A nitrification inhibitor and its application

Abstract

The present invention relates to the fields of environmental engineering, agricultural chemistry, or microbiology, and provides a nitrification inhibitor and its use. The nitrification inhibitor is N-ethylpiperazine, which can inhibit the growth of AOB and thus nitrification at a relatively low dose (10 mg / L or 0.1 g / kg). Furthermore, its Material Safety Data Sheet (SDS) does not report its ecotoxicity or potential bioaccumulation, demonstrating its environmental friendliness and its application in water treatment and soil to inhibit nitrification.

Description

Technical Field

[0001] The present invention relates to the technical fields of environmental engineering, agricultural chemistry or microbiology, and in particular to a nitrification inhibitor and application thereof. Background Art

[0002] Nitrification refers to the process of ammonium nitrogen (NH4 + -N) is gradually oxidized to nitrate (NO) through the metabolic activity of microorganisms. 3- -N) is one of the key links in the nitrogen cycle in the natural environment. This process is mainly completed by two types of nitrifying microorganisms: ammonia oxidizing bacteria (AOB) converts ammonium nitrogen (NH4 + -N) is oxidized to nitrite (NO 2- -N), nitrite oxidizing bacteria (NOB) further convert nitrite (NO 2- -N) is oxidized to nitrate (NO 3- -N). Nitrification is widely present in soil, freshwater, marine and other ecosystems, and has a significant impact on the ecological environment and agricultural production.

[0003] Excessive nitrification can lead to a range of environmental problems, such as groundwater contamination caused by nitrate leaching and reduced nitrogen fertilizer utilization efficiency. Therefore, effectively regulating the nitrification process to minimize its adverse effects has become a critical issue in agriculture and environmental management. In recent years, the research and development of novel nitrification inhibitors that selectively inhibit the activity of ammonia-oxidizing bacteria has become a key focus for achieving efficient nitrogen utilization and environmental protection.

[0004] Traditional nitrification inhibitors primarily include nitrothiazoles, dicyandiamide, 3,4-dimethylpyrazole phosphate, and nitroacetamide compounds. 3,4-dimethylpyrazole phosphate (DMPP) is the least commonly used of these nitrification inhibitors, typically at a concentration of 0.5%-1% (by weight) of nitrogen fertilizer applied to soil. It exhibits excellent inhibitory effects and low toxicity. Further reductions in nitrification inhibitor dosages would be crucial for improving agricultural economic efficiency. The N-ethylpiperazine-based nitrification inhibitors of the present invention can inhibit ammonia oxidation, the first step in nitrification, at relatively low dosages, thereby effectively regulating the nitrification process. Summary of the Invention

[0005] In view of this, the present invention proposes a nitrification inhibitor that has a small dosage and is highly effective in inhibiting nitrification, and its application.

[0006] The technical solution of the present invention is achieved as follows: In a first aspect, the present invention provides a nitrification inhibitor, which is N-ethylpiperazine (CAS: 5308-25-8).

[0007] In a second aspect, the present invention provides a method for treating sewage using a nitrification inhibitor.

[0008] Nitrification converts ammonium nitrogen into nitrate, a water-soluble compound that, at high concentrations, can seep into rivers, lakes, and groundwater with rainwater or irrigation, leading to eutrophication. Similarly, inhibiting nitrification can reduce nitrate loss, thereby minimizing negative impacts on aquatic ecosystems.

[0009] In addition, in intensive aquaculture, controlling nitrification in water can reduce the accumulation of nitrates and nitrites, avoid toxicity to aquatic organisms, and thus improve aquaculture production efficiency and ecological balance.

[0010] Based on the above technical solution, preferably, the minimum usage dosage of the nitrification inhibitor in sewage is 10 mg / L.

[0011] In a third aspect, the present invention provides a method for inhibiting the nitrification of agricultural soils.

[0012] In a fourth aspect, the present invention provides a fertilizer comprising a nitrification inhibitor.

[0013] Nitrification causes nitrogen fertilizer to be lost as nitrates, which are easily leached into the deep soil or surface runoff, making them ineffective for crops to utilize, resulting in nitrogen fertilizer waste. Inhibiting nitrification in water can preserve nitrogen as ammonium nitrogen, increasing the effectiveness of nitrogen fertilizers, thereby reducing fertilizer costs and improving agricultural economic benefits.

[0014] In addition, hydrogen ions (H + ), accelerating soil acidification and thus reducing the fertility of farmland soil. By inhibiting nitrification, soil acidification can be delayed, playing a positive role in protecting soil quality.

[0015] Based on the above technical solution, preferably, the minimum dosage of the nitrification inhibitor in the soil is 0.1 g / kg.

[0016] The nitrification inhibitor of the present invention and its application have the following beneficial effects compared with the prior art:

[0017] The nitrification inhibitor N-ethylpiperazine provided by the present invention can inhibit the growth of AOB and thus inhibit nitrification at a relatively low dose (0.01‰w / v or 0.01‰w / w), and its chemical safety data sheet (SDS) does not report its ecotoxicity and potential bioaccumulation, indicating its environmental friendliness and can be used in water treatment and soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 The effect of different concentrations of N-ethylpiperazine on the growth of Nitrosomonas sp. CZ-4.

[0020] Figure 2 The effect of different concentrations of N-ethylpiperazine on the growth of autotrophic ammonia oxidizing bacteria (Nitrosomonas nitrosa) WH-1.

[0021] Figure 3 The effect of 0.01‰w / w N-ethylpiperazine on the growth of heterotrophic microorganisms in soil.

[0022] Figure 4 The effect of different concentrations of N-ethylpiperazine on ammonia nitrogen and nitrate nitrogen in soil. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] Ammonia oxidation pathway (NH4 + -N is converted to NO 2- Ammonia oxidation (NH-N) is the first and rate-limiting step in the nitrification reaction. Nitrification can be inhibited by inhibiting the ammonia oxidation process. Therefore, the present invention dissolves N-ethylpiperazine (CAS: 5308-25-8, purchased from Adamas / Titan) in an aqueous solution to study its effect on the growth of two typical ammonia-oxidizing microorganisms in an inorganic salt culture medium, thereby evaluating the nitrification inhibitory effect of N-ethylpiperazine.

[0025] The two ammonia oxidizing microorganisms used in the present invention are Nitrosomonas sp. CZ-4 (hereinafter referred to as "CZ-4", derived from the invention patent with patent application publication number CN109402026A) and autotrophic ammonia oxidizing bacteria (Nitrosomonas nitrosa) WH-1 (hereinafter referred to as "WH-1", derived from the invention patent with patent application publication number CN112625940A).

[0026] The solution preparation method used in the embodiment of the present invention is as follows:

[0027] AOB culture medium: FeSO4·7H2O 0.4 g / L, MgSO4·7H2O 0.5 g / L, K2HPO4 0.1 g / L, NaHCO3 1.5 g / L, CaCl2·2H2O 0.5 g / L, NaCl 2.0 g / L, NH4Cl 0.4 g / L, trace element solution 1 mL / L, pH 7.5, sterilize at high temperature and high pressure (121°C, 20 min) and store until use.

[0028] Trace element solution: EDTA 0.5 g / L, CuSO4·5H2O 0.075 g / L, ZnSO4·7H2O 0.3 g / L, CoCl2·6H2O 0.375 g / L, MnCl2·2H2O 0.3 g / L, H3BO4 0.014 g / L, NaMoO4·2H2O 0.22 g / L.

[0029] Beef peptone medium (BPM) was purchased from Qingdao Haibo Biological with a concentration of 18 g / L and stored for future use after high temperature and high pressure sterilization (121°C, 20 min).

[0030] N-Ethylpiperazine mother solution (10000 mg / L): Weigh 1 g of N-ethylpiperazine using an analytical balance, add a small amount of water to mix, transfer to a 100 mL volumetric flask, make up to volume, filter through a 0.22 μm filter membrane to sterilize, and store until ready for use.

[0031] All the indicators involved were tested using national standard methods, among which the ammonia nitrogen detection method adopted the "Water quality - Determination of ammonia nitrogen - Nessler's reagent spectrophotometric method" (HJ 535-2009); the nitrite nitrogen was determined using the "Water quality - Determination of nitrite nitrogen - Spectrophotometric method" (GB / T 7493-1987), and the nitrate nitrogen content was determined using the "Water quality - Determination of nitrate nitrogen - Ultraviolet spectrophotometric method" (HZ / T 346-2007); the amount of dry matter and moisture in the soil were determined using the "Soil dry matter and moisture determination gravimetric method" (HJ 613-2011), and the concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in the soil were extracted with potassium chloride according to the "Soil ammonia nitrogen, nitrite nitrogen and nitrate nitrogen - Potassium chloride solution extraction-spectrophotometric method" (HJ 634-2012), and then determined in accordance with the national water quality standard.

[0032] The nitrification inhibition rate was calculated using the following formula:

[0033]

[0034] Example 1: Inhibition of strain CZ-4 by N-ethylpiperazine

[0035] Cultivation of CZ-4 inoculum: 1 mL of CZ-4 culture was inoculated into fresh AOB medium and cultured in a shaking incubator at 30°C (140 rpm). Samples were taken every 24 h to detect ammonia nitrogen and nitrite nitrogen until the nitrite nitrogen growth rate reached 2 mg / L / h.

[0036] According to the experimental system in Table 1, the CZ-4 culture system with different concentrations of N-ethylpiperazine (0.001‰w / v, 0.01‰w / v, 0.1‰w / v, 1‰w / v) was prepared and cultured in a shaking incubator at 30℃ (140r / min). After 4 days, samples were taken to determine ammonia nitrogen and nitrite nitrogen. The results are shown in the table. Figure 1 .

[0037] Table 1 CZ-4 experimental system

[0038] comparison 1‰ 0.1‰ 0.01‰ 0.001‰ AOB medium 90mL 90mL 90mL 90mL 90mL N-Ethylpiperazine mother liquor - 10mL 1mL 0.1mL 0.01mL Sterile water 10mL - 9mL 9.9mL 9.99mL CZ-4 inoculum 1mL 1mL 1mL 1mL 1mL

[0039] Figure 1 As shown, the nitrite nitrogen content of the control group CK increased sharply after 4 days, indicating good bacterial growth. After the addition of N-ethylpiperazine, the increase of nitrite nitrogen in the culture system was inhibited to varying degrees, indicating that the growth of CZ-4 was inhibited to varying degrees. As the concentration of N-ethylpiperazine decreased, the inhibition ability decreased. At a concentration of 0.001‰ w / v, there was no inhibition. At a concentration of 0.01‰ w / v, the nitrification inhibition rate was 10.52%, and at a concentration of 1‰ w / v, the nitrification inhibition rate was 98.15%, indicating that the optimal concentration of N-ethylpiperazine is 1‰ w / v.

[0040] Example 2: Inhibition of strain WH-1 by N-ethylpiperazine

[0041] Culture of WH-1 inoculum: Take 1 mL of WH-1 bacterial solution and inoculate it into fresh AOB medium. Incubate it in a shaking incubator at 30°C (140 rpm). Take samples every 24 hours to test ammonia nitrogen and nitrite nitrogen until the nitrite nitrogen growth rate reaches 2 mg / L / h.

[0042] According to the experimental system in Table 2, WH-1 culture system with different concentrations (0.001‰w / v, 0.01‰w / v, 0.1‰w / v, 1‰w / v) of N-ethylpiperazine was prepared and cultured in a shaking incubator at 30℃ (140r / min). Ammonia nitrogen and nitrite nitrogen were sampled and determined every day. The results are shown in the table. Figure 2 .

[0043] Table 2 WH-1 experimental system

[0044] comparison 1‰ 0.1‰ 0.01‰ 0.001‰ AOB medium 90mL 90mL 90mL 90mL 90mL N-Ethylpiperazine mother liquor - 10mL 1mL 0.1mL 0.01mL Sterile water 10mL - 9mL 9.9mL 9.99mL WH-1 ​​inoculum 1mL 1mL 1mL 1mL 1mL

[0045] Figure 2 As shown, the nitrite nitrogen content in the control group CK increased sharply after 3 days, then leveled off on the 4th day, entering a stable phase. After the addition of N-ethylpiperazine, the increase in nitrite nitrogen in the SN-6 culture system was inhibited to varying degrees, indicating that the growth of the SN-6 strain was inhibited to varying degrees. The inhibitory ability decreased with decreasing N-ethylpiperazine concentrations. SN-6 was weakly inhibited at concentrations of 0.001‰ w / v and 0.01‰ w / v N-ethylpiperazine, with nitrification inhibition rates of 4.94% and 5.19%, respectively. At a concentration of 1‰ w / v N-ethylpiperazine, the nitrification inhibition rate was 96.85%, indicating that the optimal concentration was 1‰ w / v.

[0046] Example 3: Inhibition of heterotrophic microorganisms in soil by 0.01‰ N-ethylpiperazine

[0047] Add 20g of soil and 20 glass beads to 200mL of normal saline, shake at 180r / min for 30min, let settle for 5min, and collect the supernatant as the soil microbial inoculum. Take 20mL of BPM medium in a sterile conical flask and inoculate 20μL of soil microorganisms. Add 0.01‰w / v N-ethylpiperazine to the experimental group and leave it untreated in the control group. After incubation at 30℃ in a shaker overnight, use a visible spectrophotometer to measure the absorbance at 600nm. The results are shown in the table. Figure 3 .

[0048] Figure 3As shown in the figure: after adding 0.01‰ N-ethylpiperazine, the growth degree of microorganisms in the soil extract was consistent with that of the blank control group, indicating that 0.01‰ N-ethylpiperazine had no inhibitory effect on other heterotrophic microorganisms in the soil. Combined with the above results, it can be seen that 0.01‰ N-ethylpiperazine selectively inhibits ammonia oxidizing bacteria.

[0049] Example 4: Comparison of Nitrification Inhibitors DMPP and N-Ethylpiperazine

[0050] According to the search results, there are few research reports on the inhibition of DMPP on the growth of ammonia oxidizing bacteria (AOB). Existing studies have shown that when DMPP is added in an amount of 0.1‰ (w / v), the nitrite inhibition rate during the growth of AOB is about 14% (Guo Changqing, Study on the Evaluation Method of Nitrification Inhibitors and the Synthesis and Application Effect of New Nitrification Inhibitors, Doctoral Dissertation of Shenyang Agricultural University, 2021). In contrast, Examples 1 and 2 of the present invention show that under the same addition amount (0.1‰), the nitrite inhibition rate of N-ethylpiperazine reached 84.41% and 19.03%, respectively. This result shows that compared with DMPP, N-ethylpiperazine exhibits a significantly higher inhibitory effect at the same dosage, and thus may have a lower dosage requirement in practical applications.

[0051] Example 5: Fertilizer retention effect of N-ethylpiperazine in soil

[0052] The soil used in this experiment was taken from the sandy soil in Xinzhou District, Wuhan City. After the soil was retrieved, the soil dry matter and water content were measured, impurities were removed, and it was naturally air-dried until the moisture content was less than 10%, and passed through a 2mm sieve. 50g of dry soil was added with ammonium chloride (added at 300mg / kg) and N-ethylpiperazine (added at 1‰, 0.1‰, 0.01‰w / w, respectively) and mixed thoroughly. Pour it into a glass bottle, adjust the moisture to 60% of the field water holding capacity, and seal it with a breathable membrane to ensure oxygen supply during the cultivation process. Cultivate it at 30°C for 3 days, and add volatile water by weighing during the cultivation process. On the fourth day of cultivation, soil samples were taken destructively, the soil in the glass bottle was fully mixed, and samples were taken to determine the soil ammonium nitrogen, nitrite nitrogen and nitrate nitrogen. The results are shown in the table. Figure 4 .

[0053] like Figure 4 The results show that 0.1‰w / w and 1‰w / w N-ethylpiperazine can increase the ammonium nitrogen content in the soil and reduce the nitrate nitrogen content in the soil, indicating that a dosage of 0.1g / kg (0.1‰w / w) in the soil can reduce nitrogen fertilizer loss. Therefore, in actual production, N-ethylpiperazine can be used as a nitrogen fertilizer additive, mixed with nitrogen fertilizer (ammonium chloride or urea) and applied to the soil together to prevent nitrogen fertilizer loss and thus improve nitrogen fertilizer utilization efficiency.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of a nitrification inhibitor in sewage treatment, characterized by: The nitrification inhibitor is N-ethylpiperazine.

2. The use according to claim 1, characterized in that: The minimum dosage of the nitrification inhibitor in sewage is 10 mg / L.

3. Application of a nitrification inhibitor in agricultural soil, characterized by: The nitrification inhibitor is N-ethylpiperazine.

4. The use according to claim 3, characterized in that: The minimum dosage of the nitrification inhibitor in soil is 0.1 g / kg.

5. Use of a nitrification inhibitor in fertilizer, characterized in that: The nitrification inhibitor is N-ethylpiperazine.

Images