With the increasing demand for renewable energy and sustainable waste treatment, biogas production is expanding. Approximately four billion litres of bio-ethanol are produced annually for vehicle fuel in Europe, resulting in the production of large amounts of stillage residues. This stillage is energy-rich and can be used for biogas production, but is a challenging substrate due to its high levels of nitrogen and sulphate. At the full-scale biogas production plant in Norrköping, Sweden (Svensk Biogas i Linköping AB), thin grain stillage is used as a biogas substrate. This paper describes the plant operation and strategies that have been implemented to digest thin stillage successfully. High ammonia concentrations in the digester have resulted in syntrophic acetate oxidation (SAO) becoming the major pathway for acetate degradation. Therefore, a long hydraulic retention time (HRT) (40–60 days) is used to allow the syntrophic acetate-oxidising bacteria time to grow. The high sulphate levels in thin stillage result in high levels of hydrogen sulphide following degradation of protein and the activity of sulphate-reducing bacteria (SRB), the presence of which has been confirmed by quantitative polymerase chain reaction (qPCR) analysis. To optimise biogas production and maintain a stable process, the substrate is diluted with tap water and co-digested with grain residues and glycerine to keep the ammonium nitrogen (NH4
-N) concentration below 6 g L−1
. Combined addition of iron, hydrochloric acid and cobalt successfully precipitates sulphides, reduces ammonia toxicity and supplies microorganisms with trace element. Mesophilic temperature (38 °C) is employed to further avoid ammonia toxicity. Together, these measures and doubling the digester volume have made it possible to increase annual biogas production from 27.7 TJ to 69.1 TJ.