CN106315868A - Application of degrading bacterium capable of metabolizing various hydrocarbons to petroleum pollutant disposal - Google Patents
Application of degrading bacterium capable of metabolizing various hydrocarbons to petroleum pollutant disposal Download PDFInfo
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- CN106315868A CN106315868A CN201610934776.XA CN201610934776A CN106315868A CN 106315868 A CN106315868 A CN 106315868A CN 201610934776 A CN201610934776 A CN 201610934776A CN 106315868 A CN106315868 A CN 106315868A
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
Abstract
The invention relates to an application of a degrading bacterium capable of metabolizing various hydrocarbons to petroleum pollutant disposal. The degrading bacterium is applied to degrading and removing pollutants generated by diesel, diesel products, petroleum and petroleum products in soil and wastewater as a bioremediation agent. The degrading bacterium has removal effects on various petroleum hydrocarbons, comprises straight-chain alkanes (long-chain alkanes, such as dotriacontane), branched alkanes, polyaromatic hydrocarbons (phenanthrene, anthracene) and heterocyclic hydrocarbons (carbazole, dibenzothiophene) and has the advantages of high degradation rate, high metabolizable concentration and a wide range of application to polluted environments.
Description
Technical field
The present invention relates to a strain can the application in petroleum pollution processes of the degradation bacteria of the multiple hydro carbons of metabolism, belong to biological
Technical field.
Background technology
Oil is the multicomponent mixture being made up of organic and inorganic compound of a kind of complexity, including hydro carbons (alkane, ring
Alkane, aromatic hydrocarbon) and non-hydrocarbon component (oxygen-containing, sulfur, nitrogen compound, resin and asphalt).Different regions, the oil of different layers position
Physicochemical properties also exist obvious difference.Hydrocarbon compound in oil i.e. Hydrocarbon, accounts for absolutely in oil
Major part, and the structure of various hydro carbons and shared ratio differ greatly.
Petroleum-type compound is that in environment, harm is serious, pollute the industrial pollutants of cycle length.Oil exploitation, accumulating,
Inevitable that reveal oil accident and occur during processing and use, the life to water body, soil, atmospheric environment and the mankind
Cause loss difficult to the appraisal.Petroleum pollution has stronger biology toxicity, can carcinogenic, teratogenesis, mutagenesis, Qi Zhongfang
Fragrant hydrocarbon compound is relatively big to the toxicity of humans and animals, and especially with dicyclo and three rings, the polycyclic aromatic hydrocarbon toxicity as representative is bigger,
Have confirmed that it has stronger carcinogenesis, human or animal body can be entered by modes such as breathing, contact skin, diet absorptions
In, cell membrane, interferases system can be dissolved, affect the normal function of the organ such as liver, kidney, even cause canceration.Some of which
Compound can also be accumulated by bioconcentration, hides some dangers for for the existence of other biological and the health of the mankind.
The recovery technique of oil pollution mainly includes peripheral doses, chemical redemption, physical chemistry reparation and biological restoration.Mesh
Before, the research to physics and chemical restoration is the most ripe, and treatment effect is preferable, but high, easily owing to there is processing cost
Produce the shortcoming such as secondary pollution to make it apply to be restricted.Biological restoration is to utilize microorganism, animal or plant by soil, water body
In contaminant degradation or enrichment, the biotechnology of absorption, wherein microorganism remediation technology is mainly by microorganism
Poisonous complicated petroleum substance is resolved into simple innocuous substance by metabolism, and then makes oil polluted environment obtain only
Change.Microorganism remediation technology is owing to low cost, efficiency are high, little to environment negative effect and the advantage such as non-secondary pollution, at oil
The recovery technique of pollutant has the biggest development potentiality and is widely applied prospect.
In recent years, the microorganism remediation technology that petroleum hydrocarbon pollutes at home and abroad has obtained developing faster, but due to
Most of microbe available hydrocarbons limitednumber, relatively slow to petroleum hydrocarbon degradation speed, governance process required time
Longer, therefore screening obtains the efficient degrading bacteria of the multiple hydrocarbons of energy metabolism and is still focus and the weight of current bioremediation technology
Point.
Summary of the invention
For the deficiencies in the prior art, the present invention provide a strain can the degradation bacteria of the multiple hydro carbons of metabolism at petroleum pollution
Application in reason, can remove petroleum pollution with efficient degradation, have degradation rate fast, can metabolite concentration be high, be suitable for and pollute ring
The advantages such as border is wide.
The present invention is achieved through the following technical solutions:
One strain can the multiple hydro carbons of metabolism degradation bacteria petroleum pollution process in application, it is characterised in that degradation bacteria
It is applied to degraded as bioremediation agents and removes diesel oil in soil and waste water, diesel product, oil, the dirt of oil product generation
Dye.
Currently preferred, the pollution that described diesel oil, diesel product, oil, oil product produce is linear paraffin, side chain
One of which or any two or more mixing in alkane, aromatic hydrocarbon or heterocyclic arene.
It is further preferred that the linear paraffin that described linear paraffin is C12～C32.
Currently preferred, removal condition of degrading: temperature is 25～42 DEG C, and pH is 6.0～8.0, salinity is 0.5～3%.
It is further preferred that degraded removal condition: temperature is 26～32 DEG C, pH is 6.0～7.0, salinity be 0.5～
2.0%.
Currently preferred, it is 2～6 × 10 that bacterium solution makes bacterium cell concentration in petroleum-contaminated water after adding7CFU/ml, stone
In soil contaminated by crude oil, bacterium cell concentration is 1～2 × 107CFU/g。
Currently preferred, the entitled Rhodococcus fascians of described degradation bacteria (Rhodococcus sp.) P52, May 23 in 2011
Being preserved in China typical culture collection center (Wuhan University, China, Wuhan) day, deposit number is CCTCCNo:
M2011181。
Currently preferred, described degraded removal is that to add Rhodococcus fascians in oil-polluted soils or petroleum-contaminated water abundant
Stand after stirring, through 2～11 days, the degradation rate of linear paraffin (C12～C32) is reached 66～87%, to branched paraffin
Degradation rate reaches 63.9%, to the utilization rate of polycyclic aromatic hydrocarbon 55～100%, to the utilization rate of heterocyclic arene 61～82%,
Contaminated by diesel oil thing clearance in soil and waste water is reached 45～63.7%.
It is further preferred that described stirring condition is 150rpm vibration training in 30 DEG C of constant-temperature tables under dark condition
Support.
Oil content 100mg/L～500mg/L in petroleum-contaminated water, oil content 1～5g/kg in soil pollution；Bavin
Determination of Diesel Oil 4～5g/L in oil polluted water, Determination of Diesel Oil 1～5g/kg in soil pollution.
Currently preferred, described Rhodococcus fascians is to cultivate production by the following method to obtain:
(1) bacterial strain selects: selecting Rhodococcus fascians (Rhodococcus sp.) P52, deposit number is CCTCC No:
M2011181；
(2) bacterial strain activation: by Rhodococcus fascians streak inoculation on LB flat board, in 25～42 DEG C of constant incubators cultivate 20～
32h；
(3) seed culture: picking step (2) activation after single colony inoculation in LB fluid medium, 30～42 DEG C, bar
Under part, shaken cultivation 18～30h prepares seed liquor；
(4) amplification culture: seed liquor is accessed in LB fluid medium with the inoculum concentration of volume ratio 0.5～4% (v/v),
Isothermal vibration is cultivated, and treats that bacterium solution cell concentration reaches 108～109During CFU/ml, the centrifugal cell collecting amplification culture；
(5) washed cell: with pH 7.0 be 0.01～0.1mol/L phosphate buffer or minimal medium washing thin
Born of the same parents, repeat 2～3 times, use centrifugal with under step (4) the same terms, collect cell, finally by cell suspension in phosphate-buffered
Liquid or minimal medium, 4 DEG C save backup.
Currently preferred, step (3) vibration rotating speed is 150～200rpm.
Currently preferred, the centrifugal collection described in step (4) is in 4～10 DEG C by culture fluid, rotating speed 4000～
8000rpm is centrifuged the 4～8min cells being collected amplification culture.
Currently preferred, the formula of described phosphate buffer is:
Na2HPO41.4～12.5g, KH2PO40.5～4.5g, distilled water 1L, pH 7.0, high pressure steam sterilization 121 DEG C,
20min。
Currently preferred, the formula of described minimal medium is:
K2HPO42.7～15g, KH2PO40.8～14g, Na2SO42.0g, MgSO4·7H2O 0.2g, NH4NO33g,
Trace meter saline solution 0.1～1mL, distilled water 1L, pH 7.0, high pressure steam sterilization 121 DEG C, 20min.
Wherein the formula of trace meter saline solution (1mL) is:
FeCl2·4H2O 0.1～0.5g, CoCl2·6H2O 0.038g, MgCl2·4H2O 0.02g, ZnCl2
0.014g, H3BO30.0124g, Na2MO4·H2O 0.04g, CaCl2·2H2O 0.001～0.01g.
The present invention is raw materials used and LB flat board, LB fluid medium are prior art.
Advantages of the present invention is as follows:
1. the application efficient degrading bacteria that the present invention provides removes petroleum pollution, can produce for different kinds of petroleum hydrocarbon and remove
Effect, including linear paraffin (especially long chain alkane, such as n-dotriacontane), branched paraffin, polycyclic aromatic hydrocarbon (luxuriant and rich with fragrance, anthracene), heterocycle virtue
Hydrocarbon (carbazole, dibenzothiophenes), therefore can produce removal effect for the multiclass petroleum pollution containing different hydrocarbon compositions.
2. the application efficient degrading bacteria that the present invention provides removes petroleum pollution, have degradation rate fast, can metabolite concentration
The advantages such as pollution environment high, applicable is wide.
Accompanying drawing explanation
Fig. 1 a is that the degraded of n-tetracosane and utilization are supported the growth of thalline by Rhodococcus fascians P52.(■): experimental group positive two
The concentration change of the tetradecane；(●): the concentration change of bacteria control group n-tetracosane；(): thalli growth amount changes；
Fig. 1 b is that the degraded of n-tetradecane and utilization are supported the growth of thalline by Rhodococcus fascians P52.(■): experimental group positive 14
The concentration change of alkane；(●): the concentration change of bacteria control group n-tetradecane；(): thalli growth amount changes；
Fig. 1 c is that the degraded of n-dotriacontane and utilization are supported the growth of thalline by Rhodococcus fascians P52.(■): experimental group positive three
The concentration change of dodecane；(●): the concentration change of bacteria control group n-dotriacontane；(): thalli growth amount changes；
Fig. 2 is that the degraded of branched paraffin and utilization are supported the growth of thalline by Rhodococcus fascians P52.(■): experimental group different 19
The concentration change of alkane；(●): the concentration change of bacteria control group norphytane；(): thalli growth amount changes.
Fig. 3 a is that the degraded of naphthalene and utilization are supported the growth of thalline by Rhodococcus fascians P52.(■): the concentration of experimental group naphthalene becomes
Change；(●): the concentration change of bacteria control group naphthalene；(): thalli growth amount changes；
Fig. 3 b is that luxuriant and rich with fragrance degraded and utilization are supported the growth of thalline by Rhodococcus fascians P52.(■): the concentration change that experimental group is luxuriant and rich with fragrance
Change；(●): the concentration change that bacteria control group is luxuriant and rich with fragrance；(): thalli growth amount changes；
Fig. 4 is that Rhodococcus fascians P52 is to 0#The degraded of diesel oil.
Detailed description of the invention
Below by specific embodiment, the present invention will be further described, but is not limited to this.
The production of embodiment 1 Rhodococcus fascians (Rhodococcus sp.) P52CCTCC No:M2011181
(1) bacterial strain selects: select Rhodococcus fascians (Rhodococcus sp.) P52CCTCC No:M2011181；
(2) bacterial strain activation: taking out the dry powder preservation pipe one being stored in ultra cold storage freezer, streak inoculation is in LB flat board
On, it is inverted in 30 DEG C of constant incubators and cultivates 24h；
(3) seed culture: the mono-colony inoculation of picking Rhodococcus fascians P52 in LB fluid medium, 30 DEG C, under the conditions of 180rpm
Shaken cultivation 20h prepares seed liquor；
(4) amplification culture: seed liquor is accessed in the conical flask of LB fluid medium by the inoculum concentration with 1%, isothermal vibration
Cultivate, treat that bacterium solution cell concentration is about 109During CFU/ml, centrifugal collection thalline；
(5) collecting cell: by the culture fluid of step (4) in 4 DEG C, 4000rpm is centrifuged 8min, collects thalline；
(6) washed cell: with the 0.05M phosphate buffer wash cell of pH 7.0, be repeated 2 times, under the same terms from
The heart, collects cell, is suspended in phosphate buffer, and 4 DEG C save backup.
The formula of the phosphate buffer used is:
Na2HPO45.6g, KH2PO42g, distilled water 1L, pH 7.0, high pressure steam sterilization 121 DEG C, 20min.
The LB culture medium prescription used is:
Tryptone 10g, yeast extract 5g, NaCl 10g, distilled water 1L, natural pH, high pressure steam sterilization 121 DEG C,
20min。
Above-mentioned solid culture based formulas is the agar of addition 1.5% in described fluid medium.
The production of embodiment 2 Rhodococcus fascians (Rhodococcus sp.) P52CCTCC No:M2011181 and to linear paraffin
The degraded of (positive 14,24, dotriacontane)
(1) bacterial strain selects: select Rhodococcus fascians (Rhodococcus sp.) P52CCTCC No:M2011181；
(2) bacterial strain activation: take out the glycerol stocks Guan Yizhi that is stored in-70 DEG C of refrigerators, streak inoculation on LB flat board,
20h is cultivated in 35 DEG C of calorstats；
(3) seed culture: the mono-colony inoculation of picking Rhodococcus fascians P52 in 5mL LB fluid medium, 30 DEG C, 180rpm bar
Under part, shaken cultivation 20h prepares seed liquor；
(4) amplification culture: seed liquor is accessed the 500mL taper containing 100mL LB fluid medium by the inoculum concentration with 1%
In Ping, constant-temperature shaking culture, cultivation to bacterium solution cell concentration is about 109CFU/ml, centrifugal collection thalline；
(5) collecting cell: by the culture fluid of step (4) in 4 DEG C, 5000rpm is centrifuged 6min, collects thalline；
(6) washed cell: with the minimal medium washed cell of pH 7.0, be repeated 2 times, centrifugal under the same terms, receive
Collection cell, is suspended in minimal medium, prepares bacteria suspension standby.
LB culture medium: with embodiment 1；
Minimal medium: K2HPO412g, KH2PO411g, Na2SO42.0g, MgSO4·7H2O 0.2g, NH4NO3
3g, trace meter saline solution 1mL, distilled water 1L, pH 7.0, high pressure steam sterilization 121 DEG C, 20min.
Wherein 1ml trace meter saline solution contains: FeCl2·4H2O 0.3g, CoCl2·6H2O 0.038g, MgCl2·
4H2O 0.02g, ZnCl20.014g, H3BO30.0124g, Na2MO4·H2O 0.04g, CaCl2·2H2O 0.0034g, distillation
Water 0.1mL.
Rhodococcus fascians (Rhodococcus sp.) P52CCTCC No:M2011181 to linear paraffin (positive 14,24,
Dotriacontane) degraded:
Degraded system is arranged: is inoculated in respectively by bacteria suspension equipped with in the ground conical flask of 50mL minimal medium, adjusts
Ganglion cell's concentration, makes initial cell density 2.5 × 107CFU/ml, be separately added into substrate n-tetradecane to be degraded, 24
Alkane, dotriacontane so that it is final concentration is about 200mg/L, under dark condition in 30 DEG C of constant-temperature tables 150rpm shaken cultivation；With
It is not added with the minimal medium (containing equivalent degraded substrate) of bacteria suspension and only adds the minimal medium of bacteria suspension (without the degraded end
Thing) as blank group, experimental group respectively set with blank group 3 parts parallel, different time to degraded system sampling, use gas phase
The degraded situation of chromatography detection linear paraffin, dilution is coated with flat board detection thalline and increases.
Sample treatment: by adding the dichloromethane with institute's water sampling sample culturing liquid phase same volume in sample, fully shake
After swinging, stratification, takes off a layer organic facies, extracts three times, merge organic facies, through anhydrous Na2SO4After drying, organic by 0.45 μm
Membrane filtration, stores in brown sample bottle, to be measured.Sample uses chromatogram ration analysis hydrocarbon after pre-treatment (extraction process)
The concentration change of class material
The analytical conditions for gas chromatography of hydrocarbons in sample: chromatographic column is HP-5 capillary column (30m × 250 μ m
0.25μm)
N-tetradecane: fid detector, injector, detector temperature be 280 DEG C, and column temperature is 260 DEG C；
N-tetracosane: fid detector, injector, detector temperature be respectively 290 DEG C, and column temperature is 270 DEG C；
N-dotriacontane: fid detector, injector, detector temperature be 300 DEG C, and column temperature is 300 DEG C.
To the degradation results of linear paraffin (positive 14,24, dotriacontane) as it is shown in figure 1, from Fig. 1 a, Fig. 1 b, figure
It can be seen that after 48h cultivates, the n-tetradecane of 85.7% is degraded in 1c；In 11 days, the n-tetracosane of 79.4%,
The n-dotriacontane of 66.4% is degraded, and with degradation process, cell concentration rises to original 4～5 times.
The embodiment 3 degraded to branched paraffin (norphytane)
The production of Rhodococcus fascians (Rhodococcus sp.) P52CCTCC No:M2011181 is with embodiment 1；
Degraded system is arranged: the bacteria suspension of preparation is inoculated in ground conical flask equipped with 50mL minimal medium respectively
In, regulate cell concentration, make initial cell density be about 4 × 107CFU/ml, adds degraded substrate norphytane so that it is the denseest
Degree be about 200mg/L, under dark condition in 30 DEG C of constant-temperature tables 150rpm shaken cultivation；To be not added with the inorganic salt training of bacteria suspension
Support base (degrading substrate containing equivalent) and only add the minimal medium (without degraded substrate) of bacteria suspension as blank group, in fact
Test group respectively set with blank group 3 parts parallel, different time, to degraded system sampling, detects norphytane with gas chromatography
Degraded situation, dilution is coated with flat board detection thalline and increases；
Sample treatment: with embodiment 2；
The analytical conditions for gas chromatography of hydrocarbons in sample: chromatographic column is HP-5 capillary column (30m × 250 μ m
0.25 μm), fid detector；Injector, detector temperature are 260 DEG C, and column temperature is 280 DEG C；
The formula of involved culture medium: LB culture medium, minimal medium are with embodiment 2.
To the degradation results of branched paraffin (norphytane) as in figure 2 it is shown, from figure 2 it can be seen that in 9 days,
The norphytane of 63.9% is degraded utilization.
The embodiment 4 degraded to aromatic hydrocarbon
The production of Rhodococcus fascians (Rhodococcus sp.) P52CCTCC No:M2011181 is with embodiment 1；
Degraded system is arranged: the bacteria suspension of preparation is inoculated in ground conical flask equipped with 50mL minimal medium respectively
In, regulate cell concentration, make initial cell density be about 3～4 × 107CFU/ml, be separately added into degraded substrate naphthalene, phenanthrene, anthracene,
Carbazole, dibenzothiophenes so that it is final concentration is about 200～250mg/L, under dark condition, in 30 DEG C of constant-temperature tables, 150rpm shakes
Swing cultivation；To be not added with the minimal medium (containing equivalent degraded substrate) of bacteria suspension and only to add the minimal medium of bacteria suspension
(without degraded substrate) as blank group, experimental group respectively set with blank group 3 parts parallel, different time to degrade system
Sampling, by gas chromatography detection naphthalene and luxuriant and rich with fragrance degraded situation, dilution is coated with flat board detection thalline and increases；
Sample treatment: with embodiment 2；
The analytical conditions for gas chromatography of hydrocarbons in sample: chromatographic column is HP-5 capillary column (30m × 250 μ m
0.25 μm), fid detector；
Naphthalene: injector, detector temperature are 280 DEG C, column temperature is 260 DEG C；
Luxuriant and rich with fragrance, anthracene: injector, detector temperature are 290 DEG C, and column temperature is 270 DEG C.
Carbazole and dibenzothiophenes: injector, detector temperature are 280 DEG C, column temperature is 165 DEG C.
The formula of involved culture medium: LB culture medium, minimal medium are with embodiment 2.
To naphthalene, luxuriant and rich with fragrance degradation results as shown in Figure 3 a, 3 b, from figure 3, it can be seen that after processing 11 days, concentration is
The naphthalene of 175mg/L 100% is degraded for thalli growth；After processing 9 days, concentration is that the phenanthrene 55.3% of 180mg/L is degraded profit
With.
Additionally process after 60 hours, the anthracene of 250mg/L, carbazole, the degradation rate of dibenzothiophenes respectively reach 57%,
61%, 82%.
Embodiment 5 processes the waste water Han diesel oil
The production of Rhodococcus fascians (Rhodococcus sp.) P52CCTCC No:M2011181 is with embodiment 1；
System for handling is arranged: add appropriate 0 in phosphate buffer#Diesel oil, is configured to the waste water containing diesel oil so that it is just
Beginning concentration is about 4～5g/L, adds the bacteria suspension of preparation, makes cell concentration be about 3 × 107CFU/ml；With under the same terms with
Be not added with the sample of bacteria suspension as comparison, experimental group respectively set with matched group 3 parts parallel.System is placed in constant-temperature table 30 DEG C,
180rpm lucifuge processes 96 hours, is spaced 12h sample analysis total hydrocarbon concentration.
Sample treatment: add the HCl solution acidifying of 3mol/L after every sub-sampling in sample, add isopyknic four chloromethanes
Alkane extracts, and fully stands after vibration, treats that solution is thoroughly layered collection lower floor organic facies, extracts three times, merge organic facies, with
Anhydrous Na2SO4After drying, the concentration of diesel oil is measured with infrared spectrometric oil detector.
The formula of involved culture medium: LB culture medium, minimal medium are with embodiment 2.
To the result containing diesel oil waste water as shown in Figure 4, figure 4, it is seen that according to Fig. 4, initial when diesel oil
When concentration is 4.4g/L, the diesel oil of 63.7% is had to be degraded removal in the process of 4 days, the waste water containing diesel oil of preparation.
Embodiment 6 degrades in soil 0#Diesel oil, wherein 0#The concentration of diesel oil is 3g/L
The production of Rhodococcus fascians (Rhodococcus sp.) P52CCTCC No:M2011181 is with embodiment 1；
By the bacteria suspension cell of preparation and containing 0#The pedotheque of diesel oil 3g/kg wet soil is thoroughly mixed, and makes cell dense
Degree is about 106CFU/g wet soil, water content is about 15%, 30 DEG C of standings, soil sample of stirring every day once, suitable moisturizing, persistently locate
Managing 7 days, different time sampling infrared spectrometric oil detector measures the concentration of diesel oil in soil sample.
During processing, taken pedotheque 0.5g adds 1mL tetrachloromethane, the HCl solution of appropriate 3mol/L, airtight appearance
Device is placed in ultrasound wave bath, 500 watts of supersound process 10S, is repeated 5 times, and is centrifuged 20 minutes with 2000rpm rotating speed, collects organic
Phase, repeats above-mentioned extraction step 1 time, merges the organic facies collected, adds anhydrous sodium sulfate and survey with infrared spectrometric oil detector after drying
The concentration of diesel oil in amount soil sample.
The formula of involved LB culture medium, the phosphate buffer used are with embodiment 1.
Testing result shows, after treated 7 days, in soil, the diesel oil of 3g/kg wet soil decreases 45.4%.
Claims (10)
1. a strain can the multiple hydro carbons of metabolism degradation bacteria petroleum pollution process in application, it is characterised in that degradation bacteria make
It is applied to degraded for bioremediation agents and removes diesel oil in soil and waste water, diesel product, oil, the pollution of oil product generation.
The most according to claim 1 can the application in petroleum pollution processes of the degradation bacteria of the multiple hydro carbons of metabolism, it is special
Levying and be, the pollution that diesel oil, diesel product, oil, oil product produce is linear paraffin, branched paraffin, aromatic hydrocarbon or heterocycle
One of which or any two or more mixing in aromatic hydrocarbons, described linear paraffin is the linear paraffin of C12～C32.
The most according to claim 1 can the application in petroleum pollution processes of the degradation bacteria of the multiple hydro carbons of metabolism, it is special
Levy and be, removal condition of degrading: temperature is 25～42 DEG C, and pH is 6.0～8.0, salinity is 0.5～3%, it is preferred that degraded is gone
Except condition: temperature is 26～32 DEG C, pH is 6.0～7.0, salinity is 0.5～2.0%.
The most according to claim 1 can the application in petroleum pollution processes of the degradation bacteria of the multiple hydro carbons of metabolism, it is special
Levying and be, it is 2～6 × 10 that bacterium solution makes bacterium cell concentration in petroleum-contaminated water after adding7CFU/ml, bacterium in oil-polluted soils
Cell concentration is 1～2 × 107CFU/g。
The most according to claim 1 can the application in petroleum pollution processes of the degradation bacteria of the multiple hydro carbons of metabolism, it is special
Levy and be, the entitled Rhodococcus fascians of described degradation bacteria (Rhodococcus sp.) P52, it is preserved in China's allusion quotation on May 23rd, 2011
Type culture collection center (Wuhan University, China, Wuhan), deposit number is CCTCC No:M2011181.
The most according to claim 1 can the application in petroleum pollution processes of the degradation bacteria of the multiple hydro carbons of metabolism, it is special
Levying and be, described degraded removal is to add in oil-polluted soils or petroleum-contaminated water to stand after Rhodococcus fascians is sufficiently stirred for, warp
Spend 2～11 days, the degradation rate of linear paraffin (C12～C32) is reached 66～87%, the degradation rate of branched paraffin is reached
63.9%, to the utilization rate of polycyclic aromatic hydrocarbon 55～100%, to the utilization rate of heterocyclic arene 61～82%, to soil and useless
Contaminated by diesel oil thing clearance in water reaches 45～63.7%, it is preferred that described waste water stirring condition is under dark condition
150rpm shaken cultivation in 30 DEG C of constant-temperature tables.
The most according to claim 1 can the application in petroleum pollution processes of the degradation bacteria of the multiple hydro carbons of metabolism, it is special
Levy and be, oil content 100mg/L～500mg/L in petroleum-contaminated water, oil content 1～5g/kg in soil pollution；Diesel oil
Determination of Diesel Oil 4～5g/L in polluted-water, Determination of Diesel Oil 1～5g/kg in soil pollution.
The most according to claim 1 can the application in petroleum pollution processes of the degradation bacteria of the multiple hydro carbons of metabolism, it is special
Levying and be, described Rhodococcus fascians is to cultivate production by the following method to obtain:
(1) bacterial strain selects: selecting Rhodococcus fascians (Rhodococcus sp.) P52, deposit number is CCTCC No:M2011181；
(2) bacterial strain activation: by Rhodococcus fascians streak inoculation on LB flat board, cultivates 20～32h in 25～42 DEG C of constant incubators；
(3) seed culture: picking step (2) activation after single colony inoculation in LB fluid medium, 30～42 DEG C, under the conditions of
Shaken cultivation 18～30h prepares seed liquor；
(4) amplification culture: with the inoculum concentration of volume ratio 0.5～4% (v/v), seed liquor is accessed in LB fluid medium, constant temperature
Concussion is cultivated, and treats that bacterium solution cell concentration reaches 108～109During CFU/ml, the centrifugal cell collecting amplification culture；
(5) washed cell: be 0.01～0.1mol/L phosphate buffer or minimal medium washed cell with pH 7.0, weight
Multiple 2～3 times, use centrifugal with under step (4) the same terms, collect cell, finally by cell suspension in phosphate buffer or
Minimal medium, 4 DEG C save backup.
The most according to claim 8 can the application in petroleum pollution processes of the degradation bacteria of the multiple hydro carbons of metabolism, it is special
Levying and be, step (3) vibration rotating speed is 150～200rpm.Centrifugal collection described in step (4) is in 4～10 DEG C by culture fluid,
Rotating speed 4000～8000rpm is centrifuged the 4～8min cells being collected amplification culture.
The most according to claim 8 can the application in petroleum pollution processes of the degradation bacteria of the multiple hydro carbons of metabolism, it is special
Levying and be, the formula of described phosphate buffer is:
Na2HPO41.4～12.5g, KH2PO40.5～4.5g, distilled water 1L, pH 7.0, high pressure steam sterilization 121 DEG C,
20min；
The formula of described minimal medium is:
K2HPO42.7～15g, KH2PO40.8～14g, Na2SO42.0g, MgSO4·7H2O 0.2g, NH4NO33g, trace gold
Belong to saline solution 0.1～1mL, distilled water 1L, pH 7.0, high pressure steam sterilization 121 DEG C, 20min；
Wherein the formula of trace meter saline solution (1mL) is:
FeCl2·4H2O 0.1～0.5g, CoCl2·6H2O 0.038g, MgCl2·4H2O 0.02g, ZnCl20.014g,
H3BO30.0124g, Na2MO4·H2O 0.04g, CaCl2·2H2O 0.001～0.01g.
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| CN115488141A (en) * | 2022-10-24 | 2022-12-20 | 常州大学 | Deep degradation method of oil sludge scum microbial agent in crude oil exploitation and refining processes |
| CN113583908B (en) * | 2021-07-28 | 2023-08-04 | 东朝科技发展(上海)有限公司 | Degreasing strain Alcaligenes sp capable of degrading greasy dirt and application thereof |
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| CN111595922A (en) * | 2020-04-29 | 2020-08-28 | 中国石油天然气股份有限公司 | Method for judging biodegradation degree of thick oil according to petromics |
| CN113583908B (en) * | 2021-07-28 | 2023-08-04 | 东朝科技发展(上海)有限公司 | Degreasing strain Alcaligenes sp capable of degrading greasy dirt and application thereof |
| CN115488141A (en) * | 2022-10-24 | 2022-12-20 | 常州大学 | Deep degradation method of oil sludge scum microbial agent in crude oil exploitation and refining processes |
| CN115488141B (en) * | 2022-10-24 | 2023-10-27 | 常州大学 | Deep degradation method for sludge scum microbial agent in crude oil exploitation and refining process |
| CN117165483A (en) * | 2023-09-06 | 2023-12-05 | 山东碧蓝生物科技有限公司 | Petroleum degrading bacterium and application thereof in petroleum pollution repairing field |
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