Defining the toxicity limits on microbial range in a metal-contaminated aquifer Running Title: Inorganic ion toxicity limits on microbial range

Abstract In extreme environments, toxic compounds restrict which microorganisms persist. However, in complex mixtures of inhibitory compounds, it is challenging to determine which specific compounds cause changes in abundance and prevent some microorganisms from growing. We focused on a contaminated aquifer in Oak Ridge, Tennessee, U.S.A. that has low pH and high concentrations of uranium, nitrate and many other inorganic ions. In the most contaminated wells, the microbial community is enriched in the Rhodanobacter genus. Rhodanobacter relative abundance is positively correlated with low pH and high concentrations of U, Mn, Al, Cd, Zn, Ni, Co, Ca, NO3−, Mg, Cl, SO42−, Sr, K and Ba and we sought to determine which of these correlated parameters are selective pressures that favor the growth of Rhodanobacter over other taxa. Using high-throughput cultivation, we determined that of the ions correlated high Rhodanobacter abundance, only low pH and high U, Mn, Al, Cd, Zn, Co and Ni (a) are selectively inhibitory of a sensitive Pseudomonas isolate from a background well versus a representative resistant Rhodanobacter isolate from a contaminated well, and (b) reach toxic concentrations in the most contaminated wells that can inhibit the sensitive Pseudomonas isolate. We prepared mixtures of inorganic ions representative of the most contaminated wells and verified that few other isolates aside from Rhodanobacter can tolerate these 8 parameters. These results clarify which toxic inorganic ions are causal factors that impact the microbial community at this field site and are not merely correlated with taxonomic shifts.