Two trichloroethylene assimilating and degrading bacteria and their applications

Abstract

The present invention belongs to the field of environmental microbial technology, and specifically discloses two trichloroethylene-assimilating and degrading bacteria, Cytobacillus oceanisediminis SH48 and Lysinibacillus fusiformis SH13, and their applications. The strains of Cytobacillus oceanisediminis SH48 and Lysinibacillus fusiformis SH13 are deposited in the China Center for Type Culture Collection, with the deposit numbers being CCTCC M 2023370 and CCTCC M 2023371 respectively, and the deposit date being March 21, 2023. The growth of these two strains has low requirements for environmental conditions. They can use trichloroethylene as the sole carbon source and energy source for their own growth without adding additional growth substrates, effectively degrade chlorinated hydrocarbon pollutants, release inorganic free chloride ions, and there is no accumulation of toxic intermediate products. The strain or the microbial agent prepared therefrom can be used for the bioremediation of the soil or groundwater in industrial polluted sites contaminated with chlorinated hydrocarbons, and has important application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of environmental microorganisms, and in particular to two strains of trichloroethylene assimilation and degradation bacteria and applications thereof. Background Art

[0002] At the beginning of the last century, short-chain chlorinated hydrocarbons were manufactured in large quantities due to their stable chemical properties, ease of use and low price. With its widespread application and the continuous development of industry, a large number of chlorinated hydrocarbons have been released into the environment, among which trichloroethylene (TCE) is the most important pollutant. Because it can be absorbed into the human body through the respiratory tract and skin and has DNA damage, trichloroethylene is listed as a "three-hazard" (carcinogenic, teratogenic, mutagenic) compound by the International Agency for Research on Cancer. Therefore, it is urgent to develop economical, efficient and sustainable pollution remediation technology.

[0003] Compared with physical and chemical methods, bioremediation uses the metabolism of microorganisms for remediation, which can achieve harmlessness, no secondary pollution, and low treatment cost. It is an important method for controlling environmental pollution and has the advantages of economy, effectiveness, sustainability and environmental friendliness.

[0004] Current bioremediation methods focus on the reductive dechlorination process under anaerobic conditions. High-chlorinated olefins (tetrachloroethylene, trichloroethylene) are gradually reduced and dechlorinated to low-chlorinated olefins (dichloroethylene, vinyl chloride) under the action of anaerobic dechlorinating microorganisms. Under the action of certain microorganisms, they can continue to be reduced and completely dechlorinated to produce harmless ethylene. As the reductive dechlorination proceeds, the dechlorination rate slows down, and the process of dechlorinating vinyl chloride to ethylene is very slow, which is the key rate-limiting step in this process. However, most anaerobic dechlorinating microorganisms cannot completely reduce dechlorination, resulting in the accumulation of more toxic intermediates, accompanied by problems such as low remediation efficiency and long cycle. The types of microorganisms that can fully reduce dechlorination are rare and have strict requirements for the growth environment, which has limitations and high costs. Summary of the invention

[0005] To solve the above problems, one of the purposes of the present invention is to provide two strains of trichloroethylene assimilation-degrading bacteria, both of which can assimilate trichloroethylene under aerobic conditions, thereby achieving its degradation.

[0006] The present invention achieves the above object through the following technical solutions:

[0007] The two trichloroethylene assimilatory and degradative bacteria described in the present invention are Cytobacillus oceanisediminis SH48 and Lysinibacillus fusiformis SH13 from marine sediments, which are derived from soil contaminated with high concentrations of chlorinated hydrocarbons. Both belong to the family Bacillaceae and are deposited in the China Center for Type Culture Collection at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is March 21, 2023, and the deposit numbers are CCTCC M 2023370 and CCTCC M 2023371 respectively. Among them, the 16S rRNA sequence of the Cytobacillus oceanisediminis SH48 strain is shown in SEQ ID NO:1, and the 16S rRNA sequence of the Lysinibacillus fusiformis SH13 strain is shown in SEQ ID NO:2.

[0008] The two strains of the present invention are both trichloroethylene heterotrophic assimilatory bacteria, which can grow using chlorinated hydrocarbons such as trichloroethylene (TCE) as the sole carbon source and energy source, and completely mineralize it into CO2 and environmentally acceptable water-soluble substances. They have strong adaptability, fast growth rate, do not require the addition of extra growth substrates, and there is no accumulation of toxic intermediate products, and can effectively carry out chlorinated hydrocarbon remediation, providing a new option for the remediation of polluted sites.

[0009] The screening methods for the Cytobacillus oceanisediminis SH48 strain and the Lysinibacillus fusiformis SH13 strain include the following steps:

[0010] S1. Anaerobic enrichment: Collect soil samples from trichloroethylene-polluted sites, add the soil samples to sterile water and mix, and externally add trichloroethylene and methanol as stimulants to enrich and culture the flora under anaerobic conditions, and supplement trichloroethylene during the period;

[0011] S2. Isolation and purification: Obtain gradient dilution suspensions of the enriched soil by gradient dilution method, and spread them on an inorganic salt solid medium supplemented with acetate, trichloroethylene and methanol, and carry out isolation culture under aerobic conditions. The grown single colonies are purified by streaking to obtain multiple purified strains;

[0012] S3. Molecular identification: Extract DNA from the strains isolated in step S2, and perform ERIC-PCR typing. Select representative strains for PCR amplification and sequencing of the full-length 16S rRNA gene, and identify their taxonomic status by Blast comparison using the NCBI database;

[0013] S4. Degradation test: The identified strains were separately inoculated into the inorganic salt basal liquid medium with trichloroethylene as the sole carbon source for cultivation. After the cultivation, the strains that could effectively degrade trichloroethylene were screened out by observing the growth status, detecting the trichloroethylene content in the headspace of the culture solution, and the chloride ion release amount in the culture solution.

[0014] The second object of the present invention is to provide the application of the above Cytobacillus oceanisediminis SH48 strain and Lysinibacillus fusiformis SH13 strain in the degradation of chlorinated hydrocarbons. The strain or the microbial inoculum prepared therefrom can be used for the bioremediation of the soil or groundwater of industrial polluted sites contaminated with chlorinated hydrocarbons such as trichloroethylene.

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

[0016] The Cytobacillus oceanisediminis SH48 and Lysinibacillus fusiformis SH13 strains are derived from the in-situ soil environment of chlorinated hydrocarbon contaminated sites, and both can achieve the assimilatory degradation of chlorinated hydrocarbons. They are the first reported chlorinated hydrocarbon heterotrophic assimilatory bacteria of the genera Cytobacillus and Lysinibacillus. Both strains can utilize trichloroethylene without adding additional growth substrates, grow and metabolize while degrading it. The growth of the strains has loose requirements for environmental conditions and low nutritional requirements, and there is no accumulation of toxic intermediate products during the degradation process, so it will not cause secondary pollution to the site in practical applications. Therefore, it has good application prospects in the bioremediation of industrial sites contaminated with chlorinated olefins. Description of the Drawings

[0017] Figure 1 : Colony morphology of Cytobacillus oceanisediminis SH48 strain (A) and Lysinibacillus fusiformis SH13 strain (B) in the examples.

[0018] Figure 2 : Microscopic cell morphology (1000×) of Cytobacillus oceanisediminis SH48 strain (A) and Lysinibacillus fusiformis SH13 strain (B) in the examples.

[0019] Figure 3: Phylogenetic tree diagrams of Cytobacillus oceanisediminis strain SH48 and Lysinibacillus fusiformis strain SH13 in the examples and other strains of the same genus in the database.

[0020] Figure 4 : Diagrams of the dynamic changes of trichloroethylene and chloride ions during the growth of Cytobacillus oceanisediminis strain SH48 and Lysinibacillus fusiformis strain SH13 in the inorganic salt basic liquid medium containing trichloroethylene in the examples.

[0021] Figure 5 : Flow cytometry counting results of the two strains before and after cultivation in the inorganic salt basic liquid medium containing trichloroethylene in the examples. In the figure, A represents the blank medium control containing TCE without inoculation; B represents the initial moment of cultivation of Lysinibacillus fusiformis strain SH13; C represents the end moment of cultivation of Lysinibacillus fusiformis strain SH13; D represents the initial moment of cultivation of Cytobacillus oceanisediminis strain SH48; E represents the end moment of cultivation of Cytobacillus oceanisediminis strain SH48. Detailed implementation manners

[0022] The present invention will be further described below in conjunction with the accompanying drawings and specific examples.

[0023] Examples

[0024] In this example, two trichloroethylene assimilating and degrading bacteria, Cytobacillus oceanisediminis SH48 and Lysinibacillus fusiformis SH13, were screened out. The specific process is as follows:

[0025] I. Medium preparation

[0026] Prepare the required media and related reagents according to the following formula:

[0027] (1) Inorganic salt solid medium: 0.2 g / L (NH4)2SO4, 0.62 g / L MgSO4·7H2O, 0.023 g / L CaSO4·2H2O, 0.82 g / L phosphate buffer, 0.82 g / L sodium acetate, 0.5 g / L yeast extract, 14 g / L agar, sterilized at high temperature and high pressure at 121 °C; after sterilization, add 1 mL / L trace element solution A, 1 mL / L trace element solution B, 5 mL of 200× vitamin mother liquor, 100 μM TCE, and 202 μL / L methanol;

[0028] (2) Inorganic salt basic liquid medium: 0.2 g / L (NH4)2SO4, 0.62 g / L MgSO4·7H2O, 0.023 g / L CaSO4·2H2O, 0.82 g / L phosphate buffer, sterilized at high temperature and high pressure at 121 °C for 20 min. After sterilization, add 1 mL / L trace element solution A, 1 mL / L trace element solution B, 5 mL / L of 200× vitamin mother liquor, and 100 μM TCE;

[0029] (3) Phosphate buffer: 0.348 g / L K2HPO4 and 0.262 g / L KH2PO4, filtered through a 0.22 μm filter membrane and stored;

[0030] (4) Trace element solution A: 10 mL / L HCl (25%, w / w), 1.5 g / L FeCl2·4H2O, 0.19 g / L CoCl2·6H2O, 0.1 g / L MnCl2·4H2O, 70 mg / L ZnCl2, 6 mg H3BO3, 36 mg / L Na2MoO4·2H2O, 24 mg / L NiCl2·6H2O, 2 mg / L CuCl2·2H2O, sterilized at high temperature and high pressure at 121 °C for 20 min;

[0031] (5) Trace element solution B: 6 mg / L Na2SeO3·5H2O, 8 mg / L Na2WO4·2H2O, 0.5 g / L NaOH, sterilized at 121 °C for 20 min;

[0032] (6) 200× vitamin mother liquor: 20 mg biotin, 20 mg folic acid, 100 mg pyridoxine hydrochloride, 50 mg riboflavin, 50 mg thiamine, 50 mg nicotinic acid, 50 mg pantothenic acid, 1 mg vitamin B12, 50 mg p-aminobenzoic acid, 50 mg lipoic acid dissolved in 1 L of pure water, adjust the pH value to 7.5 with NaOH to obtain a 1000× vitamin stock solution, and then dilute it 5 times with pure water to obtain a 200× vitamin mother liquor, filtered and sterilized with a 0.22 μm filter membrane before use;

[0033] (7) TSA medium: 15 g / L peptone, 5 g / L soybean peptone, 5 g / L NaCl, 15 g / L agar, sterilized at 121 °C for 20 min.

[0034] II. Strain screening method

[0035] The strain screening is carried out according to the following steps:

[0036] S1. Anaerobic enrichment: Soil samples from different contaminated sites were collected from an industrial site contaminated with a mixture of trichloroethylene and benzene in Suzhou. 50 g of contaminated soil was weighed and mixed with 10 mL of sterile water and placed in a 120 mL serum bottle and sealed. 1 mL of trichloroethylene and 1 mL of methanol were added exogenously as stimulants for anaerobic enrichment of the in-situ contaminated soil flora for up to five months, and 0.5 mL of trichloroethylene was replenished once during this period.

[0037] S2. Isolation and purification: 1 g of the TCE-enriched soil sample was weighed and added to 10 mL of 0.85% NaCl solution, and vortexed to obtain a soil suspension. The soil suspension was serially diluted with 0.85% NaCl solution. 200 μL of the serially diluted suspension and 10 μL of TCE were respectively pipetted and spread on a solid medium for strain isolation and culture, and cultured at 30 °C under aerobic conditions for 2 weeks. The newly grown single colonies on the isolation plate were continuously observed and recorded daily, and various different bacterial isolates were selected as much as possible according to the size, color, and morphology of the colonies. The strains were continuously streaked and purified on TSA medium until a single colony was obtained, and multiple purified strains were obtained.

[0038] S3. Molecular identification: DNA was extracted from each isolated strain in step S2, and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) was performed. The typing of the strains was achieved using the DNA fingerprint pattern provided by gel electrophoresis. After removing the repeated strains, the full-length fragment of the 16S rRNA gene was amplified from the DNA of the selected strains and sequenced, and its taxonomic status was identified by Blast alignment using the NCBI database and a phylogenetic tree was constructed.

[0039] S4. Degradation test: The identified strains were separately inoculated into the basic inorganic salt medium containing 100 μM trichloroethylene and cultured. After thorough mixing and shaking, enough headspace was left in the flask for the aerobic respiration growth of microorganisms, and the bottle cap was sealed to prevent the volatilization of trichloroethylene. The system was cultured in an incubator at 30 °C for 8 days. 10 mL of samples were collected at the initial moment and the end of the culture for subsequent analysis. Among them, 5 mL of the culture solution was centrifuged at 14000×g for 5 min to collect the bacteria for cell counting by flow cytometry; 1 mL of the culture solution was used for headspace gas chromatography (HS-GC) to detect the remaining content of trichloroethylene and the presence of low-chlorinated toxic intermediates, dichloroethylene and vinyl chloride. The remaining samples were used to detect the chloride ion content by using a chloride ion selective composite electrode with the standard curve method. The strains that could effectively degrade trichloroethylene were screened out by comparing the growth status, the content of trichloroethylene in the culture solution, and the chloride ion release amount in the culture solution at the initial and end moments of the culture.

[0040] III. Screening results of strains

[0041] After screening by comparing the degradation effect of trichloroethylene and the growth of bacteria by the above method, the strains Cytobacillus oceanisediminis SH48 and Lysinibacillus fusiformis SH13 showed the best growth status and degradation ability and were determined as highly efficient degradation strains. Specifically, as Figure 4 shown, the two strains released free chloride ions while degrading trichloroethylene, and the degradation effect was obvious. The degradation rates of Cytobacillus oceanisediminis SH48 and Lysinibacillus fusiformis SH13 for trichloroethylene in 8 days were 58.6% and 42.42% respectively; as Figure 5 shown, after 8 days of culture, the flow cytometry results showed that the cell concentration of Cytobacillus oceanisediminis SH48 increased from 1×10 5 cell / ml to 5.97×10 5 cell / ml, and the cell concentration of Lysinibacillus fusiformis SH13 increased from 1.22×10 5 cell / ml to 5.26×10 5cells / ml, indicating that both can use trichloroethylene in the culture medium as a carbon source for growth and metabolism; in addition, according to the GC-MS results, there is no peak of intermediate metabolites, indicating that there is no accumulation of more toxic low-chlorinated olefin intermediate products during the degradation process. Thus, the two strains can heterotrophically assimilate trichloroethylene for degradation without the need to add additional growth substrates, and this characteristic reveals their application value in chlorinated hydrocarbon remediation, contributing to the development of new environmental microbial resources and the expansion of available resources for in-situ remediation of contaminated sites.

[0042] The morphological observation results of the two strains are as follows: After culturing the Cytobacillus oceanisediminis SH48 strain and the Lysinibacillus fusiformis SH13 strain on TSA solid medium for 2 days, as Figure 1-2 shown, the colonies of Cytobacillus oceanisediminis SH48 are light yellow, semi-turbid, round and convex, with a relatively transparent and irregular edge, the surface of the colony is moist and smooth, and the cell morphology is short rod-shaped; the colony morphology of Lysinibacillus fusiformis SH13 is milky white and round, with a neat and semi-transparent edge, and the cell morphology is short rod-shaped. The Gram staining results of both strains show that they are Gram-positive bacteria.

[0043] The molecular identification results of the two strains are as follows: The 16S rRNA sequence of Cytobacillus oceanisediminis SH48 is shown in SEQ ID NO:1, and the 16S rRNA sequence of the Lysinibacillus fusiformis SH13 strain is shown in SEQ ID NO:2; After Blast alignment with the information in the NCBI database, based on the homology of the 16S rRNA gene sequences of the Cytobacillus oceanisediminis SH48 and Lysinibacillus fusiformis SH13 strains, the phylogenetic tree constructed using MEGAX software is as Figure 3 shown to complete the taxonomic identification.

[0044] The Cytobacillus oceanisediminis SH48 and Lysinibacillus fusiformis SH13 screened in this example have been deposited in the China Center for Type Culture Collection at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is March 21, 2023, and the deposit numbers are CCTCC M 2023370 and CCTCC M 2023371 respectively.

[0045] The nucleotide sequences involved in the present invention are as follows:

[0046] SEQ ID NO:1 (16S rRNA sequence of Cytobacillus oceanisediminis SH48):

[0047] TGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGAAGTCAGCGGCGGA

[0048] CGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTCCGG

[0049] GAAACCGGGGCTAATACCGGATAATTCTTTCCCTCACATGAGGGAAAGCT

[0050] GAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGCGCATTAGCT

[0051] AGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAG

[0052] AGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGG

[0053] AGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAA

[0054] CGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGG

[0055] AAGAACAAGTACCGGAGTAACTGCCGGTACCTTGACGGTACCTAACCAGA

[0056] AAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAA

[0057] GCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTCCTTAAG

[0058] TCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGG

[0059] GAACTTGAGTGCAGAAGAGAAGAGTGGAATTCCACGTGTAGCGGTGAAA

[0060] TGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTTTGGTCTG

[0061] TAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATAC

[0062] CCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCC

[0063] GCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACA

[0064] GCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT

[0065] GGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTG

[0066] ACATCTCCTGACAACCCTAGAGATAGGGCGTTCCCCTTCGGGGGACAGGA

[0067] TGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTA

[0068] AGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGG

[0069] GCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGAC

[0070] GTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGG

[0071] ATGGTACAAAGGGCTGCGAGACCGCGAGGTTAAGCGAATCCCATAAAAC

[0072] CATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAAT

[0073] CGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTT

[0074] GTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTG

[0075] GGGTAACCTTTTGGAGCCAGCCGCCAGGT

[0076]

[0077] This specific implementation manner is only an interpretation of the present invention and not a limitation thereof. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A trichloroethylene-assimilating and degrading bacterium, characterized in that: The bacterium is Cytobacillus oceanisediminis SH48, and its preservation number is: CCTCC NO: M 2023370. The 16S rRNA sequence is as shown in SEQ ID NO:

1.

2. A trichloroethylene-assimilating and degrading bacterium, characterized in that: The bacterium is Lysinibacillus fusiformis SH13, and its preservation number is: CCTCC NO: M 2023371. The 16S rRNA sequence is as shown in SEQ ID NO:

2.

3. Use of the trichloroethylene assimilating and degrading bacterium according to claim 1 or 2 in the remediation of chlorinated hydrocarbon contaminated sites, characterized in that, The chlorinated hydrocarbon is trichloroethylene.

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