CN103865820B - A kind of rattan Flavimonas and Synthesis and applications thereof - Google Patents
A kind of rattan Flavimonas and Synthesis and applications thereof Download PDFInfo
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- CN103865820B CN103865820B CN201210528104.0A CN201210528104A CN103865820B CN 103865820 B CN103865820 B CN 103865820B CN 201210528104 A CN201210528104 A CN 201210528104A CN 103865820 B CN103865820 B CN 103865820B
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Abstract
The present invention relates to a kind of rattan Flavimonas and application thereof; Rattan Flavimonas (Luteimonas? sp.) HB-2CGMCC? No.6457 is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and the preservation time is on 08 17th, 2012; This rattan Flavimonas is used for oil reservoir oil displacement, and bacterial strain is adopted 12m under 45 DEG C of conditions
3industrial fermentation tank ferments 48 hours, and the bacterium liquid after fermentation is injected into oil reservoir according to the mass concentration of 1% and the nutritive medium of quality 1% from water injection well, and injection rate is 0.07PV; Viscosity of crude declines 53%, and improve recovery ratio and be greater than 5.0%, input-output ratio is 1:6.4.
Description
Technical field
The present invention relates to microbe oil production field, be specifically related to a strain rattan Flavimonas and preparation thereof and the application in petroleum production engineering.
Background technology
Microbial Enhanced Oil Recovery is by the microorganism of screening is together injected oil reservoir with nutritive medium, or inject the endogenous microbes that activator activates oil reservoir, utilize the interaction of liquid phase and solid phase in the useful activity of microorganism in oil reservoir and meta-bolites thereof and oil reservoir, improve the flowing property of crude oil, thus improve the integrated technology of oil recovery factor.Microbial Enhanced Oil Recovery is through development for many years, and becoming a current development raising oil recovery factor technology rapidly gradually, is also 21 century High biotechnology.
Microbe Oil Recovery mainly contains microorganism self to the degraded of crude oil and meta-bolites thereof to the effect of crude oil, it take oil as carbon source that the Degradation of Microbes On Crude Oil is embodied in microorganism, long chain alkane is degraded by microorganisms as short chain alkanes, thus increases crude oil fluidity; And meta-bolites effect mainly comprises bio-surfactant, organic acid, biogas and micromolecular organism, these components can reduce viscosity of crude to a certain extent, increase crude oil fluidity, thus start the effect improving recovery ratio.Obviously, improve the size of recovery ratio action effect and the kind of microorganism and function here and have much relations.
Middle low temperature heavy crude reservoir due to the viscosity of crude oil own large, and reservoir temperature is lower, result causes the non-constant of crude oil fluidity, and conventional waterflood development effectiveness is undesirable, by screening efficient viscosity reduction bacterial classification and to improve recovery ratio research for low temperature heavy crude reservoir significant.
According to consulting domestic and international patent and document discovery, the bacterial classification filtered out at present for microbial oil displacement has Xanthomonas campestris, pseudomonas putida, Pseudomonas aeruginosa, Bacillus licheniformis, thermophilic denitrifying bacillocin, enterobacteria, bacillus brevis, bacillus cereus, splits hydrocarbon bar bacterium, hare bar bacterium, yeast addicted to oil bacillus, Bacillus subtilus, candida tropicalis, torulopsis, viscous Serratia, enterobacter cloacae, Shandong epidemic disease suis, Bacillus fusiforms, Potsdam bacillus brevis, acinetobacter calcoaceticus, bright string coccus.Desk research shows, these bacterial strains all have reduction fermented liquid surface tension and the oil water interfacial tension of certain reduction, part can degrading crude oil, there is the function reducing viscosity of crude, but there is certain limitation, such as formation temperature, the aspects such as the adaptability of salinity and oil property, and seldom have through the evaluation of thing mould oil displacement experiment and effect of field application analysis.
Summary of the invention
The object of the invention is: provide a strain can effectively to degrade high wax and high resin crude, reduce viscosity of crude and also improve a kind of rattan Flavimonas of oil recovery factor and Synthesis and applications thereof.
Technical scheme of the present invention is: rattan Flavimonas (Luteimonassp.) HB-2CGMCCNo.6457 provided by the present invention is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, the preservation time is on 08 17th, 2012.
Rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 is separated and obtains from Bao Lige produced liquid in oil well, and its bacterium colony is faint yellow, and circular, smooth surface is opaque, neat in edge, and diameter is 1.5mm.
Cell is shaft-like, and size is 0.5 μm × 1.0 ~ 2.0 μm, single or arrange in pairs.Gram-negative, not raw spore, produces β-polyhydroxybutyrate salt particle.Concrete bio-chemical characteristics is in table 1.
Table 1 rattan Flavimonas part physiological and biochemical property
Note: "+" represents growth or reacting positive, "-" expression does not grow or reaction negative
Experiment proves, rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 can effective degrading crude oil improve oil property, viscosity of crude after effect drops to 700mPa.S by 2300mPa.S, fall glutinous rate and reach 69.6%, in oil component, light component increases, heavy component reduces, and crude oil fluidity waits until obvious improvement.This bacterial strain can produce a certain amount of organic acid and bio-surfactant, and the surface tension of fermented liquid reduces by 58%, and oil water interfacial tension reduces 92%, can make crude oil and the complete emulsification of water, form typical O/w emulsion.This bacterial strain can be widely used in petroleum production engineering and petroleum transportation field, particularly microbial enhanced waterflooding increase oily field.
The screening of rattan Flavimonas (Luteimonassp.) CGMCCNo.6457, is made up of the following step:
(1) sampling and enrichment
(2) screening and purifying
(3) bacterial strain Displacement Efficiency
(4) on-the-spot oil displacement experiment
Concrete technology step and processing condition are:
Step (1) sampling and enrichment: sample from Bao Lige oilfield produced fluid and excessive fuel consumption hole, then adopt and carry out enrichment with enrichment medium.
Enrichment medium mass percent consists of: glucose 2%, peptone 0.05%, yeast powder 0.05%, urea 0.05%, ammonium sulfate 0.05%, potassium primary phosphate 0.5%, 7 water magnesium sulfates 0.02%, sodium-chlor 0.01%, and tap water is prepared.
The processing condition preparing enrichment medium are: pH value 6 ~ 8, and 115 DEG C of sterilizings 20 minutes, culture temperature is 25-45 DEG C, and incubation time is 8-48 hour.
Step (2) bacteria selection and purifying:
The Bao Lige mixing oil of the mixed bacteria liquid of quality 2% and quality 5% is joined in enrichment medium, shaking table constant temperature culture 48 hours under 45 DEG C of conditions, observe the emulsification situation of crude oil and measure the surface and interface tension value of fermented liquid, treat that crude oil dispersion and emulsion and fermented liquid table/interfacial tension value can reduce the separation and purification that the sample being greater than more than 50% carries out next step, separation substratum: nutrient agar medium+5% defiber sheep blood completely.To the experiment of the single strain repeating step (2) of separation and purification, finishing screen selects a plant height effect edge-water encroachment fungoid, determines that this bacterial strain is rattan xanthomonas through 16srDNA sequential analysis in conjunction with bio-chemical characteristics.
Step (3) bacterial strain Displacement Efficiency
Displacement Efficiency is carried out to the rattan Flavimonas of screening, comprises crude oil total hydrocarbon proximate analysis before and after microbial process, microbial metabolites analysis, glutinous emulsifying effectiveness analysis and thing mould oil displacement experiment are fallen to crude oil.
Step (4) on-the-spot oil displacement experiment
Bacterial strain is adopted 12m under 45 DEG C of conditions
3industrial fermentation tank ferments 48 hours, and the bacterium liquid after fermentation is injected into oil reservoir according to the mass concentration of 1% and the nutritive medium of quality 1% from water injection well, and injection rate is 0.07PV, drives effect carry out tracking monitor from corresponding oil well to microorganism.
The invention has the beneficial effects as follows:
Rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 of the present invention has excellent reduction viscosity of crude and improves the performance of oil recovery factor, indoor logistics organizations improves recovery ratio and reaches 15.4%, on-the-spot oil displacement efficiency experiment also serves positive effect, the object bacteria monitored in production fluid becomes dominant microflora, and bacterium is dense reaches 10
6individual/mL, water_bearing escalating rate is effectively controlled, and viscosity of crude declines 53%, and improve recovery ratio and be greater than 5.0%, input-output ratio is 1:6.4.This invention can be widely used in microbial oil displacement field, and the use that suits large area to popularize, have broad application prospects.
Accompanying drawing explanation
Fig. 1 rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 sequencing sequence;
Crude oil gas chromatogram (before microbial process) before Fig. 2 A microbial process
Crude oil gas chromatogram (after microbial process) after Fig. 2 B microbial process
Fig. 3 rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 thing mould oil displacement efficiency.
Fig. 4 rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 thing mould oil displacement efficiency figure
Embodiment
In subordinate's embodiment, method therefor is ordinary method if no special instructions, and all percentage concentrations are mass percent, and the solvent in all substratum is distilled water.
The screening of embodiment 1, rattan Flavimonas (Luteimonassp.) CGMCCNo.6457, cultivation and the treatment effect to crude oil
The screening of rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 and cultivation:
In 250mL triangular flask, add 99mL produced liquid in oil well sewage, and then add the substratum of 1%, at temperature is 45 DEG C, constant temperature vibration 18-24 hour.The each constituent mass per-cent of substratum is:
Glucose 2%, peptone 0.05%, yeast powder 0.05%, urea 0.05%, ammonium sulfate 0.05%, potassium primary phosphate 0.5%, 7 water magnesium sulfates 0.02%, sodium-chlor 0.01%, all the other are production fluid sewage.Cultivate 3 ~ 5 days at temperature is 25 ~ 30 DEG C, then draw the above-mentioned bacterium liquid of 1mL with aseptic rifle head and add to fill in 9mL sterilized water test tube and fully mix, be mixed with 10 with this
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6different diluent.Respectively from 10
-2, 10
-3, 10
-4in three pipe diluents, each 100ul that draws is on solid LB culture plate, the each constituent mass per-cent of solid LB media: extractum carnis 5%, peptone 1%, NaCl1%, agar 2%, all the other are distilled water, be cultivate 24h in 30 DEG C of incubators in temperature, occur faint yellow thalline, pick out this thalline with transfering loop from flat board, solid LB media to be rule purifying 2-3 time, obtain rattan Flavimonas (Luteimonassp.) CGMCCNo.6457.Above-mentioned substratum is all 121 DEG C, sterilizing 20 minutes under 0.1Mpa.
Rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 is to oil degradation effect analysis:
Process of the test: bacterial strain rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 is activated 24 hours with LB substratum, then enrichment medium (glucose 2% is transferred to by the inoculum size of 5%, peptone 0.05%, yeast powder 0.05%, urea 0.05%, ammonium sulfate 0.05%, potassium primary phosphate 0.5%, 7 water magnesium sulfates 0.02%, sodium-chlor 0.01%, tap water is prepared) in, add the crude oil of 5%, at temperature 45 C, rotating speed 150rpm shaking table is cultivated 7 days, laggard for dehydrating of crude oil row gas-chromatography total hydrocarbon is analyzed, and do blank with crude oil before microbial process, measure petroleum concentration by Infrared Oil Determination Instrument simultaneously, calculate petroleum degradation rate, the results are shown in Figure 2.Experimental result shows, rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 has good Degradation to crude oil, and degradation rate reaches more than 50%, and the crude oil light component relative content after effect increases, and heavy component reduces, and pristane/C
17, phytane/C
18all obviously increase, the mobility of illustration and crude oil increases.
Rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 falls glutinous emulsifying effectiveness analysis to crude oil:
Process of the test: bacterial strain rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 is activated 24 hours with LB substratum, then enrichment medium (glucose 2% is transferred to by the inoculum size of 5%, peptone 0.05%, yeast powder 0.05%, urea 0.05%, ammonium sulfate 0.05%, potassium primary phosphate 0.5%, 7 water magnesium sulfates 0.02%, sodium-chlor 0.01%, tap water is prepared) in, add the crude oil of 5%, at temperature 45 C, rotating speed 150rpm shaking table carries out cultivation 2 days, the emulsification situation of crude oil was not observed every 2 hours, experiment finds that rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 just can by complete for crude oil emulsification at 8 hours, and form O/W type emulsion, microbial process after 2 days viscosity of crude drop to 700mPa.S by 2300mPa.S, fall glutinous rate and reach 69.6%.The surface tension of fermented liquid reduces by 58%, and oil water interfacial tension reduces 92%.
Embodiment 2, Methanogenesis
Bacterial strain rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 is activated 24 hours, is then transferred to enrichment medium (glucose 2%, peptone 0.05% by the inoculum size of 5%, yeast powder 0.05%, urea 0.05%, ammonium sulfate 0.05%, potassium primary phosphate 0.5%, 7 water magnesium sulfates 0.02%, sodium-chlor 0.01%, tap water is prepared) in, temperature 45 C, rotating speed 150rpm shaking table carry out cultivation 2 days, carry out Methanogenesis to fermented liquid, analytic process is as follows:
(1) mensuration of tensio-active agent
Get 50mL fermented liquid, use 100mL extracted with diethyl ether after filtering 3 times, extract is collected by after ether evaporate to dryness, add the hydrochloric acid soln of 2mL6mol/L, sealing hydrolysis, evaporate to dryness, Silylation reagent solution 0.500mL is added 60 degrees Celsius of reactions 20 minutes after drying, measure with GC-MS, identify amino acid and the hydroxy fatty acid of derivatize, according to the mass-to-charge ratio in total ion chromatogram and amino acid area, qualitative and quantitative analysis is carried out to product, the results are shown in Figure 3, the surfactant component that rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 produces by analysis is lipopeptid, molecular weight is 1035, output is 1.25g/L.
(2) organic acid analysis
Get fermented liquid 10mL bactofugation body, dry by ammoniacal liquor adjust ph 10, add butanols 0.5mL, add 3 vitriol oils again and make catalyzer, supersound process mixing 2min, 90 ° of C lower seal reaction regular hours, in reaction process, interval 10min or 20min rocks. and reaction terminates, cooling, add 5.0mL deionized water, mixing, 3 (1mL are extracted with dodecane, 0.5mL, 0.5mL), after stratification, upper organic phase is drawn in 10.0mL colorimetric cylinder with dropper, add extraction agent be dissolved to 5.00mL. add anhydrous sodium sulphate concussion 2min, carry out gas chromatographic analysis.
GC conditions: 250 DEG C; Detector temperature: 250 DEG C; Carrier gas: High Purity Nitrogen, sample size: 2.0 μ L; Column temperature: starting temperature 100 DEG C (5min), temperature rise rate 10 ° of C/min, stop temperature 250 DEG C (3min); Flow rate of carrier gas: 15.00mL/min; Hydrogen flow rate: 26.50mL/min; Air velocity: 252.31mL/min; Carrier gas flux: 3.5 circles; Splitting ratio: 1.5 circles; Hydrogen flowing quantity: 4.5 circles, air flow quantity: 6 circles; GC-MS analytical procedure: column temperature: starting temperature 60 DEG C (1min); Temperature rise rate 15 ° of C/min; Stop temperature 150 DEG C (5min).According to GC-MS qualitative and quantitative analysis, rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 mainly produces formic acid, acetic acid, 2 hydroxy propanoic acid and butyric acid, and its output is respectively 18.5,72.1,9.5 and 15.8mg/L.
Embodiment 3, indoor thing mould oil displacement experiment
Experimental model: testing by artificial back-up sand basket model length is 100cm, internal diameter 2.5cm.
Experimental water, oil, bacterium liquid:
Experimental water is Bao Lige oil field production water, belongs to NaHCO
3type, salinity is 7105mg/L.
Experiment oil is Bao Lige oil field bar 51-34 well crude oil, and viscosity of crude is 525mP.S.
Experiment with bacterium liquid be rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 experiment with micro-ly driving that nutritive medium is 0.6% glucose, 0.1% peptone, 0.08% yeast extract paste, 0.1% ammonium chloride, 0.1% phosphoric acid hydrogen two receives, 0.02% potassium primary phosphate, total concn is 1.0%.
Experimental implementation is followed successively by: rock core is found time saturated local water, injects saturated oil, aging 5 days, injects 1PV " bacterium liquid (5 × 10 after water drive reaches 95%
6individual/mL)+1.0% nutrition " slug; then close basket two ends valves; carry out water drive in fermentation after 2 days; stop experiment when water ratio reaches more than 98%; micro-driving in process monitors the dense and oil recovery factor of bacterium; parallel 3 groups of oil displacement experiment, whole process control injection speed is 0.2mL/min, and experimental result is shown in Fig. 4.
As can be seen from Figure 4, rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 can improve recovery ratio 15.4%, and the bacterium of exit end is dense can reach 4 × 10
7individual/mL, and production fluid Crude Oil viscosity degradation more than 50%, show rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 can under reservoir media effective growth and breeding, by microorganism itself, obvious displacement of reservoir oil effect is played to the degraded of crude oil and the effect of meta-bolites.
Example 4, field experiment
At Bao Lige scene, bar 51 fault blocks select two mouthfuls of representational well groups to carry out rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 oil displacement experiment, and bacterial strain is adopted 12m under 45 DEG C of conditions
3industrial fermentation tank ferments 48 hours, and the bacterium liquid after fermentation is injected into oil reservoir according to the concentration of 1% and the nutritive medium of 1% from water injection well, and injection rate is 0.07PV, drives effect carry out tracking monitor from corresponding oil well to microorganism.Analytical results shows, rattan Flavimonas (Luteimonassp.) CGMCCNo.6457 of injection can growth and breeding well at oil reservoir, and production fluid bacterium is dense reaches 10
6individual/more than mL, water_bearing escalating rate is effectively controlled, and viscosity of crude declines 53%, and micro-validity period of driving is 8 months, and improve recovery ratio and be greater than 5.0%, input-output ratio reaches 1:6.4.
Claims (2)
1. rattan Flavimonas (Luteimonassp.) HB-2 bacterial strain, it is characterized in that: described bacterial strain is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, its deposit number is CGMCCNo.6457, and the preservation time is on 08 17th, 2012.
2. an application for rattan Flavimonas according to claim 1, is characterized in that: this rattan Flavimonas is used for oil reservoir oil displacement, and described bacterial strain is adopted 12m under 45 DEG C of conditions
3industrial fermentation tank ferments 48 hours, bacterium liquid after fermentation and nutritive medium are all injected into oil reservoir according to the mass concentration of 1% from water injection well, injection rate is 0.07PV, each composition of described nutritive medium and component content as follows: glucose 0.6%, peptone 0.1%, yeast powder 0.08%, ammonium chloride 0.1%, Sodium phosphate dibasic 0.1%, potassium primary phosphate 0.02%, wherein the total mass concentration of each composition is 1.0%.
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CN110922011A (en) * | 2019-12-06 | 2020-03-27 | 朱玉波 | Microbial agent for degrading oily sludge and COD (chemical oxygen demand) and application method thereof |
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CN114574388B (en) * | 2022-03-03 | 2023-05-02 | 云南农业大学 | Gamboge pseudomonas LC202103 strain for degrading alpha-solanine and preparation method and application thereof |
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