CN103540519A - Double-layer flat plate and preparation method thereof - Google Patents

Double-layer flat plate and preparation method thereof Download PDF

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CN103540519A
CN103540519A CN201310441319.3A CN201310441319A CN103540519A CN 103540519 A CN103540519 A CN 103540519A CN 201310441319 A CN201310441319 A CN 201310441319A CN 103540519 A CN103540519 A CN 103540519A
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yeast
double
solution
candida
digboiensiszby
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CN103540519B (en
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刘学端
梁伊丽
尹华群
恩刚姆
胡琪
马丽媛
肖云花
邱冠周
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Central South University
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
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    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/06—Plates; Walls; Drawers; Multilayer plates
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Abstract

The invention discloses a double-layer flat plate and a preparation method thereof. By using Candida digboiensis ZBY with a preservation number of CCTCC NO: M2013311, a bottom culture medium in the double-layer flat plate is manufactured, and autotrophic leaching microorganisms are screened. Compared with traditional methods, the preparation method disclosed by the invention greatly improves the separating and screening efficiency of leaching microorganisms, and provides a feasible way for the inheritance and metabolization of leaching microorganisms.

Description

A kind of double-layer plate and preparation method thereof
Technical field
The invention belongs to the product technical field of leaching microbacteria screening, be specifically related to a kind of double-layer plate for leaching microbacteria screening and preparation method thereof, and bacterial strain.
Background technology
Biological metallurgy utilizes iron and the sulphur in the mineral such as microbiological oxidation sulphide ores, makes to be combined in valuable metal in ore and is discharged into and in solution, is beneficial to further extraction.Leaching microbacteria mainly comprises iron-oxidizing bacteria and the sulfur oxidizing bacterium of autotrophy, and they utilize the Fe in sulphide ores 2+or the sulfide of reduced form is (as pyrite Fe 2s) as energy growth, the leaching that produces sulfuric acid sulphide ores, therefore, filters out the key that highly effective ore leaching microorganism is biological metallurgy.
Solid medium is generally used for the separated of leaching microbacteria and cultivates, yet leaching microbacteria is mainly autotrophic bacteria, growth has restraining effect to leaching microbacteria for organic substance in the peptizer such as agar and hydrolysate thereof, acid solid medium is difficult to make, so leaching microbacteria is difficult to produce a large amount of distinctive bacterium colonies on solid plate conventionally for bacterial strain screening, seed selection and hereditary property research.Therefore, highly active leaching microbacteria microorganism resource lacks, and ore soaking machine reason is not clear, causes Bioleaching technology leaching rate slow, limits its heavy industrialization application.
For improving leaching microbacteria recall rate and shortening culture cycle, double-layer plate method invention for the separation screening of this quasi-microorganism.In double-layer plate, adopt prime-coating to have a liking for sour heterotrophic bacteriums such as (Candida digboiensis sp.) to remove the toxic organic compound matter in substratum, upper strata is for the screening of chemoautotrophic bacteria.Medium component in double-layer plate is also updated, but the method still exists very large challenge at present.
The present invention has screened a kind of acidophilic yeast, utilize it to design the double-layer plate that ore deposit bacterium is extremely soaked in a kind of novel separation and purification, it is obvious that this yeast is removed the effect of the toxic organic compound matter in substratum, successfully realized the solid plate that soaks ore deposit bacterium and cultivated and purifying.
Summary of the invention
The object of the invention is to soak the acidophilic yeast in ore body system by screening acidity, a kind of double-layer plate that utilizes the rapid screening leaching microbacteria that this bacterium sets up and preparation method thereof is provided, to enrich leaching microbacteria resource, successfully realize the solid plate cultivation and the purifying that soak ore deposit bacterium.
A kind of double-layer plate, be that inoculation has acidophilic yeast (Candida digboiensisZBY) in the bottom platform of Double-Medium flat board, described acidophilic yeast's (Candida digboiensisZBY) deposit number is: CCTCC NO:M2013311.
The preparation method of described double-layer plate, is characterized in that, comprises the following steps:
1) selecting the single bacterium colony of acidophilic yeast (Candida digboiensisZBY) is placed in 5mL9k liquid nutrient medium containing the pancreas peptone soybean broth substratum (TSB) of the glucose of 1wt% and 0.025wt% and is activated to bacterial concentration and reaches 10 9individual/more than mL, to get the good nutrient solution of activation that accounts for volume ratio 10% and add containing in the 9K liquid nutrient medium of 1% glucose, obtain yeast nutrient solution;
2) prepare respectively following three kinds of solution: (a) 2 * 9K liquid nutrient medium of pH=2.0 (be in substratum, the concentration of every kind of composition is 2 times), (b) copperas solution of the 250mM of pH=2.0, wherein containing 10mM K 2s 4o 6; (c) the 3wt% agarose solution of pH=4.5; In the time of 50 ℃ by (a) (b) (c) three kinds of solution by volume 4:1:5 mix, prepared composition from 2 parts of substratum for purifying;
3) the yeast nutrient solution 1 part of inoculation step 1 immediately wherein) obtaining, inoculum size is 1% of culture volume ratio, is then poured over and in sterile petri dish, forms gel, another part of insulation, gel in culture dish solidifies, then pours second part of unleavened substratum into, makes double-layer plate.
Described yeast is acidophilic yeast (Candida digboiensisZBY), and deposit number is: CCTCC NO:M2013311.Depositary institution: Chinese Typical Representative culture collection center, depositary institution address: China. Wuhan. Wuhan University, preservation date: on July 2nd, 2013.
The present invention has screened a kind of acidophilic yeast, utilize it to design the double-layer plate that ore deposit bacterium is extremely soaked in a kind of novel separation and purification, it is obvious that this yeast is removed the effect of the toxic organic compound matter in substratum, successfully realized the solid plate that soaks ore deposit bacterium and cultivated and purifying.
Accompanying drawing explanation
Fig. 1 is yeast (Candida digboiensisZBY) microscopic morphology;
Fig. 2 is the double-layer plate that the present invention utilizes yeast (Candida digboiensisZBY) to make;
Fig. 3 is leaching microbacteria colonial morphology and electron scanning micrograph (SEM) image of double-layer plate screening of the present invention;
(a) bottom inoculation yeast, the double-layer plate of upper strata inoculation leaching microbacteria;
(b) the SEM image of Z1 bacterium;
(c) the SEM image of Z2 bacterium.
Embodiment
Below in conjunction with embodiment, be intended to further illustrate the present invention, and unrestricted the present invention.
Embodiment 1 acidophilic yeast's (Candida digboiensisZBY) separation screening and evaluation:
Gather acidic mine water 10mL, by the 9K liquid nutrient medium 90mL enrichment culture containing 1% glucose, 9K medium component is (g/L): (NH4) 2sO 43.0, KCl0.1, K 2hPO 40.5, MgSO 47H 2o0.5, Ca (NO 3) 20.01, H 2o1000mL, pH=2.5,35 ℃ of culture temperature, rotating speed 200rpm.After 2 days, when bacterial concentration reaches 10 9individual/during mL, to get 10mL nutrient solution enrichment culture in the fresh 9K liquid nutrient medium of 90mL (containing 1% glucose), this process repetition four times.
Acidity is soaked heterotrophic microorganism separation and purification in ore body system and is adopted individual layer dull and stereotyped.3% agarose solution of 2 * 9K liquid nutrient medium of pH=2.0 and pH=4.5 is pressed to 1:1 after 121 ℃ of high-temperature sterilization 30min and mix, add 1% glucose and the 0.025%TSB of filtration sterilization, adjust pH is 2.5, pours in sterile petri dish after being cooled to 50 ℃; The nutrient solution dilution spread of above-mentioned enrichment is dull and stereotyped, cultivate in 35 ℃; The picking list bacterium colony separation and Culture of ruling on same agar plate.This process repeats four times, obtains the pure culture of yeast.
Containing the yeast (Candida digboiensisZBY) of growing on the 9K flat board of 1% glucose, bacterium colony oyster white, bacterium colony is smooth, and surface is smooth with edge; With ordinary optical microscope, observe and find that isolate cell is oval, gemmation, has thecaspore to form (Fig. 1).Extract acidophilic yeast's genomic dna, amplification 18S rDNA sequence, combining form feature, 18S rDNA gene order are accredited as yeast (Candida digboiensisZBY).Bacterial classification, by the center preservation of Chinese Typical Representative culture collection, is numbered: CCTCC NO:M2013311.
Embodiment 2 utilizes acidophilic yeast (Candida digboiensisZBY) to make double-layer plate
Select yeast list bacterium colony and containing being activated to bacterial concentration in glucose (1%) and 9k liquid nutrient medium TSB(0.025%), reach 10 in 5mL 9individual/during mL, to get 5mL nutrient solution in the fresh 9K liquid nutrient medium of 45mL (containing 1% glucose), make it there is good activity.
The following three kinds of solution of preparation are for the making of double-layer plate respectively: (a) 2 * 9K substratum of pH=2.0,121 ℃ of high-temperature sterilizations 30 minutes; (b) the 250mM copperas solution of pH=2.0 is (containing K 2s 4o 610mM), the nylon membrane of 0.2 μ m filters; (c) 3% agarose solution of pH=4.5,121 ℃ of high-temperature sterilizations 30 minutes.In the time of 50 ℃ by (a) (b) (c) three kinds of solution by volume 4:1:5 mix, preparation separation and 2 parts of solid mediums of purifying, 1 part of yeast (inoculum size 1%) of having activated of inoculation immediately wherein, is then poured over and in sterile petri dish, forms thin gel; Another part of 50 ℃ of insulations, the gel in culture dish solidifies, then pours second part of unleavened nutrient solution into, treats that it solidifies.Make double-layer plate (as shown in Figure 2).
Embodiment 3 utilizes double-layer plate screening leaching microbacteria:
Get enrichment culture in the 9K substratum that 10mL acidic mine water contains 250mM ferrous sulfate to 90ml, choose the suitableeest pH value and the temperature of bacterial classification to be separated, be placed on the shaking table that rotating speed is 200rpm/min and cultivate.When solution reddens, the nutrient solution of getting 10ml is transferred in the substratum above-mentioned of the same race that 90ml is fresh and is continued enrichment culture, and this process repeats four times.By the dilution of enrichment culture liquid, and coat on the double-layer plate of embodiment 2 preparations, be placed in suitable incubator and cultivate.Choosing colony is rule separated on agar plate, under suitable condition, cultivates again, obtains pure culture.
In nonvaccinated culture dish, only have yeast bacterium colony to occur in bottom substratum.In the culture dish of upper strata inoculation enriched substance, there is the bacterium colony (shown in Fig. 3 a) of yeast and iron-oxidizing bacteria.According to the morphological specificity of microorganism, separated and be purified into two kinds of ore-leaching bacterias, respectively called after Z1 and Z2 on upper strata.In double-deck agar plate, two kinds of bacterium colonies all occurred in one week, and they cover the surface of upper strata substratum, and culture dish is reddened, and the speed of Z1 iron oxidation is fast compared with Z2.The time that Z1 bacterium detects on double-layer plate, than the dull and stereotyped 5d ahead of time of individual layer, detects approximately 800 of colony numbers, and recall rate, than 25 times of the dull and stereotyped raisings of individual layer, improves 2 times than existing double-layer plate.
By SEM(scanning electronic microscope) observe the form (shown in Fig. 3) of new bacterial strain.Z1 is rod-shaped bacterium, and diameter is 0.40-0.70 μ m, long 1.00-2.50 μ m; And Z2 cell is curved, diameter is 0.25-0.50 μ m, long 0.80-2.15 μ m.Opticmicroscope discovery, Z2 mobility is strong compared with Z1.The morphological specificity of Z1 and Z2 is similar with Leptospirillum to thiobacillus ferrooxidant.16S rDNA sequential analysis shows, Z1 and Z2 belong to respectively thiobacillus ferrooxidant and Leptospirillum.

Claims (3)

1. a double-layer plate, it is characterized in that, be that inoculation has acidophilic yeast (Candida digboiensisZBY) in the bottom platform of Double-Medium flat board, described acidophilic yeast's (Candida digboiensisZBY) deposit number is: CCTCC NO:M2013311.
2. the preparation method of double-layer plate claimed in claim 1, is characterized in that, comprises the following steps:
1) selecting the single bacterium colony of acidophilic yeast (Candida digboiensisZBY) is placed in 5mL9k liquid nutrient medium containing the pancreas peptone soybean broth substratum of the glucose of 1wt% and 0.025wt% and is activated to bacterial concentration and reaches 10 9individual/more than mL, to get the good nutrient solution of activation that accounts for volume ratio 10% and add containing in the 9K liquid nutrient medium of 1% glucose, obtain yeast nutrient solution;
2) prepare respectively following three kinds of solution: (a) 2 * 9K liquid nutrient medium of pH=2.0, (b) copperas solution of the 250mM of pH=2.0, wherein containing 10mM K 2s 4o 6; (c) the 3wt% agarose solution of pH=4.5; In the time of 50 ℃ by (a) (b) (c) three kinds of solution by volume 4:1:5 mix, prepared composition from 2 parts of substratum for purifying;
3) the yeast nutrient solution 1 part of inoculation step 1 immediately wherein) obtaining, inoculum size is 1% of culture volume ratio, is then poured over and in sterile petri dish, forms gel, another part of insulation, gel in culture dish solidifies, then pours second part of unleavened substratum into, makes double-layer plate.
3. a Yeasts, is characterized in that, described yeast is acidophilic yeast (Candida digboiensisZBY), and deposit number is: CCTCC NO:M2013311.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107446826A (en) * 2017-09-12 2017-12-08 佛山市海天调味食品股份有限公司 A kind of screening and culturing medium of filamentous fungi and preparation method and application
CN113957118A (en) * 2021-10-29 2022-01-21 播恩集团股份有限公司 Detection method for viable count of bacillus coagulans

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1746315A (en) * 2005-09-19 2006-03-15 南京农业大学 Improvement of plate detection ratio of self-culture sulbacillus with chemical energy

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1746315A (en) * 2005-09-19 2006-03-15 南京农业大学 Improvement of plate detection ratio of self-culture sulbacillus with chemical energy

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
PATEL ET AL: "Isolation and identification of a Candida digboiensis strain from an extreme acid mine drainage of the Lignite Mine,Gujarat", 《JOURNAL OF BASIC MICROBIOLOGY》 *
SOOD AND LAL: "Isolation of a novel yeast strain Candida digboiensis TERI ASN6 capable of degrading petroleum hydrocarbons in acidic conditions", 《JOURNAL OF ENVIRONMENTAL MANAGEMENT》 *
王世梅和周立祥: "提高氧化亚铁硫杆菌和氧化硫硫杆菌平板检出率的方法:双层平板法", 《环境科学学报》 *
龚文琪等: "氧化亚铁硫杆菌的分离培养及其浸磷效果", 《过程工程学报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107446826A (en) * 2017-09-12 2017-12-08 佛山市海天调味食品股份有限公司 A kind of screening and culturing medium of filamentous fungi and preparation method and application
CN113957118A (en) * 2021-10-29 2022-01-21 播恩集团股份有限公司 Detection method for viable count of bacillus coagulans

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