Updated project metadata. Biofilms are structured communities of tightly associated cells that constitute thepredominant state of bacterial growth in naturaland human-madeenvironments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilishas been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, weperformed a time-resolved analysisof the metabolic changes associated with pellicle biofilm formation and development inB. subtilisby combining metabolomic, transcriptomic, and proteomic analyses. We report a surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hithertounrecognized as biofilm-associated.For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was generally controlled at the transcriptional level. Our resultsalsoprovide insights into the transcription factors and regulatory networks involved in thiscomplexmetabolic remodeling. Following upon these results, we demonstrate that acetoin production via acetolactate synthase is essential for robust biofilm growthand has the dual role of conservingredox balance and maintaining extracellularpH.This study represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilisbiofilm development that will serve as a useful roadmap for future studies on biofilm physiology.