The effect of isolated Bacillus ureolytic bacteria in improving the bio-healing of concrete cracks

Reinforcement corrosion and the concrete strength reduction are critical problems that resulted from crack creation in concrete. Very expensive and hazardous technologies based on chemical materials have been provided for repairing the cracks. Recently, crack repair using bio-catalysis precipitating bacteria has been developed as a viable and ecofriendly alternative technique. The main target of this study was to select and identify bacterial isolates with high urease activity to use in filling the cracks by the precipitation of CaCO3. Two endospore-forming and alkali-resistant ureolytic bacteria were combined with concrete to tolerate the mechanical stresses generated by mixing. The two isolates designated as (B1 and B2) were selected and identified as Bacillus wiedmannii strain FSL W8-0169 and Bacillus paramycoides strain MCCC 1A04098, respectively, using 16SrDNA gene sequencing. Both bacterial species completely heal cracks in fully destructed concrete and significant enhancement in compressive strength was illustrated. The calcite filling of cracks and CaCO3 crystals that were screened using a scanning electron microscope may explain the crack healing and the enhancement in concrete strength. Bacillus wiedmannii strain FSL W8-0169 and Bacillus paramycoides strain MCCC 1A04098 can be inserted with the concrete to improve the compressive strength and the self-healing of cracks. The two ureolytic bacterial strains can be used to protect water buildings from exposure to frequent cracks.


Background
The creation of cracks in concrete is highly undesirable as it provides an open pathway for the water ingress leading to reinforcement corrosion and the concrete strength reduction. Various methods have been provided for repairing the cracks based on chemical materials that are very expensive and hazardous to environment [1]. Recently, crack repair using bio-catalysis precipitating bacteria has been developed as a viable alternative. This bacterial curing technique is preferable over other technologies as it is ecofriendly, bio-based, durable, and cost-effective [2]. Urease-positive bacteria affect CaCO 3 precipitation (calcite) by producing an enzyme called urease. This enzyme converts urea to CO 2 and ammonia, leading to a rise in pH and CaCO 3 precipitation in the bacterial environment [3]. The precipitation of calcite was promoted by bacteria as Bacillus sp was found to be effective in concrete crack healing and increasing the concrete and mortar compressive strength [4]. In various studies, it has been shown that adding specific bacterial species to cement-sand concrete or mortar causes calcite deposition within matrix pores, allowing it to be used as a filling material to repair cracks in concrete structures [5]. It was also noticed that the mixing of bacterial spores or cells with growth medium, consisting of 20 g/l urea and 25 Mm calcium chloride with the concrete could increase the strength of the material by 20-35%

Open Access
Beni-Suef University Journal of Basic and Applied Sciences compared to control [6,7]. Castanier et al. [8] showed that many factors influence microbial-induced CaCO 3 precipitation (MICP), including (a) the pH of the atmosphere, (b) carbonate concentration, (c) the presence of nucleation sites, and (d) calcium concentration. Because of the widespread existence of ureolytic bacteria, MICP by ureolytic activity has been used in several studies [9,10]. Due to the fact that it is easy to control and generates large quantities of carbonate in a short period of time, urea hydrolysis is the most powerful of the calcitegenerating reactions. Moreover, it does not only maintain an alkaline pH, but it also produces a plentiful supply of calcite by providing bio-based minerals [11]. The current research focuses on isolating and identifying endospore-forming, calcite-precipitating, and ureasepositive bacteria, as well as determining their suitability for use in concrete. The impact of bacteria on compressive strength and crack healing has also been studied EDX-SEM was used to visually inspect and quantify the calcite precipitation. For the first time, we detected the stalactite deposits in cracks after 90 days water curing of bacterial concrete specimens.

Bacterial isolation
With minor modifications, the method defined by Ishaka et al. [12] was used. From the Sewage Water Treatment Company, Fayoum, Egypt, a sample was collected from the active sludge. Ten milliliters from the sample was diluted in 100 mL sterilized distilled water. Fifty microliters from the diluted mixture was inoculated in Petri dishes of the previously sterilized alkaline nutrient agar (ANA). ANA was prepared by adding NaOH solution droplets till reaching pH 14. Incubation for 36 h at 30 °C was performed and the alkali-tolerant bacterial species that could grow were purified and preserved at -80 for further tests.

Urease activity assay (was detected using two methods)
Method one: Seven alkali-tolerant bacterial isolates were inoculated into urea broth and incubated at 30 °C for 24 h. A pink color was produced with bacteria that rapidly hydrolyze urea using Phenol red as indicator [13]. Method two: The electric conductivity approach was used as Omoregie et al. [14] for the bacterial isolates that rapidly hydrolyze urea. The urease reaction involved the hydrolysis of the non-ionic substrate urea to ionic compounds, resulting in a proportionate increase in conductivity under normal conditions. Urea-nutrient broth was used for bacterial culturing. One millimeter of bacterial broth culture was mixed with 9.0 mL of 1 M urea solution. By electric conductivity meter, the conductivity was recorded after 0, 10, 60, and 100 min of incubation at 25 °C. The rate of increase in conductivity, measured in mS/m.

CaCo 3 (calcium carbonate) precipitation
For the measurement of CaCO 3 precipitation, a method showed by Krishnapriya et al. [1] was followed. Thirty millimeters of NB-U/Ca (Nutrient broth implemented with 2% urea and CaCl 2 ) was injected with 2% bacterial inoculum then incubated in shaking incubator (130 rpm) for 7 days at 30 °C. Using a Whatman filter paper, precipitated CaCO 3 was filtered and dried in an oven of 55 °C for 8 h and then weighed. From the following equation, weight of precipitated CaCO 3 (W c ) was calculated.
where (Wf ) is the empty filter paper weight and Wfc is the weight of filter paper containing CaCO 3 precipitant.

Endospore staining
By Schaeffer-Fulton endospore staining procedure as per Hussey [15], the ability to synthesize endospores was determined in the selected bacterial isolates. Heat-fixed bacterial isolate smears were prepared. The smears were then immersed with malachite green after being coated with absorbent paper. Steam started to rise from the slide surface as they were heated. Drops of malachite green were supplied on the paper as it started to dry to maintain it moist. Using tweezers, the paper was taken away from the slide, and then washed thoroughly under running water. The slide was drained, counterstained, polished, and tested under a light microscope (100 x) after being incubated for 60 s with 0.5 percent safranin stain. The endospores are green and the vegetative cells are red or pink.

Molecular identification
For the molecular identification, the genomic DNA has been extracted using standard bacterial procedures described by Molecular Cloning [16]. The PCR blend was prepared as follow: 10 μL (10x) PCR buffer, 3 μL (50 mM) MgCl 2 , 1 μL (20 pmol/μL) of each primer, 1 μL (10 mM) Mixture of dNTPs, 0.5 μL (2.5U) Taq polymerase, 2 μL gross DNA extract, and volume completed by sterilized distilled H 2 O to 100 μL. Under the conditions set out below, PCR was performed for 35 cycles: denaturation stage at 90-94 °C for 40 s, the annealing step was controlled for 1 min at 55 °C, for the extension step, 72 °C for 2 min was adjusted, and the final expansion for 10 min at 72 °C. 10 µL from the products of PCR was added to 2 μL of DNA the gel containing 0.5 μg/mL ethidium bromide in the Tris-Borate-EDTA (TBE) buffer is then visualized using a UV transilluminator by the buffer loading and electrophoresis analysis on 0.7% horizontal agarose (60 min at 15 V/cm,). The sequencing of the amplified fragments was completed at GATC Biotech, Constance, Germany. DNA sequences were aligned at NCBI Data Base (www. ncbi. nlm. nlh. gov). The phylogenetic trees were established using a neighbor-joining technique using TREEVIEW software (1.6.6) derived from gene sequences of 16S rDNA of some phylogenetic close strains to the isolated strain. The sequences had accession numbers by their submission in GenBank NCBI database.

Bio-healing agent preparation
Bacterial cultures were prepared in alkaline nutrient broth. Turbidity was conducted under spectrophotometric conditions giving optical density (OD) 0.6 at 600 nm. For the bacterial spore harvest, several times of centrifugation in dual sterilized tap water were made after heating the bacterial cultures for 35 min at 80 °C to inactivate bacterial vegetative cells and obtain a high number of spores. The bacterial spores were adjusted to be 1.39 × 10 7 cells/cm 3 of the total concrete mixture, in sterilized tap water for preparing the bio-based concrete, the nutrients, including 2 g/L yeast extract, 40 g/L calcium chloride anhydrous, and 65 g/L urea were dissolved.

Experimental design
Following Egyptian Code Practice (ECP) and American Society for Testing and Materials (ASTM) standards, the bio-based agent was added in the final step of concrete constituents mixing. Control concrete specimens (C) were prepared using all medium nutrients except spores, B1 was inoculated by bacterial spores of B. wiedmannii, and B2 was inoculated by bacterial spores of B. paramycoides. Concrete mixture design is illustrated in Table 1. Immediately after mixing, the test concrete specimens were cast. After 24 h, the specimens were removed from the molds, treated in water for 28 days, and then checked for compressive strength. To conduct the test, a 2000 KN (ADR 2000) compression testing system was used. Following the ISO 4012 standard requirements, specimens were positioned on a rigid bottom bearing block with a spherical bearing block attached to the compressive testing unit. The compression load was applied to the specimen at a rate of 0.6 N/mm 2 per second, which was within the ECP's specified range. The max compressive strength (σ) was calculated by the division of the peak load (P) by the cross-sectional area (A) of each specimen. The compressive strength was examined at 7, 14, and 28 days and calculated using the following formula (average for three cubes).

SEM calcite precipitates characterization
Full destruction was applied on concrete beams for cracks creation. For a total of 90 days, the beams were submerged in water. After 28 and 90 days period, photographs were taken for detecting calcite appearance in cracks. The calcite precipitation by bacterial isolates in the cracks of concrete specimens was screened and quantified using EDX-SEM analysis. EDX-SEM analysis was obtained using a Carl Zeiss sigma 500 VP Jeol JSM -6390 apparatus.

Statistical analysis
Data were statistically analyzed using a two-way analysis of variance (ANOVA test) using SPSS Statistical Package Program [17] version 23. Mean of treatments was compared by Duncan multiple range test when the differences were significant [18]. Level of significance in all tests was P ≤ 0.05. The results are expressed as means ± standard error (SE).

Isolation endospore-forming and alkali-tolerant bacteria
From the isolation step, seven bacterial isolates could tolerate till pH 14. All isolates are gram negative and endospore-forming (see Additional file 1: Figure S1). Figure 1 shows the green color of B1 and B2 endospores as dark black colored spherical dots and the vegetative cells seem light colored rods.

Urease activity
From the seven bacterial isolates, six of them gave positive urease results. Two isolates (B1 and B2) gave dark pink color after 24 h as shown in Fig. 1. The other types gave positive results after 48 h. Electric conductivity was measured only for (B1 and B2). B1 exceeded B2 in its activity as recorded in Table 2. After 100 min, electric conductivity was 191.5 mS/m and 186mS/m for B2.

CaCO 3 (calcium carbonate) precipitation
After bacterial inoculation of NB-U/Ca, a white powder immediately appeared in the medium and its intensity increased with incubation period. After 7 days from incubation, CaCO 3 precipitants were harvested and weighed. The two bacterial isolates precipitated calcite. The highest amount of calcite (1.18 g) was precipitated by B. wiedmannii strain FSL W8-0169 and 0.98 g for B. paramycoides strain MCCC 1A04098 as shown in Table 2.

Molecular identification
The     Figure 4 shows the photographs of control and bacterial spore beam specimens' cracks. After 28 and 90 days from water curing, white precipitates were screened in the cracks of bio-concrete specimens. Complete crack repair was recorded in both bacterial beam specimens.

Crack healing appearance
In control crack specimens, no observed crack healing was detected. Calcite quantification using EDX-SEM microanalysis showed significant increase percentages in CaCO 3 amount and ettringite like structure was observed (Figs. 5, 6, 7). Figure 4 shows the formation of vertical outgrowths of calcium carbonate that look like stalactite in their appearance (were detected after 90 days from water curing). These outgrowths were observed in bio-concrete specimens but were not observed for control (see also Additional file 1: Figure S2). Ettringite, calcite, and stalactite deposits were screened in bio-concrete specimens and that was the reason for compressive strength improvements.

Compressive strength
The values for the 7, 14, and 28 days of the curing period were determined using three concrete cubes for each period. The values of compressive strength and the percent from 28 days for each period (7, 14, and28 days) were shown in Table 3. It is seen from the compressive strength data in Table 3 that the bio-based specimens of strength are greater than those of the control sample. The maximum increase in compressive strength at 28 days (33.7 MPa) was obtained for concrete specimens inoculated with B. wiedmannii strain FSL W8-0169 and 28.5 MPa for B. paramycoides strain MCCC 1A04098, the strength of control was 23 MPa indicating that there were strength improvements by 46.52% and 23.91%, respectively more than control.

Discussion
Bacteria can withstand harsh mechanical-and chemical-induced stresses during the mixing of concrete by their capability for endospore formation [19] and these endospores can live for up to 200 years [20]. As cracks occur, water will ingress into them causing the activation of the incorporated bacterial spores, and by microbial nutrients metabolism, CaCO 3 is formed by germination of spores into vegetative cells [21]. Because of their ability to hydrolyze urea into ammonia and carbonate, both bacterial isolates may precipitate CaCO 3 . Ammonia causes a rise in pH, which creates a favorable climate for calcium carbonate precipitation. CaCO 3 precipitates when carbonate combines with calcium in a mixture [3]. The improvement in compressive strength caused by the inclusion of bacteria is likely due to the deposition of calcite on the surfaces of the microorganism cells and in the concrete pores [1]. The insignificant increase in the compressive strength of control specimens of media alone without spore inoculation shows that the medium nutrients do not have any effective impact on strength improvement. Chahal et al. [22]; Al-Thawadi and Cord-Ruwisch [23] showed the improvement in compressive strength through the bacterial based materials that is possibly due to the deposition of calcite on the surfaces of the microorganism cells and through the pores of the concrete.  [25] who also illustrated that specific calcite crystals were screened in the SEM analysis of bio-based concrete and the matrix of untreated specimens seemed to be amorphous, exhibiting no apparent growth of crystals.

Conclusions
B. wiedmannii and B. paramycoides can act as biohealing agents as they were examined for calcite-precipitating in concrete to fill the pre-cracked specimens and the improvement in compressive strength was also illustrated. Ettringite, calcite, and stalactite deposits formation were illustrated. From our findings, the two bacterial species can be included in water buildings and those are built on the shores of the seas and rivers to protect them against cracks they suffer from. More studies related to the healing effect of new bacterial species on slabs and columns are recommended for future work.