CN104046580B - Sphingobacterium bacterial strain and its application for degrading polycyclic aromatic hydrocarbons class organic pollution - Google Patents
Sphingobacterium bacterial strain and its application for degrading polycyclic aromatic hydrocarbons class organic pollution Download PDFInfo
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- CN104046580B CN104046580B CN201410221389.2A CN201410221389A CN104046580B CN 104046580 B CN104046580 B CN 104046580B CN 201410221389 A CN201410221389 A CN 201410221389A CN 104046580 B CN104046580 B CN 104046580B
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Abstract
The invention belongs to the biological prosthetic field of organic pollution, a kind of Sphingobacterium (Sphingobacterium sp.) bacterial strain W144 is specifically disclosed, the bacterial strain is deposited in China typical culture collection center, and deposit number is：CCTCC NO：M2014151.Bacterial strain disclosed in this invention has efficiently quickly degradation capability to organic pollutions such as phenanthrene, biphenyl, pyrene, fluorenes or fluoranthene, the bacterial strain has extensive pH value, salinity and Acclimation temperature ability, are to carry out biological prosthetic excellent material to soil or environment multiring aromatic hydrocarbon organic pollution.
Description
Technical field
The invention belongs to environmental organic pollutant repairing and treating field.Specifically related to one plant organic for degrading polycyclic aromatic hydrocarbons class
The Sphingobacterium bacterial strain of pollutant and its application.
Background technology
Polycyclic aromatic hydrocarbon (Polycyclic Aromatic Hydrocarbons, PAHs) be a class contain two or two with
The organic matter of upper benzene ring structure, because of its in the environment widely distributed Yu Haiyang, lake and soil in, it is and water-soluble low, do not wave
Hair, Stability Analysis of Structures, and turn into typical persistence organic pollutant.PAHs has " carcinogenic, teratogenesis, mutagenesis " effect, to ring
Border ecological safety and human health have great potential hazard.Since eighties of last century the eighties, countries in the world Environmental Protection Agency is all
PAHs is classified as screen priority pollutants in environment.PAHs in environment is mainly derived from Petroleum Production transport, oil refining
System, organic industry, fossil energy burning, motor vehicle exhaust emission etc.；The volcanic eruption of nature, plant and microorganism itself synthesize
With the Environment release that metabolism also results in part PAHs.Its petrochina is one of main source of PAHs in environment.Contain in oil
There are the PAHs mixtures of complexity；PAHs containing about 30-40% in diesel oil；PAHs in creasote is up to 85-90%.Due to
PAHs indissolubles or insoluble in water, during the PAHs in most of environment can deposit to soil in environment migration course.In soil
PAHs is essentially from atmospheric sedimentation, sewage irrigation, oil exploitation or leakage, and trade effluent or waste discharge etc..Soil
In PAHs pollutions on the one hand will destroy ecological environment of soil, influence soil productivity, reduce crop yield；Also result in agriculture
PAHs accumulation in product, directly threatens human body health.
The biological prosthetic of soil is to utilize biological especially microorganism, is two by poisonous and hazardous pollutant mineralising in soil
Carbonoxide and water, or innocuous substance is converted into, recover the engineering technology system of the ecological functions of pollution environment.It is biological prosthetic
Through in petroleum pollution, the removal of soil organic agricultural chemicals, the environment remediation mistake such as Heavy Metal Pollution Control and nitrogen and phosphorus regulation
Found broad application in journey.The oil pipeline leakage oil in guest's Western method Leah state is removed using bioremediation technology in the U.S. in 1972
It is that first time Successful utilization biotechnology carries out soil organic pollutant reparation, subsequent EPA in 1989 is in Alaska
One at occur Oil spills bay carry out microbial bacterial agent dispensing, completed in the short time leakage oil removing.From
Between 10 years of 1991 to 1999, the U.S. completes 104 site remediations of heavily polluted area using bioremediation technology,
Wherein 40% belongs to polycyclic aromatic hydrocarbons contaminated.China Zhongyuan Oil Field area uses microorganism remediation technology, with reference to phytoremediation and thing
Physicochemical method, completes the biological prosthetic practice in more than 40 mus of oil pollution region, and its petroleum pollution degradation rate reaches
85%, the soil after arrangement recovers cultivated land function substantially.
The essence of microorganism remediation is, using the organic pollution in edaphon selectivity metabolism soil, to be converted
The process of other carbon-based macromoleculars and carbon dioxide for cell.Therefore implement and set up the microorganism of a set of soil organic pollutant
The core of renovation technique is fast accretion rate, the screening and identification of the strong contaminant degradation bacterium of environmental suitability.Because biology is pacified
Full the reason for, genetic engineering bacterium is difficult to the microorganism remediation that large area applies to true environment soil, and this is caused from contaminated soil
The middle native bacterium for obtaining that separates turns into the candidate material of optimal microorganism remediation microbial inoculum.Separated from soil simultaneously and obtained
Microorganism also has the features such as colonization ability is strong, and environmental suitability is wide, is to carry out biological prosthetic core carrier and pollutant drop
The direct executor of solution.Therefore, screen and identify that the microbial resources with organic pollutant degradation ability are to carry out ring
The biological prosthetic premise of border organic pollution and basis.
The content of the invention
It is an object of the invention to provide one plant be used for degrading polycyclic aromatic hydrocarbons class organic pollution Sphingobacterium bacterial strain and
Its application.
Applicant separates from oil-polluted soils and obtains one plant of bacterial strain for being capable of fast degradation polyaromatic hydrocarbon pollutant,
Identified by Physiology and biochemistry and ribosomes 16S rDNA gene sequencings are accredited as Sphingobacterium
(Sphingobacterium sp.) bacterial strain W144.The bacterial strain delivers China typical culture collection on April 24th, 2014
Center (CCTCC) preservation, deposit number is：CCTCC NO：M2014151.
The bacterial strain can efficient degradation multiring aromatic hydrocarbon organic pollution, especially phenanthrene, biphenyl, pyrene, fluorenes or fluoranthene etc. have
Organic pollutants.
The present invention further provides a kind of method of degrading polycyclic aromatic hydrocarbons class organic pollution, the method is included to contaminated
Sample in the step of be inoculated with described Sphingobacterium (Sphingobacterium sp.) bacterial strain W144.
Preferably, be additionally included in inoculation after by the temperature adjustment of sample in the range of 16-37 DEG C the step of.
It is further preferred that the step of adjusting the pH value of sample after being additionally included in inoculation in the range of 5-9.
The present invention further provides a kind of microbial bacterial agent for degrading polycyclic aromatic hydrocarbons class organic pollution, its work
Property composition be described Sphingobacterium (Sphingobacterium sp.) bacterial strain W144.
Bacterial strain provided by the present invention has efficiently quickly degraded to organic pollutions such as phenanthrene, biphenyl, pyrene, fluorenes or fluoranthene
Ability.The bacterial strain also has extensive pH value, salinity and Acclimation temperature ability, is to soil or the organic dirt of environment multiring aromatic hydrocarbon
Dye thing carries out biological prosthetic outstanding microbial material.
More detailed technical scheme is shown in《Specific embodiment》It is described.
Brief description of the drawings
Fig. 1 is the Gram's staining micro-imaging photo of bacterial strain W144.
Fig. 2 is degradation capabilities of the bacterial strain W144 to different polycyclic aromatic hydrocarbon pollutants.In figure NC be negative control,
Phenanthrene is that naphthalene, Fluorene are that fluorenes, Fluoranthrene are that fluoranthene, Biphenyl are for luxuriant and rich with fragrance, naphthalene
Biphenyl, Pyrene are pyrene, Benzo (a) pyrene are BaP.
Fig. 3 is growth curves and luxuriant and rich with fragrance degradation curve of the bacterial strain W144 under the conditions of with phenanthrene for sole carbon source.
Fig. 4 is sensitivity curves of the bacterial strain W144 to pH value.
Fig. 5 is sensitivity curves of the bacterial strain W144 to Na ion concentration.
Specific embodiment
The present invention is described in detail with reference to embodiments.It should be noted that embodiments of the invention are only limited
Illustrated in for the present invention, without restriction effect.Involved relevant screening technique in embodiment, buffer and
Common culture medium formula etc. can refer to Zhao Bin, He Shaojiang chief editors'《Microbiology Experiment》And《Molecular Cloning:A Laboratory guide》Institute
Description content (referring to J. Pehanorm Brookers etc., 2002, Molecular Cloning:A Laboratory guide, the third edition, (the translating), science such as golden winter wild goose
Publishing house, Beijing).Other involved various experimental implementations in the present invention, are the ordinary skill in the art, without spy in text
Bright part is not mentionleted alone, one of ordinary skill in the art is referred to various common tool books, section before the present patent application day
Specification, handbook of skill document or correlation etc. are practiced.
The separation identification of the W144 bacterial strains of embodiment 1
It is screening soil to use from the long-term petroleum-polluted soil of Hubei Qianjiang Jianghan Oil-field, and specific screening scheme is such as
Under：
1) 10g soil is taken, is added in the minimal medium MSM of not carbonaceous sources, be subsequently adding phenanthrene
(Phenanthrene) final concentration of 100mg/L, is made；28 DEG C of concussion and cultivates 5 days, then it is connected to corresponding fresh with 5% amount of connecing
In culture medium；Continuous switching 5 times；
2) the enrichment culture liquid of 0.1ml is taken, is applied to containing on luxuriant and rich with fragrance MSM solid plates, 28 DEG C of static gas wave refrigerators are after 5 days,
The bacterium colony grown on flat board is transferred to containing on luxuriant and rich with fragrance flat board, growth is cultivated in continuation；Then the fireballing bacterium of growth selection
Drop into the purifying of row plate streaking；
3) bacterium colony of picking stabilization growth, is inoculated into the luxuriant and rich with fragrance MSM culture mediums containing 100mg/L, 28 DEG C of concussion and cultivates 5
My god, obtain bacterial strain of the invention.
Morphology and molecular biology identification further have been carried out to bacterial strain.
First, the morphological feature to bacterial strain has carried out preliminary observation, as a result shows, the bacterial strain is Gram-negative
Bacterium, spherical (Fig. 1).
Meanwhile, inventor separates the genomic DNA for obtaining bacterial strain, then with the universal primer of bacterial 16 S rRNA genes
27F and 1492R primer pairs enter performing PCR amplification；The pcr amplification product of acquisition is sequenced, its sequence such as sequence table SEQ ID
NO：Shown in 1.Then, using NCBI nucleic acid databases, BLAST analyses have been carried out according to bacterial strain 16S rRNA gene orders, as a result
Show the 16S rRNA gene orders and Sphingobacterium Sphingobacterium siyangense strain SY1 of bacterial strain,
The 16S rRNA gene orders of many plants of bacteriums such as Sphingobacterium sp.IN42 all have 99% similitude, with reference to leather
The Strain Designation is Sphingobacterium (Sphingobacterium sp.) bacterial strain by blue Albert'stain Albert and morphological observation, inventor
W144。
Degraded feature of the W144 bacterial strains of embodiment 2 to polycyclic aromatic hydrocarbon
Inventor is studied common degrading polycyclic aromatic hydrocarbons ability W144 bacterial strains.Trained with the MSM for not containing carbon source
Support based on base, different polycyclic aromatic hydrocarbons are added respectively as sole carbon source, make its final concentration of 100mg/L；Then with 1%
Inoculum concentration W144 culture of the inoculation in exponential phase, was placed in 28 DEG C of concussion and cultivates after 5 days, determined culture in OD600's
Absorbance.It is negative control with the culture medium without any carbon source.Due to being not added with appointing in addition to polycyclic aromatic hydrocarbon in culture medium
What carbon source, therefore the speed of growth and biomass of bacterial strain are the utilization and degradation capability for embodying bacterial strain to polycyclic aromatic hydrocarbon.
As shown in Fig. 2 W144 has maximum Utilization ability to phenanthrene, there is stronger degraded energy to biphenyl, pyrene, fluorenes and fluoranthene
Power；Poor Utilization ability is shown to BaP, naphthalene.This shows that W144 bacterial strains have degraded energy to various polycyclic aromatic hydrocarbons simultaneously
Power.
The W144 bacterial strains of embodiment 3 are to luxuriant and rich with fragrance degraded feature
In order to further analyze degraded feature of the W144 bacterial strains provided by the present invention to polycyclic aromatic hydrocarbon, inventor is with phenanthrene
Sole carbon source, the speed of growth and degradation speed to W144 bacterial strains are tested.By the W144 bacterial strains in exponential phase
It is inoculated into the luxuriant and rich with fragrance MSM culture mediums as sole carbon source with 500mg/L with 1% inoculum concentration, is placed in 28 DEG C of concussion and cultivates.Meanwhile,
Every 12 hours determine the OD of a subculture600Value and the luxuriant and rich with fragrance concentration of residual.
As shown in figure 3, W144 bacterium start just possess luxuriant and rich with fragrance degradation capability since inoculation, and after 12h degradation capability with
Strain growth all increases sharply, also, with the reduction of luxuriant and rich with fragrance concentration is remained in culture, strain growth progresses into plateau.
When W144 bacterial strains 84 hours are inoculated with, the phenanthrene in culture has 95% to be degraded.This shows that W144 bacterial strains have and rapidly and efficiently drops
The luxuriant and rich with fragrance ability of solution organic pollution, can completely drop the phenanthrene of the 500mg/L in fluid nutrient medium within the time of five days or so
Solution.
The environment meeting market's demand of the W144 bacterial strains of embodiment 4
W144 bacterial strains are tested the adaptability of temperature, pH value and salt ionic concentration.
Using R2A culture mediums, in W144 bacterial strains of the inoculation in exponential phase to fresh R2A culture mediums, it is inoculated with dense
Spend is 1%；28 DEG C of concussion and cultivates take 0.1ml cultures and are applied on R2A solid plates after 12 hours, be respectively placed in 4 DEG C, 16
DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 37 DEG C, cultivate 48 hours in 42 DEG C of constant incubators, as a result show：W144 bacterial strains are in 16 DEG C, 20
DEG C, 25 DEG C, 30 DEG C, well-grown in 37 DEG C, and be not significantly different from；Can also be grown under the conditions of 4 DEG C, can not in 42 DEG C of conditions
Growth.This shows that W144 bacterial strains have extensive thermal adaptability, can be carried out under Various Seasonal and different year-round average temperatures
Growth and metabolism.
It is similar to above-mentioned condition of culture, it is same to use R2A culture mediums, it is utilized respectively MOPS, Tris.Cl, PBS are by R2A liquid
The pH value of body culture medium is adjusted to 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；Then fresh W144 bacterial strains are inoculated with the culture medium of above-mentioned different pH value, 28 DEG C of concussion and cultivates are determined after 24 hours
Its OD600, as a result as shown in Figure 4：Growth of the W144 bacterial strains in the range of pH5 to pH9 is not significantly different from, in pH3 to pH5, with
And the growth of 60-80% can be maintained within pH10, show that there is W144 bacterial strains provided by the present invention extensive pH to adapt to
Property, it is adapted to growth and metabolism under salt-soda soil and acidified environment.
It is similar to above-mentioned condition of culture, it is same to use R2A culture mediums, NaCl is added in its culture medium, make its end
Concentration is respectively 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%；Then fresh W144 bacterial strains are inoculated with to upper
State in the culture medium of different Na ion concentrations, 28 DEG C of concussion and cultivates determine its OD after 24 hours600, as a result as shown in Figure 5.W144
Its growth is unaffected under conditions of sodium ion of the bacterial strain within addition 4%；Addition Na ion concentration is able to maintain that less than 7%
50% growth is not less than, but when the sodium ion of addition more than 9% in the medium, W144 will be difficult to remain effective
Growth.This show when using W144 carry out organic pollution it is biological prosthetic when, in order to realize the efficient field planting of its bacterial strain
With its metabolic activity of maintenance, effective salinity should be controlled within 9% in environment.
Claims (1)
1. a kind of method of degrading polycyclic aromatic hydrocarbons class organic pollution, including to being inoculated with Sphingobacterium in contaminated sample
The step of (Sphingobacterium sp.) bacterial strain W144, the Sphingobacterium bacterial strain W144 is deposited in Chinese Typical Representative training
Thing collection is supported, deposit number is：CCTCC NO：M2014151, the multiring aromatic hydrocarbon organic pollution be phenanthrene, biphenyl,
Pyrene, fluorenes, fluoranthene, be additionally included in inoculation after by the temperature adjustment of sample in the range of 16-37 DEG C and regulation sample pH value exist
The step of 5-9.
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| CN112852660A (en) * | 2021-01-05 | 2021-05-28 | 西南科技大学 | Microbial agent for repairing coking soil and application thereof |
| CN113583899B (en) * | 2021-07-19 | 2022-08-23 | 河南师范大学 | Sphingosine strain JT-M9-H as polycyclic aromatic hydrocarbon degrading strain |
| CN113717902B (en) * | 2021-10-11 | 2022-10-04 | 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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| CN102120976A (en) * | 2010-12-21 | 2011-07-13 | 中国农业大学 | Sphingobium yanoikuyae and application thereof in degrading polycyclic aromatic hydrocarbon |
| CN102277312A (en) * | 2011-01-10 | 2011-12-14 | 辽宁石油化工大学 | Strain for degrading polycyclic aromatic hydrocarbons at low temperature and application in bioremediation of groundwater in petroleum hydrocarbon polluted places thereof |
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