Phenomena of Bacillus sphaericus LMG 22257 Activity and Its Influence on Properties of Portland Cement Mortar Exposed to Different Curing Media

Abstract

This study determined the influences of Bacillus sphaericus Laboratorium voor Microbiologie Gent (LMG) 22257 bacteria activity on mortar samples cured in various media regarding compressive strength, porosity, water absorption, and water permeability. Three types of curing media were utilized, namely distilled water (D.W.), deposition water (D.M.), and run-off water (R.W.). The compressive strength was measured using 100 mm mortar cubes. The water porosity, water absorption, and water permeability were analyzed using the Leeds permeability cell with dimensions of the mortar cylindrical specimens of 55 mm diameter and 40 mm thickness. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDX) were utilized, respectively, for microstructure analysis and quantifying the elements with atomic numbers. The results indicated the presence of calcium carbonate and more calcium silicate hydrate (CSH) depositions on bacterial mortars. The inclusion of Bacillus sphaericus LMG 22257 bacteria activity and curing media type affected mortar properties through compressive strength and durability improvements, as well as the reduction in water porosity, water absorption, and water permeability of mortar. The comparison of CaCO₃ precipitation, such as a sufficient growth nutrient requirement and hostile bacteria environment, was observed. Curing in R.W. produced the most significant bio-based cement (BBC) mortar improvement, followed by D.M. BBC curing in runoff water had a 40% improvement in strength compared to normal curing. As a conclusion, runoff water is a highly promising sufficient nutrient to bacteria for the biomineralization process to produce CaCO₃. This work also aims to apply this approach in the field, especially in sewerage and drainage systems.

1. Introduction

Concrete is the most commonly used building material worldwide. It is robust, rigid, and fire-resistant and can be molded to any size and shape. It is also considered a low-cost material that requires a low maintenance cost. Although concrete has its advantages, it can deteriorate when exposed to certain harsh environments. Hence, concrete with varying properties and performance has been prescribed and used for different types of concrete structures, exposure conditions, and expected service life. In the design and construction of concrete structures, a service life of 100 years or more is sometimes required [1,2]). Therefore, the quality of the concrete structure is vital and needs to be preserved or protected to ensure adequate durability. Thus, the concrete structures must be protected from facing pressing durability issues such as chloride and sulphate attacks, which could induce premature degradation of concrete structures.

Cracks are one of the initial physical phenomena of concrete degradation. During the service life of concrete structures, cracks profoundly affect the properties and durability performance. In practice today, detailed investigations followed by appropriate diagnosis are required to identify these defects, then proper repair techniques and scheduled maintenance are undertaken [3,4]. Cracking of concrete is a regular occurrence. However, if concrete structures crack without instant and proper treatment, they tend to grow further and eventually necessitate expensive repairs. Thus, the degree of cracking can be minimized by the available modern technology, which enables the remediation of cracks in concrete by utilizing suitable repair materials and techniques [5,6,7]. A bio-based cement technology is one of the means to repair cracks and fissures in concrete buildings [8,9,10].

The utilization of specific microbes in cement-sand mortar or concrete has been shown in previous research to deposit inorganic substances as the filling materials of pore matrices, thereby healing structural fissures [11,12]. Compressive strength is increased by 20–35% when bacterial cells with growth media, which consists of 25 mM calcium chloride and 20 g/L of urea, are added to the mortar or concrete compared to the control mix [13,14,15]. However, despite the tiny amount of calcium chloride in the growth media, cement mortar’s strength did not significantly increase when growth media without bacterial cells was employed [16,17]. The strength and ability to mend cracks were enhanced compared to conventional concrete when biologically induced cement was used [18,19,20,21]. The deposition of CaCO₃ by the microorganisms into the pores helps to plug the pores of mortar. The refinement of the pores of mortar improves the transport properties, such as reducing the porosity, water absorption, and permeability of the mortar or concrete, which contributes positively toward better durability performance.

Bacillus sphaericus is the selected bacteria for this study due to its natural extreme mophilic bacteria, which can live for more than 1000 years and is widely used as a standard spore-forming calcite bacteria in crack treatment in concrete [22]. The bacteria will hibernate in the absence of proper nutrients and revive due to the presence of abundant sustenance. The self-healing concept is to heal by itself and regain its strength from low-level damage such as micro-cracks, which consists of specific types of material designed to perform for the long term as effective crack sealing and preferably during the total construction’s life time and over the course of its service life. Furthermore, from the perspective of human health and safety for a life-long duration of service period, Bacillus sphaericus is unlikely to cause human diseases, and it is safe to practice on the development of sustainable infrastructure. Therefore, this study evaluates how the biological processes affect the transport and mechanical properties of Portland Cement (PC) mortar. A biochemical healing agent is used as an additive for the mortar mixture to evaluate the self-healing potential of bacterial mortar to improve the sustainability and durability of mortar or concrete structures.

Factors influencing the activity of living organisms are expected in the biological processes, particularly nutrients, pH, and temperature. These environmental conditions are vital for the survival of bacteria-intended activity in the mortar or concrete structures. This research focuses on CaCO₃ precipitations, such as the sufficient growth nutrient requirement and hostile bacteria environment in high pH, which are yet to be materialized. The objectives are to develop a bio-based cement mortar (BBC mortar) and to determine microbial activity influences on immobilized Bacillus sphaericus LMG 22257 in terms of PC mortar properties cured in various curing media. For the Microbiologically Induced Calcite Precipitation (MICP) process, several different curing media are utilized as an alternative method to supply nutrients. These curing media include distilled water as normal water, run-off water (uncontrolled nutrient), and deposition medium (controlled nutrient). Meanwhile, the Diatomaceous Earth (D.E.) acts as an agent to immobilize the Bacillus sphaericus.

2. Materials and Methods

2.1. Portland Cement (PC)

The PC used as the main binder in this study complied with the requirements of ASTM C150/C150M-15 [23]. The physical properties of cement included a dark grey color, a bulk density of 1.561 g/cm³, fineness characterized by 85% passing a 40 µm sieve, and a specific gravity of 3.162. The chemical compositions of the PC are provided in

Table 1. Chemical compositions of the PC Mortar.

Composition	(%)
Al2O3	5.64
Fe2O3	4.07
CaO	63.42
MgO	2.08
SO3	2.92
Na2O + K2O	2.2
LOI	1.52

.

2.2. Distilled Water (D.W.)

Distilled water was used as the mixing water throughout the experiments. Distilled water was used to avoid any contamination in the mixtures.

2.3. Fine Aggregates

Fine aggregates from natural river sand with a maximum size of 4.75 mm were used as fine aggregates in the mortar mixtures. The natural river sand was wet-sieved, and materials passing through a 75 µm sieve were discarded to remove excess silts, impurities, and dust. Subsequently, the natural river sand was oven-dried for 24 ± 0.5 h at 105 ± 5 °C. The fine aggregate’s specific gravity and absorption coefficient were determined following the ASTM C33 [24], where the specific gravity obtained was 2.15, and the water absorption was 1.02%. The physical properties of natural river sand were tested using the ASTM C128 standard, which included a fineness modulus of 2.58 [25]. The physical properties of the fine aggregate are provided in

Table 2. Physical properties of fine aggregates.

Properties	Values
Bulk density, kg/m3	1672
Specific gravity	2.15
Water absorption (%)	1.02
Material finer than 75 µ (%)	0.5
Fineness modulus	2.58

.

2.4. Bacillus Sphaericus (B.S.)

Bacillus sphaericus was selected as it is commonly used for standard-forming calcite bacteria in concrete. Based on the previous research by Dick et al. [26], Bacillus sphaericus LMG 22257 in the freeze-dried strain was obtained from the Belgian Co-ordinated Collections of Microorganisms (BCCMTM), Ghent.

2.5. Diatomaceous Earth (D.E.)

Diatomaceous Earth, as shown in Figure 1, was used as an immobilization agent because the Bacillus sphaericus cell could not be directly added to concrete [27,28]. D.E. was purchased from B.G. Oil Sdn. Bhd. with the Sigma-Aldrich brand. The properties and chemical compositions of the D.E. are given in

Table 3. Physical properties and Chemical Composition of Diatomaceous Earth.

Physical Properties
Particle size	Less than 3 µm to more than 1 mm, but typically 10 to 200 µm
Uniformity coefficient	1.75
Bulk density	266 kg/m3
Color	Brownish-grey
BET surface area	15 m2/g
Specific gravity	2.33
Chemical composition	Percentage (%)
Silica	80–90%
Alumina	2–4%
Iron	0.5–2%
Calcium	5%
Sodium	5%
Type	Calcined

.

2.6. Mix Proportions and Specimens Preparation

The mixture proportions of the mortar mixtures used in this study are shown in

Table 4. Mix proportions of cement mortar specimens for the different mixtures.

Mixtures	Cement (g)	Sand (g)	Water (g)	D.E. (g)	B.S. (g)	Nutrients (g)
NC mortar	900	2700	450	0	0	0
BBC mortar	900	2700	225	45	225	69.75 *

* Nutrients included 2.25 g of yeast extract, 22.5 g of urea, and 45 g of Ca(NO₃)₂.4H₂O.

. Two types of cement–mortar mixtures, namely normal cement (NC) mortar and bio-based cement mortar (BBC), were produced. The specimens were prepared following ASTM 403 [29], where the mortar compositions constitute a cement: sand ratio of 1:3 with a water/binder ratio of 0.5.

These mortar mixtures were prepared to assess the effect of bio-based cement on compressive strength, porosity, water absorption, and permeability. A total of 90 pieces of 50 mm cube samples were prepared for the compressive strength test after 3, 7, 28, 56, and 90 days of curing. The samples were de-molded 24 h after casting. The specimens were cured in a closed container in a room subjected to different types of curing media with a temperature of 25 ± 2 °C and a relative humidity of 95% until the day of testing. All curing media were sterilized by autoclaving for 20 min at 120 °C before the curing process to ensure that the MICP process during this study was subjected to Bacillus sphaericus and not due to any outsourcing bacteria. Meanwhile, 60 pieces of cylindrical mortar samples with a diameter of 55 mm and thickness of 40 mm were used for porosity, water absorption, and permeability testing.

Deposition medium (D.M.), distilled water (D.W.), and run-off water (R.W.) were the three curing media used in this study. D.W. was employed as one of the curing media as it was free of contaminants and chemicals. The D.M. consisted of 20 gL⁻¹ of yeast extract, 20 gL⁻¹ of urea, and 79 gL⁻¹ of Ca(NO₃)₂.4H₂O prepared as a controlled nutrients medium. The proportion was calculated using previous studies [30]. Run-off water (R.W.) from the drain at Universiti Sains Malaysia was employed as a natural nutrient-rich media. Chemical analysis was used to determine the characteristics of the run-off water, which are reported in

Table 5. The characteristics of the Run-off water.

Parameters	Results
Color and appearance	Turbid
Total suspended solids (TSS) (mgL−1)	225
Total dissolved solids (TDS) (mgL−1)	968
pH	8.4
Oxygen consumed from N/8 KMnO4 in:
3 min (mgL−1)	24
4 h (mgL−1)	32
Biochemical oxygen demand	60
(5th day at 20C) (mgL−1)
Chemical oxygen demand (mgL−1)	121
Chlorides as Cl− (mgL−1)	248

.

2.7. Test Procedures

2.7.1. Compressive Strength

The cubes specimens of 50 mm were tested using a universal test machine (Model Shimadzu UH-F1000) according to ASTM C 109 [31] for a compressive strength test for each mix at each testing age.

2.7.2. Porosity and Absorption

The cylindrical mortar specimens were tested for porosity and absorption using the vacuum saturation apparatus [32]. First, the specimens were dried at 105 °C for 24 h in an oven. Then, the weight of the dried specimens was measured (W4). The samples were then placed in the vacuum saturation device for three hours at a vacuum pressure of one bar. Then, de-aired water was introduced under vacuum until the specimens were submerged at least one centimeter below the water level. The vacuum state was maintained for three hours after the introduction of de-aired water. After removing the vacuum, the samples were placed in the de-aired water for an additional 1 h to achieve full saturation. The specimens were then extracted from the water, and their surfaces were wiped with a dry cloth. The specimen weights in the air (W2) and water (W3) were determined. The following Equations (1) and (2) were used to calculate porosity (P) and water absorption (A), respectively.

$$P \left(\%\right)=\left.\left(\frac{W2-W4}{W2-W3} \right.\right)\ast 100$$

(1)

$$A \left(\%\right)=\left.\left(\frac{W2-W4}{W4} \right.\right)\ast 100$$

(2)

2.7.3. Water Permeability

The water permeability test was performed using cylindrical specimens with a diameter of 55 mm and 40 mm thickness. Before applying the water pressure on the specimens, the specimens were weighed. Water was run through the specimen for three hours at a pressure of three bars because water penetration on the top surface is required. The specimens were weighed to determine the differences in weight increment. The specimen was then cut in half, and the water penetration through the sample was measured. The depth of penetration was estimated as the average of three readings. The water permeability coefficient (Kw) was computed using Equation (3) [33,34,35,36].

$$Kw=\frac{d^{2}v }{2Th }$$

(3)

where:

d = the depth of water penetration, meter (m);

T = the time of penetration, second, (s);

h = the applied pressure, meter (m);

v = the total porosity.

Porosity v was calculated using Equation (4):

$$v=\frac{m }{Ad\rho }$$

(4)

where:

m = gain in mass, (kg);

A = cross-sectional area of specimen, meter (m²);

ρ = density of water, (1000 kg/m³).

2.7.4. Microstructure and Composition

The microstructural effect on BBC mortar cured in different curing media was assessed using Scanning electron microscopy (SEM). Meanwhile, the presence of elements on the BBC mortar specimens was determined by examining the SEM of freshly fractured samples with energy-dispersive X-ray spectrometry (EDX) analysis. Furthermore, the type of mineral in BBC mortar fluctuates significantly when exposed to varying environmental conditions. A variety of curing media can alter the efficiency and viability of the precipitation process. Calcite precipitation was measured with an X-ray Diffraction (XRD) instrument. The Shimadzu XD-D1 X-ray diffractometer was used to assess the mineral alterations and to identify the crystalline mineral. Specimens were air-dried and grinded using a pestle and mortar and the surfaces of the specimens were left smooth to prevent any disturbance from the XRD data. The sample was mounted on the aluminum holder and placed in the diffraction instrument with the angle scan ranging from 10° to 90° with a scan rate of 2° per minute.

3. Results and Discussions

3.1. Compressive Strength

3.1.1. Effect of BBC Mortar on Compressive Strength in Curing Period

The effect of BBC mortar on compressive strength, compared with a similar mix proportion of NC mortar, was studied. The immobilized Bacillus sphaericus was used as a self-healing agent in this investigation. To provide controlled and uncontrolled nutrient supplies for the healing agent, three curing media were used: deposition medium (D.M.), distilled water (D.W.), and run-off water (R.W.). For each curing media, the compressive strength was measured at 3, 7, 28, 56, and 90 days, and the results obtained are provided in

Table 6. Compressive strength of NC mortar and BBC mortar subjected to different curing media.

Curing Media	Mixed of Mortar I.D.	Curing Periods (Days/Compressive Strength (MPa)
		3 Days	7 Days	28 Days	56 Days	90 Days
Distilled water (D.W.)	NC-DW	23.0	26.5	33.0	33.5	41.5
	BBC-DW	26.5	28.0	35.0	36.5	43.5
Deposition water (D.M.)	NC-DM	20.5	25.5	32.5	33.0	40.0
	BBC-DM	29.0	34.0	37.5	42.5	48.0
Run-off water (R.W.)	NC-RW	24.5	26.0	32.0	34.0	40.5
	BBC-RW	34.0	39.5	45.5	46.0	58.0

. Any improvement in the strength of cubes specimens was observed due to the activity of Bacillus sphaericus in the cement mortar. In curing media of BBC-RW, no significant improvement in strength was noticed after 7 days up to 56 days, whereas it suddenly shot up at 90 days. This is believed to occur due to the bacterial growth phase [37,38]. The strength of the mortar cube specimens was examined using a universal testing machine with a loading rate of 21 MPa/min according to ASTM C 109. As described in Table 4, the different curing media did not significantly affect the compressive strength of NC mortar series among NC-DM and NC-RW mortar compared with NC-DW, except for the 3rd day, where the NC-DM showed slightly lower compressive strength.

3.1.2. Effect of BBC Mortar on Compressive Strength in Different Curing Media

Figure 2 shows the strength gradually increasing from the 3rd to 28th days, and the strength for all curing media seemed to increase slightly up to 56 days. A slight increase after 28 days was noticed in NC-DW and BBC-DW mortars due to a lack of nutrients, but a progressive increase in strength occurred after 56 days for BBC-DM mortar and BBC-RW mortar. The gradual increase in the strength of BBC-DM mortar over 90 days may be due to the continuous precipitation of CaCO₃ in the mortar cubes as there were sufficient nutrients from Bacillus sphaericus. This improvement was probably due to deposition of CaCO₃ on the microorganism cell surfaces and within the pore of cement-sand matrix, which plug the pores within the mortar [39]. The overall trend of an increase in compressive strength up to 90 days was attributed to the behavior of microbial cells within the cement mortar matrix and the influence of nutrient for survival. Furthermore, it was concluded that the increase in compressive strength was mainly due to consolidation of CaCO₃ that was induced by bacteria in the pores of the cement mortar cubes.

In the case of the BBC-RW mortar, there was a noticeable increment from 7 to 56 days, but there was a significant improvement in strength from 56 to 90 days. This is supposed to occur due to the new phase of Bacillus sphaericus due to the adaptation to a new environment [37,38]. The optimal bacteria population growth occurred during the log phase after 56 days. The microbes multiplied exponentially to the upper limit known as the carrying capacity. The population growth beyond this stage had insufficient nutrients, leading to a reduced growth rate in which the microbial death and new cell formation were balanced, eventually stabilizing the population. The phase is known as the stationary phase, where metabolic activities for surviving cells slow down. Waste and dead cells begin to accumulate, leading to the dead phase.

The Bacillus sphaericus growth phase directly reflects the strength improvement of BBC-RW. During the log phase, between 3 and 28 days, the compressive strength increased gradually due to considerable bacteria activity. During the stationary phase, a minimal improvement was recorded between 28 and 56 days. A sudden compressive strength improvement after 56 days related to the bacteria growth phase. In the next stage, during the exponential phase, the highest Bacillus sphaericus activity multiplied the cell, causing a gradual strength increment. A slight strength increment between 28 and 56 days was due to insufficient nutrients for survival during exponential bacteria multiplication, causing the bacteria to start dying. However, after the bacteria amount reduced to a certain number, allowing for sufficient nutrients and resumed bacterial activity, the strength was re-enhanced between 56 and 90 days.

The survival of both BBC-DM and BBC-RW was ensured by controlled nutrients obtained from deposition solution consisting of yeast extract, urea, and Ca (NO₃)₂.4H₂O, and uncontrolled nutrients from run-off water. BBC-RW specimens had a higher strength improvement than BBC-DM due to the increased nutrients in run-off water. Table 5 shows the chemical analysis of run-off water characteristics, which proved the presence of rich nutrient sources. Therefore, the bacteria obtained sufficient nutrients for activity, leading to increased strength. The utilization of bacteria to enhance the strength and durability through CaCO₃ precipitation in mortar is known as Microbiologically Induced Calcite Precipitation (MICP). The total dissolved solids and total suspended solids present in run-off water are significant for bacteria survival. Furthermore, less oxidation means that more oxygen is available to ensure the existence of bacteria in mortar.

A two-way analysis of variance (ANOVA) for the compressive strength test for concrete mixed with Bacillus sphaericus is summarized in

Table 7. Two-way analysis of variance (ANOVA) results for compressive strength test for concrete mixed with Bacillus sphaericus.

ANOVA
Source of Variation	Sum off Squares	df	MS	FCAL	p-Value	Partial Eta Squared
Curing days	1289.87	4	322.46	279.39	1.26 × 10−8	0.993
Curing media	148.52	2	74.26	64.34	1.17 × 10−5	0.941
Error	9.233	8	1.154
Total	1447.62	14

. From the partial eta squared results, it can be concluded that the effect size for curing days was very large, while the effect size for curing media was small for the compressive strength. These results matched the p-values shown in the output of the ANOVA in Table 7. The p-value for the effect of curing days (1.26 × 10⁻⁸) was much smaller than the p-value for the effect of curing media (1.17 × 10⁻⁵), which indicates that the effect of curing days was much more significant in compressive strength for self-healing Bacillus sphaericus in cement mortar. The results show a comparison with the previous researcher, where the effect of curing days on the compressive strength was statistically significant for curing days, while the effect of Bacillus coagulans suspension density on the compressive strength was not statistically significant [40].

3.2. Water Absorption and Porosity

3.2.1. Effect of BBC Mortar on Absorption and Porosity in Curing Period

The effect of BBC mortar on water absorption and porosity was also investigated. The purpose was to understand the terms of pore formation based on exposure to the different curing media that act as a nutrient-supplying medium for the metabolic activities of bacteria. According to a previous study [41], the metabolic activity of the bacteria caused calcium carbonate to precipitate and decreased water absorption and porosity. The water absorption and porosity percentage of BBC mortar in different curing media are shown in

Table 8. Water absorption of NC and BBC mortars subjected to different media (%).

Curing Media	Mixed of Mortar I.D.	Testing Ages (Days)
		3 Days	7 Days	28 Days	56 Days	90 Days
Distilled water (D.W.)	NC-DW	8.4	7.9	7.8	7.7	7.6
	BBC-DW	7.8	6.6	5.4	4.3	4.2
Deposition water (D.M.)	NC-DM	8.9	8.7	8.4	7.9	7.8
	BBC-DM	6.5	5.2	3.8	2.8	2.6
Run-of-water (R.W.)	NC-RW	8.4	8.2	7.8	7.7	7.6
	BBC-RW	6.1	4.9	3.5	2.7	2.5

and

Table 9. Water porosity of NC and BBC mortars subjected to different media (%).

Curing Media	Mixed of Mortar I.D.	Testing Ages (Days)
		3	7	28	56	90
Distilled water (D.W.)	NC-DW	15.1	13.5	13.2	12.8	12.9
	BBC-DW	14.5	12.0	10.0	7.9	7.8
Deposition water (D.M.)	NC-DM	16.5	16.2	15.0	14.0	13.6
	BBC-DM	12.0	9.0	6.5	5.1	4.7
Run-of-water (R.W.)	NC-RW	15.8	14.6	14.3	13.9	13.4
	BBC-RW	11.2	8.8	6.0	4.6	4.5

, respectively. The results portray that the inclusion of a self-healing agent significantly influenced the BBC mortar. The absorption and porosity of the NC mortar series were not much different among NC-DM and NC-RW mortar compared with NC-DW, so the influence of the various curing media in the case of the NC mortars was not significant [37,38].

The effect of BBC mortar on water absorption is described in Figure 3, and the effect on porosity is described in Figure 4 for the different curing media. A decrease in water absorption and porosity was observed as the curing period increased for all curing media. The reduction percentage was found to vary for the different samples. BBC reduced water absorption and porosity by incorporating a self-healing agent compared to NC specimens. For curing periods of 3, 7, 28, 56, and 90 days, water absorption and porosity in BBC-RW decreased by 27.1%, 42.3%, 56.3%, 64.1%, and 66.1%, and reduced by 22.2%, 37.5% 57.0%, 64.7%, and 65.4% for BBC-DW mortar, respectively, compared to the NC mortar specimens.

3.2.2. Effect of BBC Mortar on Absorption and Porosity in Different Curing Media

Different curing media affecting the water absorption and porosity of BBC mortar are described in Figure 5 and Figure 6. The results showed that the various curing media directly impacted the water absorption and porosity of BBC mortar. There was a significant difference in the reduction in water absorption percentage between BBC-RW mortar and BBC-DM mortar with BBC-DW mortar. In contrast, it is substantial that BBC-RW mortar had a more significant decrease in water absorption and porosity percentage. The respective water absorption and porosity of BBC-RW at three days were 27.4% and 28.9% before it further reduced to 65.4% and 66.3% after 90 days. These results showed a more significant water absorption and porosity reduction than D.M. and D.W. curing media. BBC-DM exhibited a similar trend with a respective water absorption and porosity at three days of 26.9% and 27.3% before reducing to 66.7% and 65.4% after 90 days. However, BBC-DW produced a higher water absorption and porosity than the other curing media at 7.5% and 3.8%, respectively, at an early stage, and continuously reduced to 45.0% and 39.9%, respectively, in the long term. In conclusion, the water absorption and porosity rate of BBC-DW were higher than those of both BBC-RW and BBC-DM.

The self-healing agent involvement positively affected mortar properties by reducing the water absorption and porosity of cement mortar. The absorption and porosity of BBC mortar decreased, as shown in Figure 5 and Figure 6. The results were caused by the curing period and sufficient nutrients during bacterial metabolic activities precipitating CaCO₃. CaCO₃ fills the pores, thus reducing the porosity of mortar and possibly blocking the water penetration path. Calcite produced from bacterial metabolic activity clogs the pores on the surface, hence reducing water absorption and porosity.

A two-way analysis of variance (ANOVA) for the water absorption test for concrete mixed with Bacillus sphaericus is summarized in

Table 10. Two-way analysis of variance (ANOVA) results for water absorption test for concrete mixed with Bacillus sphaericus.

ANOVA
Source of Variation	Sum off Squares	df	MS	FCAL	p-Value	Partial Eta Squared
Curing days	22.27	4	5.668	279.90	1.26 × 10−8	0.993
Curing media	3.51	2	1.752	86.54	3.81 × 10−6	0.956
Error	0.162	8	0.02
Total	25.94	14

. From the partial eta squared results, it can be concluded that the effect size for curing days was very large, while the effect size for curing media was not too massive for the water absorption test. These results matched the p-values shown in the output of the ANOVA of Table 10. The p-value for the effect of curing days (1.26 × 10⁻⁸ ) was much smaller than the p-value for the effect of curing media (3.81 × 10⁻⁶ ), which indicates that effect of curing days was much more significant at water absorption for self-healing Bacillus sphaericus in cement mortar.

A two-way analysis of variance (ANOVA) for the water porosity test for concrete mixed with Bacillus sphaericus is summarized in

Table 11. Two-way analysis of variance (ANOVA) results for water porosity test for concrete mixed with Bacillus sphaericus.

ANOVA
Source of Variation	Sum off Squares	df	MS	FCAL	p-Value	Partial Eta Squared
Curing days	92.641	4	23.16	256.387	1.78 × 10−8	0.992
Curing media	7.981	2	3.990	44.173	4.75 × 10−5	0.917
Error	0.723	8	0.09
Total	101.345	14

. From the partial eta squared results, it can be concluded that the effect size for curing days was very large, while the effect size for curing media was smaller than the compressive strength for the water porosity test. These results matched the p-Values shown in the output of the ANOVA of Table 11. The p-Value for the effect of curing days (1.78 × 10⁻⁸ ) was much smaller than the p-Value for the effect of curing media (4.75 × 10⁻⁵ ), which indicates that effect of curing days was much more significant at water porosity for self-healing Bacillus sphaericus in cement mortar.

Other researchers found that there was a maximum improvement of 22% in compressive strength, and a reduction in water absorption by a factor of four was observed with 10⁵ cells/mL of Sporoscarcina pasteurii bacteria [42]. Their results showed that an increase in compressive strength was brought about as a result of deposition on the cell surfaces of the bacteria that lived within the pores. In general, through its self-healing function, BBC concrete mortar gained in both strength and durability when Bacillus sphaericus was used in this experiment.

3.3. Water Permeability

3.3.1. Effect of BBC Mortar on Water Permeability in Different Curing Periods

The evaluation of mortar quality is necessary for service life and proper maintenance. In terms of durability, the permeability of mortar is the most important of its performance. Penetration of liquids and gases such as carbon dioxide, chloride ions, and water are the primary causes of harmful effects in mortar. The pore structure of the mortar governs its penetration into the mortar. Thus, water permeability can indicate the ease with which liquid can penetrate the mortar. In this investigation, the resistance of BBC mortar was tested by penetrating deleterious elements. The purpose was to determine the efficiency of BBC mortar to resist environmental conditions. The effects of BBC mortar on water permeability were evaluated in terms of the water permeability coefficient. The water permeability of NC and BBC mortar in different curing media is shown in Table 8 and the water permeability of the BBC mortar effect is plotted in Figure 7.

The water permeability of BBC mortar using various curing media was compared to NC mortar cured with the same curing media. The results showed that the water permeability of BBC significantly dropped in 7 days, slowly decreased to 28 days after the water exposure, and remained constant afterward. As described in Table 11,

Table 12. Water permeability coefficient (×10⁻¹² m/s).

Curing Media	Mixed of Mortar I.D.	Testing Ages (Days)
		3	7	28	56	90
Distilled water (D.W.)	NC-DW	8.31	7.84	3.57	3.25	3.04
	BBC-DW	7.13	5.36	2.38	2.03	1.85
Deposition water (D.M.)	NC-DM	8.14	6.46	3.24	3.2	3.17
	BBC-DM	6.52	4.25	2.16	1.86	1.54
Run-of-water (R.W.)	NC-RW	8.09	6.33	3.16	3.04	2.99
	BBC-RW	6.01	3.12	1.97	1.32	1.17

, Table 13, the permeability decreased as the curing time increased in all curing media for both samples, with different reduction percentages. The respective reduction in water permeability coefficient of BBC mortar cured in distilled water (D.W.) for 3, 7, 28, 56, and 90 days was 14.2%, 31.6%, 33.3%, 37.5%, and 39.1%. The samples cured in deposition medium (D.M.) and run-off water (R.W.) had a very positive effect on water permeability at an early stage, short term, and long term, where the coefficients of water permeability were decreasing in the range of 19.9–25.7%, 34.2–50.7%, 33.3–37.7%, 41.9–56.6%, and 51.4–60.9%, after 3, 7, 28, 56, and 90 days, respectively. It is noticeable that with prolonged curing time, there was a significant decrease in the coefficient of water permeability of the mortars. Figure 7 illustrate the reduction in permeability of BBC mortar due to the curing period and different curing media. The bacterial metabolic activity produced calcite, which clogs the surface pores, decreasing water permeability. Similarly, as discussed earlier, sufficient nutrients during the bacterial metabolic activities precipitate the CaCO₃, which fills and reduces mortar pores, causing blockage of the water penetration path [37,38,39].

3.3.2. Effect of BBC Mortar on Water Permeability in Different Curing Media

The coefficient of water permeability of BBC mortar in various curing media is shown in Figure 8. Curing media application directly affected water permeability. Compared to D.W. media, D.M. and R.W. media exhibited a positive water permeability coefficient of BBC mortar at the early stage, short-term, and long-term. Different curing media with different nutrients affected the characteristic of water permeability of BBC mortar. Water permeability coefficients obtained after the 90-day curing period were 1.54 × 10⁻¹² m/s for BBC-DM, 1.17 × 10⁻¹² m/s for BBC-RW mortar, and 1.85 × 10⁻¹² m/s for BBC-DW mortar. Therefore, D.M. and R.W. curing media had the highest water permeability reduction.

In conclusion, using a self-healing agent in cement mortar reduces its permeability. Self-healing occurs after a certain period due to sufficient nutrients for BBC mortar samples. This advantageous effect depends on the amount of CaCO₃ precipitation relating to bacterial metabolic activities. The bacteria with higher ureolytic and carbonatogenesis activity curing in media containing nutrients produce more CaCO₃ to fill the pores. Self-healing performance improves significantly after seven days of curing, possibly due to additional hydration of the un-hydrated cementitious components. Thus, more precipitation is possible in the early stage as more voids are present, which enhances the minerals and media reactions to produce CaCO₃.

3.3.3. Microstructure and Composition

Energy-dispersive X-ray spectrometry (EDX) was used to quantitatively determine the elements with atomic numbers. Using scanning electron microscopy (SEM) for microstructure analysis on BBC mortar with different curing media, selected specimens were compared using scanning electron microscopy (SEM). Figure 9 shows the surface of BBC mortar specimens with the presence of carbonate crystals after being subjected to different curing media. Figure 9a shows SEM images of BBC mortar cured in distilled water. The SEM magnification was set between 100 and 20,000 times. SEM could clearly observe the whitish precipitates or organic material through the mixture on sample surfaces. Immobilized Bacillus sphaericus precipitates were probably an organic compound due to their high organic carbon content. Main elements such as calcium (Ca) 21.91%, oxygen (O) 46.02%, silica (Si) 12.63%, carbon (C) 15.32%, aluminum (Al) 2.74%, and magnesium (Mg) 1.38% were determined through EDX analysis.

Figure 9b shows SEM micrographs of BBC mortar surface particles cured in a deposition medium. The formation of white lumps on the surface was evident, indicating calcite compounds. These new materials, calcite precipitates of BBC mortar rich in organic carbon, filled the pores in the cement matrix. These highly potential nutrient-derived compounds were urea, yeast extract, and calcium nitrate. The formation of these compounds is expected to enhance the strength gained from the Bacillus sphaericus activity. From the previous studies by Norfaniza et al. [43], the results are supported by the determination of primary elements, namely calcium (Ca) 45.7%, oxygen (O) 35.48%, silica (Si) 8.22%, carbon (C) 7.86%, aluminum (Al) 11.87%, and magnesium (Mg) 0.87% through EDX analysis.

Figure 9c illustrates a large variety of crystal layers on the BBC mortar surface cured in R.W. Two main shapes of crystalline CaCO₃, namely lamellar rhombohedral and needle-like shapes, were visible on the SEM images. The former shape produces the most stable characteristic of calcium carbonate in calcite [44]. Meanwhile, the latter was a vaterite type, a metastable form of calcium carbonate [45]. The absorption of organic and inorganic matter inhibited or altered the crystal growth to certain crystallographic planes of the growing crystal. In addition, crystal morphology differences were probably due to the actual urease activity levels linked to cell activity.

The existence of several crystal types proved the carbonate precipitation of bacterial mediation. The density and strength of the particles can be observed due to the interlocking arrangement of calcite materials. The CaCO₃ precipitate can be observed based on bacteria activity in certain curing media. The results show that Bacillus sphaericus assisted calcite precipitation in mortar specimens [46]. EDX analysis supported evidence that the main elements obtained were calcium (Ca) 49.62%, oxygen (O) 32.24%, silica (Si) 9.07%, carbon (C) 6.32%, aluminum (Al) 1.95%, and magnesium (Mg) 0.80%. In conclusion, BBC mortar in R.W. curing media produced more CaCO₃ precipitates than other curing media.

The direct influence of curing media on the amount of calcite precipitation on the surface was very significant, as observed by the SEM micrograph. The results were proven quantitatively by the elements with an atomic number through EDX analysis. BBC mortar in D.M. and R.W. curing media had faster biomineralization than in D.W. The biomineralization that occurred in D.W. was caused by the dissolution of pore solution at atmospheric CO₂ and CaCO₃ formation from portlandite. However, biomineralization in D.M. and R.W. occurred through microbiology hydrolysis of urea using an external nutrients supply from calcium ions, producing carbonate ions.

Biomineralization is technically a two-component coating system with pore-blocking capabilities [12]. The incorporation of Bacillus sphaericus resulted in pores plugging, as evident from water absorption analysis and CaCO₃ formation on the surface. The biological process of calcite formation occurred in a supersaturation condition caused by nutrient supply. Heterogenous CaCO₃ precipitation occurred in the internal pores as specimens were exposed to the solution [13]. The presence of carbonate crystals on the surface, especially inside the porous matrix, decreased the permeability of BBC mortar.

The MICP procedure was used to identify and categorize the mineralogical changes brought on by the various curing mediums. Furthermore, the mineralogical alterations of BBC and any newly produced mineral due to the different curing media were determined by X-ray diffraction (XRD). Figure 10 depicts the results of a series of XRD analyses performed to investigate the cement matrix’s mineralogical modification and compound formation in response to exposure to various curing conditions. Different curing media did not appear to add a new peak to the XRD spectrum of BBCM, and they did not alter the peaks’ characteristics of the two theta scales or their relative positions. The results demonstrated the efficiency of various curing media in maintaining the pozzolanic reaction during hydration and the lack of inorganic material in the curing media.

The existence of two prominent peaks, at 18.1° and 23.3°, verified the production of ettringite, which is typically caused by the interaction of C₃A with gypsum in the presence of CaCO₃ during the earliest stage of setting. All samples showed ettringite production, although the BBCM-DM had the most pronounced peak. There was a less prominent peak in the BBC-DW and BBC-RW. The free hydroxyl ions present in the cement matrix after curing in D.M. created a conducive environment for ettringite production [8]. There were several prominent primary peaks at two theta scales of 21°, 26.8°, 29.5°, 39.2°, 48.4°, 50.1°, and 60° in XRD spectra, indicating the presence of calcite. The strain’s effectiveness in the precipitation of CaCO₃ by producing carbonate ions and using calcium ions was demonstrated by a notable rise in the strength of peaks attributable to calcite for the BBC matrix cured in RW. Minimal calcite precipitation and CH production were observed in the XRD spectra for D.W. curing. X-ray diffraction analysis showed that nutrients played a significant role in cement mortar. D.M. and R.W. curing media appeared more efficient than the others at transforming CH into C-S-H and CaCO₃. The ability of the strain to precipitate a high amount of CH at 27.7°, 40.4°, and 47.2° in the presence of D.M. and R.W. was indicative of the usual reactive chemistry that prevailed in the cement mortar, suggesting that there was enough buffer to facilitate further hydration of the cement.

3.4. Comparative Study

Most studies have focused on bacterial spore germination and precipitation; however, there is still a significant knowledge gap in regard to the kinetics characteristics and biochemical aspects of the ureolysis process. This research aimed to identify the factors contributing to CaCO₃’s optimal precipitation using different curing media. Compressive strength and transport qualities are major determinants of healing efficacy; however, the results do not consider the impact of nutrients on bacterial viability and their ability to precipitate. There is still a lack of knowledge regarding the effect of providing nutrients to the bacteria that incorporate with the concrete and mortar. To ensure accurate interpretation of the nutrient delivery method, the curing procedure was tested using a variety of mediums. In light of these problems, it is clear that more research into the mechanism of microbial-induced calcite precipitation is required, specifically into its kinetics characteristics and biochemical properties, in order to develop a product that is less harmful to the environment. Biomineralization research draws from several fields of study and many types of expertise. Although it has shown promise in various areas, there are still many research and development problems that must be answered before it can be used on a large basis in the commercial market.

Bacillus group bacteria were used for urease production and calcite precipitation. As the concrete matrix is oxygenic due to oxygen ingress, the bacteria must be oxygen-brilliant, resistant to high calcium ion concentration, and able to produce more CaCO₃. Bacillus bacteria were non-pathogenic in high-alkaline environments. The significant findings by other researchers using the bacteria species B. sphaericus and their potential for biomineralization in concrete/mortar are presented in Table 13.

Table 13. Recent findings of bacteria B. Sphaericus population and their potential for biomineralization.

Proposed Mechanism	Major Findings	Reference
Silica sol-incorporated urease metabolism	Incorporation with a calcium source; Effective in sealing cracks of 0.01 mm–0.6 mm; Reduced concrete permeability.	[46]
Encapsulation of bacteria, nutrients, and PU in the glass rod	The higher the concentration of microbes, the greater the precipitation; PU contributed to an increase in strength.	[47]
Autonomous healing-microbial-induced calcite precipitation (MICP)	Improved mortar compressive strength and durability; Reduced mortar water porosity, absorption, and permeability; Bio-based cement (BBC) in runoff water curing had a 40% enhancement in strength compared to normal curing (deposition water); SEM analysis revealed calcite crystals in BBC mortar, proving calcite exists in calcium carbonate form.	This study

Table 13. Recent findings of bacteria B. Sphaericus population and their potential for biomineralization.

Proposed Mechanism	Major Findings	Reference
Silica sol-incorporated urease metabolism	Incorporation with a calcium source; Effective in sealing cracks of 0.01 mm–0.6 mm; Reduced concrete permeability.	[46]
Encapsulation of bacteria, nutrients, and PU in the glass rod	The higher the concentration of microbes, the greater the precipitation; PU contributed to an increase in strength.	[47]
Autonomous healing-microbial-induced calcite precipitation (MICP)	Improved mortar compressive strength and durability; Reduced mortar water porosity, absorption, and permeability; Bio-based cement (BBC) in runoff water curing had a 40% enhancement in strength compared to normal curing (deposition water); SEM analysis revealed calcite crystals in BBC mortar, proving calcite exists in calcium carbonate form.	This study

However, the microbial cementation process is typically slower than chemical approaches, which is one of the main drawbacks of the microbial cementation method. As microbial activity is affected by a wide range of environmental factors, including temperature, pH, concentrations of donors and acceptors of electrons, concentrations, diffusion of rare nutrients, and metabolic factors, this technique is more intricate than its chemical counterpart. Inconvenient application procedures, the potential for the growth of other unwanted microbes due to the repeated application of nutrients, and the requirement of in-depth microbial ecology studies to ascertain the effects of introducing new bacteria are some of the other issues when using calcinogenic microbes. However, this technique is hampered by the high cost of using laboratory-grade nutrient sources in real-world applications.

4. Conclusions

Bacillus Sphaericus LMG 2257-precipitated calcium carbonate significantly enhances compressive strength, improving BBC mortar’s mechanical, durability, and microstructure properties. Reduced mean expansion and improved concrete durability performances result from a higher matrix strength for the BBC-DM and BBC-RW series. Self-healing agents added to cement mortar have been shown to increase surface absorption initially and over time, regardless of the curing medium used. The initial water absorption of BBC specimens was noticeably lower than that of NC. BBC mortar cured in D.M. and R.W. produced the highest improvement, most probably due to CaCO₃ deposition on cell surfaces and internal pores of microorganisms during the MICP process. The findings suggested the existence of rich organic compounds throughout the curing process. Sufficient nutrients during bacterial activity were shown to improve the characteristics of BBC mortar because pores were filled with the production of CaCO₃, thus resulting in a decrease in water absorption and permeability. CaCO₃ formation was firmly plugged into pores, as shown in the SEM micrographs earlier. SEM analysis revealed distinct calcite crystals in BBC mortar samples, indicated by the calcium amount in all BBC mortar samples, proving that calcite existed in calcium carbonate form. X-ray diffraction analysis showed that nutrients played a significant role in cement mortar. D.M. and R.W. curing media appeared more efficient than others at transforming CH into C-S-H and CaCO₃. Bacteria associated with crystalline calcite and served as nucleation sites during the mineralization process. Calcite crystal growth in BBC mortar samples from various curing media was analyzed. BBC-DW mortar samples possessed fewer crystal growths in the matrix than those cured in nutrient media. The current work demonstrates that this approach has great promise as a novel biological metabolic product that can be applied in the sewerage and drainage system, as this study has proven that run-off water is able to supply sufficient nutrients to bacteria for the biomineralization process to produce CaCO₃. At once, it may reduce the maintenance and rehabilitation cost. Further research can be carried out with the various species of bacteria and comparing the performance concerning healing efficiency, enhancement of durability, practical feasibility in the service period, and treatment under different aggressive environments.