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

applied sciences

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

Gunjo Kim 1, Janghwan Kim 2 ID and Heejung Youn 1,* ID 1 Department of Civil Engineering, Hongik University, Seoul 04066, Korea; [email protected] 2 Civil Design Team, Daelim Industrial Corp. Ltd., Seoul 08826, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-2-320-3071

Received: 24 May 2018; Accepted: 30 July 2018; Published: 1 August 2018

Featured Application: This work can be used to determine the optimal conditions for microbially induced calcite precipitation (MICP).

Abstract: 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.

Keywords: MICP; optimal condition; Staphylococcus saprophyticus; Sporosarcina pasteurii

1. 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 [3–14]. Temperature, pH of the environment, and the reaction duration are some of the major factors influencing calcite precipitation [15–18]. The influence of temperature and pH on MICP is complicated because they affect various processes including microbial activity/growth, urease activity, and the ◦ CaCO3 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

Appl. Sci. 2018, 8, 1277; doi:10.3390/app8081277 www.mdpi.com/journal/applsci Appl. Sci. 2018, 8, 1277 2 of 10 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 CaCO3 solubility, which varies with temperature and pH [25]. The CaCO3 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(NH2)2) is hydrolyzed by the urea + 2− 2− enzyme into ammonium (NH4 ) and carbonate ions (CO3 ). This CO3 is combined with calcium 2+ ions (Ca ) from the supplied medium to form calcium carbonate (CaCO3).

+ 2− CO(NH2)2 + 2H2O → 2NH4 + CO3

The urea enzyme is optimally active at a speciﬁc range of pH; the optimum pH for active enzyme ranges between 7 and 8.0 [9,27–29]. 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 [27,32–34]. 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-CaCl2 medium of different pH and cultured at various temperatures. The effects of the pH of the urea-CaCl2 medium, the temperature during precipitation, and the reaction duration on the amount of calcite precipitated were investigated.

2. Materials and Methods

2.1. 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 (OD620) of S. saprophyticus was much higher than that of S. pasteurii. Appl. Sci. 2018, 8, x FOR PEER REVIEW 3 of 9

2.2. Experimental Procedures Figure 1 shows the experimental process to measure the amount of calcite precipitation. The 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. Appl. Sci.The2018 measurement, 8, 1277 of calcite precipitation was performed according to the Standard Test Method3 of 10 for Rapid Determination of Carbonate Content of Soils [42]. A calibration curve was plotted using 1 N2.2. hydrochloric Experimental acid Procedures (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 reactedFigure with1 HCl,shows and the the experimental carbon dioxide process (CO2) to pressure measure generated the amount by this of calcite reaction precipitation. was measured The to ploturea-CaCl the calibration2 medium curve. was prepared It was assumed using 500 that mL the of pressure distilled of water, CO2 was 1.5 gproportional nutrient broth to the (Difco), amount 5 g ofNH calcite4Cl, and precipitated. 1.06 g NaHCO Using3 (equivalent this method, to 25.2 calcite mM). Theprecipitation initial pH ofwas the mediummeasured was and varied the betweenoptimal ◦ conditions6–10 with hydrochloric for precipitation acid were and buffer determined solution. by Aftervarying autoclaving different parameters at 121 C for like 15 pH, min, temperature, the medium andwas reaction cooled and duration. 14 g CaCl 2·2H2O and 1 g urea were added.

Step 1 Step 2 Step 3

Add microbe 2 mL into Precipitation and Measurement of CaCO3 amount urea-CaCl2 medium 50 mL dry for 2 days using HCl

50 40 60 30 70 20 80 Pressure 10 90 100 gauge Top cover Teflon beaker

Urea-CaCl2 medium Reaction + microbe 2 mL Calcite cylinder precipitation

HCl 20 mL

Figure 1. Experimental process for measuring the calcite precipitation. Figure 1. Experimental process for measuring the calcite precipitation.

The testing conditions included different pHs (6, 7, 8, 9, and 10) of the medium, temperatures (20, 30,The 40, measurement and 50 °C), of and calcite reaction precipitation durations was (2, performed 3, and 4 according days). First, to the the Standard two species Test Method under investigationfor Rapid Determination were activated of Carbonate by culturing Content in 35 ofmL Soils of tryptic [42]. A soy calibration broth (TSB; curve Difco was plottedLaboratories, using Detroit,1 N hydrochloric MI, USA, pH acid 7.2) (80 for mL 2–3 HCl days and at 72028 °C mL (Figure distilled 2a)). water) The microbes and 0.2, were 0.4, 0.6, then 0.8, incubated and 1.0 for g of 3 calcite powder (CaCO : Showa Chemicals Inc. (Tokyo, Japan)). The calcite powder in the reaction days at 30 °C before injecting3 them into the urea‐CaCl2 medium (Figure 2b). For this, 50 mL of urea‐ cylinder reacted with HCl, and the carbon dioxide (CO ) pressure generated by this reaction was CaCl2 medium with a pH ranging from 6–10 was prepared2 in Teflon beakers, and inoculated with 2 mLmeasured of 108–109 to plot CFU/mL the calibration microbes. curve. Then It wasthe beaker assumed was that covered the pressure and placed of CO 2inwas the proportional incubators, temperaturesto the amount of of which calcite were precipitated. set to 20, Using 30, 40, this and method, 50°C. calcite For twenty precipitation different was sets measured of pH and temperatures,the optimal conditions the tests for were precipitation performed were for determineddifferent reaction by varying durations different of parameters2, 3, and like4 days. pH, Subsequently,temperature, and the reactionbeaker containing duration. the medium was oven‐dried at 105 °C for 2 days (Figure 2c). It was placedThe testing in the conditions reaction includedcylinder differentas in Figure pHs 1, (6, and 7, 8, 20 9, mL and of 10) 1 ofN the HCl medium, was poured temperatures into it. The (20, 30, 40, and 50 ◦C), and reaction durations (2, 3, and 4 days). First, the two species under investigation calcite that had precipitated at the bottom of the beaker chemically reacted with HCl, emitting CO2 were activated by culturing in 35 mL of tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA, gas. According to the ASTM D4373 [42], the pressure of the CO2 gas was measured to determine the ◦ ◦ amountpH 7.2) forof calcite 2–3 days precipitation. at 28 C (Figure 2a)). The microbes were then incubated for 3 days at 30 C before injecting them into the urea-CaCl2 medium (Figure2b). 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 (Figure2c). It was placed in the reaction cylinder as in Figure1, 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. Appl. Sci. 2018, 8, 1277 4 of 10 Appl. Sci. 2018, 8, x FOR PEER REVIEW 4 of 9

(a) (b) (c)

Figure 2. Testing procedure: (a) insert strain into 35 mL TSB; (b) urea‐CaCl2 medium and microbes; Figure 2. Testing procedure: (a) insert strain into 35 mL TSB; (b) urea-CaCl2 medium and microbes; and (c) oven‐dried sample. and (c) oven-dried sample.

3. Test Results 3. Test Results Sixty different combinations of pH, temperature, and reaction duration were used for each 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 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 test results are tabulated in Table1. Figure3 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 precipitates for S. saprophyticus and S. pasteurii. It is clear that under all the different testing conditions, conditions, the S. saprophyticus precipitates considerably more than S. pasteurii. The amount of the S. saprophyticus precipitates considerably more than S. pasteurii. The amount of precipitation precipitation is approximately five times more in the majority of conditions used for testing in S. is approximately five times more in the majority of conditions used for testing in S. saprophyticus. saprophyticus. Although the amount of precipitation significantly differs between the two microbes, Although the amount of precipitation significantly differs between the two microbes, the optimal the optimal condition for maximum precipitation was identical. Maximum precipitation was condition for maximum precipitation was identical. Maximum precipitation was observed at a observed at a temperature of 30 °C and a pH 7 of the urea‐CaCl2 medium. At higher temperatures, temperature of 30 ◦C and a pH 7 of the urea-CaCl medium. At higher temperatures, the precipitation the precipitation significantly dropped for both 2microbes. Figure 4 shows calcite precipitation at a significantly dropped for both microbes. Figure4 shows calcite precipitation at a specific condition specific condition relative to that of the optimal condition at reaction duration of 3 days. Both S. relative to that of the optimal condition at reaction duration of 3 days. Both S. saprophyticus and saprophyticus and S. pasteurii were found to be very sensitive to the change in temperature during S. pasteurii were found to be very sensitive to the change in temperature during precipitation, showing precipitation, showing a considerable drop of approximately 60%. On the other hand, the effect of a considerable drop of approximately 60%. On the other hand, the effect of pH was found to be pH was found to be different between the microbes. The productivity of S. saprophyticus is less different between the microbes. The productivity of S. saprophyticus is less influenced by a change in influenced by a change in pH, while that of S. pasteurii is strongly influenced. The difference in the pH, while that of S. pasteurii is strongly influenced. The difference in the precipitation was at the most precipitation was at the most around 25% for S. saprophyticus (see Figure 4c) while it was around 60% around 25% for S. saprophyticus (see Figure4c) while it was around 60% for S. pasteurii (see Figure4d). for S. pasteurii (see Figure 4d). It should be noted that the measurement tolerance is 0.0018 g, which It should be noted that the measurement tolerance is 0.0018 g, which is about 1.2% and 4.5% of the is about 1.2% and 4.5% of the maximum calcite precipitation of 3 days for S. saprophyticus and S. maximum calcite precipitation of 3 days for S. saprophyticus and S. pasteurii, respectively. pasteurii, respectively. TheTable calcite 1. The precipitations estimated calcite for precipitation both the species at different appear pHs, to temperatures, reach a maximum and reaction value durations. 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 Amountduration of Calcite was changed Precipitation from (g) 2 to 3 days. An increase of Reaction Staphylococcus saprophyticus Sporosarcina pasteurii 14% wasDuration seen when optimal pH (7) and temperature (30 °C) were used. However, precipitation pH (Day) 6 7 8 9 10 6 7 8 9 10 significantly increasedTemp. for S. pasteurii, with an average increase of 37% and 22% for the optimal condition, indicating20 that 0.1102 reaction 0.1193 occurred 0.1065 more 0.1010 slowly 0.0991 for 0.0184 S. pasteurii 0.0220 0.0147 than for 0.0147 S. saprophyticus 0.0147 . 30 0.1193 0.1285 0.1230 0.1157 0.1120 0.0220 0.0330 0.0220 0.0165 0.0165 Although S. 2pasteurii reaches exponential phase of growth within 12 hours, the increase in the calcite 40 0.0918 0.1010 0.0918 0.0863 0.0826 0.0129 0.0202 0.0147 0.0092 0.0092 precipitation was measured50 0.0551 at a reaction 0.0661 0.0569 duration 0.0496 of 2 0.0477 days because 0.0110 0.0165 cell viability 0.0129 0.0092is not solely 0.0055 related with urease activity 20and calcite 0.1138 precipitation 0.1230 0.1120. In 0.1065 fact, 0.1010urease 0.0220and its 0.0275 activity 0.0239 might 0.0184 not be 0.0184 associated 30 0.1432 0.1469 0.1285 0.1285 0.1249 0.0330 0.0404 0.0312 0.0220 0.0220 3 with an active microbial40 population 0.1010 0.1138 (cell 0.1010 viability) 0.0955 since 0.0918 there 0.0202 was no 0.0294 significant 0.0184 correlation 0.0147 0.0129 between urease activity and respiration50 0.0643 in 0.0734 soil bacteria 0.0606 0.0551 [43]. This 0.0551 is because 0.0165 0.0239 urease 0.0147 is a cytoplasmic 0.0092 0.0092 protein [30,44]. For example,20 cell fractionation 0.1120 0.1249 studies 0.1102 0.1065generally 0.1065 demonstrate 0.0239 0.0294 that 0.0239urease 0.0184 partitions 0.0184 with the 30 0.1322 0.1432 0.1285 0.1230 0.1193 0.0312 0.0422 0.0294 0.0239 0.0220 4 cytoplasmic fraction 40[44]. Thus, 0.1047 the 0.1102 urease 0.1010 enzyme 0.0955 activity 0.0973 could 0.0184 increase 0.0312 after 0.0202 cell 0.0147 lysis, 0.0147which might result in an increase 50in calcite 0.0643 precipitation 0.0753 0.0588 even 0.0569 after 0.0551 2 days. 0.0165 0.0220 0.0129 0.0129 0.0092 Appl. Sci. 2018, 8, 1277 5 of 10

0.20 0.20 (a) 2 days (d) 2 days pH 6 pH 6 ) S. saprophyticus S. pasteurii

g pH 7 pH 7

(

3 pH 8 pH 8 0.15 0.15

O pH 9 pH 9 C

a pH 10 pH 10

t 0.10 0.10

m A 0.05 0.05

0.00 0.00 20 30 40 50 20 30 40 50

0.20 0.20 (b) 3 days (e) 3 days pH 6 pH 6

) S. saprophyticus pH 7 S. pasteurii

g pH 7 ( pH 8 3 pH 8 0.15 0.15 O pH 9 pH 9

C pH 10 pH 10

t 0.10 0.10

o m A 0.05 0.05

0.00 0.00 20 30 40 50 20 30 40 50

0.20 0.20 (c) 4 days (f) 4 days pH 6 pH 6 S. saprophyticus pH 7 S. pasteurii pH 7 ) pH 8 g pH 8 0.15 ( 0.15 pH 9 3 pH 9

O pH 10 pH 10

f 0.10 0.10

u o

m 0.05 0.05 A

0.00 0.00 20 30 40 50 20 30 40 50 Temperature during precipitation (°C) Temperature during precipitation (°C)

FigureFigure 3. Effect 3. Effect of temperature of temperature on onprecipitation precipitation..

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

Appl. Sci. 2018, 8, x; doi: FOR PEER REVIEW www.mdpi.com/journal/applsci Appl. Sci. 2018, 8, 1277 6 of 10

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 Appl.precipitation Sci. 2018, 8, x FOR even PEER after REVIEW 2 days. 2 of 2

1.0 1.0 (a) (b)

n 0.8 0.8 o i t a t i

p 0.6

i 0.6 c e r p

e 0.4 0.4 v i

t S. saprophyticus, pH=6 S. pasteurii, pH=6 a l S. saprophyticus, pH=7 S. pasteurii, pH=7 e S. saprophyticus, pH=8 S. pasteurii, pH=8 R 0.2 0.2 S. saprophyticus, pH=9 S. pasteurii, pH=9 S. saprophyticus, pH=10 S. pasteurii, pH=10 0.0 0.0 20 30 40 50 20 30 40 50 Temperature during precipitation (oC) Temperature during precipitation (oC)

1.0 1.0 (c) (d)

n 0.8 0.8 o i t a t i p

i 0.6 0.6 c e r p

e 0.4 0.4 v i t a l

e S. saprophyticus, T=20°C S. pasteurii, T=20°C

R 0.2 S. saprophyticus, T=30°C S. pasteurii, T=30°C 0.2 S. saprophyticus, T=40°C S. pasteurii, T=40°C S. saprophyticus, T=50°C S. pasteurii, T=50°C 0.0 0.0 6 7 8 9 10 6 7 8 9 10 pH of medium pH of medium

FigureFigure 4. 4.RelativeRelative calcite calcite precipitation precipitation of the of the two two microbial microbial species species at a at reaction a reaction duration duration of 3 of days: 3 days: ◦ (a,(ba), bthe) the precipitation precipitation relative relative to to that that at at a atemperature temperature of of 3 300 °CC;; (c (c,d,d) )the the precipitation precipitation relative relative to to that that at at a pH of 7 7..

9.5 Previous research along with this study reveal that80 the optimum pH and temperature for urease

activity, calcite precipitation, and bacterialWhiffingrowth 2004 are) the values as shown in Figure5[ 19–22,27,29,31,

9.0 C 70 Whiffin 2004 o

S. pasteurii (

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r Sahrawat 1984 8.5 * Negligible effect 60

difference reported by Lauchnor et al. [45]. On the otheru hand, the pH required for optimum bacterial t

H Liang et al. 2005

p r growth was nearly 9. Calcite precipitation is a complex process resulting from various factors, with the Arunachalam et al. 2010 e

m 8.0 50 Nemati & Voordouw 2003 p Dhami 2014 u * B. sphaericus

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* B. megaterium i t

Whiffin 2004 p environment. Since CaCO3 Tsolubilityhis study decreases with increasingNemati pH,et al. 2 calcite005 T precipitationhis study is likely to 6.5 Balan et al. 2012 * S. pasteurii O 20 * S. pasteurii increase* withKlebsei increasingla spp. pH.* S However,. saprophyticu thes pH was measured to be optimum* S at. sa 7pro becausephyticus increasing the6.0 pH makes the medium alkaline during ureolysis.10 The optimum temperature for urease activity varies fromU 30rea tose 70 ◦CC foralcitS.e pasteuriiBacter.ia Previousl research has shownUrease thatC thealcit optimume Bacte temperaturerial precipitation activity precipitation growth for calcite precipitationactivity is 50 ◦C[22gro],w whichth 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 optimumFigure 5. temperature Optimal pH and was temperature not thoroughly for urease investigated. activity, calcite precipitation, and bacterial growth. Appl. Sci. 2018, 8, x FOR PEER REVIEW 2 of 2

1.0 1.0 (a) (b)

n 0.8 0.8 o i t a t i

p 0.6

i 0.6 c e r p

e 0.4 0.4 v i

1.0 1.0 (c) (d)

n 0.8 0.8 o i t a t i p

i 0.6 0.6 c e r p

e 0.4 0.4 v i t a l

e S. saprophyticus, T=20°C S. pasteurii, T=20°C

Figure 4. 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 Appl. Sci. 2018, 8, 1277 7 of 10 at a pH of 7.

9.5 80

Whiffin 2004 )

9.0 C 70 Whiffin 2004

o (

Lauchnor et al. 2015 Bang et al. 2001 e

r Sahrawat 1984

8.5 * Negligible effect 60

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Whiffin 2004 p This study Nemati et al. 2005 This study 6.5 Balan et al. 2012 * S. pasteurii O 20 * S. pasteurii * Klebseila spp. * S. saprophyticus * S. saprophyticus 6.0 10 Urease Calcite Bacterial Urease Calcite Bacterial activity precipitation growth activity precipitation growth Figure 5. Optimal pH and temperature for urease activity, calcite precipitation, and bacterial growth. Figure 5. Optimal pH and temperature for urease activity, calcite precipitation, and bacterial growth.

4. 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 CO2 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-CaCl2 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-CaCl2 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.

Author Contributions: G.K. performed the laboratory experiments; J.K. analyzed testing results; H.Y. designed the study and prepared for the manuscript. Funding: National Research Foundation of Korea: 2016R1C1B2013478. Acknowledgments: This work was supported by National Research Foundation of Korea (NRF) funded by Ministry of Science, ICT & Future Planning (NRF-2016R1C1B2013478), and by 2017 Hongik University Research Fund. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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