CN104726370A - Enterobacter with crude oil degradation effect and application of enteric bacilli - Google Patents
Enterobacter with crude oil degradation effect and application of enteric bacilli Download PDFInfo
- Publication number
- CN104726370A CN104726370A CN201510084360.9A CN201510084360A CN104726370A CN 104726370 A CN104726370 A CN 104726370A CN 201510084360 A CN201510084360 A CN 201510084360A CN 104726370 A CN104726370 A CN 104726370A
- Authority
- CN
- China
- Prior art keywords
- enterobacteria
- crude oil
- oil
- degradation
- contaminated soil
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Environmental & Geological Engineering (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Soil Sciences (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention relates to a crude oil degradation bacterium and application thereof, provides a method for rapidly degrading crude oil by virtue of the crude oil degradation bacterium, and belongs to the technical field of environmental pollution. The strain is capable of rapidly decomposing the crude oil, the crude oil can be decreased from 50g/L to about 20g/L at proper conditions, the total removal rate of petroleum is above 80%, and the degradation rate of the strain to the crude oil is not less than 60%.
Description
Art
The present invention relates to a kind of oil degradation enterobacteria and application thereof, and provide a kind of method utilizing oil degradation bacterium fast degradation crude oil, belong to environment pollution technology field.
Background technology
Tensio-active agent is the material that a kind of existing hydrophilic radical has again lipophilic group, it has wetting or anti-stick, emulsification or breakdown of emulsion, foaming or froth breaking and solubilising, dispersion, washing, the effect such as anticorrosion, antistatic, is a kind of purposes fine chemical product widely.Tensio-active agent is except in daily life as washing composition, and other application almost can cover all field of fine chemical.
But a large amount of uses of chemical surfactant may cause the secondary pollution of soil or water body, and most of chemical surfactant is all difficult to again be utilized thus can be detained in the environment.Therefore, environment amenable bio-surfactant more and more receives the concern of people.
Bio-surfactant is the one of natural surface active agent, and some mainly referring to that microorganism produces under certain culture condition have and obviously reduce capillary meta-bolites.Compared with the tensio-active agent of chemosynthesis, bio-surfactant is except having identical characteristics such as reducing surface tension, stable emulsion and foaming, also there is the environmental friendliness characteristic not available for general chemical surfactant, as nontoxic, can the advantage such as natural biology degraded, ecological safety.Bio-surfactant is also normal in addition has higher surfactivity than chemical surfactant, and emulsifying capacity is also stronger, has potential using value in industrial aspect such as medicine, makeup, washing composition and food.Particularly at difficult degradation hydrophobic organic compound as in the reparation of polycyclic aromatic hydrocarbons, polychlorobiphenyl, petroleum hydrocarbon, bio-surfactant plays an important role, and it makes hydrophobic organic compound emulsification and obvious solubilising, and degradation bacteria is more easily degraded these materials.
In addition, bio-surfactant still can play a role in extreme environment, and it is not by the impact of the external environments such as temperature, and general chemical surfactant is strict to requirement for environmental conditions, can not get due effect in extreme circumstances.And bio-surfactant can be utilized by microorganism, can not extended residual in the environment, can not harmful effect be produced to environment.But bio-surfactant is still difficult to commercialization at present and utilizes, mainly because its productive rate is low and substrate cost is high, purge process investment is large.Obtain the bacterial classification of the bio-surfactant of metabolism to be solve the high a kind of approach of current bio-surfactant cost in a large number.
Publication number is that the Chinese patent of CN 102978135A discloses a kind of oil degradation bacterium and application of producing lipidic biomass tensio-active agent.The name of this oil degradation bacterium is called Pseudomonas aeruginosa MZ01, be preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center being positioned at Chaoyang District, city of BeiJing, China great Tun road Institute of Microorganism, Academia Sinica on July 12nd, 2012, deposit number is CGMCC No.6354.This bacterial strain mainly produces lipid, and its CMC value is 0.1g/l, and the surface tension of water can be down to 29.9mN/m from 72.0mN/m, and it has obvious emulsification and solublization to hydrophobic organic compound, and persistent; Its in 25 DEG C, pH value is 7, rotating speed is that under the condition of 150rpm, crude oil can be down to about about 92mg/l from 200mg/l by 5d, oil total removal rate for reaching more than 52%, wherein this bacterial strain to the degradation rate of crude oil more than 30%.
Publication number is that the Chinese patent of CN 102911892A discloses a strain oil degradation bacteria and the application in ternary oil recovery treatment thereof.Belong to petroleum microorganism field.It solve the problem lacking the microbial strains that may be used in ternary Treatment technology of oil extraction wastewater at present.Oil degradation bacteria of the present invention is pseudomonas (Pseudomonassp.) JNS01, is deposited in China typical culture collection center, and preserving number is CCTCCNO:M2012021.Pseudomonas JNS01 of the present invention can be well-grown in the polyacrylamide basic solution of 10.5 up to 1800mg/L, pH value in concentration, crude content in Produced Liquid after pseudomonas JNS01 process can be reduced to 25mg/L, and degradation rate can reach 92%.
Publication number is that the Chinese patent of CN 103160452A discloses and is a kind ofly separated from petroleum-contaminated soil fast and screens the method for the pure bacterial strain of product tensio-active agent petroleum hydrocarbon degradation.First gather petroleum-contaminated soil from oil field, the amount by 5% is added into crude oil and is sole carbon source and in the alkane degradation substratum that improves gradually of content, constant-temperature table carries out enrichment culture.Bacterium liquid after enrichment is diluted to coating cultivation on blood agar to a certain degree, cultivate 1-3d for 30 DEG C, get transparent circle edge clear on blood agar and large bacterium colony, the dibbling of bacterium liquid is dipped on oily flat board with sterilizing toothpick is blunt nosed after liquid meat peptone culture medium culturing, cultivate 3d for 30 DEG C, get bite oil mark obviously and larger colony inoculation to save backup and after using meat peptone culture medium culturing in meat peptone inclined-plane, successively inoculation alkane degradation substratum is with in product tensio-active agent substratum, cultivate 7d and 3d for 30 DEG C, measure the ability of alkane degradation ability and product tensio-active agent, obtain the petroleum hydrocarbon degradation bacterial strain producing tensio-active agent.
Publication number is the separating screening method that the Chinese patent of CN 102839124A discloses a kind of extreme environment oil degradation bacterium, solves the operating process complexity of existing oil degradation bacterium separating screening method existence, the shortcoming of low, the separating obtained microorganism effect difference of separation efficiency.Its technical characterstic is one, adopts minimal medium enrichment oil degradation bacterium from extreme environment contaminated soil; Two, by after gained enrichment bacterium liquid gradient dilution, carry out batch test and investigate oil degradation situation; Three, adopt group's composition of DGGE technical Analysis each extent of dilution bacterium liquid, thus obtain crude oil removal effect with enrichment bacterium liquid phase is similar, form the most simply bacterium liquid, be efficient oil degradation function yeast.The method has simply, efficiently, feature fast, separating obtained oil degradation bacterium has higher removal effect usually.
Publication number is a kind of method that the Chinese patent of CN 101607120 discloses degrading crude oil with bacillus, by the proportions degradation solution of 80 ~ 150ml crude oil nutrient solution inoculation, 0.8 ~ 1.5ml genus bacillus bacterium liquid, by degradation solution 30 ~ 37 DEG C of temperature, under the shake condition of 150 ~ 200r/min, degrade 10 ~ 30 days, the concentration of genus bacillus bacterium liquid is 107 ~ 109/ml, and the mass concentration of crude oil nutrient solution is 0.5 ~ 5%; The present invention is the genus bacillus utilizing single kind, eliminate the trouble will being prepared degradation bacteria liquid at present by two kinds and above bacterial classification, more convenient to operate, the present invention does not need to prepare nutritive medium in addition yet, also without additional surfactants, degraded cost can be lower, and the present invention is better to the effect of oil degradation, about 20 days substantially can be degradable by crude oil.Therefore the present invention is a kind of easy to operate, and cost is lower, and degradation efficiency uses the method for degrading crude oil with bacillus preferably.
Publication number is that the Chinese patent of CN 103436464A discloses a kind of low temperature resistant oil degradation bacterial strain, cultural method and application thereof.The cultural method of low temperature-resistant strains genus bacillus of the present invention comprises: petroleum pollution soil sample is utilized physiological saline solution, stratification after shaking table vibration, bacteria suspension is accessed in crude oil inorganic salt liquid substratum, shaking table gets appropriate lower floor fermented liquid in liquid nutrient medium after cultivating, be seeded in fresh inorganic salt liquid substratum and continue to cultivate, so repeatedly carry out 5 times.Gradient dilution is carried out to gained bacterium liquid, is applied on crude oil inorganic salt solid medium and cultivates, observe substratum colony counts, select the substratum that quantity is moderate, carry out streak culture; Repeatedly tame, streak culture, until obtain the pure bacterium of many strains.Middle long-chain saturated alkane in low temperature-resistant strains genus bacillus preferential degradation oil component of the present invention, this bacterium can be applicable to, in the biological restoration of petroleum hydrocarbon degradation and soil or petroleum polluting water body, especially can be used for low temperature environment.
Publication number is the biostimulation method that the Chinese patent of CN 101947544A discloses the reparation of a kind of oil-polluted soils.The method is under the prerequisite not adding petroleum pollution biological degradation microbial inoculum, by adding soil conditioner, improvement edatope, reduce soil stickiness, increase soil permeability, regulate retention capacity of soil, regulate the necessary inorganic and Organic nutrient of indigenous microbes growth simultaneously, maintain soil pH value near neutral, promote soil PetroChina Company Limited. contaminant degradation microbial growth and breeding, improve indigenous microbes to the degradation efficiency of petroleum pollution; By planting plants, improve the Soil Micro-environment near root system of plant, plant, by stimulating the amount reproduction of indigenous microbes to low molecule organic matters such as root system secretion amino acid, promotes the rhizosphere microbial degraded of petroleum pollution indirectly; Accelerate the reparation of oil contaminated soil, recover soil original state.
Soil, as acceptor maximum in environment, is a kind of organic and inorganic combination, containing different kinds of petroleum hydrocarbon pollutant degrading microorganism, therefore has powerful buffering and self-repairing capability to pollution.Soil do not have petroleum-polluted before, its natural community's structure or microbial population are also not suitable for petroleum pollution; After edatope is by petroleum pollution, biological community structure can change, the population that can adapt to petroleum pollution continues development or can develop population better, and these indigenous microbes achieve the degraded of petroleum pollution while utilizing the oil in soil and the hydrocarbon polymer in derivative thereof as its carbon source and energy growth and breeding.Large quantity research shows; when flora is in oil polluted environment, utilize the microbe population sharp increase of hydrocarbon compound, Atlas is reported in 1% of the general Zhi Zhan microflora of degradation bacteria under home; and when environment is subject to petroleum pollution, degradation bacteria ratio then brings up to 10%.
Utilize petroleum hydrocarbon degradation microorganism in native country in soil can carry out the reparation of oil-polluted soils, but after soil particle is combined with petroleum pollution, the native separating difficulty of oil is large, under the prerequisite not adding external source petroleum hydrocarbon degradation microbial inoculum, the impact of environmental factors is more remarkable, thus slow down the degradation speed of these pollutents.These environmental impact factors mainly comprise following several: oxygen concentration, soil pH value and soil penetrability etc. in envrionment temperature, soil humidity, soil Middle nutrition levels of substance, electron acceptor(EA) supply situation, soil.Therefore, improve the character of oil-polluted soils, regulate and control these external influence factors affecting remediation efficiency, for raising repairing effect, there is extremely important effect.Edatope is improved by taking engineering measure, increase the bioavailability of petroleum pollution, the edatope condition of more suitable native country petroleum hydrocarbon degradation microorganism is provided, native country petroleum hydrocarbon degradation microbial growth and breeding can be stimulated, improve the degradation capability of native country petroleum hydrocarbon degradation microorganism self, thus accelerate the degraded of petroleum pollution, improve the remediation efficiency of oil-polluted soils.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming overcoming prior art is with not enough, provides a kind of oil degradation enterobacteria producing lipidic biomass tensio-active agent.
Another object of the present invention is to the application of the oil degradation enterobacteria that described product lipidic biomass tensio-active agent is provided.
Object of the present invention is achieved through the following technical solutions: a kind of oil degradation bacterium producing lipidic biomass tensio-active agent, name is called enterobacteria (Enterobacter sp.) ODB01, be preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center being positioned at Chaoyang District, city of BeiJing, China great Tun road Institute of Microorganism, Academia Sinica on December 23rd, 2014, deposit number is CGMCC No.10233.
The form of described enterobacteria ODB01 is: bacterium colony is smooth, ball-type, flush edge, and in faint yellow, belong to Gram-negative bacteria, the form of this thalline of observed under electron microscope is tyrothricin;
The 16S rDNA of described enterobacteria ODB01 is as shown in SEQ ID NO.3;
The oil degradation bacterium of described product lipidic biomass tensio-active agent is preparing the application in bio-surfactant and oil degradation.
Wherein, the screening process of described enterobacteria: first by sample mixture diluted samples to 10 such as the water and soils of crude oil pollution
-5coat on solid separation and Culture flat board, 30 DEG C of constant temperature culture, separation obtains single bacterium colony totally 453, picking list bacterium colony is in the test tube of 10mL filling 3mL liquid separation culture medium, after 30 DEG C of constant-temperature tables cultivate 12h, be forwarded in the test tube containing 5mL crude oil liquid substratum, 30 DEG C, cultivate under 150rpm condition, the changing conditions of Real Time Observation nutrient solution Crude Oil, be that leading indicator is selected by the ability of oil degradation, obtain the bacterial strain that oil degradation ability is higher, and carry out strain identification, its 16S rDNA gene sequencing the results are shown in SEQ ID NO.3.Its expert's conclusion of data analysis such as cultural characteristic, microscopic features, rRNA gene order of breeding species is enterobacteria (Enterobacter sp.).
The formula of solid separation and Culture is copper sulfate 0.5mg, ferrous sulfate 5mg, zinc chloride 5mg, manganous sulfate 5mg, calcium chloride 20mg, dipotassium hydrogen phosphate 4g, potassium primary phosphate 0.5g magnesium sulfate 1g, sodium-chlor 1g, ammonium nitrate 2g, crude oil 1mL, agar 10g, with distilled water constant volume 1000mL, pH nature, 121 DEG C of sterilizing 20min.
The formula of liquid separation culture medium is copper sulfate 0.5mg, ferrous sulfate 5mg, zinc chloride 5mg, manganous sulfate 5mg, calcium chloride 20mg, dipotassium hydrogen phosphate 4g, potassium primary phosphate 0.5g magnesium sulfate 1g, sodium-chlor 1g, ammonium nitrate 2g, crude oil 1mL, with distilled water constant volume 1000mL, pH nature, 121 DEG C of sterilizing 20min.
The formula of crude oil liquid substratum is copper sulfate 0.5mg, ferrous sulfate 5mg, zinc chloride 5mg, manganous sulfate 5mg, calcium chloride 20mg, dipotassium hydrogen phosphate 4g, potassium primary phosphate 0.5g magnesium sulfate 1g, sodium-chlor 1g, ammonium nitrate 2g, crude oil 5mL, with distilled water constant volume 1000mL, pH nature, 121 DEG C of sterilizing 20min.
Summary of the invention two. provide described enterobacteria (Enterobacter sp.), the extracellular enzyme that CGMCC No.10233 fermentation is standby.Described bacterial strain extracellular enzyme is one group of prozyme, comprising lipase, polygalacturonase, proteolytic enzyme, rennin etc.
In a specific embodiment, described enterobacteria fermented liquid also comprises: this bacterium culture medium matter and this bacterium meta-bolites.
The present invention's second goal of the invention is enterobacteria purposes for rapid cleaning in crude oil pollution area.
Wherein said purposes comprises: the treatment process spraying oil contaminated soil with enterobacteria starter (solid formulation).
In a specific embodiment, use the treatment process of above-mentioned enterobacteria starter (solid powder) appropriate amounts of sterilized water dilution spraying oil contaminated soil, can fast processing crude oil pollution problem, compare with conventional cleaning process, the clean time can be shortened, decrease opportunities for contamination simultaneously, improve the degree of cleaning after crude oil pollution.
Wherein, with enterobacteria fermented liquid spice (oil contaminated soil) zymotechnique: adopt enterobacteria (solid powder), every gram containing microbe population 1 × 10
10more than cfu, first with sterilized water by Dilution for powder, spray inoculation on oil contaminated soil, carry out after inoculation fermentation clean.Adopt enterobacteria solid powder to utilize certain sterilized water furnishing inoculation liquid (liquid preparation), every milliliter containing microbe population 1 × 10
7more than cfu, is seeded in fermented liquid on oil contaminated soil, ferments.Consumption is every mu of contaminated soil enterobacteria 0.5kg ~ 1.0kg/ mu.
In addition, directly use enterobacteria fermented liquid, shorten clean oil contaminated soil cycle 10-15 days, reduce crude oil pollution risk.
Accompanying drawing explanation
Fig. 1: screen the major microorganisms bacterial strain with degrading crude oil effect obtained.Left 1 is contrast; Left 2-9 is that different bacterial strains is cultivated in containing the minimal medium of crude oil, and left 2 is ODB01 bacterial strain.
Fig. 2: the drug susceptibility test result figure of enterobacteria (Enterobacter sp.) ODB01.1-11 represents the result figure of the antibiotic treatment such as paraxin, amikacin, Liu Suanyan NEOMYCIN SULPHATE, Streptomycin sulphate, many glutinous mycin B, Nalidixic Acid, Ampicillin Trihydrate, erythromycin, penicillin, Vulkamycin. PA-93 and tsiklomitsin respectively.
Technique effect
(1) bacterial strain provided by the invention mainly produces lipid, has obvious emulsification and solublization to hydrophobic organic compound, and persistent.
(2) bacterial strain provided by the invention 25 DEG C, pH value are 7, and rotating speed is that under the condition of 150rpm, crude oil can be down to about about 20g/L from 50g/L by 5d, oil total removal rate for reaching more than 80%, wherein this bacterial strain to the degradation rate of crude oil more than 60%.
Embodiment
Below, the present invention will be further detailed by embodiment, but it is not limited to any one or similar example of these embodiments.
Embodiment 1: enterobacteria ODB01 strains separation
First by sample mixture diluted samples to 10 such as the water and soils of crude oil pollution
-5coat on solid separation and Culture flat board, 30 DEG C of constant temperature culture, separation obtains single bacterium colony totally 453, picking list bacterium colony is in the test tube of 10mL filling 3mL liquid separation culture medium, after 30 DEG C of constant-temperature tables cultivate 12h, be forwarded in the test tube containing 5mL crude oil liquid substratum, 30 DEG C, cultivate under 150rpm condition, the changing conditions of Real Time Observation nutrient solution Crude Oil, therefrom filter out the bacterial strain that 49 strains have oil degradation ability, determine that 109 bacterial strains (being enterobacteria ODB01) are for the strongest bacterial strain of these bacterial strain Crude Oil degradation capabilities by repeatedly repeating screening, further employing weighting method learns that 109 bacterial strains are at 150rpm, after 30 DEG C of shaking tables cultivate 7,45% is reached to the degradation rate of crude oil.
Embodiment 2: identification of strains
One, form and biochemical identification
1, form is enterobacteria, and carry out Physiology and biochemistry qualification according to Bacteria Identification handbook to it, result is as follows:
"+" represents positive; "-" represents negative
2, drug susceptibility test result shows: enterobacteria (Enterobacter sp.) ODB01 is the most responsive to paraxin, secondly be amikacin, Liu Suanyan NEOMYCIN SULPHATE, Streptomycin sulphate, many glutinous mycin B and Nalidixic Acid, but insensitive to Ampicillin Trihydrate, erythromycin, penicillin, Vulkamycin. PA-93 and tsiklomitsin etc.
Two, Molecular Identification
1, design and select appropriate primer
According to the 16S rDNA gene order of bacterium, design PCR primer, is respectively:
| SEQ ID No：1 | 5-AGAGTTTGATCCTGGCTCAG-3(SEQ ID No：1) |
| SEQ ID No：2 | 5-GGTTACCTTGTTACGACTT-3(SEQ ID No：2) |
Relevant primer synthesizes in Sangon Biotech (Shanghai) Co., Ltd..
2, universal primer amplification
To the DNA of enterobacteria, use ABI 9700 type PCR instrument, carry out biological experiment operation.
(1) reaction system of PCR is:
| PCR reaction buffer (10x PCR Buffer with 20mM MgCl 2) | 2.5μL |
| dNTP mix(25mM each) | 1μL |
| High-fidelity Taq enzyme (PCR Enzyme, 5U/ μ L) | 1μL |
| SEQ ID No：1(1μM) | 1μL |
| SEQ ID No：2(1μM) | 1μL |
| DNA of bacteria | 1μL |
| Ultrapure water | 17.5μL |
Above reagent, except DNA of bacteria, ultrapure water, primer, all comes from Sequenom company of the U.S..
(2) loop parameter of PCR is: 95 DEG C, 4 minutes, 1 circulation, 95 DEG C, 20 seconds, 56 DEG C, 30 seconds, 72 DEG C, 60 seconds, 45 circulations, 72 DEG C, 3 minutes, 1 circulation, 4 DEG C, preserves.
3, pcr amplification product checks order through Sangon Biotech (Shanghai) Co., Ltd., obtains the 16S rDNA sequencing result of enterobacteria ODB01: see SEQ ID No:3.
Embodiment 3: the degrading crude oil characteristic of enterobacteria ODB01
The formula of preparation substratum is copper sulfate 0.5mg, ferrous sulfate 5mg, zinc chloride 5mg, manganous sulfate 5mg, calcium chloride 20mg, dipotassium hydrogen phosphate 4g, potassium primary phosphate 0.5g magnesium sulfate 1g, sodium-chlor 1g, ammonium nitrate 2g, crude oil 10g, with distilled water constant volume 1000mL, pH nature, every bottled 400mL, 121 DEG C of sterilizing 20min.The ODB01 bacterium liquid of inoculation 1mL, 30 DEG C, carry out after shaking table cultivates 7 days under 200rpm condition, by 400mL fermented liquid after the centrifugal 10min of 8000rpm, use sherwood oil extracting, get petroleum ether extraction liquid after removing enterobacteria body, concentrating under reduced pressure removal sherwood oil is weighed.Found that and crude oil can be down to about about 2g/L from 10g/L through 7d process Enterobacter sp.ODB01, crude oil total removal rate reaches about 80%.
Embodiment 4: enterobacteria ODB01 repairs oil contaminated soil effect
The liquid nutrient medium composition of preparation enterobacteria ODB01 is peptone 10g, yeast extract 5g, sodium-chlor 10g, ammonium chloride 60mg, glucose 300mg, potassium primary phosphate 60mg, crude oil 1g, distilled water 1000mL, pH nature, 121 DEG C of sterilizing 20min.The ODB01 bacterium liquid of inoculation 1mL, 30 DEG C, carry out after shaking table cultivates 2 days under 150rpm condition, enterobacteria ODB01 fermented liquid is for subsequent use.Load the soil of orcharding with 10L nutrition pot, be divided into three groups, in first group and second group, every alms bowl adds 200g crude oil, 3rd group of every alms bowl adds 200g distilled water in contrast, and first group adds 200ml enterobacteria ODB01 bacterium liquid, and second group adds 200ml nutrient solution (peptone 10g, yeast extract 5g, sodium-chlor 10g, ammonium chloride 60mg, glucose 300mg, potassium primary phosphate 60mg, crude oil 1g, distilled water 1000mL, pH nature, 121 DEG C of sterilizing 20min).The greenhouse that each process is placed in 30 DEG C is respectively cultivated.Every day quantitatively keeps the skin wet, and is taken out by soil in each alms bowl, add 60 DEG C of water and carry out immersion 30min after 30d, after standing 10min after the centrifugal 10min of 8000rpm, go precipitation, use sherwood oil extracting, get petroleum ether extraction liquid, concentrating under reduced pressure is weighed for each group after removing sherwood oil.First group of weight in average is 99g, second group of weight in average is 197g, the 3rd group of weight in average is 0.2g, and crude oil total removal rate reaches about 50%, repairing effect highly significant.
The SEQ ID No:3 sequence of enterobacteria ODB0116S rDNA is as follows:
GTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCCGATGGAGGG GGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGTTGTGGTTAATAACCGCAGCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAAGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCCTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCTGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACC。
Claims (7)
1. the invention provides the separation screening qualification that one comprises enterobacteria (Enterobacter sp.), wherein said bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number on December 23rd, 2014: CGMCC No.10233.
2. method according to claim 1, enterobacteria also comprises: this bacterium culture medium matter and this bacterium meta-bolites.
3. method according to claim 1, enterobacteria in crude oil pollution area for the purposes of rapid cleaning.
4. method according to claim 3, wherein said enterobacteria purposes comprises: the treatment process spraying oil contaminated soil with the agent of enterobacteria solid state fermentation.
5. method according to claim 4, wherein said enterobacteria application comprises: microorganism enlarged culturing mechanism; Cadmium pollution rehabilitating soil manages; Product development management and quality control.
6. method according to claim 5, the treatment process of wherein said enterobacteria starter (solid powder) appropriate amounts of sterilized water dilution spraying oil contaminated soil.
7. method according to claim 6, wherein saidly mixes oil contaminated soil zymotechnique with enterobacteria fermented liquid: adopt enterobacteria solid powder to utilize certain sterilized water furnishing inoculation liquid, every milliliter containing microbe population 1 × 10
7more than cfu, is seeded in fermented liquid on oil contaminated soil, ferments.Consumption is every mu of contaminated soil enterobacteria 0.5kg ~ 1.0kg/ mu.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510084360.9A CN104726370A (en) | 2015-02-16 | 2015-02-16 | Enterobacter with crude oil degradation effect and application of enteric bacilli |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510084360.9A CN104726370A (en) | 2015-02-16 | 2015-02-16 | Enterobacter with crude oil degradation effect and application of enteric bacilli |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104726370A true CN104726370A (en) | 2015-06-24 |
Family
ID=53450789
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510084360.9A Pending CN104726370A (en) | 2015-02-16 | 2015-02-16 | Enterobacter with crude oil degradation effect and application of enteric bacilli |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104726370A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106544395A (en) * | 2016-11-03 | 2017-03-29 | 西安建筑科技大学 | A kind of measure method of the soil microorganism to petroleum hydrocarbon degradation ability |
| CN107629976A (en) * | 2016-07-18 | 2018-01-26 | 中国石油天然气集团公司 | A kind of microorganism and its microbial inoculum and application |
| CN109370950A (en) * | 2018-11-29 | 2019-02-22 | 重庆工商大学 | A kind of light petroleum alkane degradation enterobacteria and its preparation method and application |
| CN109694837A (en) * | 2019-03-01 | 2019-04-30 | 中国科学院烟台海岸带研究所 | A kind of electricity production bacterial strain of degradable organic pollutant and its application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104177A (en) * | 2007-08-01 | 2008-01-16 | 中国石油化工股份有限公司 | In-situ biological repairing method for biomass intensified petroleum contaminative soil |
| CN101130754A (en) * | 2007-08-01 | 2008-02-27 | 中国石油化工股份有限公司 | High-density ferment method for petroleum hydrocarbon degradation bacterium |
| CN102659252A (en) * | 2012-06-05 | 2012-09-12 | 山东大学 | Treatment method of wastewater containing petroleum |
| CN103523928A (en) * | 2013-07-16 | 2014-01-22 | 中国人民解放军海军医学研究所 | Bacterial agent for bioremediation of oil-polluted water area and preparation method |
-
2015
- 2015-02-16 CN CN201510084360.9A patent/CN104726370A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104177A (en) * | 2007-08-01 | 2008-01-16 | 中国石油化工股份有限公司 | In-situ biological repairing method for biomass intensified petroleum contaminative soil |
| CN101130754A (en) * | 2007-08-01 | 2008-02-27 | 中国石油化工股份有限公司 | High-density ferment method for petroleum hydrocarbon degradation bacterium |
| CN102659252A (en) * | 2012-06-05 | 2012-09-12 | 山东大学 | Treatment method of wastewater containing petroleum |
| CN103523928A (en) * | 2013-07-16 | 2014-01-22 | 中国人民解放军海军医学研究所 | Bacterial agent for bioremediation of oil-polluted water area and preparation method |
Non-Patent Citations (4)
| Title |
|---|
| TIAN Y 等: "CP015227.1", 《GENBANK》 * |
| ZHAO Y G: "KJ730211.1", 《GENBANK》 * |
| 徐圆圆 等: "肠杆菌X-11对石油烃污染物的降解特性", 《辽宁化工》 * |
| 徐圆圆 等: "阴沟肠杆菌分泌生物表面活性剂的合成研究", 《辽宁石油化工大学学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107629976A (en) * | 2016-07-18 | 2018-01-26 | 中国石油天然气集团公司 | A kind of microorganism and its microbial inoculum and application |
| CN106544395A (en) * | 2016-11-03 | 2017-03-29 | 西安建筑科技大学 | A kind of measure method of the soil microorganism to petroleum hydrocarbon degradation ability |
| CN109370950A (en) * | 2018-11-29 | 2019-02-22 | 重庆工商大学 | A kind of light petroleum alkane degradation enterobacteria and its preparation method and application |
| CN109694837A (en) * | 2019-03-01 | 2019-04-30 | 中国科学院烟台海岸带研究所 | A kind of electricity production bacterial strain of degradable organic pollutant and its application |
| CN109694837B (en) * | 2019-03-01 | 2022-05-06 | 中国科学院烟台海岸带研究所 | Electricity-generating strain for degrading organic pollutants and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101580808B (en) | Rhodococcus ruber and application thereof in degradation of hydrocarbon compounds | |
| CN102250798B (en) | Pseudomonas and microbial agent as well as applications thereof | |
| CN109679858A (en) | The pseudomonas fluorescens strain and its cultural method of one plant of phosphorus decomposing salt tolerant and application | |
| CN102250794B (en) | Pseudoxanthomonasjaponensis and microorganism microbial inoculum as well as applications thereof | |
| CN106167776A (en) | A kind of can bacillus cereus (Bacillus cereus) TH 35 of heavy metal cadmium and application thereof in activating soil | |
| US20090029442A1 (en) | Estrogenic substance degradable microorganism and use thereof | |
| CN102250801B (en) | Microbial agent, and its preparation method and application | |
| WO2023050513A1 (en) | Fungus ljd-6 capable of efficiently degrading benzo[a]pyrene and application thereof | |
| CN104726370A (en) | Enterobacter with crude oil degradation effect and application of enteric bacilli | |
| CN101935631B (en) | Ralstoniasp. and application thereof in bioremediation of petroleum-contaminated saline-alkali soil | |
| CN106978367A (en) | A kind of urea Bacillus strain TB42 and its application in compost maturity is promoted | |
| CN113231462A (en) | Method for stimulating indigenous flora to rapidly degrade cypermethrin in soil | |
| CN113215033A (en) | Sulfonamide antibiotic degrading bacteria and application thereof | |
| Ugochukwu et al. | Lipase activities of microbial isolates from soil contaminated with crude oil after bioremediation | |
| CN102876621B (en) | Mesorhizobium ZY1 and application thereof in soil remediation | |
| CN114854626A (en) | Pseudomonas strain for degrading polycyclic aromatic hydrocarbon pollutants and application thereof | |
| RU2509150C2 (en) | Association of strains of bacteria-oil decomposers, and remediation method of oil-contaminated objects | |
| CN104818234A (en) | Streptomycete with cadmium tolerant characteristic and application thereof | |
| CN102492637A (en) | Atrazine degrading bacterium | |
| CN102250797B (en) | Gordoniaamicalis and microbial agent and applications thereof | |
| CN102250770B (en) | Microbial agent and preparation method and application thereof | |
| CN102250796B (en) | Rhodococcusruber and microbial agent and application of Rhodococcusruber and microbial agent | |
| CN114410508B (en) | Grease degrading bacterium and screening method and application thereof | |
| CN102250795B (en) | Gordoniaalkanivorans, microbial agent and use thereof | |
| CN102864086B (en) | Strain capable of degrading polycyclic aromatic hydrocarbons (PAHs) and application thereof in soil remediation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150624 |
|
| WD01 | Invention patent application deemed withdrawn after publication |