CN111792661B - Submicron spherical biological calcium carbonate and preparation method and application thereof - Google Patents
Submicron spherical biological calcium carbonate and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01F—COMPOUNDS OF THE METALS BERYLLIUM, MAGNESIUM, ALUMINIUM, CALCIUM, STRONTIUM, BARIUM, RADIUM, THORIUM, OR OF THE RARE-EARTH METALS
- C01F11/00—Compounds of calcium, strontium, or barium
- C01F11/18—Carbonates
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- C—CHEMISTRY; METALLURGY
- C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
- C04B—LIME, MAGNESIA; SLAG; CEMENTS; COMPOSITIONS THEREOF, e.g. MORTARS, CONCRETE OR LIKE BUILDING MATERIALS; ARTIFICIAL STONE; CERAMICS; REFRACTORIES; TREATMENT OF NATURAL STONE
- C04B18/00—Use of agglomerated or waste materials or refuse as fillers for mortars, concrete or artificial stone; Treatment of agglomerated or waste materials or refuse, specially adapted to enhance their filling properties in mortars, concrete or artificial stone
- C04B18/04—Waste materials; Refuse
- C04B18/16—Waste materials; Refuse from building or ceramic industry
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- C—CHEMISTRY; METALLURGY
- C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
- C04B—LIME, MAGNESIA; SLAG; CEMENTS; COMPOSITIONS THEREOF, e.g. MORTARS, CONCRETE OR LIKE BUILDING MATERIALS; ARTIFICIAL STONE; CERAMICS; REFRACTORIES; TREATMENT OF NATURAL STONE
- C04B20/00—Use of materials as fillers for mortars, concrete or artificial stone according to more than one of groups C04B14/00 - C04B18/00 and characterised by shape or grain distribution; Treatment of materials according to more than one of the groups C04B14/00 - C04B18/00 specially adapted to enhance their filling properties in mortars, concrete or artificial stone; Expanding or defibrillating materials
- C04B20/10—Coating or impregnating
- C04B20/12—Multiple coating or impregnating
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/91—Use of waste materials as fillers for mortars or concrete
Abstract
The invention discloses a submicron spherical biological calcium carbonate and a preparation method and application thereof. The preparation method of the submicron spherical biological calcium carbonate comprises the following steps: inoculating the microorganism into the sterilized liquid culture medium, performing shake culture in a constant temperature shaking table to obtain a microorganism liquid, and preserving in a refrigerator at 4 ℃ for later use; adding the crystal seeds and the crystal form control agent into sterile water, and stirring to obtain a crystal seed suspension; spraying the cultured microbial liquid on the surface of a microbial propagation carrier or a film-coated object, then transferring the microbial liquid to a nutrient solution for immersion, culturing in an environment of 30-35 ℃, adding a seed crystal suspension and a calcium source into the nutrient solution in the culturing process, and continuing for 3-7 days to obtain the submicron spherical biological calcium carbonate. The biological calcium carbonate is tried to repair the waste cement concrete recycled aggregate, and test tests show that the water absorption of the recycled aggregate is reduced by 98%, harmful pores with the pore diameter of more than 50nm are repaired, the porosity is reduced by more than 45%, and pores and cracks are repaired.
Description
Technical Field
The invention belongs to the interdisciplinary science, relates to the fields of microbiology, crystallography and physical and chemical science, and particularly relates to submicron (100-1000 nm) spherical biological calcium carbonate and a preparation method and application thereof.
Background
Microbial mineralization refers to mineral precipitation caused by microbial physiological activity, and includes three mechanisms of microbial control mineralization, microbial influence mineralization and microbial induction mineralization. Wherein microbial controlled mineralization refers to mineralization synthesized directly at a specific location within or on a cell under specific circumstances. Whereas the microbial influence on mineralization refers to the process of mineral precipitation caused by organic matter on the cell surface. Microbial induction of mineralization is a major concern and refers to the process of mineral satiety and subsequent precipitation through metabolic activity of microorganisms. The application of microbe induced mineralization is wide, and the microbe induced mineralization attracts the extensive attention of experts and scholars all over the world. At present, the minerals generated by the microorganism induction are various, including ferric hydride, hematite, goethite, metal sulfate, phosphate, carbonate, phosphorite, iron-aluminosilicate, metal sulfide and the like.
Microbial induction of calcium carbonate precipitation is the most widespread and common phenomenon in biomineralization, and according to existing research reports, at least more than 200 microorganisms can induce calcium carbonate to biomineralize. Because the metabolism of microorganism such as photosynthesis, urea hydrolysis and nitrification, etc. causes the change of chemical components in the biological membrane and the environment around the organism, and further changes the saturation degree of calcium carbonate in the environment, and causes the precipitation of carbonate crystals. The microorganism induced calcium carbonate precipitation has the characteristics of easy control, environmental protection, gelling capability of reaction products and the like, and is expected to be widely applied to the fields of biotechnology, ancient biology, environmental engineering, geotechnical engineering, hydraulic engineering, civil engineering and the like. The prior literature indicates that biological deposited calcium carbonate is expected to play an important role in the following practical engineering: 1) the gel effect and the hole filling effect are utilized to repair and reinforce historical and cultural ancient buildings, and the original appearances of the buildings, such as new buildings and old buildings, are kept; 2) the neutral and environment-friendly performance of the soil is utilized to improve the performance of the soil body, and the soil loss, the soil acidification and the alkalization consolidation are prevented; 3) the stability protection is carried out on the side slope and the dam by utilizing the gelling property and the hole filling effect of the material; 4) the gelatinization property of the sand fixing agent is used as a sand fixing agent to fix the surface of the flowing sand dune; 5) fixing and removing heavy metal ions in water and soil by using the ion exchange function of biological calcium carbonate; 6) the gelling property of the self-repairing agent can be utilized to obtain microbial cement or realize self-repairing of a concrete structure.
Currently, there are mainly four crystal morphologies of microbially precipitated calcium carbonate: calcite, aragonite, vaterite and amorphous calcium carbonate. Among them, calcite is the most thermodynamically stable crystal form of calcium carbonate, and vaterite and aragonite are secondary, metastable, transition-stage crystal forms of calcium carbonate in calcite form, and can be converted into calcite under certain conditions. Amorphous calcium carbonate is the most unstable and often exists as a transition state that is then converted to calcite, aragonite or vaterite. It is noted that although calcite has the most thermodynamically stable characteristic, existing research (wuhaowun, bio-coating anti-seepage research based on microbial-induced calcium carbonate deposition, master thesis at tianjin university, 2016) indicates that the crystal form of microbial-deposited calcite generally appears as rhombohedral, rhombohedral or complex trigonal, has a single size distribution, and is not favorable for close packing (as shown in fig. 1). In contrast, vaterite is spherical, but has high solubility and poor thermodynamic stability. Moreover, the size of the crystals, whether calcite or vaterite, is large, generally 10 to 200 μm, and some are even more than 200 μm. The large particle size has the following disadvantages: 1) the calcium carbonate can not penetrate into the deep part of cracks and holes, and the repairing and enhancing effects of the biological deposited calcium carbonate are not obvious; 2) small specific surface area and weak binding power. As is known from surface interface science and physical chemistry, the larger the particle size of a material, the smaller the specific surface area, and the weaker the van der waals force. Therefore, the existing microorganism deposited calcium carbonate is difficult to effectively repair buildings, fix sandy soil and the like. Moreover, according to the existing literature, the efficiency of inducing and depositing calcium carbonate by microorganisms is low, the particles are not uniformly distributed, the requirement on mineralization environment is strict, and the selected nutrient solution such as beef extract and the like can pollute the repair materials, thereby hindering the industrial production and the practical application of the repair materials.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a preparation method of submicron (100-1000 nm) spherical biological calcium carbonate.
The invention also aims to provide the submicron (100-1000 nm) spherical biological calcium carbonate prepared by the method.
The invention further aims to provide an application of the submicron (100-1000 nm) spherical biological calcium carbonate.
The purpose of the invention is realized by the following scheme:
a preparation method of submicron (100-1000 nm) spherical biological calcium carbonate comprises the following steps:
(1) inoculating the microorganism into the sterilized liquid culture medium, performing shake culture in a constant temperature shaking table to obtain a microorganism liquid, and storing in a refrigerator at 4 ℃ for preservation and standby;
(2) adding the crystal seeds and the crystal form control agent into sterile water, and stirring to obtain uniformly distributed crystal seed suspension;
(3) spraying the microbial liquid cultured in the step (1) on the surface of a microbial propagation carrier or a film-coated object, then transferring the microbial liquid to a nutrient solution to immerse, culturing in an environment of 30-35 ℃, adding a seed crystal suspension and a calcium source into the nutrient solution in the culturing process, and continuing for 3-7 days to obtain the submicron (100-1000 nm) spherical biological calcium carbonate.
The microorganism in the step (1) is ureolytic bacillus pasteurii;
the formula of the liquid culture medium in the step (1) is as follows: 10-30 g/L of sucrose, 5-20 g/L of soybean peptone, 20-50 g/L of urea, 3-8 g/L of salt, deionized water and 8-10 of pH value;
the sterilization in the step (1) is to sterilize for 20-40 min at 110-130 ℃ by adopting an autoclave.
The using amount of the microorganisms and the liquid culture medium in the step (1) meets the condition that 0.3-0.8 part by mass of microorganisms are inoculated to every 100 parts by mass of the liquid culture medium; it is preferable to inoculate 0.5 parts by mass of the microorganism per 100 parts by mass of the liquid medium.
The temperature of the shaking culture in the constant-temperature shaking table in the step (1) is 30-35 ℃, the shaking frequency is 150r/min, and the culture time is 24-36 h;
in order to obtain a higher-concentration microbial liquid so as to shorten the time for generating the spherical biological calcium carbonate, the step (1) further comprises a centrifugal operation after the shaking culture in the constant-temperature shaking table, preferably the centrifugal operation is carried out for 5-10 min at 6000r/min, and then the microbial liquid is stored in a refrigerator at 4 ℃ for later use;
the seed crystal in the step (2) is homologous zero-unique nano calcium carbonate, preferably calcite type calcium carbonate with the average particle size of 20nm, and the dosage of the seed crystal in the step (3) is 0.01-0.5 part by mass of the seed crystal per 100 parts by mass of sterile water;
the crystal form control agent in the step (2) is an amino acid surfactant, wherein the amino acid surfactant is preferably at least one of lysine salt, sarcosine salt, glutamate and N-cocoyl glutamate; the dosage of the crystal form control agent in the step (2) meets the requirement that 0.02-0.3 part by mass of the crystal form control agent is added into every 100 parts by mass of sterile water.
The microorganism propagation carrier or the film covering matter in the step (3) is preferably cement mortar particles, wherein the pH value of the cement mortar particles is 9.0-9.5, the fineness modulus is 1.5-2.2, and the cement mortar particles are wetted with sterilized water before use. The microbial propagation carrier or coating serves to immobilize the microorganisms to increase local concentration and to allow the crystalline deposition of biological calcium carbonate at predetermined locations.
The formula of the nutrient solution in the step (3) is as follows: 3-5 g/L peptone, 5-10 g/L glucose, 30-90 g/L urea, deionized water and pH value of 8.0-9.0.
The calcium sources in the step (3) are all preferably 100g/L CaCl2An aqueous solution;
the dosages of the microbial bacteria liquid and the nutrient solution in the step (3) meet the following requirements: in the step (1), 100 parts by weight of nutrient solution is used for every 0.3-0.8 part by weight of microorganism solution obtained after the microorganism culture.
The seed crystal suspension and the calcium source are added in the culture process in the step (3) and refer to the following steps: during the culture process, adding the seed crystal suspension and the calcium source every 2 hours in the first 12 hours, and then adding the seed crystal suspension and the calcium source every 6 hours; wherein the seed crystal suspension and the calcium source are used in amounts that: when the weight part of the nutrient solution in the step (3) is 100 parts, the weight part of the seed crystal suspension added each time is 1-4 parts, and the weight part of the calcium source added each time is 5-15 parts;
the submicron (100-1000 nm) spherical biological calcium carbonate prepared by the method.
The submicron (100-1000 nm) spherical biological calcium carbonate has the characteristics of large specific surface area, high surface energy, good stacking effect, excellent gradation, good hydrophobicity, strong water resistance, good cementing property and the like, and can be widely used in the projects of solid waste residue resource utilization, sand solidification, building maintenance reinforcement, soil body modification and the like.
The mechanism of the invention is as follows:
chelating Ca for negatively charged functional groups on the cell wall of microorganisms such as Bacillus pasteurii2+Acting as nucleation sites, bacterial respiration and urease hydrolysis cause local crystal anion CO3 2-The concentration increases, thereby further attracting more Ca again2+Until the crystal precursor concentration is increased to favor nucleation of the deposited crystals. The deposition rate is closely related to cell growth and urease activity, and kinetic research shows that pH value has obvious influence on urease activity during deposition and is favorable for inducing biological calcium carbonate deposition in alkaline environment. However, existing studies have shown that the biologically induced calcium carbonate deposition rate is generally very low, and the particle size of the deposited calcium carbonate is large, usually from tens of microns to hundreds of microns, which limits the application of the deposited calcium carbonate in engineering.
Based on solid phase reaction kinetics, heterogeneous nucleation of crystals has lower activation energy. On the other hand, as seen from surface interface science, the finer the material particles are, the larger the specific surface area is, the higher the surface energy is, and the stronger the interface acting force is. Based on the principle, the invention designs the calcium carbonate crystallization process from the molecular scale, thereby achieving the purposes of reducing the particle size of the calcium carbonate and improving the deposition efficiency. For this purpose, we have used a method of incorporating homonuclei (calcite crystallites) and a crystal form control agent. Specifically, sodium sarcosinate or sodium N-cocoyl glutamate which is amino acid surfactant is adopted as a crystal form control agent. The amino acid surfactant mainly has the following functions: 1) accelerate the formation of calcium carbonate crystal nucleus because amino acid surfactant such as sodium sarcosinate or N-cocoyl sodium glutamate is a bionic mineralized template and forms functional groups with negative charges after hydrolysis, and the functional groups and Ca in solution2+A series of interactions such as static electricity and coordination are generated, which act as nucleation sites and regulate the growth of crystals. 2) The amino acid surfactant is also oneThe protein can provide nutrients for the growth and the propagation of microorganisms, thereby improving the activity of the microorganisms and accelerating the nucleation and the crystallization of calcium carbonate. 3) Because the amino acid surfactant has the directional arrangement characteristic, the amino acid surfactant can be selectively adsorbed on a specific crystal face of a mineral during the process of depositing calcium carbonate by microorganisms, and the function of controlling the composition and the shape and the size of a crystal phase (a crystal form control agent) is realized. 4) The surface of the nano calcium carbonate particles is coated by the hydrophobic surfactant, so that the solubility of the biological calcium carbonate can be reduced, and the uniform dispersion of sediments can be promoted. 5) The amino acid surfactant can also prevent microorganisms from aggregating and adhering to form a biological film, and improve the activity of the microorganisms. This is because the microbial cells forming the biofilm are often in a relatively quiescent or semi-dormant state, and various physiological activities and metabolism are slow, and the ability to mineralize to form biological calcium carbonate is weak.
In addition, the invention also adds a proper amount of nano calcium carbonate as a homologous crystal nucleus (seed crystal). According to crystallography, the existence of the seed crystal is beneficial to reducing the nucleation activation energy of the biological calcium carbonate and improving the deposition rate of the biological calcium carbonate. On the other hand, the externally doped nano calcium carbonate also has pH stability, and when the biological calcium carbonate is not formed in a large amount, the solution can be weakly alkaline. For the bacillus pasteurii, the alkalescent environment is beneficial to keeping activity and breeding growth of the bacillus pasteurii, thereby providing conditions for improving mineralization efficiency.
The biological calcium carbonate has the characteristics of high mineralization efficiency, easily obtained raw materials, simple process, environmental protection and the like, and lays conditions for engineering application. And the generated microbial calcium carbonate has a submicron (100-1000 nm) structure, high surface energy and strong binding power. The biological calcium carbonate can close and fill various pores and cracks, and has high repair efficiency. More importantly, the sub-nanometer biological calcium carbonate obtained by the invention is insoluble in water, and provides conditions for improving the durability of the material.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention tries to repair the waste cement concrete recycled aggregate by adopting the method, and test tests show that the water absorption of the recycled aggregate is reduced by 98 percent, harmful holes with the aperture more than 50nm are repaired, the porosity is reduced by more than 45 percent, and holes and cracks are repaired.
Drawings
Figure 1 is a topographical map of microbial deposit of calcite.
Fig. 2 is a topographical view of the microbial calcium carbonate prepared in example 1 and comparative example 1, wherein the three figures on the left represent topographical views of the microbial calcium carbonate prepared in example 1, and the three figures on the right represent topographical views of the microbial calcium carbonate prepared in comparative example 1.
FIG. 3 is a TEM particle size distribution diagram of microbial calcium carbonate prepared in example 1 and comparative example 1; wherein the left side is a TEM particle size distribution diagram of the microbial calcium carbonate prepared in example 1, and the right side is a TEM particle size distribution diagram of the microbial calcium carbonate prepared in comparative example 1.
FIG. 4 is a comparison between before and after the repair of the reclaimed mineral aggregate, wherein (a) represents a physical diagram and an SEM diagram of an acid aggregate before the repair, and (b) represents a physical diagram and an SEM diagram of an aggregate after the repair by using the submicron biological calcium carbonate of the present invention.
FIG. 5 is a graph of pore size porosity test results before and after reclaimed mineral restoration.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
The reagents used in the examples are commercially available without specific reference.
In the examples, parts are by weight unless otherwise specified.
Example 1
A submicron spherical biological calcium carbonate mainly comprises the following components in parts by weight: 100 parts of culture medium (20 g/L of sucrose, 10g/L of soybean peptone, 30g/L of urea, 5g/L of salt and deionized water); 0.5 part of microbial strain (Bacillus pasteurianus, Bacillus pateurii, from DSMZ, Germany Collection of microbial strains); 32 parts of seed crystal suspension (the concentration of the nano calcium carbonate is 0.05 wt%, and the concentration of the crystal form control agent is 0.1 wt%); 100g/L CaCl2160 parts of a solution; nutrient solution (3g/L peptone, 10g/L glucose)60g/L urea and deionized water).
A preparation method of submicron spherical biological calcium carbonate mainly comprises the following steps:
(1) weighing soybean peptone, sucrose and salt according to the formula of a culture medium (20 g/L of sucrose, 10g/L of soybean peptone, 30g/L of urea and 5g/L of salt), dissolving in deionized water, sterilizing at 125 deg.C for 20min, and adding urea to obtain a liquid culture medium;
(2) inoculating 0.5 weight part of Bacillus pasteurii into 100 weight parts of liquid culture medium, performing shake culture in a constant temperature shaking table at 35 deg.C and 150r/min, and centrifuging the obtained product (6000r/min, 5min) after culturing for 24 hr to obtain high concentration Bacillus pasteurii seed (10)9cells/mL) stored in a refrigerator at 4 ℃ for preservation and standby;
(3) dissolving nano calcium carbonate (Ruicheng Huaner nano material Co., Ltd.) with the average particle size of 20nm in sterile water, adding a crystal form control agent (sodium lysinate amino acid surfactant), and stirring for 30 minutes by using a stirrer at the rotation speed of 1000r/min to obtain a seed crystal suspension (the concentration of the nano calcium carbonate in the suspension is 0.05 wt%, and the concentration of the crystal form control agent is 0.1 wt%;
(4) preparing nutrient solution according to the proportion of 3g/L peptone, 10g/L glucose, 60g/L urea and deionized water; CaCl is prepared according to 100g/L2And (3) solution.
(5) Transplanting all the high-concentration bacterial liquid cultured in the step (2) to the surface (the dosage is 5 ml/cm) of a carrier (cement mortar test block)2). After standing for 5 minutes, slowly moving into and immersing in the nutrient solution (100 parts) obtained in the step (4), and then spraying 2 parts of seed crystal suspension and 10 parts of CaCl into the nutrient solution every 2 hours for 12 hours2A solution; then, 2 parts of seed crystal suspension and 10 parts of CaCl were sprayed every 6 hours2And culturing the solution in an environment of 30-35 ℃ for 3 days.
Comparative example 1
A spherical biological calcium carbonate mainly comprises the following components in parts by weight: 100 parts of culture medium (20 g/L of sucrose, 10g/L of soybean peptone, 30g/L of urea, 5g/L of salt and deionized water)(ii) a 0.5 part of microbial strain (Bacillus pasteurii); 100 parts of nutrient solution (3g/L peptone, 10g/L glucose, 60g/L urea and deionized water). 100g/L CaCl2160 parts of the solution.
The preparation method of the spherical biological calcium carbonate mainly comprises the following steps:
(1) weighing soybean peptone, sucrose and salt according to the formula of a culture medium (20 g/L of sucrose, 10g/L of soybean peptone, 30g/L of urea and 5g/L of salt), dissolving in deionized water, sterilizing at 125 deg.C for 20min, and adding urea to obtain a liquid culture medium;
(2) inoculating 0.5 weight part of Bacillus pasteurii into 100 weight parts of liquid culture medium, performing shake culture in a constant temperature shaking table at 35 deg.C and 150r/min, and centrifuging the obtained product (6000r/min, 5min) after culturing for 24 hr to obtain high concentration Bacillus pasteurii seed (10)9cells/mL). Storing in a refrigerator at 4 deg.C for storage;
(3) preparing nutrient solution (3g/L peptone, 10g/L glucose, 60g/L urea and deionized water) according to the proportion;
(4) coating all the high-concentration bacterial liquid cultured in the step (2) on the surface of the cement mortar block (the dosage is 5 ml/cm)2) Standing for 5 minutes, slowly transferring into the nutrient solution (100 parts) obtained in the step (4), and then spraying 10 parts of CaCl every 2 hours for 12 hours2A solution; then, 10 parts of CaCl was sprayed every 6 hours2And culturing the solution in an environment of 30-35 ℃ for 3 days.
The morphology and particle size of the calcium carbonate prepared in example 1 and the calcium carbonate prepared in comparative example 1 were measured, and the results are shown in fig. 2 and 3, wherein 1) the solution in example 1 is a white suspension, while comparative example 1 is a clear solution, indicating that the amount of calcium carbonate deposited by the microorganisms in example 1 is much higher than in comparative example 1. 2) Submicron spherical biological calcium carbonate is obtained in example 1, and the particle size of the calcium carbonate is less than 1000nm through SEM and TEM observation; whereas the calcium carbonate particles of comparative example 1 are in the micron range, the particle size is generally over 5 μm. 3) The test result of the laser particle sizer further shows that the particle size of the submicron biological calcium carbonate obtained in the example 1 is between 100nm and 1000nm, and the median value of the particle size is 284.2 nm; in contrast, the calcium carbonate particles of comparative example 1 had a size distribution of 3000nm to 18000nm and a median particle size of 12591.6 nm.
The application example is as follows:
the invention tries to adopt a method of mineralizing nano biological calcium carbonate by microorganisms to repair the waste cement concrete recycled aggregate, which mainly comprises the following steps:
(1) weighing soybean peptone, sucrose and salt according to a culture medium formula (20 g/L of sucrose, 10g/L of soybean peptone, 30g/L of urea and 5g/L of salt), dissolving in deionized water, sterilizing at 125 ℃ for 20min, and adding 30 parts of urea to obtain a liquid culture medium;
(2) inoculating 0.5 weight part of Bacillus pasteurii into 100 weight parts of liquid culture medium, performing shake culture in a constant temperature shaking table at 35 deg.C and 150r/min, and centrifuging the obtained product (6000r/min, 5min) after culturing for 24 hr to obtain high concentration Bacillus pasteurii seed (10)9cells/mL) stored in a refrigerator at 4 ℃ for preservation and standby;
(3) preparing nutrient solution according to the proportion of 3g/L peptone, 10g/L glucose, 60g/L urea and deionized water; CaCl is prepared according to 100g/L2And (3) solution.
(4) Dissolving nano calcium carbonate (20nm, Ranchenne nanometer material Co., Ltd) (concentration of 0.05 wt%) and a crystal form control agent (sodium lysinate amino acid surfactant) (concentration of 0.1 wt%) in sterile water, and stirring for 30 minutes by using a stirrer at the rotation speed of 1000r/min to obtain a seed crystal suspension liquid with uniform distribution;
(5) the method comprises the steps of adopting cement mortar particles as microorganism precipitated calcium carbonate carriers or tectorial membranes, wherein the pH value of the cement mortar particles is 9-9.5, the fineness modulus is 1.5-2.2, and wetting the cement mortar particles with sterilized water before inoculation. And (3) spraying the bacterial liquid cultured in the step (2) to the wetted cement mortar particles (the bacterial liquid: the mortar is 1:2), uniformly stirring, and covering the materials to be repaired (the waste cement concrete recycled aggregate). Spraying 6 parts of the nutrient solution prepared in the step (3) and 2 parts of the seed crystal suspension prepared in the step (4) every 2 hours in 12 hours (containing nano calcium carbonate with the concentration of 0.05 wt% and 0.1 wt% of crystal form control)Formulation) and 10 parts of CaCl2A solution; then, 6 parts of nutrient solution, 2 parts of seed crystal suspension and 10 parts of CaCl are sprayed once every 6 hours2And culturing the solution in an environment of 30-35 ℃ for 3 days.
The topography before and after the restoration of the recycled aggregate of the waste cement concrete is shown in fig. 4, wherein (a) represents a physical map and an SEM image of an acid aggregate before the restoration, and (b) represents a physical map and an SEM image of an aggregate after the restoration by the submicron biological calcium carbonate. As can be seen from FIG. 4, the recycled aggregate before being repaired by the submicron-scale microbial calcium carbonate of the invention has a large number of cracks and pores and uneven surface chemical properties; the cracks and holes of the recycled aggregate repaired by the submicron-grade microbial calcium carbonate are repaired, the surface of the recycled aggregate is covered by a layer of biological calcium carbonate, and the recycled aggregate has uniform chemical properties. Through test tests, the water absorption of the recycled aggregate is reduced by 98%, harmful pores with the pore diameter of more than 50nm are repaired, the porosity is reduced by more than 45% (the pore diameter distribution is shown in figure 5), and pores and cracks are repaired.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. A preparation method of submicron spherical biological calcium carbonate is characterized by comprising the following steps:
(1) inoculating the microorganism into the sterilized liquid culture medium, performing shake culture in a constant temperature shaking table to obtain a microorganism liquid, and storing in a refrigerator at 4 ℃ for preservation and standby;
(2) adding the crystal seeds and the crystal form control agent into sterile water, and stirring to obtain uniformly distributed crystal seed suspension;
(3) spraying the microbial liquid cultured in the step (1) on the surface of a microbial propagation carrier or a film-coated object, then transferring the microbial liquid to a nutrient solution to immerse, culturing in an environment of 30-35 ℃, adding a seed crystal suspension and a calcium source into the nutrient solution in the culturing process, and continuing for 3-7 days to obtain submicron spherical biological calcium carbonate;
the crystal form control agent in the step (2) is an amino acid surfactant;
the seed crystal in the step (2) is homologous zeroth nanometer calcium carbonate;
the dosage of the seed crystal in the step (2) meets the requirement that 0.01-0.5 part by mass of the seed crystal is added into every 100 parts by mass of the sterile water;
the dosage of the crystal form control agent in the step (2) meets the requirement that 0.02-0.3 part by mass of the crystal form control agent is added into every 100 parts by mass of sterile water;
the formula of the nutrient solution in the step (3) is as follows: 3-5 g/L peptone, 5-10 g/L glucose, 30-90 g/L urea, deionized water and a pH value of 8.0-9.0;
the calcium source in the step (3) is 100g/L CaCl2An aqueous solution.
2. The method for preparing submicron spherical biological calcium carbonate according to claim 1, wherein:
the microorganism in the step (1) is bacillus pasteurii;
the formula of the liquid culture medium in the step (1) is as follows: 10-30 g/L of sucrose, 5-20 g/L of soybean peptone, 20-50 g/L of urea, 3-8 g/L of salt, deionized water and 8-10 of pH value;
the sterilization in the step (1) is to sterilize for 20-40 min at 110-130 ℃ by adopting an autoclave;
the using amount of the microorganisms and the liquid culture medium in the step (1) meets the condition that 0.3-0.8 part by mass of microorganisms are inoculated to every 100 parts by mass of the liquid culture medium;
the temperature of the shaking culture in the constant-temperature shaking table in the step (1) is 30-35 ℃, the shaking frequency is 150r/min, and the culture time is 24-36 h.
3. The method for preparing submicron spherical biological calcium carbonate according to claim 1, wherein:
and (2) after the shaking culture in the constant-temperature shaking table in the step (1), centrifuging at 6000r/min for 5-10 min, and then storing the microbial liquid in a refrigerator at 4 ℃ for later use.
4. The method for preparing submicron spherical biological calcium carbonate according to claim 1, wherein:
the seed crystal in the step (2) is calcite type calcium carbonate with the average grain diameter of 20 nm;
the crystal form control agent in the step (2) is at least one of lysine salt, sarcosine salt, glutamate and N-cocoyl glutamate.
5. The method for preparing submicron spherical biological calcium carbonate according to claim 1, wherein:
the microorganism propagation carrier or the film covering matter in the step (3) refers to cement mortar particles, wherein the pH value of the cement mortar particles is 9.0-9.5, the fineness modulus is 1.5-2.2, and the cement mortar particles are wetted with sterilized water before use.
6. The method for preparing submicron spherical biological calcium carbonate according to claim 1, wherein:
the dosages of the microbial bacteria liquid and the nutrient solution in the step (3) meet the following requirements: in the step (1), every 0.3-0.8 parts by mass of microbial liquid obtained after microbial culture is used for correspondingly using 100 parts by weight of nutrient solution;
the seed crystal suspension and the calcium source are added in the culture process in the step (3) and refer to the following steps: during the culture process, adding the seed crystal suspension and the calcium source every 2 hours in the first 12 hours, and then adding the seed crystal suspension and the calcium source every 6 hours;
the dosage of the seed crystal suspension and the calcium source added in each time in the step (3) meets the following requirements: when the weight part of the nutrient solution in the step (3) is 100 parts, the weight part of the seed crystal suspension added each time is 1-4 parts, and the weight part of the calcium source added each time is 5-15 parts.
7. A submicron spherical biological calcium carbonate prepared according to the method of any one of claims 1 to 6.
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