Advanced Manufacturing and Microenvironment Control for Bioengineering Complex Microbial Communities

Micro- and millifluidic, or lab-on-a-chip, devices are superior platforms for cell culture applications where replicating complex microenvironments or spatial interactions are important. Unlike traditional cell culture techniques, such as agar plates, liquid culture flasks, or microwell plates, they are able to more accurately reproduce the micro-scale geometry of actual environments. Furthermore, lab-on-a-chip microhabitats enable researchers to have better control over environmental conditions, such as oxygen, nutrient, or other chemical gradients, on a cellular scale. This research project aims to develop tools to enable replication of the oxygen and chemical gradients found within the termite digestive tract in order to culture and study the complex microbial community inhabiting termites in nature. Reproducing the physiochemical environment inside the termite gut requires a number of engineering solutions. One particular design requirement is maintaining nearly anoxic conditions in much of the device, with a slightly oxic gradient near the exterior boundary. In order to accomplish this, a procedure was developed to fabricate microelectrodes in situ along the interior side walls of a microfluidic channel. Theoretically, at these microelectrodes, oxygen could be produced and used to supply the cell culture area of the device. While ultimately oxygen and nitrogen saturated liquids were used for establishing oxygen gradients within test devices due to ease of use, the surface chemistry and specific photopatterning techniques developed can be used for a variety of other applications. Additionally, a 3D-printed cell culture millifluidic device was designed and manufactured to enable long-term microbial cell culture. Methods were developed to test and minimize adverse biotoxicity effects of 3D-printed polymer, which had not previously been done with bacterial cells. Hydrogel polymer barriers were engineered to form a boundary between the substrate supply flow channel and the cell culture area of the designed device. These UVpatterned hydrogels allowed diffusion of oxygen, carbon substrates, and other important chemical species into the culture area, but successfully prevented cell motility out of the specified region when tested with the bacteria Pseudomonas putida KT2440. The P. putida cells were successfully cultured in this proof-of-concept device for 44 h, which had not been documented with a bacteria in a stereolithography 3D-printed channel before. The incorporation of additive manufacturing in this project has identified 3D-printed as an enabling technology for scaling up from microfluidic to millifluidic devices and making lab-on-a-chip technology more attainable for a wider variety of users. The techniques and engineering tools developed in this project may be applied to design, fabricate, and control conditions with a wide variety of cell types and synthetic microhabitat systems.