An innovative method for directly and explicitly quantifying the maintenance energy requirements of pure cultures growing on volatile organic compound (VOC) substrates in a two-phase partitioning bioscrubber is described. Direct evidence of maintenance energy requirements of Achromobacter xylosoxidans Y234 is provided both through observed reductions in the macroscopic biomass-to-substrate yield with decreasing specific growth rates, but more remarkably through achievement of steady-state operation. The data conclusively show that maintenance activities do occur in the two-phase partitioning bioscrubber and clearly illustrate the importance of this phenomenon to the operation of this process, and similar bioreactor systems. While benzene was selected as the principal, sole substrate of interest in this study, ethylbenzene degradation experiments were also subsequently performed to illustrate and confirm the general applicability of the proposed technique, as well as the potential capabilities of the two-phase partitioning bioscrubber for the continuous treatment of waste gases containing various VOCs. The proposed method has been shown to generate maintenance energy estimates that are consistent with those obtained while employing more widely recognized estimation strategies, further validating its capabilities. The proposed steady-state mode also offers key operational advantages in terms of decreased disposal requirements in two-phase partitioning bioscrubbers. (c) 2005 Wiley Periodicals, Inc.