3D printing of microbial communities: A new platform for understanding and engineering microbiomes.

3D printing has emerged as a powerful way to produce complex materials on-demand. These printing technologies are now being applied in microbiology, with many recent examples where microbes and matrices are co-printed to create bespoke living materials. Here, we propose a new paradigm for microbial printing. In addition to its importance for materials, we argue that printing can be used to understand and engineer microbiome communities, analogous to its use in human tissue engineering. Many microbes naturally live in diverse, spatially structured communities that are challenging to study and manipulate. 3D printing offers an exciting new solution to these challenges, as it can precisely arrange microbes in 3D space, allowing one to build custom microbial communities for a wide range of purposes in research, medicine, and industry.

Droplet printing reveals the importance of micron-scale structure for bacterial ecology.

Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology. However, a test of this hypothesis requires manipulation at the fine scales at which spatial structure naturally occurs. Here we develop a droplet-based printing method to arrange bacterial genotypes across a sub-millimetre array. We print strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins. Our experiments reveal that toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge where susceptible strains can persist in large numbers. Moving to competitions between toxin producers reveals that spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between competing strains to understand how ecological refuges form, which shows that cells closest to a toxin producer mop up the toxin and protect their clonemates. Our work provides a method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in ecology.

Controlled packing and single-droplet resolution of 3D-printed functional synthetic tissues.

3D-printing networks of droplets connected by interface bilayers are a powerful platform to build synthetic tissues in which functionality relies on precisely ordered structures. However, the structural precision and consistency in assembling these structures is currently limited, which restricts intricate designs and the complexity of functions performed by synthetic tissues. Here, we report that the equilibrium contact angle (θDIB) between a pair of droplets is a key parameter that dictates the tessellation and precise positioning of hundreds of picolitre-sized droplets within 3D-printed, multi-layer networks. When θDIB approximates the geometrically-derived critical angle (θc) of 35.3°, the resulting networks of droplets arrange in regular hexagonal close-packed (hcp) lattices with the least fraction of defects. With this improved control over droplet packing, we can 3D-print functional synthetic tissues with single-droplet-wide conductive pathways. Our new insights into 3D droplet packing permit the fabrication of complex synthetic tissues, where precisely positioned compartments perform coordinated tasks.

Self-transformation and structural reconfiguration in coacervate-based protocells.

A functionalized small-molecule dipeptide capable of structural adaptation is used to prepare coacervate-based protocells that exhibit a pH-triggered process of self-transformation and structural reconfiguration. Polymer-dipeptide coacervate micro-droplets are prepared at pH 8.5 from aqueous mixtures of poly(diallyldimethylammonium chloride) and deprotonated N-(fluorenyl-9-methoxycarbonyl)-d-Ala-d-Ala, and transform into discrete aster-like micro-architectures by controlled lowering of the pH to 4.5. Reconfiguration of the micro-droplets results in entanglement and formation of an interpenetrating fibrous network that subsequently develops into a polymer-containing dipeptide hydrogel. Our results provide a step towards the assembly of synthetic protocells exhibiting rudimentary aspects of metamorphosis, and should offer a new approach to the design and construction of soft reconfigurable chemical micro-ensembles.

Cerium oxide nanoparticle-mediated self-assembly of hybrid supramolecular hydrogels.

Hybrid supramolecular hydrogels are prepared by non-enzymatic dephosphorylation of N-fluorenylmethyloxycarbonyl tyrosine-(O)-phosphate (FMOC-Tyr-P) using catalytic cerium oxide nanoparticles. The organic-inorganic hydrogels exhibit enhanced viscoelastic properties compared with analogous materials prepared using alkaline phosphatase.