Background Determination of the volumetric oxygen transfer coefficient (k(L)a) plays an important role in the design and characterization of bioreactors. Among all methods available, few techniques are suitable for the determination of k(L)a in pneumatic bioreactors during the course of cultivations. The present work proposes a new aeration step method for k(L)a evaluation in pneumatic bioreactors during biological consumption of oxygen. Results The aeration step method consisted in promoting step changes in the specific air flow rate (phi(air)) to change the dissolved oxygen concentration during cultivations, allowing the measurement of both overall oxygen transfer coefficient (k(L)a) and specific oxygen uptake rate (Q(O2)). These parameters were determined experimentally by employing two microorganisms, one a strict aerobe (Bacillus subtilis) and the other a facultative aerobe (Saccharomyces cerevisiae), for phi(air) values ranging from 2.0 to 5.0 vvm. For the purpose of comparison, k(L)a was also determined in experiments using water as the liquid phase, employing a dynamic gassing-out method. The k(L)a values during the cultivations ranged from 0.0137 to 0.0545 s(-1), while Q(O2) varied from 0.44 to 10.06 mol kg(-1) h(-1). The results obtained are shown to be reliable, exhibited excellent reproducibility and are in agreement with other values reported in the literature by different methodologies. Conclusion The new aeration step method proved to be reliable as an alternative technique for the determination of k(L)a and Q(O2) during microbial cultivation in pneumatic bioreactors. (c) 2019 Society of Chemical Industry