The acclimatization of methanogens to two-phase olive mill wastes (TPOMW) was investigated in pilot fermenters started up with cattle excreta (37°C) and after changing their feed to excreta plus TPOMW (37°C or 55°C) or TPOMW alone (37°C) until a steady state was reached (28 days). Methanogenic diversity was screened using a phylogenetic microarray (AnaeroChip), and positive targets were quantified by real-time PCR. Results revealed high phylogenetic richness, with representatives of three out of the four taxonomic orders found in digesters. Methanosarcina dominated in the starting excreta (>96% of total 16S rRNA gene copies; over 45 times more abundant than any other methanogen) at high acetate (0.21 g liter(-1)) and ammonia N concentrations (1.3 g liter(-1)). Codigestion at 37°C induced a 6-fold increase of Methanosarcina numbers, correlated with CH(4) production (r(Pearson) = 0.94; P = 0.02). At 55°C, the rise in temperature and H(2) partial pressure induced a burst of Methanobacterium, Methanoculleus, Methanothermobacter, and a group of uncultured archaea. The digestion of excreta alone resulted in low but constant biogas production despite certain oscillations in the methanogenic biomass. Unsuccessful digestion of TPOMW alone was attributed to high Cu levels inducing inhibition of methanogenic activity. In conclusion, the versatile Methanosarcina immediately adapted to the shift from excreta to excreta plus TPOMW and was responsible for the stimulated CH(4) production at 37°C. Higher temperatures (55°C) fostered methanogenic diversity by promoting some H(2) scavengers while yielding the highest CH(4) production. Further testing is needed to find out whether there is a link between increased methanogenic diversity and reactor productivity.