(542f) Culturing Spatially Separated Anaerobes in Porous Microfluidic Culture Chambers

Title: Culturing Spatially Separated Anaerobes in Porous Microfluidic Culture Chambers

Authors: Peter G. Shankles, M. J. Doktycz, J. Morrell-Falvey, M. Podar, S. T. Retterer

Microbial communities are found throughout our environment and adapt unique architectures that enhance their survival and function. Their impact on our ecosystems and our individual health is tremendous.  Current studies often rely on the use of non-culture based methods to study natural community composition. Such metagenomic and single-cell genomic techniques give rise to a more complete understanding of the individuals that comprise these communities and their metabolic potential. However, these techniques fail to capture the spatial dependencies and heterogeneities that shape community structure and function.  Moreover, such techniques do not yield organisms for physiological studies, screening of specific microbe-microbe interactions, or the construction and testing of synthetic communities that can be used to recapitulate the function of natural communities.

The oral microbiome consists of over 1000 distinct species of microbes, and has a much higher culture rate than other natural environments. Nonetheless, only about half of the microbes catalogued using omics analysis have been successfully cultured.   Even fewer of those associated with periodontal disease have been successfully isolated. Coevolution has caused the microbes associated with this disease to develop a complex system of relationships that are crucial to the growth of the microbes. As a result, the microbes do not grow when isolated individually. In order to culture uncultured bacteria from the oral microbiome, a co-culture approach, where bacteria are cultured together based on predicted dependencies, can be used.

In this study, we look at cultured strains of bacteria associated with periodontal disease in a microfluidic culture chamber to better understand the effects that co-culture has on growth of these bacteria. The goal of this study is to develop a platform through which sustainable culture of microbes associated with periodontitis can be achieved. To this end, we have developed a PDMS-based microfluidic device that incorporates culture chambers, media channels, and gas diffusion to allow for the culture of anaerobic bacteria. Multiple chambers are linked through micro- and nanofabricated pores that constrict the movement of microbes between chambers, but allow for cell-to-cell communication through molecular diffusion. The device is kept in an anaerobic state with a gas diffusion layer in the PDMS. CO₂ and N₂ are mixed at a ratio of 20:80 and flowed through the system. A thin, 100 mm separation, allows for gas diffusion that maintains an anaerobic environment. In earlier work, operation of the microfluidic culture chamber array, and transport across the porous membranes was characterized using fluorescent small molecules, nanoparticles and sender and receiver strains of E. coli.  In the latter case, the production of AHL by the sender strains and transport of the small molecule signal across the connecting membranes was characterized by observing the fluorescence of the receiver strain in response to the diffusing AHL signal.  Ongoing tests are intended to quantify the effects of co-culture on oral microbiome species such as Synergistetes and Desulfobulbus.  Effects of co-culture on community health will be determined using these cultured microbes. In future studies, the demonstrated device can be applied to uncultured oral microbes as well as microbes from other environments.