CN112322519B - Microbial composite flora for biomineralization and preparation and application thereof - Google Patents

Abstract

The invention belongs to the field of biomineralization application, and discloses a microbial composite flora for biomineralization and preparation and application thereof, wherein the microbial composite flora for biomineralization comprises a first microorganism with high urease activity and a second microorganism with high carbonic anhydrase activity, and the first microorganism and the second microorganism are both preserved in China center for type culture Collection with the preservation numbers of CCTCC M2020469 and CCTCC M2020468. According to the invention, the microorganism composite flora is obtained by selecting the specific first microorganism and the specific second microorganism, the microorganism composite flora has high urease activity and high carbonic anhydrase activity, and the composite flora liquid obtained by compounding can be especially used for biological mineralization, so that the defects and the defects of low consolidation rate of natural biological cement strain consolidated loose particles and low strength of the obtained consolidated body in the prior art are effectively solved.

Description

Microbial composite flora for biomineralization and preparation and application thereof

Technical Field

The invention belongs to the field of biomineralization application, and particularly relates to a microbial composite flora for biomineralization, as well as preparation and application thereof.

Background

Calcium carbonate precipitation induced by microorganisms is a biological consolidation phenomenon commonly existing in the nature, and some microorganisms can form calcite crystals outside cells through continuous enzymatic action between the metabolism of the microorganisms and surrounding environment media, so that loose sandy soil particles are cemented and mineralized. At present, calcium carbonate precipitation induced by microorganisms is widely applied to the research fields of building foundation reinforcement, concrete crack repair, water conservancy project seepage prevention and the like due to good ecological compatibility. The invention patent CN101644047 discloses a method for cementing loose sand particles by using mineralization of wild microorganism Bacillus pasteurianus, which comprises the steps of sequentially injecting cultured bacteria liquid and mixed solution of urea and calcium chloride into the sand particles, and circulating for several times to form consolidated sand bodies with the highest compression strength of about 2 MPa. In addition, the chinese patent specification CN103725288A discloses a reagent for solidifying loose particles by using bacillus mucilaginosus with carbonic anhydrase activity and a use method thereof, wherein concentrated bacterial liquid of the bacillus mucilaginosus and the loose particles are uniformly stirred according to a certain proportion and then filled into a test mold for compaction, carbon dioxide is continuously introduced from the top of the test mold, and the compressive strength of a consolidated soil sample obtained after mold removal can reach 5 MPa.

However, the microorganisms used for biological consolidation at present are all wild strains, and the urease activity and the carbonic anhydrase activity which are closely related to the consolidation capability in vivo are relatively low, so that the rate of the microorganisms in the consolidation application process is low, and the strength of the formed consolidation body is not high, so that substantial engineering application is not obtained, and the application development of the microorganism induced calcium carbonate precipitation is limited.

Disclosure of Invention

Aiming at the technical defects or improvement requirements of low consolidation rate and low strength of consolidated bodies during biomineralization in the prior art, the invention aims to provide a microbial composite flora for biomineralization and preparation and application thereof, wherein the microbial composite flora is obtained by selecting specific first microorganisms and second microorganisms, the microbial composite flora has high urease activity and high carbonic anhydrase activity, and the composite flora liquid obtained by compounding is particularly applicable to biomineralization, so that the defects and defects of low consolidation rate of natural biological cement strain consolidated loose particles and low strength of the obtained consolidated bodies in the prior art are effectively overcome; the microbial composite flora is used for biological consolidation, the speed of consolidating loose particles can be increased, and the strength of the obtained consolidated body is also obviously improved.

To achieve the above object, according to one aspect of the present invention, there is provided a complex microbial flora for biomineralization, comprising a first microorganism having high urease activity and a second microorganism having high carbonic anhydrase activity, wherein the first microorganism and the second microorganism are both preserved in the chinese typical culture collection with the preservation numbers of CCTCC M2020469 and CCTCC M2020468, respectively.

As a further preferred aspect of the present invention, the microbial complex bacteria is obtained by compounding a first microbial solution and a second microbial solution according to a volume ratio of 1:1 to 10:1, and adding glycerol to a final concentration of 10.0 to 20.0 ml/L; the urease activity of the microbial composite flora liquid obtained after the compounding is 1173-1832U/ml, and the carbonic anhydrase activity is 121-435U/ml.

According to another aspect of the present invention, there is provided a method for preparing a microbial complex flora liquid based on the above-described microbial complex flora for biomineralization, comprising the steps of:

(1) preparing a first microbial liquid with high urease activity: inoculating a first microorganism having high urease activity to the sterilized medium solution, andcontrolling pH to 7.0-7.5, adding chloramphenicol to a final concentration of 5.0-25.0mg/L, and adding NiCl₂Culturing under shaking at 30-37 deg.C for 28-30h to obtain first microorganism solution with high urease activity;

(2) preparing a second microbial liquid with high carbonic anhydrase activity: inoculating a second microorganism with high carbonic anhydrase activity to the sterilized culture medium solution, adding ampicillin to a final concentration of 10.0-100.0mg/L, and adding ZnCl₂Until the final concentration is 5.0-10.0mg/L, shake culturing at 35-37 deg.C for 4-6h, and lowering temperature to 20-28 deg.C for further shake culturing for 5-7 days to obtain second microorganism solution with high carbonic anhydrase activity;

(3) and compounding the first microbial liquid and the second microbial liquid according to the volume ratio of 1:1 to 10:1, adding glycerol to the final concentration of 10.0-20.0ml/L, and uniformly mixing to obtain the microbial composite flora liquid with high urease activity and high carbonic anhydrase activity.

As a further preferred aspect of the present invention, in both of the step (1) and the step (2), the culture medium solution satisfies: the culture medium solution contains tryptone 8.0-12.0g, yeast extract 4.0-6.0g, (NH)₄)₂SO₄ 3.0-3.5g、KH₂PO₄ 6.5-7.0g、Na₂HPO₄6.9-7.3g, glucose 0.5-1.0g, lactose 1.0-3.0g, MgSO₄0.1-0.2g and 3-5ml of glycerol.

As a further preferred aspect of the present invention, in the step (1) and the step (2), the rotation speed for the shaking culture is 100-220 rpm.

According to a further aspect of the present invention, there is provided the use of a complex population of microorganisms for biomineralisation as defined above, in immobilising loose particles characterised in that the loose particles are sand or soil particles.

According to still another aspect of the present invention, there is provided a use of the complex microbial flora liquid obtained by the above preparation method for solidifying loose particles, wherein the use comprises the steps of:

(S1) filling the loose particles into a mould, compacting, adding the microbial composite flora solution obtained by the preparation method, and standing and culturing for 6-12h at 30-37 ℃; the loose particles are sand or soil particles; every 1000g of the loose particles are correspondingly added with 200-500ml of the microorganism composite flora solution;

(S2) adding the compound solution of urea and calcium acetate from the top of the mould each time, repeating for 14-21 days for 2-3 times per day, and removing the mould to obtain a consolidated product; the concentration of urea in the composite solution is 0.5-1.0mol/L, and the concentration of calcium acetate is 0.5-2.0 mol/L; every 1000g of the loose particles are added with 180 ml of the composite solution correspondingly.

Compared with the prior art, the technical scheme of the invention has the advantages that the specific first microorganism and the specific second microorganism are selected, so that the obtained microorganism composite flora has high urease activity and high carbonic anhydrase activity, is obviously higher than that of a natural strain, and can be particularly used for biomineralization. Specifically, the present invention can achieve the following advantageous effects:

(1) according to the invention, specific first microorganisms and specific second microorganisms are selected, wherein the urease activity of an independent first microorganism liquid can reach 2000U/ml, the carbonic anhydrase activity of an independent second microorganism liquid can reach 500U/ml, the urease activity and the carbonic anhydrase activity of the independent first microorganism liquid and the independent second microorganism liquid are preferably compounded according to the volume ratio of the first microorganism liquid to the second microorganism liquid of 1:1 to 10:1, glycerol is added until the final concentration is 10.0 to 20.0ml/L, the urease activity of the compounded bacterial liquid is 1173-1832U/ml, and the carbonic anhydrase activity is 121-435U/ml, so that higher enzyme activity can be kept, and the enzyme activity is obviously higher than that of a common wild strain;

(2) the microbial composite flora based on the first microorganisms and the second microorganisms, which is obtained by the invention, can be particularly used for biologically mineralizing loose particles (such as sand, stone or soil and the like), the speed of consolidating the loose particles can be obviously increased, and the strength of the obtained consolidated body is also obviously improved.

In conclusion, the invention uses the well-constructed engineering bacteria with high urease activity and high carbonic anhydrase activity to complete the preparation of the composite flora, the activity of the composite flora is higher than that of natural strains, the composite flora has better cementing effect (correspondingly, the cementing rate is higher) in the same time when being applied to curing loose particles, and in addition, the obtained cementing body has higher strength.

Drawings

Fig. 1 is a diagram showing a standard sand consolidation body prepared by using a composite bacterial flora and a loose particle object before consolidation, wherein (a) in fig. 1 corresponds to a standard sand loose particle, (b) in fig. 1 corresponds to a loading mold, and (c) in fig. 1 corresponds to a consolidation body sand column.

Fig. 2 is a stress-strain diagram of a standard sand consolidation body prepared using composite flora.

FIG. 3 is a diagram showing a sample of a standard sand solidification body prepared by using a common wild strain and complex bacteria, respectively, wherein (a) in FIG. 3 corresponds to a Pasteurella sarcina solidification body, and (b) in FIG. 3 corresponds to a complex bacteria solidification body.

FIG. 4 is a graph showing the comparison of the flexural strength and compressive strength of a standard sand consolidation body prepared by using common wild strains and complex flora respectively.

Fig. 5 is a microscopic structure view of the inside of a standard sand solidification body prepared using a common wild strain and complex bacteria, respectively, wherein (a) in fig. 5 corresponds to a sporosarcina pasteurii solidification body and (b) in fig. 5 corresponds to a complex bacteria solidification body.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The microorganisms (A) with high urease activity and the microorganisms (B) with high carbonic anhydrase activity used in the application are constructed by the inventor team of the invention based on molecular biology technology, are preserved in China Center for Type Culture Collection (CCTCC), and have the preservation numbers of CCTCC M2020469 and CCTCC M2020468, which are named as Bacillus subtilis and Escherichia coli respectively; the preservation time is 9 months and 4 days in 2020; the preservation unit is China center for type culture Collection; and (4) storage address: wuhan university Collection, Lodoku mountain, Wuchang, Wuhan, Hubei province. The microorganism (A) having high urease activity is a first microorganism, and the microorganism (B) having high carbonic anhydrase activity is a second microorganism.

In general, the present invention is based on the synergistic effect of the above-mentioned microorganisms (A) with high urease activity and microorganisms (B) with high carbonic anhydrase activity in solidifying loose particles of sand, soil, etc., thereby realizing solidifying loose particles of sand, soil, etc. Either one of these alone or the original wild strains of Bacillus subtilis and Escherichia coli used alone had no or poor solidifying effect. The specific curing operation method comprises the following steps:

(1) preparing a microorganism (A) bacterial solution with high urease activity: inoculating microorganism (A) with high urease activity into sterilized culture medium solution containing tryptone 8.0-12.0g, yeast extract 4.0-6.0g, (NH) per liter₄)₂SO₄3.0-3.5g、KH₂PO₄ 6.5-7.0g、Na₂HPO₄6.9-7.3g, glucose 0.5-1.0g, lactose 1.0-3.0g, MgSO₄0.1-0.2g, 3-5ml of glycerol, and controlling pH to 7.0-7.5, and adding chloramphenicol to a final concentration of 5.0-25.0mg/L and NiCl₂Culturing under shaking at 30-37 deg.C for 28-30 hr at 200 rpm until the final concentration is 5.0-10.0mg/L to obtain bacterial solution of microorganism (A) with high urease activity;

(2) preparing a microorganism (B) bacterial solution with high carbonic anhydrase activity: inoculating the microorganism (B) having high carbonic anhydrase activity to the sterilized medium solution in the same manner as in the step (1), and adding ampicillin to a final concentration of 10.0-100.0mg/L and ZnCl₂Until the final concentration is 5.0-10.0mg/L, performing shake culture at 35-37 deg.C for 4-6h at 200 rpm, and lowering the temperature to 20-28 deg.C for further shake culture for 5-7 days to obtain the bacterial solution of microorganism (B) with high carbonic anhydrase activity;

(3) compounding the microbial bacterium liquid of the microorganism (A) and the microbial bacterium liquid of the microorganism (B) according to the volume ratio of 1:1 to 10:1, adding glycerol to the final concentration of 10.0 to 20.0ml/L, uniformly mixing, and storing at the temperature of minus 4 ℃ for later use;

(4) filling 1000g of loose particles such as sand, soil and the like into a mold for compaction, slowly adding 500ml of 200-fold composite bacterial liquid obtained in the step (3), and standing and culturing for 6-12h at 30-37 ℃;

(5) adding 180-200ml of urea with the concentration of 0.5-1.0mol/L and 0.5-2.0mol/L of calcium acetate composite solution from the top of the mold each time, repeating for 14-21 days 2-3 times per day, and obtaining a consolidated sample after demolding.

The following are specific examples:

example 1:

weighing tryptone 12.0g, yeast extract 4.0g and (NH)₄)₂SO₄ 3.0g、KH₂PO₄ 7.0g、Na₂HPO₄7.3g, glucose 1.0g, lactose 3.0g, MgSO₄0.1g and 3ml of glycerol, adding purified water to dissolve, fixing the volume to 1L, measuring the pH value to 7.5, evenly subpackaging into 10 triangular flasks of 500ml, sterilizing at 118 ℃ for 20min, taking out and cooling to room temperature for later use. Taking a bottle of the culture medium, sequentially adding the chloramphenicol with suction filtration sterilization to a final concentration of 20.0mg/L and NiCl₂Inoculating the microorganism (A) strain with high urease activity until the final concentration is 5.0mg/L, and performing shaking culture at 37 ℃ for 30h at 200 rpm to obtain the microorganism (A) strain with high urease activity. Taking a bottle of the sterilized culture medium, and sequentially adding suction filtration sterilized ampicillin to a final concentration of 50.0mg/L and ZnCl₂Inoculating the microorganism (B) strain with high carbonic anhydrase activity until the final concentration is 5.0mg/L, performing shake culture at 37 deg.C for 6h at 200 rpm, and then lowering the temperature to 24 deg.C for further shake culture for 5 days to obtain the bacterial liquid of the microorganism (B) with high carbonic anhydrase activity. And compounding 100ml of microbial (A) bacterial liquid and 10ml of microbial (B) bacterial liquid, quickly adding 2ml of glycerol, uniformly mixing, and sampling to obtain the urease and carbonic anhydrase activities of the compound bacterial liquid of 1832U/ml and 121U/ml respectively.

Filling 60g of Chinese standard sand into a mold with the inner diameter of 29mm and the height of 100mm, compacting, slowly adding 30ml of the composite bacterial liquid, and standing and culturing at 37 ℃ for 6 hours. Slowly adding 30ml of urea with the concentration of 0.5mol/L and 2.0mol/L of calcium acetate composite solution from the top of the mold, repeating for 14 days at intervals of 8 hours three times a day, obtaining a solidified sample after demolding (shown in figure 1), and measuring the unconfined compressive maximum strength to be 7.9MPa (shown in figure 2) after washing and drying.

Example 2:

weighing tryptone 10.0g, yeast extract 5.0g and (NH)₄)₂SO₄ 3.5g、KH₂PO₄ 6.8g、Na₂HPO₄7.1g, glucose 0.5g, lactose 2.0g, MgSO₄0.2g and 5ml of glycerol, adding purified water to dissolve, fixing the volume to 1L, subpackaging in 10 triangular flasks of 500ml on average, sterilizing at 118 ℃ for 20min, taking out and cooling to room temperature for later use. Taking a bottle of the culture medium, sequentially adding the chloramphenicol with suction filtration sterilization to a final concentration of 5.0mg/L and NiCl₂Inoculating the microorganism (A) strain with high urease activity until the final concentration is 10.0mg/L, and performing shaking culture at 37 ℃ for 28h at 200 r/min to obtain the microorganism (A) bacterial liquid with high urease activity. Taking a bottle of sterilized culture medium, sequentially adding suction filtration sterilized ampicillin to a final concentration of 100.0mg/L and ZnCl₂Inoculating the microorganism (B) strain with high carbonic anhydrase activity until the final concentration is 10.0mg/L, performing shake culture at 37 deg.C for 4h at 200 rpm, and then lowering the temperature to 24 deg.C for further shake culture for 6 days to obtain the bacterial liquid of the microorganism (B) with high carbonic anhydrase activity. 100ml of microorganism (A) bacterial liquid and 100ml of microorganism (B) bacterial liquid are compounded, 2ml of glycerol is immediately added, the urease and the carbonic anhydrase activities are respectively 1173U/ml and 435U/ml (shown in table 1) after the mixture is uniformly mixed and sampled, and the rest is temporarily stored in a refrigerator at the temperature of-4 ℃.

TABLE 1 measurement of urease and carbonic anhydrase activities in different combinations

460g of Chinese standard sand is filled into a mold with the thickness of 160mm x 40mm x 60mm for compaction, 92ml of the composite bacterial liquid is slowly added, and the mixture is kept stand and cultured for 6 hours at the temperature of 37 ℃. 90ml of urea with the concentration of 0.5mol/L and 2.0mol/L of calcium acetate composite solution are added from the top of a mold, the process is repeated for 21 days in the morning and at the evening each day, a consolidated sample is obtained after the mold is removed (shown as (b) in figure 3), after washing and drying, the three-point flexural strength is measured to be 4.8MPa, the unconfined compressive strength is 17.3MPa (shown as figure 4), and the fracture surface structure is observed by using an electronic scanning electron microscope.

Comparative example:

weighing tryptone 15.0g, soybean peptone 5.0g and NaCl5.0g, adding purified water to dissolve, fixing the volume to 900ml, sterilizing at 121 ℃ for 20min, taking out and cooling to room temperature for later use. Weighing 20.0g of urea, adding purified water to dissolve, fixing the volume to 900ml, performing suction filtration and sterilization, and adding the urea into the culture medium which is sterilized at high temperature before use. Aseptically, 100ml was taken into 500ml Erlenmeyer flasks, inoculated with Sporosarcina pasteurii (ATCC 11859), and cultured at 30 ℃ for 24 hours with shaking at 170 rpm. Samples were taken and assayed for urease and carbonic anhydrase activity of 651U/ml and 10U/ml, respectively (as shown in Table 1), the remainder being stored in a freezer at-4 ℃.

460g of Chinese standard sand is filled into a mold with the thickness of 160mm x 40mm x 60mm for compaction, 92ml of Sporosarcina pasteurii bacterial liquid is slowly added, and the mixture is kept stand and cultured for 6 hours at the temperature of 30 ℃. 90ml of urea with the concentration of 0.5mol/L and 2.0mol/L of calcium acetate composite solution are added from the top of a mold, the process is repeated for 21 days in the morning and at the evening of each day, a consolidated sample is obtained after the mold is removed (as shown in (a) in figure 3), after washing and drying, the three-point rupture strength is measured to be 3.2MPa, the unconfined compressive strength is 10.3MPa, and the consolidation effect of two strains is compared, so that the strength of a consolidated body obtained by induced mineralization of the composite flora prepared by the method is remarkably improved (as shown in figure 4). The fracture surface structure of the sarcina pasteurii is observed by using an electronic scanning electron microscope, the number of calcite crystals generated by induction of sarcina pasteurii is small, the volume is small, the packing density among sand grains is low, and large gaps still exist among the sand grains (as shown in figure 5). In comparison, in the same time, the quantity and the volume of calcite crystals generated by the induction of the composite flora prepared by the method are larger, so that a more compact solidification structure is formed among sand grains, and the speed of consolidating loose particles can be effectively improved by the composite flora prepared by the method.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (7)

1. The microbial composite flora for biomineralization is characterized by consisting of a first microorganism with high urease activity and a second microorganism with high carbonic anhydrase activity, wherein the first microorganism is named as Bacillus subtilis 2019/7-UE, is preserved in China center for type culture Collection (CCTCC NO) in 9 and 4 months 2020, and has the preservation number of CCTCC NO: m2020469; the second microorganism is named as Escherichia coli (Escherichia coli)2018/6-CA, is preserved in China Center for Type Culture Collection (CCTCC) in 9 months and 4 days in 2020, and has a preservation number of CCTCC NO: m2020468.

2. The microbial complex bacteria of claim 1, wherein the microbial complex bacteria is obtained by compounding a first microbial solution and a second microbial solution according to a volume ratio of 1:1 to 10:1 and adding glycerol to a final concentration of 10.0-20.0 ml/L; the urease activity of the microbial composite flora liquid obtained after the compounding is 1173-1832U/ml, and the carbonic anhydrase activity is 121-435U/ml.

3. The method for preparing a microbial consortium liquid based on the microbial consortium for biomineralization according to claim 1 or 2, comprising the steps of:

(1) preparing a first microbial liquid with high urease activity: inoculating the first microorganism with high urease activity into the sterilized culture medium solution, controlling pH to 7.0-7.5, adding chloramphenicol to a final concentration of 5.0-25.0mg/L, and adding NiCl₂Culturing under shaking at 30-37 deg.C for 28-30h to obtain first microorganism solution with high urease activity;

(2) preparing a second microbial liquid with high carbonic anhydrase activity: inoculating a second microorganism having high carbonic anhydrase activity to the sterilized culture mediumAdding ampicillin to a final concentration of 10.0-100.0mg/L, and adding ZnCl₂Until the final concentration is 5.0-10.0mg/L, shake culturing at 35-37 deg.C for 4-6h, and lowering temperature to 20-28 deg.C for further shake culturing for 5-7 days to obtain second microorganism solution with high carbonic anhydrase activity;

(3) and compounding the first microbial liquid and the second microbial liquid according to the volume ratio of 1:1 to 10:1, adding glycerol to the final concentration of 10.0-20.0ml/L, and uniformly mixing to obtain the microbial composite flora liquid with high urease activity and high carbonic anhydrase activity.

4. The method according to claim 3, wherein the medium solution satisfies, in both of the step (1) and the step (2): the culture medium solution contains tryptone 8.0-12.0g, yeast extract 4.0-6.0g, (NH)₄)₂SO₄ 3.0-3.5g、KH₂PO₄ 6.5-7.0g、Na₂HPO₄6.9-7.3g, glucose 0.5-1.0g, lactose 1.0-3.0g, MgSO₄0.1-0.2g and 3-5ml of glycerol.

5. The method according to claim 3, wherein the rotation speed for the shaking culture in the steps (1) and (2) is 100-220 rpm.

6. Use of a complex population of microorganisms for biomineralization according to claim 1 or 2, for solidifying loose particles, characterized in that said loose particles are sand or soil particles.

7. The use of a complex microbial consortium fluid obtained by the method of any one of claims 3 to 5 for consolidating loose granules, comprising the steps of:

(S1) filling the loose particles into a mould, compacting, adding the microbial composite flora solution obtained by the preparation method, and standing and culturing for 6-12h at 30-37 ℃; the loose particles are sand or soil particles; every 1000g of the loose particles are correspondingly added with 200-500ml of the microorganism composite flora solution;

(S2) adding the compound solution of urea and calcium acetate from the top of the mould each time, repeating for 14-21 days for 2-3 times per day, and removing the mould to obtain a consolidated product; the concentration of urea in the composite solution is 0.5-1.0mol/L, and the concentration of calcium acetate is 0.5-2.0 mol/L; every 1000g of the loose particles are added with 180 ml of the composite solution correspondingly.

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