Bioelectrochemistry 业 ELSEVIER Bioelectrochemistry 55(2002)107-112 www.elsevier.com/locate/bioelechem Recent biotechnological developments of electropulsation. A prospective review J.Teissie*,N.Eynard,M.C.Vernhes,A.Benichou,V.Ganeva,B.Galutzovb, P.A.Cabanes "IPBS CNRS (UMR 5089).205 Route de Narbonne,31077 Toulouse Cedex.France bUniversity of Sofia,Sofia.Bulgaria SEM EDF Paris,France Received 31 July 2001;received in revised form 12 October 2001;accepted 15 October 2001 Abstract During the last 25 years,basic research has improved our knowledge on the molecular mechanisms triggered at the membrane level by electric pulses.Applied aspects may now be used under safe conditions. Electropulsation is known as a very efficient tool for obtaining gene transfer in many species to produce genetically modified organisms (GMO).This is routinely used for industrial purposes to transfer exogenous activities in bacteria,yeasts and plants.The method is simple and of a low cost. But electropulsation is not limited to this application for biotechnological purposes.It is known that the field-associated membrane alterations can be irreversible.The pulsed species cannot recover after the treatment.Their viability is strongly affected.This appears as a very promising technology for the eradication of pathogenic microorganisms.Recent developments are proposed for sterilization purposes. New flow technologies of field generation allow the treatment of large volumes of solution.When high flow rates are used,microorganisms are submitted both to a hydromechanical and to an electrical stress.The synergy of the two effects may be present when suitable pulsing conditions are chosen.Several examples for the treatment of domestic water and in the food industry are described. Walled microorganisms are affected not only at the membrane level.We observed that alterations are present on the cell wall.A very promising technology is the associated controlled leakage of the cytoplasmic soluble proteins.Large dimeric proteins such as B- galactosidases can be extracted at a high yield.High volumes can be treated by using a flow process.Extraction of proteins is obtained with many systems including mammalian cells.2002 Elsevier Science B.V.All rights reserved. Keywords:Electropulsation;Biotechnology;Microorganisms;Yeast;Amoebae;Legionella 1.Introduction use in other biotechnological fields is somewhat limited with the exception of its key function in animal cloning [2]. When during the Szeged BES meeting in 1987,the Electrotransformation (electrically mediated gene transfer) principal investigator was asked to give a survey of the is routinely used at the bench to obtain genetically modified effects of electric fields and currents on living cells and their organisms (GMO)[3-5]. potential use in biotechnology,two main emerging fields But no significant progress at the industrial scale has were present [1].Taking benefit of the basic research results been made to obtain by such technologies: of the early 1980s,electrofusion and electrotransformation were starting to be used not only in academic laboratories (a)an increase in the growth of the microorganism while cell electroactivation was expected to be a fruitful (b)an increase in the metabolic activity of micro- technology.More than 10 years later,while electrofusion is organisms used for the formation of plant hybrids rather routinely,its while this was expected in 1987. In fact biotechnological applications remain focused on ”Corresponding author..Tel:+33-5-6117-5812;fax:+33-5-6117-5994. small-scale experiments.Getting a limited number of trans- E-mail address:justin@ipbs.fr (J.Teissie). formed microorganisms is enough to prepare the availability 1567-5394/02/S-see front matter 2002 Elsevier Science B.V.All rights reserved. P正：S1567-5394(01)00138-4
Recent biotechnological developments of electropulsation. A prospective review J. Teissie´ a,*, N. Eynard a , M.C. Vernhes a , A. Be´nichou a , V. Ganeva b , B. Galutzov b , P.A. Cabanes c a IPBS CNRS (UMR 5089), 205 Route de Narbonne, 31077 Toulouse Cedex, France b University of Sofia, Sofia, Bulgaria c SEM EDF, Paris, France Received 31 July 2001; received in revised form 12 October 2001; accepted 15 October 2001 Abstract During the last 25 years, basic research has improved our knowledge on the molecular mechanisms triggered at the membrane level by electric pulses. Applied aspects may now be used under safe conditions. Electropulsation is known as a very efficient tool for obtaining gene transfer in many species to produce genetically modified organisms (GMO). This is routinely used for industrial purposes to transfer exogenous activities in bacteria, yeasts and plants. The method is simple and of a low cost. But electropulsation is not limited to this application for biotechnological purposes. It is known that the field-associated membrane alterations can be irreversible. The pulsed species cannot recover after the treatment. Their viability is strongly affected. This appears as a very promising technology for the eradication of pathogenic microorganisms. Recent developments are proposed for sterilization purposes. New flow technologies of field generation allow the treatment of large volumes of solution. When high flow rates are used, microorganisms are submitted both to a hydromechanical and to an electrical stress. The synergy of the two effects may be present when suitable pulsing conditions are chosen. Several examples for the treatment of domestic water and in the food industry are described. Walled microorganisms are affected not only at the membrane level. We observed that alterations are present on the cell wall. A very promising technology is the associated controlled leakage of the cytoplasmic soluble proteins. Large dimeric proteins such as bgalactosidases can be extracted at a high yield. High volumes can be treated by using a flow process. Extraction of proteins is obtained with many systems including mammalian cells. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Electropulsation; Biotechnology; Microorganisms; Yeast; Amoebae; Legionella 1. Introduction When during the Szeged BES meeting in 1987, the principal investigator was asked to give a survey of the effects of electric fields and currents on living cells and their potential use in biotechnology, two main emerging fields were present [1]. Taking benefit of the basic research results of the early 1980s, electrofusion and electrotransformation were starting to be used not only in academic laboratories while cell electroactivation was expected to be a fruitful technology. More than 10 years later, while electrofusion is used for the formation of plant hybrids rather routinely, its use in other biotechnological fields is somewhat limited with the exception of its key function in animal cloning [2]. Electrotransformation (electrically mediated gene transfer) is routinely used at the bench to obtain genetically modified organisms (GMO) [3– 5]. But no significant progress at the industrial scale has been made to obtain by such technologies: (a) an increase in the growth of the microorganism (b) an increase in the metabolic activity of microorganisms while this was expected in 1987. In fact biotechnological applications remain focused on small-scale experiments. Getting a limited number of transformed microorganisms is enough to prepare the availability 1567-5394/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S1567-5394(01)00138-4 * Corresponding author. Tel.: +33-5-6117-5812; fax: +33-5-6117-5994. E-mail address: justin@ipbs.fr (J. Teissie´). www.elsevier.com/locate/bioelechem Bioelectrochemistry 55 (2002) 107 – 112
108 J.Teissie et al.Bioelectrochemistry 55 (2002)107-112 of GMO for the market.The selected microorganisms can sive pulses but not with the delay between pulses if delay is be grown and expanded under selective pressure. larger than 1 ms and shorter than 10 s. New developments of electropulsation in biotechnology These conclusions on spherical cells can be used with are obtained when large volumes can be treated.Metabolites ellipsoidal cells (rod-like bacteria)but one must take into can be extracted or introduced as a result of electropermea- account the orientation of their long axis relative to the bilization.They can be small sized but cytoplasmic proteins field [8]. can be the target by using suitable electrical parameters [6]. Microorganisms can be eradicated when stringent pulse conditions are used,which bring an irreversible electro- 3.Technological problems linked to large volume permeabilization [7]. treatment Working on large volumes can be obtained by an up- 2.Theory sizing of the present laboratory-scale processes.Batch technology is always limited by the amount of energy that When applied on a cell suspension,an external field can be delivered by the power generators.The volume Vol induces a time-and position-dependent membrane potential that can be treated with a pulse of duration T at a field E difference modification AV in a buffer with a conductance A requires an available energy: AV(M)=fgrEcos 0(M)(1-exp(-t/t)) (1) W=E2AVol T (5) where f is dependent on the cell shape,g on the conduc- tance of the outer and inner buffers and of the cell that is,15 kJ is needed to pulse 1 I of phosphate buffer membrane,2r is the length of the cell in the direction of saline (PBS)at 1 kV/cm during 1 ms.This is clearly a the field,E is the field strength,0 is the angle between the technical limit in the design because with a width between direction of the field and the normal to the membrane plane the electrodes of 1 cm (to limit the voltage to 1 kV),the at the point of interest M and t is the membrane capacitance current would reach 15 kA! loading time.The field pulse is supposed to be a square Other methodologies are clearly needed.Flow processes wave. appear to be a suitable approach.We introduced the tech- The loading time is described by nology in the mid-1980s [9]for an up-scaling of electro- fusion [10,11]and showed some years later that it can be t=rCmg (2) used for electrotransformation but the plasmid cost was high [12].The possibility to treat blood samples was proposed where Cm is the membrane unit capacitance and g*a for clinical applications [13]. complex function of the conductances.For microorganisms, is in the microsecond range.It increases with a decrease in the conductance of the external buffer and with the cell size. 4.Flow electropulsation The resulting membrane potential difference is the sum of the resting membrane potential difference(assumed to be The basic concept is to apply calibrated pulses as in independent of the external field)and of the field-dependent batch process but at a delivery frequency that is linked to modulation the flow rate(Fig.1).The relationship between frequency Electropermeabilization is triggered as soon as locally the and flow is such that the desired number of pulses is resulting membrane potential difference reaches a critical actually delivered on each cell during its residency in the value (between 200 and 300 mV).This means that for a pulsing chamber.The geometry of the chamber is chosen to (spherical)given cell,this is obtained when give a homogeneous field distribution and a uniform flow rate (turbulent flow conditions may be advantageous). Ecos 0(M)(1-exp(-t/))=Ep (3) Therefore,the residency time Tres of a given cell in the chamber is: The conclusion is that for long pulses (duration larger than several t)for a field intensity E(E>Ep),a cap on the Tres Vollo (6) cell surface is in the permeabilized state and its surface is where Vol is the volume of the pulsing flow chamber and O A perm 2n(1-Ep/E) (4) is the flow rate.The number of pulses delivered per cell is: N=TresF (7) but clearly its size will depend on the pulse duration when this duration is in the order of t. F being the frequency of the pulses,which is set by the The density of local defects supporting the permeabiliza- pulse generator,which also controls the pulse duration T tion is increased with pulse duration and number of succes- and the voltage U
of GMO for the market. The selected microorganisms can be grown and expanded under selective pressure. New developments of electropulsation in biotechnology are obtained when large volumes can be treated. Metabolites can be extracted or introduced as a result of electropermeabilization. They can be small sized but cytoplasmic proteins can be the target by using suitable electrical parameters [6]. Microorganisms can be eradicated when stringent pulse conditions are used, which bring an irreversible electropermeabilization [7]. 2. Theory When applied on a cell suspension, an external field induces a time- and position-dependent membrane potential difference modification DV DVðMÞ ¼ fgrEcos hðMÞð1 expðt=sÞÞ ð1Þ where f is dependent on the cell shape, g on the conductance of the outer and inner buffers and of the cell membrane, 2r is the length of the cell in the direction of the field, E is the field strength, h is the angle between the direction of the field and the normal to the membrane plane at the point of interest M and s is the membrane capacitance loading time. The field pulse is supposed to be a square wave. The loading time is described by s ¼ rCmg* ð2Þ where Cm is the membrane unit capacitance and g* a complex function of the conductances. For microorganisms, s is in the microsecond range. It increases with a decrease in the conductance of the external buffer and with the cell size. The resulting membrane potential difference is the sum of the resting membrane potential difference (assumed to be independent of the external field) and of the field-dependent modulation. Electropermeabilization is triggered as soon as locally the resulting membrane potential difference reaches a critical value (between 200 and 300 mV). This means that for a (spherical) given cell, this is obtained when Ecos hðMÞð1 expðt=sÞÞ ¼ Ep ð3Þ The conclusion is that for long pulses (duration larger than several s) for a field intensity E (E > Ep), a cap on the cell surface is in the permeabilized state and its surface is A perm ¼ 2pr 2 ð1 Ep=EÞ ð4Þ but clearly its size will depend on the pulse duration when this duration is in the order of s. The density of local defects supporting the permeabilization is increased with pulse duration and number of successive pulses but not with the delay between pulses if delay is larger than 1 ms and shorter than 10 s. These conclusions on spherical cells can be used with ellipsoidal cells (rod-like bacteria) but one must take into account the orientation of their long axis relative to the field [8]. 3. Technological problems linked to large volume treatment Working on large volumes can be obtained by an upsizing of the present laboratory-scale processes. Batch technology is always limited by the amount of energy that can be delivered by the power generators. The volume Vol that can be treated with a pulse of duration T at a field E in a buffer with a conductance K requires an available energy: W ¼ E2 KVol T ð5Þ that is, 15 kJ is needed to pulse 1 l of phosphate buffer saline (PBS) at 1 kV/cm during 1 ms. This is clearly a technical limit in the design because with a width between the electrodes of 1 cm (to limit the voltage to 1 kV), the current would reach 15 kA! Other methodologies are clearly needed. Flow processes appear to be a suitable approach. We introduced the technology in the mid-1980s [9] for an up-scaling of electrofusion [10,11] and showed some years later that it can be used for electrotransformation but the plasmid cost was high [12]. The possibility to treat blood samples was proposed for clinical applications [13]. 4. Flow electropulsation The basic concept is to apply calibrated pulses as in batch process but at a delivery frequency that is linked to the flow rate (Fig. 1). The relationship between frequency and flow is such that the desired number of pulses is actually delivered on each cell during its residency in the pulsing chamber. The geometry of the chamber is chosen to give a homogeneous field distribution and a uniform flow rate (turbulent flow conditions may be advantageous). Therefore, the residency time Tres of a given cell in the chamber is: Tres ¼ Vol=Q ð6Þ where Vol is the volume of the pulsing flow chamber and Q is the flow rate. The number of pulses delivered per cell is: N ¼ TresF ð7Þ F being the frequency of the pulses, which is set by the pulse generator, which also controls the pulse duration T and the voltage U. 108 J. Teissie´ et al. / Bioelectrochemistry 55 (2002) 107–112
J.Teissie et al.Bioelectrochemistry 55 (2002)107-112 109 U,T,F electropulsator Pulsing chamber Flow control pump reservoir Collecting reservoir Fig.1.Flow electropulsation.(A)Cells are taken from the reservoir.(B)They flow through the pulsing chamber where a controlled number of calibrated pulses is applied.The pulsing chamber is connected to the high-power pulse electropulsator where the voltage U,the pulse duration Tand the pulse frequency F are under control.(C)The flow o is obtained by a pump and controlled.(D)Pulsed cells are collected and processed in a collecting reservoir. The field strength is solvent,membrane disintegration,heat,pH and osmotic E=U/d (8) shock)or enzymatic (zymolase,lyticase)methods are used. A critical drawback is presently due to the nonspecificity of d being the width between the two electrodes that are flat these methods:the vacuoles are destroyed allowing the and parallel. proteases to have a free access to the cytoplasmic enzymes. The description of the flow electropulsation device is Another limit is that these methods are energy consuming cartooned in Fig.1. because the treatment is most of the time operated at high The electrical requirements are reasonable.To treat 1 I of temperature. PBS per minute with 10 pulses of 1 ms requests a power of Taking into account our recent observations with batch 2.5 kW from Eq.(5). processes [15,16],flow processes were developed to bring electroextraction at an industrial scale [17]. A simple procedure is applied.Yeasts cells are washed 5.Protein extraction and suspended in pure water,a low-conductance medium.A limited number of pulses is applied with pulse duration in the Yeasts (Saccharomyces,Kluyveromyces,Picha)are a millisecond time range.Field intensities are less than 4 kV/ well-established cell factory for the production of endoge- cm.Pulsed cells are then incubated in 0.105 M salt solution nous proteins (alcohol dehydrogenase,aldehyde dehydro- (PBS and glycerol as osmotic protector)at room temper- genase,glucose-6-phosphate dehydrogenase,hexokinase,3- ature.A slow release of cytoplasmic proteins is obtained,but phosphoglyceric phosphokinase,B-galactosidase,alcohol up to 90%of the cell content can be recovered within 6-8 h oxidase).Their electrotransformation to produce exogenous (100%being assumed to be obtained by the bead mill proteins follows an easy-to-perform protocol on intact process or the enzyme lysis procedures).A key feature is systems [14]. that the specific activity of the recovered proteins is higher A technological bottle neck is the extraction of proteins by a factor of 1.5-2 than with the mechanical extraction. from the cytoplasm under conditions where the protein Electrophoretic characterization of the extracted proteins integrity (i.e.,activity)is preserved.Many approaches are does not indicate a size limit in the recovered proteins. proposed and already used in the biotech industry.As the Indeed dimeric B-galactosidase(250 kDa)is extracted. cell wall must be degradated,drastic mechanical (bead mill The electric conditions that are requested are easily homogenization,rotor stator homogenization,high-pressure obtained due to the low current intensity that is needed as homogenization,US disintegration),chemical(autolysis by the experiments are run on a suspension in pure water
The field strength is E ¼ U=d ð8Þ d being the width between the two electrodes that are flat and parallel. The description of the flow electropulsation device is cartooned in Fig. 1. The electrical requirements are reasonable. To treat 1 l of PBS per minute with 10 pulses of 1 ms requests a power of 2.5 kW from Eq. (5). 5. Protein extraction Yeasts (Saccharomyces, Kluyveromyces, Picha) are a well-established cell factory for the production of endogenous proteins (alcohol dehydrogenase, aldehyde dehydrogenase, glucose-6-phosphate dehydrogenase, hexokinase, 3- phosphoglyceric phosphokinase, b-galactosidase, alcohol oxidase). Their electrotransformation to produce exogenous proteins follows an easy-to-perform protocol on intact systems [14]. A technological bottle neck is the extraction of proteins from the cytoplasm under conditions where the protein integrity (i.e., activity) is preserved. Many approaches are proposed and already used in the biotech industry. As the cell wall must be degradated, drastic mechanical (bead mill homogenization, rotor stator homogenization, high-pressure homogenization, US disintegration), chemical (autolysis by solvent, membrane disintegration, heat, pH and osmotic shock) or enzymatic (zymolase, lyticase) methods are used. A critical drawback is presently due to the nonspecificity of these methods: the vacuoles are destroyed allowing the proteases to have a free access to the cytoplasmic enzymes. Another limit is that these methods are energy consuming because the treatment is most of the time operated at high temperature. Taking into account our recent observations with batch processes [15,16], flow processes were developed to bring electroextraction at an industrial scale [17]. A simple procedure is applied. Yeasts cells are washed and suspended in pure water, a low-conductance medium. A limited number of pulses is applied with pulse duration in the millisecond time range. Field intensities are less than 4 kV/ cm. Pulsed cells are then incubated in 0.105 M salt solution (PBS and glycerol as osmotic protector) at room temperature. A slow release of cytoplasmic proteins is obtained, but up to 90% of the cell content can be recovered within 6 –8 h (100% being assumed to be obtained by the bead mill process or the enzyme lysis procedures). A key feature is that the specific activity of the recovered proteins is higher by a factor of 1.5 –2 than with the mechanical extraction. Electrophoretic characterization of the extracted proteins does not indicate a size limit in the recovered proteins. Indeed dimeric b-galactosidase (250 kDa) is extracted. The electric conditions that are requested are easily obtained due to the low current intensity that is needed as the experiments are run on a suspension in pure water. Fig. 1. Flow electropulsation. (A) Cells are taken from the reservoir. (B) They flow through the pulsing chamber where a controlled number of calibrated pulses is applied. The pulsing chamber is connected to the high-power pulse electropulsator where the voltage U, the pulse duration T and the pulse frequency F are under control. (C) The flow Q is obtained by a pump and controlled. (D) Pulsed cells are collected and processed in a collecting reservoir. J. Teissie´ et al. / Bioelectrochemistry 55 (2002) 107–112 109
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 electrical 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 microorganisms 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 treatment will bring a safety level. Electropulsation has been shown in Toulouse to be a suitable approach when used in a batch approach. A lowfield long-duration square wave repetitive pulse approach (20 times 10 ms at 550 V/cm) was designed to cause irreversible electropermeabilization. The electrical parameters (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, permeabilization 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 treatment. 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 cytoplasm 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
J.Teissie et al.Bioelectrochemistry 55 (2002)107-112 111 A Electropulsation was then used on a flow process.In a lab pilot system,it was observed than the direction of the pulse field versus the flow was very important.Eradication was not possible at high flow rate when the perpendicular orientation was used while it was obtained with a parallel orientation [18].A pilot set up was recently tested on a power plant [24].Results are encouraging.A 2 log erad- Q(Flow) ication was obtained for less than 1 MW when treating 1 m'/s. Fig.3.The hydrodynamic drag affects the cell shape in the flow process The field is applied in the perpendicular orientation to the flow.(A)There is no flow;the cell is spherical.For a given field intensity,two large caps on 7.Consequences for basic research the cell surface are brought to the electropermeabilized state.(B)A flow is present.Being a viscoelastic body,the cell shape changes and becomes an ellipsoid with a decrease of its size in the direction of the field.The Most of the results are explained by an irreversible electropermeabilized cap size is decreased for the same applied field permeabilization.Nevertheless in the case of protein extrac- intensity.(C)A high flow is present.The hydrodynamic cell deformation is tion,leakages of species with molecular weights larger than even higher.Almost no electropermeabilization can be induced by the 200 kDa are evidences that defects are present in the yeast electric field pulse. wall.The outflow was slow (several hours)suggesting that no large defects were present.This was confirmed by electron microscopy studies [7].The creation of these change in the shape of the vesicle results.In a solution with defects as a result of membrane electropermeabilization a low conductivity,this is associated to its elongation in the remains unexplained. direction of the field,that is,for small values of 0,would Electropermeabilization results from low-field long-last- result in lower critical values of the damaging field inten- ing pulses and is easily monitored by the associated leakage sities.The mechanical stress on the membrane organization of small compounds.But optimized eradication in the food should have direct damaging effects and clearly facilitate industry and in the case of amoebae is obtained under the outflow of cell content.This would explain the obser- different electrical conditions.Indeed the effect of short vations on microorganism lysis obtained in the reported pulses is not supported by such a model. biotech applications.Basic study investigations are needed Conditions for permeabilization are not energy optimal. to confirm these predictions.Therefore,observations at the The membrane charging time is always shorter than the single-cell level are strongly needed. pulse duration.As a consequence,the field-induced mem- The dependence of eradication on the flow rate and on brane potential difference does not reach its steady-state the orientation of the field relative to the flow suggests that value.As the membrane-permeabilized part is under the the hydrodynamic drag may: control of the induced potential difference,a smaller fraction is brought to the permeabilized state with a lower density of 1.prevent the reorientation of rod-like bacteria during defects than obtained with long-lasting pulses.This con- the field pulse,a step which facilitates electro- clusion opens the question of the origin of the microorgan- permeabilization [30] ism death under these pulsing conditions.Other physical 2. induce a deformation of cells in the direction of the factors are present when a field pulse is applied on a vesicle flow,resulting in a decrease of its size in the direc- [25,26].Electrical fields induce mechanical forces.As it is a tion of the field and of its sensitivity to the field field effect on a field induced dipole,the general expression (Fig.3) of the force F is given by F=UE2 The physical conditions present in the flow conditions (9) cannot therefore be described by taking into account exclu- sively the electrical stress associated to the external field but where the 0 parameter is dependent on the frequency of the the mechanical tensions associated to the flow are other field and on the membrane state [27].As due to electro- parameters affecting the stability of the membrane. permeabilization,which is triggered within less than I us, the membrane state is affected and as Uis time dependent,F changes during the pulse in a complex way.But the final result is that a time-dependent strain is applied on a cell with 8.Conclusions a time-dependent membrane organization. One should consider that a cell is a viscoelastic body and Electropulsation can be in the coming years a powerful that a change in its geometry would result.This is clearly tool for the biotech industry.But such a development,as observed on liposomes [28,29].Elongation is predicted to already observed during the last 20 years for the obtainment be present in the early steps of the field pulse [25,27].A of GMOs,needs an improvement of our basic knowledge on
Electropulsation was then used on a flow process. In a lab pilot system, it was observed than the direction of the pulse field versus the flow was very important. Eradication was not possible at high flow rate when the perpendicular orientation was used while it was obtained with a parallel orientation [18]. A pilot set up was recently tested on a power plant [24]. Results are encouraging. A 2 log eradication was obtained for less than 1 MW when treating 1 m3 /s. 7. Consequences for basic research Most of the results are explained by an irreversible permeabilization. Nevertheless in the case of protein extraction, leakages of species with molecular weights larger than 200 kDa are evidences that defects are present in the yeast wall. The outflow was slow (several hours) suggesting that no large defects were present. This was confirmed by electron microscopy studies [7]. The creation of these defects as a result of membrane electropermeabilization remains unexplained. Electropermeabilization results from low-field long-lasting pulses and is easily monitored by the associated leakage of small compounds. But optimized eradication in the food industry and in the case of amoebae is obtained under different electrical conditions. Indeed the effect of short pulses is not supported by such a model. Conditions for permeabilization are not energy optimal. The membrane charging time is always shorter than the pulse duration. As a consequence, the field-induced membrane potential difference does not reach its steady-state value. As the membrane-permeabilized part is under the control of the induced potential difference, a smaller fraction is brought to the permeabilized state with a lower density of defects than obtained with long-lasting pulses. This conclusion opens the question of the origin of the microorganism death under these pulsing conditions. Other physical factors are present when a field pulse is applied on a vesicle [25,26]. Electrical fields induce mechanical forces. As it is a field effect on a field induced dipole, the general expression of the force F is given by F ¼ UE2 ð9Þ where the U parameter is dependent on the frequency of the field and on the membrane state [27]. As due to electropermeabilization, which is triggered within less than 1 ms, the membrane state is affected and as U is time dependent, F changes during the pulse in a complex way. But the final result is that a time-dependent strain is applied on a cell with a time-dependent membrane organization. One should consider that a cell is a viscoelastic body and that a change in its geometry would result. This is clearly observed on liposomes [28,29]. Elongation is predicted to be present in the early steps of the field pulse [25,27]. A change in the shape of the vesicle results. In a solution with a low conductivity, this is associated to its elongation in the direction of the field, that is, for small values of h, would result in lower critical values of the damaging field intensities. The mechanical stress on the membrane organization should have direct damaging effects and clearly facilitate the outflow of cell content. This would explain the observations on microorganism lysis obtained in the reported biotech applications. Basic study investigations are needed to confirm these predictions. Therefore, observations at the single-cell level are strongly needed. The dependence of eradication on the flow rate and on the orientation of the field relative to the flow suggests that the hydrodynamic drag may: 1. prevent the reorientation of rod-like bacteria during the field pulse, a step which facilitates electropermeabilization [30] 2. induce a deformation of cells in the direction of the flow, resulting in a decrease of its size in the direction of the field and of its sensitivity to the field (Fig. 3) The physical conditions present in the flow conditions cannot therefore be described by taking into account exclusively the electrical stress associated to the external field but the mechanical tensions associated to the flow are other parameters affecting the stability of the membrane. 8. Conclusions Electropulsation can be in the coming years a powerful tool for the biotech industry. But such a development, as already observed during the last 20 years for the obtainment of GMOs, needs an improvement of our basic knowledge on Fig. 3. The hydrodynamic drag affects the cell shape in the flow process. The field is applied in the perpendicular orientation to the flow. (A) There is no flow; the cell is spherical. For a given field intensity, two large caps on the cell surface are brought to the electropermeabilized state. (B) A flow is present. Being a viscoelastic body, the cell shape changes and becomes an ellipsoid with a decrease of its size in the direction of the field. The electropermeabilized cap size is decreased for the same applied field intensity. (C) A high flow is present. The hydrodynamic cell deformation is even higher. Almost no electropermeabilization can be induced by the electric field pulse. J. Teissie´ et al. / Bioelectrochemistry 55 (2002) 107–112 111
112 J.Teissie et al.Bioelectrochemistry 55 (2002)107-112 the molecular processes affecting the cell organization [15]V.Ganeva,B.Galutzov,J.Teissie,Electric field mediated loading of during the field pulses. macromolecules in intact yeast cells is critically controlled at the wall level,Biochim.Biophys.Acta 1240(1995)229-236. [16]V.Ganeva,B.Galutzov,N.Eynard,J.Teissie,Electroinduced extrac- tion of B-galactosidase from Kluyveromyces lactis,Appl.Microbiol. Acknowledgements Biotechnol.56(2001)411-413. [17]French patent 0013415. Financial supports from EDF and NATO are acknowl- [18]M.C.Vernhes,These d'Universite,Universite Toulouse 3.1999. edged. [19]D.Knorr,Novel approaches in food processing technology:new tech- nologies for preserving foods and modifying function,Cur.Opin. Biotechnol.10(1999)485-491. [20]C.H.King,E.B.Shotts,R.E.Wooley,K.G.Porter,Survival of coli- References forms and bacterial pathogens within protozoa during chlorination, Appl.Environ.Microbiol.54 (1998)3023-3033. [1]J.Teissie,Effects of electric fields and currents on living cells and [21]C.N.Haas,D.Aturaliye,Semi-quantitative characterization ofelectro- their potential use in biotechnology:a survey,Bioelectrochem.Bio- poration-assisted disinfection processes for inactivation of Giardia energ..20(1988)133-142. and Cryptosporidium,J.Appl.Microbiol.86 (1999)899-905. [2]P.Aldhous,'Dolly'team wins further patents,Nature 405 (2000)986 [22]M.C.Vernhes,A.Benichou,P.Pernin,P.A.Cabanes,J.Teissie,De- [3]N.Eynard,J.Teissie,"Electrotransformation of Bacteria",Springer, struction of free-living amoebae in fresh water by pulsed electric fields Berlin(Germany),2000. combined with chloramine or UV treatment,in:S.Billot-Bonef, [4]J.A.Nickoloff,Electroporation Protocols for Microorganisms,Meth- P.A.Cabanes,F.Marciano-Cabral,P.Pernin,E.Pringuez (Eds.),Pro- ods Mol.Biol.,vol.47,Humana Press,Totowa (USA),1995. ceedings 9th Intemational Meeting on the Biology and Pathology of [5]J.A.Nickoloff,Plant Cell Electroporation and Electrofusion Protocols, Free Living Amoebae,J.Libbey Eurotext,Montrouge (France),2001, Methods Mol.Biol.,vol.55,Humana Press,Totowa (USA),1995. Pp.315-322. [6]M.P.Rols,J.Teissie,Electropermeabilization of mammalian cells to [23]M.C.Vemhes,P.A.Cabanes,J.Teissie,CHO cell sensitivity to local- macromolecules:control by pulse duration,Biophys.J.75 (1998) ized electrical stresses,Bioelectrochem.Bioenerg.48(1999)17-25. 1415-1423. [24]P.Baraton,P.A.Cabanes,M.C.Vernhes,Eradication of Naegleria [7]G.V.Barbosa-Canovas,et al,Preservation of Foods with Pulsed Elec. lovaniensis amoebae by pulsed electric fields,Validation of the effec- tric Fields,Academic Press,San Diego (USA),1999. tiveness of a high-flow process,in:S.Billot-Bonef,P.A.Cabanes,F. [8]T.Kotnik.D.Miklavcic,Analytical description of transmembrane Marciano-Cabral,P.Pernin,E.Pringuez (Eds.),Proceedings 9th In- voltage induced by electric fields on spheroidal cells,Biophys.J.79 ternational Meeting on the Biology and Pathology of Free Living (2000)670-679. Amoebae,J.Libbey Eurotext,Montrouge(France),2001,pp.323- [9]J.Teissi,Electropulsation de cultures cellulaires,Biofutur 45(1986) 329. 64-67. [25]E.Neumann,S.Kakorin,K.Toensing.Membrane electroporation and [10]J.Teissie,P.Conte,Electrofusion of large volumes of cells in culture: electromechanical deformation of vesicles and cells,Faraday Discuss 1,Anchorage dependent strains,Bioelectrochem.Bioenerg.19(1988) 111(1998)111-125. 49-57. [26]N.Asgharian,Z.A.Schelly,Electric field-induced transient birefrin- [11]J.Teissie,P.Conte,Electrofusion of large volumes of cells in culture: gence and light scattering of synthetic liposomes,Biochim.Biophys. 2.Cells growing in suspension,Bioelectrochem.Bioenerg.19(1988) Acta1418(1999)295-306 59-66. [27]H.Mussauer,V.L.Sukhorukov,A.Haase,U.Zimmermann,Resistiv- [12]M.P.Rols,D.Coulet,J.Teissie,Highly efficient transfection of mam- ity of red blood cells against high-intensity,short-duration electric malian cells by electric field pulses:application to large volumes of field pulses induced by chelating agents,J.Membr.Biol.170 cell culture by using a flow system,Eur.J.Biochem.206 (1992) (1999)121-133. 115-121. [28]W.Helfrich,Deformation of lipid bilayer spheres by electric fields,Z. [13]S.Sixou,J.Teissie,Specific electropermeabilization of leucocytes in a Naturforsch.,C 29 (1974)182-183. blood sample and application to large volumes of cells,Biochim. [29]M.Kummrow,W.Helfrich,Deformation of giant lipid vesicles by Biophys.Acta1028(1990)154-160. electric fields,Phys.Rev.A 44(1991)8356-8360 [14]E.Meilhoc,J.Teissie,J.M.Masson,High efficiency transformation of 30]N.Eynard,F.Rodriguez,J.Trotard,J.Teissi,Electrooptics studies of intact yeast cells by electric field pulses,Bio/Technology 8(1990) E.coli electropulsation:orientation,permeabilization and gene trans- 223-227. fer,Biophys..J.75(1998)2587-2596
the molecular processes affecting the cell organization during the field pulses. Acknowledgements Financial supports from EDF and NATO are acknowledged. References [1] J. Teissie´, Effects of electric fields and currents on living cells and their potential use in biotechnology: a survey, Bioelectrochem. Bioenerg. 20 (1988) 133 – 142. [2] P. Aldhous, ‘Dolly’ team wins further patents, Nature 405 (2000) 986. [3] N. Eynard, J. Teissie´, "Electrotransformation of Bacteria", Springer, Berlin (Germany), 2000. [4] J.A. Nickoloff, Electroporation Protocols for Microorganisms, Methods Mol. Biol., vol. 47, Humana Press, Totowa (USA), 1995. [5] J.A. Nickoloff, Plant Cell Electroporation and Electrofusion Protocols, Methods Mol. Biol., vol. 55, Humana Press, Totowa (USA), 1995. [6] M.P. Rols, J. Teissie´, Electropermeabilization of mammalian cells to macromolecules: control by pulse duration, Biophys. J. 75 (1998) 1415 – 1423. [7] G.V. Barbosa-Canovas, et al., Preservation of Foods with Pulsed Electric Fields, Academic Press, San Diego (USA), 1999. [8] T. Kotnik, D. Miklavcic, Analytical description of transmembrane voltage induced by electric fields on spheroidal cells, Biophys. J. 79 (2000) 670 – 679. [9] J. Teissie´, Electropulsation de cultures cellulaires, Biofutur 45 (1986) 64 – 67. [10] J. Teissie´, P. Conte, Electrofusion of large volumes of cells in culture: 1, Anchorage dependent strains, Bioelectrochem. Bioenerg. 19 (1988) 49 – 57. [11] J. Teissie´, P. Conte, Electrofusion of large volumes of cells in culture: 2, Cells growing in suspension, Bioelectrochem. Bioenerg. 19 (1988) 59 – 66. [12] M.P. Rols, D. Coulet, J. Teissie´, Highly efficient transfection of mammalian cells by electric field pulses: application to large volumes of cell culture by using a flow system, Eur. J. Biochem. 206 (1992) 115 – 121. [13] S. Sixou, J. Teissie´, Specific electropermeabilization of leucocytes in a blood sample and application to large volumes of cells, Biochim. Biophys. Acta 1028 (1990) 154 – 160. [14] E. Meilhoc, J. Teissie´, J.M. Masson, High efficiency transformation of intact yeast cells by electric field pulses, Bio/Technology 8 (1990) 223 – 227. [15] V. Ganeva, B. Galutzov, J. Teissie´, Electric field mediated loading of macromolecules in intact yeast cells is critically controlled at the wall level, Biochim. Biophys. Acta 1240 (1995) 229 – 236. [16] V. Ganeva, B. Galutzov, N. Eynard, J. Teissie´, Electroinduced extraction of b-galactosidase from Kluyveromyces lactis, Appl. Microbiol. Biotechnol. 56 (2001) 411 – 413. [17] French patent 0013415. [18] M.C. Vernhes, The`se d’Universite´, Universite´ Toulouse 3, 1999. [19] D. Knorr, Novel approaches in food processing technology: new technologies for preserving foods and modifying function, Curr. Opin. Biotechnol. 10 (1999) 485 – 491. [20] C.H. King, E.B. Shotts, R.E. Wooley, K.G. Porter, Survival of coliforms and bacterial pathogens within protozoa during chlorination, Appl. Environ. Microbiol. 54 (1998) 3023 – 3033. [21] C.N. Haas, D. Aturaliye, Semi-quantitative characterization of electroporation-assisted disinfection processes for inactivation of Giardia and Cryptosporidium, J. Appl. Microbiol. 86 (1999) 899 – 905. [22] M.C. Vernhes, A. Benichou, P. Pernin, P.A. Cabanes, J. Teissie´, Destruction of free-living amoebae in fresh water by pulsed electric fields combined with chloramine or UV treatment, in: S. Billot-Bonef, P.A. Cabanes, F. Marciano-Cabral, P. Pernin, E. Pringuez (Eds.), Proceedings 9th International Meeting on the Biology and Pathology of Free Living Amoebae, J. Libbey Eurotext, Montrouge (France), 2001, pp. 315 – 322. [23] M.C. Vernhes, P.A. Cabanes, J. Teissie´, CHO cell sensitivity to localized electrical stresses, Bioelectrochem. Bioenerg. 48 (1999) 17 – 25. [24] P. Baraton, P.A. Cabanes, M.C. Vernhes, Eradication of Naegleria lovaniensis amoebae by pulsed electric fields, Validation of the effectiveness of a high-flow process, in: S. Billot-Bonef, P.A. Cabanes, F. Marciano-Cabral, P. Pernin, E. Pringuez (Eds.), Proceedings 9th International Meeting on the Biology and Pathology of Free Living Amoebae, J. Libbey Eurotext, Montrouge (France), 2001, pp. 323 – 329. [25] E. Neumann, S. Kakorin, K. Toensing, Membrane electroporation and electromechanical deformation of vesicles and cells, Faraday Discuss 111 (1998) 111 – 125. [26] N. Asgharian, Z.A. Schelly, Electric field-induced transient birefringence and light scattering of synthetic liposomes, Biochim. Biophys. Acta 1418 (1999) 295 – 306. [27] H. Mussauer, V.L. Sukhorukov, A. Haase, U. Zimmermann, Resistivity of red blood cells against high-intensity, short-duration electric field pulses induced by chelating agents, J. Membr. Biol. 170 (1999) 121 – 133. [28] W. Helfrich, Deformation of lipid bilayer spheres by electric fields, Z. Naturforsch., C 29 (1974) 182 – 183. [29] M. Kummrow, W. Helfrich, Deformation of giant lipid vesicles by electric fields, Phys. Rev. A 44 (1991) 8356 – 8360. [30] N. Eynard, F. Rodriguez, J. Trotard, J. Teissie´, Electrooptics studies of E. coli electropulsation: orientation, permeabilization and gene transfer, Biophys. J. 75 (1998) 2587 – 2596. 112 J. Teissie´ et al. / Bioelectrochemistry 55 (2002) 107–112
