CN105861362B - The actinomycetes strain of one plant of resistance to heavy metal and its application - Google Patents
The actinomycetes strain of one plant of resistance to heavy metal and its application Download PDFInfo
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- CN105861362B CN105861362B CN201610229295.9A CN201610229295A CN105861362B CN 105861362 B CN105861362 B CN 105861362B CN 201610229295 A CN201610229295 A CN 201610229295A CN 105861362 B CN105861362 B CN 105861362B
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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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
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Abstract
The present invention provides the actinomycetes strains of one plant of resistance to heavy metal and its application, the classification naming of the actinomycetes strain of resistance to heavy metal to beLechevalieria nanjingensis NJ07-19, in the deposit number of China typical culture collection center are as follows: CCTCC No:M 2015737.The bacterial strain is through obtained by separation, screening, identification and mutagenesis.Actinomyces of the invention can be resistant to and adsorb various heavy, can be used for the improvement of water body and soil of heavy metal pollution etc..
Description
Technical field
The invention belongs to field of biotechnology or technical field of environmental management, be related to the discovery of microorganism, mutagenesis and
The removing of heavy metal is carried out using the microorganism.
Background technique
With the development of industry, people to heavy metal using more and more (such as lead, copper, zinc, nickel), bring therewith
The problem of heavy metal environmental pollution, is also increasing.Heavy metal is not easy 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 are that pollutant Poisoning is very big and a kind of heavy without any physiological function and based on neurotoxicity to body
Metallic element.Some researches show that the intracorporal safe concentration range of the human body of lead is 0.3-1mg, when to absorb lead content daily super for human body
This range is crossed, lead can accumulate in blood, lead to anaemia, hypertension, neurasthenia, myocardial damage etc..Lead is to children's intelligence
Power development has a significant impact, and children blood lead levels are excessively high to will lead to children's intelligence decline.
Copper, it is considered that copper toxicity very little itself, copper lack the decline and essence that will lead to anaemia, osteoporosis, Muscle tensility
Mind movement sexual dysfunction.Although copper is important essential trace element, misapplication also easily causes toxic reaction.Intake is excessive
Copper can lead to the damage of liver cell and red blood cell, symptom is nausea,vomiting,diarrhea, will stupor when serious.
Currently, the governance way of heavy metal pollution mostly uses physical method, chemical method and bioanalysis.Physical method mainly includes
Soil moved in improve the original, flushing complexometry, electronic chemical method etc., it is quick but at high cost, it is easy to produce secondary pollution；Chemical method usually passes through
Foreign substance is added to change Heavy Metals, so that soluble ion is formed insoluble sediment and is removed, as chemistry changes
Good dose, precipitating reagent etc., but additional substance cannot be biodegradable substantially, will cause secondary pollution.Bioanalysis is a kind of emerging
Processing heavy metal pollution method, mainly include plant and microorganism, especially microorganism, due to its is at low cost, without secondary
It pollutes, be suitable for the advantages such as the reparation of large area and be concerned.Microorganism will be applied to just must in the improvement of heavy metal pollution
There must be strong tolerance to heavy metal, and secondary pollution cannot be generated to environment.Existing research at present shows actinomyces to more
Heavy metal species have tolerance and are used in the improvement of heavy metal pollution, such as streptomyces (Streptomyces) small monospore
Pseudomonas (Micromonospora), Neurospora (Catellatospora), Streptosporangium
(Streptosporangium), actinoplanes (Actinoplanes) and corynebacterium (Corynebacterium)
Deng.It there is no the report of column contract Giovanni and Bordetella (Lechevalieria) tolerance, absorption heavy metal at present.
Summary of the invention
Technical purpose of the invention is to provide one plant of novel tolerance and adsorbs the actinomycetes strain of heavy metal, for inhaling
Attached heavy metal.
Specifically, technical scheme is as follows:
The actinomycetes strain of one plant of resistance to heavy metal, wherein the strain classification is named as Lechevalieria
Nanjingensis NJ07-19, is preserved in China typical culture collection center, and deposit number is CCTCC No:M
2015737。
Wherein, original strain of the invention is obtained by separating, screening from the soil of heavy metal pollution；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) gradient dilution is carried out after the soil from heavy metal pollution area being dissolved in sterile saline, chooses 10-2、
10-3、10-4Dilution is respectively coated on the isolation medium plate containing heavy metal, and 25 DEG C of trainings are set in 3 repetitions of each gradient
It supports.The bacterium colony difference streak inoculation that picking shape, size, color etc. are different after growing bacterium colony is in corresponding plate, until nothing
Miscellaneous bacteria falls.
(2) the NJ07 bacterial strain determined after culture screening observes form, substrate mycelium branch hair in 25 DEG C of culture 6-7d
It reaches, be broken, gas silk is white.Morphology is carried out to NJ07 bacterial strain referring to " primary Jie Shi systematic bacteriology handbook " and Physiology and biochemistry is surveyed
It is fixed.
(3) the 16S rDNA sequence that NJ07 bacterial strain is obtained by PCR, through sequencing and BLAST homology alignment with into
Change analysis, the results showed that the homology of NJ07 and Lechevalieria xinjianggensis sp.nov.16S rDNA is
98.8%, the homology with Lechevalieria flanava NRRL B-1613116S rDNA is 97.5%, determines NJ07
The novel species belonged to for Lechevalieria.
Mutagenic strain LechevalieriaNJ07 obtains the specific of Lechevalieria nanjingensis NJ07-19
Operating procedure are as follows:
(1) actinomyces starting strain LechevalieriaNJ07 is subjected to ion implanting physical mutagenesis, obtains mutant strain,
Wherein, ion implanting conditions are using 15KeV N+Mutagenesis is injected, fluence is 50~200*1013/cm2, target chamber vacuum degree is 10- 3Pa。
(2) step (1) resulting mutant strain is coated on the selective solid containing high concentration heavy metal lead, mercury respectively
Base plane is cultivated, culture obtains the actinomyces of heavy metal tolerance.
(3) resistant stability of the bacterial strain of step (2) resulting heavy metal tolerance is investigated, final selected one plant anti-
The stable bacterial strain of property, is named as Lechevalieria nanjingensis NJ07-19.
Bacterial strain of the present invention is resistant to heavy metal lead, copper, zinc, nickel, wherein the highest tolerable concentration to lead and copper is respectively
1500mg/L、600mg/L。
Bacterial strain of the invention can achieve 83%, 56% to the attached rate of heavy metal lead and copper respectively.
Application of the bacterial strain of the present invention in water body and heavy metal pollution of soil are administered.
Specifically, the bacterial strain of resistance to heavy metal Lechevalieria nanjingensis NJ07-19 is in terms of adsorbing heavy metal
Application process are as follows:
Lechevalieria nanjingensis NJ07-19 is inoculated in fermentation medium, in 25 DEG C, revolving speed
Shake culture 6-7d under the conditions of 150-200rpm, obtains fermentation liquid.Fermentation liquid is centrifuged, culture medium is removed, obtains thallus.By bacterium
Body is placed in the heavy metal-containing polluted water respectively containing lead, copper, zinc, nickel etc., adsorbs 36-48h.
The beneficial effects of the present invention are:
The present invention screens the actinomyces NJ07 of one plant of resistance to heavy metal, and carries out ion implanting physical mutagenesis to it, obtains
Mutant strain NJ07-19 is named as Lechevalieria nanjingensis NJ07-19.Lechevalieria
Nanjingensis NJ07-19 is more stronger than the tolerance of original strain, absorption heavy metal 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, classification naming are Lechevalieria nanjingensis NJ07-19,
It is preserved in China typical culture collection center (abbreviation CCTCC, address: the Chinese Wuhan Wuhan University), the deposit date is
On December 11st, 2015, deposit number are CCTCC No:M 2015737.
Specific embodiment
Embodiment 1: this example demonstrates that the culture medium that the present invention uses
Isolation medium: glucose 5g/L, sour hydrolyzed casein 2g/L, yeast extract 1g/L, beef extract 1g/L, agar powder 15g/
L, the heavy metals such as lead, copper (0-1800mg/L).
Fermentation medium: glucose 5g/L, sour hydrolyzed casein 2g/L, yeast extract 1g/L, beef extract 1g/L, agar powder 15g/
L。
Embodiment 2: separation, screening, the identification of actinomyces Lechevalieria NJ07
It weighs 1g and is dissolved in 100ml sterile saline rear progress gradient dilution, choosing from the soil sample in heavy metal pollution area
Take 10-2、10-3、10-4Dilution is respectively coated on the isolation medium plate containing heavy metal, and 3 repetitions of each gradient set 25
DEG C culture.The bacterium colony difference streak inoculation that picking shape, size, color etc. are different after growing bacterium colony is in corresponding plate, directly
It is fallen to no miscellaneous bacteria.The NJ07 bacterial strain determined after culture screening observes form, substrate mycelium branch hair in 25 DEG C of culture 6-7d
It reaches, be broken, gas silk is white.Morphology is carried out to NJ07 bacterial strain referring to " primary Jie Shi systematic bacteriology handbook " and Physiology and biochemistry is surveyed
It is fixed.
The main physiological and biochemical property of table 1.NJ07 bacterial strain
Note: "+" is positive reaction in table 1, and "-" is negative reaction, and " w " is weakly positive reaction
The 16S rDNA sequence that NJ07 bacterial strain is obtained by PCR, through sequencing and BLAST homology alignment and evolution point
Analysis, the results showed that the homology of NJ07 and Lechevalieria xinjianggensis sp.nov.16S rDNA is
99.1%, the homology with Lechevalieria flanava NRRL B-16131 16S rDNA is 98.5%, determines NJ07
The novel species belonged to for Lechevalieria.
Embodiment 3: mutagenesis, the screening of bacterial strain NJ07
One, the inclined-plane actinomyces NJ07 for covering with spore is taken, spore is eluted with physiological saline, fills glass loaded on sterile
In the triangular flask of pearl, 25 DEG C, 160rpm 30min fullys shake, keep spore evenly dispersed, filter, spore suspension is made, dilute
Concentration to spore is 107A/ml, the spore suspension even spread for taking 0.1ml to dilute air-dry, and are put into ion implantation apparatus
It is interior, using 15KeV N+, fluence is 50~200*1013/cm2, target chamber vacuum degree is 10-3Pa handles spore.
The spore after mutagenesis is eluted with physiological saline, is made 107The spore suspension of a/ml takes 200ul to be coated on highly concentrated
On the isolation medium of the heavy metal ion containing various concentration of degree, 25 DEG C are cultivated 6-7 days.Period observes the bacterium on plate
Form is fallen, and counts total plate count, picking is resistant to lead, the highest bacterium colony of copper concentration, finally selects bacterial strain NJ07-19.
Bacterial strain NJ07-19 is named as 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 energy of resistance to heavy metal of bacterial strain NJ07-19
By NJ07 and NJ07-19 bacterial strain be respectively coated on containing lead 800,1000,1200,1400,1600,1800mg/L and
Containing on copper 200,300,400,500,600,700, the isolation medium plate of 800mg/L, 25 DEG C are cultivated 6-7 days.NJ07 bacterium
Strain is being higher than sterile length of being born on 1200mg/L and the plate for being higher than 400mg/L containing copper concentration containing lead concentration；NJ07-19 bacterium
Strain containing lead concentration be higher than 1600mg/L and containing copper concentration higher than 600mg/L plate on sterile length of being born, to lead with
The tolerance of copper is respectively the 133.3% of original strain, 150%.
Embodiment 5: absorption property of the bacterial strain NJ07-19 to heavy metal
NJ07 and NJ07-19 bacterial strain is inoculated in fermentation medium respectively, training is shaken under the conditions of revolving speed 150-200rpm
6-7d is supported, fermentation liquid is obtained.Fermentation liquid is centrifuged, culture medium is removed, obtains thallus.It takes 1g thallus to be placed in and contains 50mg/L respectively
Lead and 50mg/L copper 1L heavy metal-containing polluted water in, absorption 48h reach balance.NJ07 is respectively to the adsorption rate of lead, copper
72%, 38.5%；NJ07-19 is respectively 72%, 38.5% to the adsorption rate of lead, copper, and the adsorption rate to lead and copper is respectively
115.2%, the 145.5% of original strain.
The calculation method of adsorption rate (R):
R (%)=(C0-Ct)/C0* 100%
Wherein, C0For the initial concentration of metal ion in solution, CtThallus be added adsorb be within t hours metal in solution from
The concentration of son.
Claims (4)
1. the actinomycetes strain of one plant of resistance to heavy metal, which is characterized in that the strain classification is named asLechevalieria nanjingensis NJ07-19, is preserved in China typical culture collection center, and deposit number is CCTCC No:M
2015737。
2. bacterial strain as described in claim 1, which is characterized in that the bacterial strain is resistant to heavy metal lead, copper, zinc, nickel, wherein
Highest tolerable concentration to lead and copper is respectively 1500mg/L, 600mg/L.
3. bacterial strain as described in claim 1, which is characterized in that the bacterial strain respectively can be with to the adsorption rate of heavy metal lead and copper
Reach 83%, 56%.
4. application of the bacterial strain as described in claim 1 in water body or heavy metal pollution of soil are administered.
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| 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 |
| 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 |
| 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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| 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 |
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| 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 |
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Address after: 211161 No. 712 Shengan Road, Binjiang Economic Development Zone, Jiangning District, Nanjing, Jiangsu. Applicant after: Nanjing Beishengrong Energy Technology Co., Ltd. Address before: 210019 2, building 2, 8 Taishan Road, Jianye District, Nanjing, Jiangsu. Applicant before: Nanjing Beishengrong Energy Technology Co., Ltd. |
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