Bacterial EPS in Agarose Hydrogels Directs Mineral Organization in Calcite Precipitates: Species-Specific Biosignatures of Bacillus subtilis, Mycobacterium phley, Mycobacterium smagmatis, and Pseudomonas putida EPS

Abstract

Hydrogels present model systems for biopolymer matrices in biological structural materials, as their fabric and physicochemical properties can be tailored to mimic characteristics of polymer matrices in biological hard tissues. However, hydrogels comprise synthetic compounds and lack attributes of native biopolymers, in contrast to extracellular polymeric substance (EPS) actively secreted by microbes for protection and enhancement of physiological activities. For testing the directing influence of native biopolymers on mineralization, we precipitated calcite/gel composite aggregates from agarose gels containing EPS of Bacillus subtilis, Mycobacterium phley, Mycobacterium smagmatis, or Pseudomonas putida, respectively. We characterized the aggregates with Fourier transform infrared spectroscopy, field-emission scanning electron microscopy imaging, and electron backscatter diffraction. Relative to reference aggregates devoid of EPS, aggregates containing EPS are reduced in size and show distinctive morphologies directed by the EPS of a specific bacterium. In P. putida and M. phley composites, occluded polymers are present as membranes, for M. smagmatis and B. subtilis occluded polymers are mainly developed as networks of fibrils. Precipitate crystal subunit formation in EPS-containing composites is extensive compared to the reference crystal; subunits vary in shape, size, and organization: for M. smagmatis and B. subtilis, subunit organization is radial to spherulitic; for P. putida, it is random; for M. phley, it is coaligned in all three dimensions (single-crystal-like). Bacterial EPS changes mineral microstructure/texture in a species-specific manner, a characteristic that, when developed further, might be used as an identification tool for bacterial calcification in present/past environments.