Effect of a Bacterial Consortium on the Mechanical and Durability Properties of Self-Healing Concrete at Different Water–Cement Ratios

Abstract

Concrete, when used in construction, is prone to internal micro cracks that compromise its strength, flexibility, durability and lifespan. To address this, self-healing concrete technologies using microbial-induced calcium carbonate precipitation (MICP) have gained significant attention. The objective of this study was to focus on the preparation of a bacterial consortium (BV) composed of Bacillus cereus and Vibrio natriegens, selected for their specific characteristics to produce calcium carbonate under alkaline conditions. These bacterial strains with nutrients were added in optimised proportions to the concrete mixes and evaluated their healing potential. The effectiveness of the bacterial consortium on the self-healing potential of concrete was investigated. Similarly, the performance of this consortium was assessed across three different water–cement (w/c) ratios: 0.40, 0.45, and 0.50. These variations were selected to investigate the influence of moisture availability and mixed porosity on bacterial activation and crack healing efficiency. Mechanical tests like flexural strength, split tensile strength and compressive strength were performed to assess the structural recovery. Durability tests such as acid resistance, water absorption, and non-destructive tests like ultrasonic pulse velocity were also performed. Based on these investigations, a 0.40 w/c ratio of bacterial consortia (0.40 BV) showed the best performance. These results indicate that the bacterial consortium can significantly improve the self-healing properties of concrete, particularly at low w/c ratios.

1. Introduction

Concrete is a versatile material widely used in construction projects around the world. By altering its characteristics and components, concrete can be adapted for various applications [1,2,3]. While cement concrete exhibits high compressive strength, it has relatively low tensile strength. Loading conditions, chemical reactions, and environmental stresses can compromise its performance [4,5]. When the tensile stress exceeds the concrete’s tensile capacity, cracks form, leading to a decline in its structural integrity [6]. Cracks are failures that can occur in parts of a structure either before or after being subjected to service loads. In some cases, they lead to sudden structural collapse due to unforeseen circumstances. Certain cracks can be detected and managed in advance [7,8,9,10], while others remain unnoticed until a significant structural issue arises. Once cracking begins, the overall durability and reliability of the concrete structure deteriorate [11,12]. To address this issue, engineers have developed self-healing concrete. One innovative type is bio-concrete, which uses microorganisms to facilitate the self-healing process [13,14,15]. Bio-based concrete is manufactured in the same way as conventional concrete. The treatment involves introducing harmless bacteria that are activated upon contact with water [16,17]. Bacterial plaster is particularly effective for crack repair, as it promotes the microbial-induced precipitation of calcium carbonate (CaCO₃), sealing the cracks. One major research challenge is identifying suitable bacterial strains that can survive and remain active in the harsh environment of concrete over long periods [18,19,20]. Concrete is a very dry and inhospitable material, making it difficult for some bacteria to survive in such an environment [21].

Bio mineralization—the process by which microorganisms synthesise minerals—has emerged as a promising approach for concrete repair. This method is known for its long-term, rapid, and active crack healing capabilities on concrete surfaces [22,23,24,25,26]. It offers strong adhesion and compatibility with aged concrete components, while also being environmentally friendly and non-polluting. The bio mineralization technique has shown significant potential due to its positive effects on compressive strength and other mechanical properties, such as tensile and flexural strength. These improvements result from the filling of pores in the concrete with calcium carbonate (CaCO₃) deposits [27,28,29]. Self-healing concrete (SHC) is primarily used to mend cracks and enhance the durability of concrete structures. Various types of self-healing processes exist, including natural, chemical, and biological mechanisms. Among these, biological processes involving mineral precipitation have received considerable attention [30,31].

One particularly effective genus of bacteria used in this context is Bacillus, which can thrive in alkaline environments like concrete. These bacteria can survive for years without food or oxygen by remaining dormant until activated by moisture entering the cracks [32,33,34,35,36]. When calcium lactate is added as a nutrient during mixing, it provides a food source for the bacteria, enabling them to initiate healing when cracks occur. The bacteria and nutrients are incorporated into fresh concrete through various techniques [37,38,39,40,41]. Once moisture infiltrates the cracks, the bacteria become active, consume the lactate, and begin to multiply. This process results in the precipitation of limestone or calcite, effectively sealing the cracks [42,43]. This method is highly desirable, as mineral precipitation via microbial activity is natural, sustainable, and non-toxic. Damaged concrete specimens can be reinforced using this technique [44,45,46,47]. The bacterial activity alters the chemical composition of the surrounding solution, leading to super saturation and subsequent mineral precipitation [48,49]. When such biological concepts are applied to construction materials, a new class of material bacterial concrete is created. While cracking does not directly cause infrastructure failure, it allows aggressive environmental agents to penetrate and initiate deterioration [50]. The inability to block such external agents leads to structural damage and the weakening of load-bearing elements. Therefore, developing concrete with self-healing properties is crucial for extending the service life of infrastructure [51,52].

Self-healing concrete has the potential to autonomously cure cracks and reduce the need for costly repairs and inspections, without requiring external intervention. It minimises structural deterioration and reinforcement corrosion, thereby enhancing durability and reducing maintenance costs. Developing an effective method to combine two types of bacteria to address both minor and major concrete and mortar issues could significantly reduce repair expenses, while also being environmentally and economically beneficial [53,54]. In this study, bio mineralization was applied to concrete to enhance its performance. The investigation focused on assessing the impact of bacterial concrete on various mechanical, strength, and microstructural properties.

2. Materials and Methods

2.1. Cement

A binder is a construction material that adheres, hardens, and sets to other substances to tight them together. In this study, PPC was purchased from the sreekumar stores, a local merchant (chathamangalam, Kozhikode, India) and the chemical compositions of cement are shown in

Table 1. Elemental composition of cement.

Components	SiO2	Al2O3	Fe2O3	CaO	MgO	SO3
%	21.28	5.60	2.85	64.18	1.66	2.57

and physical properties of cement in

Table 2. Physical properties of cement.

Properties	Values
Specific Gravity	2.76
Consistency	31%
Fineness	0.09%
Initial Setting Time	60 min
Final Setting Time	220 min
Compressive strength 3rd day	18.78
Compressive strength 3rd day	25.96
Compressive strength 3rd day	35.01

. Cement, a fine material obtained from lime and clay, is one such binder. It is typically not used on its own but rather to bind fine and coarse aggregates, and the cement is shown in Figure 1. Concrete, which incorporates with cement, is the most widely used construction material in the world, featuring in more than 25% of building projects.

2.2. Fine Aggregate

The fine aggregate used in this study composed natural sand, manufactured sand, or a combination of both. The physical property of fine aggregate (

Table 3. (a) Physical properties of fine aggregates. (b) Sieve analysis.

(a)
Physical Properties	Value
Bulk Density	1.69
Specific Gravity	2.60
Water Absorption	3.85%
Void Ratio	0.5
Fineness Modulus	2.84
Coefficient of Uniformity	6.43
Grade of Fine Aggregate	Grade II
(b)
Sieve Size (mm)	Percentage (%) Passing (Approx)
4.75	100
2.36	90
1.18	70
0.6	45
0.3	25
0.15	10
0.075	0–5

) selection was carried out in accordance with the criteria mentioned in IS 2386-2016 [55]. The fine aggregate should not have more than 45% retained between any two consecutive sieves. The particle size distribution curves were shown in Figure 2.

2.3. Coarse Aggregate

The qualities of coarse aggregate play a significant impact in determining the strength of concrete, with coarse aggregate regarded as a primary component of the mix. In accordance with IS 2386 guidelines and ASTM C33 [56] requirements, coarse aggregate may consist of air-cooled blast furnace slag, gravel, crushed hydraulic cement concrete, crushed stone, or a combination thereof. In this study, 12.5 mm aggregates were selected as the coarse aggregate material for concrete preparation. The physical properties of coarse aggregate are displayed in

Table 4. Physical properties of coarse aggregate.

Physical Properties	Value
Bulk Density	1.54
Specific Gravity	2.53
Water Absorption	1%
Void Ratio	0.78
Fineness Modulus	6.038

.

2.4. Admixture

In this study, Auramix 400 super plasticizer was used. It is a chloride-free super softening additive material based on synthetic polymers. It comes as a brown liquid that dissolves quickly in water, provide significant water reduction, and helps in cement dispersion. This leads to a substantial strength gain while preserving workability, making installation process easier.

2.5. Water

Water is the least expensive yet one of the most critical ingredients in concrete preparation. For this study, we used sterile water (free from harmful contaminants such as acids, alkalis, tar, and other deleterious substances). Potable water is typically used for various construction activities, including mixing and curing. According to IS 456:2000, the pH value of the water must be at least 6.

2.6. Bacterial Agent

The bacterial strains Bacillus cereus (ATCC-14579) and Vibrio natriegens (ATCC-14048) were used in this study. The cultures were procured as a kind gift from Prof. H. Shakila, Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamilnadu (India). Bacillus cereus, a non-pathogenic bacteria normally present in food, soil, and marine sponges [57], is rod-shaped, spore-producing, Gram-positive, and motile and can be confirmed by the predefined protocol. Vibrio natriegens is a non-toxigenic marine bacterium with a double growth rate within a short duration and can survive in hostile environment to enhance pH tolerance. The growth of V. natriegens requires additional amount of sodium chloride in LB medium. V. natriegens is rod-shaped, Gram-negative, has no spore formation activity and the novel host has no reports available for a self-healing mechanism with cement mortar. Figure 3 represents the cultivation of bacteria strains of Bacillus cereus and Vibrio natriegens.

2.6.1. Cultivation of B. cereus

Bacillus cereus was cultured using Luria-Bertani (LB) broth under aseptic conditions. A single colony of B. cereus was initially isolated from a nutrient agar plate and transferred into 5 mL of sterile LB broth in a 15 mL culture tube. The culture was incubated at 30 ± 2 °C under shaking conditions at 150 rpm for 16–18 h to obtain an actively growing overnight culture. For larger-scale cultivation, 1% (v/v) of the overnight culture was inoculated into fresh LB broth (e.g., 100 mL in a 250 mL Erlenmeyer flask) and incubated under the same conditions. The bacterial growth was monitored by measuring the optical density at 600 nm (OD₆₀₀) using a spectrophotometer. The culture was harvested at the desired growth phase, typically mid-log phase (OD₆₀₀ ~ 0.6–0.8), for further experimental use. All procedures were performed under sterile conditions to avoid contamination.

2.6.2. Cultivation of V. natriegens

The cultivation of V. natriegens procedure was done with slight modification [58]. The culture was recovered from the glycerol stock and the bacteria were initially cultivated in the preculture using LB medium. The LB medium consists of 5 g L⁻¹ yeast extract, 20 g L⁻¹ NaCl, and 10 g L⁻¹ tryptone with pH 7.0 and sterilised using autoclave under standard procedures. For this culture preparation, 10 μL of bacterial suspension was collected from glycerol stock and inoculated into 10 mL of sterile LB medium and incubated at 27 °C in a shaking incubator with 160 rpm for 5–6 h. This preculture was used to prepare fresh culture for this study.

2.6.3. Preparation of B. cereus and V. natriegens Consortia Preparation

In this study, the bacterial consortia (Figure 4) were prepared using Bacillus cereus and Vibrio natriegens in LB medium for concrete applications. Bacterial cell densities were examined by measuring optical density (OD) and bacterial concentration was determined using the serial dilution method. The concentration of 10⁴ was fixed for V. natriegens and 10⁶ for Bacillus cereus. The compatibility between the two strains were examined by streaking the strains in the LB agar plate, incubated and kept at optimum temperature for 12 h. Based on the Complexometric titration method, the optimal mixing ratio was fixed at 1:1.25 [45]. The most effective bacterial consortia preparation was analysed to be 3% based on bacterial viability assessments and compressive strength.

3. Experimental Investigation

An experimental investigation (Figure 5) was performed to evaluate the effect of incorporating a bacterial consortium into concrete by replacing water content with a bacterial solution. Concrete cubes of M30 grade were prepared using a mix proportion of 1:1.9:3.58 and a water–cement (w/c) ratio of 0.45 as per the mix design. As per IS 10262:2019, the lower and upper limits were set to maintain the assessment of microbial activity and mechanical performance, without introducing excessive variation in hydration or harming bacteria. For this purpose, 150 × 150 × 150 mm concrete cubes were cast using both normal water and a bacterial solution composed of a combination of Bacillus cereus and Vibrio natriegens. The control specimens (denoted as C) were prepared without a bacterial consortium, while the test specimens containing the bacterial consortium were denoted as BV. In this study, the bacterial species were introduced into liquid form, replacing a 3% of bacterial solution with water and, to assess the influence of bacterial incorporation on concrete properties, the experiment maintained a constant bacterial percentage of 3% (BV) [59] and tested three different water–cement ratios (0.4 BV, 0.45 BV, and 0.5 BV) in comparison with corresponding control specimens (0.4 C, 0.45 C, and 0.5 C). The bacteria were mixed in a 1:1.25 ratio to form the consortia fixed by the Complexometric titration method. The specimens were cured using the ponding method for 28 days. The impact of bacterial addition on workability, durability, and microstructural characteristics was analysed as part of the evaluation. Sample details of the concrete mix are illustrated in

Table 5. Details of the concrete specimen for casting.

S.No	Specimens	Test	Formula
1		Compressive strength (Equation (1))	$f_{ck}={\displaystyle \frac{P}{a}}$ P—Load a—Coss sectional area of specimen
2		Split tensile strength (Equation (2))	$f_{ct}={\displaystyle \frac{2P}{\pi DL}}$ P—Maximum applied load D—Specimen’s diameter L—Specimen’s length
3		Flexural strength (Equation (3))	$f_b={\displaystyle \frac{3PL}{2b{d}^2}}$ P—Maximum applied load b—Specimen’s width d—Specimen’s depth L—Span length between the two supports
4		Water absorption (Equation (4))	$W_a={\displaystyle \frac{W_s-w_d}{W_d}}$ × 100 Wa—Water absorption ws—Weight of saturated specimen wd—Weight of dry specimen
5		Acid attack (Equations (5) and (6))	$\mathrm{Mass}\mathrm{loss}={\displaystyle \frac{{W_I-W}_F}{W_F}}$ × 100 Wi—Initial weight before immersion Wf—Final weight after immersion$\mathrm{Compressive}\mathrm{strength}={\displaystyle \frac{Strengthbeforeimmersion-Strengthafterimmersion}{Strengthbeforeimmersion}}$ × 100
6		Ultrasonic pulse velocity (Equation (7))	$V={\displaystyle \frac{L}{T}}$ V—Pulse velocity L—Distance travelled T—Time taken
7		Compressive strength regain	$f_{CSR}={\displaystyle \frac{C_b{-C}_{nb}}{C_b}}$ × 100% fCSR—Compressive strength regain Cb—Compressive strength of bacterial cube Cnb—Compressive strength of control cube

.

3.1. Slump Cone

The concrete slump cone test is a widely used method to assess the workability and consistency of freshly mixed concrete. The test utilises a metallic conical mould, commonly known as the slump cone, which is 300 mm in height with a base diameter of 200 mm and a top diameter of 100 mm. For this experiment, three different w/c were performed on both control (0.4, 0.45 and 0.5) and bacterial consortia (0.4, 0.45 and 0.5) concrete. All the six concrete mixes were tested in accordance with ASTM C143 [60]. To perform the test, the slump cone is placed vertically on a rigid, non-absorbent base plate. Concrete was filled in three equal layers, each comprising roughly one-third of the mould’s volume. To prevent the concrete mixture from sticking to the mould, the inner surface of slump cone was uniformly oiled, and the mould was placed on a horizontal, flat surface. Each layer was tapped 25 times with a tamping rod, ensuring equal distribution of strokes across the cross-section. After successful tapping, the surface was levelled off using the tamping rod. Finally, the mould was carefully lifted in a vertical position without damage (Figure 6). Then the slump was measured by determining the vertical distance between the top of the mould and the highest point of the slumped concrete at its original centre.

3.2. Compressive Strength Test

The compressive test was performed as per IS 516-1959 [61]. As per code, 150 mm × 150 mm dimension cubes were used in this study. For this test, the cubes were collected from the curing tank and wiped off the surface moisture completely. Then the cubes were placed centrally on 300-ton capacity UTM (Universal Testing Machine), and applied uniform load distribution to the cube properly. During the load distribution, the maximum load at which each specimen passed and failed were recorded respectively. The compressive strength for each specimen was examined using the formula in Equation (1).

3.3. Split Tensile Strength

The split tensile strength was done according to the guidelines of IS 5816:1999 [62]. The cylindrical specimens with a length of 300 mm and diameter of 150 mm were cast and kept for curing for 7 and 28 days under standard conditions. Prior to testing, the cylindrical specimens were wiped off clearly and the surface moisture was removed completely. The specimens were placed in a 300-ton capacity Universal Testing Machine (UTM), and ensured the even stress distribution with 3 mm neoprene strips or thick plywood placed along the length of the specimen. The cylinder specimens were then positioned in a horizontal manner between the loading platens so that the compressive load was given diametrically along the vertical plane of the cylinder. The maximum load applied was recorded using the standard formula in Equation (2).

3.4. Flexural Strength Test

The flexural strength test was done according to the standard guidelines IS 516:1959 [61]. The beam specimens with the dimension of 100 mm × 100 mm × 500 mm were used in this study. The specimens were prepared, and cast with appropriate mix proportions, and cured in water at normal room temperature for 7 and 28 days. After curing, the specimens were collected from the curing tank, wiped off completely without surface moisture. Each specimen was placed in a horizontal position on two supports with an appropriate span based on its size. Gradually, the load pressure was increased until the specimen shows visible cracking and the specimen failing and passing test was recorded. The flexural strength is then calculated using the following formula in Equation (3).

3.5. Water Absorption Test

The water absorption test was performed based on ASTM C143 [60] guidelines. This analysis is used to test the amount of water absorbed by the specimen. This test shows the indication of the concrete permeability and porosity, which are important factors in determining the resistance and durability to moisture related issues. High water absorption values indicate higher porosity and permeability, which can lead to problems such as freeze thaw damage, chemical attacks, and reduced durability. For this experiment, concrete cubes were cast and cured for 28 days in normal water at room temperature. After curing process, the cube specimens were collected and completely removed the surface moisture and dried to a constant weight before the test to ensure reliable and accurate measurements. For this test, the specimens were immersed in water for a period of 24 h of incubation. After the immersion period, the specimens were collected from the water and excess water was gently removed by using a damp cloth. The wet specimen was immediately measured in a weighing balance, and the weight was recorded as the saturated weight (W_s). Then the specimen was placed in an oven for drying at a temperature between 100° C and 115° C for 24 h. After the drying period, the specimens were collected and recorded as the dry weight of the specimen (W_d). Equation (4) states that the water absorption of the concrete specimen was calculated.

3.6. Acid Attack

The acid attack test for concrete is conducted to analyse the materials’ resistance and durability in acidic environment. As per recommended standards ASTM C 1898-20, cubical concrete specimens of size 100 mm × 100 mm × 100 mm were prepared and cured for 28 days under stable conditions to conduct the acid test. The specimens were typically dried to a constant weight before the test to ensure accurate measurements and measure the initial dry weight of all the specimens denoted as W_I. Dried specimens were completely immersed in the aggressive acidic solution (Figure 7) and were diluted with 5% H₂SO₄ (prepared using concentrated sulphuric acid) solution for 28 days and kept at room temperature. The experiment was performed in accordance with standard and safety laboratory guidelines. The sulphuric acid solution was recurrently replaced with a freshly prepared solution. During the immersion period, the specimens were inspected at intervals to assess any visible signs of degradation, such as surface deterioration, colour changes, and cracking (Figure 8).

After the specified exposure period, the specimens were removed from the acid solution and carefully rinsed with clean water to remove any residual acid. Then the specimens were weighed to determine the mass loss caused by the acid attack. The mass loss of the concrete specimens was calculated by comparing the initial weight (W_I) before immersion with the final weight (W_F) after immersion. The percentage of mass loss was calculated using the formula in Equation (5). The mass loss indicates the concrete’s resistance to acid attack. Higher mass loss values indicate greater susceptibility to chemical degradation and reduced durability in acidic environments. Strength loss can be evaluated by the (Equation (6)) variation in compressive strength of the specimens.

3.7. Ultrasonic Pulse Velocity Test

The ultrasonic pulse velocity (UPV) test is a non-destructive testing method used to assess the quality, uniformity, and integrity of concrete. It measures the speed at which ultrasonic pulses travel through the concrete, providing information about its homogeneity and potential defects. The test was performed based on IS 516 (5)-2018 guidelines. Initially, the transmitting transducers are placed on the surface of the specimen, typically opposite to each other. One transducer acts as a sender which emits high-frequency ultrasonic pulses, while the other transducer acts as a receiver that detects the pulse. The sender transducer emits a short pulse of ultrasonic waves into the concrete, which then travel through the material. These waves propagate through the concrete until they reach the receiver transducer. The time taken for the waves to travel between these two transducers was measured.

The UPV test was performed on different orientations of the concrete specimen (Figure 9), such as longitudinal, transverse, or diagonal, to assess the anisotropic properties of the concrete. The pulse velocity was typically reported in metres per second (m/s) or kilometres per second (km/s). The test was conducted for each mix with varying water–cement ratios in both bacterial and control specimens. The UPV test was calculated by the formula in Equation (7).

3.8. Self-Healing

Bacteria-based self-healing concrete is a relatively new technique (Figure 10); therefore, it is important to gather more results in simulated real conditions before applying it on a bigger scale. To assess the quality of concrete, UPV testing was done before and after cubes were kept for self-healing. Concrete cubes of control mix and bacteria mix were prepared. To simulate the real state of a concrete crack, the specimens were loaded on the compression test machine. When the surface appears visibly with micro cracks, the loading is stopped. For the crack healing quantification, the cracked specimens were immersed in tap water in a plastic basin, which was kept open to the atmosphere for 28 days. Progress on the cracked surface for conventional and bacterial concrete was noted frequently at 3,7,14, and 28 days.

3.9. X-Ray Diffraction

X-ray diffraction (XRD) is a method used in materials research to ascertain a material’s crystal structure. By exposing a material to X-rays, XRD can determine the substance’s X-ray emission intensity and scattering angle (2 theta). Cementitious materials have been characterised in several investigations using XRD methods. The PANalytical X’Pert3 powder X-ray diffractometer was the apparatus (Figure 11) utilised in this investigation. A wide range of solids and powders can be used to analyse the phase. The machine has a PIXel1D scanner and a Bragg–Brentano HD mirror for improved results. Cement hydration outcomes at various hardness levels and material inclusions can be studied to the peak regions and intensity of the XRD pattern. Cementitious materials have been characterised in some investigations using XRD methods. To comprehend the composition, XRD patterns of both bacterial and regular concrete were created for this study. Additionally, the precipitate that was produced during self-healing was examined.

4. Results and Discussion

4.1. Slump Cone Test

The water-to-cement (w/c) ratio, which impacts the mix’s workability and flowability, has an impact on the slump values of concrete. According to IS 10262, the maximum water-to-cement ratio is 0.45; in this study, 0.40, 0.45 and 0.50 for control and bacterial concrete were chosen to examine the impact on concrete strength and durability parameters. There is no recommended IS code specifying precise slump values for bacterial concrete at different w/c ratios when comparing the slump values of concrete on concrete with different water cement ratios according to ASTM C 143. By forming calcium carbonate precipitates that fill pores and may decrease workability, bacteria can change the characteristics of bacterial concrete. Based on specific mix designs and bacterial strains used, bacterial concrete may have slightly lower slump values due to reduced workability, as illustrated in the graph (Figure 12).

4.2. Compressive Strength Test

Based on the slump cone test, the concrete specimens were cast into cube moulds of 100 mm and tested for 7, 14, and 28 days of water curing to calculate the compressive strength and the variations are plotted in Figure 13. It is evident from the graph that even though increased water content improves the workability, it reduces the strength of the concrete. Out of three conventional mixes, the 0.4 C mix shows higher strength on the 7th, 14th and 28th day. But it is also noticeable that, while adding bacterial solution, irrespective of the w/c, there is an increment in strength.

The 3rd-day strength of the 0.45 BV mix is more than that of 0.4 BV. When it reaches the 28th day, the compressive strength of the 0.4 BV mix increases. In the conventional mix, the strength of the 0.4 C mix and 0.45 C mix show a significant variation. Also, their workability is extremely different. A 0.4 BV mix at 28th day shows maximum compressive strength of 56.81 MPa with +32.085% than that of 0.4 C mix. A 0.45 BV mix shows +24.61% than 0.45 C with 50.78 MPa. Increased strength with better workability was achieved in the case of 0.40 w/c. So, an average of +4.97% on the 3rd day, +11.47 on the 7th day and +26.85% on the 28th day was estimated when compared to normal control.

4.3. Split Tensile Strength

The variation in split tensile strength is shown in Figure 14. Split tensile strength for normal concrete varies by the w/c. The more the w/c, the lesser the split tensile strength was observed. So, 0.4 C has a value of 4.3 MPa at 28 days, while it is 4.08 MPa for 0.45 C. The value obtained for 0.5 C was 3.59 Mpa, even less than both control mixes at the end of 28 days. However, when analysing bacterial concrete, the 0.4 BV mix shows the maximum split tensile strength of 5.68 MPa, which is +24.01%, when compared to the 0.4 C mix. An increment of 20.09% and 10.97% is shown by 0.45 BV and 0.50 BV mixes than corresponding normal concrete mix. The addition of bacterial solution significantly contributes to the improvement in the split tensile strength in the concrete.

4.4. Flexural Strength

The flexural strength of concrete is plotted in Figure 15. Strength was calculated for 7 and 28 days of curing and it was found that flexure value decreased with increased w/c for normal concrete mix. The minimum w/c of 0.4 C mix for normal concrete exhibits a maximum strength of 6.11 MPa during bending while a 0.5 C mix yielded a strength of 5.89 MPa. It can be seen that bacterial concrete shows an average of +18.65% increment in bending. As the bacteria starts depositing calcite, the voids inside the concrete get filled and reduced which results in improvement in mechanical strength characteristics. From the results, the effect of bacterial growth is more visible in the 0.4 w/c. An adequate amount of water in the 0.4 BV mix enhances the workability, which in turn requires less amount of mechanical strength to mix and place the concrete into moulds. With more mechanical forces, the chances are there that the bacteria could be harmed. This can be the reason for improved strength in 0.4 w/c for bacterial concrete. When compared to 0.45 C, 0.45 BV shows +17.32% strength.

When it comes to 0.5 BV, the water content is way too much, which creates greater space between the aggregate materials, thus the voids will fill with air after the moisture evaporates. Even in this condition, 0.5 BV shows a result similar to that of 0.4 C which is an advantage. Overall, the mechanical properties of bacterial concrete are better and more than that of conventional mix.

4.5. Water Absorption

The test results of the water absorption and variation is plotted in Figure 16. It can be observed that the water absorption varies from 3.43% to 4.12% for conventional concrete. For bacterial concrete, maximum water absorption is limited up to 2.56% variation. The maximum water absorption is exhibited by 0.5 C which is due to the porosity in the concrete microstructure and it is mainly affected by w/c. As could be expected, the experimental results show that both the concrete mix and its w/c ratio affect concrete absorption.

4.6. Acid Attack

The acid attack test was performed on six mixes of concrete, including three control mixes, 0.4 C, 0.45 C, 0.5 C, and three bacterial mixes, 0.4 BV, 0.45 BV, and 0.5 BV. Based on mass loss and variation in compressive strength, acid resistance can be analysed. It (

Table 6. Percentage weight loss and compressive strength loss for all the mixes.

Mix Designation	Control Concrete			Mix Designation	Bacterial Concrete
	% Weight Loss	Compressive Strength	% Loss in Compressive Strength		% Weight Loss	Compressive Strength	% Loss in Compressive Strength
0.4 C	2.64	26.25	38.88	0.4 BV	2.06	42.55	23.90
0.45 C	2.75	26.09	35.31	0.45 BV	2.24	37.21	26.71
0.5 C	3.35	23.03	35.93	0.5 BV	2.41	28.61	35.75

) can be noted that the percentage weight loss can be calculated by comparing the weights of the specimens after 28 days of 5% sulphuric acid immersion of all the mixes.

For bacterial concrete, the weight loss percentage (Figure 17a) is minimal, while it is maximum for conventional concrete. Mix 0.4 BV has the minimum weight loss as bacterial concrete with a 0.4 water cement ratio. Lowering the water-to-cement ratio leads to a reduction in the porosity of the concrete specimen, which is potentially resistant more to acid attack. When concrete containing cement, gravel and sand reacts with sulphuric acid it results in the occurrence of gypsum and degradation. Cracking and expansion occur on the concrete due to the formation of gypsum. When introducing bacteria into conventional concrete with different water–cement ratios it reacts with sulphuric acid, reducing the percentage mass loss which increases the stability [13].

The residual compressive strength (Figure 17b) of the conventional mix is less than that of the bacterial mix. The bacterial concrete with lower water–cement ratio stability was enhanced due to the increased amount of calcite content precipitated by the bacteria inside the pores. The concrete cube strength varies based on the exposure time; after immersing the cubes in an acidic solution for 28 days percentage loss of compressive strength can be noted. In both control and bacterial concrete mix, the water–cement ratio increases then the residual compressive strength decreases. In the bacterial concrete mix w/c ratio increases with the strength loss percentage increases; mix 0.4 BV is the optimal mix that has more resistance in an acidic environment due to the bacterial action.

4.7. Ultrasonic Pulse Velocity Test

Ultrasonic pulse velocity rest results are shown for different mixes in

Table 7. UPV test results.

Concrete Mix	Time (µm/s)	Velocity (km/s)	Remarks
0.4 C	20.7	4.81	Excellent
0.45 C	20.5	4.878	Excellent
0.5 C	21.7	4.608	Excellent
0.4 BV	20.5	5.135	Excellent
0.45 BV	19.47	4.878	Excellent
0.5 BV	21	4.878	Excellent

. After 28 days all the mixes show excellent quality of concrete. The pulse velocity of bacterial concrete is better than normal mix. When the voids inside the concrete fill with calcite precipitation, the quality of concrete also get improved.

4.8. Self-Healing

The healing of cracks in the bacterial specimen can be studied. After the 28 days of curing, the load was applied to the concrete specimen to initiate the artificial cracks and measure the residual compressive strength and UPV values. The cracked specimen was placed in a water tank for curing and observed the crack filling activity continuously based on the calcite precipitation by visual examination for 3, 7, 14 and 28 days. While the water curing process was used in the bacterial concrete, bacteria enzymatically start to react with the moisture condition. It precipitates the mineral as calcium carbonate in the pores and cracks to fill them. Mix 0.4 BV chosen for crack healing process and Figure 18 depicts the crack fills at the age of 3, 7, 14 and 28 days.

4.8.1. Compressive Strength Recovery Test

Cubic specimens of triplicates of 150 mm × 150 mm × 150 mm were pre-compressed and measured the compressive strength with cracks, and placed the specimen for incubation. The residual strength of control and bacterial concrete was experimented with after 28 days to calculate the strength regain. The strength regain of the concrete specimen was calculated by the equation. There was an increase of 7.90% for control concrete (

Table 8. Compressive strength and UPV values of before and after healing process.

Mix Specification	Before Keeping for Self-Healing		After 28 Days of Healing
	UPV (km/s)	Residual Comp. Strength (Mpa)	UPV (km/s)	Residual Comp. Strength (Mpa)	% Increment
Damaged Control Concrete	3.041	31.25	3.452	33.72	7.90
Damaged Bacterial Concrete	4.621	38.18	5.321	43.08	12.83

) and a 12.83% increase in the bacterial concrete.

4.8.2. Ultrasonic Pulse Velocity Test

As per IS 13311:1992, the internal crack healing can be studied by the ultrasonic pulse velocity for both the control and conventional mixes. UPV values taken for the conventional and bacterial specimens before and after healing. An increase in the pulse velocity was observed at the 0.4 BV mix of bacterial specimens, which results in bacterial addition enhancing the concrete quality. As the day increased, pores and voids were filled by calcite precipitation, and the pulse velocity increased steadily. An increment in the velocity implied reduced porosity and voids, resistance to water penetration and improvement in concrete properties.

4.9. X-Ray Diffraction Analysis

The precipitates that occurred over the bacterial specimens were collected and oven-dried for some time (Figure 19a) and taken for XRD analysis. Powder samples were collected from bacteria and also normal concrete tested for analysing the microstructural variation. Bacterial concrete has more calcite peaks than conventional concrete, proving that it has more calcite precipitation (Figure 19b). The content of calcium silicate gel, which is an important factor in providing concrete strength, is also more in bacterial concrete (Figure 19c). Calcite content is more abundant in the bacterial concrete, which leads to filling the cracks and ensuring more strength, as confirmed by XRD analysis. Calcite precipitation’s highest peak was spotted on the two theta values of 29.8770 shown in (Figure 19d).

5. Conclusions

The fundamental idea behind bacterial concrete is that microorganisms in the cement particle react with water to produce calcium carbonate, which serves as the healing agent. This sets bacterial concrete apart from other self-healing techniques. The following summarises the key findings.

• The slump cone test yielded workability findings for concrete with varying water-to-cement ratios of 0.40, 0.45, and 0.50. The w/c ratio is correlated to the slump values and workability, but the higher workability affects the durability and porosity. Due to the presence of calcite, bacterial concrete has lower workability values at the water cement ratio of 0.4 than the control.
• Mechanical strength tests were done for different curing ages. In the case of compressive strength test, an average of +4.97% on the 3rd day, +11.47% on the 7th day and +26.85% at the 28th day was observed when compared to normal control. For the flexural strength test, an average of +17.32% increment was observed at the end of 28 days. When analysing split tensile strength, an average of 8.56% increment was observed after 7 days and +14.04% after 28 days of curing.
• Water absorption results for bacterial concrete show superior results than normal concrete. This is mainly due to the calcite precipitation by the activity of bacterial culture inside the concrete. Minimum water absorption was shown by 0.4 BV mix, which was 1.98% of weight of concrete.
• Acid attack tests and water absorption tests conducted for six concrete mixes revealed that the amount of water absorption is less for bacterial specimens. This may be due to calcite-filled voids inside the specimen. In the case of the acid attack test, less weight loss was observed in the bacterial specimen. However, the residual compressive strength was much less for conventional concrete.
• The self-healing ability of the bacterial specimen is proved by keeping the specimen for 28 days after a crack was initiated. XRD results indicated a high calcite content in bacterial concrete, which may explain the improved mechanical and durability properties. So, it can be concluded that the bacteria B. cereus and V. natriegens can be used simultaneously to improve the self-healing property of concrete as a consortia mix.