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

B4 is the most common medium used in general organomineralization studies and has been used to assay or to characterize mineral precipitation potential. In an exercise for the optimization of the laboratory conditions of crystal precipitation in vitro, we used Bacillus subtilis 168 as a type strain and its isogenic mutants. While literature is mainly focused on observing generic precipitation, we investigated the requirement to obtain large crystals (≥1mm), which could be advantageous in wide-ranging implications for bioconsolidation of soil, sand, stone, and cementitious materials. Calcite crystals are visible on B4 agar plates within 7 days at 37˚C after inoculum of B. subtilis 168 strain. 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 a circular shaped spot. 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 formation of large calcite crystal