Sulfidogenic denitrifying thauera strain and its application

Abstract

This invention relates to the field of microbial technology, and more particularly to a sulfur-resistant denitrifying Zobelella strain and its applications. The strain is Zobelella denitrifyingus HZ-F-004, and its classification name is... Zobellella denitrificans The strain, with accession number CGMCC No. 35796, can efficiently reduce nitrite in aquaculture wastewater to nitrogen gas without accumulating acidic substances. It also exhibits good denitrification capabilities even in wastewater environments with a salinity of 3 wt% and containing 100 mg / L hydrogen sulfide. Strain HZ-F-004 can not only grow normally in water bodies with high salinity, low C / N ratio, and hydrogen sulfide pollution, but also achieves efficient nitrite degradation, making it particularly suitable for denitrification in old ponds, black-bottomed, and foul-smelling ponds with extremely high sulfide content.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to a sulfur-resistant denitrifying Zobel bacterium and its applications. Background Technology

[0002] Aquaculture is one of the earliest productive activities of humankind and an ancient traditional industry. Currently, with the continuous development of history, aquaculture is deeply integrated into all aspects of people's production and life, playing a vital role. However, due to factors such as excessive feed input and overcrowding, large amounts of nitrite accumulate in aquaculture water. If denitrification is not carried out in time, it will cause decreased immunity, anorexia, slow growth, tissue hypoxia, and even death in fish and shrimp, affecting the income of aquaculture farmers. Furthermore, untreated wastewater with high nitrite levels entering natural water bodies will cause serious marine nitrogen pollution, eutrophication, and frequent red tides, which is detrimental to the healthy and sustainable development of aquaculture. High concentrations of nitrite in drinking water can irritate the human gastrointestinal tract, increase the risk of cardiovascular disease, and raise the risk of digestive system cancers. Therefore, excessive nitrite levels in water bodies seriously endanger aquatic ecosystems and human health, making efficient denitrification and harmless treatment of nitrite in aquaculture wastewater crucial.

[0003] Currently, the main methods for treating wastewater with high nitrite levels include water exchange and dilution, increased aeration, application of sodium thiosulfate, application of activated carbon powder, oxidation-reduction methods, or the use of chelating agents such as sodium humate and sodium lignosulfonate. However, these methods have drawbacks such as short duration of action and high investment and operating costs, and can only temporarily solve the problem of high nitrite levels. To maintain long-term compliance with nitrite standards, a sustainable solution is needed. Biological treatment methods have received much attention in recent years due to their advantages such as low cost, simple operation, environmental friendliness, sustainability, high efficiency, and convenience. How to use microorganisms to treat nitrite-laden aquaculture wastewater has also become one of the current research hotspots.

[0004] However, using microorganisms to treat nitrite-laden aquaculture wastewater also faces some challenges. Some microorganisms cannot efficiently degrade nitrite in the harsh environment of aquaculture wastewater, and some cannot even grow normally. Traditional nitrifying bacteria treatment of nitrite in wastewater suffers from problems such as slow proliferation rate, long growth cycle, difficulty in maintaining high biological concentration, and acid production during the nitrification process.

[0005] Therefore, there is an urgent need for a strain and application method that can grow in harsh wastewater environments and achieve high-efficiency degradation of nitrite. Summary of the Invention

[0006] In view of this, the present invention proposes a sulfur-resistant denitrifying Zobelella strain and its application. The present invention relates to a denitrifying Zobelella strain (… Zobellella denitrificansHZ-F-004 strain can efficiently reduce nitrite in aquaculture wastewater to nitrogen gas without accumulating acidic substances. It also exhibits good denitrification capabilities even in environments with a salinity of 3 wt% and containing 100 mg / L hydrogen sulfide. This strain not only grows normally in water bodies with high salinity, low C / N ratio, and hydrogen sulfide pollution, but also achieves efficient nitrite degradation. It is suitable for treating nitrite in aquaculture wastewater with high salinity, low C / N ratio, and hydrogen sulfide pollution, and is particularly suitable for denitrification in old ponds, black-bottomed ponds, and odorous ponds with extremely high sulfide content.

[0007] The technical solution of this invention is implemented as follows: In a first aspect, the present invention provides a sulfur-resistant strain of *Zobelia denitrifica*, wherein the strain is *Zobelia denitrifica* HZ-F-004, and its classification name is... Zobellella denitrificans The accession number is CGMCC No. 35796.

[0008] It was deposited on September 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the 16S rRNA sequence of the strain is shown in SEQ NO.1:

[0009] Based on the above technical solutions, preferably, the denitrifying Zobel bacteria can achieve complete degradation of nitrite (nitrite nitrogen concentration of 100 mg / L) within 16 hours under the conditions of optimal carbon source sucrose, carbon-nitrogen ratio of 10 / 1, salt content not exceeding 40 g / L, inoculum amount of 1% (v / v), and hydrogen sulfide concentration of 100 mg / L.

[0010] Secondly, the application of denitrifying Zobel bacteria as described above in the degradation of nitrite in wastewater is provided.

[0011] Based on the above technical solutions, preferably, the wastewater contains sulfides.

[0012] Based on the above technical solutions, preferably, the sulfide includes hydrogen sulfide.

[0013] Based on the above technical solution, a further preferred embodiment is that the concentration of the sulfide is 50~150 mg / L.

[0014] Based on the above technical solution, a further preferred embodiment is that the concentration of the sulfide is 50~100mg / L.

[0015] Based on the above technical solutions, preferably, the salt concentration in the wastewater is 20~60 mg / L.

[0016] Based on the above technical solutions, a further preferred embodiment is that the salt concentration in the wastewater is 20~40 mg / L.

[0017] Based on the above technical solutions, preferably, the concentration threshold of nitrite nitrogen in the wastewater reaches 100 mg / L.

[0018] Thirdly, a microbial agent is provided, which includes Zobelella denitrificans as described above.

[0019] Fourthly, a method for degrading nitrite in wastewater is provided, comprising the step of inoculating the wastewater with the denitrifying Zobel bacteria as described above, or the microbial agent as described above.

[0020] Based on the above technical solutions, preferably, the inoculation volume is 1~2 vol%.

[0021] Based on the above technical solution, a further preferred embodiment is that the inoculation amount is 1 vol%.

[0022] The denitrifying Zobelella of the present invention has the following advantages over the prior art: 1. The denitrifying Zobelella of the present invention ( Zobellella denitrificansHZ-F-004 strain can efficiently reduce nitrite in aquaculture wastewater to nitrogen gas without accumulating acidic substances. It also exhibits good denitrification capabilities even in environments with a salinity of 3 wt% and containing 100 mg / L hydrogen sulfide. This strain not only grows normally in water bodies with high salinity, low C / N ratio, and hydrogen sulfide pollution, but also achieves efficient nitrite degradation. It is suitable for treating nitrite in aquaculture wastewater with high salinity, low C / N ratio, and hydrogen sulfide pollution, and is particularly suitable for denitrification in old ponds, black-bottomed ponds, and odorous ponds with extremely high sulfide content.

[0023] 2. This invention optimizes fermentation conditions to achieve high-density cultivation of denitrifying Zobelella, thereby preparing a biological agent to achieve low-cost, high-efficiency, and pollution-free degradation of nitrite in field applications. The strain uses sucrose as the sole carbon source during degradation, with an optimal carbon-to-nitrogen ratio of 10:1. Sucrose is cheaper than traditional carbon sources like glucose and sodium acetate, significantly reducing the cost of additional carbon sources and increasing farmers' profits. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a scanning electron microscope image of the denitrifying Zobelella HZ-F-004 of this invention; Figure 2 This is a graph showing the effect of the type of carbon source of the present invention on the denitrifying Zobelella HZ-F-004; Figure 3 This is a graph showing the effect of the carbon-to-nitrogen ratio of the culture medium of this invention on denitrifying Zobelella HZ-F-004; Figure 4 This is a graph showing the effect of the salt concentration in the culture medium of the present invention on the denitrifying Zobelella HZ-F-004; Figure 5 This is a graph showing the effect of the inoculum size of the present invention on *Zobelia denitrifica* HZ-F-004. Figure 6 The figure shows the experimental results of the tolerance of denitrifying Zobelella HZ-F-004 to hydrogen sulfide in this invention. Figure 7 The graph shows the degradation curve of nitrite nitrogen by the denitrifying Zobelella HZ-F-004 of this invention. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] The culture medium used in this invention is as follows: Activation medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, sea salt 30 g / L, initial pH 7.0. Solid medium is prepared by adding 2 wt% agar powder to the original formulation.

[0028] Enrichment medium (inorganic salt medium): sea salt 30 g / L, K₂HPO₄ 0.25 g / L, MgSO₄ 0.125 g / L, FeSO₄ 2.5 mg / L, MnSO₄ 2.5 mg / L, sucrose 0.12 g / L, sodium nitrite 0.5 g / L, hydrogen sulfide 100 mg / L, initial pH 7.2. Solid medium is prepared by adding 2 wt% agar powder to the above formula.

[0029] After the culture medium is prepared, it must be sterilized in a high-pressure steam sterilizer at 121°C for 30 minutes. Sucrose and hydrogen sulfide are added after sterilization.

[0030] Example 1: Enrichment, Isolation and Purification of Strains Strain enrichment: 20g of activated sludge from a shrimp farm was mixed with 300ml of enrichment medium and placed in a 500ml Erlenmeyer flask for enrichment. 100mg / L nitrite was used as the sole nitrogen source, and the mixture was cultured at 30℃ and 150r / min on a shaker. The nitrite content in the supernatant was measured every two days. If degradation was complete, the supernatant was completely replaced with fresh enrichment medium, and enrichment was continued. This process was repeated seven times. The resulting enriched strain was then separated and purified.

[0031] Strain isolation and purification: Take 1 ml of the well-mixed enrichment and perform serial dilutions using sterilized enrichment medium. Dilute the enrichment to 10⁻⁶ ppm. -2 10 -3 10 -4 10 -5 and 10 -6 Five gradients were prepared, with 100 μL of each gradient spread onto solid enrichment medium. Five replicates of each gradient were performed. The plates were incubated at 30°C under aerobic conditions for approximately 72 hours. Different colonies that grew on the plates were then streaked for purification. After 4-5 rounds of streak purification, a single strain, HZ-F-004, was finally obtained. The pure colonies were inoculated onto slant agar plates and stored at 4°C.

[0032] Example 2: Strain Identification (1) Strain morphology: Strain HZ-F-004 was streaked on solid activation medium and cultured at 30℃ under microaerobic conditions for 48 h. The colony diameter was 2 mm, the colonies were round, off-white, slightly raised, smooth, moist, glossy, opaque and viscous, odorless and tasteless, with regular edges. Gram staining confirmed that the strain was Gram-negative. Scanning electron microscopy revealed that the strain was a bacillus, 4-5 μm long. The scanning electron microscopy results are as follows. Figure 1 As shown.

[0033] (2) Molecular biological identification: The strain was identified by 16S rRNA sequencing, and the sequence results are shown in SEQ NO.1. Sequence comparison results showed that strain HZ-F-004 is... Zobellella denitrificans (Denitrifying Zobelella).

[0034] (3) Physiological and biochemical tests of the strain: The physiological and biochemical identification of the strain was performed in accordance with the 8th edition of Bergey's Manual of Bacteriological Identification. The identification result was Gram-negative bacteria. Oxidase was positive, VP test was negative, starch hydrolysis was negative, gelatin liquefaction was negative, citric acid, malic acid, and D-mannitol were available, glucose fermentation test was positive, methyl red test was negative, and indole production test was negative. The specific experimental results are shown in Table 1.

[0035] Table 1

[0036] Note: + positive reaction; - negative reaction.

[0037] (4) Preservation of strain HZ-F-004 was deposited on September 1, 2025 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35796, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0038] Example 3: Optimization of strain culture conditions (1) Optimization of carbon source types Strain activation: Streaking of strain HZ-F-004 onto a solid plate of activation medium and incubating upside down at 30°C for 24 hours. Single colonies were picked and inoculated onto enrichment medium and incubated at 30°C and 150 r / min on a shaker for 40 hours.

[0039] Sodium acetate, glycerol, sucrose, sodium citrate, and glucose were added at 1000 mg / L as initial carbon sources to the inorganic salt medium. The activated bacterial strains were then inoculated into the medium at a ratio of 1%, and the medium was incubated on a shaker (30℃, 150 r / min). Samples were taken after 16 hours to detect the concentrations of nitrite and nitrate nitrogen. The results are as follows: Figure 2 As shown.

[0040] Data analysis shows that the strain utilized sucrose and glucose most effectively. In both groups, nitrite nitrogen levels decreased to 0 mg / L after 16 hours. Furthermore, no nitrite nitrogen accumulated in the sucrose group, while the glucose group had a residual nitrite nitrogen level of 1 mg / L. This indicates that when sucrose is used as a carbon source, the strain's degradation of nitrite nitrogen and decomposition of nitrite nitrogen occur almost simultaneously. Therefore, sucrose is the optimal carbon source for the strain.

[0041] (2) Optimization of the carbon-nitrogen ratio of the culture medium Sucrose at concentrations of 400 mg / L, 700 mg / L, 1000 mg / L, 1300 mg / L, and 1600 mg / L was added to an inorganic salt culture medium as an initial carbon source. The activated bacterial strains were inoculated into the medium at a ratio of 1% (v / v), and the medium was incubated on a shaker (30℃, 150 r / min). Samples were taken after 16 hours to detect the concentrations of nitrite and nitrate nitrogen. The results are as follows: Figure 3 As shown.

[0042] Data analysis shows that when the carbon-to-nitrogen ratio (C / N ratio) of the culture medium is less than or equal to 7:1, nitrite nitrogen remains after 16 hours of fermentation. When the C / N ratio is greater than or equal to 10:1, no nitrite nitrogen remains after 16 hours of fermentation, the nitrite nitrogen degradation rate is 100%, and there is no accumulation of nitrate nitrogen. From an economic perspective, a C / N ratio of 10:1 can save more costs. Therefore, the optimal C / N ratio for this strain is 10:1, which is lower than that of most strains, and it has great application potential.

[0043] (3) Optimization of culture medium salt concentration The salt concentrations of the inorganic salt culture medium were adjusted to 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, and 60 mg / L, respectively. The activated bacterial strains were inoculated into the culture medium at a ratio of 1% (v / v), and the medium was incubated on a shaker (30℃, 150 r / min). Samples were taken after 16 hours to detect the concentrations of nitrite and nitrate nitrogen. The results are as follows: Figure 4 As shown.

[0044] Data analysis shows that the strain achieved 100% degradation of nitrite nitrogen at salt concentrations of 40 g / L or less, with no accumulation of nitrate nitrogen. Degradation was partially inhibited at 50 g / L, and severely suppressed at 60 g / L. Therefore, the strain can rapidly degrade nitrite nitrogen in water bodies with salt concentrations of 40 g / L or less without accumulating nitrate nitrogen, making it suitable for various applications.

[0045] (4) Optimization of strain inoculum The activated bacterial strains were inoculated into an inorganic salt medium with sucrose as the carbon source and a carbon-to-nitrogen ratio of 10:1 at volume ratios of 0.5%, 1%, 2%, 3%, and 4%, respectively. The medium was then incubated on a shaker (30℃, 150 r / min), and samples were taken after 16 hours to determine the concentrations of nitrite and nitrate nitrogen. The results are as follows: Figure 5 As shown.

[0046] from Figure 5 It can be seen that an inoculum size of 0.5% (v / v) is too low, and the strain cannot degrade 100 mg / L of nitrite nitrogen within 16 hours. At inoculum sizes of 1% (v / v) and 2% (v / v), the nitrite nitrogen in the culture medium can be completely degraded within 16 hours without the accumulation of nitrate nitrogen. At inoculum sizes of 3% (v / v) and 4% (v / v), the nitrite nitrogen also cannot be completely reduced within 16 hours, which may be due to an excessively large inoculum size and insufficient carbon source. Therefore, the optimal inoculum size for the strain is 1% (v / v).

[0047] (5) Verification of tolerance to hydrogen sulfide concentration Hydrogen sulfide was added to inorganic salt culture medium at final concentrations of 50 mg / L, 75 mg / L, 100 mg / L, 125 mg / L, and 150 mg / L, respectively, to investigate the strain's tolerance to hydrogen sulfide concentrations. The activated strain was inoculated at a ratio of 1% (v / v) into inorganic salt culture medium containing different concentrations of hydrogen sulfide, and cultured at 30℃ and 150 r / min on a shaker. Samples were taken after 16 h to detect nitrite and nitrate nitrogen concentrations. The results are as follows: Figure 6 As shown.

[0048] from Figure 6 It can be seen that in an environment with hydrogen sulfide concentrations of 100 mg / L or less, the strain can degrade 100 mg / L of nitrite nitrogen in 16 hours, while simultaneously consuming the product nitrate nitrogen, achieving nitrogen removal without residue. Therefore, if this strain is applied to wastewater treatment, it will not inhibit the strain as long as the hydrogen sulfide concentration in the treated water does not exceed 100 mg / L.

[0049] (6) Nitrite degradation curve Under the optimized conditions of sucrose as the carbon source, a carbon-to-nitrogen ratio of 10 / 1, a salt concentration of 40 mg / L, an inoculum size of 1% (v / v), and a hydrogen sulfide concentration of 100 mg / L, the activated strain was inoculated into an inorganic salt medium. The medium was incubated at 30°C with shaking at 150 rpm. Samples were taken at 0 h, 4 h, 8 h, 12 h, and 16 h to detect OD. 600 And nitrite concentration, the results are as follows Figure 7 As shown in the figure, the data indicates that strain HZ-F-004 entered the logarithmic growth phase 4 hours after inoculation, with a faster cell growth rate and a significantly increased nitrite degradation rate. After 16 hours of cultivation, the nitrite concentration decreased to 0 mg / L.

[0050] Example 4: Application of bacterial strains in treating high-sulfur, high-saline aquaculture wastewater A simulation experiment was conducted to treat high-sulfur, high-salinity aquaculture wastewater from a livestock farm using the HZ-F-004 strain. The wastewater from this wastewater treatment plant had a salt concentration of 3.8 wt%, sulfide pollution of 82 mg / L, and nitrite concentration of 9.96 mg / L, resulting in nitrite levels significantly exceeding wastewater discharge standards. In the experimental group, activated strain HZ-F-004 was inoculated into the wastewater at a 1% (v / v) inoculation rate and cultured at 30℃ with shaking at 150 rpm. The control group received no inoculum, but all other conditions were identical. Samples were taken at 0h and 8h to measure nitrite concentration and OD in both groups. 600 The pH value changes were observed, and the results are shown in Table 2.

[0051] Table 2

[0052] Analysis of the data in Table 2 leads to the conclusion that after inoculating wastewater with 1% (v / v) of the nitrite-degrading strain HZ-F-004 and incubating with shaking for 8 hours, the nitrite concentration in the wastewater decreased to 0 mg / L, meeting the discharge standards. The nitrite degradation rate reached 100% after 8 hours. This confirms that the nitrite-degrading strain HZ-F-004 can rapidly degrade nitrite in wastewater with high salinity, high sulfide pollution, and excessive nitrite concentrations without causing secondary metabolite pollution. No additional chemical additives are required throughout the wastewater treatment process, significantly reducing wastewater treatment costs. This demonstrates the significant application potential of the HZ-F-004 strain in the rapid treatment of aquaculture wastewater with high salinity, high sulfide pollution, and excessive nitrite concentrations.

[0053] 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 within the protection scope of the present invention.

Claims

1. A sulfur-resistant denitrifying Zobel bacterium, characterized by: The strain is Zobellia denitrificans HZ-F-004, which is classified and named as Zobellella denitrificans , and the preservation number is CGMCC No. 35796.

2. The application of Zobelella denitrifyingis as described in claim 1 in the degradation of nitrite in wastewater.

3. Use according to claim 2, wherein: The wastewater contains sulfides.

4. Use according to claim 3, wherein: The concentration of the sulfide is 50~150 mg / L.

5. The use according to claim 4, characterized in that: The concentration of the sulfide is 50~100 mg / L.

6. The use according to claim 2, characterized in that: The salt concentration in the wastewater is 20~60 mg / L.

7. Use according to claim 6, wherein: The salt concentration in the wastewater is 20~40 mg / L.

8. The use according to claim 2, characterized in that: The concentration threshold of nitrite nitrogen in the wastewater is 100 mg / L.

9. A microbial inoculant characterized by: Includes the denitrifying Zobel bacteria as described in claim 1.

10. A method of degrading nitrite in wastewater, characterized by: The method includes the step of inoculating wastewater with the denitrifying Zobel bacteria of claim 1 or the microbial agent of claim 9.

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