(462d) Cell Culture of the Termite Gut Microbiome Using a 3D-Printed Synthetic Microhabitat

A community of microbes living in association with a host organism is sometimes called a microbiome. The interactions among members of a microbiome can give rise to important communal functionality that cannot be achieved by its constituent organisms alone. Examples of microbiome function include normal human digestion, productive and resilient plant root systems, and the degradation of lignocellulose in lower termites such as Reticulitermes flavipes. A microbiome lives in a complex and often dynamically changing microhabitat. No single set of cell culture conditions is suitable for the entire microbiome. As a result, microbiomes are often studied in ways that destroy the community such as the use of metagenomic analysis. However, emerging technology in microfabrication is leading to novel cell culture systems that better emulate complex features of microhabitats. Here, the steep oxygen gradient that naturally exists in the lower termite hindgut was emulated in a microfluidic device using a rapid prototyping and iterative design approach. 3D-printed devices with a 200-µm resolution were used as casting molds for oxygen-permeable, polydimethylsiloxane (PDMS) cell culture chambers. The microbiota from Reticulitermes flavipes hindguts were inserted into synthetic microhabitats under anaerobic conditions before allowing diffusive flux from an 8 ppm dissolved oxygen reservoir to reach one side of the culture chamber, thereby closely emulating oxygen gradients in the real hindgut. Initial results indicate spatial reorganizations of protists within this synthetic microbiome as micro-oxygen gradients evolve. Several enabling technologies are under development that will further enhance complex microhabitat cell culture systems. One such technology is semipermeable membranes incorporated directly into disposable and easy to use 3D printed cell culture chambers for the delivery of oxygen and other nutrients. Our work contributes to a better understanding of the Reticulitermes flavipes hindgut microbiome by enabling hypothesis-driven research of how culture conditions and perturbations influence community structure and microbiome function. Eventually, gradient bioengineering technology may enable efficient, adaptive conversion of waste biomass into valuable chemicals and fuels.