Production of alcohol-free beer by limited fermentation is optimally performed in a packed-bed reactor operating in downflow. This ensures a highly controllable system with optimal reactor design. In the present study, we report on changes in the physiology of immobilized yeast cells in the reactor. During production, a simultaneous increase in the activity of alcohol acetyl transferase, and formation of the esters ethyl acetate and isoamyl acetate were observed. In addition, the amount of unsaturated fatty acids decreased significantly. Since these phenomena coincided with a significant decrease in growth rate, we conclude that the anaerobic conditions, and the absence of substantial levels of unsaturated fatty acids in wort limit cell growth during production and stimulate formation of acetate esters. Low temperatures (2oC) appeared to suppress production of -acetolactate whereas at 12oC, substantial levels were obtained. An optimal and constant flavor profile of the alcohol-free beer can be achieved by introduction of regular aerobic periods to stimulate yeast growth. Temperature can be used to control the rate of growth as well as the rate of flavor formation.