Short-cut nitrification bacteria and its application in wastewater denitrification treatment

Abstract

This invention relates to the field of wastewater treatment technology, specifically to a short-range nitrifying bacterium and its application in wastewater denitrification treatment. The short-range nitrifying bacterium is taxonomically classified as *Klebsiella grimonella* (…). Klebsiella grimontii The strain, named A5, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 2025642, dated March 31, 2025. This invention's short-cut nitrifying bacteria can oxidize ammonia nitrogen to nitrite nitrogen, preventing further oxidation of nitrite nitrogen to nitrate nitrogen. It can be used for short-cut nitrification treatment of wastewater, achieving efficient conversion of ammonia nitrogen to nitrite nitrogen even at high ammonia nitrogen concentrations and over a wide temperature and pH range, demonstrating promising application prospects in wastewater denitrification.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment technology, and in particular to a short-range nitrifying bacterium and its application in wastewater denitrification treatment. Background Technology

[0002] Nitrogen is one of the most common pollutants in wastewater, widely present in various types of wastewater including domestic sewage, industrial wastewater, and agricultural wastewater. Excessive discharge can cause ecological and environmental problems and harm human health. Biological denitrification technology utilizes the metabolic activities of microorganisms such as nitrifying bacteria and denitrifying bacteria to treat wastewater for nitrogen removal. Compared with physicochemical methods, it has advantages such as convenient operation, no secondary pollution, and high denitrification efficiency. Traditional biological denitrification technology uses nitrifying bacteria to remove ammonia nitrogen (NH3-N or NH4+). + -N) is oxidized to nitrite nitrogen (NO2). - -N), and further oxidizes nitrite nitrogen to nitrate nitrogen (NO3). - After the nitrogen is converted into nitrogen gas by denitrifying bacteria, it requires a large amount of oxygen supply and external carbon source, resulting in low wastewater treatment efficiency, high operating energy consumption and cost, and also generates a lot of greenhouse gas emissions.

[0003] Short-cut nitrification-denitrification (LTD) and short-cut nitrification-anaerobic ammonium oxidation (ANAO) technologies, as alternatives to traditional nitrification-denitrification processes, are currently a research hotspot in novel wastewater denitrification technologies. LTD utilizes ammonia-oxidizing bacteria (AOB) to convert ammonia nitrogen into nitrite nitrogen, then directly converts nitrite nitrogen into nitrogen gas using denitrifying bacteria, bypassing the nitrate nitrogen stage. ANAO, on the other hand, involves AOB converting ammonia nitrogen into nitrite nitrogen, followed by anaerobic ammonium oxidation bacteria using ammonia nitrogen as the electron donor and nitrite nitrogen as the electron acceptor, to convert both ammonia nitrogen and nitrite nitrogen into nitrogen gas. These novel biological denitrification technologies, by omitting the nitrite-to-nitrate nitrogen conversion process, can improve wastewater treatment efficiency, reduce oxygen and carbon source requirements, and thus lower energy consumption and costs. The core of short-cut nitrification-denitrification and short-cut nitrification-anaerobic ammonia oxidation technologies lies in keeping the oxidation process of ammonia nitrogen at the nitrite stage, thus preventing nitrite from being further oxidized to nitrate. Developing microbial strains that can achieve this function will help promote the development of new biological nitrogen removal technologies.

[0004] Klebsiella grimontii It is Klebsiella spp. Klebsiella This species is a type of *Klebsiella grimonis*, and currently there is no universally accepted Chinese translation for it. This invention will temporarily translate it as "Klebsiella grimonis". Currently, strains of this species that can reduce sulfates have been discovered (e.g., those in patent CN116622541A). Klebsiella grimontii YWC001), strains that can prevent soybean root rot (such as those in patent CN118207111A) Klebsiella grimontiiDR11), etc., but no strains with short-range nitrification function have been found in this species. Summary of the Invention

[0005] In view of this, the present invention provides a short-range nitrifying bacterium and its application in wastewater denitrification treatment. This short-range nitrifying bacterium is *Klebsiella grimonella* (… Klebsiella grimontii A new strain discovered in the laboratory can oxidize ammonia nitrogen to nitrite nitrogen, avoiding further oxidation of nitrite nitrogen to nitrate nitrogen. It can be used for short-cut nitrification treatment of wastewater. It can achieve efficient conversion of ammonia nitrogen to nitrite nitrogen even at high ammonia nitrogen concentrations and over a wide range of temperature and pH. In particular, it has strong alkali resistance and has good application prospects in wastewater denitrification treatment.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] First, this invention provides a short-range nitrifying bacterium, which taxonomically belongs to Klebsiella grimonella (Klebsiella). Klebsiella grimontii The strain, named A5, is deposited at the China Center for Type Culture Collection (CCTCC, located at Wuhan University, Wuhan, China), with accession number CCTCC No: M 2025642 and deposit date of March 31, 2025.

[0008] The 16S rRNA sequence of the short-range nitrifying bacteria is shown in SEQ ID NO.1.

[0009] Second, the present invention provides a live bacteria preparation for short-range nitrification, wherein the live bacteria preparation contains the aforementioned short-range nitrifying bacteria.

[0010] Preferably, the live bacteria preparation also contains a short-range nitrifying bacteria culture medium.

[0011] More preferably, the short-range nitrifying bacteria culture medium is TSA medium (Tryptic Soy Agar) or TSB medium (Tryptic Soy Broth).

[0012] Preferably, the live bacteria preparation is a powder, granules, liquid, or tablet.

[0013] Third, the present invention provides the application of the short-range nitrifying bacteria in wastewater denitrification treatment, wherein the wastewater denitrification treatment process includes: using the short-range nitrifying bacteria to convert ammonia nitrogen in wastewater into nitrite nitrogen.

[0014] Preferably, in the process of using the short-range nitrifying bacteria to convert ammonia nitrogen in wastewater into nitrite nitrogen, the dissolved oxygen concentration in the wastewater is controlled at 0.4~0.5 mg / L, the water temperature at 10~40℃, the initial pH of the wastewater at 5~9, and the initial ammonia nitrogen concentration at 5~2000 g / L.

[0015] More preferably, in the process of using the short-range nitrifying bacteria to convert ammonia nitrogen in wastewater into nitrite nitrogen, the dissolved oxygen concentration in the wastewater is controlled at 0.4~0.5 mg / L, the water temperature at 37~40℃, the initial pH of the wastewater at 7~9, and the initial ammonia nitrogen concentration at 5~2000 g / L.

[0016] Preferably, the wastewater denitrification treatment method is short-cut nitrification-denitrification or short-cut nitrification-anaerobic ammonium oxidation.

[0017] Preferably, the wastewater denitrification treatment device consists of a detection module, a microbial culture module for simultaneous short-range nitrification and denitrification, and an intelligent remote feedback module. The wastewater denitrification treatment process includes: in the detection module, using microbial electrodes to detect the toxicity and biochemical oxygen demand (BOD) of the wastewater; in the microbial culture module for simultaneous short-range nitrification and denitrification, after the short-range nitrifying and denitrifying bacteria are cultured in a large-scale manner to obtain a bacterial solution, which is then added to the wastewater for short-range nitrification and denitrification; the intelligent remote feedback module adjusts one or more of the following based on the toxicity and BOD measured in the detection module: influent flow rate, aeration rate, carbon source dosage, and dosage of the bacterial solution obtained from the large-scale culture.

[0018] In the aforementioned device and wastewater denitrification process: the microbial electrode mainly utilizes the principle that electrogenic microorganisms (such as Shewanella) can discharge electricity. The different electrical signals released by the microbial electrode in different states of wastewater influent (toxic, non-toxic) indicate the presence or absence of toxicity in the influent, helping to ensure timely emergency measures for wastewater treatment. Simultaneously, the microbial electrode can also exhibit different electrical values ​​based on the influent BOD level. After long-term simulation (approximately 3 months) with real BOD data, a fitting curve between the electrical signal and BOD can be generated. This allows for real-time monitoring of the influent BOD data using the microbial electrode. Through these methods, the detection module can predict the toxicity and biodegradability of the influent in advance, and then the wastewater treatment system can be controlled in real-time via an intelligent remote feedback module. The integrated microbial culture and short-cut nitrification / denitrification module allows for the timely and continuous addition of cultured microorganisms to the wastewater treatment system.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) Klebsiella grimonis of the present invention ( Klebsiella grimontiiA5 can convert ammonia nitrogen into nitrite nitrogen and can limit the oxidation process of ammonia nitrogen to the nitrite nitrogen stage, without further oxidizing nitrite nitrogen into nitrate nitrogen. Therefore, it can be used for short-cut nitrification of wastewater. After short-cut nitrification is completed using the strain of this invention, nitrite nitrogen can be further converted into nitrogen gas for removal through denitrification or anaerobic ammonia oxidation.

[0021] (2) The Klebsiella grimonis of the present invention ( Klebsiella grimontii A5 strain can adapt to high ammonia nitrogen concentrations and a wide pH range (especially high alkalinity) and temperature range, and can achieve high short-cut nitrification efficiency, showing good application prospects in wastewater denitrification treatment. Experiments have shown that this strain can perform short-cut nitrification in environments with pH 5.0–9.0 and temperatures of 10–40℃, and can still achieve complete removal of ammonia nitrogen even when the initial ammonia nitrogen concentration reaches 2000 mg / L, with a conversion rate of ammonia nitrogen to nitrite nitrogen reaching 95%. Attached Figure Description

[0022] Figure 1 This is a graph showing the results of the Griess test in Example 1. Wherein: Figure 1 Photo A shows the negative control group after Griess reagent was added; Figure 1 B is a photograph of the positive control group after Griess' reagent was added; Figure 1 C is a photograph of the experimental group after Griess reagent was added.

[0023] Information on the preservation of biological materials

[0024] The strain of this invention, Klebsiella grimonis ( Klebsiella grimontii A5 is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC No: M2025642, and deposited on March 31, 2025. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments. These embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way.

[0026] The strain involved in this invention is Klebsiella grimonis. Klebsiella grimontii A5 was isolated from municipal sewage sludge samples, and its 16S rRNA sequence is shown in SEQ ID NO.1. The specific sequence is as follows:

[0027]

[0028] Example 1: Validation of short-range nitrification function of Klebsiella griseus A5 (Griess test)

[0029] Prepare aerobic ZPM14 liquid culture medium according to the following formula: (NH4)2SO4 2.0 g / L, K2HPO4 1.0 g / L, MgSO4 0.5 g / L, CaCO3 1.0 g / L, NaCl 10.0 g / L, solvent is purified water; pH=7.0; sterilize at 121℃ for 15 min.

[0030] Griess reagent can be used to detect nitrite. When nitrite ions are present in the system, Griess reagent reacts with nitrite ions in a diazotization reaction to generate a red azo compound. Based on this, this example uses Griess reagent to detect whether the strain can convert ammonia nitrogen into nitrite nitrogen. The experimental group design is as follows:

[0031] (1) Negative control group: 400 μL of Griess reagent was added to a centrifuge tube containing 5 mL of aerobic ZPM14 liquid culture medium.

[0032] (2) Positive control group: 400 μL of Griess reagent was added to a container containing 5 mL of 6.4×10 -6 In a centrifuge tube containing mol / L sodium nitrite solution.

[0033] (3) Experimental group: Single colonies of Klebsiella grimonis A5 were picked and inoculated into aerobic ZPM14 liquid medium. The culture was carried out at 37°C for 2 days under aerobic conditions to obtain the initial culture. The initial culture was then transferred to a centrifuge tube containing 5 mL of aerobic ZPM14 liquid medium and incubated at 37°C for 3 days under aerobic conditions. Then, 400 μL of Griess reagent was added, and the color change of the liquid in the centrifuge tube was observed.

[0034] The experimental results are as follows: In the negative control group, the liquid in the centrifuge tube remained colorless and transparent after the addition of Griess' reagent, with no color change (e.g., Figure 1 As shown in Figure A); in the positive control group, the liquid in the centrifuge tube changed from colorless and transparent to purple-red after the addition of Griess' reagent (as shown in Figure A). Figure 1 (As shown in B); In the experimental group, after adding Griess' reagent, the liquid in the centrifuge tube changed from colorless and transparent to purple-red (as shown in B). Figure 1 (As shown in C). The above results indicate that Klebsiella grimonis A5 can convert ammonia nitrogen into nitrite nitrogen.

[0035] Example 2: Effect of initial ammonia nitrogen concentration on the short-range nitrification function of Klebsiella griseus A5

[0036] Prepare preparations with different ammonia nitrogen (NH4) concentrations according to the following formula. + Aerobic ZPM14 liquid medium with (NH4)2SO4 concentrations of 24, 142, 283, 472, 944, and 9439 mg / L (equivalent to ammonia nitrogen concentrations of 5, 30, 60, 100, 200, and 2000 mg / L in the medium), K2HPO4 1.0 g / L, MgSO4 0.5 g / L, CaCO3 1.0 g / L, and NaCl 10.0 g / L, in purified water as solvent; pH=9.0; sterilized at 121℃ for 15 min using moist heat.

[0037] Single colonies of Klebsiella grimonis A5 were picked and inoculated into TSB medium. The cultures were then incubated aerobically at 37°C until the logarithmic growth phase to obtain Klebsiella grimonis A5 bacterial suspension. The Klebsiella grimonis A5 bacterial suspension was then inoculated at a rate of 3% into aerobically cultured ZPM14 liquid medium with different ammonia nitrogen concentrations. After incubation at 37°C for 3 days aerobically, the concentrations of ammonia nitrogen, nitrite nitrogen, and total nitrogen in the culture medium were measured. The results are shown in Table 1.

[0038] Table 1. Results of the detection of the effect of initial ammonia nitrogen concentration on the short-range nitrification function of the strains

[0039]

[0040] Analysis of test results: As shown in Table 1, the Klebsiella grimonella A5 strain of the present invention can tolerate and effectively treat high concentrations of ammonia nitrogen. When the initial ammonia nitrogen concentration reaches 2000 mg / L, the strain can still perform good short-cut nitrification, efficiently converting ammonia nitrogen into nitrite nitrogen. The ammonia nitrogen removal rate can reach 100% within 3 days, and 95% of the ammonia nitrogen can be converted into nitrite nitrogen.

[0041] Example 3: Effect of pH on the short-range nitrification function of Klebsiella griseus A5

[0042] Prepare aerobic ZPM14 liquid culture media with different pH values ​​according to the following formula: (NH4)2SO4 9439 mg / L (equivalent to 2000 mg / L ammonia nitrogen concentration in the culture medium), K2HPO4 1.0 g / L, MgSO4 0.5 g / L, CaCO3 1.0 g / L, NaCl 10.0 g / L, with purified water as the solvent; pH = 5.0, 6.0, 7.0, 8.0, 9.0; sterilize at 121℃ for 15 min using moist heat.

[0043] Single colonies of Klebsiella grimonis A5 were picked and inoculated into TSB medium. The cultures were then incubated aerobically at 37°C until the logarithmic growth phase, yielding Klebsiella grimonis A5 bacterial suspension. The Klebsiella grimonis A5 bacterial suspension was then inoculated at a rate of 3% into aerobically cultured ZPM14 liquid media with different pH values. After incubation at 37°C for 3 days aerobically, the concentrations of ammonia nitrogen, nitrite nitrogen, and total nitrogen in the media were measured. The results are shown in Table 2.

[0044] Table 2 Results of the detection of the effect of pH on the short-range nitrification function of bacterial strains

[0045]

[0046] Analysis of test results: As shown in Table 2, the Klebsiella grimonella A5 of the present invention can perform short-cut nitrification in an environment with pH 5.0~9.0, converting ammonia nitrogen into nitrite nitrogen. Its ammonia nitrogen removal rate can reach more than 50% within 3 days, and the conversion rate of ammonia nitrogen to nitrite nitrogen can reach more than 48%. In particular, it has strong alkali resistance. When pH=9.0, the ammonia nitrogen removal rate can reach 100% within 3 days.

[0047] Example 4: Effect of temperature on the short-range nitrification function of Klebsiella griseus A5

[0048] Prepare aerobic ZPM14 liquid culture medium with different pH values ​​according to the following formula: (NH4)2SO4 9439 mg / L (equivalent to 2000 mg / L ammonia nitrogen concentration in the culture medium), K2HPO4 1.0 g / L, MgSO4 0.5 g / L, CaCO3 1.0 g / L, NaCl 10.0 g / L, solvent is purified water; pH=9.0; sterilize at 121℃ for 15 min by high temperature moist heat.

[0049] Single colonies of Klebsiella grimonis A5 were picked and inoculated into TSB medium. The culture was then incubated aerobically at 37°C until the logarithmic growth phase to obtain Klebsiella grimonis A5 bacterial suspension. The Klebsiella grimonis A5 bacterial suspension was then inoculated into aerobically cultured ZPM14 liquid medium at a 3% inoculum. After incubation at 10°C, 20°C, 37°C, and 40°C for 3 days under aerobically controlled conditions, the concentrations of ammonia nitrogen, nitrite nitrogen, and total nitrogen in the culture medium were measured. The results are shown in Table 3.

[0050] Table 3. Results of the test on the effect of temperature on the short-range nitrification function of the strains

[0051]

[0052] Analysis of test results: As shown in Table 3, the Klebsiella grimonella A5 of the present invention can survive and play a short-range nitrification role in an environment of 10~40℃. The optimal temperature is around 37℃. When the temperature is 37~40℃, the ammonia nitrogen removal rate can reach 78% or more, and the conversion rate of ammonia nitrogen to nitrite nitrogen can reach 74% or more.

[0053] Example 5: Application of Klebsiella grimonis A5 in wastewater denitrification treatment

[0054] The experimental setup used in this embodiment consists of an inlet tank, an aerobic tank, an anaerobic tank, and an effluent tank connected in sequence. The aerobic and anaerobic tanks are filled with polyurethane biological packing material, with a filling rate of 20%. Klebsiella pneumoniae A5 was inoculated into TSB medium and cultured at 37°C for 3 days under aerobic conditions. The resulting bacterial solution was then inoculated into the aerobic tank for biofilm formation. Denitrifying bacteria powder purchased from Zelin Environmental Protection was mixed with anaerobic system wastewater (pH adjusted to 7.5) at a mass ratio of 1:10, with dissolved oxygen (DO) content controlled to be no higher than 0.2 mg / L. After culturing at 32°C for 24 hours, the resulting bacterial solution was inoculated into the anaerobic tank for biofilm formation. The experimental water was urban domestic sewage, and the water quality is shown in Table 4. The test water was sequentially passed through an influent tank, an aerobic tank, and an anaerobic tank before being temporarily stored in an effluent tank. Sodium acetate was added to the aerobic tank at a dosage 20 times the mass of nitrate nitrogen in the influent. The dissolved oxygen concentration in the anaerobic tank was kept below 0.2 mg / L. Aeration was used to control the dissolved oxygen content in the aerobic tank at 0.4–0.5 mg / L. The hydraulic retention times in the aerobic and anaerobic tanks were 15 h and 10 h, respectively. During stable operation of the device, the effluent quality was monitored, and the results are shown in Table 4 (average values).

[0055] Table 4. Water quality of the test water

[0056]

[0057] Analysis of test results: As can be seen from Table 4, the Klebsiella grimonella A5 of the present invention can be used in combination with denitrifying bacteria to effectively reduce the ammonia nitrogen and total nitrogen content in wastewater, while degrading organic matter in wastewater and reducing COD and BOD values.

[0058] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of this invention should be considered within the scope of protection of this invention.

Claims

1. A short-range nitrifying bacterium, characterized in that, The short-range nitrifying bacteria are taxonomically classified as Klebsiella grimonis. Klebsiella grimontii The strain, named A5, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 2025642 and a deposit date of March 31, 2025.

2. A live bacteria preparation for short-cut nitrification, characterized in that, The live bacteria preparation contains the short-range nitrifying bacteria as described in claim 1.

3. The live bacteria preparation as described in claim 2, characterized in that, The live bacteria preparation also contains a short-range nitrifying bacteria culture medium.

4. The live bacteria preparation as described in claim 3, characterized in that, The short-range nitrifying bacteria culture medium is either TSA medium or TSB medium.

5. The live bacteria preparation according to any one of claims 3 to 4, characterized in that, The live bacteria preparation is in the form of powder, granules, liquid, or tablets.

6. The application of short-range nitrifying bacteria as described in claim 1 in wastewater denitrification treatment, characterized in that, The wastewater denitrification process includes: using the short-range nitrifying bacteria to convert ammonia nitrogen in the wastewater into nitrite nitrogen.

7. The application according to claim 6, characterized in that, In the process of using the aforementioned short-range nitrifying bacteria to convert ammonia nitrogen in wastewater into nitrite nitrogen, the dissolved oxygen concentration in the wastewater is controlled at 0.4~0.5 mg / L, the water temperature at 10~40℃, the initial pH of the wastewater at 5~9, and the initial ammonia nitrogen concentration at 5~2000 g / L.

8. The application according to claim 6, characterized in that, The wastewater denitrification treatment method is either short-cut nitrification-denitrification or short-cut nitrification-anaerobic ammonium oxidation.

9. The application according to claim 6, characterized in that, The wastewater denitrification treatment device consists of a detection module, a microbial culture module for simultaneous short-range nitrification and denitrification, and an intelligent remote feedback module. The wastewater denitrification process includes: in the detection module, microbial electrodes are used to detect the toxicity and biochemical oxygen demand (BOD) of the wastewater; in the microbial culture module for simultaneous short-range nitrification and denitrification, the short-range nitrifying and denitrifying bacteria are cultured in a large-scale manner to obtain a bacterial solution, which is then added to the wastewater for short-range nitrification and denitrification; the intelligent remote feedback module adjusts one or more of the following based on the toxicity and BOD measured in the detection module: influent flow rate, aeration rate, carbon source dosage, and dosage of the bacterial solution obtained from the large-scale culture.

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