阅读说明：本技术 一种测定沉积物全程硝化反应速率及对氨氮去除率的方法 (Method for measuring whole-course nitration reaction rate of sediment and ammonia nitrogen removal rate ) 是由 王衫允 祝贵兵 王晓敏 于 2020-04-01 设计创作，主要内容包括：本发明公开了一种测定水体沉积物全程硝化反应速率及其对氨氮去除贡献率的方法,属湿地氮污染控制领域。其原理是利用氯酸盐对亚硝酸盐氧化酶和辛炔对氨氧化细菌和古菌的选择性抑制,测定沉积物中全程硝化微生物的反应速率,并计算其对氨氮去除的贡献率。主要步骤包括：(1)待测沉积物的抑制剂处理及培养：①不添加,②添加氯酸钾,③添加氯酸钾和辛炔；(2)测培养过程中沉积物NO<Sub>3</Sub><Sup>-</Sup>和NO<Sub>2</Sub><Sup>-</Sup>浓度；(3)与培养时间线性回归得全程硝化反应速率；(4)计算全程硝化对氨氧化去除的贡献率。该方法能模拟原位温度、营养元素、含水率等,可精确、高效地测定自然界多种水体沉积物中全程硝化反应速率及其对氨氮去除率的研究,推动全程硝化研究的发展。(The invention discloses a method for measuring the whole-course nitration reaction rate of water body sediment and the contribution rate of the water body sediment to ammonia nitrogen removal, belonging to the field of wetland nitrogen pollution control, wherein the principle is that the chlorate is utilized to selectively inhibit nitrite oxidase and octyne from ammonia oxidizing bacteria and archaea, the reaction rate of the whole-course nitration microorganisms in the sediment is measured, and the contribution rate of the whole-course nitration microorganisms to ammonia nitrogen removal is calculated 3 ‑ And NO 2 ‑ Concentration; (3) linearly regressing with the culture time to obtain the whole nitration reaction rate; (4) and calculating the contribution rate of the whole-course nitration to the ammonia oxidation removal. The method can simulate in-situ temperature and operationThe method can accurately and efficiently measure the whole-course nitration reaction rate in various water sediments in the nature and the research on the ammonia nitrogen removal rate, and promotes the development of the whole-course nitration research.)

1. A method for measuring the whole-course nitration reaction rate of water sediments and the removal rate of ammonia nitrogen by the water sediments comprises the following steps:

(1) double-inhibitor culture: placing a fresh sediment sample in a serum bottle, covering a rubber plug and an aluminum cover, sealing and avoiding light, respectively treating the serum bottle with different inhibitors, and culturing under in-situ temperature and DO conditions;

(2) and (3) product analysis: extraction and determination of sediment samples at different incubation timesNO as the intermediate product₃ ^-And NO₂ ^-And calculating the corresponding accumulation rate;

(3) and (3) regression calculation: by production of NO between three different treatments₃ ^-And NO₂ ^-The total nitrification reaction rate in the sediment and the contribution rate to ammonia nitrogen removal are calculated.

2. According to claim 1, the method is preferably used for double inhibitor cultures in 60 ml serum bottles.

3. The method according to claim 1, preferably adds 1.5ml of sterile water to each serum bottle as a treatment 1-blank.

4. The method according to claim 1, preferably adding 1.5ml of 0.13mol/L potassium chlorate to the serum bottle as treatment group of 2-chlorate inhibitor.

5. According to claim 1, the method preferably adds 1.5ml of 0.13mol/L potassium chlorate and 2k Pa octyne to the serum bottle as the treatment group of 3-chlorate + octyne dual inhibitors.

6. The method of claim 1, wherein each treatment is performed in 3 parallel groups.

7. According to claim 1, the method preferably comprises placing a serum bottle on a constant temperature shaker at 150rpm and in situ temperature for culturing, and stopping culturing on days 0, 1, 2 and 4 after the start of culturing.

8. According to the method of claim 1, 20ml of 2mol/L KCl is preferably added into a serum bottle with corresponding culture time, shaking is carried out on a shaking table at 150rpm for 15min, nitrite and nitrate in sediments are extracted into a liquid phase, centrifugal filtration treatment is carried out, and a supernatant is collected for testing.

9. The method of claim 1, wherein the step of passing NO is required₃ ^-(treatment 1) and NO₂ ^-The cumulative rate difference of (process 2) is calculated to obtain the global nitrification rate.

10. The method of claim 1, wherein the linear regression coefficient (R2) is greater than 0.80.

11. According to claim 1, the contribution rate of total nitrification to ammonia nitrogen removal is calculated according to the metabolic rates of AOB and AOA.

Technical Field

The invention relates to the field of wetland nitrogen pollution control, and discloses novel nitrogen-circulating microorganism whole-course nitrifying bacteria which avoid the speed-limiting step of a nitrification process, namely an ammonia oxidation process, can realize the rapid removal of ammonia nitrogen pollutants in a water ecosystem, and avoid the generation of a toxic intermediate product nitrite; meanwhile, compared with the traditional two-step method, the method has lower reaction potential, so that the method can be more widely applied to the removal of ammonia nitrogen in the natural environment, and a new idea is provided for the treatment of ammonia nitrogen in the water environment ecology.

Background

The nitrogen (N) cycle is driven primarily by microorganisms that convert to different nitrogen compounds through a series of redox reactions. The interface between two phases characterized by different redox gradients is often a hot zone of the biogeochemical N cycle. Nitrification is the rate-limiting step of nitrogen circulation, is a biological process for continuously oxidizing ammonia into nitrate by microorganisms, is a key process in biogeochemical nitrogen circulation and biological wastewater treatment processes, and also plays an important role in many aspects of agriculture. Since its discovery in the 90 s of the 19 th century, nitrification has been thought to be caused by two different classes of chemoautotrophic bacteria: ammonia oxidizing microorganisms (ammonia-oxidizing bacteria, AOB) and ammonia-oxidizing Archaea (AOA)) and nitrite-oxidizing bacteria (NOB). Although studies have shown that ammonia-oxidizing archaea (AOA) plays an important role in the nitrification of soil and aquatic systems, this does not change the traditional understanding of nitrification, i.e., ammonia is ultimately oxidized to nitrate by microorganisms through a two-step nitrogen cycle. Subsequently, the discovery of anammox (anammox) indicated the actual presence of the ammoxidation process under anoxic and anaerobic conditions, which is quite different from the conventional ammoxidation process. The continued discovery of the course of new nitrogen cycles indicates that our knowledge of the total nitrogen cycle is not yet complete. Costa et al believe that, from a microbial energy metabolism point of view, there are theoretically fully nitrifying bacteria, but no direct evidence can be obtained. Recently, it has been shown that microorganisms in the genus Nitrospira can independently complete the nitrogen metabolism process of ammonia oxidation to nitrate and that such microorganisms are defined as whole-course nitrifying microorganisms. The discovery of the process makes a completely new understanding and comprehension of nitrification. In recent years, whole-process nitrifying bacteria have been widely detected in artificial aquatic and cultivated soil ecosystems, but their contribution to the ammonia oxidation process and total nitrogen cycle in natural ecosystems is still unclear.

Disclosure of Invention

The invention aims to solve the problems and provides a double-inhibitor method for determinationThe method for calculating the contribution rate of the whole course nitrifying microbe in the deposit to ammonia oxidation utilizes the selective inhibition action of chlorate to nitrite oxidase and octyne to ammonia oxidizing bacteria and ammonia oxidizing archaea, and adds inhibitor into the deposit to treat and culture the productAndand (3) measuring the whole-course nitration reaction rate of the water/sediment system, and further calculating to obtain the contribution rate of the whole-course nitration to the removal of the ammonia nitrogen.

The present invention is achieved by the following sample processing and data analysis protocol:

(1) dual inhibitor culture

Weighing fresh sediment samples with equal mass into a plurality of 60 ml serum bottles, covering a rubber plug and an aluminum cover, sealing and culturing in a dark place. Serum bottles were randomly selected for treatment with three different inhibitors. Treat 1-blank group, add 1.5ml of sterile water to each serum bottle; treating the 2-chlorate inhibitor treated group, adding 1.5ml of 0.13mol/L potassium chlorate to the serum bottle; the 3-chlorate + octyne dual inhibitor treatment group was treated, and 1.5ml of 0.13mol/L potassium chlorate and 2kPa octyne were added to the serum bottles. Each treatment was done in 3 replicates. All serum bottles were placed on a constant temperature shaker at 150rpm and in situ temperature for culture, and the culture was stopped on days 0, 1, 2 and 4 after the start of the culture. Adding 20ml of 2mol/L KCl into a serum bottle with corresponding culture time, shaking the serum bottle at 150rpm for 15min by a shaking table, extracting nitrite and nitrate in sediments into a liquid phase, performing centrifugal filtration treatment, and collecting supernatant to be tested.

(2) Analysis of the product

Full range nitration rate pass(treatment 1) andthe accumulated rate difference of (process 2) is calculated, in which,is mainly because the nitrous acid oxidation process is inhibited; with respect to AOA and AOB rates, it is based mainly on treatment 2Produced from AOA and AOB without consumption of NOB; both AOB and NOB were inhibited in treatment 3, and NO 2-was derived from the AOA reaction alone. The AOA rate is calculated(process 2) accumulation rate; the AOA plus AOB rate is determined byThe difference between the accumulation rates in the processes 2 and 3; AOB rate is obtained by subtracting AOA rate from AOA plus AOB rate.

In each culture flaskThe concentration calculation method is as the formula 1:

in each culture flaskThe concentration calculation method is as the formula 2:

in the formula 1 and the formula 2,finger-shapedThe concentration of the active ingredients in the mixture is,finger-shapedThe concentration of the active ingredients in the mixture is,refers to the concentration of the nitrogen element in the nitrite,means the concentration of nitrogen element in nitrate, V_KClRefers to the volume of KCl solution added to leach the sediment,refers to the addition of sterile water or KClO to the serum bottle sediment of Process 1 and Process 2₃Volume of solution, M_sedimentRefers to the quality of the fresh sediment in each serum bottle. In the present invention, V_KClIt is preferably set to 20mL,preferably 1.5mL, M_soilPreferably 5 g.

Based on the three treatments, the reaction rates of the three nitrifying microorganisms including full-course nitrification, ammonia oxidizing archaea and ammonia oxidizing bacteria in the water body sediment respectively pass throughAndthe concentration accumulation rate is obtained. In the present invention, the linear regression coefficient (R)²) Greater than 0.80 is required.

(3) Regression calculation

In treatment 3, since the activities of both AOB and NOB were inhibited,generated by AOA only, as in equation 3. Thus, the AOA rate can be determined fromThe rate of accumulation over time is given as shown in equation 4.

In the present invention,finger-shapedThe amount of accumulation in the process 3,representsAccumulation rate of, K_AOARepresenting the reaction rate of the AOA,representsThe accumulated amount of (3).

In treatment 2, since NOB activity was inhibited, the NOB was produced by AOA and AOBAnd is no longer degraded by NOB metabolism, as shown in equation 5. Therefore, the temperature of the molten metal is controlled,can indicate the reaction rate of AOA and AOBAnd the sum is as in equation 6.

From equations 4 and 6, the reaction rate of AOB can be derived, as in equation 7.

In the present invention,finger-shapedThe amount of accumulation in the process 2,representsAccumulation rate of, K_AOBRepresenting the reaction rate of AOB.

In treatment 1, no inhibitor was added,produced by both AOA and AOB and then acting as substrates for continued metabolic degradation by NOB. Therefore, the temperature of the molten metal is controlled,the accumulated amount of (2) is the difference between the generated amount and the consumed amount, as shown in equation 8.

Linear regression with incubation time can be used to obtain treatment 1The accumulation rate of (c) as in equation 9.

From equations 6 and 9, the NOB reaction rate can be derived, as in equation 10.

In the present invention,finger-shapedThe amount of accumulation in the process 1 is,representsAccumulation rate of, K_NOBRepresenting the reaction rate of NOB.

In the process I, the process is carried out,the source of (A) has two main parts, one isBy oxidation of NOB, the other part is derived fromBy oxidation of comamox, from whichThe accumulation rate of (c) as in equation 11.

Linear regression with incubation time to obtain treatment ISuch as equation 12.

From equations 9 and 12, the reaction rate of comamox can be derived, as in equation 13.

In the present invention,finger-shapedThe amount of accumulation in the process 1 is,representsAccumulation rate of, K_comammoxRepresenting the reaction rate of comamox.