- Escherichia coli attachment to a surface initiates a complex series of interconnected signaling and regulation pathways that promote biofilm formation and maturation. The present work investigates the effect of deposited cell density on E. coli cell physiology, metabolic activity, and gene expression in the initial stages of biofilm development. Deposited cell density is controlled by exploiting the relationship between ionic strength and bacterial attachment efficiency in a packed bed column. Distinct differences in cell transcriptome are analyzed by comparing sessile cultures at two different cell surface densities and differentiating ionic strength effects by analyzing planktonic cultures in parallel. Our results indicate that operons regulating trypotophan production and the galactitol phosphotransferase system (including dihydroxyacetone phosphate synthesis) are strongly affected by cell density on the surface. Additional transcriptome and metabolomic impacts of cell density on succinate, proline, and pyroglutamic acid systems are also reported. These results are consistent with the hypothesis that surface cell density plays a major role in sessile cell physiology, commencing with the first stage of biofilm formation. These findings improve our understanding of biofilm formation in natural and engineered environmental systems and will contribute to future work ranging from pathogen migration in the environment to control of biofouling on engineered surfaces.