A salt-tolerant triethylamine-degrading bacterial strain and its agent and its application in chemical wastewater treatment

Abstract

The present invention discloses a salt-resistant triethylamine-degrading bacterial strain and its bacterial agent and its application in chemical wastewater treatment. The bacterial strain is Mycobacterium ( Mycobacterium sp. ), classified as Mycobacterium sp. CY-1, deposited in China Center for Type Culture Collection on April 16, 2025, with the deposit number: CCTCC NO: M 2025797. The strain of the present invention Mycobacterium sp. CY‑1 can use triethylamine as the sole carbon and nitrogen source to degrade triethylamine, and can achieve efficient degradation of high-concentration triethylamine in a high-salt environment with excellent degradation efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a salt-resistant triethylamine-degrading bacterial strain and a bacterial agent thereof, and application thereof in chemical wastewater treatment. Background Art

[0002] Triethylamine (TEA) is widely used in the chemical, pharmaceutical, and dye industries as an acid-binding agent, polymerization inhibitor, catalyst, purification solvent, biocide, and preservative. However, due to its high volatility, irritation, and biotoxicity, it poses a serious threat to the environment and ecosystems. Therefore, the development of efficient TEA wastewater treatment technologies is urgently needed.

[0003] Currently, treatment methods for triethylamine wastewater primarily include physical methods (such as distillation, adsorption, and membrane separation), chemical methods (such as neutralization, oxidation, and coagulation), and biological treatment. Physical methods offer the advantages of simple operation, high treatment efficiency, and no secondary pollution. However, the high cost of adsorbents for adsorption, the high energy consumption of distillation, and the limitations of membrane fouling in membrane separation restrict their practical application for triethylamine wastewater treatment. Chemical methods primarily aim to remove and convert triethylamine. Neutralization removes triethylamine by adding acidic substances to triethylamine wastewater. However, this process inevitably produces high-salinity wastewater, increasing subsequent treatment costs. Oxidation removes triethylamine by adding strong oxidants to the wastewater, offering the advantages of simple operation and high efficiency. However, oxidation methods are costly, require strict control of reaction conditions, and may generate secondary pollution. Therefore, biological methods remain at the core of triethylamine wastewater treatment and, in general, water treatment.

[0004] Studies have reported that due to the toxicity of triethylamine to microorganisms, biological treatment of high-concentration triethylamine wastewater (>50 mg / L) remains unsatisfactory. Furthermore, traditional biological treatments have limited tolerance to salt, as excessive salt levels can inhibit microbial activity. It is important to note that triethylamine wastewater, widely used in the chemical industry, can contain significant amounts of salt, which can significantly inhibit and toxic microbial growth. Therefore, identifying a triethylamine-degrading strain with high degradation efficiency and strong environmental tolerance is of great value in improving the treatment rate of toxic and difficult-to-degrade triethylamine wastewater. Summary of the Invention

[0005] To address the deficiencies of the prior art, the present invention aims to provide a salt-tolerant triethylamine-degrading bacterial strain and a bacterial agent thereof, as well as applications thereof in chemical wastewater treatment. The degrading bacterial strain can use triethylamine as the sole carbon and nitrogen source to degrade triethylamine, and can efficiently degrade high-concentration triethylamine in a high-salt environment, with excellent degradation efficiency.

[0006] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0007] A salt-tolerant triethylamine-degrading strain, the strain is Mycobacterium ( Mycobacterium sp. ), classified as Mycobacterium sp. CY-1 was deposited in the China Center for Type Culture Collection on April 16, 2025, with the deposit number: CCTCC NO: M 2025797.

[0008] Preferably, the concentration of triethylamine in the aforementioned chemical wastewater is 0.5-2 g / L, the pH is 6.0-8.0, and the NaCl concentration is 0-40 g / L.

[0009] A bacterial agent produced by utilizing the above-mentioned degradation strain.

[0010] The invention relates to an application of the bacterial agent in degrading triethylamine in chemical wastewater, wherein the concentration of triethylamine in the chemical wastewater is 0.5-2 g / L, the pH is 6.0-8.0, and the NaCl concentration is 0-40 g / L.

[0011] A method for treating chemical wastewater with the above-mentioned bacterial agent comprises adding 2% to 6% of the volume ratio of the bacterial agent into the chemical wastewater for degradation treatment.

[0012] A method for preparing the above-mentioned bacterial agent comprises the following specific steps:

[0013] The triethylamine-degrading strain was picked from the LB plate, inoculated into the LB liquid culture medium, and cultured with shaking to obtain a bacterial agent.

[0014] Preferably, the components of the aforementioned LB medium are: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 5 g / L, sterilized by high-pressure steam at 121 ° C for 30 min, and culture conditions are: speed 200 rpm, culture temperature 25~35 ° C, and shaking culture for 48~60 h.

[0015] The present invention is beneficial in that: the strain of the present invention Mycobacterium sp. CY-1 can use triethylamine as the only carbon and nitrogen source to achieve the degradation of triethylamine, and can achieve efficient degradation of high-concentration triethylamine in a high-salt environment with excellent degradation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a strain Mycobacterium sp. Colony morphology of CY-1;

[0017] Figure 2 It is a strain Mycobacterium sp. Phylogenetic tree of CY-1;

[0018] Figure 3 Is pH effective for strains Mycobacterium sp. Effects of CY-1 growth;

[0019] Figure 4 The effect of NaCl concentration on the strain Mycobacterium sp. Effects of CY-1 growth;

[0020] Figure 5 It is a strain Mycobacterium sp. The degradation effect of CY-1 on triethylamine at different concentrations;

[0021] Figure 6 strains under different pH conditions Mycobacterium sp. The degradation effect of CY-1 on triethylamine;

[0022] Figure 7 The strains under different NaCl concentrations Mycobacterium sp. The degradation effect of CY-1 on triethylamine;

[0023] Figure 8 It is a strain Mycobacterium sp. Gas chromatography peak diagram of triethylamine in triethylamine wastewater after CY-1 degradation for 72 hours. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] Example 1, Isolation and identification of strains:

[0026] (1) Source of strain

[0027] The strains were screened from the biochemical treatment unit of a sewage treatment plant in an industrial park.

[0028] (2) Culture medium formula

[0029] The components of the enrichment medium are: triethylamine 0.5 g / L, Na2HPO4•12H2O 9 g / L, KH2PO4 1.5 g / L, and MgSO4•7H2O 0.2 g / L.

[0030] The components of LB liquid culture medium are: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, and the solvent is pure water.

[0031] (3) Isolation of strains

[0032] 1 mL of activated sludge was taken from the biochemical pool and inoculated into the enrichment medium; the culture was shaken in a shaker at 30°C and 200 rpm for 48 h; 1 mL of the cultured bacterial liquid was transferred to fresh enrichment medium, and the culture was continuously transferred 20 times according to the above culture conditions; the strains were isolated by the plate dilution and spreading method, and 100 μL of the culture liquid with different dilution gradients was respectively taken and spread onto an inorganic salt medium plate containing 1 g / L triethylamine, and cultured in a constant temperature incubator at 30°C for 72 h.

[0033] Through enrichment culture, separation and purification, a strain with triethylamine degradation function was finally screened out from the wastewater and named CY-1. Its colony morphology on LB solid medium is as follows Figure 1 As shown in the figure, the main physiological characteristics are: the colonies are light yellow, round, with clear edges, a slight bulge in the middle and a smooth surface.

[0034] (4) Identification of strains

[0035] The species of triethylamine-degrading bacteria were identified by bacterial 16S rDNA sequencing, the sequence of which is shown in SEQ ID NO. 1. The sequencing results were compared with the sequences in the NCBI website database for homology analysis, and a phylogenetic tree was constructed, see Figure 2 , the results showed that strain CY-1 belongs to the genus Mycobacterium ( Mycobacterium sp. ).

[0036] The above strain was deposited in China Center for Type Culture Collection on April 16, 2025, with the deposit number: CCTCCNO: M 2025797, and the classification name is Mycobacterium sp. CY-1, deposited at: Wuhan University, Wuhan, China.

[0037] Example 2: A method for preparing a microbial agent, comprising the following specific steps:

[0038] A colony of the triethylamine-degrading strain CY-1 was picked and inoculated into LB liquid medium. The medium was shaken at 200 rpm and 30°C for 48–60 h to obtain a triethylamine-degrading bacterial agent. The LB medium consisted of 5 g / L yeast extract, 10 g / L tryptone, and 5 g / L sodium chloride. The medium was sterilized by high-pressure steam at 121°C for 30 min.

[0039] Example 3: Effect of pH on bacterial growth

[0040] The triethylamine-degrading agent was inoculated into LB liquid medium at different pH values ​​at an inoculum size of 0.2%. The LB liquid medium consisted of 5 g / L yeast extract, 10 g / L tryptone, and 5 g / L sodium chloride. The pH was adjusted to 5, 6, 7, and 8, respectively, using 50% hydrochloric acid or 200 g / L sodium hydroxide. The culture was shaken at 200 rpm and 30°C for 48 h. The bacterial concentration (OD value) was measured at a wavelength of 600 nm. The results are shown in Table 1. Figure 3 .

[0041] pass Figure 3 It can be seen that the strain can grow in the pH range of 6 to 9. After 48 hours of culture, the cell density was the highest (OD600 nm = 3.79 ± 0.098) under neutral conditions (pH = 7), indicating that the strain CY-1 had the strongest metabolic activity and the best growth under this pH condition.

[0042] Example 4: Effect of NaCl concentration on strain growth

[0043] The triethylamine-degrading agent was inoculated into LB liquid medium with different pH values ​​at an inoculum size of 0.2%. The components of the LB liquid medium were: yeast extract 5 g / L, tryptone 10 g / L, pH 7. The sodium chloride concentration in the medium was adjusted to 0, 5, 10, 15, 20, 30, 40, and 50 g / L using 300 g / L sodium chloride solution. The culture was shaken at 200 rpm and 30°C for 48 h. The bacterial concentration (OD value) was measured at a wavelength of 600 nm. The results are shown in Figure 2. Figure 4 .

[0044] pass Figure 4 As can be seen, there was no significant difference in the biomass of the strain after 48 hours of incubation between sodium chloride concentrations of 0 and 15 g / L (p>0.05), indicating that the strain is highly adaptable within this range and that salinity fluctuations do not affect its normal growth. However, when the concentration exceeds 15 g / L, the growth of the strain is inhibited as the sodium chloride concentration increases. Notably, the strain can still grow at a sodium chloride concentration of 50 g / L, indicating its potential for adaptation to high-salinity wastewater.

[0045] Example 5: Degradation Effect of Triethylamine-Degrading Bacteria on Triethylamine at Different Concentrations

[0046] The triethylamine-degrading bacterial agent was inoculated at an inoculum size of 4% into an inorganic salt medium containing different concentrations of triethylamine (0.5, 1.0, 1.5, and 2.0 g / L). The inorganic salt medium composition was as follows: Na2HPO4•12H2O 9 g / L, KH2PO4 1.5 g / L, MgSO4•7H2O 0.2 g / L, H3BO3 0.3 g / L, CoCl2•6H2O 0.2 g / L, ZnSO4•7H2O 0.1 g / L, MnSO4•5H2O 0.03 g / L, (NH4)6Mo7O 24 •4H2O 0.03 g / L, NiCl2•6H2O 0.02 g / L, CuSO4•5H2O 0.01 g / L, pH 7, shake culture at 200 rpm, 30 ℃ for 48 h, and measure the concentration of triethylamine every 6 h. The results are shown in Figure 5 .

[0047] Depend on Figure 5 It can be seen that after inoculation with 4% triethylamine-degrading bacteria, 0.5-2 g / L of triethylamine can be completely degraded within 48 hours. This shows that the strain has strong tolerance to fluctuations in substrate concentration and has the potential to treat high-load industrial wastewater.

[0048] Example 6: Degradation effect of strain CY-1 on triethylamine under different pH conditions

[0049] The triethylamine-degrading bacteria agent was inoculated into an inorganic salt medium containing 1 g / L triethylamine at an inoculum size of 4%. The pH of the medium was adjusted to 5, 6, 7, and 8, respectively. The culture was shaken at 200 rpm and 30°C for 48 h. The concentration of triethylamine was measured and the degradation rate of triethylamine was calculated. The results are shown in Figure 6 .

[0050] Depend on Figure 6 It can be seen that when the pH is in the range of 6-8, the strain can completely remove 1 g / L triethylamine after 48 hours of cultivation, indicating that strain CY-1 can maintain efficient degradation of triethylamine in a neutral to weakly acidic / weakly alkaline environment (pH 6-8).

[0051] Example 7: Degradation Effect of Strain CY-1 on Triethylamine at Different NaCl Concentrations

[0052] The triethylamine-degrading bacterial agent was inoculated into an inorganic salt medium containing 1 g / L triethylamine at an inoculum size of 4%. The NaCl concentration was adjusted to 0 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 30 g / L, and 40 g / L. The temperature was 30 °C, the pH was 7, and the shaking culture was carried out at 200 rpm for 72 h. The concentration of triethylamine was determined and the degradation rate of triethylamine was calculated. The results are shown in Table 2. Figure 7.

[0053] Depend on Figure 7 As shown, after 48 hours of cultivation, the strain achieved a triethylamine removal efficiency exceeding 99% at sodium chloride concentrations ≤15 g / L. With increasing sodium chloride concentrations, the triethylamine removal efficiency decreased significantly. At sodium chloride concentrations of 20, 30, and 40 g / L, the strain achieved removal efficiencies of 86.7%, 75.4%, and 52.1% for 1 g / L of triethylamine within 48 hours, respectively. However, after extending the cultivation time to 72 hours, the strain was able to completely remove triethylamine in a sodium chloride concentration range of ≤30 g / L. Even at higher salinity levels (40 g / L), strain CY-1 demonstrated excellent triethylamine degradation (>98%). Although the high-salt environment inhibited the strain's degradation efficiency of triethylamine in the short term (48 hours), the strain was still able to completely degrade triethylamine after a longer period of time, indicating that the strain may have a salt-tolerant adaptation mechanism. Although the high-salt environment (≤30 g / L) affects the strain's degradation rate of triethylamine, it does not completely block its ability to degrade triethylamine.

[0054] Example 8: Degradation effect of strain CY-1 on triethylamine in actual wastewater

[0055] Using the influent from a chemical park wastewater treatment plant as the background solution (pH 7.8 ± 0.3, salinity 1.2%), triethylamine was added to 1.0 g / L, and sodium chloride was added to a salinity of 3.0% to simulate actual industrial wastewater containing triethylamine. A triethylamine-degrading bacterial agent was inoculated into 100 mL of wastewater at a 4% inoculum. A control group was inoculated with 4% activated sludge from the biochemical treatment unit of the chemical park wastewater treatment plant. The cultures were shaken at 30°C and 200 rpm for 48 hours, and the triethylamine concentration was measured. The results are shown in Table 1. Figure 8 .

[0056] Depend on Figure 8 As can be seen, after inoculation with the inoculum, the experimental group was able to completely degrade triethylamine at a concentration of 1.0 g / L within 72 hours. In contrast, the triethylamine concentration in the control group only decreased slightly. This demonstrates that the strain can grow and reproduce in certain triethylamine-containing industrial wastewaters and effectively remove it. Therefore, this strain has great potential for application.

[0057] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of protection of the present invention.

Claims

1. A salt-tolerant triethylamine-degrading bacterial strain, characterized in that: The strain is Mycobacterium ( Mycobacterium sp. ), classified as Mycobacterium sp. CY-1 was deposited in the China Center for Type Culture Collection on April 16, 2025, with the deposit number: CCTCC NO: M 2025797.

2. Use of the degradation strain according to claim 1 in degrading triethylamine in chemical wastewater, characterized in that: The concentration of triethylamine in the chemical wastewater is 0.5-2 g / L, the pH is 6.0-8.0, and the NaCl concentration is 0-40 g / L.

3. A bacterial agent produced using the degradation strain according to claim 1.

4. Use of the bacterial agent according to claim 3 in degrading triethylamine in chemical wastewater, characterized in that: The concentration of triethylamine in the chemical wastewater is 0.5-2 g / L, the pH is 6.0-8.0, and the NaCl concentration is 0-40 g / L.

5. A method for treating chemical wastewater using the bacterial agent according to claim 3, characterized in that: Add 2% to 6% bacterial agent by volume into chemical wastewater for degradation treatment.

6. A method for preparing the microbial agent according to claim 3, characterized in that: The specific steps include: The triethylamine-degrading strain was picked from the LB plate, inoculated into the LB liquid culture medium, and cultured with shaking to obtain a bacterial agent.

7. The method for preparing the microbial agent according to claim 6, characterized in that: The LB medium contains 5 g / L yeast extract, 10 g / L tryptone, and 5 g / L sodium chloride, and is sterilized with high-pressure steam at 121°C for 30 min. The culture conditions are: a rotation speed of 200 rpm, a culture temperature of 25-35°C, and a shaking culture for 48-60 h.

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