The main materials used in the microbial experiments include Bacillus pasteurii (North Bio Company, strain ATCC 11859; Gram-positive; rod-shaped cells, 2–3 µm in length; spherical spores, 0.5–1.5 µm in length), beef extract, urea, peptone, calcium nitrate, and deionized water. All reagents used in the experiments are sourced from the China National Pharmaceutical Group Chemical Reagents Platform. Before utilizing the microorganisms, they need to be cultured in advance. The culture medium components include beef extract (6 g/L), soybean peptone (10 g/L), urea (30 g/L), and deionized water. The prepared medium is then aliquoted into wide-mouth triangle flasks and subjected to sterilization in a high-pressure steam autoclave at 121°C for 20 min. After sterilization, under sterile conditions in an ultra-clean table, the medium in the triangle flasks is inoculated with Bacillus pasteurii. Finally, the inoculated medium is incubated in a shaking incubator at 30°C, with a rotation speed of 150 rpm, for 48 h.

The primary materials used in the cement-based material experiments include Ordinary Portland Cement (Grade 52.5), limestone powder, calcined clay, sand and fiber (PVA). The Ordinary Portland Cement (OPC) is supplied by Shandong Kang Jing New Materials Technology Co., Ltd. The limestone powder is a fine, super-white heavy calcium carbonate produced by Tianjin Yan Dong Mineral Products Co., Ltd. The calcined clay, sourced from Yichang, Hubei, has its particle size distribution analyzed using a laser particle size analyzer (JL—1155), with results provided in Table 1. Chemical composition analysis of the cement, limestone, and calcined clay is conducted using X-ray Fluorescence, with the chemical compositions listed in Table 2.

The experiment initially focused on determining the optimal growth conditions for B. pasteurii by varying pH, temperature, shaking speed, and inoculation volume. Based on these optimal conditions, the microbial mineralization technique was employed, using B. pasteurii as the mineralizing agent to modify LC3. The effectiveness of microbial mineralization in enhancing the self-healing properties of low-carbon LC3 materials was investigated through compressive strength tests, capillary water absorption tests, micro-crack self-healing rate tests, and microstructural characterization using SEM-EDS. Three cement mortar mix ratios were designed for the experiment: M1 represents the OPC mortar group; M2 represents the LC3 mortar group; and M3 represents the Microbe-LC3 mortar group. The composition ratios for each experimental group are detailed in Table 3. It should be noted that the quantities of bacterial solution, calcium nitrate, and urea have been optimized for effective microbial-induced calcite precipitation, resulting in enhanced performance of the modified LC3 materials. However, further experiments could explore varying these amounts to achieve additional improvements in self-healing, durability, or mechanical properties.

The experiment utilized a planetary mixer to prepare the specimens. Initially, the solid materials were dry mixed for 3 min. Subsequently, water and bacterial solution were gradually and uniformly added, and mixing continued for an additional 3 min. The prepared cement mortar was then poured into molds and vibrated on a vibration table for approximately 2 min to remove any air bubbles. Three types of test specimens were prepared: cylindrical specimens with a diameter of 5 cm and a height of 1.5 cm for crack observation; cylindrical specimens with a diameter of 10 cm and a height of 5 cm for water absorption tests; and cubic specimens measuring 50 cm × 50 cm × 50 cm for compressive strength testing. After casting, the specimens were cured at room temperature (25°C) and relative humidity of 40%–50%. To prevent excessive moisture evaporation, the specimens were covered with plastic film. After 24 h of curing, the specimens were demolded and then maintained at room temperature until performance testing.

The growth of B. pasteurii is influenced by various factors. Through optimizing the cultivation conditions of this strain, a deeper understanding of its physiological characteristics, adaptation mechanisms, growth curve, and metabolic pathways can be achieved. This optimization aims to enhance the strain’s activity and application effectiveness, thereby maximizing resource utilization efficiency and reducing economic costs.

The pH regulates the physiological characteristics, metabolic processes, and activity of the strain (Gat et al., 2017). Figure 3A shows the impact of pH on the growth of B. pasteurii. It can be observed that, within the alkaline pH range of 7–12, the OD600 value of B. pasteurii initially increases and then decreases as pH increases. This indicates that the growth rate of B. pasteurii initially increases and then declines. Specifically, the growth of B. pasteurii is optimal at a pH of 9, where bacterial metabolic activity is most vigorous and the growth rate is the highest. However, at a pH of 12, the growth of B. pasteurii is significantly inhibited. While B. pasteurii can grow in alkaline environments, extreme alkaline conditions may adversely affect its growth, including enzyme activity and cell membrane stability (Mobley et al., 1995). Additionally, studies have shown that both acidic and strongly alkaline conditions can disrupt the metabolism of B. pasteurii and its production of urease, potentially rendering the produced enzyme irreversibly inactive (Mobley et al., 1995; Ciurli et al., 1996).

Temperature is also a crucial factor affecting strain activity, stability, spore formation, and growth rate (Sun et al., 2019; Yi et al., 2021). Figure 3C illustrates the impact of temperature on the growth of B. pasteurii. The growth curve of the strain shows that as the incubation temperature increases from 25°C to 35°C, the OD600 value of B. pasteurii initially increases and then decreases. The OD600 value peaks at 30°C, indicate that this temperature is most suitable for the growth of B. pasteurii. The experimental results are consistent with previous studies, which shows that B. pasteurii exhibits optimal growth and metabolic activity around 30°C (Yi et al., 2021; Kim et al., 2018; Omoregie et al., 2017).

The inoculation amount of microorganisms in the culture medium is crucial, as both excessively high and low inoculation levels can affect microbial growth. Figure 3B illustrates the impact of inoculation amounts on the growth of B. pasteurii. When the inoculation volume is 5% of the total culture medium, compared to 10%, 15%, and 20%, the bacterial concentration and growth rate are significantly lower. A lower inoculation amount results in insufficient microbial colonies, leading to slower growth rates due to the reduced concentration of bacteria (Panikov, 2023). Conversely, a higher initial bacterial concentration can accelerate growth and reproduction. However, an excessively high inoculation amount may lead to resource competition, thereby inhibiting microbial growth (Guo L. et al., 2024). Considering the adverse effects of both extreme cases on B. pasteurii growth, an inoculation level of 10% is deemed appropriate.

For microbial growth in liquid culture media, mechanical shaking techniques can create a favorable environment for microorganisms, enhancing their growth and metabolic processes. Continuous shaking increases the surface area of contact between the culture medium and air, which is especially crucial for aerobic microorganisms, facilitating their faster and more complete growth. Additionally, shaking helps to evenly distribute nutrients and microorganisms in the culture medium, preventing issues such as cell sedimentation and nutrient unevenness that can occur in static cultures. Figure 3D illustrates the effect of shaking speed on the growth of B. pasteurii. In the figure, 1 mL and 1.5 mL refer to the volumes of Bacillus spores inoculated into the culture medium, while the corresponding rpm indicates the shaking speed of the medium in the shaker. As shown in Figure 3D, the optical density of the bacterial culture is highest at a shaking speed of 150 rpm, indicating that B. pasteurii exhibits optimal growth under this condition.

Through the optimization study of B. pasteurii cultivation conditions, it was found that the optimal growth environment for B. pasteurii is achieved when the pH of the culture medium is maintained at 9, the inoculation amount is 10%, the shaking speed in the incubator is set to 150 rpm, and the temperature is at 30°C. The growth curve of B. pasteurii under these conditions is shown in Figure 4.

Figure 5 presents the compressive strength of 50 mm × 50 mm × 50 mm cubic samples at curing ages of 7, 14, and 28 days. It is evident from the figure that the compressive strength of the OPC mortar samples (M1 group) is higher at 7, 14, and 28 days compared to the LC3 mortar samples (M2 and M3 groups). The partial replacement of cement with calcined clay and limestone powder has led to a decrease in mechanical performance. Specifically, the compressive strength of the LC3 mortar samples decreased by approximately 20% compared to the OPC mortar samples. The decrease in compressive strength is primarily attributed to the partial replacement of ordinary cement with calcined clay and limestone in LC3. The addition of calcined clay has led to a significant refinement of the pore structure in the cementitious system (Tironi et al., 2014). Research indicates that the hydration rate of clinker is related to the internal pore structure of the material; refined pores are less conducive to the hydration reactions (Avet and Scrivener, 2018). Consequently, the refined pore structure slows down the hydration rate of the clinker, resulting in reduced long-term hydration of C₃S and C₂S, and a suppression of the formation of AFm phases, carbonate-aluminate phases, and other hydration products (Krishnan et al., 2019; Antoni et al., 2012). Additionally, among the two types of LC3 mortar specimens, the Microbe-LC3 mortar specimen in the M3 group showed improved compressive strength at both 14 and 28 days compared to the LC3 mortar specimen in the M2 group. This enhancement is primarily due to the positive effect of microbial incorporation on strength development. The calcium carbonate precipitation induced by B. pasteurii mineralization improves the internal pore structure, resulting in a denser and more cohesive matrix, thereby increasing the compressive strength (Mondal and Ghosh, 2023).

Figure 6 shows the water absorption regression curves of cement mortar specimens at specific time intervals. From the figure, it is observed that the M1 group of OPC mortar specimens has the lowest water absorption rate, while the M2 group of LC3 mortar specimens exhibits significantly higher water absorption compared to both M1 and M3. The increased water absorption rate is likely due to the slower early hydration rate and incomplete hydration of LC3 mortar, which results in a less dense internal pore structure. Consequently, the higher capillary porosity in the LC3 mortar leads to a higher water absorption rate compared to OPC mortar.

Furthermore, the capillary water absorption rate of the M3 group (Microbe-LC3 mortar) is significantly lower than that of the M2 group (LC3 mortar). The presence of microorganisms in the M3 group introduces a dual-component coating system with capillary pore-blocking characteristics. First, microorganisms can deposit on the surface of the cement matrix and form a biofilm, which blocks a certain number of capillary pores. Additionally, microorganisms act as nucleation sites, leading to the formation of a carbonate coating under the MICP process. This dual-component coating system, consisting of microorganisms and carbonate deposits, plays a crucial role in reducing the material’s water absorption rate by effectively blocking capillary pores (De Muynck et al., 2008; Tiano et al., 1999).

Figure 7 illustrates the surface crack self-healing results for specimens from the M1 group (OPC mortar). Figure 8 presents the surface crack self-healing observations for specimens from the M2 group (LC3 mortar). Figure 9 shows the surface crack self-healing results for specimens from the M3 group (Microbe-LC3 mortar). A comparison of the observed images of surface crack self-healing across the different groups reveals that the M2 group (LC3 mortar) exhibits the poorest self-healing performance. Compared to the other two groups, the width of the repaired cracks is significantly smaller, and the formation of crystalline deposits within the cracks is minimal. The M1 group (OPC mortar) shows somewhat better repair performance, although complete repair is not achieved, with larger areas remaining unfilled and gaps present. In contrast, the M3 group (Microbe-LC3 mortar) demonstrates nearly complete crack repair, with dense and fully covering crystalline deposits, showing the best results. For the crystalline deposits, SEM results (Figure 10) reveal that the deposits at cracks P1 and P2 in the M2 group appear as spherical aggregates. In comparison, the deposits at cracks P3 and P4 in the M3 group display a significantly different morphology. The particles are smaller and more densely connected. The differences in the morphology and distribution of CaCO₃ crystals are attributed to the presence of B. pasteurii. The MICP process enhances mineralization efficiency, leading to increased CaCO₃ production and a denser arrangement of the deposits. Additionally, the microorganisms themselves serve as nucleation sites, and factors such as microbial metabolic activity, cell surface structure, and biofilm formation influence the crystal morphology of the precipitates. During metabolism, microorganisms also produce organic substances that bind the mineral precipitates together, resulting in an irregular and dense distribution (Liu H. et al., 2024).

Additionally, as shown in Figure 10B, PVA fibers are covered by calcium carbonate crystals. This indicates that PVA fibers can promote the precipitation of calcium carbonate by providing preferred nucleation sites. The impact of PVA fibers on improving the precipitation of self-repair products in LC3 materials can be attributed to the unique molecular structure of their hydroxyl groups (Feng et al., 2019a). PVA fiber molecules interact with ions in the surrounding environment through intermolecular forces such as van der Waals forces, ionic bonds, and hydrogen bonds. This interaction aids in the aggregation and precipitation of calcium ions, thereby promoting the formation of calcium carbonate (Homma et al., 2009). Additionally, in aqueous solutions, hydroxyl groups can form hydrogen bonds with water molecules, enhancing their affinity for water. This strong interaction can increase the ionic concentration around the hydroxyl groups (Guan et al., 2017). Hydroxyl radicals can serve as nucleation centers, further promoting the binding of calcium ions and carbonate ions, and synergistically accelerating the repair of cracks in LC3 cement-based materials alongside microbial mineralization.

To further investigate the crack self-healing capabilities of the different cement mortar specimens, the self-healing performance was quantified by measuring the crack widths before and after the repair process. As shown in Figure 11, the self-healing rate of specimens in the M2 group is significantly lower than that of the other two groups. The M3 group exhibits the highest self-healing rate, achieving up to approximately 97% repair efficiency for cracks narrower than 100 μm, while for cracks with widths between 100 and 300 μm, the repair rate remains above 80%. Notably, the M3 group does not experience a significant decrease in repair efficiency as crack width increases, unlike the other two groups. The differences in crack self-healing capabilities between the M1 and M2 groups are likely due to the characteristics of LC3 compared to OPC. LC3 has a lower clinker content, which results in a reduced amount of Ca(OH)₂ produced during the hydration process. This lower concentration of Ca(OH)₂ limits the availability of calcium ions for mineralization and thus impairs the ability to repair cracks. Therefore, the self-healing capability of LC3 based materials is less than that of OPC based materials.

The superior crack self-healing ability of the M3 group, consisting of Microbe-LC3 mortar specimens, can be attributed to both inherent self-healing processes and the accelerated self-healing effects facilitated by microorganisms (De Muynck et al., 2008). Inherent self-healing primarily involves the reaction of CO₂ from the air, which dissolves in water to form unstable carbonic acid. In an alkaline environment provided by the cement matrix, carbonic acid readily decomposes into carbonate ions. These carbonate ions react with calcium ions released from the LC3 matrix, leading to the precipitation of CaCO₃ within the cracks and reducing their width. Compared to inherent self-healing, the accelerated self-healing facilitated by microorganisms plays a more crucial role. When urease-producing bacteria are incorporated into LC3 based materials, the lack of oxygen and moisture in the matrix initially causes the bacteria to enter a dormant state. Upon the formation of cracks, water and oxygen infiltrate, reactivating the bacterial spores near the cracks. The bacteria produce a significant amount of carbonate ions through the action of urease. The inclusion of external calcium sources during the mixing of Microbe-LC3 mortar enhances the dissolution of calcium ions, which further accelerates the precipitation of calcium carbonate, thus improving the efficiency of crack repair (Turner et al., 2023).

In Microbe-LC3 materials, the self-healing mechanism of cracks driven by MICP technology is influenced by several critical factors, including urease, dissolved calcium ions, and nucleation sites (Feng et al., 2019b). The primary role of microorganisms is to produce urease, which catalyzes the hydrolysis of urea to provide the carbonate ions necessary for calcium carbonate precipitation. Additionally, microorganisms serve as nucleation sites for the continuous and stable deposition of calcium carbonate (Wang et al., 2021; Zhang et al., 2024c). As illustrated in Figure 12, urease-producing bacteria are encased by multiple layers of peptidoglycan in their cell walls, where small particulate precipitates can be induced and retained (Liu H. et al., 2024; De Belie and De Muynck, 2008). Furthermore, the presence of fatty acids containing functional groups such as phosphate and carboxyl groups imparts a negative charge to the cell walls, facilitating the attraction and binding of metal cations (calcium ions) to the bacterial surface (Scott and Barnett, 2006; Castro-Alonso et al., 2019). The mechanism of MICP is related to formulas 4–7. N H 2 − CO − N H 2 + H 2 O → u r e a s e 2 N H 3 + C O 2 (4) N H 3 + H 2 O ↔ NH 4 + + OH − (5) C O 2 + OH − → HC O 3 − (6) Cell − C a 2 + + HC O 3 2 − + O H − → Cell − CaC O 3 + H 2 O (7)