Fig. 7

Schematic diagram of engineered calcium carbonate precipitation in Bacillus subtilis W168. The cellular processes and features are shown that drive biomineralisation in the engineered ureolytic precipitator strain (left) and native B. subtilis W168 (right). In the precipitator strain, heterologously produced urease catalyses the breakdown of urea leading to an increased pH of the microenvironment (pink gradient) which favours calcium carbonate precipitation. The presence of accessory proteins UreE, UreF, UreG, and UreD as well as nickel and urea transporters contribute to increased ureolysis and precipitation. At sufficiently high pH, precipitates may even form in the micro-environment surrounding the cell. The presence of exopolysaccharides within the biofilm matrix amplifies precipitation promoted by the increased pH and may act as additional nucleation templates. Proteinaceous fibres were not found to promote precipitation. Negatively charged sites on the cell surface likely act as nucleation sites in both native and engineered B. subtilis, but are by themselves insufficient to drive detectable biomineralisation. Native B. subtilis W168 increases the localised pH only slightly from metabolic activities (such as amino acid degradation), which is not sufficient to reach conditions required for precipitation. Low levels of undetected precipitates are still likely to form on the cell surface and extracellular matrix