CN103540519B - Double-layer flat plate and preparation method thereof - Google Patents

Double-layer flat plate and preparation method thereof Download PDF

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CN103540519B
CN103540519B CN201310441319.3A CN201310441319A CN103540519B CN 103540519 B CN103540519 B CN 103540519B CN 201310441319 A CN201310441319 A CN 201310441319A CN 103540519 B CN103540519 B CN 103540519B
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yeast
double
solution
candida
digboiensiszby
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CN103540519A (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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    • C—CHEMISTRY; METALLURGY
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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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    • C12R2001/00—Microorganisms ; Processes using microorganisms
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    • C12R2001/72—Candida

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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 in the mineral such as microbiological oxidation sulphide ores and sulphur, the valuable metal be combined in ore is discharged in solution and 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, produce the leaching of sulfuric acid sulphide ores, therefore, filter out the key that highly effective ore leaching microorganism is biological metallurgy.
Solid medium is generally used for separation and the cultivation of leaching microbacteria, but leaching microbacteria mainly autotrophic bacteria, organic substance in the peptizer such as agar and hydrolysate thereof have restraining effect to leaching microbacteria growth, acid solid medium is difficult to make, so leaching microbacteria is difficult to produce a large amount of distinctive bacterium colony for bacterial strain screening, seed selection and hereditary property research on solid plate usually.Therefore, highly active leaching microbacteria microorganism resource lacks, and bioleaching mechanism is failed to understand, causes Bioleaching technology leaching rate slow, limits the application of its heavy industrialization.
For improving leaching microbacteria recall rate shorten culture cycle, double-layer plate method invention for the separation screening of this quasi-microorganism.Adopt prime-coating in double-layer plate addicted to heterotrophic bacteriums such as (the Candida digboiensis sp.) of acid to remove the toxic organic compound matter in substratum, upper strata is used 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 devise double-layer plate that ore deposit bacterium is extremely soaked in a kind of novel separation and purification, the effect of the toxic organic compound matter in this yeast removing substratum is obvious, and the solid plate successfully achieving leaching ore deposit bacterium is cultivated and purifying.
Summary of the invention
The object of the invention is the acidophilic yeast by screening in acid leaching ore body system, double-layer plate of a kind of rapid screening leaching microbacteria utilizing this bacterium to set up and preparation method thereof is provided, to enrich leaching microbacteria resource, the solid plate successfully realizing leaching ore deposit bacterium is cultivated and purifying.
A kind of double-layer plate, be that inoculation has acidophilic yeast (Candida digboiensisZBY) in the bottom platform of Double-Medium flat board, the deposit number of described acidophilic yeast (Candida digboiensisZBY) is: CCTCC NO:M2013311.
The preparation method of described double-layer plate, is characterized in that, comprises the following steps:
1) select acidophilic yeast (Candida digboiensisZBY) single bacterium colony 5mL9k liquid nutrient medium be placed in containing the glucose of 1wt% and the pancreas peptone soybean broth substratum (TSB) of 0.025wt% to be activated to bacterial concentration and to reach 10 9individual/more than mL, the nutrient solution getting the activation that accounts for volume ratio 10% good adds in the 9K liquid nutrient medium containing 1% glucose, obtains yeast nutrient solution;
2) following three kinds of solution are prepared respectively: 2 × 9K liquid nutrient medium (namely in substratum, the concentration of often kind of composition is 2 times) of (a) pH=2.0, the copperas solution of the 250mM of (b) pH=2.0, wherein containing 10mM K 2s 4o 6; The 3wt% agarose solution of (c) pH=4.5; By the 4:1:5 mixing by volume of (a) (b) (c) three kinds of solution when 50 DEG C, be prepared into abstraction and purification substratum 2 parts;
3) wherein 1 part of inoculation step 1 immediately) the yeast nutrient solution that obtains, inoculum size is 1% of culture volume ratio, is then poured in sterile petri dish and forms gel, another part is incubated, when the gel sets in culture dish, then pour second part of unleavened substratum into, make double-layer plate.
Described yeast is acidophilic yeast (Candida digboiensisZBY), and deposit number is: CCTCC NO:M2013311.Depositary institution: China typical 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 devise double-layer plate that ore deposit bacterium is extremely soaked in a kind of novel separation and purification, the effect of the toxic organic compound matter in this yeast removing substratum is obvious, and the solid plate successfully achieving leaching ore deposit bacterium is 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) and makes;
Fig. 3 is leaching microbacteria colonial morphology and electron scanning micrograph (SEM) image of double-layer plate of the present invention screening;
(a) bottom inoculation yeast, the double-layer plate of upper strata inoculation leaching microbacteria;
The SEM image of (b) Z1 bacterium;
The SEM image of (c) Z2 bacterium.
Embodiment
Be intended to further illustrate the present invention below in conjunction with embodiment, and unrestricted the present invention.
The separation screening of embodiment 1 acidophilic yeast (Candida digboiensisZBY) and qualification:
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, culture temperature 35 DEG C, rotating speed 200rpm.After 2 days, when bacterial concentration reaches 10 9individual/mL time, get 10mL nutrient solution enrichment culture in the fresh 9K liquid nutrient medium (containing 1% glucose) of 90mL, this process repeats four times.
In acid leaching ore body system, heterotrophic microorganism separation and purification adopts individual layer dull and stereotyped.2 × 9K liquid nutrient medium of pH=2.0 and 3% agarose solution of pH=4.5 are pressed after 121 DEG C of high-temperature sterilization 30min 1:1 mixing, 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 DEG C; The nutrient solution dilution spread of above-mentioned enrichment is dull and stereotyped, cultivate in 35 DEG C; Picking list bacterium colony is rule separation and Culture on same agar plate.This process repeats four times, obtains the pure culture of yeast.
Containing the yeast (Candida digboiensisZBY) of the 9K grow on plates of 1% glucose, bacterium colony oyster white, bacterium colony is smooth, surface and flush edge; Observe with ordinary optical microscope and find that isolate cell is oval, gemmation, have 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 China typical culture collection center preservation, is numbered: CCTCC NO:M2013311.
Embodiment 2 utilizes acidophilic yeast (Candida digboiensisZBY) to make double-layer plate
Select yeast list bacterium colony and reach 10 in 5mL containing being activated to bacterial concentration in glucose (1%) and 9k liquid nutrient medium TSB(0.025%) 9individual/mL time, get 5mL nutrient solution in the fresh 9K liquid nutrient medium (containing 1% glucose) of 45mL, make it have good activity.
The following three kinds of solution of preparation are used for the making of double-layer plate respectively: 2 × 9K substratum of (a) pH=2.0,121 DEG C 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 μm filters; 3% agarose solution of (c) pH=4.5,121 DEG C of high-temperature sterilizations 30 minutes.By the 4:1:5 mixing by volume of (a) (b) (c) three kinds of solution when 50 DEG C, preparative separation and purifying solid medium 2 parts, wherein inoculate the yeast (inoculum size 1%) activated immediately for 1 part, be then poured in sterile petri dish and form thin gel; Another part 50 DEG C insulation, when the gel sets in culture dish, 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 to screen leaching microbacteria:
Get 10mL acidic mine water and contain enrichment culture in the 9K substratum of 250mM ferrous sulfate to 90ml, choose the suitableeest pH value and the temperature of bacterial classification to be separated, be placed on shaking table that rotating speed is 200rpm/min and cultivate.When solution reddens, the nutrient solution getting 10ml is transferred in the fresh substratum above-mentioned of the same race of 90ml and is continued enrichment culture, and this process repeats four times.Enrichment culture liquid is diluted, and coats on the double-layer plate of embodiment 2 preparation, be placed in suitable incubator and cultivate.Choosing colony is rule separation on agar plate, cultivates again, obtain pure culture under suitable condition.
In nonvaccinated culture dish, yeast bacterium colony is only had to occur in bottom substratum.In the culture dish of upper strata inoculation enriched substance, there is the bacterium colony of yeast and iron-oxidizing bacteria (shown in Fig. 3 a).According to the morphological specificity of microorganism, go out two kinds of ore-leaching bacterias at upper strata abstraction and purification, respectively called after Z1 and Z2.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 comparatively Z2 is fast for the speed of Z1 Fe forms.The time that Z1 bacterium detects on double-layer plate ahead of time 5d more dull and more stereotyped than individual layer, detect colony number about 800, recall rate raising 25 times more dull and more stereotyped than individual layer, improves 2 times than existing double-layer plate.
By SEM(scanning electronic microscope) observe the form (Fig. 3 shown in) of new strains.Z1 is rod-shaped bacterium, and diameter is 0.40-0.70 μm, long 1.00-2.50 μm; And Z2 cell is curved, diameter is 0.25-0.50 μm, long 0.80-2.15 μm.Opticmicroscope finds, Z2 mobility comparatively Z1 is strong.The morphological specificity of Z1 with Z2 is similar with Leptospirillum to thiobacillus ferrooxidant.16S rDNA sequential analysis shows, Z1 and Z2 belongs to thiobacillus ferrooxidant and Leptospirillum respectively.

Claims (2)

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, the deposit number of described acidophilic yeast (Candida digboiensisZBY) is: CCTCC NO:M 2013311;
The preparation method of described double-layer plate, comprises the following steps:
1) select acidophilic yeast (Candida digboiensisZBY) single bacterium colony 5mL 9k liquid nutrient medium be placed in containing the glucose of 1wt% and the pancreas peptone soybean broth substratum of 0.025wt% to be activated to bacterial concentration and to reach 10 9individual/more than mL, the nutrient solution getting the activation that accounts for volume ratio 10% good adds in the 9K liquid nutrient medium containing 1% glucose, obtains yeast nutrient solution;
2) following three kinds of solution are prepared respectively: 2 × 9K liquid nutrient medium of (a) pH=2.0, the copperas solution of the 250mM of (b) pH=2.0, wherein containing 10mM K 2s 4o 6; The 3wt% agarose solution of (c) pH=4.5; By the 4:1:5 mixing by volume of (a) (b) (c) three kinds of solution when 50 DEG C, be prepared into abstraction and purification substratum 2 parts;
3) wherein 1 part of inoculation step 1 immediately) the yeast nutrient solution that obtains, inoculum size is 1% of culture volume ratio, then be poured in sterile petri dish and form gel, another part is incubated, when the gel sets in culture dish, pour second part of unleavened substratum again into, make double-layer plate.
2. a Yeasts, is characterized in that, described yeast is acidophilic yeast (Candida digboiensisZBY), and deposit number is: CCTCC NO:M 2013311.
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Citations (1)

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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
Isolation and identification of a Candida digboiensis strain from an extreme acid mine drainage of the Lignite Mine,Gujarat;Patel et al;《Journal of Basic Microbiology》;20091231;第49卷;全文 *
Isolation of a novel yeast strain Candida digboiensis TERI ASN6 capable of degrading petroleum hydrocarbons in acidic conditions;Sood and Lal;《Journal of Environmental Management》;20081225;第90卷;摘要 *
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;王世梅和周立祥;《环境科学学报》;20051031;第25卷(第10期);全文 *
氧化亚铁硫杆菌的分离培养及其浸磷效果;龚文琪等;《过程工程学报》;20070630;第7卷(第3期);全文 *

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