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A new tray bioreactor was developed for the production of diosgenin from Dioscorea zingiberensis with Trichoderma reesei and Aspergillus fumigatus. The influence of initial moisture content, temperature, tray bed depth and mixing times was investigated. The best fermentation condition is initial moisture content of 75%, bioreactor temperature of 35°C, solid bed depth of 1.5 cm and three mixings carrying out on the first, third and fifth day. Under the optimized fermentation conditions, after 144 h incubation, maximum diogenin concentration of 68.2 μmol/g was detected.