Effect of Microbial Calcite Precipitation on Physical and Mechanical Properties of Cold Asphalt Emulsion Mixtures

Abstract

In this study, the effects of Microbial Carbonate Precipitation (MCP), on physical and mechanical properties of Cold Asphalt Emulsion mixtures (CAEMs) have been investigated. MCP method was performed on CAE mixtures by two scenarios: (i) Bio-deposition treatment of aggregates (3 days for bio-deposition treatment and 28 days for CaCO₃ fixation), and, (ii) Using a medium culture containing bacteria and urea-CaCl₂ nutrient solution as a replacement for optimum water content, in the manufacturing process. Additionally, in the second scenario, the effect of different curing durations (7, 14, 28 and 56 days) on the mixtures’ properties has been examined. In order to ensure the formation of CaCO₃ precipitation on the aggregates (first scenario), X-Ray Diffraction (XRD) and Fourier-Transform Infrared Spectroscopy (FT-IR) tests were used. Atomic Force Microscopy (AFM) and FE-SEM technique were also employed to evaluate the micro-roughness of aggregates’ surface and the effect of MCP on the structure of CMA mixtures, respectively. Their results indicated that the CaCO₃ precipitations were formed on the aggregates, and therefore, the roughness was increased on the surface of treated aggregates. Ultrasonic Pulse Velocity (UPV), Indirect Tensile Strength (ITS) and Semi-Circular Bending tests (SCB) were performed to investigate the mechanical properties of the cold asphalt mixtures. In order to assess the significance of the tests’ results, analysis of variance (ANOVA) was done at 95% confidence level. Results indicate that the cold asphalt mixtures containing bio-deposition treated aggregates (first scenario) have significantly higher dynamic modulus, indirect tensile strength, fracture energy and resistance to crack propagation, compared with the control sample. In cold asphalt mixtures that are cured during the mixing process (second scenario), significant improvements were also observed in the mechanical properties compared with control samples, after 28 days of curing, but this improvement is still lower than that of asphalt mixtures with bio-deposition treated aggregates. Findings revealed that MCP method can significantly improve the mechanical properties of cold asphalt mixtures. But, it doesn’t significantly affect their physical properties.

Fig. 1. Bio-deposition treatment process on the surfaces of aggregates

Fig. 2. The Surfaces of aggregates (A) before bio-deposition treatment (B) after bio-deposition treatment

2.4. Sample preparation and testing program

Table 3. Testing program for manufacturing of CMA mixtures by different scenarios of treatment

                                  *At-least, 3-5 samples were tested.

Fig. 4. CMA, AT-CMA and BS-CMA manufactured by Gyratory Compactor device

2.5. Testing procedures

2.5.1. Fourier-Transform Infrared Spectroscopy (FT-IR) and X-Ray Diffraction (XRD)

To prepare specimens for XRD and FT-IR tests, a number of coarse aggregates were selected. A layer of aggregates was removed using abrasive device, and scenario I was performed on these aggregates. After bio-deposition treatment on aggregates’ surfaces, they were washed by distilled water, to remove extra materials, and then were placed in the oven to dry. Next, a thin layer was again removed from aggregates’ surfaces, and, two remaining powders were prepared for the above-mentioned tests. FT-IR test was performed for identifying chemical changes on aggregates’ surfaces, before and after bio-deposition treatment. This test is based on absorbance and transmitted radiation intensity of molecules and multi-atomic ions. Fourier-transform infrared spectroscopy test is a useful tool in identifying mineral compounds and their functional groups [26-28]. Around 95% of the spectroscope’s application is qualitative which is based on the peak absorbance. IR spectra were obtained in the absorption mode with a resolution of 400 cm^-1 and 4000 cm^-1 scans. The purpose of the XRD test is to identify the formed precipitation on aggregates, before and after bio-deposition treatment. This test is performed utilizing an Explorer GNR X-ray Diffractometer with Dectris detector Cu-K radiation operating at 40 kV and 30 mA. The scan was conducted in fixed time intervals. The time interval and the step length were 1 second and 0.01  respectively. The scan step for the scattering angle was ( ) set between  and .

2.5.2. Atomic force microscopy method

Surface roughness for each material can be evaluated using AFM method in the non-contact mode. Non-contact mode is the mode in which the probe is not in direct contact with the surface of specimen, and vibrates in top of it during the scan process. The AFM method can provide roughness data more precise than the surface roughness tester device, but, this scan can only be performed on small areas of surface. Average roughness (Ra) and root mean square roughness (Rq) obtained from AFM method, are used as specimen surface specifications. Also, 3D photos with high resolution can be obtained from the surface of specimens, using AFM. In this study, 2D roughness profile, 3D surface changes and surface roughness specifications of aggregates were compared using AFM, before and after treatment by scenario I.

 2.5.3. Indirect tensile strength test

 Indirect tensile strength test is employed to determine tensile specifications of asphalt mixtures that are related to cracking specifications of the mixture [29]. Specimens manufactured for this purpose, which were mentioned in Table 3, were subject to loading, utilizing a UTM-14P device, and according to AASHTO T283 standard, with a 500 mm/min   displacement rate [30]. This test induces a relatively uniform tensile strength along the vertical diametrical plane, by applying a vertical load on the cylindrical specimen, and fracture usually occurs in the same plane [31]. Tensile strength of the specimen can be calculated using Equation 1.

                                                                                                                                                                                                                                                                                               (1)

where S_t; indirect tensile strength (kPa), P; maximum load (N), t; thickness of the specimen (mm), and D is diameter of the specimen (mm).   The area beneath the stress-strain curve on the left side of the maximum point was also measured, so, the fracture energy (U_ITS) can be obtained in kJ/m³   units [32, 33].

2.5.4. Semi-circular bending test

The semi-circular bending test can measure the asphalt mixture’s long term resistance to cracking in intermediate temperature. One of the parameters related to this resistance is the fracture energy (G_f) of asphalt specimens, according to AASHTO TP 105-13 standard [34]. Specimens manufactured for this purpose were manufactured as indicated in Table 3, using a gyratory compactor with a 101mm diameter and a 25mm notch depth. After preparation, the specimens were placed in UTM-14P device in  temperature. When the asphalt mixture reached a constant temperature, to ensure the sample is seated properly on the support, preloading with a 45N load was done for a maximum 30 seconds duration. Then, loading of the specimens was initiated in displacement-control, with a 0.5 mm/min rate. Work of fracture can be calculated by measuring the area under the load versus average load line displacement curve, and the fracture energy can be calculated using Equation 2.

                                                                                                                                                                                                                                                                                                         (2)

 A_lig = (r – a) × t                                                                                                                                                                                                                                                                                                        

where G_f; fracture energy (J/m²), W_f; work of fracture (J), A_lig; ligament area (m²), r; specimen radius (m), a; notch length (m), and t is specimen thickness (m).

 2.5.5. Ultrasonic pulse velocity test

Ultrasonic device produces pulses of longitudinal stress waves with high frequencies, which can be used for determining different functional characteristics in materials. This test is utilized to investigate cracks and voids in the specimens which shows their uniformity and quality, and, can be used to evaluate the effects of different factors on crack healing in concretes. Compatibility of this method with asphalt mixtures have been tested and assessed, and the results were acceptable [35, 36]. Dynamic modulus of elasticity, or, the stiffness can also be estimated by this method. In this research, an ultrasonic device has been used to investigate transit time, and dynamic modulus of elasticity of asphalt specimens. The ultrasonic device includes: a pulse generator, a pair of transducers (transmitter and receiver), an amplifier, a time measuring circuit, a time display unit and connecting cables. At first, the time was calibrated on  using a special metal shaft, then, the pulse transit time was measured by connecting the transducers to specimen’s surface on both sides (Figure 5). Test precision is dependent on transducers’ connection method and the distance between transmitter to receiver (center to center distance). To reduce the measurement error, some grease was applied on the surface between the transducer and the specimen. The connection surface and the pressure of transducers on the specimen’s surface needs to be enough so that the pulse transit time reaches a constant value. The measurement criteria in this test is pulse transit time. Having the pulse transit time, the pulse velocity can be calculated in 54 kHz frequency, using Equation 3.

                                                                                                                                                                                                                                                                                                      (3)

where V denotes pulse velocity (m/s), L denotes the specimen length (m), and T is pulse transit time (s). Having the pulse velocity obtained, dynamic modulus of elasticity can be calculated in MPa by Equation 4.

(4)                                                                                                                                                                                                                                                

Density of the CMA is calculated in kg/m³. Dynamic Poission’s ratio ( ) was assumed to be 0.35 for the cold asphalt mixture.

Fig. 5. The method of performing an ultrasonic test to obtain the transit time of CMA

3. Results

3.1. Effect of bio-deposition treatment on aggregate properties

3.1.1. Weight increase

Bio-deposition treated aggregates that remain on No. 50 sieve, were washed so that non-adhesive remains are eliminated from their surfaces. They were then placed in the oven with 75 ºC temperature, until the weight of the specimens didn’t fluctuate significantly anymore, and then dried aggregates were weighed. In the classification presented by MS-19, the weight of aggregates that remain on No. 50 sieve must be equal to 79% of the total aggregate weight, which is equal to 918.75 g (0.79 1163 g). After performing bio-deposition treatment on three aggregate samples, changes in weight are obtained as shown in Figure 6. According to this figure, the amount of produced precipitation in the mixture of aggregates can be estimated to be around 7.65 g. The observed increase in the specimens’ weight is caused by the CaCO₃ precipitated by bacteria on the surfaces’ aggregates and can be used as a measure for the amount of CaCO₃ produced on aggregates [11, 12]. Surfaces with higher roughness lead to more fixation of precipitations produced on aggregates due to harder slipping of them on aggregates.

Fig. 6. Weight increase of aggregates after before and after bio-deposition treatment

3.1.2. FT-IR test

In the past, FT-IR spectroscopy has been well employed to identify calcite, and researchers have utilized this method to compare the CaCO₃ precipitations, before and after the bacteria agent is applied. In Figure 7, FT-IR spectrum for the aggregates’ surfaces before and after bio-deposition treatment, are compared. Spectrum with wavenumbers close to 712, 875, and 1432 cm^-1 are associated with CaCO₃. Comparing the aggregate’s FT-IR spectrum before and after bio-deposition treatment, it can be seen that CaCO₃ related transmittance in treated aggregate is higher than in the ordinary aggregate. The spectrum associated with  includes spectra with wavenumbers equal to 473 cm^-1 and 468 cm^-1, which are related to  out of plane vibration, and with wavenumbers equal to 1082 cm^-1 and 1094 cm^-1, which are related to  stretching vibration. Spectrum associated with SiO₂ in treated aggregates, show lower transmittance which is due to the CaCO₃ surface layer that reduces the silica phase in this layer.

Fig. 7. FT-IR spectra of the aggregates’ surfaces before and after bio-deposition treatment

3.1.3. XRD test

Compounds that form the surface layer of aggregates play a critical role in adhesion between asphalt and aggregates. Therefore, to better understand the effects of produced bio-deposition on the aggregates’ surfaces, mineral compounds on aggregates’ surfaces, were investigated before and after bio-deposition treatment, using XRD test. Calcium carbonate precipitations have different forms: Calcite which forms the main phase in CaCO₃, vaterite and aragonite.  Among these, calcite is trigonal and shows maximum strength, while vaterite is hexagonal and aragonite is cubic [37]. In Figure 8, different compounds such as Calcite, Dolomite and Silicon dioxide are shown before and after bio-deposition treatment on aggregates. The diffraction peak intensity associated with each compound has a direct relation with the amount of that compound [38].Therefore, calcite’s diffraction peak intensity on aggregates’ surfaces after treatment is more than its intensity before treatment, while lower amounts of dolomite and silicon dioxide can be observed in the specimen, after the treatment. So, the presence of calcite crystals on aggregates’ surfaces can be found out after bio-deposition treatment. Results of FT-IR test in Figure 7, and XRD test in Figure 8, indicate a favorable overlap between these two tests, meaning that FT-IR results can confirm XRD results.

Fig. 8. XRD analysis before and after MCP on the aggregates’ surfaces

3.1.4. AFM test

Materials’ surface roughness was scanned on a  area. Aggregates’ surface roughness properties before and after the treatment, are presented in Table 4. Aggregates’ average surface roughness was equal to 13.34 nm before the treatment and, 27.24 nm after the treatment. In other words, surface roughness of aggregates has increased by 104% after the treatment. Figure 9, shows the aggregates’ surface fluctuations in 3D form, which indicates smoother surface before the treatment. Previous research suggest that bio-deposition fills the voids in aggregates [12] and aggregates take a more uniform texture, as shown in Figure 10. But, conforming to the results of AFM test, the surface roughness increases in microscopic scales, due to the formation of polyhedral calcite crystals. The increased roughness on the aggregates can be attributed to CaCO₃ precipitations on aggregates’ surface, which prevents asphalt from slipping on the surface of aggregates during mixing process, and in turn, increases the aggregate coating. In addition, the roughness of the aggregates’ surface texture has a direct relation with the asphalt mixture’s stiffness [39]. On the other hand, filling the aggregates’ voids with MCP decreases asphalt absorbance, and consequently, effective asphalt and aggregate coating increases.

Table 4. Roughness properties on aggregates’ surface before and after bio-deposition treatment

(a)                                                                                                                                                              (b)      

Fig. 9. The 3D aggregates’ surface profile (a) before and (b) after bio-deposition treatment

                                                                                                                          (a)                                                                                                                                                                                                              (b)

Fig. 10. Void properties and effective asphalt (a) before MCP and (b) after MCP, on aggregates’ surfaces

3.2. Effect of bio-deposition treatment on the properties of cold asphalt emulsion mixtures

3.2.1. Change in physical properties

To investigate the effects of bacteria on mechanical properties of asphalt, maximum specific gravity (G_mm) was measured for three mixtures. After that the asphalt mixtures were manufactured using gyratory compactor device according to the mixture design, weight in air (W_a) and bulk specific gravity (G_mb) was measured for these mixtures. Figure 11 shows changes in physical properties of CMA, AT-CMA and BS-CMA mixtures in comparison with each other. According to Figure 11-a, weight of AT-CMA mixture has increased by 7.06 g higher than CMA mixture which can be attributed to the formation of CaCO₃ precipitations on aggregates’ surfaces. Figure 11-b shows that G_mm for AT-CMA mixture has increased by 0.54% and Figure 11-c indicates that bulk specific gravity has increased by 1.05% compared with the control specimens. This can be attributed to the increased aggregate weight which is directly related to the specific gravity of mixtures. According to Figure 11-d, the air void percentage (P_a) in AT-CMA mixture was reduced in 0.44% compared with the control specimens which is not a significant change. The weight of the BS-CMA specimens had also increased by 3 g higher than CMA mixture. In addition, G_mm and G_mb of the specimens had increased by 0.12% and 0.5%-0.8% respectively, while the air void percentage negligibly reduced.

                                                                                            (a)                                                                                                                                                                                                     (b)

                                                                                          (c)                                                                                                                                                                                                (d)                            

Fig. 11. Comparison of physical properties of asphalt mixtures of CMA, AT-CMA and BS-CMA. a) G_mm,  b) W_a,  c) P_a and d) G_mb

3.2.3. ITS and fracture energy test

 3.2.3.1. Optimum curing time for BS-CMA mixtures

Figure 12, shows average ITS and U_ITS for BS-CMA mixtures. These parameters for BS-CMA mixtures with 7 days of curing were 115 kPa and 0.7 kJ/m³ respectively. These values increased by 29% when the curing duration was 14 days, but, according to Table 5 (one-way ANOVA), these increases are not significant (P-value>0.05). The values of tensile strength and fracture energy for 28-day curing duration were 63% and 75% higher than the values of 7-day curing duration respectively. These increases on the other hand, are significant according to Table 5 (P-value<0.05). The above mentioned properties for 56-day cured specimens were not significantly different from those of 28-day sample. Thus, 28 days is a proper curing duration for evaluating these mixtures based on their indirect tensile strength and fracture energy.

Fig. 12. Effect of curing time on ITS and U_ITS of BS-CMA

Table 5. ANOVA and Dunnett comparison at 95% confidence level for the effect of curing time on ITS and U_ITS of BS-CMA

Means labeled with the (*) are significantly different from the control level mean.

 3.2.3.2. Comparison of ITS and fracture energy (U_ITS)

As mentioned before, in the literature, the proper curing time for reaching constant values of indirect tensile strength for common CMA mixtures is 7 days, but, in this research, the optimal curing duration for BS-CMA mixtures was estimated 28 days. CMA specimens were manufactured with proper curing durations associated with them, and were subject to loading in UTM-14P device (Figure 13), then, as shown in Figure 14,  and  of these mixtures were compared with each other.  and  for AT-CMA mixtures were 38% and 46% higher than CMA mixtures (control specimens) respectively. These values were 20% and 43% higher than the control specimens, for BS-CMA mixtures, respectively. Thus, AT-CMA specimens has the highest  and  among all mixtures. Figure 15, shows an example of stress-strain curve for CMA mixtures in ITS test.

Fig. 13. ITS test with universal testing machine (UTM)

Fig. 14. ITS and fracture energy (U_ITS) of CMA, AT-CMA and BS-CMA asphalt mixtures during optimal(proper) curing of each mixture

Fig. 15. Stress-strain curve of CMA mixtures related to the ITS test

3.2.4. Semi-circular bending test

In this test, the fracture energy (G_f) represents the resistance of specimen to cracking under bending. Specimens were subject to loading in UTM-14P device (Figure 16), after preparation and cutting. Figure 17, shows the obtained fracture energy (G_f) for each mixture, in semi-circular bending test.  for AT-CMA and BS-CMA mixtures were 292% and 104% higher than the control specimens, respectively. Therefore, AT-CMA mixture has the highest resistance to cracking at  temperature, amongst all mixtures. Based on the results of ITS and SCB tests, it can be concluded that MCP increases ITS, U_ITS and G_f in AT-CMA mixtures, by inducing adhesion between aggregates and asphalt. Also, the above-mentioned properties were enhanced in BS-CMA mixtures, due to crystallization of CaCO₃. Figure 18 shows an instance of load-displacement curve for CMA mixtures in SCB test.

Fig. 16. SCB test with UTM-14P

Fig. 17. Fracture energy (G_f) measured by SCB test in CMA, AT-CMA and BS-CMA asphalt mixtures

Fig. 18.   An example of the load-displacement curve of CMA mixtures related to the SCB test

3.2.5. Ultrasonic pulse velocity

This test expresses the stiffness of asphalt specimens. As mentioned before, rougher aggregates’ surfaces and more adhesion between asphalt and aggregates result in higher stiffness in asphalt mixtures. Figure 19, shows pulse velocity and dynamic modulus of elasticity in asphalt mixtures. Regarding this figure, pulse transit time and dynamic modulus of elasticity in AT-CMA mixtures were 23% and 54% higher than CMA mixture, respectively. For BS-CMA mixtures, these values were higher than CMA mixture by 9% and 20% respectively.

Fig. 19. Pulse velocity and dynamic modulus of elasticity of CMA, AT-CMA and BS-CMA asphalt mixtures

3.2.6. Field Emission Scanning Electron Microscopy (FE-SEM)

In order to evaluate the effect of MCP on the structure of CMA mixtures, the SEM technique was used. Since the mechanical properties are dependent on the structure of mixtures, analysis of these images can be very useful in identifying and determining the causes of changes in tests results. Figure 20 show the FE-SEM images for the CMA mixtures with 5000X and 500X magnifications. Figure 20-a and 20-b show the structure of CMA specimen (control specimen).

According to Figure 20-d small particles were formed on the surface of the BS-CMA specimen. The size of the particles was around 0.5-2 µm with cubic shapes that are probably aragonites (Figure 20-e). The non-active bacteria are also completely visible in the BS-CMA mixture. In Figure 20-h, which shows the AT-CMA specimen, the particle sizes were much larger (around 5-10 µm) with spherical and hexagonal shapes. Bacterial imprints can be clearly seen on the particles. The chemical composition of the particles was calcium carbonate, indicated by the EDS spectra in Figure 20-f for BS-CMA and Figure 20-i for AT-CMA. By comparing the CMA, AT-CMA and BS-CMA mixtures in Figure 20, it is observed that the surface of untreated CMA specimen was relatively smooth compared with that of the treated, namely, AT-CMA and BS-CMA.

Fig. 20. FE-SEM images of untreated and treated CMA mixtures (a and b: CMA surface; d and e: BS-CMA surface; g and h: AT-CMA surface at 5000 and 500 magnification; c, f and i: EDS spectra of the particles in the red rectangle in a, d and g).

3.2.7. Analysis of variance

To evaluate the significance of both types of treatment scenarios mentioned in this study, an analysis of variance was performed at 95% confidence level, on CMA mixtures containing B.pasteurii. Table 6. Indicates that scenario I for AT-CMA and scenario II for BS-CMA, induce significant changes (P-value<0.05) in their weights, while other physical characteristics were not significantly changed. Results of the ultrasonic test (pulse velocity and dynamic modulus of elasticity) for AT-CMA mixtures show significant changes compared with control samples, but, for BS-CMA mixtures these results were not significantly fluctuated.

Table 6. ANOVA test at 95% confidence level on physical and mechanical properties of the different type of treated CMA mixtures

Means labeled with the (*) are significantly different from the control level mean.

4. Discussion

In this study, Effects of MCP has been investigated on physical and mechanical properties of CMA mixtures in 2 different scenarios: (i) treatment of aggregates’ surfaces by creating a bio-deposition layer, and then manufacturing the CMA, (ii) utilizing a medium culture containing bacteria and urea-CaCl₂ nutrient solution, as a replacement for water, during mixing process of CMA. Results of XRD and FT-IR tests indicate the formation of a CaCO₃ precipitation layer on the surfaces of aggregates, by B.pasteurii. The AFM test show that the aggregates’ surface roughness was increased by formation of CaCO₃ on surface. Generally, from the results of AFM, ITS, SCB and ultrasonic for AT-CMA, it can be concluded that the bio-deposition treatment on aggregates’ surfaces can improve the mechanical properties of these mixtures in 3 ways: (i) bio-deposition fills the voids in aggregates, and as shown in Figure 10, aggregates take a more uniform texture (macro texture), but, conforming with the AFM test results, in microscopic scales, surface roughness increases due to formation of polyhedral calcite crystals. This change in roughness is caused by the formation of bio-deposition by bacteria, and can prevent asphalt from slipping on the surfaces of aggregates, and therefore, increase the aggregate coating. In addition, rougher aggregates’ surfaces micro texture lead to higher stiffness in asphalt. (ii) Filling the aggregates’ voids by the bio-deposition causes the asphalt absorbance to reduce, which in turn, increases the effective asphalt and the aggregate coating. (iii) The produced CaCO₃ improves the adhesion between asphalt and aggregates, due to its calcareous characteristic. Results of the ITS and SCB experiments similar to work of Pan et. al. [9] confirm this statement. Since the roughness of BS-CMA samples undergo lower variations, and also, the pH of the mixture is low in the presence of cationic asphalt, the bacterial activity and consequently MCP functions are weakened compared with AT-CMA specimens. Thus, changes in mechanical properties of the BS-CMA asphalt mixtures, especially stiffness and dynamic modulus of elasticity, are lower than changes in AT-CMA, in which the treated aggregates make the surfaces rougher and the pH is more favorable for bacterial activity. But, BS-CMA mixtures still show improved mechanical properties compared with the control specimens, except for dynamic modulus of elasticity. This can be attributed to enhanced rheological properties of the asphalt due to production of CaCO₃ precipitation which makes better asphalt mastic compared with the control sample, which also improves the aggregate coating. Due to acidity of cationic asphalt emulsion, using anionic asphalt emulsion is recommended since more CaCO₃ precipitation will likely be produced and the bacterial death rate will reduce, but, the mixture must be prevented from getting extremely alkali and the pH must remain in the allowed range ( ).

5. Conclusion

Based on the experiments conducted in this research, and treatment scenarios (scenario I for AT-CMA and scenario II for BS-CMA using B.pasteurii), the following conclusions can be drawn:

• Results of the FT-IR test indicate increased transmittance for CaCO₃ bands and decreased transmittance for  bands. This variation is due to formation of CaCO₃ and reduction of silica compounds on the surfaces of aggregates. Results of XRD test show higher calcite related diffraction peaks on the surfaces of aggregates, after bio-deposition treatment. Therefore, results of both these tests show great overlap to prove CaCO₃ precipitation on aggregates’ surfaces, which suggests that the bio-deposition treatment was successfully done.
• In microscopic scales, surface roughness of bio-deposition treated aggregates was higher than the roughness before treatment in the AFM test. Therefore, this rougher surface leads to higher stiffness, lower asphalt absorbed, higher effective asphalt, lower slipping of asphalt emulsion during mixing, and consequently, better aggregate coating, in cold asphalt mixtures.
• After the treatment was performed on the aggregates, their weight was increased by 7.65 g which is due to CaCO₃ precipitation on the surfaces of aggregates. The weight increase in BS-CMA mixtures is also caused by CaCO₃ precipitation produced by bacteria.
• MCP treatment did not cause significant changes (at 95% confidence level) in physical properties of AT-CMA and BS-CMA mixtures.
• In BS-CMA mixtures,  and  of 28-day cured specimens were 63% and 75% higher than 7-day cured specimens, respectively, which is a significant variation (P-value<0.05). These variations were not significant for curing durations more than 28 days. Thus, 28 days is a proper curing duration for the  and  to reach constant amounts. This phenomenon is caused by the fixation of precipitations produced by bacteria in 28-days, which remains constant after that.
• , , and,  which express tensile strength, fracture energy caused by tension in  test, and, resistance to cracking, respectively, were increased by 38%, 46% and 292% for AT-CMA mixtures(P-value<0.05). these values for BS-CMA mixtures were 20%, 43% and 104% compared with the control specimens, in that order (P-value<0.05).(at??)
• Ultrasonic test results revealed that the pulse velocity and dynamic modulus of elasticity in AT-CMA samples increased by 23% and 54% respectively, compared with CMA (P-value0.05). Thus, stiffness and as a result, ultrasonic pulse velocity in AT-CMA mixtures were increased significantly, while these variations were not significant for BS-CMA mixtures.
• The FE-SEM images for the CMA mixtures with 5000X and 500X magnifications were used. The size of the particles for CaCO₃ in BS-CMA specimen was around 0.5-2 µm with cubic shapes that are probably aragonites. the particle sizes of CaCO₃ in AT-CMA specimen were much larger (around 5-10 µm) with spherical and hexagonal shapes. Also, the surface of untreated CMA specimen was relatively smooth compared with that of the treated, namely, AT-CMA and BS-CMA.

6. Future research

Using anionic asphalt is recommended for MCP method, with the condition that the mixture must not get excessively alkali ( ). The reason for this recommendation is the higher possibility of CaCO₃ precipitation, and lower bacterial death rate in anionic asphalt. The MCP method can also be used in concrete to heal or prevent Alkali-Silica Reaction (ASR).

7. Acknowledgement

The authors would like to acknowledge Ferdowsi University of Mashhad for financial support by Grant NO. 3/44800. Also, we would like to thank Pavement laboratory technicians (Mr. Hajinezhad and Fanoudi) and Microbiology laboratory technician (Ms. Pordeli), for their support and Ms. Khamar for her invaluable contribution in microbiological works in this study.

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