Bacteria commonly live in spatially structured assemblages encased by an extracellular matrix, termed biofilms. Metabolic activity of the cells inside biofilms causes gradients in local environmental conditions, which leads to the emergence of subpopulations with different metabolism. Basic information about the spatial arrangement of such metabolic subpopulations, as well as their interaction strength and interaction length scales are lacking, even for model systems like biofilms of

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... coli grown as colonies on agar-solidified media. Here, we use an unbiased approach based on temporal and spatial transcriptome and metabolome data during E. coli colony biofilm growth to identify many potential cross-feeding interactions. The strongest signature for cross-feeding in these data was displayed by alanine metabolism, and we discovered that alanine is indeed a cross-fed metabolite between two spatially segregated subpopulations: Alanine is secreted primarily via the transporter AlaE by anaerobically growing cells that are saturated with carbon and nitrogen, whereas alanine is utilized as a carbon and nitrogen source via DadA and DadX in the aerobic nutrient-deprived region at mid-height of the colony. We demonstrate that alanine cross-feeding influences cellular viability and growth in the cross-feeding-dependent region, which shapes the overall colony morphology. More generally, our methodology enables an unbiased path to the identification and characterization of spatially organized metabolic interactions in microbial communities, which are essential for understanding community structure and stability.