# Zinc and hydroxyapatite co-localize during in vitro E. coli biofilms mineralization

## TL;DR

This study shows that zinc accumulates in mineralized E. coli biofilms and affects hydroxyapatite crystal structure, suggesting a role for zinc in biofilm mineralization.

## Contribution

The study introduces X-ray fluorescence to analyze mineralized biofilms and demonstrates zinc's role in hydroxyapatite distortion.

## Key findings

- Zinc accumulates in mineralized regions of E. coli biofilms alongside calcium and phosphate.
- Zinc partially substitutes calcium in hydroxyapatite, causing crystal lattice shortening.
- Higher zinc concentrations in abiotic models increase hydroxyapatite distortion and reduce crystal size.

## Abstract

Biofilms are biological materials that form as bacteria protect themselves from environmental challenges secreting extracellular matrix and accumulating minerals under specific conditions. 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 introduced X-ray fluorescence techniques as powerful tools to analyze the composition of mineralized biofilms 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.

The online version contains supplementary material available at 10.1038/s41598-025-33942-3.