KR102616660B1 - Microbial agent available as spontaneous crack healing agent and spontaneous crack healing agent using same - Google Patents
Microbial agent available as spontaneous crack healing agent and spontaneous crack healing agent using same Download PDFInfo
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- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 29
- 230000000813 microbial effect Effects 0.000 title claims abstract description 12
- 230000002269 spontaneous effect Effects 0.000 title description 8
- 239000013003 healing agent Substances 0.000 title description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims abstract description 82
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 50
- 229910000019 calcium carbonate Inorganic materials 0.000 claims abstract description 41
- 230000015572 biosynthetic process Effects 0.000 claims description 16
- 102000016943 Muramidase Human genes 0.000 claims description 6
- 108010014251 Muramidase Proteins 0.000 claims description 6
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 6
- 239000004615 ingredient Substances 0.000 claims description 6
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
- C04B—LIME, MAGNESIA; SLAG; CEMENTS; COMPOSITIONS THEREOF, e.g. MORTARS, CONCRETE OR LIKE BUILDING MATERIALS; ARTIFICIAL STONE; CERAMICS; REFRACTORIES; TREATMENT OF NATURAL STONE
- C04B24/00—Use of organic materials as active ingredients for mortars, concrete or artificial stone, e.g. plasticisers
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- C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
- C04B—LIME, MAGNESIA; SLAG; CEMENTS; COMPOSITIONS THEREOF, e.g. MORTARS, CONCRETE OR LIKE BUILDING MATERIALS; ARTIFICIAL STONE; CERAMICS; REFRACTORIES; TREATMENT OF NATURAL STONE
- C04B40/00—Processes, in general, for influencing or modifying the properties of mortars, concrete or artificial stone compositions, e.g. their setting or hardening ability
- C04B40/06—Inhibiting the setting, e.g. mortars of the deferred action type containing water in breakable containers ; Inhibiting the action of active ingredients
- C04B40/0633—Chemical separation of ingredients, e.g. slowly soluble activator
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- C04B2103/0001—Living organisms, e.g. microorganisms, or enzymes
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract
본 발명은 탄산칼슘을 생성하여 자발적으로 콘크리트의 균열을 치유할 수 있는 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)를 포함하는 미생물 제제에 관한 것이다. The present invention relates to a microbial agent containing Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05 strain (KACC 92401P), which can spontaneously heal cracks in concrete by producing calcium carbonate.
Description
본 발명은 자발적 균열 치유제로 이용가능한 미생물 제제 및 이를 이용한 자발적 균열 치유제에 관한 것이다. The present invention relates to a microbial agent that can be used as a spontaneous crack healing agent and a spontaneous crack healing agent using the same.
콘크리트는 건축 등 다양한 구조물을 만들기 위한 필수불가결한 재료이다. 콘크리트는 다루기가 쉽고 강도가 높고, 수명도 60 ~ 100 년으로 매우 길다는 장점이 있다. 하지만 콘크리트는 균열이 발생하기 쉽다는 단점이 있으며, 콘크리트에 균열이 발생할 경우 구조물의 내구성이 급격히 감소하게 되어 심각한 문제를 유발할 수 있다. Concrete is an essential material for making various structures such as buildings. Concrete has the advantage of being easy to handle, having high strength, and having a very long lifespan of 60 to 100 years. However, concrete has the disadvantage of being prone to cracks, and when cracks occur in concrete, the durability of the structure is drastically reduced, which can cause serious problems.
콘크리트에 발생한 균열을 치유하기 위해 탄산칼슘 형성능을 가지는 미생물을 보수제로 사용하는 기술들이 알려져 있다. 하지만 탄산칼슘을 형성하는 미생물은 많이 알려져 있지만, 탄산칼슘을 형성하는 양이 부족하여 제대로 상용화가 되지 못하고 있는 실정이다. There are known technologies that use microorganisms with the ability to form calcium carbonate as a repair agent to heal cracks in concrete. However, although many microorganisms that form calcium carbonate are known, they are not properly commercialized due to the insufficient amount of calcium carbonate formed.
따라서 상용화할 수 있을 정도로 뛰어난 탄산칼슘 형성이 가능한 신규의 미생물이 필요하다. Therefore, a new microorganism capable of forming calcium carbonate excellent enough to be commercialized is needed.
본 발명의 일 목적은 탄산칼슘 형성능이 뛰어난 신규의 미생물을 포함하여 자발적 균열 치유제로 이용가능한 미생물 제제를 제공하는 것이다. One object of the present invention is to provide a microbial preparation that can be used as a spontaneous crack healing agent, including a novel microorganism with excellent calcium carbonate forming ability.
본 발명의 또 다른 목적은 탄산칼슘을 형성할 수 있는 신규의 미생물의 엔도스포어(endospore)를 포함하는 균열 치유제를 제공하는 것이다. Another object of the present invention is to provide a crack healing agent containing novel microbial endospores capable of forming calcium carbonate.
한편, 본 발명의 명시되지 않은 또 다른 목적들은 하기의 상세한 설명 및 그 효과로부터 용이하게 추론할 수 있는 범위 내에서 추가적으로 고려될 것이다.Meanwhile, other unspecified purposes of the present invention will be additionally considered within the scope that can be easily inferred from the following detailed description and its effects.
이상에서 설명한 문제를 해결하기 위해 다음과 같은 해결수단을 제안한다. To solve the problems described above, we propose the following solutions.
본 발명의 일 실시예에 따른 미생물 제제는 탄산칼슘을 생성하여 자발적으로 콘크리트의 균열을 치유할 수 있는 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)를 포함한다.The microbial agent according to an embodiment of the present invention includes Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05 strain (KACC 92401P), which can spontaneously heal cracks in concrete by producing calcium carbonate.
일 예에 있어서, 상기 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)가 엔도소포어(endospore) 상태인 것을 특징으로 할 수 있다. In one example, the Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05 strain (KACC 92401P) may be characterized as being in an endospore state.
본 발명의 다른 실시예에 따른 균열 치료제는 탄산칼슘을 생성하여 자발적으로 콘크리트의 균열을 치유할 수 있는 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)를 포함한다. A crack treatment agent according to another embodiment of the present invention includes Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05 strain (KACC 92401P), which can spontaneously heal cracks in concrete by producing calcium carbonate.
다른 실시예에 있어서, 상기 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)가 엔도소포어(endospore) 상태인 것을 특징으로 할 수 있다. In another embodiment, the Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05 strain (KACC 92401P) may be characterized as being in an endospore state.
다른 실시예에 있어서, 상기 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)의 생장 및 탄산칼슘 형성에 필요한 성분을 더 포함할 수 있다. In another embodiment, components necessary for the growth and calcium carbonate formation of the Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05 strain (KACC 92401P) may be further included.
본 발명의 또 다른 실시예에 따른 균열 치료제를 제조하는 방법은 탄산칼슘을 생성하여 자발적으로 콘크리트의 균열을 치유할 수 있는 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)에 대하여 lysozyme 처리 및 열처리하여 영양세포를 분해하고 다른 균주를 사멸시킴으로써 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)의 엔도소포어를 포함하는 것을 특징으로 한다. A method for manufacturing a crack treatment agent according to another embodiment of the present invention is for Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05 strain (KACC 92401P), which can spontaneously heal cracks in concrete by producing calcium carbonate. It is characterized by containing endosophores of Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05 strain (KACC 92401P) by decomposing vegetative cells and killing other strains through lysozyme treatment and heat treatment.
본 발명의 일 실시예에 따른 자발적 균열 치유제로 이용가능한 미생물 제제는 다른 Bacillus 균주 대비 탄산칼슘 형성능이 50 ~ 100% 높은 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)를 포함한다. Microbial preparations that can be used as a spontaneous crack healing agent according to an embodiment of the present invention include Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05 strain (KACC 92401P), which has 50 to 100% higher calcium carbonate forming ability than other Bacillus strains. .
본 발명의 다른 실시예에 따른 자발적 균열 치료제는 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)를 포함하며, 탄산칼슘 형성능이 뛰어난 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)에 의해 콘크리트의 균열을 치유할 수 있다는 효과가 있다. A treatment agent for spontaneous cracking according to another embodiment of the present invention includes Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05 strain (KACC 92401P), and Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05, which has excellent calcium carbonate forming ability. The strain (KACC 92401P) has the effect of healing cracks in concrete.
또한, 본 발명의 다른 실시예에 따른 자발적 균열 치료제는 Bacillus velezensis MCE03 균주(KACC 92400P) 또는 Bacillus subtilis MCE05 균주(KACC 92401P)의 엔도소포어를 이용함으로써 콘크리트 구조물을 제작한 후 오랜 시간이 지난 후에 발생한 균열도 자가 치유할 수 있다. In addition, the spontaneous crack treatment according to another embodiment of the present invention uses endospores of Bacillus velezensis MCE03 strain (KACC 92400P) or Bacillus subtilis MCE05 strain (KACC 92401P) to prevent cracks that occur a long time after manufacturing a concrete structure. Cracks can also heal themselves.
한편, 여기에서 명시적으로 언급되지 않은 효과라 하더라도, 본 발명의 기술적 특징에 의해 기대되는 이하의 명세서에서 기재된 효과 및 그 잠정적인 효과는 본 발명의 명세서에 기재된 것과 같이 취급됨을 첨언한다.Meanwhile, it is to be added that even if the effects are not explicitly mentioned herein, the effects described in the following specification and their potential effects expected from the technical features of the present invention are treated as if described in the specification of the present invention.
도 1a는 Bacillus velezensis MCE03 균주의 계통수이며, 도 1b는 Bacillus subtilis MCE05 균주의 계통수이다.
도 2는 시료에서 분리하여 영양배지에 배양한 여러가지 종류의 Bacillus 균주를 촬영한 사잔이다.
도 3으 탄산칼슘 형성 고체 배지에서 콜로니 주변으로 탄산칼슘을 형성한 여러가지 종류의 Bacillus 균주를 촬영한 사잔이다.
도 4는 여러가지 종류의 Bacillus 균주에 대한 Urease 테스트 결과를 촬영한 사진이다.
도 5는 여러가지 종류의 Bacillus 균주에 의해 형성된 탄산칼슘의 양을 측정하는 과정을 촬영한 사진이다.
도 6은 Bacillus velezensis MCE03 균주(KACC 92400P) 및 Bacillus subtilis MCE05 균주(KACC 92401P)의 엔도소포어 형성을 촬영한 사진이며, 배양시간에 따른 엔도소포어 형성률 그래프를 도시한 것이다.
도 7a는 Bacillus velezensis MCE03 균주(KACC 92400P) 및 Bacillus subtilis MCE05 균주(KACC 92401P)의 엔도소포어의 몰탈 내부에서의 생존 테스트 결과를 촬영한 것이며, 도7 b는 생존 테스트 결과에서 확인한 엔도소포어가 영양배지에서 영양세포로 분화하여 콜로니를 형성하는 것을 촬영한 것이다.
도 8은 Bacillus velezensis MCE03 균주(KACC 92400P) 및 Bacillus subtilis MCE05 균주(KACC 92401P)의 엔도소포어를 포함하는 균열치료제를 몰탈 자체에 첨가한 후 균열을 유발한 후에 누수 조건 하에서 균열치유 여부를 실험한 결과를 촬영한 것이다.
첨부된 도면은 본 발명의 기술사상에 대한 이해를 위하여 참조로서 예시된 것임을 밝히며, 그것에 의해 본 발명의 권리범위가 제한되지는 아니한다.Figure 1a is a phylogenetic tree of the Bacillus velezensis MCE03 strain, and Figure 1b is a phylogenetic tree of the Bacillus subtilis MCE05 strain.
Figure 2 is a photograph of various types of Bacillus strains isolated from samples and cultured on nutrient medium.
Figure 3 is a photograph of various types of Bacillus strains that formed calcium carbonate around colonies on a calcium carbonate forming solid medium.
Figure 4 is a photograph of the urease test results for various types of Bacillus strains.
Figure 5 is a photograph of the process of measuring the amount of calcium carbonate formed by various types of Bacillus strains.
Figure 6 is a photograph of endosophore formation in Bacillus velezensis MCE03 strain (KACC 92400P) and Bacillus subtilis MCE05 strain (KACC 92401P), and shows a graph of endosophore formation rate according to culture time.
Figure 7a shows the results of a survival test inside the mortar of endosophores of Bacillus velezensis MCE03 strain (KACC 92400P) and Bacillus subtilis MCE05 strain (KACC 92401P), and Figure 7b shows that the endospores confirmed in the survival test results are nutritional. This was filmed as they differentiate into vegetative cells in the medium and form colonies.
Figure 8 shows a test for crack healing under water leakage conditions after adding a crack treatment agent containing endosophores of Bacillus velezensis MCE03 strain (KACC 92400P) and Bacillus subtilis MCE05 strain (KACC 92401P) to the mortar itself to induce cracks. The results were filmed.
The attached drawings are intended as reference for understanding the technical idea of the present invention, and are not intended to limit the scope of the present invention.
이하, 도면을 참조하여 본 발명의 다양한 실시예가 안내하는 본 발명의 구성과 그 구성으로부터 비롯되는 효과에 대해 살펴본다. 본 발명을 설명함에 있어서 관련된 공지기능에 대하여 이 분야의 기술자에게 자명한 사항으로서 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략한다.Hereinafter, with reference to the drawings, we will look at the configuration of the present invention guided by various embodiments of the present invention and the effects resulting from the configuration. In describing the present invention, if it is determined that related known functions may unnecessarily obscure the gist of the present invention as they are obvious to those skilled in the art, the detailed description thereof will be omitted.
본 발명은 탄산칼슘 형성능이 뛰어나며, 동시에 엔도소포어 형성률이 높은 신규한 미생물에 관한 것이다. The present invention relates to a novel microorganism that has excellent calcium carbonate forming ability and at the same time has a high endosophore formation rate.
본 발명의 신규한 균주는 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주이다. The new strains of the present invention are Bacillus velezensis MCE03 strain and Bacillus subtilis MCE05 strain.
Bacillus velezensis MCE03 균주는 KACC(Korean Agricultural Culture Collection)에 서열번호(들)의 정보를 KACC 92400P로 등록하였으며, Bacillus subtilis MCE05 균주는 KACC에 서열번호(들)의 정보를 KACC 92401P로 등록하였다. For the Bacillus velezensis MCE03 strain, the sequence number(s) information was registered with KACC (Korean Agricultural Culture Collection) as KACC 92400P, and for the Bacillus subtilis MCE05 strain, the sequence number(s) information was registered with KACC as KACC 92401P.
도 1a는 Bacillus velezensis MCE03 균주의 계통수이며, 도 1b는 Bacillus subtilis MCE05 균주의 계통수이다. Figure 1a is a phylogenetic tree of the Bacillus velezensis MCE03 strain, and Figure 1b is a phylogenetic tree of the Bacillus subtilis MCE05 strain.
확보한 균주는 16s rRNA 분석을 수행하였으며, Bacillus velezensis MCE03 균주는 Bacillus velezensis 균주와 99% 유사한 신규한 균주이며, Bacillus subtilis MCE05 균주는 Bacillus subtilis 균주와 99% 유사한 신규한 균주임을 확인하였다. 16s rRNA analysis was performed on the obtained strain, and it was confirmed that the Bacillus velezensis MCE03 strain is a new strain that is 99% similar to the Bacillus velezensis strain, and the Bacillus subtilis MCE05 strain is a new strain that is 99% similar to the Bacillus subtilis strain.
각 균주의 서열은 다음과 같다. The sequence of each strain is as follows.
미생물을 콘크리트의 균열치료제로 이용하는 방법은 균열치료제를 적용하는 시점에 따라 2가지로 구분할 수 있다. Methods of using microorganisms as a crack treatment agent for concrete can be divided into two types depending on the time of application of the crack treatment agent.
첫번째는 균열이 발생한 콘크리트에 사후적으로 균열치료제를 적용하는 것이고, 두번째는 콘크리트 조성물에 첨가제 형태로 균열치료제를 적용하는 것이다. The first is to apply a crack treatment agent ex post to cracked concrete, and the second is to apply a crack treatment agent in the form of an additive to the concrete composition.
이처럼 미생물을 콘크리트의 균열치료제로 이용하기 위해서는 알칼리 환경에서 미생물의 생장이 가능하여야 하며, 무엇보다 탄산칼슘 형성능이 뛰어나야 한다. In order to use microorganisms as a crack treatment agent for concrete, the microorganisms must be able to grow in an alkaline environment and, above all, must have an excellent ability to form calcium carbonate.
또한, 균열이 발생한 콘크리트에 사후적으로 균열치료제를 적용하는 보수제 형태가 아닌, 콘크리트 조성물에 첨가제 형태로 균열치료제를 적용하기 위해서는 가혹한 환경에서 견딜 수 있는 엔도소포어 형성률이 높아야만 한다. In addition, in order to apply a crack treatment agent in the form of an additive to a concrete composition, rather than in the form of a repair agent that is applied ex post to cracked concrete, the endosophore formation rate that can withstand harsh environments must be high.
본 발명에서 제시하는 신규한 균주는 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주는 위와 같은 요건을 들을 모두 만족하기 때문에 첫번째 형태와 같이 콘크리트에 균열이 발생 후에 사후적으로 적용가능함은 물론, 콘크리트 조성물에 첨가제 형태로 적용하는 것도 가능하다. The new strains presented in the present invention, Bacillus velezensis MCE03 strain and Bacillus subtilis MCE05 strain, satisfy all of the above requirements, so they can be applied ex post after a crack occurs in concrete like the first type, and can also be applied as an additive to the concrete composition. It is also possible to apply it in the form.
콘크리트 조성물에 첨가제 형태로 본 발명의 균열치료제를 적용할 경우 균열이 발생하기 전까지 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주가 엔도소포어로 존재하다가, 균열이 발생하여 그 균열로 물이 침투할 경우 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주가 깨어나 탄산칼슘을 형성하여 균열을 치유하게 된다. When applying the crack treatment agent of the present invention in the form of an additive to a concrete composition , Bacillus velezensis MCE03 and Bacillus subtilis MCE05 strains exist as endospores until cracks occur, and when cracks occur and water penetrates into the cracks, Bacillus velezensis The MCE03 strain and the Bacillus subtilis MCE05 strain wake up and form calcium carbonate to heal the crack.
특히, Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주는 탄산칼슘 형성능이 뛰어나기 때문에 균열 발생후 2일 이내에 간격 0.5 mm 수준의 균열을 치유할 수 있다. In particular, the Bacillus velezensis MCE03 strain and Bacillus subtilis MCE05 strain have excellent calcium carbonate forming ability, so they can heal cracks with a spacing of 0.5 mm within 2 days after crack occurrence.
한편, 콘크리트에서 발생하는 균열은 짧게는 양생후 수일 이내에 발생하기도 하지만, 길게는 몇 년 후에 발생하는 경우도 있다. Meanwhile, cracks that occur in concrete may occur within a few days after curing, but in some cases, they may occur after several years.
본 발명의 균열치료제는 lysozyme 처리와 고온 열처리를 통해 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주의 엔도소포어 외에 영양세포 및 다른 균주를 제거하게 된다. The crack treatment agent of the present invention removes vegetative cells and other strains in addition to endospores of Bacillus velezensis MCE03 and Bacillus subtilis MCE05 strains through lysozyme treatment and high-temperature heat treatment.
lysozyme 처리와 고온 열처리를 통해 본 발명의 균열 치료제는 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주의 생장 및 탄산칼슘 형성에 필요한 성분이 영양세포 또는 다른 균주에 의해 소모되는 것을 방지함으로써, 수년후에 콘크리트에 균열이 발생한 경우에도 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주가 깨어나 탄산칼슘을 형성할 수 있게 된다. Through lysozyme treatment and high-temperature heat treatment, the crack treatment agent of the present invention prevents the ingredients required for the growth and calcium carbonate formation of Bacillus velezensis MCE03 and Bacillus subtilis MCE05 strains from being consumed by vegetative cells or other strains, preventing cracks in concrete after several years. Even in this case, the Bacillus velezensis MCE03 strain and Bacillus subtilis MCE05 strain can wake up and form calcium carbonate.
Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주의 생장 및 탄산칼슘 형성에 필요한 성분으로는 yeast extract, calcium ion, NaCl, Amino acid, Sodium alginate, LB broth로 이루어진 군에서 선택되는 적어도 어느 하나일 수 있으며, 이 성분을 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주의 엔도소포어와 함께 균열치료제로 포함할 수 있다. The ingredients required for the growth and calcium carbonate formation of Bacillus velezensis MCE03 and Bacillus subtilis MCE05 strains may be at least one selected from the group consisting of yeast extract, calcium ion, NaCl, amino acid, sodium alginate, and LB broth. The ingredient can be included as a crack treatment agent along with endospores of Bacillus velezensis MCE03 strain and Bacillus subtilis MCE05 strain.
Bacillus velezensis MCE03 균주는 후술하는 바와 같이 Urease를 보유하고 있는바, 균주의 생장 및 탄산칼슘 형성에 필요한 성분으로 Urea를 더 포함할 수 있다. As described later, the Bacillus velezensis MCE03 strain possesses urease, and may further contain urea as a necessary ingredient for the growth of the strain and the formation of calcium carbonate.
균열치료제에 포함되는 균주의 생장 및 탄산칼슘 형성에 필요한 성분의 함량은 각 균주의 생장조건에 맞춰 적절히 선택될 수 있다. The content of ingredients required for the growth of strains and the formation of calcium carbonate included in the crack treatment agent can be appropriately selected according to the growth conditions of each strain.
아래에서는 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주에 대해서 보다 더 상세하게 설명하도록 한다. Below, the Bacillus velezensis MCE03 strain and Bacillus subtilis MCE05 strain will be described in more detail.
먼지, 흙, 메주가루 등의 시료에서 Bacillus 균주를 분리했다. 시료에서 Bacillus 균주를 분리하는 것은 알칼리성(pH 9 ~ 12) 완충용액에서 30분 ~ 120분 동안 150 ~ 200 rpm으로 진탕 배양하여 수행될 수 있으며, 여기서는 시료 1g(흙, 메주가루 등)을 완충용액(0.1M Tris-HCl pH 9.0) 100ml에 1시간동안 상온에서 200rpm으로 진탕배양을 실시하였다. Bacillus strains were isolated from samples such as dust, dirt, and meju powder. Isolation of Bacillus strains from samples can be performed by shaking and culturing at 150 to 200 rpm for 30 to 120 minutes in an alkaline (pH 9 to 12) buffer solution, where 1 g of sample (soil, meju powder, etc.) is mixed with the buffer solution. Culture was performed in 100 ml of (0.1M Tris-HCl pH 9.0) for 1 hour at room temperature with shaking at 200 rpm.
배양 후 20 ~ 30분간 이물질을 가라앉힌다. After incubation, allow foreign substances to settle for 20 to 30 minutes.
이물질이 가라앉은 후 상층액을 채취하여 90 ~ 100 ℃에서 5 ~ 10분간 열처리를 할 수 있으며, 여기서는 상층액 채취한 후 끓는 물에 10분간 중탕하여 열처리를 수행하였다. After the foreign matter has settled, the supernatant can be collected and heat treated at 90 to 100°C for 5 to 10 minutes. In this case, the supernatant was collected and then heat treated by boiling in boiling water for 10 minutes.
상층액을 희석시켜 영양배지에 30
미생물 콜로니에 대해 염기서열 분석을 수행하여 엔도소포어(Endospore)를 형성할 수 있는 Bacillus 균주를 선별하였다. Base sequence analysis was performed on microbial colonies to select Bacillus strains capable of forming endospores.
다음으로 선별한 Bacillus 균주를 탄산칼슘 형성을 위한 고체배지에 접종하여 30 ℃에서 5 ~ 10일간 배양하였다.Next, the selected Bacillus strains were inoculated onto a solid medium for calcium carbonate formation and cultured at 30°C for 5 to 10 days.
도 3에서 보는 바와 같이 콜로니 주변으로 흰색의 탄산칼슘이 축적되어 선별한 모든 Bacillus 균주가 탄산칼슘 형성능을 가짐을 확인하였다.As shown in Figure 3, white calcium carbonate accumulated around the colonies, confirming that all selected Bacillus strains had the ability to form calcium carbonate.
한편, 탄산칼슘형성이 확인된 균주들의 urease 보유 여부를 파악하기 위해 urease 실험 배지에 균주를 접종하여 30
그 결과 도 4에서 보는 바와 같이 urease를 갖고 있는 균주인 Bacillus velezensis MCE03 균주의 배지에서는 노란색 배지가 분홍색으로 변하게 되는 것을 확인하여, urease를 가지고 있음을 확인하였다. As a result, as shown in Figure 4, it was confirmed that the yellow medium changed to pink in the medium of the Bacillus velezensis MCE03 strain, which is a strain containing urease, confirming that it contains urease.
위에서 선별한 6가지 Bacillus 균주의 탄산칼슘 형성능을 측정하였다. The calcium carbonate forming ability of the six Bacillus strains selected above was measured.
선별한 선별한 6가지 Bacillus 균주의 탄산칼슘형성 액체배지(urease 보유 균주는 urea를 첨가하여 배지 제작)에 접종하여 30 ℃, 200 rpm 조건으로 60시간동안 진탕배양을 실시하였다. Six selected Bacillus strains were inoculated into calcium carbonate-forming liquid medium (urea was added for urease-possessing strains) and cultured with shaking at 30°C and 200 rpm for 60 hours.
순수한 탄산칼슘만을 분리하기 위해 lysozyme 처리를 통해 배양액 내 영양세포를 분해하고 원심분리를 수행하였다. To isolate only pure calcium carbonate, vegetative cells in the culture medium were decomposed through lysozyme treatment and centrifugation was performed.
상대적으로 무거운 탄산칼슘은 침전물이 되어 가라앉고 세포 잔해물이나 배지 성분은 상층액에 부유하게 된다. The relatively heavy calcium carbonate settles as a precipitate, and cell debris and medium components float in the supernatant.
원심분리 수행 후 상층액을 버리고 침전물을 증류수에 풀어준 뒤 동일한 조건으로 원심분리를 돌리는 방식으로 2 ~ 5회 세척을 실시하여 이물질을 최대한 제거하였다. After centrifugation, the supernatant was discarded, the precipitate was dissolved in distilled water, and the mixture was washed 2 to 5 times by centrifuging under the same conditions to remove as much contaminants as possible.
세척이 끝나면 도 5의 사진처럼 흰색 침전물이 형성되고, 이를 건조시켜 미세저울로 무게를 측정하여 탄산칼슘 형성량을 측정하였다. After washing, a white precipitate was formed as shown in the photo in Figure 5, which was dried and weighed on a microbalance to measure the amount of calcium carbonate formed.
6가지 Bacillus 균주의 탄산칼슘 형성량은 다음의 표 3 과 같다. The amount of calcium carbonate formed by the six Bacillus strains is shown in Table 3 below.
표 3에서 보는 바와 같이, 본 발명의 미생물 제제로 이용하는 실시예 1 및 2의 균주는 탄산칼슘 형성량이 배양액 100 ml 당 60 mg 이상으로써 비교예 2 ~ 5에 기재된 다른 Bacillus 균주에 비해 50 ~ 100% 이상 높은 것을 확인할 수 있다. As shown in Table 3, the strains of Examples 1 and 2 used as microbial preparations of the present invention have a calcium carbonate formation amount of 60 mg or more per 100 ml of culture medium, which is 50 to 100% compared to other Bacillus strains described in Comparative Examples 2 to 5. You can see that it is higher than above.
이와 같은 뛰어난 탄산칼슘 형성능을 가지는 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주를 포자형성 배지에서 배양하여 엔도소포어 형성률을 측정하여 그 결과를 도 5에 표시하였다. Bacillus velezensis MCE03 and Bacillus subtilis MCE05 strains, which have such excellent calcium carbonate forming ability, were cultured in spore formation medium, and the endosophore formation rate was measured, and the results are shown in Figure 5.
도 6에서 확인할 수 있는 바와 같이, Bacillus velezensis MCE03 균주의 경우 48 시간 경과 후 약 90 % 수준의 엔도소포어 형성률을 나타냈으며, Bacillus subtilis MCE05 균주는 72시간 경과 후 약 90% 수준의 엔도소포어 형성률을 나타내었다. As can be seen in Figure 6, the Bacillus velezensis MCE03 strain showed an endosophore formation rate of about 90% after 48 hours, and the Bacillus subtilis MCE05 strain showed an endosophore formation rate of about 90% after 72 hours. indicated.
포자형성 배지에서 엔도소포어를 형성시킨 후 영양세포 및 기타 균주를 사멸시켜 엔도소포어를 수득하게 된다. After forming endospores in a sporulation medium, endospores are obtained by killing vegetative cells and other strains.
즉, Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주의 엔도소포어 외에 영양세포와 다른 균주를 사멸시키기 위해서 lysozyme 처리를 진행하였으며, 고온 열처리(75 ℃, 10분)를 수행하였다. That is, lysozyme treatment was performed to kill vegetative cells and other strains in addition to the endosophores of Bacillus velezensis MCE03 and Bacillus subtilis MCE05 strains, and high-temperature heat treatment (75°C, 10 minutes) was performed.
이후 원심분리를 통해 세포 잔해물, 배지성분 및 기타 이물질을 제거하여 순도 높은 엔도소포어를 획득하였다. Afterwards, cell debris, media components, and other foreign substances were removed through centrifugation to obtain highly pure endospores.
Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주가 실제 콘크리트에 대해 내성과 균열치유 성능을 가지는지 여부를 확인하기 위한 실험을 수행하였다. An experiment was conducted to determine whether the Bacillus velezensis MCE03 strain and Bacillus subtilis MCE05 strain had resistance and crack healing performance against actual concrete.
세포 잔해물, 배지성분 및 기타 이물질을 제거하여 획득한 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주의 엔도소포어를 원형 시멘트 파레트의 제작과정에 첨가하여 경화시켰다. Endosophores of Bacillus velezensis MCE03 and Bacillus subtilis MCE05 strains, obtained by removing cell debris, medium components, and other foreign substances, were added to the manufacturing process of a circular cement pallet and hardened.
경화된 후 24시간 간격으로 몰탈의 일부를 채취하여 완충용액(1x PBS pH 7.4)에 상온에서 200rpm으로 1시간동안 진탕 배양하고 현미경으로 관찰하고 영양배지에 접종하여 배양하였다. After curing, a portion of the mortar was collected at 24-hour intervals, incubated in a buffer solution (1x PBS pH 7.4) at room temperature with shaking at 200 rpm for 1 hour, observed under a microscope, and inoculated into a nutrient medium for cultivation.
그 결과 도 7a에서 보는 바와 같이 엔도소포어가 온전한 상태로 생존하고 있는 것을 확인할 수 있었다. As a result, as shown in Figure 7a, it was confirmed that the endosophore was surviving in an intact state.
생존을 확인한 엔도소포어는 도 7b에서 보는 바와 같이 영양배지에서 영양세포로 분화하여 콜로니를 형성하였다. Endosophores whose survival was confirmed differentiated into vegetative cells in the nutrient medium to form colonies, as shown in Figure 7b.
즉, Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주는 시멘트가 경화되는 환경에서 대해 내성을 가지고 있음을 확인할 수 있었다. In other words, it was confirmed that Bacillus velezensis MCE03 strain and Bacillus subtilis MCE05 strain were resistant to the environment where cement hardens.
마지막으로 세포 잔해물, 배지성분 및 기타 이물질을 제거하여 획득한 Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주의 엔도소포어를 하이드로젤 형태로 제작하여 균열치료제를 형성하였다. Finally, endospores of Bacillus velezensis MCE03 and Bacillus subtilis MCE05 strains obtained by removing cell debris, medium components, and other foreign substances were fabricated in the form of hydrogel to form a crack treatment agent.
먼저, 제1액을 준비하였다. 제1액은 alginate solution (1 L)이며, 5 ~ 20 g {0.5 ~ 2 %[weight(kg)/volume(L)]의 sodium alginate}, 2.5 g의 LB broth를 증류수 1,000.00 g과 혼합하여 제조하였으며, 여기에 엔도소포어가 후술하는 몰탈 1 mm3 당 1 X 107개 포함되도록 하였다. First, the first liquid was prepared. The first solution is alginate solution (1 L), prepared by mixing 5 to 20 g {0.5 to 2 %[weight(kg)/volume(L)] of sodium alginate} and 2.5 g of LB broth with 1,000.00 g of distilled water. The endospores were included in an amount of 1
다음으로 제2액인 Calcium solution으로서 0.01~0.05M의 Calcium ion을 증류수 1.000.00 g과 혼합하여 제조하였다. Next, the second liquid, Calcium solution, was prepared by mixing 0.01~0.05M Calcium ion with 1.000.00 g of distilled water.
제조한 제1액을 제2액에 방울 형태로 떨어트려 방울 형태의 하이드로젤 입자를 제조하였으며, 이를 세척 및 건조하여 균열치료제를 제조하였다. The prepared first liquid was dropped into the second liquid to prepare drop-shaped hydrogel particles, which were washed and dried to prepare a crack treatment agent.
Bacillus velezensis MCE03 균주의 경우에는 제1액에 20g의Urea를 함께 혼합하여 균열치료제를 형성하였다. In the case of Bacillus velezensis MCE03 strain, 20 g of Urea was mixed with the first solution to form a crack treatment agent.
물:시멘트:모래를 1:2:3 비율로 혼합하여 50 X 50 X 50 mm 크기의 몰탈을 제작하였다. Mortar measuring 50
물, 시멘트, 모래를 혼합할 때 균열치료제를 함께 혼합하였다. When mixing water, cement, and sand, a crack treatment agent was mixed together.
제작한 몰탈을 3일 동안 상온에서 굳히고 물리적으로 균열을 유발시킨 후에 누수 상황을 재현하기 위해서 1cm 높이로 증류수에 침지해두었다. The manufactured mortar was hardened at room temperature for 3 days, physically cracked, and then immersed in distilled water to a height of 1 cm to reproduce the water leakage situation.
증류수는 수위가 낮아지지 않도록 지속적으로 공급하였으며, 그 결과를 도 8에 나타내었다. Distilled water was continuously supplied to prevent the water level from dropping, and the results are shown in Figure 8.
도 8에서 보는 바와 같이, Bacillus velezensis MCE03 균주와 Bacillus subtilis MCE05 균주 모두 48 시간 경과 후에 균열을 자가 치유하였으며, 최대 0.5 mm 수준의 균열을 자가 치유하였음을 확인할 수 있었다. As shown in Figure 8, both the Bacillus velezensis MCE03 strain and the Bacillus subtilis MCE05 strain self-healed cracks after 48 hours, and it was confirmed that cracks of up to 0.5 mm were self-healed.
본 발명의 보호범위가 이상에서 명시적으로 설명한 실시예의 기재와 표현에 제한되는 것은 아니다. 또한, 본 발명이 속하는 기술분야에서 자명한 변경이나 치환으로 말미암아 본 발명이 보호범위가 제한될 수도 없음을 다시 한번 첨언한다.The scope of protection of the present invention is not limited to the description and expression of the embodiments explicitly described above. In addition, it is once again added that the scope of protection of the present invention may not be limited due to changes or substitutions that are obvious in the technical field to which the present invention pertains.
Claims (6)
상기 수탁번호 KACC 92400P인 Bacillus velezensis MCE03 균주가 엔도소포어(endospore) 상태인 것을 특징으로 하는 미생물 제제. According to paragraph 1,
remind A microbial preparation characterized in that the Bacillus velezensis MCE03 strain, accession number KACC 92400P, is in an endosopore state.
상기 수탁번호 KACC 92400P인 Bacillus velezensis MCE03 균주가 엔도소포어(endospore) 상태인 것을 특징으로 하는 균열 치료제. According to paragraph 3,
A crack treatment agent, characterized in that the Bacillus velezensis MCE03 strain, accession number KACC 92400P, is in an endospore state.
상기 수탁번호 KACC 92400P인 Bacillus velezensis MCE03 균주의 생장 및 탄산칼슘 형성에 필요한 성분을 더 포함하는 것을 특징으로 하는 균열 치료제. According to paragraph 3,
A crack treatment agent, characterized in that it further contains ingredients necessary for the growth and calcium carbonate formation of the Bacillus velezensis MCE03 strain, accession number KACC 92400P.
The Bacillus velezensis MCE03 strain, accession number KACC 92400P, which can spontaneously heal cracks in concrete by producing calcium carbonate, is treated with lysozyme and heat treatment to decompose vegetative cells and kill other strains. Method for producing a crack treatment agent containing endosophore.
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