CN110484485A - A method of the sulfate reducing bacteria of the high resistance to cadmium of separation - Google Patents
A method of the sulfate reducing bacteria of the high resistance to cadmium of separation Download PDFInfo
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- CN110484485A CN110484485A CN201910836792.9A CN201910836792A CN110484485A CN 110484485 A CN110484485 A CN 110484485A CN 201910836792 A CN201910836792 A CN 201910836792A CN 110484485 A CN110484485 A CN 110484485A
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- 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
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- 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
- C12N1/02—Separating microorganisms from their culture media
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- 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
- C12N1/36—Adaptation or attenuation of cells
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Abstract
The invention discloses a kind of method of the sulfate reducing bacteria of the high resistance to cadmium of separation, include the following steps: that step 1) will be activated from the sulfate reducing bacteria sample in cadmium pollution place；Step 2) isolates and purifies sulfate reducing bacteria sample；The bacterial screening of step 3) tolerance cadmium；Step 4) carries out oxytolerant domestication to the bacterium of high tolerance cadmium；Step 5) laboratory lab scale.
Description
Technical field
The present invention relates to strain separating technical field, in particular to the side of the sulfate reducing bacteria of a kind of high resistance to cadmium of separation
Method.
Background technique
Sulfate reducing bacteria (Soulfate-Reducing Bacteria, abbreviation SRB) is a kind of microorganism of anaerobism.Extensively
It is general to be present in the anaerobic environments such as soil, seawater, river water, underground piping and oil/gas well.Research shows that in anaerobic or few oxygen
In the case of, it can be using the organic matter of metal surface as carbon source, and utilizes the hydrogen generated in bacterial biof iotalm, also by sulfate
Original obtains the energy of existence at hydrogen sulfide from redox reaction.
Southern region of China heavy metals in farmland cadmium pollution is serious, and agricultural product cadmium content severely exceeds.Since cadmium is toxic member
Element, toxicity of compound are bigger.In nature, the compound of cadmium has different toxicity.Cadmium sulfide, selenium sulfonic acid cadmium toxicity compared with
Low, cadmium oxide, caddy, sulfuric acid cadmium toxicity are higher.But there are problems for modifying agent product now, and reclamation result is bad, changes
Good effect is affected by outside environmental elements, cannot reduce sulfuric acid cadmium content in soil well.It therefore can be using biology
The method of reparation carries out soil remediation and improvement to cadmium pollution place using the sulfate reducing bacteria of high resistance to cadmium.
The method that the method for existing separation sulfate reducing bacteria mostly uses inflated with nitrogen, keeps in cultivating system being strictly to detest
Oxygen, or cultivated in carbon dioxide incubator.The bacterium for isolating and purifying out is being inoculated into the training containing metal ion
It supports in base, carries out selective screening.Screening is divided into primary dcreening operation and secondary screening, and the purpose of primary dcreening operation is to filter out the bacterial strain with the function,
The purpose of secondary screening is for the stronger representational bacterial strain of screening function.But the condition of current strictly anaerobic is in laboratory
It is easily done, but bad realization in actual production, so to carry out the improvement of method, and certain oxytolerant is done to bacterial strain and is tamed and dociled
Change.
Summary of the invention
The objective disadvantage of the prior art: when cultivating and isolating and purifying sulfate reducing bacteria, the condition of strictly anaerobic is in reality
Test interior to be easily done, but bad realization in actual production, if increase bacterium it is not in place if be easy to allow other dominant bacteria occupy-places,
Sulfate reducing bacteria just can not be grown.Therefore the invention proposes following technical solutions:
A method of the sulfate reducing bacteria of the high resistance to cadmium of separation includes the following steps:
Step 1, it in the sulfate reducing bacteria sample inoculation culture bottle in cadmium pollution place, will carry out increasing bacterium training three times
It supports；
Step 2, increase sampling in the culture bottle of bacterium to third time and carry out plate streaking separation, constant temperature is trained in an anaerobic environment
It supports, then picking black colonies carry out isolating and purifying 3-5 times again, tentatively judge purity by microscope；
Step 3, the sulfate reducing bacteria of purifying is inoculated into constant-temperatureanaerobic anaerobic culture in fluid nutrient medium, is then inoculated in one
In the sulfate reducing bacteria fluid nutrient medium of serial difference cadmium concentration gradient, constant temperature stationary culture；
Step 4, it is cultivated under the anaerobic environment gradually weakened；
Step 5, it uses the soil of cadmium pollution as culture medium, culture solution is embedded in contaminated soil, results of regular determination soil
Cadmium content in sample.
Preferably, in the step 1, used culture medium is the dedicated culture of sulfate reducing bacteria in the culture bottle
Base.
Preferably, the step 1, further are as follows: in the Zengjing Granule three times, use unsterilised sulfuric acid for the first time
Salt reducing bacteria special culture media, and inoculum concentration is not less than 10%；Second special using the sulfate reducing bacteria of sterilizing with third time
Use culture medium.
Preferably, in the step 2, the anaerobic environment is by anaerobism bag and produces gas bag acquisition
Preferably, in the step 2, the temperature of the constant temperature incubation is 30-35 DEG C.
Preferably, in the step 3, the difference cadmium concentration gradient is 25mg/L~100mg/L.Preferably, described
Constant-temperatureanaerobic anaerobic culture described in step 3 when it is 7 days a length of.
Preferably, the step 3, further are as follows: the bacterium solution after the constant-temperatureanaerobic anaerobic culture is inoculated in 5% inoculum concentration
In a series of sulfate reducing bacteria fluid nutrient medium of difference cadmium concentration gradients.
Preferably, the step 4, further are as follows: pass through removal anaerobism bag and reduce the body of fluid nutrient medium in culture bottle
Product is gradually to weaken anaerobic environment.
Preferably, the fluid nutrient medium volume accounting in the culture bottle is 80%.
Compared with prior art, the method for the sulfate reducing bacteria of the high resistance to cadmium of one-kind of separation of the present invention, reaches
Following effect:
(1) method of the sulfate reducing bacteria of the high resistance to cadmium of a kind of separation provided by the invention, for sulfate reducing bacteria
Increase bacterium part to improve, in the growth for guaranteeing to inhibit other advantage miscellaneous bacterias in the case where sulfate reducing bacteria steady growth.
(2) method of the sulfate reducing bacteria of the high resistance to cadmium of a kind of separation provided by the invention, for sulphur
Hydrochlorate reducing bacteria has carried out oxytolerant domestication, so that it is more preferably realized in actual industrial production.
(3) method of the sulfate reducing bacteria of the high resistance to cadmium of a kind of separation provided by the invention, the bacterial strain after screening is to cadmium
Tolerance is very strong, is conducive to biological prosthetic progress.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.
Fig. 1 is a kind of flow chart of the method for the sulfate reducing bacteria of the high resistance to cadmium of separation described in embodiment.
Specific embodiment
The application is described in further detail below in conjunction with attached drawing, but not as the restriction to the application.
As shown in Figure 1, firstly, sample source of the present invention is in cadmium pollution place.The first step is to swash sample in separation process
It is living, that is, increase bacterium.The enriched medium that this step present invention uses is sulfate reducing bacteria special culture media.Increase bacterium in three times, the
Primary to increase bacterium using being unsterilised sulfate reducing bacteria special culture media, inoculum concentration is not less than 10%, and second and the
Increase bacterium use three times is all the sulfate reducing bacteria special culture media by sterilization treatment.
It finds to generate black precipitate in culture bottle after increasing bacterium, bottleneck has bubble generation, and bottleneck has stink generation, explanation
There is a large amount of sulfate reducing bacteria to generate.
Second step is exactly the sampling progress plate streaking separation out of third time increases bacterium culture bottle.Tentatively want to obtain purebred sulphur
Hydrochlorate reducing bacteria is also to ensure that and carries out in the environment of opposite anaerobism.The plate isolated and purified, the anaerobism that uses in the present invention
The method that method is anaerobism bag and production gas bag.After (30 DEG C~35 DEG C) of constant temperature are cultivated 7 days, anaerobism bag is opened, can be generated in sack
Stink, picking planar surface black colonies.Isolating and purifying so to carry out 3-5 times, tentatively judge purity by sediments microscope inspection.
Third step is exactly the bacterial screening for being resistant to cadmium.The sulfate reducing bacteria of purifying is inoculated into hydraulically full culture first
In the culture tube of base, constant temperature (30 DEG C~35 DEG C Anaerobic culturel 7 days, culture tube blackening has stink generation, culture can be terminated, to
With.Bacterium solution is inoculated in a series of sulfate of different cadmium concentration gradients (25mg/L^100mg/L) respectively with 5% inoculum concentration
In reducing bacteria fluid nutrient medium, constant temperature (30 DEG C~35 DEG C) stationary culture observes culture medium color change situation, it can be observed that
Culture medium has and pale yellow becomes black.
4th step carries out oxytolerant domestication to the bacterium of high tolerance cadmium.Gradually weaken anaerobic environment, first removal anaerobism bag, just
Normal CMC model, and fluid nutrient medium is made to be full of the 80% of culture bottle.
5th step is laboratory lab scale, is used as culture in the soil of use for laboratory pollution cadmium, culture solution is embedded in dirt
It contaminates in soil, the cadmium content in results of regular determination pedotheque.
Compared with prior art, the method for the sulfate reducing bacteria of the high resistance to cadmium of a kind of separation of the present invention, reaches
Following effect:
(1) method of the sulfate reducing bacteria of the high resistance to cadmium of a kind of separation provided by the invention, for sulfate reducing bacteria
Increase bacterium part to improve, in the growth for guaranteeing to inhibit other advantage miscellaneous bacterias in the case where sulfate reducing bacteria steady growth.
(2) method of the sulfate reducing bacteria of the high resistance to cadmium of a kind of separation provided by the invention, for sulfate reducing bacteria into
Oxytolerant domestication is gone, so that it is more preferably realized in actual industrial production.
(3) method of the sulfate reducing bacteria of the high resistance to cadmium of a kind of separation provided by the invention, the bacterial strain after screening is to cadmium
Tolerance is very strong, is conducive to biological prosthetic progress.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replaces and deforms.
Claims (9)
1. a kind of method of the sulfate reducing bacteria of the high resistance to cadmium of separation, includes the following steps:
Step 1, by the sulfate reducing bacteria sample inoculation culture bottle in cadmium pollution place, Zengjing Granule is carried out；
Step 2, plate streaking separation is carried out to sampling in the culture bottle after increasing bacterium, constant temperature incubation, picking are black in an anaerobic environment
Color bacterium colony tentatively judges purity by microscope；
Step 3, the sulfate reducing bacteria of purifying is inoculated into constant-temperatureanaerobic anaerobic culture in fluid nutrient medium, be then inoculated in a series of
In the fluid nutrient medium of different cadmium concentration gradients, constant temperature stationary culture；
Step 4, it is cultivated under the anaerobic environment gradually weakened；
Step 5, it uses the soil of cadmium pollution as culture medium, culture solution is embedded in contaminated soil, results of regular determination pedotheque
In cadmium content.
2. the method for the sulfate reducing bacteria of the high resistance to cadmium of separation according to claim 1, which is characterized in that in the step
In 1, used culture medium is sulfate reducing bacteria special culture media in the culture bottle.
3. the method for the sulfate reducing bacteria of the high resistance to cadmium of separation according to claim 2, which is characterized in that the step 1,
Further are as follows: in the Zengjing Granule three times, use unsterilised sulfate reducing bacteria special culture media for the first time, and be inoculated with
Amount is not less than 10%；Second with third time using the sulfate reducing bacteria special culture media of sterilizing.
4. the method for the sulfate reducing bacteria of the high resistance to cadmium of separation according to claim 1, which is characterized in that the step 2
In, the anaerobic environment is by anaerobism bag and produces gas bag acquisition.
5. the method for the sulfate reducing bacteria of the high resistance to cadmium of separation according to claim 4, which is characterized in that the step 2
In, the temperature of the constant temperature incubation is 30-35 DEG C.
6. the method for the sulfate reducing bacteria according to the high resistance to cadmium of separation described in claim 5, which is characterized in that step 2 weight
It carries out 3-5 times again.
7. the method for the sulfate reducing bacteria of the high resistance to cadmium of separation according to claim 1, which is characterized in that the step 3
In, the difference cadmium concentration gradient is 25mg/L~100mg/L.
8. the method for the sulfate reducing bacteria of the high resistance to cadmium of separation according to claim 7, which is characterized in that in the step
Constant-temperatureanaerobic anaerobic culture described in 3 when it is 7 days a length of.
9. the method for the sulfate reducing bacteria of the high resistance to cadmium of separation according to claim 1, which is characterized in that the step 4,
Further are as follows: pass through removal anaerobism bag and reduce the volume of fluid nutrient medium in culture bottle gradually to weaken anaerobic environment.
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| CN201910836792.9A CN110484485A (en) | 2019-09-05 | 2019-09-05 | A method of the sulfate reducing bacteria of the high resistance to cadmium of separation |
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| CN201910836792.9A CN110484485A (en) | 2019-09-05 | 2019-09-05 | A method of the sulfate reducing bacteria of the high resistance to cadmium of separation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110937692A (en) * | 2019-12-02 | 2020-03-31 | 江苏艾特克环境工程设计研究院有限公司 | Basalt fiber-based sulfate reducing bacteria fixing device and method |
Citations (2)
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| CN103523909A (en) * | 2013-11-01 | 2014-01-22 | 南昌航空大学 | Process method and reactor for recycling elemental sulfur from organic waste water containing sulfate |
| US20180051309A1 (en) * | 2016-08-18 | 2018-02-22 | Exxonmobil Research And Engineering Company | Methods to assess monitor and control bacterial biofilms |
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2019
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103523909A (en) * | 2013-11-01 | 2014-01-22 | 南昌航空大学 | Process method and reactor for recycling elemental sulfur from organic waste water containing sulfate |
| US20180051309A1 (en) * | 2016-08-18 | 2018-02-22 | Exxonmobil Research And Engineering Company | Methods to assess monitor and control bacterial biofilms |
Non-Patent Citations (4)
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110937692A (en) * | 2019-12-02 | 2020-03-31 | 江苏艾特克环境工程设计研究院有限公司 | Basalt fiber-based sulfate reducing bacteria fixing device and method |
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Application publication date: 20191122 |