During wastewater treatment, biofilm-coated sand particles stratified in a fluidized bed bioreactor (FBB); particles coated by thicker biofilm segregated toward the top of the bed. Stratification was so well developed that at least two co-existing regions of significantly different mean biofilm thickness were visually distinct within the operating FEB. The observed stratification is attributed to differences in forces of drag, buoyancy, shear, and collisional impact, as well as differences of collision rate within the different regions. Particles with thick biofilm (thickness >100 mu m) near the top of the bed consumed substrate at significantly lower rates per unit biomass than particles with thin biofilm (10-20 mu m) near the bottom of the bed, thereby suggesting that substrate mass-transfer resistance through biofilm may limit biodegradation rates in the upper portion of the FEB. Large agglomerates of biomass flee and sand, which formed at the top of the fluidized bed, and sand particles with thick biofilm were susceptible to washout from the FEB, causing operational and treatment instability. Radial injection of supplemental liquid feed near the top of the bed increased shear and mixing, thereby preventing formation and washout of agglomerates and thickly coated sand particles. Supplemental liquid injection caused the mean specific biomass loading on the sand to increase and also increased the total biomass inventory in the FEB. Rates of biodegradation in the FEB appeared to be limited by penetration of substrates into the biofilm and absorption of oxygen from air into the wastewater.