Whole stillage, as sole substrate or co-digested with cattle manure, was evaluated as substrate for biogas production in five mesophilic laboratory-scale biogas reactors, operating semi-continuously for 640 days. The process performance was monitored by chemical parameters and by quantitative analysis of the methanogenic and acetogenic population. With whole stillage as sole substrate the process showed clear signs of instability after 120 days of operation. However, co-digestion with manure clearly improved biogas productivity and process stability and indicated increased methane yield compared with theoretical values. The methane yield at an organic loading rate (OLR) at 2.8 g VS/(L x day) and a hydraulic retention time (HRT) of 45 days with a substrate mixture 85% whole stillage and 15% manure (based on volatile solids [VS]) was 0.31 N L CH4/g VS. Surprisingly, the abundance of the methanogenic and acetogenic populations remained relatively stable throughout the whole operation and was not influenced by process performance. (C) 2012 Elsevier Ltd. All rights reserved.