A Bacteria Acclimation Technology Based on Nitrogen Source Regulation and Its Application in the Reinforcement of Island and Reef Slopes

Abstract

Microbially Induced Calcium Carbonate Precipitation (MICP) technology has garnered significant attention in geotechnical engineering and environmental remediation due to its environmentally friendly and cost-effective advantages. However, the current MICP technology faces challenges in practical engineering applications, particularly the prolonged cementation time, which makes it difficult to meet the requirements for coastal slope reinforcement. Therefore, this study designed novel cultivation conditions for Sporosarcina pasteurii by regulating external nitrogen source concentration and evaluated its environmental adaptability by measuring OD600, urease activity, and bacterial length. By monitoring the changes in Ca²⁺ concentration, pH, and precipitation rate over time during the mineralization process, rapid cementation under MICP conditions was achieved. The engineering applicability of this approach in slope reinforcement was comprehensively assessed through simulated on-site scouring and penetration tests. The reinforcement mechanism and the microstructure of the cementation under novel cultivation conditions were analyzed using scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), and X-ray diffraction (XRD). The results indicated that the activity of Sporosarcina pasteurii in the modified NH₄-YE medium significantly improved in freshwater environments, and the MICP mineralization reaction was rapid, completing within 4 h. The primary crystal form of the generated precipitate was rhombohedral calcite, which formed a tightly bonded microstructure with calcareous sand, achieving a maximum reinforcement strength of 13.61 MPa. The penetration strength increased by at least 20%, and the precipitation rate improved by at least 2-fold. The scouring morphology remained essentially unchanged within 6 h. The findings of this study provide foundational and theoretical data for the application of MICP reinforcement technology to coastal calcareous sand models.

1. Introduction

Coastal areas are transitional areas where the interaction between the sea and the land is of remarkable ecological and economic importance. With the increasing demand for infrastructure and ecological engineering in coastal areas, the stability and sustainability of coastal slopes have become a key research focus. However, due to the vulnerability of calcium sand particles, sandy slopes, especially those composed of calcium sand, are prone to structural damage under normal stress levels. This vulnerability poses a challenge to maintaining slope integrity, requiring advanced reinforcement technology to ensure the long-term stability and resilience of the coastal environment [1]. For example, in the coastal areas of southern China, the combination of wave action and biological erosion caused by typhoons causes the atoll slope to drop by 12–15% per year [2]. Similarly, in Rocky Reef, Florida, ship damage and eutrophication synergy led to slope collapse, causing about 12.4% of habitat loss in five years [3,4]. This shows that under the combined action of ocean dynamics (such as waves and tides) and human interference, coastal areas have become highly vulnerable to erosion, leading to coastline retreat, ecosystem degradation, and even threatening the safety of coastal infrastructure [5,6,7]. In order to solve the problem of coastal slope erosion, traditional reinforcement measures include cruel protection methods (such as coastal groin [8,9], coastal breakwaters [10], and seawalls [11,12]) and soft protection methods (such as seawall design based on ecological restoration [13], innovative water permeability wave dissipation breakwater structure [14], artificial reef sinking breakwater that imitates the shape of natural reefs [15,16], and beach maintenance technology replenished by artificial sand [17,18]). Traditional protective measures, such as the traditional Gabe shore protection, may fail within only two years of construction due to the low cohesion characteristics of calcium sand [19,20]. Similarly, under the condition of climate change, soft protection measures such as artificial reef slopes in the South China Sea have experienced accelerated biological erosion rate, and 38.2% of engineering structures need to be repaired within three years after installation [21,22]. These cases show that although existing stabilization methods can partially mitigate erosion, their high cost, complex construction requirements, ecological impact, and potential threats to human safety and economic security seriously limit their wide application [23,24,25]. Therefore, there is an urgent need to develop sustainable and stable solutions that can cope with mechanical degradation and ecological decline at the same time.

In recent years, with the rise of sustainable engineering concepts, microbially induced calcium carbonate precipitation (MICP) technology, as an environmentally friendly and cost-effective soil improvement method, has gradually attracted the attention of academia and engineering circles [26,27]. MICP technology utilizes microbial metabolic activities to induce calcium carbonate precipitation through the hydrolysis of urea [28]. The precipitates generated by MICP can significantly enhance the erosion resistance of sandy slopes by filling soil pores and cementing particles [29,30]. The specific reinforcement mechanism is as follows.

$$\mathrm{CO}(\mathrm{N}{\mathrm{H}}_2{)}_2+2{\mathrm{H}}_2\mathrm{O}\stackrel{\mathrm{Urease}}{\to }{2\mathrm{NH}}_3+{\mathrm{CO}}_2$$

(1)

$${\mathrm{NH}}_3+{\mathrm{H}}_2\mathrm{O}\to {\mathrm{NH}}_4^{+}+{\mathrm{OH}}^{-}$$

(2)

$${\mathrm{CO}}_{2\left(\mathrm{g}\right)}+{\mathrm{OH}}^{-}\to {\mathrm{HCO}}_3^{-}$$

(3)

$${\mathrm{HCO}}_3^{-}{+\mathrm{OH}}^{-}\leftrightarrow {\mathrm{CO}}_3^{2-}+{\mathrm{H}}_2\mathrm{O}$$

(4)

$${\mathrm{CO}}_3^{2-}+{\mathrm{Ca}}^{2+}\to \mathrm{CaC}{\mathrm{O}}_3\downarrow$$

(5)

This paper involves enzyme-induced carbonate precipitation (EICP) technology, and EICP relies on urease from plant sources or microorganisms to catalyze the hydrolysis of urea, which is different from MICP. MICP depends on urease secreted by urease-producing bacteria (UPB) [31]. However, many studies have incorporated other substances to increase its strength due to its low nuclear site. However, since this article focuses on coastal protection, only the urease obtained from bacterial centrifugation is used for research. Currently, research on MICP reinforcement technology in engineering geology mainly focuses on soil solidification [20,32], anti-cracking and seepage [33], wind proofing and anti-erosion [34,35], and remediation of contaminated water/soil [36,37]. Compared to traditional coastal erosion protection technologies, MICP technology is conducive to ecological protection and improving land resource utilization [38]. This technology avoids using traditional high-energy-consuming materials (such as concrete) and reduces material waste and energy loss. It reduces carbon emissions, minimizes environmental impact, and eliminates the need for harmful chemicals, thus helping protect marine and coastal ecosystems [26,39].

Both domestic and international scholars have explored the application of MICP technology to mitigate erosion in coastal slopes. For instance, Cheng et al. [40] found that cementation samples produced during durability testing of MICP-treated slopes exhibited high resistance to freeze–thaw erosion and the impact of raindrops and runoff erosion. Daryono et al. [41] utilized MICP to cement beach sediments into artificial beach rock, demonstrating that the artificial beach rock shared similar properties with natural beach rock, suggesting that MICP could be an eco-friendly solution for beach erosion. Nayantara et al. [42] observed that MICP significantly enhanced the erosion resistance of beaches without negatively affecting hydrological parameters such as soil permeability. Salifu et al. [43] applied MICP to selected slopes under simulated tidal cycles and found that the technology significantly improved slope stability and erosion resistance. Kou et al. [44] conducted a series of small-scale flume erosion tests to quantitatively study the erosion resistance of MICP-treated slopes. Shashin et al. [45] prepared 45° inclined sand samples in acrylic flume devices and tested them under erosive and accretive surge waves, finding that MICP limited soil erosion to less than 5% for both wave types. Tsai et al. [46] noted that appropriate MICP treatment could mitigate sand slope erosion under various wave conditions. Ding et al. [47] combined different enzyme precursors to strengthen coastal sediments and conducted a sink-flushing test. Their research shows that the seawater’s domesticated strains of the same bacteria showed excellent enhancement effects and enhanced flush resistance. In coastal calcareous sands, the MICP reaction depends on microbial metabolic activity and is influenced by environmental conditions such as salinity, temperature, and pH [48,49,50]. Overall, research on the fundamental reaction processes has primarily focused on the properties of mineralization products [51] and factors influencing the reaction [52]. However, studies have shown that the OD600 values of Sporosarcina pasteurii under MICP conditions are generally low [53,54]. Incorporating additional materials to enhance reinforcement effects may lead to varying degrees of ecological pollution. Furthermore, while there is research on the application of MICP technology for slope erosion protection, studies focusing on increasing the concentration of Sporosarcina pasteurii for slope reinforcement, combined with flume models to simulate real-world environments, remain relatively limited. This highlights the need for further exploration into optimizing MICP conditions and scaling up its application for coastal slope stabilization. In addition, this study is consistent with SDG 13 (climate action) and SDG 14 (underwater life), providing a low-carbon solution to deal with coastal erosion while balancing infrastructure needs and ecological protection.

This study designed novel cultivation conditions for Sporosarcina pasteurii to address these challenges by regulating external nitrogen source concentrations. The environmental adaptability of Sporosarcina pasteurii was evaluated by measuring OD600, urease activity, and bacterial length. The mineralization process was investigated by monitoring changes in Ca²⁺ concentration, pH, and precipitation rate over time. Additionally, a customized flume model was used to simulate on-site scouring and penetration tests, comprehensively assessing the engineering applicability of MICP in slope reinforcement. The microstructure of the cementation under the new cultivation conditions was analyzed using SEM–EDS and XRD. This study explores the potential of MICP technology in reinforcing sandy slopes by combining laboratory experiments with simulated scouring tests. The goal is to evaluate its engineering applicability and long-term stability, providing foundational and theoretical data for applying MICP reinforcement technology to coastal calcareous sand models. Ultimately, this research offers a new solution for ecological protection in coastal zones, contributing to developing sustainable and environmentally friendly slope stabilization methods.

2. Materials and Methods

2.1. Test Materials

(1) The bacteria selected for the experiment were Sporosarcina pasteurii (NO. 337394, BNCC), purchased from the Guangdong Microbial Culture Collection Center. The bacteria were activated from freeze-dried powder to the first generation, then expanded and subcultured for 2–3 generations before being stored in a freezer for future use. Based on experimental requirements, the liquid culture environment for the bacterial suspension was divided into deionized water and seawater. The seawater was collected from Dadonghai, Sanya, with a salinity of 36‰ and a pH of 8.25. The culture media were prepared in both seawater and deionized water. The configuration of the liquid culture medium is shown in Figure 1a, while the seawater ion composition, concentration, and experimental procedures are detailed in Figure 1b.

(2) The calcareous sand samples were collected from a reef in the South China Sea, with a specific gravity (Gs) of 2.69, a void ratio ranging from 0.63 to 1.29, and a dry density ranging from 1.22 g/cm³ to 1.65 g/cm³ [55]. Figure 1c shows the sand’s particle size distribution curve. The specimen models were made of acrylic material and divided into two types: (1) A transparent hollow cylinder with a diameter of 39.1 mm and a height of 88 mm, as shown in Figure 1d. (2) A transparent flume model with overall dimensions of 520 mm (length) × 140 mm (width) × 140 mm (height) and individual compartment dimensions of 114 mm (length) × 110 mm (width) × 110 mm (height), as shown in Figure 1e.

2.2. Test Methods

2.2.1. Sporosarcina pasteurella Test Protocol

This study used an improved culture medium and a traditional urea-based culture medium to conduct a comparative analysis of the culture of Bacillus Pasteurella. The purpose is to determine the culture conditions that promote bacterial growth, enhance the surface resistance in the experiment, and improve the corrosion resistance. Bacteria are cultured under freshwater and seawater conditions. The culture scheme is shown in

Table 1. Experimental Scheme for Cultivating Sporosarcina pasteurii.

Culture Medium	Group	Liquid Medium Volume (mL)
		Deionized Water Seawater
The traditional urea medium with deionized water	OFM	250      --
The traditional urea medium with seawater water	OSM	--      250
The modified medium with deionized water	NFM	250      --
The modified medium with seawater water	NSM	--      250

Note: Old freshwater medium is abbreviated as OFM, old seawater medium is abbreviated as OSM, new freshwater medium is abbreviated as NFM, new seawater medium is abbreviated as NSM.

[56]. The experimental process is shown in Figure 2a.

Preparation and cultivation of freshwater bacterial suspension: Take a 2 mL cryotube from the −80 °C freezer and let it warm up to room temperature (25–30 °C) until it completely melts. Pour the melted bacterial liquid into the traditional urea and the modified liquid medium prepared with deionized water. Subsequently, place a 250 mL conical flask in a shaker and cultivate it at a controlled temperature of 30 °C with a rotation speed of 220 r/min for 36 h. Thus, the deionized water bacterial suspension of the traditional urea medium and the modified medium (OFM/NFM) used in the experiment is obtained.

Preparation and cultivation of seawater bacterial suspension: Take a 2 mL cryotube from the −80 °C freezer and let it warm up to room temperature (25–30 °C) until it completely melts. Pour the melted bacterial liquid into the traditional urea and the modified liquid medium prepared with seawater. Then, place a 250 mL conical flask in a shaker and cultivate it at a controlled temperature of 30 °C with a rotation speed of 220 r/min for 36 h. As a result, the seawater bacterial suspension of the traditional urea medium and the modified medium (OSM/NSM) used in the experiment is obtained.

Observation of bacterial morphology: After cultivation, the bacterial suspension was treated with a Gram stain reagent to differentiate and visualize the bacteria. The stained samples were then observed under an inverted biological microscope to analyze the morphology of Sporosarcina pasteurii. This step helps assess bacterial health, growth patterns, and morphological changes under different culture conditions.

Measurement of urease activity: The conductivity meter was used to measure urease activity by monitoring changes in conductivity resulting from urea hydrolysis. This process is illustrated in the flowcharts in Figure 2b,c, which detail the steps from bacterial cultivation to morphological observation and urease activity measurement.

2.2.2. MICP/EICP Aqueous Solution Experiments

This experiment was carried out by mixing the bacterial solution, urease, and deionized water cementing solution in a 250 mL beaker at a ratio of 1:4, and the deionized water cementing solution was obtained by 1 mol/L calcium chloride and urea mixed solution. In order to maintain the accuracy of the experiment, three parallel samples were set up and sterilized with deionized water. Because calcium chloride is dissolved in water and released in large quantities, it is cooled and mixed with urea solution to a specific volume when the cementing liquid is configured. The setting of the experimental value is based on the optimal value of mineral precipitation.

Urease was extracted using low-temperature high-speed centrifugation, and its reaction with the cementation solution is referred to as Enzyme-Induced Carbonate Precipitation (EICP). Preparation of urease by centrifugation: Add the bacterial suspensions with consistent concentrations (OFM/NFM/OSM/NSM) to a 50 mL centrifuge tube and place it in a low-temperature high-speed centrifuge for centrifugation. After centrifuging at 4 °C and 7830 rpm for 25 min, the obtained urease (OFU/NFU/OSU/NSU) is placed in a freezer for later use. To maintain the consistency of urease activity, it should be used within 2 days after the centrifugation is completed. In this experiment, the abbreviations are as follows: old freshwater urease is abbreviated as OFU, old seawater urease as OSU, new freshwater urease as NFU, and new seawater urease as NSU.

The experiment investigated the mineralization reaction process of bacterial solution, urease, and cementation solution over 72 h to deeply explore the reaction mechanism, with the precipitation formation time set at 4 h. The specific test scheme is detailed in

Table 2. Experimental scheme for MICP aqueous solution tests under different culture media.

Group	Bacterial Volume (mL)	Cemented Volume (mL)	$\mathbf{C}{\mathbf{c}\mathbf{a}\mathbf{c}\mathbf{l}}_2$$\text{:} \mathbf{C}\mathbf{u}\mathbf{r}\mathbf{e}\mathbf{a}$
OFM	40 mL	160 mL	1:1
OSM
NFM
NSM

Note: This article will be referred to as the traditional urea medium; the modified medium deionized water/seawater is OFM, NFM, OSM, and NSM.

and

Table 3. Experimental scheme for EICP aqueous solution tests under different culture media.

Group	Bacterial Volume (mL)	Cemented Volume (mL)	$\mathbf{C}{\mathbf{c}\mathbf{a}\mathbf{c}\mathbf{l}}_2$$: \mathbf{C}\mathbf{u}\mathbf{r}\mathbf{e}\mathbf{a}$
OFU	40 mL	160 mL	1:1
OSU
NFU
NSU

Note: This article will be referred to as the traditional urea medium; the modified medium deionized water/seawater urease is OFU, NFU, OSU, and NSU.

, and the flow chart is shown in Figure 3a.

2.2.3. MICP/EICP Sand Column and Scouring Experiments

Sand column reinforcement penetration test: Pour the washed and dried calcareous sand with mixed particle sizes into a transparent hollow tube with a diameter of 39.1 mm and a height of 88 mm. Before pouring, one end of the hollow tube should be wrapped with gauze and adhesive tape, and a layer of filter paper should be placed at the bottom to prevent the sand grains from falling off. The pouring is carried out three times, and the mold should be shaken each time after pouring to ensure the uniform mixing of the calcareous sand.

The reinforcement test is mainly divided into single reinforcement and double reinforcement. Determine the parameter setting according to the pore saturation threshold obtained in the preliminary experiment. Manually drip the bacterial suspension and the cementing solution onto the surface of the sand column. The cementing solution is mainly the deionized water cementing solution. For the single bacterial injection reinforcement, 10 mL of bacteria and 20 mL of nutrient salts are added. For the double bacterial injection reinforcement, 5 mL of bacteria and 10 mL of nutrient salts are added two times. There are 12 h between the bacterial suspension and the nutrient salts, and an interval of 4 h between the additions of the nutrient salts. After the reinforcement is completed, put it into an oven to dry.

Table 4. MICP sand column cementation test scheme.

Group	Number of Injections	Inject Volume (mL)	Number of Times the Cementation Solution Is Injected	Cemented Volume (mL)
OFM	1	10	2	20
	2	5
OSM	1	10
	2	5
NFM	1	10
	2	5
NSM	1	10
	2	5

and

Table 5. EICP sand column cementation test scheme.

Group	Number of Injections	Inject Volume (mL)	Number of Times the Cementation Solution Is Injected	Cemented Volume (mL)
OFU	1	10	2	20
	2	5
OSU	1	10
	2	5
NFU	1	10
	2	5
NSU	1	10
	2	5

detail the test schemes for the cementation of the sand columns.

The penetration test aims to study the bearing capacity of loose calcareous sand surface cementation under different culture conditions using bacterial suspension or urease. To minimize experimental errors, the midpoint of the sand column is selected as the measurement point, and two parallel samples are tested for each condition. The average values are used to plot the penetration depth vs. penetration resistance curve.

Based on the penetration test results, the condition corresponding to the optimal surface bearing capacity is selected for the scouring test. The scouring test involves setting different scouring power levels and comparing reinforced vs. unreinforced conditions. The changes in the scouring profile are observed over 6 h, providing valuable insights for reinforcing calcareous sand in South China Sea reefs. Penetration test: Measure the penetration resistance at the midpoint of the sand column. Use two parallel samples for each condition to ensure accuracy. Plot the penetration depth vs. penetration resistance curve to evaluate the bearing capacity. Scouring test: Select the optimal condition based on the penetration test results. Set different scouring power levels and compare reinforced vs. unreinforced sand columns. Observe and record changes in the scouring profile over 6 h. During the flushing and photography process, the unfixed profile was photographed at the exact location with a magnification of 4.5 times and a light–width ratio of 4:3. The fixed profile was photographed with a magnification of 2 times and the same aspect ratio of 4:3. Only the profile of calcium sand was photographed. This experiment aims to verify the engineering applicability of microbially induced carbonate precipitation (MICP) solidification technology under nitrogen source regulation and domestication conditions. Therefore, only the macroscopic observation of using the degree of profile depression to evaluate the feasibility of applying this technology was carried out. The detailed experimental process is shown in Figure 3b. During the sample preparation process, 721 g of mixed particle-size calcium sand was poured into each compartment three times, and the height was controlled at 6.5 cm. During the flushing process, we placed the sink compartment in the sink for inclined flushing. This approach provides a systematic evaluation of the effectiveness of MICP in enhancing the bearing capacity and erosion resistance of calcareous sand, offering practical guidance for coastal slope stabilization and reef protection.

2.2.4. SEM–EDS and XRD Tests

To explore the microstructure and chemical composition of the cementation between calcareous sands, analyses were performed using a JSM-7610F PLUS SEM manufactured in Japan and an EDS system from Oxford, UK. The state of the calcareous sands after cementation was analyzed by SEM. Before sample preparation, the sample was dried in an oven at 60 °C, and randomly selected parts of the dried sample were used for sample preparation. Gold plating enhanced all samples’ conductivity before analysis [57]. When conducting the analysis, the observation voltage was set at 10 KV or 15 KV, and the magnification options allowed for detailed observation ranging from 500 to 2000 times. The Empyrean experimental instrument produced by PANalytical was used. The scanning angle and step size were 5–80 degrees and 0.01, respectively. The scanning rate is 1.0°/min based on the diffraction angle 2θ [58], and the scanning was carried out indoors for 1 h. Before scanning, the precipitates were rolled and ground until they could pass through a 300-mesh sieve. The JSM-7610F PLUS field emission scanning electron microscope used in the experiment is an original imported device from JEOL Ltd., Tokyo, Japan. The X-MaxN 80 mm² energy-dispersive spectrometer (EDS) detector is manufactured by Oxford Instruments plc, Oxfordshire, UK. The Empyrean X-ray diffractometer is produced by Malvern Panalytical B.V., Almelo, Netherlands. The specific operation process is shown in Figure 3c,d.

3. Results

3.1. Analysis of the Results of Bacterial Acclimation Under Novel Nitrogen Source Regulation

In order to study the effects of different culture conditions on the growth status and urease activity of the cultured Bacillus spores, the inoculated bacterial solutions were uniformly shaken continuously in a shaker for 72 h. The bacterial concentration (OD600) and urease activity (UA) were measured during the shaking period.

The curves of the change in bacterial solution concentration over time under different culture conditions are shown in Figure 4a. According to the figure, OFM and NFM, under deionized water conditions, both grow in the logarithmic phase in the initial stage. The growth of OSM under seawater conditions is delayed in the initial stage. Seawater likely inhibits the growth of the bacterial solution, and the bacteria need to adapt to the environment before showing growth. In the initial stage, the value of NSM under the seawater condition increases more rapidly than that under the deionized water condition, and then there is a brief decrease. This may be because the culture medium provides more nutrients and minerals, promoting the growth of bacteria. In addition, the extra minerals, salts, and trace elements provided by seawater may promote Bacillus pasteurii’s growth, making it grow more rapidly in the culture medium containing seawater [56]. The subsequent brief decrease may be due to the consumption of nutrients or the accumulation of metabolic waste, resulting in a temporary decrease in the concentration of the bacterial solution. Subsequently, the concentration of the bacterial solution gradually rises and reaches a stable state, indicating that Bacillus pasteurii has gone through the stationary and decline phases.

From Figure 4b, the following can be observed: For Bacillus pasteurii cultured in the traditional culture medium under both freshwater and seawater conditions, its urease activity increases exponentially within 18 h. The activity gradually stabilizes between 18 and 36 h. After 36 h, it gradually decreases. In contrast, the urease activity of Bacillus pasteurii cultured in the improved culture medium under deionized water conditions gradually increases over time. The reason for this may be that urea persists continuously under the deionized water condition. Urea serves as the primary nitrogen source for the bacteria. During the culture process, the bacteria continuously require urease to decompose urea. This helps optimize the utilization of urea, and other nitrogen sources do not immediately replace the use of urea. Under seawater conditions, the urease activity shows a brief decrease. This may be due to the consumption of urea and the influence of the salts in seawater. It could also be attributed to the abundance of other nitrogen sources in the culture medium and the metabolic adaptability of the bacteria. The brief decrease in urease activity indicates that bacteria may no longer rely on urea as the primary nitrogen source within 3 to 6 h. Instead, they may choose other metabolic pathways during this period.

Overall, the urease activity under the deionized water condition is better than that under the seawater condition. The urease activity of Bacillus pasteurii is lower under seawater conditions. This is because the salts in seawater affect the metabolic activities of the bacteria. As a result, the urease activity is inhibited. In addition, bacteria may change their metabolic pathways in seawater. They may preferentially utilize other nitrogen sources instead of urea. In deionized water, without the interference of salts, the metabolic activities of bacteria are relatively stable. Under these conditions, the urease activity is higher. By observing the length of the bacteria in the bacterial solution under a microscope, it is evident that the bacterial length in the improved culture medium is greater than that in the traditional culture medium, with a length range of 0.04–0.105 μm. This indicates that the improved medium provides a more suitable environment for the growth of Bacillus pasteurii.

3.2. Analysis of MICP and EICP Aqueous Solution Test Results

This experiment studies the reaction mechanisms of the aqueous solutions of MICP and EICP under different culture conditions by measuring the changes in pH and Ca²⁺ over time during the mineralization processes of MICP and EICP under different culture conditions, as well as the amount and formation rate of the produced calcium carbonate.

As seen from Figure 5a, the pH values of EICP and MICP under different culture medium conditions all increase first, and then gradually decrease and tend to be stable over time. The probable reason is that the bacterial solution contains urease, which interacts with the cementing solution. This interaction causes urea to decompose, producing carbonate and ammonium ions. As a result, the pH of the solution increases. In the initial stage of the experiment, the carbonate ions produced by the decomposition of urea by the bacterial solution and urease react with the calcium ions in the solution to form precipitates. As time progresses, the amount of precipitate gradually increases, and the pH value of the solution approaches a stable level. The trends of the calcium ion concentrations in Figure 5b,c are gradually decreasing over time. The calcium ion concentration of NFU decreases rapidly, and the precipitation formation rate is the highest, indicating that its mineralization reaction rate is fast. It can be seen from the figures that the urease production rates are not high, and the calcium ion concentration decreases and tends to be between 0.2 and 0.4 mol/L over time. Likely, the components in the cementing solution (such as calcium ions, etc.) have an inhibitory effect on the catalytic action of urease, resulting in a low production rate. NSM’s precipitation production rate is higher than OSM’s, but the calcium ion concentrations tend to be the same. The formation of other precipitates leads to a higher production rate. The low yield of OSM might be due to the fact that seawater inhibits bacterial activity, resulting in a lower rate of precipitation [56].

3.3. Analysis of the Results of MICP and EICP Solidified Shore Slope Sand and Simulated Scouring Tests

In order to study the surface reinforcement effect of Bacillus pasteurii on calcareous sand under different culture conditions, a penetration test was carried out on calcareous sand with mixed particle sizes. As shown in Figure 6a, under the technical conditions of MICP and EICP, the penetration resistance increases with the increase of penetration depth. Under the MICP technical condition, the peak penetration resistance of Bacillus pasteurii cultured in a deionized water environment is superior to that of Bacillus pasteurii cultured in a seawater environment. The penetration strength value of NFM is the highest, reaching 13.614 MPa, and the change in its penetration strength is better than that of OFM. Under seawater conditions, NSM’s penetration peak is higher than OSM’s. Under the EICP technical condition, the peak penetration strength of urease centrifuged from the culture in a seawater environment is higher than that of urease centrifuged from the culture in a deionized water environment. The reason may be that the ion concentration in seawater is more conducive to the catalytic action of urease than that in deionized water, and it can enhance the surface cohesion of calcareous sand, thus improving its strength. The low ion concentration in deionized water and the relatively low stability of urease result in a weaker effect of urease, leading to a lower strength of the reinforced calcareous sand. Overall, as shown in Figure 6b, the effect of single reinforcement on penetration resistance is not as good as that of double reinforcement, and most of them reach the maximum peak at about 6 MPa. This may be because, under mixed particle size reinforcement, a relatively stable structure and strength have been formed after the first reinforcement, and the second reinforcement improves the penetration strength within its stable space [53,59]. According to the penetration peak graph, the reinforcement effects of OFU and NFU under the EICP conditions are not ideal. The reason may be that the reaction between urease activity and the cementing solution cannot reach the ideal condition. The peak differences between OSU and NSU are not noticeable. Based on this, double reinforcement is selected for the scouring test, and the bacterial solutions under different culture conditions are mainly selected to study the scouring effects of different bacterial solutions applied to the flume model. In order to study the surface reinforcement effect of Bacillus pasteurii on calcareous sand under different culture conditions, a penetration test was carried out on calcareous sand with mixed particle sizes.

As shown in Figure 6a, under the technical conditions of MICP and EICP, the penetration resistance increases with the increase of penetration depth. Under the MICP technical condition, the peak penetration resistance of Bacillus pasteurii cultured in a deionized water environment is superior to that of Bacillus pasteurii cultured in a seawater environment. The penetration strength value of NFM is the highest, reaching 13.614 MPa, and the change in its penetration strength is better than that of OFM. Under seawater conditions, NSM’s penetration peak is higher than OSM’s. Under the EICP technical condition, the peak penetration strength of urease centrifuged from the culture in a seawater environment is higher than that of urease centrifuged from the culture in a deionized water environment. The reason may be that the ion concentration in seawater is more conducive to the catalytic action of urease than that in deionized water, and it can enhance the surface cohesion of calcareous sand, thus improving its strength. The low ion concentration in deionized water and the relatively low stability of urease result in a weaker effect of urease, leading to a lower strength of the reinforced calcareous sand. Overall, as shown in Figure 6b, the effect of single reinforcement on penetration resistance is not as good as that of double reinforcement, and most of them reach the maximum peak at about 6 MPa. This may be because, under mixed particle size reinforcement, a relatively stable structure and strength have been formed after the first reinforcement, and the second reinforcement improves the penetration strength within its stable space. According to the penetration peak graph, the reinforcement effects of OFU and NFU under the EICP conditions are not ideal. The reason may be that the reaction between urease activity and the cementing solution cannot reach the ideal condition. The peak differences between OSU and NSU are not noticeable. Based on this, double reinforcement is selected for the scouring test, and the bacterial solutions under different culture conditions are mainly selected to study the scouring effects of different bacterial solutions applied to the flume model.

The collected scouring images intuitively observe the changing effects of the profiles of the reinforced and unreinforced coastal calcareous sand over time. Since inclined scouring generates corresponding shear stress, the calcareous sand close to the water flow wall surface will undergo significant changes.

From the sectional views with different scouring intensities in Figure 7 and Figure 8, it can be seen that the scouring effect is pronounced within 1 h under the unreinforced condition. A concave surface is formed on the side close to the water flow wall surface. Due to the relatively strong scouring intensity of 15 W, it can be seen from the image that the concave surface is more profound, and cracks appear. After that, the changing effect gradually tends to be stable with time, and it reaches a stable state after 6 h, and there is not much change in the profile.

The reinforced groups are divided into a freshwater group and a seawater group. The samples in the freshwater group are OFM and NFM in sequence from left to right, and the samples in the seawater group are OSM and NSM in sequence from left to right. By comparing the 6 h scouring sectional images and front views under different scouring intensities and different bacterial solutions, as shown in Figure 7, Figure 8 and Figure 9, it can be observed that the scouring effects of NFM and NSM are better than those of OFM and OSM.

Under the scouring of 10 W and 15 W, the surface of OSM is eroded to different degrees, while NSM changes little and its morphology remains unchanged. Under the scouring condition of 10 W, there is not much change in the sectional view. Under the scouring condition of 15 W, a concave surface is formed along the water flow wall surface in the sectional view of OSM.

Horizontally speaking, as the scouring force increases, the changing effect of the sectional view of the seawater bacteria is more evident than that of the freshwater bacteria. Based on the comprehensive analysis of the front views, it can be seen that the reinforcement effect of the improved NH₄-YE medium is better than that of the traditional urea medium; its scouring effect, when applied to the scouring model, is also the best.

3.4. Microstructure Characterization Analysis

In order to study the enhancing effect of MICP and EICP technology on calcareous sand under different culture conditions and to deeply understand the microstructure of precipitation products in aqueous solution, SEM–EDS techniques were employed, and XRD techniques were used to characterize the precipitation products.

It can be seen from the SEM image in Figure 10 that the morphology of the sediment under the MICP process conditions is mainly spherical and diamond (more diamonds), and the morphology of the sediment under the EICP process conditions is spherical and diamond (more spherical). The crystal morphology of the precipitate generated by NFM is composed of a rhombic morphology formed by multiple irregularly arranged clusters. OFM, OSM, and NFM comprise rhombic calcite, OFU, and OSU, mainly vanadium iron ore. At the same time, NFU and NSU are mainly made of vanadium iron ore transformed into calcite morphology.

Based on the EDS analysis, it can be observed that the freshwater group primarily contains C, O, and Ca elements, while the seawater group, in addition to these three elements, also contains trace amounts of S, Cl, Mg, and other elements. The main reason for the higher elemental diversity in the seawater group’s precipitates is likely the high salinity and complex composition of seawater itself, as well as differences in microbial metabolic activity and crystal structure under seawater conditions. These factors collectively result in a greater variety of elements in the seawater group’s precipitates compared to the freshwater group. Since the SEM–EDS images of bacterial precipitates show superior crystal structure compared to those of urease precipitates, XRD analysis was chosen for the bacterial precipitates.

Figure 11 displays the XRD patterns of the bacterial precipitates. By comparing the crystal structures of the precipitates with standard reference cards, it was found that both OFM and NFM primarily consist of calcite. However, OFM contains a certain proportion of hexagonal calcite, while NFM contains trigonal calcite. Since the structure of trigonal calcite is more stable than that of hexagonal calcite, the precipitates in NFM exhibit higher thermodynamic and structural stability [60]. On the other hand, OSM contains some magnesium-containing calcite, while NSM contains some hexagonal calcite. Although magnesium-containing calcite is thermodynamically more stable, the superior cementation microstructure of the NSM group is mainly attributed to the combined advantages of precipitate density, uniform distribution, cementation strength, environmental adaptability, and precipitation rate. These factors collectively contribute to the more significant cementation effect of the NSM group.

Since the penetration test results demonstrated superior performance of MICP over EICP, this study focused exclusively on investigating the cementation effects of MICP-reinforced calcareous sand columns under microscopic conditions. For representative analysis, the midpoint region of the sand column’s penetration surface was selected for SEM sample preparation, with microscopic structure observations conducted at a depth of 1 cm thickness. As shown in Figure 12, the microscopic images of cemented calcareous sand columns in solidified shore slopes demonstrate that the modified culture medium significantly enhances the cementation effect by improving the bonding between particles, resulting in a denser and more uniform microstructure with improved mechanical properties. The schematic diagram of the mechanism is shown in Figure 13. As a result, the surface strength of the consolidated sand columns is greatly improved. Measurements of surface penetration strength also show that NFM and NSM outperform OFM and OSM. This demonstrates that the density of the cementation structure is positively correlated with erosion and scouring resistance: the denser the structure, the stronger the resistance to erosion and scouring; conversely, the looser the structure, the weaker the resistance.

4. Discussion

In the study of MICP technology, the OD600 value is generally low, which may be attributed to the growth characteristics of Sporosarcina pasteurii. However, this experiment successfully optimized the culture conditions of Sporosarcina pasteurii by regulating the nitrogen source concentration, significantly enhancing its activity and mineralization efficiency. Concurrently, the mineralization reaction time was substantially reduced to within 4 h, significantly improving the feasibility of engineering applications and providing important theoretical and experimental foundations for applying MICP technology in coastal calcareous sand models. Through SEM–EDS and XRD analyses, the reinforcement mechanism of the bacteria under the new culture conditions and the microstructure of the cementation products were further elucidated, confirming the formation of rhombic calcite and its critical role in enhancing material strength. Therefore, based on the experimental results, staged grouting could be adopted during construction to enhance reinforcement. Shortening the curing time and using a cementation solution at a 1:4 ratio improved the solidification strength and curing efficiency. Additionally, periodic penetration tests and SEM microscopic observations could be conducted to evaluate the durability of the cementation layer before and after the typhoon season.

However, this study still has some limitations. For instance, experiments have been conducted mainly in seawater under controlled laboratory conditions. In contrast, engineered environments may involve complex conditions such as pH changes, organic contamination, microbial competition, and the coupling effects of multiple factors. These factors may affect the activity and MICP mineralization efficiency of sporangium. Additionally, penetration tests only reflect localized strength and cannot assess the overall continuity of the reinforced mass. Moreover, SEM analysis involves destructive sampling, making long-term in situ monitoring challenging. Therefore, future studies can be conducted under conditions more similar to the actual engineering environment, simulating the interaction of various factors such as salinity, pH, and organic matter to evaluate the adaptability and stability of the MICP technique comprehensively.

5. Conclusions

This study aims to regulate the external nitrogen source concentration to design new culture conditions for Bacillus pasteurii. According to the peak value of penetration strength, the MICP technology is applied to the coastal model, providing the fundamental and theoretical data for applying the MICP reinforcement technology to the coastal calcareous sand model. The results are as follows:

(1) By monitoring the changes in OD600 and urease activity over time, it was found that the improved NH4-YE medium is more suitable for the growth of Bacillus pasteurii. The OD600 value becomes higher and gradually stable as the culture time prolongs. Under the condition of culturing the bacteria for four generations, the highest OD600 value can reach 3.6 in the freshwater environment, and the change in urease activity can reach 18.48 mmol/L·min. In the seawater environment, the highest OD600 value can reach 3.2, and the change in urease activity can reach 13.56 mmol/L·min.

(2) By detecting the changes in Ca²⁺, pH, and the precipitation formation rate over time during the mineralization reaction process, it was found that under the condition of the improved NH4-YE medium in the freshwater environment, the mineralization reaction time is rapid, the precipitation rate is high, and the amount of precipitation is increased by at least 1.26 times. The reaction is completed within 4 h.

(3) Using the automatic multifunctional penetrometer, it was found that under the technical conditions of MICP/EICP, a hardened shell with a particular strength can be formed on the surface of the calcareous sand, which improves the mechanical properties of the treated specimens and endows them with elemental erosion resistance. However, according to the penetration peak values, the reinforcement effect of the bacterial solution is better than that of urease. That is, the effect of MICP is higher than that of EICP. When scouring with the bacterial solution for reinforcement, it was found that the scouring morphology of NFM did not change within 6 h. According to the SEM images, the NFM and NSM cemented areas were tightly cemented and wrapped by calcite composed of multiple irregularly arranged clusters of rhombohedral morphology. The cementation state of OFM was not ideal, and it was not tightly wrapped by calcite. Aragonite was also present in the cemented area of OSM. The joints of NFM and NSM were better than those of OFM and OSM. The tighter the cementation, the better the penetration resistance and the stronger the erosion resistance.

The flushing model in this experiment was washed under ideal conditions without considering a series of other flushing factors, such as wind erosion. The experiments were only conducted under laboratory conditions without simulating harsh weather conditions, and no slope stability analysis or durability tests were performed. Therefore, the results of this study only provide fundamental and theoretical data for applying MICP reinforcement technology to coastal calcium sand models. Future research could focus on these aspects to address the issues. To further advance the translation of this technology into engineering applications, future research should prioritize field-scale validation and practical implementation, long-term ecological impact assessments, and the development of intelligent construction systems. These efforts will provide a new technological paradigm for coral reef ecological engineering.