LB 2-9 - Building a synthetic microbiome for Lemna minor: A system for investigating nitrogen removal and plant-microbe communication during water remediation

Duckweeds are small, fast-growing, aquatic plants that can remediate the water they inhabit by accumulating nutrients and environmental pollutants. Duckweeds are already utilized in constructed wetlands to remove excess nitrogen from agricultural, municipal, and factory wastewaters and some have a demonstrated potential to take up polluting antibiotics. Laboratory experiments suggest that the relationships between duckweeds and their microbiomes can be harnessed for improved bioremediation. This possibility can be explored by characterizing natural duckweed microbiomes, developing synthetic duckweed microbiomes, and challenging duckweed-microbiome systems with pollutants of concern to select for a maximally remediating community. We are interested in remediating agricultural wastewaters and urban runoff contaminated with excess ammonia and the antibiotic sulfamethoxazole and aim to establish a duckweed-microbe community that remediates these pollutants with high efficiency. The first steps in this process are to isolate duckweed-inhabiting bacteria and to develop synthetic microbiomes that stably colonize duckweed plants. We have isolated more than 70 bacterial strains from duckweed microbiomes collected in and near New York state and here present our initial effort to construct minimal synthetic microbiomes stably cohabiting duckweed plants. Our work along these lines includes 1) investigating the development and stability of a five-member synthetic microbiome using 16s sequencing to compare microbiome composition at different time points 2) comparing a microbiome of closely related bacteria to one composed of more distantly related bacteria to test the hypothesis that closely related bacteria will produce less stable communities than groups of distantly related bacteria and 3) challenging a stable synthetic microbiome with ammonia and sulfamethoxazole. Sterile duckweeds (Lemna minor) were co-inoculated with five of our bacterial isolates representing five families (Pseudomonadaceae, Micrococcaceae, Oxalobacteraceae, Bacillaceae and Sphingobacteriaceae) and grown for nine days in modified Hoagland’s growth media supplemented with Ammonium Nitrate to simulate agricultural wastewater. After nine days, bacteria collected from duckweed surfaces formed colonies with four different morphologies when plated, suggesting that at least four of the five bacterial strains remained throughout the duration of the experiment. 16s amplicon sequencing is in progress to confirm which strains survived on the duckweed surfaces and in what proportions across the elevated-nitrogen treatment groups.