CN102417891A - Bacterium with crude oil degradation function and bioremediation agent suitable for crude oil contaminated soil in low-temperature and saline-alkali areas - Google Patents
Bacterium with crude oil degradation function and bioremediation agent suitable for crude oil contaminated soil in low-temperature and saline-alkali areas Download PDFInfo
- Publication number
- CN102417891A CN102417891A CN2011104099826A CN201110409982A CN102417891A CN 102417891 A CN102417891 A CN 102417891A CN 2011104099826 A CN2011104099826 A CN 2011104099826A CN 201110409982 A CN201110409982 A CN 201110409982A CN 102417891 A CN102417891 A CN 102417891A
- Authority
- CN
- China
- Prior art keywords
- crude oil
- soil
- sjhx
- saline
- acinetobacter haemolyticus
- 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.)
- Granted
Links
- 239000002689 soil Substances 0.000 title claims abstract description 63
- 239000010779 crude oil Substances 0.000 title claims abstract description 38
- 241000894006 Bacteria Species 0.000 title claims abstract description 22
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 21
- 230000015556 catabolic process Effects 0.000 title claims abstract description 14
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 14
- 239000003513 alkali Substances 0.000 title abstract description 7
- 241001148231 Acinetobacter haemolyticus Species 0.000 claims abstract description 29
- 239000003921 oil Substances 0.000 claims abstract description 29
- 230000001580 bacterial effect Effects 0.000 claims abstract description 28
- 235000015097 nutrients Nutrition 0.000 claims abstract description 21
- 239000000243 solution Substances 0.000 claims abstract description 16
- 244000005700 microbiome Species 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims abstract description 4
- 230000000050 nutritive effect Effects 0.000 claims description 25
- 239000002054 inoculum Substances 0.000 claims description 19
- 241000589291 Acinetobacter Species 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 7
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 5
- 238000004321 preservation Methods 0.000 abstract description 4
- 150000003839 salts Chemical class 0.000 abstract description 4
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 abstract description 3
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 abstract description 3
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 abstract description 3
- 244000241235 Citrullus lanatus Species 0.000 abstract description 3
- 244000088415 Raphanus sativus Species 0.000 abstract description 3
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 abstract description 3
- 235000011274 Benincasa cerifera Nutrition 0.000 abstract 1
- 239000012880 LB liquid culture medium Substances 0.000 abstract 1
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 28
- 230000012010 growth Effects 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000012258 culturing Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 239000004215 Carbon black (E152) Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 229930195733 hydrocarbon Natural products 0.000 description 3
- 150000002430 hydrocarbons Chemical class 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical class CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 230000003020 moisturizing effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000005067 remediation Methods 0.000 description 2
- 238000009418 renovation Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000588621 Moraxella Species 0.000 description 1
- 241000589289 Moraxellaceae Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004026 adhesive bonding Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012543 microbiological analysis Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A bacterium with crude oil degradation function and a bioremediation agent applicable to crude oil polluted soil in low-temperature and saline-alkali areas. Belongs to the field of microorganisms. The method solves the problem that no repairing agent suitable for repairing the crude oil polluted soil in low-temperature and saline-alkali areas exists at present. The acinetobacter haemolyticus SJHX-2 is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 5458. The bioremediation agent consists of a bacterial culture solution and a nutrient solution, wherein the bacterial culture solution is a culture solution obtained by inoculating the acinetobacter haemolyticus SJHX-2 into an LB liquid culture medium for culture; the nutrient solution is an aqueous solution containing inorganic salts. The bioremediation agent can reduce the oil content of the soil to be less than 0.3 percent within 2 years after the soil polluted by crude oil is treated, and the radish, Chinese cabbage and watermelon planted in the restored soil grow well.
Description
Technical field
The invention belongs to microorganism field, be specifically related to a kind of acinetobacter haemolyticus and a kind of soil-repairing agent.
Background technology
In field produces, processes such as mud oil, wellsite operation, oil-gas gathering and transportation all can produce ground oil spilling or ground crude oil.These crude oil can corrode soil layer under the effect of self gravitation, surface tension and capillarity, and then change the normal physico-chemical property of soil, influence the eubiosis of poluted region.At present, annual about 700,000 tons of the ground crude oil that produces of China, individual well ground crude oil contaminated area on average can reach 0.5 ~ 2.1m
2, after recycling, still have 70,000 tons and get into soil.At northern oil-producing area, enlarged year by year by the area of crude oil pollution soil.Crude oil infiltrates the harm that produces behind the soil and mainly shows the following aspects: (1) soil particle gluing is agglomerating, influences the permeability of soil, reduces soil quality, hinders the breathing and the absorption of root system of plant, influences the root growth of plant; (2) content of Soil Nitrogen, phosphorus is reduced relatively, nutrient imbalance influences the dietetic alimentation of crop; (3) polycyclic aromatic hydrocarbons in the crude oil (PAHs) have teratogenesis, carcinogenic and cause transgenation " three cause " effect; (4) be difficult in the crude oil by the component of adsorption by soil meeting polluted underground water; (5) also possibly contain a small amount of heavy metal element in the crude oil, also can cause influence to a certain degree plant and microbial growth.Therefore, in order to protect the oil field surrounding enviroment to repair to crude oil pollution soil.
The central and north of China's song-Nen plain and area, Huanghe delta hydrocarbon resources are abundant, but the salinization of You Qu soil is serious, and winter temperature is very low, and the reparation difficulty of crude oil pollution soil is big.Existing treatment process to crude oil pollution soil mainly contains two kinds, and the one, concentrate to stack natural degradation, the 2nd, be transported to that sump oil recycle bin, sludge treatment station carry out heat sinkly falling, modified physics and chemistry such as centrifugal is handled, the oleaginousness of handling the back residue is about 2%.The crude oil pollution soil natural be degraded to state capable of using for up to 5 ~ 10 years, be prone to polluted underground water; And to reclaim facility investment and the running cost height that crude oil is master's physics and chemistry processing.At present, Shang Weiyou is applicable to the renovation agent of low temperature and saline and alkaline geographic crude oil pollution soil remediation.
Summary of the invention
The objective of the invention is to have the bacterium of oil degradation function and be applicable to low temperature and the bioremediation agents of saline and alkaline geographic crude oil pollution soil and provide a kind of in order to solve the present problem that the renovation agent that is applicable to low temperature and saline and alkaline geographic crude oil pollution soil remediation is not arranged as yet.
Bacterium with oil degradation function of the present invention be acinetobacter haemolyticus SJHX-2 (
Acinetobacter haemolyticusSJHX-2), this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.5458.
This acinetobacter haemolyticus SJHX-2 (
Acinetobacter haemolyticusSJHX-2) belong to the Moraxella Cordycepps (
Moraxellaceae) acinetobacter (
Acinetobacter).
This acinetobacter haemolyticus SJHX-2 is from being obtained by separation the normal soil of crude oil pollution and the saline-alkali soil, purifying, screening; Its 30 ~ 37 ℃ of cultivation 18 ~ 24h on the LB plate culture medium promptly form the circular bacterium colony of White-opalescent; Diameter 2 ~ 4mm; Be Gram-negative bacteria, thalli morphology is a thicker quarter butt or spherical, the about 1.2 μ m of diameter; Oxidase negative, catalase, gelatin hydrolysis, hemolytic are all the positive; It is faint that glucose produces acid; Long chain hydrocarbon, ethanol, oxysuccinic acid etc. do not utilize formate, lactic acid salt and propionic salt etc. in capable of using.Through detecting, the oil degradation rate of acinetobacter haemolyticus SJHX-2 of the present invention can reach 9.39%, and the TR that can tolerate is-30 ~ 60 ℃; This bacterial classification best incubation time in the time of 37 ℃ is 9 ~ 10h, the OD of LB nutrient solution
600=1.5 ~ 1.6.
Acinetobacter haemolyticus SJHX-2 of the present invention is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation address is No. 3 institutes of microbiology of the Chinese Academy of Sciences in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date is on November 14th, 2011, and preserving number is CGMCC No.5458.
The bioremediation agents of low temperature and saline and alkaline geographic crude oil pollution soil that is applicable to of the present invention is made up of inoculum and nutritive medium, and described inoculum is inoculated into the nutrient solution of cultivating in the LB liquid nutrient medium for acinetobacter haemolyticus SJHX-2; Described nutritive medium is the aqueous solution that contains inorganic salt, this nutritive medium by mole per-cent contain N40 ~ 45%, P20 ~ 24%, K23 ~ 30%, Na6 ~ 10%, Mg0.03 ~ 0.07%, Ca0.01 ~ 0.03%, nutritive medium pH value is 6.6 ± 0.3.This nutritive medium is formulated to microthermal climate condition and saline and alkaline geographic soil property, can regulate pH in soil and carbon N/P ratio example, promote the metabolic effect of strain growth.Acinetobacter haemolyticus SJHX-2 can be in being added with this nutritive medium of 2g/l glucose normal growth.Inoculum and nutritive medium are united use, and the addition of inoculum is each every kilogram of soil 10 ~ 20ml, and the addition of nutritive medium is each every kilogram of soil 35 ~ 45ml.The TR of oil producing area, north physical environment is between-25 ~ 35 ℃, and saline-alkali soil pH value generally is not higher than 9.Experiment shows, utilizes bioremediation agents of the present invention to handle crude oil pollution soil and in 2 years, can the oleaginousness of soil be reduced to below 0.3%, repairs back soil plantation radish Chinese cabbage watermelon and grows fine.
Description of drawings
Fig. 1 be acinetobacter haemolyticus SJHX-2 of the present invention (
Acinetobacter haemolyticusSJHX-2) aspect graph;
Fig. 2 is the crude oil pollution soil oleaginousness variation diagram of embodiment three.
Embodiment
Embodiment one: the bacterium with oil degradation function of this embodiment be acinetobacter haemolyticus SJHX-2 (
Acinetobacter haemolyticusSJHX-2), this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.5458.
This acinetobacter haemolyticus SJHX-2 is from being obtained by separation the normal soil of crude oil pollution and the saline-alkali soil, purifying, screening.Concrete steps are following: the separation and purification of (one) bacterial classification: take from during 5 ℃ of left and right sides of external temperature in the fall and landed the thin solum that sump oil and oil spilling polluted near Daqing oil field well site or the sump oil recycle bin, at room temperature carry out 3 ~ 4 days activation with the additional zero(ppm) water of watering can with bacterium source mixing with soil, after pulverizing; Get 2 ~ 3g soil and add in the 100ml triangular flask that 15ml LB substratum is housed, under 37 ℃, the condition of 200rpm, shaking table concussion incubated overnight.Get elementary nutrient solution, be seeded to the cultivation of going down to posterity in the bottle of shaking that 100ml LB substratum is housed with pipettor with 1% ~ 2% inoculum size, the nutrient solution that is cultured to exponential growth latter stage is as inoculum, with the cultivation of transferring of 1% ~ 2% inoculum size; As stated above continuous 5 generations of enrichment culture, get last nutrient solution 100 μ l and be applied on the solid LB plate culture medium, be coated with 5 ~ 6 flat boards continuously; Flat board is placed incubator, cultivate down, treat the dull and stereotyped single bacterium colony that forms high dispersing of going up, to the LB slant medium, the inclined-plane placed under 37 ℃, be cultured to the formation lawn in the incubator according to bacterium colony mode of appearance picking list bacterium colony at 37 ℃; The inclined-plane is placed 4 ℃ of refrigerator preservations; The dyed microscopy of resulting slant strains is the laggard row filter of pure bacterium;
(2) primary dcreening operation of bacterial classification: the picking bacterium is inoculated into the LB liquid nutrient medium and cultivated for 1 generation from the inclined-plane; With the nutrient solution striped on the selectivity plate culture medium; Cultivate 2 ~ 3d for 30 ~ 33 ℃, observe on the plate culture medium tangible, well-grown bacterium colony whether occurs; Other picking bacterium from the inclined-plane is inoculated in the LB liquid nutrient medium to be cultivated for 1 generation, and nutrient solution inserts in the LB liquid nutrient medium that dilutes four times and cultivates, with 2 generation nutrient solution (OD
600=1.1 ~ 1.2) insert in the selected liq substratum under 35 ~ 37.5 ℃, the condition of 180 ~ 200r/min and cultivate 144h; The selected liq substratum is extracted with industrial naptha; Detect oleaginousness with spectrophotometer; Calculate the oil degradation rate, oil degradation rate (%)=(initial oil mass-Jia bacterium is handled the back oil-contg)/initial oil mass * 100%; Described selected liq substratum is for adding MgSO in sterile distilled water
4.7H
2O 0.2%, KH
2PO
40.07 ~ 0.12%, K
2HPO
40 ~ 0.05%, NH
4NO
30.25%, outer oil transportation 0.3 ~ 1%; The selectivity plate culture medium is in the selected liq substratum, to add agar 1.8%, pours the cooling of sterile petri dish room temperature behind the boiling sterilization into;
(3) the multiple sieve of bacterial classification: behind primary dcreening operation, obtain 4 bacterial strains that the degrading crude oil ability is strong; After microscopy dyeing is confirmed to be pure bacterium; Carry out the bacterial classification Adaptability Evaluation: the optimum culturing temperature test: bacterial classification is inoculated into respectively in the LB substratum cultivates 9h, measure the OD of bacterial classification under the different culture temperature in 10 ~ 50 ℃ of scopes
600Value, measuring the bacterial classification optimum culturing temperature is 35 ~ 40 ℃; The test of growth curve: bacterial classification is inoculated into respectively in the LB substratum, measures the growth curve of third generation list bacteria culture fluid respectively, probe temperature is respectively 33 ℃, 35 ℃, 37 ℃, shaking speed 180r/min; Heat resistance test: bacterial classification is placed-40 ~ 60 ℃ environment 7d respectively, be seeded to the OD of the different incubation times of test in the LB liquid nutrient medium then
600Value.After above-mentioned test result carried out comprehensive evaluation, from 4 bacterial strains, pick out a thick bacillus and prepare inoculum, through measuring, the oil degradation rate of this bacterial strain is 9.39%, is higher than other three bacterial strains; The optimum culturing temperature of this bacterial strain is 37 ~ 40 ℃; The TR that this bacterial classification can tolerate is-30 ~ 60 ℃; This bacterial strain best incubation time in the time of 37 ℃ is 9 ~ 10h, the OD of LB nutrient solution
600=1.5 ~ 1.6.
The above-mentioned bacterial strain that optimizes is delivered biotechnology institute of University Of Science and Technology Of Tianjin carry out strain identification and dna sequencing.Through identifying, this bacterial strain on the LB plate culture medium 30 ~ 37 ℃ cultivate 18 ~ 24h and promptly form the circular bacterium colony of White-opalescent, diameter 2 ~ 4mm is Gram-negative bacteria, thalli morphology is a thicker quarter butt or spherical, the about 1.2 μ m of diameter; Oxidase negative, catalase, gelatin hydrolysis, hemolytic are all the positive; It is faint that glucose produces acid; Long chain hydrocarbon, ethanol, oxysuccinic acid etc. do not utilize formate, lactic acid salt and propionic salt etc. in capable of using.Bacterial strain aspect graph as shown in Figure 1.Qualification result does, this bacterial strain be acinetobacter haemolyticus (
Acinetobacter haemolyticus), the present invention with this acinetobacter haemolyticus (
Acinetobacter haemolyticus) called after acinetobacter haemolyticus SJHX-2 (
Acinetobacter haemolyticusSJHX-2).Acinetobacter haemolyticus SJHX-2 16S rDNA is shown in SEQ ID NO:1.Acinetobacter haemolyticus SJHX-2 homology is compared, and the BLAST comparison of this sequence in GenBank does
Acinetobacter haemolyticus(acinetobacter haemolyticus), close bacterial classification has Accession No:NR_026207.1, homology degree: 99%.Read Biolog microbiological analysis system detected result data, the result show acinetobacter haemolyticus SJHX-2 with
Acinetobacter haemolyticusThe possibility of (acinetobacter haemolyticus) is 100%, and similarity is 0.837 (in these identification systems, if likelihood is greater than 0.5, and the result is comparatively credible).
Through test, the oil degradation rate of this embodiment acinetobacter haemolyticus SJHX-2 can reach 9.39%, has good oil degradation ability; Its TR that can tolerate is-30 ~ 60 ℃, can adapt to the northern cold cold weather, is suitable for the cold district oil field by the reparation of crude oil pollution soil.
Embodiment two: the bioremediation agents that is applicable to low temperature and saline and alkaline geographic crude oil pollution soil of this embodiment is made up of inoculum and nutritive medium, described inoculum be acinetobacter haemolyticus SJHX-2 (
Acinetobacter haemolyticusSJHX-2) be inoculated into the nutrient solution of cultivating in the LB liquid nutrient medium, training method is each bacterial strain single culture of 1,2 generations, the 3rd pickup go into a collection of shake the bottle or fermentor tank in large scale culturing.Optimum culturing temperature is 35 ~ 37 ℃, and best incubation time is 10h.
Described nutritive medium is the aqueous solution that contains inorganic salt, this nutritive medium by mole per-cent contain N40 ~ 45%, P20 ~ 24%, K23 ~ 30%, Na6 ~ 10%, Mg0.03 ~ 0.07%, Ca0.01 ~ 0.03%, nutritive medium pH value is 6.6 ± 0.3.This nutritive medium is formulated to microthermal climate condition and saline and alkaline geographic soil property, can regulate pH in soil and carbon N/P ratio example, promote the metabolic effect of strain growth.Acinetobacter haemolyticus can be in being added with this nutritive medium of 2g/l glucose normal growth.Inoculum and nutritive medium are united use, and the addition of inoculum is each every kilogram of soil 10 ~ 20ml, and the addition of nutritive medium is each every kilogram of soil 35 ~ 45ml.The TR of oil producing area, north physical environment is between-25 ~ 35 ℃, and saline-alkali soil pH value generally is not higher than 9.Concrete method of use is following:
(1) the outdoor day lowest temperature is in the time of 0 ~ 5 ℃: by the inoculum that 10 ~ 15ml/kg begins to add, a small amount of moisturizing is digged; Behind 3 ~ 4d, add nutritive medium by 40ml/kg, dig, moisturizing to soil humidity is about 50%; The every 30d of inoculum adds once, and the every 30d of nutritive medium adds once;
(2) the outdoor day lowest temperature is in the time of 5 ~ 15 ℃: the every 30d of inoculum adds once, and the per 20 ~ 25d of nutritive medium adds once;
When (3) lowest temperature was greater than 15 ℃ in outdoor day: the every 30d of inoculum added once, and the per 15 ~ 20d of nutritive medium adds once;
When (4) outdoor daily maximum temperature was lower than 0 ℃: stop to add bioremediation agents, spring next year, recovery added;
(5) the daily need maintenance of watering of digging is to keep soil humidity about 40 ~ 60% and enough ventilation properties.
Experiment shows, utilizes bioremediation agents of the present invention to handle crude oil pollution soil and in 2 years, can the oleaginousness of soil be reduced to below 0.3%, repairs back soil plantation radish Chinese cabbage watermelon and grows fine.
Embodiment three: this embodiment the bioremediation agents that is applicable to low temperature and saline and alkaline geographic crude oil pollution soil identical with the bioremediation agents of embodiment two.With this bioremediation agents be applied to Daqing oil field recover the oil near nine factory's well heads by the salt affected soil of crude oil pollution, the soil area is 2 * 2m, thickness of soil 0.25m, oleaginousness 9.81%, pH8.4.Reparation is since in September, 2009, adds nutritive medium 14 times totally 1480 L during the reparation altogether, adds time altogether 517L of inoculum, in June, 2011 this plot soil oleaginousness be 0.29%, oleaginousness changes as shown in Figure 2 in the soil.Can find out that by above-mentioned experimental data the bioremediation agents of this embodiment can effectively decompose the crude oil in the soil, reduce crude content in the soil, thereby repair by the soil of crude oil pollution.
Claims (2)
1. bacterium with oil degradation function, its be acinetobacter haemolyticus SJHX-2 (
Acinetobacter haemolyticusSJHX-2), this bacterial strain is deposited in Chinese common micro-organisms culture presevation administrative center, and preserving number is CGMCC No.5458.
2. bioremediation agents that is applicable to low temperature and saline and alkaline geographic crude oil pollution soil, it is characterized in that: this bioremediation agents is made up of inoculum and nutritive medium, described inoculum be acinetobacter haemolyticus SJHX-2 (
Acinetobacter haemolyticusSJHX-2) be inoculated into the nutrient solution of cultivating in the LB liquid nutrient medium; Described nutritive medium is the aqueous solution that contains inorganic salt, this nutritive medium by mole per-cent contain N40 ~ 45%, P20 ~ 24%, K23 ~ 30%, Na6 ~ 10%, Mg0.03 ~ 0.07%, Ca0.01 ~ 0.03%, nutritive medium pH value is 6.6 ± 0.3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110409982 CN102417891B (en) | 2011-12-09 | 2011-12-09 | Bacterium with crude oil degradation function and bioremediation agent suitable for crude oil contaminated soil in low-temperature and saline-alkali areas |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110409982 CN102417891B (en) | 2011-12-09 | 2011-12-09 | Bacterium with crude oil degradation function and bioremediation agent suitable for crude oil contaminated soil in low-temperature and saline-alkali areas |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102417891A true CN102417891A (en) | 2012-04-18 |
| CN102417891B CN102417891B (en) | 2012-12-05 |
Family
ID=45942535
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110409982 Active CN102417891B (en) | 2011-12-09 | 2011-12-09 | Bacterium with crude oil degradation function and bioremediation agent suitable for crude oil contaminated soil in low-temperature and saline-alkali areas |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102417891B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102718597A (en) * | 2012-06-07 | 2012-10-10 | 中国人民解放军海军医学研究所 | Oleophylic fertilizer for bioremediation of oil polluted environment and method for preparing oleophylic fertilizer |
| CN103865820A (en) * | 2012-12-10 | 2014-06-18 | 中国石油天然气股份有限公司 | Tenghuang yellow unicellular bacterium and preparation and application thereof |
| CN105586294A (en) * | 2016-01-07 | 2016-05-18 | 温州大学 | Acinetobacter and application of acinetobacter in removal of nitrogen and phosphorus from wastewater |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101130684A (en) * | 2006-08-25 | 2008-02-27 | 上海中油企业集团有限公司 | Complex microorganism preparations for oil production |
| CN101597576A (en) * | 2009-06-30 | 2009-12-09 | 山东省科学院生物研究所 | A kind of solid microbial agent to remedy soil contaminated by petroleum and its production and application |
| CN101890426A (en) * | 2010-07-19 | 2010-11-24 | 南开大学 | Method for restoring petroleum-polluted saline alkali soil by ryegrass and high efficiency microbes |
-
2011
- 2011-12-09 CN CN 201110409982 patent/CN102417891B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101130684A (en) * | 2006-08-25 | 2008-02-27 | 上海中油企业集团有限公司 | Complex microorganism preparations for oil production |
| CN101597576A (en) * | 2009-06-30 | 2009-12-09 | 山东省科学院生物研究所 | A kind of solid microbial agent to remedy soil contaminated by petroleum and its production and application |
| CN101890426A (en) * | 2010-07-19 | 2010-11-24 | 南开大学 | Method for restoring petroleum-polluted saline alkali soil by ryegrass and high efficiency microbes |
Non-Patent Citations (1)
| Title |
|---|
| Z. BIHARI ET AL.: "Isolation and Characterization of a Novel n-Alkane-Degrading Strain,Acinetobacter haemolyticus AR-46", 《Z. NATURFORSCH》, vol. 62, no. 34, 30 April 2007 (2007-04-30), pages 285 - 295 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102718597A (en) * | 2012-06-07 | 2012-10-10 | 中国人民解放军海军医学研究所 | Oleophylic fertilizer for bioremediation of oil polluted environment and method for preparing oleophylic fertilizer |
| CN102718597B (en) * | 2012-06-07 | 2013-12-25 | 中国人民解放军海军医学研究所 | Oleophylic fertilizer for bioremediation of oil polluted environment and method for preparing oleophylic fertilizer |
| CN103865820A (en) * | 2012-12-10 | 2014-06-18 | 中国石油天然气股份有限公司 | Tenghuang yellow unicellular bacterium and preparation and application thereof |
| CN103865820B (en) * | 2012-12-10 | 2016-01-20 | 中国石油天然气股份有限公司 | Tenghuang yellow unicellular bacterium and preparation and application thereof |
| CN105586294A (en) * | 2016-01-07 | 2016-05-18 | 温州大学 | Acinetobacter and application of acinetobacter in removal of nitrogen and phosphorus from wastewater |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102417891B (en) | 2012-12-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105567597B (en) | One plant height imitates Atrazine degradation bacterium and its application and screening technique | |
| CN109182178B (en) | Strain with chromium tolerance and Cr (VI) removal capacity and application thereof in-situ remediation of moderately and slightly chromium-polluted soil | |
| CN102277312A (en) | Strain for degrading polycyclic aromatic hydrocarbons at low temperature and application in bioremediation of groundwater in petroleum hydrocarbon polluted places thereof | |
| CN102250789B (en) | Acinetobacter baumannii capable of efficiently degrading imazamox | |
| CN109055261A (en) | Bacillus subtilis capable of degrading crude oil at medium and high temperature and application thereof | |
| CN103436464A (en) | Low temperature-resistant petroleum-degrading bacillus sp. strain, culture method and application thereof | |
| CN109055282A (en) | One Klebsiella pneumoniae new strains and its separation method and application | |
| CN101643707A (en) | Microbial inoculum for degrading polycyclic aromatic hydrocarbons | |
| CN109486726A (en) | The bacterial strain of one plant of degradable petroleum hydrocarbon and its application | |
| CN102220246A (en) | Blueberry mycorrhizal fungi (coprinus micaceus) and separation method and application thereof | |
| CN104673715A (en) | Enteric bacilli with fixing effect on cadmium capable of promoting plant growth and application of enteric bacilli | |
| CN103756928A (en) | Bacterial strain for degradation of p-xylene and culture method and application thereof | |
| CN102417891B (en) | Bacterium with crude oil degradation function and bioremediation agent suitable for crude oil contaminated soil in low-temperature and saline-alkali areas | |
| CN104745506A (en) | Petroleum hydrocarbon degrading bacteria and application thereof | |
| CN117397610B (en) | Method for repairing polluted bottom mud of cage culture | |
| CN101531974A (en) | Arctic bacteria strain for highly efficiently degrading crude oil and application thereof | |
| CN105316269A (en) | Micro-aerobic and hyper-saline environment tolerant pseudomonas aeruginosa and application thereof in degrading petroleum | |
| CN104694439B (en) | A kind of degrading crude oil bacterium and its application | |
| CN110229764B (en) | Bacillus belgii YC666 and application thereof | |
| CN102827786A (en) | Lysinibacillus sp. for degrading S<2-> in petrochemical wastewater | |
| CN104745515A (en) | Acinetobacter sp. for degrading polycyclic aromatic hydrocarbon and application of acinetobacter sp. | |
| KR102665552B1 (en) | Novel Novosphingobium sp. strain, and uses thereof | |
| CN105483026A (en) | Mucor circinelloides H3 and application thereof in pyrene degradation | |
| CN113388545B (en) | Bacillus belgii and application thereof in cleaning low-concentration petroleum-polluted soil | |
| CN105154353A (en) | Bacillus subtilis and application thereof in greenhouse 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 | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |