CN107384796A - The method of concentration and separation microorganism, corresponding culture medium and preparation method in a kind of sample from extreme acid condition - Google Patents
The method of concentration and separation microorganism, corresponding culture medium and preparation method in a kind of sample from extreme acid condition Download PDFInfo
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- CN107384796A CN107384796A CN201710776805.9A CN201710776805A CN107384796A CN 107384796 A CN107384796 A CN 107384796A CN 201710776805 A CN201710776805 A CN 201710776805A CN 107384796 A CN107384796 A CN 107384796A
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Abstract
The invention discloses method, corresponding culture medium and the preparation method of concentration and separation microorganism in a kind of sample from extreme acid condition.The invention discloses a kind of microbial liquid enriched medium and a kind of microbial solid plating medium, both culture mediums can be used for the separate microorganism from extreme acid condition sample.The present invention is the enrichment of simulation extreme acid condition multiple trace element culture medium, separation and identification extreme acid condition microorganism, by enrichment and separation method of the present invention, some rare microbe groups can be made the increase of individual amount occur, so as to significantly improve its separated probability, the microbial diversity of separating resulting is added, the identification and research to microorganism in extreme acid condition have important value and meaning.
Description
Technical field
The invention belongs to Situation of Microorganism Under Extremity Environment separation technology field, more specifically to a kind of acidic mine waste water
Method, corresponding culture medium and the preparation method of middle concentration and separation microorganism.
Background technology
Acidic mine waste water (acid mine drainage, AMD) is commonly referred to as the leaching water of abandoned mine and mine tailing, presents
Extremely low acidity (pH<3), the metal containing high concentration (Pb, Zn, Cu, Mn, Cd, As, Fe, Al etc.) and nonmetallic ion, quilt
It is considered as a kind of extreme sour environment, and the serious pollution problem that global mining industry generally faces.This kind of acid waste water
Destructive destruction can be caused to the land of surrounding and water ecosystem, cause most animals and plants, fish, algae etc. raw
Thing is dead, directly endangers human survival.The acidic mine waste water pollution problem in China is also quite serious, and the whole nation tills the land area
About 10% by heavy metal pollution (about 1.5 hundred million mu), every year because the grain of heavy metal pollution is up to 12,000,000 tons, caused by it is straight
Economic loss is connect more than 20,000,000,000 yuan.Guangdong is as economic development forefront, and seriously pollution and ecological disruption problem are existing in mining area
More than 20 years, acid wastewater in mine largely containing toxic heavy metal ion is unprocessed be just directly discharged into downstream river among, make
The ecosystem of mining area and its river alongshore is obtained by destructive destruction.Therefore, it has been one global to administer AMD pollutions
Great environmental problem urgently to be resolved hurrily, and China's current environment administers faced urgent task.
The microbiological treatment technology of environmental pollution for this kind of Extreme acid of acid wastewater in mine and other treatment technologies
(artificial swamp method and neutralisation) is compared, and is had low cost, non-secondary pollution and can be reached control soil or useless from source
The advantages that water is acidified, in existing practical application example, more using iron-reducing bacteria (FRB) and sulfate reducing bacteria (SRB)
To handle acidifying problem.Therefore, the more microorganism with such biological function is isolated from class extreme acid condition to provide
Source contributes to us to further speed up the solution to such environmental problem.
Due to the Extreme acid of acidic mine waste water environment, its biological community structure is simple, is given up in early stage Acid mine
Microbiologic population's research of water finds that microorganism main groups include Proteobacteria (Proteobacteria), nitre in this environment
Change spirillum door (Nitrospira), Firmicutes (Fimicutes) and acidfast bacilli door (Acidobacteria).For acidity
The research of mine wastewater middle ancient times bacterium is relatively fewer, the Gu Jun branches being related to be mainly Thermoplasmatales and
Sulfolobales.Some researchs are found in recent years, in some extreme sour environments Eukaryotic diversity apparently higher than
The diversity of prokaryotes.Such as Hispanic R í o Tinto River are a typical acidic mine waste water environment, and its is true
Core bio-diversity and biomass are all more than prokaryotes.These microbial associations act on, and accelerate the oxidation of ore, cause
The huge pollution of environment, and the maximally efficient method of current same for treating acidic mine wastewater is biological preventative strategies.Therefore, deeply
Understanding microbe groups and its diversity in grasp acidic mine waste water environment contributes to later-stage utilization related microorganisms to be given birth to
Thing reparation, there is great actual production meaning.
Traditional sour environment microbe research method, i.e. pure culture technigne, including select, separate, cultivating and physiology mirror
Fixed etc., its method is simple, but has great limitation.Due to the extremely special (pH of the life condition of this environment<3), can
Category is a small number of in fact for the species of the microorganism of pure culture.First, heterotrophic microorganism largely needs to carry out by Metals in Environments ion
Redox potential is changed, and the pure culture, nutritional condition, temperature, humidity etc. under laboratory condition is different from the original ecology of its growth
Environment, the type and quantity of the microorganism of acquisition also change.Secondly, the growth of fungi and algae (autotrophic microbe) is delayed
Slowly, it is strong for the sensitiveness of organic matter, it is difficult to be obtained during pure culture.In addition, conventional solid isolation medium is extremely low
It can not be separately cultured under the conditions of pH.At present, this kind of extreme acid condition microbiologic population of acidic mine waste water is tied
The understanding of structure is still extremely limited, is based primarily upon grand genome, grand transcript profile and a small amount of pure culture microorganism, but this environment
In microbe groups still have and largely do not obtain pure culture, it studies and applied far behind other Situation of Microorganism Under Extremity Environment.
Therefore, the technical problem of its pure culture of urgent need to resolve instantly, to which the microorganism pure culture of the environment can be obtained as much as possible
And microbiologic population's composition research of the environment is promoted with this, so the reparation problem of acidic mine waste water environment.
The content of the invention
It is an object of the invention to：The Situation of Microorganism Under Extremity Environment such as existing acidic mine waste water structure of community understanding is overcome to have
Limit, separates the problems such as difficult, there is provided it is isolated that one kind can significantly improve extreme acid condition in existing pure culture level
The method of microbe species and quantity.
In order to realize foregoing invention purpose, the present invention provides a kind of microbial liquid enriched medium, and it includes such as the following group
Point：
Every liter of A liquid：MgSO4·7H2O 0.70g, (NH4)2SO41.80g K2S4O60.76g, TSB 0.25g, pH2.5；
Every liter of B liquid：FeSO4·7H2O 7g, MnCl2-4H2O 1.8mg, Na2B4O7·10H2O 4.5mg, ZnSO4-7H2O
0.22mg, CuCl2-2H2O 0.05mg, NaMoO4-2H2O 0.03mg, VOSO4-2H2O 0.03mg, CoSO40.01mg,
pH2.0。
The preparation method of microbial liquid enriched medium of the present invention comprises the following steps：By 121 DEG C of the A fluid-tights mouth
Sterilize 25min, adds the mixing of the B liquid after filtration sterilization, obtains microbial liquid enriched medium；The body of the A liquid and B liquid
Product is than being 17：1；It is the acidic mine waste water after filtration sterilization to prepare the water used in the A liquid and B liquid.
As a kind of optimal technical scheme of the preparation method of microbial liquid enriched medium of the present invention, the mistake filters out
Bacterium is to use 0.22 μm of membrane filtration.
In order to realize foregoing invention purpose, the present invention also provides a kind of microbial solid plating medium, and it includes as follows
Component：
Every liter of A liquid：MgSO4·7H2O 0.70g, (NH4)2SO41.80g K2S4O60.76g, TSB 0.25g, pH2.5；
Every liter of B liquid：FeSO4·7H2O 7g, MnCl2-4H2O 1.8mg, Na2B4O7·10H2O 4.5mg, ZnSO4-7H2O
0.22mg, CuCl2-2H2O 0.05mg, NaMoO4-2H2O 0.03mg, VOSO4-2H2O 0.03mg, CoSO40.01mg,
pH2.0；
Every liter of C liquid：Agarose 80g.
The preparation method of microbial solid plating medium of the present invention comprises the following steps：The A liquid and C liquid are distinguished
121 DEG C of sterilizing 25min of sealing, are cooled to the mixing of the B liquid after filtration sterilization is added after 55 DEG C；The body of the A liquid, B liquid and C liquid
Product is than being 73：2：25；It is the acidic mine waste water after filtration sterilization to prepare the water used in the A liquid and B liquid；Configure the C liquid
Water used is distilled water.
As a kind of optimal technical scheme of the preparation method of microbial solid plating medium of the present invention, the mistake filters out
Bacterium is to use 0.22 μm of membrane filtration.
In order to realize foregoing invention purpose, the present invention also provides separate microorganism in a kind of sample from extreme acid condition
Method, it comprises the following steps：
(1) extreme acid condition sample is enriched with using the microbial liquid enriched medium, after obtaining enrichment
Sample；
(2) it is dilute to carry out point gradient to the sample after step (1) described enrichment for the use microbial solid plating medium
Release and be coated with, be subsequently placed in different incubators and persistently cultivate；
(3) taxonomic identification is carried out to gained microorganism, obtains the type and quantity of the microorganism of final separation.
As a kind of optimal technical scheme of present invention method of separate microorganism from extreme acid condition sample, step
(1) in, the enrichment is to add extreme acid condition sample in the microbial liquid enriched medium, is respectively placed in 28 DEG C
With 37 DEG C of shaking tables, 180rpm not lucifuge culture 1 week；In step (2), used when being corresponding be enriched with 28 DEG C of the lasting culture and
37 DEG C of two temperature, flat board is inverted in incubator not lucifuge culture 14~28 days.
As a kind of optimal technical scheme of present invention method of separate microorganism from extreme acid condition sample, step
(2) it is described to divide gradient dilution and coating to be with 10 in-1、10-2Two dilution gradients, are respectively coated.
It is described as a kind of optimal technical scheme of present invention method of separate microorganism from extreme acid condition sample
Extreme acid condition sample is acidic mine waste water.
Gained microorganism is classified and identify selected tri- kinds of universal primers of 18S, ITS and 16S rDNA expanded and
Sequencing, wherein being extracted for Eukaryotic gene, the method that liquid nitrogen grinding can be used.
Compared with prior art, the present invention has the advantages that：
The present invention is that the enrichment of simulation extreme acid condition multiple trace element culture medium, separation and identification extreme acid condition are micro-
Biology, by enrichment and separation method of the present invention, some rare microbe groups can be made the increase of individual amount occur, so as to significantly
Its separated probability is improved, adds the microbial diversity of separating resulting, the identification to microorganism in extreme acid condition
There is important value and meaning with research.
Brief description of the drawings
With reference to the accompanying drawings and detailed description, the present invention and beneficial effect are described in detail.
Fig. 1 is the result that the inventive method is enriched with to sample.
Flat board separating resulting and its repetition of Fig. 2~3 for the inventive method.
Embodiment
In order that goal of the invention, technical scheme and the advantageous effects of the present invention become apparent from, with reference to embodiments,
The present invention will be described in further detail.It should be appreciated that the embodiment described in this specification is just for the sake of explanation
The present invention, being not intended to limit the present invention, the formula of embodiment, ratio etc. can suit measures to local conditions to make a choice and have no reality to result
Matter influences.
Embodiment 1
Prepare microbial liquid enriched medium：
Every liter of A liquid：MgSO4·7H2O 0.70g, (NH4)2SO41.80g K2S4O60.76g, TSB 0.25g, pH2.5；Take
17mLA liquid is placed in conical flask and sealed, and 121 DEG C sterilize 25 minutes；
Every liter of B liquid：FeSO4·7H2O 7g, MnCl2-4H2O 1.8mg, Na2B4O7·10H2O 4.5mg, ZnSO4-7H2O
0.22mg, CuCl2-2H2O 0.05mg, NaMoO4-2H2O 0.03mg, VOSO4-2H2O 0.03mg, CoSO40.01mg,
pH2.0；Take 1mLB liquid to be added with 0.22 μm of membrane filtration after degerming in the A liquid after sterilizing, and 2 repetitions are set.
Water used is by 0.22 μm of filter membrane of acidic mine waste water of all mouth Pb-Zn deposits in Guangdong province, China Shaoguan Renhua County
The water of gained after filtering.
Prepare microbial solid plating medium：
Every liter of A liquid：MgSO4·7H2O 0.70g, (NH4)2SO41.80g K2S4O60.76g, TSB 0.25g, pH2.5；Take
730mLA liquid is placed in conical flask and sealed, and 121 DEG C sterilize 25 minutes；
Every liter of B liquid：FeSO4·7H2O 7g, MnCl2-4H2O 1.8mg, Na2B4O7·10H2O 4.5mg, ZnSO4-7H2O
0.22mg, CuCl2-2H2O 0.05mg, NaMoO4-2H2O 0.03mg, VOSO4-2H2O 0.03mg, CoSO40.01mg,
pH2.0；Take 20mL B liquid degerming with 0.22 μm of membrane filtration；
Every liter of C liquid：Agarose 80g, 1L distilled water；250mL C liquid is taken to be placed in conical flask and seal, 121 DEG C of sterilizings 25
Minute.
B liquid after A liquid after sterilizing, C liquid and filtration sterilization is mixed and is down flat plate.
Embodiment 2
Using the acidic mine waste water of all mouth Pb-Zn deposits in Guangdong province, China Shaoguan Renhua County as sample (pH<3) it is, big on middle mountain
Learn Life Science College microbial resources and ecological laboratory and carry out microorganism enrichment with microbial liquid enriched medium, 1
It is coated with and is inverted in incubator after week and is separately cultured, single bacterium colony is obtained after 14-28 days, and carry out 18S, ITS and 16S
RDNA sequencing identifications are to count the microorganism pure culture type and quantity in all mouth samples.Completion ITS, 18S rRNA and
In 16S rRNA identified for genes results, 26 kinds of bacterium is obtained, 6 kinds of fungi, a kind of algae, data are also in continuous renewal.Pass through
This sample pure culture micro organism quantity that this method obtains is much larger than other culture mediums delivered and its method, also much larger than not
The direct spread plate cultivation being enriched with.
Isolated microorganism in the Fankou Lead-zinc Deposit, Guangdong acidic mine waste water of table 1
Above-listed detailed description is illustrating for one of present invention possible embodiments, and the embodiment simultaneously is not used to limit
The scope of the claims of the present invention, all equivalence enforcements or change without departing from carried out by the present invention, it is intended to be limited solely by the scope of the claims of this case
In.
Claims (10)
1. a kind of microbial liquid enriched medium, it is characterised in that including following component：
Every liter of A liquid：MgSO4·7H2O 0.70g, (NH4)2SO41.80g K2S4O60.76g, TSB 0.25g, pH2.5；
Every liter of B liquid：FeSO4·7H2O 7g, MnCl2-4H2O 1.8mg, Na2B4O7·10H2O 4.5mg, ZnSO4-7H2O
0.22mg, CuCl2-2H2O 0.05mg, NaMoO4-2H2O 0.03mg, VOSO4-2H2O 0.03mg, CoSO40.01mg,
pH2.0。
2. the preparation method of microbial liquid enriched medium described in claim 1, it is characterised in that comprise the following steps：Will
Described 121 DEG C of sterilizing 25min of A fluid-tights mouth, add the mixing of the B liquid after filtration sterilization, obtain microbial liquid enriched medium；
The volume ratio of the A liquid and B liquid is 17：1；It is that the Acid mine after filtration sterilization gives up to prepare the water used in the A liquid and B liquid
Water.
3. the preparation method of microbial liquid enriched medium according to claim 2, it is characterised in that the filtration sterilization
It is to use 0.22 μm of membrane filtration.
4. a kind of microbial solid plating medium, it is characterised in that including following component：
Every liter of A liquid：MgSO4·7H2O 0.70g, (NH4)2SO41.80g K2S4O60.76g, TSB 0.25g, pH2.5；
Every liter of B liquid：FeSO4·7H2O 7g, MnCl2-4H2O 1.8mg, Na2B4O7·10H2O 4.5mg, ZnSO4-7H2O
0.22mg, CuCl2-2H2O 0.05mg, NaMoO4-2H2O 0.03mg, VOSO4-2H2O 0.03mg, CoSO40.01mg,
pH2.0；
Every liter of C liquid：Agarose 80g.
5. the preparation method of microbial solid plating medium described in claim 4, it is characterised in that comprise the following steps：Will
The A liquid and C liquid seal 121 DEG C of sterilizing 25min respectively, are cooled to the mixing of the B liquid after filtration sterilization is added after 55 DEG C；It is described
The volume ratio of A liquid, B liquid and C liquid is 73：2：25；It is the Acid mine after filtration sterilization to prepare the water used in the A liquid and B liquid
Waste water；It is distilled water to configure the water used in the C liquid.
6. the preparation method of microbial solid plating medium according to claim 5, it is characterised in that the filtration sterilization
It is to use 0.22 μm of membrane filtration.
7. a kind of method of separate microorganism in sample from extreme acid condition, it is characterised in that comprise the following steps：
(1) usage right requires that the 1 microbial liquid enriched medium is enriched with to extreme acid condition sample, obtains richness
Sample after collection；
(2) usage right requires that the 4 microbial solid plating mediums carry out a point ladder to the sample after step (1) described enrichment
Degree dilutes and is coated with, and is subsequently placed in different incubators and persistently cultivates；
(3) taxonomic identification is carried out to gained microorganism, obtains the type and quantity of the microorganism of final separation.
8. the method for separate microorganism in the sample according to claim 7 from extreme acid condition, it is characterised in that step
(1) in, the enrichment is to add extreme acid condition sample in the microbial liquid enriched medium, is respectively placed in 28 DEG C
With 37 DEG C of shaking tables, 180rpm not lucifuge culture 1 week；In step (2), used when being corresponding be enriched with 28 DEG C of the lasting culture and
37 DEG C of two temperature, flat board is inverted in incubator not lucifuge culture 14~28 days.
9. the method for separate microorganism in the sample according to claim 7 from extreme acid condition, it is characterised in that step
(2) it is described to divide gradient dilution and coating to be with 10 in-1、10-2Two dilution gradients, are respectively coated.
10. the method for separate microorganism in the sample according to claim 7 from extreme acid condition, it is characterised in that institute
It is acidic mine waste water to state extreme acid condition sample.
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Cited By (1)
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| CN111334435A (en) * | 2020-01-22 | 2020-06-26 | 华南师范大学 | Separation and identification method of acidophilic fungus with biological induction mineralization effect |
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| CN111334435A (en) * | 2020-01-22 | 2020-06-26 | 华南师范大学 | Separation and identification method of acidophilic fungus with biological induction mineralization effect |
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