Microbial Surfaces and their Effects on Carbonate Mineralization

Geologic carbon dioxide sequestration, the underground storage of carbon dioxide (CO₂), will be an essential component of climate change mitigation. Carbonate minerals are a promising form of stable CO₂ storage, but their geologic formation is slow. Many microbes can increase the rate of carbonate mineral formation; however the mechanisms of such mineralization are largely unknown. Hypothesized mechanisms include metabolic processes that alter pH and supersaturation, as well as cell surface properties that induce mineral nucleation. This work systematically investigates these mechanisms by allowing calcium carbonate (CaCO₃) to form in the presence or absence of microbes with various surfaces features included Escherichia coli, Synechocystis sp. PCC 6803, Caulobacter vibrioides, and Lysinibacilllus sphaericus. Surprisingly, formation of stable crystalline CaCO₃ was accelerated by the presence of all microbes relative to abiotic solutions. This rate acceleration also occurred for metabolically inactive bacteria, indicating that metabolic activity was not the operating mechanism. Rather, since the CaCO₃ crystals increased in number as the cell density increased, these results indicate that many bacterial species accelerate the nucleation of CaCO₃ crystals. To understand the role of specific biomolecules on nucleation, we used genetic mutants with altered lipopolysaccharides (LPS) and crystalline surface layer proteins (S-layers). Bacterial surface charge and cation binding was assessed using zeta potential measurements and correlated to the bacterial surface chemistry and biomineralization experiments with varying Ca²⁺ concentrations. From these results, we postulate that the S-layer surfaces can selectively attract Ca²⁺ ions, serving as nucleation sites for CaCO₃, thereby accelerating crystal formation. These observations provide substantive evidence for a non-specific nucleation mechanism, and stress the importance of microbes, even dead ones, on the rate of formation of carbonate minerals. This work also indicates that additional microbial engineering specifically targeted to S-layer proteins could optimize these interactions and be used to implement the sequestration of CO₂ as stable mineral carbonates on an accelerated timescale.