阅读说明：本技术 一种亚微米级球状生物碳酸钙及其制备方法和应用 (Submicron spherical biological calcium carbonate and preparation method and application thereof ) 是由 李庚英 王林彬 谢攀 雷思捷 何春保 陆金驰 于 2020-07-29 设计创作，主要内容包括：本发明公开了一种亚微米球形生物碳酸钙及其制法和应用。该亚微米球形生物碳酸钙的制法包括以下步骤：将微生物接种于灭菌后的液体培养基中,在恒温摇床中震荡培养制得微生物菌液,于4℃冰箱保藏和备用；将晶种和晶型控制剂加入到无菌水中,搅拌获得晶种悬浮液；将培养后的微生物菌液喷洒到微生物繁殖载体或覆膜物表面,然后再转移至营养液中浸没,置于30～35℃环境中培养,培养过程中向营养液中加入晶种悬浮液和钙源,共持续3～7天,即得亚微米级球状生物碳酸钙。尝试采用该生物碳酸钙修复废旧水泥混凝土再生骨料,经试验测试发现再生骨料的吸水率降低98％,孔径超过50nm的有害孔被修复,孔隙率降低45％以上,孔洞和裂缝被修复。(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.)

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 the submicron spherical biological calcium carbonate.

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 preservation and standby.

4. The method for preparing submicron spherical biological calcium carbonate according to claim 1, wherein:

the seed crystal in the step (2) is homologous zeroth nanometer calcium carbonate;

the crystal form control agent in the step (2) is an amino acid surfactant.

5. 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.

6. The method for preparing submicron spherical biological calcium carbonate according to claim 1, wherein:

the dosage of the seed crystal in the step (3) 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 (3) 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.

7. 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;

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.

8. 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.

9. A submicron spherical biological calcium carbonate prepared according to the method of any one of claims 1 to 8.

10. The use of the submicron spherical biological calcium carbonate according to claim 9 in solid waste recycling, sandy soil solidification, building repair reinforcement and soil modification.

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 interface is madeThe stronger the force. 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 a protein, and can provide nutrients for the growth and reproduction of microorganisms, so that the activity of the microorganisms is improved, and the nucleation and crystallization of calcium carbonate are accelerated. 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.