Comparative Analysis of Autogenous and Microbial-Based Calcite Precipitation in Concrete: State-of-the-Art Review

Abstract

Cracks in concrete are a persistent issue that compromises structural durability, increases maintenance costs, and poses environmental challenges. Self-healing concrete has emerged as a promising innovation to address these concerns by autonomously sealing cracks and restoring integrity. This review focuses on two primary healing mechanisms: autogenous healing and microbial-induced calcite precipitation (MICP), the latter involving the biomineralization activity of bacteria, such as Bacillus subtilis and Sporosarcina pasteurii (formerly known as B. pasteurii). This review explores the selection, survivability, and activity of these microbes within the alkaline concrete environment. Additionally, the review highlights the role of fiber-reinforced cementitious composites (FRCCs), including high-performance fiber-reinforced cement composites (HPFRCCs) and engineered cement composites (ECCs), in enhancing crack control and enabling more effective microbial healing. The hybridization of natural and synthetic fibers contributes to both improved mechanical properties and crack width regulation, key factors in facilitating bacterial calcite precipitation. This review synthesizes current findings on self-healing efficiency, fiber compatibility, and the scalability of bacterial healing in concrete. It also evaluates critical parameters, such as healing agent integration, long-term performance, and testing methodologies, including both destructive and non-destructive techniques. By identifying existing knowledge gaps and performance barriers, this review offers insights for advancing sustainable, fiber-assisted microbial self-healing concrete for resilient infrastructure applications.

1. Introduction

Concrete is widely used in construction but is prone to cracking, which can compromise its structural integrity and longevity. Cement, aggregates, and water are widely utilized to produce concrete. Concrete plays a crucial role in infrastructure design and improvement, and it offers both advantages and disadvantages. The strength, durability, and permeability of concrete make it highly desirable in the construction industry [1]. Additionally, due to its availability, low cost, and ability to be made in any form or shape, its demand is also rising. Normally, concrete lasts for about 50 years before developing cracks due to weather effects (such as rain and sunlight). However, it is impossible to prevent cracks entirely. Concrete becomes less durable when it cracks, exposing the steel rebar reinforcements to air, facilitating corrosion and weakening the bars. Both the plastic and hardened states exhibit cracks. In the plastic state, shrinkage, settlement, and rapid water loss are the causes of cracks, while in the hardened state, weather conditions, thermal effects, a lack of water content (dryness), and numerous other factors are the causes of cracks [2,3].

However, its inherent brittleness and susceptibility to cracking pose significant challenges, particularly regarding durability and maintenance. These cracks allow the infiltration of water and other aggressive agents, accelerating deterioration and increasing repair costs [4]. To mitigate this issue, research has provided diverse innovative solutions, one of which is the advent of bacterial self-healing concrete, which has huge potential as a leading solution [5]. Traditional repair methods are costly and time-consuming, stoking interest in self-healing concrete that can autonomously repair cracks [4]. The use of microbial-induced calcium precipitation (MICP) for self-healing bio-concrete has shown promise, with bacteria like B. megaterium and B. subtilis being used to precipitate calcium carbonate to seal cracks [5,6,7,8]. Bacterial concrete leverages microbial-induced calcium precipitation (MICP) to autonomously seal cracks, restoring structural integrity and reducing the need for external intervention. This self-healing mechanism aims to reduce cracking development, mitigate reinforcement corrosion, and increase the service life of concrete structures, reducing maintenance requirements [9]. The potential impact extends beyond the immediate structural benefits, further aligning with sustainable construction and infrastructure resilience goals [10]. Self-healing concrete is crucial for addressing the issues of global urbanization and infrastructure demand [11]. In the 1980s, with researchers in both biotechnology and civil engineering showing significant interest in the effectiveness of bacterial self-healing concrete, there was a dearth of research articles about the ability of concrete to cure itself [9]. However, after the 1990s, several papers were published on the subject. Despite its potential, the mechanical performance of bacterial concrete often requires enhancement to meet the demands of modern construction projects. The mechanical properties and healing efficiency of bio-concrete can be further enhanced by incorporating fibers [12]. Natural fibers, such as coir, flax, and jute, are environmentally sustainable and cost-effective, offering excellent crack resistance. Synthetic fibers, including polypropylene and glass fibers, provide superior tensile strength and durability, ensuring long-term performance [13].

This review aims to delve into microbial-induced calcium precipitation (MICP), a key process that enables bacterial self-healing in concrete [14,15]. It will explore various bacterial species used in this process, their selection criteria, and survivability within the harsh alkaline environment of concrete. The selection criteria for these bacteria include their ability to induce calcium carbonate precipitation, survivability in harsh conditions, and their overall effectiveness in repairing concrete cracks [16,17]. Additionally, the review will examine the mechanisms through which bacteria contribute to sealing cracks and restoring the structural integrity of concrete, supported by insights from recent studies [18,19,20].

Extensively, this review indicates that fiber-reinforced cement composites (FRCCs), high-performance fiber-reinforced cement composites (HPFRCCs), and engineered cement composites (ECCs) can integrate high-performance concrete characteristics with enhanced crack width control and energy absorption capabilities. The combination of these fibers results in a composite material exhibiting improved mechanical properties and crack management. Research by Rajesh et al. [21] notably demonstrates the effectiveness of natural fibers, such as coir, flax, and jute, as carriers for B. paramycoides in self-healing concrete, leading to enhanced mechanical properties and durability. Moreover, studies in [21,22,23] have shown that hybrid fibers can facilitate bacterial activity by maintaining optimal crack widths for healing.

By synthesizing current literature, this review aims to identify key challenges and knowledge gaps, such as bacterial survivability in concrete, fiber compatibility, and the large-scale application of hybrid fiber-reinforced bacterial concrete. It will also discuss critical parameters, including the compatibility of healing agents with cementitious materials, self-healing effectiveness, and long-term reliability. Furthermore, this review will explore the latest approaches for developing high-performance self-healing concrete, evaluating various testing methods used to measure healing efficiency. By addressing these aspects, this review aims to provide practical insights into advancing self-healing concrete technologies and promoting their adoption in modern construction practices. In the following sections, the paper examines both autogenous and microbial-induced healing mechanisms in concrete, outlining their underlying chemical principles, associated fiber enhancements, and recent developments in evaluation techniques. These insights frame the critical performance indicators necessary for scaling self-healing concrete toward sustainable infrastructure applications.

2. Review Methodology

This systematic literature review on self-healing concrete followed a rigorous and structured methodology to ensure the inclusion of high-quality and relevant studies. A comprehensive search was conducted across multiple reputable academic databases—namely, Scopus, Web of Science, ScienceDirect, and Google Scholar—to capture a wide array of peer-reviewed journal articles, conference papers, and reviews published between 2010 and 2025. These databases were selected for their extensive coverage of scientific and engineering research in civil and materials engineering. A Boolean search strategy was employed using targeted keywords and combinations, such as “self-healing concrete,” “autonomous concrete repair,” “microbial-induced calcite precipitation (MICP),” “autogenous healing,” “crack prevention,” “fiber-reinforced concrete,” and “structural durability.” This search string was designed to extract studies focusing specifically on mechanisms, materials, and evaluation techniques related to self-healing technologies in cementitious systems.

Studies were included based on the following criteria: (1) original research or review papers on autogenous or MICP-based self-healing in concrete, (2) incorporation of natural, synthetic, or hybrid fiber reinforcements, and (3) experimental evaluation of healing performance using techniques such as UPV, SEM, XRD, FTIR, or mechanical testing. Studies that did not focus on self-healing mechanisms, lacked methodological transparency, or did not report evaluation outcomes were excluded. This review aims to synthesize current progress in microbial and autogenous healing, highlight critical research gaps, and propose practical pathways for advancing durable and resilient self-healing concrete systems. The overall structure and thematic flow of the manuscript is illustrated in Figure 1.

3. Self-Healing Mechanisms in Concrete

The mechanisms of self-healing in concrete represent a fascinating convergence of materials science and engineering innovation, encompassing both natural and engineered processes that enable concrete to repair internal damage autonomously. These healing mechanisms can be broadly categorized into two fundamental approaches: autogenous healing, which leverages the inherent ability of concrete to seal minor cracks, and autonomous healing, which incorporates engineered solutions to enhance and accelerate the repair process. However, it is thought that both autogenous and autonomous healing techniques can only fix cracks that are a few hundred micrometers in size, which means that major structural damage cannot be fixed [24].

3.1. Autogenous Healing

Concrete cracks can be naturally repaired by autogenous healing when moisture is present and tensile stress is absent. The repair process consists of the deposition of calcium carbonate from the cementitious material and mechanical blocking by particles that are carried into the crack with the water [25]. This intrinsic mechanism leverages the inherent properties of the cementitious matrix to fill cracks with hydration products like calcium silicate hydrate (CaH₂O₄Si or C-S-H) gel. During the early hydration stages, unhydrated cement particles remain within the matrix. When exposed to water through a crack, these particles further react and form new hydration products, effectively filling the voids and promoting crack closure [26]. The effectiveness of autogenous healing depends on several factors, including crack width (generally limited to hairline cracks), availability of unreacted cement particles, and adequate moisture conditions [27]. Several factors influence this process, including crack size, environmental conditions, and the composition of the cementitious material.

Another important aspect impacting the efficacy of autogenous healing is the composition of the concrete mixture. Studies have shown that incorporating mineral additives, such as fly ash, silica fume, and ground granulated blast-furnace slag (GGBFS), can enhance the self-healing properties of concrete. These materials contribute to the formation of additional C-S-H phases, which are essential for restoring the integrity of the concrete matrix after cracking [18,19,28]. For instance, research indicates that the presence of GGBFS in concrete can lead to superior self-healing capabilities due to the significant amount of unhydrated particles that remain available for hydration [18].

Several studies have investigated these mechanisms and their enhancement through mineral additives and tailored curing methods.

Table 1. Autogenous healing additives, reinforcement, curing methods, and outcomes.

Research Objective	Mineral Additives	Specimen Size	Reinforcement	Specimen Count	Additive Ratio	Curing Method	Type of Damage Test	Crack Sealing (%)/ Time	Conclusions	References
Investigates the self-healing potential of mortar using CSA and CA additives. Explores hydration and CaCO3 precipitation mechanisms.	CSA, CA, Fly Ash	Not specified	Galvanized wire mesh	6	Not specified	Water	Splitting Tensile Test	150 μm in 28 days (M1); 200–250 μm (M2–M5); 400 μm (M6)	M6 achieved the best sealing performance. CA and CSA improved permeability and crack closure significantly.	[19]
Examines MgO and silica fume for self-healing composites, focusing on strength recovery and crack closure.	MgO, Silica, Fume	50 mm cement paste cube	None	4	M0 (OPC), M1 (OPC + 5% SF), M2 (OPC + 5% MgO), M3 (OPC + 5% SF + 5% MgO)	Water	Compressive Strength Test	M3: 26% (28 days); MX: 46% (56 days)	Silica fume enhances strength. MgO promotes self-healing, especially in blends.	[28]
Studies crack width as an indicator of healing in mortars with fly ash, silica fume, and crystalline additives.	Fly Ash, Silica Fume, CA	50 × 50 × 50 mm	Two 10 mm deformed bars	4	M1 (OPC only), M2 (30% FA), M3 (10% SF), M4 (1% CA)	Water	Splitting Test	CA: 80% in 7 days; SF: complete at 19 days (cracks 0.1–0.2 mm)	CA works best for small cracks, while SF is best for wider cracks. Performance depends on cracking age and additive type.	[20]
Evaluates self-healing using zeolite as an internal curing agent, enhancing compressive strength and sealing efficiency.	Zeolite, Fly Ash, Sky Cement	Not specified	PVA fibers	5	MZ_0% Zeolite, MZ_7.5%, MZ_15%, MZ_22.5%, MZ_30%	Wet/dry cycles	Splitting Tensile Test	Not available	Zeolite enhances strength and self-healing significantly in composites under wet/dry conditions.	[29]
Investigates CA effects on self-healing in normal-strength and high-performance fiber-reinforced cementitious composites.	CA, Microsilica	500 × 100 × 50 mm (NSC Size); 500 × 100 × 30 mm (HPFRCC size)	None (NSC); dispersed straight steel fibers (HPFRCC)	4	NCS1 (No CA), NCS2 (CA), HPFRCC1 (No CA), HPFRCC2 (CA)	Water, open air	Bending Tests	NCS (60%) under air exposure; HPFRCC showed higher recovery	CA improves sealing and mechanical recovery in both normal and high-performance composites. Fiber-CAs synergistically enhance performance.	[30]

summarizes the mechanisms, materials, and outcomes of autogenous healing in cementitious composites.

3.1.1. Chemical Reactions and the Healing Mechanism

When concrete develops cracks, unhydrated cement particles interact with infiltrating water, reinitiating the hydration process and producing hydration products that seal the cracks [31,32,33,34,35]. The continuous hydration of unreacted cement and the precipitation of calcium carbonate, which fills in spaces and cracks in the concrete matrix, are the main chemical processes for autogenous healing.

Hydration of Unreacted Cement Particles: Unhydrated cement particles remaining in the concrete matrix react with water to form additional C-S-H gel and calcium hydroxide (Ca(OH)₂ or C-H). This secondary hydration not only seals cracks but also strengthens the surrounding matrix. Studies indicate that this reaction is more effective in cracks smaller than 0.3 mm, where capillary forces enhance water ingress and reaction efficiency [36]. The continued hydration of the cement particles consists of either the primary cement minerals, alite (C₃S) or belite (C₂S), with water, forming calcium silicate hydrate (C-S-H) and calcium hydroxide (CH), which are critical to strength and durability in cementitious materials:

2C₃S + 6H → C₃S₂H₃ + 3CH,

(1)

2C₂S + 4H → C₃S₂H₃ + CH.

(2)

The calcium silicate hydrate (C-S-H) and C-H that are produced when unhydrated cement particles react with water efficiently fill microcracks.

A review by Xue C. et al. [26] observed that the autogenous self-healing products formed by continued hydration primarily consist of 78 wt% C-H and 17 wt% C-S-H [37]. This highlights a notable difference in the composition of hydration products in cracks compared to the matrix. Specifically, the matrix typically has a higher mass content of C-S-H compared to C-H [38]. These findings strongly suggest that the C-H crystals forming in cracks arise from the hydration of unreacted cement particles [39] and the recrystallization of leached portlandite [40].

Precipitation of Calcium Carbonate: Calcium hydroxide, a product of cement hydration, reacts with atmospheric or dissolved carbon dioxide (CO₂) in water to form calcium carbonate (CaCO₃). This process, known as carbonation, produces a dense precipitate that seals cracks and reduces the permeability of the concrete matrix. Moist environments with moderate CO₂ availability enhance this reaction, making it a critical mechanism in the autogenous healing of cracks [18,19,24].

The formation of calcite, a natural mineral that is composed primarily of CaCO₃, within cracks is a primary contributor to autogenous healing, with the growth rate of crystals influenced by crack width and water pressure. Interestingly, factors such as concrete composition and water hardness have minimal impact on this process [25]. The pH of the water that enters the fissures is normally between 5.5 and 7.5, and it contains CO₂ and Ca²⁺ ions. The pH of the water rises as more Ca²⁺ ions from the C-H and C-S-H phases dissolve as CO₂-rich water seeps through the hardened cement. At pH levels above 8, bicarbonate (CHO₃⁻) ions in the water are converted to carbonate (CO₃²⁻) ions, facilitating the precipitation of calcite. The fundamental reactions governing the formation of CaCO₃ and C-H include:

• Dissolution of CO₂ in water:

H₂O + CO₂ → H₂CO₂ → H⁺ + HCO₃⁻ → 2H⁺ + CO₃²⁻.

(3)

• Formation of calcium carbonate:

Ca²⁺ + CO₃²⁻ → CaCO₃.

(4)

• Reaction involving bicarbonates:

Ca²⁺ + HCO₃⁻ → CaCO₃ + H⁺.

(5)

These reactions occur predominantly at pH levels above 8, where the conversion of HCO₃⁻ ions to CO₃²⁻ ions is favored. CaCO₃ crystals grow along crack surfaces, gradually filling the gaps and restoring structural integrity.

Research indicates that carbonation plays a dominant role in self-healing for older concrete, whereas the further hydration of anhydrous cement components is more significant in younger concrete [31]. Self-healing through calcite precipitation is particularly effective for cracks up to 0.1–0.2 mm wide [15,41]. As the crystals develop both within and on the surface of cracks, they create a robust seal that enhances the durability of the concrete structure. The precipitation efficiency is influenced by environmental factors, such as temperature, humidity, and CO₂ concentration. Moderate CO₂ availability in moist conditions optimizes the rate of CaCO₃ formation, whereas excessive CO₂ can lead to over-carbonation, reducing the structural integrity of the precipitate [31].

Research has shown that CaCO₃ precipitation is more pronounced in cracks less than 0.3 mm wide, as smaller cracks provide better capillary action and confinement for the chemical reactions to occur [24,42]. Together, the hydration of unreacted cement particles and the precipitation of calcium carbonate form the backbone of autogenous self-healing in cementitious systems [18,31]. These processes, while effective in sealing microcracks, are constrained by environmental dependency and crack width limitations, which will be discussed in the next section, and these setbacks create opportunities for bioengineered approaches, such as microbial-induced calcite precipitation.

3.1.2. Crack Geometry and Sizes

Crack geometry, including shape, width, and depth, plays a pivotal role in determining the efficiency of self-healing processes in concrete. Autogenous healing is most effective for cracks narrower than 0.3 mm in width [24,43,44]. Larger widths are more likely to achieve complete sealing due to the availability of reactive compounds and the increased capillary action that facilitates the transportation of healing agents. Conversely, larger cracks require additional mechanisms, such as the incorporation of healing additives or bacterial activity, to bridge and seal the gaps effectively [24,31].

In autogenous healing, narrow cracks allow unhydrated cement particles to react with water, forming secondary hydration products that close the voids [26]. Crack depth is also a critical parameter, as deeper cracks are less accessible to healing agents, thereby reducing the likelihood of complete healing [31]. Autogenous healing in concrete is most effective for crack widths ranging from less than 0.2 mm to 0.3 mm [45,46]. Autogenous crack healing can be improved by limiting crack widths through compressive forces, incorporating fiber reinforcements in ECC [46,47] using superabsorbent polymers (SAPs) or other internal water reservoirs to supply water for continued hydration, and promoting the continuous hydration process [48,49].

3.1.3. Effect of Additives on Autogenous Healing

The incorporation of additives, such as fly ash, silica fume, and crystalline admixtures, has proven to significantly enhance the autogenous healing of concrete [50,51,52,53,54,55]. These additives improve the availability of reactive compounds and modify the microstructure of the concrete matrix, enabling the self-healing process to seal cracks, reduce porosity, and enhance the durability and lifespan of concrete structures.

Table 2. Autogenous healing additives and their mechanisms of action.

Additive	Mechanism of Autogenous Healing	Effects on Concrete Properties	Effectiveness in Healing	References
Calcium-based minerals (e.g., calcium hydroxide, calcium carbonate, calcium sulfoaluminate)	Enhances self-healing by reacting with CO2 and water to form healing compounds (CaCO3).	Improves crack sealing, enhances durability, and reduces permeability.	Effective for healing cracks up to 0.5 mm, particularly in wet environments.	[41]
Crystalline admixtures (CAs)	Reacts with moisture and unhydrated cement particles to form insoluble crystals (C-S-H and CaCO3), filling cracks and reducing porosity.	Improves crack closure (up to 0.4 mm), enhances strength, reduces water permeability, and ensures long-term durability.	Effective in healing cracks up to 0.4 mm and continues healing in the presence of water.	[48,49,53,54]
Silica fume	Promotes the formation of additional C-S-H, refining microstructure and sealing cracks. Works well in combination with MgO.	Improves durability, reduces permeability, and enhances self-healing of small cracks (<0.3 mm).	Best for small cracks (<0.3 mm). Helps enhance matrix densification.	[50,51,55]
Fly ash	Rich in silica and alumina, it combines with calcium hydroxide to produce more C-S-H, which densifies the concrete matrix.	Enhances crack healing, improves durability, reduces water absorption, and supports bacterial activity for self-healing.	Moderately effective in improving long-term healing and reducing porosity.	[51,52]
Magnesium oxide (MgO)	Expands upon hydration, forming MgO, which fills cracks and voids. Works synergistically with silica fume and fly ash.	Improves autogenous healing, reduces shrinkage, and enhances long-term durability. However, it may slightly reduce initial strength.	Moderate to high effectiveness. Works best in moist conditions. Effective in reducing shrinkage cracks.	[50,56,57]
Ground granulated blast furnace slag (GGBFS)	Enhances latent hydraulic activity, contributing to crack closure.	Improves durability and sulfate resistance.	Enhances self-healing over extended periods, particularly in humid environments.	[58]
Superabsorbent polymers (SAPs)	Retains water to promote continuous hydration and healing.	Improves internal curing, reduces shrinkage, and enhances self-healing.	Moderate to high effectiveness. Works best in cyclic wet/dry conditions. Enhances healing of cracks up to 0.4 mm.	[56,57]

summarizes autogenous healing additives and their mechanisms of action.

Crystalline Admixtures

Crystalline admixtures (CAs) are some of the most effective additives for improving autogenous healing. These admixtures react with moisture and unhydrated cement particles to form insoluble crystals within the pores and cracks of the concrete. Compounds such as L-aspartic acid and EDTA are commonly used crystalline agents that stimulate the formation of C-S-H gel and CaCO₃, filling cracks and reducing porosity [50,59,60]. As a result, the concrete matrix becomes denser, more compact, and has better strength and impermeability. Research has demonstrated that CAs enhance the self-healing capacity of concrete by significantly improving crack closure rates and reducing water permeability. For instance, cracks up to 0.4 mm wide have been shown to heal effectively in the presence of crystalline admixtures, even under aggressive environmental conditions. Additionally, these admixtures ensure long-term durability by continuously activating in the presence of water, sustaining the healing process [51,52].

Silica Fume

Silica fume is a byproduct of silicon metal or ferrosilicon alloy production and is a highly reactive pozzolanic substance, which enhances the self-healing capacity of concrete by refining its microstructure [61]. A natural siliceous substance known as pozzolanic material has a pozzolanic reaction with Ca(OH)₂ at room temperature when water is present. When combined with MgO or other complementary additives, silica fume creates a tightly packed microstructure, improving the durability and self-healing capacity of cement composites. Silica fume promotes the formation of additional C-S-H, which is crucial for sealing cracks and voids. Furthermore, it enhances the mechanical and durability properties of concrete by reducing its permeability and porosity. Studies have indicated that silica fume is particularly effective in self-healing smaller cracks (<0.3 mm), as its fine particle size allows it to penetrate and react within microcracks [53,54].

Fly Ash

Another common addition to improve autogenous healing in concrete is fly ash, a byproduct of burning coal in power plants. It contains a lot of silica and alumina, which combine with calcium hydroxide to create more C-S-H, strengthening the concrete matrix and repairing cracks. Fly ash also contributes to the densification of concrete, reducing porosity and improving durability. Fly ash, used in conjunction with biomineral additives, enhances the self-healing of concrete by lowering the pH, which is favorable for bacterial activity that aids in crack healing [55]. It contributes to the formation of a denser microstructure, which is essential for reducing water absorption and improving the durability of self-healing concrete [54].

Magnesium Oxide (MgO)

MgO is an expansive additive that improves autogenous healing by reacting with water to form magnesium hydroxide (Mg(OH)₂), which fills cracks and voids [62,63]. While MgO can slightly reduce the initial strength of concrete, its inclusion in combination with silica fume or fly ash mitigates these effects and enhances the overall self-healing capacity. In addition, MgO aids in reducing shrinkage and improving long-term durability, particularly in moist curing conditions [64]. While these additives significantly enhance the self-healing properties of concrete, it is important to consider the potential trade-offs, such as the initial reduction in strength. Additives like MgO can lower the initial compressive strength of concrete, though this can be counteracted by optimizing mix proportions [53]. A study by Noor et al. [53] specifically evaluated the impact of silica fume as an additive in MgO blended mortar, indicating that it slightly improved the self-healing ability of the mortar. The study confirms that incorporating silica fume with MgO improves the autogenous self-healing process without compromising strength development. This suggests that additives like silica fume can provide additional reactive compounds that contribute positively to the healing mechanism in cement-based composites.

Additionally, the effectiveness of these additives depends on environmental factors, such as temperature, humidity, and curing methods, and can vary based on the dosage of each additive, which must be carefully calibrated to ensure optimal performance.

3.1.4. Factors Impacting the Autogenous Process

The autogenous process in cementitious materials is influenced by several factors that impact the self-healing capabilities of these materials. Secondary hydration and the creation of reaction products that fill cracks are the main drivers of this process, which increases the resilience and longevity of concrete structures. Various parameters and mechanisms impact the autogenous self-healing process, including the kind of materials used, environmental conditions, and the presence of some specific additives.

Material Composition and Additives: The components of autogenous self-healing reactions are directly impacted by the matrix’s composition [65,66]. Young concrete exhibits superior autogenous self-healing ability than older concrete due to the higher availability of unhydrated binders, which are critical for continued hydration [37,67,68]. This difference is evident when autogenous healing relies on secondary hydration mechanisms. Low water/binder (w/b) ratio cementitious composites, like those found in ultra-high-performance fiber-reinforced concrete (UHPFRC) and ECC, retain more unhydrated binders, increasing the potential for autogenous self-healing [69]. The addition of silica fume speeds up secondary hydration and increases the formation of Si-containing healing phases. These phases are essential for crack closure and fiber–matrix interface regeneration in UHPFRC [70].

Environmental Conditions: Environmental factors, particularly water availability and temperature, significantly influence the autogenous healing process [71]. The effectiveness of these reactions depends on factors such as temperature, humidity, and the availability of reactive compounds. For instance, higher temperatures accelerate hydration reactions, while controlled moisture levels prevent desiccation, which could halt the healing process. Ions are transported into cracks by water, which makes it easier for reaction products like portlandite and C-S-H to dissolve and precipitate. Negligible self-healing is observed in specimens exposed to air, whereas water-rich environments promote crack healing. Wet/dry cycles, in particular, accelerate CaCO₃ precipitation by enhancing the transfer of CO₃²⁻ ions and the leaching of Ca²⁺ ions into cracks [72,73]. Water migration from cracks into the bulk paste facilitates further hydration, densifying the microstructure and reducing the ingress of aggressive agents. However, excessive water migration may limit the distribution of healing products into cracks [74]. Higher water temperatures enhance the hydration of unhydrated binders, accelerating the self-healing process. For instance, crack closure is accelerated when the healing temperature of high-performance concrete (HPC) is raised from 20 °C to 80 °C [64].

In summary, the effectiveness of autogenous self-healing depends heavily on crack dimensions, mix composition, environmental exposure, and the incorporation of targeted additives. Because the volume of healing products formed is less proportional to the initial crack width, narrower cracks (<0.3 mm) have higher crack closure ratios than wider cracks. While promising for small cracks and favorable curing conditions, this approach has inherent limitations in scope and responsiveness, paving the way for microbial-induced strategies to overcome such constraints. Fiber addition can restrict the crack width [75], which is a natural benefit of fiber-reinforced composites, which exhibit the best possible ability to completely self-heal cracks [76].

3.2. Microbial-Induced Calcium Precipitation (MICP)

While autogenous healing relies primarily on hydration-driven mechanisms, microbial-induced calcite precipitation (MICP) introduces a bioengineered alternative that extends healing capacity to wider cracks and more variable environments. This section delves into the biochemical processes, bacterial strains, and practical variables influencing MICP-based healing systems.

The ability of microorganisms to produce CaCO₃ extracellularly through metabolic activity is known as MICP. Biomineralization is the process by which living organisms produce minerals as a result of their metabolic products reacting with their surroundings [25]. This bio-inspired approach utilizes bacteria specifically chosen for their ability to precipitate CaCO₃ as a mineral binding agent. Spores of these bacteria are encapsulated within the concrete matrix. When water enters a crack, the capsules break open, releasing the bacterial spores. These spores germinate in the presence of moisture and nutrients (often provided alongside the bacteria), and the bacteria start metabolizing readily available substrates like Ca²⁺ and HCO₃⁻ (often present in the pore water of concrete) to precipitate CaCO₃ within the crack, promoting healing [5].

3.2.1. Mechanisms of MICP

MICP is an innovative process that leverages the metabolic activity of specific bacteria to precipitate CaCO₃, which enhances the mechanical properties of concrete and facilitates self-healing. This process is predominantly mediated by ureolytic bacteria, which hydrolyze urea to produce CO₃²⁻ ions that react with Ca²⁺ ions, resulting in the formation of CaCO₃ [77,78]. It has been shown that microbial self-healing may effectively seal cracks up to 1 mm in width [79,80].

The MICP process is driven by the enzymatic activity of urease, which catalyzes the hydrolysis of urea into ammonia and carbamate. The reaction sequence is as follows:

Equations (6) and (7) show that 1 mole of urea is first hydrolyzed intracellularly to produce 1 mol of ammonia and 1 mol of carbamate, which then hydrolyzes spontaneously to produce 1 mol of ammonia and carbonic acid:

$$\mathrm{CO}{\left({\mathrm{NH}}_2\right)}_2 +{ \mathrm{H}}_2{\mathrm{ONH}}_2\stackrel{\mathrm{Bacteria}}{\to } \mathrm{COOH} +{ \mathrm{NH}}_3,$$

(6)

$${\mathrm{NH}}_2\mathrm{COOH} +{ \mathrm{H}}_2\mathrm{O}\to { \mathrm{NH}}_3+{ \mathrm{H}}_2{\mathrm{CO}}_3,$$

(7)

Then, the products further equilibrate in water to form bicarbonate and 2 mol of ammonium and hydroxide ions (Equations (8) and (9)):

$$2{\mathrm{NH}}_3+ 2{\mathrm{H}}_2\mathrm{O}\leftrightarrow 2{ \mathrm{NH}}_{4^{+}}+ 2\mathrm{OH},$$

(8)

$$2{\mathrm{OH}}^{-}+2{\mathrm{H}}_2{\mathrm{CO}}_3\leftrightarrow {\mathrm{CO}}_{3^{2-}}+2{\mathrm{H}}_2\mathrm{O},$$

(9)

When urea is hydrolyzed to produce ammonia, the pH rises and an alkaline microenvironment surrounds the bacterial cell [25]. When supersaturation is reached, the presence of Ca²⁺ ions around the bacterial cell wall causes CaCO₃ to precipitate, as indicated by Equation (5):

$${\mathrm{CO}}_{3^{2-}}+{ \mathrm{Ca}}^{2+} \leftrightarrow {\mathrm{CaCO}}_3$$

(10)

The bacterial cell wall, characterized by negatively charged groups, serves as a nucleation site for positively charged cations, such as Ca²⁺, facilitating the precipitation of CaCO₃ [81]. The bacterial cell surface serves as a nucleation site in the precipitation of CaCO₃, as shown in Equations (11)–(13):

$${\mathrm{Ca}}^{2+} + \mathrm{Cell}\to { \mathrm{Cell} \mathrm{Ca}}^{2+},$$

(11)

$${\mathrm{Cl}}^{-}+{ \mathrm{HCO}}_{3^{-}}+{ \mathrm{NH}}_3\to { \mathrm{NH}}_4\mathrm{Cl}+{\mathrm{CO}}_{3^{2-}},$$

(12)

$$\mathrm{Cell}-{ \mathrm{Ca}}^{2+}+{ \mathrm{CO}}_{3^{2-}}\to { \mathrm{Cell} \mathrm{CaCO}}_3,$$

(13)

Bacteria used in self-healing concrete can be classified based on their ability to thrive in different pH environments, ranging from acidophiles that prefer highly acidic conditions (pH 1–5.5) to neutrophiles that grow in neutral to slightly alkaline environments (pH 5.5–8.5), and alkaliphiles that survive in highly alkaline conditions (pH 7.5–12) [82]. Among these, Gram-positive alkaliphilic bacteria are the most commonly employed in MICP for self-healing concrete. In particular, species from the Bacillus genus, such as Lysinibacillus sphaericus (previously known as B. sphaericus), B. subtilis, and S. pasteurii, have demonstrated high resilience in extreme pH and temperature conditions. These bacteria are widely used due to their ability to enhance crack healing by precipitating CaCO₃, thereby improving the durability and longevity of concrete. As highlighted in

Table 3. Bacillus bacterial strains most commonly used in self-healing concrete.

Bacillus Bacterial Strains	Advantages	References
B. cereus	Adapts to harsh environmental conditions, such as high pH and temperature. Enhances crack healing and reduces water permeability.	[51,83]
L. sphaericus	Precipitates CaCO3, leading to strength gain and reduced water absorption. Forms resilient endospores under extreme conditions.	[82,84,85]
B. subtilis	Exhibits high urease activity up to pH 9. Produces constant CaCO3 and withstands harsh environmental conditions.	[52,86]
S. pasteurii	Exhibits high efficiency in urea hydrolysis, making it highly effective in CaCO3 precipitation.	[77,78]

below, research has primarily focused on these alkaliphilic Bacillus strains because of their effectiveness in self-healing mechanisms.

When it comes to crack healing, ureolytic bacteria like S. pasteurii, B. subtilis, and L. sphaericus are superior to non-ureolytic bacteria. These bacteria have the ability to repair cracks up to 0.85–0.97 mm, while non-ureolytic species, such as B. halodurans and B. licheniformis, can only repair cracks up to 0.45 mm and only regain 65% of their initial strength [78,80,87,88,89,90].

3.2.2. Crack Geometry and Sizes

The efficiency of bacterial healing largely depends on the size and geometry of cracks. Smaller cracks, particularly those with widths between 0.1 and 0.5 mm, demonstrate the highest efficiency in MICP. The bacterial calcite precipitation process diminishes for larger cracks due to limitations in the dispersion of bacterial solutions and nutrients within the crack matrix [91]. Research indicates that MICP-based bacterial healing is significantly more effective than autogenous healing for larger cracks. While autogenous healing is limited to sealing cracks up to 0.1 mm in width, microbial self-healing techniques have been shown to effectively seal cracks up to 1 mm. For example, microencapsulated bacteria-based concrete has effectively sealed cracks as large as 970 μm, while bio-hydrogels can repair cracks up to 500 μm wide [92].

The efficiency of healing also varies depending on the material composition and environmental conditions. For example, in recycled aggregate concrete, MICP has been observed to seal cracks up to 0.58 mm within 56 days, accompanied by a significant recovery in compressive strength, ranging from 94% to 56% depending on the initial crack size [93]. The orientation and shape of cracks also influence the healing process. Straighter and more uniform cracks exhibit higher healing efficiency compared to irregular or tortuous crack paths. Irregular cracks pose challenges to the uniform distribution of bacterial solutions, reducing the overall effectiveness of the self-healing mechanism [94]. Both the bacterial viability and the efficacy of crack healing are significantly influenced by the curing conditions. Wet–dry cycles, surface exposure with tin foil wrapping, immersion in water, bacterial solution, and curing at 20 °C with more than 95% relative humidity were some of the parameters Wu et al. [82] used to assess bacterial healing. Cracks healed under all curing conditions, but wet–dry cycles left a sediment layer on the surface in addition to filling them with mineralized products. Those submerged in water had the highest healing percentages, followed by those cured in soil over a 28-day incubation period, according to Souid et al. [95]. Wang et al. [80] achieved full healing even with limited water immersion by immersing specimens in tap water for one hour per day and then exposing them to humid air (60% relative humidity) for eleven hours at 20 °C. Xu et al. [96] showed that cracks up to 400 μm may be completely healed in 28 days by culturing bacterial specimens in a mixture that contained calcium, yeast extract, and bacterial spores. Crack healing up to 417 μm under wet/dry cycle circumstances was reported in another investigation by Xu et al. [97].

These findings underscore the importance of understanding crack geometry and optimizing environmental and curing conditions to maximize the self-healing potential of MICP-based concrete systems. By tailoring these parameters, researchers can enhance the efficiency and practicality of bacterial self-healing for real-world applications.

3.2.3. Influencing Factors

Several environmental factors affect the activity and survival of bacteria within concrete. Sporulation is influenced by a number of variables, including temperature changes, oxygen concentrations, pH variations, and mineral concentrations. High alkalinity (pH 12) in fresh concrete can inhibit bacterial growth. Alkaliphilic bacteria, however, are adapted to survive under such conditions [98]. Oxygen availability influences bacterial metabolism and sporulation. Ca²⁺ ion availability directly affects the efficiency of CaCO₃ precipitation. Under conditions that are unfavorable for growth, to preserve genetic material, bacteria enter a dormant state until favorable conditions return [42]. Wet–dry cycles, heat cycles, chemical exposure, and radiation are all situations that bacterial spores may tolerate because of this decrease in metabolic activity. While autogenous healing in cement-based materials can repair small cracks (up to 100 µm) in the presence of water, it is ineffective for larger cracks [19]. In contrast, microbial self-healing has demonstrated the ability to repair cracks up to 1 mm in width [79,80]. Below, we discuss the specific factors that affect the activity and survival rate of bacteria within the concrete.

3.2.4. Bacterial Strains Used in MICP

Different bacterial strains, such as B. subtilis, S. pasteurii, and L. sphaericus, have been studied for their effectiveness in MICP. Concrete contains an alkaline medium that these bacteria may tolerate [84,85]. By transforming urea into ammonium and CO₃²⁻ ions, L. sphaericus can precipitate CaCO₃ in this extremely alkaline environment [25]. As shown in

Table 4. Bacillus bacterial strains most commonly used in microbial-induced calcite precipitation.

Bacterial Species	References
Bacillus	[15,79,98,99]
L. sphaericus	[86,100,101]
B. pasteurii	[81,102]
B. subtilis	[103]
B. megaterium	[104,105]
S. pasteurii	[102,106,107]
B. cereus	[51,83,108]

from earlier research, a variety of bacterial species can mediate CaCO₃ generation in accordance with metabolic pathways.

3.2.5. Concentrations of Bacteria Used in MICP

The concentration of bacteria used in self-healing concrete plays an important role in determining the efficiency and extent of MICP. An optimal bacterial concentration ensures that sufficient metabolic activity is maintained to facilitate the hydrolysis of urea, the production of CO₃²⁻ ions, and the subsequent precipitation of CaCO₃ [80,89]. However, a concentration too high or too low can adversely affect the self-healing process.

Research indicates that bacterial concentrations within the range of 10⁵ to 10⁸ cells/mL solution are most effective for self-healing applications. Concentrations below this range fail to produce adequate urease enzyme activity, leading to insufficient CaCO₃ precipitation, while concentrations above this range can result in nutrient depletion, reduced bacterial viability, and diminished healing efficiency [109]. Tie et al. [110] demonstrated that a bacterial concentration of 2.5 optical density (OD) is optimal when using B. subtilis in self-healing concrete. This concentration improved mechanical properties, including compressive and flexural strength, more effectively than 2.0 OD or 3.0 OD concentrations.

Different bacterial concentrations have demonstrated varying levels of success in repairing cracks and restoring strength. S. pasteurii and L. sphaericus at 10⁸ cells/mL were shown to successfully precipitate CaCO₃, sealing cracks and recovering compressive strength in damaged specimens [111]. On the other hand, 10⁶ and 10⁷ cells/mL, respectively, produced about 30% and 45% of urea hydrolysis, with the maximum hydrolysis taking place at 10⁸ cells/mL, leading to better healing efficacy. The lower concentrations showed incomplete crack healing and lower mechanical property recovery. In a separate study, B. megaterium at 10⁵ cells/mL improved compressive, split tensile, and flexural strength in fly ash concrete. This concentration allowed for effective CaCO₃ deposition, highlighting its potential in structural applications [112].

Bacterial concentration also influences strength development in different concrete grades. When added to higher-grade concrete, B. megaterium at 30 × 10⁵ CFU/mL showed a faster rate of strength development than when added to lower-grade concrete. In 50 MPa concrete, a 24% increase in strength was achieved due to enhanced calcite precipitation [104]. S. pasteurii at 10⁵ cells/mL, combined with a 10% replacement of cement with fly ash, enhanced the compressive strength of structural concrete by 20%. Microstructural analysis confirmed CaCO₃ deposition on bacterial cell surfaces [113,114].

The specifics of the bacterial strains, concentrations, and compressive strength values obtained are compiled in

Table 5. Summary of Bacillus strains, compressive strength values, and concentrations.

Bacteria Strain Used	Best Result	Concentration	References
B. subtilis	12% improvement in compressive strength compared to controlled lightweight aggregate concrete.	2.8 × 108 cells/mL	[103]
B.megaterium	Maximum strength development of 24% achieved in 50 MPa concrete.	30 × 105 cells/mL	[104]
S. pasteurii and L. sphaericus	Sealed cracks and recovered compressive strength in damaged specimens.	108 cells/mL	[111]
B. megaterium	Improved compressive, split tensile, and flexural strength in fly ash concrete.	105 cells/mL	[112]
S. pasteurii	35% increase in compressive strength compared to control concrete.	105 cells/mL	[114]
B. aerius	Increase in compressive strength by 11.8% in bacterial concrete compared to control with 10% dosage of rice husk ash.	105 cells/mL	[115]

, which vary based on the calcium source that the bacteria were fed.

These findings reinforce the need for precision in bacterial concentration, as both under- and over-dosing can negatively impact healing performance. The next sections explore how nutrients and delivery systems further influence microbial effectiveness in concrete matrices.

3.3. Bacterial Nutrients

Bacterial nutrients also play a pivotal role in supporting bacterial activity within self-healing concrete and are the essential substrates required for MICP, ensuring sustained metabolic activity and efficient crack healing. This section talks about the selection and concentration of nutrients and how they directly influence bacterial viability and CaCO₃ precipitation.

Urea as a nutrient: Urea is one of the most commonly used nutrient sources in MICP due to its role in the ureolytic process. Bacteria, such as S. pasteurii, hydrolyze urea to produce ammonium and CO₃²⁻ ions, which react with Ca²⁺ ions to precipitate CaCO₃. Optimal urea concentrations enhance bacterial activity and maximize the efficiency of crack healing [16,25,109]. For example, B. subtilis utilizes urea to produce CaCO₃, which is vital for the self-healing process [16].

Calcium salts, such as calcium chloride, calcium lactate, and calcium nitrate, serve as Ca²⁺ ion sources for the precipitation of CaCO₃. Among these, calcium lactate is particularly effective due to its high solubility and compatibility with bacterial systems. Studies have shown that calcium lactate facilitates efficient crack healing by promoting the deposition of calcium carbonate on crack surfaces [15,101,116]. Research also highlights the synergistic effect of combining urea and calcium salts. For instance, a nutrient solution comprising urea and calcium lactate improved the healing of cracks within 28 days, demonstrating the importance of a balanced nutrient composition [117]. According to Achal et al. [118], concrete specimens treated with ureolytic bacterial strain B. megaterium demonstrated improved strength and permeability properties, as did fly-ash-amended mortar.

Additional organic nutrients, such as yeast extract and peptone, can enhance bacterial growth and activity. These compounds support the long-term viability of bacteria by providing essential proteins and amino acids. Wong et al. [17] demonstrated that including yeast extract in nutrient solutions significantly improved bacterial survival and CaCO₃ precipitation over time. Insufficient nutrients can limit bacterial urease activity, while excessive concentrations may lead to unwanted side effects, such as increased porosity and reduced mechanical strength. Proper nutrient formulations are essential for achieving efficient crack healing and maintaining the structural integrity of self-healing concrete systems. By fine-tuning nutrient levels, researchers have successfully enhanced the mechanical recovery and the healing efficiency of damaged concrete.

3.4. Bacterial Carriers

Bacterial carriers play an important role in protecting bacteria and ensuring their effective distribution within the concrete matrix. These carriers provide a microenvironment that shields bacterial spores from the harsh alkaline conditions of concrete, facilitates their activation upon crack formation, and enhances their self-healing efficacy [119]. The choice of carriers directly impacts bacterial viability and the efficiency of crack sealing. Methods such as polymer encapsulation and impregnation enhance the durability and functionality of carriers

Figure 2 illustrates the autonomous self-healing approach achieved through microencapsulation techniques. In this method, healing agents are encapsulated within microcapsules and integrated in the concrete matrix [120]. The healing agent is released and enters the crack faces through capillary action when a crack develops and ruptures these implanted microcapsules. This self-healing system has been predominantly developed for use in polymers and composites and demonstrates the potential to provide comprehensive protection for bacteria. Researchers often utilize microcapsules to isolate bacterial spores from external environmental stress. This isolation allows the spores to remain in a dormant state until favorable conditions for activation arise. When cracks appear, the rupture of the capsules exposes the spores to nutrients, triggering their transformation from a dormant to an active state. This process initiates self-healing under the right environmental conditions [119].

Lightweight aggregates (LWAs), such as expanded clay, perlite, and pumice, are widely utilized as bacterial carriers. These materials encapsulate bacterial spores and nutrients, gradually releasing them into cracks upon exposure to moisture. LWA improves the distribution of bacteria throughout the concrete, ensuring consistent healing across the matrix. In earlier research, Jonkers [104] immobilized bacterial spores and calcium lactate using porous, expanded clay as a prelude to mineral precipitation. When soft, light clay aggregates rupture, bacteria in the air cause the CaCO₃ to precipitate. The specimens with the maximum healed crack width, 0.46 mm, were those that were analyzed after being submerged in tap water for two weeks. After six months, there was no discernible decline in the viability of bacteria. Furthermore, the use of graphite nanoplatelets (GNPs) as additives in LWA enhances bacterial survival and distribution [104]. Impregnating LWA with bacteria and subsequently encapsulating them in a polymer coating improves the overall performance of self-healing concrete. This technique protects the bacteria from premature activation and enhances crack sealing [121].

Silica gel is another effective carrier due to its high surface area and porous structure, which enable silica gel to retain and gradually release bacteria and nutrients in a controlled manner, ensuring their long-term viability. This controlled release mechanism is critical for maintaining bacterial activity over extended periods and optimizing the self-healing process. Research indicates that silica-gel-encapsulated bacteria significantly enhance CaCO₃ precipitation, which plays a vital role in crack sealing and the mechanical recovery of concrete. For example, a study by Van Tittelboom et al. [122,123] utilized L. sphaericus immobilized within silica gel. For three days, the encapsulated bacteria were submerged in a urea and calcium source solution. Cracks measuring 0.3 mm in breadth and 10.0 mm and 20.0 mm in depth were effectively repaired during this time. This demonstrates the effectiveness of silica gel in not only preserving bacterial activity but also in promoting robust healing performance.

Hydrogels are increasingly gaining attention as carriers for self-healing applications due to their water-retention capabilities. These materials swell upon contact with water, releasing encapsulated bacteria and nutrients into cracks. Hydrogels have been shown to heal cracks up to 1 mm wide and enhance CaCO₃ deposition in the crack matrix. Their ability to improve bacterial survivability and precipitation efficiency has made them a preferred choice in advanced self-healing systems [124].

Research has highlighted the advantages of using carriers to enhance autonomous self-healing. Encapsulation techniques improve bacterial survivability in harsh alkaline environments, enabling their functionality over extended periods. Studies have shown that bacterial spores embedded in hydrogels exhibit increased healing efficiency, both in terms of CaCO₃ precipitation and crack sealing [124]. Direct application of bacteria in concrete, such as Shewanella bacteria, the sole genus included in the marine bacteria family Shewanellaceae, has shown up to a 25% increase in compressive strength after 28 days, emphasizing the importance of carrier compatibility and nutrient availability [125]. By optimizing carrier materials and encapsulation methods, researchers have achieved significant improvements in self-healing efficiency and mechanical property recovery in concrete. This highlights the potential of advanced carrier systems in scaling up self-healing concrete technologies for practical applications. Carriers like LWA and silica gel protect bacteria and enhance their distribution within the concrete matrix [119,121].

Table 6. Summary of the carriers used with Bacillus bacterial strains.

Carrier Material	Microorganism	Crack Healing	References
Bacteria immobilized in silica gel	L. sphaericus	Crack width of 0.3 mm and depths of 10.0 and 20.0 mm	[123]
Spores with calcium lactate are embedded in expanded clay	B. alkalinitrilicus	Crack width ranging from 0.05 to 1.0 mm	[126]
Hydrogel-encapsulated spores with nutrients and calcium source	L. sphaericus	Crack width of 0.5 mm	[127]
Bacteria externally applied on cracked concrete structures	B. cohnii	Crack width ranging 0.1–0.4 mm	[128]
Spores encapsulated in microcapsules	L. sphaericus	Maximum crack width healed is 0.97 mm	[80]

gives a summary of carriers used with Bacillus bacterial strains in other studies. The choice of encapsulation method and carrier material must balance bacterial protection with release efficiency and compatibility with concrete processing. The synergy between nutrient supply and carrier design is critical to MICP effectiveness in practical applications.

3.5. Environmental Factors Influencing Microbial-Induced Self-Healing in Concrete

The success of microbial-induced calcium carbonate precipitation (MICP) in self-healing concrete relies on a delicate interplay between bacterial density, nutrient availability, and environmental conditions, such as temperature, pH, moisture, and oxygen. These factors directly influence bacterial viability, enzymatic activity, and ultimately the rate and quality of calcium carbonate (CaCO₃) deposition within cracks, which governs healing efficiency and durability enhancement in concrete structures [17].

The complex reactions (Equations (6)–(13)) described in Section 3.2.1 depend on these outside variables. One of the main factors influencing the generation of CaCO₃ in the pH setting is the urease enzyme’s capacity to initiate urea hydrolysis. The availability of urease varies with pH, which has an immediate effect on the self-healing process. Similarly, urease catalysis and other enzymatic reactions are impacted by temperature. The kinetics of CaCO₃ generation are directly impacted by the temperature sensitivity of these processes. Therefore, the delicate balance needed for concrete to effectively self-heal is highlighted by the interdependence of pH, temperature, and bacterial viability. The effects of temperature, pH, and other variables on microbial viability will be covered in the sections that follow.

3.5.1. pH Levels

The alkaline environment of concrete, with pH values typically ranging between 9 and 12, provides a suitable habitat for alkaliphilic bacteria, such as B. subtilis and S. pasteurii. These bacteria demonstrate urease activity, which facilitates urea hydrolysis and subsequent calcium carbonate precipitation. However, extreme pH fluctuations can impair bacterial survival and metabolic efficiency, thereby reducing healing performance. Maintaining a stable pH within the optimal range is essential for sustaining bacterial activity and ensuring effective self-healing [129]. According to Gauvry et al. [130], B. subtilis can be seen to grow at a pH between pH 4.8 and 9.2. According to Luhar and Gourav [131], L. sphaericus grew at pH ranges between 8 and 9. Over the course of five days, L. sphaericus displayed the greatest urease degradation at pH 7. However, only on the fourth day of observation did S. pasteurii exhibit the highest urea breakdown at pH 7. Javeed et al.’s paper [42] compiles research results on the urease activity of several bacteria at various pH values. Different bacterial types have different associations with pH in terms of urease activity, as shown in Figure 3 below by [42]. At varying pHs, the same bacterial species may even have the highest urease activity.

3.5.2. Temperature

Temperature plays a crucial role in regulating bacterial metabolic activity and the precipitation of calcium carbonate, which are essential for microbial-induced calcium precipitation (MICP) in self-healing concrete. Optimal bacterial performance generally occurs within a temperature range of 20 °C to 40 °C. At low temperatures, bacterial activity slows down significantly, reducing the healing efficiency, while excessively high temperatures can denature critical enzymes like urease, impairing the MICP process. Studies indicate that maintaining curing temperatures within this range accelerates self-healing and enhances crack-sealing effectiveness [129].

Different bacterial strains used in self-healing concrete demonstrate varying tolerances and optimal growth temperatures. For instance, L. sphaericus exhibits an optimal growth temperature between 35 °C and 37 °C [58,132]. B. subtilis, known for its resilience, can survive temperatures as high as 70 °C, with Reddy et al. [133] reporting its survival in a wide range of temperatures from −30 °C to 70 °C.

Further studies highlight the temperature-dependent growth behavior of various bacterial species. Durga et al. [134] investigated the effect of temperatures ranging from 25 °C to 60 °C on four bacterial strains: B. cereus, B. subtilis, B. halodurans, and B. licheniformis. Their findings revealed that B. subtilis exhibited the highest growth within 30 °C to 37 °C, while B. licheniformis thrived best at 37 °C. Interestingly, B. cereus demonstrated superior growth at 60 °C in relation to other strains, indicating its potential suitability for high-temperature applications.

A review by Javeed et al. [42] presents a graphical analysis, Figure 4, illustrating the relationship between bacterial growth and temperature. The analysis confirms that temperature significantly influences bacterial development, with most species achieving optimal growth up to 40 °C. These findings underscore the importance of maintaining appropriate temperature conditions to maximize the efficiency and longevity of microbial self-healing mechanisms in concrete.

3.5.3. Moisture Content and Oxygen Availability

Adequate humidity and moisture levels are essential for activating bacteria and facilitating nutrient transport within the concrete matrix. Insufficient moisture can hinder bacterial activity, reducing the efficiency of the self-healing process. Conversely, controlled wet–dry cycles have been shown to enhance calcite precipitation by improving nutrients and ion transport into cracks. Concrete specimens subjected to these cycles demonstrate better healing performance compared to those immersed continuously in water [135]. The availability of oxygen influences bacterial growth and metabolic activity. Aerobic bacteria, commonly used in MICP-based self-healing systems, require sufficient oxygen for optimal urease production and metabolic function. In low-oxygen environments, bacterial activity may be suppressed, leading to reduced calcite precipitation and incomplete crack sealing [17].

Overall, microbial healing presents a significant advancement over traditional methods, with the ability to heal cracks up to 1 mm wide and adapt to harsh environmental conditions. However, its success depends on careful selection of bacterial strains, compatible nutrient systems, and effective curing strategies. These insights form the foundation for evaluating the field readiness of microbial self-healing concrete.

4. Self-Healing Performance Evaluation

The development of self-healing technologies marks a significant advancement in improving the durability and longevity of construction materials, particularly in infrastructure applications like pavements and concrete structures. The evaluation of self-healing performance involves assessing the recovery of microstructure, durability, and mechanical properties after damage. When assessing the effectiveness of self-healing, parameters like crack width, length, depth, and number are crucial. Cracks for testing self-healing are typically induced using methods such as mechanical loading, the introduction of thin copper plates [136], or treatment with corrosive solutions. Experimental studies have demonstrated that healing products—such as continuous hydration or CaCO₃ precipitation—are key contributors to crack closure. The mechanisms for crack sealing in systems containing capsules or vascular networks may vary depending on the type of healing agent. Fully sealed cracks have been reported in some self-healing systems, showcasing the potential of these technologies [32,109,120,121,137].

Surface cracks are typically assessed using cameras or optical microscopes, though these methods are limited to external features. Non-destructive methods, such as X-ray computed tomography (XCT), allow for the measurement of crack healing, the observation of internal and external features, and the tracking of the behavior of healing materials like vascular systems and capsules. XCT observation has been widely used to track the condition and rupture behavior of healing materials like capsules, as well as to measure the crack-healing efficiency inside self-healing concrete [138,139,140]. Ultrasonic pulse velocity (UPV) has also been found to improve the evaluation of autonomous self-healing by detecting changes in material density and integrity [81]. The efficacy of healing has been evaluated using a variety of test techniques, many of which are common concrete testing techniques. Based on the evaluated qualities of the healed concrete, Nodehi [141] and Van Tittelboom [142] provided a comprehensive classification of assessment methods for self-healing concrete in their extensive study of self-healing processes in cementitious materials. The three distinct assessment methods are visualization and determination, restoration of mechanical properties, and restoration of resistance. The applicability of the several assessment techniques for self-healing strategies discussed in the literature is compiled in

Table 7. Summary of the applicability of various assessment methods for self-healing approaches.

Approaches	Test	Assessments	References
Visual Appearance	X-ray diffraction (XRD)	Chemical composition of healing substances	[48,143,144]
	X-ray computed tomography (XCT)	3D visualization of crack healing	[145,146]
	Environmental scanning electron microscopy (ESEM)	Surface morphology; microstructure of the healing substance	[147]
	Scanning electron microscope (SEM)		[144,148,149,150]
	Image analysis and camera/optical microscope	Rate of healing and crack characterization	[144,145,150,151,152]
	Isothermal calorimetry	Hydration procedure	[48,153]
	Transmission electron microscopy (TEM)	Healing material morphology	[153]
Durability Enhancement	Sorptivity test/capillary water absorption test	Water tightness	[154,155]
	Water permeability test	Water tightness	[139,146,147,156,157]
	Ultrasonic pulse velocity test	Degree of damage	[96,158,159]
	Gas permeability	Gas tightness	[152,155]
	Corrosion test and chloride diffusion test	Resistance to chloride incursion	[160]
	Rapid chloride permeability test		[113,144,158]
	Electrochemical measurements	Electrodeposition E-passivity of steel bar	[161]
	Electrical impedance test	Microstructural characteristics	[144]
Mechanical Properties Improvement	Compression test	Fragmentation of capsules with a cracking effect Generating new cracks as opposed to reopening old ones Strength, modulus, stiffness, fracture energy, and toughness all increased in the reloaded, healed specimen	[151,158,162]
	Impact loading test		[137]
	Fatigue test		[163,164]
	Nanoscale mechanical measurements		[128]
	Tensile test		[145,148,165]
	Three-point bending test		[153,154,166,167]
	Dynamic mechanical analysis		[166]
	Four-point bending test		[128,151]
	Cyclic four-point bending test		[168,169]
	Bond strength test	Bond strength between capsules and matrix	[166]

.

The comprehensive evaluation of self-healing materials ensures their effectiveness in extending the service life of construction materials. By leveraging advanced testing methods, optimizing healing conditions, and understanding the interaction between crack geometry and self-healing mechanisms, researchers can further refine these technologies to meet the demands of modern infrastructure. Evaluating the self-healing performance is crucial for understanding how effectively these materials recover their properties after damage, ensuring their practical application in real-world conditions. The following section focuses on the methodologies, parameters, and experimental setups employed to assess the self-healing performance efficiency of bio-concrete.

4.1. Pre-Cracking Test Methods for Evaluating Healing Efficiency and Mechanical Properties

Pre-cracking methods are a fundamental step in evaluating the self-healing performance of concrete. These methods involve creating controlled cracks within the concrete to facilitate the healing process, allowing for the integration of self-healing agents. By introducing controlled damage, these methods ensure uniformity and reproducibility in experimental setups, providing a consistent baseline for evaluating the efficiency of self-healing mechanisms. The recovery of mechanical characteristics and alterations in the microstructure of the fractured cementitious materials must be evaluated qualitatively and quantitatively in order to fully evaluate the efficacy and resilience of self-healing systems in cementitious materials [166]. These methods simulate real-world damage scenarios and allow researchers to test the ability of self-healing systems to restore structural integrity. The following sections detail various pre-cracking techniques and their implications for self-healing concrete.

4.1.1. Flexural Bending Tests

Flexural bending tests are performed on beam specimens subjected to a load at the center or multiple points to induce bending stresses. This method mimics the loading conditions of structural elements, such as beams, girders, and floor slabs, in buildings, as well as flexural members in bridges and pavements. Cementitious materials with crystalline and expanding admixtures [63,170], bacteria [96,110,171], grass tubes or lightweight aggregate impregnated with sodium silicates [113,154], adhesive agents [155], and strain-hardening fibers [145,148,157] or SMP tendons [157] are among the materials whose healing ability is most commonly assessed using three-point and four-point bending tests. Three-point bending tests makes a single crack at a defined position, often pre-notched, and are employed to measure the load recovery rate, stiffness, and post-cracking load-bearing capacity.

In order to evaluate the healing potential of concrete with mineral admixtures and encapsulated glass tubes to estimate load capacity, Kanellopoulos et al. [172] and Qureshi et al. [155] employed three-point bending tests. With an ideal load recovery rate of 20–25%, all experimental groups with mineral admixtures had better load recovery rates than the control; nevertheless, this did not account for the high recovery value for whole cracks. Alghamri et al. [171] demonstrated improved healing efficiency using lightweight aggregate infused with sodium silicate, showing up to 80% recovery of pre-cracking strength and enhanced stiffness recovery. Ferrara et al. [154] measured the recovery of stiffness and load-bearing capacity and assessed the healing of concrete with crystalline admixtures using the three-point bending test. Figure 5 shows our experimental setup for the three-point bending test.

The researchers introduced indices like the index of load recovery (ILR) and index of damage recovery (IDR) for assessing recovery in concrete with crystalline admixtures.

• Index of load recovery (ILR):

$$ILR={\displaystyle \frac{P_{max,reloading}-P_{unloading} }{P_{max,uncracked }-P_{unloading}}},$$

(14)

where P_{max,reloading} is the maximum reloading load, P_unloading is the load at unloading, and P_{max,uncracked} is the maximum load for uncracked material.

• Index of damage recovery (IDR):

$$IDR={\displaystyle \frac{K_{reloading}-K_{unloading} }{K_{loading, uncracked }-K_{unloading}}},$$

(15)

where K_reloading and K_{loading,uncracked} are the stiffness values after reloading and initial loading, respectively.

Four-point bending tests are an effective method for assessing the healing efficiency of materials prone to multiple cracking behaviors, such as strain-hardening cementitious composites (SHCCs). Sisomphon et al. [154] investigated SHCC incorporating calcium sulfoaluminate (CSA) and crystalline admixtures (CAs) by analyzing the deflection capacity, stiffness, and recovery of flexural strength. The study revealed that specimens containing 1.5% CA exhibited optimal recovery of flexural stiffness and strength, while those with a combination of 10% CSA and 1.5% CA demonstrated the best deflection capacity under wet–dry cycles.

Ferrara et al. [150,173] extended the analysis to high-performance fiber-reinforced cement composites (HPFRCCs), introducing novel mechanical recovery indices to establish a connection between crack healing and strength restoration. The studies included the use of load versus crack-opening displacement (COD) curves, as shown in the Figure 6, to evaluate specimens in their virgin, pre-cracked, and post-healing states. Parameters such as f_peak (maximum stress or strain of a material) and f_unloading (unloading load) were employed to calculate the index of strength recovery, demonstrating the relationship between strength recovery and crack-healing efficiency. This approach underscores the versatility of four-point bending tests in evaluating the mechanical recovery of advanced cementitious composites and offers valuable insights into optimizing material compositions and healing strategies.

4.1.2. Splitting Tests (Indirect Tension)

The splitting test, also known as the Brazilian test, includes applying a compressive load along the diameter of cylindrical specimens to generate tensile stress and induce a single, controlled crack. This method is particularly valuable for investigating localized healing performance in bacterial and fiber-reinforced concrete. The uniform cracks produced by this technique make it ideal for comparative studies on healing efficiency. Despite the strong unsteadiness observed after the post-cracking response [174], splitting tests have been effectively used to assess the mechanical properties of cement-based composites. For example, some researchers utilized splitting tests to create controlled cracks in specimens [175]. Ozbay et al. [176] explored the healing potential of ECC with high-volume fly ash under various curing conditions. Their findings showed that preloaded specimens exposed to continuous moisture and cyclic wet–dry conditions for 60 days exhibited significant recovery in mechanical properties, including load-carrying capacity, deflection capacity, and stiffness. Notably, the healed specimens maintained mechanical properties comparable to their virgin states. Figure 7 shows the experimental setup of the tensile splitting test used by Neves et al. [177]. Four internal linear variable differential transducers (LVDTs) were installed at opposite faces of each core to measure the crack width along the fracture surface, and one external LVDT.

Choi et al. [178] used splitting tests to assess tensile strength recovery in pre-cracked cylindrical samples treated with urea hydrolysis bacteria. The tensile stress recovery ranged from 32 to 386 kPa (up to 8% of the virgin specimens), with strain increments between 0.22% and 2.17%. Some specimens exhibited an upward stress–strain curve. The optimal recovery of tensile stress and the most significant precipitation of CaCO₃ occurred when the average crack width ranged from 0.52 to 1.1 mm. These results highlight the suitability of splitting tests for evaluating the mechanical recovery and localized healing efficiency in cementitious materials, providing insights into the effectiveness of various healing agents and conditions.

4.1.3. Compression Test

Compression tests are a method mostly used for evaluating concrete’s compressive strength, reflecting the material’s integrity, internal porosity, and compactness. These tests are instrumental in assessing the healing efficiency, recovery of mechanical properties, and durability of self-healing cementitious materials. Specifically, compression tests have been employed to study bacteria-based self-healing mechanisms, where bacteria induce CaCO₃ precipitation within pores and cracks, enhancing matrix density and compressive strength recovery. Compression tests are employed to measure the recovery of compressive strength and evaluate the effectiveness of healing strategies, such as bacteria-based self-healing, superabsorbent polymers (SAPs), and encapsulation materials.

Biochar was shown by Gupta et al. [179] to be a transporter for bacterial spores that precipitate carbonate in cement mortar. This method improved crack sealing (up to 700 μm) and enhanced compressive strength by 38% while reducing permeability by 65%. Bhaskar et al. [180] and Bundur et al. [181] used bacteria immobilized by porous materials like iron oxide nanoparticles and graphite nanoplates. Their findings indicated substantial improvements in healing efficiency and strength recovery. Polypropylene fibers and SAP particles have also been incorporated into cement mortar to retain moisture and prevent crack formation. Specimens cured for 14 days and subjected to compression tests at 50% and 70% of their peak strength demonstrated significant healing and strength recovery. Xu and Wang [97] explored calcium sulfoaluminate cement as a matrix material, observing a 130% increase in compressive strength recovery after healing for 28 days, with nearly complete closure of cracks up to 417 μm. De Nardi et al. [182] studied lime mortars with and without crystalline admixtures (CA) under varying aging conditions, highlighting their self-healing potential. The impact of several self-healing systems, such as bacterial concentrations, microcapsule doses, fibers, and encapsulating materials, on the hardening characteristics of cementitious materials have also been thoroughly studied using compression tests. These studies affirm the efficacy of compression tests in evaluating the performance of diverse self-healing strategies [170].

4.1.4. Uniaxial Tensile Test

The uniaxial tensile test has demonstrated efficacy in assessing the healing efficiency of cementitious materials, despite its generally restricted capacity to provide reliable tensile stress data. It is particularly useful for assessing the recovery of tensile stiffness, tensile strength, and first-crack strength [170,183]. Wang et al. [124,127] investigated bacteria-based self-healing of numerous cracks by applying uniaxial tensile tests on prismatic concrete specimens. Transport tests and visual inspection were used to evaluate crack closure, demonstrating the method’s suitability for microcrack-healing assessments. Liu et al. [184] used uniaxial tensile tests to evaluate the healing of ECC comprising various materials, with an emphasis on stiffness recovery, first-crack strength, and durability in sulfate and chloride-rich environments. While regular mortar disintegrated dramatically, ECC showed strain-hardening behavior after 420 days, highlighting its improved hydraulic performance. The best tensile strength recovery in sulfate solutions was achieved by enhanced fiber–matrix bonding caused by ettringite and gypsum formation rather than continuous hydration. The role of environmental exposure was further explored by Zhang and Zhang [185] and Sisomphon et al. [150], who highlighted its influence on ECC healing properties. Deng and Liao [186] investigated ECC with SAP particles, finding that a 4% SAP dosage yielded optimal tensile property recovery and complete crack closure, particularly with larger SAP particles. Ranade et al. [187] used uniaxial tensile tests to create cracks of varying patterns and developed analytical models linking crack patterns and single-crack electrical responses to the composite’s overall behavior, offering potential for self-sensing damage monitoring.

The uniaxial compression test experimental setup used by Neves et al. [177] is depicted in Figure 8. Four LVDTs were used to perform the test, three internal LVDTs were placed in the space between plates in the sample, and one exterior LVDT was used. In this test, the LVDTs were used to monitor and control the cracks. This test provides valuable insights into the tensile properties of self-healing materials, supporting advancements in material composition and environmental resilience, while also paving the way for real-time structural health monitoring applications.

4.2. Healing Efficiency, Visualization, and Determination

The analyses in this category focus on qualitative techniques like the release of an encapsulated healing agent and crystal deposition. Crack closure falls under this category as well. The techniques in this category can be used to perform certain quantitative studies. Three categories can be used to view and analyze cementitious materials’ ability to cure themselves: spectroscopy, microscopy, and imaging. Thermal gravimetric analysis (TGA) [100,184,185] is one of the less well-known techniques for characterizing post-healing items.

4.2.1. Ultrasonic Pulse Velocity (UPV)

The ultrasonic pulse velocity (UPV) test is a non-destructive evaluation method widely used to monitor the self-healing efficiency of cementitious materials. This technique involves transmitting short ultrasonic waves through a material to detect small defects, discontinuities, or cracks. The transit time of the waves is measured, allowing for the calculation of wave velocity. Figure 9 illustrates how simple the setup is: An emitter and a receiver are placed on either side of the crack. A longitudinal vibration pulse produced by the specimen’s emitter makes its way to the receiver via the fracture. The transit time and distance between the emitter and receiver are used to determine the average vibration velocity. The velocity difference between the cracked and self-healed cracked specimens is used to evaluate the self-healing.

Since ultrasonic waves propagate slower in liquids or gases than in a solid matrix, changes in transit time serve as indicators of crack healing and recovery of material integrity [96,188]. UPV tests are particularly effective for evaluating crack depth, recovery of strength, and durability properties of cementitious materials. As cracks heal, their closure reduces the ultrasonic wave propagation time, reflecting the efficiency of the self-healing mechanism.

Ahn et al. [188] provided an extensive review of UPV principles and applications, emphasizing its utility in evaluating self-healing cementitious materials. UPV tests have been used to evaluate natural self-healing in cementitious materials, demonstrating significant improvements in wave velocity after healing [173,182]. The study conducted by Alghamri et al. [154] utilized UPV to evaluate the healing process in systems in which silicate solutions were injected into lightweight aggregates coated with PVA or double-walled polyurethane/urea microcapsules. Non-ureolytic bacteria-based healing agents were evaluated using UPV tests, showing measurable recovery of ultrasonic wave velocity due to crack sealing [96]. Cementitious materials containing crystalline and expansive admixtures, such as MgO agents, showed effective healing and recovery of wave velocities [63,189].

4.2.2. Imaging (BSEI, DIC, XCT, and Neutron Radiography/Tomography)

Imaging is a powerful and intuitive method for observing the healing process and microstructural changes in cracked specimens [170]. These techniques provide both qualitative and quantitative insights into crack closure, distribution of healing products, and microstructural transformations, making them essential for evaluating the effectiveness of self-healing mechanisms. The primary imaging methods include backscattered electron imaging (BSEI), X-ray computed tomography (XCT), digital image correlation (DIC), and neutron radiography/tomography, each contributing to a multi-scale understanding of self-healing systems and advancing material development. These methods enable researchers to observe crack closure at various scales, analyze the distribution of healing agents, and correlate healing products with mechanical recovery. Three-dimensional computed tomography was used to visualize the pine needle-length steel fibers that were mixed in to liquid concrete. A 10 cm-long core sample of steel fiber-reinforced concrete was analyzed using software developed at the Fraunhofer Institute for Industrial Mathematics ITWM in Kaiserslautern, which uses probability calculations to determine the distribution of all the steel fibers within the sample. The sample was X-rayed using an industrial CT scanner with a resolution a thousand times finer than a medical scanner. This revealed steel fibers in the concrete that were µm in size. A high-resolution 3D dataset with 8 billion pixels was created for the sample, as shown in Figure 10. All of the fibers in the image eventually became visible as the software processed the dataset by evaluating contrast differences and assigning each pixel to a specific structure within the sample [190].

Backscattered electron imaging (BSEI), a variation of scanning electron microscopy (SEM), is widely utilized to examine the microstructure of healed materials. By generating high-contrast images based on density differences, BSEI effectively distinguishes healed regions from unhealed ones and identifies healing products like CaCO₃ and hydrated compounds. Digital image correlation (DIC) measures strain and deformation on the surface of specimens by comparing images captured before and after healing. This optical technique provides detailed information on crack closure, mechanical recovery, and strain fields during loading and healing processes [170]. By incorporating digital geometry processing, DIC creates 3D images from 2D photographs, enabling precise analysis of crack development, closure, and displacement [31].

A non-destructive imaging method called X-ray computed tomography (XCT) produces high-resolution 2D and 3D images of a specimen’s interior and exterior features. Volumetric study of crack shape and healing progression is made possible by XCT. Visualizing the distribution of microcapsules, capsule breakage, healing agent release, and crack closure are examples of recent applications. Snoeck et al. [56] used XCT to study healing in concrete containing superabsorbent polymers (SAPs), observing that cracks were predominantly sealed near their mouths (up to 800–1000 µm in depth). Using XCT, Wang et al. [124] investigated the healing capabilities of bacteria trapped in hydrogel in concrete. The XCT data demonstrated that mineral precipitation is present throughout the cylinder specimens, not only in the cracks. Neutron radiography and tomography are particularly suited for studying water-based healing systems like SAPs and mineral admixtures. These techniques utilize the attenuation of neutron beams to visualize water movement, moisture distribution, and healing efficiency. Neutron imaging provides critical data on moisture content and water permeability in cementitious materials. In their analysis of SAP performance in sealing cracks, Snoeck et al. [57] used neutron radiography, which confirmed through imaging that SAPs successfully sealed cracks up to 100 µm wide and decreased water permeability. The effects of crack healing over time on the water absorption of cracked strain-hardening cement-based composites (SHCCs) were quantitatively investigated by Zhang et al. [191] using neutron radiography. After 90 days of healing, the cracked specimen’s water absorption was much less than it was both before and after five days. The fracture-healing efficiency was 70%, which matched the permeability test results.

These imaging methods collectively enhance our ability to study self-healing processes, offering multi-scale, detailed analyses of crack behavior, healing agent distribution, and water interaction. The integration of these techniques significantly advances the development of more efficient and sustainable self-healing materials.

4.2.3. Microscopy (OM, SEM, and ESEM)

Microscopy provides detailed insights into the microstructure, crack closure, and the formation of healing products, allowing for both qualitative and quantitative evaluation of healing efficiency. Microscopy techniques are used to visualize the extent of crack closure, the morphology of healing products, and the interaction between healing agents and the cementitious matrix. The three most widely utilized testing techniques in this group are environmental scanning electron microscopy (ESEM), optical microscopy (OM), and scanning electron microscopy (SEM), respectively.

Optical microscopy is used to observe surface-level changes in cracks, such as crack closure and deposition of healing products. It provides a straightforward and cost-effective method for visualizing healing at a macroscopic level but is limited in its ability to capture internal structures or nanoscale changes. Researchers often use optical microscopy to quantify and check the area and width of surface cracks before and after healing so as to evaluate healing efficiency [155,192,193]. However, because polymer adhesives, especially microcapsules with a little amount of agent, are only released and polymerize inside the crack, an OM is ineffective for assessing the healing effectiveness of cementitious materials containing polymer adhesives. Interestingly, Wang et al. [194] applied OM to examine how intrinsic carbonate and mineral admixtures influence self-healing behavior. Crack areas were quantified using imaging software, revealing that area-based measurements had lower standard deviation than width-based ones (Figure 11). This suggested that crack area provided a more consistent and representative indicator of overall healing performance. Similarly, Rong et al. [195] utilized a digital camera to capture pre- and post-healing crack images, which were then analyzed using an ultra-depth-of-field microscope to count pixel numbers corresponding to the damaged area. Despite its utility, OM should be complemented with other evaluation techniques, as it is limited to assessing visible surface cracks and cannot quantify healing occurring within the interior of the concrete matrix.

SEM is one of the most commonly used techniques for examining the microstructure of healed materials. It allows for high-resolution imaging of crack interfaces, healing products (e.g., calcium carbonate and ettringite), and microstructural changes. SEM images provide detailed insights into how healing materials interact with cracks and the distribution of precipitated products. Another microscopy technique for viewing and evaluating healing products is scanning electron microscopy–energy dispersive X-ray spectroscopy (SEM-EDX), which provides a higher magnification and allows for stereoscopic vision in comparison to optical microscopy. Backscattered electron imaging (BSEI) is frequently used in conjunction with SEM [196]. The idea behind this technique is to scan the sample with a high-energy electron beam that is extremely tightly focused in order to extract physical information. The surface topography of the test sample can be determined by receiving, amplifying, and presenting the data. Thus, SEM is often used to determine crystal structure, shape, size, and distribution.

Researchers, including Pang et al. [48] and Wang et al. [194], utilized SEM to analyze the healing efficiency of cementitious composites. Their studies identified the deposition of healing products and quantified their distribution within cracks. The shape, structure, and distribution of the crystallization products were examined by Wang et al. [194] using SEM up to 10 mm (2 mm) from the crack surface (Figure 12). Their findings revealed significant calcite accumulation not only at the crack mouth but also deep within the fissure, particularly in specimens with intrinsic carbonate content—highlighting the role of inherent carbonate in enhancing internal crack sealing. In a related study, Rong et al. [195] investigated the microstructure of bacterial mortar and identified precipitated calcite with rhombohedral and orthorhombic crystal shapes. Scanning electron microscopy–energy dispersive X-ray spectroscopy (SEM-EDX) confirmed that these crystals were composed of calcium, carbon, and oxygen, consistent with calcite’s elemental makeup.

Similarly, Wiktor and Jonkers [194] reported two distinct morphologies of calcite in healed bacterial concrete: needle-like clustered formations and distorted lamellar rhombohedral crystals, emphasizing the variability in crystal formation depending on healing conditions. Extending this investigation, Feng et al. [15] used SEM to examine the healing products formed when Bacillus subtilis was combined with polyvinyl alcohol (PVA) fibers in a cement matrix. Their analysis revealed notable adhesion of calcite crystals to the surface of PVA fibers, as illustrated in Figure 13a, suggesting that fiber–matrix interfaces can act as nucleation sites for crystal growth. Notably, the study also found microstructural variation in calcite morphology across the crack depth: the rear surface, closer to the fracture, exhibited coarse crystal structures, while the front surface featured finer hexahedral calcite formations (Figure 13b). Additionally, Wang et al. [194] encapsulated bacterial spores within hydrogel carriers and evaluated their self-healing potential. Post-healing SEM-EDS analyses identified spherical and cubic calcium carbonate crystals, typically ranging from 10 to 50 nm in size, confirming successful microbial precipitation and microstructural integration of the healing products. ESEM is an advanced variant of SEM that enables imaging under controlled environmental conditions, such as varying humidity and temperature. This allows for real-time monitoring of self-healing processes and the behavior of healing agents under conditions mimicking their operational environment.

4.2.4. Spectroscopy and Thermoanalysis (XRD, FTIR, and TGA)

Spectroscopy techniques, combined with thermoanalysis methods, provide invaluable insights into the evaluation of self-healing efficiency in cementitious materials. In order to uncover important details regarding the molecular or atomic structure, crystallinity, crystal structure, bonding state, and the content of different crystalline components of the healing products, these techniques entail shining a light beam through a specimen and examining the spectrum that results, as shown in Figure 14 [196]. The most commonly used spectroscopy techniques include X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy. Additionally, thermoanalysis methods like TGA and differential scanning calorimetry (DSC) are often used to complement these spectroscopic techniques, providing a deeper understanding of the thermal behavior and chemical properties of healing products. These techniques help identify the types and quantities of healing compounds, analyze crystalline structures, and monitor thermal stability. They offer detailed insights into the mechanisms of self-healing and contribute significantly to the development of advanced materials with improved healing efficiency. By integrating these methods, researchers can better understand the interplay between healing agents and cementitious matrices, driving innovations in sustainable and durable construction materials.

X-ray diffraction (XRD) is a powerful technique used to identify the crystalline structure and phase composition of healing products in cementitious materials. It is particularly effective for differentiating between polymorphs of calcium carbonate, such as calcite, aragonite, and vaterite, which play key roles in self-healing. As one of the most widely used methods for analyzing crystalline morphology, XRD provides detailed insights into the crystal phases and structural characteristics of healing compounds. Wang et al. [194] used XRD to confirm that calcite was the predominant healing product both within the interior and on the surface of healed cracks, underscoring the importance of carbonation in autogenous and microbial healing processes. Similarly, Pang et al. [48] employed XRD to investigate healing in concrete incorporating carbonated steel slag aggregates, identifying major products such as calcium carbonate (CaCO₃), calcium silicate hydrate (C-S-H), and amorphous silica. Rong et al. [195] analyzed white precipitates formed in cracks of bio-mortars inoculated with varying bacterial concentrations (10⁷–10⁹ cells/mL), confirming via XRD that the primary healing product was calcite. In another study, Shaheen et al. [88] observed the formation of multiple mineral phases, including calcite, quartz, calcium hydroxide, and calcium oxide, within the healed zones of bacterial concrete (Figure 15), providing further evidence of the complex mineralogy involved in the self-healing process.

Fourier transform infrared spectroscopy (FTIR) is a valuable analytical technique for identifying the chemical composition and functional groups of healing products through vibrational frequency detection. It is particularly effective in characterizing key self-healing compounds, such as calcium carbonate (CaCO₃) and calcium silicate hydrate (C-S-H), as well as tracking changes in organic healing agents over time [170]. Figure 16 shows the FTIR spectral study of calcium carbonate precipitation caused by microbes (MICP) [156]. Alghamri et al. [154] identified C-S-H and calcite as the main healing chemicals by using FTIR to detect healing products on the surface of sodium-silicate-encapsulated concrete. The specimens’ FTIR spectra for the two mixes showed that the bands in the two spectra were remarkably similar, and the precipitate of the self-healing material (SHM) specimens which contained sodium silicate was a combination of calcite and C-S-H. The enhanced C-S-H precipitation on the cracked surfaces of the SHM specimens was caused by the sodium silicate contained in the lightweight aggregates, as indicated by the growing shift of the Si-O asymmetric stretching band to a higher wavenumber. In a related study by Tan et al. [197], FTIR was used to compare reference and healed samples incorporating calcium nitrate or encapsulated calcium acetate with microbial nutrients. Characteristic peaks confirmed the presence of calcite and C-S-H across all specimens. Notably, shifts in Si-O stretching bands suggested a reduction in the Ca/Si ratio, particularly in samples treated with calcium acetate. This observation implies that microbial-induced calcium carbonate precipitation (MICP) may extract calcium from C-S-H gels, not just from portlandite, leading to nanoscale decalcification and the development of silica-rich gel structures. These alterations resemble early-stage carbonation, indicating that MICP can induce localized internal transformations distinct from atmospheric carbonation effects.

TGA measures weight changes in materials as a function of temperature to determine the thermal stability and composition of healing products. This technique provides quantitative data on the amount of carbonate precipitated during the self-healing process. Wang et al. [127] used TGA to analyze white precipitates in cracks of specimens containing hydrogel-encapsulated spores. The results confirmed that the crystals were composed of CaCO₃, and the mass loss observed prior to calcium carbonate decomposition was attributed to hydrogel degradation. In a more comprehensive study [197], TGA was used to assess the thermal decomposition behavior of self-healing mortars. In reference CEM II samples, distinct mass losses were observed corresponding to known phase changes, including dehydration of C-S-H and ettringite, portlandite decomposition, and CaCO₃ decarbonation. In calcium-nitrate-based samples, increased mass loss indicated enhanced hydration, while the stability of nitrate AFm phases suggested limited bacterial influence. In mortars containing calcium acetate and aerated cement granules, TGA revealed a notable reduction in hydration products after healing implying their conversion into self-healing compounds. Despite overlapping peaks complicating exact quantification, TGA effectively demonstrated the impact of both chemical additives and microbial activity on healing-related phase transformations.

4.3. Durability Properties Testing Methods

Concrete deterioration and steel rebar corrosion are caused by water and harsh chemicals seeping through concrete cracks. In this regard, fixing or caulking cracks is important since it increases the concrete composite’s resistance to gas and water, which in turn lengthens its lifespan [157,192]. Durability testing methods are important in evaluating the long-term performance and reliability of self-healing concrete systems. These tests assess the material’s ability to resist degradation caused by environmental and mechanical stressors, such as freeze–thaw cycles, chloride ion penetration, and sulfate attack. Durability tests also provide insights into how self-healing mechanisms contribute to maintaining or restoring concrete’s protective properties.

4.3.1. Freeze–Thaw Resistance Tests

Freeze-thaw resistance tests are a critical method for assessing the durability and self-healing efficiency of bio-concrete, particularly in cold climates where cyclic freezing and thawing can cause significant damage to concrete structures. These tests evaluate the material’s ability to withstand the internal stress generated by the expansion of water as it freezes, which can lead to cracking, scaling, and spalling [198,199]. By measuring the extent of damage and the recovery of mechanical and physical properties after self-healing, freeze-thaw resistance tests provide valuable insights into the effectiveness of microbial-induced calcium carbonate precipitation (MICP) and other self-healing mechanisms [200]. Freeze-thaw damage occurs when water within the concrete pores’ freezes, expanding by approximately 9% in volume. This expansion generates internal stresses that can exceed the tensile strength of the concrete, leading to the formation and propagation of microcracks Repeated cycles exacerbate damage, especially in the presence of de-icing salts. Freeze-thaw resistance tests, conducted per standards like ASTM C666, involve subjecting specimens to repeated freezing and thawing cycles, followed by measurements of mass loss, dynamic modulus of elasticity, and surface scaling [201].

Research has shown that when compared to traditional concrete, bio-concrete has better freeze-thaw resistance. For example, regardless of treatment settings, all biotreated samples showed a strength drop of less than 5% and 10% after 5 and 10 FT cycles [202]. The incorporation of SCMs, such as silica fume and fly ash, further enhanced the freeze-thaw resistance of bio-concrete by promoting the formation of dense CaCO₃ layers and gel-like healing products [203]. The efficiency of self-healing in bio-concrete can be quantified using the recovery ratio (RR), defined as:

$$RR={\displaystyle \frac{P_{healed} - P_{damaged} }{P_{initial }- P_{damaged}}},$$

(16)

where P_initial, P_damaged, and P_healed represent the measured property (e.g., RDME or compressive strength) of the specimen before damage, after damage, and after self-healing, respectively. A higher RR indicates more effective self-healing and greater recovery of mechanical properties [13].

4.3.2. Chloride Ion Penetration Tests

Chloride ingress is a significant threat to reinforced concrete durability, as it induces steel reinforcement corrosion and compromises structural integrity. Tests like the Rapid Chloride Permeability Test (RCPT) and ponding tests assess chloride penetration depth and concentration. Crack closure via calcium carbonate precipitation in self-healing systems significantly reduces chloride permeability, providing enhanced corrosion protection. The rate of chloride ion penetration into concrete primarily depends on its pore structure, which is influenced by curing conditions, mix design, hydration degree, supplementary cementitious materials (SCMs), and construction methods. RCPT measures concrete permeability by monitoring the electrical charge passed through a specimen, correlating the charge to the concrete’s resistance to chloride penetration.

Incorporating bacterial agents into concrete has shown promise in enhancing chloride resistance. Studies revealed that bacterial concrete exhibited a 12% reduction in chloride infiltration compared to conventional concrete. Specifically, the inclusion of B. subtilis and S. pasteurii reduced chloride penetration and improved resistance to sulfate and acid attacks [204]. Bacterial concrete also displayed lower electrical charges at all curing ages, with reductions of 54.6%, 48.7%, and 47.5% at 7, 28, and 56 days, respectively [115]. Silica fume-based bacterial concrete further enhanced chloride resistance. For instance, adding S. pasteurii (10⁵ cells/mL) to 15% silica fume concrete resulted in a permeability rating of 410 coulombs [113]. Similarly, fly ash concrete with 30% fly ash and bacterial agents showed significantly reduced chloride ion penetration, as low as 759 coulombs [114]. These improvements are attributed to bacterial action, which enhances the microstructure by filling internal pores, promoting calcium carbonate formation, and encapsulating unhydrated materials.

The durability of concrete structures in marine environments or deicing salt exposure relies on resistance to chloride infiltration. Bacteria-based treatments have demonstrated substantial improvements in this regard. Studies indicate that bacterial surface treatments can reduce chloride permeability by over 15% within 7 days of curing, while treatments in urea solutions for 28 days have achieved reductions exceeding 45% [82,205]. Additionally, B. cohnii has been effective in generating dense calcium carbonate precipitates (20–80 μm thick) on crack surfaces, enhancing crack healing and chloride resistance [81]. Calcium lactate and calcium glutamate have been identified as alternative calcium sources to mitigate the risks associated with calcium chloride, such as ammonia production and reinforcement corrosion [206]. Research by De Muynck et al. [121]. highlighted that microbial-induced calcium carbonate precipitation (MICP) significantly decreased water and gas permeability, thereby enhancing carbonation resistance and reducing chloride migration coefficients by 10–40%. These bacterial treatments performed comparably to acrylic coatings and water-repellent silanes in reducing chloride permeability, with superior durability against chloride ingress. In conclusion, the integration of MICP in concrete offers a cost-effective, eco-friendly solution to improve durability by reducing chloride permeability, enhancing strength, and extending the lifespan of reinforced concrete structures.

4.3.3. Water Permeability Tests

Water permeability tests evaluate the efficiency of crack sealing in preventing water ingress. These tests measure the rate of water flow or pressure required to pass through healed cracks. The results are critical for evaluating the performance of self-healing mechanisms, particularly in environments with high humidity or submersion conditions. A significant reduction in water permeability is often regarded as a direct indicator of successful self-healing. Resistance to water penetration is a fundamental parameter in determining the long-term durability of concrete. It prevents the infiltration of harmful substances responsible for concrete degradation. The permeability of cement-based materials is largely influenced by the characteristics of the pore system, which includes variables such as porosity, tortuosity, connectivity, pore size distribution, microcracks, and specific surface area [207]. Microbial-induced calcite precipitation (MICP) has shown remarkable potential in reducing water penetration in cementitious and other construction materials.

Permeability is used to assess the infiltration of harmful materials that cause concrete wear and tear under pressure, and it is regarded to be an important factor in determining concrete durability. This is predicated on the pore system properties of binding materials, as assessed by surface area, permeability, size distribution, and microcracking. The water-to-binder ratio (w/b), the infiltration of hazardous substances, the curing age of hardened binding materials, and the particle size distribution are some of the factors that affect these limitations [207]. Concrete samples’ permeability and water absorption were decreased by the buildup of calcium carbonate. According to research [208], the permeability and perviousness of silica-fume-based concrete were enhanced by the addition of the bacterial agent Subtilis pasteurii. All reference concrete specimens showed increased perviousness to mild perviousness following a 28-day curing period. However, when the pores were filled with CaCO₃, the bacterial-agent-containing specimen displayed increased perviousness, as opposed to decreased perviousness [208]. Concrete is a permeable substance, and its porosity is determined by the kind and size of its pores. According to the results of recent tests on porosity, when bacterial material is added to cement mortar, the porosity decreases by 27% [209] and 48% [205] for L. sphaericus and B. cereus, respectively, when compared to the control mortar. The formation of calcium carbonate and the filling behavior of biomaterials in the matrix may be responsible for this [210]. The addition of bacterial spores decreases the water permeability and absorption of the bio-based concrete, as previous research has shown. The stimulated autogenous self-healing of mortars affected by the addition of different additives was assessed using the relative water permeability (Rp), which is expressed in Equation (17), where P_self-healed is the water permeability of the self-healed specimen after self-healing for a period of t and P_cracked is the water permeability of the cracked specimen [19]:

$$R_p={\displaystyle \frac{P_{self-healed} }{ P_{cracked}}}.$$

(17)

4.3.4. Water Absorption Tests

In addition to permeability, capillary absorption is another way that water can enter concrete. The water absorption test is usually carried out in accordance with ASTM C1585, where the mass change of the specimen is used to calculate both initial absorption (within the first 6 h) and secondary absorption (beyond 6 h). Advanced methods such nuclear magnetic resonance (NMR), X-ray radiation attenuation, and neutron radiography can be used to measure water absorption in addition to the conventional gravimetric method [57]. The water absorption test assesses the self-healing capability of concrete by measuring changes in water absorption through cracks. This test serves as a direct indicator of crack closure effectiveness and the impermeability of the concrete after healing.

The primary mechanism of self-healing in bio-concrete involves the precipitation of calcium carbonate (CaCO₃) by bacteria, which seals cracks and reduces water absorption. However, even in cases where cracks are fully sealed, the water absorption of self-healed specimens often remains higher than that of uncracked specimens. For example, research has shown that the secondary water absorption of self-healed cracked specimens can be up to five times greater than that of uncracked specimens, even after complete crack closure [211,212]. This suggests that while self-healing improves impermeability, it may not fully restore the concrete to its original state. Incorporating supplementary cementitious materials (SCMs) such as fly ash has been shown to enhance self-healing efficiency. In bio-concrete with high volumes of fly ash, the formation of gel-like products within cracks can reduce water absorption to levels comparable to uncracked specimens [26]. This improvement is attributed to the additional binding and pore-filling effects of the gel-like products, which further restrict water movement. While water absorption tests provide valuable insights into self-healing efficiency, several factors can influence the results. The degree of saturation and the saturation technique used during testing can significantly affect water absorption measurements, necessitating consistent experimental conditions for reliable comparisons [26]. Additionally, water absorption tests may not fully capture the long-term durability of self-healed concrete, as they primarily focus on short-term changes in permeability.

Therefore, to ensure reliable assessment of self-healing, water absorption measurements for cracked specimens must be conducted under consistent conditions before and after healing.

4.3.5. Gas Permeability

Gas permeability tests serve as a crucial method for assessing the self-healing efficiency of bio-concrete. These tests provide insights into the material’s ability to resist gas infiltration through cracks and pores, offering a direct measure of its impermeability and durability. In self-healing concrete, particularly those incorporating microbial-induced calcium carbonate precipitation (MICP), gas permeability testing is instrumental in evaluating the effectiveness of crack-sealing mechanisms and their impact on long-term structural performance [213]. The effect of carbonation, in which calcium hydroxide and carbon dioxide combine to form calcium carbonate (CaCO₃) when moisture is present, is one of the main issues with concrete durability. While CaCO₃ contributes to autogenous healing, excessive carbonation can accelerate reinforcement corrosion, particularly in the presence of oxygen at the steel–concrete interface [26]. Similar to water ingress, gas penetration occurs through cracks, creating pathways that facilitate deterioration. Self-healing mechanisms are expected to mitigate gas permeability by sealing these micro-channels, thereby improving the overall integrity of the structure.

Gas permeability testing is typically conducted using a standardized apparatus, such as the Cembureau device, which applies controlled gas pressure to the specimen and measures the resulting flow rate [213]. The setup for cementitious composite materials’ gas permeability is depicted in Figure 17. Through the comparison of cracked and healed specimens under various exposure settings, this technique allows researchers to measure the efficiency of self-healing on permeability decrease.

The permeability of concrete is significantly influenced by crack characteristics, including width, density, and roughness. Studies on high-performance concrete (HPC), high-performance fiber-reinforced concrete (HPFC), and normal strength concrete (NSC) have shown that internal dispersed fractures brought on by uniaxial compressive loading result in different levels of permeability increase. A correlation has been established between the reduction in modulus of elasticity and the increase in permeability, providing a basis for assessing damage–permeability relationships across different concrete types [214]. The index g_gas can be used to measure the recovery of gas permeability brought on by self-healing. It is defined as:

$$g_{gas}={\displaystyle \frac{K_{cracked} - K_{self-healed} }{ K_{cracked}}},$$

(18)

where K_cracked and K_self-healed stand for the specimen’s gas permeability coefficients prior to and following self-healing, respectively. This index accounts for both surface crack closure and the formation of internal self-healing products, with higher values indicating more effective healing [194].

Self-healing mechanisms, particularly those involving bacterial activity and supplementary cementitious materials (SCMs), have been shown to significantly reduce gas permeability in concrete. Experimental comparisons between cracked and healed ECCs have revealed a notable reduction in gas permeability, regardless of the mixture composition, exposure environment, or initial crack width [214]. Bio-concrete specimens treated with bacterial-based self-healing agents have demonstrated significantly lower gas permeability coefficients compared to untreated samples. For example, De Muynck et al. [121] reported a reduction in gas permeability in bacterial-treated mortar, attributing this improvement to the formation of a dense CaCO₃ layer that sealed cracks and reduced internal porosity. Also, Achal et al. [118] observed a 25% reduction in gas permeability in bio-concrete specimens treated with Sporosarcina pasteurii, further supporting the effectiveness of microbial precipitation in enhancing impermeability.

Gas permeability tests complement other standard durability assessments, such as water absorption and chloride penetration tests, by offering a sensitive measure of crack sealing and pore connectivity. While water absorption tests evaluate a material’s resistance to liquid ingress, gas permeability testing provides a more refined analysis of early-stage self-healing performance. The ability of self-healing concrete to reduce gas permeability underscores its potential to enhance long-term durability and resistance to environmental degradation [36].

5. Fiber Application in Self-Healing Concrete

High-performance fiber-reinforced cement composites (HPFRCCs) and standard fiber-reinforced cement composites (FRCCs) are two examples of the fibers that have long been used to reinforce concrete. In a review done by Meraz et al. [36], as depicted in Figure 18, polyvinyl alcohol (PVA) fibers have been identified as the most widely utilized, followed by steel [31], polypropylene [215], and natural fibers, each contributing about 12% of the cases observed in the literature [4]. Significant benefits come from combining FRCCs, HPFRCCs, and ECCs. For example, improved fracture width control and energy absorption capacity boost concrete’s mechanical qualities, increasing its durability and improving its resistance to cracks [216,217]. These composites, when combined with self-healing mechanisms, produce extremely durable concrete that can fix itself over time, increasing the longevity of buildings and lowering maintenance expenses. Concrete’s durability and sustainability in construction could be greatly increased by this fiber–self-healing technology synergy.

However, several challenges remain in this synergistic relationship between fibers and self-healing technologies. One major issue is the use of natural fibers as self-healing facilitators. While they offer a cost-effective replacement to synthetic fibers and steel, their long-term performance, especially in harsh environments, is still under evaluation. Cement-based composites reinforced with natural fibers, such as sisal, coir, jute, and kraft pulp fibers, have been the subject of numerous investigations. These studies found that fiber reinforcement increases cement-based materials’ flexural, impact, tensile, and compressive strength, and toughness [218,219], which is similar to the outcomes of using polymer fibers as reinforcement.

The integration of fibers into self-healing concrete enhances structural resilience while supporting crack width control and microbial activation. The long-term durability of these reinforced materials is called into question by the use of natural fibers in cement-based matrices, as they may lose their toughness and strength [220]. Because of the embrittlement linked to the mineralization of the fibers, natural fibers are particularly vulnerable to the alkalinity of the cementitious matrix, which causes fiber degradation and lowers the fibers’ flexibility and deformation capacity [220]. Furthermore, the effectiveness of natural fibers in environments with extreme corrosion or other environmental factors remains a concern, suggesting a need for hybrid or modified fiber systems.

6. Limitations and Challenges

Despite the promising advancements in bacterial self-healing concrete, several limitations and challenges hinder its widespread application in real-world infrastructure. The efficiency of this technology is influenced by factors such as bacterial survivability, compatibility with cementitious materials, continuous healing ability, and scalability for large-scale applications. Addressing these challenges is crucial for optimizing self-healing efficiency and ensuring that bacterial concrete meets the durability and cost-effectiveness required for modern construction. This section addresses the challenges and limitations.

6.1. Autogenous Healing Limitation

Autogenous healing in concrete primarily relies on the continued hydration of unreacted cement particles and the precipitation of calcium carbonate under favorable conditions. Several studies have reported its effectiveness in sealing cracks up to 0.2–0.3 mm in width under prolonged water exposure, with healing ratios exceeding 60–80% in some cases [24,45,220]. Mechanical recovery, particularly compressive and flexural strength regain, has been shown to reach 30–50% of original strength values depending on exposure conditions, curing duration, and crack geometry [47,175,176]. The incorporation of supplementary cementitious materials (SCMs), such as fly ash and slag, improves healing by promoting pozzolanic reactions that produce additional calcium hydroxide, enhancing the availability of reactants for carbonation-based sealing [53,182]. Furthermore, the addition of superabsorbent polymers (SAPs) supports internal curing, maintains moisture at crack sites, and increases autogenous healing efficiency, particularly in dry environments [46,55]. Despite its environmental friendliness and simplicity, autogenous healing is limited by environmental dependency, variability in crack sealing across specimens, and slower kinetics compared to MICP. Unlike bacterial methods, autogenous healing does not provide targeted biochemical activity and is highly sensitive to the presence of water and cementitious matrix composition [71,142]. Therefore, while effective for microcracks, autogenous healing remains a passive mechanism that may benefit most from hybridization with engineered self-healing strategies [72].

6.2. MICP Limitations

6.2.1. Bacterial Survivability for MICP

A major limitation in using bacterial self-healing concrete is ensuring the long-term survivability of the bacteria within concrete’s highly alkaline environment. Fresh concrete typically has a pH between 12 and 13, which is lethal to most microorganisms. However, some alkaliphilic bacteria, particularly those from the Bacillus genus, such as L. sphaericus, B. subtilis, and S. pasteurii, have demonstrated the ability to survive and induce microbial-induced calcite precipitation (MICP) even in such extreme conditions [79,84,85,98]. Nutrients play a pivotal role in supporting bacterial activity within self-healing concrete. Insufficient nutrients can limit bacterial urease activity, while excessive concentrations may lead to unwanted side effects, such as increased porosity and reduced mechanical strength. They provide the essential substrates required for MICP, ensuring sustained metabolic activity and efficient crack healing. The selection and concentration of nutrients directly influence the effectiveness of bacterial systems in concrete. For example, B. subtilis utilizes urea to produce calcium carbonate, which is vital for the self-healing process [16].

Despite these promising findings, maintaining the viability of these bacteria over the long lifespan of concrete remains a significant challenge. The bacteria need to stay dormant until cracks form and moisture activates them. Encapsulating materials like hydrogels and microcapsules have been used to combat this issue by shielding the bacteria from the hostile concrete environment. These carriers provide a microenvironment that shields bacterial spores from the harsh alkaline conditions of concrete, facilitates their activation upon crack formation, and enhances their self-healing efficacy [119]. However, the long-term effectiveness of these carriers in preserving bacterial viability is still under investigation.

6.2.2. Compatibility Issues

Compatibility issues between bacterial agents, their nutrients, and the cementitious matrix remain a significant challenge in the development of bacterial self-healing concrete. The complex chemical composition of concrete, including its high alkalinity, presence of calcium ions, and reactive compounds, can interfere with bacterial metabolic processes essential for calcium carbonate (CaCO₃) precipitation. This interaction can lead to the premature reaction of bacterial activators with cementitious materials, reducing their efficiency and limiting the self-healing capacity of the system [17,65,66,68,98]. One major compatibility concern is the interaction between bacterial nutrients and the cement matrix. Bacteria rely on compounds such as urea and calcium lactate to facilitate MICP, yet these additives must be carefully optimized to ensure they do not disrupt the hydration process or compromise the structural integrity of concrete [16,17,25]. Excessive urea content, for instance, can lead to an increased ammonia byproduct, which may alter the pH balance and negatively impact both bacterial viability and reinforcement durability [129]. Similarly, the addition of calcium lactate must be controlled to prevent early reactions with cementitious compounds that could hinder bacterial activation when cracks form [116].

Furthermore, incorporating fibers to enhance the mechanical properties of self-healing concrete introduces additional compatibility challenges. Natural fibers such as coir, flax, and jute are increasingly being used as bacterial carriers, allowing spores to remain dormant until activation [22]. However, these fibers are biodegradable, and their long-term degradation within the concrete matrix may affect the sustained performance of self-healing mechanisms. Studies have shown that the decay of natural fibers can lead to localized voids and microstructural instability, potentially reducing the mechanical integrity of the healed concrete [13,21]. The low degradation resistance of lignin and hemicellulose in highly alkaline environments can cause a decrease in integrity and stability of the cell wall of natural fibers in cement-based materials [221]. On the other hand, synthetic fibers such as polypropylene and glass fibers are non-biodegradable, offering superior tensile strength and durability. However, they may not always provide an ideal environment for bacterial colonization. Some synthetic fibers exhibit hydrophobic properties, limiting water retention in the crack zone, which is necessary for bacterial activation and the subsequent healing process [13]. Additionally, the surface properties of synthetic fibers may hinder bacterial attachment, thereby reducing the efficiency of bio-based crack sealing [42,141].

To address these compatibility challenges, researchers are investigating encapsulation techniques and alternative carrier materials to enhance bacterial survivability and optimize nutrient delivery. Microencapsulation of bacteria and nutrients in silica gel, hydrogel, or lightweight aggregates (LWA) has shown promise in protecting bacterial spores from premature activation while maintaining a conducive environment for self-healing [119]. Additionally, the development of bio-compatible synthetic fibers with improved surface roughness and moisture retention capabilities is being explored to enhance bacterial adhesion and crack-sealing efficiency [222].

Overall, ensuring the compatibility of bacterial agents, nutrients, and fiber reinforcements with the cementitious matrix is critical for maximizing the effectiveness of bacterial self-healing concrete. Further research is needed to refine material formulations, optimize nutrient integration, and develop novel encapsulation techniques that enhance bacterial survivability while maintaining structural integrity over extended periods.

6.2.3. Scaling Challenges

Scaling microbial self-healing concrete from laboratory research to practical, field-scale applications presents a complex array of biological, material, and operational challenges. A primary concern is the diminished survivability of microbial agents under fluctuating real-world conditions, where exposure to drying, temperature extremes, and mechanical stress can compromise bacterial viability before activation [17]. Achieving uniform distribution of bacteria and nutrients across large structural elements remains difficult—unlike in small-scale laboratory specimens, large pours often result in settling, aggregation, or inconsistent bacterial activation. Furthermore, the bacteria must remain dormant until crack formation occurs, necessitating sophisticated delivery systems, such as microencapsulation, to enable controlled release triggered by water ingress [222]. In arid or dry conditions, spores may remain inactive for prolonged periods, leading to delayed or ineffective healing. Additionally, incorporating live biological components can interfere with traditional mixing, curing, and casting processes [16,17,223]. Cost is another barrier, particularly in producing carriers that are both biologically protective and chemically compatible with cementitious systems. These limitations underscore the need for moisture-responsive carriers, the selection of robust alkaliphilic bacterial strains, and the development of standardized protocols to facilitate field implementation at scale [17].

The long-term durability and regulatory approval of self-healing concrete remain unresolved issues. Bacterial spores must survive extreme conditions, such as high alkalinity, fluctuating temperatures, and prolonged dry periods, to ensure long-term crack-sealing performance [222]. Moreover, large-scale applications require compliance with industry regulations and safety standards before widespread adoption is feasible. Research into bio-compatible encapsulation materials and field performance studies is crucial to addressing these uncertainties. Finally, integrating bacterial-based self-healing technology into real-world infrastructure demands extensive field testing and collaboration between researchers, construction companies, and policymakers. Pilot projects in bridges, tunnels, and pavements have shown promising results, but further validation is necessary to ensure scalability and reliability in diverse environmental conditions [16,17,25,26]. By overcoming these obstacles, bacterial self-healing concrete has the potential to transform the building sector by increasing the longevity of buildings, lowering repair costs, and encouraging environmentally friendly construction methods.

6.3. Economic Parameters

The production cost of bacterial self-healing concrete has been estimated at approximately USD 260 per cubic meter, primarily due to the inclusion of multiple self-healing agents, such as bacterial spores, nutrient compounds (e.g., calcium lactate), and encapsulating carriers [26]. This price, also reported in [224], represents nearly double the cost of conventional concrete, as previously highlighted by Khushnood et al. [224] and Jonkers [225]. The key contributors to this cost differential include the bacterial suspension and the materials required to ensure bacterial viability and controlled activation during the healing process. Despite the higher initial cost, the long-term economic benefits of self-healing concrete are significant. Traditional crack remediation methods, such as epoxy injection, grouting, and surface sealing, are labor-intensive and often need to be repeated multiple times throughout a structure’s lifespan. These interventions can cost up to USD 160 per cubic meter, according to Bravo [226], not including indirect costs associated with service disruption, labor, and equipment. Moreover, such reactive repair methods may not address damage promptly, as microcracks are often undetected until substantial corrosion or material degradation has already occurred, particularly in reinforcing steel [227].

In contrast, microbial self-healing concrete offers a proactive solution, enabling early-stage crack sealing through the autonomous precipitation of calcium carbonate. This not only reduces the need for recurrent maintenance but also extends the functional lifespan of concrete infrastructure. Over time, the reduced frequency of repair, improved durability, and lower material replacement demand can offset the initial investment in self-healing technologies. From a sustainability standpoint, the adoption of self-healing systems can lead to reduced consumption of raw materials, lower construction and demolition waste, and a decline in carbon emissions associated with frequent repairs and reconstruction. As global infrastructure shifts toward long-term resilience and environmental responsibility, the integration of cost-effective microbial self-healing concrete is becoming an increasingly viable option, especially in structures exposed to aggressive environmental conditions.

7. Conclusions and Knowledge Gaps

This review has provided a comprehensive analysis of the mechanisms, influencing factors, and performance evaluations of both autogenous and microbial-induced calcium precipitation (MICP) in self-healing concrete. Autogenous healing primarily relies on continued hydration and calcium carbonate precipitation, which is effective for sealing microcracks but limited by factors such as cement composition and moisture availability. While useful for minor crack closure, its effectiveness declines with increasing crack width, necessitating the incorporation of supplementary materials, such as mineral admixtures and superabsorbent polymers (SAPs), to enhance healing performance [127,228].

In contrast, microbial-based self-healing through MICP has shown greater potential in crack sealing, particularly for larger cracks. The metabolic activity of alkaliphilic bacteria, such as B. subtilis and S. pasteurii, facilitates the precipitation of calcium carbonate, improving the mechanical properties and impermeability of concrete [79,84,85,98,142]. However, key challenges remain, including bacterial survivability in highly alkaline environments, nutrient availability, and material compatibility. Advances in encapsulation techniques, such as fibers, hydrogels, silica gel, and lightweight aggregates, have been explored to protect bacterial spores and ensure controlled activation upon crack formation [119,129]. However, long-term durability studies are needed to validate these methods under real-world conditions.

Additionally, the interaction between fibers and self-healing mechanisms warrants further investigation. The incorporation of natural and synthetic fibers affects crack width control and bacterial activation, yet their impact on the fiber–matrix bond and overall mechanical performance of self-healing concrete is not fully understood [13,21,22,23,125]. While steel and polypropylene fibers have been widely studied, their effectiveness in aggressive environments where corrosion, moisture exposure, and chemical attacks may occur remains a topic for future research. Addressing these uncertainties will help optimize fiber-reinforced self-healing systems and improve their practical application in construction [145,146,229]. This includes investigating the sustained healing capabilities of fibers, their contribution to overall matrix durability, and the efficiency of various fiber types under different environmental conditions. Addressing these challenges will allow researchers to optimize fiber technology for self-healing concrete, ensuring its practical implementation in real-world infrastructure. By integrating optimized fiber reinforcement techniques with microbial and autogenous healing mechanisms, self-healing concrete can become a viable and sustainable solution for improving the resilience and longevity of modern construction materials.

Despite these promising advancements, scaling self-healing concrete for large-scale infrastructure projects presents logistical and economic challenges. Ensuring uniform bacterial distribution, optimizing encapsulation techniques, and reducing production costs are essential for its widespread adoption [16,148]. Moreover, regulatory approval and industry acceptance require further field validation to demonstrate the long-term reliability and economic feasibility of self-healing concrete.

Overall, this review highlights that autogenous healing is a cost-effective, naturally occurring process, whereas microbial-based healing offers enhanced crack-sealing capabilities. Future research should focus on hybrid self-healing approaches that integrate both mechanisms for improved durability and resilience in concrete structures. Advancements in material science, bioengineering, and structural optimization will play a crucial role in overcoming existing challenges and realizing self-healing concrete as a sustainable and innovative solution for modern infrastructure.