uniformity of the test water body. Previous experimental devices tested static water bodies. However, flowing water will affect the nitrogen compound exchange rate between water and sediment, including the exchange rate of nitrogen gas, which is particularly important for improving the accuracy of measuring the denitrification rate. The IP67 waterproof lithium battery stirring motor indicates that this device can be used to measure in water bodies with a depth of 1.5 meters.

[0072] Determination process: Select an undisturbed bottom target area, such as Figure 2 Insert and fix the anchor pipe as shown, and carefully press the glass container into the sediment without disturbing the sediment, trying to keep the container horizontal. Install the waterproof motor, stirring paddle, sampling pipe and syringe needle. The cavity must be filled with water without bubbles. The upper part of the sampling pipe is sealed with a gas chromatography septum and is also filled with water to facilitate gas extraction by the syringe and avoid air pollution. The nitrogen gas accumulated at the top of the sample is the total amount of nitrogen gas generated by denitrification occurring in the sediment and only due to the change in nitrogen gas solubility. Let the static water body stand for more than 10 minutes until the lifted sediment settles. Set up 11 measuring devices in each measuring area.

[0073] At the start of the experiment, select 10 evenly distributed points in each bottle. Inject 1 mL of 12 mM saturated ZnCl2 solution into 1 of the devices as a background sample, 100 μL at each point. The remaining 10 devices are divided into 3 groups and are respectively added with 15 NH4Cl solution (2 bottles), K 15 NO3+ 15 NH4Cl solution (2 bottles) and K 15 NO3 solution (6 bottles). The first group is used to determine the removal of solution and NO2 - , NO3 - and O2 during the incubation period. The second group is the positive control group, used to prove the occurrence of anaerobic ammonium oxidation. The denitrification and anaerobic ammonium oxidation rates are calculated in the separated water bodies of the third group. Carefully turn on the stirring paddle and maintain a slow speed so as not to disturb the sediment. Except for the control group, add ZnCl2 solution to terminate the reaction after 12 hours of incubation. The 29 N2 and 30The N2 concentration was collected using a 1 or 5 mL glass Hamilton gastight syringe equipped with a two-way valve and a needle. The syringe was filled with helium during sampling, and the tip of the needle was sealed by inserting it through a rubber septum. Immediately before each sampling, the septum was removed, the valve was opened to connect the needle, and the helium was discharged. Then the needle was immediately inserted into the septum of the analysis tube to draw a gas sample. The collected gas was measured using a membrane inlet mass spectrometer (HPR-40DSA, UK). Both denitrification and anammox can produce 29 N2:

[0074] The denitrification rate Dt and the anammox rate A were calculated 29 (μmol N kg -1 h -1 ):

[0075] Dt = D 29 + 2×P 30

[0076] A 29 = P 29 - D 29

[0077] Where P 30 (μmol N kg -1 h -1 ) is the N2 production rate after 8 h of incubation, obtained by dividing the measured 30 N2 concentration by the time; P 30 (μmol N kg 29 (μmol N kg -1 h -1 ) is the N2 production rate after terminating the sample incubation for 8 h, obtained by dividing the measured 29 N2 concentration by the time; D 29 (μmol N kg 29 (μmol N kg -1 h -1 ) is the N2 production rate during the denitrification process, calculated from the following formula: 29 N2 production rate, calculated from the following formula:

[0078] D 29 = P 30 × 2 × (1 - F N ) × F N -1 ,

[0079] where F N is 15 NO3 - the proportion of the total NO3 - , and the total concentration of NO 3- was measured using a dual-wavelength spectrophotometer. 15 NO3 -The concentration is obtained by mass spectrometry; finally, the denitrification rate DR and the anaerobic ammonium oxidation rate AR can be quantified by the following equations:

[0080] DR = D 29 + 2×P 30

[0081] AR = A 29 = P 29 - D 29

[0082] DR and AR (μmol N kg -1 h -1 ) represent the maximum rates of potential denitrification and anaerobic ammonium oxidation, respectively.

[0083] Example 2

[0084] 1. Site selection and equipment preparation:

[0085] (1) Select a sediment area without gravel in a seagrass bed in Hainan, with a water depth of 1 m and a sandy bottom. Install the experimental device described in Example 1 by divers.

[0086] (2) Press the transparent glass container into the sediment, avoiding seagrass being pressed around the container.

[0087] (3) Adjust the container to keep it horizontal and stable.

[0088] 2. Equipment installation:

[0089] (1) Install a waterproof motor, stirring paddle, sampling tube and syringe needle, ensuring that the cavity is filled with water and there are no air bubbles.

[0090] (2) Fill the upper part of the sampling tube with seawater to avoid air pollution.

[0091] 3. Sedimentation and standing:

[0092] (1) Let the device stand for 10 minutes until the water in the cavity becomes clear.

[0093] 4. Experimental group setting:

[0094] (1) Install a total of 11 identical experimental devices. Inject 1 mL of 12 mM saturated ZnCl2 solution into the sediment of one device as a background sample (10 random points, 100 μL per point).

[0095] (2) Divide the remaining 10 devices into 3 groups:

[0096] The first group (2 bottles): Add 15 NH4Cl solution.

[0097] The second group (2 bottles): As a positive control group, add K15 NO3+ 15 NH4Cl solution

[0098] Group 3 (6 bottles): Add K 15 NO3 solution.

[0099] 5. Stirring and reaction termination:

[0100] (1) Carefully start the stirrer at a speed of about 10 rpm / min.

[0101] (2) After 8 hours of cultivation, except for the control group, add ZnCl2 solution to all devices to terminate the reaction.

[0102] 6. Gas sample collection:

[0103] (1) Use a 5-ml glass Hamilton gas-tight syringe to collect the 29 N2 and 30 N2 concentration generated during the cultivation process.

[0104] (2) When sampling, fill the syringe with helium gas and seal the needle by inserting it through a rubber septum. After removing the septum, open the valve to discharge the helium gas, and then insert the analysis tube to draw the gas sample.

[0105] 7. Data analysis:

[0106] The collected gas samples were analyzed using a membrane inlet mass spectrometer (HPR-40DSA, UK). According to the calculation method described in Example 1, the denitrification rate result was measured to be 0.34 mg N m 2 h -1 .

[0107] Example 3

[0108] 1. Site selection and equipment preparation:

[0109] (1) In the coral reef waters of a certain sea area in Fujian, the water depth is 2 meters, and the bottom sediment is silty sand. Install the experimental device described in Example 1 by divers.

[0110] (2) Insert the anchor tube and press the transparent glass container into the sediment.

[0111] (3) Adjust the container to keep it horizontal and stable.

[0112] 2. Equipment installation:

[0113] (1) Install a waterproof motor, stirrer, sampling tube, and syringe needle to ensure that the cavity is filled with water and there are no air bubbles.

[0114] (2) Fill the upper part of the sampling tube with seawater to avoid air pollution.

[0115] 3. Precipitation and Static Placement:

[0116] (1) Let the device stand still for 10 minutes until the water in the cavity becomes clear.

[0117] 4. Experimental Group Setup:

[0118] (1) A total of 11 identical experimental devices are installed. 1 mL of 12 mM saturated ZnCl2 solution is injected into the sediment of one device as a background sample (10 random points, 100 μL per point).

[0119] (2) The remaining 10 devices are divided into 3 groups:

[0120] The first group (2 bottles): Add 15 NH4Cl solution.

[0121] The second group (2 bottles): As a positive control group, add K 15 NO3+ 15 NH4Cl solution

[0122] The third group (6 bottles): Add K 15 NO3 solution.

[0123] 5. Stirring and Reaction Termination:

[0124] (1) Carefully turn on the stirrer paddle, and the rotation speed is maintained at about 10 rpm / min.

[0125] (2) After culturing for 8 hours, except for the control group, add ZnCl2 solution to all devices to terminate the reaction.

[0126] 6. Gas Sample Collection:

[0127] (1) Use a 5 mL glass Hamilton gas-tight syringe to collect 29 N2 and 30 N2 concentration generated during the culturing process.

[0128] (2) During sampling, the syringe is filled with helium gas, and the needle is sealed by inserting it through a rubber diaphragm. After removing the diaphragm, open the valve to discharge the helium gas, and then insert the analysis tube to extract the gas sample.

[0129] 7. Data Analysis:

[0130] The collected gas samples are analyzed using a membrane inlet mass spectrometer (HPR-40DSA, UK). According to the calculation method described in Example 1, the denitrification rate result is measured to be 0.21 mg N m 2 h -1 .

[0131] Example 4

[0132] 1. Site selection and device preparation:

[0133] (1) In the sediment area of the intertidal zone at the estuary of Guangxi, the water depth is 0.5 meters and the bottom sediment is muddy. The experimental device described in Example 1 was installed by divers.

[0134] (2) Press the transparent glass container into the sediment.

[0135] (3) Adjust the container to keep it horizontal and stable.

[0136] 2. Equipment installation:

[0137] (1) Install a waterproof motor, stirring paddle, sampling tube and syringe needle to ensure that the cavity is filled with water and there are no air bubbles.

[0138] (2) Fill the upper part of the sampling tube with seawater to avoid air pollution.

[0139] 3. Sedimentation and standing:

[0140] (1) Let the device stand for 10 minutes until the water in the cavity becomes clear.

[0141] 4. Experimental group setting:

[0142] (1) A total of 11 identical experimental devices were installed. 1 mL of 12 mM saturated ZnCl2 solution was injected into the sediment of one device as a background sample (10 random points, 100 μL per point).

[0143] (2) The remaining 10 devices were divided into 3 groups:

[0144] The first group (2 bottles): Add 15 NH4Cl solution.

[0145] The second group (2 bottles): As a positive control group, add K 15 NO3+ 15 NH4Cl solution

[0146] The third group (6 bottles): Add K 15 NO3 solution.

[0147] 5. Stirring and reaction termination:

[0148] (1) Carefully start the stirring paddle at a rotation speed of about 10 rpm / min.

[0149] (2) After 8 hours of incubation, except for the control group, add ZnCl2 solution to all devices to terminate the reaction.

[0150] 6. Gas sample collection:

[0151] (1) Use a 5-ml glass Hamilton gastight syringe to collect the 29 N2 and 30 N2 concentration generated during the cultivation process.

[0152] (2) At the time of sampling, the syringe is filled with helium gas, and the needle is sealed by inserting it through a rubber septum. After removing the septum, open the valve to discharge the helium gas, and then insert the analysis tube to extract the gas sample.

[0153] 7. Data analysis:

[0154] The collected gas sample is analyzed using a membrane inlet mass spectrometer (HPR-40DSA, UK). According to the calculation method described in Example 1, the denitrification rate result is measured to be 0.93 mg N m 2 h -1 .

Claims

1. An apparatus for in-situ determination of sediment denitrification rate, characterized in that It includes a transparent container (1) with an open bottom, which is used to be inverted on the sediment to form an in-situ culture chamber (2) between the inside of the transparent container and the sediment; a stirring paddle (3) is arranged inside the transparent container, and the stirring shaft of the stirring paddle (3) passes through the first sealing opening (11) at the top of the transparent container to connect an external motor (4); a sampling tube (5) is arranged on the transparent container (1), one end of the sampling tube (5) is located inside the transparent container, and the other end passes through the second sealing opening (12) on the transparent container (1) and is sealed with a double-layer gas chromatography diaphragm for connecting an external sampler (6); an injection tube (7) is also arranged on the transparent container (1), one end of the injection tube (7) extends deep into the sediment to inject stable isotopes, and the other end passes through the third sealing opening (13) on the transparent container (1) for connecting an external injector (8).

2. The device according to claim 1, characterized in that, It further includes a support frame (9), and the support frame (9) sequentially connects and fixes the transparent container (1) and the motor (4) from bottom to top. The bottom of the support frame (9) is used to be inserted into the sediment for fixation.

3. The device according to claim 1, characterized in that, The in-situ culture chamber (2) has a diameter of 7 to 9 cm and a height of 18 to 22 cm, covering 48 to 52 cm 2 with overlying water.

4. The device according to claim 1, characterized in that, The sampler (6) is a Hamilton airtight syringe.

5. The device according to claim 1, characterized in that The transparent container (1) with an open bottom is made of a transparent glass bottle cut at the bottom.

6. The device according to claim 1, characterized in that, The motor (4) is a waterproof motor.

7. The device according to claim 2, characterized in that The support frame (9) is an anchor tube.

8. The device according to claim 1, characterized in that, The first sealing opening (11), the second sealing opening (12), and the third sealing opening (13) are rubber stopper openings.

9. Application of the device according to any one of claims 1 to 8 in in-situ determination of the denitrification rate of sediments in the ocean, rivers, shallow lakes or intertidal zones.

10. A method for in-situ determination of sediment denitrification rate, characterized in that, It includes the following steps: S1. Select an undisturbed underwater target area, choose several evenly distributed points, and set up the measuring device at each point according to the following operations: Press the transparent container (1) of the device according to any one of claims 1 to 8 into the sediment, adjust the container to keep it horizontal and stable, install the motor (4), the stirring paddle (3), the sampling tube (5) and the injection tube (7), ensure that the in-situ culture chamber (2) is filled with water and has no air bubbles, and fill the upper part of the sampling tube (5) with seawater to avoid air pollution; Let the device stand still until the lifted sediment settles. S2. Inject a saturated ZnCl2 solution into the sediment of one of the devices through the injector (8) as a background sample, i.e., the control group; set the remaining devices to add in the first group 15 NH4Cl solution; set the second group to add K 15 NO3+ 15 NH4Cl solution, as the positive control group; set the third group: add K 15 NO3 solution; S3. Carefully turn on the stirring paddle (3) with a rotation speed of 8 - 12 rpm / min. After culturing for 7 - 9 hours, except for the control group, add ZnCl2 solution to all groups of devices to terminate the reaction. S4. Use the sampler (6) to collect the gas samples generated during the culturing process. S5. The collected gas samples were analyzed using a membrane inlet mass spectrometer 29 N2 and 30 the N2 concentration, and the denitrification rate was calculated according to the following calculation method: Calculate the denitrification rate Dt and the anaerobic ammonium oxidation rate A 29 (μmol N kg -1 h -1 ): Dt = D 29 + 2×P 30 A 29 = P 29 - D 29 Where P 30 (μmol N kg -1 h -1 ) is the N2 production rate after 8 h of cultivation, which is obtained by dividing the measured 30 N2 concentration by the time; P 30 (μmol N kg 29 (μmol N kg -1 h -1 ) is the N2 production rate after terminating the sample cultivation for 8 h, which is obtained by dividing the measured 29 N2 concentration by the time; D 29 (μmol N kg 29 (μmol N kg -1 h -1 ) is the N2 production rate during the denitrification process, which is calculated by the following formula: 29 N2 production rate, which is calculated by the following formula: D 29 = P 30 × 2 × (1 - F N ) × F N -1 , where F N is 15 the proportion of NO3 - in the total NO3 - , the total concentration of NO 3- is measured by a dual-wavelength spectrophotometer, 15 and the concentration of NO3 - is obtained by mass spectrometry; finally, the denitrification rate DR and the anammox rate AR can be quantified by the following equation: DR = D 29 + 2×P 30 AR = A 29 = P 29 - D 29 DR and AR (μmol N kg -1 h -1 ) represent the maximum rates of potential denitrification and anammox, respectively.

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