CN101603022B - Pseudomonas stutzeri strain and application thereof in degrading polycyclic aromatic hydrocarbon with high molecular weight - Google Patents
Pseudomonas stutzeri strain and application thereof in degrading polycyclic aromatic hydrocarbon with high molecular weight Download PDFInfo
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- CN101603022B CN101603022B CN 200910303045 CN200910303045A CN101603022B CN 101603022 B CN101603022 B CN 101603022B CN 200910303045 CN200910303045 CN 200910303045 CN 200910303045 A CN200910303045 A CN 200910303045A CN 101603022 B CN101603022 B CN 101603022B
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Abstract
The invention relates to a novel strain of bacteria and application thereof in degrading polycyclic aromatic hydrocarbon with high molecular weight. The strain is identified and named as Pseudomonas stutzeri MT-5 strain which has bacteriological characteristic of Gram-negative, has cells in a straight or bent rod shape but not spiral shape and not in a chain, has the size of 0.60-0.8*1.4-2.0mu m,has polar flagellum, can move, does not form spore or have sheath or protrusion, has no poly beta hydroxybutyrate observed, has round bacterial colony, not irregular edge, rough surface and irregularbump shape, and is white and opaque. The Pseudomonas stutzeri MT-5 strain can be applied to degrading PAHs, BAP/BGP with high molecular weight. The Pseudomonas stutzeri MT-5 strain has the degradation efficiency of 65 percent on the BGP and 59.6 percent on the BAP in 168h, and has the degradation capacity higher than related bacteria for degrading the BAP/BGP reported at home and abroad.
Description
Technical field
The present invention relates to the application of the new bacterial strain of a kind of bacterium and this bacterial strain at the degraded macromolecular polycyclic aromatic hydrocarbons.
Background technology
The microorganism that polycyclic aromatic hydrocarbons (PAHs) pollutes is repaired and is meant the PAHs pollutent that utilizes in the PAHs degradation bacteria degraded environment or PAHs is converted into the process of innoxious substance, has efficient low-consume, advantages of environment protection enjoys people to pay close attention to.The core of this technology is to obtain the microbial strains of efficient degradation PAHs, because the good and bad repair time and the repairing effect of directly influencing of bacterial classification.So far, in numerous separated and PAHs degradation bacteria of identifying, most of bacterial strains lower molecular weight PAHs that can only degrade, and the bacterial classification of tool degraded Fourth Ring and the above high molecular PAHs ability in Fourth Ring is few.The influence that lacked by high molecular PAHs degradation bacteria, existing microorganism recovery technique often is confined to remove the lower molecular weight PAHs in the environment aspect repairing effect, and it is extremely low that high-molecular weight PAHs is removed efficient.Therefore, further research and develop the germ plasm resource of high molecular PAHs efficient degrading bacteria, significant to the development that promotes this technology.
Summary of the invention
For solving the low problem of degradation rate of high molecular PAHs, new bacterial strain MT-5 (Pseudomonas stutzeri) MT-5 that a kind of Pseudomonas stutzeri is provided of the present invention, this bacterial strain is sole carbon source and can carries out efficient degradation to it as naphthalene, phenanthrene, BAP and BGP etc. with high molecular PAHs.This bacterial strain is preserved in " Chinese typical culture collection center (CCTCC) " on May 8th, 2009, and preserving number is CCTCC M 209101.
This bacterial strain is derived from and is subjected to for a long time to obtain by specific training method and isolation technique in the harbour bed mud that PAHs pollutes.
Bacteriology characteristic:
Gram-negative; Cell presents straight or crooked shaft-like, but is not spirrillum, not chaining; Size 0.6～0.8 * 1.4～2.0 μ m; Polar flagella is arranged, can move; Do not form gemma, no sheath or thrust; Do not observe poly--β hydroxybutyric acid particle.
The bacterium colony characteristic:
Circular, the edge is irregular, surface irregularity, projecting shape are irregular, white, opaque.
The present invention also provides Pseudomonas stutzeri MT-5 bacterial strain at degraded macromolecular amount PAHs, and the application among the BAP/BGP is with the nutrient solution effect and the solution that contains BAP or BGP of (Pseudomonas stutzeri) MT-5 bacterial strain (CCTCC M 209101).
Pseudomonas stutzeri MT-5 of the present invention degradation efficiency to BGP in 168h reaches 65%, and the degradation efficiency of BAP is reached 59.6%, and degradation capability is higher than both at home and abroad relevant bacterium to the report of BAP/BGP degraded.It is reported that existing bacterium is 1.4%-40% to the degradation rate of BAP, degradation time is 168h-70 days; Degradation efficiency to BGP is 0-37%, and degradation time is 60h-7 month.
Description of drawings
The colony characteristics of Fig. 1 MT-5 (reverse side scan image)
The colony characteristics of Fig. 2 bacterial strain MT-5 (front scan image)
Embodiment
The preparation of embodiment 1 Pseudomonas stutzeri MT-5
SMS nutrient solution composition (g/L) is: (NH
4)
2SO
40.5, NaNO
30.5, CaCl
20.02, MgSO
40.2, KH
2PO
41.0, NaH
2PO
4.H
2O 1.0, and pH=7.5, chemical reagent are analytical pure available from Guangzhou Chemical Reagent Factory.
(1) enrichment culture
Utilize in the therefrom big harbour bed mud of sediment sampler and take a sample, and bed mud is placed aseptic sampling bottle, deliver to the laboratory.Preserve standby down for 4 ℃.Get bed mud sample 10g and be mixed with 20% suspension, the inoculum size with 10% is inoculated in the SMS liquid nutrient medium that contains phenanthrene (0.05g/L).30 ℃ of constant temperature, shaking table was cultivated seven days.Get 1% bacterium liquid and insert in the same substratum, continue shaking table and cultivate, repeated multiple times like this is up to the enrichment culture thing that obtains to have remarkable degradation property.
(2) strains separation
After obtaining that the enrichment culture thing of the luxuriant and rich with fragrance performance of remarkable degraded is arranged, utilize dull and stereotyped culture technique, culture is inoculated in the SMS plate culture medium that contains the 0.005g/L phenanthrene, cultivated 7-12 days under 30 ℃ of conditions.
(3) bacterial strain purifying
After treating that bacterium colony is grown well, on flat board, choose the bacterium colony of different shape feature, be forwarded to again in the SMS nutrient solution that contains the 0.005g/L phenanthrene, whether have the luxuriant and rich with fragrance ability of degraded to verify it.After treating that nutrient solution becomes muddiness, separation, purifying are three times again, at last the purifying bacterial strain are lined on the solid medium, preserve under 4 ℃ of conditions, are used for further screening.
(4) screening of BAP/BGP degradation bacteria strains
With the bacterial strain behind the aforementioned purifying, be forwarded in the nutrient solution that contains BAP/BGP (5g/L), further screening has the microorganism strains of degrading polycyclic aromatic hydrocarbon with high molecular weight.After treating that nutrient solution becomes muddiness, separation, purifying line the purifying bacterial strain on the solid medium at last again, and 4 ℃ preservation is standby down, as the aimed strain of further research.
(5) evaluation of Pseudomonas stutzeri MT-5
1, morphologic observation and physiological and biochemical test
According to the standard of division bacteria handbook, degradation bacteria strains is fallen in the polymer polycyclic aromatic hydrocarbons that is separated to carried out preliminary morphologic observation, gramstaining and conventional physiological and biochemical test.The colony morphology characteristic of bacterial strain MT-5 as shown in Figure 1 and Figure 2, physiological and biochemical test the results are shown in Table 1.According to " common bacteria system identification handbook ", determine that tentatively MT-5 belongs to the Rhodopseudomonas bacterium.
The physicochemical characteristics of table 1MT-5 bacterial strain
| The physics and chemistry test | Feature | The physics and chemistry test | Feature |
| Individual morphology | Rod-short | Glucose | + |
| Gramstaining | - | Gluconic acid sodium salt | + |
| Pod membrane has or not | - | Melampyrum | + |
| The physics and chemistry test | Feature | The physics and chemistry test | Feature |
| Gemma has or not | - | Wood sugar | + |
| Mobility | + | Sorbose | + |
| Oxydase | + | Hexanodioic acid | - |
| Catalase | + | Lactose | + |
| Nitrate reduction | + | Raffinose | + |
| Utilization of carbon source: Trisodium Citrate | + | Sodium tartrate | + |
| Fructose | + | Saligenin | + |
| Inositol | + |
2,16srRNA order-checking
(1) template DNA extracts
Press the operation steps of TIANamp Bacteria DNA Kit bacterial genomes DNA extraction test kit (centrifugal column type) and extract bacterial genomes DNA.
(2) primer
Adopt the 16S rDNA universal primer of bacterium: upstream primer F27 (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and (sequence 2) and downstream primer R1522 (5 '-AAGGAGGTGATCCAGCCGCA-3 ') (sequence 3).
(3) pcr amplification
Pcr amplification system cumulative volume is 50 μ L: ultrapure water 37.5 μ L, 10 * PCR Buffer, 5 μ L, 2.5mmol/L dNTP4 μ L, 10 μ mol/L upstream primers, 1 μ L, 10 μ mol/L downstream primers, 1 μ L, 5U/ μ L Taq DNA Polymerase0.5 μ L, template DNA are diluted to proper concn and (get 1 μ L behind the 0.01ng～1ng).Replace template DNA to carry out blank test with 1 μ L ultrapure water in addition.During the PCR reaction, enter circulation behind 94.0 ℃ of sex change 4.5min: 94 ℃ of sex change 30s, 60 ℃ of annealing 40s, 72 ℃ are extended 2min.Extend 10min after 30 circulations.
Adopt the 16S rDNA universal primer of bacterium, check order by the 16sRNA to strains tested, obtain the 16sRNA sequence of MT-5, the sequence of MT-5 is 1428bp.Shown in sequence 1.
Compare by listed gene order among the 16S rRNA gene order of the long 1428bp of bacterial strain and the Genbank and to learn that the Pseudomonas stutzeri of this bacterial strain and Rhodopseudomonas (Pseudomonas stutzeri) has 99% homology.The colonial morphology of comprehensive sample MT-5 and the comparison result of physio-biochemical characteristics and 16S rRNA gene order identify that bacterial strain MT-5 is Pseudomonas stutzeri (Pseudomonas stutzeri).
The preparation of embodiment 2 Pseudomonas stutzeri MT-5 bacterial suspensions
Pseudomonas stutzeri MT-5 is inoculated in the SMS nutrient solution, and 30 ℃ are cultured to logarithmic phase.Behind the centrifugal receipts bacterium, make certain density bacterial suspension, the absorbance of suspension at the 600nm place is OD600=0.68.
The experiment of embodiment 3 Pseudomonas stutzeri MT-5 degraded BAP/BGP
BAP, BGP are all available from U.S. Fisher reagent company, and purity is 99%; The SPME micro-extracting head is available from SUPELCO (100 μ m) company.
In the time of embodiment 3-1:30 ℃ to the degradation experiment of BAP (9.125mg/L)
(1) preparation of BAP-SMS nutrient solution and packing: be added to by a certain percentage in the volumetric flask that 100ml is housed with the BAP solution of filtration sterilization with through autoclaved SMS nutrient solution, constant volume, the concentration that makes BAP in the nutrient solution is 9.125mg/L.Under aseptic condition, BAP-SMS nutrient solution branch is installed in the culturing bottle of 25ml (sterilizing) every bottle of 10ml.
(2) inoculation and cultivation: inoculation 1ml Pseudomonas stutzeri MT-5 suspension in the culturing bottle of BAP-SMS nutrient solution with layer 2-3 tinfoil parcel culturing bottle, and is placed on 30 ℃ of lucifuges cultivation 168h.
(3) degradation effect detects: (technology of solid-phase microextraction-GC-MS) contains bacteria culture fluid analysis to what cultivate 168h, detects the concentration level of BAP in the solution to utilize SPME-GC-MS.
(4) result and analysis: the result shows that behind 30 ℃ of cultivation 168h, BAP concentration is 3.695mg/L in the nutrient solution, and promptly Pseudomonas stutzeri MT-5 reaches 59.56% to the degradation efficiency of BAP.
In the time of embodiment 3-2:15 ℃ to the degradation experiment of BAP (9.125mg/L)
(1) preparation of BAP-SMS nutrient solution and packing: described in method such as the embodiment 2-1.
(2) inoculation and cultivation: inoculation 1ml Pseudomonas stutzeri MT-5 suspension in the culturing bottle of BAP-SMS nutrient solution with layer 2-3 tinfoil parcel culturing bottle, and is placed on 15 ℃ of lucifuges cultivation 168h.
(4) degradation effect detects: method is with embodiment 2-1
(5) result and analysis: the result shows that behind 15 ℃ of cultivation 168h, BAP concentration is 6.539mg/L in the nutrient solution, and promptly Pseudomonas stutzeri MT-5 reaches 28.34% to the degradation efficiency of BAP.
In the time of embodiment 3-3:30 ℃ to the degradation experiment of BAP (18.25mg/L)
(1) preparation of BAP-SMS nutrient solution and packing: be added to by a certain percentage in the volumetric flask that 100ml is housed with the BAP solution of filtration sterilization with through autoclaved SMS nutrient solution, constant volume, the concentration that makes BAP in the nutrient solution is 18.25mg/L.Under aseptic condition, BAP-SMS nutrient solution branch is installed in the culturing bottle of 25ml (sterilizing) every bottle of 10ml.
(3) inoculation and cultivation: inoculation 1ml Pseudomonas stutzeri MT-5 suspension in the culturing bottle of BAP-SMS nutrient solution, with layer 2-3 tinfoil parcel culturing bottle, one bottle places lucifuge cultivation 168h under 30 ℃ of conditions, and another bottle places 15 ℃ of lucifuges to cultivate 168h.
(4) degradation effect detects:.
(5) result and analysis: the result shows that after 168h cultivates (under 30 ℃ of conditions), BAP concentration is 10.705mg/L in the nutrient solution, and promptly Pseudomonas stutzeri MT-5 reaches 41.37% to the degradation efficiency of BAP.
In the time of embodiment 3-4:30 ℃ to the degradation effect of BGP (7.5mg/L)
(1) preparation of BAP-SMS nutrient solution and packing: be added to by a certain percentage in the volumetric flask that 100ml is housed with the BGP solution of filtration sterilization with through autoclaved SMS nutrient solution, constant volume, the concentration that makes BGP in the nutrient solution is 7.5mg/L.Under aseptic condition, BAP-SMS nutrient solution branch is installed in the culturing bottle of 25ml (sterilizing) every bottle of 10ml.
(2) inoculation with cultivate: inoculation 1ml Pseudomonas stutzeri MT-5 suspension (OD600=0.68) with layer 2-3 tinfoil parcel culturing bottle, and is placed under 30 ℃ of conditions lucifuge and cultivates 168h after divide in the culturing bottle that the BGP-SMS nutrient solution is housed.
(4) degradation effect detects: (technology of solid-phase microextraction-GC-MS) contains bacteria culture fluid analysis to what cultivate 168h, detects the concentration level of BGP in the solution to utilize SPME-GC-MS.
(5) result and analysis: the result shows that after 168h cultivates (under 30 ℃ of conditions), BGP concentration is 2.618mg/L in the nutrient solution, and promptly Pseudomonas stutzeri MT-5 reaches 65.09% to the degradation efficiency of BAP.
Under the case study on implementation 3-5:(30 ℃ condition to the degradation effect of BGP (15mg/L))
(1) preparation of BAP-SMS nutrient solution and packing: be added to by a certain percentage in the volumetric flask that 100ml is housed with the BGP solution of filtration sterilization with through autoclaved SMS nutrient solution, constant volume, the concentration that makes BGP in the nutrient solution is 15mg/L.Under aseptic condition, BAP-SMS nutrient solution branch is installed in the culturing bottle of 25ml (sterilizing) every bottle of 10ml.
(2) inoculation with cultivate: inoculation 1ml Pseudomonas stutzeri MT-5 suspension (OD600=0.68) with layer 2-3 tinfoil parcel culturing bottle, and is placed under 30 ℃ of conditions lucifuge and cultivates 168h after divide in the culturing bottle that the BGP-SMS nutrient solution is housed.
(3) degradation effect detects: (technology of solid-phase microextraction-GC-MS) contains bacteria culture fluid analysis to what cultivate 168h, detects the concentration level of BGP in the solution to utilize SPME-GC-MS.
(4) result and analysis: the result shows that after 168h cultivates (under 30 ℃ of conditions), BGP concentration is 9.746mg/L in the nutrient solution, and promptly Pseudomonas stutzeri MT-5 reaches 35.03% to the degradation efficiency of BAP.
SEQUENCE LISTING
＜110〉South China Institute of Environmental Sciences. MEP
＜120〉a kind of pseudomonas stutzeri strain and the application in degrading polycyclic aromatic hydrocarbon with high molecular weight thereof
<130>None
<160>3
<170>PatentIn version 3.5
<210>1
<211>1428
<212>DNA
<213>Pseudomonas stutzeri MT-5
<400>1
gcagtcgagc ggcagcgggt ccttcgggat gccggcgagc ggcggacggg tgagtaatgc 60
ctaggaatct gcctggtagt gggggataac tcggggaaac tcgagctaat accgcatacg 120
tcctacggga gaaagcgggg gatcttcgga cctcgcgcta ccagatgagc ctaggtcgga 180
ttagctagtt ggtgaggtaa aggctcacca aggcgacgat ccgtagctgg tctgagagga 240
tgatcagcca cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga 300
atattggaca atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag aaggtcttcg 360
gattgtaaag cactttaagt tgggaggaag gacagtaagt taataccttg ctgttttgac 420
gttaccgaca gaataagcac cggctaactt cgtgccagca gccgcggtaa tacgaagggt 480
gcaagcgtta atcggaatta ctgggcgtaa agcgcgcgta ggtggtttga taagttggat 540
gtgaaagccc cgggctcaac ctgggaattg catccaaaac tgtctgacta gagtatggca 600
gagggtggtg gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt 660
ggcgaaggcg accacctggg ctaatactga cactgaggtg cgaaagcgtg gggagcaaac 720
aggattagat accctggtag tccacgccgt aaacgatgtc gactagccgt tgggatcctt 780
gagatcttag tggcgcagct aacgcattaa gtcgaccgcc tggggagtac ggccgcaagg 840
ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 900
aagcaacgcg aagaacctta ccaggccttg acatgcagag aactttccag agatggattg 960
gtgccttcgg gaactctgac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1020
gttgggttaa gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgttaaggtg 1080
ggcactctaa ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcat 1140
catggccctt acggcctggg ctacacacgt gctacaatgg tcggtacaaa gggttgccaa 1200
gccgcgaggt ggagctaatc ccataaaacc gatcgtagtc cggatcgcag tctgcaactc 1260
gactgcgtga agtcggaatc gctagtaatc gtgaatcaga atgtcacggt gaatacgttc 1320
ccgggccttg tacacaccgc ccgtcacacc atgggagtgg gttgctccag aagtagctag 1380
tctaaccttc ggggggacgg ttaccacgga gtgattcatg actggggt 1428
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<400>2
agagtttgat cctggctcag 20
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<400>3
aaggaggtga tccagccgca 20
Claims (3)
1. a pseudomonas stutzeri strain (Pseudomonasstutzeri) MT-5, its preserving number is CCTCCM209101, the bacteriology characteristic of this bacterial strain is a Gram-negative; Cell presents straight or crooked shaft-like, but is not spirrillum, not chaining; Size 0.6～0.8 * 1.4～2.0 μ m; Polar flagella is arranged, can move; Do not form gemma, no sheath or thrust; Do not observe poly--β hydroxybutyric acid particle, the bacterium colony characteristic be circular, the edge is irregular, surface irregularity, projecting shape are irregular, white, opaque.
2. the application of the described a kind of Pseudomonas stutzeri MT-5 bacterial strain of claim 1 in degrading polycyclic aromatic hydrocarbon with high molecular weight.
3. the described application of claim 2 is characterized in that described high molecular Polycyclic aromatic hydrocarbons is BAP, BGP.
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| CN102827785B (en) * | 2011-06-17 | 2013-10-23 | 华东理工大学 | Pseudomonas degrading phenanthrene with high efficiency, and application thereof |
| CN103031256B (en) * | 2011-10-09 | 2013-12-18 | 中国科学院沈阳应用生态研究所 | Polycyclic aromatic hydrocarbon degrading bacterium suitable for electric field condition and application thereof |
| CN103013859B (en) * | 2012-11-28 | 2014-07-16 | 新疆大学 | Contaminated soil phenanthrene and application thereof in contaminated soil restoration |
| CN102978140B (en) * | 2012-12-04 | 2014-04-09 | 天津科技大学 | N-butyl alcohol tolerant strain and screening and identifying method thereof |
| CN103981119B (en) * | 2014-02-28 | 2017-09-05 | 天津科技大学 | The application of oily sludge petrochina efficient degrading bacteria and bacterium group |
| CN104388352B (en) * | 2014-11-24 | 2017-06-09 | 中国农业科学院研究生院 | It is a kind of can chlorpyrifos degradation and naphthalene simultaneously Pseudomonas stutzeri |
| CN105969760B (en) * | 2016-06-29 | 2020-02-18 | 华南理工大学 | Embedded nano iron/single microorganism composite microbial agent and preparation method thereof |
| CN108546659B (en) * | 2018-04-12 | 2021-08-13 | 陕西省微生物研究所 | Alkane and polycyclic aromatic hydrocarbon degradation complex microbial inoculum and preparation method thereof |
| CN114292775B (en) * | 2021-12-17 | 2024-03-29 | 黄河三角洲京博化工研究院有限公司 | Toluene degradation strain and application thereof |
| CN114703222B (en) * | 2022-03-15 | 2024-02-02 | 上海市农业科学院 | Cultivation method of plant capable of completely degrading polycyclic aromatic hydrocarbon |
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