CN1519312A - Erythro micrococcus Em and usage for generating biologic emulsifier as well as degrading polycyclic aromatic hydrocarbon - Google Patents
Erythro micrococcus Em and usage for generating biologic emulsifier as well as degrading polycyclic aromatic hydrocarbon Download PDFInfo
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Abstract
A Rhodococcus ruber Em (CGMCC No.0868) features that its cells, cell suspension, or immobilized cells can degradate the paraffin and polycyclic arylhydrocarbon because the paraffin and polycyclic arylhydrocarbon are used as its only carbon source and energy source for growing to generate the bioemulsifier of ester. Said emulsifier can obviously decrease the surface tension of aqueous solution, has strong emulsifying power to ester substance, and can increase the solubility of paraffin and polycyclic arylhydrocarbon in water, so it can be used for treating the oil-contained sewage and repairing the petroleum polluted soil.
Description
Technical field
The invention belongs to microorganism biological technology and Environmental Biotechnology field, specifically, it relates to a Rhodococcus ruber (Rhodococcus ruber) Em CGMCC No.0868 bacterial strain, polycyclic aromatic hydrocarbons such as alkane such as the grown cell of this bacterial strain, cell suspending liquid, the equal degradable n-Hexadecane of immobilized cell and anthracene, phenanthrene, pyrene; This bacterial strain can be sole carbon source and energy growth with alkane or polycyclic aromatic hydrocarbons, produces the lipid biological emulsifier simultaneously; This emulsifying agent can obviously reduce the aqueous solution surface tension, have very strong oil substances emulsifying capacity, improve solubleness in water of alkane and polycyclic aromatic hydrocarbons, obviously promote the degraded of active bacterial strain alkane and polycyclic aromatic hydrocarbons.The biological emulsifier of this bacterial strain and generation thereof is applicable to the biological treatment of oily(waste)water and the biological restoration of oil-polluted soils (biological regulation).
Technical background
Oil is one of important energy, all occupies important status in countries in the world, and the whole world consumes 2.76 * 10 every day approximately according to statistics
9The crude oil of gallon.Because the exploitation of oil, store, transportation, processing, the leakage of oil of processes such as petrochemical industry, reason such as oil spilling and sudden accident, annual nearly more than 1,800 ten thousand tons oil or the petroleum hydrocarbon in the whole world enters environment, cause serious pollution, one of global public hazards have been become, oil field particularly, soil around pipe laying and the petrochemical factory, the petroleum pollution of environment such as water body is particularly serious, petroleum hydrocarbon has become the second largest source of pollution of pollution of the sea, pollute as ocean and shoreline that Alaska crude oil leakages in 1989 cause, the crude oil pollution that the Gulf War causes, and not long ago the Crude oil from CNOOC that causes of the oil tanker accident in the Spain marine site polluted, caused the serious environmental ecological problem, cause people's fear, the extensive concern of countries in the world and attention petroleum pollution problem.The harm of petroleum pollution mainly shows the destruction to the structure and the function of soil ecosystem, has had a strong impact on the ventilation property, water permeability of soil etc., causes that soil fertility descends, army provisions are not long, and the good farmland is lain waste; Oil enters lithic drainage, directly the polluted underground water source; Form oil film in water surface, oxygen in water is sharply descended, cause hydrobiological mass mortality etc.; The accident of marine petroleum exploitation and transportation often has generation, causes the pollution in wide area of ocean; Contain a large amount of toxic ingredients in the petroleum pollution, have the teratogenesis carcinogenesis, (as food chain) enters human body through various channels, directly the healthy of people constituted a threat to.Therefore, the petroleum pollution of removing in the environment has become the pressing problem that present countries in the world are paid close attention to.
China is oil production, one of petrochemical industry big country, Oil spills and oil, the petrochemical industry sewage pollution, become one of primary pollution source of the at present serious environmental pollution of China, account for 5% of annual thousands of accident environmental pollutions, there is more than 60 ten thousand tons crude oil every year in the statistics whole nation because of recovering the oil, transportation, former thereby leakage such as accident, in addition, rate of loss in the China's oil refining process is 1.54%, 200,000,000 tons of crude oil of the annual processing in the whole nation now, also there are every year 3000000 tons of petroleum hydrocarbons to enter environment, therefore amounting to the whole nation has more than 360 ten thousand tons of petroleum hydrocarbons to enter environment every year, this shows that petroleum pollution is big to China's environment amount of being, wide pollution (China Economic Times, in May, 1998), severe contamination and threatening the oil field, environmental quality and river around the petrochemical plant, the water quality in lake, as but the North China Oilfield that is located in the vast crop ped location has more than 4000 mouthful of oil well, Shengli Oil Field has more than 11000 mouthful of oil well, every mouthful of oil well to less contamination 5 mu farmland, total has at least the farmland more than 80000 mu to become the long wasteland of army provisions because of petroleum pollution, the whole nation in advance in respect of the arable land of ten thousand mu of 100-200 owing to petroleum pollution is fallen into disuse; In addition, the Yellow River water system annual oil-containing reaches as high as 4.82mg/L, and Liaohe River water system annual reaches as high as 7.68mg/L, surpassed significantly three grades of surface water standards of country (＜1mg/L), directly be discharged into the offshore waters of China; That have even pollute groundwater resource (having reached 100mg/L) as the phreatic oil content in Zibo area, influenced urbanite water consumption and field irrigation.Recent years is along with the exploitation of China's offshore oilfield, petroleum pollution in ocean is also serious day by day, according to National Bureau of Oceanography " Chinese ocean environment annual report in 1998 ", petroleum pollution has become the primary pollution source of China's ocean environment, a small-sized oil spill accident surplus large-scale together oil spill accident and 20 has just taken place in China's immediate offshore area in 1998, coastal waters over half, comprise the Bohai Sea and the Huanghai Sea, and the petroleum pollution in nearly Haihe River, national fishing shelf had a strong impact on ecotope and fishing resources, arrived must improvement stage.
Methods such as the mechanical frizing of the present still main employing of China, chemisorption are come the petroleum pollution in the processing environment, these method cost height, investment are greatly, as one square kilometre Polluted area is handled with chemiadsorption, the chemosorbent that needs the supreme kiloton of hundreds of ton, need the investment of millions of units, and waste time and energy, also have secondary pollution, the processing of the sorbent material after the absorption and regeneration is a big problem especially, so these methods are not the best practice that solves petroleum pollution.Successful experience according to the pollution of foreign biomolecule reparation (Bioremediation) technology governance environment PetroChina Company Limited., and advantage such as the cost that bioremediation technology itself is had is low, the same Polluted area of handling one square kilometre, use bioremediation technology only need add several tons to tens tons microbiobacterial agent and nutritional additive, only need more than the investment hundreds of thousands of to hundred ten thousand yuan, can save a large amount of investments, and this method also have time saving and energy saving, there are not advantages such as secondary pollution, can think that biological restoration is the comparatively ideal method that solves the present petroleum pollution of China, bioremediation technology will be made significant contribution in the improvement that China's oil is polluted, and have broad application prospects.Therefore present technique will be that China administers the optimization technique that environment PetroChina Company Limited. pollutes from now on.
Biological restoration (Bioremediation) or claim that biological regulation is to have occurred and the removing of development and the biotechnology of curbing environmental pollution since 20th century the mid-80, biological restoration is to utilize the metabolism of microorganism in the treatment system to reduce the concentration of polluting on-the-spot objectionable impurities or make it innoxious fully, it is the important method of curbing environmental pollution, principal feature is to administer large-area environmental pollution, make it to be recovered fully, reach the situation before polluting, the main process object of bioremediation technology is petroleum pollution and the pesticidal contamination in the environment at present.The most successful example is that the offshore petroleum of removing Alaska Exxon Valdez is polluted.The distinguishing feature that bioremediation technology is different from other technologies just is that it is the technology for eliminating of pollutent but not separated from contaminants technology, therefore it has advantages such as non-secondary pollution, in addition, bioremediation technology also has the low advantage of cost, generally is 1/3～1/5 of other technologies.Biological restoration is to adopt such as improving ventilation efficiency, extra-nutrition (for petroleum pollution, mainly be to replenish N, P), add excellent species, improve ways such as envrionment conditions and improve metabolism and the degrading activity level of microorganism, with the degradation speed of promotion, thereby reach the purpose of administering contaminate environment to pollutent.The various countries, Europe pay much attention to bioremediation technology as Germany, Denmark, Holland, all Europe is engaged in this Study on Technology mechanism and commercial company nearly more or less a hundred, they studies have shown that, the bioremediation technology that utilizes the microbiological degradation hazardous and noxious substances is a kind of valuable method of administering the pollution in wide area zone.American National Environmental Protection Agency (USEPA) actively pushes forward the research and the application of bioremediation technology.Positive attitude is also held to bioremediation technology in some states of the U.S., as New Jersey, state of Wisconsin regulation this technology is classified as and is purified the prefered method that is subjected to storage tank leakage pollution soil remediation.USDOE (EPA) has been formulated soil and the phreatic biological restoration plan nineties, and has organized one " biological restoration Action Committee " by federal government, science and business circles' composition of personnel (Bioremediation Action Committee) to be responsible for the research and the concrete application implementation of bioremediation technology.The most basic basis in the biological restoration of biological treatment that contains petroleum waste water and oil-polluted soils is the degraded of microorganism to petroleum hydrocarbon, and therefore being applied in of highly active degradation bacteria and raising degradation bacteria and oil droplet active area----biological emulsifier administered very key of petroleum pollution.At different all over the world petroleum pollution situations, people have adopted different biological restoration measures, handle soil petroleum pollution running after 2 months as application efficient degradation microorganisms such as Gruize with the method that ventilation combines, and the pollutent in the soil obviously reduces; The petroleum pollution that U.S. Chakrabarty adopts the genetically engineered bacteria processing Gulf War to cause obtains unusual effect; (Chianelli such as Chianelli RR, R.R.et al.1991) uses the microorganism add nutrition and decomposing petroleum hydrocarbon the Exxon Valdez bay petroleum of Alaska in 1989 is leaked the processing that the pollution that causes is carried out, obtain very obvious effects, make nearly hundred kilometers seashores environmental quality be improved significantly, the most successful example that is biological restoration in removing petroleum pollution.The bioremediation technology of relevant petroleum pollution, the achievement patent applied for that has, some company has developed various oil removing microbial inoculums and hydrophobicity nutritional additive, but does not form the industrialization of petroleum pollution bioremediation technology as yet.China also once introduced external microbial inoculum and nutritional additive, but effect is also bad, major cause is that composition difference, the envrionment conditions difference of petroleum pollution causes, therefore to solve the petroleum pollution of China, must be conceived to the petroleum pollution situation of China, walk the road of self-reliance, self-development, open up our petroleum pollution evaluation method, bioremediation technology and technology, microbial strains resource.
Summary of the invention
The objective of the invention is to the serious harm that environment caused at petroleum pollution, provide a kind of Rhodococcus ruber Em and this bacterium to be used to administer the bioremediation technology of petroleum pollution, this bacterium is the Rhodococcus ruber with the multiple petroleum alkane of degraded and polycyclic aromatic hydrocarbons and generation biological emulsifier, can use in administering the environment petroleum pollution.Specifically, relate to a Rhodococcus ruber (Rhodococcusruber) Em CGMCC No.0868 bacterial strain, polycyclic aromatic hydrocarbons such as alkane such as the grown cell of this bacterial strain, cell suspending liquid, the equal degradable n-Hexadecane of immobilized cell and anthracene, phenanthrene, pyrene; This bacterial strain can be sole carbon source and energy growth with alkane or polycyclic aromatic hydrocarbons, produces the lipid biological emulsifier simultaneously; This emulsifying agent can obviously reduce the aqueous solution surface tension, have very strong oil substances emulsifying capacity, improve solubleness in water of alkane and polycyclic aromatic hydrocarbons, obviously promote the degraded of active bacterial strain alkane and polycyclic aromatic hydrocarbons.The biological emulsifier of this bacterial strain and generation thereof is applicable to the biological treatment of oily(waste)water and the biological restoration of oil-polluted soils (biological regulation).
Rhodococcus ruber provided by the invention (Rhodococcus rubber) Em bacterial strain is preserved on December 20th, 2002 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number are CGMCC No.0868; It is characterized in that:
The colony characteristics of Rhodococcus ruber (Rhodococcus ruber) Em CGMCC No.0868: the bacterium colony size of cultivating 2 days on the LB flat board is diameter 3～4mm, and bacterium colony is rounded, and surface drying is coarse, and neat in edge is protruded, and is orange.
The cell morphological characteristic of Rhodococcus ruber (Rhodococcus ruber) Em CGMCC No.0868: be suitable for normal temperature cultivation in pH5～9 scopes, its cell is shaft-like, but the cell of different cell ages has club to change, children's cell in age is shaft-like, aged cell (cultivating after 4 days) becomes sphere, Gram-positive, the cell size is diameter 0.8 μ m, random arrangement.
The physiological and biochemical property of Rhodococcus ruber (Rhodococcus ruber) Em CGMCC No.0868: acid-fast stain feminine gender, do not produce gemma or spore, the catalase positive, reduction nitrate, can utilize glucose, sucrose, maltose, D-fructose, lactose, D-semi-lactosi, rhamnosyl, trehalose, N.F,USP MANNITOL, sorbyl alcohol, D-wood sugar, L-arabinose, cellobiose, raffinose, glycerol, and all fermentation and acid can also utilize seminose, L-L-glutamic acid, L-Xie Ansuan, L-proline(Pro); Can not utilize inositol, succsinic acid, glycine, lactic acid, L-arginine.Degraded alkane and polycyclic aromatic hydrocarbons produce biological emulsifier.
The 16S rRNA gene sequence characteristic of Rhodococcus ruber (Rhodococcus ruber) Em CGMCC No.0868: CGMCC No.0868 is inoculated in the LB substratum; 30 ℃ of shaking tables were cultivated (200rpm) 18 hours; centrifugal collecting cell; again suspend; add the N,O-Diacetylmuramidase broken wall; extract DNA by phenol-chloroform method; and this carries out pcr amplification to universal primer to its 16SrDNA to adopt forward primer 27f (5 '-GAGAGTTTGATCCTGGCTCAG-3 ') and reverse primer 1541r (5 '-AAGGAGGTGATCC AGCC-3 '), amplified production is served sea base Kanggong department check order.The PCR condition is: 95 ℃, and 10min; 94 ℃, 1min, 48 ℃, 1min, 72 ℃, 1min; 30 circulations, 72 ℃, 5min, 4 ℃ of preservations.16S rDNA sequence length is 1398bp, and the sequence accession number in GenBank is AF529079, with the similarity of 16S rDNA (accession number the is X80625) sequence of bacterial strain Rhodococcus rubber DSM43338T be 99%.The sequence of its 16S rDNA is shown in Fig. 1 and sequence table 1.
Content with reference to " Bergey ' s Mannual of Systematic Bacteriology " Vol.VIII, according to its morphological specificity and physiological and biochemical property, and according to the Search Results of its 16S rRNA gene order in GenBank, identify that the Em bacterium is a new bacterial strain, belongs to Rhodococcus ruber (Rhodococcus ruber).
Rhodococcus ruber (Rhodococcus ruber) Em CGMCC No.0868 can be at nutritional medium, as: cultivate in common beef broth, LB, the nutrient agar medium, also can be with petroleum hydrocarbon, as: alkane, aromatic hydrocarbons and polycyclic aromatic hydrocarbons are to cultivate in the basic medium of sole carbon source.As tame then better effects if in the basic medium that with the polycyclic aromatic hydrocarbons is substrate, bacterial strain carries out aerobic growth under the proper temperature between 25～35 ℃ before using.
This bacterium has higher capability of decomposing petroleum hydrocarbon, and degraded substrate scope is wide, the alkane of degradable C10～C24, aromatic hydrocarbons and polycyclic aromatic hydrocarbons (benzene, naphthalene, anthracene, phenanthrene, pyrene).The result who carries out degradation experiment with Em bacteria growing cell shows, the OD during benzene of degraded 0.3 and 0.4g/L
460Can reach 0.15 and 0.13, OD when the naphthalene of degraded 0.2 and 0.3g/L
460Can reach 0.16 and 0.17.In initial pH value is 7.0, and cell extract concentration is 0.025g/L, and the concentration of anthracene is 0.1g/L, cultivates 72 hours with 200 rev/mins of rotating speed shaking tables under 30 ℃, and degradation rate can reach 39%.
CGMCC No.0868 bacterium provided by the invention can be that fermentation culture is produced biological emulsifier in the basic medium of sole carbon source with the petroleum hydrocarbon, and when wherein preferably being carbon source with the n-Hexadecane, substratum is formed (%): n-hexadecane, 8g; Yeast extract, 1g; K
2HPO
43H
2O, 1g; KH
2PO
4, 1g; MgSO
47H
2O, 0.5g; NH
4NO
3, 1g; CaCl
2, 0.02g; FeCl
3Trace; PH7.0,121 ℃ of sterilization 30min, the effect of cultivating the biological emulsifier that produces is best.The emulsifying agent that produces can make the surface tension of nutrient solution reduce to 30mN/m in back 24 hours in cultivation, and emulsifier concentration can reach 68 times of maximum values in the time of 5 days, after having diluted 68 times, is to have very high surfactivity promptly, can make the surface tension of water drop to 56mN/m.The emulsifying capacity of thick emulsifying agent can reach 100% in 24 hours in cultivation, and stable emulsifying (Fig. 2).Adopt the surface tension of the Auto-tensiometerZL-2 type surface tension apparatus mensuration nutrient solution of Shandong Zibo Ke Sen company, dilution nutrient solution concentration of emulsifying agent in nutrient solution reaches below the CMC (critical micelle concentration), the surface tension rapid drawdown of nutrient solution is represented emulsifier concentration (Fig. 3) in the nutrient solution with this extension rate.
The biological emulsifier that CGMCC No.0868 bacterium is produced is done the a-naphthol test and is judged carbohydrate, with Bradford colorimetric method for determining albumen, shows that tentatively this emulsifying agent does not contain protein, do not contain polysaccharide, and its chemical property is a lipid material.
The biological emulsifier that CGMCC No.0868 bacterium is produced has very strong surfactivity effect, can obviously reduce the surface tension of water, the surface tension of water is 72.1mN/m when not adding emulsifying agent, and during the overstriking emulsifying agent, as long as the extension rate of thick emulsifying agent is below 41 times, the surface tension of water can drop to about 30mN/m, and the thick emulsifying agent that has diluted 51 times still can make the surface tension of water drop to 50.3mN/m.This result shows the performance of the biological emulsifier that CGMCC No.0868 bacterium is produced and the content of emulsifying agent, and the height of its level is not seen the report of correlation technique both domestic and external so far.
CGMCC No.0868 bacterium produces the thing emulsifying agent and has tangible emulsifying capacity.At present in the world to the mensuration of emulsifying capacity, usually with kerosene the emulsification object, emulsifying agent mixes by equal volume with kerosene, fierce concussion 30min, leave standstill 2hr, measure the amount that residue does not have emulsified kerosene then, with per-cent (%) the expression emulsifying capacity of emulsive amount with added kerosene total amount.The result shows, at emulsifying agent: under the condition of kerosene=1: 1, the Em bacterial strain can reach 100% emulsification through 1 day cultivation; At emulsifying agent: under the condition of kerosene=1: 10, cultivate 1 day can emulsification 13% kerosene, cultivate and reached the highest emulsifying capacity in 2 days basically.
CGMCC No.0868 bacterium produces the thing emulsifying agent to have and significantly makes polycyclic aromatic hydrocarbons, lipid material solubilising in water, as shown in Figure 4, the emulsifying agent fermented liquid that adds 1/30 (V/V), the solubleness of anthracene in water from 0.06mg/L be increased to 0.83mg/L, luxuriant and rich with fragrance from 0.84mg/L be increased to 9.97mg/L, pyrene is increased to 2.32mg/L from 0.2mg/L, all improved more than 10 times.CGMCC No.0868 bacterium produces the effect that the thing emulsifying agent can obviously promote the active bacteria decomposing petroleum hydrocarbon.In initial pH value is 7.0, yeast extract concentration is 25mg/L, the concentration of anthracene is 100mg/L, cultivate with 200 rev/mins of rotating speed shaking tables under 30 ℃, the residual content of different time sampling and measuring anthracene, the result as shown in Figure 5, the emulsifying agent that adds 1/30 (V/V) can make the degradation rate of anthracene increase about 30 percentage points.Degradation amount when cultivating 3 days is brought up to 46mg/L from 19mg/L, has increased about 1.5 times.
Description of drawings
The 16S rDNA sequence of Fig. 1 Rhodococcus ruber Em CGMCC No.0868 bacterium
Fig. 2 Rhodococcus ruber Em CGMCC No.0868 bacterial strain is carbon with the n-hexadecane
The surface tension of the nutrient solution in source is change curve in time
Fig. 3 Rhodococcus ruber Em CGMCC No.0868 bacterial strain is the emulsifier concentration of nutrient solution of carbon source and emulsifying capacity change curve in time with the n-hexadecane
Fig. 4 Rhodococcus ruber Em CGMCC No.0868 emulsifying agent that bacterial strain produces is to the solublization of the luxuriant and rich with fragrance pyrene of anthracene
Fig. 5 Rhodococcus ruber Em CGMCC No.0868 emulsifying agent that bacterial strain produces is to the promoter action of microbiological deterioration degraded anthracene
Fig. 6-1 Rhodococcus ruber Em CGMCC No.0868 bacterial strain is to the degraded of benzene
Fig. 6-2 Rhodococcus ruber Em CGMCC No.0868 bacterial strain is to the degraded of naphthalene
Fig. 6-3 Rhodococcus ruber Em CGMCC No.0868 bacterial strain is to the degraded of anthracene
Fig. 6-4 Rhodococcus ruber Em CGMCC No.0868 bacterial strain is to the degraded of pyrene
The body embodiment
In order to understand the present invention better, further specified by following embodiment, but be not limitation of the invention.
Embodiment 1: the separation screening of Rhodococcus ruber Em bacterial strain provided by the invention
Crude oil pollution soil around a North China Oilfield oil recovery oil well is gathered pedotheque, sample thief 50 grams are suspended in the 50mL stroke-physiological saline solution, shaking table vibration 30min makes abundant suspension, leaves standstill 30min, gets 10mL upper strata suspension and is inoculated in (substratum composition: K in the 100mL minimal medium
2HPO
43H
2O, 1g; KH
2PO
4, 1g; MgSO
47H
2O, 0.5g; NH
4NO
3, 1g; CaCl
2, 0.02g; FeCl
3Trace; Alkanes, 1g or aromatic hydrocarbons, 0.1g; Distilled water, 1000mL; PH7.0; 121 ℃ of sterilization 30min), 30 ℃ of shaking tables (200rpm) were cultivated 7 days, get 10mL nutrient solution inoculation fresh culture, press same operation condition continuous domestication 3 times, line separates in the minimal medium flat board then, and behind single bacterium colony to be formed, picking list colony inoculation is in corresponding inclined-plane, slant culture is purifying on LB flat board or common beef broth flat board, till microscopy obtains pure culture.The pure bacterial strain that obtains is inoculated minimal medium that contains different petroleum hydrocarbons (alkanes 2000mg/L, aromatic hydrocarbons and polycyclic aromatic hydrocarbons are 100mg/L) and the substratum that produces the thing emulsifying agent (substratum composition: n-hexadecane, 8g respectively; Yeast extract, 1g; K
2HPO
43H
2O, 1g; KH
2PO
4, 1g; MgSO
47H
2O, 0.5g; NH
4NO
3, 1g; / L CaCl
2, 0.02g; FeCl
3Trace; PH7.0,121 ℃ of sterilization 30min) cultivated respectively 4 days and 2 days.With the n-hexane extraction nutrient solution, the HPLC method detects the residual content of petroleum hydrocarbon, weigh the emulsifying activity of fermentation culture with the surface tension slippage of measuring water, choosing the bacterial strain that degrading activity is the highest, emulsifying capacity is the strongest is the further experiment bacterial strain, obtains Rhodococcus ruber Em bacterial strain.
The pcr amplification and the sequencing of the 16S rRNA gene of embodiment 2:Rhodococcus ruber Em bacterial strain
Rhodococcus ruber Em inoculation LB substratum; 30 ℃ of shaking tables were cultivated (200rpm) 18 hours; centrifugal collecting cell; again suspend; add the N,O-Diacetylmuramidase broken wall; extract DNA by phenol-chloroform method, and this carries out pcr amplification to universal primer to its 16S rDNA to adopt forward primer 27f (5 '-GAGAGTTTGATCCTGGCTCAG-3 ') and reverse primer 1541r (5 '-AAGGAGGTGATCC AGCC-3 '), amplified production is served sea base Kanggong department check order.The PCR condition is: 95 ℃, and 10min; 94 ℃, 1min, 48 ℃, 1min, 72 ℃, 1min; 30 circulations, 72 ℃, 5min, 4 ℃ of preservations.16S rDNA sequence length is 1398bp, and the sequence accession number in GenBank is AF529079, with bacterial strain Rhodococcus rubber DSM43338
TThe similarity of 16S rDNA (accession number is X80625) sequence be 99%.The sequence of its 16S rDNA is seen sequence table 1 and Fig. 1.
Embodiment 3: Rhodococcus ruber Em bacterial strain provided by the invention is to the degraded of polycyclic aromatic hydrocarbons
Get the fresh slant strains of this bacterial strain, inoculate a transfering loop and contain in the minimal medium that different petroleum hydrocarbons the are carbon source triangular flask of (substratum is formed with embodiment 1) in 100mL is housed, 30 ℃ of shaking tables were cultivated (200rpm) 3 days, measured the increment (OD of bacterium
460Expression) and the degraded (HPLC) of hydro carbons, the result as shown in Figure 6, Fig. 6-1 shows that to the benzene of 200mg/L, through 3 days cultivation, degradation rate can reach 95%; Fig. 6-2 shows that to the naphthalene of 100mg/L, degradation rate can reach 99%; Fig. 6-3 shows that to the anthracene of 50mg/L, degradation rate can reach 67%; Fig. 6-4 shows that to the pyrene of 50mg/L, degradation rate can reach 13%.
Embodiment 4: Rhodococcus ruber Em bacterial strain provided by the invention produces the condition of biological emulsifier
By changing kinds of culture medium and component, culture condition tests such as (temperature, pH, air flows etc.), the optimal medium that obtains this Rhodococcus ruber Em bacterial strain generation thing emulsifying agent is: n-hexadecane, 8g; Yeast extract, 1g; K
2HPO
43H
2O, 1g; KH
2PO
4, 1g; MgSO
47H
2O, 0.5; NH
4NO
3, 1g; CaCl
2, 0.02g; The FeCl of trace
3Optimal culture condition is: initial pH value is 7.0,30 ℃, cultivates 48 hours for 200 rev/mins.Under this culture condition, the cultivation through 3 days, 60 times of nutrient solution dilutions still can make the surface tension of water drop to about 50mN/m.
Embodiment 5: the surfactivity effect of the biological emulsifier that Rhodococcus ruber Em bacterial strain provided by the invention is produced
The surfactivity effect of the biological emulsifier that Rhodococcus ruber Em bacterial strain is produced, can reduce the surface tension of water significantly, adopt the surface tension of the Auto-tensiometerZL-2 type surface tension apparatus mensuration nutrient solution of Shandong Zibo Ke Sen company, as shown in table 1, the surface tension of water is 72.1mN/m when not adding emulsifying agent, and during the overstriking emulsifying agent, as long as the extension rate of thick emulsifying agent is below 41 times, the surface tension of water can drop to about 30mN/m, the thick emulsifying agent that has diluted 51 times still can make the surface tension of water drop to 50.3mN/m, the biological emulsifier that Rhodococcus ruber Em bacterial strain is produced is described, no matter be that the performance of emulsifying agent and the content of emulsifying agent are what not appeared in the newspapers.
Embodiment 6: Rhodococcus ruber Em bacterial strain provided by the invention produces the emulsifying capacity of thing emulsifying agent to kerosene
According in the world to the mensuration of emulsifying capacity, usually with kerosene the emulsification object, emulsifying agent mixes by equal volume with kerosene, fierce concussion 30min, leave standstill 2hr, measure the amount that residue does not have emulsified kerosene then, with per-cent (%) the expression emulsifying capacity of emulsive amount with added kerosene total amount.As shown in table 2, the result shows, at emulsifying agent: under the condition of kerosene=1: 1, the Em bacterial strain can reach 100% emulsification through 1 day cultivation; At emulsifying agent: under the condition of kerosene=1: 10, cultivate 1 day can emulsification 13% kerosene, cultivate and reached the highest emulsifying capacity in 2 days basically.
The emulsifying capacity of the biological emulsifier that table 1 Rhodococcus ruber Em bacterial strain is produced
| Incubation time (my god) | ????0 | ??0.5 | ????1 | ????2 | ????8 |
| Emulsifying activity (emulsifying agent: kerosene=1: 1) | ????0 | ????0 | ??100％ | ??100％ | ??100％ |
| Emulsifying activity (emulsifying agent: kerosene=1: 10) | ????0 | ????0 | ??13％ | ??15％ | ??16％ |
Embodiment 7: the chemical property of the biological emulsifier that Rhodococcus ruber Em bacterial strain provided by the invention is produced
Rhodococcus ruber Em bacterial strain is (with embodiment 4) cultivation and fermentation under the substratum and condition that produce the thing emulsifying agent, the fermented liquid n-hexane extraction of equating volume, the a-naphthol test is judged carbohydrate, is used Bradford colorimetric method for determining albumen and lipid qualitative test, this biological emulsifier of presentation of results does not contain polysaccharide, does not contain albumen, is a kind of lipoid substance.Further use thin-layer chromatography (TLC) method to analyze the component of this biological emulsifier, concrete operations are as follows: get the 5mL n-hexane extract, point sample is in silica gel column chromatography plate (subsidiary factory of Haiyang Chemical Plant, Qingdao, thickness is 0.2～0.25mm, size is on 150 * 100mm), through chloroform: methyl alcohol: the developing agent of water=18: 6: 1 launches, dry back iodine staining, can find to be respectively the band of Rf=0.12 and Rf=0.8 in mobility, scrape this two band respectively, use n-hexane dissolution, the volatilization normal hexane, be dissolved in the 30mL water, measure the surface tension (distilled water is contrast) of solution, the result: the surface tension of distilled water is 72.2mN/m, and the surface tension of the aqueous solution of the component of Rf=0.12 is 63.7mN/m, the surface tension of the aqueous solution of the component of Rf=0.8 is 56.4mN/m, illustrates that these two kinds of components all have certain surfactivity effect.Through qualitative analysis (method is the same), these two kinds of components all do not contain polysaccharide and albumen, are lipid materials.
Embodiment 8: Rhodococcus ruber Em bacterial strain provided by the invention produces the promoter action of thing emulsifying agent to the strains for degrading polycyclic aromatic hydrocarbons.
The various petroleum hydrocarbons that add different concns at minimal medium (moiety is with embodiment 1), compare test to add emulsifying agent (dividing different dosages) and not add emulsifying agent, the sampling of cultivation different time is with the petroleum hydrocarbon residual content in the HPLC method mensuration nutrient solution.With the anthracene is representative, and the result shows (Fig. 5), and the emulsifying agent that adds 1/30 (V/V) can make the degradation rate of anthracene increase about 30 percentage points.Degradation amount when cultivating 3 days is brought up to 46mg/L from 19mg/L, has increased about 1.5 times.
SEQUENCE?LISTING
＜110〉Institute of Microorganism, Academia Sinica
＜120〉a kind of Rhodococcus ruber Em and purposes that produces biological emulsifier and degrading polycyclic aromatic hydrocarbons
<160>1
<170>PatentIn?version?3.1
<210>1
<211>1398
<212>DNA
<213>Rhodococcus?ruber?Em?CGMCC?No.0868
<400>1
tgagccgtgc?ttacacatgc?gagtcgaacg?atgcagccca?gcttgctggg?tggattagtg???60
gcgaacgggt?gagtaacacg?tgggtgatct?gccctgcact?tcgggataag?cctgggaaac??120
tgggtctaat?accggatagg?acctcgggat?gcatgttccg?gggtggaaag?gttttccggt??180
gcaggatggg?cccgcggcct?atcagcttgt?tggtggggta?acggcccacc?aaggcgacga??240
cgggtagccg?gcctgagagg?gcgaccggcc?acactgggac?tgagacacgg?cccagactcc??300
tacgggaggc?agcagtgggg?aatattgcac?aatgggcgca?agcctgatgc?agcgacgccg??360
cgtgagggat?gacggccttc?gggttgtaaa?cctctttcag?taccgacgaa?gcgcaagtga??420
cggtaggtac?agaagaagca?ccggccaact?acgtgccagc?agccgcggta?atacgtaggg??480
tgcgagcgtt?gtccggaatt?actgggcgta?aagagctcgt?aggcggtttg?tcgcgtcgtc??540
tgtgaaaacc?cgcagctcaa?ctgcgggctt?gcaggcgata?cgggcagact?tgagtactgc??600
aggggagact?ggaattcctg?gtgtagcggt?gaaatgcgca?gatatcagga?ggaacaccgg??660
tggcgaaggc?gggtctctgg?gcagtaactg?acgctgagga?gcgaaagcgt?gggtagcgaa??720
caggattaga?taccctggta?gtccacgccg?taaacggtgg?gcgctaggtg?tgggtttcct??780
tccacgggat?ccgtgccgta?gctaacgcat?taagcgcccc?gcctggggag?tacggccgca??840
aggctaaaac?tcaaaggaat?tgacgggggc?ccgcacaagc?ggcggagcat?gtggattaat??900
tcgatgcaac?gcgaagaacc?ttacctgggt?ttgacataca?ccggaccgcc?ccagagatgg??960
ggtttccctt?gtggtcggtg?tacaggtggt?gcatggctgt?cgtcagctcg?tgtcgtgaga???1020
tgttgggtta?agtcccgcaa?cgagcgcaac?ccttgtcctg?tgttgccagc?acgtaatggt???1080
ggggactcgc?aggagactgc?cggggtcaac?tcggaggaag?gtggggacga?cgtcaagtca???1140
tcatgcccct?tatgtccagg?gcttcacaca?tgctacaatg?gccggtacag?agggctgcga???1200
taccgcgagg?tggagcgaat?cccttaaagc?cggtctcagt?tcggatcggg?gtctgcaact???1260
cgaccccgtg?aagtcggagt?cgctagtaat?cgcagatcag?caacgctgcg?gtgaatacgt???1320
tcccgggcct?tgtacacacc?gcccgtcacg?tcatgaaagt?cggtaacacc?cgaagccgga???1380
gacctaactc?ctcctcaa?????????????????????????????????????????????????1398
Claims (10)
1. a strain has Rhodococcus ruber (Rhodococcus ruber) the Em CGMCC No.0868 bacterium of degraded alkane and polycyclic aromatic hydrocarbons and generation biological emulsifier.
2. bacterium according to claim 1, the nucleotide sequence that it is characterized in that its 16S rDNA is shown in sequence table 1.
3. bacterium according to claim 1, grown cell, cell suspending liquid, its immobilized cell that it is characterized in that this bacterium all can degrade alkane and anthracene, phenanthrene, pyrene polycyclic aromatic hydrocarbons.
4. bacterium according to claim 1 is characterized in that this bacterium can be sole carbon source and energy growth with alkane or polycyclic aromatic hydrocarbons, produces the lipid biological emulsifier simultaneously.
5. the lipid biological emulsifier that bacterium according to claim 4 produces is when being carbon source with the n-Hexadecane, and substratum is formed (%): n-hexadecane, 8g; Yeast extract 1g; K
2HPO
43H
2O1g; KH
2PO
41g; MgSO
47H
2O 0.5g; NH
4NO
31g; CaCl
20.02g; FeCl
3Trace; PH7.0 cultivates the biological emulsifier that CGMCC No.0868 produces.
6. can in emulsification, raising alkane and the solubleness of polycyclic aromatic hydrocarbons in water of the surface tension that reduces the aqueous solution, oil substances, use according to the lipid biological emulsifier of claim 4 or 5 described bacterium generations.
7. the lipid biological emulsifier according to claim 4 or 5 described bacterium generations is used for the biological treatment of oily(waste)water and the biological restoration of oil-polluted soils.
8. bacterium according to claim 1 is characterized in that the bacterium colony size that its colony characteristics was cultivated 2 days is diameter 3～4mm on the LB flat board, bacterium colony is rounded, and surface drying is coarse, and neat in edge is protruded, and is orange; Cell morphological characteristic children cell in age is that bar is strong, and aged cell is cultivated and become sphere after 4 days, Gram-positive, and the cell size is diameter 0.8 μ m, random arrangement; Physiological and biochemical property has the acid-fast stain feminine gender, do not produce gemma or spore, the catalase positive, reduction nitrate, can utilize glucose, sucrose, maltose, D-fructose, lactose, D-semi-lactosi, rhamnosyl, trehalose, N.F,USP MANNITOL, sorbyl alcohol, D-wood sugar, L-arabinose, cellobiose, raffinose, glycerol, and all fermentation and acid can also utilize seminose, L-L-glutamic acid, L-Xie Ansuan, L-proline(Pro); Can not utilize inositol, succsinic acid, glycine, lactic acid, L-arginine.
9. bacterium according to claim 1, Rhodococcus ruber (Rhodococcus ruber) EmCGMCC No.0868 uses in emulsification, raising alkane and the solubleness of polycyclic aromatic hydrocarbons in water of the surface tension that reduces the aqueous solution, oil substances.
10. bacterium according to claim 1, Rhodococcus ruber (Rhodococcus ruber) EmCGMCC No.0868 carries out a biological disposal upon or/and use in the biological restoration of oil-polluted soils at oily(waste)water.
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