CN111575210B - Bacillus amyloliquefaciens ZJB19161 and application thereof - Google Patents

Bacillus amyloliquefaciens ZJB19161 and application thereof Download PDF

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CN111575210B
CN111575210B CN202010454857.6A CN202010454857A CN111575210B CN 111575210 B CN111575210 B CN 111575210B CN 202010454857 A CN202010454857 A CN 202010454857A CN 111575210 B CN111575210 B CN 111575210B
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郑裕国
徐建妙
泮佳佳
程峰
王远山
贾东旭
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Zhejiang University of Technology ZJUT
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Abstract

The invention discloses bacillus amyloliquefaciens ZJB19161 and application thereof in concrete crack repair, belonging to the technical field of microorganisms. The bacillus amyloliquefaciens ZJB19161 is preserved in China center for type culture Collection (China center for type culture Collection) in 2020, 1 month and 13 months, and the preservation number is CCTCC NO: M2020044. The bacillus amyloliquefaciens ZJB19161 provided by the invention is separated from a soil sample at the ancient canal side of the morning glory of Zhejiang industrial university, and the metabolite of the bacillus amyloliquefaciens can be efficiently combined with calcium ions in the environment to form calcite, so that concrete microcracks can be effectively repaired. In addition, the strain has good thermal stability and acid-base stability, and can adapt to different pH value environments when being used for repairing concrete cracks. The strain has the potential and application prospect of being used as a bioremediation preparation for self-repairing of concrete cracks.

Description

Bacillus amyloliquefaciens ZJB19161 and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus amyloliquefaciens ZJB19161 and application thereof in concrete crack repair.
Background
With the rapid development of economy, buildings such as high-rise buildings, asphalt roads and the like are widely seen, concrete is widely applied to building materials due to high compressive strength, good ductility and low price, but after the traditional cement buildings are used for a period of time, the concrete can be subjected to micro-cracking or local damage due to plastic shrinkage, thermal stress, sedimentation, drying shrinkage, weathering, natural corrosion and steel bar corrosion or due to the action of multiple factors such as load application, so that the mechanical property and the durability of the concrete can be influenced, the normal use and the service life of the concrete can be further influenced, and the steel bars in the concrete can be exposed and corroded, thereby bringing potential safety hazards to the buildings.
The traditional repair technology is that the artificial high polymer material is used for repairing the crack once and again, which wastes time and labor. The existing novel repair technology is to add microbial spores which can adapt to alkaline environment and carry out biomineralization into cement, once cracks appear, the microbes are activated, calcium lactate is used as a calcium source to carry out biomineralization, and therefore the cracks are repaired.
For example, patent document CN 109610658A discloses a method for repairing concrete cracks by using microorganism-induced calcium carbonate deposition, which comprises washing concrete cracks with clean water, filling with common sand or quartz sand; mixing the cultured bacillus pasteurianus with urea, and standing for 1-1.5 hours to obtain a mixed solution; and simultaneously dripping the mixed solution and the calcium salt solution into the concrete crack until the concrete crack is repaired. Compared with the traditional concrete crack repairing method, the microbial repairing method has the advantages of fast aging and high strength when used for treating the concrete crack.
Patent document CN 104403959A discloses a highly efficient calcium mineralized Bacillus alcalophilus sp.H4 strain capable of being cultured at low cell concentrations (5X 10)7One/ml) is used for converting calcium ions, the mineralization is carried out at the temperature of 20-37 ℃ and the pH value of 9.0-11.0, and the calcium mineralization rate is up to 96.4%.
The method for repairing the concrete cracks by utilizing the microorganism to induce the calcium carbonate deposition saves energy sources and reduces the carbon emission, and is undoubtedly an environment-friendly repairing technology, so that the development of a more efficient concrete crack self-repairing biological agent has positive significance in the technical field of microorganism and building material intersection.
Disclosure of Invention
The invention aims to provide a high-efficiency calcium ion mineralized bacterium, and the metabolite of the high-efficiency calcium ion mineralized bacterium can be combined with calcium ions in the environment to form calcite, so that the calcite can be used for repairing concrete microcracks, and a new theoretical basis and technical support can be provided for the development of a concrete self-repairing biological preparation.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention separates the bacterial strain ZJB19161 with high urease activity from the soil sample at the ancient canal side of the morning glory of Zhejiang industrial university, and the 16S rDNA sequence is shown in SEQ ID NO. 1. The measured 16S sequence is compared with a National Center for Biological Information (NCBI) database, the ZJB19161 and a known strain Bacillus amyloliquefaciens strain NBRC 15535 are clustered in the same branch, the genetic relationship is recent, and the similarity is as high as 99.71 percent, so that the ZJB19161 belongs to Bacillus amyloliquefaciens (B. amyloliquefaciens) in classification and is named as the Bacillus amyloliquefaciens ZJB19161(Bacillus amyloliquefaciens ZJB 19161).
The invention provides a bacillus amyloliquefaciens ZJB19161, which is preserved by a preservation unit: china center for type culture Collection, collection address: wuhan, Wuhan university, China, preservation date: year 2020, 13/1, accession number: CCTCC NO: M2020044.
The invention also provides application of the bacillus amyloliquefaciens ZJB19161 in repairing concrete cracks.
The application can be passive repair by adding thalli to concrete microcracks, and specifically comprises the following steps: and mixing the bacillus amyloliquefaciens ZJB19161 thallus with a nutrient solution containing calcium salt and urea, adding the mixed solution into the concrete crack, and curing until the concrete crack is repaired.
Preferably, the nutrient solution is prepared by adding 10g of peptone, 5g of yeast extract, 10g of NaCl and CaCl into per liter of water27-27 g, 5-14 g of urea and 7.0-7.2 of pH value. More preferably, the nutrient solution is prepared by adding 10g of peptone, 5g of yeast extract, 10g of NaCl and CaCl into per liter of water222g of urea and 12g of pH 7.0-7.2.
Preferably, the concentration of the cells of Bacillus amyloliquefaciens ZJB19161 in the mixed solution is 1 × 107cfu/mL～1×109cfu/mL。
The application can also be that spores are directly mixed in the concrete preparation process, and when cracks appear, the spores are activated to rapidly germinate into nutriments to finish the self-repairing of the concrete cracks. Specifically, the method comprises the following steps: mixing the bacillus amyloliquefaciens ZJB19161 thallus spores with mixed nutrient solution containing calcium salt and urea to obtain a microbial inoculum, replacing water in the concrete preparation process with the microbial inoculum, and performing standard curing after mixing to obtain the self-repairing concrete.
The influence of the nutrient additives on the mechanical performance of the concrete needs to be considered in the active repair, and preferably, the components of the mixed nutrient solution comprise yeast extract, inosine, calcium lactate and urea, and the concentrations of the yeast extract, the inosine, the calcium lactate and the urea are 1-2 g/L, 2-3 g/L, 20-25 g/L and 8-14 g/L respectively. More preferably, the concentrations of yeast extract, inosine, calcium lactate and urea in the mixed nutrient solution are 1.5g/L, 2.68g/L, 20.8g/L and 10g/L respectively.
Preferably, the concentration of the spores of the bacillus amyloliquefaciens ZJB19161 in the microbial inoculum is 4 multiplied by 107cfu/mL～1×109cfu/mL。
The invention also provides a concrete crack self-repairing biological preparation which comprises the bacillus amyloliquefaciens ZJB 19161.
The invention has the beneficial effects that:
the bacillus amyloliquefaciens ZJB19161 provided by the invention is separated from a soil sample at the ancient canal side of the morning glory of Zhejiang industrial university, and the metabolite of the bacillus amyloliquefaciens can be efficiently combined with calcium ions in the environment to form calcite, so that concrete microcracks can be effectively repaired. In addition, the strain has good thermal stability and acid-base stability, and can adapt to different pH value environments when being used for repairing concrete cracks. The strain has the potential and application prospect of being used as a bioremediation preparation for self-repairing of concrete cracks.
Drawings
FIG. 1 is a scanning electron micrograph of the precipitates of the experimental group obtained by adding Bacillus amyloliquefaciens ZJB19161 in example 3.
FIG. 2 is a graph of the energy spectrum analysis of the mineralized precipitate of FIG. 1.
FIG. 3 is a scanning electron micrograph of the precipitates of the control group to which no bacteria were added in example 3.
FIG. 4 is a graph of the spectrum analysis of the control precipitate in example 3.
FIG. 5 is a graph showing the results of passively repairing concrete microcracks using the ZJB19161 strain in example 4, where YJ is the test group containing ZJB19161 and "none" is the control group containing equal amounts of nutrient solution without added cells.
FIG. 6 is a graph showing the results of actively repairing concrete microcracks by the strain ZJB19161 in example 9.
FIG. 7 is a graph showing the results of actively repairing concrete microcracks by the strain ZJB19161 in example 10.
FIG. 8 is a graph showing the results of actively repairing concrete microcracks by the strain ZJB19161 in example 11.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The media formulations in the examples are as follows:
the urease-producing strain screening culture medium comprises the following components: peptone 1g/L, sodium chloride 5g/L, potassium dihydrogen phosphate 2g/L, urea 2g/L, glucose 0.1g/L, phenol red solution (0.2%) 4ml/L, agar 20g/L, pH 7; wherein, the urea and the glucose are respectively and independently prepared into solution for sterilization.
The LB medium comprises the following components: 10g of peptone, 5g of yeast extract, 10g of NaCl, and 1000mL of distilled water, wherein the pH value is 7.0-7.2.
The conditions for sterilization of each medium were: sterilizing at 121 deg.C under 0.10-0.15MPa for 20 min.
Example 1 screening and identification of strains prepared from adapted concrete self-repairing bacterial agents
1) Adding 1g of soil sample at the ancient canal side of an morning glory district of Zhejiang industrial university into a sterilized conical flask with small glass beads and 100mL of sterile water, shaking at 30 ℃ at 200r/min for 30min, standing for 10min, and removing bottom sediment to obtain a soil suspension. And (3) placing the soil suspension in water of 100 ℃ for heating in a boiling water bath for 3-5min to obtain a soil sample stock solution.
The soil sample stock solution is subjected to gradient dilution in a super clean bench, and the concentration gradient is 10-1、10-2、10-3、10-4、10-5、10-6Sucking 200 μ L bacterial suspension with various concentrations, inoculating on urease-producing strain screening culture medium plate, sealing with sealing film, inverting, culturing in 37 deg.C constant temperature incubator for 24 hr, observing colony edge on plate with proper colony numberTaking typical colonies with dark red edges, separating and purifying, inoculating to LB culture medium, culturing for 24h, taking appropriate amount of bacterial liquid, and storing in a refrigerator at-80 deg.C with 30% glycerol; and taking part of the bacterial liquid to perform physiological and biochemical and molecular biological identification.
2) Extracting strain genome DNA by using a FastDNATM Spin Kit for Soil Kit, and carrying out PCR amplification by using the strain genome DNA as a template, wherein a bacterial universal primer:
27F：5'-AGAGTTTGATCCTGGCTCA-3'；
1492R：5'-AAGGAGGTGATCCAGCCGCA-3'。
PCR procedure: performing pre-denaturation at 95 deg.C for 5 min; 30 cycles of extension at 95 ℃ for 40s, 55 ℃ for 30s and 72 ℃ for 2 min; 10min at 72 ℃.
The product is subjected to PCR product sequencing by Oncorhynchs Biotechnology Limited, the obtained DNA sequence is input into GenBank, the sequence is compared with all sequences in a database by using Blast program, a proper DNA sequence is selected to establish a phylogenetic tree, and the species of the strain obtained by screening is determined.
3) Screening 23 new strains from about 3 ten thousand possible strains (screening in sequence, not screening at one time), inoculating the separated single colony into a spore forming culture medium, shaking and culturing at 30 ℃ for 24h in a shaking table, centrifuging 2ml of culture solution at 4 ℃ and 12000r/min for 3min, removing the supernatant to obtain thalli, and determining urease activity.
The urease activity is measured by adopting an indophenol blue colorimetric method, and the activity is defined as follows: at 37 ℃ per 1. mu.g NH produced3-N is defined as one unit of enzyme activity. Indicated by U. In the screening process, the enzyme activities of some strains are shown in table 1.
TABLE 1
Figure BDA0002508854360000051
And finally, screening 23 strains to obtain a Bacillus amyloliquefaciens with high urease activity, wherein the 16S rDNA nucleotide sequence of the Bacillus amyloliquefaciens is shown as SEQ ID NO.1, comparing the Bacillus amyloliquefaciens with a National Center for Biological Information (NCBI) database, clustering the Bacillus amyloliquefaciens strain NBRC 15535 with a known strain in the same branch, and determining that the ZJB19161 belongs to the Bacillus amyloliquefaciens (B. amyloliquefaciens) in classification due to the fact that the genetic relationship is recent and the similarity is as high as 99.71%. Therefore, the Bacillus amyloliquefaciens ZJB19161(Bacillus amyloliquefaciens ZJB19161) is named and preserved in China center for type culture Collection of Wuhan university in China in 1-13 th 2020, and the preservation center is tested to survive in 28 th 1-28 th 2020, and the preservation number is CCTCC NO: M2020044.
Example 2 Effect of different conditions on the urease Activity of the Strain ZJB19161
1) Thermal stability
The temperature is raised to more than 40 ℃ due to heat release in the hydration process in concrete pouring, so that the bacterial suspension of the strain ZBL19161 with the concentration of 10mg/mL is treated in a water bath at 60 ℃ for 60min, after the bacterial suspension is cooled, the dyed bacterial suspension is counted by using a blood counting chamber, and the ratio of live cells to dead cells is calculated.
The result shows that the survival rate of the strain ZJB19161 is still maintained above 90% when the survival rate is below 60 ℃, which indicates that the strain ZJB19161 has better thermal stability.
2) Stability to alkali
Since the pH value is between 12 and 13 due to the hydration process in concrete pouring, the pH value of the bacterial suspension of the strain ZBL19161 with the concentration of 10mg/mL is adjusted to 12.5 by using 1mol/L sodium hydroxide, the solution is filtered by a microfiltration membrane with the diameter of 0.22 mu m after standing for 24h, and the stained bacterial suspension is counted by using a hemocytometer by using the solution with the unadjusted pH value as a control.
The results show that the survival rate of the strain ZJB19161 is still kept above 75% at the pH of 12.5, which indicates that the alkali stability of the strain ZJB19161 is better.
Example 3 metabolites of strain ZJB19161 combine with environmental calcium ions to produce calcite
The strain ZJB19161 is inoculated into LB liquid medium with the bottling amount of 200mL, and is placed in a constant temperature shaking table at 37 ℃ for shaking culture at 150r/min for 12 h. The thalli is obtained after the fermentation liquor is centrifuged for 10min at the temperature of 4 ℃ and the speed of 8000 r/min. The centrifuged cells were added to a mixture (volume ratio of 2:1:1) containing LB liquid medium, 15g/L calcium chloride and 10g/L urea. After being mixed uniformly, the mixture is placed in a 37 ℃ culture room for static culture, and the mixed solution without adding bacteria is used as a control group.
And 7d, observing the generation condition of the sediment on the bottom surface by naked eyes, centrifuging the sediment, drying the sediment in an oven at 80 ℃, observing the sediment under a scanning electron microscope, and simultaneously performing energy spectrum test on solid particles, wherein the results are as follows:
1) mineralized precipitates are formed on the surfaces of the microorganisms after the bacterium adding group is cured, and are shown in figure 1. The result of the energy spectrum analysis shows that the solid only contains calcium ions and does not contain chloride ions, and can be presumed to be calcium carbonate, as shown in figure 2 and table 2, namely, mineralized bacteria can generate calcium carbonate precipitation in a concrete environment to realize the healing of concrete cracks.
TABLE 2
Figure BDA0002508854360000061
Figure BDA0002508854360000071
2) As seen by the scanning electron microscope results (FIG. 3) of the control without the added cells, no cells were attached to this region. The energy spectrum analysis (fig. 4 and table 3) shows that both calcium and chloride ions are deposited as calcium chloride.
TABLE 3
Element Wt％ At％
CK 22.78 40.68
OK 19.40 26.02
ClK 33.69 20.38
CaK 24.13 12.91
Matrix Correction ZAF
Example 4 Passive repair of concrete microcracks by Strain ZJB19161
Standard concrete test pieces containing micro-cracks with the length, width and depth of 15mm, 0.3mm and 20mm are prepared by a pre-crack method through small iron sheets according to a standard program, are wrapped by preservative films and are placed at room temperature, corresponding cracks are marked as 'none' and 'YJ', wherein the group 'YJ' is a ZJB19161 bacterial solution mixed with nutrient solution, and the group 'none' is a control group which is only added with the same amount of nutrient solution and is not added with bacteria. And 3 cracks in each group are injected into the concrete cracks at intervals, the surface healing condition is observed, and meanwhile, the permeation time is timed to indirectly indicate the crack repairing efficiency. Wherein the nutrient solution comprises the following components: 10g of peptone, 5g of yeast extract, 10g of NaCl and CaCl27g, 5g of urea, 1000mL of distilled water and 7.0-7.2 of pHs; the concentration of the bacteria is 3 multiplied by 107cfu/mL。
After 5d, the fractures of group "YJ" were mostly filled, while the fractures of group "no" were not healed, as shown in fig. 5. In addition, the results of the water permeation experiments in Table 4 show that the addition of the bacteria can effectively prolong the complete permeation time. The results show that the strain ZJB19161 can repair the concrete microcracks.
TABLE 4
Figure BDA0002508854360000072
Figure BDA0002508854360000081
Example 5 Strain ZJB19161 repair concrete microcrack nutrient solution condition optimization
The difference between the embodiment and the embodiment 4 is that the nutrient solution for self-repairing of concrete cracks in the embodiment comprises the following components in parts by weight: 10g of peptone, 5g of yeast extract, 10g of NaCl and CaCl212g, 8g of urea and 1000mL of distilled water, and the pH value is 7.0-7.2.
The fracture healing was observed after 5 days as an index of determination, and the first complete penetration time exceeded 50min of repair time, the results are shown in table 5.
Example 6
The difference between the embodiment and the embodiment 5 is that the nutrient solution for self-repairing of concrete cracks in the embodiment comprises the following components in parts by weight: 10g of peptone, 5g of yeast extract, 10g of NaCl and CaCl217g, 10g of urea and 1000mL of distilled water, and the pH value is 7.0-7.2.
Example 7
The difference between the embodiment and the embodiment 5 is that the nutrient solution for self-repairing of concrete cracks in the embodiment comprises the following components in parts by weight: 10g of peptone, 5g of yeast extract, 10g of NaCl and CaCl222g, 12g of urea and 1000mL of distilled water, and the pH value is 7.0-7.2.
Example 8
The difference between the embodiment and the embodiment 5 is that the nutrient solution for self-repairing of concrete cracks in the embodiment comprises the following components in parts by weight: 10g of peptone, 5g of yeast extract, 10g of NaCl and CaCl227g, 14g of urea and 1000mL of distilled water, and the pH value is 7.0-7.2.
TABLE 5
Figure BDA0002508854360000091
As can be seen from Table 5, the concrete repairing nutrient solution provided by the invention has a good repairing effect, greatly reduces the water seepage capability of cracks, and is the best embodiment.
Example 9 active repair of concrete microcracks by spores of strain ZJB19161
Separately, centrifuged spores (4X 10. sup. th cell concentration) were weighed7cfu/ml) and mixed nutrient components are evenly mixed to obtain the microbial inoculum. According to a standard procedure, the microbial inoculum replaces water in the preparation process of the concrete test piece, the standard concrete test piece containing micro cracks with the length multiplied by the width multiplied by the height of 35mm multiplied by 0.3mm multiplied by 15mm is prepared by a small iron sheet through a pre-crack preparation method, and the standard concrete test piece is wrapped by a preservative film and then is placed in a constant temperature and humidity incubator with the temperature of 30 ℃ and the humidity of 90 percent for maintenance. And observing the healing rate and the water permeability of the microcracks. Wherein the nutrient solution comprises the following components: yeast extract, inosine, calcium lactate and urea at concentrations of 1.5g/L, 2.68g/L, 20.8g/L and 10g/L, respectively. After 28 days of curing, the fracture was observed to heal, and the results are shown in FIG. 6.
Example 10
This example is the same as example 9, except that the bacterial concentration of the bacterial agent for self-repairing of concrete cracks in this example is 4 × 108cfu/ml. The results are shown in FIG. 7.
Example 11
This example is the same as example 9, except that the bacterial concentration of the bacterial agent for self-repairing of concrete cracks in this example is 1 × 109cfu/ml. The results are shown in FIG. 8.
From FIGS. 6-8, it is shown that most of the concrete cracks of examples 9-11 have healed, indicating that the spore of the strain ZJB19161 has the capability of actively repairing concrete microcracks. The embodiment 11 is the best embodiment, which shows that the optimal bacteria concentration for self-repairing of concrete cracks is 1 multiplied by 109cfu/ml。
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Sequence listing
<110> Zhejiang industrial university
<120> bacillus amyloliquefaciens ZJB19161 and application thereof
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ggaacgtatt caccgcggca tgctgatccg cgattactag cgattccagc ttcacgcagt 120
cgagttgcag actgcgatcc gaactgagaa cagatttgtg ggattggctt aacctcgcgg 180
tttcgctgcc ctttgttctg tccattgtag cacgtgtgta gcccaggtca taaggggcat 240
gatgatttga cgtcatcccc accttcctcc ggtttgtcac cggcagtcac cttagagtgc 300
ccaactgaat gctggcaact aagatcaagg gttgcgctcg ttgcgggact taacccaaca 360
tctcacgaca cgagctgacg acaaccatgc accacctgtc actctgcccc cgaaggggac 420
gtcctatctc taggattgtc agaggatgtc aagacctggt aaggttcttc gcgttgcttc 480
gaattaaacc acatgctcca ccgcttgtgc gggcccccgt caattccttt gagtttcagt 540
cttgcgaccg tactccccag gcggagtgct taatgcgtta gctgcagcac taaggggcgg 600
aaacccccta acacttagca ctcatcgttt acggcgtgga ctaccagggt atctaatcct 660
gttcgctccc cacgctttcg ctcctcagcg tcagttacag accagagagt cgccttcgcc 720
actggtgttc ctccacatct ctacgcattt caccgctaca cgtggaattc cactctcctc 780
ttctgcactc aagttcccca gtttccaatg accctccccg gttgagccgg gggctttcac 840
atcagactta agaaaccgcc tgcgagccct ttacgcccaa taattccgga caacgcttgc 900
cacctacgta ttaccgcggc tgctggcacg tagttagccg tggctttctg gttaggtacc 960
gtcaaggtgc cgccctattt gaacggcact tgttcttccc taacaacaga gctttacgat 1020
ccgaaaacct tcatcactca cgcggcgttg ctccgtcaga ctttcgtcca ttgcggaaga 1080
ttccctactg ctgcctcccg taggagtctg ggccgtgtct cagtcccagt gtggccgatc 1140
accctctcag gtcggctacg catcgtcgcc ttggtgagcc gttacctcac caactagcta 1200
atgcgccgcg ggtccatctg taagtggtag ccgaagccac cttttatgtc tgaaccatgc 1260
ggttcagaca accatccggt attagccccg gtttcccgga gttatcccag tcttacaggc 1320
aggttaccca cgtgttactc acccgtccgc cgctaacatc agggagcaag ctcccatc 1378

Claims (9)

1. Bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) ZJB19161, wherein the preservation number of the Bacillus amyloliquefaciens is CCTCC NO: M2020044.
2. The use of bacillus amyloliquefaciens ZJB19161 according to claim 1 for repairing concrete cracks.
3. The use of claim 2, comprising: and mixing the bacillus amyloliquefaciens ZJB19161 thallus with a nutrient solution containing calcium salt and urea, adding the mixed solution into the concrete crack, and curing until the concrete crack is repaired.
4. The use according to claim 3, wherein the nutrient solution is peptone added per liter of water10g, yeast extract 5g, NaCl 10g, CaCl27-27 g, 5-14 g of urea and 7.0-7.2 of pH value.
5. The use according to claim 4, wherein the concentration of Bacillus amyloliquefaciens ZJB19161 in the mixture is 1 x 107cfu/mL～1×109cfu/mL。
6. The use of claim 2, comprising: and mixing the spores of the bacillus amyloliquefaciens ZJB19161 with the mixed nutrient solution containing calcium salt and urea to obtain a microbial inoculum, replacing water in the concrete preparation process with the microbial inoculum, and performing standard curing after mixing to obtain the self-repairing concrete.
7. The use of claim 6, wherein the mixed nutrient solution comprises yeast extract, inosine, calcium lactate and urea at concentrations of 1-2 g/L, 2-3 g/L, 20-25 g/L and 8-14 g/L, respectively.
8. The use of claim 7, wherein the concentration of spores of Bacillus amyloliquefaciens ZJB19161 in the preparation is 4 x 107cfu/mL～1×109cfu/mL。
9. A biological preparation for self-repairing concrete cracks, which is characterized by comprising the bacillus amyloliquefaciens ZJB19161 according to claim 1.
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