Alkali-resistant urease-producing microbial flora for biomineralization of construction joints and preparation method of alkaline-resistant urease-producing microbial flora

Abstract

The invention relates to the technical field of biomineralization, and particularly discloses an alkali-resistant urease-producing microbial flora for biomineralization of construction joints and a preparation method of the alkali-resistant urease-producing microbial flora. The microbial flora disclosed by the invention is prepared from the following strains with relative abundance: 4.4 to 5.2 percent of bacillus, 0.39 to 0.46 percent of enterococcus, 4.8 to 5.7 percent of alcaligenes, 0.50 to 0.58 percent of cellulomonas, 22.4 to 24.2 percent of lysobacter, 1.7 to 2.4 percent of pseudomonas, 10.8 to 11.9 percent of Savagea, 15.1 to 16.2 percent of sarcina, 30.8 to 32.2 percent of chloridomonas and 0.41 to 0.48 percent of bacillus. The alkali-resistant microbial compound flora with high urease activity in the limited oxygen and normal environment can effectively overcome the defects that urease microorganisms are low in mineralization efficiency under the anoxic condition, and the strength of an obtained consolidated body is not high.

Description

Technical Field

[0001] The present application relates to the technical field of biomineralization, and in particular to an alkali-resistant urease-producing microbial flora for construction joint biomineralization and a preparation method thereof. Background Art

[0002] At present, how to repair construction joints and concrete cracks has become a research focus in engineering construction. Traditional treatment methods mainly include the following: surface repair method, pressure injection method, filling method, structural reinforcement method, and concrete replacement method. These treatment methods may have the following disadvantages: the workload of on-site wet work is large, which increases the complexity and time cost of the work; for crack treatment methods that require filling or grouting, the maintenance period is long, which may cause delays in the progress of the project; the repair effect is often affected by multiple factors such as the original structural stress level, the structural treatment of the joint surface, the construction process, the material properties, and whether it is unloaded, so it is difficult to ensure that each reinforcement can achieve the expected effect.

[0003] At present, the use of microbial mineralization technology for construction joints has attracted the attention of civil engineering and materials science, and can effectively solve these problems. This application can not only extend the service life of buildings, but also reduce maintenance costs and improve the economic and social benefits of projects.

[0004] However, the urease activity of urease-producing microorganisms in the prior art is relatively low, which limits further application, and there are defects and shortcomings such as low mineralization efficiency under anaerobic conditions and low strength of the resulting consolidated body. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides an alkali-resistant urease-producing microbial flora for construction joint biomineralization and a preparation method thereof.

[0006] In the first aspect, the present application provides an alkali-resistant urease-producing microbial flora for biomineralization of construction joints, specifically including the following strains with relative abundance: 4.4-5.2% of Trichoderma, 0.39-0.46% of Enterococcus, 4.8-5.7% of Alcaligenes, 0.50-0.58% of Cellulomonas, 22.4-24.2% of Lysobacter, 1.7-2.4% of Micromonas, 10.8-11.9% of Savagea, 15.1-16.2% of Sarcina, 30.8-32.2% of Chlorobacter, and 0.41-0.48% of Bacillus; the remainder is other strains.

[0007] The present application discloses a microbial composite flora for biomineralization of construction joints, and its preparation and application. The alkali-resistant urease-producing microbial flora for biomineralization includes both aerobic and anaerobic urease-producing bacteria. The present application selects garden soil as a source of microbial screening to screen out an alkali-resistant microbial composite flora with high urease activity for use in oxygen-limited and normal environments, especially in construction joints in engineering. The composite flora liquid obtained through various urease activity tests, aerobic type tests, and mineralization effect tests can be particularly used in construction joints, effectively solving the defects and shortcomings of the prior art in which the mineralization efficiency of urease-producing microorganisms under anaerobic conditions is low and the strength of the resulting consolidated body is not high.

[0008] Preferably, the alkali-resistant urease-producing microbial flora used for biomineralization of construction joints specifically includes the following strains with relative abundance: 4.6-5.2% of Trichoderma, 0.41-0.46% of Enterococcus, 5.0-5.7% of Alcaligenes, 0.54-0.58% of Cellulomonas, 22.4-23.5% of Lysobacter, 1.8-2.4% of Micromonas, 11.1-11.9% of Savagea, 15.1-15.9% of Sarcina, 31.9-32.2% of Chlorella, and 0.46-0.48% of Bacillus; the remainder is other strains.

[0009] Preferably, the alkali-resistant urease-producing microbial flora used for biomineralization of construction joints specifically includes the following strains with relative abundance: 4.6-5.0% of Trichoderma, 0.42-0.44% of Enterococcus, 5.0-5.3% of Alcaligenes, 0.54-0.56% of Cellulomonas, 22.8-23.5% of Lysobacter, 1.8-2.2% of Micromonas, 11.2-11.6% of Savagea, 15.5-15.9% of Sarcina, 31.9-32.1% of Chlorella, and 0.46-0.48% of Bacillus; the remainder is other strains.

[0010] Among them, Savagea in 16S analysis refers to a genus of bacteria, and its classification status is: Savagea belongs to Firmicutes, Bacillus, Bacillales, and Plancoccaceae; the morphological and physiological characteristics are: the bacteria of the genus Savagea are Gram-positive, rod-shaped, non-motile, and can form endospores. For example, the cells of Savagea faecisuis are rod-shaped, with typical morphological characteristics of Bacillus, and its growth temperature range is wide. It can grow between 25℃-45℃, and the optimal growth temperature is about 37℃℃. It can form round, raised, and neatly edged colonies on nutrient agar medium; the gene sequence characteristics are: through 16S rRNA gene sequence analysis, the bacteria of the genus Savagea have a low similarity with species of other genera of Plancoccaceae, and have the highest similarity with Savagea faecisuis con12t; for example, the 16S The rRNA gene sequence similarities were 99.2% and 99.3%, respectively.

[0011] In a second aspect, the present application provides a method for preparing the above-mentioned alkali-resistant urease-producing microbial flora, which specifically comprises the following steps in sequence: (1) Take a garden soil sample, mix the topsoil and deep soil, and make a soil suspension as a bacterial source; (2) adding 2-4 mol / L urea solution to the soil suspension, placing it at 37°C and 100-200 r / min for 66-78 h; after standing, pouring out the supernatant, using a strain screening liquid culture medium containing a urea concentration of (3.5+2n)-(4.5+2n) g / L to perform alkali resistance screening culture 2-4 times according to a gradient, and obtaining a liquid culture medium fermentation bacterial liquid; wherein n is the number of screening times; the conditions of each alkali resistance screening culture are 37°C, 100-200 r / min, and the culture time is 66-78 h; (3) inoculating the fermented bacterial liquid from the liquid culture medium into a solid culture medium for strain screening at a pH of 6.8 to 7.2 by a dilution coating method, and culturing at a constant temperature of 35° C. for 66 to 78 hours; (4) Pick the strain part with obvious red color in the solid culture medium, culture it in the strain culture liquid medium for 66 to 78 hours, replace the culture medium every three days, verify the bacterial solution by urease activity detection method, obtain the second generation of purified urease-producing bacterial flora, and then store it for a long time.

[0012] Preferably, in step (1), the garden soil sample is sampled by taking the floating soil at 1 to 2 cm on the surface and the deep soil at 8 to 12 cm, respectively, and mixing them in a weight ratio of 0.8 to 1.2:0.8 to 1.2.

[0013] Preferably, in step (1), the soil suspension is prepared by air-drying and crushing the soil sample in a ventilated place, passing it through a 0.5 mm soil sieve, and washing it 3 to 4 times with 10 times the weight of deionized water to obtain the soil suspension.

[0014] Preferably, in step (2), the volume ratio of the soil suspension to the urea solution is 1:4.5-5.5; the strain screening liquid culture medium is composed of the following components in concentration: soy peptone 0.8-1.2 g / L, sodium chloride 1.8-2.2 g / L, potassium dihydrogen phosphate 1.8-2.2 g / L, urea (3.5+2n)-(4.5+2n) g / L.

[0015] Preferably, in step (3), the solid culture medium for strain screening is composed of the following components at the following concentrations: 0.8-1.2 g / L soy peptone, 1.8-2.2 g / L sodium chloride, 1.8-2.2 g / L potassium dihydrogen phosphate, 9-11 g / L urea, 3.5-4.5 ml / L 2% phenol red solution, 16-18 g / L agar; pH is 6.8-7.2; In the step (4), the strain culture liquid medium is composed of the following components in concentrations: soy peptone 9-11 g / L, sodium chloride 4.5-5.5 g / L, urea 9-11 g / L, and beef extract 2.8-3.2 g / L.

[0016] Preferably, in step (4), the specific steps of long-term preservation are: inoculating the strain to be preserved on a solid medium for long-term preservation of the strain by a dilution coating method, culturing in a 35°C constant temperature incubator, and transferring to a 0-4°C refrigerator for preservation after 66-78 hours of growth; or using a glycerol preservation method or a liquid paraffin preservation method; The solid culture medium for long-term preservation of the strain is composed of the following components in concentrations: 11-13 g / L of soy peptone, 11-13 g / L of sodium chloride, 2.8-3.2 g / L of beef extract, and 16-18 g / L of agar powder.

[0017] In a third aspect, the present application provides the use of alkali-resistant urease-producing biomineralizing bacteria in repairing concrete cracks.

[0018] In summary, the technical solution of this application has the following effects: The present application selects garden soil as a source of microorganism screening, and the alkali-resistant urease-producing microbial flora screened out has both aerobic and anaerobic urease-producing bacteria, which can be used for biomineralization, and has a high urease activity in oxygen-limited and normal environments, especially for construction joints in engineering. The composite flora liquid obtained through various urease activity tests, aerobic type tests, and mineralization effect tests can be especially used for construction joints, effectively solving the defects and shortcomings of the prior art in which the mineralization efficiency of urease-producing microorganisms under anaerobic conditions is low and the strength of the resulting consolidated body is not high. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The growth and metabolism of the alkali-resistant urease-producing microbial flora in Example 1 at different pH levels.

[0020] Figure 2 The growth conditions of the alkali-resistant urease-producing microbial flora in Example 1 under oxygen-limited and oxygen-unlimited conditions. DETAILED DESCRIPTION

[0021] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in the present application. Example

[0022] Example 1 Example 1 provides an alkali-resistant urease-producing microbial flora for construction joint biomineralization and a preparation method thereof.

[0023] The preparation method of the alkali-resistant urease-producing microbial flora in this embodiment is specifically as follows.

[0024] Culture medium: Liquid culture medium for strain screening: soy peptone 1 g / L, sodium chloride 2 g / L, potassium dihydrogen phosphate 2 g / L, urea (4+2n) g / L, n is the number of screening times.

[0025] Solid culture medium for strain screening: 1g / L soy peptone, 2g / L sodium chloride, 2g / L potassium dihydrogen phosphate, 10g / L urea, 4ml / L phenol red solution (2%), 17g / L agar, PH=7. When preparing the solid culture medium, an appropriate amount of sodium hydroxide (0.1mol / L) solution should be added to keep the culture medium at PH=7. When the last drop of sodium hydroxide (0.1mol / L) solution is added, the solution turns from yellow to red and fades after half a minute of shaking. Stop adding sodium hydroxide solution and the solid culture medium is ready.

[0026] Liquid culture medium for strain culture: soy peptone 10g / L, sodium chloride 5g / L, urea 10g / L, beef extract 3g / L.

[0027] Solid culture medium for long-term preservation of strains: soy peptone 12g / L, sodium chloride 12g / L, beef extract 3g / L, agar powder 17g / L (1) Soil samples were obtained from the garden downstairs of the Civil Engineering and Transportation Experimental Building of Hebei University of Technology. The floating soil at 1.5 cm on the surface and the deep soil at 10 cm (to ensure that urea-producing bacteria including aerobic and anaerobic species were screened out) were respectively taken and mixed in a weight ratio of 1:1. The soil samples were air-dried and crushed in a ventilated place, passed through a 0.5 mm soil sieve, and washed three times with 10 times the weight of deionized water. The washing liquid was used to prepare a soil suspension as a bacterial source.

[0028] (2) Take 150 ml of soil suspension and divide it equally into three conical flasks A, B, and C for parallel experiments. Add 3 mol / L urea solution to 300 ml and seal the conical flasks. Place them in a constant temperature shaker at 37°C and 150 r / min and culture for 72 h.

[0029] After standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 6 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for one screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 8 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for a second screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 10 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for a third screening to obtain a liquid culture medium fermentation bacterial liquid.

[0030] (3) The liquid culture medium fermentation liquid was inoculated into the solid culture medium for strain screening at pH 7.0 by the dilution spreading method, and cultured at 35°C for 72 h.

[0031] (4) Pick the strain part with obvious red color in the solid culture medium, culture it in the strain culture liquid medium for 72 hours, replace the culture medium every three days, verify the bacterial solution by urease activity detection method, obtain the second generation purified urease-producing bacterial flora, and then store it for a long time.

[0032] The specific steps for long-term preservation are: inoculate the strain to be preserved on the solid culture medium for long-term preservation of the strain by the dilution coating method, place it in a 35°C constant temperature incubator for culture, and after 72 hours of growth, transfer it to a 0-4°C refrigerator for preservation, which can be preserved for about one month; or use the glycerol preservation method or liquid paraffin preservation method, which can be preserved for about one year.

[0033] Embodiment 2-3 Examples 2 to 3 respectively provide an alkali-resistant urease-producing microbial flora for construction joint biomineralization and a preparation method thereof.

[0034] The difference between the above embodiment and embodiment 1 is that the specific steps in step (1) are different, as shown below.

[0035] In Example 2: The floating soil at 1 cm from the surface and the deep soil at 12 cm were taken separately, and mixed in a weight ratio of 0.8:1.2. The soil sample was air-dried and crushed in a ventilated place, passed through a 0.5 mm soil sieve, and washed three times with 10 times the weight of deionized water. The washing liquid was used to prepare a soil suspension as a bacterial source.

[0036] In Example 3: the floating soil at 2 cm from the surface and the deep soil at 8 cm were taken respectively, and mixed in a weight ratio of 1.2:0.8. The soil sample was air-dried and crushed in a ventilated place, passed through a 0.5 mm soil sieve, and washed three times with 10 times the weight of deionized water. The washing liquid was used to prepare a soil suspension as a bacterial source.

[0037] The remaining process parameters in the above embodiment are the same as those in embodiment 1.

[0038] Embodiments 4 to 8 Examples 4 to 8 respectively provide an alkali-resistant urease-producing microbial flora for construction joint biomineralization and a preparation method thereof.

[0039] The difference between the above embodiment and embodiment 1 is that the specific steps in step (2) are different, as shown below.

[0040] In Example 4: 150 ml of soil suspension was equally divided into three conical flasks A, B, and C for parallel experiments, 2 mol / L urea solution was added to 300 ml, the conical flasks were sealed, and placed in a constant temperature shaker at 37°C and 150 r / min for 72 h.

[0041] After standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 6 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for one screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 8 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for a second screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 10 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for a third screening to obtain a liquid culture medium fermentation bacterial liquid.

[0042] In Example 5: 150 ml of soil suspension was equally divided into three conical flasks A, B, and C for parallel experiments, 4 mol / L urea solution was added to 300 ml, the conical flasks were sealed, and placed in a constant temperature shaker at 37°C and 150 r / min for 72 h.

[0043] After standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 6 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for one screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 8 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for a second screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 10 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for a third screening to obtain a liquid culture medium fermentation bacterial liquid.

[0044] In Example 6: 150 ml of soil suspension was equally divided into three conical flasks A, B, and C for parallel experiments, 3 mol / L urea solution was added to 300 ml, the conical flasks were sealed, and placed in a constant temperature shaker at 37°C and 150 r / min for 72 h.

[0045] After standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 6 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for primary screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 8 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for secondary screening to obtain liquid culture medium fermentation bacteria liquid.

[0046] In Example 7: 150 ml of soil suspension was equally divided into three conical flasks A, B, and C for parallel experiments, 3 mol / L urea solution was added to 300 ml, the conical flasks were sealed, and placed in a constant temperature shaker at 37°C and 150 r / min for 72 h.

[0047] After standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 5.5 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for 72 hours for primary screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 8.5 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for 72 hours for secondary screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 9.5 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for 72 hours for tertiary screening to obtain liquid culture medium fermentation bacteria liquid.

[0048] In Example 8: 150 ml of soil suspension was equally divided into three conical flasks A, B, and C for parallel experiments, 3 mol / L urea solution was added to 300 ml, the conical flasks were sealed, and placed in a constant temperature shaker at 37°C and 150 r / min for 72 h.

[0049] After standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 6.5 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for primary screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 7.5 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for secondary screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 10.5 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for tertiary screening to obtain liquid culture medium fermentation bacterial liquid.

[0050] The remaining process parameters in the above embodiment are the same as those in embodiment 1.

[0051] Example 9 Example 9 provides an alkali-resistant urease-producing microbial flora for construction joint biomineralization and a preparation method thereof.

[0052] The difference between this embodiment and embodiment 1 is that the culture medium is different, as shown below.

[0053] In Example 9: In step (2), the strain screening liquid culture medium is: 1 g / L tryptone, 2 g / L sodium chloride, 2 g / L potassium dihydrogen phosphate, and (4+2n) g / L urea, where n is the number of screening times.

[0054] In step (3), the solid culture medium for strain screening is: 1 g / L soy peptone, 2 g / L sodium chloride, 2 g / L potassium dihydrogen phosphate, 10 g / L urea, 4 ml / L phenol red solution (2%), 17 g / L agar, pH = 7.5.

[0055] The liquid culture medium for strain culture in step (4) includes: 8 g / L soy peptone, 6 g / L sodium chloride, 10 g / L urea, and 4 g / L beef extract.

[0056] The remaining process parameters in this embodiment are the same as those in Embodiment 1.

[0057] Comparative Example Comparative Examples 1 to 3 Comparative Examples 1 to 3 respectively provide a microbial flora and a preparation method thereof.

[0058] The differences between the preparation method of the microbial flora in the above comparative example and the example are specifically as follows.

[0059] In comparative example 1: the specific steps of step (1) are to take a soil sample from the garden downstairs of the Civil Engineering and Transportation Experimental Building of Hebei University of Technology, take the floating soil 1.5 cm from the surface, air-dry and crush the soil sample in a ventilated place, pass it through a 0.5 mm soil sieve, and wash it three times with 10 times the weight of deionized water, and take the washing liquid to prepare a soil suspension as a bacterial source.

[0060] In comparative example 2: the specific steps of step (1) are to take a soil sample from the garden downstairs of the Civil Engineering and Transportation Experimental Building of Hebei University of Technology, take the deep soil 10 cm from the surface, air-dry and crush the soil sample in a ventilated place, pass it through a 0.5 mm soil sieve, and wash it three times with 10 times the weight of deionized water, and take the washing liquid to prepare a soil suspension as a bacterial source.

[0061] In comparative example 3: the specific steps of step (2) are as follows: 150 ml of soil suspension is equally divided into three conical flasks A, B, and C for parallel experiments; deionized water is added to 300 ml, the conical flasks are sealed, and the flasks are placed in a constant temperature shaker at 37°C and 150 r / min and cultured for 72 h.

[0062] After standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 6 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for one screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 8 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for a second screening; after standing, pour out the supernatant, leaving about 75 ml, add a strain screening liquid culture medium containing a urea concentration of 10 g / L to 300 ml, seal the conical flask, and place it in a constant temperature shaker at 37°C and 150 r / min for culturing 72 hours for a third screening to obtain a liquid culture medium fermentation bacterial liquid.

[0063] The remaining process parameters in the above comparative example are the same as those in Example 1.

[0064] Performance testing (1) Relative abundance ratio of alkali-resistant urease-producing microorganisms The samples prepared in the example were taken by a third party, Shanghai Fuda Testing Technology Group Co., Ltd., to detect the relative abundance ratio of the bacterial flora based on "16S rRNA gene sequence analysis".

[0065] Table 1 Detection results of relative abundance ratio of strains in microbial flora It can be seen from 1 that the alkali-resistant urease-producing microbial flora for construction joint biomineralization obtained by using the technical solution of the present application specifically includes the following strains with relative abundance: 4.4-5.2% of Trichoderma, 0.39-0.46% of Enterococcus, 4.8-5.7% of Alcaligenes, 0.50-0.58% of Cellulomonas, 22.4-24.2% of Lysobacter, 1.7-2.4% of Micromonas, 10.8-11.9% of Savagea, 15.1-16.2% of Sarcina, 30.8-32.2% of Chlorella, and 0.41-0.48% of Bacillus; the remainder is other strains.

[0066] (2) Urease activity of urea-producing bacteria Add 20 ml of the bacterial solution obtained in step (4) into a small beaker and measure its conductivity σ 1 , add 20ml, 1mol / L urea solution, and measure its conductivity σ after 5min 2 , and through the formula U=(σ 1 ~σ 2 )×11.11×10 / t to calculate the urease activity of the bacterial solution. The results are shown in Table 2.

[0067] Where: Table 2 Detection results of bacterial urease activity The urease activity test results in Table 2 show that the urease-producing bacteria selected by this screening method have high urease activity.

[0068] (3) Prepare 250 ml of liquid culture medium with pH values ​​of 9, 10, 11, 12, and 13 respectively, take 10 ml of the cultured bacterial solution obtained in step (4) of Example 1 and add it to the culture medium of each pH value, and place it in a constant temperature shaker at 37°C and 150 r / min for cultivation. Measure the OD600 value of each group of bacteria at 8, 16, 24, 32, 40, and 48 hours to characterize the growth and metabolism of the bacterial community at different pH levels. The results are as follows: Figure 1 The figure shows the growth and metabolism of the alkali-resistant urease-producing microbial flora in Example 1 at different pH levels.

[0069] Figure 1 The results showed that the selected bacterial community could grow and metabolize in a high alkaline environment because of the presence of different types of bacteria.

[0070] (4) Aerobic demand test The purpose of testing the aerobicity of the bacterial flora is to study whether the cultured bacterial flora can survive normally and carry out normal growth and metabolic activities in the anaerobic environment of the construction joint. The growth of the alkali-resistant urease-producing microbial flora in Example 1 under oxygen-limited and oxygen-unlimited conditions is as follows: Figure 2 As shown, the results show that by comparing the growth of the bacterial flora in step (4) of Example 1 under oxygen-limited and oxygen-unlimited conditions, it was found that the growth of the bacterial flora was relatively inhibited under oxygen-limited conditions compared with that under oxygen-unlimited conditions. However, after 48 hours, under oxygen-limited conditions, the OD600 value of the bacterial flora also reached 1.772, and the growth was good, which indicates that this screened bacterial flora can be used in anoxic environments.

[0071] (5) Add 10 ml of the bacterial solution of step (4) in Example 1 to a small beaker, then add 20 ml of 1 mol / L urea solution, and then add 20 ml of 1 mol / L calcium chloride solution. It is observed that a large amount of flocculent precipitation is rapidly generated after the addition of calcium chloride solution. After standing for a period of time, the precipitation at the bottom of the beaker is observed, and the mineralization capacity of the bacterial community is observed by adding hydrochloric acid solution, generating carbon dioxide gas and weighing the weight loss before and after. The experiment found that after about 30 seconds of reaction, all the added calcium ions were precipitated as calcium carbonate.

[0072] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An alkali-resistant urease-producing microbial flora for construction joint biomineralization, characterized in that: Specifically, the strains with relative abundance are as follows: Trichoderma 4.4~5.2%, Enterococcus 0.39~0.46%, Alcaligenes 4.8~5.7%, Cellulomonas 0.50~0.58%, Lysobacter 22.4~24.2%, Micromonas 1.7~2.4%, Savagea 10.8~11.9%, Sarcina 15.1~16.2%, Chlorobacter 30.8~32.2%, Bacillus 0.41~0.48%; the rest are other strains.

2. The alkali-resistant urease-producing microbial flora for construction joint biomineralization according to claim 1, characterized in that: Specifically, the strains with relative abundance are as follows: Trichoderma 4.6~5.2%, Enterococcus 0.41~0.46%, Alcaligenes 5.0~5.7%, Cellulomonas 0.54~0.58%, Lysobacter 22.4~23.5%, Micromonas 1.8~2.4%, Savagea 11.1~11.9%, Sarcina 15.1~15.9%, Chlorobacter 31.9~32.2%, Bacillus 0.46~0.48%; the rest are other strains.

3. The alkali-resistant urease-producing microbial flora for construction joint biomineralization according to claim 1, characterized in that: Specifically, the strains with relative abundance are as follows: Trichoderma 4.6~5.0%, Enterococcus 0.42~0.44%, Alcaligenes 5.0~5.3%, Cellulomonas 0.54~0.56%, Lysobacter 22.8~23.5%, Micromonas 1.8~2.2%, Savagea 11.2~11.6%, Sarcina 15.5~15.9%, Chlorobacter 31.9~32.1%, Bacillus 0.46~0.48%; the rest are other strains.

4. The method for preparing the alkali-resistant urease-producing microbial flora according to any one of claims 1 to 3, characterized in that: Specifically, the following steps are performed in sequence: (1) Take a garden soil sample, mix the topsoil and deep soil, and make a soil suspension as a bacterial source; (2) adding 2-4 mol / L urea solution to the soil suspension, and culturing the suspension at 37°C and 100-200 r / min for 66-78 h; after standing, pouring out the supernatant, and using a strain screening liquid culture medium containing a urea concentration of (3.5+2n)-(4.5+2n) g / L to perform alkali resistance screening and culturing for 2-4 times according to a gradient to obtain a liquid culture medium fermentation bacterial liquid; wherein n is the number of screening times; The conditions for each alkali resistance screening culture were 37°C, 100-200 r / min, and the culture time was 66-78 h; (3) inoculating the fermented bacterial liquid from the liquid culture medium into a solid culture medium for strain screening at a pH of 6.8 to 7.2 by a dilution spreading method, and culturing at a constant temperature of 35° C. for 66 to 78 hours; (4) Pick the strain part with obvious red color in the solid culture medium, culture it in the strain culture liquid culture medium for 66-78 hours, replace the culture medium every three days, verify the bacterial solution by urease activity detection method, obtain the second-generation purified urease-producing bacterial flora, and then store it for a long time.

5. The method for preparing the alkali-resistant urease-producing microbial flora according to claim 4, characterized in that: In the step (1), the garden soil sample is sampled by taking the floating soil at 1-2 cm on the surface and the deep soil at 8-12 cm, respectively, and mixing them in a weight ratio of 0.8-1.2:0.8-1.

2.

6. The method for preparing the alkali-resistant urease-producing microbial flora according to claim 4, characterized in that: In the step (1), the soil suspension is prepared by air-drying and crushing the soil sample in a ventilated place, passing it through a 0.5 mm soil sieve, and washing it 3 to 4 times with 10 times the weight of deionized water to obtain the soil suspension.

7. The alkali-resistant urease-producing microbial flora for construction joint biomineralization according to claim 4, characterized in that: In the step (2), the volume ratio of the soil suspension to the urea solution is 1:4.5-5.5; The strain screening liquid culture medium is composed of the following components in concentrations: 0.8-1.2 g / L soy peptone, 1.8-2.2 g / L sodium chloride, 1.8-2.2 g / L potassium dihydrogen phosphate, and (3.5+2n)-(4.5+2n) g / L urea.

8. The alkali-resistant urease-producing microbial flora for construction joint biomineralization according to claim 4, characterized in that: In the step (3), the solid culture medium for strain screening is composed of the following components at the following concentrations: 0.8-1.2 g / L soy peptone, 1.8-2.2 g / L sodium chloride, 1.8-2.2 g / L potassium dihydrogen phosphate, 9-11 g / L urea, 3.5-4.5 ml / L 2% phenol red solution, and 16-18 g / L agar; the pH is 6.8-7.2; In the step (4), the strain culture liquid medium is composed of the following components in concentrations: soy peptone 9-11 g / L, sodium chloride 4.5-5.5 g / L, urea 9-11 g / L, and beef extract 2.8-3.2 g / L.

9. The alkali-resistant urease-producing microbial flora for construction joint biomineralization according to claim 8, characterized in that: In step (4), the specific steps of long-term preservation are: inoculating the strain to be preserved on a solid culture medium for long-term preservation of strains by dilution coating method, culturing in a 35°C constant temperature incubator, and transferring to a 0~4°C refrigerator for preservation after 66~78h of growth; or using a glycerol preservation method or a liquid paraffin preservation method; The solid culture medium for long-term preservation of the strain is composed of the following components in concentrations: 11-13 g / L of soy peptone, 11-13 g / L of sodium chloride, 2.8-3.2 g / L of beef extract, and 16-18 g / L of agar powder.

10. Use of the alkali-resistant urease-producing biomineralizing bacteria according to any one of claims 1 to 3 in repairing construction joints and concrete cracks.

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