To search for evidence of syntrophic acetate oxidation by cluster II Spirochaetes with hydrogenotrophic methanogens, batch reactors seeded with five different anaerobic sludge samples supplemented with acetate as the sole carbon source were operated anaerobically. The changes in abundance of the cluster II Spirochaetes, two groups of acetoclastic methanogens (Methanosaetaceae and Methanosarcinaceae), and two groups of hydrogenotrophic methanogens (Methanomicrobiales and Methanobacteriales) in the reactors were assessed using qPCR targeting the 16S rRNA genes of each group. Increase in the cluster II Spirochaetes (9.0 +/- 0.4-fold) was positively correlated with increase in hydrogenotrophic methanogens, especially Methanomicrobiales (5.6 +/- 1.0-fold), but not with acetoclastic methanogens. In addition, the activity of the cluster II Spirochaetes decreased (4.6 +/- 0.1-fold) in response to high hydrogen partial pressure, but their activity was restored after consumption of hydrogen by the hydrogenotrophic methanogens. These results strongly suggest that the cluster II Spirochaetes are involved in syntrophic acetate oxidation in anaerobic digesters. (C) 2015 Elsevier Ltd. All rights reserved.