1. Zeta Potential of Bacterial Cells: Effect of Wash Buffers Effect of Wash Buffers Wenfa NG and Yen-Peng TING * Department of Chemical and Biomolecular Engineering National University of Singapore, 4 Engineering Drive 4, Singapore 117576 *Corresponding author: chetyp@nus.edu.sg chetyp@nus.edu.sgINTRODUCTION The charge on bacteria cell surface is of interest in many biotechnology fields such as biosorption, cell surface adhesion, and biological wastewater treatment. Zeta potential, defined as the electrical charge at the shear plane, is often used as the proxy for bacterial cell surface charge, due to ease of measurement via microelectrophoresis. Nevertheless, bacteria are grown in various growth media with varying salt and organic compound composition, which would inevitably result in the nonspecific adsorption of ions and organic metabolites on the cell surface. This nonspecific adsorption may alter the polarity and value of the measured zeta potential; thus, leading to erroneous results. Hence, sample preparation is critical in zeta potential analysis, with removal of the nonspecifically adsorbed ions and charged molecules as the key goal. Typically, multiple centrifugation and washing steps are used in preparing the cell sample. Wash buffers (such as 9 g/L NaCl, 0.001M KCl and 0.1M NaNO 3 ) are routinely used for removing (via charge screening) ions and charged molecules that bind nonspecifically to the cell surface. The effectiveness of the various wash buffers in removing nonspecifically adsorbed ions and metabolites, however, has not been systematically evaluated. OBJECTIVE The goal of this study was to conduct a systematic evaluation of the effectiveness of various wash buffers in removing ions and charged molecules from the surface of a Gram-negative model bacterium, Escherichia coli DH5α (ATCC 53868) grown in two different media. The two media, LB Lennox (with 2 g/L glucose) and a semi-defined formulated medium with a high capacity phosphate buffer system, were chosen to simulate two very different nutrient growth environment for the cell, and had implications for the extent and types of ions and charged molecules nonspecifically adsorbed to the cell surface. MATERIALS AND METHODS  Escherichia coli DH5α (ATCC 53868) was grown in LB Lennox (with 2 g/L glucose) and a semi- defined formulated medium for 15 hours at 37 o C and 230 rpm prior to sample preparation for zeta potential measurement. Figure 1: Schematic diagram showing the workflow for zeta potential analysis 17 th Regional Symposium on Chemical Engineering (RSCE2010), 22-23 November 2010, Bangkok, Thailand  An aliquot of the cell broth was diluted 16 times (final OD 600nm = 0.30) with the respective wash buffers and centrifuged at 3300 x g for 10 minutes at 25 o C. After centrifugation, the supernatant was carefully decanted, and the cell pellet resuspended in the respective wash buffer. This process was repeated twice with deionized water as the final resuspension buffer. The pH of the samples were adjusted using 0.1M HNO 3 and 0.1M NaOH (Figure 1). Ionic strength of the wash buffers were estimated by the Debye-Huckel approximation, and zeta potential was measured using Malvern’s ZetaSizer Nano ZS. RESULTS AND DISCUSSION  Zeta potential is the electrical charge at the plane of shear, located a distance away from the actual surface (Figure 2), where the extent and types of nonspecifically adsorbed ions and charged molecules would change the measured value. Figure 2: The electrical double layer and the definition of zeta potential Figure 3: Effect of different wash buffers on zeta potential – pH profiles of E. coli grown in LB Lennox (with 2 g/L glucose) medium Figure 4: Effect of different wash buffers on zeta potential – pH profiles of E. coli grown in semi-defined formulated medium Figure 5: High ionic strength wash buffer caused the removal of native cations, resulting in cell surface damage and erroneous zeta potential readings.  Figure 3 revealed significant overlap of the zeta potential – pH profiles for phosphate buffered saline (PBS) and 9 g/L NaCl. Deviation of the two profiles with that of deionized water suggested charge screening played an important role in removing nonspecifically adsorbed ions and molecules. 0.1M sodium citrate caused a drastic change in the cell surface’s point of zero charge (pH zpc ) and this might be due to the adsorption of sodium and citrate ions on the cell surface.  Small differences in the zeta potential – pH profiles for DI water, PBS and 9 g/L NaCl (Figure 3) suggested that the amount of nonspecifically adsorbed ions was small, probably due to the low salt content of LB Lennox + 2 g/L glucose.  Figure 4 showed that the zeta potential – pH profiles could be clustered into 2 distinct sets; (DI water, 0.1M sodium acetate, 0.1M sodium nitrate), and (PBS, 9 g/L NaCl) which suggested that an ionic strength of at least 0.15M was needed to remove nonspecifically adsorbed ions by charge screening.  Figure 5 showed a drastic change in the pH zpc of cells washed with 0.1M sodium citrate, probably due to the removal of intrinsic cations from the cell membrane. CONCLUSIONS  Charge screening played an important role in removing nonspecifically adsorbed ions from bacterial cell surface, and a minimum ionic strength of 0.15M might be required for a wash buffer to be effective.  Nevertheless, wash buffers with excessive ionic strength (e.g., 0.6M) might result in error due to removal of intrinsic cations important to cell envelope stability.