Inactivation of Bacteria in Oil-Field Reinjection Water by Pulsed

Oct 31, 2008 - The effects on inactivation of process parameters including electric field strength, process temperature, initial bacteria concentratio...
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Ind. Eng. Chem. Res. 2008, 47, 9644–9650

Inactivation of Bacteria in Oil-Field Reinjection Water by Pulsed Electric Field (PEF) Process Qing Xin, Xingwang Zhang, and Lecheng Lei* Institute of EnVironmental Pollution Control Technologies, Xixi Campus, Zhejiang UniVersity, Hangzhou 310028, P.R. China

Microbial pollution commonly causes serious pipe corrosion in oil-field reinjection water systems. As an alternative and environmentally friendly inactivation technology, the pulsed electric field (PEF) process is employed to inactivate microorganisms in oil-field reinjection water. The effects on inactivation of process parameters including electric field strength, process temperature, initial bacteria concentration, and conductivity were investigated. The results showed that electric field intensity is the most important parameter in PEF inactivation. After a 20-min treatment, the inactivation efficiency was a 2.8 log reduction for saprophytic bacteria, a 3.6 log reduction for iron bacteria, and a 3.9 log reduction for sulfate-reducing bacteria. Transmission electron microscopy observations of the bacteria confirmed that PEF technology can lead to severe surface damage and rupture to the cells. Moreover, a possible mechanism of inactivation is discussed and used to explain the influence of process parameters on PEF treatment. 1. Introduction Water injection for pressure balance is a key element in oilfield operations; as a byproduct, oily wastewater is discharged from oil fields. After separation from the oil, the produced water is a promising source for reinjection.1,2 Microbiologically influenced corrosion commonly occurs in the produced water reinjection system. Saprophytic bacteria, iron bacteria, and sulfate-reducing bacteria are the three main microorganisms resulting in corrosion problems, and sulfate-reducing bacteria are recognized as a major group of microorganisms linked to anaerobic corrosion.3 The biocorrosion phenomenon brings consequent energy and efficiency losses and structural failures resulting from corrosion of pipes and equipments. The most common method for controlling pollution caused by microorganisms is the use of biocides.4,5 However, microbial resistance to chemical biocides and the environmental toxicity of these chemicals restricts their use.6,7 Consequently, it is necessary to explore effective and environmentally friendly bactericidal technology. Pulsed electric field (PEF) technology is the application of pulses with high field strength (microseconds) to water flowing between electrodes of a treatment chamber for a short time. The high electric field is responsible for the lethality of PEF against microorganisms and spores. It is well-established that pulsed electric fields can induce a transmembrane potential exceeding a threshold value to result in electroporation.8,9 When the electric field intensity and pulse duration are sufficient, irreversible breakdown of cell membranes occurs as a result of a large number of pores or pores of sufficiently large size. The irreversible electroporation process leads to inactivation. PEF technology has the advantages of short treatment times, no undesirable effects on the liquid component, and no addition of chemical agents. As an effective, nonthermal inactivation technology, PEF is intensively studied in food preservation, and successful results have been achieved.10-13 Until now, there are few studies focusing on the application of pulsed electric fields to inactivate bacteria in industrial wastewater. The * To whom correspondence should be addressed. Tel.: +86-57188273090. Fax: +86-571-88273916. E-mail: [email protected].

examination of the effects of operating parameters on inactivation can provide performance data for large-scale applications. In this study, we attempted to apply PEF technology in a batch treatment chamber to inactivate bacteria in oil-field reinjection water. The effects on inactivation of process parameters including electric field intensity, conductivity, temperature, and initial bacteria concentrations were investigated. The effect of electric field intensity was investigated in two reactors with different electrode distances. The surface structure and morphology of E. coli before and after PEF treatment were examined by transmission electron microscope. 2. Experimental Methods 2.1. Microorganisms and Growth Conditions. The microorganisms used in the experiments were isolated from wastewater of a municipal sewer treatment plant in Hangzhou, China. The growth media for the bacteria were prepared according to the National Standard methods in China. The artificial medium for saprophytic bacteria growth used in the experiments contained14 3.0 g of beef extract, 5.0 g of sodium chloride, and 10.0 g of peptone per liter of distilled water. The pH was adjusted to 7.0-7.4 with 10% sodium hydroxide. The artificial medium for iron bacteria growth contained15 0.5 g of magnesium sulfate heptahydrate, 0.5 g of ammonium sulfate, 0.5 g of potassium dihydrogen phosphate, 0.5 g of calcium chloride, 0.5 g of sodium nitrate, and 10.0 g of ferric ammonium citrate per liter of distilled water. The pH was adjusted to 6.6-6.8 with 10% sodium hydroxide. The artificial medium for sulfatereducing bacteria growth contained15 0.5 g of dipotassium hydrogen phosphate, 1.0 g of ammonium chloride, 2.0 g of magnesium sulfate heptahydrate, 0.5 g of sodium sulfate, 0.1 g of calcium chloride, 1.0 g of yeast extract, and 4.0 mL of sodium lactate per liter of distilled water. The pH was adjusted to 7.4-7.6 with 10% sodium hydroxide. Before use, 1.2 g of ferrous ammonium sulfate hexahydrate that had been sterilized by ultraviolet radiation for 30 min was added. Beef extract, peptone, and yeast extract were purchased from Hangzhou Microbial Reagent Co. Ltd., Hangzhou, China. Sodium chloride, sodium hydroxide, magnesium sulfate heptahydrate, ammonium sulfate, potassium dihydrogen phosphate, calcium chloride,

10.1021/ie8000524 CCC: $40.75  2008 American Chemical Society Published on Web 11/01/2008

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Figure 1. Schematic of the circulation system for pulsed electric field treatment: (1) pulsed power supply, (2) treatment chamber, (3) water pump, (4) reservoir, (5) cooling water bath.

Figure 2. Typical wave forms of the two reactors.

sodium nitrate, ferric ammonium citrate, dipotassium hydrogen phosphate, ammonium chloride, sodium sulfate, sodium lactate, and ferrous ammonium sulfate hexahydrate were purchased from China National Pharmaceutical Group Shanghai Chemical Reagent Company, Shanghai, China. All media were sterilized for 20 min at a pressure of 1.2 × 104 kPa in an autoclave (DSX280B; Shenan Medical Instrument Factory, Shanghai, China). 2.2. PEF Equipment. The experimental apparatus consisted of a pulsed power supply and a PEF treatment chamber, shown in Figure 1. All experiments were conducted in batch mode. A ZH-2006 pulsed power supply (ETL Company, Jinhua, China) was used in all tests. The pulsed high-voltage source used a thyratron switch and a Blumlein pulse-forming network (BPFN). The main advantages of the power supply were (1) increasing safety, (2) avoiding spark noise and keeping pulse frequency stable, and (3) enabling easy adjustment of the BPFN to match the pulsed voltage source of the reactor. It was designed to allow an adjustable pulse frequency (from 0.5 to 5 Hz), and the energy input per pulse was set at 5, 10, and 15 J. The input peak voltage was 3400, 4400, and 5400 V when the single-pulse energy was 5, 10, and 15 J, respectively. The two treatment chambers used in this study were made of plexiglass. The two reactors contained a coaxial electrode system. The thyratron switch power source had a 10-µs pulse width and a 1-µF storage capacitance. The typical waveforms were measured on an oscilloscope (Rigol, DS1022C, Beijing, China), shown in Figure 2. The main difference between the two reactors was the electrode distance. The electrode distance of reactor 1 was 20 mm, and that of reactor 2 was 3 mm. The input energy per pulse of reactor 1 was 15 J and that of reactor 2 was 5 J. The

volume of each of the two treatment chambers was 150 mL. A total volume of 1000 mL of solution was circulated through the treatment chamber by a water pump. A cooling water bath was used to control the increase in temperature due to joule heating. 2.3. Microbial Inactivation Treatments. The microorganisms inoculated in artificial media were diluted in 1000 mL of sterile water to the desired microbial concentration, and the conductivity was adjusted with potassium chloride (China National Pharmaceutical Group, Shanghai Chemical Reagent Company, Shanghai, China). The pH of the samples was 6.5 ( 0.4. Conductivity and pH were measured with a conductivity meter (DDS-11A; Shanghai Rex Xinjing Instrument Co., Ltd., Shanghai, China) and a pH meter (pHS-25; Shanghai Rex Xinjing Instrument Co., Ltd., Shanghai, China), respectively. Before treatment, 75% ethanol (Changzheng Chemical Reagent Co. Ltd., Hangzhou, China) was run through the chamber to sterilize the chamber interior, and then the chamber was rinsed three times using sterile deionized water to remove any residual ethanol. Nitrogen was used to remove oxygen in the system before inactivation tests of sulfate-reducing bacteria because it is an anaerobe. The experiments regarding temperature, initial bacteria concentration, and conductivity were conducted in reactor 1. The experimental conditions for investigating the effect of temperature were as follows: 5 Hz pulse frequency, 1.5 × 103 CFU/mL initial concentration, and 5 ms/cm conductivity. The temperature of oil-field-produced water is commonly less than 65 °C;16 hence, initial temperatures were set at 30, 38, 47, and 55 °C. The experimental conditions for investigating the effect of initial bacteria concentration were 5 Hz pulse frequency and 5 ms/cm conductivity, with initial concentrations of 1.7 × 103, 3.6 × 104, 3.6 × 106, and 1.5 × 107 CFU/mL. The experimental conditions for investigating the effect of conductivity were 5 Hz frequency and 3.9 × 103 CFU/mL initial cell concentration, with values of conductivity ranging from 0.03 to 10 ms/cm as adjusted by KCl. The experimental conditions for inactivation in different reactors were 5 Hz pulse frequency and 5 ms/cm conductivity, with initial concentrations of saprophytic bacteria, iron bacteria, and sulfate-reducing bacteria of 1.8 × 106, 4.5 × 105, and 4.5 × 105 CFU/mL, respectively. For the transmission electron microscopy (TEM) analysis, Escherichia coli (purchased from Zhejiang Microbial Research Institute, Hangzhou, China) suspended in samples before and after 20 min of PEF treatment in reactor 2 were concentrated by centrifugation (TDL-40B, Shanghai Anting Scientific Instrument Factory, Shanghai, China) at 4000 rpm for 10 min. After the supernatant had been discarded, the samples were fixed with

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2.5% glutaraldehyde (Xingfa Reagent Development Co., Ltd., Shaoxing, China) overnight, buffered with 0.1 M phosphate buffer, and then fixed with 1% osmium tetroxide (Beijing Zhong Jingkeyi Technology Co., Ltd., Beijing, China) for 2 h. After being rinsed by phosphate buffer, the samples were dehydrated in a graded ethanol series and acetone (Hangzhou Chemical Reagent Co., Ltd., Hangzhou, China). Then, the samples were embedded in EPON 812 (Sigma). Ultrathin sections (70-90 nm) were made on an ultramicrotome Ultracut (Reichert Jung). After being dyed with lead citrate solution and uranyl acetate (Beijing Zhong Jingkeyi Technology Co., Ltd., Beijing, China) for 15 min, the ultrathin sections were investigated by TEM (JEM-1230, JEOL, Tokyo, Japan). The preparation for scanning electron microscopy (SEM) was similar to that for TEM. After dehydration and critical-point drying with liquid carbon dioxide, the samples were coated with gold-palladium and viewed with a scanning electron microscope (XL-30-ESEM, Philips, Eindhoven, The Netherlands). 2.4. Microorganism Viable Counts. The inactivation efficiency of the technology described above was assessed by the number of viable microorganisms before and after treatment. One milliliter of treated sample was taken after each PEF treatment and diluted with sterile 0.85% sodium chloride solution. Enumeration of saprophytic bacteria before and after PEF treatment was done by the plate count method. After being incubated for 48 h at 37 °C, colonies of saprophytic bacteria on agar plates were calculated from average of at least three plates. Populations of sulfate-reducing bacteria and iron bacteria in the samples were estimated using the serial dilution method, the tree-tube regime, and the most probable number (MPN) technique. The tubes containing sulfate-reducing bacteria and iron bacteria were incubated at 37 °C for 14 and 7 days, respectively. The MPN method was based on the application of probability theory to the numbers of observed positive growth responses to a standard dilution series of sample inoculated into culture media tubes, whereas the plate count method involved direct counts of living organisms expressed in colony forming units per milliliter (CFU/mL). The medium for saprophytic bacteria enumeration contained 3.0 g of beef extract, 5.0 g of sodium chloride, 10.0 g of peptone, and 15 g of agar (Quangang Chemical Reagent Co. Ltd., Quanzhou, China) per liter of distilled water. The pH was adjusted to 7.0-7.4 with 10% sodium hydroxide. The growth media for enumeration of iron bacteria and sulfate-reducing bacteria were the same as described in section 2.1. Medium control plates or tubes were always prepared to eliminate error due to medium contamination. Every treatment was repeated three times in our work, and the average values are reported. The relative standard deviations of the data were less than 10%. The relative standard deviations were calculated using Microsoft Excel 2003, and the data were plotted using Origin 7.5. The inactivation efficiency was expressed as the base-10 logarithm of the survival fraction and inactivation rate, which were calculated as follows log S ) log η)

Nt N0

N0 - Nt × 100% N0

(1)

(2)

where S is the survival fraction; η is the inactivation rate of bacteria; and N0 and Nt are the number concentrations of bacteria at times 0 and t, respectively [in colony forming units per milliliter (CFU/mL)].

Figure 3. Inactivation curves of saprophytic bacteria as a function of time at different initial temperatures (30, 38, 47, and 55 °C). Treatment conditions: reactor 1, σ ) 5 ms/cm, f ) 5 Hz, and initial cell concentration ) 1.5 × 103 CFU/mL.

3. Results and Discussion 3.1. Effect of Temperature on Inactivation of Saprophytic Bacteria. Saprophytic bacteria were used in this investigation to detect the effect of temperature on inactivation. The results shown in Figure 3 reveal that, as the temperature was increased, there was an increase in inactivation for all treatment times. It was observed that, after 24 min of treatment, a 1.8 log reduction was obtained at 30 °C, and a 2.2 log reduction was obtained at 55 °C. Although these temperatures are below the inactivation temperature of bacteria, the instant temperature increase might add an extra stress to the cells. Many studies have reported the positive effect of a moderate initial temperature on PEF treatment.17 Fox et al. drew a similar conclusion for a PEF microreactor.18 It seems that slightly higher temperature reduces the relaxation time of the cell membrane and thus favors destabilization and death of microorganisms.19 The critical potential of the membrane damage is on the order of um ≈ 0.2-1 V. It depends on the membrane system and is known to decrease with increasing temperature or membrane tension. The effect of temperature on damage time reflects possible structural transition inside the membrane for temperatures near 55 °C.20 Electroporation and PEF-induced damage can be very sensitive to the structural changes in membranes, and it is known that, for a single membrane, the breakdown voltage decreases significantly with increasing temperature.9 The enhanced electroporation of membranes in plant tissue at elevated temperatures has also been reported.21 3.2. Effect of Initial Concentration on Inactivation of Saprophytic Bacteria. In actual injection water, the initial concentration of saprophytic bacteria is comparatively high. Hence, the effect of initial concentration is an important factor in inactivating bacteria in oil-field reinjection water using PEF. From Figure 4, it can be seen that initial concentration seems to have a negative effect on the inactivation of saprophytic bacteria. After the same treatment time, the inactivation efficiency for an initial concentration of 1.7 × 103 CFU/mL is higher than those for initial concentrations of 3.6 × 106 and 1.5 × 107 CFU/mL. That is because high bacteria concentration favors the formation of clusters. Figure 5 shows SEM images of saprophytic bacteria, and the formation of clusters is clearly visible. The bacteria inside the clusters might be protected by external layers of cells and, therefore, do not experience the

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Figure 4. Effect of initial concentration on inactivation of saprophytic bacteria. Treatment conditions: reactor 1, f ) 5 Hz, σ ) 5 ms/cm.

Figure 5. SEM image of saprophytic bacteria. Table 1. Concentration of Saprophytic Bacteria before and after 24-min PEF Treatment at Different Conductivities (ms/cm) conductivity (ms/cm) 0.03 0.1 0.5 2 5 10

concentration (CFU/mL) before

after

inactivation rate (%)

1950 1700 1650 1300 1700 4400

243 28 13 10 26 740

87.5 98.4 99.2 99.2 98.5 83.2

same electric field strength as the external bacteria.22 The influence of the initial concentration of cells on the effectiveness of the PEF process is still a matter of concern. Only a few studies have included investigations of the effects of initial cell concentration on the survival population, and the results are contradictory. Zhang et al.23,24 and Damar et al.25 demonstrated that the initial microbial concentration is inversely correlated with the survival fraction, regardless of the treatment conditions utilized. However, the results disagree with the influence of cell concentration on the lethal efficiency of Salmonella senftenberg ´ lvarez and Raso, which indicated that the obtained by A inactivation of microorganisms was independent of the initial concentration.26 Further study is needed to explain the influence of the initial bacteria concentration. 3.3. Effect of Conductivity on Inactivation of Saprophytic Bacteria. Injection water is a high-salinity liquid, so it is necessary to detect the effect of conductivity on the PEF process.

Figure 6. Inactivation of (a) saprophytic bacteria, (b) iron bacteria, and (c) sulfate-reducing bacteria with the two reactors. Treatment conditions: σ ) 5 ms/cm, f ) 5 Hz. The initial cell concentrations of saprophytic bacteria, iron bacteria, and sulfate-reducing bacteria were 1.8 × 106, 4.5 × 105, and 4.5 × 105 CFU/mL, respectively.

Table 1 reports the inactivation rates of saprophytic bacteria after 24 min of treatment for various conductivities. As can be seen from Table 1, the inactivation rate is 87.5% (t ) 24 min) for the sample with 0.03 ms/cm conductivity (without KCl). The inactivation rate is above 98% as the solution conductivities range from 0.1 to 5 ms/cm. In contrast, when the conductivity increases to 10 ms/cm, the effect of treatment diminishes. According to the results, as conductivity increases above 10 ms/cm or decreases below 0.1 ms/cm, the effectiveness of the PEF treatment weakens. Conductivity values ranging from 0.1 to 5 ms/cm are favorable for the inactivation of saprophytic bacteria. There is no clear explanation as to why a conductivity

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Figure 7. TEM images of E. coli (a,c) before and (b,d) after PEF treatment. Treatment conditions: reactor 2, f ) 5 Hz, σ ) 5 ms/cm. The original magnifications of were (a) 40000×, (b) 40000×, (c) 150000×, and (d) 100000×.

of 0.03 ms/cm has a weak effect on bacteria inactivation. It might be related to the power supply used in our study, which needs a somewhat salty liquid to match. Some authors have suggested that the number of membrane-permeabilized cells increased as the conductivity of the solution decreased at a constant energy input.27 For a given energy input, chamber configuration, and treatment time, the solution conductivity determines the maximum achievable electric field,10 as well as the maximum temperature rise during processing. Low conductivity allows lower power input and will result in lower temperature rises, especially in low-resistance chambers. Low conductivity increases the difference in electrical conductivity between the medium and the microbial cytoplasm. This increased difference in conductivity weakens the membrane structure of the microorganisms as a result of an increased flow of ionic substances across the membrane during PEF treatment.28 The explanation from an electrical point of view is that the overall resistance depends on the sample conductivity, and the higher the conductivity, the lower the resistance. A lower resistance could lead to a negative reflection of the pulse shape and a lower efficiency of the treatment because the system is underloaded. On the other hand, if the resistance is higher, the decreasing phase of the pulse can be approximated as an exponential, leading to an apparently larger pulse width and thus an increased treatment efficiency.29 3.4. Effect of Electrode Distance on Bacteria Inactivation. The values of electric field strength determined by the pulse power supply and the reactor used are analyzed in this section. The electric field strength of a coaxial reactor is defined as12 E)

V r ln

R2 R1

(3)

where r is the radius at which electric field is measured; R1 and R2 are the radii of the inner and outer electrode surfaces, respectively; and V is the input voltage. The electric field in coaxial chambers is not uniform and depends on the location. The uniformity of the electric field is improved when (R2 R1) e R1. Reactors 1 and 2 were used in this investigation to study the effect of electrode distance on inactivation efficiency. The electric field intensity of reactor 2 is much higher than that of reactor 1. Figure 6 shows the inactivation efficiencies of (a) saprophytic bacteria, (b) iron bacteria, and (c) sulfate-reducing bacteria in the two reactors. The inactivation efficiency clearly increases as the field intensity increases and the treatment time prolongs. As seen in Figure 6a, after 20 min of treatment for saprophytic bacteria, a 0.5 log reduction was attained in reactor 1, and a 2.8 log reduction was achieved in reactor 2. Similar results were obtained for the inactivation of iron bacteria and sulfate-reducing bacteria. After 16 min of treatment, the inactivation efficiencies of iron bacteria and sulfate-reducing bacteria in reactor 1 exhibited a 1.3 log reduction and a 1.6 log reduction, whereas in reactor 2, the inactivation achieved a 3.6 log reduction and a 3.9 log reduction, respectively. However, under the conditions of reactor 1, the inactivation was very fast within the first few minutes of treatment, and then the number of survivor decreased slowly. For instance, the inactivation efficiencies of iron bacteria and sulfate-reducing bacteria achieved maximum values after 16 and 12 min of treatment, respectively, and increasing pulses did not benefit the inactivation. The same conclusions were drawn by Aronsson and Ro¨nner30 and Sampedro et al.31 This can be explained by the transmembrane potential theory. For cells of spherical shape, the membrane potential (Um) can be calculated by the equation32

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Um ) 0.75dcE cos θ (4) where dc is the cell diameter and θ is the angle between the external field (E) direction and the radius-vector on the membrane surface. When the induced transmembrane voltage exceeds a certain critical value, which depends on the nature of the cell, pores are formed. The value of Um is proportional to the cell diameter dc, so smaller cells need higher field intensities to undergo electroporation. That is, at constant field intensity, large cells are easily inactivated by PEF.33,34 With the reduction in the viability of large cells, the subsequent pulses are not as effective as the earlier ones. Figure 6 also indicates that the three bacteria exhibit different sensitivities to PEF treatment in the two reactors. After 20 min of treatment in reactor 2, the inactivation efficiency is a 2.8 log reduction for saprophytic bacteria, a 3.6 log reduction for iron bacteria, and a 3.9 log reduction for sulfate-reducing bacteria. A comparison of these results shows that saprophytic bacteria are more resistant to pulsed electric field treatment. Figure 6 also verifies that PEF is able to inactivate iron bacteria and sulfate-reducing bacteria. As Gram-negative bacteria, sulfatereducing bacteria are easier to kill through treatment. Because the mechanism of inactivation by PEF seems to be related to effects on the cytoplasmic cell membrane, the thicker and more rigid cell envelops of the Gram-positive bacteria have been identified as being responsible for their higher PEF resistance.35 This implies that the PEF technology can readily inactivate various microbes in oil-field-produced water. 3.5. TEM Examination of Inactivated Bacteria. TEM was used to more closely examine the bacteria both before and after treatment. Clues on the inactivation mechanism are vital for further understanding the impact of PEF on cells. To obtain clear TEM images, a pure strain of E. coli was chosen in this investigation to indicate the destructive action of PEF on cells. E. coli bacteria are Gram-negative, calalase-positive, oxidasenegative, anaerobic short rods. The cell walls of Gram-negative bacteria are thinner in structure and have distinct layers. There is an outer layer that is more like cytoplasmic membrane. The main component of the Gram-negative cell wall is lipopolysaccharide. A sample of E. coli was treated with PEF and then observed by TEM. At that time, most of microorganisms are already dead. Figure 7 shows electron microscope images obtained after treating, embedding, and cutting the microorganisms. Figure 7a,c shows two different sections of an original cell, whereas Figure 7b,d displays corresponding sections of a treated cell. Observation of the original cell allows a clear view of flagella and the body of the cell. However, observations of the treated cell reveal that enlarged lyses of the cell wall and the outflow of cytoplasm have occurred. The cell membranes or walls remain to a certain degree and are only partially disrupted. Clear leakage can be detected. Theoretically, as the voltage across the cell increases and a certain critical value is reached, pores are formed in the bilayer liquid membrane. As the electric filed increases further, these pores are formed irreversibly, and the contents of the cell leak out.36 This is also verified by the TEM images, which show that the main shape is kept after the treatment and cytoplasm flows out of the broken cell membrane. However, further investigation is needed to explain the exact mechanism of inactivation. 4. Conclusions As a novel inactivation technology, PEF was employed to inactivate bacteria in oil-field reinjection water. The experimental results show that the electric field strength, pulse

frequency, and process temperature have positive effects on the inactivation efficiency, whereas the initial bacteria concentration has a negative effect. At the same time, it was found that the electric field strength plays the most important role in inactivation. Conductivity values ranging from 0.1 to 5 ms/cm are favorable for the inactivation of saprophytic bacteria. It was verified that PEF treatment could greatly inactivate saprophytic bacteria, iron bacteria, and sulfate-reducing bacteria. From the micrographs of treated bacteria investigated by TEM, it was seen that PEF technology results in drastic damage of the cell surface. Thus, PEF is a promising technology for inactivating bacteria in oil-field reinjection water. Acknowledgment The authors acknowledge financial support for this work provided from the National Science Foundation of China (Nos. 20576120, 90610005, and U0633003), Project of Zhejiang Province (No. 2007C13061), and “863” project of China (No. 2007AA06Z339). Literature Cited (1) Bader, M. S. H. Sulfate scale problems in oil fields water injection operations. Desalination 2006, 201, 100. (2) Ma, H.; Wang, B. Electrochemical pilot-scale plant for oil field produced wastewater by M/C/Fe electrodes for injection. J. Hazard. Mater. B 2006, 132, 237. (3) Sariog˘lu, F.; Javaherdashti, R.; Akso¨z, N. Corrosion of a drilling pipe steel in an environment containing sulphate reducing bacteria. Int. J. Pressure Vessels Piping 1997, 73, 127. (4) Videla, H. A.; Herrera, L. K. Microbiologically influenced corrosion: looking to the future. Int. Microbiol. 2005, 8, 169. (5) Gardner, L. R.; Stewart, P. S. Action of glutaraldehyde and nitrite against sulfate-reducing bacterial biofilms. J. Ind. Microbiol. Biotechnol. 2002, 29, 354. (6) Chapman, J. S. Biocide resistance mechanisms. Int. Biodeterior. Biodegrad. 2003, 51, 133. (7) Morton, L. H. G.; Greenway, D. L. A.; Gaylarde, C. C.; Surman, S. B. Consideration of some implications of the resistance of biofilms to biocides. Int. Biodeterior. Biodegrad. 1998, 41, 247. (8) Tsong, T. Y. Electropration of cell membranes. Biophys. J. 1991, 60, 297. (9) Zimmermann, U. Electric breakdown, electropermeabilization and electrofusion. ReV. Physiol. Biochem. Pharmacol. 1986, 105, 176. (10) Go´ngora-Nieto, M. M.; Sepu´lveda, D. R.; Pedrow, P.; BarbosaCa´novas, G. V.; Swanson, B. G. Food processing by pulsed electric fields: Treatment delivery, inactivation level and regulatory aspects. LWTsFood Sci. Technol. 2002, 35, 375. (11) Woulters, P. C.; Dutreux, N.; Smelt, J. P. P. M.; Lelieveld, H. L. M. Effects of pulsed electric fields on inactivation kinetics of Listeria innocua. Appl. EnViron. Microb. 1999, 65, 5364. (12) Zhang, Q. H.; Barbosa-Ca´novas, G. V.; Swanson, B. G. Engineering aspect of pulsed electric field pasteurization. J. Food Eng. 1995, 25, 261. (13) Ohshima, T.; Sato, K.; Sato, M. Physical and chemical modifications of high-voltage pulse sterilization. J. Electrostat. 1997, 42, 159. (14) The determination of saprophytic bacteria in industrial circulating cooling water: Plate counting method. GB/T14643.1-93. (15) Oil and gas industry standards of People’s Republic of China. Analysis method for bacteria in oil filed injection water: Serial dilution method. SY/T 0532-93. (16) Bader, M. S. H. Seawater versus produced water in oil-fields water injection operations. Desalination 2007, 208, 159. (17) Guyot, G.; Ferret, E.; Boehm, J. B.; Gervais, P. Yeast cell inactivation related to local heating induced by low-intensity electric field with long-duration pulses. Int. J. Food Microbiol. 2007, 113, 180. (18) Fox, M. B.; Esveld, D. C.; Mastwijk, H.; Boom, R. M. Inactivation of L. plantarum in a PEF microreactor the effect of pulsed width and temperature on the inactivation. InnoVatiVe Food Sci. Emerging Technol. 2008, 9, 101. (19) Korolczuk, J.; Keag, J. R. M.; Fernandez, J. C.; Baron, F.; Grosset, N.; Jeantet, R. Effect of pulsed electric field processing parameters on Salmonella enteritidis inactivation. J. Food Eng. 2005, 75, 11.

9650 Ind. Eng. Chem. Res., Vol. 47, No. 23, 2008 (20) Mouritsen, O. G.; Jorgensen, K. Small-scale lipid-membrane structure: Simulation versus experiment. Curr. Opin. Struct. Biol. 1997, 7, 518. (21) Lebovka, N. I.; Praporscic, I.; Ghnimi, S.; Vorobiev, E. Temperature enhanced electroporation under the pulsed electric field treatment of food tissue. J. Food Eng. 2005, 69, 177. (22) Donsı`, G.; Ferrari, G.; Pataro, G. Inactivation kinetics of Saccharomyces cereVisiae by pulsed electric field in a batch treatment chamber: The effect of electric field unevenness and initial cell concentration. J. Food Eng. 2007, 78, 784. (23) Zhang, Q. H.; Monsalve-Gonzalez, A.; Barbosa-Ca´novas, G. V.; Swanson, B. G. Inactivation of S. cereVisiae in apple juice by square-wave and exponential-decay pulsed electric fields. J. Food Process Eng. 1994, 17, 469. (24) Zhang, Q. H.; Monsalve-Gonzalez, A.; Barbosa-Ca´novas, G. V.; Swanson, B. G. Inactivation of E. coli and S. cereVisiae by pulsed electric fields under controlled temperature conditions. Trans. ASAE 1994, 37, 581. (25) Damar, S.; Bozoglu, F.; Hizal, M.; Bayindirih, A. Inactivation and injury of Escherichia coli O157:H7 and Staphylococcus aureus by pulsed electric field. World J. Microb. Biotechnol. 2002, 18, 1. ´ lvarez, I.; Raso, J.; Palop, A.; Sala, F. J. Influence of different (26) A factors on the inactivation of Salmonella senftenberg by pulsed electric fields. Int. J. Food Microbiol. 2000, 55, 143. (27) Wouters, P. C.; Bos, P.; Ueckert, J. Membrane permeabilization in relation to inactivation kinetics of Lactobacillus species due to pulsed electric fields. Appl. EnViron. Microb. 2001, 67, 3092. (28) Pothakamury, U. R.; Vega, H.; Zhang, Q.; Barbosa-Ca´novas, G. V.; Swanson, B. G. Effect of growth stage and processing temperature on the inactivation of E. coli by pulsed electric fields. J. Food Prot. 1996, 11, 1167.

(29) Zhao, W.; Yang, R. The effect of pulsed electric fields on the inactivation and structure of lysozyme. Food Chem. 2008, 110, 334. (30) Aronsson, K.; Ro¨nner, U. Influence of pH, water activity and temperature on the inactivation of Escherichia coli and Saccharomyces cereVisiae by pulsed electric fields. InnoVatiVe Food Sci. Emerging Technol. 2001, 2, 105. (31) Sampedro, F.; Rivas, A.; Rodrigo, D.; Martı´nez, A.; Rodrigo, M. Pulsed electric fields inactivation of Lactobacillus plantarum in an orange juice-milk based beverage: Effect of process parameters. J. Food Eng. 2007, 80, 931. (32) Zakhem, H. E.; Lanoiselle´, J. L.; Lebovka, N. I.; Nonus, M.; Vorobiev, E. The early stages of Saccharomyces cereVisiae yeast suspensions damage in moderate pulsed electric fields. Colloids Surf., B 2006, 47, 189. ´ lvarea, I.; Angersbach, A.; Knorr, D. Preservation of (33) Heinz, V.; A liquids foods by food high intensity pulsed electric fields, basic concepts for process design. Trends Food Sci. Technol. 2001, 12, 103. (34) Qin, B. L.; Barbosa-Ca´novas, G. V.; Swanson, B. G.; Pedrow, P. D.; Olsen, R. G. Inactivating microorganisms using a Pulsed electric field continuous treatment system. IEEE Trans. Ind. Appl. 1998, 34, 43. (35) Garcı´a, D.; Go´mez, N.; Raso, J.; Paga´n, R. Bacterial resistance after pulsed electric fields depending on the treatment medium pH. InnoVatiVe Food Sci. Emerging Technol. 2005, 6, 388. (36) Angersbach, A.; Heinz, V.; Knorr, D. Effect of pulsed electric fields on cell membranes in real food systems. InnoVatiVe Food Sci. Emerging Technol. 2000, 1, 135.

ReceiVed for reView January 13, 2008 ReVised manuscript receiVed September 22, 2008 Accepted September 22, 2008 IE8000524