CN103160448A - Petroleum hydrocarbon degradation bacteria being suitable for electric field condition and application thereof - Google Patents
Petroleum hydrocarbon degradation bacteria being suitable for electric field condition and application thereof Download PDFInfo
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- CN103160448A CN103160448A CN2011104156354A CN201110415635A CN103160448A CN 103160448 A CN103160448 A CN 103160448A CN 2011104156354 A CN2011104156354 A CN 2011104156354A CN 201110415635 A CN201110415635 A CN 201110415635A CN 103160448 A CN103160448 A CN 103160448A
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Abstract
The present invention discloses petroleum hydrocarbon degradation bacteria being suitable for electric field condition and application thereof. Bacillus licheniformis PS5 of the present invention is isolated from petroleum contaminated soil, can degrade petroleum hydrocarbons, and is preserved in the China Type Culture Collection center on July 19, 2011, wherein the preservation registration number is CCTCC No: M 2011261. The bacillus licheniformis PS5 is used for the remediation of contaminated soil. The strain isolated by the invention has high activity under the condition of 0.5-2.0 V/cm field, and can improve removal of alkanes, aromatics, colloids and mixed petroleum hydrocarbon. By applying the electric field, the degradation activity of the bacillus licheniformis PS5 is increased. After 28 days the removal rates for petroleum hydrocarbon single components and mixed petroleum hydrocarbons are increased by 4.19-21.91% and 7.64-23.32%.
Description
Technical field
The present invention relates to Environmental Biotechnology field, specifically a kind of petroleum hydrocarbon degradation bacterium and application thereof that is suitable under current field condition.
Background technology
Along with the mankind increase the demand of oil, approach and the chance of Oil spills increase, and oil has become one of large pollutent in soil.The oil complicated component, contain the organism such as stable hydrocarbon, aromatic hydrocarbon, after these materials enter soil, first be adsorbed on the surface of soil macropore and various organic granular and inorganic particle, then diffuse into gradually in the soil micropore, perhaps distribute to advance in the soil organism, also have small part to exist with pure solid or nonaqueous phase material.When the oil infiltration capacity surpass soil after net capacity, the oily substance of accumulation is extended residual, the spoiled soil structure, to edaphophyte and Ecological Distribution of Soil Microorganisms system even underground water all produce and have a strong impact on, the mankind are worked the mischief.
The oil-polluted soils reparation has been subject to the great attention of Chinese scholars, and biological renovation method is widely used with advantages such as environmental influence are little because its treatment cost is cheap at present.This technology is to utilize petroleum hydrocarbon to grow as carbon source and the energy by means of a large amount of microorganisms, by metabolism, petroleum pollution degradation is become the innoxious substances such as carbonic acid gas and water.Extensively there is the microorganism of energy decomposing petroleum hydrocarbon in occurring in nature, it is reported kind more than 200 to belong to respectively bacterium, yeast and mould etc. different classes of.But the impact of the series of factors such as the composition of the contaminated thing of biological restoration, soil property and external environment usually causes the biological degradation Efficiency Decreasing in actual applications, has limited biodegradable effect.Therefore, how the envrionment conditions of enhancement microbiological, accelerate microorganism and the degradation efficiency of petroleum pollution become the focus of current research.
Electronic-microorganism recovery technique is a kind of eco-friendly organic polluted soil restorative procedure that grew up in recent years, at present the reparation that is applied to the contaminated soils such as petroleum hydrocarbon, PAHs of success.Electronic-microorganism recovery technique is mainly to utilize the various electro kinetic effects that DC electric field produces to strengthen the degradation property of microorganism, but the motion of microorganism, activity and diversity etc. are subjected to the impact of external environment factor very large in actual applications, the quantity of indigenous microorganism or microbe inoculation and active reduction rapidly can't produce a desired effect.Therefore, in order to set up electronic-microorganism recovery technique efficiently, just must screen the high efficient petroleum degrading bacteria that is suitable under current field condition.
Summary of the invention
The present invention seeks to for oil-polluted soils electronic-problem that the microorganism recovery technique exists, a kind of petroleum hydrocarbon degradation bacterium and application thereof that is suitable under current field condition is provided.
For achieving the above object, the technical solution used in the present invention is:
A kind of petroleum hydrocarbon degradation bacterium that is suitable under current field condition, petroleum hydrocarbon degradation bacterium is Bacillus licheniformis (Bacillus licheniformis) PS5, be preserved in Chinese Typical Representative culture collection center on July 19th, 2011, its preservation registration number is CCTCC No:M 2011261.
Be suitable for the application of the petroleum hydrocarbon degradation bacterium under current field condition, described Bacillus licheniformis (Bacillus licheniformis) PS5 is used for the reparation of contaminated soil.Described Bacillus licheniformis (Bacillus licheniformis) PS5 is used for the reparation of petroleum hydrocarbon contaminated soil.Apply unidirectional and switch electric field at contaminated soil, and described Bacillus licheniformis (Bacillus licheniformis) PS5 is applied contaminated soil, carry out the reparation of contaminated soil under the current field condition of 0.5-2.0V/cm exists.
Bacillus licheniformis of the present invention (Bacillus licheniformis) PS5 CCTCC No:M2011261 can grow under different current field conditions.Described current field condition specifically can refer to 0.5V/cm, 1V/cm, the unidirectional electric field of 2V/cm and switch at least a in electric field.
Bacillus licheniformis of the present invention (Bacillus licheniformis) PS5 CCTCC No:M2011261 can be used for the degraded oil hydrocarbon compound.Described petroleum hydrocarbon compound can be petroleum component alkane, aromatic hydrocarbons and colloid and mixes at least a in petroleum hydrocarbon.
Bacillus licheniformis of the present invention (Bacillus licheniformis) PS5 CCTCC No:M2011261 can be used for the environmental pollution reparation.Described environmental pollution can be petroleum hydrocarbon contaminated.Described petroleum hydrocarbon compound specifically can be alkane, aromatic hydrocarbons and colloid and mixes a kind of of petroleum hydrocarbon.Described environment is soil.
The petroleum hydrocarbon degradation bacterium that is suitable under current field condition provided by the present invention screens from the oil-polluted soils of Liaohe Oil Field and obtains.Confirm to belong to bacillus according to its morphological specificity, cultural characteristic, physio-biochemical characteristics and 16SrRNA gene sequencing, with its called after Bacillus licheniformis (Bacillus licheniformis) PS5.Bacillus licheniformis (Bacillus licheniformis) PS5 has been preserved in Chinese Typical Representative culture collection center (abbreviation CCTCC on July 19th, 2011, the address is: wuchang, wuhan Luo Jiashan, Wuhan University), preservation registration number is CCTCC No:M 2011261.
Bacillus licheniformis of the present invention (Bacillus licheniformis) PS5 CCTCC No:M2011261 is Gram-positive, cell is straight shaft-like, and size is 0.6-0.8 * 1.2-2.5 μ m, and single or short chain is arranged, the tool nose circle moves with peritrichous; Give birth in gemma or hold living, oval; Be aerobic bacteria, the pH scope of can growing is 5.7-8.5, and the most suitable growth pH value is 7.2; But growth temperature range is 15-50 ℃, and optimum growth temperature is 30 ℃; Can utilize glucose, catalase is positive.Bacterium colony on bouillon media be flat, the edge is irregular, white, surface irregularity gauffer.
The present invention has advantages of:
Bacillus licheniformis of the present invention (Bacillus licheniformis) PS5 CCTCC No:M2011261 can be under current field condition with petroleum hydrocarbon as sole carbon source and energy growth and breeding, simultaneously with its degraded.The present invention provides new Microbial resources for electronic-microorganism remedying oil-polluted soils, has great value for the improvement of petroleum pollution, has objective economic benefit and good social benefit.
2. the present invention separates and to obtain bacterial strain have higher activity under the current field condition effect, can improve the degradation rate of alkane under current field condition, aromatic hydrocarbons, colloid and mixing petroleum hydrocarbon.After applying 0.5V/cm, 1.0V/cm, 2.0V/cm electric field, the degrading activity of Bacillus licheniformis strengthens, and after 28 days, the clearance of alkane, aromatic hydrocarbons and colloid and mixing petroleum hydrocarbon has increased respectively 4.19-21.91% and 7.64-23.32%.The present invention can be used for the original position reparation of oil-polluted soils, has advantages of quick, convenient, environmental protection.
Description of drawings
Fig. 1 is that Bacillus licheniformis (Bacillus licheniformis) PS5 CCTCC No:M2011261 is at the form photo (100 *) of microscopically.
Fig. 2 is the bacterial number temporal evolution curve of Bacillus licheniformis (Bacillus licheniformis) PS5 CCTCC No:M2011261 under different current field conditions.
Fig. 3 is Bacillus licheniformis (Bacillus licheniformis) PS5 CCTCC No:M2011261 degradation rate to alkane, aromatic hydrocarbons and colloid under different current field conditions.
Fig. 4 is Bacillus licheniformis (Bacillus licheniformis) PS5 CCTCC No:M2011261 degradation rate to the mixing petroleum hydrocarbon under different current field conditions.
Embodiment
Embodiment 1: separation and the screening of Bacillus licheniformis (Bacillus licheniformis) PS5 CCTCC No:M2011261
Sample comes from Liaohe Oil Field periphery petroleum hydrocarbon contaminated soil, takes 5g soil, adds that to draw supernatant liquor to the enrichment medium that contains the 1000mg/L petroleum hydrocarbon after 50ml sterilized water vibration 30min be that inorganic salt add the (composition of substratum: MgSO in the petroleum hydrocarbon substratum
4.7H
2O 2.6g; CaCl
2.H
2O 0.16g; K
2HPO
46.0g; KH
2PO
46.0g; (NH
4)
2SO
46.0g; NaCl 12.0g; Distilled water 1000ml; PH 7.2-7.4,121 ℃ of sterilization 30min), after cultivating 3d, on the solid inorganic salt culture medium of petroleum hydrocarbon, line separates containing, and after selecting single bacterium colony line separation and purification of growing fine three times, bacterial strain is kept on slant medium.
With the pure inoculation that obtains in the liquid minimal medium that contains alkane (550mg/L), aromatic hydrocarbons (350mg/L) and three kinds of petroleum hydrocarbon components of colloid (200mg/L), 28 ℃, 150rpm/min cultivated after 30 days, measured the residue content of alkane, aromatic hydrocarbons and colloid.Separate and to obtain a strain alkane, aromatic hydrocarbons and colloid are all had bacterial strain than high degradation rate, be that Bacillus licheniformis (Bacilluslicheniformis) PS5 is preserved in Chinese Typical Representative culture collection center on July 19th, 2011, its preservation registration number is CCTCC No:M 2011261.
Bacillus licheniformis (Bacillus licheniformis) PS5 Morphology and physiology biochemical characteristic: Gram-positive, cell are straight shaft-like, and size is 0.6-0.8 * 1.2-2.5 μ m, and single or short chain is arranged, and the tool nose circle moves with peritrichous; Give birth in gemma or hold living, oval; Be aerobic bacteria, the pH scope of can growing is 5.7-8.5, and the most suitable growth pH value is 7.2; But growth temperature range is 15-50 ℃, and optimum growth temperature is 30 ℃; Can utilize glucose, catalase is positive.Bacterium colony on bouillon media be flat, the edge is irregular, white, surface irregularity gauffer.
Embodiment 2: the biological activity of Bacillus licheniformis (Bacillus licheniformis) PS5 CCTCC No:M2011261 under current field condition
Above-mentioned Bacillus licheniformis (Bacillus licheniformis) PS5 is inoculated in the beef extract-peptone liquid nutrient medium, then 30 ℃, 160r/min constant-temperature table shaking culture 3 days; The centrifugal collection thalline of 8000r/min under room temperature, then use phosphate buffered saline buffer diluent (storage liquid KH
2PO
434.0g; 1mol/LNaOH solution 175ml; Distilled water 825ml; PH7.2; Diluent: get storage liquid 1.25ml, with distilled water diluting to 1000ml, 121 ℃ of autoclaving 30min) thalline is prepared into bacteria suspension.The bacteria suspension of Bacillus licheniformis (Bacillus licheniformis) PS5 for preparing is joined cleaning meadow burozem (ecological station, Chinese Academy of Sciences Shenyang soil) through sterilization, and in soil, the concentration of Bacillus licheniformis is 6.8 * 10
7The CFU/g dry ground.Apply unidirectional in for examination soil simultaneously and switch electric field, voltage is respectively 0.5V/cm, 1.0V/cm, 2.0V/cm, and interelectrode distance is 24cm, and soil humidity is 25%, experiment operation 28 days.Not apply reactive system that electric field adds separately microorganism in contrast.Bacillus licheniformis (Bacillus licheniformis) PS5 bacterial number alive in soil is measured in every 7d sampling 1 time, and in soil, bacterial number adopts colony counting method.
As can be seen from Figure 2, through processing after a while, Bacillus licheniformis (Bacilluslicheniformis) PS5 quantity all increases to some extent, but apply Bacillus licheniformis in electric field treatment (Bacillus licheniformis) PS5 quantity apparently higher than blank, illustrate that extra electric field is conducive to the growth of Bacillus licheniformis in soil (Bacillus licheniformis) PS5.Respectively process Bacillus licheniformis in soil (Bacilluslicheniformis) PS5 quantity when comparing 7d, 14d, 21d and 28d, find that the strength of electric field size can produce certain influence to the quantity of Soil Microorganism, 2.0V/cm strength of electric field is more conducive to the growth of Bacillus licheniformis (Bacilluslicheniformis) PS5, population size is sequentially: 2.0V/cm＞1.0V/cm＞0.5V/cm; Simultaneously, can find out also that from figure in switching electric field treatment soil, Bacillus licheniformis (Bacilluslicheniformis) PS5 quantity is higher than not switching electric field treatment.From above result, Bacillus licheniformis (Bacillus licheniformis) PS5 can grow under suitable current field condition, the current field condition optimum of 2.0V/cm, and the effect of switching electric field treatment will be higher than not switching the electric field treatment effect.
Embodiment 3: Bacillus licheniformis (Bacillus licheniformis) PS5 CCTCC No:M2011261 under different current field conditions to the Degradation of alkane, aromatic hydrocarbons and colloid
Bacillus licheniformis (Bacillus licheniformis) PS5 is inoculated in the beef extract-peptone liquid nutrient medium, then 30 ℃, 160r/min constant-temperature table shaking culture 3 days; The centrifugal collection thalline of 8000r/min under room temperature, then with phosphate buffered saline buffer, thalline is prepared into bacteria suspension.The bacteria suspension for preparing is inoculated in respectively in the contaminated soil that contains alkane (550mg/kg), aromatic hydrocarbons (350mg/kg), colloid (200mg/kg), and in soil, the concentration of Bacillus licheniformis is 6.8 * 10
7The CFU/g dry ground.Apply unidirectional in for examination soil simultaneously and switch electric field, voltage is respectively 0.5V/cm, 1.0V/cm, 2.0V/cm, and interelectrode distance is 24cm, and soil humidity is 25%, experiment operation 28 days.Not apply electric field but the reactive system that adds microorganism in contrast.Every 7d sampling 1 time, sample are compound sample middle, two ends soil, adopt the concentration of each pollutent in gravimetric determination soil, calculate each removal of pollutants rate.
As can be seen from Figure 3, each processes alkane, aromatic hydrocarbons and colloid all in various degree removal, and wherein the clearance of alkane will be higher than aromatic hydrocarbons and colloid.Relatively extra electric field is processed and independent microbiological treatment, the clearance that applies alkane after electric field, aromatic hydrocarbons and colloid all increases to some extent, the clearance increase of alkane is more obvious, the maximum material removal rate of alkane, aromatic hydrocarbons and colloid has increased respectively 21.91%, 12.51% and 4.19%, the biological activity of explanation Bacillus licheniformis (Bacillus licheniformis) PS5 under electric field action strengthens, and electric field has promoted its degraded to pollutent.Simultaneously, can find out also that from figure the mode of action of strength of electric field and electric field affects the activity of Bacillus licheniformis (Bacilluslicheniformis) PS5, each size order of processing clearance is: 2.0V/cm＞1.0V/cm＞0.5V/cm, in switching electric field treatment soil, each removal of pollutants rate will be higher than not switching electric field treatment.
Embodiment 4: Bacillus licheniformis (Bacillus licheniformis) PS5 CCTCC No:M2011261 under different current field conditions to mixing the Degradation of petroleum hydrocarbon
The preparation of Bacillus licheniformis (Bacillus licheniformis) PS5 bacteria suspension is with reference to embodiment 3.In oil-polluted soils, the total concn of mixing petroleum hydrocarbon in soil is 1200.00mg/kg with the bacterial suspension inoculation for preparing, and in soil, the concentration of Bacillus licheniformis is 6.8 * 10
7The CFU/g dry ground.Apply unidirectional in for examination soil simultaneously and switch electric field, voltage is respectively 0.5V/cm, 1.0V/cm, 2.0V/cm, and interelectrode distance is 24cm, and soil humidity is 25%, experiment operation 28 days.Not apply electric field but the reactive system that adds microorganism in contrast.Every 7d sampling 1 time, sample are compound sample middle, two ends soil, adopt the concentration of gravimetric determination soil Petroleum Hydrocarbon, calculate the clearance of soil Petroleum Hydrocarbon.
As can be seen from Figure 4, during processing, mixes extra electric field the clearance of petroleum hydrocarbon apparently higher than independent microbiological treatment, illustrate that extra electric field has improved the biological activity of Bacillus licheniformis (Bacilluslicheniformis) PS5, promoted its Degradation to petroleum hydrocarbon, its clearance has improved respectively 7.64%, 10.02%, 9.20%, 13.73%, 17.00% and 23.32%.Simultaneously, can find out also that from figure strength of electric field and the mode of action have also affected the activity of Bacillus licheniformis (Bacilluslicheniformis) PS5, each size order of processing to removal rate of petroleum hydrocarbons is: 2.0V/cm＞1.0V/cm＞0.5V/cm, the clearance that switches electric field treatment soil Petroleum Hydrocarbon will be higher than not switching electric field treatment.
Claims (4)
1. petroleum hydrocarbon degradation bacterium that is suitable under current field condition, it is characterized in that: petroleum hydrocarbon degradation bacterium is Bacillus licheniformis (Bacillus licheniformis) PS5, be preserved in Chinese Typical Representative culture collection center on July 19th, 2011, its preservation registration number is CCTCC No:M 2011261.
2. application that is suitable for the petroleum hydrocarbon degradation bacterium under current field condition claimed in claim 1 is characterized in that: described Bacillus licheniformis (Bacillus licheniformis) PS5 is used for the reparation of contaminated soil.
3. by the application that is suitable for the petroleum hydrocarbon degradation bacterium under current field condition claimed in claim 2, it is characterized in that: described Bacillus licheniformis (Bacillus licheniformis) PS5 is used for the reparation of petroleum hydrocarbon contaminated soil.
4. by the described application that is suitable for the petroleum hydrocarbon degradation bacterium under current field condition of claim 2 or 3, it is characterized in that: apply unidirectional and switch electric field at contaminated soil, and described Bacillus licheniformis (Bacillus licheniformis) PS5 is applied contaminated soil, under existing, the current field condition of 0.5-2.0V/cm carries out the reparation of contaminated soil.
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| CN104588405A (en) * | 2015-01-19 | 2015-05-06 | 中国科学院沈阳应用生态研究所 | Microorganism-electric power two-segment repairing method for petroleum contaminated soils |
| CN104762210A (en) * | 2014-12-23 | 2015-07-08 | 中国科学院沈阳应用生态研究所 | Alkane degrading enzyme preparation, and preparation method and application thereof |
| CN104907324A (en) * | 2015-06-26 | 2015-09-16 | 西安工程大学 | Oil polluted soil degrading method based on combination of zoogloearesiniphila, bacillus and electric field |
| CN104923559A (en) * | 2015-06-26 | 2015-09-23 | 西安工程大学 | Oily soil degradation method based on combination of pseudomonas, zoogloea and electric field |
| CN105524859A (en) * | 2015-12-14 | 2016-04-27 | 中国海洋石油公司 | Water-base drilling cutting petroleum hydrocarbon degrading bacterium |
| CN105670956A (en) * | 2015-10-29 | 2016-06-15 | 常州大学 | Medium thermophilic bacillus licheniformis strain and application thereof in treatment on high-temperature oil-bearing wastewater |
| CN107619826A (en) * | 2017-11-01 | 2018-01-23 | 天津科技大学 | A kind of biodegradable screening technique for using microorganism |
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| CN107893041A (en) * | 2017-12-04 | 2018-04-10 | 中国科学院沈阳应用生态研究所 | A kind of petroleum hydrocarbon degradation mix bacterium agent for being suitable for medium temperature aerobe heap repair system and application |
| CN108004163A (en) * | 2017-12-04 | 2018-05-08 | 中国科学院沈阳应用生态研究所 | Mix bacterium agent and its preparation and the application of petroleum hydrocarbon contaminated soil are repaired suitable for depth in medium temperature aerobe shut-down system |
| CN107999530A (en) * | 2017-11-30 | 2018-05-08 | 南通国盛环境修复有限责任公司 | A kind of method that electrodynamics enhancement microbiological removes soil petrochina hydrocarbon |
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| CN110193512A (en) * | 2019-05-21 | 2019-09-03 | 中电建路桥集团有限公司 | The device and method that two-dimentional AC field enhancement microbiological repairs petroleum-type organic material contaminated soil |
| CN115069756A (en) * | 2022-06-07 | 2022-09-20 | 常州大学 | Method and device for degrading petroleum hydrocarbon polluted soil |
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| CN104907324A (en) * | 2015-06-26 | 2015-09-16 | 西安工程大学 | Oil polluted soil degrading method based on combination of zoogloearesiniphila, bacillus and electric field |
| CN104923559A (en) * | 2015-06-26 | 2015-09-23 | 西安工程大学 | Oily soil degradation method based on combination of pseudomonas, zoogloea and electric field |
| CN104907324B (en) * | 2015-06-26 | 2018-03-23 | 西安工程大学 | Based on dynamic glue bacterium, bacillus and the united petroleum-contaminated soil biodegrading process of electric field |
| CN104923559B (en) * | 2015-06-26 | 2018-03-23 | 西安工程大学 | Based on pseudomonad, dynamic glue bacterium and the united petroleum-contaminated soil biodegrading process of electric field |
| CN105670956A (en) * | 2015-10-29 | 2016-06-15 | 常州大学 | Medium thermophilic bacillus licheniformis strain and application thereof in treatment on high-temperature oil-bearing wastewater |
| CN105524859A (en) * | 2015-12-14 | 2016-04-27 | 中国海洋石油公司 | Water-base drilling cutting petroleum hydrocarbon degrading bacterium |
| CN107619826B (en) * | 2017-11-01 | 2023-10-10 | 天津科技大学 | Screening method of microorganisms for biodegradation |
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| CN107828684A (en) * | 2017-11-13 | 2018-03-23 | 中国科学院沈阳应用生态研究所 | Cooperateed with suitable for electronic microorganism and repair mix bacterium agent and its preparation and application petroleum hydrocarbon contaminated soil |
| CN107828684B (en) * | 2017-11-13 | 2020-01-24 | 中国科学院沈阳应用生态研究所 | Mixed microbial inoculum suitable for electric-microbial synergistic remediation of petroleum hydrocarbon contaminated soil and preparation and application thereof |
| CN107999530A (en) * | 2017-11-30 | 2018-05-08 | 南通国盛环境修复有限责任公司 | A kind of method that electrodynamics enhancement microbiological removes soil petrochina hydrocarbon |
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| CN108160702A (en) * | 2017-12-18 | 2018-06-15 | 中国科学院沈阳应用生态研究所 | A kind of method for repairing high concentration petroleum pollution salt affected soil |
| CN108160702B (en) * | 2017-12-18 | 2020-02-21 | 中国科学院沈阳应用生态研究所 | Method for restoring saline-alkali soil polluted by high-concentration petroleum |
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| CN115069756A (en) * | 2022-06-07 | 2022-09-20 | 常州大学 | Method and device for degrading petroleum hydrocarbon polluted soil |
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