CN105861362A - Heavy metal resisting actinomycete strain and application thereof - Google Patents

Heavy metal resisting actinomycete strain and application thereof Download PDF

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CN105861362A
CN105861362A CN201610229295.9A CN201610229295A CN105861362A CN 105861362 A CN105861362 A CN 105861362A CN 201610229295 A CN201610229295 A CN 201610229295A CN 105861362 A CN105861362 A CN 105861362A
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heavy metal
strain
bacterial strain
copper
lead
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CN105861362B (en
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张志平
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Nanjing Beishengrong Energy Technology Co Ltd
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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
    • C12R2001/04—Actinomyces
    • 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
    • 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
    • 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
    • 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
    • C02F2101/10—Inorganic compounds
    • C02F2101/20—Heavy metals or heavy metal compounds

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Abstract

The invention provides a heavy metal resisting actinomycete strain and an application thereof. The heavy metal resisting actinomycete strain is named as Lechevalieria nanjingensis NJ07-19, and is preserved in China Center for Type Culture Collection with the preservation number of CCTCC No:M 2015737. The strain is obtained through separation, screening, identification and mutagenesis. The actinomyce can tolerate and adsorb multiple heavy metals, and can be used to treat heavy metal polluted water and soil.

Description

The actinomycetes strain of the one resistance to heavy metal of strain and application thereof
Technical field
The invention belongs to biological technical field or technical field of environmental management, relate to the discovery of microorganism, mutagenesis and This microorganism is utilized to carry out the removing of heavy metal.
Background technology
Along with industrial expansion, the use of people's heavy metal gets more and more (such as lead, copper, zinc, nickel etc.), brings therewith The problem of heavy metal environmental pollution is the most increasing.Heavy metal is difficult to be degraded by organism, and can be enriched with by food chain, passes through Number of ways eventually enters into human body, is detrimental to health.
Lead, lead is that pollutant Poisoning is very big and heavy without any physiological function and the one based on neurotoxicity to body Metallic element.There are some researches show that the safe concentration scope in the body of lead is 0.3-1mg, surpass when human body absorbs lead content every day Crossing this scope, lead just can accumulate in blood, causes anaemia, hypertension, neurasthenia, myocardial damage etc..Lead is to children's intelligence Power is grown and is had a significant impact, and the too high meeting of children blood lead levels causes children's intelligence to decline.
Copper, it is considered that the toxicity of copper own is the least, copper lack by cause anaemia, osteoporosis, Muscle tensility go down and essence God's motility obstacle.Although copper is important essential trace element, but misapplication, the most easily cause toxic reaction.Take in too much Copper may result in liver cell and erythrocytic damage, symptom is nausea,vomiting,diarrhea, time serious will stupor.
At present, the governance way of heavy metal pollution mostly uses Physical, chemical method and bioanalysis.Physical mainly includes Soil moved in improve the original, flushing complexometry, electronic chemical method etc., instant effect, but cost is high, easily produces secondary pollution；Chemical method generally passes through Add foreign substance and change Heavy Metals, make the ion of solubility form insoluble sediment and be removed, as chemistry changes Good dose, precipitating reagent etc., but additional material can not be biodegradable substantially, can cause secondary pollution.Bioanalysis is a kind of emerging Process heavy metal pollution method, mainly include plant and microorganism, especially microorganism, due to its low cost, without secondary Pollute, be suitable for the advantages such as large-area reparation and receive much concern.The improvement of microorganism heavy metal pollution to be applied to just must Heavy metal must have strong tolerance, and environment can not be produced secondary pollution.There are some researches show at present, actinomyces are to many Heavy metal species have tolerance and use it for heavy metal pollution improvement in, such as streptomyces (Streptomyces) little monospore Pseudomonas (Micromonospora), Neurospora (Catellatospora), Streptosporangium (Streptosporangium), actinoplanes (Actinoplanes) and corynebacterium (Corynebacterium) Deng.There is no row contract Giovanni at present and Bordetella (Lechevalieria) tolerance, the report of Adsorption of Heavy Metals.
Summary of the invention
The technical purpose of the present invention is tolerance the actinomycetes strain of Adsorption of Heavy Metals providing a strain novel, is used for inhaling Attached heavy metal.
Concrete, technical scheme is as follows:
The actinomycetes strain of the one resistance to heavy metal of strain, wherein, the named Lechevalieria of this strain classification Nanjingensis NJ07-19, is preserved in China typical culture collection center, and deposit number is CCTCC No:M 2015737。
Wherein, the original strain of the present invention is to obtain by separating from the soil of heavy metal pollution, screen；Then pass through Ion implanting physical mutagenesis obtains Lechevalieria nanjingensis NJ07-19.
The separation of the bacterial strain of resistance to heavy metal Lechevalieria nanjingensis NJ07-19 and cultural method, including with Lower step:
(1) carry out gradient dilution after being dissolved in SPSS by the soil from heavy metal pollution district, choose 10-2、 10-3、10-4Dilution is respectively coated on the isolation medium flat board containing heavy metal, each gradient 3 repetition, puts 25 DEG C of trainings Support.The bacterium colony that picking shape, size, color etc. are different after growing bacterium colony streak inoculation respectively is in corresponding flat board, until nothing Miscellaneous bacteria falls.
(2) the NJ07 bacterial strain determined after cultivating screening cultivates 6-7d in 25 DEG C, observes form, and substrate mycelium branch is sent out Reaching, rupture, gas silk is white.With reference to " primary Jie Shi systematic bacteriology handbook ", NJ07 bacterial strain is carried out morphology and Physiology and biochemistry is surveyed Fixed.
(3) obtained the 16S rDNA sequence of NJ07 bacterial strain by PCR, through sequencing and BLAST homology alignment with enter Fractional analysis, result shows that the homology of NJ07 with Lechevalieria xinjianggensis sp.nov.16S rDNA is 98.8%, it is 97.5% with the homology of Lechevalieria flanava NRRL B-1613116S rDNA, determines NJ07 The novel species belonged to for Lechevalieria.
Mutagenic strain LechevalieriaNJ07 obtains the concrete of Lechevalieria nanjingensis NJ07-19 Operating procedure is:
(1) actinomyces starting strain LechevalieriaNJ07 is carried out ion implanting physical mutagenesis, obtains mutant strain, Wherein, ion implanting conditions is to use 15KeV N+Injecting mutagenesis, fluence is 50～200*1013/cm2, target chamber vacuum is 10- 3Pa。
(2) mutant strain of step (1) gained is coated contain the selective solid of high concentration heavy metal lead, mercury respectively Culture medium plane, cultivates, obtains the actinomyces of heavy metal tolerance.
(3) investigating the resistant stability of the bacterial strain of the heavy metal tolerance of step (2) gained, a final selected strain resists The bacterial strain that property is stable, named Lechevalieria nanjingensis NJ07-19.
Bacterial strain of the present invention is resistant to heavy metal lead, copper, zinc, nickel, wherein, is respectively the highest tolerable concentration of lead with copper 1500mg/L、600mg/L。
The bacterial strain heavy metal lead of the present invention and the attached rate of copper can reach 83%, 56% respectively.
The bacterial strain of the present invention application in water body and heavy metal pollution of soil are administered.
Concrete, the bacterial strain of resistance to heavy metal Lechevalieria nanjingensis NJ07-19 is in terms of Adsorption of Heavy Metals Application process be:
Lechevalieria nanjingensis NJ07-19 is inoculated in fermentation medium, at 25 DEG C, rotating speed Under the conditions of 150-200rpm, 6-7d is cultivated in concussion, obtains zymotic fluid.Zymotic fluid is centrifuged, removes culture medium, it is thus achieved that thalline.By bacterium Body is placed in the heavy metal-containing polluted water containing lead, copper, zinc, nickel etc. respectively, adsorbs 36-48h.
The beneficial effects of the present invention is:
The present invention screens the actinomyces NJ07 of a resistance to heavy metal of strain, and it is carried out ion implanting physical mutagenesis, it is thus achieved that Mutant strain NJ07-19, named Lechevalieria nanjingensis NJ07-19.Lechevalieria Nanjingensis NJ07-19 is more higher than the tolerance of original strain, Adsorption of Heavy Metals ability, can be used for the soil of heavy metal pollution Earth, water body improvement in.
The explanation of biomaterial:
Biomaterial of the present invention, its Classification And Nomenclature is Lechevalieria nanjingensis NJ07-19, Be preserved in China typical culture collection center (be called for short CCTCC, address: China. Wuhan. Wuhan University), preservation date is On December 11st, 2015, deposit number is CCTCC No:M 2015737.
Detailed description of the invention
Embodiment 1: this example demonstrates that the culture medium that the present invention uses
Isolation medium: glucose 5g/L, acid hydrolysis casein 2g/L, yeast extract 1g/L, beef extract 1g/L, agar powder 15g/ L, the heavy metal such as lead, copper (0-1800mg/L).
Fermentation medium: glucose 5g/L, acid hydrolysis casein 2g/L, yeast extract 1g/L, beef extract 1g/L, agar powder 15g/ L。
Embodiment 2: the separation of actinomyces Lechevalieria NJ07, screen, identify
Weigh after 1g is dissolved in 100ml SPSS from the soil sample in heavy metal pollution district and carry out gradient dilution, choosing Take 10-2、10-3、10-4Dilution is respectively coated on the isolation medium flat board containing heavy metal, each gradient 3 repetition, puts 25 DEG C cultivate.Streak inoculation is in corresponding flat board respectively, directly for the bacterium colony that picking shape, size, color etc. are different after growing bacterium colony To falling without miscellaneous bacteria.The NJ07 bacterial strain determined after cultivating screening cultivates 6-7d in 25 DEG C, observes form, and substrate mycelium branch is sent out Reaching, rupture, gas silk is white.With reference to " primary Jie Shi systematic bacteriology handbook ", NJ07 bacterial strain is carried out morphology and Physiology and biochemistry is surveyed Fixed.
The physiological and biochemical property that table 1.NJ07 bacterial strain is main
Note: in table 1 "+" it is positive reaction, "-" is negative reaction, and " w " is weak positive reaction
Obtained the 16S rDNA sequence of NJ07 bacterial strain by PCR, divide with evolving through sequencing and BLAST homology alignment Analysis, result shows that the homology of NJ07 with Lechevalieria xinjianggensis sp.nov.16S rDNA is 99.1%, it is 98.5% with the homology of Lechevalieria flanava NRRL B-16131 16S rDNA, determines NJ07 The novel species belonged to for Lechevalieria.
Embodiment 3: the mutagenesis of bacterial strain NJ07, screening
Take one, the actinomyces NJ07 inclined-plane covering with spore, elute spore with physiological saline, be loaded on and aseptic fill glass In the triangular flask of pearl, 25 DEG C, 160rpm fully shake 30min, make spore dispersed, filter, make spore suspension, dilution Concentration to spore is 107Individual/ml, takes spore suspension even spread that 0.1ml diluted, air-dries, put into ion implantation apparatus In, use 15KeV N+, fluence is 50～200*1013/cm2, target chamber vacuum is 10-3Spore is processed by Pa.
With the spore after physiological saline wash-out mutagenesis, make 107The spore suspension of individual/ml, takes 200ul and coats highly concentrated Degree containing on the isolation medium of the heavy metal ion of variable concentrations, cultivate 6-7 days for 25 DEG C.Period, observe the bacterium on flat board Fall form, and adds up total plate count, picking tolerance lead, the bacterium colony that copper concentration is the highest, finally selects bacterial strain NJ07-19.
By named for bacterial strain NJ07-19 Lechevalieria nanjingensis NJ07-19, December 11 in 2015 Day is preserved in China typical culture collection center (CCTCC), and deposit number is CCTCC No:M 2015737.
Embodiment 4: the resistance to heavy metal performance of bacterial strain NJ07-19
NJ07 and NJ07-19 bacterial strain is respectively coated in containing lead 800,1000,1200,1400,1600,1800mg/L and Containing copper 200,300,400,500,600,700, on the isolation medium flat board of 800mg/L, cultivate 6-7 days for 25 DEG C.NJ07 bacterium Strain and is containing on the copper concentration flat board higher than 400mg/L without colony growth higher than 1200mg/L containing lead concentration；NJ07-19 bacterium Strain containing lead concentration higher than 1600mg/L and containing on the copper concentration flat board higher than 600mg/L without colony growth, its to lead and The tolerance of copper is respectively the 133.3% of original strain, 150%.
Embodiment 5: the absorption property of bacterial strain NJ07-19 heavy metal
NJ07 and NJ07-19 bacterial strain is inoculated in fermentation medium respectively, concussion training under the conditions of rotating speed 150-200rpm Support 6-7d, obtain zymotic fluid.Zymotic fluid is centrifuged, removes culture medium, it is thus achieved that thalline.Take 1g thalline to be placed in and contain 50mg/L respectively Lead and 50mg/L copper 1L heavy metal-containing polluted water in, absorption 48h reach balance.The adsorption rate of lead, copper is respectively by NJ07 72%, 38.5%；NJ07-19 is respectively 72%, 38.5% to the adsorption rate of lead, copper, and the adsorption rate of lead with copper is respectively by it The 115.2% of original strain, 145.5%.
The computational methods of adsorption rate (R):
R (%)=(C0-Ct)/C0* 100%
Wherein, C0For the initial concentration of metal ion in solution, CtAdd thalline absorption within t hour, be metal in solution from The concentration of son.

Claims (4)

1. the actinomycetes strain of a resistance to heavy metal of strain, it is characterised in that the named Lechevalieria of this strain classification Nanjingensis NJ07-19, is preserved in China typical culture collection center, and deposit number is CCTCC No:M 2015737。
2. bacterial strain as claimed in claim 1, it is characterised in that this bacterial strain is resistant to heavy metal lead, copper, zinc, nickel, wherein, The highest tolerable concentration of lead Yu copper is respectively 1500mg/L, 600mg/L.
3. bacterial strain as claimed in claim 1, it is characterised in that this bacterial strain heavy metal lead can reach respectively with the attached rate of copper To 83%, 56%.
4. the bacterial strain as claimed in claim 1 application in water body and heavy metal pollution of soil are administered.
CN201610229295.9A 2016-04-13 2016-04-13 The actinomycetes strain of one plant of resistance to heavy metal and its application Active CN105861362B (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106244471A (en) * 2016-10-08 2016-12-21 南京工业大学 The fungal bacterial strain of a kind of resistance to heavy metal Hg and application thereof
CN106701613A (en) * 2016-10-25 2017-05-24 广州大学 Actinomycete strain YF-64 and application thereof
CN110106108A (en) * 2019-04-25 2019-08-09 黄山市益天士生物科技有限公司 Method for improving purifying water body breeding colony speed
CN115634924A (en) * 2022-09-30 2023-01-24 四川农业大学 Tea garden polluted soil remediation and treatment method

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102250769A (en) * 2011-06-15 2011-11-23 江苏新琦环保有限公司 Biological agent for organic waste composting and preparation method thereof
CN102267787A (en) * 2011-07-27 2011-12-07 福建科佳奇迈生物工程有限公司 Method for treating livestock and poultry culture polluted marsh solution
CN102816009A (en) * 2012-09-14 2012-12-12 熊汉夫 Formula and production method of special sugarcane microorganism organic fertilizer in household garbage fertilizer production

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102250769A (en) * 2011-06-15 2011-11-23 江苏新琦环保有限公司 Biological agent for organic waste composting and preparation method thereof
CN102267787A (en) * 2011-07-27 2011-12-07 福建科佳奇迈生物工程有限公司 Method for treating livestock and poultry culture polluted marsh solution
CN102816009A (en) * 2012-09-14 2012-12-12 熊汉夫 Formula and production method of special sugarcane microorganism organic fertilizer in household garbage fertilizer production

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106244471A (en) * 2016-10-08 2016-12-21 南京工业大学 The fungal bacterial strain of a kind of resistance to heavy metal Hg and application thereof
CN106244471B (en) * 2016-10-08 2019-07-02 南京工业大学 A kind of fungal bacterial strain of resistance to heavy metal Hg and its application
CN106701613A (en) * 2016-10-25 2017-05-24 广州大学 Actinomycete strain YF-64 and application thereof
CN106701613B (en) * 2016-10-25 2020-03-27 广州大学 Actinomycete strain YF-64 and application thereof
CN110106108A (en) * 2019-04-25 2019-08-09 黄山市益天士生物科技有限公司 Method for improving purifying water body breeding colony speed
CN115634924A (en) * 2022-09-30 2023-01-24 四川农业大学 Tea garden polluted soil remediation and treatment method
CN115634924B (en) * 2022-09-30 2024-04-12 四川农业大学 Tea garden polluted soil restoration and treatment method

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