CN104004691B - A kind of bacterial strain repaired for As polluted soil and application process thereof - Google Patents
A kind of bacterial strain repaired for As polluted soil and application process thereof Download PDFInfo
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- CN104004691B CN104004691B CN201410266798.4A CN201410266798A CN104004691B CN 104004691 B CN104004691 B CN 104004691B CN 201410266798 A CN201410266798 A CN 201410266798A CN 104004691 B CN104004691 B CN 104004691B
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Abstract
The invention discloses a kind of bacterial strain Brevibacterium sp.YZ 1 repaired for As polluted soil and application process thereof, this bacterial strain preserving number is CGMCC No.8329；It reaches more than 1000mg/L to the toleration of As (III), can be by As (III) solution oxide more than 75% that concentration is 100mg/L.This bacterial strain can make water-soluble state As (III) of As polluted soil reduce by 82.6%, and available state As (III) reduces by 84.1%.Significantly reduce the toxicity of arsenic in environment.Research shows that the bacterial strain of the present invention has a good application prospect in terms of environment As pollution improvement.
Description
Technical field
The invention belongs to arsenic contaminated soil microorganism remediation field, be specifically related to what a kind of As polluted soil was repaired
Bacterial strain and application thereof.
Background technology
There are Xinjiang, Inner Mongol, Qinghai, Hunan, Yunnan and Guangxi etc. in China by the with serious pollution province of arsenic.Arsenic
Pollute mainly because artificially causing, such as exploitation and smelting, the processing of arsenic product and use, the coal of metallic ore
Burning etc..Arsenic is a kind of severe toxicity metalloid, main with arsenite (As (III)) and arsenic acid in natural environment
The inorganic form of salt (As (V)) exists and passes through oxidation and the reduction mutually conversion of microorganism.As (III) is not charged
Mobility is higher bigger to organism harm, and As (III) is higher than As (V) toxicity about 100 times, by As (III)
Being oxidized to As (V) is a detoxification processes.
It is serious, in the urgent need to the water and soil arsenic pollution of some economical and efficients of research and development that China is polluted persecution by arsenic
Purification techniques.The at present reparation of arsenium contaminated environment has traditional chemical oxidation, precipitation, ion exchange, absorption
Etc. method, these methods have cost high, easily bring the shortcomings such as secondary pollution.Occurred in that phytoremediation side later
Method, as Herba pteridis vittatae can pass through the arsenic of root absorption high concentration and be enriched in internal, safety high cost is relatively low,
But the cycle is oversize, there is also and how to dispose a difficult problem for plant after harvesting.
Newly occur in that microbial process, such as activated sludge process, microbial oxidation.Along with research
Progress, increasing As (III) oxidation bacteria is found.Study more typical strain and have Alcaligenes
faeealis NCIB8687、Rhizobium sp.NT-26、Agrobacrerium tumefaciens5A、
Herminiimonas arsenicoxvdans ULPAsl, Thiomonas sp.3As, these bacterial strains can be by poison
The As (III) that property is strong is oxidized to the weak As of toxicity (V).The most not yet it is related to Brevibacterium sp.
The report of oxidation As (III).The present invention is that arsenic contaminating microorganisms reparation provides new microbial resources.
Summary of the invention
It is an object of the invention to provide a kind of new arsenic oxidative function bacterial strain and for As polluted soil reparation
Application process, this bacterial strain has the function that trivalent arsenic is oxidized to pentavalent arsenic, this bacterial strain is used for arsenic pollute
The improvement of soil, low cost, simple to operate.
A kind of bacterial strain repaired for As polluted soil, described bacterial strain is brevibacterium (Brevibacterium sp.)
YZ-1, preserving number is CGMCC No.8329.
The application process of the described bacterial strain repaired for As polluted soil, adds described bacterial strain in soil
Trivalent arsenic is oxidized to pentavalent arsenic.Specifically As polluted soil crushing grinding is added Brevibacterium sp.
YZ-1 bacterium solution, in its bacterium solution, bacterial number reaches 104～105/ml.Bacterium solution addition is soil and bacterium solution
Volume ratio is 1g:2mL, and mix homogeneously soaks, stirs, and processes at least 10 days, filters.
The process screening, separate, tame this bacterial strain in the present invention is: gather Realgar Ore District, Shimen County, Hunan Province
As polluted soil, cultivates 2～3 days in LB culture medium, takes top mud and uses gradient dilution method, and coating contains
The solid culture of 100mg/LAs (III) is cultivated 3～4 days based on 30 DEG C, is continuously separated purification and obtains different shape
Single bacterium colony, uses AgNO3Solution is identified, colony edge is brown, can tentatively predicate arsenic oxidizing bacteria.Choose
Take isolated single colony inoculation to cultivate 3 days in the fluid medium containing As (III), step up As (III)
Concentration, carries out gradient domestication from 50～200mg/L, and every kind of culture medium is repeated 2 times.In detection fluid medium
Remaining As (III) content, determines the bacterial strain that arsenic oxidability is optimal.
Solid medium consists of: the LB culture medium of 15g/L, 2g/L sodium lactate, 2g/L potassium dihydrogen phosphate,
The magnesium sulfate of 2g/L and 15g/L agar, pH=7～8.
Fluid medium consists of: the LB culture medium of 15g/L, 2g/L sodium lactate, 2g/L potassium dihydrogen phosphate,
The magnesium sulfate of 2g/L, pH=7～8.
Gradient domestication time As (III) Concentraton gradient be respectively as follows: 50mg/L, 80mg/L, 100mg/L, 150mg/L,
200mg/L。
The present invention gathers Realgar Ore District, Shimen County, Hunan Province As polluted soil, through screening, separate, taming,
Obtain a kind of bacterial strain with oxidation trivalent arsenic function.It is Brevibacterium through 16S rRNA gene identification
Sp. belong to.
This Strain Designation is brevibacterium (Brevibacterium sp.) YZ-1；
Preservation date: on October 12nd, 2013；
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)；
Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；
Preserving number: CGMCC No.8329.
The advantage of the present invention:
1.As (III) is higher than As (V) toxicity about 100 times, and As (III) not charged mobility is higher endangers to organism
Evil is bigger.As (III) can be aoxidized by the bacterial strain Brevibacterium sp.YZ-1 that separation screening of the present invention obtains
Become As (V), As (III) solution oxide more than 75% of 100mg/L can be significantly reduced arsenic in environment
Harm.
Bacterial strain the most of the present invention is that separation screening obtains from the As polluted soil that experienced by many decades, to As (III)
Toleration reach more than 1000mg/L.This bacterial strain can make water-soluble state As (III) in As polluted soil reduce
80%, available state As (III) reduces by 84.1%.There are some researches show that this bacterial strain is expected in terms of purifying arsenic contamination from now
Play significant role.
The most existing arsenic oxidizing bacteria is mostly acidophilia bacterium and acid-producing bacteria.Bacterial strain of the present invention is arsenious same in oxidation
Time can metabolism produce alkaline matter, bacterium solution pH is increased to about 10 by initial value 7～8.The product of this bacterial strain
Alkali characteristic is more conducive to repair As polluted soil.The alkaline matter that this bacterial strain metabolism produces can increase Arsenic in Soil
As (III) in mobility, beneficially solid phase of soil moves in liquid phase, it is simple to the As (III) in bacterial strain oxidation soil.
Accompanying drawing explanation
The Phylogenetic Analysis tree of Fig. 1: bacterial strain of the present invention；
Fig. 2: strain morphology figure of the present invention；
The oxidation effectiveness figure of Fig. 3: bacterial strain of the present invention oxidation As (III)；
Fig. 4: the bacterial strain of the present invention toleration to As (III)；
Fig. 5: bacterial strain of the present invention processes the design sketch of As (III) in As polluted soil.
Detailed description of the invention:
Below by way of case study on implementation, the invention will be further described, and the unrestricted present invention.
Embodiment 1: the isolated and purified and domestication of bacterial strain of the present invention
Gather Realgar Ore District, Shimen County, Hunan Province As polluted soil, take 5g soil in the LB liquid of 50mL sterilizing
Body culture medium is cultivated 2 days, takes 1mL top mud and be diluted to 10 by gradient dilution method-5、10-6、10-7,
With liquid-transfering gun from 10-6、10-7Diluent takes the 0.1mL coating solid culture containing 100mg/L As (III) respectively
Cultivate 3～4 days based on 30 DEG C.Solid medium consists of: the LB culture medium of 15g/L, 2g/L sodium lactate,
2g/L potassium dihydrogen phosphate, the magnesium sulfate of 2g/L and 15g/L agar, pH=7～8.With aseptic inoculation pin picking not
With the bacterium colony of form, streak inoculation is cultivated 3 days based on 30 DEG C in the solid culture of 100mg/L As (III).Even
Continuous isolated and purified more than 3 times, obtain the single bacterium colony of different shape.
With the AgNO of 2mL1%3Solution covers culture medium flat plate, and colony edge is brown, tentatively predicates anti-
Arsenic oxidizing bacteria.The anti-arsenic oxidizing bacteria list bacterium colony that picking Preliminary screening goes out is transferred, and steps up As (III) dense
Degree carries out gradient domestication, from 50mg/L, 80mg/L, 100mg/L, 150mg/L, 200mg/L, every kind of training
Foster base is repeated 2 times.In detection fluid medium, remaining As (III) content, determines the bacterium that arsenic oxidability is optimal
Strain.
Embodiment 2: bacterial strain Biology identification of the present invention and morphological characteristic
Use bacterial genomes DNA extraction agent box that the genomic DNA of bacterial strain is extracted, then adopt
With 16S rRNA gene universal primer, 16S rRNA gene is expanded and checks order.
16S rRNA gene order is utilized to construct phylogenetic tree, this bacterial strain and Brevibacterium
Yomogidense gathers in cluster, and similarity reaches 99%, with Brevibacterium sp.JC4316S rRNA
Gene similarity reaches 100%, named Brevibacterium sp.YZ-1.The phylogeny of this bacterial strain divides
Analysis tree is as shown in Figure 1.
This bacterial strain thalline is shaft-like, belongs to brevibacterium.The scanning electron microscope (SEM) photograph of this bacterial strain, is shown in Fig. 2.
Embodiment 3: bacterial strain of the present invention is to the oxidation efficiency of As (III) and toleration
By the bacterium colony of inoculating loop picking purification shaken cultivation 24h in the fluid medium of 100mL sterilizing.
Then pipette 5mL bacterium solution and be inoculated into the sterilizing that As (III) concentration is respectively the 50mL of 50mg/L, 100mg/L
In culture medium, in 30 DEG C, shaken cultivation 72h.Period, cultivating 24,36 and 72h respectively, measuring it
Remaining As (III) content.Concentration is As (III) solution of 100mg/L oxidized more than 75%, and concentration is
As (III) solution of 50mg/L is fully oxidized substantially.As shown in Figure 3.
With identical inoculum concentration be inoculated in respectively containing As (III) 200mg/L, 400mg/L, 600mg/L, 800mg/L,
In 1000mg/L, 1200mg/L, 1500mg/L fluid medium, 30 DEG C of shaken cultivation 24h, use UV, visible light
Light spectrophotometer measures absorbance OD600 at 600nm.The toleration of As (III) is reached by this bacterial strain
More than 1000mg/L.Result is as shown in Figure 4.
Embodiment 4: As polluted soil repairing test
Shimen Hunan As polluted soil crushing grinding crosses 18 mesh sieves, weighs 100g soil, by soil liquor ratio (g:mL)
Add culture medium bacterium solution, mix homogeneously for 1:2, soak and agitation as appropriate, be placed in 30 DEG C of constant incubators
After cultivating 10 days, sucking filtration, measure water-soluble state and the concentration of available state As (III) in soil.The water-soluble state of soil
As (III) is reduced to 2.86mg/kg by original 16.46mg/kg, and the available state As (III) of soil is by original
84.33mg/kg is reduced to 13.47mg/kg, and water-soluble state As (III) reduces 82.6%, and available state As (III) drops
Low 84.1% (as shown in Figure 5).
Claims (2)
1. one kind for As polluted soil repair bacterial strain, it is characterised in that described bacterial strain be brevibacterium (BrevibacteriumSp.) YZ-1, preserving number is CGMCC No.8329.
2. the application process of the bacterial strain repaired for As polluted soil described in claim 1, it is characterised in that described bacterial strain is added in soil trivalent arsenic is oxidized to pentavalent arsenic；
In application process, As polluted soil crushing grinding is addedBrevibacteriumSp.YZ-1 bacterium solution, in its bacterium solution, bacterial concentration reaches 104～105Individual/ml, bacterium solution addition is soil and the mass volume ratio of bacterium solution is 1g:2mL, mix homogeneously, soaks, stir, processes at least 10 days, filtration.
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| CN105483038B (en) * | 2015-12-17 | 2018-06-08 | 南京农业大学 | The aerobic arsenic of one plant of thermophilic fiber Cordycepps methylates bacterium SM-1 and its application |
| CN109158420B (en) * | 2018-08-27 | 2019-10-18 | 湖北师范大学 | A kind of As polluted soil renovation agent and application thereof |
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| CN101735997A (en) * | 2009-12-10 | 2010-06-16 | 中国科学院地理科学与资源研究所 | Microorganism bactericide capable of reinforcing plants to restore fields polluted by arsenic and preparation and application methods thereof |
| CN102206494A (en) * | 2011-03-30 | 2011-10-05 | 云南省农业科学院质量标准与检测技术研究所 | Modifying agent capable of passivating vegetable garden soil heavy metal and preparation method and applying method thereof |
| CN103773375A (en) * | 2014-01-07 | 2014-05-07 | 中南大学 | Arsenic-contaminated soil repairing and retaining agent based on microbe catalytic synthesis, and preparation and application thereof |
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|---|---|---|---|---|
| CN101735997A (en) * | 2009-12-10 | 2010-06-16 | 中国科学院地理科学与资源研究所 | Microorganism bactericide capable of reinforcing plants to restore fields polluted by arsenic and preparation and application methods thereof |
| CN102206494A (en) * | 2011-03-30 | 2011-10-05 | 云南省农业科学院质量标准与检测技术研究所 | Modifying agent capable of passivating vegetable garden soil heavy metal and preparation method and applying method thereof |
| CN103773375A (en) * | 2014-01-07 | 2014-05-07 | 中南大学 | Arsenic-contaminated soil repairing and retaining agent based on microbe catalytic synthesis, and preparation and application thereof |
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| ARSENIC-TOLERANT PLANT-GROWTH-PROMOTING BACTERIA ISOLATED FROM ARSENIC-POLLUTED SOILS IN SOUTH KOREA;CHARLOTTE C.SHAGOL ET AL;《ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH》;20140416;第21卷(第15期);摘要、第9358页右栏第1-2段、第9362页左栏第1段 * |
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