Addition of Co and Ni often improves the production of biogas during digestion of organic matter, i.e. increasing CH4-production, process stability and substrate utilization which often opens for higher organic loading rates (OLRs). The effect of Co and Ni addition was evaluated by measuring methane production, volatile solids reduction, pH and concentration of volatile fatty acids (VFAs). A series of six lab-scale semi-continuously fed biogas tank reactors were used for this purpose. The chemical forms and potential bioavailability of Co and Ni were examined by sequential extraction, acid volatile sulfide extraction (AVS) and simultaneously extracted metals. Furthermore, the sulfur speciation in solid phase was examined by sulfur X-ray absorption near edge structure spectroscopy. The effect of Co and Ni deficiency on the microbial community composition was analyzed using quantitative polymerase chain reaction and 454-pyrosequencing. The results showed that amendment with Co and Ni was necessary to maintain biogas process stability and resulted in increased CH4-production and substrate utilization efficiency. 10-20% of the total Co concentration was in dissolved form and should be regarded as easily accessible by the microorganisms. In contrast, Ni was entirely associated with organic matter/sulfides (mainly AVS) and regarded as very difficult to take up. Still Ni had stimulatory effects suggesting mechanisms such as dissolution of NiS to be involved in the regulation of Ni availability for the microorganisms. The microbial community structure varied in relation to the occurrence of Ni and Co. The acetate-utilizing Methanosarcinales dominated during stable process performance, i.e. when both Co and Ni were supplied, while hydrogenotrophic Methanomicrobiales increased together with VFA concentrations under Co or Ni deficiency. The increase was more pronounced at Co limitation. This study demonstrates that there are good possibilities to improve the performance of biogas processes digesting sulfur-rich substrates by supplementation of Co and Ni. (C) 2013 Elsevier Ltd. All rights reserved.