WO2022012695A1 - 一种矿化微生物增量制备方法 - Google Patents

一种矿化微生物增量制备方法 Download PDF

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WO2022012695A1
WO2022012695A1 PCT/CN2021/113378 CN2021113378W WO2022012695A1 WO 2022012695 A1 WO2022012695 A1 WO 2022012695A1 CN 2021113378 W CN2021113378 W CN 2021113378W WO 2022012695 A1 WO2022012695 A1 WO 2022012695A1
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cells
mineralized
incremental
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钱春香
芮雅峰
周横一
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东南大学
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    • 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
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    • 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
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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  • the invention belongs to the interdisciplinary science and technology in the fields of microbiology and civil engineering materials, in particular to a method for incremental preparation of mineralized microorganisms, which can be used in the fields of geotechnical engineering, environmental geotechnical engineering, waste residue utilization and cement-based materials.
  • microbial mineralization technology can effectively consolidate sand particles, reduce the porosity and permeability coefficient of sand bodies, and have significant effects in slope protection and soil cementation.
  • microbial mineralization technology can effectively precipitate heavy metal ions or form a co-precipitation combined state, thereby realizing the remediation of heavy metal-contaminated soil and water bodies, with remarkable results.
  • microbial mineralization technology can effectively enhance the performance of recycled aggregates; for the utilization of steel slag in building materials, microbial methods can effectively improve the utilization efficiency of calcium and magnesium ions in steel slag.
  • microbial-induced calcium carbonate deposition can effectively block concrete cracks, improve durability, and realize concrete self-repair; at the same time, microbial mineralization technology can be used to prepare self-cleaning concrete, improve the apparent quality of concrete, and make concrete materials Both beautiful and functional.
  • the present invention proposes a method for incremental preparation of mineralized microorganisms, so as to promote the application of mild and green microbial technology in the field of engineering materials. Promote the application.
  • the method for incremental preparation of mineralized microorganisms described in the present invention by inoculating the mineralized microorganisms into the medium for incremental culture, the total number of cells in the bacterial liquid is (1-3) ⁇ 10 8 cells/mL , spray-drying to prepare mineralized microbial powder, and verify the mineralization ability; wherein, the medium components include beef extract, peptone, ammonium salt, sodium salt, and metal ions.
  • the mineralized microorganism can produce spores, can induce the generation of mineralized products, is harmless to human body and has good ecological properties. Further, the mineralizing microorganisms are one or more of Bacillus colloids, Bacillus alkalophiles, and the like.
  • the components of the culture medium include beef extract, peptone, ammonium salt, sodium salt, and metal ions, and the addition of metal ions can promote the transformation of alkali-resistant mineralized microorganisms into spores; wherein, each liter of culture medium contains 2-5 g of beef extract, Peptone 3-8g, ammonium salt 0-3g, sodium salt 3-8g.
  • the metal ions include one or more of manganese ions, magnesium ions, and zinc ions.
  • manganese ions are provided by 5-10 mg of manganese sulfate monohydrate
  • magnesium ions are provided by 0.10-0.15 g of magnesium chloride hexahydrate
  • zinc ions are provided by 0.1-0.2 g of zinc chloride.
  • the incremental culture process is as follows: firstly culture in a small amount in 500mL medium, inoculate it in a sterilized small-capacity seed tank after culturing for 24 hours, and when the total number of cells reaches (1-3) ⁇ 108 cells/mL Inoculated into sterilized bulk fermenters. I.e., first in small volume fermenter to about the total number of cells in the bacterial suspension (1 ⁇ 3) ⁇ 10 8 / mL, and then a small capacity as a seed fermenter tank, to remove the required number of cells in the culture broth, a large Inoculated and cultured in a volume fermenter until the total number of cells in the bacterial liquid was about (1-3) ⁇ 10 8 cells/mL.
  • the sterilization process of the fermenter is as follows: firstly, the air filter is sterilized, the pressure is kept at 0.10-0.11MPa, and the sterilization is 20-30min; then the fermenter is sterilized at a temperature of 115-125°C.
  • the cooling method of the fermentation tank can be natural cooling or accelerated cooling by cooling water.
  • the method for testing the total number of cells includes hemocytometer, flow cytometry or OD value method.
  • the drying technology is preferably spray drying technology, which is characterized by instant drying, good product quality, and product quality indicators that can be adjusted and controlled as required.
  • the method for verifying the microbial mineralization ability is as follows: adding mineralizing microorganisms and a calcium source into the simulated pore solution, placing it in a 25-30° C. shaking incubator for cultivation, taking it out after 3-5 days, and verifying the precipitated product.
  • the calcium source is preferably one or more of calcium chloride, calcium nitrate, etc.
  • the calcium ion concentration in the solution is preferably 10-80 mmol/L.
  • the mineralized microorganism adopted in the present invention can produce spores, is suitable for the field of engineering materials, is harmless to human body and has good ecological properties;
  • the present invention optimizes the culture medium, especially adds metal salt ions, to achieve the purpose of promoting spore transformation, and the effect is excellent;
  • the present invention proposes a method for incremental preparation of mineralized microorganisms, which does not affect the activity of mineralized microorganisms, is easy to preserve, can be directly added to engineering materials for use, and can be used in geotechnical engineering and environmental geotechnical engineering , waste residue utilization and cement-based materials.
  • Fig. 1 is the topography of mineralized microorganisms observed by inverted fluorescence microscope
  • Figure 2 shows the growth and reproduction curves of microorganisms obtained by different culture methods
  • FIG. 3 SEM image of mineralized microbial spores
  • Fig. 4 is a schematic diagram of a precipitated product
  • Figure 5 is the SEM image and EDS result of the precipitated product.
  • the invention provides a method for incremental preparation of mineralized microorganisms, and the specific operation process is as follows:
  • Mineralized microorganisms are cultured in a small amount in 500mL medium, and then inoculated and cultivated in a small-capacity fermenter; the medium components include beef extract, peptone, ammonium salt, sodium salt, and metal ions. Metal ions can promote the mineralization of microorganisms.
  • Transform into spores the medium needs to be sterilized at 121°C for 30 minutes; samples are taken from the fermenter every 2 hours during the culture process, and the total number of cells in the bacterial liquid is tested until the total number of cells is about (1 ⁇ 3) ⁇ 10 8 Stop the culture after /mL;
  • step (2) spores and calcium chloride to the simulated pore solution so that the total number of cells is 10 8 cells/mL, and the calcium ion concentration is 25 mmol/L, and placed in a shaking incubator at 30°C for 4 days.
  • the precipitation product formed is shown in Figure 4. It can be seen that the mineralized microorganisms in the pore solution can induce the formation of precipitation, and finally the precipitation product is verified. It can be seen from the SEM/EDS results in Figure 5 that the main elements of the precipitation product are C, O, and Ca, and the precipitation product is biological calcium carbonate, which shows that the microorganism has the ability to induce the deposition of mineralized products.
  • step (2) spores and calcium chloride to the simulated pore solution, so that the total number of cells is 10 8 cells/mL, and the calcium ion concentration is 50 mmol/L, and placed in a shaking incubator at 30°C for 4 days. After taking out, the precipitated product was verified. It was verified to be calcium carbonate by SEM/EDS.
  • step (2) spores and calcium nitrate to the simulated pore solution, so that the total number of cells is 10 8 cells/mL, and the calcium ion concentration is 25 mmol/L, and cultured in a shaking incubator at 30°C for 4 days. Take out and verify the precipitated product. It was verified to be calcium carbonate by SEM/EDS.
  • step (2) spores and calcium nitrate to the simulated pore solution, so that the total number of cells is 10 8 cells/mL, and the calcium ion concentration is 50 mmol/L, and placed in a shaking incubator at 30°C for 4 days. Take out and verify the precipitated product. It was verified to be calcium carbonate by SEM/EDS.

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Abstract

提供了一种矿化微生物增量制备方法。该方法首先将矿化微生物接种到种子罐中培养，种子罐中每升培养基含有牛肉膏2～5g，蛋白胨3～8g、铵盐0～3g、钠盐3～8g，金属离子选用锰离子、镁离子、锌离子等的一种或几种，以达到促进矿化微生物向芽孢转化的目的；当种子罐中细胞总数达到(1～3)×10 8个/mL时，接种到大容量发酵罐中增量培养；当增量培养的细胞总数达到(1～3)×10 8个/mL后，通过喷雾干燥技术制备矿化微生物菌粉。该矿化微生物增量培养后活性未发生变化，便于保存，可直接使用。

Description

一种矿化微生物增量制备方法 技术领域
本发明属于微生物学和土木工程材料领域的交叉科学技术，具体涉及一种矿化微生物增量制备方法，可用于岩土工程、环境岩土工程、废渣利用和水泥基材料领域。
背景技术
研究者受自然界中微生物成岩现象启发，提出一种绿色、温和的微生物矿化技术，可用于量大面广的工程材料领域，如岩土工程、环境岩土工程、废渣利用和水泥基材料。在岩土工程领域，微生物矿化技术可有效固结砂粒，降低砂体孔隙和渗透系数，在边坡防护、土壤胶结等方面具有显著效果。在环境岩土工程领域，利用微生物矿化技术，可有效沉淀重金属离子或形成共沉淀结合态，从而实现重金属污染土壤和水体的修复，效果显著。在废渣利用领域，微生物矿化技术可有效增强再生骨料性能；针对钢渣在建材中的利用，微生物方法可有效提高钢渣中钙镁离子利用效率。在水泥基材料领域，微生物诱导碳酸钙沉积可对混凝土裂缝进行有效封堵，提高耐久性，实现混凝土自修复；同时微生物矿化技术可用于制备自清洁混凝土，提高混凝土表观质量，使得混凝土材料既美观又实用。
近年来微生物矿化技术在世界范围内得到了广泛关注，但仍存在诸多问题急需解决，如矿化微生物的增量制备方法，以获得便于保存的矿化微生物并提高其存活率，促进微生物矿化技术在工程材料领域的推广应用。
发明内容
发明目的：为了矿化微生物能更好的保存和提高微生物在工程材料中的存活率，本发明提出一种矿化微生物增量制备方法，以促进温和、绿色的微生物技术在工程材料领域中的推广应用。
技术方案：本发明所述的一种矿化微生物增量制备方法，通过将矿化微生物接种至培养基中增量培养，至菌液中细胞总数为(1～3)×10 8个/mL，喷雾干燥制备矿化微生物菌粉，并验证矿化能力；其中，所述培养基成分包括牛肉膏、蛋白胨、铵盐、钠盐、金属离子。
所述矿化微生物可产芽孢，能诱导矿化产物生成，对人体无害且生态性良好。 进一步的，所述矿化微生物为胶质芽孢杆菌、嗜碱芽孢杆菌等一种或多种。
所述培养基成分包括牛肉膏、蛋白胨、铵盐、钠盐、金属离子，通过添加金属离子可促进耐碱性矿化微生物向芽孢进行转化；其中，每升培养基含有牛肉膏2～5g，蛋白胨3～8g、铵盐0～3g、钠盐3～8g。
所述金属离子包括锰离子、镁离子、锌离子等的一种或几种。其中优选的，每升培养基中，锰离子由5-10mg一水合硫酸锰提供，镁离子由0.10-0.15g六水合氯化镁提供，锌离子由0.1-0.2g氯化锌提供。
进一步的，所述增量培养过程为：先在500mL培养基中小量培养，培养24h后接种在灭菌小容量种子罐中培养，当细胞总数达到(1～3)×10 8个/mL后接种到灭菌大容量发酵罐中。即先在小容量发酵罐培养至菌液中细胞总数约为(1～3)×10 8个/mL，然后将小容量发酵罐作为种子罐，取出培养至要求细胞数量的菌液，在大容量发酵罐中接种培养，培养至菌液中细胞总数约为(1～3)×10 8个/mL。
其中，所述发酵罐灭菌处理过程为：首先对空气过滤器灭菌，保持压力为0.10～0.11MPa，灭菌20-30min；随后对发酵罐灭菌，温度为115-125℃。发酵罐冷却方式可为自然冷却或冷却水加速冷却。
本申请中，所述细胞总数测试方法包括血球计数板计数法、流式细胞仪法或OD值法。
所述干燥技术优选喷雾干燥技术，其特点是：瞬间干燥，产品质量好，可根据需要调节和控制产品质量指标等。
所述微生物矿化能力验证方法为：在模拟孔隙溶液中掺加矿化微生物和钙源，并置于25-30℃振荡培养箱中培养，3-5d后取出，对沉淀产物进行验证。其中，所述钙源优选的为氯化钙、硝酸钙等的一种或多种，溶液中钙离子浓度优选为10～80mmol/L。
有益效果：与现有技术相比，本发明的优点包括：
(1)本发明采用的矿化微生物可产芽孢，适用于工程材料领域，对人体无害且生态性良好；
(2)本发明通过培养基优化，特别是添加金属盐离子，以达到促进芽孢转化的目的，且效果优异；
(3)本发明提出了一种矿化微生物增量制备方法，且该方法不影响矿化微 生物活性，便于保存，可直接掺加到工程材料中使用，可用于岩土工程、环境岩土工程、废渣利用和水泥基材料领域。
附图说明
图1为采用倒置荧光显微镜观测的矿化微生物形貌图；
图2为不同培养方式获取微生物的生长繁殖曲线；
图3矿化微生物芽孢的SEM图像；
图4为沉淀产物示意图；
图5为沉淀产物的SEM图像和EDS结果。
具体实施方式
为更好的理解本发明，将结合具体实施例进一步阐述本发明所述内容。
本发明提供一种矿化微生物增量制备方法，具体操作过程如下：
1.对小容量发酵罐进行灭菌处理，包括空气过滤器和空发酵罐的灭菌，灭菌后通过自然冷却或冷却水加速冷却后备用；
2.将矿化微生物在500mL培养基中小量培养后，在小容量发酵罐中接种培养；其中培养基成分包括牛肉膏、蛋白胨、铵盐、钠盐、金属离子，金属离子可促进矿化微生物向芽孢进行转化；培养基需在121℃条件下灭菌30min；培养过程中每间隔2h从发酵罐中取样，测试菌液中细胞总数，直至细胞总数约为(1～3)×10 8个/mL后停止培养；
3.对大容量发酵罐进行灭菌处理，包括空气过滤器和空发酵罐的灭菌，灭菌后通过自然冷却或冷却水加速冷却后备用；
4.将小容量发酵罐作为种子罐，取出培养至要求细胞数量的菌液，在大容量发酵罐中接种培养，培养至菌液中细胞总数约为(1～3)×10 8个/mL后停止培养；
5.将增量制备的矿化微生物菌液通过喷雾干燥技术制备为菌粉保存；
6.在模拟孔隙溶液中掺加矿化微生物和钙源，并置于30℃振荡培养箱中培养，4d后取出，对沉淀产物进行验证。
实施例1
(1)称取牛肉膏3g/L，蛋白胨5g/L，NaCl5g/L，MgCl 2·6H 2O0.1g/L，配置成液体培养基，在121℃条件下灭菌30min；
(2)在500mL培养基中接种胶质芽孢杆菌(CCTCC No:M 2012406)进行 小量培养，取培养24h的菌液在小容量发酵罐中接种培养，每间隔2h从发酵罐中取样，测试菌液中细胞总数，当培养时间为24h时，测得细胞总数为1.59×10 8个/mL，采用倒置荧光显微镜观测的矿化微生物形貌如图1所示，可见试验选取的矿化微生物外形为杆状。停止培养接种至大容量发酵罐；在大容量发酵罐中每间隔2h从发酵罐中取样，测试菌液中细胞总数，当培养时间为24h时，测得细胞总数为1.52×10 8个/mL，停止培养，由图2中不同培养方式获取的微生物生长繁殖曲线可见，增量制备方法不影响矿化微生物活性。将获得的发酵液通过喷雾干燥技术制备成芽孢，微结构如图3所示。
(3)在模拟孔隙溶液中掺加步骤(2)芽孢和氯化钙，使得细胞总数为10 8个/mL，钙离子浓度为25mmol/L，并置于30℃振荡培养箱中培养，4d后取出，形成沉淀产物如图4所示，可见孔隙溶液中矿化微生物可诱导沉淀生成，最后对沉淀产物进行验证。由图5中SEM/EDS结果可见，沉淀产物主要元素为C、O、Ca，沉淀产物为生物碳酸钙，可见该微生物具有诱导矿化产物沉积的能力。
实施例2
(1)称取牛肉膏3g/L，蛋白胨5g/L，NH 4Cl2.8g/L，NaCl5g/L，MgCl 2·6H 2O0.12g/L，配置成液体培养基，在121℃条件下灭菌30min；
(2)在500mL培养基中接种胶质芽孢杆菌(CCTCC No:M 2012406)进行小量培养，取培养24h的菌液在小容量发酵罐中接种培养，每间隔2h从发酵罐中取样，测试菌液中细胞总数，当培养时间为24h时，测得细胞总数为1.65×10 8个/mL，停止培养接种至大容量发酵罐；在大容量发酵罐中每间隔2h从发酵罐中取样，测试菌液中细胞总数，当培养时间为24h时，测得细胞总数为1.71×10 8个/mL，停止培养，通过喷雾干燥技术制备成芽孢；
(3)在模拟孔隙溶液中掺加步骤(2)芽孢和氯化钙，使得细胞总数为10 8个/mL，钙离子浓度为50mmol/L，并置于30℃振荡培养箱中培养，4d后取出，对沉淀产物进行验证。通过SEM/EDS验证其为碳酸钙。
实施例3
(1)称取牛肉膏3g/L，蛋白胨5g/L，NaCl5g/L，MnSO 4·H 2O5mg/L，配置成液体培养基，在121℃条件下灭菌30min；
(2)在500mL培养基中接种嗜碱芽孢杆菌(中国工业微生物菌种保藏管理 中心菌株保藏编号为CICC 23037)进行小量培养，取培养24h的菌液在小容量发酵罐中接种培养，每间隔2h从发酵罐中取样，测试菌液中细胞总数，当培养时间为24h时，测得细胞总数为1.75×10 8个/mL，停止培养接种至大容量发酵罐；在大容量发酵罐中每间隔2h从发酵罐中取样，测试菌液中细胞总数，当培养时间为24h时，测得细胞总数为1.77×10 8个/mL，停止培养，通过喷雾干燥技术制备成芽孢；
(3)在模拟孔隙溶液中掺加步骤(2)芽孢和硝酸钙，使得细胞总数为10 8个/mL，钙离子浓度为25mmol/L，并置于30℃振荡培养箱中培养，4d后取出，对沉淀产物进行验证。通过SEM/EDS验证其为碳酸钙。
实施例4
(1)称取牛肉膏3g/L，蛋白胨5g/L，NaCl5g/L，MnSO 4·H 2O10mg/L，配置成液体培养基，在121℃条件下灭菌30min；
(2)在500mL培养基中接种嗜碱芽孢杆菌(中国工业微生物菌种保藏管理中心菌株保藏编号为CICC 23037)进行小量培养，取培养24h的在小容量发酵罐中接种培养，每间隔2h从发酵罐中取样，测试菌液中细胞总数，当培养时间为24h时，测得细胞总数为1.81×10 8个/mL，停止培养接种至大容量发酵罐；在大容量发酵罐中每间隔2h从发酵罐中取样，测试菌液中细胞总数，当培养时间为24h时，测得细胞总数为1.82×10 8个/mL，停止培养，通过喷雾干燥技术制备成芽孢；
(3)在模拟孔隙溶液中掺加步骤(2)芽孢和硝酸钙，使得细胞总数为10 8个/mL，钙离子浓度为50mmol/L，并置于30℃振荡培养箱中培养，4d后取出，对沉淀产物进行验证。通过SEM/EDS验证其为碳酸钙。

Claims (10)

  1. 一种矿化微生物增量制备方法，其特征在于，将矿化微生物接种至培养基中增量培养，至菌液中细胞总数为(1～3)×10 8个/mL，喷雾干燥制备成矿化微生物菌粉，并验证矿化能力；其中，所述培养基成分包括牛肉膏、蛋白胨、铵盐、钠盐、金属离子。
  2. 根据权利要求1所述的矿化微生物增量制备方法，其特征在于，所述矿化微生物为胶质芽孢杆菌、嗜碱芽孢杆菌等一种或多种。
  3. 根据权利要求1所述的矿化微生物增量制备方法，其特征在于，每升培养基含有牛肉膏2～5g，蛋白胨3～8g、铵盐0～3g、钠盐3～8g。
  4. 根据权利要求1所述的矿化微生物增量制备方法，其特征在于，所述金属离子包括锰离子、镁离子、锌离子等的一种或几种其中，每升培养基中，所述锰离子由5-10mg一水合硫酸锰提供，镁离子由0.10-0.15g六水合氯化镁提供，锌离子由0.1-0.2g氯化锌提供。
  5. 根据权利要求1所述的矿化微生物增量制备方法，其特征在于，所述增量培养过程为：先在培养基中小量培养，然后取培养液在灭菌小容量种子罐中接种培养，当细胞总数达到(1～3)×10 8个/mL后接种到灭菌大容量发酵罐中。
  6. 根据权利要求5所述的矿化微生物增量制备方法，其特征在于，发酵罐灭菌处理过程为：首先对空气过滤器灭菌，保持压力为0.10～0.11MPa，灭菌20-30min；随后对发酵罐灭菌，温度为115-125℃。
  7. 根据权利要求1所述的矿化微生物增量制备方法，其特征在于，所述细胞总数测试方法包括血球计数板计数法、流式细胞仪法或OD值法。
  8. 根据权利要求1所述的矿化微生物增量制备方法，其特征在于，所述干燥技术优选喷雾干燥技术。
  9. 根据权利要求1所述的矿化微生物增量制备方法，其特征在于，所述微生物矿化能力验证方法为：在模拟孔隙溶液中掺加矿化微生物和钙源，并置于25-30℃振荡培养箱中培养，3-5d后取出，对沉淀产物进行验证。
  10. 根据权利要求9所述的矿化微生物增量制备方法，其特征在于，所述钙源为氯化钙、硝酸钙中的一种或多种。
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