Chitinophage CB-L with chlorobenzene degradation performance and application of chitophage CB-L

Abstract

The invention discloses Chitinophaga sp. CB-L with chlorobenzene degradation capacity and application of the Chitinophaga sp. CB-L. The Chitinophaga sp. CB-L has the preservation number of CCTCC NO: M 2025557, the Chitinophaga sp. CB-L is used for biodegradation of chlorobenzene, the Chitinophaga sp. CB-L can degrade chlorobenzene with the initial concentration of 200 mg.L <-1 > within 12 h after the adaptation period of 1-2 d, and the degrading bacterium has important significance on treatment of chlorobenzene in industrial waste water and waste gas.

Description

Chitin-eating bacteria CB-L with chlorobenzene degradation capabilities and their applications Technical Field

[0001] This invention belongs to the field of biological treatment technology for environmental pollutants, specifically relating to a chitinophaga sp. CB-L strain with chlorobenzene degradation capabilities and its applications. Background Technology

[0002] Chlorobenzene is a typical volatile organic compound, widely used as a solvent and intermediate in the chemical, pharmaceutical and pesticide industries. Its volatility and lipid solubility cause it to migrate and accumulate extensively in the atmosphere, water bodies, soil and biosphere.

[0003] Current research indicates that inhalation of chlorobenzene irritates the respiratory tract and may trigger lung inflammation. It has also been shown to induce hepatotoxicity, neurotoxicity, and carcinogenicity in mammalian systems. Furthermore, due to its high volatility and lipid solubility, chlorobenzene tends to persist and accumulate in the environment, posing a significant risk to ecosystem integrity.

[0004] Chlorobenzene removal technologies encompass emission source treatment and environmental remediation technologies. Emission source treatment technologies include adsorption, photochemical oxidation, incineration, bioremediation, and advanced oxidation technologies. Environmental remediation technologies include gas-phase thermal desorption remediation, bioremediation, and advanced oxidation technologies. Bioremediation technologies have demonstrated applicability in both emission control and environmental remediation scenarios. Compared to traditional methods, biological processes can convert toxic compounds into harmless final products (CO2 and H2O) through microbial mineralization, offering advantages in environmental friendliness and cost-effectiveness.

[0005] The known uses of chitinophagafiliformis are: 1. Chitinophagafiliformis YT5 (GDMCC No:62178) promotes arsenic methylation and volatilization.

[0006] 2. Chitinophaga oryzae Cas201 (CGMCC No:28361) has good inhibitory activity against Solanaceae Raulella, and also has the ability to produce hepatophiles and proteases, and can promote tobacco seed germination and tobacco plant growth.

[0007] 3. Chitinophagavarians C13 (GDMCC No:63479) can produce α-L-rhamnosidase, which can catalyze the hydrolysis of neohesperidin. Summary of the Invention

[0008] To address the aforementioned technical problems, the present invention aims to provide a chitinophaga sp. strain with chlorobenzene degradation capabilities, CB-L, and its applications.

[0009] The specific technical solution is as follows: On the one hand, the present invention provides a chitinophaga sp. CB-L, with accession number: CCTCC NO:M 2025557, accession date: March 21, 2025, depositary institution: China Center for Type Culture Collection; deposit address: Wuhan University, Wuhan, China.

[0010] The chitinous bacterium CB-L of this invention is characterized by: yellow colony color, small single colonies, translucent interior, moist surface, and smooth edges. Under transmission electron microscopy, the morphology of this bacterium is that of a bacillus. Its optimal growth pH is 7.0, and its optimal temperature is 30°C. The 16S rDNA sequence of this strain is shown in SEQ ID NO. 1.

[0011] On the other hand, the present invention also provides the application of chitosan CB-L in the biodegradation of chlorobenzene; that is, the use of chitosan CB-L is to biodegrade chlorobenzene.

[0012] Furthermore, the application is as follows: the seed liquid obtained after culturing chitinous bacteria CB-L seeds is centrifuged, washed and resuspended with inorganic salt solution, and inoculated into a liquid selective medium containing chlorobenzene. Chlorobenzene is used as a carbon source and cultured at 20-40℃ (preferably 30℃) and (160±30) rpm to achieve the degradation of chlorobenzene.

[0013] The final concentration of the chlorobenzene liquid selective medium is: Na₂HPO₄·12H₂O 4500 mg·L⁻¹ -1 KH2PO4 1000 mg·L -1 (NH4)2SO4 1800 mg·L -1 Chlorobenzene 50-500 mg·L -1 (preferably 100 mg·L) -1 ), MgSO4·7H2O 200mg·L -1 CuSO4·5H2O 0.02 mg·L -1 FeSO4·7H2O 1mg·L -1 H3BO3 0.02 mg·L -1 MnSO4·4H2O 0.1 mg·L -1 ZnSO4·4H2O 0.1 mg·L -1 Na₂MoO₄·2H₂O 0.02 mg·L⁻¹ -1 The solvent is ultrapure water.

[0014] Preferably, the bacterial suspension of the present invention is prepared according to the following steps: (1) Seed culture: a single colony of 1 inoculation loop is picked from the LB solid culture medium and inoculated into 50 mL of LB liquid culture medium (seed culture medium), and cultured at 30°C for 30 h to obtain seed liquid; the final concentration of the seed culture medium is: NaCl 10 g·L -1 5g·L yeast extract -1 10 g / L of peptone -1 The solvent is ultrapure water with a pH of 7.0; (2) Fermentation culture and acclimatization period of chlorobenzene: The seed culture is centrifuged and washed and resuspended with inorganic salt solution, inoculated into fermentation medium, and cultured at 30°C to obtain the fermentation culture medium containing bacteria; The final concentration composition of the fermentation medium is: Na2HPO4·12H2O4 500mg·L -1 KH2PO4 1000 mg·L -1 (NH4)2SO4 1800 mg·L -1 Chlorobenzene 100 mg·L -1 MgSO4·7H2O 200mg·L -1 CuSO4·5H2O 0.02 mg·L -1 FeSO4·7H2O 1mg·L -1 H3BO3 0.02 mg·L -1 MnSO4·4H2O 0.1 mg·L -1 ZnSO4·4H2O 0.1 mg·L -1 Na₂MoO₄·2H₂O 0.02 mg·L⁻¹ -1 The solvent is ultrapure water with a pH of 7.0. After the chlorobenzene has been completely degraded, it is used as a bacterial suspension for later use.

[0015] The beneficial effects of this invention are mainly reflected in the following: This invention provides a chitinogenic bacterium CB-L with chlorobenzene degradation capabilities and its application in degrading chlorobenzene. Currently, no chitinogenic bacterium species has been found to degrade chlorobenzene. After an adaptation period of 1-2 days, chitinogenic bacterium CB-L can degrade an initial concentration of 200 mg·L⁻¹ within 12 hours. -1 This invention provides a novel method for the treatment of chlorobenzene, specifically for screening usable bacterial strains to biodegrade chlorobenzene in industrial wastewater and exhaust gases. This method is of significant importance. Attached Figure Description

[0016] Figure 1 is a transmission electron microscope image of strain CB-L; Figure 2 is a phylogenetic tree of strain CB-L; Figure 3 is a growth curve of strain CB-L; Figure 4 is a comparison of the chlorobenzene degradation performance of strain CB-L at different pH values; Figure 5 is a comparison of the chlorobenzene degradation performance of strain CB-L at different temperatures; Figure 6 is a comparison of the chlorobenzene degradation performance of strain CB-L at different initial chlorobenzene concentrations. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0018] Example 1: Isolation, purification and identification of Chitinophaga sp. CB-L (1) Isolation and purification of Chitinophaga sp. CB-L Chitinophaga sp. CB-L was obtained by screening from the sludge of the wastewater treatment pond of a sewage treatment plant in Zhejiang Province. The specific steps are as follows: Chlorobenzene liquid basic culture medium: Na2HPO4·12H2O 4500mg·L -1 KH2PO4 1000 mg·L -1 (NH4)2SO4 1800 mg·L -1 MgSO4·7H2O 200mg·L -1 CuSO4·5H2O 0.02 mg·L -1 FeSO4·7H2O 1mg·L -1 H3BO3 0.02 mg·L -1 MnSO4·4H2O 0.1 mg·L -1 ZnSO4·4H2O 0.1 mg·L -1 Na₂MoO₄·2H₂O 0.02 mg·L⁻¹ -1 The solvent is ultrapure water, and the product is sterilized using conventional methods (i.e., sterilized at 110°C for 40 minutes).

[0019] Chlorobenzene solid selective medium: Na₂HPO₄·12H₂O 4500 mg·L⁻¹ -1 KH2PO4 1000 mg·L -1 (NH4)2SO4 1800 mg·L -1 MgSO4·7H2O 200mg·L -1 CuSO4·5H2O 0.02 mg·L -1 FeSO4·7H2O 1mg·L -1 H3BO3 0.02 mg·L -1 MnSO4·4H2O 0.1 mg·L -1ZnSO4·4H2O 0.1 mg·L -1 Na₂MoO₄·2H₂O 0.02 mg·L⁻¹ -1 20g·L -1 2 μL of agar and chlorobenzene were dropped onto sterile defatted cotton and placed in the center of the culture medium. The mixture was then sterilized at 110°C for 40 min.

[0020] Sludge from the wastewater treatment pond of Zhejiang Shangyu Water Treatment Development Co., Ltd. was mixed with culture medium in equal proportions and aerated for two months. Chlorobenzene was added as a carbon source. The culture medium was changed every 3-4 days. Before changing the medium, the sludge was allowed to stand, and the liquid culture medium of the supernatant was replaced. The pH of the sludge was measured daily. After a significant decrease in pH, 50 mL of sludge was inoculated into a culture flask containing 50 mL of chlorobenzene liquid basal medium. The culture was incubated at 30°C with shaking at 160 rpm, and 30 mg·L⁻¹ of chlorobenzene was added. -1 After the chlorobenzene is completely degraded, it is transferred to another culture flask with an increased chlorobenzene concentration for a new round of enrichment culture. After 5-6 cycles, 100 μL of the bacterial culture is spread onto chlorobenzene solid selective medium. Colonies are then picked from the solid medium and purified by repeated streak plating to obtain single colonies, i.e., chlorobenzene-degrading bacteria, which are designated as strain CB-L.

[0021] The preservation information of the chitinophaga sp. CB-L of the present invention is as follows: Preservation name: Chitinophaga sp. CB-L; Preservation institution: China Center for Type Culture Collection; Preservation address: Wuhan University, Wuhan, China; Preservation number: CCTCC NO: M 2025557; Preservation date: March 21, 2025.

[0022] (2) Identification of strain CB-L a. Physiological and biochemical characteristics of strain CB-L: The colony color is yellow, the colonies are small single colonies, the surface is moist, and the edges are smooth. As shown in Figure 1, the morphology of the bacteria observed under a transmission electron microscope is that of a bacillus. Its optimal growth pH is 7.0 and the optimal temperature is 30℃.

[0023] b. 16S rRNA sequence analysis of strain CB-L Through 16S rRNA sequence analysis and physiological and biochemical experiments, strain CB-L was identified as Chitinophaga sp. The specific steps are as follows: DNA of strain CB-L was extracted and purified using a 3S column centrifugal environmental sample DNA recovery kit (V2 2, Zhejiang Tianke Biotechnology Co., Ltd.) and stored at 4℃. Universal primers F27 and 1492R were used to amplify the purified DNA by PCR. The primer sequences were: F27: 5'-AGA GTT TGA TCC TGG CTC AG-3'; 1492R: 5'-GGT TAC CTT GTT ACG ACT T-3'. The PCR reaction system (50 μL) consisted of: 1.75 μL template DNA, 1 μL each of primer F27 and primer R1492, and MgCl2 (25 mmol·L⁻¹). -1 3 μL, Taq enzyme (5 U·μL) -1 0.25 μL, 5 μL of 10×PCR buffer, dNTPs (2.5 mmol·L⁻¹) -1 4 μL, redistilled water 34 μL.

[0024] The PCR reaction program was set as follows: pre-denaturation at 94℃ for 4 min; followed by denaturation at 94℃ for 1 min, annealing at 59℃ for 1 min, extension at 72℃ for 1.5 min, for 35 cycles; then extension at 72℃ for 10 min; and finally hold at 4℃ for 10 min. The PCR products were sequenced (Zhejiang Tianke), and the sequencing results are shown in SEQ ID NO: 1.

[0025] The 16S rDNA sequence of CB-L was compared with the gene sequence uploaded to the same Genbank and found to belong to the genus Chitinophaga, with the highest homology to Chitinophagasp MM2321. Figure 2 shows the phylogenetic tree of this strain.

[0026] Example 2: Determination of the growth curve of *C. phage* CB-L. A single colony of *C. phage* CB-L (1 inoculation loop) was inoculated into 50 ml of LB broth and cultured in a shaker at 30°C and 160 rpm. The final concentration of the LB broth was: NaCl 10 g·L⁻¹. -1 5g·L yeast extract -1 10 g / L of peptone -1 The solvent was ultrapure water, pH 7.0; at regular intervals (as shown in Figure 3), the bacterial culture was sampled and its OD value was measured. 600 A growth curve was plotted. Figure 3 shows the growth curve of this strain. It can be seen that the strain was in the lag phase from 0-24h, the logarithmic phase from 24-36h, and the stationary phase from 36-48h during LB culture.

[0027] Example 3: Chitinophyte CB-L fermentation broth (1) Seed culture: A single colony of 1 inoculation loop was picked from the LB solid culture medium and inoculated into 50 mL of LB liquid culture medium (seed culture medium). The culture was carried out at 30 °C for 30 h to obtain the seed culture. The final concentration of the seed culture medium was: NaCl 10 g·L -1 5g·L yeast extract -1 10 g / L of peptone -1 The solvent is ultrapure water, pH 7.0; (2) Fermentation culture and acclimatization period of chlorobenzene: Centrifuge the seed culture (centrifuge at 8000 rpm for 5 minutes), remove the supernatant obtained by centrifugation, wash the centrifuged cells with inorganic salt solution (fermentation medium without chlorobenzene) and resuspend; according to OD 600 The inoculum was 0.6 g / L and inoculated into the fermentation medium. The culture was incubated at 30°C with shaking at 160 rpm for 2 days to obtain the fermentation culture broth, which is the bacterial suspension. The final concentration of the fermentation medium was: Na₂HPO₄·12H₂O 4500 mg·L⁻¹. -1 KH2PO4 1000 mg·L -1 (NH4)2SO4 1800 mg·L -1 Chlorobenzene 100 mg·L -1 MgSO4·7H2O 200mg·L -1 CuSO4·5H2O 0.02 mg·L -1 FeSO4·7H2O 1mg·L -1 H3BO3 0.02 mg·L -1 MnSO4·4H2O 0.1 mg·L -1 ZnSO4·4H2O 0.1 mg·L -1 Na₂MoO₄·2H₂O 0.02 mg·L⁻¹ -1 The solvent is ultrapure water with a pH of 7.0. After the chlorobenzene in the solution is completely degraded, it is used as a bacterial suspension for later use.

[0028] 100 mg·L⁻¹ of chlorobenzene was removed from the above fermentation medium. -1 This is an inorganic salt solution.

[0029] The removal rate of chlorobenzene was determined using a Shimadzu GC-2014 gas chromatograph (GC). 0.8 mL of gas was taken from the headspace of the culture flask, and the removal rate of chlorobenzene was determined by gas chromatography. The results showed that after an adaptation period of 1-2 days, *C. cyclophosphamide* CB-L could degrade an initial concentration of 200 mg·L⁻¹ within 12 hours. -1 Chlorobenzene.

[0030] Example 4: Degradation Performance of Chlorobenzinibacterium CB-L for Chlorobenzin 1. Investigation of the Chlorobenzinibacterium CB-L Degradation Performance under Different Initial pH Conditions Experiments were conducted on the degradation of chlorobenzene by chitosan-based CB-L at different initial pH conditions. It was found that pH = 7.0 was the optimal pH, at which the degradation rate was the highest. The specific implementation steps are as follows: Chlorobenzinibacterium was used as the carbon source (concentration 200 mg·L⁻¹). -1 Centrifuge the seed culture, wash and resuspend it with inorganic salt solution, and then adjust the concentration according to OD. 600 An inoculum of 0.05 (prepared by the method in Example 3) was inoculated into chlorobenzene liquid selective medium with different pH values ​​(pH values ​​of 5.0, 6.0, 7.0, 8.0, and 9.0, respectively), and cultured at 30°C with shaking at 160 rpm for 1 day. That is, the pH values ​​of the fermentation medium in step (3) of Example 3 were set to 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, and the rest were the same as in Example 3.

[0031] As shown in Figure 4, strain CB-L has relatively poor adaptability to strongly alkaline environments. When the pH is 7.0, strain CB-L has the best degradation effect on chlorobenzene and grows the fastest at pH 7.0. Strong acid and strong alkaline environments will inhibit the growth of strains.

[0032] 2. The performance of chitosan-eating bacteria CB-L in degrading chlorobenzene was investigated at different temperatures. Experiments were conducted at different temperatures to degrade chlorobenzene using chitosan-eating bacteria CB-L. The results showed that the optimal temperature was 30℃. The specific implementation plan was as follows: chlorobenzene was used as the carbon source (concentration 200 mg·L⁻¹). -1 Centrifuge the seed culture, wash and resuspend it with inorganic salt solution, and then adjust the concentration according to OD. 600 An inoculum of 0.05 (prepared by the method in Example 3) was inoculated into chlorobenzene liquid selective medium and cultured for 1 day with shaking at 160 rpm at five temperature gradients of 20°C, 25°C, 30°C, 35°C and 40°C, respectively.

[0033] That is, the fermentation culture temperature of step (3) in Example 3 is set to 20℃, 25℃, 30℃, 35℃ and 40℃ respectively, and the rest is the same as in Example 3.

[0034] The results are shown in Figure 5. The data indicate that the optimal temperature for the degradation of chlorobenzene by strain CB-L is around 30℃, and the optimal temperature for strain growth is around 30-35℃. High and low temperatures will inhibit the growth of the strain.

[0035] 3. The degradation performance of chitosan-eating bacteria CB-L on chlorobenzene under different initial chlorobenzene concentrations was investigated. Degradation experiments were conducted with chitosan-eating bacteria CB-L at different initial chlorobenzene concentrations. The results showed that its degradation performance was good at chlorobenzene concentrations ranging from 50 to 500 mg·L⁻¹. -1 The following methods can degrade chlorobenzene: Using chlorobenzene as a carbon source, the seed culture is centrifuged and washed and resuspended with an inorganic salt solution; OD...600 An inoculum of 0.05 (prepared by the method in Example 3) was inoculated into plants with initial chlorobenzene concentrations of 100 mg·L⁻¹. -1 200 mg·L -1 300mg·L -1 400 mg·L -1 500mg·L -1 800mg·L -1 1000mg·L -1 1200mg·L -1 Eight liquid selective culture media were used, and the cultures were incubated at 30°C and 160 rpm for 1 day with shaking. Specifically, the chlorobenzene concentration in the fermentation medium from step (3) of Example 3 was set at 100 mg·L⁻¹. -1 200 mg·L -1 300mg·L -1 400 mg·L -1 500mg·L -1 800mg·L -1 1000mg·L -1 1200mg·L -1 The rest is the same as in Example 3.

[0036] The results are shown in Figure 6. After the adaptation period, the strain was able to stably degrade 100-500 mg·L⁻¹. -1 High concentrations of chlorobenzene inhibit the growth of the strain, but strain CB-L can grow under high concentrations of chlorobenzene and can degrade 1200 mg·L⁻¹. -1 Chlorobenzene.

Claims

1. A chitinophaga sp. CB-L strain, accession number: CCTCC NO:M 2025557, accession date: March 21, 2025, depositary institution: China Center for Type Culture Collection.

2. An application of the chitosan-eating bacterium CB-L as described in claim 1, characterized in that, The application is biodegradable chlorobenzene.

3. The application of the chitinophytic bacterium CB-L according to claim 2, characterized in that, The process includes the following steps: centrifuging the seed culture obtained after culturing chitinous bacteria CB-L, washing and resuspending it with inorganic salt solution, inoculating it into a liquid selective medium containing chlorobenzene, using chlorobenzene as a carbon source, and culturing it at 20-40℃ and 160±30rpm to achieve the degradation of chlorobenzene.

4. The application of the chitin-eating bacterium CB-L as described in claim 3, characterized in that, The final concentration of the liquid selective medium containing chlorobenzene was: Na₂HPO₄·12H₂O 4500 mg·L⁻¹ -1 KH2PO4 1000 mg·L -1 (NH4)2SO4 1800 mg·L -1 Chlorobenzene 50-500 mg·L -1 MgSO4·7H2O 200mg·L -1 CuSO4·5H2O 0.02 mg·L -1 FeSO4·7H2O 1 mg·L -1 H3BO3 0.02 mg·L -1 MnSO4·4H2O 0.1 mg·L -1 ZnSO4·4H2O 0.1 mg·L -1 Na₂MoO₄·2H₂O 0.02 mg·L⁻¹ -1 The solvent is ultrapure water.

5. The application of the chitinophytic bacterium CB-L according to claim 4, characterized in that, Specifically, the process includes the following steps: (1) Seed culture: a single colony of 1 inoculation loop is picked from the LB solid medium of chitinous bacteria CB-L and inoculated into 50 mL of LB liquid medium. The culture is carried out at 30°C for 30 h to obtain the seed culture. The final concentration of the LB liquid medium is: NaCl 10 g·L⁻¹. -1 5g·L yeast extract -1 10 g / L of peptone -1 The solvent is ultrapure water with a pH of 7.0; (2) Fermentation culture: the seed culture is centrifuged and washed and resuspended with inorganic salt solution, inoculated into fermentation medium, and cultured at 30℃ and 160±30rpm to obtain the fermentation culture medium containing bacteria; the final concentration composition of the fermentation medium is: Na2HPO4·12H2O4 500mg·L -1 KH2PO4 1000 mg·L -1 (NH4)2SO4 1800 mg·L -1 Chlorobenzene 100 mg·L -1 MgSO4·7H2O 200mg·L -1 CuSO4·5H2O 0.02 mg·L -1 FeSO4·7H2O 1mg·L -1 H3BO3 0.02 mg·L -1 MnSO4·4H2O 0.1 mg·L -1 ZnSO4·4H2O 0.1 mg·L -1 Na₂MoO₄·2H₂O 0.02 mg·L⁻¹ -1 The solvent is ultrapure water, and the pH value is 7.0.

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