CN108949627B - Microbial oil recovery bacterium W-Y7 and application thereof - Google Patents
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Abstract
The invention discloses a microbial oil recovery bacterium W-Y7 and application thereof. The microbial oil production bacteria W-Y7 is Eiseniicola sp.W-Y7, and the preservation number of the microbial oil production bacteria in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 15904. Experiments prove that Eiseniicola sp.W-Y7 not only has the function of degrading petroleum and can be used for treating and repairing petroleum pollution, but also can be used for microbial oil recovery and improving the oil recovery rate. The invention has important application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a microbial oil recovery bacterium W-Y7 and application thereof.
Background
The petroleum pollution is widely distributed, and relates to various industries such as oil extraction, oil refining, chemical engineering, machinery and the like, and various polluted environments such as petroleum leakage and the like. The petroleum hydrocarbon components are complex and diverse, and have strong hydrophobicity, so that the petroleum hydrocarbon components are difficult to be contacted, degraded and utilized by common microorganisms.
The microbial oil recovery technology is to inject nutrients or microbes into the stratum and to utilize the growth and metabolism of microbes in oil deposit to raise the yield and recovery rate of crude oil. The traditional petroleum functional strains are mostly separated from water and soil. The existing research shows that the microorganisms are widely distributed in the petroleum oil phase besides water and soil, so that the separation of related functional strains from the petroleum oil phase has important significance in the microbial oil recovery technology.
Disclosure of Invention
The object of the invention is to degrade petroleum.
The invention firstly protects Eiseniicola sp.W-Y7, the strain is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 of Xilu No.1 of Beijing Kogyo area of Chaoyang) in 2018 at 06.05.month, and the preservation number is CGMCC No. 15904. Eiseniicola sp.W-Y7 CGMCC No.15904 is abbreviated as Eiseniicola sp.W-Y7.
The invention also protects a microbial inoculum which contains the Eiseniicola sp.W-Y7. The microbial inoculum is A1) or A2) or A3) or A4): A1) microbial oil recovery; A2) emulsifying the crude oil; A3) degrading petroleum; A4) and (3) treating and/or repairing petroleum pollution.
The preparation method of the microbial inoculum comprises the following steps: eiseniicola sp.W-Y7 was inoculated into a bacterial culture medium and cultured to give Eiseniicola sp.W-Y7 at a concentration of 107-109CFU/mL (e.g., 10)7-108CFU/mL、108-109CFU/mL、107CFU/mL、108CFU/mL or 109CFU/mL) to obtain the microbial inoculum.
The bacterial culture medium can be LB liquid culture medium.
In the preparation method of the microbial inoculum, the culture conditions can be as follows: 28-32 deg.C (such as 28-30 deg.C, 30-32 deg.C, 28 deg.C, 30 deg.C or 32 deg.C), 100-.
The microbial inoculum may include a carrier in addition to the active ingredient. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymeric compound. The mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth. The plant material may be at least one of corn flour, bean flour and starch. The high molecular compound may be polyvinyl alcohol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water. The organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and a filtrate. The composition can be prepared into various dosage forms, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.
According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.
The invention also protects the application of the Eiseniicola sp.W-Y7 or any microbial inoculum described above, which can be a1) or a 2): a1) microbial oil recovery; a2) preparing the product of microbial oil recovery.
The invention also protects the application of the Eiseniicola sp.W-Y7 or any microbial inoculum described above, which can be b1) or b 2): b1) emulsifying the crude oil; b2) the product of emulsified crude oil is prepared.
The invention also protects the application of the Eiseniicola sp.W-Y7 or any microbial inoculum described above, which can be c1) or c 2): c1) degrading petroleum; c2) preparing petroleum degrading products.
The invention also protects the application of the Eiseniicola sp.W-Y7 or any microbial inoculum described above, which can be d1) or d 2): d1) treating and/or repairing petroleum pollution; d2) preparing products for treating and/or repairing petroleum pollution.
The invention also protects a product containing the Eiseniicola sp.W-Y7 or any of the microbial agents described above; the product may have at least one of the following functions a1) -a 4): A1) microbial oil recovery; A2) emulsifying the crude oil; A3) degrading petroleum; A4) and (3) treating and/or repairing petroleum pollution.
The invention also provides a microbial oil recovery method, which comprises the following steps: adding the Eiseniicola sp.W-Y7 or any microbial inoculum described above in the oil extraction process.
The present invention also protects a method of emulsifying and/or degrading crude oil, which may include the steps of: adding the Eiseniicola sp.W-Y7 or the microbial inoculum described in any one of the above to crude oil or a liquid phase system containing crude oil.
The invention also provides a method for treating and/or restoring petroleum pollution, which comprises the following steps: treating petroleum contaminants with the Eiseniicola sp.W-Y7 or the microbial inoculum of any one of the above.
Experiments prove that the Eiseniicola sp.W-Y7 CGMCC No.15904 provided by the invention not only has the function of degrading petroleum and can be used for treating and repairing petroleum pollution, but also can be used for microbial oil recovery and improving the oil recovery rate. The invention has important application value.
Drawings
FIG. 1 shows the effect of W-Y7 strain on emulsifying crude oil.
FIG. 2 is a graph of the displacement results of the model oil.
Deposit description
Latin name: eiseniicola sp.
The strain number is as follows: W-Y7
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 06 and 05 months in 2018
Registration number of the preservation center: CGMCC No.15904
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Liquid inorganic salt culture medium: 5g NaCl, 1g NH4H2PO4、1g(NH4)2SO4、1g K2HPO4And 3g KNO3Dissolving in deionized water, then using the deionized water to fix the volume to 1L, and adjusting the pH value to 7-8; sterilizing at 121 deg.C for 30 min.
LB liquid medium: dissolving 10g of NaCl, 10g of peptone and 5g of yeast powder in deionized water, then using the deionized water to fix the volume to 1L, and adjusting the pH value to 7-8; sterilizing at 121 deg.C for 30 min.
LB solid medium: dissolving 10g of NaCl, 10g of peptone, 5g of yeast powder and 15g of agar powder in deionized water, then using the deionized water to fix the volume to 1L, and adjusting the pH value to 7-8; sterilizing at 121 deg.C for 30 min.
LB solid plate: LB solid medium at about 55 ℃ is poured into a sterile culture dish, and an LB solid plate is obtained after cooling.
Example 1 isolation, identification and preservation of the W-Y7 Strain
Isolation of the first, W-Y7 Strain
Taking 1g of crude oil (collected from oil wells of Jilin oil fields in China in 8 months in 2017), adding 100mL of liquid inorganic salt culture medium, performing shaking culture at 30 ℃ and 150rpm for 30d, diluting and coating the crude oil on an LB solid plate, performing culture at 30 ℃ for 7d, selecting a single colony, streaking, purifying, storing, and naming the separated and purified isolate as W-Y7 strain.
Identification of the two, W-Y7 Strain
1. Morphological identification
The W-Y7 strain is inoculated on an LB solid plate, dark culture is carried out at 30 ℃, the morphology of a colony is observed after 5 days, and the morphological characteristics of the thallus are observed through high-resolution transmission electron microscope analysis.
The experimental result shows that the bacterial colony of the W-Y7 bacterial strain is a circular bulge; the W-Y7 strain is short rod-shaped and does not produce spores.
2. Physiological and biochemical characteristic analysis
The physiological and biochemical characteristics of the W-Y7 strain were determined with reference to "Manual of identification of common bacterial System" (Dongxu bead, Chuia Miaoying. Manual of identification of common bacterial System. Beijing: scientific Press, 2011.) and "microbiological experiments" (Shenping, Fangxiong capacity, Liguanwu. microbiological experiments (third edition); Beijing: advanced education Press, 1999.).
The results show that the physiological and biochemical characteristics of the W-Y7 strain are as follows: gram staining was negative, oxidase reaction was positive, catalase reaction was positive.
3. 16s rDNA sequence homology analysis
(1) The W-Y7 strain is inoculated in LB liquid culture medium, and is subjected to shaking culture at 30 ℃ and 150rpm for 24 hours to obtain a culture solution.
(2) After the step (1) is finished, taking fresh culture bacteria liquid, centrifuging at 8000rpm and 4 ℃ for 5min, collecting thalli in a centrifuge tube (the specification is 2mL), and then extracting DNA by adopting a DNA extraction kit.
(3) And (3) after the step (2) is finished, taking DNA of the thalli, carrying out electrophoresis detection, and carrying out PCR amplification by adopting a universal primer 8F/1492R to obtain a PCR product containing a 16S rDNA conserved region of the W-Y7 strain. The primer sequences are as follows: 8F: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively; 1492R: 5'-GGTTACCTTGTTACGACTT-3' are provided.
The PCR product was electrophoretically detected and sequenced. The sequencing result shows that the nucleotide sequence of the 16S rDNA of the W-Y7 strain is shown as a sequence 1 in a sequence table.
The W-Y7 strain isolated and purified in step (A) was identified as Eiseniicola sp, in view of the above results of morphological, physiological and biochemical characterization and sequence homology analysis of 16S rDNA.
Deposit of the three, W-Y7 Strain
The W-Y7 strain has been preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 Xilu No.1 Beijing, Chaoyang, China) in 2018, No. 05 month, 06 and year 05, and the preservation number is CGMCC No. 15904. The W-Y7 strain is fully called Eiseniicola sp.W-Y7 CGMCC No. 15904.
Example 2 application of W-Y7 Strain in emulsification of crude oil and degradation of Petroleum
Application of W-Y7 bacterial strain in emulsifying crude oil
1. Inoculating W-Y7 strain in LB liquid medium, performing shaking culture at 30 deg.C and 150rpm for 24 hr to obtain W-Y7 bacterial liquid (W-Y7 bacterial liquid, W-Y7 strain concentration is 10%8CFU/mL)。
2. Inoculating the W-Y7 bacterial liquid obtained in the step 1 into a crude oil inorganic salt culture medium according to the proportion of 5% (v/v), and carrying out shaking culture at 30 ℃ and 150rpm for 7 d.
The viscosity of the crude oil was 25mpa.s at a temperature of 30 ℃.
Crude oil inorganic salt culture medium: 1g of crude oil, 5g of NaCl and 1g of NH4H2PO4、1g(NH4)2SO4、1g K2HPO4And 3gKNO3Dissolving in deionized water, then using the deionized water to fix the volume to 1L, and adjusting the pH value to 7-8; sterilizing at 121 deg.C for 30 min.
3. LB liquid culture medium was inoculated into crude oil inorganic salt culture medium at a ratio of 5% (v/v), and cultured at 30 ℃ for 7d with shaking at 150 rpm. As a control.
The experimental results are shown in FIG. 1 (the left panel shows crude oil treated with LB liquid medium, and the right panel shows crude oil treated with W-Y7 bacterial liquid). The results show that the W-Y7 strain has obvious emulsification effect on crude oil at 30 ℃.
Application of W-Y7 bacterial strain in petroleum degradation
1. The same as step 1.
2. The same as step 2 in the first step.
3. And (3) taking the system which finishes the step (2), measuring the residual petroleum amount according to a method in the literature 'separation of crude oil degrading bacteria and degradation performance thereof', and calculating the petroleum degradation rate. Petroleum degradation rate (petroleum addition-petroleum residue)/petroleum addition.
The calculation result shows that the petroleum degradation rate is 61.5%. The result shows that the W-Y7 bacterial strain has the function of degrading petroleum and can be used for treating and repairing petroleum pollution.
Example 3 application of the W-Y7 Strain in microbial oil recovery technology
Evaluating the oil displacement effect of the microbial strains through an indoor physical model oil displacement experiment, wherein the experimental steps are as follows:
1. according to the permeability of the reservoir block (0.250 μm)2) Using high pressure die type pipe (specification)
Purchased from haian oil research instruments ltd) are filled with quartz sand with different meshes to form sand filling mold pipes.
2. Measuring the permeability of the model pipe by adopting a permeability measuring device, and selecting the model pipe which meets the permeability; the sand pack parameters of the model tubes are shown in table 1.
TABLE 1 model pipe Sand filling parameters
3. And vacuumizing to saturate formation water and calculating the porosity.
4. Bound water is established by oil-driving water, and the original oil saturation is measured; and aged at reservoir temperature (30 ℃) for 3 days.
5. And (4) displacing the oil by using the injected water, stopping water drive until the outlet produced liquid reaches the limit water content (98 percent), and calculating the water drive recovery ratio.
6. Inoculating W-Y7 strain in LB liquid medium, performing shaking culture at 30 deg.C and 150rpm for 24 hr to obtain W-Y7 bacterial liquid (W-Y7 bacterial liquid, W-Y7 strain concentration is 10%8CFU/mL)。
7. Adding NH to the injection water4H2PO4、(NH4)2SO4、K2HPO4、KNO3Peptone, yeast powder and W-Y7 bacteria liquid to obtain mixed liquid containing W-Y7. NH in the mixed solution containing W-Y74H2PO4、(NH4)2SO4And K2HPO4The concentration of (B) is 1g/L, KNO3The concentration of (3) is 3g/L, the concentration of peptone is 2g/L, the concentration of yeast powder is 1g/L, and the concentration of W-Y7 bacterial liquid is 5% (v/v). The concentration of W-Y7 strain in the mixed solution containing W-Y7 was 108CFU/mL。
8. Injecting the mixed solution containing W-Y7 into a mold tube according to the injection amount of 1.0 PV; the culture was carried out at reservoir temperature (30 ℃) for 15 days.
9. And (3) performing secondary water drive, wherein the water content is more than 98%, and respectively calculating the recovery ratio of the subsequent water drive under the action of the microorganisms (the mixed liquor containing W-Y7) and the enhanced recovery ratio of the W-Y7 strain in the process of injecting the microorganisms (the mixed liquor containing W-Y7).
The results of the recovery in different stages of water flooding, microbial injection, microbial action followed by water flooding are shown in table 2. The comparison of the recovery ratios in different stages of water flooding, microbial injection, subsequent water flooding under the action of microbes and the like shows that: the W-Y1 bacterial strain is used for oil recovery, and the recovery ratio is improved by 9.41%. The physical model displacement result curve is shown in figure 2.
TABLE 2 recovery ratio in water flooding, microbial injection, and subsequent water flooding
| Stage of material model | Recovery ratio (%) | Enhanced recovery (%) |
| Water drive | 41.19 | / |
| Microbial injection process | 42.99 | 1.8 |
| Follow-up water drive of microbial action | 50.60 | 9.41 |
Note: "/" indicates absence.
<110> Beijing Ruichi energy technology Limited
<120> microbial oil recovery bacterium W-Y7 and application thereof
<160>1
<170>PatentIn version 3.5
<210>1
<211>1338
<212>DNA
<213> Artificial sequence
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ggcgagtggc gaacgggtga gtaatatatc ggaacgtgcc cagtagcggg ggataactac 60
gcgaaagcgt ggctaatacc gcatacgccc tacgggggaa agggggggat tcttcggaac 120
ctctcactat tggagcggcc gatatcagat tagctagttg gtgaggtaaa ggctcaccaa 180
ggcgacgatc tgtagctggt ttgagaggac gaccagccac actgggactg agacacggcc 240
cagactccta cgggaggcag cagtggggaa ttttggacaa tgggggaaac cctgatccag 300
ccatcccgcg tgtgcgatga aggccttcgg gttgtaaagc acttttggca gggaagaaac 360
ggcgtgccct aatacgggac gtcactgacg gtacctgcag aataagcacc ggctaactac 420
gtgccagcag ccgcggtaat acgtagggtg caagcgttaa tcggaattac tgggcgtaaa 480
gcgtgcgcag gcggttcgga aagaaagatg tgaaatccca gagcttaact ttggaactgc 540
atttttaact cccgagctag agtatgtcag aggggggtag aattccacgt gtagcagtga 600
aatgcgtaga gatgtggagg aataccgatg gcgaaggcag ccccctggga taatactgac 660
gctcatgcac gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgcccta 720
aacgatgtca actagctgtt ggggccttcg ggccttagta gcgcagctaa cgcgtgaagt 780
tgaccgcctg gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg gggacccgca 840
caagcggtgg atgatgtgga ttaattcgat gcaacgcgaa aaaccttacc tacccttgac 900
atgtctggaa gctacaagag attgaagcgt gctcgcaaga gaaccggaac acaggtgctg 960
catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1020
cttgtcatta gttgctacga aagggcactc taatgagact gccggtgaca aaccggagga 1080
aggtggggat gacgtcaagt cctcatggcc cttatgggta gggcttcaca cgtcatacaa 1140
tggtcgggac agagggtcgc caacccgcaa gggggagcca atcccagaaa cccgatcgta 1200
gtccggatcg cagtctgcaa ctcgactgcg tgaagtcgga atcgctagta atcgcggatc 1260
agaatgtcgc ggtgaatacg ttcccgggtc ttgtacacac cgcccgtcac accatgggag 1320
tgggttttac cagaagta 1338
Claims (10)
- Eiseniicola sp.W-Y7 with the preservation number of CGMCC No.15904 in China general microbiological culture Collection center.
- 2. A microbial inoculum, which is characterized in that: the microbial inoculum comprises Eiseniicola sp.W-Y7 according to claim 1.
- 3. The use of the Eiseniicola sp.W-Y7 of claim 1 or the microbial inoculum of claim 2 as a1) or a 2): a1) microbial oil recovery; a2) preparing the product of microbial oil recovery.
- 4. The use of the Eiseniicola sp.W-Y7 according to claim 1 or the microbial inoculum according to claim 2 as b1) or b 2): b1) emulsifying the crude oil; b2) the product of emulsified crude oil is prepared.
- 5. The use of the Eisenicicola sp.W-Y7 of claim 1 or the microbial inoculum of claim 2 as c1) or c 2): c1) degrading petroleum; c2) preparing petroleum degrading products.
- 6. The use of the Eisenicicola sp.W-Y7 of claim 1 or the microbial inoculum of claim 2 as d1) or d 2): d1) treating and/or repairing petroleum pollution; d2) preparing products for treating and/or repairing petroleum pollution.
- 7. A product containing the microbial agent of claim 1 or claim 2 in an Eiseniicola sp.w-Y7; the product has at least one of the following functions A1) -A4): A1) microbial oil recovery; A2) emulsifying the crude oil; A3) degrading petroleum; A4) and (3) treating and/or repairing petroleum pollution.
- 8. A method of microbial oil recovery comprising the steps of: in the oil extraction process, the microbial inoculum described in Eisenicicola sp.W-Y7 in claim 1 or 2 is added.
- 9. A method of emulsifying crude oil and/or degrading petroleum oil comprising the steps of: adding the microbial agent of Eiseniicola sp.W-Y7 described in claim 1 or the microbial agent described in claim 2 to crude oil or a liquid phase system containing crude oil.
- 10. A method for treating and/or remediating petroleum pollution, comprising the steps of: treating petroleum pollutants with the microbial inoculum of Eiseniicola sp.W-Y7 as defined in claim 1 or 2.
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| CN108130299A (en) * | 2018-01-24 | 2018-06-08 | 西北民族大学 | The microbial strains and joint renovation agent of one plant of decomposing petroleum hydrocarbon |
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| WO2016156260A1 (en) * | 2015-03-27 | 2016-10-06 | Fonds De Développement Des Filières Des Oléagineux Et Des Proteagineux Fidop | Microorganisms and use thereof for the production of diacids |
| CN108130299A (en) * | 2018-01-24 | 2018-06-08 | 西北民族大学 | The microbial strains and joint renovation agent of one plant of decomposing petroleum hydrocarbon |
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| CN108949627A (en) | 2018-12-07 |
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