CN109735575B

CN109735575B - Method for preparing calcium carbonate by directly extracting plant urease from soil

Info

Publication number: CN109735575B
Application number: CN201910047416.1A
Authority: CN
Inventors: 缪林昌; 孙潇昊
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2019-01-18
Filing date: 2019-01-18
Publication date: 2022-04-22
Anticipated expiration: 2039-01-18
Also published as: CN109735575A

Abstract

The invention relates to a method for preparing calcium carbonate by directly extracting plant urease from soil, which comprises the steps of collecting a soil sample with the organic matter content of 20-40 g/kg on site, separating the plant urease from the soil by using a mixed solution of acetone and pure ethanol as an extraction solvent, adding urea after dilution, and hydrolyzing the urea into CO₃ ^2‑And NH₄ ¹⁺Maintaining at constant temperature for more than 5 days, adding calcium acetate solution, spraying the obtained mixed solution onto a plate paved with sand, and adding Ca in the solution²⁺With hydrolysed CO₃ ^2‑Binding to form calcium carbonate precipitate. The method is simple to operate, does not need complex bacterial culture and propagation, directly utilizes plant urease to catalyze necessary reactions so as to induce carbonate precipitation, has high calcium carbonate extraction efficiency, has low requirement on the environment, and is convenient to popularize and apply.

Description

Method for preparing calcium carbonate by directly extracting plant urease from soil

Technical Field

The present invention belongs to the field of organic induction of calcium carbonate solidification, and it can be used for sand-proofing and sand-fixing, and can improve environment.

Background

The biological diagenetic mineralization is a natural phenomenon, plays an important role in the history of the formation of the earth, is the research result of geochemistry in the last thirty years, realizes the role and the status of the biological diagenetic mineralization again, and has important significance for the technical development of geotechnical engineering. The principle is that urease is produced in the growth and reproduction process of microbe to decompose urea continuously to form CO₃ ^2-In the aqueous solution with negative charges at the interface of the thallus cell membrane, the Ca in the environment is chelated with the organic matter continuously²⁺Inducing local crystal anions (CO)₃ ^2-) With CO₃ ^2-The concentration is further increased to further attract more Ca²⁺Up to the crystal surface CaCO₃The concentration is increased to be continuously nucleated, and CaCO is finally deposited₃And (3) granules. It is the principle of microbial induction of calcium carbonate that is used to solidify sand.

Chinese patent CN201610828703.2 discloses a method for preparing calcium carbonate solidified sandy soil by using microorganisms, sand is poured into a bottle to uniformly cover the bottom of the bottle, then a gelling liquid and a cultured bacterial liquid are poured, shaking culture is carried out at a constant temperature, then a part of mixed liquid in the bottle is quantitatively absorbed every day, an equal amount of gelling liquid is added, a small amount of sand is uniformly added, and finally, a solidified sand column is obtained after lasting for several days; however, the culture of the strain by the method usually needs to consume a lot of manpower and material resources, and the temperature and the pH value of the strain growth need to be strictly controlled in the culture process, which is difficult to meet in the actual engineering field, and the solidification effect is directly influenced.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the method for preparing the calcium carbonate by directly extracting the plant enzyme from the soil, which has simple operation, does not need complicated bacterial culture and propagation, directly utilizes the plant urease to catalyze necessary reactions so as to induce carbonate precipitation, has high calcium carbonate extraction efficiency, has low requirement on the environment and is convenient to popularize and apply.

In order to solve the problems, the technical scheme of the invention is as follows:

a method for preparing calcium carbonate by directly extracting plant enzymes from soil comprises the following steps:

step 1, soil sample collection: collecting soil sample with depth of 5-30cm and organic matter content of 20-40 g/kg, sealing in sterilized sample bag, and refrigerating (4 deg.C).

Step 2, separating plant urease from soil: taking 200g of a soil sample, fully shaking the soil sample with 200ml of a mixed solution (V acetone: V ethanol = 32: 100) of 32% acetone and pure ethanol to form a suspension, standing the suspension at room temperature for 4 hours, and filtering the suspension to obtain a soil supernatant;

pouring the filtered soil supernatant into 200ml of a 32% acetone and alcohol mixed solution again, shaking and mixing uniformly, standing at room temperature, and filtering to obtain the urease solution.

Step 3, taking 1mL of the urease solution obtained in the step 2, adding 99 mL of water to dilute the urease solution to 100mL, and then adding 6g of urea to hydrolyze the urea to obtain hydrolyzed urea solution;

step 4, standing the hydrolyzed urea solution obtained in the step 3 in a constant temperature box at 25 ℃ for more than 5 days, adding 100mL of calcium acetate solution with the equal volume and the molar concentration of 0.5-1.0mol/L, and stirring for later use;

and 5, spraying the mixed solution obtained in the step 4 onto a plate paved with sand with the thickness of 5-10cm, and standing for 4-5 days to be cemented by calcium carbonate.

The invention directly extracts plant urease from soil to prepare calcium carbonate, selects the mixed solution of acetone and alcohol as the extract, has high extraction efficiency, and after the extracted enzyme solution is cured, the diluted enzyme solution is diluted and added with urea, and the urea is hydrolyzed into CO in the curing time₃ ^2-At this point, Ca in the solution is added after the calcium acetate solution is added²⁺With hydrolysed CO₃ ^2-Combining to generate calcium carbonate precipitate; the method is economical and environment-friendly, and is simple and easy to operate.

Drawings

FIG. 1 is a diagram showing calcium carbonate precipitates obtained by mixing the enzyme solution extracted in example 1 with urea and an equal volume of calcium acetate solution, and it can be seen that calcium carbonate precipitates can be obtained by the method and the precipitation rate is high.

Detailed Description

The technical solution of the present invention will be specifically described below with reference to examples:

in the following examples, the alcohol is pure ethanol.

Example 1:

a method for preparing calcium carbonate by directly extracting plant urease from soil comprises the following steps:

1) collecting soil sample with organic matter content of 20-40 g/kg from Nanjing local (in garden of small school district of four-Town building of southeast university), collecting soil sample with depth of 5-30cm, placing the soil sample into sterilized sample bag, sealing, and storing in refrigerator at 4 deg.C.

2) Extracting and isolating plant urease from the soil sample:

a) weighing 200g of soil sample, pouring into 200ml of 32% acetone and alcohol mixed solution, fully shaking to enable the soil sample to be in a suspension state, and standing for 4 hours at room temperature;

b) the filtered soil supernatant was poured into a beaker containing 200ml of a 32% acetone-alcohol mixture, shaken to mix the solution uniformly, and then allowed to stand at room temperature for 4 hours, followed by 2 times of filtration to obtain an enzyme solution.

3) The urease solution separated in step 2 was diluted with 99 mL of water (1 mL) and then decomposed with 6g of urea.

4) Standing the hydrolyzed urea solution obtained in the step (3) in a constant temperature box at 25 ℃ for more than 5 days, adding a calcium acetate solution with the equal volume molar concentration of 0.5mol/L, and stirring;

5) and (3) spraying the mixed solution obtained in the step (4) onto a plate paved with sand with the thickness of 5-10cm, and standing for 4-5 days to obtain the yield of the microorganism-induced calcium carbonate precipitation of 33%.

Example 2:

the difference from example 1 is that in step (4), the molar concentration of the calcium acetate solution is 0.75mol/L, and the yield of microorganism-induced calcium carbonate precipitation is 57%.

Example 3:

the difference from example 1 is that in step (4), the molar concentration of the calcium acetate solution is 1mol/L, and the yield of microorganism-induced calcium carbonate precipitation is 49%.

Example 4 examination of extraction procedure and different extraction solvents

The method of example 1 was used to extract plant urease to obtain final enzyme solution, except that different extraction solvents (group 1: 32% acetone and alcohol mixed solution; group 2: alcohol; group 3: 32% acetone; group 4: water) were added during the extraction process, and the influence of different extraction solvents on the calcium carbonate precipitation yield was compared. Then 6g of urea was added to the prepared enzyme solution, and an equal volume of the solution was mixed with 0.75mol/L calcium acetate solution to induce precipitation of calcium carbonate. Maintaining in 25 deg.c constant temperature air bath incubator for 5 days, filtering the mixed liquid, and detecting to obtain the microbial induced calcium carbonate precipitation yield shown in Table 1.

TABLE 1

EXAMPLE 5 examination of different mixing ratios of acetone and alcohol

The experimental conditions were the same as in example 2, except that the extraction solvent was acetone and ethanol at different volume ratios, and the results are shown in Table 2:

TABLE 2

	Group 1	Group 2	Group 3	Group 4
					Acetone: alcohol	32%	20%	25%	35%
CaCO₃Precipitation yield/%	57%	31%	49%	50%

Claims

1. A method for preparing calcium carbonate by directly extracting plant enzymes from soil is characterized by comprising the following steps:

step 1, soil sample collection: collecting soil samples on site, sealing and refrigerating;

the soil sample is taken from a soil sample with the depth of 5-30cm and the organic matter content of 20-40 g/kg;

step 2, separating plant urease from soil: fully oscillating a soil sample and an extraction solvent to form a suspension, standing at room temperature, filtering to obtain a soil supernatant, pouring the soil supernatant after the first filtration into a mixed solution of acetone and ethanol under the same conditions again, oscillating and mixing uniformly, standing at room temperature, and filtering again to obtain a urease solution;

the extraction solvent is a mixed solution of acetone and pure ethanol; v_{Acetone (II)}：V_Ethanol=20-35：100；

step 4, standing the hydrolyzed urea solution obtained in the step 3 at constant temperature for more than 5 days, adding calcium acetate solution with the equal volume molar concentration of 0.5-1.0mol/L, and stirring for later use;

and 5, spraying the mixed solution obtained in the step 4 to a plate paved with sand, standing for 4-5 days, and separating out the calcium carbonate by cementation.

2. The method for preparing calcium carbonate by directly extracting plant enzymes from soil according to claim 1, wherein in the step (2), the extraction solution is a mixture of acetone and pure ethanol; v acetone: v ethanol = 32: 100.