The effects of a biodegradable chelating agent, Ethylenediamine-N, N'-disuccinic acid (EDDS), on the speciation and bioavailability of iron (Fe2+) in anaerobic digestion were examined. Fe2+ supplementation at 10 mg/L increased methane yield, but the presence of 8 mg/L sulfide led to the precipitation of Fe2+ as FeS which limited its bioavailability. The results confirmed that the EDDS could replace common chelating agents with low biodegradability (EDTA and NTA), and improve the bioavailability of Fe2+ by forming an Fe-EDDS complex, thereby protecting Fe2+ from sulfide precipitation. Experimental findings from sequential extraction using the Community Bureau of Reference (BCR) method, and quantification of free EDDS and Fe-EDDS complex using UHPLC, confirmed that 29.82% of Fe2+ was present in bioavailable forms, i.e. soluble and exchangeable, when EDDS was added at 1: 1 molar ratio to Fe2+. As a result, the methane production rate increased by 11.17%, and the methane yield increased by 13.25%. (C) 2017 Elsevier Ltd. All rights reserved.