CN104232708A - Culture medium for metabolism production of rhamnolipid and application of culture medium - Google Patents
Culture medium for metabolism production of rhamnolipid and application of culture medium Download PDFInfo
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- CN104232708A CN104232708A CN201310558200.4A CN201310558200A CN104232708A CN 104232708 A CN104232708 A CN 104232708A CN 201310558200 A CN201310558200 A CN 201310558200A CN 104232708 A CN104232708 A CN 104232708A
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- rhamnolipid
- substratum
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Abstract
The invention relates to a culture medium of microbial enhanced oil recovery, and particularly relates to a culture medium for metabolism production of rhamnolipid and an application of the culture medium. The culture medium takes glycerinum as a carbon source and NaNO3 as an inorganic nitrogen source. According to the culture medium provided by the invention, an anaerobic strain for producing the rhamnolipid can be effectively promoted to carry out quick metabolism to produce the rhamnolipid under an anoxic condition, and an effective nutrient activator formula is provided for activating in-situ related functional strains of the reservoir to carry out microbial oil displacement.
Description
Technical field
The present invention relates to the substratum of microbe oil production, particularly relate to substratum and application thereof that rhamnolipid is produced in a kind of metabolism.
Background technology
Rhamnolipid is a kind of bio-surfactant produced by microbial metabolism, and improving oil recovery factor (MEOR) technical field in microorganism has important application potential.To the existing a lot of research of rhamnolipid aerobic fermentation condition optimization, but also research is lacked to the substratum that relevant bacteria species under anaerobic metabolism produces rhamnolipid used.
At present, in the technology utilizing rhamnolipid raising recovery ratio, mostly adopts fermentative production rhamnolipid on the ground, and then inject oil reservoir, but the cost of the operation and maintenance of aerobic fermentation equipment and the extraction of product and transport is higher in oil field; And underground aerobic fermentation, then need to inject air, the problems such as this brings again operability poor, poor stability, and equipment requirements is higher, the oxidizable corrosion of air injection pipeline to oil reservoir.Therefore, obtain a kind of metabolism and produce the substratum producing rhamnolipid, above-mentioned bottleneck problem can be solved.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, the invention provides substratum and application thereof that rhamnolipid is produced in a kind of metabolism.
For achieving the above object, the technical solution used in the present invention is:
A substratum for rhamnolipid is produced in metabolism, and in substratum, carbon source is glycerine, and inorganic nitrogen-sourced is NaNO
3do.
Described substratum by massfraction is: glycerine 3-4%, NaNO
30.2-0.4%, K
2hPO
43H
2o0.35-0.45%, KH
2pO
40.4-0.5%, MgSO
47H
2o0.003-0.005%, CaCl
20.01-0.02%, KCl0.08-0.14%, NaCl0.08-0.14%, yeast powder 0.1-0.2%, surplus is water.
Further, described substratum by massfraction is: glycerine 3.6%, NaNO
30.3%, K
2hPO
43H
2o0.42%, KH
2pO
40.47%, MgSO
47H
2o0.004%, CaCl
20.013%, KCl0.1%, NaCl0.1%, yeast powder 0.16%, surplus is water.
An application for the substratum of rhamnolipid is produced in metabolism, and described substratum produces the substratum of rhamnolipid tensio-active agent as metabolism.
Described substratum metabolism is utilized to produce rhamnolipid tensio-active agent under anaerobic condition.
The advantage that the present invention has:
Rhamnolipid bacterial classification will be produced in substratum of the present invention, under anaerobic rhamnolipid is produced in metabolism rapidly, utilize good water solubility in substratum and very easily utilized glycerine for carbon source by microbial metabolism, shorten anaerobism and produce the growth of rhamnolipid bacterium cell and the time needed for rhamnolipid synthesis, compensate for the defect slowly of cellular metabolism under anoxic conditions to a certain extent.The anaerobism existed in oil reservoir anoxic habitat can be activated simultaneously and produce rhamnosyl lipid function yeast, thus improve oil recovery factor, and then the microbial oil displacement to oil reservoir original position, reduce Infrastructure and production unit expense; Reduce cost for oil production.
Substratum of the present invention can promote anaerobism produce rhamnolipid bacterial classification under anoxic conditions rapidly metabolism produce rhamnolipid, shorten fermentation period.Wherein substratum provides effective nutrition activator, and the anaerobism that can activate origin produces rhamnolipid, and anaerobic condition bottom fermentation produces rhamnolipid simultaneously, can avoid the many unfavorable factors in aerobic fermentation process.
Accompanying drawing explanation
The rhamnosyl typical curve that Fig. 1 provides for the embodiment of the present invention.
Embodiment
Embodiment 1
Substratum by massfraction is: glycerine 3.6%, NaNO
30.3%, K
2hPO
43H
2o0.42%, KH
2pO
40.47%, MgSO
47H
2o0.004%, CaCl
20.013%, KCl0.1%, NaCl0.1%, yeast powder 0.16%, surplus is distilled water, pH nature.Namely cultivate basigamy complete after without the need to adjusting pH.
NaNO is chosen in above-mentioned substratum
3as inorganic nitrogen-sourced, the anaerobism function yeast ensureing to produce rhamnolipid under anaerobic carries out the energy of nitrate respiration acquisition needed for growth metabolism.
Embodiment 2
Utilize above-mentioned substratum fermentative production rhamnolipid in anaerobism bottle:
1) bacterial strain is Pseudomonas sp ANBIOSURF-1 (the relevant record in Pseudomonas sp ANBIOSURF-1 is see Albino et al.2011Partial characterization of biosurfactant produced under anaerobic conditions by Pseudomonas sp ANBIOSURF-1).
2) anaerobic culture medium preparation method: the above-mentioned substratum prepared is boiled about 15min, to resazurin indicator by the thin out redness of blueness; Then, under high pure nitrogen protection, be dispensed in anaerobism bottle; 121 DEG C, sterilizing 20min; Inject 2.5% (w/v) Na of filtration sterilization
2s9H
2o reductive agent, to final concentration 0.02%, removes residual oxygen.
3) fermentation condition: by the cultivation after above-mentioned for 200mL sterilizing based in 250mL anaerobism bottle, then by above-mentioned bacterial strains by the inoculum size inoculation of 6%, in the rotating speed, the constant incubator of 40 DEG C of 80r/min, cultivate 7d, pH nature.
4) quantitative assay of rhamnolipid in fermented liquid:
Adopt orcinol-sulfuric acid process, glycosyl molecule dewaters under strongly acidic conditions and generates furfural or derivatives thereof, reacts and can generate band color substance, have maximum absorption at 421nm place with orcinol.
Sample preparation: fermented liquid first 10,000 × g, centrifugal 20min, to remove somatic cells; Get 0.5mL supernatant liquor 1mL extracted with diethyl ether three times, then merge upper organic phase in clean tube; Test tube is placed in ventilating kitchen, ether is vapored away completely; Add 0.5mL distilled water.
The preparation of standard substance: the L-rhamnosyl solution preparing a series of concentration (0-0.05g/L), gets L-rhamnosyl and dries to weight at 105 DEG C, take 25mg and be dissolved in distilled water, be settled to 250mL, obtain mass concentration 0.10g/L rhamnolipid standardized solution.Get 2.5mL respectively, 5.0mL, 7.5mL, 10.0mL, 12.5mL, 15.0mL, 17.5mL, 20.0mL, 22.5mL, 25.0mL rhamnosyl standardized solution, is settled to 50mL to distilled water, obtain mass concentration and be respectively 0.005,0.010,0.015,0.020,0.025,0.030, the serial rhamnosyl solution of 0.035,0.0400.045,0.050g/L.
The standardized solution of above-mentioned acquisition or sample are carried out mixing being placed in test tube with the volume ratio (ml) of orcinol solution according to 1:9; Wherein, orcinol solution is 0.19%(mass concentration) orcinol be dissolved in 53%(volumetric concentration) H
2sO
4in.
By above-mentioned test tube in 80 DEG C of water-bath 30min, under room temperature, cool 15min; Measure its OD
421.The blank of standard solution replaces rhamnosyl standard substance with distilled water, and the blank of fermented liquid replaces fermented liquid with nonvaccinated substratum.Obtain the mass concentration (see figure 1) of rhamnosyl according to typical curve, be multiplied by relation conefficient 3, obtain the mass concentration of rhamnolipid, finally draw rhamnolipid mass concentration in fermented liquid.
Under anaerobism bottle culture condition, utilize this anaerobism to produce rhamnolipid substratum, rhamnolipid output is 3.12 ± 0.11g/L.
Embodiment 3
The physical simulation core oil displacement experiment that above-mentioned anaerobism produces rhamnolipid substratum is evaluated:
1) artificial cores process: vacuumize saturated local water, saturated oil, aging.Calculate volume of voids PV=rock core weight in wet base-rock core dry weight; Artificial cores volume of voids PV=2338.7g-2233.3g)/1g*mL
-1=105.4mL.
2) water drive: utilize local water to carry out a water drive to core, reach more than 98% to water ratio, calculates waterflood recovery efficiency factor; Calculate waterflood recovery efficiency factor=oil pump capacity/saturated oil mass × 100%;
Concrete waterflood recovery efficiency factor=(43mL/68mL) × 100%=63.6%.
3) above-described embodiment substratum is injected core: by 0.3PV sterilizing and this anaerobism of deoxygenation produce rhamnolipid substratum and inject core, wherein, contrast core injects the local water of 0.3PV.
4) cultivate: close rock core two ends, simulated formation temperature cultivates one month.
5) secondary water drive: method is with a water drive.Calculate the recovery ratio of secondary recovery factor and raising; Wherein,
Secondary recovery factor=(intermediate water displaces oily total amount/saturated oil mass) × 100%;
The recovery ratio improved=(secondary recovery factor of the secondary recovery factor-contrast core of experiment core)
Be respectively through the secondary recovery factor of secondary water drive and the recovery ratio of raising:
Secondary recovery factor=(8mL/68mL) × 100%=11.7%
Recovery ratio=the 11.7%-0.9%=10.8% improved.
In the process that simulated formation temperature is cultivated, this anaerobism is produced rhamnolipid substratum in artificial cores, is activated anaerobism product rhamnolipid function yeast relevant in local water, and promote that it produces rhamnolipid tensio-active agent, act on the crude oil in core, thus on a water drive basis, improve oil recovery factor reach 10.8 percentage points.
Claims (5)
1. a substratum for rhamnolipid is produced in metabolism, and it is characterized in that: in substratum, carbon source is glycerine, inorganic nitrogen-sourced is NaNO
3.
2. produce the substratum of rhamnolipid by metabolism according to claim 1, it is characterized in that: described substratum by massfraction is: glycerine 3-4%, NaNO
30.2-0.4%, K
2hPO
43H
2o0.35-0.45%, KH
2pO
40.4-0.5%, MgSO
47H
2o0.003-0.005%, CaCl
20.01-0.02%, KCl0.08-0.14%, NaCl0.08-0.14%, yeast powder 0.1-0.2%, surplus is water.
3. produce the substratum of rhamnolipid by metabolism according to claim 2, it is characterized in that: described substratum by massfraction is: glycerine 3.6%, NaNO
30.3%, K
2hPO
43H
2o0.42%, KH
2pO
40.47%, MgSO
47H
2o0.004%, CaCl
20.013%, KCl0.1%, NaCl0.1%, yeast powder 0.16%, surplus is water.
4. an application for the substratum of rhamnolipid is produced in metabolism according to claim 1, it is characterized in that: described substratum produces the substratum of rhamnolipid tensio-active agent as metabolism.
5. produce the application of the substratum of rhamnolipid by metabolism according to claim 4, it is characterized in that: under anaerobic condition, utilize described substratum metabolism to produce rhamnolipid tensio-active agent.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108298780A (en) * | 2018-01-19 | 2018-07-20 | 中国科学院沈阳应用生态研究所 | A kind of biological cleaner and application method of processing oily sludge |
| CN113016800A (en) * | 2021-03-11 | 2021-06-25 | 辽宁大学 | Oil field sulfate reducing bacteria enrichment culture inhibitor and inhibition method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000029604A1 (en) * | 1998-11-18 | 2000-05-25 | The University Of Akron | Production of biological materials by simultaneous aerobic and anaerobic respiration |
-
2013
- 2013-11-08 CN CN201310558200.4A patent/CN104232708A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000029604A1 (en) * | 1998-11-18 | 2000-05-25 | The University Of Akron | Production of biological materials by simultaneous aerobic and anaerobic respiration |
Non-Patent Citations (2)
| Title |
|---|
| 钱欣平: "利用不同碳源合成生物表面活性剂的研究", 《日用化学工业》 * |
| 钱欣平: "营养和环境因素对甘油发酵生产鼠李糖的影响", 《食品与发酵工业》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108298780A (en) * | 2018-01-19 | 2018-07-20 | 中国科学院沈阳应用生态研究所 | A kind of biological cleaner and application method of processing oily sludge |
| CN108298780B (en) * | 2018-01-19 | 2021-07-06 | 中国科学院沈阳应用生态研究所 | Biological cleaning agent for treating oily sludge and use method |
| CN113016800A (en) * | 2021-03-11 | 2021-06-25 | 辽宁大学 | Oil field sulfate reducing bacteria enrichment culture inhibitor and inhibition method |
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Application publication date: 20141224 |