This study characterizes the response of the microbial communities of a laboratory-scale mesophilic biogas process, fed with a synthetic substrate based on cellulose and egg albumin, to single pulses of glucose overloading (15 or 25 times the daily feed based on VS). The microbial biomass and community structure were determined from analyses of membrane phospholipids. The ratio between phospholipid fatty acids (PLFAs; eubacteria and eucaryotes) and di-ethers (PLEL; archaea) suggested that methanogens constituted 4-8% of the microbial biomass. The glucose addition resulted in transient increases in the total biomass of eubacteria while there were only small changes in community structure. The total gas production rate increased, while the relative methane content of the biogas and the alkalinity decreased. However, the biomass of methanogens was not affected by the glucose addition. The results show that the microbial communities of biogas processes can respond quickly to changes in the feeding rate. The glucose overload resulted in a transient general stimulation of degradation rates and almost a doubling of eubacterial biomass, although the biomass increase corresponded to only 7% of the glucose C added.