In recent years, the interest in obtaining microbial cement has gained its popularity along with such problems as fractures and fissures in concrete structures which is created by weathering, land subsidence, faults, earthquakes and human activities.Synthetic biology has proposed a novel way to repair and remediate problems.

One of the possible solution is biomineralization of calcium carbonate using microbes such as Bacillus species. The application of microbial concrete in construction may simplify some of the existing construction processes and revolutionize them.

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Biomineralization is a natural process in living organism by which they are able to produce minerals. Production of microbial calcium carbonate (CaCO3) is a widely studied and a promising technology with many uses in various engineering applications, for example: treatment of concrete,construction materials such as building bricks and as fillers for rubber, plastics and ink.

There are three distinct pathways of bacterial calcium carbonate precipitation:

1) biologically controlled - cellular specific control of formation of the mineral (exoskeleton, bone or teeth),

2) biologically - influenced - passive mineral precipitation caused through the presence on the surface of the cell of organic matter and

3) biologically- induced - which is the chemical alteration of an environment by biological activity.

Fig. 3: Alizarin Red staining for detection of calcium carbonate composites in the precipitated proteins: A) stained calcium carbonate powder - positive control, B) stained sample of BL21 extracted protein precipitation in CaCl 1M solution - negative control , C) stained sample of CARPs extracted protein precipitation in CaCl 1M solution..

The most commonly found mechanism in bacteria for calcite precipitation has been to generate an alkaline environment through different physiological actions. Precipitation of carbonates via urea hydrolysis by ureolytic bacteria is the most straightforward and most easily controlled mechanism of microbially induced calcium carbonate precipitation with the potential to produce high amounts of carbonates in short period of time.

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Instead of calcite precipitation from natural microbes, many other organisms also have the power to produce calcium carbonate, such as corals. In the stony coral, Stylophora pistillata, 4 acid-rich proteins (CARPs 1–4; GenBank accession numbers KC148537–KC148539 and KC493647) were identified to be responsible for calcium carbonate precipitation. These proteins were found in the study of changes in the growth of corals with increasing of acidity in the ocean.

As such, bioreaction of calcite formation is far from the thermodynamic equilibrium. It may even compromise with acidification and very low mineral saturation state (E. Tambutté & A. A. Venn et al. 2015). In our project all coral acid-rich proteins (CARPs) was cloned and expressed in E.coli BL21 strain and characterized for ability of calcium carbonate precipitation.

Fig. 1: The pathway of calcium carbonate precipitation through production of coral acid-rich proteins in E.Coli.

The putative mechanism of calcium carbonate nucleation is that highly acidic pockets of CARPs localize with the substrate and buffer thus catalyzing the reaction between calcium ion and carboxylate (2). Through high-resolution magnetic resonance spectroscopy analysis, evidence have been shown that the calcification in stony coral is mainly biologically controlled and relatively robust, due to template-induced nucleation mediated by the skeleton organic matrix, in particular, acid-rich proteins like CARPs.

Fig. 2: The highly acidic regions of the proteins interact with calcium ions (grey spheres) via coordination chemistry allowing the carboxylate groups to attract and localize calcium ions in a microenvironment, enhancing the local ionic strength. This local interaction results in a shift in pKa, favoring the formation of carbonate. Being a stronger Lewis base, with greater negative charge, carbonates displace carboxyl groups from the proteins to form stable coordination bonds with the calcium on the protein scaffold.

The positive aspect of this method is that we can increase the production of microbial calcium carbonate by reducing its sensitivity to changes in the level of acidity in the environment, as well as get rid of side products in our system such as urea. Furthermore, this is a one enzyme pathway, allowing the cost of the cells to be greatly reduced.

Team:Paris Bettencourt/Biomaterials

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