CN108467840A - The method for preparing calcium carbonate using the microorganism fungus kind extracted in soil - Google Patents
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Abstract
The invention belongs to microorganism solidification, more particularly to a kind of method preparing calcium carbonate using the microorganism fungus kind extracted in soil includes the following steps：Step 1, it is extracted from the high soil of the content of organic matter and isolates bacillus category strain；Step 2, breeding culture is carried out to the bacillus category strain that step 1 is separated；Step 3, bacillus category bacterium solution and gelled fluid that step 2 obtains are mixed in equal volume, is placed in constant temperature gas bath incubator after conserving a period of time, mixed liquor is filtered, microorganism induction precipitation of calcium carbonate is obtained；The present invention directly extracts microorganism fungus kind from soil and prepares calcium carbonate, select urea and bean powder peptone as the solid selection medium of separation bacillus category strain, separative efficiency is high, the bacillus category strain isolated is mixed with gelled fluid, after being conserved under felicity condition, microorganism induction precipitation of calcium carbonate is obtained；This method is economic and environment-friendly, and operation is simple and calcium carbonate yield is high.
Description
Technical field
It is the invention belongs to microorganism solidification, more particularly to a kind of to prepare calcium carbonate using the microorganism fungus kind extracted in soil
Method.
Background technology
Biomineralization is a kind of very universal natural phenomena, and almost each biology can synthetic mineral.It is wherein close
Three/second is that calcium mine, and quite a few is with cementing function.They are by the vital movement of its own, with ambient enviroment
Enzymeization effect constantly occurs between medium, gradually forms calcium carbonate.In recent years, with the research and development of microorganism in the soil body,
In soil the product of the vital movement activity of energy and carbon (mainly obtain) of already existing microorganism be used in engineering can
Energy property conceptually has been achieved for widely approving.Under such overall situation, microorganism curing technology comes into being.
So-called microorganism solidification is exactly that the metabolite processing sand etc. generated using the vital movement of microorganism in soil is dissipated
Dress or weak material, to meet requirement of engineering.Compared with traditional reinforcement technique, biological energy source during microorganism curing technology
Instead of mechanical energy, the product of bacteria metabolism is instead of chemicals, and with clean environment firendly, energy consumption is low, is disturbed to the soil body
It is dynamic small, the advantages that uniform is handled, the strategic objective of sustainable development is extremely agreed with.
Chinese patent CN201610828703.2 discloses a kind of method preparing calcium carbonate solidification sand using microorganism,
Sand is poured into bottle, its uniform fold bottom of bottle is made, is subsequently poured into gelled fluid and cultured bacterium solution, isothermal vibration culture, it
It is quantitative daily afterwards to draw part mixed liquor in bottle, then add the gelled fluid of equivalent, be uniformly added into a small amount of sand, last last from days
The sand column being cured afterwards；But the selection and breeding of the strain of this method usually require to consume many manpower and materials, and calcium carbonate produces
Rate needs to be further increased.
Invention content
The present invention solves the above-mentioned technical problems in the prior art, provides and a kind of utilizing the microorganism extracted in soil
The method that strain prepares calcium carbonate.
To solve the above problems, technical scheme is as follows：
A method of calcium carbonate being prepared using the microorganism fungus kind extracted in soil, is included the following steps：
Step 1, it is extracted from the soil of content of organic matter 20-30g/kg and isolates bacillus category strain；Detach bud
The solid selection medium of spore bacillus class strain is urea and bean powder peptone；
Step 2, breeding culture is carried out to the bacillus category strain that step 1 is separated, using spectrophotometer 600nm
The absorbance value that wavelength measurement obtains bacillus category bacterium solution is 1.4-1.7；
Step 3, bacillus category bacterium solution and gelled fluid that step 2 obtains are mixed in equal volume, is placed in 30 DEG C of constant temperature gas baths
After conserving a period of time in incubator, mixed liquor is filtered, microorganism induction precipitation of calcium carbonate is obtained；The gelled fluid by
Urea, calcium acetate and water form, and the molar concentration of urea is 0.5-1.0mol/L, the molar concentration of calcium acetate in the coagulant liquid
For 0.5-1.0mol/L.
Preferably, soil collection described in step 1 is from the soil sample in depth 5-30cm, the store method of soil sample：It is put into and goes out
The sample sack interior sealing of bacterium is stored in 4 DEG C of refrigerators.
Preferably, it is extracted described in step 1 and the specific method for isolating bacillus category strain is：
A) soil sample 5g is weighed, is poured into load weighted soil sample in 90ml sterile waters in superclean bench, fully shaking makes
Soil sample is in suspension shape, sets and stands 4 hours at room temperature, and as 10-1Dilution；
B) it is shaken in equipped with the test tube in 4.5ml sterile waters with the micropipettor soil supernatant 0.5ml that takes that treated
It swings, bacterium solution is allowed to be uniformly mixed, as 10-2Dilution；Draw 10-2Dilution 0.5ml is moved into equipped with the examination in 4.5ml sterile waters
In pipe, oscillation, allow bacterium solution be uniformly mixed, 10-3Dilution；Serial dilution is made 10-2、10-3、10-4、10-5Dilution；
C) 10 are drawn with liquid-transfering gun-2、10-3、10-4、10-5Each 0.05ml of dilution, is seeded in the agar accordingly numbered respectively
On tablet；The tablet for being coated with dilution is lain against on desktop, standing 10-20min makes bacterium solution penetrate into culture medium, then will
Culture medium is inverted, and is placed in 30 DEG C of constant incubators and is cultivated for 24 hours；
D) urea and bean powder peptone are added in the medium and is used as solid selection medium, in the culture medium urea with
The concentration of bean powder peptone is 10-20g/L, is used in combination spreader to be coated with phenolphthalein solution in media surface, then uses platinum wire
The strain suspension after culture is dipped in media surface streak inoculation, sets in 30 DEG C of constant incubators and cultivates for 24 hours, finally take week
Enclose the bacterial strain as purifying next time there are the bacterial plaque of red area；It purifies 3 times repeatedly, finally obtains purifying bacterial strain.
Preferably, bacillus category strain described in step 2 carries out breeding and cultivates selected culture medium to include following weight
The component of part：
Yeast extract：5 parts；
Peptone：10 parts；
Sodium chloride：10 parts；
Urea：10 parts；
Potassium dihydrogen phosphate：1.4 part；
Disodium hydrogen phosphate：5.3 part；
Magnesium sulfate：0.2 part；
Powdered glucose：10 parts；
Agar：15 parts；
Water：1000 parts；
The pH of the culture medium is 7.
Preferably, the curing time in 30 DEG C of constant temperature gas bath incubators of mixture described in step 3 is 2 days or more.
Preferably, the curing time in 30 DEG C of constant temperature gas bath incubators of mixture described in step 3 is 5 days or more.
Compared with the existing technology, advantages of the present invention is as follows,
The present invention prepares calcium carbonate using the microorganism fungus kind extracted in soil, select urea and bean powder peptone as point
Solid selection medium from bacillus category strain, separative efficiency is high, the bacillus category strain and gelled fluid that will be isolated
It mixes, after being conserved under felicity condition, obtains microorganism induction precipitation of calcium carbonate；This method is economic and environment-friendly, operation it is simple and
Calcium carbonate yield is high.
Description of the drawings
Fig. 1 is addition urea (10g/L) and bean powder peptone (10g/L) in Selective agar medium, using isometric bacterium solution
The precipitation of calcium carbonate obtained with gelled fluid (formula is shown in Table 2).
Specific implementation mode
Embodiment 1：
A method of preparing calcium carbonate, the key step of this method using the microorganism fungus kind extracted in soil：
1) soil sample of acquisition content of organic matter 20-30g/kg, takes the soil sample in depth 5-30cm several, soil sample is put into and is gone out
The sample sack interior sealing of bacterium is stored in 4 DEG C of refrigerators.
2) it is extracted from soil sample and isolates bacillus category strain.
A) soil sample 5g is weighed respectively, pours into load weighted soil sample in 90ml sterile waters in superclean bench, is fully shaken
Swinging makes soil sample be in suspension shape, sets and stands 4 hours at room temperature, and as 10-1Dilution.
B) it is shaken in equipped with the test tube in 4.5ml sterile waters with the micropipettor soil supernatant 0.5ml that takes that treated
It swings, bacterium solution is allowed to be uniformly mixed, as 10-2Dilution.Draw 10-2Dilution 0.5ml is moved into equipped with the examination in 4.5ml sterile waters
In pipe, oscillation, allow bacterium solution be uniformly mixed, 10-3Dilution；Serial dilution is made 10-2、10-3、10-4、10-5Dilution
C) 10 are drawn with liquid-transfering gun-2、10-3、10-4、10-5Each 0.05ml of dilution, is seeded in the agar accordingly numbered respectively
On tablet.The tablet for being coated with dilution is lain against on desktop, standing 10-20min makes bacterium solution penetrate into culture medium, then will
Culture medium is inverted, and is placed in 30 DEG C of constant incubators and is cultivated for 24 hours.
D) addition urea (10g/L) and bean powder peptone (10g/L) are used as solid selection medium in the medium, are used in combination
Spreader is coated with phenolphthalein solution in media surface, and the strain suspension after then dipping culture with platinum wire is in media surface
Streak inoculation is set in 30 DEG C of constant incubators and is cultivated for 24 hours, finally takes the bacterial plaque that around there is red area as purifying next time
Bacterial strain.It purifies 3 times repeatedly, finally obtains purifying bacterial strain.
3) corresponding culture medium is prepared, formula is shown in Table 1, is bred to the bacillus category strain separated；By 3
The secondary Spawn incubation 36h isolated and purified obtains the suction of bacillus category bacterium solution using spectrophotometer 600nm wavelength measurements
Shading value reaches 1.7.
4) it prepares gelled fluid and induces strain winnofil together with strain liquid.Isometric bacterium solution and gelled fluid (are matched
Fang Jianbiao 2) mixing be added 50ml centrifuge tubes in, centrifuge tube is placed in 30 DEG C of constant temperature gas bath incubators and is conserved 5 days, mixed liquor into
Row filtering, it is 83% to obtain microorganism induction precipitation of calcium carbonate yield.
1 culture medium prescription of table (1000mL)
The gelling formula of liquid of table 2 (1000mL)
Embodiment 2：
Using bacillus category strain in the method extraction purification soil of same embodiment 1, the difference is that adding in the medium
Add urea (20g/L) and bean powder peptone (20g/L) to be used as solid selection medium, finally obtains purifying bacterial strain.Prepare such as table 1
Shown in culture medium the bacillus category strain separated is bred；The Spawn incubation isolated and purified by 3 times
36h, absorbance reach 1.4.It finally prepares gelled fluid and induces strain winnofil together with strain liquid.By isometric bacterium solution
It mixes and is added in 50ml centrifuge tubes with gelled fluid (formula is shown in Table 2), centrifuge tube is placed in 30 DEG C of constant temperature gas bath incubators and conserves 2
It, is filtered mixed liquor, and it is 56% to obtain microorganism induction precipitation of calcium carbonate yield.
Embodiment 3：
Using bacillus category strain in the method extraction purification soil of same embodiment 1, the difference is that adding in the medium
Add urea (20g/L) and bean powder peptone (20g/L) to be used as solid selection medium, finally obtains purifying bacterial strain.Prepare such as table 1
Shown in culture medium the bacillus category strain separated is bred；The Spawn incubation isolated and purified by 3 times
36h, absorbance reach 1.4.It finally prepares gelled fluid and induces strain winnofil together with strain liquid.By isometric bacterium solution
It mixes and is added in 50ml centrifuge tubes with gelled fluid (formula is shown in Table 3), centrifuge tube is placed in 30 DEG C of constant temperature gas bath incubators and conserves 2
It, is filtered mixed liquor, and it is 42% to obtain microorganism induction precipitation of calcium carbonate yield.
The gelling formula of liquid of table 3 (1000mL)
Comparative example 1：
Using bacillus category strain in the method extraction purification soil of same embodiment 1, the difference is that solid selection culture
Without addition urea and bean powder peptone in base.Culture medium as shown in Table 1 is prepared to the bacillus category strain separated
It is bred；The Spawn incubation 36h isolated and purified by 3 times, absorbance reach 1.0.Finally prepare gelled fluid and strain
Liquid induces strain winnofil together.Isometric bacterium solution and gelled fluid (formula is shown in Table 2) are mixed, 50ml centrifuge tubes are added
In, centrifuge tube is placed in 30 DEG C of constant temperature gas bath incubators and is conserved 2 days, mixed liquor is filtered, microorganism induction carbon is obtained
Sour calcium precipitate yield is 20%.
Comparative example 2：
Using bacillus category strain in the method extraction purification soil of same embodiment 1, the difference is that solid selection culture
Urea (20g/L) is only added in base.It is numerous to the bacillus category strain progress separated to prepare culture medium as shown in Table 1
It grows；The Spawn incubation 36h isolated and purified by 3 times, absorbance reach 1.1.Gelled fluid is finally prepared together with strain liquid
Induce strain winnofil.Isometric bacterium solution and gelled fluid (formula is shown in Table 2) is mixed and is added in 50ml centrifuge tubes, it will be from
Heart pipe is placed in 30 DEG C of constant temperature gas bath incubators and conserves 5 days, is filtered to mixed liquor, obtains microorganism induction precipitation of calcium carbonate
Yield is 25%.
Comparative example 3：
Using bacillus category strain in the method extraction purification soil of same embodiment 1, the difference is that solid selection culture
Bean powder peptone (20g/L) is only added in base.Prepare culture medium as shown in Table 1 to the bacillus category strain separated into
Row breeding；The Spawn incubation 36h isolated and purified by 3 times, absorbance reach 1.0.Finally prepare gelled fluid and strain liquid
Strain winnofil is induced together.Isometric bacterium solution and gelled fluid (formula is shown in Table 2) are mixed and are added in 50ml centrifuge tubes,
Centrifuge tube is placed in 30 DEG C of constant temperature gas bath incubators and is conserved 5 days, mixed liquor is filtered, microorganism induction calcium carbonate is obtained
It is 21% to precipitate yield.
Comparative example 4：
Using bacillus category strain in the method extraction purification soil of same embodiment 1, the difference is that adding in the medium
Add cellulose powder (10g/L) and hydrolyzed casein (10g/L) to be used as solid selection medium, prepares culture medium pair as shown in Table 1
The bacillus category strain separated is bred；The Spawn incubation 36h isolated and purified by 3 times, absorbance reach
1.2.It finally prepares gelled fluid and induces strain winnofil together with strain liquid.By isometric bacterium solution and gelled fluid (formula
Be shown in Table 2) mixing be added 50ml centrifuge tubes in, centrifuge tube is placed in 30 DEG C of constant temperature gas bath incubators and is conserved 5 days, to mixed liquor into
Row filtering, it is 23% to obtain microorganism induction precipitation of calcium carbonate yield.
It should be noted that above-described embodiment is only presently preferred embodiments of the present invention, there is no for the purpose of limiting the invention
Protection domain, the equivalent replacement or replacement made on the basis of the above all belong to the scope of protection of the present invention.
Claims (6)
1. a kind of method preparing calcium carbonate using the microorganism fungus kind extracted in soil, which is characterized in that include the following steps：
Step 1, it is extracted from the soil of content of organic matter 20-30g/kg and isolates bacillus category strain；Detach gemma bar
The solid selection medium of mushroom strain is urea and bean powder peptone；
Step 2, breeding culture is carried out to the bacillus category strain that step 1 is separated, using spectrophotometer 600nm wavelength
It is 1.4-1.7 to measure and obtain the absorbance value of bacillus category bacterium solution；
Step 3, bacillus category bacterium solution and gelled fluid that step 2 obtains are mixed in equal volume, is placed in 30 DEG C of constant temperature gas bath cultures
After conserving a period of time in case, mixed liquor is filtered, microorganism induction precipitation of calcium carbonate is obtained；The gelled fluid is by urinating
Element, calcium acetate and water form, and the molar concentration of urea is 0.5-1.0mol/L in the coagulant liquid, and the molar concentration of calcium acetate is
0.5-1.0mol/L。
2. the method for preparing calcium carbonate using the microorganism fungus kind extracted in soil as described in claim 1, which is characterized in that
Soil collection described in step 1 is from the soil sample in depth 5-30cm, the store method of soil sample：It is put into the sample sack interior sealing of sterilizing
It is stored in 4 DEG C of refrigerators.
3. the method for preparing calcium carbonate using the microorganism fungus kind extracted in soil as described in claim 1, which is characterized in that
It is extracted described in step 1 and the specific method for isolating bacillus category strain is：
A) soil sample 5g is weighed, is poured into load weighted soil sample in 90ml sterile waters in superclean bench, fully shaking makes soil sample
In suspension shape, sets and stand 4 hours at room temperature, as 10-1Dilution；
B) with the micropipettor soil supernatant 0.5ml that takes that treated in equipped with the test tube in 4.5ml sterile waters, oscillation,
Bacterium solution is allowed to be uniformly mixed, as 10-2Dilution；Draw 10-2Dilution 0.5ml is moved into equipped with the test tube in 4.5ml sterile waters
It is interior, oscillation, allow bacterium solution be uniformly mixed, 10-3Dilution；Serial dilution is made 10-2、10-3、10-4、10-5Dilution；
C) 10 are drawn with liquid-transfering gun-2、10-3、10-4、10-5Each 0.05ml of dilution, is seeded in the agar plate accordingly numbered respectively
On；The tablet for being coated with dilution is lain against on desktop, standing 10-20min makes bacterium solution penetrate into culture medium, then will culture
Base is inverted, and is placed in 30 DEG C of constant incubators and is cultivated for 24 hours；
D) urea and bean powder peptone are added in the medium as solid selection medium, urea and bean powder in the culture medium
The concentration of peptone is 10-20g/L, is used in combination spreader to be coated with phenolphthalein solution in media surface, is then dipped with platinum wire
Strain suspension after culture is set in 30 DEG C of constant incubators and is cultivated for 24 hours in media surface streak inoculation, and surrounding is finally taken to deposit
In the bacterial strain that the bacterial plaque of red area is purified as next time；It purifies 3 times repeatedly, finally obtains purifying bacterial strain.
4. the method for preparing calcium carbonate using the microorganism fungus kind extracted in soil as described in claim 1, which is characterized in that
Bacillus category strain described in step 2 carries out breeding and cultivates the component that selected culture medium includes following parts by weight：
Yeast extract：5 parts；
Peptone：10 parts；
Sodium chloride：10 parts；
Urea：10 parts；
Potassium dihydrogen phosphate：1.4 part；
Disodium hydrogen phosphate：5.3 part；
Magnesium sulfate：0.2 part；
Powdered glucose：10 parts；
Agar：15 parts；
Water：1000 parts；
The pH of the culture medium is 7.
5. the method for preparing calcium carbonate using the microorganism fungus kind extracted in soil as described in claim 1, which is characterized in that
The curing time in 30 DEG C of constant temperature gas bath incubators of mixture described in step 3 is 2 days or more.
6. the method for preparing calcium carbonate using the microorganism fungus kind extracted in soil as described in claim 1, which is characterized in that
The curing time in 30 DEG C of constant temperature gas bath incubators of mixture described in step 3 is 5 days or more.
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| CN109459364A (en) * | 2018-10-30 | 2019-03-12 | 河海大学 | A kind of experimental rig and method for reinforcing calcareous coarse-grained soil based on MICP |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109459364B (en) * | 2018-10-30 | 2021-04-09 | 河海大学 | Test device and method for reinforcing calcareous coarse-grained soil based on MICP |
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| CN110665961A (en) * | 2019-10-22 | 2020-01-10 | 武汉科技大学 | Method for restoring chromium-polluted land |
| CN110665961B (en) * | 2019-10-22 | 2021-05-25 | 武汉科技大学 | Method for restoring chromium-polluted land |
| CN111875306A (en) * | 2020-08-10 | 2020-11-03 | 中国科学院武汉岩土力学研究所 | Microorganism enhancement method for three-dimensional printing of solid cement-based solid model |
| CN114605166A (en) * | 2022-03-07 | 2022-06-10 | 上海市地江建筑科技有限公司 | Carbon-immobilized biological foam concrete and preparation method thereof |
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