Biofilm formation and plasmid segregational instability in biofilm cultures of Escherichia coli DH5 alpha (pMJR1750) were investigated under different medium carbon-to-nitrogen (CIN) ratios. At CIN ratios of 0.07 and 1, net accumulation of both biofilm plasmid-bearing and plasmid-free cells continued throughout the entire experiment without attaining any apparent steady state. At CIN ratios of 5 and 10, net biofilm cell accumulation for the two populations reached apparent steady states after 84 and 72 h, respectively. At CIN ratios of 0.07 and 1, polysaccharide production increased slowly and reached about 2 mu g alginate equivalent/cm(2) by the end of both experiments. At a C/N ratio of 5, polysaccharide increased significantly after 84 h, reaching about 7 mu g alginate equivalent/cm(2) prior to termination. At a C/N ratio of 10, polysaccharide increased significantly after 72 h and reached 21 mu g alginate equivalent/cm(2) at 108 h. At C/N ratios of 0.07 and 1, protein production reached 6.5 and 4 mu g/cm(2), respectively. At C/N ratios of 5 and 10, protein production increased slightly for the first 84 h and reached a maximum at 108 h, at 3 and 2 mu g/cm(2), respectively, then decreased over the last 12 h of the experiment. Ratios of polysaccharide to protein increased with increasing C/N ratios. At C/N ratios of 0.07 and 1, the ratios between extracellular polysaccharide (EP) and protein were no more than 2.5 mu g polysaccharide/mu g protein, whereas those at CIN ratios of 5 and 10 increased to about 7 and 12 mu g polysaccharide/mu g protein, respectively. Probabilities, of plasmid loss in the biofilm cultures increased with increasing CIN ratios. At CIN ratios of 0.07, 1, and 5, the probabilities of plasmid loss were 0.013 +/- 0.011, 0.020 +/- 0.006 and 0.122 +/- 0.021, respectively. At a C/N ratio of 10, the probability of plasmid loss was significantly higher, reaching 0.388 +/- 0.125. The increase of probability of plasmid loss at higher CIN ratios results from competition between cell replication and extracellular polysaccharide production.