Bacteria capable of degrading o-dichlorobenzene and its application

Abstract

The present invention relates to the field of microbial engineering technology, and in particular to a strain for degrading o-dichlorobenzene and its application. The strain of the present invention belongs to Gram-negative bacteria, is identified as Pseudomonas stutzeri at the molecular level, and the deposit number of the degrading bacteria is CGMCC No.24318. The strain or its bacterial agent is put into a sewage or waste gas treatment system containing o-dichlorobenzene to perform harmless treatment on it. At the same time, the strain also has a certain ability to treat chlorobenzene and phenol. The application of the strain can avoid the disadvantages of high cost and secondary pollution caused by physical and chemical methods, has no side effects on operators, and lays a material foundation for the research on biological treatment of chlorobenzene-containing compounds, and has good application value in actual production.

Description

Technical Field

[0001] The invention relates to the technical field of microbial engineering, in particular to a strain for degrading o-dichlorobenzene and application thereof. Background Art

[0002] o-Dichlorobenzene is one of the chlorobenzene compounds and a class of hydrophobic and persistent organic pollutants. o-Dichlorobenzene is widely present in the environment and has varying degrees of toxicity, bioaccumulation and persistence in the air, soil, groundwater, surface water and oceans, posing a potential hazard to the environment.

[0003] Research on chlorobenzene pollutants has gradually become a new field and a growth point in the study of environmental science and pollution ecology. Due to the extremely stable chemical properties of o-dichlorobenzene, there is currently no particularly effective, economical, and widely applicable physical and chemical treatment method for it. Biological degradation is recognized as the most ideal treatment method because of its simple operation and management, low treatment cost, no secondary pollution, and is suitable for wastewater with various complex components and has a significant treatment effect. Biological treatment mainly degrades organic pollutants in soil, groundwater or ocean into carbon dioxide and water or converts them into harmless substances through the action of microorganisms. According to current relevant literature, the organisms that repair pollution are mainly microbial bacteria and fungi, plants and mycorrhizae.

[0004] At the same time, o-dichlorobenzene is generally considered to be an artificially synthesized, environmentally foreign compound. Microorganisms in nature lack the corresponding degradation enzyme system and are difficult to be utilized by microorganisms. Therefore, there are not many studies on the biodegradation of o-dichlorobenzene, and not much work has been done on the isolation and screening of o-dichlorobenzene-degrading bacteria. Therefore, it is particularly important to isolate and breed strains with high tolerance and degradation capabilities for use in bioaugmentation technology. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a strain for degrading o-dichlorobenzene and its application, so as to solve the problem that o-dichlorobenzene is poor in biodegradability and difficult to degrade.

[0006] The screening method of the strain of the present invention comprises: collecting samples from the sewage treatment system of the petrochemical sewage plant, enriching them, and further obtaining them by gradient enrichment, separation and purification in an inorganic salt medium with o-dichlorobenzene as the sole carbon source. The concentration of o-dichlorobenzene in the medium is gradually increased to 200 mg / L with a gradient of 50 mg / L, 75 mg / L, 100 mg / L, 125 mg / L, 150 mg / L, and 200 mg / L. The above-mentioned inorganic salt culture medium is composed of KH2PO4 0.5g / L, Na2HPO4 0.5g / L, (NH4)2SO42g / L, ZnCl2 0.23mg / L, CoCl2·6H2O 0.42mg / L, MnSO4·H2O 0.13mg / L, Na2MoO4·2H2O0.15mg / L, AlCl3·6H2O 0.05mg / L, CuSO4·H2O 0.03mg / L, CaCl2 0.01g / L, sterilized for use, natural pH, prepared with distilled water, sterilized at 121°C for 20min, 20g of agar is added to the solid culture medium, and o-dichlorobenzene is added before use.

[0007] The present invention provides Pseudomonas stutzeri with a deposit number of CGMCC No.24318.

[0008] The Pseudomonas stutzeri with a deposit number of CGMCC No. 24318 provided by the present invention is named YJY22-09, is a Gram-negative bacterium, has a round light pink colony with neat edges, a convex middle, and a dry surface, is identified as Pseudomonas stutzeri by 16S rDNA, and the nucleotides of its 16S rDNA have a sequence as shown in SEQ ID No: 1.

[0009] The invention also provides the use of Pseudomonas stutzeri with the deposit number of CGMCC No. 24318 in degrading organic pollutants.

[0010] The organic pollutant described in the present invention is at least one of o-dichlorobenzene, chlorobenzene and phenol.

[0011] The invention provides a product for degrading organic pollutants, which comprises Pseudomonas stutzeri with a deposit number of CGMCC No.24318.

[0012] The product for degrading organic pollutants described in the present invention is a bacterial suspension or dry powder.

[0013] In some embodiments, the product for degrading organic pollutants of the present invention has an effective viable bacterial count of 5×10 8 ~2×10 10 cfu / mL. In some specific embodiments, the viable count of the bacterial agent is 1×1010 cfu / mL.

[0014] In some embodiments, the product of the present invention is used in a fermentation culture, wherein the fermentation temperature is 33°C ± 1°C, the dissolved oxygen is 30%, the culture is continued for 20 h to 30 h, and the fermentation is stopped after the dissolved oxygen rises and the pH drops.

[0015] The present invention also provides a preparation method of the product, which comprises culturing Pseudomonas stutzeri with a preservation number of CGMCC No. 24318 and collecting the bacteria.

[0016] In the preparation method of the product of the present invention, after collecting the bacterial cells, the step of resuspending the bacterial cells to prepare a bacterial suspension is also included; or the step of drying the bacterial suspension is also included. The present invention also provides a method for preparing the bacterial suspension, centrifuging the bacterial liquid to obtain the bacterial cells, washing and suspending the bacterial cells with a sterilized culture medium to obtain a bacterial suspension.

[0017] In the preparation method of the present invention, the culture medium comprises water and 7% to 9% glycerol, 0.5% to 1.2% yeast powder, 0.5% to 1.2% corn steep liquor powder, 0.5% to 1.2% sodium chloride, 0.1% to 1% sodium citrate and 0.05% to 0.2% ammonium sulfate; the medium is sterilized at 121° C. and used after 20 minutes.

[0018] In some embodiments, the preparation method of the present invention may include high-density fermentation culture at 32°C to 37°C for 20h to 30h; wherein, the culture medium is used for fermentation, the balance is water, and the pH is 6.8 to 7.5.

[0019] In some specific embodiments, the strain of the present invention is used to prepare an o-dichlorobenzene-degrading bacterial agent, and the specific preparation method is as follows:

[0020] The first step is to move the test tube slant culture stored on the nutrient agar medium at 4°C to room temperature at 20°C to 25°C and activate it for 4h to 8h. No additional culture medium is required during activation.

[0021] The second step is to inoculate the single colony obtained in the first step into LB liquid medium (100 ml), culture it overnight at 35°C and 185 r / min to obtain a primary seed solution, inoculate the primary seed solution into LB medium at 10% (V / V), culture it for 16 hours under the same conditions, obtain a secondary seed solution, and store it at 4°C;

[0022] In the third step, the secondary seed liquid of Pseudomonas stutzeri is inoculated into the fermentation medium at 5% (V / V) for fermentation culture, and the fermentation temperature is controlled to be 33°C ± 1°C, the dissolved oxygen is about 30%, and the culture is carried out for 20h to 30h. When the dissolved oxygen rises and the pH drops, the fermentation is stopped to obtain the o-dichlorobenzene degrading bacterial agent. In this embodiment, the number of viable bacteria of the dichlorobenzene degrading bacterial agent is 1×10 10 cfu / mL.

[0023] The fermentation medium is calculated by mass percentage as follows: 8.5% glycerol, 0.5% yeast powder, 0.75% corn steep liquor powder, 1% sodium chloride, 0.15% sodium citrate, 0.05% ammonium sulfate, 0.1% polyether defoamer, pH 7-7.5; high pressure sterilization at 121°C for 30 minutes before use. Each liter of LB medium is prepared by adding 10.0g of peptone, 5.0g of yeast powder, 10.0g of sodium chloride, and water to 1.0L, adjusting the pH value to 7.0, and sterilizing at 121°C for 20 minutes before use.

[0024] The invention also provides a method for degrading organic pollutants, which comprises treating the organic pollutants with the product.

[0025] In the method of the present invention, the organic pollutant is at least one of o-dichlorobenzene, chlorobenzene and phenol.

[0026] In some embodiments, in the method described in the present invention, the treatment temperature is 25°C to 35°C, pH is 6.0 to 8.0, and the dissolved oxygen is above 2 mg / L.

[0027] In the method described in the present invention, the organic pollutants exist in wastewater, waste gas and / or soil, and the strain and / or the product prepared by using the strain are put into the treatment system. The strain can effectively withstand the impact of pollutants such as o-dichlorobenzene, chlorobenzene and phenol, and can use the organic matter in the biochemical system as nutrients for its own growth, thereby quickly degrading the organic matter.

[0028] In the method of the present invention, the content of o-dichlorobenzene is below 500 mg / L, and the amount of bacterial agent added is V / V 0.1% to 0.5%.

[0029] In some embodiments, the amount of bacterial agent added is 1‰ to 5‰ of the volume of the biochemical system, calculated by volume ratio, and the concentration of o-dichlorobenzene in the system is below 300 mg / L. Preferably, the conditions of the biochemical system are a temperature of 25°C to 35°C, a pH of 6.0 to 8.0, and dissolved oxygen ≥ 2 mg / L.

[0030] The invention provides an o-dichlorobenzene degrading bacterium, which is identified as Pseudomonas stutzeri and has a deposit number of CGMCC No. 24318. Under the condition of using o-dichlorobenzene as the only carbon source and energy source, the strain can quickly degrade o-dichlorobenzene; and the strain or its bacterial agent is put into a sewage or waste gas treatment system containing o-dichlorobenzene, and o-dichlorobenzene can be harmlessly treated; at the same time, the strain also has a certain ability to treat chlorobenzene and phenol, and the application of the strain can avoid the disadvantages of high cost and secondary pollution caused by physical and chemical methods, and has no side effects on operators, laying a material foundation for the research on biological treatment of chlorobenzene-containing compounds. It also has the characteristics of simplicity and high efficiency in application, and the strain has strong tolerance and a wide range of adaptability to conditions such as temperature and pH. In addition, the bacteria have a certain broad spectrum in degrading pollutants, and have a good application prospect in the biological purification of industrial wastewater and waste gas.

[0031] Biological Deposit Description

[0032] Biological material: YJY22-09, classification name: Pseudomonas stutzeri, deposited on January 17, 2022 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No.24318. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation or the prior art description are briefly introduced below. Obviously, the drawings in the following description are some implementations of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work:

[0034] Figure 1 The degradation rate of dichlorobenzene by Pseudomonas stutzeri YJY22-09 at different temperatures is shown;

[0035] Figure 2 The degradation rate of dichlorobenzene by Pseudomonas stutzeri YJY22-09 under different pH conditions is shown;

[0036] Figure 3 The degradation of o-dichlorobenzene with different initial concentrations by Pseudomonas stutzeri YJY22-09 is shown;

[0037] Figure 4 The degradation rate of o-dichlorobenzene with different initial concentrations by Pseudomonas stutzeri YJY22-09 is shown;

[0038] Figure 5The removal rates of benzene, chlorobenzene and phenol by o-dichlorobenzene-degrading bacteria are shown;

[0039] Figure 6 The device for treating o-dichlorobenzene gas with o-dichlorobenzene-degrading bacteria is shown;

[0040] Figure 7 The figure shows the degradation rate of o-dichlorobenzene by other bacterial species. DETAILED DESCRIPTION

[0041] The present invention provides strains for degrading o-dichlorobenzene and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] Firstly, the o-dichlorobenzene-degrading bacteria of the present invention are obtained by collecting samples from a sewage treatment system of a petrochemical sewage plant, enriching the samples, and further gradient enriching, separating and purifying the samples in an inorganic salt culture medium with o-dichlorobenzene as the sole carbon source.

[0043] After testing, the o-dichlorobenzene degrading bacteria of the present invention is a Gram-negative bacteria, the colony is round and light pink, the edges are neat, the middle is convex, the surface is dry, and it is identified as Pseudomonas stutzeri by 16S rDNA, and is deposited in the General Microbiology Center of China Microbiological Culture Collection Committee with a deposit number of CGMCC No.24318.

[0044] As a further embodiment of the present invention, the present invention provides a bacterial agent of the o-dichlorobenzene-degrading bacteria, which is prepared by fermentation:

[0045] The fermentation medium is calculated by mass percentage as follows: glycerol 7%-9%, yeast powder 0.5%-1.2%, corn steep liquor dry powder 0.5%-1.2%, sodium chloride 0.5%-1.2%, sodium citrate 0.1%-1%, ammonium sulfate 0.05%-0.2%, pH 6.8-7.5; high pressure sterilization at 121°C for 20 minutes before use.

[0046] The o-dichlorobenzene-degrading bacteria are subjected to high-density fermentation culture at 30°C to 37°C for 15h to 30h, and after the fermentation is completed, a liquid bacterial agent is obtained, and the effective viable bacterial count of the bacterial agent is (5×10 8 )~(2×10 10 ) cfu / mL. It can also be further prepared into a solid bacterial agent, etc., and the preparation method can be prepared using conventional techniques.

[0047] Furthermore, the fermentation temperature is 33°C ± 1°C, the dissolved oxygen is about 30%, and the culture is carried out for 20h to 30h. When the dissolved oxygen rises and the pH drops, the fermentation is stopped.

[0048] The above-mentioned bacterial agent can be applied to the bio-enhanced treatment of wastewater, waste gas and soil containing o-dichlorobenzene, phenol, chlorobenzene, etc. The bacterial agent prepared by the strain is put into the treatment system, which can effectively withstand the impact of such pollutants, and can use the organic matter in the biochemical system as nutrients for its own growth and quickly degrade the organic matter.

[0049] The bacterial agent obtained above is applied to a system containing o-dichlorobenzene, the amount of the bacterial agent added (by volume) is 1‰ to 5‰ of the volume of the biochemical system, the o-dichlorobenzene concentration in the system is below 300 mg / L, and preferably, the biochemical system conditions are a temperature of 25°C to 35°C, a pH of 6.0 to 8.0, and dissolved oxygen ≥ 2 mg / L.

[0050] Furthermore, the o-dichlorobenzene-degrading bacteria of the present invention can be continuously cultured and applied in o-dichlorobenzene wastewater, and the degradation effect is stable.

[0051] The present invention provides a method for detecting the degradation characteristics of o-dichlorobenzene-degrading bacteria:

[0052] The prepared o-dichlorobenzene degrading bacterial agent was inoculated into 2 mL to 50 mL of inorganic salt culture medium, in which o-dichlorobenzene was used as the only carbon source and energy source, and the degradation ability was tested. In order to reduce the volatilization of o-dichlorobenzene and ensure the dissolved oxygen content of the bacteria, the following degradation experiments were all carried out in 250 mL frosted glass bottles.

[0053] 1. Biodegradation characteristics of o-dichlorobenzene by Pseudomonas stutzeri YJY22-09 at different temperatures

[0054] The biodegradation characteristics of Pseudomonas stutzeri YJY22-09 on o-dichlorobenzene at different temperatures were tested and found that it has a good degradation ability for o-dichlorobenzene at 25-35°C. From the practical application point of view, its degradation ability is best at 33°C. The specific implementation steps are as follows:

[0055] Seven groups were set up, with 3 replicates in each group. 120 mg / L o-dichlorobenzene was added to the inorganic salt culture medium, and the pH was natural. After inoculation, different culture temperatures were set, and the experimental temperature was set to 20℃~40℃ (set to 20℃, 25℃, 28℃, 30℃, 33℃, 35℃, 40℃ respectively). The culture was placed in a shaker with a shaking speed of 160r / min. After 20h, samples were taken for extraction and gas phase detection, and the optimal action temperature was found to be 25℃~35℃. For details, see Figure 1 .

[0056] 2. Biodegradation characteristics of o-dichlorobenzene by Pseudomonas stutzeri YJY22-09 under different pH conditions

[0057] Eleven groups were set up, and 120 mg / L o-dichlorobenzene was added to the inorganic salt culture medium with 3 replicates in each group. Different pH values ​​were set, and the pH experimental range was set to 5-10 (set to 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10, respectively). After inoculation, the culture was shaken in a shaker at 30°C and 160 r / min. After 20 hours, samples were taken for extraction and detection, and the most suitable pH value was 6.5-8.0. It has a wide range of adaptability. For details, see Figure 2 .

[0058] 3. Degradation of o-dichlorobenzene at different initial concentrations by Pseudomonas stutzeri YJY22-09

[0059] A total of 4 groups of experiments were set up, each with multiple replicates. 50 mL of inorganic salt culture medium was added to a ground-mouth Erlenmeyer flask, and o-dichlorobenzene was used as the only carbon source and energy source. The initial concentrations of o-dichlorobenzene were 60 mg / L, 120 mg / L, 180 mg / L, and 240 mg / L. The pH was natural, and the culture was carried out at 30 °C and 160 r / min. Samples were taken at intervals to detect the residual amount of o-dichlorobenzene. When the substrate concentration was 240 mg / L, it could be degraded by more than 99% within 72 h. For details, see Figure 3 .

[0060] 4. Degradation of o-dichlorobenzene at different initial concentrations by Pseudomonas stutzeri YJY22-09

[0061] A total of 4 groups of experiments were set up, each with multiple replicates. 50 mL of inorganic salt culture medium was added to a ground-mouth Erlenmeyer flask, and o-dichlorobenzene was used as the only carbon source. The initial concentrations of o-dichlorobenzene were 240 mg / L, 360 mg / L, 480 mg / L, and 600 mg / L. Samples were taken after 48 hours to detect the residual amount of o-dichlorobenzene. For details, see Figure 4 .

[0062] The present invention also provides a method for detecting the biodegradation performance of o-dichlorobenzene-degrading bacteria on benzene, chlorobenzene and phenol: a total of 4 groups of experiments are set up, each group is set up with three replicates, 50 mL of inorganic salt culture medium is added to a ground-mouth triangle bottle, benzene, chlorobenzene and phenol are used as the only carbon source, the initial concentration of the substrate is 50 mg / L, and the residual amount of the substrate is detected after 30 hours of sampling. It can be seen that the removal rates of benzene, chlorobenzene and phenol in 30 hours are 88%, 97% and 59% respectively. For details, see Figure 5 .

[0063] The "dissolved oxygen" in the bacterial fermentation process involved in the following embodiments refers to the air saturation (%), which is a common method of expressing dissolved oxygen concentration in the fermentation industry.

[0064] The "dissolved oxygen" in the aeration process of sewage treatment involved in the following embodiments refers to the absolute concentration of dissolved oxygen in water, expressed in mg / L, which is a commonly used method for expressing dissolved oxygen in sewage treatment in the environmental protection industry.

[0065] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0066] Example 1 Enrichment, screening and separation of o-dichlorobenzene-degrading bacteria

[0067] The sludge from the sewage treatment system of a petrochemical wastewater treatment plant was enriched and screened, and the o-dichlorobenzene-degrading bacteria were enriched by gradually increasing the concentration of o-dichlorobenzene.

[0068] The specific operation method is: take 10mL of activated sludge sample, inoculate it in 100mL of enrichment culture medium (initial concentration of o-dichlorobenzene is 10mg / L), and culture it in a shaking table at 30℃ and 160r / min for 2d to 5d for enrichment culture. Take samples to detect the content of o-dichlorobenzene in the system. When it is degraded to less than 2mg / L, take out 10mL of bacterial liquid and inoculate it into fresh inorganic salt culture medium (o-dichlorobenzene concentration is 20mg / L). Repeat the above steps, with 50mg / L, 75mg / L, 100mg / L, 125mg / L, 150mg / L, 200mg / L as the gradient, and gradually increase the concentration of o-dichlorobenzene in the culture medium to 200mg / L.

[0069] The final enriched culture fluid is spread on an inorganic salt medium plate with o-dichlorobenzene as the sole carbon source after gradient dilution, and cultured in a 30°C incubator for 2d to 3d. The single colonies with different morphologies grown on the plate are streaked on the inorganic salt medium plate for purification at least 3 times, and after purification, the single colonies are picked and inoculated into 10mL LB liquid culture medium, and the next day, they are inoculated into 100mL inorganic salt liquid culture medium containing 50mg / L o-dichlorobenzene at an inoculum amount of 2% (V / V), and cultured at 30°C and 160r / min for 36h, and then the content of o-dichlorobenzene is detected by gas phase sampling. 12 strains of bacteria are isolated by this method, among which the bacterium numbered 09 can completely degrade 50mg / L o-dichlorobenzene within 36h, and it is determined to be the o-dichlorobenzene degrading bacteria of the present invention, and it is preserved.

[0070] The above-mentioned inorganic salt culture medium is composed of KH2PO4 0.5g / L, Na2HPO4 0.5g / L, (NH4)2SO4 2g / L, ZnCl20.23mg / L, CoCl2·6H2O 0.42mg / L, MnSO4·H2O 0.13mg / L, Na2MoO4·2H2O 0.15mg / L, AlCl3·6H2O 0.05mg / L, CuSO4·H2O 0.03mg / L, CaCl20.01g / L, sterilized for use, natural pH, prepared with distilled water, sterilized at 121°C for 20min, 20g of agar is added to the solid culture medium, and o-dichlorobenzene is added before use.

[0071] The addition of o-dichlorobenzene to the solid culture medium is performed by sticking a sterile filter paper on the cover of the culture dish, dropping o-dichlorobenzene on the filter paper, and using the vapor of o-dichlorobenzene to culture the bacteria.

[0072] The enrichment medium is prepared by adding water to 1.0 L of peptone, yeast powder 5.0 g, sodium chloride 10.0 g, and adjusting the pH value to 7.0. Add 20.0 g of agar to the solid medium and sterilize at 121° C. for 20 min.

[0073] Example 2 Identification and Colony Characteristics of O-Dichlorobenzene Degrading Bacteria

[0074] Bacterial species identification: The obtained o-dichlorobenzene-degrading bacteria were subjected to 16S rDNA identification. The 16S rDNA sequence was compared with the NCBI database. The result of molecular identification showed that the bacteria belonged to Pseudomonas stutzeri. The nucleotide sequence of its 16S rDNA is shown in SEQ ID NO.1 in the sequence table.

[0075] The o-dichlorobenzene degrading bacteria Pseudomonas stutzeri is a Gram-negative bacterium, with round light pink colonies, neat edges, a convex middle, a moist surface, and is easy to pick up. It is named YJY22-09 and has been biologically preserved, with a biological preservation number of CGMCC No. 24318.

[0076] Example 3 Preparation of o-dichlorobenzene degradation bacterial agent

[0077] The above strains were used to prepare an o-dichlorobenzene degradation bacterial agent, and the specific preparation method was as follows:

[0078] The first step is to move the test tube slant culture stored on the nutrient agar medium at 4°C to room temperature (20°C to 25°C) for activation for 4h to 8h. No additional culture medium is required during activation.

[0079] The second step is to inoculate the single colony obtained in the first step into LB liquid medium (100 ml), culture it overnight at 35°C and 185 r / min to obtain a primary seed solution, inoculate the primary seed solution into LB medium at 10% (V / V), culture it for 16 hours under the same conditions, obtain a secondary seed solution, and store it at 4°C;

[0080] The third step is to inoculate the secondary seed liquid of Pseudomonas stutzeri into the fermentation medium at 1‰ (V / V) for fermentation culture, control the fermentation temperature to 33°C ± 1°C, the dissolved oxygen to about 30%, culture for 20h to 30h, the dissolved oxygen rises, the pH drops, and the fermentation is stopped to obtain the o-dichlorobenzene degradation bacterial agent. It can also be further prepared into solid bacterial agents, bacterial powders, etc., and the preparation method can be prepared using conventional techniques.

[0081] The viable bacterial count of the basic degradation agent prepared in this example is 1×10 10 cfu / mL.

[0082] The fermentation medium comprises, by mass percentage, 8.5% glycerol, 0.5% yeast powder, 0.75% corn steep liquor powder, 1% sodium chloride, 0.15% sodium citrate, 0.05% ammonium sulfate, 0.1% polyether defoamer, pH 7-7.5; and is sterilized at high pressure at 121° C. for 30 minutes before use.

[0083] The LB medium was prepared by adding water to 1.0 L of 10.0 g of peptone, 5.0 g of yeast powder, and 10.0 g of sodium chloride, adjusting the pH value to 7.0, and sterilizing at 121° C. for 20 min before use.

[0084] Example 4 Detection of degradation characteristics of o-dichlorobenzene-degrading bacteria

[0085] The o-dichlorobenzene degradation bacterial agent obtained in Example 3 was inoculated into 2 mL to 50 mL of an inorganic salt culture medium, in which o-dichlorobenzene was used as the sole carbon source and energy source, to test the degradation ability. In order to reduce the volatilization of o-dichlorobenzene and ensure the dissolved oxygen content of the bacteria, the following degradation experiments were all carried out in a 250 mL frosted glass bottle.

[0086] 1. Biodegradation characteristics of o-dichlorobenzene by Pseudomonas stutzeri YJY22-09 at different temperatures

[0087] The biodegradation characteristics of Pseudomonas stutzeri YJY22-09 at different temperatures for o-dichlorobenzene were tested and found to have good degradation ability for o-dichlorobenzene at 25℃~35℃. From the practical application, its degradation ability was best at 33℃. The specific implementation steps are as follows:

[0088] Seven groups were set up, with 3 replicates in each group. 120 mg / L o-dichlorobenzene was added to the inorganic salt culture medium, and the pH was natural. After inoculation, different culture temperatures were set, and the experimental temperature was set to 20℃~40℃ (set to 20℃, 25℃, 28℃, 30℃, 33℃, 35℃, 40℃ respectively). The culture was placed in a shaker with a shaking speed of 160r / min. After 20h, samples were taken for extraction and gas phase detection, and the optimal action temperature was found to be 25℃~35℃. For details, see Figure 1 ;

[0089] 2. Biodegradation characteristics of o-dichlorobenzene by Pseudomonas stutzeri YJY22-09 under different pH conditions

[0090] Eleven groups were set up, and 120 mg / L o-dichlorobenzene was added to the inorganic salt culture medium with 3 replicates in each group. Different pH values ​​were set, and the pH experimental range was set to 5-10 (set to 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10, respectively). After inoculation, the culture was shaken in a shaker at 30°C and 160 r / min. After 20 hours, samples were taken for extraction and detection, and the most suitable pH value was 6.5-8.0. It has a wide range of adaptability. For details, see Figure 2 .

[0091] 3. Degradation of o-dichlorobenzene at different initial concentrations by Pseudomonas stutzeri YJY22-09

[0092] A total of 4 groups of experiments were set up, each with multiple replicates. 50 mL of inorganic salt culture medium was added to a ground-mouth Erlenmeyer flask, and o-dichlorobenzene was used as the only carbon source. The initial concentrations of o-dichlorobenzene were 60 mg / L, 120 mg / L, 180 mg / L, and 240 mg / L. The pH was natural, and the culture was carried out at 30 °C and 160 r / min. Samples were taken at intervals to detect the residual amount of o-dichlorobenzene. When the substrate concentration was 240 mg / L, it could be degraded by more than 99% within 72 h. For details, see Figure 3 .

[0093] 4. Degradation of o-dichlorobenzene at different initial concentrations by Pseudomonas stutzeri YJY22-09

[0094] A total of 4 groups of experiments were set up, each with multiple replicates. 50 mL of inorganic salt culture medium was added to a ground-mouth Erlenmeyer flask, and o-dichlorobenzene was used as the only carbon source. The initial concentrations of o-dichlorobenzene were 240 mg / L, 360 mg / L, 480 mg / L, and 600 mg / L. Samples were taken after 48 hours to detect the residual amount of o-dichlorobenzene. For details, see Figure 4 .

[0095] Example 5 Biodegradation performance test of o-dichlorobenzene-degrading bacteria on benzene, chlorobenzene and phenol

[0096] A total of 4 groups of experiments were set up, each with three replicates. 50 mL of inorganic salt culture medium was added to a ground-mouth conical flask, with benzene, chlorobenzene, and phenol as the only carbon source. The initial concentration of the substrate was 50 mg / L. After 30 hours, samples were taken to detect the residual amount of the substrate. It can be seen that the removal rates of benzene, chlorobenzene, and phenol in 30 hours were 88%, 97%, and 59%, respectively. For details, see Figure 5 .

[0097] Example 6 Application of o-dichlorobenzene-degrading bacteria in o-dichlorobenzene gas treatment

[0098] The o-dichlorobenzene degradation bacterial agent was prepared according to the method of Example 3. The culture temperature was 32°C ± 1°C, the dissolved oxygen was controlled at 30%, and the viable bacterial count of the obtained bacterial agent was 8×10 9 cfu / mL.

[0099] The waste gas degradation experiment of o-dichlorobenzene, chlorobenzene and benzene was carried out in a simulated waste gas biological treatment device. The device consists of three parts: a biological treatment device, an aeration device and a gas generating device. The o-dichlorobenzene bacterial agent was added to the biological treatment device filled with ceramsite for bacterial treatment, mainly to cover the ceramsite as much as possible, and the bacterial treatment was carried out for about 18h to 36h. During this period, watering was continuously carried out to allow the strain to be fully hung on the carrier; a certain amount of benzene, chlorobenzene and o-dichlorobenzene were added to the gas generating device respectively, and the substrate was supplemented once every 24h, and the inorganic salt culture medium in Example 1 was sprayed. The aeration device was turned on for ventilation to carry out the experiment. o-dichlorobenzene, chlorobenzene and benzene entered the bottom of the biological treatment device through the gas pressure, and were discharged from the device after passing through the ceramsite with bacteria. The experimental device is shown in FIG. Figure 6 After two days of continuous operation, the air intake volume was about 50mg / m 3 , 50mg / m 3 and 120 mg / m 3 When the reactor was operated for about 12 hours, samples were taken to detect the substrate contents in the gases at the outlet and inlet. The removal rates of benzene, chlorobenzene and o-dichlorobenzene were all above 99%.

[0100] Example 7 Application of o-dichlorobenzene-degrading bacteria in o-dichlorobenzene wastewater treatment 1

[0101] The o-dichlorobenzene degradation bacterial agent was prepared according to the method of Example 3. The culture temperature was 33°C ± 1°C, the dissolved oxygen was controlled at 30%, and the viable bacterial count of the obtained bacterial agent was 5×10 8 cfu / mL.

[0102] Simulation application: The treated water source is the influent of a pesticide wastewater treatment plant, with a treatment capacity of 5L. The addition amount of o-dichlorobenzene degradation bacteria is 0.3% (V / V). The dissolved oxygen used in aeration treatment is above 2mg / L. The pH value of sewage is 6.0-8.0. The COD of raw sewage is about 4500mg / L, ammonia nitrogen is about 480mg / L, and organic amines are about 300mg / L. The concentration of o-dichlorobenzene is artificially added to 150mg / L, chlorobenzene is 50mg / L, and benzene is 20mg / L. After 48h, the contents of o-dichlorobenzene, chlorobenzene and benzene in sewage are <1mg / L, and the degradation rate is more than 99.9%.

[0103] Example 8 Application of o-dichlorobenzene-degrading bacteria in o-dichlorobenzene wastewater treatment 2

[0104] The o-dichlorobenzene degradation bacterial agent was prepared according to the method of Example 3. The culture temperature was 35±1°C, the dissolved oxygen was controlled at 30%, and the viable bacterial count of the obtained bacterial agent was 1.5×10 10 cfu / mL.

[0105] The source of treated water is the influent of the aerobic pool of the petrochemical wastewater treatment plant, with a treatment capacity of 10L. The amount of o-dichlorobenzene degradation bacteria added is 0.2% (V / V). Aeration treatment makes the dissolved oxygen above 2mg / L, the sewage pH 6.0-8.0, the o-dichlorobenzene concentration below 5mg / L, COD about 2500mg / L, volatile phenol about 280mg / L, ammonia nitrogen about 100mg / L, sulfide about 10mg / L. The content of o-dichlorobenzene in the influent is adjusted at any time during the treatment process. The water is continuously inlet and outlet for 24h, the water inlet speed is about 17mL / min, the residence time is about 10h, and the o-dichlorobenzene content in the effluent is detected by sampling. The detailed process and test results are shown in the table below:

[0106] Table 1:

[0107] 0d 1d 2d 3d 4d 5d 6d 7d Influent (mg / L) 90 90 120 150 200 200 200 200 Outlet water (mg / L) / ＜1.0 ＜1.0 ＜1.0 ＜1.0 ＜1.0 ＜1.0 ＜1.0 Degradation rate / >99.9％ >99.9％ >99.9％ >99.9％ >99.9％ >99.9％ >99.9％

[0108] The above-mentioned experiments and various application simulation results of the present invention show that the o-dichlorobenzene-degrading bacteria YJY22-09 has significant effects in the sewage and waste gas treatment process.

[0109] Comparative Example 1

[0110] Table 2: Degradation rate of o-dichlorobenzene by other strains

[0111] serial number Degradation rate / % 01 69.71 02 59.09 03 23.32 04 54.46 05 15.86 06 29.52 07 33.24 08 41.31 09 90.68 10 22.24 11 45.02 12 30.34

[0112] The enrichment system obtained in Example 1 was separated into strains to obtain a total of 12 strains of bacteria of different sizes and shapes, which were numbered according to the sequence numbers, as shown in Table 2, and were recorded as 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, and 12, respectively, and inoculated in 100 mL of inorganic salt liquid culture medium containing 50 mg / L o-dichlorobenzene at an inoculum size of 2% (V / V). The blank group was not inoculated with strains, and cultured at 30° C. and 160 r / min for 36 h. After that, samples were taken to detect the content of o-dichlorobenzene by gas phase, and its degradation rate relative to the blank group was calculated.

[0113] As shown in Table 2 and Figure 7 The numbers shown are the serial numbers after the strains were separated from the enrichment system, and the degradation rates are the degradation rates relative to the blank group.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Pseudomonas stutzeri with a deposit number of CGMCC No. 24318 ( Pseudomonas stutzeri )YJY22-09.

2. Use of Pseudomonas stutzeri with a deposit number of CGMCC No. 24318 in degrading organic pollutants; the organic pollutants are at least one of o-dichlorobenzene, benzene, chlorobenzene and phenol.

3. A product for degrading organic pollutants, characterized in that: Including Pseudomonas stutzeri with the deposit number of CGMCC No.24318.

4. The method for preparing the product according to claim 3, characterized in that: Pseudomonas stutzeri with a preservation number of CGMCC No. 24318 was cultured and the bacterial cells were collected.

5. The preparation method according to claim 4, characterized in that: After collecting the bacterial cells, the method further includes the step of resuspending the bacterial cells to prepare a bacterial suspension; or further includes the step of drying the bacterial suspension.

6. The preparation method according to claim 4 or 5, characterized in that: The culture medium comprises water and glycerol 7% to 9%, yeast powder 0.5% to 1.2%, corn steep liquor dry powder 0.5% to 1.2%, sodium chloride 0.5% to 1.2%, sodium citrate 0.1% to 1% and ammonium sulfate 0.05% to 0.2%.

7. A method for degrading organic pollutants, characterized in that: The organic pollutant is treated with the product according to claim 3; the organic pollutant is at least one of o-dichlorobenzene, chlorobenzene and phenol.

8. The method according to claim 7, characterized in that The treatment temperature is 25°C to 35°C, and the pH is 6.0 to 8.0.

Images