Klebsiella hy-99b and its use

Abstract

This invention discloses Klebsiella HY-99B and its applications. This strain is currently deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, China, on September 20, 2022, with accession number CCTCC No: M 20221448. Klebsiella HY-99B can utilize organic pollutants such as chlorobenzene as its sole carbon and energy source for growth and reproduction, mineralizing these pollutants into CO2 and H2O. Under pure culture conditions, the degradation efficiency of chlorobenzene by this bacterium is above 85% within a pH range of 6.0–9.0 and a temperature range of 20–35℃. This strain has a wide growth temperature and pH range and strong environmental adaptability, providing strong support for the engineering application of biological purification of chlorobenzene waste gas. Furthermore, this strain can utilize toluene, ethylbenzene, and o-dichlorobenzene as its sole carbon and energy source to varying degrees for growth and degradation of these substrates.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Klebsiella HY-99B and its applications. Background Technology

[0002] Chlorobenzene is a colorless, transparent liquid that is highly toxic, chemically stable, and difficult to degrade, with a bitter almond odor. As a chemical solvent, chlorobenzene is mainly used in flavorings, herbicides, pesticides, and as a chemical intermediate in the production of dyes and pharmaceuticals. Due to its wide range of applications, it is widely used in industry.

[0003] Chlorobenzene possesses characteristics of both benzene and chlorine compounds, contributing to photochemical smog and PM2.5 formation. Due to its persistent nature, it remains in the environment for extended periods, causing severe pollution to surface water, soil, and the atmosphere. Furthermore, it accumulates in animals and plants, gradually damaging organs such as the liver and kidneys, and has an anesthetic effect on the nervous system. Exposure to high concentrations of chlorobenzene can cause acute poisoning, leading to anesthetic symptoms such as headache, dizziness, weakness, and even coma. It is irritating to the skin and mucous membranes; long-term exposure can cause chronic poisoning, resulting in skin erythema or mild superficial necrosis, and neurasthenia symptoms such as headache, loss of appetite, insomnia, conjunctival congestion, and memory loss, posing a serious threat to human health. Therefore, developing efficient, green, and safe methods for chlorobenzene degradation is urgently needed.

[0004] Biological treatment technology features high purification efficiency, no secondary pollution, and low operating costs. Microorganisms are highly adaptable to various environments and can handle multi-component organic pollutants under complex conditions. Therefore, for the microbial degradation of chlorobenzene, highly efficient degrading strains are crucial. Previous researchers have screened Klebsiella bacteria for efficient degradation of nitrobenzene compounds, but no reports, either domestically or internationally, have documented the screening of Klebsiella bacteria that achieve efficient degradation of chlorobenzene using chlorobenzene as the sole carbon source. If a Klebsiella bacterium could be screened to achieve efficient degradation of chlorobenzene using chlorobenzene as the sole carbon source, it would not only effectively solve the problem of chlorobenzene biodegradation but also provide another new example of efficient VOCs treatment using Klebsiella bacteria.

[0005] This invention screened a highly efficient degrading strain of chlorobenzene from the environment, providing strong support for bioremediation engineering to treat pollutants containing this type of contaminant. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for Klebsiella HY-99B and its application.

[0007] This invention is specifically achieved through the following technical solutions:

[0008] The first aspect of this invention provides Klebsiella sp. HY-99B, which is deposited at the China Center for Type Culture Collection (CCTCC) on September 20, 2022, with accession number CCTCC No: M 20221448, located at Wuhan University, Wuhan, China, 430072, China.

[0009] The second aspect of the present invention provides the application of the above-mentioned Klebsiella HY-99B in the degradation of organic pollutants.

[0010] Furthermore, the organic pollutant is chlorobenzene, toluene, ethylbenzene, or o-dichlorobenzene.

[0011] A third aspect of the present invention provides a method for degrading organic pollutants using the above-mentioned Klebsiella HY-99B, characterized by comprising the following steps:

[0012] 1) Use the bacterial culture obtained by expanding Klebsiella HY-99B as an enzyme source;

[0013] 2) Using organic pollutants as substrates, degradation reactions are carried out in inorganic salt culture media at pH 3–9 and 20℃–40℃ to achieve the degradation of organic pollutants.

[0014] Furthermore, step 1) specifically includes the following steps:

[0015] a. Slant culture: Klebsiella HY-99B was inoculated onto LB slant solid medium and cultured at 30℃ for 24-36 h to obtain slant cells;

[0016] b. Expanded Culture: Using an inoculation loop, pick up the slant cells obtained in step 1) and inoculate them into LB liquid medium. Incubate at 30°C for 24–36 h to obtain OD. 600 =0.1~0.2% bacterial solution.

[0017] Furthermore, in step 1), the amount of enzyme source used is based on the OD content of the inorganic salt culture medium. 600 The value is calculated to be 0.01 to 0.5.

[0018] Furthermore, the organic pollutant in step 2) is chlorobenzene, toluene, ethylbenzene, or o-dichlorobenzene.

[0019] Furthermore, in step 2), the initial concentration of organic pollutants in the inorganic salt culture medium is 20–1600 mg / L.

[0020] Furthermore, the final concentration of the inorganic salt culture medium in step 2) is as follows: Na2HPO4·12H2O 4.5g / L, KH2PO4 1.0g / L, (NH4)2SO4 2.5g / L, MgSO4·7H2O 0.2g / L, anhydrous CaCl2 0.023g / L, trace element stock solution 1mL / L, pH 7.0, and the solvent is deionized water;

[0021] The trace element mother liquor concentration composition is as follows: FeSO4·7H2O 1.0 g / L, CuSO4·5H2O 0.02 g / L, H3BO3 0.014 g / L, MnSO4·4H2O 0.10 g / L, ZnSO4·7H2O 0.10 g / L, Na2MoO4·2H2O 0.02 g / L, CoCl2·6H2O 0.02 g / L, with deionized water as the solvent.

[0022] Furthermore, the final concentration composition of the LB slant solid medium is: NaCl 10g / L, tryptone 10g / L, yeast extract 5g / L, agar 18-20g / L, with deionized water as the solvent and a natural pH value; the final concentration composition of the LB liquid medium is: NaCl 10g / L, tryptone 10g / L, yeast extract 5g / L, with deionized water as the solvent and a natural pH value.

[0023] This invention provides a highly efficient degrading bacterium for organic pollutants, particularly chlorobenzene—Klebsiella HY-99B. This Gram-negative bacterium can grow and reproduce using organic pollutants such as chlorobenzene as its sole carbon and energy source, and efficiently degrade these substrates. The initial bacterial count (expressed as OD...) is... 600 (Calculation) is only for OD 600 At a concentration of 0.02, this strain can completely degrade organic pollutants such as chlorobenzene up to 660 mg / L. It exhibits a wide growth temperature and pH range, showing good degradation of chlorobenzene within the range of 20–35℃ and pH 6.0–9.0, with a degradation rate exceeding 85% after 22 hours (reaching over 99% at 25–30℃ and pH 7.0–8.0). This has significant implications for the engineering application of biological methods for chlorobenzene purification. Furthermore, Klebsiella HY-99B can also degrade toluene, o-dichlorobenzene, and ethylbenzene to varying degrees. Attached Figure Description

[0024] Figure 1 Transmission electron micrograph of Klebsiella HY-99B;

[0025] Figure 2 Phylogenetic tree;

[0026] Figure 3 The graph shows the cell growth and chlorobenzene degradation curves of Klebsiella HY-99B.

[0027] Figure 4 Bar graph showing the effect of different pH culture media on chlorobenzene degradation (A), growth (B), and mineralization (C) of Klebsiella HY-99B;

[0028] Figure 5 Bar graph showing the effect of different temperatures on the chlorobenzene degradation (A) and mineralization (B) of Klebsiella HY-99B;

[0029] Figure 6 Degradation curves of chlorobenzene at different initial concentrations by Klebsiella HY-99B;

[0030] Figure 7 The growth curves of Klebsiella HY-99B at different initial chlorobenzene concentrations are shown.

[0031] Figure 8 To simulate the inlet and outlet concentrations and removal rates of chlorobenzene waste gas treated by a bio-trickling filter;

[0032] Figure 9 To simulate the inlet and outlet concentrations and removal rates of mixed waste gas treated by a bio-trickling filter. Detailed Implementation

[0033] 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:

[0034] The inorganic salt culture medium used in the examples consisted of: Na₂HPO₄·12H₂O 4.5 g / L, KH₂PO₄ 1.0 g / L, (NH₄)₂SO₄ 2.5 g / L, MgSO₄·7H₂O 0.2 g / L, anhydrous CaCl₂ 0.023 g / L, trace element stock solution 1 mL / L, pH 7.0, and deionized water as the solvent. The trace element stock solution consisted of: FeSO₄·7H₂O 1.0 g / L, CuSO₄·5H₂O 0.02 g / L, H₃BO₃ 0.014 g / L, MnSO₄·4H₂O 0.10 g / L, ZnSO₄·7H₂O 0.10 g / L, Na₂MoO₄·2H₂O 0.02 g / L, CoCl₂·6H₂O 0.02 g / L, and deionized water as the solvent.

[0035] The LB solid culture medium consists of: 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.

[0036] The LB liquid culture medium consists of: NaCl 10g / L, tryptone 10g / L, yeast extract 5g / L, deionized water as solvent, and natural pH.

[0037] The room temperature refers to 25-30℃.

[0038] Example 1: Isolation, purification and identification of Klebsiella HY-99B

[0039] (1) Isolation and purification of Klebsiella HY-99B

[0040] Activated sludge was collected from a wastewater treatment pond of a petrochemical company in Zhejiang Province. The lower layer of sludge after settling was mixed with inorganic salt culture medium at a ratio of 1:2 (v / v). 3L of the mixture was added to a 5L sludge acclimatization tank (see Jin Xiaojun. Isolation, Identification, Degradation Characteristics and Preparation of New Dioxane-Degrading Strains [D]. Zhejiang University of Technology, 2012.). 500mg / L of chlorobenzene substrate was added daily as the sole carbon and energy source. The acclimatization culture was carried out at room temperature. After nearly 20 days, 5mL of sludge was taken from the acclimatization tank and added to a shake flask containing 50mL of inorganic salt. It was found that the acclimatized sludge could stably degrade 108mg / L of chlorobenzene per day in the shake flask, with a degradation rate of 80-90% (shake flask experimental conditions: 30℃, 160r / min). The acclimatized sample was obtained.

[0041] Sludge samples of 1 and 2 mL were transferred to other shake flasks for further performance testing. It was found that the transferred sludge could stably degrade 108 mg / L chlorobenzene per day in the shake flasks. This sludge sample was then further transferred and enriched for 6 generations (1 mL each time). The sludge enriched for 6 generations was then processed at 10... -1 ~10 -6 The bacteria were spread on LB solid medium plates at multiples, and single colonies were picked. Then, using chlorobenzene at a final concentration of 108 mg / L as a substrate, the degradation activity was determined. After isolation and purification, a strain HY-99B with chlorobenzene degradation activity was obtained.

[0042] (2) Identification of strain HY-99B

[0043] Morphological characteristics of strain HY-99B (e.g.) Figure 1 As shown): The bacteria are short rods, measuring (0.85–0.91) μm × (1.27–1.49) μm; they are non-spore-forming, non-flagellated, and have a relatively thick capsule; the colonies are round, milky white, opaque, plump, smooth, and moist, and are easy to pick up, with the bacterial growth following the streaks.

[0044] The physiological and biochemical characteristics of strain HY-99B are: aerobic, Gram-negative.

[0045] PCR amplification and sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd. The 16S rDNA sequence of the strain (SEQ ID NO.1) is as follows (Genebank accession number OP389117):

[0046]

[0047]

[0048] The 16S rDNA sequence of strain HY-99B was uploaded to the Ezbiocloud.net website and compared with standard strains on the website. A phylogenetic tree was constructed using the Neighbor-Joining method with MEGA 5.05 software, and evaluated using the Bootstrap method (repeated 1000 times). The constructed phylogenetic tree is shown below. Figure 2 Thus, the strain HY-99B was identified as Bacillus and named Klebsiella sp. HY-99B. It is deposited at the China Center for Type Culture Collection (CCTCC) on September 20, 2022, with accession number CCTCC No: M 20221448, located at Wuhan University, Wuhan, China, 430072, China.

[0049] Example 2: Preparation of seed culture of Klebsiella HY-99B

[0050] 1) Slant culture: Klebsiella HY-99B was inoculated onto LB slant solid medium and cultured at 30℃ for 18-36 h to obtain slant cells.

[0051] 2) Seed culture: Use an inoculation loop to pick up the slant cells obtained in step 1) and inoculate them into LB liquid medium. Incubate at 30℃ for 24–36 h to obtain OD. 600 =0.1~0.2% bacterial solution.

[0052] Example 3: Performance of Klebsiella HY-99B in degrading chlorobenzene

[0053] Using chlorobenzene as the sole carbon source for Klebsiella HY-99B, the OD prepared by the method in Example 2 was used. 600 =0.1 bacterial suspension, inoculated into 50 mL of fresh inorganic salt medium containing a final concentration of 108 mg / L chlorobenzene, so that the initial bacterial concentration is expressed as OD0.1. 600 The concentration was 0.02, and the pH was 7. The cells were incubated in a shaker at 30℃ and 160 rpm. Samples were taken every 5 hours to determine the chlorobenzene degradation rate. A portion of the bacterial culture was also extracted using a 5 mL syringe to determine the bacterial cell OD value. The results are shown below. Figure 3 During the experiment, two parallel samples and a blank control group without bacterial inoculation were designed. As time progressed, the bacterial concentration gradually increased, reaching a maximum of approximately 0.108 (OD0.05) at 24 hours. 600(Calculated), the degradation rate of chlorobenzene reaches over 99%. This example demonstrates that Klebsiella HY-99B can utilize chlorobenzene as its sole carbon and energy source for growth and reproduction, and possesses a stable and efficient ability to degrade chlorobenzene.

[0054] Example 4: Effect of initial pH of inorganic salt culture medium on the degradation of chlorobenzene by Klebsiella HY-99B

[0055] The inorganic salt culture medium was adjusted to different pH values ​​(3.0, 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 suspension prepared according to the method in Example 2 was inoculated under the condition that the initial chlorobenzene concentration was 108 mg / L, so that the initial bacterial concentration in each parallel sample was expressed as an OD value. 600 The value was calculated to be 0.02. The sample was incubated at 30℃ and 160 rpm in a constant temperature shaker for 22 hours. Samples were taken after incubation, and the chlorobenzene degradation rate and OD value of the bacterial culture were measured. 600 The experiment included (calculation) and CO2 values. Two to three parallel samples and one blank control group without inoculation were designed. Results were as follows: Figure 4 As shown, Klebsiella HY-99B exhibits a high degradation rate of chlorobenzene within the pH range of 6–9; especially at pH = 7–8, the degradation rate of chlorobenzene by Klebsiella HY-99B reaches its optimal level (above 99%). Simultaneously, the growth rate of Klebsiella HY-99B and the CO2 production also show a trend consistent with the chlorobenzene degradation rate (the lower CO2 production at pH = 9 may be due to the absorption of some CO2 by the alkaline solution).

[0056] Example 5: Effect of temperature on the degradation of chlorobenzene by Klebsiella HY-99B

[0057] In an inorganic salt culture medium with an initial chlorobenzene concentration of 108 mg / L, the bacterial suspension prepared according to the method in Example 2 was inoculated, so that the initial bacterial concentration in each parallel sample was expressed as OD0. 600 The concentration was 0.02, and the pH was 7. Each sample was placed in a shaker at 20℃, 25℃, 30℃, 35℃, and 40℃ for constant temperature shaking (shaking speed 160 r / min). After 24 h of incubation, samples were taken to measure the chlorobenzene degradation rate and OD value in the reaction solution. Two parallel samples and one blank control group without inoculation were designed during the experiment. Figure 5 It can be seen that Klebsiella HY-99B has a high degradation rate of chlorobenzene in the temperature range of 25-35℃; especially in the temperature range of 25-30℃, the degradation rate of chlorobenzene by Klebsiella HY-99B reaches the best (above 99%). At the same time, the growth of Klebsiella HY-99B and the CO2 value produced also show a trend consistent with the degradation rate of chlorobenzene.

[0058] Example 6: Effect of substrate concentration on the degradation of chlorobenzene by Klebsiella HY-99B

[0059] The degradation of chlorobenzene by Klebsiella HY-99B strain was studied under suitable culture conditions (pH = 7, t = 30℃). Different concentrations of chlorobenzene substrate were added to fresh inorganic salt medium to achieve initial concentrations of 110, 220, 330, 440, 550, and 660 mg / L. Bacterial suspensions prepared according to the method in Example 2 were then inoculated, and the initial bacterial concentrations in each parallel sample were determined by OD0.05. 600 The value was calculated as 0.02, with no bacterial inoculation as a blank. The bacteria were cultured in a shaker at 30℃ and 160 rpm, and samples were taken periodically to determine the OD of Klebsiella HY-99B. 600 Regarding the concentration of chlorobenzene, the experiment included two parallel samples and a blank control group without inoculation. The final results are shown below. Figure 6 and Figure 7 ,Depend on Figure 6 It is known that Klebsiella HY-99B can almost completely convert chlorobenzene to CO2 and H2O after 56 hours at a chlorobenzene concentration of 440 mg / L. However, at a chlorobenzene concentration of 660 mg / L, it takes 80 hours for Klebsiella HY-99B to completely degrade chlorobenzene. The degradation rate of chlorobenzene by Klebsiella HY-99B decreases with increasing chlorobenzene concentration, and the degradation time also increases. Figure 7 It can be seen that when the degradation rate of Klebsiella HY-99B reaches its highest level at different chlorobenzene concentrations, the bacterial count also tends to stabilize.

[0060] Example 7: Degradation capacity of Klebsiella HY-99B for different carbon source substrates

[0061] The Klebsiella HY-99B bacterial suspension prepared according to the method in Example 2 was inoculated into other substrates except chlorobenzene (concentrations shown in Table 1) to achieve an initial bacterial concentration expressed as OD0.05. 600 The concentration was 0.02, pH = 7, and t = 30℃. This strain was found to have varying degrees of degradation ability for toluene, o-dichlorobenzene, and ethylbenzene, as detailed in Table 1 below.

[0062] Table 1: Degradation capacity of HY-99B for different carbon sources

[0063]

[0064] Example 8: Simulated biological trickling filter treatment of chlorobenzene waste gas inlet and outlet concentrations and removal rates

[0065] OD prepared by the method in Example 2 600Klebsiella HY-99B bacterial suspension with a concentration of 0.2 g / L was inoculated into a biotrickling filter (see Zhang Dingfeng, Fang Junyi, Ye Jiexu, et al. Study on purification of multi-component waste gas by biotrickling filter [J]. Environmental Science, 2013.), with an inoculation volume of 2 L, a temperature of 30 °C, and a nutrient solution (i.e., inorganic salt culture medium) spray rate of 6 L·h. -1 The nutrient solution was changed every 2 days, and the pH was adjusted using a 0.5 mol / L NaOH aqueous solution to maintain it between 6 and 8. Raschig rings were used as the packing material. The residence time during biofilm formation was 36 seconds, and the chlorobenzene inlet gas concentration was 200 mg / m³. 3 .

[0066] After 20 days of biofilm formation, the chlorobenzene removal rate reached over 92%, as shown in the results. Figure 8 As shown. On day 21, mixed waste gas was started to be added (the mixed waste gas composition was 100 mg / m³). 3 Toluene, 100 mg / m 3 o-Dichlorobenzene, 100 mg / m 3 The removal rates of ethylbenzene and chlorobenzene can reach over 90%, while the removal rates of toluene and ethylbenzene can reach over 90%, and the removal rate of o-dichlorobenzene can reach over 80%. The results are as follows... Figure 9 As shown in A, B, C, and D.

Claims

1. Klebsiella pneumoniae ( Klebsiella sp.) HY-99B, its accession number is: CCTCC No: M 20221448.

2. The application of Klebsiella HY-99B as described in claim 1 in the degradation of organic pollutants, wherein the organic pollutants are chlorobenzene, toluene, ethylbenzene or o-dichlorobenzene.

3. The method for degrading organic pollutants using Klebsiella HY-99B according to claim 1, wherein the organic pollutant is chlorobenzene, toluene, ethylbenzene, or o-dichlorobenzene, characterized in that, Includes the following steps: 1) Use the bacterial culture obtained by expanding Klebsiella HY-99B as an enzyme source; 2) Using organic pollutants as substrates, degradation reactions are carried out in inorganic salt culture media at pH 3–9 and 20℃–40℃ to achieve the degradation of organic pollutants.

4. The method as described in claim 3, characterized in that, Step 1) specifically includes the following steps: a. Slant culture: Klebsiella HY-99B was inoculated onto LB slant solid medium and cultured at 30℃ for 24-36 h to obtain slant cells; b. Expanded Culture: Using an inoculation loop, pick up the slant cells obtained in step a and inoculate them into LB liquid medium. Incubate at 30°C for 24–36 h to obtain OD. 600 =0.1~0.2% bacterial solution.

5. The method as described in claim 3 or 4, characterized in that, In step 1), the amount of enzyme source used is based on the OD content in the inorganic salt culture medium. 600 The value is calculated to be 0.01 to 0.

5.

6. The method as described in claim 3 or 4, characterized in that, In step 2), the initial concentration of organic pollutants in the inorganic salt culture medium is 20–1600 mg / L.

7. The method as described in claim 3 or 4, characterized in that, The final concentration of the inorganic salt culture medium in step 2) is as follows: Na2HPO4·12H2O 4.5g / L, KH2PO4 1.0g / L, (NH4)2SO4 2.5g / L, MgSO4·7H2O 0.2g / L, anhydrous CaCl2 0.023g / L, trace element stock solution 1mL / L, pH 7.0, and deionized water as the solvent. The concentration of the trace element mother liquor Composition: FeSO4·7H2O 1.0g / L, CuSO4·5H2O 0.02g / L, H3BO3 0.014g / L, MnSO4·4H2O 0.10g / L, ZnSO4·7H2O 0.10g / L, Na2MoO4·2H2O 0.02g / L, CoCl2·6H2O 0.02g / L, solvent is deionized water.

8. The method as described in claim 4, characterized in that, The final concentration of the LB slant solid medium is: NaCl 10g / L, tryptone 10g / L, yeast extract 5g / L, agar 18-20g / L, with deionized water as the solvent and a natural pH value; the final concentration of the LB liquid medium is: NaCl 10g / L, tryptone 10g / L, yeast extract 5g / L, with deionized water as the solvent and a natural pH value.

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