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Rice straw is an abundant and sustainable substrate for anaerobic digestion (AD), but it is often deficient in essential trace elements (TEs) for proper microbial growth and metabolism. A lack of TEs leads to AD imbalances and suboptimal biogas yields. However, the total TE concentration is not a sufficient indicator of the amount of TEs available to the microorganisms. Therefore, this study investigated the degree of bioavailability of iron (Fe) and cobalt (Co) during the AD of rice straw, and correlated it to the biomethane yields and volatile fatty acids (VFAs) produced. When the two TEs were dosed at 205 &micro, g Fe/g TS and 18 &micro, g Co/g TS of rice straw, the biomethane production was approximately 260 mL CH4/g VS, i.e., similar to that obtained when Fe and Co were not added. Despite an increased bioavailable fraction of 23 and 48% for Fe and Co, respectively, after TEs addition, the AD performance was not enhanced. Moreover, VFAs did not exceed 250 mg HAc/L both in the presence and absence of added TEs, confirming no enhancement of the methanogenesis step. Therefore, the bioavailability of Fe and Co was not a limiting factor for the biomethane production at low total VFAs concentration.