Factors affecting formation of large calcite crystals (≥1mm) in Bacillus subtilis 168 biofilm

Organimoneralization is the capability of organisms to precipitate minerals. Previous studies have used Bacillus subtilis 168 as a type strain to study calcite organomineralization on bacterial biofilms while growing on the calcinogenic maximum medium B4. Calcite crystals are visible on B4 agar plates within 7 days at 37˚C after inoculum of B. subtilis 168 strain. The phenomenon is finely tuned by the net pH, buffer conditions of the precipitation medium and the bacterial metabolism. To further optimize the in vitro conditions of the system, in this study we show that to form large crystals with a diameter ≥1mm several conditions must be met: i) Reduced amount of B4 medium into the Petri plate improve crystal formation. 55mm Petri plates contained only 4mL of B4 agar medium reached a plateau in 6 days at 37ºC. High moisture and presence of water condense would decrease crystal formation. ii) Inoculation of cells using a rod instead of spotering. When the same number of B. subtilis cells was streaked, rod-shape biofilm significantly fostered crystal precipitation, while spot-shape prevented precipitation. iii) When more than one biofilm is present within the same plate, mutual interactions can affect precipitation in each biofilm. iv) Spherical nucleation sites are identified as initial step during the 29 formation of large calcite crystal. These results show that when precipitation potential of bacterial biofilms is measured in cultivation conditions, specific physical traits must also be considered in addition to the chemical environment.