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The comparative performance of an inverse fluidized bed reactor (IFBR) having high density polyethylene beads as carrier materials for biofilm formation and a continuous stirred tank reactor (CSTR), both maintaining autotrophic denitrification using biogenic sulphur (ADBIOS) in the absence and presence of nickel (Ni2+), was studied. The reactors were compared in terms of NO3−-N and NO2−-N removal and SO42--S production throughout the study. A simulated wastewater with an inlet NO3−-N concentration of 225 mg/L and a decreasing concentration of biogenic sulphur (bio-S) from 1.5 to 0.375 g/L was used. Both reactors were operated at a hydraulic retention time (HRT) of 48 h for 140 days and at an HRT of 42 h for the following 68 days. A more efficient ADBIOS was observed in the CSTR than IFBR throughout the study due to a better mixing of the feed wastewater in the bulk liquid and a higher availability of bio-S to the suspended cells. The NO3−-N removal efficiency in the IFBR decreased by approximately 41% when the feed bio-S was reduced to 0.375 g/L, while it remained unaffected in the CSTR. Conversely, the presence of Ni2+ did not significantly affect NO3−-N removal in both reactors even at a feed Ni2+ concentration of 120 mg/L. The highest NO3−-N removal rates achieved were 86 and 108 mg NO3−-N/(L·day) in the IFBR and CSTR, respectively, in the presence of 120 mg/L of feed Ni2+ at an HRT of 42 h. Batch studies conducted with acclimatized biomass showed that the continuous-flow operation mode in both reactors played a major role in helping the autotrophic denitrifiers to tolerate Ni2+ toxicity.