Pseudomonas nitroreducens ZKJ and application thereof in degrading organic pollutants

Abstract

The application discloses a nitrate-reducing pseudomonas ZKJ and application thereof in degradation of organic pollutants. The nitrate-reducing pseudomonas ZKJ has high degradation efficiency on linalyl acetate pollutants, and can convert the pollutants into harmless substances such as CO2 and H2O, thus having wide application prospects in biological purification of industrial waste gas and waste water. The nitrate-reducing pseudomonas ZKJ can completely degrade linalyl acetate into inorganic substances and cell biomass, realizes mineralization, and the removal rate of linalyl acetate within 216 mg / L is higher than 95%.

Description

(I) Technical Field

[0001] This invention relates to a nitrate-reducing Pseudomonas nitritireducens ZKJ and its application in the degradation of organic pollutants such as linalyl acetate. (II) Background Technology

[0002] Linaloyl acetate is an organic compound found in natural bergamot, lavender, salvia miltiorrhiza, and other essential oils. It is also found in cocoa seeds, celery, grapes, peaches, and kelp. It is obtained by esterification of linalool with a mixture of acetic anhydride and phosphoric acid (the phosphoric acid and acetic anhydride form a complex catalyst) at a relatively low temperature. After esterification, the product is washed with water, then with brine until neutral, dried with anhydrous sodium carbonate, and then fractionated under reduced pressure. This yields a product with the highest ester content (≥95%) and a purer aroma. Alternatively, linalool can be added to solvent-diluted acetic anhydride and anhydrous sodium acetate for esterification, followed by fractional distillation. It is a colorless, transparent liquid with a pleasant floral and fruity aroma. It is volatile, slightly soluble in water, insoluble in glycerol, slightly soluble in water, and soluble in ethanol, propylene glycol, ether, diethyl phthalate, benzyl benzoate, non-volatile oils, and mineral oil fragrances. It is chemically stable and does not change color, and is often used in mid- to high-end perfume products and soap fragrances.

[0003] Although linalyl acetate is classified as low-toxicity, its volatility still poses a risk to human health. Acute exposure to high concentrations of linalyl acetate can cause symptoms such as skin allergies, eye inflammation, and respiratory tract inflammation.

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

[0005] The purpose of this invention is to provide a nitrate-reducing Pseudomonas ZKJ bacterium and its application in the degradation of organic pollutants. This nitrate-reducing Pseudomonas ZKJ can degrade linaloyl ester pollutants, such as linalyl acetate, using it as the sole carbon source. It exhibits particularly high efficiency and strong linalyl acetate degradation capacity, and its growth environment is mild, making it easy to scale up. The discovery of this degrading bacterium is of great significance for the efficient purification of linaloyl ester pollutants in industrial wastewater and exhaust gases.

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

[0007] This invention provides a novel linaloacetate-degrading bacterium—nitrate-reducing Pseudomonas nitritireducens ZKJ, deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M2023672, deposit date: May 4, 2023, address: Wuhan University, Wuhan, China, 430072.

[0008] The basic characteristics of the nitrate-reducing Pseudomonas ZKJ of this invention are as follows: the colonies are pale yellow, smooth, and without flagella. The edges are relatively regular, opaque, and easy to pick up; the bacterial growth generally follows the streaks, and a halo is evenly distributed around the colonies; it is aerobic and Gram-negative.

[0009] The present invention also provides an application of the aforementioned nitrate-reducing Pseudomonas ZKJ in the degradation of organic pollutants. Specifically, the application involves adding the bacterial culture or resting cells of nitrate-reducing Pseudomonas ZKJ obtained through expanded culture to an inorganic culture medium containing organic pollutants at pH 4-9, and culturing at 15-30℃ and 100-200 rpm to achieve the degradation of organic pollutants.

[0010] Furthermore, the organic pollutants include linalool acetate and toluene.

[0011] Furthermore, the initial concentration of organic pollutants in the inorganic salt culture medium is 54-270 mg / L, preferably 108 mg / L.

[0012] Furthermore, the amount of resting nitrate-reducing Pseudomonas ZKJ cells added is 20-80 mg / L on a dry weight basis, preferably 50 mg / L; the concentration is determined by the OD of the bacterial culture. 600 The value is calculated to be 0.01-0.05, with 0.02 being preferred.

[0013] Furthermore, the culture conditions were 15℃ and 160rpm.

[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 cells of the nitrate-reducing Pseudomonas ZKJ were prepared according to the following steps:

[0016] (1) Slant culture:

[0017] Nitrate-reducing Pseudomonas ZKJ was inoculated onto LB slant medium and cultured at 30°C for 24–36 h to obtain slant cells. The final concentration of the LB slant 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] The slant cells obtained in step (1) were picked up with an inoculation loop and inoculated into LB liquid medium. The culture was carried out at 15°C and 160 rpm for 24–36 h to obtain a bacterial suspension with OD600 = 0.1–0.2. The suspension was centrifuged, the wet cells were collected, and the suspension was washed with inorganic salt culture medium to obtain Pseudomonas ZKJ resting cells. The final concentration of the LB liquid medium was: NaCl 10 g / L, peptone 10 g / L, yeast extract 5 g / L, deionized water as solvent, and natural pH.

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

[0021] The nitrate-reducing Pseudomonas ZKJ provided by this invention is taken from sewage sludge from wastewater treatment plants. It has a high efficiency in degrading linalool pollutants 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 nitrate-reducing Pseudomonas ZKJ strain described in this invention can completely degrade linalyl acetate into inorganic substances (CO2, H2O) and cell biomass, achieving complete mineralization, and the removal rate of linalyl acetate up to 216 mg / L is higher than 95%. (iv) Description of the attached drawings

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

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

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

[0026] Figure 4 The degradation curves of strain ZKJ for different concentrations of linalool acetate are shown.

[0027] Figure 5 The degradation rate of 108 mg / L linalool acetate by strain ZKJ at different pH values ​​is shown.

[0028] Figure 6 The degradation rate of 108 mg / L linalool acetate by strain ZKJ at different pH values ​​at 22 h is given. (V) Detailed Implementation

[0029] 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:

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Example 1: Isolation, purification and identification of strain ZKJ.

[0035] 1. Isolation and purification of strain ZKJ.

[0036] Strain ZKJ is a Gram-negative bacterium that was domesticated and isolated from activated sludge collected from a municipal wastewater treatment plant. The specific steps are as follows:

[0037] Add 50 mL of inorganic salt culture medium to a 300 mL shake flask, then add 10 mL of activated sludge and 54 mg / L linalyl acetate. Seal the flask and incubate at 15 °C and 160 rpm for enrichment. The linalyl acetate concentration is determined using the method described in Example 3. When the linalyl acetate concentration reaches 50% of the initial concentration, 5 mL of the enriched solution is taken and added to 50 mL of fresh inorganic salt culture medium. The same amount of linalyl acetate (54 mg / L) is added, and the enrichment process is repeated 5 times. Finally, the last enriched solution is serially diluted with sterile water to a concentration of 10. -6 The culture medium was spread on LB solid medium and incubated at 15°C for 1-2 days. Single colonies were selected and inoculated again onto LB solid medium for isolation and plate streaking purification. The selected candidate bacteria were inoculated into an inorganic salt culture medium with 54 mg / L linalyl acetate as the sole carbon and energy source and incubated at 15°C for 2 days. The concentration of linalyl acetate was detected using the method in Example 3. The candidate bacteria corresponding to the culture medium with the highest linalyl acetate degradation rate were selected and recorded as strain ZKJ.

[0038] 2. Identification of strain ZKJ

[0039] (1) Characteristics of strain ZKJ: Strain ZKJ was inoculated onto LB solid medium and cultured at 15°C for 1 day. Colony photos are shown below. Figure 1 The colonies are pale yellow, disc-shaped, with neat edges, opaque, and easy to pick up. (Transmission electron microscopy) Figure 2 The morphology of the bacteria observed under light was elliptic, without flagella, measuring 4600×3500 nm in size, and Gram-negative.

[0040] (2) 16S rRNA sequence analysis

[0041] DNA from strain ZKJ 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 result (SEQ ID NO.1) was uploaded to NCBI, obtaining accession number OQ829420. This sequence was also compared with gene sequences in the NCBI database using BLAST. It was found to belong to the genus *Pseudomonas*, and showed 99% homology with the 16Sribosomal RNA gene of *Pseudomonas entomophila str. L48chromosome*, *Pseudomonas hamedanensis* strain SWRI65chromosome, and *Pseudomonas scrudulactis* strain UCMA17988. From the results, 10 representative *Pseudomonas* 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 *Pseudomonas nitritireducens*.

[0042] 16S rRNA sequence

[0043] 1tggggagcttgctcccggattcagcggcggacgggtgagtaatgcctaggaatctgcctgg

[0044] 61tagtgggggacaacgtttcgaaaggaacgctaataccgcatacgtcctacgggagaaagc

[0045] 121aggggaccttcgggccttgcgctatcagatgagcctaggtcggattagctagttggtgggg

[0046] 181taaaggcctaccaaggcgacgatccgtaactggtctgagaggatgatcagtcacactgga

[0047] 241actgagacacggtccagactcctacgggaggcagcagtggggaatattggacaatgggcg

[0048] 301aaagcctgatccagccatgccgcgtgtgtgaagaaggtcttcggattgtaaagcacttta

[0049] 361agttgggaggaagggcagtaagttaataccttgctgttttgacgttaccaacagaataag

[0050] 421caccggctaacttcgtgccagcagccgcggtaatacgaagggtgcaagcgttaatcggaa

[0051] 481ttactgggcgtaaagcgcgcgtaggtggtttggtaagatggatgtgaaatccccgggctc

[0052] 541aacctgggaactgcatccataactgcctgactagagtacggtagagggtggtggaatttc

[0053] 601ctgtgtagcggtgaaatgcgtagatataggaaggaacaccagtggcgaaggcgaccacct

[0054] 661ggactgatactgacactgaggtgcgaaagcgtggggagcaaacaggattagataccctgg

[0055] 721tagtccacgccgtaaacgatgtcgactagccgttgggatccttgagatcttagtggcgca

[0056] 781gctaacgcgataagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaat

[0057] 841tgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaacc

[0058] 901ttacctggccttgacatgtccggaatcttgcagagatgcgagagtgccttcgggaatcgg

[0059] 961aacacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgt

[0060] 1021aacgagcgcaacccttgtccttagttaccagcacgttatggtgggcactctaaggagact

[0061] 1081gccggtgacaaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacggcca

[0062] 1141gggctacacacgtgctacaatggtcggtacagagggttgccaagccgcgaggtggagcta

[0063] 1201atcccataaaaccgatcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcgga

[0064] 1261atcgctagtaatcgtgaatcagaatgtcacggtgaatacgttcccgggccttgtacacac

[0065] 1321cgcc.

[0066] (3) The ability of strain ZKJ to utilize 47 carbon sources on bioMérieux GN card.

[0067] The metabolic activity of strain ZKJ to 47 different carbon sources was investigated using a bioMérieux 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, strain ZKJ showed strong utilization of 11 carbon sources, but could not utilize the other 36.

[0068] Table 1. Biochemical reaction results of strain ZKJ using the VITEK automated identification system (GN card).

[0069]

[0070]

[0071] Note: + indicates a positive reaction; - indicates a negative reaction.

[0072] Based on colony morphology, 16S rRNA sequence analysis, and physiological and biochemical experiments, strain ZKJ was identified as Pseudomonas nitritireducens and named Pseudomonas nitritireducens ZKJ. It is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, China, accession number: CCTCC NO: M 2023672, deposit date: May 4, 2023.

[0073] Example 2: Obtaining resting cells of nitrate-reducing Pseudomonas ZKJ

[0074] 1. Slant culture:

[0075] Nitrate-reducing Pseudomonas ZKJ 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. 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.

[0076] 2. Expand training

[0077] The slant cells from step 1 were inoculated into LB liquid medium and cultured at 15°C and 160 rpm for 24–36 h to obtain a bacterial suspension with OD600 = 0.1–0.2. The suspension was centrifuged, the wet cells were collected, and the suspension was washed with inorganic salt culture medium to obtain resting cells of nitrate-reducing Pseudomonas ZKJ.

[0078] Example 3: Degradation performance of nitrate-reducing Pseudomonas ZKJ on linalool acetate at different concentrations.

[0079] 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). Linaloyl acetate was then added as the sole carbon source to achieve final concentrations of 54, 108, 162, 216, and 270 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 included. The concentration of linaloyl acetate in the gas phase above the shake flasks was determined using an Agilent gas chromatograph under the following conditions: HP-INNOWax Polyethylene Glycol column (30 m × 320 μm × 0.50 μm), column temperature 240 °C, and flow rate 0.5 mL / min. -1 The injection port temperature was 250℃, the split ratio was 40:1, the detector was a flame ionization detector (FID) at 250℃, and the hydrogen flow rate was 40 mL / min. -1 Air flow rate 450 mL·min -1 The injection volume was 0.8 mL. The concentration of residual linalyl acetate in the shake flask was measured periodically, and the removal rate curves of linalyl acetate at different initial concentrations for the strain over time were plotted. The results are shown in [Figure number missing]. Figure 4 As shown in the figure. The results indicate that when the concentration of linaloacetate is below 216 mg / L, strain ZKJ can rapidly degrade all added substrates; when the concentration of linaloacetate is 270 mg / L, strain ZKJ achieves a degradation rate of approximately 80% of the substrate within the tested time.

[0080] Example 4: Degradation performance of nitrate-reducing Pseudomonas ZKJ on 108 mg / L linalool acetate under different initial pH conditions.

[0081] 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. Bacterial suspensions prepared according to the method in Example 2 were inoculated under the condition that the initial linaloacetate concentration was 108 mg / L. The initial bacterial suspension concentrations in each parallel sample were expressed as OD0. 600 The value was calculated to be 0.02. The shake flasks were sealed, and the samples were cultured in a constant temperature shaker at 15°C and 160 rpm, with a blank control without bacteria. The concentration of residual linalyl acetate in the shake flasks was measured periodically using the method described in Example 3. Removal rate curves of linalyl acetate over time under different pH conditions were plotted, as well as the degradation rate of 108 mg / L linalyl acetate at different pH conditions at 22 h. The results are shown in [Figure Number]. Figure 5 and Figure 6As shown in the figure. The results indicate that Pseudomonas ZKJ can degrade linalyl acetate at various pH values, with the best degradation effect observed at pH 7 and 8.

[0082] Example 5: Degradation performance of nitrate-reducing Pseudomonas ZKJ on different organic compounds.

[0083] The 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, linaloacetate, toluene, chlorobenzene, and dichloromethane were added as the sole carbon source to achieve a final concentration of 80 mg / L, respectively. 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 concentrations of the four contaminants in the gas phase of the shake flasks were determined using the Agilent gas chromatography method described in Example 3.

[0084] The results are shown in Table 2. It can be seen that, in addition to being able to degrade linalyl acetate, Pseudomonas ZKJ has a certain ability to degrade toluene, but it has almost no degradation effect on chlorinated substrates such as chlorobenzene and dichloromethane.

[0085] Table 2 Test results of various substrates

[0086] Linaloyl acetate 4.8 94 Toluene 37.6 53 chlorobenzene 72 10 dichloromethane 73.6 8

[0087] 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. Nitrate-reducing Pseudomonas ( Pseudomonas nitritireducens ZKJ, deposited at the China Center for Type Culture Collection, accession number: CCTCC NO: M 2023672, deposit date: May 4, 2023, address: Wuhan University, Wuhan, China, 430072, China.

2. The application of the nitrate-reducing Pseudomonas ZKJ as described in claim 1 in the degradation of organic pollutants, characterized in that, The organic pollutants are linaloacetate and toluene.

3. The application as described in claim 2, characterized in that, The application involves adding the bacterial culture or resting cells of nitrate-reducing Pseudomonas ZKJ obtained through expanded culture to an inorganic salt culture medium containing organic pollutants at pH 4-9, and culturing at 15-30℃ and 100-200 rpm to achieve the degradation of organic pollutants.

4. The application as described in claim 3, characterized in that, The initial concentration of linaloacetate in the inorganic salt culture medium was 54-270 mg / L.

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

6. The application as described in claim 3, characterized in that, The amount of resting nitrate-reducing Pseudomonas ZKJ cells added was 20-80 mg / L on a dry weight basis.

7. The application as described in claim 3, characterized in that, The resting cells of the nitrate-reducing Pseudomonas ZKJ were prepared according to the following steps: (1) Slant culture: Nitrate-reducing Pseudomonas ZKJ was inoculated onto LB slant medium and cultured at 30°C for 24–36 h to obtain slant cells. The final concentration of the LB slant 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 The bacterial suspension was diluted to 0.1-0.2 g / L, centrifuged, and the wet cells were collected. The cells were washed with inorganic salt medium to obtain resting nitrate-reducing Pseudomonas ZKJ cells. The final concentration of the LB liquid medium was: NaCl 10 g / L, peptone 10 g / L, yeast extract 5 g / L, with deionized water as the solvent and natural pH.

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