The common methods for inactivation of bacteria involve heating or exposure to toxic chemicals. These methods are not suitable for heat-sensitive materials, food, and pharmaceutical products. Recently, a complete inactivation of many microorganisms was achieved with high-pressure carbon dioxide at ambient temperature and in the absence of organic solvent and irradiation. The inactivation of spores with CO2 required long residence time and high temperatures, such as 60degreesC. In this study the synergistic effect of pulsed electric field (PEF) in combination with high-pressure CO2 for inactivation was investigated. The bacteria Escherichia coli, Staphylococcus aureus, and Bacillus cereus were suspended in glycerol solution and treated in the first step with PEF (up to 25 KV/cm) and then with high-pressure CO2 not higher than 40degreesC and 200 bar. The inactivation efficiency was determined by counting the colony formation units of control and sample. Samples of the cells subjected to PEF treatment alone and in combination with CO2 treatment were examined by scanning electron microscopy to determine the effect of the processes on the cell wall. Experimental results indicate that the viability decreased with increasing electrical field strength and number of pulses. A further batch treatment with supercritical CO2 lead to complete inactivation of bacterial species and decreased the count of the spores by at least three orders of magnitude, the inactivation being enhanced by an increase of contact time between CO2 and the sample. A synergistic effect between the pulsed electric field and the high-pressure CO2 was evident in all the species treated. The new low temperature process is an alternative for pasteurization of thermally labile compounds such as protein and plasma and minimizes denaturation of important nutrient compounds in the liquid media. (C) 2003 Wiley Periodicals, Inc.