Microbial self-repairing concrete crack repairing material and preparation method thereof

Abstract

The application discloses a microbial self-repairing concrete crack repairing material and a preparation method thereof, and belongs to the technical field of concrete repairing materials. The repairing material comprises a fungus component and a bacteria component, the fungus is selected from filamentous fungi capable of forming a three-dimensional mycelium network, and the bacteria are selected from urease-producing bacteria capable of inducing calcium carbonate precipitation. The preparation method comprises respectively preparing a fungus spore suspension and a bacteria suspension, and inoculating the mixture in a simulated concrete pore solution containing a nutrient component to perform co-culture. The fungus mycelium network provides an attachment site and a protective microenvironment for the bacteria, and the two cooperate in metabolism to generate calcium carbonate precipitation, which significantly improves mineralization efficiency and crack repairing width, enhances adaptability in a high alkaline environment, and can effectively plug 0.5-2.0mm cracks, and has a good engineering application prospect.

Description

Technical Field

[0001] A self-healing microbial concrete crack repair material and its preparation method are disclosed, belonging to the field of concrete repair material technology. Background Technology

[0002] Concrete is the most widely used building material in modern civil engineering. However, during long-term service, microcracks inevitably develop due to factors such as load, temperature changes, shrinkage, creep, and environmental erosion. These cracks provide channels for the rapid penetration of corrosive media such as water, chloride ions, and sulfate ions, accelerating the corrosion of internal steel reinforcement and the carbonation of concrete, ultimately leading to a decrease in structural load-bearing capacity and a shortened service life. Therefore, effective crack repair is a key issue in ensuring the durability of concrete structures.

[0003] Traditional crack repair methods mainly include surface coating, pressure grouting, and cement mortar grouting. These methods are all passive repairs, requiring treatment only after cracks appear, and have inherent drawbacks such as high cost, poor compatibility with the original substrate, and difficulty in repairing deep microcracks. In recent years, inspired by the phenomenon of microbial mineralization in nature, microbially induced calcium carbonate precipitation (MICP) technology has been proposed and rapidly developed into a research hotspot in the field of intelligent self-healing concrete. This technology utilizes the metabolic activities of specific microorganisms to induce the in-situ formation of calcium carbonate crystals with cementing effects at the location of cracks and other defects, thereby achieving self-healing of cracks and offering significant advantages such as being environmentally friendly and providing in-situ repair.

[0004] In MICP (Microbial Injection Processing) technology, urease-producing bacteria such as *Sporosarcina pasteurii* are the most commonly used model organisms due to their high metabolic efficiency and ease of regulation. However, remediation systems based on single bacteria face numerous challenges in practical applications. On the one hand, the pore solution in concrete is highly alkaline (pH usually exceeds 10), and coupled with high osmotic pressure and limited nutrients, this leads to low bacterial survival rates and a rapid decline in metabolic activity. On the other hand, single-bacterial systems can typically effectively repair cracks less than 0.5 mm wide, while cracks commonly encountered in engineering practice often reach the mm level, making it difficult to effectively seal wide cracks using only a single-bacterial system.

[0005] To overcome these limitations, researchers have begun exploring strategies for multi-species synergistic repair. Among these, the synergistic system of fungi and bacteria has shown unique potential. Filamentous fungi can grow three-dimensional, multi-scale hyphal networks. These networks not only serve as a physical framework providing anchors and support for wide cracks, but also secrete extracellular polymers (EPS), providing attachment sites and a protective microenvironment for bacteria. However, current research on the construction methods of fungal-bacterial synergistic systems, the quantitative evaluation of mineralization efficiency, and the elucidation of deep synergistic mechanisms is still insufficient, and mature technical solutions have not yet been developed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a microbial self-healing concrete crack repair material with strong environmental adaptability, high mineralization efficiency, and based on the synergistic effect of fungi and bacteria, as well as its preparation method.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a microbial self-healing concrete crack repair material, characterized in that it comprises a fungal component and a bacterial component; the fungal component is selected from filamentous fungi capable of forming a three-dimensional hyphal network, and the bacterial component is selected from urease-producing bacteria capable of inducing calcium carbonate precipitation through metabolic activity.

[0008] This invention is the first to combine filamentous fungi with urease-producing bacteria for the repair of concrete cracks. The three-dimensional hyphal network formed by the fungi serves as a physical framework, providing support and anchor points for wide cracks, while simultaneously providing attachment sites and a protective microenvironment for the bacteria, significantly improving the survival rate and metabolic activity of the bacteria in highly alkaline and high-osmotic pressure environments. This synergistic system overcomes the limitation of single-bacterial systems in repairing cracks less than 0.5 mm wide, achieving effective sealing of millimeter-level cracks, significantly improving mineralization efficiency, and increasing calcium carbonate production by more than 49% compared to single-bacterial groups.

[0009] Preferably, the fungal component is *Rhizopus oryzae*, and the bacterial component is *Sporosarcina pasteurii*. *Rhizopus oryzae* is a common, safe, and easily culturable filamentous fungus with rapid hyphal growth and a well-developed network, capable of stable growth in strongly alkaline, high-calcium environments. *Sporosarcina pasteurii* is a recognized highly efficient urease-producing strain with strong urease activity and rapid mineralization. When combined, the organic acids secreted by the fungus can neutralize the OH- produced by bacterial metabolism. - This stabilizes the system pH within the optimal mineralization range of 6.5–7.5, while simultaneously reducing the NH4 produced by bacterial metabolism. + It provides fungi with a high-quality nitrogen source, forming a metabolic mutualism and further enhancing the synergistic effect.

[0010] Preferably, this remediation material also contains nutrient components, including a calcium source, urea, a carbon source, and a nitrogen source. The addition of these nutrients provides essential substrates for the growth and metabolism of fungi and bacteria. Calcium and urea are core raw materials for the MICP reaction, while the carbon and nitrogen sources maintain bacterial viability. This optimized nutrient formula meets the nutritional needs of both bacterial species in the co-culture system without causing side reactions or environmental pollution due to excessive addition, ensuring the continuous and efficient mineralization reaction. Within 14 days, the calcium content... 2+ The consumption rate can reach 45%.

[0011] Preferably, the calcium source is anhydrous calcium chloride, the carbon source is glucose, and the nitrogen source is selected from one or more of yeast extract, beef extract, and peptone. Anhydrous calcium chloride has high solubility and releases calcium. 2+ The combination of these ingredients is fast, facilitating the rapid formation of a supersaturated calcium carbonate environment; glucose, as an readily available carbon source, can be utilized by both fungi and bacteria; yeast extract, beef extract, and peptone are rich in amino acids, vitamins, and growth factors, comprehensively promoting cell proliferation and metabolism. This optimized combination enabled the co-culture system to achieve a calcium carbonate precipitation yield of 1.89 g / 100 mL within 14 days, representing a 49.2% increase compared to the single bacterial group and a 117.9% increase compared to the single fungal group.

[0012] A method for preparing the above-mentioned microbial self-healing concrete crack repair material includes the following steps: preparing fungal spore suspension and bacterial suspension respectively; mixing the fungal spore suspension and bacterial suspension, inoculating them into a simulated concrete pore solution containing nutrient components, and co-culturing them.

[0013] This method is simple to operate, operates under mild conditions, and requires no complex equipment. By pre-preparing high-concentration spore suspensions and bacterial suspensions, the inoculation amount is precisely controllable. After mixing the two, they are directly inoculated into the simulated well solution for co-cultivation, realizing the in-situ growth of fungal hyphae networks and the simultaneous mineralization of bacteria. The one-step method completes the preparation of remediation materials, which is convenient for on-site engineering applications or prefabrication.

[0014] Preferably, the concentration of the fungal spore suspension is 1×10⁻⁶. 6 The bacterial suspension had a concentration of 1 × 10⁶ spores / mL. 8 Cells / mL. This concentration ratio was systematically optimized, ensuring a moderate number of fungal spores to guarantee a sufficiently dense hyphal network after germination while avoiding excessive competition for nutrients; and a sufficient number of bacteria to ensure urease activity and mineralization efficiency. At this ratio, the growth curves of fungi and bacteria matched well, the symbiotic period was prolonged, and the OD of the co-culture system increased significantly after 14 days. 600 The value remains stable above 1.5, indicating sustained metabolic activity.

[0015] Preferably, the volume ratio of the fungal spore suspension to the bacterial suspension is 1:1. A volume ratio of 1:1 is the optimal inoculation ratio, at which point the initial ratio of fungi to bacteria is approximately 1:100, ensuring both rapid expansion and space occupation by fungal hyphae, and sufficient bacterial density for colonization on the hyphal surface. Experiments have shown that at this ratio, Ca... 2+ It has the fastest consumption rate, reaching 45% in 14 days, and the highest calcium carbonate production. Moreover, the mycelium-bacteria-calcite in the precipitate product are the most densely cemented.

[0016] Preferably, the co-culture temperature is 25℃~35℃, the oscillation speed is 60r / min~100r / min, and the culture time is 10d~18d. This temperature range is close to room temperature, has low energy consumption, and is easy to control, making it suitable for engineering site conditions. Moderate oscillation can increase dissolved oxygen levels and promote mass transfer, while avoiding excessive hyphal entanglement that could affect bacterial attachment. A culture period of 10~18 days is sufficient for the fungi to form a complete hyphal network and complete the main mineralization process; too long a time may lead to nutrient depletion or cell autolysis. This range balances efficiency and product quality.

[0017] Preferably, the pH value of the simulated concrete pore solution is 10.5~12.0, with a calcium ion concentration of 200mmol / L~400mmol / L and a urea concentration of 0.1mol / L~0.5mol / L. This parameter range simulates the strongly alkaline and high-calcium environment of real concrete pore solutions, giving the prepared repair material excellent in-situ adaptability. Under these conditions, the fungal-bacterial synergistic system can stabilize the pH value at 6.5~7.5, the calcium ion concentration gradient drives the rapid deposition of calcium carbonate, and the moderate urea concentration ensures urease catalytic efficiency without the risk of ammonia gas escape. The generated calcium carbonate is all thermodynamically stable calcite crystals with excellent durability.

[0018] Preferably, after the co-cultivation is completed, the resulting culture system is directly used to repair concrete cracks with a width of 0.5 mm to 2.0 mm. The repair material prepared by this invention can be directly injected or applied to the cracks without separation and purification. The fungal hyphae network continues to grow within the cracks and bridges both sides of the cracks, while bacteria continuously mineralize and fill the gaps. Experiments show that for cracks up to 1.5 mm wide, this system can achieve complete sealing, and the impermeability of the repaired specimens recovers to more than 85% of their original state, far superior to a single bacterial system.

[0019] Preferably, this preparation method further includes injecting or coating the co-cultured product onto concrete cracks and allowing it to cure statically at room temperature and pressure for 7–21 days, maintaining a relative humidity of 80%–85% during the curing period. The room temperature and pressure curing conditions are simple, requiring no special equipment, and the high humidity environment prevents excessive evaporation of moisture, ensuring the water and ion transport needed for bacterial metabolism. During the 7–21 day curing period, calcium carbonate continuously precipitates and densifies, eventually forming a dense sealing layer. After 21 days of curing, the compressive strength recovery rate of the repaired area can reach 72%, and the repair product adheres firmly to the concrete substrate without secondary cracking.

[0020] Preferably, each liter of the simulated concrete pore solution contains: 32.0g~35g anhydrous calcium chloride, 9.5g~10.5g urea, 4.7g~5.3g glucose, 0.8g~1.2g yeast extract, 1.2g~1.8g beef extract, and 4.5g~5.5g peptone. The 33.3g / L anhydrous calcium chloride provides approximately 300mmol / L of calcium ions, and combined with 10g / L urea, a 45% calcium ion conversion rate can be achieved within 14 days. The 5g / L glucose serves as a sufficient carbon source to support dual-strain metabolism without residue. The combination of yeast extract, beef extract, and peptone provides comprehensive amino acids, vitamins, and trace elements, enabling vigorous growth of both fungi and bacteria. This formulation is low-cost, uses readily available raw materials, and is suitable for large-scale production.

[0021] The repair material of this invention can be directly applied to repair cracks in existing concrete structures, or it can be pre-embedded in fresh concrete to achieve intelligent self-healing. Its application is flexible, construction is simple, and the durability of the repaired structure is significantly improved, extending the service life of buildings by more than 50%, reducing maintenance costs, and thus possessing extremely high economic and social benefits.

[0022] The width of the concrete cracks ranges from 0.5 to 2.0 mm. This width range covers the most common moderate cracks in engineering projects and is also the area where traditional single-bacterial systems struggle to repair effectively. The repair material of this invention exhibits the best repair effect within this width range, with a calcium carbonate filling rate exceeding 90%, no visible traces on the crack surface, and both waterproofing performance and carbonation resistance restored to their original levels.

[0023] A method for repairing concrete cracks using the aforementioned repair material involves injecting or coating the material onto the concrete cracks and allowing it to cure statically at room temperature and pressure. This allows fungi and bacteria to metabolize synergistically, generating calcium carbonate precipitate that fills and seals the cracks. This method provides a standard operating procedure for on-site construction. The injection or coating operation is simple and requires no specialized tools; the room temperature and pressure curing reduces energy consumption and the construction threshold. Fungal hyphae first grow along the crack walls and overlap to form a three-dimensional network support. Subsequently, bacteria multiply extensively on the surface of the hyphae and induce calcium carbonate to deposit directionally along the hyphae, ultimately forming a dense cementitious structure of "hyphae-bacteria-calcite," achieving permanent sealing of the cracks.

[0024] The static curing period is 7 to 21 days, during which the relative humidity is maintained at 80% to 85%. Curing time is a key parameter for ensuring the repair effect. After 7 days, calcium carbonate has basically filled the cracks, but a small amount of porosity remains in the microstructure; after 21 days, the porosity drops to its minimum, and the impermeability coefficient of the repair layer reaches 10. -11 The flow rate is on the order of m / s, comparable to that of intact concrete. A high humidity environment (≥80%) can maintain the metabolic activity of the microorganisms, prevent early drying that could lead to cracking or peeling of the repair layer, and ensure the long-term stability of the repair quality.

[0025] The fungal-bacterial synergistic repair mechanism of this invention is mainly reflected in two aspects: metabolic complementarity and physical template coupling. At the metabolic complementarity level, *Rhizopus oryzae* secretes organic acids during its growth, which can effectively neutralize the OH- produced by *Pasteurella multocida* urease hydrolysis of urea. - This stabilized the system pH within the optimal mineralization range of 6.5–7.5, avoiding the inhibitory effect of drastic pH fluctuations on the mineralization reaction in a single bacterial system; simultaneously, the NH4 produced by bacterial metabolism... + This provides a high-quality nitrogen source for the fungi, promoting mycelial growth and extracellular polymer secretion, thus forming a positive metabolic feedback loop. At the physical template coupling level, the three-dimensional hyphal network formed by *Rhizopus oryzae* provides numerous attachment sites for *Pasteurella spp.*, and the negatively charged extracellular polymers on the hyphal surface can efficiently chelate and enrich Ca in the solution. 2+ This lowers the energy barrier for heterogeneous nucleation of calcium carbonate; bacteria attached to the hyphal surface efficiently hydrolyze urea in situ, generating high concentrations of CO3 at the hyphal-bacterial interface. 2- , forming Ca 2+ With CO3 2- The localized supersaturated region guides the calcite crystals to deposit and grow directionally along the long axis of the hyphae. This ultimately forms a dense cemented structure integrating hyphae, bacteria, and calcite, which combines the flexibility of the hyphal network with the rigidity of the calcite crystals, significantly improving the width, density, and mechanical recovery performance of the crack repair.

[0026] Compared with existing technologies, the beneficial effects of the microbial self-healing concrete crack repair material and its preparation method of the present invention are as follows: The present invention is the first to combine filamentous fungi with urease-producing bacteria, utilizing the three-dimensional hyphal network of the fungi to provide attachment sites and a protective microenvironment for the bacteria. The two synergistically metabolize to generate calcium carbonate precipitate, thus... 2+ The consumption rate reached 45%, and the calcium carbonate production increased by 49.2% compared to the single bacterial group and 117.9% compared to the single fungal group. It broke through the limitation of single bacterial systems in repairing cracks less than 0.5 mm wide, effectively sealing cracks of 0.5–2.0 mm. The compressive strength recovery rate after repair reached 72%–76%, and the water permeability coefficient decreased to 1.0 × 10⁻⁶. -11 The system is on the order of m / s; it can stabilize the pH of highly alkaline environments within the optimal mineralization range of 6.5 to 7.5, generate thermodynamically stable calcite crystals, and form a dense "hyphae-bacteria-calcite" cemented structure, which significantly improves environmental adaptability and repair durability. Attached Figure Description

[0027] Figure 1 This is the morphology of the strain after revival.

[0028] Figure 1 In the middle (a), it is Pasteurella spp., and in (b), it is Rhizopus oryzae.

[0029] Figure 2 The graph shows the pH value of each group changing over time.

[0030] Figure 2 This indicates that the pH value of the co-culture group (FB group) remained stable within the ideal range of 6.5 to 7.5.

[0031] Figure 3 For each group of Ca 2+ Concentration versus time curve.

[0032] Figure 3 This indicates that the co-culture group (FB group) Ca 2+ It has the fastest consumption rate, with a final consumption rate of 45%.

[0033] Figure 4 The X-ray diffraction (XRD) patterns of each group of calcium carbonate precipitates are shown.

[0034] Figure 4 This indicates that the precipitate products are mainly calcite crystals.

[0035] Figure 5 Scanning electron microscope (SEM) image of calcium carbonate precipitation in the co-culture group.

[0036] Figure 5 It shows a dense cemented structure of hyphae-bacteria-calcite as a unified whole. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, wherein Embodiment 1 is the best embodiment, and the process conditions in other embodiments and comparative examples are as described in Embodiment 1. All raw materials used in the embodiments are commercially available, the preparation conditions are mild and the operation is simple, and they are suitable for crack repair construction in engineering sites. Example 1

[0038] First, the strain was activated and a suspension was prepared. Rhizopus oryzae was inoculated onto PDA slant agar and incubated at 30°C for 6 days. Once a large number of black spores appeared on the colony surface, 10 mL of sterile physiological saline (containing 0.05% v / v Tween-80) was added to the slant. The spores were gently scraped off with an inoculation loop, and the spore suspension was transferred to sterile test tubes. The suspension was then filtered through sterile absorbent cotton to remove mycelial debris, yielding a concentration of 1×10⁻⁶. 6 A fungal spore suspension of 1 spore / mL was prepared. Separately, *Sporosarcina pasteurii* was inoculated into nutrient broth and cultured at 30°C and 150 rpm for 36 h with shaking until the logarithmic growth phase. The cells were collected by centrifugation at 8000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with sterile physiological saline and resuspended, adjusting the concentration to 1×10⁻⁶. 8 A bacterial suspension of cells / mL.

[0039] Next, prepare the simulated concrete hole solution. Prepare each liter of simulated hole solution according to the following formula: Weigh 33.3g anhydrous calcium chloride, 10.0g urea, 5.0g glucose, 1.0g yeast extract, 1.5g beef extract, and 5.0g peptone, and add them sequentially to deionized water while stirring to dissolve. Adjust the pH to 11.35 with 1mol / L sodium hydroxide solution, and bring the volume to 1L. Dispense the prepared solution into 100mL 250mL Erlenmeyer flasks, sterilize each flask in an autoclave at 121℃ for 15 minutes, and cool to room temperature before use.

[0040] A co-culture system was then constructed in a sterile operating room. 0.5 mL of *Rhizopus oryzae* spore suspension and 0.5 mL of *Pasteurella spores* bacterial suspension were added sequentially to a conical flask containing 100 mL of sterile simulated concrete hole solution, ensuring a fungal spore to bacterial suspension volume ratio of 1:1, with a total inoculum of 1 mL / 100 mL. After tightening the stopper, the flask was gently shaken to evenly disperse the bacteria. The conical flask was then placed in a constant temperature air bath shaking incubator at 30°C and a shaking speed of 80 rpm for 14 days. During the incubation period, samples were taken every 24 hours to measure the pH and Ca content of the system. 2+ Changes in concentration and turbidity.

[0041] After cultivation, the resulting culture system is the microbial self-healing concrete crack repair material of this invention. This culture system was directly used in concrete crack repair experiments. Standard cement mortar blocks with dimensions of 40mm × 40mm × 160mm were selected, and a through crack with a width of 1.5mm and a depth of 20mm was pre-cast in the center of the block. The co-cultured repair material was slowly injected into the crack using a syringe until it overflowed from the crack surface, and then the excess material was smoothed off with a scraper. The repaired blocks were placed in a constant temperature and humidity curing chamber at 25±2℃ and 82% relative humidity for 21 days for static curing. Example 2

[0042] First, the bacterial strain was activated and the suspension was prepared using the same method as in Example 1, resulting in a concentration of 1×10⁻⁶. 6 A suspension of Rhizopus oryzae spores per mL and a concentration of 1×10 8 A suspension of *Pasteurella salina* cells / mL was prepared. A simulated concrete well solution was prepared with the following formula per liter: 35g anhydrous calcium chloride, 9.5g urea, 5.3g glucose, 0.8g yeast extract, 1.8g beef extract, and 4.5g peptone. The pH was adjusted to 10.5 with 1mol / L sodium hydroxide solution, and the volume was brought to 1L before aliquoting and sterilization. In an aseptic environment, 0.5mL of *Rhizopus oryzae* spore suspension and 0.5mL of *Pasteurella salina* suspension were added to conical flasks containing 100mL of sterile simulated well solution, resulting in a total inoculum volume of 1mL / 100mL. The conical flasks were placed in a constant temperature air bath shaking incubator at 25℃ and a shaking speed of 60 rpm for 10 days. After the culture was completed, the culture system was used to repair precast cracked cement mortar test blocks with a width of 0.5 mm and a depth of 15 mm. The injection method was the same as in Example 1. The blocks were left to cure at room temperature and pressure for 7 days, and the relative humidity was kept at 80% during the curing period. Example 3

[0043] The bacterial strain activation and suspension preparation were the same as in Example 1, yielding a concentration of 1×10⁻⁶. 6 A suspension of Rhizopus oryzae spores per mL and a concentration of 1×10 8A suspension of *Pasteurella salina* cells / mL was prepared. A simulated concrete well solution was prepared with the following formula per liter: 32.0 g anhydrous calcium chloride, 10.5 g urea, 4.7 g glucose, 1.2 g yeast extract, 1.2 g beef extract, and 5.5 g peptone. The pH was adjusted to 12.0 with 1 mol / L sodium hydroxide solution, and the solution was brought to a final volume of 1 L before being dispensed and sterilized. In an aseptic environment, 0.5 mL of *Rhizopus oryzae* spore suspension and 0.5 mL of *Pasteurella salina* suspension were added to conical flasks containing 100 mL of sterile simulated well solution. The flasks were placed in a constant temperature air bath shaking incubator at 35°C and a shaking speed of 100 rpm for 18 days. After cultivation, the resulting culture system was used to repair precast cracked cement mortar test blocks with a width of 2.0 mm and a depth of 25 mm. The injection method was the same as in Example 1. The blocks were statically cured at room temperature and pressure for 21 days, maintaining a relative humidity of 85% during the curing period. Results showed that the pH value of the co-culture system decreased rapidly from an initial 12.0, stabilizing in the range of 6.5–7.4 from day 5 to 10. Ca... 2+ The consumption rate reached 47%, and the calcium carbonate precipitation yield was 1.92 g / 100 mL. XRD analysis showed that the precipitate was pure calcite crystals. SEM observation revealed that the hyphal network extended fully within the cracks, and calcite crystals were deposited directionally along the hyphae to form a dense cement. After crack repair, the cracks on the sample surface were completely sealed. Example 4

[0044] The activation and suspension preparation of the bacterial strain were the same as in Example 1. Two simulated concrete hole solutions were prepared: the calcium ion concentration of the solution was 200 mmol / L (corresponding to 22.2 g / L of anhydrous calcium chloride), and the urea concentration was 0.1 mol / L (corresponding to 6 g / L of urea); the carbon and nitrogen source formulations of the solutions were the same, each containing 5 g of glucose, 1 g of yeast extract, 1.5 g of beef extract, and 5 g of peptone per liter. The pH was adjusted to 11.35 with 1 mol / L sodium hydroxide solution, and after making up to volume, the solutions were dispensed and sterilized. In an aseptic operating table, 0.5 mL of Rhizopus oryzae spore suspension and 0.5 mL of Pasteurella multocida bacterial suspension were added to a 100 mL Erlenmeyer flask containing solution A. The Erlenmeyer flask was placed in a constant temperature air bath shaking incubator, with the temperature set at 30°C and the shaking speed at 80 r / min, and continuously cultured for 14 days. After the culture was completed, the culture system was used to repair precast cracked cement mortar test blocks with a width of 1.0 mm and a depth of 20 mm. The injection and curing methods were the same as in Example 1. Example 5

[0045] Same as Example 4, except that the calcium ion concentration of the solution is 400 mmol / L (corresponding to 44.4 g / L of anhydrous calcium chloride) and the urea concentration is 0.5 mol / L (corresponding to 30 g / L of urea). Example 6

[0046] The activation and suspension preparation of the microbial strain were the same as in Example 1. A simulated concrete hole solution was prepared: the nitrogen source for solution C was solely yeast extract (6 g / L); the calcium, urea, and carbon source formulations were 33.3 g anhydrous calcium chloride, 10 g urea, and 5 g glucose per liter. The pH was adjusted to 11.35 with 1 mol / L sodium hydroxide solution, and the solution was dispensed and sterilized after volume adjustment. In a sterile operating table, 0.5 mL of *Rhizopus oryzae* spore suspension and 0.5 mL of *Pasteurella spp.* bacterial suspension were added to a 100 mL Erlenmeyer flask containing the solution. The Erlenmeyer flask was placed in a constant temperature air bath shaking incubator, set at 30°C and a shaking speed of 80 r / min, and continuously incubated for 14 days. After incubation, the culture system was used to repair precast cracked cement mortar test blocks with a width of 1.5 mm and a depth of 20 mm. Two parallel samples were prepared and cured for 14 days and 21 days respectively. Example 7

[0047] Similar to Example 6, except that when preparing the simulated concrete hole solution, the nitrogen source of the solution is only peptone (7.5 g / L). Example 8

[0048] The bacterial strain activation and suspension preparation were the same as in Example 1, yielding a concentration of 1×10⁻⁶. 6 A suspension of Rhizopus oryzae spores per mL and a concentration of 1×10 8 A suspension of *Pasteurella spores* cells / mL was prepared. The solution for simulating concrete pores was prepared as in Example 1. In a sterile operating room, the volume ratio of fungal spore suspension to bacterial suspension was 1:1, and the total inoculum was fixed at 1 mL / 100 mL. The conical flasks were incubated at 30°C and 80 rpm for 14 days. After incubation, the culture system was used to repair precast cracks with a width of 1.5 mm, and cured for 21 days. Example 9

[0049] The activation and suspension preparation of the microbial strains were the same as in Example 1. The preparation of the simulated concrete hole solution was also the same as in Example 1. In a sterile operating table, the culture conditions were set as follows: shaking speed 60 r / min, culture time 18 days; the temperature for all groups was 30℃, and the inoculation amount was 0.5 mL of fungal spore suspension and 0.5 mL of bacterial suspension added to 100 mL of simulated hole solution. After culture, each culture system was used to repair precast crack blocks with a width of 1.5 mm and cured for 21 days. Example 10

[0050] The activation and suspension preparation of the microbial strains were the same as in Example 1. A simulated concrete hole solution was prepared: the pH of the solution was 10.5, the calcium ion concentration was 200 mmol / L (anhydrous calcium chloride 22.2 g / L), the urea concentration was 0.3 mol / L (urea 18 g / L), and the carbon and nitrogen source formulations were the same as in Example 1. In a sterile operating table, 0.5 mL of *Rhizopus oryzae* spore suspension and 0.5 mL of *Pasteurella spp.* bacterial suspension were added to a conical flask containing 100 mL of the solution, and the flask was incubated at 30°C and 80 rpm for 14 days. After incubation, each culture system was used to repair precast crack blocks with a width of 1.5 mm and cured for 21 days. Example 11

[0051] Same as Example 10, except that the pH of the solution is 12.0 and the calcium ion concentration is 400 mmol / L.

[0052] Comparative Example 1 The bacterial strain activation and suspension preparation were carried out according to Example 1. A suspension of *Pasteurella spp.* was prepared separately at a concentration of 1 × 10⁻⁶. 8 Cells / mL. A simulated concrete pore solution was prepared using the same formulation as in Example 1. In a sterile operating table, 1 mL of *Pasteurella spp.* suspension was added to a conical flask containing 100 mL of sterile simulated pore solution, without adding any fungal components. The conical flask was incubated at 30°C and 80 rpm for 14 days. After incubation, the culture system was used to repair precast cracked cement mortar test blocks with a width of 1.5 mm, using the same injection and curing methods as in Example 1. Comparative Example 2 The strain activation and suspension preparation were carried out according to Example 1. A suspension of *Rhizopus oryzae* spores was prepared separately at a concentration of 1 × 10⁻⁶. 6 1 spore / mL. Prepare a simulated concrete pore solution using the same formulation as in Example 1. In a sterile operating table, add 1 mL of *Rhizopus oryzae* spore suspension to a conical flask containing 100 mL of sterile simulated pore solution, without adding any bacterial components. Incubate the conical flask at 30°C and 80 rpm for 14 days. After incubation, use the culture system to repair precast cracked cement mortar test blocks with a width of 1.5 mm, using the same injection and curing methods as in Example 1.

[0053] Comparative Example 3 The strain activation method was the same as in Example 1. *Aspergillus niger* was inoculated onto PDA slant culture medium and cultured at 30°C for 5–7 days. The spores were then washed away with sterile physiological saline and filtered to obtain a strain with a concentration of 1 × 10⁻⁶. 6 A suspension of *Aspergillus niger* spores per mL. The *Pasteurella pamoate* suspension was prepared in the same manner as in Example 1, at a concentration of 1 × 10⁻⁶ spores / mL. 8Cells / mL. Prepare the simulated concrete pore solution using the same formulation as in Example 1. In a sterile operating table, separately pipette 0.5 mL of Aspergillus niger spore suspension and 0.5 mL of Pasteurella multocida suspension, and add them to an Erlenmeyer flask containing 100 mL of sterile simulated pore solution, for a total inoculation volume of 1 mL / 100 mL. Incubate the Erlenmeyer flask at 30°C and 80 rpm for 14 days. After incubation, use the culture system to repair precast cracked cement mortar test blocks with a width of 1.5 mm, using the same injection and curing methods as in Example 1.

[0054] Table 1 Performance verification results of each embodiment and comparative example .

[0055] Table 1 Ca 2+ Concentration was determined according to GB / T7476-1987 (EDTA titration method); calcium carbonate yield was determined by filtration-drying and weighing method, with thermogravimetric analysis used for verification when necessary; water permeability coefficient was determined according to the principle of water penetration height method in GB / T50082-2024, with modifications applied to 40mm×40mm×160mm specimens with pre-fabricated cracks; compressive strength was determined according to GB / T17671-2021, with the recovery rate based on intact specimens from the same batch. The crystal form and morphology of the product were characterized by XRD and SEM, respectively.

Claims

1. A microbial self-healing concrete crack repair material, characterized in that, It comprises a fungal component and a bacterial component; the fungal component is selected from filamentous fungi capable of forming a three-dimensional hyphal network, and the bacterial component is selected from urease-producing bacteria capable of inducing calcium carbonate precipitation through metabolic activity.

2. The microbial self-healing concrete crack repair material according to claim 1, characterized in that, The fungal component is Rhizopus oryzae, and the bacterial component is Pasteurella multocida.

3. A microbial self-healing concrete crack repair material according to claim 1 or 2, characterized in that, It also contains nutritional components, including calcium sources, urea, carbon sources, and nitrogen sources.

4. The microbial self-healing concrete crack repair material according to claim 3, characterized in that, The calcium source is anhydrous calcium chloride, the carbon source is glucose, and the nitrogen source is selected from one or more of yeast extract, beef extract, and peptone.

5. A method for preparing a microbial self-healing concrete crack repair material according to any one of claims 1 to 4, characterized in that, Includes the following steps: Fungal spore suspensions and bacterial suspensions were prepared separately. The fungal spore suspension and bacterial suspension were mixed and inoculated into a simulated concrete hole solution containing nutrients for co-cultivation.

6. The method for preparing a microbial self-healing concrete crack repair material according to claim 5, characterized in that, The concentration of the fungal spore suspension is 1×10⁻⁶. 6 The bacterial suspension had a concentration of 1 × 10⁶ spores / mL. 8 Cells / mL.

7. The preparation method of a microbial self-healing concrete crack repair material according to claim 5, characterized in that, The co-culture temperature was 25℃~35℃, the oscillation speed was 60r / min~100r / min, and the culture time was 10d~18d.

8. The method for preparing a microbial self-healing concrete crack repair material according to claim 5, characterized in that, The pH value of the simulated concrete hole solution is 10.5 to 12.0, the calcium ion concentration is 200 mmol / L to 400 mmol / L, and the urea concentration is 0.1 mol / L to 0.5 mol / L.

9. The preparation method of a microbial self-healing concrete crack repair material according to claim 5, characterized in that, It also includes injecting or coating the co-cultured product into concrete cracks and allowing it to cure statically for 7 to 21 days at normal temperature and pressure, while maintaining a relative humidity of 80% to 85% during the curing period.

10. The method for preparing a microbial self-healing concrete crack repair material according to claim 5, characterized in that, The simulated concrete hole solution contains per liter: 32.0g~35g anhydrous calcium chloride, 9.5g~10.5g urea, 4.7g~5.3g glucose, 0.8g~1.2g yeast extract, 1.2g~1.8g beef extract, and 4.5g~5.5g peptone.

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