CN104745515A - Acinetobacter sp. for degrading polycyclic aromatic hydrocarbon and application of acinetobacter sp. - Google Patents
Acinetobacter sp. for degrading polycyclic aromatic hydrocarbon and application of acinetobacter sp. Download PDFInfo
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- CN104745515A CN104745515A CN201510159230.7A CN201510159230A CN104745515A CN 104745515 A CN104745515 A CN 104745515A CN 201510159230 A CN201510159230 A CN 201510159230A CN 104745515 A CN104745515 A CN 104745515A
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- Prior art keywords
- polycyclic aromatic
- aromatic hydrocarbons
- acinetobacter
- acinetobacter calcoaceticus
- degrading
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
Abstract
The invention belongs to the biotechnical field and discloses acinetobacter sp. W145. The strain is preserved in CCTCC, wherein the preservation number is CCTCC NO: M2014192. The strain disclosed by the invention has an efficient and rapid degradation capacity for phenanthrene, fluoranthene, pyrene and naphthalene and is an excellent material for biologically repairing soil or environments.
Description
Technical field
The invention belongs to biological technical field, relate to a strain acinetobacter calcoaceticus bacterial strain, the invention still further relates to the application of described bacterial strain in degrading polycyclic aromatic hydrocarbons and a kind of method of degrading polycyclic aromatic hydrocarbons.
Background technology
Polycyclic aromatic compound (PAHs) is a class by two or more phenyl ring with linear array, curvedly connects or the different modes such as bunch poly-arranges the organic compound formed.According to the size of PAHs molecular weight, polycyclic aromatic hydrocarbons can be divided into the PAHs (as fluoranthene, pyrene, benzopyrene etc.) of low-molecular-weight PAHs (as naphthalene, phenanthrene, fluorenes etc.) and high molecular.The source of polycyclic aromatic hydrocarbons mainly comprises two aspects: the activity being on the one hand nature self, as hill fire, animals and plants remains rot, the formation of oil coal, volcanic eruption etc.; Another one aspect is mankind's activity, as the discharge etc. of the burning of the incomplete combustion of fossil, municipal solid wastes, oil production, processing transport and Chemical Manufacture and vehicle exhaust.Polycyclic aromatic hydrocarbons contaminated most to ambient contribution of mankind's activity is generally acknowledged main pollution source.PAHs is extensively present in air, the settling of water body and soil and rivers and lakes thereof, along with the continuous aggravation of the continuous growth of population in the world quantity and urbanization, process of industrialization, the control of the polycyclic aromatic hydrocarbons in environment for human survival and repair and become very urgent.
Polycyclic aromatic hydrocarbons is a class environmental persistence pollutent.Due to the chemical structure of its uniqueness, polycyclic aromatic hydrocarbons has low water solubility, high hydrophobicity and extensive chemical stability, is difficult to be utilized by organism at occurring in nature.Meanwhile, polycyclic aromatic hydrocarbons is also had carcinogenic, teratogenesis, mutagenic " three cause " effect environmental pollutant by what extensively confirm.Due to the chemical stability of its uniqueness, traditional physico-chemical process (volatilization, photodissociation, chemical oxidation, electricity are repaired and the method such as coagulation-flocculation) is difficult to remove, and processing cost is high simultaneously, easily produces secondary pollution.
Microorganism remediation is the process utilizing microorganism organic pollutant to be converted into carbon back macromole and carbonic acid gas, the core of the microorganism remediation technique implemented and set up a set of organic pollutant is that searching accretion rate is fast, the contaminant degradation bacterium that environmental compatibility is strong, directly from environment, enrichment isolation microorganism is the most effective technique means of one, it is strong that it has colonization ability, the features such as environmental compatibility is wide.Current people have screened and have obtained multiple polycyclic aromatic hydrocarbon-degrading bacteria from all kinds of ecotope, comprise pseudomonas (Pseudomonas), rhodococcus (Rhodococcus), mycobacterium (Mycobacterium), genus bacillus (Bacillus), Sphingol single-cell (Sphingomonas), Aeromonas (Aeromonas), Bai Yelin kirschner bacterium (Beijerinckia), nocardia (Nocardioides), to unlink bacterium (Cycloclasticus), Burkholderia (Burkholderia), the Pseudomonas of comamonas (Polaromonas) and some thermophilic anaerobics is as addicted to oily bacterium (Neptunomonas), Janus bacterium (Janibacter), Nocardia bacteria (Nocardia), sex change bacterium (Deltaproteobacteria) and Alcaligenes (Alcaligenes) etc.
Summary of the invention
The object of this invention is to provide the acinetobacter calcoaceticus of a strain for degrading polycyclic aromatic hydrocarbons.
Applicant be separated from the soil of petroleum pollution obtain a strain can the bacterial strain of fast degradation polycyclic aromatic hydrocarbons, acinetobacter (Acinetobacter sp.) is accredited as, called after W145 by Physiology and biochemistry qualification and rrna 16S rDNA gene sequencing.This bacterial strain delivers China typical culture collection center (CCTCC) preservation being positioned at Wuhan City, Hubei Province Wuhan University on May 9th, 2014, deposit number is: CCTCC NO:M2014192.
This bacterial strain can efficient degradation polycyclic aromatic hydrocarbons, especially phenanthrene, pyrene, fluoranthene, naphthalene.This bacterial strain is in pH5.5 ~ 9.0 simultaneously, and salt ionic concentration≤5% time, can grow fast, and having Salt And Alkali Tolerance and potential of hydrogen adaptive capacity to environment widely, is the outstanding biomaterial of degrading to the polycyclic aromatic hydrocarbons in soil, sewage, mud.
When degrading the polycyclic aromatic hydrocarbons in soil, sewage, mud, inoculate 10 by every 1Kg pollutent
5~ 10
7the acinetobacter calcoaceticus W145 of CFU, can reach best degradation effect.
Before inoculation, regulate the pH of pollutent to be 5.5 ~ 9.0, and salt ionic concentration≤5%, preferably degradation effect can be reached.
Before inoculation, regulate the pH of pollutent to be 7.2, and salt ionic concentration is 2%, best degradation effect can be reached.
More detailed technical scheme is shown in described in " specific embodiments ".
Accompanying drawing explanation
Fig. 1 is the degradation capability of acinetobacter calcoaceticus W145 to different polycyclic aromatic hydrocarbons.
The growth that Fig. 2 is acinetobacter calcoaceticus W145 under the luxuriant and rich with fragrance concentration of 250mg/L and degradation curve.
Fig. 3 is the susceptibility histogram of acinetobacter calcoaceticus W145 to pH value.
Fig. 4 is the susceptibility histogram of acinetobacter calcoaceticus W145 to Na ion concentration.
Specific embodiments
Below in conjunction with embodiment, the present invention is described in detail.Should be noted that embodiments of the invention are only limitted to be described for the present invention, and there is no restriction.Other various operation involved in the present invention, be the ordinary skill in the art, the part be not particularly illustrated 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 acinetobacter calcoaceticus W145
Adopt the soil from Shengli Oil Field is petroleum-polluted to be for a long time screening soil, concrete screening scheme is as follows:
1) get 10g soil, join 100ml and contain in the minimal medium MSM of 250mg/L phenanthrene, 28 DEG C of concussion cultivations 7 days, get 10ml culture and be transferred to another 100ml and contain in the minimal medium MSM of 250mg/L phenanthrene; Continuous switching 5 times, obtains the nutrient solution of enriched microorganism.
2) 0.1ml step 1 is got) nutrient solution that obtains, be applied on the MSM solid plate containing 250mg/L phenanthrene, 28 DEG C of static gas wave refrigerator are after 5 days, the bacterium colony that flat board grows is transferred on the MSM solid plate containing 250mg/L phenanthrene, continue incubation growth, until the bacterium colony that dull and stereotyped upper appearance is single.
3) single bacterium colony picking MSM solid plate grown out, is inoculated in the MSM liquid nutrient medium containing 250mg/L phenanthrene, and 28 DEG C shake cultivation 5 days, obtain bacterial strain of the present invention.
Further morphology and molecular biology identification are carried out to bacterial strain.
First, carried out preliminary observation to the morphological feature of bacterial strain, result shows, this bacterial strain is Gram-negative bacteria, shaft-like.
Meanwhile, contriver is separated the genomic dna obtaining bacterial strain, then carries out pcr amplification with the universal primer 27F of bacterial 16 S rRNA gene and 1492R primer pair DNA; Checked order by the pcr amplification product of acquisition, its sequence is as shown in sequence table SEQ ID NO:1.Then, utilize NCBI nucleic acid database, BLAST analysis has been carried out according to bacterial strain 16S rRNA gene order, result show the 16SrRNA gene order of bacterial strain and the homology of acinetobacter up to 99% similarity, in conjunction with gramstaining and morphological observation, this Strain Designation is acinetobacter calcoaceticus (Acinetobacter sp.) W145 by contriver.
Embodiment 2 acinetobacter calcoaceticus W145 verifies the degradation capability of polycyclic aromatic hydrocarbons
Contriver is studied common degrading polycyclic aromatic hydrocarbons ability W145 bacterial strain.By 100ml not containing based on the MSM substratum of carbon source, add different polycyclic aromatic hydrocarbonss respectively as sole carbon source; Then 10 are accessed
6the acinetobacter calcoaceticus W145 of CFU, is placed in 28 DEG C of concussion cultivations after 5 days, measures culture at OD
600absorbance.Not add the substratum of any carbon source for negative contrast.Owing to not adding any carbon source except polycyclic aromatic hydrocarbons in substratum, therefore namely the speed of growth of bacterial strain and biomass embody bacterial strain to the degraded of polycyclic aromatic hydrocarbons and Utilization ability.
As shown in Figure 1, acinetobacter calcoaceticus W145 has stronger degradation capability to fluoranthene, pyrene, phenanthrene, to naphthalene, also there is certain degradation capability, and degradation capability is not almost had to other polycyclic aromatic hydrocarbons such as biphenyl, fluorenes, benzopyrene etc., show that the degraded of acinetobacter calcoaceticus W145 to polycyclic aromatic hydrocarbons has certain specificity.
Embodiment 3 acinetobacter calcoaceticus W145 is to the degradation efficiency of polycyclic aromatic hydrocarbons
In order to analyze the degradation efficiency of acinetobacter calcoaceticus W145 to polycyclic aromatic hydrocarbons further, take phenanthrene as sole carbon source, the speed of growth of acinetobacter calcoaceticus W145 and degradation speed are tested.The acinetobacter calcoaceticus W14 being in logarithmic phase is inoculated into 250mg/L luxuriant and rich with fragrance in the MSM substratum of sole carbon source with 1% inoculum size, is placed in 28 DEG C of concussions and cultivates.Meanwhile, within every 12 hours, the OD of a subculture is measured
600value and residual luxuriant and rich with fragrance concentration.
As shown in Figure 2, acinetobacter calcoaceticus W145 has just possessed luxuriant and rich with fragrance degradation capability from inoculation, and degradation capability and strain growth all increase sharply after 12h, and along with the minimizing of luxuriant and rich with fragrance concentration residual in culture, strain growth progresses into plateau.In inoculation after 100 hours, the phenanthrene in culture is almost degraded completely.This shows that acinetobacter calcoaceticus W145 has the ability of rapidly and efficiently degrading polycyclic aromatic hydrocarbons, can be degradable in five days by the phenanthrene of the 250mg/L in liquid nutrient medium.
The environmental adaptation feature of embodiment 4 acinetobacter calcoaceticus W145
Acinetobacter calcoaceticus W145 is tested the adaptability of pH value and salt ionic concentration.
Adopt R2A substratum, utilize MES (2-(N-morpholine) ethyl sulfonic acid) respectively, TAPSO (3-[N-trihydroxymethylaminomethane]-2-hydroxy-propanesulfonic acid), the pH value of R2A liquid nutrient medium is adjusted to 5.0,5.5,6.0,6.5,7.0,7.2,8.0,8.5,9.0 by Tris-HCl; Then inoculate fresh acinetobacter calcoaceticus W145 in the substratum of above-mentioned different pH value, 28 DEG C of concussion cultivations measured its OD after 24 hours
600, result as shown in Figure 3: the growth of acinetobacter calcoaceticus W145 in pH6.0 to pH9.0 scope, pH lower than 5.5 time there is no obvious growth tendency.This shows that acinetobacter calcoaceticus W145 provided by the present invention has pH adaptability widely, has the potentiality of repairing acid and alkaline soil.
Similar to above-mentioned culture condition, same employing R2A substratum, adds NaCl, makes its final concentration be respectively 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% in its substratum; Then inoculate fresh acinetobacter calcoaceticus W145 in the substratum of above-mentioned different Na ion concentration, 28 DEG C of concussion cultivations measured its OD after 24 hours
600, result as shown in Figure 4.Under the condition of the sodium ion of acinetobacter calcoaceticus W145 within interpolation 4%, its growth is unaffected; Interpolation Na ion concentration reaches 5% and can maintain the growth being not less than 50%, and in time adding the sodium ion of more than 6% in the medium, W145 still has the trend of growth.This shows in time adopting acinetobacter calcoaceticus W145 to carry out organic pollutant biological restoration, and in order to realize the efficient field planting of its bacterial strain and maintain its metabolic activity, the salt ionic concentration in pollutent should control within 5%.
The degraded of embodiment 5 polycyclic aromatic hydrocarbons organic pollutant
Fetch earth earth or sewage or mud, and first regulate pH to be 7.2, salt ion weight concentration is 2%, then by inoculation 10 in every 1Kg soil or sewage or mud
5~ 10
7the acinetobacter calcoaceticus W145 of CFU, mixing.
Claims (6)
1. acinetobacter calcoaceticus (Acinetobacter sp.) W145 of a strain degrading polycyclic aromatic hydrocarbons, be deposited in China typical culture collection center, deposit number is: M2014192, and described polycyclic aromatic hydrocarbons is phenanthrene, fluoranthene, pyrene and naphthalene.
2. the application of acinetobacter calcoaceticus W145 according to claim 1 in degrading polycyclic aromatic hydrocarbons.
3. a method for degrading polycyclic aromatic hydrocarbons, is characterized in that: comprise to containing the step inoculating acinetobacter calcoaceticus W145 described in claim 1 in the pollutent of polycyclic aromatic hydrocarbons.
4. the method for degrading polycyclic aromatic hydrocarbons according to claim 3, is characterized in that: contain to every 1Kg in the pollutent of polycyclic aromatic hydrocarbons and inoculate 10
5~ 10
7the acinetobacter calcoaceticus W145 of CFU, described pollutent is soil or sewage or mud.
5. the method for the degrading polycyclic aromatic hydrocarbons according to claim 3 or 4, is characterized in that: regulate the pH of pollutent to be 5.5 ~ 9.0 before being also included in inoculation, and the step of salt ionic concentration≤5%.
6. the method for degrading polycyclic aromatic hydrocarbons according to claim 5, is characterized in that: regulate the pH of pollutent to be 7.2 before being also included in inoculation, and salt ionic concentration is the step of 2%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105695360A (en) * | 2016-03-18 | 2016-06-22 | 中国科学院广州地球化学研究所 | Phenanthrene-degrading strain Acinetobacter tandoii LJ-5 and application thereof |
| CN107306532A (en) * | 2017-06-13 | 2017-11-03 | 南京农业大学 | A kind of method for removing USEPA PAHs in plant simultaneously using compound PAHs degradation bacterias |
| CN113388542A (en) * | 2021-06-09 | 2021-09-14 | 中国科学院沈阳应用生态研究所 | Polyhalogenated aromatic hydrocarbon degrading bacteria and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695360A (en) * | 2016-03-18 | 2016-06-22 | 中国科学院广州地球化学研究所 | Phenanthrene-degrading strain Acinetobacter tandoii LJ-5 and application thereof |
| CN107306532A (en) * | 2017-06-13 | 2017-11-03 | 南京农业大学 | A kind of method for removing USEPA PAHs in plant simultaneously using compound PAHs degradation bacterias |
| CN107306532B (en) * | 2017-06-13 | 2021-09-07 | 南京农业大学 | Method for simultaneously removing USEPA PAHs in plant body by using composite PAHs degrading bacteria |
| CN113388542A (en) * | 2021-06-09 | 2021-09-14 | 中国科学院沈阳应用生态研究所 | Polyhalogenated aromatic hydrocarbon degrading bacteria and application thereof |
| CN113388542B (en) * | 2021-06-09 | 2022-05-31 | 中国科学院沈阳应用生态研究所 | Polyhalogenated aromatic hydrocarbon degrading bacteria and application thereof |
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