Sporosarcina pasteurii in a freeze-dried form from China General Microbiological Culture Collection Center (CGMCC) was used as ureolytic bacteria for catalyzing urea hydrolysis. The strain was transferred to a liquid medium which consists of yeast extract (Oxoid Ltd., United Kingdom) of 20 g/L, urea (Damao Chemical Reagent Factory, China) of 20 g/L, NH₄Cl (Chengdu Chron Chemicals Co. Ltd., China) of 10 g/L, MnSO₄·H₂O (Shanghai Aladdin Biochemical Technology Co. Ltd., China) of 10 mg/L, and NiCl·6H₂O (Tianli Chemical Reagent Co. Ltd., China) of 24 mg/L and was cultured at 180 rpm and 30°C for 24 h. pH for the liquid medium was measured being 8.8. The activated ureolytic bacteria were mixed with glycerol using a ratio of 7:3 and stored at −20°C (Achal et al., 2012). They were used as the inoculum in subsequent test tube experiments.

The purpose for implementing the test tube experiments is to reproduce the biomineralization process using the ureolytic bacteria for Pb and Cu immobilization. The testing scheme for the test tube experiments under the effect of bacterial culture (i.e., yeast extract) and calcium source (i.e., CaCl₂) addition is summarized in Table 1. Bacterial solution from the frozen stock was first inoculated (0.1% (v/v)) into the liquid medium containing Cu(NO₃)₂ or Pb(NO₃)₂ at concentrations varying in a 0–50 mM range, 0.5 M urea, 2 g/L yeast extract, and 0.25 M CaCl₂. There were three replicates for each testing set. Urease activity (UA) and pH measurements were conducted at 0, 4, 12, 24, and 48 h, while NH₄ ⁺ and Pb²⁺ or Cu²⁺ concentration were measured at 0, 24, and 48 h, respectively. pH was measured using a bench pH meter (Hanna Instruments Inc. HI 2003), while UA was measured on a basis of the ureolysis rate, as recommended by Whiffin et al. (2007); 2 ml reaction solution is mixed with 18 ml 1.11 M urea, and EC is measured at 0 and 5 min after mixing. UA can be evaluated using the equation below: U A = E C 5 − E C 0 5 × 10 × 1.11 ( mM   Urea   min − 1 ) , (8) where EC ₀ and EC ₅ are electrical conductivity at 0 and 5 min, respectively. Furthermore, NH₄ ⁺ concentration in solution was determined using the modified Nessler method (Whiffin et al., 2007). Moreover, Pb²⁺ or Cu²⁺ concentration was measured using an atomic spectrophotometer (Beijing Purkinje General Instrument TAS-990). The remediation efficiency can be evaluated as follows: R e m e d i a t i o n   e f f i c i e n c y = C 1 − C 0 C 0 × 100 % , (9) where C ₀ and C ₁ are Pb²⁺ or Cu²⁺ concentration before and after remediation, respectively. NH₄ ⁺ concentration of the reaction solution is measured at 0.1, 24, and 48 h, and the method for measuring NH₄ ⁺ concentration corresponds to the modified Nessler method (Whiffin et al., 2007). Prior to the measurement, a calibration line was set up, and the absorbance was measured using a spectrophotometer 10 min after adding Nessler’s reagent and potassium sodium tartrate. It was substituted into the calibration line, yielding NH₄ ⁺ concentration. The more the NH₄ ⁺ produced, the higher the degree of urea hydrolysis. For this reason, the NH₄ ⁺ concentration was adopted here toward assessing the degree of urea hydrolysis.

Given that the speciation and sequence of carbonate precipitation cannot be revealed by the experimental results, they were reproduced using the Visual MINTEQ software package. In the present work, urea hydrolysis was modeled using the ratio of NH₄ ⁺ to CO₃ ²⁻ being 2:1, although the bacterial culture and inoculation were omitted in the numerical simulation (Gat et al., 2017). NH₄ ⁺ and CO₃ ²⁻ concentrations that are extracted upon the completion of urea hydrolysis aim to backanalyze the speciation and sequence of carbonate precipitation. The details in terms of the numerical simulation are summarized in Table 2. The simulated results are beneficial to analyze how the speciation and sequence of carbonate precipitation vary under the sole effect of yeast extract and the effect of yeast extract and calcium source addition.

UA and pH are deemed as the key indicators that reflect the degree of urea hydrolysis and remediation efficiency while introducing the microbial-induced carbonate precipitation (MICP) technology for heavy metal immobilization (Stocks-Fischer et al., 1999; Zhu and Maria, 2016; Omoregie et al., 2017). This section aims to analyze the effect of bacterial culture and calcium source addition on the degree of urea hydrolysis during the MICP process. As shown in Figures 1A–C, UA generally decreases with the increase of Pb(NO₃)₂ concentration. The temporal relationships, when not subjected to yeast extract and CaCl₂ addition, can be characterized as UA ascending initially and descending after reaching the peak (Figure 1A). Furthermore, the temporal relationships of UA are divided into two curve groups; the temporal relationships of UA under the Pb(NO₃)₂ concentration ≤20 mM lie above those under the Pb(NO₃)₂ concentration >20 Mm. When subjected to the effect of yeast extract, the temporal relationships of UA are found to occur in an ascending manner throughout (Figure 1B). The temporal relationships under the effect of yeast extract and CaCl₂ addition can be characterized as UA descending initially and ascending after reaching the minimum value (Figure 1C).

The secretion of urease by the ureolytic bacteria is determined by whether the availability of sufficient nutrients can be assured for bacterial growth and reproduction. Higher concentrations of Pb²⁺ prevent the ureolytic bacteria from secreting and producing enough extracellular polysaccharides (EPS), aggravating the effect of Pb²⁺ and deactivating the ureolytic bacteria (Wu et al., 2019). On the other hand, higher Pb²⁺ concentrations can also cause urease denaturation, leading to its deactivation. These results, in turn, impede the development of UA. The consumption of nutrients over time is the main contributor to the formation of the descending tendency of bacterial activity. For this reason, UA descends slowly after 24 h (Figure 1A). In contrast, the ureolytic bacteria under the effect of yeast extract continuously secrete urease, and therefore, UA increases all along (Figure 1B). Considering CaCl₂ is a calcium source addition and the yeast extract aids the ureolytic bacteria to secrete urease, the negatively charged ureolytic bacteria adsorb Pb²⁺ and Ca²⁺ and precipitate with CO₃ ^2- when using the ureolytic bacteria as nucleation sites (Rui Zhang et al., 2021; Yin et al., 2021). To this end, the ureolytic bacteria settle together with the carbonate precipitation toward the bottom of the test tube, reducing the bacterial concentration in the supernatant and subsequently reducing UA (Figure 1C). Thus, UA shows a decreasing tendency from 0.1 to 4 h . Ca²⁺ form competitive adsorption with Pb²⁺ and cause the ureolytic bacteria to preferentially bind themselves together with Ca²⁺, which also indicates an enhancement of the resistance against Pb²⁺. Given the effect of CaCl₂ addition, the ureolytic bacteria that commence showing their resistance against the effect of Pb²⁺ continue to grow and secrete urease, and therefore, UA increases after reaching the minimum from 4 to 48 h. This shows a difference in the curve slopes from those we have seen in Figure 1B.

The temporal relationships of pH in the mixture against Pb(NO₃)₂ concentrations are shown in Figure 2. They can be characterized with the pH increasing rapidly at the very beginning of the MICP process and then remaining constant or decreasing slightly after reaching the peak. Furthermore, similar to the temporal relationships of UA, pH decreases with the increase of Pb(NO₃)₂ concentration. Moreover, when subjected to no yeast extract and CaCl₂ addition, pH reaches its maximum value being approximately 9.5 at 12 h after the commencement of the MICP process (Figure 2A). pH, when subjected to the effect of yeast extract, reaches its maximum of about 8.5 after 12 h, which is close to that under the effect of yeast extract and CaCl₂ addition (Figures 2B,C). These results can help infer that under the sole effect of yeast extract, a small number of ureolytic bacteria remain active when subjected to lower Pb²⁺ concentrations and the majority of the ureolytic bacteria lose their activity when subjected to higher Pb²⁺ concentrations. The smaller the number of ureolytic bacteria remaining active, the lower the degree of ureo hydrolysis. Therefore, UA distributes in a scattered manner at the end of the process, and due to the lack of NH₄ ⁺ resulting from the reduced degree of urea hydrolysis, pH reduces to 8.5. pH reducing to 8.5 could also be ascribed to bacteria metabolism. Bacteria metabolism consumes carbon from the yeast extract, yielding organic and carbonic acids and lowering pH (Jimenez-Lopez et al., 2008). In contrast, UA is beyond 10 at the end of the process under the effect of yeast extract and CaCl₂ addition. This is because the majority of the ureolytic bacteria commence showing their resistance against the effect of Pb²⁺ (Peng et al., 2021). The higher degree of urea hydrolysis facilitates the formation of carbonate precipitation, which also indicates consumption of CO₃ ²⁻ and further decrease in pH.

Figures 3, 4 show the temporal relationships of UA and pH against Cu(NO₃)₂ concentrations, respectively. UA = 5 mM U min⁻¹ (maximum) under the effect of Cu²⁺ increases to UA = 13 mM U min⁻¹ (maximum) under the effect of Pb²⁺ (Figures 1, 3), indicating the effect of Cu²⁺ on the bacterial activity outweighing the effect of Pb²⁺. For this reason, UA under the effect of Cu²⁺ could be as low as 0.5 mM U min⁻¹ (Figure 3A). The effect of Cu²⁺ depresses the ureolytic bacteria and then UA, as indicated by the descending tendency from 0 to 4 h (Figure 3B). The effect of yeast extract aids the ureolytic bacteria to secrete urease and promotes notably the development of UA. This especially holds true when subjected to lower Cu²⁺ concentrations and is considered the main cause, leading to a discrepancy in the curve slope after 24 h. As indicated by the measurements of UA, when the ureolytic bacteria provide nucleation sites for precipitating CaCO₃, the concentration of the ureolytic bacteria in the supernanant is reduced and the development of UA is depressed, forming the descending tendency from 0 to 4 h (Figure 3C). The addition of CaCl₂ not only promotes the formation of CaCO₃ but also develops resistance of the ureolytic bacteria against the effect of Cu²⁺. Such resistance is especially significant when subjected to lower Cu²⁺ concentrations, which also indicates the main cause leading to the difference of the curve slope after 4 h. On the other hand, the lower the Cu²⁺ concentration, the higher the degree of urea hydrolysis, and the higher the concentration of NH₄ ⁺ and CO₃ ^2-. The higher concentrations of NH₄ ⁺, induced by Cu²⁺ = 5 mM, turn pH surrounding conditions into a strong alkaline environment (i.e., pH = 9), while due to Cu²⁺ = 50 mM, the lower concentrations of NH₄ ⁺ turn pH surrounding conditions to a weak acid environment (i.e., pH below 6) (Figure 4A). The effect of yeast extract differentiates pH under higher Cu²⁺ concentrations from those under lower Cu²⁺ concentrations (Figure 4B). There appear two groups; one is pH above 7 and the other is pH below 6. The effect of yeast extract and CaCl₂ addition, in turn, eases such difference in pH (Figure 4C).

Figures 5, 6 show the variations of NH₄ ⁺ concentration against Pb²⁺ and Cu²⁺ concentrations, respectively, after 0.1, 24, and 48 h. It is evident that when subjected to no yeast extract and calcium source addition, the higher the concentration of Pb²⁺ or Cu²⁺, the lower the degree of urea hydrolysis, and the lower the concentration of NH₄ ⁺ (Figure 5A and Figure 6A). Furthermore, the concentration of NH₄ ⁺ remains nearly unchanged throughout the process, indicating that the majority of the ureolytic bacteria lose their activity when subjected to the effect of Pb²⁺ or Cu²⁺, and this leads to the inability of discharging NH₄ ⁺ and CO₃ ²⁻ while catalyzing urea hydrolysis. Under the effect of yeast extract, the concentration of NH₄ ⁺ shows a significant change with time, indicating a continuous discharge of NH₄ ⁺ and CO₃ ²⁻ (Figures 5B, 6B). This result also indicates that the yeast extract can ease the effect of Pb²⁺ or Cu²⁺ and prevent the ureolytic bacteria from losing their activity. It is worth noting that the more significant change in NH₄ ⁺ concentration under the effect of Pb²⁺ than under the effect of Cu²⁺ provides testimony of the effect of Cu²⁺ outweighing the effect of Pb²⁺. When the ureolytic bacteria, under the effect of yeast extract and CaCl₂ addition, start showing their resistance against the effect of Pb²⁺ or Cu²⁺, the concentration of NH₄ ⁺ after 0.1 h differs notably from that after 24 and 48 h (Figures 5C, 6C); the concentration of NH₄ ⁺, under Pb²⁺ = 5 mM, increases quickly from approximately 110 mM after 0.1 h to about 800 mM after 48 h, while under Cu²⁺ = 5 mM, the concentration of NH₄ ⁺ increases from about 190 mM after 0.1 h to approximately 600 mM after 48 h. Although the addition of CaCl₂ promotes the precipitation of CaCO₃ and depresses the ureolytic bacteria at the very beginning of the process (i.e., after 0.1 h), it enhances the resistance of the ureolytic bacteria against the effect of Pb²⁺ or Cu²⁺ after 24 and 48 h. This is deemed as the main cause leading to the exaggerated difference between the concentration of NH₄ ⁺ after 0.1 h and the concentration of NH₄ ⁺ after 24 and 48 h.

It is widely known that the lower the remaining heavy metal concentration after catalyzing urea hydrolysis and precipitating carbonate, the higher the remediation efficiency. Figure 7 presents the relationships of the remediation efficiency versus Pb(NO₃)₂ concentration after 0.1, 24, and 48 h. The remediation efficiency could be as low as 80% when subjected to no yeast extract and calcium source addition. It is increased to 92% under the effect of yeast extract and further to 100% under the effect of yeast extract and CaCl₂ addition. It is worth noting that Pb²⁺ > 20 mM starts depressing the ureolytic bacteria, and therefore, there appears a sudden decline in the remediation efficiency when subjected to no yeast extract and calcium source addition. In contrast, a similar decline cannot be observed when subjected either to the effect of yeast extract or to the effect of yeast extract and CaCl₂ addition. On the other hand, for a given concentration of Pb(NO₃)₂, the remediation efficiency is increased notably from 0.1 to 24 h, and its increase from 24 to 48 h becomes insignificant as before. These results are especially significant when subjected to no yeast extract and calcium source addition and are in good agreement with the measurements of NH₄ ⁺ concentration, as shown in Figure 6. Figure 8 shows the relationships of the remediation efficiency versus Cu(NO₃)₂ concentration after 0.1, 24, and 48 h. The remediation efficiency of about 20% is measured when Cu(NO₃)₂ concentration = 5 mM, and it sharply increases to higher than 80% when Cu(NO₃)₂ concentration = 10 mM (see Figure 8A). The remediation efficiency continues to increase to as high as 92% when Cu(NO₃)₂ concentration = 30 mM, and then it decreases and remains constant when Cu(NO₃)₂ concentration falls in a 40–50 mM range. Under the effect of yeast extract, the remediation efficiency below 10% is measured when Cu(NO₃)₂ concentration falls in a 5–10 mM range, and it is increased up to approximately 80% when Cu(NO₃)₂ concentration = 50 mM (Figure 8B). The remediation efficiency under the effect of yeast extract and CaCl₂ addition is measured to be below 10% when Cu(NO₃)₂ concentration falls in a 5–30 mM range (Figure 8C). It increases to about 77% when Cu(NO₃)₂ concentration = 40 mM and further to 84% when Cu(NO₃)₂ concentration = 50 mM. These results indicate that the remediation efficiency against Cu²⁺ is not as good as that against Pb²⁺, and its reduction appears to present in some specific ranges of Cu(NO₃)₂ concentration. Further exploration to identify the speciation and sequence of carbonate precipitation is considered of great necessity in order to reveal the mechanisms leading to the reduction in the remediation efficiency. In light of this, biomineralization simulations were conducted in the present work, and the simulated results are presented and analyzed as follows.

Carbonate precipitations present in the test tube experiments were simulated by Visual MINTEQ software toward investigating their speciation and sequence of precipitation. NH₄ ⁺ and CO₃ ^2- concentrations that are extracted upon the completion of urea hydrolysis in the test tube experiments are inputted in the biomineralization simulations. Furthermore, when the effect of yeast extract has already increased the degree of urea hydrolysis, the biomineralization simulations were simplified to those considering the effect of calcium source addition only. Figure 9A shows the relationships of the remediation efficiency versus the hydrolyzed urea concentration against Pb(NO₃)₂ concentrations varying within a 5–50 mM range, when subjected to no calcium source addition, in which the hydrolyzed urea concentration can be equivalent to the concentration of CO₃ ²⁻ (i.e., degree of urea hydrolysis). When the hydrolyzed urea concentration is 500 mM, the degree of urea hydrolysis is 100% (Table 2). In contrast, the degree of urea hydrolysis corresponds to 0% when the hydrolyzed urea concentration is reduced to 0 mM. Given that the concentration of Pb(NO₃)₂ is 10 mM and the speciation of carbonate precipitation is recognized as Pb₃(CO₃)₂(OH)₂, 10 mM CO₃ ²⁻ can precipitate 15 mM Pb²⁺ (= 10 × 3/2), which is in excess of 10 mM Pb²⁺ and also indicates the remediation efficiency being 100% (Figure 9A). When the discharge of CO₃ ^2- does not reach a concentration that is required to precipitate Pb²⁺, the remediation efficiency is degraded where PbCl(OH), (PbCl)₂CO₃, and Pb₃(CO₃)₂(OH)₂ are precipitated (Figure 9A). In contrast, when subjected to the effect of CaCl₂ addition, PbCl₂, PbCl(OH), (PbCl)₂CO₃, and Pb₃(CO₃)₂(OH)₂ are precipitated when a lower concentration of the CO₃ ²⁻ present causes difficulty in precipitating Pb²⁺ (Figure 9B). As the concentration of CO₃ ^2- in the biomineralization process is high enough, the speciation of carbonate precipitation corresponds to PbCO₃ under no calcium source addition and PbCO₃ and CaCO₃ under the effect of CaCl₂ addition (see Eqs 6, 7). These results show that the speciation of carbonate precipitation varies not only with the concentration of CO₃ ^2- but also with the calcium source addition. Figure 9C shows the relationships of the remediation efficiency versus the hydrolyzed urea concentration against Cu(NO₃)₂ concentrations varying in a 5–50 mM range, when subjected to no calcium source addition. They behave in an ascending manner in the first place and then in a descending manner, which differs from what we have seen in Figure 9A. Cu₂Cl(OH)₃ and Cu₃(CO₃)₂(OH)₂ are precipitated when low CO₃ ²⁻ concentration precipitates a small number of Cu²⁺. In contrast, high CO₃ ²⁻ precipitates the majority of Cu²⁺, and Cu₂CO₃(OH)₂ is precipitated. Cu₂Cl(OH)₃, Cu₃(CO₃)₂(OH)₂, and Cu₂CO₃(OH)₂, when under the effect of CaCl₂ addition, are transformed with the increasing concentration of CO₃ ²⁻ to CaCO₃ (Figure 9D). The degradation in the remediation efficiency against Pb²⁺ presents under lower CO₃ ²⁻ concentrations (corresponding to higher Pb²⁺ concentrations), whereas it vanishes or becomes minimal when subjected to higher CO₃ ²⁻ concentrations (corresponding to lower Pb²⁺ concentrations) (Figures 9A,B). However, the degradation in the remediation efficiency against Cu²⁺ presents not only under lower CO₃ ²⁻ concentrations but also under higher ones, indicating that it does not rely only on the concentrations of CO₃ ²⁻ but on other influencing factors. They are worthy of investigation, revealing the mechanism affecting the remediation efficiency against Cu²⁺. As indicated by the measurements of pH, the higher the degree of urea hydrolysis, the closer the pH surrounding conditions to approximately 9, and the more significant the degradation in the remediation efficiency against Cu²⁺ (Figure 4B). In other words, the degradation in the remediation efficiency against Cu²⁺ is also attributed to pH surrounding conditions. However, the degradation in the remediation efficiency, under the effect of CaCl₂ addition, appears not as significant as that under no calcium source addition. The effect of CaCl₂ addition seems to ease the impact of pH surrounding conditions because in most cases, the remediation efficiency of approximately 100% is attained when the hydrolyzed urea concentration falls within a 0–300 mM range (corresponding to Cu²⁺ concentration range of 30–50 mM). This is due to the fact that Ca²⁺ which ionized from CaCl₂ can react with CO₃ ²⁻ to form CaCO₃ precipitation, where CO₃ ²⁻ is obtained by the reaction of CO₂ produced by urea hydrolysis with OH⁻. A large amount of OH⁻ is consumed, reducing the pH value of the solution (see Eqs 1–6). The reducing pH causes a difficulty in promoting the formation of copper–ammonia complexes.

Pb²⁺ or Cu²⁺ can notably affect the ureolytic bacteria and urease activity in the biomineralization process, leading to some difficulty in elevating the degree of urea hydrolysis and attaining a satisfactory remediation efficiency. Generally, the higher the concentration of Pb²⁺ or Cu²⁺, the lower the degree of urea hydrolysis, and lower the remediation efficiency. Furthermore, the effect of Cu²⁺ outweighing the effect of Pb²⁺ explains why the remediation efficiency against Cu²⁺ is much lower than that against Pb²⁺. On the other hand, the effect of yeast extract is proven effective in reducing the effect of Pb²⁺ or Cu²⁺ and causing the ureolytic bacteria to secrete urease for catalyzing urea hydrolysis. The experimental results indicate that the degradation in the remediation efficiency against Cu²⁺ is not only counted upon the degree of urea hydrolysis but also upon pH surrounding conditions. In the present work, a pH of approximately 9 leads to the lowest remediation efficiency. In light of this, attaining satisfactory remediation efficiency relies upon the degree of urea hydrolysis and pH surrounding conditions, while remedying Cu²⁺. When the degree of urea hydrolysis is high enough but the remediation efficiency is not as high as expected, a modification of pH surrounding conditions is considered to be of great necessity. Notwithstanding that, the simulated results indicate that the degradation in the remediation efficiency against Cu²⁺ presents when subjected not only to lower degrees of urea hydrolysis but also to higher degrees of urea hydrolysis. Such a high degree of urea hydrolysis turns pH surrounding conditions into highly alkaline environments, thereby modifying the speciation of carbonate precipitation (i.e. copper–ammonia complexes). The formation of copper–ammonia complexes turns Cu²⁺ into a free state toward degrading the remediation efficiency. The effect of CaCl₂ addition eases the degradation in the remediation efficiency. It is worth noting that there appears a discrepancy in the remediation efficiency between the experimental and simulated results, most likely because of the neglection of dissolution of carbonate precipitation by the biomineralization simulations.

On the whole, attaining a satisfactory remediation efficiency relies generally upon the degree of urea hydrolysis. pH surrounding conditions, while remedying Cu²⁺, can significantly degrade the remediation efficiency. The effect of yeast extract prevents the ureolytic bacteria from losing their activity under heavy metal ion stress. The degradation in the remediation efficiency against Cu²⁺ also presents under higher degrees of urea hydrolysis. This is due to the fact that such a high degree of urea hydrolysis turns pH surrounding conditions into highly alkaline environments, thereby promoting the formation of copper–ammonia complexes and degrading the remediation efficiency. The effect of CaCl₂ addition modifies pH surrounding conditions and further eases the degradation in the remediation efficiency. These results shed light on the importance of modifying pH surrounding conditions in capsulizing Cu²⁺ using the bioinspired calcium carbonate precipitation.