Microbial-induced calcite precipitation by indigenous alkaliphilic bacteria: a dual-enzyme strategy for crack-healing in cementitious materials

Microbial-induced calcite precipitation (MICP) offers a sustainable strategy for extending the service life of concrete through autonomous crack healing, yet the high alkalinity of cementitious environments restricts microbial viability. In this study, more than 200 indigenous bacterial isolates collected from extreme environments across Iran were systematically screened for urease and carbonic anhydrase (CA) activities. A dual-enzyme activity index (EAI) was developed to quantitatively rank their calcification potential. Four robust spore-forming strains— Bacillus subtilis , Sporosarcina pasteurii , Bacillus sphaericus , and the environmental isolate E10.2—were identified as top candidates based on high EAI values, sporulation capacity, and survival at pH 13.5. These strains retained at least 70% of their enzymatic activity after alkaline exposure and precipitated up to 89% more CaCO 3 than controls. When incorporated into mortar, bio-treated specimens reached strength levels slightly exceeding the uncracked control under the tested conditions (46.8 MPa at 28 days compared to 34.2 MPa in cracked controls). Ultrasonic pulse velocity, SEM, and XRD analyses confirmed dense CaCO 3 bridging within healed cracks. This study establishes a performance-based framework for selecting dual-enzyme-producing alkaliphilic bacteria for durable, self-healing concrete.