CN106754539A - One plant of anaerobism produces bacillus subtilis and its application of lipopeptide surfactant - Google Patents
One plant of anaerobism produces bacillus subtilis and its application of lipopeptide surfactant Download PDFInfo
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Abstract
Bacterial strain of the invention is one plant can under anaerobic produce bacillus subtilis (Bacillus subtilis) AnPL 1 of lipopeptide surfactant, and the deposit number of the bacterial strain is CGMCC NO.13461.Strains A nPL 1 disclosed by the invention is facultative anaerobic bacteria, and the temperature range that lipopeptid is efficiently produced under anaerobic is 30 DEG C 40 DEG C, and pH value range is 0 5% for the tolerance range of 6.0 7.5, NaCl.The bacterial strain anaerobic fermented liquid is more than 56% to the emulsification coefficient of crude oil, and oil extraction loop diameter is more than 15mm, and strains A nPL 1 reaches 9.9% in the oil recovery factor that physical analogy rock core situ product lipopeptid surfactant is improved.Bacterial strain of the invention repairs field in microbe oil production and environmental pollution, and there are important promotion and application to be worth.
Description
Technical field
The invention belongs to Environmental Biotechnology and microbe oil production field, specifically, it is related to one plant of anaerobism to produce lipopeptid
Bacillus subtilis (Bacillus subtilis) AnPL-1 of biosurfactant and its dirty in microbe oil production and environment
The application in dye reparation field.
Background technology
Biosurfactant is, by Microbe synthesis and the metabolite with surface-active that secrets out of, mainly to include
Lipopeptid, glycolipid, phosphatide etc..Compared with synthetic surfactant, biosurfactant has high surface, safe nothing
The advantage such as malicious, biodegradable, thus it is widely used in environmental pollution reparation, oil exploitation, food, medicine and other fields.
In the biosurfactant studied, lipopeptide surfactant has relatively low critical micelle concentration and surface work higher
Property.
In petroleum hydrocarbon contaminated environment remediation, biosurfactant can be with emulsion dispersion petroleum hydrocarbon, and it is dirty to strengthen hydro carbons
The dissolubility and bioavailability of thing are contaminated, and then promotes degraded of the microorganism to petroleum hydrocarbon class pollutant to utilize.Lack at some
The petroleum hydrocarbon contaminated in-situ immobilization of the pollution amelioration application field of oxygen environment, such as bed mud in river, it is desirable to be able in anaerobic condition
The microorganism resource of lower biosurfactant production.The surfactant producing strains for being screened at present are most for aerobic micro- life
Thing；The microorganism resource that can under anaerobic produce surfactant reported is also fewer.
Microbial Enhanced Oil Recovery displacement of reservoir oil potentiality are big, technique is easy, low cost, environment-friendly, Produced Liquid are not required to specially treated,
It is the oil recovery technique for receiving much concern at present.But the anaerobic environment of oil reservoir influence surfactant producing strains metabolic activity so as to
Weaken its oil displacement efficiency；Oil reservoir air injection can increase cost of winning again.Screening obtains the bacterial strain that anaerobism produces surfactant, can
To realize that producing surfactant in oil reservoir anaerobic environment situ carries out the displacement of reservoir oil, further save oil extraction cost, simplification and open
Excavating technology.
The content of the invention
Patent of the present invention aims to provide the bacillus subtilis (Bacillus that one plant of anaerobism produces lipopeptid surfactant
Subtilis) AnPL-1 and the bacterium repair the application in field in microbe oil production and environmental pollution.
The anaerobism that the present invention is provided produces the bacillus subtilis (Bacillus subtilis) of lipopeptid surfactant
AnPL-1, hereinafter referred to as AnPL-1, are preserved in that " China Committee for Culture Collection of Microorganisms is common on December 23rd, 2016
Microorganism " center ", deposit number is CGMCC NO.13461.
The anaerobism that the present invention is provided produces the bacillus subtilis AnPL-1 of lipopeptide surfactant, is from Chinese certain oil field
In profit sample, enriched culture, the screening of flat board separation method are obtained, and have investigated the generation of bacterial strain anaerobism by Anaerobic culturel
The characteristic of surfactant.
Anaerobism produces the morphological characteristic and genetics characteristic of the bacillus subtilis AnPL-1 of lipopeptide surfactant such as
Under：24h is cultivated on aerobic LB solid mediums, bacterium colony is slightly yellow, rough surface is opaque；Somatic cells form (such as accompanying drawing
Shown in 1) it is shaft-like, size is 1.7-1.9 μm of 0.5-0.7 μ ms；16S rDNA sequence analyses show strains A nPL-1 and withered grass
Bacillus is similar, but is different from bacillus other monoids (phylogenetic tree of strains A nPL-1 is as shown in Figure 2).
Anaerobism produces the growth metabolism characteristic of the bacillus subtilis AnPL-1 of lipopeptide surfactant：Bacterial strain is in anaerobism bar
The temperature range of high yield Lipopeptide Biosurfactants is 30 DEG C -40 DEG C under part, and pH value range is 6.0-7.5, and NaCl's is resistance to
It is 0-5% by scope.
Bacillus subtilis AnPL-1 is facultative anaerobic bacteria, and the bacterial strain is equal under aerobic, anoxic or complete anaerobic condition
Biosurfactant can be produced, zymotic fluid surface tension is reduced to below 32mN/m；Through thin-layer chromatography (such as the institute of accompanying drawing 3
Show) and the analysis of infrared spectrum (as shown in Figure 4) initial characterization, the biosurfactant produced by strains A nPL-1 is lipopeptid
Class surfactant.
Bacillus subtilis AnPL-1 enters in the anaerobism bottle equipped with fermentation medium under the conditions of 37 DEG C, 80rpm/min
Row Anaerobic culturel 8-10 days, anaerobic fermented liquid was more than for 56% (as shown in Figure 5) to the emulsifying activity of hydrocarbons, and oil extraction circle is straight
Footpath is more than 15mm.Fermentative medium formula is：Sucrose 30.0, NaNO32.0, K2HPO4·3H2O 2.0, KH2PO41.7,
MgSO4·7H2O 0.3, CaCl20.1, KCl 0.8, NaCl 0.5, deionized water 1L.
The physical analogy core oil-displacement test evaluation method of bacillus subtilis AnPL-1 is as follows：
(1) preparation of rock core weighed including rock core, vacuumized, saturation water, determine rock pore volume (PV) and porosity
(2) rock core saturation crude oil, rock core is aging, calculates crude oil saturation degree.
(3) water drives, the moisture content more than 99% of water drive to rock core port of export Produced Liquid calculates a water drive harvesting
Rate.
(4) to the mixed liquor that bacillus subtilis AnPL-1 and its anaerobic fermentation culture medium are injected in rock core, injected slurry volume
0.5PV；Seal core two ends, 10d is cultivated at rock core is placed on into 39 DEG C in constant incubator, makes bacillus subtilis AnPL-1
Lipopeptid surfactant can be produced in rock core situ growth metabolism.
(5) secondary water drive, the moisture content more than 99% of water drive to rock core port of export Produced Liquid calculates secondary waterflood recovery efficiency factor
(6) Data Processing in Experiment：Using a waterflood recovery efficiency factor and secondary waterflood recovery efficiency factor, bacillus subtilis is calculated
AnPL-1 anaerobism produces the oil recovery factor that lipopeptid surfactant is improved.
In physical analogy core oil-displacement test, bacillus subtilis AnPL-1 can give birth in rock core situ growth metabolism
Lipopeptid surfactant is produced, the oil recovery factor of raising is 9.9%.
Anaerobism produces lipopeptid surfactant bacillus subtilis AnPL-1 in microbe oil production and environmental pollution are repaired
Using.Bacillus subtilis AnPL-1 can produce lipopeptid surfactant under aerobic, anoxic or complete anaerobic condition,
The bacterium can effectively reduce oil water interfacial tension, emulsified crude oil, improve oil recovery factor, increase the dissolubility of petroleum hydrocarbon.In reality
In the application of border, oil recovery factor can be improved in the reservoir media situ production lipopeptid surfactant of anoxic using the bacterium；
And using the emulsifying activity of bacterial strain lipopeptid surfactant products, it is applied to the environmental organism reparation of hydrocarbons pollutant.
Beneficial effects of the present invention：
Bacillus subtilis AnPL-1 of the invention can synthesize lipopeptid under aerobic, anoxic or complete anaerobic condition
Surfactant, especially its anaerobism produce the characteristic of lipopeptid surfactant so that the bacterial strain can be applied to anaerobic environment
Pollution amelioration and the oil reservoir surfactant that produces in situ improve oil recovery factor.Can be realized in oil reservoir anaerobic environment based on the bacterial strain
Situ produces surfactant and employs crude oil, it is not necessary to which air injects, and further saves oil extraction cost and simplifies exploitation work
Skill.The anaerobic fermented liquid of the bacterial strain has boundary's surface-active and stronger emulsification of crude oil activity higher, can meet microorganism and adopt
The demand that oil tech and petroleum hydrocarbon contaminated environmental organism are repaired.
Brief description of the drawings
Accompanying drawing 1 is the stereoscan photograph of strains A nPL-1；
Accompanying drawing 2 is the systematic growth tree graph of strains A nPL-1；
Accompanying drawing 3 is the thin-layer chromatogram of strains A nPL-1 lipopeptid surfactant products；
Accompanying drawing 4 is the infrared spectrogram of strains A nPL-1 lipopeptid surfactant products；
Accompanying drawing 5 is the emulsifying activity figure of the anaerobic fermented liquid to different hydrocarbons material of strains A nPL-1.
Specific embodiment
With reference to following specific embodiments, the present invention will be described in detail, makes one of ordinary skill in the art more comprehensively
Understanding the present invention, but do not limit the invention in any way.In the following example, unless otherwise specified, material used
Can be obtained from the purchase of biochemical reagents company with reagent；In addition to the method for specified otherwise, it is normal that test method used is laboratory
Rule experimental technique.
Culture medium prescription used is as follows in following embodiments：
Fermentation medium：Sucrose 30g, NaNO31.5g, KH2PO41.0g, MgSO40.25g, KCl 0.8g, NaCl
0.5g, adds deionized water to 1L, pH 6.5-7.0.
Enriched medium：With fermentation medium
LB culture mediums：Peptone 10g, dusty yeast 5g, NaCl 10g, adds deionized water to 1L, pH 6.5-7.0
LB solid mediums：In LB culture mediums, the agar of 17-20g/L is added.
Embodiment 1：The Screening and Identification of strains A nPL-1
The separation screening process of strains A nPL-1 is as follows：
The profit sample 10ml in Chinese certain oil field is taken, is added in the anaerobism bottle containing 190ml enriched mediums, 37 DEG C,
80rpm carries out anaerobism enrichment culture 15 days.
The anaerobism pregnant solution for taking 1ml carries out gradient dilution with sterile distilled water, is diluted to 104Again, 105Again, 106Again, 107
Times volume, the sample that the different extension rates of 0.1ml are taken respectively is applied to the LB solid mediums containing the aseptic sheep blood of 5% (v/v)
On flat board, flat board is in inversion culture at 37 DEG C.The big bacterium colony of picking haemolysis circle is in the new LB solid cultures containing 5% aseptic sheep blood
Line is carried out on base flat board to isolate and purify, single bacterium colony after purification is scoring in LB culture medium slants, carry out temporarily
Culture presevation.
By in the inoculation on LB inclined-planes to the anaerobism pipe for containing 20ml fermentation mediums, anaerobism is trained under 37 DEG C, 80rpm
Support 10 days.The above-mentioned anaerobic fermented liquid for taking 10ml is centrifuged 10min under 8000rpm, takes supernatant and determines surface tension, and selection is detested
The bacterial strain of aerobe fermentation liquid surface tension minimum (30.5mN/m), numbering is strains A nPL-1.
The authentication method of strains A nPL-1 is as follows：
Colonial morphology is described：Strains A nPL-1 cultivates 24h on LB solid mediums, and bacterium colony is slightly yellow, rough surface,
It is opaque.
Somatic cells morphologic description：Strains A nPL-1 is described for bacillus long using scanning electron microscopic observation, size is 0.5-0.7
1.7-1.9 μm of μ m, as shown in Figure 1.
Molecular biology identification：16S rDNA sequence analyses show that strains A nPL-1 is similar to bacillus subtilis, but
Bacillus other monoids are different from, the phylogenetic tree of strains A nPL-1 is as shown in Figure 2.
Embodiment 2：The extraction of strains A nPL-1 surfactant products under anaerobic and identification
Strains A nPL-1 is facultative anaerobic bacteria, and the bacterial strain can produce Bio-surface active under aerobic and anaerobic conditions
Agent.By strains A nPL-1 in LB culture mediums, 37 DEG C, 24h is cultivated under 160rpm.LB medium centrifugals are taken, supernatant is discarded, so
Washed and resuspended bacterial sediment with the NaCl solution of isometric mass concentration 0.9% afterwards, be made inoculation seed liquor；With 1:20
Volume ratio be inoculated into containing 200ml fermentation mediums anaerobism bottle in, Anaerobic culturel 10 days under 37 DEG C, 80rpm.
Above-mentioned anaerobic fermented liquid is taken, bactofugation body adjusts supernatant pH value to 2.0, in 4 with the HCl solution of 6mol/L
DEG C overnight place；Then isometric chloroform/methanol (2 is utilized:1, v/v) mixture is extracted, lower floor's organic phase of extraction
Rotary evaporation in vacuo (45 DEG C, 50rpm) is carried out, strains A nPL-1 surfactant products under anaerobic are obtained.
Surfactant products to obtaining carry out the qualitative analysis of thin-layer chromatography and infrared spectrum respectively.
As shown in Figure 3, under ninhydrin developer, the thin-layer chromatography point display of surface-active product is purple for thin-layer chromatogram
Color, shows to contain component polypeptides in the surface-active product.
Infrared spectrogram as shown in Figure 4, in 1650cm-1And 1541cm-1Respectively acid amides bands of a spectrum I and bands of a spectrum II,
3386cm-1Place is that the N-H that hydrogen bond causes in strand shrinks vibrational band, and these characteristic absorptions show the surfactant molecule
Containing peptide bond, the hydrophilic group for illustrating the surfactant molecule that strains A nPL-1 is produced is peptide chain.2959-2927cm on spectrogram-1
And 1464-1385cm-1Absworption peak is the C-H stretching vibration peaks of aliphatic carbon chain, 1716cm-1And 1210cm-1It is the feature of lactone
Absworption peak, the hydrophobic group for showing the surfactant molecule is fatty acid chain.
Summary thin-layer chromatography and the result of infrared spectrum analysis, are inferred to strains A nPL-1 and are produced under anaerobic
Raw biosurfactant is lipopeptide surfactant.
Embodiment 3：The anaerobism of strains A nPL-1 efficiently produces the environmental condition evaluation of lipopeptid surfactant
Be equipped with 20ml fermentation mediums anaerobism pipe investigated bacillus subtilis AnPL-1 different temperatures, pH and
Anaerobism produces the ability of lipopeptid surfactant under Variation of Salinity Condition, and each treatment 3 is parallel, is connect with 3% (v/v) inoculum concentration
Kind, at 37 DEG C, Anaerobic culturel 8 days.Strains A nPL-1 is characterized with the surface tension of anaerobic fermented liquid and oil extraction loop diameter to detest
Oxygen produces the ability of lipopeptid surfactant, and zymotic fluid surface tension is more than 10mm, that is, shows bacterium less than 35mN/m, oil extraction loop diameter
Strain AnPL-1 can effectively produce lipopeptid surfactant.
Temperature experiment process group：Set respectively 30 DEG C, 35 DEG C, 40 DEG C and 45 DEG C totally 4 temperature experimental groups (initial pH value is
7.0, NaCl mass concentrations are for 0.1%), every group of Setup Experiments three are parallel.In the range of 30 DEG C -40 DEG C of temperature, strains A nPL-
The surface tension of 1 anaerobic fermented liquid is more than 10mm less than 35mN/m, oil extraction loop diameter；And under other temperature conditionss (45 DEG C),
The surface tension of strains A nPL-1 anaerobic fermented liquids is less than 8mm higher than 40mN/m, oil extraction loop diameter.Result shows, strains A nPL-
1 under anaerobic efficiently produce lipopeptid surfactant temperature range be 30 DEG C -40 DEG C；Under strains A nPL-1 anaerobic conditions
The optimal cultivation temperature for producing lipopeptid surfactant is 37 DEG C.
PH experiment process groups：The initial pH value for setting culture medium respectively is 5.0,6.0,6.5,7.0,7.5,8.0 and 9.0
(37 DEG C of temperature, NaCl mass concentrations are for 0.1%), every group of Setup Experiments three are parallel, in pH 6.0-7.5 scopes for seven groups of experiments
Interior, the surface tension of strains A nPL-1 anaerobic fermented liquids is more than 10mm less than 35mN/m, oil extraction loop diameter；And in other pH conditions
Under, the surface tension of strains A nPL-1 anaerobic fermented liquids is less than 8mm higher than 40mN/m, oil extraction loop diameter.Result shows, bacterial strain
It is 6.0-7.5 that AnPL-1 efficiently produces the pH scopes of lipopeptid surfactant under anaerobic；Strains A nPL-1 anaerobism is produced
The optimal ph of lipopeptid surfactant is 7.
Salinity experiment process group：It is 0.1%, 1%, 3%, 5%, 7%, 9% and 11% to set NaCl mass concentrations respectively
Seven groups of experiments (37 DEG C of temperature, initial pH value is that 7.0), every group of Setup Experiments three are parallel, NaCl mass concentrations be less than 5%
In the range of, the surface tension of strains A nPL-1 anaerobic fermented liquids is more than 10mm less than 35mN/m, oil extraction loop diameter；And in NaCl matter
Under the conditions of amount concentration is higher than 5%, the surface tension of strains A nPL-1 anaerobic fermented liquids is less than higher than 40mN/m, oil extraction loop diameter
8mm.Result shows that efficiently the NaCl mass concentration scopes of generation lipopeptid surfactant are strains A nPL-1 under anaerobic
Less than 5%..
The temperature range that strains A nPL-1 anaerobism efficiently produces lipopeptide surfactant is 30 DEG C -40 DEG C, pH value range
It is 6.0-7.5, the tolerance range of NaCl is 0-5%.Bacterial strain anaerobism efficiently produces the wider environment of lipopeptide surfactant
Adaptability scope, contains the reservoir media condition of most of onshore oil fields and the environmental condition in hydrocarbon contamination place.The present invention
Strains A nPL-1 repair field in microbe oil production and environmental pollution there are important promotion and application to be worth.
Embodiment 4：The characteristic of strains A nPL-1 anaerobic fermented liquids
Bacillus subtilis AnPL-1 equipped with 200ml fermentation mediums anaerobism bottle in, in 37 DEG C, 80rpm/min bars
Under part, Anaerobic culturel 10 days, emulsifying activity and oil extraction characteristic to strains A nPL-1 anaerobic fermented liquids are analyzed and evaluated.
Emulsifying activity of the strains A nPL-1 anaerobic fermented liquids to various hydrocarbons：Emulsifying activity is emulsifying coefficient EI24Come
Characterize, EI24Emulsify the percent value of layer height and mixed liquor total height.The assay method of emulsifying activity is as follows：Take one
The graduated test tube of 10ml, plus 3ml hydrocarbons and the strains A nPL-1 anaerobic fermented liquids of 3ml；In miniature whirlpool mixed instrument
On, vibrate 2min；In at 40 DEG C, after standing 24h, emulsification coefficient EI is calculated24.Certain crude oil, liquid are have chosen in the present embodiment
The hydrocarbons such as body paraffin, benzene, petroleum ether, kerosene, normal heptane and hexamethylene are detested as emulsification object, analysis strains A nPL-1
The emulsifying activity of aerobe fermentation liquid.
Strains A nPL-1 anaerobic fermented liquid oil extractions loop diameter is determined：The operating procedure that oil extraction loop diameter is determined is as follows：Take 2ml
Strains A nPL-1 anaerobic fermented liquids obtain supernatant after 10min is centrifuged under the conditions of 5 000rpm；By the culture dish of a diameter of 90mm
Be put on graph paper, add 30ml distilled water, then in culture dish the water surface center drop 20 μ l certain crude oil (viscosity is less than
20mPas), one layer of oil film is formed on the water surface；The sample to be determined for taking 10 μ l drips to oil film center, records oil extraction loop diameter.
The oil extraction loop diameter of strains A nPL-1 anaerobic fermented liquids is more than 15mm；Strains A nPL-1 anaerobic fermented liquids are to various hydrocarbon
The emulsifying activity of class material was all higher than for 56% (as shown in Figure 5), has shown emulsified crude oil and has improved recovery ratio and emulsification solubilising
Hydrocarbons pollutant and then strengthen its biodegradable application value.
Embodiment 5：Strains A nPL-1 anaerobism produces the thing mould displacement of reservoir oil Potential Evaluation of lipopeptid surfactant
The experimental arrangement of core physical simulation experiment is as follows：
(1) preparation of rock core：Rock core is weighed, the entrance point and the port of export of rock core is set.
(2) rock core is vacuumized and saturation water process：The ready rock core port of export in (1) is connected with vavuum pump, is entered
Row vacuumize process；Then, the port of export of rock core is closed, entrance point is connected into the rustless steel container equipped with reservoir flooding water, entered
Row saturation water process.
(3) calculating of rock pore volume (PV)：Rock core after saturation water is weighed for the second time, hole is calculated
Volume is 64.8mL.
(4) rock core saturation crude Treatment：Saturation oil processing is carried out to rock core with constant-flux pump, the rock core port of export places one
100ml graduated cylinders, advection flow rate pump is 0.5ml/min, as fuel-displaced more than the 20mL of the rock core port of export, that is, thinks saturated oils process knot
Beam.
(5) rock core burin-in process：Seal core two ends, being placed under simulation reservoir temperature (39 DEG C) carries out burin-in process 24h.
(6) water-drive oil recoveries：A water drive treatment is carried out to rock core with constant-flux pump, the rock core port of export places one
100ml graduated cylinders, advection flow rate pump is 0.5ml/min, when rock core port of export Produced Liquid moisture content more than 99%, that is, is thought once
Water drive process terminates, and it is 45.7% to calculate a waterflood recovery efficiency factor.
(7) microbial bacterial agent injection：After water drive, with constant-flux pump to the bacillus subtilis that 0.5PV is injected in rock core
The mixed liquor of AnPL-1 seed liquors and its anaerobic fermentation culture medium；Seal core two ends, constant temperature training at rock core is placed on into 39 DEG C
10d is cultivated in foster case.
(8) secondary water drive：Water drive calculates secondary waterflood recovery efficiency factor to the moisture content more than 99% of rock core port of export Produced Liquid
55.6%.
(9) Data Processing in Experiment：Using a waterflood recovery efficiency factor 45.7% and secondary waterflood recovery efficiency factor 55.6%, calculate withered
Careless bacillus AnPL-1 employs the recovery ratio that crude oil is improved in rock core.
Rock core used is artificial consolidated core (rock core is in an airtight chamber with inlet and outlet) in experiment,
Crude oil used is the dehydration degassed crude of certain factory of oil extraction in oil field two, and saturation water and driven water-replacing are the oil reservoir of certain factory of oil extraction in oil field two
Injection water.In this experiment, in order to ensure anaerobic condition as far as possible, culture medium before injection, have passed through boil, nitrogen charging gas disposal；
And inoculation seed liquor have passed through centrifugation and thalline carrying out washing treatment.The bacterial sediment of strains A nPL-1 is accessed into corresponding culture medium,
To exclude interference of the lipopeptid surfactant generated in seed liquor to oil displacement efficiency, so as to ensure that improved recovery ratio comes from
The result of crude oil is employed in the lipopeptid surfactant that rock core situ is produced in strains A nPL-1.In core experiment, a water
The recovery ratio of drive is 45.7%, and after growth metabolism in rock core, the recovery ratio of secondary water drive is bacillus subtilis AnPL-1
It is 9.9% that 55.6%, i.e. bacillus subtilis AnPL-1 employ the recovery ratio that crude oil improved in rock core.
In physical analogy core oil-displacement test, bacillus subtilis AnPL-1 produces fat in rock core situ growth metabolism
Peptide surfactant, the oil recovery factor of raising is 9.9%.Strains A nPL-1 of the invention has in microbe oil production application field
There are important promotion and application to be worth.
Claims (8)
1. one plant of anaerobism produces the bacillus subtilis of lipopeptide surfactant, bacillus subtilis (Bacillus
Subtilis) AnPL-1, is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms center ", and deposit number is
CGMCC NO.13461。
2. the application of the bacillus subtilis described in a kind of claim 1, it is characterised in that：
The AnPL-1 bacterial strains can be used for the production of Lipopeptide Biosurfactants.
3. the application of bacillus subtilis according to claim 2, it is characterised in that：
The AnPL-1 bacterial strains are facultative anaerobic bacteria, and strains A nPL-1 is equal under aerobic, amphimicrobian or complete anaerobic condition
Lipopeptide Biosurfactants can be produced.
4. the application of the bacillus subtilis according to Claims 2 or 3, it is characterised in that：The bacterial strain is in anaerobic condition
The temperature range of lower high yield Lipopeptide Biosurfactants is 30 DEG C -40 DEG C, and pH value range is 6.0-7.5, the quality of NaCl
Concentration range is 0-5%.
5. the application of bacillus subtilis according to claim 4, it is characterised in that：The bacterial strain is equipped with fermented and cultured
In the anaerobism bottle of base, Anaerobic culturel is carried out 8-10 days under the conditions of 37 DEG C, 80rpm/min.
6. the application of bacillus subtilis according to claim 4, it is characterised in that：Fermentation medium：Sucrose 20-35g,
NaNO31.0-2.5g, KH2PO41.0-2.0g, MgSO40.20-0.30g, KCl 0.5-1.0g, NaCl 0.5-1.0g, mend
Plus deionized water is to 1L, pH 6.5-7.0.
7. the application of the bacillus subtilis described in a kind of claim 1, it is characterised in that：The bacillus subtilis is in crude oil
Application in harvesting, that is, be applied to during microbe oil production,
The bacillus subtilis AnPL-1 being capable of the growth in situ metabolism life (i.e. in reservoir media) in the rock stratum containing crude oil
Lipopeptid surfactant is produced, can be used for the oil recovery factor for improving.
8. the application of the bacillus subtilis described in a kind of claim 1, it is characterised in that：Bacillus subtilis AnPL-1 passes through
In anaerobic environment (anaerobic environment is bed mud in river etc.) situ production lipopeptid surfactant, hydro carbons is can apply to dirty
The environmental pollution for contaminating thing is biological prosthetic.
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