CN109593687A - One pseudomonas and its application in oil degradation - Google Patents

One pseudomonas and its application in oil degradation Download PDF

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CN109593687A
CN109593687A CN201910051201.7A CN201910051201A CN109593687A CN 109593687 A CN109593687 A CN 109593687A CN 201910051201 A CN201910051201 A CN 201910051201A CN 109593687 A CN109593687 A CN 109593687A
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microbial inoculum
pseudomonad
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戴柏平
张晓林
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Hangzhou Zaopin St Co ltd
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    • C—CHEMISTRY; METALLURGY
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Abstract

The present invention discloses a pseudomonasPseudomonadaceae sp.GR5, China typical culture collection center, preservation address, China, Wuhan, Wuhan University, deposit number CCTCC M 2018488 are preserved on July 16th, 2018.It is obtained with being screened in petroleum-contaminated soil in the tower of Kuerle, bacterial strain GR5 is in club-shaped, and gram result is negative, and is accredited as pseudomonad through 16sRNA.By domestication by the strain domestication to crude oil at concentrations up to 60g/L when still can normal growth breeding.Oil degradation bacteria of the invention is degraded 20 days under conditions of 30 DEG C of optimum temperature, best PH7.0, reaches 84.4% to the fluid nutrient medium Central Plains oil degradation rate of the 20g/L containing crude oil, oil degradation rate is high, has great application prospect.It can be applied to the fields such as the crude oil pollution in microorganism remediation removal soil and water body environment.

Description

One pseudomonas and its application in oil degradation
Technical field
The present invention relates to a kind of pseudomonad GR5 and its applications in oil degradation.
Background technique
In crude oil production, refining, transport and use process, the leakage of petroleum inevitably occurs, into ring Border pollutes.The environment such as river, soil, underground water and ocean are caused seriously to pollute.On land, crude oil pollution generally by Exploitation drilling cuttings greasy filth, oil pipeline rupture etc. cause；In ocean, oil well oil spilling, oil carrier leakage are caused.The whole world is flat according to statistics There are ten thousand tons of crude oil of 600-1000 to enter environment by all means every year.Expanded later by physics chemical action mode to surrounding It is dissipated to wider.The chemical composition of crude oil is complicated, including many kinds of substance Toxic such as alkane, cycloalkane, aromatic hydrocarbon, asphalitine Matter.Some of polycyclic aromatic hydrocarbons have carcinogenic, teratogenesis, mutagenesis, and human body can be eventually entered by food chain, endanger Human health.
Currently, there are mainly three types of the minimizing technologies of oil pollution: physical method, chemical method and biological method.Physics side Method is mainly incineration method, isolation method and soil replacement method, at high cost, cannot completely remove pollutant.Chemical method mainly extracts Method, washing method and chemical oxidization method, pollutant removal are not thorough, and easily cause secondary pollution.Biological method, which refers to, utilizes biology Vital metabolic activity degraded oil pollutant (mainly microorganism), high treating effect, at low cost, effect on environment compared with It is small, with good application prospect.
Although bioanalysis, which administers oil polluted environment, a large amount of research, currently, being applied to petroleum wastewater The degradation bacteria growth rate of dye processing is slow, degradation rate is low, and degradation rate is slow, cannot reach ideal degradation effect.In order to Efficient, thorough biological prosthetic effect is obtained, the biological prosthetic certainty of the efficient degrading bacteria of petroleum pollution is separated and screen It is required that.
Summary of the invention
It is an object of the invention to be to provide bacterial strain GR5 (the pseudomonad Pseudomonas of one plant of decomposing petroleum hydrocarbon Sp. GR5), China typical culture collection center, preservation address, China, Wuhan, force are preserved on July 16th, 2018 Chinese university, deposit number CCTCC N0:M2018488.
The purpose of the present invention is to provide the applications of above-mentioned oil degradation bacteria GR5.
The application is specially willPseudomonadaceaeSp. GR5 bacterium solution is added directly the water body of oil pollution Or in soil；Or it willPseudomonadaceaeSp. GR5 is fixed on carrier and is prepared into microbial inoculum, then microbial inoculum is administered to In the water body or soil of oil pollution.
It is a further object to provide a kind of solid fungicide of decomposing petroleum hydrocarbon, the solid fungicide activity at Divide includes bacterial strain GR5, deposit number CCTCC M 2018488.
The solid fungicide is willPseudomonadaceaeSp. GR5 is fixed to the microbial inoculum being prepared on carrier.
Further, the carrier is corn stover, the sterile stalk of powder particle diameter≤200 micron.
Further, the processing method of the carrier is high pressure steam sterilization processing, by the stalk after sterilizing in 40-50 DEG C Drying to constant weight, and pulverizer crushed 40 meshes, and 2h is impregnated in distilled water, adjusts pH7-8, is dried to perseverance as 40-50 DEG C Weight.
Further in the microbial inoculumPseudomonadaceaeSp. GR5 living bacteria count and stalk ratio are 0.5x 109- 2x109A/gram.
Further the bacterial preparation process is to incite somebody to actionPseudomonadaceaeSp. GR5 bacterium solution and stalk are directly mixed It closes uniformly, obtains mix bacterium agent.
Further, freeze-drying microbial inoculum, preparation method are made into favor of storing and transporting to reduce the moisture of microbial inoculum It is as follows: willPseudomonadaceaeSp. 150-300g/L glycerol is added in GR5 bacterium solution to mix, then is directly mixed with stalk Uniformly, it is placed in vacuum freeze drier and is dried, obtain freeze-drying microbial inoculum.
Further, the parameter of the vacuum freeze drying is -44 DEG C of pre-cooling 2h, is warming up to -34 DEG C of distillation 10h, is continued 31 DEG C are warming up to get freeze-drying microbial inoculum is arrived.
Further, it is applied to oil degradation bacteriaPseudomonadaceaeSp. GR5 bacterium solution be in logarithmic growth phase or Stationary phase, density 1x1010- 2x1010A/ml.
Further, describedPseudomonadaceaeSp. the culture of GR5 uses LB liquid medium, formula are as follows: chlorine Change sodium 10g/L, 10 g/L of peptone, 5 g/L of yeast powder.
Further, describedPseudomonadaceaeSp. the condition of culture of GR5 is 30 DEG C of temperature, pH7.0, culture Time 60h, revolving speed 150rpm.
Further, the microbial inoculum is used for the usage mode of oil-polluted soils reparation are as follows: is kept for containing for soil 30%-35% Water carries out the sowing of microbial inoculum according to the dosage of every cubic metre of soil 3kg microbial inoculum, turns over to soil, be allowed to fill with microbial inoculum Divide mixing.
Compared with the prior art, the present invention has following advantage:
(1) provided by the inventionPseudomonadaceaeSp. GR5 bacterial strain can be grown by sole carbon source of crude oil, therefore can With the degradation of petroleum pollution in the degradation reparation for oil pollution, especially water body, soil；Its cultural method is simple, raw Long period is short, biomass is big, and tolerance petroleum hydrocarbon concentration is high, has a wide range of application；48hOD is cultivated in LB culture medium600Absorption value can Up to 6.0 or more, high concentration thallus can be obtained in a short time.
(2) present invention is prepared into microbial inoculum as fixation support material using corn stover, and corn stover is agriculture waste Object, price is low, is easy to get, nutrition rich in, degradable to introduce secondary pollution, on the one hand provides money for agricultural wastes The outlet that source utilizes, another aspect corn stover improves soil environment, ventilated, provides more polyoxy for microorganism growth Gas.
Process for fixation used in microbial inoculum of the present invention is absorption method, it is only necessary to be uniformly mixed bacterium solution and stalk, prepare work Skill is simple, and producing cost is low, high treating effect.
The present invention discloses a pseudomonasPseudomonadaceae sp.GR5 with oil field Polluted Soil in the tower of Kuerle Screening obtains in earth, and bacterial strain GR5 is in club-shaped, and gram result is negative, and is accredited as pseudomonad through 16sRNA.By artificial Domestication by the strain domestication to crude oil at concentrations up to 60g/L when still can normal growth breed, oil resistivity is strong, pH6-8's Range can well-grown.Oil degradation bacteria of the invention is anti-in degradation under conditions of 30 DEG C of optimum temperature, best PH7.0 After answering 20 days, 84.4% is reached to the fluid nutrient medium Central Plains oil degradation rate of the 20g/L containing crude oil, oil degradation rate is high, has very Big application prospect.Microbial inoculum of the present invention has apparent repairing effect to low concentration (less than 2%) oil-polluted soils, can be used for big Area oil-polluted soils it is biological prosthetic.
Detailed description of the invention
Fig. 1 is increment of the bacterial strain under condition of different pH in crude oil inorganic salt liquid culture medium in experimental example.
Fig. 2 is the increment in experimental example under condition of different pH in crude oil inorganic salt liquid culture medium.
Fig. 3 is strain under the conditions of optimal pH temperature, to the oil degradation rate of crude oil minimal medium.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described.
It is culture medium prescription used in embodiment below:
Enriched medium: KH2PO41.0 g, (NH4)2SO41.0 g, MgSO40.5 g, CaCl22H2O 0.04g, FeSO4·7H2O 0.01 g, Na2HPO40.5 g, 1.0 g of peptone, 1.0 g of yeast extract, 1000 ml(culture medium of distilled water PH is adjusted according to the pH of original soil sample).
Minimal medium (using petroleum as sole carbon source when experiment): KH2PO41.0 g, (NH4)2SO41.0 g, MgSO4 0.5 g, CaCl22H2O 0.04g, FeSO4·7H20.001 g of O, 1000 ml(medium pH of distilled water is according to original soil sample PH is adjusted, and purifying needs solid medium Shi Kejia agarose 15 g).
LB culture medium: peptone 10 g, NaCl 10 g, 5 g of yeast extract, 1000 ml(medium pH root of distilled water It is adjusted according to the pH of original soil sample, needs solid medium Shi Kejia agar 20 g).
Crude oil culture medium: the crude oil of concentration 2% is added in minimal medium.
Screening and culturing medium: being added 0.5% diesel oil in minimal medium, adds 2% agar.
The above culture medium uses preceding in 0.1MPa, 121 DEG C of sterilizing 20min.
Embodiment 1PseudomonadaceaeSp. the acquisition of GR5 bacterial strain
Screening, separation and the purifying of oil degradation bacteria
(1) bacterium source: oil degradation bacteria is separated from the auspicious oil field contaminated soil of grand celebration lattice and is obtained.
(2) the domestication enrichment of bacterial strain
A. the greasy filth sample for taking 10g oil pollution is added in the minimal medium containing 0.4% crude oil, 30 DEG C of constant-temperature tables Cultivate 5d, revolving speed 150rpm.
B. 5ml supernatant is taken from previous step culture solution, is transferred to containing in 0.6% crude oil minimal medium, 150rpm, 30 DEG C constant-temperature table culture 5 days.
C. step b is repeated, increases by 0.2% oil content every time until be transferred in the crude oil minimal medium containing 1%, 150rpm, 30 DEG C constant-temperature table culture 5 days.
(3) purifying of bacterial strain
By finally obtained culture solution in step (2) according to 10-4、10-5、10-6Multiple be diluted and be respectively coated difference LB Solid media for plates in, after 30 DEG C of inversion 48h, the single colonie that grows of observation plate, the bacterium colony of picking different shape Scribing line separation is then seeded into crude oil minimal medium to there is single colonie.In 150rpm, 30 DEG C of constant-temperature table cultures 5d chooses well-grown bacterial strain.
(4) bacterial strain is tamed
Tame step: by the strain inoculated screened in (3) to containing in 1% crude oil minimal medium, in 150rpm, 30 DEG C of perseverances Warm shaking table culture 5d, what is grown fine is transferred to containing in 2% crude oil minimal medium, and in 150rpm, 30 DEG C of constant-temperature tables are trained Support 5d.Above step is repeated, increases by 1% oil content every time, until bacterial strain is not being grown.
Final domestication obtains the oil degradation bacterial strain of plant height tolerance oil-containing, and tolerance oil content is 5%, and the bacterium is through 16SrRNA It is accredited as pseudomonadPseudomonadaceae sp .GR5。
The bacterium uses glycerol tube preserving process, and LB culture medium culture 12h is mixed in equal volume with 50% sterile glycerol, and sealing is protected It is stored in -20 DEG C of refrigerators, every plant fungi preservation 5.
What the present embodiment was separated toPseudomonadaceae spIt .GR5 can be using petroleum as sole carbon source and the energy Carry out growth and breeding.
Embodiment 2Pseudomonadaceae sp.GR5 optimum growing condition
By the way that different pH is arranged, different temperature is determinedPseudomonadaceae sp.GR5 optimum growing condition.
2-1pH pairs of experimental examplePseudomonadaceae sp.GR5 the influence of growth
Minimal medium 100ml, sodium hydroxide or salt acid for adjusting pH using 1mol/L are 5,6,7,8,9,121 DEG C of sterilizings 30min, cooling inoculation 5%Pseudomonadaceae sp.GR5,150rpm, 30 DEG C of constant-temperature table cultures.With UV, visible light Light spectrophotometer measures light absorption value A value at wavelength 600nm, and the increment of thallus is indicated with the turbidity measured, for 24 hours afterPseudomonadaceae sp.GR5 growing state is shown in Fig. 1.As can be seen that the bacterium can be between 5-9 in pH in figure Growth, and under conditions of pH is 7, the increment highest of the bacterium.
Minimal medium (using petroleum as sole carbon source when experiment): KH2PO41.0 g, (NH4)2SO41.0 g, MgSO4 0.5 g, CaCl22H2O 0.04g, FeSO4·7H20.001 g of O, 1000 ml of distilled water
This experimental example showsPseudomonadaceae sp.GR5 growth has wider pH adaptation range, optimal pH 7- 8。
Such as Fig. 1Pseudomonadaceae sp.GR5 the increment under condition of different pH
Experimental example 2-2 temperature pairPseudomonadaceae sp.GR5 the influence grown
Minimal medium 100ml, sodium hydroxide or salt acid for adjusting pH using 1mol/L are 7,121 DEG C of high-temperature sterilizations 30min, cooling inoculation 5%Pseudomonadaceae sp .GR5。
It is placed in shaking table and cultivates under the conditions of 150rpm, temperature is respectively set to 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C.With Ultraviolet-visible spectrophotometer measures absorbance A value at wavelength 600nm, indicates the increment of thallus to measure turbidity, 5d it AfterwardsPseudomonadaceae sp.GR5 growing state is shown in Fig. 2, and it is equal between 20-35 DEG C to can be seen that the bacterium in chart Can well-grown, and temperature be 30 DEG C under conditions of, the increment highest of the bacterium.
Minimal medium: KH2PO41.0 g, (NH4)2SO41.0 g, MgSO40.5 g, CaCl2·2H2O 0.04g, FeSO4·7H20.001 g of O, 1000 ml of distilled water.
This experimental example showsPseudomonadaceae sp.GR5 growth has wider Acclimation temperature range, and most suitable Growth temperature is between 20-35 DEG C.
In 3 crude oil minimal medium of embodiment,Pseudomonadaceae sp.GR5 degradation property.
It willPseudomonadaceae sp.GR5 be inoculated into 100ml 20g/L containing crude oil, the inorganic salts culture of pH=7.0 In base, then 15d is cultivated in shaking table under the conditions of 150rpm for 30 DEG C, do blank and strain control, measure degradation rate.
Petroleum hydrocarbon measuring method uses infrared spectrophotometer, with isometric carbon tetrachloride extraction crude oil, vibrates 30 minutes, Extract liquor is taken to measure oil content.Degradation rate=(blank group-test group)/blank group × 100%, test strain degradation rate are shown in Fig. 3.
This experimental example shows separating obtainedPseudomonadaceae sp.GR5 under the conditions of 30 DEG C, using petroleum Growth and breeding, oil degradation ability with higher are carried out for sole carbon source.
In 4 place oil contaminated soil of embodiment,Pseudomonadaceae sp.GR5 degradation property
The oil degradation environment of simulation under field conditions (factors), willPseudomonadaceae sp.GR5 it is added to place oil dirt It contaminates in soil, probes into its degradation property under field conditions (factors).
Test example 4-1: inorganic salt liquid culture medium is added in place oil contaminated soil, then willPseudomonadaceae sp.GR5 it being seeded in the culture medium, does not add surfactant, Tu Shui ratio 2:1 is normally cultivated under room temperature, by 15d, with Blank is compared,Pseudomonadaceae sp.GR5 oil content is made to reduce by 18%.Measurement result is shown in Table 1
Petroleum hydrocarbon measuring method uses infrared spectrophotometer, with isometric carbon tetrachloride extraction crude oil, vibrates 30 minutes, takes extraction Liquid is taken to measure oil content.Degradation rate=(blank group-test group)/blank group × 100%, test strain degradation rate are shown in Fig. 3.
Add petroleum hydrocarbon degradation effect after bacterial strain
Processing Blank Bacterial strain GR5 Degradation rate
Soil petroleum hydrocarbon content (g/kg) 48.62 41.35 15.05%
Embodiment 5Pseudomonadaceae sp.GR5 the preparation of microbial inoculum
1、Pseudomonadaceae sp.GR5 the preparation of bacterium solution
It will isolate and purify and tamePseudomonadaceae sp.GR5 it is inoculated into LB culture medium, 150rpm, 30 DEG C of items 3d is cultivated under part, measures OD600Absorption value 6.0 or so.Cell density is 2 × 1010A/ml.
2, the pretreatment of corn stover
Processing step
(1) it crushes: corn stover being dried, crushed 40 meshes with pulverizer；
(2) it sterilizes: the corn stover of crushing being subjected to high pressure steam sterilization (121 DEG C, 30min), 50 DEG C drying to constant weight；
(3) impregnate: the corn stover after drying impregnates 1h in distilled water, adjusts pH to 7 or so；
(4) dry: to be placed in 50 DEG C of baking ovens that drying to constant weight.
3 Pseudomonadaceae sp.GR5 immobilization
(1) microbial inoculum is directly mixed
It willPseudomonadaceae sp.GR5 bacterium solution and the ratio of treated corn stover mass ratio 1:1 are mixed with At straight mixed microbial inoculum.
(2) microbial inoculum is lyophilized
The microbial inoculum of above method preparation is aqueous higher, is easy pollution microbes, toPseudomonadaceae sp.GR5 bacterium solution It is mixed well again with corn stover after middle addition 200g/L glycerol, mixed microbial inoculum is placed in vacuum freeze drier It is dried, obtains freeze-drying microbial inoculum.
Embodiment 6 directly mixes the soil remediation experiment of microbial inoculum
Straight mixed microbial inoculum prepared by embodiment 5 is applied to protect in tower in oil field oil-polluted soils (petroleum concentration 40g/kg) Holding soil moisture content is 30% or so, and every cube of oil soil directly mixes microbial inoculum with 3kg, is mixed well, and keep the good ventilation of soil Property, repair time 30d, the petroleum degradation rate for measuring oil-polluted soils is 53.6%.
The soil remediation experiment of microbial inoculum is lyophilized in embodiment 7
Freeze-drying microbial inoculum prepared by every cube of oil soil application embodiment 5, repair time 30d measure soil as different from Example 6 The degradation rate of petroleum is 51.3%.

Claims (16)

1. a kind of pseudomonad GR5, deposit number is CCTCC M 2018488.
2. application of the pseudomonad GR5 described in claim 1 in degraded oil.
3. the application of pseudomonad GR5 according to claim 2, it is characterised in that: the application is specially by false unit cell Bacterium bacterium solution is added directly in the water body or soil of oil pollution；Or pseudomonad is fixed on carrier and is prepared into microbial inoculum, then Microbial inoculum is administered in the water body or soil of oil pollution.
4. application of the pseudomonad GR5 described in claim 1 in biological prosthetic oil contaminated soil.
5. application of the pseudomonad GR5 described in claim 1 in the water body of biological prosthetic crude oil pollution.
6. a kind of microbial inoculum of decomposing petroleum hydrocarbon, it is characterised in that: the active constituent of the microbial inoculum is pseudomonad GR5, deposit number CCTCC M 2018488。
7. microbial inoculum according to claim 6, it is characterised in that: the microbial inoculum is that pseudomonad GR5 is fixed on carrier The solid fungicide being prepared into.
8. microbial inoculum according to claim 7, it is characterised in that: the carrier is corn stover, powder particle diameter≤200 micron Sterile stalk.
9. microbial inoculum according to claim 7, it is characterised in that: the processing method of the carrier is at high pressure steam sterilization Reason, by the stalk after sterilizing, drying to constant weight in 40-50 DEG C, and pulverizer crushed 40 meshes, and 2h is impregnated in distilled water, adjusts PH7-8, as 40-50 DEG C, drying to constant weight.
10. microbial inoculum according to claim 7, it is characterised in that: pseudomonad GR5 living bacteria count and stalk ratio in microbial inoculum Example is 0.5x109- 2x109A/gram.
11. microbial inoculum according to claim 7, it is characterised in that: the preparation method of the solid fungicide is by pseudomonad GR5 bacterium solution is mixed directly with stalk, obtains mix bacterium agent.
12. microbial inoculum according to claim 6, it is characterised in that: microbial inoculum is freeze-drying microbial inoculum, the preparation method is as follows: by false single 150-300g/L glycerol is added in born of the same parents' bacterium GR5 bacterium solution to mix, then is mixed directly with stalk, is placed in vacuum freeze drier It is dried, obtains freeze-drying microbial inoculum.
13. microbial inoculum according to claim 12, it is characterised in that: the parameter of the vacuum freeze drying is -44 DEG C of pre-coolings 2h is warming up to -34 DEG C of distillation 10h, continues to be warming up to 31 DEG C to get freeze-drying microbial inoculum is arrived.
14. the usage mode that any microbial inoculum of claim 6-13 is used for oil-polluted soils reparation are as follows: keep soil 30%- 35% water content carries out the sowing of microbial inoculum according to the dosage of every cubic metre of soil 3kg microbial inoculum, turns over to soil, be allowed to Microbial inoculum is sufficiently mixed.
15. the bacterium solution that pseudomonad GR5 described in claim 1 is applied to oil degradation is in logarithmic growth phase or stationary phase, Density is 1x1010- 2x1010A/ml.
16. the cultural method of pseudomonad GR5 described in claim 1 are as follows: use LB liquid medium, formula are as follows: sodium chloride 10g/L, 10 g/L of peptone, 5 g/L of yeast powder；Condition of culture is 30 DEG C of temperature, pH7.0, incubation time 60h, revolving speed 150rpm。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111906140A (en) * 2020-07-08 2020-11-10 西安建筑科技大学 Method for degrading large amount of crude oil in soil through oleophilic organisms and activator thereof
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CN111906140A (en) * 2020-07-08 2020-11-10 西安建筑科技大学 Method for degrading large amount of crude oil in soil through oleophilic organisms and activator thereof
CN113583908A (en) * 2021-07-28 2021-11-02 东朝科技发展(上海)有限公司 Oil removing strain Alcaligenes sp capable of degrading oil stains and application thereof
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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