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• Studied the distribution characteristics of six heavy metals in four anaerobic digestion system. • Effects of substrate types on the transformation of heavy metal speciation was investigated. • The mechanism leading to the transformation of heavy metal speciation was hypothesized. Chemical speciation can fundamentally affect the potential toxicity and bioavailability of heavy metals. The transformation of heavy metal speciation and change of bioavailability were investigated in an anaerobic digestion (AD) system using four different substrates (pig manure (PM), cattle manure (CAM), chicken manure (CHM) and rice straw (RS)). The results obtained indicated that the total contents of heavy metals in PM, CHM and CAM were higher than in RS and decreased in the order Zn > Cu > Ni > Pb > As > Cd in all substrates. Moreover, the speciation with the largest proportion for each heavy metal was the same both in the different substrates and the biogas residues. Among them, Zn, Ni, Cd and As were mainly in the reducible fraction (F2), while Cu was mainly in the oxidizable fraction (F3) and Pb occurred predominantly in the residual fraction (F4). Our results further indicated that the AD process had a greater effect on the speciation of heavy metals in CHM and PM, but less on CAM and RS. The rates of change in bioavailability followed the order PM > CHM > CAM > RS. Changes in organic matter, humic acid or local metal ion environment as a result of AD were inferred as likely mechanisms leading to the transformation of heavy metal speciation. These results enhanced our understanding of the behavior of heavy metals in AD and provided a new perspective for the treatment and disposal of the substrates. Image, graphical abstract [ABSTRACT FROM AUTHOR]