CN104109641A - Method for rapidly screening silicate bacteria - Google Patents
Method for rapidly screening silicate bacteria Download PDFInfo
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- CN104109641A CN104109641A CN201310138722.9A CN201310138722A CN104109641A CN 104109641 A CN104109641 A CN 104109641A CN 201310138722 A CN201310138722 A CN 201310138722A CN 104109641 A CN104109641 A CN 104109641A
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- bauxite
- caco
- powder
- bacteria
- free agar
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Abstract
The invention relates to a method for rapidly screening silicate bacteria, which comprises the following steps: (1) obtaining soil bacterium suspension; (2) enrichment culture, namely, adopting a method of gradually increasing the adding amount of bauxite to carry out enrichment culture on silicate bacteria; (3) screening and culturing, namely picking out transparent colonies with mucus-shaped bulges from a culture medium flat plate according to the growth time of the colonies and the morphology of the colonies, and culturing again until pure culture is obtained; (4) and (4) re-screening. The invention adopts a method of gradually increasing the adding amount of bauxite, so that the bacteria are more easily adapted to the silicate environment, thereby enriching more silicate bacteria. By applying CaCO3Method for applying powder to surface of flat plate3The bottom of the sedimentation flat plate is not reached, so that the acid-producing bacteria can be screened visually. Solid FeCl added in a small amount in a culture medium3Instead using FeCl3The solution makes it more readily available to bacteria. The invention provides a rapid and convenient method for rapidly screening and domesticating silicate bacteria in a laboratory.
Description
Technical field
The present invention relates to the screening method of a kind of silicate bacteria, particularly relate to a kind of in laboratory the method for rapid screening silicate bacteria.
Background technology
Along with a large amount of exploitations of people to Mineral resources, higher-grade, easily select Mineral resources day by day to reduce, legislations of environmental protection is increasingly severe simultaneously, and the development scheme of the Mineral resources that some are traditional has not been suitable for low-grade Mineral resources.Biological ore dressing demonstrates good application prospect with the advantage such as less demanding to ore grade, cost is low, less energy consumption, Production Flow Chart are simple, environmentally friendly in mineral products processing.Biological ore dressing is by the interaction of microorganism or its meta-bolites and mineral, thereby removes the impurity in ore or reclaim the technology of valuable metal wherein, utilizes the process of the impurity silicon in microorganism removal mineral to be called as biological desiliconization.
Silicate bacteria is not the name on taxonomy, and having of referring to can analysing silicon Barbiturates mineral and discharged a bacterioid of the elements such as phosphorus, potassium.According to its name cause and separation method, scholar isolates silicate bacteria from different soil all over the world, and their form, Physiology and biochemistry are identified and named and done research, there is a lot of disputes in the name for silicate bacteria in classification always.The kind of report mainly contains bacillusmusilaginosiengineering (or colloid bacillus cereus) (Bacillus mucilaginosus), Bacillus circulans (Bacillus circulans) and Soil Bacillus (B.edaphicus) at present.
Technology screening silicate bacteria method is traditional dilution spread method at present: be roughly divided into 1. soil diluent preparations; 2. coating inoculation: be inoculated into and carry out constant temperature culture on culture machine by the nutrient solution of gradient dilution; 3. screening for the first time: the bacterium colony that picking has certain feature on flat board carries out purifying; 4 multiple sieves: carry out repeatedly purifying and obtain pure culture.In traditional method, the harsh gained bacterial classification of primary dcreening operation condition kind is few, not directly perceived visual for producing acids advantage silicate bacteria in multiple sieve.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of in laboratory the method for rapid screening silicate bacteria.
For reaching above-mentioned purpose, the method for a kind of rapid screening silicate bacteria of the present invention, comprises the following steps:
(1) soil bacteria suspension obtains: alum clay sample ore is joined in sterilized water, and standing after vibration, get supernatant liquor and join in liquid nitrogen-free agar;
(2) enrichment culture: will cultivate the cultivation of transferring of gained bacterial classification by liquid nitrogen-free agar in liquid nitrogen-free agar in step (1), corotation connects 4 times, by volume per-cent meter is measured in switching, and 10% of each nutrient solution is inoculated in the liquid nutrient medium that bauxite content increases gradually;
(3) sub-sieve is cultivated: the nutrient solution dilution 10 that the last switching in step (2) is cultivated
6-10
8doubly, coat in solid nitrogen-free agar, be placed in 30 ℃ of constant incubators and cultivate 24h, when planar surface no longer includes water droplet, be inverted flat board, cultivate 3-5 days, observe at any time bacterial colony growing state; The time growing according to bacterium colony and the form of bacterium colony, choose the transparent bacterium colony of mucus shape projection from culture medium flat plate, then cultivate, until obtain pure culture;
(4) multiple sieve: the pure strain obtaining in step (3) is inoculated in the nitrogenous substratum of liquid, can analysing silicon hydrochlorate and the bacterium of the rock forming mineral that forms of aluminosilicate be silicate bacteria.Authentication method is: by silicon molybdenum blue method, measure silicone content in solution.
Method of the present invention, wherein the alum clay sample ore in preferred described step (1) is illite powder, crosses 200 mesh sieves.
Method of the present invention, wherein the liquid nitrogen-free agar in preferred described step (2) contains: sucrose 3-5g, Na
2hPO
42-4g or Na
2hPO
4h
2o 5-10g, MgSO
47H
2o 0.5-1g, CaCO
30.1-0.3g, bauxite 1g, water 1L, the FeCl that is 1% by the weight percentage that sulfuric acid modulation is dissolved
3solution 2ml, the pH value of substratum is 6-7.
Method of the present invention, wherein in preferred described step (2), each switching culture condition is cultivated 3 days for being placed in 180rpm on 30 ℃ of constant-temperature tables.
Method of the present invention, wherein in preferred described step (2) liquid nitrogen-free agar bauxite first add-on be 200mg/L, to increase the speed of 200mg/L at every turn, increase gradually afterwards the concentration of bauxite in liquid nitrogen-free agar.
Method of the present invention, wherein in preferred described step (3), solid nitrogen-free agar contains: sucrose 3-5g, Na
2hPO
42-4g or Na
2hPO
4h
2o 5-10g, MgSO
47H
2o 0.5-1.0g, CaCO
30.1-0.3g, bauxite 1g, water 1L, the FeCl that is 1% by the weight percentage that sulfuric acid modulation is dissolved
3solution 2ml, CaCO
3powder, 10g agar, the pH value of substratum is 6-7.
Method of the present invention, wherein in preferred described step (4), the nitrogenous substratum of liquid contains: sucrose 3-5g, Na
2hPO
42-4g or Na
2hPO
4h
2o 5g, MgSO
47H
2o 0.5-1g, (NH
4)
2sO
31.5-3.8g, CaCO
30.1-0.3g, bauxite 1g, water 1L, the FeCl that is 1% by the weight percentage that sulfuric acid modulation is dissolved
3solution 2ml, the pH value of substratum is 6-7.
Method of the present invention, wherein CaCO in preferred described solid nitrogen-free agar
3powder is: first in mortar, be ground to powder, after 200 mesh sieves; Afterwards by CaCO
3powder is 121 ℃ of high-pressure sterilizing pots, after sterilizing 20min, stand-by; Deng solid medium, be down flat after plate condensation, with sticky a little CaCO that gets of glass stick
3powder, is applied in planar surface.
The invention difference from existing technology is, adopts the method that progressively improves bauxite concentration in liquid nitrogen-free agar, makes the easier accommodate silicon hydrochlorate of bacterium environment, is enriched to more silicate bacteria.CaCO is smeared in employing
3powder allows CaCO in the method for planar surface
3the dull and stereotyped bottom of unlikely precipitation, makes to screen acid-producing bacteria visual.The solid FeCl of indium addition in substratum
3change into and use FeCl
3solution, is more easily utilized it by bacterium.The present invention tames silicate bacteria for rapid screening in laboratory a kind of means is quickly and easily provided.
Accompanying drawing explanation
Fig. 1 is SiO
2solution canonical plotting.
Embodiment
Below in conjunction with embodiment and testing data, to the present invention is above-mentioned, be described in more detail with other technical characterictic and advantage.
Embodiment 1
(1) gather alum clay mining area (bauxite can pick up from county magistrate stockaded village town, Changshun, Bouyei-Miao Autonomous Prefecture of Qiannan, Guizhou Province, Yun Biao town, Heng County, Nanning City, Guangxi Zhuang Autonomous Region or Meng Gong township, Guigang, Guangxi) top layer 0-20cm soil as sample, 4 ℃ of Refrigerator stores.Take for examination pedotheque 10g, join in the sterilized water of 100ml, with the quick vortex mixer 5min that vibrates, standing 1h, gets supernatant liquor 3ml and joins in the illitic liquid nitrogen-free agar of 200mg/L, is placed on 30 ℃ of constant-temperature tables 180rpm enrichment culture 3 days.After enrichment culture, pipette the volume ratio of nutrient solution 10% for the first time and access in the illitic liquid nutrient medium of 200mg/L, adopt the method that progressively improves bauxite concentration to silicate bacteria enrichment culture; Each illite concentration that increases 200mg/L, making last illite concentration is 1g/L, corotation connects to be cultivated 4 times.
Wherein, aforesaid liquid nitrogen-free agar contains: sucrose 5g, Na
2hPO
44g, MgSO
47H
2o 1g, CaCO
30.3g, bauxite 1g, water 1L, the FeCl that is 1% by the weight percentage that sulfuric acid modulation is dissolved
3solution 2ml, the pH value of substratum is 6-7.
(2) above-mentioned enrichment culture liquid (nutrient solution obtaining) is diluted to 10 with sterilized water under aseptic condition after 4 switchings are cultivated
6-10
8doubly, coat respectively in solid nitrogen-free agar, be placed in 30 ℃ of constant incubators and cultivate 24h, when planar surface no longer includes water droplet, be inverted flat board, cultivate 3-5 days, observe at any time bacterial colony growing state; The time growing according to bacterium colony and the form of bacterium colony, choose the transparent bacterium colony of mucus shape projection from culture medium flat plate, then cultivate, until obtain pure culture.
Wherein solid nitrogen-free agar contains: sucrose 5g, Na
2hPO
4h
2o 5g, MgSO
47H
2o 1.0g, CaCO
30.1g, bauxite 1g, water 1L, the FeCl that is 1% by the weight percentage that sulfuric acid modulation is dissolved
3solution 2ml, CaCO
3powder, 10g agar, the pH value of substratum is 6-7.CaCO
3powder is first ground to powder in mortar, after 200 mesh sieves; Afterwards by CaCO
3powder is 121 ℃ of high-pressure sterilizing pots, after sterilizing 20min, stand-by; Deng solid medium, be down flat after plate condensation, with sticky a little CaCO that gets of glass stick
3powder, is applied in planar surface.
(3) by obtaining pure strain, by 1% volume ratio, be inoculated in the nitrogenous liquid nutrient medium of 100ml respectively, every 24h, by silicon molybdenum blue method (this area ordinary method), survey silicon concentration in a solution.
Wherein the nitrogenous substratum of liquid contains: sucrose 3g, Na
2hPO
42g, MgSO
47H
2o 1g, (NH
4)
2sO
33.8g, CaCO
30.2g, bauxite 1g, water 1L, the FeCl that is 1% by the weight percentage that sulfuric acid modulation is dissolved
3solution 2ml, the pH value of substratum is 6-7.
(4) take < < People's Republic of China (PRC) city-building industry standard: the mensuration silicon molybdenum blue spectrophotometry > > of Urban water supply silicon-dioxide is as with reference to measuring SiO in solution
2content, typical curve is shown in Fig. 1.
Get solution 1ml after desiliconization, dilute 50 times, measure its light absorption value and be up to 1.241.From data measured, calculated SiO in solution
2content be up to 68.82mg/L.
(5) by the morphological specificity to this bacterium, observe: it grows bacterium colony on the flat board of solid nitrogen-free agar is that water white transparency projection degree is large, as half granulated glass sphere, bacterium colony surface wettability is and smooth, and quality thickness is also flexible.Further with scanning horizontal glass, observe ne ar, observe bacterium thalline stock shape, the blunt circle in two ends, general size is 4～7 microns * 1～1.2 micron, atrichia.
(6) screening obtained strains is shown to different capacity of decomposition to different potassium-bearing minerals, under same culture condition, take potassium felspar sand and white mica respectively as unique potassium source, and connecing bacterium processing increases respectively 67.4% and 54.5% than the potassium content connecing in inactivated bacteria contrast solution.With atomic absorption spectrochemical analysis and high performance liquid chromatography, the product in silicate bacteria nutrient solution is measured.Result shows, this silicate bacteria has the ability of decomposing potassium in glass.At shake-flask culture, after 72 hours, the content of water-soluble potassium has improved 55% than contrast.This has just proved that this strain silicate bacteria has the performance of potassium decomposing really.
Above-described embodiment is described the preferred embodiment of the present invention; not scope of the present invention is limited; design under the prerequisite of spirit not departing from the present invention; various distortion and improvement that those of ordinary skills make technical scheme of the present invention, all should fall in the definite protection domain of the claims in the present invention book.
Claims (8)
1. a method for rapid screening silicate bacteria, is characterized in that, comprises the following steps:
(1) soil bacteria suspension obtains: bauxite powder is joined in sterilized water, and standing after vibration, get supernatant liquor and join in liquid nitrogen-free agar;
(2) enrichment culture: will cultivate the cultivation of transferring of gained bacterial classification by liquid nitrogen-free agar in liquid nitrogen-free agar in step (1), corotation connects 4 times, by volume per-cent meter is measured in switching, and 10% of each nutrient solution is inoculated in the liquid nitrogen-free agar that bauxite addition increases gradually;
(3) sub-sieve is cultivated: the nutrient solution dilution 10 that the last switching in step (2) is cultivated
6-10
8doubly, coat in solid nitrogen-free agar, be placed in 30 ℃ of constant incubators and cultivate 24h, when solid nitrogen-free agar planar surface no longer includes water droplet, be inverted flat board, cultivate 3-5 days, observe at any time bacterial colony growing state; The time growing according to bacterium colony and the form of bacterium colony, choose the transparent bacterium colony of mucus shape projection from culture medium flat plate, then cultivate, until obtain pure culture;
(4) multiple sieve: the pure strain obtaining in step (3) is inoculated in the nitrogenous substratum of liquid, can analysing silicon hydrochlorate and the bacterium of the rock forming mineral that forms of aluminosilicate be silicate bacteria.
2. method according to claim 1, is characterized in that: the bauxite powder in described step (1) is illite powder, crosses 200 mesh sieves.
3. method according to claim 1, is characterized in that: the liquid nitrogen-free agar in described step (2) contains: sucrose 3-5g, Na
2hPO
42-4g or Na
2hPO
4h
2o 5-10g, MgSO
47H
2o 0.5-1.0g, CaCO
30.1-0.3g, bauxite 1g, water 1L, the FeCl that is 1% by the weight percentage that sulfuric acid modulation is dissolved
3solution 2ml, the pH value of substratum is 6-7.
4. method according to claim 1, is characterized in that: in described step (2), each switching culture condition is cultivated 3 days for being placed in 180rpm on 30 ℃ of constant-temperature tables.
5. method according to claim 1, is characterized in that: in described step (2) in liquid nitrogen-free agar bauxite first add-on be 200mg/L, each increasing amount is 200mg/L afterwards.
6. method according to claim 1, is characterized in that: in described step (3), solid nitrogen-free agar contains: sucrose 3-5g, Na
2hPO
42-4g or Na
2hPO
4h
2o 5-10g, MgSO
47H
2o 0.5-1.0g, CaCO
30.1-0.3g, bauxite 1g, water 1L, the FeCl that is 1% by the weight percentage that sulfuric acid modulation is dissolved
3solution 2ml, CaCO
3powder, 10g agar, the pH value of substratum is 6-7.
7. method according to claim 6, is characterized in that: CaCO in described solid nitrogen-free agar
3powder is: first in mortar, be ground to powder, after 200 mesh sieves; Afterwards by CaCO
3powder is 121 ℃ of high-pressure sterilizing pots, after sterilizing 20min, stand-by; Deng solid medium, be down flat after plate condensation, with sticky a little CaCO that gets of glass stick
3powder, is applied in planar surface.
8. method according to claim 1, is characterized in that: in described step (4), the nitrogenous substratum of liquid contains: sucrose 3-5g, Na
2hPO
42-4g or Na
2hPO
4h
2o 5g, MgSO
47H
2o 0.5-1g, (NH
4)
2sO
31.5-3.8g, CaCO
30.1-0.3g, bauxite 1g, water 1L, the FeCl that is 1% by the weight percentage that sulfuric acid modulation is dissolved
3solution 2ml, the pH value of substratum is 6-7.
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| CN201310138722.9A CN104109641A (en) | 2013-04-19 | 2013-04-19 | Method for rapidly screening silicate bacteria |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894003A (en) * | 2015-04-15 | 2015-09-09 | 中国科学院过程工程研究所 | Method for screening silicate bacteria from manganese nodule |
| CN105347320A (en) * | 2015-12-08 | 2016-02-24 | 中南大学 | Bacterial leaching agent and method for leaching phosphorus from apatite floatation tailings |
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| JP2002355552A (en) * | 2001-05-30 | 2002-12-10 | Yasunori Marubayashi | Material and method for treating nitrogen oxide, and method of treating flue gas |
| CN1546392A (en) * | 2003-12-08 | 2004-11-17 | 宾 连 | Method for preparing composite silicate bacterium flocculant |
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2013
- 2013-04-19 CN CN201310138722.9A patent/CN104109641A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2002355552A (en) * | 2001-05-30 | 2002-12-10 | Yasunori Marubayashi | Material and method for treating nitrogen oxide, and method of treating flue gas |
| CN1546392A (en) * | 2003-12-08 | 2004-11-17 | 宾 连 | Method for preparing composite silicate bacterium flocculant |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894003A (en) * | 2015-04-15 | 2015-09-09 | 中国科学院过程工程研究所 | Method for screening silicate bacteria from manganese nodule |
| CN105347320A (en) * | 2015-12-08 | 2016-02-24 | 中南大学 | Bacterial leaching agent and method for leaching phosphorus from apatite floatation tailings |
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