CN101724579A - Rhodococcus sp., lipopeptid extra-cellular bio-demulsifier, preparation method and application thereof - Google Patents
Rhodococcus sp., lipopeptid extra-cellular bio-demulsifier, preparation method and application thereof Download PDFInfo
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- CN101724579A CN101724579A CN200810201236A CN200810201236A CN101724579A CN 101724579 A CN101724579 A CN 101724579A CN 200810201236 A CN200810201236 A CN 200810201236A CN 200810201236 A CN200810201236 A CN 200810201236A CN 101724579 A CN101724579 A CN 101724579A
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Abstract
The invention relates to Rhodococcus sp., lipopeptid extra-cellular bio-demulsifier produced by culturing the Rhodococcus sp. and an application thereof. The dosage of the bio-demulsifier is 100-200mg/L, and the demulsification rate of the W/O type kerosene model emulsion can be 72.7-89.1% within 24h when the demulsification test temperature is 35 DEG C. When the bio-demulsifier is used in thin oil extraction liquid in ocean extraction plants of Shengli Oil Field (SLOF) and the dosage is 90-140mg/L, the demulsification rate can achieve 60.0-62.5% within 6.5h which is further superior to the chemical demulsifier used on site. The extra-cellular bio-demulsifier has the advantages of convenient extraction, low production cost, easy storage and transportation, and can solve the problems of influenced oil product quality and high extraction cost of cell wall combined products when full culture liquid is added.
Description
Technical field
The invention belongs to petrochemical complex and industrial microorganism field.
Background technology
China adopts in the oil field mode crude oil extraction of water filling mostly at present, therefore in the Oil extraction process, often contains a certain amount of water in the extraction crude oil.The method of dehydrating of crude oil has a lot, as centrifugal, heating, electro-dewatering and interpolation chemical demulsifier etc.Since easy to operate, and do not need special equipment, be that the crude oil dehydration method of core has obtained using the most widely in the petroleum industry at home with the chemical demulsification.But problems such as use that chemical demulsifier exists that the emulsion splitter dosage is big, demulsification is not good enough, bad adaptability, environmental pollution are serious.Therefore, from reducing the angle consideration of polluting and protecting environment, need find a kind of emulsion splitter of cleaning more to replace chemical demulsifier.It is exactly a feasible approach that the bio-surfactant that utilization has a demulsification carries out breakdown of emulsion.
Adopt the bio-surfactant breakdown of emulsion that following advantage is arranged: the bio-surfactant that (1) microorganism metabolism is produced has particular structure and function, and has easy degraded, characteristics that environmental pollution is little; (2) add the effect that biological demulsifying agent can improve breakdown of emulsion greatly; (3) microorganism fermentation process is easy, and is with low cost, will the working cost of dewatering process be reduced; (4) wide adaptability of biological demulsifying agent can be used down in extreme environmental conditions (high temperature, high salt, strong acid and strong base etc.).
Past two during the last ten years, the foreign study person has carried out careful research to microorganism and the demulsification performance thereof that produces the thing tensio-active agent.They are from the bacterial classification collection, progressively filter out bacterial classification by separation and purification, enrichment culture, and many influence factors such as spawn culture condition, breakdown of emulsion operational condition, emulsion water content and emulsion kind have been carried out experimental study with strong breakdown of emulsion ability from nature.From present result of study, the bio-surfactant with demulsification has two big classes: a class is the extracellular products that is secreted in culturing process in the substratum, and another kind of is product with cell wall-bound.In laboratory study, carry out the breakdown of emulsion test with the full nutrient solution of microorganism that contains biological demulsifying agent usually, study its demulsification and influence factor.Can save the cost that product extracts although adopt full nutrient solution to carry out breakdown of emulsion, the residue substratum that wherein contains, other meta-bolites etc. may exert an influence to the quality of deviating from water and oil.In addition, undressed full nutrient solution volume is big and be difficult for preservation, and its transportation and preservation also are more scabrous problems.And product is extracted, will increase production cost.Comparatively speaking, the cost for purification of the outer biological emulsion splitter of born of the same parents will be far below the biological demulsifying agent of cell wall mating type.
Summary of the invention
At above problem, the present invention is intended to study a kind of lipopeptid extra-cellular bio-demulsifier, when adding full nutrient solution with solution to the influence and the high problem of cell wall mating type product extraction cost of oil quality.
The present invention is by the following technical solutions to realize the foregoing invention purpose.
One of the object of the invention relates to a kind of rhodococcus (Rhodococcus sp.), the existing mode of freezing with very low temperature of this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, No. 3, A, DaTun Road, Chaoyang District, BeiJing City, Institute of Microorganism, Academia Sinica, postcode 100101, preservation date on September 12nd, 2008, deposit number CGMCC No.2667, classification name: rhodococcus.
Cultivate the method for the described rhodococcus of claim 1, be characterised in that the employing broth culture, the mass ratio of each component is extractum carnis substratum 4.5-5.5 part, peptone 9.5-10.5 part, glucose 4.5-5.5 part, NaCl 4.5-5.5 part, 1000 parts in water, pH7.0 perhaps adopts the paraffin substratum, and the mass ratio of each component is: NH
4NO
33.5-4.5 part, K
2HPO
43.5-4.5 part, KH
2PO
45.5-6.5 part, MgSO
47H
2O 0.1-0.3 part, CaCl
22H
2O 0.8-1.2 part, FeSO
47H
2O 0.8-1.2 part, EDTA 0.8-1.2 part, whiteruss 4% (V/V), 1000 parts in water, pH 7.0; In temperature is 30-40 ℃, and rotating speed is to cultivate in the shaking table of 120-160rpm.
The full nutrient solution of above-mentioned rhodococcus is as the application of lipopeptid extra-cellular bio-demulsifier.
The method for preparing lipopeptid extra-cellular bio dry powder emulsion splitter, full nutrient solution centrifugal 15-25min under 8000-12000rpm with above-mentioned rhodococcus, remove thalline, clear liquid is transferred pH to 1.5-2.5 with acid solution, under 0-4 ℃, leave standstill, obtain flocks, centrifugal 15-25min under 8000-12000rpm, discard clear liquid, the flocks that obtains is head product; Head product obtains biological dry powder emulsion splitter after lyophilize.
The application of above-mentioned biological dry powder emulsion splitter is dissolved biological dry powder emulsion splitter with basic solution, and with pH transfer to 7.0 the back be made into working concentration 90～200mg/L with distilled water, use temperature is between 35～60 ℃, duration of service is between 18-36 hour.
The invention still further relates to the screening method of this rhodococcus.
The screening of bacterial strain: as the screening source, add (L in the following substratum with the oily sludge of Shengli Oil Field
-1): NH
4NO
34.0g, K
2HPO
44.0g, KH
2PO
46.0g, crude oil 4% (V/V), MgSO
47H
2O 0.2g, CaCl
22H
2O 1.0g, MnSO
41.0g, EDTA 1.4g, pH=7.0.With 7 days be one-period, 3 all after dates of transferring therefrom sieve to such an extent that a strain can produce the rhodococcus of lipopeptid extra-cellular bio-demulsifier.
The invention still further relates to the application method of this lipopeptid extra-cellular bio-demulsifier.Biological demulsifying agent is dissolved with basic solution, and pH is transferred to 7.0 backs be made into working concentration with distilled water, the temperature of breakdown of emulsion test is 35～60 ℃, and dosage is 90～200mg/L.
Advantage of the present invention is as follows:
1. the leaching process of the outer biological emulsion splitter of born of the same parents is convenient, and cost is lower, has solved the high problem of cell wall mating type biological demulsifying agent extraction cost.
2. it is little to take volume after the outer biological emulsion splitter drying of born of the same parents that extraction obtains, and has solved the difficult problem in full nutrient solution transportation and the preservation.
3. the rhodococcus that screens is indigenous bacterial classification, and the breakdown of emulsion ability is strong.
Description of drawings
Fig. 1 is the microphotograph of rhodococcus of the present invention behind gramstaining;
Fig. 2 is the breakdown of emulsion test-results of the broth culture of rhodococcus of the present invention to w/o type kerosene model milk sap;
Fig. 3 is the breakdown of emulsion test-results of the broth culture of rhodococcus of the present invention to O/W moulded coal oil model milk sap;
Fig. 4 is the breakdown of emulsion test-results of the paraffin culture of rhodococcus of the present invention to w/o type kerosene model milk sap;
Fig. 5 is the breakdown of emulsion test-results of the paraffin culture of rhodococcus of the present invention to O/W moulded coal oil model milk sap;
The biological demulsifying agent that Fig. 6 generates in broth culture for rhodococcus of the present invention is to the breakdown of emulsion test-results of w/o type kerosene model milk sap;
The biological demulsifying agent that Fig. 7 generates in the paraffin substratum for rhodococcus of the present invention is to the breakdown of emulsion test-results of w/o type kerosene model milk sap;
Fig. 8 is applied to the breakdown of emulsion test-results of ocean extraction factory thin oil extraction liquid for biological demulsifying agent of the present invention.
Embodiment
The screening of rhodococcus:
The oily sludge of getting the 5g Shengli Oil Field adds (L to the following substratum of 100mL
-1): NH
4NO
34.0g, K
2HPO
44.0g, KH
2PO
46.0g, crude oil 4% (V/V), MgSO
47H
2O 0.2g, CaCl
22H
2O 1.0g, MnSO
41.0g, EDTA 1.4g, pH=7.0.In temperature is 35 ℃, rotating speed is to cultivate in the shaking table of 140rpm, with 7 days was one-period, 3 all after dates are cultivated in switching, therefrom successfully be separated to the rhodococcus that a strain can produce lipopeptid extra-cellular bio-demulsifier by the dilution coating method, microphotograph behind its gramstaining is seen Fig. 1 (magnification of bacterial strain is 1000 times among the figure), this rhodococcus be numbered S-SL-2.
The cultivation of rhodococcus:
This strain rhodococcus can both be grown in broth culture and paraffin substratum, and produces the outer biological emulsion splitter of born of the same parents.Following (the L of the prescription of broth culture
-1): extractum carnis 5.0g, peptone 10.0g, glucose 5.0g, NaCl 5.0g, pH 7.0.Following (the L of the prescription of paraffin substratum
-1): NH
4NO
34.0g, K
2HPO
44.0g, KH
2PO
46.0g, MgSO
47H
2O 0.2g, CaCl
22H
2O 1.0g, FeSO
47H
2O 1.0g, EDTA 1.4g, whiteruss 4% (V/V), pH 7.0.It is 35 ℃ that culture condition is in temperature, and rotating speed is to cultivate in the shaking table of 140rpm.
The extraction of biological demulsifying agent and evaluation:
With full nutrient solution centrifugal 20min under 10000rpm, remove thalline, supernatant liquor is transferred pH to 2.0 with the HCl of 6mol/L, leaves standstill 24h under 4 ℃, centrifugal 20min under 10000rpm again, abandoning supernatant, the flocks that obtains is head product.Head product is behind lyophilize 24h, and mass loss 80%-90% obtains final biological demulsifying agent.
Biological demulsifying agent is made into the chloroformic solution that concentration is 6.25g/L, is that developping agent carries out thin-layer chromatography with chloroform-methanol-water (65: 25: 4), can make triketohydrindene hydrate developer displaing amaranth, is accredited as lipopeptid class biological demulsifying agent.
The preparation of kerosene model milk sap:
With kerosene and distilled water mixed preparing kerosene model milk sap.Kerosene mixes with volume ratio with water at 2: 3, is emulsifying agent with the sorbester p17, and dosage is 1.67% (V/V) of kerosene, stirs 3min under 10000r/min, makes w/o type milk sap.Kerosene mixes with volume ratio with water at 3: 2, is emulsifying agent with sorbester p18 and polysorbate60, and dosage is respectively 0.075%, 0.175% (V/V) of water, stirs 3min under 10000r/min, and restir 3min behind the time-out 30s makes O/W type milk sap.
Embodiment 1:
Broth culture is to the demulsification of kerosene model milk sap:
The rhodococcus S-SL-2 that screening is obtained inserts broth culture, in temperature is 35 ℃, rotating speed is to cultivate 3 days in the shaking table of 140rpm, its full nutrient solution is at the centrifugal 20min of 10000rpm, pour out supernatant liquor, centrifugal thalline is made bacteria suspension with distilled water suspended reduction to initial volume, at W/O, add full nutrient solution respectively in the O/W moulded coal oil model milk sap, supernatant liquor, bacteria suspension, the demulsification that compares the three, and compare with the demulsification that does not add any sample and blank broth culture, the temperature of breakdown of emulsion test is 35 ℃, and dosage is 10% (V/V), the results are shown in Figure 2, Fig. 3.
As can be seen from Figure 2, full nutrient solution, supernatant liquor, bacteria suspension are respectively 94.1%, 98.6% and 18.2% to the demulsification efficiency of w/o type kerosene model milk sap 24h, and the demulsification efficiency of blank and broth culture 24h is all not high, be respectively 12.0% and 55.5%, contain the outer biological emulsion splitter of the born of the same parents that can make kerosene model emulsion breakdown in the supernatant liquor of visible S-SL-2.
As can be seen from Figure 3, the S-SL-2 broth culture does not all surpass 40% to the demulsification efficiency of O/W moulded coal oil model milk sap 24h.S-SL-2 is better than O/W moulded coal oil model milk sap to the demulsification of w/o type kerosene model milk sap.
Embodiment 2:
The paraffin culture is to the demulsification of kerosene model milk sap:
The rhodococcus S-SL-2 that screening is obtained inserts the paraffin substratum, in temperature is 35 ℃, rotating speed is to cultivate 7 days in the shaking table of 140rpm, its full nutrient solution is at the centrifugal 20min of 10000rpm, remove upper strata residue paraffin, behind the liquid of middle level, centrifugal thalline is made bacteria suspension with distilled water suspended reduction to initial volume, at W/O, add full nutrient solution respectively in the O/W moulded coal oil model milk sap, middle level liquid, bacteria suspension, the demulsification that compares the three, and compare with the demulsification that does not add any sample, the temperature of breakdown of emulsion test is 35 ℃, and dosage is 10% (V/V), the results are shown in Figure 4, Fig. 5.
As can be seen from Figure 4, full nutrient solution, middle level liquid, bacteria suspension are respectively 88.2%, 93.2% and 49.7% to the demulsification efficiency of w/o type kerosene model milk sap 24h, and the demulsification efficiency of blank 24h is 12.5%, contains the outer biological emulsion splitter of the born of the same parents that can make kerosene model emulsion breakdown in the middle level liquid of visible S-SL-2.
As can be seen from Figure 5, full nutrient solution, middle level liquid, bacteria suspension are respectively 73.2%, 40.5% and 49.6% to the demulsification efficiency of O/W moulded coal oil model milk sap 24h, compare with w/o type kerosene model milk sap, and the demulsification efficiency of middle level liquid has descended 52.7%.
Embodiment 3:
Biological demulsifying agent is to the demulsification of w/o type kerosene model milk sap:
Supernatant liquor and middle level liquid that S-SL-2 is obtained in broth culture and paraffin substratum, HCl with 6mol/L transfers pH to 2.0 respectively, under 4 ℃, preserve and spend the night, centrifugal 20min under 10000rpm again, abandoning supernatant, obtain the head product of separating out, behind lyophilize 24h, obtain final biological dry powder emulsion splitter with the flocks form.With a small amount of basic solution biological demulsifying agent is dissolved, and with NaOH with pH transfer to 7.0 the back be made into working concentration with distilled water.The demulsification of biological demulsifying agent when testing different dosage the results are shown in Figure 6, Fig. 7.
As can be seen from Figure 6, the biological demulsifying agent that extracts from broth culture is along with the increase of dosage, and to the demulsification efficiency raising of w/o type kerosene model milk sap, when dosage was 100mg/L, the 24h demulsification efficiency reached 89.1%.
As can be seen from Figure 7, the biological demulsifying agent that extracts from the paraffin substratum also increases along with the increase of dosage the demulsification efficiency of w/o type kerosene model milk sap, when dosage surpasses 100mg/L, it is not obvious to the effect that demulsification efficiency improves to continue to strengthen dosage, the maximum demulsification efficiency of 24h is 73.6%, and demulsification is worse than the biological demulsifying agent that extracts in the broth culture.
Embodiment 4:
Biological demulsifying agent of the present invention is applied to the breakdown of emulsion of ocean extraction factory thin oil extraction liquid:
The extraction liquid of Shengli Oil Field ocean extraction factory belongs to w/o type thin oil milk sap, and water ratio is 10%.Under 60 ℃ of conditions, in extraction liquid, add the biological demulsifying agent that from the full nutrient solution of meat soup, extracts, the biological demulsifying agent that from the full nutrient solution of paraffin, extracts respectively, and the on-the-spot polyethers chemical demulsifier that uses, dosage is followed successively by 90mg/L, 140mg/L and 110mg/L, wherein the dosage of chemical demulsifier is 2 times of the actual dosage of ocean extraction factory, and test-results is seen Fig. 8.
As can be seen from Fig. 8,6.5h after, the blank sample that does not add any emulsion splitter is highly stable, do not deviate from any water, though and the dosage of chemical demulsifier has been increased to 2 times of actual dosage, but demulsification efficiency only is 26.7%, and the demulsification of the biological demulsifying agent that extracts from meat soup and the full nutrient solution of paraffin obviously is better than chemical demulsifier, and the demulsification efficiency of 6.5h reaches 62.5% and 60.0% respectively.
Claims (5)
1. a rhodococcus Rhodococcus sp. is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date on September 12nd, 2008, deposit number CGMCC No.2667.
2. " cultivate the method for the described rhodococcus of claim 1; be characterised in that the employing broth culture; the mass ratio of each component is extractum carnis 4.5-5.5 part; peptone 9.5-10.5 part, glucose 4.5-5.5 part, NaCl 4.5-5.5 part; 1000 parts in water; pH 7.0 are perhaps adopted the paraffin substratum, and the mass ratio of each component is: NH
4NO
33.5-4.5 part, K
2HPO
43.5-4.5 part, KH
2PO
45.5-6.5 part, MgSO
47H
2O 0.1-0.3 part, CaCl
22H
2O 0.8-1.2 part, FeSO
47H
2O 0.8-1.2 part, EDTA0.8-1.2 part, whiteruss 4% (V/V), 1000 parts in water, pH 7.0; In temperature is 30-40 ℃, and rotating speed is to cultivate in the shaking table of 120-160rpm.
3. the full nutrient solution of the described rhodococcus of claim 1 is as the application of lipopeptid extra-cellular bio-demulsifier.
4. the method for preparing lipopeptid extra-cellular bio dry powder emulsion splitter, be characterised in that: with full nutrient solution centrifugal 15-25min under 8000-12000rpm of the described rhodococcus of claim 1, remove thalline, clear liquid is transferred pH to 1.5-2.5 with acid solution, under 0-4 ℃, leave standstill, obtain flocks, centrifugal 15-25min under 8000-12000rpm, discard clear liquid, the flocks that obtains is head product; Head product obtains biological dry powder emulsion splitter after lyophilize.
5. the application of biological dry powder emulsion splitter according to claim 4, be characterised in that: biological dry powder emulsion splitter is dissolved with basic solution, and pH is transferred to 7.0 backs use with the solution that distilled water is made into concentration 90～200mg/L, use temperature is between 35～60 ℃, and duration of service is between 18-36 hour.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286536A (en) * | 2011-07-14 | 2011-12-21 | 同济大学 | Method for extracting cell wall combined biological demulsifiers |
| CN105368875A (en) * | 2015-12-19 | 2016-03-02 | 仇颖超 | Method for preparing bio-demulsifier based on rapeseed dreg cultured microorganisms |
| CN105694952A (en) * | 2016-03-15 | 2016-06-22 | 常州市蓝勖化工有限公司 | Preparation method of compound microorganism demulsifying agent |
| CN105727599A (en) * | 2016-02-23 | 2016-07-06 | 同济大学 | Method for adopting magnetic nanometer particles for modifying demulsifying bacteria to reinforce lactescence demulsification |
| CN111876353A (en) * | 2020-07-30 | 2020-11-03 | 董丁 | Bacterial strain for producing biological demulsifier and biological demulsifier produced by bacterial strain |
| CN112159055A (en) * | 2020-10-20 | 2021-01-01 | 山东理工大学 | Electrochemical response type demulsifier for polymer-containing oil sludge sand and preparation and application methods thereof |
| CN112830650A (en) * | 2021-03-04 | 2021-05-25 | 榕知(杭州)信息技术有限公司 | Process for treating oily sludge generated in crude oil exploitation process by microorganisms |
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2008
- 2008-10-15 CN CN200810201236A patent/CN101724579A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286536A (en) * | 2011-07-14 | 2011-12-21 | 同济大学 | Method for extracting cell wall combined biological demulsifiers |
| CN102286536B (en) * | 2011-07-14 | 2014-04-02 | 同济大学 | Method for extracting cell wall combined biological demulsifiers |
| CN105368875A (en) * | 2015-12-19 | 2016-03-02 | 仇颖超 | Method for preparing bio-demulsifier based on rapeseed dreg cultured microorganisms |
| CN105727599A (en) * | 2016-02-23 | 2016-07-06 | 同济大学 | Method for adopting magnetic nanometer particles for modifying demulsifying bacteria to reinforce lactescence demulsification |
| CN105727599B (en) * | 2016-02-23 | 2017-11-07 | 同济大学 | The method that demulsifying bacteria is used to strengthen emulsion breakdown is modified using magnetic nano-particle |
| CN105694952A (en) * | 2016-03-15 | 2016-06-22 | 常州市蓝勖化工有限公司 | Preparation method of compound microorganism demulsifying agent |
| CN111876353A (en) * | 2020-07-30 | 2020-11-03 | 董丁 | Bacterial strain for producing biological demulsifier and biological demulsifier produced by bacterial strain |
| CN111876353B (en) * | 2020-07-30 | 2022-03-29 | 董丁 | Bacterial strain for producing biological demulsifier and biological demulsifier produced by bacterial strain |
| CN112159055A (en) * | 2020-10-20 | 2021-01-01 | 山东理工大学 | Electrochemical response type demulsifier for polymer-containing oil sludge sand and preparation and application methods thereof |
| CN112830650A (en) * | 2021-03-04 | 2021-05-25 | 榕知(杭州)信息技术有限公司 | Process for treating oily sludge generated in crude oil exploitation process by microorganisms |
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Open date: 20100609 |