In biotechnical processes the availability of real-time information about the biological activity is of crucial importance, and this is represented by the oxygen uptake rate (OUR). The aim of this paper is to design and evaluate a real-time estimator both for this quantity and for the related oxygen transfer rate coefficient. The design is based on linear approximations of these unknown variables and nonlinear system dynamics, and relies on a careful choice of probing signals. It is found that a maximum length binary sequence (MLBS) can be successfully used as an alternative to a switching sequence resulting from an on/off control. Later, estimation in the closed loop is considered, coupling the previous estimator with a self-tuning PID regulator. Given the nonlinear nature of the system the analysis is carried out mostly by simulation, and the results confirm that in all cases efficient estimates can be obtained. The performance compares very well with previous literature results.