Salt-tolerant strain for degrading 2, 4-di-tert-butylphenol and application of salt-tolerant strain

Abstract

The invention discloses a salt-tolerant strain for degrading 2, 4-di-tert-butylphenol, which is classified and named as Acinetobacter junii, has the strain number of GXGD-YZ-2506 (D1), is preserved in the Guangdong Microbial Culture Collection Center on July 4, 2025, has the preservation number of GDMCC No: 66641, and is preserved in the 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou City, Guangdong Province. The strain can grow under the condition that the salinity is 7wt%, can efficiently degrade 2, 4-di-tert-butylphenol, is suitable for treating high-salt organic wastewater containing 2, 4-di-tert-butylphenol in the industries of petrifaction, metallurgy, chemical engineering and the like, and has the advantages of being high in treatment efficiency, high in environmental adaptability and the like. The invention provides a novel microbial resource and an effective technical support for biological treatment of refractory phenol pollutants, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to a salt-tolerant strain that degrades 2,4-di-tert-butylphenol and its applications. Background Technology

[0002] High-salinity wastewater from refining mainly originates from reverse osmosis membrane reuse systems and is widely distributed in petrochemical, metallurgical, and chemical industries. It is characterized by high total dissolved solids (TDS) concentrations, persistent organic pollutants, and high heavy metal content. Direct discharge without effective treatment not only harms the growth and reproduction of aquatic organisms but may also affect the quality of agricultural water, thus posing a serious threat to human health. In addition to conventional pollutants, a noteworthy phenolic derivative—2,4-di-tert-butylphenol (2,4-DTBP)—has been detected in the high-salinity wastewater. As an important intermediate in the production of antioxidants for rubber, plastics, coatings, and fuels, this substance is continuously released into the wastewater system during production and use. It exhibits strong toxicity and endocrine disruption, adversely affecting the aquatic ecosystem and urgently requires proper treatment. Studies have shown that 2,4-DTBP is highly lipophilic (logKow = 5.3) and environmentally persistent (half-life of approximately 900 hours), easily accumulating in organisms and migrating through the food chain. It significantly inhibits and damages the growth of aquatic algae and the liver and kidney function of fish. It also has anti-estrogenic and endocrine-disrupting effects. It has been detected in fish liver, human milk, and human urine, and is widely present in surface water and sewage treatment plant sediment. Therefore, it has been listed as a key controlled toxic substance by the European Chemicals Agency (ECHA).

[0003] Existing methods for treating 2,4-DTBP mainly include coagulation and persulfate oxidation. However, these methods often face limitations in high-salt environments, such as high dosage, high treatment costs, low removal efficiency, and the potential for secondary pollution, making them insufficient to meet treatment needs. Biological methods have attracted significant attention due to their low investment costs, environmental friendliness, and strong sustainability. However, high salt concentrations and toxicity in wastewater can inhibit the activity of conventional microorganisms. Therefore, developing a microbial strain that can tolerate high salt levels and possesses efficient 2,4-DTBP degradation capabilities is of great importance for treating wastewater containing 2,4-DTBP, including high-salt refining wastewater. Summary of the Invention

[0004] The purpose of this invention is to address the problem that existing biological treatment technologies have poor treatment effects on wastewater containing 2,4-DTBP, and to provide a microbial strain that can efficiently degrade 2,4-DTBP in a high-salt environment, thus providing effective technical support for the biological treatment of high-salt organic wastewater.

[0005] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0006] A salt-tolerant strain that degrades 2,4-di-tert-butylphenol, classified as *Acinetobacter junii*, strain number GXGD-YZ-2506(D1), was deposited on July 4, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 66641), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0007] The 16S rDNA nucleotide sequence of the salt-tolerant strain that degrades 2,4-di-tert-butylphenol is shown in SEQ ID NO. 1; the colonies of the salt-tolerant strain that degrades 2,4-di-tert-butylphenol are milky white to pale yellow, opaque, with neat edges and a smooth, moist surface; under an optical microscope, the individual morphology of the salt-tolerant strain that degrades 2,4-di-tert-butylphenol is short rod-shaped, about 1.5–2.0 μm long and 0.7–0.9 μm wide.

[0008] The salt-tolerant strain that degrades 2,4-di-tert-butylphenol has a salt tolerance range of 0-7% (the mass fraction of total salt in wastewater calculated as NaCl content). It has the ability to survive in a high-salt environment with a salinity of 0-7 wt% and to efficiently degrade 2,4-di-tert-butylphenol (2,4-DTBP).

[0009] The present invention also claims protection for the use of the salt-tolerant strains that degrade 2,4-di-tert-butylphenol in the treatment of wastewater containing 2,4-di-tert-butylphenol.

[0010] The total salt content in the wastewater containing 2,4-di-tert-butylphenol is 0-7% by mass.

[0011] The present invention also provides a method for treating wastewater containing 2,4-di-tert-butylphenol, wherein the salt-tolerant strain that degrades 2,4-di-tert-butylphenol is inoculated into the wastewater containing 2,4-di-tert-butylphenol to carry out a biological reaction.

[0012] The total salt content in the wastewater containing 2,4-di-tert-butylphenol is 0-7% by mass.

[0013] Preferably, the content of 2,4-di-tert-butylphenol in the wastewater containing 2,4-di-tert-butylphenol is less than 1 g / L; more preferably, the content of 2,4-di-tert-butylphenol in the wastewater containing 2,4-di-tert-butylphenol is less than 0.6 g / L.

[0014] The inoculum size of the salt-tolerant strain that degrades 2,4-di-tert-butylphenol is 10. 6 ~10 8 CFU / mL.

[0015] The biological reaction is carried out under aerobic conditions with a temperature of 28–37°C, a pH of 6.5–8.5, and a carbon-to-nitrogen ratio of 10–15:1.

[0016] The salt-tolerant strains that degrade 2,4-di-tert-butylphenol exist in either a free or immobilized state.

[0017] Preferably, the salt-tolerant strain that is immobilized to degrade 2,4-di-tert-butylphenol is embedded in an immobilization carrier or attached to the surface of a packing material.

[0018] Preferably, the salt-tolerant strain that degrades 2,4-di-tert-butylphenol exists in the form of an adhesive attached to the surface of the packing material. The strain forms a biofilm on the surface of the packing material, thereby improving its salt tolerance and the degradation efficiency of 2,4-di-tert-butylphenol.

[0019] Preferably, the filler is an inert filler; more preferably, the filler is a carbon-based carrier.

[0020] The present invention also provides a microbial agent comprising the salt-tolerant strain that degrades 2,4-di-tert-butylphenol.

[0021] The microbial agent exists in liquid, powder or granule form.

[0022] Preferably, the microbial agent is prepared into powder or granules by fermentation and expansion followed by freeze-drying or spray drying, and stored at room temperature for 6 months, with a cell survival rate of more than 85%.

[0023] Beneficial effects:

[0024] The *Acinetobacter junii* GXGD-YZ-2506(D1) provided by this invention exhibits excellent growth activity and 2,4-DTBP degradation performance under high-salt conditions, with a salt tolerance range of 0–7 wt%. Under conditions of 1 wt% NaCl and an initial 2,4-DTBP concentration of 600 mg / L (COD concentration of 1800 mg / L), the above strain can reduce COD to 120 mg / L within 7 days, with a 2,4-DTBP removal rate as high as 93%, while simultaneously reducing OD... 600The concentration remained stable at approximately 0.2. Under conditions of 7 wt% NaCl and an initial 2,4-DTBP concentration of 600 mg / L (COD concentration of 1800 mg / L), the above-mentioned strain could reduce COD to 480 mg / L within 7 days, with a 2,4-DTBP removal rate as high as 73%. The strain *Acinetobacter junii* GXGD-YZ-2506(D1) not only exhibits high salt tolerance but also effectively degrades 2,4-DTBP under high-salt conditions, making it suitable for the treatment of organic pollutants in high-salt environments and demonstrating significant potential in salt tolerance mechanism research and industrial applications. Attached Figure Description

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0026] Figure 1 This is a colony photograph of Acinetobacter junii GXGD-YZ-2506(D1), a strain screened in this invention.

[0027] Figure 2 The image shows the degradation effect of strain Acinetobacter junii GXGD-YZ-2506(D1) on 2,4-DTBP at a salinity of 1wt%.

[0028] Figure 3 This is a growth curve of strain Acinetobacter junii GXGD-YZ-2506(D1) in Example 2 under a salinity of 1wt%.

[0029] Figure 4 This is a growth curve of strain Acinetobacter junii GXGD-YZ-2506(D1) in Example 4 under different salinity conditions. Detailed Implementation

[0030] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0031] The *Acinetobacter junii* GXGD-YZ-2506(D1) provided in this invention is a Gram-negative bacterium isolated from high-salt wastewater from a chemical refinery. Under LB agar culture conditions, its colony morphology is characterized as follows: milky white to pale yellow, opaque, with regular edges, and a smooth, moist surface. Under an optical microscope, the individual cells of this strain are short rod-shaped, approximately 1.5–2.0 μm in length and 0.7–0.9 μm in width.

[0032] The salt tolerance range of the Acinetobacter junii GXGD-YZ-2506(D1) is 0-7% (mass fraction of total salt in wastewater calculated as NaCl content).

[0033] The MSM (Mineral Salts Medium) used in the following examples has the following formulation: Na₂HPO₄ 2.5 g / L, KH₂PO₄ 1.5 g / L, NH₄Cl 0.5 g / L, NaCl 10 g / L, MgSO₄·7H₂O 0.2 g / L, CaCl₂·2H₂O 0.02 g / L, FeSO₄·7H₂O 0.01 g / L, deionized water 1000 mL, pH adjusted to 7.0, and sterilized at 121°C for 20 minutes. This medium is a basic inorganic salt medium, containing no organic carbon source, and is suitable for screening and enriching degrading strains that use the target contaminant as their sole carbon source.

[0034] Unless otherwise specified, the experimental methods used in the following examples follow standard experimental procedures in the field of biology; and the reagents and materials used are commercially available unless otherwise specified.

[0035] Example 1: Isolation, Screening, Domestication, and Identification of Strains

[0036] 10 mL of high-salt wastewater from a wastewater treatment plant in Jieyang, Guangdong Province, originating from the petroleum refining process, was added to 90 mL of MSM medium containing 50 mg / L 2,4-DTBP. The medium was incubated at 30℃ and 180 rpm for 72 hours with shaking to enrich the microbial community capable of degrading 2,4-DTBP. After incubation, the medium was serially diluted 10-fold. The diluted samples were then taken from each of the 10-fold dilutions. -5 10 -6 10 -7 0.15 mL of bacterial suspension was evenly spread onto the surface of MSM solid agar plates containing 100 mg / L 2,4-DTBP and incubated at 30°C for 3–7 days. Colony growth was observed. Obvious colonies on the plates were picked and purified again using the dilution plating method in multiple rounds until homogeneous pure cultures were obtained. Ultimately, 12 strains capable of growing on MSM solid agar plates containing 100 mg / L 2,4-DTBP were successfully isolated, providing candidate bacterial sources for subsequent domestication and identification.

[0037] Since 2,4-DTBP is a strongly hydrophobic compound and insoluble in water, it is added as follows: Dissolve 2,4-DTBP in anhydrous ethanol. After thorough stirring, prepare a 200 g / L 2,4-DTBP ethanol solution stock solution. As needed, add this stock solution directly to MSM liquid culture medium to obtain the culture system with the required 2,4-DTBP concentration.

[0038] To further improve the tolerance and degradation capacity of the strains to 2,4-DTBP, the 12 isolated strains were subjected to four rounds of gradient acclimatization culture. The acclimatization process used MSM basal medium, with 2,4-DTBP added to concentrations of 50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L, simulating the environmental pressure of gradually increasing pollutant concentrations. Each round of culture lasted 3 days, with the temperature maintained at 30℃, a shaking speed of 180 r / min, pH controlled at 7.0, and salinity adjusted to 1% (NaCl content 10 g / L) to adapt to the high-salt environment of the refining wastewater. This method induced the strains to adapt to 2,4-DTBP and enhanced their 2,4-DTBP degradation capacity.

[0039] After four rounds of gradient acclimatization, a dominant strain exhibiting good growth ability and outstanding degradation effect on 2,4-DTBP was screened from 12 strains and named GXGD-YZ-2506(D1). GXGD-YZ-2506(D1) was identified as *Acinetobacter junii*. This strain was mixed with 25% v / v glycerol protective solution and stored in an ultra-low temperature freezer at -80℃ for long-term preservation and subsequent research. The colony morphology of strain GXGD-YZ-2506(D1) is as follows: Figure 1 As shown.

[0040] Example 2: 2,4-DTBP Degradation Experiment

[0041] The strain *Acinetobacter junii* GXGD-YZ-2506(D1) obtained in Example 1 was inoculated into an inorganic salt medium (MSM) with 2,4-di-tert-butylphenol (2,4-DTBP) as the sole carbon source to verify its degradation ability for typical recalcitrant pollutants. Specifically, the strain culture was centrifuged at 4000 rpm and 4°C for 10 min, the supernatant was discarded, and the bacterial cells were collected and inoculated into prepared MSM liquid medium, resulting in an initial bacterial concentration of 10⁻⁶. 7 The medium contains 600 mg / L 2,4-DTBP and has a salinity of 1 wt%. The initial chemical oxygen demand of the medium is 1800 mg / L, all derived from 2,4-DTBP, and is used to simulate a high-concentration organic pollution environment.

[0042] The experimental culture conditions were set at a constant temperature of 30℃ with shaking at 180 rpm to ensure sufficient contact between the bacteria and the contaminants and an aerobic degradation environment. The total culture period was 7 days, during which samples were taken daily at regular intervals to measure the COD value and optical density (OD) of the culture medium. 600 COD (Chemical Oxygen Demand) is used as a measure of the concentration of the organic pollutant 2,4-DTBP to assess the 2,4-DTBP degradation capacity of bacterial strains; OD (Oxygen Demand) is used to assess the 2,4-DTBP degradation capacity of bacterial strains. 600 The COD concentration reflects the microbial cell density in the culture medium and is used to characterize bacterial growth. COD was determined using the potassium dichromate standard method (Determination of Chemical Oxygen Demand in Water - Dichromate Method GB 11914-89) to ensure the scientific validity and comparability of the data. To verify the reliability of COD changes, a blank control group without inoculated strains was set up, with the same culture conditions as the experimental group, to eliminate interference from factors such as the autodegradation or volatilization of 2,4-DTBP on COD. The COD concentration in the control group remained essentially unchanged over 7 days, indicating that pollutant degradation mainly originates from the biodegradation process of the strains.

[0043] The experimental results are shown in Table 1. Figure 2 and Figure 3 As shown, the COD value in the culture medium decreased day by day with the extension of the culture time, and by the 7th day, the COD value had dropped to 120 mg / L. Figure 2 This indicates that the pollutants have been largely degraded, with a degradation rate exceeding 93%. (Bacterial growth curve) Figure 3 The results show that the bacteria were in an adaptation period for the first two days, with OD... 600 Little change; days 3 to 5 are the logarithmic growth phase, OD 600 It rises significantly, reaching a maximum of around 0.2; it enters a stable period from day 6 to 7, OD 600 The fact that the levels remained essentially unchanged indicates that the carbon source was gradually depleted and the bacterial growth tended to stabilize. Under the condition that 2,4-DTBP was the only carbon source, although the strain could maintain metabolic activity and continue to grow, the overall biomass growth was limited. It is speculated that although 2,4-DTBP can serve as a carbon source, its toxicity still has a certain inhibitory effect on bacterial growth.

[0044] Table 1. Degradation effect of strain Acinetobacter junii GXGD-YZ-2506(D1) on 2,4-DTBP

[0045]

[0046] To more accurately assess the degradation kinetics, COD removal rate (η) was used for quantification, and the calculation formula is as follows:

[0047] Wherein, COD0 is the initial COD concentration (1800 mg / L), CODt Let be the COD concentration on day t. Taking day 7 data as an example...

[0048] The above experimental results indicate that strain GXGD-YZ-2506(D1) has a significant 2,4-DTBP removal capacity throughout the entire culture cycle.

[0049] Example 3: Salt Tolerance Growth Test of Strains

[0050] To evaluate the adaptability and application potential of strain *Acinetobacter junii* GXGD-YZ-2506(D1) in high-salt environments, a salinity tolerance analysis experiment was conducted. Under standard nutrient culture conditions, the growth of the strain at different NaCl concentrations was preliminarily evaluated. Specifically, strain GXGD-YZ-2506(D1) was streaked onto LB solid medium containing 0%, 1%, 3%, 5%, 7%, 8%, and 9% NaCl, and incubated upside down at 30°C for 48 hours. Colony formation and indicators of bacterial viability, such as size, color, and edge condition, were observed and recorded. The results are shown in Table 2. This step was used to rapidly screen the strain's sensitivity range to salt stress, providing a reference for subsequent evaluation under conditions closer to actual wastewater environments.

[0051] Experimental results show that the salt tolerance range of Acinetobacter junii GXGD-YZ-2506(D1) is 0–7 wt%.

[0052] Table 2. Results of salinity gradient test for strain Acinetobacter junii GXGD-YZ-2506(D1)

[0053]

[0054] Note: "++++" is good, "+++" is relatively good, "++" is average, "+" can grow, and "-" cannot grow.

[0055] Example 4: Degradation effect of strain Acinetobacter junii GXGD-YZ-2506(D1) on 2,4-DTBP under high salt conditions

[0056] Furthermore, to investigate the growth of strain *Acinetobacter junii* GXGD-YZ-2506(D1) under different salt concentrations and its degradation effect on 2,4-DTBP, a high-salt selective culture system containing 600 mg / L 2,4-DTBP was constructed by adding 2,4-DTBP as the sole carbon source to inorganic salt medium (MSM) containing different mass fractions of NaCl (0%, 1%, 3%, 5%, 7%, 8%, and 9%). The strain was inoculated into the MSM liquid medium with different salinities, and the initial bacterial concentration in the MSM medium was 10... 7 CFU / mL, cultured at 30℃ and 180 rpm with shaking for 7 days. No additional carbon source was added during the experiment, ensuring 2,4-DTBP was the sole energy source. During the culture period, the OD of the bacterial cells in MSM medium at various salt concentrations was measured daily using a spectrophotometer. 600 The values ​​and COD concentrations were used to quantitatively assess the growth of the strain under different salinity conditions and its ability to degrade 2,4-DTBP under different salinity conditions. Cell OD... 600 The value directly reflects the cell density and biological activity level, and can be used as a basis for judging the strain's ability to adapt to salinity stress. All COD in MSM medium comes from 2,4-DTBP; therefore, COD can be used as a measure of the concentration of the organic pollutant 2,4-DTBP to assess the strain's ability to degrade 2,4-DTBP.

[0057] The experimental results of the growth status of strain Acinetobacter junii GXGD-YZ-2506(D1) are as follows: Figure 4 As shown, strain GXGD-YZ-2506(D1) exhibited good growth ability within a salinity range of 1%–5%, OD 600 The OD value fluctuates somewhat with increasing salinity but does not decrease significantly. When the salinity is in the range of 5% to 7%, the OD value of the strain is [not specified]. 600 The value decreased, but it could still grow under the salt stress conditions at this time, indicating that the strain has strong salt tolerance; when the salinity was higher than 7%, the growth of strain GXGD-YZ-2506(D1) was inhibited and almost stopped.

[0058] The degradation effect of the strain on 2,4-DTBP is shown in Table 3. In the salinity range of 0-7%, Acinetobacterjunii GXGD-YZ-2506(D1) showed good 2,4-DTBP degradation ability. After being cultured for seven days under a salinity of 7%, the removal rate of 2,4-DTBP by this strain could still reach 73.33%.

[0059] The above results demonstrate that strain Acinetobacter junii GXGD-YZ-2506(D1) has the potential to survive in high-salt organic wastewater environments and continuously exert 2,4-DTBP degradation activity, providing theoretical support and experimental basis for its engineering application in complex water bodies such as high-salt refining wastewater.

[0060] Table 3. Degradation effect of strain Acinetobacter junii GXGD-YZ-2506(D1) on 2,4-DTBP under different salinities.

[0061]

[0062] This invention provides a salt-tolerant strain for degrading 2,4-di-tert-butylphenol and its application, along with a method and approach. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A salt-tolerant strain that degrades 2,4-di-tert-butylphenol, classified as Acinetobacter junii, strain number GXGD-YZ-2506(D1), and preservation number GDMCC No:66641.

2. The application of the salt-tolerant strain for degrading 2,4-di-tert-butylphenol as described in claim 1 in the treatment of wastewater containing 2,4-di-tert-butylphenol.

3. The application according to claim 2, characterized in that, The total salt content in the wastewater containing 2,4-di-tert-butylphenol is 0-7% by mass.

4. A method for treating wastewater containing 2,4-di-tert-butylphenol, characterized in that, The salt-tolerant strain that degrades 2,4-di-tert-butylphenol as described in claim 1 was inoculated into wastewater containing 2,4-di-tert-butylphenol to carry out a biological reaction.

5. The method according to claim 4, characterized in that, The total salt content in the wastewater containing 2,4-di-tert-butylphenol is 0-7% by mass.

6. The method according to claim 4, characterized in that, The inoculum size of the salt-tolerant strain that degrades 2,4-di-tert-butylphenol is 10. 6 ~10 8 CFU / mL.

7. The method according to claim 4, characterized in that, The biological reaction was carried out under aerobic conditions with a temperature of 28–37°C, a pH of 6.5–8.5, and a carbon-to-nitrogen ratio of 10–15:

1.

8. The method according to any one of claims 4 to 7, characterized in that, The salt-tolerant strains that degrade 2,4-di-tert-butylphenol exist in free or immobilized form.

9. A microbial inoculant, characterized in that, The salt-tolerant strain comprising the 2,4-di-tert-butylphenol degrading strain of claim 1.

10. The microbial agent according to claim 9, characterized in that, The microbial agent exists in liquid, powder, or granular form.

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