Experimental study on biological solutions for anchoring earthen heritage sites

Abstract

In projects with earthen cultural heritage sites, selecting appropriate grouting materials is essential and challenging. This study examines the soil from the Zhouqiao site, with the objective of developing an innovative grouting material. The Enzyme-Induced Calcium Carbonate Precipitation (EICP) technique was utilized to modify soil-fly ash mixed grouting material. Mechanical performance tests and microanalysis were performed under different material ratios, concentrations of cementing solution, and freeze-thaw cycles. The results demonstrate that optimal mechanical performance of the grouting material is achieved with the soil-ash ratio of 1:1, water-cement ratio of 0.52 and concentration of cementing solution of 0.8 mol/L. Multiple freeze–thaw cycles lead to a decrease in compressive and shear strengths, causing a reduction in the internal friction angle by up to 46.42%. Microanalysis indicates that EICP treatment enhances cementation and bridging between particles. These findings provide a scientific foundation for optimizing EICP technology in earthen heritage site anchoring.

Introduction

Earthen sites are significant remnants and ruins made from soil, reflecting extended human habitation¹. A significant quantity of earthen sites has been identified globally, with approximately one thousand situated in China. Earthen sites are susceptible to damage from natural factors, including weathering and ultraviolet radiation, as well as external influences such as biological environments and human activities. These circumstances result in multiple problems, such as surface spalling, erosion, cracking, and microbiological deterioration. The original buildings and characteristics are progressively altered, resulting in possible instability or destruction, hence posing a substantial threat to the preservation and restoration of earthen sites².

In recent years, numerous investigations, design initiatives, experimental research, and preservation projects have concentrated on earthen sites, getting substantial research outcomes³. The application of anchoring technology has emerged as a significant focus in the investigation of the mechanical stability of earthen sites. Based on the principles of minimal intervention and preservation of original conditions in heritage conservation, the compatibility of grouting materials with site soil presents a significant research challenge. As early as 1984, Stillborg demonstrated that the interface bearing capacity is affected by different additives⁴. Benmokrane investigated the effects of incorporating aluminum-based materials, silica powder, and sand into standard grouts through a series of physical and mechanical tests, demonstrating that these additives can effectively improve the shear strength of the anchoring body⁵. Recently, Murat demonstrated that an increase in sodium silicate and boric acid content resulted in an accelerated setting rate, a decrease in grout fluidity, and an increase in hardening shrinkage⁶. Erminio assessed the setting rate, viscosity, and shear strength of colloidal silica nanoparticle-enhanced grouts, demonstrating their performance-enhancing effects while emphasizing the need to balance cost-effectiveness with practical usability⁷. Zhang revealed the bond failure mechanisms at the soil-grout interface in earthen site anchoring and investigated the compatibility issues of using fired aggregate as the primary cementing grout material with the site soil⁸. Zhang investigated the reinforcement effects of different materials, specifically potassium silicate solution with high modulus (PS) combined with powdered soil and fly ash, as grouting agents. The findings demonstrated that the anchoring system delivers adequate anchoring force to satisfy the reinforcement requirements of earthen sites⁹. Cui et al. sought to diversify anchoring grout types and enhance their compatibility by examining grouts made from traditional materials, including rice paste, SH (modified polyvinyl alcohol), and fly ash¹⁰. However, most researches originate from arid regions in the northwest, characterized by distinct environmental conditions, resulting in insufficient data regarding their compatibility in humid regions.

As an industrial by-product, fly ash is widely used in soil improvement and consolidation projects due to its abundance of reactive components, fine particle size, and excellent filling properties^11,12,13. Numerous studies^14,15,16 have demonstrated that fly ash can react with moisture and calcium ions in soil to form cementitious products, thereby enhancing mechanical properties, significantly reducing shrinkage and permeability, and improving overall structural stability. It also presents benefits including reduced weight, enhanced flowability, and superior workability. Moreover, incorporating fly ash can enhance the freeze–thaw resistance and salt corrosion resistance of samples, thereby reducing the risk of secondary deterioration caused by environmental erosion^17,18. The use of fly ash–based grouting is recommended for anchoring and protecting earthen sites due to its superior cementation potential, durability, and environmental benefits.

Recent breakthroughs in grouting technology have led to the development of an innovative microbially-induced grouting technique. A widely studied biostabilization technique is Microbially Induced Calcium Carbonate Precipitation (MICP), which improves the engineering properties of soil by employing urease-producing bacteria and adding urea along with various calcium source solutions¹⁹. Prior research indicates that integrating fly ash with MICP technology markedly enhances the mechanical properties of soil²⁰. Nonetheless, the stringent requirements for bacterial cultivation conditions and compatibility challenges in natural environments present considerable limitations to this technology²¹. A further approach to urea hydrolysis involves enzyme-assisted calcium carbonate mineralization, referred to as Enzyme-Induced Calcium Carbonate Precipitation (EICP). It is economically efficient and provides multiple extraction methods for urease, effectively mitigating the application limitations of MICP²². Additionally, the urease used is on a nanoscale, providing better options for reinforcing finer soils²³, and it also has been applied in areas such as slope erosion control, dust control, and cultural heritage protection²⁴. Gao combined EICP technology with XG to repair structural cracks in the soil of the Liangzhu ancient city. The calcium carbonate crystals generated by this combination effectively served as a binding agent, achieving a favorable composite effect²⁵. Yuan demonstrated the enhancing effect of EICP technology on the dynamic performance of site soils²⁶. While existing research has thoroughly investigated the potential of EICP technology to improve the mechanical properties of site soils, there is a scarcity of studies regarding its application in grouting materials. Therefore, investigating the potential application of the material as an innovative grouting agent in earthen heritage anchorage engineering presents significant research merit.

Building on this foundation, this study pioneers a laboratory evaluation of the engineering performance of EICP-Fly ash grouting anchorage materials. It explores the strength characteristics across different soil-ash ratios, water-cement ratios, concentration of cementing solution (CCS), and freeze-thaw cycles through sonic testing, unconfined compressive strength tests, direct shear tests, and freeze-thaw cycling tests, analyzes the microscopic morphology, and finally evaluates the anchoring grout’s applicability.

Methods

Materials

The soil utilized in this experiment was obtained from the exposed surface layer at the Zhouqiao site in Kaifeng, Henan Province. Following oven-drying and screening to eliminate particles over 1 mm, the soil underwent a routine Proctor compaction test and was categorized as silty clay based on its physical characteristics. X-ray diffraction (XRD) analysis further confirmed the mineralogical composition, indicating the presence of quartz, albite, muscovite, and kaolinite. X-ray fluorescence (XRF) analysis revealed that the main chemical components were SiO₂, Al₂O₃ and a small amount of CaO. The grout mixture incorporated Class F fly ash, with its primary chemical composition, ascertained by XRF, comprising SiO₂, Al₂O₃, Fe₂O₃, and CaO. All pertinent physical and chemical parameters of the soil and fly ash are encapsulated in Fig. 1.

Fig. 1: Characteristics of the test materials.

a Basic physical properties of the site soils; b photograph of the site soils; c chemical composition of the site soils; d photograph of fly ash; e chemical composition of the fly ash; f X-ray diffraction pattern of the site soils.

The soybeans utilized in the experiments were sourced locally in Kaifeng. Urea (CO(NH₂)₂) and anhydrous calcium chloride (CaCl₂) were both produced by China National Pharmaceutical Group. Deionized water was used in the experiment.

Sample preparation

Preparation of bioreinforcement solutions

The urease was extracted using the method of Zhang²⁷, with the following steps:(a) Soybeans were processed into soybean flour through grinding and subsequently subjected to thorough drying. (b) The dried soybean flour was combined with deionized water at a 1:10 ratio to create a soybean flour solution with a concentration of 100 g/mol. The mixture was stirred on a magnetic stirrer for 0.5–1 h, then allowed to stand to facilitate the precipitate of the soybean dregs. (c) The soybean flour solution was placed in a refrigerator at 4°C and left to stand for 2 h. (d) Finally, the solution was centrifuged at 4 °C with a speed of 4000 rpm for 15 min. The supernatant obtained after centrifugation was filtered to obtain the urease solution.

The activity of soybean urease was quantitatively evaluated based on conductivity as proposed by Whiffin VS²⁸. The conductivity value measured with a conductivity meter is used to determine the soybean urease activity U_a as given by the following formula:

$${U}_{a}=\frac{(b-a)\times 0.111}{15}$$

(1)

In the formula, ‘a’ represents the conductivity value of the solution at 0 min, and ‘b’ represents the conductivity value at 15 min. The average activity of the soybean urease used in this study is 20.3 mmol/(L·min) at 30 °C. Compared with domestic and international tests on EICP (Enzyme-Induced Carbonate Precipitation) reinforced soils, this urease activity meets the requirements.

The cementing solution is prepared by mixing calcium chloride (CaCl₂) and urea (CO(NH₂)₂) in equal molar amounts (1:1) and dissolving them in deionized water. During the experiment, ensure that the volume ratio of the grout solution to the soybean urease solution is 1:1.

Sample preparation method

According to the Standard of JGJ/T70–2009²⁹, the mixing method was used to ensure uniformity in the reinforcement. To assess the impact of different grout concentrations, water-cement ratios, and soil-ash ratios (by mass) on the test results, a controlled variable approach was employed to set up different conditions for comparative analysis. For each condition, three parallel samples were prepared for a series of macro- and micro-experiments. The test data were averaged, with sample errors controlled within approximately 3%.

The experimental procedure is outlined as follows: Initially, in accordance with ASTM testing standards, the urease solution, cementing solution, fly ash, and site soil are combined in the designated proportions. The mixture is subsequently transferred to a mixer and agitated rapidly until uniformity is achieved. The mixed slurry is subsequently poured into molds and subjected to vibration to ensure complete filling of the mold and to minimize the presence of air bubbles. The specimens are subsequently cured in the molds for a minimum duration of six days. Following removal from the molds, the specimens undergo natural curing for 28 days at an ambient temperature of 25 °C and a relative humidity of 30%. The specimens utilized in the tests have dimensions of 70.7 mm × 70.7 mm × 70.7 mm for elastic wave velocity and unconfined compressive strength testing, and Φ61.8 mm × 20 mm for shear testing. Figure 2 illustrates the experimental flowchart. To ensure uniform reinforcement of each sample, it is essential to control the mixing time and speed meticulously to prevent the formation of uneven voids and defects. The total preparation time must not exceed 30 minutes. Following the curing process, the specimens must be measured using a caliper to determine edge length and weight. Samples exhibiting surface damage, cracks, or other potential issues that could influence the test results must be remade.

Fig. 2

The experimental flowchart.

Elastic wave velocity test

An RSM-type ultrasonic device was used to measure high-frequency elastic waves through the transmission method. By analyzing the wave velocities, the integrity, hardness, and internal defects of the specimens were assessed. The analysis focused on the mechanical properties and internal compactness of the specimens to assess their quality and safety.

Freeze-thaw cycle resistance test

The freeze-thaw cycle resistance test provides insights into the variations in mechanical properties of grouting materials under low temperature conditions. The experiment employed a rapid freeze-thaw technique to replicate low-temperature conditions in the laboratory. Informed by current research and the climatic conditions of the archeological site, various freeze-thaw cycles (N_i, i = 0,1,3,6,10,15) were established for analysis^30,31. The freeze-thaw temperature ranged from −15 to 15 °C, with each cycle comprising a 12-hour freezing phase followed by a 12-hour thawing phase³². The specimens were encased in sealed plastic film to minimize moisture loss during the freeze-thaw cycle.

Compressive strength test

To study the deformation and failure characteristics of anchoring materials under different pressure conditions, the standard (GB/T50081-2002) was referenced³³, The universal testing machine was used to determine the unconfined compressive strength of the specimens under different conditions and after multiple freeze-thaw cycles, with an axial displacement loading rate of 2 mm/min. According to previous studies³⁴, the CCS of 0.75 mol/L demonstrated a better curing effect. Therefore, this experiment uses a cementing solution concentration of 0.75 mol/L as a constant variable and sets the soil-ash ratio (by mass) to 1:1, 1:2, and 2:1, as well as the water-cement ratio to 0.48, 0.5, and 0.52 to analyze and compare the failure strength and stress-strain trends of the samples^35,36,37.

Shear strength test

Rapid shear tests were performed to assess the performance and stability of the samples under shear stress, specifically to determine their shear strength. Normal stresses were established at 100, 200, 300, and 400 kPa, accompanied by a shear rate of 0.8 mm/min. The test was halted upon reaching a shear displacement of 6 mm. The shear strength of the sample was characterized as the maximum shear stress or the shear stress associated with a displacement of 6 mm. This study established a water-cement ratio of 0.52 to evaluate the impact of CCS on urease activity³⁸. Shear strength tests were performed using six concentrations of cementing solution and three soil-cement ratios. Table 1 delineates the various experimental conditions.

Table. 1 Proportion of shear strength test

XRD and SEM analysis

Fifty grams of samples that experienced 0 and 15 freeze-thaw cycles, respectively, were selected, thoroughly dried, and ground into powder. The powder was subjected to sieving through a 0.075 mm screen until no discernible granules remained, and was subsequently utilized as test samples. A Bruker D8 Advance X-ray diffractometer was employed to determine the mineralogical composition of the samples under different conditions. The experimental parameters were set to a voltage of 40 kV, a current of 40 mA, a constant step size of 0.02°, a scanning rate of 8°/min, and a scanning range of 5° to 90°.

To elucidate the morphological characteristics, size distribution, and complex interactions among particles in the samples, an FEI Quanta 250 environmental scanning electron microscope (SEM) was used. The samples underwent multiple freeze-thaw cycles and were subsequently processed through drying, gold coating, and vacuuming to prepare for scanning electron microscopy. Imaging was conducted in selected representative areas. Additionally, to further obtain microscopic structural information of the samples, observations were made at magnifications of 500x, 1000x, and 2000x.

Results

Elastic wave velocity

The elastic wave experiment offers notable benefits for investigating mortar solidification, ensuring quality control, and detecting internal defects³⁹. By analyzing parameters such as wave propagation speed, amplitude, and waveform, important evidence is provided for engineering quality and safety assessment⁴⁰.

Figure 3a illustrates that the wave velocity of the specimens increased in direct proportion to the water-cement ratio. The wave velocity of samples with a soil-ash ratio of 1:1 exhibited the most significant increase, surpassing other ratios, with an overall enhancement of up to 30%. Thus, an increase in the water-cement ratio improves the density of the anchoring grout. The SEM images (Fig. 3b, c) demonstrate that increased water content enhances the flowability of fly ash and facilitates the EICP process, leading to the generation of significant calcium carbonate precipitates that occupy and adhere within the micropores. The wave velocity increases with an increase in the water-cement ratio. Optimal performance of the samples was attained through a balanced water-cement ratio and soil-ash ratio. The highest velocity values were recorded for the conglomerate samples at a soil-ash ratio of 1:1 and a water-cement ratio of 0.52, corroborating existing literature on the topic⁴¹. The experimental results elucidate the rationale behind the selection of working conditions in the shear resistance tests.

Fig. 3: Wave velocity values of samples at different mixing ratios.

a Wave velocity at different water–cement and soil–ash ratios; b, c SEM images of the anchoring grout at ×500 and ×2000.

Improving the cementitious property of calcium carbonate crystal means that the cohesion and consolidation between soil particles and the crystals are stronger, and the reaction factor CCS significantly affects the cementitious property of calcium carbonate crystals produced by the EICP technology⁴². To investigate the impact of cementation liquid concentration on the wave speed of the specimens, optimal testing conditions were selected, specifically a soil-ash ratio of 1:1 and a water-cement ratio of 0.52.

To investigate the influence of CCS on the wave velocity of EICP-treated specimens, experiments were performed under optimal conditions by varying the CCS. Figure 4 illustrates that wave velocity increases significantly with the rise in CCS concentration, achieving a maximum enhancement of 42.6%. When the concentration surpasses 0.8 mol/L, a decline in wave velocity is observed. Therefore, the optimal CCS is determined to be between 0.6 and 0.8 mol/L. The observed variations in wave velocity result from the progressive decline in urease activity associated with increasing CCS levels. The presence of free Ca²⁺ with CO₃²⁻, leading to a reduction in pH and subsequent inhibition of urease activity. This disrupts the dynamic equilibrium of the urea hydrolysis reaction, resulting in changes in the quantity and morphology of the calcium carbonate crystals produced⁴³, ultimately leading to a decrease.

Fig. 4

Effect of CCS on wave velocity (m/s).

Unconfined compressive strength

The unconfined compressive strength test assesses the stability and load-bearing capacity of grout materials subjected to vertical pressure. Figure 5a analysis indicates that the strength of the specimens generally increases with a higher water-cement ratio. Under a soil-ash ratio of 1:1 and a water-cement ratio of 0.52, the strength peaked, representing a 22.8% increase compared to specimens with a water-cement ratio of 0.48. The maximum strength variation among specimens with differing soil-ash ratios is 58.5%. Thus, an optimal ratio can enhance the uniformity of the mixing process, subsequently improving the overall performance of the specimens at a macroscopic scale. The unconfined compressive strength values peak at a soil-ash ratio of 1:1 and decline at a ratio of 2:1, supporting the findings from the elastic wave velocity tests.

Fig. 5: Analysis of UCS and failure modes.

a UCS strength at different water–cement and soil–ash ratios; b variations in stress and strain of anchoring grout under several freeze–thaw cycles; c failure mode under one freeze–thaw cycle (N₁); d failure mode under three freeze–thaw cycles (N₃); e failure mode under six freeze–thaw cycles (N₆); f failure mode under ten freeze–thaw cycles (N₁₀); g, h failure mode under fifteen freeze–thaw cycles (N₁₅).

The axial deformation properties of mortar are essential for the deformation analysis of masonry and anchor rods⁴⁴. The optimal mixing ratio determined by the UCS experiment was chosen for the freeze-thaw experiment. Figure 5 illustrates the failure mode of the specimen. Following a single freeze-thaw cycle, the specimen exhibited several cracks resulting from expansion and contraction, as illustrated in Fig. 5c. With an increasing number of cycles, both the size and quantity of these cracks expand under pressure, as illustrated in Fig. 5d, ultimately exposing inclined cracks that extend vertically and unstable elements, as shown in Fig. 5e. As the cracks progress, the specimen demonstrates localized spalling, as illustrated in Fig. 5g.

Figure 5b presents the stress-strain characteristics of samples subjected to varying processing cycles. The soil’s strength is comparatively low, posing challenges for engineering construction. The incorporation of fly ash and the application of EICP technology resulted in a peak axial stress of 431.1 kPa, representing an increase of approximately 129%, thereby enhancing the deformation characteristics of the silt significantly. After N₁₅ cycles, the sample stress measured 221.5 kPa, indicating a 48.6% reduction relative to N₀, with water variation having a significant impact on strength. The periodic interaction of ice-water phase changes causes an expansion of the soil’s pore volume, resulting in localized stress concentration within micropores and a reduction in intermolecular forces between particles. Concurrently, the freezing forces exerted by ice crystals compromise the original structural stability of the soil, hindering recovery post-thawing. As internal damage increases, there is a progressive loss of strength, resulting in a sustained decline in compressive strength.

The differing stress-strain curves suggest that the samples underwent distinct failure modes. Typically, the failure modes in unconfined compressive strength tests include plastic failure and brittle failure. The differing stress-strain curves suggest that the samples underwent distinct failure modes. The failure modes observed in unconfined compressive strength tests are primarily plastic failure and brittle failure. The initial mode of change was noted in samples with 0 and 1 cycles (red and blue curves in Fig. 5b), where the stress-strain curve exhibited a three-stage pattern: gradual increase, swift increase, and rapid decline, signifying brittle failure. The second trend was observed at N₃, N₆, N₁₀, and N₁₅, where the slope of the curve gradually flattened, sustaining the peak stress for an extended period. This behavior indicates a two-stage change characterized by a slow rise followed by a slow decline, consistent with the characteristics of plastic failure. The probable cause of this phenomenon is the deformation due to expansion and contraction of the specimen following initial freeze-thaw cycles, which leads to the formation of micro-cracks. The effects of calcium carbonate and fly ash in filling and cementing these cracks preserved the integrity of the samples, culminating in characteristics indicative of brittle failure. Following several freeze–thaw cycles, the soil skeleton collapses, leading to a deterioration of cementation between particles, which causes particle displacement or fragmentation and ultimately results in a decrease in overall soil volume⁴⁵. The material’s internal structure experiences rearrangement, leading to energy dissipation and the formation of irregularly distributed cracks within the specimen⁴⁶ (Fig. 5h). The cracks systematically divide the initially intact specimen into multiple “loosened” cores, which progressively deform under axial stress, resulting in plastic deformation instead of abrupt brittle failure.

Shear strength

The variation in shear strength of the specimens is shown in Fig. 6a–c. With increasing vertical stress, the shear strength of Groups A, B, and C all exhibited an upward trend, though peak values varied among the groups. Specimens without CCS exhibited the lowest shear strength across all soil-cement ratios. In Groups B and C, the peak shear strength occurred at a CCS concentration of 0.8 mol/L, showing increases of approximately 73% and 61% compared to specimens without CCS. The B-0.8 mixture demonstrated the highest reinforcement performance. Nonetheless, additional increases in CCS resulted in a decrease in shear strength. The inhibitory effect of elevated Ca²⁺ concentrations on urea hydrolysis is the main factor contributing to strength reduction, as it diminishes calcium carbonate production, consequently decreasing soil shear strength.

Fig. 6: Sample shear strength and strength parameter changes.

a–c Variation in the shear strength of the anchoring grout; d effect of CCS on shear strength parameters.

Figure 6d illustrates the impact of CCS on the shear strength parameters of the samples. Cohesion demonstrated a trend of initial increase followed by a decrease in relation to the independent variable, reaching a maximum at a concentration of 0.8 mol/L, with corresponding increases of approximately 98%, 140%, and 71% compared to the sample without CCS. Group B achieved a maximum pressure of 186 kPa, surpassing the values recorded for Groups A and C, thereby demonstrating the most significant improvement effect. The cohesion of all three groups significantly decreased when CCS surpassed 0.8 mol/L, indicating the influence of CCS on EICP technology, which aligns with the observed reduction in shear strength. Consequently, the optimal soil-ash ratio and CCS for cohesion were determined to be 1:1 and 0.8 mol/L, respectively. The internal friction angle exhibited variability, with maxima observed at 0.6 and 0.8 mol/L. The internal friction angles for Groups A, B, and C were 40.2°, 54.5°, and 43.5°, respectively, indicating improvements of approximately 63%, 68%, and 42% relative to the sample without CCS. The analysis reveals that the ideal soil-ash ratio and CCS for the internal friction angle are 1:1 and 0.8 mol/L, consistent with the optimal conditions for cohesion.

The freeze-thaw cycle causes structural changes internally, subsequently affecting shear strength, cohesion, and internal friction angle. Analysis of Fig. 6b indicates that B-0.8 showed better results; therefore, this condition was chosen for the freeze-thaw cycle tests. Figure 7a illustrates the shear strength following multiple cycles. Under consistent normal stress, the strength shows an overall decreasing trend, indicating that the freeze-thaw cycle has led to a deterioration of soil strength. The initial freeze-thaw cycle resulted in a notable decline in strength, though this trend progressively diminished and ultimately reached a state of stability. The strength loss rates were 35%, 37.3%, 31.3%, and 41.5%, remaining generally below 50%.

Fig. 7: Evolution curve and degradation analysis of shear strength strength index c and φ with cycle number N.

a Anchoring grout shear strength variations under several freeze–thaw cycles; b variation of shear strength parameters (c and φ) under freeze–thaw cycles; c degradation of c and φ under freeze–thaw cycles.

The shear strength parameters c and φ of the specimen body under freeze-thaw action are derived from the Mohr–Coulomb strength criterion, as illustrated in Fig. 7b. A degradation formula for c and φ values is established through a nonlinear fitting method, based on the variation patterns of the parameter values. The degradation trends follow exponential and power law functions concerning the number of cycles N, as illustrated in the subsequent equations:

$$c=63.81* {e}^{-\frac{N}{7.43}}+122.55$$

(2)

$$\varphi ={(0.019+0.003* {N}^{0.535})}^{-1}$$

(3)

To further assess the impact of the freeze-thaw cycle on the strength parameters of the samples, the strength degradation index ${{YG}}_{C(\varphi )}$ is introduced as follows:

$${{YG}}_{{Nc}(\varphi )}=\frac{{Y}_{0c\left(\varphi \right)}-{Y}_{{Nc}\left(\varphi \right)}}{{Y}_{0c\left(\varphi \right)}}$$

(4)

In the equation, ${{YG}}_{0c(\varphi )}$ and ${{YG}}_{{Nc}(\varphi )}$ represent the cohesion (internal friction angle) after 0 cycles and N cycles, respectively.

Research generally indicates that the cohesion of soil is primarily influenced by the bonding forces between particles, molecular forces, and the cementation between binding materials, while the internal friction angle is mainly affected by the shape, density, and contact methods of the particles⁴⁷. Analysis of Fig. 7c reveals that the degradation of cohesion exhibits an approximately progressive change with respect to the number of cycles, with a rapid decline in cohesion after the first six cycles, reaching a degradation level of 19.46%. The subsequent decline becomes more gradual, which is attributed to the lateral constraint effect of the ring cutter; the changes in particle bonding gradually slow down, resulting in a diminishing impact on cohesion. This observation is consistent with phenomena revealed in similar experiments^48,49. Ultimately, the degradation level reaches 29.19%, which is relatively low; in contrast, the internal friction angle φ varies almost linearly with the number of cycles, and the degradation level changes significantly, reaching 29.36% after six cycles. The final degradation level reaches 46.42%. The freeze-thaw cycle causes damage to the internal structure, leading to the decomposition of particle aggregates and changes in the contact methods between particles, which increases the variation in the internal friction angle and significantly affects its degradation level.

Micro-scale analysis

The X-ray diffraction patterns are shown in Fig. 8. A comparative analysis of the diffraction curves of samples subjected to 15 cycles and those that did not undergo freeze-thaw cycles reveals that the positions of the diffraction peaks for both are essentially in agreement. This indicates that no new phases have formed, and the application of fly ash and EICP technology does not have biochemical effects on the soil itself. Induced calcium carbonate generally exhibits various crystal types, such as calcite, aragonite, and vaterite. Among these, calcite is the most stable calcium carbonate mineral found in nature and possesses high strength. Fortunately, the main components of the two samples are quartz, calcite, illite, and feldspar, and no thermodynamically unstable aragonite or vaterite has formed.

Fig. 8

X-ray diffraction pattern.

The alterations in macroscopic mechanical properties are probably associated with modifications in the microscopic structure. Scanning electron microscopy (SEM) facilitated direct inspections of untreated soil and treated samples to examine the variations in internal structures pre- and post-treatment. Figure 9a illustrates that the soil particles in the untreated sample are loosely organized, exhibiting visible pores of differing dimensions between them. The limited surface attachments and weak interparticle bonding account for its significantly worse physical and mechanical properties. Figure 9b illustrates that the soil skeleton in the reinforced sample predominantly displays a stacked structure. The sample exhibits no microcracks or fractures, and the interparticle interactions are robust. A substantial quantity of fly ash particles of diverse sizes is visible occupying the pores, illustrating an exceptional micro-aggregate filling action. Calcium carbonate precipitates attach to soil particles and fly ash, contributing to coating, binding, and filling interstitial spaces between particles. This produces a more compact structure, hence augmenting the soil’s mechanical strength.

Fig. 9: Reinforcement mechanism and SEM image analysis.

a SEM image of untreated site soils; b SEM image of the anchoring grout.

The interactions among calcium carbonate, soil particles, and fly ash allow for the categorization of strengthening mechanisms induced by EICP treatment as follows (Fig. 9): (1) Coating effect. Fly ash acts as a nucleation site, characterized by a loose and porous surface structure. It experiences charge adsorption resulting from the hydrolysis of Si–O bonds, which promotes the adhesion of significant quantities of Ca²⁺ and CaCO₃ crystals to the particle surfaces, consequently enhancing their volume and surface area⁵⁰. (2) Bonding effect. Fly ash particles containing significant amounts of attached CaCO₃ exhibit a tendency to aggregate, which facilitates the formation of cementitious products and leads to the development of flocculated structures. (3) Bridging effect. Calcium carbonate crystals precipitate and develop between neighboring soil particles that lack direct contact, ultimately creating “bridges” that connect them. The observed strengthening effects lead to a reduction in soil porosity, as indicated by the increased elastic wave velocity recorded in tests. The bonding and bridging functions of calcium carbonate are the main contributors to the structural reinforcement of the soil⁵¹.

Figure 10 presents scanning electron microscopy images of samples exposed to 3, 6, and 15 freeze-thaw cycles, displayed at various magnifications. Following three freeze-thaw cycles, the agglomeration of soil particles within the sample exhibited alterations. The cyclical freezing and thawing of water within soil pores resulted in expansion and tensile stresses, leading to displacements among particles. This caused damage to the soil structure, resulting in increased looseness and the formation of numerous localized micro-pores (Fig. 10a, b), which sharply contrasts with the samples that did not experience freeze-thaw cycles.

Fig. 10: Microscopic analysis of samples at different cycle times.

a–c SEM images of specimens after three freeze–thaw cycles (×500, ×1000, ×2000); d–f SEM images of specimens after six freeze–thaw cycles (×500, ×1000, ×2000); g–i SEM images of specimens after fifteen freeze–thaw cycles (×500, ×1000, ×2000).

Following 6 and 15 freeze-thaw cycles, water that infiltrated the pores and cracks during thawing expanded the pore sizes upon refreezing (Fig. 10d–f). This diminished the point-to-surface contact among particles, ultimately resulting in the degradation of the bio-cemented soil structure (Fig. 10g), and a reduction in both compressive and shear strength of the sample was observed.

As shown in Fig. 10i, calcium carbonate (white particles in the image) remains largely deposited at the contact points between soil and fly ash particles, as well as on the surface of the fly ash particles. These calcium carbonate deposits effectively contribute to the cementation and bonding functions. However, after freeze-thaw cycles, the bridging of calcium carbonate within the sample is significantly reduced, which is suspected to be related to the formation of microcracks between calcite clusters³⁰. The main reason for this is that microcracks raise porosity, which has an immediate impact on water flow and infiltration. Water migration and the continuous filling of pore spaces exert pressure on the soil matrix, resulting in crack propagation.

Limitations and future work

This study demonstrates the feasibility of enzyme-induced calcium carbonate precipitation (EICP) technology for preserving earthen heritage sites. Nonetheless, other obstacles and challenges persist before this strategy can be broadly adopted in real conservation initiatives. Existing studies have shown that freeze–thaw cycles alter the pore structure of treated samples, thereby undermining the effectiveness of the reinforcement³⁰. This underscores the necessity for quantitative investigation of microstructural alterations to provide a precise comprehension of the fundamental degradation mechanisms. Furthermore, in contrast to controlled laboratory environments, the intricate and fluctuating conditions in the field—such as intense winds⁵², precipitation⁵³, temperature variations⁵⁴, salt crystallization⁵⁵, and biological degradation⁵⁶—present considerable challenges to in situ application. This is directly linked to the long-term durability of the biomineralization process. To attain more reliable and scientifically validated outcomes, subsequent research should focus on developing testing methodologies associated with long-term durability and addressing the logistical, legal, and sociocultural challenges related to large-scale field implementation.

Discussion

This study presents a strategy for formulating a novel anchoring grout utilizing EICP technology in conjunction with fly ash and site soil, grounded on the unique protection principles for earthen architectural heritage sites. We assessed the effects of several material ratios and freeze-thaw (F-T) cycles on the mechanical properties of the treated specimens, identified the optimal mixture ratio, and employed microstructural analysis to elucidate the reinforcing mechanisms involved. Fly ash, a prevalent industrial byproduct, when integrated with crude urease-extracted EICP technology, attains a synergy between economic viability and environmental sustainability, demonstrating its practical applicability in the anchoring protection of earthen cultural monuments. The experimental data led to the following conclusions:

A detailed analysis was carried out on the enhancement effects on mechanical parameters, such as wave velocity, unconfined compressive strength (UCS), and shear strength. The results indicate that the optimal ratio is a soil-ash ratio of 1:1, a water-cement ratio of 0.52 and a CCS of 0.8 mol/L, corresponding to maximum wave velocity, UCS, and shear strength. Furthermore, there was a significant enhancement in cohesiveness and internal friction angle. The concentration of cementing solution significantly influenced the enhancement of mechanical performance.

Freeze–thaw cycles caused phase transitions between ice and water, resulting in numerous internal cracks, which significantly diminished the mechanical characteristics of the specimens and altered their failure modes. The reduction in internal friction angle (up to 46.42%) was more significant than that of cohesion (up to 29.19%), demonstrating that freeze-thaw cycles considerably affected interparticle bonding and contact mechanisms.

The microscopic analysis revealed that the calcium carbonate and fly ash particles were securely integrated into the soil matrix, facilitating cementation and pore filling. The noted reduction in mechanical properties was exacerbated by the structural degradation inflicted by freeze-thaw cycles in the grouted material.