Biofilms are biological living tissues that occur after bacteria colonize a surface and synthesize an extracellular matrix, as a survival strategy in challenging environments. In addition to the organic matrix, some biofilms precipitate mineral particles such as calcium phosphates and calcium carbonates. Previously, we investigated the role of alkaline phosphatase (ALP) in the precipitation of calcium phosphates in the form of hydroxyapatite in biofilms produced by the model strain E. coli K-12 W3110 (1). From cryo-focused ion beam-scanning electron microscopy, we could identify both mineralized bacteria and mineralized portions of the extracellular matrix. With few exceptions, microbial mineralization usually results from adventitious precipitation of inorganic compounds led by their interactions with different metabolic processes (2). Such biologically induced mineralization results directly from microbial activity, tends to accumulate minerals on the surface of the bacteria, and eventually embed them in the growing crystals (2). Moreover, biominerals can also be influenced by interactions of their precursors with the environment, e.g. the extracellular matrix (3). In this context, we investigated the influence of different macromolecules in the biofilm on the formation of calcium phosphate crystals. On nutritive agar substrates inducing mineralization, we cultivated diverse E. coli strains, which produce as extracellular matrix only curli fibers, only phosphoethanolamine-cellulose fibers, both or none of them (4). We estimated crystalline lattice parameters using wide-angle x-ray scattering and assessed crystal strain at the nanoscale. Microindentation was exploited to study the influence of the extracellular matrix on the mechanical properties of the mineralized and unmineralized biofilms. Uncovering the influence of the biomolecules present in the extracellular matrix is an important piece of knowledge to engineer living composites.

1. L. Zorzetto, E. Scoppola, E. Raguin, K. G. Blank, P. Fratzl, C. M. Bidan, Induced Mineralization of Hydroxyapatite in Escherichia coli Biofilms and the Potential Role of Bacterial Alkaline Phosphatase. Chem. Mater. 35, 2762–2772 (2023).
2. S. Mann, Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry (Oxford University Press, New York, NY, 2001; https://doi.org/10.1021/cg020033l).
3. D. N. Azulay, M. Fraenkel, L. Chai, A Bacterial Biofilm Polysaccharide Affects the Morphology and Structure of Calcium Oxalate Crystals. Cryst. Growth Des. 23, 7853–7862 (2023).
4. Di. O. Serra, R. Hengge, Bacterial Multicellularity: The Biology of Escherichia coli Building Large-Scale Biofilm Communities. Annu. Rev. Microbiol. 75, 269–290 (2021).