KR101310634B1 - Bacillus thuringiensis knuc2103 and a cement additive for durability improvement and crack reparing of cement paste or concrete containing the same - Google Patents

Abstract

PURPOSE: Bacillus thuringiensis KNUC2103 (KCTC12149BP) is provided to seal cracks of a cement or concrete structure by forming calcium carbonate, thereby repairing the cracks. CONSTITUTION: A novel microorganism, Bacillus thuringiensis KNUC2103 (KCTC12149BP) has an antifungal activity and forms calcium carbonate. The strain contains 16S rDNA gene with a base sequence of sequence number 1. A cement additive for enhancing durability of cement paste or concrete and repairing cracks contains the strain.

Description

New Bacillus thuringiensis KNUC2103 and a cement additive for durability improvement and crack reparing of cement paste or concrete containing the same

The present invention is a novel microbial Bacillus Turingis and Bacillus thuringiensis ) relates to KNUC2103, and more specifically to the novel microbial Bacillus thuringiensis KNUC2103 (KCTC12149BP) having antifungal activity and calcium carbonate forming ability, and cement additives for enhancing durability and crack repair of cement pastes containing the same It is about.

The cement or concrete building surface repair materials currently used are mostly used for synthetic compounds. These compounds are the cause of sick house syndrome (SBS, Sick Building Syndrom), and it has been proved that the cause of hypersensitivity reactions, respiratory diseases, skin diseases, atopic diseases. Also, the effect is never lasting and expensive.

Apart from this, mold derived from the building causes respiratory diseases in a closed building, and also causes sick house syndrome.In the allergy diagnosis kit, allergens (allergens) of the fungus are included in the hospital's allergy diagnosis kit. have. In addition, antigens have been developed for the diagnosis of diseases caused by them. In order to inhibit the growth of the fungus, chemical antifungal substances and pesticide-based substances were included in cement, and the antifungal ability of cement mortar was also tested. However, the inclusion of antifungal agents has never been permanently effective because of their resistance to antimicrobial agents.

Until now, research on the improvement of compressive strength and crack repair of buildings using bacteria metabolism has been conducted. In addition, the development of concrete that can not grow mold in a separate area has been made. In addition, there is a case study where bacteria were used to repair cracks in stone cultural properties and old buildings.

However, there has never been a single study to repair cracks by inhibiting fungal growth in actual building cracks by using bacteria that have anti-molding ability and calcium carbonate crystallization ability at the cracking site of the building where the mold is actively growing. The application of biochemical mechanisms of bacteria isolated from buildings has the advantage of being environmentally friendly and lasting.

If only the nutrients that bacteria can grow are provided continuously, the cost of the material is very low because of the infinitely multipliable nature. Although there have been previous studies to make cement-based cement structures such as vegetation-grown concrete and water-purifying concrete into environmentally friendly materials, microorganisms can be used to repair the cracks of eroded cement, improve strength, and suppress mold growth. No research on the technology has been reported yet.

Accordingly, the present inventors have diligently researched to overcome the problems of the prior arts, such as antifungal activity, crack repair, water permeation rate reduction, etc. when treating or manufacturing cement paste or concrete using Bacillus thuringiensis KNUC2103 strain. Through the effects it was confirmed that it can be effectively used for improving the durability and crack repair of cement paste or concrete structure, and completed the present invention.

Therefore, the main object of the present invention is to provide a Bacillus Turingiansis KNUC2103 (KCTC12149BP) strain having antifungal activity and calcium carbonate forming ability.

It is another object of the present invention to provide a cement additive or cement additive for improving the durability of cement paste or concrete containing the Bacillus thuringiensis KNUC2103 strain.

Still another object of the present invention is to provide a crack repair method and a method for improving durability of cement paste or concrete structure using the Bacillus thuringiensis KNUC2103 strain.

Still another object of the present invention is to provide a method of producing concrete having improved durability using the Bacillus thuringiensis KNUC2103 strain.

According to one aspect of the invention there is provided novel Bacillus microbe having an antifungal activity, and calcium carbonate-forming tube Lindsay and cis (Bacillus thuringiensis ) KNUC2103 (KCTC12149BP).

Bacillus Turingis and Bacillus thuringiensis ) KNUC2103 has antifungal activity against major contaminant molds in cement buildings that can resist degradation due to various pollutants and forms calcium carbonate.

The present inventors searched for spore forming bacteria that can be easily found in soil, sand, natural minerals, etc. related to buildings for the study of the interaction between microorganisms and building materials, and as a result spore forming bacteria isolated from Dokdo Among them, strains having antifungal activity and calcium carbonate forming ability were selected. DNA was isolated from the selected strains and the 16s rDNA sequence was amplified to perform molecular partial identification using NCBI public domain site and EzTaxon server. Identification results, were identified as in Bacillus (Bacillus) Bacillus 99.86% homology with the thuringiensis strain. The strain is Bacillus Turingiansis ( Bacillus) thuringiensis ) was named KNUC2103, and was deposited on February 23, 2012 by the Korea Institute of Bioscience and Biotechnology (KCTC) and was assigned the accession number KCTC12149BP. Preferably, the Bacillus thuringiensis KNUC2103 strain comprises a 16S rDNA gene having the nucleotide sequence of SEQ ID NO: 1.

In a preferred embodiment of the present invention, the Bacillus thuringiensis KNUC2103 strain was confirmed to exhibit antifungal activity and calcium carbonate forming ability (see FIGS. 1 to 3).

According to another aspect of the present invention, the present invention provides a cement paste including Bacillus thuringiensis KNUC2103 (KCTC12149BP) or cement additive for durability improvement and crack repair of concrete.

The cement additive for durability improvement and crack repair of the cement paste or concrete refers to a repair material or a filler used for durability improvement and crack repair of a cement building (concrete). Can be improved.

The cement paste is a cement mixture prepared by mixing cement and water to form a structure by itself, or may be used as an adhesive or a filler of a cement structure (eg, a concrete product). In addition, the concrete is a cement mixture prepared by mixing cement, water, aggregate, and includes cement mortar, ready-mixed concrete, and post-cured concrete structures.

The cement additive refers to a composition that is added to improve a specific function in the preparation of the cement mixture, in the present invention, in addition to the Bacillus Turingiansis KNUC2103 strain, a medium component for the growth of the strain, diluent, strain culture solution, etc. are further included. Can be.

If previous studies on cement structures using microorganisms are to add environmentally friendly elements to cement structures, the present invention has a multifunctional effect of coating the eroded cement surface to repair cracks, enhance strength and inhibit the growth of mold. . This is a multifunctional biomaterial concept, which has not been tried in Korea or abroad. In the future, the use of microorganisms to improve concrete mechanical performance will continue to develop.

In the present invention, the Bacillus thuringiensis KNUC2103 is characterized by having antifungal activity. Neutralized cement is seriously contaminated by mold, and the cement additive containing the strain inhibits the growth of such mold in cement paste or concrete. When the strain is applied to the cement paste in an embodiment of the present invention, Cladosporium reported as a contaminant mold of major buildings (Park et al., J. Microbiol . Biotechnol . 2011, 3: 287293) sphaerospermum KNUC253 was found to inhibit the proliferation (see FIG. 4).

In the present invention, the Bacillus thuringiensis KNUC2103 is characterized by showing the effect of improving durability and cracks through the formation of calcium carbonate (mineral crystal) on the surface of the cement paste or concrete. The Bacillus Turingiansis KNUC2103 strain is a genus of Bacillus belonging to the high G + C group, Gram positive, and is the first strain isolated by the present inventors. The new application of the improvement of the durability of the cement structure and the crack repair through the function of coating the eroded cement surface with calcium carbonate crystal by using this strain to repair cracks, reduce the moisture penetration rate, increase the strength and suppress the growth of mold. I would like to propose.

In a preferred embodiment of the present invention, it was confirmed that the cement cracks can be filled by treating the crack site of the cement structure using the Bacillus Turingiansis KNUC2103 (see FIG. 4). In addition, when treating the culture medium of the strain on the surface of the cement structure it was confirmed that the calcium carbonate crystals to form an effect of coating the surface to prevent moisture infiltration from the outside and to improve the durability of the cement structure (Fig. 5 Reference).

According to another aspect of the present invention, the present invention provides a Bacillus touring cracks in the cement paste or concrete structure ( Bacillus thuringiensis ) KNUC2103 (KCTC12149BP) Provides a method for crack repair of cement pastes or concrete structures comprising treating a culture.

In accordance with another aspect of the invention, the invention is Bacillus on the surface of the cement paste or concrete tube Lindsay and cis (Bacillus thuringiensis ) KNUC2103 (KCTC12149BP) Provides a method for enhancing the durability of a cement paste or concrete structure comprising treating a culture solution.

Due to the calcium carbonate forming action of the Bacillus thuringiensis and KNUC2103 strain, the crack of the structure can be repaired by filling the crack of the cement paste or the concrete structure during the treatment of the strain culture medium, and also the calcium carbonate crystals are formed on the surface thereof and formed. Minerals can grow to cover the strain itself and to enhance durability by preventing water penetration from the outside.

Previously, crack repair using bacterial metabolism and anti-fungal concrete added with antimicrobial agents were developed as separate areas, and the present invention has the effect of improving the durability of concrete buildings by repairing cracks on cement surfaces without mold growth. It can be said. In the future, it can be used not only for crack repair of concrete structures, but also for old buildings and stone cultural properties.

According to another aspect of the present invention, the present invention comprises the steps of preparing concrete by combining water with cement and aggregate; Injecting the concrete into a mold to shape the concrete; And curing the concrete after the shaping step, at the time of curing Bacillus Turingiansis ( Bacillus thuringiensis ) KNUC2103 (KCTC12149BP) provides a method for producing a durable concrete, including;

Aggregate used in the concrete production is preferably sand, including all the aggregates for concrete production known in the art such as sand, gravel, crushed stone.

The method for producing concrete having improved durability of the present invention has its technical feature in adding a process of treating the strain of the present invention during curing in a general concrete manufacturing process. Therefore, any method for producing concrete known in the art can easily add the strain treatment process of the present invention to any method. Thus prepared concrete can be improved in durability through the antifungal, calcium carbonate crystal formation and water permeability reduction effect compared to the untreated strain.

As described above, according to the present invention, reports of repairing cracks in cement using microorganisms are gradually increasing. In addition, research is ongoing on cements that inhibit the growth of mold using chemicals. In addition, there is a domestic patent for cement having antimicrobial efficacy by including sewage sludge in cement. However, there has never been a single report that a multifunctional microorganism that combines these two functions, namely fungal growth inhibition and repair, can be repaired. In addition, even the cements that have been researched and developed for inhibiting mold have been tested for their efficacy against molds derived from food-based molds, and have not been tested for molds that actually degrade structures.

Separation of Cement Structure Cracks in Buildings Bacteria can be used to repair cracks on the surface while preventing the growth of reported mold using bacteria. Therefore, the present invention is not only a new invention at home and abroad, but also has a large ripple effect. In addition, compared to many synthetic compounds currently used for crack repair of concrete, it has the advantage of being cost-saving and environmentally friendly. In the future, various characteristics of this strain in the multifunctional aspect of Bacillus thuringiensis KNUC2103 strain will be applied to the cement structure as it is to become a key material for smart concrete development.

1 is an optical microscope image of Bacillus thuringiensis KNUC2103 strain to form calcium carbonate crystals on urea-CaCl2 agar medium and B4 agar medium.
FIG. 2 is an image showing antifungal activity against C. sphaerospermum KNUC253 in which the Bacillus thuringiensis KNUC2103 strain was reported as a major architectural contamination fungus.
FIG. 3 is an image showing antifungal activity against A. niger KCTC6906, in which the Bacillus thuringiensis KNUC2103 strain is widely known as a major deteriorating fungal strain.
4 shows the appearance of Bacillus thuringiensis KNUC2103 strain repairing the crack in the cement paste surface crack.
Figure 5 shows the effect of reducing the water permeability (cement surface permeability) of the cement surface when Bacillus Turingiansis KNUC2103 strain to the cement paste.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1.Isolation of Bacillus thuringiensis KNUC2103 strain

Sampling was carried out using a sterile bag for sampling in the soil of Dong-do and Do-do in Dokdo. Samples were diluted in 0.85% physiological saline and shaken at 100 rpm for 24 hours, then plated on pre-prepared TSB (tryptic soy broth, Difco, USA) agar medium and incubated at 30 ° C. for 24 to 48 hours. The isolated strains were plated on B4 agar medium and cultured for 4-5 days to isolate the strains that form the crystals of calcium carbonate and were subjected to deep storage for laboratory preservation.

In order to identify the molecular part of the isolated bacteria, 16S rDNA of the strain was amplified by colony PCR technique after one colony of cells cultured at 30 ° C. for 24 to 48 hours. The primer used was (5'-CCAGCAGCCGCGGTAATACG-3 ') as the 518F (Forward) primer and (5'-TACCAGGGTATCTAATCC-3') as the 800R (Reverse) primer. The obtained 16S rDNA sequence (SEQ ID NO: 1) was determined using the EzTaxon server in comparison with the highest homology with the type strain (type strain).

Identification result, pure isolated strains were identified as belonging to the genus Bacillus high G + C group, Gram positive exhibited a Bacillus thuringiensis strain and 99.86% homology. The present inventors named this strain Bacillus thuringiensis KNUC2103, and deposited it on February 23, 2012 at the Korea Research Institute of Bioscience and Biotechnology, and received the accession number KCTC12149BP. Table 1 below shows the bacteriological properties of the isolated strains.

Taxonomy,
Morphological features It is an aerobic bacillus and taxonomically gram-positive mesophilic strain. Physiological features When spread on B4 agar medium and incubated at 30 ° C. for 4 to 5 days, crystals of calcium carbonate (CaCO ₃ ) form. The 16S rDNA of this strain was amplified by PCR, and when the base sequence was aligned with the type strain using EzTaxon, it was not completely identical as 99.86%. It is a new microorganism because it is separated from the site completely different from the type strain. Cultural characteristics It was separated on TSB agar medium and showed optimal growth at 30 ° C. It is a bacterium isolated from the soil of Dongdo, Dokdo, Korea.

Example  2. Bacillus Turing G & S KNUC2103  Calcium carbonate crystal formation test by strain

B4 agar medium (yeast extract 4.0 g, glucose 5.0 g, calcium acetate 15.0 g, agar 15.0 g in 1L of deionized water) and Urea-CaCl, named after a colony of Bacillus thuringiensis KNUC2103 strain grown on TSB agar medium ₂ Inoculated with agar on agar medium (nutrient broth 3 g, urea 20 g, NaHCO ₃ 2.12 g, NH ₄ Cl 10 g, CaCl ₂ 3.7 g, agar powder 15 g in 1 L of deionized water) and in a 30 ° C incubator Incubated for 4-5 days. In the case of urea-CaCl ₂ medium, heat sensitive components were sterilized by filter sterilization, and in the case of B4 medium, sterilization was performed separately to prevent condensation of calcium acetate with other components in the medium. Calcium carbonate crystal formation on the medium was confirmed through an optical microscope 40x magnification. An optical microscope (Sw 804425, Samwon, Seoul, Korea) was used for the observation of the crystal, and a Zentech digital camera was used for the imaging of the formed calcium carbonate crystal.

1 is an optical microscope image of Bacillus thuringiensis KNUC2103 strain to form calcium carbonate crystals on urea-CaCl ₂ agar medium and B4 agar medium. The scale bar is 500 μm.

Example  3. Bacillus Turing G & S KNUC2103  Analysis of Antifungal Activity on Building Contaminant Fungi of Strains

To obtain probiotics of the Bacillus thuringiensis KNUC2103 strain, their single colonies were inoculated into 5 ml of TSB (tryptic soy broth, Difco, USA) liquid medium and shaken cultured at 30 ° C. at 180 rpm. After inoculating 1 ml each of the previous culture in 100 ml TSB liquid medium, shaking culture (shaking culture) for 7 days at 180 rpm in a 30 ℃ incubator. The culture solution was centrifuged at 3,000 rpm for 10 minutes at 4 ° C, the supernatant was removed with a 0.22 μm filter (nitrocellulose membrane filter), and the culture supernatant was concentrated 30 times through a freeze-drying device (Bondiro Co, Korea), followed by antifungal It was used as a prophylactic material for testing.

In addition, in order to obtain a susceptible fungal strain spore suspension for antifungal testing, in the case of Cladosporium sphaerospermum KNUC253, V8 juice agar medium (300 ml of V8 juice (Campbell Soup Company, Camden, NJ), 4.5 g of calcium carbonate (Mallinckrodt Baker, Inc., Phillipsburg, NJ), and 15 g of Bacto agar (Becton, Dickson and Co., Sparks, and from 18 ℃ in MD)) were obtained 4 weeks incubation the spores with a sterile distilled water, Aspergillus In the case of niger KCTC6906, spores were obtained using sterile distilled water by culturing for 2 weeks on PDB (potato dextrose broth) agar medium. A. niger KCTC6906 was used from the Korea Institute of Bioscience and Biotechnology (KCTC).

The spores obtained were placed in a liquid 50 ° C. PDB (potato dextrose broth, Difco, USA) agar medium, suspended well, and then plated onto plates. Sterile 8 mm diameter disk paper (Advantec, Japan) was placed on this medium and inoculated with 100 μl of the probiotic material prepared above to confirm antifungal activity against A. niger KCTC6906 and C. spaherospermum KNUC253 strains. . At this time, the growth conditions were confirmed after incubation for 48 hours at 25 ℃ for A. niger KCTC6906 and 96 hours at 18 ℃ for C. sphaerospermum KNUC253. At this time, cycloheximide (Sigma-Aldrich Co., US), an antifungal agent, was adjusted to concentrations of 100 μg / ml, 300 μg / ml and 500 μg / ml as a positive control. In the same proportion, TSB and sterile distilled water (SDW) were used as negative controls.

Figure 2 is an image showing the antifungal activity against C. sphaerospermum KNUC253 strain Bacillus Turingiansis KNUC2103 is reported as a major building contaminant fungi, Figure 3 is a Bacillus Turingiansis KNUC2103 strain is widely used as a major building deterioration mold strain This image shows antifungal activity against known A. niger KCTC6906.

As a result, it was confirmed that Bacillus thuringiensis KNUC2103 strain had antifungal activity against susceptible fungi C. sphaerospermum KNUC253 and A. niger KCTC6906 when compared with negative control TSB, sterile distilled water. Compared with mead, it showed better antifungal activity.

Example  4. Bacillus Turing G & S KNUC2103  Confirmation of crack recovery and mold growth inhibition on cement surface by strain

As an experiment to find out whether the cracks can be repaired while suppressing the growth of mold on the cracks on the cement, an artificial method is used to create cracks on the cement paste to recover the cracks of the cement paste having a crack size of about 0.4 mm. The test body was selected for. Crack size was taken / measured through a microscope. A single colony of Bacillus thuringiensis KNUC2103 strain was taken and then inoculated into 5 ml of B4 liquid medium and pre-cultured with shaking in a 30 ° C. incubator. The 250 μl Bacillus thuringiensis KNUC2103 strain pre-culture suspension was centrifuged at 1000 rpm for 10 minutes at 4 ° C., mixed with 250 μl B4 liquid medium and initially inoculated into the cracked site. Was then inoculated into liquid medium and B4 10 ⁵ ~ 10 ⁶ / ml concentration in a 12-hour interval that contains the spores of C. sphaerospermum KNUC253 concentrated in 9 days cracks. Also, as a negative control, Escherichia without mineral crystal forming ability coli K12 (KCTC2441) was inoculated in the same manner, and only the negative B4 liquid medium was inoculated.

4 shows the appearance of Bacillus thuringiensis KNUC2103 strain repairing the crack in the cement paste surface crack. In FIG. 4, the left side is a photograph immediately after fabrication of the surface of the cement paste, and the center is a state of repairing the cracks after treatment of each control and KNUC2103, and the right side is a result of testing mold growth inhibition at the cracked portion. . A: When the B4 liquid medium was treated as a negative control, cracks could not be repaired (middle), and mold growth was clear after 10 days of culture (right). B: The same result as A was obtained when E. coli K12, which is known to have no calcium carbonate crystal forming ability, was treated as a negative control. C: As a result of treatment of the Bacillus thuringiensis KNUC2103 strain, it can be seen that while restoring the crack (middle) it inhibits mold growth compared to negative control (right). Therefore, it can be seen that when the Bacillus Turingiansis KNUC2103 strain is treated, the cracks can be recovered while inhibiting the growth of mold in the cracks on the cement paste.

Example  5. Bacillus Turing G & S KNUC2103 Reduction of Cement Surface Water Penetration Rate by

In order to measure the moisture permeability, cement paste disc test bodies were prepared. Cement powder and sterile distilled water are mixed in a 10: 4 weight ratio, and then dispensed in 35 × 10 mm Petri dishes (style petri-dish; Falcon, Becton Dickinson Labware Co., USA) in equal amounts and cured at room temperature for 24 hours. ) The cement paste disks were dried at a high temperature for 24 hours in an oven at 75 ° C. to obtain dry weights. The weights of the cement paste disks were selected to select six disks each having a difference in weight of 0.3 g or less and used as a test specimen for moisture permeability test. A single colony of Bacillus thuringiensis KNUC2103 strain was inoculated into 50 ml of B4 liquid medium and shaken at 180 rpm in a 30 ° C incubator for 24 hours to preculture (pre-culture) after pre-culture. Culture suspension) The whole amount was inoculated into 450 ml B4 liquid medium containing 6 paste specimens and shaken culture for 5 days at 30 rpm incubator at 50 rpm. The treated cement paste was dried in an oven at 75 ° C. for 24 hours to obtain a constant value (W ₁ ), which was completely immersed in sterilized distilled water for 24 hours and then weighed (W ₂ ). Moisture penetration rate was measured as N (water penetration rate) = W ₂ -W ₁ / W ₁ × 100 (%). Escherichia, a negative control used, known for its ability to form B4 liquid media and calcium carbonate crystals Cement paste discs treated only with coli K-12 were used for the measurement of water penetration.

Figure 5 shows the effect of reducing the water permeability (cement surface permeability) of the cement surface when Bacillus Turingiansis KNUC2103 strain to the cement paste. In Fig. 5, the cement paste treated with Bacillus thuringiensis KNUC2103 has a water permeation rate of 18.02%, compared with 24.06% when treated with control E. coli K12, and 24.88% when treated with B4 liquid medium alone. Bacillus thuringiensis KNUC2103 strain significantly lowered the water penetration rate of the cement paste.

The penetration of moisture into the cement structure accelerates the corrosion of concrete due to the chemical action of moisture and cement components, which affects the durability of the building by corroding the reinforcing bars in the deep concrete. Therefore, lowering the moisture permeation rate can increase the durability of the cement structure.

Korea Biotechnology Research Institute KCTC12149BP 20120223

Attach an electronic file to a sequence list

Claims (8)

Novel Microbial Bacillus Turingiansis with Antifungal Activity and Calcium Carbonate Formation Capacity thuringiensis ) KNUC2103 (KCTC12149BP).
The Bacillus thuringiensis KNUC2103 (KCTC12149BP) according to claim 1, comprising a 16S rDNA gene having a nucleotide sequence of SEQ ID NO: 1.
Bacillus thuringiensis ) Cement paste or cement additive for the durability improvement and crack repair of cement containing KNUC2103 (KCTC12149BP).
According to claim 3, Bacillus thuringiensis ( Bacillus thuringiensis ) KNUC2103 (KCTC12149BP) is a cement additive for improving the durability and crack repair of cement paste or concrete characterized in that it has antifungal activity.
The method of claim 3, wherein the Bacillus Turingiansis ( Bacillus) thuringiensis ) KNUC2103 (KCTC12149BP) is a cement additive or cement additive for improving durability and crack repair of cement paste or concrete, which is characterized by the improvement of durability and crack repair effect by forming calcium carbonate crystals on the surface of cement paste or concrete.
A method of crack repair of a cement paste or concrete structure comprising treating Bacillus thuringiensis KNUC2103 (KCTC12149BP) culture solution to a cracked portion of the cement paste or concrete structure.
A method of enhancing the durability of a cement paste or concrete structure comprising treating Bacillus thuringiensis KNUC2103 (KCTC12149BP) culture on the surface of the cement paste or concrete structure.
Preparing concrete by mixing water with cement and aggregate;
Injecting the concrete into a mold to shape the concrete; And
Curing the concrete after the shaping step, at the time of curing Bacillus Turingiansis ( Bacillus thuringiensis ) KNUC2103 (KCTC12149BP) after the sprinkling of the culture solution and curing; manufacturing method of the improved concrete comprising a.

Images