Accumulation of zinc during E. coli biofilm mineralization: within or surrounding the hydroxyapatite?

Biofilms are biological materials that form as bacteria secrete extracellular matrix to protect themselves from environmental challenges. Specific conditions lead to biofilm mineralization, like in dental calculus or kidney stones. To understand biofilm formation and mineralization, we grew Escherichia coli on agar plates containing a nutritive and mineralizing medium. Previous studies showed that the alkaline phosphatase (ALP) present in E. coli biofilms leads to hydroxyapatite precipitation in such conditions. Here, we analyzed the composition of mineralized biofilms with X-ray fluorescence techniques in two and three dimensions. In addition to calcium and phosphate, we found that the traces of zinc introduced via the nutrients and bacteria, also accumulates in the mineralized regions. Wide-angle X-ray scattering performed on dry mineralized biofilm powder also revealed a shortening of the hydroxyapatite crystal lattice, which could indicate partial substitution of calcium by zinc. Using an abiotic model, we reproduced enzymatic hydroxyapatite mineralization with pure ALP, without bacteria nor extracellular matrix. In this model, hydroxyapatite distortion increased with zinc concentration, whereas crystal size decreased. Since zinc is a ubiquitous element that is essential to bacteria metabolism but also antibacterial, these results are fundamental for biofilm research and call for further studies on zinc management in biofilms.