CN106811426A - Bacillus thermopile fertilizer strain for emulsifying crude oil, culture method and application - Google Patents
Bacillus thermopile fertilizer strain for emulsifying crude oil, culture method and application Download PDFInfo
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- 238000011084 recovery Methods 0.000 claims abstract description 21
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- 238000000034 method Methods 0.000 claims description 12
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- 239000004317 sodium nitrate Substances 0.000 claims description 12
- 235000010344 sodium nitrate Nutrition 0.000 claims description 12
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 8
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 7
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
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- Fertilizers (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a Bacillus thermopile copriae (BLG 69) strain for emulsifying crude oil, a culture method and application, and belongs to the technical field of microbial oil recovery. The bacillus thermopile BLG69 strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 11441. The bacillus thermopile BLG69 strain can take crude oil as a unique carbon source, metabolize at the temperature of 37-60 ℃ to generate an emulsifier, emulsify the crude oil, and reduce the viscosity of the crude oil, so that the recovery efficiency of the crude oil is improved, and the application temperature range is wide. Meanwhile, the bacillus thermopile BLG69 strain has good adaptability to oil reservoir environment, and when the bacillus thermopile fertilizer is used for oil displacement, the bacillus thermopile fertilizer does not need to be fermented on the ground, and the effect of improving the crude oil recovery rate can be achieved by injecting an activator capable of promoting the growth and metabolism of the bacillus thermopile fertilizer into an oil reservoir.
Description
Technical Field
The invention relates to the technical field of microbial oil extraction, in particular to a bacillus thermopile fertilizer strain for emulsifying crude oil, a culture method and application.
Background
Microbial Enhanced Oil Recovery (MEOR) is a biological technology for improving crude Oil yield and Recovery efficiency by using life activities and metabolites of microbes in Oil reservoirs, and has the characteristics of wide application range, low implementation cost, remarkable economic benefit and the like, so that the MEOR is widely concerned. The metabolic characteristics of different microbial strains are different, the produced metabolites are different, and the oil displacement mechanisms of the microbial strains are different, so that different microbial oil recovery processes are formed. The mechanism of microbial oil displacement mainly comprises the following steps: gas generated in the microbial growth and metabolism process increases formation pressure and reduces the viscosity of crude oil; producing organic acid to improve the permeability of reservoir rock; the biosurfactant and the organic solvent are generated to reduce the oil-water interfacial tension and improve the oil washing efficiency; generating biopolymer to control fluidity selective plugging; degrading heavy components of the crude oil to reduce the viscosity of the crude oil, improve the fluidity of the crude oil, and the like. The microbial oil recovery technology for reducing the viscosity of the crude oil by emulsifying the crude oil by using microorganisms can obviously improve the recovery rate of the crude oil.
The key of the microbial oil recovery technology for reducing the viscosity of crude oil by emulsifying the crude oil by using microorganisms is the selection of strains. Patent applications publication nos. CN 104371940 a and CN 102757994 a disclose pseudomonas and bacillus, respectively, which are capable of metabolically producing biosurfactants, but the growth temperature of these two strains is 37 ℃, and the applicable temperature range is narrow. Bacillus licheniformis (Bacillus licheniformis) is a thermophilic Bacillus, the culture temperature is 25-40 ℃ (see the journal of ecology 2013, 32(3): 779-786; 25 th volume of 25, No. 7, 16-18 and the Chinese patent application with the application number of 201380012425), and the applicable temperature range is still narrow. The patent application of publication No. CN 102399847A discloses a high temperature bacillus LEY11, the fermentation temperature of which reaches 45-70 ℃, but the fermentation is needed on the ground, the operation is complex, and the field requirement can not be met.
In conclusion, in the process of implementing the invention, the inventor finds that the prior art at least has the problems that the prior bacterial strain capable of emulsifying the crude oil can not simultaneously meet the requirements of wide applicable temperature range and no on-site fermentation.
Disclosure of Invention
In order to solve the technical problems, the invention provides a bacillus thermopile fertilizer strain for emulsifying crude oil, a culture method and application, wherein the bacillus thermopile fertilizer strain is wide in application temperature range and does not need field fermentation.
Specifically, the method comprises the following technical scheme:
a Bacillus thermocompostae (Bacillus thermocompare) BLG69 strain for emulsifying crude oil, which is deposited in China general microbiological culture Collection center (CGMCC) at 9-24 months in 2015 and has the classification name: bacillus thermopile fertilizer (Bacillus thermocyprae), the preservation number is CGMCC No.11441, the preservation address is: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
The 16S rRNA gene sequence of the bacillus thermopile BLG69 strain is shown as SEQ ID No.1 in a sequence table, and the accession number of 16S rRNA in GenBank is KT 807465.
The colony morphology of the bacillus thermopile BLG69 strain is characterized in that the diameter of the colony cultured for 2 days on a solid fermentation medium is 2-4 mm, the colony is circular, the surface is smooth, the edge is neat, and the colony is milky yellow; the cell morphology is characterized in that: the cell is rod-shaped, has a length of 2-5 um and a width of 0.5-0.6 um, belongs to gram-positive bacteria and can form terminal spores; facultative anaerobic growth, positive catalase and oxidase, unable to utilize glucose. The growth environment of the bacillus thermopile BLG69 strain is as follows: the temperature is 37-60 ℃, the pH value is 6-9, and the concentration of sodium chloride is 0-3% (w/v, mass volume concentration); the optimal growth environment is as follows: the temperature is 45-50 ℃, the pH value is 7.5, and the concentration of sodium chloride is 1-2% (w/v, mass volume concentration).
The bacillus thermopcompost BLG69 strain is obtained by culturing the following culture method, wherein the culture method comprises the following steps:
step a, placing an oil-water sample collected from an oil reservoir at 45-60 ℃ in a North China oil field in a sterilized enrichment medium, carrying out shake culture in a shaker at 45-60 ℃ and a rotating speed of 120-200 rpm, and separating and purifying to obtain a strain taking crude oil as a unique carbon source;
b, inoculating the strain which is obtained in the step a and takes the crude oil as the only carbon source into a fermentation medium, and carrying out shake culture for 72 hours in a shaking table with the temperature of 45-60 ℃ and the rotating speed of 120-200 rpm to obtain fermentation liquor; detecting the surface tension of the fermentation liquor, wherein the strain corresponding to the fermentation liquor with the surface tension of less than 40mN/m is the thermal compost bacillus BLG69 strain;
wherein,
in the step a, the enrichment medium comprises the following components in parts by mass:
100 parts of clear water; 8-10 parts of crude oil; ammonium chloride (NH)4Cl) 0.09-0.12 part; sodium nitrate (NaNO)3) 0.18 to 0.21 portion; dipotassium hydrogen phosphate (K)2HPO4) 0.04 to 0.06 portion; sodium sulfate (Na)2SO4) 0.18 to 0.21 portion; magnesium sulfate (MgSO)4) 0.02-0.03 part; sodium carbonate (Na)2CO3) 0.04 to 0.06 portion; 0.02-0.03 part of sodium acetate (NaAc); 0.04-0.06 part of yeast powder; 0.09-0.10 part of corn steep liquor; the pH value of the enrichment medium is 7.5-8.0;
in the step b, the fermentation medium comprises the following components in parts by weight:
100 parts of oil reservoir produced water; 0.08-0.11 part of ammonium chloride; 0.14-0.18 part of sodium nitrate; 0.04-0.06 part of dipotassium hydrogen phosphate; 0.01-0.02 part of magnesium sulfate; 0.8-1.1 parts of sodium chloride; 0.04-0.06 part of yeast powder; 0.1-0.12 part of corn steep liquor; 5-8 parts of crude oil; the pH value of the fermentation medium is 7.5-8.0.
Preferably, in the step a, an oil-water sample collected from an oil reservoir at 55 ℃ in the North China oilfield is placed in a sterilized enrichment medium.
Preferably, in step b, the liquid loading amount of the fermentation medium is 100ml in a 250ml triangular flask or 10ml in a 50ml test tube.
The method for improving the recovery ratio of crude oil by using the bacillus thermocomposter BLG69 strain comprises the steps of injecting an activator into an oil reservoir containing the bacillus thermocomposter BLG69 strain, closing a well for a preset time and then extracting the crude oil; the activating agent comprises the following components in parts by mass: 100 parts of oil reservoir produced water; 0.08-0.11 part of ammonium chloride; 0.14-0.18 part of sodium nitrate; 0.04-0.06 part of dipotassium hydrogen phosphate; 0.01-0.02 part of magnesium sulfate; 0.8-1.1 parts of sodium chloride; 0.04-0.06 part of yeast powder; 0.1-0.12 part of corn steep liquor; 5-8 parts of crude oil.
Preferably, the temperature of the oil reservoir is 37 ℃ to 60 ℃.
The technical scheme provided by the embodiment of the invention has the beneficial effects that:
the bacillus thermopile BLG69 strain provided by the embodiment of the invention can take crude oil as a unique carbon source, and metabolize at the temperature of 37-60 ℃ to generate an emulsifier, so that the crude oil is emulsified to reduce the viscosity of the crude oil, and the recovery ratio of the crude oil is improved. The surface tension of fermentation liquor of the bacillus thermopile BLG69 strain cultured for 72 hours at 45 ℃ is 29-32 mN/m, the emulsifying power is more than 90%, and the bacillus thermopile BLG69 strain has strong crude oil emulsifying capacity. Meanwhile, the bacillus thermopile BLG69 strain has good adaptability to oil reservoir environment, so that when the bacillus thermopile fertilizer is used for oil displacement, the fermentation on the ground is not needed, and the effect of improving the crude oil recovery rate can be achieved by injecting an activator capable of promoting the growth and metabolism of the bacillus thermopile fertilizer into an oil reservoir. In conclusion, the bacillus thermopile BLG69 strain provided by the embodiment of the invention has a wide application temperature range, does not need field fermentation, and has a good application prospect in the technical field of microbial oil recovery of medium-high temperature oil reservoirs.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below.
FIG. 1 is a phylogenetic tree constructed based on the 16S rRNA gene sequence;
FIG. 2 is a graph showing the emulsification of crude oil after the thermal composting Bacillus strain BLG69 in example 4 was cultured in a fermentation medium;
FIG. 3 shows the fermentation medium of example 4 without inoculated strain.
Biological preservation information: the bacillus thermopile BLG69 strain provided by the embodiment of the invention has been preserved in China general microbiological culture Collection center (CGMCC) in 2015 at 9-24 months, and the classification name is as follows: bacillus thermocompostis (Bacillus thermocyprae) with the preservation number of CGMCC No. 11441; the preservation address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
Detailed Description
In order to make the technical solutions and advantages of the present invention clearer, the following describes embodiments of the present invention in further detail.
The embodiment of the invention provides a Bacillus thermopile fertilizer BLG69 strain for emulsifying crude oil, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 11441.
The 16S rRNA gene sequence of the bacillus thermopile BLG69 strain is shown as SEQ ID No.1 in a sequence table, contains 1402bp, and has a GenBank accession number of KT807465, and concretely comprises the following steps:
GCAGTCGAGCGGACCAATAGAAGCTTGCTTCTGTTGGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAATTCATCCCCTCGCATGAGGGGGTGCTGAAAGTCGGTTCACGCTGACACTTACAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGCGATGAAGGCCTTCGGGTCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGGAGTAACTGCCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGGACTTGAGTGCAGAAGAGGAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTACGACCTCCCTAGAGATAGGGATTTCCCCTTCGGGGGACGGAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCAGCGAAACCGCGAGGTCGAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACC。
bacillus thermocompostus (Bacillus thermocopriae) is a new species of Bacillus thermophilus established in 2013 based on phylogenetic analysis and other characteristics of 16S rRNA genes (see International Journal of Systematic and evolution Microbiology (2013),63, 3024-3029). At present, an SgZ-7(JX113681) strain is reported, and the SgZ-7 strain is obtained by separating from Dongguan compost samples in China. The SgZ-7 strain can ferment glucose to produce acid, but cannot produce a biosurfactant to emulsify crude oil.
The 16S rRNA gene sequence of the BLG69 strain of the bacillus thermopile fertilizer provided by the embodiment of the invention is compared with the sequence in GenBank, and the sequence similarity with the bacillus members is found to be higher. The phylogenetic tree (FIG. 1) constructed by the Neighbor2Joining method was shown to be a new member of Bacillus, having 99.86% homology with the above Bacillus thermoplasticae SgZ-7 strain, and to be a new strain different from Bacillus thermoplasticus SgZ-7 strain.
The bacillus thermopile BLG69 strain provided by the embodiment of the invention grows well on a TSA (soybean casein) solid fermentation medium without crude oil, the bacterial colony is creamy yellow and round, has different sizes, has the diameter of 2-4 mm, has a smooth and moist surface, and has a flat and transparent edge. The cells are rod-shaped, the length is 2-5 um, the width is 0.5-0.6 um, spores are produced, the cells have motility, gram staining is positive, facultative anaerobic growth, catalase and oxidase are positive, and glucose can not be utilized. The growth environment is as follows: the temperature is 37-60 ℃, the pH value is 6-9, and the concentration of sodium chloride is 0-3% (w/v, mass volume concentration); the optimal growth environment is as follows: the temperature is 45-50 ℃, the pH value is 7.5, and the concentration of sodium chloride is 1-2% (w/v, mass volume concentration).
The bacillus thermopile BLG69 strain can take crude oil as a unique carbon source, metabolizes at the temperature of 37-60 ℃ to generate a biological emulsifier to emulsify the crude oil, reduces the viscosity of the crude oil, has good adaptability to the oil reservoir environment, does not need to ferment on the ground when the bacillus thermopile fertilizer is used for oil displacement, and can achieve the effect of improving the recovery rate of the crude oil by injecting an activator capable of promoting the growth and metabolism of the bacillus thermopile fertilizer into the oil reservoir. Therefore, the bacillus thermopile fertilizer BLG69 strain has a good application prospect in the technical field of microbial oil recovery of medium-high temperature oil reservoirs.
The bacillus thermopile BLG69 strain provided by the embodiment of the invention is separated from an oil-water sample of an oil reservoir at 45-60 ℃ in a North China oilfield, and the specific culture and screening method comprises the following steps:
step 1, placing an oil-water sample collected from an oil reservoir at 45-60 ℃ in a North China oil field in a sterilized enrichment medium, carrying out shake culture in a shaking table at 45-60 ℃ and 120-200 rpm, and separating and purifying to obtain a strain taking crude oil as a unique carbon source; since there are many strains that can use crude oil as a sole carbon source in an oil-water sample, it is necessary to further screen the obtained strains that can use crude oil as a sole carbon source to obtain strains with strong ability to emulsify crude oil.
Step 2, respectively inoculating the strains which are obtained in the step 1 and take the crude oil as the only carbon source into a fermentation medium, and carrying out shake culture for 72 hours in a shaking table with the temperature of 45-60 ℃ and the rotating speed of 120-200 rpm to obtain fermentation liquor; detecting the surface tension of the obtained fermentation liquor, wherein the bacterial strain which can enable the surface tension of the fermentation liquor to reach below 40mN/m is the Bacillus thermopile fertilizer BLG69 bacterial strain provided by the embodiment of the invention;
wherein,
the enrichment medium used in the step 1 comprises the following components in parts by mass: 100 parts of clear water; 8-10 parts of crude oil; 0.09-0.12 part of ammonium chloride; 0.18-0.21 part of sodium nitrate; 0.04-0.06 part of dipotassium hydrogen phosphate; 0.18-0.21 part of sodium sulfate; 0.02-0.03 part of magnesium sulfate; 0.04-0.06 part of sodium carbonate; 0.02-0.03 part of sodium acetate; 0.04-0.06 part of yeast powder; 0.09-0.10 part of corn steep liquor; the pH value of the enrichment medium is 7.5-8.0;
the fermentation medium used in the step 2 comprises the following components in parts by weight: 100 parts of oil reservoir produced water; 0.08-0.11 part of ammonium chloride; 0.14-0.18 part of sodium nitrate; 0.04-0.06 part of dipotassium hydrogen phosphate; 0.01-0.02 part of magnesium sulfate; 0.8-1.1 parts of sodium chloride; 0.04-0.06 part of yeast powder; 0.1-0.12 part of corn steep liquor; 5-8 parts of crude oil; the pH value of the fermentation medium is 7.5-8.0.
In the above culture method, in step 1, preferably, the oil-water sample collected from the 55 ℃ oil reservoir in the north China oil field is separated and purified to obtain the bacillus thermopile BLG69 strain provided by the embodiment of the present invention.
In the above culture method, in step 2, the amount of the fermentation medium is preferably 100ml in a 250ml Erlenmeyer flask or 10ml in a 50ml test tube.
In the above-mentioned culture method, the temperature for culturing in the enrichment medium in step 1 may be 45 ℃, 50 ℃, 55 ℃, 60 ℃ or the like.
In the above-mentioned culture method, the temperature for culturing in the fermentation medium in step 2 may be 45 ℃, 50 ℃, 55 ℃, 60 ℃ or the like.
Because the Bacillus thermopile BLG69 strain provided by the embodiment of the invention exists in the oil reservoir, the growth and metabolism speed of the Bacillus thermopile BLG69 strain can be accelerated by injecting an activator capable of promoting the growth and metabolism of the Bacillus thermopile BLG69 strain into the oil reservoir, and the metabolism of the Bacillus thermopile BLG69 strain is promoted to generate a biological emulsifier, so that the crude oil is emulsified, the viscosity of the crude oil is reduced, and the recovery rate of the crude oil is improved. The well can be shut in for a period of time after the activator is injected into the oil reservoir, for example, the well can be shut in for more than 3 days, and then crude oil extraction is carried out, so that the bacillus thermopile BLG69 strain can fully grow and metabolize. The used activating agent can adopt the following formula (in parts by mass): 100 parts of oil reservoir produced water; 0.08-0.11 part of ammonium chloride; 0.14-0.18 part of sodium nitrate; 0.04-0.06 part of dipotassium hydrogen phosphate; 0.01-0.02 part of magnesium sulfate; 0.8-1.1 parts of sodium chloride; 0.04-0.06 part of yeast powder; 0.1-0.12 part of corn steep liquor; 5-8 parts of crude oil.
The bacillus thermopile BLG69 strain provided by the embodiment of the invention is particularly suitable for oil reservoirs with the temperature of 37-60 ℃.
The culture screening method of the bacillus thermopile BLG69 strain and the performance of the emulsified crude oil thereof are further detailed by specific experimental data.
The experimental methods in the following examples are all conventional in the art unless otherwise specified; the experimental materials used in the following examples are all conventional biochemical reagents unless otherwise specified.
In the following examples, the crude oil is crude oil from a 55 ℃ oil reservoir in the North China oil field, and the produced water of the oil reservoir is produced water from the 55 ℃ oil reservoir in the North China oil field.
The surface tension measurements in the following examples were carried out using an instrument: model JYW-200 surface tension meter, available from dingsheng testing machine, chengde, north Hebei.
Example 1
The embodiment provides a method for separating and screening a bacillus thermopile BLG69 strain, which comprises the following steps:
step 1, sterilizing an enrichment medium at 121 ℃ for 30min by high-pressure steam, then placing an oil-water sample collected from an oil reservoir at 55 ℃ in a North China oilfield in the sterilized enrichment medium, and carrying out shake culture in a shaking table at 60 ℃ and 200rpm to enrich a strain taking crude oil as a unique carbon source. Then, carrying out plate streaking separation and purification on a solid culture medium without crude oil to obtain a pure strain taking the crude oil as a unique carbon source; the formula of the enrichment medium is as follows:
clear water, 1000ml (i.e. 1000 g); ammonium chloride, 1 g; 2g of sodium nitrate; dipotassium hydrogen phosphate, 0.5 g; sodium sulfate, 2 g; magnesium sulfate, 0.2 g; sodium carbonate, 0.5 g; 0.2g of sodium acetate; 0.5g of yeast powder; 1g of corn steep liquor; crude oil, 100 g; the pH of the enriched medium was 7.5.
Step 2, respectively inoculating the strains which are obtained in the step 1 and take the crude oil as the only carbon source into a fermentation culture medium, wherein the liquid loading of the culture medium is 100ml of that of a 250ml triangular flask; carrying out shake culture for 72 hours in a shaking table with the temperature of 45 ℃ and the rotating speed of 200rpm to obtain fermentation liquor; detecting the surface tension of the obtained fermentation liquor, wherein the strain corresponding to the fermentation liquor with the surface tension of 32mN/m is the Bacillus thermopile BLG69 strain; the formula of the fermentation medium is as follows:
reservoir produced water, 1000ml (i.e. 1000 g); ammonium chloride, 1 g; 1.5g of sodium nitrate; dipotassium hydrogen phosphate, 0.5 g; magnesium sulfate, 0.1 g; sodium chloride, 10 g; 0.5g of yeast powder; 1g of corn steep liquor; crude oil, 50 g; the pH of the fermentation medium was 7.5.
Example 2
The present embodiment provides a method for separating and screening bacillus thermopile BLG69 strain, which is different from the method provided in embodiment 1 in that:
the liquid loading of the medium in step 2 was 10ml in a 50ml test tube.
Example 3
The present embodiment provides a method for separating and screening bacillus thermopile BLG69 strain, which is different from the method provided in embodiment 1 in that:
in step 2, the strain obtained in step 1 and using crude oil as a sole carbon source is inoculated into a TSA culture medium containing crude oil. The formulation of TSA medium containing crude oil is as follows:
sodium chloride, 5 g; soybean peptone, 5 g; casein peptone, 10 g; 1000ml of water is produced from the oil reservoir; crude oil, 50 g; the pH of the TSA medium was 7.5.
Example 4
In this example, the performance of emulsified crude oil by bacillus thermopile BLG69 strain was tested, and the specific steps were as follows:
inoculating a mother solution inoculated by a bacillus thermopile BLG69 strain into a fermentation culture medium, wherein the inoculation amount is 1%, and the liquid loading amount of the culture medium is 10ml in a 50ml test tube; shaking and culturing in shaking table at 60 deg.C and 200rpm for 3 days; the formula of the fermentation medium is as follows:
producing water from the oil reservoir, 1000 ml; ammonium chloride, 1 g; 1.5g of sodium nitrate; dipotassium hydrogen phosphate, 0.5 g; magnesium sulfate, 0.1 g; sodium chloride, 10 g; 0.5g of yeast powder; 1g of corn steep liquor; crude oil, 50 g; the pH was 7.5.
For comparison, a blank fermentation medium not inoculated with the Bacillus thermoplasticus BLG69 strain was also placed on a shaker at 200rpm and 60 ℃ for 3 days.
As shown in FIGS. 2 and 3, the crude oil in the fermentation medium inoculated with the strain of Bacillus thermocomposted BLG69 was significantly emulsified, the viscosity was reduced, and the fluidity was improved, whereas the crude oil in the fermentation medium not inoculated with the strain of Bacillus thermocomposted BLG69 was not emulsified.
Example 5
In this example, the performance of emulsified crude oil of bacillus thermopile BLG69 strain was tested, and the difference between this example and example 4 is that: the culture temperature was 55 ℃.
Similar to the results of example 4, the crude oil in the fermentation medium inoculated with the strain of Bacillus thermocomposted BLG69 was significantly emulsified, the viscosity was reduced, and the fluidity was improved, whereas the crude oil in the fermentation medium not inoculated with the strain of Bacillus thermocomposted BLG69 was not emulsified.
Example 6
In this example, the performance of emulsified crude oil of bacillus thermopile BLG69 strain was tested, and the difference between this example and example 4 is that: the incubation temperature was 50 ℃.
Similar to the results of example 4, the crude oil in the fermentation medium inoculated with the strain of Bacillus thermocomposted BLG69 was significantly emulsified, the viscosity was reduced, and the fluidity was improved, whereas the crude oil in the fermentation medium not inoculated with the strain of Bacillus thermocomposted BLG69 was not emulsified.
Example 7
In this example, the performance of emulsified crude oil of bacillus thermopile BLG69 strain was tested, and the difference between this example and example 4 is that: the culture temperature was 45 ℃.
Similar to the results of example 4, the crude oil in the fermentation medium inoculated with the strain of Bacillus thermocomposted BLG69 was significantly emulsified, the viscosity was reduced, and the fluidity was improved, whereas the crude oil in the fermentation medium not inoculated with the strain of Bacillus thermocomposted BLG69 was not emulsified.
Example 8
In this example, the performance of emulsified crude oil of bacillus thermopile BLG69 strain was tested, and the difference between this example and example 4 is that: all media were TSA media containing crude oil, at 45 ℃ for 2 days. The formula of the TSA culture medium containing the crude oil is as follows:
sodium chloride, 5 g; soybean peptone, 5 g; casein peptone, 15 g; producing water from the oil reservoir, 1000 ml; crude oil, 50 g; the pH was 7.5.
Similar to the results of example 4, the crude oil in the medium inoculated with the strain of Bacillus thermopcompost BLG69 was significantly emulsified, the viscosity was reduced, and the fluidity was improved, whereas the crude oil in the medium not inoculated with the strain of Bacillus thermopcompost BLG69 was not emulsified.
The surface tension of the culture solution in examples 4 to 8 was 29 to 32 mN/m.
From the above examples 4-8, it can be seen that the bacillus thermopile BLG69 strain can use crude oil as a carbon source for growth and metabolism, generate a biological emulsifier during the metabolism process, emulsify the crude oil, reduce the viscosity of the crude oil, thereby facilitating the exploitation of the crude oil and increasing the recovery ratio of the crude oil.
Example 9
In this example, the emulsifying capacity of the culture solution obtained in examples 4 to 8 was measured by the following specific steps:
respectively mixing the culture solution obtained in the example 4-8 with liquid paraffin at 25 ℃, adding the mixture into a 10ml centrifuge tube, wherein the using amount of the culture solution is 4ml, the using amount of the liquid paraffin is 1ml, violently shaking for 1min, standing for 24 hours, and observing the layering condition of an emulsion layer. The results show that the emulsion layer is slightly layered, and the emulsifying power is more than 90%.
In summary, the embodiment of the invention provides a bacillus thermopile BLG69 strain which can be metabolized by taking crude oil as a carbon source to generate a biological emulsifier to emulsify the crude oil under the condition of 37-60 ℃ so as to improve the recovery rate of the crude oil. The bacillus thermopile BLG69 strain has wide application temperature range, simple culture method and high growth speed, does not need ground fermentation when being used for oil displacement, and has good application prospect in the technical field of medium-high temperature oil reservoir microbial oil recovery.
The above description is only for facilitating the understanding of the technical solutions of the present invention by those skilled in the art, and is not intended to limit the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A Bacillus thermophoresis BLG69 strain for emulsifying crude oil is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 11441.
2. The bacillus thermopastus BLG69 strain according to claim 1, wherein the 16S rRNA gene sequence of the bacillus thermopastus BLG69 strain is shown in SEQ ID No. 1.
3. The method for culturing the bacillus thermopile BLG69 strain of claim 1, comprising the steps of:
step a, placing an oil-water sample collected from an oil reservoir at 45-60 ℃ in a North China oil field in a sterilized enrichment medium, carrying out shake culture in a shaking table at 45-60 ℃ and 120-200 rpm, and separating and purifying to obtain a strain taking crude oil as a unique carbon source;
b, inoculating the strain which is obtained in the step a and takes the crude oil as the only carbon source into a fermentation medium, and carrying out shake culture for 72 hours in a shaking table with the temperature of 45-60 ℃ and the rotating speed of 120-200 rpm to obtain fermentation liquor; detecting the surface tension of the fermentation liquor, wherein the strain corresponding to the fermentation liquor with the surface tension of less than 40mN/m is the thermal compost bacillus BLG69 strain;
wherein,
in the step a, the enrichment medium comprises the following components in parts by mass:
100 parts of clear water; 8-10 parts of crude oil; 0.09-0.12 part of ammonium chloride; 0.18-0.21 part of sodium nitrate; 0.04-0.06 part of dipotassium hydrogen phosphate; 0.18-0.21 part of sodium sulfate; 0.02-0.03 part of magnesium sulfate; 0.04-0.06 part of sodium carbonate; 0.02-0.03 part of sodium acetate; 0.04-0.06 part of yeast powder; 0.09-0.10 part of corn steep liquor; the pH value of the enrichment medium is 7.5-8.0;
in the step b, the fermentation medium comprises the following components in parts by weight:
100 parts of oil reservoir produced water; 0.08-0.11 part of ammonium chloride; 0.14-0.18 part of sodium nitrate; 0.04-0.06 part of dipotassium hydrogen phosphate; 0.01-0.02 part of magnesium sulfate; 0.8-1.1 parts of sodium chloride; 0.04-0.06 part of yeast powder; 0.1-0.12 part of corn steep liquor; 5-8 parts of crude oil; the pH value of the fermentation medium is 7.5-8.0.
4. The culture method according to claim 3, wherein in the step a, the oil-water sample collected from the 55 ℃ oil reservoir in North China oil field is placed in the sterilized enrichment medium.
5. The culture method according to claim 3, wherein the amount of the fermentation medium contained in the step b is 100ml in a 250ml Erlenmeyer flask or 10ml in a 50ml test tube.
6. The use of the bacillus thermopile BLG69 strain of claim 1 for enhanced oil recovery, wherein an activator is injected into an oil reservoir containing the bacillus thermopile BLG69 strain, and oil recovery is performed after a shut-in for a predetermined period of time;
the activator comprises the following components in parts by mass:
100 parts of oil reservoir produced water; 0.08-0.11 part of ammonium chloride; 0.14-0.18 part of sodium nitrate; 0.04-0.06 part of dipotassium hydrogen phosphate; 0.01-0.02 part of magnesium sulfate; 0.8-1.1 parts of sodium chloride; 0.04-0.06 part of yeast powder; 0.1-0.12 part of corn steep liquor; 5-8 parts of crude oil.
7. The use according to claim 6, wherein the temperature of the reservoir is 37 ℃ to 60 ℃.
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