Strong alkaline condition tolerant sporosarcina pasteurii and application thereof

Abstract

The invention discloses a sporosarcina pasteurii DA13 (sporosarcina pasteurii DA13) strain capable of tolerating a strong alkaline condition, and an application of the sporosarcina pasteurii DA13 strain. The bacterial strain is obtained by carrying out mutagenesis on a sporosarcina pasteurii DSM33 host by adopting an ARTP mutagenesis technology, and carrying out primary screening by utilizing a strong alkaline screening plate, carrying out high-throughput secondary screening by utilizing a 96-pore plate and carrying out shake flask fermentation screening verification. Compared with an original strain, the mutant strain DA13 can tolerate the pH of 10.0, and the enzyme activity of urease in a unit cell is improved by 16.3%. When the sporosarcina pasteurii mutant strain DA13 is used for curing 0.5 mol / L cementing fluid, the CaCO3 precipitation amount is increased by 4.7%, and the sporosarcina pasteurii mutant strain DA13 has important practical and economic values.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and relates to a mutant strain of Sporosarcina pasteurii that can tolerate strong alkaline conditions and application of the mutant strain in inducing calcium carbonate deposition to prepare bio-bricks. Background Art

[0002] Microbial induced calcium carbonate precipitation (MICP) is a biomineralization phenomenon that is ubiquitous in nature. It uses the mineralization behavior of microbial metabolism to induce calcium carbonate precipitation, forming mineralized structures such as calcite outside the microbial cells, thereby cementing the loose rock and soil particles in the environment to form a solid body with a certain hardness. Microbial solidifiers provide an economical and sustainable option in soil improvement. They are a new type of liquid soil solidifier that can treat soil naturally in a non-toxic, harmless and pollution-free manner. They are dissolved in the soil to make the soil dense and meet relevant technical requirements.

[0003] During the MICP process, Sporosarcina pasteurianum ( Sporosarcina pasteurii ) and other microorganisms express urease to hydrolyze urea, thereby rapidly increasing the pH value and carbonate concentration of the cell microenvironment, forming an alkaline environment required to induce calcium carbonate precipitation.

[0004] Since the activity of urease produced by Sporosarcina pasteurianum is currently the highest, it has been studied in depth as a representative of MICP functional microorganisms. However, its mineralization rate still cannot reach the expected intensity in a short period of time to meet the application of large-scale practical engineering. The optimal environmental pH value of Sporosarcina pasteurianum is 9.25. When the external pH is higher than this value, the bacteria will reduce the urease activity through their own metabolism to prevent the occurrence of conditions that are not conducive to bacterial growth caused by excessively high pH values. This leads to Sporosarcina pasteurii The decrease of urease activity in the late logarithmic phase induced Ca 2+ Therefore, screening for high pH-tolerant mutants of Sporosarcina pasteurianus and improving the pH tolerance range of the Sporosarcina pasteurianus process are of great significance for the application of Sporosarcina pasteurianus in actual MICP engineering. Summary of the invention

[0005] In view of the above problems, the present invention uses ARTP mutagenesis technology to (Sporosarcina) pasteurii) DSM33 host was induced to mutagenesis. First, it was screened on alkaline screening plates with different pH values, then transferred to 96-well plates with corresponding pH values ​​for high-throughput screening, and finally a strain of Sporosarcina pasteurianus mutant DA13 was obtained by shake flask fermentation verification screening, which could tolerate pH 10.0, had a 16.3% increase in urease activity per unit cell, and a 4.7% increase in the amount of CaCO3 precipitate produced by solidifying 0.5 mol / L binder.

[0006] The first object of the present invention is to provide a strain of Sporosarcina pasteurii ( Sporosarcina pasteurii ) DA13 strain, its preservation number is CGMCC No.31423, the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation date is July 22, 2024.

[0007] The second objective of the present invention is to provide the use of the above-mentioned Sporosarcina pasteurianum DA13 in inducing calcium carbonate deposition.

[0008] Preferably, the application comprises: inoculating the pasteurian sporosarcina DA13 in an induction medium containing urea, and after culturing, adding the bacterial solution to a medium containing Ca 2+ The deposition of calcium carbonate is achieved in the cementing liquid of cement and urea.

[0009] Preferably, the induction medium is CASO medium containing urea.

[0010] More preferably, the CASO medium is a liquid culture medium or a solid culture medium, the formula of the liquid culture medium is: 15 g / L casein peptone, 5 g / L soy peptone, 5 g / L sodium chloride and 20 g / L urea; the formula of the solid culture medium is: 15 g / L casein peptone, 5 g / L soy peptone, 5 g / L sodium chloride, 20 g / L urea and 15 g / L agar.

[0011] Preferably, the culture conditions are: 30°C, 36h.

[0012] Preferably, the formula of the binder is: 0.5 mol / L CaCl2 and 0.5 mol / L urea.

[0013] The present invention provides a strain of Sporosarcina pasteurii ( Sporosarcina pasteurii ) DA13 strain, from Sporosarcina pasteurii (Sporosarcina pasteurii) ) The DSM33 host was obtained by ARTP mutagenesis technology.

[0014] The sporosarcina pasteurii provided by the present invention ( Sporosarcina pasteurii ) The DA13 strain can tolerate pH 10.0, the urease activity per unit cell is increased by 16.3%, and the amount of CaCO3 precipitation produced by the solidified cementing fluid is increased by 4.7%. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a trend chart of the mutagenesis time and the lethality rate in Example 1 (3) of the present invention.

[0016] Figure 2 It is a comparison chart of pH resistance of DSM33 and DA13 in Example 1 of the present invention.

[0017] Figure 3 This is a comparison chart of the urease activity per unit cell of DSM33 and DA13 in Example 2 of the present invention.

[0018] Figure 4 This is a comparison chart of the calcium carbonate production of DSM33 and DA13 in Example 2 of the present invention.

[0019] Description of biological deposit: The biological material of the present invention is classified and named as: Sporosarcina pasteurii ( Sporosarcoma pasteurii )DA13, depository: General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, Yard 1, Beichen Road, Chaoyang District, Beijing, deposit date: July 22, 2024, deposit number: CGMCC No. 31423. DETAILED DESCRIPTION

[0020] The present invention utilizes synthetic biology to generate a novel Sporosarcina pasteurii ) DSM33 was used as the host, and by combining atmospheric pressure and room temperature plasma mutagenesis with high-throughput screening, a mutant strain of Sporosarcina pasteurianum DA13 was obtained, which could tolerate pH 10.0, had a 16.3% increase in urease activity per unit cell, and a 4.7% increase in the amount of CaCO3 precipitate produced by solidifying 0.5 mol / L cementing fluid.

[0021] Materials used: 1. Sporosarcina pasteurii (Sporosarcina pasteurii) )DSM33 (Ningbo Mingzhou Biotechnology Co., Ltd.).

[0022] 2. CASO liquid medium (g / L): casein peptone 15, soy peptone 5, sodium chloride 5, urea 20.

[0023] 3. CASO solid medium (g / L): casein peptone 15, soy peptone 5, sodium chloride 5, urea 20, agar 15.

[0024] 4. Normal saline: 0.9% sodium chloride solution.

[0025] 5. 5% glycerol water.

[0026] 6. ARTP mutagenesis instrument (Wuxi Yuanqing Tianmu Biotechnology Co., Ltd.).

[0027] 7. 96-well plate (Thermo Fisher Scientific).

[0028] 8. Conductivity meter (Shenzhen Kedida Electronics Co., Ltd.).

[0029] 9. 0.5mol / L cementing fluid: Mix equal volumes of 1.0mol / L CaCl2 solution and 1.0mol / L urea solution.

[0030] Sporosarcina pasteurii (Sporosarcina pasteurii) ) DSM33 was streaked on CASO plates (containing 20 g / L urea), and a single colony on the plate was picked and inoculated into CASO liquid culture medium (containing 20 g / L urea), and the cells were collected by culturing at 30°C overnight.

[0031] The collected cell bodies were diluted to OD between 0.6 and 0.8, and the cells were washed twice with sterile saline and resuspended with 5% glycerol water. 10 μL of the resuspended bacterial solution was evenly applied on the slide and placed under the ARTP mutagenizer treatment source for ARTP mutagenesis. The ARTP mutagenizer was sterilized with UV for 30 minutes, with high-purity helium as the working gas, the mutagenesis power was set to 120W, the ventilation volume was set to 10L / min, the temperature was controlled at 20°C using a condensing water circulation machine, the distance between the plasma emission source and the slide was 2mm, and different mutagenesis treatment times were set (0, 10, 20, 30, 40s).

[0032] After the mutagenesis, the bacterial solution was immediately transferred to fresh CASO medium (containing 20g / L urea) and cultured for 2-3h. The cultured bacteria were diluted to OD between 0.6 and 0.8 and spread on CASO plates (containing 20g / L urea). Cultured at 30℃ for 48h, the number of colonies was recorded and the lethality was calculated to obtain the optimal mutagenesis time. The results showed that when the ARTP mutagenesis time was 10s, the lethality reached 61.4%, when the mutagenesis time was 20s, the lethality reached 95.2%, and when the mutagenesis time was 30s, the lethality reached 99.1%. In order to obtain strains with more mutagenesis damage and still have sufficient growth, the lethality of 95.2% and the mutagenesis time of 20s were selected as the mutagenesis time of the pasteurian sporosarcina strain, see Figure 1 .

[0033] Overnight culture S. pasteuriiThe DSM33 bacterial cells were diluted to OD between 0.6 and 0.8, and the cells were washed twice with sterile saline and resuspended with 5% glycerol water. 10 μL of the resuspended bacterial solution was evenly applied on the slide, and placed under the ARTP mutagenizer treatment source for 20 seconds of ARTP mutagenesis. The ARTP mutagenizer was sterilized with ultraviolet light for 30 minutes, with high-purity helium as the working gas, the mutagenesis power was set to 120W, the ventilation volume was set to 10L / min, the temperature was controlled at 20°C using a condensing water circulation machine, the distance between the plasma emission source and the slide was 2mm, and the mutagenesis treatment time was set to 20 seconds.

[0034] The bacterial liquid obtained after the mutagenesis was immediately transferred to fresh CASO medium (containing 20 g / L urea) and cultured for 2 to 3 hours. The cultured bacteria were diluted to an OD between 0.6 and 0.8 and spread on CASO plates (containing 20 g / L urea) at pH 7.5 to pH 10.0, and cultured at 30°C for 48 hours.

[0035] The growth spots on the plates with different pH values ​​were transferred to the 96-well CASO medium (containing 20 g / L urea) with the corresponding pH and cultured at 30°C for 36 h. The urease activity of the strains in each well was determined by monitoring and recording the change in conductivity within 5 min in each well using a conductivity meter, and 10 strains with much improved urease activity were obtained.

[0036] Ten strains with much improved urease activity were inoculated into 50 mL (packed in 300 mL shake flask) of fresh CASO medium (containing 20 g / L urea) of the same pH, and cultured at 30°C for 36 h. The biomass during the culture period and the urease activity at the end of the culture were measured. By comparison, a mutant strain DA13 of Sporosarcina pasteurii with a stable growth state and the highest improvement in urease activity was obtained. The results showed that the mutant strain DA13 of Sporosarcina pasteurii obtained by the present invention can tolerate a pH value increased from 9.5 to 10.0, as shown in FIG. Figure 2 .

[0037] (1) Culture medium: Seed liquid culture medium (g / L): casein peptone 15, soy peptone 5, sodium chloride 5, urea 20.

[0038] Fermentation medium (g / L): casein peptone 15, soy peptone 5, sodium chloride 5, urea 20.

[0039] (2) Fermentation experiment: Pick the activated DA13 strain from the plate and inoculate it into 5 ml of liquid CASO medium, and culture it overnight at 30°C and 200 rpm to obtain seed liquid; take an appropriate amount of pasteurian sporosarcina seed liquid from the CASO test tube and add it to 50 ml of fermentation medium, so that the initial OD of the shake flask medium is 0.5-1. Culture it at 30°C and 200 rpm for 36 hours.

[0040] (3) Determination of urease activity per unit cell: 1 mL of bacterial solution after the culture was completed was taken and added to a glass beaker containing 9 mL of 1.1 mol / L urea solution and mixed thoroughly. Under an environment in which the temperature change was guaranteed to be ±0.1°C, a conductivity meter was used to monitor the change in solution conductivity within 5 minutes. After the monitoring was completed, the average conductivity change value per minute within 5 minutes (mS / cm·min) was calculated and then multiplied by the dilution factor (10 times) to obtain the urease activity (mS / cm·min). The measurement results showed that the urease activity per unit cell of the mutant strain DA13 of sporogenous Sarcina pasteurii obtained by the present invention increased from 86 mS / cm·min to 100 mS / cm·min, an increase of 16.3%. Figure 3 .

[0041] (1) Culture medium: Seed liquid culture medium (g / L): casein peptone 15, soy peptone 5, sodium chloride 5, urea 20.

[0042] Fermentation medium (g / L): casein peptone 15, soy peptone 5, sodium chloride 5, urea 20.

[0043] (2) Fermentation experiment: Pick the activated DA13 strain from the plate and inoculate it into 5 ml of liquid CASO medium, and culture it overnight at 30°C and 200 rpm to obtain seed liquid; take an appropriate amount of pasteurian sporosarcina seed liquid from the CASO test tube and add it to 50 ml of fermentation medium, so that the initial OD of the shake flask medium is 0.5-1. Culture it at 30°C and 200 rpm for 36 hours.

[0044] (3) Determination of the content of calcium carbonate that can be generated by cross-linking with the cementing fluid: Take 10mL of bacterial liquid after the culture is completed, add it to a 50mL centrifuge tube filled with 30mL of 0.5mol / L cementing liquid and mix thoroughly. After keeping it at a constant temperature of 30℃ for 24h, take out the centrifuge tube, remove the upper liquid in the tube, and place it in a drying oven for drying for 24h. After drying, weigh the centrifuge tube to calculate the amount of CaCO3 precipitation generated by the bacterial solidification cementing liquid. The measurement results show that the content of calcium carbonate generated by cross-linking the pasteurized sporosarcina mutant strain DA13 obtained by the present invention with the cementing liquid increased from 3.14g to 3.29g, an increase of 4.7%, Figure 4 。

Claims

1. A strain of Sporosarcina pasteurii ( Sporosarcina pasteurii ) DA13 strain, the deposit number is CGMCC No.31423.

2. Use of the strain described in claim 1 in inducing calcium carbonate deposition.

3. The use according to claim 2, characterized in that: include: The pasteurian sporosarcina DA13 was inoculated into an induction medium containing urea, and after culturing, the bacterial solution was added to a medium containing Ca 2+ The deposition of calcium carbonate is achieved in the cementing liquid of cement and urea.

4. The use according to claim 3, characterized in that: The induction medium is a CASO medium containing urea.

5. The use according to claim 4, characterized in that The CASO culture medium is a liquid culture medium or a solid culture medium. The formula of the liquid culture medium is: 15 g / L casein peptone, 5 g / L soy peptone, 5 g / L sodium chloride and 20 g / L urea; the formula of the solid culture medium is: 15 g / L casein peptone, 5 g / L soy peptone, 5 g / L sodium chloride, 20 g / L urea and 15 g / L agar.

6. The use according to claim 3, characterized in that: The culture conditions are: 30° C., 36 h.

7. The use according to claim 3, characterized in that: The formula of the cementing fluid is: 0.5 mol / L CaCl2 and 0.5 mol / L urea.

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