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Elemental sulfur (S⁰) autotrophic denitrification (SAD) has been proved feasible for nitrate removal from aquatic environments. The long start-up period up to weeks of the SAD reactor impedes its industrial application. To accelerate the start-up process, this study employed S⁰ powder packed sequencing batch reactor operated for 10 days to obtain a seed biofilm, which was inoculated into a regular S⁰ flake packed bed reactor afterwards. Merely two days after inoculation, the reactor inoculated with seed biofilm was well started up and outperformed the control reactor, which was inoculated with regular anaerobic sludge and operated for more than 10 days, delivering much increased denitrification rate of 126 ± 0.68 mg N/(L·d) and a high nitrate removal efficiency of 93.0%. Batch tests during the start-up period showed that the seed biofilm developed well on S⁰ flakes and delivered improved nitrate removal …