SubTap, a Versatile 3D Printed Platform for Eavesdropping on Extracellular Interactions.

Communication within the microbiome occurs through an immense diversity of small molecules. Capturing these microbial interactions is a significant challenge due to the complexity of the exometabolome and its sensitivity to environmental stimuli. Traditional methods for acquiring exometabolomic data from interacting microorganisms are limited by their low throughput or lack of sampling depth. To address this challenge, we introduce subtapping (short for substrate tapping), a technique for tapping into extracellular metabolites that are being transferred through the growth substrate during coculture. High-throughput subtapping is made possible by a new coculturing platform, named SubTap, that we engineered to resemble a 96-well plate. The three-dimensional (3D) printed SubTap platform captures the exometabolome in an agar compartment that connects physically separated growth chambers, which permits cell growth without competition for space. We show how SubTap facilitates replicable and quick detection of exometabolites via direct infusion mass spectrometry analysis. Using bacterial isolates from the soil, we apply SubTap to characterize the effects of growth medium, growth duration, and mixed versus unmixed coculturing on the exometabolome. Finally, we demonstrate SubTap's versatility by interrogating microbial interactions in multicultures with up to four strains.