ExFAB automated metabolite extraction from microbial cultures

DOI #10.17504: The ExFAB biofoundry seeks to develop automated workflows to discover, phenotype, and prototype biology from extreme and exceptional fungi, archaea, and bacteria. To this end, ExFAB has active metabolomics workflows that enable metabolite discovery and analysis. This protocol describes a semi-automated method for the methanol extraction of metabolites from microbial cell cultures. The protocol leverages the automated liquid handling and centrifugation capabilities of a Tecan Fluent 780 system equipped with 96- and 8-channel pipetting heads and an associated Rotanta 460 Robotic centrifuge. The protocol begins with plate-based microbial culture samples, isolates cell pellet and spent media samples, and processes these samples for metabolite extraction. First, spent media samples are isolated and prepared for solid phase extraction. These samples are processed offline using a multi-sample vacuum manifold, producing 100 μL of methanol extracted metabolites. Next, cell pellets are processed using the Tecan Fluent 780 and associated equipment, ultimately producing 300 μL metabolite samples in 80% methanol that are ready for downstream analysis by mass spectrometry. The protocol is designed to process two 96-deepwell plates per run with a runtime of approximately 6 hours and requires user intervention for cell pellet disruption and spent media extraction. The protocol was validated by extracting metabolites from each isolate of 304-member library of the basidiomycetes Rhodoturula.