110 J.Teissie et al.Bioelectrochemistry 55 (2002)107-112 Optimization of the extraction procedure can be obtained 6.2.Legionella in domestic water by playing on the electrical parameters(field intensity,pulse duration,number of pulses)in such a way as to obtain a A health problem is present in developed countries:how high flow rate.The cellular load can be high (up to 20%dry to get rid of pathogens with a technology preserving the w/v). environment.Since the epidemic in 1976,Legionella are The present results are mostly focused on the yeast known to be present in domestic water and its inhalation system,but we were able to obtain analogous results with through aerosol is deleterious for human beings.Legionella mammalian cells [18]and other walled systems may be are present in many systems:water heater,shower,central targets(plant cells,molds)[19].While proteins are products heating,air cooler.Its growth in domestic water must be with a high added value,the electro-assisted extraction is eliminated.Several methodologies have been proposed: valid for small metabolites as illustrated with the use of ATP chlorine treatment or heat shock but only an online treat- leakage to quantitatively assay cell electropermeabilization ment will bring a safety level. in batch experiments. Electropulsation has been shown in Toulouse to be a suitable approach when used in a batch approach.A low- field long-duration square wave repetitive pulse approach (20 times 10 ms at 550 V/cm)was designed to cause 6.Pathogen eradication irreversible electropermeabilization.The electrical parame- ters (50 Hz frequency)were suitable to develop a flow Electropulsation is known for many years to cause technology able to treat suitable volumes of domestic water irreversible membrane permeabilization when drastic elec- (which conductivity is low,less than 1 mS/cm).The cost in trical conditions are used(Fig.2).This offers a new physical energy is limited as the current to be delivered is with a low approach for the elimination of microorganisms. intensity and that moderate field intensities(applied voltage) are needed.A 6 log efficiency in eradication has been obtained with a lab pilot system at a flow rate of 24 ml/ 6.1.Food industry min.A scaled-up system is under development. Cold sterilization is supposed to eliminate the micro- organisms in food(milk,fruit juices)while preserving the 6.3.Amoeba downstream of power plants "real"taste of the product [7].The idea is that the field is able to disrupt the cell envelope but is too weak to inactivate The presence of pathogenic amoebae(Naegleria fowleri) enzymes [19].Among the two technologies presently under is detected at increasing level in the closed looped cooling development:high pressure(HP)and high-intensity electric systems of power plants that use water for cooling.This is field pulses (HELP),the electropulsation approach is due to the facilitated growth of protozoa above 40 C.A already on the market with a prototype of pilot scale continuous treatment system of the cooling water at the equipment able to work a flow rate of 300 I/h.Electrical system drain appears necessary.Different methods have parameters are always using strong electric pulses (more been proposed when chlorine treatment of the water [20] than 20 kV/cm)with microsecond pulse duration with a was forbidden by councils for Public Health.Pilot studies capacitor discharge technology. are under evaluation using different physical methods such as UV light,ultrasound treatment or electropulsation. Electropulsation can be applied for batch eradication either alone or with a concomitant chemical or UV treatment [21,22].The results show that eradication can be obtained under low-field long-pulse duration conditions by inducing an irreversible permeabilization.Interestingly,permeabiliza- tion was obtained under conditions that were more stringent than for smaller mammalian cells such as Chinese ovary cells (CHO)while it is assumed that the field effects are larger on larger cells.Another interesting result was,that as already observed on CHO cells [23],the eradicating effect A of the field pulses was not directly linked to the energy that was delivered to the cell.This observation is very important Fig.2.Irreversible electropermeabilization.(A)Intact cells.Their cyto- for industrial developments to reduce the cost of the treat- plasm content is pictured in dark grey.(B)Electropulsation.(C)Cell ment.Short pulses with a high field intensity(microseconds, membranes are permeabilized.The cytoplasm content leaks out as shown by the light grey colour and the small arrows.(D)The cell membrane is more than 10 kV/cm)were the most cost-effective for irreversibly permeabilized and cannot be repaired.All the cytoplasmic eradication.Death was then not due to a classical process content leaks out. of irreversible permeabilization.Optimization of the extraction procedure can be obtained by playing on the electrical parameters (field intensity, pulse duration, number of pulses) in such a way as to obtain a high flow rate. The cellular load can be high (up to 20% dry w/v). The present results are mostly focused on the yeast system, but we were able to obtain analogous results with mammalian cells [18] and other walled systems may be targets (plant cells, molds) [19]. While proteins are products with a high added value, the electro-assisted extraction is valid for small metabolites as illustrated with the use of ATP leakage to quantitatively assay cell electropermeabilization in batch experiments. 6. Pathogen eradication Electropulsation is known for many years to cause irreversible membrane permeabilization when drastic elec￾trical conditions are used (Fig. 2). This offers a new physical approach for the elimination of microorganisms. 6.1. Food industry Cold sterilization is supposed to eliminate the micro￾organisms in food (milk, fruit juices) while preserving the ‘‘real’’ taste of the product [7]. The idea is that the field is able to disrupt the cell envelope but is too weak to inactivate enzymes [19]. Among the two technologies presently under development: high pressure (HP) and high-intensity electric field pulses (HELP), the electropulsation approach is already on the market with a prototype of pilot scale equipment able to work a flow rate of 300 l/h. Electrical parameters are always using strong electric pulses (more than 20 kV/cm) with microsecond pulse duration with a capacitor discharge technology. 6.2. Legionella in domestic water A health problem is present in developed countries: how to get rid of pathogens with a technology preserving the environment. Since the epidemic in 1976, Legionella are known to be present in domestic water and its inhalation through aerosol is deleterious for human beings. Legionella are present in many systems: water heater, shower, central heating, air cooler. Its growth in domestic water must be eliminated. Several methodologies have been proposed: chlorine treatment or heat shock but only an online treat￾ment will bring a safety level. Electropulsation has been shown in Toulouse to be a suitable approach when used in a batch approach. A low￾field long-duration square wave repetitive pulse approach (20 times 10 ms at 550 V/cm) was designed to cause irreversible electropermeabilization. The electrical parame￾ters (50 Hz frequency) were suitable to develop a flow technology able to treat suitable volumes of domestic water (which conductivity is low, less than 1 mS/cm). The cost in energy is limited as the current to be delivered is with a low intensity and that moderate field intensities (applied voltage) are needed. A 6 log efficiency in eradication has been obtained with a lab pilot system at a flow rate of 24 ml/ min. A scaled-up system is under development. 6.3. Amoeba downstream of power plants The presence of pathogenic amoebae (Naegleria fowleri) is detected at increasing level in the closed looped cooling systems of power plants that use water for cooling. This is due to the facilitated growth of protozoa above 40 C. A continuous treatment system of the cooling water at the system drain appears necessary. Different methods have been proposed when chlorine treatment of the water [20] was forbidden by councils for Public Health. Pilot studies are under evaluation using different physical methods such as UV light, ultrasound treatment or electropulsation. Electropulsation can be applied for batch eradication either alone or with a concomitant chemical or UV treatment [21,22]. The results show that eradication can be obtained under low-field long-pulse duration conditions by inducing an irreversible permeabilization. Interestingly, permeabiliza￾tion was obtained under conditions that were more stringent than for smaller mammalian cells such as Chinese ovary cells (CHO) while it is assumed that the field effects are larger on larger cells. Another interesting result was, that as already observed on CHO cells [23], the eradicating effect of the field pulses was not directly linked to the energy that was delivered to the cell. This observation is very important for industrial developments to reduce the cost of the treat￾ment. Short pulses with a high field intensity (microseconds, more than 10 kV/cm) were the most cost-effective for eradication. Death was then not due to a classical process of irreversible permeabilization. Fig. 2. Irreversible electropermeabilization. (A) Intact cells. Their cyto￾plasm content is pictured in dark grey. (B) Electropulsation. (C) Cell membranes are permeabilized. The cytoplasm content leaks out as shown by the light grey colour and the small arrows. (D) The cell membrane is irreversibly permeabilized and cannot be repaired. All the cytoplasmic content leaks out. 110 J. Teissie´ et al. / Bioelectrochemistry 55 (2002) 107–112