Halophilic fast-growing bacillus for degrading chlorinated persistent organic pollutants and application of halophilic fast-growing bacillus

Abstract

The invention discloses a halophilic fast-growing bacillus for degrading chlorinated persistent organic pollutants and application of the halophilic fast-growing bacillus. The bacterial strain is a halophilic fast-growing bacterium TBC005, and the preservation number of the halophilic fast-growing bacterium TBC005 is GDMCC No: 65236. The invention further discloses a preparation method of the halophilic fast-growing bacterium TBC005. The halophilic fast-growing bacillus TBC005 disclosed by the invention has an excellent degradation capability on chlorinated persistent organic pollutants, and the degradation rates of C10Cl6, TECP (Tetraethyl Cyclohexyl Phosphate) and TCPP (Transmission Control Protocol) can respectively reach 21.5 percent, 16.7 percent and 16.9 percent after the halophilic fast-growing bacillus TBC005 is cultured for 14 days under an anaerobic condition; after the strain is cultured for 35 days, the degradation rates of C10Cl6, TECP and TCPP can reach 50.7%, 59.2% and 61.3% respectively, and the strain can be applied to the fields of environmental pollution abatement and bioremediation of chlorinated persistent organic pollutants.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a halophilic fast-growing bacillus strain that degrades chlorinated persistent organic pollutants and its applications. Background Technology

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

[0003] Chlorinated POPs are particularly difficult to treat due to the stability of their chemical structure and the 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, developing an economical, efficient, and environmentally friendly technology for treating chlorinated POPs is of significant practical importance.

[0004] In recent years, microbial degradation technology has received widespread attention due to its environmental friendliness and cost-effectiveness. Microorganisms can transform complex organic pollutants into harmless or less 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 demonstrated excellent pollutant degradation capabilities under extreme environmental conditions. However, current research on the degradation of chlorinated POPs by halophilic microorganisms is relatively limited, particularly lacking efficient, rapid, and adaptable halophilic strains. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a novel halophilic microbial strain and its application in the degradation of chlorinated pollutants (POPs). This strain can effectively degrade chlorinated POPs and exhibits rapid growth and high salt tolerance. Furthermore, the present invention also relates to methods for applying this strain in environmental pollution control and bioremediation, providing a novel solution for the treatment of chlorinated POPs.

[0007] The first objective of this invention is to provide a strain of halophilic fast-growing bacillus (Celeribacter baekdonensis) TBC005, with accession number GDMCC No:65236.

[0008] A second objective of the present invention is to provide a bacterial agent comprising the aforementioned halophilic fast-growing bacillus 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 C l6 ) 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 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 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 graph shows the degradation rate of chlorinated POPs by Celeris baekdonensis TBC005. Detailed Implementation

[0019] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the embodiments described in this specification are merely illustrative of the invention and are not intended to limit the invention.

[0020] Example 1: Enrichment, isolation, purification and identification of Halophyte TBC005

[0021] (1) Enrichment culture of chlorinated POPs degrading bacteria: 1g of sediment sample collected from the cold seep of the seahorse in the South China Sea was added to 100mL of anaerobic enrichment medium (containing 100ppb of 55.5% Cl short-chain chlorinated paraffin) and cultured for 28 days under low temperature, light-free and anaerobic conditions for the first enrichment culture; 5mL of the first enrichment culture was transferred to a new 100mL of anaerobic enrichment medium (containing 1000ppb of 55.5% Cl short-chain chlorinated paraffin) and cultured for the second enrichment culture under the same conditions; 5mL of the second enrichment culture was transferred to a new 100mL of anaerobic enrichment medium (containing 1000ppb of 55.5% Cl short-chain chlorinated paraffin) and cultured for the third enrichment culture under the same conditions. This process was repeated for the fourth and fifth enrichment cultures. The fourth and fifth enrichment cultures contained 5000ppb of 55.5% Cl short-chain chlorinated paraffin.

[0022] Preparation of anaerobic enrichment medium: Purge nitrogen into 50 mL of basal salt medium for 15 min, add 0.05 g of cysteine, purge nitrogen again for 5 min, add the corresponding concentration of 55.5% Cl short-chain chlorinated paraffin, and sterilize at 121℃ for 20 min. After sterilization and cooling to room temperature, add 50 μL of filtered and sterilized vitamin solution, 50 μL of trace element solution, and 400 μL of K2HPO4 solution (5%, g / mL) to a laminar flow hood.

[0023] The basic salt culture medium consists of the following components: 0.8g NH4NO3, 0.5g KCl, 0.5g CaCl2·2H2O, 3.0g MgCl2·6H2O, 22g NaCl, 3.0g Na2SO4, 5g PIPES buffer, 1mL 1g / L resazurin, and 1L ultrapure water, adjusted to pH 6.7.

[0024] Preparation of vitamin solution: Biotin (VH, VB7) 2.0mg, folic acid (VB9) 2.0mg, pyridoxine·HCl (VB6) 10.0mg, riboflavin (VB2) 5.0mg, thiamine (VB1) 5.0mg, niacin (VB3) 5.0mg, pantothenic acid (VB5) 5.0mg, VB-12 (VB12) 0.1mg, para-aminobenzoic acid 5.0mg, lipoic acid 5.0mg and ultrapure water 1L. The vitamin mixture is filtered through a 0.22μm filter membrane for sterilization.

[0025] The trace element solution consists of the following components: 1.50 g of hyponitrotriacetic 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. The trace element mixture is sterilized by filtration through a 0.22 μm filter membrane.

[0026] (2) Isolation and purification of bacteria: The culture medium after the fifth enrichment culture was serially diluted 10 times, spread on MA plates, and cultured at 4°C under dark and anaerobic conditions. After the colonies grew, single colonies with different morphologies were picked from the plates in a clean bench and purified by streaking multiple times on MA plates to obtain halophilic fast-growing bacillus TBC005.

[0027] Preparation of MA plates: Add 15g of agar and 37.4g of Marine Broth 2216 to 1L of ultrapure water to prepare a solution. Sterilize at 121℃ for 20min, and after cooling to about 60℃, pour into petri dishes and let solidify overnight before use.

[0028] Bacterial identification: DNA was extracted from *Celeribacter baekdonensis* TBC005, and the 16S rRNA gene fragment was amplified and sequenced using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5'-GGTTACCTTGTTACGACTT-3'). 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, followed by 30 cycles of denaturation, annealing, and extension, and then 70℃ for 10 min. The sequence of *Celeribacter baekdonensis* TBC005 is shown in SEQ ID NO.1. The 16S rRNA gene sequence was compared on the EzBiocloud website, and it was found to have the highest similarity (100%) to *Celeribacter baekdonensis* DSM 27375. The results showed that the strain TBC005 of this invention belongs to the genus Celeris, and it was named Celeris baekdonensis TBC005. This strain 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.

[0029] SEQ ID NO.1

[0030] CTTCGGATCTAGCGGCGGACGGGTTAGTAACGCGTGGGAACGTACCCAGATCTACGGAATAG

[0031] CCTCGGGAAACTGAGAGTAATACCGTATACGCCCTTCGGGGGAAAGATTTATCGGATTTGGA

[0032] TCGGCCCGCGTAAGATTAGATAGTTGGTGGGGTAATGGCCTACCAAGTCTACGATCTTTAGCT

[0033] GGTTTGAGAGGATGATCAGCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCA

[0034] GCAGTGGGGAATCTTAGACAATGGGCGCAAGCCTGATCTAGCGATGCCGCGTGAGTGATGAA

[0035] GGCCTTAGGGTCGTAAAGCTCTTTCGCCTGTGAAGATAATGACGGTAGCAGGTAAAGAAACC

[0036] CCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGTTAGCGTTGTTCGGAATTAC

[0037] TGGGCGTAAAGCGCACGTAGGCGGACTAGTCAGTCAGAGGTGAAATCCCAGGGCTCAACCCT

[0038] GGAACTGCCTTTGATACTGCTAGTCTTGAGTTCGAGAGAGGTAAGTGGAATTCCGAGTGTAG

[0039] AGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGCTCGATAC

[0040] TGACGCTGAGGTGCGAAAGTGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACACCG

[0041] TAAACGATGAATGCCAGACGTCGGGTAGCATGCTATTCGGTGTCACACCTAACGGATTAAGC

[0042] ATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACA

[0043] AGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAACCCTTGACATCC

[0044] TGATCGCGGATCGTAGAGATACTTTCCTTCAGTTCGGCTGGATCAGTGACAGGTGCTGCATGG

[0045] CTGTCGTCAGCTCGTGTCGTGAGATGTTCGGTTAAGTCCGGCAACGAGCGCAACCCACATCCT

[0046] TAGTTACCAGCGGTTTGGCCGGGGACTCTAGGGAAACTGCCCGTGATAAGCGGGAGGAAGGT

[0047] GTGGATGACGTCAAGTCCTCATGGCCCTTACGGGTTGGGCTACACACGTGCTACAATGGCAGT

[0048] GACAATGGGTTAATCCCAAAAAGCTGTCTCAGTTCGGATTGGGGTCTGCAACTCGGCCCCATG

[0049] AAGTCGGAATCGCTAGTAATCGCGTAACAGCATGACGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGGTCTACCCGACGGCCGTGCGCCAA.

[0050] Example 2: Detection of chlorinated POPs degradation by Halophyte TBC005

[0051] Degradation culture experiment: 10 mM sodium acetate was added to the anaerobic basic salt medium (formula same as the basic salt medium in Example 1), and 5 mL was dispensed into 40 mL glass bottles. 1000 ppb of C was added to each bottle. 10 Cl6, TCEP, and TCPP were used as the sole carbon sources, and then 1 mL of methane gas was added and connected to the OD. 600 A 0.5 μL suspension of Celeris baekdonensis TBC0055 was cultured at 4°C under dark and anaerobic conditions. Each experimental group was replicated in triplicate. Samples were collected on days 14 and 35 of culture, pretreated, and the content was determined by gas chromatography.

[0052] Preparation of bacterial suspension: Celeris baekdonensis TBC0055 was inoculated on MA plates and cultured at 28°C under dark and aerobic conditions for 2-3 days. After colony growth, the bacterial cells were collected and washed 2-3 times with basal salt medium. Then, a bacterial suspension with OD=0.5 was prepared using basal salt medium.

[0053] Sample pretreatment: Add 5 mL of a hexane-dichloromethane (1:1, v / v) mixture to the collected sample, vortex, sonicate for 20 min, centrifuge at 5000 r / min for 5 min, and collect the supernatant. Extract twice more with the hexane-dichloromethane (1:1, v / v) mixture, and combine the three extracts. Transfer 300 μL of the extract to a vial, concentrate to near dryness under nitrogen, air dry, and then dilute to volume with 900 μL of hexane. Results are as follows: Figure 1 As shown, Celeris baekdonensis TBC0055 is effective against C 10 On day 14 of culture, the degradation rates of Cl6, TECP, and TCPP reached 21.5%, 16.7%, and 16.9%, respectively; and on day 35 of culture, the degradation rates reached 50.7%, 59.2%, and 61.3%, respectively.

[0054] Sample determination: C 10 Cl6, TECP, and TCPP samples were all determined by gas chromatography, with each sample measured three times. 10 The determination conditions for Cl6 were as follows: the chromatographic column was an HP-5 capillary column (30m × 0.32mm, 0.25μm); the column temperature was: initial temperature 60℃, increased to 120℃ at 20℃ / min and held for 3min; increased to 320℃ at 15℃ / min and held for 5min; the detector temperature was 280℃; the injection port temperature was 280℃; the carrier gas was N2, the carrier gas flow rate was 2.0mL / min; the split ratio was 5:1; and the injection volume was 1μL.

[0055] The TECP determination conditions were as follows: the chromatographic column was an HP-5 capillary column (30m × 0.32mm, 0.25μm); the column temperature was initially 90℃, increased to 140℃ at 20℃ / min, then increased to 320℃ at 15℃ / min and held for 6 min; the detector temperature was 280℃; the injection port temperature was 280℃; the carrier gas was N2, and the carrier gas flow rate was 2.0mL / min; splitless; and the injection volume was 5μL.

[0056] The TCPP determination conditions were as follows: the chromatographic column was an HP-5 capillary column (30m × 0.32mm, 0.25μm); the column temperature was: initial temperature 90℃, increased to 100℃ at 20℃ / min, increased to 255℃ at 10℃ / min, increased to 320℃ at 15℃ / min, and held for 5 min; the detector temperature was 280℃; the injection port temperature was 280℃; the carrier gas was N2, and the carrier gas flow rate was 2.0mL / min; splitless; and the injection volume was 5μL.

[0057] Figure 1The results showed that the halophilic fast-growing bacterium TBC0055 exhibited excellent degradation ability against chlorinated POPs, and after 14 days of anaerobic culture, it showed significant degradation ability against C60 POPs. 10 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 The degradation rates of Cl6, TECP, and TCPP can reach 50.7%, 59.2%, and 61.3%, respectively.

[0058] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Celeribacter baekdonensis TBC005, deposit number: GDMCC No: 65236.

2. A bacterial agent, characterized in that It comprises the halophilic fast-growing bacillus TBC005 described in claim 1.

3. Use of the halophilic fast-growing bacillus TBC005 according to claim 1 or the bacterial agent according to claim 2 in degrading chlorinated persistent organic pollutants.

4. The use according to claim 3, characterized in that: The chlorinated persistent organic pollutants include short-chain chlorinated paraffins and chlorinated organic phosphates.

5. The use according to claim 4, characterized in that: The chlorinated organic phosphates include tris(2-chloroethyl) phosphate and tris(1-chloro-2-propyl) phosphate.

6. A method for degrading chlorinated persistent organic pollutants, characterized in that: The halophilic fast-growing bacillus TBC005 described in claim 1 or the bacterial agent described in claim 2 is placed in an environment containing chlorinated persistent organic pollutants for growth.

7. The method according to claim 6, characterized in that The environment includes soil, water, atmosphere or organisms.

8. The method according to claim 6, characterized in that The growth includes aerobic growth, anaerobic growth or facultative growth.

9. The method according to claim 6, characterized in that The chlorinated persistent organic pollutants include short-chain chlorinated paraffins and chlorinated organic phosphates.

10. The method according to claim 9, characterized in that The chlorinated organic phosphates include tris(2-chloroethyl) phosphate and tris(1-chloro-2-propyl) phosphate.

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