CN102943052B - Heavy metal-resistant polycyclic aromatic hydrocarbon (PAHs) degrading bacteria and application thereof in remediation of composite contaminated soil - Google Patents
Heavy metal-resistant polycyclic aromatic hydrocarbon (PAHs) degrading bacteria and application thereof in remediation of composite contaminated soil Download PDFInfo
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- 239000002689 soil Substances 0.000 title claims abstract description 53
- 239000002131 composite material Substances 0.000 title claims abstract description 8
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 title claims description 54
- 229910001385 heavy metal Inorganic materials 0.000 title claims description 39
- 241000894006 Bacteria Species 0.000 title claims description 23
- 230000000593 degrading effect Effects 0.000 title claims description 10
- 238000005067 remediation Methods 0.000 title abstract description 3
- 241000588747 Klebsiella pneumoniae Species 0.000 claims abstract description 26
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Abstract
The present invention belongs to the technical field of environmental engineering and microbial engineering, and especially relates to klebsiella pneumoniae tzyx1 for degradation of PAHs and application thereof in remediation of composite contaminated soil. The accession number of klebsiella pneumoniae tzyx1 strain in the center of the Chinese Typical Culture Collection Center is CCTCC M2012239. The klebsiella pneumoniae tzyx1 strain has the ability to degrade PAHs. The strain can be applied in the governance of PAHs and heavy metal-contaminated soils.
Description
Technical field
The invention belongs to environmental engineering and microbial engineering field.Relate in particular to K. pneumonia (Klebsiella pneumoniae tzyx1) and the application in combined contamination soil is repaired thereof of a strain degrading polycyclic aromatic hydrocarbons.
Background technology
Polycyclic aromatic hydrocarbons (PAHs) is a class persistence organic pollutant (POPs).The source that PAHs pollutes mainly come from the lubricating oil of waste and old lubricant, electric equipment products and from waste metal, disassemble in the material of the hydrocarbon-containiproducts such as timber, organic high molecular compound, paper through incomplete combustion or in reducing atmosphere, through thermolysis, generate.Because Polycyclic aromatic hydrocarbons and Derivatives has carinogenicity, mutagenicity and teratogenecity, and acute toxicity and sub-lethal effect, people's very big concern caused at present.
The reparation of polycyclic aromatic hydrocarbons (PAHs) contaminated soil is the focus of research during current environment is repaired.Biological degradation is one of important channel that PAHs contaminated soil is repaired.For polycyclic aromatic hydrocarbons contaminated, carried out the screening operation of a large amount of degrading polycyclic aromatic hydrocarbons microorganisms both at home and abroad.Up to the present, isolate a plurality of bacterial strains with degrading polycyclic aromatic hydrocarbons ability, as Rhodopseudomonas, Mycobacterium, bacillus etc., but had no pneumobacillus for the report of degrading polycyclic aromatic hydrocarbons.
Investigation shows, many contaminated soils all present the trend of heavy metal and polycyclic aromatic hydrocarbon composite pollution.This two class pollutent of different nature can produce the interaction of chemistry, biochemistry and the physical and chemical process three aspects: of series of complex while coexisting, cause the environmental behaviour of the pollutent under compound condition more complicated with comparing in non-combined pollution environment, and heavy metal ion also can not be ignored the bio-toxicity impact of microorganism species, when heavy metal ion content in soil is higher, the physiological structure of meeting destroy microorganisms, makes it thoroughly lose the function of decomposing organic matter, rehabilitating soil.
Therefore, prior art lacks a kind of polycyclic aromatic hydrocarbon-degrading bacteria strain that can tolerate heavy metal environment
Summary of the invention
The present invention is directed in prior art the not weak point of resistance to heavy metal of polycyclic aromatic hydrocarbon-degrading bacteria, provide a kind of degrading polycyclic aromatic hydrocarbons bacterial strain, this bacterial strain can apply in administering polycyclic aromatic hydrocarbons and heavy-metal contaminated soil.
For achieving the above object, the present invention is achieved by the following technical programs:
A kind of polycyclic aromatic hydrocarbon-degrading bacteria of resistance to heavy metal, it is characterized in that, this bacterial strain is K. pneumonia (Klebsiella pneumoniae) Tzyx1, and the preserving number at Chinese Typical Representative culture collection center is CCTCC M 2012239, possesses the ability of degrading polycyclic aromatic hydrocarbons.
Described bacterial strain has take the ability that polycyclic aromatic hydrocarbons grows as sole carbon source and the energy.
The 16SrDNA sequence of described bacterial strain is as shown in SEQ NO.1.
Described heavy metal is selected from Cu
2+, Zn
2+, Pb
3+in a kind of or its mixture.
The application of the polycyclic aromatic hydrocarbon-degrading bacteria of described resistance to heavy metal in the biological restoration of polycyclic aromatic hydrocarbons contaminated soil.
The application of the polycyclic aromatic hydrocarbon-degrading bacteria of described resistance to heavy metal in heavy metal and the reparation of polycyclic aromatic hydrocarbon composite pollution soil organisms.
The method of utilizing the polycyclic aromatic hydrocarbon-degrading bacteria of described resistance to heavy metal to carry out soil organisms reparation, it is characterized in that the method is comprised of following steps: choose heavy metal and polycyclic aromatic hydrocarbons contaminated soil 100g, 2.0% adds K. pneumonia (Klebsiella pneumoniae) tzyx1 by mass percentage, even with mixing with soil, adding water makes soil moisture content remain on 15%, put into constant temperature oscillator, control culture temperature under the constant temperature of 25 ℃, shaking culture is repaired 15d, can realize the biological restoration to combined contamination soil.
K. pneumonia provided by the invention (Klebsiella pneumoniae), called after, in tzyx1, is preserved in " Chinese Typical Representative culture collection " center ", its preserving number CCTCC NO:M 2012239 on June 24th, 2012.
Tzyx1 disassembles separation certain contaminated soil from City of Taizhou solid waste, and screening obtains.Bacterial strain can be at 15～35 ℃, better growth under pH value 6.0～8.5 culture condition; Strain morphology is characterized as gramstaining and is negative, and observed under electron microscope is the bacillus compared with tubbiness, separately, become two or short chain shape, without brood cell and flagellum, has pod membrane and pili.It is circular that bacterial strain LB cultivates 24h colony characteristics, white, and that with transfering loop, chooses easily draws wire; Bacterium colony smooth surface, neat in edge.
As preferably, bacterial strain of the present invention can be take polycyclic aromatic hydrocarbons as sole carbon source and energy growth.
The screening method of the above-mentioned polycyclic aromatic hydrocarbon-degrading bacteria of resistance to heavy metal, can implement successively as follows:
(1) from solid waste, disassemble and to gather apart from 5～20cm topsoil sample under earth's surface, as bacterium source, carry out nature enrichment culture; Described enrichment medium contains: glucose, extractum carnis, peptone, MgSO
4;
(2) get enrichment culture liquid described in (1) and be inoculated in inorganic salt and select to select to cultivate in substratum, institute's inorganic salt selection substratum contains: Trisodium Citrate, (NH
4)
2sO
4, MgSO
4, K
2hPO
4, KH
2pO
4; Add polycyclic aromatic hydrocarbons and heavy metal.(concentration of polycyclic aromatic hydrocarbons naphthalene, phenanthrene, fluoranthene, pyrene, benzanthrene, benzopyrene is 150mg/L～250mg/L; Heavy metal Cu
2+, Zn
2+, Pb
3+concentration is 150mg/L～250mg/L);
(3) get and described in (2), select culture dilution spread to select on substratum in solid inorganic salt, carry out separation and carry out separation and obtain polycyclic aromatic hydrocarbon-degrading bacteria Tzyx1.
Above-mentioned polycyclic aromatic hydrocarbon-degrading bacteria Tzyx1 can be used for the biological restoration of polycyclic aromatic hydrocarbons and heavy-metal contaminated soil.
Tzyx1 of the present invention can be at 15～35 ℃, better growth under pH value 6.0～8.5 culture condition; Strain morphology is characterized as gramstaining and is negative, and observed under electron microscope is the bacillus compared with tubbiness, separately, become two or short chain shape, without brood cell and flagellum, has pod membrane and pili.It is circular that bacterial strain LB cultivates 24h colony characteristics, white, and that with transfering loop, chooses easily draws wire; Bacterium colony smooth surface, neat in edge.
Tzyx1 is at LB(Tryptones 1.0%, yeast extract 0.5%, sodium-chlor 1.0%) cultivate 24 hours colony characteristicses for circular, colony diameter 2～3mm, white, smooth surface, neat in edge.The physiological and biochemical property of tzyx1, in Table 1.
The physiological and biochemical property of table 1tzyx1
| Certified variety | Qualification result |
| Indoles produces | - |
| M.R experiment | - |
| V.P experiment | + |
| Gelatin hydrolysis | + |
| The experiment of urine enzyme | + |
| Casein hydrolysis | - |
| Phenylalanine decarboxylase | - |
| Arginine dihydrolase | - |
| Lecithinase | - |
| KCN | + |
| KNO 3 | + |
| Malonate utilizes | + |
| Citrate trianion utilizes | + |
| Glucose produces sour aerogenesis | + |
| Lactose produces sour aerogenesis | + |
| Catalase | + |
| Glucose utilization | + |
| Maltose utilizes | + |
| Pectinose utilizes | - |
| Xylose utilization | - |
| N.F,USP MANNITOL utilizes | + |
| Raffinose utilizes | + |
| Sucrose utilizes | + |
The pcr amplification of the 16SrDNA gene of tzyx1, order-checking are completed by Beijing San Bo polygala root company, the sequence that to have obtained length be 1430bp.Select homology to be greater than 97% gene order and carry out Phylogenetic Analysis, show that bacterial strain and Klebsiella pneumoniae homology are the highest, in conjunction with strain morphology, colony characteristics and physio-biochemical characteristics, identify that bacterial strain belongs to K. pneumonia (Klebsiella pneumoniae).
Tzyx1 of the present invention both can be at nutritional medium, as: in common beef extract-peptone, common LB, nutrient agar medium, grow, can grow take in the inorganic salt basic medium that polycyclic aromatic hydrocarbons is sole carbon source, the energy again.
In sum, the present invention has the following advantages: tzyx1 provided by the invention has fast degradation ability to the polycyclic aromatic hydrocarbons in heavy metal and polycyclic aromatic hydrocarbon composite pollution soil, and degraded substrate scope is wide, and degradation rate is high at short notice.Because this bacterium has resistance to heavy metal effect, make it in heavy metal and polycyclic aromatic hydrocarbon composite pollution soil remediation, have wide application potential.
Classification And Nomenclature: K. pneumonia tzyx1
Latin literary fame: Klebsiella pneumoniae tzyx1
Depositary institution: Chinese Typical Representative culture collection center
Address: Wuhan, China Wuhan University
Preservation date: on June 28th, 2012
Deposit number: CCTCC NO:M 2012239.
Accompanying drawing explanation
Fig. 1 is naphthalene kinetics degradation curve figure in time in soil.
Fig. 2 is the kinetics degradation curve figure in time of soil China and Philippines.
Fig. 3 is fluoranthene kinetics degradation curve figure in time in soil.
Fig. 4 is pyrene kinetics degradation curve figure in time in soil.
Fig. 5 is benzanthrene kinetics degradation curve figure in time in soil.
Fig. 6 is benzopyrene kinetics degradation curve figure in time in soil.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail, but the present invention is not limited to these embodiment.
The separating screening method of tzyx1 provided by the invention.
From solid waste, disassemble and to gather apart from 5～20cm topsoil sample under earth's surface, as bacterium source, carry out nature enrichment culture, by glucose 2g, Tryptones 4g, extractum carnis 4g, MgSO
40.8g joins in the distilled water of 400mL, the nutrient solution preparing is sterilising treatment 20min under the temperature condition of 121 ℃, on this substratum, cultivate three days, prepare again inorganic salt basic medium, the polycyclic aromatic hydrocarbon compounds that adds pyrene, benzanthrene, benzopyrene, add again heavy metal ion, be mixed with after solution, and then add distilled water to be settled to 500mL, in the nutrient solution that the above-mentioned amount that adds pyrene, benzanthrene, benzopyrene will make to prepare, the ultimate density of pyrene, benzanthrene, benzopyrene is 200mg/mL, the above-mentioned heavy metal Cu that adds
2+, Zn
2+, Pb
3+the amount nutrient solution that will make to prepare in heavy metal Cu
2+, Zn
2+, Pb
3+ultimate density be 200mg/mL, then, by the nutrient solution preparing sterilising treatment 20min under the temperature condition of 121 ℃, obtain required nutrient solution.On this substratum, cultivate three days, dilution spread was isolated a strain bacterium called after Tzyx1, and this bacterium is preserved in " its preserving number of Chinese Typical Representative culture collection " center " CCTCC M 2012239 on June 24th, 2012.
The pcr amplification of the 16SrDNA gene of tzyx1, order-checking are completed by Beijing San Bo polygala root company.According to sequencing result phylogenetic tree construction.Phylogeny shows that the evolutionary distance of bacterial strain and Klebsiella pneumoniae is nearest, and combining form, colony characteristics and physio-biochemical characteristics identify that bacterial strain belongs to K. pneumonia (Klebsiella pneumoniae).
Embodiment 2
Polycyclic aromatic hydrocarbons in tzyx1 degraded combined contamination soil.
Choose heavy metal and polycyclic aromatic hydrocarbons contaminated soil 100g, tzyx11 presses 2.0%(mass percent) consumption and mixing with soil even, adding a small amount of water makes soil moisture content remain on 15% left and right, put into constant temperature oscillator, control culture temperature under the constant temperature of 25 ℃, shaking culture is repaired 15d, can realize combined contamination soil is repaired, the soil after being repaired.
Fig. 1-Fig. 6 has represented respectively PAHs naphthalene, phenanthrene, fluoranthene, pyrene, benzanthrene, benzopyrene kinetics degradation curve in time in different treatment soil.Three different treatment are respectively polycyclic aromatic hydrocarbons contrast and pollute soil sample (CK), do not add heavy metal, inoculate; Polycyclic aromatic hydrocarbons contaminated soil sample does not add heavy metal, inoculation PAHs degradation bacteria strains (KL); Polycyclic aromatic hydrocarbons contaminated soil sample adds heavy metal, inoculation PAHs degradation bacteria strains (KL+M).As shown in Figure 2,6 kinds of PAHs naphthalenes, phenanthrene, fluoranthene, pyrene, benzanthrene, benzopyrenes have all obtained degraded in various degree after cultivating 15d.
CK soil sample PAHs degraded is very slow.Heavy metal Cu
2+, Zn
2+, Pb
3+the K. pneumonia of test group and control group is not remarkable to degrading polycyclic aromatic hydrocarbons difference, illustrates that K. pneumonia is to heavy metal Cu
2+, Zn
2+, Pb
3+all there is stronger tolerance.Data monitoring demonstration, after 15d, the degradation rate of KL+M processing soil polycyclic aromatic hydrocarbons is: naphthalene 86.6%, phenanthrene 91.0%, fluoranthene 54.7%, pyrene 91.7%, benzanthrene 64.7%, benzopyrene 49.7%.
Utilize the repairing effect of the soil polycyclic aromatic hydrocarbons after method of the present invention is repaired combined contamination soil good, degradation effect to polycyclic aromatic hydrocarbons is obvious, the resistance to heavy metal of bacterial strain is described and combined contamination soil reparation is reached to the effect of good degrading polycyclic aromatic hydrocarbons, compared with prior art, improve the repairing effect to polycyclic aromatic hydrocarbon composite pollution soil, be more conducive to the reparation to combined contamination soil in reality.
Specific embodiment described in the present invention is only to the explanation for example of the present invention's spirit.Those skilled in the art can make various modifications or supplement or adopt similar mode to substitute described specific embodiment; but can't depart from spirit of the present invention or surmount the defined scope of appended claims; within the spirit and principles in the present invention all; any modification of doing, be equal to replacement, improvement etc., within protection scope of the present invention all should be included in.
SEQUENCE LISTING
<110> Taizhou Vocational & Technical College
The polycyclic aromatic hydrocarbon-degrading bacteria of the resistance to heavy metal of <120> mono-strain and the application in combined contamination soil is repaired thereof
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1430
<212> DNA
<213> Klebsiella pneumoniae
<400> 1
aggcacctgc agtcgagcgg tagcacagag agcttgctct cgggtgacga gcggcggacg 60
ggtgagtaat gtctgggaaa ctgcctgatg gagggggata actactggaa acggtagcta 120
ataccgcata acgtcgcaag accaaagtgg gggaccttcg ggcctcatgc catcagatgt 180
gcccagatgg gattagctag taggtggggt aacggctcac ctaggcgacg atccctagct 240
ggtctgagag gatgaccagc cacactggaa ctgagacacg gtccagactc ctacgggagg 300
cagcagtggg gaatattgca caatgggcgc aagcctgatg cagccatgcc gcgtgtatga 360
agaaggcctt cgggttgtaa agtactttca gcggggagga aggcgataag gttaataacc 420
ttgtcgattg acgttacccg cagaagaagc accggctaac tccgtgccag cagccgcggt 480
aatacggagg gtgcaagcgt taatcggaat tactgggcgt aaagcgcacg caggcggtct 540
gtcaagtcgg atgtgaaatc cccgggctca acctgggaac tgcattcgaa actggcaggc 600
tagagtcttg tagagggggg tagaattcca ggtgtagcgg tgaaatgcgt agagatctgg 660
aggaataccg gtggcgaagg cggccccctg gacaaagact gacgctcagg tgcgaaagcg 720
tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg tcgatttgga 780
ggttgtgccc ttgaggcgtg gcttccggag ctaacgcgtt aaatcgaccg cctggggagt 840
acggccgcaa ggttaaaact caaatgaatt gacgggggcc cgcacaagcg gtggagcatg 900
tggtttaatt cgatgcaacg cgaagaacct tacctggtct tgacatccac agaacttagc 960
agagatgctt tggtgccttc gggaactgtg agacaggtgc tgcatggctg tcgtcagctc 1020
gtgttgtgaa atgttgggtt aagtcccgca acgagcgcaa cccttatcct ttgttgccag 1080
cggttcggcc gggaactcaa aggagactgc cagtgataaa ctggaggaag gtggggatga 1140
cgtcaagtca tcatggccct tacgaccagg gctacacacg tgctacaatg gcatatacaa 1200
agagaagcga cctcgcgaga gcaagcggac ctcataaagt atgtcgtagt ccggattgga 1260
gtctgcaact cgactccatg aagtcggaat cgctagtaat cgtagatcag aatgctacgg 1320
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgggagtg ggttgcaaaa 1380
gaagtaggta gcttaacctt cggagggcgc ttaccacctt ggagtgacgg 1430
Claims (4)
1. a polycyclic aromatic hydrocarbon-degrading bacteria for resistance to heavy metal, is characterized in that, this bacterial strain be K. pneumonia (
klebsiella pneumoniae) tzyx1, preserving number at Chinese Typical Representative culture collection center is CCTCC NO:M 2012239, the ability that possesses degrading polycyclic aromatic hydrocarbons, described polycyclic aromatic hydrocarbons is selected from a kind of or its mixture of naphthalene, phenanthrene, fluoranthene, pyrene, benzanthrene, benzopyrene, described bacterial strain has take the ability that polycyclic aromatic hydrocarbons grows as sole carbon source and the energy, the 16SrDNA sequence of described bacterial strain is as shown in SEQ NO.1, and described heavy metal is selected from Cu
2+, Zn
2+, Pb
3+in a kind of or its mixture.
2. the application of bacterial strain according to claim 1 in the biological restoration of the soil by polycyclic aromatic hydrocarbons contaminated, is characterized in that described polycyclic aromatic hydrocarbons is selected from a kind of or its mixture of naphthalene, phenanthrene, fluoranthene, pyrene, benzanthrene, benzopyrene.
3. bacterial strain according to claim 1, by the application of heavy metal and polycyclic aromatic hydrocarbon composite pollution degraded in soil polycyclic aromatic hydrocarbons, is characterized in that described heavy metal is selected from Cu
2+, Zn
2+, Pb
3+in a kind of or its mixture; Described polycyclic aromatic hydrocarbons is selected from a kind of or its mixture of naphthalene, phenanthrene, fluoranthene, pyrene, benzanthrene, benzopyrene.
4. utilize the method that the polycyclic aromatic hydrocarbon-degrading bacteria of resistance to heavy metal carries out soil organisms reparation described in claim 1, it is characterized in that the method is comprised of following steps: choose heavy metal and polycyclic aromatic hydrocarbons contaminated soil 100g, 2.0% adds K. pneumonia by mass percentage
(Klebsiella pneumoniae) tzyx1, even with mixing with soil, add water and make soil moisture content remain on 15%, put into constant temperature oscillator, control culture temperature under the constant temperature of 25 ℃, shaking culture is repaired 15d, can realize the biological restoration to combined contamination soil.
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| CN104607458A (en) * | 2015-01-14 | 2015-05-13 | 苏忠 | Method for restoring chemically contaminated soil through Klebsiella pneumoniae |
| CN104673683B (en) * | 2015-02-27 | 2017-10-31 | 台州职业技术学院 | One plant of salt tolerant polycyclic aromatic hydrocarbon-degrading bacteria Tzyjm and its application in marine pollution environment |
| CN104877934B (en) * | 2015-02-27 | 2018-01-09 | 台州职业技术学院 | One plant of salt tolerant polycyclic aromatic hydrocarbon-degrading bacteria Tzymx and its application in marine pollution environment |
| CN105861366B (en) * | 2016-04-19 | 2019-06-14 | 南京大学 | Application of the polycyclic aromatic hydrocarbon-degrading bacteria FA1 in absorption heavy metal Pb ion |
| CN106282057B (en) * | 2016-08-15 | 2019-05-28 | 台州职业技术学院 | A kind of bacterial strain of degrading polyaromatic hydrocarbon and its application |
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