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.
