Building 3D printed synthetic biology to explore biofilm lifestyle

Abstract

A predominant proportion of the microbial biosphere has been naturally selected to live and grow as biofilms. Biofilms are communities of microorganisms where cells live attached to surfaces and/or other cells and are embedded in self-produced extracellular polymeric substances. Biofilms are spatially structured, often in three dimensions, and small-scale heterogeneity has important consequences for community-level behavior and ecology. However, biofilms are usually studied as self-assembled systems, and the physical forces that drive biofilm structural development are difficult to manipulate at small scales. 3D hydrogel bioprinting is an emerging technology directly addressing the need for small-scale control over biofilm structure and has been used to great effect for building synthetic biofilms with desirable functional characteristics. However, the selective environment of a hydrogel matrix is often different from where biofilms were naturally selected to grow. The intrinsic behavioral responses of microorganisms to encapsulation in hydrogel matrices are not well understood. To effectively design synthetic biofilms for diverse functional purposes, predicting and understanding encapsulated microbial behavior are salient goals. We integrated 3D bioprinting with quantitative microscopy and flow cell reactors to observe spatiotemporal population dynamics in synthetic biofilms. We varied initial population density, varied external transport and nutrient conditions, and compared population growth of synthetic and self-assembled biofilms among Pseudomonas isolates. Results suggest that (i) length scale of biovolume development can be controlled by altering initial population density, (ii) hydrogel carrying capacity can be altered by changing external fluid velocity but not initial population density, (iii) growth and behavior of self-assembled biofilms in fluctuating transport and nutrient environments does not predict growth and behavior of synthetic biofilms, (iv) encapsulation in a hydrogel matrix acts as a selection bottleneck where only a subset of the initial population is detectable with fluorescence microscopy, and (v) encapsulation in the hydrogel matrix homogenizes growth rate averages compared to self-assembled biofilms under identical fluid velocity conditions. Altogether, spatiotemporal population dynamics of synthetic 3D printed biofilms were distinct from self-assembled biofilms. Integrating observations of hydrogel- encapsulated cell behavior with synthetic biofilm design will aid efforts in developing biofilm- based biotechnology and aid in vitro structural-functional mimicry of natural biofilms.