Growth environment optimization for inducing bacterial mineralization and its application in concrete healing

Construction and Building Materials 209 (2019) 631–643

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Growth environment optimization for inducing bacterial mineralization and its application in concrete healing Mingyue Wu a, Xiangming Hu a,b,⇑, Qian Zhang a, Di Xue a, Yanyun Zhao c a Key Lab of Mine Disaster Prevention and Control, College of Mining and Safety Engineering, Shandong University of Science and Technology, No. 579, Qianwangang Road, Xin’an Street, Huangdao District, Qingdao, Shandong, China b Department of Chemical Engineering and Safety, Binzhou University, Yellow River 5 Road, Pengli Street, Bincheng District, Binzhou City, Shandong, China c College of Chemical and Environmental Engineering, Shandong University of Science and Technology, No. 579, Qianwangang Road, Xin’an Street, Huangdao District, Qingdao, Shandong, China

h i g h l i g h t s
Bacterial treatment reduced the water absorption and chloride permeability of concrete. 2+


B. cereus CS1 tolerates high pH, Ca

and mineralizes CaCO3; it may heal concrete.


The first study of B. cereus CS1 used for concrete crack healing.

a r t i c l e

i n f o

Article history: Received 19 October 2018 Received in revised form 3 March 2019 Accepted 13 March 2019

Keywords: Microbially induced carbonate precipitation Alkali tolerance Bacillus cereus Urea decomposition Crack healing

a b s t r a c t Bacterial-based healing is a promising solution for sustainable concrete maintenance. The purpose of this research was to heal the concrete cracks with Bacillus cereus as a healing agent. The bacteria strain was a dominant strain isolated from carbide slag. The effects of different environments on the growth and urease activities of bacteria were studied first. Secondly, healing capacity was evaluated by water absorption, water permeability, rapid chloride permeability test (RCPT) and optical photographs of cracks, and bacterial mineralization products were analyzed by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), and Differential Scanning Calorimetry (DSC). The results indicated that the B. cereus is alkali tolerant, and grows well at pH 7–10. The optimum concentrations of Ca2+ and urea are 0.9 mol/L and 0.75 mol/L, respectively. The minimum amount of yeast extract required for mineralization is 7 g/L. The water absorption and chloride permeability rate of the samples incubated in the bacterial liquid can be reduced by 12.0% and 10.9% and it can heal 100– 800 lm cracks when incubation for 28 d. The water permeability of the healed specimens decreased about two orders of magnitude. Based on phase analysis, the product of microbial mineralization was confirmed to be calcite CaCO3. The mineralized product has a good compatibility with concrete and can prevent further deterioration of concrete. Ó 2019 Elsevier Ltd. All rights reserved.

1. Introduction Owing to high compressive strength, low cost, good durability, and ease of pouring, concrete has become one of the most commonly used building materials, and plays an indispensable role in many fields [1]. However, concrete structures are often subject to erosion by physical, chemical, and biological agents that cause ⇑ Corresponding author at: Key Lab of Mine Disaster Prevention and Control, College of Mining and Safety Engineering, Shandong University of Science and Technology, No. 579, Qianwangang Road, Xin’an Street, Huangdao District, Qingdao, Shandong, China. E-mail address: [email protected] (X. Hu). https://doi.org/10.1016/j.conbuildmat.2019.03.181 0950-0618/Ó 2019 Elsevier Ltd. All rights reserved.

microcracks and thereby reduce mechanical strength and structural performance [2]. Although microcracks do not initially reduce strength, such cracks will eventually and severely compromise service life [3–6]. The direct cost of crack repair and maintenance is as high as several billion USD each year, while indirect maintenance costs are probably higher but more difficult to estimate [7]. Therefore, it is critical to prevent early cracks from continuous expanding. Self-repair techniques, in which a healing agent added during concrete preparation is released when cracks develop, have attracted increasing research attention as an alternative to traditional methods. Accordingly, various types of crack sealing

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treatments such as the manual application of chemical [8–10], polymeric sealers [11,12], and microbially induced carbonate precipitation (MICP) [13–16,19] have been introduced. Of these, MICP have attracted much attention due to environmental friendliness, sustainability, and good compatibility with the inorganic concrete matrix [14,18]. MICP for self-healing of concrete cracks has been used in many concrete-based projects [13–19]. In such projects, bacterial strains and their substrate are added to the concrete matrix during preparation. When cracks form in the concrete, the dormant bacteria are activated, and will mineralize and precipitate calcium carbonate at the surface of the crack to repair the crack in situ. In the microbially induced precipitation process, suitable strains are an important guarantee to ensure that calcium carbonate can be mineralized on the surface of concrete cracks. In order to adapt to the high pH in concrete, researchers have isolated bacteria mostly from alkaline environments. For example, Stabniko et al. [20] isolated a salt-tolerant, basophilic, urease-producing bacterium (Bacillus sp. VS1) from tropical beach sand and used it to form an anti-seepage layer on the surface of cement. Achal et al. [21] isolated three strains from alkaline soil and found that these strains can produce CaCO3 through mineralization; Belie et al. [22] isolated Sporosarcina pasteurii from alkaline soil and found that its ability to produce urease was strong. In addition to the high pH environment, the Ca2+ concentration in concrete is also high. However, attention has been paid mostly to the adaptability of the strains to high-alkaline environments, while the tolerance of the bacteria to Ca2+ has been neglected. If bacteria are isolated from an environment that has both a high pH and a high Ca2+ concentration, are these bacteria more adaptable to the environment in concrete? Based on idea above, the bacteria is isolated from carbide slag, which is composed mainly of CaO and Ca(OH)2, and has pH of up to 12.5. Studies have found that the strain isolated from carbide slag was Bacillus cereus. So far, the bacteria used in most studies included Sporosarcina pasteurii [23,24], Bacillus sphaericus [25,26], Bacillus subtilis [27–30], etc., while the use of B. cereus to heal concrete cracks has not been reported. In this study, the alkali tolerance of B. cereus CS1, the effect of pH on bacterial growth, and bacterial urease activity, was first investigated. The concentration of both free Ca2+ ions and urea are key factors for inducing calcium

carbonate biomineralization when B. cereus CS1 is used for concrete crack healing. In the view of practical application, temperature tolerance of this specific bacterial strain was considered. Therefore, the growth of B. cereus CS1 was characterized taking into account, the urease activity at different temperatures, Ca2+ and urea concentrations. The amount of available nutrient in the crack zone is a limiting factor because the dosage of added nutrient has to be restricted due to its negative effect on concrete strength. Furthermore, the B. cereus CS1 was used for concrete crack healing, the healing effect of concrete cracks under different curing methods was studied. And this study will lay a foundation for the further application of B. cereus CS1 in the self-healing of concrete cracks. 2. Materials and methods 2.1. Bacterial strain and cultivation conditions B. cereus CS1 (GenBank accession number: MH985305) is a dominant strain isolated from carbide slag. Fig. 1a shows this strain on solid medium. B. cereus CS1 were cultivated in sterile liquid medium, which was composed of peptone (10.0 g/L, purchased from Beijing Aoboxing Bio-Tech Co., Ltd., China), yeast extract powder (7.0 g/L, purchased from Beijing Aoboxing Bio-Tech Co., Ltd., China), and sodium chloride (10.0 g/L, purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd., China). The solid medium was prepared by adding agar (20 g/L, purchased from Beijing Aoboxing Bio-Tech Co., Ltd., China) to the above components. For the purposes of this study, accurately weigh the above nutrients and mix them evenly with distilled water, then sterilize the mixture in an autoclave (G180T, Shanghai Shenan Medical Instrument Factory, China) at 121 °C for 30 min, cool to room temperature (25 °C). The inoculate a liquid medium with an OD value of 0.1 with a pipette (inoculation amount is 1% of the volume of the liquid medium), and the inoculated medium was placed in a constant temperature shaking incubator at 28 °C and 180 rpm, and after culturing for a predetermined time, the OD600 value of the bacterial liquid was measured by an ultraviolet and visible light spectrophotometer (T6 New Century, Beijing Persee General Instrument, China). The bacterial concentration is expressed by the OD value. If urea is required for the test, it is sterilized by filtration using a microporous membrane having a pore size of 0.2 lm. pH was adjusted to 8 with 2 mol/L NaOH and 1 mol/L HCl, unless otherwise specified. Note that the whole culturing process was performed under sterile condition. 2.2. Urease activity Bacterial decomposition of urea to produce mineralized calcium carbonate critically depends on urease. Indeed, the amount of precipitated calcium carbonate can be calculated from the amount of urea decomposed by urease per unit time.

Fig. 1. B. cereus CS1 growing on solid media (a), solid media with phenol red before (top) and after (bottom) inoculation with B. cereusCS1 (b).

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The B. cereus CS1 urease activity was qualitatively evaluated after 24 h from inoculation by the color change of the solid medium containing phenol red reagent (Sinopharm Chemical Reagent Co., Ltd.). The solid medium turned red after 24 h in the presence of ureolytic activity (Fig. 1b). This result indicates an increase in pH due to the microbial breakdown of urea into ammonia. In this paper, two methods were used to characterize bacterial urease activity.

2.2.1. Electrical conductivity method In some experiments, urease activity was quantified based on electrical conductivity as described by Whiffin [31]. Briefly, 1 vol of bacterial suspension was mixed with 9 volumes of 1.1 mol/L sterile-filtered urea, and the change in electrical conductivity over 5 min was measured on a DDB-11A meter (Hangzhou Orion Instruments, China). The mean conductivity change in mS/cm/min was then multiplied by the dilution factor (10) to calculate urease activity in the bacterial suspension, also in mS/cm/min. Then, according to the method of Whiffin, the amount of urea decomposition per unit time (mmol/Lmin1) is calculated to characterize the ureolytic activity. This method cannot be used in the presence of Ca2+ or other cations.

2.2.2. p-Dimethylaminobenzaldehyde colorimetry In the presence of Ca2+ or other cations, urease activity was quantified by pdimethylaminobenzaldehyde colorimetry [32]. The ureolytic activity was quantitatively evaluated by spectrophotometric measurements at 430 nm by using pdimethylaminobenzaldehyde (Sinopharm Chemical Reagent Co., Ltd.). Under acidic conditions the p-dimethylaminobenzaldehyde reacts with urea to produce pdimethylaminobenzaldehyde urea, a lemon yellow compound. The color intensity is proportional to the content of urea. During the test, the optical density value of the solution to be tested before adding bacteria solution was measured first, and then add the bacterial solution to the urea solution to be tested according to the ratio of urea to bacterial solution volume ratio of 9:1. The optical density value of the solution was tested again after 5 min. Calculate the urea concentrations N1 and N2 in the bacterial solution in the two measurements based on the relationship between the obtained urea concentrations and the OD value. Samples with initial urea concentration >100 mg/mL were diluted as needed to collect measurements within the calibration curve, and results were then multiplied by corresponding dilution factors. The urease activity (mmol/Lmin1) of the bacterial solution is calculated by using the following formula:

Urea decomposed ¼

N1  N2 1000  t 60

ð1Þ

N1 — Urea concentration before decomposed N2 — Urea concentration after decomposed

Fig. 2. Microphotograph of B. cereus CS1 (Gram-positive) strain.

2.5. Applying B. cereus CS1 for healing cracks 2.5.1. Preparation of the specimens Eight groups of prismatic specimens (40 mm  40 mm  160 mm, n = 3) were prepared to study the healing of cracks by bacteria. Five groups cylinders specimens (U = 80 mm, H = 22 mm, n = 3) for measuring water permeability. And five groups cylinders specimens (U = 100 mm, H = 200 mm, n = 1) for measuring RCPT. The cement mortar specimens with w/c ratio of 0.65 consisted of 310 kg/m3 ordinary Portland cement (CEM I-42.5N, purchased from Binzhou Shanshui Cement Co., Ltd.), 930 kg/m3 fine aggregate (sand 0.15–3.35 mm), and 201.5 kg/m3 water. Fineness modulus and bulk specific gravity of the fine aggregate were 2.23 and 2.15, respectively. After casting, the molds were placed in the curing room (20 °C, N 95%RH). The specimens were de-molded after 24 h and were placed in the same curing room until the time of testing.

2.3. Gram staining Gram staining is a bacteriological laboratory method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the physical properties of their cell walls [33]. A Gram-positive results in a purpleblue color, while a Gram-negative results in a pink-red color. In Gram staining, the cells are first heat fixed and then stained with a basic dye, crystal violet, which is taken up in similar amounts by all bacteria. The slides are then treated with an I2KI mixture (mordant) to fix the stain, washed briefly with 95% alcohol (destained), and finally counterstained with safranin. Gram-positive organisms retain the initial violet stain, while Gram-negative organisms are decolorized by the organic solvent and, hence, show the pink counterstain. The difference between Gram-positive and Gram-negative bacteria lies in the ability of the cell wall of the organism to retain the crystal violet [34]. Gram method helped the authors to ensure other types of microorganisms have not infected the cultures. Fig. 2 shows Gram-stained B. cereus CS1 observed by microscope.

2.4. Effects of environmental factors on bacterial growth and urease activity To increase mineralization yield and maximize concrete healing, optimal growth conditions would have to be determined for B. cereus CS1. In order to avoid the influence of the bacterial concentration on the test results, if not specified, the bacterial inoculum amount was 1% of the medium volume, and the initial concentration for the experiments have the same OD600 value. The bacterial cultures were grown in nutrient medium at 28 °C and 180 rpm. Optical density value of bacteria at 600 nm (OD600) was measured at 1 h intervals for 25 h after inoculation. Urease activity was quantified according to section 2.2 at 24 h. Data were collected from triplicates and averaged. The bacterial growth and urease activity was assessed (a) at initial pH of 7, 8, 9, 10, 11, and 12; (b) at 0.3, 0.6, 0.9, 1.2, and 1.5 mol/L Ca2+; (c) at 0, 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 mol/L urea; (d) at 10, 28, and 40 °C; and (e) at 1, 3, 5, 7, 9, and 11 g/L yeast extract. The bacteria were inoculated in the above experiment, the initial OD value of the bacteria solution was 0.1, and the bacterial concentration was 3.52  107 cells/mL.

2.5.2. Creation of cracks and incubation conditions Artificial cracks were induced by using two methodologies on two different shapes: i) three-point bending test on prismatic specimens, and ii) splitting test on cylindric specimens. The three-point bending test was carried out on five groups of prismatic specimens to create cracks at 28 days after casting, as shown in Fig. 3a. Crack width was measured by means of a linear variable differential transformer (LVDT) that was attached to the side of the specimens [35]. The crack width was increased with a velocity of 1 lm/s until a crack of 1500 lm was obtained. After unloading, the remaining crack width ranged from 750 to 970 lm. Since there is no bar in the center of the specimens, three rubber bands are carefully placed on the specimens to fix the cracks after the crack is created, as shown in Fig. 3b. After the specimens are fixed with rubber bands, the remaining crack width ranged from 100 to 800 lm. The cylinders (U = 80 mm, H = 22 mm, n = 3) were cured for 28 days as well, and the cracks were created by splitting test, as shown in Fig. 4. The crack width was controlled by the average value of the crack opening measured by two LVDTs attached at both sides of the cylinders. The crack width was increased with a velocity of 1 lm/s until a crack of 1000 lm was obtained. After unloading, the remaining crack width ranged from 620 to 750 lm. And three rubber bands are also carefully placed on the specimens to fix the cracks after the cracks are created. After the specimens are fixed with rubber bands, the remaining crack width ranged from 370 to 430 lm. The cracked prismatic specimens (recorded as group R, Sw, Sb, Sb-WD, and Sn, respectively) were subjected to five incubation conditions: a. 20 °C, N 95%RH; b. in water; c. immersion in the bacterial solution containing deposition medium (BSDM); d. wet–dry cycles with BSDM; e. The specimen was wrapped with tin foil and exposed only to the top. The deposition medium and mature bacteria solution were injected with a syringe every 24 h and placed in 20 °C, >95%RH airconditioned room. The deposition medium consists of urea and calcium lactate, the mass of each component is 0.75 mol/L and 0.9 mol/L, and the pH is 8. During one wet–dry cycle, the specimens were immersed in BSDM for 16 h and were then exposed to air for 8 h. The incubation of b, c and d was performed in an airconditioned room (20 °C, 60%RH).

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Fig. 3. Three-point bending produces cracks (a) and rubber bands are used to fix the specimens (b).

the rubber sleeve containing the specimens were installed into the test cells. Then, one cell was filled with 3% NaCl solution and was connected to the cathode terminal of the power supply while the other cell was filled with 0.3 mol/L NaOH solution and was connected to anode terminal. The potential of 60 V was applied between the opposite surfaces of the concrete circular ends as driving force for permeation of chloride. Current and temperature were recorded every 5 min over 6 h test period by software. The conductivity of the saturated specimens was measured measuring the passed coulombs at the end of the test. The larger the coulomb number is, which is representing the transferred charge during the test, the greater the permeability of the specimen is. If the current is recorded at 30 min intervals, the following formula was used to calculate the total charge passed over 6 h.

Q ¼ 900ðI0 þ 2I30 þ 2I60 þ    þ 2I300 þ 2I330 þ 2I360 Þ

ð3Þ

Q — the charge passed (C); I0 — the current immediately after voltage is applied (A); It — It is the current at t time-in after voltage is applied (A).

Fig. 4. Schematic diagram of cracks generated by splitting test. 2.5.3. Capillary water absorption Three groups of prismatic specimens without cracks (recorded as group R-a, Sba, and Sb-WD-a, respectively) were also placed in the above three curing conditions: a, c and d for the water absorption test (based on the RILEM 25 PEM (II-6) [36]). After 7 d, the specimens were put in the 40 °C oven until the mass changes were less than 0.1% at 24 h intervals. The four sides of the specimens adjacent to the top surface were then wrapped in an aluminum tape to prevent water evaporation through the sides during the water absorption test. The prismatic specimens were brought into a water bath with a water level of 10 ± 1 mm and the top surface facing downwards. At regular time intervals, the prismatic specimens were taken out from the water bath and weighed after removing the surface water. The test was done in an air-conditioned room (20 °C, 60%RH). The water absorption coefficient k (g cm2 h1/2) [15] was determined by Eq. (2).

pffiffi Q ¼k t S

ð2Þ

2.5.5. Water permeability test The cracked cylinders specimens (U = 80 mm, H = 22 mm, n = 3) were in different incubation conditions (a. 20 °C, N95%RH; b. in water; c. wd + in water; d. in BSDM; e. wd + in BSDM, recorded as group R-p, Sw-p, Sw-WD-p, Sb-p, and SbWD-p, respectively) for permeability test. The detailed description of the test process and the calculation of the water permeability coefficient (k) can be seen in the previous research [18]. 2.6. Specimens crack healing and the phase analysis of healing materials The cracked specimens were observed for crack healing at regular curing time, and the healing effect was recorded by an optical camera. At the end of the test, the filler in the crack area was taken out and subjected to SEM [39,40], EDS [41], XRD [42,43], TG-DSC [44] and FTIR [45–47] analysis to determine the composition of the filler.

3. Results 3.1. Effects of pH on bacterial growth and urease activity

Q — the weight of water absorbed at different time intervals (g); S — the area in contact with water (cm2); K — the slope of a plot of water absorbed per square centimeter in function of the square root of time. 2.5.4. Rapid chloride permeability test (RCPT) The influence of bacterial surface treatment on reducing permeability of concrete was assessed by RCPT [37]. The five groups of cylindrical specimens were cut into specimens with a height of 50 mm accordingly to the ASTM C 1202 standard [38], and the RCPT test was conducted in different curing environments (a. 20 °C, N95%RH; b. in water; c. wd + in water; d. in BSDM; e. wd + in BSDM, recorded as group R-c, Sw-c, Sw-WD-c, Sb-c, and Sb-WD-c, respectively) after 7 days of curing. During the test, the side of the cylindrical specimens were wrapped with a silicone rubber sleeve and sealed with epoxy resin, and then they were immersed in water under vacuum condition (1 mmHg) for 3 h. After soaking for 1 h, normal pressure were used, the samples being let to soak for other 18–20 h. In next step,

As can be seen in Fig. 5a, the OD value of the bacterial solution generally shows a decreasing trend with increasing pH during the whole test period. The lag phase lasted around 4 h when the pH of the growth medium was 7–9. With the pH increased to 10, 11, and 12, the lag phase increased to 5.5 h, 10 h and 10.5 h, respectively. The propagation rate (slope of the tangent line of the graph) during log phase was almost the same when the pH was 7 to 8 but greatly decreased when the pH increased from 9 to 12. No further growth was observed beyond 21 h in all cultures. The urea decomposition under different pH conditions by bacteria is shown in Fig. 5b. The highest urease activity was seen at the pH of 8, which was 43.11 mmol/L min1. The urease activity at the pH of 7 and 9 was slightly lower, around 40.66 and 40 mmol/Lmin1,

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Fig. 5. Bacterial growth and urease activity at different pH.

respectively. The urease activity further decreased to 26.66 and 11.33 mmol/Lmin1 with pH increase to 11 and 12. 3.2. Effects of Ca2+ and urea on bacterial growth and urease activity As can be seen in Fig. 6a, growth gradually decreased with increasing Ca2+, although the effect was small at 0–0.9 mol/L

Ca2+, with lag phase lasting about 4 h at these concentrations. However, growth was significantly inhibited at 1.2–1.5 mol/L. Similarly, urease activity gradually decreased with increasing Ca2+, the effect being significant at 1.2–1.5 mol/L (Fig. 6b). With the increase of urea concentrations, the bacterial growth volume generally showed a decreasing trend, but the decrease was not significant (Fig. 6c), and the lag time about 3 h in all cultures. Indeed, growth

Fig. 6. Bacterial growth and urease activity at different Ca2+ and urea concentrations.

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at logarithmic phase was comparable at 0–0.75 mol/L urea but only slightly lower at 1–1.5 mol/L. The stationary phase was reached at around 18 h. The optimal concentration was 0.75 mol/ L, and when the urea concentration was 1.5 mol/L, it starts to inhibit bacteria and affects their life activity. Further, ureolytic activity modestly increased with urea concentration to 45.77 mmol/ Lmin1 at 0.75 mol/L urea (Fig. 6d) but diminished thereafter to significantly lower levels at 1–1.5 mol/L. 3.3. Effects of temperature on bacterial growth and urease activity Fig. 7a shows bacterial growth at 10, 28 and 40 °C in the growth medium of pH 8. It can be seen that low temperature greatly slowed down the bacterial growth. The lag phase during bacterial growth was about 10 h at 10 °C and only 4 h at 28 and 40 °C. The growth rate in the log phase was also considerably lower at 10 °C. In addition, the OD value after 10 h was much higher at 40 °C than at 28 °C. At a temperature of 10 °C, the OD value of the bacteria was significantly reduced, although growth was still observed. This indicates that the bacteria not only have good tolerance to high temperature but also can grow in low-temperature environments. In addition to growth, bacterial ureolytic activity was also greatly affected by temperature. As shown in Fig. 7b, the higher the temperature was, the higher the urease activity obtained. Low temperature greatly slowed down the ureolytic activity which can be attributed to slower growth of bacterial and related production of urease enzyme by vegetative cells. Of note, Wang et al. [26] demonstrated that at low temperatures, increasing the bacterial concentration effectively compensates for the loss of ureolytic activity.

extract. As shown in Fig. 8b, bacterial in the media with yeast extract concentrations of 7–11 g/L had a good ureolytic activity. This implied that yeast extract greatly facilitated the ureolytic activity due to the outgrowth of bacterial. 3.5. Effect of different curing methods on water absorption Fig. 9 shows the water absorption of specimens under different curing conditions. The relationship of water absorption is always: R-a series > Sb-a series > Sb-WD-a series. Compared with the R-a series of specimens, the Sb-WD-a series and the Sb-a series decreased by 12.0% and 3.9% after 72 h, respectively. The water absorption of the Sb-a series decreased slightly, while the water absorption of the Sb- WD-a series showed a significant decrease due to the precipitation of more bacterial mineralization on the surface of the specimens cured by the wet and dry cycle in the BSDM. These mineralized products form a dense film on the surface of the specimens, which can effectively block the moisture in the environment from entering the specimens. The Sb-a series has a higher water absorption than the Sb-WD-a series because the Sb-WD-a series can obtain sufficient oxygen on the surface of the specimens for metabolism in the dry curing, while the bacteria of Sb-a series are always immersed in BSDM and lack enough oxygen, resulting in fewer bacterial mineralization products on the surface. The decrease in water absorption of specimens cured by the wet and dry cycle in the BSDM can be found to effectively reduce the erosion of concrete by harsh external environments and increase its useful life, which also indicates that B. cereus CS1 be used to repair concrete cracks. 3.6. Effect of different curing methods on rapid chloride permeability

3.4. Effects of nutrient concentration on bacterial growth and urease activity The growth of bacterial was greatly influenced by the concentration of the nutrient (yeast extract) in the medium. As shown in Fig. 8a, the higher the concentration of yeast extract, the higher the OD value. When the nutrient concentrations were 1 g/L and 3 g/L, the inhibition period of bacterial growth was about 4 h, and the growth was relatively slow. When the concentration of nutrients was 5–11 g/L, the inhibition period of bacterial growth was about 3 h. The growth rate of bacteria increases, especially when the nutrient concentration is 9 g/L and 11 g/L. The ureolytic activity of bacterial was also affected by the amount of yeast

RCPT is another indicator of concrete durability. Fig. 10 shows the RCPT results of specimens under different curing conditions. The results showed that the average charge of specimens was 7.8% and 10.9% lower in the case of Sb-c and Sb-WD-c groups than that in the R-c group, which suggest a significantly improvement of their resistance to chloride penetration due to bacterial treatment. In this part, the conclusion is similar to the water absorption test, that is, the specimens which have been cured in the dry-wet circulation in the bacterial solution were more resistant to chloride ion permeability than the samples kept in the bacterial solution. This is because the surface of the specimens subjected to a dry-wet circulation curing in microbial bacteria mineralizing more products

Fig. 7. Bacterial growth and urease activity at different temperatures.

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Fig. 8. Bacterial growth and urease activity at different nutrient concentrations.

solution can further increase the resistance of the specimen to chloride permeability. 3.7. Water permeability

Fig. 9. Water absorption of concrete specimens under different incubation conditions.

The water permeability was tested for the cracked cylinders after the incubation under different conditions for 4 weeks. During the 30 d of testing, the water permeability coefficient k of the specimens gradually reached a stable value. The final k values of each series are shown in Fig. 11. The k values of the reference specimens ranged from 7.40  106 to 9.87  106 m/s. The Sw-p and SwWD-p specimens were similar to the reference specimens and the final k value was about 106 m/s which indicated that the specimens cured in water also partially healed. One specimen of SwWD-p had higher water permeability than the Sw-WD-p specimens. The reason was because this specimen had a larger crack width (the initial crack width was 620 lm and decreased to 370 lm after de-loading; the other specimens had an initial crack width of 430 lm). This demonstrates that crack width has a profound influence on water permeability. The water permeability of the Sb-p specimens decreased about two orders of magnitude compared with the reference ones. The final k values of this series varied from 2.51  108 to 4.47  108 m/s. The final k values were the lowest in the Sb-WD-p specimens, varied from 9.86  10–9 – 1.25  10–8 m/s. This is due to the filling effect of CaCO3 produced

Fig. 10. Rapid chloride permeability test of concrete specimens under different incubation conditions.

induced a clogging of the surface pores, enhancing therefore its resistance to chloride ion penetration. The research made by Farzaneh et al. [48] states that increasing the curing time in the bacterial

Fig. 11. Water permeability of the cracked cylinders after being repaired by the different incubation conditions.

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by bacteria and the specimens in Sb-p and Sb-WD-p show lower water permeability. The reason why the Sb-WD-p specimens are lower than the final k value of the Sb-p specimens is that the specimens in the dry-wet cycle in BSDM can be exposed to more O2, which is beneficial to bacterial growth and ureolytic activity, thereby more CaCO3 is produced to fill the crack. 4. Discussion 4.1. Alkali tolerance of B. cereus CS1 B. cereus CS1 has a good alkali tolerance. It can grow in a broad range of alkaline pH values from 7 to 12. The optimal pH range for bacterial growth is 7 to 8. Under higher alkaline conditions (pH 9– 12), bacteria can still grow, but at a much-retarded rate. Although the pH is relatively high in fresh concrete, the pH at cracks may drop to 8–11 due carbonation, exposure, and humidity [26]. This indicates that B. cereus CS1 can be used to heal concrete cracks. Furthermore, in the pH range of 7–11, bacteria had a remarkable ureolytic activity, which ensures the decomposition of urea and the precipitation of CaCO3. Overall, it can be concluded that the appropriate working pH of B. cereus CS1 is 7–11. Above pH 11, the bacteria have limited capacity to precipitate CaCO3. This implies that bacterial spores will keep dormant after being embedded in the concrete matrix (pH > 12), and only start to become active after cracks appear and crack surface pH drops. 4.2. Ca2+ and urea tolerance B. cereus CS1 is a carbonate-precipitating bacterium. It produces urease, which catalyzes urea hydrolysis resulting in the formation of carbonate and ammonium ions. In the presence of Ca2+, CaCO3 can be formed due to the precipitation reaction of calcium and carbonate ions. Bacterial urease activity directly determines the productivity of biogenic CaCO3, which is the main crack healing material in the bacteria-based healing system. The amount and

rate of urea that can be decomposed were influenced by the urea, and Ca source [26]. In this system, urea is the source of carbonate. The more urea is supplied, the more CaCO3 can be formed, provided that a sufficient amount of calcium ions is available. However, it is shown that when the content of urea is excessive, bacterial growth and ureolytic activity are inhibited. For instance, when the urea concentration was 0.75 mol/L. Above this limit, the amount of urea decomposition was decreased and thus appears inhibitory. The reason could be due to too high urea molecule transportation over the cell membrane into the cell at elevated urea concentrations, inhibiting other cellular processes. Therefore, a certain amount of bacteria can only metabolize a certain amount of urea hydrolysis. Calcium ion concentration had also a profound effect on biogenic CaCO3 precipitation activity. On the one hand, Ca2+ is essential for the formation of CaCO3. On the other hand, Ca2+ influences bacterial ureolytic activity as demonstrated in this study. Too much Ca2+ will become toxic to bacteria since they only need a limited amount to regulate their life activities [26]. It can be concluded in this research that B. cereus CS1 had a good Ca tolerance. The low concentrations of Ca2+ and urea (0–0.9 mol/L and 0– 0.75 mol/L, respectively) have little effect on bacterial growth, but with a great influence on the urease activity. Therefore, the concentration of calcium ions and urea concentration on the enzyme activity were determined (0.9 mol/L and 0.75 mol/L, respectively). These concentrations were higher than that ones used by Farzaneh et al. [49] and Wang et al. [50] (0.33 mol/L and 0.5 mol/L, respectively). On the one hand, the calcium source used in this work is calcium lactate, and the lactate ion is known to be conducive to the life activities of bacteria. On the other hand, the B. cereus CS1 was isolated from carbide slag (its main components are CaO and Ca(OH)2). Therefore, B. cereus CS1 is more resistant to high concentrations of Ca2+. In addition, the nutrient medium used in this study does not contain urea, unlike Farzaneh et al. and Wang et al. The results showed that the resistance of B. cereus CS1 to urea may be due to the nitrogen source that activates the urease.

Fig. 12. Crack healing process of specimens under different curing conditions (Sb: immersed in the BSDM; Sb-WD: dry-wet cycle with the BSDM; Sn: the precipitation medium and the mature bacterial solution are separately injected every 24 h with a syringe. In each curing mode, a: images of the crack just after water injection, b: images after 30 min of water injection.)

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4.3. Effect of temperature and yeast extract Bacterial ureolysis is an urease-controlled reaction, which was greatly affected by temperature. Temperature affects bacterial activity, urease activity and therefore reaction rate, and hence the rate of formation of biogenic CaCO3 and crack healing efficiency. Urease activity was stable between 15 and 25 °C, and an increase in temperature (until 60 °C) results in increased urease activity [31]. It was found in this research that B. cereus CS1 not only tolerates high temperatures, but also grows at low temperatures. Although it inhibits bacterial growth and ureolytic activity at low temperatures. However, this retardation can be compensated by using a higher bacterial concentration what can be obtained by supplementing additional yeast extract. The higher the concentration of yeast extract, the shorter the retardation [26].

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Yeast extract has an overall positive effect on the germination of spores and formation of bio-precipitation. Without yeast extract, spore germination is only moderate (but without significant outgrowth) resulting in limited precipitation of CaCO3 [51]. Therefore, particularly under low temperature conditions, the enhanced addition of yeast extract is required. To assure a significant self-healing, a considerable amount of yeast extract (7 g/L) should be used. 4.4. Mortar specimen crack healing The development of the crack healing process can be seen from the images of cracks taken at certain time intervals. As is shown in Fig. 12. It was clearly observed that the crack area gradually decreased as time went on. By 28 d, the crack was almost completely healed. Although the same methodology was applied to

Fig. 13. SEM images of minerals obtained (Sb: a, b; Sn: c, d.)

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create cracks, the cracking behavior was different due to the different mechanical properties of the specimens. In other words, the particle size and shape, the strength, the volume percentage and the random distribution of the fine aggregate will directly affect the strength of the specimens at the microscopic level. Aggregate distribution is an important mesoscopic structural feature of the specimens, and uneven aggregate distribution will lead to irregular internal distribution, affecting therefore its mechanical properties. In this view, the crack propagation and size are not consistent when the same methodology is applied. Crack numbers varied on the specimens and the crack widths varied from 100 to 800 lm. There was no obvious crack healing was observed in the specimens cured at 20 °C >95%RH and immersed in water. However, it is known from the water permeability that the specimens incubation in water are also healed. The healing that occurred was due to autogenic sources. Cementitious materials have a certain capacity of autogenous healing depending on the composition of the matrix and the environment they are exposed to [52]. In general, two main mechanisms are responsible for the autogenous healing: secondary hydration of the unhydrated cement particles and precipitation of calcium carbonate. Besides that, the swelling of the hydration products also contributes to the decrease of the crack area. As can be seen from Fig. 12, no apparent mineralized precipitation was observed on the crack surface at 5 d, but when distilled water was injected into the crack surface with a syringe, the distilled water did not immediately flow down the crack. Thus, it can be proved that the internal crack of the specimen has healed, and the amount of distilled water on the crack surface by 30 min is: Sn series > Sb-WD series > Sb series. This indicates that among the three types of curing methods, the Sn series of specimens have the best repair effect, followed by Sb-WD series, and the cracks inside the Sb series specimens are not healed. The reason why the healing effect of dry and wet circulation curing is better is that the mineralization reaction in the crack area must have an aqueous medium, which serves as a place for bacterial metabolic reaction and a carrier for Ca2+ diffusion transport, but immersing in water for a long time is not conducive to O2 in the air. Medium diffusion transport results in lower bacterial urease activity and less mineralized products. When the specimen was cured for 14 d, it was observed that mineralized products accumulated on the surface of the crack, but the cracks did not completely heal (Fig. 12). At this

time, the amount of mineralized product at the crack was still Sn series > Sb-WD series > Sb series. When curing to 28 d, no matter which kind of curing method is used, the cracks on the surface of the test piece are basically healed. Especially the specimens of Sb-WD series, not only the crack is filled with mineralized products, but also there is a layer of sediment at other positions on the surface. The results were consistent with the water absorption test. This also indicates that B. cereus CS1 can be used for the healing of concrete cracks. The healed specimens were manually broken, and the mineralized products in the cracks were taken out for SEM and EDS analysis. As can be seen from Fig. 13b–e, the bacterial mineralization product is a spherical calcite [53], and it had rough surfaces. Moreover, the crystals showed evidence of bacterial involvement. Rodshaped and round holes were found on the surface of the crystals (Fig. 13c and e), which presumably occurred in the space occupied by the bacterial cells or spores. These holes in the crystals also suggested that bacteria served as nucleation sites during the mineralization process, and Fig. 14 is a diagram of the mineralization mechanism of B. cereus CS1. Element composition analysis via EDS revealed that the crystal is primarily composed of calcium, carbon, and oxygen with a weight ratio closely matching that of CaCO3, indicating that the crystal is CaCO3 (Fig. 13a). It can also be seen from Fig. 13b and c that these spherical calcites are tightly bonded together, which proves that the capillary water absorption rate of the specimens which is immersed for a long time in the bacterial liquid is largely decreased. The mineralized product in the crack area was taken out, and the phase analysis was carried out, and it was found that the mineralized product was calcite-type calcium carbonate. Fig. 15a is an FTIR analysis of mineralized products, as seen in Fig. 15a, C–O bonds in calcite produced chemically and biologically all contain anti-symmetric stretch vibration peaks at 2513.2 cm1, 1402.25 cm1, 1086.69 cm1, 876 cm1, 731.24 cm-1 and 712.57 cm1. The results obtained are consistent with the study by Rong et al. [54,55]. Fig. 15b shows XRD analysis of mineralized products, peaks at 2h = 29.4, 36.0, 39.4, 43.1, 47.6, 48.5, 57.3, and 64.6° are characteristic of calcite-type CaCO3 [55]. Fig. 15c shows TG-DSC analysis of mineralized products, a distinct endothermic peak near 750–820 °C [56] was observed in TG-DSC spectra collected from mineralized products. Drastic loss of mass near this temperature indicates CaCO3 decomposition, small and early loss

Fig. 14. Process of bacterial CaCO3 mineralization.

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Fig. 15. Phase analysis of minerals obtained (a: FTIR; b: XRD; c: TG-DSC).

of mass in Fig. 15c may be due to residual moisture or organic matter decomposition. Taken together, the data suggest that B. cereus CS1 is suitable as a healing agent for concrete, although its ability to do so has yet to be demonstrated. Finally, a method to add B. cereus CS1 to concrete without affecting survival or mineralization potential has yet to be established. These are the focus of our future research.

Bacterial remediation of cracks has shown very promising results. After the bacterial repair, the water permeability of the specimens was about two orders of magnitude lower than that of the reference specimens, and the width of cracks that the bacteria can heal is 100–800 lm. Phase analysis showed that the product in crack area was calcite. Conflict of interest

5. Conclusions B. cereus CS1 is an alkali tolerant strain, and grows well at pH 7– 10. The concentrations of Ca2+ and urea, which have little influence on bacterial growth and urease activity, and can guarantee the mineralization effect, are 0.9 mol/L and 0.75 mol/L respectively. The growth of bacteria is inhibited at 10 °C, and the difference is small in the first 10 h of culture at 28 °C and 40 °C. When the time exceeds 10 h, the 40 °C is the best temperature to promote bacterial growth. The minimum quantity of yeast extract necessary to ensure mineralization is 7 g/L. The water absorption and chloride permeability of the specimens can be significantly reduced after bacterial treatment. The best treatment method was wet-dry cycles curing in the bacterial solution. The water absorption and chloride permeability of the specimens treated in this way were 12% and 10.9% lower than those of the reference specimens, respectively.

The authors declared that they have no conflicts of interest to this work. Acknowledgments This study was funded by the National Natural Science Foundation of China (grant number: 51674038 and 51874193); the National Key R&D Program of China (grant number: 2018YFC0807900); the Shandong Province Natural Science Foundation (grant number: ZR2018JL019 and ZR2017PEE024); the Shandong Province Science and Technology Development Plan (grant number: 2017GSF220003); the State Key Program for Coal Joint Funds of the National Natural Science Foundation of China (grant number: U1261205); Scientific Research Foundation of Shandong University of Science and Technology for Recruited Talents (grant number: 2017RCJJ010 and 2017RCJJ037); Shandong

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Province First Class Subject Funding Project (grant number: 01AQ05202); the SDUST Research Fund (grant 2018TDJH102); Taishan Scholar Talent Team Support Plan for Advantaged & Unique Discipline Areas.

[24]

[25]

References [1] H. Van Damme, Concrete material science: past, present, and future innovations, Cem. Concr. Res. (2018), https://doi.org/10.1016/j. cemconres.2018.05.002. [2] M. Seifan, A.K. Samani, A. Berenjian, Bioconcrete: next generation of selfhealing concrete, Appl. Microbiol. Biotechnol. 100 (6) (2016) 2591–2602, https://doi.org/10.1007/s00253-016-7316-z. [3] N. Chahal, R. Siddique, A. Rajor, Influence of bacteria on the compressive strength, water absorption and rapid chloride permeability of fly ash concrete, Constr. Build. Mater. 28 (1) (2012) 645–651, https://doi.org/10.1016/ j.conbuildmat.2011.07.042. [4] F.T. Pacheco, J.A. Labrincha, Biotech cementitious materials: Some aspects of an innovative approach for concrete with enhanced durability, Constr. Build. Mater. 40 (7) (2013) 1136–1141, https://doi.org/10.1016/ j.conbuildmat.2012.09.080. [5] A.K. Samani, M.M. Attard, A stress–strain model for uniaxial and confined concrete under compression, Eng. Struct. 41 (3) (2012) 335–349, https://doi. org/10.1016/j.engstruct.2012.03.027. [6] K.V. Tittelboom, N.D. Belie, W.D. Muynck, W. Verstraete, Use of bacteria to repair cracks in concrete, Cem. Concr. Res. 40 (1) (2010) 157–166, https://doi. org/10.1016/j.cemconres.2009.08.025. [7] F.B.D. Silva, N. Boon, N.D. Belie, V. Willy, Industrial application of biological self-healing concrete: challenges and economical feasibility, J. Commer. Biotechnol. 21 (1) (2015) 31–38, https://doi.org/10.5912/jcb662. [8] T.H. Ahn, T. Kishi, Crack self-healing behavior of cementitious composites incorporating various mineral admixtures, J. Adv. Concr. Technol. 8 (2) (2010) 171–186, https://doi.org/10.3151/jact.8.171. [9] K. Sisomphon, O. Copuroglu, E.A.B. Koenders, Self-healing of surface cracks in mortars with expansive additive and crystalline additive, Cem. Concr. Compos. 34 (4) (2012) 566–574, https://doi.org/10.1016/j.cemconcomp.2012.01.005. [10] D. Snoeck, K.V. Tittelboom, S. Steuperaert, P. Dubruel, N.D. Belie, Self-healing cementitious materials by the combination of microfibres and superabsorbent polymers, J. Intel. Mater. Syst. Struct. 25 (1) (2012) 13–24, https://doi.org/ 10.1177/1045389X12438623. [11] C.A. Issa, P. Debs, Experimental study of epoxy repairing of cracks in concrete, Constr. Build. Mater. 21 (1) (2007) 157–163, https://doi.org/10.1016/ j.conbuildmat.2005.06.030. [12] K.V. Tittelboom, K. Adesanya, P. Dubruel, P.V. Puyvelde, N.D. Belie, Methyl methacrylate as a healing agent for self-healing cementitious materials, Smart Mater. Struct. 20 (12) (2011) 1–12, https://doi.org/10.1088/0964-1726/20/12/ 125016. [13] J.Y. Wang, J. Dewanckele, V. Cnudde, V. Vlierberghee, W. Verstraeteb, N.D. Belie, X-ray computed tomography proof of bacterial-based self-healing in concrete, Cem. Concr. Compos. 53 (10) (2014) 289–304, https://doi.org/ 10.1016/j.cemconcomp.2014.07.014. [14] J.Y. Wang, D. Snoeck, S.V. Vlierberghe, W. Verstraete, N.D. Belie, Application of hydrogel encapsulated carbonate precipitating bacteria for approaching a realistic self-healing in concrete, Constr. Build. Mater. 68 (68) (2014) 110–119, https://doi.org/10.1016/j.conbuildmat.2014.06.018. [15] J.Y. Wang, H. Soens, W. Verstraete, N.D. Belie, Self-healing concrete by use of microencapsulated bacterial spores, Cem. Concr. Res. 56 (2) (2014) 139–152, https://doi.org/10.1016/J.CEMCONRES.2013.11.009. [16] J.Y. Wang, A. Mignon, D. Snoeck, V. Wiktor, S. Van Vliergerghe, N. Boon, Application of modified-alginate encapsulated carbonate producing bacteria in concrete: a promising strategy for crack self-healing, Front. Microbiol. 6 (2015) 1088–1101, https://doi.org/10.3389/fmicb.2015.01088. [17] J.Y. Wang, N.D. Belie, W. Verstraete, Diatomaceous earth as a protective vehicle for bacteria applied for self-healing concrete, J. Ind. Microbiol. Biotechnol. 39 (4) (2012) 567–577, https://doi.org/10.1007/s10295-011-1037-1. [18] J.Y. Wang, K.V. Tittelboom, N.D. Belie, W. Verstraete, Use of silica gel or polyurethane immobilized bacteria for self-healing concrete, Constr. Build. Mater. 26 (1) (2012) 532–540, https://doi.org/10.1016/ j.conbuildmat.2011.06.054. [19] N. De Belie, J. Wang, Z. Bundur, K. Paine, Bacteria-based concrete, in: Ecoefficient Repair and Rehabilitation of Concrete Infrastructures, Woodhead Publishing, Cambridge, 2018, pp. 531–567, https://doi.org/10.1016/B978-008-102181-1.00019-8. [20] V. Stabnikov, M. Naeimi, V. Ivanov, J. Chu, Formation of water-impermeable crust on sand surface using biocement, Cem. Concr. Res. 41 (11) (2011) 1143– 1149, https://doi.org/10.1016/j.cemconres.2011.06.017. [21] V. Achal, X.L. Pan, N. Zyurt, Improved strength and durability of fly ashamended concrete by microbial calcite precipitation, Ecol. Eng. 37 (4) (2011) 554–559, https://doi.org/10.1016/j.ecoleng.2010.11.009. [22] N.D. Belie, J.Y. Wang, Bacteria-based repair and self-healing of concrete, J. Sustain. Cement-Based. Mater. 5 (1–2) (2015) 35–56, https://doi.org/10.1080/ 21650373.2015.1077754. [23] N.H. Balam, D. Mostofinejad, M. Eftekhar, Effects of bacterial remediation on compressive strength, water absorption, and chloride permeability of

[26]

[27]

[28]

[29] [30]

[31] [32]

[33]

[34]

[35]

[36] [37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

lightweight aggregate concrete, Constr. Build. Mater. 145 (2017) 107–116, https://doi.org/10.1016/j.conbuildmat.2017.04.003. S. Bhaskar, K.M.A. Hossain, M. Lachemi, G. Wolfaardt, M.O. Kroukamp, Effect of self-healing on strength and durability of zeolite-immobilized bacterial cementitious mortar composites, Cem. Concr. Compos. 82 (2017) 23–33, https://doi.org/10.1016/j.cemconcomp.2017.05.013. M. Seifan, A.K. Samani, A. Berenjian, Induced calcium carbonate precipitation using Bacillus species, Appl. Microbiol. Biotechnol. 100 (23) (2016) 9895–9906, https://doi.org/10.1007/s00253-016-7701-7. J.Y. Wang, H.M. Jonkers, N. Boon, D.B. Belie, Bacillus sphaericus LMG 22257 is physiologically suitable for self-healing concrete, Appl. Microbiol. Biotechnol. 101 (12) (2017) 5101–5114, https://doi.org/10.1007/s00253-0178260-2. N.N.T. Huynh, N.M. Phuong, N.P.A. Toan, N.K. Son, Bacillus subtilis HU58 immobilized in micropores of diatomite for using in self-healing, Concrete. Proc. Eng. 171 (2017) 598–605, https://doi.org/10.1016/j. proeng.2017.01.385. W. Khaliq, M.B. Ehsan, Crack healing in concrete using various bio influenced self-healing techniques, Constr. Build. Mater. 102 (1) (2016) 349–357, https:// doi.org/10.1016/j.conbuildmat.2015.11.006. S. Mondal, P. Das, A.K. Chakraborty, Application of bacteria in concrete, Mater. Today 4 (9) (2017) 9833–9836, https://doi.org/10.1016/j.matpr.2017.06.276. M.V.S. Rao, V.S. Reddy, C. Sasikala, Performance of microbial concrete developed using Bacillus subtilis JC3, J. Inst. Eng. 98 (4) (2017) 1–10, https:// doi.org/10.1007/s40030-017-0227-x. V.S. Whiffin, Microbial CaCO3 precipitation for the production of biocement Ph.D thesis, Murdoch University, Western Australia, 2004. R.X. Wang, Microbial Regulation of Calcium Carbonate Formation and its Application in Defect Repair of Cement-based Materials Ph.D. thesis, Southeast University, Nanjing, China, 2009. N. Farzaneh, M. Davood, Reducing permeability of concrete by bacterial mediation on surface using treatment gel, ACI Mater. J. 113 (3) (2016) 287– 294, https://doi.org/10.14359/51688701. H.B. Nafise, M. Davood, E. Mohamadreza, Use of carbonate precipitating bacteria to reduce water absorption of aggregates, Constr. Build. Mater. 141 (2017) 565–577, https://doi.org/10.1016/j.conbuildmat.2017.03.042. Z.X. Hu, X.M. Hu, W.M. Cheng, Y.Y. Zhao, M.Y. Wu, Performance optimization of one-component polyurethane healing agent for self-healing concrete, Constr. Build. Mater. 179 (2018) 151–159. RILEM 25 PEM. Commission, ‘‘Experimental methods”, Test n.II-6: Water absorption coefficient (capillarity), 1980. T. Bahareh, M. Davood, Penetrability, corrosion potential, and electrical resistivity of bacterial concrete, J. Mater. Civ. Eng. 31 (3) (2019), https://doi. org/10.1061/(ASCE)MT.1943-5533.0002618 04019002. ASTM, Standard Test Method for Electrical Indication of Concrete’s Ability to Resist Chloride Ion Penetration ASTM C1202-17a, ASTM, West Conshohocken, PA, 2017. J. Wang, X.Y. Zhan, J.X. Ding, Y. Zhao, Y.J. Jiang, Synthesis of organic-inorganic hybrid perovskite CH3NH3PbI3 crystal and tests of its gas sensitivity, J. Shandong Sci. 37 (2) (2018) 88–92. G.H. Ni, H.C. Xie, Shang Li, Q. Sun, D.M. Huang, Y.Y. Cheng, N. Wang, The effect of anionic surfactant (SDS) on pore-fracture evolution of acidified coal and its significance for coalbed methane extraction, Adv. Powder Technol. (2019), https://doi.org/10.1016/j.apt.2019.02.008. X.F. Ren, H. Dong, X.M. Hu, W.M. Cheng, Y.Y. Zhao, L. Xin, W. Lu, Novel sodium silicate/polymer composite gels for the prevention of spontaneous combustion of coal, J. Hazard. Mater. (2019), https://doi.org/10.1016/j. jhazmat.2019.03.041. S. Huang, B.Y. Liu, X.J. Tian, L.F. Zhao, Effect of polyphosphoester flame retardant on crystallization behavior and morphology, J. Shandong Sci.-Tech. Univ. (Nat. Sci.) 37 (5) (2018) 45–51. Q. Zhang, X.M. Hu, M.Y. Wu, Y.Y. Zhao, C. Yu, Effects of different catalysts on the structure and properties of polyurethane/water glass grouting materials, J. Appl. Polym. Sci. 135 (27) (2018), https://doi.org/10.1002/app.46460. W.M. Cheng, X.M. Hu, J. Xie, Y.Y. Zhao, An intelligent gel designed to control the spontaneous combustion of coal: Fire prevention and extinguishing properties, Fuel 210 (2017) 826–835. Z.W. Wang, J. Zhang, K.L. Zhang, Q. Yu, L. Song, Synthesis of 2,4-di-tertbutylphenol diphenylphosphinate and its antioxidant performance in polyolefin, J. Shandong Sci.-Tech. Univ. (Nat. Sci.) 37 (2) (2018) 59–65. Q. Zhang, X.M. Hu, M.Y. Wu, M.M. Wang, Y.Y. Zhao, T.T. Li, Synthesis and performance characterization of poly(vinyl alcohol)-xanthan gum composite hydrogel, React. Funct. Polym. 136 (2019) 34–43. Z.X. Hu, X.M. Hu, W.M. Cheng, W. Lu, Influence of synthetic conditions on the performance of melamine-phenol-formaldehyde resin microcapsules, High Perform. Polym. 31 (2) (2019) 197–210. N. Farzaneh, M. Davood, H. Hasti, Influence of biodeposition treatment on concrete durability in a sulphate environment, Biosyst. Eng. 133 (2015) 141– 152, https://doi.org/10.1016/j.biosystemseng.2015.03.008. N. Farzaneh, M. Davood, H. Hasti, Concrete durability improvement in a sulfate environment using bacteria, J. Mater. Civ. Eng. 28 (1) (2015) 04015064, https:// doi.org/10.1061/(ASCE)MT.1943-5533.0001337. J.Y. Wang, K.V. Tittelboom, N.D. Belie, et al., Potential of Applying Bacteria to Heal Cracks in Concrete., in: International Conference on Sustainable Construction Materials & Technologies, UWM Center for By-Products Utilization, 2010, pp. 1807–1818.

M. Wu et al. / Construction and Building Materials 209 (2019) 631–643 [51] J.Y. Wang, Self-healing Concrete by Means of Immobilized Carbonate Precipitating Bacteria PhD thesis, Ghent University, Ghent, Belgium, 2013. [52] Y.Z. Yang, M.D. Lepech, E.H. Yang, V.C. Li, Autogenous healing of engineered cementitious composites under wet–dry cycles, Cem. Concr. Res. 39 (5) (2009) 382–390, https://doi.org/10.1016/j.cemconres.2009.01.013. [53] H. Rong, C.X. Qian, Cementation of loose sand particles based on bio-cement, J. Wuhan Univ. Technol. Mater. Sci. Ed. 29 (6) (2014) 1208–1212.

643

[54] H. Rong, C.X. Qian, L.Z. Li, Study on infra-red spectra of microbe cementitious materials, J. Funct. Mater. 44 (23) (2013) 3408–3411, https://doi.org/10.3969/ j.issn.1001-9731.2013.23.011. [55] H. Rong, C.X. Qian, Binding functions of microbe cement, Adv. Eng. Mater. 17 (3) (2015) 334–340, https://doi.org/10.1002/adem.201400030. [56] C.X. Qian, J.Y. Wang, R.X. Wang, et al., Yield of microbial deposited calcite, J. Ceram. Soc. 34 (2006) 618–621.