High-salt-resistant high-COD (Chemical Oxygen Demand) strain and application thereof

Abstract

The invention discloses a high-salt-resistant and high-COD (Chemical Oxygen Demand) strain and application thereof, the high-salt-resistant and high-COD strain is Pseudomonas stutzeri, the name of the high-salt-resistant and high-COD strain is NY-4, and the preservation number of the high-salt-resistant and high-COD strain is CCTCC (China Center for Type Culture Collection) NO: M 2025631. The high-salt-resistant and high-COD strain provided by the invention can be used for removing COD in wastewater. An efficient strain source is provided for high-salt high-COD pharmaceutical wastewater treatment, the biochemical treatment effect is remarkably improved, and the COD removal rate is increased. The high-salt-resistant and high-COD strain can enhance the salt resistance and COD removal efficiency of the existing activated sludge system, expands the application of pseudomonas stutzeri in pharmaceutical wastewater treatment, and has high practical application value.

Description

Technical Field

[0001] The present invention relates to the field of environmental microorganisms, and in particular to a high-salt and high-chemical oxygen demand (COD) tolerant bacterial strain and applications thereof. Background Art

[0002] Today, organic pollution mainly comes from chemical substances used in industrial production, such as organic dissolved matter and chemical residues in industrial wastewater.

[0003] The degree of organic wastewater pollution is primarily assessed based on COD. High-concentration organic wastewater refers to wastewater with a COD of several thousand or even tens of thousands of milligrams per liter. Improperly treated, high-concentration organic wastewater can cause oxygen depletion in the water due to biodegradation, leading to the death of aquatic organisms and deteriorating water quality.

[0004] Due to the diversity of pharmaceutical raw materials and production processes, the types and contents of pollutants in wastewater vary, but common problems include high organic matter content, high toxicity and high salinity.

[0005] High concentrations of salt and organic matter in pharmaceutical wastewater create high osmotic pressure on microorganisms, causing cell damage and inhibiting their activity. The treatment of high-salt organic pharmaceutical wastewater is not only a matter of environmental protection, but also of public health and social stability.

[0006] Currently, pharmaceutical wastewater treatment methods include flotation sedimentation, Fenton catalytic oxidation, and double-membrane processes, but these processes present challenges such as high energy consumption, low treatment efficiency, and the generation of numerous byproducts. Biological treatment methods offer lower costs, but due to the complex composition of pharmaceutical wastewater, which, in addition to high COD and salt content, also contains heavy metals, alcohols, nitrogen and phosphorus, halides, and various antibacterial drugs. This makes it difficult for microorganisms to survive in this complex environment, resulting in poor results for activated sludge and biofilm reactor processes when treating high-salt, high-COD wastewater.

[0007] Patent specification CN106635857A discloses a strain of Pseudomonas stutzeri FSTB-5 and its use in removing COD from saline wastewater. The strain is tolerant to one or more of lincomycin, minocycline, rifamycin SV, troleandomycin, vancomycin, aztreonam, and nalidixic acid. The saline wastewater in this patented technical solution is pretreated chemical synthesis wastewater, coal chemical wastewater, or concentrated brine produced by a reverse osmosis reduction unit from wastewater generated by oil refining, coal chemical, and chemical synthesis processes. The salt content is 0.5wt%-10wt% and the COD content is 200-20,000 mg / L.

[0008] However, pharmaceutical wastewater generally exhibits high salinity, high organic matter concentrations, and complex composition, posing significant challenges to traditional biological treatment processes. For this type of high-salinity, high-organic-load wastewater, it is necessary to identify and domesticate superior bacterial strains that exhibit strong salt tolerance, efficient degradation of complex organic matter, and stable growth in pharmaceutical wastewater environments. Further incorporating these functional strains into enhanced biological treatment systems can significantly improve treatment efficiency, achieving optimized and cost-effective biological treatment of high-salinity, high-organic-load wastewater. Summary of the Invention

[0009] To address the aforementioned technical issues and shortcomings in the art, the present invention provides a functional bacterial strain tolerant to high salt and high COD, and its application. This strain not only exhibits excellent salt tolerance but also maintains good physiological activity in environments with extremely high organic matter concentrations. It can survive stably and efficiently metabolize organic pollutants even in pharmaceutical wastewater containing antibacterial components (such as rifampicin). Application of this strain significantly reduces the COD content of the biochemical system effluent, effectively enhancing the salt tolerance and organic matter treatment capacity of the existing activated sludge system, and significantly improving the stability and efficiency of the existing biochemical treatment system in treating high-salt, high-COD pharmaceutical wastewater.

[0010] The specific technical solutions are as follows: In the first aspect, the present invention provides a high-salt and high-COD tolerant strain, wherein the high-salt and high-COD tolerant strain is Pseudomonas stutzeri ( Stutzerimonas stutzeri ), named NY-4, with a deposit number of CCTCC NO: M 2025631.

[0011] The high-salt and high-COD-tolerant strain was deposited in the China Center for Type Culture Collection (CCTCC) on March 31, 2025, at Wuhan University, Wuhan, China.

[0012] The high-salt and high-COD tolerant strain can survive, grow, metabolize and reproduce normally in pharmaceutical wastewater containing high concentrations of salt (such as sodium chloride, etc.), organic matter and antibacterial drugs (such as rifampicin, etc.), and has the ability to degrade high concentrations of organic matter.

[0013] The high-salt and high-COD resistant strain can tolerate antibacterial drugs such as rifampicin.

[0014] In a second aspect, the present invention provides a bacterial agent comprising the high-salt and high-COD tolerant strain described in the first aspect.

[0015] In a third aspect, the present invention provides the use of the high-salt and high-COD tolerant strain described in the first aspect or the bacterial agent described in the second aspect for removing COD in wastewater.

[0016] In some embodiments, the application described in the third aspect is to inoculate the high-salt and high-COD tolerant strain described in the first aspect or the bacterial agent described in the second aspect into the wastewater to achieve COD removal. In the present invention, the definition of inoculation concentration depends on the inoculation method: if bacterial powder is used for inoculation, it refers to the mass concentration of the bacteria in the culture medium; if bacterial liquid or bacterial suspension is used for inoculation, it refers to its volume concentration. For example, 1% bacterial liquid inoculation means that every 1 mL of bacterial liquid corresponds to 99 mL of wastewater to be treated. For example, the bacterial liquid inoculation amount of the high-salt and high-COD tolerant strain is 1%~2%, and the working viable bacteria count concentration is 10 8 ~10 10 CFU / mL.

[0017] In some embodiments, the application described in the third aspect is to inoculate the high-salt and high-COD tolerant strain described in the first aspect or the bacterial agent described in the second aspect into the biochemical system for wastewater treatment to improve the treatment capacity and daily treatment ratio of wastewater, especially high-salt and high-COD wastewater. For example, the COD content of the original biochemical influent of the biochemical system is 1065 mg / L, and the chloride ion content is 1900.84 mg / L. High-salt and high-COD wastewater can be introduced into the original biochemical influent. The COD content of the high-salt and high-COD wastewater is 15000 mg / L, the chloride ion content is 196939 mg / L, and the B / C ratio is 0.4. It has good biodegradability, and the volume proportion of the high-salt and high-COD wastewater in the total influent is 1% to 3.5%.

[0018] In some embodiments, the application described in the third aspect, the wastewater is pharmaceutical wastewater. Further, the wastewater contains rifampicin.

[0019] In some embodiments, in the application described in the third aspect, the pH of the wastewater is 7.1~7.2, for example, 7.15.

[0020] In some embodiments, in the application described in the third aspect, the salt content in the wastewater is ≤120 g / L, for example, 40 g / L, 80 g / L, 100 g / L, etc. Furthermore, the salt content in the wastewater is greater than 100 g / L and does not exceed 120 g / L, and the COD content is 30,000 to 40,000 mg / L. For example, the chloride ion content in the wastewater is 17,856 mg / L, and the COD content is 32,528 to 39,873.5 mg / L.

[0021] In a fourth aspect, the present invention provides a method for removing COD in wastewater, comprising: adding the high-salt-tolerant and high-COD-tolerant strain described in the first aspect or the bacterial agent described in the second aspect to the wastewater to remove COD in the wastewater.

[0022] In some embodiments, the method according to the fourth aspect, the wastewater is pharmaceutical wastewater. Further, the wastewater contains rifampicin.

[0023] In some embodiments, in the method described in the fourth aspect, the pH of the wastewater is 7.1~7.2, for example, 7.15.

[0024] In some embodiments, in the method of the fourth aspect, the salt content in the wastewater is ≤120 g / L, for example, 40 g / L, 80 g / L, 100 g / L, etc. Further, the salt content in the wastewater is greater than 100 g / L and does not exceed 120 g / L, and the COD content is 30,000 to 40,000 mg / L. For example, the chloride ion content in the wastewater is 17,856 mg / L, and the COD content is 32,528 to 39,873.5 mg / L.

[0025] The present invention carries out gradient domestication on the activated sludge in the biochemical pool of the pharmaceutical factory wastewater treatment, and isolates and screens a high-salt, high-COD tolerant Pseudomonas stutzeri ( Stutzerimonas stutzeri ), named NY-4. This strain can tolerate sodium chloride concentrations up to 120 g / L and maintains strong degradation capabilities in pharmaceutical wastewater with COD concentrations as high as 40,000 mg / L, achieving a COD removal rate exceeding 60%. When NY-4 was inoculated into a pharmaceutical wastewater bioreactor, it significantly improved the COD degradation efficiency and salt tolerance of the activated sludge process.

[0026] The strain NY-4 obtained in this study exhibits excellent salt tolerance and high COD tolerance. Compared to the previously reported Oligotrophomonas M02 (CN113604397A) and Manglobacter yixingensis T3 (CN113913329A), while M02 and T3 can tolerate higher sodium chloride concentrations (up to 200 g / L) and achieve removal rates of 90-95% at a COD concentration of 15,000 mg / L, NY-4's advantage lies in its tolerance to COD concentrations as high as 40,000 mg / L and its ability to stably remove higher COD equivalents (removal rate of 60%). This makes NY-4 practical for the pretreatment of high-COD wastewater, such as high-concentration pharmaceutical wastewater.

[0027] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a highly efficient bacterial strain source for treating high-salt, high-COD pharmaceutical wastewater, significantly improving biochemical treatment efficiency and COD removal rates. Furthermore, the strain can enhance the salt tolerance and COD removal efficiency of existing activated sludge systems, expanding the application of Pseudomonas stutzeri in pharmaceutical wastewater treatment and possessing significant practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure is a growth curve of the Pseudomonas stutzeri NY-4 strain provided by the present invention in liquid culture medium with different sodium chloride concentrations.

[0029] Figure 2 A photo of the colony morphology of the Pseudomonas stutzeri NY-4 strain provided by the present invention; Figure 3 A phylogenetic tree diagram of the Pseudomonas stutzeri NY-4 strain provided by the present invention; Figure 4 This is a diagram showing the COD degradation effect of the Pseudomonas stutzeri NY-4 strain provided by the present invention on pharmaceutical wastewater; Figure 5 This is an analysis diagram of the COD removal effect of the biochemical system after being strengthened with the Pseudomonas stutzeri NY-4 strain provided by the present invention and without strengthening. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The operating methods in the following examples where no specific conditions are specified are generally performed under conventional conditions or as recommended by the manufacturer.

[0031] Example 1: Directed domestication of salt-tolerant and COD-tolerant bacteria.

[0032] The specific experimental steps are as follows: 1. Directed domestication of salt-tolerant and COD-tolerant bacteria.

[0033] (1) Sample Collection: This example collected activated sludge from a biochemical pond at a pharmaceutical factory in Shaoxing City. The method of gradually increasing the proportion of raw pharmaceutical wastewater was used to domesticate a bacterial community tolerant to high salt and high COD. The wastewater from this pharmaceutical factory, originating from antibiotic production and synthetic drug manufacturing, is rifampicin pharmaceutical wastewater, containing rifampicin residues and an extremely high organic pollution load, with COD levels typically reaching 20,000-50,000 mg / L. The wastewater contains organic solvents such as methanol, ethanol, acetone, and dichloromethane, and has a high chroma, a deep red color, which increases the difficulty of treatment.

[0034] (2) Enrichment culture and acclimation: Activated sludge was placed in 100 mL acclimation medium and cultured in a shaker at 30°C and 150 rpm. The acclimation medium was a mixture of LB medium and pharmaceutical wastewater in proportion. During the acclimation process, the wastewater ratio was gradually increased (the wastewater ratio was set to 20%, 40%, 60%, 80%, and 100%, respectively. This ratio is a volume ratio, that is, 20% is 20 mL pharmaceutical wastewater + 80 mL LB medium). The ratio was increased every three days until the final concentration was 100% pharmaceutical wastewater. After transferring to the raw pharmaceutical wastewater, activated sludge was added every three days. After continuous culture for one month, separation and screening were performed.

[0035] LB medium composition (per liter of water): 10 g / L peptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, sterilized before use, pH = 7.0-8.0. For solid medium, add 1.5 wt% agar.

[0036] 2. Strain screening and purification.

[0037] After the acclimation is completed, the shake flask is left to stand for a period of time, and 10 mL of the enrichment solution is taken for gradient dilution (10 1 , 10 2 , 10 3 , 10 4 ), take 100 μL of different gradient dilutions and spread them on LB solid medium, invert it and culture it in a constant temperature medium at 30℃. After single colonies grow on the solid plate, select different single colonies for isolation and purification.

[0038] 3. Salt tolerance test.

[0039] Prepare screening culture media with gradient sodium chloride concentrations (sodium chloride addition amounts are 40 g / L, 80 g / L, 100 g / L and 120 g / L), and culture the purified strains in liquid (absorbance value at 600 nm OD 600 =1.0), inoculated into the screening medium at a 1.0% inoculum, cultured in a shaking incubator at 30°C and 150 rpm, and the OD was measured every 12 h. 600 , and draw growth curves to screen out high-salt-tolerant strains. Figure 1 The growth curves of the Pseudomonas stutzeri NY-4 strain provided by the present invention in liquid culture media with different sodium chloride concentrations are shown.

[0040] 4. COD degradation capacity test.

[0041] Single colonies of the salt-tolerant bacteria screened above were selected for liquid culture. The liquid fermentation broth was inoculated into the screening medium at a 2% inoculum size and cultured in a shaking incubator at 30°C and 150 rpm for 4 days. COD concentrations were sampled and tested every 24 h to screen out high-salt-tolerant COD-reducing strains.

[0042] Screening culture medium components: glucose 1.88 g / L, NaNO3 1.0 g / L, KH2PO4 0.05 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 0.01 g / L, NaCl 60~120 g / L, add 20%~30% pharmaceutical wastewater, control the COD concentration at 10000 mg / L, add water to 1 L, and adjust the pH to 7.0~7.5.

[0043] Based on the results of the degradation experiment, a highly efficient COD-degrading bacterium was screened and named NY-4. The isolated NY-4 strain was inoculated onto LB agar plates and cultured in a 30°C incubator for 48 h to obtain a single colony. Figure 2 As shown, the strain formed irregular, dry, rough, yellow-brown colonies on LB medium. Subsequently, NY-4 was cultured in liquid form, glycerol was added, and stored at -80°C for subsequent research.

[0044] The strain NY-4 was deposited in the China Center for Type Culture Collection (CCTCC) on March 31, 2025, at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 2025631.

[0045] 5. Molecular biological identification of the strain.

[0046] The genomic DNA of the NY-4 strain screened above was extracted and the 16S rDNA fragment was amplified using bacterial universal primers 27F and 1492R.

[0047] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1) 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2) The product was recovered from the gel and sent to a sequencing company for sequencing. The obtained sequence was submitted to the NCBI database for BLAST comparison. The results showed that it was consistent with Pseudomonas stutzeri ( Stutzerimonas stutzeri ) with a similarity of 99%. Subsequently, MEGA X software was used to perform phylogenetic analysis on the sequences of NY-4 and its closely related strains, and an evolutionary tree was drawn (e.g. Figure 3 Combined with the sequencing results, the functional strain was finally identified as Pseudomonas stutzeri ( Stutzerimonas stutzeri ).

[0048] The 16S rDNA gene sequencing results of the NY-4 strain are shown in SEQ ID NO: 3.

[0049] Example 2: Application of strain NY-4 in treating pharmaceutical wastewater.

[0050] This example evaluates the Stutzerimonas stutzeri ) NY-4 degradation performance of organic matter in high-salt pharmaceutical wastewater. The water quality characteristics of this wastewater are as follows: COD concentration is 39873.5 mg / L, pH is 7.15, and chloride ion content is 17856 mg / L. The experimental steps are as follows: First, NY-4 is cultured in liquid culture until OD 600 = 1.0, collect the bacteria by centrifugation, and wash them with 0.9% sterile saline. Subsequently, the bacterial suspension was inoculated into pharmaceutical wastewater at a 2% inoculum and incubated in a shaker at 30°C and 120 rpm. Samples were taken every 12 hours to measure COD concentration changes.

[0051] Results see Figure 4 After 48 h of cultivation, the COD concentration dropped from 39873.5 mg / L to 15550.6 mg / L, and the COD removal rate was about 61%.

[0052] Example 3: Application of strain NY-4 in an enhanced biochemical treatment system.

[0053] In this example, Pseudomonas stutzeri ( Stutzerimonas stutzeri NY-4 is used to bio-augment the biochemical system of an existing sewage treatment plant. By gradually increasing the proportion of high-salt wastewater in the biochemical system's influent and raising the influent salinity, while ensuring the effluent COD is below 200 mg / L (the standard for inlet), the maximum amount of high-salt wastewater that can be introduced, the salt tolerance limit of the biochemical system, and the effectiveness of NY-4 in enhancing the system's salt tolerance are evaluated. The original biochemical influent COD concentration is 1065 mg / L, with a chloride ion concentration of 1900.84 mg / L; the high-salt wastewater COD is ≥15,000 mg / L, with a chloride ion concentration of 196,938.94 mg / L. During the experimental phase of this project, the influent proportion of high-salt wastewater was set at 1.0% to 3.5%. The main treatment process is as follows: 1) Design two biochemical reactors with a 5 L effective volume: one fortified reactor for salt-tolerant bacteria (bioaugmentation group) and one for verification of a biochemical system (control group). The biochemical system was augmented with 1‰ (v / v) NY-4, while the control group was the biochemical system. Aeration pumps were started to control dissolved oxygen to ≤6 mg·L. -1 The hydraulic retention time of the reactor was set to 3 days.

[0054] 2) The initial influent ratio of the reactor was 99% raw water and 1% high-salt wastewater. The proportion of high-salt wastewater in the biochemical system was gradually increased in stages, and the chloride concentration was increased from 3000 mg / L to approximately 9000-10000 mg / L. The salt tolerance of the biochemical sludge was tested in small-scale tests, and the treatment effect of the reactor was verified by continuous flow. The maximum salt tolerance of the biochemical system and the COD conversion efficiency were tested.

[0055] like Figure 5 As shown in the figure, the degradation experiment results are as follows: when the chloride ion concentration in the reactor is low (less than 5000 mg / L, the volume ratio of high-salt wastewater is about 1%), the effluent of the bio-augmentation group and the control group is stable at below 200 mg / L; when the chloride ion concentration in the reactor reaches 10000±300 mg / L, the COD concentration of the effluent of the control group has risen to 285~293.85 mg / L, and although the enhanced group with NY-4 addition has a short-term fluctuation (COD concentration rises to 210-230 mg / L), it has steadily dropped to below 200 mg / L after several days of acclimation. This result shows that NY-4 effectively improves the salt tolerance and COD removal capacity of the original biochemical system.

[0056] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A high salt and high COD tolerant bacterial strain, characterized in that: The high salt and high COD resistant strain is Pseudomonas stutzeri ( Stutzerimonas stutzeri ), named NY-4, with a deposit number of CCTCC NO: M 2025631.

2. A bacterial agent, characterized in that The invention comprises the high-salt and high-COD tolerant strain according to claim 1.

3. Use of the high-salt and high-COD tolerant strain according to claim 1 or the bacterial agent according to claim 2 for removing COD from wastewater.

4. The use according to claim 3, characterized in that The wastewater is pharmaceutical wastewater and contains rifampicin.

5. The use according to claim 3, characterized in that The pH of the wastewater is 7.1-7.

2.

6. The use according to claim 3, characterized in that The salt content in the wastewater is ≤120 g / L, and the COD content is 30,000-40,000 mg / L.

7. A method for removing COD from wastewater, characterized in that: include: The high-salt and high-COD tolerant strain according to claim 1 or the bacterial agent according to claim 2 is added to the wastewater to remove the COD in the wastewater.

8. The method according to claim 7, characterized in that The wastewater is pharmaceutical wastewater and contains rifampicin.

9. The method according to claim 7, characterized in that The pH of the wastewater is 7.1-7.

2.

10. The method according to claim 7, characterized in that The salt content in the wastewater is ≤120 g / L, and the COD content is 30,000-40,000 mg / L.

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