Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Effect of Microorganism Sporosarcina pasteurii on the Hydration of Cement Paste

  • Lee, Jun Cheol (School of Architecture and Civil Engineering, Kyungpook National University) ;
  • Lee, Chang Joon (Department of Architectural Engineering, Chungbuk National University) ;
  • Chun, Woo Young (School of Architectural, Civil, Environmental, and Energy Engineering, Kyungpook National University) ;
  • Kim, Wha Jung (School of Architectural, Civil, Environmental, and Energy Engineering, Kyungpook National University) ;
  • Chung, Chul-Woo (Department of Architectural Engineering, Pukyong National University)
  • Received : 2014.11.14
  • Accepted : 2015.04.14
  • Published : 2015.09.28
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Abstract

Years of research have shown that the application of microorganisms increases the compressive strength of cement-based material when it is cured in a culture medium. Because the compressive strength is strongly affected by the hydration of cement paste, this research aimed to investigate the role of the microorganism Sporosarcina pasteurii in hydration of cement paste. The microorganism's role was investigated with and without the presence of a urea-CaCl2 culture medium (i.e., without curing the specimens in the culture medium). The results showed that S. pasteurii accelerated the early hydration of cement paste. The addition of the urea-CaCl2 culture medium also increased the speed of hydration. However, no clear evidence of microbially induced calcite precipitation appeared when the microorganisms were directly mixed with cement paste.

Keywords

Introduction

Concrete has been widely used as a construction and building material. During the service life of concrete, it experiences weathering, including abrasion, shrinkage, and expansion cracking associated with freeze–thaw cycles, sulfate attack, alkali silica reaction, biological degradation, and so on. To remediate the integrity of concrete, a broad range of organic and inorganic products have been proposed as self-healing materials [8, 9].

Microbially induced calcite (CaCO3) precipitation (MICP) is one of the processes that drives the self-healing of concrete. The concept is to use the urease activity of microorganisms [9]. When microorganisms are in contact with urea, they hydrolyze urea into CO2 and ammonia. The alkalinity increases as a result. Because the negatively charged bacterial cells favor binding of divalent cations such as Ca2+, the HCO3- (CO2 that is produced during urease activity and dissolved in solution) reacts with Ca2+ in the solution and forms a heterogeneous calcite (CaCO3) nucleus on the bacterial cell [18]. This becomes a nucleation site for a continuous MICP process [5-8] that repairs damaged concrete.

MICP has also been shown to increase the compressive strength of cementitious materials [1, 4, 7, 13, 18, 19]. The increase in the compressive strength was associated with a reduction of the porosity by filling of the pore space with calcite when the cementitious specimens were cured in a culture medium. MICP has been used to coat the surface of cement-based materials to increase their durability [16, 17]. It was clearly shown that MICP can be successfully induced in the open pore spaces to remediate damage when it is applied after cracking [9].

Note that it is cumbersome to apply a urea-CaCl2 culture medium in the curing of cementitious materials. The medium is known to drive the increase in the compressive strength, but it is economically infeasible to use urea in mortar or concrete because the same curing condition is unlikely to be applied for real concrete structures. Therefore, research has been conducted to improve the strength and durability of mortar or concrete using microorganisms without a urea-CaCl2 culture medium. Ghosh et al. [5] reported that the compressive strength of cement mortar was positively affected, although no clear evidence of calcite precipitation was observed. Note that the available literature is still limited with respect to the role of microorganisms on hydration of cement paste with and without a urea-CaCl2 culture medium, so further investigation is necessary.

In this study, the role of the microorganism Sporosarcina pasteurii (ATCC 11859) in hydration of cement paste with and without the presence of a culture medium was investigated. S. pasteurii was chosen because it has been known to survive in a cementitious environment (high-pH, calcium-rich environment) and was also shown to increase the compressive strength in the presence of a culture medium [9, 7, 13].

 

Materials and Methods

Preparation of Microorganisms

As mentioned, S. pasteurii (ATCC 11859) was used for this study. The strain was purchased from the Korean Biological Resource Center (Daejon, Korea). Tryptic soy broth (TSB) culture medium was used to grow the microorganisms. The TSB culture medium was stored in autoclave conditions for 15 min at 121℃ to eliminate other microorganisms that may be present. The microorganisms were inoculated in the TSB culture medium, and the medium with the microorganisms was shaken at 170 rpm for 24 h at 30℃ under microaerobic conditions to facilitate rapid growth [14]. Then the TSB culture medium with the microorganisms was centrifuged at 8,000 rpm, washed twice with distilled water, and diluted in distilled water to obtain a mixing water with an optical density of 1.0 at 600 nm (approximately 107 cells/ml). This diluted solution of 107 cells/ml was used as an original source. Mixing water with cell concentrations of 105 , 103 , and 101 cells/ml was obtained by diluting the original source.

Observation of S. pasteurii

Before the cement paste samples were prepared, the presence and activity of the microorganisms that were grown in TSB culture were verified using a urea-CaCl2 culture medium. The medium was prepared using 3 g/l nutrient broth, 20 g/l urea, 2.12 g/l NaHCO3, 10 g/l NH4Cl, and 3.7 g/l CaCl2·2H2O [6, 14]. The pH of the urea-CaCl2 medium was adjusted to 6.0 using 6 N HCl solution [14]. The urea-CaCl2 culture medium was used to facilitate MICP after the microorganisms were grown. The MICP process caused by the microorganisms under atmospheric conditions was observed using a light transmission optical microscope (biological microscope model NSB-50T/B; Samwon Ltd., Korea) and a field emission scanning electron microscope (FE-SEM model SU8200; Hitachi Ltd., Japan).

Semi-Adiabatic Calorimetry

Distilled water containing no microorganisms was used to make a plain cement paste sample. Note that no culture medium solution was used to mix the cement paste samples with the microorganisms. Two cell concentrations, 103 and 107 cells/ml, were added directly to the cement paste to investigate the effect of the cell concentration on the hydration of cement paste without the urea-CaCl2 culture medium.

To make the specimens, 2,000 g of type I Portland cement that conforms to the American Society for Testing and Materials (ASTM) C 150 specification and 700 ml of distilled water (w/c = 0.35) containing the targeted cell concentration were used. The chemical composition of type I cement is shown in Table 1. The materials were mixed by following the American Society for Testing and Materials (ASTM) C 305 specification.

Table 1.Chemical composition of type I Portland cement.

After a cement paste sample of w/c 0.35 was prepared, it was immediately poured into a container having a diameter of 60 mm and a height of 72 mm encapsulated within polystyrene foam. A thermocouple was immersed inside each cement paste sample to measure the temperature rise of the cement paste during the early hydration period. The temperature rise of specimens with and without the microorganisms can be used to understand their effect on early hydration of cement paste.

Hydration Study

To investigate the role of the microorganisms in the hydration kinetics of cement paste, 10 g of type I Portland cement and 10 ml of distilled water (or urea-CaCl2 culture medium) containing 107 cells/ml (w/c = 1) were poured into a 50 ml plastic tube. The cap of the tube was sealed, and the tube was vigorously shaken to mix the cement, water, and microorganisms. A water-to-cement ratio yielding a loose concentration was chosen to improve the effectiveness of the evaluation by reducing the effect of microorganisms being captured within the pores and becoming inactive. Only one concentration level, 107 cells/ml, was chosen for this experiment. Cement paste mixed with distilled water containing no microorganisms was also prepared to provide a reference guideline. After shaking, the lid of the tube was opened again, and the plastic tube was filled with N2 gas to limit further ingress of ambient air into the specimen.

To understand the reaction kinetics of cement paste with the microorganisms, quantitative X-ray diffraction (XRD) and differential scanning calorimetry/thermogravimetric analysis (DSC/ TGA) were used at sample ages of 1, 3, 7, and 28 days. Because it was difficult to directly measure the amount of C-S-H during hydration, the amount of cal cium hydroxide was measured to estimate the calcium silicate hydration.

X-Ray Diffraction

The crystalline structure of w/c = 1 cement paste samples with and without the microorganisms was examined by XRD, using a Rigaku D/Max-2500 instrument (Rigaku, Tokyo, Japan). For quantitative Rietveld analysis, 10% of TiO2 (rutile) was added to the specimens as an internal standard, and the specimens were gently ground to equally disperse the rutile into the cement paste. The scanning angle 2θ was varied from 5° to 90° with a step size of 0.02° and a dwell time of 1.5 sec. The working voltage was 40 kV, and the electric current was 200 mA. The scanned data at the ages of 1, 3, 7, and 28 days were first analyzed using the EVA software. The data were compared with the Inorganic Crystal Structure Database (ICSD) to obtain the phase analysis. A quantitative Rietveld analysis was conducted using TOPAS 4.2 to determine the amount of calcium hydroxide in the cement paste samples. The following ICSD entries were used: 1841, tricalcium aluminate; 1956, anhydrite; 9197, brownmillerite (tetracalcium aluminoferrite); 27039, ettringite; 31330, rutile; 59327, monocarbonate; 62363, Friedel's salt; 63250, hydrocalumite; 64759, hatrurite (tricalcium silicate); 79550, larnite (dicalcium silicate); 79674, calcite; 100138, monosulfate; 29210, quartz; 9863, periclase; and 202220, portlandite (calcium hydroxide). Note that no ICSD entry was available for hemicarbonate, so the amount of hemicarbonate could not be investigated.

Differential Scanning Calorimetry/Thermogravimetric Analysis

To verify the amount of calcium hydroxide in the w/c = 1 cement paste samples with the microorganisms, the samples were analyzed at the ages of 1, 3, 7, and 28 days using DSC/TGA equipment (SDT Q600; TA Instrument, Japan). For the analysis, the temperature was raised from 25℃ to 1,000℃ at a heating rate of 10℃/min. The weight loss and endothermic DSC peak at about 450℃ were used to calculate the amount of calcium hydroxide in the samples. The amount of calcite in the specimens was also verified using DSC/TGA data.

 

Results

Observation of S. pasteurii

Fig. 1 shows optical microscopy images of the culture medium before and during inoculation. Before inoculation with the microorganisms (Fig. 1A), the urea-CaCl2 culture medium showed no evidence of MICP. However, as the microorganisms were inoculated into the medium, dark spherical material clearly appeared (Fig. 1B). The dark color was associated with a lack of light transmission, indicating that the observed material was solid. A round shape appears in Fig. 1B because the microorganisms tend to gather and form spheres when they are active. Note that each sphere cannot be directly related to an individual calcite crystal. In fact, it is a group of calcite crystals being precipitated onto the shell of the microorganisms.

Fig. 1.Light transmission optical microscopy images of samples. (A) Without inoculation of S. pasteurii into urea-CaCl2 culture medium, and (B) S. pasteurii being inoculated into urea-CaCl2 culture medium.

The observed SEM images are presented in Fig. 2. At 1,000× (Fig. 2A), the surface of the sphere was rough with some open spaces. The SEM image at 6,000× (Fig. 2B) shows that the crust of the sphere consisted of many small crystals. The microscopy observations clearly showed that the microorganisms used in this experimental work actively formed calcite under ambient conditions when the urea-CaCl2 culture medium was available.

Fig. 2.Scanning electron microscopy images of the shell of S. pasteurii at (A) 1,000× magnification and (B) 6,000× magnification.

Semi-Adiabatic Calorimetry

The temperature rise of the specimens containing 103 and 107 cells/ml is presented in Fig. 3. Although the differences in the temperature rise were minimal, the addition of the microorganisms clearly increased the early temperature rise of the cement paste samples. The temperature rise of the samples also increased when the cell concentration was increased. The results indicate that the incorporation of the microorganisms into cement paste accelerated hydration of the cement paste.

Fig. 3.Temperature rise of cement paste with and without S. pasteurii. Gray line: plain cement paste; black line: cement paste with 103 cell/ml of S. pasteurii; black dotted line: cement paste with 107 cell/ml of S. pasteurii.

Hydration Study

X-ray diffraction. The XRD patterns of hydrated cement paste (w/c = 1) with the microorganisms are presented in Fig. 4. The XRD pattern of unhydrated cement powder is also shown in Fig. 4A. The observed phases in the hydrated cement paste samples and their amounts identified by Rietveld quantitative analysis are shown in Tables 2–5. Although the XRD scan was performed from 5° to 90° for quantitative Rietveld analysis, Fig. 4 shows only the XRD patterns from 5° to 35° to facilitate the identification of phases associated with calcium silicate and aluminate hydration.

Fig. 4.XRD patterns of hydrated cement paste with S. pasteurii after (A) 1 day of hydration, (B) 3 days of hydration, (C) 7 days of hydration, and (D) 28 days of hydration. (E) Same as (D) but focused on 5 to 15°. □: cement paste, ◇: cement paste with S. pasteurii, △: cement paste with urea-CaCl2 culture medium, ○: cement paste with S. pasteurii and urea-CaCl2 culture medium, ☆: unhydrated cement paste

Table 2.C = Plain cement paste; CS = cement paste with S. pasteurii; CU = cement paste with urea-CaCl2; and CSU = cement paste with urea-CaCl2 and S. pasteurii.

Table 3.C = Plain cement paste; CS = cement paste with S. pasteurii; CU = cement paste with urea-CaCl2; and CSU = cement paste with urea-CaCl2 and S. pasteurii.

Table 4.C = Plain cement paste; CS = cement paste with S. pasteurii; CU = cement paste with urea-CaCl2; and CSU = cement paste with urea-CaCl2 and S. pasteurii.

Table 5.C = Plain cement paste; CS = cement paste with S. pasteurii; CU = cement paste with urea-CaCl2; and CSU = cement paste with urea-CaCl2 and S. pasteurii.

According to Fig. 4A, the XRD patterns of all the 1-day- old specimens showed hydrated phases of ettringite (at 9.1°) and calcium hydroxide (portlandite, at 18.1°). In the cement paste mixed with urea-CaCl2 or urea-CaCl2 and the microorganisms, a clear indication of Friedel’s salt (AFm structure that incorporates a Cl- ion in the crystal structure) was observed. Because Friedel’s salt was not observed in the absence of urea-CaCl2, the results indicate that the available Cl- ions in the urea-CaCl2 culture medium entered the AFm structure. Gypsum (at 11.6°) in the unreacted cement disappeared when the cement was hydrated. Some unreacted ferrite (brownmillerite: C4AF), C3S, C2S, and C3A was also observed (at 2θ angles above 30°) in all the samples after 1 day. However, the presence of C3A was slightly unclear. The peak at 29.4° (after 1 day) was calcite that originated from unreacted cement powder. The peak at 27.4° was the rutile internal standard used for quantitative Rietveld analysis. Quartz was observed in all the samples at 26.5° at all ages. A very small peak of monosulfate was identified at 9.8°, except in the cement paste mixed with urea-CaCl2 and the microorganisms. Although it was not shown in Fig. 4A, periclase (MgO) was also identified in all the samples at all ages.

After 3 days (Fig. 4B), cement paste and cement paste with the microorganisms started to develop hemicarbonate (at 10.6°). Monosulfate completely disappeared in all the samples. Ettringite and portlandite were still observed, and the peak intensity of portlandite increased after 3 days. However, the cement paste with urea-CaCl2 and urea-CaCl2 plus the microorganisms still showed Friedel’s salt as the dominant form of AFm. No hemicarbonate was observed in these samples. Unreacted C3S, C2S, and ferrite were still observed after 3 days. After 7 days (Fig. 4C), the XRD patterns were similar to those of the 3-day-old specimens. The peak intensity of hemicarbonate and Friedel’s salt continued to increase, and ettringite still existed. The peak intensity of portlandite also increased after 7 days. The calcium silicate peaks (C3S and C2S) clearly showed a reduction in XRD intensity. The peak intensity of ferrite decreased after 7 days.

After 28 days (Fig. 4D), the peak intensity of portlandite dominated that of all the other peaks, so Fig. 4E shows the patterns redrawn for easy identification of the hydrated calcium aluminate phases. According to Fig. 4E, the XRD peak pattern was similar to that of the 7-day-old specimens, but the development of monocarbonate (at 11.62°) was observed in the plain cement paste and cement paste with the microorganisms. In the samples with urea-CaCl2 and urea-CaCl2 plus the microorganisms, the dominant form of the AFm phase was still Friedel’s salt.

Differential scanning calorimetry/thermogravimetric analysis. Fig. 5 shows the thermal behavior of the unreacted cement powder, hydrated cement paste with and without the microorganisms, and hydrated cement paste with urea-CaCl2 and urea CaCl2 plus the microorganisms. The unreacted cement (Fig. 5A) showed a very small amount of thermal activity at about 110℃ and 380℃. This is most likely related to the thermal transition of calcium sulfate phases (gypsum to hemihydrate and to anhydrite). A small exothermic reaction was observed at about 680℃. This can be related to the decomposition of calcite. The measured total weight loss of calcite was about 1.1%.

Fig. 5.DSC/TGA data from cement pastes with S. pasteurii after (A) no hydration, (B) 1 day of hydration, (C) 3 days of hydration, (D) 7 days of hydration, and (E) 28 days of hydration. Red line: heat flow; black line: weight loss; solid line: plain cement paste; dotted line: cement paste with S. pasteurii; bold dotted line: cement paste with urea-CaCl2 culture medium; double line: cement paste with S. pasteurii and urea-CaCl2 culture medium.

After 1 day (Fig. 5B), thermal activity appeared in the DSC curves at around 60–110℃ in all the cement paste samples. The thermal activity can be associated with the decomposition of ettringite and also possibly with desorption of water from the C-S-H phases. The data show a noticeabl endothermic DSC peak at 440℃ with accompanying weight loss in the TGA curve. This thermal activity is known to indicate the decomposition of portlandite (calcium hydroxide) [15]. At about 680℃, some amount of weight loss was observed. This can be associated with calcite decomposition. However, the weight loss in the TGA curve was not clearly accompanied by thermal activity in the DSC curve. After 3 days (Fig. 5C), 7 days (Fig. 5D), and 28 days (Fig. 5E), the thermal behaviors of the cement paste samples were similar to those of the 1-day-old specimens. The thermal analysis data were further used to derive the amount of calcium hydroxide in the hydrated cement paste samples in order to support the results of the quantitative XRD analysis.

Quantitative analysis. The amounts of the phases observed in hydrated cement pastes with and without the microorganisms are shown in Tables 2–5. The amount of C3S clearly decreased as hydration progressed, and C3A seemed to react immediately during the first day of hydration, but the amount of C2S did not show a clear trend. Therefore, C2S did not seem to react significantly during the 28-day hydration period with or without the presence of microorganisms. The amount of C4AF showed an overall decrease as hydration progressed.

In all the cement paste samples, the amount of ettringite generally decreased as hydration proceeded. In the plain cement paste and plain cement paste with the microorganisms, the amount of monosulfate was about 0.9% after 1 day, and then it disappeared. As mentioned, the amount of hemicarbonate could not be characterized because of its absence in the ICSD. After 28 days, the amount of monocarbonate in plain cement paste with and without the microorganisms was about 7.2–7.4%. In the cement paste with urea-CaCl2 , the amount of Friedel’s salt increased from 2.7% after 1 day to 9.6% after 7 days and then decreased to 7.2% after 28 days. In the cement paste with urea-CaCl2 and the microorganisms, the amount of Friedel’s salt also increased from 2.0% after 1 day to 10.3% after 7 days and decreased to 6.5% after 28 days.

Figs. 6A-6D show the amount of calcium hydroxide (obtained by Rietveld quantitative analysis) in the cement paste with and without the microorganisms. The amount of calcium hydroxide generally increased as hydration progressed. The specimens with the microorganisms had more calcium hydroxide after 3 and 7 days, but the amount of calcium hydroxide decreased between 7 and 28 days.

Fig. 6.Calcium hydroxide content of cement pastes with and without S. pasteurii. (A) Calcium hydroxide content of plain cement paste obtained by Rietveld quantitative analysis, (B) calcium hydroxide content of cement paste with S. pasteurii obtained by Rietveld quantitative analysis, (C) calcium hydroxide content of cement paste with urea-CaCl2 culture medium obtained by Rietveld quantitative analysis, (D) calcium hydroxide content of cement paste with S. pasteurii and urea-CaCl2 culture medium obtained by Rietveld quantitative analysis, (E) calcium hydroxide content of plain cement paste obtained by DSC/TGA, (F) calcium hydroxide content of cement paste with S. pasteurii obtained by DSC/TGA, (G) calcium hydroxide content of cement paste with urea-CaCl2 culture medium obtained by DSC/TGA, and (H) calcium hydroxide content of cement paste with S. pasteurii and urea-CaCl2 culture medium obtained by DSC/TGA.

The amount of calcium hydroxide in the cement paste samples was also verified using DSC/TGA (Figs. 6E-6H). The weight loss at around 400-450℃ was quantified because calcium hydroxide is known to decompose in this temperature range [15]. From the TGA results shown in Figs. 6E-6H, the cement paste with the microorganisms always showed a higher amount of calcium hydroxide for the first 7 days. After 28 days, although the plain cement paste with the microorganisms showed slightly more calcium hydroxide than the plain cement paste, the cement paste with urea-CaCl2 and the microorganisms showed slightly less calcium hydroxide than the cement paste with only urea-CaCl2 . According to the quantitative phase analysis of the cement paste with and without the microorganisms, the presence of S. pasteurii affected the hydration of the cement paste by reducing the amount of calcium hydroxide after 28 days.

 

Discussion

The microorganism S. pasteurii was found to increase the early hydration rate of cement paste (Fig. 3). This finding can be related to an early increase in the amount of calcium silicate hydration. Because it is difficult to characterize the amount of C-S-H owing to its amorphous nature, the amount of calcium silicate hydration was evaluated using the amount of calcium hydroxide. When the microorganisms were used, the amount of calcium hydroxide clearly increased after 3 and 7 days regardless of whether the urea-CaCl2 culture medium was used. This tendency seemed to be maintained until 28 days, but the increase in the amount of calcium hydroxide was not clearly observed after 28 days when urea-CaCl2 was present. The results of XRD and DSC/TGA in this research do not clearly verify the formation of calcite by MICP when the microorganisms were directly incorporated during mixing. To remove the problem of a lack of nutrition and to promote MICP, the urea-CaCl2 culture medium was incorporated into the cement paste during mixing (cement paste samples were mixed with urea-CaCl2 culture medium at w/c = 1). This was done to investigate whether the microorganisms can be active when sufficient nutrition is available, even though they were captured within the pore structure of the cement paste with lack of oxygen. Note that the specimen was not cured in the urea-CaCl2 culture medium, but the amount of nutrition for MICP when hydration began was sufficient considering the 1:1 ratio of water (in this case, urea-CaCl2 culture medium) to cement.

The XRD patterns of cement paste with 107 cells/ml (Fig. 4) were similar to that of plain cement paste. The decrease in the calcite peak and the peak widening at 29.4° (angle 2θ) were observed in all the cement paste samples after 3, 7, and 28 days. The peak intensity at 29.4° also decreased as a function of the hydration time. This result may indicate that small or poorly crystalline calcite was formed, but amorphous calcite usually does not form because calcite is very crystalline, with a strong preferred orientation that yields a sharp XRD peak. Therefore, after 1 day, the observed XRD pattern at 29.4° was calcite, but the calcite was consumed to form hemicarbonate after 3 and 7 days and later to form monocarbonate after 28 days. The peak widening at 29.4° after 3, 7, and 28 days is expected to be better correlated to the formation of C-S-H, as indicated in other reports [2].

It is still possible to consider that the calcite produced by MICP was consumed to form hemicarbonate and monocarbonate. The transition from hemicarbonate to monocarbonate after 28 days can be related to the results of MICP. However, no clear differences between the XRD patterns of cement paste with and without the microorganisms were observed, and the thermal analysis (DSC/TGA) provided no clear evidence for MICP. In fact, the TGA curve gave some indication of calcite decomposition, but it was not clearly associated with the DSC peak. In addition, the amount of calcite in the samples with the microorganisms did not differ from the amount in the plain cement paste sample.

The main difference between the XRD patterns of plain cement paste and that with the urea-CaCl2 culture medium was the formation of Friedel’s salt. However, as mentioned earlier, the formation of Friedel’s salt was associated with the presence of CaCl2 in the urea-CaCl2 culture medium. Other than that, no clear differences between cement paste with or without the microorganisms and with or without urea-CaCl2 were observed. Further research is necessary to understand the role of the microorganisms in the hydration kinetics of cement paste when they are directly incorporated into the system.

According to the results, it can be concluded that calcite formation (MICP) was not observed when the microorganisms were directly incorporated into the cement paste during mixing, regardless of whether the urea-CaCl2 culture medium was used. It seems that the microorganisms were active during the first day of hydration (as evidenced by the calorimetry peak). After hardening, continuous MICP did not seem to occur; thus, it is possible that the metabolism of the microorganisms ceased after they were captured within the pore structure of the cement paste. In other words, the inactivity of the microorganisms might have been associated with the lack of available oxygen and carbon dioxide for continuous MICP (metabolism of S. pasteurii microorganisms). In addition, the microorganisms might have occupied the available pore space, which could inhibit the growth of hydration products. It is not clear whether the microorganisms can recover their activity when they are open to the ambient conditions. It is also not clear whether the produced calcites are incorporated into the AFm structure as hemi- or monocarbonate. Further research is necessary to answer these questions. However, it is certain from our microscopy observation that MICP occurred under ambient air conditions (Figs. 1 and 2). The results from other studies also indicate that MICP occurs when the microorganisms are supplied from outside with sufficient nutrition (urea-CaCl2 culture medium) [10, 12, 13]. We found that the microorganisms only accelerated the early hydration of cement paste.

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