A method for regulating microbial induced growth of calcium carbonate crystals into calcite using glucuronic acid

Abstract

The present application relates to a method for regulating the growth of calcium carbonate crystals into calcite by glucose uronic acid, so as to improve the mechanical strength of the solidified body. The specific steps of the method are as follows: (1) screening and identification of acetate mineralization bacteria with calcium acetate as substrate; (2) culture of carbonate mineralization strain; (3) growth and regulation of calcium carbonate crystals induced by microorganisms; (4) characterization of calcium carbonate crystals. The microbial solidification of the uranium tailings beach is a green solidification method with great application prospect. The carbonate mineralization strain provided by the present application has no secondary pollution in the process of microbial induction of calcium carbonate precipitation, has strong environmental friendliness, and can regulate the growth of calcium carbonate crystals into calcite by glucose uronic acid, so as to improve the mechanical strength of the calcium carbonate crystals, and has potential application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomineralization regulation technology. It screens out a strain of bacteria with carbonate mineralization ability and specifically provides a method for using glucuronic acid to regulate microbial induction of calcium carbonate crystal growth into calcite. Background Technology

[0002] Uranium tailings ponds are the largest potential source of radioactive pollution in the nuclear industry. How to solidify the surface of uranium tailings ponds and isolate the radioactive waste therein from the human living environment in a long-term, stable and safe manner has become the most urgent task in the safety management of uranium tailings ponds.

[0003] Microbial-induced carbonate precipitation is an environmentally friendly biosolidification technology for uranium tailings pond surfaces that is widely found in nature. However, current methods using urea-decomposing bacteria and sulfate-reducing bacteria to induce calcium carbonate precipitation present problems such as secondary pollution from ammonia and sulfur, and insufficient mechanical strength of the solidified body.

[0004] Recent studies have shown that microbial-induced calcium carbonate precipitation under oxidative reaction conditions produces no secondary pollution, and the reaction mechanism is as follows: Calcium acetate, as a substrate, can be decomposed and utilized by bacteria. Therefore, finding a biomineralizing bacterium capable of decomposing calcium acetate is fundamental to the application of this technology. CaCO3 includes three crystal types: calcite, aragonite, and aragonite, with the thermodynamic stability decreasing sequentially among these three calcium carbonate crystal types. In the application of solidified sand based on microbial-induced calcium carbonate precipitation technology, calcite exhibits good thermodynamic stability, strong bonding, and good shear resistance. Therefore, calcite is the most ideal calcium carbonate crystal type for practical applications of sand solidification. Controlling the crystal type of calcium carbonate to grow into calcite crystals has become a key factor in improving the mechanical properties of the solidified body.

[0005] In summary, there is an urgent need to screen out carbonate mineralizing bacteria that use calcium acetate as a substrate, and to use control methods to completely transform the calcium carbonate crystal type from aragonite to calcite, so as to improve the mechanical strength of the solidified body and provide a technical prototype for in-situ green biological solidification of uranium tailings pond surfaces. Summary of the Invention

[0006] The purpose of this invention is to provide a carbonate mineralizing bacterium that uses calcium acetate as a substrate, and to improve the mechanical strength of the solidified body by using glucuronic acid to regulate the microbial induction of calcium carbonate crystal growth into calcite.

[0007] Specific measures include:

[0008] 1. Screening and identification of carbonate mineralizing bacteria using calcium acetate as a substrate;

[0009] 2. Cultivation of carbonate mineralizing strains;

[0010] 3. Microbial-induced growth and regulation of calcium carbonate crystals;

[0011] 4. Characterization of calcium carbonate crystals.

[0012] Further measures are as follows:

[0013] The specific method for screening and identifying carbonate mineralizing bacteria using calcium acetate as a substrate is as follows:

[0014] Accurately weigh 2 g of the collected slope soil sample and add it to a prepared high-concentration sodium acetate liquid culture medium under aseptic conditions for enrichment culture. The high-concentration sodium acetate liquid culture medium containing the soil sample is placed at 30℃ and allowed to stand for 48 h. The prepared high-concentration sodium acetate liquid culture medium consists of 80 g / L sodium acetate and 4 g / L yeast extract, with the pH adjusted to 7, and sterilized at 121℃ for 20 min. After enrichment culture, in a sterile workbench, take 1 mL of the supernatant of the enriched culture medium and add it to 100 mL of low-concentration sodium acetate liquid culture medium for re-culture. The medium is then incubated at 30℃ and 100 rpm for 24 h using a constant temperature shaker. The sodium acetate liquid culture medium consists of 20 g / L sodium acetate and 4 g / L yeast extract, with the pH adjusted to 7, and sterilized at 121℃ for 20 min. Take 0.01–0.1 mL of the supernatant from the re-cultured medium, dilute it 10–1,000,000 times with deionized water, spread it on a plate purification medium, and then incubate it upside down in a 30°C incubator for 18–48 h. Collect colonies of varying morphology and size, and repeatedly spread them on the plate purification medium until a single strain with consistent morphology is obtained. Identify the bacteria using 16S rDNA sequencing, and preserve the bacterial culture. Expand the obtained strain using a shaker, then transfer 1% of the cultured bacterial culture to calcium acetate medium (17.5 g / L calcium acetate, 4 g / L yeast extract, pH 7). If a white precipitate forms in the calcium acetate medium, separate the precipitate and analyze it using X-ray diffraction. If the precipitate is identified as calcium carbonate, the bacterial strain has been successfully screened. DNA sequencing was performed on the screened carbonate-mineralized strains, and the strain genome was extracted. Polymerase chain reaction (PCR) amplification was then performed using universal primers (27f: 5'-AGAGTTTGATC-MTGGCTCAG-3', 1492r: 5'-TACCT-TGTTACGACTT-3'). The PCR system contained: Buffer (with Mg...) 2+2.5 μl of reagents, 1 μl of dNTPs, 0.5 μl of template, 0.2 μl of Taq DNA polymerase, and 25 μl of ultrapure water. The polymerase chain reaction products were sequenced using the dideoxy chain termination method. Sequence analysis was performed using BLASTN software to identify 16S rDNA sequences with high homology, and these sequences were compared with gene banks to confirm that the screened bacterial strain belonged to Klebsiella pneumoniae.

[0015] The specific method for culturing the aforementioned carbonate mineralizing strains is as follows:

[0016] Adjust the pH of the culture medium to 7, and set the Ca... 2+ Experimental groups were established with concentrations of 0.05 mol / L, 0.1 mol / L, 0.25 mol / L, and 0.5 mol / L, and each conical flask was inoculated with 1% bacterial suspension (OD). 600 >1), and cultured in a shaker at 100 rpm / min and 30℃ for 13 days. Samples were taken on days 1, 3, 5, 7, 9, 11, and 13 to determine the amount of calcium carbonate precipitate formed. The calcium ion concentration in the culture medium was adjusted to 0.1 mol / L, and experimental groups were set up with pH values ​​of 3, 5, 7, and 9. 1% (OD) solution was inoculated into each conical flask. 600 >1) bacterial suspension was cultured in a shaker at 100 rpm / min and 30℃ for 13 days. Samples were taken on days 1, 3, 5, 7, 9, 11, and 13 to determine the amount of calcium carbonate precipitate formed. The pH of the culture medium was adjusted to 7, and the calcium ion concentration was 0.1 mol / L. Experimental groups with bacterial inoculum amounts of 1%, 2%, 4%, and 6% were set up and cultured in a shaker at 100 rpm / min and 30℃ for 13 days. Samples were taken on days 1, 3, 5, 7, 9, 11, and 13 to determine the amount of calcium carbonate precipitate formed.

[0017] The specific method for inducing and regulating the growth of calcium carbonate crystals by microorganisms is as follows:

[0018] Prepare 100 mL of calcium acetate culture medium in a 250 mL Erlenmeyer flask, add 1% activated bacterial solution, and incubate at 30 ℃ in a shaker at 100 rpm / min for 3 days. Then, in a sterile environment, add filtered and sterilized glucuronic acid solution to each Erlenmeyer flask. The concentration of each monosaccharide in each experimental group is 1 g / L. A control group without added monosaccharides or proteins is used. The control group is incubated at 30 ℃ in a shaker at 100 rpm / min for 5 days. Each experiment has three replicates.

[0019] The specific method for characterizing the calcium carbonate crystals is as follows:

[0020] At the end of the 9th day of cultivation, the suspension containing calcium carbonate precipitate was poured into 50 ml centrifuge tubes and centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded, and the lower precipitate was dried in a 60°C oven for 48 hours to obtain a dry white calcium carbonate precipitate. X-ray diffraction was used to determine the crystal type of the white calcium carbonate precipitate to identify whether it was calcite. Scanning electron microscopy was used to observe whether the surface morphology of the white calcium carbonate precipitate exhibited the morphological characteristics of calcite.

[0021] This invention screened a carbonate mineralizing bacterium that uses calcium acetate as a substrate, and by adding glucuronic acid, it achieved the transformation of unstable aragonite into stable calcite, regulated the transformation direction of calcium carbonate crystals, and improved the mechanical strength of the solidified body, providing a technical prototype for in-situ green biological solidification of uranium tailings pond surfaces. Attached Figure Description

[0022] Figure 1 This is a plate isolation diagram of bacterial strains.

[0023] Figure 2 This is a Gram staining image of a bacterial strain.

[0024] Figure 3 It is a white precipitate that appears in calcium acetate culture medium.

[0025] Figure 4 It refers to the proportion of genes in the strain.

[0026] Figure 5 It is an X-ray diffraction analysis of the precipitated products.

[0027] Figure 6 This is the growth curve of Klebsiella pneumoniae.

[0028] Figure 7 Calcium carbonate precipitation yield at different calcium acetate concentrations

[0029] Figure 8 Calcium carbonate precipitation yield under different pH conditions

[0030] Figure 9 Calcium carbonate precipitation yield at different bacterial concentrations

[0031] Figure 10 SEM image of calcium carbonate without glucuronic acid.

[0032] Figure 11 SEM image of calcium carbonate regulated by glucuronic acid.

[0033] Figure 12 XRD patterns of calcium carbonate in the control group and under glucuronic acid regulation. Detailed Implementation

[0034] When the strain of this invention induces calcium carbonate precipitation using the principle of oxidation reaction, the substrate calcium acetate can be decomposed and utilized by the bacteria as a nutrient, and the CO2 and H2O produced by the substrate reaction will not cause secondary pollution to the surrounding environment. This strain exhibits strong environmental friendliness during the microbial induction of calcium carbonate precipitation. Furthermore, by adding glucuronic acid, it achieves the transformation of unstable aragonite into stable calcite, regulating the transformation direction of calcium carbonate crystals and improving the mechanical strength of the solidified body, demonstrating potential application prospects. The specific implementation of this invention will be described in detail below with reference to specific embodiments.

[0035] Example:

[0036] Screening of microbial strains. Accurately weigh 2 g of soil sample collected from the slope. Under aseptic conditions, add the soil sample to a prepared high-concentration sodium acetate liquid medium for enrichment culture. The high-concentration sodium acetate liquid medium containing the soil sample is placed at 30℃ and allowed to stand for 48 h. The prepared high-concentration sodium acetate liquid medium consists of 80 g / L sodium acetate and 4 g / L yeast extract, adjusted to pH 7, and sterilized at 121℃ for 20 min. After enrichment culture, in a sterile workbench, take 1 mL of the supernatant of the enriched culture medium and add it to 100 mL of low-concentration sodium acetate liquid medium. Incubate in a constant temperature shaker at 30℃ and 100 rpm for 24 h. The sodium acetate liquid medium consists of 20 g / L sodium acetate and 4 g / L yeast extract, adjusted to pH 7, and sterilized at 121℃ for 20 min. Take 0.01–0.1 mL of the supernatant from the microbial-enriched culture medium, dilute it 10–1,000,000 times with deionized water, spread it on a plate purification medium, and then incubate it upside down in a 30°C incubator for 18–48 h. Collect colonies of varying morphology and size, and repeatedly spread them on the plate purification medium until a single strain with consistent morphology is obtained. Identify the bacteria using 16S rDNA sequencing, and preserve the bacterial culture. Expand the obtained strain using a shaker, then transfer 1% of the cultured bacterial culture to calcium acetate medium (17.5 g / L calcium acetate, 4 g / L yeast extract, pH 7). If a white precipitate forms in the calcium acetate medium, separate the precipitate and analyze it using X-ray diffraction. If the precipitate is identified as calcium carbonate, the bacterial strain has been successfully screened.

[0037] Identification of bacterial strains. Specific methods include: (1) Morphological identification, ① Slide fixation. Under aseptic conditions, pick up a small amount of bacteria with an inoculation loop and spread it evenly on a clean slide. Heat it over a flame to kill the bacteria and make them adhere and fix. ② Stain with ammonium oxalate crystal violet for 1 minute. ③ Rinse with tap water to remove excess stain. ④ Mordant with iodine-potassium iodide solution for 1 minute and pour off excess solution. ⑤ Decolorize with a neutral decolorizing agent such as ethanol (95%) or pyruvic acid for 30 seconds. Gram-positive bacteria will not be decolorized and will appear purple, while Gram-negative bacteria will be decolorized and will appear colorless. ⑥ Counterstain with safranin or safranin for 30 seconds. Gram-positive bacteria will still appear purple, while Gram-negative bacteria will appear red. (2) Molecular identification: The genome of the strain was extracted and amplified by polymerase chain reaction using universal primers (27f: 5'-AGAGTTTGATC-MTGGCTCAG-3', 1492r: 5'-TACCT-TGTTACGACTT-3'). The polymerase chain reaction system contained: Buffer (with Mg 2+ 2.5 μl of dNTPs, 1 μl of template, 0.5 μl of Taq DNA polymerase, and 25 μl of ultrapure water were added. The polymerase chain reaction products were sequenced using the dideoxy chain termination method, and the sequences were analyzed using BLASTN software to identify 16S rDNA sequences with high homology, which were then compared with gene banks. The strain screened in this invention was identified as Klebsiella. According to the BLAST results of the 16S rRNA gene sequencing of the strain by Shanghai Bioengineering Co., Ltd. (see Table 1), the strain was identified as Klebsiella. The gene proportion of the strain was identified by Beijing Novogene Technology Co., Ltd. (see Table 1). Figure 4 The strain is Klebsiella aerogenes.

[0038] Preservation of microbial strains. The strains screened in this invention are preserved at the China Center for Type Culture Collection.

[0039] Address of the repository: Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (opposite to the First Affiliated Primary School of Wuhan University).

[0040] Preservation period: December 1, 2021

[0041] Category Naming: Klebsiella sp. 11027

[0042] Accession number: M20211523

[0043] Determination of microbial growth curves and the effects of different factors on calcium carbonate precipitation. Microbial growth was determined using OD (Organic Dioxide) assay. 600The absorbance was measured using a UV spectrophotometer, with the wavelength adjusted to 600 nm. A blank culture medium was used as the blank sample, and the bacterial culture medium was used as the test sample. Higher absorbance indicates a higher concentration of bacterial cells. Measurements showed that between 6 and 96 hours, the number of Klebsiella pneumoniae steadily increased; this period is the logarithmic growth phase, also known as the exponential phase. During this period, the bacteria exhibit strong biological activity, and microorganisms in the logarithmic phase are often used for inoculation and other experiments. From 96 to 122 hours thereafter, as the available carbon sources and nutrients in the culture medium are continuously consumed, the total number of bacteria increases and decreases, and the overall bacterial population gradually stabilizes; this period is called the stationary phase. After 122 hours, Klebsiella pneumoniae enters the decline phase, with a significant decrease in bacterial reproduction and a near cessation of physiological metabolic activity. Figure 7 This represents the amount of calcium carbonate precipitate corresponding to initial calcium acetate concentrations of 0.05 mol / L, 0.1 mol / L, 0.25 mol / L, and 0.5 mol / L. The pH of the culture medium was adjusted to 7, and the Ca... 2+ The concentrations were 0.05 mol / L, 0.1 mol / L, 0.25 mol / L, and 0.5 mol / L, and each conical flask was inoculated with 1% bacterial suspension (OD). 600 >1), incubate in a shaker at 100 rpm / min and 30℃ for 13 days. Samples were taken on days 1, 3, 5, 7, 9, 11, and 13 to determine the amount of calcium carbonate precipitate formed. The effect of calcium acetate concentration on the amount of calcium carbonate precipitate formed is shown in [reference needed]. Figure 7 As time progressed, the amount of calcium carbonate precipitate formed increased continuously, stabilizing around day 9. The amount of calcium carbonate precipitate formed also increased with increasing calcium acetate concentration, indicating that increasing calcium acetate concentration is beneficial for increasing calcium carbonate precipitation yield. However, excessively high calcium ion concentrations reduced the amount of calcium precipitate. This may be because when the calcium ion concentration in the solution exceeds a certain limit, the concentration difference between the inside and outside of the cell wall becomes too large, causing an imbalance in cell wall pressure, reducing cell activity, and consequently affecting the activity of enzymes during bacterial metabolism, ultimately impacting calcium precipitation efficiency. The results show that the highest calcium carbonate precipitate formation (1.23 g) was observed at a calcium acetate concentration of 0.25 mol / L, with a calcium acetate utilization rate of 49.25%. Therefore, it is speculated that a calcium acetate concentration of 0.25 mol / L is the optimal concentration for Klebsiella pneumoniae to induce the highest amount of calcium carbonate precipitation. Figure 8 This represents the amount of calcium carbonate precipitation corresponding to initial pH values ​​of 3, 5, 7, and 9. The calcium ion concentration in the culture medium was adjusted to 0.1 mol / L, and experimental groups were set up with pH values ​​of 3, 5, 7, and 9. 1% (OD) solution was then inoculated into each conical flask. 600>1) bacterial suspension was cultured in a shaker at 100 rpm / min and 30℃ for 13 days. Samples were taken on days 1, 3, 5, 7, 9, 11, and 13 to determine the amount of calcium carbonate precipitate formed. The effect of the initial pH of the culture medium on the amount of calcium carbonate precipitate formed is shown in [reference needed]. Figure 8 As time progressed, calcium carbonate precipitation increased continuously, stabilizing around day 9. After day 9, calcium carbonate production decreased with further time. No calcium carbonate precipitate was formed at pH 3, indicating that the bacteria could not survive at this pH. The amount of calcium carbonate precipitate formed increased with increasing initial pH of the culture medium. When the pH further increased to 9, the amount of calcium carbonate precipitate formed decreased. The highest amount of calcium carbonate precipitate was formed at pH 7, at 0.8417 g, with a calcium acetate utilization rate of 84.73%. Therefore, it is inferred that pH 7 is the optimal pH for Klebsiella pneumoniae to induce the highest amount of calcium carbonate precipitation. Figure 9 This describes the amount of calcium carbonate precipitate corresponding to bacterial concentrations of 1%, 2%, 4%, and 6%. The pH of the culture medium was adjusted to 7, and the calcium ion concentration was 0.1 mol / L. Experimental groups with inoculum amounts of 1%, 2%, 4%, and 6% were set up and cultured in a shaker at 100 rpm / min and 30 ℃ for 13 days. Samples were taken on days 1, 3, 5, 7, 9, 11, and 13 to determine the amount of calcium carbonate precipitate formed. Part of the generated calcium carbonate was at the bottom of the conical flask, and part was on the wall of the flask. The entire liquid in the conical flask was poured into a 50 mL centrifuge tube and centrifuged using a high-speed centrifuge at 8000 rpm / min for 10 min. The supernatant was filtered off, and the precipitate was dried in a 60 ℃ oven, weighed, and the mass data recorded. Then, it was immersed and washed several times in 0.1 M hydrochloric acid solution until no obvious bubbles were generated. It was then dried again in the oven, and the mass data was recorded again. The difference between these two mass data is the mass of CaCO3 produced. The effect of bacterial inoculum size on the amount of calcium carbonate precipitate formed is shown in the figure. Figure 9 As time progressed, calcium carbonate precipitation increased continuously, stabilizing around day 9. At an inoculum size of 4%, the maximum calcium carbonate precipitate produced was 0.96 g, with a calcium acetate utilization rate of 96.6%. The amount of calcium carbonate precipitate increased with increasing inoculum size, but decreased at 6%. This may be due to the limited nutrients in the culture medium at excessively high inoculum sizes, leading to reduced bacterial activity. Increasing the inoculum size is not only uneconomical but also fails to improve calcium carbonate production. Therefore, it is speculated that an inoculum size of 4% is the optimal inoculum size for inducing the highest calcium carbonate precipitation by Klebsiella pneumoniae.

[0044] Regulation of calcium carbonate crystals by glucuronic acid. 100 mL of calcium acetate culture medium was prepared in a 250 mL Erlenmeyer flask, and 1% activated bacterial solution was added. The flasks were incubated at 30 ℃ and 100 rpm / min for 3 days with constant temperature shaking. Then, filtered and sterilized glucuronic acid solution was added to the Erlenmeyer flasks in a sterile operating room. The glucuronic acid concentration in each experimental group was 1 g / L. A control group without added glucuronic acid was used. The flasks were incubated at 30 ℃ and 100 rpm / min for 5 days with constant temperature shaking.

[0045] Characterization of calcium carbonate crystals. At the end of day 9 of cultivation, the solution was collected and poured into 50 mL centrifuge tubes. The tubes were centrifuged at 10000 r / min for 10 min, and the supernatant was discarded. The lower precipitate was dried in a 60℃ oven for 48 h to obtain a dry white precipitate. SEM and XRD techniques were used for further analysis of the precipitate. X-ray diffraction was used to determine the crystal type of the white calcium carbonate precipitate to identify whether it was calcite. Scanning electron microscopy was used to observe whether the surface morphology of the white calcium carbonate precipitate exhibited calcite-like characteristics. Figure 10 Scanning electron micrograph of calcium carbonate without glucuronic acid. Figure 11 Scanning electron micrograph of calcium carbonate regulated by glucuronic acid. Figure 12 X-ray diffraction patterns of calcium carbonate under control and glucuronic acid regulation are shown in the figures. It is clear from the figures that almost all formed calcium carbonate samples are composed of calcite. By comparing with the PDF card, the principal diffraction 2θ angles of 29.45°, 35.96°, 39.42°, 43.17°, and 48.57° correspond to the (104), (110), (113), (202), and (116) crystal planes of calcite, respectively, and are labeled with C. Analysis of the X-ray diffraction patterns using Jade 6.0 software shows that under the action of glucuronic acid, only calcite diffraction peaks are observed. The X-ray diffraction patterns indicate that, compared to the control group, glucuronic acid is more conducive to the nucleation and growth of calcite.

[0046] The above are merely preferred embodiments of the present invention. Based on the above concept, those skilled in the art can make various modifications and variations. For example, changing the soil sampling location and method, using different microbial screening and identification methods, changing the type of culture medium, and changing the concentration and addition time of glucuronic acid. However, such modifications and variations are all within the scope of the present invention.

[0047] type Matching score Overall score Coverage E value Similarity sequence name aerogenes strain HNHF1 chromosome, complete genome 2651 21180 100% 0 100.00% CP047669.1 aerogenes strain Y1 chromosome,complete genome 2651 21075 100% 0 100.00% CP045870.1 aerogenes strain Y3 chromosome,complete genome 2651 21075 100% 0 100.00% CP045869.1 aerogenes strainY6 chromosome,complete genome 2651 21075 100% 0 100.00% CP045868.1 aerogenes strain FDAARGOS_641 chromosome, complete genome 2651 21103 100% 0 100.00% CP044083.1 aerogenes strain Ka37751 chromosome,complete genome 2651 21070 100% 0 100.00% CP041925.1 aerogenes strain NCTC10006 genome assembly, chromosome: 6 2651 15404 100% 0 100.00% LR134126.1 aerogenes strain RHBSTW-00898 chromosome, complete genome 2651 21191 100% 0 100.00% CP056260.1 aerogenes DNA, complete genome, strain: WP5-W18-CRE-01 2651 21068 100% 0 100.00% AP022108.1

Claims

1. A carbonate mineralizing bacterium using calcium acetate as a substrate, characterized in that, The carbonate mineralizing bacteria belong to Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella aerogenes It has been deposited at the China Center for Type Culture Collection on December 1, 2021, with accession number CCTCC NO: M20211523.

2. A method for regulating the growth of calcium carbonate crystals into calcite by microorganisms using glucuronic acid, characterized in that, The cultured carbonate mineralizing bacteria solution was added to calcium acetate culture medium for reaction, and then glucuronic acid solution was added to regulate the generated calcium carbonate crystals. Finally, the type and surface morphology of calcium carbonate crystals were characterized. The carbonate mineralizing bacteria are the strains described in claim 1.

3. The method for regulating microbial-induced growth of calcium carbonate crystals into calcite using glucuronic acid according to claim 2, characterized in that, On the third day of culture of the carbonate mineralizing strain, filtered and sterilized glucuronic acid solution was added to make the glucuronic acid concentration 1 g / L. After adding the glucuronic acid solution, the strain was cultured for 5 days to carry out the growth and regulation of calcium carbonate crystals.

4. The method for regulating the growth of calcium carbonate crystals into calcite by microorganisms using glucuronic acid according to claim 2, characterized in that, At the end of the 9th day of cultivation, the suspension containing calcium carbonate precipitate was poured into a 50 ml centrifuge tube and centrifuged at 10,000 rpm / min for 10 minutes in a high-speed centrifuge. The supernatant was discarded, and the lower precipitate was dried in a 60℃ oven for 48 hours to obtain a dry white calcium carbonate precipitate. The precipitate was analyzed by SEM and XRD techniques.

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