CN116640682A - Microbial strain and microbial agent suitable for degrading petroleum and application thereof - Google Patents
Microbial strain and microbial agent suitable for degrading petroleum and application thereof Download PDFInfo
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- 239000003208 petroleum Substances 0.000 title claims abstract description 23
- 230000000593 degrading effect Effects 0.000 title claims abstract description 17
- 230000000813 microbial effect Effects 0.000 title claims description 22
- 239000003795 chemical substances by application Substances 0.000 title claims description 7
- 239000002689 soil Substances 0.000 claims abstract description 21
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 229930195733 hydrocarbon Natural products 0.000 claims description 9
- 150000002430 hydrocarbons Chemical class 0.000 claims description 9
- 238000005067 remediation Methods 0.000 claims description 5
- 241000187562 Rhodococcus sp. Species 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims 1
- 239000003209 petroleum derivative Substances 0.000 abstract description 19
- 241000316848 Rhodococcus <scale insect> Species 0.000 abstract description 15
- 244000005700 microbiome Species 0.000 abstract description 15
- 241000894006 Bacteria Species 0.000 abstract description 8
- 230000002906 microbiologic effect Effects 0.000 abstract description 4
- 241000209527 Arum Species 0.000 abstract description 3
- 235000006481 Colocasia esculenta Nutrition 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- 230000015556 catabolic process Effects 0.000 description 15
- 238000006731 degradation reaction Methods 0.000 description 15
- 239000010779 crude oil Substances 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 230000012010 growth Effects 0.000 description 12
- 239000001963 growth medium Substances 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000004215 Carbon black (E152) Substances 0.000 description 7
- 229910017053 inorganic salt Inorganic materials 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
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- 229910052799 carbon Inorganic materials 0.000 description 6
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- 238000012258 culturing Methods 0.000 description 5
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- 238000011161 development Methods 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 2
- 241000187563 Rhodococcus ruber Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
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- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
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- 229920001817 Agar Polymers 0.000 description 1
- WZPBZJONDBGPKJ-UHFFFAOYSA-N Antibiotic SQ 26917 Natural products O=C1N(S(O)(=O)=O)C(C)C1NC(=O)C(=NOC(C)(C)C(O)=O)C1=CSC(N)=N1 WZPBZJONDBGPKJ-UHFFFAOYSA-N 0.000 description 1
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- HEBKCHPVOIAQTA-NGQZWQHPSA-N D-Arabitol Natural products OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 1
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- HEBKCHPVOIAQTA-QWWZWVQMSA-N D-arabinitol Chemical compound OC[C@@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-QWWZWVQMSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
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- 238000003794 Gram staining Methods 0.000 description 1
- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 description 1
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- AAWZDTNXLSGCEK-ZHQZDSKASA-N Quinic acid Natural products O[C@H]1CC(O)(C(O)=O)C[C@H](O)C1O AAWZDTNXLSGCEK-ZHQZDSKASA-N 0.000 description 1
- 241001425152 Rhodococcus aetherivorans Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- WZPBZJONDBGPKJ-VEHQQRBSSA-N aztreonam Chemical compound O=C1N(S([O-])(=O)=O)[C@@H](C)[C@@H]1NC(=O)C(=N/OC(C)(C)C(O)=O)\C1=CSC([NH3+])=N1 WZPBZJONDBGPKJ-VEHQQRBSSA-N 0.000 description 1
- 229960003644 aztreonam Drugs 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- QQWGVQWAEANRTK-UHFFFAOYSA-N bromosuccinic acid Chemical compound OC(=O)CC(Br)C(O)=O QQWGVQWAEANRTK-UHFFFAOYSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- WOWBFOBYOAGEEA-UHFFFAOYSA-N diafenthiuron Chemical compound CC(C)C1=C(NC(=S)NC(C)(C)C)C(C(C)C)=CC(OC=2C=CC=CC=2)=C1 WOWBFOBYOAGEEA-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
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- 239000012535 impurity Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229940116298 l- malic acid Drugs 0.000 description 1
- OJMMVQQUTAEWLP-KIDUDLJLSA-N lincomycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@@H](C)O)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 OJMMVQQUTAEWLP-KIDUDLJLSA-N 0.000 description 1
- 229960005287 lincomycin Drugs 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- CWKLZLBVOJRSOM-UHFFFAOYSA-N methyl pyruvate Chemical compound COC(=O)C(C)=O CWKLZLBVOJRSOM-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
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- 239000000243 solution Substances 0.000 description 1
- 229960002920 sorbitol Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 108010050327 trypticase-soy broth Proteins 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
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- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Soil Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Business, Economics & Management (AREA)
- Emergency Management (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application relates to a microorganism strain suitable for degrading petroleum, which is Rhodococcus sp C83 with a preservation number of CCTCC No. M2023510. According to the application, the soil near the oil field in the arid region of south ARUM is selected as a research object, the petroleum hydrocarbon degrading bacteria are cultivated, and separated, purified and identified, and finally the strain with degrading capability is screened out, so that the problem of environmental pollution caused by petroleum hydrocarbon substances is solved by a microbiological technology, a reference is provided, a basic material is provided for developing the microbiological restoration of the petroleum contaminated soil, and a support is provided for constructing indigenous microorganism petroleum hydrocarbon degrading bacteria.
Description
Technical Field
The application relates to the technical field of materials, in particular to a microbial strain and microbial agent suitable for degrading petroleum and application thereof.
Background
With the rapid development of the petroleum industry, pollution of soil and groundwater by spilled and leaked petroleum during exploitation, collection, transportation and storage has become an important pollution problem subsequent to pesticide pollution. The predicted amount of the Xinjiang petroleum resources reaches 208.6 hundred million tons, the predicted amount of the Xinjiang petroleum resources accounts for 30 percent of the total amount of the land petroleum resources in China, the development of crude oil exploitation and downstream industries brings challenges to ecological environment protection, the restoration of petroleum hydrocarbon pollution becomes a research hot spot, and the petroleum hydrocarbon pollution can be roughly divided into a physical chemistry technology, a chemical technology, a thermal technology and a biological technology according to the working principle of the restoration technology. The biotechnology comprises phytoremediation, animal remediation and microbial remediation, wherein the microbial remediation technology is the most promising remediation technology with development potential and application prospect due to the characteristics of low cost, easy implementation, environmental friendliness and the like.
Disclosure of Invention
In order to solve the problems in the prior art, the application provides a microorganism strain and a microorganism microbial inoculum which are suitable for degrading petroleum and application thereof.
The application provides a microorganism strain suitable for degrading petroleum, which is Rhodococcus sp and is named as C83, and the preservation number is CCTCC No. M2023510.
The microbial strain C83 is gram-positive bacteria, has oxygen, no acid resistance and no motility. Cells exhibit a rod-like coccoid cycle during growth. The colonies are round, opaque, convex, grey pink and irregular in edge. Growth occurs at 15-40℃and pH 6.0-9.0, and optimal growth occurs at 30℃and pH7.0. Can grow up to 4% NaCl. The strain is subjected to 16S rDNA sequencing, and the nucleotide sequence of the strain is as shown in Seq ID No: 1. And determining as rhodococcus.
The microbial strain provided by the application is preserved in China center for type culture collection (China) for 4 months and 10 days, and the preservation address is No. 299 of Wuchang district in Wuhan, hubei province.
The strain is obtained by screening and separating from the oil-polluted soil in the arid area of south ARUM. Inorganic salt culture medium with petroleum as unique carbon source is used, and the culture medium is concentrated, separated and screened.
The application also provides a microbial agent prepared from the microbial strain.
The microorganism strain or the microorganism microbial inoculum is applied to degrading petroleum hydrocarbon.
The microbial strain or the microbial agent is applied to the treatment of greasy dirt soil.
According to the application, the soil near the oil field in the arid region of south ARUM is selected as a research object, the petroleum hydrocarbon degrading bacteria are cultivated, and separated, purified and identified, and finally the strain with degrading capability is screened out, so that the problem of environmental pollution caused by petroleum hydrocarbon substances is solved by a microbiological technology, a reference is provided, a basic material is provided for developing the microbiological restoration of the petroleum contaminated soil, and a support is provided for constructing indigenous microorganism petroleum hydrocarbon degrading bacteria.
Drawings
FIG. 1 is a photograph of the effect of a first enrichment of a soil sample;
FIG. 2 is a photograph of the effect of a second enrichment of a soil sample;
FIG. 3 is a photograph of the effect of the third enrichment of a soil sample;
FIG. 4 is a photograph of the effect of the fourth enrichment of a soil sample;
FIG. 5 is a photograph showing colony morphology of strain C83;
FIG. 6 is a photograph of the purification of strain C83;
FIG. 7 is a phylogenetic tree for the strain C83 16 SrRNA.
Detailed Description
The technical scheme of the present application will be further described with reference to specific examples, but the present application is not limited to the examples in any way. The experimental methods described in the examples are all conventional methods unless otherwise specified; the reagents and biomaterials are commercially available unless otherwise specified.
Example 1: screening and isolation of Strain C83
(1) And (3) collecting: selecting a petroleum pollution-bearing area, arranging 5 sample points according to an S shape, collecting soil samples of a surface soil layer (0-20 cm), removing impurities such as stones, fully and uniformly mixing the soil samples, reserving 2kg by a quartering method, and marking the soil samples as C; and selecting a soil sample CK1 in a plant growth area and a soil sample CK2 in a plant growth area in an uncontaminated area which is more than 500m away from the petroleum pollution area. And (5) storing at 4 ℃ and bringing the product back to a laboratory for later use.
(2) Culture medium: enrichment medium: na (Na) 2 HPO 4 2.44g；KH 2 PO 4 1.52g；(NH 4 ) 2 SO 4 1g; 3g of NaCl; 10ml of inorganic salt mother liquor; 1L of water; mother liquor formula (1L): mgSO (MgSO) 4 50g，FeSO 4 0.2g，ZnSO 4 0.2g，MnSO 4 0.2g，CaCl 2 5g; the pH value is 7.2+/-0.2.
The slant preservation medium was R2A medium, available from Qingdao sea Bo biotechnology Co.
(3) Enrichment and separation: weighing 5g of collected soil sample, inoculating into a nutrient-deficient inorganic salt hydrocarbon degrading bacteria enrichment medium taking 1% petroleum as a unique carbon source, and performing 180r min at 28 DEG C -1 After shaking culture at constant temperature for 5 days, transferring to fresh hydrocarbon degrading bacteria enrichment culture medium at a ratio of 5%, and sequentially carrying out enrichment culture for 4 times.
The results of domestication culture of microorganisms in petroleum-contaminated soil and non-contaminated soil samples using an enrichment medium with a petroleum concentration of 1% are shown in fig. 1-4, and strain growth is evident by enrichment treatment of each sample, but there is a significant difference between different samples. During the first enrichment, the strain growth concentration is lower, and the separation limit of petroleum and a culture medium is obvious; the second enrichment is obviously different from the first enrichment, CK1 is in a bulk shape, and C and CK2 are obviously emulsified. And the CK1 bulk is decomposed into smaller blocks during the third enrichment, the shape of the CK2 is not changed, the emulsification effect is still obvious, and the C is obviously changed into bulk from emulsification. And in the fourth enrichment, the CK1 agglomerate is obviously aggregated, the culture medium is clearer, the C and CK2 emulsification effects are obvious, and the C and CK2 emulsification effects are obviously changed compared with the previous ones.
After enrichment and domestication for 20 days in 4 periods, gradient dilution is adopted, the culture solution is coated on nutrient-deficient inorganic salt hydrocarbon degradation agar, and the culture is carried out for 1-2 days at 28 ℃, so that the obvious growth of bacterial colonies can be observed in a plate. After culturing for 7 days, single colonies with different colors and morphologies were selected, and purified by repeated streaking on a solid medium, and the C83 bacteria were isolated, with the strain morphology shown in FIG. 5.
Example 2: morphological observation and identification of strains
(1) The dominant strain selected was isolated and purified, and subjected to gram staining and microscopic observation, see FIG. 6. Rhodococcus C83 is a gram positive bacterium, aerobic, acid free, and motile free. Cells exhibit a rod-like coccoid cycle during growth. The colonies are round, opaque, convex, grey pink and irregular in edge. Growth occurs at 15-40℃and pH 6.0-9.0, and optimal growth occurs at 30℃and pH7.0. Can grow up to 4% NaCl.
The Rhodococcus C83 (Rhodococcus sp.) phylogenetic tree of the present application is shown in FIG. 7, and the nucleotide sequence of the strain is as shown in Seq ID No:1, the homology with the 16S rRNA gene sequence of Rhodococcus (Rhodococcus) is highest, the similarity is 99.43%, the homology with the 16S rRNA gene sequence of Rhodococcus electrodiphilus and Rhodococcus (Rhodococcus ruber) strains is 99.995%, the homology with the 16S rRNA gene sequence of Rhodococcus aetherivorans strain is 99.99%, and the Rhodococcus C83 (Rhodococcus sp.) is determined to belong to Rhodococcus (Rhodococcus) by combining with the morphological characteristics, growth conditions and physiological biochemical identification results, but the obvious difference with the Rhodococcus (Rhodococcus ruber) strain exists, and the evolution tree of FIG. 7 shows that the C83 and the Rhodococcus are initially determined to be potential new species on different branches, and named as Rhodococcus sp.C83.
Seq ID No：1
The rhodococcus C83 can grow on an inorganic salt culture medium with petroleum as the only carbon source, the optimal temperature is 28-32 ℃, and the pH value which is most suitable for the growth environment is 6.0-9.0.
Single carbon source demand: glycerol, alpha-D-glucose, D-sorbitol, D-mannitol, D-fructose, D-arabitol, L-lactic acid, beta-hydroxy-D, L-butyric acid, citric acid, acetoacetic acid, propionic acid, quinic acid, L-malic acid, bromo-succinic acid, methyl pyruvate are the only carbon sources for energy and growth, and the strain can normally grow at pH6.0,1% and 4% NaCl concentration, and has tolerance to both antibiotics of aztreonam and lincomycin.
Example 3: degradation of Petroleum hydrocarbons by Strain C83
(1) Inoculating the strain into an R2A liquid culture medium for culturing for 72 hours to obtain bacterial liquid (R2A is purchased from Qingdao high-tech industrial park Haibo biotechnology Co., ltd.);
preparation of inorganic salt selective liquid Medium (Na 2 HPO 4 2.44g，KH 2 PO 4 1.52g，(NH 4 ) 2 SO 4 1g, 3g of NaCl, 10ml of inorganic salt mother liquor, 1L of water, 1% of crude oil and 7.2+/-0.2 of pH value. Mother liquor formula (1L): mgSO (MgSO) 4 50g，FeSO 4 0.2g，ZnSO 4 0.2g，MnSO 4 0.2g，CaCl 2 5g) Packaging into test tubes every 5mL, and sterilizing with 121 deg.C high pressure steam for 30min;
sequentially adding 50 microliter of crude oil and 100 microliter of bacterial liquid, culturing for 21d in a constant temperature shaking table at 30 ℃ and 180r/min, and taking the same steps as blank control;
determination of Petroleum hydrocarbon Components (C) of Experimental group, control group and crude oil Using gas chromatography-Mass Spectrometry 13 -C 34 ) The content is as follows.
Assay results (see tables 1 and 2): wherein C is 13 -C 34 The total degradation rate of saturated hydrocarbon is as follows: 87.1% of the experimental group and 0.5% of the control group, and the degradation effect of the microorganism used in the experiment on the crude oil is obvious. C (C) 13 -C 20 Short of (2)The total degradation rate of the chain petroleum hydrocarbon is as follows: 89.2% of experimental group and 3.86% of control group; c (C) 21 -C 34 The total degradation rate of the medium-long chain petroleum hydrocarbon is as follows: 84.66% of the experimental group and-3.35% of the control group. The microorganism used in the experiment has obvious degradation effects on short-chain petroleum hydrocarbon and medium-long-chain petroleum hydrocarbon, and has obvious advantages in repairing crude oil pollution, especially treating medium-long-chain hydrocarbon components in crude oil because the biodegradability of medium-long-chain hydrocarbon in crude oil is much lower than that of short-chain hydrocarbon.
Table 1:
table 2:
(2) Inoculating the strain into an R2A liquid culture medium for culturing for 72 hours to obtain bacterial liquid (R2A is purchased from Qingdao high-tech industrial park Haibo biotechnology Co., ltd.);
preparing TSB culture medium (trypticase soy peptone liquid culture medium) with concentration of 1/5, subpackaging into test tubes every 5mL, and sterilizing with steam at 121deg.C for 30min;
sequentially adding 50 microliter of crude oil and 50 microliter of bacterial liquid, culturing for 7d in a constant temperature shaking table at 30 ℃ and 180r/min, and taking the same steps as blank comparison;
the petroleum hydrocarbon component (C13-C34) content of the experimental, control and crude oils was determined using gas chromatography-mass spectrometry.
Assay results (see tables 3 and 4): c (C) 13 -C 34 The total degradation rate of saturated hydrocarbon is as follows: 97.0% of the experimental group, 15.89% of the control group, and the degradation effect of the microorganism used in the experiment on the crude oil is more obvious, although the value is basically consistent with the first scheme after the degradation rate of the control group is deducted, in the practical concentration, the degradation effect of the microorganism in the scheme is more thorough, the time (7 d) is obviously shortened, and the degradation capability of the microorganism can be further improved by additionally adding a carbon source and a nitrogen source. C (C) 13 -C 20 The total degradation rate of short-chain petroleum hydrocarbon is as follows: 97.0% of experimental group and 16.3% of control group; c (C) 21 -C 34 The total degradation rate of the medium-long chain petroleum hydrocarbon is as follows: 97.1% of experimental group and 15.4% of control group. The microorganism used in the experiment has obvious degradation effects on short-chain petroleum hydrocarbon and medium-long-chain petroleum hydrocarbon, and has obvious advantages in repairing crude oil pollution, especially treating medium-long-chain hydrocarbon components in the crude oil.
Table 3:
table 4:
while the application has been described in terms of the preferred embodiment, it is not intended to limit the scope of the claims, and any person skilled in the art can make many variations and modifications without departing from the spirit of the application, so that the scope of the application shall be defined by the claims.
Claims (4)
1. A microbial strain suitable for degrading petroleum, characterized in that the microbial strain is Rhodococcus sp.c83, with a preservation number of cctccc No. m2023510.
2. A microbial agent prepared by using the microbial strain of claim 1.
3. Use of the microbial strain of claim 1 or the microbial agent of claim 2 for degrading petroleum hydrocarbons.
4. Use of the microbial strain of claim 1 or the microbial inoculant of claim 2 in the remediation of greasy soil.
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