Applicability of Two-Step Models in Estimating Nitrification Kinetics from Batch Respirograms Under Different Relative Dynamics of Ammonia and Nitrite Oxidation Kartik Chandran, 1 Barth F. Smets 1,2 1 Environmental Engineering Program, University of Connecticut, 261 Glenbrook Road, Storrs, Connecticut 06269-2037. telephone: 860-486-2270, e-mail: barth.smets@uconn.edu 2 Department of Civil & Environmental Engineering, University of Connecticut, Storrs, Connecticut Received 17 September 1999; accepted 30 April 2000 Abstract: A mechanistically based nitrification model was formulated to facilitate determination of both NH 4 + -N to NO 2 - -N and NO 2 - -N to NO 3 - -N oxidation ki- netics from a single NH 4 + -N to NO 3 - -N batch-oxidation profile by explicitly considering the kinetics of each oxi- dation step. The developed model incorporated a novel convention for expressing the concentrations of nitrogen species in terms of their nitrogenous oxygen demand (NOD). Stoichiometric coefficients relating nitrogen re- moval, oxygen uptake, and biomass synthesis were de- rived from an electron-balanced equation. A parameter identifiability analysis of the developed two-step model revealed a decrease in correlation and an increase in the precision of the kinetic parameter esti- mates when NO 2 - -N oxidation kinetics became increas- ingly rate-limiting. These findings demonstrate that two- step models describe nitrification kinetics adequately only when NH 4 + -N to NO 3 - -N oxidation profiles contain sufficient information pertaining to both nitrification steps. Thus, the rate-determining step in overall nitrifica- tion must be identified before applying conventionally used models to describe batch nitrification respirograms. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 54–64, 2000. Keywords: nitrification; biokinetics; extant respirometry; two-step model; parameter identifiability INTRODUCTION Nitrification involves the sequential oxidation of ammo- nium-nitrogen (NH 4 + -N, N(-III)) to nitrite-nitrogen (NO 2 - - N, N(+III)) and nitrate-nitrogen (NO 3 - -N, N(+V)) by two distinct bacterial groups. The stoichiometry of these reac- tions is described by the following Mole-balanced equations (adapted from Grady et al., 1999). 55NH 4 + + 76O 2 + 109HCO 3 - → C 5 H 7 NO 2 + 54NO 2 - + 57H 2 O + 104H 2 CO 3 (129 400NO 2 - + NH 4 + + 195O 2 + 4H 2 CO 3 + HCO 3 - → C 5 H 7 NO 2 + 3H 2 O + 400NO 3 - (229 Though many bacterial genera can perform nitrification, we collectively represent NH 4 + -N oxidizing bacteria using the subscript ns and NO 2 - -N oxidizing bacteria, by the sub- script nb, for brevity. Traditionally, NH 4 + -N to NO 3 - -N oxidation has been de- scribed as one composite biochemical process using single- step nitrification models that assume NH 4 + -N to NO 2 - -N oxidation to be the sole rate-limiting step throughout the oxidation sequence (Grady et al., 1999). Consequently, single-step models are not valid when both NH 4 + -N to NO 2 - -N and NO 2 - -N to NO 3 - -N oxidation limit overall nitrification because they do not consider the dynamics of NO 2 - -N oxidation. Two-step models expressed in terms of nitrogen species (Gee et al., 1990; Knowles et al., 1965; Mauret et al., 1996), or oxygen uptake measurements (Brou- wer et al., 1998; Ossenbruggen et al., 1996; Ossenbruggen et al., 1991) have been used to estimate kinetic parameters under dual-rate limitation of nitrification by NH 4 + -N and NO 2 - -N oxidation. However, in some of these formulations, biochemical phenomena involved in the overall nitrification process are depicted by expressions that do not portray the electron-flow coupling between NH 4 + -N and NO 2 - -N oxi- dation accurately (Ossenbruggen et al., 1996; Ossenbruggen et al., 1991). On the other hand, some two-step models fail to account for NH 4 + -N assimilation by NH 4 + -N oxidizing bacteria (Gee et al., 1990; Knowles et al., 1965; Mauret et al., 1996). In this study, a two-step nitrification model expressed solely in terms of oxygen-uptake data was derived and used to describe batch NH 4 + -N to NO 3 - -N oxidation respiro- grams obtained with a continuously cultivated highly en- riched nitrifying culture. The specific objectives of this study were: Correspondence to: B. F. Smets © 2000 John Wiley & Sons, Inc.