The effects of a biodegradable chelating agent, Ethylenediamine-N,N'-disuccinic acid (EDDS), on the speciation and bioavailability of iron (Fe(2+)) in anaerobic digestion were examined. Fe(2+) supplementation at 10mg/L increased methane yield, but the presence of 8mg/L sulfide led to the precipitation of Fe(2+) 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 Fe(2+) by forming an Fe-EDDS complex, thereby protecting Fe(2+) 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 Fe(2+) was present in bioavailable forms, i.e. soluble and exchangeable, when EDDS was added at 1:1 molar ratio to Fe(2+). As a result, the methane production rate increased by 11.17%, and the methane yield increased by 13.25%.