Automated substrate supply by three types of pH-mediated control methods for ethanol fermentation using Zymomonas mobilis was investigated. Method 1 involved continuous substrate feeding with pH control using alkali. The system was stabilized by the growth rate of the microorganisms depending on a limiting substrate concentration; however, the growth rate could not be maintained at the maximum specific growth rate (mu(max)) value. Method 2 involved pH self-stabilization without alkali. When the system was operated under this method, a stopped-flow situation occurred and the growth rate became substrate-limited due to the extremely low glucose concentration. In Method 3, pH-stat culture with separate inflows of alkali and feed medium was carried out. The rate of medium input in this system was determined by the growth rate of the culture in response to a change of pH and it was possible to culture the microorganism at the maximum growth rate without the risk of washout. In this method, the dilution rate and biomass concentration were influenced by the pH set-points, the control interval time and the ratio of inflow of feed medium to alkali. A small difference between the higher pH set-point limit and the culture pH as well as a short control interval time for a computer-controlled digital set point resulted in a large specific growth rate (mu) and higher ethanol productivity. A setup involving a digital set-point control (DSC) system demonstrated advantages over a setup involving a feedback control loop using an analog signal. Under optimum operation conditions of DSC, high ethanol productivity of over of 11 g l-1 h-1 was obtained.