Cultivation of bacterial strain
Bacillus subtilis (supplied by Philip Harris, UK) was utilised for the experimental programme. It was selected based on previous literature [29, 30] using similar strains and was driven largely by the ability of this genus to form resistant, long-lived spores. The bacterial strains were cultivated in Basal medium 121 and its derivatives 121A and 121B, as described by Sonenshein et al. . The culture was incubated in a shaker at 125 rpm at a temperature of 36 °C for 72 h until the formation of spores was observed. This was confirmed under a microscope (LABOPHOT-2, Nikon) using the spore stain method. To minimise the presence of vegetative cells, spores were harvested using a centrifuge machine, where the culture was spun at high speed (3390 RCF) for 10 min and then washed twice using distilled water. Vegetative cells have been reported  to have an average density of 1.135 g/cm3, whereas the average spore density is 1.305 g/cm3. Therefore, the centrifugal force causes heavier particles to move away from the axis of rotation, resulting in the deposition of spores (forming what is known as a pellet) at the bottom of the test tube.
Encapsulation of spores into perlite
The main point of using capsules is to protect the healing agents (bacterial spores) from the harsh environment of fresh concrete such as high pH and temperature and also to prevent the agent from undesirable release during the mix. There are many approaches for encapsulation reported in the literature [33,34,35]. In this study, perlite was used to encapsulate the bacteria (with nutrients) due to its highly porous structure, which creates a suitable host environment for the healing agent. The properties of the used perlite were tested in accordance with British Standards [36, 37]; it had a unit weight of 128 kg/m3, porosity of 65% and moisture content of 22%. The particle size distribution of the perlite is illustrated in Fig. 1, which can be considered well-graded.
The perlite was first sterilised in the oven at a temperature of 160 °C for two days to destroy any microbes or bacteria present and remove moisture. To impregnate the perlite with bacterial spores, it was soaked in the bacterial suspension for 2 h until the suspension was absorbed. The surface of the perlite was then sprayed with a nutrient solution containing calcium acetate (60 g l−1) and yeast extract (6 g l−1). After each treatment, the perlite was dried in the oven at 40 °C for two days until a constant weight was obtained. These produced capsules containing approximately 0.3% of nutrients by perlite weight. The percentage of the nutrients was calculated by taking the weight of the perlite before and after the impregnation.
The viability of bacterial spores in the perlite capsules was measured in terms of Colony Forming Unit (CFU). This was conducted according to the procedures described in Microbiology: laboratory manual . Accordingly, the spore concentration contained in perlite capsules was approximately 6.4 × 107 CFU g−1.
Preparation of specimens and crack creation
Control Mortar Specimens (CMS) and Bio-Mortar Specimens (BMS) were prepared according to BS EN 196-1. This was achieved by mixing Hanson Sulphate Resisting Cement (CEM III/A + SR), sand, tap water, and self-healing agent impregnated into perlite. The proportions of the mixture are given in Table 1. The water to cement ratio for all mixes was 0.5.
Two specimen geometries were prepared: (1) cylindrical specimens (with a diameter of 100 mm and a height of 40 mm) were used for the visual inspection of crack sealing, and (2) prismatic specimens (40 × 40 × 160 mm) were used for water absorption testing. To avoid complete failure during crack propagation, the prisms specimens were reinforced by a fibre mesh placed at the centre of the specimens during casting. After 24 h, the mortar specimens were removed from their moulds and kept in water for a curing period of 28 days until they were tested.
After curing, the specimens were removed from the water and dried at room temperature in preparation for crack generation using standard mechanical testing. A three-point-bending test was used for the prisms and a splitting test for the cylinders. The prism was installed on two parallel beams at the bottom side, with the distance between them measuring approximately two-thirds of the total length of the sample. The top surface of the sample was compressed by one central beam. The induced cracks were controlled via Linear Variable Differential Transducers (LVDT) attached to the bottom of the specimens. The load was then applied gradually at a velocity of 0.001 mm s−1 until a crack was formed. The velocity was then decreased carefully to allow the crack to be formed around the specimen without failing it. The final load level was found to be in the range of 1.6–1.8 kN. The specimen was then unloaded, resulting in a decrease in crack width.
For the splitting test, the cylindrical specimens were wrapped with carbon fibre adhesive tape to prevent collapse during the formation of cracks under indirect tensile stresses. After placing the specimen horizontally between the upper and bottom plates in the uniaxial compressive strength test machine, the load was applied at low speed until a crack was observed on both sides of the cylinder. At this point, the loading was immediately stopped, and the specimen was removed for visual inspection under a light microscope (Nikon) where the widths of cracks were measured at regular intervals of 1 cm using Shuttlepix Editor software.
The inspection indicated that various cracks were generated with widths ranging between 60 and 350 µm as a result of the mechanical loading. Measuring and classifying these cracks were necessary to ensure each incubation environment would have similar ranges of crack widths.
Characterisation of soil and incubation process
The soil used in this study was sand with high-quality premium and free chemical with a sub-rounded grain shape. Typical sand was used for the incubation of bio and control mortar specimens. To characterise this soil, Particle Size Distribution (PSD) analysis was conducted using a sieve test in accordance with BS EN 933 . The test confirmed that the soil was medium to fine sand BS 5930 , with 95–97% of the material between 100 and 700 μm and only around 3–5% smaller than 63 μm (Fig. 1). This sand is considered a permeable material with quick water drainage. Therefore, any induced change in water content or saturation degree (e.g. fully and partially saturated cycles required during the test) is expected to take effect quickly. In contrast to clay soils, such a change in water content could take an inordinately long time due to low permeability .
The chemical composition of the sand was also investigated under SEM and EDX. The results indicated that the sand was mainly composed of (SiO2) Silicon dioxide, (K2O) potassium oxide, (Al2O3) aluminium oxide, (Fe2O3) iron oxide, and (MgO) Magnesium oxide. This was similar to the SEM and EDX results conducted on the sand used for preparing the mortar mixture.
The sand was poured dry into plastic boxes (with dimensions of 35 × 60 × 40 cm) using a flexible hose attached to a sand hopper. The sand density (of 20.4 ± 0.5 kN/m3) was controlled by maintaining a constant drop height and flow rate. The sand was poured and levelled into two layers, the first one was below the specimens and another layer on top and between the specimens.
Up to 6 specimens were placed within each plastic box, which was previously outfitted with a filtration system containing a porous sheet and thin gravel layer, with an outlet at the base of these boxes for controlled drainage. This setup (see Fig. 2) facilitated the creation of fully and partially saturated cycles during the incubation stage of the cracked specimens.
The pH value of the soil was adjusted to match the aggressive chemical environments with three categories X0, XA1, and XA3 classified in accordance with BS EN 206:2013 + A1:2016 . X0 was the control class with a neutral pH value of around 7; whereas the pH values for classes XA1 and XA3 were adjusted to be approximately 6 (moderately acidic environment) and 4.5 (extremely acidic), respectively. As stated in BS 1377-3:1990 , Calcium sulphate is considered the sulphate salt that is most commonly found in soil. Therefore, the soil pH was reduced to the required level by mixing the soil with calcium sulphate (supplied by Sigma-Aldrich Ltd, UK) in the percentages shown in Table 2. For further confirmation, the soil pH was measured using a portable pH meter.
The cracked specimens were first visually inspected and tested for water absorption and then incubated at a depth of 20 cm from the bottom surface. The BMS and CMS specimens were incubated in separate boxes to avoid cross-contamination, i.e. immigrating the spores and nutrients from the crack zone of BMS specimens to CMS through water seepage within the soil. All incubations were conducted at a room temperature of approximately 23 °C.
The cracked specimens were then incubated either in water or conditioned soil. The water incubation was used as a controlled environment where specimens were fully immersed during the entire incubation period. However, the conditioned soil was subjected to fully and partially saturated cycles for 120 days, as shown in Fig. 3. At the beginning of each cycle, the soil was made fully saturated by raising the water level above the mortar specimens up to the soil surface. Any loss of water (due to the natural evaporation) was compensated by regularly adding some water from the soil surface. This fully saturated stage lasted for 20 days, following which the test moved to the second stage (partially saturated stage) by allowing water to drain away from the soil at the bottom of the incubation box (Fig. 2). Because of the high permeability of sand, water drained away relatively fast (within 12 h) leaving the soil in a moist condition where the voids space became partially occupied by water. Based on the balance between the added and drained water, the degree of saturation was estimated at approximately 50 ± 8%. This period of partially saturated condition was stopped after 10 days, and the soil was subjected to another fully and partially saturated cycles until the end of the incubation period (120 days).
During these cycles, the moisture content and pH level of the soil were regularly checked to ensure these were within the required range (Table 2). When the soil was partially saturated, we noticed some drop in pH level and moisture content, therefore it was necessary to recondition the soil by spraying its surface with some of the water previously drained from the soil (mixed with calcium sulphate if necessary).