Exploring the Potential of Polypropylene Fibers and Bacterial Co-Culture in Repairing and Strengthening Geopolymer-Based Construction Materials ^†

Abstract

This study explored self-healing in geopolymer mortar cured at ambient temperature using polypropylene fibers and bacterial co-cultures of Bacillus subtilis and Bacillus megaterium. Damage degree, compressive strength, ultrasonic pulse velocity (UPV), strength-regain percentage, and self-healing percentage were evaluated. A full factorial design was used, which resulted in an eight-run complete factorial design with four levels in the first factor (polypropylene content: 0%, 0.25%, 0.5%, and 0.75%) and two levels in the second factor (bacteria concentration: 0 (without) and 1 (with)). The results indicate that increasing the polypropylene fiber content enhanced strength regains up to 199.97% with 0.75% fibers and bacteria. The bacteria alone improved strength-regain percentages by 11.22% through mineral precipitation. The analysis of variance (ANOVA) showed no interaction between fibers and bacteria, but both independently improved the compressive strength. Only bacterial samples exhibited positive self-healing, ranging from 16.77 to 147.18%. The analysis using a scanning electron microscope with energy dispersive X-ray (SEM-EDX) and X-ray fluorescence (XRF) also revealed greater calcite crystal formation in bacterial samples, increasing the strength-regain and self-healing percentages. The results demonstrate that polypropylene fibers and bacteria cultures could substantially enhance the strength, durability, and self-healing percentage of geopolymer mortars. The findings present the potential of a bio-based self-healing approach for sustainable construction and repair materials.

1. Introduction

Concrete is considered the most commonly used construction material around the world. It is a heterogeneous mixture formed by combining water, cement, and aggregates to form a rock-like material with varying applications in the industry. However, cement production is energy-intensive and has a large carbon footprint [1]. Cement production is responsible for 7% of the total greenhouse gas emission in the environment, which can induce a 1.4-to-5.8-degree Celsius increase in temperature in the earth over the next 100 years [2], hence, the need for developing alternative binders for concrete.

Geopolymer concretes (GPC) present a more eco-friendly, sustainable, and economical alternative to cement in future construction [3]. The preparation of GPC contributes 20–50% lower CO₂ gas emissions than ordinary Portland cement (OPC) concrete [4]. A geopolymer can be formed by mixing aluminosilicate source materials with alkaline activators; fine and coarse aggregates are combined with water to create a material that resembles concrete and has comparable mechanical qualities [5]. Aluminosilicate materials are rich in aluminum, silicon, and oxygen, which can be found in industrial waste materials such as fly-ash, silica fume, and ground granulated blast furnace slag (GGBS). The commonly used alkaline activators are sodium silicate (Na₂SiO₃) and sodium hydroxide (NaOH). Since geopolymer concrete requires raw materials that have high aluminosilicates, the consumption of industrial materials such as fly ash, rice husk ash, and ground granulated blast furnace slag in the production of geopolymer concrete reduce the pollution burden on the environment [6]. Thus, GPC is an emerging sustainable building material that can replace the Portland cement concrete (PCC) in the construction industry.

Like PCCs, GPCs are prone to microcracks due to its low tensile strength and brittleness. Fibers have been added into the cementitious matrix as reinforcement to alleviate the shortcomings caused by brittleness, resulting in fiber-reinforced cementitious composites (FRCCs) [7]. The fiber’s function is to act as a bridge between the stress-induced microcracks and prevent them from spreading throughout the composite material; increasing the geopolymer matrix’s ductility and improving its mechanical properties such as durability, stiffness, and residual impact strength [8]. The types of fiber reinforcement such as synthetic and natural fibers, can be differentiated by variations in shape, mechanical attributes, content, and aspect ratio [9]. Polypropylene is one of the fibers that can be utilized to strengthen the geopolymer concrete. A significant increase in strength and toughness can be achieved by using polypropylene fiber (PPF), which provide a bridge effect across pores and flaws and alter the ways that cracks expand [10]. A study by Ranjbar et al. [11] shows that incorporating by up to 3% by weight into the geopolymer paste reduces the shrinkage and enhances the energy absorption of the composites. Mohseni [12] showed that using PPF in geopolymer samples increased the flexural strength by 28%, while demonstrating that using 1% PPF had an acceptable outcome in improving GPC performance based on environmental, structural, and economic aspects. Chindaprasirt and Rattanasak [13] also proved that the addition of PPF in composites led to improved tensile strength, crack control, and resistance to acid solutions of fly-ash-based geopolymers. Thus, PPF can enhance the mechanical properties and minimize the cracks within the geopolymer composite material.

The varying load combinations to be encountered by the construction material throughout its lifespan will result in micro-cracks inside the cementitious composites that tend to spread and cause macro-cracks that compromises strength and longevity of construction materials. Traditional methods of repairing cracks are often complex, expensive, and labor-intensive [14]. Hence, the concept of self-healing using bacteria is a promising solution to address these problems.

Little research has been conducted to investigate the healing capability of bacteria in geopolymers. Most studies employ non-destructive testing, where bacilli are directly added as a microbiological agent, while fly ash is used as a precursor. De Koster et al. [15] utilized a geopolymer covering to enclose the bacteria and nutrients. They observed that the metakaolin covering material had a compressive strength of 29.85 MPa and could interact adequately with the concrete mixture. This coating allowed non-encapsulated bacteria to thrive in the concrete mixture, as supported by the study of Jadhav et al. [16] using Sporosarcina pasteurii (previously known as Bacillus pasteurii). Fly ash is another option for a precursor along with metakaolin. Microbial agents can be used in both fly-ash-based geopolymer and Portland cement paste [17]. Moreover, Bacillus subtilis can be incorporated in fly-ash geopolymer matrix with a compressive strength of 34 MPa in an ambient curing environment [18]. Wulandari [19] used a combination of Sporosarcina pasteurii and Rhizopus oligosporus in a fly-ash-based geopolymer and reported that it can increase the compressive strength up to 43.75% relative to the control specimen. Chatterjee et al. [20] developed a 100% fly-ash-based polymer with Bacillus subtilis. In this study, authors found out that the properties of the designed geopolymer not only significantly increased the compressive strength but also improved the following properties: flexural, tensile, water absorption, durability, and sulfate and chloride resistance. Rautray et al. [21] used Bacillus licheniformis in fly ash (30%) and GGBS (70%) as a precursor in geopolymer self-compacting concrete. They achieved a maximum compressive strength of 55.78 MPa after a 56-day curing period. The self-healing effectiveness of the geopolymer material was found to be greatly improved by co-culturing Bacillus sphaericus and Bacillus thuringiensis as studied by Doctolero et al. [22], with the observed maximum fracture width being 0.65 mm. Tian et al. [23] directly applied Sporosarcina pasteurii in fly-ash-based geopolymer mortar and noticed a strength improvement of 16 to 45% with appropriate MICP formulations. Ganesh et al. [24] used GGBS as a precursor with glass fiber and a combination of two bacteria (Bacillus subtillis and Bacillus sphaericus) found that the incorporation of 1% glass fiber with the two bacteria can increase the compressive strength by 27.75% and the split tensile strength by 46.15% relative to the control geopolymer batch. Wulandari et al. [19] utilized fly ash and bottom ash as precursors, with Rhizopus oligosporus and Sporosarcina pasteurii as the microbial agents. They observed that the improvement in compressive strength of 43.75% was attributed to the discovery that most geopolymers supplemented with bacteria cultures had higher levels of closed porosity than comparable specimens without microbial supplementation. Tanyildizi et al. [25] compared the different curing methods, such as submersion, spray and injection in metakaolin geopolymer mortar using Sporosarcina pasteurii as the microbial agent. They discovered that the injection method was the most effective compared to the other curing methods, as it could improve compressive strength by 3.5% and flexural strength by 2.4%. When Sporasarcina pasteurii was used as a microbial agent along with metakaolin and slag as precursors, Polat [26] discovered that the bacterial cells could sporulate directly on the enlarged perlite aggregate, increasing the crack closure rate by 100%. Ekinci et al. [27] used granulated blast furnace slag (GBFS) as a precursor with Bacillus subtilis and found that compressive strength increased by 118% relative to the control samples. The identical precursor and microbial agent were employed by Ekinci et al. [28], who proposed that the direct addition of bacteria to a self-healing process can only be achieved by using a precipitating curing solution consisting of calcium, urea, and bacteria.

Calcium carbonate and sodium carbonate formation occur naturally when calcium and sodium present in a concrete mixture react with hydrated carbon dioxide, in a process called carbonation. This process, however, takes time and requires certain factors such as the continuous hydration of CO₂. Bacillus produces an enzyme called carbonic anhydrase that serves as a transducer in regulating internal CO₂ levels during their metabolism. This process occurs using the byproducts of cellular respiration, CO₂ and H₂O. The enzyme utilizes these byproducts by hydrating CO₂ to produce protons, carbonates, and bicarbonates. This continuous hydration increases the pH through the production of ammonia and supersaturates the minerals present in the concrete due to the continuous production of carbonates and bicarbonates, even when the number of bacterial cells decline. This reaction, in turn, encourages the mineralization of sodium and calcium carbonates that fill the cracks and holes present in the concrete [29,30,31,32,33,34,35].

Based on the literature reviews, it is evident that self-healing geopolymer is generally conducted using a single species or a consortium of bacteria and fiber and, in most cases, without considering the actual application to a building structure. Geopolymer materials are increasingly being considered as repair materials for structures. Using materials that can self-heal is one technique that can reduce costs and increase the longevity of a structural element in a building. The addition of fiber can provide enough reinforcement to enhance the mechanical properties and resistance to deformation of the geopolymer in a composite structure. Meanwhile, the inclusion of bacteria can react with geopolymer material through the process of mineral precipitation. Some bacteria have the ability to induce the formation of calcium carbonate, which can fill in the cracks inside the geopolymer composite material to rapidly heal minor cracks. However, larger cracks cannot be easily repaired by them because it takes a longer time to do so. To overcome this issue, researchers used a combination of bacteria and polypropylene fiber in geopolymer mortar. Geopolymer with bacteria has the potential to heal the cracks, whereas polypropylene fiber can minimize the crack width and improve the strength of the geopolymer matrix. This makes fiber-reinforced geopolymer composites with bacteria suitable for a range of applications, including but not limited to high-performance composite materials, infrastructure repair and maintenance, and building materials.

To the authors’ knowledge, no research has been carried out on the application of geopolymer mortar as a strengthening and repair material when combined with bacteria and polypropylene fiber. For this study, a full factorial design for the design of experiment (DOE) was employed, which resulted in an eight-run complete factorial design with four levels in the first factor (polypropylene content: 0%, 0.25%, 0.5%, and 0.75%) and two levels in the second factor (bacteria concentration: 0 (without) and 1 (with)). In order to better understand these factors, the study investigates fly-ash-based geopolymer mortar’s compressive strength, strength-regain ratio, damage degree, ultrasonic pulse velocity (UPV), and self-healing percentage. SEM-EDX, XRF, and a stereoscopic microscope were also used to examine the mineralogy and morphology of the geopolymer mortar.

2. Materials and Methods

2.1. Materials

Pozzolanic Philippines Incorporated (PPI) provided the fly ash, which was categorized as Class F with low calcium content, in accordance with the ASTM C618-19 [36] test specification. As per manufacturer specifications, the fly ash contains 57.2% silicon dioxide, 21.8% aluminum trioxide, 4.73% ferric oxide, 6.9% calcium oxide, 9.9% magnesium oxide, and 1.23% sulfur trioxide with a density of 2.27 g/cm³.

Sodium silica solution (Na₂SiO₃), sodium hydroxide (NaOH), and potassium hydroxide (KOH) were the alkaline activator (AA) solutions used in the study. Alysons’ Chemical Enterprises Incorporated provided sodium hydroxide flakes (NaOH) and potassium hydroxide flakes (KOH), while Chemline Scientific Corporation supplied the sodium silicate solution (Na₂SiO₃). As per the manufacturer’s specifications, the sodium silicate contains 34.13% SiO₂, 14.65% Na₂O, and 51.22% H₂0 with a silica modulus of 2.33. Sodium silicate (Na₂SiO₃) contains sodium oxide (Na₂O) and silica (silicon dioxide, SiO₂), and it can further help in enhancing the compressive strength and accelerating the geopolymerization process [37]. This mainly occurs as the polycondensation process proceeds much faster when soluble sodium silicate is coupled with alkaline hydroxides rather than using alkaline hydroxides alone [38]. A molar concentration of 12 M was used for NaOH and KOH in all geopolymer batch mixtures. The basis for setting the molar concentration of the alkali activator was in accordance with the previous study by Nagaraj and Venkatesh Babu [39], in which they reported that the optimized molar concentration is 12 M for geopolymer concrete cured at ambient conditions. The results of the experiments carried out by Herwani et al. [40] and Azzahran Abdullah et al. [41] demonstrated the same findings.

Vibro sand and water. The fine aggregates (vibro sand) were bought from a local hardware store and complied with the ASTM C778 [42] criteria and requirements for graded standard sand. The particle size distribution analysis was determined by standard procedures, as described by ASTM C136 [43], as shown in Figure 1, with a fineness modulus of 2.57. For mixing the mortar, ordinary tap water was utilized.

Bacteria. The Philippine National Collection of Microorganisms (PNCM) BIOTECH at the University of the Philippines Los Baños (UPLB) in Laguna, Philippines, provided the Bacillus subtilis and Bacillus megaterium. These are Gram-positive, spore-forming bacteria that naturally thrives in soil environments, hence having a certain level of tolerance in natural stressors such as changes in temperature and pH [44,45]. The two strains were selected due to their alkali tolerance, spore-forming ability, high potential for precipitating calcite, and non-pathogenicity. In addition, Doctolero et al. [22] employed these bacteria and found that co-culturing bacteria significantly enhanced the healing efficiency of the geopolymer mortar.

Polypropylene fiber. A polypropylene fiber of 12 mm in length was purchased from Tertex International Philippines Incorporated. This fiber was used since it was lighter relative to other fibers, able to resist bacteria or microorganism attacks, and naturally rot-proof, moth-proof, and mildew-resistant [46]. The study by Nematollahi et al. [47] served as the basis for the polypropylene fiber percentage range.

2.2. Preparation of Geopolymer Samples

As illustrated in

Table 1. Batch formulation of the geopolymer mortar with varying amounts of polypropylene and bacteria.

Batch Code	Fly-Ash (g)	Sand (g)	Polypropylene (g)	NaOH (g)	KOH (g)	Na2SiO3 (g)	Water (mL)	Bacteria (Cells/mL)
PP0	1469	1039	0	165	55	441	220	0
PP1	1469	1039	36.73	165	55	441	220	0
PP2	1469	1039	73.45	165	55	441	220	0
PP3	1469	1039	110.18	165	55	441	220	0
BP0	1469	1039	0	165	55	441	220	1.5 × 108
BP1	1469	1039	36.73	165	55	441	220	1.5 × 108
BP2	1469	1039	73.45	165	55	441	220	1.5 × 108
BP3	1469	1039	110.18	165	55	441	220	1.5 × 108

, the batch formulation of the various geopolymer combinations resulted in varying amounts of polypropylene and bacteria. The researchers utilized a full factorial design for the design of experiment (DOE), which resulted in an eight-run complete factorial design with four levels in the first factor (polypropylene content: 0%, 0.25%, 0.5%, and 0.75%) and two levels in the second factor (bacteria concentration: 0 (without) and 1 (with)). The findings of the previous research by Quiatchon et al. [48] served as an initial basis for determining the values of the three (3) parameters: activator/precursor ratio = 0.45, water/solid ratio = 0.2, and (NaOH + KOH)/sodium silicate ratio = 0.5. In that study, they varied the three (3) parameters (activator/precursor, water/solids, and sodium hydroxide/sodium silicate ratio) in order to determine the effects to the following response parameters (initial, final setting time and compressive strength).

The following raw materials (polypropylene fiber, sand, fly ash, potassium hydroxide (KOH), and sodium hydroxide (NaOH) flakes) were weighed beforehand with an automated balance for the production of geopolymer mortar samples. In order to have a 12 M for both NaOH and KOH, 343.78 mL of water was used to dilute 165 g of NaOH flakes and 81.69 mL of water for 55 g of KOH flakes. Separately, the flakes of NaOH and KOH were dissolved in water and mixed for ten (10) minutes in a plastic bowl over an ice bath. To make the alkaline solution, the sodium silicate solution was mixed with the dissolved NaOH and KOH flakes and stirred for fifteen (15) minutes.

Polypropylene fiber, fly ash, sand, water, and alkaline solution (NaOH, sodium silicate, and KOH) were combined as raw ingredients and then mixed for ten (10) minutes in a cement mortar mixer. It was then followed by three (3) minutes of manual mixing using a spatula to ensure that the polypropylene fibers were evenly distributed throughout the homogenous geopolymer mixture and that all the fly-ash particles at the bottom had completely reacted with the geopolymer mixture. The last minute was allotted to directly add the two types of bacterial solutions, as shown in Figure 2. In this research, McFarland Standard was used to standardize the number of cells per mL based on its turbidity [49]. The number of cells per mL varies based on the cell size, and in this case, a 0.5 McFarland Standard turbidity equates to 1.5 × 10⁸ bacterial cells/mL [50]. This number of cells per mL is suitable for most bacterial assays, as a higher number of cells tends to increase the errors in an experiment due to, but not limited to, nutrient availability.

After properly mixing all the raw materials, the geopolymer mortar was poured into plastic square molds with three compartments with a dimension of 50 mm by 50 mm by 50 mm. Using a tamping rod, the mixture was compacted in two layers in order to ensure that there were no air bubbles inside the mold. Finally, a trowel was used to flatten the top surface of the cubical mold with geopolymer mortar.

The geopolymer batch mixture inside the cubical mold was left undisturbed for 24 h prior to demolding. After demolding, a plastic cling wrap was used to entirely wrap all the cubical specimens in order to keep the level of moisture inside the geopolymer matrix. The demolded samples were stored at an ambient temperature of 25 to 30 °C and a relative humidity of 50 ± 15% in an undisturbed environment that is free from sunlight and rain.

The researchers used the ASTM C109/C109M-20 [51] standard as a reference for the preparation, casting, quality control, and compressive strength of geopolymer mortars; thus, considered the existing criteria for ordinary Portland cement (OPC).

Figure 3 illustrates the flowchart outlining the test procedure carried out in this study. After molding eight (8) batches of geopolymer mixtures, each in sextuplicate, the specimens were cured for 28 days in an ambient environment. Three samples were allotted for pre-cracking at 60% of the ultimate capacity, whereas the other three samples were used to determine the maximum compressive strength. The UPV values before and after the inducing of cracks were obtained by a direct method using Pundit Lab+ CT-133 UPV equipment. Then, after a healing period of fourteen (14) days, the UPV test was conducted again before subjecting all the geopolymer batches to a final compressive strength test.

2.3. Bacterial Culture Maintenance and Spore Induction Assay

It is important that the microbial cultures used in each experiment be fresh and standardized in terms of their age, underscoring the importance of culture maintenance. In this research, a mother culture was used in all experiments as further passages, or subculturing of bacteria, could lead to genetic drifts and cause certain traits of the bacteria to disappear, explaining the presence of atypical colonies on multiple passages.

The geopolymer mixture used in this research contains high amounts of KOH, which increases the pH levels. Though the bacteria can tolerate and grow in alkaline conditions, it is not recommended to immediately subject them to a harsh environment in their vegetative state as they may fail to recover from the stress and perish. Thereby, the number of cells or spores present in each geopolymer mortar will greatly vary, and the margin of error increases. On the other hand, Bacillus species are well-known spore-forming bacteria. Bacterial spores are tolerant of high-stress environments such as extremely low and high pH levels, extremely high temperatures, and lack of oxygen. By inducing sporulation, the researchers achieved a standardization of the cell count within the geopolymer specimen, effectively reducing the margin of error. This dormant state also served to naturally preserve the bacteria, as they are meant to be embedded within the geopolymer matrix where oxygen levels might be low or absent. Subsequent bacterial germination can then take place upon the introduction of oxygen, often occurring within the gaps in the solidified geopolymer.

The flowchart of the process used to prepare bacterial samples for the geopolymer mortar is shown in Figure 4. Bacterial samples were inoculated onto pre-prepared nutrient agar (NA) plates for 24 h at 37 °C to serve as the mother culture for future experiments. The mother culture was initially subjected to 16S rRNA sequencing and BLAST analysis to confirm its identity. Once confirmed, a loopful of bacteria from the mother culture was inoculated in NA slants and incubated for 18 h under the same temperature setting. A 7-day-old culture in NA slants from the mother plate was standardized at 0.5 McFarland Standard before being subjected to heat- and cold-shock treatments. The use of 7-day-old cultures in fast-growing bacteria ensures that the bacterium of interest is at the late stage of the stationary growth phase and nutrients are scarce or rapidly depleting. The ability of Bacillus species to sporulate assures the researcher that most of the bacteria in the culture have already sporulated or are preparing for sporulation due to the stress of having low nutrients. Adding additional stressors to these bacteria, such as heat- and cold-shock treatments, hastened this process and ensured that most of the bacteria sporulated.

Figure 5 illustrates how bacterial cultures were streaked onto nutrient agar plates, incubated for 18 to 24 h at 37 degrees Celsius, and then maintained cold at 4 to 8 degrees Celsius. Seven (7) day old cultures were inoculated with a sterile saline solution standard at 0.5 McFarland Standard, and 1 mL was then aliquoted to microcentrifuge tubes. To stimulate sporulation, the samples were placed in an ice bath for 5 min after being heated to 80 °C in a dry bath for 10 min. Schaeffer-Fulton staining was used to confirm sporulation while spore concentration was measured using a hemocytometer. All samples were utilized immediately to avoid germination [22].

2.4. pH and Temperature Tolerance Assay

The main objective of this research was to formulate the optimized geopolymer mixture with bacteria and polypropylene, which can be used as a repair material in composite structures. Since microorganisms are ubiquitous, other microorganisms will contaminate the mixture when aseptic conditions are not met.

Thus, it is important to introduce limiting factors, such as high pH and temperature, to prevent or limit contamination in non-aseptic environments. To make this possible, it is important to determine if the bacterium of interest can tolerate and grow in the presence of the stressors, namely, high pH and temperature. It is also worth noting that Bacillus species, in their vegetative states, have varying levels of stress tolerance depending on their strain and chromosomal and extrachromosomal makeup. Hence, B. subtilis isolated from point A may be susceptible to high pH, while B. subtilis isolated from point B may be tolerant.

The temperature and alkalinity tolerance of B. subtilis and B. megaterium were analyzed using the protocol of Sahoo and colleagues [52] with modifications. Briefly, 18 h. cultures of bacterial samples were inoculated in 0.9% NaCl standardized at 0.5 McFarland Standard. An 18-h incubation period before experimentation ensured that fast-growing bacteria, such as Bacillus sp., are within the exponential growth phase and would produce the best possible results when tested. Then, 100 µL of standardized bacteria were aliquoted into two sets of NB with increasing alkalinity levels, wherein the pH of nutrient broth (NB) (TM Media) was adjusted to 8, 9, 10, 11, 12, 13, and 14 using 1 M NaOH before autoclaving. A set of NB tubes was incubated at 37 °C and the other at 50 °C for 24 h. Uninoculated NB at increasing alkalinity levels and inoculated NB at pH 7 were used as controls per set. After the initial growth observation, the cultures were allowed to incubate for 10 days under the same conditions.

2.5. Test Procedure

2.5.1. Ultrasonic Pulse Velocity (UPV)

After 28 days of ambient curing, the UPV test was carried out using UPV (Pundit Lab+ CT-133, Proceq) equipment with 54 kHz transducers. ASTM recommends utilizing transducers with resonance frequencies ranging from 20 to 100 kHz in UPV measurements. The researchers used a frequency of 54 kHz since this value is typically used in UPV for testing concrete and geopolymer materials.

For each batch of geopolymer mortar, an average of three readings was determined using the direct UPV technique. The UPV values were measured both before and after inducing the cracks. Finally, after 14 days, when the geopolymer mortar had healed, the UPV values were also determined.

In this study, the damage degrees of fly-ash-based geopolymer mortars were calculated using Equation (1). Tanyildizi et al.’s [25] research provided the basis for Equations (1) and (2).

$$\mathrm{DD}=1-(\frac{{\mathrm{V}}_1}{{\mathrm{V}}_2})$$

(1)

DD is the damage degree in Equation (1). V₁ is the UPV value after the cracking, and V₂ is the UPV value before the cracking. Moreover, the self-healing percentages of the geopolymer material were calculated using Equation (2).

$$\mathrm{H}=\frac{({\mathrm{V}}_3-{\mathrm{V}}_2)}{{\mathrm{V}}_2} \times 100$$

(2)

where V₃ is the UPV value after the healing period of geopolymer material.

2.5.2. Compressive Strength

One of the key mechanical characteristics to take into account when evaluating the performance of geopolymer is its compressive strength. According to ASTM C109/C109M [51], the unconfined compressive strength (UCS) test was carried out on a MATEST S.p.A. Treviolo with a capacity of 250 kN and a loading rate of 1.2 kN/s. This standard specifies the requirements and procedure for determining the compressive strength of hydraulic cement and other mortars.

Similar to ordinary Portland cement (OPC)-based mortar, compressive strength is the capacity of the material to carry the maximum applied load before failure. The unconfined compressive strength of each individual specimen is equal to the maximum compressive force recorded by the testing machine divided by the perpendicular surface area of the specimen, which can be computed using Equation (3). In Figure 6, the actual compressive strength test of the geopolymer mortar (a) before and (b) after the test can be seen.

$$Unconfined Compressive strength=\frac{P}{A}$$

(3)

P is the maximum compressive load, and A is the perpendicular area of the specimen.

2.5.3. Microstructural Observation

The microstructural test was selectively conducted on a subset of specimens, as the primary goal was to assess the variations in chemical composition between the inoculated and non-inoculated geopolymer samples. Two specimens were chosen from the two batches of geopolymer mortar (PP0 to PP3 and BP0 to BP3) for SEM-EDX and XRF. The optimal specimens are the most suitable specimens with the highest compressive strength after the healing period and strength-regain percentages.

Prior to imaging of the specimens via SEM, all non-conductive materials, such as geopolymer mortar, must be coated with a conductive element for enhanced imaging. This procedure lessens the illumination from the sample, which damages the filament of the source, and prohibits the samples from being burned by the high-intensity beams of the equipment. The geopolymer samples were, thus, coated with gold using a magnetron-type coater (JEOL JFC-1200 auto-fine coater) before being subjected to Phenom XL Desktop scanning electron microscopy (SEM) equipment.

Geopolymer specimens were also subjected to X-ray Fluorescence (XRF) using Horiba MESA50 equipment. The XRF spectra were obtained by scanning the geopolymer samples, and the components of the sample were identified. A Nikon Stereoscopic Zoom Microscope C-LEDS was used to examine the microscopic photos of inoculated and non-inoculated geopolymer mortar specimens.

3. Results and Discussion

3.1. pH and Temperature Tolerance

Microbial growth was observed up to pH 9 for both cultures during incubation at 37 °C. Notably, B. subtilis exhibited growth at pH 8, exclusively at 50 °C, whereas no growth was detected in the case of B. megaterium. However, on the seventh day of incubation, bacterial growth was observed across all pH levels and temperatures, as demonstrated in

Table 2. pH and temperature tolerance of Bacillus subtilis and Bacillus megaterium after 24 h and 7 days of incubation.

pH	24 h of Incubation				7 Days of Incubation
	B. megaterium		B. subtilis		B. megaterium		B. subtilis
	37 °C	50 °C	37 °C	50 °C	37 °C	50 °C	37 °C	50 °C
8	G	N/G	G	G	G	G	G	G
9	G	N/G	G	N/G	G	G	G	G
10	N/G	N/G	N/G	N/G	G	G	G	G
11	N/G	N/G	N/G	N/G	G	G	G	G
12	N/G	N/G	N/G	N/G	G	G	G	G
13	N/G	N/G	N/G	N/G	G	G	G	G
14	N/G	N/G	N/G	N/G	G	G	G	G

N/G means no growth, G means growth.

. Following a seven-day incubation period, both B. subtilis and B. megaterium displayed signs of bacterial growth. This suggests that both bacterial strains may experience an extended lag growth phase within the geopolymer but exhibit tolerance to the high pH and temperature conditions it presents.

3.2. Compressive Strength

Compressive strength is one of the most important properties of mortar and concrete. This strength represents the quality and structural performance of mortar, which can be applied as part of any structural element. A compressive strength test was performed following the procedures stated in ASTM C109/C109M [51]. A total of twenty-four (24) cubical geopolymer mortar samples were tested using MATEST S.p.A. Treviolo. A pre-cracking percentage of 60% was used in order to induce cracks in all the geopolymer mortar samples. Immersion curing in water was used as a healing period for 14 days before subjecting all the samples to the ultimate compressive strength test.

The compressive strength test result of geopolymer mortar samples with varying amounts of polypropylene fibers after 28 days of ambient curing is seen in Figure 7. Among the non-inoculated samples, PP1 obtained the highest average compressive strength of 33.85 ± 0.66 MPa (95% confidence interval (CI)), while PP2 recorded the lowest average compressive strength value of 25.37 ± 0.34 MPa (95% CI). Statistically, this implies that there is a 95% probability that the true average compressive strength for PP1 falls within the range of 33.20 to 34.51 MPa, whereas for PP2, it is expected to be between 25.04 and 25.71 MPa. The confidence interval for PP2 is narrower since the standard deviation of PP2 (0.30) is less than PP1 (0.58). The compressive test results agree with those of Nematollahi et al. [47], who reported that the maximum compressive strength can be obtained from geopolymer mortar with 0.25% polypropylene fibers. It indicates that when the compressive force is applied perpendicularly to the geopolymer material, a significant percentage of the fibers can help to maintain the geopolymer matrix intact within the microcracks. In comparison to PP1, the compressive strength decreased by 25% for PP2 and 12% for PP3 as the fiber concentration increased. One possible reason for the decrease in compressive strength is the polypropylene fiber’s hydrophobic characteristics, which resulted in poor fiber attachment to the geopolymer matrix. Furthermore, this might be attributed to a fiber-induced increase in entrapped air, which results in a larger porosity of the mixture [53].

PP3 obtained the highest average strength-regain ratio of 179.79% (coefficient of variation (CoV) = 3.09%), while PP1 recorded the lowest average strength-regain ratio of 97.21% (CoV = 51.07%). Increasing the polypropylene fiber content increases the strength-regain ratio because of the bridging effect of the fiber in the geopolymer matrix. The polypropylene fibers serve as a bridge that transfers the stress across the geopolymer matrix, improving the material’s strength, durability, and self-healing ability. Thus, increasing the amount of fiber would increase the contact area of the fiber with the geopolymer matrix, which can minimize crack propagation and enhance the strength recovery and durability of the material. Similar findings were also observed by other authors, such as Feng [54] and Zhu [55]. Feng et al. [54] found that adding polypropylene fibers can improve the self-healing ability of cement-based materials, whereas Zhu et al. [55] revealed that PP fibers can improve the compressive strength recovery rate and the self-healing behavior of micro-cracks.

The compressive strength test result of the geopolymer mortar samples with varying amounts of polypropylene fibers and bacteria after 28 days of ambient curing are seen in Figure 8. BP0 obtained the highest average compressive strength of 21.59 ± 5.83 MPa (95% CI), while BP2 recorded the lowest average compressive strength value of 17.03 ± 2.41 MPa (95% CI). Statistically, this implies that there is a 95% probability that the true average compressive strength for BP0 falls within the range of 15.76 to 27.42 MPa, whereas for BP2, it is expected to be between 14.62 and 19.44 MPa. The confidence interval for BP0 is wider since the standard deviation of BP0 (5.83) is greater than BP2 (2.41).

Meanwhile, the average strength regain of BP3 and BP1 was 199.97% (CoV = 32.31%) and 142.27% (CoV = 15.26%), respectively, with BP3 being the highest among the geopolymer samples for both inoculated and non-inoculated batches. Comparing the results from non-inoculated batches (PP0-PP3), it can be observed that the strength-regain percentage of BP3 is 11.22% higher than PP3. It only shows that the inclusion of bacteria can improve the strength-regain value of the geopolymer mortar through the process of mineral precipitation.

A two-way ANOVA was used using Microsoft Excel to investigate the influence of polypropylene fibers and the presence of bacteria on the compressive strength of fly-ash-based geopolymer mortar. The result of the two-way ANOVA can be seen in

Table 3. Two-way ANOVA results for the interaction of polypropylene fibers and the presence of bacteria on the compressive strength of the geopolymer mortar.

ANOVA
Source of Variation	SS	df	MS	F	p-Value	F Crit
Bacteria	773.6162	1	773.6162	64.83539	5.1 × 10−7	4.493998
PP	150.0091	3	50.00304	4.190666	0.022846	3.238872
Interaction	23.33088	3	7.776959	0.651773	0.593333	3.238872
Within	190.9121	16	11.932
Total	1137.868	23

. It revealed that there was no statistically significant interaction between the effects of the polypropylene fiber and the presence of bacteria (F(3,16) = 0.65, p = 0.5933). A simple main effect analysis showed that the inclusion of bacteria has a significant effect on the compressive strength of geopolymers. Similarly, polypropylene fibers have a statistically significant effect on the compressive strength of geopolymers since the p-value is less than 0.05.

Therefore, a geopolymer mortar containing polypropylene and bacteria has the potential to be used as a repair mortar with an R2 classification, according to Ducman et al. [56] since its compressive strength is at least 15 MPa ranging from 17.03 to 21.59 MPa. According to the European Standard EN 1504-3:2006 [57], which defines the materials that may be used for the preservation and repair of concrete structures, a geopolymer mortar containing polypropylene fiber and bacteria may be used as a non-structural and aesthetic repair. Meanwhile, geopolymer with polypropylene fiber only has the potential to be used as a structural repair material in severe environments since its compressive strength value is at least 25 MPa with an R3 classification.

3.3. Ultrasonic Pulse Velocity (UPV)

The ultrasonic pulse velocity (UPV) is a non-destructive test to assess the quality (porosity/solidness/compactness) of a material, and this method was utilized in order to assess the homogeneity of the geopolymer mortar samples after 28 days of ambient curing. It was conducted following the procedures stated in ASTM C 597 [58] using Pundit Lab equipment with a frequency of 54 kHz, pulse voltage of 500V, and receiver gain of 10x. A total of twenty-four (24) geopolymer mortar samples were tested before, after inducing cracks and after a healing period of 14 days.

Figure 9 illustrates the variation in ultrasonic pulse velocity (UPV) values of geopolymer mortar samples containing polypropylene fiber after 28 days. The largest average UPV value obtained by PP0 (0% PP fiber) was 2736.33 ± 356.82 m/s (CoV = 9.41%), whereas the lowest average UPV value obtained by PP2 (0.5% PP fiber) was 2161 ± 109.90 m/s (CoV = 3.67%). Statistically, this implies that there is a 95% probability that the true average UPV value for PP0 falls within the range of 2379.52 to 3093.15 m/s, whereas for PP2, it is expected to be between 2051.10 and 2270.90 m/s. The confidence interval for PP0 is wider since the standard deviation of PP0 (315.32) is greater than PP2 (97.12). Higher ultrasonic pulse velocity values indicate better material quality in terms of homogeneity and uniformity [59]. A lower UPV value indicates a weaker pulse intensity that, as a result of the discontinuity, propagates through the material for a longer period of time. The discontinuity might be found as cracks, voids, or fissures that could obstruct the transmission of the pulse.

The results show that the velocity of the ultrasonic wave is slower after applying 60% of the ultimate load capacity, and the UPV values improved after the healing period for the PP2 and PP3 batches only. After a healing period, the UPV values increased significantly, by 167.46%, for the PP3 batch.

PP3 obtained the highest average damage degree of 67.20%, while PP1 recorded the lowest value of 19.78%. PP3 (0.75% PP) had the greatest average self-healing percentage of −12.05%, while PP0 (0%) had the lowest value of −38.66%. Clearly, polypropylene plays a crucial role in reducing the damage degree and enhancing the self-healing percentages of geopolymer mortar.

The ultrasonic pulse velocity (UPV) values of the geopolymer mortar samples with varying amounts of polypropylene fibers and bacteria after 28 days can be seen in Figure 10. The greatest average UPV value achieved by BP2 (0.5% PP fiber) was 1,794.67 ± 195.78 (95% CI) m/s, whereas the lowest average UPV value obtained by BP1 (0.25% PP fiber) was 686.33 ± 147.81 m/s (95% CI). Statistically, this implies that there is a 95% probability that the true average UPV value for BP2 falls within the range of 1598.88 to 1990.45 m/s, whereas for BP1, it is expected to be between 538.53 and 834.14 m/s. The confidence interval for BP2 is wider since the standard deviation of BP2 (173.02) is greater than BP1 (130.62).

The results show that the velocity of the ultrasonic wave is reduced after applying 60% of the ultimate load capacity, and the UPV values improved significantly after the healing process for all the batches. A lower UPV measurement indicates that the cracks in the material caused a delay in the transmission of the ultrasonic pulse velocity. After a healing period, a percentage increase of 34.68% to 481.73% in the UPV values was observed in the geopolymer mortar with polypropylene and bacteria batches. Indications suggest that the inclusion of bacteria has a significant effect on improving the quality of the geopolymer mortar with polypropylene fiber.

PP3 (0.75% PP) obtained the highest average damage degree of 67.20 ± 2.75 (95% CI), whereas BP3 (0.75% PP) recorded the lowest value of −39.94 ± 53.96 (95% CI). BP1 obtained the highest average self-healing percentage of 147.18 ± 32.0% (95% CI), while the lowest value was −38.66 ± 23.49% (95% CI), as seen in

Table 4. Damage degree and self-healing percentages of the geopolymer mortar with polypropylene and bacteria.

Batch Code	UPV after Cracking, V1 (m/s)	UPV before Cracking, V2 (m/s)	UPV after Healing, V3 (m/s)	Damage Degree, DD (%)	Self-Healing, H (%)
PP0 (0%)	1672 ± 866.12	2736.33 ± 356.82	1637.33 ± 481.83	38.61 ± 32.23	−38.66 ± 23.49
PP1 (0.25%)	1980 ± 485.01	2591.33 ± 603.70	1789.33 ± 709.03	19.78 ± 32.65	−28.43 ± 36.38
PP2 (0.5%)	1671 ± 88.66	2161 ± 109.90	1761.33 ± 98.29	22.63 ± 3.77	−18.50 ± 1.31
PP3 (0.75% PP)	817.33 ± 36.43	2503 ± 274.43	2186 ± 100.02	67.20 ± 2.75	−12.05 ± 11.19
BP0 (0%)	951.33 ± 753.72	1187.67 ± 148.78	1365.33 ± 753.14	20.47 ± 57.92	16.77 ± 67.99
BP1 (0.25%)	288.33 ± 268.95	686.33 ± 147.81	1677.33 ± 245.59	58.36 ± 37.62	147.18 ± 32.0
BP2 (0.5%)	1695.33 ± 370.91	1794.67 ± 195.78	2281.67 ± 262.53	4.21 ± 29.60	28.77 ± 28.76
BP3 (0.75% PP)	1634.67 ± 193.40	1261.67 ± 489.36	2234 ± 148.68	−39.94 ± 53.96	90 ± 65.49

± Confidence interval (95%).

. Clearly, combining polypropylene with bacteria has a major impact on reducing the damage and increasing the healing percentages in a geopolymer mortar. It is also worthwhile to mention that only geopolymer with bacteria batches ranging from BP0 to BP3 exhibited acceptable self-healing percentages. This means that incorporating bacteria into geopolymer mortar with polypropylene fiber improves the overall quality of the composite material.

In order to establish the relationship between the compressive strength and the ultrasonic pulse velocity of the geopolymer mortar, a linear correlation was used. It can be observed from Figure 11 that there is a very strong positive relationship between the compressive strength and the ultrasonic pulse velocity, since the R2 value is 0.9086. A higher value of the UPV means that the compressive strength of the geopolymer mortar is also higher. From the given linear equation below, it can be used as a prediction tool for the compressive strength of the geopolymer mortar containing polypropylene fiber and bacteria. Srivastava et al. [60] demonstrated that the UPV technique is feasible for rapid estimation of coal bottom ash (CBA) mortar characteristics, both in the laboratory and at the site. Moreover, Sitarz et al. [61] stated that ultrasonic pulse velocity measurements are an effective method for evaluating the maturity of a geopolymer and correlate well with the evolution of its mechanical properties over time. The result of this study is similar to the output of Gupta et al. [62], in which they demonstrated that UPV can be used to accurately predict the compressive strength of geopolymer composites with a linear correlation coefficient value of 0.9843.

3.4. Material Characterization

For SEM and XRF analyses, PP3 and BP3 were selected since they represent the optimized specimens for the inoculated and non-inoculated geopolymer batches. An increase in mineral components was observed in the inoculated geopolymer mortar samples compared to the non-inoculated control, suggesting that inoculated samples have undergone increased geopolymerization activity, as shown in Figure 12. However, aggregates of unreacted fly ash were observed in sample BP3, while only small amounts were observed in its uninoculated counterpart, PP3, implying a correlation between bacterial inoculation and polypropylene quantity.

The elemental composition using XRF analysis of the two geopolymer specimens, as shown in

Table 5. Elemental composition of geopolymer samples (% w/w).

Specimen Code	Ca	Si	Fe	K	Ti	Rh	Sr
PP3	78.84	--	14.71	2.74	1.31	0.99	0.72
BP3	56.11	19.74	16.58	3.55	1.75	0.85	--

, shows that they are mainly composed of calcium (Ca), iron (Fe), and potassium (K). The majority of the calcium content was evident in the PP3 (with 0.75% PP but no bacteria)specimen relative to the other sample, which is the BP3 (with 0.75% PP and bacteria)specimen. The amount of calcium content in the fly ash was found to have a significant impact on the resulting hardened geopolymer [63]. Calcium oxide is believed to form minerals such as calcium silicate hydrate (CSH), along with the aluminosilicate geopolymer gel [64]. Thus, it is expected that the PP3 (geopolymer with PP but no bacteria) has a higher mechanical property relative to BP3 (geopolymer with PP and bacteria) since the calcium content of PP3 (78.84%) is higher than BP3 (56.11%). It can be observed that PP3 obtained a compressive strength of 29.60 MPa, while BP3 only obtained a compressive strength value of 17.32 MPa, as seen in Figure 7 and Figure 8.

Patches of calcite crystals were also prominent in inoculated samples such as BP2 (Figure 13b), which can be attributed to the inoculation of Bacillus. Similar findings were observed by Sahoo et al. [52], in which the presence of calcite was the main reason for the improvement in the compressive strength of the cement mortar with bacteria. Bacillus is known to be responsible in the biomineralization phenomena by precipitating ample amounts of minerals such as calcium carbonates, and this occurs as a byproduct of its metabolic process. In aerobic microorganisms such as Bacillus, CO₂ is produced as a byproduct of its metabolic activity. The increasing presence of CO₂, however, disrupts cellular homeostasis when improperly handled. By producing an enzyme called carbonic anhydrase from Bacillus, it can regulate the internal CO₂ levels during metabolism by converting CO₂ into HCO₃ and H+ using another metabolic byproduct, H₂O [32]. Bacillus can then use the protons and bicarbonates to regulate its intracellular and extracellular pH levels and support physiological functions [31]. Calcium anhydrases are also found to be secreted by Bacillus species. The secretion of this enzyme entails the continuous hydration of CO₂ to produce bicarbonates, which then readily react to the components of the concrete mixture, such as calcium oxides, to produce calcium carbonates, hence, the increase in calcite crystals and other mineral components in inoculated samples.

4. Conclusions and Recommendations

The current study demonstrates that the self-healing technique has notable potential as a novel biological metabolic product created by co-culturing bacteria (B. subtilis and B. megaterium) in geopolymer mortar cured at an ambient temperature. Polypropylene fiber and bacteria can be used in fly-ash-based geopolymer mortar to improve its strength, durability, and self-healing percentages. The compressive strength, ultrasonic pulse velocity, strength-regain percentage, damage degree, and self-healing percentage of geopolymer mortars with polypropylene fibers and bacteria were investigated. The following conclusions can be drawn:

• Geopolymer mortar containing polypropylene and bacteria has the potential to be utilized as a non-structural and aesthetic repair material due to its compressive strength of at least 15 MPa, whereas the combination of geopolymer and polypropylene fiber has the potential to be employed as a structural repair material in severe environments.
• Increasing the amount of polypropylene fiber would increase the contact area of the fiber with the geopolymer matrix, which can minimize crack propagation and enhance the strength-regain ratio of the geopolymer material. Meanwhile, the strength-regain percentage of the inoculated geopolymer is 11.22% higher than that of the non-inoculated geopolymer. This only shows that the inclusion of bacteria can improve the strength-regain value of the geopolymer mortar through the process of mineral precipitation. The inclusion of 0.75% polypropylene fiber in the geopolymer mortar with bacteria had a strength-regain percentage value of 199.97%.
• For the damage degree and self-healing percentages, polypropylene with bacteria has a significant effect on minimizing the damage degree and improving the healing percentages of geopolymer mortar. It is worth noting that only geopolymers containing bacteria have shown positive self-healing percentages, ranging from 16.77% to 147.18%.
• The results of the SEM-EDX and XRF revealed that the amount of calcite crystals was more evident in the bacterial samples than in the samples without bacteria, which resulted in an increase in the strength-regain ratio and self-healing percentages.
• A two-way ANOVA revealed that there was no statistically significant interaction between the effects of the polypropylene fiber and the presence of bacteria. However, co-culturing of bacteria and polypropylene has a significant effect on the compressive strength of the geopolymer mortar.

The future work will further investigate the potential of polypropylene fibers and bacteria co-culture inclusion in fly-ash geopolymer mortar for repair applications. This includes comprehensive tests on its adhesive bonding and bending strength. The consideration of how diverse bacteria affect natural fibers as reinforcements under various curing conditions in the cementitious matrix is also a study of interest. This exploration will contribute to our knowledge of microbial self-healing and fiber-reinforced concrete, with implications for sustainable construction practices.