696 Research Article Received: 9 September 2008 Revised: 29 October 2008 Accepted: 3 November 2008 Published online in Wiley Interscience: 12 January 2009 (www.interscience.wiley.com) DOI 10.1002/jctb.2101 Using viscosity-time plots of Escherichia coli cells undergoing chemical lysis to measure the impact of physiological changes occurring during batch cell growth Simyee Kong, a Andrew F. Day, a Ronan D. O’Kennedy, b Parviz A. Shamlou c and Nigel J. Titchener-Hooker a, * Abstract BACKGROUND: Viscosity–time plots for plasmid-bearing E. coli cells undergoing alkaline lysis are reported in this study. The plots demonstrate generic features that reﬂect the progress of fermentation and allow an assessment of the genomic DNA denaturation following cellular release into the alkaline solution. This rheological analysis could offer useful insights to the state of fermentation or the selection of operational speciﬁcations and predictions of the performance of subsequent downstream operations. RESULTS: Studies showed a distinct change in the rheological proﬁle throughout the batch fermentation, with different viscosity versus time proﬁles for lag, exponential and stationary microbial growth phases. The DNA denaturation time was found to increase with fermentation time from about 120 s after 3 h of fermentation to about 180 s after 7 h of fermentation. CONCLUSION: The increase of denaturation time was mainly caused by a rise in the genomic content of cells during the exponential growth phase. The viscosity–time proﬁles were found to provide a good indication of the cellular contents, reﬂecting the physiological changes occurring during a batch fermentation process. c  2009 Society of Chemical Industry Keywords: alkaline lysis; plasmid DNA; chromosomal DNA; cell disruption; batch fermentation INTRODUCTION The potential use of DNA-based vectors for medical therapeutic applications and to formulate global vaccines such as those against inﬂuenza, HIV and malaria would lead to rising demand and a need for industrial scale manufacturing. 1–5 Resolving issues related to the processing of plasmid DNA are crucial 4,5 and concurrently, the development of methodologies that could rapidly provide information to aid process decision making are becoming increasingly important. In fermentation, E. coli is at present a common production host for plasmid DNA. 6 It has been observed that the presence of plasmids have an effect on the physiology of their host cells 7 and different modes of culture systems and growth medium used in fermentation can inﬂuence the ratio of genomic contamination and product yield. 8 Following cell harvest, the initial recovery step involves cell lysis and often a variation of the two-step chemical lysis originally described by Birnboim and Doly. 9 During the ﬁrst step of this lysis procedure, a suspension of bacterial cells is typically mixed with an alkaline solution such as NaOH that contains a detergent like SDS, which solubilises cell membranes causing the release of intracellular contents. 10–12 The intracellular contents and denatured proteins form complexes with SDS and the alkaline environment causes the denaturation of plasmids and chromosomal DNA; the alkaline lysate is neutralised after a brief incubation period with a high ionic strength acidic reagent such as potassium acetate. 6,10 – 12 Previously it has been demonstrated that there is a characteristic viscosity versus time proﬁle for the alkaline lysis of E. coli within a laminar ﬂow scaled down reactor using constant shear rate conditions. 13 In this present paper, viscosity – time proﬁles during cell lysis (without neutralisation) were investigated to examine the physiological changes occurring during fermentation. The aim here was to determine and relate the proﬁles to the cellular components as they change during fermentation. Ultimately such insights may allow process decisions to be made quickly about the state of fermentation or predictions of the performance of ∗ Correspondence to: Nigel J. Titchener-Hooker, The Advanced Centre for Bio- chemical Engineering, Department of Biochemical Engineering, University Col- lege London, Torrington Place, London WC1E 7JE, UK. E-mail: nigelth@ucl.ac.uk a The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK b GlaxoSmithKline, South Eden Park Road, Beckenham, Kent BR3 3BS, UK c Present address: Eli Lilly and Company, Indianapolis, Indiana 46285, USA J Chem Technol Biotechnol 2009; 84: 696–701 www.soci.org c  2009 Society of Chemical Industry