Iterative Prototyping of Bioengineered Microenvironments Using 3D Printing

Microbial consortia may have metabolic capabilities that far exceed those of pure cultures. For example, the microbiome present in the hindgut of the lower termite Reticulitermes flavipes can efficiently and adaptively produce biomolecules from lignin. This consortium could be enormously valuable for biofuel production, but it has evolved to live within steep oxygen gradients and cannot be cultured using standard methods. Here we describe a process of iterative microenvironment bioengineering to create and optimize gradient microhabitats for support of this microbial consortia. Stereolithography printing was used to create a photopolymer resin cavity mold master, designed for soft lithography with polydimethylsiloxane. A practical x-y resolution of 500 µm and a z-plane resolution of 300 µm was achieved with our Formlabs Form 1+ stereolithography printer. Culture experiments with R. flavipes gut contents confirm stereolithography can produce cavity mold masters with suitable features to support this microbial consortium. Most importantly, the iteration cycle time including simulation-enabled design, 3D printing the master, and device production could be completed at low cost and in as little as 24 hours (compared with 1-2 weeks for traditional photolithography). Cheap, quick turnaround is essential when nearly everything related to culturing a microbial consortium must be learned by trial and error. Faster, cheaper, and more accurate printing options, paired with expanding material choices, will continue to transform the way microenvironments are engineered. One day, gradient bioengineering may allow society to make full use of the extraordinary capabilities of microbial consortia.