Use of 5-methylisoxazole nitration inhibitors and methods of inhibiting nitration thereof

Abstract

The application belongs to the technical field of soil treatment and sewage treatment, and particularly relates to application of 5-methylisoxazole nitrification inhibitor and a method for inhibiting nitrification. The application comprises application in inhibiting nitrification activity of ammonia-oxidizing bacteria and in inhibiting nitrification activity of nitrite-oxidizing bacteria. Through a large number of researches and experiments, the application finds nitrification inhibition activity of 5-methylisoxazole, and realizes full-path regulation of nitrification. Experiments show that the compound can inhibit ammonia oxidation activity of a typical nitrifying strain at a low dose (>=0.01‰w / v), and can effectively inhibit activity of nitrite-oxidizing bacteria at a concentration lower than 0.1‰m / v. The aqueous solution of the compound has a neutral pH, and has no significant inhibitory effect on non-target heterotrophic microorganisms, and has high selectivity and environmental friendliness.

Description

Technical Field

[0001] This invention belongs to the field of soil remediation and wastewater treatment technology, specifically relating to the application of 5-methylisoxazole nitrification inhibitors and methods for inhibiting nitrification. Background Technology

[0002] Nitrification, as one of the core processes in the nitrogen biogeochemical cycle, refers to the process of nitrification of ammonium nitrogen (NH4+). + -N) is converted into nitrate nitrogen (NO3) through a multi-step oxidation reaction mediated by microorganisms. - The biochemical pathway of NH4+ (-N). This process is sequentially catalyzed by two types of chemoautotrophic nitrifying microorganisms: ammonia-oxidizing bacteria (AOB) first oxidize NH4+... + -N is oxidized to nitrite nitrogen (NO2). - Nitrite-oxidizing bacteria (NOB) then further convert it into NO3 (NO3-). - -N. As a key step in nitrogen form transformation, nitrification is widely distributed in habitats such as soil, freshwater and marine ecosystems. Its metabolic intensity directly affects global nitrogen cycle flux, greenhouse gas emissions and nitrogen use efficiency in agricultural production systems.

[0003] Currently, the intensified nitrification caused by excessive nitrogen input poses a significant environmental threat, with its risk chain reaction manifesting as follows: (1) nitrate nitrogen leaching through soil leads to nitrate pollution in groundwater; (2) intensified denitrification induces the release of the strong greenhouse gas N2O; and (3) reduced nitrogen fertilizer utilization leads to watershed-scale non-point source pollution. Based on this, how to achieve a dynamic balance between agricultural production and ecological protection through precise regulation of the nitrification process has become a core issue to be addressed in the collaborative development of environmental microbiology and sustainable agriculture. Further reducing the dosage of inhibitors and enriching the types of inhibitors are of great significance for improving agricultural economic benefits. Summary of the Invention

[0004] The purpose of this invention is to provide an application and method for inhibiting nitrification by providing a 5-methylisoxazole nitrification inhibitor that can suppress the growth of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria and effectively reduce the production of nitrification.

[0005] To address the problems existing in the prior art, the present invention adopts the following technical solution:

[0006] The use of 5-methylisoxazole nitration inhibitors includes any of the following:

[0007] (1) Application in inhibiting the nitrification activity of ammonia-oxidizing bacteria;

[0008] (2) Application in inhibiting the nitrification activity of nitrite-oxidizing bacteria.

[0009] The aforementioned application of the 5-methylisoxazole nitrification inhibitor includes ammonia-oxidizing bacteria such as Nitrosomonas sp. CZ-4 or Nitrosomonas nitrosa strain SN-6.

[0010] In the application of the aforementioned 5-methylisoxazole nitrification inhibitor, the nitrite-oxidizing bacteria is the nitrifying bacillus enrichment culture N-winogradskyi WY-8.

[0011] A method for inhibiting nitrification using 5-methylisoxazole nitrification inhibitor, comprising: treating soil or water containing ammonia-oxidizing bacteria and / or nitrite-oxidizing bacteria with 5-methylisoxazole, wherein the concentration of 5-methylisoxazole is greater than or equal to 0.01‰ w / v, based on 100% of the volume of the soil or water.

[0012] The ammonia-oxidizing bacteria mentioned above in the method for inhibiting nitrification with 5-methylisoxazole nitrification inhibitors include Nitrosomonas sp. CZ-4 and Nitrosomonas nitrosa strain SN-6.

[0013] The nitrite-oxidizing bacteria mentioned above in the method for inhibiting nitrification with the 5-methylisoxazole nitrification inhibitor is the nitrite-oxidizing bacterium enrichment culture N-winogradskyi WY-8.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] This invention, through extensive research and experimentation, has discovered the nitrification inhibitory activity of 5-methylisoxazole. Testing showed that 5-methylisoxazole can inhibit the growth of ammonia-oxidizing bacteria, such as *Nitrosomonas*, at a low dose (0.01‰ w / v), thereby effectively inhibiting nitrification in soil or water, reducing nitrogen loss from farmland and N2O emissions from soil and water. Furthermore, at a concentration of 0.1‰ w / v, 5-methylisoxazole achieved an inhibition rate of over 98% against the ammonia oxidation activity of *Nitrosomonas* CZ-4. It also showed good inhibitory effects on the nitrification activity of *Nitrobacterium* enrichment cultures at a concentration of 0.1‰ w / v. Its aqueous solution is neutral and does not significantly inhibit other bacteria without affecting the pH of water / soil, demonstrating high environmental friendliness. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The effect of different concentrations of 5-methylisoxazole on the growth of Nitrosomonas sp. CZ-4.

[0018] Figure 2 The effect of different concentrations of 5-methylisoxazole on the growth of Nitrosomonas nitrosa SN-6.

[0019] Figure 3 The effect of low concentrations of 5-methylisoxazole on the growth of Nitrosomonas sp. CZ-4.

[0020] Figure 4 This represents the nitrite consumption of N-winogradskyi WY-8 treated with different concentrations of 5-methylisoxazole on day 4.

[0021] Figure 5 This represents the accumulation of nitrate nitrogen in N-winogradskyi WY-8 treated with different concentrations of 5-methylisoxazole on day 4.

[0022] Figure 6 The effect of 5-methylisoxazole at a concentration of 0.05‰ m / v on the growth of heterotrophic microorganisms in activated sludge.

[0023] Figure 7 The study investigated the effect of different concentrations of 5-methylisoxazole on ammonia nitrogen concentration in domestic sewage.

[0024] Figure 8 The study investigated the effects of different concentrations of 5-methylisoxazole on nitrite concentration in domestic sewage.

[0025] Figure 9 The study investigated the effect of different concentrations of 5-methylisoxazole on nitrate nitrogen concentration in domestic sewage.

[0026] Figure 10 The effect of 5-methylisoxazole on ammonia nitrogen concentration in soil. Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0030] The culture medium and 5-methylisoxazole stock solution used in the following examples were prepared as follows:

[0031] AOB medium: , , , , , Trace element solution , Trace element solution: , , , , , , After high-temperature and high-pressure sterilization (121℃, 20 min), add an appropriate amount of filtered and sterilized HEPES stock solution and Mother liquor, and with The pH of the culture medium was adjusted to 7.8 with NaOH and HCl before use.

[0032] NOB culture medium: , , 1 mL of trace elements. Trace element solution: , , , , , , No water Add an appropriate amount of NaNO2 mother liquor, and use... The pH of the culture medium was adjusted to 7.8 with NaOH and HCl before use.

[0033] Beef extract peptone medium (BPM): purchased from Qingdao Haibo Biotechnology, prepared at a concentration of [missing information]. After high-temperature and high-pressure sterilization (121℃, 20 min), it is stored for later use.

[0034] 5-Methylisoxazole stock solution: Weigh 0.5 g of 5-methylisoxazole stock solution (CAS: 5765-44-6, purchased from Aladdin) using an analytical balance, dissolve it in a small amount of water, transfer it to a 50 mL volumetric flask, make up to volume, and filter it through a 0.22 μm organic filter membrane to sterilize and store it for later use.

[0035] It should be noted that in this invention, the method for detecting ammonia nitrogen concentration in water is "Determination of Ammonia Nitrogen in Water - Nessler's Reagent Spectrophotometric Method" (HJ 535-2009); the method for detecting nitrite nitrogen concentration is "Determination of Nitrite Nitrogen in Water - Spectrophotometric Method" (GB / T 7493-1987); and the method for detecting nitrate nitrogen concentration is "Determination of Nitrite Nitrogen in Water - Spectrophotometric Method" (HJ / T346-2007). The method for detecting ammonia nitrogen concentration in soil is to extract with 1 mol / L KCl solution and then measure using a continuous flow apparatus. Further, it should be noted that in the embodiments of this invention, the nitrification inhibition rate is calculated using the following formula:

[0036] (1) Nitrite accumulation (mg / L):

[0037]

[0038] Where C1 is the initial nitrite concentration, C t-1 This represents the concentration of nitrite nitrogen on that day.

[0039] (2) Nitrite consumption (mg / L):

[0040]

[0041] Where C1 is the initial nitrite concentration, C t-1 This represents the concentration of nitrite nitrogen on that day.

[0042] (3) Nitrate accumulation (mg / L):

[0043]

[0044] Where C1 is the initial nitrate concentration, C t-1 This represents the nitrate concentration for that day.

[0045] (4) Inhibition rate of ammonia oxidation activity (%)

[0046]

[0047] Among them, C k C represents the nitrite accumulation in the control group at a certain time. t This represents the amount of nitrite accumulated in the experimental group at the same time.

[0048] Example 1

[0049] This embodiment relates to an experiment evaluating the inhibitory effect of 5-methylisoxazole nitrification inhibitor on the ammonia-oxidizing bacterium Nitrosomonas sp. CZ-4 (hereinafter referred to as CZ-4).

[0050] Take 1 mL of CZ-4 bacterial culture into AOB medium and culture it in a shaker at 30℃ and 140 r / min. Take samples every 24 h to detect the concentrations of ammonia nitrogen and nitrite nitrogen until the nitrite nitrogen growth rate is greater than 30 mg / L / d. Use this as the inoculum.

[0051] Figure 1 The implementation method as shown in Table 2 is as follows: 5-methylisoxazole stock solution was added to AOB medium with an initial ammonia nitrogen concentration of 100 mg / L at different concentrations, and CZ-4 inoculum was added at 1% v / v. The medium was then cultured in a shaker at 30℃ and 140 r / min. Specific operating procedures are shown in Table 1. The CZ-4 culture system without 5-methylisoxazole served as the control group, while the CZ-4 culture systems with different concentrations of 5-methylisoxazole served as the experimental groups.

[0052] Table 1. CZ-4 experimental system of control and experimental groups in Example 1

[0053]

[0054] Samples were taken every 24 hours to measure the concentrations of ammonia nitrogen and nitrite nitrogen in the control group and each experimental group. Figure 1 Table 2 shows the trend of nitrite nitrogen changes in each group, and the nitrite nitrogen concentration and ammonia oxidation inhibition rate of each group at 3 days.

[0055] Table 2. Nitrite concentration and ammonia oxidation activity inhibition rate of each group on day 3 in Example 1

[0056]

[0057] From Table 2 and Figure 1It was found that the nitrite nitrogen concentration in the control group reached 101.26 mg / L on day 3, indicating good bacterial growth. The nitrite nitrogen concentrations in the experimental groups with added 5-methylisoxazole were all lower than those in the control group, indicating that the growth of CZ-4 was inhibited to varying degrees. Furthermore, the inhibitory effect decreased with decreasing concentration; at a concentration of 0.01‰ w / v, the inhibition rate of ammonia oxidation activity was 15.00%. At concentrations of 0.1‰ and 1‰ w / v, the inhibition rates of ammonia oxidation activity reached 95.85% and 98.12%, respectively, indicating that ammonia oxidation activity was almost completely inhibited. Example 1 shows that 5-methylisoxazole can effectively inhibit the ammonia oxidation activity of CZ-4 at concentrations greater than 0.1‰ w / v (inhibition rate > 95%), and also shows a slight inhibitory effect at a concentration of 0.01‰ w / v.

[0058] Example 2

[0059] This embodiment relates to an experiment evaluating the inhibitory effect of 5-methylisoxazole nitrification inhibitor on the ammonia-oxidizing bacteria species Nitrosomonas nitrosa SN-6 (hereinafter referred to as SN-6) in water.

[0060] Figure 2 The implementation method was the same as in Table 3: the bacterial strain was replaced with SN-6, and the experimental system and culture method were the same as in Example 1. Samples were taken every 24 hours to measure the concentrations of ammonia nitrogen and nitrite nitrogen in the control group and each experimental group. Figure 3 Table 3 shows the trends of nitrite nitrogen in each group, and the nitrite nitrogen concentration and ammonia oxidation inhibition rate in each group on day 3.

[0061] Table 3. Nitrite concentration and ammonia oxidation activity inhibition rate of each control group and experimental group on day 3 in Example 2.

[0062]

[0063] From Table 3 and Figure 2 It was found that the nitrite nitrogen concentration in the control group reached 112.33 mg / L on day 3, indicating good bacterial growth. The nitrite nitrogen concentrations in the experimental groups with added 5-methylisoxazole were all lower than those in the control group, indicating that SN-6 growth was inhibited to varying degrees. Furthermore, the inhibitory effect decreased with decreasing concentration; no inhibition was observed at a concentration of 0.01‰ w / v. At concentrations of 0.1‰ and 1‰ w / v, the inhibition rates of ammonia oxidation activity reached 86.65% and 98.74%, respectively, indicating high inhibition rates. Example 2 shows that 5-methylisoxazole can effectively inhibit the ammonia oxidation activity of SN-6 at concentrations greater than 0.1‰ w / v, and can also inhibit SN-6 growth at concentrations of 0.1‰-0.01‰ w / v.

[0064] Example 3

[0065] The purpose of this study was to investigate the inhibitory effect of 5-methylisoxazole on CZ-4 at low concentrations (0.1‰-0.01‰ w / v).

[0066] 1 mL of CZ-4 bacterial culture was added to AOB medium and cultured in a shaker at 30℃ and 140 r / min. Ammonia nitrogen and nitrite nitrogen concentrations were measured every 24 h until the nitrite nitrogen growth rate exceeded 30 mg / L / d, at which point this was used as the inoculum. The purpose of this study was to investigate the inhibitory effect of 5-methylisoxazole on CZ-4 at lower concentrations (0.1‰-0.01‰ w / v).

[0067] Figure 3 The implementation method as shown in Table 4 was as follows: Each group was inoculated with 1% (v / v) of logarithmic-phase CZ-4 culture medium in 100 mL of AOB medium with an initial ammonia nitrogen concentration of 200 mg / L. The experimental groups were then supplemented with 5-methylisoxazole stock solution to achieve concentrations of 0.075‰, 0.05‰, 0.025‰, and 0.01‰ (w / v), respectively. The control group did not receive 5-methylisoxazole. Ammonia nitrogen and nitrite nitrogen concentrations were monitored every 24 h. Figure 3 Table 4 shows the trend of nitrite nitrogen changes in each group, and the relationship between nitrite nitrogen concentration and ammonia oxidation activity inhibition rate in each group at 3 days.

[0068] Table 4. Nitrite concentration and ammonia oxidation activity inhibition rate of each control group and experimental group on day 3 in Example 3.

[0069]

[0070] Depend on Figure 3 As shown in Table 4, the nitrite concentration in the control group reached 156.04 mg / L on day 3. In the experimental group, the inhibition rates at concentrations of 0.075‰, 0.05‰, 0.025‰, and 0.01‰ w / v were 86.75%, 76.72%, 48.08%, and 7.93%, respectively. Example 3 demonstrates that even at a low concentration of 0.01‰ w / v, a weak inhibitory effect can still be detected, and the inhibition rate increases with increasing concentration.

[0071] Example 4

[0072] This embodiment relates to an experiment evaluating the inhibitory effect of 5-methylisoxazole on the N-winogradskyi WY-8 (hereinafter referred to as WY-8) enrichment culture of Nitrifying Bacteria.

[0073] Take 1 mL of WY-8 bacterial culture with an initial nitrite nitrogen concentration of 300 mg / L in NOB medium and incubate it at 30℃ and 140 r / min. Take samples every 24 h to detect the nitrite nitrogen concentration and nitrate nitrogen concentration until the nitrite nitrogen concentration increases at a rate greater than 50 mg / L / d. Use this as the inoculum.

[0074] Figure 4 The implementation method as shown in Table 5 is as follows: 5-methylisoxazole stock solution is added to NOB medium at different concentrations, and an appropriate amount of sterile water is added. WY-8 culture is then inoculated at a ratio of 1%. The WY-8 culture system is prepared according to the formula in Table 4. The CZ-4 culture system without 5-methylisoxazole serves as the control group, and the other CZ-4 culture systems serve as the experimental groups. The cultures are then incubated in a shaker at 30℃ and 140 r / min.

[0075] Table 4. WY-8 experimental system of control group and experimental group in Example 4

[0076]

[0077] Samples were taken at 0 and 4 days to measure the concentrations of nitrite and nitrate in the control group and each experimental group, respectively. Figure 4 The nitrite consumption of each group at 4 days. Figure 5 Table 5 shows the accumulation of nitrate nitrogen in each group on day 4. The table also shows the consumption of nitrite nitrogen and the accumulation of nitrate nitrogen in each group on day 4.

[0078] Table 5. Nitrite consumption and nitrate accumulation in the control and experimental groups on day 4 in Example 4.

[0079]

[0080] From Table 5 and Figure 4 , Figure 5 The consumption of nitrite and the accumulation of nitrate indicate that 5-methylisoxazole also has an inhibitory effect on WY-8, and the inhibitory effect is directly proportional to the concentration. When the concentration of 5-methylisoxazole is 1‰ and 0.1‰ w / v, the accumulation of nitrate is 7.3% and 68.8% of that in the control group, respectively. No inhibitory effect was observed at 0.01‰ w / v. In conclusion, 5-methylisoxazole also has a certain inhibitory ability on WY-8.

[0081] Example 5

[0082] The purpose of this study was to investigate the inhibitory effect of 5-methylisoxazole on heterotrophic microorganisms in water.

[0083] Figure 6The implementation method was as follows: 20 mL of beef extract peptone medium was taken and activated sludge (from a municipal wastewater aerobic tank) was inoculated at a ratio of 1 / 1000. The experimental group received 0.05‰ w / v 5-methylisoxazole, while the control group received no 5-methylisoxazole. The mixture was cultured in a shaker at 30℃ and 140 r / min. OD was measured at 0 h and 24 h. 600 .

[0084] Depend on Figure 6 It can be seen that after adding 0.05‰ w / v of 5-methylisoxazole, the OD of the experimental group was... 600 There was no significant difference compared to the control group, indicating that 0.05‰ of 5-methylisoxazole had no inhibitory effect on other heterotrophic microorganisms in the soil. Example 5 shows that the target compound effectively inhibits nitrifying bacteria while not interfering with the metabolic activity of non-target heterotrophic microorganisms, highlighting the selective advantage of its mechanism of action.

[0085] Example 6

[0086] The purpose of this study is to explore the specific application effects of 5-methylisoxazole in complex scenarios (domestic sewage).

[0087] Figure 7 , Figure 8 , Figure 9 The implementation method was as follows: 20 mL of domestic sewage was taken and inoculated with activated sludge from an aerobic tank at a ratio of 1%. The experimental group received 5-methylisoxazole at a ratio of 0.5 / 0.2 / 0.1 / 0.05‰ w / v, while the control group received no 5-methylisoxazole. The mixture was cultured in a shaker at 30℃ and 140 r / min. Ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen were measured every 24 hours. Figure 7 The changing trend of ammonia nitrogen Figure 8 The changing trend of nitrite nitrogen, Figure 9 This represents the changing trend of nitrate nitrogen.

[0088] Depend on Figure 7 , Figure 8 , Figure 9 It was found that the addition of 5-methylisoxazole significantly inhibited nitrification. Ammonia nitrogen consumption slowed down, and the accumulation rate of nitrate nitrogen was also suppressed. For example, at 48 hours, the experimental group treated with 0.05‰ w / v methylisoxazole showed ammonia nitrogen consumption of 53.7% and nitrate nitrogen accumulation of only 14.63% of the control group. Furthermore, the effects of 5-methylisoxazole concentrations between 0.5‰ and 0.05‰ w / v were comparable. These results indicate that in complex scenarios, concentrations of 5-methylisoxazole below 0.05‰ w / v can reduce ammonia nitrogen consumption and nitrate nitrogen accumulation.

[0089] Example 7

[0090] The purpose of this study is to investigate the specific application effects of 5-methylisoxazole in soil. Figure 10 The implementation method was as follows: Take 50g of soil and supplement it with ammonium sulfate to 100 mg / kg. The experimental group was given 0.1‰ w / v concentration of 5-methylisoxazole, and the control group was given an equal amount of sterile water. The soil was kept moist by replenishing water regularly every day. Ammonia nitrogen in the soil was measured on day 0 and day 14.

[0091] Depend on Figure 10 It was found that the ammonia nitrogen consumption in the experimental group after adding 5-methylisoxazole was significantly less than that in the control group on day 14. On day 14, the ammonia nitrogen consumption in the control group was 27.66 mg / kg, while that in the experimental group was 45% of that in the control group. These results indicate that 5-methylisoxazole can not only exert a good effect in water bodies, but also slow down the loss of ammonia nitrogen in soil.

[0092] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

The application of 1,5-methylisoxazole nitration inhibitors is characterized by, Applications that include any of the following: (1) Application in inhibiting the nitrification activity of ammonia-oxidizing bacteria; (2) Application in inhibiting the nitrification activity of nitrite-oxidizing bacteria.

2. The application of the 5-methylisoxazole nitration inhibitor according to claim 1, characterized in that, The ammonia-oxidizing bacteria include Nitrosomonas sp. CZ-4 or Nitrosomonas nitrosa strain SN-6.

3. The application of the 5-methylisoxazole nitration inhibitor according to claim 1, characterized in that, The nitrite-oxidizing bacteria are enriched cultures of nitrifying bacteria. N-winogradskyi WY-8. A method for inhibiting nitration using 4,5-methylisoxazole nitration inhibitors, characterized in that, include: Soil or water containing ammonia-oxidizing bacteria and / or nitrite-oxidizing bacteria is treated with 5-methylisoxazole, wherein the concentration of 5-methylisoxazole is greater than or equal to 0.01‰ w / v, based on 100% of the volume of the soil or water.

5. The method for inhibiting nitrification using the 5-methylisoxazole nitrification inhibitor according to claim 4, characterized in that, The ammonia-oxidizing bacteria include Nitrosomonas sp. CZ-4 and Nitrosomonas nitrosa strain SN-6.

6. The method for inhibiting nitrification with the 5-methylisoxazole nitrification inhibitor according to claim 4, characterized in that, The nitrite-oxidizing bacteria are enriched cultures of nitrifying bacteria. N-winogradskyi WY-8.

Images