A method of promoting microbial induced calcium carbonate precipitation

Abstract

The application discloses a method for promoting microbial induced calcium carbonate deposition, and relates to the technical field of microbial mineralization and microbial carbon fixation, and comprises adding zinc sulfate in the process of microbial mineralization. In the method, zinc sulfate is added into a culture medium when culturing microorganisms capable of realizing mineralization, and then the cultured bacteria and the culture medium containing zinc sulfate are added into a mineralization system, and the mineralization is completed after waiting. Compared with a conventional microbial mineralization method, after adding zinc sulfate, the deposition conversion rate of calcium ions in the mineralization system during the microbial mineralization can be effectively improved, the calcium ion consumption can be increased, and more calcium carbonate can be generated.

Description

Technical Field

[0001] The invention relates to the technical field of microbial mineralization and microbial carbon fixation, and in particular to a method for promoting microbial-induced calcium carbonate deposition. Background Art

[0002] Microbially induced calcium carbonate deposition (MICP) is a common bioinduced mineralization process characterized by rapid reaction speed, low environmental requirements, wide application range, and significant greenhouse gas emission reduction. It is widely used in various fields including geology, civil engineering, water conservancy, and the environment. Microbial-induced carbonate deposition pathways include urea decomposition, amino acid ammoniation, denitrification, reduction by dissimilatory sulfate bacteria, photosynthesis, and methane oxidation. This method can fix carbon dioxide in the form of carbonate minerals such as calcite, dolomite, and vaterite without causing harmful effects on the environment. Its mechanism of action also has important implications for mitigating the greenhouse effect and can be applied to tailings fixation, sand consolidation, and concrete remediation.

[0003] When urease-producing microorganisms currently used induce calcium carbonate deposition and mineralization, the added urea cannot be fully hydrolyzed, resulting in a waste of raw materials. At the same time, the generated NH+4 is likely to cause new environmental pollution. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for promoting microbial-induced calcium carbonate deposition.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] A method for promoting microbial induction of calcium carbonate deposition is provided. The method comprises culturing microorganisms capable of inducing calcium carbonate deposition by adding a culture medium containing zinc sulfate, and contacting a mixture of the cultured microorganisms and the culture medium with a calcium source, thereby increasing the deposition efficiency of calcium carbonate.

[0007] Furthermore, the microorganism is a combination of one or more of Bacillus cereus and Serratia marcescens.

[0008] Furthermore, the concentration of zinc sulfate in the culture medium containing zinc sulfate is 0.02-0.06 g / L.

[0009] Furthermore, the concentration of zinc sulfate in the culture medium containing zinc sulfate is 0.04 g / L.

[0010] Furthermore, the calcium source in the calcium source solution is a combination of one or more of calcium acetate and calcium chloride.

[0011] Furthermore, the mixture of the cultured microorganisms and the culture medium is contacted with a calcium source and then continuously shaken at 30° C. for mineralization.

[0012] The present invention also provides an accelerator for promoting microbial-induced calcium carbonate deposition, wherein the accelerator comprises zinc sulfate.

[0013] The beneficial effects of the present invention are:

[0014] The present invention involves adding zinc sulfate to the culture medium while culturing microorganisms capable of mineralization, then adding the cultured bacteria and the zinc sulfate-containing culture solution to the mineral source solution, and waiting for mineralization to complete. Compared to conventional microbial mineralization methods, the addition of zinc sulfate effectively improves the deposition and conversion rate of calcium ions in the mineralization system during microbial mineralization, increasing calcium ion consumption and producing more calcium carbonate.

[0015] Using zinc sulfate to cultivate microorganisms can rapidly catalyze the hydration of carbon dioxide, prompting the rapid deposition of calcium carbonate, thereby accelerating the matrix consolidation process and making it suitable for applications requiring rapid repair. Furthermore, microorganisms cultivated in a medium containing zinc sulfate increase the concentration of inorganic carbon in the matrix, promoting the deposition of more calcium carbonate and enhancing matrix stability. The primary byproduct produced is calcium carbonate, an environmentally friendly mineral with no other toxic byproducts, preventing new soil contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure shows the comparison of mineralization results between Bacillus cereus cultured with zinc sulfate and the control;

[0017] Figure 2 This is a comparison chart of the changes in calcium ion concentration during the mineralization process of Bacillus cereus cultured with zinc sulfate;

[0018] Figure 3 This is a comparison chart of the mineralization results of Serratia marcescens cultured with zinc sulfate;

[0019] Figure 4 This is a comparison chart of the changes in calcium ion concentration during the mineralization process of Serratia marcescens cultured with zinc sulfate;

[0020] Figure 5 This is the X-ray detection image of the mineralization products of Bacillus cereus and Serratia marcescens;

[0021] FIG6 is a scanning electron micrograph of the mineralization products of Bacillus cereus and Serratia marcescens;

[0022] Figure 7 This is a photo of the sample prepared in Example 2;

[0023] FIG8 is a stress variation diagram of Example 3;

[0024] Figure 9 is the XRD pattern of the sample prepared in Example 2;

[0025] Figure 10 This is the SEM image of the sample prepared in Example 2. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0027] Example 1

[0028] In this example, Bacillus cereus ATCC 14579 purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) and Serratia marcescens ATCC 13880 purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) were selected as experimental bacterial strains. The experimental bacteria were inoculated onto solid plates and cultured at 30°C for 24 hours. Single colonies were then picked and inoculated into liquid culture medium to obtain a bacterial suspension for later use.

[0029] The promotion culture medium is prepared according to the formula in Table 1. Table 1 includes liquid culture medium and solid culture medium. In specific implementation, either one can be selected.

[0030] Table 1

[0031]

[0032] A calcium source solution was prepared. In this embodiment, calcium acetate solution was selected as the calcium source solution. 26.43 g of calcium acetate was weighed and dissolved in water in a beaker. The volume was then fixed to 1000 mL to obtain a calcium source solution with a concentration of 150 mmol / L for later use.

[0033] Experimental group (BC0.04) was set up: 5 mL of cultured bacterial solution ATCC 14579 was mixed with 200 mL of promotion liquid medium and 100 mL of calcium source solution, added to a conical flask, and placed in an incubator at 120 rpm and 30°C for 120 h.

[0034] Experimental group (SM0.04) was set up: 5 mL of cultured bacterial solution ATCC 13880 was mixed with 200 mL of promotion liquid medium and 100 mL of calcium source solution, added to a conical flask, and placed in an incubator at 120 rpm and 30°C for 120 h.

[0035] A control group (BC0) was also set up: the difference between the control group (BC0) and the experimental group (BC0.04) was that zinc sulfate was not added to the 200 mL liquid culture medium, and the other proportions were the same as those of the 200 mL promotion liquid culture medium;

[0036] Set up a control group (SM0): The difference between the control group (SM0) and the experimental group (SM0.04) is that zinc sulfate is not added to the 200 mL liquid culture medium, and other proportions are the same as those of the 200 mL promotion liquid culture medium;

[0037] In culture, the Ca 2+ The concentration change trend is measured by:

[0038] Determination of Ca2+ in bacterial mineralization by EDTA (disodium ethylenediaminetetraacetic acid) method 2+ The concentration trend is as follows: after centrifuging 5.0 mL of the mineralized solution, take the supernatant and place it in a 250 mL conical flask. Add deionized water to dilute it to 100 mL, then add 2 mL of 20% triethanolamine solution and shake well. Then add 5.0 mL of 10% NaOH solution and shake well. Immediately add 2 drops of calcium indicator solution, controlling the amount to just enough to see its color. Add EDTA standard solution until the solution changes from red to purple-blue. Measure the amount of EDTA standard solution and use the following formula to calculate the Ca in the mineralized solution. 2+ concentration .

[0039] ;

[0040] in, is the concentration of standard EDTA solution, mmol / L; is the volume of EDTA solution consumed, mL; is the volume of the mineralization fluid, mL.

[0041] The results of the experimental group (BC0.04) and the control group (BC0) are shown in Figure 2. Figure 1 shown by Figure 1 It can be seen that after the addition of zinc sulfate, the amount of calcium carbonate mineralized by Bacillus cereus ATCC 14579 increased from 0.55243 g to 0.68523 g, and the overall efficiency increased by about 24.04%.

[0042] After 24 hours from the start of the mineralization experiment, the mineralization solution Ca 2+ Concentration determination, the results are as follows Figure 2 As shown, Figure 2 (+) ZnSO4 is the experimental group (BC0.04), (-) ZnSO4 is the control group (BC0); Figure 2It can be seen that during the 120-h mineralization process, the calcium ion concentration of the mineralized solution of the experimental group (BC0.04) with the addition of zinc sulfate was always lower than that of the control group (BC0), and the calcium ion concentration curve of the experimental group (BC0.04) began to slow down after 120 h of mineralization, while the calcium ion concentration curve of the control group (BC0) began to slow down after 100 h of mineralization.

[0043] The results of the experimental group (SM0.04) and the control group (SM0) are shown in Figure 2. Figure 3 shown by Figure 3 It can be seen that after the addition of zinc sulfate, the amount of calcium carbonate mineralized by Serratia marcescens ATCC 13880 increased from 0.82873 g to 0.97137 g, and the overall efficiency increased by about 17.21%.

[0044] After 24 hours from the start of the mineralization experiment, the mineralization solution Ca 2+ Concentration determination, the results are as follows Figure 4 As shown, the calcium ion concentration in the experimental group (SM0.04) with zinc sulfate added decreased at a faster rate, reaching the mineralization endpoint around the 72nd hour, compared to around the 96th hour for the control group (SM0) without zinc sulfate. During the 70-80 hour period, the microbial mineralization reaction reached its final stage, with nutrients in the system essentially depleted and the microbial population reaching the maximum capacity. At this point, all microbial metabolic activities were inhibited, the microbial mineralization reaction reached its endpoint, and the calcium ion concentration in the system remained essentially constant.

[0045] The mineralized sediments of the experimental group (BC0.04) and the experimental group (SM0.04) were filtered, and the organic matter in the sediments was removed with 30% hydrogen peroxide. Then, the sediments were washed with distilled water, dried, and ground into a fine powder with a mortar until there was no particle feeling. The phase composition of the mineralized product was determined by X-ray diffractometer. The results are as follows: Figure 5 The mineralization of the experimental group (BC0.04) and the experimental group (SM0.04) was detected using a scanning electron microscope, and the results are shown in Figure 6. Figure 6A The mineralization of the experimental group (SM0.04) Figure 6B This is the mineralization of the experimental group (BC0.04).

[0046] Depend on Figure 5 -6 shows that the main phase composition of the mineralized product of Bacillus cereus ATCC 14579 is vaterite-type calcium carbonate, followed by calcite-type calcium carbonate. However, the main phase composition of the mineralized product of Serratia marcescens ATCC 13880 is vaterite-type calcium carbonate, and no calcite-type calcium carbonate appears.

[0047] Example 2

[0048] A promotion liquid culture medium was prepared using the method of Example 1. A loopful of Bacillus cereus ATCC 14579 was inoculated into the seed liquid culture medium and cultured in a shaking incubator at 30°C and 120 rpm for 24 hours to prepare a seed liquid. Subsequently, 1 mL of the seed liquid was pipetted at a 1% (v / v) inoculum volume and inoculated into the promotion liquid culture medium. The culture was further incubated in a shaking incubator at 30°C and 120 rpm for 24 hours to prepare a promotion liquid.

[0049] Take 100g of standard sand and place it in a 5.5×5.5cm mold for later use. Take 100mL each of the cultured bacterial solution and 0.5M calcium acetate solution, mix them in a beaker, and use a plastic dropper to draw it into the mold as described above. Inject it once every 12 hours and cure it in a 30℃ constant temperature incubator for 7 days. After curing, dry it in a 60℃ oven. Remove the cured specimen from the mold and dry it for observation. Figure 7 As shown, Figure 7 A is loose sand before solidification, Figure 7 B is a mixed grouting solidification specimen. Before solidification, the specimen was dry loose sand. Figure 7 It can be seen that this method can solidify loose sand into a whole. The surface of the specimen is covered with a calcium carbonate deposition layer up to 4 mm thick. The specimen is well consolidated as a whole, with no obvious loose sand scattering phenomenon. A clearly visible microbial cementation layer is formed on the entire surface of the solidified specimen.

[0050] The sand used in the experiment was Xiamen standard sand (containing 99.8% quartz). This sand had an uneven gradation, a gradation coefficient Cu = 6.4, a curvature coefficient Cc = 0.5, a median particle size (D50) of 0.67 mm, and a maximum dry density of 1.478 for dry bulk sand. Before the experiment, the sand was soaked in 1M HCl solution. When no bubbles were generated, it was repeatedly washed with deionized water 4-5 times and dried for use.

[0051] Example 3

[0052] Take the test piece prepared in Example 2 and set up a control group. The difference between the control group and Example 2 is that the seed liquid is inoculated in a liquid culture medium without zinc sulfate. A shearing experiment is carried out on Example 2 and the control group, specifically: a ZJ strain-controlled direct shear apparatus is used to place the sample in a shear box, and shearing is carried out at a shear rate of 0.8 mm per minute under a vertical pressure of 50 kPa. The hand wheel is rotated once to record the dynamometer reading until a peak value appears in the dynamometer reading, and the shearing is continued until the shear displacement reaches 4 mm. The shear strength is the peak value in the shear displacement and shear stress diagram. If there is no peak value, the shear stress value at the shear displacement of 4 mm is taken. The specimen size is 61.8 mm in diameter and 20 mm in height. The initial cross-sectional area of ​​the specimen is 30 m 2 , the dynamometer calibration coefficient is 1.87N / 0.01mm.

[0053] The stress change diagram is obtained by statistical integration, and the results are shown in Figure 8. Figure 8A is the stress change diagram of the control group, Figure 8B FIG8 is a stress change diagram of Example 2. As can be seen from FIG8 , both Example 2 and the control group show a trend of first increasing and then decreasing. Since there is no calcium carbonate in the control group, the pores between the sand particles are large, and the resulting shear strength mainly comes from the interaction force between the sand particles. Although there is calcium carbonate deposition inside Example 2, the distribution is uneven. When the specimen is subjected to stress, it immediately cracks into blocks, and some parts become loose sand. However, since the surface sand particles of Example 2 are coated with calcium carbonate, it still has a certain cohesive force.

[0054] Example 4

[0055] The specimen prepared in Example 2 and destroyed in Example 3 was recovered and weighed to obtain the total weight of calcium carbonate and sand, which was recorded as m0. After repeated washing with sterile water for 4 to 5 times, 1M hydrochloric acid was added for pickling. After no bubbles were generated, it was washed with sterile water and dried in an oven at 60°C. It was weighed again to obtain the net sand weight, which was recorded as m1. The amount of calcium carbonate deposition was m c = m1 - m0. Calculations show that the calcium carbonate deposited in the specimen of Example 2 reached 16.18 g. Because the entire specimen was immersed in the deposition system, the entire process from chemical deposition to decomposition of the chemical deposit followed by biological deposition was completed, resulting in minimal calcium carbonate loss. The maximum dry density of the cured specimen was measured using the water displacement method, and the maximum dry density of the cured specimen was 1.974.

[0056] Example 5

[0057] A small amount of the sample prepared in Example 2, which was destroyed in Example 3, was ground until there was no particle feeling. X-ray diffraction experiments were performed using a Japanese Rigaku D / max2200VPC X-ray diffractometer (XRD) with a diffraction angle of 3° to 80°. After obtaining the diffraction pattern, the powder diffraction card (PDF card) edited and published by the Joint Conference on Powder Diffraction Standards (JCPDS) was used for phase analysis to obtain the crystal form of calcium carbonate obtained by deposition. The XRD pattern is shown in FIG. Figure 9 As shown by Figure 9 It can be seen that quartz is the main component of the standard sand used in the experiment. By comparing with the standard card, it was found that calcium carbonate with calcite and vaterite crystal forms was generated in the solidified specimen.

[0058] Take small particles from the specimen after shear failure and observe the bonding between sand particles, between sand particles and calcium carbonate particles, and between calcium carbonate particles in the solidified specimen under a high and low variable vacuum field emission scanning electron microscope. Figure 10 As shown by Figure 10It can be seen that the strain induced the formation of calcium carbonate after being cultured with zinc sulfate, which was deposited in the gaps of the sand samples and attached to the surface of the sand samples. The calcium carbonate is mostly granular in shape, with a very small part being layered rhombohedral. At the same time, bacterial pits left by the strain can also be seen on the sediment. In the process of strain-induced calcium carbonate deposition, calcium carbonate crystals use mineralizing bacteria as nucleation sites and are deposited on the surface of sand particles and in the gaps between particles. Their size gradually increases due to the continuous stacking of calcium carbonate crystals, which eventually leads to the cementation of calcium carbonate crystal clusters on the surface of adjacent sand particles, thereby cementing the originally adjacent but non-contacting particles into a whole. In the process, calcium carbonate crystals first have a filling effect and then a cementing effect, thereby improving the integrity and strength of the sand sample.

Claims

1. A method for promoting microbial induction of calcium carbonate deposition, characterized in that: By adding a culture medium containing zinc sulfate, culturing microorganisms capable of inducing calcium carbonate deposition, and contacting a mixture of the cultured microorganisms and the culture medium with a calcium source, the deposition efficiency of calcium carbonate can be increased; The microorganism is Bacillus cereus ( Bacillus cereus ) or Serratia marcescens ( Serratia marcescens ) or a combination of one or more thereof; the Bacillus cereus is Bacillus cereus with a deposit number of ATCC 14579, and the Serratia marcescens is Serratia marcescens with a deposit number of ATCC 13880; The concentration of zinc sulfate in the zinc sulfate-containing culture medium is 0.04 g / L.

2. The method for promoting microbial-induced calcium carbonate deposition according to claim 1, wherein: The calcium source in the microbial mineralization process is a combination of one or more of calcium acetate and calcium chloride.

3. The method for promoting microbial-induced calcium carbonate deposition according to claim 1, wherein: The mixture of the cultured microorganisms and the culture medium is brought into contact with a calcium source and then mineralized at 30°C.

Images