(639a) Microwell Arrays for Screening Interactions Between Root-Associated Microbes

Authors: 
Hansen, R., Kansas State University
Retterer, S. T., Oak Ridge National Laboratory
McGinley, L., Kansas State University
Timm, A., Oak Ridge National Lab
Timm, C. M., University of Wisconsin-Madison
Doktycz, M. J., Oak Ridge National Laboratory
Plant growth promoting rhizobacteria (PGPR) associate with root surfaces in order to provide benefits to the plant host, some of which include improved nutrient uptake, pathogen protection, and plant hormone regulation. An improved understanding of the relationships between PGPR and root hosts has spurred recent interest in developing constructed, artificial communities of PGPR for use as a low-energy, sustainable alternative to chemical fertilizers. However, wide-spread application of constructed communities has been limited due to the fact that the vast majority of microbe-microbe interactions occurring between PGPR in the rhizosphere are unknown or poorly characterized. This limitation stems from the fact that traditional microbiological assays designed to characterize microbial interactions do so using co-culturing methods that are qualitative and low-throughput. Additionally, these assays fail to re-create the physical and chemical complexities of natural ecosystems, such as soil or rhizospere, which are spatially confined microenvironments. To address these types of limitations, we have developed a microfabricated cell-screening platform termed the â??microbe arrayâ? which uses a novel biofabrication method to randomly assemble thousands of independent bacterial meta-populations into microscale wells with controlled physicochemical properties. It was recently shown that by tuning well dimensions, interacting bacteria can be combined into wells at low dispersity, allowing for the assembly of replicate initial populations; or at high dispersity, allowing for thousands of unique meta-populations to be assembled across a single array of wells. After this, communities can be simultaneously screened for growth or decay to identify combinations of bacteria and environmental parameters that promote or inhibit colonization. In development of this platform, we investigated the effect of spatial confinement on the growth of Pseudomonas bacteria. Here, populations were assembled into wells at high dispersity, and growth screening experiments revealed that successful colonization of microbial communities in a spatially confined, diffusion limited environment is highly dependent on inoculum levels. Our current work focuses on screening rhizobacterium populations in this platform to identify pathogenic, competitive or symbiotic interactions that inform the development of constructed communities for biofertilizer and biocontrol applications.