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To elucidate the role of Fe(III) reduction in mining lake sediments amended with organic substrates, we performed a large (10 m diameter) enclosure experiment in which sediments were amended with Carbokalk, a waste product from sugar industry containing organic carbon and lime. Fe(III) reduction rates were determined monthly by measuring the accumulation of Fe(II) in the sediments in the field. Fe(III) reduction rates were also determined by incubating sediment samples with synthetic Fe(III) oxyhydroxide under in situ temperature in the laboratory. Sulfate reduction was selectively inhibited in the Fe(III) reduction experiments by addition of sodium molybdate. Sulfate reduction was measured by accumulation of reduced inorganic sulfides in the field and by 35S radiotracer using a core injection technique. Sediment incubation and determination of sulfate reduction rates with radiotracer showed that sulfate reduction and direct microbial Fe(III) reduction occured simultaneously in the upper centimeters of the sediments and that both processes contributed to alkalinity generation. However, Fe(III) reduction was the initial process and rates were at least 3.5 fold higher than sulfate reduction rates. The results indicate that the presence of suitable anions for Fe(II) precipitation as carbonate or sulfide is needed in order to prevent loss of potential alkalinity by Fe(II) diffusion and reoxidation in the water column.