Bacterial strain for degrading phenol-polluted wastewater and application of bacterial strain
By using Acinetobacter pittii TJ2 strain, the wastewater treatment problem in high concentration of phenol, low temperature and high salt environments was solved, and efficient phenol degradation effect was achieved.
Patent Information
- Application Number
- CN202510653798.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-21
- Publication Date
- 2025-08-08
AI Technical Summary
The existing microbial technology has low efficiency in treating wastewater under high concentrations of phenol and low temperature conditions, and the high-salt environment puts pressure on microbial activity, making it difficult to achieve effective degradation.
Acinetobacter pittii TJ2 strain is used, which has medium and high concentrations of phenol, low temperature and high salt tolerance, and is used to degrade phenol contaminated wastewater.
The degradation rate of phenol in high concentrations is as high as 99.42%, and the degradation capacity remains high at low temperatures. The degradation rate in high-salt environments is still 55.71%, breaking through the efficiency bottleneck of traditional strains and reducing treatment time and cost.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial wastewater treatment, and in particular relates to a bacterial strain for degrading phenol-contaminated wastewater and application thereof. Background Art
[0002] Phenol pollution in industrial wastewater is one of the major challenges in the current field of environmental governance. As a highly toxic and difficult-to-degrade organic pollutant, phenol is widely present in wastewater from the petrochemical, pharmaceutical, and coking industries. Its high-concentration discharge not only causes serious damage to the aquatic ecosystem, but may also threaten human health through enrichment in the food chain and even cause risks such as carcinogenesis and teratogenicity. Therefore, it is of great significance to develop efficient and economical phenol wastewater treatment technologies. Microbial degradation technology is considered to be the preferred solution for phenol pollution control due to its environmental friendliness, ease of operation and cost-effectiveness. However, existing microbial treatment technologies still face many challenges in practical application, especially the treatment efficiency issues under high phenol concentration and low temperature conditions.
[0003] However, current microbial phenol removal technologies face multiple challenges in practical application. First, a significant limitation is the microbial tolerance to high phenol concentrations. When phenol concentrations in wastewater exceed a certain threshold, the microbial cell membrane structure is easily damaged, leading to an imbalance in the exchange of substances inside and outside the cell. Simultaneously, the activity of key metabolic enzymes (such as monooxygenases and dioxygenases) is inhibited, thereby affecting the microbial degradation capacity. This inhibition can lead to a decline in microbial metabolic function or even death. Especially at phenol concentrations above 2000 mg / L, the degradation efficiency of many microorganisms drops significantly, making it difficult to meet the requirements of high-concentration wastewater treatment.
[0004] Secondly, low temperatures pose a severe challenge to the adaptability of microorganisms. Below 20°C, the metabolic rate of microorganisms decreases significantly, and enzymatic reactions become sluggish, resulting in a significant reduction in phenol removal efficiency and difficulty in achieving operational stability.
[0005] Furthermore, industrial wastewater is often accompanied by high salinity, which puts additional pressure on microbial activity. High-salt environments can cause dehydration of microbial cells, osmotic imbalance, and even protein denaturation and enzyme inactivation, further weakening their ability to degrade phenol.
[0006] Therefore, finding new strains that can tolerate complex environments is a problem that needs to be solved currently. Summary of the Invention
[0007] The purpose of the present invention is to provide a bacterial strain for degrading phenol-contaminated wastewater and its application, thereby achieving effective degradation of phenol in complex environments.
[0008] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a strain for degrading phenol-contaminated wastewater, wherein the strain is Acinetobacter pituitus ( Acinetobacter pittii ) TJ2 was deposited on December 10, 2024 in the General Microbiology Center of the China Culture Collection Administration, abbreviated as CGMCC, with its address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33016.
[0009] Preferably, the 16S rRNA gene sequence of the strain is shown as SEQ ID NO.1.
[0010] Preferably, the strain has the ability to tolerate medium to high initial phenol concentrations, low temperature tolerance and high salt tolerance.
[0011] Preferably, the medium-high initial phenol concentration is 1000 mg / L-2000 mg / L; the low temperature tolerance temperature is 10° C.-20° C.; and the high salt tolerance is 2%-6% NaCl.
[0012] In a second aspect, the present invention provides the use of the strain in degrading phenol-containing wastewater.
[0013] In a third aspect, the present invention provides a method for treating phenol-containing wastewater, the method comprising the following steps: (1) Acinetobacter pituitus ( Acinetobacter pittii ) was inoculated into phenol-containing wastewater; (2) Degradation treatment of phenol-containing wastewater is carried out at a temperature of 10-30°C.
[0014] Preferably, the strain concentration in the phenol-containing wastewater is ≥10 8 CFU / mL.
[0015] In a fourth aspect, the present invention provides a use of a strain of Acinetobacter pitei in preparing a degrading bacterial agent for degrading phenol-contaminated wastewater, wherein the Acinetobacter pitei is Acinetobacter pitei ( Acinetobacter pittii )TJ2, the deposit number is CGMCC No. 33016.
[0016] Preferably, in the degradation bacterial agent, the Acinetobacter pituitus ( Acinetobacter pittii ) TJ2 strain concentration ≥10 8 CFU / mL.
[0017] Preferably, the amount of the degradation bacterial agent used in phenol-contaminated wastewater is 5%-10%.
[0018] Preferably, the degradation bacterial agent is a degradation bacterial agent for low-temperature and high-salt phenol-contaminated wastewater; The low temperature is 10-20° C., and the high salt concentration is 2%-6% NaCl.
[0019] The beneficial effects of the present invention are: First, the strain provided by this invention is highly efficient at degrading high-concentration phenol: at an initial concentration of 1000 mg / L, the degradation rate reached 99.42% within 48 hours (Table 1), virtually completely removing phenol. Even at a high concentration of 2000 mg / L, the degradation rate reached 76.54% within 48 hours (Table 3). This characteristic overcomes the efficiency bottleneck of traditional strains in treating high-concentration phenol wastewater, significantly reducing treatment time and costs.
[0020] Secondly, the strain provided by the present invention has excellent low-temperature tolerance and can still maintain a high phenol degradation ability under low-temperature environment. Compared with ordinary strains whose activity drops sharply under low temperature, TJ2 has significant advantages in cold environment.
[0021] In addition, the strain provided by the present invention has strong salt tolerance and adaptability, and the strain TJ2 has outstanding phenol degradation ability in saline wastewater (2-6% NaCl). BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is a morphological diagram of the strain TJ2 provided by the present invention; Figure 2 This is the phylogenetic tree diagram of the strain TJ2 provided by the present invention; Figure 3 The phenol tolerance and degradation effect of strain TJ2 at an initial concentration of 1000 mg / L; Figure 4 The phenol tolerance and degradation effect of strain TJ2 at an initial concentration of 1500 mg / L; Figure 5 The phenol tolerance and degradation effect of strain TJ2 at an initial concentration of 2000 mg / L; Figure 6 The bar graph shows the comparison of the degradation rates of strain TJ2 at different temperatures (10°C, 15°C, and 20°C); Figure 7 The bar graph shows the comparison of the degradation rates of strain TJ2 under different NaCl concentrations (2%, 4%, and 6%). DETAILED DESCRIPTION
[0023] The following embodiments of the technical solution of the present invention are described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only provided as examples and are not intended to limit the scope of protection of the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.
[0024] The culture medium used in the examples is as follows: LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 1 L distilled water. Solid medium: add 15 g agar to the liquid medium.
[0025] Basal medium: 2.25 g dipotassium hydrogen phosphate, 2.75 g potassium dihydrogen phosphate, 1.00 g ammonium sulfate, 0.20 g magnesium chloride hexahydrate, 0.10 g sodium chloride, 0.02 g ferric chloride hexahydrate, 0.01 g calcium chloride, 1 L distilled water, pH 6.8-7.0. After sterilization, add varying volumes of phenol stock solution depending on the experimental purpose.
[0026] Example 1 Isolation and screening of strains (1) The samples of the present invention were collected from the wastewater and activated sludge (winter) of the regulating tank of a coking wastewater treatment plant in Changzhi, Shanxi Province, and phenol was added to adjust the phenol concentration therein.
[0027] (2) Acclimation was performed for a total of 6 cycles, of which the first 2 cycles were 24 h each, with a phenol concentration of 2000 mg / L, and acclimation and enrichment culture was performed at 30°C and 180 rpm; (3) In the middle two cycles, each cycle was 48 h, the phenol concentration was 2000 mg / L, and the acclimation was continued at 20°C and 180 rpm.
[0028] (4) The last two cycles, each 48 h, continued acclimation at 20°C and 180 rpm with a phenol concentration of 2000 mg / L and a NaCl concentration of 4%.
[0029] (5) After each cycle, all samples are centrifuged and the supernatant is discarded. New wastewater from the equalization tank is added to continue the next cycle of enrichment culture.
[0030] (6) Take the mixed sample of wastewater and activated sludge after the last cycle, dilute it with sterile water in a gradient manner, and spread 100 μL of the dilution liquid on LB solid culture medium for each gradient. Place it in a biochemical incubator at 30℃ and culture it for 48 hours. Observe the growth of the colonies, select single colonies with different morphologies, and streak them on LB solid culture medium using the plate streak method. Then culture them under the same conditions for 48 hours.
[0031] (7) Then, single colonies were selected and purified by multiple partitioning and streaking until a pure strain was obtained. The purified strain was placed in 20% glycerol and stored at -80°C in a refrigerator for future use.
[0032] Example 2 (1) A phenol-degrading bacterium was obtained through the separation and purification process of Example 1. The obtained strain was subjected to molecular biological identification and sequencing comparison after PCR amplification of the bacterial 16S rRNA gene sequence (SEQ ID NO. 1).
[0033] (2) The amplification primers used in this example are: 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO. 2), 1492R: TACGGYTACCTTGTTACGACTT (SEQ ID NO. 3); The reaction system used in this example is: 10× Buffer 2 μL, 2.5 mM dNTP 1.5 μL, Primer11 μL, Primer2 1 μL, Template 1 μL, Enzyme 0.3 μL, Water 13.2 μL, Total volume 20 μL; The reaction conditions used in this example were: pre-denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1.5 min, extension at 72°C for 10 min, and incubation at 4°C (until removal).
[0034] The PCR products were detected by agarose gel electrophoresis and then sequenced.
[0035] The 16S rRNA gene sequence of the bacterium was obtained by forward and reverse sequencing and splicing (SEQ ID NO.1). The sequence was compared with the NCBI database by Blast. The results showed that it was similar to Acinetobacter pituitii ( Acinetobacter pittii )FDAARGOS had the highest homology, reaching 99.72%, and the strain was named Acinetobacter pitei ( Acinetobacter pittii ) TJ2, hereinafter referred to as strain TJ2 in this application.
[0036] Acinetobacter pituitus ( Acinetobacter pittii ) TJ2 was deposited on December 10, 2024 in the General Microbiology Center of the China Culture Collection Administration, abbreviated as CGMCC, with its address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33016.
[0037] Example 3 Strain expansion culture (1) Inoculate strain TJ2 into 50 mL LB medium at 30°C with a shaker at 150 rpm. Cultivate the bacterial seed solution until the logarithmic growth phase, with an OD600 of 0.6-0.8. (2) Inoculate the seed solution into fresh LB medium at a volume ratio of 1:10, incubate at 30°C, shake at 150 rpm, and culture the strain to an OD600 of 1.2-1.5. (3) Centrifuge the expanded cultured strain at 5000 rpm for 10 minutes at 4°C, discard the supernatant, and collect the bacterial precipitate; (4) Wash the cells twice with sterile saline (0.9% NaCl solution), then resuspend them in PBS and adjust the concentration of the bacterial suspension to 10 9 CFU / mL, and the phenol-degrading bacterial agent was obtained.
[0038] Example 4 Detection of strain TJ2's tolerance and degradation of medium and high concentrations of phenol Phenol was added to the basal medium to adjust the final concentrations of phenol to 1000 mg / L, 2000 mg / L, and 2500 mg / L, respectively; The obtained phenol-degrading bacterial agent was inoculated into a basal culture medium containing different concentrations of phenol at an inoculum size of 5%; The culture was carried out in a shaker at 30°C and 180 rpm for 72 h. The phenol concentration was measured at 6 h, 12 h, 24 h, and 48 h. The results are shown in Tables 1-3 and Figure 3-Figure 5 shown.
[0039] Table 1 Phenol tolerance and degradation effect of strain TJ2 at an initial concentration of 1000 mg / L
[0040] Table 2 Phenol tolerance and degradation effect of strain TJ2 at an initial concentration of 1500 mg / L
[0041] Table 3 Phenol tolerance and degradation effect of strain TJ2 at an initial concentration of 2000 mg / L
[0042] From Table 1 and Figure 3The results show that strain TJ2 exhibited exceptionally high degradation capacity at an initial concentration of 1000 mg / L. After 48 hours, the phenol concentration had dropped to 5.76 ± 2.89 mg / L, with a degradation rate of 99.42%, virtually complete. This demonstrates that strain TJ2 possesses excellent tolerance and degradation performance at 1000 mg / L.
[0043] From Table 2 and Figure 4 The results show that strain TJ2 maintained significant degradation capabilities even at an initial concentration of 1500 mg / L. After 48 hours, the phenol concentration had dropped to 191.30 ± 39.02 mg / L, with a degradation rate of 87.25%. This demonstrates that strain TJ2 maintained excellent tolerance and degradation performance at 1500 mg / L.
[0044] From Table 3 and Figure 5 The results show that at an initial concentration of 2000 mg / L, the degradation rate was 4.54% after 6 hours, and the difference was not statistically significant. However, as time continued, the degradation rate reached 76.54% at 48 hours. This result suggests that although the high concentration of phenol initially inhibited strain TJ2 to some extent, resulting in lower degradation efficiency, strain TJ2 possessed excellent tolerance and adaptability, ultimately achieving excellent degradation results in a relatively short period of time.
[0045] Example 5 Detection of strain TJ2's tolerance to low temperature Phenol was added to the basal medium to adjust the final concentration of phenol to 1000 mg / L; The obtained phenol-degrading bacterial agent was inoculated into a basic culture medium containing phenol at an inoculum size of 5%; The basal culture medium of the inoculated strain was cultured in a low-temperature incubator at 10°C, 15°C and 20°C, at 180 rpm for 48 h, and the concentration of phenol was measured. The results are shown in Table 4 and Figure 6 shown.
[0046] Table 4 Phenol degradation effect of strain TJ2 under different treatment temperatures
[0047] From Table 4 and Figure 4The results show that the phenol degradation efficiency of the strain decreases with decreasing treatment temperature. At 20°C, the degradation rate reached 87.83%, while at 15°C and 10°C, the degradation rates dropped to 67.27% and 41.14%, respectively. This indicates that low temperatures significantly inhibit the metabolic activity of microorganisms, but the strain provided by the present invention maintains a high degradation capacity under low temperature conditions, significantly outperforming existing common strains. This characteristic makes it of great application value in industrial wastewater treatment under low-temperature environments.
[0048] Example 6 Detection of strain TJ2's tolerance to high salt Phenol was added to the basal medium to adjust the final concentration of phenol to 1000 mg / L, and basal medium containing 2%, 4%, and 6% NaCl was also set up; The obtained phenol-degrading bacterial agent was inoculated into a basic culture medium containing phenol at an inoculum size of 5%; The basal culture medium of the inoculated strain was cultured in a shaker at 30°C and 180 rpm for 48 h, and the concentration of phenol was measured. The results are shown in Table 5 and Figure 5 shown.
[0049]
[0050] From Table 5 and Figure 7 The results show that strain TJ2 exhibited significant salt tolerance under treatment with different concentrations of NaCl while maintaining a high phenol degradation efficiency. Specifically: Even after 48 hours of cultivation at 2% NaCl, strain TJ2 maintained a phenol degradation rate of 96.34%. This result demonstrates that low concentrations of NaCl have no significant effect on the phenol degradation ability of strain TJ2, indicating that the strain's metabolic activity and degradation performance are very stable in a low-salt environment.
[0051] While strain TJ2's degradation capacity decreased under high-salt conditions of 4% and 6% NaCl, it still demonstrated excellent tolerance. Under 4% NaCl treatment, the degradation rate reached 74.07% after 48 hours, while under 6% NaCl, the degradation rate remained at 55.71%. This demonstrates that despite the inhibitory effect of high-salt conditions on the strain's metabolic activity, strain TJ2 was still able to adapt and effectively degrade phenol, demonstrating strong salt tolerance. This outstanding salt tolerance gives strain TJ2 significant potential for treating saline industrial wastewaters, such as coking and chemical wastewater.
Claims
1. A strain for degrading phenol-contaminated wastewater, characterized in that: The strain is Acinetobacter pituitus ( Acinetobacter pittii ) TJ2 was deposited on December 10, 2024 in the General Microbiology Center of the China Culture Collection Administration, abbreviated as CGMCC, with its address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33016.
2. The strain according to claim 1, characterized in that The 16S rRNA gene sequence of the strain is shown as SEQ ID NO.
1.
3. The strain according to claim 2, characterized in that The strain has the ability to tolerate medium and high initial phenol concentrations, low temperature tolerance and high salt tolerance.
4. The strain according to claim 3, characterized in that The medium-high concentration initial phenol concentration is 1000 mg / L-2000 mg / L; the low temperature tolerance temperature is 10°C-20°C; and the high salt tolerance is 2%-6% NaCl.
5. Use of the strain according to any one of claims 1 to 4 in degrading phenol-containing wastewater.
6. A method for treating phenol-containing wastewater, characterized in that: The processing method comprises the following steps: (1) Acinetobacter pituitus ( Acinetobacter pittii ) was inoculated into phenol-containing wastewater; (2) Degradation treatment of phenol-containing wastewater is carried out at a temperature of 10-30°C.
7. The processing method according to claim 6, characterized in that The strain concentration in the phenol-containing wastewater is ≥10 8 CFU / mL.
8. Use of a strain of Acinetobacter pitei in preparing a degrading agent for degrading phenol-contaminated wastewater, characterized in that: The Acinetobacter Pittii is Acinetobacter Pittii ( Acinetobacter pittii )TJ2, the deposit number is CGMCC No.33016.
9. The use according to claim 8, characterized in that In the degrading bacterial agent, the Acinetobacter pituitus ( Acinetobacter pittii ) TJ2 strain concentration ≥10 8 CFU / mL; The dosage of the degradation bacterial agent in phenol-contaminated wastewater is 5%-10%.
10. The use according to claim 9, characterized in that The degradation bacterial agent is a degradation bacterial agent for low-temperature and high-salt phenol-contaminated wastewater; The low temperature is 10-20° C., and the high salt concentration is 2%-6% NaCl.