ARTICLE Fast Dynamic Response of the Fermentative Metabolism of Escherichia coli to Aerobic and Anaerobic Glucose Pulses Alvaro R. Lara, 1 Hilal Taymaz-Nikerel, 2,3 Mlawule R. Mashego, 2,3 Walter M. van Gulik, 2,3 Joseph J. Heijnen, 2,3 Octavio T. Ramı´rez, 4 Wouter A. van Winden 2,3 1 Departamento de Procesos y Tecnologı´a, Universidad Auto ´ noma Metropolitana-Cuajimalpa Artificios No. 40, Col. Miguel Hidalgo, Del. A ´ lvaro Obrego ´n, Mexico DF, C P 01120, Mexico; telephone: 52-55-26363800; fax: 52-777-3138811; e-mail: alara@correo.cua.uam.mx 2 Department of Biotechnology, Delft University of Technology, Delft, the Netherlands 3 Kluyver Centre for Genomics of Industrial Fermentation, Delft, the Netherlands 4 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologı´a, Universidad Nacional Auto ´ noma de Mexico Cuernavaca, Morelos, Mexico Received 26 May 2009; revision received 22 July 2009; accepted 4 August 2009 Published online 14 August 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22503 ABSTRACT: The response of Escherichia coli cells to tran- sient exposure (step increase) in substrate concentration and anaerobiosis leading to mixed-acid fermentation metabo- lism was studied in a two-compartment bioreactor system consisting of a stirred tank reactor (STR) connected to a mini-plug-flow reactor (PFR: BioScope, 3.5 mL volume). Such a system can mimic the situation often encountered in large-scale, fed-batch bioreactors. The STR represented the zones of a large-scale bioreactor that are far from the point of substrate addition and that can be considered as glucose limited, whereas the PFR simulated the region close to the point of substrate addition, where glucose concentration is much higher than in the rest of the bioreactor. In addition, oxygen-poor and glucose-rich regions can occur in large- scale bioreactors. The response of E. coli to these large-scale conditions was simulated by continuously pumping E. coli cells from a well stirred, glucose limited, aerated chemostat (D ¼ 0.1 h 1 ) into the mini-PFR. A glucose pulse was added at the entrance of the PFR. In the PFR, a total of 11 samples were taken in a time frame of 92 s. In one case aerobicity in the PFR was maintained in order to evaluate the effects of glucose overflow independently of oxygen limitation. Accu- mulation of acetate and formate was detected after E. coli cells had been exposed for only 2 s to the glucose-rich (aerobic) region in the PFR. In the other case, the glucose pulse was also combined with anaerobiosis in the PFR. Glucose overflow combined with anaerobiosis caused the accumulation of formate, acetate, lactate, ethanol, and suc- cinate, which were also detected as soon as 2 s after of exposure of E. coli cells to the glucose and O 2 gradients. This approach (STR-mini-PFR) is useful for a better understanding of the fast dynamic phenomena occurring in large-scale bioreactors and for the design of modified strains with an improved behavior under large-scale conditions. Biotechnol. Bioeng. 2009;104: 1153–1161. ß 2009 Wiley Periodicals, Inc. KEYWORDS: Escherichia coli; fermentation metabolism; aerobic-anaerobic metabolism; mixing; BioScope Introduction Imperfect mixing in large-scale bioreactors leads to the emergence of spatial gradients. In the case of large-scale fed-batch cultures, substrate gradients are commonly encountered (Enfors et al., 2001; Schmalzriedt et al., 2003; Lara et al., 2006c), and can cause significant effects in cell physiology (Lara et al., 2006c). Cells can travel from glucose-limited conditions in the bulk of the bioreactor, to zones of high glucose concentrations at the substrate feeding point. This in turn, may create an anaerobic region, as the respiration rate of the cells is importantly increased during exposure to glucose-concentrated regions (Enfors et al., 2001; Schmalzriedt et al., 2003; Lara et al., 2006c). This is particularly important in high-cell density cultures. The presence of glucose or dissolved oxygen gradients can have a negative impact on cells, decreasing their productivity. It has also been reported to be a main source of failure during the scale-up of cultures (Bylund et al., 1999, 2000). The Alvaro R. Lara and Hilal Taymaz-Nikerel contributed equally to this work. Correspondence to: A.R. Lara ß 2009 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 104, No. 6, December 15, 2009 1153