Effect of Temperature, pH, and Reaction Duration on Microbially Induced Calcite Precipitation

In this study, the amount of calcite precipitate resulting from microbially induced calcite precipitation (MICP) was estimated in order to determine the optimal conditions for precipitation. Two microbial species (Staphylococcus saprophyticus and Sporosarcina pasteurii) were tested by varying certain parameters such as (1) initial potential of hydrogen (pH) of urea-CaCl2 medium, (2) temperature during precipitation, and (3) the reaction duration. The pH values used for testing were 6, 7, 8, 9, and 10, the temperatures were 20, 30, 40, and 50 °C, and the reaction durations were 2, 3, and 4 days. Maximum calcite precipitation was observed at a pH of 7 and temperature of 30 °C. Most of the precipitation occurred within a reaction duration of 3 days. Under similar conditions, the amount of calcite precipitated by S. saprophyticus was estimated to be five times more than that by S. pasteurii. Both the species were sensitive to temperature; however, S. saprophyticus was less sensitive to pH and required a shorter reaction duration than S. pasteurii.


Introduction
Microbially induced calcite precipitation (MICP) is a bio-grouting method that can improve geotechnical properties by precipitating calcite using microbes. MICP is considered an eco-friendly technology used in the field of soil improvement as it involves biological treatment rather than mechanical or chemical ones. However, this technology is not completely applicable to geotechnical practice because the field condition is not favorable for cultivating microbes. Furthermore, injecting nutrients and medium, and utilizing microbes requires a high level of skilled manpower and involves high costs. Nevertheless, the technology looks promising with the researchers solving some of the problems that they have encountered. Microbial activity and reproduction rate are governed by many factors including availability of nutrients, water, or other environmental factors. The environmental factors consist of pH, redox potential, temperature, presence of predatory microorganisms, which may limit bacterial population, and space limitations [1,2]. These factors have been studied extensively to determine the optimum environmental conditions for soil improvement and to verify the applicability of the technique in the field of geotechnical engineering [3][4][5][6][7][8][9][10][11][12][13][14].
Temperature, pH of the environment, and the reaction duration are some of the major factors influencing calcite precipitation [15][16][17][18]. The influence of temperature and pH on MICP is complicated because they affect various processes including microbial activity/growth, urease activity, and the CaCO 3 solubility. Urease was found to be active at temperatures ranging from 10-60 • C; the activity usually peaked at a temperature around 60 • C [9,19,20]. However, precipitation was not found to be at its peak at this temperature. It was reported that the microbial activity and growth was slightly higher at 30 • C than at 20 • C [21]. Likewise, an increased calcite production was reported when the temperature was increased from 20 to 50 • C, while there was no precipitation at a temperature of 60 • C or higher [22,23]. Some researchers indicate that urease activity does not have a strong correlation with the engineering properties of MICP treated soil. For instance, the strength of MICP treated coarse sand was found to be higher when soil improvement was performed at a relatively lower temperature of 20 • C [15]. Interestingly, even with the same amount of calcium carbonate precipitated in soil, the strength of MICP treated soil would differ. Cheng et al. (2016) measured the unconfined compressive strength of the soil at temperatures of 4, 25, and 50 • C, and reported that soil improvement was most effective at 25 • C for the same calcium carbonate content in the treated soil [12]. Moreover, more calcite precipitation does not necessarily result in higher strength if the precipitation occurs at a different temperature [24]. This is likely due to the crystal size resulting from a change in CaCO 3 solubility, which varies with temperature and pH [25]. The CaCO 3 solubility decreases with an increase in temperature and pH, thus affecting calcite precipitation.
Another factor that highly influences MICP is the pH, with an alkaline environment known to be favorable to the process [24,26]. Through hydrolysis, urea (CO(NH 2 ) 2 ) is hydrolyzed by the urea enzyme into ammonium (NH 4 + ) and carbonate ions (CO 3 2−  The urea enzyme is optimally active at a specific range of pH; the optimum pH for active enzyme ranges between 7 and 8.0 [9,[27][28][29]. Its activity peaked at a pH close to 8.0 and gradually decreased at a pH of 8 or higher. It was reported that the optimum pH for the action of most of the microbial ureases is around 7 [30], while that for Sporosarcina pasteurii, which is the most common microbe for MICP, is 8 [27,31]; and the precipitation amount increased with pH and converged to a certain value at a pH range of 8.7 to 9.5 [27,[32][33][34]. Other microbes such as Bacillus megaterium and Bacillus sphaericus have an optimum pH value of 7 and 8, respectively [29,35]. Although an optimal pH has been reported for various microbial species, the initial pH of the medium increased during precipitation, thus changing the environment for optimum precipitation. The increase in pH was due to the hydroxide ions (OH − ) generated during ureolysis, which changed the pH of the soil to alkaline [36,37]. When a source of ammonia was added, a temporal increase in pH was found in slightly acidic soil, but the difference was insignificant in slightly alkaline soil [38].
In this study, Staphylococcus saprophyticus, the microbe isolated from calcareous beach sand, and S. pasteurii, (ATCC 11859), the most commonly used microbe in MICP, were used to determine the optimal conditions for calcite precipitation. Each microbe was injected into the urea-CaCl 2 medium of different pH and cultured at various temperatures. The effects of the pH of the urea-CaCl 2 medium, the temperature during precipitation, and the reaction duration on the amount of calcite precipitated were investigated.

Microorganisms
One of the microbes studied was S. saprophyticus, which was isolated from the calcareous sand of Daechon beach located at the western coast of South Korea. The other microbe was S. pasteurii (ATCC 11859), which was purchased from the Korean Collection for Type Cultures in South Korea. S. pasteurii has been extensively used with great success in MICP research [16,39,40]. On the other hand, MICP using S. saprophyticus has rarely been reported, even though its urease activity was estimated to be higher than that of S. pasteurii in Reference [41]. In the aforementioned study, the urease activities of both the microbial species were measured using the phenol-hypochlorite urease assay, and the optical density at a wavelength of 620 nm (OD 620 ) of S. saprophyticus was much higher than that of S. pasteurii. Figure 1 shows the experimental process to measure the amount of calcite precipitation. The urea-CaCl 2 medium was prepared using 500 mL of distilled water, 1.5 g nutrient broth (Difco), 5 g NH 4 Cl, and 1.06 g NaHCO 3 (equivalent to 25.2 mM). The initial pH of the medium was varied between 6-10 with hydrochloric acid and buffer solution. After autoclaving at 121 • C for 15 min, the medium was cooled and 14 g CaCl 2 ·2H 2 O and 1 g urea were added. urea-CaCl2 medium was prepared using 500 mL of distilled water, 1.5 g nutrient broth (Difco), 5 g NH4Cl, and 1.06 g NaHCO3 (equivalent to 25.2 mM). The initial pH of the medium was varied between 6-10 with hydrochloric acid and buffer solution. After autoclaving at 121 °C for 15 min, the medium was cooled and 14 g CaCl2•2H2O and 1 g urea were added.

Experimental Procedures
The measurement of calcite precipitation was performed according to the Standard Test Method for Rapid Determination of Carbonate Content of Soils [42]. A calibration curve was plotted using 1 N hydrochloric acid (80 mL HCl and 720 mL distilled water) and 0.2, 0.4, 0.6, 0.8, and 1.0 g of calcite powder (CaCO3: Showa Chemicals Inc. (Tokyo, Japan)). The calcite powder in the reaction cylinder reacted with HCl, and the carbon dioxide (CO2) pressure generated by this reaction was measured to plot the calibration curve. It was assumed that the pressure of CO2 was proportional to the amount of calcite precipitated. Using this method, calcite precipitation was measured and the optimal conditions for precipitation were determined by varying different parameters like pH, temperature, and reaction duration.
Step 1 Add microbe 2 mL into urea-CaCl 2 medium 50 mL Step 2 Precipitation and dry for 2 days Step 3 Measurement of CaCO 3 amount using HCl  The testing conditions included different pHs (6,7,8,9, and 10) of the medium, temperatures (20,30,40, and 50 °C), and reaction durations (2, 3, and 4 days). First, the two species under investigation were activated by culturing in 35 mL of tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA, pH 7.2) for 2-3 days at 28 °C (Figure 2a)). The microbes were then incubated for 3 days at 30 °C before injecting them into the urea-CaCl2 medium ( Figure 2b). For this, 50 mL of urea-CaCl2 medium with a pH ranging from 6-10 was prepared in Teflon beakers, and inoculated with 2 mL of 108-109 CFU/mL microbes. Then the beaker was covered and placed in the incubators, temperatures of which were set to 20, 30, 40, and 50°C. For twenty different sets of pH and temperatures, the tests were performed for different reaction durations of 2, 3, and 4 days. Subsequently, the beaker containing the medium was oven-dried at 105 °C for 2 days (Figure 2c). It was placed in the reaction cylinder as in Figure 1, and 20 mL of 1 N HCl was poured into it. The calcite that had precipitated at the bottom of the beaker chemically reacted with HCl, emitting CO2 gas. According to the ASTM D4373 [42], the pressure of the CO2 gas was measured to determine the amount of calcite precipitation. The measurement of calcite precipitation was performed according to the Standard Test Method for Rapid Determination of Carbonate Content of Soils [42]. A calibration curve was plotted using 1 N hydrochloric acid (80 mL HCl and 720 mL distilled water) and 0.2, 0.4, 0.6, 0.8, and 1.0 g of calcite powder (CaCO 3 : Showa Chemicals Inc. (Tokyo, Japan)). The calcite powder in the reaction cylinder reacted with HCl, and the carbon dioxide (CO 2 ) pressure generated by this reaction was measured to plot the calibration curve. It was assumed that the pressure of CO 2 was proportional to the amount of calcite precipitated. Using this method, calcite precipitation was measured and the optimal conditions for precipitation were determined by varying different parameters like pH, temperature, and reaction duration.
The testing conditions included different pHs (6,7,8,9, and 10) of the medium, temperatures (20, 30, 40, and 50 • C), and reaction durations (2, 3, and 4 days). First, the two species under investigation were activated by culturing in 35 mL of tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA, pH 7.2) for 2-3 days at 28 • C (Figure 2a)). The microbes were then incubated for 3 days at 30 • C before injecting them into the urea-CaCl 2 medium (Figure 2b). For this, 50 mL of urea-CaCl 2 medium with a pH ranging from 6-10 was prepared in Teflon beakers, and inoculated with 2 mL of 108-109 CFU/mL microbes. Then the beaker was covered and placed in the incubators, temperatures of which were set to 20, 30, 40, and 50 • C. For twenty different sets of pH and temperatures, the tests were performed for different reaction durations of 2, 3, and 4 days. Subsequently, the beaker containing the medium was oven-dried at 105 • C for 2 days (Figure 2c). It was placed in the reaction cylinder as in Figure 1, and 20 mL of 1 N HCl was poured into it. The calcite that had precipitated at the bottom of the beaker chemically reacted with HCl, emitting CO 2 gas. According to the ASTM D4373 [42], the pressure of the CO 2 gas was measured to determine the amount of calcite precipitation.

Test Results
Sixty different combinations of pH, temperature, and reaction duration were used for each microbial species, and the amount of calcite precipitation was measured for each combination. The test results are tabulated in Table 1. Figure 3 shows the effect of temperature on the amount of calcite precipitates for S. saprophyticus and S. pasteurii. It is clear that under all the different testing conditions, the S. saprophyticus precipitates considerably more than S. pasteurii. The amount of precipitation is approximately five times more in the majority of conditions used for testing in S. saprophyticus. Although the amount of precipitation significantly differs between the two microbes, the optimal condition for maximum precipitation was identical. Maximum precipitation was observed at a temperature of 30 °C and a pH 7 of the urea-CaCl2 medium. At higher temperatures, the precipitation significantly dropped for both microbes. Figure 4 shows calcite precipitation at a specific condition relative to that of the optimal condition at reaction duration of 3 days. Both S. saprophyticus and S. pasteurii were found to be very sensitive to the change in temperature during precipitation, showing a considerable drop of approximately 60%. On the other hand, the effect of pH was found to be different between the microbes. The productivity of S. saprophyticus is less influenced by a change in pH, while that of S. pasteurii is strongly influenced. The difference in the precipitation was at the most around 25% for S. saprophyticus (see Figure 4c) while it was around 60% for S. pasteurii (see Figure 4d). It should be noted that the measurement tolerance is 0.0018 g, which is about 1.2% and 4.5% of the maximum calcite precipitation of 3 days for S. saprophyticus and S. pasteurii, respectively.
The calcite precipitations for both the species appear to reach a maximum value at a reaction duration of 3 days under most of the tested conditions. An average increase of about 10% was observed in S. saprophyticus when the reaction duration was changed from 2 to 3 days. An increase of 14% was seen when optimal pH (7) and temperature (30 °C) were used. However, precipitation significantly increased for S. pasteurii, with an average increase of 37% and 22% for the optimal condition, indicating that reaction occurred more slowly for S. pasteurii than for S. saprophyticus. Although S. pasteurii reaches exponential phase of growth within 12 hours, the increase in the calcite precipitation was measured at a reaction duration of 2 days because cell viability is not solely related with urease activity and calcite precipitation. In fact, urease and its activity might not be associated with an active microbial population (cell viability) since there was no significant correlation between urease activity and respiration in soil bacteria [43]. This is because urease is a cytoplasmic protein [30,44]. For example, cell fractionation studies generally demonstrate that urease partitions with the cytoplasmic fraction [44]. Thus, the urease enzyme activity could increase after cell lysis, which might result in an increase in calcite precipitation even after 2 days.

Test Results
Sixty different combinations of pH, temperature, and reaction duration were used for each microbial species, and the amount of calcite precipitation was measured for each combination. The test results are tabulated in Table 1. Figure 3 shows the effect of temperature on the amount of calcite precipitates for S. saprophyticus and S. pasteurii. It is clear that under all the different testing conditions, the S. saprophyticus precipitates considerably more than S. pasteurii. The amount of precipitation is approximately five times more in the majority of conditions used for testing in S. saprophyticus. Although the amount of precipitation significantly differs between the two microbes, the optimal condition for maximum precipitation was identical. Maximum precipitation was observed at a temperature of 30 • C and a pH 7 of the urea-CaCl 2 medium. At higher temperatures, the precipitation significantly dropped for both microbes. Figure 4 shows calcite precipitation at a specific condition relative to that of the optimal condition at reaction duration of 3 days. Both S. saprophyticus and S. pasteurii were found to be very sensitive to the change in temperature during precipitation, showing a considerable drop of approximately 60%. On the other hand, the effect of pH was found to be different between the microbes. The productivity of S. saprophyticus is less influenced by a change in pH, while that of S. pasteurii is strongly influenced. The difference in the precipitation was at the most around 25% for S. saprophyticus (see Figure 4c) while it was around 60% for S. pasteurii (see Figure 4d). It should be noted that the measurement tolerance is 0.0018 g, which is about 1.2% and 4.5% of the maximum calcite precipitation of 3 days for S. saprophyticus and S. pasteurii, respectively.

Staphylococcus saprophyticus
Sporosarcina pasteurii pH Temp.    The calcite precipitations for both the species appear to reach a maximum value at a reaction duration of 3 days under most of the tested conditions. An average increase of about 10% was observed in S. saprophyticus when the reaction duration was changed from 2 to 3 days. An increase of 14% was seen when optimal pH (7) and temperature (30 • C) were used. However, precipitation significantly increased for S. pasteurii, with an average increase of 37% and 22% for the optimal condition, indicating that reaction occurred more slowly for S. pasteurii than for S. saprophyticus. Although S. pasteurii reaches exponential phase of growth within 12 hours, the increase in the calcite precipitation was measured at a reaction duration of 2 days because cell viability is not solely related with urease activity and calcite precipitation. In fact, urease and its activity might not be associated with an active microbial population (cell viability) since there was no significant correlation between urease activity and respiration in soil bacteria [43]. This is because urease is a cytoplasmic protein [30,44]. For example, cell fractionation studies generally demonstrate that urease partitions with the cytoplasmic fraction [44]. Thus, the urease enzyme activity could increase after cell lysis, which might result in an increase in calcite precipitation even after 2 days.   Previous research along with this study reveal that the optimum pH and temperature for urease activity, calcite precipitation, and bacterial growth are the values as shown in Figure 5 [19][20][21][22]27,29,31,35,[45][46][47][48][49][50]. The optimum pH of S. pasteurii for urease activity ranges widely from 7 to 9, with a negligible difference reported by Lauchnor et al. [45]. On the other hand, the pH required for optimum bacterial growth was nearly 9. Calcite precipitation is a complex process resulting from various factors, with the optimum pH not coinciding with either urease activity or bacterial growth. For the two microbes used in this study, the optimum pH for calcite precipitation was 7 (neutral), which is same for B. megaterium and lower than B. sphaericus. Urease activity and bacterial growth tend to favor a highly alkaline environment. Since CaCO 3 solubility decreases with increasing pH, calcite precipitation is likely to increase with increasing pH. However, the pH was measured to be optimum at 7 because increasing the pH makes the medium alkaline during ureolysis. The optimum temperature for urease activity varies from 30 to 70 • C for S. pasteurii. Previous research has shown that the optimum temperature for calcite precipitation is 50 • C [22], which is higher than 30 • C obtained from this study. However, it should be noted that the previous research adopted only two temperatures (22 and 50 • C), so the optimum temperature was not thoroughly investigated. . Relative calcite precipitation of the two microbial species at a reaction duration of 3 days: (a,b) the precipitation relative to that at a temperature of 30 °C ; (c,d) the precipitation relative to that at a pH of 7.

Conclusions
In this study, the effects of temperature, pH of medium, and the reaction duration on calcite precipitation were evaluated for two microbial species (S. saprophyticus and S. pasteurii). The amount of precipitated calcite was quantified indirectly by measuring the pressure of CO 2 gas, which is generated as a result of the reaction between calcite and HCl. The following conclusions were drawn from the test results: 1.
The optimal conditions for calcite precipitation for both the species were identical: the optimum pH of the urea-CaCl 2 medium was 7 and the optimum temperature during precipitation was 30 • C. The precipitation of S. saprophyticus was five times more than that of S. pasteurii under identical conditions. 2.
Both microbial species were strongly influenced by the temperature of the environment during precipitation, showing a significant decrease with varying temperatures. The least precipitation was measured at a temperature of 50 • C, which was as little as 30-40% of that seen at the optimal temperature (30 • C).

3.
S. saprophyticus was found to be less sensitive to a change in pH of the urea-CaCl 2 medium than S. pasteurii; the decrease in precipitation by varying the pH was less than 20% for S. saprophyticus. On the other hand, pH significantly affected the precipitation of S. pasteurii, reducing it by 60%. 4.
For S. saprophyticus, a meaningful increase in precipitation was observed between the reaction durations of 2 and 3 days, but little difference was measured for longer durations, indicating that maximum precipitation occurred within 3 days. Likewise, most of the precipitation occurred within 3 days for S. pasteurii.