The weathered tufa used in the experiment was collected from Zhangza Town in Jiuzhaigou County, Sichuan Province, China. According to the GB/T 50123-2019 standard, the natural particles' moisture content was measured by the drying method, the dry density was determined by the cutting ring method, the specific gravity was measured by the density pycnometer method, the particle size distribution was determined by sieving analysis, the permeability coefficient was measured by the constant head test and the pH value was measured using a portable pH meter [26].

The basic physical and chemical properties are summarized in Table S1. The tufa particles were primarily composed of grains with diameters of 0.1–2.0 mm, accounting for 90.66% of the total particle size distribution, which belonged to the typical particle size range. The content of fine particles (d ≤ 0.075 mm) was 7.09%. According to the GB/T 50145-2007 classification, these particles were categorized as sand particles containing fine particles (SF) [27].

10 g of tufa were added to 50 mL of 0.85% sterile NaCl solution and shaken at 150 rpm for 2 h to ensure even dispersion. After 2 h, it was allowed to settle for sedimentation, getting a sample suspension. From this suspension, 10 mL of the supernatant was inoculated into 90 mL of sterile Nutrient Broth Urea (NBU) containing 10 g/L of peptone, 3 g/L of beef extract, 5 g/L of sodium chloride, and 20 g/L of urea, with the pH adjusted to 6.5. The samples were then incubated at 30°C with shaking (150 rpm) for 72 h, after which 10 mL of the culture was sub-cultured twice under the same conditions. Target colonies were obtained by serial dilution and plating in NBU plates with phenol red at 0.012 g/L as a pH indicator. The plates were incubated at 30°C for 72 h and the uninoculated mediums were set as controls. Colonies that exhibited a red coloration after this incubation period were identified as urease-producing bacteria (UPB). Throughout the cultivation period, characteristics like colony size, shape, surface morphology (smooth or rough), moisture level, and edge neatness were monitored. Selected red colonies were transferred to new media, with this purification process repeated until uniformity was achieved. Ultimately, four strains were isolated and designated U1, U2, U3 and U4. Urease activity, an important indicator for evaluating MICP [28], was assessed using the phenol hypochlorite method [29]. The purified UPB was inoculated into 100 mL of NBU and cultured at 30°C with shaking (150 rpm) for 72 h, with readings taken every 24 h. Urease activity (UV) was defined as the amount of enzyme that releases 2 μmol of ammonium per minute per 1 μmol of hydrolyzed urea. The two strains with the highest urease activity were designated as U1 and U2, which were subsequently cultured both separately and together (designated Mc) for further experiments.

Genomic DNA was extracted using a DNA Extraction Kit (Beijing Tsingke Biotech Co., Ltd). The 16S rRNA gene was amplified through Polymerase Chain Reaction (PCR) using universal primers 27F (AGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTG TTACGACTT), sequenced by Beijing Tsingke Biotech Co. The obtained sequences were compared with the NCBI database via BLAST. Phylogenetic relationships among species were established based on the neighbor-joining method using MEGA 7.0 evolutionary analysis software, and evolutionary distances were calculated based on 1000 bootstrap replicates.

To better understand the roles of U1 and U2 in MICP, it is significant to study their growth patterns and urease activity of U1 and U2. The cells were cultured at 30°C with shaking (150 rpm) for 72 h and the uninoculated mediums were set as controls. OD₆₀₀ and pH values were continuously measured using a UV-480 ultraviolet-visible spectrophotometer and pH meter for 72 h. Urease activity was assessed using the phenol hypochlorite method, with measurements taken at 24 h, 48 h, and 72 h.

For the evaluation of bio-mineralization characteristics of strains, U1, U2, and Mc were inoculated into sterilized NBU medium containing 14.5 g/L calcium acetate at 2% inoculum concentration and the uninoculated mediums served as controls. The cultures were incubated at 30°C with shaking (150 rpm) for 7 d, with samples taken every 24 h. After cultivation, the cultures were centrifuged at 8000 rpm for 5 min, and the concentration of Ca²⁺ was measured using inductively coupled plasma optical emission spectrometry (ICP-OES). The precipitated products were filtered, washed, air-dried, and weighed to determine the quantity of calcium carbonate precipitate formed.

In this experiment, calcium acetate was chosen as the calcium source for MICP to minimize environmental pollution and reduce costs [30]. The submerse method was employed to improve the effectiveness of the cementing solution (urea and calcium acetate), in solidifying the weathered tufa. The solidifying test of the weathered tufa was carried out in a fully mixed reactor (Fig. 1), which consisted of a plastic box to hold the tufa columns and the cementing solution, and an air pump to supply oxygen to the UPB. The cementing solution was composed of 1 mol/L calcium acetate [31, 32], 1 mol/L urea, 3.0 g/L Nutrient Broth, 10 g/L ammonium chloride, and 2.12 g/L sodium bicarbonate, with the pH adjusted to 6.5. The solidification test adopted a full-contact flexible mold, which had the characteristics of isolation, filtration, drainage, and reinforcement. This geotextile fabric is easy to cut and provides good permeability [33].

Based on the studies by Tang et al. [20] and Qian et al. [33], tufa columns with an initial dry density of 1.63 g/cm³, a mold diameter of 52 mm, and a height of 102 mm were prepared for the unconfined compressive strength (UCS) and direct shear tests. According to the initial dry density and the height, 320 g of tufa was determined to fill each sample. Three groups of tufa columns correspond to different bacterial solutions (U1, U2, and Mc). Each sample was thoroughly mixed with a bacterial solution cultured for 48 h (OD₆₀₀ > 1) and allowed to stand for 4 h to ensure bacteria adhered to the tufa surface. Tufa columns without bacteria were set as control group. The filled molds were placed into the reactors, where the cementing solution was slowly poured until the samples completely submersed. The air pump was used to inject air into the cementation solution, ensuring the bacteria received sufficient oxygen.

After the solidifying period, UCS and direct shear tests were performed on the cured columns to determine their strength according to ASTM D2166 and ASTM D3080, respectively [34, 35]. After the UCS test, the columns were broken, and their internal fragments were collected and ground for microscopic analysis of the mineralized products. Scanning electron microscopy (SEM) was applied to observe their microstructure, morphology, and size. X-ray diffraction (XRD) analysis was applied to identify their main constituents and phases.

All tests were performed in triplicate and the results are reported as mean±standard deviation. One-way analysis of variance (ANOVA) was used to examine the impacts of treatment with single and mixed cultures on urease activities, the amount of CaCO₃ precipitation, and the mechanical responses of tufa columns. Statistical analyses and graphs were performed with GraphPad (Inc., San Diego CA. USA) and Origin 9.6 (Inc., Northampton, MA, USA), respectively. p < 0.05 was reported as statistically significant.

In the NBU medium, a distinct red color appeared around the strains (Fig. 2a, b), indicating their urease-producing capabilities. The two strains with the highest urease activity were designated U1 and U2. Their 16S rRNA gene sequences were subjected to a BLAST sequence similarity search in the NCBI database. The results confirm that U1 belongs to Stenotrophomonas sp. and U2 to Lysinibacillus boronitolerans (Fig. 2c, d). Notably, Stenotrophomonas sp. has been documented to inhibit fungi growth and mitigate the corrosion of repair materials [18], while Lysinibacillus sp. can grow and precipitate CaCO₃ in highly alkaline conditions [36].

The growth characteristics, pH levels, urease activity, concentration of Ca²⁺, and CaCO₃ precipitation of the three groups of strains and the control group (CK) are shown in Fig. 3(a, b, c) and Fig. 4(a, b). UPB produces ammonia and carbonic acid by hydrolyzing urea through the production of urease. Ammonia is dissolved in the solution, forming ammonium and hydroxide ions. This process creates an alkaline environment that promotes the growth of UPB [37] and disturbs the carbonic acid equilibrium, which facilitates the formation of CaCO₃ [38]. Among the strains, Mc showed the highest growth and urease activity, peaking at a cell count of OD₆₀₀=3.9±0.1 after 51 h. At different time points (24 h, 48 h, 72 h), Mc consistently demonstrated significantly higher urease activity compared to U1 and U2, with U2 significantly higher than U1 (p < 0.05). At 72 h, urease activity for Mc, U2, and U1 reached their maximum values of 865±17 U/ml, 760±20 U/ml, and 741±11 U/ml, respectively. As bacterial populations increased, both pH levels and urease activity rose. Conversely, the concentration of Ca²⁺ in the culture medium decreased, leading to the formation of CaCO₃ precipitates. Notably, Mc produced a significantly greater amount of CaCO₃ (0.92±0.02 g) compared to U1 (0.59±0.03 g) and U2 (0.53±0.02 g) (p < 0.05). In the control groups, both pH and Ca²⁺ values remained stable and the urease activity consistently at 0. These results demonstrated that urease activity and pH increase were caused by the strains, which induced the production of CaCO₃ and achieved the MICP process. Meanwhile, mixed cultivation produced more CaCO₃ precipitation, which is beneficial for the stability of weathered tufa.

A high pH environment can improve the biomineralization ability of the strains [39]. U1 exhibited higher biomass and solution pH than U2 (Fig. 3a, b), which could provide more nucleation sites for CaCO₃ and create an alkaline environment for its formation. The level of urease activity is key to achieving MICP [40]. U2, with its superior urease activity (Fig. 3c), could increase urea hydrolysis rates, accelerating CaCO₃ formation. Therefore, the synergistic effects of mixed cultures between U1 and U2 proved beneficial: U2 generated more carbonate ions while U1 effectively adsorbed Ca²⁺ and provided a high pH.

CO₂ is produced during urea hydrolysis by urease, dissolving in water to form H₂CO₃, which dissociates into HCO₃⁻ and CO₃²−. The Ca²⁺ in solution reacts with CO₃²− to produce insoluble CaCO₃ precipitate. The quantity of CaCO₃ formed is influenced by factors such as pH, CO₂ concentration, Ca²⁺ concentration, and temperature. As shown in Fig. 4(a), the concentration of Ca²⁺ in the cultures with individual bacteria remained relatively stable after the 5th day, with the final concentration of 1.8±0.3 g/L for U1 and 2.3±0.2 g/L for U2. In contrast, Mc fully consumed Ca²⁺ by the 4th day and maintained the highest pH value, eventually stabilizing at 9.1±0.1 (Fig. 3b). Therefore, the pH had the greatest effect on the amount of CaCO₃ precipitate when the temperature, CO₂ concentration, and concentration of Ca²⁺ were consistent. Interestingly, U1 exhibited lower urease activity than U2, while it appeared a higher growth rate and CaCO₃ precipitation. This indicates that the CaCO₃ formation during the MICP process also depends on cell concentration. Previous research has emphasized that cell concentration significantly affects CaCO₃ yield [41]. These results verify the feasibility of using MICP to stabilize weathered tufa and Mc have a positive effect. For practical applications, it is important to evaluate and adjust the quantities of calcium source and urea according to the specific situation.

To evaluate the efficacy of the UPB in stabilizing weathered tufa, three groups of columns were treated using a submersion method and designated as SCU-1, SCU-2, and SCM. Since the control samples remained loose after mold removal, they were excluded from further testing. This suggests the crucial role of microorganisms in the MICP process.

Fig. 5(a) shows the axial stress-strain of tufa columns derived from unconfined compression tests. Stress reflects the internal forces acting within the material due to external factors, leading to deformation, while strain quantifies this deformation. As axial pressure rose, both stress and strain increased correspondingly.

The point of failure occurred when the tufa column could no longer withstand greater pressure, making its peak stress, known as the unconfined compressive strength (UCS). The UCS of SCM was 656±76 kPa, significantly higher than the 276±34 kPa and 396±41 kPa of SCU-1 and SCU-2, respectively (p < 0.05) (Fig. 5b). The enhanced strength of SCM is likely attributed to Mc inducing a greater formation of CaCO₃ crystals, which strengthens the tufa columns [42]. The strain value (%) corresponding to the peak was the strain of the tufa column. The strain values at peak stress for SCM, SCU-1, and SCU-2 were 5.6%, 4.2%, and 5.9%, respectively. Following the peak, the curves exhibited either a gradual decline or vertical drop. The former indicates plastic failure, while the latter suggests brittle failure [43]. It can be concluded from the stress-strain curves that SCM, SCU-1, and SCU-2 all experienced brittle failure.

Fig. 5(b) compares the UCS and stiffness of the treated samples. Stiffness, defined as the secant modulus at a stress level corresponding to half the peak stress, measures the material's resistance to elastic deformation. The stiffness of SCM was 8.9±2.7 MPa, significantly higher than the 3.3±0.4 MPa and 3.4±0.9 MPa of SCU-1 and SCU-2, respectively (p < 0.05). These results indicate that the treatment significantly enhanced the UCS and stiffness of the weathered tufa, demonstrating its improved stability following cementation by CaCO₃, consistent with findings from previous studies [44].

To further assess the strength improvements resulting from biological treatments, direct shear tests were conducted on treated tufa columns using a direct shear apparatus. Fig. 6 (a, b, c, d) shows the shear stress versus horizontal shear displacement under different normal stress levels (100, 200, 300, and 400 kPa). The shear stress of SCM surpassed that of SCU-1 and SCU-2, indicating enhanced resistance to weathering damage. Cohesion reflects a measure of the effectiveness of MICP in enhancing tufa strength and stability [19]. The friction angle reflects the overall mechanical behavior of the columns, influenced by the deformation pattern, density, and stress level of the columns [45, 46]. As shown in Fig.6(e), the cohesion and internal friction angle were calculated based on the peak shear stresses from the tests. The cohesion for SCM, SCU-1, and SCU-2 were recorded at 50.23, 26.20, and 31.74 kPa, respectively, with corresponding friction angles of 33.6°, 24.9°, and 29.9°. SCM exhibited higher cohesion and internal friction angle than SCU-1 and SCU-2, indicating that MICP's application through mixed bacteria is a more effective stabilization method for weathered tufa.

In the solidification test, SCU-2 exhibited stronger mechanical properties than SCU-1, which contrasted with the growth and mineralization performance of strains U1 and U2. This result arose from the limited nutrients supplied to the strains during solidification to ensure their survival, which aimed to better fit their natural microbial environment. Thus, the strains were in minimal growth and consistent biomass on the weathered tufa. At this stage, urease activity was the primary determinant influencing MICP effectiveness. Therefore, U2 with a higher urease activity led to induce more CaCO₃ precipitation and enhanced the mechanical properties for SCU-2.

The UCS and shear strength of the cured tufa columns were lower than Li et al. [47]. This discrepancy may be attributed to the loose and porous nature of the weathered tufa, as well as the submersion method employed in the solidification process. The inherent characteristics of tufa make it easily damaged after curing compared to other materials. To enhance its stability, supplementing NH₄ ⁺ and NaHCO₃ into the cementing solution can elevate the pH and provide additional CO₃ ²⁻ for CaCO₃ formation. Additionally, the submersion of tufa columns in cementing solution may raise tufa's moisture content, leading to softening and subsequently reducing compressive and shear strengths [31, 48]. To improve outcomes, the solidification process could be optimized by shortening the submersing period and employing a combined submersion and grouting technique. This approach would enhance contact between the solution and tufa, promoting a more uniform distribution of the cementing agent. Despite the tufa columns showing relatively low mechanical performance, the application of MICP successfully improved their compressive and shear strengths. It demonstrates the feasibility of using MICP to stabilize weathered tufa.

Typical images of SCU-1, SCU-2, SCM, and untreated columns provided by SEM are used to interpret the results. These images revealed a notable coverage of CaCO₃ crystals on the surfaces of the treated tufa, highlighting the cementation between tufa particles (Fig. 7a). In the columns with the single treatment, the particles displayed partial wrap by CaCO₃ crystals, with visible pores between them (Fig. 7b, c). The CaCO₃ precipitation bonded the particles, which led to increased strength in the treated columns. In contrast, the untreated samples had no CaCO₃ crystal bonding to particles (Fig. 7d), resulting in loose weathered tufa that lacked cohesion. Similar observations have been reported in previous studies [44, 49].

Further analysis was conducted using X-ray diffraction (XRD) on the CaCO₃ formed in the solidified tufa, covering a scanning range of 5° to 85°. As shown in Fig. 8, the tufa in Jiuzhaigou consists of a calcite structure. SCU-1, SCU-2, and SCM showed an additional characteristic diffraction peak of calcite at the 2θ angle of 32.778°, suggesting that the strains had induced CaCO₃ precipitation on the weathered tufa surface. The SCM sample also presented a distinctive peak for vaterite at 2θ angle of 24.900°, which is likely related to the cell concentration. At low bacterial cell concentrations, the crystals formed were primarily calcite, while higher concentrations resulted in vaterite formation [20]. Mixed bacteria exhibited higher growth rates than single bacteria, so a small amount of vaterite was present in the SCM. Nevertheless, the SEM of the SCM sample predominantly revealed a significant quantity of calcite, while the vaterite was not observed due to its few quantity. The strongest diffraction peak in SCM indicates that it produced the most CaCO₃ crystals, serving as a filler and gelling agent for the weathered tufa. Overall, these findings illustrate that the mechanical properties of weathered tufa are significantly influenced by UPB, which induces the production of CaCO₃. The CaCO₃ crystals function as a binding agent, effectively stabilizing the weathered tufa.