CN104450569B - The ultrahigh concentration bacterial strain of resistance to cadmium and its separating screening method in a kind of mining soil - Google Patents
The ultrahigh concentration bacterial strain of resistance to cadmium and its separating screening method in a kind of mining soil Download PDFInfo
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- CN104450569B CN104450569B CN201410723803.XA CN201410723803A CN104450569B CN 104450569 B CN104450569 B CN 104450569B CN 201410723803 A CN201410723803 A CN 201410723803A CN 104450569 B CN104450569 B CN 104450569B
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Abstract
The invention discloses the ultrahigh concentration bacterial strain of resistance to cadmium and its separating screening method in a kind of mining soil, the entitled enterobacteria Enterobacter sp of the ultrahigh concentration bacterial strain of resistance to cadmium in mining soil of the invention, deposit number is CGMCC NO.9795.The ultrahigh concentration bacterial strain of resistance to cadmium has good heavy metal resistance cadmium tolerable concentration up to 1800mg/l in the mining soil of the present invention, and in the mining soil of the present invention ultrahigh concentration bacterial strain of resistance to cadmium separating screening method it is low to instrument requirements, it is easy to operate, it is safe and efficient.The ultrahigh concentration bacterial strain of resistance to cadmium has great importance to mining area polluted soil reparation in the mining soil of the present invention, has great society and economic value.
Description
Technical field
The present invention relates to the ultrahigh concentration bacterial strain of resistance to cadmium in technical field of soil remediation more particularly to a kind of mining soil and its
Separating screening method.
Background technology
Southern Jiangxi is known as the name of " world's tungsten is all ", and tungsten ore is numerous.While tungsten resource is exploited, also to mining area and locality
The ecosystem leaves some ecological problems, such as tungsten ore tailings stone, CHARACTERISTICS OF TAILINGS SAND cover arable land, destroy Landscape with planting
The pollutants such as the cadmium (Cd) of association in quilt, especially tungsten ore recovery process, lead (Pb), arsenic (As) give local soil and water resource band
Carry out serious heavy metal pollution.Biological prosthetic is considered as that one kind derives from a wealth of sources, processing cost is low, pollution-free and remediation efficiency is high
Method, particularly microorganism remediation technology increasingly favored by scholar.
The microorganism of repairing heavy metal pollution is mainly indigenous fungi and bacterium, and different microorganisms are to the resistance to of heavy metal pollution
Property is also different, usually fungi>Bacterium>Actinomyces.Indigenous microorganism in contaminated soil often there is higher heavy metal to resist
Property, the microorganism therefrom detached is due to adapting to local natural environment, so can effective repairing heavy metal pollution.But at present also
Do not report the bacterial strain of fine heavy metal resistance, so find a kind of bacterial strain of high heavy metal resistance, just to soil remediation with
Environmental protection has great importance.
Invention content
Base what this, the present invention provides the ultrahigh concentration bacterial strain of resistance to cadmium and its separating screening methods in a kind of mining soil, adopt
It is detached from heavy metals mining area cadmium (Cd) contaminated soil with dilution plate method, filtering out resisting high-concentration Cd, (tolerable concentration reaches
1800mg/l) microbial strains E learns that the bacterial strain is cloaca Escherichia coli Enterobacter by 16SrRNA serial analysis
sp。
The present invention adopts the following technical scheme that：
The entitled enterobacteria Enterobacter sp of the ultrahigh concentration bacterial strain of resistance to cadmium in the mining soil of the present invention；Preservation
Organization：China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address：BeiChen West Road, Chaoyang District, BeiJing City
No. 1 institute 3, Institute of Microorganism, Academia Sinica；Preservation date：On October 17th, 2014；Deposit number is CGMCC
NO.9795。
The separating screening method of the ultrahigh concentration bacterial strain of resistance to cadmium is as follows in the mining soil of the present invention：
(1) soil sample acquires：
The soil sample at 0~20cm under mining soil surface layer is acquired, then plus soil sample dilution is made in water；
(2) isolation and purification for the bacterial strain of resistance to cadmium：
10 are drawn respectively with Sterile pipette-1、10-2、10-3、10-4Each soil sample dilution 0.2mL is in Cd2+It is a concentration of
It is uniform with sterile glass rod coating in the tablet of 3 kinds of concentration cultures of 10mg/L, 30mg/L, 60mg/L, it is each concentration, every
A gradient is parallel to do 3, cultivates 2d on beef-protein medium at room temperature, and 3d is cultivated on potato culture, separation
With oese, picking is placed in Cd to the single bacterium colony grown afterwards a little respectively2+On the culture medium of the same race of concentration 60mg/L, at room temperature in ox
2d is cultivated on meat extract peptone culture medium, 3d is cultivated on potato culture, after bacterium colony is grown, is checked it is characterized in that no one
It causes, while is carried out microscopy, if for single microorganism, if finding there is miscellaneous bacteria, need to be detached, purified again, directly
To the purebred culture of acquisition；
(3) the high concentration screening of Cd resistant strains：
Purifying obtained strains are inoculated into successively containing Cd2+Concentration is respectively the same of the series concentration gradient of 60~1800mg/L
On kind culture medium, 2d is cultivated on beef-protein medium at room temperature, 3d is cultivated on potato culture, picks out anti-height
Concentration C d2+Bacterial strain, obtain the present invention the ultrahigh concentration bacterial strain of resistance to cadmium.
It in step (1), acquires in the soil sample of mining area, chooses representational irrigating region and micro organism quantity is more, type is enriched
Vegetable plot or beach acquisition soil sample (being poured for a long time with polluted source that upstream is FIX CHARGE RATE ABOUT THE WASTE ditch), because of the microorganism in soil sample
By specific environment Natural Selection.Then Cd is detected through ICP for the soil of screening2+Content reaches 5.07mg/kg.
In step (2) and step (3), the composition of the potato culture is as follows：Contain in per 1000ml water：Peeling
Potato 200g, glucose 20g, agar 20g；The composition of beef-protein medium is as follows：Contain in per 300ml water：Ox
Meat extract 1.5g, peptone 3g, NaCl1.5g；The pH value of culture medium is 7.0~7.5.
In step (2) and step (3), the preparation of the culture medium sterile mixing that need to individually sterilize again could solidify.
The positive effect of the present invention is as follows：
There is the ultrahigh concentration bacterial strain of resistance to cadmium good heavy metal resistance cadmium tolerable concentration to reach in the mining soil of the present invention
1800mg/l, and the present invention mining soil in the ultrahigh concentration bacterial strain of resistance to cadmium separating screening method it is low to instrument requirements, behaviour
Make simply, it is safe and efficient.The ultrahigh concentration bacterial strain of resistance to cadmium has weight to mining area polluted soil reparation in the mining soil of the present invention
The meaning wanted has great society and economic value.
Specific embodiment
The following examples are that the present invention is described in further detail.
Embodiment 1
1) test material
(1) experiment soil sample picks up from soil for screening of the Ganzhou Chongyi tungsten ore Kuang Ji areas through sampling and detecting in advance
Earth detects Cd through ICP2+Content reaches 5.07mg/kg.Under surface layer at 0~20cm.
(2) potato culture：Peeled potatoes 200g, glucose 20g, agar 20g, water 1000ml, nature pH.
(3) beef-protein medium：Beef extract 1.5g, peptone 3g, NaCl1.5g, water 300ml, pH7.0~7.5.
2) separation, purifying, the screening and identification for the bacterial strain of resistance to cadmium
(1) the isolation and purification for the bacterial strain of resistance to cadmium：10 are drawn respectively with Sterile pipette-1、10-2、10-3、10-4Each soil sample dilution
Liquid 0.2mL is in Cd2+In the tablet of 3 kinds of concentration cultures of a concentration of 10mg/L, 30mg/L, 60mg/L, with sterile glass rod coating
Cloth is uniformly (each concentration, each gradient are parallel to do 3).It is cultivated on beef-protein medium under room temperature (25 DEG C or so)
2d cultivates 3d on potato culture.With oese, picking is placed in Cd to the single bacterium colony grown after separation a little respectively2+Concentration
On the culture medium of the same race of 60mg/L, 2d is cultivated on beef-protein medium at room temperature, 3d is cultivated on potato culture.
After bacterium colony is grown, check it is characterized in that no consistent, while is carried out microscopy, if for single microorganism.If it was found that have
Miscellaneous bacteria need to be detached, be purified again, until obtaining purebred culture.
(2) the high concentration screening of Cd resistant strains：Purifying obtained strains are inoculated into successively containing Cd2+Concentration is respectively 60~
On the culture medium of the same race of the series concentration gradient of 1800mg/L, 2d, Ma Ling are cultivated on beef-protein medium at room temperature
3d is cultivated on potato culture medium.Pick out anti-high concentrations of Cd2+Strain excellent.In the process, with the height of what screening separate use
The preparation of the concentration cultures sterile mixing that need to individually sterilize again could solidify.Screen the bacterial strain maximal tolerable concentration Cd of gained2+
1800mg/l。
(3) morphology Preliminary Identification and molecular biology identification：Activated spawn 12h takes the strain dilution 10 of activation-1、
10-2、10-3, it is connected on fresh culture, growth form is observed by Gram's stain after culture 3d at room temperature.And it samples
Carry out 16srRNA serial analysis.3) qualification result of bacterial strain
The ultrahigh concentration bacterial strain of resistance to cadmium is Gram-negative in the mining soil of the present invention, rod-short, binding molecule life
Object 16SrRNA serial analysis understands one unnamed kind that the bacterial strain is cloaca Escherichia coli Enterobacter sp subordinates.
4) bacterial strain studies the adsorption effect of cadmium in simulated wastewater
Activation thalline is inoculated into 2% inoculum concentration in fresh culture medium, pH7.0~7.5,30 DEG C, moderate rotation
Shaking flask culture is for 24 hours.10000r centrifuges 5min, collects thalline, and thalline is washed twice through deionized water, discarded supernatant.Weigh wet bacterium
The bacteria suspension of 200mg/L is made in body.
It takes containing Cd2+Each 20mL of solution of 50mg/L, 100mg/L, 200mg/L, are respectively put into 250mL triangular flasks, then divide
Not plus the above-mentioned bacteria suspensions of people 20mL (being repeated 3 times per sample), be placed on 30 DEG C of shaking bath, after moderate rotation oscillation 30min,
10000r centrifuges 5min, pipettes certain supernatant, and ICP method measures concentration of metal ions.Blank control for 20mL solions and
30min is vibrated in the mixed liquor of 20mL deionized waters, similary water-bath.
Adsorption rate=(C0-Ct)/C0× 100%
In formula:C0Concentration (mg/L) for blank control；
CtFor the supernatant (mg/L) after absorption；
Cd of the wet thallus to various concentration is understood with calculating after testing2+Solution adsorption rate is respectively 69.65%, 73.45%,
78.70%.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace
And modification, the scope of the present invention is defined by the appended.
Claims (1)
1. one plant of resistance to cadmium enterobacteria (Enterobacter sp.) for being isolated from mining soil, deposit number CGMCC
No.9795, bacterial strain maximum tolerance Cd2+A concentration of 1800mg/L.
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607248B (en) * | 2018-06-16 | 2021-01-01 | 华中农业大学 | Fogerella strain A6 for repairing soil cadmium pollution and application |
| CN108753667B (en) * | 2018-07-03 | 2021-07-06 | 湖南大学 | Heavy metal tolerant enterobacter populus YG-14 |
| CN109266575B (en) * | 2018-09-17 | 2021-06-11 | 广西师范大学 | Cadmium-resistant intestinal strain and method for adsorbing cadmium |
| CN109909294B (en) * | 2019-04-18 | 2021-03-23 | 山东省食品药品检验研究院 | Microbial remediation method for heavy metal contaminated soil |
| CN111925950A (en) * | 2020-03-17 | 2020-11-13 | 徐州工程学院 | High-cadmium-resistance strain and separation and screening method thereof |
| CN114774319B (en) * | 2022-04-24 | 2023-10-27 | 湖南农业大学 | Enterobacter strain for passivating cadmium and arsenic |
| CN114766118B (en) * | 2022-04-24 | 2024-06-04 | 湖南农业大学 | Application of enterobacter M5 in inhibiting rice from absorbing and accumulating cadmium and arsenic |
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Non-Patent Citations (3)
| Title |
|---|
| Biosorption of lead, copper and cadmium by an indigenous isolate Enterobacter sp. J1 possessing high heavy-metal resistance;Wei-Bin Lu et al.;《Journal of Hazardous Materials》;20051128;第B134卷;摘要，第81页左栏第2段，右栏最后1段至第82页左栏第1段，第85页左栏4.总结 * |
| Uptake of some heavy metals by metal resistant Enterobacter sp. isolate from Egypt;Abd El-Raheem R. El-Shanshoury et al.;《African Journal of Microbiology Research》;20130604;第7卷(第23期);摘要，第2877页右栏倒数第2段，第2879页右栏第2段-2881页左栏第1段 * |
| 耐镉肠杆菌的分子鉴定和抗性分析;侯进慧 等;《食品工业科技》;20131231;第34卷(第7期);第149页摘要，第150页1.2筛选耐镉细菌，第150右栏2.结果与分析，图3 * |
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