CN101914471B - Bacillus engineering bacteria and application thereof in oil deposit tracking and oil extraction - Google Patents

Bacillus engineering bacteria and application thereof in oil deposit tracking and oil extraction Download PDF

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CN101914471B
CN101914471B CN2010102350887A CN201010235088A CN101914471B CN 101914471 B CN101914471 B CN 101914471B CN 2010102350887 A CN2010102350887 A CN 2010102350887A CN 201010235088 A CN201010235088 A CN 201010235088A CN 101914471 B CN101914471 B CN 101914471B
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oil
bacillus
engineering bacteria
bacteria
bacterial
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CN101914471A (en
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李尔炀
蔡志强
杨广花
李亮
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CHANGZHOU WOOLEN TOPS FACTORY Co Ltd
Changzhou University
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CHANGZHOU WOOLEN TOPS FACTORY Co Ltd
Changzhou University
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Abstract

The invention relates to bacillus engineering bacteria (Bacillus sp.). The bacillus engineering bacteria are characterized in that: the bacterial strain is preserved in the CGMCC on July 2nd, 2010, and the collection number is CGMCC No.3767; the bacillus engineering bacteria belong to bacillus, and the bacterial strain LEY11 is Gram's strain; the bacteria are good facultative aerobic bacteria and are rod-like; according to the body size, the length is 2.5 to 3.5 microns, and the width is 0.6 micron; and a milk white bacterial colony is formed on a bacterial culture medium, and the bacterial colony is circular or oval or terminal spore. The bacillus engineering bacteria can be applied to microbial flooding in oil extraction. The bacillus engineering bacteria can be applied to oil well tracking. The pollution of an oilfield chemical tracking agent to the environment and the crude oil is improved, and the yield of crude oil of an oilfield is improved.

Description

Bacillus engineering bacteria and in the application of oil deposit tracking and oil recovery
Technical field
The present invention relates to a kind ofly can be applied to oil deposit tracking and recover the oil dual-purpose bacillus engineering bacteria, belong to mikrobe recover the oil and the oil deposit tracking method on application.
Background technology
Microbe oil production is that useful activity such as degrading crude oil or meta-bolites such as the bio-surfactant, organic acid, the alcohol that utilize mikrobe self wait and improves crude oil fluidity, and reaches the oil recovery technique that improves RF.
At present, the oil mass of oil production common process technology extraction only accounts for the 1/3-1/2 of original oil in place, and the technology (MEOR) of using microbe raising oil recovery is 3 intensified oil reduction technology with good development prospect.The bacterial classification that is used for microbe oil production must adapt to the formation condition of oil reservoir, so the different condition oil reservoir must adopt the bacterial classification of different qualities, this is that mechanism of action by MEOR determines.
The oil well tracer technique is in order to investigate the permeability of rock stratum between WIW and the producing well, generally to adopt water-soluble chemical tracer agent or ri; Advance to inject from water filling, detect tracer concentration, thereby judge the situation that is communicated with between oil well and the WIW from adjacent oil well water sampling; And the rate of permeation and the duct radius of each substratum of calculating oil well; The oil well tracer technique adopts chemical tracer, can cause certain pollution to oil well and environment, and subsequent disposal is difficult.
The present invention has selected the Zhongyuan Oil Field, lasts 4 years, has carried out screening, cultivation, evaluation, the assignment of genes gene mapping and the clone of microbe oil production bacterial classification, polygenic expression, finally obtains microbe oil production, the dual-purpose bacterium of spike.The present invention adopts the oil well endogenous microbes; Selectivity clone foreign gene degraded n-hexadecane gene, degraded naphthyl are because of, produced high-temperature amylase and bio-surfactant gene H-1 and N-3; Be a kind of environment amenable endogenous tracer agent, do not have any pollution and toxic side effect.
Summary of the invention
The present invention is directed to the deficiency that above-mentioned prior art exists; Make up a strain oil deposit tracking, oil recovery dual-purpose engineering bacteria LEY11 (Bacillus sp.); This bacterial strain has been preserved in the Chinese common micro-organisms culture presevation administrative center (CGMCC) that the address is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on July 2nd, 2010; Deposit number is CGMCC No.3767, belongs to bacillus.
Bacterial strain LEY11 of the present invention is a gram, and facultative property aerobic bacteria is shaft-like, individual size, and length is 2.5～3.5 μ m, and wide is 0.6 μ m, on bacteria culture medium, forms the oyster white bacterium colony, and bacterium colony is circular; Avette, terminal spore.
This bacterial strain is a strain transgenic microorganism that adopts the polygene transformation technology to be built into.Donor is: 10# bacterial strain, high temperature alkane genus bacillus (Bacillus thermoleovorans 10#) and N-3 bacterial strain, high temperature alkane genus bacillus (Bacillusthermoleovorans N-3).Acceptor is: 1-1 bacterial strain, genus bacillus (Bacillus sp.1-1); Operate according to the relevant chapters and sections in " polygene transformation technology " (Li Eryang, polygene transformation technology [M], Chemical Industry Press, Beijing, 2006.8).
Engineering bacillus growth temperature range: 45 ℃-71 ℃; Optimum growth temperature scope: 55 ℃-65 ℃; Ph optimum is 6.5-8.0.
The used substratum of engineering bacillus is: Carnis Bovis seu Bubali cream 0.5g, peptone 1.0g, NaCl 0.5g, agar 1.8-2.0g, water 100ml.
Engineering bacillus can be used LEY of the present invention in the application of recovering the oil 11Engineering bacteria carries out microbial oil displacement.
Engineering bacillus can be used oil deposit tracking LEY of the present invention in the application of oil well spike 11Bacterium carries out the oil well spike.
Advantage of the present invention, engineering bacteria LEY 11Can improve of the pollution of oilfield chemistry tracer agent simultaneously to the oil field displacement of reservoir oil and spike, improve the output of crude oil environment and crude oil.
Description of drawings
MM+N among Fig. 1: for the naphthalene being the detection flat board of sole carbon source.LEY 11Growth above that; MM+Hex: for the n-hexadecane being the detection flat board of sole carbon source.LEY 11Growth above that.
MM+amy among Fig. 2: for being the detection flat board of sole carbon source with starch, develop, prove LEY through iodine liquid 11High temperature shallow lake enzyme is produced in growth simultaneously above that.
Fig. 3. engineering bacteria LEY 11The bio-surfactant H that produces -1Infrared spectrum;
Fig. 4. engineering bacteria LEY 11The bio-surfactant N that produces -3Infrared spectrum; Can confirm bio-surfactant H according to ir spectra -1And N -3Be lipopeptide compound.
Embodiment
Embodiment one: this experiment method oil deposit tracking engineering bacteria belongs to bacillus, and bacterial strain LEY11 is a gram, facultative aerobe; Shaft-like, individual size, length is 2.5～3.5 μ m; Wide is 0.6 μ m, on bacteria culture medium, forms the oyster white bacterium colony, and bacterium colony is circular; Avette, terminal spore.Functions such as oil deposit tracking of the present invention, the dual-purpose engineering bacteria LEY11 that recovers the oil carry five kinds of foreign genes, have degraded n-hexadecane, degraded naphthalene, produce alpha-amylase, bio-surfactant H-1 and N-3, and be high temperature resistant.
Table 1. high temperature bacterium proterties table
Figure BSA00000202820100021
The cultural characteristic of table 2. on bacteria culture medium
Figure BSA00000202820100031
Cultivate used substratum
1. perfect medium (100mL)
Peptone 1g; Carnis Bovis seu Bubali cream 0.5g; NaCl 0.5g; PH 7.0-7.2; Agar 2.0g.
2. basic medium (100mL)
K2HPO4 0.57g; KH2PO4 0.17g; NH4Cl 0.214g; Salts solution 1mL;
Agar 2.0g.
Salts solution (1000mL)
MgSO4?19.5g；MnSO4·H2O?5g；FeSO4·7H2O?5g；CaCl2·2H2O?0.3g。
3. the high substratum (100mL) that oozes
Peptone 1g; Carnis Bovis seu Bubali cream 0.5g; NaCl 3.2g; PH 7.0-7.2; Agar 2.0g.Transgeneic procedure is following in the engineering bacillus building process:
1. material
1.1. bacterial strain:
Donor bacterium: 10# bacterial strain, high temperature alkane genus bacillus (Bacillus thermoleovorans 10#).
The N-3 bacterial strain, high temperature alkane genus bacillus (Bacillus thermoleovorans N-3).
Recipient bacterium: 1-1 bacterial strain, genus bacillus (Bacillus sp.1-1).
1.2. substratum
4. perfect medium (100mL)
Peptone 1g; Carnis Bovis seu Bubali cream 0.5g; NaCl 0.5g; PH 7.0-7.2; Agar 2.0g.
5. basic medium (100mL)
K2HPO4 0.57g; KH2PO4 0.17g; NH4Cl 0.214g; Salts solution 1mL;
Agar 2.0g.
Salts solution (1000mL)
MgSO4?19.5g；MnSO4·H2O?5g；FeSO4·7H2O?5g；CaCl2·2H2O?0.3g。
6. the high substratum (100mL) that oozes
Peptone 1g; Carnis Bovis seu Bubali cream 0.5g; NaCl 3.2g; PH 7.0-7.2; Agar 2.0g.
1.3. medicine
The phenol analytical pure, the chloroform analytical pure,
The absolute ethyl alcohol analytical pure, the PEG6000 analytical pure,
Sodium lauryl sulphate (SDS) analytical pure, the ether analytical pure,
The N,O-Diacetylmuramidase analytical pure, the EDTA analytical pure,
The RNase analytical pure,
2. method
2.1DNA extraction, separation and purifying
2.1.1 solution
(1) sucrose damping fluid sucrose 10g/100mL, Tris 50mmol/L, pH 8.0.
(2) SDS liquid SDS 10g/100mL, Tris 50mmol/L, pH 8.0.
(3) lysate N,O-Diacetylmuramidase 10mg/mL prepares with the sucrose damping fluid.
(4) sodium-chlor liquid NaCl 5mol/L.
(5) phenol/chloroform solution phenol: chloroform=1: 1.
(6) TE liquid Tris 10mmol/L, EDTA 1mmol/L, pH 8.0.
(7) RNase liquid RNase 100 μ g/mL prepare with TE liquid.
(8) chloroform (9) ether (analytical pure) (10) ethanol (analytical pure) [11] saline water 0.85g NaCl is dissolved in the 100mL zero(ppm) water.
2.1.2DNA extraction, separation and purifying (DNA that from donor, extracts has those? Its dna sequence dna? All at " polygene transformation technology " (Li Eryang; Polygene transformation technology [M]; Chemical Industry Press, Beijing, 2006.8) earlier donor bacterium switching slant activation is cultivated; And then, put 60 ℃ of shaking table overnight cultures with in the ring thalline access 50ml completely liq substratum.Get culture 20ml, under 6000r/min, collected thalline in centrifugal 10 minutes, abandon supernatant, open deposition, and wash twice with saline water; Deposition is opened, and adds sucrose damping fluid 0.1mL and mixes, and adds the 2mL lysate again, and it is even to be mixed, and puts 30 ℃ of water-baths 10 minutes; Taking-up adding SDS liquid 0.1mL is mixed even, puts ice-water bath 10 minutes, adds 5mol/l NaCl liquid 0.55ml again, and it is even to be mixed, and puts ice-water bath 40 minutes; Use phenol/chloroform solution deproteinated then, add isopyknic phenol/chloroform solution, it is even to be mixed, centrifugal 10 minutes of 6000r/min; Upper strata water clear liquid is got in the liquid in pipe layering, changes another centrifuge tube over to, adds equal-volume phenol/chloroform solution again.Repeat the operation of above-mentioned deproteinated repeatedly, till water and organic phase interface do not have albumen.Slough proteic aqueous phase, adding the cold ethanol of two volumes, be mixed and put into refrigerator (20 ℃) freezing one hour after even, taking out 6000r/min centrifugal 10 minutes, collecting precipitation; Abandon supernatant, add RNase liquid 0.1mL, treat resolution of precipitate after, put 30 ℃ of water bath heat preservations 40 minutes, take out then and add equal-volume phenol/chloroform; It is even to be mixed, centrifugal 10 minutes of 6000r/min, deproteinated once, supernatant adds the equal-volume chloroform again; It is even to be mixed, centrifugal 10 minutes of 6000r/min, and dephenolize once goes to supernatant in another centrifuge tube again, adds ether dephenolize, chloroform; It is even to be mixed, and centrifugal 10 minutes of 6000r/min changes water over to another centrifuge tube, again centrifuge tube is put into 37 ℃ of water bath heat preservations 10 minutes, takes off ether; After insulation finishes, take out the cold ethanol that in centrifuge tube, adds two volumes again, be mixed and put after even under the refrigerator-20 ℃, be incubated 1 hour; Took out 6000r/min centrifugal 10 minutes, and abandoned supernatant, deposition is with the dissolving of 0.5ml TE liquid, and it is subsequent use to put refrigerator after the dissolving.
2.2 the preparation of recipient bacterium spheroplast, conversion and regeneration
2.2.1 solution
[1] NSM solution: NaCl 0.55mol/L, sodium succinate 0.2mol/L, MgCl26H2O 0.02mol/L, pH 6.7.
[2] PEG solution PEG6000 0.3g/mL (with the preparation of NSM liquid).
[3] lysate N,O-Diacetylmuramidase 5mg/mL (with the preparation of NSM liquid).
[4] CaCl2 liquid CaCl2 liquid concentration is 1mol/L.
2.2.2 the preparation of recipient bacterium spheroplast, conversion and regeneration
The 1-1 bacterial strain inserts in the 50mL perfect medium, and 60 ℃ of following shaking table overnight cultures are got the centrifugal collection thalline of culture 1mL, and thalline is washed twice with saline water; Add NSM liquid 400 μ L then, lysate 600 μ L are put 30 ℃ of water bath heat preservations after being mixed, and make it to ooze at height that (NSM) enzymolysis takes off wall in the environment; The centrifugal supernatant of abandoning of low speed (about 2500 rev/mins) adds PEG6000 solution 800 μ L in spheroplast then, adds dna solution 100 μ L (from three donors, the mixing solutions of the DNA of extraction) again; Add CaCl2 liquid 100 μ L again, be mixed, put into 30 ℃ of water-bath insulations and carried out the conversion of DNA in 10 minutes, after the conversion; Insert in the hypertonic liquid substratum and carry out regenerating and culturing, after 24 hours, get the 1mL culture, centrifugal 10 minutes of 6000r/min; Abandon supernatant, open deposition, wash twice with saline water, (the 100mL basic medium adds n-Hexadecane 0.1g to be applied to selection with every ware 0.1mL then; Naphthalene 0.1g) flat board is put 60 ℃ of incubators and was cultivated 4-5 days, grows bacterium colony on the flat board and is transformant.
2.2.3 transformant detects and stable
With the regeneration bacterium colony that obtains; On the basic medium that with n-hexadecane and naphthalene is sole carbon source, rule respectively; Separate single bacterium colony, with the repeated screening on the basic medium that with n-hexadecane, naphthalene and starch is sole carbon source respectively of these single bacterium colonies, obtain engineering bacteria LEY11 at last again.Engineering bacteria LEY11 can utilize n-hexadecane, naphthalene and starch growth, produces two kinds of different bio-surfactants (H-1 and N-3) and alpha-amylase simultaneously.
3. engineering bacteria LEY11 proterties
3.1 growth temperature proterties and pH
Growth temperature range: 45 ℃-71 ℃.
Optimum growth temperature scope: 55 ℃-65 ℃.
Ph optimum is 6.5-8.0
3.2 utilize the carbon source proterties
Need not any growth factor, can be sole carbon source and energy growth with n-hexadecane, naphthalene, starch or glucose, can in the minimal medium that only contains n-hexadecane, naphthalene, starch or glucose (phosphoric acid salt, inorganic nitrogen and trace element), grow.
3.3. produce thing tensio-active agent and alpha-amylase
Can under the inducing of n-hexadecane or naphthalene, produce two kinds of different biology table promoting agent H-1 and N-3 respectively.Under the inducing of starch, can produce alpha-amylase.
3.3 engineering bacteria LEY11 proterties characterizes
3.3.1 utilize n-hexadecane and naphthalene proterties
Embodiment 1: this embodiment oil deposit tracking bacterium is to adopt genetically engineered, and fermentative medium formula is: Carnis Bovis seu Bubali cream 0.5g, peptone 1.0g, NaCl 0.5g, agar 1.8-2.0g, water 100ml.
The application that bacillus engineering bacteria is recovering the oil: use LEY of the present invention 11Engineering bacteria carries out " microbial oil displacement "; Step is following: 1. select the water flood recovery block of a suitable microbial oil displacement, the condition of the water flood recovery block that this enforcement selective bottom-up is selected is: the oil reservoir degree of depth is 400～2500m, temperature＜70 ℃, salinity＜30000mg/L, rate of permeation＞0.11u m 2, Residual oil saturation ratio＞27%, the oily content of wax＞30%, contain glue＞25%, output liquid moisture>=80%, individual well day produce oil≤5 tons;
2. time-out displacement of reservoir oil by filling water is with LEY 11It is inner that fermented liquid injects oil reservoir, and its consumption can be estimated by the volume of voids in this oil well oil reservoir inside;
3. close well head after 7～15 days, recover former displacement of reservoir oil by filling water work, but can not add any sterilant;
4. detected the pH value once annotate output liquid behind the bacterium, surface tension, bacteria concentration, and project such as oil yield at least in every month, with inject the oil deposit tracking bacterium before data relatively, reach and follow the tracks of the purpose that detects;
5. wait petroleum production to return to the preceding level of microbial flooding, a collection of new fermented liquid more as stated above can reinject.
Bacillus engineering bacteria is in the application of oil well spike: with oil deposit tracking LEY of the present invention 11Bacteria preparation carries out the oil well spike, and step is following:
1. select a bite oil well, the oily ingoing silver spare that the practical implementation condition is as described above;
2. estimate the biomass of injection according to the volume of voids in this oil well and oil well oil reservoir inside that communicates with it;
3. with clear water that the thalline injection oil well of q.s is inner;
4. characteristics and the residing environment according to oil well calculates the time of water injection manifold to the oil well that communicates with it, after this time, carries out the oil well sampling;
5. multiplication culture, and broken wall detects the electrophoresis detection intracellular protein, if intracellular protein is consistent with the standard protein electrophoresis band, explains that then this oil well communicates with the oil well that injects thalline; If inconsistent, then explanation does not communicate.

Claims (2)

1. genus bacillus (Bacillus sp.) LEY11, deposit number is CGMCC No.3767.
2. the described genus bacillus of claim 1 can be used engineering bacillus and carry out microbial oil displacement in the application of recovering the oil.
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CN102399847A (en) * 2011-07-29 2012-04-04 常州大学 Method for preparing lipopeptid biosurfactant by utilizing high temperature Bacillus spp
CN106434798A (en) * 2016-08-30 2017-02-22 江苏金茂源生物化工有限责任公司 Method for preparing biological flocculant from high-temperature-resistant bacillus subtilis and application of method

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CN101412980A (en) * 2008-12-01 2009-04-22 大庆油田有限责任公司 Spindle bacillus and use thereof
CN101493003A (en) * 2009-04-14 2009-07-29 大庆油田有限责任公司 Microbe oil production method after polymer drive

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CN101493003A (en) * 2009-04-14 2009-07-29 大庆油田有限责任公司 Microbe oil production method after polymer drive

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