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In mine water passive treatment systems, biological sulfate (SO42-) reduction in vertical flow bioreactors (VFBRs) is often utilized to precipitate trace metals as metal sulfides and to generate alkalinity. Sulfate removal is not typically targeted and is trivial in bioreactors compared to the removal of targeted metals. However, utilizing biological sulfate reduction in VFBRs to specifically remove sulfate can be an effective strategy to decrease elevated SO42- concentration from mine drainage. In this study, a laboratory bench-scale continuous flow-through column study simulating mine water vertical flow bioreactors was conducted over 370 days evaluating the effectiveness of three locally available waste organic substrates (Norman Aged Compost (NAC), Murphy Compost (MC), and Spent Mushroom Compost (SMC)), on biological SO42- removal by sulfate reducing bacteria (SRB). The substrates were tested in triplicate columns, constructed in opaque PVC pipes (0.019 m3), filled with a 2:1 mixture by volume of the organic substrate to washed river rock, and were fed with a solution containing 1000 mg SO42- L-1 + 10%. Conditions at the start and termination of the study were optimal for SO42- reduction: circumneutral pH, reducing oxidation-reduction potential (ORP), and appropriate temperatures. During the first 305 days, SO42 removal rates and percent removal of SO42 decreased significantly (p < 0.01). On Day 306, the hydraulic retention time (HRT) was decreased from eight days to four days for 30 days and then again to two days on Day 336 for an additional 30 days in order to observe changes in SO42- removal rates. At HRT = 8 days, the SMC treatment consistently produced the lowest effluent sulfate concentrations (median = 221 mg L-1), greatest sulfate removal rates (mean = 548 mmol m-3 day-1), percent removal of sulfate (median =77.3%), and effluent sulfide concentrations (median = 123.6 mg L-1) (p < 0.01). The SMC and MC treatments did not show significant differences in sulfate removal across all HRTs, however the NAC treatment had significantly lower sulfate removal with shorter HRTs (p < 0.05). Sulfide production decreased significantly with shorter HRTs in all treatments (p < 0.05). The results of this study confirm that mine water passive treatment system VFBRs are appropriate for the removal of elevated sulfate given that optimal conditions for bacterial sulfate reduction are created and maintained. The selected waste organic substrates showed similar sulfate removal capabilities within a range of different organic carbon contents in the substrates and in the effluents. SMC has been demonstrated for its ability to serve as the media in VFBRs targeting metals removal and retention as metal sulfides and can also be utilized in those systems aiming to specifically remove sulfate.