Identification of Genes in Bacterial Strains (Bacillus australimaris and Bacillus safensis) and Prediction of the Pathway Involved in Microbial Induced Calcite Precipitation (MICP) for Concrete Self-healing

Abstract

Microbially Induced Calcite Precipitation (MICP), secreted through bacterial metabolic activity has assisted in providing restorative measures within the construction industry and reduced the negative impact on the economy as well as the environment. The bacterial strains precipitate calcite through active and passive pathways. The passive pathway includes urea hydrolysis, amino acid ammonification, denitrification, and dissimilatory sulphate reduction, whereas the active pathway involves oxidation of organic matter. The process of CaCO3 precipitation also enhances the mechanical and flexural strength of cement matrix. However, the repairing efficiency of MICP is limited by its adverse effects, such as accumulation of new products owing to the chemical reactions between the bacterial metabolic by-products and the cement minerals, and formation of stained patches due to fungal growth as a consequence of nutrient availability, in cement matrix. Therefore, in the present study calcite precipitating genes in the already isolated bacterial strains were identified to investigate the mechanism at molecular and genetic level. For this purpose, 16S rRNA sequencing was performed to identify the isolates capable of CaCO3 production, followed by phylogenetic analysis through MEGA X. The bacterial strains were identified as species of Bacillus, Arthrobacter, Planococcus, Chryseomicrobium and, Corynebacterium. Furthermore, lcfA operon (lcfA, ysiA, ysiB, etfB, and etfA) in Bacillus subtilis was reported to be involved in CaCO3 precipitation. Local alignment was performed between the calcifying gene sequences of Bacillus subtilis and other strains of calcite precipitating bacteria. Consequently, gene sequences of bacterial strains (Bacillus australimaris and Bacillus safensis) with highest sequence homology were retrieved from NCBI, followed by primer designing (using Primer3) and PCR. Moreover, for the confirmation of genes, gradient PCR and Gel electrophoresis were performed. The results indicated the presence of calcifying genes. Besides, the gene sequences were translated to amino acid sequences that were used for the modelling of protein structure via Swiss-Model Web Tool. In addition, the non-ureolytic pathway was also first time predicted that is likely to be involved in the precipitation of calcium carbonate by these bacterial strains. A link between the fatty acid metabolism and calcite precipitation was reported as the genes responsible for β oxidation of fatty acids were similar to the genes of calcite precipitation. Moreover, etfA encodes a membrane associated flavoprotein that is involved in the exchange of ions across the cell that are essential for the formation of calcium carbonate, outside the cell.