WO2023216674A1 - 具有脲酶活性的多孔生物碳酸钙钝化材料及其制备方法和应用 - Google Patents

具有脲酶活性的多孔生物碳酸钙钝化材料及其制备方法和应用 Download PDF

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WO2023216674A1
WO2023216674A1 PCT/CN2023/077190 CN2023077190W WO2023216674A1 WO 2023216674 A1 WO2023216674 A1 WO 2023216674A1 CN 2023077190 W CN2023077190 W CN 2023077190W WO 2023216674 A1 WO2023216674 A1 WO 2023216674A1
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urease
passivation material
calcium carbonate
heavy metal
urease activity
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PCT/CN2023/077190
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English (en)
French (fr)
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成亮
王胜
方龙洋
由天艳
俞洋洋
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江苏大学
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/08—Reclamation of contaminated soil chemically
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/52—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/52—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities
    • C02F1/5236—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities using inorganic agents
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • 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)
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/10—Inorganic compounds
    • C02F2101/20—Heavy metals or heavy metal compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the invention belongs to the field of environmental engineering materials. Specifically, it relates to a preparation method of a heavy metal passivation material, a porous biological calcium carbonate passivation material with urease activity obtained by the preparation method, and its application in environmental engineering, especially in Applications in heavy metal remediation.
  • heavy metal pollution has become one of the important environmental problems that humans need to solve. Because heavy metals can persist in the environment and can cause permanent damage to multiple organs due to their chronic toxicity, non-biodegradability and bioaccumulation in the food chain, heavy metals are extremely harmful environmental pollutants. Among them, heavy metal pollution such as cadmium, lead, nickel, copper and zinc has received special attention.
  • Passivation refers to the process of converting heavy metals into an inactive state or passivation state after treatment, thereby effectively reducing the poisonous ability of heavy metals to organisms in water and soil.
  • Traditional passivation technologies for heavy metal pollution mainly include pH adjustment, mineralization technology, adsorption technology, ion exchange technology, etc.
  • the passivation material provided by the present invention is a porous biological calcium carbonate with urease activity. Specific components thereof not only exert the heavy metal adsorption performance of the porous biological calcium carbonate, but also can still perform in extremely high-concentration heavy metal environments. Maintain urease activity and continue to biomineralize residual heavy metals.
  • a method for preparing porous biological calcium carbonate passivation material with urease activity which includes the following steps:
  • step S1 the added amounts of urea and soluble calcium salt are controlled so that their concentrations are 0.1 mol/L to 0.8 mol/L.
  • the bacterial liquid containing urease bacteria mentioned above refers to the entire culture system containing culture medium residues, metabolites and other components in which urease bacteria have been cultured; that is, the urease bacteria used here do not need to be separated from the culture system first.
  • the urease bacteria can be Bacillus pasteurianus ATCC11859; accordingly, the specific composition of the culture medium includes: 20g/L ⁇ 30g/L yeast powder, 15g/L ⁇ 30g/L ammonium chloride, 0 ⁇ 1mmol/L chlorine nickel, and the pH is 9.2 ⁇ 9.3.
  • the above-mentioned bacterial liquid containing urease bacteria can be obtained in the following manner: inoculating the urease bacteria mother liquid after sterilization In the culture medium, culture it with shaking for about 1.5 days at a temperature of about 30°C, and then place it in an environment of 4°C for later use.
  • step S1 before adding urea and soluble calcium salt, the activity of urease bacteria needs to be fixed to 10U/mL to 30U/mL using physiological saline.
  • 1U represents the amount of urease bacteria that can produce the urease required to hydrolyze 1 ⁇ mol of urea per minute.
  • the above-mentioned soluble calcium salt may be calcium chloride, calcium acetate, or calcium nitrate.
  • step S1 the biomineralization reaction can be achieved by stirring the mixed system of the above bacterial liquid, urea and soluble calcium salt at a rotation speed of 100 rpm to 600 rpm for 2 h to 24 h.
  • step S2 the preferred conditions for the freeze-drying operation are: controlling the temperature of the freeze-dried sample (ie, the cleaned filter residue) to -40°C to -30°C.
  • freeze-drying operation time can be 24h to 48h.
  • the filter residue is obtained by suction filtration of the calcium carbonate slurry suspension; for example, vacuum filtration is performed under the conditions of controlling the pressure of the vacuum pump to 0.6MPa to 0.8MPa and the filter paper being medium-speed qualitative filter paper.
  • the passivation material is a porous calcium carbonate including vaterite crystal form and urease bacteria and the urease produced by it, and has urease activity. It is a composite material with 150U/g ⁇ 1000U/g.
  • Another object of the present invention is to provide an application of the above-mentioned porous biological calcium carbonate passivation material with urease activity in heavy metal repair.
  • the present invention has the following beneficial effects:
  • the passivation material prepared by the above method provided by the present invention has a porous structure, which not only provides more nucleation sites, but also has an extremely high heavy metal adsorption capacity. Among them, the adsorption capacity of heavy metal Cd 2+ is about 1000mg/g, which is far above the average level.
  • the passivation material prepared by the above method provided by the present invention has significantly improved resistance to heavy metal toxicity, solving the limitations of traditional MICP technology to remove heavy metal pollution and limit heavy metal concentration.
  • its unique urease activity can be used to effectively combine adsorption and biomineralization, which not only can effectively and completely remove heavy metals, but also can wrap heavy metals inside calcium carbonate by adding an additional calcium source again to prevent the secondary release of heavy metals. pollute.
  • the above preparation method provided by the present invention is simple and controllable, low in price, safe and pollution-free, therefore It is easy to realize industrial production and has good promotion potential and application value.
  • Figure 1 is a physical diagram of the biomineralization reaction of urease bacteria in Example 1 of the present invention
  • Figure 2 is a physical diagram after completion of the biomineralization reaction of urease bacteria in Example 1 according to the present invention
  • Figure 3 is a physical diagram of a passivation material according to Embodiment 1 of the present invention.
  • Figure 4 is an SEM image of the passivation material according to Embodiment 1 of the present invention.
  • Figure 5 is an XRD pattern of the passivation material according to Embodiment 1 of the present invention.
  • Figure 6 is the stability test result of the passivation material according to Embodiment 1 of the present invention.
  • Figure 7 is the toxicity resistance test result of the passivation material according to Embodiment 1 of the present invention.
  • Figure 8 is the stability test result of the comparative passivation material according to Comparative Example 1 of the present invention.
  • Figure 9 is a comparison of the activity test results of the comparative passivation material of Comparative Example 2 according to the present invention and the passivation material of Example 1;
  • Figure 10 is an SEM image of the precipitates during the removal of cadmium from the passivation material in Application Example 1 according to the present invention.
  • Figure 11 is an XRD pattern of the precipitate after removing cadmium from the passivation material in Application Example 1 according to the present invention.
  • Figure 12 is an SEM image of the precipitate after removing cadmium from the passivation material in Application Example 1 according to the present invention.
  • Figure 13 is a diagram showing the removal effect of passivation material on heavy metal cadmium in Application Example 1 according to the present invention.
  • Figure 14 is the durability test result of the passivation material in Application Example 2 according to the present invention.
  • This embodiment provides a porous biological calcium carbonate passivation material with urease activity, which is prepared by the following method.
  • the bacterial solution was obtained by inoculating the urease bacteria stock solution into the sterilized culture medium and culturing it with shaking at a temperature of 30°C for 1.5 days.
  • the composition of the culture medium is: 20g/L yeast powder, 15g/L ammonium chloride, 1mmol/L nickel chloride are mixed, and water is added to make up to 1L; the pH of the culture medium is controlled to 9.25.
  • Figure 1 shows the state during the above-mentioned biomineralization reaction.
  • the reaction system is chaotic.
  • Figure 2 shows the state after the biomineralization reaction has been left to stand for a period of time. It can be seen that , the suspension gradually settles into layers.
  • Figure 3 shows the actual product of the above product. It can be seen that it is in the form of white-gray powder.
  • the products obtained above were analyzed by scanning electron microscope (SEM) and X-ray diffraction (XRD) respectively, and the characterization results are shown in Figure 4 and Figure 5 respectively.
  • the product has a porous structure.
  • the main crystal is calcium carbonate, and it is in the vaterite crystal form. The specific morphology of the crystal component ensures its heavy metal adsorption performance.
  • the urease activity of the product obtained above was tested, and its urease activity was 350 U/g. This property ensures its heavy metal mineralizing properties.
  • the above passivation materials were tested for their stability. Specifically, 10 g of the above-mentioned passivation materials were placed in environments with temperatures of 4°C and 25°C respectively, and their urease activities were tested on days 1, 3, 14, 21, and 28. Take 0.1g of the passivation material each time and place it in 1.8mol/L urea, and test the change rate of ammonia nitrogen concentration within 30 minutes to express its urease activity, the unit is U/g.
  • the passivation materials mentioned above were tested for their resistance to toxicity. Specifically, take 1g of the above passivation materials and put them into 150mL Erlenmeyer flasks with heavy metal Cd concentrations of 0, 10mg/L, 20mg/L, 50mg/L, 100mg/L, 250mg/L, and 500mg/L respectively. ; Which all contain urea at a concentration of 100mmol/L. In a shaker at 30°C and 180 rpm, react for 24 hours and test the changes in ammonia nitrogen concentration produced in the aqueous solution at this stage.
  • Example 2 In the description of Embodiment 2, the similarities with Embodiment 1 will not be repeated here, and only the differences from Embodiment 1 will be described.
  • the difference between Example 2 and Example 1 is:
  • Example 2 In the preparation method of the passivation material provided in Example 2, 1.5015g urea and 11.098g calcium chloride were added to 250 mL of bacterial liquid; and lyophilized at -35°C for 42 hours; the rest were as described in Example 1 , to obtain porous biological calcium carbonate passivation materials with urease activity.
  • the urease activity of the passivation material provided in this example is 307 U/g.
  • Example 3 In the description of Embodiment 3, the similarities with Embodiment 1 will not be repeated here, and only the differences from Embodiment 1 will be described.
  • the difference between Example 3 and Example 1 is:
  • the urease activity of the passivation material provided in this example is 151 U/g.
  • the bacterial liquid containing urease bacteria used is diluted with physiological saline to a urease activity of about 30 U/mL; for the rest, refer to the description in Example 1 to obtain urease activity.
  • the urease activity of the passivation material provided in this example is 298 U/g.
  • Example 5 In the description of Embodiment 5, the similarities with Embodiment 1 will not be repeated here, and only the differences from Embodiment 1 will be described.
  • the difference between Example 5 and Example 1 is:
  • the bacterial liquid containing urease bacteria used is diluted with physiological saline to a urease activity of about 10 U/mL; 6.006g urea and 2.7746g chloride are added to 250mL of bacterial liquid. calcium; and, lyophilize at -40°C for 24 hours; refer to the rest as described in Example 1 to obtain a porous biological calcium carbonate passivation material with urease activity.
  • the urease activity of the passivation material provided in this embodiment is 998 U/g.
  • Example 6 the similarities with Embodiment 1 will not be repeated here, and only the differences from Embodiment 1 will be described.
  • the difference between Example 6 and Example 1 is:
  • the bacterial liquid containing urease bacteria is used, and the composition of the culture medium used is: 20g/L yeast powder, 30g/L ammonium chloride, and adding water It was obtained by supplementing to 1 L, and the pH of the medium was controlled to 9.2; for the rest, refer to the description in Example 1 to obtain a porous biological calcium carbonate passivation material with urease activity.
  • the urease activity of the passivation material provided in this embodiment is 301 U/g.
  • Example 7 is:
  • the composition of the culture medium used for the bacterial liquid containing urease bacteria is: 30g/L yeast powder, 25g/L ammonium chloride, 0.5mmol /L nickel chloride was mixed, and water was added to make up to 1L, and the pH of the culture medium was controlled to 9.3; for the rest, refer to the description in Example 1 to obtain a porous biological calcium carbonate passivation material with urease activity.
  • the urease activity of the passivation material provided in this example is 303 U/g.
  • the urease bacteria are not provided as a single strain, but the bacterial liquid that has cultivated the urease bacteria is mixed together; on the other hand, the filter residue obtained from the suspension is dried All methods have a significant impact on obtaining the above-mentioned products with specific morphological components and excellent application properties.
  • the calcium carbonate crystal form finally obtained by drying and depositing is only calcite, not the calcium carbonate in the present invention. vaterite.
  • the calcium carbonate slurry suspension with urease activity prepared in this comparative example was divided into two parts after vacuum filtration and filter residue cleaning, and was placed at 4°C and 25°C for natural drying, instead of using the method in Example 1.
  • the freeze-drying method corresponds to obtaining the first comparative passivation material and the second comparative passivation material.
  • the calcium carbonate slurry suspension with urease activity prepared in this comparative example was subjected to vacuum filtration and filter residue cleaning, and then dried at about 60°C instead of using the freeze-drying method in Example 1, corresponding to the third Contrast passive materials.
  • Example 1 The urease activity in the comparative passivation material was tested, and the comparison with the results in Example 1 is shown in Figure 9. Among them, “freeze drying” refers to the activity data in Example 1, and “high temperature drying” refers to the activity data in this comparative example. It can be seen that when the drying operation is performed at a temperature of about 60°C, the urease bacteria in the obtained product are almost inactivated. It can be seen from this that the product obtained by this drying method cannot be used in applications Then exert the effect of biomineralization.
  • the above-mentioned passivation material provided by the present invention can produce dual effects of adsorption and mineralization on heavy metals, and can be used in the field of heavy metal repair.
  • the following application examples are provided.
  • the passivation material provided in the above embodiment 1 is applied to the removal of heavy metal cadmium.
  • This application example tests the durability of the passivation material provided in Example 1.
  • 0.1g of passivation material was placed in a 150mL Erlenmeyer flask in which the concentrations of heavy metal cadmium were 0, 10mg/L, 50mg/L, 150mg/L, 300mg/L, and 500mg/L respectively.
  • concentrations of heavy metal cadmium were 0, 10mg/L, 50mg/L, 150mg/L, 300mg/L, and 500mg/L respectively.
  • urea is added to the environment to a concentration of 0.1 mol/L.
  • react for 48 hours and test the changes in ammonia nitrogen concentration produced during this stage In a shaker at 30°C and 180 rpm, react for 48 hours and test the changes in ammonia nitrogen concentration produced during this stage.
  • the passivation material can still effectively retain its urease activity under low concentrations of heavy metal cadmium, and can continue to undergo biomineralization reactions after adding urea, indicating that it has good toxicity resistance and durability.

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Abstract

提供了一种重金属钝化材料的制备方法、以及由此获得的具有脲酶活性的多孔生物碳酸钙钝化材料。基于其中特定的组分，该钝化材料发挥了多孔生物碳酸钙的重金属吸附性能，还在高浓度重金属环境中仍能保持脲酶活性，持续对残留的重金属发挥生物矿化作用，在对重金属修复中体现吸附和矿化双重钝化作用。明显提升了对重金属耐毒性，解决了传统MICP技术限制重金属浓度的局限性，且对重金属具有超过平均水平的高吸附量。该钝化材料可应用于重金属修复中，且可在钝化重金属后通过额外添加钙源而防止重金属的二次污染。

Description

具有脲酶活性的多孔生物碳酸钙钝化材料及其制备方法和应用
相关申请的交叉引用
本申请要求于2022年5月10日提交的题目为“具有脲酶活性的多孔生物碳酸钙钝化材料及其制备方法和应用”的中国发明专利申请CN202210504664.6的权益，其全部内容通过引用并入本文。
技术领域
本发明属于环境工程材料领域，具体来讲，涉及一种重金属钝化材料的制备方法、以及经该制备方法获得的具有脲酶活性的多孔生物碳酸钙钝化材料，和其在环境工程、尤其在重金属修复中的应用。
背景技术
目前，随着工业化和城市化的快速发展，重金属污染已经成为人类所需要解决的重要环境问题之一。因为重金属在环境中能够持续存在，由其慢性毒性、不可生物降解性及食物链中的生物积累性，可对多个器官造成永久损害，所以重金属是危害极其严重的环境污染物。其中，镉、铅、镍、铜和锌等重金属污染更是受到重点关注。
截止目前，针对重金属污染的各种修复材料与修复方法层出不穷，但大多数都存在着成本高、去除效果不彻底、可回收利用率低、操作复杂、修复时间久的问题。就目前的情况来看，钝化技术是一种可行、有效的修复重金属污染技术，同时更符合现阶段我国在环境保护发展水平和国情的所需。
钝化，即是指对重金属经处理后，使其转换成为不活泼态即钝态的过程，进而有效降低重金属在水体、土壤中对生物的毒害能力。传统钝化重金属污染技术主要有调节pH值、矿化技术、吸附技术、离子交换技术等。
现在存在一种新兴的微生物诱导碳酸盐沉积技术，该技术是利用脲酶菌产生的脲酶分解出的碳酸根，在存在金属离子如钙离子、镉离子等的环境中，与碳酸根生成更稳定的碳酸盐形态，从而达到有效降低重金属毒害的作用。如此，在水体重金属污染中，利用一定活性的脲酶菌和尿素反应生成碳酸根离子和铵 根离子，重金属离子与碳酸根离子会以沉淀分离出溶液。在土壤重金属污染中，脲酶菌和尿素反应生成碳酸根离子和铵根离子，重金属离子与碳酸根离子会以更为稳定的碳酸盐形态存在，同时加速重金属向更稳定的形态转变，降低土壤中重金属的可迁移性，进而降低重金属对植物、微生物等的毒害能力。
然而，目前对于以上碳酸盐沉积技术的研究中，存在无法应用于较高浓度重金属环境的弊端。这是由于较高浓度的重金属即可导致脲酶菌失活而导致无法产生脲酶，继而无法将尿素分解产生碳酸根，如在Cd2+浓度为225mg/L左右可能即导致失活。虽然目前有针对如何提高脲酶菌耐毒性的改进研究，但仍无法高效解决上述问题。
发明内容
为解决上述现有技术存在的问题，本发明的发明人在基于对微生物诱导碳酸盐沉积技术的长期研究的基础上，提供了一种高效解决上述技术问题的改进方案。本发明提供的该钝化材料，其是一种具有脲酶活性的多孔生物碳酸钙，其中特定的组分，不仅发挥多孔生物碳酸钙的重金属吸附性能，并且在极高浓度的重金属环境中仍能保持脲酶活性，持续对残留的重金属发挥生物矿化作用。
为了达到上述发明目的，本发明采用了如下的技术方案：
一种具有脲酶活性的多孔生物碳酸钙钝化材料的制备方法，其包括下述步骤：
S1、向具有脲酶菌的菌液中添加尿素和可溶性钙盐，脲酶菌发生生物矿化反应，生成具有脲酶活性的碳酸钙浆状悬浮液；
S2、将该碳酸钙浆状悬浮液进行过滤后，滤渣经清洗和冻干处理，获得具有脲酶活性的多孔生物碳酸钙钝化材料。
具体地，在步骤S1中，尿素和可溶性钙盐的添加量均控制其浓度为0.1mol/L～0.8mol/L。
上述具有脲酶菌的菌液指已经培养出脲酶菌的带有培养基残留物、代谢物等组分的整个培养体系；也即，此处使用的脲酶菌无需先行从培养体系中分离。其中，脲酶菌可以为巴氏芽孢杆菌ATCC11859；相应地，其培养基的具体组成包括：20g/L～30g/L酵母粉、15g/L～30g/L氯化铵、0～1mmol/L氯化镍，且pH为9.2～9.3。
上述具有脲酶菌的菌液可通过下述方式获得：将脲酶菌母液接种于灭菌后 的培养基中，在30℃左右的温度下振荡培养1.5d左右，即得，将其置于4℃的环境下备用。
在步骤S1中，在添加尿素和可溶性钙盐前，需先利用生理盐水将其中脲酶菌的活性固定至10U/mL～30U/mL。
其中，1U表示能产生每分钟水解1μmol尿素所需脲酶的脲酶菌菌量。
上述可溶性钙盐可以是氯化钙、醋酸钙、硝酸钙。
在步骤S1中，生物矿化反应的发生可以是在100rpm～600rpm的转速下搅拌上述菌液、尿素与可溶性钙盐的混合体系2h～24h来实现。
在步骤S2中，冻干操作的优选条件为：控制冻干样品(即经过清洗的滤渣)温度为-40℃～-30℃。
进一步地，冻干操作的时间可以是24h～48h。
优选地，在步骤S2中，滤渣是通过将碳酸钙浆状悬浮液进行抽滤来获得的；如控制真空泵的压强0.6MPa～0.8MPa、滤纸为中速定性滤纸的条件下进行真空抽滤。
本发明的另一目的在于提供一种具有脲酶活性的多孔生物碳酸钙钝化材料，该钝化材料是一种包括球霰石晶型的多孔碳酸钙和脲酶菌及其所产脲酶、且脲酶活性为150U/g～1000U/g的复合材料。
本发明的再一目的在于提供一种上述具有脲酶活性的多孔生物碳酸钙钝化材料在重金属修复中的应用。
本发明相比现有技术，具有以下有益效果：
1)本发明提供的上述方法制得的该钝化材料，具有多孔结构，不仅可提供更多的成核位点，而且还具有极高的重金属吸附量。其中，对重金属Cd2+的吸附量约为1000mg/g，远超平均水平。
2)本发明提供的上述方法制得的该钝化材料，对重金属耐毒性明显提升，解决了传统MICP技术去除重金属污染限制重金属浓度的局限性。同时，利用其特有的脲酶活性，可以将吸附和生物矿化有效地结合，不仅能够有效彻底地去除重金属，而且能够通过再次额外加钙源，将重金属包裹在碳酸钙内部，防止重金属的二次污染。
3)本发明提供的上述制备方法，简单可控、价格低廉、安全无污染，因此 易于实现工业化生产，具有很好的推广潜力和应用价值。
附图说明
通过结合附图进行的以下描述，本发明的实施例的上述和其它方面、特点和优点将变得更加清楚，附图中：
图1是根据本发明的实施例1中脲酶菌的生物矿化反应时的实物图；
图2是根据本发明的实施例1中脲酶菌的生物矿化反应结束后的实物图；
图3是根据本发明的实施例1的钝化材料的实物图；
图4是根据本发明的实施例1的钝化材料的SEM图；
图5是根据本发明的实施例1的钝化材料的XRD图；
图6是根据本发明的实施例1的钝化材料的稳定性测试结果；
图7是根据本发明的实施例1的钝化材料的耐毒性测试结果；
图8是根据本发明的对比例1的对比钝化材料的稳定性测试结果；
图9是根据本发明的对比例2的对比钝化材料与实施例1中钝化材料的活性测试结果对比；
图10是根据本发明的应用例1中钝化材料在去除镉过程中沉淀物的SEM图；
图11是根据本发明的应用例1中钝化材料在去除镉后沉淀物的XRD图；
图12是根据本发明的应用例1中钝化材料在去除镉后沉淀物的SEM图；
图13是根据本发明的应用例1中钝化材料对重金属镉的去除效果图；
图14是根据本发明的应用例2中钝化材料的耐久性测试结果。
具体实施方式
以下，将参照附图来详细描述本发明的实施例。然而，可以以许多不同的形式来实施本发明，并且本发明不应该被解释为限制于这里阐述的具体实施例。相反，提供这些实施例是为了解释本发明的原理及其实际应用，从而使本领域的其他技术人员能够理解本发明的各种实施例和适合于特定预期应用的各种修改。
实施例1
本实施例提供一种具有脲酶活性的多孔生物碳酸钙钝化材料，其通过下述方法制备。
首先，取500mL具有脲酶菌的菌液，用电导率仪测量其脲酶活性，再通过生理盐水稀释至20U/mL左右的脲酶活性。
具体来讲，该菌液是通过将脲酶菌母液接种至灭菌后的培养基，并在30℃的温度条件下振荡培养1.5d获得的。
其中培养基的组成为：20g/L酵母粉、15g/L的氯化铵、1mmol/L氯化镍混合，并加水补足至1L而获得；该培养基的pH控制为9.25。
然后，取250mL上述菌液，向其中加入6.006g尿素和11.098g氯化钙，以300rpm的转速搅拌该混合液12h，脲酶菌发生生物矿化反应，生成具有脲酶活性的碳酸钙浆状悬浮液。
如图1和图2所示，图1示出了上述生物矿化反应过程中的状态，反应体系呈混沌状，图2示出了生物矿化反应后静置一段时间的状态，可以看出，悬浮液逐渐沉淀分层。
最后，将上述悬浮液倒入装有真空抽滤装置中进行过滤，过滤出的固体物质使用去离子水进行清洗3次，然后放入冷冻干燥机中于-30℃下进行冻干48h，最后进行研磨，即得。
图3示出了上述产物的实物，可见其呈白灰色粉体状。
对上述获得的产物分别进行了扫描电子显微镜(SEM)和X射线衍射(XRD)分析，表征结果分别如图4和图5所示。从图4中可以看出，该产物具有多孔结构。而从图5可以看出，其中主要晶体为碳酸钙，且呈球霰石(vaterite)晶型。该特定形貌的晶体组分保证了其具有重金属吸附性能。
对上述获得的产物进行脲酶活性的测试，其脲酶活性为350U/g。该性能保证了其具有重金属矿化性能。
由此可见，本实施例获得的是一种具有脲酶活性的多孔生物碳酸钙钝化材料。
对上述钝化材料的稳定性进行了测试。具体来讲，分别取10g上述钝化材料置于温度分别为4℃和25℃的环境中，测试它们在第1、3、14、21、28天时的脲酶活性。每次取0.1g该钝化材料置于1.8mol/L的尿素中，通过测试其30min内氨氮浓度变化率，来表示其脲酶活性，单位为U/g。
稳定性测试结果如图6所示。可以看出，在近一个月内，该钝化材料的脲酶活性没有明显的降低，尤其是在4℃条件下，一个月内基本是维持在其90％以上的原始活性；同时，在25℃条件下，其活性仍留存较好的脲酶活性，体现出了其具有较好的热稳定性，这为其运用在实际情况中提供了很好的应用前景。
对上述钝化材料的耐毒性进行了测试。具体来讲，分别取1g上述钝化材料放入重金属Cd浓度分别为0、10mg/L、20mg/L、50mg/L、100mg/L、250mg/L、和500mg/L的150mL锥形瓶中；其中均含有浓度100mmol/L的尿素。在30℃、180rpm的摇床中，反应24h并测试该阶段水溶液中产生的氨氮浓度变化情况。
耐毒性测试结果如图7所示。可以看出，在不超过500mg/L的重金属Cd浓度范围内，虽反应强度受到重金属影响，但仍具有一定脲酶活性，而且均在24h左右充分地反应掉了100mol/L的尿素。表明该钝化材料具有很强重金属耐受能力，同时也表现出了具有强大的去除重金属的潜力，表明该复合的钝化材料具有比单纯的钝化剂具有更优的应用前景。
实施例2
在实施例2的描述中，与实施例1的相同之处在此不再赘述，只描述与实施例1的不同之处。实施例2与实施例1的不同之处在于：
在实施例2提供的钝化材料的制备方法中，向250mL菌液中加入1.5015g尿素和11.098g氯化钙；并且，于-35℃下进行冻干42h；其余参照实施例1中所述，获得具有脲酶活性的多孔生物碳酸钙钝化材料。
本实施例提供的该钝化材料的脲酶活性为307U/g。
实施例3
在实施例3的描述中，与实施例1的相同之处在此不再赘述，只描述与实施例1的不同之处。实施例3与实施例1的不同之处在于：
在实施例3提供的钝化材料的制备方法中，向250mL菌液中加入12.012g尿素和22.1968g氯化钙；并且，于-40℃下进行冻干36h；其余参照实施例1中所述，获得具有脲酶活性的多孔生物碳酸钙钝化材料。
本实施例提供的该钝化材料的脲酶活性为151U/g。
实施例4
在实施例4的描述中，与实施例1的相同之处在此不再赘述，只描述与实施例1的不同之处。实施例4与实施例1的不同之处在于：
在实施例4提供的钝化材料的制备方法中，所采用的具有脲酶菌的菌液通过生理盐水稀释至30U/mL左右的脲酶活性；其余参照实施例1中所述，获得具有脲酶活性的多孔生物碳酸钙钝化材料。
本实施例提供的该钝化材料的脲酶活性为298U/g。
实施例5
在实施例5的描述中，与实施例1的相同之处在此不再赘述，只描述与实施例1的不同之处。实施例5与实施例1的不同之处在于：
在实施例5提供的钝化材料的制备方法中，所采用的具有脲酶菌的菌液通过生理盐水稀释至10U/mL左右的脲酶活性；向250mL菌液中加入6.006g尿素和2.7746g氯化钙；并且，于-40℃下进行冻干24h；其余参照实施例1中所述，获得具有脲酶活性的多孔生物碳酸钙钝化材料。
本实施例提供的该钝化材料的脲酶活性为998U/g。
实施例6
在实施例6的描述中，与实施例1的相同之处在此不再赘述，只描述与实施例1的不同之处。实施例6与实施例1的不同之处在于：
在实施例6提供的钝化材料的制备方法中，所采用的具有脲酶菌的菌液，其所使用的培养基的组成为：20g/L酵母粉、30g/L的氯化铵，并加水补足至1L而获得，且该培养基的pH控制为9.2；其余参照实施例1中所述，获得具有脲酶活性的多孔生物碳酸钙钝化材料。
本实施例提供的该钝化材料的脲酶活性为301U/g。
实施例7
在实施例7的描述中，与实施例1的相同之处在此不再赘述，只描述与实施例1的不同之处。实施例7与实施例1的不同之处在于：
在实施例7提供的钝化材料的制备方法中，所采用的具有脲酶菌的菌液，其所使用的培养基的组成为：30g/L酵母粉、25g/L的氯化铵、0.5mmol/L氯化镍混合，并加水补足至1L而获得，且该培养基的pH控制为9.3；其余参照实施例1中所述，获得具有脲酶活性的多孔生物碳酸钙钝化材料。
本实施例提供的该钝化材料的脲酶活性为303U/g。
本发明提供的该钝化材料的制备过程中，一方面，脲酶菌的提供并非单一的菌种，而是培养出脲酶菌的菌液一并混合；另一方面，悬浮液获得的滤渣的干燥方式，均对获得上述特定形貌组分及优异应用性能的产物有重大影响。
针对上述影响因素一，基于发明人的前期研究，若将提取出的脲酶菌直接与尿素、可溶性钙盐混合，最终经干燥后沉积获得的碳酸钙晶型仅为方解石，而非本发明中的球霰石。
针对上述影响因素二，进行了下述对比实验。
对比例1
在本对比例中，与实施例1的相同之处在此不再赘述，只描述与实施例1的不同之处。本对比例与实施例1的不同之处在于：
本对比例制备获得的具有脲酶活性的碳酸钙浆状悬浮液，经真空抽滤、滤渣清洗后，分成两份，分别置于4℃和25℃下进行自然干燥，而并非采用实施例1中的冻干法，对应获得第一对比钝化材料和第二对比钝化材料。
采用同样的测试方法分别对两种对比钝化材料的稳定性进行了测试，测试结果如图8所示。从图8中可以看出，不采用冻干法制得的产品，其稳定性均出现了大幅下降。尤其在室温下(25℃)自然干燥的方式所获得的产品，其脲酶活性下降迅猛，甚至在第7天左右几乎丧失了脲酶活性，显然无法正常使用。即便是在4℃较低温度下干燥获得的产品，其脲酶活性也在超过第7天后即出现了迅猛下降。
对比例2
在本对比例中，与实施例1的相同之处在此不再赘述，只描述与实施例1的不同之处。本对比例与实施例1的不同之处在于：
本对比例制备获得的具有脲酶活性的碳酸钙浆状悬浮液，经真空抽滤、滤渣清洗后，进行60℃左右烘干处理，而并非采用实施例1中的冻干法，对应获得第三对比钝化材料。
测试该对比钝化材料其中的脲酶活性，其与实施例1中的结果对比如图9所示。其中“冷冻干燥”即实施例1中活性数据，而“高温烘干”系本对比例中活性数据。可以看出，当采用60℃左右的温度下的烘干操作时，所获得的产物中脲酶菌即已几乎失活。由此可知，该干燥方式获得的产物在应用中，无法 再发挥生物矿化作用。
本发明提供的上述钝化材料，基于其特定的结构和性能，能够对重金属产生吸附作用和矿化作用的双重作用，为此可应用于重金属修复领域中。为此，提供了下述应用例。
应用例1
将上述实施例1提供的钝化材料应用于重金属镉的去除中。
取0.1g钝化材料放入体积为300mL、浓度为400mg/L的重金属镉的溶液中，使其对重金属镉的去除率达到平衡。
在上述去除过程中(10h左右)，取部分沉淀物，并进行扫描电镜测试，其SEM图如图10所示。待达到去除平衡(10d左右)后，取部分沉淀物，分别进行X射线衍射测试和扫描电镜测试，其XRD图和SEM图分别如11和图12所示。
从图10中可以看出，图4中所展现的多孔球体外生成部分块状晶体；对比图12可以看出，该块状晶体越来越多，并逐渐布满多孔球体的外表面。结合图11的结果，可以发现，上述位于多孔碳酸钙球体外表面的块状晶体即为钝化重金属镉而生成的碳酸镉，且其晶型为菱镉矿。
通过ICP-OES仪器测试上述过程中重金属镉的浓度变化，去除效果如图13所示。从图13中可以看出，该钝化材料对该高浓度重金属镉具有非常优异的去除效果。
应用例2
本应用例对实施例1提供的钝化材料的耐久性进行了测试。
具体来讲，分别取0.1g钝化材料置于其中重金属镉的浓度分别为0、10mg/L、50mg/L、150mg/L、300mg/L、及500mg/L的150mL锥形瓶中，在重金属镉离子环境中毒害24h后，再向其中加入尿素至浓度为0.1mol/L。在30℃、180rpm的摇床中，反应48h并测试该阶段产生的氨氮浓度变化情况。
该耐久性测试结果如图14所示。从图14中可以看出，该钝化材料在低浓度重金属镉条件下仍然能够有效地保留其脲酶活性，同时在加入尿素后还能继续进行生物矿化反应，说明其具有很好的耐毒性和耐久性。
基于该优异的耐久性，能够获知，若继续向其中补充可溶性钙盐作为钙源， 其展现出来的脲酶活性能够继续分解尿素产生碳酸根，继而与这些新添加的钙离子生成碳酸钙，同时已完成重金属钝化后生成的沉淀颗粒提供生长位点，即可使这些新生成的碳酸钙包覆在重金属碳酸盐外，从而防止重金属的二次污染，这为其在实际应用场景中创造了即为有利的使用条件。
虽然已经参照特定实施例示出并描述了本发明，但是本领域的技术人员将理解：在不脱离由权利要求及其等同物限定的本发明的精神和范围的情况下，可在此进行形式和细节上的各种变化。

Claims (10)

  1. 一种具有脲酶活性的多孔生物碳酸钙钝化材料的制备方法，其特征在于，包括步骤：
    S1、向具有脲酶菌的菌液中添加尿素和可溶性钙盐，所述脲酶菌发生生物矿化反应，生成具有脲酶活性的碳酸钙浆状悬浮液；
    S2、将所述碳酸钙浆状悬浮液进行过滤后，滤渣经清洗和冻干处理，获得具有脲酶活性的多孔生物碳酸钙钝化材料。
  2. 根据权利要求1所述的制备方法，其特征在于，所述尿素和所述可溶性钙盐的添加量均控制为添加后的浓度为0.1mol/L～0.8mol/L。
  3. 根据权利要求2所述的制备方法，其特征在于，所述可溶性钙盐选自氯化钙、醋酸钙、硝酸钙。
  4. 根据权利要求1所述的制备方法，其特征在于，所述具有脲酶菌的菌液为脲酶菌母液接种于培养基中经培养及脲酶活性调整而获得的混合体系，且其中脲酶菌活性为10U/mL～30U/mL；其中，所述培养基包括：20g/L～30g/L酵母粉、15g/L～30g/L氯化铵、0～1mmol/L氯化镍，且所述培养基的pH为9.2～9.3。
  5. 根据权利要求4所述的制备方法，其特征在于，所述脲酶菌为巴氏芽孢杆菌。
  6. 根据权利要求1～5任一所述的制备方法，其特征在于，在步骤S2中，控制样品温度为-40℃～-30℃下进行冻干处理。
  7. 根据权利要求6所述的制备方法，其特征在于，在步骤S2中，冻干时间为24h～48h。
  8. 一种具有脲酶活性的多孔生物碳酸钙钝化材料，其特征在于，采用权利要求1～7任一所述的制备方法获得。
  9. 根据权利要求8所述的具有脲酶活性的多孔生物碳酸钙钝化材料，其特征在于，包括球霰石形貌的多孔碳酸钙骨架，且其中脲酶活性为150U/g～1000U/g。
  10. 如权利要求8或9所述的具有脲酶活性的多孔生物碳酸钙钝化材料在重金属修复中的应用。
PCT/CN2023/077190 2022-05-10 2023-02-20 具有脲酶活性的多孔生物碳酸钙钝化材料及其制备方法和应用 WO2023216674A1 (zh)

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