CN102559194A - Method for controlling process of degrading crude oil by microbes for oil extraction and application of method - Google Patents
Method for controlling process of degrading crude oil by microbes for oil extraction and application of method Download PDFInfo
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Abstract
The invention relates to a method for controlling the process of degrading crude oil by microbes. The method comprises the step of: determining parameters of variety, concentration, using amount of a surfactant by studying the influence of the surfactant on the growth of microbes and the degradation of the crude oil. The invention also provides a degradant for petroleum pollutants, and the degradant comprises oil extraction bacteria and the determined surfactant. The invention also provides a method for degrading the petroleum pollutants by utilizing the microbes, and the method comprises the step of pouring the determined degradant for the petroleum pollutants into polluted environment. By the method, the process of degrading the crude oil by the microbes can be controlled, and the pollution of petroleum to the environment is reduced or eliminated under the combined action of the surfactant and degradation bacteria.
Description
Technical field
The invention belongs to biological treating petroleum pollution technical field, particularly relate to control method and application thereof in a kind of microbiological deterioration petroleum pollution process.
Background technology
Along with going deep into of industrialization development, urbanization process, the usage quantity of oil strengthens, and the environmental pollution that brings thus constantly aggravates.Petroleum substance gets into the destruction that soil causes structure of soil ecosystem and function, has a strong impact on soil permeability, and soil fertility is descended, thereby the poisonous plant growth destroys surface vegetation, causes grain drop in production; Can cause serious harm to human health through food chain enrichment in human body behind the petroleum pollution ocean, simultaneously, petroleum pollution gets into the ocean can produce considerable damage to hydrobiological growth, growth, breeding and whole ecological system.At present, petroleum pollution problem becomes the countries in the world question of common concern.
Oil is the mixture that various alkane, naphthenic hydrocarbon and aromatics are formed, and wherein also contains heterogeneous ring compound; Polycyclc aromatic compound wherein (PAHs) and some heterocyclic materials (like carbazoles, thiophene-based, furans etc.) have persistence and carinogenicity, are the environmental poisonous substances of generally acknowledging.
At present, mainly rely on physics, chemical processes such as mechanical frizing and chemisorption to carry out the improvement that petroleum-type is polluted.But physics, chemical process cost height, investment are greatly, and have secondary pollution, and therefore, this is not the best practice that solves petroleum pollution.In recent years, utilize mikrobe to repair the attention that contaminated environment causes people.Have many good qualities because utilize mikrobe to repair the pollution of allos biomass, such as: pollutent is degraded in the original place; On-the-spot disposal is easy and simple to handle, surrounding environment is disturbed few; The reparation funds are less; The chance that the mankind are directly exposed under these pollutents reduces; Less or do not produce secondary pollution, leftover problem is few etc.Yet it also is not obvious especially that simple microbial treatment pollutes on the scheme effect, therefore has the dealer to study tensio-active agent and the shared improvement of carrying out petroleum pollution of mikrobe.
There is document to show (solublization of surface-active substance confrontation polycyclic aromatic hydrocarbons and the influence of mikrobe utilizability; Wu Yingqin, 2007), tensio-active agent can promote the release of PAHs; Make PAHs be assigned in the surfactant micelle or be adsorbed onto on the surfactant monomer; Improve the PAHs apparent solubility, thereby moving of promotion and PAHs improves the biological efficient that PAHs is degraded.
Tensio-active agent is directly related with the kind of the tensio-active agent that is adopted to the solublization and the biological degradation of pollutent.(Journal of Hazardous Materials such as Doong; 2003) tested of the influence of different surfaces promoting agent to polycyclic aromatic hydrocarbons solubilising and biological degradation; In the tensio-active agent of finding to be tested; The less tensio-active agent Tritonx-100 of HLB value is stronger to the solublization of polycyclic aromatic hydrocarbons, and the SDS solublization that the HLB value is bigger is less; TritonX-100 can strengthen the biodegradability of polycyclic aromatic hydrocarbons, and the different surfaces promoting agent is relevant with the biological degradability of tensio-active agent to the biodegradable influence of pollutent.
TWeen-80 is the non-ionics that is formed by anhydrous sorbitol oleic acid simple substance and oxyethane adduction, has good solubilising and emulsifying effect, and its toxicity and micelle-forming concentration are also less, is ideal degradation of promoter comparatively.(Chinese Journal of Applied Ecology such as Song Yufang; 1999) studied nonionogenic tenside TWeen-80 to the biodegradable influence of polycyclic aromatic hydrocarbons in the soil; Think that tensio-active agent can improve the bioavailability of polycyclic aromatic hydrocarbons; Accelerate the degradation rate of polycyclic aromatic hydrocarbons, but too high mikrobe (referring to original mikrobe in the soil) activity that then can suppress of surfactant concentration.Poplar is built firm (Chinese Journal of Applied Ecology such as grade; 2004) studied the influence of nonionogenic tenside Tween-20 to the polycyclic arene compound biodegradation process; The result shows; Tween-20 not only has solubilizing effect preferably to phenanthrene, and in the employed concentration range of experiment, TWeen-20 can strengthen the removal effect of degradation bacteria to phenanthrene.Though the tensio-active agent of chemosynthesis is very effective to the solublization of hydrophobic organic pollutant; But seeing that toxicity and biodegradability that some chemical surfactants had are poor; Might introduce secondary pollution at contaminated site, thereby people have turned to sight the bio-surfactant of natural green.
In the process of microbiological deterioration crude oil; Bio-surfactant has played important effect; There is report to think that bio-surfactant can impel hydrocarbons emulsification, dispersion, and changes the hydrophobicity on microorganism cells surface, reach the avidity of strengthening between mikrobe and hydrocarbons; Simultaneously also play the effect that impels hydrocarbons to diffuse into cell, thereby enhancement microbiological is to the degraded of petroleum pollution.
Yet, how to utilize these chemistry or bio-surfactant to impel it in microbiological deterioration petroleum pollution process, to play a role and remain the difficult problem that need capture.
Summary of the invention
The object of the invention is to provide a kind of oil extraction microbial degrading crude oil course control method for use and application, and the method for active substance and the shared degraded oil pollutent of mikrobe.
Microbiological deterioration crude oil course control method for use of the present invention; Be used for confirming the kind and the concentration of petroleum pollution degradation agent active substance, comprise through the research active substance and the impact analysis of microorganism growth and oil degradation is confirmed parameters such as used activated species, concentration, consumption.
Wherein, the impact analysis to microorganism growth may further comprise the steps:
1) the LB slant medium is cultivated 24h, activation bacterial strain;
2) seed is inoculated into the LB liquid nutrient medium after the activation, and behind the cultivation 12h, centrifugal collection thalline is washed with phosphoric acid buffer, and is resuspended in this damping fluid as seed liquor;
3) 5% inoculum size is inoculated in minimal medium with seed liquor, adds crude oil respectively as carbon source, and active substance that add to set concentration respectively is as activator, shaking culture post analysis bacterium liquid value, with the substratum not inoculating and do not add active substance as contrast.
The impact analysis of said oil degradation is to carry out determination and analysis to the AV of crude oil before and after handling with the flow curve of shear rate change.Said processing is meant crude oil is contacted jointly and keeps in touch for some time with active substance with microbial strains.
One of aforesaid method directly is applied as and is used for confirming petroleum pollution degradation agent assembly.The petroleum pollution degradation agent that the present invention utilizes aforesaid method to confirm; Comprise microbial bacteria and active substance, said active substance is for being selected from sulfonated alkylbenzene, triton x-100 (a kind of or biological table agent lipopeptid alive or yeast powder in the agent alive of chemistry such as (TritonX-100), tween (TWeen20), carboxylate salt, sulfonated petro-leum, tween (tween80) table.
Wherein, Said bacterium is for being selected from bacterium 004 (Potsdam short gemma bar (Brevibacillus borstelensis) CGMCC No.2441), bacterium shp (bacillus cereus (Bacillus cereus) CGMCC No.1141) and the bacterium 019 (pseudomonas aeruginosa strains (Pseudomonas aeruginosa) CCTCC No.208114) any; Bacteria concentration is 2% (v/v)～10% (v/v), preferred 5% (v/v).Said chemistry table agent alive concentration in the petroleum pollution degradation agent is 100mg/l～500mg/l, preferred 500mg/l; Said yeast powder weight concentration in the petroleum pollution degradation agent is 0.01%～0.2%, preferred 0.2%; Said biological table agent lipopeptid alive concentration in the petroleum pollution degradation agent is 10mg/l～200mg/l, preferred 50mg/l.
Concrete, it comprises the component of following final concentration in the said petroleum pollution degradation agent: bacterium 004,2～10% (v/v), and chemistry table agent 500mg/l alive or yeast powder 0.2wt% or lipopeptid 50mg/l, the petroleum crude oil of 1wt% is a carbon source, KH
2PO
41g/l, CaCL
2.2H
2O 0.05g/l, Na
2HPO
41g/l, FeSO
40.01g/l, MgSO
4.7H
2O 0.16g/l, MnCL
20.01g/l, NH
4NO
31g/l, NaCL 3g/l, KCL 1g/l, water preparation, pH7-8.
More specifically, be to comprise that the petroleum crude oil of 1wt% is a carbon source, KH
2PO
41g/l, CaCL
2.2H
2O0.05g/l, Na
2HPO
41g/l, FeSO
40.01g/l, MgSO
4.7H
2O 0.16g/l, MnCL
20.01g/l, NH
4NO
31g/l, NaCL 3g/l, KCL 1g/l, the aqueous solution of pH value 7-8, also contain one of following listed assembly:
Assembly one: sulfonated alkylbenzene 500mg/l; Bacterium 004,5%;
Assembly two: triton x-100 ((TritonX-100) 100mg/l; Bacterium 019,2%;
Assembly three: tween (TWeen20) 200mg/l; Bacterium 004,4%;
Assembly four: carboxylate salt 300mg/l; Bacterium 004,8%;
Assembly five: sulfonated petro-leum 400mg/l; Bacterium shp, 10%;
Assembly six: tween (tween80) 500mg/l; Bacterium 004,5%;
Assembly seven: tween (tween80) 100mg/l; Bacterium 004,5%;
Assembly eight: yeast powder 0.2%; Bacterium 004,5%;
Assembly nine: yeast powder 0.1%; Bacterium 004,5%;
Assembly ten: yeast powder 0.01%; Bacterium 004,5%;
Assembly 11: lipopeptid 50mg/l; Bacterium 004,5%.
A kind of degradation method of petroleum pollution also belongs to the present invention, comprises the above degradation agents adding is contained in the environment of petroleum pollution, to contaminated soil, can add at 1: 50 by degradation agents and soil volume ratio.
Through the inventive method, make microbiological deterioration crude oil process be able to control, reduce or eliminate the petroleum pollution in the environment through active substance and the shared mode of degradation bacteria.
Description of drawings
Figure 1A shows the effect of interpolation yeast powder to bacterium 004, and Figure 1B shows the effect of interpolation yeast powder to bacterium 019;
Fig. 2 A shows the effect of adding alkyl benzene sulphonate(ABS) salt pair bacterium 004, and Fig. 2 B shows the effect of adding alkyl benzene sulphonate(ABS) salt pair bacterium 019;
Fig. 3 A, Fig. 3 B and Fig. 3 C show the effect of Tween80 to bacterium 004, bacterium 019 and bacterium shp respectively;
Fig. 4 A, Fig. 4 B and Fig. 4 C show the effect of Tween20 to bacterium 004, bacterium 019 and bacterium shp respectively;
Fig. 5 A, Fig. 5 B and Fig. 5 C show the effect of petroleum carboxylate to bacterium 004, bacterium 019 and bacterium shp respectively;
Fig. 6 A, Fig. 6 B and Fig. 6 C show the effect of sulphonate to bacterium 004, bacterium 019 and bacterium shp respectively;
Fig. 7 A and Fig. 7 B show the effect of Triton X100 to bacterium 004 and bacterium 019 respectively;
Fig. 8 A, Fig. 8 B and Fig. 8 C show the effect of lipopeptid biological table agent alive to bacterium 004, bacterium shp and bacterium 019 respectively;
Fig. 9 A, Fig. 9 B and Fig. 9 C show petroleum carboxylate bacterium combination 004, bacterium 019 and the bacterium shp influence to rheological characteristic of crude oil respectively;
Figure 10 A, Figure 10 B and Figure 10 C show lipopeptid bacterium combination 004, bacterium 019 and the bacterium shp influence to rheological characteristic of crude oil respectively;
Figure 11 A, Figure 11 B and Figure 11 C show Tween20 combination bacterium 004, bacterium 019 and the bacterium shp influence to rheological characteristic of crude oil respectively;
Figure 12 A, Figure 12 B and Figure 12 C show Tween80 combination bacterium 004, bacterium 019 and the bacterium shp influence to rheological characteristic of crude oil respectively;
Figure 13 A, Figure 13 B show the influence of yeast powder combination bacterium 004,019 pair of rheological characteristic of crude oil of bacterium respectively;
Figure 14 A, Figure 14 B show the influence of sulfonated alkylbenzene combination bacterium 004,019 pair of rheological characteristic of crude oil of bacterium respectively;
Figure 15 A, Figure 15 B show the influence of Triton X100 combination bacterium 004,019 pair of rheological characteristic of crude oil of bacterium respectively;
Figure 16 A, Figure 16 B and Figure 16 C show sulphonate combination bacterium 004, bacterium 019 and the bacterium shp influence to rheological characteristic of crude oil respectively.
Embodiment
The invention reside in the method that a kind of active substance and the shared degraded oil pollutent of mikrobe are provided.
The present invention has at first selected seven kinds of tensio-active agents for use: wherein the agent alive of chemistry table has sulfonated petro-leum, petroleum carboxylate, sulfonated alkylbenzene and tween 80 (Tween80), polysorbas20 (Tween20), triton x-100 (TritonX-100) commonly used in the oil recovery field; The agent (Daqing Vertex Chemical Co., Ltd. provides) alive of the biological table of a kind of lipopeptid class; And a kind of nutritive substance yeast powder (as shown in table 1) is studied their influences to bacterial growth and oil degradation as active substance.
Table 1 active substance type
1 active substance is to microorganism growth and rheological characteristic of crude oil influence experiment
1.1 main laboratory apparatus:
Whizzer, shaking table, spectrophotometer, constant temperature shaking table, spectrophotometer, rheometer etc.
1.2 main experiment material:
Experimental drug:
Yeast powder, sulfonated alkylbenzene, triton x-100 ((TritonX-100), lipopeptid, tween (TWeen20), carboxylate salt, sulfonated petro-leum, tween (tween80) etc.
Crude oil: four factory's joint station dewatered oils (apricot three) recover the oil.
Bacterial strain: bacterium 004 (culture presevation is numbered CGMCC No.2441), bacterium shp (culture presevation number be CGMCC № 1141), bacterium 019 (culture presevation is numbered CCTCC M 208114).
Substratum:
LB substratum: contain in the 1L zero(ppm) water: 10g peptone, 5g yeast powder, 10gNaCl, solid LB substratum add mass volume ratio on the basis of above-mentioned prescription be 1.6% agar powder, and sterilization is 20 minutes under 121 ℃ of conditions.
Minimal medium: KH
2PO
41g/l, CaCL
2.2H
2O 0.05g/l, Na
2HPO
41g/l, FeSO
40.01g/l, MgSO
4.7H
2O 0.16g/l, MnCL
20.01g/l, NH
4NO
31g/l, NaCL 3g/l, KCL 1g/l debug pH7-8, and sterilization is 20 minutes under 121 ℃ of conditions.
1.3 add the impact analysis of nutrient yeast powder to microorganism growth
1) the LB slant medium is cultivated 24h, activation bacterial strain;
2) seed inoculation LB liquid nutrient medium after the activation, behind the cultivation 12h, centrifugal collection thalline washes twice with pH 8.0 phosphoric acid buffers, and is resuspended in this damping fluid as seed liquor;
3) 5% inoculum size; Seed liquor is inoculated in minimal medium; Add 10% crude oil respectively as carbon source, the yeast powder that adds weight percent 0.01%, 0.1%, 0.2% respectively is as activator, and temperature is 45 ℃; Rotating speed is 120～130rpm shaking culture 15d post analysis, with the substratum not inoculating and do not add active substance as contrast.
The result: nutrient yeast powder is to the impact analysis of microorganism growth
Yeast powder contains multiple amino acids (L-glutamic acid, l-arginine, Methionin) and growth-stimulating factor (ergosterol), and the growth of bacterium is had promoter action, the growth of bacterium apparently higher than blank with reference to (like Figure 1A and Figure 1B).Preparation contains the minimal medium of 0.01%, 0.1%, 0.2% yeast powder; Other experimental programs are the same, and the bacterium liquid of cultivation is taken a sample at regular intervals, measure bacterium liquid value in 600 places with ultraviolet spectrophotometer; Test-results shows that yeast powder has promoter action to thalli growth; In the minimal medium that contains yeast powder 0.01%, 0.1%, 0.2%, maximum growth amount 0.2% maximum of thalline, and 0.1% and 0.01% is suitable.
1.4 add the impact analysis of chemistry table agent alive to microorganism growth
1) the LB slant medium is cultivated 24h, activation bacterial strain;
2) seed inoculation LB liquid nutrient medium after the activation, behind the cultivation 12h, centrifugal collection thalline washes twice with pH 8.0 phosphoric acid buffers, and is resuspended in this damping fluid as seed liquor;
3) 5% inoculum size; Seed liquor is inoculated in minimal medium, adds 10% crude oil respectively, add 100mg/l, 250mg/l, 500mg/l chemistry table mainly commonly used respectively and live agent (like table 1) as activator as carbon source; Temperature is 45 ℃; Rotating speed is 120～130rpm, shaking culture 15d post analysis, with the substratum not inoculating and do not add activator as contrast.
The result: chemical surfactant is to the impact analysis of the growth of bacterium
Tensio-active agent can increase the solubleness of petroleum hydrocarbon, but it possibly utilized by preferential as microbial growth matrix again.Result of study like Tiemh (1994) shows, the tensio-active agent of being tested can both increase luxuriant and rich with fragrance solubleness, but SDS has suppressed luxuriant and rich with fragrance degraded as the preferential matrix of utilizing of mikrobe, and SDS the increase with its concentration increases to the restraining effect of phenanthrene.
Fig. 2 A, Fig. 2 B, Fig. 3 A-Fig. 3 C result show that it is bigger that the bacterial growth activity is influenced by tensio-active agent.The dense bacterium during all apparently higher than lower concentration and blank of sulfonated alkylbenzene and the bacterium of Tween80 concentration when 500mg/l is dense; Contain in the minimal medium of sulfonated alkylbenzene; The maximum growth amount basic trend of thalline is 500mg/l＞250mg/l＞100mg/l, and contains in the minimal medium of Tween80, and the maximum growth amount trend of thalline is 500mg/l＞100mg/l＞250mg/l; In addition; In this pilot system, the pH value can guarantee that bacterium keeps good growth activity between 7-8.Therefore, the pH value is little to the growth activity influence of bacterium in this test.
Show that through experiment the live different mass concentration of agent of these several kinds chemistry tables of Tween-20 (Fig. 4 A-4C), petroleum carboxylate (Fig. 5 A-5C), sulfonated petro-leum (Fig. 6 A-6C), Triton X100 (Fig. 7 A and Fig. 7 B) is not too obvious to the growth effect of oil recovery bacterium, but the height that adds the high-biomass (the dense OD value of ordinate zou bacterium) that reached behind the tensio-active agent when not adding; Simultaneously, can explain that by experimental result adding tensio-active agent all can reach higher oil degradation effect and (see Fig. 9-Figure 14).
1.5 add the impact analysis of lipopeptid biological table agent alive to microorganism growth
1) the LB slant medium is cultivated 24h, activation bacterial strain;
2) seed inoculation LB liquid nutrient medium after the activation, behind the cultivation 12h, centrifugal collection thalline washes twice with pH 8.0 phosphoric acid buffers, and is resuspended in this damping fluid as seed liquor;
3.) 5% inoculum size; Seed liquor is inoculated in minimal medium, adds 10% crude oil respectively, add 10mg/l, 50mg/l, 100mg/l, 200mg/l lipopeptid biological table agent alive (like table 1) respectively as activator as carbon source; Temperature is 45 ℃; Rotating speed is 120～130rpm, shaking culture 15d post analysis, with the substratum not inoculating and do not add activator as contrast.
The result: bio-surfactant is to the impact analysis of the growth of bacterium
Bio-surfactant be one type by microorganisms, the polymer substance of molecular structure possess hydrophilic property and hydrophobic group comprises glycolipid, lipopeptid, polysaccharide-protein complex compound, phosphatide, lipid acid and neutral fat etc.Compare with other tensio-active agents, bio-surfactant not only has can reduce IT and the organic effect of emulsification insoluble, is easy to the advantage of biological degradation and toxicological harmless simultaneously in addition.
It is different with concentration to the growth effect of bacterium to add the lipopeptid type biological surfactant; Show among Fig. 8 A-Fig. 8 C; Add of the increment not influence of the bio-surfactant of low concentration to bacterium; Wherein add concentration at 50mg/l, bacterium 004 bacterium is dense to reach the highest, and all apparent in view to the action effect of bacterium 019 and bacterium shp when adding concentration greater than 50mg/l.
1.6 the determination and analysis of rheological characteristic of crude oil
With the rotary viscosity measuring of the HAAKE company rheological in the higher shear rates scope, the shearing rate useful range is 0.01～3000S
-1, the apparent viscosity useful range is 0.001～10000Pa.s, adopts on line mode to continuously change shearing rate.After measurement finished, instrument provided relation curve automatically.
The result: the AV of oil sample is seen Fig. 9 A-Figure 16 C with the flow curve of shear rate change before and after handling.Can find out that from figure the AV of crude oil shows as the characteristic of pseudoplastic fluid before handling; After the processing, the AV of crude oil then is tending towards the characteristic of Newtonian fuid.This description of test adds mikrobe and the agent alive of various table simultaneously in minimal medium, can improve the apparent viscosity of crude oil, shows the oil degradation effect that can reach higher.
1.7 brief summary
More than experiment is in order further to inquire into the influence of tensio-active agent to degradation by bacteria crude oil; Selected agent alive of the biological table of AS sulfonated petro-leum, petroleum carboxylate, sulfonated alkylbenzene and nonionogenic tenside TritonX-100, tween 80 (Tween80) and polysorbas20 (Tween20) and a kind of lipopeptid class and nutritive substance yeast powder research experiment respectively for use, adopted the spectrophotometer counting process to investigate the influence of tensio-active agent to the growth activity of bacterium.Test-results shows that yeast powder contains growth-stimulating factors such as L-glutamic acid, l-arginine, Methionin, ergosterol, not only can promote bacteria growing but also can quicken the degraded of crude oil, and when yeast powder concentration is 0.2%, increment is maximum; The chemistry table is lived agent under identical condition; Sulfonated alkylbenzene and TW80 action effect are more obvious relatively; Wherein Triton X100 is high when the bacterial growth amount of 250mg/l activity compares at the 500mg/l activity, and the result shows that the tensio-active agent of higher concentration possibly have inhibition or toxic action to the growth of bacterium.
Rheological characteristic of crude oil curve before and after the effect can find out that effect back viscosity of crude reduces, and rheological obviously improves.Wherein the variation of the rheological behind yeast powder, sulfonated alkylbenzene, tw80, Triton, petroleum carboxylate, several kinds of effect crude oil of sulfonated petro-leum is certain rules with the increment variation of bacterial classification.
Utilize above-mentioned experimental result, the present invention proposes to utilize the shared scheme of coming the degraded oil pollutent of suitable active substance and mikrobe, promptly through preparation and use the petroleum pollution degradation agent and reach and alleviate and the purpose of eliminating petroleum pollution.
Following result according to experiment 1 provides the preferred embodiment of the invention.
2. the preparation of petroleum pollution degradation agent
Petroleum pollution degradation agent provided by the invention comprises microbial bacteria and active substance, also comprises microbial bacteria necessary base substratum.Wherein:
The petroleum crude oil of basic medium: 1wt% is a carbon source, KH
2PO
41g/l, CaCL
2.2H
2O 0.05g/l, Na
2HPO
41g/l, FeSO
40.01g/l, MgSO
4.7H
2O 0.16g/l, MnCL
20.01g/l, NH
4NO
31g/l, NaCL 3g/l, KCL 1g/l, the water preparation is regulated pH7-8 with Hydrogen chloride or sodium hydroxide, and sterilization is 20 minutes under 121 ℃ of conditions.
Microbial bacteria: for being selected from bacterium 004, bacterium shp, the bacterium 019 any; Bacteria concentration is 2% (v/v)-10% (v/v), preferred 5% (v/v).
Bacterium shp; Open in patent ZL200410038054.3; Be bacillus cereus (Bacilluscereus) HP, this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 04 08th, 2004, and preserving number is CGMCC № 1141.
Active substance: for being selected from sulfonated alkylbenzene, triton x-100 (live a kind of in the agent of chemistry such as (TritonX-100), tween (TWeen20), carboxylate salt, sulfonated petro-leum, tween (tween80) table; Final concentration is 100mg/l～500mg/l in the petroleum pollution degradation agent, preferred 500mg/l; Or be biological table agent lipopeptid alive, final concentration is 10mg/l～200mg/l in the petroleum pollution degradation agent, preferred 50mg/l; Or be yeast powder, weight concentration is 0.01%～0.2% in the petroleum pollution degradation agent, preferred 0.2%.
Table 2 petroleum pollution degradation agent assembly
| Numbering | Active substance | Concentration | Oil recovery bacterium and concentration (volume percent) | ||
| 1 | Sulfonated alkylbenzene | 500mg/ | Bacterium | 004,5% | |
| 2 | Triton x-100 ((TritonX-100) | 100mg/ | Bacterium | 019,2% | |
| 3 | Tween (TWeen20) | 200mg/ | Bacterium | 004,4% | |
| 4 | Carboxylate salt | 300mg/ | Bacterium | 004,8% | |
| 5 | Sulfonated petro-leum | 400mg/l | Bacterium shp, 10% |
| 6 | Tween (tween80) | 500mg/ | Bacterium | 004,5% | |
| 7 | Tween (tween80) | 100mg/ | Bacterium | 004,5% | |
| 8 | Yeast powder | 0.2 | Bacterium | 004,5% | |
| 9 | Yeast powder | 0.1 | Bacterium | 004,5% | |
| 10 | Yeast powder | 0.01 | Bacterium | 004,5% | |
| 11 | Lipopeptid | 50mg/ | Bacterium | 004,5% |
Carry out following operation preparation by above-mentioned assembly and form serial petroleum pollution degradation agent:
1) bacterial classification is added preparation bacterium liquid in the basic medium (liquid), wherein bacteria concentration requires greater than 10
7Individual/ml;
2) establish concentration value calculated activity material consumption by table, the preparation active substance aqueous solution;
3) with above-mentioned bacterium liquid and active substance aqueous solution, the water adjustment makes each component final concentration reach requirement.3. the degradation experiment of petroleum pollution
Get near the oily factory by the soil of petroleum pollution as sample, use gas chromatographic detection, mensuration is the content of several kinds of important component phenanthrene, fluoranthene, pyrene, the tetradecane and fluorenes wherein.
1: 50 by volume ratio will join in the pedotheque by the petroleum pollution degradation agent that table 2 prepares, simultaneously with the dense single bacterium liquid of same bacterium as contrast.The room temperature held was measured the wherein content of each polycyclic aromatic hydrocarbons at 2 days, 7 days and 12 days respectively once more, calculated degradation rate.Experimental result is seen table 3.
The degradation effect of table 3 soil PetroChina Company Limited. pollutent
This experiment shows that the petroleum pollution degradation agent of the present invention's preparation is for concentrating main petroleum pollution by tangible Degradation in the soil.
Claims (10)
1. petroleum pollution degradation agent; Comprise microbial bacteria and active substance, it is characterized in that: said active substance is for being selected from sulfonated alkylbenzene, triton x-100 (a kind of or biological table agent lipopeptid alive or yeast powder in the agent alive of chemistry such as (TritonX-100), tween (TWeen20), carboxylate salt, sulfonated petro-leum, tween (tween80) table.
2. according to the said petroleum pollution degradation agent of claim 1; It is characterized in that: said bacterium is for being selected from bacterium 004 (Potsdam short gemma bar (Brevibacillus borstelensis) CGMCC No.2441), bacterium shp (bacillus cereus (Bacillus cereus) CGMCC No.1141) and the bacterium 019 (pseudomonas aeruginosa strains (Pseudomonas aeruginosa) CCTCC No.208114) any; Bacteria concentration is 2% (v/v)～10% (v/v), preferred 5% (v/v).
3. according to claim 1 or 2 said petroleum pollution degradation agent, it is characterized in that: said chemistry table agent alive concentration in the petroleum pollution degradation agent is 100mg/l～500mg/l, preferred 500mg/l; Said yeast powder weight concentration in the petroleum pollution degradation agent is 0.01%～0.2%, preferred 0.2%; Said biological table agent lipopeptid alive concentration in the petroleum pollution degradation agent is 10mg/l～200mg/l, preferred 50mg/l.
4. according to claim 1 or 2 or 3 said petroleum pollution degradation agent; It is characterized in that: it comprises the component of following final concentration in the said petroleum pollution degradation agent: bacterium 004; 2～10% (v/v); Chemistry table agent 500mg/l alive or yeast powder 0.2wt% or lipopeptid 50mg/l, the petroleum crude oil of 1wt% is a carbon source, KH
2PO
41g/l, CaCL
2.2H
2O 0.05g/l, Na
2HPO
41g/l, FeSO
40.01g/l, MgSO
4.7H
2O 0.16g/l, MnCL
20.01g/l, NH
4NO
31g/l, NaCL 3g/l, KCL 1g/l, water preparation, pH7-8.
5. according to the said petroleum pollution degradation agent of claim 4, it is characterized in that: be to comprise that the petroleum crude oil of 1wt% is a carbon source, KH
2PO
41g/l, CaCL
2.2H
2O 0.05g/l, Na
2HPO
41g/l, FeSO
40.01g/l, MgSO
4.7H
2O 0.16g/l, MnCL
20.01g/l, NH
4NO
31g/l, NaCL 3g/l, KCL 1g/l, the aqueous solution of pH value 7-8, also contain one of following listed assembly:
Assembly one: sulfonated alkylbenzene 500mg/l; Bacterium 004,5%;
Assembly two: triton x-100 ((TritonX-100) 100mg/l; Bacterium 019,2%;
Assembly three: tween (TWeen20) 200mg/l; Bacterium 004,4%;
Assembly four: carboxylate salt 300mg/l; Bacterium 004,8%;
Assembly five: sulfonated petro-leum 400mg/l; Bacterium shp, 10%;
Assembly six: tween (tween80) 500mg/l; Bacterium 004,5%;
Assembly seven: tween (tween80) 100mg/l; Bacterium 004,5%;
Assembly eight: yeast powder 0.2%; Bacterium 004,5%;
Assembly nine: yeast powder 0.1%; Bacterium 004,5%;
Assembly ten: yeast powder 0.01%; Bacterium 004,5%;
Assembly 11: lipopeptid 50mg/l; Bacterium 004,5%.
6. the degradation method of a petroleum pollution is characterized in that: comprise the arbitrary said degradation agents adding of claim 1 to 5 is contained in the environment of petroleum pollution, to contaminated soil, can add at 1: 50 by degradation agents and soil volume ratio.
7. microbiological deterioration crude oil course control method for use; Be used for confirming the kind and the concentration of the arbitrary said petroleum pollution degradation agent active substance of claim 1 to 5, comprise through the research active substance and the impact analysis of microorganism growth and oil degradation is confirmed parameters such as used activated species, concentration, consumption.
8. according to the said method of claim 7, it is characterized in that, the impact analysis of microorganism growth may further comprise the steps:
1) the LB slant medium is cultivated 24h, activation bacterial strain;
2) seed is inoculated into the LB liquid nutrient medium after the activation, and behind the cultivation 12h, centrifugal collection thalline is washed with phosphoric acid buffer, and is resuspended in this damping fluid as seed liquor;
3) 5% inoculum size is inoculated in minimal medium with seed liquor, adds crude oil respectively as carbon source, and active substance that add to set concentration respectively is as activator, shaking culture post analysis bacterium liquid value, with the substratum not inoculating and do not add active substance as contrast.
9. according to the said method of claim 7, it is characterized in that the impact analysis of said oil degradation is to carry out determination and analysis to the AV of crude oil before and after handling with the flow curve of shear rate change.
10. according to the said method of claim 9, it is characterized in that said processing is meant crude oil is contacted jointly and keeps in touch for some time with active substance with microbial strains.
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| CN104353667A (en) * | 2014-10-10 | 2015-02-18 | 浙江大学 | United degradation method for dehydrated crude in water by petroleum degradation bacterial flora and surfactant |
| CN104907331A (en) * | 2015-05-27 | 2015-09-16 | 山东康诚石油化工科技股份有限公司 | Soil biological repair method |
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| CN110052493A (en) * | 2019-03-26 | 2019-07-26 | 中国矿业大学 | A kind of the biological mud restorative procedure and corollary equipment of polycyclic aromatic hydrocarbon pollution |
| CN114472503A (en) * | 2020-11-13 | 2022-05-13 | 新疆大学 | Composite remediation method for soil polluted by leaky petroleum |
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Effective date of registration: 20200929 Address after: 100007 Dongcheng District, Dongzhimen, China, North Street, No. 9 Oil Mansion, No. Patentee after: PetroChina Co.,Ltd. Patentee after: Daqing Oilfield Co.,Ltd. Address before: 163453 Heilongjiang Province, Daqing City Ranghulu District Central Plains Road No. 99 Patentee before: Daqing Oilfield Co.,Ltd. |