CN110396486A - A kind of bacterial strain for biological prosthetic Petroleum concentration water body, composite bacteria agent and the preparation method and application thereof - Google Patents
A kind of bacterial strain for biological prosthetic Petroleum concentration water body, composite bacteria agent and the preparation method and application thereof Download PDFInfo
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- CN110396486A CN110396486A CN201910540800.5A CN201910540800A CN110396486A CN 110396486 A CN110396486 A CN 110396486A CN 201910540800 A CN201910540800 A CN 201910540800A CN 110396486 A CN110396486 A CN 110396486A
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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
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
- C12R2001/40—Pseudomonas putida
Abstract
The bacterial strain that the invention discloses a kind of for biological prosthetic Petroleum concentration water body, composite bacteria agent and the preparation method and application thereof.The invention particularly discloses one plant of pseudomonas putida (Pseudomonas putida), pollute for biological prosthetic petroleum and oil product.On the one hand a kind of composite bacteria agent A is disclosed, the composite bacteria agent is using above-mentioned pseudomonas putida as activity core, it is equipped with bacillus pumilus, bacillus subtilis and bacillus licheniformis, pass through synergistic effect, petroleum hydrocarbon degradation rate is substantially increased, there is the application potential for efficiently repairing petroleum and oil product pollution.On the other hand a kind of composite bacteria agent B is disclosed, includes above-mentioned pseudomonas putida, and the Nitrosomonas europaea with nitrification function, Nitrobacter winogradskyi, cooperative degradation of petroleum and oil product while improve ammonium removal rate.
Description
Technical field
The present invention relates to pollutant abatement technology fields more particularly to a kind of for biological prosthetic Petroleum concentration water body
Bacterial strain, composite bacteria agent and the preparation method and application thereof.
Background technique
Petroleum is as a kind of fossil energy, complicated component multiplicity, once it leaks into environment, it is difficult to it thoroughly removes, has
There are high toxicity and carcinogenicity.Traditional oil pollution restorative procedure rely primarily on contain, adsorb, dispersing agent and surfactant
Physical chemistry means are waited to recycle the petroleum of leakage, reparation energy quick start is the advantage is that, is suitable for emergency processing, however but
Remaining low concentration petroleum can not be thoroughly removed.And microbial remediation method can make up this deficiency, using oil degradation bacteria with
Petroleum is the characteristic of carbon source and the energy, achievees the purpose that eliminate pollution, restores the ecological balance.Microbial method have it is efficient, low at
Originally, advantage without secondary pollution, is one of the technological means in current oil pollution control field more forward position.But micro- life
There is also certain limitations, such as low efficiency, period length etc. for the method for object degraded oil pollution.
In a kind of patent of invention of complex function petroleum hydrocarbon degradation microbial bacterial agent and the preparation method and application thereof
(CN201811356566.2), disclose a kind of petroleum hydrocarbon degradation microbial inoculum, main effectively viable bacteria ingredient be phosphate solubilizing bacteria, nitrogen-fixing bacteria,
Petroleum hydrocarbon degradation bacterium, but the microbial inoculum is primarily adapted for use in the soil remediation of high salinity environment of oil pollution, and oil degradation
Rate is not greatly improved.It is special in a kind of invention of the biomass microorganism microbial inoculum of degraded oil and preparation method thereof
In benefit (CN201710528958.1), discloses a kind of mix bacterium agent and improve petroleum hydrocarbon degradation rate to a certain extent, but bacterium
Agent complex manufacturing technology, preparation cost is high, is primarily adapted for use in soil remediation.Petroleum polluting water body and soil petroleum pollution are administered
Difference, water have mobility, and processing can be such that pollution range constantly expands with cracking speed not in time.In addition, oil pollution
Ammonium polluted by nitrogen is usually associated in water body.Currently, that there is nitrogen removal rates is low for the ammonium nitrogen processing of oil pollution waste water, water outlet
Not up to standard, the problems such as reagent consumption amount is big and mud handling capacity is big.In a kind of patent of invention of oil refining waste water treatment system
In (CN201320752408.5), disclose a kind of device and method of biological denitrificaion, but there is no very big for nitrogen removal rate
Raising, and the biodegrade of petroleum hydrocarbon cannot be carried out simultaneously.
With the exploitation and extensive use of petroleum-based energy, environment oil pollution problem is got worse, and is studied and is found height
The oil degradation bacterial strain and composite bacteria agent of effect become petroleum and oil product pollutes a biological prosthetic important research direction.
Summary of the invention
The purpose of the present invention is being got worse for current environment petroleum and oil product pollution, biological prosthetic method
The problem of low efficiency, specifically provides a kind of efficient oil degradation bacterial strain and its two kinds of composite bacteria agents, improves petroleum hydrocarbon degradation
Rate, for efficiently repairing petroleum and oil product pollution；And ammonium nitrogen is improved while cooperative degradation of petroleum and oil product
Removal rate.
Technical scheme is as follows:
The first aspect of the present invention, discloses a kind of petroleum hydrocarbon degradation bacterial strain, and the bacterial strain is pseudomonasputidasp
(Pseudomonas putida), the entitled China typical culture collection center of depositary institution, address is Wuhan City, Hubei Province
No. 299 Wuhan Universitys of Wuchang District Bayi Road, the deposit date is 2018.07.30, deposit number is CCTCC NO:M 2018507.
The bacterial strain can still keep higher activity under lower environment temperature (10-15 DEG C), and degradation rate is with respect to optimum temperature condition
Under only decline 19.4%, with environmental applications potential advantages.
The second aspect of the present invention is provided for biological prosthetic composite bacteria agent A and preparation method thereof.Wherein, compound
Microbial inoculum A is equipped with bacillus pumilus, bacillus subtilis and bacillus licheniformis using above-mentioned pseudomonas putida as core, association
Same degraded oil, for efficiently repairing petroleum and oil product polluted-water and soil.Wherein, bacillus pumilus preservation is compiled
Number be CGMCC No.3411；Bacillus subtilis deposit number is CCTCC NO:M 2015688；Bacillus licheniformis preservation
Number is CGMCC No.6677.
The composite bacteria agent A is solid product, and preparation method includes the following steps: in cultivating system independent
It is middle to cultivate respectively, and it is dried to obtain bacterium powder, various bacterium powder are mixed in a certain ratio, so that total viable count is 1 × 109~1 ×
1011Cfu/g, the wherein ratio of pseudomonas putida, bacillus pumilus, four kinds of bacillus subtilis, bacillus licheniformis bacterium
For 10~70%:10~50%:10~50%:10~50%.
Preferably, pseudomonas putida, bacillus pumilus, bacillus subtilis, lichens gemma in composite bacteria agent A
The ratio of four kinds of bacterium of bacillus is 30~50%:10~30%:10~30%:10~30%.
The third aspect of the present invention is provided for biological prosthetic composite bacteria agent B and preparation method thereof.Wherein, this is multiple
Combined bacteria agent B includes above-mentioned pseudomonas putida, and the Nitrosomonas europaea with nitrification function, Vickers nitrify bar
Bacterium, petroleum and oil product in Synergistic degradation polluted-water while, improve ammonium removal rate.A kind of nitrobacteria preservation volume
Number be CCTCC NO:M2014202, preservation date are as follows: on May 14th, 2014, depositary institution's title: Chinese Typical Representative culture protect
Hiding center (CCTCC), preservation address: wuchang, wuhan Luo Jia Shan Wuhan University；Nitrobacter winogradskyi deposit number is CCTCC
NO:M2014203, preservation date are as follows: on May 14th, 2014, depositary institution's title: China typical culture collection center
(CCTCC), preservation address: wuchang, wuhan Luo Jia Shan Wuhan University.
The composite bacteria agent B is liquid form product, and preparation method includes the following steps: in cultivating system independent
It is middle to cultivate respectively, it is mixed in a certain ratio after obtaining bacterium solution, so that total viable count is 1 × 107~5 × 109Cfu/mL, wherein disliking
Smelly pseudomonad, Nitrosomonas europaea, three kinds of bacterium of Nitrobacter winogradskyi ratio be 60~90%:5~20%:5~
20%.
Preferably, three kinds of pseudomonas putida, Nitrosomonas europaea, Nitrobacter winogradskyi bacterium in the composite bacteria agent
Ratio be 70~90%:5~15%:5~15%.
The fourth aspect of the present invention provides petroleum hydrocarbon degradation bacterial strain, composite bacteria agent A or composite bacteria agent B in degraded oil
Application in hydrocarbon；And the application in biological prosthetic petroleum and oil product pollution.
Preferably, the oil product includes but is not limited to gasoline, diesel oil, kerosene, heavy oil, lubricating oil, fuel oil,
Residual oil, tar, paraffin oil, petroleum solvent, pitch, crude oil, one of petrobenzene class or a variety of.
Preferably, the composite bacteria agent A described in gives birth in microbe oil production, washing oil technique, containing petroleum and oil product sewage
Application in object degradation, application method include: S1, microbial inoculum activation: by pseudomonas putida, bacillus pumilus, withered grass bud
It after spore bacillus and Bacillus licheniformis powder mix in proportion, is dissolved in the brown sugar water containing 1%, is aerated 30min, obtains compound
Microbial inoculum activating solution A；It is S2, biological prosthetic: to take composite bacteria agent activating solution A, be added to Petroleum concentration water body in the ratio of 1-5 ‰
In, and nitrogen and phosphorus is supplemented, it carries out biological prosthetic.
Preferably, the composite bacteria agent B described in is applied to while repairing the Petroleum concentration in water body and ammonium polluted by nitrogen,
Its application method includes: to take composite bacteria agent activating solution B, is added in the ratio of 0.1-0.5 ‰ containing petroleum hydrocarbons and ammonium nitrogen
In exceeded sewage, biological prosthetic, guarantee Dissolved Oxygen in Water 2.0-6.0mg/L is carried out.
Of the invention is technically characterized in that
The main cooperation mechanism of composite bacteria agent A: pseudomonas putida disclosed by the invention be composite bacteria agent A core at
Point, there is efficient petroleum hydrocarbon and phenol degrading ability.In addition, the bacillus pumilus in microbial inoculum can generate lipopeptid class biology
The property of crude oil can be changed during growth and breeding, make emulsification of crude oil fine particle for surfactant, be convenient for microorganism
Contact degradation.Bacillus subtilis can be that sole carbon source carries out growth metabolism using petroleum hydrocarbon, and substrate kind is abundant, crude oil
Degradation rate is very fast.Bacillus licheniformis have petroleum hydrocarbon metabolic capability, to alkane, aromatic hydrocarbons, colloid and mixing petroleum hydrocarbon have compared with
Good degradation effect.Therefore, the Pseudomonas putida that will there are different degradation properties and different role to petroleum hydrocarbon different component
Bacterium, bacillus pumilus, bacillus subtilis, bacillus licheniformis are compounded, using different strains to variety classes petroleum
The synergistic effect of alkane degradation accelerates the degradation rate of petroleum hydrocarbon contaminant, all kinds of petroleum hydrocarbon contaminants of more thoroughly degrading, and
The Bacterial community of different strains composition is more stable, and conformability is stronger, can better conform to the dirt of all kinds of petroleum and oil product
Environment is contaminated, to greatly enhance biological degradation rate, and more preferable to the conformability of environment.It can be applied to micro- life
Object oil recovery, washing oil technique, the biological prosthetic technique of oily wastewater.
The main cooperation mechanism of composite bacteria agent B: Nitrosomonas europaea is a kind of autotrophic ammonia oxidizing bacterium, is being aoxidized
In the process using oxygen as final electron acceptor.Nitrobacter winogradskyi belongs to stringent chemosynthetic autotroph, can only be sub- by oxidation
Nitrate obtains energy, and growth needs stringent aerobic.Phenol type substances in petroleum components have very strong bio-toxicity, In
The activity of nitrobacteria will receive the serious inhibition of the pollutant in petroleum-contaminated water, and stench disclosed by the invention is false single
Born of the same parents bacterium ensure that the activity of nitrobacteria, Synergistic degradation contaminant water by the concentration of phenol type substances in reduction petroleum pollution
Ammonium removal rate is improved while petroleum and oil product in body.
Beneficial effect
1. petroleum hydrocarbon degradation bacterial strain pseudomonas putida disclosed in the present invention, should compared with known petroleum hydrocarbon degradation bacterium
Bacterial strain can still keep higher activity under lower environment temperature (10-15 DEG C), under the conditions of degradation rate is with respect to optimum temperature
Only decline 19.4%, with environmental applications potential advantages.
2. composite bacteria agent A produced by the present invention utilizes different strains using pseudomonas putida as the composite bacteria agent of core
To the synergistic effect of variety classes petroleum hydrocarbon degradation, accelerate the degradation rate of petroleum hydrocarbon contaminant, all kinds of stones of more thoroughly degrading
Petroleum hydrocarbon pollutant, and the Bacterial community of different strains composition is more stable, conformability is stronger, can better conform to all kinds of petroleum
And the pollution environment of oil product, so that biological degradation rate is greatly improved, for efficiently repairing petroleum and oil product contaminant water
Body and soil overcome the defect of the prior art.
3. composite bacteria agent B produced by the present invention, petroleum and oil product in Synergistic degradation polluted-water while, is improved
Ammonium removal rate solves the problems, such as that the rate of ammonium removal containing petroleum wastewater is low, overcomes the defect of the prior art.
4. composite bacteria agent A produced by the present invention, in the water body of low concentration pollution, the present invention has better effect, stone
Petroleum hydrocarbon degradation rate reaches 60% or more, illustrate the composite bacteria agent be more applicable in/reparation of slight petroleum-contaminated water.
5. composite bacteria agent B produced by the present invention, of the invention in the waste water containing oil product pollution and ammonium polluted by nitrogen
Ammonium removal rate reaches 95% or more, illustrates that the composite bacteria agent is more applicable for the reparation of petroliferous ammonium nitrogen wastewater.
6. the water body that composite bacteria agent produced by the present invention pollutes petroleum and oil product has good degradation effect,
It is biological prosthetic to illustrate that composite bacteria agent is polluted suitable for petroleum and oil product, has great application prospect.
Detailed description of the invention
Fig. 1 is treatment effect of the petroleum hydrocarbon degradation microbial inoculum in low concentration petroleum-contaminated water.
Fig. 2 is treatment effect of the petroleum hydrocarbon degradation microbial inoculum in middle concentration petroleum-contaminated water.
Fig. 3 is treatment effect of the petroleum hydrocarbon degradation microbial inoculum in high concentration petroleum pollution water body.
Specific embodiment
Clear, complete description is carried out below with reference to technical solution of the attached drawing to various embodiments of the present invention, it is clear that institute
The embodiment of description is only a part of the embodiments of the present invention, instead of all the embodiments；Based on implementation of the invention
Example, those of ordinary skill in the art's obtained all other embodiment without making creative work, all belongs to
In the range that the present invention is protected.
Strain in the present invention is purchased from China General Microbiological culture presevation administrative center (CGMCC) and Chinese Typical Representative
Culture collection (CCTCC), it is CGMCC No.3411 that number, which is respectively as follows: bacillus pumilus deposit number,；Withered grass
Bacillus deposit number is CCTCC NO:M 2015688；Bacillus licheniformis deposit number is CGMCC No.6677.
Experimental example 1
It is low dense at a temperature of the pseudomonas putida activating solution 5mL that effective bacteria concentration is 10,000,000,000 cfu/g is put into 8 DEG C
Oil pollution simulation water body (the natural lake water of 0.5g crude oil+1L) is spent, and supplements nitrogen and phosphorus, is stored at room temperature 2 weeks.
Experimental example 1-1
It is low dense at a temperature of the common pseudomonad activating solution 5mL that effective bacteria concentration is 10,000,000,000 cfu/g is put into 8 DEG C
Oil pollution simulation water body (the natural lake water of 0.5g crude oil+1L) is spent, and supplements nitrogen and phosphorus, is stored at room temperature 2 weeks.
Experimental example 2-5
Experimental example 2-5 is that polluted-water temperature is different from the difference of experimental example 1, in addition to above-mentioned difference, other operation with
Embodiment 1 is all the same.
The difference of experimental example 2-1,3-1,4-1,5-1 and experimental example 1-1 are the difference of polluted-water temperature, except above-mentioned
Difference is outer, other operations are all the same with embodiment 1-1.
Table 1
| Experimental example | Temperature DEG C | Degradation rate % |
| Experimental example 1 | 8 | 24.3±3.2 |
| Experimental example 1-1 | 8 | 10.1±4.2 |
| Experimental example 2 | 10 | 32.9±3.8 |
| Experimental example 2-1 | 10 | 15.6±3.1 |
| Experimental example 3 | 15 | 34.2±3.2 |
| Experimental example 3-1 | 15 | 23.9±4.1 |
| Experimental example 4 | 20 | 37.6±2.9 |
| Experimental example 4-1 | 20 | 32.2±2.7 |
| Experimental example 5 | 25 | 40.8±3.2 |
| Experimental example 5-1 | 25 | 41.5±3.6 |
Interpretation of result, as shown in upper table, the present invention disclosed in petroleum hydrocarbon degradation bacterial strain pseudomonas putida with it is known general
Logical petroleum hydrocarbon degradation bacterium is compared, which can still keep higher activity under lower environment temperature (10-15 DEG C), drop
Solution rate is with respect to only decline 19.4% under the conditions of optimum temperature, the potential advantages with environmental applications.
Embodiment 1
It is a kind of for biological prosthetic bacterial strain, composite bacteria agent and preparation method thereof, specifically comprise the following steps:
The preparation of S1 microbial inoculum: it takes 2g pseudomonas putida to be dissolved in 100mL containing in 1% brown sugar water, is aerated 30 min, obtains
Pseudomonas putida bacteria agent activating solution.Wherein, the effective bacteria concentration of pseudomonas putida is 10,000,000,000 cfu/g.
The reparation of S2 low concentration oil pollution simulation water body: 5mL Pseudomonas putida bacteria agent activating solution is taken to be added to totality
Water body (the natural lake water of 0.5g crude oil+1L) is simulated in the low concentration oil pollution that product is 1L, and supplements nitrogen and phosphorus, is stored at room temperature 2
Week.
Embodiment 2
It is a kind of for biological prosthetic bacterial strain, composite bacteria agent and preparation method thereof, specifically comprise the following steps:
The preparation of S1 microbial inoculum: bacillus pumilus, bacillus subtilis and bacillus licheniformis 2:1:2 in mass ratio are mixed
After conjunction, takes 5g to be dissolved in 100mL containing in 1% brown sugar water, be aerated 30min, obtain composite bacteria agent activating solution a.Wherein, short and small bud
Effective 10,000,000,000 cfu/g of bacteria concentration of spore bacillus, effective 20,000,000,000 cfu/g of bacteria concentration of bacillus subtilis, bacillus licheniformis are effective
10,000,000,000 cfu/g of bacteria concentration.
The reparation of S2 low concentration oil pollution simulation water body: taking 5mL composite bacteria agent activating solution a to be added to total volume is 1L
Low concentration oil pollution simulate water body (the natural lake water of 0.5g crude oil+1L), and supplement nitrogen and phosphorus, be stored at room temperature 2 weeks.
Embodiment 3
It is a kind of for biological prosthetic bacterial strain, composite bacteria agent and preparation method thereof, specifically comprise the following steps:
The preparation of S1 microbial inoculum: pseudomonas putida, bacillus pumilus, bacillus subtilis and bacillus licheniformis are pressed
After mass ratio 2:2:1:2 mixing, takes 7g to be dissolved in 100mL containing in 1% brown sugar water, be aerated 30 min, obtain composite bacteria agent activation
Liquid A.Wherein, effective 10,000,000,000 cfu/g of bacteria concentration of pseudomonas putida, effective 10,000,000,000 cfu/g of bacteria concentration of bacillus pumilus,
Effective 20,000,000,000 cfu/g of bacteria concentration of bacillus subtilis, effective 10,000,000,000 cfu/g of bacteria concentration of bacillus licheniformis.
The reparation of S2 low concentration oil pollution simulation water body: taking 5mL composite bacteria agent activating solution A to be added to total volume is 1L
Low concentration oil pollution simulate water body (the natural lake water of 0.5g crude oil+1L), and supplement nitrogen and phosphorus, be stored at room temperature 2 weeks.
Comparative example 1
Referring to embodiment 1-3, one group of setting does not add low concentration oil pollution simulation water body (the 0.5g original of any microbial inoculum
The natural lake water of oil+1L) comparative example as embodiment 1-3, and nitrogen and phosphorus is supplemented, it is stored at room temperature 2 weeks.
The difference for being distinguished as polluted-water of embodiment 4 and embodiment 1, the polluted-water of embodiment 4 are 1g crude oil+1L
Sanitary sewage.
The difference for being distinguished as polluted-water of embodiment 5 and embodiment 2, the polluted-water of embodiment 5 are 1g crude oil+1L
Sanitary sewage.
The difference for being distinguished as polluted-water of embodiment 6 and embodiment 3, the polluted-water of embodiment 6 are 1g crude oil+1L
Sanitary sewage.
The difference for being distinguished as polluted-water of comparative example 2 and comparative example 1, the polluted-water of comparative example 2 are 1g crude oil+1L
Sanitary sewage.
The difference for being distinguished as polluted-water of embodiment 7 and embodiment 1, the polluted-water of embodiment 7 be 1.5g crude oil+
1L sanitary sewage.
The difference for being distinguished as polluted-water of embodiment 8 and embodiment 2, the polluted-water of embodiment 8 be 1.5g crude oil+
1L sanitary sewage.
The difference for being distinguished as polluted-water of embodiment 9 and embodiment 3, the polluted-water of embodiment 9 be 1.5g crude oil+
1L sanitary sewage.
The difference for being distinguished as polluted-water of comparative example 3 and comparative example 1, the polluted-water of comparative example 3 be 1.5g crude oil+
1L sanitary sewage.
Table 2 is embodiment 1-9, the removal rate of petroleum hydrocarbons data of comparative example 1-3.
Table 2
| Embodiment | Polluted-water (1L) | It puts into microbial inoculum (5mL) | Removal rate of petroleum hydrocarbons (%) |
| Embodiment 1 | 0.5g crude oil | Pseudomonas putida | 40.8% (± 3.2%) |
| Embodiment 2 | 0.5g crude oil | Composite bacteria agent activating solution a | 28.2% (± 4.5%) |
| Embodiment 3 | 0.5g crude oil | Composite bacteria agent activating solution A | 60.4% (± 4.0%) |
| Comparative example 1 | 0.5g crude oil | / | 23.0% (± 6.1%) |
| Embodiment 4 | 1g crude oil | Pseudomonas putida | 38.2% (± 3.9%) |
| Embodiment 5 | 1g crude oil | Composite bacteria agent activating solution a | 29.1% (± 4.1%) |
| Embodiment 6 | 1g crude oil | Composite bacteria agent activating solution A | 54.1% (± 4.6%) |
| Comparative example 2 | 1g crude oil | / | 22.5% (± 5.2%) |
| Embodiment 7 | 1.5g crude oil | Pseudomonas putida | 31.6% (± 5.3%) |
| Embodiment 8 | 1.5g crude oil | Composite bacteria agent activating solution a | 27.5% (± 4.9%) |
| Embodiment 9 | 1.5g crude oil | Composite bacteria agent activating solution A | 46.6% (± 4.2%) |
| Comparative example 3 | 1.5g crude oil | / | 23.2% (± 4.6%) |
Embodiment 1-3 and comparative example 1 are compared analysis, as a result as shown in Fig. 1 and table 2, comparative example 1, which does not add, appoints
The petroleum hydrocarbon degradation rate of what microbial inoculum is 23.0% (± 6.1%), and the petroleum hydrocarbon degradation rate for individually adding pseudomonas putida group is
40.8% (± 3.2%), the petroleum hydrocarbon degradation rate for adding composite bacteria agent a group is 28.2% (± 4.5%), adds composite bacteria agent A
The petroleum hydrocarbon degradation rate of group is 60.4% (± 4.0%).As can be seen that in the reparation of low concentration petroleum-contaminated water, individually
Pseudomonas putida itself there is the ability of certain degraded oil crude oil, and using oil degradation bacteria as the compound bacteria of core
Agent A substantially increases petroleum hydrocarbon degradation rate by synergistic effect, has the application potential for repairing polluted-water.
Embodiment 4-6 and comparative example 2 are compared analysis, as a result as shown in Fig. 2 and table 2, comparative example 2, which does not add, appoints
The petroleum hydrocarbon degradation rate of what microbial inoculum is 22.5% (± 5.2%), and the petroleum hydrocarbon degradation rate for individually adding pseudomonas putida group is
38.2% (± 3.9%), the petroleum hydrocarbon degradation rate for adding composite bacteria agent a group is 29.1% (± 4.1%), adds composite bacteria agent A
The petroleum hydrocarbon degradation rate of group is 54.1% (± 4.6%).As can be seen that in the reparation of middle concentration petroleum-contaminated water, individually
Pseudomonas putida still there is the ability of certain degraded oil crude oil, and using oil degradation bacteria as the compound bacteria of core
Agent substantially increases petroleum hydrocarbon degradation rate by synergistic effect, has the application potential for repairing polluted-water.
Embodiment 7-9 and comparative example 3 are compared analysis, as a result as shown in figure 3 and table 2, comparative example 3, which does not add, appoints
The petroleum hydrocarbon degradation rate of what microbial inoculum is 23.2% (± 4.6%), and the petroleum hydrocarbon degradation rate for individually adding pseudomonas putida group is
31.6% (± 5.3%), the petroleum hydrocarbon degradation rate for adding composite bacteria agent a group is 27.5% (± 4.9%), adds composite bacteria agent A
The petroleum hydrocarbon degradation rate of group is 46.6% (± 4.2%).As can be seen that in the reparation of high concentration petroleum pollution water body, individually
Pseudomonas putida petroleum hydrocarbon degradation rate there was no significant difference with control group (P > 0.05), and using oil degradation bacteria as core
The composite bacteria agent of the heart still significantly improves petroleum hydrocarbon degradation rate by synergistic effect, has the application for repairing polluted-water
Potentiality.
Interpretation of result: pseudomonas putida disclosed by the invention is the core component of composite bacteria agent A, has efficient stone
Petroleum hydrocarbon and phenol degrading ability.In addition, the bacillus pumilus in microbial inoculum can generate Lipopeptide Biosurfactants, in life
The property that crude oil can be changed in long reproductive process, makes emulsification of crude oil fine particle, convenient for microorganism contact degradation.Withered grass bud
Spore bacillus can be that sole carbon source carries out growth metabolism using petroleum hydrocarbon, and substrate kind is abundant, oil degradation rate is very fast.Ground
Clothing bacillus has petroleum hydrocarbon metabolic capability, has preferable degradation effect to alkane, aromatic hydrocarbons, colloid and mixing petroleum hydrocarbon.Cause
This, by the pseudomonas putida to petroleum hydrocarbon different component with different degradation properties and different role, bacillus pumilus,
Bacillus subtilis, bacillus licheniformis are compounded, and are made using different strains to the collaboration of variety classes petroleum hydrocarbon degradation
With, accelerate the degradation rate of petroleum hydrocarbon contaminant, all kinds of petroleum hydrocarbon contaminants of more thoroughly degrading, and different strains composition
Bacterial community is more stable, and conformability is stronger, the pollution environment of all kinds of petroleum and oil product can be better conformed to, thus very
Biological degradation rate is improved in big degree, and more preferable to the conformability of environment.It can be applied to microbe oil production, washing oil work
The biological prosthetic technique of skill, oily wastewater biodegrade and petroleum-contaminated soil.
The petroleum degradation rate of embodiment 10-15 composite bacteria agent A different proportion
Embodiment 10-15 compared with Example 3, the difference is that: the mass ratio of each strain is different in composite bacteria agent A.
In addition to above-mentioned difference, other operations are all the same with embodiment 3.
Table 3
Under embodiment 10-15 composite bacteria agent A different proportion, as shown in table 3, very high petroleum hydrocarbon degradation rate is all had, demonstrate,proved
The Synergistic degradation ability of bright composite bacteria agent A is excellent.
The degradation rate of embodiment 16-19 composite bacteria agent A difference oil product pollution
Embodiment 16-19 compared with Example 3, the difference is that: the source of polluted-water is different.Except above-mentioned difference
Outside, other operations are all the same with embodiment 3.
Table 4
| Embodiment | Oil product minuent polluted-water | Degradation rate % |
| Embodiment 16 | Gasoline | 63.7±3.2 |
| Embodiment 17 | Diesel oil | 60.8±4.3 |
| Embodiment 18 | Kerosene | 62.2±2.9 |
| Embodiment 19 | Heavy oil | 59.5±3.1 |
The result of embodiment 16-19 is as described in Table 4, the degradation of composite bacteria agent A of the invention to oil product polluted-water
Rate is up to 63.7 ± 3.2%.This also illustrates composite bacteria agent A of the invention to have not to petroleum and oil product pollution
Wrong biological prosthetic effect, has great application prospect.
Embodiment 20-28 is the biological prosthetic ability of composite bacteria agent B preparation and different polluted-waters
Embodiment 20
It is a kind of for biological prosthetic bacterial strain, composite bacteria agent and preparation method thereof, specifically comprise the following steps:
The preparation of S1 microbial inoculum: it takes 2g pseudomonas putida to be dissolved in 100mL containing in 1% brown sugar water, is aerated 30 min, obtains
Pseudomonas putida bacteria agent activating solution.Wherein, the effective bacteria concentration of pseudomonas putida is 10,000,000,000 cfu/g.
The reparation of the nitrogenous simulation water body of S2 low concentration oil pollution: 5mL Pseudomonas putida bacteria agent activating solution is taken to add
To the nitrogenous simulation water body of the low concentration oil pollution that total volume is 1L, ((ammonium nitrogen is 50- to 0.5g crude oil+1L sanitary sewage
60mg/L)), for 24 hours, dissolved oxygen is controlled in 2.0-6.0mg/L lasting aeration.
Embodiment 21
The preparation of S1 microbial inoculum: 1:1 is mixed by volume for Nitrosomonas europaea, Nitrobacter winogradskyi, obtains compound bacteria
Agent b.Wherein, effective 1,000,000,000 cfu/mL of bacteria concentration of Nitrosomonas europaea, 1,000,000,000 cfu/mL of Nitrobacter winogradskyi.
The reparation of the nitrogenous simulation water body of S2 low concentration oil pollution: it takes 0.4mL composite bacteria agent b to be added to total volume to be
The nitrogenous simulation water body (0.5g crude oil+1L sanitary sewage (ammonium nitrogen is 50-60mg/L)) of the low concentration oil pollution of 1L, continues
For 24 hours, dissolved oxygen is controlled in 2.0-6.0mg/L aeration.
Embodiment 22
The preparation of S1 microbial inoculum: pseudomonas putida, Nitrosomonas europaea, Nitrobacter winogradskyi 1:2:2 by volume
Mixing, obtains composite bacteria agent B.Wherein, effective 10,000,000,000 cfu/mL of bacteria concentration of pseudomonas putida, Nitrosomonas europaea have
Imitate 1,000,000,000 cfu/mL of bacteria concentration, 1,000,000,000 cfu/mL of Nitrobacter winogradskyi.
The reparation of the nitrogenous simulation water body of S2 low concentration oil pollution: it takes 0.5mL composite bacteria agent B to be added to total volume to be
The nitrogenous simulation water body (0.5g crude oil+1L sanitary sewage (ammonium nitrogen is 50-60mg/L)) of the low concentration oil pollution of 1L, continues
For 24 hours, dissolved oxygen is controlled in 2.0-6.0mg/L aeration.
The difference for being distinguished as polluted-water of embodiment 23 and embodiment 20, the polluted-water of embodiment 23 be 1g crude oil+
1L sanitary sewage (ammonium nitrogen is 50-60mg/L).
The difference for being distinguished as polluted-water of embodiment 24 and embodiment 21, the polluted-water of embodiment 24 be 1g crude oil+
1L sanitary sewage (ammonium nitrogen is 50-60mg/L).
The difference for being distinguished as polluted-water of embodiment 25 and embodiment 22, the polluted-water of embodiment 25 be 1g crude oil+
1L sanitary sewage (ammonium nitrogen is 50-60mg/L).
The difference for being distinguished as polluted-water of embodiment 26 and embodiment 20, the polluted-water of embodiment 26 are 1.5g former
Oil+1L sanitary sewage (ammonium nitrogen is 50-60mg/L).
The difference for being distinguished as polluted-water of embodiment 27 and embodiment 21, the polluted-water of embodiment 27 are 1.5g former
Oil+1L sanitary sewage (ammonium nitrogen is 50-60mg/L).
The difference for being distinguished as polluted-water of embodiment 28 and embodiment 22, the polluted-water of embodiment 28 are 1.5g former
Oil+1L sanitary sewage (ammonium nitrogen is 50-60mg/L).
Table 5
| Embodiment | Polluted-water (1L) | Removal rate of petroleum hydrocarbons (%) | Ammonia nitrogen removal frank (%) |
| Embodiment 20 | 0.5g crude oil (ammonium nitrogen is 50-60mg/L) | 40.3±3.3 | 6.1±0.4 |
| Embodiment 21 | 0.5g crude oil (ammonium nitrogen is 50-60mg/L) | 23.2±5.4 | 64.2±0.6 |
| Embodiment 22 | 0.5g crude oil (ammonium nitrogen is 50-60mg/L) | 39.9±4.1 | 98.5±0.5 |
| Embodiment 23 | 1g crude oil (ammonium nitrogen is 50-60mg/L) | 38.2±3.5 | 5.8±0.5 |
| Embodiment 24 | 1g crude oil (ammonium nitrogen is 50-60mg/L) | 22.6±4.8 | 50.5±0.3 |
| Embodiment 25 | 1g crude oil (ammonium nitrogen is 50-60mg/L) | 38.6±5.7 | 97.6±0.7 |
| Embodiment 26 | 1.5g crude oil (ammonium nitrogen is 50-60mg/L) | 31.9±4.8 | 5.9±0.4 |
| Embodiment 27 | 1.5g crude oil (ammonium nitrogen is 50-60mg/L) | 22.1±5.1 | 37.7±0.5 |
| Embodiment 28 | 1.5g crude oil (ammonium nitrogen is 50-60mg/L) | 31.5±5.0 | 95.1±0.4 |
Embodiment 20-22, embodiment 23-25, embodiment 25-28 pollute petroleum/ammonium polluted by nitrogen in various concentration respectively
In water body, the comparison of removal rate of petroleum hydrocarbons and ammonium removal rate is carried out, the results are shown in Table 5, in various concentration polluted-water
In, the biological prosthetic effect of composite bacteria agent B not only ensure that the removal rate of petroleum hydrocarbon, and ammonium removal rate is compared to composite bacteria agent
B has obtained significantly being promoted, it was demonstrated that pseudomonas putida, Nitrosomonas europaea, Vickers nitrification in composite bacteria agent B
Three kinds of bacterium of bacillus have synergistic effect.Reason is analyzed, is that there is very strong biology poison due to the phenol type substances in petroleum components
Property, the activity of nitrobacteria will receive the serious inhibition of the pollutant, stench disclosed by the invention in petroleum-contaminated water
Pseudomonad ensure that the activity of nitrobacteria, Synergistic degradation by the concentration of phenol type substances in reduction petroleum pollution
Ammonium removal rate is improved while petroleum and oil product in polluted-water.
The biological prosthetic efficiency of embodiment 29-35 composite bacteria agent B different proportion
Embodiment 29-35 compared with embodiment 22, the difference is that: the ratio of each strain is different in composite bacteria agent B.
In addition to above-mentioned difference, other operations are all the same with embodiment 22.
Table 6
Embodiment 29-35 composite bacteria agent B is under different proportion, as shown in table 6, all has very high petroleum hydrocarbon degradation rate
With ammonium removal rate, it was demonstrated that the Synergistic degradation ability of composite bacteria agent B is excellent.Pseudomonas putida disclosed by the invention passes through drop
The concentration of phenol type substances in low petroleum pollution ensure that the activity of nitrobacteria, the petroleum in Synergistic degradation polluted-water
And ammonium removal rate is improved while oil product.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited to
This, anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention
And its inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (12)
1. a kind of petroleum hydrocarbon degradation bacterial strain, it is characterised in that: the bacterial strain is pseudomonas putida (Pseudomonas
Putida), the entitled China typical culture collection center of depositary institution, address are Wuhan City, Hubei Province Wuchang District Bayi Road
No. 299 Wuhan Universitys, the deposit date is 2018.07.30, deposit number is CCTCC NO:M 2018507.
2. a kind of composite bacteria agent A comprising petroleum hydrocarbon degradation bacterial strain described in claim 1, it is characterised in that: the composite bacteria agent
Using pseudomonas putida described in claim 1 as core, it is equipped with bacillus pumilus, bacillus subtilis and lichens gemma bar
Bacterium, cooperative degradation of petroleum and oil product.
3. composite bacteria agent according to claim 2, it is characterised in that: bacillus pumilus deposit number is CGMCC
No.3411；Bacillus subtilis deposit number is CCTCC NO:M 2015688；Bacillus licheniformis deposit number is CGMCC
NO.6677。
4. composite bacteria agent A according to claim 2, it is characterised in that: the composite bacteria agent is solid product, preparation
Method includes the following steps: to cultivate respectively in cultivating system independent, and is dried to obtain bacterium powder, and various bacterium powder are pressed one
Certainty ratio mixing, so that total viable count is 1 × 109~1 × 1011Cfu/g, wherein pseudomonas putida, bacillus pumilus, withered
Careless bacillus, four kinds of bacterium of bacillus licheniformis ratio be 10~70%:10~50%:10~50%:10~50%.
5. composite bacteria agent A according to claim 2, it is characterised in that: pseudomonas putida, short and small bud in the composite bacteria agent
Spore bacillus, bacillus subtilis, four kinds of bacterium of bacillus licheniformis ratio be 30~50%:10~30%:10~30%:10~
30%.
6. a kind of composite bacteria agent B comprising petroleum hydrocarbon degradation bacterial strain described in claim 1 is it is characterized by: the composite bacteria agent packet
Pseudomonas putida described in claim 1 is included, and the Nitrosomonas europaea with nitrification function, Vickers nitrify bar
Ammonium removal rate is improved while bacterium, cooperative degradation of petroleum and oil product.
7. composite bacteria agent B according to claim 6, it is characterised in that: the Nitrosomonas europaea deposit number
For CCTCC NO:M2014202；Nitrobacter winogradskyi deposit number is CCTCC NO:M2014203.
8. composite bacteria agent B according to claim 6, it is characterised in that: the composite bacteria agent is liquid form product, preparation
Method includes the following steps: to cultivate respectively in cultivating system independent, is mixed in a certain ratio after obtaining bacterium solution, so that
Total viable count is 1 × 107~5 × 109Cfu/mL, wherein pseudomonas putida, Nitrosomonas europaea, Nitrobacter winogradskyi
The ratio of three kinds of bacterium is 60~90%:5~20%:5~20%.
9. composite bacteria agent B according to claim 6, it is characterised in that: pseudomonas putida, Europe are sub- in the composite bacteria agent
Nitrify monad, the ratio of three kinds of bacterium of Nitrobacter winogradskyi is 70~90%:5~15%:5~15%.
10. described in petroleum hydrocarbon degradation bacterial strain described in claim 1, composite bacteria agent A as claimed in claim 2 or claim 6
Composite bacteria agent B decomposing petroleum hydrocarbon and biological prosthetic petroleum and oil product pollution in application, it is characterised in that: stone
Oil product includes but is not limited to gasoline, diesel oil, kerosene, heavy oil, lubricating oil, fuel oil, residual oil, tar, paraffin oil, and petroleum is molten
Agent, pitch, crude oil, one of petrobenzene class or a variety of.
11. application described in any one of claim 10, it is characterised in that: the composite bacteria agent A described in it is in microbe oil production, washing oil work
Skill, containing petroleum and oil product saprobia degradation in application, application method include: S1, microbial inoculum activation: will be mixed to prepare
Composite bacteria agent A press solid-to-liquid ratio 1:10, be dissolved in the brown sugar water containing 1%, be aerated 30min, obtain composite bacteria agent activating solution A；S2,
It is biological prosthetic: to take composite bacteria agent activating solution A, be added in Petroleum concentration water body in the ratio of 1-5 ‰, and supplement nitrogen phosphorus battalion
It supports, carries out biological prosthetic.
12. application described in any one of claim 10, it is characterised in that: the composite bacteria agent B described in it is applied to while repairing in water body
Petroleum concentration and ammonium polluted by nitrogen, application method includes: to take composite bacteria agent activating solution B, is added in the ratio of 0.1-0.5 ‰
It in containing petroleum hydrocarbons and the exceeded sewage of ammonium nitrogen, carries out biological prosthetic, guarantees Dissolved Oxygen in Water 2.0-6.0mg/L.
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Denomination of invention: A kind of strain and compound bacterial agent used for bioremediation of petroleum polluted water and its preparation method and application Effective date of registration: 20221011 Granted publication date: 20210316 Pledgee: Wuhan area branch of Hubei pilot free trade zone of Bank of China Ltd. Pledgor: WUHAN SHUIZHIGUO ENVIRONMENTAL PROTECTION TECHNOLOGY Co.,Ltd. Registration number: Y2022420000345 |