CN117305145A - Bacterial strain capable of effectively degrading benzene series and alkane under high-salt condition and acquisition method and application thereof - Google Patents
Bacterial strain capable of effectively degrading benzene series and alkane under high-salt condition and acquisition method and application thereof Download PDFInfo
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- 230000000593 degrading effect Effects 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 21
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 15
- 125000001997 phenyl group Chemical class [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 title abstract 3
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 37
- 239000001963 growth medium Substances 0.000 claims abstract description 35
- 239000007788 liquid Substances 0.000 claims abstract description 32
- 239000003208 petroleum Substances 0.000 claims abstract description 30
- 239000002689 soil Substances 0.000 claims abstract description 30
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 25
- 239000003344 environmental pollutant Substances 0.000 claims abstract description 11
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- 244000005700 microbiome Species 0.000 claims abstract description 10
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- 238000004321 preservation Methods 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 4
- 230000001105 regulatory effect Effects 0.000 claims abstract description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 38
- 241000589516 Pseudomonas Species 0.000 claims description 34
- 150000001555 benzenes Chemical class 0.000 claims description 32
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 31
- QMVPMAAFGQKVCJ-UHFFFAOYSA-N citronellol Chemical compound OCCC(C)CCC=C(C)C QMVPMAAFGQKVCJ-UHFFFAOYSA-N 0.000 claims description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- QMVPMAAFGQKVCJ-SNVBAGLBSA-N (R)-(+)-citronellol Natural products OCC[C@H](C)CCC=C(C)C QMVPMAAFGQKVCJ-SNVBAGLBSA-N 0.000 claims description 15
- JGQFVRIQXUFPAH-UHFFFAOYSA-N beta-citronellol Natural products OCCC(C)CCCC(C)=C JGQFVRIQXUFPAH-UHFFFAOYSA-N 0.000 claims description 15
- 235000000484 citronellol Nutrition 0.000 claims description 15
- 239000008096 xylene Substances 0.000 claims description 13
- 150000003839 salts Chemical class 0.000 claims description 11
- 239000011780 sodium chloride Substances 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 125000003944 tolyl group Chemical group 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 10
- 239000003209 petroleum derivative Substances 0.000 abstract description 4
- 230000015556 catabolic process Effects 0.000 description 13
- 238000006731 degradation reaction Methods 0.000 description 13
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- 239000002609 medium Substances 0.000 description 8
- 230000008439 repair process Effects 0.000 description 7
- 238000010170 biological method Methods 0.000 description 5
- 238000000354 decomposition reaction Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
- RZJRJXONCZWCBN-UHFFFAOYSA-N octadecane Chemical compound CCCCCCCCCCCCCCCCCC RZJRJXONCZWCBN-UHFFFAOYSA-N 0.000 description 4
- YCOZIPAWZNQLMR-UHFFFAOYSA-N pentadecane Chemical compound CCCCCCCCCCCCCCC YCOZIPAWZNQLMR-UHFFFAOYSA-N 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 3
- 235000011470 Adenanthera pavonina Nutrition 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000428378 Lopa Species 0.000 description 2
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- 238000011081 inoculation Methods 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
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- 239000011707 mineral Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010028400 Mutagenic effect Diseases 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 231100000693 bioaccumulation Toxicity 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
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- 238000006243 chemical reaction Methods 0.000 description 1
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- 231100000590 oncogenic Toxicity 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C12N1/02—Separating microorganisms from their culture media
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/38—Pseudomonas
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Abstract
The invention discloses a bacterial strain for effectively degrading benzene series and alkane under a high-salt condition, and an acquisition method and application thereof, wherein the bacterial strain is named as SJBP16, and the preservation number of the bacterial strain is CCTCCNo. M2020780. The method for obtaining the strain comprises the following steps: collecting soil polluted by petroleum as a microorganism source; preparing a liquid inorganic salt culture medium containing carbon sources, regulating the pH value to 7.0-7.4, and sterilizing at high temperature; inoculating soil into a liquid inorganic salt culture medium under an ultra-clean bench, and carrying out enrichment culture in a constant-temperature shaking table; repeatedly culturing for multiple times, repeatedly streaking and culturing on a solid inorganic salt culture medium plate until single strain is separated, inoculating the single strain into the liquid inorganic salt culture medium, and domesticating; repeating the operations of scribing, separating and domesticating for 3-5 times. The strain of the invention has the characteristic of efficiently degrading benzene series and petroleum hydrocarbon substances under the high-salt condition, and can be applied to the soil environment restoration containing the petroleum pollutants.
Description
Technical Field
The invention relates to the technical fields of environmental engineering and microbial engineering, in particular to a bacterial strain for effectively degrading benzene series and alkane under a high-salt condition, and an acquisition method and application thereof.
Background
Petroleum pollution refers to pollution caused by leakage and discharge of petroleum during petroleum exploitation, transportation, loading and unloading, processing and use. On the one hand, petroleum pollution is caused by the fact that petroleum pollutants undergo a physical-chemical reaction after being irradiated by solar ultraviolet rays to generate photochemical smog, pollute the atmosphere, generate cancerogenic substances and greenhouse effect and destroy an ozone layer. On the other hand, petroleum pollutants can enter water or soil to cause salinization and poisoning of the soil, so that the soil is destroyed and destroyed; the benzene compounds, hydrocarbons and aromatic compounds in petroleum have strong teratogenic, oncogenic and mutagenic effects, have bioaccumulation effects, can enter organisms and human bodies through food chains, and threaten the health of the organisms and the human bodies. Therefore, research on the control and repair of petroleum pollution is increasingly receiving attention from governments and researchers of various countries.
At present, the petroleum polluted soil repairing method mainly comprises a physical method, a chemical method and a biological method. Since the 80 s of the last century, bioremediation technology of petroleum-contaminated soil has attracted more and more attention and has made great progress. Bioremediation is a process of restoring contaminated soil to a healthy state by reducing the concentration of toxic and harmful substances in the soil environment using the life metabolic activity of organisms. In comparison, biological methods have advantages that are not comparable to physical and chemical methods. Firstly, the treatment cost of the biological method is low, and the treatment cost is only about one third of that of the physical and chemical method; secondly, the biological method has simple repair operation and can realize in-situ repair; and thirdly, the secondary pollution to the environment caused by the biological method is small. At present, bioremediation technologies for treating petroleum hydrocarbon contaminated soil mainly include two types: one is a microbial repair technology, and the repair sites can be divided into in-situ bioremediation and ectopic bioremediation; another class is phytoremediation. Because the microorganism resources are rich, the culture is easy, and the tolerance to the environment is strong, the microorganism repair is a main method in the biological repair, and has wide application prospect. The core of the microorganism for repairing petroleum polluted environment is to screen or enrich functional strains which can efficiently and stably degrade petroleum pollutants from the environment. Since benzene series and hydrocarbons (saturated hydrocarbon and aromatic hydrocarbon) are main components of petroleum pollutants, bacterial strains with degradation efficiency on the two compounds are screened and identified, so that effective restoration of petroleum-polluted soil is possible. Meanwhile, the problems of easy hardening, high salt and alkali exist in the petroleum-polluted soil, so that the microorganisms applied to the actual petroleum-polluted soil restoration are required to have certain adaptability to conditions such as high salt and the like.
Disclosure of Invention
In view of the above problems, the present invention has an object of: the citronellol pseudomonas can be used for stably and efficiently decomposing benzene series and long-chain alkane in a high-salt environment, and can be used for biodegradation of petroleum pollutants in a soil environment and environment restoration.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention discloses a bacterial strain for effectively degrading benzene series and alkane under a high-salt condition, which is named as SJBP16 and has a preservation number of CCTCCNo. M2020780.
Preferably, the sequence of the 16SrDNA fragment of the strain is shown as SEQ ID NO. 1.
The invention discloses a method for obtaining a bacterial strain capable of effectively degrading benzene series and alkane under a high-salt condition, which comprises the following steps:
collecting soil polluted by petroleum as a microorganism source;
preparing a liquid inorganic salt culture medium containing carbon sources, regulating the pH value to 7.0-7.4, and sterilizing at high temperature;
inoculating a petroleum-polluted soil sample into a liquid inorganic salt culture medium under an ultra-clean bench, and carrying out enrichment culture in a constant-temperature shaking table;
repeatedly culturing for multiple times, repeatedly streaking on a solid inorganic salt culture medium plate until single strain is separated, and inoculating the single strain into the liquid inorganic salt culture medium for domestication; the scribing, separating and domesticating operations are repeated for 3 to 5 times to obtain pure citronellol pseudomonas which is named as SJBP16 and has the preservation number of CCTCCNo. M2020780.
Preferably, the carbon source is an alkane or a benzene series, and the benzene series is xylene or toluene; when the carbon source is alkane, the concentration of the alkane in the liquid inorganic salt culture medium is 500mg/L-2g/L; when the carbon source is dimethylbenzene, the concentration of dimethylbenzene in the liquid inorganic salt culture medium is 10mg/L-50mg/L; when the carbon source is toluene, the concentration of toluene in the liquid inorganic salt culture medium is 10mg/L-50mg/L.
Preferably, the inorganic components and concentrations of the liquid inorganic salt medium are as follows: k (K) 2 HPO 4 ，1g/L；KH 2 PO 4 ，2.25g/L；NaCl，2.0g/L；(NH 4 ) 2 HPO 4 ，1g/L；MgCl 2 ·6H 2 O，25mg/L；FeCl 3 ，0.3mg/L；CaCl 2 ，2.5mg/L；KNO 3 ，1.25g/L。
Preferably, the conditions of the high temperature sterilization are: sterilizing at 121deg.C for 15-20min.
Preferably, the pH value is adjusted to 7.0-7.4 by adopting hydrochloric acid.
Preferably, the petroleum-polluted soil sample is inoculated into the liquid inorganic salt culture medium under an ultra-clean bench, and enrichment culture is carried out in a constant-temperature shaking table as follows:
after the temperature of the liquid inorganic salt culture medium after high-temperature sterilization is reduced to 25-30 ℃, inoculating the soil under an ultra-clean bench according to the inoculum size of 5W/V percent, and carrying out enrichment culture in a constant-temperature shaking incubator at 28 ℃ and 180-200rpm for 14-16 days.
The invention discloses an application of a strain for effectively degrading benzene series and alkane under a high-salt condition, which is used for decomposing and removing petroleum pollutants in a high-salt environment, wherein the strain is named as SJBP16, and the preservation number of the strain is CCTCC No. M2020780.
Preferably, the petroleum pollutants comprise benzene series and/or alkane, wherein the benzene series is one or two of dimethylbenzene and toluene; alkane is C 16 －C 18 Is an alkane of (a).
Preferably, the high salt environment is a sodium chloride-containing environment and the concentration of sodium chloride is not less than 2.0g/L.
The invention has the beneficial effects that:
the strain (citronellol pseudomonas SJBP 16) can normally grow in a culture medium with benzene series (xylene or toluene) as the only carbon source up to 50mg/L, and can tolerate a high-salt environment of 2.0g/L. 10mg/L of dimethylbenzene can be completely decomposed in 48 hours, and 20mg/L of dimethylbenzene can be completely decomposed in 72 hours; the decomposition rate of 50mg/L xylene exceeds 70% for seven days.
The citronellol pseudomonas SJBP16 can normally grow in a culture medium with long-chain alkane of up to 2g/L as the only carbon source, and can tolerate a high-salt environment of 2.0g/L. Can store 500mg/L of C in 48 hours 18 Alkane is completely decomposed, and C with the concentration of 1g/L can be completely decomposed in 72 hours 18 An alkane; seven days to 2g/L C 18 The alkane decomposition rate exceeds 70%.
To sum up: as the citronellol pseudomonas SJBP16 strain has the characteristic of efficiently degrading benzene series and petroleum hydrocarbon substances under the high-salt condition, the citronellol pseudomonas SJBP16 strain can be applied to the soil environment restoration containing the petroleum pollutants. Compared with the existing petroleum degradation strain, the degradation efficiency of the invention is obviously improved, the degradation period is obviously shortened, and the environmental adaptability is better.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a plate drawing of a Pseudomonas citronellosis SJBP16 strain in accordance with the present invention;
FIG. 2 shows the concentration of C in Pseudomonas citronellosis SJBP16 strain 16 The growth curve of the strain when alkane is a single carbon source, wherein ■ mg/L; ● 250mg/L; 500mg/L; 1g/L; 2g/L of the solid;
FIG. 3 shows the concentration of C in Pseudomonas citronellosis SJBP16 strain 18 The growth curve of the strain when alkane is a single carbon source, wherein ■ mg/L; ● 250mg/L; 500mg/L; 1g/L; 2g/L of the solid;
FIG. 4 shows the growth curves of the strain of Pseudomonas citronellosis SJBP16 according to the invention, with different concentrations of xylene as the sole carbon source, wherein ■ mg/L; ● 20mg/L; 50mg/L;
FIG. 5 shows the growth curves of the strain of Pseudomonas citronellosis SJBP16 according to the invention, with toluene of different concentrations as the sole carbon source, wherein ■ mg/L; ● 20mg/L; 50mg/L;
FIG. 6 shows degradation curves of Pseudomonas citronellosis SJBP16 strain according to the invention, with respect to different concentrations of n-octadecane, wherein ∈500mg/L; 1g/L; 2g/L;
FIG. 7 is a graph showing the degradation curve of Pseudomonas citronellosis SJBP16 strain according to the invention, in which ■ mg/L, for various concentrations of xylene; ● 20mg/L; 50mg/L.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The citronellol pseudomonas SJBP16 provided by the invention is obtained from petroleum polluted soil in a petroleum refinery through artificial enrichment, screening and purification. SJBP16 colony is round, raised, light yellow, semitransparent, gram-negative, straight or bent bacillus, single or paired, no spore, good aerobic and good growth at 30 ℃. The identification and sequence analysis of the 16SrDNA of the strain show that the strain is citronellol pseudomonas. The citronellol pseudomonas SJBP16 strain is preserved in China center for type culture collection (address: eight paths of Lopa nationality in Wuhan, hubei province) on 11/27 days in 2020, and registered with the registration number of CCTCC No. M2020780.
The invention is further described below in connection with examples, but the practice of the invention is not limited thereto.
1. Acquisition and preservation of Pseudomonas citronellosis (Pseudomonas) SJBP16 Strain
Collecting petroleum polluted soil of a certain oil refinery as a microorganism source, and refrigerating and transporting the petroleum polluted soil back to a laboratory. 200mL of liquid inorganic salt culture medium containing 500mg/L alkane or 50mg/L xylene or 50mg/L toluene is prepared, and inoculated into a 500mL triangular flask containing 100mL of liquid inorganic salt culture medium according to a soil inoculation amount of 5W/V% (5W/V% is that the mass of soil accounts for 5% of the volume of the liquid inorganic salt culture medium), specifically, the inorganic components and the concentration of the liquid inorganic salt culture medium are as follows: k (K) 2 HPO 4 ，1g/L；KH 2 PO 4 ，2.25g/L；NaCl，2.0g/L；(NH 4 ) 2 HPO 4 ，1g/L；MgCl 2 ·6H 2 O，25mg/L；FeCl 3 ，0.3mg/L；CaCl 2 ，2.5mg/L；KNO 3 1.25g/L, adjusting pH to 7.0-7.4 with hydrochloric acid, sterilizing at 121deg.C for 15-20min, and inoculating under an ultra clean bench when the temperature of the culture medium is reduced to about 28deg.C (25-30deg.C); enrichment culture was performed in a constant temperature shaker incubator at 28℃and 200rpm (where the rotation speed was 180-200 rpm) for 15 days (where the time was 14-16 days) after soil inoculation. After repeating the culture five times, streaking culture was repeated on a solid inorganic salt medium plate (the solid inorganic salt medium was the above-mentioned liquid inorganic salt medium+20.0 g/L agar) until a single strain was isolated. Then inoculating the strain into the liquid inorganic salt culture medium for domestication, repeating the above operations (streaking, separating and domestication) for a plurality of times to obtain purified SJBP16 strain capable of degrading petroleum substances, wherein the streaking diagram of the plate is shown in figure 1. SJBP16 colony is round, raised, light yellow, semitransparent, gram-negative, straight or bent bacillus, single or paired, no spore, aerobic and good growth at 30 ℃. The identification and sequence analysis of the 16SrDNA of the strain show that the strain is citronellol pseudomonas. The citronellol pseudomonas SJBP16 strain is preserved in China center for type culture collection (address: eight paths of Lopa nationality in Wuhan, hubei province) on 11/27 days in 2020, and registered with the registration number of CCTCC No. M2020780.
2. Identification of Pseudomonas citronellosis (Pseudomonas sp.) SJBP16 Strain
The isolated SJBP16 strain was subjected to amplification of 16SrDNA sequence, sequencing and database alignment. Genomic DNA of SjBP16 strain was extracted using a genomic extraction kit (TIANGEN, beijing), and then 16SrDNA fragment of the strain was amplified using a 16SrDNA fragment amplification and detection kit (Takara, dalian) and sequenced. The 16SrDNA sequence of the SJBP16 strain is shown as SEQ ID NO. 1. Comparison with NCBI database (http:// www.ncbi.nlm.nih.gov /), shows that the SJBP16 strain is Pseudomonas citronellosis, the strain is Bacillus cereus, single, belongs to gram-negative bacteria, aerobic and metatrophic.
SEQIDNO.1
1GACGTCAATCTCGTGGTACCGTCCCCTTGCGGTTAGACTAGCTACTTCTGGAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTCTGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTCTGTACCGACCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCTTAACGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCCGATTCCCGAAGGGCACTCCCGCATCTCTGCAGGATTCCGGACATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTATCGCGTTAGCTGCGCCACTAAGATCTCAAGGATCCCAACGGCTAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATCAGTCCAGGCGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCACCCTCTACCGTACTCTAGTCAGGCAGTTATGGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTCATCTTACCAAACCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCAGATGCTTATTCTGTTGGTAACGTCAAAACAGCAAGGTATTAACTTACTGCCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCGGCATGACTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGAACGAGTCTCAGTTCCAGTGTGACTGATCATCCTCTCAGACCAGTTACGGATCGTCGCCTAGGTGAGCCATTACCCCACCTACTAGCTAATCCGACCTAGGCTCATCTGATAGCGTGAGGTCCGAAGATCCCCCACTTTCTCCCGTAGGACGTATGCGGTATTAGCGCTCCTTTCGGAACGTTGTCCCCCACTACCAGGCAGATTCCTAGGCATTACTCACCCGTCCGCCGCTGAATCCGGGAGCAAGCTCCCTCATCCGCTCGACTGCATGGTAGCTGCCGCACCCTA 1439
3. Degradation ability of benzene series and long-chain alkane by citronellol pseudomonas (pseudoomnascidiphyllalis) SJBP16 strain
(1) Degradation ability of Pseudomonas citronellosis (Pseudomonas) SJBP16 strain on benzene series
Pseudoomonaasccontrol sjbp16 strain was streaked onto solid mineral salts medium plates and incubated overnight at 30 ℃. The single clone was selected and the mixture was then used to select,respectively inoculating to 500ml liquid inorganic salt culture medium (inorganic components and concentrations of the liquid inorganic salt culture medium are as follows: K) with benzene series (toluene, xylene) of 10mg/L-50mg/L as unique carbon source 2 HPO 4 ，1g/L；KH 2 PO 4 ，2.25g/L；NaCl，2.0g/L；(NH 4 ) 2 HPO 4 ，1g/L；MgCl 2 ·6H 2 O，25mg/L；FeCl 3 ，0.3mg/L；CaCl 2 ，2.5mg/L；KNO 3 1.25g/L. ) In the above, the culture was carried out in a shaking incubator at a constant temperature of 28℃and 200rpm for 7 days. 2 3ml replicates were collected at different time points.
One sample was measured for 600nm absorbance by ultraviolet spectrophotometer (UV-2100 spectrophotometer, unic) to show the growth of microorganisms, and the results are shown in FIGS. 4 to 5, and FIG. 4 shows the growth curves of Pseudomonas citronellosis SJBP16 strain of the present invention when various concentrations of xylene are used as a single carbon source, wherein ■ mg/L; ● 20mg/L; 50mg/L; FIG. 5 shows the growth curves of the strain of Pseudomonas citronellosis SJBP16 according to the invention, with toluene of different concentrations as the sole carbon source, wherein ■ mg/L; ● 20mg/L; 50mg/L.
Adding 1/6 volume of n-hexane (HPLC pure) into the other sample, mixing, ultrasonic treating for 5min, centrifuging for 5min at 3000g, and collecting the upper organic phase; the extracts were combined and repeated twice. Adding anhydrous Na 2 SO 4 After mixing, the internal standard n-pentadecane (final concentration 100 ng/. Mu.L) was added and the volume was set to 3mL. The benzene and petroleum hydrocarbon concentrations were determined by gas chromatography (GC, agilent 6850) using a DB-5 MScalillary column (0.25 mm. Times.30 m. Times.0.25 μm). The concentration measurement results of different petroleum substances (benzene series) are shown in FIG. 7, and FIG. 7 shows the degradation curve of the citronellol pseudomonas SJBP16 strain of the invention on xylene with different concentrations, wherein ■ mg/L; ● 20mg/L; 50mg/L.
From the growth curves of fig. 4 and 5 and the degradation curves of fig. 7, it can be seen that:
the strain SJBP16 of the invention can normally grow in a liquid inorganic salt culture medium which takes benzene series (dimethylbenzene or methylbenzene) with concentration of 50mg/L as the only carbon source and contains sodium chloride with concentration of 2.0g/L. The strain can completely decompose 10mg/L of xylene within 48 hours, and can completely decompose 20mg/L of xylene within 72 hours; the decomposition rate of 50mg/L xylene exceeds 70% for seven days.
(2) Degradation ability of citronellol Pseudomonas (Pseudomonas) SJBP16 strain on long-chain alkane
Pseudoomonaasccontrol sjbp16 strain was streaked onto solid mineral salts medium plates and incubated overnight at 30 ℃. Picking up the monoclonal and inoculating to 500mg/L-2g/L long-chain alkane (C) 16 Alkane, C 18 Alkane) is the only carbon source 500ml of liquid inorganic salt medium (the inorganic components and concentrations of the liquid inorganic salt medium are as follows: k (K) 2 HPO 4 ，1g/L；KH 2 PO 4 ，2.25g/L；NaCl，2.0g/L；(NH 4 ) 2 HPO 4 ，1g/L；MgCl 2 ·6H 2 O，25mg/L；FeCl 3 ，0.3mg/L；CaCl 2 ，2.5mg/L；KNO 3 1.25g/L. ) In the above, the culture was carried out in a shaking incubator at a constant temperature of 28℃and 200rpm for 7 days. 2 3ml replicates were collected at different time points.
A sample was assayed for 600nm absorbance by an ultraviolet spectrophotometer (UV-2100 spectrophotometer, unic) to show the growth of microorganisms, and the results are shown in FIGS. 2 to 3, FIG. 2 shows the C at various concentrations of Pseudomonas citronellosis SJBP16 strain of the present invention 16 The growth curve of the strain when alkane is a single carbon source, wherein ■ mg/L; ● 250mg/L; 500mg/L; 1g/L; 2g/L of the solid; FIG. 3 shows the concentration of C in Pseudomonas citronellosis SJBP16 strain 18 The growth curve of the strain when alkane is a single carbon source, wherein ■ mg/L; ● 250mg/L; 500mg/L; 1g/L; 2g/L.
Adding 1/6 volume of n-hexane (HPLC pure) into the other sample, mixing, ultrasonic treating for 5min, centrifuging for 5min at 3000g, and collecting the upper organic phase; the extracts were combined and repeated twice. Adding anhydrous Na 2 SO 4 After mixing, the internal standard n-pentadecane (final concentration 100 ng/. Mu.L) was added and the volume was set to 3mL. Determination of long chain alkane (C) by gas chromatography (GC, agilent 6850) 18 Alkane) concentration, the column was a DB-5 MScalillary column (0.25 mm. Times.30 m. Times.0.25 μm). The results of the concentration measurement of different petroleum-based substances (long-chain alkanes) are shown in FIG. 6FIG. 6 shows the degradation curves of Pseudomonas citronellosis SJBP16 strain according to the invention, with respect to different concentrations of n-octadecane, wherein ∈500mg/L; 1g/L; 2g/L.
From the growth curves of fig. 2 and 3 and the degradation curves of fig. 6, it can be seen that:
the strain SJBP16 of the invention can normally grow in a culture medium which takes up to 2g/L of long-chain alkane as the sole carbon source and contains 2.0g/L of sodium chloride. Can store 500mg/L of C in 48 hours 18 Alkane is completely decomposed, and C with the concentration of 1g/L can be completely decomposed in 72 hours 18 An alkane; seven days to 2g/L C 18 The alkane decomposition rate exceeds 70%.
Although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> national institute of Petroleum institute for safety and environmental protection technology
<120> a strain for effectively degrading benzene series and alkane under high salt condition, and its obtaining method and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1439
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gacgtcaatc tcgtggtacc gtccccttgc ggttagacta gctacttctg gagcaaccca 60
ctcccatggt gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gtgacattct 120
gattcacgat tactagcgat tccgacttca cgcagtcgag ttgcagactg cgatccggac 180
tacgatcggt tttctgggat tagctccacc tcgcggcttg gcaaccctct gtaccgacca 240
ttgtagcacg tgtgtagccc tggccgtaag ggccatgatg acttgacgtc atccccacct 300
tcctccggtt tgtcaccggc agtctcctta gagtgcccac cttaacgtgc tggtaactaa 360
ggacaagggt tgcgctcgtt acgggactta acccaacatc tcacgacacg agctgacgac 420
agccatgcag cacctgtgtt ccgattcccg aagggcactc ccgcatctct gcaggattcc 480
ggacatgtca agaccaggta aggttcttcg cgttgcttcg aattaaacca catgctccac 540
cgcttgtgcg ggcccccgtc aattcatttg agttttaacc ttgcggccgt actccccagg 600
cggtcgactt atcgcgttag ctgcgccact aagatctcaa ggatcccaac ggctagtcga 660
catcgtttac ggcgtggact accagggtat ctaatcctgt ttgctcccca cgctttcgca 720
cctcagtgtc agtatcagtc caggcggtcg ccttcgccac tggtgttcct tcctatatct 780
acgcatttca ccgctacaca ggaaattcca ccaccctcta ccgtactcta gtcaggcagt 840
tatggatgca gttcccaggt tgagcccggg gatttcacat ctcatcttac caaaccacct 900
acgcgcgctt tacgcccagt aattccgatt aacgcttgca cccttcgtat taccgcggct 960
gctggcacga agttagccag atgcttattc tgttggtaac gtcaaaacag caaggtatta 1020
acttactgcc cttcctccca acttaaagtg ctttacaatc cgaagacctt cttcacacac 1080
gcggcatgac tggatcaggc tttcgcccat tgtccaatat tccccactgc tgcctcccgt 1140
aggagtctgg aacgagtctc agttccagtg tgactgatca tcctctcaga ccagttacgg 1200
atcgtcgcct aggtgagcca ttaccccacc tactagctaa tccgacctag gctcatctga 1260
tagcgtgagg tccgaagatc ccccactttc tcccgtagga cgtatgcggt attagcgctc 1320
ctttcggaac gttgtccccc actaccaggc agattcctag gcattactca cccgtccgcc 1380
gctgaatccg ggagcaagct ccctcatccg ctcgactgca tggtagctgc cgcacccta 1439
Claims (11)
1. A bacterial strain for effectively degrading benzene series and alkane under high-salt condition is characterized by being named as SJBP16, and the preservation number of the bacterial strain is CCTCCNo. M2020780.
2. A strain effective for degrading benzene series and alkane under high salt conditions according to claim 1, wherein the sequence of the 16SrDNA fragment of the strain is shown in SEQ ID No. 1.
3. The method for obtaining the bacterial strain capable of effectively degrading benzene series and alkane under the high-salt condition is characterized by comprising the following steps of:
collecting soil polluted by petroleum as a microorganism source;
preparing a liquid inorganic salt culture medium containing carbon sources, regulating the pH value to 7.0-7.4, and sterilizing at high temperature;
inoculating a petroleum-polluted soil sample into a liquid inorganic salt culture medium under an ultra-clean bench, and carrying out enrichment culture in a constant-temperature shaking table;
repeatedly culturing for multiple times, repeatedly streaking on a solid inorganic salt culture medium plate until single strain is separated, and inoculating the single strain into the liquid inorganic salt culture medium for domestication; the scribing, separating and domesticating operations are repeated for 3 to 5 times to obtain pure citronellol pseudomonas which is named as SJBP16 and has the preservation number of CCTCCNo. M2020780.
4. The method for obtaining a strain effective for degrading benzene series and alkane under high salt conditions according to claim 3, wherein the carbon source is alkane or benzene series, and the benzene series is xylene or toluene; when the carbon source is alkane, the concentration of the alkane in the liquid inorganic salt culture medium is 500mg/L-2g/L; when the carbon source is dimethylbenzene, the concentration of dimethylbenzene in the liquid inorganic salt culture medium is 10mg/L-50mg/L; when the carbon source is toluene, the concentration of toluene in the liquid inorganic salt culture medium is 10mg/L-50mg/L.
5. According to claim 3The method for obtaining the bacterial strain capable of effectively degrading benzene series and alkane under the high-salt condition is characterized in that the inorganic components and the concentration of the liquid inorganic salt culture medium are as follows: k (K) 2 HPO 4 ，1g/L；KH 2 PO 4 ，2.25g/L；NaCl，2.0g/L；(NH 4 ) 2 HPO 4 ，1g/L；MgCl 2 ·6H 2 O，25mg/L；FeCl 3 ，0.3mg/L；CaCl 2 ，2.5mg/L；KNO 3 ，1.25g/L。
6. The method for obtaining a strain effective for degrading benzene series and alkane under high salt conditions as claimed in claim 3, wherein the conditions of high temperature sterilization are: sterilizing at 121deg.C for 15-20min.
7. The method of claim 3, wherein the pH is adjusted to 7.0-7.4 by hydrochloric acid.
8. The method for obtaining the bacterial strain capable of effectively degrading benzene series and alkane under high-salt conditions according to claim 3, wherein the specific process of inoculating the petroleum-polluted soil sample into a liquid inorganic salt culture medium under an ultra-clean bench and carrying out enrichment culture in a constant-temperature shaking table is as follows:
after the temperature of the liquid inorganic salt culture medium after high-temperature sterilization is reduced to 25-30 ℃, inoculating the soil under an ultra-clean bench according to the inoculum size of 5W/V percent, and carrying out enrichment culture in a constant-temperature shaking incubator at 28 ℃ and 180-200rpm for 14-16 days.
9. The application of the strain for effectively degrading benzene series and alkane under the high-salt condition is characterized in that the strain is used for decomposing and removing petroleum pollutants in the high-salt environment, wherein the strain is named as SJBP16, and the preservation number of the strain is CCTCC No. M2020780.
10. A method according to claim 9The application of the bacterial strain for effectively degrading benzene series and alkane under the high-salt condition is characterized in that the petroleum pollutants comprise benzene series and/or alkane, wherein the benzene series is one or two of dimethylbenzene and methylbenzene; alkane is C 16 －C 18 Is an alkane of (a).
11. The use of a strain effective for degrading benzene series and alkanes under high salt conditions according to claim 9, wherein said high salt environment is a sodium chloride-containing environment and the concentration of sodium chloride is not less than 2.0g/L.
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