Aquatic microorganisms TLB and their application in degrading organic pollutants

Abstract

This invention discloses an aquatic microbacterium TLB and its application in degrading organic pollutants. The aquatic microbacterium TLB provided by this invention is derived from sewage sludge from wastewater treatment plants and exhibits highly efficient degradation effects on organic pollutants such as tert-butyl acetate, acetone, and benzene. It can relatively completely convert pollutants into harmless substances such as CO2 and H2O, thus showing broad application prospects in the biological purification of industrial waste gas and wastewater. The aquatic microbacterium TLB described in this invention can completely degrade tert-butyl acetate into inorganic matter and cellular biomass, achieving complete mineralization, and the removal rate of tert-butyl acetate up to 260 mg / L is as high as 100%.

Description

(I) Technical Field

[0001] This invention relates to an aquatic microbacterium TLB and its application in the degradation of organic pollutants such as tert-butyl acetate. (II) Background Technology

[0002] tert-butyl acetate, also known as tert-butyl ester, is an organic compound belonging to the aliphatic ester class. It is a colorless, transparent, flammable liquid with an ester-like odor. Tert-butyl acetate is volatile, insoluble in water, miscible with alcohols and ethers, and readily soluble in acetic acid and some other organic solvents. The synthesis of tert-butyl acetate generally involves the reaction of tert-butanol with acetic acid or acetic anhydride. Tert-butyl acetate is an excellent solvent for nitrocellulose and a shock-absorbing additive in gasoline, with a wide range of applications.

[0003] Although tert-butyl acetate is classified as low-toxicity, its high volatility and lipid solubility allow it to accumulate in the body and produce neurotoxicity, thus classifying it as a highly dangerous poison. Tert-butyl acetate is a neurotoxin that can cause nerve fiber degeneration. Acute inhalation of high concentrations of tert-butyl acetate can cause symptoms such as nosebleeds, hoarseness, cough, chest tightness, headache, and dizziness. Eye and skin contact is irritating, and repeated long-term exposure may cause rashes. Oral ingestion can cause nausea, vomiting, and bronchial and gastrointestinal irritation; severe cases can lead to central respiratory depression. Ingestion of approximately 50g can be fatal to humans.

[0004] Therefore, it is necessary to study the efficient degradation of tert-butyl acetate in the environment for human health. However, a literature search did not find any reports on the efficient degradation of Aquamicrobium lusatiense using tert-butyl acetate as the sole carbon source. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a strain of aquamicrobium lusatiense (TLB) that degrades organic pollutants such as tert-butyl acetate, and its application in degrading organic pollutants. This strain can degrade organic pollutants such as tert-butyl acetate using them as the sole carbon source, and it grows in a mild environment, making it easy to scale up. It exhibits a strong removal capacity for organic pollutants and can efficiently degrade them. This is of great significance for the efficient purification of aliphatic ester pollutants in industrial wastewater and exhaust gases.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a novel tert-butyl acetate degrading bacterium—Aquamicrobium lusatiense TLB, deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 2023953, deposit date: June 6, 2023, address: Wuhan University, Wuhan, China, postcode 430072.

[0008] The basic characteristics of the aquatic microbacterium TLB provided by this invention are: the colonies are orange, disc-shaped, non-spore-forming, and non-flagellated; the edges are neat, opaque, easy to pick up, and the bacterial growth follows the streaks; it is aerobic and Gram-positive.

[0009] The present invention also provides an application of the aquatic microbacterium TLB in the degradation of organic pollutants. The application involves adding the bacterial culture obtained by expanding the culture of the aquatic microbacterium TLB or the resting cells obtained by centrifuging the bacterial culture to an inorganic salt culture medium containing organic pollutants at pH 5-9, and culturing it at 10-20℃ and 100-200rpm to achieve the degradation of organic pollutants.

[0010] Furthermore, the organic pollutants are tert-butyl acetate, acetone, benzene, and toluene.

[0011] Furthermore, in the inorganic salt culture medium, the amount of resting cells added is 20-80 mg / L based on the dry weight of the cells, preferably 50 mg / L; the amount of bacterial solution added is 0.01-0.1 based on the OD value, preferably 0.02.

[0012] Furthermore, the initial concentration of organic pollutants in the inorganic salt culture medium is 52-260 mg / L, preferably 104 mg / L.

[0013] Furthermore, the culture conditions are: 15℃, 160rpm reaction; the pH of the inorganic salt culture medium is preferably 5-9.

[0014] Furthermore, the inorganic salt culture medium comprises: K₂HPO₄·3H₂O 0.942 g / L, KH₂PO₄ 0.234 g / L, NaNO₃ 1.7 g / L, NH₄Cl 0.98 g / L, MgCl₂·6H₂O 0.2033 g / L, CaCl₂·2H₂O 0.011 g / L, FeCl₃ 0.0162 g / L, and a trace element stock solution of 5 ml / L, with deionized water as the solvent and pH 7.0; wherein the trace element stock solution comprises: CuSO₄·5H₂O 0.02 g / L, FeSO₄·7H₂O 1.0 g / L, MnSO₄·4H₂O 0.1 g / L, NaMoO₄·2H₂O 0.02 g / L, CoCl₂·6H₂O 0.02 g / L, H₃BO₃ 0.014 g / L, ZnSO4·7H2O 0.10 g / L, solvent is deionized water.

[0015] Furthermore, the resting TLB cells of the aquatic microorganisms were prepared according to the following steps:

[0016] (1) Slant culture:

[0017] The aquatic microorganism TLB was inoculated onto LB solid medium slant and cultured at 30°C for 24–36 h to obtain slant cells. The final concentration of the LB solid medium was: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, agar 18–20 g / L, deionized water as solvent, and natural pH.

[0018] (2) Expanded cultivation:

[0019] Using an inoculation loop, pick up the slant cells obtained in step (1) and inoculate them into LB liquid medium. Incubate at 15°C and 160 rpm for 24–36 h to obtain OD. 600 The bacterial suspension with a concentration of 0.1-0.2 was centrifuged, and the wet bacterial cells were collected. The cells were washed with inorganic salt culture medium to obtain resting TLB cells of the aquatic microorganism. The final concentration of the LB liquid culture medium was: NaCl 10g / L, peptone 10g / L, yeast extract 5g / L, with deionized water as the solvent and natural pH.

[0020] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0021] The aquatic microbacterium TLB provided by this invention is taken from sewage sludge from wastewater treatment plants. It has a highly efficient degradation effect on organic pollutants such as tert-butyl acetate and can completely convert pollutants into harmless substances such as CO2 and H2O. Therefore, it has broad application prospects in the biological purification of industrial waste gas and wastewater.

[0022] The aquatic microbacterial TLB described in this invention can completely degrade tert-butyl acetate into inorganic substances (CO2, H2O) and cellular biomass, achieving complete mineralization. Furthermore, it achieves a 100% removal rate for tert-butyl acetate concentrations up to 260 mg / L. In addition, this aquatic microbacterial TLB also exhibits certain degradation effects on acetone, benzene, toluene, and other substances. (iv) Description of the attached drawings

[0023] Figure 1 This is a photograph of the colony morphology of strain TLB on LB medium.

[0024] Figure 2 This is a transmission electron microscope image of strain TLB.

[0025] Figure 3 This is a phylogenetic tree diagram of strain TLB.

[0026] Figure 4 The degradation curves of strain TLB for different concentrations of tert-butyl acetate are shown.

[0027] Figure 5 The degradation curves of strain TLB for 104 mg / L tert-butyl acetate at different pH values ​​are shown. (V) Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] The inorganic salt culture medium consisted of: K₂HPO₄·3H₂O 0.942 g / L, KH₂PO₄ 0.234 g / L, NaNO₃ 1.7 g / L, NH₄Cl 0.98 g / L, MgCl₂·6H₂O 0.2033 g / L, CaCl₂·2H₂O 0.011 g / L, FeCl₃ 0.0162 g / L, and a trace element stock solution of 5 ml / L. The solvent was deionized water, and the pH was 7.0. The trace element stock solution consisted of: CuSO₄·5H₂O 0.02 g / L, FeSO₄·7H₂O 1.0 g / L, MnSO₄·4H₂O 0.1 g / L, NaMoO₄·2H₂O 0.02 g / L, CoCl₂·6H₂O 0.02 g / L, and H₃BO₃. 0.014 g / L, ZnSO4·7H2O 0.10 g / L, solvent is deionized water.

[0031] The final concentration of the LB solid culture medium is as follows: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, agar 18-20 g / L, deionized water as solvent, and natural pH value.

[0032] The final concentration of the LB liquid culture medium is: NaCl 10g / L, peptone 10g / L, yeast extract 5g / L, with deionized water as the solvent and a natural pH value.

[0033] Example 1: Isolation, purification and identification of strain TLB.

[0034] 1. Isolation and purification of strain TLB.

[0035] Strains TLB are Gram-positive bacteria that were domesticated and isolated from activated sludge collected from a municipal wastewater treatment plant. The specific steps are as follows:

[0036] Add 50 mL of inorganic salt culture medium to a 300 mL shake flask, along with 10 mL of activated sludge and 30 mg / L tert-butyl acetate. Incubate at 15 °C. When the tert-butyl acetate concentration reaches 50% of the initial concentration, take 5 mL of the enriched solution and add it to 50 mL of fresh inorganic salt culture medium. Add the same amount of tert-butyl acetate (30 mg / L). Repeat the enrichment process 5 times. Finally, serially dilute the last enriched solution with sterile water. -5The diluted bacterial suspension was spread onto LB agar and incubated at 15°C for 24 hours. Single colonies were then streaked onto LB agar and incubated at 15°C for isolation and purification. Figure 1 The selected bacteria were added to an inorganic salt culture medium, and 30 mg / L tert-butyl acetate was added as the sole carbon and energy source. The cultures were incubated at 15°C and 160 rpm for 36 h. The degradation rate of tert-butyl acetate was detected using the method described in Example 3. The target strain with the highest degradation rate was screened and designated as strain TLB.

[0037] 2. Identification of strain TLB

[0038] (1) Characteristics of strain TLB: Colonies are orange, disc-shaped; edges are neat, opaque, and easy to pick up. Under transmission electron microscopy, the bacteria are observed to be elliptic bacilli, without flagella, and Gram-positive. Its morphology was confirmed by transmission electron microscopy. Figure 2 ).

[0039] (2) Analysis of 16S rRNA sequence

[0040] DNA from bacterial strain TLB was extracted and purified using the Ezup column-based bacterial genomic DNA extraction kit and stored at 4°C. The purified DNA was amplified by PCR using universal primers for bacteria: 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). The PCR reaction program was set as follows: 94°C pre-denaturation for 4 min, followed by 94°C denaturation for 45 s, 55°C annealing for 45 s, and 72°C extension for 1 min, for 30 cycles, and a final 72°C repair extension for 10 min. The PCR product was purified and recovered, and then sequenced (Zhejiang Tianke High-Tech Development Co., Ltd. (formerly Zhejiang Institute of Microbiology)). The 16S rRNA sequencing results (nucleotide sequence as shown in SEQ ID NO.1) were uploaded to NCBI, obtaining accession number OR144353. This sequence was also compared with gene sequences in the NCBI database using BLAST. It was found to belong to the genus *Aquamicrobium*, and showed 99% homology with *Aquamicrobium lusatiense* strain S1, *Aquamicrobium defluvii* strain DSM 11603, and *Aquamicrobiuma erolatum* strain Sa14. From the results, 10 representative *Aquamicrobium* strains were selected, and a phylogenetic tree was constructed using MEGA 7.0 software based on 16S rRNA gene sequence homology. Figure 3 Based on genetic distance and 16S rRNA sequence comparison, it was identified as *Aquamicrobium lusatiense*.

[0041] (3) The ability of strain TLB to utilize 63 carbon sources on bioMérieux GN card.

[0042] The metabolic activity of the strain to 63 different carbon sources was investigated using the bioMérieux fully automated identification system (contracted to Zhejiang Tianke High-Tech Development Co., Ltd. (formerly Zhejiang Institute of Microbiology)). The identification results are shown in Table 1. Using the VITEK biochemical reaction system of the bioMérieux fully automated identification system, strain TLB showed strong utilization of 10 carbon sources, but could not utilize the other 53 carbon sources.

[0043] Table 1. Biochemical reaction results of strain TLB using the VITEK automated identification system (GN card) on bioMérieux.

[0044]

[0045]

[0046]

[0047] Note: + indicates a positive reaction; - indicates a negative reaction.

[0048] The strain was identified as *Aquamicrobium lusatiense* TLB through 16S rRNA sequence analysis and physiological and biochemical experiments. It is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 2023953, deposit date: June 6, 2023, address: Wuhan University, Wuhan, China, 430072, China.

[0049] Example 2: Obtaining resting TLB cells from aquatic microorganisms

[0050] 1. Slant culture:

[0051] The aquatic microorganism TLB was inoculated into LB liquid medium and cultured at 15°C and 160 rpm for 24–36 h. The activated bacteria were then streaked onto solid LB plates and incubated at 30°C for 24–36 h. Single colonies were taken and streaked again to test the purity of the bacteria. The bacteria were then stored in LB slant tubes at 4°C using standard procedures.

[0052] 2. Expand training

[0053] The slant culture cells from step 1 were inoculated into LB liquid medium and cultured at 15℃ and 160 rpm for 24–36 h to obtain OD. 600 Centrifuge the bacterial suspension at a concentration of 0.1–0.2, collect the wet cells, wash with inorganic salt culture medium, and obtain resting TLB cells of the aquatic microorganism.

[0054] Example 3: Degradation performance of aquatic microbacterium TLB on different concentrations of tert-butyl acetate.

[0055] Inorganic salt culture medium was dispensed into 300 mL shake flasks (50 mL per flask) and sterilized at 110 °C for 40 min. After sterilization, the flasks were left at room temperature for 2 days to confirm the absence of bacterial growth. Resting cells obtained in Example 2 were added to a final concentration of 50 mg / L (based on cell dry weight). Tert-butyl acetate was then added as the sole carbon source to final concentrations of 52, 104, 156, 208, and 260 mg / L. The shake flasks were sealed and incubated at 15 °C on a shaker at 160 rpm. A blank control without bacteria was also included. The residual tert-butyl acetate concentration in the shake flasks was measured every 24 h. Removal rate curves of different initial tert-butyl acetate concentrations over time were plotted. The results are shown in [Figure number missing]. Figure 4 As shown.

[0056] Gas chromatographic column parameters for the determination of tert-butyl acetate: HP-INNOWax Polyethylene Glycol (30m × 320μm × 0.50μm); column temperature: 120℃, column pressure: 5.68psi, column flow rate: 0.8mL·min -1 Inlet temperature 210℃, split ratio 18:1, carrier gas:nitrogen:carrier gas flow rate: 15.0 mL·min -1 Pressure: 5.68 psi; Detector: Flame Ionization Detector (FID), 230 °C, Hydrogen Flow Rate: 40 mL / min -1 Air flow rate: 450 mL / min -1 Tail purge: 45 mL / min -1 Injection volume: 0.8 mL.

[0057] The results showed that when the concentration of tert-butyl acetate was below 260 mg / L, strain TLB could rapidly degrade all added substrates.

[0058] Example 4: Degradation performance of 104 mg / L tert-butyl acetate by aquatic microbacterium TLB under different initial pH conditions.

[0059] The inorganic salt culture medium was adjusted to different pH values ​​(4.0, 5.0, 6.0, 7.0, 8.0, 9.0) using 1 mol / L NaOH or 1 mol / L H2SO4 aqueous solution. The bacterial culture prepared according to the method in Example 2 was inoculated under the condition that the initial bacterial concentration in each parallel sample was 104 mg / L. 600The value was calculated to be 0.02. The sample was cultured in a constant temperature shaker at 15℃ and 160 rpm, with a blank control without bacteria. The concentration of residual tert-butyl acetate in the shake flask was measured periodically using the method described in Example 3. The removal rate curves of 104 mg / L tert-butyl acetate by the strain under different pH conditions over time were plotted. The results are shown in [Figure 1]. Figure 5 As shown in the figure. The results indicate that the aquatic microorganism TLB can degrade tert-butyl acetate at pH 5 and above, and the degradation effect is the worst at pH 4, where it is almost impossible to degrade tert-butyl acetate.

[0060] Example 5: Degradation performance of water microbacteria TLB on other pollutants.

[0061] Inorganic salt culture medium was dispensed into 300 mL shake flasks (50 mL per flask) and sterilized at 110°C for 40 min. After sterilization, the flasks were left at room temperature for 2 days to confirm the absence of bacterial growth. Resting cells obtained in Example 2 were added to a final concentration of 50 mg / L (based on cell dry weight). Then, common industrial contaminants such as tert-butyl acetate, acetone, benzene, toluene, chlorobenzene, and dichloromethane were added as the sole carbon source, each at a concentration of 100 mg / L. The shake flasks were sealed and incubated at 15°C and 160 rpm on a shaker. A blank control without bacteria was also prepared. The residual concentrations of tert-butyl acetate, acetone, benzene, toluene, chlorobenzene, and dichloromethane in the shake flasks were measured periodically.

[0062] Gas chromatographic column parameters for the determination of tert-butyl acetate, acetone, benzene, and toluene: HP-INNOWax Polyethylene Glycol (30m × 320μm × 0.50μm); column temperature: 120℃ for tert-butyl acetate and acetone, 90℃ for benzene and toluene; column pressure: 5.68 psi; column flow rate: 0.8 mL·min -1 Inlet temperature 210℃, split ratio 18:1, carrier gas:nitrogen:carrier gas flow rate: 15.0 mL·min -1 Pressure: 5.68 psi; Detector: Flame Ionization Detector (FID), 230 °C, Hydrogen Flow Rate: 40 mL / min -1 Air flow rate: 450 mL / min -1 Tail purge: 45 mL / min -1 Injection volume: 0.8 mL. Gas chromatographic column parameters for the determination of chlorobenzene and dichloromethane: J&W122-5531 capillary column (30 m × 0.25 mm × 0.1 μm), carrier gas N2 (20 mL / min), injector temperature 250 °C, GC column oven temperature program 50 °C, increased to 80 °C at 5 °C / min, then increased to 100 °C at 20 °C / min, held for 2 minutes, and injected in splitless mode (0.5 min).

[0063] The results are shown in Table 2. The results indicate that strain TLB can rapidly degrade tert-butyl acetate, and has a certain degradation effect on acetone, benzene, and toluene, but has almost no degradation effect on chlorobenzene and dichloromethane.

[0064] Table 2. Degradation effect of aquatic microorganisms TLB on different pollutants.

[0065]

[0066]

[0067] Note: "+" indicates the degree of degradation; more "+" indicates easier degradation, and "-" indicates no degradation.

[0068] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the scope of protection of the present invention. Any modifications and refinements made by those skilled in the art without departing from the concept and scope of the present invention shall fall within the scope of protection of the present invention.

Claims

1. Aquatic microbes Aquamicrobium lusatiense TLB, characterized in that, It is deposited at the China Center for Type Culture Collection, accession number: CCTCC NO: M 2023953, deposit date: June 6, 2023, deposit address: Wuhan University, Wuhan, China, 430072, China.

2. The aquatic microbacteria of claim 1 Aquamicrobium lusatiense The application of TLB in the degradation of organic pollutants is characterized by, The organic pollutants are tert-butyl acetate, acetone, benzene, and toluene.

3. The application as described in claim 2, characterized in that, The application described is to use aquatic microorganisms Aquamicrobium lusatiense The bacterial suspension obtained by expanding TLB culture or the resting cells obtained by centrifuging the bacterial suspension are added to an inorganic salt culture medium containing organic pollutants at pH 5-9, and cultured at 15-20℃ and 100-200rpm to achieve the degradation of organic pollutants.

4. The application as described in claim 3, characterized in that, In the inorganic salt culture medium, the amount of resting cells added is 20-80 mg / L based on the dry weight of the cells; the amount of bacterial solution added is 0.01-0.1 based on the OD600 value; and the initial concentration of the organic pollutant, tert-butyl acetate, is 52-256 mg / L.

5. The application as described in claim 3, characterized in that, The culture conditions were: 15℃ and 160 rpm.

6. The application as described in claim 3, characterized in that, The water microbacteria Aquamicrobium lusatiense TLB resting cells were prepared according to the following steps: (1) Slant culture: Water microbacteria Aquamicrobium lusatiense TLB was inoculated onto LB solid medium slant and cultured at 30℃ for 24-36 h to obtain slant cells; the final concentration of the LB solid medium was: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, agar 18-20 g / L, deionized water as solvent, and natural pH. (2) Expanded cultivation: Using an inoculation loop, pick up the slant cells obtained in step (1) and inoculate them into LB liquid medium. Incubate at 15°C and 160 rpm for 24–36 h to obtain OD. 600 Centrifuge the bacterial suspension at a concentration of 0.1-0.2 mg / L, collect the wet bacterial cells, wash with inorganic salt culture medium, and obtain aquatic microbes. Aquamicrobium lusatiense TLB resting cells; the final concentration of the LB liquid culture medium is: NaCl 10 g / L, peptone 10 g / L, yeast extract 5 g / L, deionized water as solvent, and natural pH value.

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