Bacteria-powered self-healing concrete: Breakthroughs, challenges, and future prospects

Abstract

 

In a world where concrete structures face constant degradation from environmental forces, a revolutionary solution has emerged: bio-self-healing concrete. This innovation involves embedding dormant bacteria within the concrete mix, poised to spring into action when cracks form. As moisture seeps into the cracks, these bacterial agents are activated, consuming nutrients and converting them into calcium carbonate, a natural substance that fills and repairs the fractures, restoring the material’s integrity. This fascinating process represents a cutting-edge approach to maintaining concrete infrastructure, turning once-vulnerable materials into self-sustaining systems capable of healing themselves. The ongoing research into bio-self-healing concrete is focused on selecting bacterial strains that can withstand the extreme conditions within concrete, including its highly alkaline environment. The bacteria must also form resilient spores, remaining viable until they are needed for repair. Additionally, the study explores various challenges associated with this technology, such as the cost of production, the bacteria’s long-term viability, and their potential environmental impact. Advancements in genetic engineering and smart technology are being explored to enhance these bacterial strains, making them more efficient and robust in their role as microscopic repair agents. This review delves into the potential of bio-self-healing concrete to revolutionize how we approach infrastructure maintenance, offering a glimpse into a future where concrete structures not only endure but actively repair themselves, extending their lifespan and reducing the need for costly repairs.

One-Sentence Summary

Bio-self-healing concrete utilizes bacteria that activate upon crack formation to repair structures by producing calcium carbonate, offering a sustainable solution to prolong the lifespan of concrete infrastructure.

Graphical Abstract

Introduction

Concrete is a cornerstone of modern infrastructure, used in everything from residential buildings to bridges, dams, and highways. Its popularity stems from its strength, durability, and relatively low cost. However, like all materials, concrete has its limitations, particularly its susceptibility to cracking. These cracks, even when minor, can lead to significant structural problems over time. They allow water, chlorides, and other aggressive agents to penetrate the concrete, leading to the corrosion of embedded steel reinforcement, further deterioration, and ultimately, costly repairs or even premature failure of the structure (Luhar et al., 2022; Mahmood et al., 2022; Seifan et al., 2016). The conventional methods for repairing cracked concrete typically involve labor-intensive and expensive processes such as injecting synthetic resins, applying surface sealants, or installing patch materials (Ghaffary & Moustafa, 2020). While these methods can be effective in the short term, they often require repeated applications and may not fully restore the material’s integrity. Additionally, many of these repair methods involve the use of nonrenewable resources and generate waste, raising concerns about their environmental impact (Raza & Arsalan Khushnood, 2022).

In recent years, the concept of self-healing concrete has emerged as an innovative solution to these challenges. Self-healing concrete is designed to repair its own cracks without the need for external intervention, effectively prolonging the life of the structure and reducing the need for maintenance. This capability can be achieved through various mechanisms, including chemical, physical, and biological processes. Among these, the biological approach particularly the use of bacteria and fungi to heal cracks has gained significant attention due to its unique advantages. Bacteria and fungi both offer promising potential in enhancing the self-healing capabilities of concrete. While this review primarily focuses on bacterial methods, fungi also contribute by producing substances that enhance concrete repair and by thriving in diverse environments, further improving the effectiveness of self-healing concrete (Amran et al., 2022).

Bio-self-healing concrete, as this biologically driven approach is known, involves incorporating specific strains of bacteria into the concrete mix. These bacteria, typically from the Bacillus genus, are capable of surviving in the harsh environment of concrete. They remain dormant until cracks form and water seeps into the structure, at which point they become active. Once activated, these bacteria begin to metabolize available nutrients, leading to the production of calcium carbonate (CaCO₃). The CaCO₃ precipitates within the cracks, effectively sealing them and restoring the integrity of the concrete (Benjamin et al., 2023). The primary mechanism that enables this process is the bacteria’s ability to hydrolyze urea into ammonia and carbon dioxide, a process known as urea metabolism. The ammonia and carbon dioxide subsequently react with calcium ions present in the concrete mix to form CaCO₃. The use of bacteria for self-healing is particularly appealing because it leverages a natural process to address a long-standing problem in civil engineering, aligning with broader efforts to develop sustainable construction practices (Mahmod et al., 2021).

The effectiveness of bio-self-healing concrete is influenced by several factors, including the type of bacteria used, the availability of nutrients, the size and nature of the cracks, and the environmental conditions (Mahmod et al., 2021). Different bacterial strains have been explored for their potential to enhance the self-healing process, with some showing greater resilience and efficiency than others. In addition to naturally occurring bacteria, advances in biotechnology have opened new possibilities for enhancing the self-healing capabilities of concrete through genetic modification. For example, genetic engineering can be used to increase the production of urease, the enzyme responsible for urea hydrolysis, thereby enhancing the overall healing process (Mobley et al., 1986; Veaudor et al., 2018). Moreover, the genetic engineering of specific genes, such as the ureC gene responsible for urease production, holds promise for further improving the efficiency and effectiveness of bio-self-healing concrete. By enhancing the expression of this gene, it may be possible to increase the rate of CaCO₃ production, thereby accelerating the healing process and making it more reliable across a range of conditions (Mobley et al., 1986; Veaudor et al., 2018). In addition to genetic modifications, research has focused on the use of protective carriers to ensure the viability and longevity of the bacteria within the concrete matrix. These carriers, which can include materials like microcapsules, hydrogels, or porous aggregates, protect the bacteria from the harsh alkaline environment of the concrete ,and provide a sustained release of nutrients. The use of protective carriers not only improves bacterial survival but also enhances the overall effectiveness of the self-healing process, making it more consistent and scalable for real-world applications (Amer Algaifi et al., 2020).

This review aims to provide a comprehensive overview of the current state of research on bio-self-healing concrete, with a focus on the various bacterial strains used, the metabolism of urea that underpins the healing process, and the factors that influence the effectiveness of this technology. Furthermore, the review will explore recent advances in bacterial engineering, including genetic modifications, biofilm development, and the use of protective carriers, all of which hold the potential to enhance the performance of bio-self-healing concrete. By examining these aspects, this review seeks to highlight the potential of bio-self-healing concrete as a revolutionary material in the construction industry and to identify the key areas where further research and development are needed to realize its full potential.

Mechanism of Bio-Self-Healing Concrete

Selection of Bacteria

With its excellent capacity to repair cracks, bacterial-based self-healing concrete (BSHC) is a well-known healing technology that has been studied for several decades (Huang et al., 2022). Bacteria are alive, single-cell organisms with a wide range of uses (Su et al., 2021). Their ecological diversity is high, and they can be found in a variety of natural settings. Only about half of the phyla of bacteria have species that can be grown in the laboratory, although there are approximately 51,030 bacteria on the planet. Bacillus has been chosen as the biological component of the self-healing agent in the majority of studies (Dinarvand & Rashno, 2022). Because they are commonly found in soil and can produce spores in unfavorable conditions, those over 50 are dormant in the highly alkaline environment of concrete (pH value up to 13). They produce enough amount of urease enzyme resulting in CaCO₃ precipitation through urea hydrolysis (Nguyen et al., 2019).

Bacillus pasteurii is a bacterium found in the soil, not pathogenic or toxic (Yatish Reddy et al., 2020). Bacillus pasteurii DSM33, a Gram-positive aerobic bacterium ubiquitous in soil and producing a large amount of intracellular urease, is the most used strain for biomineralization. Bacillus pasteurii can repair cracks of 0.1- to 2.0-mm width under certain conditions (Chen et al., 2019). Also, Bacillus megaterium, which is a rod-shaped, aerobic, spore-producing gram-positive bacteria, can be used for self-healing. Polysaccharides on the cell wall link cells together to form pairs and chains. It has good temperature resistance and can reproduce at temperatures ranging from 3 to 45°C (Su et al., 2021). Another species is Bacillus subtilis, which is a Gram-positive, rod-shaped bacterium that produces dormant, heat-resistant spores. It is not pathogenic and is commonly used as a healing agent for crack repair because this bacterium forms spores with specialized cells capable of withstanding high mechanical forces and harsh environments. With low metabolic activity, some strains are known to produce extremely long-life spores viable for up to 200 years when used as a model endospore-forming bacterium with the ability to grow both aerobically and anaerobically by fermentation. The use of B. subtilis M9 in self-healing concrete is still being researched, but it indicates remarkable promise for future building applications. More research is being done to improve the performance and efficiency of this bacteria strain in self-healing concrete systems. A production with 0.3-mm crack sealing demonstrated the best alkali-resistant character, which is required for self-healing concrete preparation. CaCO₃ particles were generated in size of 8 μm by microbially induced CaCO₃ precipitation (MICCP) (Qian, Ren, et al., 2021). Bacillus cereus CS1 is an alkali-tolerant strain that grows at pH 7–10. The width of cracks that the bacteria can heal is 100–800 µm (Wu et al., 2019). Moreover, Bacillus sphaericus is a Gram-positive, mesophilic, rod-shaped bacterium common in soil. It exhibits the ability to resist endospores tolerant to high temperatures, chemicals, and ultraviolet (UV) light and can survive for an extended period and the healed crack length is 0.970 mm (Yatish Reddy et al., 2020). Bacillus alcalophilus is a facultative aerobic Gram-positive bacillus with a bar shape that lives in soil and is harmless to humans, highly adaptable to a highly alkaline environment, and can be added directly to highly alkaline cement-based materials (Qian, Ren, et al., 2021). Finally, Sporosarcina pasteurii (ATCC 11859) due to its high urease activity, good salt tolerance, and ability to survive in relatively high pH environments has been used to induce CaCO₃ precipitation. Pasteurii could survive at a pH of about 11.2 environments. The crack at the bottom of the beam was about 0.4 mm wide (Chen et al., 2021). Other bacterial species with concrete healing criteria and their optimum healing conditions are listed in Table 1.

Table 1.

Self-Healing Ratio of Cracked Concrete Using Bacteria

Type of Bacteria	Healing Ratio	Condition of Healing	Reference
Bacillus pasteurii	Repair the 0.1–2.0 mm	The calcium carbonate crystal filled the crack when the curing age reached 91 days.	Chen et al. (2019)
Bacillus megaterium		Polysaccharides on the cell wall link cells together to form pairs and chains. It has good temperature resistance and can reproduce at temperatures ranging from 3 to 45°C.	Su et al. (2021)
Bacillus subtilis	Fractures up to 0.81 mm	Rod-shaped bacterium that produces dormant, heat-resistant spores. Bioencapsulation by bacterial carrier and organic salt oxidation using nanoplatelets and lightweight aggregates.	Qian, Ren, et al. (2021)
Bacillus cereus CS1	The width of cracks that the bacteria can heal is 100–800 µm	It exhibits the ability to resist endospores tolerant to high temperatures, chemicals, and UV light and can survive for an extended period.	Wu et al. (2019)
Bacillus sphaericus	Healed crack length is 0.970 mm	Mesophilic, rod-shaped bacterium	Yatish Reddy et al. (2020)
Bacillus alcalophilus		Facultative aerobic Gram-positive bacillus with a bar shape, highly adaptable to a highly alkaline environment	Qian, Ren, et al. (2021)
Sporosarcina pasteurii (ATCC 11859)	Healed crack up to 0.4 mm	Has high urease activity, good salt tolerance, and ability to survive in relatively high pH environments (could survive at a pH of about 11.2). The microbial-induced calcium carbonate precipitations produced by S. pasteurii were mainly vaterite. The operating pH would affect precipitation morphology, i.e., the higher pH resulted in smoother and smaller particles. The optimum pH to make heal for these bacteria is 10. Grow at 30oC.	Chen et al. (2021)
Bacillus pseudofirmus and Bacillus cohnii (case study)	0.497 (56 days)	Healed cracks up to 0.13 mm in high-pH cement, retaining viability within encapsulated form	Ivaškė et al., (2023)
Sporosarcina koreensis and Bacillus	S. koreensis restored a crack with a width of 288.7 µm, while B. flexus bacterial concrete recuperated a crack with a width of 218.8 µm in 28 days	The filling of the cracked sections is attributed to the formation and deposition of calcite via microbial activity. Effective in at temperatures of 38°C and alkaline conditions (pH 9.5), significant crack closure.	Mohammed et al. (2024)
Bacillus pseudomycoides strain HASS3	Crack width of 0.4 mm in 68 days	Urea-based MICCP (microbially induced calcium carbonate precipitation) repaired a 0.4-mm crack.	Algaifi et al. (2021) and Wong et al. (2024)
Bacillus sphaericus LMG 22 557	Cracks up to 0.97 mm in 8 weeks	Urea-based MICCP with bacterial spore bioencapsulation achieved 48%–80% healing with nutrients.	Wang, Snoeck, et al. (2014) and Wang, Soens, et al. (2014)
Bacillus subtilis PTCC 1254	Fractures up to 0.2 mm	Organic salt oxidation effectively sealed fractures with a maximum width of 0.2 mm.	Kalhori and Bagherpour (2017)
Bacillus alkalinitrilicus	Cracks up to 0.46 mm in 100 days	Organic salt oxidation with bioencapsulated spores doubled healing width compared to control (0.18 mm).	Wiktor and Jonkers (2011)
Diaphorobacter nitroreducens	Cracks up to 0.40 mm	NO3 reduction healed over 90% of cracks ≤0.35 mm in 14 days, achieving full closure for smaller cracks.	Erşan et al. (2016)
Pseudomonas aeruginosa	Cracks from 0.35 to 0.40 mm	NO3 reduction significantly outperformed abiotic controls in healing.	Erşan et al. (2016)
Synechococcus PCC8806	Precipitated layer 0.2–0.27 mm	Photosynthesis resulted in a precipitated calcium layer, surpassing abiotic control by 38%.	Zhu et al. (2015)
Spore-forming bacteria species (case study)	Concrete cracks with the width of 0.3–0.5 mm	Concrete cracks repair based on microbial-induced calcium carbonate precipitation	Qian, Zheng, et al. (2021)

Selecting bacterial strains for bio-self-healing concrete requires considering several key factors to ensure their survival and effectiveness within the concrete’s harsh environment. The ability to form endospores is crucial, as it allows bacteria to remain dormant under extreme conditions and activate when cracks appear. Alkaliphilic properties are also important, enabling bacteria to thrive in the high pH levels typical of concrete. High urease production is essential for efficiently precipitating CaCO₃, which seals cracks. Additionally, the bacteria must be compatible with the concrete matrix, ensuring they do not negatively affect its strength or durability, and should have minimal nutrient requirements to sustain their activity over the long term. These considerations are vital for developing effective and reliable bio-self-healing concrete (Esaker et al., 2021; Griño et al., 2020; Ivaškė et al., 2023).

Metabolism of Urea

In the highly alkaline or acidic environment of concrete, bacteria survive the process and can adapt and even form spores. The spores remain inactive and can endure high temperatures and pressure, as well as dehydration and chemical processes, but they can become metabolically active when water and nutrients become available (Hossain et al., 2022). Once the bacteria are active, they create the urease enzyme, which catalyzes the conversion of urea (CO(NH₂)₂) into carbonate (CO₃²⁻) and ammonium ions (NH⁴⁺) (Huseien et al., 2022). Initially, 1 mol of urea undergoes intracellular hydrolysis to produce 1 mol of ammonia and 1 mol of carbamate (Equation (1)). Equation (2) shows that carbamate hydrolyzes spontaneously to produce an extra 1 mol of ammonia and carbonic acid. Equations (3) and (4) show that these products then create 1 mol of bicarbonate and 2 mol of ammonium and hydroxide ions. The last two reactions give rise to a pH increase, which in turn shifts the bicarbonate equilibrium, resulting in the formation of carbonate ions (Equation (5))

The bacteria take up cations from the surrounding environment, such as Ca²⁺, to deposit on their cell surface because their cell wall is negatively charged. Equations (6) and (7) describe how the Ca²⁺ ions combine with the CO₃²⁻ ions to precipitate CaCO₃ at the cell surface, which acts as a nucleation site (Van Tittelboom et al., 2010).

$$\begin{eqnarray} {\rm{CO}}{\left( {{\rm{N}}{{\rm{H}}_2}} \right)_2} + {{\rm{H}}_2}{\rm{O}} \to {\rm{N}}{{\rm{H}}_2}{\rm{COOH}} + {\rm{N}}{{\rm{H}}_3} \end{eqnarray}$$

(1)

$$\begin{eqnarray} {\rm{N}}{{\rm{H}}_2}{\rm{COOH}} + {{\rm{H}}_2}{\rm{O}} \to {\rm{N}}{{\rm{H}}_3} + {{\rm{H}}_2}{\rm{C}}{{\rm{O}}_3} \end{eqnarray}$$

(2)

$$\begin{eqnarray} {\rm{H}}_2{\rm{CO}}_3 \rightleftharpoons {\rm{HCO}}_3^- + {\rm{H}}^+ \end{eqnarray}$$

(3)

$$\begin{eqnarray} 2{\rm{NH}}_3 + 2{\rm{H}}_2{\rm{O}} \rightleftharpoons 2{\rm{NH}}_4^+ + 2 {\rm{OH}}^- \end{eqnarray}$$

(4)

$$\begin{eqnarray} {\rm{HCO}}_3^- + {\rm{H}}^+ + 2{\rm{NH}}_4^+ + 2{\rm{OH}}^- \rightleftharpoons {\rm{CO}^{2-}_3}+ 2{\rm{NH}}_4^+ + 2{\rm{H}}_2{\rm{O}} \end{eqnarray}$$

(5)

$$\begin{eqnarray} {\rm{C}}{{\rm{a}}^{2 + }} + {\rm{Cell}} \to {\rm{Cell}} - {\rm{C}}{{\rm{a}}^{2 + }} \end{eqnarray}$$

(6)

$$\begin{eqnarray} {\rm{Cell - C}}{{\rm{a}}^{2 + }} + {\rm{C}}{{\rm{O}}^{2-}_3} \to {\rm{Cell - CaC}}{{\rm{O}}_3} \downarrow \end{eqnarray}$$

(7)

Healing Process

The healing process of cracks in concrete through bacterial activity begins when cracks form and allow water to infiltrate the concrete structure. This water acts as a trigger for the dormant bacteria embedded within the concrete matrix, typically spore-forming and alkaliphilic strains. These bacteria remain inactive until they come into contact with moisture, which prompts them to germinate and become metabolically active. Once active, the bacteria start to metabolize available nutrients within the concrete, often supplied in the form of calcium lactate or similar compounds. As the bacteria metabolize these nutrients, they produce metabolic by-products, including carbonate ions. These carbonate ions react with calcium ions present in the concrete environment to precipitate CaCO₃. This CaCO₃ forms a crystalline structure that gradually fills the cracks in the concrete, effectively sealing them and restoring the material’s structural integrity. This process is driven by a key chemical reaction where urea hydrolysis, catalyzed by the enzyme urease, results in the formation of carbonate ions that combine with calcium to produce the CaCO₃ needed for healing (Griño et al., 2020; Mahmood et al., 2022).

Factors Affecting Biological Healing in Concrete

Microbial population is defined as the number of microbes in concrete has an impact on its ability to heal biologically because urease productivity and CaCO₃ precipitation are both related to bacterial cell concentration. A sufficient number of cells should be required to ensure high self-healing efficiency (Amer Algaifi et al., 2020). Because the quantity of urease enzymes generated by bacteria is a major factor in urea hydrolysis, CaCO₃ would not arise if urea were not digested, which is impossible in the absence of bacteria cells (Amer Algaifi et al., 2020; Zhao et al., 2014). Therefore, the proper concentration of bacteria in the concrete must be appropriate because high and very low cell concentrations cause lower calcite precipitation (Abdelgalil et al., 2022). The amount of hydrolyzed urea at low concentrations of bacterial cells (10⁶ and 10⁷) is insufficient to precipitate calcite because of the small amount of urease enzyme. The highest concentration of bacterial cells in concrete was found to be 10⁸ cells/cm³, which precipitated the highest amount of calcite. This concentration of cells results in the best concrete healing because it produces the most urease enzyme (Abdelgalil et al., 2022; Amer Algaifi et al., 2020). As a result, during urea hydrolysis, high cell density is effective in increasing the diversity and size of CaCO₃ crystals; however, once the concentration of bacterial cells exceeds 10⁸ cells, calcite precipitation does not increase; instead, this increase influences the sedimentation rate to decrease (Abdelgalil et al., 2022; Son et al., 2018). It was observed that cell density significantly influenced the decline in the rate of sedimentation. This is because a high cell density alters the pH value, which in turn influences the process of sedimentation (Xu, Tang, & Wang, 2020).

Bacterial cells functioned as nucleation sites, adding more of them to the substrate would undoubtedly speed up MICCP process (Abdelgalil et al., 2022). Calcium ions were adsorbed onto negatively charged bacterial cell walls when the urea broke down. It has been noted that certain macromolecules on the surface of bacterial cells, such as proteins and exopolysaccharides, may act as nucleation sites, which reduces the net negative electric potential of the bacteria cell surface. This is because Ca²⁺ is adsorbed on the cell surface, resulting in a higher concentration of Ca²⁺ around the bacteria than in the surrounding environment (Son et al., 2018; Xu, Tang, & Wang, 2020; Zhang et al., 2016). As a result, the carbonate ion surrounded the cell calcium, which precipitated the microbial CaCO₃ that encircled the bacterial cell wall. Due to the CaCO₃ and Ca²⁺ coating on the bacterial cell wall, this action may further restrict the breakdown of urea. The bacterial cell’s encapsulation by an overabundance of calcium ions, which prevents the release of the urease enzyme, is another reason for the decrease in urea decomposition (Amer Algaifi et al., 2020). Therefore, the presence of nucleation sites is essential for the growth of CaCO₃ crystals, as the absence of sufficient cells to serve as nucleation sites prevents the formation of CaCO₃ crystals (Xu, Tang, & Wang, 2020; Zhang et al., 2016). Factors that affect the self-healing are outlined in Fig. 1.

Fig. 1.

Factors affecting the bacterial healing process.

pH

The growth of bacteria and the activity of the urease enzyme in concrete are significantly influenced by the pH of the concrete; microbial growth and urease activity were suppressed when the initial pH of the medium was either strongly alkaline and acidic. In contrast, the urease activity was stronger, and the microbial growth was superior in the pH range of 6.0–10.0. More specifically, the pH range of 7.5–8.0 is ideal for the urease enzyme (Abdelgalil et al., 2022; Yi et al., 2021). High pH values (12–13) prevented the bacterial cell from undergoing binary fission. These same pH values inhibited bacterial growth and lowered the efficiency of urea hydrolysis by 75%–80%. Because urease activity decreases at pH values of 11 and 13, exposure to high pH momentarily halts the breakdown of urea. This occurs because, at pH values higher than 11, the bacteria’s ability to hydrolyze urea into carbonate and ammonia is hindered. This restricted ureolysis prevents CaCO₃ precipitation from continuing inside the concrete matrix, which is necessary to fill the pores and microcracks in the concrete (Amer Algaifi et al., 2020; Williams et al., 2017). The cause might be because the pH level influences the permeability of the microbial cell membrane, which in turn affects how the bacteria absorb and transform nutrients. Later, there was an inability for the bacterial metabolism process to continue as usual, which had an impact on microbial growth and maybe resulted in bacterial mortality (Yi et al., 2021).

Temperature

The efficiency of concrete’s bio-self-healing can be significantly impacted by temperature (Jonkers et al., 2010). The main reasons could be attributed to bacterial growth had a suitable temperature range. Temperature influences bacterial activity, enzymatic activity, and reaction rate, which in turn influences the rate at which biogenic CaCO₃ is formed and the effectiveness of crack healing (Wang et al., 2017; Yi et al., 2021). The bacteria were less successful in generating CaCO₃ at temperatures of 40°C or higher, most likely as a result of thermal stress on the cells, which lowers cell viability, and decreased urea hydrolysis because of a drop in urease activity (Williams et al., 2017; Zhang et al., 2015). High temperature would cause the structure of the bacterium to become inactive, which could have an impact on the metabolism of the bacteria. Thus, the microorganisms might have died (Yi et al., 2021).

At low temperatures, spore germination and bacterial growth are significantly slowed, extending the lag phase to 48 hours at 10°C compared to 6 hours at 28°C (Wang et al., 2017). Also, the urease activity of the bacteria would also be impacted and significantly decreased, making it difficult for the bacteria to carry out regular metabolism. At this time the bacteria remain dormant, which resulted in a decrease in CaCO₃ production and a slower rate of healing. So, maintaining an ideal temperature range is crucial to encouraging bacterial activity and maximizing the self-healing process’ efficacy (Jonkers et al., 2010; Yi et al., 2021). Therefore, a temperature between 30°C and 35°C is optimal for biohealing, as it enhances urease activity and bacterial growth, leading to increased CaCO₃ production (Yi et al., 2021; Zhang et al., 2015).

Calcium Concentration

The concentration of calcium ions is also a critical factor affecting the self-healing efficiency of bacteria in concrete because it impacts on bacterial calcium precipitation (Van Tittelboom et al., 2010; Zhang et al., 2016). The precipitation of CaCO₃ depends on the concentration of Ca²⁺. Although it is probably not used by metabolic processes, it accumulates outside the cell and becomes easily accessible for the precipitation of CaCO₃. A low precipitation rate is caused by both high and low calcium source concentrations (Abdelgalil et al., 2022). High calcium concentrations have been shown to restrict the growth of bacterial cells. Calcium ions can alter the permeability of cellular membranes, which in turn affects how cells metabolize and lowers the rate of precipitation (Zhang et al., 2016). Low calcium concentration decreases the amount of dissolved urea, and it results in less CaCO₃ being formed and precipitated (Abdelgalil et al., 2022; Wang et al., 2017). To encourage bio-healing, the concentration of calcium ions in concrete must be optimized. This is because the ideal concentration of calcium ions causes all of the urea to hydrolyze completely (Van Tittelboom et al., 2010; Wang et al., 2017).

As a cofactor for the urease enzyme, calcium ions can either increase or reduce urease activity. This is because calcium ions that bind to the active site of the enzyme improve the interaction between the enzyme and the substrate. The activity of the urease enzyme is affected by different calcium salts and each calcium salt has a unique urease activity (Gorospe et al., 2013). The most effective calcium supply for the MICCP process is calcium chloride, which also increases urease activity (Achal & Pan, 2014). The mixtures’ source of calcium ions should be more soluble and not have an adverse effect on the materials used to make concrete (Putri et al., 2020). One of the main factors influencing the morphology of CaCO₃ and the creation of its many forms is the source of calcium (Jung et al., 2020). There are three anhydrous polymorphs of CaCO₃: vaterite, aragonite, and calcite. Calcite exhibits greater thermodynamic stability than aragonite, while vaterite exhibits the least amount of stability (Putri et al., 2020). Rhombohedral CaCO₃ (calcite) was precipitated when calcium chloride was utilized as a source of Ca²⁺. A rhombohedral shape is a characteristic of the most stable form of CaCO₃. Another form of vaterite, a metastable form of CaCO₃, was generated by calcium acetate in the form of a lettucelike or lamellar structure. Spherical vaterite was produced by using calcium lactate and calcium gluconate as sources of calcium ions. The largest crystals were produced by using calcium lactate, which was followed by calcium acetate, calcium chloride, and calcium gluconate, which produced the smallest crystals (Gorospe et al., 2013). The calcium source that produces more CaCO₃ is calcium chloride, while calcium oxide produces the least amount of CaCO₃ (Achal & Pan, 2014).

Urea

Urea concentration plays a great role in bio-self-healing in concrete and effect on precipitation on CaCO₃ (Amer Algaifi et al., 2020). The quantity and rate of CaCO₃ formation, which depend on the quantity and rate of urea breakdown, are directly related to the crack healing efficiency (Wang et al., 2017). Urea is considered the substrate for urease enzyme, so the hydrolysis of urea mainly depends on the urea concentration (Abdelgalil et al., 2022). Urea is broken down by the urease enzyme into ammonium and carbonate ions (Mirshahmohammad et al., 2022). Chemical reactions are as follows:

$$\begin{eqnarray} {\rm{CO}}{\left( {{\rm{N}}{{\rm{H}}_2}} \right)_2} + 2{{\rm{H}}_2}{\rm{O}} \to 2{\rm{N}}{{\rm{H}}^ +_4} + {\rm{C}}{{\rm{O}}^{2 - }_3} \end{eqnarray}$$

$$\begin{eqnarray} {\rm{C}}{{\rm{a}}^{2 + }} + {\rm{C}}{{\rm{O}}^{2 - }_3} \to {\rm{CaC}}{{\rm{O}}_3} \end{eqnarray}$$

Urea decomposition is influenced by urea concentration (Amer Algaifi et al., 2020). When urea concentration rises, the rate of urea decomposition also rises, which causes CaCO₃ to precipitate more frequently until it reaches its maximum rate at which point the rate of urea decomposition and CaCO₃ precipitation are negatively impacted by the increased urea concentration (Abdelgalil et al., 2022; Xu et al., 2018). This is because the urease enzyme hydrolyzes urea, causing a rise in pH due to an increase in ammonium ion concentration. This changes the viability of the cells, which in turn changes the rate at which CaCO₃ precipitates (Amer Algaifi et al., 2020).

Additionally, the bacterial concentration and the rate of urea breakdown are connected. More urea was broken down in proportion to the amount of bacteria present (Wang et al., 2017). Precipitation of carbonate on the bacterial cells was observed to impact the surface available for nutrient uptake, interfere with the proton motive force, and ultimately result in cell death. This led to a decrease in the concentration of bacteria, and a reduction in the number of cells means a reduction in ureolytic activity (De Muynck et al., 2010). As previously stated, temperature and pH level both affect cell concentration, which can have a favorable or negative effect on urea decomposition (Xu et al., 2018).

Improvement in Bacterial Strains

The viability of bacteria is a critical factor in applying MICP to self-healing concrete technology (Pawar & Parekar, 2018) because microbial precipitation is affected by several factors including the concentration of bacteria, presence of nucleation sites, pH, temperature, calcium, and urea concentration. Also, when bacteria are used to work for the healing of cracks in concrete, it must face the harsh environment of the concrete such as high alkalinity of concrete, restricting the growth of the bacteria, and limit the efficiency of bio-self-healing. Therefore, in recent years, researchers and engineers have been working to improve and optimize bacterial strains in order to increase their capacity to seal cracks and increase the longevity of concrete (Zhang et al., 2019) (Fig. 2).

Fig. 2.

Improvement of bacterial strains.

Genetic Modification

Genetic modification involves changing the genetic composition of the bacteria employed in concrete in an effort to increase their capacity for survival, activity, and efficacy in challenging environments. This would eventually aid in the concrete’s ability to mend cracks. Researchers have identified several bacterial strains from hot springs, including BKH1 and BKH2. These bacteria can strengthen and extend the durability of concrete by producing a particular secretary protein called bioremediase (Chattopadhyay, 2020; Sarkar, Adak, et al., 2015). The bioremediase protein is unique thermostable, high pH tolerant protein and possesses silica leaching activity that forms gehlenite (calcium aluminum silicate) in concrete (Sarkar, Alam, et al., 2015). Through its catalytic activity, the bioremediase protein functions as an enzyme and forms nano-silica particles. When the highly active silica nanoparticles are combined with the calcium and aluminum oxides found in the cementitious matrix, a nanocrystalline material is created that fills in the gaps in the concrete structures, increasing their compressive strength and longevity (Chattopadhyay, 2020). Due to the laborious nature of maintaining and cultivating the bacteria BKH2, genetic engineering techniques were utilized to transfer the bioremediase-releasing gene to Escherichia coli (JMJ107). The sample containing JMJ107 has a higher compressive strength, but due to the high alkalinity and aerobic conditions within the concrete, it was found that E. coli cannot survive there (Sarkar, Alam, et al., 2015). On the other hand, the alkaliphilic spore Bacillus sp. has the ability to produce CaCO₃ in concrete and can live in the concrete for a long time (Sarkar, Adak, et al., 2015). By adding the biosilification activity to the B. subtilis bacterial strain that forms spores, the capacity for self-healing may be strengthened (Chattopadhyay, 2020). The introduction of a bioremediase-like gene to B. subtilis from BKH2 resulted in the observation that genetically modified B. subtilis is more effective than nonmodified B. subtilis because it forms gehlenite and calcite precipitation inside the concrete. Additionally, genetically modified Bacillus sp. forms during self-healing crystalline matrix, whereas nonmodified Bacillus sp. has an amorphous matrix (Sarkar, Adak, et al., 2015). In a different study, S. pasteurii was exposed to UV radiation in order to cause mutations that would enhance the organism’s growth and urease production, which would cause more calcite to precipitate. Results demonstrate that, in comparison to the wild-type strain of S. pasteurii, the mutant strain’s growth and urease enzyme output both increased (Achal et al., 2009).

Development of Biofilm

Most microbial communities exist in biofilms, which are made up of living cells embedded in extracellular polymers like proteins and polysaccharides. These polymers can shield cells from environmental stresses, encourage cell adherence to various substrates, and create an environment that is favorable for biomineralization (Li et al., 2022). The biofilm formed when microbial cells attach themselves to solid surfaces, multiply, and use the extracellular polymeric material (EPS) they excrete to bind themselves securely to the surface (Salifu et al., 2021). Numerous biofilm features are determined by EPS, and depending on the specific bacterial strain and the growth conditions during biofilm development, its composition can vary significantly (Hayta et al., 2021). A composite biofilm was created using genetic engineering technology. This biofilm can function as a sustainable cellular building biomaterial to produce uniform and controlled biological construction, while also fostering uniform bacterial distribution and viability and offering mineralization sites to improve cementation. Xanthan gum, an anionic polysaccharide extracted from Xanthomonas extracellular polymeric substances, is added to composite biofilms to improve the homogeneity and mechanical properties of the cementitious materials (Li et al., 2022). Transposon mutagenesis results in stronger biofilm development, which increases the amount of CaCO₃ that bacteria generate in stable environments (Zhang et al., 2019). In another study on S. pasteurii, the strain was exposed to UV in order to create a mutant strain. This mutant strain was able to produce more EPS and biofilm than the wild-type strain (Achal et al., 2009).

Genetic Engineering of the ureC Gene to Increase Urease Production

The ureC gene encodes for the alpha subunit of urease, an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia. This gene is part of the urease gene cluster, which typically includes other genes such as ureA, ureB, ureD, ureE, ureF, and ureG (Koper et al., 2004; Zhang et al., 2020). Carbon dioxide produced from ammonia hydrolysis increases the local pH and provides carbonate ions, which can react with calcium ions present in the concrete matrix to form CaCO₃ (Ali et al., 2023).

Commonly used bacteria for this purpose include Bacillus sp., which are known for their robust spore-forming ability and resilience in harsh environments. Bacteria are incorporated into the concrete mix. These bacteria remain dormant as spores until cracks form and water ingress occurs. When cracks appear in the concrete, water penetrates the cracks and reactivates the dormant bacterial spores. The bacteria then start metabolizing urea present in the concrete environment, leading to the production of urease (Benjamin et al., 2023). To increase urease production, bacterial strains must be genetically engineered at first to overexpress the ureC gene, so the urease enzyme produced by the overexpressed ureC gene catalyzes the conversion of urea into ammonia and carbon dioxide. The resulting increase in pH and the availability of carbonate ions facilitate the precipitation of CaCO₃, which fills and seals the cracks. The CaCO₃ crystals grow within the cracks, effectively healing the concrete. This process can significantly extend the lifespan of the concrete structure and reduce maintenance costs.

Strong and constitutive promoter is essential to drive the expression of the ureC gene such as the λp_R T7 promoter. Make sure that a strong ribosome binding site exists to ensure efficient translation initiation. A transcription terminator sequence downstream of the gene ensures proper termination of transcription . A plasmid vector is needed that has a high copy number and a suitable antibiotic resistance marker. Broad-host-range plasmids RSF1010 and pBR322 were used (Mobley et al., 1986; Veaudor et al., 2018).

Overexpression of the gene began with gene synthesis and cloning using primers that were designed with restriction sites for cloning. Then, the ureC gene was amplified using PCR (Koper et al., 2004). Digestion of both the PCR product (ureC gene) and the plasmid vector occurred with the same restriction enzymes to create compatible ends. The next step is purification and ligation of the ureC gene into the plasmid vector using DNA ligase. Recent advancements in ligation techniques, such as the PRESSO method, have improved the efficiency and accuracy of DNA cloning by enabling the rapid and efficient assembly of multiple DNA fragments in a specific order and orientation. Then, transformation of the ligated plasmid into a suitable host strain. After that, platting the transformed cells on agar plates containing the appropriate antibiotic for selecting transformants. Then, screening of the antibiotic-resistant colonies by colony PCR or restriction digestion to confirm the presence of the ureC gene. Sequencing of the insert was made to ensure there are no mutations and that the gene is correctly inserted. Finally, cells were harvested and lysed to extract proteins. Purification of proteins was done by gel filtration chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting using specific antibodies against the ureC protein was done to analyze the expression of urease (Sarkar, Adak, et al., 2015; Zhang, 2013).

Protective Carriers for Bacteria

The success of bio-self-healing concrete depends significantly on the survival and activity of the bacteria within the harsh environment of the concrete matrix. To ensure the bacteria remain viable and functional, protective carriers are employed. These carriers shield the bacteria from extreme conditions such as high pH, desiccation, and mechanical stresses, while also providing a controlled release of nutrients to sustain bacterial activity over time (Amer Algaifi et al., 2020). Protective carriers include many types such as expanded perlite (EP), diatomaceous earth (DE), expanded clay (EC), microcapsules, pelelith, nanometer material, lightweight aggregates, polyurethane foams, porous ceramsite, limestone particles, and siliceous sand. They have been discussed in this section.

The use of EP to immobilize bacteria within concrete shields them from high pH levels, enhancing self-healing properties and bending strength. Unlike surface-level bacterial treatments, EP enables deeper crack healing. Field emission scanning electron microscopy (FESEM) and X-ray diffraction (XRD) analyses reveal that EP particles have numerous cavities, providing oxygen and attachment sites for bacterial activity. EP’s high porosity and water absorption ensure bacteria receive water when cracks form. Concrete with EP particles and B. cohnii can heal cracks up to 0.79 mm, outperforming EC particles. Recent advancements include sugar-coated EP, achieving crack healing up to 1.24 mm (Jiang et al., 2020; Shen et al., 2021; Zhang et al., 2017).

Diatomaceous earth particles demonstrate a protective effect in high-pH cement environments due to their porous nature. They exhibit a strong capacity to adsorb bacterial cells on their surfaces, creating a new microenvironment that facilitates sustained bacterial urea degradation. Experimental results indicate that DE-immobilized bacteria maintain enzymatic activity and can completely repair cracks measuring 0.15 to 0.17 mm, even in a cement environment with a pH of 12.5. However, the amount of DE used is crucial for practical applications because DE particles significantly absorb water, which can dry out the mortar paste and negatively impact its workability (Shen et al., 2021).

Microcapsules protect the bacteria from harsh conditions and release them when the concrete cracks, triggered by environmental changes such as moisture ingress. Microcapsules, used primarily in polymers and composites, offer comprehensive protection to bacteria by isolating spores from external conditions. When cracks occur, these capsules rupture, exposing spores to nutrients and triggering the self-healing process. Authors found that microcapsule specimens with microorganisms had a higher healing rate (48%–80%) compared to bacteria-free specimens (18%–50%). The median crack healing width was 0.97 mm, nearly four times greater than nonbacterial specimens, and the permeability was 10 times lower (Wang, Soens, et al., 2014). Another study evaluated the encapsulation of Bacillus pseudofirmus spores in calcium alginate (CaAlg) capsules, assessing spore survival, retention, and CaCO₃ precipitation. Most spores survived encapsulation and remained in the capsules under simulated cement mixing conditions. Although CaAlg capsules degraded after exposure to cement filtrate, they remained stable when embedded in cement samples. The encapsulated bacteria effectively precipitated CaCO₃ in alkaline conditions, initiating self-healing when water and oxygen seeped through cracks. Despite an initial reduction in flexural strength, the capsules healed small cracks after 56 days of wet–dry cycles, recovering 39.6% strength in cement mortars and 32.5% in cement paste. Future research should focus on optimizing capsule use to balance self-healing efficacy with minimal strength reduction and explore applications in marine environments (Fahimizadeh et al., 2020).

Pelelith serves as an effective bacterial carrier for protecting bacterial spores and facilitating concrete crack repair. Unlike other carriers, pelelith’s positively charged surface and stable physical and chemical properties make it highly compatible with microorganisms and supportive of CaCO₃ deposition. Researchers examined concrete crack repair using pelelith as a bacterial vehicle by testing concrete specimens’ unregulated compressive strength and ultrasonic wave response. They compared the performance of the immobilized pelelith bacterial system with traditional methods. The study found that cracks measuring 1.5 mm and 2.0 mm were fully sealed within 20 days, achieving resistance recovery rates of 19.68% and 15.51%, respectively, with greater repair efficacy observed near the upper surface of the specimens (Shen et al., 2021).

Lightweight aggregates such as EP or pumice can be used as carriers. They provide physical protection and have a porous structure that allows bacteria to reside within the aggregates, shielding them from the high pH and other adverse conditions of the concrete matrix. Unlike lightweight aggregate carriers, graphene nanosheets increase the flexural strength of concrete, thus reducing cracks. Nanometer materials, which include artificial organic substances, offer advantages in enhancing the healing efficiency and performance of concrete due to their nanoscale size. It was demonstrated that graphite nanoplatelets significantly improve concrete’s self-repair potential. These nanoplatelets uniformly deliver the curing agent, enhancing the self-healing mechanism. However, the high cost of nanometer materials poses a limitation for their commercial application (Khaliq & Ehsan, 2016).

Polyurethane foams provide a protective matrix that can be infused with bacterial spores. Polyurethane foams offer excellent mechanical stability and can withstand the high pH environment of concrete. Polyurethan is commonly used for waterproofing. The potential of using polyurethane and silica gel as carriers for bacterial protection was investigated. Bacterial activity, indicated by the amount of CaCO₃ precipitated in the carriers, showed that silica gel-immobilized bacteria exhibited higher activity. However, specimens using polyurethane as the carrier demonstrated superior self-healing performance, significantly surpassing the recovery capacity of silica gel-immobilized microbial specimens (Shen et al., 2021).

Expanded clay is used as a carrier for bacteria in concrete to promote carbonatogenesis. Expanded clay protects bacteria from the harsh concrete environment, enhancing CaCO₃ precipitation in cracks. For EC to be effective, the YS11 bacterial strain must be able to enter or be immobilized within its channels, shielding the bacteria from mechanical stress, high pH, and temperature. Although EC has limited space for bacterial immobilization, it offers significant advantages in withstanding concrete’s internal heat and pressure. The viability of EC-immobilized bacteria was nearly equivalent to free bacteria with a concentration of 1.0 × 10⁶ cells/cm³, likely because the broken EC pieces exposed the bacteria more directly to the environment (Han et al., 2019).

Another study examined the ability of reinforced concrete containing ureolytic-type microbial self-healing agents immobilized in porous ceramsite particles to heal cracks. The research investigated the impact of healing on the mechanical and electrochemical properties of the reinforced concrete. They found that the bacteria could completely heal cracks up to 450 μm wide within 120 days. Tafel polarization results indicated that the closure of cracks due to microbial precipitation effectively inhibited reinforcement corrosion, with no observed adverse effects (Xu, Tang, Wang, et al., 2020).

Another study investigated the potential of using Gram-positive B. subtilis microorganisms to effectively heal nano-/microscale cracks in cement mortar (Khushnood et al., 2019). The study addressed concerns about the survival of these microorganisms in a cementitious environment by developing an efficient immobilization scheme. Various immobilizing media, including iron oxide nano-sized particles, micro-sized limestone particles, and milli-sized siliceous sand, were explored. The effect of immobilized B. subtilis microorganisms on these additives was assessed by quantifying the average compressive resistance of specimens and evaluating the healing process. The results indicate that B. subtilis microorganisms significantly improve the compressive strength and healing process of precracked cement mortar. Iron oxide nano-sized particles were found to be the most effective immobilizer, followed by siliceous sand and limestone particles. The use of iron oxide nanoparticles (IONPs) in smaller particle sizes ensures effective filling of pores and even distribution of bacteria throughout the cement matrix. This smaller size also enhances the retention of B. subtilis microbes in a dormant stage until cracks develop. The compressive strength regain of specimens treated with IONPs was measured at 91.3%. In comparison, samples treated with limestone particles as a protective material showed a regain of 77.6%, and those treated with sand reached 72.9%. Control samples without microbial treatment exhibited lower levels of compressive strength regain than those with microbes (Khushnood et al., 2019).

Hydrogels are hydrophilic polymer networks that can retain a significant amount of water. They provide a moist environment for the bacteria, protecting them from desiccation and allowing for the gradual release of nutrients. Alginate is the common type of hydrogels. In a study introduced a novel hydrogel crosslinked with alginate, chitosan, and calcium ions, the addition of chitosan enhanced the swelling properties of calcium alginate. Notably, when the chitosan content reached 1.0%, an opposite pH response to that of calcium alginate was observed. Incorporating 1.0% chitosan into hydrogel beads resulted in a 10.28% increase in compressive strength and a 13.79% increase in flexural strength compared to the control. These findings demonstrate the self-healing properties of concrete, evidenced by the healing of a 4-cm long and 1-mm wide crack in cement PO325. This healing was achieved with bacterial spores (2.54–3.07 × 10⁵/cm³ concrete) encapsulated in a hydrogel without chitosan (Gao et al., 2020).

Protective carriers include many benefits. They ensure that bacteria survive the hostile conditions of concrete, maintaining their ability to precipitate CaCO₃ when needed. By providing a controlled release of nutrients, carriers can sustain bacterial activity over extended periods, allowing for long-term self-healing properties. Also, the autonomous crack-healing capability reduces the ingress of harmful substances, such as chlorides and sulfates, thereby enhancing the durability and lifespan of concrete structures (Shen et al., 2021). Future research is focused on developing more cost-effective carriers, enhancing the efficiency of nutrient release, and improving the overall compatibility of the carriers with the concrete matrix. Advances in biotechnology and materials science are likely to yield new types of protective carriers that can further enhance the performance and affordability of bio-self-healing concrete.

Challenges and Limitations of Bio-Self-Healing Concrete

While bio-self-healing concrete presents a promising solution for enhancing the durability and longevity of concrete structures, several challenges and limitations must be addressed to make this technology more practical and widely applicable. The incorporation of bacteria and nutrients into the concrete mix increases the overall production cost. This cost can be a significant barrier, especially for large-scale projects or in regions with limited budgets. Also, the time it takes for bacteria to become active and initiate the healing process may not always align with the immediate need to repair cracks, potentially compromising the integrity of the structure in the short term. Moreover, there is a need to assess the long-term environmental impact of introducing bacteria into concrete, especially concerning leaching of bacteria or their metabolic by-products into surrounding environments. Another challenge is adequate moisture, which is necessary for bacterial activation and the subsequent healing process. In arid climates or in structures where moisture exposure is limited, the self-healing capability may be compromised. Finally, there is no guarantee that the bacteria will completely fill every crack, especially larger or more complex cracks. This could lead to partial healing, leaving some areas vulnerable to further damage (Lee & Park, 2018; Sharma et al., 2024).

Conclusion and Future Prospective

The future of bio-self-healing concrete is promising, with advancements in bacterial engineering, smart technologies, sustainability, and economic viability expected to drive the technology forward. Genetic engineering could lead to the development of bacteria specifically tailored for concrete environments, enhancing their ability to repair cracks and withstand harsh conditions. Additionally, the integration of bio-self-healing concrete with smart technologies, such as self-sensing mechanisms, could allow real-time detection and response to damage, further extending the lifespan of concrete structures. Sustainability is another key area of focus, with research exploring the use of waste materials as nutrient sources for bacteria and the potential for bio-self-healing concrete to contribute to carbon sequestration. These innovations could reduce the environmental impact of concrete production and support global efforts to combat climate change. As the technology matures, cost reductions through improved production methods and economies of scale could make bio-self-healing concrete more economically viable, leading to its widespread adoption in the construction industry.

Regulatory developments and the establishment of industry standards will be crucial for the broader application of bio-self-healing concrete, ensuring consistent quality and safety across projects. In the future, this technology could be used not only in new construction but also in retrofitting existing structures, offering a valuable solution for extending the lifespan of aging infrastructure. Future research may focus on selecting thermotolerant bacterial strains and developing genetic modifications to improve heat resistance in bacteria used for bioconcrete. These approaches aim to enhance microbial survival and activity under high temperature conditions within concrete, potentially improving self-healing performance in warmer climates and expanding the applicability of bioconcrete to various environmental settings. Overall, the future prospects of bio-self-healing concrete are bright, with the potential to revolutionize construction practices and contribute to more durable, sustainable, and resilient infrastructure.

Acknowledgments

All the authors thank and acknowledge their respective universities for support, and the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research (KFU242759), King Faisal University, Saudi Arabi for financial support

Author Contributions

All authors participated in conceptualization, writing, review, and editing the manuscript; Y.A.-G.M, project administration; and K.M. and M.A.M., fund acquisition.

Funding

This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research (KFU242759), King Faisal University, Saudi Arabia.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Data Availability

Data are available upon reasonable request from the corresponding author.