Clostridium hathewayi capable of anaerobically degrading nitrogen-containing heterocyclic compounds and use thereof

Abstract

The present application relates to a Clostridium sinovenosum capable of anaerobically degrading nitrogen-containing heterocyclic compounds and its application, and is suitable for the field of environmental microorganisms and wastewater treatment technology. Clostridium huakuii ) FL017, which has been preserved in the China Center for Type Culture Collection on December 1, 2025, with the preservation number CCTCC NO: M20252755, and the strain is detected to be alive. The Clostridium sinovenosum FL017 provided by the present application is an anaerobic functional strain, which can grow normally and play a degrading role in a completely anaerobic / hypoxic environment, and can be directly applied to an anaerobic treatment unit of a sewage system, solving the technical pain point that the existing aerobic degrading bacteria cannot adapt to the anaerobic section, and perfecting the pollutant removal function of the anaerobic section of wastewater.

Description

Technical Field

[0001] This invention relates to a Clostridium kanehirae strain capable of anaerobic degradation of nitrogen-containing heterocyclic compounds and its applications. It is applicable to the fields of environmental microbiology and wastewater treatment technology. Background Technology

[0002] Nitrogen-containing heterocyclic compounds are common and difficult-to-degrade substances in wastewater. Most of them have a foul odor and are highly toxic, exhibiting a strong inhibitory effect on microorganisms. Common nitrogen-containing heterocyclic compounds in wastewater treatment include pyrrole, pyridine, quinoline, methylquinoline and its derivatives, benzothiazole, and benzimidazole. These are the main components of organic pollutants in coking wastewater and are also abundant in chemical, pharmaceutical, and pesticide wastewater. The long-term accumulation of these substances in the environment not only pollutes the environment but also affects human life and harms human health.

[0003] Common treatment processes for nitrogen-containing heterocyclic compounds include catalytic wet oxidation, biodegradation, and physicochemical methods. Among these, biodegradation utilizes the metabolism of different types of microorganisms to degrade and transform nitrogen-containing heterocyclic compounds into inorganic substances, thereby removing the pollutants. Microbial methods are economical, efficient, produce no secondary pollution, and operate under relatively mild conditions, making them an effective way to remove and transform nitrogen-containing heterocyclic compounds. In recent years, some functional bacterial strains capable of degrading nitrogen-containing heterocyclic compounds have been isolated and identified, such as Rhodococcus and Bacillus. However, these strains are all aerobic microorganisms and can only exert their degradation effect under aerobic conditions.

[0004] In practical wastewater biological treatment systems, nitrogen-containing heterocyclic compounds first enter the anaerobic treatment unit. However, there is a severe shortage of functional bacterial strains capable of efficiently degrading nitrogen-containing heterocyclic compounds under anaerobic / anoxic conditions. This results in extremely low removal efficiency of such pollutants in the anaerobic stage, leading to excessively high treatment loads in subsequent aerobic units and making it difficult for the overall effluent quality to consistently meet standards. Therefore, screening for functional bacterial strains that possess both high degradation capabilities and anaerobic adaptability is a key technological requirement for improving the treatment effect of nitrogen-containing heterocyclic wastewater. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a Clostridium kanehirae strain capable of anaerobic degradation of nitrogen-containing heterocyclic compounds and its applications, in view of the above-mentioned problems.

[0006] The technical solution adopted in this invention is: In a first aspect, the present invention provides a Clostridium kanehirae strain capable of anaerobic degradation of nitrogen-containing heterocyclic compounds (Clostridium kanehirae). Clostridium huakuii FL017, this strain was deposited on December 1, 2025 at the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China), with accession number CCTCCNO:M20252755, and the strain was found to be viable.

[0007] Secondly, the present invention provides the application of the aforementioned Clostridium huaguiens FL017 in the anaerobic degradation of nitrogen-containing heterocyclic compounds in water.

[0008] Furthermore, the nitrogen-containing heterocyclic compound is selected from one or more of quinoline, pyridine, indole, benzothiazole, and methylquinoline.

[0009] Furthermore, when the concentration of nitrogen-containing heterocyclic compounds in the water body is not higher than 300 mg / L, the degradation rate of nitrogen-containing heterocyclic compounds by Clostridium huacanthii FL017 is greater than 80%.

[0010] Thirdly, the present invention provides a method for anaerobic degradation of nitrogen-containing heterocyclic compounds in water, characterized by comprising the following steps: inoculating the aforementioned Clostridium huacanthii FL017 into the water to be treated containing nitrogen-containing heterocyclic compounds, and performing static cultivation under anaerobic conditions to achieve the degradation and removal of the nitrogen-containing heterocyclic compounds.

[0011] Furthermore, the nitrogen-containing heterocyclic compound is selected from one or more of quinoline, pyridine, indole, benzothiazole, and methylquinoline.

[0012] Furthermore, when the concentration of nitrogen-containing heterocyclic compounds in the water body is not higher than 300 mg / L, the degradation rate of nitrogen-containing heterocyclic compounds by Clostridium huacanthii FL017 is greater than 80%.

[0013] Furthermore, the *Clostridium huaguiens* FL017 was inoculated in the form of activated seed liquid.

[0014] The beneficial effects of this invention are: the Clostridium huaguiens FL017 provided by this invention is an anaerobic functional strain that can grow normally and play a degradation role in a completely anaerobic / anoxic environment. It can be directly applied to the anaerobic treatment unit of the sewage system, solving the technical pain point that existing aerobic degrading bacteria cannot be adapted to the anaerobic section, and improving the pollutant removal function of the anaerobic section of wastewater.

[0015] The Clostridium huaguiensis FL017 provided by this invention can simultaneously degrade a variety of typical nitrogen-containing heterocyclic pollutants, including quinoline, pyridine, indole, benzothiazole, and methylquinoline. At an initial substrate concentration of 100 mg / L, the highest degradation rate for all of the above pollutants can reach over 99%, demonstrating thorough degradation and excellent treatment effect.

[0016] The Clostridium huaguiens FL017 provided by this invention has good tolerance to high concentrations of nitrogen-containing heterocyclic compounds, and can tolerate a maximum pollutant concentration of 700 mg / L. When the substrate concentration is not higher than 300 mg / L, the degradation rate is stably maintained at over 80%, which can be adapted to industrial wastewater treatment scenarios with different loads and has strong resistance to water quality shocks.

[0017] The degradation method of the present invention does not require additional aeration and oxygen supply. It only requires inoculating the activated strain into the water body to be treated, and the pollutant degradation can be completed under anaerobic static conditions. It can be directly connected to existing sewage treatment structures without large-scale equipment modification. It has low operating energy consumption and no secondary pollution, making it suitable for industrial promotion and application. Attached Figure Description

[0018] Figure 1 The degradation rate of different nitrogen-containing heterocyclic compounds by Clostridium huacanthii FL017 varies with culture time. Figure 2 This is a comparison chart showing the degradation rates of nitrogen-containing heterocyclic compounds by Clostridium huacanthii FL017 at different concentrations. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Non-substantial improvements and adjustments made by those skilled in the art in conjunction with the content of the present invention should all fall within the scope of protection of the present invention.

[0020] The present invention relates to Clostridium huaguinifolium (Clostridium huaguinifolium) Clostridium huakuii FL017 was deposited at the China Center for Type Culture Collection (CCTCC) on December 1, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20252755. The viability of this strain was tested on December 8, 2025, and the result was positive.

[0021] Example 1: Isolation, screening and identification of Clostridium huaguilloché FL017 1. Sample Source The original sample was taken from anaerobic activated sludge from Jiangxia Wastewater Treatment Plant in Wuhan.

[0022] 2. Enrichment and domestication culture 2.1 Prepare the liquid synthetic culture medium, the formula of which is as follows: The solution contains K₂HPO₄ (0.025), KH₂PO₄ (0.025), NH₄Cl (0.10), KCl (0.1), FeCl₃·6H₂O (0.02), CaCl₂ (0.05), and 1 mL of trace element solution. The trace element solution composition (g / L) is: MgSO₄·7H₂O (0.5), EDTA (1.0), ZnSO₄ (0.2), MnCl₂·4H₂O (0.1), FeSO₄·7H₂O (0.5), CuSO₄·5H₂O (0.5), and CoCl₂·6H₂O (0.2).

[0023] Simultaneously, nitrogen heterocyclic compounds (quinoline 0.02, pyridine 0.02, indole 0.02) were added to obtain a liquid synthesis medium containing nitrogen heterocyclic compounds. Unit: g / L.

[0024] 2.2 Weigh 50g of fresh sludge and place it in a 500mL Erlenmeyer flask. Add 150mL of liquid synthesis culture medium containing nitrogen heterocyclic compounds. 2.3 Place the Erlenmeyer flask in a 28℃ constant temperature incubator and let it stand for 3 days.

[0025] 2.4 After 3 days, discard the supernatant in the Erlenmeyer flask and add an equal amount of synthetic culture medium.

[0026] 2.5 After 3 days, discard the supernatant in the Erlenmeyer flask and add an equal amount of synthetic culture medium.

[0027] 2.6 After 3 days, discard the supernatant in the Erlenmeyer flask and add an equal amount of synthetic culture medium.

[0028] 2.7 After 3 days, use a pipette to remove the sediment from the 0.1 mL Erlenmeyer flask and place it in a glass petri dish. Then pour in the melted and cooled solid synthetic culture medium, mix well, and place in a 28℃ constant temperature incubator for 3 days. After single colonies grow on the plate, pick out the uniformly shaped single colonies and repeatedly streak them for purification to obtain a pure strain, named FL017.

[0029] The solid synthetic medium is prepared by adding 15 g / L of agar powder to the liquid synthetic medium prepared in step 2.1.

[0030] 3. Molecular identification of strains Pick a single colony from a petri dish, place it in liquid synthetic medium, and incubate at 28°C for about 48 hours. After the culture becomes turbid, identify the bacterial 16S rDNA. Then, prepare the bacterial strain into glycerol tubes and place them at -80°C.

[0031] Microbial 16S rDNA amplification and sequencing were performed using primers 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R: 5'-TACGGYTACCTTGTTAYGACTT-3'.

[0032] Its sequence is as follows: The obtained 16S rDNA sequence was compared with the NCBI database using BLAST, and a phylogenetic tree was constructed for analysis. The results showed that this strain is related to *Clostridium huaguiens* (…). Clostridium huakuii Based on the highest homology with *Clostridium huagui*, and combined with morphological and physiological-biochemical characteristics, it was identified as *Clostridium huagui* and named *Clostridium huagui* (…). Clostridium huakuii FL017.

[0033] Example 2: Degradation effect of Clostridium huaguillii FL017 on different nitrogen-containing heterocyclic compounds 1. Seed liquid preparation Prepare LB liquid culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, adjust pH to 7.0, and sterilize for later use.

[0034] Pure colonies of FL017 were picked and inoculated into LB medium, and then anaerobically cultured at 28°C for 48 hours to obtain activated seed culture.

[0035] 2. Simulated wastewater preparation Based on the inorganic salt culture medium in Example 1, 100 mg / L of quinoline, pyridine, indole, benzothiazole, and methylquinoline were added respectively to prepare simulated nitrogen-containing heterocyclic wastewater with five single substrates.

[0036] 3. Degradation experiment Take 1 mL of FL017 seed culture and inoculate it into 100 mL of each of the five simulated wastewater samples mentioned above. Place the samples in an anaerobic static incubator at 28℃. Take samples every 24 hours and determine the concentration of residual nitrogen-containing heterocyclic compounds in the water using high performance liquid chromatography to calculate the degradation rate.

[0037] High-performance liquid chromatography (HPLC) detection conditions: Agilent C18 column, Agilent UV detector, mobile phase of methanol and water (6:4), flow rate of 1 mL / min, column temperature of 30 °C. The detection wavelength and elution time vary for different nitrogen heterocyclic compounds.

[0038] The degradation effects of FL017 on different nitrogen heterocyclic compounds are as follows: Figure 1 As shown in the table below, the highest degradation rates of FL017 for different nitrogen heterocyclic compounds are as follows:

[0039] Example 3: Degradation performance of Clostridium huaguillii FL017 on nitrogen-containing heterocyclic compounds of different concentrations 1. Preparation of wastewater with gradient concentration Based on the inorganic salt culture medium in Example 1, simulated wastewater with different loads was prepared by setting concentration gradients of quinoline, pyridine, indole, benzothiazole, and methylquinoline at 100 mg / L, 300 mg / L, 500 mg / L, and 700 mg / L, respectively.

[0040] 2. Degradation experiment FL017 seed culture was inoculated into wastewater of various concentrations at a volume ratio of 1%. After anaerobic static culture at 28℃ for 96 hours, samples were taken, and the residual concentration of pollutants was determined by high performance liquid chromatography to calculate the degradation rate.

[0041] 3. Experimental Results like Figure 2 As shown, when the pollutant concentration is 100 mg / L, the degradation rate of various substrates by the strain is over 99%; when the concentration is not higher than 300 mg / L, the degradation rate of various nitrogen-containing heterocyclic compounds is greater than 80%; when the concentration increases to 500 mg / L, the degradation rate decreases but still has a significant degradation effect; when the concentration reaches 700 mg / L, the strain still maintains a certain degradation activity.

[0042] The results showed that Clostridium huaguiens FL017 has a strong tolerance to high concentrations of nitrogen-containing heterocyclic compounds and can be adapted to wastewater treatment scenarios with a wide range of concentrations.

[0043] Example 4: Method for anaerobic degradation of nitrogen-containing heterocyclic wastewater This embodiment provides a specific method for anaerobic treatment of nitrogen-containing heterocyclic wastewater using Clostridium huaguiens FL017, the steps of which are as follows: 1. Activation of strain: Clostridium huaguiensis FL017 was inoculated into LB liquid medium and anaerobicly cultured at 28°C for 48 h to prepare active seed liquid.

[0044] 2. Wastewater inoculation treatment: The activated seed liquid is inoculated into the wastewater containing nitrogen-containing heterocyclic compounds at a volume ratio of 0.5~2%. The wastewater substrate is a mixed system of quinoline, pyridine and indole.

[0045] 3. Anaerobic degradation reaction: The inoculated wastewater was anaerobically cultured at 28℃.

[0046] 4. Treatment effect: After 72 hours of cultivation, the degradation rate of total nitrogen-containing heterocyclic compounds in the wastewater reached over 85%, achieving efficient removal under anaerobic conditions.

Claims

1. A Clostridium kanehirae species capable of anaerobic degradation of nitrogen-containing heterocyclic compounds (Clostridium kanehirae) Clostridium huakuii FL017 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20252755.

2. The application of Clostridium huaguierii FL017 as described in claim 1 in the anaerobic degradation of nitrogen-containing heterocyclic compounds in water.

3. The application according to claim 2, characterized in that, The nitrogen-containing heterocyclic compound is selected from one or more of quinoline, pyridine, indole, benzothiazole, and methylquinoline.

4. The application according to claim 3, characterized in that, When the concentration of nitrogen-containing heterocyclic compounds in the water body is not higher than 300 mg / L, the degradation rate of nitrogen-containing heterocyclic compounds by Clostridium huacanthii FL017 is greater than 80%.

5. A method for anaerobic degradation of nitrogen-containing heterocyclic compounds in water, characterized in that, Includes the following steps: The *Clostridium difficile* FL017 of claim 1 is inoculated into the water containing nitrogen-containing heterocyclic compounds and then statically cultured under anaerobic conditions to achieve the degradation and removal of the nitrogen-containing heterocyclic compounds.

6. The method according to claim 5, characterized in that, The nitrogen-containing heterocyclic compound is selected from one or more of quinoline, pyridine, indole, benzothiazole, and methylquinoline.

7. The method according to claim 5, characterized in that, When the concentration of nitrogen-containing heterocyclic compounds in the water body is not higher than 300 mg / L, the degradation rate of nitrogen-containing heterocyclic compounds by Clostridium huacanthii FL017 is greater than 80%.

8. The method according to claim 5, characterized in that, The Clostridium huaguilloché FL017 was inoculated in the form of activated seed liquid.

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