A strain of denitrifying Achromobacterium LJC-02 capable of degrading PAEs and its application

Abstract

The present invention discloses a strain of Achromobacter denitrificans LJC‑02 capable of degrading PAEs and its application. The Achromobacter denitrificans LJC‑02 provided by the present invention has been preserved in the Guangdong Provincial Microbial Culture Collection Center on September 30, 2021, with a preservation number of GDMCC No: 61971. The LJC‑02 strain has good PAEs removal ability and aerobic denitrification performance, can effectively degrade a variety of PAEs pollutants, and the degradation rate can reach more than 80%, and is an efficient PAEs degrading bacteria. The LJC‑02 bacteria can also use nitrate nitrogen or DEHP as the only carbon source for aerobic denitrification and denitrification, and can achieve the simultaneous removal of DEHP and nitrate nitrogen in the aerobic stage. Therefore, it can be widely used in the repair and control of PAEs pollution at estuaries.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and more specifically relates to a strain of denitrifying achromobacterium LJC-02 capable of degrading PAEs and its application. Background Art

[0002] Plasticizers are widely used as plasticizers and softeners in the plastics industry and are important components of plastics. PAEs, as external plasticizers, are bonded to the plastic matrix by hydrogen bonds and van der Waals forces rather than chemical bonds, so they are easily released from plastic products and are widely detected in water bodies, sediments, and animal and plant samples at estuaries. PAEs have a stable structure and are not easily degraded. They have toxic and cumulative properties such as "teratogenicity, carcinogenicity, and mutagenicity", which seriously damage the structure and functional diversity of microbial communities in water bodies and inhibit the self-purification ability of various pollutants in the water environment.

[0003] Microbial degradation is an important way for PAEs to self-purify. It is well known that PAEs-contaminated environments contain a variety of degrading microorganisms. The reported PAEs-degrading microorganisms are mainly bacteria and fungi, such as Providencia, Bacillus subtilis, Mycobacterium and Mucor, Fusarium Subglutinans, Penicillium Funiculosum, etc. Bacteria play a major role in PAEs biodegradation due to their strong adaptability and diversity. At present, a certain amount of PAEs-degrading bacteria have been isolated and reported (isolated from urban wastewater, soil, sludge, etc.). However, there are few reports on PAEs-degrading microorganisms in the land-sea junction area. At present, the only known degrading bacteria isolated under high salt stress are Sphingomonas yanoikuyae DOS01, Rhodotorula mucilaginosa Mar-Y3 and marine filamentous fungus Aspergillus versicolor IR-M4. However, the way microorganisms degrade PAEs is different under different environmental conditions. Under aerobic conditions, PAEs are hydrolyzed to form monoesters and corresponding alcohol compounds, and then the intermediates are metabolized to phthalic acid (PA) under enzyme catalysis, and alcohol substances are generated at the same time (upstream pathway). Phthalic acid is ring-opened to form the corresponding organic acid, which is then converted into pyruvate, succinate and fumarate, etc., and enters the tricarboxylic acid cycle, and finally degraded into CO2 and H2O (downstream pathway). Little is known about the anaerobic degradation of PAEs, and it is difficult to achieve in the natural environment. Although the obtained degrading bacteria can use PAEs as the sole carbon source for growth, their degradation capacity is affected by different environmental factors, such as temperature, pH, salt ion concentration, pollutant concentration, etc. The biodegradation of PAEs at the estuary faces relatively more complex or extreme conditions (such as the interface between salt and fresh water, too high or too low pH, and the presence of multiple pollutants, etc.), resulting in poor degradation of PAEs at the estuary, long residual time, and severe inhibition of the growth of denitrifying bacteria and denitrification rate, which has a significant negative effect on denitrification in the aquatic ecological environment.

[0004] Organic pollution at the estuaries is often accompanied by eutrophication problems such as excessive ammonia nitrogen. For a long time, anaerobic denitrifying bacteria have been considered the only bacteria capable of denitrification. Due to the complexity of the actual environment, anaerobic denitrification is difficult to achieve in real ecosystems. Aerobic denitrifying bacteria are functional microorganisms that can simultaneously breathe oxygen and nitrate, which were discovered in the 1980s. Aerobic denitrifying bacteria break the barriers of traditional anaerobic denitrification and can metabolize nitrite nitrogen and nitrate nitrogen into nitrogen gas under the condition of dissolved oxygen in water. Aerobic denitrifying bacteria are one of the common bacteria in the natural environment. They have strong environmental adaptability, fast growth rate, short reproduction cycle, high genetic adaptability, and play a key role in the denitrification and self-purification process of various natural water ecological environments. At present, the aerobic denitrifying bacteria isolated from the environment mainly include Pseudomonas, Acinetobacter, Bacillus, Alcaligenes faecalis, Thiobacillus, etc. In recent years, more efficient aerobic denitrifying strains have been isolated and have been widely used in the remediation of large-scale polluted waters due to their simple process, good denitrification effect, and no need for acid and alkali supplementation.

[0005] So far, a large number of studies have been reported on colorless bacteria that can degrade PAEs or perform denitrification. However, there are relatively few reports on degradation bacteria that can simultaneously remove PAEs and nitrate nitrogen from water bodies under aerobic conditions and can tolerate higher salt concentrations. In view of the coexistence of PAEs and nitrate nitrogen in water bodies at estuaries, how to effectively remove these two pollutants at the same time has become a focus of attention. Therefore, discovering bacteria that have both PAEs removal capabilities and aerobic denitrification properties and applying them to domestic and industrial sewage outlets at estuaries will effectively reduce the toxicity of the two to environmental pollution and provide scientific support for the coordinated land and sea pollution prevention and control and ecological environmental protection for the ongoing total amount of pollutants entering the sea. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the above-mentioned defects and shortcomings and provide a strain of Achromobacter denitrifying LJC-02 capable of degrading PAEs and its application.

[0007] The first object of the present invention is to provide a strain of Achromobacter denitrifying LJC-02.

[0008] The second object of the present invention is to provide a new application of the denitrifying Achromobacterium LJC-02 strain.

[0009] The third object of the present invention is to provide a bacterial agent for degrading phthalates and / or denitrifying wastewater.

[0010] A fourth object of the present invention is to provide a method for degrading phthalates and / or removing nitrogen.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention isolated a strain of Achromobacter denitrificans LJC-02 with both DEHP removal and aerobic denitrification capabilities from the sediments of the estuary in Xiashan District, Zhanjiang City, Guangdong Province. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 30, 2021. The deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, and the deposit number is GDMCC No: 61971. The colony morphology of the LJC-02 strain is round, with a raised surface, milky white, opaque, moist, smooth and protruding. The strain was negative by Gram staining; catalase activity, oxidase activity, starch hydrolase activity, gelatin liquefaction and urease reaction were all positive; glucose and maltose hydrolysis, and indole reaction were all positive. Suitable growth conditions for the LJC-02 strain are: temperature range of 20-40°C, pH range of 5-8, salt concentration range of 0-4%, and inoculum range of 1-7%. The nucleotide sequence of 16S rDNA of the LJC-02 strain is shown in SEQ ID NO:1.

[0013] The present invention shows that the strain LJC-02 can perform aerobic denitrification under high salinity and aerobic conditions. When the nitrate content is 100-400 mg / L, LJC-02 has good denitrification performance, and the removal rates of NO3-N and NO2-N are both above 80%. The LJC-02 strain can efficiently degrade a variety of phthalic acid esters (PAEs), including diester phthalate (DEHP), dimethyl phthalate (DMP), dibutyl phthalate (DBP), diallyl phthalate (DAP), dioctyl phthalate (DOP), and diethyl phthalate (DEP), with degradation rates of 97.20%, 88.02%, 76.08%, 94.73%, 91.84% and 82.59%, respectively; at the same time, the LJC-02 strain can simultaneously remove DEHP and denitrify under high nitrate conditions, with a DEHP degradation rate of more than 90%, and NO3-N and NO2-N removal rates of more than 80%.

[0014] Therefore, the present invention provides the use of the Achromobacter denitrificans LJC-02 strain in degrading phthalates and / or denitrifying, in repairing a phthalate-contaminated environment, or in preparing a bacterial agent for degrading phthalates and / or denitrifying wastewater.

[0015] Furthermore, the phthalate polluted environment is soil or water pollution.

[0016] Preferably, it is pollution at the estuary of a river entering the sea.

[0017] Preferably, the phthalate ester is one or more of diester phthalate, dimethyl phthalate, dibutyl phthalate, diallyl phthalate, dioctyl phthalate or diethyl phthalate.

[0018] The invention provides a bacterial agent for degrading phthalates and / or denitrifying wastewater, comprising achromobacter denitrifying LJC-02 strain or its bacterial solution.

[0019] Preferably, the bacterial solution concentration is not less than 2.0×10 8 CFU / mL.

[0020] The present invention provides a method for degrading phthalates and / or removing nitrogen, which is characterized in that a sample to be treated is treated with a denitrifying Achromobacterium LJC-02 strain.

[0021] Preferably, the inoculation amount of the LJC-02 strain is 1-7%, the salt concentration during treatment ranges from 0-4%, the nitrate content is 100-400 mg / L, and the phthalate diester concentration is 100-500 mg / L.

[0022] The present invention has the following beneficial effects:

[0023] (1) The Achromobacter denitrificans LJC-02 provided by the present invention can effectively degrade different PAEs (DEHP, DMP, DBP, DAP, DOP, DEP) pollutants, and the degradation rate can reach more than 80%. Among them, the degradation effect of long ester chain PAEs is the best. It is a highly efficient degradation bacterium and has important practical significance and value for the degradation of PAEs and the repair of PAEs-polluted environment.

[0024] (2) The LJC-02 strain provided by the present invention is pollution-free and harmless during use, can use nitrate nitrogen or DEHP as the sole carbon source for aerobic denitrification, and can remove DEHP at the same time, and can achieve the simultaneous removal of DEHP and nitrate nitrogen in the aerobic stage. At the same time, it has good tolerance to salinity, so that it still has a high degradation efficiency in a high-salt, high-nitrate nitrogen and high-DEHP environment. The LJC-02 strain provided by the present invention has good PAEs removal ability and aerobic denitrification performance, can be widely used in the repair and treatment of PAEs pollution at estuaries, and has good economic value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The morphological characteristics of the LJC-02 strain;

[0026] Figure 2 is the phylogenetic tree of the LJC-02 strain;

[0027] Figure 3 The optimal growth conditions for the LJC-02 strain;

[0028] Figure 4 The denitrification performance of LJC-02 strain under different nitrate concentrations;

[0029] Figure 5 The degradation performance of LJC-02 strain on different PAEs;

[0030] Figure 6 The degradation performance of LJC-02 strain on different concentrations of DEHP;

[0031] Figure 7 The performance of LJC-02 strain in simultaneous removal of DEHP and denitrification under high nitrate conditions. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0034] The culture medium used in the following examples is as follows:

[0035] Denitrification medium: 4.72 g / L CH3COONa, 0.61 g / L NaNO3, 1.5 g / L K2HPO4·1 2H2O, 0.20 g / L MgSO4·7H2O, 0.01 g / L CaCl2, 0.005 g / L FeSO4·7H2O, pH = 7. 0±0.2.

[0036] High nitrate culture medium: 4.72g / L CH3COONa, 2.44g / L NaNO3, 1.5g / L K2HP O4·12H2O, 0.20g / L MgSO4·7H2O, 0.01g / L CaCl2, 0.005g / L FeSO4·7H2O, pH=7.0±0.2.

[0037] Inorganic salt culture medium: (NH4)2SO3 1.0 g, NaCl 0.5 g, NH4NO3 1.0 g, KH2PO4 0.5 g, CaCl2 0.01 g, K2HPO4·12H2O 1.5 g, yeast extract 0.005 g, pH = 7.0±0.2.

[0038] The composition of LB medium was as follows: 10.0 g / L peptone, 5.0 g / L NaCl, 10.0 g / L yeast extract, pH = 7.0 ± 0.2.

[0039] The solid plate culture medium used was a corresponding culture medium with 20 g of agar added.

[0040] The nucleotide sequence of 16S rDNA of LJC-02 strain is as follows:

[0041] SEQ ID NO:1:TTATGTTTGATCCTGGCTCAGATTGAACGCTAGCGGGA TGCCTTACACATGCAAGTCGAACGGCAGCACGGACTTCGGTCTGGTGGCGAGTGGCGAACGGGTGAGTAATGTATCGGAACGTGCCCAGTAGCGGGGGA TAACTACGCGAAAGCGTAGCTAATACCGCATACGCCCTACGGGGGAAAGC AGGGGATCGCAAGACCTTGCACTATTGGAGCGGCCGATATCGGATTAGCTAGTTGGGGTAACGGCTCACCAAGGCGACGATCCGTAGCTGGTTTGAG AGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGAAACCCTGATCCAGCCAT CCCGCGTGTGCGATGAAGGCCTTCGGGTTGTAAAGCACTTTTGGCAGGAA AGAAACGTCGCGGGTTAATACCTCGCGAAACTGACGGTACCTGCAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAG CGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTCGGAAAGAAAGATGTGAAATCCCAGAGCTTAACTTTGGAACTGCATTTTTAACTACCGGGCTAGAGTGTGTCAGAGGGAGGTGGAATTCCGCGTGTAGCAGTGAAATGC GTAGATATGCGGAGGAACACCGATGGCGAAGGCAGCCTCCTGGGATAACACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCT GGTAGTCCACGCCCTAAACGATGTCAACTAGCTGTTGGGGCCTTCGGGCCTTGGTAGCGCAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGC AAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTCTGGAATGCCGAAGAGATTTGGCAGTGCTCGCAAGAGAACCGGAACACA GGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACGAAAGGGCACTCTAAT GAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCT CATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGGACAGAGGGTCGCCAACCCGCGAGGGGGAGCCAATCCCAGAAACCCGATCGTAGTC CGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGC CCGTCACACCATGGGAGTGGGTTTTACCAGAAGTAGTTAGCCTAACCGCAAGGGGGGCGATTACCACGGTAGGATTCATGACT

[0042] Example 1 Enrichment, separation, purification and characterization of strains

[0043] (1) Enrichment of degrading bacteria

[0044] Sediment samples were collected from the sediments of the estuary in Xiashan District, Zhanjiang City, Guangdong Province. Under aseptic operation, 10g of sediment samples were accurately weighed and placed in a 250mL triangular flask containing 90mL of sterile water (with glass beads). The samples were shaken for 30min to fully mix the samples with the water, disperse the cells, and form a uniform bacterial suspension. After standing for 20-30s, 2mL of supernatant was drawn into the inorganic salt medium containing 100mg / L DEHP (with DEHP as the only nitrogen source), and cultured at 30℃ and 180rpm in the dark. After 5 days of culture, the inoculation was again inoculated in the inorganic salt medium containing 200mg / L DEHP at a 2% inoculation amount, and cultured under the same conditions, and so on, until the DEHP concentration in the inorganic salt medium was 500mg / L. Each enrichment culture has a blank control without adding bacterial solution.

[0045] (2) Isolation and purification of degradation bacteria

[0046] The enrichment culture at the DEHP acclimation concentration of 500 mg / L was diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 were taken for each treatment -4 , 10 -5 , 10 -6 100 μL of the dilution was spread on a solid culture medium plate with the corresponding DEHP concentration (500 mg / L) and placed in a 30°C constant temperature incubator in the dark for incubation. Repeat 3 times until purified highly efficient DEHP-degrading bacteria were isolated and obtained.

[0047] (3) Denitrification performance measurement

[0048] The purified single efficient DEHP degrading bacteria were inoculated into the denitrification identification medium and cultured for 2-3 days. The denitrification of the strain caused the pH of the medium to increase. Due to the acid-base indicator characteristics of BTB, the medium will change from green to blue, and the strains with denitrification performance can be preliminarily screened out, and the denitrification capacity can be preliminarily inferred based on the degree of color change. In addition, the nitrate nitrogen and nitrite nitrogen of BTB were tested, and the highest nitrate nitrogen and nitrite nitrogen removal rates were used as the main indicators to screen out the strains with both DEHP removal and efficient aerobic denitrification capabilities, and the strain was named LJC-02.

[0049] (3) Identification of strain characteristics

[0050] 3.1 Morphological characteristics of strains

[0051] The LJC-02 strain is a Gram-negative bacterium with colony characteristics such as Figure 1 As shown, the colonies formed after culturing on LB medium for 24 h were round, with raised surfaces, milky white, opaque, moist, smooth and protruding.

[0052] 3.2 Physiological and biochemical characteristics of strains

[0053] The LJC-02 strain was negative by Gram staining; catalase activity, oxidase activity, starch hydrolase activity, gelatin liquefaction and urease reaction were all positive; glucose and maltose hydrolysis, indole reaction were all positive.

[0054] 3.3 Molecular biological characteristics

[0055] The total bacterial DNA was extracted using a kit. The bacterial 16S rDNA universal primers 27F and 1492R were used to PCR amplify the bacterial 16S rDNA, and a clear band appeared near 1400 bp. The PCR amplification product was recovered and sequenced. The nucleotide sequence of the 16S rDNA of the LJC-02 strain is shown in SEQ ID NO: 1. The obtained DNA sequence was subjected to Blast comparison analysis on the NCBI website (http: / / www.ncbi.nlm.nih.gov), and the phylogenetic tree was constructed using MEGA software (version: 7.0) as shown in Figure 2 As shown, it was found that the 16S rDNA sequence of the strain of the present invention has a high homology with Achromobacter denitrificans in the genus Achromobacter sp. in GenBank. Combined with the above-mentioned morphological characteristics and the results of the 16S rDNA sequence, the strain isolated by the present invention was identified as Achromobacter denitrificans, named LJC-02, and preserved in the Guangdong Provincial Microbial Culture Collection Center on September 30, 2021, with a preservation number of GDMCC No: 61971, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0056] Example 2 Experiment on the optimal growth conditions of LJC-02 strain

[0057] (1) Effect of temperature on strain growth

[0058] The seed solution (OD 600 =0.8) was inoculated into the inorganic salt medium containing DEHP at a 1% inoculum, and 3 replicates were set up. The culture was cultured at 10, 20, 30, 40, and 50°C and 180 rpm / min under constant temperature shaking conditions. After culturing for 3 days, samples were taken to determine the growth of the strain (OD 600 ).

[0059] (2) Effect of pH on strain growth

[0060] The seed solution (OD 600 =0.8) was inoculated into an inorganic salt medium containing DEHP at a 1% inoculum, and the pH was set to 4, 5, 6, 7, 8, 9, and 10, respectively. A total of 3 replicates were set and cultured at 30°C and 180 rpm / min under constant temperature shaking. After culturing for 3 days, samples were taken to determine the growth of the strain (OD 600 ).

[0061] (3) Effect of salt concentration on strain growth

[0062] The seed solution (OD 600 =0.8) was inoculated into the inorganic salt medium containing DEHP at a 1% inoculum, and the salt concentrations were set to 0, 2, 4, 6, 8, and 10%, respectively. A total of 3 replicates were set and cultured at 30°C and 180 rpm / min under constant temperature shaking conditions. After culturing for 3 days, samples were taken to determine the growth of the strain (OD 600 ).

[0063] (4) Effect of inoculum size on strain growth

[0064] The seed solution (OD 600 =0.8) were inoculated into the inorganic salt medium containing DEHP at an inoculum of 1, 3, 5, 7, and 9%, with a total of 3 replicates, and cultured at 30°C and 180 rpm / min under constant temperature shaking conditions. After culturing for 3 days, samples were taken to determine the growth of the strain (OD 600 ).

[0065] The results are as follows Figure 3 As shown, the suitable culture conditions for strain LJC-02 are: temperature range of 20-40°C, pH range of 5-8, salt concentration range of 0-4%, and inoculum range of 1-7%.

[0066] Example 3 Denitrification performance experiment of LJC-02 under different nitrate concentration conditions

[0067] The seed solution (OD 600 =0.8) was inoculated at 3% into denitrification culture media with nitrate contents of 100, 200, 300, 400 and 500 mg / L, respectively, and the salt concentration was set to 4%. A total of 3 replicates were set and cultured at 30°C, 180 rpm / min under constant temperature shaking conditions. After culturing for 5 days, samples were taken to determine the residual contents of NO3-N and NO2-N in the culture fluid. The culture fluid without inoculation of the strain was used as the blank control, and the removal rates of NO3-N and NO2-N were calculated.

[0068] The results are as follows Figure 4 As shown in the figure, when the nitrate content is 100-400 mg / L, strain LJC-02 has a good denitrification performance, and the removal rates of NO3-N and NO2-N are both above 80%. When the nitrate content is greater than 400 mg / L, strain LJC-02 has a poor denitrification performance. Therefore, this strain can effectively remove NO3-N and NO2-N under the condition of nitrate content of 100-400 mg / L.

[0069] Example 4 Experiment on the degradation performance of LJC-02 on different PAEs

[0070] The seed solution (OD 600 =0.8) was inoculated at 3% into an inorganic salt culture medium containing 100 mg / L of DEHP, DMP, DBP, DAP, DOP, and DEP, respectively, and the salt concentration was set to 4%. A total of 3 replicates were set and cultured at 30°C, 180 rpm / min under constant temperature shaking conditions. After culturing for 5 days, samples were taken to determine the residual concentration of each PAEs in the culture medium. The culture medium without the inoculated strain was used as the blank control, and the degradation rate of each PAEs was calculated.

[0071] The results are as follows Figure 5 As shown in the results, strain LJC-02 had a good degradation effect on DEHP, DOP and DAP, with degradation rates of 97.20%, 91.84% and 94.73% respectively, and a slightly poorer degradation effect on DMP, DBP and DEP, with degradation rates of 88.02%, 76.08% and 82.59% respectively. Therefore, it is speculated that strain LJC-02 prefers to degrade PAEs with long ester chains.

[0072] Example 5 Experiment on the degradation performance of LJC-02 to different concentrations of DEHP

[0073] The seed solution (OD 600 =0.8) was inoculated at 3% into inorganic salt culture medium with DEHP concentrations of 100, 200, 300, 400 and 500 mg / L, respectively, and the salt concentration was set to 4%. There were 3 replicates in total, and cultured at 30°C, 180 rpm / min under constant temperature shaking conditions. After culturing for 5 days, samples were taken to determine the residual DEHP concentration in the culture medium. The culture medium without the inoculated strain was used as the blank control, and the DEHP degradation rate was calculated.

[0074] The results are as follows Figure 6 As shown in the figure, when the DEHP concentration is 100-300 mg / L, the strain LJC-02 has a good ability to degrade DEHP, and the DEHP degradation rate is above 80%. When the DEHP concentration is greater than 300 mg / L, the strain LJC-02 has a poor performance in degrading DEHP. Therefore, the strain LJC-02 can effectively degrade DEHP in the presence of a DEHP concentration of 100-300 mg / L.

[0075] Example 6: Simultaneous removal of DEHP and denitrification performance of LJC-02 under high nitrate conditions

[0076] The seed solution (OD 600=0.8) were inoculated at 3% in a high nitrate medium containing 100, 200, 300, 400, and 500 mg / L DEHP, respectively, and the salt concentration was set to 4%. A total of 3 replicates were set and cultured at 30°C, 180 rpm / min under constant temperature shaking conditions. After culturing for 5 days, samples were taken to determine the residual concentration of each DEHP in the culture solution, the residual contents of NO3-N and NO2-N, and the culture solution without inoculation was used as a blank control to calculate the degradation rate of each DEHP and the removal rates of NO3-N and NO2-N.

[0077] The results are as follows Figure 7 As shown in the figure, when the DEHP concentration is 100-200 mg / L DEHP, the strain LJC-02 has good DEHP removal and denitrification performance, the DEHP degradation rate is above 90%, and the NO3-N and NO2-N removal rates are above 80%. When the DEHP concentration is greater than 200 mg / L, the strain LJC-02 has poor removal effects on DEHP and nitrate. Therefore, the strain LJC-02 can effectively degrade DEHP and has efficient denitrification performance under the condition of DEHP concentration of 100-200 mg / L.

[0078] Example 7 Experiment on simultaneous removal of DEHP and denitrification performance of LJC-02 at an actual estuary

[0079] The strain LJC-02 has good PAEs removal ability and aerobic denitrification performance, and is used in the restoration and management of polluted water bodies at the estuary with high salt, high nitrate nitrogen and high DEHP. The polluted water bodies at the estuary used in this example were taken from the sea area near the Nanliu River estuary in Zhanjiang City, Guangdong Province. The seed liquid (OD 600 =0.8) was inoculated at 3% into the polluted water (100 mL) of the estuary with high salt, high nitrate nitrogen and high DEHP. The properties of the polluted water are as follows: pH 7.8, salinity 3.3%, nitrate nitrogen content 327 mg / L, DEHP concentration 184 mg / L. A blank control and experimental treatment were set at the same time.

[0080] The results are shown in Table 1. In the blank treatment, the denitrification effect was greatly limited in the presence of DEHP. On the contrary, in the treatment after the strain LJC-02 was added, the nitrate nitrogen removal rate of the polluted water entering the estuary after 96 hours was 89.62%, the total nitrogen removal rate was 77.85%, and the DEHP degradation rate was 90.38%.

[0081] Table 1 Treatment of polluted water bodies in estuaries with and without inoculation

[0082]

[0083] In summary, the denitrifying colorless bacteria (Achromobacter denitrificans) LJC-02 provided by the present invention can effectively degrade different PAEs (DEHP, DMP, DBP, DAP, DOP, DEP) pollutants, and the degradation rate can reach more than 80%, among which the degradation effect of long ester chain PAEs is the best, and it belongs to efficient degradation bacteria, which has important practical significance and value for the degradation of PAEs and the repair of PAEs polluted environment. The LJC-02 strain is pollution-free and pollution-free during use, and can use nitrate nitrogen or DEHP as the only carbon source for aerobic denitrification, denitrification, and removal of DEHP at the same time, and can achieve the simultaneous removal of DEHP and nitrate nitrogen in the aerobic stage. At the same time, it has a good tolerance to salinity, so that it still has a high degradation efficiency in a high-salt, high-nitrate nitrogen and high-DEHP environment. The LJC-02 strain provided by the present invention has good PAEs removal ability and aerobic denitrification performance, can be widely used in the restoration and treatment of PAEs pollution at estuaries, and has good economic value and application prospects.

[0084] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Achromobacter denitrificans ( Achromobacter denitrificans ) LJC-02 strain, characterized in that The strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 30, 2021, with the collection number GDMCC No: 61971.

2. The use of the denitrifying Achromobacter denitrifying strain LJC-02 according to claim 1 in simultaneously degrading di(2-ethylhexyl) phthalate in polluted water and removing nitrate nitrogen in polluted water, characterized in that: The nitrate content in the polluted water is 100-400 mg / L, and the concentration of di(2-ethylhexyl) phthalate is 100-500 mg / L.

3. Application of the denitrifying Achromobacterium LJC-02 strain described in claim 1 in simultaneously degrading di(2-ethylhexyl) phthalate, dimethyl phthalate, dibutyl phthalate, diallyl phthalate, dioctyl phthalate and diethyl phthalate in polluted water.

4. Use of the denitrifying Achromobacter LJC-02 strain according to claim 1 in removing nitrate nitrogen or nitrite nitrogen from polluted water.

5. The use of the Achromobacter denitrificans LJC-02 strain according to claim 1 in repairing a water environment contaminated by phthalates, characterized in that: The phthalate is one or more of di(2-ethylhexyl)phthalate, dimethyl phthalate, dibutyl phthalate, diallyl phthalate, dioctyl phthalate or diethyl phthalate; and the concentration of the di(2-ethylhexyl)phthalate is 100-500 mg / L.

6. The use according to claim 5, characterized in that: The water environment polluted by the phthalates is pollution at the river estuary.

7. The use of the denitrifying Achromobacter denitrificans LJC-02 strain according to claim 1 in the preparation of a bacterial agent for degrading di(2-ethylhexyl) phthalate in polluted water and removing nitrate nitrogen in polluted water, characterized in that: The nitrate content in the polluted water is 100-400 mg / L, and the concentration of di(2-ethylhexyl) phthalate is 100-500 mg / L.

8. A bacterial agent for simultaneously degrading di(2-ethylhexyl) phthalate in polluted water and removing nitrate nitrogen in polluted water, characterized in that: Contains the denitrifying Achromobacter LJC-02 strain as described in claim 1.

9. The bacterial agent according to claim 8, characterized in that: A bacterial solution containing the denitrifying Achromobacterium LJC-02 strain.

10. The bacterial agent according to claim 9, characterized in that The concentration of the bacterial solution is not less than 2.0×10 8 CFU / mL.

11. A method for simultaneously degrading di(2-ethylhexyl) phthalate in polluted water and removing nitrate nitrogen from polluted water, characterized in that: The denitrifying Achromobacter LJC-02 strain described in claim 1 is used to treat the sample to be treated; the nitrate content in the polluted water is 100-400 mg / L, and the concentration of di(2-ethylhexyl) phthalate is 100-500 mg / L.

12. The method according to claim 11, characterized in that: The inoculation amount of the LJC-02 strain is 1-7%, and the salt concentration range during treatment is 0-4%.

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