CN101948786B - Pseudomonas aeruginosa for producing rhamnolipid with high yield and application thereof - Google Patents
Pseudomonas aeruginosa for producing rhamnolipid with high yield and application thereof Download PDFInfo
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Abstract
The invention provides a bacteria strain for degrading crude oil and producing rhamnolipid with high yield and application thereof, and in particular relates to a Pseudomonas aeruginosa with a preservation number of CGMCC No.4002 and a Pseudomonas aeruginosa preparation containing the same. The invention also relates to application of the Pseudomonas aeruginosa or preparation thereof to producing a biological surface-active substance by fermentation and culture, application to treating heavy metals in wastewater, application to degrading the crude oil and application to fuel scavenge and oil extraction. The Pseudomonas aeruginosa can be used for producing glycolipid biosurfactants with high yield by using waste oil as a carbon source. The biosurfactant can remarkably reduce the surface tension of a water solution, has strong emulsion capacity, effectively improves the solubility of alkane or crude oil in the water, and obviously promotes the degradation of the strain to the crude oil. The strain and fermentation liquor thereof are used for treating lead in the wastewater and have higher lead removal rate.
Description
Technical field
The invention belongs to energy biotechnology and Environmental Biotechnology field; Specifically; It relates to a Pseudomonas aeruginosa strain Pseudomonas aeruginosa SLQT-6 bacterial strain and an application thereof; This bacterial strain can be carbon source high yield rhamnolipid type biological surfactant with waste oil, and has higher plumbous clearance for the lead of handling in the waste water, has the ability of higher degrading crude oil simultaneously.
Background technology
Pseudomonas aeruginosa (Pseudomonas aeruginosa) is widely used in the organic biological degradation of former wet goods; Its excretory rhamnolipid (RL) is to study more a kind of bio-surfactant at present; Have functions such as emulsification, solubilising, reduction IT; Low toxicity, readily biodegradable have very big application prospect in fields such as petrochemical complex, oil production, biological medicine, food and environment protection.Pseudomonas aeruginosa is generally believed it is the effective efficiency bacterium of microbe oil production.Other has the bibliographical information rhamnolipid, and for example Cd and Pb have certain function for removing heavy metal in the settling.
The composition of its excretory tensio-active agent of the Pseudomonas aeruginosa of different sources, performance, poor are sought suitable Pseudomonas aeruginosa and are had important value apart from all bigger.
Summary of the invention
The object of the present invention is to provide the new pseudomonas aeruginosa strains of a strain, this bacterial strain has the ability of high yield bio-surfactant, and be used for degrading crude oil, change rheological characteristic of crude oil, and for the particularly plumbous effect of removing in the waste water with uniqueness of heavy metal.
Another object of the present invention is to provide the application of described pseudomonas aeruginosa strains in producing bio-surfactant, and the bio-surfactant that obtains of producing.
Another object of the present invention is to provide the particularly application in the lead of heavy metal in handling waste water of described pseudomonas aeruginosa strains and fermented liquid thereof.
Another object of the present invention is to provide the particularly plumbous method of heavy metal in described pseudomonas aeruginosa strains or its fermentation liquor treatment waste water of utilizing.
The invention provides the pseudomonas aeruginosa strains that a strain can high yield comprises the tensio-active agent of rhamnolipid, called after Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 of the present invention.This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), deposit number: CGMCC No.4002 on 07 13rd, 2010.This bacterial strain can with various carbon sources particularly crude oil, vegetables oil or even abendoned oil be carbon source for growth, high yield glycolipid type biological surfactant; This bio-surfactant can obviously reduce the aqueous solution surface tension, have very strong emulsifying capacity, effectively improve solubleness in water of alkane or crude oil, obviously promote the degraded of bacterial strain to crude oil, the bio-surfactant of this bacterial strain and generation thereof can be used for microbe oil production.And this Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 bacterial strain and fermented liquid thereof have significant effect in handling the particularly plumbous application of waste water heavy metal.Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 bacterial strain among the present invention also is called Pseudomonas aeruginosa Pseudomonas aeruginosa CGMCC No.4002.
Pseudomonas aeruginosa SLQT-6 of the present invention separates to obtain from the profit appearance of North China Mongolia woods.With reference to " ubiquitous system system identification handbook ", according to the physiological and biochemical property of SLQT-6, and the result of 16S rDNA gene order BLAST comparison in the Genbank DB, can identify that SLQT-6 is a pseudomonas aeruginosa Pseudomonas aeruginosa.
The present invention also provides a kind of Pseudomonas aeruginosa preparation, contains the Pseudomonas aeruginosa that deposit number is CGMCC No.4002 in this Pseudomonas aeruginosa preparation, and this Pseudomonas aeruginosa preparation is solid-state or liquid bacteria preparation.
The present invention also provides described Pseudomonas aeruginosa or described Pseudomonas aeruginosa preparation with various carbon sources particularly with the abendoned oil being the application in the carbon source through fermentation cultivating and producing Bio-surface active material.
The present invention also provides a kind of bio-surfactant, and it utilizes described Pseudomonas aeruginosa or the cultivation of described Pseudomonas aeruginosa preparation fermentation and obtains.
The present invention also provides a kind of method of producing bio-surfactant, and this method comprises: utilizing described Pseudomonas aeruginosa or described Pseudomonas aeruginosa preparation particularly is that carbon source is carried out fermentation culture, the centrifugal thalline of removing with the abendoned oil with various carbon sources; Supernatant is transferred below the pH to 2.0 with sulfuric acid, and added ammonium sulfate and leave standstill, the extraction liquid with chloroform and methyl alcohol extracts supernatant again; Behind the standing demix; Take off layer, remove wherein solvent, prepare bio-surfactant.Concrete, in this method, said fermentation culture conditions is preferably: fermention medium is: carbon source is preferably abendoned oil 60～120g/L, NaNO3 8～10g/L, yeast extract paste 1～1.5g/L; K
2HPO
40.1 CaCl～0.5%,
20～0.1%, MgSO
40～0.1%, FeSO
40～0.1%, Na
2MO
40～0.1%, 7.0～7.5,34～37 ℃ of 100～300rpm of the initial pH of substratum cultivated 72～180 hours.The present invention also provides the bio-surfactant for preparing according to this method.The bio-surfactant that SLQT-6 bacterial strain of the present invention produces, the sample diluting liquid after acid hydrolysis carry out the TLC experiment, with being blue-greenish colour after sulfuric acid-anthrone colour developing, explain and contain carbohydrate content in the surfactant samples; Be purple yellow with the triketohydrindene hydrate colour developing, explain and contain protein component in the surfactant samples; The butanone extract of surfactant samples is indigo plant-black splotch, explains and contain lipid material in the surfactant samples with ammonium molybdate-perchloric acid colour developing.The tensio-active agent that the SLQT-6 metabolism produces is main with the glycolipid class mainly.Through the glycolipid in the IR spectroscopy tensio-active agent is rhamnolipid.That is, the bio-surfactant that SLQT-6 bacterial strain of the present invention produces is a kind of mixed fermentation product that mainly comprises sugar component, fat component and protein ingredient, and wherein contains rhamnolipid.
The present invention also provides the particularly application in the lead of heavy metal in removing waste water of described Pseudomonas aeruginosa or described Pseudomonas aeruginosa preparation.That is, the present invention also provides a kind of particularly plumbous method of heavy metal in described pseudomonas aeruginosa strains or its fermented liquid or the described Pseudomonas aeruginosa treated waste water of utilizing.According to the preferred embodiments of the invention; Utilizing Pseudomonas aeruginosa of the present invention to handle method plumbous in the waste water comprises: in pending lead waste water, add Pseudomonas aeruginosa of the present invention or described Pseudomonas aeruginosa the preparation form of seed liquor (can add); Regulate wastewater pH neutral (7.0～7.5); 30～38 ℃ of cultivations (shaking culture) 72～180 hours can effectively reduce the lead concentration in the waste water.During practical implementation; Can with Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 of the present invention in the LB substratum 30～38 ℃ cultivated 8～24 hours; Make seed liquor, put in the lead waste water according to 0.5%～15% inoculum size then and (can adjust lead content 10～200mg/L in the said waste water in advance.In addition, can add suitable nutrition agent in the waste water, movable for bacterial metabolism), shaking culture, the lead content of cultivating in the waste water of removing thalline in the back promptly significantly reduces.
The present invention also provides the application in degrading crude oil and/or the displacement of reservoir oil, oil recovery of described Pseudomonas aeruginosa or described Pseudomonas aeruginosa preparation or described bio-surfactant.
SLQT-6 bacterium of the present invention is in the ability with good degraded different viscosity crude oil.Carry out different viscosity oil degradation result of experiment is shown with SLQT-6 strain growth cell: SLQT-6 can effectively degrade and emulsification viscous crude and thin oil; The water that does not insert after the crude oil substratum constant temperature culture of thalline remains limpid; Oil phase is large stretch of oil clot and swims in aqueous solution upper strata or be attached on a bottle wall; Two isolate mutually fully, and water-oil interface are clear.After inserting the crude oil culture medium culturing 7d of thalline, oil reservoir is dispersed in aqueous phase and is the smalls shape, does not stick crude oil on the bottle wall, and water is brownish black, and water-oil interface are unclear, and static back is not stratified.The degradation rate that SLQT-6 pacifies 415 thin oils to Mongolian woods, Xinjiang Liu Zhongqu viscous crude and capital 9-11, middle fork is respectively 65.51%, 56.25%, 45.45%, 61.98%, and the degradation rate of viscous crude is higher than thin oil.The key distinction of viscous crude and thin oil is that the gum asphaltic content of viscous crude is big, and visible SLQT-6 is better to the degradation capability of number of carbons component.
SLQT-6 bacterial strain provided by the invention can also be that fermentation culture is produced bio-surfactant in the basic medium of sole carbon source with crude oil.The substratum of optimizing consists of (g/L): crude oil 8～10, NaNO
31.0, K
2HPO
41.0, NaH
2PO
41.0, MgSO
47H
2O 0.5, and NaCl 1.0, FeSO
47H
2O 0.01, CaCl
20.01, ZnCl
20.01, Na
2MoO
42H
2O 0.01, and pH 7.2～7.5.The bio-surfactant that is produced can make cell go the surface tension of the nutrient solution of irreducible oil after cultivating 48 hours, to reduce to 29.58mN/m; Cultivate that surfactant concentration reaches peak after 90 hours; After 1000 times of its dilutions, still has good surfactivity.96 hours emulsification coefficient EI of tensio-active agent
96Still can reach 90%, and emulsification is highly stable.
Pseudomonas aeruginosa SLQT-6 of the present invention can improve the character of crude oil effectively; Gas chromatographic analysis and column chromatography analysis revealed: before and after the oil degradation; The stable hydrocarbon of crude oil divides obviously to be increased, and fragrance, nonhydrocarbon and bitum content obviously reduce.Explanation bacterium effect back crude oil light component content increases, and heavy components content reduces, and rheological characteristic of crude oil improves.
Rheological characteristic of crude oil changes before and after the Pseudomonas aeruginosa SLQT-6 effect crude oil of the present invention, and viscosity changes before and after the measurement effect, and the result shows that effect back rheological characteristic of crude oil obviously improves, and viscosity reduces.
SLQT-6 bacterial strain of the present invention and fermented liquid carry out the physical simulation oil displacement experiment, and the result shows: the oil displacement efficiency of rock core is 9.06% behind the injection SLQT-6 bacterium liquid.It is thus clear that tensio-active agent of the present invention can effectively improve oil recovery factor, very powerful application potential is arranged in microbe oil production, oil displacement process.
In sum; The invention provides Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6; It can be that carbon source is effectively produced glycolipid class tensio-active agent with the abendoned oil, and thin-layer chromatography and IR spectroscopy must this glycolipid be rhamnolipid, and output is up to 60.86g/L; Significantly improve rhamnolipid output, had unforeseeable technique effect; This bacterium has good degraded emulsifying capacity to different crude oil, and the mensuration through gc, column chromatography, oil degradation rate shows that SLQT-6 can improve oil property, and its heavy constituent are reduced, and light constituent increases, and crude oil fluidity improves; The tensio-active agent that this bacterium generates can reduce surface tension effectively, and diesel oil is had good emulsifying effectiveness, has increased hydrocarbon and the crude oil solubleness in water, obviously promotes the degraded of active bacterial strain to alkane and crude oil; Physical Experiment shows that SLQT-6 and tensio-active agent thereof can be applied to the petroleum production engineering field and especially have great potential in the mikrobe raising petroleum recovery technology.And Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 of the present invention has higher plumbous clearance when handling lead waste water, brought significant technique effect.
Description of drawings
Fig. 1 is that Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 of the present invention produces the rhamnolipid content comparative analysis chart in the tensio-active agent with different carbon source through fermentation.
Fig. 2 is that Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 of the present invention is with the influence of different thalline inoculum sizes to clearance plumbous in the waste water.
Fig. 3 is a Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 of the present invention clearance effect when handling the waste water of different concns lead content.
Fig. 4 A～Fig. 4 D is respectively the saturated hydrocarbon gas chromatography analytical results that Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 of the present invention obtains in degraded different crude oils separate front and back.
Fig. 5 is the bio-surfactant surface tension figure that Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 produces.
Fig. 6 is the bio-surfactant infrared spectrogram that Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 produces.
Fig. 7 is the bio-surfactant CMC figure that Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 produces.
The mikrobe that is used for patented procedure preserves:
Preservation date: on 07 13rd, 2010;
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC);
Deposit number: CGMCC No.4002;
Classification name: Pseudomonas aeruginosa (Pseudomonas aeruginosa).
Embodiment
Technique effect through being had in specific embodiment further explain Pseudomonas aeruginosa Pseudomonas of the present invention aeruginosa SLQT-6 and characteristics and the application below, but therefore the present invention does not receive any restriction.
The separation of embodiment 1 Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6
The screening of Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 mainly comprises sample circuit enrichment culture, screening and purifying and evaluation with separating, and concrete grammar is following:
(1) sample circuit enrichment culture
(the Ge Er group of rising that is collected in North China Mongolia woods on November 3rd, 2009 is risen the local water 10ml of 2,3 thin layers of a pars infrasegmentalis the 3rd substratum (removing the visible mechanical impurity of naked eyes), is transferred in the 90ml enrichment medium and cultivates to get the profit appearance of an amount of North China Oilfield Mongolia woods oil reservoir.That enrichment medium is used is minimal medium (g/L): NaNO
33.5, Na
2HPO
41.0, MgSO
47H
2O 0.5, NaCl 1.5, FeCl
20.5, yeast powder 0.3, whiteruss 10, green dense plain 10, pH7.2～7.5,121 ℃ sterilization, 37 ℃ of 200rpm shaking tables were cultivated 7～14 days.
(2) Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 screening and purifying
With the enrichment culture liquid gradient dilution in the step (1), getting extension rate respectively is 10
4, 10
5Bacterium liquid be uniformly coated on the haemolysis plate culture medium (beef peptone 0.2%, yeast extract paste 0.01%, sodium-chlor 0.1%, agar 1.8%, sheep blood 5%).Cultivated 1～2 day for 37 ℃, observe colonial morphology, select the bacterial classification that periphery of bacterial colonies after the cultivation forms tangible haemolysis transparent circle; Especially select bacterium colony with following characteristic: beige, to put down partially, the edge is irregular; And often be mutual fusion state; Periphery of bacterial colonies has water colo(u)r, makes substratum dyed blue-greenish colour or yellow-green colour, and bacterium colony has metalluster or the ginger smell is arranged.Can be with the dyeing observation down of single colony microscope of being selected; If it is impure; Utilize above-mentioned substratum to separate again, obtain pure bacterium colony (the pure bacterium colony that obtains is switchable to plate culture medium, is that carbon source is cultivated its degraded hydrocarbon characteristic of maintenance with the whiteruss) up to separation.
Single colony inoculation that the transfering loop picking obtains through aforesaid method screening is in fermention medium (soya-bean oil 60, NaNO
36, yeast extract paste 1.5; K
2HPO
40.3%, CaCl
20.010%, MgSO
40.024%, FeSO
40.01%, Na
2MO
40.01%, pH7.0～7.5), the 250ml triangular flask, liquid amount 100ml, 34 ℃ of 200r/min shaking tables are cultivated.Take out fermented liquid behind the 80h, 60 ℃ of heating in water bath 10min, the centrifugal 20min of 10000*g gets supernatant respectively with method of discharge, circle surface tension SF and emulsification coefficient EI
24Test surfaces is active, selects the oil extraction loop diameter to be lower than 30mN/m greater than 10cm, surface tension, and EI
24Be more than 90% bacterial classification preserve as the high yield bacterium.To go cell fermentation liquid supernatant kapillary point sample to analyze on the silica-gel plate in thin-layer chromatography TLC; Developping agent is a chloroform: methane: acetate (V/VV)=65: 15: 2 is in chromatography cylinder; The silica-gel plate of point sample is put into chromatography cylinder; Take out when treating developping agent apart from silica-gel plate top 1cm, (developer: the 2g anthrone is dissolved into 80%H to treat evenly to spray developer with the glass atomizer to silica-gel plate in developping agent volatilization back
2SO
4In, with 80%H
2SO
4Constant volume is to 1000ml) 110 ℃ of times heating 5min colour developing.Select to occur after the colour developing on the silica-gel plate pale brown color spot point and the subzero 80 ℃ of glycerine preservation of bacterial classification that the shade of colour is too deep.
(3) evaluation of Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6
In the present embodiment, from the bacterium colony that is screened, pick out a strain fermented liquid and have the active bacterial strain of high surfaces, called after SLQT-6.
The colony characteristics of SLQT-6: well-grown on common Pseudomonas aeruginosa substratum, bacterium colony is not of uniform size, and mean diameter＜2mm is flat, and the edge is irregular, and often is mutual fusion state.This bacterium produces water colo(u)r, makes substratum dyed blue-greenish colour or yellow-green colour.Bacterium colony is bigger on blood agar plate, and metalluster and ginger smell are arranged, and periphery of bacterial colonies forms transparent zone of hemolysis.In meat soup, form mycoderm, meat soup clarification or little muddiness, it is green that bacterium liquid upper strata is.It is directly or bacillus curved slightly, the blunt circle in two ends that microscopically is observed form, big or small 0.5～0.8 μ m * 1.5～2.0 μ m.1 single-ended flagellum is arranged, and motion is active.This bacterium physiological and biochemical property is as shown in table 1 below.
Table 1 Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 physiological and biochemical property
| Test subject | The result | Test subject | The result |
| Glucose sugar | + | Gramstaining | - |
| Sucrose | + | The starch hydrolysis | - |
| Wood sugar | + | Gelatine liquefication | + |
| Utilize the glucose aerogenesis | + | Catalase | + |
| Utilize Citrate trianion | + | Oxydase | + |
| Nitrate reduction | + | PHB | - |
| Autotrophy H2 | + | Anaerobic growth | - |
| Lipase | +- | Growth temperature | 5-45 |
Annotate: "+" expression growth or reacting positive; "-" expression is not grown or reaction negative
The 16S rDNA gene sequence characteristic of bacterial strain SLQT-6: SLQT-6 is inoculated in LB substratum (beef peptone 0.5%, yeast powder 1%, sodium-chlor 0.2%; PH7.0～7.2), 37 ℃ of shaking tables (200rpm) were cultivated 18 hours, the 10000rpm centrifugal collecting cell; Again suspend; The DNA rapid extraction test kit of producing with the precious biotech firm in Dalian extracts (operation is carried out to specifications) total DNA, carries out pcr amplification, obtains the 16SrDNA fragment of bacterial strain SLQT-6; Dna sequencing (order-checking of the precious biotech firm in Dalian) length is 1613bp, and concrete sequence is referring to SEQ ID NO.1.BLAST comparison in the Genbank DB; ClusterX software is combined with MEGA software; Use the neighbor-joining method to make up the phylogenetic tree that is the basis with 16S rDNA sequence, the highest homology property of bacterial strain and pseudomonas aeruginosa bacterial strain is 99.0%.
With reference to " ubiquitous system system identification handbook "; Physiological and biochemical property according to SLQT-6; And the result of 16S rDNA gene order BLAST comparison in the Genbank DB; Identify that SLQT-6 is a pseudomonas aeruginosa Pseudomonas aeruginosa, be called Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 among the present invention, this bacterial strain was preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on 07 13rd, 2010; Institute of Microorganism, Academia Sinica), deposit number: CGMCC No.4002.
Pseudomonas aeruginosa SLQT-6 can be at nutritional medium; As: grow in common beef tea, LB, the nutrient agar medium; Also can in the minimal medium that adds glucose or sucrose, grow, can be carbon source for growth with the for example various alkane oil of various carbon sources or crude oil, vegetables oil even waste gas oil also.Bacterial strain is aerobic growth under the optimal temperature between 5～40 ℃.
Pseudomonas aeruginosa SLQT-6 of the present invention, it has the character that high yield comprises the bio-surfactant of rhamnolipid, is being that carbon source carries out having produced unforeseeable technique effect aspect the fermentation to produce biological tensio-active agent with the abendoned oil particularly.
Abendoned oil has another name called waste oil, mainly can be divided into production and life and use abendoned oil.The productivity abendoned oil mainly refers to the pin dregs of fat that diesel oil common in the industrial production, gasoline, machine oil and chemical industry grease etc. are remaining, comprises production machinery washing oil, transmission lubrication wet goods, all belongs to productivity waste oil; And life abendoned oil mainly refers to the discarded dregs of fat and the Gan Zhishui of common vegetables oil, animal oil two big residues and discharging, comprises frying waste oil, waste cooking oil and sewer oil.
In the present embodiment, respectively with sewer oil, frying waste oil, waste cooking oil, seed oil of sunflower waste material, oil, 0# diesel oil, gasoline as carbon source, carry out the fermentative prodn rhamnolipid respectively.Concrete grammar is following:
Bacterial strain SLQT-6 of the present invention is inoculated in the LB substratum, the 100ml triangular flask, liquid amount 30ml, 34 ℃ of 200r/min cultivate; Bacterium liquid is inoculated in the fermentation one-level substratum (yeast extract paste 0.3%, SODIUMNITRATE 0.6%, sodium-chlor 0.5%, pH is transferred to 7.2-7.5) behind the 10h, the 250ml triangular flask, and liquid amount 100ml, 34 ℃ of 200r/min shaking tables are cultivated; Change fermentation secondary medium (yeast extract paste 0.5%, SODIUMNITRATE 1.0%, sodium-chlor 0.5%, calcium chloride 0.01%, ferrous sulfate 0.004%, Sodium orthomolybdate 0.05% behind the 10h over to.PH is transferred to 7.2-7.5) in, the 2L triangular flask, liquid amount 500ml, 34 ℃ of 200r/min shaking tables are cultivated; Change fermentation three-stage culture medium (carbon source 80g/L, NaNO behind the 10h over to
310g/L, yeast extract paste 1.5g/L; K
2HPO
40.3%, CaCl
20.010%, MgSO
40.024%, FeSO
40.01%, Na
2MO
40.01%, pH7.0～7.5), 10L intelligence fermentor tank, liquid amount 5L, preceding 10h are 34 ℃ of 300r/min, the later stage is that 37 ℃ of 200r/min cultivate.Behind the 90h, take out 60 ℃ of heating in water bath 10min of fermented liquid, 10000*g is centrifugal, and 20min removes thalline, gets fermented liquid supernatant liquid, adopt colourimetry detect rhamnolipid content.In the present embodiment, be that the synthetic rhamnolipid content detection data of carbon source through fermentation are respectively 54.88,45.25,60.86,49.57,13.48,10.05,9.68 (g/L) (please combine referring to Fig. 1) with sewer oil, frying waste oil, waste cooking oil, seed oil of sunflower waste material, oil, 0# diesel oil, gasoline respectively.
The result of present embodiment can show; Although Pseudomonas aeruginosa SLQT-6 of the present invention is being that rhamnolipid content is not very high in the carbon source through fermentation production tensio-active agent with gasoline, diesel oil, oil; But, in view of the above, use bacterial classification of the present invention being that carbon source carries out having produced unforeseeable technique effect aspect the fermentation to produce biological tensio-active agent with the abendoned oil; Can be that carbon source through fermentation is produced rhamnolipid with the abendoned oil, can reduce cost and turn waste into wealth.
Embodiment 3 Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 are the application aspect the heavy metal in handling waste water
In the present embodiment, utilize Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 fermentation of the present invention to remove the heavy metal lead in the waste water.
1. experiment material
Lead waste water: according to the actual industrial wastewater parameter, this experiment is simulated lead waste water concentration between 10～200mg/L with the plumbic acid potassium dihydrogen.Add suitable nutrition agent, movable for bacterial metabolism.Lead waste water parameter (g/L): Pb (NO
3)
20.01～0.2, NaNO
33.0, Na
2HPO4 1.0, KH
2PO4 1.0, MgSO
47H
2O 0.5, NaCl 1.5, FeCl
20.5, yeast powder 0.3, glucose 5, pH7.2～7.5.
Mikrobe: Pseudomonas aeruginosa SLQT-6, in LB (beef peptone 0.5%, yeast powder 1%, sodium-chlor 0.2%, pH7.0～7.2) substratum; The 100ml triangular flask; Liquid amount 30ml, 34 ℃ of 200r/min cultivate 10h, and zymocyte liquid is subsequent use as liquid Pseudomonas aeruginosa preparation.
2. plumbous measuring method
Adopt standard GB 5009.12-2010 analytical procedure.
3. biochemical processing method
Get the certain volume lead waste water and place the 250mL Erlenmeyer flask, add the bacterium liquid formulation, regulate wastewater pH, place on the vibrator and vibrate, fully contact.Cultivate to get after 5 days and measure plumbous content in an amount of nutrient solution, calculate its clearance.Clearance can be removed efficient with biochemistry and represent.
The biochemical efficient E that removes can use computes:
E=G/G
0, wherein: biochemical efficient, the % of removing of E-; The biochemical removal amount of G-, mg; G
0--initial mass, mg.
4. the different vaccination amount is to the treatment effect experiment of lead waste water
Contain in the Erlenmeyer flask of waste water that lead concentration is 100mg/L filling 100mL, it be neutral regulating wastewater pH, adds the bacterium liquid of different vaccination amount, places on the vibrator 37 ℃ of 200rpm shaking tables cultivations of vibration 5 days, calculates its clearance.
In the present embodiment, different thalline inoculum sizes are that the influence of 100mg/L lead waste water effect sees also Fig. 2 to concentration of treatment.The result shows: in concentration is that the thalline inoculum size is 0.5% o'clock, clearance about 53% in the laboratory simulation lead waste water experiment of 100mg/L; The thalline inoculum size is 2% o'clock, and plumbous clearance is 58.06%; Along with the increase of inoculum size, the clearance of lead waste water increases to some extent.
5. the treatment effect of different concns waste water experiment
In filling the Erlenmeyer flask of lead waste water that the 100mL lead concentration is respectively 10mg/L, 20mg/L, 30mg/L, 40mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L; It is neutral regulating wastewater pH; Add 10% bacterium liquid successively; Place the 37 ℃ of 200rpm shaking tables that vibrate on the vibrator to cultivate 5 days, calculate its clearance.
In the present embodiment, plumbous different concns sees also shown in Figure 3 to the influence of removal effect in the waste water.The result shows; When the concentration of leaded nutrient solution is increased to 200mg/L by 40mg/L; Clearance reduces to 60.09% by 62.64% gradual slow, and when the concentration of lead waste water was reduced to 10mg/L by 30mg/L, clearance sharply increased to 82.16% by 62.64%.It is thus clear that SLQT-6 has good result to the processing of leaded nutrient solution.There is a critical biochemical removal amount (critical absorption) in mikrobe to the clearance of lead in this experiment; Two tangent lines doing curve meet at a bit as shown in the figure; The concentration that this point is corresponding is 35.2mg/L; Clearance is 62.5%, and critical biochemical removal amount is 35.2mg/L * 62.5%=22mg/L.
In order to study the character of bacterial strain SLQT-6 degrading crude oil; Present embodiment utilizes this bacterial strain that the crude oil of the different viscosity in oil fields such as Xinjiang, middle fork, grand celebration Jiang Qiao, Mongolian woods has been carried out the microbiological deterioration experiment, has analyzed degradation by bacteria to crude oil stable hydrocarbon, aromatic hydrocarbons and colloid, each component of bituminous matter relative content and the inner influence of forming in crude oil.
Tensio-active agent is analyzed
From 4 kinds of different crude oils (fork (58 ℃ of viscosity 18.5mPas), grand celebration Jiang Qiao (50 ℃ of viscosity 307.2mPas), Mongolian woods viscous crude (37 ℃ of viscosity 116mPas) Xinjiang Liu Zhongqu viscous crude (20.6 ℃ of viscosity 80mPas), the North China) degradation solution (crude oil 10, NaNO
31.0, K
2HPO
41.0, NaH
2PO
41.0, MgSO
47H
2O 0.5, and NaCl 1.0, FeSO
47H
2O 0.01, CaCl
20.01, ZnCl
20.01, Na
2MoO
42H
2O 0.01, and pH 7.2～7.5, and 37 ℃ of 200rpm cultivated 96 hours) in the tensio-active agent that obtains be respectively BS-1, BS-2, BS-3, BS-4.
Answer qualitative and quantitative analysis (referring to table 2) to know through TLC with developing the color to send out: the component of BS-1, BS-2, BS-3, BS-4 and each components contents are different.The research report is arranged, different nutraceutical matrixes, mikrobe is just carved can have different substrate utilization modes, thereby forms different meta-bolitess.The crude oil of different viscosity; The composition of stable hydrocarbon wherein, aromatic hydrocarbon, gum asphalt, nonhydrocarbon and content are different; Make bacterial strain SLQT-6 have different metabolic waies to different crude oil, particularly the content difference of rhamnolipid is very big thereby make the component of BS-1, BS-2, BS-3, BS-4.Can know 4 kinds of different crude oils with Mongolian woods crude oil to be that carbon source institute synthetic rhamnolipid content is higher from table 2.
Table 2 different crude oils lower surface promoting agent is analyzed
The crude oil group composition changes
Geographic crude oil such as the Xinjiang Liu Zhongqu before and after respectively bacterial strain SLQT-6 being handled, Mongolian woods, grand celebration Jiang Qiao, Liaohe Oil Field carry out PONA analysis.Since the gum asphalt in the crude oil more than 90% by the non-hydrocarbons component, so in analytical data, non-hydrocarbons, gum asphalt in the crude oil are merged into non-hydrocarbons consider together.After the microbiological treatment, the stable hydrocarbon of crude oil, aromatic hydrocarbons, bituminous matter and gelationus content all have different variations (table 3), and this is because the bacterium selectivity consumes the result of crude oil different components.Bacterial classification SLQT-6 handles the crude oil of different oil wells, and crude oil group composition variation tendency is inconsistent.Generally, it is the most obvious to make after the microbiological deterioration that crude oil non-hydrocarbons, colloid, asphalt content reduce ground relatively, and saturated hydrocarbon content but increases relatively, crude oil property be improved significantly.Aromaticity content changes fluctuation greatly.Therefore, microbe oil production must be according to different oil reservoirs and base oil properties screen effective MEOR bacterium thereof.
Oil component analysis before and after table 3 microbial process
Crude oil stable hydrocarbon, aromatic hydrocarbons gas chromatographic analysis
In order further to understand the situation of microbiological deterioration crude oil, carry out gas chromatographic analysis with separating the stable hydrocarbon, the aromatic hydrocarbons that obtain.Crude oil saturated hydrocarbon gas chromatography analytical results (seeing Fig. 4 A～Fig. 4 D) shows: 4 kinds of different crude oils are after bacterium SLQT-6 effect; The stable hydrocarbon of crude oil distributes considerable change (table 4) has taken place; The short chain normal paraffin content increases relatively, and long-chain n-praaffin content then reduces relatively.The w of different viscosity crude oil (nC21-)/w (nC22+) value rises to 1.64,1.5,1.43,1.35 by effect preceding 1.31,1.2,1.22,1.13; Increased by 25.19%, 25%, 17.21%, 19.46% respectively; The SLQT-6 high carbon chain alkane of preferentially degrading is described; Improve the rheological of crude oil, thereby help improving RF.Pristane (Pr) and phytane (Ph) are the biomarkers in the crude oil, and structure is more stable, and bacteriological action does not generally influence its content.See that from the gc conclusion variation has in various degree taken place for 4 kinds of crude oil w (pr)/w (nC17), w (pr)/w (nC18), w (pr)/w (nC27), w (pr)/w (nC28) value after the SLQT-6 degraded.W (pr)/w (nC17), w (pr)/w (nC18) are worth minimizing, explain that the normal paraffin of low carbon chain in the crude oil stable hydrocarbon is increasing; W (pr)/w (nC27), w (pr)/w (nC28) value are increasing, and the normal paraffin of explanation crude oil stable hydrocarbon medium high carbon chain is reducing, and isoparaffin increases relatively; This shows that SLQT-6 effect back lower alkanes hydrocarbon content increases, the high-carbon paraffinicity reduces; Normal paraffin reduces, and isoparaffin increases, and the result makes the physical properties of crude oil change, the mobile enhancing.
Table 4 crude oil saturated hydrocarbon gas chromatography
After microbiological deterioration, the content of the Sino-Philippines series of crude oil aromatic hydrocarbons has all produced bigger variation (table 5) with forming.The SLQT-6 different components in the luxuriant and rich with fragrance series of optionally having degraded causes MPI, DPI index variation.Luxuriant and rich with fragrance series in the crude oil is commonly considered as the cracking from steroid terpene compound.Because alkyl position on phenanthrene ring is different; Stability is also variant, generally is in the methyl of β position, and is more stable like 3-base and 2-MP; At the 1-of α position methyl, 4-MP and to be in the 9-MP of meta more active; Dimethylphenanthrene also has similarity rules, and to β-β type, stability increases successively from α-α type, alpha-beta type.After the bacterium SLQT-6 effect, the MPI of crude oil and DPI index value all improve a lot, and explain that the comparatively active component of chemical property is prone to be degraded in the luxuriant and rich with fragrance series.
Aromatic hydrocarbon GC analyzes before and after table 5 microbial process
Crude oil property changes
After microbiological treatment, crude oil property changes more obvious, and experiment adopts the Brook-VISCO viscosity apparatus to measure the viscosity (referring to table 6) of crude oil fungi degradation front and back.Measure former oil freezing point with reference to the SY/T0541-94 method.The result shows: 4 kinds of crude oil after the SLQT-6 bacteriological action, viscosity and depression of the freezing point significantly, heavy component reduces in the crude oil, improves crude oil property well, has strengthened crude oil fluidity.The conclusion of this and front is all coincide.
Crude oil property f analyzes behind table 6 microbial process
Conclusion: be carbon source with different viscosity crude oil; In the SLQT-6 degradation experiment, through meta-bolites surfactant content before and after the degraded is measured, crude oil family component and gas chromatographic analysis, the result shows that SLQT-6 is main pathways metabolism with the long carbochain alkane of degrading, gum asphalt; Make short chain stable hydrocarbon, the isoparaffin relative content of crude oil increase; Improve the crude oil physical properties, strengthened the flowability of crude oil, further help mikrobe and improve oil recovery factor.Simultaneously, experimental result also shows: the content of same strain degradation crude oil, the degree that changes crude oil and meta-bolites is different, and this is that promptly different nutrient substrates causes different metabolic waies because the rerum natura of crude oil itself is different.So select different mikrobes to different reservoir conditions, specific aim optionally improves microbe oil production efficient.
Embodiment 5 Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 are that carbon source produces surfactant properties research with crude oil
1. produce the condition of bio-surfactant
Through kinds of culture medium and composition and culture condition (temperature, Ph, air flow, rotating speed etc.) single factor analysis and orthogonal experiment; The indoor optimal medium that obtains this Pseudomonas aeruginosa SLQT-6 bacterial strain generation bio-surfactant is: crude oil 8, NaNO
31.0, K
2HPO
41.0, NaH
2PO
41.0, MgSO
47H
2O 0.5, and NaCl 1.0, FeSO
47H
2O 0.01, CaCl
20.01, ZnCl
20.01, Na
2MoO
42H
2O 0.01, and pH 7.2～7.5.Under the optimal culture condition, 7.5,37 ℃ of 200rpm of original ph cultivated 96 hours.
2. the thalline oleophilicity is measured
Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 is under optimal medium and culture condition; The 200rpm shaking table was cultivated 7 days; With the BATH method hydrophobicity on mycetocyte surface is measured: get quantitative bacterial strain fermentation liquor 10ml, the centrifugal thalline that obtains is mixed with 5mL bacteria suspension (mensuration OD under the 8000rpm
600Add 200 μ L n-hexadecanes quantitatively), mixing 2min leaves standstill 25min, thereby the OD value of mensuration 600nm calculates the dense calculating of the bacterium oleophilicity coefficient CSL of lower floor's aqueous phase.
CSL(cell?surface?lipophilicity)＝[(a-b)/a]×100％
A: the OD value of initial bacterium liquid cell concn
B: the OD value of bacterium liquid cell concn during termination
Through measuring; Thalline surface oleophilicity rises earlier in the strain fermentation process; Arrive vertex CSL=[(0.195-0.016)/0.195] * 100%=91.7% behind the 70h; Begin again subsequently to descend, this explanation cell possibly taked different pattern picked-up hydrocarbon at different growth phases, this hydrophobic variation possibly contain that the hydrophobicity composition changes with cell walls in its process of growth and metabolic tensio-active agent attached to relevant on the cell walls.
3. the fermented liquid surface tension is analyzed
Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 is under optimal medium and culture condition; The 200rpm shaking table was cultivated 7 days; Resulting bio-surfactant; Can reduce the surface tension of the aqueous solution significantly, adopt POWEREACH JK99C type automatic surface tensiometer plate method to detect, surface tension is reduced to 21.58 ± 0.3mN/m from 71.59 ± 0.3mN/m.Under the situation that the surfactivity dilution agent is different 10 times, 100 times, 1000 times, still keep aqueous solution low surface tension 30mN/m (referring to Fig. 5).This explanation the SLQT-6 bacterial strain well metabolism produce and to have surface-active bio-surfactant, effectively improved interfacial activity, reduce IT.
4. the oil extraction circle is measured
Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 is under optimal medium and culture condition; The 200rpm shaking table was cultivated 7 days; Resulting bio-surfactant is mixed with certain density solution; Getting a diameter is the 150mm petridish, adds the 20mL deionized water, adds whiteruss and forms oil film.Add the 0.1ml water phase surfactant mixture at the oil film center, form the oil extraction circle around the center oil film is pressed to, the diameter of record oil extraction circle.The oil extraction loop diameter increases to 14.6cm from 0.1cm, and the tensio-active agent that this explanation SLQT-6 produces has good surfactivity.
5. emulsification coefficient E
96Mensuration
Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 is containing NaNO
33.5, Na
2HPO
41.0, MgSO
47H
2O 0.5, NaCl 1.5, FeCl
20.5, yeast powder 0.3, vegetables oil 10.In the substratum of pH7.2～7.5,37 ℃, the 200rpm shaking table was cultivated after 7 days; In the band scale test tube, add 6mL test hydrocarbon (kerosene) and 4mL emulsifying activity material solution, the vortex vibrator 2min that fully vibrates; Leave standstill 96h, with emulsification index (Emusification index, EI
96) expression emulsifying agent emulsifying activity.EI
96Account for the percentage ratio of total height for the water-oil interface upper level; The result can see that the emulsification coefficient of SLQT-6 fermented liquid is up to 99%; This explanation contains surfactant in the SLQT-6 bacterial strain fermentation liquor; Water-insoluble organism is had the effect of dispersion and emulsion, improved the contact area between thalline and the water-insoluble organism greatly.
The purification purifying and the qualitative analysis of 5 bio-surfactants
(1) preliminary evaluation of Bio-surface active
Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 is under optimal medium and culture condition, and the 200rpm shaking table was cultivated 7 days, is that 10000r/min, temperature are that centrifugal 20min removes thalline under 4 ℃ the condition with fermented liquid at rotating speed.Supernatant is transferred below the pH to 2.0 with the sulfuric acid of massfraction 10%, and to add mass concentration be the ammonium sulfate of 320mg/L that the refrigerator of putting into 4 ℃ leaves standstill 12h.Use the extraction liquid equal-volume extraction supernatant of V (chloroform): V (methyl alcohol)=2: 1 again, behind the standing demix, take off layer.The middle level is extracted again, leave standstill and make the white precipitate layer compression, take off layer again.Merging lower floor's extraction liquid, place rotatory evaporator, is that 0.05～0.07MPa, temperature are that solvent evaporated gets the thick product of tensio-active agent under 40 ℃ the condition in vacuum tightness, and the thick product that obtains carries out TLC thin-layer chromatography and coupling reaction.The result shows, the bio-surfactant that the SLQT-6 bacterial strain produces, and the sample diluting liquid after acid hydrolysis with being blue-greenish colour after sulfuric acid-anthrone colour developing, is explained and is contained carbohydrate content in the surfactant samples; Be purple yellow with the triketohydrindene hydrate colour developing, explain and contain protein component in the surfactant samples; The butanone extract of surfactant samples is indigo plant-black splotch, explains and contain lipid material in the surfactant samples with ammonium molybdate-perchloric acid colour developing.Adopt the phenolsulfuric acid method, as standard substance preparation standard curve, (% is 78.26 w/w) through repeatedly measuring contents of saccharide with rhamnosyl; Adopt said methylene chloride methods such as Han Ju, (% is 8.49 w/w) through repeatedly measuring lipid content; Adopt the crystallization bovine serum albumin as standard substance preparation standard curve, (% is 8.25 w/w) through repeatedly measuring protein contnt.It is thus clear that the tensio-active agent that the SLQT-6 metabolism produces is main with the glycolipid class mainly.
(2) IR spectroscopy of bio-surfactant
The material ir spectra that obtains after SLQT-6 purifies is as shown in Figure 6, and analysis revealed: 2954cm-1,2925cm-1,2855cm-1 and 1464cm-1 are in the lipophilic hydrocarbon chain due to the C-H stretching vibration, show existence-CH in the molecule
2-; The wide absorption peak at 1054cm-1 and 3370cm-1 place is due to sugar-OH stretching vibration; 1736cm-1 and 1601cm-1 are the C=O stretching vibration peaks that typically is connected with saturated aliphatic chain; 1459cm-1 and 1378cm-1 are caused by C-H or O-H formation vibration; 1283cm-1 place broad peak is typical ester absorption peak.Confirm that further this bio-surfactant is a rhamnosyl lipid tensio-active agent.Record glycolipid content up to 48.4g/L through sulfuric acid-anthrone method, have good industrial production and use prospect.
(3) cmc (micelle-forming concentration) measures
Bio-surfactant behind the gained purifying is configured to the solution of different concns ratio; Content is: 0,0.02,0.04,0.06,0.08,0.1,0.2,0.4,0.6,0.8 (g/L); Adopt POWEREACH JK99C type automatic surface tensiometer plate method to detect its surface tension; Rendered surface surfactant concentration and surface tension relation curve, thus confirm cmc.The result sees Fig. 7.Along with the increase of this bio-surfactant (bullion) concentration, surface tension is more and more littler, and when concentration was increased to certain value, surface tension no longer included noticeable change, and the flex point place is apparent CMC value among the figure, is 12mg/L.The CMC of the existing rhamnolipid that produces about microbial fermentation of this concentration ratio is much lower.
The evaluation of embodiment 6 thing moulds experiment oil displacement efficiency
(1) laboratory apparatus and material
The 2PB20C constant-flux pump, FY-3 type thermostat container, 0.1MPa tensimeter, ZXZ-0.5 type rotary-vane vaccum pump, the silica sand of core barrel, all size.
Silica sand, Daqing oil field block local water and crude oil, bacterial strain SLQT-6 fermented liquid.
(2) experimental technique
1. three core diameters that use are 2.5cm, pipe range 20cm, and wherein 3# is the contrast core.Silica sand simulated formation situation mixes in proportion, and manual work is suppressed, tamped;
The logical nitrogen of three core barrels that 2. will prepare is surveyed the gas-phase permeation rate;
3. three core barrels are with vacuumizing in the Daqing oil field block local water saturated 24 hours, and the core barrel before and after the more saturated local water is of poor quality to confirm volume of voids PV, calculates porosity then;
4. measure the pressure reduction of whole core section under constant flow rate, confirm absolute permeability through Darcy's law;
5. moisture less than 2% in outlet output liquid with the water in the Daqing oil field block mother oil displacement core barrel, it is the crude oil saturated volume that volume of water is displaced in metering, calculates initial oil saturation;
6. with substratum, add SLQT-6 bacterium liquid, inject the thermostat container that air and 3# blank core be placed on 37 ℃ together then when being injected into 1# and 2# core and leave standstill 7d through sterilization;
7. use the follow-up water drive of local water.
Pseudomonas aeruginosa Pseudomonas aeruginosa SLQT-6 is containing (g/L) NaNO
33.5, Na
2HPO
41.0, KH
2PO4 1.0, MgSO
47H
2O 0.5, NaCl 1.5, FeCl
20.5, in the substratum of yeast powder 0.3, VT 18 10, pH7.2～7.5,37 ℃, the 200rpm shaking table was cultivated after 7 days, and its fermented liquid is carried out the physical simulation oil displacement experiment, the result sees table 7, shows to inject that the oil displacement efficiency of rock core is 9.06% behind the SLQT-6 bacterium liquid.It is thus clear that the SLQT-6 fermented liquid can effectively improve oil recovery factor.
Table 7 is annotated the physical simulation parameter of SLQT-6 bacterium liquid and polymkeric substance
Claims (11)
1. deposit number is the Pseudomonas aeruginosa (Pseudomonas aeruginosa) of CGMCC No.4002.
2. a Pseudomonas aeruginosa preparation contains the Pseudomonas aeruginosa that deposit number is CGMCC No.4002 in this Pseudomonas aeruginosa preparation, and this Pseudomonas aeruginosa preparation is solid-state or liquid bacteria preparation.
3. the described Pseudomonas aeruginosa preparation of described Pseudomonas aeruginosa of claim 1 or claim 2 is produced the application in the Bio-surface active material in fermentation culture.
4. the described Pseudomonas aeruginosa preparation of described Pseudomonas aeruginosa of claim 1 or claim 2 with the abendoned oil is being the application in the carbon source through fermentation cultivating and producing Bio-surface active material.
5. application according to claim 4; Wherein, Said abendoned oil is selected from the remaining pin dregs of fat of diesel oil, gasoline, machine oil and chemical industry grease in the industrial production; And the discarded dregs of fat and in the swill fat water one or more of vegetables oil, animal oil in the life, the content of abendoned oil in fermention medium is 60～120g/L.
6. method of producing bio-surfactant, this method comprises:
Utilize the described Pseudomonas aeruginosa preparation of described Pseudomonas aeruginosa of claim 1 or claim 2 to carry out fermentation culture, the centrifugal thalline of removing is transferred supernatant below the pH to 2.0 with sulfuric acid; And add ammonium sulfate and leave standstill, the extraction liquid with chloroform and methyl alcohol extracts supernatant again, behind the standing demix; Take off layer; Remove wherein solvent, prepare bio-surfactant, this bio-surfactant contains rhamnolipid.
7. method according to claim 6, wherein, said fermentation culture conditions is:
Fermention medium is: abendoned oil 60～120g/L, NaNO
38～10g/L, yeast extract paste 1～1.5g/L; K
2HPO
40.1 CaCl～0.5%,
20～0.1%, MgSO
40～0.1%, FeSO
40～0.1%, Na
2MO
40～0.1%, 7.0～7.5,34～37 ℃ of 100～300rpm of the initial pH of substratum cultivated 72～180 hours.
8. the application of the described Pseudomonas aeruginosa preparation of described Pseudomonas aeruginosa of claim 1 or claim 2 in degrading crude oil and/or the displacement of reservoir oil, oil recovery.
9. the described Pseudomonas aeruginosa preparation of described Pseudomonas aeruginosa of claim 1 or claim 2 application in the heavy metal in removing waste water.
10. the described application of claim 9, wherein said heavy metal are plumbous.
11. one kind is utilized described Pseudomonas aeruginosa of claim 1 or the described Pseudomonas aeruginosa treated of claim 2 lead waste water to remove method plumbous in the waste water; This method comprises: in pending lead waste water, add described Pseudomonas aeruginosa of claim 1 or the described Pseudomonas aeruginosa preparation of claim 2; Regulating wastewater pH cultivated 72～180 hours for 7.0～7.5,30～38 ℃.
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