Enzyme and Microbial Technology 32 (2003) 92–98 The double substrate growth kinetics of Pseudomonas aeruginosa Haluk Beyenal a , Suet Nee Chen a , Zbigniew Lewandowski a,b,∗ a Center for Biofilm Engineering, Montana State University, P.O. Box 173980, Room 366 EPS, Bozeman, MT 59717, USA b Department of Civil Engineering, Montana State University, Bozeman, MT 59717, USA Received 14 June 2001; received in revised form 19 September 2002; accepted 23 September 2002 Abstract Growth parameters of Pseudomonas aeruginosa were quantified based on steady-state concentrations, utilization rates of glucose and dissolved oxygen, and microorganism concentration in a chemostat that was operated at 25 ◦ C, pH 7.2, and an agitation rate 350 rpm. The results showed that the microbial growth was limited by the concentration of glucose and the concentration of oxygen. A dual-substrate, Tessier growth kinetics for oxygen and glucose, was in good agreement with the experimental data using the fol- lowing biokinetic parameters: μ max = 0.29 h -1 , K g = 26.9 mg/l, K o = 1.18 mg/l, Y x/g = 0.628 g microorganism/g glucose and, Y x/o = 0.635 g microorganism/g oxygen. Maintenance factors for glucose and oxygen were: m g = 0.0078 g glucose consumed/g microorganism h, and m o = 0.014 g oxygen consumed/g microorganism h. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Pseudomonas aeruginosa; Growth kinetics; Multiple-substrate; Biokinetics 1. Introduction To our best knowledge, there are not any available multiple-substrate growth kinetic model developed for Pseudomonas aeruginosa. The goal of this study is to de- velop such a model and to calculate biokinetic parameters associated with the model. P. aeruginosa is often used in biofilm studies and modeling biofilm accumulation, proba- bly because microbial geneticists have been studying this organism intensively and its physiology and genetics are well known [1–3]. Biokinetic parameters for microbial growth of P. aeruginosa have been determined in biofilms by Bakke et al. [4], and in planktonic cultures by Robinson et al. [5]. However, in both papers the growth parameters of P. aeruginosa have been determined at relatively low glucose concentrations, less than 7.5 mg/l in the chemostat [4], and less than 1.4 mg/l in the biofilm reactor [5]. We can only guess that the reason for using such low glucose con- centrations was to assure that glucose—not oxygen—was the limiting substrate. In biofilms, however, there is little control over the substrates that may act as growth-limiting factors [6,7]. Concentrations of electron donors and electron acceptors in biofilms decrease toward the bottom because of mass transfer limitations and microbial consumption, and it may be difficult to assess which of them is exhausted ∗ Corresponding author. Tel.: +1-406-994-5915; fax: +1-406-994-6098. E-mail address: zl@erc.montana.edu (Z. Lewandowski). first in the deeper biofilm layers. In the excess of glucose in solution, it is reasonable to assume that oxygen rather than glucose will be the growth-limiting factor. However, to quantify biofilm accumulation rate, models accounting for multiple-substrate utilization by the microorganisms should be used to judge whether the electron donor or the electron acceptor is the growth-limiting factor [8]. The inherent difficulty associated with developing rele- vant multiple-substrate growth models stems from the ne- cessity of providing relevant experimental data and solving non-linear equations. Appropriate techniques to build such models are available [5,9]. We have generated experimen- tal data and constructed a multiple-substrate growth model for P. aeruginosa. To acquire experimental data we used a chemostat, measured concentrations, consumption rates of glucose and dissolved oxygen, and concentration of the mi- croorganism. All measurements were made at steady states. 2. Materials and methods 2.1. Microorganism and growth conditions A pure culture of P. aeruginosa (ATCC 700829) was used throughout the study. To grow the microorganism we used an artificial growth medium containing Na 2 HPO 4 (1.83 g/l); K 2 HPO 4 (0.35 g/l); MgSO 4 ·7H 2 O (0.01 g/l); yeast extract (0.001 g/l); (NH 4 ) 2 SO 4 (0.1 g/l) and glucose 0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII:S0141-0229(02)00246-6