ABSTRACTThe syntrophic cooperation between hydrogen‐producing acetogens and hydrogenotrophic methanogens relies on a critical balance between both partners. A recent study, provided several indications for the dependence of the biomass‐specific growth rate of a methanogenic coculture on the acetogen. Nevertheless, final experimental proof was lacking since biomass‐specific rates were obtained from a descriptive model, and not from direct measurement of individual biomass concentrations. In this study, a recently developed quantitative PCR approach was used to measure the individual biomass concentrations in the coculture of Desulfovibrio sp. G11 and Methanospirillum hungatei JF1 on lactate, formate or both. The model‐derived growth yields and biomass‐specific rates were successfully validated. Experimental findings identified the acetogen as the growth‐limiting partner in the coculture on lactate. While the acetogen was operating at its maximum biomass‐specific lactate consumption rate, the hydrogenotrophic methanogen showed a significant overcapacity. Furthermore, this study provides experimental evidence for different growth strategies followed by the syntrophic partners in order to maintain a common biomass‐specific growth rate. During syntrophic lactate conversion, the biomass‐specific electron transfer rate of Methanospirillum hungatei JF1 was three‐fold higher compared to Desulfovibrio sp. G11. This is to compensate for the lower methanogenic biomass yield per electron‐mole of substrate, which is dictated by the thermodynamics of the underlying reaction. Biotechnol. Bioeng. 2016;113: 560–567. © 2015 Wiley Periodicals, Inc.