CN105624061A - Bacillus amyloliquefaciens subsp.amyloliquefaciens as well as preparation and application of solid bacterial preparation of bacillus amyloliquefaciens subsp.amyloliquefaciens - Google Patents
Bacillus amyloliquefaciens subsp.amyloliquefaciens as well as preparation and application of solid bacterial preparation of bacillus amyloliquefaciens subsp.amyloliquefaciens Download PDFInfo
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- 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
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K8/00—Compositions for drilling of boreholes or wells; Compositions for treating boreholes or wells, e.g. for completion or for remedial operations
- C09K8/58—Compositions for enhanced recovery methods for obtaining hydrocarbons, i.e. for improving the mobility of the oil, e.g. displacing fluids
- C09K8/582—Compositions for enhanced recovery methods for obtaining hydrocarbons, i.e. for improving the mobility of the oil, e.g. displacing fluids characterised by the use of bacteria
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- E21—EARTH DRILLING; MINING
- E21B—EARTH DRILLING, e.g. DEEP DRILLING; OBTAINING OIL, GAS, WATER, SOLUBLE OR MELTABLE MATERIALS OR A SLURRY OF MINERALS FROM WELLS
- E21B43/00—Methods or apparatus for obtaining oil, gas, water, soluble or meltable materials or a slurry of minerals from wells
- E21B43/16—Enhanced recovery methods for obtaining hydrocarbons
Abstract
The invention belongs to the technical field of the tertiary oil recovery for improving crude oil recovery rate by using microbes and metabolism products thereof and discloses a bacillus which is preserved in China Center for Type Culture Collection with the preservation number being CCTCC M2015740 and named as bacillus amyloliquefaciens subsp.amyloliquefaciens, a solid bacterial preparation taking bacillus amyloliquefaciens subsp.amyloliquefaciens 6-2c as an active component and a preparation method thereof. The solid bacterial preparation prepared by a liquid-solid two-step fermentation method disclosed by the invention is small in volume, easy to preserve and low in transportation cost; meanwhile, the main raw materials adopted by the preparation method are agricultural and sideline products; the raw materials are wide in sources and low in costs. The invention also discloses application of the solid bacterial preparation to degradation of crude oil or solid paraffin; the application result shows that the solid bacterial preparation simultaneously has the capabilities of producing acid, production gas and producing surface active materials, is capable of greatly reducing the adhesion of the crude oil, and serves as an oil displacement bacterium with multiple functions.
Description
Technical field
The invention belongs to the technical field of tertiary oil recovery utilizing microorganism and metabolite thereof to improve oil recovery factor, be specifically related to the preparation and application of bacillus amyloliquefaciens solution starch subspecies and solid-state bacteria preparation thereof.
Background technology
Oil is the blood of industrial economy, petroleum reserves and Relationship with Yield to national economy, social sustainable development and national defense safety. At present, by once and secondary oil recovery, oil recovery factor is about about the 30% of oil in place, still has substantial amounts of residual crude oil to need to be exploited by new technique. Utilize physical chemistry technology to improve oil recovery factor and have big quantity research and application, but prior art is to special oil reservoir poor effect such as a large amount of hypotonic, viscous crude existed.
The principal element that restriction oil recovery factor improves is: one is that the higher hydrocabon viscosity such as Colophonium in crude oil, colloid, waxiness are big, poor fluidity; Two is that crude oil is adhering closely on the porous carrier of oil reservoirs, not easily flows; Three is containing a large amount of paraffin in crude oil, and along with crude oil production, when paraffin flows from depths, stratum to top layer, along with the reduction of temperature, its coagulating sedimentation, on the borehole wall and tubing wall, forms blocking, affects yield and the pipeline transportation of oil extraction. Therefore can by high molecular component in degrading crude oil, reduce viscosity of crude, alleviate paraffin deposit blocking and improve crude oil fluidity and improve oil recovery factor.
Microbial Enhanced Oil Recovery is the biology and the physical chemistry complex art that include the complex processes such as growth of microorganism, migration and metabolism, there is multiple action and resultant effect, mainly include the fermentation of microorganism ground and the microorganism ground big class of bottom fermentation two, its ground bottom fermentation is as huge bioreactor using oil reservoir, good for compatibility inoculating microbe is injected wherein, utilizes the comprehensive function of microorganism growth and breeding and metabolite thereof in oilbearing stratum to improve recovery ratio. Ground bottom fermentation need to reservoir formation inject a large amount of microbial bacterial agents, inoculating microbe used is antibacterial, its product be liquid fermentation produce liquid bacteria preparation, it is necessary to antibacterial microbial inoculum high number. But liquid bacteria preparation can not long storage periods, volume is big, transport inconvenience, seriously constrains the extensive use on producing of the displacement of reservoir oil antibacterial.
Summary of the invention
For problems of the prior art, it is an object of the invention to provide the preparation and application of bacillus amyloliquefaciens solution starch subspecies and solid-state microbial inoculum thereof, can be used for the degraded to crude oil or paraffin, and then promote the recovery ratio of crude oil.
In order to reach object above, the present invention is achieved by the following technical solutions.
(1) one bacillus, it is isolatable from Ansai Oilfield, the crude oil of Zhidan oil field and petroleum-contaminated soil, Wuhan University of Wuhan City China typical culture collection center within 11st, it is preserved in December in 2015, preserving number is CCTCCM2015740, called after bacillus amyloliquefaciens solution starch subspecies 6-2c (Bacillusamyloliquefacienssubsp.Amyloliquefaciens6-2c).
The 16SrDNA sequence analysis of bacillus amyloliquefaciens solution starch subspecies 6-2c: reacted by PCR, from the 16SrDNA sequence of bacillus amyloliquefaciens solution starch subspecies 6-2c, amplification obtains the genetic fragment of 1.6kb, after PCR primer purification, Nanjing Genscript Biotechnology Co., Ltd. check order. By being analyzed with other bacterial strains 16SrDNA sequence in Genebank, find that the 16SrDNA sequence homology of this bacillus cereus 6-2c and Bacillusamyloliquefacienssubsp.amyloliquefaciensFN597644 reaches 99.78%, confirm that this bacillus cereus is bacillus amyloliquefaciens solution starch subspecies (Bacillusamyloliquefacienssubsp.amyloliquefaciens).
The morphological characteristic of bacillus amyloliquefaciens solution starch subspecies 6-2c: colony diameter is relatively big, white, and circular, edge is irregular, and smooth surface moistens, opaque, swells slightly up, and under Electronic Speculum, cell is elongated rod shape, wide 0.5��1.0 ��m, long 2.0��3.0 ��m.
(2) a kind of spore bacteria preparation, it is characterised in that with bacillus amyloliquefaciens solution starch subspecies 6-2c for active component.
Described spore bacteria preparation is solid-state bacteria preparation.
(3) a kind of preparation method with the bacillus amyloliquefaciens solution starch subspecies 6-2c spore bacteria preparation being active component, it is characterised in that adopt liquid-solid two-step fermentation, specifically include following steps:
Step (1), the preparation of liquid fermentation medium: liquid fermentation medium include Carnis Bovis seu Bubali cream that mass percent is 0.3%, 1% peptone, 0.5% NaCl, 1% brown sugar, all the other be tap water, pH value is natural, mixing, standby;
Step (2), the preparation of liquid seed liquor: bacillus amyloliquefaciens inclined-plane is linked into equipped with in the tissue culture bottle of liquid fermentation medium, 30 DEG C, 120r/min when, 3d cultivated by shaking table, it is thus achieved that liquid seed liquor, standby;
Step (3), the preparation of solid-state fermentation culture medium: solid-state fermentation culture medium includes wheat bran, crude oil and inorganic salt solution; According to inorganic salt solution: wheat bran: crude oil is the ratio of 50��80mL: 90��110g: 1.5��2.5g, first that wheat bran is uniform with churning up the raw oil, then add inorganic salt solution, continuation mix homogeneously, sterilizing 30min under 121 DEG C of conditions, standby;
Step (4), the preparation of solid-state bacteria preparation: the liquid seed liquor taking step (2) is transferred in the solid-state fermentation culture medium of step (3), wherein, the ratio of liquid seed liquor and solid-state fermentation culture medium is 3mL: 5g, then under 28��30 DEG C of conditions constant temperature quiescent culture 5��6d; After cultivation terminates, culture is taken out, 40 DEG C of drying, pulverize, cross 0.10mm sieve, obtain solid-state spore bacteria preparation.
Wherein, in described step (2), the cultural method on bacillus amyloliquefaciens inclined-plane is: be inoculated in beef extract-peptone agar (composition is known) slant medium by bacillus amyloliquefaciens solution starch subspecies 6-2c, cultivates 48h, to obtain final product for 28��30 DEG C.
Wherein, in described step (3), described inorganic salt solution includes the composition of following mass fraction: 0.2%NaNO3, 0.1% (NH4)2SO4, 0.03%MgSO4��7H2O, 0.5%KH2PO4, 1%K2HPO4��3H2O, 0.5%NaCl, all the other are tap water, and pH value is natural.
The viable count utilizing spore bacteria preparation prepared by the method is 1.77 �� 1010CFU/g��
(4) present invention also offers the application in crude oil or hard paraffin are degraded of the above-mentioned spore bacteria preparation.
The application in crude oil or hard paraffin are degraded of the above-mentioned spore bacteria preparation, it is characterized in that, using method is as follows: solid-state spore bacteria preparation is joined in the tap water of 30��35 DEG C by the ratio of 1: 30 in mass ratio, vibration 0.5h, glass cotton filters, and gained filtrate is solid-state Bacteria dilution agent bacteria suspension; This dilution bacteria suspension is mixed homogeneously by 15: 1 with crude oil or solid paraffin, 40 DEG C of reaction 96h.
The application in crude oil or hard paraffin are degraded of the above-mentioned spore bacteria preparation, it is characterized in that, using method is as follows: solid-state spore bacteria preparation is joined in the tap water of 30��35 DEG C by the ratio of 1: 30 in mass ratio, mixing, stirring, filtration, gained filtrate is for examination solid-state Bacteria dilution agent bacteria suspension; This dilution bacteria suspension and water are mixed homogeneously injection oil well by 1: 100��200, with the solid paraffin that the borehole wall of degrading condenses; Maybe this dilution bacteria suspension and water are mixed homogeneously injection oil reservoir by 1: 200��500, with degrading crude oil.
Compared with prior art, the invention have the advantages that
(1) in the existing technology utilizing microorganism to improve oil recovery factor, all use liquid culture, however liquid bacteria preparation can not long storage periods, volume is big, transport inconvenience, seriously constrains the extensive use on producing of the displacement of reservoir oil antibacterial. The present invention adopts liquid-solid two-step fermentation to prepare solid-state bacteria preparation, and prepared bacteria preparation volume is little, easy preservation, and cost of transportation is low, and during use, dilute filters, and fundamentally solves the problems referred to above.
(2) preparation method of solid-state bacteria preparation of the present invention, the primary raw material of employing is agricultural byproducts, and raw material sources are extensive, cheap, and the practical application improving oil recovery factor technology for microorganism provides feasible scheme.
(3) have now found that bacillus amyloliquefaciens plant subspecies (B.amyloliquefacienssubsp.plantarum) has controlling plant diseases, promotes plant growing function, but do not find that it has the function of degrading crude oil and paraffin. The present invention proposes bacillus amyloliquefaciens solution starch subspecies 6-2c application in crude oil or hard paraffin are degraded, it has the ability producing acid aerogenesis and product surfactant simultaneously, the tack of crude oil can be greatly lowered, be the strain displacement of reservoir oil antibacterial with several functions.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is described in further details.
Fig. 1 is colonial morphology and the cell microscopic morphology figure of bacillus amyloliquefaciens solution starch subspecies 6-2c.
Fig. 2 is the phyletic evolution tree graph of bacillus amyloliquefaciens solution starch subspecies 6-2c.
Fig. 3 is that bacillus amyloliquefaciens solution starch subspecies 6-2c solid-state bacteria preparation processes solid paraffin solute effect comparison diagram in normal hexane. Wherein, a figure left side is that blank organizes distilled water to the deliquescent effect of solid paraffin, and the figure right side is that bacillus amyloliquefaciens solution starch subspecies 6-2c solid-state bacteria preparation dilution bacterium solution is to the deliquescent effect of solid paraffin.
Fig. 4 is the bacillus amyloliquefaciens solution starch subspecies 6-2c solid-state bacteria preparation microbial inoculum dilution bacterium solution desorption effect comparison diagram to Adsorption of Filter Paper crude oil. Wherein, a figure left side is blank group effect, schemes right dilution bacterium solution desorption test group effect.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further details, but following example are not intended as limiting the scope of the invention.
The qualification of embodiment 1 strain
(1) morphological characteristic
Colony diameter is relatively big, white, and circular, edge is irregular, and smooth surface moistens, opaque, swells slightly up, and under Electronic Speculum, cell is elongated rod shape, wide 0.5��1.0 ��m, and long 2.0��3.0 ��m, concrete form is Fig. 1 such as.
(2) 16SrDNA sequence analysis
Being reacted by PCR, from the 16SrDNA sequence of bacillus amyloliquefaciens solution starch subspecies 6-2c, amplification obtains the genetic fragment of 1.6kb, after PCR primer purification, Nanjing Genscript Biotechnology Co., Ltd. checks order. By being analyzed with other bacterial strains 16SrDNA sequence in Genebank, find that the 16SrDNA sequence homology of this bacillus cereus 6-2c and Bacillusamyloliquefacienssubsp.amyloliquefaciensFN597644 reaches 99.78%, confirm that this bacillus cereus is bacillus amyloliquefaciens solution starch subspecies (Bacillusamyloliquefacienssubsp.amyloliquefaciens). Systematic evolution tree such as Fig. 2 of bacillus amyloliquefaciens solution starch subspecies 6-2c.
16SrDNA expands sequencing result such as shown in SEQIDNo.1, as follows:
CGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCC
The preparation of embodiment 2 solid-state spore bacteria preparation
Adopt liquid-solid two-step fermentation to cultivate, specifically include following steps:
Step (1), the preparation of liquid fermentation medium: Carnis Bovis seu Bubali cream 3.00g, peptone 10.00g, NaCl5.00g, brown sugar 10.00g, tap water 1000mL, pH value is natural, mixing, standby;
Step (2), the preparation of liquid seed liquor: bacillus amyloliquefaciens solution starch subspecies 6-2c inclined-plane is linked into equipped with in the 600mL tissue culture bottle of 100mL liquid fermentation medium, 30 DEG C, 120r/min when, 3d cultivated by shaking table, obtain liquid seed liquor, standby;
Wherein, the cultural method on bacillus amyloliquefaciens inclined-plane is: be inoculated in slant medium by bacillus amyloliquefaciens solution starch subspecies 6-2c, cultivates 48h, to obtain final product for 30 DEG C.
Step (3), the preparation of solid-state fermentation culture medium: include wheat bran, crude oil and inorganic salt solution, first that 100g wheat bran is uniform with 2g churning up the raw oil, then add 60mL inorganic salt solution, and continuation mix homogeneously, sterilizing 30min under 121 DEG C of conditions, standby; Wherein, inorganic salt solution includes NaNO32.00g��(NH4)2SO41.00g��MgSO4��7H2O0.30g��KH2PO45.00g��K2HPO4��3H2O10.00g, NaCl5.00g and tap water 1000mL, pH value is natural.
Step (4), the preparation of solid-state bacteria preparation: the liquid seed liquor taking 30mL step (2) is transferred in the solid-state fermentation culture medium of 50.00g step (3), wherein, the ratio of liquid seed liquor and solid-state fermentation culture medium is 3mL: 5g, then under 0 DEG C of condition constant temperature quiescent culture 5d; After cultivation terminates, being taken out by culture, 40 DEG C of drying, pulverize, cross 0.10mm sieve, obtain solid-state spore bacteria preparation, its bacterium number is 1.77 �� 1010CFU/g��
The application of embodiment 3 solid-state spore bacteria preparation
A solid-state spore bacteria preparation consumption and usage
(1) preparation of solid-state spore bacteria preparation diluent bacteria suspension: weigh 1.500g solid-state spore bacteria preparation, the ratio in 1: 30 adds 45mL distilled water, in 30 DEG C, vibration 0.5h in the shaking table of 120r/min, then with the filtration of 0.500g glass cotton, to obtain final product.
(2) crude oil or hard paraffin degraded: add 2.000g crude oil in the thin mouth vial of 100mL, add 30mL solid-state spore bacteria preparation diluent bacteria suspension, adds bottle closure of rubber and seals the reinforcing of blend compounds band, in case pressure of the inside of a bottle increases washes bottle stopper open. Standing and reacting 96h under 40 DEG C of conditions, shakes 1 time for every 4 hours.
This test to add 30mL distilled water for matched group in the thin mouth vial of 100mL.
After above-mentioned test reaction terminates, after measuring the degraded of solid-state spore bacteria preparation, in crude oil, saturated hydrocarbons, aromatic hydrocarbon, colloid and asphalt content change; Viscosity of crude is affected by solid-state spore bacteria preparation; Degrading crude oil process is produced acid, aerogenesis quantification and qualification.
Replacing crude oil to repeat above incubation with hard paraffin, question response measures hard paraffin dissolubility in normal hexane and produces acid gas production and composition after terminating, its solute effect is as shown in Figure 3.
The application result of B solid-state spore bacteria preparation
(1) the solid-state spore bacteria preparation degraded to crude oil
1. to the degraded of saturated hydrocarbons, aromatic hydrocarbon, resin and asphalt in crude oil: degradation results is as shown in table 1, as can be seen from Table 1, solid-state spore bacteria preparation diluent bacteria suspension is to saturated hydrocarbons, aromatic hydrocarbon, colloid and bitum degradation rate in crude oil respectively 15.1%, 23.7%, 24.2% and 56.2%, and solid-state spore bacteria preparation diluent bacteria suspension degradation treatment all reaches significant level (P < 0.05) with compareing the difference of 4 kinds of components in crude oil.
The table 1 solid-state spore bacteria preparation diluent bacteria suspension degradation rate in crude oil 4 kinds of components
2. the dissolubility to Crude viscosity and paraffin: result is table 2 such as, from table 2 it can be seen that when 30 DEG C, after solid-state spore bacteria preparation diluent bacteria suspension degrading crude oil, viscosity of crude relatively compares viscosity of crude and reduces by 29.4%; When 40 DEG C, relatively comparison reduction by 26.5%. Hard paraffin is after solid-state spore bacteria preparation diluent bacteria suspension is degraded, there is notable change in the dissolubility in normal hexane, solubilized paraffin amount has reached the 77.2% of paraffin total amount, crystallization paraffin amount is the 20.7% of paraffin total amount, result is shown in Fig. 3, it is shown that this solid-state spore bacteria preparation diluent bacteria suspension can reduce viscosity of crude, and paraffin solvability in normal hexane can be increased substantially, increase the little molecule paraffin amount that normal hexane is solvable.
Crude viscosity and paraffin dissolubility are affected by table 2 solid-state Bacteria dilution agent bacteria suspension
3. gas production crude oil and hard paraffin degraded and gas componant: having gas to produce in solid-state spore bacteria preparation diluent bacteria suspension degrading crude oil and hard paraffin process, through gas chromatographic analysis institute, aerogenesis body is mainly CO2And H2. As seen from Table 3, in degradation process, total gas production reaches 69.0��70.0mL/ bottle, CO2Yield be 24.0��24.3mL/ bottle, H2Being 45.0��45.7mL/ bottle, factor of created gase is 230.0%��233.3%.
Aerogenesis kind and gas production in table 3 solid-state antibacterial microbial inoculum bacterium dilution bacteria suspension degradation process
4. product acid amount crude oil and hard paraffin degraded and acid composition: as seen from Table 4, after solid-state spore bacteria preparation diluent bacteria suspension is degraded, reactant liquor pH relatively compares decline 36.0%��36.8%, and total acid content is 25.8��26.0mmol/L, and acid number reaches 1548��1560mg/L. Through organic acid chromatography, the short chain organic acid of generation is mainly oxalic acid and propanoic acid.
Culture fluid pH and product acid when table 4 solid-state Bacteria dilution agent bacteria suspension degrading crude oil and hard paraffin are measured
5. solid-state bacteria preparation dilutes degreasing activity and the desorption of bacteria suspension: as seen from Table 5, this solid-state spore bacteria preparation diluent bacteria suspension has certain degreasing activity, and oil extraction loop diameter is 18.9cm, for 63.0 times of comparison; The crude oil of attachment on filter paper is had good desorption by this solid-state spore bacteria preparation diluent bacteria suspension, in the desorption test being adsorbing medium with filter paper processes, the crude oil of 94.20% splits away off from filter paper, the amount of coming off is 8.37 times of comparison, reclaim solid-state spore bacteria preparation diluent bacteria suspension liquid and still crude oil is had the desorption rate of 92.47%, little for solid-state spore bacteria preparation diluent bacteria suspension difference with brand-new, result is shown in Fig. 4, it was shown that solid-state spore bacteria preparation diluent bacteria suspension has good desorption and recycling is worth.
Table 5 solid-state bacteria preparation dilution bacteria suspension oil extraction loop diameter and desorption of crude oil rate
Above example result shows, the bacillus amyloliquefaciens solution starch subspecies 6-2c of the present invention is the multi-functional displacement of reservoir oil antibacterial that 1 strain is new, there are following 4 kinds of functions: 1. Degradation: alkane in degradable crude oil, aromatic hydrocarbon, paraffin, colloid and Colophonium, the heavy component in crude oil is made to be decomposed into light components, reduce Crude viscosity, the macromolecular components that minimizing can deposit, is effectively improved the mobility of crude oil, it is prevented that paraffin deposit blocking pipeline and the borehole wall. 2. aerogenesis: microbial inoculum produces a large amount of CO in degrading crude oil and paraffin process2And H2, these gases can increase oil reservoir internal pressure, and gas dissolves in crude oil can reduce Crude viscosity, improves crude oil fluidity. 3. acid is produced: microbial inoculum produces the organic acid such as a large amount of oxalic acid, propanoic acid in degrading crude oil and paraffin process, can effectively dissolve the carbonate in oil reservoir rock hole, increase the porosity and permeability of oil reservoir, the interfacial tension between profit can also be reduced simultaneously, form oil-water emulsion, thus improving oil recovery factor. 4. surfactant is produced, contribute to desorption of crude oil, the paraffinic components in crude oil is produced certain emulsifying, peptizaiton simultaneously, make wax recovery attenuate and diminish and flow out oil well with Produced Liquid, alleviate or eliminate wax deposit blocking, and then improving oil recovery factor and pipeline transportation effect.
Although, in this specification, the present invention is described in detail with a general description of the specific embodiments, but on basis of the present invention, it is possible to it is made some modifications or improvements, and this will be apparent to those skilled in the art. Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. a bacillus, it is characterized in that, being preserved in China typical culture collection center, preserving number is CCTCCM2015740, called after bacillus amyloliquefaciens solution starch subspecies (Bacillusamyloliquefacienssubsp.amyloliquefaciens) 6-2c.
2. bacillus amyloliquefaciens solution starch subspecies 6-2c according to claim 1, it is characterised in that the 16SrDNA sequence of described bacillus amyloliquefaciens solution starch subspecies 6-2c is such as shown in SEQIDNo.1.
3. a spore bacteria preparation, it is characterised in that with bacillus amyloliquefaciens solution starch subspecies 6-2c for active component.
4. spore bacteria preparation according to claim 3, it is characterised in that described spore bacteria preparation is solid-state bacteria preparation.
5. the preparation method of a spore bacteria preparation according to claim 3, it is characterised in that comprise the following steps:
Step (1), the preparation of liquid fermentation medium: liquid fermentation medium include Carnis Bovis seu Bubali cream that mass percent is 0.3%, 1% peptone, 0.5% NaCl, 1% brown sugar, all the other be tap water, pH value is natural, mixing, standby;
Step (2), the preparation of liquid seed liquor: bacillus amyloliquefaciens inclined-plane is linked into equipped with in the tissue culture bottle of liquid fermentation medium, 30 DEG C, 120r/min when, 3d cultivated by shaking table, it is thus achieved that liquid seed liquor, standby;
Step (3), the preparation of solid-state fermentation culture medium: solid-state fermentation culture medium includes wheat bran, crude oil and inorganic salt solution; According to inorganic salt solution: wheat bran: crude oil is the ratio of 50��80mL: 90��110g: 1.5��2.5g, first that wheat bran is uniform with churning up the raw oil, then add inorganic salt solution, continuation mix homogeneously, sterilizing 30min under 121 DEG C of conditions, standby;
Step (4), the preparation of solid-state bacteria preparation: the liquid seed liquor taking step (2) is transferred in the solid-state fermentation culture medium of step (3), wherein, the ratio of liquid seed liquor and solid-state fermentation culture medium is 3mL: 5g, then under 28��30 DEG C of conditions constant temperature quiescent culture 5��6d; After cultivation terminates, culture is taken out, 40 DEG C of drying, pulverize, cross 0.10mm sieve, obtain solid-state spore bacteria preparation.
6. the preparation method of spore bacteria preparation according to claim 5, it is characterized in that, in step (2), the cultural method on bacillus amyloliquefaciens inclined-plane is: be inoculated in beef extract-peptone agar slant culture-medium by bacillus amyloliquefaciens solution starch subspecies 6-2c, cultivate 48h, to obtain final product for 28��30 DEG C.
7. the preparation method of spore bacteria preparation according to claim 5, it is characterised in that in step (3), described inorganic salt solution includes the composition of following mass fraction: 0.2%NaNO3, 0.1% (NH4)2SO4, 0.03%MgSO4��7H2O, 0.5%KH2PO4, 1%K2HPO4��3H2O, 0.5%NaCl, all the other are tap water, and pH value is natural.
8. the application in crude oil or hard paraffin are degraded of the spore bacteria preparation described in claim 3 or 4.
9. the spore bacteria preparation according to claim 8 application in crude oil or hard paraffin are degraded, it is characterized in that, using method is as follows: solid-state spore bacteria preparation is joined in the tap water of 30��35 DEG C by the ratio of 1: 30 in mass ratio, vibration 0.5h, glass cotton filters, and gained filtrate is solid-state spore bacteria preparation diluent bacteria suspension; This bacteria suspension and crude oil or solid paraffin being mixed homogeneously at 15: 1 in mass ratio, 40 DEG C of reactions 96h, every 3��5h shake once.
10. the spore bacteria preparation according to claim 8 application in crude oil or hard paraffin are degraded, it is characterized in that, using method is as follows: solid-state spore bacteria preparation is joined in the tap water of 30��35 DEG C by the ratio of 1: 30 in mass ratio, mixing, stirring, filtration, gained filtrate is for examination solid-state Bacteria dilution agent bacteria suspension; This dilution bacteria suspension and water are mixed homogeneously injection oil well by 1: 100��200, with the solid paraffin that the borehole wall of degrading condenses; Maybe this dilution bacteria suspension and water are mixed homogeneously injection oil reservoir by 1: 200��500, with degrading crude oil.
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