The Effect Of Pulsed Electric Fields On Biological Cells: Experiments

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IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 25, NO. 2, APRIL 1997

The Effect of Pulsed Electric Fields on Biological Cells: Experiments and Applications Karl H. Schoenbach, Fellow, IEEE, Frank E. Peterkin, Raymond W. Alden, III, and Stephen J. Beebe (Invited Paper)

Abstract— The effect of pulsed electric fields with amplitudes in the range of 100 V/cm–100 kV/cm on bacteria and aquatic nuisance species has been explored. The pulse duration was so short that heating of the biological matter could be neglected. The electrical energy required for lysing of bacteria, or stunning of aquatic species, decreases when the pulse duration is reduced. For lysing of Eschericia coli, this tendency has been proven to hold for pulsewidths as short as 60 ns. For macroorganisms, however, it was found that for pulsewidths of less than 5 s, the tendency is reversed: the energy required to affect the macroorganisms increases again. This minimum in energy, or maximum in efficiency, respectively, can be understood by taking the time required for electrical charging of the cell membrane into account. Applications of the pulsed electric field technique (PEFT) are in biofouling prevention, debacterialization of liquids, and in the field of medicine. A series of field tests on biofouling prevention in a cooling system with untreated water as coolant has demonstrated the economic feasibility of the electrotechnology.

I. INTRODUCTION

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HE application of electric fields to biological cells in a conducting medium, e.g., water, causes buildup of electrical charges at the cell membrane, and consequently a change in the voltage across the membrane. For low electric fields, this causes voltage-dependent gating, the voltage-induced opening of channels in the cell membrane. A flux of ions through the channels, e.g., sodium and potassium ions, changes the ion concentrations close to the cell membrane and causes cell stress. The stress for short-duration, low-electric-field electrical signals lasts on the order of milliseconds, and does not cause irreparable damage. At higher electric fields, and a correspondingly higher voltage across the cell membrane, the permeability of the membrane increases to such a level that either the cell needs from seconds to hours to recover (reversible breakdown), or Manuscript received September 12, 1996; revised February 26, 1997. The work of K. H. Schoenbach and R. W. Alden, III was supported by CASRM, T. Fox, Program Monitor. K. H. Schoenbach is with the Department of Electrical and Computer Engineering, Physical Electronics Research Institute, Old Dominion University, Norfolk, VA 23529-0246 USA (e-mail: [email protected]). F. E. Peterkin was with the Department of Electrical and Computer Engineering, Physical Electronics Research Institute, Old Dominion University, Norfolk, VA 23529-0246 USA. He is now with the Naval Surface Warfare Center, Dahlgren, VA 22448-5100 USA. R. W. Alden, III is with the Applied Marine Research Laboratory, Old Dominion University, Norfolk, VA 23529-0246 USA. S. J. Beebe is with the Center for Pediatric Research, Eastern Virginia Medical School, Norfolk, VA 23501-1980 USA. Publisher Item Identifier S 0093-3813(97)04072-1.

cell death may occur (irreversible breakdown). The mechanism of this membrane breakdown is not well understood. The most common hypothesis is that pores are generated, openings in the membrane of sizes which allow the exchange of macro molecules. Applications of electroporation, the reversible opening of pores which allows for example DNA to enter the cell, are in medicine and biotechnology [1]. The pores may close again after times which could last hours [2] or the damage may, at very high fields, become irreparable, and cell death occurs. The magnitude of the critical voltage across the membrane which causes the onset of a certain effect on the cell—voltage-dependent gating, poration, or lysing—is dependent on the type and the size of cell, and on pulse duration. For pulse durations in the range from tens of microseconds to milliseconds, typical values of required for lysing of Eschericia coli are on the order of 1 V [3]; for voltagedependent gating, the required transmembrane voltage is 100 mV and less. The corresponding electric field in the medium containing the cells is given as (1) where is the radius of the cell and is a form factor which depends on the shape of the cell [4]. For spherical cells, is 1.5; for cylindrical cells of length with hemispheres of diameter at each end, the form factor is (2) The critical field strength for lysing of bacteria (prokaryotic cells), with dimensions of approximately 1 m and a critical voltage of 1 V across the cell membrane is therefore on the order of 10 kV/cm for pulses of tens of microsecond to millisecond duration. Organisms other than bacteria have eukaryotic cells as their basic building blocks. Dimensions of these cells are in the range of 10–40 m, making them much more vulnerable to electric fields than bacteria. For electric fields above the critical field the survivability , the fraction of surviving microorganisms, decreases exponentially with the field amplitude and linearly with the pulse duration [3]. An empirical law which describes this dependency is valid for pulses of 50 s duration and electric fields 8 kV/cm

0093–3813/97$10.00  1997 IEEE

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SCHOENBACH et al.: EFFECT OF PULSED ELECTRIC FIELDS ON BIOLOGICAL CELLS

where is the pulse duration, is the strength of the applied field, is a threshold field below which no effect is observed, and and are constants which depend on the type of cell and suspension. From this empirical law, an important conclusion can be drawn. The required electrical energy density for lysing which depends on electric field strength resistivity and pulse duration (4) decreases with reduced pulse duration, at least in the pulse duration range 50 s. This is not only true for bacteria. Experimental studies on aquatic nuisance species such as Zebra mussels indicate that similar scaling laws hold for more complicated biological systems [5]. Since the energy density is a measure for the cost of a process, these empirical laws indicate that reducing the duration of the high-field pulses serves to increase the efficiency of the process. This is, however, as recent experiments on aquatic nuisance species have shown [6], not necessarily true for pulses short compared to 50 s. For brine shrimp immersed in sea water, the energy required for lysing or stunning actually increased again when the pulses were shortened below values of 5 s. We have not observed such a minimum in energy in the effect of pulsed electric fields on bacteria. But, as will be shown later, this is probably due to the fact that our pulses, when applied to bacteria, were not short enough to show the reversal in efficiency. For optimum performance, any system which is based on field-induced membrane effects needs to be operated in or close to the minimum in energy density. Optimization with respect to the two variables, electric field and pulse duration, generally requires a vast amount of experimental studies. In order to reduce the number of experiments, we have tried to use a simple electrical model of cells in suspension to predict the position of the energy minimum in the field amplitude and duration space. The results of our model are compared with experimental results obtained with E.coli and brine shrimp. Applications of the electro technology are discussed, particularly biofouling prevention in cooling water systems, where field studies already have demonstrated the feasibility of this technology [7]. II. EXPERIMENTAL SETUP In order to explore the effect of electric fields with pulse durations shorter than those used in previous experiments [3], [8], we have used pulse power systems which allowed us to generate high-voltage pulses of 60 ns, 300 ns, and 2 s duration (FWHM). The 60 and 300 ns electrical pulses were generated by means of a line-type pulser, with coaxial cables used as pulse-forming networks (PFN). Five 50 cables of m and m length each, respectively, are charged to a voltage by means of a dc-power supply. The energy stored in the cables was transferred into the load by closing a laser-triggered spark gap with a rise time of approximately 10 ns. The pulse amplitude is for a matched load, where the resistance of the suspension (in parallel with a resistor) is identical to the impedance of the five parallel cables

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With an applied voltage of up to 20 kV, the maximum pulse amplitude across the load was 10 kV. The third pulse generator is also a line-type pulser, but with an network as PFN. The switch is an electrically triggered spark gap. The system can be charged up to 40 kV, providing the possibility to generate 2 s long, 20 kV pulses at a 1.4 load. Experiments with pulsewidths much greater than 2 s required lower electric fields. Here, we have used solid-state high-voltage power MOSFET’s, capable of generating electric fields of up to 2 kV/cm in the load. The load consisted of aqueous suspension containing the test organisms. The medium was placed in commercially available cuvettes between two plane aluminum electrodes with an area on the order of 1 cm The electric field in the sample is given as where is the distance between the electrodes in the cuvette. With a minimum distance of 1 mm, maximum electric fields of 100 kV/cm could be obtained in the 60 and 300 ns systems. However, surface flashover at the suspension surface has limited the maximum field to about 80 kV/cm. The study of the effect of 2 s pulses was performed at a field strength far below the surface flashover limit. III. RESULTS Experiments on two types of organisms have been performed: one on bacteria (E. coli) and one on an aquatic species, (Artemia salina, also known as brine shrimp). Both sets of experiments were performed at the Physical Electronics Research Institute; the first one was in cooperation with S. J. Beebe from the Eastern Virginia Medical School, and the second one with R. W. Alden, III, from the Applied Marine Research Laboratory at Old Dominion University. Eschericia coli Experimental studies on the effect of pulsed electric fields were mainly performed on XL1Blue E. coli (Stratagene, San Diego, CA). In a small number of experiments, we have also used a second strain of E. coli: BL21DE3 (Novagene, Madison, WI). The volume of the medium in the cuvette for the bacteria experiments was 0.1 cm experiments were performed in nutrient broth where the resistivity was 100 cm, or in tap water of resistivity 1.9 k cm. Measured was the viability of the bacteria (ratio of the concentration of surviving bacteria to the total concentration) using standard procedures: the bacteria were diluted in a serial fashion, spread onto 1.5% LB Agar plates in triplicate, grown overnight, and counted. Because of the relatively small number of bacterial counts (50–100), the statistical error is on the order of 20%. Experimental results with E. coli in tap water are shown in Fig. 1. The measured viability is plotted versus electric field intensity for the three pulse durations: 60 ns, 300 ns, and 2 s The results can be described by an equation which is identical to the one used to represent the effect of electric fields on E. coli in the 50 s domain (3). However, the characteristic parameters in this equation differ considerably from those used to describe the E. coli survivability at 50 s pulses. Whereas for pulses with duration 50 s the measured survivability can be best described with an of 4.9 kV/cm, an of 6.3

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Fig. 1. The effect of electric field pulses of 60 ns, 300 ns, and 2 width on the survivability of E. coli in tap water.

s pulse

kV/cm, and a of 12 s [3], for the results obtained with pulses of less than 2 s duration, in (3) would need to be changed to 40 kV/cm, to 80 kV/cm, and to 10 ns. The value of the electric field required for one order of magnitude reduction of the E. coli population in tap water with 1.9 k cm resistivity is estimated (through extrapolation of the curves in Fig. 1) as 164 kV/cm for 60 ns pulses, 107 kV/cm for 300 ns pulses, and 66 kV/cm for 2 s pulses. The corresponding energies are 0.85, 1.8, and 4.6 J/cm We have compared our results with those obtained by Huelsheger et al. [3] in a suspension with 600 cm resistivity for pulse durations in excess of 50 s (Fig. 2). Although Huelsheger’s results were obtained under different conditions (exponentially decaying pulses versus rectangular pulses, different strains of E. coli, different suspensions), our results, obtained with pulses of almost three orders of magnitude shorter duration, clearly follow the trend: shorter pulses are more effective than longer ones. The electrical field and the energy density (required for one order of magnitude reduction of the E. coli population) dependence on the pulse duration can be described by power laws (Fig. 2). The electric field required to reduce the E.coli population by one order of magnitude decreases with increasing pulse duration in the range from 10 to 6 10 s with (5) with the electric field expressed in volts per centimeter, and (4) the pulse duration in seconds. The energy density increases consequently with The electric field strength required for lysing also was found to be dependent on the resistivity of the suspension. For 60 required for lysing ns pulses, the reduced electric field of E. coli in the 100 cm suspension was approximately 50% less than for E. coli in tap water (1.9 k cm) as suspending

(b) Fig. 2. Experimentally obtained values of electric field and of energy expenditure for one order of magnitude reduction in E. coli population versus pulse duration (open circles: values obtained from [3]; closed circles: results obtained in this study).

medium. For example, for 60 ns pulses, electric fields of 100 kV/cm are needed to reduce the E. coli population in tap water by one order of magnitude, compared to 70 kV/cm is in both cases in nutrient broth. The critical electric field approximately 40 kV/cm. That means that the viability of E. coli is mainly determined by the electric field, rather than by in the suspension. the current density The effect of multiple shots on the viability of E. coli was explored in nutrient broth and in tap water. For an applied electric field of 69 kV/cm and a pulse duration of 60 ns in tap water, the viability is 58% for one pulse, 36% for five pulses, and 21% for ten pulses. The ratio is similar for a nutrient broth suspension. Although the results have a relatively large margin of error, they indicate that subsequent pulses have increasingly less effect on the viability of E. coli. An interesting result was obtained when electrical pulses were applied to E. coli at different phases of their development. The effect of high electric fields on the viability of E. coli in their logarithmic phase of growth, where they rapidly divide,


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was much stronger than on E. coli in the stationary phase, where the net growth rate is near zero. This effect has been observed previously, but for tens of microsecond exposure [8]. Our experiments with E. coli XL1Blue have shown that rapidly dividing cells are also more susceptible to electric fields down to the nanosecond range than cells which are not proliferating. For 100 pulses at a relatively low electric field of 50 kV/cm, the concentration of E. coli in the stationary phase was reduced to 32.5% of its initial value, and that of E. coli in the logarithmic phase to 12.7%. Artemia salina In order to determine the effect of pulsed electric fields on more complex biological systems, we have used brine shrimp (Artemia salina). Brine shrimp are adapted to highly variable environments, and therefore are resistant to extreme conditions, including ionic stress. Artificial sea water with a salinity of 15 ppt corresponding to a resistivity of 50 cm was used as the medium for the brine shrimp. The cuvettes containing the medium had an electrode separation of 2 and 4 mm, and allowed treatment of a volume of 0.4 and 0.8 cm , respectively. Maximum electric fields were 40 kV/cm in this experiment. The quantitative evaluation of the effect of electric fields on complex organisms is more difficult than that for single cells. The evaluation technique used so far consists of observing the motion of brine shrimp with a microscope for up to 10 min after field exposure. Results are shown in Fig. 3 for a two-min stunning effect and a greater than 10 min stunning effect [6]. In the latter case, the mortality was high. Plotted in the upper diagram is the required electric field intensity versus the time of exposure for single-shot operation. The results (for stunning, but similarly for killing of brine shrimp) show that for pulse durations exceeding 10 s, the electric field increases slightly with inverse pulse duration for pulse durations below 10 s, the slope changes to a much larger value The energy density (4) required for stunning (Fig. 3, lower diagram) for pulses longer than 10 s increases with pulse duration, as expected from emHowever, the tendency is pirical laws [3], [5]: reversed for shorter pulses: The minimum of the energy expenditure, the maximum of efficiency, for stunning of brine shrimp is at pulse durations of approximately 5 s IV. DISCUSSION The “resonance effect,” the optimum in efficiency at a pulse duration of approximately 5 s in the results of the brine shrimp experiments (Fig. 3), can be understood by considering the electrical equivalent circuit (Fig. 4) of a cell embedded in a conducting medium [9]. The capacitance and the resistances (parallel to the cell) and (in series to the cell) describe the capacitive and resistive properties of the suspension. Generally, for a low concentration of organisms in the suspension, the time scale of the polarization of the suspension (dielectric relaxation time) determines the lower limit in pulse duration for membrane effects. The time constant for polarization is given as the

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(b) Fig. 3. The electric field required for two-min stunning (closed circles) and greater than 10-min stunning (open circles), and the corresponding electrical energy expenditure.

product of the dielectric constant and the resistivity Water, which is often used as suspension, has a relative dielectric constant of The resistivity of fresh water is several k cm. Assuming a resistivity of 5 k cm, the dielectric relaxation time is 35 ns. For sea water with resistivities of about 50 cm, the dielectric relaxation time is 0.35 ns. The cell immersed in the suspension is described by the of the cell membrane, in series with the capacitance resistance of the cell interior Typical capacitances per unit area for the cell membranes are 1 F/cm [10]. The cell interior has a resistivity on the order of 100 [11]. The membrane consists of a lipid bilayer with proteins embedded into the bilayer. Some of the proteins act as voltagegated channels that provide a pathway for the flux of ions. This effect is modeled by voltage-dependent conductances in series with driving voltage sources (Nernst potential) for each ion species [12]. Up to a threshold voltage which is on the order of several tens of millivolts above the resting voltage of the membrane, the conductance is constant. Increasing the voltage above this threshold causes a nonlinear increase in the conductance, first, of one particular

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Fig. 4. Electrical equivalent circuit for cells in suspension.

type of channel, and later, of other ion channels. Further increase of voltage causes more and more channels of the same type to open. The increased molecular exchange between cells and their environment and the cell stress due to chemical imbalances causes cell death [13]. The large increase in current due to multiple opening of ion channels is analogous to a dielectric breakdown of the membrane. In some of the literature, this massive breakdown effect is termed electroporation, the formation of large pores in the membrane. However, there is only scarce evidence of the formation of large openings [2]. Some researchers therefore prefer to use the term “electropermeabilization” or “electric breakdown” [14]. This electric breakdown may be reversible, and allows the injection of macromolecules such as nucleic acids or proteins and/or pharmaceutical agents. It also inactivates the cell, making it nonreceptive to any other stimulus, an effect which could be described as stunning. The breakdown also may be irreversible, and therefore can be used to eliminate unwanted species, e.g., bacteria in drinking water or food. Voltage-dependent gating and “electropermeabilization” is not in instantaneous effect. It takes time to establish the required voltage across the membrane. This time is, according to the model depicted in the equivalent circuit (Fig. 4), the charging time of the membrane capacitance. The time constant for cell membrane charging is [11] (6) is the resistivity of the cell interior, is the where resistivity of the suspension, is the capacitance of the cell membrane per unit area, and is the cell radius. This equation is valid only if the product of membrane resistivity and membrane capacitance is very large compared to Since the “breakdown” is possibly initiated or even identical to voltagedependent gating at high voltages, a voltage-induced increase in permeability, the “leakage current” across the membrane, might cause the time constant required for membrane charging to exceed the value of in (6). Assuming that the required voltage across the membrane necessary to achieve a certain effect, e.g., lysing of bacteria, is constant for times on the order of and less than the charging time the condition for the required electric field is (7)

is the electric field required to obtain to reduce the viability of a certain species by a certain amount at times long compared to the charging time. In the case of E. coli, for the case of one order of magnitude reduction in E. coli population, is determined by (5). The electric field dependence on the pulse duration for lysing of E.coli, including the membrane charging effect, is therefore (8) given as 585 s V/cm. The charging with the constant time of the cell membrane is assumed to be 50 ns, a value which was obtained from (6), with the radius of the E. coli cell assumed to be 0.5 m, and a suspension resistivity of 1.9 k cm. The computed curves for electric field (8) and energy density versus pulse duration are plotted in Fig. 5. At approximately 100 ns, just above the shortest pulse duration in our experiments on E. coli, the tendency predicted by the empirical laws is reversed: the modeling results predict an increase in energy when the pulse duration is reduced below this value. Whereas this reversal in energy consumption has not been experimentally demonstrated yet for bacteria (pulses with durations small compared to 100 ns would need to be applied), this effect could be shown for another class of cells. For eukaryotic cells, the basic building blocks of macroorganisms, the same equation for the charging time (6) holds, but with different values of the dimension of the cell. Assuming a cell radius of m, the charging time for these cells in a suspension with a resistivity of 50 cm is 0.125 s. Computed electric fields and energy densities, required for stunning of macroorganisms, are shown in Fig. 6(a) and (b). The calculations which take the membrane charging effect into account (7) are based on experimental electric field values for stunning of brine shrimp at long pulse durations 10 s in Fig. 3), values which are almost identical with those obtained on Zebra mussels and Daphnia [5]. As for bacteria, there is an optimum in energy efficiency. It is shifted to longer pulse duration compared to that for bacteria, and the minimum energy is less. Comparing the modeling results (which depend on experimental values obtained at long pulse durations) with our experimental results for short pulses shows a qualitative agreement. The membrane-charging correction term explains the


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Fig. 5. Measured (Fig. 2) and computed values of electric field and energy density required to reduce the E. coli population by one order of magnitude dependent on the pulse duration. The empirical law for the survivability of E. coli exposed to pulses of several tens of microseconds (3) is shown for comparison (upper diagram: dashed line).

Fig. 6. Computed electric field and energy density required for stunning/killing of larval macro-organisms, in sea water (solid line) and fresh water (dashed line).

observed “resonance” on the energy curve. The fact that the computed minimum in energy is shifted toward shorter times, compared to the experimental results, might be due to “leakage currents” in the membrane, an effect not considered in the calculation of the charging time constant (6). The brine shrimp experiments were performed in tidal water cm. An important application of with a resistivity of 50 the technology is the biofouling prevention in fresh water, particularly the prevention of Zebra mussel infestation. The effect of the suspension on the optimum in energy consumption is according to the electrical model only contained in the charging time constant for the cell membrane (6). Changing from tidal water with 50 cm resistivity to fresh water with values on the order of 5 k cm changes the time constant by a factor of 20.8. The corresponding energy curves for larval macroorganisms in fresh water are shown in Fig. 6 as dashed curves. Applying the pulsed electric field technique to organisms in suspensions with higher resistivity clearly results

in a reduction of energy as shown in the lower diagram of Fig. 6 and consequently, in a reduction of cost. V. APPLICATIONS Biofouling Control As shown in our laboratory experiments, the application of electric field pulses can stun aquatic species or, at increased fields, induce mortality. Attachment of the larvae of fouling organisms to surfaces (e.g., pipes in cooling systems of power plants) therefore can be prevented with this method. In addition, the ballast water of ships can be treated to kill the larvae of nuisance species (e.g., Zebra mussels) before they are introduced into a different ecosystem. In order to explore the preventive effect of pulsed electric fields on biofoulants, we have performed field tests with a pulse power system which was designed to provide microsecond pulses of up to 40 kV into a low impedance 10 ) load. It consists of an 8 KJ/s Maxwell CCDS power supply which

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(b) Fig. 7. Schematics of the field test system, including a Blumlein type pulse generator; and, below, a voltage pulse measured across the load.

charges a Blumlein PFN made of 160 TDK 2 nF capacitors depending on the with an impedance between 6 and 7 inductance in the PFN. When discharged through a thyratron (EG&G HY-3190), the circuit provides a pulse of 770 ns FWHM to a matched resistive load which is connected to the PFN by means of a stripline. A schematic of the field system and a typical voltage pulse is shown in Fig. 7. The load is water from the Elizabeth River in Norfolk with a cm. The water was pumped resistivity of approximately 50 through the rectangular treatment cell with titanium electrodes on top and bottom to generate a homogeneous electric field perpendicular to the direction of the water flow. A second cell with identical dimensions, but without fields applied, was used in the control part of the system. After passing through the cells, the water flowed through 4.5 m long, 0.6 in ID PVC pipes, and was then discharged back into the river. The flow of the water was adjusted to a velocity such that it was exposed at least once to the pulsed electric field between the electrodes. The first test [15] was performed in November 1995, over a duration of 20 days. It was terminated due to failure of the thyratron. In the 20 days of operation, the pulsed electric field system was operated with a repetition rate of 12 Hz. A voltage of 12 kV was applied to a layer of water of 1 cm thickness kV/cm) between two planar electrodes of 5 cm length and 1.5 cm width. The instantaneous electrical power of the 0.7 s long pulse was 24 MW, but the average power delivered to the load was only 200 W. The flow rate of the water was set to 1 gal/min. The pipes were analyzed for biofouling three days after the thyratron failure. In the control tubes, more than 600 barnacles were counted, about 30 polyacheate worms, 12 hydrazoans, and eight unknown organisms. In the pipes where

the electrically treated water had circulated, no macroscopic organism was observed. The second test [7] was performed in May 1996 with an electric field, half the value of that used in the first test: 6.45 kV/cm. The second test was terminated after 23 days, with all pulse power system components working properly. The load in this test was slightly mismatched, 9 versus 7 the PFN impedance. This was done deliberately to prevent voltage reversal in the case of a strong increase of the salt content, and consequently the conductivity, of the river water. The gap between the electrodes was increased to 2 cm, and 2.6 cm. With the voltage the electrode area was 5 cm slightly higher compared to the first test series (due to the mismatch of the load), the electric field in the water was 6.45 kV/cm. The peak power was slightly less than in the first run: 18.5 MW; the average power delivered to the load was 170 W at a repetition rate of 12 Hz. The flow rate was set to 4 gal/min. A total of 140 000 gal was treated over the duration of the experiment. The results were the same as in test 1: biofouling was prevented completely by the treatment with pulsed electric fields. The composition and the densities of the fouling organisms (barnacles, tunicates, and mussels) in the control pipes, however, were different from that obtained in the Fall of 1995 (dozens of individuals versus hundreds) due to a very high suspended solids load in the water at this time of the year. The efficiency of the pulsed electric field method can be defined as the volume of successfully treated water per electrical energy unit, e.g., number of gallons of treated water per kilowatthour electrical energy. Using the results of the second experiment, where an electrical field of 6.45 kV/cm


of 770 ns duration was sufficient to prevent blue mussel, barnacle, and hydrazoan biofouling in pipes, the efficiency is 1400 gal/kWh. This value is still too small to make the pulsed electric field method competitive with chemical methods used to prevent biofouling in very large systems in saline environments (e.g., cooling systems of power plants). However, we feel that it can be increased considerably by operating the pulse power system closer to the minimum for energy dissipation, and by applying the method to fresh water biofoulants, such as Zebra mussels. An increase in efficiency by two orders of magnitude seems to be achievable by optimizing the system for fresh water treatment [16]. Even if the predicted efficiency cannot be reached, the use of pulsed electric field systems at the present state of development already might be economically feasible for small cooling water systems, which are difficult to clean. The following are advantages over other techniques used for biofouling prevention: • independence from chemicals; • the fact that it is possible to stun, rather than kill, unwanted biofouling species; • it allows us to preserve valuable species, such as fish and shellfish larvae, from being destroyed; • it does not generate shock waves which could affect the structure of the cooling system; • it can be installed like a filter in front of an existing cooling system, without requiring any changes in the cooling system. These make the pulsed electric field technology (PEFT) a strong contender to existing biofouling prevention methods.

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VI. SUMMARY The application of pulsed electric fields to suspensions containing micro- or macroorganisms has been proven to be a means of controlling the population of these organisms, or to stun them over a certain field-dependent time interval. This effect can be utilized for environmental or medical applications. The efficiency of the pulsed electric field effect increases with decreasing pulse duration. This holds down to pulse durations which correspond to the charging time of the cell membrane. The optimum range for biofouling prevention therefore seems to be in the pulse length range of microseconds, and that for debacterialization in the tens of nanoseconds range. For biofouling protection, field studies already have proven the applicability of this technique in large-scale cooling systems. The duration of the high-power electrical pulses is such that thermal effects on micro- and macroorganisms can be neglected. The study of the effect of extremely short pulsed electric fields on an organism allows us to separate nonlinear electrical effects from thermal effects. These nonlinear effects might open possibilities to modify the cell structure in a controlled way without lysing the cell, beyond the electroporation method [1], and to open a new field of applications for pulse power technology. ACKNOWLEDGMENT The authors appreciate the technical support of A. AbouGhazala, R. Allen, T. Vithoulkas, T. Turner, P. Adolphson, and D. Byars. REFERENCES

Debacterialization Attempts to utilize pulsed electric fields for debacterialization in the area of food preservation are in progress [17], [18]. Studies on the debacterialization of drinking water mainly have concentrated on the use of ionizing radiation. Energies required to reduce the initial population by one order of magnitude are 3–5 J/cm [19]. Using 20 s electrical pulses to reduce the population of E. coli in tap water by one order of magnitude would require, according to the results of Huelsheger [3], more than 10 J/cm By extending the electrical exposure to higher electric field strength at shorter pulse duration, debacterialization of a medium is possible with much reduced energy, as shown in Figs. 2 and 5. For E. coli XL1Blue, the energy required to debacterialize a medium with a resistivity of 1.9 k cm (tap water) with a 300 ns pulse requires only 1.5 J/cm This value corresponds to an energy expenditure of 1 kWh for the debacterialization of 2400 L of tap water. Medical Applications The observed electric field response of E. coli in different stages of their growth indicates that rapidly dividing cells are more susceptible to electric fields than cells which are not proliferating. If this also holds true for mammalian cells, then ultrashort high-electric-field pulses could affect tumor or cancer development significantly.

[1] G. A. Hofmann and G. A. Evans, “Electronic genetic—Physical and biological aspects of cellular electromanipulation,” IEEE Eng. Med. Biol. Mag., p. 6, Dec. 1986. [2] D. C. Chang and T. S. Reese, “Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy,” Biophys. J., vol. 58, p. 1, 1990. [3] H. Huelsheger, J. Potel, and E. G. Niemann, “Killing of bacteria with electric pulses of high electric field strength,” Radiat. Environ. Biophys. vol. 20, p. 53, 1981. [4] A. J. H. Sale and W. A. Hamilton, “Effects of high electric fields on micro-organisms: III. Lysis of erythrocytes and protoplasts,” Biochim. Biophys. Acta, vol. 163, p. 37, 1968. [5] J. R. Duncan, M. Rader, S. Levy, I. Alexeff, J. A. Drake, and J. Johnson, “The use of pulse power to control Zebra mussels,” in Proc. 3rd Int. Zebra Mussel Conf., Toronto, Ont., Canada, Feb. 1993. [6] K. H. Schoenbach, F. E. Peterkin, S. J. Beebe, D. Byars, R. W. Alden, III, P. Adolphson, and T. Turner, “Effect of pulsed electric fields on micro-organisms: experiments and applications,” in Proc. 10th IEEE Int. Pulsed Power Conf., Albuquerque, NM, June 1995, p. 25. [7] K. H. Schoenbach, R. W. Alden, III, and T. J. Fox, “Biofouling prevention with pulsed electric fields,” in Conf. Rec., 1996 22nd Int. Power Modulator Symp., Boca Raton, FL, June 1996, p. 75. [8] H. Huelsheger, J. Potel, and E.-G. Niemann, “Electric field effects on bacteria and yeast cells,” Radiat. Environ. Biophys., vol. 22, p. 149, 1983. [9] H. P. Schwan, “Dielectric properties of cells and tissues,” in Interactions Between Electromagnetic Fields and Cells, A. Chiabrera, C. Nicolini, and H. P. Schwan, Eds. New York and London: Pergamon, 1985, p. 75. , Biophys., vol. 1, p. 198, 1963. [10] [11] K. S. Cole, “Electric impedance of marine egg membranes,” Trans. Faraday Soc., vol. 23, p. 966, 1937. [12] A. L. Hodgkin and A. F. Huxley, “A quantitative description of membrane current and its application to conduction and excitation in nerve,” J. Physiol., vol. 117, p. 500, 1952.

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[13] C. Weaver and Y. Chizmadzhev, “Electroporation,” in Handbook of Biological Effects of Electromagnetic Fields, 2nd ed., C. Polk and E. Postow, Eds. Boca Raton, FL: CRC, 1996, p. 247. [14] U. Zimmermann, “The effect of high intensity electric field pulses on eukaryotic cell membranes: Fundamentals and applications,” in Electromanipulation of Cells, U. Zimmermann and G.A. Neil, Eds. Boca Raton, FL: CRC, 1996, p. 1. [15] K. H. Schoenbach, R. W. Alden, III, and T. J. Fox, “Effect of pulsed electric fields on aquatic nuisance species,” in Zebra Mussels and Aquatic Nuisance Species, F. D’Itri, Ed. Chelsea, MI: Ann Arbor Press, 1996, p. 399. [16] An increase in efficiency in tidal water by one order of magnitude already has been demonstrated since this paper was submitted. [17] A. H. Bushnell, J. E. Dunn, R. W. Clark, and J. S. Pearlman, “High pulsed voltage systems for extending the shelf life of pumpable food products,” U.S. Patent 5 235 905, Aug. 17, 1993. [18] G. S. Mittal, S. Ho, and J. Cross, “High voltage electric pulses for food pasteurization,” in Proc. Agricultural Inst. Can. Annu. Conf., Regina, Sask., Canada, 1994. [19] R. Curry, Univ. Missouri, Columbia, private communication.

Karl H. Schoenbach (F’94) received the Diploma degree in physics and the Dr.rer.nat. degree in physics in 1966 and 1970, respectively, both from the Technische Hochschule in Darmstadt, Germany. During his tenure at the Institute for Applied Physics at the Technische Hochschule in Darmstadt, from 1966 to 1979, he was working in the areas of gas discharge physics and the dense plasma focus. From 1979 to 1985, he held a faculty position at Texas Tech University, Lubbock, where he was involved in research on fast high-power switches, especially high-pressure glow discharge opening switches. Since 1985, after accepting a faculty position at Old Dominion University, Norfolk, VA, he has been active in research on pulse power photoconductive switches and in modeling and experimental studies on hollow cathode gas discharges. Since 1993, he has concentrated on exploring the potential of pulse power technology for medical and environmental applications. He has organized several workshops and conferences on plasma science, was guest editor of the IEEE TRANSACTIONS ON ELECTRON DEVICES (1990) and is presently associate editor of the IEEE TRANSACTIONS ON PLASMA SCIENCE.


Frank E. Peterkin, (M’96) for a biography, see this issue, pp. 137.

Raymond W. Alden, III received the B.S. degree in biology from Stetson University, DeLand, FL, in 1971 and the Ph.D. degree in zoology/marine biology from University of Florida, Gainesville, in 1976. He served in a postdoctoral position at University of North Carolina (Chapel Hill) 1975–1976 and has been at Old Dominion University (ODU), Norfolk, VA, since 1976. He is a Professor of Biological Sciences and Director of the Applied Marine Research Laboratory at ODU. His areas of research include investigations into the effects of pollution on aquatic organisms and in exploring innovative, nonpolluting technologies for applications in aquatic ecosystems. He has served as PI on over $20 million of research grants and contracts, is a member of numerous professional societies, and has been a technical advisor to over 40 municipal, state, federal and international agencies. Recently, he was selected for the position of Dean of the College of Sciences of University of Nevada, Las Vegas, which he will fill beginning in August, 1997.

Stephen J. Beebe received the B.S. degree in zoology from Ohio University, Athens in 1971, and the Ph.D. degree from the Medical College of Ohio, Toledo, in 1982. Prior to graduate studies, he was a U.S. Peace Corps Volunteer in the British West Indies (1973–1975). He was a Post-Doctoral Fellow at the Howard Hughes Medical Institute, Vanderbilt University Medical Center, Nashville, TN, (1982–1987) and a Fulbright Scholar and Professor at the Institute for Medical Biochemistry and the National Hospital, Oslo, Norway (1987–1988). He is presently an Associate Professor of Pediatrics and Physiology at the Eastern Virginia Medical School, Norfolk, VA. His areas of research include molecular and cellular Biochemistry of cell signal transduction. Specific areas of interest include structure-function relationships of protein kinases, the regulation of gene transcription and programmed cell death (apoptosis), and the effects of short duration, high energy electrical pulses on cell proliferation, differentiation, and function in normal and abnormal human cells. Dr. Beebe is a member of the American Association for the Advancement of Science, the New York Academy of Sciences, and the American Society of Biochemistry and Molecular Biology.