CN104046580A - Sphingobacterium strain for degrading polycyclic aromatic hydrocarbon organic pollutant and application thereof - Google Patents
Sphingobacterium strain for degrading polycyclic aromatic hydrocarbon organic pollutant and application thereof Download PDFInfo
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- CN104046580A CN104046580A CN201410221389.2A CN201410221389A CN104046580A CN 104046580 A CN104046580 A CN 104046580A CN 201410221389 A CN201410221389 A CN 201410221389A CN 104046580 A CN104046580 A CN 104046580A
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Abstract
The invention belongs to the field of bioremediation of organic pollutants, and specifically discloses a Sphingobacterium sp. strain W144. The strain is preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC NO:M2014151. The strain disclosed by the invention has rapid and efficient degradation ability on phenanthrene, biphenyl, pyrene, fluorene, fluoranthene and other organic pollutants, has a wide range of pH value, and salinity and temperature adaptation ability, and is a good material for bioremediation of polycyclic aromatic hydrocarbons in soil or the environment.
Description
Technical field
The invention belongs to environmental organic pollutant repairing and treating field.Be specifically related to a strain for Sphingobacterium bacterial strain and the application thereof of degrading polycyclic aromatic hydrocarbons class organic pollutant.
Background technology
Polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbons, PAHs) be the organism that a class contains two or more benzene ring structures, because it is distributed widely in ocean, lake and soil in environment, and water-soluble low, non-volatile, Stability Analysis of Structures, and become typical persistence organic pollutant.PAHs has " carcinogenic, teratogenesis, mutagenesis " effect, and Environment Ecological Safe and human health are had to great potential hazard.Since eighties of last century the eighties, countries in the world Environmental Protection Agency has all classified PAHs as screen priority pollutants in environment.PAHs in environment is mainly derived from oil production transportation, refining of petroleum, organic industry, fossil energy burning, motor vehicle exhaust etc.; Natural volcanic eruption, plant and microorganism self is synthetic also can cause the environment of part PAHs to discharge with metabolism.Its PetroChina Company Limited. is one of the main source of PAHs in environment.In oil, contain complicated PAHs mixture; In diesel oil, contain the PAHs of the 30-40% that has an appointment; PAHs in creosote is up to 85-90%.Due to PAHs indissoluble or insoluble in water, the PAHs in most of environment can deposit in soil in environment migration course.PAHs in soil is mainly from atmospheric precipitation, sewage irrigation, oil production or leakage, and industrial sewage or waste discharge etc.PAHs in soil pollutes one side by spoiled soil ecotope, affects soil productivity, reduces crop yield; Cause PAHs accumulation in agricultural-food simultaneously, directly threaten human body healthy.
The biological restoration of soil is to utilize especially microorganism of biology, is carbonic acid gas and water, or is converted into innoxious substance by poisonous and hazardous pollutent mineralising in soil, recovers the engineering system of the ecological functions of contaminate environment.Biological restoration is at petroleum pollution, and soil organic pesticide is removed, and in the environment remediation processes such as Heavy Metal Pollution Control and the adjusting of nitrogen phosphorus nutrition, finds broad application.It is successfully to use for the first time biotechnology to carry out soil organic pollutant reparation that the U.S. in 1972 utilizes the oil pipeline in bioremediation technology removing guest's Western method Leah state to leak oil, within 1989 subsequently, EPA carries out microbiobacterial agent input in the bay of the place generation Oil spills of Alaska, has completed the removing of leaking oil in the short period of time.From 1991 to 1999 10 years, the U.S. adopts bioremediation technology to complete the site remediation of 104 heavily polluted areas, and wherein 40% belongs to polycyclic aromatic hydrocarbons contaminated.Area, China Zhongyuan Oil Field adopts microorganism recovery technique, in conjunction with phytoremediation and physico-chemical process, completed the biological restoration practice in the petroleum pollution region of more than 40 mu, its petroleum pollution degradation rate reaches 85%, and the soil after arrangement recovers cultivated land function substantially.
The essence that microorganism is repaired is the organic pollutant utilizing in soil microorganisms specificity metabolism soil, is translated into the process of other carbon back macromole of cell and carbonic acid gas.Therefore the core of implementing and setting up the microorganism renovation technique of a set of soil organic pollutant is that accretion rate is fast, the screening and identification of the contaminant degradation bacterium that environmental compatibility is strong.Due to Biosafety, genetic engineering bacterium is difficult to the microorganism that big area applies to true environment soil to be repaired, and this makes from the contaminated soil separated native bacterium obtaining become the candidate material of optimal microorganism remediation microbial inoculum.Simultaneously the separated features such as also to have colonization ability strong, and environmental compatibility is wide that obtain microorganism from soil, are the direct executives who carries out core carrier and the contaminant degradation of biological restoration.Therefore, to have the microbial resources of organic pollutant degradation ability be prerequisite and the basis of carrying out environmental organic pollutant biological restoration to screening and identification.
Summary of the invention
The object of this invention is to provide a strain for Sphingobacterium bacterial strain and the application thereof of degrading polycyclic aromatic hydrocarbons class organic pollutant.
Applicant is the separated bacterial strain that obtains a strain can fast degradation polyaromatic hydrocarbon pollutant from oil-polluted soils, by Physiology and biochemistry, is identified with rrna 16S rDNA gene sequencing and is accredited as Sphingobacterium (Sphingobacterium sp.) bacterial strain W144.This bacterial strain is delivered Chinese Typical Representative culture collection center (CCTCC) preservation on April 24th, 2014, and deposit number is: CCTCC NO:M2014151.
This bacterial strain can the organic pollutant such as efficient degradation polycyclic aromatic hydrocarbons organic pollutant, especially phenanthrene, biphenyl, pyrene, fluorenes or fluoranthene.
The present invention further provides a kind of method of degrading polycyclic aromatic hydrocarbons class organic pollutant, the method comprises to the step of described Sphingobacterium (Sphingobacterium sp.) the bacterial strain W144 of inoculation in contaminated sample.
Preferably, the temperature of sample is adjusted in to the step within the scope of 16-37 ℃ after being also included in inoculation.
Further preferably, be also included in the step of pH value within the scope of 5-9 that regulates sample after inoculation.
The present invention further provides a kind of microbiobacterial agent for degrading polycyclic aromatic hydrocarbons class organic pollutant again, and its activeconstituents is described Sphingobacterium (Sphingobacterium sp.) bacterial strain W144.
Bacterial strain provided by the present invention has efficiently degradation capability fast to organic pollutants such as phenanthrene, biphenyl, pyrene, fluorenes or fluoranthene.This bacterial strain also has pH value widely, and salinity and thermal adaptation ability are soil or environment polycyclic aromatic hydrocarbons organic pollutant to be carried out to the outstanding microbial material of biological restoration.
More detailed technical scheme is shown in described in < < specific embodiments > >.
Accompanying drawing explanation
Fig. 1 is the gramstaining micro-imaging photo of bacterial strain W144.
Fig. 2 is the degradation capability of bacterial strain W144 to different polycyclic aromatic hydrocarbon pollutants.In figure, NC contrasts for bearing, Phenanthrene is phenanthrene, naphthalene is that naphthalene, Fluorene are that fluorenes, Fluoranthrene are that fluoranthene, Biphenyl are that biphenyl, Pyrene are that pyrene, Benzo (a) pyrene are benzopyrene.
Fig. 3 is that bacterial strain W144 take growth curve and the luxuriant and rich with fragrance degradation curve of phenanthrene under sole carbon source condition.
Fig. 4 is the sensitivity curves of bacterial strain W144 to pH value.
Fig. 5 is the sensitivity curves of bacterial strain W144 to Na ion concentration.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.Should be noted that embodiments of the invention only limit to describe for the present invention, and there is no restriction.Relevant screening method, damping fluid preparation and common culture medium prescription etc. related in embodiment can be with reference to Zhao Bin, He Shaojiang chief editor's < < Microbiology Experiment > > and the described content of < < molecular cloning experiment guide > > (referring to J. Pehanorm Brooker etc., 2002, molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), Science Press, Beijing).Related other various experimental implementation in the present invention, be the ordinary skill in the art, the part that there is no special instruction in literary composition, those of ordinary skill in the art can be implemented with reference to the various common tool books before the present patent application day, scientific and technical literature or relevant specification sheets, handbook etc.
The isolation identification of embodiment 1 W144 bacterial strain
Employing is screening soil from the long-term petroleum-polluted soil of Qianjiang, Hubei Jianghan Oil-field, and concrete screening scheme is as follows:
1) get 10g soil, join in the minimal medium MSM of carbonaceous sources not, then add phenanthrene (Phenanthrene), making final concentration is 100mg/L; 28 ℃ of concussions are cultivated 5 days, then receive in corresponding fresh culture with 5% the amount of connecing; Switching is 5 times continuously;
2) get the enrichment culture liquid of 0.1ml, be applied to and contain on luxuriant and rich with fragrance MSM solid plate, 28 ℃ of static cultivations, after 5 days, are transferred to bacterium colony longer on flat board to contain on luxuriant and rich with fragrance flat board, continue incubation growth; Then select the bacterium colony of fast growth to carry out plate streaking purifying;
3) bacterium colony of picking stable growth, is inoculated in the luxuriant and rich with fragrance MSM substratum that contains 100mg/L, and 28 ℃ of concussions are cultivated 5 days, obtain bacterial strain of the present invention.
Further bacterial strain has been carried out to morphology and molecular biology identification.
First, the morphological feature of bacterial strain has been carried out to preliminary observation, result shows, this bacterial strain is Gram-negative bacteria, spherical (Fig. 1).
Meanwhile, the separated genomic dna that obtains bacterial strain of contriver, then universal primer 27F and the 1492R primer pair with bacterial 16 S rRNA gene carries out pcr amplification; The pcr amplification product of acquisition is checked order, and its sequence is as shown in sequence table SEQ ID NO:1.Then, utilize NCBI nucleic acid database, according to bacterial strain 16S rRNA gene order, carried out BLAST analysis, result shows 16S rRNA gene order and the Sphingobacterium Sphingobacterium siyangense strain SY1 of bacterial strain, the 16S rRNA gene order of many strains bacteriums such as Sphingobacterium sp.IN42 all has 99% similarity, in conjunction with gramstaining and morphological observation, contriver is by this bacterial strain called after Sphingobacterium (Sphingobacterium sp.) bacterial strain W144.
The degraded feature of embodiment 2 W144 bacterial strains to polycyclic aromatic hydrocarbons
Contriver is studied common degrading polycyclic aromatic hydrocarbons ability W144 bacterial strain.Take do not contain carbon source MSM substratum as basis, add respectively different polycyclic aromatic hydrocarbonss as sole carbon source, making its final concentration is 100mg/L; Then with 1% inoculum size, inoculate in the W144 of logarithmic phase culture, be placed in 28 ℃ of concussions and cultivate after 5 days, measure culture at OD
600absorbance.The substratum that does not add any carbon source of take is negative contrast.Due to any carbon source of not adding in substratum except polycyclic aromatic hydrocarbons, so the speed of growth of bacterial strain and biomass embodied bacterial strain to the utilization of polycyclic aromatic hydrocarbons and degradation capability.
As shown in Figure 2, W144 has maximum to phenanthrene and utilizes ability, and biphenyl, pyrene, fluorenes and fluoranthene are had to stronger degradation capability; Benzopyrene, naphthalene are shown to the poor ability of utilizing.This shows that W144 bacterial strain has degradation capability to multiple polycyclic aromatic hydrocarbons simultaneously.
Embodiment 3 W144 bacterial strains are to luxuriant and rich with fragrance degraded feature
In order further to analyze the degraded feature of W144 bacterial strain provided by the present invention to polycyclic aromatic hydrocarbons, contriver be take phenanthrene as sole carbon source, and the speed of growth of W144 bacterial strain and degradation speed are tested.W144 bacterial strain in logarithmic phase is inoculated into and is take in the MSM substratum that the phenanthrene of 500mg/L is sole carbon source with 1% inoculum size, be placed in 28 ℃ of concussions and cultivate.Meanwhile, within every 12 hours, measure the OD of a subculture
600the concentration of value and residual phenanthrene.
As shown in Figure 3, W144 bacterium starts just to have possessed luxuriant and rich with fragrance degradation capability from inoculation, and degradation capability and strain growth all increase sharply after 12 hours, and along with the minimizing of residual luxuriant and rich with fragrance concentration in culture, strain growth progresses into plateau.At inoculation W144 bacterial strain, in the time of 84 hours, the phenanthrene in culture has 95% to be degraded.This shows that W144 bacterial strain has the rapidly and efficiently ability of degradable organic pollutant phenanthrene, can the phenanthrene of the 500mg/L in liquid nutrient medium is degradable in the time about five days.
The environmental adaptation feature of embodiment 4 W144 bacterial strains
W144 bacterial strain is tested the adaptability of temperature, pH value and salt ionic concentration.
Adopt R2A substratum, inoculation is in the W144 of logarithmic phase bacterial strain in fresh R2A substratum, and inoculum density is 1%; 28 ℃ of concussions were cultivated after 12 hours, getting 0.1ml culture is applied on R2A solid plate, being placed in respectively 4 ℃, 16 ℃, 20 ℃, 25 ℃, 30 ℃, 37 ℃, 42 ℃ constant incubators cultivates 48 hours, result shows: W144 bacterial strain is at 16 ℃, 20 ℃, 25 ℃, 30 ℃, well-grown in 37 ℃, and there is no significant difference; Under 4 ℃ of conditions, also can grow, 42 ℃ of conditions, can not grow.This shows that W144 bacterial strain has thermal adaptability widely, can under Various Seasonal and different year-round average temperature, carry out metabolism and growth.
Similar to above-mentioned culture condition, the same R2A substratum that adopts, utilize respectively MOPS, Tris.Cl, PBS is adjusted into 3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10.0 by the pH value of R2A liquid nutrient medium; Then inoculate fresh W144 bacterial strain in the substratum of above-mentioned different pH values, 28 ℃ of concussions are cultivated after 24 hours and are measured its OD
600result is as shown in Figure 4: W144 bacterial strain does not have significant difference at pH5 to the growth within the scope of pH9, at pH3 to pH5, and pH10 is with the interior growth that can both maintain 60-80%, show that W144 bacterial strain provided by the present invention has pH adaptability widely, be applicable to metabolism and growth under saltings and acidifying environment.
Similar to above-mentioned culture condition, adopt equally R2A substratum, in its substratum, add NaCl, make its final concentration be respectively 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%; Then inoculate fresh W144 bacterial strain in the substratum of above-mentioned different Na ion concentrations, 28 ℃ of concussions are cultivated after 24 hours and are measured its OD
600, result as shown in Figure 5.W144 bacterial strain add 4% with the condition of interior sodium ion under its growth unaffected; Add Na ion concentration and can maintain lower than 7% the growth that is not less than 50%, but when adding more than 9% sodium ion in substratum when, W144 will be difficult to maintain effective growth.This shows that, when adopting W144 to carry out organic pollutant biological restoration, in order to realize the efficient field planting of its bacterial strain and to maintain its metabolic activity, in environment, effectively salinity should be controlled in 9%.
Claims (7)
1. Sphingobacterium (Sphingobacterium sp.) bacterial strain W144, is deposited in Chinese Typical Representative culture collection center, and deposit number is: CCTCC NO:M2014151.
2. the application of Sphingobacterium claimed in claim 1 (Sphingobacterium sp.) bacterial strain W144 in degrading polycyclic aromatic hydrocarbons class organic pollutant.
3. application according to claim 2, is characterized in that: described polycyclic aromatic hydrocarbons organic pollutant is phenanthrene, biphenyl, pyrene, fluorenes, fluoranthene.
4. a method for degrading polycyclic aromatic hydrocarbons class organic pollutant, comprises to the step of inoculating Sphingobacterium claimed in claim 1 (Sphingobacterium sp.) bacterial strain W144 in contaminated sample.
5. method according to claim 4, is characterized in that: after being also included in inoculation, the temperature of sample is adjusted in to the step within the scope of 16-37 ℃.
6. method according to claim 4, is characterized in that: be also included in the step of pH value within the scope of 5-9 that regulates sample after inoculation.
7. for a microbiobacterial agent for degrading polycyclic aromatic hydrocarbons class organic pollutant, its activeconstituents is Sphingobacterium claimed in claim 1 (Sphingobacterium sp.) bacterial strain W144.
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| CN113717902A (en) * | 2021-10-11 | 2021-11-30 | 中国热带农业科学院热带作物品种资源研究所 | Sphingobacterium multivorum and application thereof |
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| CN115717116A (en) * | 2022-11-11 | 2023-02-28 | 浙江海洋大学 | Sphingobacterium HY-100C and application thereof |
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