Method for preparing calcareous sand reinforced by MICP combined with waste molasses

Abstract

The application discloses a preparation method of MICP combined with molasses reinforced calcareous sand. The preparation method comprises the following steps: activating and culturing mineralization bacteria, preparing a molasses culture medium by using waste molasses, inoculating the activated mineralization bacteria liquid on the molasses culture medium, and after the OD600 value of the mineralization bacteria in the molasses culture medium is between 1.0 and 1.5, injecting the molasses culture medium containing the mineralization bacteria into the calcareous sand for reinforcement, so that the reinforced calcareous sand is obtained. The preparation method of the MICP combined with molasses reinforced calcareous sand disclosed by the application directly uses waste molasses as a carbon source to culture mineralization bacteria, greatly reduces the preparation cost, and is green and environment-friendly in the preparation process; by adding sugarcane residue with a proper proportion and length in the waste molasses, the viscous sugar silk and the sugarcane residue are uniformly distributed in the reinforced calcareous sand, so that the generation amount of the microbial-induced calcium carbonate crystal is improved, and the prepared calcareous sand has more excellent reinforcement effect, and the unconfined compressive strength can reach 2 MPa at most.

Description

Technical Field

[0001] This invention relates to a method for preparing calcareous sand reinforced with MICP combined with waste molasses, belonging to the field of calcareous sand reinforcement. Background Technology

[0002] Microbial-induced calcium carbonate precipitation (MICP) is an emerging soil reinforcement method with significant application potential in various fields, including soil mechanical modification, embankment erosion resistance, and prevention of heavy metal ion pollution. Currently, the mainstream method involves injecting urea-hydrolyzing bacteria and a cementing solution containing calcium ions into the soil. Through bacterial metabolism, carbonate ions are produced and combine with calcium ions to form calcium carbonate in the soil pores, thus bonding the soil. The process is simple, low-cost, and non-biochemically toxic. It has also achieved good results in improving the mechanical properties of calcareous sand, meeting the stringent requirements of island and reef construction in the South China Sea.

[0003] Molasses is a byproduct of the sugar industry and serves as a low-cost, sustainable hydrophilic carbon source. In 2020, China's annual molasses production reached 3.59 million tons. Molasses has a complex composition, primarily containing approximately 40-60% (by mass) total sugars (sucrose, glucose, and fructose), along with some suspended colloids, nitrogenous compounds, and the remainder being water. Its rich carbon source content and relatively low price make molasses a raw material for the production of many important industrial chemicals.

[0004] Existing technologies, particularly those involving microbial coagulation (MICP) solidification, struggle to balance matrix toughness and overall strength. Furthermore, microorganisms require specific nutrients to function effectively, and the expensive carbon sources in conventional MIP reinforcement methods add to the cost. Waste molasses, however, can serve as a low-cost carbon source, and certain types of waste molasses possess physical properties beneficial for calcareous sand reinforcement. Therefore, the combination of microorganisms and waste molasses in reinforcement methods holds promise for future applications. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for preparing MICP combined with waste molasses to reinforce calcareous sand.

[0006] Technical solution: The preparation method of MICP combined with waste molasses to reinforce calcareous sand of the present invention includes the following steps: activating and culturing mineralizing bacteria, preparing waste molasses culture medium using waste molasses, inoculating the activated mineralizing bacteria solution onto the waste molasses culture medium, culturing until the OD600 value of the mineralizing bacteria in the waste molasses culture medium is between 1.0 and 1.5, and then injecting the waste molasses culture medium containing mineralizing bacteria into calcareous sand for reinforcement, thereby obtaining reinforced calcareous sand.

[0007] This invention's preparation method utilizes waste molasses directly as a carbon source to cultivate mineralizing bacteria, reducing costs. Furthermore, the liquid waste molasses can be more fully injected into the pores of calcareous sand, simplifying the injection process. The waste molasses used in this invention is sugarcane molasses, which is low in cost and rich in calcium ions, beneficial for the induction of calcium carbonate. The alkaline environment of the waste molasses is also more suitable for the growth of mineralizing bacteria. When injected into the pores of the calcareous sand, the waste molasses can absorb water in the flow path due to osmotic pressure, thereby improving the overall resistance to liquefaction. As a green and environmentally friendly emerging process, it can, to some extent, replace cement-based chemical binders, meeting engineering requirements while reducing carbon dioxide emissions. When the reinforced calcareous sand prepared according to this invention is used to construct coastal calcareous sand foundations, heavy metal ions in seawater can stress the growth of microorganisms. However, waste molasses is rich in reducing substances that have a reducing effect on heavy metal ions (such as toxic hexavalent chromium ions, which are easily reduced to harmless trivalent chromium ions by waste molasses), indirectly enhancing microbial activity and thus improving the ability of microorganisms to induce calcium carbonate, achieving two benefits at once. Furthermore, the viscous liquid waste molasses coating the surface of the generated calcium carbonate can also form an antioxidant film, improving the antioxidant capacity of calcium carbonate in the initial stage of induction. In the preparation method of this invention, the waste molasses is diluted to a low degree and is more viscous. Its viscous sugar strands and bagasse are evenly distributed in the MICP-reinforced calcareous sand, allowing it to interweave into a network within the calcareous sand, providing more attachment sites for bacteria. Microbial cells can more easily colonize the biocompatible bagasse surface. Calcium carbonate grows around the surface of the cells, providing more locations for microorganisms to colonize after entering the soil, thereby increasing the amount of microbially induced calcium carbonate crystals and improving the strength of the solidified calcareous sand. Microbial-induced calcium carbonate crystals adhere to the surface of fibrous bagasse and molasses, making it rougher. This improves the interfacial mechanical properties between the bagasse and calcareous sand particles, enhancing their tensile strength. However, since the internal pore volume of the calcareous sand is fixed, a suitable size of bagasse is needed to ensure a sufficient living environment for the microorganisms and achieve a superior reinforcement effect. Therefore, the bagasse used in this invention has a length of 15mm-35mm.

[0008] Furthermore, the activation culture medium used for the activation culture of mineralizing bacteria includes yeast extract, peptone, sodium chloride, beef extract, and inorganic salts.

[0009] Furthermore, the waste molasses is sugarcane molasses.

[0010] Furthermore, it also includes a pretreatment step for waste molasses: filtering out the sugarcane bagasse from the waste molasses, diluting it, using activated carbon for decolorization, adding flocculants to remove ash, and then adding sugarcane bagasse.

[0011] Furthermore, the waste molasses is diluted by 2 to 5 times.

[0012] Furthermore, the flocculant is polyacrylamide.

[0013] Furthermore, the amount of sugarcane bagasse added accounts for 0.3%-0.7% of the mass of waste molasses, and the length of the sugarcane bagasse is 15mm-35mm.

[0014] Furthermore, the preparation of the waste molasses culture medium is as follows: calcium chloride, ammonium chloride, and urea are added to the waste molasses, mixed evenly, placed in an anaerobic bottle, N2 is poured in, and the mixture is autoclaved at 120°C for 20 minutes to obtain the culture medium.

[0015] Furthermore, the amount of the mineralizing bacteria solution added to the waste molasses culture medium is 30-50 mL / 100 cm^3 of the total amount.

[0016] Furthermore, the amount of nutrients added is 0.5 to 3.0 mol / L of the total water content in the waste molasses culture medium, and the nutrients include sodium chloride, calcium chloride, ammonium chloride, urea, dipotassium hydrogen phosphate, and magnesium sulfate.

[0017] Furthermore, the urease activity of the activated mineralized bacterial solution is 7.0 U / ml ± 0.2.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The preparation method of MICP combined with waste molasses for strengthening calcareous sand disclosed in the present invention utilizes waste molasses directly as a carbon source to cultivate mineralizing bacteria, which greatly reduces the preparation cost and the preparation process is green and environmentally friendly; by adding an appropriate proportion and length of sugarcane bagasse to the waste molasses, the viscous sugar threads and sugarcane bagasse are evenly distributed in the strengthened calcareous sand, thereby increasing the amount of calcium carbonate crystals generated by microorganisms, so that the prepared calcareous sand has a better strengthening effect and the unconfined compressive strength can reach up to 2 MPa. Attached Figure Description

[0019] Figure 1 Figures showing the unconfined compressive strength and calcium carbonate formation of calcareous sand reinforced with different sugarcane bagasse content;

[0020] Figure 2 Figures showing the unconfined compressive strength and calcium carbonate formation of calcareous sand reinforced with different bagasse lengths;

[0021] Figure 3 The effect of different dilution ratios of waste molasses on urease content. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0023] Example 1: Optimization of Sugarcane Bagasse Addition Amount

[0024] 1. Bacterial activation culture

[0025] Preparation of solid activated culture medium: Dissolve 6g yeast extract, nutrient broth (8g peptone, 5g sodium chloride, 3g beef extract), 20g inorganic salts (18g NaCl and 2g K2HPO4), and 15g agar powder in 1L of water.

[0026] The solid culture medium was sterilized at 121℃ for 30 min and then allowed to cool naturally to room temperature. A single colony of mineralizing bacteria (Bacillus pasteurellii, ATCC11859, Wuhan Huana Biotechnology Co., Ltd.) was then streaked onto the solid culture medium and incubated at 30℃ for 12-14 h. Then, an inoculum of 10... 4 -10 6 CFU was inoculated into liquid seed culture medium (containing 3g beef extract, 10g peptone, 5g NaCl, and 1000mL sterile water) and cultured at 30℃ and 200 rpm for 24 hours in a biochemical incubator, then stored at low temperature for later use. When needed, the mineralized bacterial culture was removed from the low-temperature storage, and the inoculated bacterial colony was picked up using an inoculation loop and cultured on solid activated culture medium at room temperature for 48 hours using the streak plate method.

[0027] 2. Preparation of waste molasses and liquid culture medium for waste molasses

[0028] Waste molasses pretreatment:

[0029] The waste molasses used in this embodiment is sugarcane molasses (Guimi Biotechnology Co., Ltd.), whose composition is controlled as 48% sucrose, 8% reducing sugar, 10% inorganic salts (mainly calcium, sodium and potassium ions), 8% colloid and 26% water.

[0030] The sugarcane bagasse from the waste molasses is filtered out, and the waste molasses is diluted twice using aseptic techniques and then decolorized using activated carbon.

[0031] Add 0.3% polyacrylamide (PAM) as a flocculant to accelerate clarification and sedimentation and quickly remove ash.

[0032] Sugarcane bagasse was then added to the waste molasses, resulting in a sugarcane bagasse mass percentage of 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, and 0.9%, respectively, with the long sugarcane bagasse having a length of 25 mm.

[0033] Preparation of waste molasses culture medium:

[0034] For a 1L culture medium, add 60g of calcium chloride, 20g of ammonium chloride, and 40g of urea to 1L of pretreated liquid waste molasses, adjusting the pH to above 8.5 (pH adjustment was performed using a sterilized 1mol / L sodium hydroxide solution). Place the culture medium in an anaerobic flask and add N2 to optimize the carbon-to-nitrogen ratio. Finally, autoclave the waste molasses at 120℃ for at least 20 minutes.

[0035] 3. Screening mineralizing bacteria

[0036] Take out the bacteria prepared in step 1, pick a single colony and inoculate it into liquid seed culture medium. Shake and culture in an air bath shaker at 200 rpm / min and 35℃. After 24 h of culture, measure the urease content in the bacterial solution and select the bacterial solution with the highest unit urease activity (7.0 U / ml ± 0.2) for the next step of growth culture.

[0037] The pH of the bacterial culture obtained above was adjusted to above 9.5 using a strong alkaline aqueous solution (sterilized 1 mol / L sodium hydroxide solution), and the solution was allowed to stand for ≥30 min. Then, the culture was carried out using the waste molasses culture medium prepared in step 2. The amount of bacterial culture added to the waste molasses culture medium was 45 mL / 100 cm³. 3 The amount of nutrients (sodium chloride, calcium chloride, ammonium chloride, urea, dipotassium hydrogen phosphate, and magnesium sulfate in a mass ratio of 0.45:0.27:0.09:0.18:0.001:0.001) added is 2.5 mol / L of the total water content in the waste molasses culture medium. Incubate at room temperature for 24–36 h until the OD600 value of the mineralizing bacteria in the culture medium is between 1.0 and 1.5.

[0038] 4. Grouting reinforcement

[0039] The prepared bacterial solution is injected into the residual soil particles using a peristaltic pump at a rate of 1 ml / min. After the reaction is complete, the reinforced calcareous sand is obtained. During grouting, care should be taken that the diameter of the pipe for transporting the mixed bacterial solution should not be too small, generally 50 mm; the diameter of the pipe for transporting and loading / unloading molasses should be above 100 mm.

[0040] The results are as follows Figure 1 As shown, when the sugarcane bagasse content is in the range of 0.3%-0.7%, the calcium carbonate production of 24%-26% and its unconfined compressive strength of 1.55-2 MPa are both at a relatively high level.

[0041] Example 2: Optimization of sugarcane bagasse length

[0042] The steps are basically the same as in Example 1, except that the amount of bagasse added is 0.5%, and the variable is the length of the added bagasse (bagasse length is 0mm, 5mm, 10mm, 15mm, 20mm, 25mm, 30mm, 35mm, 40mm). The results are as follows. Figure 2 As shown, when the bagasse length is in the range of 15mm-35mm, the calcium carbonate production of 23%-26% and its unconfined compressive strength of 1.5-2MPa are both at a relatively high level.

[0043] Example 3: Optimization of the dilution ratio of waste molasses

[0044] The steps are basically the same as in Example 1, except that the amount of sugarcane bagasse added is 0.5%, and the variable is the dilution factor of the waste molasses (0, 1, 2, 3, 4, and 5 times, respectively). The results of the urease activity test are as follows. Figure 3 As shown, mineralizing bacteria were cultured on a medium made from liquid waste molasses at different dilutions. Urease activity was low at dilutions of 0-1 times, and approximately the same at dilutions of 2-5 times. To maintain the physical properties of the waste molasses, a lower dilution should be selected while ensuring high urease activity; therefore, the optimal dilution is 2 times.

[0045] Example 4: Optimization of bacterial culture addition amount

[0046] The steps are basically the same as in Example 1, with the amount of sugarcane bagasse added being 0.5%. The variable is the amount of bacterial solution added to the waste molasses culture medium (10 mL / 100 cm^3, 30 mL / 100 cm^3, 50 mL / 100 cm^3, and 70 mL / 100 cm^3, respectively). When the amount of bacterial solution added accounts for less than 30 mL / 100 cm^3 or more than 50 mL / 100 cm^3 of the total waste molasses, the OD600 of the mineralizing bacteria is less than 1.0, and the growth of the strains is not ideal.

[0047] Example 5: Optimization of Nutrient Salt Addition Amount

[0048] The steps are basically the same as in Example 1, with the amount of sugarcane bagasse added being 0.5%, and the variable being the amount of nutrient salt added (0.5 mol / L, 1 mol / L, 1.5 mol / L, 2.0 mol / L, 2.5 mol / L, 3.0 mol / L, and 3.5 mol / L, respectively). When the amount of nutrient salt added is less than 0.5 mol / L or greater than 3.0 mol / L of the total water content in the waste molasses, the OD600 of the mineralizing bacteria is less than 1.0, and the growth of the strains is not ideal.

Claims

1. A method for preparing MICP combined with waste molasses-reinforced calcareous sand, characterized in that, Includes the following steps: The mineralizing bacteria are activated and cultured. A molasses culture medium is prepared using waste molasses. The activated mineralizing bacteria solution is inoculated onto the molasses culture medium and cultured until the OD600 value of the mineralizing bacteria in the molasses culture medium is between 1.0 and 1.

5. Then, the molasses culture medium containing the mineralizing bacteria is injected into calcareous sand for reinforcement, thus obtaining reinforced calcareous sand. The waste molasses is sugarcane molasses. The preparation method also includes a waste molasses pretreatment step: filtering out the sugarcane bagasse from the waste molasses, diluting, decolorizing with activated carbon, adding a flocculant to remove ash, and then adding sugarcane bagasse. The amount of sugarcane bagasse added is 0.3%-0.7% of the mass of the waste molasses, and the length of the sugarcane bagasse is 15mm-35mm. The amount of the mineralizing bacteria solution added to the waste molasses culture medium is 30-50 mL / 100cm of the total volume. 3 .

2. The preparation method according to claim 1, characterized in that, The activation culture medium used for the activation culture of mineralizing bacteria includes yeast extract, peptone, sodium chloride, beef extract, and inorganic salts.

3. The preparation method according to claim 1, characterized in that, The waste molasses is diluted by a factor of 2 to 5.

4. The preparation method according to claim 1, characterized in that, The flocculant is polyacrylamide.

5. The preparation method according to claim 1, characterized in that, Preparation of the waste molasses culture medium: Add 60g-100g of calcium chloride, 20g-30g of ammonium chloride, and 40g-60g of urea to every 1L of waste molasses, mix well, put into an anaerobic bottle, pour in N2, and autoclave at 120℃ for 20 minutes to obtain the culture medium.

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