Salt-tolerant denitrifying vibrio fluvialis JH-3 and application thereof

Abstract

The invention discloses a vibrio fluvialis JH-3 which is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number of the vibrio fluvialis JH-3 is GDMCC (China General Microbiological Culture Collection Center) NO: 66094. The invention further discloses an application of the vibrio fluvialis JH-3 in nitrogen removal by denitrification, and the vibrio fluvialis JH-3 can be used for preparing a nitrogen removal agent for nitrogen removal by denitrification. The invention also discloses a denitrification nitrogen removal method of the salt-containing wastewater, which comprises the following steps: uniformly mixing the Vibrio fluvialis JH-3 with the salt-containing wastewater to obtain a mixed solution, and culturing to perform denitrification nitrogen removal. According to the invention, the Vibrio fluvialis JH-3 is screened out, and the Vibrio fluvialis JH-3 has relatively strong salt-resistant denitrification capability and can be used for biological nitrogen removal of high-salt wastewater.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a salt-tolerant and denitrifying Vibrio fluvialis JH-3 and an application thereof. Background Art

[0002] Nitrogen pollutants are widely present in agricultural production, industrial manufacturing, municipal sewage discharge, livestock breeding, and atmospheric dry and wet deposition. Such pollutants pose multiple threats to ecological security and human health: ① causing eutrophication of water bodies, leading to algal blooms and the disruption of aquatic biological chains; ② destroying soil aggregate structure, causing hardening of cultivated land and crop yield reduction; ③ accumulating in the food chain and inducing human health risks. Nitrate nitrogen (NO3 - -N) is one of the main forms of nitrogen pollution in water bodies, and its toxic effects are particularly prominent. Studies have shown that nitrate can be reduced to nitrite (NO2 - Nitrate (-N) binds to hemoglobin to form methemoglobin, significantly reducing the blood's oxygen-carrying capacity. Epidemiological studies have confirmed that long-term ingestion of water with nitrate concentrations exceeding 10 mg / L can lead to methemoglobinemia, which can cause death from asphyxiation when blood oxygen saturation drops below 70%.

[0003] The current mainstream denitrification technologies cover two major systems: physical and chemical methods (ion exchange, membrane separation, 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 no secondary pollution. However, with the continuous increase in the salinity of industrial wastewater (the proportion of high-salt industrial wastewater in my country exceeds 5%, with an annual growth rate of 3%-5%), traditional biological treatment systems face severe challenges. Wastewater discharged by industries such as petroleum refining, dye synthesis, and marine aquaculture has the characteristics of high nitrate nitrogen and high salinity. Conventional denitrifying bacteria are prone to osmotic pressure imbalance in such extreme environments, causing cell wall separation, inactivation of functional enzyme systems, and blockage of metabolic pathways, ultimately resulting in 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 and denitrifying Vibrio fluvialis JH-3 and its application. The present invention screened out a strain of Vibrio fluvialis JH-3 with strong salt-tolerant and denitrifying ability, which can be used for biological denitrification of high-salt wastewater.

[0005] The invention provides a Vibrio fluvialis JH-3, which is preserved in Guangdong Province Microbial Culture Collection Center with a preservation number of GDMCC NO: 66094.

[0006] The inventors took activated sludge from the biochemical treatment unit of an epoxy resin wastewater plant in Huangshan, Anhui, enriched it in a high-salt environment, separated it using a solid plate, and screened its denitrification and denitrification ability. Finally, they obtained a new strain, Vibrio fluvialis JH-3, which has good salt-tolerant denitrification and denitrification effects. The inventors deposited this strain on April 3, 2025 in the Guangdong Provincial Microbial Culture Collection Center on the 5th floor of the Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and its preservation number is GDMCC NO: 66094.

[0007] The present invention also proposes the application of the Vibrio fluvialis JH-3 in denitrification.

[0008] Preferably, the method is used for denitrification in a saline environment.

[0009] Preferably, the method is used for denitrification in a saline and oxygen-containing environment.

[0010] Preferably, the salinity of the saline environment is 2-6 wt % in terms of NaCl content.

[0011] The present invention also proposes a denitrification and denitrification method for saline wastewater, comprising the following steps: mixing the above-mentioned Vibrio fluvialis JH-3 with saline wastewater to obtain a mixed solution, and culturing the solution to perform denitrification and denitrification.

[0012] Preferably, the culture temperature is 23-28°C and the culture time is 70-80 hours.

[0013] Preferably, adjust the OD of the mixture 600 It is 0.15-0.25.

[0014] Preferably, the pH of the mixed solution 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 formula of the culture medium is: LB medium containing NaCl.

[0017] Preferably, the content of NaCl is 2-6 wt%.

[0018] The present invention screened out a strain of Vibrio fluvialis JH-3 with strong salt-tolerant denitrification ability, which can efficiently remove NO3-N in water bodies under high-salt 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 being used for biological denitrification of high-salt wastewater, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the colony morphology of Vibrio fluvialis JH-3.

[0020] Figure 2 This is the Gram staining image of Vibrio fluvialis JH-3.

[0021] Figure 3 This is the phylogenetic tree of Vibrio fluvialis JH-3.

[0022] Figure 4 OD during denitrification by Vibrio fluvialis JH-3 under different salinity stresses 600 Value and the change results of nitrogen concentration of each valence state, where a is OD 600 value, b is NO3 - -N concentration, c is NO2 - -N concentration, d is TN concentration.

[0023] Figure 5 The denitrification effect of Vibrio fluvialis JH-3 on pickle factory wastewater. DETAILED DESCRIPTION

[0024] Hereinafter, the technical solutions of the present invention will be described in detail through specific embodiments. However, it should be clearly stated that these embodiments are provided for illustration only and are not to be construed as limiting the scope of the present invention.

[0025] Example 1

[0026] Strain isolation and screening

[0027] The inventors took activated sludge from the biochemical treatment unit of an epoxy resin wastewater plant in Huangshan, Anhui, and inoculated it into LB medium containing 4wt% NaCl to obtain a salt-tolerant strain. The salt-tolerant strain was inoculated into a denitrification medium for cultivation, and a single strain capable of denitrification, Vibrio fluvialis JH-3, was screened out.

[0028] The denitrification culture medium is as follows: 1L of water contains 1.08g KNO3, 4.1g CH3COONa, 6.26g K2HPO4·3H2O, 1.24g KH2PO4, 0.2g MgSO4, 2mL trace element solution and NaCl, wherein the content of NaCl is 4wt%.

[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] Take the above single strain and streak culture on solid separation plate for 48 hours, observe the colony, the result is as follows Figure 1 As shown; and the strain was subjected to Gram staining, the results were as follows Figure 2 shown.

[0032] Figure 1 This is the colony morphology of Vibrio fluvialis JH-3.

[0033] Figure 2 This is the 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, with a smooth and moist surface and complete edges; after Gram staining, the bacteria are red, indicating that they are Gram-negative bacteria.

[0035] Molecular biology identification

[0036] Prokaryotes have three types of ribosomal DNA: 23S, 16S, and 5S. These are directly related to protein translation. Ribosomes are present in all cellular organisms and are highly conserved in sequence, yet they also contain regions of greater variability within their molecules. Of the three ribosomal molecules, 16S rDNA has been chosen as a benchmark for biological evolution and for systematic classification of organisms due to its high information content and moderate sequence size (1.5 KB). It is currently the best marker known for phylogenetic studies. Currently, bacteriologists generally believe that when 16S rRNA sequences share greater than 97% identity, they can be considered conspecifics within the genus. Sequences obtained are then compared in NCBI, and the sequence with the greatest similarity is selected as the species identification result.

[0037] The 16S rRNA sequence of Vibrio fluvialis JH-3 detected by the inventors is shown in SEQ ID NO.1.

[0038] The inventors used the NCBI Blast program to compare the sequenced 16S rRNA sequences with the data in the NCBI 16S database and constructed a phylogenetic tree. The results are as follows: Figure 3 shown. Figure 3 This is the phylogenetic tree of Vibrio fluvialis JH-3.

[0039] Depend on Figure 3 It can be seen that this single strain belongs to the genus Vibrio, and the inventor named it Vibrio fluvialis JH-3. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 3, 2025, and its deposit number is GDMCC NO: 66094.

[0040] Example 2

[0041] Study on Denitrification and Nitrogen Removal by Vibrio fluvialis JH-3 at Different Salins

[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 a seed solution;

[0043] Then the seed solution was added to denitrification medium with different NaCl contents (0 wt%, 2 wt%, 4 wt%, 6 wt%, 8 wt%), the pH was adjusted to 7.0 ± 0.2, and the initial OD was adjusted. 600 0.2, and then cultured at 25 ° C, 180 rpm shaking for 3 days; during this period, samples were taken every 12 hours to determine NO3 - -N, NO2 - -N、TN、OD 600 , the results are as follows Figure 4 As shown;

[0044] Among them, NO3 - -N test uses UV spectrophotometry, NO2 - -N test uses α-naphthylamine hydrochloride spectrophotometry, and TN test uses alkaline potassium persulfate digestion UV spectrophotometry;

[0045] The denitrification medium was as follows: 1 L of water contained 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 different amounts of NaCl.

[0046] Figure 4 OD during denitrification by Vibrio fluvialis JH-3 under different salinity stresses 600 Value and the change results of nitrogen concentration of each valence state, where a is OD 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 of Vibrio fluvialis JH-3 in different salinity environments is related to the NO3 - -N reduction process basically showed a positive correlation trend; when the salinity was 2wt%-6wt%, the growth of strain JH-3 was good, and NO3 - -N removal rate is also quite impressive; especially when the salinity is 2wt%-4wt%, NO3 - The removal rate of -N can reach 90%.

[0048] In an environment with a salinity of 0%, due to the low external osmotic pressure, a large amount of water flows into the cells, causing the cell volume to expand and the cell membrane to be damaged by excessive pressure, which seriously affects the normal growth and reproduction of the strain JH-3. The growth of the strain JH-3 is at a low level, so the denitrification effect is poor.

[0049] When the salinity was increased to 2wt%, the concentration of NaCl just compensated for the problem of low osmotic pressure at 0% salinity, and the strain JH-3 was able to grow normally. The growth ability of the strain JH-3 and its resistance to NO3 - -N removal ability was significantly enhanced; for example, the growth of strain JH-3 in the stable phase (i.e., OD 600 ) increased significantly from 0.201 to 1.463, NO3 - -N removal rate is as high as 90.8%; when the salinity is further increased to 4wt%, the growth of strain JH-3 and NO3 - There was no significant difference in the removal effect of -N compared with that at 2 wt% salinity;

[0050] However, when the salinity continued to rise to above 6wt%, the growth hysteresis period of strain JH-3 was significantly prolonged, the growth of strain JH-3 in the stable period also decreased significantly, and the denitrification efficiency decreased accordingly. However, after 72h, the NO3 - The removal rate of -N can still reach 83.7%;

[0051] It is worth noting that strain JH-3 produces NO2 during the denitrification process. - -N accumulation phenomenon, but when the reaction was carried out for 72h, NO2 - -N accumulation was significantly reduced, indicating that the denitrification process was relatively more thorough under these two salinities.

[0052] In summary, Vibrio fluvialis JH-3 can grow in an environment with a salinity of 2wt%-6wt% and successfully carry out the denitrification process; among them, it performs best under the condition of salinity of 2wt%-4wt%, and can remove about 90% of NO3 within 72h. - -N, and NO2 - The accumulation of -N is low, and the denitrification process is more thorough. Therefore, Vibrio fluvialis JH-3 has great potential for application in the field of denitrification of 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 denitrification method for saline wastewater comprises the following steps:

[0055] Vibrio fluvialis strain JH-3 was cultured in a seed medium (LB medium containing 4 wt% NaCl) at 25°C and 180 rpm under aerobic conditions for 24 hours to the late logarithmic phase, then centrifuged and resuspended in PBS to prepare a seed solution.

[0056] Take the wastewater from the pickle factory (its water quality is: NO3 - -N 137.82mgN / L, NO2 - -N 1.22mgN / L, NH4 + -N0.03mgN / L, TN 148.64mgN / L, TOC 1230.80mg / L, salinity 3.8wt%) was sterilized, and then the seed liquid was inoculated into it, and the initial inoculation amount was controlled to be OD 600 =0.2, cultured in a constant temperature shaking incubator at 25°C and 180 rpm, and samples were taken regularly to test OD 600 Value, NO3 --N, NO2 - -N、NH4 + -N and TN concentrations were used to evaluate the denitrification effect of Vibrio fluvialis strain JH-3 on actual wastewater. The results are shown in Figure 5 shown.

[0057] NO3 - -N test uses UV spectrophotometry, NO2 - -N test was performed by α-naphthylamine hydrochloride spectrophotometry, and TN test was performed by alkaline potassium persulfate digestion UV spectrophotometry.

[0058] Figure 5 The denitrification effect of Vibrio fluvialis JH-3 on pickle factory wastewater.

[0059] Depend on Figure 5 It can be seen that Vibrio fluvialis JH-3 grew slowly in 0-12h, and the change of nitrogen concentration was not significant; it grew rapidly and reached a plateau in 12-36h, and NO3 - -N was also basically removed, with a removal rate of 86.8%; during this period, although there would be NO2 - -N accumulates, but as the reaction proceeds, NO2 - The concentration of -N also gradually decreased, and denitrification was more thorough. It can be seen that Vibrio fluvialis JH-3 has an ideal denitrification effect on pickle factory wastewater, and the cultivation method is simple. Therefore, Vibrio fluvialis JH-3 has great potential in the actual application of high-salt wastewater denitrification.

[0060] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A Vibrio fluvialis JH-3, characterized in that It is deposited in Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC NO: 66094.

2. Use of Vibrio fluvialis JH-3 as claimed in claim 1 in denitrification.

3. The application according to claim 2, characterized in that: Application in denitrification and denitrification in saline environments; preferably, application in denitrification and denitrification in saline and oxygen-containing environments.

4. The application according to claim 3, characterized in that The salinity of the saline environment is 2-6 wt % in terms of NaCl content.

5. A denitrification method for saline wastewater, characterized in that: The method comprises the following steps: mixing the Vibrio fluvialis JH-3 as claimed in claim 1 with saline wastewater to obtain a mixed solution, and culturing the solution to perform denitrification and denitrification.

6. The denitrification method for saline wastewater according to claim 5, characterized in that: The culture temperature is 23-28°C and the culture time is 70-80h.

7. The denitrification method for saline wastewater according to claim 5 or 6, characterized in that: Adjust the OD of the mixture 600 is 0.15-0.25; preferably, the pH of the mixed solution is adjusted to 6.8-7.

2.

8. The denitrification method for saline wastewater according to any one of claims 5 to 7, characterized in that: 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.

9. The denitrification method for saline wastewater according to claim 8, characterized in that: The formula of the culture medium is: LB medium containing NaCl.

10. The denitrification method for saline wastewater according to claim 9, characterized in that: The content of NaCl is 2-6 wt%.

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