WO2022227239A1 - 一株嗜盐石油烃降解菌及其应用 - Google Patents
一株嗜盐石油烃降解菌及其应用 Download PDFInfo
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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
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Images
Classifications
-
- 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 NOT OTHERWISE PROVIDED FOR
- 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.
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Description
编号 | 组分 | 体积(μL) |
1 | 2×Pfu PCR MasterMix | 25 |
2 | 27F | 2 |
3 | 1492R | 2 |
4 | DNA模板 | 1 |
5 | 加ddH 2O至 | 50 |
Claims (10)
- 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。
- 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: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;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;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。
- 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:(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;(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;(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。
- 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: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;ii.步骤(1)中所述活化培养的条件为:28~32℃倒置培养1~2天;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;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;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。
- 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。
- 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。
- 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;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,所述石油降解固体菌剂的活菌浓度为(2~5)×10 9cfu/g。
- 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;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;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;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,所述石油烃降解复合菌剂的活菌浓度为(1~10)×10 9cfu/g。
- 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。
- 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。
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