WO2023123082A1 - 一种双菌菌剂的制备方法和一种土壤修复方法 - Google Patents

一种双菌菌剂的制备方法和一种土壤修复方法 Download PDF

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WO2023123082A1
WO2023123082A1 PCT/CN2021/142592 CN2021142592W WO2023123082A1 WO 2023123082 A1 WO2023123082 A1 WO 2023123082A1 CN 2021142592 W CN2021142592 W CN 2021142592W WO 2023123082 A1 WO2023123082 A1 WO 2023123082A1
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bacteria
soil
carbonate
carbonic anhydrase
mineralizing
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French (fr)
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郭丽莉
刘亚茹
高艳丽
李书鹏
王祺
熊静
郝弟
李嘉晨
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北京建工环境修复股份有限公司
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Priority to PCT/CN2021/142592 priority Critical patent/WO2023123082A1/zh
Publication of WO2023123082A1 publication Critical patent/WO2023123082A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K17/00—Soil-conditioning materials or soil-stabilising materials
    • C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
    • 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
    • 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
    • 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

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  • the invention relates to the field of soil remediation, in particular to a preparation method of a dual-bacteria agent and a soil remediation method.
  • Desertification soil control methods can be divided into three categories: physical methods, chemical methods and biological methods.
  • the implementation process of physical methods is more complicated and requires a lot of manpower and material resources.
  • the chemical method is costly and has the problem of causing secondary pollution.
  • Biological methods are divided into plant methods and microbial methods.
  • the restoration process of plant methods has a long cycle and slow results.
  • Microbially Induced Carbonate Precipitation (MICP) technology has received extensive attention in the control of desertified soils due to its advantages of low cost, sustainability, and no secondary pollution.
  • MICP technology is based on a series of biochemical reactions of carbonate mineralizing bacteria, that is, cations such as Ca 2+ and Mg 2+ adsorbed on microbial extracellular polymers and CO 3 2- produced during microbial metabolism form calcium carbonate crystals , These calcium carbonate crystals fill the gaps between soil particles, increase the compactness of the soil and the friction and bonding between soil particles, and improve the aggregation of the soil.
  • MICP technology can form a hard shell on the surface of sandy soil that can effectively resist wind and water erosion. However, it takes a long time to fix desertified soil with carbonate mineralizing bacteria alone under natural conditions, and a large amount of urea is required as a substrate, which limits the application of carbonate mineralizing bacteria in desertification soil remediation.
  • the technical problem to be solved by the present invention is to overcome the defects that the existing MICP technology is slow to fix under natural conditions and needs to use a large amount of urea as a substrate, thereby providing a dual-bacteria agent and its application in desertification soil restoration.
  • the invention provides a dual-bacteria agent, the dual-bacteria agent includes carbonate mineralizing bacteria and carbonic anhydrase-producing bacteria, the carbonic anhydrase-producing bacteria in the dual-bacteria agent
  • the live bacteria ratio is 1-5:1.
  • the present invention also provides a preparation method of the above-mentioned dual-bacteria agent, which comprises separately fermenting the carbonate mineralizing bacteria and the carbonic anhydrase-producing bacteria to obtain a bacterial liquid, and then mixing the bacterial liquid evenly to obtain the dual-bacterial agent.
  • the method for cultivating the carbonate mineralizing bacteria is to pick a single colony of the carbonate mineralizing bacteria into the first culture medium, and culture the activated bacteria at 25-35°C and 200-250rpm overnight to obtain Overnight culture: absorb the overnight culture and add it to the first medium whose volume is 100-200 times its volume, and then culture it at 25-35° C. at 200-250 rpm for 24 hours to obtain a carbonate mineralizing bacteria liquid.
  • the cultivation method of the carbonic anhydrase-producing bacteria is as follows: pick a single colony of the carbonic anhydrase-producing bacteria to the second culture medium, place the activated bacteria overnight at 25-35°C and 200-250rpm to obtain an overnight culture; The overnight culture is added to the second culture medium whose volume is 100-200 times its volume, and cultured at 25-35° C. at 200-250 rpm for 24 hours to obtain a carbonic anhydrase-producing bacteria liquid.
  • the first medium contains 20 g of yeast extract, 10 g of ammonium sulfate, and 15.748 g of Tris per liter, and the pH is adjusted to 9 with dilute hydrochloric acid;
  • the second medium contains 10 g of peptone, 5 g of yeast powder, and 10 g of sodium chloride per liter.
  • the present invention also provides the application of the above-mentioned dual-bacteria agent, which is applied to desertification soil remediation.
  • the present invention also provides a method for remediating desertification soil, comprising the following steps:
  • Steps S1-S3 are repeated 1-5 times to obtain repaired soil.
  • fixative in step S2 is a calcium chloride solution with a concentration of 0.01M-0.05M;
  • the cementing liquid in step S3 is a mixed solution of calcium chloride and urea, and the molar concentration of the calcium chloride and urea is the same, which is 0.5M-1.5M.
  • step S1 the volume ratio of the dual-bacteria agent to the soil to be repaired is 1-1.5:1, and the standing time is 2-5h;
  • step S2 the volume ratio of the fixative to the soil to be repaired is 0.1-0.3:1;
  • step S3 the volume ratio of the cementing liquid to the soil to be repaired is 5-10:1.
  • the carbonate mineralizing bacteria can secrete urease in a large amount, and urease can decompose molecules such as urea in the environment to form carbonate ions, which can be metabolized into calcium carbonate precipitation through its growth
  • urease can decompose molecules such as urea in the environment to form carbonate ions, which can be metabolized into calcium carbonate precipitation through its growth
  • the microorganisms are negatively charged, and will adsorb positively charged calcium ions and other cations, causing Ca 2+ to be enriched, and carbonate ions and calcium ions form carbonic acid with carbonate mineralizing bacteria as crystallization nuclei.
  • Calcium and magnesium carbonate crystallization, the induced calcium carbonate crystallization plays a bridging role around the loose gravel and between particles, improving the mechanical properties of the medium;
  • Carbonic anhydrase-producing bacteria can accelerate the dissolution rate of CO 2 in the system, provide enough carbonate ions for carbonate mineralizing bacteria, and accelerate the effect of carbonate mineralizing bacteria while utilizing carbon dioxide in the environment.
  • the present invention applies the dual-bacteria agent to desertification soil remediation. Compared with the process of immobilizing desertification soil with carbonate mineralizing bacteria only, the dual-bacteria system greatly improves the curing rate of the system. Under these conditions, the soil with more than 2 times the strength of the single-bacteria system can be obtained.
  • the carbonic anhydrase-producing bacteria use part of the carbon dioxide produced in the atmosphere and by the carbonate mineralizing bacteria to provide Licarbonate ions for the curing system.
  • Using CO 2 as part of the carbon source reduces CO 2 emissions and reduces the amount of urea used.
  • the fixative is used before the cementation fluid, so that the microbial cells are fixed on the surface of the soil particles and are not easily washed away by the subsequent cementation fluid.
  • the desertification soil restoration provided by the present invention is non-toxic, harmless, and has no secondary pollution, low cost, simple preparation of the dual-bacteria agent, and the urea left in the soil during the solidification process also makes the soil more fertile.
  • Fig. 1 is a diagram of the metabolic relationship of the dual-bacteria agent in the process of soil remediation in a specific embodiment of the present invention.
  • the carbonate mineralizing bacterium used in the present invention is Bacillus Pasteur, purchased from the American Culture Collection, numbered ATCC 11859;
  • the carbonic anhydrase-producing bacterium used in the present invention is an engineering bacterium E-22b-IRLS, and its preparation method is described in the master's thesis of the School of Chemical Engineering, Tianjin University, "Research on Carbonic Anhydrase Displayed on Cell Surface and Its Enzymatic Properties".
  • the carbonate mineralizing bacteria can secrete a large amount of urease, which can decompose molecules such as urea in the environment to form carbonate ions, and pass Its growth and metabolism are calcium carbonate precipitation to create a suitable alkaline environment.
  • the microorganisms are negatively charged and will absorb positively charged calcium ions and other cations, resulting in the enrichment of Ca 2+ .
  • Carbonate ions and calcium ions form carbonate minerals.
  • the bacteria form calcium carbonate and magnesium carbonate crystals as crystallization nuclei, and the induced calcium carbonate crystals act as bridges around the loose gravel and between particles, improving the mechanical properties of the medium; at the same time, the carbonic anhydrase-producing bacteria can accelerate the CO 2
  • the dissolution rate in the system provides sufficient carbonate ions for the carbonate mineralizing bacteria, and accelerates the solidification rate of desertified soil while utilizing carbon dioxide in the environment.
  • Present embodiment provides a kind of antibacterial agent, preparation method is as follows:
  • the first culture medium contained 20g of yeast extract, 10g of ammonium sulfate, and 15.748g of Tris per liter, and the pH was adjusted with dilute hydrochloric acid to 9;
  • the present invention also provides a method for remediating desertification soil, comprising the following steps:
  • Steps (1)-(3) were repeated every 48 hours for a total of 4 times to obtain the restored desertified soil.
  • Present embodiment provides a kind of antibacterial agent, preparation method is as follows:
  • the present invention also provides a method for remediating desertification soil, comprising the following steps:
  • Steps (1)-(3) were repeated every 48 hours for a total of 3 times to obtain the restored desertified soil.
  • Present embodiment provides a kind of antibacterial agent, preparation method is as follows:
  • the first medium contains 20g of yeast extract, 10g of ammonium sulfate, and 15.748g of Tris per liter, and the pH is adjusted with dilute hydrochloric acid to 9;
  • the present invention also provides a method for remediating desertification soil, comprising the following steps:
  • Steps (1)-(3) were repeated every 48 hours for a total of 5 times to obtain the restored desertified soil.
  • Example 1 Compared with Example 1, this Comparative Example 1 only uses the carbonate mineralizing bacteria liquid, and does not use the carbonic anhydrase-producing bacteria liquid, and the rest of the steps are the same as in Example 1.
  • Example 2 Compared with Example 1, this Comparative Example 2 only uses the carbonic anhydrase-producing bacterium liquid, and does not use the carbonic anhydrase-producing bacterium liquid, and the rest of the steps are the same as in Example 1.
  • Restoration experiments are recorded as experimental group, comparison group 1 and comparison group 2, and steps (1)-(3) of the desertification soil restoration method are recorded as a batch of grouting, and each sample is filled with a batch of grouting every 48 hours. A total of 4 grouting. From the second day after the grouting started, two samples were taken from the experimental group and the control group every 48 hours to test their compressive strength and shear strength, and the average value of the results was taken.
  • Table 1 The results are shown in Table 1:
  • Example 1 Under the same grouting conditions, compared with Comparative Example 1 in Example 1, under the same time and urea consumption, the compressive strength and shear strength of Example 1 are the same as those of Comparative Example 1 More than 2 times, and the compressive strength and shear strength in Comparative Example 2 cannot be compared with Example 1. It shows that to obtain the same strength of soil, the technical solution in Example 1 of the present application can greatly save time and reduce the consumption of urea compared with the comparative example.

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Abstract

一种包括碳酸盐矿化菌和产碳酸酐酶菌的双菌菌剂，以及该双菌菌剂的制备方法和在荒漠化土壤修复中的应用。双菌菌剂中的碳酸盐矿化菌和产碳酸酐酶菌相互配合，应用于荒漠化土壤修复，提高了体系的固化速率，固化时间和单独使用碳酸盐矿化菌相比大幅减少，以CO 2为部分碳源，减少了CO 2的排放并降低了尿素的用量；同时该方法无毒、无害、无二次污染，成本低，双菌菌剂制备简单、且固化过程中留在土壤里的尿素也使土壤更加肥沃。

Description

一种双菌菌剂的制备方法和一种土壤修复方法 技术领域
本发明涉及土壤修复领域，具体涉及一种双菌菌剂的制备方法和一种土壤修复方法。
背景技术
由于人类活动、如乱砍滥伐、乱采滥挖、超载过牧等行为，导致土壤荒漠化日益严重，由此出现的草地退化、水土流失、植被覆盖度降低、频繁出现沙尘暴等严重威胁了人类的生存空间。荒漠化土壤的治理问题在全球范围内受到了越来越多的关注，“治沙止漠”刻不容缓。
荒漠化土壤治理的办法主要可分为三大类：物理方法、化学方法和生物方法。物理方法实施过程较复杂，需要耗费较高的人力物力。化学方法成本较高，并且存在会造成二次污染的问题。生物法分为植物法和微生物法，植物法的修复过程周期长、见效慢。近年来，微生物诱导碳酸盐沉淀技术(Microbially Induced Carbonate Precipitation,MICP)因其成本低、可持续、无二次污染等优势在荒漠化土壤的治理中受到了广泛的关注。MICP技术是基于碳酸盐矿化菌的一系列生物化学反应，即吸附于微生物胞外聚合物的Ca 2+、Mg 2+等阳离子与微生物代谢过程中产生的CO 3 2-形成碳酸钙晶体，这些碳酸钙晶体填充土颗粒之间的缝隙，增加土体的密实程度以及土颗粒之间的摩擦和粘结，提高土壤的聚合。MICP技术可在沙化土壤表面形成可有效抵抗风、水侵蚀的硬壳。但在自然条件下单独使用碳酸盐矿化 菌固定荒漠化土壤时间较慢，且需使用大量的尿素为底物，限制了碳酸盐矿化菌在荒漠化土壤修复中的应用。
发明内容
因此，本发明要解决的技术问题在于克服现有MICP技术在自然条件下固定慢，需使用大量的尿素为底物的缺陷，从而提供一种双菌菌剂及在荒漠化土壤修复的应用。
为此，本发明采用如下技术方案：
本发明提供一种双菌菌剂，所述双菌菌剂包括碳酸盐矿化菌和产碳酸酐酶菌，所述双菌菌剂中碳酸盐矿化菌和产碳酸酐酶菌的活菌比为1-5：1。
本发明还提供上述双菌菌剂的制备方法，包括将碳酸盐矿化菌和产碳酸酐酶菌分别发酵得到菌液，然后将菌液混合均匀，既得所述双菌菌剂。
进一步地，所述碳酸盐矿化菌的培养方法为，挑取碳酸盐矿化菌单菌落至第一培养基，置于25-35℃200-250rpm条件下过夜培养活化菌体，得到过夜培养物；吸取过夜培养物加入其体积100-200倍的第一培养基中，再于25-35℃200-250rpm下培养24h，得到碳酸盐矿化菌菌液。
所述产碳酸酐酶菌的培养方法为，挑取产碳酸酐酶菌单菌落至第二培养基，置于25-35℃200-250rpm条件下过夜培养活化菌体，得到过夜培养物；吸取过夜培养物加入其体积100-200倍的第二培养基中，再于25-35℃200-250rpm下培养24h，得到产碳酸酐酶菌菌液。
优选地，所述第一培养基每升含酵母提取物20g、硫酸铵10g、Tris15.748g，用稀盐酸调整pH至9；
所述第二培养基每升含蛋白胨10g、酵母粉5g、氯化钠10g
进一步地，使用第一培养基将碳酸盐矿化菌菌液稀释至OD 600＝1，使用第二培养基将产碳酸酐酶菌菌液稀释至OD 600＝1，然后将碳酸盐矿化菌菌液和产碳酸酐酶菌菌液的按体积比为1-5：1混合均匀
本发明还提供上述双菌菌剂的应用，其应用于荒漠化土壤修复。
本发明还提供一种荒漠化土壤修复方法，包括如下步骤：
S1：将上述双菌菌剂与待修复土壤混合静置，得到第一混合土壤；
S2：向第一混合土壤中加入固定液，混合后得到第二混合土壤；
S3：向第二混合土壤中缓慢加入胶结液，混合后得到第三混合土壤；
S4：将步骤S1-S3重复进行1-5次，得到修复后土壤。
进一步地，步骤S2中所述固定液为氯化钙溶液，浓度为0.01M-0.05M；
步骤S3中所述胶结液为氯化钙和尿素的混合溶液，所述氯化钙和尿素的摩尔浓度相同，为0.5M-1.5M。
优选地，步骤S1中，所述双菌菌剂和待修复土壤的体积比为1-1.5:1，静置时间为2-5h；
步骤S2中，所述固定液和待修复土壤的体积比为0.1-0.3:1；
步骤S3中，所述胶结液和待修复土壤的体积比为5-10:1。
本发明技术方案，具有如下优点：
(1)本发明提供的双菌菌剂，其中的碳酸盐矿化菌可以大量分泌脲酶，脲酶可将环境中的尿素等分子分解，形成碳酸根离子，并通过其生长代谢为碳酸钙沉淀造成适合的碱性环境，同时微生物体带负电荷，会吸附带正电荷的钙离子等阳离子，引起Ca 2+富集，碳酸根离子与钙离子以碳酸盐矿化菌为结晶核形成碳酸钙、碳酸镁结晶，诱导形成的碳酸钙结晶在松散砂 砾周围和颗粒之间起到桥连作用，提高介质的力学性能；
产碳酸酐酶菌可加速CO 2在体系内的溶解速度，为碳酸盐矿化菌提供足量的碳酸根离子，利用了环境中二氧化碳的同时加速了碳酸盐矿化菌的效果。
(2)本发明将双菌菌剂应用于荒漠化土壤修复，相比于只用碳酸盐矿化菌进行荒漠化土壤固定化的过程缓慢，双菌体系固化大大提高了体系的固化速率，在相同条件下可以得到单菌体系强度2倍以上的土壤，产碳酸酐酶菌利用大气中和由碳酸盐矿化菌产生的部分二氧化碳为固化体系提供里碳酸根离子，与单菌固化相比，以CO 2为部分碳源，减少了CO 2的排放并降低了尿素的用量。
(3)本发明荒漠化土壤修复中，在胶结液前使用固定液，使微生物细胞固定到土壤颗粒表面，不容易被后续的胶结液冲掉。
(4)本发明提供的荒漠化土壤修复，无毒、无害、无二次污染，成本低，双菌菌剂制备简单、且固化过程中留在土壤里的尿素也使土壤更加肥沃。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明具体实施方式中双菌菌剂在土壤修复过程中的代谢关系图。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明，并不局限于所述最佳实施方式，不对本发明的内容和保护范围构成限制，任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品，均落在本发明的保护范围之内。
本发明使用的碳酸盐矿化菌为巴斯德芽孢杆菌，购自美国菌种保藏中心，编号ATCC 11859；
本发明使用的产碳酸酐酶菌为工程菌E-22b-IRLS，其制备方法记载于天津大学化工学院硕士毕业论文《细胞表面展示碳酸酐酶及其酶学性质研究》中。
实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
如图1所示，本发明提供的双菌菌剂应用于土壤修复过程时，碳酸盐矿化菌可以大量分泌脲酶，脲酶可将环境中的尿素等分子分解，形成碳酸根离子，并通过其生长代谢为碳酸钙沉淀造成适合的碱性环境，同时微生物体带负电荷，会吸附带正电荷的钙离子等阳离子，引起Ca 2+富集，碳酸根离子与钙离子以碳酸盐矿化菌为结晶核形成碳酸钙、碳酸镁结晶，诱导形成的碳酸钙结晶在松散砂砾周围和颗粒之间起到桥连作用，提高介质的力学性能；同时，产碳酸酐酶菌可加速CO 2在体系内的溶解速度，为碳酸盐矿化菌提供足量的碳酸根离子，在利用了环境中二氧化碳的同时加速了荒漠化土壤的固化速度。
实施例1
本实施例提供一种双菌菌剂，制备方法如下：
(1)制备单菌菌液：
挑取碳酸盐矿化菌单菌落至5mL第一培养基中，置于30℃220rpm条件下过夜培养活化菌体，得到过夜培养物；吸取过夜培养物至5瓶第一培养基中，每瓶100mL，置于30℃220rpm条件下培养24h，得到碳酸盐矿化菌菌液；所述第一培养基每升含酵母提取物20g、硫酸铵10g、Tris 15.748g，用稀盐酸调整pH至9；
挑取产碳酸酐酶菌单菌落至5mL第二培养基中，置于30℃220rpm条件下过夜培养活化菌体，得到过夜培养物；吸取过夜培养物至5瓶第二培养基中，每瓶100mL，置于30℃220rpm条件下培养24h，得到产碳酸酐酶菌菌液；所述第二培养基中每升含蛋白胨10g、酵母粉5g、氯化钠10g。
(2)制备双菌菌剂：
使用第一培养基将碳酸盐矿化菌菌液稀释至OD 600＝1，使用第二培养基将产碳酸酐酶菌菌液稀释至OD 600＝1，然后将碳酸盐矿化菌菌液和产碳酸酐酶菌菌液的按体积比为1：1混合均匀，既得双菌菌剂。
本发明还提供一种荒漠化土壤修复方法，包括如下步骤：
(1)取上述双菌菌剂与待修复土壤体积比为1:1混合静置，静置时间为3h，得到第一混合土壤；
(2)向第一混合土壤中加入0.05M氯化钙溶液，所述氯化钙溶液和待修复土壤的体积比为0.2:1，混合后得到第二混合土壤；
(3)向第二混合土壤中加入氯化钙和尿素浓度均为1M的混合溶液， 所述混合溶液和待修复土壤的体积比为8:1，混合后得到第三混合土壤；
(4)每48h重复一次步骤(1)-(3)，一共4次，得到修复后的荒漠化土壤。
实施例2
本实施例提供一种双菌菌剂，制备方法如下：
(1)制备单菌菌液：
挑取碳酸盐矿化菌单菌落至5mL第一培养基中，置于25℃250rpm条件下过夜培养活化菌体，得到过夜培养物；吸取过夜培养物至10瓶第一培养基中，每瓶100mL，置于25℃200rpm条件下培养24h，得到碳酸盐矿化菌菌液；所述第一培养基每升含酵母提取物20g、硫酸铵10g、Tris 15.748g，用稀盐酸调整pH至9；
挑取产碳酸酐酶菌单菌落至5mL第二培养基中，置于25℃200rpm条件下过夜培养活化菌体，得到过夜培养物；吸取过夜培养物至10瓶第二培养基中，每瓶100mL，置于25℃200rpm条件下培养24h，得到产碳酸酐酶菌菌液；所述第二培养基中每升含蛋白胨10g、酵母粉5g、氯化钠10g。
(2)制备双菌菌剂：
使用第一培养基将碳酸盐矿化菌菌液稀释至OD 600＝1，使用第二培养基将产碳酸酐酶菌菌液稀释至OD 600＝1，然后将碳酸盐矿化菌菌液和产碳酸酐酶菌菌液的按体积比为5：1混合均匀，既得双菌菌剂。
本发明还提供一种荒漠化土壤修复方法，包括如下步骤：
(1)取上述双菌菌剂与待修复土壤体积比为1.5:1混合静置静置时间为5h，得到第一混合土壤；
(2)向第一混合土壤中加入0.01M氯化钙溶液，所述氯化钙溶液和待修复土壤的体积比为0.3:1，混合后得到第二混合土壤；
(3)向第二混合土壤中加入氯化钙和尿素浓度均为0.5M的混合溶液，所述混合溶液和待修复土壤的体积比为10:1，混合后得到第三混合土壤；
(4)每48h重复一次步骤(1)-(3)，一共3次，得到修复后的荒漠化土壤。
实施例3
本实施例提供一种双菌菌剂，制备方法如下：
(1)制备单菌菌液：
挑取碳酸盐矿化菌单菌落至5mL第一培养基中，置于35℃200rpm条件下过夜培养活化菌体，得到过夜培养物；吸取过夜培养物至7瓶第一培养基中，每瓶100mL，置于35℃200rpm条件下培养24h，得到碳酸盐矿化菌菌液；所述第一培养基每升含酵母提取物20g、硫酸铵10g、Tris 15.748g，用稀盐酸调整pH至9；
挑取产碳酸酐酶菌单菌落至5mL第二培养基中，置于35℃200rpm条件下过夜培养活化菌体，得到过夜培养物；吸取过夜培养物至7瓶第二培养基中，每瓶100mL，置于35℃200rpm条件下培养24h，得到产碳酸酐酶菌菌液；所述第二培养基中每升含蛋白胨10g、酵母粉5g、氯化钠10g。
(2)制备双菌菌剂：
使用第一培养基将碳酸盐矿化菌菌液稀释至OD 600＝1，使用第二培养基将产碳酸酐酶菌菌液稀释至OD 600＝1，然后将碳酸盐矿化菌菌液和产碳酸酐酶菌菌液的按体积比为3：1混合均匀，既得双菌菌剂。
本发明还提供一种荒漠化土壤修复方法，包括如下步骤：
(1)取上述双菌菌剂与待修复土壤体积比为1:1混合静置，静置时间为2h，得到第一混合土壤；
(2)向第一混合土壤中加入0.04M氯化钙溶液，所述氯化钙溶液和待修复土壤的体积比为0.1:1，混合后得到第二混合土壤；
(3)向第二混合土壤中加入氯化钙和尿素浓度均为1.5M的混合溶液，所述混合溶液和待修复土壤的体积比为5:1，混合后得到第三混合土壤；
(4)每48h重复一次步骤(1)-(3)，一共5次，得到修复后的荒漠化土壤。
对比例1
本对比例1和实施例1相比，只使用碳酸盐矿化菌菌液，不使用产碳酸酐酶菌菌液，其余步骤和实施例1相同。
对比例2
本对比例2和实施例1相比，只使用产碳酸酐酶菌菌液，不使用产碳酸酐酶菌菌液，其余步骤和实施例1相同。
试验例
分别各取8个相同来源的待修复土壤试样，使用实施例1得到的双菌菌剂和对比例1、2得到的单菌菌剂作为修复试剂，按照本发明提供的荒漠化土壤修复方法进行修复实验，记为实验组、对比组1和对比组2，将其中荒漠化土壤修复方法步骤(1)-(3)记为一个批次的灌浆，每个试样每48h进行一批次灌浆，共计4次灌浆。开始灌浆后的第2天起每48h从实验组和对比组中各取两个试样检测其抗压强度和抗剪强度，取其结果的平均值， 结果如表1所示：
表1实验组和对比组实验结果
Figure PCTCN2021142592-appb-000001
由上表可知，在相同的灌浆条件下，实施例1和对比例1相比，在同样时间和尿素使用量的情况下，实施例1的抗压强度和抗剪强度均为对比例1的2倍以上，而对比例2中抗压强度和抗剪强度更是无法和实施例1进行比较。说明要得到相同强度的土壤，本申请实施例1中的技术方案和对比例相比，可以大幅节省时间，同时减少尿素的用量。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可 以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种双菌菌剂，其特征在于，所述双菌菌剂包括碳酸盐矿化菌和产碳酸酐酶菌，所述双菌菌剂中碳酸盐矿化菌和产碳酸酐酶菌的活菌比为1-5：1。
  2. 权利要求1所述的双菌菌剂的制备方法，其特征在于，包括将碳酸盐矿化菌和产碳酸酐酶菌分别发酵得到菌液，然后将菌液混合均匀，既得所述双菌菌剂。
  3. 根据权利要求2所述的制备方法，其特征在于，所述碳酸盐矿化菌的培养方法为，挑取碳酸盐矿化菌单菌落至第一培养基，置于25-35℃ 200-250rpm条件下过夜培养活化菌体，得到过夜培养物；吸取过夜培养物加入其体积100-200倍的第一培养基中，再于25-35℃ 200-250rpm下培养24h，得到碳酸盐矿化菌菌液。
  4. 根据权利要求3所述的制备方法，其特征在于，所述产碳酸酐酶菌的培养方法为，挑取产碳酸酐酶菌单菌落至第二培养基，置于25-35℃ 200-250rpm条件下过夜培养活化菌体，得到过夜培养物；吸取过夜培养物加入其体积100-200倍的第二培养基中，再于25-35℃ 200-250rpm下培养24h，得到产碳酸酐酶菌菌液。
  5. 根据权利要求4所述的制备方法，其特征在于，所述第一培养基每升含酵母提取物20g、硫酸铵10g、Tris 15.748g，用稀盐酸调整pH至9；
    所述第二培养基每升含蛋白胨10g、酵母粉5g、氯化钠10g。
  6. 根据权利要求5所述的制备方法，其特征在于，使用第一培养基将碳酸盐矿化菌菌液稀释至OD 600＝1，使用第二培养基将产碳酸酐酶菌菌液稀 释至OD 600＝1，然后将碳酸盐矿化菌菌液和产碳酸酐酶菌菌液的按体积比为1-5：1混合均匀。
  7. 权利要求1所述的双菌菌剂或权利要求2-6任一项所述的制备方法制得的双菌菌剂的应用，其特征在于，应用于荒漠化土壤修复。
  8. 一种荒漠化土壤修复方法，其特征在于，包括如下步骤：
    S1：将权利要求1所述的双菌菌剂或权利要求2-6任一项所述的制备方法制得的双菌菌剂与待修复土壤混合静置，得到第一混合土壤；
    S2：向第一混合土壤中加入固定液，混合后得到第二混合土壤；
    S3：向第二混合土壤中加入胶结液，混合后得到第三混合土壤；
    S4：将步骤S1-S3重复进行1-5次，得到修复后土壤。
  9. 根据权利要求8所述的修复方法，其特征在于，步骤S2中所述固定液为氯化钙溶液，浓度为0.01M～0.05M；
    步骤S3中所述胶结液为氯化钙和尿素的混合溶液，所述氯化钙和尿素的摩尔浓度相同，为0.5M～1.5M。
  10. 根据权利要求8或9所述的修复方法，其特征在于，步骤S1中，所述双菌菌剂和待修复土壤的体积比为1-1.5:1，静置时间为2-5h；
    步骤S2中，所述固定液和待修复土壤的体积比为0.1-0.3:1；
    步骤S3中，所述胶结液和待修复土壤的体积比为5-10:1。
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CN108220380A (zh) * 2018-02-08 2018-06-29 天津科技大学 利用两种微生物共同矿化制备碳酸钙的方法
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CN108220380A (zh) * 2018-02-08 2018-06-29 天津科技大学 利用两种微生物共同矿化制备碳酸钙的方法
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