CN1844361A - Polycyclic aromatic hydrocarbon degrading bacteria and use thereof - Google Patents
Polycyclic aromatic hydrocarbon degrading bacteria and use thereof Download PDFInfo
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- CN1844361A CN1844361A CN 200610034169 CN200610034169A CN1844361A CN 1844361 A CN1844361 A CN 1844361A CN 200610034169 CN200610034169 CN 200610034169 CN 200610034169 A CN200610034169 A CN 200610034169A CN 1844361 A CN1844361 A CN 1844361A
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Abstract
This invention discloses a polycyclic-aromatic degrading bacterial strain and the application thereof. The strain, GY2B, belonging to Sphingomonas sp., can efficiently degrade phenanthrene in the aerobic condition, and can also utilize naphthalene, 2-naphthol, 1-hydroxy-2-naphthoate, benzene, toluene, salicylic acid, catechol, fenol and many other aromatic compounds. Sphingomonas sp. GY2 is sensitive to the four common antibiotics of cefuroxime, alficetin, alficetin and tetracycline, and is moderately sensitive to amoxicillin, which provides safety insurance for its application in the biological treatment of waste water and the restortion of environmental pollution.
Description
Technical field
The invention belongs to environmental pollutant biologic treating technique field, be specifically related to the bacterium and the application in biological wastewater treatment and environmental pollution reparation thereof of a strain degrading polycyclic aromatic hydrocarbons.
Background technology
Polycyclic aromatic hydrocarbons (PAHs) is meant that two or more phenyl ring with the compound that chain, horn shape or string shape rearrange, are the byproducts of organic incomplete combustion or Pintsch process.PAHs in the environment is mainly derived from mankind's activity, as wood working, petroleum refining, transport trade, coal gas manufacturing, military installations, hazardous material processing and landfill etc.Though the content trace of PAHs in environment is still widely distributed, all detects the existence of multiple PAHs in atmosphere, water body, soil, settling and food.Many PAHs not only have intensive " three cause " toxicity---and carcinogenic, teratogenesis and mutagenicity also have short carcinogenesis.EPA (USEPA) eighties of last century eighties just with 16 kinds of Black Lists of having listed the environment priority pollutant with ramose PAHs in.The low water solubility of PAHs and highly lipophilic make it than being easier to be assigned in the organism, enter the ecosystem by food chain, thereby the safety of the human health and the ecosystem is constituted harm greatly.
Owing to the PAHs chemical stability is fine, be difficult under the natural condition decompose by chemical actions such as hydrolysis, a part takes place the photochemistry decomposition except having seldom, and the polycyclic aromatic hydrocarbons of the overwhelming majority mainly slowly disappears from environment by biodegradation pathway.Therefore, utilize the Degradation of microorganism that PAHs is converted into the best means that innoxious substance is considered to remove PAHs in the environment.
At present, people are by technology such as artificial enrichment culture, have isolated many bacterium, fungi and actinomycetes with ability of degraded PAHs, and bacterium is because the multiple adaptive faculty on its biochemistry and easily bring out mutant strain and in the highest flight.But high-efficiency strain is few in the PAHs degradation bacteria that has been separated to, as strain separated SKY such as Tam the phenanthrene of 10mg/L is only degraded 46% in 6 days; Two kinds of bacterial strain Rhodotorula glutinis of researchs such as Romero and Pseudomonas aeruginosa take that month ability degrades 25,50,100 fully, the phenanthrene of several concentration of 200mg/L; Isolating six kinds of single bacterium all need the 6-8 days 5mg/L phenanthrene of could degrading fully in the research mixed bacterium such as Yuan.On the other hand, great majority seldom relate to the research of its degraded substrate scope only at single pollutent in the PAHs degradation bacteria that has been separated to.Therefore, the degradation bacteria of searching high-efficiency broad spectrum is the hot issue of present Study on Environmental Protection.
Summary of the invention
The objective of the invention is a difficult problem, a kind of bacterial classification of energy efficient degradation polycyclic aromatic hydrocarbons is provided at above-mentioned existing degrading polycyclic aromatic hydrocarbons existence.
Another object of the present invention provides the application of above-mentioned bacterial classification in the various organic compound of degraded.
Efficient degrading bacteria Sphingomonas sp.GY2B provided by the present invention is subjected to the petroleum chemicals Contaminated soil near deriving from Guangzhou oil gas factory, obtains through artificial enrichment culture, separation and purification.This bacterium is that the Sphingol single-cell of Gram-negative belongs to the bacterial strain GY2B of (Sphingomonas sp.), biological characteristics is a catalase-positive, oxidase negative, non-fermented type, obligate is aerobic, and thalli morphology is a sporeless bacterium, bacterium colony is milk yellow, circle, neat in edge, smooth moistening, the GenBank number of landing of bacterial strain 16S rDNA is DQ139343, on February 24th, 2006 in China's typical culture collection center preservation, be numbered CCTCC M 206019.
The optimum growing condition of this bacterium is: pH=6.5～9.5,25～35 ℃ of temperature.
This bacterium can utilize luxuriant and rich with fragrance as sole carbon source and energy growth and breeding, and luxuriant and rich with fragrance permineralization is become CO
2And H
2O.Under the pure culture condition, this bacterium 48h can degrade the phenanthrene of 100mg/L in the minimal medium more than 99.1%.Can improve the luxuriant and rich with fragrance efficient of this bacterium degraded if add nutritive substances such as an amount of glucose, peptone.In addition, this bacterium can utilize polycyclic aromatic hydrocarbonss such as naphthalene, beta naphthal, 1-hydroxyl-2-naphthoic acid and substituent as sole carbon source and energy growth and breeding, simultaneously with its degraded respectively.For the material that contains phenyl ring, this bacterium can utilize benzene series things such as benzene, toluene, Whitfield's ointment, pyrocatechol, phenol as sole carbon source and energy growth and breeding, simultaneously with its degraded respectively.
This bacterium is to cephalofruxin, paraxin, erythromycin, four kinds of common antibiotics sensitivities of tsiklomitsin, to the amoxycillin medium sensitivity.
Compared with prior art, Sphingomonas sp.GY2B of the present invention can be under aerobic condition respectively with benzene series things such as polynuclear compounds such as phenanthrene, naphthalene, beta naphthal, 1-hydroxyl-2-naphthoic acid and benzene, toluene, Whitfield's ointment, pyrocatechol, phenol as sole carbon source and energy growth and breeding, simultaneously with its degraded.Under the pure culture condition, it can degrade more than 99.1% the phenanthrene of 100mg/L in 48h.This bacterium, can be thought when thalline is released in the physical environment to the amoxycillin medium sensitivity to four kinds of common antibiotics sensitivities such as cephalofruxin, paraxin, erythromycin, tsiklomitsins, can not carry resistance determining factor simultaneously and propagate between indigenous bacterium.Compare with existing bacterial strain, this bacterium has the ability of efficient degradation phenanthrene, and can utilize multiple substrate, does not carry resistance determining factor, can obtain safety, use widely.This bacterium will play a significant role in the biological restoration of the biological treatment of trade effluent and contaminate environment.
Description of drawings
Fig. 1 is Sphingomonas sp.GY2B growth and luxuriant and rich with fragrance degradation curve figure;
Fig. 2 is the UV-Vis collection of illustrative plates of the different substrates of Sphingomonas sp.GY2B degraded 25mg/L.Wherein, the meaning of each bar curve representative is among Fig. 1:
-◆ the residual rate of-nutrient solution China and Philippines, the nectar degree of GY2B in--nutrient solution;
The meaning of each bar curve representative is among Fig. 2:
---substrate adds the scanning spectra behind the bacterium degraded 2d;---substrate does not add the scanning spectra of bacterium.
Embodiment
The invention will be further described below in conjunction with specific embodiment.
The separation of embodiment 1:Sphingomonas sp.GY2B and the luxuriant and rich with fragrance performance of degraded
Collection in worksite Guangzhou oil gas factory periphery is subjected to the petroleum chemicals Contaminated soil, adopts water-silicone oil (volume ratio 4: 1) diphasic system to tame luxuriant and rich with fragrance degradation bacteria, and luxuriant and rich with fragrance initial concentration keeps 100mg/L.Water inorganic salt nutrient solution (MSM) composition: 5.0ml/L phosphate buffered saline buffer (8.5g/L KH
2PO
4, 21.75g/L K
2HPO
4H
2O, 33.4g/L Na
2HPO
412H
2O, 5.0g/L NH
4) C), 3.0ml/LMgSO
4The aqueous solution (22.5g/L), 1.0ml/LCaCl
2The aqueous solution (36.4g/L), 1.0ml/L FeCl
3The aqueous solution (0.25g/L), 1.0ml/L trace element solution (39.9mg/L MnSO
4H
2O, 42.8mg/L ZnSO
4H
2O, 34.7mg/L (NH
4)
6Mo
7O
244H
2O).
Passing through some generations tames repeatedly, streak culture in solid medium (NR), obtain the pure bacterium of many strains, pure bacterium is through being seeded to the luxuriant and rich with fragrance ability of its degraded of checking in the liquid nutrient medium, obtain the efficient degrading bacteria GY2B of a strain phenanthrene at last, this bacterium is accredited as Sphingol single-cell through 16S rDNA and belongs to (Sphingomonas sp.).The NR composition: peptone 10g, extractum carnis 5g, NaCl 5g, agar strip 15g, distilled water 1L, transferring pH is 7.0.
Sphingomonas sp.GY2B is seeded in the MSM substratum that contains luxuriant and rich with fragrance 100mg/L, lucifuge shaking culture in 150r/min, 30 ℃ of shaking tables, the 0th, 6,12,24,30,36,48h nectar degree and luxuriant and rich with fragrance residual rate in the sampling and measuring nutrient solution respectively, experimental result is seen Fig. 1.By the luxuriant and rich with fragrance degradation curve of Fig. 1 as can be seen, under the starting point concentration of 100mg/L, luxuriant and rich with fragrance degraded of initial stage (0-6h) is slower, and degraded luxuriant and rich with fragrance between the ensuing 6-30h is very fast, and to 48h time, the degradation rate of phenanthrene has reached 99.1%.In addition, the cell growth curve of Fig. 1 shows that the growth of degraded incipient cell has a bit slowly, and growth afterwards is very fast, and the nectar degree reaches 4.2 * 10 in the time of 48h
8Individual/mL.Calculating luxuriant and rich with fragrance degradation rate at linear decrement phase (6-30h) from luxuriant and rich with fragrance degradation curve is 3.45mg/Lh; Calculating GY2B from growth curve of bacteria is 0.135h at the specific growth rate μ of logarithmic phase (6-24h)
-1, cell doubling time t is 5.1h.
The Sphingomonas sp.GY2B of present embodiment explanation resulting separation can utilize phenanthrene to carry out growth and breeding as the sole carbon source and the energy, and has the ability of efficient degradation phenanthrene.
Embodiment 2: add nutritive substance to the influence of Sphingomonas sp.GY2B cell growth with luxuriant and rich with fragrance degradation rate
Sphingomonas sp.GY2B is seeded in the MSM substratum that contains luxuriant and rich with fragrance 100mg/L, in nutrient solution, add the glucose of 100mg/L or peptone then as nutritive substance, do simultaneously and do not add nutritive substance, only inoculate bacterium and do not add carbon source, do not inoculate bacterium and only add control experiments such as phenanthrene.Lucifuge shaking culture in 150r/min, 30 ℃ of shaking tables is measured nectar degree and luxuriant and rich with fragrance residual quantity in the nutrient solution behind the 36h, calculate luxuriant and rich with fragrance degradation rate, and experimental data sees Table 1.As known from Table 1, Fei non-biodegradation effect is very little; Added that the nectar degree significantly increases in the nutrient solution of peptone or glucose, luxuriant and rich with fragrance degradation rate also is improved.
The growth that the adequate nutrition material can promote Sphingomonas sp.GY2B is added in the present embodiment explanation, improves its degradation rate to phenanthrene simultaneously.
Table 1 adds nutritive substance to the influence of Sphingomonas sp.GY2B cell growth with luxuriant and rich with fragrance degradation rate
| Substratum | The nectar degree (individual/ml) | Luxuriant and rich with fragrance degradation rate (%) |
| MSM substratum+bacterium+phenanthrene+glucose MSM substratum+bacterium+phenanthrene+peptone MSM substratum+bacterium+luxuriant and rich with fragrance MSM substratum+bacterium MSM substratum+phenanthrene | 2.2×10 8 1.9×10 8 3.3×10 7 1.9×10 6 | 97.8 95.1 91.7 3.8 |
The degraded substrate experiment of embodiment 3:Sphingomonas sp.GY2B
Sphingomonas sp.GY2B is seeded to contains different concns (25～2000mg/L, see Table 2) the MSM substratum of substrate such as Whitfield's ointment, pyrocatechol, 1-hydroxyl 2-naphthoic acid, phenol, beta naphthal, naphthalene, benzene, toluene in, establish substrate blank (only add substrate and do not add bacterium) simultaneously, the lucifuge shaking culture is measured biomass and is detected the degraded situation of substrate after 2 days in 150r/min, 30 ℃ of shaking tables.The growing state of Sphingomonas sp.GY2B sees Table 2, the result shows that Sphingomonas sp.GY2B can utilize certain density experiment substrate to carry out growth and breeding, to and the tolerance concentration difference of different substrates, except that beta naphthal tolerance concentration was low, the tolerance concentration of all the other substrates reached 100mg/L at least.Cultivate and observe the naphthalene, benzene, the toluene that swim in the nutrient solution with solid or oily when initial after 2 days and disappear, initial Whitfield's ointment, pyrocatechol, 1-hydroxyl 2-naphthoic acid, phenol, the beta naphthal that just is dissolved in the MSM substratum then detects its existence in the nutrient solution by UV-Vis, and detected result is seen Fig. 2.Starting point concentration is that the Whitfield's ointment, pyrocatechol, 1-hydroxyl 2-naphthoic acid, phenol, beta naphthal of 25mg/L cultivates that the UV-Vis absorption peak has all disappeared after 2 days, shows that they by Sphingomonas sp.GY2B degraded fully.
Present embodiment shows that Sphingomonas sp.GY2B has the ability of the multiple substrate of degraded, is the degradation bacteria of a strain wide spectrum.
The diversity of table 2 bacterial strain GY2B degraded substrate
| Concentration of substrate (mg/L) | 25 | 50 | 75 | 100 | 150 | 200 | 500 | 1000 | 2000 |
| Salicylic acid catechol 1-hydroxyl-2-naphthoic acid phenol beta naphthal naphthalene benzene toluene | ++ ++ ++ ++ ++ ++ ND ND | ++ ++ ++ ++ + ++ ND ND | ++ ++ ++ ++ + ++ ND ND | ++ ++ ++ ++ - ++ ND ND | - + ++ + ND ND ND ND | - +/- - + ND ++ ++ ++ | ND ND ND +/- ND ++ + ++ | ND ND ND - ND ++ + +/- | ND ND ND ND ND ND +/- - |
Annotate: ++ expression is grown, and quantity is higher one more than the order of magnitude than initial nectar degree;
+ expression can be grown, and quantity and initial nectar degree are at the same order of magnitude;
Only there is a small amount of growth in+/-, and quantity is lower one more than the order of magnitude than initial nectar degree;
-expression can not be grown;
ND represents not do the experiment of this concentration.
The antibiotics sensitivity experiment of embodiment 4:Sphingomonas sp.GY2B
Antibiotic sensitivity test adopts paper disk method, select five kinds of representative microbiotic commonly used such as amoxycillin, cephalofruxin, paraxin, erythromycin, tsiklomitsin, with the bacteriostatic diameter size of each microbiotic scraps of paper on cultivation degradation bacteria NR flat board, as responsive or drug-fast criterion.The experimental result that table 3 provides shows that bacterial strain GY2B is to four kinds of representative antibiotic sensitive commonly used such as cephalofruxin, paraxin, erythromycin, tsiklomitsin, to the amoxycillin medium sensitivity.
Present embodiment explanation can not propagated between indigenous bacterium because of carrying resistance determining factor when thalline is released in the physical environment, for the practical application of Sphingomonas sp.GY2B provides theoretical basis and safety assurance.
The resistant proof result of table 3 bacterial strain GY2B
| Microbiotic | The antibiotic content of the scraps of paper (μ g/ sheet) | Medium sensitivity scope (mm) | Suppress loop diameter (mm) | Result of determination ① |
| Amoxycillin cephalofruxin paraxin erythromycin tsiklomitsin | 25 30 30 15 30 | 22-27 14-22 23-25 23-25 23-25 | 22 37 39 36 40 | ○ - - - - |
Annotate: 1. the diameter of inhibition circle is responsive (-) greater than the higher limit of medium sensitivity scope, is anti-medicine (+) less than lower value, is medium sensitivity (zero) between this value.
Claims (5)
1. a polycyclic aromatic hydrocarbon degrading bacteria, it is characterized in that: this bacterium is the bacterial strain GY2B that the Sphingol single-cell of Gram-negative belongs to, biological characteristics is a catalase-positive, oxidase negative, non-fermented type, obligate is aerobic, thalli morphology is a sporeless bacterium, and bacterium colony is milk yellow, circle, neat in edge, smooth moistening, and the GenBank number of landing of bacterial strain 16SrDNA is DQ139343, in on February 24th, 2006 in China's typical culture collection center preservation, be numbered CCTCC M 206019.
2. the application of the described polycyclic aromatic hydrocarbon-degrading bacteria of claim 1 in the degraded organic aromatic compound.
3. application according to claim 2 is characterized in that described organic compound is polycyclic arene compound or benzene series thing.
4. application according to claim 3 is characterized in that described polycyclic arene compound is phenanthrene, naphthalene, beta naphthal or 1-hydroxyl-2-naphthoic acid.
5, application according to claim 3 is characterized in that described benzene series thing is benzene, toluene, Whitfield's ointment, pyrocatechol or phenol.
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| CN101948772A (en) * | 2010-08-27 | 2011-01-19 | 华南理工大学 | High-efficiency strain for degrading polycyclic aromatic hydrocarbon and construction method and application thereof |
| CN101948772B (en) * | 2010-08-27 | 2012-08-08 | 华南理工大学 | High-efficiency strain for degrading polycyclic aromatic hydrocarbon and construction method and application thereof |
| CN102614619A (en) * | 2011-03-01 | 2012-08-01 | 南京大学 | Application of Sphingomonas strains in polycyclic aromatic hydrocarbons degradation |
| CN102250788A (en) * | 2011-05-27 | 2011-11-23 | 清华大学 | Stenotrophomonas maltophilia for degrading high-concentration methylbenzene and application of stenotrophomonas maltophilia |
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| CN109331391A (en) * | 2018-10-22 | 2019-02-15 | 陈先锐 | Utilize the method for antibiotic in microorganism and infra-red radiation degrading cultivation field biogas slurry |
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