A strong alkali-resistant carbonic anhydrase-producing bacterium and its application and method

Abstract

The present invention belongs to the technical field of microbial mineralization, and discloses a strong alkali-resistant carbonic anhydrase-producing bacterium, whose name is X27.1, classification name is Halalkalibacterium halodurans, preservation number is CGMCCNo.29094, preservation date is November 22, 2023, preservation unit is General Microbiological Center of China Microbiological Culture Collection Administration Committee, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The present invention obtains a strong alkali-resistant carbonic anhydrase-producing bacterium X27.1 by domestication, and its alkali resistance range is pH10-12, and carbonic anhydrase can be produced within the range of pH10-12, and its alkali resistance is relatively strong. The strain has good reproduction ability and calcium carbonate precipitation ability in a strong alkaline environment, and has broad application prospects in the direction of repairing cracks produced in concrete buildings in marine environments.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial mineralization, in particular to a strong alkali-resistant carbonic anhydrase-producing bacterium and an application and method thereof. Background Art

[0002] Microbial induced calcium carbonate deposition (MICP) technology has the characteristics of being green, environmentally friendly and highly efficient. It has now become a hot topic in the field of crack repair in concrete building materials. Concrete cracks in the marine environment provide channels for chloride ions, harmful liquids and gases in the environment to enter deep into the concrete cracks, accelerating the aging of the concrete and seriously affecting the durability and safety of the building. Many existing studies have shown that MICP technology performs well in the field of concrete crack repair, but microbial remediation under marine port environmental conditions faces a high-salt and high-alkali environment (pH 12, NaCl 35‰), which seriously affects the survival of the strain. Halalkalibacterium halodurans is a mineralizing strain that produces carbonic anhydrase and can be used in concrete self-repair. Concrete self-repair is to mix the strain into the concrete. When the concrete cracks, the strain in the concrete comes into contact with oxygen and water to revive, using CO in the air. 2 At the same time, Halalkalibacteriumhalodurans can also be used for passive repair of concrete, that is, adding bacterial liquid and calcium source into the cracked concrete gap, which uses CO in the air to 2 It reacts with calcium sources to produce calcium carbonate precipitation to seal the cracks. Halalkalibacteriumhalodurans itself has adapted to the environment of 35‰ NaCl, but has not yet adapted to the alkaline environment inside the concrete with a pH of 12. In order to solve its survival problem as a concrete self-repairing strain in concrete, it is of great significance to adapt it to the high alkaline environment of concrete.

[0003] The process of calcium carbonate deposition by Halalkalibacterium halodurans is as follows:

[0004] (1) The zinc ion (Zn) in the active center of carbonic anhydrase 2+ ) acts on the water molecules connected to it, reducing the acidolysis constant of the water molecules, causing the water molecules to deprotonate from the active area to form regenerated active ions E·ZnOH - ;

[0005] (2) Due to E·ZnOH - The presence of hydrogen bonds in E·ZnOH - The oxygen atom in the solution has strong nucleophilicity and can combine with hydrated CO2 in the solution to generate E·ZnHCO 3- ;

[0006] (3) Then, H 2 O replaces E·ZnHCO 3- HCO 3- , forming E·ZnOH 2 and HCO 3- ;

[0007] (4) In alkaline solution, HCO 3- With OH - The reaction produces CO 3 2- and H 2 O;

[0008] (5) Since microorganisms and bacteria are negatively charged, Ca 2+ Can be adsorbed and accumulated on its surface;

[0009] (6) Microorganisms act as nucleation sites, promoting the deposition of mineralized products on their surfaces.

[0010] In the prior art, there is no carbonic anhydrase-producing bacterial strain with similar functions that can adapt to the environment with NaCl of 35‰ and pH of 12. 2 Mineralization can reduce carbon emissions and is a more environmentally friendly mineralizing strain. Summary of the invention

[0011] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a strong alkali-resistant carbonic anhydrase-producing bacterium and its application and method.

[0012] The technical solution adopted by the present invention to solve its technical problem is:

[0013] A strain of alkali-resistant carbonic anhydrase-producing bacteria, named X27.1, classified as Halalkalibacterium halodurans, with a deposit number of CGMCC No.29094, a deposit date of November 22, 2023, and a depository unit: General Microbiology Center of China National Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0014] Furthermore, after the carbonic anhydrase-producing bacteria were cultured on a nutrient agar medium at 30 degrees Celsius for 24 hours, the colonies were light yellow, translucent and round, with a moist and smooth surface, and a small single colony; the bacteria were rod-shaped under a scanning electron microscope.

[0015] Furthermore, the carbonic anhydrase-producing bacteria can tolerate a pH value of 10-12 under aerobic conditions.

[0016] Furthermore, the carbonic anhydrase-producing bacteria can survive in a strongly alkaline condition with a pH value of 10-12 and utilize CO in the air.2 Calcium ions formed by dissolution in water produce calcium carbonate.

[0017] An application of the alkali-resistant carbonic anhydrase-producing bacteria as described above in microbial mineralization.

[0018] An application of the alkali-resistant carbonic anhydrase-producing bacteria as described above in active microbial concrete repair.

[0019] Furthermore, the alkali-resistant carbonic anhydrase-producing bacteria is added into concrete and stirred.

[0020] An application of the alkali-resistant carbonic anhydrase-producing bacteria as described above in passive repair of concrete cracks.

[0021] An application of the alkali-resistant carbonic anhydrase-producing bacteria as described above in repairing concrete cracks in a marine environment.

[0022] A method for repairing concrete cracks in a marine environment using the alkali-resistant carbonic anhydrase-producing bacteria as described above.

[0023] The advantages and positive effects achieved by the present invention are:

[0024] 1. The present invention obtains a strong alkali-resistant carbonic anhydrase-producing bacterium X27.1 through domestication. The bacterium is a carbonic anhydrase-producing bacterium with an alkali resistance range of pH 10-12. It can produce carbonic anhydrase within the range of pH 10-12 and has strong alkali resistance. The strain has good reproduction ability and calcium carbonate precipitation ability in a strong alkali environment, and has broad application prospects in the direction of repairing cracks produced in concrete buildings in marine environments.

[0025] 2. The strain of the present invention has good ability to reproduce and produce calcium carbonate precipitation in a strong alkaline environment, and has broad application prospects in concrete self-repair applications. The strain can carry out microbial mineralization in a strong alkaline environment, solving the problem of the strong alkaline environment of concrete adapted to the marine environment inhibiting its growth, thereby affecting the carbonic anhydrase-producing bacteria in concrete self-repair, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a colony diagram of the alkali-resistant carbonic anhydrase-producing bacteria in the present invention;

[0027] Figure 2 A scanning electron microscope (SEM) of the alkali-resistant carbonic anhydrase-producing bacteria of the present invention;

[0028] Figure 3 This is a comparison chart of OD600 of the carbonic anhydrase-producing bacteria starting strain and the alkali-resistant acclimated strain in a pH 12, NaCl 35g / L medium in the present invention;

[0029] Figure 4It is a pH comparison diagram of the carbonic anhydrase-producing strain and the alkali-resistant strain in the original culture medium and the pH 12, NaCl 35g / L culture medium;

[0030] Figure 5 It is a comparison chart of the relative enzyme activity of carbonic anhydrase of the carbonic anhydrase-producing strain and the strong alkali-resistant acclimated strain in the original culture medium and the pH 12, NaCl 35g / L culture medium;

[0031] Figure 6 It is a comparison chart of the amount of calcium carbonate precipitation of the carbonic anhydrase-producing strain and the alkali-resistant strain in the original culture medium and the pH 12, NaCl 35g / L culture medium;

[0032] Figure 7 The figure is a scanning electron microscope (SEM) of calcium carbonate of the carbonic anhydrase-producing strain and the alkali-resistant domesticated strain in the original culture medium and the culture medium with pH 12 and NaCl 35 g / L; wherein, the left figure is an electron microscope image of calcium carbonate precipitation of the starting strain; and the right figure is an electron microscope image of calcium carbonate precipitation of the domesticated strain.

[0033] A strain of alkali-resistant carbonic anhydrase-producing bacteria, named X27.1, classified as Halalkalibacteriumhalodurans, deposited on November 22, 2023, deposited at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0035] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0036] A strain of alkali-resistant carbonic anhydrase-producing bacteria, named X27.1, classified as Halalkalibacteriumhalodurans, deposited on November 22, 2023, deposited at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0037] Preferably, after the carbonic anhydrase-producing bacteria are cultured on a nutrient agar medium at 30 degrees Celsius for 24 hours, the colonies are light yellow, translucent and round, with a moist and smooth surface, and a small single colony; the bacteria are rod-shaped under a scanning electron microscope.

[0038] Preferably, the carbonic anhydrase-producing bacteria tolerates a pH value of 10-12 under aerobic conditions.

[0039] Preferably, the carbonic anhydrase-producing bacteria can survive in a strongly alkaline pH of 10-12 and utilize CO in the air. 2 Calcium ions formed by dissolution in water produce calcium carbonate.

[0040] An application of the alkali-resistant carbonic anhydrase-producing bacteria as described above in microbial mineralization.

[0041] An application of the alkali-resistant carbonic anhydrase-producing bacteria as described above in active microbial concrete repair.

[0042] Preferably, the alkali-resistant carbonic anhydrase-producing bacteria is added into concrete and stirred.

[0043] An application of the alkali-resistant carbonic anhydrase-producing bacteria as described above in passive repair of concrete cracks.

[0044] An application of the alkali-resistant carbonic anhydrase-producing bacteria as described above in repairing concrete cracks in a marine environment.

[0045] The method for repairing cracks in marine environment concrete using the alkali-resistant carbonic anhydrase-producing bacteria as described above comprises the following steps:

[0046] The alkali-resistant carbonic anhydrase-producing bacteria are mixed into the concrete, and the concrete is revived after cracking, using CO in the air. 2 The calcium ions produced in the dissolved water are deposited as calcium carbonate, which is used to repair concrete cracks.

[0047] Specifically, the relevant preparation and detection are as follows:

[0048] In the examples, the OD600 value was measured by Eppendorf BioPhotometer D30; and the pH was measured by Shanghai Leici PHSJ-4A.

[0049] Example 1 Alkali-resistance acclimation of carbonic anhydrase-producing bacteria

[0050] 1) The activation medium for carbonic anhydrase-producing bacteria is as follows: 5 g yeast extract, 10 g glucose, K 2 H 4 1g, 10g of peptone was dissolved in 900ml of distilled water and sterilized at high temperature, 45ml of culture medium was added to a 250ml conical flask, and then 5ml of 10% Na2CO3 sterilized by membrane was added.

[0051] 2) In a clean bench, a single colony of carbonic anhydrase-producing bacteria numbered ATCC 27557 purchased from ATCC was selected and inoculated into 30 ml of sterilized culture medium for activation.

[0052] 3) The alkali-resistant acclimatization medium for carbonic anhydrase-producing bacteria is as follows: 5 g yeast extract, 10 g glucose, K 2 HPO 4 1g, 10g peptone, 35g / L NaCl dissolved in 900ml distilled water and sterilized at high temperature, 45ml culture medium was added to a 250ml conical flask, and then 5ml of 10% NaCl sterilized by membrane was added. 2 Co 3 , and then adjusted the pH (=9, 9.5, 10, 10.5, 11, 12) with 10 M NaOH.

[0053] 4) Transfer 5% of the activated bacterial solution into culture media with different pH values, place in a shaker (30°C, 200 r / min) and culture for 24 h. Measure the OD to determine the initial concentration of strong base acclimation.

[0054] 5) The initial pH of the strong alkaline acclimation of carbonic anhydrase-producing bacteria is 10. After the OD of the gradient-acclimated bacteria reaches stability, it is transferred to the next stage.

[0055] 6) Observation of bacterial morphology: Scanning electron microscopy (SEM) observation, such as Figure 1 , Figure 2 As shown, it can be seen that after the carbonic anhydrase-producing bacteria of the present invention are cultured on a nutrient agar medium at 30° C. for 24 hours, the colonies are light yellow, translucent and round, with a moist and smooth surface, and the single colony is small; the bacteria are rod-shaped under a scanning electron microscope.

[0056] 7) Determination of OD and pH values ​​of carbonic anhydrase-resistant bacteria before and after acclimation, such as Figure 3 , Figure 4 As shown, the OD value of the strain after domestication in the stress environment is 7.49, and the OD value of the starting strain in the non-stress environment is 7.46, with little difference between the two, while the OD value of the starting strain in the stress environment is 0.329; the pH value of the domesticated strain at the end of fermentation in the stress environment is 9.235, and the pH value of the strain before domestication at the end of fermentation in the non-stress environment is 9, also with little difference, while the pH of the starting strain in the stress environment is 9.9; This indicates that the carbonic anhydrase-producing bacteria after domestication can survive normally in an environment with a pH of 12.

[0057] Example 2 Strain Identification

[0058] The method for determining the activity of carbonic anhydrase-producing bacteria is as follows:

[0059] 1) The culture medium of X27.1, a strong alkali-resistant carbonic anhydrase-producing bacterium, is as follows: 5 g yeast extract, 10 g glucose, and K 2 H 4 1g, 10g peptone, 35g / L NaCl dissolved in 900ml distilled water and sterilized at high temperature, 45ml culture medium was added to a 250ml conical flask, and then 5ml of 10% NaCl sterilized by membrane was added. 2 CO 3 , and then adjusted to pH 12 with 10 M NaOH.

[0060] 2) The culture medium for the carbonic anhydrase-producing strain is as follows: 5 g yeast extract, 10 g glucose, K 2 HPO 4 1g, 10g of peptone was dissolved in 900ml of distilled water and sterilized at high temperature, 45ml of culture medium was added to a 250ml conical flask, and then 5ml of 10% Na 2 CO 3 .

[0061] 3) The domesticated and undomesticated carbonic anhydrase-producing bacteria were transferred to a sterilized fresh culture medium and cultured at 200 r / min and 30° C. for 48 h, and the supernatant was obtained by centrifugation.

[0062] 4) Carbonic anhydrase has esterase activity and can catalyze the reaction of p-nitrophenyl acetate to generate p-nitrophenol. The activity of carbonic anhydrase can be reflected by detecting the rate of increase of absorbance at 400 nm. The activity of carbonic anhydrase is determined by colorimetry: 1mM 3-NPA is dissolved in 1ml acetone, 0.156g ethylenedimethylmalonic acid is added to PB at pH 6.8, and mixed to make a working solution.

[0063] 5) Use 0.5-3 mmol p-nitrophenol culture medium to prepare a standard curve.

[0064] 6) The working solution and the supernatant were reacted at 30°C for 5 min in a volume ratio of 1:1, and the absorbance was measured at a wavelength of 400. The relative enzyme activity was calculated with the highest enzyme activity as 100%, and the changes in carbonic anhydrase of the strain before and after acclimation were compared, such as Figure 5 As shown, the relative enzyme activity of the carbonic anhydrase-producing bacteria after acclimation in the stress environment was 60%, the relative enzyme activity of the starting strain of carbonic anhydrase-producing bacteria in the non-stress environment was 100%, and no esterase activity was detected in the starting strain in the stress environment.

[0065] Example 3 Method for preparing calcium carbonate using carbonic anhydrase producing bacteria

[0066] The steps for preparing calcium carbonate using the alkali-resistant carbonic anhydrase-producing bacteria X27.1 are as follows:

[0067] 1) The culture medium for alkaline-resistant carbonic anhydrase-producing bacteria is as follows: 5 g yeast extract, 10 g glucose, K2 HPO 4 1 g of peptone, 10 g of peptone, 35 g / L of NaCl are dissolved in 900 ml of distilled water and sterilized at high temperature. 45 ml of the medium is added to a 250 ml conical flask, and then 5 ml of 10% Na 2 Co 3 is added. Then, the pH is adjusted to 12 with 10 M NaOH.

[0068] 2) The medium for the starting strain producing carbonic anhydrase is as follows: 5 g of yeast extract, 10 g of glucose, K 2 HPO 4 1 g of peptone, 10 g of peptone are dissolved in 900 ml of distilled water and sterilized at high temperature. 45 ml of the medium is added to a 250 ml conical flask, and then 5 ml of 10% Na 2 CO 3 is added.

[0069] 3) The strong alkali-resistant carbonic anhydrase-producing bacteria are transferred to a sterilized fresh medium for cultivation.

[0070] 4) Weigh 1.5 mol of calcium acetate and dissolve it in 1 L of distilled water to prepare a mineralization mixture.

[0071] 5) Take X27.1 and the starting strain (i.e., the carbonic anhydrase-producing bacteria numbered ATCC 27557) at a volume ratio of 1:1 and mix them evenly with calcium acetate. Place them at 30 °C and let them stand for mineralization reaction. After 72 h, take out the precipitate, wash it 3 times with distilled water, place it in an oven at 60 °C and dry it for 3 h, then take it out to obtain calcium carbonate product. The results are as Figure 6 、 Figure 7 shown. It can be seen that the amount of calcium carbonate precipitate generated by the starting strain cultured in a non-stress environment and reacted with calcium acetate is about 0.25 g, and the precipitate generated by the domesticated strain in a pH 12 environment is 0.21 g.

[0072] Example 4 Method for actively repairing concrete cracks in the marine environment using carbonic anhydrase-producing bacteria

[0073] The steps for repairing marine concrete cracks using strong alkali-resistant carbonic anhydrase-producing bacteria X27.1 are as follows:

[0074] 1) Medium for strong alkali-resistant carbonic anhydrase-producing bacteria X27.1: 5 g of yeast extract, 10 g of glucose, K 2 HPo 4 1 g of peptone, 10 g of peptone, 35 g / L of NaCl are dissolved in 900 ml of distilled water and sterilized at high temperature. 45 ml of the medium is added to a 250 ml conical flask, and then 5 ml of 10% Na 2 Co 3 is added. Then, the pH is adjusted to 12 with 10 M NaOH.

[0075] 2) premixing the alkali-resistant carbonic anhydrase-producing bacteria into concrete to make test blocks, and inserting metal sheets when the concrete is not fully hydrated;

[0076] 3) There is a lack of water medium and oxygen in the concrete, and the alkali-resistant carbonic anhydrase-producing bacteria are dormant in the concrete. When cracking occurs, external water and oxygen enter, and the dormant bacteria revive;

[0077] 4) Alkali-resistant carbonic anhydrase-producing bacteria and CO in the air 2 A metabolic reaction occurs, producing calcium carbonate to seal the cracks.

[0078] Example 5 Method for passively repairing concrete cracks in marine environment using carbonic anhydrase-producing bacteria

[0079] The steps for passively repairing marine concrete cracks using alkali-resistant carbonic anhydrase-producing bacteria X27.1 are as follows:

[0080] 1) Alkali-resistant carbonic anhydrase-producing bacteria culture medium: 5g yeast extract, 10g glucose, K 2 HPO 4 1g, 10g peptone, 35g / L NaCl dissolved in 900ml distilled water and sterilized at high temperature, add 45ml culture medium in a 250ml conical flask, and then add 5ml of 10% NaCl sterilized by membrane 2 CO 3 , and then adjusted to pH 12 with 10 M NaOH.

[0081] 2) Transfer the carbonic anhydrase-producing bacteria to a sterilized fresh culture medium for cultivation.

[0082] 3) Weigh 1.5 mol of calcium acetate and dissolve it in 1 L of distilled water to prepare a mineralization solution.

[0083] 4) Inject the cultured X27.1 bacterial solution into the concrete cracks, and then add the mineralizing solution mentioned in 3) into the cracks. X27.1 utilizes CO in the air to 2 The two are mixed to produce calcium carbonate, and bacterial liquid and mineralizing liquid are added multiple times according to the crack blocking situation to block the cracks.

[0084] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A strong alkali-resistant carbonic anhydrase-producing bacterium, characterized in that: Its name is X27.1 and its classification name is: Halalkalibacterium halodurans , the deposit number is: CGMCC No.29094, the deposit date is: November 22, 2023, the deposit unit is: General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. Use of the alkali-resistant carbonic anhydrase-producing bacteria as claimed in claim 1 in microbial mineralization.

3. Use of the alkali-resistant carbonic anhydrase-producing bacteria as claimed in claim 1 in active repair of microbial concrete.

4. The use according to claim 3, characterized in that: The alkali-resistant carbonic anhydrase-producing bacteria are added into concrete and stirred.

5. Use of the alkali-resistant carbonic anhydrase-producing bacteria as claimed in claim 1 in passive repair of concrete cracks.

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