AU2021229252B2 - Halophilic degrading bacterium of petroleum hydrocarbon and applications thereof - Google Patents
Halophilic degrading bacterium of petroleum hydrocarbon and applications thereof Download PDFInfo
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
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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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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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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
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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
- C12N1/26—Processes using, or culture media containing, hydrocarbons
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
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- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Abstract
The present invention relates to a halophilic degrading bacterium of petroleum hydrocarbon
and applications thereof. Provided is a halophilic degrading bacterium of petroleum hydrocarbon
Halomonas elongate ZQ1-3; the bacterium was deposited in the China General Microbiological
Culture Collection Center on October 26, 2020 with the Accession No. CGMCC No. 20953 and
address of Institute of Microbiology, Chinese Academy of Sciences, No.3 of No.1 Courtyard, West
Beichen Road, Chaoyang District, Beijing. The present invention discloses a halophilic degrading
bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 obtained by natural screening for
the first time. The bacterial strain can grow normally and degrade petroleum hydrocarbon under a
condition of 1-20 wt% salinity; compared with the existing Halomonas meridiana, the strain has
significant advantages in the aspect of petroleum hydrocarbon degradation under a high salinity
condition. Therefore, the bacterium can be applied for the removal of petroleum hydrocarbon from
petroleum-polluted soil and/or a water body under a high saline-alkaline environment.
Description
Halophilic Degrading Bacterium of Petroleum Hydrocarbon and Applications Thereof
Technical Field
[0001] The present invention relates to a halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 and applications thereof, and belongs to the technical field of microorganisms and biodegradation. Background
[0002] Most of oil fields in China are located in saline-alkaline regions, and the salinization of oil fields in coastal regions is particularly serious. During the restoration of petroleum-contaminated soil in high saline-alkaline and other special habitats, the degrading microorganism of petroleum hydrocarbon has a good remediation effect in initial stage after being added to petroleum hydrocarbon-contaminated soil in high-salinity environment, but the proportion of the added microorganism in the microbial community structure of soil decreases rapidly in a very short time, a month and even half a month to rapidly recover to a salt-tolerant or halophilic microorganism stable structure before adding the microorganism, which seriously affects the remediation effect of the petroleum hydrocarbon-contaminated soil.
[0003] Researches show that native microorganisms has a maximum potential to degrade pollutants during the bioremediation of petroleum-contaminated soil, foreign microorganisms difficultly maintain higher metabolic activity in the environment. Efficient foreign microorganisms for degrading polutants inoculated to the polluted environment are competed by the native microorganisms; and such kind of competitive relation is particularly obvious in hyperhaline special habitats.
[0004] Microbial remediation is a kind of important method for the remediation of petroleum hydrocarbon-contaminated soil. Researches show that Pseudomonas, Arthrobacter, Alcaligenes, Corynebacterium, Flavobacterium, Achromobacter, Micrococcus, Nocardia and Mycobacterium have efficient degradation properties of petroleum hydrocarbon. However, the above microorganisms are mainly non-salt-tolerant microorganisms and thus, cannot normally exert degradation in saline environment. The bioremediation in high-salinity environment is only completed by salt-tolerant or halophilic microorganisms having a biodegradability of petroleum hydrocarbon compounds. A Chinese patent literature CN110669700A (application number: 201911082757.9) discloses an efficient degrading bacterium of petroleum hydrocarbon (Halomonas titanicae) HTPA16-9; in an anaerobic medium, n-Hexadecane serves as a sole carbon source, with an initial volume of addition of 0.07734 g, the bacteria are standing in dark place at a constant temperature of 33C for anaerobic culture, 3 months later, the residual amount of n-Hexadecane is measured to calculate the degradation rate which is about 76.7%-86.5%. A Chinese patent literature CN101838616A (application number: 200910080012.9) discloses a Halomonas xianhensis A-i capable of degrading polycyclic aromatic hydrocarbon; and Halomonas xianhensis is a halophilic microorganism, and can grow and reproduce within a salinity range of 0.05%-27.5%, and can degrade phenanthrene, anthracene or fluoranthene and other various kinds of polycyclic aromatic hydrocarbons, and the salinity range most suitable for growth is 4%-10%. At present, there are reports on the degradation of petroleum hydrocarbon by salt-tolerant or halophilic bacteria; it is believed that different sources of bacterial strains have different adaptability to the environment and degradation of petroleum hydrocarbon. Moreover, through the natural screening on the environmental deviation of soil in different regions, it will cause different degrees of influence on the salt tolerance adaptability and biodegradability of petroleum hydrocarbon of different bacterial strains from the same source of species. Therefore, the bacterial strain from the same species screened from the different regional environment will have a greater difference on the biodegradability to petroleum hydrocarbon in different environment.
[0005] In the present invention, a halophilic Halomonas elongate ZQ1-3 having a high degradation efficiency of petroleum hydrocarbon is obtained with self-screening of the environment as means and by means of the particularity of the coastal saline environment of the Shengli Oil Field, and the culture and application methods are further studied. Summary
[0006] Directed to the shortcomings of the prior art, the present invention provides a halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQi-3 and applications thereof.
[0007] The present invention has the following technical solution: A halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQI-3, was deposited in the China General Microbiological Culture Collection Center (CGMCC) on October 26, 2020 with the Accession No.: CGMCC No. 20953 and an address of Institute of Microbiology, Chinese Academy of Sciences, No.3 of No.1 Courtyard, West Beichen Road, Chaoyang District, Beijing.
[0008] According to the present invention, preferably, the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ-3 has a nucleotide sequence of 16S rDNA as shown in SEQ ID NO.1.
[0009] According to the present invention, preferably, the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 grows and reproduces under a condition having a salinity of 1-20 wt%.
[0010] Further preferably, According to the present invention, preferably, the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 grows and reproduces under a condition having a salinity of 9-18 wt%.
[0011] According to the present invention, preferably, the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 degrades C1 0 -C 4 0 saturated hydrocarbon.
[0012] The culture method of the above halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 includes the following steps of: (1) taking the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1 3 and lining onto a solid activated medium for activated culture, thus obtaining an activated strain; (2) taking and inoculating the activated strain obtained in the step (1) onto a fluid medium for shake culture to prepare a seed solution; (3) taking the seed solution prepared in the step (2), transferring and inoculating the seed solution onto an expanded medium according to a volume percent of 1-10% for enlarged culture, thus preparing a Halomonas elongate ZQ1-3 bacterial solution.
[0013] According to the present invention, preferably, the solid activated medium in the step (1) has the following components: g/L peptone, 5 g/L yeast extract, 20 g/L potassium chloride, 15 g/L magnesium sulfate heptahydrate, 130 g/L sodium chloride, 20 g/L agar, and the balance being water, and pH natural.
[0014] According to the present invention, preferably, the solid activated medium in the step (1) has the following condition: inverted culture is performed for 1-2 d at 28-32°C.
[0015] According to the present invention, preferably, the fluid medium in the step (2) and the enlarged medium in the step (3) are high-saline fluid media; and the components thereof are as follows: g/L peptone, 5 g/L yeast extract, 20 g/L potassium chloride, 15 g/L magnesium sulfate heptahydrate, 130 g/L sodium chloride, and the balance being water, and pH natural.
[0016] According to the present invention, preferably, the shake culture in the step (2) has the following conditions: the shake culture is performed for 2-5 d at 28-32C and a rotating rate of
100-200 rpm.
[0017] According to the present invention, preferably, the enlarged culture in the step (3) has the following conditions: the enlarged culture is performed for 1-2 d at 28-32C under a condition of
-40% dissolved oxygen.
[0018] A degrading bacterium preparation of petroleum hydrocarbon containing the above halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 is provided.
[0019] In the present invention, the above degrading bacterium preparation of petroleum hydrocarbon may be a liquid bacterium preparation or a soild bacterium preparation; the degrading bacterium preparation of petroleum hydrocarbon may only contain a Halomonas elongate ZQ1-3 bacterium and other bacterial strains. In a preferred technical solution of the present invention, the degrading bacterium preparation of petroleum hydrocarbon is a liquid bacterium preparation, and is a Halomonas elongate ZQ1-3 bacterial solution obtained by culturing the bacterial strain. In a preferred technical solution of the present invention, the degrading bacterium preparation of petroleum hydrocarbon is a solid bacterium preparation, and is obtained by mixing the Halomonas elongate ZQ1-3 bacteria solution obtained by the culture with a solid organic matter carrier.
[0020] A liquid degrading bacterium preparation of petroleum hydrocarbon, is a bacterial solution of the above halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3.
[0021] A solid degrading bacterium preparation of petroleum hydrocarbon, is prepared by mixing the bacterial solution of the above halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 with an organic matter carrier according to a mass ratio of 1:(10-20).
[0022] Further preferably, the organic matter carrier is turfy soil, saw dust and wheat bran grass having a mass ratio of (1-3): (1-3): (1-3); preferably, the turfy soil, saw dust and the wheat bran has a mass ratio of 3:1:1.
[0023] Further preferably, the solid degradation bacterium preparation of petroleum hydrocarbon has a viable bacteria concentration of (2-5)x109cfu/g.
[0024] A composite degrading bacterium preparation of petroleum hydrocarbon includes the above halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 and Ochrobactrumdaejeonense MG35.
[0025] According to the present invention, preferably, the Ochrobactrum daejeonense MG35 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on April 27, 2020 with the Accession No. CGMCC No. 19745 and address of Institute of Microbiology, Chinese Academy of Sciences, No.3 of No.1 Courtyard, West Beichen Road, Chaoyang District, Beijing.
[0026] According to the present invention, preferably, the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 and Ochrobactrum daejeonense MG35 has a concentration ratio of living bacteria of (1-3):(1-3); and preferably, 1:1.
[0027] According to the present invention, preferably, the composite degrading bacterium preparation of petroleum hydrocarbon is prepared by mixing the Halomonas elongate ZQ1-3 bacterial solution and Ochrobactrum daejeonense MG35 bacterial solution with the organic matter carrier according to a mass ratio of 1:(10-20).
[0028] Further preferably, the organic matter carrier is turfy soil, saw dust and wheat bran grass having a mass ratio of (1-3): (1-3): (1-3); preferably, the turfy soil, saw dust and the wheat bran has a mass ratio of 3:1:1.
[0029] In a preferred technical solution of the present invention, the Halomonas elongate ZQ1-3 bacterial solution and Ochrobactrum daejeonense MG35 bacterial solution in the above bacterium preparation are obtained by the culture method of the above halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3.
[0030] According to the present invention, preferably, the composite degrading bacterium preparation of petroleum hydrocarbon has a viable bacteria concentration of (1-10)xc109 fu/g.
[0031] An application of the above halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 in repairing a petroleum-contaminated water body and/or soil.
[0032] An application of the above bacterium preparation in repairing a petroleum-contaminated water body and/or soil.
[0033] According to the present invention, preferably, the application has the following steps of: inoculating the above bacterium preparation onto petroleum-contaminated soil having an oil content of 3-7%, where the mass ratio of the bacterium preparation to the petroleum-contaminated soil is (1 ): 100, water content is adjusted to 20-25%, and mixing evenly, and naturally pilling for degradation.
[0034] An application of the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 in repairing a petroleum-contaminated water body and/or soil in a saline environment.
[0035] The present invention has the following technical features and beneficial effects: The present invention discloses a halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 obtained by natural screening for the first time. The bacterial strain can grow normally and degrade petroleum hydrocarbon under a condition of 1-20wt% salinity; and the bacterial strain can grow much better under a condition of 9-18wt% salinity. Compared with the existing Halomonas meridiana, the strain has significant advantages in the aspect of the degradation of petroleum hydrocarbon under a high salinity condition (especially, C 10-C 4 0 saturated hydrocarbon). Therefore, the bacterium can be applied for the removal of petroleum hydrocarbon from petroleum-polluted soil and/or a water body under a high saline-alkaline environment.
[0036] Compared with the Halomonas titanicae HTPA16-9 in the patent literature CN110669700A, the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 has a stronger salt tolerance and a higher degradation efficiency of petroleum hydrocarbon. Halomonas elongate ZQ1-3 grows better under a condition of 9-18 wt% salinity, while during the screening and degradation of Halomonas titanicae HTPA16-9, the content of sodium chloride in the medium is only 22 g/L. Halomonas elongate ZQ1-3 has a 65.7% degradation rate to petroleum hydrocarbon after being degraded for 15 d under a condition of sodium chloride content of 5%; Halomonas titanicae HTPA16-9 is standing in dark place at a constant temperature for anaerobic culture for 3 months with n-Hexadecane as a sole carbon source under a condition of sodium chloride content of 22 g/L; where the degradation rate to n-Hexadecane is about 76.7%-86.5%, that is, the degradation time is long, efficiency is low, and degradation conditions are complex. Besides having a higher degradation efficiency to N-hexadecane, Halomonas elongate ZQ1-3 of the present invention has a better degradation effect on C1 0 -C 4 0 saturated hydrocarbon as well.
[0037] Compared with the Halomonas xianhensis A-1 in the patent literature CN101838616A, the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 in the present invention has a stronger salt tolerance, and the petroleum hydrocarbon has different types of degradation. Halomonas elongate ZQ1-3 of the present invention can grow and reproduce within a salinity range of 1-20%, and the salinity range suitable for growth is 9-18%; Halomonas xianhensis A-1 can grow and reproduce within a salinity range of 0.05-27.5%, and the salinity range suitable for growth is 4-10%. Through the analysis on the salinity range suitable for growth, Halomonas elongate ZQ1-3 of the present invention has a better salt tolerance. Saturated hydrocarbon (alkane) is a major component of petroleum; hydrocarbon substances leaked are poured into the gap of soil to influence the soil permeability, damage the water phase, gas phase and solid phase structures of the crude oil soil, and influence the growth of microorganisms in soil; and may be usually migrated with the water flow to continuously expand the contaminated areas. As a kind of hazardous substance in petroleum, polycyclic aromatic hydrocarbon can cause carcinogenesis, teratogenesis and mutagenesis, and can enter to organisms even to human body through a food chain, thereby directly damaging human health. Saturated hydrocarbon and polycyclic aromatic hydrocarbon are major polluting components in petroleum. Halomonas elongate ZQ1-3 of the present invention mainly degrades saturated hydrocarbon, especially, C1 0 -C 4 0 saturated hydrocarbon; Halomonas xianhensis A-1 mainly degrades polycyclic aromatic hydrocarbon, especially phenanthrene, anthracene and fluoranthene.
[0038] The present invention further provides a composite degrading bacterium preparation of petroleum hydrocarbon mainly consisting of Halomonas elongate ZQ1-3 and Ochrobactrum daejeonense MG35. Under the condition of the same concentration of the living bacteria, the degradation efficiency of the composite degrading bacterium preparation consisting of Halomonas elongate ZQ1-3 and Ochrobactrum daejeonense MG35 to petroleum hydrocarbon is significantly greater than that of the single Halomonas elongate ZQ1-3 or Ochrobactrum daejeonense MG35. It indicates that the composite degrading bacterium preparation formed by the two strains has synergistic effect and acceleration to the degradation of petroleum hydrocarbon. Brief Description of the Drawings
[0039] FIG. 1 is an agarose gel electrophoretogram of 16S rDNA of Halomonas elongate ZQ1-3.
[0040] FIG. 2 is a growth curve of Halomonas elongate ZQ1-3 at different NaCl concentrations.
[0041] FIG. 3 is a bar graph of degradation rate of Halomonas elongate ZQ1-3 to petroleum hydrocarbon having different carbon number.
[0042] FIG. 4 is a degradation rate curve of the composite degrading bacterium preparation of petroleum hydrocarbon to petroleum hydrocarbon. Detailed Description of the Embodiments
[0043] The technical solution of the present invention will be further specified in combination with examples and drawings of the description, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, drugs and reagents in the examples are common products available in the market; the experimental operation and steps involved in the examples are conventional operations of the art.
[00441 Source of biological materials: A halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3, was deposited in the China General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms (CCCCM) on October 26, 2020 with the Accession No.: CGMCC No. 20953 and an address of Institute of Microbiology, Chinese Academy of Sciences, No.3 of No.1 Courtyard, West Beichen Road, Chaoyang District, Beijing.
[0045] An Ochrobactrumdaejeonense MG35 was deposited in the China General Microbiological Culture Collection Center of China Committee for Culture Collection of Microorganisms(CCCCM) on April 27, 2020 with the Accession No. CGMCC No. 19745 and address of Institute of Microbiology, Chinese Academy of Sciences, No.3 of No.1 Courtyard, West Beichen Road, Chaoyang District, Beijing. The bacterial strain has been disclosed in a patent CN202010443089.4 and does not relate to microbial preservation in the present invention.
[0046] Medium: In an inorganic salt medium, per liter of component includes as follows: KNO3 1.5 g, (NH 4 ) 2 SO4 1.5 g, K 2HP04 1 g, KH 2 PO4 1 g, MgSO 4 -7H 2 O 0.5 g, NaCl 130 g, FeSO4 -7H2 0.01 g,, and dH 20 is filled to a volume of 1 L.
[0047] In a petroleum-solid inorganic salt medium, per liter of component includes as follows: KNO3 1.5 g, (NH 4 ) 2 SO4 1.5 g, K2HPO4 1 g, KH 2 PO 4 1 g, MgSO4-7H 2 O 0.5 g, NaCl 130 g, FeSO4 -7H 2 0.01 g, petroleum 20 g, agar 20 g, and dH 20 is filled to a volume of1 L.
[0048] In a petroleum-inorganic salt fluid medium, per liter of component includes as follows:
KNO3 1.5 g, (NH4 ) 2 SO4 1.5 g, K2HPO4 1 g, KH 2 PO4 1 g, MgSO4-7H 2 O 0.5 g, NaCl 130 g, FeSO4-7H 2 0.01 g, petroleum 20 g, and dH2 0 is filled to a volume of 1 L.
[0049] Example 1 Isolation and identification of a halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 2 g high-concentration petroleum-contaminated soil from a coastal area of Shengli Oil Field were collected, and put to a 150 mL sterile conical flask, 50 mL sterile inorganic salt medium was added for culture for 3 d at 30°C and 150 rpm; then bacterial solution was sucked up and gradiently
diluted by sterile water respectively to 10-1, 10-2, 10-3, 10-4, and 10-5 folds, 100 L diluted bacterial solution was coated on a sterile petroleum-solid inorganic salt medium for static culture for 3 d at °C; the large bacterial colonies growing fast were picked and cloned onto a 50 mL petroleum
inorganic salt fluid medium for culture for 3 d at 30°C and 150 rpm; then 100 L bacterial solution
was sucked onto a sterile petroleum-solid inorganic salt medium, and then single colonies were picked.
[0050] The single colonies picked after culture were taken and sent to a sequencing company for sequencing; through detection, 16S rDNA sequence contained 1392bp having a nucleotide sequence as shown in SEQ ID NO.1.
[0051] Identification process of the species was as follows: Sample: bacterial solution screened in the present invention; Kit for the genome DNA extraction of the bacterium: Sangon Biotech (Shanghai); TAE buffer solution (50x, 1 L): water wadded to 242 g Tris, 57.1 mL glacial acetic acid, 37.2 g Na2EDTA-2H 2 O to 1 L; agarose: BIOWET, AGAROSE G-10; 2xPfu PCR MasterMix, D2000 DNA Marker, nucleic acid dye, loading buffer and the like: Sangon Biotech (Shanghai); DNA purification and recovery kit: Sangon Biotech (Shanghai); Centrifuge tube, hammerhead and other consumables: Gene Era Biotech; Primer was synthesized by Qingdao TSINGKE Zixi Biological Technology, and ddH20 was added alone to prepare into a 10 M solution.
[0052] 1. Genome DNA extraction was performed by the kit for the genome DNA extraction of the bacterium.
[0053] 2. PCR amplification
[0054] 2.1. Universal primer information was shown in Table 1. Table 1 Universal primer information of the bacterium
Primer name Primer sequence (5'-3') Amplified sequence Amplification length
27F AGAGTTTGATCMTGGCTCAG 16S rDNA 1500bp around 1492R TACGGYTACCTTGTTACGACTT
[0055] 2.2. Components and composition of the PCR amplification system, as shown in Table 2.
Table 2 PCR amplification system No. Component Volume (pL)
1 2xPfu PCR MasterMix 25 2 27F 2 3 1492R 2 4 DNA template 1 5 ddH20 was added to 50
[0056] 2.3. PCR procedure
Predegeneration: the bacterium was denatured for 3 min at 94C, for 30 s at 94C, annealed for 30 s
at 55C, extended for 1.5 min at 72C (35 cycles), and then extended for 10 min at 72C, and
preserved at 4C.
[0057] 3. Agarose gel electrophoresis detection
1.0% agarose gel was prepared, where electrophoresis voltage was set to 18 V/cm and electrophoresis time was 20 min; a nucleic acid dye was used for agarose electrophoresis dying, UV gel-imaging system was used for taking pictures, and the results were shown in FIG. 1; there was one and only one bright target band in the sample lane, and the band had a size of 1400 bp around.
[0058] 4. Purification and recovery
Target fragments were subjected to agarose gel recovery by a common agarose gel DNA recovery kit, and the recovery product was sent to Qingdao TSINGKE Zixi Biological Technology for sequencing, and the blast alignment result of a sequencing splice sequence was shown in Table 3.
Table 3 Blast alignment result of the sequencing splice sequence
Description Max Total Query E value Ident Accession score score cover Halomonaselongatastrain BK-AB8 2571 2571 100% 0.0 100% KJ185379.1 16S ribosomal RNA gene, partial sequence
Through the sequence alignment of a sequence 16S rDNA, it was found that the bacterial strain having a proximal genetic relationship was Halomonas elongata BK-AB8, NCBI accession number was KJ185379.1.
[0059] Through the identification of the above bacterial strain, the bacterial strain screened in the present invention falls with the category of Halomonas elongata, and is named Halomonas elongate ZQ1-3. The bacterial strain was deposited in the China General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms(CCCCM) on Monday, October 26, 2020 with the Accession No. CGMCC No. 20953 and address of Institute of Microbiology, Chinese Academy of Sciences, No.3 of No.1 Courtyard, West Beichen Road, Chaoyang District, Beijing.
[0060] Example 2 The culture method of the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 had the following steps: (1) the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 was taken and lined onto a solid medium for activated culture for 2 d at 32C, thus finally obtaining an activated strain; (2) the activated strain obtained in the step (1) was taken and inoculated onto a fluid medium for shake culture for 2 d at a condition of 150 rmp at 32C to prepare a seed solution; (3) the seed solution prepared in the step (2) was taken, transferred and inoculated onto an expanded medium according to a volume percent of 2% for enlarged culture for 2 d at 32C and 30% dissolved oxygen, thus preparing a Halomonas elongate ZQ1-3 bacterial solution, where the viable bacteria concentration in the bacterial solution was 2x109 cfu/mL.
[0061] The medium used was as follows: In the fluid medium, per liter of component included as follows: 10 g/L peptone, 5 g/L yeast extract, g/L potassium chloride, 15 g/L magnesium sulfate heptahydrate, 130 g/L sodium chloride, and water was filled to a volume of 1 L at a natural pH value.
[0062] In the solid medium, per liter of component included as follows: 10 g/L peptone, 5 g/L yeast extract, 20 g/L potassium chloride, 15 g/L magnesium sulfate heptahydrate, 130 g/L sodium chloride, 20 g agar and water was filled to a volume of 1 L at a natural pH value.
[0063] A degrading bacterium preparation of petroleum hydrocarbon was provided; a solid degrading bacterium preparation of petroleum hydrocarbon was prepared by mixing the Halomonas elongate ZQ1-3 bacterial solution prepared above with an organic matter carrier (turfy soil: saw dust: bran=3:1:1, a mass ratio) according to a mass ratio of 1:10, and the naturally put for 5 d, then detected; where the viable bacteria concentration in the bacterium preparation was 3.3x109 cfu/g.
[0064] Example 3 The salt-tolerance performance test of the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 had the following steps:
(1) NaCl content in a LB medium was adjusted to respectively prepare into LB fluid media having NaCl mass concentrations of 3%, 6%, 9%, 12%, 15%, and 18%; (2) the seed solution prepared in the step (2) of Example 2 was taken and inoculated onto LB fluid media containing different NaCl content prepared in the step (1) by an inoculum size of 2% volume percent; (3) the culture solution inoculated in the step (2) was cultured for 5 d at a condition of 32°C and
% dissolved oxygen.
[0065] During the culture process, OD 6 0 0of the bacterial solution was measured every 12 h to draw a growth curve of the bacterial strain, and the results were shown in FIG. 2; the growth of Halomonas elongate ZQ1-3 exhibited a trend of climbing up and then decline at a low concentration (3 wt% and 6 wt%) of NaCl, and exhibited a trend of climbing up and being stable at a high concentration (9 wt%, 12 wt%, 15 wt% and 18 wt%) of NaCl, which indicated that low salinity condition was against the stable growth of Halomonas elongate ZQ1-3, and Halomonas elongate ZQ1-3 has halophilic characteristics.
[0066] Viable bacteria were counted 5 d after culture; the viable counts in fermentation broth of LB fluid media at NaCl mass concentrations of 3%, 6%, 9%, 12%, 15%, and 18% were respectively 3.1x108, 3.3x108, 2.9x109, 3.2x109, and 3.1x109 cfu/mL; and the above results also indicated that Halomonas elongate ZQ1-3 grew better in high-salinity condition than low-salinity condition, and had halophilic characteristics.
[0067] Example 4 An application of the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 in repairing a petroleum-contaminated water body and/or soil in a high-salinity environment had the following steps: (1) mass concentration of NaCl in a petroleum-inorganic salt fluid medium was respectively adjusted to 1%, 5%, 10%, 15%, and 20%; and then the seed solution prepared in the step (2) of Eaxmple 2 was inoculated according to a ratio of 2% volume percent; (2) the culture solution inoculated in the step (1) was cultured and degraded for 15 d at a condition of 32C and 150 rpm.
[0068] Gas phase method (HJ 1021-2019) was used to detect the components of the residual C10 C 4 0 petroleum hydrocarbon and calculate a degradation rate, and the results were shown in FIG. 3. The halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 respectively had a degradation rate to petroleum hydrocarbon of 31.2%, 65.7%, 29.8%, 28.6%, and 27.3% at the conditions of NaCl mass concentration of 1%, 5%0, 10%,15%, and 20%, and had a better degrading effect on petroleum hydrocarbon with different carbon number in petroleum.
[0069] In a petroleum-inorganic salt fluid medium of this example, per liter of component included as follows: KNO3 1.5 g, (NH 4 ) 2 SO4 1.5 g, K2HPO4 1 g, KH 2 PO4 1 g, MgSO4-7H 2 0 0.5 g, NaCl 130 g, FeSO4 -7H20 0.01 g, petroleum 20 g, and dH2 0 is filled to a volume of 1 L. NaCl concentration in the media was adjusted according to the demands.
[0070] Example 5 An application of the degrading bacterium preparation of petroleum hydrocarbon prepared by the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 in repairing a petroleum hydrocarbon-contaminated water body and/or soil in a high-salinity environment had the following steps: (1) petroleum-contaminated saline alkali soil (soluble salt had a content of 1.08%) from coastal areas and having an oil content of 4.58% was evenly mixed with the degrading bacterium preparation of petroleum hydrocarbon prepared in Example 2 according to a mass ratio of 50:1, and water content of the bacterium-soil mixture was adjusted to 25% with sterile purified water, and at this time, the viable bacteria concentration was 4.8x108 CFU/mL; (2) water content of the bacterium-soil mixture was kept to 25%, and naturally piled up at a height of 75 cm and degraded for 30 d at room temperature (25±5°C).
[0071] A gas phase method (HJ 1021-2019) was used to detect the components of the residual petroleum hydrocarbon and calculate the degradation rate; 30 d later, petroleum-contaminated saline alkali soil had an oil content of 2.44%; the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 had a degradation rate of 46.7% to petroleum in the petroleum-contaminated saline alkali soil having an oil content of 4.58%; 30 d later, the viable bacteria concentration was 3.8x1O 8 CFU/mL, indicating that the petroleum-contaminated saline alkali environment basically had little impact on the growth of Halomonas elongate ZQ1-3, maintained the growth activity of Halomonas elongate ZQ1-3, and continued to exert the ability to repair petroleum hydrocarbon-contaminated soil.
[0072] Example 6 A composite degrading bacterium preparation of petroleum hydrocarbon, included the halophilic degradation bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 and Ochrobactrum daejeonense MG35; and the composite preparation was prepared by mixing the Halomonas elongate ZQ1-3 bacterial solution and Ochrobactrum daejeonense MG35 bacterial solution with an organic matter carrier.
[0073] An application of the above composite degrading bacterium preparation of petroleum hydrocarbon in repairing a high-salinity petroleum hydrocarbon-contaminated soil had the following steps: (1) the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 and Ochrobactrum daejeonense MG35 were respectively cultured according to the culture method in Example 2, and the obtained bacterial solution was centrifuged, and then bacteria were washed by sterile purified water for twice, and resuspended, and the resuspended bacterial solution was adjusted to OD6 0 0 =1.0; (2) three sets of bacterium preparations were set below: mL Ochrobactrumdaejeonense MG35 resuspended baterial solution (OD6 0 0 =1.0) was taken and added to 50 g organic matter carrier (turfy soil: saw dust: bran=3:1:1, amass ratio), placed for 3 d at room temperature (25±5 °C ) to prepare a bacterium preparation 1, and the viable bacteria concentration was 3.52x10 9cfu/g; mL Halomonas elongate ZQ1-3 resuspended bacterial solution (OD6 0 0 =1.0) was taken and added to 50 g organic matter carrier (turfy soil: saw dust: bran=3:1:1, amass ratio), placed for 3 d at room temperature (25±5C) to prepare a bacterium preparation 2, and the viable bacteria concentration was 3.38x10 9cfu/g; mL Ochrobactrum daejeonense MG35 resuspended baterial solution (OD 600=1.0) and 5 mL Halomonas elongate ZQ1-3 resuspended bacterial solution (OD6 0 0 =1.0) were respectively taken and added to 50 g organic matter carrier (turfy soil: saw dust: bran=3:1:1, amass ratio), placed for 3 d at room temperature (25±5C) to prepare a bacterium preparation 3, and the viable bacteria concentration was 3.47x10 9cfu/g; water content in the 50 g organic matter carrier (turfy soil: saw dust: bran=3:1:1, amass ratio) was adjusted to 20% around, and placed for 3 d at room temperature (25±5C) as a control group CK;
(3) the organic matter carrier CK and bacterium preparations 1, 2, and 3 were respectively added to 500 g petroleum-contaminated soil (soluble salt content was 1.03%) having an oil content (measured by a gravimetric method) of 3.23%, the water content was adjusted and maintained to %, then the mixture was stirred and ventilated for once, and degraded for 25 d.
[0074] A gravimetric method was used to monitor the residuals of the petroleum hydrocarbon in different treatment groups, and to calculate the degradation rate, and the results were shown in FIG. 4. 25 d later, the CK group had a degradation rate of petroleum hydrocarbon of 14.3%; the bacterium preparation 1 had a degradation rate of petroleum hydrocarbon of 29.9%, the bacterium preparation 2 had a degradation rate of petroleum hydrocarbon of 32.4%; and the bacterium preparation 3 had a degradation rate of petroleum hydrocarbon of 42.2%. Thus, it can be seen that the degradation rate of the bacterium preparations 1, 2 and 3 is higher than that of the control group. The composite bacterium preparation formed by the two bacterial strains has a maximum degradation efficiency to petroleum hydrocarbon, which indicates that the two strains have synergistic effect and acceleration to the degradation of petroleum hydrocarbon, thus achieving the unexpected technical effect.
[0075] Result analysis: It can be seen from the salt-tolerant growth data and degradation data of salt-tolerant petroleum hydrocarbon of the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 in Examples 3 and 4 that the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 can be cultured normally at a NaCl content of 18%, and has a good degradating effect on petroleum hydrocarbon at a condition of 10%-20% NaCl content. Therefore, the present invention has a wide application scope and good application value.
[0076] It can be seen from the degradation data of the composite bacterium preparation formed by the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 and the salt-tolerant strain Ochrobactrum daejeonense MG35 in Example 6 that the composite bacterium preparation formed by the two strains has synergistic effect and acceleration to the degradation of petroleum hydrocarbon. Moreover, the composite bacterium preparation has a better application than the effect of a single bacterium, and thus has a better popularization and application value.
3306195_1 Sequence Listing
<110> Ecology Institute of Shandong Academy of Sciences(China-Japan Friendship Biotechnology Research Center of Shandong Academy of Sciences)
<120> Halophilic Degrading Bacterium of Petroleum Hydrocarbon and Applications Thereof
<130> QCNFPCT2102
<150> CN202110482736.7 <151> 2021-04-30
<160> 1
<170> PatentIn version 3.5
<210> 1 <211> 1392 <212> DNA <213> Halomonas elongate
<400> 1 accgtggtga tcgccctccg aagttaggct aaccacttct ggtgcagtcc actcccatgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgtgccattc tgatgcacga 120
ttactagcga ttccgacttc acggagtcga gttgcagact ccgatccgga ctgagaccgg 180
ctttatgaga ttagctccac gtcgccgctt tgcaacccat tgtaccggcc attgtagcac 240
gtgtgtagcc ctacccgtaa gggccatgat gacttgacgt cgtccccacc ttcctccggt 300
ttgtcaccgg cagtctccct agagttcccg accgaatcgc tggcaaatag ggacaagggt 360
tgcgctcgtt acgggactta acccaacatt tcacaacacg agctgacgac agccatgcag 420
cacctgtctg tgcgctcccg aaggcaccaa tccatctctg gaaagttcgc acgatgtcaa 480
gggtaggtaa ggttcttcgc gttgcatcga attaaaccac atgctccacc gcttgtgcgg 540
gcccccgtca attcatttga gttttaacct tgcggccgta ctccccaggc ggtcgactta 600
gtgcgttaac tgcgccacaa aggtctcgag gaccccaacg gctagtcgac atcgtttacg 660
gcgtggacta ccagggtatc taatcctgtt tgctacccac gctttcgtac ctcagcgtca 720
gtgtcagtcc agaaggccgc cttcgccact ggtattcctc ccgatctcta cgcatttcac 780
cgctacaccg ggaattctac cttcctctcc tgcactctag cctaacagtt ccggatgccg 840
ttcccaggtt gagcccgggg ctttcacaac cggcttatca agccgcctac gcacgcttta 900 Page 1
3306195_1
cgcccagtaa ttccgattaa cgctcgcacc ctccgtatta ccgcggctgc tggcacggag 960
ttagccggtg cttcttctgc gagtgatgtc tcccttgccg ggtattaacc gacaagcatt 1020
cttcctcgct gaaagtgctt tacaacccga gggccttctt cacacacgcg gcatggctgg 1080
atcagggttg cccccattgt ccaatattcc ccactgctgc ctcccgtagg agttcgggcc 1140
gtgtctcagt cccgatgtgg ctgatcatcc tctcagacca gctacggatc gtcgccttgg 1200
tgagccgtta cctcaccaac cagctaatcc gacataagct catccgatag cgcaaggtcc 1260
gaagatcccc tgctttcccc cgtagggcgt atgcggtatt agcttgagtt tcctcaagtt 1320
atcccccact accgggcaga ttcctatgca ttactcaccc gtccgccgct cgacgcctcc 1380
tagcaagcta gg 1392
Page 2
Claims (10)
- CLAIMS 1. A halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3, characterized in that the bacterium was deposited in the China General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms (CCCCM) on October 26, 2020 with the Accession No.: CGMCC No. 20953 and an address of Institute of Microbiology, Chinese Academy of Sciences, No.3 of No.1 Courtyard, West Beichen Road, Chaoyang District, Beijing.
- 2. The halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 of claim 1, characterized in that one or more of the following conditions are satisfied: i. the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 has a nucleotide sequence of 16S rDNA as shown in SEQ ID NO.1; ii. the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 grows and reproduces under a condition having a salinity of 1-20 wt%; iii. the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 degrades C10-C 4 0 saturated hydrocarbon.
- 3. A culture method of the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 of claim 1, characterized by comprising the following steps of: (1) taking the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1 3 and lining onto a solid activated medium for activated culture, thus obtaining an activated strain; (2) taking and inoculating the activated strain obtained in the step (1) onto a fluid medium for shake culture to prepare a seed solution; (3) taking the seed solution prepared in the step (2), transferring and inoculating the seed solution onto an expanded medium according to a volume percent of 1%-10% for enlarged cultivation, thus preparing a Halomonas elongate ZQ1-3 bacterial solution.
- 4. The culture method of claim 3, characterized in that one or more of the following conditions are satisfied: i. the solid activated medium in the step (1) has the following components: 10 g/L peptone, 5 g/L yeast extract, 20 g/L potassium chloride, 15 g/L magnesium sulfate heptahydrate, 130 g/L sodium chloride, 20 g/L agar, and the balance being water, and pH natural; ii. the activated culture in the step (1) has the following conditions: inverted culture is performed for 1-2 d at 28-32°C; iii. the fluid medium in the step (2) and the enlarged medium in the step (3) are high-saline fluid media; and the components thereof are as follows: 10 g/L peptone, 5 g/L yeast extract, 20 g/L potassium chloride, 15 g/L magnesium sulfate heptahydrate, 130 g/L sodium chloride, and the balance being water, and pH natural; iv. the shake culture in the step (2) has the following conditions: the shake culture is performed for 2-5 d at 28-32C and a rotating rate of 100-200 rmp; v. the enlarged culture in the step (3) has the following conditions: the enlarged culture is performed for 1-2 d at 28-32C under a condition of 20-40% dissolved oxygen.
- 5. A degrading bacterium preparation of petroleum hydrocarbon comprising the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 of claim 1.
- 6. A liquid degrading bacterium preparation of petroleum hydrocarbon, characterized in that the preparation is a bacterial solution of the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 of claim 1.
- 7. A solid degrading bacterium preparation of petroleum hydrocarbon, characterized in that the solid preparation is prepared by mixing the degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 of claim 1 with an organic matter carrier according to a mass ratio of 1:(10-20); preferably, the organic matter carrier is turfy soil, saw dust and wheat bran grass having a mass ratio of (1-3): (1-3):(1-3); preferably, the solid degrading bacterium preparation of petroleum hydrocarbon has a viable bacteria concentration of (2-5)x10 9cfu/g.
- 8. A composite degrading bacterium preparation of petroleum hydrocarbon, characterized by comprising the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 of claim 1 and Ochrobactrum daejeonense MG35; wherein, the Ochrobactrumdaejeonense MG35 was deposited in the China General Microbiological Culture Collection Center of China Committee for Culture Collection of Microorganisms(CCCCM) on April 27, 2020 with the Accession No. CGMCC No. 19745 and address of Institute of Microbiology, Chinese Academy of Sciences, No.3 ofNo.1 Courtyard, West Beichen Road, Chaoyang District, Beijing. Preferably, the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 and Ochrobactrumdaejeonense MG35 has a concentration ratio of living bacteria of (1 3):(1-3); preferably, the composite degrading bacterium preparation of petroleum hydrocarbon is prepared by mixing the Halomonas elongate ZQ1-3 bacterial solution and Ochrobactrum daejeonense MG35 bacterial solution with the organic matter carrier according to a mass ratio of 1:(10-20); preferably, the organic matter carrier is turfy soil, saw dust and wheat bran grass having a mass ratio of (1-3): (1-3):(1-3); preferably, the composite degrading bacterium preparation of petroleum hydrocarbon has a viable bacteria concentration of (1-1O)x109cfu/g.
- 9. An application of the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ1-3 of claim 1 or the bacterial preparation any one of claims 5-8 in repairing a petroleum-polluted water body and/or soil.
- 10. An application of the halophilic degrading bacterium of petroleum hydrocarbon Halomonas elongate ZQ-3 of claim 1 or the bacterial preparation any one of claims 5-8 in repairing a petroleum-polluted water body and/or soil in a saline environment.
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