Research on the Preparation of Microbial Capsules by Epoxy Resin-Coated Bacillus pasteurii

Abstract

With the increasing number of underground engineering construction projects such as coal mining, tunnel, and subway, water inrush disasters occur more and more frequently. Inspired by the phenomenon of microbial mineralization and diagenesis, microbial-induced calcium carbonate precipitation (MICP) is used to repair cracks in cement-based materials, which provides a new idea to solve the problem of water inrush. To investigate the self-healing properties of microbial capsules, this paper selected epoxy resin E-51 cured by DMP-30 as the wall material and Bacillus pasteurii as the core materials for experiments. In this paper, a single-factor method was adopted to determine the optimal preparation process of microbial capsules and the oil-phase separation method to prepare the microbial capsules. The effects of various factors on the experimental results under different core-wall ratios, reaction time, reaction temperatures, and agitation rates were analyzed. Microbial capsules were analyzed by Fourier transform infrared spectroscopy and optical microscopy to explore the functional groups and features of microbial capsules. The experimental results showed that the microbial capsules achieved the best performance with a core-to-wall ratio of 1 : 3, a reaction temperature of 50°C, a reaction time of 40 min, and a stirring rate of 300 rpm. Meanwhile, we determined the spore survival rate of microbial capsules and finally studied the waterproofness, storage stability, and rupture under the pressure of microbial capsules. We concluded that microbial capsules have high-efficiency and self-healing properties.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

In the underground engineering, microcracks often occur in concrete layer, which not only reduces the bearing capacity of the concrete structure but also affects its durability and waterproofness [1–8]. Currently, a more effective method for repairing concrete cracks is microcapsule technology. This technique involves coating the desired material with a suitable material to form a core-wall structure. The material used for coating is called a wall material, and the material to be coated is called a core material. The wall material is usually selected from organic polymer materials and inorganic materials, and the selection of the core material is determined according to the application of the microbial capsules. When microcapsule technology applies to the crack repair of concrete, its essence is the use of microorganisms to induce the formation of calcium carbonate. A specific microorganism is coated as a core material and embedded in concrete [9–13]. When the concrete cracks, the microbial capsule will break, and microbial metabolism will produce calcium carbonate, thereby repairing the crack.

At present, microcapsule technology has made some progresses in concrete repair. To improve cemented coral sand’s self-healing efficiency, Xu et al. [14] fabricated the urea-formaldehyde (UF) microcapsule with in situ polymerization using controlled particles size distributions. They confirmed that the specimen’s crack or compression triggered the microcapsules, which then reacted with the contribution from the healing agent to self-heal the internal cracks. Singh et al. [15] found that the crack healing capacity of the calcium lactate-prepared samples significantly improved, in spite that the expanded clay aggregates-based samples featured a considerable decrease in strength. Also, the use of Bacillus sp. bacterial solution in the concrete mix enhances its mechanical properties. Wang et al. [16] studied the physical properties of microcapsules and the microstructure of self-healing concrete. They also analyzed the influence of microcapsules on the strength, permeability, and long-term shrinkage of self-healing concrete. Al-Tabbaa et al. [17] focused on microcapsules, which contain a healing agent. This healing agent will be released upon crack propagation. Han et al. [18] pointed out that bacteria with expanded clay (EC) are very active in generating CaCO₃. Liu [19] prepared a kind of microcapsule and manufactured a new type of self-healing asphalt. To do so, he mixed asphalt, microcapsule, and curing agent with proper ratio. Mohamed et al. [20] found that efficient self-healing capacity not only requires sufficient healing compounds (e.g., calcium acetate) but also demands a minimal number of bacterial spores. Tripathi et al. [21] studied the effect of microcapsule shell material on the mechanical behavior of self-healing epoxy composites. They encapsulated the liquid epoxy healant in melamine-formaldehyde (MF) and urea-formaldehyde (UF), using the emulsion polymerization technique to prepare microcapsules of different shell walls. Singh and Gupta [22] produced self-healing mortar by using alkaline bacteria producing minerals. Shahid et al. [23] focused on the microcracks’ self-healing capability in concrete by using different Bacillus strains. They achieved a 16% increase in compressive strength in concrete and significant healing with Bacillus subtilis. Hu et al. [24–26] considered three novel types of healing agent encapsulation systems, namely, body-contact double capsule, parallel-style double capsule, and concentric-style capsule. They examined the compatibility of the PU healing agent with a dynamic crack using different accelerants. They also investigated the crack’s adhesion area for different healing systems in terms of the capsule type, capsule spacing, accelerant, and crack width. Al-Ansari et al. [27] characterized the average diameter and shell thickness of the produced microcapsules. Souza and Al-Tabbaa [28] found that the aqueous core microcapsules presented a thicker shell which precluded the rupture upon crack. Cheng [29] researched into the epoxy resin microcapsules which contain both solid particles and cured epoxy resin. He finally obtained the thickness of the shell coated with alkali-resistant Bacillus H₄.

Wang et al. [30, 31] used alginate as the wall material and successfully prepared microbial capsules by using Bacillus sphaericus fixed in hydrogel as the core material, thus effectively protecting the bacterial activity. Van Tittelboom et al. [32] adopted epoxy resin as the wall material and Bacillus coli as the core material to make spore bio-microbial capsules, which solved the problem of spore burial and dormancy in concrete. Cheng [33] prepared microbial capsules with ethyl cellulose and epoxy resin as the wall material, respectively, and Bacillus alkalisediminis H₄ as the core material. The experimental results showed that microbial capsules have an excellent repair effect on concrete cracks [34, 35]. There are still some issues in the existing research that require further in-depth research. Among them, one of the critical issues is how to reduce the preparation cost and efficiently utilize the material to prepare microbial capsules with the best biocompatibility and mechanical triggering performance.

This paper investigated the application of epoxy resin as a bacterial carrier in the self-repairing system of Bacillus pasteurii. First, we designed and successfully prepared Bacillus pasteurianum microcapsules, optimized the microcapsule preparation process through experiments, and obtained the process parameters such as the core-wall ratios, reaction temperatures, reaction time, and agitation rate required to achieve the best performance. Next, we characterized the performance of the prepared microcapsules and studied the biocompatibility of epoxy resin and Bacillus pasteurii by measuring the spore survival rate of the microcapsules. In addition, the strength test of the prefabricated cracks in this paper confirms that the prepared microbial capsules have high-efficiency self-healing properties. The research results of this paper provide new ideas for studying a wider range of self-repairing bacterial carriers and improving the repair activity and strength repair rate of bacteria in harsh environments such as mines.

2. Experiments and Materials

2.1. Selection of Wall Materials

In the preparation of microbial capsules, in order to achieve the best results, the wall material needs to meet specific requirements. First, to prevent the core material from oxidizing and volatilizing during storage, the wall material needs to have good airtightness. Meanwhile, the wall material must have a specific mechanical strength to ensure the isolation and protection of the core material. The wall material also needs to have suitable brittleness to guarantee that the core material can be released typically when the triggering condition is reached.

On the other hand, in the preparation of microbial capsules, attention should be paid to the adverse effects of concrete conditions on microorganisms. In other words, we should prevent outside air, moisture, and oxygen from entering the microbial capsules to cause spores to germinate in advance. Furthermore, the wall material needs to be biocompatible and low in toxicity to maintain the activity of the core material.

Epoxy resin is not only dense, waterproof, and mechanically robust and brittle but also nontoxic and has excellent biocompatibility. Therefore, in all aspects, this paper chooses epoxy resin E-51 as the wall material.

Table 1 shows the experimental instruments. This paper utilized the ultraclean workbench and Chinese medicine pellet machine for the preparation of bacterial core material. Meanwhile, we used a digital constant temperature water bath and drying box to complete the wall material to cover the core material. We observed the microbial capsule’s core-wall structure with an optical microscope and the absorption peak in the infrared spectrum of the microbial capsule by Fourier transform infrared spectroscopy. Additionally, a television microscope was used to explore the waterproofness, storage stability, and rupture ability under the pressure of the microbial capsules. The survival rate of the spores was measured using the biochemistry incubators.

Table 1

Experimental instruments.

Experimental instruments	Model	Manufacturer
Electronic balance	BSA223S	Sartorius Instrument Co., Ltd
Television microscope	SA3300	Beijing Tech Instrument Co., Ltd
Digital constant speed electric mixer	JJ-1A	Jintan Hongye Experimental Instrument Factory
Digital constant temperature water bath	HH-1	Changzhou Jintan Kexing Instrument Factory
Electric blast drying box	Model 101	Beijing Ever Bright Medical Treatment Instrument Co., Ltd
Chinese medicine pellet machine	HBZ-201	Ruian Hanbo Electromechanical Co., Ltd
Fourier transform infrared spectrometer	NICOLET380	Thermo Nicolet Co., Ltd
Powder tablet press	PC-24	Tianjin JingTuo Instrument Technology Co., Ltd
Biochemical incubator	HPX-80	Shanghai Hengyue Medical Equipment Co., Ltd

2.2. Selection of Core Materials

The microbial capsule core must be a microorganism with life activity and mineralization. Microbial mineralization types fall into two types: microbial-controlled mineralization and microbial-induced mineralization. Microbial-controlled mineralization depends on the physiological processes of microorganisms and has nothing to do with changes in the external environment, while microbial-induced mineralization is produced by activities such as cell metabolism.

This research selects Bacillus pasteurii as the core microbe that adopts the microbially induced mineralization. More specifically, we make microbes into dry spore powder and mix the powder with the required nutrients and auxiliary materials such as microcrystalline cellulose to make core particles. Meanwhile, we induce calcium carbonate by cell metabolism to self-repair the concrete.

2.3. Measurement of Curing Agent Effect

To explore the effect of the curing agent, the epoxy resin E-51 was precured separately in advance with three curing agents: 2,4,6-trisphenol (DMP-30), m-xylylenediamine (MXDA), and silane coupling agent (KH-151). The amount of the curing agent MXDA is usually calculated based on the active hydrogen equivalent of the amine curing agent. Figure 1 illustrates the structural formula of MXDA.

[figure omitted; refer to PDF]

The molecular weight of MXDA is 136.19, and the number of active hydrogens is four. Accordingly, its active hydrogen equivalent is 34.05. The epoxy value of epoxy resin E-51 is 0.51, and the theoretical amount of MXDA is 17.37 g per 100 g of epoxy resin E-51. Its calculation formula is shown as follows:$$\begin{array}{}\text{(1)}& W=\frac{M}{N}\times E,\end{array}$$where W is the amount of curing agent required per 100 g of epoxy resin E-51, M is the molecular weight of MXDA, N is the number of active hydrogens of MXDA, and E is the epoxy value of the epoxy resin.

The amounts of curing agents DMP-30 and KH-151 were added according to empirical values. That is, the amount of DMP-30 is 10 g per 100 g of epoxy resin E-51, and the amount of KH-151 is 15 g.

In order to determine the curing effect, we placed 20 g of epoxy resin E-51 in a 50°C water bath for 10 minutes, diluted, and added with a curing agent. The bubbles were uniformly discharged after we stirred it, and we then placed it in a water bath for heat preservation. Afterward, we placed it in a beaker and observed the curing phenomenon. Table 2 shows the control experiments.

Table 2

Control experiments.

Number	Experimental conditions
	Curing agents	Water bath temperature °C	Duration of heat preservation min	Curing agent amount g
1	DMP-30	50	10	2
2	MXDA	50	10	3.5
3	KH-151	50	10	3
4	DMP-30	20	10	2
5	MXDA	20	10	3.5
6	KH-151	20	10	3

2.4. Microbial Capsule Preparation

2.4.1. Core Material Preparation

The microcapsule core material is prepared by mixing dry spore powder, auxiliary materials, and nutrients and then granulating by a granulation apparatus that is shown in Figure 2. The specific preparation method is as follows:

  Step 1. Mixing stage: Weigh nutrients according to the proportion of the medium components of the optimized strains, i.e., weigh 1 g of urea, 0.25 g of soy peptone, 0.75 g of casein, and 0.25 g of sodium chloride. Add 50 g of microcrystalline cellulose for expanding the core volume and 1.5 g of hydroxypropyl methylcellulose to promote adhesion of microcrystalline cellulose to spores and nutrients.

  Step 2. Grouping stage: Weigh 0.5 g of dry spore powder in a beaker and add 150 g of distilled water to stir. Add the liquid solution to the mixture and stir to form a paste.

  Step 3. Granulation stage: Place the obtained paste from the previous step in the extrusion port of the Chinese medicine pelletizing machine and obtain a flat body after being extruded appropriately. Take out the flat body and place it at the mouth of the purlin to obtain a strip. The strips are separated into individual pieces and placed at the pellet opening to roll out the granules. Place the pellets in a rounder, take out, and dry in a low-temperature drying oven at 40°C for 24 hours. Finally, we obtain core particles with single-particle size.

[figure omitted; refer to PDF]

The flowchart of preparing the core material is shown in Figure 3. The nutrients contain urea, soy peptone, casein, and sodium chloride; and the auxiliary materials encompass microcrystalline cellulose and hydroxypropyl methylcellulose.

[figure omitted; refer to PDF]

The specific preparation steps are as follows:

(1) Weigh the required materials according to the required amount, add cement, sand, and water in sequence, mix well, and finally add the microcapsules and mix well.

(2) Brush a layer of grease on the inside of the mold, pour the evenly mixed mixture into the mold, and shake it up.

(3) After leaving the mixture at room temperature for one day, demold. Then, put it in a standard constant temperature and humidity curing box (temperature 25°C; air humidity 90%), and stay until a specific time.

2.6.2. Prefabricating Crack for Test Pieces

This paper took the cement mortar test pieces within the curing period and manually prefabricated cracks. Based on a comprehensive analysis of various prefabricated cracking methods, this paper adopted a universal pressure testing machine to prefabricate cracks for cylindrical specimens, as shown in Figure 6. The specific steps are as follows:

(1) Place the test piece in the center of the pressure-bearing plate of the testing machine, and adjust the bottom disc to a balanced state.

(2) Apply the axial stress at a displacement speed of 0.01 mm/min, and prefabricate the crack using a concentrated load in the center.

(3) When the obvious cracking sound occurs, stop the pressurization. By then, we can obtain a striped crack that diverges from the center of the test piece to the surroundings.

(4) Record the failure load and calculate the compressive strength according to the following formula:$$\begin{array}{}\text{(3)}& f=\frac{F}{A},\end{array}$$

  where f is the compressive strength of cubes of similar rock text pieces (MPa); F is the failure load of the test piece (N); A is the pressure area of the test piece (mm²).

(5) Record the stress-strain curve and the test force-deformation curve, and analyze the uniaxial compression curve of the test piece.

[figure omitted; refer to PDF]

The color of the core material particles changes from white to pale yellow due to the residual suspension in the added spore dry powder (as shown in Figure 8). Currently, the texture of the core material particles is hard, and the pressing does not break, which ensures the integrity of the core material particles in the process of preparing the microbial capsule.

[figure omitted; refer to PDF]

As can be seen from the figure, round white colony spots appear on the surface of the culture dish, and the number of colonies and diameters vary. After statistics, the average number of colonies per dish was 40. That is, after 1 × 10⁸ times dilution, the survival spores in the microbial capsule were 4 × 10⁹ cfu/ml.

According to the core-wall ratio, 4 × 10⁹ cfu/ml was converted to 5.33 × 109 cfu/ml of the spore survival per gram of core material in the microbial capsule. The spore survival per gram of the core material before preparation of the microbial capsule was 8 × 10⁹ cfu/ml. Thus, the spore survival rate in the microbial capsule was 66.7%.

After the preparation of the core material and the microbial capsule, the Bacillus pasteurii remains active. The selected wall material has excellent biocompatibility with the spores, thus ensuring that the microbial capsule can be self-repaired when being embedded in the concrete.

3.6. Experimental Analysis of Self-Healing Performance

3.6.1. Analysis of Water Absorption Test

In the mortar test piece, the calcium carbonate precipitate generated by microbial mineralization binds the sand particles and effectively fills the pores of the mortar test piece. Consequently, the porosity is reduced to a certain extent, and the water absorption of the test piece reduces accordingly. The change trend of water absorption of the test piece with time is shown in Figure 16.

[figure omitted; refer to PDF]

As shown in the figure, with a fixed water-to-binder ratio, the water absorption rate of the cement mortar test pieces showed a downward trend with the extension of the curing age. This is due to the continuous increase in hydration products caused by microbial reactions, resulting in a tighter internal structure of the mortar. In addition, the addition of microbial capsules increased the water absorption of the mortar test piece, and the water absorption rate of the test piece showed an upward trend as the number of microbial capsules increased. Since the density of the microcapsules is relatively low, the density of the cement mortar test pieces also decreases with the increase in the microcapsule content, resulting in an increase in the internal porosity of the test pieces.

3.6.2. Analysis of Uniaxial Compression Test

Table 7 shows the compressive strength of the three groups of test pieces. The compressive strength of group A increased with the extension of maintenance age (48 days). The compressive strength of Group B was higher than that of standard test pieces in the initial stage of curing, but there was no significant change in the overall situation. When bacteria were added, the CO₂ hydration reaction in the crack area was accelerated. However, because the cement-based material is more alkaline and the bacteria have poor tolerance, the survival time inside the test pieces became shorter. With the increase in the curing time, few bacteria survived. To sum up, the compression resistance of Group C is significantly improved compared with that of the other two groups. Using epoxy resin as the wall material can achieve long-term survival of bacteria in cement-based materials.

Table 7

Comparison of compression strength under various curing ages.

Repair agent	Strength (MPa)
	3(d)	5(d)	7(d)
N/A	2.311	2.432	2.517
Bacterial body	2.436	2.317	2.336
Microbial capsule	2.316	2.425	2.618

4. Conclusion

In this paper, microbial capsules were prepared by using Bacillus pasteurii coated with epoxy resin E-51, and the optimal conditions for preparing microbial capsules were determined according to single-factor experiments. We conclude that

(1) The optimum process conditions for microbial capsules are 1 : 3 in the core-wall ratio, 50°C in the reaction temperature, 40 min in the reaction time, and a stirring rate of 300 rpm.

(2) Epoxy resin E-51 was coated on the surface of the core material particles to form an epoxy resin E-51 coated microbial capsule. The test piece mixed with microbial capsules was cured for a certain period after predamage. Its compressive strength was enhanced, indicating that the microbial capsules played a repairing role.

(3) Microbial capsules have excellent waterproofness and high storage stability. The microbial capsules are embedded in the concrete to achieve mechanical triggering properties and thus rupture. The spore survival rate in the microbial capsules is 66.7%. The wall material is biocompatible with the core material.

Acknowledgments

This work was funded by the National Natural Science Foundation of China (Grant nos. 51904032, 51874192, and 41807211), Research Fund of Binzhou University (Grant no. BZXYLG1914), Open Fund of Key Laboratory of Mine Disaster Prevention and Control (Grant no. MDPC201920), Natural Science Foundation of Shandong Province (Grant no. ZR2019MEE084), and SDUST Research Fund (Grant no. 2018TDJH102).

References

[1] S. Joshi, S. Goyal, A. Mukherjee, M. S. Reddy, "Microbial healing of cracks in concrete: a review," Journal of Industrial Microbiology and Biotechnology, vol. 44 no. 11, pp. 1511-1525, DOI: 10.1007/s10295-017-1978-0, 2017.

[2] M. Wu, X. Hu, Q. Zhang, D. Xue, Y. Zhao, "Growth environment optimization for inducing bacterial mineralization and its application in concrete healing," Construction and Building Materials, vol. 209, pp. 631-643, DOI: 10.1016/j.conbuildmat.2019.03.181, 2019.

[3] M. Seifan, A. K. Samani, A. Berenjian, "Bioconcrete: next generation of self-healing concrete," Applied Microbiology and Biotechnology, vol. 100 no. 6, pp. 2591-2602, DOI: 10.1007/s00253-016-7316-z, 2016.

[4] J. Wang, A. Mignon, D. Snoeck, V. Wiktor, S. Van Vliergerghe, N. Boon, N. De Belie, "Application of modified-alginate encapsulated carbonate producing bacteria in concrete: a promising strategy for crack self-healing," Frontiers in Microbiology, vol. 6,DOI: 10.3389/fmicb.2015.01088, 2015.

[5] S. Bhaskar, K. M. Anwar Hossain, M. Lachemi, G. Wolfaardt, M. Otini Kroukamp, M. O. Kroukamp, "Effect of self-healing on strength and durability of zeolite-immobilized bacterial cementitious mortar composites," Cement and Concrete Composites, vol. 82, pp. 23-33, DOI: 10.1016/j.cemconcomp.2017.05.013, 2017.

[6] J. Hommel, E. Lauchnor, A. Phillips, R. Gerlach, A. B. Cunningham, R. Helmig, A. Ebigbo, H. Class, "A revised model for microbially induced calcite precipitation: improvements and new insights based on recent experiments," Water Resources Research, vol. 51 no. 5, pp. 3695-3715, DOI: 10.1002/2014wr016503, 2015.

[7] M. Seifan, A. K. Samani, A. Berenjian, "Induced calcium carbonate precipitation using Bacillus species," Applied Microbiology and Biotechnology, vol. 100 no. 23, pp. 9895-9906, DOI: 10.1007/s00253-016-7701-7, 2016.

[8] J. Wang, H. M. Jonkers, N. Boon, N. De Belie, "Bacillus sphaericus LMG 22257 is physiologically suitable for self-healing concrete," Applied Microbiology and Biotechnology, vol. 101 no. 12, pp. 5101-5114, DOI: 10.1007/s00253-017-8260-2, 2017.

[9] J. Y. Wang, H. Soens, W. Verstraete, N. De Belie, "Self-healing concrete by use of microencapsulated bacterial spores," Cement and Concrete Research, vol. 56 no. 2, pp. 139-152, DOI: 10.1016/j.cemconres.2013.11.009, 2014.

[10] S. Rathore, P. W. S. Heng, L. W. Chan, "Feasibility study on microencapsulation of anaerobic Clostridium acetobutylicum ATCC 824 by emulsification method for application in biobutanol production," Journal of Microencapsulation, vol. 31 no. 5, pp. 469-478, DOI: 10.3109/02652048.2013.879928, 2014.

[11] X. Wu, S. Zhao, J. Zhang, P. Wu, C. Peng, "Encapsulation of EV71-specific IgY antibodies by multilayer polypeptide microcapsules and its sustained release for inhibiting enterovirus 71 replication," RSC Advances, vol. 4 no. 28, pp. 14603-14612, DOI: 10.1039/c3ra46943c, 2014.

[12] M. G. Sierra-Beltran, H. M. Jonkers, E. Schlangen, "Characterization of sustainable bio-based mortar for concrete repair," Construction and Building Materials, vol. 67, pp. 344-352, DOI: 10.1016/j.conbuildmat.2014.01.012, 2014.

[13] Q. Zhang, G. Zhou, Y. Hu, S. Wang, B. Sun, W. Yin, F. Guo, "Risk evaluation and analysis of a gas tank explosion based on a vapor cloud explosion model: a case study," Engineering Failure Analysis, vol. 101, pp. 22-35, DOI: 10.1016/j.engfailanal.2019.03.003, 2019.

[14] D. Xu, W. Chen, X. Fan, "Experimental investigation of particle size effect on the self-healing performance of microcapsule for cemented coral sand," Construction and Building Materials, vol. 256,DOI: 10.1016/j.conbuildmat.2020.119343, 2020.

[15] P. Singh, I. Kaur, N. Singh, "A review of different bacteria carriers used in self-healing mechanism," Materials Today: Proceedings, vol. 32, pp. 955-960, DOI: 10.1016/j.matpr.2020.05.100, 2020.

[16] X. Wang, Y. Huang, Y. Huang, J. Zhang, C. Fang, K. Yu, Q. Chen, T. Li, R. Han, Z. Yang, P. Xu, G. Liang, D. Su, X. Ding, D. Li, N. Han, F. Xing, "Laboratory and field study on the performance of microcapsule-based self-healing concrete in tunnel engineering," Construction and Building Materials, vol. 220, pp. 90-101, DOI: 10.1016/j.conbuildmat.2019.06.017, 2019.

[17] A. Al-Tabbaa, C. Litina, P. Giannaros, A. Kanellopoulos, L. Souza, "First UK field application and performance of microcapsule-based self-healing concrete," Construction and Building Materials, vol. 208, pp. 669-685, DOI: 10.1016/j.conbuildmat.2019.02.178, 2019.

[18] S. Han, E. K. Choi, W. Park, C. Yi, N. Chung, "Effectiveness of expanded clay as a bacteria carrier for self-healing concrete," Applied Biological Chemistry, vol. 62 no. 1,DOI: 10.1186/s13765-019-0426-4, 2019.

[19] Z. Liu, "Preparation of microcapsule and its influence on self-healing property of asphalt," Petroleum Science and Technology, vol. 37 no. 9, pp. 1025-1032, DOI: 10.1080/10916466.2019.1570253, 2019.

[20] A. Mohamed, T. Sharma, A. Heath, C. Richard, K. Paine, "Application of expanded perlite encapsulated bacteria and growth media for self-healing concrete," Construction and Building Materials, vol. 160, pp. 610-619, DOI: 10.1016/j.conbuildmat.2017.11.086, 2018.

[21] M. Tripathi, D. K. Rahamtullah, D. Kumar, C. Rajagopal, P. Kumar Roy, "Influence of microcapsule shell material on the mechanical behavior of epoxy composites for self-healing applications," Journal of Applied Polymer Science, vol. 131 no. 15,DOI: 10.1002/app.40572, 2014.

[22] H. Singh, R. Gupta, "Cellulose fiber as bacteria-carrier in mortar Self-healing quantification using UPV," Journal of Building Engineering, vol. 28,DOI: 10.1016/j.jobe.2019.101090, 2020.

[23] S. Shahid, M. A. Aslam, S. Ali, M. Zameer, M. Faisal, "Self‐healing of cracks in concrete using Bacillus strains encapsulated in sodium alginate beads," Chemistry, vol. 5 no. 1, pp. 312-323, DOI: 10.1002/slct.201902206, 2020.

[24] M. Wu, X. Hu, Z. Hu, Y. Zhao, W. Cheng, W. Lu, "Two-component polyurethane healing system: effect of different accelerators and capsules on the healing efficiency of dynamic concrete cracks," Construction and Building Materials, vol. 227,DOI: 10.1016/j.conbuildmat.2019.116700, 2019.

[25] W. Mingyue, H. Xiangming, Z. Qian, L. Wei, Z. Yanyun, H. Zhenglong, "Study on preparation and properties of environmentally-friendly dust suppressant with semi-interpenetrating network structure," Journal of Cleaner Production, vol. 259,DOI: 10.1016/j.jclepro.2020.120870, 2020.

[26] 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," Construction and Building Materials, vol. 179, pp. 151-159, DOI: 10.1016/j.conbuildmat.2018.05.199, 2018.

[27] M. Al-Ansari, A. G. Abu-Taqa, M. M. Hassan, A. Senouci, J. Milla, "Performance of modified self-healing concrete with calcium nitrate microencapsulation," Construction and Building Materials, vol. 149, pp. 525-534, DOI: 10.1016/j.conbuildmat.2017.05.152, 2017.

[28] L. Souza, A. Al-Tabbaa, "Microfluidic fabrication of microcapsules tailored for self-healing in cementitious materials," Construction and Building Materials, vol. 184, pp. 713-722, DOI: 10.1016/j.conbuildmat.2018.07.005, 2018.

[29] W. Cheng, Study on the Performance of Micro Encapsulated Microbe Self Repairing concrete Cracks, 2016.

[30] J. Y. Wang, D. Snoeck, S. Van Vlierberghe, W. Verstraete, N. De Belie, "Application of hydrogel encapsulated carbonate precipitating bacteria for approaching a realistic self-healing in concrete," Construction and Building Materials, vol. 68, pp. 110-119, DOI: 10.1016/j.conbuildmat.2014.06.018, 2014.

[31] J. Wang, K. Van Tittelboom, N. De Belie, W. Verstraete, "Use of silica gel or polyurethane immobilized bacteria for self-healing concrete," Construction and Building Materials, vol. 26 no. 1, pp. 532-540, DOI: 10.1016/j.conbuildmat.2011.06.054, 2012.

[32] K. Van Tittelboom, N. De Belie, W. De Muynck, W. Verstraete, "Use of bacteria to repair cracks in concrete," Cement and Concrete Research, vol. 40 no. 1, pp. 157-166, DOI: 10.1016/j.cemconres.2009.08.025, 2010.

[33] W. Cheng, X. Hu, J. Xie, Y. Zhao, "An intelligent gel designed to control the spontaneous combustion of coal: fire prevention and extinguishing properties," Fuel, vol. 210, pp. 826-835, DOI: 10.1016/j.fuel.2017.09.007, 2017.

[34] D. O. George, L. Jin, "Optimum conditions for microbial carbonate precipitation," Chemosphere, vol. 81 no. 9, pp. 1143-1148, DOI: 10.1016/j.chemosphere.2010.09.066, 2010.

[35] E. Y. Cagatay, S. E. Hernandez, B. Nico, D. B. Nele, "Enhanced crack closure performance of microbial mortar through nitrate reduction," Cement and Concrete Composites, vol. 70, pp. 159-170, DOI: 10.1016/j.cemconcomp.2016.04.001, 2016.