Diversification of biofilm architecture among freshwater Pararheinheimera isolates.

UNLABELLED: Extracellular matrices that scaffold microbial biofilms offer a rich source of polymeric materials with a range of potential applications. However, our understanding of these matrices is largely restricted to model organisms that form biofilms with specific architectures. Examining biofilms from understudied niches has the potential to identify extracellular matrices with novel properties. Here, we targeted a resource-rich boundary at the top of the water column known as the air-liquid interface (ALI). ALIs lack a solid substrate for cellular attachment, suggesting that microbes utilize specialized biofilm architectures to persist at this boundary. We used samples from a lake to enrich for microbes that colonize the ALI. Mixed-species pellicle biofilms formed rapidly in these enrichments and displayed a pronounced ecological succession. We isolated 31 members of the genus Pararheinheimera from early stages of pellicle maturation that formed five phylogenetically distinct clades. We used representative isolates to show that only one Pararheinheimera clade formed adherent films resembling classical pellicles. Isolates from the remaining clades formed floating structures that could be categorized either as non-adhesive films or viscous masses (VMs). VM pellicle formation was a polyphyletic trait that correlated with a mucoid appearance on agar plates, suggesting that the process is driven by copious extracellular matrix secretion. Matrices from VM biofilms were largely non-adhesive, contained a mixture of acidic polysaccharides and proteins, and formed thermally stable, shear-thinning hydrogels. Our results demonstrate that ALI colonization strategies vary widely even among closely related aquatic bacteria and identify VM pellicles as a promising platform for biomaterials development. IMPORTANCE: Microbial biofilms contain specialized polymers with immense industrial potential, yet only a subset of biofilm architectures has been studied in detail. Many aquatic microbes live within a boundary at the surface of lakes, rivers, and oceans known as the air-liquid interface (ALI). The strategies microbes use to form biofilms at this boundary remain poorly characterized. Our study investigated how bacteria from a freshwater lake accumulate at the ALI. Lake water samples incubated in nutrient medium formed a floating pellicle biofilm, and we isolated 31 bacteria from the genus Pararheinheimera that were abundant during the early stages of pellicle formation. Only a subset of Pararheinheimera isolates formed traditional pellicle biofilms. Most formed either thin, non-adhesive films or large, gelatinous aggregates that appeared to persist at the ALI due to buoyancy. These findings expand our understanding of biofilm diversity in aquatic systems and suggest that the production of buoyant hydrogels could be useful scaffolds for engineering new materials.