The sequential statistical experimental design (Plackett-Burman, factorial, response surface and steepest ascent experiment) was applied to optimize the culture medium of nitrite oxidizing bacteria for improving the nitrite oxidizing rate. Estimated optimum medium composition of the nitrite oxidizing rate was as follows: NaHCO3, 1.86 g l(-1): NaNO2, 2.04 g l(-1); Na2CO3, 0.2 g l(-1); NaCl, 0.2 g l(-1); K(H)2PO(1), 0.1 g l(-1); MgSO4 center dot 7H(2)O, 0.1 g l(-1): and FeSO4 center dot 7H(2)O, 0.01 g l(-1). The nitrite oxidizing rate was increased by 48.0% and reached a maximum at 859.5 +/- 8.4 mgNO(2)-N/gMLSS.d as compared to 580.7 +/- 25.8 mgNO(2)-N/gMLSS.d. In the Held trial, 50 L of nitrite oxidizing bacteria concentrate (1.99 gVSS/L) with 8 50 mgNO(2)N/gMLSS.d were added to 0.6 ha of the aquaculture water. Nitrite level in all treated ponds remained very low compared to the steady increase observed in all of the control ponds during 7 days. (C) 2008 Elsevier Ltd. All rights reserved.