WO2022227239A1 - Halophilic petroleum-hydrocarbon-degrading bacterium and use thereof - Google Patents

Halophilic petroleum-hydrocarbon-degrading bacterium and use thereof Download PDF

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WO2022227239A1
WO2022227239A1 PCT/CN2021/099990 CN2021099990W WO2022227239A1 WO 2022227239 A1 WO2022227239 A1 WO 2022227239A1 CN 2021099990 W CN2021099990 W CN 2021099990W WO 2022227239 A1 WO2022227239 A1 WO 2022227239A1
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petroleum hydrocarbon
halophilic
petroleum
bacterial
hydrocarbon degrading
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French (fr)
Chinese (zh)
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张强
翟栓丽
侯心然
李天元
季蕾
傅晓文
王加宁
李琪
邢颖娜
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山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心)
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Priority to AU2021229252A priority Critical patent/AU2021229252B2/en
Publication of WO2022227239A1 publication Critical patent/WO2022227239A1/en

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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
    • 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
    • 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
    • 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
    • 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
    • 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
    • C12N1/26—Processes using, or culture media containing, hydrocarbons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • 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
    • 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
    • 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

Definitions

  • the invention relates to a halophilic petroleum hydrocarbon degrading bacterium Halomonas elongata ZQ1-3 and an application thereof, belonging to the technical field of microorganisms and biodegradation.
  • microorganisms have the greatest potential to degrade pollutants during the bioremediation of oil-contaminated soils, and it is difficult for foreign microorganisms to maintain high metabolic activity in the environment.
  • microorganisms are subject to competition from indigenous microorganisms, especially in special habitats with high salinity.
  • Microbial remediation is an important method for remediation of petroleum hydrocarbon contaminated soil.
  • Pseudomonas Pseudomonas
  • Arthrobacter Arthrobacter
  • Alcaligenes Alcaligenes
  • Corynebacterium Corynebacterium
  • Flavobacterium Flavobacterium
  • Achromobacter Achromobacter
  • Micrococcus Micrococcus
  • Nocardia Nocardia
  • Mycobacterium Mycobacterium
  • the invention utilizes the particularity of the coastal saline-alkali environment of Shengli Oilfield, and takes the self-screening of the environment as a means to obtain a Halomonas elongate ZQ1-3 which is halophilic and has high petroleum hydrocarbon degradation efficiency, and studies its culture and application. method.
  • the present invention provides a halophilic petroleum hydrocarbon degrading bacterium Halomonas elongate ZQ1-3 and its application.
  • nucleotide sequence of the 16S rDNA of the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 is shown in SEQ ID NO.1.
  • the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 grows and reproduces under the condition that the salt content is 1-20wt%.
  • the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 grows and reproduces under the condition that the salt content is 9-18wt%.
  • the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 degrades C 10 -C 40 saturated hydrocarbons.
  • the culture method of above-mentioned halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 comprises the steps:
  • step (2) get the activated bacterial strain obtained in step (1) and inoculate it into a liquid medium, and shake it to cultivate to obtain seed liquid;
  • step (3) Take the seed liquid obtained in step (2), transfer it to the expansion medium according to the volume percentage of 1% to 10%, and expand the culture to obtain the Halomonas elongate ZQ1-3 bacterial liquid.
  • the components of the solid activation medium described in step (1) are as follows:
  • the conditions for activation and cultivation in step (1) are: inverted cultivation at 28-32° C. for 1-2 days.
  • the liquid medium described in step (2) and the expansion medium described in step (3) are high-salt liquid medium, and the components are as follows:
  • the conditions of the shaker culture in step (2) are: shaker culture for 2 to 5 days at 28 to 32°C and a rotational speed of 100 to 200 rpm.
  • the conditions for expanding the culture in step (3) are: expanding the culture for 1-2 days under the conditions of 28-32° C. and dissolved oxygen of 20-40%.
  • the above-mentioned petroleum hydrocarbon-degrading bacterial agent can be a liquid bacterial agent or a solid bacterial agent; the petroleum hydrocarbon-degrading bacterial agent can only contain Halomonas elongate ZQ1-3 one kind of bacteria and can also contain other bacterial strains.
  • the petroleum hydrocarbon degrading bacterial agent is a liquid bacterial agent, which is the Halomonas elongate ZQ1-3 bacterial liquid obtained after strain culture.
  • the petroleum hydrocarbon degrading bacterial agent is a solid bacterial agent, obtained by mixing the Halomonas elongate ZQ1-3 bacterial liquid obtained by culturing with an organic solid carrier.
  • a petroleum hydrocarbon degrading liquid bacterial agent is the above-mentioned halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 bacterial liquid.
  • a petroleum hydrocarbon degrading solid bacterial agent is prepared by mixing the above-mentioned halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 bacterial liquid and an organic matter carrier in a mass ratio of 1:(10-20).
  • the organic matter carrier is turf soil, sawdust and bran grass, and the mass ratio is (1-3):(1-3):(1-3); The mass ratio is 3:1:1.
  • the viable bacteria concentration of the petroleum-degrading solid bacterial agent is (2-5) ⁇ 10 9 cfu/g.
  • a petroleum hydrocarbon degrading compound bacterial agent comprising the above-mentioned halomonas elongate ZQ1-3 and Ochrobactrum daejeonense MG35.
  • the Ochrobactrum daejeonense MG35 was deposited in the General Microorganism Center of the China Microorganism Culture Collection Management Committee on April 27, 2020, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Academy of Sciences, strain collection number: CGMCC No.19745.
  • the effective bacterial concentration ratio of the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 and Ochrobactrum daejeonense MG35 is (1 ⁇ 3): (1 ⁇ 3); preferably 1:1 .
  • the described petroleum hydrocarbon degrading compound bacterial agent is prepared by mixing Halomonas elongate ZQ1-3 bacterial liquid and Bacillus pallidum MG35 bacterial liquid with organic matter carrier in a ratio of 1:(10 ⁇ 20) by mass.
  • the organic matter carrier is turf soil, sawdust and bran grass, and the mass ratio is (1-3):(1-3):(1-3); The mass ratio is 3:1:1.
  • the Halomonas elongate ZQ1-3 bacterial liquid and the pale bacillus MG35 bacterial liquid in the above bacterial agent are both obtained by culturing the above-mentioned halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3.
  • the viable bacteria concentration of the petroleum hydrocarbon-degrading compound bacterial agent is (1-10) ⁇ 10 9 cfu/g.
  • the application steps are as follows:
  • the mass ratio of the bacterial agent to the oil-contaminated soil is (1-10): 100, adjust the moisture content to 20-25%, mix well, and naturally stacking degradation.
  • the present invention discloses for the first time a halophilic petroleum hydrocarbon degrading bacterium Halomonas elongate ZQ1-3 obtained through natural screening.
  • the strain can grow normally and degrade petroleum hydrocarbons under the condition that the salt content is 1-20wt%. It is more suitable for strain growth under the condition of 9-18 wt%, and compared with the existing known Halomonas, it has significant resistance to the degradation of petroleum hydrocarbons (especially C 10 -C 40 saturated hydrocarbons) under high salinity conditions. It can be applied to the removal of petroleum hydrocarbons in petroleum-contaminated soil and/or water in high salinity environments.
  • Halomonas titanicae HTPA16-9 in patent document CN110669700A Compared with Halomonas titanicae HTPA16-9 in patent document CN110669700A, the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 in the present invention has stronger salt tolerance and higher petroleum hydrocarbon degradation efficiency. high. Halomonas elongate ZQ1-3 grew better under the condition of salt content of 9-18wt%, while Halomonas titanicae HTPA16-9 had only 22g/L of sodium chloride in the medium during screening and degradation.
  • the Halomonas elongate ZQ1-3 of the present invention has a 5% sodium chloride content and is degraded for 15 days, and the degradation rate of petroleum hydrocarbons reaches 65.7%, and the Halomonas titanicae HTPA16-9 has a sodium chloride content of 22g/L and n-hexadecane is the only carbon source. After 3 months of anaerobic culture, the degradation rate of n-hexadecane is about 76.7% to 86.5%, the degradation time is long, the efficiency is low, and the degradation conditions are complicated. In the present invention, Halomonas elongate ZQ1-3 has good degradation effect on C 10 -C 40 saturated hydrocarbon in addition to high degradation efficiency of n-hexadecane.
  • Halomonas xianhensis A-1 in patent document CN101838616A the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 in the present invention has stronger salt tolerance and different types of petroleum hydrocarbon degradation.
  • the Halomonas elongate ZQ1-3 of the present invention can grow and reproduce in the range of 1-20% salinity, the suitable growth salinity range is 9-18%, and Halomonas xianhensis A-1 can be in the range of 0.05-27.5% salinity For growth and reproduction, the salinity range suitable for growth is 4-10%.
  • the Halomonas elongate ZQ1-3 of the present invention has stronger salt tolerance.
  • Saturated hydrocarbons alkanes
  • the leaked hydrocarbons are poured into the voids of the soil, affecting the permeability of the soil, destroying the soil water, gas and solid phase structures of crude oil, and affecting the growth of microorganisms in the soil. It can also often migrate with the flow of water, resulting in the continuous expansion of polluted areas; PAHs, as harmful substances in petroleum, have carcinogenic, teratogenic, mutagenic and other effects, and can enter the organism and even the human body through the food chain, directly harming the human body. healthy.
  • Halomonas elongate ZQ1-3 of the present invention mainly degrades saturated hydrocarbons, especially C 10 -C 40 saturated hydrocarbons, and Halomonas xianhensis A-1 mainly degrades polycyclic aromatic hydrocarbons, Especially phenanthrene, anthracene and fluoranthene.
  • the invention also provides a petroleum hydrocarbon degrading compound bacterial agent, which is mainly composed of Halomonas elongate ZQ1-3 and Bacillus pallidum MG35. Under the same effective bacterial concentration, the compound bacteria composed of Halomonas elongate ZQ1-3 and Bacillus pallidum MG35 The degradation efficiency of petroleum hydrocarbons was significantly higher than that of Halomonas elongate ZQ1-3 alone or Bacillus pallidus MG35, indicating that the compound bacterial agent formed by the two strains had a synergistic effect on the degradation of petroleum hydrocarbons.
  • Figure 1 is an agarose gel electrophoresis image of the 16S rDNA of Halomonas elongate ZQ1-3.
  • Figure 2 shows the growth curves of Halomonas elongate ZQ1-3 at different NaCl concentrations.
  • Figure 3 is a histogram of the degradation rate of Halomonas elongate ZQ1-3 to petroleum hydrocarbons with different carbon numbers.
  • Fig. 4 is the degradation rate curve of petroleum hydrocarbon degradation compound bacterial agent to petroleum hydrocarbon.
  • Inorganic salt medium the components per liter include the following:
  • Petroleum-inorganic salt solid medium the components per liter include the following:
  • Petroleum-inorganic salt liquid medium the components per liter include the following:
  • Collect 2g of high-concentration petroleum-contaminated soil in the coastal area of Shengli Oilfield put it in a 150mL sterile conical flask, add 50mL of sterile inorganic salt medium, culture it for 3 days at 30°C and 150rpm, absorb the bacterial solution, and perform gradient dilution with sterile water. , respectively diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 times, and each was coated with 100 ⁇ L of sterile petroleum-inorganic salt solid medium, and cultured at 30°C for 3 days.
  • the single colony picked after cultivation was sent to a sequencing company for sequencing. After detection, the 16S rDNA sequence contained 1392bp, and the nucleotide sequence was shown in SEQ ID NO.1.
  • the bacteria identification process is as follows:
  • Sample the bacterial liquid screened by the present invention
  • Bacterial genomic DNA extraction kit Sangon Bioengineering (Shanghai) Co., Ltd.;
  • TAE buffer 50 ⁇ , 1L: Tris 242g, glacial acetic acid 57.1mL, Na 2 EDTA ⁇ 2H 2 O 37.2g, add water to 1L;
  • DNA purification and recovery kit Sangon Bioengineering (Shanghai) Co., Ltd.;
  • Primer synthesized by Qingdao Qingke Zixi Biotechnology Co., Ltd., and ddH 2 O was added according to the synthesis list to make a 10 ⁇ M solution.
  • Genomic DNA extraction operate according to the bacterial genomic DNA extraction kit.
  • Pre-denaturation 94°C, 3min; denaturation at 94°C, 30s, annealing at 55°C, 30s, extension, 72°C, 1.5min (35 cycles in total); extension at 72°C, 10min; storage at 4°C.
  • the target fragment was recovered by agarose gel using an ordinary agarose gel DNA recovery kit, and the recovered product was sent to Qingdao Qingke Zixi Biotechnology Co., Ltd. for sequencing.
  • the blast comparison results of the sequencing and splicing sequences are shown in Table 3.
  • the bacterial species screened in the present invention belongs to Halomonas elongata, named Halomonas elongate ZQ1-3, and was preserved in the General Microorganism Center of the China Microorganism Culture Collection on October 26, 2020, address: Beichen, Chaoyang District, Beijing Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, West Road, strain collection number CGMCC No. 20953.
  • the cultivation method of Halomonas elongate ZQ1-3 of halophilic petroleum hydrocarbon degrading bacteria the steps are as follows:
  • step (2) taking the activated bacterial strain obtained in step (1) and inoculating it into a liquid medium, and under the condition that the rotating speed at 32° C. is 150 rev/min, it is shaken for 2 days to obtain seed liquid;
  • step (3) Take the seed solution obtained in step (2), transfer it to a liquid medium by volume percentage of 2%, and expand the culture for 2 days under the conditions of 32° C. and 30% dissolved oxygen to obtain Halomonas elongate ZQ1-3 Bacterial liquid, the concentration of viable bacteria in the bacterial liquid was 2 ⁇ 10 9 cfu/mL.
  • the medium used is as follows:
  • Liquid medium the components per liter include the following: peptone 10g, yeast extract 5g, potassium chloride 20g, magnesium sulfate heptahydrate 15g, sodium chloride 130g, water to 1L, natural pH.
  • the solid medium contains the following components per liter: peptone 10g, yeast extract 5g, potassium chloride 20g, magnesium sulfate heptahydrate 15g, sodium chloride 130g, agar 20g, water to 1L, natural pH.
  • step (2) get the seed liquid obtained by step (2) in embodiment 2, inoculate in the LB liquid medium of different NaCl contents prepared by step (1) by the inoculum size of 2% by volume;
  • step (3) The culture solution after inoculation in step (2) is cultured for 5 days under the conditions of 32° C. and dissolved oxygen of 30%.
  • Halomonas elongate ZQ1-3 has halophilic properties.
  • step (1) The culture solution inoculated in step (1) was cultured and degraded in a shaker at 32° C. and 150 rpm for 15 days.
  • the gas phase method (HJ 1021-2019) was used to detect the petroleum hydrocarbon components of residual C 10 -C 40 , and the degradation rate was calculated. The results are shown in Fig. 3. The degradation rates of petroleum hydrocarbons under the conditions of better degradation effect.
  • each liter of components includes the following:
  • Example 2 (1) The coastal petroleum-polluted saline-alkali soil with an oil content of 4.58% (the soluble salt content is 1.08%) and the petroleum hydrocarbon degrading bacterial agent obtained in Example 2 were uniformly mixed according to the mass ratio of 50:1, and sterilized distilled water was used. The moisture content of the bacteria-soil mixture was adjusted to 25%, and the viable bacteria concentration at this time was 4.8 ⁇ 10 8 CFU/mL;
  • the gas phase method (HJ 1021-2019) was used to detect the residual petroleum hydrocarbon components, and the degradation rate was calculated.
  • the oil content of the petroleum-contaminated saline-alkali soil was 2.44%.
  • the degradation rate of petroleum in the oil-contaminated saline-alkali soil was 4.58%; the viable bacteria concentration was 3.8 ⁇ 10 8 CFU/mL after 30 days, indicating that the oil-contaminated saline-alkali environment basically did not affect the growth of Halomonas elongate ZQ1-3. , maintained the growth activity of Halomonas elongate ZQ1-3, and continued to exert its ability to remediate petroleum hydrocarbon-contaminated soil.
  • a petroleum hydrocarbon degrading compound bacterial agent comprising Halomonas elongate ZQ1-3, a halophilic petroleum hydrocarbon degrading bacterium and a salt-tolerant strain Ochrobactrum daejeonense MG35; the Halomonas elongate ZQ1-3 bacterial liquid and the bacterial liquid of Ochrobactrum daejeonense MG35 are combined with an organic matter carrier Made after mixing.
  • CK organic matter carrier and bacterial agent 1, bacterial agent 2, bacterial agent 3 are respectively added to 500g oil-contaminated soil (determined by gravimetric method) 3.23% oil-contaminated soil (soluble salt content is 1.03%), adjust and keep moisture The content is 25%, agitated and ventilated once a day, and degraded for 25 days.
  • the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 disclosed in the present invention It can be cultivated normally when the NaCl content is 18%, and it also has a good degradation effect on petroleum hydrocarbons under the condition of 10%-20% NaCl content. It has a wide range of adaptation and good application value.

Abstract

Provided are a strain of halophilic petroleum-hydrocarbon-degrading bacterium and the use thereof. The halophilic petroleum-hydrocarbon-degrading bacterium Halomonas elongate ZQ1-3 was deposited in the China General Microbiological Culture Collection Center on October 26, 2020, the address of which is the Institute of Microbiology, Chinese Academy of Sciences, No.3, Courtyard 1 West Beichen Road, Chaoyang District, Beijing, and the deposit number of the strain is CGMCC No. 20953. The strain is obtained by means of natural screening, can grow normally and degrade petroleum hydrocarbons under a salt content of 1-20 wt%, is advantageous in the degradation of petroleum hydrocarbons under high salinity conditions compared with existing known Halomonas, and can be used in the removal of petroleum hydrocarbons from petroleum-polluted soil and/or water bodies in high saline-alkali environments.

Description

一株嗜盐石油烃降解菌及其应用A halophilic petroleum hydrocarbon degrading bacterium and its application 技术领域technical field
本发明涉及一株嗜盐石油烃降解菌Halomonas elongata ZQ1-3及其应用，属于微生物及生物降解技术领域。The invention relates to a halophilic petroleum hydrocarbon degrading bacterium Halomonas elongata ZQ1-3 and an application thereof, belonging to the technical field of microorganisms and biodegradation.
背景技术Background technique
我国的油田多位于盐碱地区，海滨区域的油田盐碱尤为严重。高盐碱等特殊生境的石油污染土壤修复时，石油烃降解微生物投加到高盐环境的石油烃污染土壤后，初期具有良好的修复效果，但在很短的时间内，一个月甚至半个月，投加的微生物在土壤菌群结构中所占比例迅速下降，而快速恢复到以耐盐或嗜盐微生物为主的投加前的稳定结构，严重影响了石油烃污染土壤的修复效果。Most of my country's oil fields are located in saline-alkali areas, especially in coastal areas. During the remediation of petroleum-contaminated soils in special habitats such as high salinity and alkali, after adding petroleum hydrocarbon-degrading microorganisms to petroleum hydrocarbon-contaminated soils in high salinity environments, the initial remediation effect is good, but in a very short period of time, a month or even half a The proportion of the added microorganisms in the soil microflora structure decreased rapidly, and quickly recovered to the stable structure before the addition of salt-tolerant or halophilic microorganisms, which seriously affected the remediation effect of petroleum hydrocarbon-contaminated soil.
研究表明，石油污染土壤生物修复过程中土著微生物(native microorganisms)降解污染物的潜力最大，外来微生物在环境中难以保持较高的代谢活性，向污染环境接种的降解污染物的高效外来微生物(foreign microorganisms)受到土著微生物的竞争，在高盐的特殊生境中这种竞争关系尤为明显。Studies have shown that native microorganisms have the greatest potential to degrade pollutants during the bioremediation of oil-contaminated soils, and it is difficult for foreign microorganisms to maintain high metabolic activity in the environment. microorganisms) are subject to competition from indigenous microorganisms, especially in special habitats with high salinity.
微生物修复是重要的石油烃污染土壤修复方法，研究表明，Pseudomonas(假单胞菌属)、Arthrobacter(节杆菌属)、Alcaligenes(产碱杆菌属)、Corynebacterium(棒状杆菌属)、Flavobacterium(黄杆菌属)、Achromobacter(无色杆菌属)、Micrococcus(微球菌属)、Nocardia(诺卡菌属)和Mycobacterium(分枝杆菌属)均具有高效的石油烃降解特性。然而，上述微生物主要为非耐盐微生物，在盐碱环境中无法正常发挥降解作用，而高盐环境中的生物修复只能通过使用具有石油烃化合物降解能力的耐盐或嗜盐微生物来完成。中国专利文献CN110669700A(申请号201911082757.9)公开了一株高效石油烃降解菌泰坦尼克号盐单胞菌(Halomonas titanicae)HTPA16-9，在厌氧培养基中，正十六烷为唯一碳源，初始添加量为0.07734g，恒温33℃且避光静置，厌氧培养3个月后，测残余正十六烷量，计算降解率约为76.7％～86.5％。中国专利文献CN101838616A(申请号200910080012.9)公开了一株可降解多环芳烃的盐单胞菌仙河盐单胞菌(Halomonas xianhensis)A-1，属于嗜盐微生物，能在0.05％～27.5％的盐度范围内进行生长繁殖，最适宜生长的盐度范围为4％～10％，能够降解菲、蒽或荧蒽等多种多环芳烃。目前有耐盐或嗜盐菌对石油烃降解的报道，认为不同来源的菌株对环境的适应性及石油烃的降解都有所不同，而且，经过不同地区土壤环境差异的自然筛选，会对同一种属来源的不同菌株的耐盐适应性及石油烃降解能力产生不同程度的 影响。因此，不同区域环境中筛选到的同一种属的菌株在不同的环境中对石油烃的降解能力会有较大的差别。Microbial remediation is an important method for remediation of petroleum hydrocarbon contaminated soil. Studies have shown that Pseudomonas (Pseudomonas), Arthrobacter (Arthrobacter), Alcaligenes (Alcaligenes), Corynebacterium (Corynebacterium), Flavobacterium (Flavobacterium) genus), Achromobacter (Achromobacter), Micrococcus (Micrococcus), Nocardia (Nocardia) and Mycobacterium (Mycobacterium) all have efficient petroleum hydrocarbon degradation properties. However, the above-mentioned microorganisms are mainly non-salt-tolerant microorganisms and cannot degrade normally in saline-alkali environments, while bioremediation in high-salt environments can only be accomplished by using salt-tolerant or halophilic microorganisms that have the ability to degrade petroleum hydrocarbon compounds. Chinese patent document CN110669700A (application number 201911082757.9) discloses a high-efficiency petroleum hydrocarbon degrading bacterium Halomonas titanicae HTPA16-9. In the anaerobic medium, n-hexadecane is the only carbon source. The addition amount is 0.07734 g, the constant temperature is 33° C. and the product is kept in the dark. After anaerobic culture for 3 months, the residual n-hexadecane content is measured, and the calculated degradation rate is about 76.7% to 86.5%. Chinese patent document CN101838616A (application number 200910080012.9) discloses a strain of Halomonas xianhensis A-1, which can degrade polycyclic aromatic hydrocarbons. It can grow and reproduce within the salinity range, and the most suitable salinity range is 4% to 10%. It can degrade various polycyclic aromatic hydrocarbons such as phenanthrene, anthracene or fluoranthene. At present, there are reports on the degradation of petroleum hydrocarbons by salt-tolerant or halophilic bacteria. Different strains from species have different degrees of influence on the adaptability of salt tolerance and the ability to degrade petroleum hydrocarbons. Therefore, the strains of the same genus screened in different regional environments will have great differences in their ability to degrade petroleum hydrocarbons in different environments.
本发明利用胜利油田滨海盐碱环境的特殊性，以环境的自筛选为手段，得到一株嗜盐而且石油烃降解效率高的盐单胞菌Halomonas elongate ZQ1-3，并研究了其培养和应用方法。The invention utilizes the particularity of the coastal saline-alkali environment of Shengli Oilfield, and takes the self-screening of the environment as a means to obtain a Halomonas elongate ZQ1-3 which is halophilic and has high petroleum hydrocarbon degradation efficiency, and studies its culture and application. method.
发明内容SUMMARY OF THE INVENTION
针对现有技术的不足，本发明提供了一株嗜盐石油烃降解菌Halomonas elongate ZQ1-3及其应用。In view of the deficiencies of the prior art, the present invention provides a halophilic petroleum hydrocarbon degrading bacterium Halomonas elongate ZQ1-3 and its application.
本发明技术方案如下：The technical scheme of the present invention is as follows:
一株嗜盐石油烃降解菌Halomonas elongate ZQ1-3，于2020年10月26日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，菌种保藏号：CGMCC No.20953。A halophilic petroleum hydrocarbon-degrading bacteria, Halomonas elongate ZQ1-3, was deposited on October 26, 2020 in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee, address: Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Institute of Microbiology, strain collection number: CGMCC No.20953.
根据本发明优选的，所述嗜盐石油烃降解菌Halomonas elongate ZQ1-3的16S rDNA的核苷酸序列如SEQ ID NO.1所示。Preferably according to the present invention, the nucleotide sequence of the 16S rDNA of the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 is shown in SEQ ID NO.1.
根据本发明优选的，所述嗜盐石油烃降解菌Halomonas elongate ZQ1-3在含盐量为1～20wt％条件下生长繁殖。Preferably according to the present invention, the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 grows and reproduces under the condition that the salt content is 1-20wt%.
进一步优选的，所述嗜盐石油烃降解菌Halomonas elongate ZQ1-3在含盐量为9～18wt％条件下生长繁殖。Further preferably, the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 grows and reproduces under the condition that the salt content is 9-18wt%.
根据本发明优选的，所述嗜盐石油烃降解菌Halomonas elongate ZQ1-3降解C 10-C 40的饱和烃。 Preferably according to the present invention, the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 degrades C 10 -C 40 saturated hydrocarbons.
上述嗜盐石油烃降解菌Halomonas elongate ZQ1-3的培养方法，包括如下步骤：The culture method of above-mentioned halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3, comprises the steps:
(1)取嗜盐石油烃降解菌Halomonas elongate ZQ1-3划线于固体活化培养基上，活化培养，得活化后菌株；(1) get the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 and streak it on the solid activated medium, and activate the culture to obtain the activated bacterial strain;
(2)取步骤(1)得到的活化后菌株接种至液体培养基中，摇床培养，制得种子液；(2) get the activated bacterial strain obtained in step (1) and inoculate it into a liquid medium, and shake it to cultivate to obtain seed liquid;
(3)取步骤(2)制得的种子液，按体积百分比1％～10％转接至扩大培养基中，扩大培养，制得Halomonas elongate ZQ1-3菌液。(3) Take the seed liquid obtained in step (2), transfer it to the expansion medium according to the volume percentage of 1% to 10%, and expand the culture to obtain the Halomonas elongate ZQ1-3 bacterial liquid.
根据本发明优选的，步骤(1)中所述固体活化培养基的组分如下：Preferably according to the present invention, the components of the solid activation medium described in step (1) are as follows:
蛋白胨10g/L，酵母提取物5g/L，氯化钾20g/L，七水硫酸镁15g/L，氯化钠130g/L，琼脂20g/L，余量水，pH自然。Peptone 10g/L, yeast extract 5g/L, potassium chloride 20g/L, magnesium sulfate heptahydrate 15g/L, sodium chloride 130g/L, agar 20g/L, balance water, pH natural.
根据本发明优选的，步骤(1)中所述活化培养的条件为：28～32℃倒置培养1～2 天。According to a preferred embodiment of the present invention, the conditions for activation and cultivation in step (1) are: inverted cultivation at 28-32° C. for 1-2 days.
根据本发明优选的，步骤(2)中所述液体培养基与步骤(3)中所述扩大培养基均为高盐液体培养基，组分如下：Preferably according to the present invention, the liquid medium described in step (2) and the expansion medium described in step (3) are high-salt liquid medium, and the components are as follows:
蛋白胨10g/L，酵母提取物5g/L，氯化钾20g/L，七水硫酸镁15g/L，氯化钠130g/L，余量水，pH自然。Peptone 10g/L, yeast extract 5g/L, potassium chloride 20g/L, magnesium sulfate heptahydrate 15g/L, sodium chloride 130g/L, balance water, pH natural.
根据本发明优选的，步骤(2)中所述摇床培养的条件为：28～32℃转速为100～200转/分钟的条件下，摇床培养2～5天。According to a preferred embodiment of the present invention, the conditions of the shaker culture in step (2) are: shaker culture for 2 to 5 days at 28 to 32°C and a rotational speed of 100 to 200 rpm.
根据本发明优选的，步骤(3)中所述扩大培养的条件为：28～32℃、溶氧20～40％的条件下，扩大培养1～2天。According to a preferred embodiment of the present invention, the conditions for expanding the culture in step (3) are: expanding the culture for 1-2 days under the conditions of 28-32° C. and dissolved oxygen of 20-40%.
一种含有上述嗜盐石油烃降解菌Halomonas elongate ZQ1-3的石油烃降解菌剂。A petroleum hydrocarbon degrading bacterial agent containing the above-mentioned halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3.
本发明中，上述石油烃降解菌剂可以是液体菌剂也可以是固体菌剂；在石油烃降解菌剂中可以只含有Halomonas elongate ZQ1-3一种菌也可以包含有其他菌株。在本发明一种优选的技术方案中，所述石油烃降解菌剂为液体菌剂，是经菌株培养后得到的Halomonas elongate ZQ1-3菌液。在本发明一种优选的技术方案中，所述石油烃降解菌剂为固体菌剂，是将培养得到的Halomonas elongate ZQ1-3菌液与有机质固体载体混合后得到的。In the present invention, the above-mentioned petroleum hydrocarbon-degrading bacterial agent can be a liquid bacterial agent or a solid bacterial agent; the petroleum hydrocarbon-degrading bacterial agent can only contain Halomonas elongate ZQ1-3 one kind of bacteria and can also contain other bacterial strains. In a preferred technical solution of the present invention, the petroleum hydrocarbon degrading bacterial agent is a liquid bacterial agent, which is the Halomonas elongate ZQ1-3 bacterial liquid obtained after strain culture. In a preferred technical solution of the present invention, the petroleum hydrocarbon degrading bacterial agent is a solid bacterial agent, obtained by mixing the Halomonas elongate ZQ1-3 bacterial liquid obtained by culturing with an organic solid carrier.
一种石油烃降解液体菌剂，是上述嗜盐石油烃降解菌Halomonas elongate ZQ1-3菌液。A petroleum hydrocarbon degrading liquid bacterial agent is the above-mentioned halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 bacterial liquid.
一种石油烃降解固体菌剂，是上述嗜盐石油烃降解菌Halomonas elongate ZQ1-3菌液与有机质载体按质量比1︰(10～20)的比例混合制得。A petroleum hydrocarbon degrading solid bacterial agent is prepared by mixing the above-mentioned halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 bacterial liquid and an organic matter carrier in a mass ratio of 1:(10-20).
进一步优选的，所述有机质载体为草炭土、锯末和麸皮草，质量比为(1～3)︰(1～3)︰(1～3)；优选的所述炭土、锯末和麸皮的质量比为3︰1︰1。Further preferably, the organic matter carrier is turf soil, sawdust and bran grass, and the mass ratio is (1-3):(1-3):(1-3); The mass ratio is 3:1:1.
进一步优选的，所述石油降解固体菌剂的活菌浓度为(2～5)×10 9cfu/g。 Further preferably, the viable bacteria concentration of the petroleum-degrading solid bacterial agent is (2-5)×10 9 cfu/g.
一种石油烃降解复合菌剂，包括上述嗜盐石油烃降解菌Halomonas elongate ZQ1-3和苍白杆菌(Ochrobactrum daejeonense)MG35。A petroleum hydrocarbon degrading compound bacterial agent, comprising the above-mentioned halomonas elongate ZQ1-3 and Ochrobactrum daejeonense MG35.
根据本发明优选的，所述苍白杆菌(Ochrobactrum daejeonense)MG35，2020年4月27日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，菌种保藏号：CGMCC No.19745。Preferably according to the present invention, the Ochrobactrum daejeonense MG35 was deposited in the General Microorganism Center of the China Microorganism Culture Collection Management Committee on April 27, 2020, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Academy of Sciences, strain collection number: CGMCC No.19745.
根据本发明优选的，所述嗜盐石油烃降解菌Halomonas elongate ZQ1-3和苍白杆菌(Ochrobactrum daejeonense)MG35的有效菌浓比为(1～3)︰(1～3)；优选为1:1。Preferably according to the present invention, the effective bacterial concentration ratio of the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 and Ochrobactrum daejeonense MG35 is (1～3): (1～3); preferably 1:1 .
根据本发明优选的，所述石油烃降解复合菌剂是将Halomonas elongate ZQ1-3菌液和 苍白杆菌MG35菌液与有机质载体按质量比1︰(10～20)的比例混合制得。Preferably according to the present invention, the described petroleum hydrocarbon degrading compound bacterial agent is prepared by mixing Halomonas elongate ZQ1-3 bacterial liquid and Bacillus pallidum MG35 bacterial liquid with organic matter carrier in a ratio of 1:(10～20) by mass.
进一步优选的，所述有机质载体为草炭土、锯末和麸皮草，质量比为(1～3)︰(1～3)︰(1～3)；优选的所述炭土、锯末和麸皮的质量比为3︰1︰1。Further preferably, the organic matter carrier is turf soil, sawdust and bran grass, and the mass ratio is (1-3):(1-3):(1-3); The mass ratio is 3:1:1.
在本发明优选的一种技术方案中，上述菌剂中Halomonas elongate ZQ1-3菌液和苍白杆菌MG35菌液均按照上述嗜盐石油烃降解菌Halomonas elongate ZQ1-3的培养方法培养得到。In a preferred technical solution of the present invention, the Halomonas elongate ZQ1-3 bacterial liquid and the pale bacillus MG35 bacterial liquid in the above bacterial agent are both obtained by culturing the above-mentioned halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3.
根据本发明优选的，所述石油烃降解复合菌剂的活菌浓度为(1～10)×10 9cfu/g。 Preferably according to the present invention, the viable bacteria concentration of the petroleum hydrocarbon-degrading compound bacterial agent is (1-10)×10 9 cfu/g.
上述嗜盐石油烃降解菌Halomonas elongate ZQ1-3在修复石油污染的水体和/或土壤中的应用。The application of the above-mentioned halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 in remediating oil-contaminated water bodies and/or soils.
上述菌剂在修复石油污染的水体和/或土壤中的应用。The application of the above bacterial agent in remediating oil-contaminated water body and/or soil.
根据本发明优选的，所述应用步骤如下：Preferably according to the present invention, the application steps are as follows:
向含油量为3～7％的石油污染土壤中接种上述菌剂，菌剂与石油污染土壤的质量比为(1～10)︰100，调节含水率为20～25％，混合混匀，自然堆置降解。Inoculate the above-mentioned bacterial agent into the oil-contaminated soil with an oil content of 3-7%, the mass ratio of the bacterial agent to the oil-contaminated soil is (1-10): 100, adjust the moisture content to 20-25%, mix well, and naturally stacking degradation.
上述嗜盐石油烃降解菌Halomonas elongate ZQ1-3或上述菌剂在盐碱环境石油污染水体和/或土壤修复中的应用。The application of the above-mentioned halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 or the above-mentioned bacterial agent in the remediation of petroleum-contaminated water bodies and/or soils in a saline-alkali environment.
本发明的技术特点和有益效果：Technical characteristics and beneficial effects of the present invention:
本发明首次公开了一株通过自然筛选获得的嗜盐石油烃降解菌Halomonas elongate ZQ1-3，该菌株在含盐量为1～20wt％的条件下能够正常生长并降解石油烃，在含盐量为9～18wt％条件下更加适宜菌株生长，与现有已知的盐单胞菌相比，在耐高盐度条件下的石油烃(尤其是C 10-C 40饱和烃)降解方面具有显著的优势，可应用于高盐碱环境下的石油污染土壤和/或水体中石油烃的去除。 The present invention discloses for the first time a halophilic petroleum hydrocarbon degrading bacterium Halomonas elongate ZQ1-3 obtained through natural screening. The strain can grow normally and degrade petroleum hydrocarbons under the condition that the salt content is 1-20wt%. It is more suitable for strain growth under the condition of 9-18 wt%, and compared with the existing known Halomonas, it has significant resistance to the degradation of petroleum hydrocarbons (especially C 10 -C 40 saturated hydrocarbons) under high salinity conditions. It can be applied to the removal of petroleum hydrocarbons in petroleum-contaminated soil and/or water in high salinity environments.
本发明中的嗜盐石油烃降解菌Halomonas elongate ZQ1-3与专利文献CN110669700A中的泰坦尼克号盐单胞菌(Halomonas titanicae)HTPA16-9相比，其耐盐性更强，石油烃降解效率更高。Halomonas elongate ZQ1-3在含盐量为9～18wt％条件下生长更好，而Halomonas titanicae HTPA16-9在筛选及降解时培养基中氯化钠含量仅为22g/L。本发明Halomonas elongate ZQ1-3在5％氯化钠含量、降解15d后，石油烃的降解率达到65.7％，Halomonas titanicae HTPA16-9在22g/L氯化钠含量、正十六烷为唯一碳源、恒温避光静置、厌氧培养3月后，正十六烷的降解率约为76.7％～86.5％，降解时间长、效率低，降解条件复杂。本发明中Halomonas elongate ZQ1-3除了对正十六烷烃具有较高的降解效率之外，对C 10-C 40的饱和烃都具有较好的降解效果。 Compared with Halomonas titanicae HTPA16-9 in patent document CN110669700A, the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 in the present invention has stronger salt tolerance and higher petroleum hydrocarbon degradation efficiency. high. Halomonas elongate ZQ1-3 grew better under the condition of salt content of 9-18wt%, while Halomonas titanicae HTPA16-9 had only 22g/L of sodium chloride in the medium during screening and degradation. The Halomonas elongate ZQ1-3 of the present invention has a 5% sodium chloride content and is degraded for 15 days, and the degradation rate of petroleum hydrocarbons reaches 65.7%, and the Halomonas titanicae HTPA16-9 has a sodium chloride content of 22g/L and n-hexadecane is the only carbon source. After 3 months of anaerobic culture, the degradation rate of n-hexadecane is about 76.7% to 86.5%, the degradation time is long, the efficiency is low, and the degradation conditions are complicated. In the present invention, Halomonas elongate ZQ1-3 has good degradation effect on C 10 -C 40 saturated hydrocarbon in addition to high degradation efficiency of n-hexadecane.
本发明中的嗜盐石油烃降解菌Halomonas elongate ZQ1-3与专利文献CN101838616A中的仙河盐单胞菌(Halomonas xianhensis)A-1相比，其耐盐性更强，石油烃的降解类型不同。本发明的Halomonas elongate ZQ1-3能在1～20％的盐度范围内生长繁殖，适宜生长的盐度范围为9～18％，Halomonas xianhensis A-1能在0.05～27.5％的盐度范围内生长繁殖，适宜生长的盐度范围为4～10％，从菌株适宜生长的盐度范围分析，本发明的Halomonas elongate ZQ1-3耐盐性更强。饱和烃(烷烃)是石油的主要成分，泄露的烃类物质灌注入土壤的空隙中，影响土壤的通透性，破坏原油的土壤水相、气相和固相结构，影响土壤中微生物的生长，也可以常伴随水的流动而发生迁徙，导致污染区域不断扩大；多环芳烃作为石油中的有害物质具有致癌、致畸、致突变等作用，可以通过食物链进入生物体乃至人体中，直接危害人体健康。饱和烃和多环芳烃都是石油中主要的污染成分，本发明的Halomonas elongate ZQ1-3主要降解饱和烃，尤其是C 10-C 40的饱和烃，Halomonas xianhensis A-1主要降解多环芳烃，尤其是菲、蒽和荧蒽。 Compared with Halomonas xianhensis A-1 in patent document CN101838616A, the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 in the present invention has stronger salt tolerance and different types of petroleum hydrocarbon degradation. . The Halomonas elongate ZQ1-3 of the present invention can grow and reproduce in the range of 1-20% salinity, the suitable growth salinity range is 9-18%, and Halomonas xianhensis A-1 can be in the range of 0.05-27.5% salinity For growth and reproduction, the salinity range suitable for growth is 4-10%. From the analysis of the salinity range suitable for growth of the strain, the Halomonas elongate ZQ1-3 of the present invention has stronger salt tolerance. Saturated hydrocarbons (alkanes) are the main components of oil. The leaked hydrocarbons are poured into the voids of the soil, affecting the permeability of the soil, destroying the soil water, gas and solid phase structures of crude oil, and affecting the growth of microorganisms in the soil. It can also often migrate with the flow of water, resulting in the continuous expansion of polluted areas; PAHs, as harmful substances in petroleum, have carcinogenic, teratogenic, mutagenic and other effects, and can enter the organism and even the human body through the food chain, directly harming the human body. healthy. Both saturated hydrocarbons and polycyclic aromatic hydrocarbons are the main pollutant components in petroleum. The Halomonas elongate ZQ1-3 of the present invention mainly degrades saturated hydrocarbons, especially C 10 -C 40 saturated hydrocarbons, and Halomonas xianhensis A-1 mainly degrades polycyclic aromatic hydrocarbons, Especially phenanthrene, anthracene and fluoranthene.
本发明还提供了一种石油烃降解复合菌剂，主要是由Halomonas elongate ZQ1-3和苍白杆菌MG35组成，在有效菌浓相同的条件下，Halomonas elongate ZQ1-3和苍白杆菌MG35组成的复合菌剂对于石油烃的降解效率显著高于单独的Halomonas elongate ZQ1-3或苍白杆菌MG35，说明两株菌复合形成的复合菌剂对石油烃降解有协同促进作用。The invention also provides a petroleum hydrocarbon degrading compound bacterial agent, which is mainly composed of Halomonas elongate ZQ1-3 and Bacillus pallidum MG35. Under the same effective bacterial concentration, the compound bacteria composed of Halomonas elongate ZQ1-3 and Bacillus pallidum MG35 The degradation efficiency of petroleum hydrocarbons was significantly higher than that of Halomonas elongate ZQ1-3 alone or Bacillus pallidus MG35, indicating that the compound bacterial agent formed by the two strains had a synergistic effect on the degradation of petroleum hydrocarbons.
附图说明Description of drawings
图1为Halomonas elongate ZQ1-3的16S rDNA的琼脂糖凝胶电泳图。Figure 1 is an agarose gel electrophoresis image of the 16S rDNA of Halomonas elongate ZQ1-3.
图2为Halomonas elongate ZQ1-3在不同NaCl浓度下的生长曲线。Figure 2 shows the growth curves of Halomonas elongate ZQ1-3 at different NaCl concentrations.
图3为Halomonas elongate ZQ1-3对不同碳数石油烃的降解率柱状图。Figure 3 is a histogram of the degradation rate of Halomonas elongate ZQ1-3 to petroleum hydrocarbons with different carbon numbers.
图4为石油烃降解复合菌剂对石油烃的降解率曲线。Fig. 4 is the degradation rate curve of petroleum hydrocarbon degradation compound bacterial agent to petroleum hydrocarbon.
具体实施方式Detailed ways
下面结合实施例和说明书附图对本发明的技术方案做进一步阐述，但本发明所保护范围不限于此。实施例中涉及的药品及试剂，若无特殊说明，均为普通市售产品；实施例中涉及的实验操作及步骤，若无特殊说明，均为本领域常规操作。The technical solutions of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the protection scope of the present invention is not limited thereto. The medicines and reagents involved in the examples are ordinary commercial products unless otherwise specified; the experimental operations and steps involved in the examples are routine operations in the art unless otherwise specified.
生物材料来源：Source of biological material:
一株嗜盐石油烃降解菌Halomonas elongate ZQ1-3，于2020年10月26日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，菌种保藏号：CGMCC No.20953。A halophilic petroleum hydrocarbon-degrading bacteria, Halomonas elongate ZQ1-3, was deposited on October 26, 2020 in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee, address: Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Institute of Microbiology, strain collection number: CGMCC No.20953.
一株苍白杆菌(Ochrobactrum daejeonense)MG35，2020年4月27日保藏于中国微 生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，菌种保藏号：CGMCC No.19745。该菌株在专利CN202010443089.4中已经公开，在本发明中不涉及微生物保藏。A strain of Ochrobactrum daejeonense MG35, which was deposited on April 27, 2020 in the General Microbiology Center of the China Microorganism Culture Collection, Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China Species deposit number: CGMCC No.19745. The strain has been disclosed in the patent CN202010443089.4, and microorganism preservation is not involved in the present invention.
培养基：Medium:
无机盐培养基，每升组分包括如下：Inorganic salt medium, the components per liter include the following:
KNO 3 1.5g，(NH 4) 2SO 4 1.5g，K 2HPO 4 1g，KH 2PO 4 1g，MgSO 4·7H 2O 0.5g，NaCl 130g，FeSO 4·7H 2O 0.01g，dH 2O定容至1L。 KNO 3 1.5g, (NH 4 ) 2 SO 4 1.5g, K 2 HPO 4 1g, KH 2 PO 4 1g, MgSO 4 ·7H 2 O 0.5g, NaCl 130g, FeSO 4 ·7H 2 O 0.01g, dH 2 O Make up to 1L.
石油-无机盐固体培养基，每升组分包括如下：Petroleum-inorganic salt solid medium, the components per liter include the following:
KNO 3 1.5g，(NH 4) 2SO 4 1.5g，K 2HPO 4 1g，KH 2PO 4 1g，MgSO 4·7H 2O 0.5g，NaCl 130g，FeSO 4·7H 2O 0.01g，石油20g，琼脂20g，dH 2O定容至1L。 KNO 3 1.5g, (NH 4 ) 2 SO 4 1.5g, K 2 HPO 4 1g, KH 2 PO 4 1g, MgSO 4 ·7H 2 O 0.5g, NaCl 130g, FeSO 4 ·7H 2 O 0.01g, petroleum 20g , agar 20g, dH2O volume to 1L.
石油-无机盐液体培养基，每升组分包括如下：Petroleum-inorganic salt liquid medium, the components per liter include the following:
KNO 3 1.5g，(NH 4) 2SO 4 1.5g，K 2HPO 4 1g，KH 2PO 4 1g，MgSO 4·7H 2O 0.5g，NaCl 130g，FeSO 4·7H 2O 0.01g，石油20g，dH 2O定容至1L。 KNO 3 1.5g, (NH 4 ) 2 SO 4 1.5g, K 2 HPO 4 1g, KH 2 PO 4 1g, MgSO 4 ·7H 2 O 0.5g, NaCl 130g, FeSO 4 ·7H 2 O 0.01g, petroleum 20g , dH 2 O to 1L.
实施例1Example 1
一株嗜盐石油烃降解菌Halomonas elongate ZQ1-3的分离和鉴定Isolation and identification of a halophilic petroleum hydrocarbon-degrading bacterium Halomonas elongate ZQ1-3
采集胜利油田滨海高浓度石油污染土壤2g，置于150mL无菌三角瓶中，加入无菌无机盐培养基50mL，在30℃、150rpm条件下培养3d，吸取菌液，用无菌水进行梯度稀释，分别稀释至10 -1，10 -2，10 -3，10 -4，10 -5倍，各涂布100μL至无菌的石油-无机盐固体培养基，在30℃条件下静置培养3d，挑取生长快、菌落大的克隆至50mL石油-无机盐液体培养基，在30℃、150rpm条件下培养3d，再吸取100μL菌液涂布至无菌的石油-无机盐固体培养基，挑取单菌落。 Collect 2g of high-concentration petroleum-contaminated soil in the coastal area of Shengli Oilfield, put it in a 150mL sterile conical flask, add 50mL of sterile inorganic salt medium, culture it for 3 days at 30°C and 150rpm, absorb the bacterial solution, and perform gradient dilution with sterile water. , respectively diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 times, and each was coated with 100 μL of sterile petroleum-inorganic salt solid medium, and cultured at 30°C for 3 days. , pick the clones with fast growth and large colonies to 50mL petroleum-inorganic salt liquid medium, cultivate at 30°C and 150rpm for 3 days, and then draw 100 μL of bacterial liquid and apply it to sterile petroleum-inorganic salt solid medium, pick Take a single colony.
取培养后挑取的单菌落送测序公司进行测序，经检测，16S rDNA序列含有1392bp，核苷酸序列如SEQ ID NO.1所示。The single colony picked after cultivation was sent to a sequencing company for sequencing. After detection, the 16S rDNA sequence contained 1392bp, and the nucleotide sequence was shown in SEQ ID NO.1.
菌种鉴定过程如下：The bacteria identification process is as follows:
样品：本发明筛选的细菌菌液；Sample: the bacterial liquid screened by the present invention;
细菌基因组DNA提取试剂盒：生工生物工程(上海)股份有限公司；Bacterial genomic DNA extraction kit: Sangon Bioengineering (Shanghai) Co., Ltd.;
TAE缓冲液(50×，1L)：Tris 242g、冰醋酸57.1mL、Na 2EDTA·2H 2O 37.2g、加水至1L； TAE buffer (50×, 1L): Tris 242g, glacial acetic acid 57.1mL, Na 2 EDTA·2H 2 O 37.2g, add water to 1L;
琼脂糖：BIOWET，AGAROSE G-10；Agarose: BIOWET, AGAROSE G-10;
2×Pfu PCR MasterMix、D2000 DNA Marker、核酸染料，loading buffer等：生工生物工程 (上海)股份有限公司；2×Pfu PCR MasterMix, D2000 DNA Marker, nucleic acid dye, loading buffer, etc.: Sangon Bioengineering (Shanghai) Co., Ltd.;
DNA纯化回收试剂盒：生工生物工程(上海)股份有限公司；DNA purification and recovery kit: Sangon Bioengineering (Shanghai) Co., Ltd.;
离心管、枪头等耗材：美国Gene Era Biotech公司；Consumables such as centrifuge tubes and pipette tips: Gene Era Biotech, USA;
引物：由青岛擎科梓熙生物技术有限公司合成，按照合成单加入ddH 2O，制成10μM溶液。 Primer: synthesized by Qingdao Qingke Zixi Biotechnology Co., Ltd., and ddH 2 O was added according to the synthesis list to make a 10 μM solution.
1、基因组DNA提取，按细菌基因组DNA提取试剂盒操作。1. Genomic DNA extraction, operate according to the bacterial genomic DNA extraction kit.
2、PCR扩增2. PCR amplification
2.1、通用引物信息，见表1。2.1. For general primer information, see Table 1.
表1 细菌通用引物信息Table 1 Bacterial universal primer information
Figure PCTCN2021099990-appb-000001
Figure PCTCN2021099990-appb-000001
2.2、PCR扩增体系组分及构成，见表2。2.2. The components and composition of the PCR amplification system are shown in Table 2.
表2 PCR扩增体系Table 2 PCR amplification system
编号Numbering 组分component 体积(μL)Volume (μL)
11 2×Pfu PCR MasterMix2×Pfu PCR MasterMix 2525
22 27F 27F 22
33 1492R 1492R 22
44 DNA模板 DNA template 11
55 加ddH 2O至 Add ddH 2 O to 5050
2.3、PCR扩增程序2.3. PCR amplification procedure
预变性：94℃，3min；变性94℃，30s，退火55℃，30s，延伸，72℃，1.5min(共35个循环)；延伸72℃，10min；4℃保存。Pre-denaturation: 94°C, 3min; denaturation at 94°C, 30s, annealing at 55°C, 30s, extension, 72°C, 1.5min (35 cycles in total); extension at 72°C, 10min; storage at 4°C.
3、琼脂糖凝胶电泳检测3. Agarose gel electrophoresis detection
制备1.0％的琼脂糖凝胶，电泳时电压设置为18V/cm，电泳时间为20min；采用核酸染料进行琼脂糖电泳染色，采用紫外凝胶成像系统进行拍照，结果如图1所示，样品泳道中有且仅有一条明亮的目的条带，条带的大小为1400bp左右。Prepare a 1.0% agarose gel, set the voltage to 18V/cm during electrophoresis, and set the electrophoresis time to 20min; use nucleic acid dyes for agarose electrophoresis staining, and use a UV gel imaging system to take pictures. The results are shown in Figure 1. The sample lanes There is only one bright target band, and the size of the band is about 1400bp.
4、纯化回收4. Purification and recovery
使用普通琼脂糖凝胶DNA回收试剂盒对目的片段进行琼脂糖凝胶回收，回收产物送青岛擎科梓熙生物技术有限公司测序，测序拼接序列的blast比对结果见表3。The target fragment was recovered by agarose gel using an ordinary agarose gel DNA recovery kit, and the recovered product was sent to Qingdao Qingke Zixi Biotechnology Co., Ltd. for sequencing. The blast comparison results of the sequencing and splicing sequences are shown in Table 3.
表3 测序拼接序列的blast比对结果Table 3 The blast alignment results of the sequenced spliced sequences
Figure PCTCN2021099990-appb-000002
Figure PCTCN2021099990-appb-000002
通过序列16S rDNA进行序列比对，发现遗传关系最接近的菌株为Halomonas elongata BK-AB8，NCBI登录号为KJ185379.1。Through the sequence alignment of 16S rDNA, it was found that the strain with the closest genetic relationship was Halomonas elongata BK-AB8, and the NCBI accession number was KJ185379.1.
经上述菌种鉴定，本发明筛选的菌种属于Halomonas elongata，命名为Halomonas elongate ZQ1-3，2020年10月26日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，菌种保藏号CGMCC No.20953。Through the identification of the above bacterial species, the bacterial species screened in the present invention belongs to Halomonas elongata, named Halomonas elongate ZQ1-3, and was preserved in the General Microorganism Center of the China Microorganism Culture Collection on October 26, 2020, address: Beichen, Chaoyang District, Beijing Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, West Road, strain collection number CGMCC No. 20953.
实施例2Example 2
嗜盐石油烃降解菌Halomonas elongate ZQ1-3的培养方法，步骤如下：The cultivation method of Halomonas elongate ZQ1-3 of halophilic petroleum hydrocarbon degrading bacteria, the steps are as follows:
(1)取嗜盐石油烃降解菌Halomonas elongate ZQ1-3划线于固体培养基上，32℃倒置活化培养2天，得活化后菌株；(1) Take the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 and streak it on the solid medium, and invert and activate at 32°C for 2 days to obtain the activated strain;
(2)取步骤(1)得到的活化后菌株接种至液体培养基中，32℃转速为150转/分钟的条件下，摇床培养2天，制得种子液；(2) taking the activated bacterial strain obtained in step (1) and inoculating it into a liquid medium, and under the condition that the rotating speed at 32° C. is 150 rev/min, it is shaken for 2 days to obtain seed liquid;
(3)取步骤(2)制得的种子液，按体积百分比2％转接至液体培养基中，32℃、溶氧30％的条件下，扩大培养2天，制得Halomonas elongate ZQ1-3菌液，菌液中活菌浓度为2×10 9cfu/mL。 (3) Take the seed solution obtained in step (2), transfer it to a liquid medium by volume percentage of 2%, and expand the culture for 2 days under the conditions of 32° C. and 30% dissolved oxygen to obtain Halomonas elongate ZQ1-3 Bacterial liquid, the concentration of viable bacteria in the bacterial liquid was 2×10 9 cfu/mL.
其中，所用培养基如下：Among them, the medium used is as follows:
液体培养基，每升组分包括如下：蛋白胨10g，酵母提取物5g，氯化钾20g，七水硫酸镁15g，氯化钠130g，水定容至1L，pH自然。Liquid medium, the components per liter include the following: peptone 10g, yeast extract 5g, potassium chloride 20g, magnesium sulfate heptahydrate 15g, sodium chloride 130g, water to 1L, natural pH.
固体培养基，每升组分包括如下：蛋白胨10g，酵母提取物5g，氯化钾20g，七水硫酸镁15g，氯化钠130g，琼脂20g，水定容至1L，pH自然。The solid medium contains the following components per liter: peptone 10g, yeast extract 5g, potassium chloride 20g, magnesium sulfate heptahydrate 15g, sodium chloride 130g, agar 20g, water to 1L, natural pH.
一种石油烃降解菌剂，由上述制得的Halomonas elongate ZQ1-3菌液与有机质载体(草炭土︰锯末︰麸皮＝3︰1︰1，质量比)按质量比1︰10的比例混合，制得石油烃降解固体菌剂；自然放置5天后检测，固体菌剂中活菌浓度为3.3×10 9cfu/g。 A petroleum hydrocarbon degrading bacterial agent, the Halomonas elongate ZQ1-3 bacterial liquid prepared above is mixed with an organic matter carrier (turf soil: sawdust: bran=3:1:1, mass ratio) in a ratio of 1:10 by mass , the petroleum hydrocarbon-degrading solid inoculum was prepared; the concentration of viable bacteria in the solid inoculum was 3.3×10 9 cfu/g after being placed for 5 days.
实施例3Example 3
嗜盐石油烃降解菌Halomonas elongate ZQ1-3的耐盐性能实验，步骤如下：The salt tolerance experiment of Halomonas elongate ZQ1-3, the halophilic petroleum hydrocarbon degrading bacteria, the steps are as follows:
(1)调节LB培养基中的NaCl含量，分别配制NaCl质量浓度为3％、6％、9％、12％、15％、18％的LB液体培养基；(1) Adjust the NaCl content in the LB medium, and prepare LB liquid medium with NaCl mass concentrations of 3%, 6%, 9%, 12%, 15%, and 18% respectively;
(2)取实施例2中步骤(2)制得的种子液，按体积百分比2％的接种量接种至步骤(1)制备的不同NaCl含量的LB液体培养基中；(2) get the seed liquid obtained by step (2) in embodiment 2, inoculate in the LB liquid medium of different NaCl contents prepared by step (1) by the inoculum size of 2% by volume;
(3)将步骤(2)接种后的培养液在32℃、溶氧30％的条件下，培养5天。(3) The culture solution after inoculation in step (2) is cultured for 5 days under the conditions of 32° C. and dissolved oxygen of 30%.
在培养过程中，每隔12h测定菌液OD 600，绘制菌株生长曲线，结果如图2所示，Halomonas elongate ZQ1-3的生长在NaCl低浓度下(3wt％和6wt％)表现出先增加后减少的趋势，而在NaCl高浓度下(9wt％、12wt％、15wt％和18wt％)表现出先增长后趋于稳定的趋势，说明低盐条件不利于Halomonas elongate ZQ1-3的稳定生长，Halomonas elongate ZQ1-3具有嗜盐特性。 During the cultivation process, the OD 600 of the bacterial solution was measured every 12h, and the growth curve of the strain was drawn. The results are shown in Figure 2. The growth of Halomonas elongate ZQ1-3 at low NaCl concentrations (3wt% and 6wt%) showed an increase first and then decrease However, at high concentrations of NaCl (9wt%, 12wt%, 15wt% and 18wt%), it showed a trend of first increasing and then tending to be stable, indicating that low-salt conditions are not conducive to the stable growth of Halomonas elongate ZQ1-3. Halomonas elongate ZQ1 -3 has halophilic properties.
培养5天后，计活菌数，NaCl质量浓度为3％、6％、9％、12％、15％、18％的LB液体培养基的发酵液中的活菌数分别为3.1×10 8、3.3×10 8、2.9×10 9、3.2×10 9、3.1×10 9cfu/mL；上述结果同样说明了Halomonas elongate ZQ1-3在高盐条件下比在低盐条件下生长更好，具有嗜盐特性。 After culturing for 5 days, the number of viable bacteria was counted, and the number of viable bacteria in the fermentation broth of LB liquid medium with NaCl mass concentration of 3%, 6%, 9%, 12%, 15%, and 18% was 3.1×10 8 , 3.3×10 8 , 2.9×10 9 , 3.2×10 9 , 3.1×10 9 cfu/mL; the above results also show that Halomonas elongate ZQ1-3 grows better under high-salt conditions than under low-salt conditions, and has a Salt properties.
实施例4Example 4
嗜盐石油烃降解菌Halomonas elongate ZQ1-3高盐环境下在石油污染水体修复中的应用，步骤如下：The application of the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 in the remediation of petroleum-contaminated water bodies in a high-salt environment, the steps are as follows:
(1)将石油-无机盐液体培养基中NaCl的质量浓度分别调至1％、5％、10％、15％、20％，按体积百分比2％的比例接入实施例2中步骤(2)制得的种子液；(1) the mass concentration of NaCl in petroleum-inorganic salt liquid culture medium is adjusted to 1%, 5%, 10%, 15%, 20% respectively, and the ratio of 2% by volume is inserted into step (2) in Example 2 ) obtained seed liquor;
(2)将步骤(1)接种后的培养液在摇床中32℃、150rpm培养降解15d。(2) The culture solution inoculated in step (1) was cultured and degraded in a shaker at 32° C. and 150 rpm for 15 days.
采用气相法(HJ 1021-2019)检测残留C 10-C 40的石油烃组分，并计算降解率，结果如图3所示，嗜盐石油烃降解菌Halomonas elongate ZQ1-3在NaCl质量浓度1％、5％、10％、15％、20％条件下对石油烃的降解率分别为31.2％、65.7％、29.8％、28.6％、27.3％，而且对石油中的不同碳数石油烃均有较好的降解效果。 The gas phase method (HJ 1021-2019) was used to detect the petroleum hydrocarbon components of residual C 10 -C 40 , and the degradation rate was calculated. The results are shown in Fig. 3. The degradation rates of petroleum hydrocarbons under the conditions of better degradation effect.
本实施例所用石油-无机盐液体培养基，每升组分包括如下：The petroleum-inorganic salt liquid culture medium used in the present embodiment, each liter of components includes the following:
KNO 3 1.5g，(NH 4) 2SO 4 1.5g，K 2HPO 4 1g，KH 2PO 4 1g，MgSO 4·7H 2O 0.5g，NaCl 130g，FeSO 4·7H 2O 0.01g，石油20g，dH 2O定容至1L。并根据需求调整培养基中的NaCl的浓度。 KNO 3 1.5 g, (NH 4 ) 2 SO 4 1.5 g, K 2 HPO 4 1 g, KH 2 PO 4 1 g, MgSO 4 7H 2 O 0.5 g, NaCl 130 g, FeSO 4 7H 2 O 0.01 g, petroleum 20 g , dH 2 O to 1L. And adjust the concentration of NaCl in the medium as needed.
实施例5Example 5
由嗜盐石油烃降解菌Halomonas elongate ZQ1-3制备的石油烃降解菌剂在高盐石油烃污染土壤修复中的应用，步骤如下：The application of the petroleum hydrocarbon degrading bacterial agent prepared by the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 in the remediation of high-salt petroleum hydrocarbon polluted soil, the steps are as follows:
(1)将含油率4.58％的滨海石油污染盐碱土壤(可溶性盐含量为1.08％)与实施例2制得 的石油烃降解菌剂按照质量比50:1的比例混合均匀，用灭菌蒸馏水调节菌土混合物含水率为25％，此时的活菌浓度为4.8×10 8CFU/mL； (1) The coastal petroleum-polluted saline-alkali soil with an oil content of 4.58% (the soluble salt content is 1.08%) and the petroleum hydrocarbon degrading bacterial agent obtained in Example 2 were uniformly mixed according to the mass ratio of 50:1, and sterilized distilled water was used. The moisture content of the bacteria-soil mixture was adjusted to 25%, and the viable bacteria concentration at this time was 4.8×10 8 CFU/mL;
(2)保持菌土混合物含水率为25％，自然堆置高度75cm，保持室温(25±5℃)下降解30d。(2) Keep the moisture content of the fungus-soil mixture at 25%, the natural stacking height of 75cm, and keep it at room temperature (25±5°C) for 30 days of degradation.
采用气相法(HJ 1021-2019)检测残留的石油烃组分，并计算降解率，30d后石油污染盐碱土壤的含油率为2.44％，嗜盐石油烃降解菌Halomonas elongate ZQ1-3对含油量为4.58％的石油污染盐碱土壤中石油的降解率为46.7％；30天后活菌浓度为3.8×10 8CFU/mL，说明石油污染盐碱环境基本不会对Halomonas elongate ZQ1-3的生长产生影响，维持了Halomonas elongate ZQ1-3的生长活性，持续发挥对石油烃污染土壤的修复能力。 The gas phase method (HJ 1021-2019) was used to detect the residual petroleum hydrocarbon components, and the degradation rate was calculated. After 30 days, the oil content of the petroleum-contaminated saline-alkali soil was 2.44%. The degradation rate of petroleum in the oil-contaminated saline-alkali soil was 4.58%; the viable bacteria concentration was 3.8×10 8 CFU/mL after 30 days, indicating that the oil-contaminated saline-alkali environment basically did not affect the growth of Halomonas elongate ZQ1-3. , maintained the growth activity of Halomonas elongate ZQ1-3, and continued to exert its ability to remediate petroleum hydrocarbon-contaminated soil.
实施例6Example 6
一种石油烃降解复合菌剂，包括嗜盐石油烃降解菌Halomonas elongate ZQ1-3和耐盐菌株苍白杆菌(Ochrobactrum daejeonense)MG35；将Halomonas elongate ZQ1-3菌液和苍白杆菌MG35菌液与有机质载体混合后制得。A petroleum hydrocarbon degrading compound bacterial agent, comprising Halomonas elongate ZQ1-3, a halophilic petroleum hydrocarbon degrading bacterium and a salt-tolerant strain Ochrobactrum daejeonense MG35; the Halomonas elongate ZQ1-3 bacterial liquid and the bacterial liquid of Ochrobactrum daejeonense MG35 are combined with an organic matter carrier Made after mixing.
上述石油烃降解复合菌剂在高盐石油烃污染土壤修复中的应用，步骤如下：The application of the above-mentioned petroleum hydrocarbon-degrading compound bacterial agent in the remediation of high-salt petroleum hydrocarbon-contaminated soil, the steps are as follows:
(1)将嗜盐石油烃降解菌Halomonas elongate ZQ1-3和苍白杆菌MG35分别按照实施例2所述的培养方法进行培养，所得菌液离心，然后用灭菌蒸馏水洗涤菌体2次，重悬，调节重悬菌液至OD 600＝1.0； (1) The halophilic petroleum hydrocarbon-degrading bacteria Halomonas elongate ZQ1-3 and Bacillus pallidus MG35 were respectively cultivated according to the culture method described in Example 2, the obtained bacterial liquid was centrifuged, and then the bacteria were washed twice with sterilized distilled water and resuspended. , adjust the resuspended bacterial solution to OD 600 =1.0;
(2)设置以下三组菌剂：(2) Set the following three groups of bacterial agents:
取OD 600＝1.0的苍白杆菌MG35重悬菌液10mL添加至50g有机质载体(草炭土︰锯末︰麸皮＝3︰1︰1，质量比)，室温(25±5℃)放置3d制备菌剂1，活菌浓度为3.52×10 9cfu/g； Take 10 mL of Bacillus pallorus MG35 resuspended bacterial solution with OD 600 = 1.0 and add it to 50 g of organic matter carrier (powder soil: sawdust: bran = 3:1:1, mass ratio), and leave it at room temperature (25±5℃) for 3 days to prepare the inoculum 1. The concentration of viable bacteria is 3.52×10 9 cfu/g;
取OD 600＝1.0的Halomonas elongate ZQ1-3重悬菌液10mL添加至50g有机质载体(草炭土︰锯末︰麸皮＝3︰1︰1，质量比)，室温(25±5℃)放置3d制备菌剂2，活菌浓度为3.38×10 9cfu/g； Take 10 mL of Halomonas elongate ZQ1-3 resuspended bacterial solution with OD 600 = 1.0 and add it to 50 g of organic matter carrier (peat soil: sawdust: bran = 3:1:1, mass ratio), and leave it at room temperature (25±5℃) for 3d preparation Bacterial agent 2, the viable bacteria concentration is 3.38×10 9 cfu/g;
分别取OD 600＝1.0的苍白杆菌MG35重悬菌液5mL和OD 600＝1.0的Halomonas elongate ZQ1-3重悬菌液5mL一起添加至50g有机质载体(草炭土︰锯末︰麸皮＝3︰1︰1，质量比)，室温(25±5℃)放置3d制备菌剂3，活菌浓度为3.47×10 9cfu/g； Take 5 mL of Bacillus pallorus MG35 resuspended bacterial solution with OD 600 = 1.0 and 5 mL of Halomonas elongate ZQ1-3 resuspended bacterial solution with OD 600 = 1.0 and add them to 50 g of organic matter carrier (turf soil: sawdust: bran = 3:1: 1, mass ratio), placed at room temperature (25±5°C) for 3 days to prepare bacterial agent 3, and the viable bacteria concentration was 3.47×10 9 cfu/g;
50g有机质载体(草炭土︰锯末︰麸皮＝3︰1︰1，质量比)，调节水分含量20％左右室温(25±5℃)放置3d，为对照组CK；50g organic carrier (peat soil: sawdust: bran = 3:1:1, mass ratio), adjust the moisture content to about 20% and place it at room temperature (25±5°C) for 3 days, which is the control group CK;
(3)将CK有机质载体以及菌剂1、菌剂2、菌剂3分别添加至500g石油含量(重量法测 定)3.23％的石油污染土壤(可溶性盐含量为1.03％)中，调节并保持水分含量为25％，每天搅拌通风一次，降解25d。(3) CK organic matter carrier and bacterial agent 1, bacterial agent 2, bacterial agent 3 are respectively added to 500g oil-contaminated soil (determined by gravimetric method) 3.23% oil-contaminated soil (soluble salt content is 1.03%), adjust and keep moisture The content is 25%, agitated and ventilated once a day, and degraded for 25 days.
重量法监测不同处理组中石油烃的残留，并计算降解率，结果如图4所示，25d后CK组的石油烃降解率为14.3％，菌剂1的石油烃降解率为29.9％，菌剂2的石油烃降解率为32.4％，菌剂3的石油烃降解率为42.2％。可见，菌剂1、菌剂2和菌剂3的是由降解率均高于对照组，其中，两株菌组合形成的复合菌剂对石油烃的降解效率最高，说明两株菌对石油烃降解具有协同促进作用，取得了预料不到的技术效果。The residues of petroleum hydrocarbons in different treatment groups were monitored by gravimetric method, and the degradation rates were calculated. The results are shown in Figure 4. After 25 days, the degradation rate of petroleum hydrocarbons in the CK group was 14.3%, and the degradation rate of petroleum hydrocarbons in the bacterial agent 1 was 29.9%. The petroleum hydrocarbon degradation rate of 2 was 32.4%, and the petroleum hydrocarbon degradation rate of bacterial agent 3 was 42.2%. It can be seen that the degradation rates of bacterial agent 1, bacterial agent 2 and bacterial agent 3 are all higher than those of the control group. Among them, the composite bacterial agent formed by the combination of the two strains has the highest degradation efficiency of petroleum hydrocarbons, indicating that the two strains have the highest degradation efficiency to petroleum hydrocarbons. Degradation has a synergistic promotion effect and achieves unexpected technical effects.
结果分析：Result analysis:
通过实施例3和实施例4中嗜盐石油烃降解菌Halomonas elongate ZQ1-3的耐盐生长及耐盐石油烃降解数据来看，本发明所公开的嗜盐石油烃降解菌Halomonas elongate ZQ1-3在NaCl含量18％时能够正常培养，而且在NaCl含量10％-20％条件下对石油烃也有良好的降解效果。适应范围较广，具有良好的应用价值。Judging from the salt-tolerant growth and salt-tolerant petroleum hydrocarbon degradation data of the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 in Examples 3 and 4, the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 disclosed in the present invention It can be cultivated normally when the NaCl content is 18%, and it also has a good degradation effect on petroleum hydrocarbons under the condition of 10%-20% NaCl content. It has a wide range of adaptation and good application value.
通过实施例6中嗜盐石油烃降解菌Halomonas elongate ZQ1-3以及耐盐菌株苍白杆菌MG35复合形成的复合菌剂对石油烃的降解数据，可以看出两株菌复合形成的复合菌剂对石油烃降解有协同增效作用，复合菌剂的应用比单菌效果更佳，具有更好的推广应用价值。According to the degradation data of petroleum hydrocarbons by the compound inoculum formed by the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 and the halophilic strain Bacillus pallidum MG35 in Example 6, it can be seen that the compound inoculum formed by the two strains is effective against petroleum hydrocarbons. Hydrocarbon degradation has a synergistic effect, and the application of compound bacteria is better than single bacteria, and has better promotion and application value.

Claims (10)

  1. 一株嗜盐石油烃降解菌Halomonas elongate ZQ1-3，其特征在于，于2020年10月26日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，菌种保藏号：CGMCC No.20953。A halophilic petroleum hydrocarbon degrading bacterium Halomonas elongate ZQ1-3, characterized in that it was preserved in the General Microbiology Center of the China Microbial Culture Collection Management Committee on October 26, 2020, address: No. 1, Beichen West Road, Chaoyang District, Beijing No. 3 Institute of Microbiology, Chinese Academy of Sciences, strain collection number: CGMCC No.20953.
  2. 如权利要求1所述的嗜盐石油烃降解菌Halomonas elongate ZQ1-3，其特征在于，满足以下条件之一项或多项：The halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 as claimed in claim 1, wherein one or more of the following conditions are met:
    i.所述嗜盐石油烃降解菌Halomonas elongate ZQ1-3的16S rDNA的核苷酸序列如SEQ ID NO.1所示；i. The nucleotide sequence of the 16S rDNA of the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 is shown in SEQ ID NO.1;
    ii.所述嗜盐石油烃降解菌Halomonas elongate ZQ1-3在含盐量为1～20wt％条件下生长繁殖；ii. the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 grows and reproduces under the condition that the salt content is 1～20wt%;
    iii.所述嗜盐石油烃降解菌Halomonas elongate ZQ1-3降解C 10-C 40的饱和烃。 iii. The halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 degrades C 10 -C 40 saturated hydrocarbons.
  3. 权利要求1所述的嗜盐石油烃降解菌Halomonas elongate ZQ1-3的培养方法，其特征在于，包括如下步骤：The culture method of the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 according to claim 1, is characterized in that, comprises the steps:
    (1)取嗜盐石油烃降解菌Halomonas elongate ZQ1-3划线于固体活化培养基上，活化培养，得活化后菌株；(1) get the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 and streak it on the solid activated medium, and activate the culture to obtain the activated bacterial strain;
    (2)取步骤(1)得到的活化后菌株接种至液体培养基中，摇床培养，制得种子液；(2) get the activated bacterial strain obtained in step (1) and inoculate it into a liquid medium, and shake it to cultivate to obtain seed liquid;
    (3)取步骤(2)制得的种子液，按体积百分比1％～10％转接至扩大培养基中，扩大培养，制得Halomonas elongate ZQ1-3菌液。(3) Take the seed liquid obtained in step (2), transfer it to the expansion medium according to the volume percentage of 1% to 10%, and expand the culture to obtain the Halomonas elongate ZQ1-3 bacterial liquid.
  4. 如权利要求3所述的培养方法，其特征在于，满足以下条件之一项或多项：The culture method of claim 3, wherein one or more of the following conditions are met:
    i.步骤(1)中所述固体活化培养基的组分如下：蛋白胨10g/L，酵母提取物5g/L，氯化钾20g/L，七水硫酸镁15g/L，氯化钠130g/L，琼脂20g/L，余量水，pH自然；i. The components of the solid activation medium described in step (1) are as follows: peptone 10g/L, yeast extract 5g/L, potassium chloride 20g/L, magnesium sulfate heptahydrate 15g/L, sodium chloride 130g/L L, agar 20g/L, balance water, natural pH;
    ii.步骤(1)中所述活化培养的条件为：28～32℃倒置培养1～2天；ii. The conditions for the activation culture described in step (1) are: 28-32°C inversion culture for 1-2 days;
    iii.步骤(2)中所述液体培养基与步骤(3)中所述扩大培养基均为高盐液体培养基，组分如下：蛋白胨10g/L，酵母提取物5g/L，氯化钾20g/L，七水硫酸镁15g/L，氯化钠130g/L，余量水，pH自然；iii. the liquid medium described in the step (2) and the expansion medium described in the step (3) are high-salt liquid medium, and the components are as follows: peptone 10g/L, yeast extract 5g/L, potassium chloride 20g/L, magnesium sulfate heptahydrate 15g/L, sodium chloride 130g/L, balance water, natural pH;
    iv.步骤(2)中所述摇床培养的条件为：28～32℃转速为100～200转/分钟的条件下，摇床培养2～5天；iv. The conditions of the shaker culture in the step (2) are as follows: under the condition that the rotational speed is 100～200 rpm at 28～32°C, the shaker culture is carried out for 2～5 days;
    v.步骤(3)中所述扩大培养的条件为：28～32℃、溶氧20～40％的条件下，扩大培养1～2天。v. The conditions for expanding the culture in step (3) are: expanding the culture for 1-2 days under the conditions of 28-32° C. and dissolved oxygen of 20-40%.
  5. 一种含有权利要求1所述的嗜盐石油烃降解菌Halomonas elongate ZQ1-3的石油烃降解菌剂。A petroleum hydrocarbon degrading bacterial agent containing the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 according to claim 1.
  6. 一种石油烃降解液体菌剂，其特征在于，是权利要求1所述的嗜盐石油烃降解菌 Halomonas elongate ZQ1-3菌液。A petroleum hydrocarbon degrading liquid bacterial agent, characterized in that it is the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 bacterial liquid according to claim 1.
  7. 一种石油烃降解固体菌剂，其特征在于，是权利要求1所述的嗜盐石油烃降解菌Halomonas elongate ZQ1-3菌液与有机质载体按质量比1:(10～20)的比例混合制得；A petroleum hydrocarbon degrading solid bacterial agent is characterized in that, it is prepared by mixing the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 bacterial liquid and organic matter carrier according to the mass ratio of 1:(10～20) according to claim 1. have to;
    优选的，所述有机质载体为草炭土、锯末和麸皮草，质量比为(1～3)︰(1～3)︰(1～3)；Preferably, the organic matter carrier is turf soil, sawdust and bran grass, and the mass ratio is (1-3):(1-3):(1-3);
    优选的，所述石油降解固体菌剂的活菌浓度为(2～5)×10 9cfu/g。 Preferably, the viable bacteria concentration of the petroleum-degrading solid bacterial agent is (2-5)×10 9 cfu/g.
  8. 一种石油烃降解复合菌剂，其特征在于，包括权利要求1所述的嗜盐石油烃降解菌Halomonas elongate ZQ1-3和苍白杆菌(Ochrobactrum daejeonense)MG35；A petroleum hydrocarbon degrading compound bacterial agent, characterized in that, comprising the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 and Ochrobactrum daejeonense MG35 according to claim 1;
    其中，所述苍白杆菌(Ochrobactrum daejeonense)MG35，2020年4月27日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，菌种保藏号：CGMCC No.19745；Among them, the Ochrobactrum daejeonense MG35 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee on April 27, 2020, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing , strain preservation number: CGMCC No.19745;
    优选的，所述嗜盐石油烃降解菌Halomonas elongate ZQ1-3和苍白杆菌(Ochrobactrum daejeonense)MG35的有效菌浓比为(1～3):(1～3)；Preferably, the effective bacterial concentration ratio of Halomonas elongate ZQ1-3 and Ochrobactrum daejeonense MG35 is (1～3):(1～3);
    优选的，所述石油烃降解复合菌剂是将Halomonas elongate ZQ1-3菌液和苍白杆菌MG35菌液与有机质载体按质量比1:(10～20)的比例混合制得；Preferably, the petroleum hydrocarbon degrading compound bacterial agent is prepared by mixing Halomonas elongate ZQ1-3 bacterial liquid and Bacillus pallidus MG35 bacterial liquid with an organic matter carrier in a mass ratio of 1:(10-20);
    优选的，所述有机质载体为草炭土、锯末和麸皮草，质量比为(1～3):(1～3):(1～3)；Preferably, the organic matter carrier is turf soil, sawdust and bran grass, and the mass ratio is (1-3):(1-3):(1-3);
    优选的，所述石油烃降解复合菌剂的活菌浓度为(1～10)×10 9cfu/g。 Preferably, the viable bacteria concentration of the petroleum hydrocarbon-degrading compound bacterial agent is (1-10)×10 9 cfu/g.
  9. 权利要求1所述的嗜盐石油烃降解菌Halomonas elongate ZQ1-3或权利要求5～8任一项所述的菌剂在修复石油污染的水体和/或土壤中的应用。The application of the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 according to claim 1 or the bacterial agent according to any one of claims 5 to 8 in remediating oil-contaminated water bodies and/or soil.
  10. 权利要求1所述的嗜盐石油烃降解菌Halomonas elongate ZQ1-3或权利要求5～8任一项所述的菌剂在盐碱环境石油污染水体和/或土壤修复中的应用。Application of the halophilic petroleum hydrocarbon degrading bacteria Halomonas elongate ZQ1-3 according to claim 1 or the bacterial agent according to any one of claims 5 to 8 in the remediation of petroleum polluted water bodies and/or soils in saline-alkali environments.
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