The objective of the present study was to assess major chemical reactions and chemical forms contributing to solubility and speciation of Fe(II), Co(II), and Ni(II) during anaerobic digestion of sulfur (S)-rich stillage in semi-continuous stirred tank biogas reactors (SCSTR). These metals are essential supplements for efficient and stable performance of stillage-fed SCSTR. In particular, the influence of reduced inorganic and organic S species on kinetics and thermodynamics of the metals and their partitioning between aqueous and solid phases were investigated. Solid phase S speciation was determined by use of S Kedge X-ray absorption near-edge spectroscopy. Results demonstrated that the solubility and speciation of supplemented Fe were controlled by precipitation of FeS(s) and formation of the aqueous complexes of Fe-sulfide and Fe-thiol. The relatively high solubility of Co (similar to 20% of total Co content) was attributed to the formation of compounds other than Co-sulfide and Co-thiol, presumably of microbial origin. Nickel had lower solubility than Co and its speciation was regulated by interactions with FeS(s) (e.g. co-precipitation, adsorption, and ion substitution) in addition to precipitation/dissolution of discrete NiS(s) phase and formation of aqueous Ni-sulfide complexes. (C) 2014 Elsevier B.V. All rights reserved.