A halotolerant bacterium of pseudomonas sp. for degrading chlorinated persistent organic pollutants and its application

Abstract

This invention discloses a halophilic fast-growing bacillus strain that degrades chlorinated persistent organic pollutants and its applications. The strain is *Celeribacter baekdonensis* TBC005, with accession number GDMCC No:65236. The *Celeribacter baekdonensis* TBC005 of this invention exhibits excellent degradation ability against chlorinated persistent organic pollutants. After 14 days of anaerobic culture, it shows significant degradation of C... 10 H 16 The degradation rates of Cl6, TECP, and TCPP reached 21.5%, 16.7%, and 16.9%, respectively; after 35 days of cultivation, the degradation rates of C... 10 H 16 The degradation rates of Cl6, TECP, and TCPP can reach 50.7%, 59.2%, and 61.3%, respectively, and they can be applied to the environmental pollution control and bioremediation of chlorinated persistent organic pollutants.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology, in particular to a halophilic Celeribacter baekdonensis strain capable of degrading chlorinated persistent organic pollutants and its application. BACKGROUND

[0002] Persistent organic pollutants (POPs) are organic compounds that are difficult to degrade in the environment, highly toxic, can accumulate in living organisms and be amplified through the food chain. These pollutants include, but are not limited to, polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans. Due to their potential threat to the environment and human health, the degradation and treatment of POPs has become a research hotspot in the field of environmental science and engineering.

[0003] Chlorinated POPs are particularly difficult to treat due to their chemical stability and difficulty of biodegradation. Traditional physical and chemical treatment methods, such as incineration, adsorption and chemical oxidation, are often costly and may generate secondary pollution. Therefore, it is of great practical significance to develop an economic, efficient and environmentally friendly treatment technology for chlorinated POPs.

[0004] In recent years, microbial degradation technology has received widespread attention due to its environmental friendliness and cost-effectiveness. Microorganisms can convert complex organic pollutants into harmless or low-toxic substances through their metabolic pathways. However, most known microbial strains capable of degrading POPs have limited efficiency in degrading chlorinated POPs, and often require specific environmental conditions to achieve optimal results.

[0005] Halophilic microorganisms, due to their unique physiological characteristics and metabolic pathways, have shown excellent pollutant degradation ability under extreme environmental conditions. However, current research on halophilic microorganisms degrading chlorinated POPs is still relatively limited, especially the lack of efficient, fast and adaptable halophilic strains. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application aims to provide a new halophilic microbial strain and its application in degrading chlorinated POPs. The strain can effectively degrade chlorinated POPs and has the characteristics of rapid growth and high salt tolerance. In addition, the present application also relates to the application method of the strain in the field of environmental pollution control and bioremediation, providing a new solution for the treatment of chlorinated POPs.

[0007] The first object of the present application is to provide a halophilic Celeribacter baekdonensis strain TBC005, with a preservation number of GDMCC No:65236.

[0008] The second object of the present application is to provide a microbial agent comprising the above-mentioned halophilic Celeribacter baekdonensis strain TBC005.

[0009] A third objective of this invention is to provide the application of the aforementioned halophilic fast-growing Bacillus TBC005 or its agent in the degradation of chlorinated persistent organic pollutants.

[0010] Preferably, the chlorinated persistent organic pollutants include short-chain chlorinated paraffins (C... 10 H 16 Cl6) and chlorinated organophosphates.

[0011] Preferably, the chlorinated organophosphates include tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP).

[0012] The fourth objective of this invention is to provide a method for degrading chlorinated persistent organic pollutants, which involves growing the aforementioned halophilic fast-growing Bacillus TBC005 or bacterial agent in an environment containing chlorinated persistent organic pollutants.

[0013] Preferably, the environment includes soil, water, atmosphere, or living organisms.

[0014] Preferably, the growth includes aerobic growth, anaerobic growth, or facultative growth.

[0015] The present invention has the following beneficial effects:

[0016] This invention provides a halophilic fast-growing bacterium (Celeribacter baekdonensis) TBC005 strain that degrades chlorinated POPs. This strain TBC005 exhibits excellent degradation ability against chlorinated POPs, showing significant degradation of C4Ps after 14 days of anaerobic culture. 10 H 16 The degradation rates of Cl6, TECP, and TCPP reached 21.5%, 16.7%, and 16.9%, respectively; after 35 days of cultivation, the degradation rates of C... 10 H 16 The degradation rates of Cl6, TECP, and TCPP can reach 50.7%, 59.2%, and 61.3%, respectively.

[0017] Celeris baekdonensis TBC005 was deposited on October 10, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No:65236. Attached Figure Description

[0018] Figure 1The degradation rate results chart of chlorinated POPs degraded by Celeribacter baekdonensis TBC005. DETAILED DESCRIPTION

[0019] In order to make the purposes, technical solutions and beneficial technical effects of the present application clearer, the present application will be further described in detail below in combination with embodiments. It should be understood that the embodiments described in the present specification are only for explaining the present application, and are not intended to limit the present application.

[0020] Example 1: enrichment, isolation, purification and identification of Celeribacter baekdonensis TBC005

[0021] (1) Chlorinated POPs degrading bacterial community enrichment culture: 1 g of sediment sample collected from the South China Sea seahorse cold spring was added to 100 mL anaerobic enrichment culture medium (containing 100 ppb of 55.5% Cl short-chain chlorinated paraffin), and cultured under low temperature, no light and anaerobic conditions for 28 days for the first enrichment culture; 5 mL of the first enrichment culture was removed and inoculated into a new 100 mL anaerobic enrichment culture medium (containing 1000 ppb of 55.5% Cl short-chain chlorinated paraffin), and cultured under the same conditions for the second enrichment culture; 5 mL of the second enrichment culture was removed and inoculated into a new 100 mL anaerobic enrichment culture medium (containing 1000 ppb of 55.5% Cl short-chain chlorinated paraffin), and cultured under the same conditions for the third enrichment culture, and so on for the fourth and fifth enrichment cultures. The fourth and fifth enrichment culture media contained 5000 ppb of 55.5% Cl short-chain chlorinated paraffin.

[0022] Preparation of anaerobic enrichment culture medium: 50 mL of basic salt culture medium was purged with nitrogen for 15 min, 0.05 g of cysteine was added, and then purged with nitrogen for another 5 min. The corresponding concentration of 55.5% Cl short-chain chlorinated paraffin was added, and sterilized at 121°C for 20 min. After sterilization was completed and the temperature dropped to room temperature, 50 μL of filtered bacteria-free vitamin solution, 50 μL of trace element solution and 400 μL of K2HPO4 solution (5%, g / mL) were added in a clean bench.

[0023] The basic salt culture medium is composed of NH4NO30.8 g, KCl 0.5 g, CaCl2·2H2O 0.5 g, MgCl2·6H2O 3.0 g, NaCl 22 g, Na2SO4 3.0 g, PIPES buffer 5 g, 1 g / L resazurin 1 mL, ultrapure water 1 L, and the pH value is adjusted to 6.7.

[0024] The vitamin solution was prepared by mixing 2.0 mg of biotin (VBH, VB7), 2.0 mg of folic acid (VB9), 10.0 mg of pyridoxine-HCl (VB6), 5.0 mg of riboflavin (VB2), 5.0 mg of thiamine (VB1), 5.0 mg of nicotinic acid (VB3), 5.0 mg of pantothenic acid (VB5), 0.1 mg of VB-12 (VB12), 5.0 mg of p-aminobenzoic acid, 5.0 mg of lipoic acid, and 1 L of ultrapure water, and filtering the mixture through a 0.22-μm filter to sterilize it.

[0025] The trace element solution was prepared by mixing 1.50 g of nitrilotriacetic acid, 3.0 g of MgSO4-7H2O, 0.5 g of MnSO4-2H2O, 1.0 g of NaCl, 0.1 g of FeSO4-7H2O, 0.1 g of CoCl2, 0.1 g of CaCl2-2H2O, 0.1 g of ZnSO4, 0.01 g of CuSO4-5H2O, 0.01 g of AlK(SO4)2-12H2O, 0.01 g of H3BO3, 0.01 g of Na2MoO4-2H2O, and 1 L of ultrapure water, and filtering the mixture through a 0.22-μm filter to sterilize it.

[0026] (2) Isolation and purification of bacteria: The culture solution after the fifth enrichment culture was diluted by 10-fold gradient, spread on MA plates, and incubated at 4°C in the dark and anaerobic conditions. After the colonies grew, single colonies with different morphologies were picked from the plates in a clean bench, and repeatedly streaked on MA plates to obtain Halomonas sp. TBC005.

[0027] Preparation of MA plates: 15 g of agar and 37.4 g of Marine Broth 2216 were added to 1 L of ultrapure water to prepare a solution, which was sterilized at 121°C for 20 min. After cooling to about 60°C, the solution was poured into petri dishes, and used after overnight solidification.

[0028] Identification of bacteria: DNA of the halotolerant Bacterium TBC005 was extracted, and a 16S rRNA gene fragment was amplified and sequenced by using 27F / 1492R universal primers, i.e., 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction conditions were pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 57℃ for 30 s, extension at 72℃ for 90 s, and denaturation, annealing and extension for 30 cycles, followed by 70℃ for 10 min. The sequence of the halotolerant Bacterium TBC005 was shown as SEQ ID NO. 1. The 16S rRNA gene sequence was compared on the EzBiocloud website, and it was found that it had the highest similarity with the Celeribacter baekdonensis DSM 27375 strain, with a similarity of 100%. The results show that the strain TBC005 of the application belongs to the genus Celeribacter, and it is named as the halotolerant Bacterium (Celeribacter baekdonensis) TBC005. The strain was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on October 10, 2024, which is located at No. 59 Building, 5th Floor, 100 Middle Martyrs Road, Guangzhou, Guangdong, China, with a postal code of 510070, and the preservation number is GDMCC No: 65236.

[0029] SEQ ID NO. 1

[0030] CTTCGGATCTAGCGGCGGACGGGTTAGTAACGCGTGGGAACGTACCCAGATCTACGGAATAGC

[0031] CTCGGGAAACTGAGAGTAATACCGTATACGCCCTTCGGGGGAAAGATTTATCGGATTTGGATCG

[0032] GCCCGCGTAAGATTAGATAGTTGGTGGGGTAATGGCCTACCAAGTCTACGATCTTTAGCTGGTT

[0033] TGAGAGGATGATCAGCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGT

[0034] GGGGAATCTTAGACAATGGGCGCAAGCCTGATCTAGCGATGCCGCGTGAGTGATGAAGGCCTT

[0035] AGGGTCGTAAAGCTCTTTCGCCTGTGAAGATAATGACGGTAGCAGGTAAAGAAACCCCGGCTA

[0036] ACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGTTAGCGTTGTTCGGAATTACTGGGCGTA

[0037] AAGCGCACGTAGGCGGACTAGTCAGTCAGAGGTGAAATCCCAGGGCTCAACCCTGGAACTGCC

[0038] TTTGATACTGCTAGTCTTGAGTTCGAGAGAGGTAAGTGGAATTCCGAGTGTAGAGGTGAAATTC

[0039] GTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGCTCGATACTGACGCTGAGGT

[0040] GCGAAAGTGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGAATG

[0041] CCAGACGTCGGGTAGCATGCTATTCGGTGTCACACCTAACGGATTAAGCATTCCGCCTGGGGAG

[0042] TACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG

[0043] GTTTAATTCGAAGCAACGCGCAGAACCTTACCAACCCTTGACATCCTGATCGCGGATCGTAGAG

[0044] ATACTTTCCTTCAGTTCGGCTGGATCAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGT

[0045] GAGATGTTCGGTTAAGTCCGGCAACGAGCGCAACCCACATCCTTAGTTACCAGCGGTTTGGCCG

[0046] GGGACTCTAGGGAAACTGCCCGTGATAAGCGGGAGGAAGGTGTGGATGACGTCAAGTCCTCAT

[0047] GGCCCTTACGGGTTGGGCTACACACGTGCTACAATGGCAGTGACAATGGGTTAATCCCAAAAA

[0048] GCTGTCTCAGTTCGGATTGGGGTCTGCAACTCGGCCCCATGAAGTCGGAATCGCTAGTAATCGC

[0049] GTAACAGCATGACGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGGTCTACCCGACGGCCGTGCGCCAA.

[0050] Example 2: Detection of Chlorinated POPs degradation by Celeribacter baekdonensis TBC005

[0051] Degradation culture experiment: 10 mM sodium acetate was added to the anaerobic base salt medium (formula same as the base salt medium in Example 1), 5 mL per bottle, and 1000 ppb of C 10 H 16 Cl6, TCEP and TCPP as the sole carbon source, then 1 mL of methane gas was added, and the OD 600 0.5 of Celeribacter baekdonensis TBC0055 bacterial suspension was inoculated, and cultured at 4°C, in the dark and anaerobic conditions. Each experimental group was set up in triplicate, and samples were collected on the 14th and 35th days of culture, pretreated, and determined by gas chromatograph.

[0052] Preparation of bacterial suspension: Celeribacter baekdonensis TBC0055 was inoculated on MA plates and cultured at 28°C, in the dark and aerobic conditions for 2-3 days. When the colonies grew, the bacterial cells were collected and washed with base salt medium for 2-3 times, and then the base salt medium was used to prepare the bacterial suspension with OD = 0.5.

[0053] Sample pretreatment: The collected sample was added with 5 mL of a mixed solution of n-hexane-dichloromethane (1:1, by volume), vortexed, ultrasonically extracted for 20 min, centrifuged at 5000 r / min for 5 min, and the supernatant was taken; the mixed solution of n-hexane-dichloromethane (1:1, by volume) was used for ultrasonic extraction twice, and the three extraction solutions were combined; 300 μL of the extraction solution was transferred into a sample vial, concentrated to near dryness by nitrogen blowing, naturally dried, and then diluted with 900 μL of n-hexane. The results are shown in Figure 1 As shown in the results, Celeribacter baekdonensis TBC0055 had a high degradation rate on C 10 H 16 The degradation rates of C16, TECP and TCPP were 21.5%, 16.7% and 16.9% respectively on the 14th day of culture, and the degradation rates were 50.7%, 59.2% and 61.3% respectively on the 35th day of culture.

[0054] Sample determination: The samples of C 10 H 16 The samples of C16, TECP and TCPP were determined by gas chromatography, and each sample was determined three times. 10 H 16 The determination conditions of C16 were as follows: the chromatographic column was an HP-5 capillary column (30 m x 0.32 mm, 0.25 μm); the column temperature was initially 60°C, increased to 120°C at a rate of 20°C / min, maintained for 3 min, increased to 320°C at a rate of 15°C / min, and maintained for 5 min; the detector temperature was 280°C; the injection port temperature was 280°C; the carrier gas was N2, and the carrier gas flow rate was 2.0 mL / min; the split ratio was 5:1; and the injection volume was 1 μL.

[0055] The determination conditions of TECP were as follows: the chromatographic column was an HP-5 capillary column (30 m x 0.32 mm, 0.25 μm); the column temperature was initially 90°C, increased to 140°C at a rate of 20°C / min, increased to 320°C at a rate of 15°C / min, and maintained for 6 min; the detector temperature was 280°C; the injection port temperature was 280°C; the carrier gas was N2, and the carrier gas flow rate was 2.0 mL / min; there was no split; and the injection volume was 5 μL.

[0056] The determination conditions of TCPP were as follows: the chromatographic column was an HP-5 capillary column (30 m x 0.32 mm, 0.25 μm); the column temperature was initially 90°C, increased to 100°C at a rate of 20°C / min, increased to 255°C at a rate of 10°C / min, increased to 320°C at a rate of 15°C / min, and maintained for 5 min; the detector temperature was 280°C; the injection port temperature was 280°C; the carrier gas was N2, and the carrier gas flow rate was 2.0 mL / min; there was no split; and the injection volume was 5 μL.

[0057] Figure 1 It is shown that the halophilic B. rapidus TBC0055 has excellent degradation ability for chlorinated POPs, and the degradation rates of C 10 H 16 The degradation rates of C16, TECP and TCPP can reach 21.5%, 16.7% and 16.9% respectively; and the degradation rates of C 10 H 16 The degradation rates of C16, TECP and TCPP can reach 50.7%, 59.2% and 61.3% respectively.

[0058] The above is only the preferred embodiment of the present application, it should be noted that the above preferred embodiment should not be considered as a limitation of the present application, the protection scope of the present application should be limited by the scope defined by the claims. For those skilled in the art, without departing from the spirit and scope of the present application, a number of improvements and refinements can also be made, which should be considered as the protection scope of the present application.

Claims

1. Celeribacter baekdonensis TBC005, with the preservation number of GDMCC No: 65236.

2. An inoculant characterized in that, A microbial inoculum comprising the Celeribacter baekdonensis TBC005 of claim 1.

3. Use of the Celeribacter baekdonensis TBC005 of claim 1 or the microbial inoculum of claim 2 in degrading chlorinated persistent organic pollutants, including short-chain chlorinated paraffins and chlorinated organophosphates. The chlorinated organophosphates include tris(2-chloroethyl) phosphate and tris(1-chloro-2-propyl) phosphate.

4. A method of degrading a chlorinated persistent organic pollutant, characterized by, The Celeribacter baekdonensis TBC005 of claim 1 or the microbial inoculum of claim 2 is grown in an environment containing chlorinated persistent organic pollutants, including short-chain chlorinated paraffins and chlorinated organophosphates, the chlorinated organophosphates including tris(2-chloroethyl) phosphate and tris(1-chloro-2-propyl) phosphate.

5. The method of claim 4, wherein, The environment includes soil, water, air, or a living organism.

6. The method of claim 4, wherein, The growth includes aerobic growth, anaerobic growth, or facultative growth.

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