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 electropermea￾bilization. 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 electro￾permeabilization [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 conduc￾tance 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 permeabiliza￾tion is increased with pulse duration and number of succes￾sive 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 up￾sizing 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 tech￾nology in the mid-1980s [9] for an up-scaling of electro￾fusion [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