Keywords

6.1 Overview

Pulsed electric field (PEF) treatment is a physical method which exhibits specific advantages over conventional processing in various applications and was proven for feasibility on pilot and industrial scale for large mass-flow applications in energy technology, environmental technology, and biotechnology.

For bacterial inactivation of wastewater, PEF treatment was shown to exhibit high inactivation efficiency without adding harmful chemicals and proven not to cause any adverse effects to the aquatic environment. Studies on sustainability revealed that the inactivation rate is not impaired by repetitive treatments of bacterial populations already multiply exposed to PEFs. Moreover, by combining mild thermal pretreatment and PEFs, the electrical energy demand for bacterial inactivation is competitively low.

The technique was also demonstrated to be highly efficient for eradication of unwanted Cyanobacteria in surface waters. For this application, it was shown at demo-scale that an autarkic treatment facility can efficiently control Cyanobacteria populations in lakes.

Another specific advantage of PEF treatment, the comparably low heat transfer to the processed medium, is exerted in liquid food preservation, maintaining flavor and nutritional value of foods.

For component extraction, the specific advantages of PEF treatment are low heat influx, low-energy demand, and selectivity of compound release. Existing PEF processing facilities in winemaking, extraction of sugar from sugar beets, and valuable component extraction from fruits and vegetables nowadays operate on a mass-flow scale of 1–10 tons/h and larger.

Recent activities in PEF-downstream processing of microalgae have proven a high-energy efficiency of PEF treatment in comparison to conventional techniques and furthermore revealed the fractionating ability of PEF processing, enabling separation and simultaneous recovery of lipids and water-soluble compounds at low-energy demand.

These promising applications of PEF processing are introduced in more detail in the following sections.

6.2 Disinfection of Hospital Wastewater by PEF Treatment

  • Christian Gusbeth &
  • Wolfgang Frey 

6.2.1 Introduction

The latest report by the World Health Organization 2014 reveals that the dissemination of antibiotic-resistant bacteria is a global issue and that “this serious threat is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country.”

Hospital wastewaters are known to be one important source of the dissemination of infectious agents, especially antibiotic multiresistant pathogens, into the downstream aquatic systems [1–5, 44]. In contrast to chemical contaminants that were metabolized by a number of biological and physic-chemical reactions, harmful microorganisms are able to proliferate in an appropriate milieu to high densities. Moreover, when they reach the aquatic environment system, these resistant bacteria contribute to the variety of resistance genes and enable the genetic exchange between many different bacterial genera in nature [6, 7]. Beyond that, resistance determinants most probably were acquired also by pathogenic bacteria from this pool of resistance genes in other microbial genera, including antibiotic-producing organisms.

Therefore, it is essential to retard the development of antibiotic resistance and to prolong the effective lifetime of valuable antimicrobial agents until bacteria adapted to them. This can be done, on the one hand, by enhancing the overall medical care and hygiene and, on the other hand, by preventing the dissemination of resistant bacteria. Disinfection is considered to be the primary mechanism for the inactivation/destruction of pathogenic and resistant organisms to prevent the spread of waterborne diseases to downstream users and the environment [8]. Whenever antibiotic-resistant bacteria will be released in the environmental aquatic system, the chance for gene transfer and implicit antibiotic resistance transfer will be increased. During an outbreak of infections in a hospital, the hospital wastewater effluent becomes a major source for pathogens and opportunistic bacteria. As a consequence, a direct contact of humans with pathogenic agents cannot be excluded, and the risk of contamination of water resources increases with the diversity of harmful bacteria [43]. Once the pathogens have reached surface water, the bacterial dissemination is irreversible. The dissemination of antibiotic-resistant bacteria can be stopped by a variety of disinfection methods like ultraviolet (UV) radiation, thermal treatment (pasteurization), and chlorination. It is known that the disinfection techniques applied have demonstrated disadvantages like generation of toxic disinfection by-products (DBP) during chemical disinfection or reduced efficiency in liquids with high turbidity in case of UV radiation. An alternative disinfection technique for clinical wastewater is the pulsed electric field treatment (PEF) that is based on the process of electroporation in cells [9–12]. Previous work on applications of PEF demonstrated the feasibility of PEF treatment to improve the shelf life of juice, milk, soup, and other liquid foods [13–15]. Owing to the nonchemical and nonthermal character of PEF treatment, no toxic DBP can be expected. The chemical stability of flavors and vitamins of PEF-treated food was demonstrated for different kinds of fruit juices [16]. It was also shown that a large variety of undesirable microorganisms, among them Gram-negative, Gram-positive bacteria and fungi, were effectively reduced by PEF treatment [17].

6.2.2 Conventional Disinfection Methods Versus PEF Treatment

The majority of the conventional water disinfection methods are known from water purification plants. These methods have been intensively used and continuously developed for decades. Therefore, most of them are economic and effective in bacterial reduction. Owing to the different consistency of freshwater and hospital wastewater, which has an increased organic and particle load, some or most of these methods cannot be used for hospital wastewater effluents.

Water chlorination is the most widely used disinfection method for municipal water purification plants and swimming pools. It consists in adding chlorine as gas (Cl2), sodium hypochlorite solution, or other chlorine compounds to water. As a strong oxidizing agent, chlorine is highly efficient in killing pathogenic agents causing many diseases such as cholera, typhoid fever, or dysentery [18]. It was shown that the required concentrations of disinfectants were higher in the case of heavily contaminated wastewater than in uncontaminated, if similar disinfection levels have to be achieved [19]. It has, however, some drawbacks including minimum contact time and the need to handle, store, and dispense hazardous chemicals accurately in case of fast-changing flow rates; chlorine residuals can prolong and affect the environmental surface water. One significant drawback of chlorination is that chlorine reacts with organic compounds in water, causing unwanted DBP such as halogenated organics [20], which are harmful and/or carcinogenic (trihalomethanes and haloacetic acids). This excludes its use for hospital wastewater disinfection.

Ozone disinfection is an effective method to inactivate harmful microorganisms in drinking water. Ozone (O3) is an unstable inorganic molecule, decomposing in di-oxygen and one atom of oxygen providing a powerful oxidizing agent. Similar to chlorination, it consists in adding ozone directly to water. Due to the instability of ozone, it must be created on site to ensure a high level of bacterial inactivation. Owing to the short half-life of ozone and the limited production of harmful DBP in comparison to chlorination, ozone disinfection is considered a feasible disinfection technology [21, 22]. It was found that the half-life of O3 in water ranges from 14 h (without presence of phosphate and carbonate) to 8 min and strongly depends on water impurities [23, 24]. In a medium such as wastewater, O3 reacts with the complex organic compounds (e.g., saturated hydrocarbons, amines, and aromatic compounds), destroys them, and forms by-products such as acids, aldehydes, bromates, ketones, and peroxides [25–27]. In addition, it was shown [28] that the use of ozone in marine-based aquaculture systems has been limited because of the risk to form the carcinogenic bromate, which is formed during the oxidation of naturally occurring bromide by ozone. However, the presence of water impurities was found to be a major limiting factor of ozonation for wastewater applications [29].

UV disinfection is nowadays a widely used method for water disinfection and became more and more a common method in municipal drinking water treatment in the last decades. It is based on irradiation of water with UV light at a wavelength of 254 nm (generated by low- and medium-pressure mercury arc lamps), which is near the absorption peak of nucleotide bases of RNA and DNA. The UV radiation damages the bacterial DNA of microorganisms and retards their ability to reproduce. In continuous operation, an effective bacterial reduction of Gram-negative and Gram-positive bacteria such as enteric bacteria and Enterococci could be demonstrated [30]. However, some authors found a recovery of the bacterial population 24–48 h after UV treatment [31]. In addition, RNA repair mechanisms that were demonstrated in E. coli might lead to a long-term UV adaptation of bacteria. In case of hospital wastewater, the high turbidity of the wastewater impedes application of this disinfection method.

Pulsed electric field treatment is a nonthermal physical method for liquid food preservation and water disinfection. In recent years, there has been increasing interest for a potential use of PEF treatment for inactivation of pathogenic microorganisms carried by hospital wastewater effluents. Although this method was intensively investigated for food preservation over decades, there is less experience for wastewater disinfection. The benefit of PEF processing of food lies in the extended shelf life without the loss of flavor and nutrition value (fresh-like liquid food). PEF can be used for processing liquid and semiliquid (mash-like) food products with low electrical conductivity (<4 mS∙cm−1), but also for drinking water and wastewater. The fluid is disinfected on-site in the PEF-treatment zone, and there is no prolonged effect such as in case of chlorination. Rather, no systemic DBP generation is expected after PEF disinfection of hospital wastewater. Commercial scale continuous PEF systems for processing volumes ranging from 100 to 5000 l h−1 are available [32–34]. This equipment is scalable when solid-state pulse power generator modules are used, which can be stacked in series and parallel to provide the required/desired generator power. According to the state of the art, PEF processing would add only $0.10 l−1 to the final treatment costs [35]. For wastewater purification plants with a large throughput of 1 million liters of water per day, the operating and maintenance costs increase with the throughput flow, negatively affecting the economic efficiency of this method. On the other hand, due to the high efficiency in bacterial inactivation and the small equipment size, PEF processing is a safe method for local hotspot disinfection of hospital wastewater effluents (Table 6.1), especially for combating temporary outbreaks of infections.

Table 6.1 Advantages and disadvantages of different technologies for wastewater disinfection. The total equipment costs and operating and maintenance costs were calculated on the basis of a treatment volume flow of 4000 m3∙h−1 and a disinfection efficiency of at least 99.9 % (3 log). Cost estimates for PEF-treatment facilities are only available on pilot scale and are based on real prototype costs. Please note that alternative technology costs are adopted from industrial municipal water processing technology which may not be transferable to or applicable for hospital wastewater treatment
Full size table

6.2.3 Reduction Efficiencies of Bacteria During PEF Treatment of Clinical Wastewater

Since PEF treatment is a novel method in the context of disinfection of hospital wastewater, investigation of the efficiency of the PEF processing depending on the specific bacterial population in wastewater is necessary. Considering the fact that there is no “standard” wastewater, this can be done by processing wastewater, sampled from different municipal wastewater purification plant and hospital effluents. By regular sampling during the year, the variety of bacterial population and the influence of weather and season are covered. One problem is that the cell density and cell population strongly vary, complicating comparability and conclusiveness of experimental results.

Bacterial Load of Hospital Wastewater Enterococci/Streptococci and Enterobacteriaceae

The bacterial load of hospital wastewater Enterococci/Streptococci and Enterobacteriaceae can be found in all biofilms from hospital wastewater effluents. Enterococci and Enterobacteriaceae are naturally occurring microorganisms from human and animal intestines. The Enterococci/Streptococci loads and the presence of resistant bacteria are higher in hospital biofilms than in effluxes of municipal sewage. The largest number of cefazolin- and cefotaxime-resistant Enterobacteriaceae is measured in biofilms from hospital wastewater, 54 % and 17 %, respectively. Typical bacterial densities of E. faecium and P. aeruginosa being below 105 cells∙ml−1 were found by Volkman et al. [5, 43] and Schwartz et al. [4] in wastewater, sampled at the outlet of the hospital wastewater system, close to the public sewer system.

Hospital Wastewater Parameters

The conductivity of wastewater has a great influence on the configuration of a PEF system that has to be employed. Wastewater conductivity varies in the range between 0.5 and 2.0 mS∙cm−1, depending on time and site of sampling. In the case of hospital wastewater from a sewer of the university hospital of the city of Mainz, the measured conductivity was between 1.0 and 1.7 mS∙cm−1 at a temperature ranging from 7 to 14 °C [1]. For lab experiments, depending on the original wastewater parameters and the employed pulse generator, an adjustment of the conductivity of the model wastewater by dilution or addition of buffer is possible. Usually the wastewater pH is in the range of six to eight, whereas the turbidity at 600 nm is 0.7.

Performance of PEF Processing

For the evaluation of the PEF performance, the parameters used for food processing serve as a reference for performing inactivation studies on hospital wastewater. The main parameters under investigation are pulsed electric field amplitude E (5–100 kV∙cm−1), pulse duration t (0.2–100 μs), and specific energy, which can be adjusted by the number of pulses N. The pulse shape (rectangular, exponentially decaying, unipolar, or bipolar) and the frequency play a minor role in improving the inactivation performance. Bipolar pulse protocols are proven to be advantageous for pulsed electric field processing in general since it decreases electrochemical erosion of treatment electrodes [36, 37]. Based on these studies, the following standard treatment conditions have been defined: specific treatment energy 120 J∙ml−1, electric field amplitude 80 kV∙cm−1, and pulse duration 1–2 μs.

In general, the density of bacteria in hospital wastewater is not high enough to demonstrate the PEF efficiency at inactivation rates of several orders of magnitude. Therefore, for the evaluation of the PEF technique, with respect to its efficiency in reducing Enterococci and Pseudomonads as target organisms, an increased cell density is considered necessary. For this purpose, model wastewater spiked with specific bacteria strains with certain cell densities can be used. Prior to spiking with target organisms, hospital wastewater must be disinfected at 121 °C for 15 min in order to eliminate the original population that could interfere with the subsequent analysis. P. aeruginosa as a representative of Gram-negative bacteria, well-known as an opportunistic human pathogen with high incidences in hospitals, and the enteric Gram-positive bacterium E. faecium, are identified as target organisms. A main observation is that cell inactivation is not directly proportional to a specific energy input. The efficiency of PEF processing, investigated on P. putida—a widespread wastewater microorganism and nonpathogenic and safe pseudomonas species—was similar to the results obtained from food processing (Fig. 6.1). Also in this case, the inactivation kinetics deviates from the first-order kinetics, and it seems that the bacterial inactivation saturates at around 6 log reduction when pulse numbers range between 120 and 200 pulses. Furthermore, within a pulse duration range of 100–10 μs and at invariable suspension conductivity, inactivation does not depend on pulse duration in case the external field amplitude is high enough to provide fast membrane charging at the entire cell surface [38]. The dissipated energy and bacterial reduction follow a dose–response relationship, and the inactivation rate scales with the specific energy (~E∙t2) [38]. It has to be pointed out that in this case the temperature rise was below 7 K in particular due to the fact that the heat could dissipate across the aluminum electrodes into the cooling medium surrounding the treatment chamber. Therefore, the observed bacterial inactivation reflects the impact due to the electric field and is not caused by electrically induced thermal heat. For orientation, the PEF treatment with a specific electrical energy input of 40 J∙ml−1 causes heating with a temperature increase of 10 K, if adiabatic conditions are considered. For scalable systems, the temperature inside the treatment chamber will equilibrate according to treatment volume, pulse frequency, ambient temperature, and electrical energy.

Fig. 6.1
figure 1

Inactivation rate of P. putida after PEF treatment with rectangular pulses of 100 kV∙cm−1 electric field strength and pulse duration of 200 ns. The conductivity of the medium was 2.0 mS∙cm−1, at 21 °C. The temperature increase due to electrical energy dissipation was below 7 K. With increasing number of pulses/treatment energy, the bacterial inactivation approaches a climax (6 log), which is below complete inactivation (8 log; correspond to 108 colony forming units (CFU) ∙ml−1 initial cell density) [1]

Full size image

In general, Gram-negative bacteria (such as Escherichia coli or P. aeruginosa) are much more sensitive to electric field treatment than Gram-positive bacteria (such as Staphylococcus aureus or Enterococcus faecium) [17, 39, 40]. This was demonstrated also for spiked hospital wastewater, which was treated by PEF at specific energies ranging from 80 to 190 J∙ml−1 [2]. The PEF treatment was performed with a PEF system consisting of a 65-kV, 1-kJ, 10-Hz, two-stage pulse-forming generator. The pulse-forming network of the generator delivers a 1.2-μs-long rectangular output pulse. The field strength amplitude in the treatment chamber was 80 kV∙cm−1. A reduction efficiency of up to 5.5 decimal orders of magnitude was reached for the Gram-negative reference strain, P. aeruginosa, when the treatment energies exceeded 162 J∙ml−1 (Table 6.2). In contrast to P. aeruginosa, the reduction efficiency of the Gram-positive vancomycin-resistant E. faecium (strain 2) was lower (Table 6.3). Moreover, inactivation experiments on different E. faecium strains showed strongly varying inactivation efficiencies. In different independent experiments, different reduction patterns were observed. These differences are attributed, on the one hand, to a varying robustness of different species against PEF treatment and, on the other hand, to the time independent experiments and the resulting different bacterial populations in the wastewater matrices. Inactivation experiments on wastewater containing mixed bacterial populations reveal that the most robust bacteria strain dominates the resulting inactivation rate of the wastewater sample. At standard treatment conditions, W = 120 J∙ml−1, the inactivation rate varied between 1.5 and 3.4 log.

Table 6.2 Reduction efficiency of the PEF treatment of hospital wastewater spiked with Pseudomonas aeruginosa depending on specific treatment energy [2]
Full size table
Table 6.3 Reduction efficiency of the PEF treatment of hospital wastewater spiked with different E. faecium strains. Strains labeled with 2, 1265 and B7641 were isolated from hospital patients, whereas DSM strains originated from German Collection of Microorganisms
Full size table

6.2.4 Sustainability of PEF Disinfection Method

In order to establish the PEF processing for disinfection of hospital wastewater, the hazards of this method with respect to environment and humans have to be analyzed. Consequently, two important issues have to be investigated: (i) the adaptation of bacteria to electric field treatment (electro-tolerance) and (ii) the mutagenicity of PEF-treated clinical wastewater.

In the first case, an adaptation of bacteria to PEF treatment would decrease the efficiencies of this disinfection method, with possibly hazardous consequences to environment and humans. Induced electro-tolerance in reference bacteria can be proved by consecutive treatment of already treated bacteria suspensions over several cycles. A selective enrichment of electro-tolerant bacteria would finally result in increased survival rates. For these investigations, surviving bacteria from the previous treatment were cultivated in growth medium for 3 days and treated again by PEFs. In case of P. putida, no significant deviation of the survival rate from the average could be observed over 30 treatment cycles (Fig. 6.2), indicating that bacteria do not develop an adaptation against PEF treatment [1].

Fig. 6.2
figure 2

Inactivation rate of samples of P. putida after consecutive PEF treatment. For each treatment cycle, surviving bacteria from the previous treatment were cultivated in growth medium for 3 days and treated again by PEF. An average inactivation of 3.6 log for a specific treatment energy of 120 J∙ml−1 was obtained [1]

Full size image

Furthermore, the comparison of the band patterns of the variable intergenic spacer region (ISR) of ribosomal operon over 30 cycles of treatments reveals the same pattern for all samples. This result underlines that no alterations become visible within the ISR, which means that no phenotypic changes of the bacterial population were induced by repetitive PEF treatments. Thus, an enrichment of bacterial population with electro-tolerant bacteria after PEF treatment as well as the transmission of electro-tolerance from bacteria to bacterial descendants can be excluded.

Another impediment of this method could be the generation of genotoxic by-products due to the electrolytic reaction of the wastewater compounds. The formation of such genotoxic compounds during PEF treatment might provoke mutations in the bacterial and human genomes, having unforeseeable consequences for aquatic environment and human population health. Usually, to assess the genotoxicity in water, the umu-test is employed as a standard method. The test is based on the ability of genetically modified bacteria strain (Salmonella typhimurium) to induce SOS response and to express the umuC gene induced by RNA lesions. Studies performed with SOS chromotest and Salmonella fluctuation test show that the different wastewater samples exhibit a genotoxic risk, which is not unusual for hospital wastewater [41]. This was also the case for wastewater from a sewer of the university hospital of the city of Mainz; all samples were positive when umu-test was performed [42]. However, PEF treatments with specific treatment energies of more than 250 J∙ml−1 did not change the hospital wastewater genotoxicity. Moreover, in domestic water and phosphate buffer (PBS), no genotoxicity could be found before and after PEF treatment. Even PEF treatments with more than 480 J∙ml−1 generated no genotoxic by-products in PBS.

6.2.5 Economic Feasibility of Combining PEF with Thermal Treatment

This section is dedicated to the economic feasibility of the PEF technology for wastewater disinfection. In order to achieve satisfactory bacterial inactivation (>4 log), an electric treatment energy between 120 and 240 J∙ml−1 is necessary. This energy is enough to increase the fluid temperature by about 30–60 K, if adiabatic conditions are applied. Therefore, also the thermal treatment (pasteurization) might be considered as an alternative treatment.

Contrary to thermal disinfection or sterilization, PEF treatment reveals the important feature that the activity of nucleases is not affected by pulsed electric fields. Nucleases naturally digest free bacterial RNA fragments in water systems, thus preventing a spread of possibly antibiotic-resistant plasmids and transposons by uptake into other bacteria. It could be demonstrated that nuclease activity is not affected by PEF treatment [2, 42], whereas a thermal treatment to 72 °C already reduced DNA degradation by nucleases significantly. However, the degradation temperature of plasmids and DNA fragments is considerably higher (T > 100 °C).

It was shown that a combined treatment with PEF and heating increases bacterial inactivation of hospital wastewater significantly [42]. For comparison, the inactivation rate of E. faecium by PEF treatment with a specific energy input of 120 J∙ml−1 at 25 °C is 2 log, whereas at a treatment temperature of 60 °C, a complete inactivation of the bacteria (>8 log) could be achieved at the same electrical energy expenditure. At treatment temperatures below 55 °C, the combined treatment does not improve the inactivation rate.

The effectiveness of the combined thermal and electric wastewater treatment was demonstrated on a prototype plant. Using heat exchangers for preheating and heat recovery, a disinfection of 4–5 log could be achieved with PEF-treatment energy of 40 J∙ml−1. Under this premise, the PEF processing costs of $0.10 l−1 can be drastically reduced, resulting in a competitive price compared to conventional disinfection methods.

Considering all these results, it can be concluded that PEF treatment has no adverse effect on environment and humans, underlined by the facts that PEF processing does not increase the genotoxicity of hospital wastewater and also doesn’t cause phenotypic changes, in terms of electro-tolerance induced by repetitive treatments. Further, PEF treatment became very effective in bacteria inactivation in combination with thermal treatment. Thus, PEF treatment is a sustainable and suitable disinfection method, which effectively reduces bacteria from hospital wastewater effluents without any negative impact on aquatic environment.

6.3 Algae (Cyanobacteria) Treatment in Dams and Lakes

  • Takashi Sakugawa 

6.3.1 Introduction

Blue-green algal bloom is a rapid increase in the population of cyanobacterial cells in aquatic systems. In recent years, frequent occurrences of large-scale water blooms due to eutrophication of water threaten human health all over the world. Environmental problems caused by water blooms include clogging intake pipes and filter lines of water treatment facilities, loss of landscape beauty, and smell of drinking water. Moreover, when a bloom dies in a pond or shallow lake, severe oxygen depletion can produce objectionable odors and damage of fisheries.

The most serious problem is that some bloom-forming cyanobacterial genera produce toxic substances, which can cause a range of human health effects such as skin irritation, liver damage, stomach and intestinal illness, and so on [34]. Field investigations have reported that microcystins in drinking water are one major factor that resulted in locally high incidence of liver cancer. The current methods of water blooms treatment such as chemical compounds, ultrasonic, and microwave have been used to eliminate water blooms or to slow down cyanobacterial cell proliferation [46]. However, chemical compounds and ultrasonic can cause the cell membrane to be destroyed and the contents of the cells released into ambient water. And energy consumptions of ultrasonic treatment system and microwave options are too great to be used in large-scale treatments. Therefore, the development of an efficient method for large-scale water-bloom treatment becomes an urgent task of environmental protection.

It is well known that pulsed streamer discharges generate several physical phenomena and chemical phenomena and chemical reactions simultaneously. The use of pulsed streamer discharges in the gas to realize bacterial decontamination has been a firmly established method [47]. Accompanied with the development of applications of this kind of discharge in liquid, it has been used to realize water sterilization. A new method using underwater pulsed streamer-like discharges to prevent water blooms from large area generation is developed.

6.3.2 Effects of Underwater Streamer-Like Discharge

In recent years, pulsed power modulator for underwater streamer discharge has been developed. Figure 6.3 shows typical circuit of this modulator. A type of this modulator is magnetic pulse compressor (MPC) . First-stage semiconductor switching devices are used such as thyristor and insulated-gate bipolar transistor (IGBT). Usually, the ferromagnetic core of the toroidal shape is used for the magnetic switch SI1, SI2, and SI3. Input voltage and energy from the charger to C0 are 1.2 kV and 5 (J/p). A maximum output voltage of the MPC is 30 kV with pulse width 4 μs. Maximum pulse repetition rate is 180 pulses per second (pps) [48]. Figure 6.4 shows typical waveforms of the discharge voltage and the current. The voltage risetime is about 800 ns. After 800 ns, the voltage is decreased and increasing again. In this time, the current flows. After 6 μs, it can be recognized that reverse phases of the voltage and the current are almost the same from Fig. 6.4 [48].

Fig. 6.3
figure 3

Schematic diagram of pulsed power modulator for underwater streamer-like discharge

Full size image
Fig. 6.4
figure 4

Voltage and current waveforms of the underwater streamer-like discharge

Full size image

Photograph of underwater streamer-like discharge and effects of streamer discharge are shown in Fig. 6.5 [48]. It can be confirmed that streamer-like discharge branches have progressed radially from tip of the electrode. Distance from tip to tip of the streamer-like discharge branches is about 30 mm. Underwater streamer-like discharge is effective in the generation of radicals, high-electric fields, ultraviolet rays, microbubbles, and shock waves.

Fig. 6.5
figure 5

Underwater streamer-like discharge

Full size image

6.3.3 Method of Algae Treatment

Cyanobacteria , also called “blue-green algae,” are relatively simple, primitive life forms closely related to bacteria. A diameter of the cyanobacterial cell is several micrometers. There are some honeycomb shape and hollow cylindrical structures inside the cytoplasm of cyanobacterial cells named as gas vesicles. Usually, cyanobacterial cells live in the surface of the water body. We have investigated that streamer-like discharge affects the cyanobacterial cells [48]. Figure 6.6 shows the effects of the streamer-like discharges for cyanobacterial cells. We have results of three kinds of experiments. Control is only stirred for 1 min. The second treatment is applied with 60 pulses and stirred for 1 min. The third treatment is applied with 600 pulses and stirred for 1 min. Volume of each water vessel is 1 l. A half day after treatment, the cells of the control rise to the surface of the water vessel. Some of the cells sink to the bottom of the water vessel by the second treatment. And almost all the cells sink to the bottom of the water vessel by the third treatment. We consider that the effect on gas vesicles of cell is assumed to be fast rising shock wave. We had results of laboratory experiments that UV and enhanced electric field do not affect the sedimentation of cyanobacterial cells [49]. Figure 6.7 shows TEM images of cyanobacterial cells. In Fig. 6.7b, gas vesicles are collapsed by applying streamer-like discharge [48].

Fig. 6.6
figure 6

Effects of the streamer-like discharges for cyanobacterial cells

Full size image
Fig. 6.7
figure 7

TEM images of cyanobacterial cells

Full size image

6.3.4 Algae Treatment Apparatus

Figure 6.8 shows a large-sized algae treatment apparatus constructed in 2006. This system becomes a product. The MPC is used as a pulsed power modulator [50–52]. Usually, pulsed power modulator for treatment of blue-green algae was using a voltage higher than 100 kV in order to increase volume of the streamer [51, 53, 54].

Fig. 6.8
figure 8

A large-sized algae treatment system in 2006

Full size image

On the other hand, in recent study, we have developed a water intake mechanism that can treat the blue-green algae by the relatively small streamer-like discharge [48]. We were able to develop new practical blue-green algae treatment apparatus (named MIZUMORI-A2) using a low-voltage pulsed power modulator. The block diagram of the system is shown in Fig. 6.9. The MIZUMORI-A2 has photovoltaic cells as an independent power source. Maximum power generated by the photovoltaic cells is 518 W. Pulsed power system consists of the charger, IGBT switches, and MPC circuit. Pulse repetition rate of the modulator is 10 pps. Figure 6.10 shows the specifications and appearance of the MIZUMORI-A2. Photovoltaic cells are attached to the roof portion of the MIZUMORI-A2. There is a box of electrical components, including the MPC under the photovoltaic cells. In the center, streamer-like discharge generated place and algae intake of the MIZUMORI-A2 [48]. An anode of discharge electrode uses a tungsten diameter of 1 mm. It’s covered with an insulating material with a diameter of 10 mm around the anode. A cathode of discharge electrode is made of stainless steel. In cylindrical shape, inner diameter is 104 mm. As the inner diameter of section of streamer-like discharge generation is 104 mm, water flow velocity of algae intake is 86 cm/s. Treatment flow rate is 26 m3/h.

Fig. 6.9
figure 9

Block diagram of algae treatment apparatus in 2012

Full size image
Fig. 6.10
figure 10

Photograph of algae treatment apparatus in 2012

Full size image

We have demonstrated blue-green algae treatment by the MIZUMORI-A2 at the dam in Kyushu district, Japan [48]. Figure 6.11 shows the treating area by the MIZUMORI-A2. The treating area has a square of each side of 6 m. The area has been separated from the outside by a curtain fence of 1 m in depth. The MIZUMORI-A2 was carried out treating blue-green algae during 1 h at the treatment area. Pulse repetition rate of the modulator used in this experiment is 10 pps. We can recognize that blue-green alga has been lost from the surface of the water inside of the fence. Figure 6.12 shows experimental results of the treatment. In Fig. 6.12a, we found that chlorophyll concentration and turbidity decrease after the start of treatment by the MIZUMORI-A2. These results mean that Cyanobacteria are precipitated under the influence of underwater streamer-like discharge. On the other hand, Fig. 6.12b shows potential of hydrogen (pH), dissolved oxygen (DO), conductivity (COND), and water temperature (TEMP). DO and COND are decreased gradually. We consider that reduction of DO indicates inactivated cyanobacterial cells or blue-green alga disappears from surface of water, because DO is increased by photosynthesis of the plant cell. Efficacy of Cyanobacteria treatment was confirmed by demonstrated experiments of the MIZUMORI-A2 at the dam.

Fig. 6.11
figure 11

Cyanobacterial bloom treatment at dam

Full size image
Fig. 6.12
figure 12

The quality change of water by the cyanobacterial treatment apparatus (MIZUMORI-A2)

Full size image

6.4 Large-Scale PEF Processing of Sugar Beets and Grape Mash

  • Martin Sack,
  • Juergen Sigler &
  • Georg Mueller 

6.4.1 PEF Treatment of Sugar Beets

Pulsed electric field-assisted extraction of valuable substances from plant cells enables an energy-efficient processing compared to a conventional thermal process, fast processing, and a more complete extraction of ingredients.

For the production of sugar from sugar beets, saving energy is the main goal for the use of electroporation-assisted extraction. Other than for the production of sugar from sugar cane, the sugar production from sugar beets requires a considerable amount of energy covered by fossil fuels coal, gas, and oil. In the sugar beets, the sugar is stored inside vacuoles in the cells of the parenchyma tissue. The vacuole fills nearly the whole space of the cell, which has in average a diameter of 50 μm. Conventionally, the cells of the sugar beet tissue are opened thermally at a temperature of approximately 70–78 °C in order to prepare the tissue for the following extraction process. The thermal denaturation can be replaced by PEF treatment either of whole sugar beets or of cossettes [55–57]. For the extraction process, the cell membranes and the membranes of the vacuoles need to be opened. Figure 6.13 shows raw and permeabilized tissue in comparison. After PEF treatment, a part of the juice drains out of the cells due to the pressure inside the cells. The treatment of whole sugar beets has the advantage that the energy consumption for the subsequent slicing process and the wear of the blades inside the slicing machines are reduced. According to texture tests, cutting PEF-treated beets requires with 8 N approximately half of the mechanical force required for cutting fresh beets. A package of PEF-treated cossettes exhibits better perfusion properties. As single PEF-treated cossettes are more flexible than thermally treated cossettes, there is less fragmentation of the cossettes during transport and processing. The diffusion of sucrose molecules in an aqueous suspension is increased slightly. Moreover, the extracted juice exhibits a higher purity requiring less lime milk for purging. Hence, less lime stone and coke for the production of lime milk is required [57].

Fig. 6.13
figure 13

Raw (left) and PEF-treated half of a sugar beet [346]

Full size image

The combination of PEF treatment with an alkaline extraction enables more efficient dewatering. For alkaline extraction, lime milk is added to the PEF-treated cossettes before the extraction process. It causes strengthening of the cell walls and a chemical modification of the pectin of the beet tissue. Thus, the juice is able to drain better out of the cells. A dry matter content of approximately 40 % after pressing the cossettes has been achieved [57]. A typical value for thermally treated cossettes without alkaline extraction is 35 %. The increased dry matter content enables a reduction of energy required for a subsequent drying process of the cossettes.

However, the energy required for complete cell opening by pulsed electric fields depends on the temperature. Figure 6.14 shows the temperature dependence of the yield of juice after pressing for a PEF treatment with 30 pulses at Ê = 3 kV/cm. Experiments showed a significant decrease of the electroporation efficiency for a treatment temperature below 7 °C at an electric field strength of Ê = 6 kV/cm. In a sugar factory, thermal energy for warming up the sugar beets is available as waste heat. However, the surface-to-volume ratio of whole sugar beets is too small for a sufficiently fast heat transfer in the course of the production process. Hence, the PEF treatment of cossettes instead of whole sugar beets is preferred, as they can be warmed up in time. A processing temperature during PEF treatment of between approximately 10 °C and 30 °C for the cossettes is preferred, because with rising temperature also the conductivity of the suspension is increased causing a higher electric current during pulse application.

Fig. 6.14
figure 14

Temperature dependency of the yield of juice after PEF treatment and subsequent pressing of slices of sugar beets (Ê = 3 kV/cm, 30 pulses) [77]

Full size image

The temperature of the thin juice after extraction needs to be kept at approximately 60 °C in order to prevent the growth of mesophilic bacteria. In a conventional extraction process, the extraction starts at a temperature level of approximately 70 °C due to the requirements of the preceding thermal denaturation stage. The temperature is reduced during extraction due to thermal losses and increased again by adding hot water required for the extraction. For a combined process based on PEF treatment and alkaline extraction, an inverse temperature profile might be employed during the extraction process [58]. Starting with a temperature of approximately 45 °C, the cossettes are warmed up to approximately 60 °C during a countercurrent extraction process by the hot water required for the extraction. The alkaline extraction of PEF-treated cossettes requires less water. As a consequence, the draft, which is the ratio of juice to cossettes, can be reduced from approximately 105 % for the conventional process to less than 100 %. In experiments a draft of 90 % has been achieved [55, 57] Thus, less evaporation energy is required for the subsequent concentration process of the sugar solution. When using a combined process of PEF treatment and alkaline extraction, in total energy savings of more than 20 % of the primary energy consumption of a sugar factory can be expected. Moreover, the alkaline milieu due to the alkaline extraction prevents corrosion, which is an issue in the acid environment of pure sugar juice.

Apart from a conventional extraction process, a discontinuous process based on pressing of cossettes while a pulsed electric field is applied has been described [59]. An increased purity of the juice after PEF treatment has been observed.

Another approach for the sugar extraction is based on ultrafiltration [60]. After PEF treatment and pressing of the cossettes, the extracted juice is filtered by means of ultrafiltration. This process results in an undiluted juice without additional water from an extraction process. Moreover, there is no need for lime milk as for a conventional thermal process with countercurrent extraction and purging by lime milk.

Pulse shape, electric field strength, pulse length, and repetition rate of the pulse application vary depending on the employed technology. For the application of rectangular pulses with an electric field strength of 0.6 kV/cm and a pulse length between 7 and 10 ms at a temperature of 20 °C, a specific energy of 7.2–10.8 kJ/kg required for cell opening has been reported [79]. When applying an aperiodically damped pulse shape with an electric field strength of 5 kV/cm and a pulse length of approximately 2 μs at a temperature of 10–15 °C, a specific energy in the order of 4–5 kJ/kg is required for cell opening [57]. However, in both cases the cell membranes need to be charged to a value required for cell opening. Pulse length and a polarity reversal of the pulse have an influence on the electrochemical wear of the electroporation electrodes [61].

6.4.2 PEF Treatment of Grapes

For the production of wine from red and white grape varieties, valuable substances are extracted especially from the cells of the peel tissue. The pigments of most red grape varieties can be found in the grape skins only. They are stored in the cells inside vacuoles. Conventional methods for cell opening for red grape varieties comprise the fermentation on skins and thermovinification .

The fermentation on skins is a fermentation process, which takes at least several days up to 1, 2, or 3 weeks. The cells are opened by maceration, e.g., by enzymes. In the course of the fermentation, alcohol is produced, which supports the extraction of valuable substances to the must. In contrast to such an alcoholic extraction after thermovinification, an aqueous extraction takes place. For thermal denaturation, the destemmed and crushed grapes are heated up to a temperature of approximately 80 °C and kept at this temperature level for about 2 min and are subsequently cooled back to 45 °C or lower. The following extraction process is enhanced by the increased temperature. However, specific taste reminding of marmalade may occur due to the heating process. For white wines, often a light character is preferred. Therefore, gentle pressing and further processing are applied. However, added enzymes cannot always be removed completely in the course of the following production steps and can be found in the wine. The treatment of white grapes by pulsed electric fields enables fast and gentle processing at low temperature without additives.

Electroporation can be applied as self-contained method for cell permeabilization or as a supplementary processing step, especially in combination with fermentation on skins [62–65]. A comparative study of red wines produced from PEF treatment and thermovinification confirmed that equal contents of tannins and pigments for both disintegration methods could be achieved. PEF treatment of white grape varieties leads to an increased content of yeast available nitrogen, which helps in preventing the atypical aging defect in the course of the fermentation. Moreover, due to a better extraction of flavoring substances, the must has a higher content also of tannins, but less acidity compared with white wine produced by gently pressing the whole berries directly. The reason for less acidity is improved chemical buffering caused by a total increase of extracted substances [66].

A combination of PEF treatment of red grapes and subsequent fermentation on skins enables a more intense color of the must than it could be achieved by each method separately [65]. For such a combined process, the time for the fermentation on skins can be reduced to approximately 3 days without a substantial effect on the extraction result. Hence, a comparably fast and complete extraction is possible.

For an extraction of pigments and tannins from the grape skins, the cell membranes and vacuoles both need to be opened. Figure 6.15 shows a microscopic view of cells from the peel tissue of Lemberger grapes before and after cell permeabilization by pulsed electric fields. The extraction starts directly after the PEF treatment, so the must after PEF treatment is becoming red. The vacuoles of this grape variety have a diameter of approximately 5 μm. Whether a complete cell opening by PEF has been achieved can be evaluated by a comparison of the contents of pigments and tannins in the must of a PEF-treated sample with a sample denatured by thermovinification both with a subsequent extraction. The measurement of the disintegration index according to Angersbach [80] based on impedance measurements may not always correlate with the extraction result, because it is more sensitive to an opening of the cell membrane, while the extraction of pigments and tannins depends also on the degree of opening of the vacuoles [67, 68]. In a continuous process, cell opening is also caused to some extent by mechanical stress due to pumping. In order to distinguish this type of disintegration from the cell opening by pulsed electric fields, a sham treatment by pumping the crushed grapes through the treatment device with the pulse voltage being switched off should be included into the experiments [69]. Figure 6.16 shows extraction curves of must and the color of the wine for raw, untreated crushed grapes, grapes that have been pumped through the device only, and PEF-treated grapes. Processing of raw, untreated grapes results in a white wine made from a red grape variety (so-called blanc de noirs). For Pinot noir grapes , a complete cell opening has been achieved when applying an aperiodically damped pulse shape with an electric field strength of 20 kV/cm and a pulse length of approximately 1.5 μs at a temperature of 10–15 °C. It requires a specific energy in the order of 40 kJ/kg. For grape varieties in Spain, it has been reported that cell opening could be achieved with rectangular pulses at an electric field strength of Ê = 5 kV/cm and a pulse length of 3 μs at an applied specific energy of 3.67 kJ/kg [348]. For grapes grown in Portugal, a treatment with rectangular pulses at an electric field strength of approximately Ê = 0.5 kV/cm at pulse lengths between 610 μs and 1.53 ms at a flow rate between 11 and 28 t/h has been reported [70]. A specific energy of 0.4 kJ/kg has been applied to the grapes.

Fig. 6.15
figure 15

Microscopic views of cells from the peel tissue of Lemberger grapes: (a) before PEF treatment; (b) after PEF treatment; (c) extraction of pigments to the must directly after PEF treatment [65, 347]

Full size image
Fig. 6.16
figure 16

Extraction curves for crushed Trollinger grapes and color of the wine: change of color intensity during extraction after PEF treatment, after pumping through the treatment device only, and untreated

Full size image

6.4.3 Transport of Material

The transport of the material in large scale is performed as a package. The means for transport differ with the requirements of the material [71]. Whole sugar beets can be transported by means of a wheel equipped with rods made out of insulation material. Figure 6.17 shows such a wheel during assembly of the electroporation device. The distance of the rods is adapted to the size of the sugar beets in such a way that the package is moved as a whole rather than a few single beets only. A conveyor belt transports the beets to the top of the wheel. In order to establish a conductive connection to the electrodes for pulse application, the sugar beets are immersed into water, which fills the space around the lower half of the wheel. The wheel pushes the beets below the water surface and prevents them from floating.

Fig. 6.17
figure 17

Wheel equipped with rods for the transport of whole sugar beets

Full size image

Raw cossettes need to be transported gently in order to prevent them from breaking. Even in a vertical chute, the cossettes may block due to the formation of bridges, if too much pressure is applied. Therefore, the application of external pressure for the transport needs to be omitted.

Grapes also need to be transported gently through the PEF treatment device in order to keep mechanical damage as low as possible. Therefore, progressive cavity pumps or peristaltic pumps are used. Figure 6.18 shows an example for a flow scheme [72]. A minimum diameter of 40 mm is required for the grape transport . As progressive cavity pumps and peristaltic pumps do not convey the material continuously, an air chamber after the pump equalizes pressure variations and, hence, the transportation velocity. If during PEF treatment a high-electric field needs to be applied, a flashover between the electrodes inside the treatment chamber has to be prevented. Air bubbles in the crushed grapes may initiate a flashover due to their lower breakdown strength. They might be removed partly by means of a degassing valve before the grapes enter the treatment chamber. Additionally, an increase of the pressure causes the bubbles to shrink and increases their breakdown strength. For pressure regulation, a second pump after the electroporation reactor might be used. A feedback control loop for the speeds of both pumps based on a pressure gauge keeps the pressure constant.

Fig. 6.18
figure 18

Flow scheme of a PEF treatment device for crushed grapes [349]

Full size image

6.4.4 Electrode System for Pulsed Electric Field Application

The treatment chamber comprises an electrode system for applying the electric field to the material. For an energy-efficient operation to each volume element of material, approximately the same energy needs to be applied. If the material is transported through the electrode system as a package with equal velocity across the cross section, a substantially homogeneous electric field results in an equal energy distribution over the treatment volume. However, due to inhomogeneities at the borders of the electrodes near inlet and outlet of the treatment area, regions with low electric field outside the treatment area may occur, which cause additional losses [73]. A substantially homogeneous field might be established either by a plate electrode system according to Fig. 6.19a or by a collinear electrode arrangement as shown in Fig. 6.19b [74–76]. During the design, the size of the electrode system needs to be adapted, on one hand, to the requirements for the material flow and, on the other hand, to the other circuit elements of the pulse circuit to adjust the circuit to the required pulse shape. In the collinear electrode arrangement , the direction of the electric field is oriented in the direction of the material flow. So the electric resistance of the electrode system rises with the length of the PEF treatment area. For a plate electrode system, the electric resistance decreases inversely proportional to the length. When scaling the electrode system in both cases, the resistance varies inversely proportional with the scaling factor.

Fig. 6.19
figure 19

(a) Plate electrode system, (b) collinear electrode system, (c) plate electrode system with separate pairs of electrodes (1, 2) each connected to one pulse generator

Full size image

Due to the material supply at ground potential, the pulse circuit needs to be grounded at the treatment chamber in order to omit an electric current flow out of the treatment area. Additionally, for safety reasons, protective electrodes might be installed before and after the electroporation area, which are safely tied to ground potential [74, 75]. For a plate electrode system, a leakage current to ground can be prevented, if the voltage is supplied to both electrodes symmetrically to ground potential. So in the case of a homogeneous material distribution, the center of the treatment area will be at ground potential due to a resistive divider formed by both halves of the electrode system. Only in the case of an inhomogeneity in the material, there will be a considerable current flow out of the treatment zone to the protective electrodes. In order to achieve material supply at ground potential for a collinear electrode system, two electrode systems are put one after the other such that they share one common high-voltage electrode, while the other two electrodes are at ground potential. From an electric point of view, both electrode systems are switched in parallel. The voltage supply is done unsymmetrically to ground. So the insulation to ground needs to be designed for the total applied voltage. In the case of a supply symmetrically to ground, a more compact setup is possible, as the insulation to ground has to be designed for half of the total applied voltage only.

Especially for the application of higher electric field strength, the electrodes can be shaped such that the electric field at the edge is reduced. Usually, a radius or a combination of different radii is applied. The electrodes might be designed such that they provide a proper electric field distribution also inside the dielectric material surrounding the channel for material transport, where a capacitive electric field control is applied.

For large PEF treatment devices, the inductance of the pulse circuit might limit the current, which can be provided to the electrode system at a predefined pulse shape without considerable oscillations. In order to reduce the inductance pulse, generators might be connected in parallel configuration to the electrode system. Thus, the total inductance of the circuit is reduced according to the number of parallel current paths. However, if the switching time of at least one pulse generator exhibits a considerable jitter, energy oscillations between the generators may occur. In order to dampen such oscillations, the electrode system can be set up as separate pairs of electrodes with each pair connected to one pulse generator as shown in Fig. 6.19c. The pairs of electrodes are placed next to each other such that the electric field is still nearly homogeneous. The distance of two adjacent electrodes influences the electric resistance between both electrodes, which acts as damping element for the energy oscillations between different pulse generators. Each pair of electrodes has been designed for equal resistance. However, the pairs of electrodes at the inlet and at the outlet are much shorter, as they have to supply additionally the neighbored low-field regions.

Semiconductor-based pulse generators exhibit a negligibly small jitter of the switching moment. However, currently semiconductor switches for high switching power are still much more expensive than spark gap switches. Therefore, spark gap switches are for large-scale electroporation devices still a cost-effective alternative. If several Marx generators with spark gap switches are connected to a common PEF treatment chamber, they need to be synchronized to each other by triggering [73]. Marx generators equipped with spark gap switches might be advantageously triggered by means of overvoltage triggering of the first stage’s spark gap. For overvoltage triggering, one or both charging coils between the first and second stage are replaced by pulse transformers, which are connected to semiconductor-based trigger pulse generators. For triggering by this arrangement, an additional pulse voltage is superimposed to the charging voltage of the spark gap. Subsequently, the remaining spark gaps ignite in the usual manner. For a low jitter , the first stage’s spark gap is equipped with a corona wire. The high and inhomogeneous electric field in the vicinity of the wire causes the formation of a corona discharge, which emits ultraviolet light for seed electron generation in the electrode system. For an overvoltage-triggered seven-stage Marx generator, a jitter of the total switching time of less than 60 ns has been achieved [77].

6.5 Recovery of Valuable Components from Plants and Microorganisms

  • Eugene Vorobiev &
  • Nikolai Lebovka 

6.5.1 Basics of PEF-Induced Electroporation in Plants and Microorganisms

Application of pulsed electric field treatment (PEF) for recovery of valuable components from plants and microorganisms becomes more and more popular [81, 82]. The short-duration pulses (from several nanoseconds to several milliseconds) with amplitude from 100–300 V/cm to 10–50 kV/cm can affect the permeability and barrier properties of cell membranes. Usually, the effect of ohmic heating during PEF treatment is unessential, and there exists possibility of processing food materials at low temperatures. PEF treatment allows avoidance of undesirable changes in biological material, which is typical for other techniques, such as thermal, chemical, and enzymatic ones [83]. The supplementary advantage is possibility of microbial inactivation [84]. Modern PEF applications include intensification of separation, extraction, pressing, diffusion, and drying processes.

Impact of PEF on cells reflects losing of barrier functions by biomembranes. Traditionally, this phenomenon is called “electroporation,” “electropermeabilization,” or “electroplasmolysis” (see reference [85] and references cited therein). Efficient electroporation requires some threshold value of transmembrane potential, u m , typically 0.5–1.5 V. Depending on conditions of treatment, the value of u m , and PEF exposure time t PEF , a temporary (reversible) or irreversible loss of barrier function can occur. The electroporation efficiency depends upon details of pulse protocol [81, 82]. The most important parameters are the electric field strength E and the total time of treatment t PEF . For plant tissues, an important damage of rather large food tissue cells (R ≈ 30–50 μm) can be observed at E = 200–1000 V/cm and treatment time within 10−4–10−1 s [85], and for small microbial cells (R ≈ 1–10 μm), larger field (E = 20–50 kV/cm) and smaller treatment time (10−5–10−4 s) are required [84]. The experimental data evidence also that longer pulses are more effective and bipolar pulses are more advantageous [86]. Moreover, a complex protocol with adjustable long pause between the trains allows fine regulation of the disintegration of tissue without noticeable temperature elevation during the PEF treatment. The correlations between electroporation efficiency and electrical conductivity contrast k = σ d /σ i (here, σ d and σ i are electrical conductivities of completely damaged and intact tissues, respectively) were also experimentally observed and reasonably explained on the base of electroporation theory [87].

In general, the electroporation effect does not require high-power consumption, and it stipulates the industrial attractiveness of PEF treatment. Experiments and theory evidence that minimal power consumption Q is observed at certain optimum electric field strength E op . For vegetable and fruit tissues (apple, potato, cucumber, aubergine, pear, banana, and carrot), the typical values of E op were within 200–700 V/cm and PEF treatment times t PEF , required for effective damage, were within 1000 μs–0.1 s [82, 86]. The estimated power consumptions Q for PEF-treated tissues were found to be rather low and typically lying within 1–15 kJ/kg. It is rather low as compared to other methods of treatment like mechanical (20–40 kJ/kg), enzymatic (60–100 kJ/kg), and heating or freezing/thawing (>100 kJ/kg).

6.5.2 Plants

Among very promising potential applications of PEF are technologies of “cold” extraction and recovery of valuable components from fresh food plants and microorganisms, e.g., sucrose extraction from sugar beetroot, betanin extraction from red beet, inulin extraction from chicory, beta-carotene extraction from carrot, phenolics extraction from grapes, etc.

6.5.2.1 Potato and Apple

Potatoes and apples were frequently used as model systems for testing of different PEF-induced effects on plant tissues. For example, the effects of reversible electroporation, transient viscoelastic behavior, stress-induced effects, metabolic responses, and electrostimulated effects were intensively studied for these plants [87–91].

Potato was used in studies of textural and compressive properties of PEF-treated tissue [90]. PEF treatment allowed elimination of the textural strength to some extent, and the effects were more pronounced after mild thermal pretreatment at 45–55 °C. Experimental data for PEF-treated apple tissue were used for testing of the theoretical models of PEF-induced effects [89]. Potato and apple were widely used also in investigations of temperature and PEF protocol effects on characteristic damage time, dehydration, freezing, and drying [87–89, 91]. The effect of the synergy of PEF and thermal treatments on the textural properties of apple tissue and apple juice expression was demonstrated [91]. It was shown that mild thermal treatment allows increase of the damage efficiency of PEF treatment, and apple tissue preheated at 50 °C and treated by PEF at E ≈ 500 V/cm exhibits a noticeable enhancement of juice extraction by pressing.

The effects of apple tissue anisotropy and orientation with respect to the applied electric field on electropermeabilization were reported [92]. It was shown that elongated cells (taken from the inner region of the apple parenchyma) responded to the electric field in a different manner than round cells, while no field orientation dependence was observed for round cells (taken from the outer region of parenchyma). The textural relaxation data support higher apple damage efficiency at longer pulse duration.

The attempts of practical application of PEF-assisted recovery of valuable components from potato and apple were also done. PEF treatment was used for facilitation of starch extraction from potato, and enhancement of the extractability of an anthocyanin-rich pigment was reported [93]. PEF treatment is also useful for production of the apple juice with increased pressing yield without using of enzymes [94]. PEF application improved noticeably the juice yield and soluble matter content in the juice. It was demonstrated that juice characteristics and yield are directly related to the size of slices [91]. Juice yield Y increased significantly after PEF treatment of large apple mash (Y = 71.4 %) at E = 450 V/cm as compared with the check sample with small apple mash (45.6 %) [94]. The PEF pretreatment was accompanied by a noticeable improvement of the apple juice clarity, an increase of the total soluble matter and the content of polyphenols, and intensification of the antioxidant capacities of juice.

6.5.2.2 Roots

6.5.2.2.1 Sugar Beet

Traditional recovery of sugar from sugar beet utilizes a power-consuming hot water diffusion of sugar from sliced cossettes at 70–75 °C and a very complex multi-staged juice purification using big quantity of lime [95]. PEF treatment has large potential for replacement or modification of the conventional thermal technology for sugar extraction and purification. Application of PEF treatment allowed recovery of sucrose even at ambient temperature, “cold” pressing of the PEF-treated sugar beet cossettes, preservation of thermal degradation of cell walls, and binding of pectins in the cellular matrix [96–109]. It was shown that up to 82 % of the overall yield could be achieved by two-stage pressing with an intermediate PEF application. The “cold” juices, expressed from the sugar beet slices after the intermediate PEF treatment, had higher purity (95–98 %) as compared to those before PEF application (90–93 %). Additionally, the quantity of pectin was noticeably lower, and the color of juice was systematically three to four times less intensive than the color of factory juices [21]. PEF-assisted “cold” extraction resulted in lower concentration of colloidal impurities (especially pectins), lower coloration, and better filterability of juice [109–111]. It is expected that PEF treatment will simplify (or even eliminate) the very complicated and polluting carbonic purification process existing today in sugar production [109–112]. The scale-up study of PEF-assisted aqueous recovery of sugar using a pilot countercurrent extractor with 14 extraction sections was reported [109]. The estimated energy gain for cold extraction with temperature reduction from 70 to 30 °C (i.e., by ΔT = 40 °C) was ≈46.7 kW.h/t, and it was noticeably higher than the power consumption, required for PEF treatment, ≈5.4 kWh/t.

6.5.2.2.2 Red Beet

PEF treatment allowed achievement of the good recovery of betalains (≈90 %) from red beet (Beta vulgaris L.) [113, 114]. The combination of PEF (7 kV/cm) and pressing permitted 18-fold shortening of extraction time and ≈4-fold increase of betanin yield [113]. Effects of PEF (400–600 V/cm) and thermal (30–80 °C) treatments on degradation of colorants and recovery kinetics were also discussed [114, 115]. The positive effect of PEF treatment in improvement of extraction and decrease of degradation was observed. PEF-assisted “cold” recovery at 30 °C allowed reaching of the high yield of colorants (≈95 %) at lower level of colorant destruction (≈10 %).

6.5.2.2.3 Chicory

Traditional industrial production of inulin from chicory roots (Cichorium intybus) needs high-temperature diffusion. The effects of PEF treatment (100–600 V/cm) on efficiency of recovery of soluble matters (inulin, sucrose, proteins) from chicory were studied [116]. The benefits of the PEF application for “cold” recovery soluble matter extraction from chicory were demonstrated. It was shown that diffusion activation energy of the usual thermal damage was rather high (more than 200 kJ/mol). However, it could be noticeably reduced to about 20 kJ/mol by PEF treatment.

6.5.2.2.4 Rhizome

PEF treatment was applied to Podophyllum peltatum in order to enhance recovery of podophyllotoxin , which is valuable for the treatment of cancer and venereal warts [117]. Conventional extraction of this chemical is inefficient and involves mechanical fragmentation of dehydrated rhizomes followed by solvent extraction at warm temperatures. The dried rhizomes of P. peltatum were soaked in deionized water and then were PEF treated (17.7, 19.4 kV/cm). The color of the sample changed after PEF treatment from sand yellow to deep red. The data demonstrated significant increase (up to 47 %) of podophyllotoxin concentration as compared with control samples.

6.5.2.2.5 Ginseng

The effects of PEF on the drying of ginseng (at 55 °C) and hot water (95 °C) extraction of dried ginseng were studied [118]. PEF was applied to the fresh ginseng using 1- and 2-kV/cm electric field strengths, 30-μs pulse duration, 25- and 200-Hz frequencies, and a pulse number of 175. Such PEF treatment resulted in reduction (≈38 %) of the drying time. It also increased the soluble solid content and significantly reduced the sugar content of the extract as compared to samples that were not treated by PEF.

6.5.2.3 Grape

PEF treatment may be rather promising for different applications in winemaking industry. PEF-assisted pressing of wine grapes improved must expression [91] and enhanced compression kinetics [90]. For PEF-treated (E = 750 V/cm) white grapes, the final juice yield increased up to 73–78 % as compared to 49–54 % for the untreated grapes (Muscadelle, Sauvignon and Semillon) [91]. Moreover, the juices extracted from PEF-treated grapes were less turbid (clearer) and didn’t require filtration through polluting filter aids (such as diatomite).

In the case of red grapes, application of PEF treatment is important for enhancement of the recovery of colorants, aromatic compounds, and phenolic bays [119, 120]. Phenolic compounds play an important role in enology owing to their contribution to the sensory properties of wine. The effects of different pretreatments (moderate thermal, ultrasound, and PEF) on the phenolics extraction from Cabernet Franc grapes were compared [121]. The results show that all pretreatments improve phenolic extraction (content of anthocyanins and tannins ), color intensity, and scavenging activity of the samples during red fermentation. However, the PEF pretreatment was the most effective. For example, pretreatment at 0.8 kV/cm and 5 kV/cm increased the yield of phenolic extraction by 51 % and 62 %, respectively. The effects of pulsed ohmic heating (POH, 100–800 V/cm) on extraction of polyphenols from red grape pomace were studied [119]. The highest yields were obtained after POH pretreatment at 400 V/cm followed by 60 min diffusion at 50 °C and using solvent composed of 30 % of ethanol in water. PEF treatment of Merlot grapes (500–700 V/cm) has demonstrated positive impact on the evolution of color intensity and content of anthocyanins and phenolic during the alcoholic fermentation [122]. Sensory analysis indicates that PEF treatment contributes to the enhancement of the sensory attributes of wine.

The important potential application of PEF is also related with extraction of polyphenols from grape by-products (skins, seeds) [123]. The effects of PEF treatment on Cabernet Sauvignon grape skin histocytological structures and on the organization of skin cell wall polysaccharides and tannins were studied using two different protocols: PEF1 (4 kV/cm and 1 ms) and PEF2 (0.7 kV/cm and 200 ms). It was shown that PEF1 protocol had little effect on the polyphenol structure and pectic fraction, and PEF2 protocol profoundly modified the organization of skin cell walls [124, 125]. Application of PEF1 protocol resulted in alteration of the visual appearance of phenolic compounds in the skins and led to increased extraction of the anthocyanins (19 %). From the other hand, application of PEF2 protocol resulted in changes in the structure of grape skins, and produced wine was richer in tannins (34 %). It was demonstrated that changes in the operating parameters of the PEF treatment did not affect the alcohol content, total acidity, or volatile acidity in the finished wines compared to the control wine.

The polyphenol extractions from grape seeds after three different pretreatments by pulsed electric field (PEF) (8–20 kV/cm, 0–20 ms), high-voltage electrical discharges (HVED) (10 kA/40 kV, 1 ms), and grinding (180 W, 40 s) were compared [126]. The PEF efficiency was higher when the treatment was performed at 50 °C in the presence of ethanol. The subsequent solid–liquid separation was faster after PEF treatment as compared to ground and HVED treatments.

PEF treatment application is promising for reduction of the maceration time during vinification and production of wines with better characteristics [120]. The effect of PEF treatment on the cold maceration (6 days at 6 °C) of Cabernet franc and Cabernet Sauvignon grapes was investigated [127]. The wines obtained from PEF-treated musts had higher phenolic content and color intensity during the alcoholic fermentation period than wines obtained from the untreated musts. Recently, the potential of PEF treatment during winemaking was tested on industrial scale [128]. PEF treatment (4.3 kV/cm, 60 μs) was done in continuous mode using collinear treatment chamber (1900 kg/h). It was demonstrated that after 7 days of maceration, the color intensity, anthocyanin content, and polyphenol index in the tank, containing grapes treated by PEF, were higher by 12.5 %, 25 %, and 23.5 %, respectively, than in the tank containing untreated grapes. Finally, the capability of PEF to inactivate the wine spoilage microorganisms may be also important [129]. It allows the fine control of fermentation and enhancement of the quality of wines.

6.5.2.4 Mushroom

Application of PEF-assisted pressure and solvent extractions for recovery of total polyphenols, polysaccharides, and proteins from the mushrooms (Agaricus bisporus) was studied [130]. The traditional hot water or ethanol extractions resulted in cloudy extracts with low colloid stability. It was demonstrated that extracts produced by PEF-assisted pressure extraction (PE+PEF) were clear, and their colloid stability was high. In general, PE + PEF allowed production of mushroom extracts with high contents of fresh-like proteins and polysaccharides. PEF-assisted extraction technique was used to optimize conditions of extraction of exopolysaccharides (EPS) from Tibetan spiritual mushroom broth [131]. The results show that the optimal conditions of such extraction technique are 40 kV/cm electric field intensity, the number of pulses 8, and pH 7. The effect of different factors on EPS extraction increases in the following order: electric field intensity > pH > number of pulses. The optimal conditions increased the EPS extraction by 84.3 % compared to that of the control group.

6.5.2.5 Tea Leaves and Wine Shoots

Thin slices of the fresh tea leaves were subjected to PEF treatment (400–1100 V/cm) in order to recover polyphenols [132]. PEF treatment accelerated kinetics of extraction, and the maximum recovery yield (≈27 %) was reached at 900 V/cm. PEF-assisted extraction (tea leaves/water ratio of 1:16, 20 kV/cm, and pulse frequency of 125 Hz) was applied for production of high-aroma instant tea powder [132]. This method allowed avoiding of the tea aroma losses during thermal processing of tea beverage production. The product obtained using the proposed method had an excellent quality and good aromatic characteristics. The application of combined PEF and freeze concentration technology to tea soup was studied [133]. The optimal conditions of PEF extraction were obtained using electric field strength of 37 kV/cm and solid–liquid ratio of 1:30.

The effects of different physical treatments (PEF, high-voltage electrical discharges, and ultrasound US) on intensification of polyphenol and protein recovery from the vine shoots were studied [134]. It was demonstrated that HVED had the highest polyphenol and protein recovery yields with the lowest energetic prerequisite.

6.5.2.6 Herbal and Flowering Plants

The PEF-assisted extraction (20 kV/cm) by 90 % ethanol–water solution was applied for enhancement of recovery of an alkaloid (Guanfu base A , GFA) from Chinese medicinal herb Aconitum coreanum [135, 136]. It demonstrated the highest yield of GFA (3.94 mg g−1) with the shortest extraction time (0.5–1 min) and the lowest energy costs as compared to other extraction methods (cold maceration extraction, percolation extraction, heat reflux extraction (HRE), and ultrasonic-assisted extraction (UE)). PEF treatment (5 kV/cm) resulted in enhancement of recovery of the major components (crocin, color; safranal, flavor; and picrocrocin, taste) from stigma and pomace of saffron (Crocus sativus) [137]. PEF treatment (30 kV/cm) was applied for reaching the optimized extraction of polysaccharides from the corn silk [138]. Corn silk is a traditional Chinese herbal medicine, which is rich in antioxidants, polyphenols, vitamins (vitamin K, C), and minerals. The yield of polysaccharides under the optimal extraction conditions was ≈7 %. The microwave and PEF-assisted extraction of polyphenols from defatted hemp seed cake by the mixed methanol, acetone, and water solvent (MAW, 7:7:6 v/v/v) was compared. It was suggested that microwave and PEF treatment can be integrated to enhance polyphenol extraction and maximize the yield [139]. A critical review on application of different pretreatments for the efficient extraction of bioactive compounds from herbal plants was recently presented [140].

6.5.2.7 Other Plants

PEF treatment was also applied for recovery of valuable components from other plants, e.g., carrot, red bell pepper and paprika, fennel, alfalfa, and red cabbage. A possibility of PEF-assisted selective recovery of water-soluble components (soluble sugars) and production of a “sugar-free” concentrate, rich in vitamins and carotenoids, was demonstrated for carrot [90]. The positive effect of PEF treatment (0.25–1.0 kV/cm) of the carrot purees on extraction of polyacetylene and sugars, color changes, and total carotenoid content was also shown [141]. The effect of PEF treatment (0.1–1 kV/cm and frequency of 5–75 Hz) on extraction of carotenoids from the carrot pomace was examined using different vegetable oils (sunflower, soya bean, and peanut) [142]. It was demonstrated PEF effects on the extractability of carotenoids from the carrot were dependent on the electric field strength, frequency, and type of vegetable oil. The stable microemulsions were used to extract β-carotene from PEF-treated carrot pomace [143]. The content of β-carotene, extracted from PEF-treated carrot pomace using microemulsions, was higher than when it was extracted from untreated pomace. The high recovery yield (up to 96.7 %) of lycopene from tomato residual was achieved using PEF treatment with electric field strength 30 kV/cm, liquid–stuff ratio 9 ml/g, temperature 30 °C, and ethyl acetate as an extraction solvent [144]. The proposed method was proved to be a fast way for lycopene extraction from the tomato residual. PEF treatment (2.5 kV/cm) applied to mashed red cabbage in a batch treatment chamber allowed enhancement of the total anthocyanin recovery by 2.15 times [145].

6.5.3 Microorganisms

6.5.3.1 Yeast Cells

The yeast cells were used in many works as a model microorganism for testing the effects of electrical stimulation, permeabilization of cell membranes, and cell lysis. Depending on the protocol of PEF treatment, inactivation [146] or partial electropermeabilization of yeast cells [147, 148] is possible. In addition, PEF treatment can be useful for recovery of the high-quality intracellular components (ions, saccharides, enzymes, proteins, and nucleic acids) from the yeast cells [149–151]. The most effective method for recovery of valuable substances is the intensive high-pressure homogenization (HPH); however, it produces not pure extracts. It was shown that PEF treatment combined with mild HPH can be used for more efficient extraction of proteins [151]. For example, PEF treatment (10 kV/cm) of the aqueous suspension of wine yeast allowed high extraction of ionic components and low extraction of high-molecular-weight components [150]. PEF treatment (40 kV/cm) of the aqueous suspension of the same wine yeast allowed extraction of the 70 % of ionic substances, 1 % of proteins, and 16 % of nucleic acids [151]. It was demonstrated that PEF and high-voltage electrical discharge (HVED) treatments always resulted in incomplete recovery from yeast cells, though efficiency of HVED was higher than that of PEF [151]. For example, treatment at E = 40 kV/cm allowed extraction of ≈80 % and ≈70 % of ionic substances, ≈4 % and ≈1 % of proteins, and ≈30 % and ≈16 % of nucleic acids in cases of HVED and PEF treatments, respectively. Recently, PEF treatment was applied for assistance of recovery of valuable components from waste brewing yeasts [152, 153]. For PEF treatment at ≈20 kV/cm, the rate of trehalose recovery was up to 2.635 % and was 15.96 times higher than that of extraction microwave and 34.08 times higher than for ultrasound technique. For PEF treatment at 50 kV/cm, recovery of RNA in ethanol extraction was 1.69 times higher than that of water extraction.

6.5.3.2 Microalgae

Microalgae have high content of lipids, proteins, polyunsaturated fatty acids, carotenoids, valuable pigments, and vitamins and can be used in the food, feed, cosmetics, pharmaceutical, and biofuel industries. Traditional methods for extraction of these components use environmentally toxic solvents. Recently, several groups have developed PEF-assisted techniques for extraction of valuable components from microalgae. PEF treatment (23–43 kV/cm) in a flow cell was applied to microalgae Auxenochlorella protothecoides suspension [154]. It was demonstrated that PEF-assisted extraction was highly selective and allowed release of the soluble intracellular matter, while extraction of lipids required application of solvents. The opportunity of using PEF treatment at the first step of extraction and solvents at the second step of extraction was demonstrated. Continuous PEF treatment was studied, and a flow technology was proposed for extraction of the total of cytoplasmic proteins from microalgae (Nannochloropsis salina and Chlorella vulgaris) [155]. The technology was developed on the preindustrial pilot scale that allows easy treatment of large volumes. Effective extraction was observed in the case when PEF was followed by a 24-h incubation period in a salty buffer. PEF treatment was applied to enhance lipid extraction from Ankistrodesmus falcatus wet biomass using the green solvent ethyl acetate. Application of PEF allowed significant enhancement of the rate of lipid recovery. It was noted that the increase in lipid recovery was due to the electroporation and not due to temperature effects [156]. Experimental data evidence that electrically based PEF (20 kV/cm)- and HVED (40 kV/cm)-assisted techniques allow selective recovery of water-soluble ionic components and microelements, small-molecular-weight compounds, and water-soluble proteins from microalgae Nannochloropsis sp. [157]. The increase in lipid recovery for PEF-treated microalgae combined with ethyl acetate was explained by electroporation [156]. PEF treatment of microalgae also resulted in significant increase of the yield of fatty acids [158]. PEF-assisted technology was used as an effective tool for extraction of proteins from microalgae [155]. The possibility of enhancement of the extraction yield of pigments (chlorophyll and carotenoid ) from microalgae by PEF treatment was also shown [159]. PEF treatment was also proposed as an effective tool for control of predators in industrial scale microalgae cultures [160]. It was shown PEF can be used for selective elimination of such contaminant as protozoa that can highly jeopardize the productivity of the culture.

6.6 Bacterial Inactivation for Food Preservation

  • Ignacio Álvarez &
  • Javier Raso 

Pulsed electric field technology (PEF) is viewed as one of the most promising nonthermal methods for inactivating microorganisms in liquid foods. The local defects or pores created by the application of an external electric field led to the loss of the microbial membrane’s integrity abolishing its capacity to maintain the microbial homeostasis. The treatment causes the inactivation of pathogenic and spoilage microorganisms, but it also results in the retention of flavor, nutrients, and the color of the food compared to thermal processing. As bacterial spores are resistant to PEF treatments, the main applications of this technology for food preservation must be focused on pasteurization. Commercialization of PEF technology as a pasteurization process requires the estimation of its efficacy against pathogenic and spoilage foodborne microorganisms. This chapter reviews the current state of the art in microbial inactivation by PEF. Particular attention is devoted to the microbial inactivation mechanisms and the different factors influencing the microbial inactivation by PEF.

6.6.1 Introduction

Most food products constitute a rich nutrient source for microbial development. Microbial growth of microorganisms causes food spoilage as a consequence of metabolic activities leading to the production of molecules that alter the sensorial attributes of foods. The microbial deterioration of food is evidenced by slime formation and changes in the appearance, texture, color, odor, and flavor [161]. More important, foods may be sources of foodborne pathogenic microorganisms. The consumption of foods contaminated with these poisoning microorganisms and without generally presenting any sign of food spoilage may cause serious illness outbreaks. While microbial food spoilage is a huge economic problem (it is estimated that about 25 % of the world’s food supply is lost from microbial spoilage), foodborne illnesses are an enormous public health concern worldwide with severe direct and indirect economic consequences.

Food preservation technologies used in the food industry aim at combating the deleterious effects of microorganisms in foods and avoiding foodborne illnesses. These technologies act by preventing microbial growth or by microbial killing. Metabolic activity of the microorganisms may be inhibited or slowed through those factors such as temperature, water activity, preservatives, pH, or atmosphere that most effectively influence the growth and survival of microorganisms in foods. Chilling, freezing, drying, modified atmosphere packing, acidifying, and adding preservatives are examples of preservation techniques based on preventing or slowing microbial growth. Among the many techniques used to preserve foods, a much smaller number rely on killing microorganisms. These techniques that inactivate microorganisms are more effective to ensure that potentially hazardous levels of microorganisms are not present in foods at the time of consumption. Pasteurization and sterilization by heat are the methods of microbial destruction traditionally used in the food industry. These techniques are very effective for microbial inactivation. However excessive heat treatment may cause undesirable effects on foods such as protein denaturation, nonenzymatic browning, and loss of vitamins and volatile flavor compounds [162]. In order to reduce the negative effects of the heat treatments in foods, alternative technologies capable of inactivating microorganisms at temperatures below those used during thermal processing are being demanded by the food industry. These technologies, called nonthermal technologies, not only inactivate pathogenic and spoilage microorganisms but also result in the retention of flavor, nutrients, and color of foods compared to thermal processing [45].

Pulsed electric field technology (PEF) is viewed as one of the most promising nonthermal methods for inactivating microorganisms in liquid foods. Electroporation of microbial membranes has the ability to effectively inactivate vegetative forms of microorganisms, thus extending foods’ shelf life and enhancing microbial food safety without compromising the nutritional and sensory characteristics of the foods.

6.6.2 Mechanism of Microbial Inactivation by PEF

Although the mechanism underlying microbial inactivation by PEF has not been fully elucidated, it is believed that the formation of local defects or pores that leads to an increment of the cell membrane permeability to ions and macromolecules is the main cause of microbial killing.

The basic function of the cell membrane is to protect the cell from its surroundings. The cell membrane separates the microbial cytoplasm from the outside environment, acting as a selectively permeable barrier to ions and organic molecules and controlling the movement of substances in and out of the cells. The maintenance of the microbial homeostasis requires that the cytoplasmic membrane acts as an intact semipermeable barrier under fluctuating external conditions. The local defects or pores created by the application of an external electric field lead to the loss of the membrane integrity, and uncontrolled molecular transport across the membrane may occur. These events may abolish the homeostatic capacity of the cells and will eventually lead to microbial death. Membrane damage presumably becomes lethal if the damage is of a nature that precludes resealing or if irreversible effects occur as a secondary consequence of the loss of permeability control, energy conservation, or other membrane functions [163].

However, in microorganisms including bacteria and yeast, the cytoplasmic membrane is not the only barrier that separates the cytoplasm from the environment. The cytoplasmic membrane of Gram-positive bacteria is surrounded by a thick cell wall made of peptidoglycans and tectonic acids. On the other hand, the cell wall in Gram-negative bacteria is thinner, but it is surrounded by an outer membrane that differs from typical biological membrane because the main molecular constituents of the external lipid bilayer are lipopolysaccharides. This outer membrane prevents the entrance of some molecules such as antibiotics, lytic enzymes, or bacteriocins but allows low-molecular-weight nutrients to diffuse into the periplasmic space. On the other hand, yeast cells are surrounded by a cell wall similar to Gram-positive bacteria. Important differences in the effect caused by PEF in Gram-positive and Gram-negative bacteria have been described. However, how the envelopes surrounding the cytoplasmic membrane influence electroporation is an aspect that requires further research.

Different techniques such as detection of leakage of intracellular material, measurement of osmotic response, or fluorescent dye exclusion assays have been used to evidence the electroporation of microbial cytoplasmic membrane caused by PEF.

The presence in the medium surrounding the microorganisms of ultraviolet-absorbing material such as nucleic acid, proteins, and adenosine triphosphate (ATP) is one of the most commonly used indicators of the leakage of intracellular material. Leakage of ultraviolet-absorbing material from different microorganisms treated by nonlethal PEF treatments has been observed, indicating that the temporary loss of permeability control is not necessarily lethal [164]. It was observed that increasing the severity of the treatment resulted in a greater leakage of ATP, nucleic acids, and proteins [40, 164, 165].

Bacterial plasmolytic response to osmotic stress is a physical indicator of membrane integrity. The ability of microbial cells to undergo plasmolysis indicates that a semipermeable membrane is present and functioning to maintain protoplast integrity. When intact microbial cells are suspended in a hypertonic medium, water diffuses from the cell, thus causing a strong condensation of the cytoplasmic content that can be determined by the increments in the optical density of the cell suspensions. Using this procedure, early studies on microbial electroporation demonstrated that cells of Escherichia coli treated by PEF lost their ability to plasmolyze in a hypertonic medium, which supports that the treatment affected the integrity of cytoplasmic membrane [17].

One of the most often used techniques to investigate the electroporation of the microbial membranes caused by PEF is the exclusion of dyes. The hydrophilic fluorescent molecule propidium iodide (PI) with a molecular weight of 660 Daltons has been the most commonly used probe for this purpose. PI that is only able to enter permeabilized microbial cells has been used to analyze the electroporation of individual cells with epifluorescent microscopy and flow cytometry, or of the whole population, by using spectrofluorometer procedures. Using flow cytometry, it has been observed that when the PI is added after the PEF treatment, there is a linear correlation between the number of permeabilized and inactivated cells for E. coli, Listeria innocua, and Lactobacillus plantarum [40, 164]. However, in the case of the yeast Saccharomyces cerevisiae, it was observed that some treatments that increased the permeability to PI did not necessarily cause a loss of viability. The reversible and irreversible electroporation can be detected by comparing the fluorescent intensity of a microbial suspension treated by PEF when the PI was added before or after the PEF treatment [166]. Reversible electroporation was detected when the fluorescent intensity of the microbial suspension that was in contact with PI during the PEF treatment was higher than the fluorescent intensity of the microbial suspension that was put in contact with PI after the PEF treatment. Results obtained using this technique supported that reversible and irreversible electroporation was involved in the microbial inactivation by PEF, depending on the treatment medium pH and the characteristics of the microbial envelopes surrounding the cytoplasmic membrane. At pH 7 and pH 4, the loss of viability for two Gram-positive bacteria (Listeria monocytogenes and L. plantarum) was correlated with an irreversible loss of membrane integrity. However, for the two Gram-negative bacteria (E. coli and Salmonella senftenberg), inactivation was correlated with the proportion of reversible and irreversible electroporated cells. Therefore, these results indicate that the reversible electroporation of the cytoplasmic membrane of Gram-negative bacteria may cause also microbial death. For Gram-negative bacteria treated at pH 4, no correlation was observed between the loss of viability and membrane permeabilization being the proportion of permeabilized cells lower than the inactivated ones. This behavior could be a consequence of that the size of the pores caused by PEF treatments in cells suspended in a medium of pH 4 was smaller than those required for the PI uptake but big enough to abolish the homeostatic capacity of the cells.

The ability of the microorganism to recover from the damage caused by PEF in the cytoplasmic membrane has been correlated with the occurrence of sublethal injury after the PEF treatment. The sublethally injured population fails to survive and multiply in harsh environments tolerated by the untreated cells. Comparison of cell counts of PEF-treated samples on selective (harsh environment) and nonselective media is the most conventional technique for detecting the occurrence of sublethal injury. Early studies on sublethal injury caused by PEF concluded that microbial inactivation by PEF was an all-or-nothing effect because after the treatment, alive or dead cells were detected but not sublethally injured ones [165, 167]. However, at present it is well established that PEF causes sublethal injury depending on the microorganisms and pH of the treatment medium [168, 169]. Generally, a greater number of sublethally injured cells were detected in a population of Gram-negative bacteria when treated by PEF at pH 4 than at pH 7. In Gram-positive bacteria, the occurrence of sublethal injury was greater at pH 7 than at pH 4. The fact that the presence of NaCl in the recovery medium prevented the growth of sublethal injured cells of E. coli after PEF treatments and the demonstration that these damaged cells required energy and the synthesis of lipids for injury repair supports the involvement of the cytoplasmic membrane on the microbial inactivation by PEF [170].

Recovery media with bile salts added are generally used to evaluate the permeabilization of the outer membrane of Gram-negative microorganisms. Using this technique, several authors have reported that PEF did not affect the permeability barrier of the outer membrane of the bacteria surviving the treatments [17]. However, it was observed in cells of Enterobacter sakazakii treated by PEF in media of low pH the occurrence of sublethal injury in both cytoplasmic membrane and outer membrane [171]. This damage in the outer membrane facilitated the antimicrobial activity of citral in cells of this microorganism previously treated by PEF.

It can therefore be concluded that PEF may cause reversible or irreversible electroporation of the cytoplasmic membrane of microorganisms depending on the intensity of the applied treatment but also on the type of microorganism and pH of the treatment medium. Irreversible electroporation leads to microbial inactivation. However, reversible microbial electroporation may result in cells that are able to return to their original state by membrane resealing, in sublethally injured cells or dead cells. The presence of sublethally injured cells permits combining PEF with additional hurdles to improve the preservation effect of PEF. Such is the case of acid medium that inhibits the recovery of damaged cells [172] or the addition of antimicrobial substances that otherwise would not be effective in undamaged cells [173, 198, 199].

6.6.3 Factors Affecting Microbial Inactivation by Pulsed Electric Fields

The microbial inactivation by PEF has been found to depend on many factors [173, 174]. In order to define the processing conditions required to inactivate spoiling and pathogenic microorganisms, the influence of these factors must be understood. Critical factors affecting microbial inactivation can essentially be classified into three groups: processing parameters, microbial characteristics, and treatment medium characteristics (Table 6.4).

Table 6.4 Factors affecting microbial resistance by PEF. The main factors are indicated in bold letters
Full size table

6.6.3.1 Processing Parameters

The most typical processing parameters that characterize PEF technology are electric field strength, pulse shape, pulse width, number of pulses, pulse-specific energy, frequency, and temperature. From among them, electric field strength, treatment time, specific energy, and temperature are the most critical for the effectiveness of microbial inactivation by PEF.

The distance between the electrodes of the treatment chamber and the voltage delivered defines the electric field strength that is generally reported as kV/cm. In general, microbial inactivation increases by augmenting the strength of the electric field over a threshold field strength called critical electric field strength (E c ). This E c is the field strength required to exceed the transmembrane potential that varies from 0.5 to 1.0 V and is responsible for the membrane electroporation [175, 176]. The higher the field strength, the larger the electroporation phenomenon. Due to the size of microbial cells (1–10 μm), which is smaller than that of the eukaryote plant cells (40–200 μm), the critical electric field intensity to induce electroporation of microbial cells is much higher than for induce electroporation in eukaryote cells of plant or animal tissues (>5 kV/cm). This fact indicates that microbial inactivation requires more powerful equipment and higher energetic costs than the electroporation of eukaryote cells. Generally, studies on microbial inactivation by PEF have been conducted in the range of 10–30 kV/cm because the application of higher electric field strengths has technical limitations, especially at an industrial scale, and may cause the dielectric breakdown of the food material.

Treatment time is defined as a function of the duration of pulse width and the number of pulses applied. It is generally reported in μs. In square waveform pulses, pulse width corresponds to the duration of the pulse, but in exponential decay pulses, the time required for the input voltage to decay to 37 % of its maximum value has been adopted as the effective pulse width. The survival curves (Log10 of survivors along the time) at constant electric field strength are characterized by a fast inactivation in the first moments of the treatment, and then the number of survivors slowly decreases as the number of pulses applied becomes longer (Fig. 6.20). Due to this kinetics of inactivation, many investigations have been carried out in order to define the most adequate mathematical model to describe it and to develop equations to predict the PEF microbial inactivation [177].

Fig. 6.20
figure 20

Theoretical survival curves corresponding to microbial inactivation by PEF treatments at different electric field strengths

Full size image

Specific energy of the treatment (energy applied per mass unit) depends on the applied voltage, pulse width, number of pulses, and resistance of the treatment chamber that varies according to its geometry and conductivity of the treated material. Usually it is reported in kJ/kg. This parameter permits the evaluation of the energy costs of the PEF process and, consequently, the comparison of the energy efficiency of PEF with other inactivation technologies. The specific energy has been proposed as a control parameter of the PEF process together with the electric field strength mainly when exponential decay pulses are used due to the lack of precision in the measurement of the pulse width [178]. Microbial inactivation by PEF increases with specific energy, but when different treatments of the same specific energy are compared in terms of microbial inactivation, those applied at higher electric fields are more effective [179, 180].

Microbial inactivation by PEF is usually enhanced when the temperature of the treatment medium is increased, even in ranges of temperatures that are not lethal for microorganisms (Fig. 6.21) [181–183]. The higher microbial PEF sensitivity when applying PEF at moderate temperatures is the basis of current PEF pasteurization treatments of fruit juices and smoothies [173]. The sensitizing effect of temperature has been attributed to changes in the phospholipid bilayer structure of the cell membranes, from a gel-like consistency to a liquid crystalline state that is caused by the temperature increase. The improved membrane fluidity reduces its stability and facilitates the PEF electroporation. However, further studies are required to demonstrate this fact.

Fig. 6.21
figure 21

Influence of the temperature on the inactivation of Escherichia coli O157:H7 (●); Salmonella typhimurium 878 (○); Staphylococcus aureus 4459 (■); and Listeria monocytogenes 5672 (□) by PEF treatments (30 kV/cm; 0.5 Hz; 1 pulse of 3 μs) in McIlvaine buffer of pH 3.5 (■)

Full size image

It has to be pointed out that the combination of PEF with lethal temperatures has been recently investigated as a treatment to inactivate bacterial spores of Bacillus subtilis [184]. In this case, a new processing concept has been proposed based on previous research of Heinz et al. [185] for the pasteurization of apple juice or milk by Guerrero-Beltrán et al. [186] on using the heating due to the Joule effect because of the electrical energy dissipation that occurs during the PEF processing. In this approach, treatment conditions were selected (9 kV/cm, 146–178 kJ/kg) to apply treatment temperatures over 80 °C but with extremely short residence times reducing up to 3 Log10 cycles of the spore population. More research is necessary in order to identify the advantages of this process against traditional thermal processing in terms of energy requirements, impact on nutritional compounds, and sensorial properties.

Finally, there is some controversy concerning the influence of the pulse shape, width, and frequency on PEF microbial inactivation. It is generally accepted that square wave pulses are more efficient than exponential decay ones because the characteristic slow-decaying rate causes a long tail section that is ineffective in killing the microorganisms in the food material. Some authors have reported that when treatments of the same duration are applied with pulses of different width or at different frequencies, longer pulses and higher frequencies are more effective [187, 188]. However, these two parameters apparently do not exert an influence on microbial inactivation when the temperature rise of the medium caused by the application of longer pulses of higher frequencies is avoided [189].

6.6.3.2 Microbial Characteristics

Microbial inactivation by PEF depends on microbial properties such as the type of microorganism, characteristics of the cell envelopes (Gram-positive or Gram-negative), cell size and shape, growth conditions (growth temperature and phase), and recovery conditions (medium composition, temperature, recovery time, oxygen concentration, etc.). Generally, it has been reported that bacteria are more PEF resistant than yeast; Gram-negative microorganisms are more sensitive than Gram-positive microorganisms; and cocci are more resistant than rods. However, it seems that the intrinsic microbial resistance is more important than the effect of the microbial characteristics in determining the microbial sensitivity to PEF. When the PEF resistance of different microorganisms is compared under the same experimental conditions, it is observed that some yeast cells are more PEF resistant than some bacteria, some Gram-positive microorganisms are more sensitive than Gram-negative microorganisms, and some yeast species and some rod bacteria are more resistant than some coccus bacteria [177, 183].

The PEF resistance of different strains of bacterial species may vary greatly. It has been observed that depending of the strain and pH of the treatment medium, the inactivation of different strains of the same microorganism may range from hardly any inactivation to more than 4.0 Log10 CFU/ml [190]. As the PEF resistance of the different strains depended on the pH of the treatment medium, the target microorganisms to define treatment conditions for PEF pasteurization could be expected to be different for foods, depending on their pH (Fig. 6.22). This can be a limitation of the technology for industrial application.

Fig. 6.22
figure 22

Influence of the pH of the treatment medium on the resistance of different microbial species and strains to a PEF treatment (30 kV/cm; 50 pulses of 3 μs) (Adapted from [45, 205, 206])

Full size image

Culture conditions of microorganisms also influence microbial inactivation by PEF. Generally, it has been reported that microorganisms at the exponential phase of growth are more PEF sensitive than those at the stationary phase [191, 192]. The higher size of the cells in the exponential phase could explain this difference in resistance. On the other hand, microbial resistance to PEF depends on the cultivation temperature. It has been reported that cells grown at temperatures lower than the optimal one are more PEF sensitive than those grown at optimal temperature. Variations in the lipid composition of cultures grown at different temperatures could be the reason of this behavior.

6.6.3.3 Treatment Medium Characteristics

Generally, studies on microbial inactivation by PEF have been conducted with microorganisms suspended in liquid media, receiving less attention the microbial inactivation in solid media. The influence of the pH and the electrical conductivity of the substrate on microbial inactivation have been the main investigated treatment medium parameters. It has been observed that microbial PEF resistance varies considerably, depending of the pH of the treatment medium . An increment, decrease, or no effect on the PEF microbial inactivation has been reported when varying the pH of the treatment medium when applying the same PEF treatment [177]. Generally, Gram-positive microorganisms are more PEF resistant in media of neutral pH than in acidic conditions, and Gram-negative ones are more resistant in media of acidic pH than in neutral conditions [193] (Fig. 6.22). This effect of the pH on microbial resistance has been confirmed in both buffers and liquid foods. The mechanism that explains these differences seems to be related to the occurrence of sublethal membrane damage by PEF as it has been previously described [193]: when Gram-positive bacteria are treated in neutral media, their ability for repairing sublethal injury caused by PEF is higher than when treated in low pH media. On the contrary, the higher PEF ability for repairing sublethal injury in Gram-negative bacteria occurs when they are treated in acidic pH media.

Several studies have reported that the electrical conductivity of the treatment medium affects microbial inactivation. However, it is unclear if the conductivity influences electroporation or if the observed effect is a consequence of the influence of conductivity on the intensity of the PEF treatment applied. A change in conductivity modifies the resistance of the treatment chamber, and as a consequence, it may cause changes in the electric field strength and the pulse width and total specific energy of the pulses. Studying the lethality in a range from 0.05 S/m up to 0.4 S/m, which corresponds to the conductivity of most of liquid foods, it has been observed that the conductivity did not affect microbial inactivation when the input voltage and input pulse width were modified in order to obtain the same treatment (electric field strength and treatment time) in media of different conductivities [179, 180].

6.6.4 Food Pasteurization by PEF

Inactivation of vegetative cells of bacteria and yeast by PEF has widely been demonstrated. However, the few studies conducted on the inactivation of bacterial spores by PEF at moderate temperatures describe these structures as resistant to PEF treatments [194, 195]. Therefore, currently practical applications of PEF processing aim at replacing thermal pasteurization as a means of killing vegetative microorganisms rather than sterilization. Pasteurization refers to a treatment used for food preservation that aims to inactivate pathogenic forms of vegetative microorganisms. Although the main objective of PEF pasteurization is to guarantee food safety, a large proportion of the population of vegetative spoilage microorganisms is also inactivated by the treatment. Therefore, PEF treatments may extend the shelf life of foods [196, 197]. However, the treatment is not capable of achieving commercial sterility because spores or other nonpublic health significant microorganisms can survive the treatment; thus other preservation techniques, such as refrigeration, atmosphere modification, the addition of preservatives, or a combination of these techniques, will be required to preserve the quality and stability of the food during its distribution and storage [173, 198, 199].

PEF is gaining interest as a gentle method of food preservation of heat-sensitive foods such as fruit juices. Demands by consumers for fresh-like and natural taste foods have promoted the introduction in the market of non-pasteurized fruit juices produced from fresh fruit and distributed under refrigeration. As psychotropic microorganisms are able to grow at refrigeration temperatures, the shelf life of these fruit juices is very short (around 7 days). On the other hand, several outbreaks associated with the consumption of unpasteurized juices have demonstrated that these products can be a vehicle of foodborne illnesses caused by pathogens such as Salmonella spp. or E. coli O157:H7 [200].

Commercial exploitation of PEF for food pasteurization requires proof that PEF promotes a level of microbial safety that is equal to that made possible via traditional processing. A 5-Log10 reduction of the most resistant microorganism of public health significance has been established by the US Food and Drug Administration (FDA) concerning fruit juice pasteurization [201]. Studies to evaluate the application of PEF for microbial decontamination at room temperature have shown that to obtain these levels of reduction, it is necessary to apply long treatments (i.e., >100 μs) at high-electric field strengths (i.e., ≥30 kV/cm) [173, 177, 179, 180, 202]. At a commercial scale, technical and economical limitations exist in applying these intense treatments in a continuous flow. However, several studies confirmed that the application of PEF at moderate temperatures provides the possibility of obtaining substantial microbial inactivation of pathogenic microorganisms that are particularly PEF resistant with a short residence time (less than 1 s) at moderate electric field strengths (≤25 kV/cm). Under these treatment conditions, the shelf life of fruit juices and smoothies is extended up to 21 days, while maintaining a fresh-like taste and product quality. Currently such products are commercialized in the Netherlands, Germany, and the UK, using PEF equipment with a capacity in the range of 1500–8000 l/h. The total processing costs, including investment and operation, could vary in a range from 0.01 to 0.03 euros/l [203, 204].

Although the first commercial applications of PEF are available in the market, more multidisciplinary research efforts are required to identify the most PEF-resistant pathogens of concern for each specific food, to define process criteria for PEF pasteurization, to get a better mechanistic understanding of the critical parameters affecting microbial inactivation, and to combine all this knowledge with engineering aspects involving the distribution of the electric field strength in continuous flow treatment chambers or to develop suitable sensors to assess the PEF process. A deeper knowledge of these aspects is needed to satisfy regulatory agencies and to enhance the safety and stability of minimal processed foods of the future.

6.7 Microbial Decontamination Using Atmospheric-Pressure Plasma

  • Loree C. Heller &
  • Muhammad A. Malik 

6.7.1 Introduction

Adding energy to a gas breaks bonds between atoms and ionizes the atoms creating positively charged particles (ions) and negatively charged particles (electrons). When a significant number of atoms in the gas ionize, the resulting overall electrically neutral medium of free positive and negative particles can be described as plasma. When the temperature of neutral particles (gas temperature, Tg) is the same as electron temperature (Te) and ion temperature (Ti), it is called equilibrium plasma. In nonequilibrium plasma, electron temperature is much higher than gas temperature, i.e., Te>>Tg≈Ti. The plasma can be formed at reduced pressure or at atmospheric pressure. Atmospheric-pressure nonthermal plasma relevant to microbial decontamination is described in the following chapter.

Plasma is usually generated by high-voltage electrical discharges between electrodes with the interelectrode gap filled with gas. The gas discharges are usually classified on the basis of electrode configuration and power source/excitation frequencies. The main groups of discharges relevant of microbial decontamination are corona discharges powered by direct current (dc) or pulsed dc, dielectric barrier discharges (DBD) powered by alternating voltages of low frequency to several megahertz, and atmospheric-pressure plasma jets (APPJs) powered by dc to some gigahertz [207–209].

Corona discharge is formed in a diverging electrode geometry, such as needle to plate or wire to cylinder [210]. An intense electric field is formed at the needle tip or thin wire upon application of high voltage. Electrical breakdown in the gas occurs in the form of initiation of several thin plasma channels distributed along the electrode that propagate toward the counter electrode. Arcing is avoided by either limiting the current in the case of dc power or by applying pulsed dc to cut off the electric field before arcing. Inserting a dielectric layer in the discharge gap between electrodes changes the corona discharge into sliding discharge at the solid–gas interface [214, 215]. Covering at least one of the electrodes also avoids arcing in the case of DBD [213]. The electrodes in DBD can be large area plate-to-plate configuration where a large number of micro-plasma channels are formed, distributed in time and space along the electrode. Accumulation of charges at the dielectric reduces local electric field and extinguishes the discharge in a few nanoseconds. New discharges initiate and continue the process as long as the alternating voltage continues. Removing the air gap between the electrodes, i.e., placing a wire or strip electrode on the dielectric and a plate-type electrode covering the opposite side of the dielectric, transforms the volume DBD into surface DBD [214, 215].

Several versions of atmospheric-pressure plasma jets have been developed which generate plasma in open space [207, 208]. The plasma in these cases is formed in a dielectric tube with attached electrode(s) and is expelled in the open space by fast flowing gas and/or by redistribution of electric fields driving the plasma into the open space. The target to be treated with plasma may be one of the electrodes responsible for the redistribution of the electric fields. The APPJs have been extended into a brush-shaped plasma, e.g., by using an array of the jets [215] or an edge electrode to generate large area plasmas [216]. A cross-section view of a large area plasma device used in bacterial decontamination on surfaces [212] is illustrated in Fig. 6.23. It forms sliding discharges in the chamber, activating air. The activated air is expelled and targeted on the surface to be decontaminated. It can be enlarged further by stacking and operating multiple discharge chambers in parallel [211].

Fig. 6.23
figure 23

Cross-section view of a sliding discharge-based large area plasma device

Full size image

6.7.2 What Makes Plasma Reactive?

Nonthermal plasmas are usually formed by high-voltage electrical discharges. Any free electrons in the interelectrode gap are accelerated under the influence of strong electric fields. Inelastic collisions between high-energy electrons and ambient gas molecules causes ionization, dissociation, or excitation of ambient gas molecules producing ions, more free electrons, reactive free radicals , and excited states like O, N, H, OH, O2*, and N2*. A major fraction of the input electrical energy ultimately ends up as heat. Some of the excited state species emit photons that may fall in UV or VUV spectrum range. The primary reactive species ultimately produce secondary reactive species, like O3, H2O2, and nitrogen oxides (NOx). All of them affect the cell being treated by the plasma to a varying extent.

What reactive component exists in the plasma and which one plays a dominant role in microbial decontamination depends on the feed gas, experimental conditions, and type of cell being treated. For example, noble gas plasma is easier to form and sustain and efficiently produces and transmits VUV and UV radiations, but has a low concentration of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [217]. Addition of a reactive gas such as oxygen in the noble gas can generate ROS-like O, metastable oxygen, and O3. Addition of water vapor results in production of H, OH, and H2O2. Therefore, air, particularly humid air, is an excellent option because it is easily available and produces a wide range of ROS and RNS. Particularly the strongest oxidizing agents, i.e., OH and ONOO−, are produced in humid air and are considered to play a major role in microbial decontamination [218]. UV radiations are found to play in synergy with free radicals in the case of reduced pressure plasmas [209]. In atmospheric-pressure plasmas, free radicals and reactive neutrals play a dominant role, while most of the UV radiations are absorbed in air [219].

Direct exposure to plasma is generally more effective than remote exposure. In the case of direct exposure of the target to the plasma, both short-lived species like O and N and long-lived species like OH, O3, H2O2, and NOx affect the cells being treated. Heat [220, 221] and electric fields [222, 223] that are often associated with the direct plasma treatment are also known to play a significant role. In the case of remote plasma exposure, only long-lived ROS and RNS, like O3, OH, H2O2, and ONOO−, are primarily involved in the microbial decontamination.

6.7.3 The Effects of Plasma Reactive Components on Microbes

The observation that atmospheric-pressure plasma reduced vegetative bacterial numbers [224] led to a large number of studies confirming inactivation of a variety of organisms, including vegetative bacteria, bacterial spores, bacteriophages, and viruses, fungi, and fungal spores with a variety of direct and indirect plasma types in proof-of-principle or translational studies. Several excellent review articles are available for more detail [209, 225–228]; this chapter presents an overview of mechanisms and recent translational studies.

Some species of bacteria and fungi are protected by a mucoid polysaccharide capsule, which may shield the exterior of these organisms from plasma components. All bacterial and fungal cells possess a cell wall composed primarily of peptidoglycan or chitin respectively that protects the cell from mechanical damage and osmotic rupture. Microscopically observable cell surface lesions [229] commonly described as “etching” are observed microscopically after plasma application.

Although bacteria can generally be categorized as Gram-negative or Gram-positive based on the specific cell wall composition, this category does not necessarily predict a cell’s plasma sensitivity [212, 221, 230–237]. Although the cell wall may protect the cell from lysis, damage to lipids in the plasma membrane can produce loss of membrane potential [238] and leakage of cellular contents [229, 231, 239]. Plasma exposure acidifies lipid films [240] and membrane lipids [238], although this is not a primary antibacterial component [241]. Viral genomes are encased in a protein capsid that may or may not be enclosed in a protective membrane, so the sensitivity of various free viral species to plasma application should vary. Interestingly, while several groups have demonstrated free viral inactivation, intracellular virus is plasma resistant [236], an indication of the limitations of plasma inactivation.

Proteins may be imbedded in membranes or free in the cytoplasm. ROS may generate many temporary and permanent amino acid modifications, the peptide backbone can be cleaved, and the protein can aggregately react with the oxidation products fatty acids and carbohydrates. Several groups have demonstrated protein degradation [242] or a loss in enzymatic activity after plasma application [242–244]. Proteins within cells may be more sensitive to plasma application than on surfaces [245].

The interaction between superoxide and nitric oxides produces a wide array of reactive molecular species that can deaminate DNA, cause abasic sites, strand breaks, and cross-link and induce reactions with proteins and with metal cofactors [246]. Direct exposure to plasma degrades DNA [242, 245]. In addition, oxidative DNA damage markers significantly increase with plasma exposure [238].

All cells possess protective enzymes such as catalases, peroxidases, superoxide dismutase, scavengers including glutathione, and stress response genes to combat reactive species. A number of pathways are devoted to DNA repair by reversal, removal, or repair of damaged bases. Genetic studies in Escherichia coli [247], the yeast Saccharomyces cerevisiae [248], and Bacillus subtilis [245] elucidated the important antimicrobial constituents. Oxidative stress appears to be the major antimicrobial component, but osmotic stress and heat also contribute to inactivation. Damage markers for nearly all cell components, the membrane, cell wall, DNA, and protein are upregulated. Clearly, multiple plasma components participate in cell inactivation.

Potential applications of the antimicrobial properties of direct or indirect plasma delivery include surface, air, and liquid decontamination and medical and dental antimicrobial treatments. The next section highlights a small number of recent microbial decontamination studies from a considerable body of literature.

6.7.4 Microbial Decontamination on Surfaces

One primary application of surface inactivation is environmental decontamination. In healthcare environments, an important reservoir of nosocomial pathogens related to transmission is inanimate surfaces, including hospital room surfaces and medical plastics. Many planktonic clinically relevant bacteria, yeasts, and one viral model, a bacteriophage, have been inactivated by plasma application on plastics, glass, laminate, linoleum, cloth, and steel [212, 237, 249, 250]. Bacterial cells in biofilms are protected by a bacterially produced polymer matrix that makes them more environmentally resistant than planktonic cells. In spite of this, these cells are significantly inactivated by plasma application on plastics, glass, and stainless steel [234, 251–257]. Spores are particularly difficult to inactivate, producing varying results [250, 258]. The mechanisms of plasma microbial inactivation appear to be independent of bacterial antibiotic resistance status [229, 237, 255, 256, 258, 259].

Biological residues on medical instruments and consumables may also pose an environmental hazard. An interesting potential use of plasma is the decontamination of biological residues on medical instruments and consumables. Prions, or infectious misfolded proteins [260], normal protein [261], and amyloid [262] are reduced substantially by plasma application.

Other applications of plasma decontamination are reviewed elsewhere in this book. A burgeoning area of research for microbial inactivation by plasmas is in food preservation, particularly in fresh produce and meats [228, 261, 263, 264]. Since plasma application to food can cause discoloration and other undesirable effect, one group has explored “gentle” plasma application parameters [265].

Bacteria, yeasts, and molds on biological surfaces such as corneas, dentin, and skin are inactivated by various plasmas [219, 221, 233, 236, 259, 266–269]. Dental and medical applications are discussed elsewhere in this book including wound decontamination, which has reached clinical trials [270]. Decontamination of biological surfaces may be more challenging than inanimate surfaces since plasma modification of the surface itself may be more detrimental.

6.7.5 Microbial Decontamination in Air

Experimental design to confirm air decontamination is a daunting task. Aerosolized bacteria, fungal spores, bacteriophages, or viruses can be inactivated after capture on a HEPA filter [271], contained within a chamber [272] or in closed circulation systems [273–275]. In a fascinating study, nebulized airborne human pathogenic respiratory viruses were inactivated to a higher level by a combination of gas plasma and UV than by either component alone [276].

6.7.6 Microbial Decontamination in Liquids

Vegetative bacteria [277–280], bacterial spores [281], and bacteriophages [275, 282] have been significantly inactivated in medium, broth, or water. Enteropathogenic E. coli O157:H7 was in activated in apple juice [283] and an E. coli model in milk [284]. One concern of plasma inactivation in liquids is potential for a significant pH decrease with exposure, which would potentially affect beverage flavor or modify a liquid’s composition.

6.7.7 Conclusions

Multiple studies confirm that plasma has many biological and biomedical applications, including microbial inactivation on surfaces, in food and beverages, in air, and in medical and dental practices. Differences in antimicrobial sensitivity may not only be due to differences between organisms but also be due to differences in the employed plasmas and in the medium or surface used as a support for the inactivation. Inactivation on both biological and nonbiological surfaces can be less effective than on smooth agar plates; an obvious hypothesis for this lower inactivation is that a surface quality such as topography protects the organisms. For each plasma device, it is important to characterize the components that play a role in decontamination. A major future development area for plasma translation is the application-specific device design and scaling of plasma delivery devices.

6.8 Downstream Processing of Microalgae for Energetic Use

  • Wolfgang Frey &
  • Christian Eing 

6.8.1 Introduction

The depletion of petroleum-based fossil resources, the urgent need to reduce greenhouse gas emissions, and the increasing demand for fuels for the transportation and energy sector call for alternatives based on renewable sources. During the past decades, research and development has been intensified to replace fossil fuels like diesel and gasoline by biomass-derived products.

The production of first-generation biofuels, i.e., biodiesel and bioethanol, from oil- and carbohydrate-rich, agriculturally grown biomass turned out to evoke a strong competition with food industry for arable land resources. Moreover, the current amount of worldwide produced vegetable oil and fat might only cover about 10 % of the global diesel demand [285]. Second-generation biofuels are based on the conversion of energy crops, agriculture and forestry waste streams, and food industry residuals to fuels. This approach can indeed alleviate competition problematics, but also not satisfy the future demand for transportation fuels to any remarkable extent [286].

During the last decade, research and development efforts have been intensified on microalgae biomass, referred to as third-generation biomass. Although not being demonstrated on industrial scale for fuel production yet, it exhibits advantageous properties over conventional biomass that justifies its consideration as a sustainable feedstock for future biofuel production.

6.8.2 Microalgae: Third-Generation Biomass

Microalgae exhibit a high biomass yield per footprint which is two to five times higher compared to agriculturally grown biomass. Typical yields of efficient energy crops, e.g., miscanthus, are 20 t∙ha−1∙year−1 [287], whereas microalgae cultivation in closed systems, i.e., photobioreactors [288], can yield up to 70–100 t∙ha−1∙year−1 [289]. Cultivation in photobioreactors saves resources, such as water and nutrients, and enables biomass production on arid or barren lands, thus avoiding competition with food production and preventing degradation of soil as evident for intensive energy crop agriculture.

Microalgae can accumulate high amounts of lipids . The lipid content of some species, e.g., Botryococcus braunii, can exceed 70 % of weight [290] when cultivated under nitrogen depletion. Unfortunately, for these cultivation conditions, biomass production rates are poor. The lipid content of microalgae still exhibiting satisfying biomass growth is on the order of 20 % of weight. For comparison, the oil content of rape is only 5 % in maximum related to the entire biomass [291]. Thus, conservatively estimated, the possible oil yield per footprint from microalgal biomass 0.2 70 = 14 t∙ha−1∙year−1 is more than ten times higher compared to rapeseed, which allows lipid yields of 1.2 t∙ha−1∙year−1. Optimistic scenarios even estimate oil yields of more than 50 t∙ha−1∙year−1 [292].

Besides lipids, microalgae produce other valuable components as proteins, carbohydrates, antioxidants, and vitamins [293, 294]. Altogether the content of value-added products (including lipids) of microalgae biomass exceeds 80 %, which is a value much higher than for conventional biomass. Nevertheless, multiple component recovery from microalgae is still a biotechnological challenge, since all these components are stored intracellularly and they are protected by a rigid cell wall.

6.8.3 Some Energetic and Economic Numbers for Microalgae Production and Processing

Compared to agriculturally grown biomass, a photobioreactor-based production of microalgae biomass is more expensive. For northern irradiation conditions and based on production systems already operating at commercial intermediate scale, the costs for 1 kgdw of dry (dw, dry weight) microalgae biomass range between 4 and 5 € [295]. Important cost factors [296, 297] are light transfer, mixing of the microalgae suspension in the photobioreactor to prevent sedimentation, C02 supply, nutrients, and dewatering. Investment costs for photobioreactors and labor costs for maintenance and operation are noticeable and have to be kept on a minimum extent [316]. On industrial scale, for a 100 ha production site, these costs are forecasted to be reduced to 0.68 €/kgdw, which appears to be reasonable for an economic microalgae-based biofuel production [295]. This scenario already involves the use of flue gas as C02 source, wastewater for nutrient supply, and irradiation conditions being available in southern European countries.

From the energetic point of view, the production of 1 kgdw of microalgal biomass with flat panel photobioreactors consumes lowest 10 MJ of energy [298]. Depending on the lipid content, the energy (enthalpy of combustion) stored in microalgae ranges between 20.6 and 26.7 MJ/kgdw [299]. The lower value was obtained from microalgae exhibiting a lipid content of about 20 %. In consequence, almost half of stored energy of microalgae biomass is consumed by cultivation.

As a first step for efficient downstream processing , microalgae have to be concentrated from a biomass density out of the photobioreactor, i.e., 2–5 gdw/l, to a density of 150–200 gdw/l. Dewatering of cell suspensions is commonly done by batch or continuous centrifugation methods on bench scale. Some energy values for centrifugation of microalgae biomass given in literature are 0.95 kWh/kgdw [www.evodos.eu] and 8 kWh per m3 of centrifuged microalgae suspension [300], which corresponds to 6 MJ/kgdw (1 kWh = 3.6 MJ) for a cell density of 5 gdw/l and can reach up to 10 MJ/kgdw [289]. They additionally depend on strain, processing scale, targeted final density of the biomass wet paste, and, last but not least, the tolerable content of small debris in the supernatant. This makes clear that the number of biomass washing steps and concentration steps has to be restricted to a minimum extent along the complete microalgae processing chain. Furthermore, for large-scale energetic use of microalgae biomass, centrifugation is agreed to be too energy intensive [301]. Biofuel production scenarios pursue flocculation combined with pressing, optimistically requiring less than 0.5 MJ/kgdw on a 100 ha production scale [302].

After harvesting and dewatering of microalgae to a microalgae paste, which then exhibits a dry biomass content of about 200 gdw/l, subsequent processing alternatives split up into dry-route and wet-route processing. In the first case, the energy for evaporation of water, i.e., 7 MJ/kgdw, has to be expended for biomass drying. Already at this point, it immediately can be realized that wet-route processing of microalgae paste has to be the most appropriate choice for a targeted energetic use.

For efficient recovery of intracellularly stored components, a cell disintegration step is needed. Among microwave or enzymatic treatment, ultrasound sonication, and ball milling, high-pressure homogenization and ball milling were demonstrated to be efficient in conventional microalgae processing. High-pressure homogenization has developed to a standard cell disruption method for ranking the efficiency of alternative methods. Recently bead milling was reported to require less than 2.5 kWh/kgdw [303]. Energy consumption values from literature for high-pressure homogenization vary over several orders of magnitude, starting from some kWh/kgdw and ending up at values of more than 100 kWh/kgdw [304]. It has to be emphasized that the cell disruption efficiency of high-pressure homogenization and bead milling highly depends on biomass density in the suspension to be treated. For constant treatment conditions, the fraction of disrupted cells decreases at higher cell densities.

After cell disruption, microalgae-based fuel production requires a solvent extraction step for lipid recovery followed by a refinement step and subsequent transesterification to biofuel [305]. Processing data on larger scale for lipid conversion into biofuels are rare in literature. Nevertheless, fatty acid profile determination revealed that common species are suitable for refining to biodiesel allowing the use of currently available refining technologies [292]. Nowadays, the direct transesterification of microalgae biomass is discussed to be a promising conversion route and has been demonstrated on pilot scale [306]. Under realistic conditions on pilot scale, the yield on fatty acid methyl esters (FAME, biodiesel) was determined to be 15 % related to initial microalgae biomass exhibiting a lipid content of 17 %wt [307]. On pilot scale, realistic overall production costs for biodiesel by direct transesterification of microalgae biomass at current state of technology are close to 30 €/kg [300]. Projections into near future allow expecting a cost reduction by a factor of 10 for microalgae-based fuel [308, 317]. Without doubt, microalgal-based biofuels will be a sustainable future source for transportation fuels, but nowadays can only become feasible if additional products and components except lipids can be valorized [309]. Therefore and as agreed in the community, energy-efficient and fractionating downstream processing methods are mandatory.

6.8.4 PEF Processing of Microalgae Biomass

Pulsed electric field treatment, involving plasma membrane permeabilization as basic biophysical process, is an efficient wet-route processing technique exhibiting fractionating properties. When treating microalgae suspensions of A. protothecoides, pre-concentrated from cultivation density of 5 to 100 gdw/l, with rectangular 1 μs pulses of an electric field strength of 34 kV/cm, an increase of suspension conductivity due to ion release by a factor of 1.6–2 can be observed immediately at the outlet of the flow treatment chamber, indicating efficient membrane permeabilization. In this case, 15 % of the total biomass could be released into the extracellular medium right after PEF treatment [310]. This water-soluble fraction contains salts, sugars, amino acids, and water-soluble proteins. Without washing step between microalgae harvesting and PEF treatment, the conductivity of the pre-concentrated microalgae suspension was 1 mS/cm. Under these conditions, an increase in component release could already be obtained for specific treatment energy values of 50 kJ per kg of treated suspension (kJ/kgsus). A saturation of the yield of water-soluble components was observed for energies higher than 150 kJ/kgsus.

An additional waiting time of 2 h after PEF treatment further increased the yield of water-soluble components up to 20 % [313]. After that time, no further increase could be obtained for A. protothecoides.

Intracellularly stored oil consists in bodies with an average diameter of ~1 μm [311]. Mainly due to their size and their hydrophobic character, oil bodies cannot pass cell wall and permeabilized membrane and remain intracellular. After separation of the water-soluble fraction, lipids were extracted with ethanol from the residual, lipid-rich biomass fraction. At a specific treatment energy of 150 kJ/kgsus, the lipid yield from the PEF-treated residual fraction was three to four times higher, compared to the untreated sample, recovering more than 80 % of the stored lipids on average [312]. This indicates that PEF treatment not only improves component transport to the extracellular medium but also solvent access into the cell by a single PEF-treatment step.

Contrary to the curve progression of the yield of water-soluble products over treatment energy, which shows a steep initial increase at low energies and saturation behavior at values above 150 kJ/kgsus [310], the energy dependence of the lipid yield is different. Up to values of 75 kJ/kgsus, the yield increase is small and can be neglected. A steep increase in lipid yield can be observed at 100 kJ/kgsus and higher [312]. These results indicate that an improvement of intracellular solvent access requires higher treatment energies, whereas water-soluble components already can be released under milder treatment conditions.

Measurements on the influence of pulse protocol on ethanolic extraction yield of intracellular components, i.e., carotenoids and chlorophylls, from Chlorella vulgaris, revealed that pulse shape and duration can impact extraction efficiency [313]. Comparable extraction yields could be achieved with monopolar 3 μs pulses at a specific treatment energy of 17 kJ/kgsus, whereas for 1 ms bipolar pulses, 150 kJ/kgsus were required. This compares well with our findings on C. vulgaris. Compared to A. protothecoides, the required energy for increased lipid recovery from C. vulgaris is lower and amounts to 25 kJ/kgsus. In general, the required specific energy for effective PEF treatment depends on the algae strain under consideration and has to be determined individually. For instance, PEF treatment of Neochloris oleoabundans within an energy range of 25 kJ/kgsus and 150 kJ/kgsus did not influence component yield at all, neither in lipid yield nor for water-soluble component release.

Treatment energies for microalgae processing are related to one kilogram of dry biomass. In the case that the required treatment energy is 150 kJ per kilogram of treated suspension (kJ/kgsus) and the biomass density of the pre-concentrated suspension amounts to 100 gdw/l, the specific energy value related to dry biomass is 1.5 MJ/kgdw. Since treatment energy is coupled to the suspension conductivity, the specific energy consumption can be reduced by increasing the cell density in the suspension to be treated, provided that the extraction efficiency is not affected. Experiments on the influence of biomass density in the suspension to be treated on the component yield have shown that the extraction efficiency did not decrease at higher biomass densities up to 160 gdw/l [310]. Consequently, microalgae suspensions of 200 gdw/l, which are still well pumpable, require a specific treatment energy of 0.75 MJ/kgdw, which is considerably lower compared to conventional processing methods. Moreover, PEF treatment does not produce cell debris, which facilitates subsequently required separation processes.

These merits of PEF processing can satisfy the demand for a low-energy-consuming technology for cascade valorization of microalgae biomass for energetic use. PEF-assisted fractionating component recovery allows for partial compensation of the comparatively high-energy demand for cultivation by simultaneous valorization of both higher-value water-soluble products and lipid-rich residual biomass. Furthermore, the fractionating character of PEF processing opens several energetic processing alternatives for microalgae [314] to be investigated in the future.

As a first valorization step, water-soluble proteins can be separated as already demonstrated for Nannochloropsis [155], C. vulgaris, and H. pluvialis [315]. In subsequent steps, either lipids can be extracted from the residual lipid-rich biomass or the lipid-rich biomass fraction may be fed into other energetic valorization pathways, i.e., thermochemical conversion or biogas production. The challenge for future R&D work will be to identify the appropriate pathway exhibiting the highest economic and energetic gain. Since energy consumption of PEF treatment is very low, it undoubtedly will advance to an important tool promoting the energetic valorization of microalgae.

6.9 Protein Electroextraction from Microorganisms (Bacteria, Yeasts, Microalgae)

  • Justin Teissie 

It will describe that long (ms) pulses affect the cell membrane and cell wall in such a way that a slow release of proteins is induced. A flow technology is available and is described. The cell remains macroscopically intact allowing an easy separation without the formation of debris.

6.9.1 Introduction

Walled microorganisms are cell factories. Among the many systems available for heterologous protein production, the Gram-negative bacterium Escherichia coli remains one of the most attractive ones because of its ability to grow rapidly and, at high density on inexpensive substrates, its well-characterized genetics and the availability of a large number of cloning vectors and mutant host strains [318, 319].

Yeasts are widely used for industrial production of homologous proteins, most of them with intracellular location. Nowadays, Saccharomyces cerevisiae as well as yeasts from genera Kluyveromyces, Pichia, and Hansenula have become a suitable host for industrial production of recombinant proteins with high biotechnological and pharmacological values [320, 321]. Their secretion from the cell is the optimal way for isolation, but often this is impossible or of low efficiency. Thus, the newly synthesized heterologous proteins remain accumulated in the cell cytoplasm.

The simple growth requirements of microalgae made these microorganisms attractive bioreactor systems for the production of high-value heterologous proteins. The cultivation could be achieved in photobioreactors (PBRs) which provided safe control of culture environment. It was possible to produce recombinant proteins either in cytosol or chloroplast of microalgae [322]. A promising development was given by the production of unique immunotoxin cancer therapeutics in algal chloroplasts [323]. Extraction of proteins was hindered by the cell wall barrier. Only a slow release was observed on the microalgae having a rigid cell wall, and therefore, disrupting the rigid cell wall of C. vulgaris was required to obtain a complete protein release after extraction [324].

The main methods utilized for liberation of intracellular enzymes are mechanical disintegration and chemical extraction [325, 326]. Although applicable on a large scale, these are relatively drastic procedures, which affect the stability of the proteins or introduce additional impurities to be removed in the associated downstream processes. The running costs are high. Formation of debris is a problem for the downstream purification. Thus alternative methods for enzyme extraction are investigated to ensure a higher selectivity of release and mild experimental condition preserving in maximal level the enzyme activity.

Pulsed electric fields are described as one of the most promising approaches. Long pulses (ms long) appear to permeabilize the plasma membrane and to induce a structural change in the wall. As a final consequence, a slow release of soluble cytoplasmic proteins is obtained. Field conditions can be adjusted in such a way to leave the vacuole intact, to prevent the release of proteolytic enzymes. The proof of concept of the flow process protocol to treat industrially significant volumes was previously validated [149] (Fig. 6.24).

Fig. 6.24
figure 24

Flow process for electroextraction. The microorganisms are washed and pumped across the pulsing chamber. A train of pulses are delivered by the pulse generator, and their voltage and current are monitored on line. The pulsed flow is collected and incubated in the appropriate incubation buffer. The extracted proteins are treated downstream from the supernatant

Full size image

Flow process electroextraction was indeed patented to the CNRS (FR # 0013415; Euro/PCT # 1982525.6). A preindustrial pilot was developed in our group during the FP7 “Electroextraction” project ([FP7-SME-2007-1], Grant agreement n°222220) and is now commercially available.

6.9.2 Electroextraction by a Flow Process

6.9.2.1 Description

The basic concept was to apply calibrated electric pulses with a delivery delay which was linked to the flow rate (Figs. 6.24 and 6.25). The desired number of pulses was actually delivered on each cell during its residency in the pulsing chamber. The geometry of the chamber (flat parallel electrodes) was chosen to give a homogeneous field distribution. Therefore, the residency time Tres of a given cell in the chamber was

$$ \mathrm{Tres}=\mathrm{V}\mathrm{o}\mathrm{l}/\mathrm{Q} $$
(6.1)

where Vol was the volume of the pulsing flow chamber and Q, the flow rate. The number of pulses delivered per cell was

$$ \mathrm{N}=\mathrm{V}\mathrm{o}\mathrm{l}/\mathrm{Q}\mathrm{P} $$
(6.2)

P being the period of the pulses. The field strength was

$$ \mathrm{E}=\mathrm{U}/\mathrm{d} $$
(6.3)

d being the width between the two electrodes and U the voltage.This average power associated to the train of pulses was

$$ \left\langle \mathrm{P}\mathrm{o}\mathrm{w}\right\rangle =\mathrm{U}\mathrm{I}\mathrm{T}/\mathrm{P} $$
(6.4)

T being the single-pulse duration and I the current.As the chamber resistance, when filled by the sample, could be approximated by

$$ \mathrm{R}=\mathrm{d}/\left(\varLambda \mathrm{S}\right) $$
(6.5)

where Λ is the conductance of the sample and S the section of the electrodes, then

$$ \left\langle \mathrm{P}\mathrm{o}\mathrm{w}\right\rangle =\mathrm{T}{\mathrm{E}}^2\varLambda \mathrm{V}\mathrm{o}\mathrm{l}/\mathrm{P} $$
(6.6)

From Eq. 6.2, an increase in the flow rate Q while keeping the number of applied pulses N constant needs to increase Vol/P, i.e., either increase in Vol or decrease in P (or both). From Eq. 6.6, more power is needed.

Fig. 6.25
figure 25

Train of pulses. A positive voltage pulse (intensity U1, duration T1) is followed after an interval Ti by a negative voltage pulse (intensity U2, duration T2). This is repeated at a period P to limit electrochemical reactions, U1T1 = U2T2. In practice U1 = U2 and T1 = T2

Full size image

One other physical problem is that Λ increases with the temperature, meaning that due to the Joule heating, the current increases during the pulse and from Eq. 6.6 more power than predicted is needed.

6.9.2.2 Specifications of the Pulse Generator

Long square wave pulses, not an accumulation of short pulses, are needed to obtain cytoplasmic soluble protein extraction [157, 327]. The voltage to be delivered must be adjusted to the microorganism and to the pulsing chamber to obtain a field strength critical to trigger the plasma membrane permeabilization. To keep the required voltage at a high current during the pulse duration (Eq. 6.4), a significant electrical charge must be stored in the capacitors to be partly delivered in the biological sample. To obtain a high frequency in the delivery of pulses in the train needed with the flow process, the power supply that provides the charge to the capacitor needs to be designed large enough.

The use of long electric pulses is associated to a technical drawback: electrochemical reactions are occurring at the surface of the electrode. Formation of bubbles of H2 and Cl2 are observed. Electro-erosion of the electrode surface is present (details are given in Chap. 2). This can be prevented to delivering trains of pulses with alternating polarities with a short (about 10 ms or less) delay between each.

The DeexBio pilot was therefore designed to provide trains of bipolar pulses (Betatech, France). The electric system was made of two S20u generators each able to deliver up to 2 kV under 10 A (maximum for safety reason), with adjustable pulse duration of a few milliseconds. Delays between pulses were down to 30 ms. An analog switch working at 15 ms was used to connect the two generators with the pulsing chamber. All electrical settings (T1, Ti, U1, and P) were selected from the touch screen. Current and voltage were monitored online.

6.9.2.3 Pulsing Chambers (Applicators)

The pulsing chamber was built of several parts, the most important being the discharge chamber. A parallel plate configuration was chosen to obtain a homogeneous field. Two geometries were designed to obtain fields up to 6 kV/cm.

For microalgae or yeast, the distance between electrodes was 6 mm, the height was 6 mm, the length was longer than 1 cm, and the volume was up to 1.08 ml. The other applicator, used for bacteria or smaller microalgae, had a distance between electrodes reduced to 3 mm, the width was 5 mm, and the volume was 150 μl.

A laminar flow was present where the velocity of the microorganisms is not uniform. To obtain a more homogeneous treatment, several chambers are connected in series (see Fig. 6.26) with a remixing of the microorganism suspension. This feature limits the Joule heating [328].

Fig. 6.26
figure 26

The Deex Bio. The two pulse generators (yellow) deliver voltage with alternating polarities on the pulsing chambers (gray, two in series). The flow of cells is pumped at a controlled flow rate (pump on the right). The pulsed cells are collected in the reservoir on the left. Temperatures are controlled at different levels to avoid overheating. The pulses are monitored online on the PC (in the back) (Picture by courtesy of Beta tech)

Full size image

6.9.3 Electroextraction Protocols: A General Approach

To limit the current delivered along the pulse application, there is a need to work with a suspension in a low conductivity buffer. Cells are therefore washed in distilled (or at least detergent-free) water to a final conductance of about 0.2 mS/cm.

The cell concentration is limited by the electropermeabilization-induced release of the cytoplasmic ions that induces a fast increase in the suspension conductivity. This increase is a linear function of the cell concentration.

After the pulse train delivery, the cell suspension is diluted in a salty solution containing DTT (PB/DTT) to weaken the S-S bonds and the electrostatic interactions present in the wall.

The suspension is incubated at room temperature during several hours. Under the microscope, it is observed that the morphology of the pulsed cells is not affected when compared to controls. Separation of the supernatant where the released proteins are from the pulsed cells is easy as no debris are present. This can be obtained by a mild centrifugation and does not affect the downstream separation by chromatography [329].

Permeabilization was assayed by the propidium uptake and from the increase of the conductivity of the pulsed solution.

Protein extraction was assayed by the Coomassie blue assay. Due to the excretion process, protein release was observed in controls when the postpulse incubation was long (overnight).

SDS-PAGE of protein samples was performed on 12 % acrylamide slab gel as described by Laemmli [330]. The silver staining of gels as described by Nesterenko et al. [331] was made with a protein molecular weight marker (such as Page-RulerTM Prestained Protein Ladder, Thermo Scientific). Most of the bands appeared between 35 and 170 kDa. The same bands appeared both on negative controls (due to excretion) and samples, with a higher intensity for the samples incubated in water or phosphate buffer (PB/DTT)(PB 105 mM, 0.3 M Glycerol, 1 mM DTT, pH = 7). Several new proteins appeared after electroextraction in comparison to the negative controls.

Protein activity was assayed by routine-specific protocols. 3-phosphoglycerate kinase (PGK)(45 kDa) activity was determined according to Kulbe and Bojanovski [332], glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (37 kDa) according to Kirshner and Voigt [333], hexokinase (a dimer of 100 kD) according to McDonald [334], and β-D-galactosidase (a 464-kDa homotetramer) according to Mbuyi-Kalala et al. [335]. Protease activity was determined according to Meussdoerffer et al. [336].

6.9.4 Electroextraction Protocols for Bacteria

Electroextraction has been proposed as a method to recover cytoplasmic content from bacteria [337–339]. The following protocols are given for E. coli.

6.9.4.1 Microorganisms and Cultivation Conditions

Escherichia coli BL21 and DH5α strains were used.

Cells were grown in LB, 0.5 % (w/v) glucose. After reaching appropriate growth phase, the cells were collected by centrifugation at 4000 × g for 10 min, washed once with deionized water, and resuspended to a final concentration corresponding to 5 % wet weight.

Viability after the different PEF treatments was obtained by counting colony-forming units (CFU). After discharge delivery, the suspensions were removed and incubated for 1 h at 37 °C. The bacterial suspensions were diluted, and 100 μl samples from selected dilutions were spread onto Petri dishes for incubation overnight at 37 °C.

6.9.4.2 Results

Extraction of GAPDH from cells of strain BL21 in exponential growth phase was obtained when cells were treated with 15 pulses, 4 Hz, 0.5, and 1 ms duration, followed by a postpulse incubation at 30 °C. Maximal release was obtained for GAPDH at field intensity of 7 kV/cm and pulse duration of 0.5 ms. In control cells, the incubation in buffer provoked the liberation of only 25 % of GAPDH (half less than in pulsed cells).

The upper limits in the field strength were detected by formation of precipitates and a decrease of GAPDH activity tested. This may be due to an increased Joule heating during electric treatment. Similar precipitation was observed with the application of pulses with 1 ms duration at field intensities over 5.75 kV/cm or of pulses of 0.5 ms duration but over 7.25 kV/cm.

Extraction was strain dependent. The optimal conditions for electroextraction from DH5α were the same as those for BL21. But cells of this strain were less sensitive to electric fields, in comparison with BL21. A lower extraction (GAPDH and PGK) was obtained.

6.9.4.2.1 Influence of Incubation Buffer Content

The buffer content for postpulse incubation was found to be crucial for protein recovery. The common buffer for cell lysis and protein extraction with E. coli is Tris buffer pH = 8–8.5. Very often additional components such as EDTA and DTT are present.

6.9.4.2.2 Influence of the Growth Phase

The cell wall porosity strongly depends on growth phase. We checked the influence of growth phase on the release of intracellular enzymes.

The release of PGK in the middle and late exponential phase cells was considerably higher than for GAPDH. This might be due to its lower molecular weight, thus demonstrating some sieving selectivity of protein electroextraction. The electro-induced release of enzymes from cells in late exponential growth phase was only 50 % of what was obtained in the middle exponential phase. When the cells were in stationary growth phase, the electric field was completely inefficient.

6.9.4.2.3 Increase in Sensitivity to Lytic Enzymes

Electric field pulse in bacteria not only permeabilized plasma membrane but also affected other cell envelop components. Pulsed and control cells were incubated for 30 min in medium containing lysozyme, in concentrations considerably lower than that used for cell lysis . As a result of this mild lytic treatment, the release of GAPDH and PGK of pulsed cells was up to 85–86 % of total activity for both enzymes. The effect on control, unpulsed cells was present but distinctly lower—only 28 % of total activity.

6.9.5 Electroextraction Protocols for Yeasts

Cytoplasmic proteins can be extracted from yeasts by electropulsation (pulsed electric field technology, PEF) [149, 327, 340–344].

Protein release from the cells is a slow process occurring along several hours during the incubation in the specific buffer.

Flow process treatment of yeasts (Saccharomyces cerevisiae), with high-intensity electric field pulses, allows the release of the intracellular protein content on large culture volumes [149, 343].

The extraction yield is dependent on the field strength (to obtain plasma membrane permeabilization), pulse duration (several ms is more effective than subms), and the number of successive pulses. Optimization is cell strain dependent. DTT brings a significant increase in extraction due to the effect on the wall [345].

Extraction from Saccharomyces cerevisiae was obtained with 15 pulses of 2 ms at 6 Hz. Maximal yield for glyceraldehyde-3-phosphate dehydrogenase (GAPDH , 145 kDa) (85 %) was obtained at 3.2 kV/cm where all cells were permeabilized. About 58 % from cell activity was liberated within 1 h after pulsation. The maximal release of two other cytoplasmic enzymes 3-phosphoglycerate kinase (PGK, 45 kDa) and hexokinase (HK, 100 kDa) was obtained during the same time period.

6.9.5.1 Growth Phase

Cells in stationary phase are frequently used as source for production of enzymes. Previous data with a batch process showed that the growth phase determined the effect of electric pulsation. Electroextraction of protein using cells in stationary phase (5 × 108cells/ml) was obtained. When applying 15 pulses (2 ms duration, 6 Hz), maximal yield of GAPDH 4 h after pulsation (85 %) was obtained at 3.5 kV/cm. While at 3.2 kV/cm, which was optimal for cells in exponentially phase, the activity of GAPDH in the supernatant was only 65 % from the total.

6.9.5.2 Protein Stability

The extractable total protein for different pulse length/intensity/frequency combinations remained limited to about 37–45 % from the total content. But the specific activity of three electroextracted enzymes (GAPDH, PGK and HK) was about two times higher than that was obtained in cell extracts, from either after enzymatic lysis or mechanical grinding.

An increase of the intensity above the optimal value (established for a given cell concentration) provoked a decrease of enzyme activity—probably a thermal denaturation. This is a critical event as an increase of intensity from 2.7 to 3 kV/cm on a 20 % wet weight suspension resulted in about 90 % decrease of extracted GAPDH activity.

The proteins from crude extracts and supernatants of electrically treated cells were analyzed by SDS–polyacrylamide gel electrophoresis under reducing conditions. Most of the bands above 29 kDa present in the lysate, even those with highest molecular weights (proteins over 97 kDa), were quite similar in both electropulsated and mechanically broken cells.

6.9.5.3 Vacuole Preservation

In yeast the main protease activity is concentrated in the vacuole, which is affected by classical extraction methods. But when using electrical conditions giving 100 % permeabilization, and isotonic postpulse medium, the liberated protease activity at 4 and 6 h after pulsation was only about 15 % and 20 % from what was present in the extract after classical lysis. The preservation of vacuole integrity was checked by a classical Lucifer yellow (LY) staining of pulsed cells. Electropermeabilized cells incubated in isotonic medium showed fluorescence only in the cytoplasm, the vacuole remaining unstained—showing that the vacuolar membrane was intact.

6.9.5.4 Cell Concentration

The possibility for treatment of more concentrated suspensions—up to 20 % wet weight (2.2 × 109 cells/ml)—was checked by assaying GAPDH extraction. Results were that when using shorter pulses, i.e., 1 ms, one could obtain 85 % extraction at lower field intensity—2.7 kV/cm. At this intensity (15 pulses, 1 ms duration, 6 Hz), only about 40 % IP could be achieved when pulsing the more diluted 4.5 % wet weight (5 × 108 cells/ml) suspension. At the high concentration, the optimal intensity was 4.3 kV/cm, i.e., the five time increase of cell concentration is associated to an about 1.6 reduction of optimal field intensity.

These effects could be attributed to the increased conductivities of the suspensions during the pulse application, as a result of higher inorganic ion outflow. The conductivity of 4.5 % and 20 % wet weight suspensions before pulsation was about 40 and 100 μS/cm, respectively. Immediately after pulsation (30s), these values were ten times greater. Thus, the concentrated yeast suspensions were treated with higher currents. The technical limit is that the current that was delivered is higher (reaching the limits of the specifications of the pulse generator) and the Joule effect is larger (risks in thermal denaturation).

6.9.5.5 Postpulse Conditions

It was observed that the incubation of pulsed cells at room temperature did not influence the efficiency of extraction (only about 3 % decrease in GAPDH and PGK leakage as compared to 30 °C). This is a positive advantage in running costs.

The presence of glycerol and DTT in the postpulse incubation medium contributed to higher GAPDH (about 15 %) and PGK (about 20 %) activities in the supernatant of electrically treated cells but did not influence hexokinase activity, 80 % of HK activity being recovered within 4 h (data not shown).

6.9.6 Electroextraction Protocols for Microalgae

6.9.6.1 Panel of Strains

C. vulgaris and H. pluvialis are growing in freshwater, while Dunaliella salina and N. salina are marine species. All have a rigid cell wall.

Culture conditions were in photoreactors. Growth was checked by measuring the density of algae under an inverted microscope with a Malassez slide.

6.9.6.2 Results

It should be kept in mind that microalgae are indeed photosynthetic yeasts. Electroextraction protocols were rather similar. Protein release was a slow process (h) following the very fast pulse delivery (<s) [317].

6.9.6.3 Influence of the Electric Field on Protein Electroextraction

The extraction increased with the increase in the field strength (always with a single-pulse duration of 2 ms). Extraction efficiency was dependent on the field strength in a specie-dependent manner. Fields of 3 kV/cm were efficient on C. vulgaris and H. pluvialis, while 6 kV/cm was needed for N. salina due to its smaller size.

6.9.6.3.1 Influence of the Number of Pulses and of the Delay Between the Train of Pulses

A systematic evaluation of the effect of accumulating pulses showed that the amount of released proteins was increased with the number of successive pulses, whatever the field strength applied by the applicator.

A highly efficient protein release was obtained with one cycle of 15 bipolar pulses. There was no improvement in extraction when cycling again the microalgae (C. vulgaris) suspension.

6.9.6.3.2 Pulse Duration

Pulse duration is clearly a leading factor to obtain protein extraction from walled species. Microsecond long pulses were not inducing the protein release even if the cumulated application time lasts several ms. A slight modification of the single-pulse duration, while keeping the cumulated pulse duration constant, did not affect the extraction efficiency. Thirty pulses of 1 ms gave the same amount of released proteins from Chlorella as 15 pulses of 2 ms. The 30 pulses were obtained by doing two passages, while such a protocol did not improve the extraction efficiency for pulses of 2 ms. The temperature increase linked to the Joule heating was reduced.

6.9.6.3.3 Post-Zap Dilution

In our previous results obtained on yeasts, there was a need for postpulse incubation in a salty buffer containing DTT. The pulsed sample (in a low conductivity buffer) was routinely diluted in 4 volumes of an incubation buffer (dilution factor of 5). But further industrial development, a one to one dilution (with a 1.7× concentrated extraction buffer), was observed to be as efficient with C. vulgaris and N. salina, after treating under their respective optimized conditions.

6.9.6.3.4 Kinetics of Protein Leakage After PEF

Protein leakage from microalgae happened in a time-dependent manner. A massive leakage was obtained during the first 30 min following the postpulse dilution, while no release of protein was observed in the control samples during that time. To obtain a more complete extraction, an overnight postpulse incubation was required. Due to endogenous excretion processes, a substantial amount of proteins was released during that long incubation period that was run at room temperature.

6.9.6.3.5 Postpulse Temperature

No obvious difference was observed between incubation at 4 °C and at 20 °C, whatever the duration of incubation.

6.9.6.3.6 Influence of the Cell Concentration

C. vulgaris at 105 cells/ml and 106 cells/ml were exposed to nine pulses of 2 ms at 3 kV/cm. After 24 h incubation at 20 °C, results showed that the increase of cell concentration increased linearly the extracted protein concentration. Pulsing more concentrated suspension brought the problem of the increase in conductivity due to the release of cytoplasmic ions.

6.9.7 Conclusions and Tips

The effect of electric pulsation is size dependent, and stronger electric treatment should be applied to obtain the same effect on smaller cells.

Pulse duration should be long (ms). Pulses should be delivered in a low conductivity buffer. The release of ions during the pulse delivery is a limit in the cell concentration to be treated. Joule heating (Temperature increase) can be reduced by using a series of pulsing chambers and coolers, using a larger number of shorter pulses with more successive chambers.

Postpulse incubation should be operated in an ionic solution containing DTT. The release of proteins during the incubation is a slow process (h.) but occurs at room temperature. Separation of the supernatant where the released proteins are from the pulsed cells is easy as no debris are present. The downstream separation by chromatography is not subject to clogging. No ultrastructural damages are observed.