CN113717992A - Method for economically and efficiently generating calcium carbonate under high-temperature condition - Google Patents
Method for economically and efficiently generating calcium carbonate under high-temperature condition Download PDFInfo
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- CN113717992A CN113717992A CN202110817282.4A CN202110817282A CN113717992A CN 113717992 A CN113717992 A CN 113717992A CN 202110817282 A CN202110817282 A CN 202110817282A CN 113717992 A CN113717992 A CN 113717992A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01005—Urease (3.5.1.5)
Abstract
The invention belongs to curing of exogenous urease, and particularly relates to a method for economically and efficiently generating calcium carbonate under a high-temperature condition, which comprises the following steps: step 1, preparing a soybean solution and a gelling liquid, and standing at a low temperature; step 2, taking the soybean solution which is kept stand in the step 1, uniformly stirring and centrifuging, and taking supernatant liquid for low-temperature refrigeration for later use; step 3, mixing the supernatant obtained in the step 2 with the gelling liquid in equal volume, maintaining for a period of time under a high temperature condition, carrying out centrifugal filtration on the mixed liquid, and drying the filtered product in an oven; and 4, carrying out acid washing on the dried product, and drying after acid washing to obtain the content of the calcium carbonate generated by the reaction. The invention uses urease in soybean solution to promote the generation of calcium carbonate, selects urea and calcium chloride as gelling liquid, mixes the soybean solution and the gelling liquid, cultures the mixture under high temperature, centrifuges, dries, acid washes and dries the mixture to obtain the quality of the generated calcium carbonate, thereby obtaining the optimal concentration of the soybean solution under different temperatures and determining the proper reaction temperature. The method has the advantages of easy operation, high calcium carbonate yield, economy and high efficiency.
Description
Technical Field
The invention belongs to curing of exogenous urease, and particularly relates to a method for economically and efficiently producing calcium carbonate under a high-temperature condition.
Background
Biomineralization is a very common natural phenomenon, and almost every organism can synthesize minerals. Nearly two thirds of the calcium minerals are calcium minerals, and a considerable part of the calcium minerals have a cementing function. Through their own vital activities, they are constantly enzymatically active with the surrounding medium, gradually forming calcium carbonate. Microorganism-induced calcium carbonate precipitation (MICP) also becomes a new research direction for reinforcing soil, but MICP has a non-negligible negative effect on the environment. In recent years, mineralization induced by using enzyme to generate calcium carbonate precipitate (EICP) becomes a hotspot concerned by many scholars, and compared with MICP, EICP has the advantages of rapid response, environmental friendliness, wide application range and the like.
In actual conditions, the environment where soil needs to be reinforced is complicated, for example, a high-temperature environment is often encountered in the process of oil and natural gas exploitation, and how to apply the EICP in the high-temperature environment is a problem to be solved urgently. Urease is the most central substance of EICP reaction, and urease activity determines the rate of enzymatic reaction, and is finally reflected on the generation amount of calcium carbonate and the generation rate thereof. As a protein, high temperature can affect the activity of the enzyme and even inactivate the enzyme, and the application of the EICP in a high-temperature environment is affected.
The influence of temperature on urease activity was reported by Whiffinfs, a foreign scholars article "Microbial CaCO3 Precipitation for The Production of biological". The literature indicates that urease activity remains stable at 15-25 ℃, increases linearly with increasing temperature between 25-60 ℃, peaks at 75 ℃, and deactivates at a higher temperature after reaching 75 ℃ at a faster rate. To ensure the rationality of the high temperature test, the test flow design was carried out according to the data given in this document.
Disclosure of Invention
The invention provides a method for economically and efficiently generating calcium carbonate under the high-temperature condition by considering the influence of the concentration and the temperature of a soybean solution on the activity of urease.
In order to achieve the above object, the technical solution of the present invention is as follows:
a method for economically and efficiently producing calcium carbonate under high temperature conditions, comprising the steps of:
step 1, preparing a soybean solution and a gelling liquid, and standing at a low temperature; the concentration range of the soybean solution is 20-100g/L, the concentration gradient is 20g/L, the content of the gelling liquid is urea and calcium chloride, the concentration of the urea is 1mol/L, and the concentration of the calcium chloride is 0.67 mol/L;
step 2, taking the soybean solution which is kept stand in the step 1, uniformly stirring and centrifuging, and taking supernate for low-temperature refrigeration for later use after centrifuging;
step 3, mixing the supernatant obtained in the step 2 and the gelling liquid in equal volumes respectively, maintaining for a period of time under a high temperature condition, centrifuging the mixed liquid, filtering after centrifugation, and drying the filtered product in an oven;
step 4, weighing the dried centrifugal tube and filter paper m1Acid cleaning the centrifuge tube and the filter paper, drying in a drying oven after acid cleaning, and weighing the mass m of the centrifuge tube and the filter paper after acid cleaning2The difference m between the two2-m1I.e. the content of calcium carbonate produced by the reaction.
Preferably, the soybean flour used in the soybean solution of step 1 is finely ground soybean flour having a particle size of 5 to 20 μm, and the soybean solution and the gelling solution are preserved by the following steps: sealing with preservative film and storing in refrigerator at 4 deg.c.
Preferably, the specific method for preparing the solution in step 1 is as follows:
a) weighing 10-50g soybean powder with an electronic balance within + -0.0.1 g. Weighing 500ml of distilled water, placing in a clean and anhydrous beaker, dissolving the bean powder in the distilled water, and stirring for 3min by using a stirrer to ensure that the bean powder is fully dissolved;
b) 60g of urea and 105.8g of calcium acetate are weighed by using an electronic balance, and the error is required to be within the range of +/-0.0.1 g. 1000ml of distilled water is weighed and placed in a clean anhydrous beaker, the urea and the calcium chloride are dissolved in the distilled water, and the mixture is stirred for 3min by using a stirrer, so that the urea and the calcium chloride are fully dissolved.
c) Taking 15ml of the uniformly stirred gel liquid by using a micropipettor, and placing the gel liquid into a centrifugal tube for later use;
preferably, the specific operation method of the step 2 centrifugation is as follows:
a) uniformly stirring the soybean solution which is kept stand at a low temperature, pouring the soybean solution into centrifuge tubes, pouring 45ml of the soybean solution into each centrifuge tube, and using 12 centrifuge tubes in total;
b) centrifugation was performed in two groups using a tube centrifuge, with 6 centrifuge tubes per group. The centrifugation time is 10 minutes, and the centrifugation speed is 3000 r/min;
c) after centrifugation, pouring the supernatant into a clean beaker, in the process, taking care not to pour the bean dregs, pouring all the supernatant in 12 centrifuge tubes into the beaker, sealing the beaker by using a preservative film, and standing the beaker at a low temperature of 4 ℃;
preferably, 15ml of the supernatant and the gel liquid in the step 3 are taken, the curing temperature is 65/75/85 ℃, the curing time is 3h, and the centrifugation time is 5 min. The filtered filter paper and the centrifuge tube are dried to ensure the accuracy of the result.
Preferably, the drying temperature in step 3 and the drying time in step 4 are both 75 ℃ and 24 h.
Preferably, the specific steps of acid washing in step 4 are as follows:
a) adding 20ml of dilute hydrochloric acid into a centrifugal tube, standing for 2 minutes, and mashing the precipitate in the centrifugal tube by using a vibrating rod to ensure that the precipitate and the hydrochloric acid fully react for 2 minutes;
b) adding 20ml of dilute hydrochloric acid again, washing the wall-hung reaction product to the bottom in the process, and oscillating the centrifugal tube to fully react;
c) observing whether sediment exists at the bottom of the centrifuge tube which is kept stand, if so, repeating the step b) for 2-3 times until no obvious sediment exists in the centrifuge tube and no air bubble is generated, and filtering the solution in the centrifuge tube;
d) observing whether residual foam wall hanging exists in the centrifuge tube or not, if so, using dilute hydrochloric acid to be clear, and pouring the mixed solution into a funnel for filtering; and after the filtration is finished, putting the centrifuge tube and the filter paper into an oven for drying, and finishing the pickling operation. Note that the mark is made in the acid washing process to ensure that the centrifuge tubes and the filter paper are in one-to-one correspondence
In view of the current application, the advantages of the present invention are as follows,
the invention economically and efficiently generates calcium carbonate precipitation under the high-temperature condition, selects soybean as a urease source, selects urea and calcium chloride as a gelling liquid, mixes the soybean solution and the gelling liquid, cultures the soybean solution under the high-temperature condition, centrifuges, dries, acid washes and dries the mixed solution again to obtain the quality of the generated calcium carbonate, thereby obtaining the optimal concentration of the soybean solution under different temperatures and determining the proper reaction temperature. The method has the advantages of easy operation, high calcium carbonate yield, economy and high efficiency.
Drawings
FIG. 1 is a process flow for producing and measuring calcium carbonate at high temperature
Detailed Description
Example 1:
a method for economically and efficiently producing calcium carbonate under high temperature conditions mainly comprises the following steps:
1) finely ground soybean powder having a particle size of 5 to 20 μm was weighed in the amounts shown in Table 1. 500ml of distilled water was measured from a dry clean beaker, and the soybean powder was dissolved in distilled water to give a soybean solution having a concentration shown in Table 1. And stirred for 5 minutes using a magnetic stirrer. Weighing 60g of urea and 105.8g of calcium chloride, weighing 1000ml of distilled water in a dry clean beaker, dissolving the urea and the calcium chloride in the distilled water, stirring for 5 minutes by using a magnetic stirrer, sealing the soybean solution and the gel liquid, and storing in a refrigerator at 4 ℃.
2) Taking out the standing soybean solution, uniformly stirring, and pouring into 12 centrifuge tubes, wherein the volume of the solution in each centrifuge tube is 45 mL. The soybean solution was centrifuged using a centrifuge at 3000 rpm for 10 minutes in two groups of six. And (4) sealing the centrifuged supernatant in a refrigerator at 4 ℃ for storage, wherein the supernatant is the urease solution.
3) Taking out the gel liquid, taking 15ml by using a micro-pipette, placing the gel liquid in a centrifuge tube, taking out the urease solution which is kept stand, taking 15ml and mixing the gel liquid with the same volume, and placing the centrifuge tube in a constant temperature water bath box at 65 ℃ for reaction for 3 hours.
4) After the reaction is finished, centrifuging the centrifuge tube for 5 minutes at 3000 r/min. And (4) filtering the product after centrifugation, and putting filter paper and a centrifuge tube into a 75 ℃ oven for drying for 24 hours.
5) The filter paper and the centrifuge tube are taken out, and the mass of the filter paper and the centrifuge tube is weighed. And then carrying out acid washing operation, and placing the filter paper and the centrifuge tube in a 75 ℃ oven for drying for 24 hours until no precipitate exists in the centrifuge tube and no air bubbles are generated on the liquid surface.
6) And taking out the filter paper and the centrifuge tube, weighing the mass of the filter paper and the centrifuge tube to obtain the generation amount of the calcium carbonate, and calculating to obtain the yield of the calcium carbonate.
7) Two groups of parallel samples are set in the test, and the average value of the test results is taken as the final result. The carbonic acid yield/concentration of the soybean solution was used to reflect the economic benefits of a specific concentration of soybean solution at that temperature, and the quality, yield and economic benefits of the obtained calcium carbonate are shown in table 2.
TABLE 1 Soybean dose and Soybean solution concentration (500mL)
TABLE 2 calcium carbonate quality, yield and economic benefit
Example 2:
the soybean solutions and the gelling solutions were prepared in the same manner as in example 1 to prepare the soybean solutions shown in table 1, and finally, urease solutions were obtained. Mixing urease solution with the same volume of 15mL and the gel liquid, placing the mixture in a constant-temperature water bath box with the temperature of 75 ℃ for reaction for 3 hours, centrifugally filtering the product, drying the product, then carrying out acid washing operation, drying the product after acid washing, weighing the product to obtain the generation amount of calcium carbonate, and calculating the yield of the calcium carbonate and the corresponding economic benefit, which are shown in Table 3.
TABLE 3 calcium carbonate quality, yield and economic benefit
Example 3:
the soybean solutions and the gelling solutions were prepared in the same manner as in example 1 to prepare the soybean solutions shown in table 1, and finally, urease solutions were obtained. Mixing urease solution with the same volume of 15mL and the gelling solution, placing the mixture in a constant-temperature water bath box with the temperature of 85 ℃ for reaction for 3 hours, centrifugally filtering the product, drying the product, then carrying out acid washing operation, drying the product after acid washing, weighing the product to obtain the generation amount of calcium carbonate, and calculating the yield of the calcium carbonate and the corresponding economic benefit, which are shown in Table 4.
TABLE 4 calcium carbonate quality, yield and economic benefits
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and all equivalent substitutions or substitutions made on the above-mentioned embodiments are included in the scope of the present invention.
Claims (7)
1. A method for economically and efficiently producing calcium carbonate at high temperatures, comprising the steps of:
step 1, preparing a soybean solution and a gelling liquid, and standing at a low temperature; the concentration range of the soybean solution is 20-100g/L, the concentration gradient is 20g/L, the content of the gelling liquid is urea and calcium chloride, the concentration of the urea is 1mol/L, and the concentration of the calcium chloride is 0.67 mol/L;
step 2, taking the soybean solution which is kept stand in the step 1, uniformly stirring and centrifuging, and taking supernate for low-temperature refrigeration for later use after centrifuging;
step 3, mixing the supernatant obtained in the step 2 and the gelling liquid in equal volumes respectively, maintaining for a period of time under a high temperature condition, centrifuging the mixed liquid, filtering after centrifugation, and drying the filtered product in an oven;
step 4, weighing the dried centrifugal tube and filter paper m1Acid cleaning the centrifuge tube and the filter paper, drying in a drying oven after acid cleaning, and weighing the mass m of the centrifuge tube and the filter paper after acid cleaning2The difference m between the two2-m1I.e. the content of calcium carbonate produced by the reaction.
2. The method for producing calcium carbonate under high temperature conditions with high efficiency as claimed in claim 1, wherein the soybean flour used in the soybean solution of step 1 is finely ground soybean flour having a particle size of 5 to 20 μm, and the soybean solution and the gel are preserved by: sealing and storing in a refrigerator at 4 deg.C.
3. The method for economically and efficiently producing calcium carbonate at high temperature according to claim 1, wherein the solution preparation in step 1 comprises the following steps:
a) weighing 10-50g soybean powder with an electronic balance within + -0.0.1 g. Weighing 500ml of distilled water, placing in a clean and anhydrous beaker, dissolving the bean powder in the distilled water, and stirring for 3min by using a stirrer to ensure that the bean powder is fully dissolved;
b) 60g of urea and 105.8g of calcium acetate are weighed by using an electronic balance, and the error is required to be within the range of +/-0.0.1 g. 1000ml of distilled water is weighed and placed in a clean anhydrous beaker, the urea and the calcium chloride are dissolved in the distilled water, and the mixture is stirred for 3min by using a stirrer, so that the urea and the calcium chloride are fully dissolved.
c) And (3) taking 15ml of the uniformly stirred gel liquid by using a micropipette, and placing the gel liquid into a centrifuge tube for later use.
4. The method for economically and efficiently producing calcium carbonate under high temperature conditions according to claim 1, wherein the centrifugation is carried out by:
a) uniformly stirring the soybean solution which is kept stand at a low temperature, pouring the soybean solution into centrifuge tubes, pouring 45ml of the soybean solution into each centrifuge tube, and using 12 centrifuge tubes in total;
b) centrifugation was performed in two groups using a tube centrifuge, with 6 centrifuge tubes per group. The centrifugation time is 10 minutes, and the centrifugation speed is 3000 r/min;
c) and (3) after centrifugation, pouring the supernatant into a clean beaker, taking care not to pour the bean dregs in the process, pouring all the supernatant in 12 centrifuge tubes into the beaker, and sealing the beaker by using a preservative film to stand at a low temperature of 4 ℃.
5. The method for economically and efficiently producing calcium carbonate under high temperature conditions according to claim 1, wherein 15ml of the supernatant and the gel liquid are taken in the step 3, the curing temperature is 65/75/85 ℃, the curing time is 3h, and the centrifugation time is 5 min. The filtered filter paper and the centrifuge tube are dried to ensure the accuracy of the result.
6. The method for economically and efficiently producing calcium carbonate under high temperature conditions according to claim 1, wherein the drying temperature in step 3 and the drying time in step 4 are both 75 ℃ and 24 hours.
7. The method for economically and efficiently producing calcium carbonate under high temperature conditions according to claim 1, wherein the acid washing comprises the following specific steps:
a) adding 20ml of dilute hydrochloric acid into a centrifugal tube, standing for 2 minutes, and mashing the precipitate in the centrifugal tube by using a vibrating rod to ensure that the precipitate and the hydrochloric acid fully react for 2 minutes;
b) adding 20ml of dilute hydrochloric acid again, washing the wall-hung reaction product to the bottom in the process, and oscillating the centrifugal tube to fully react;
c) observing whether sediment exists at the bottom of the centrifuge tube which is kept stand, if so, repeating the step b) for 2-3 times until no obvious sediment exists in the centrifuge tube and no air bubble is generated, and filtering the solution in the centrifuge tube;
d) observing whether residual foam wall hanging exists in the centrifuge tube or not, if so, using dilute hydrochloric acid to be clear, and pouring the mixed solution into a funnel for filtering; and after the filtration is finished, putting the centrifuge tube and the filter paper into an oven for drying, and finishing the pickling operation. Note that the mark is made during the acid washing process to ensure that the centrifuge tubes and the filter paper are in one-to-one correspondence.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097061A (en) * | 2018-08-07 | 2018-12-28 | 河海大学 | A kind of agent and its preparation method and application of checking winds and fixing drifting sand of the urase containing plant |
| CN109735575A (en) * | 2019-01-18 | 2019-05-10 | 东南大学 | A method of plant urase, which is directly extracted, from soil prepares calcium carbonate |
| CN110698104A (en) * | 2019-10-24 | 2020-01-17 | 太原理工大学 | Method for repairing cement-based material cracks by using soybean urease |
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- 2021-07-20 CN CN202110817282.4A patent/CN113717992A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097061A (en) * | 2018-08-07 | 2018-12-28 | 河海大学 | A kind of agent and its preparation method and application of checking winds and fixing drifting sand of the urase containing plant |
| CN109735575A (en) * | 2019-01-18 | 2019-05-10 | 东南大学 | A method of plant urase, which is directly extracted, from soil prepares calcium carbonate |
| CN110698104A (en) * | 2019-10-24 | 2020-01-17 | 太原理工大学 | Method for repairing cement-based material cracks by using soybean urease |
Non-Patent Citations (2)
| Title |
|---|
| 任冠洲等: "大豆脲酶诱导碳酸钙沉积技术反应液配方试验", 中国科技论文, vol. 15, no. 9, pages 1085 - 1089 * |
| 吴林玉等: "植物源脲酶诱导碳酸钙固化砂土试验研究", 岩土工程学报, vol. 42, no. 4, pages 714 - 720 * |
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