H-Aquil: A Chemically Defined Cell Culture Medium for Trace Metal Studies in Vibrios and Other Marine Heterotrophic Bacteria

Abstract

Iron (Fe) is a micronutrient necessary for the growth of marine microorganisms. Well-established defined media exist to study phytoplankton Fe usage. However, a defined medium for facilitating Fe acquisition research in marine heterotrophic bacteria has not yet been developed. Studying Fe and other trace metal usage in marine bacteria is near impossible using existing complex media as these recipes utilize ill-defined chemical mixtures like tryptone, casamino acids, and yeast extract. These components can introduce unknown amounts of metal contamination and complicate trace metal speciation. Thus, a defined medium with precisely controlled trace metals is necessary for conducting Fe studies. Here we present H-Aquil (Aquil for marine heterotrophic bacteria), a new recipe for a fully defined and low Fe medium suitable for investigating Fe usage in a variety of marine bacteria. This recipe consists of artificial seawater supplemented with glycerol, phosphate, amino acids, vitamins, and Aquil trace metals, including the synthetic chelator EDTA. We used H-Aquil to culture numerous marine bacteria, including species of Vibrio, Phaeobacter, Silicibacter, and several bacterial isolates from the environment. In addition, we conducted Fe-limitation studies with these cultures in H-Aquil. We report an average decrease in growth rate of 50% between cultures grown with 1 μM Fe and no added Fe. We also explored Fe-limitation in SAR11, one of the most abundant clades of marine bacteria, by modifying its defined medium (AMS1) with Aquil trace metals and EDTA. Overall, H-Aquil is suitable for culturing and conducting Fe-acquisition studies in a variety of marine heterotrophic bacteria.