4-methyl-1-piperazineethylamine as a nitrification inhibitor and method for inhibiting nitrification

Abstract

The application provides application of 4-methyl-1-piperazine ethylamine as a nitrification inhibitor and a method for inhibiting nitrification, and relates to the technical field of soil treatment. Specifically, the application relates to application of 4-methyl-1-piperazine ethylamine as a nitrification inhibitor, in soil treatment and / or sewage treatment and inhibition of nitrifying bacteria nitrosation activity. The method for inhibiting nitrification in the application comprises: treating soil or water containing ammonium nitrogen and nitrifying bacteria with 4-methyl-1-piperazine ethylamine. The concentration of the 4-methyl-1-piperazine ethylamine is not less than 0.01‰ w / v, with the volume of the soil or water being 100%. The application finds the nitrification inhibition activity of 4-methyl-1-piperazine ethylamine, thereby enriching the selection of nitrification inhibitor types.

Description

Technical Field

[0001] This invention relates to the technical field of soil remediation, and more specifically, to the application of 4-methyl-1-piperazine ethylamine as a nitrification inhibitor and methods for inhibiting nitrification. Background Technology

[0002] Nitrification refers to the process of reacting ammonium nitrogen (NH4) with nitrogen. + -N) is gradually oxidized into nitrate (NO3) through the metabolic activities of microorganisms. - The process of nitrification (NH4+) is a key step in the nitrogen cycle in the natural environment. Nitrification is mainly accomplished by two types of nitrifying microorganisms: ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). Ammonia-oxidizing bacteria convert ammonium nitrogen (NH4+) into nitrogen. + -N) is oxidized to nitrite (NO2) - Nitrification (nitrification) further oxidizes nitrite to nitrate by nitrite-oxidizing bacteria. Nitrification is widespread in ecosystems such as soil, freshwater, and marine environments, and has a significant impact on the ecological environment and agricultural production.

[0003] Excessive nitrification can lead to a series of environmental problems, such as groundwater pollution caused by nitrate leaching and reduced nitrogen fertilizer utilization. Therefore, effectively regulating the nitrification process to reduce adverse effects has become a crucial issue that urgently needs to be addressed in agriculture and environmental management. In recent years, research and development of novel nitrification inhibitors to selectively inhibit the activity of ammonia-oxidizing bacteria has become an important direction for achieving efficient nitrogen utilization and environmental protection. Summary of the Invention

[0004] The present invention aims to develop a novel nitration inhibitor to enrich the selection of nitration inhibitors.

[0005] To address the above problems, this invention provides the application of 4-methyl-1-piperazine ethylamine as a nitration inhibitor and a method for inhibiting nitration.

[0006] As a first aspect, the present invention relates to the use of 4-methyl-1-piperazineethylamine as a nitration inhibitor.

[0007] As a second aspect, the present invention also relates to the use of 4-methyl-1-piperazine ethylamine in inhibiting the nitrification activity of ammonia-oxidizing bacteria.

[0008] As a third aspect, the present invention also relates to the use of 4-methyl-1-piperazine ethylamine in inhibiting the nitrosation activity of Nitrosomonas.

[0009] Optionally, the nitrosomonas includes Nitrosomonas sp. CZ-4 and / or Nitrosomonasnitrosa strain WH-1.

[0010] As a fourth aspect, the present invention also relates to the use of 4-methyl-1-piperazine ethylamine in soil treatment and / or wastewater treatment.

[0011] As a fifth aspect, the present invention also relates to a method for inhibiting nitrification, comprising: treating soil or water containing ammonium nitrogen and ammonia-oxidizing bacteria with 4-methyl-1-piperazine ethylamine.

[0012] Optionally, the concentration of 4-methyl-1-piperazine ethylamine is not less than 0.01‰w / v, based on 100% of the volume of the soil or water body.

[0013] Optionally, the concentration of 4-methyl-1-piperazine ethylamine is 1‰ w / v, based on 100% of the volume of the soil or water body.

[0014] As a sixth aspect, the present invention also relates to a nitration inhibitor comprising 4-methyl-1-piperazineethylamine.

[0015] As a seventh aspect, the present invention also relates to the application of the above-mentioned nitrification inhibitors in soil treatment and / or wastewater treatment.

[0016] The advantages of this invention compared to the prior art are:

[0017] This invention, through extensive research and experimentation, has discovered the nitrification inhibitory activity of 4-methyl-1-piperazine ethylamine. Testing showed that 4-methyl-1-piperazine ethylamine 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 1‰ w / v, 4-methyl-1-piperazine ethylamine achieved a nitrification inhibition rate of over 96% against *Nitrosomonas*. In addition, the 4-methyl-1-piperazine ethylamine of this invention does not exhibit ecotoxicity or potential bioaccumulation, has minimal impact on other heterotrophic microorganisms in the soil, and demonstrates good environmental friendliness. Attached Figure Description

[0018] Figure 1 This is a graph showing the changes in nitrite concentration in the control group and each experimental group on day 4 in Example 1 of the present invention;

[0019] Figure 2 This is a graph showing the changes in nitrite nitrogen concentration in the control group and each experimental group during the first 4 days in Example 2 of this invention;

[0020] Figure 3 This is a graph showing the change in absorbance values ​​at 600 nm for the mixture of the experimental group and the control group in Example 3 of the present invention. Detailed Implementation

[0021] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Although some embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the accompanying drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used in the description of this application is for the purpose of describing particular embodiments only and is not intended to limit this application.

[0023] The term "comprising" and its variations as used herein are open-ended, meaning "including but not limited to"; the term "based on" means "at least partially based on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments". Definitions of other terms will be given in the description below. It should be noted that the concepts of "first," "second," etc., mentioned in this invention are used to distinguish different objects, not to describe a specific order or hierarchy. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0024] To enrich the selection of nitration inhibitors, this invention provides an application of 4-methyl-1-piperazine ethylamine as a nitration inhibitor and a method for inhibiting nitration.

[0025] In a first aspect, the present invention relates to the use of 4-methyl-1-piperazine ethylamine as a nitrification inhibitor, specifically, to the use of 4-methyl-1-piperazine ethylamine in inhibiting the nitrification activity of ammonia-oxidizing bacteria, and more specifically, to the use of 4-methyl-1-piperazine ethylamine in inhibiting the nitrification activity of nitrosomonas bacteria. The nitrosomonas bacteria may include *Nitrosomonas* sp. CZ-4 (accession number CCTCCNO: M 2018813) and / or *Nitrosomonas nitrosastrain* WH-1 (accession number CCTCCNO: M 2020526). Furthermore, based on the activity of 4-methyl-1-piperazine ethylamine in inhibiting nitrification, the present invention also relates to the use of 4-methyl-1-piperazine ethylamine in soil treatment and / or wastewater treatment.

[0026] The present invention also specifically relates to a method for inhibiting nitrification, comprising: treating soil or water containing ammonium nitrogen and ammonia-oxidizing bacteria with 4-methyl-1-piperazine ethylamine. Specifically, the concentration of 4-methyl-1-piperazine ethylamine is not less than 0.01‰ w / v based on 100% of the volume of the soil or water, and further preferably, the concentration of 4-methyl-1-piperazine ethylamine is 1‰ w / v.

[0027] This invention also relates to a nitrification inhibitor, the component of which includes 4-methyl-1-piperazineethylamine. Specifically, 4-methyl-1-piperazineethylamine can be used as the active ingredient of this nitrification inhibitor. Based on the nitrification-inhibiting activity of the nitrification inhibitor, this invention also relates to the application of the above-mentioned nitrification inhibitor in soil treatment and / or wastewater treatment.

[0028] This invention, through extensive research and experimentation, has discovered the nitrification inhibitory activity of 4-methyl-1-piperazine ethylamine. Testing showed that 4-methyl-1-piperazine ethylamine 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 1‰ w / v, 4-methyl-1-piperazine ethylamine achieved a nitrification inhibition rate of over 96% against *Nitrosomonas*. In addition, the 4-methyl-1-piperazine ethylamine of this invention does not exhibit ecotoxicity or potential bioaccumulation, has minimal impact on other heterotrophic microorganisms in the soil, and demonstrates good environmental friendliness.

[0029] The present invention will be described in detail below with reference to specific embodiments and comparative examples:

[0030] It should be noted that the solutions used in the following examples and their preparation methods are as follows:

[0031] Trace element solution: comprising the following components at the following mass concentrations: 0.5 g / L EDTA, 0.075 g / L CuSO4·5H2O, 0.3 g / L ZnSO4·7H2O, 0.375 g / L CoCl2·6H2O, 0.3 g / L MnCl2·2H2O, 0.014 g / L H3BO4, and 0.22 g / L NaMoO4·2H2O.

[0032] Ammonia-oxidizing bacteria culture medium (AOB): FeSO4·7H2O, MgSO4·7H2O, K2HPO4, NaHCO3, CaCl2·2H2O, NaCl, NH4Cl, and trace element solution are dissolved in water. The concentrations of FeSO4·7H2O, MgSO4·7H2O, K2HPO4, NaHCO3, CaCl2·2H2O, NaCl, and NH4Cl are controlled at 0.4 g / L, 1.5 g / L, 0.5 g / L, 2.0 g / L, and 0.4 g / L, respectively. The volume concentration of the trace element solution is 1 mL / L. The pH of the ammonia-oxidizing bacteria culture medium is controlled at 7.5. The medium is then sterilized under high temperature and high pressure (121℃, 20 min) and stored for later use.

[0033] Beef extract peptone medium (BPM): purchased from Qingdao Haibo Biotechnology, prepared at a concentration of 18 g / L, sterilized by high temperature and high pressure (121℃, 20 min) and stored for later use.

[0034] 4-Methyl-1-piperazine ethylamine stock solution: Weigh 1 g of 4-methyl-1-piperazine ethylamine (CAS: 934-98-5, purchased from Maclean's) using an analytical balance, dissolve it in a small amount of water, and transfer it to a 100 mL volumetric flask and dilute to volume to obtain the 4-methyl-1-piperazine ethylamine stock solution. Then, filter the 4-methyl-1-piperazine ethylamine stock solution through a 0.22 μm filter membrane for sterilization and store for later use.

[0035] It should be further noted that in the embodiments of the present invention, the ammonia nitrogen concentration detection method adopts the Nessler's reagent spectrophotometric method for the determination of ammonia nitrogen in water quality (HJ 535-2009); the nitrite nitrogen concentration is determined by the spectrophotometric method for the determination of nitrite nitrogen in water quality (GB / T 7493-1987).

[0036] Furthermore, in this embodiment of the invention, the nitrification inhibition rate is calculated using the following formula:

[0037]

[0038] Example 1

[0039] This embodiment relates to an experiment evaluating the nitrification inhibition effect of 4-methyl-1-piperazine ethylamine on the strain Nitrosomonas sp. CZ-4 (hereinafter referred to as CZ-4).

[0040] Take 1 mL of CZ-4 bacterial culture into fresh AOB medium and culture it in a shaker at 30℃ and 140 r / min. Take samples every 24 h to detect the concentration of ammonia nitrogen and nitrite nitrogen until the nitrite nitrogen concentration increases at a rate of 2 mg / L / h.

[0041] The 4-methyl-1-piperazine ethylamine stock solution was added to AOB medium containing CZ-4 bacterial culture at different concentrations, specifically according to the formulations in Table 1. The CZ-4 culture system without the addition of 4-methyl-1-piperazine ethylamine stock solution served as the control group, while the other CZ-4 culture systems served as the experimental groups. The cultures were then incubated in a shaker at 30℃ and 140 rpm.

[0042] Table 1. Formulas of CZ-4 experimental systems for the control and experimental groups in Example 1.

[0043]

[0044] Four days later, samples were taken and the concentrations of ammonia nitrogen and nitrite nitrogen in the control group and each experimental group were measured. The nitrite nitrogen concentrations in each control group and each experimental group on day 4 are shown in Table 2 and... Figure 1 As shown:

[0045] Table 2. Nitrite concentrations in each control group and experimental group in Example 1.

[0046]

[0047] From Table 2 and Figure 1 It was found that the nitrite concentration in the control group reached 80.76 mg / L on day 4, indicating good bacterial growth. After the addition of 4-methyl-1-piperazineethylamine, the increase in nitrite concentration in the culture system was inhibited to varying degrees, indicating that the growth of CZ-4 was inhibited to varying degrees. Furthermore, the inhibitory effect decreased with decreasing concentration; at a concentration of 0.001‰ w / v, there was no inhibitory effect; at a concentration of 0.01‰ w / v, the nitrification inhibition rate was 3.38%; and at a concentration of 1‰ w / v, the nitrification inhibition rate reached 96.87%.

[0048] Example 2

[0049] This embodiment relates to an experiment evaluating the nitrification inhibition effect of 4-methyl-1-piperazine ethylamine on the strain Nitrosomonas nitrosa WH-1 (hereinafter referred to as WH-1).

[0050] Take 1 mL of WH-1 bacterial culture into fresh AOB medium and culture it in a shaker at 30℃ and 140 r / min. Take samples every 24 h to detect the concentration of ammonia nitrogen and nitrite nitrogen until the nitrite nitrogen concentration increases at a rate of 2 mg / L / h.

[0051] The 4-methyl-1-piperazine ethylamine stock solution was added to AOB medium containing WH-1 bacterial culture at different concentrations, specifically according to the formulations in Table 3 to prepare the WH-1 culture system. The WH-1 culture system without the addition of 4-methyl-1-piperazine ethylamine stock solution served as the control group, while the other WH-1 culture systems served as the experimental groups. The cultures were then incubated in a shaker at 30℃ and 140 rpm, and ammonia nitrogen and nitrite nitrogen concentrations were measured daily.

[0052] Table 3. WH-1 experimental system formulations for the control and experimental groups in Example 2.

[0053]

[0054] The nitrite concentrations of each control group and each experimental group on days 1, 2, 3, and 4 are shown in Table 4. Figure 2 As shown:

[0055] Table 4. Nitrite concentrations in each control group and experimental group in Example 2.

[0056]

[0057]

[0058] From Table 4 and Figure 2 It was observed that the nitrite concentration in the control group increased sharply after 3 days, then leveled off on the 4th day, entering a stable period. The addition of 4-methyl-1-piperazine ethylamine inhibited the increase of nitrite in the WH-1 culture system to varying degrees, indicating that the growth of WH-1 was inhibited to varying degrees, and the inhibitory effect decreased with decreasing concentration.

[0059] From Table 4 and Figure 2 Further analysis revealed that 4-methyl-1-piperazine ethylamine showed almost no inhibition in the WH-1 experimental system at a concentration of 0.001‰ w / v, and a slight inhibitory effect at 0.01‰ w / v, with a nitration inhibition rate of 2.43%. At a concentration of 0.1‰ w / v, the nitration inhibition rate of the WH-1 experimental system was 9.47%, with the inhibitory effect becoming significant starting on day 3. At a concentration of 1‰ w / v, the nitration inhibition rate of the WH-1 experimental system reached 96.85%, with the inhibitory effect becoming significant starting on day 2.

[0060] Example 3

[0061] This embodiment relates to an evaluation experiment on the inhibitory effect of 4-methyl-1-piperazine ethylamine on heterotrophic microorganisms in soil.

[0062] Specifically, 20 grams of soil and 20 glass beads were added to 200 ml of physiological saline, shaken at 180 rpm for 30 min, and then allowed to settle for 5 min. The supernatant was collected as the soil microbial inoculum. Two 20 mL BPM culture media were placed in sterile Erlenmeyer flasks, and 20 μL of soil microorganisms were inoculated into each BPM medium. One group was further treated with 0.01‰ w / v 4-methyl-1-piperazine ethylamine as the experimental group; the other group was left untreated as the control group. The experimental and control groups were cultured overnight in a shaker at 30℃ and 140 rpm. The absorbance of the mixture of the experimental and control groups at 600 nm was measured using a visible spectrophotometer. The results are as follows: Figure 3 As shown.

[0063] Depend on Figure 3 It was found that after 16 hours, the absorbance values ​​of the mixed solution of the control group and the experimental group at 600 nm were 1.5 and 1.4, respectively. This indicates that after adding 0.01‰ w / v of 4-methyl-1-piperazine ethylamine, the microbial growth in the soil leachate was relatively consistent with that of the blank control group, suggesting that 0.01‰ w / v of 4-methyl-1-piperazine ethylamine has little impact on other heterotrophic microorganisms in the soil and has good environmental friendliness.

[0064] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

Application of 1,4-methyl-1-piperazine ethylamine in inhibiting the nitrosation activity of Nitrifying Monotrophus.

2. The application according to claim 1, characterized in that, The nitrosomonas include Nitrosomonas sp. CZ-4 and / or Nitrosomonas nitrosa strain WH-1. Application of 3,4-methyl-1-piperazine ethylamine in inhibiting the nitrification activity of Nitromonas bacteria in soil and / or wastewater treatment.

4. A method for inhibiting nitrification, characterized in that, include: Treat soil or water containing ammonium nitrogen and nitrosomonas bacteria with 4-methyl-1-piperazine ethylamine.

5. The method for inhibiting nitrification according to claim 4, characterized in that, The concentration of 4-methyl-1-piperazine ethylamine is not less than 0.01‰ w / v, based on the volume of the soil or water body as 100%.

6. The method for inhibiting nitrification according to claim 4, characterized in that, The concentration of 4-methyl-1-piperazine ethylamine is 1‰ w / v, based on the volume of the soil or water body as 100%.

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