A salt-tolerant denitrifying Vibrio fluvialis JH-3 and its applications
By screening out salt-tolerant river Vibrio JH-3, the problem of low denitrification efficiency in high-salt environments was solved, achieving a highly efficient denitrification effect, which is suitable for high-salt wastewater treatment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ANHUI DUANGONG ZHILIAN ENVIRONMENTAL PROTECTION TECH CO LTD
- Filing Date
- 2025-05-09
- Publication Date
- 2026-07-24
AI Technical Summary
Traditional biological treatment systems suffer from reduced denitrification efficiency in high-salt industrial wastewater. Conventional denitrifying bacteria are easily inactivated in high-salt environments, leading to decreased denitrification efficiency.
Salt-tolerant Vibrio fluvialis JH-3 was screened for denitrification of high-salt wastewater, and culture conditions were optimized to efficiently remove NO3-N in a 2-6 wt% NaCl environment.
In a 2-6 wt% NaCl environment, Vibrio fluvibrio JH-3 exhibits highly efficient denitrification and nitrogen removal capabilities, with NO3-N removal rates reaching 90%, optimizing the operating costs of traditional processes and making it suitable for biological denitrification of high-salinity wastewater.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a salt-tolerant denitrifying Vibriofluvialis JH-3 and its applications. Background Technology
[0002] Nitrogen pollutants are widely present in agricultural production, industrial manufacturing, municipal sewage discharge, livestock farming, and atmospheric dry and wet deposition processes. These pollutants pose multiple threats to ecological security and human health: ① They cause eutrophication in water bodies, leading to algal blooms and disruption of the aquatic food chain; ② They damage soil aggregate structure, causing soil compaction and reduced crop yields; ③ They induce human health risks through bioaccumulation in the food chain. Nitrate nitrogen (NO3) - Nitrate (NO₂) is one of the main forms of nitrogen pollution in water bodies, and its toxic effects are particularly prominent. Studies have shown that nitrates can be reduced to nitrites (NO₂) in the human digestive tract. - Nitrate (N-nitrate) combines with hemoglobin to form methemoglobin, significantly reducing the blood's oxygen-carrying capacity. Epidemiological surveys have confirmed that long-term intake of water with nitrate concentrations exceeding 10 mg / L will lead to methemoglobinemia, which can cause asphyxiation and death when blood oxygen saturation drops below 70%.
[0003] Current mainstream denitrification technologies encompass two major systems: physicochemical methods (ion exchange, membrane separation, and electrochemical reduction) and biological treatment methods. Among them, biological denitrification technology has become a research hotspot in the field of sustainable water treatment due to its economic efficiency, environmental friendliness, and lack of secondary pollution. However, with the continuous increase in the salinity of industrial wastewater (high-salinity industrial wastewater accounts for over 5% in my country, with an annual growth rate of 3%-5%), traditional biological treatment systems face severe challenges. Wastewater discharged from industries such as petroleum refining, dye synthesis, and mariculture is characterized by both high nitrate nitrogen and high salinity. Conventional denitrifying bacteria are prone to cell plasmolysis, inactivation of functional enzyme systems, and metabolic pathway blockage due to osmotic pressure imbalance in such extreme environments, ultimately leading to a 50%-80% decrease in denitrification efficiency. Summary of the Invention
[0004] Based on the technical problems existing in the background technology, the present invention proposes a salt-tolerant denitrifying Vibrio fluvialis JH-3 and its application. The present invention screened a strain of Vibrio fluvialis JH-3 with strong salt tolerance and denitrification ability, which can be used for biological denitrification of high-salt wastewater.
[0005] This invention proposes a strain of Vibrio fluvialis JH-3, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 66094.
[0006] The inventors collected activated sludge from the biochemical treatment unit of an epoxy resin wastewater plant in Huangshan, Anhui Province, enriched it in a high-salt environment, separated it using solid plates, and screened its denitrification capacity. Ultimately, they obtained a new strain, Vibrio fluvialis JH-3, which exhibits excellent salt tolerance and denitrification performance. The inventors deposited this strain on April 3, 2025, at the Guangdong Provincial Microbial Culture Collection Center, 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC NO: 66094.
[0007] This invention also proposes the application of the above-mentioned Vibrio fluvialis JH-3 in denitrification.
[0008] Preferably, it is used in denitrification in saline environments.
[0009] Preferably, it is used in denitrification in saline and oxygen-containing environments.
[0010] Preferably, the salinity of the saline environment is 2-6 wt% based on the NaCl content.
[0011] The present invention also proposes a method for denitrification of saline wastewater, comprising the following steps: mixing the above-mentioned Vibrio fluvialis JH-3 with saline wastewater to obtain a mixed solution, and culturing it for denitrification.
[0012] Preferably, the culture temperature is 23-28℃ and the culture time is 70-80h.
[0013] Preferably, the OD of the mixture is adjusted. 600 It is 0.15-0.25.
[0014] Preferably, the pH of the mixture is adjusted to 6.8-7.2.
[0015] Preferably, Vibrio fluvialis JH-3 is cultured in a culture medium to obtain a culture solution, and then the culture solution is mixed with saline wastewater to obtain a mixed solution.
[0016] Preferably, the culture medium is formulated as follows: LB medium containing NaCl.
[0017] Preferably, the NaCl content is 2-6 wt%.
[0018] This invention screened a strain of Vibrio fluvialis JH-3 with strong salt tolerance and denitrification ability, which can efficiently remove NO3-N from water bodies under high salinity conditions. It can directly act on high-salt nitrate nitrogen wastewater, effectively alleviating the rigid demand for desalination pretreatment in traditional processes, optimizing operating costs, and has broad application prospects for biological denitrification of high-salt wastewater. Attached Figure Description
[0019] Figure 1 This is a colony morphology diagram of Vibrio fluvialis JH-3.
[0020] Figure 2 Gram staining image of Vibrio fluvialis JH-3.
[0021] Figure 3 Phylogenetic tree diagram of Vibrio fluvialis JH-3.
[0022] Figure 4 OD during denitrification of Vibrio fluvialis JH-3 under different salinity stress 600 Values and changes in nitrogen concentrations at various valence states, where a is the OD value. 600 Value, b is NO3 - -N concentration, c is NO2 - -N concentration, d is TN concentration.
[0023] Figure 5 The effect of Vibrio fluvialis JH-3 on denitrification of wastewater from a pickle factory was studied. Detailed Implementation
[0024] The technical solution of the present invention will be described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.
[0025] Example 1
[0026] Strains Isolation and Screening
[0027] The inventors obtained salt-tolerant strains by inoculating activated sludge from the biochemical treatment unit of an epoxy resin wastewater plant in Huangshan, Anhui Province, into LB medium containing 4wt% NaCl. The salt-tolerant strains were then inoculated into denitrification medium and cultured to screen out a single strain capable of denitrification, namely Vibrio fluvialis JH-3.
[0028] The above denitrification medium is: 1 L of water containing 1.08 g KNO3, 4.1 g CH3COONa, 6.26 g K2HPO4·3H2O, 1.24 g KH2PO4, 0.2 g MgSO4, 2 mL of trace element solution and NaCl, wherein the NaCl content is 4 wt%.
[0029] The above 1L trace element solution contains: 0.1g FeSO4·7H2O, 0.02g ZnSO4·7H2O, 0.04g CuSO4·5H2O, 0.04g MnSO4·H2O, 0.03g H3BO3, 0.04g Na2MoO4·2H2O, and 0.05g CoCl2·6H2O.
[0030] Morphological identification
[0031] The above-mentioned single strain was streaked on a solid isolation plate and cultured for 48 hours. The colonies were observed, and the results were as follows: Figure 1 As shown; and the strain was Gram-stained, the results are as follows. Figure 2 As shown.
[0032] Figure 1 This is a colony morphology diagram of Vibrio fluvialis JH-3.
[0033] Figure 2 Gram staining image of Vibrio fluvialis JH-3.
[0034] Depend on Figure 1-2 It can be seen that the colonies of Vibrio fluvialis JH-3 are milky white, round, smooth and moist, with intact edges; after Gram staining, the bacteria turn red, indicating that they are Gram-negative bacteria.
[0035] Molecular biological identification
[0036] Prokaryotic ribosomal DNA exists in three forms: 23S, 16S, and 5S, and is directly related to protein translation. Ribosomes are present in all cellular organisms and exhibit high sequence conservation, while also containing highly variable regions within the molecule. Among the three ribosomal molecules, 16S rDNA has been chosen as a benchmark for biological evolution and is used for systematic classification due to its sufficiently large information content and moderate sequence size (1.5K). It is currently considered the best marker in phylogenetic research. Bacteriologists generally agree that when the 16S rRNA sequence homology is higher than 97%, it can be considered a single species within the same genus. Sequences obtained from sequencing are compared with those in NCBI, and the sequence with the highest similarity is selected for species identification.
[0037] The inventors detected the 16S rRNA sequence of Vibrio fluvialis JH-3 as shown in SEQ ID NO.1.
[0038] The inventors used the NCBI Blast program to compare the 16S rRNA sequences obtained from sequencing with data from the NCBI 16S database and constructed a phylogenetic tree. The results are as follows: Figure 3 As shown. Figure 3 Phylogenetic tree diagram of Vibrio fluvialis JH-3.
[0039] Depend on Figure 3 It can be seen that this single strain belongs to the genus Vibrio. The inventor named it Vibriofluvialis JH-3 and deposited it at the Guangdong Provincial Center for Microbial Culture Collection on April 3, 2025, with the accession number GDMCC NO: 66094.
[0040] Example 2
[0041] Study on denitrification of Vibrio fluvialis JH-3 under different salinities
[0042] Vibrio fluvialis JH-3 was inoculated into LB medium containing 4 wt% NaCl and cultured at 25°C with shaking at 180 rpm for 24 h until the late logarithmic phase to obtain the seed culture.
[0043] The seed culture was then added to denitrification media with different NaCl contents (0 wt%, 2 wt%, 4 wt%, 6 wt%, 8 wt%), and the pH was adjusted to 7.0 ± 0.2. The initial OD was also adjusted. 600 The concentration was set at 0.2, and then the mixture was incubated at 25°C with shaking at 180 rpm for 3 days; samples were taken every 12 hours during this period to determine NO3. - -N, NO2 - -N, TN, OD 600 The result is as follows Figure 4 As shown;
[0044] Among them, NO3 - -N was measured using ultraviolet spectrophotometry. - -N was tested using the hydrochloric acid α-naphthylamine spectrophotometric method, and TN was tested using the alkaline potassium persulfate digestion ultraviolet spectrophotometric method.
[0045] The denitrification medium consists of 1.08g KNO3, 4.1g CH3COONa, 6.26g K2HPO4·3H2O, 1.24g KH2PO4, 0.2g MgSO4, 2mL of trace element solution, and different amounts of NaCl in 1L of water.
[0046] Figure 4 OD during denitrification of Vibrio fluvialis JH-3 under different salinity stress 600 Values and changes in nitrogen concentrations at various valence states, where a is the OD value. 600 Value, b is NO3 - -N concentration, c is NO2 - -N concentration, d is TN concentration.
[0047] Depend on Figure 4 It can be seen that the growth status of Vibrio fluvialis JH-3 is related to NO3 under different salinity environments. - The reduction process of NO3- showed a generally positive correlation trend; when the salinity was 2wt%-6wt%, strain JH-3 exhibited good growth, while NO3-... - The removal rate of NO3- is also quite considerable; especially when the salinity is 2wt%-4wt%, the removal rate of NO3- is also quite good. - The removal rate of -N can reach 90%.
[0048] In an environment with a salinity of 0%, due to the extremely low external osmotic pressure, a large amount of water rushes into the cells, causing the cells to swell and the cell membrane to be damaged by excessive pressure. This severely affects the normal growth and reproduction of strain JH-3, resulting in a low growth rate and poor denitrification effect.
[0049] When the salinity increased to 2 wt%, the NaCl concentration at this point perfectly compensated for the excessively low osmotic pressure at 0% salinity, allowing strain JH-3 to grow normally. This improved the growth ability of strain JH-3 and its resistance to NO3-. - The removal capacity of -N was significantly enhanced; for example, the growth rate (OD) of strain JH-3 in the stationary phase was significantly increased. 600 The NO3 content increased significantly from 0.201 to 1.463. - The removal rate of -N was as high as 90.8%; when the salinity was further increased to 4wt%, the growth of strain JH-3 and NO3... - The removal effect of -N was not significantly different compared with that at 2wt% salinity;
[0050] However, when the salinity continued to rise above 6 wt%, the lag phase of strain JH-3 was significantly prolonged, and the growth of strain JH-3 in the stationary phase also decreased significantly, resulting in a reduction in denitrification efficiency. However, after 72 hours, NO3 under 6 wt% salinity conditions... - The removal rate of -N can still reach 83.7%;
[0051] It is worth noting that strain JH-3 produces NO2 during denitrification. - The phenomenon of -N accumulation was observed, but when the reaction proceeded for 72 hours, NO2 under 2wt% and 4wt% salinity conditions... - The significant reduction in N-N accumulation indicates that the denitrification process is relatively more thorough at these two salinities.
[0052] In summary, Vibrio fluvialis JH-3 can grow and successfully undergo denitrification in environments with salinity ranging from 2 wt% to 6 wt%. Among these conditions, it performs best at a salinity of 2 wt% to 4 wt%, removing approximately 90% of NO3 within 72 hours. - -N, and NO2 - The accumulation of nitrogen-3 (-N) is relatively low, and the denitrification process is more thorough. Therefore, Vibrio fluvialis JH-3 shows great potential for application in the field of denitrification and nitrogen removal in high-salinity wastewater, and is expected to become a key technical means to solve the problem of nitrogen pollution in high-salinity wastewater.
[0053] Example 3
[0054] A method for denitrification of saline wastewater includes the following steps:
[0055] Vibrio fluvialis strain JH-3 was cultured in seed medium (LB medium containing 4 wt% NaCl) under aerobic conditions at 25 °C and 180 rpm for 24 h to the late logarithmic phase. The culture was then centrifuged and resuspended in PBS to obtain the seed culture.
[0056] Wastewater from a pickled vegetable factory (its water quality is: NO3) - -N 137.82 mgN / L, NO2 - -N 1.22 mgN / L, NH4 + The solution (N 0.03 mg N / L, TN 148.64 mg N / L, TOC 1230.80 mg / L, salinity 3.8 wt%) was sterilized, and then the seed culture was inoculated into it, controlling the initial inoculation amount to OD. 600 =0.2, cultured in a constant temperature shaking incubator at 25℃ and 180rpm, and OD was measured periodically. 600 Value, NO3 --N, NO2 - -N, NH4 + The nitrogen removal efficiency of Vibrio fluvialis strain JH-3 on actual wastewater was evaluated using -N and TN concentrations. The results are as follows: Figure 5 As shown.
[0057] NO3 - -N was measured using ultraviolet spectrophotometry. - -N was measured using the hydrochloric acid α-naphthylamine spectrophotometric method, and TN was measured using the alkaline potassium persulfate digestion ultraviolet spectrophotometric method.
[0058] Figure 5 The effect of Vibrio fluvialis JH-3 on denitrification of wastewater from a pickle factory was studied.
[0059] Depend on Figure 5 It can be seen that Vibrio fluvialis JH-3 grows relatively slowly within 0-12 hours, with no significant change in nitrogen concentration; it grows rapidly and reaches a plateau phase within 12-36 hours, with NO3... - -N was also largely removed, with a removal rate of 86.8%; during this period, although NO2 was present... - -N accumulates, but as the reaction proceeds further, NO2... - The concentration of -N gradually decreased, indicating that denitrification was more thorough. This shows that Vibrio fluvialis JH-3 has a relatively ideal effect on denitrification of wastewater from pickle factories, and the cultivation method is simple. Therefore, Vibrio fluvialis JH-3 has great potential in practical applications of denitrification of high-salt wastewater.
[0060] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A type of Vibrio fluvialis ( Vibrio fluvialis JH-3, characterized in that, It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 66094.
2. A *Vibrio rivubilis* strain as described in claim 1 (… Vibrio fluvialis Application of JH-3 in denitrification.
3. The application according to claim 2, characterized in that, Application in denitrification in saline environments.
4. The application according to claim 2, characterized in that, Application in denitrification in saline and oxygen-containing environments.
5. The application according to claim 3, characterized in that, The salinity of the saline environment, expressed as NaCl content, is 2-6 wt%.
6. A method for denitrification of saline wastewater, characterized in that, Includes the following steps: applying the Vibrio fluvialis as described in claim 1 (… Vibrio fluvialis JH-3 was mixed with saline wastewater to obtain a mixed solution, which was then cultured for denitrification.
7. The denitrification method for saline wastewater according to claim 6, characterized in that, The saline wastewater is from a pickled vegetable factory.
8. The denitrification method for saline wastewater according to claim 6, characterized in that, The incubation temperature is 23-28℃, and the incubation time is 70-80h.
9. The denitrification method for saline wastewater according to claim 6, characterized in that, Adjusting the OD of the mixture 600 It is 0.15-0.
25.
10. The denitrification method for saline wastewater according to claim 6, characterized in that, Adjust the pH of the mixture to 6.8-7.
2.
11. The denitrification method for saline wastewater according to claim 6, characterized in that, Vibrio riverine ( Vibrio fluvialis JH-3 was cultured in a culture medium to obtain a culture solution, and then the culture solution was mixed with saline wastewater to obtain a mixed solution.
12. The denitrification method for saline wastewater according to claim 11, characterized in that, The culture medium formula is: LB medium containing NaCl.
13. The denitrification method for saline wastewater according to claim 12, characterized in that, The NaCl content is 2-6 wt%.