Uncovering the Dynamics of Urease and Carbonic Anhydrase Genes in Ureolysis, Carbon Dioxide Hydration, and Calcium Carbonate Precipitation.

The hydration of CO2 suffers from kinetic inefficiencies that make its natural trapping impractically sluggish. However, CO2-fixing carbonic anhydrases (CAs) remarkably accelerate its equilibration by 6 orders of magnitude and are, therefore, "ideal" catalysts. Notably, CA has been detected in ureolytic bacteria, suggesting its potential involvement in microbially induced carbonate precipitation (MICP), yet the dynamics of the urease (Ur) and CA genes remain poorly understood. Here, through the use of the ureolytic bacteriumSporosarcina pasteurii, we investigate the differing role of Ur and CA in ureolysis, CO2 hydration, and CaCO3 precipitation with increasing CO2(g) concentrations. We show that Ur gene up-regulation coincides with an increase in [HCO3-] following the hydration of CO2 to HCO3- by CA. Hence, CA physiologically promotes buffering, which enhances solubility trapping and affects the phase of the CaCO3 mineral formed. Understanding the role of CO2 hydration on the performance of ureolysis and CaCO3 precipitation provides essential new insights, required for the development of next-generation biocatalyzed CO2 trapping technologies.

Controlling the calcium carbonate microstructure of engineered living building materials.

The fabrication of responsive soft materials that enable the controlled release of microbial induced calcium carbonate (CaCO3) precipitation (MICP) would be highly desirable for the creation of living materials that can be used, for example, as self-healing construction materials. To obtain a tight control over the mechanical properties of these materials, needed for civil engineering applications, the amount, location, and structure of the forming minerals must be precisely tuned; this requires good control over the dynamic functionality of bacteria. Despite recent advances in the self-healing of concrete cracks and the understanding of the role of synthesis conditions on the CaCO3 polymorphic regulation, the degree of control over the CaCO3 remains insufficient to meet these requirements. We demonstrate that the amount and location of CaCO3 produced within a matrix, can be controlled through the concentration and location of bacteria; these parameters can be precisely tuned if bacteria are encapsulated, as we demonstrate with the soil-dwelling bacterium Sporosarcina pasteurii that is deposited within biocompatible alginate and carboxymethyl cellulose (CMC) hydrogels. Using a competitive ligand exchange mechanism that relies on the presence of yeast extract, we control the timing of the release of calcium ions that crosslink the alginate or CMC without compromising bacterial viability. With this novel use of hydrogel encapsulation of bacteria for on-demand release of MICP, we achieve control over the amount and structure of CaCO3-based composites and demonstrate that S. pasteurii can be stored for up to 3 months at an accessible storage temperature of 4 °C, which are two important factors that currently limit the applicability of MICP for the reinforcement of construction materials. These composites thus have the potential to sense, respond, and heal without the need for external intervention.

Characterisation of CaCO3 phases during strain-specific ureolytic precipitation.

Numerous microbial species can selectively precipitate mineral carbonates with enhanced mechanical properties, however, understanding exactly how they achieve this control represents a major challenge in the field of biomineralisation. We have studied microbial induced calcium carbonate (CaCO3) precipitation (MICP) in three ureolytic bacterial strains from the Sporosarcina family, including S. newyorkensis, a newly isolated microbe from the deep sea. We find that the interplay between structural water and strain-specific amino acid groups is fundamental to the stabilisation of vaterite and that, under the same conditions, different isolates yield distinctly different polymorphs. The latter is found to be associated with different urease activities and, consequently, precipitation kinetics, which change depending on pressure-temperature conditions. Further, CaCO3 polymorph selection also depends on the coupled effect of chemical treatment and initial bacterial concentrations. Our findings provide new insights into strain-specific CaCO3 polymorphic selection and stabilisation, and open up promising avenues for designing bio-reinforced geo-materials that capitalise on the different particle bond mechanical properties offered by different polymorphs.