We studied shortening the acclimation and enhancement of the degradation rate for a xenobiotic organic pollutant using microbial populations grown in suspension as activated sludge. Soil populations cultivated as raw activated sludge in a steady-state fed-batch reactor and the raw sludge were re-cultured (energy enriched) with biogenic substrates and compared for their performances in acclimation and degradation of 2,4-D. The cells’ mass and ATP content were also measured during the 2,4-D acclimation and degradation. The optimal concentration of supplemental biogenic substrate and most suitable time point of re-cultivation that could produce the most shortening of lag phase were determined in a previous study. The purpose of this study was to find the true mechanism of this shortening based on the contents of cell internal ATP. As compared with the raw sludge, the enriched sludge contained a higher amount of energy at the start of acclimation and brought a shortened lag time during the acclimation process. External energy (biogenic substrates’s energy) invested into activated sludge cells could improve the microorganisms in acclimation to a xenobiotic by sustaining the energy needed for the difficult xenobiotic metabolism.