Changes in fermenter gas composition within a given 24 h period can cause severe bias in calculations of biogas or energy yields based on just one or two measurements of gas composition per day, as is common in other studies of two-stage fermentation. To overcome this bias, real time recording of gas composition and production were used to undertake a detailed and controlled comparison of single-stage and two-stage fermentation using a real world substrate (wheat feed pellets). When a two-stage fermentation system was used, methane yields increased from 261 L kg(-1) VS using a 20 day HRT, single-stage fermentation, to 359 L kg(-1) VS using a two-stage fermentation with the same overall retention time - an increase of 37%. Additionally a hydrogen yield of 7 L kg(-1) VS was obtained when two-stage fermentation was used. The two-stage system could also be operated at a shorter, 12 day HRT and still produce higher methane yields (306 L kg(-1) VS). Both two-stage fermentation systems evaluated exhibited methane yields in excess of that predicted by a biological methane potential test (BMP) performed using the same feedstock (260 L kg(-1) VS). (C) 2012 Elsevier Ltd. All rights reserved.