The effects of electric, magnetic and electromagnetic fields on microorganisms in the perspective of bioremediation

Some studies show how exposure to fields can enhance or reduce cell activity, with possible applicative consequences in the field of biotechnology, including biological techniques for depollution. In order to identify full-scale conditions that are suitable and potentially applicable for use in electromagnetic fields to stimulate and accelerate bioremediation processes, this paper offers an examination of the scientific literature that is available on the effects of fields on microorganisms, and a critical analysis of it. The biological effects at times contrast with each other.


Introduction
The first studies on the influence of electromagnetism on organisms dates back to the end of the nineteenth century, to then intensify in the following decades after global electrification and the diffusion of telecommunication. The massive introduction into daily life of technologies that emit electric, magnetic and electromagnetic fields in an enormous range of frequencies and intensities led institutions and the scientific community to question itself about the effects on public health and the environment. Pertinent scientific literature is very vast and includes studies that vary greatly on the type of field, intensity, exposure duration, long-/short-term effects and considered biological targets (cell, tissue, organ and organism).
In contrast to the studies indicated above, the research that investigates the field effects on microorganisms is very limited. Model microorganisms, well characterised with genetic markers, were used in medical-health research to better understand the field action mechanisms. Some of these works showed how, in some situations, exposure to electromagnetic fields tends to enhance rather than reduce cell activity, with possible applicative consequences in the field of biotechnology, including biological techniques for depollution.
In order to identify full-scale conditions that are suitable and potentially applicable for use in electromagnetic fields to stimulate and accelerate bioremediation processes, this paper offers an examination of the scientific literature that is available on the effects of fields applied on microorganisms, and a critical analysis of it. In consideration of the objective of this document, aimed at environmental bioremediation, the effects of electromagnetic fields applied on cells, bacterial cells in particular, are focused on. The information obtained from the literature that was consulted is summarised in Tables 1, 2 and 3, respectively on nominal exposure to electrostatic fields/fields generated by direct current (DC), magnetic and electromagnetic currents/fields generated by alternate current (AC). The decision was made to treat electrostatic field applications (typically generated while maintaining a constant voltage between pairs of electrodes) together with fields generated by direct current because when the sources are applied to dielectric means (soil, wastewater, etc.) they produce similar effects. These effects are, in fact, so similar that even in most of the literature that was analysed, they are treated simultaneously, without any distinction between the two situations.
In consideration of the manuscript objectives, the effects indicated in the literature were divided into four different categories (''positive'', ''negative'', ''undefinable'' or ''null'') according to their possible implications on environmental bioremediation: 1. ''positive'' (?) effects: stimulation of the degradation of contaminants, increased denitrification/ nitrification activity, acceleration of the substratum consumption kinetics, increase in the resistance to pollutants, increase of the biomass, increase in metabolic activity or in the activity of specific enzymes (e.g. dehydrogenases); 2. ''negative'' (-) effects: reduction of the degradation of pollutants and/or substratum consumption, inhibition of bacterial growth, reduction in metabolic activity or in the respiration rate, damage to the cellular membrane. At times, these effects are not tied to the direct action of the field/current on the cells, but rather to modifications in the environmental conditions (e.g. extreme pH values, electrochemical production of toxic species, radicals, etc.) (''indirect negative''); 3. undefinable (''9''): in the absence of effects (1) or (2), modifications in the activity of enzymes that are not involved in the degradative metabolism, variation in the concentration of ATP, modifications to the microbial community (structure/diversity/genotype/morphotype), effects of mutagenicity, alterations to the cell proteome, synthesis alterations of the DNA/RNA and correlated activities, variations in the transposition and production of secondary metabolites, modifications in the cell form and the characteristics of the cell wall and its electrostatic charge, increased cell hydrophobicity, increased adhesion between bacterial cells, increased or reduced resistance to antibiotics; 4. ''null'' (=): absence of significant effects on the aspects indicated above.
2 Electrostatic fields and fields generated by direct current The first experiences in using fields that are electrostatic or generated by DC current to favour microbial growth date back to more than 50 years ago. They were based on the use of water hydrolysis to produce O 2 electrochemically, as a replacement to other aeration systems, in order to grow Pseudomonas fluorescens (Sadoff et al. 1956 in Thrash and Coates 2008) or the combined production of O 2 and H 2 to grow the hydrogen-reducing aerobic microorganism Ralstonia eutropha H16 (Schlegel et al. 1965 in Thrash and Coates 2008).

Studies on microorganisms
Regarding the direct effects on microorganisms of the field that is electrostatic/generated by direct current, the literature indicates possible modifications in the physiology and form of the cells, the chemicalphysical characteristics of the cellular membrane (Zimmermann et al. 1973) and membrane permeability and potential, with repercussions on its ability to exchange with the external environment, cell metabolism and mobility (Luo et al. 2005a;Golzio et al. 2004). The entity of these phenomena is a function of the species, but generally proportional to the intensity of the field/current applied and the duration of exposure. The results are stimulation of bacterial activity and increased cell mobility, observed in light intensity fields, as well as irreversible damage to the microorganisms, with a loss of membrane integrity, in the case of exposure to more intense electric fields (Sakakibara and Kuroda 1993;Satoshi et al. 1997;Chen et al. 2002;Diao et al. 2004;Zituni et al. 2014). Even though different species present different levels of sensitivity, high intensity electric fields, for example of 1000 kV/m, or circulating direct currents of 1 A damage the cells irreversibly and cause the          Superconducting magnet In the stationary phase, significant reduction (40-80%) of the bacterial decay rate in the samples exposed to a uniform field, 2.7-3.6 times higher in the samples exposed to a non-uniform field Punctual alterations of DNA in the in vitro tests  Superconducting magnet During the first 6 h approximately (start of the log growth phase), slight reduction in the growth rate. During the stationary phase, significant reduction in the bacterial decay rate (at 30 h, number of cells in the exposed samples 2-3 times greater than the control). Effect more pronounced with exposure to non-uniform than to uniform fields ? Tsuchiya et al. (1996Tsuchiya et al. ( , 1999      Electric line 5000 V Slight alteration of the cellular proteome in the exposed samples ? Baraúna et al. (2015) The field probably acted as a stress factor The magnetic field was a stress condition Reduction in the production of citrinin (toxic metabolite) at 1.2-1.6 mT; increased at 2 mT; no effect at 0.5-0.9 mT. The production of citrinin can increase, reduce or remain constant according to magnetic flux density, duration and exposure period; inhibition more evident with exposure during the initial growth phase  (Sale and Hamilton 1967;Park et al. 2003). Indeed, membranes that are exposed to an electric field become charged, in the same manner as a condenser, and this induces a potential transmembrane that, if greater than 1 V, causes cellular death. The bacterial mortality rate grows proportionally with the intensity of the field that is applied and the overall duration of exposure.

Studies in an environmental setting
Excluding extreme intensity values, a summary is given below of the environmental experiences carried out with modest intensity fields; this determines negative and/or positive effects, even contextual, on the microorganisms.

Beneficial effects
2.2.1.1 Electrochemical reactions Electrochemical reactions, for example various electro-oxidations and water hydrolysis, can increase the availability of oxygen or hydrogen, respectively favouring aerobic biodegradation processes (Mena Ramírez et al. 2014) and anaerobic biodegradation processes (She et al. 2006). Bioelectric systems based on water electrolysis were tested, in various configurations and with different operation parameters, for the removal of nitrogen from wastewater, mainly using the production of hydrogen at the cathode to promote denitrification reactions (Cast and Flora 1998;Feleke et al. 1998;Hayes et al. 1998;Mousavi et al. 2010Mousavi et al. , 2012, or oxygen at the anode to stimulate aerobic nitrification (Goel and Flora 2005a, b), or both processes simultaneously (Kuroda et al. 1996;Zhan et al. 2012). Variations in the pH and the production of H 2 induced near the cathode by a current of 20 mA were used to stimulate the activity of sulphur-oxidising bacteria (Jackman et al. 1999). She et al. (2006), in a bioelectrochemical system with a current of 10 mA, stimulated dehydrogenase activity thanks to the simultaneous production of O 2 and H 2 at the electrodes.
In addition to water hydrolysis, other phenomena tied to electrochemical reactions were recently identified, such as the partial oxidation/reduction of pollutants, the release or removal of ions in solution, the possibility of adjusting redox potential for activating/stimulating the production of specific enzymes, as well as variations in bacterial metabolism (Aronsson et al. 2001;Li et al. 2006;Huang et al. 2012).

Electron
exchange and enzyme production Some microorganisms were able to use electroactive soluble substances (for example iron and humic substances) (Lovley et al. 1996) or solid electrodes (Bond et al. 2002;Gregory et al. 2004;Aulenta et al. 2009) as donors/acceptors of electrons for substrata oxidation/reduction. Zhang et al. (2013) affirm that in bioelectrochemical tests the electrochemical assistance provided the electrons and accelerated the electron transfer rate in the microbial reduction of 2,4-dichlorophenoxyacetic. Zhang et al. (2014) observed an increase in the mineralisation efficiency of 2-fluoroaniline by an aerobic culture exposed to a direct current of 10-15 mA, as a result of the increased activity of the catechol dioxygenase and the selection of microorganisms with specific degradative abilities. Velasco- Alvarez et al. (2011), by applying a current of 8 mA for 24 h to a culture of Aspergillus niger, observed that the bacterial growth halved but that the hexadecane degraded more, causing the supposition of transition from an assimilative metabolism without the electric field to a non-assimilative one with the electric field.

Electrokinesis
Electrostatic fields generated by constant differences in potential (of the order of 100 V/m) applied between pairs of electrodes and fields generated by direct current are already being applied full-scale for decontamination with electrokinesis, to remove pollutants in sediments or soils (both saturated and unsaturated), especially if the particles are small (Acar et al. 1995).
The benefits of applying electric fields to soils/ sediments were initially related to activated transport mechanisms (electroosmosis, electromigration, electrophoresis and dielectrophoresis) (Alshawabkeh and Bricka 2000). Electroosmosis is the movement of the liquids present in the soil pores, generally from the anode to the cathode, under the action of an electric field, which promotes the migration of pollutants towards the cathode by advection (Acar and Alshawabkeh 1993;Acar et al. 1995). Electromigration is the movement of ionic species caused directly by the electric field. Electrophoresis is the transport of solids with a charged surface, for example bacteria or clay, towards the electrode with the opposite pole, while dielectrophoresis is the movement of neutron solids with a diameter of between 1 and 1000 lm because of induced polarisation (Pohl et al. 1978). These mechanisms determine the movement of organic molecules, nutrients, fluids and bacterial cells (Luo et al. 2005b;Wick et al. 2007) with a faster recovery of pollutants, mass transfers and interactions between pollutants, bacterial cells and nutrients, which is advantageous for the bioremediation processes.
Electrostatic fields can also cause pollutants to degrade partially with an increase in their bioavailability/biodegradability (Wick et al. 2007;Yeung and Gu 2011;Gill et al. 2014;Moghadam et al. 2016). Increases in temperature because of ohmic losses can accelerate the kinetics of bioremediation (Suni et al. 2007).
Experiences of electrokinesis with electric fields having an intensity of 20-200 V/m (Wick et al. 2007;Gill et al. 2014) showed better degradation for various classes of compounds, among which petroleum hydrocarbons due to changes in the microbial community structure (Probstein and Hicks 1993;Pazos et al. 2012), polycyclic aromatic hydrocarbons due to the transport of PAH-degrading bacteria in the medium (Pazos et al. 2010), organochlorine compounds due to changes in pollutant mobility (Gomes et al. 2012), and phenols due to variation in bacteria hydrophobicity and pollutant mobility (Luo et al. 2005a, b).

Negative effects
On account of the variety of processes induced by the electrostatic fields or generated by DC currents, inhibitive effects on biological activity were reported, mostly related to: (1) important variations in the pH (Fan et al. 2007;Yeung and Gu 2011;Gill et al. 2014;Ailijiang et al. 2016), above all near the electrodes (Lear et al. 2004;She et al. 2006); (2) electrochemical reactions, with the production of reactive species of oxygen, chlorine or metallic ions, according to the species present in the system and the materials used for the electrodes (Liu et al. 1997;Li et al. 2011); (3) excessive heating because of ohmic loss (Palaniappan et al. 1992;Shi et al. 2008). Part of the research on these technologies focuses on investigating operation expedients for optimising the degradation processes and guaranteeing maintenance of optimal conditions for bioremediation (Jamshidi-Zanjani and Darban 2017). Approaches proposed for controlling the pH include, for example, the continual injection of electrolytes (Kim et al. 2005), anolyte and catholyte mixing (Rabbi et al. 2000), the use of buffer solutions (Niqui-Arroyo et al. 2006), the periodic inversion of the electric field polarity (Luo et al. 2005b;Guo et al. 2014;Mena et al. 2016a, b).

No effects
Some experiments with electrostatic fields of 100-200 V/m or applied/induced currents below 20 mA, typically bioelectrochemical and bioelectrokinetic treatments, excluded important field effects on the basis of biotransformation kinetics, evolution of the CO 2 , microbial charges or enzymatic activity (Jackman et al. 1999;Wick et al. 2004;Harbottle et al. 2009). Lear et al. (2004), for example, do not report any effect on the composition and structure of the microbial community of the soil following the application of direct current at 1 mA for 27 days, and attribute the variations observed near the electrodes only to the variations in pH. In Wick et al. (2004), exposure to DC currents did not cause Mycobacterium LB501T to have any effect on the degradation of some polycyclic aromatic hydrocarbons; in relation to the control experiment, the exposed microorganisms did, however, show levels of ATP that were higher by about 50%, even though without repercussions on the development of biomass and on the degradation speed of fluorine. Zanardini et al. (2002) refer to an increase of about 3 times the ATP content in a mixed culture in wastewater, after exposure for 10 days to direct currents of 40-200 mA. Luo et al. (2005a), studying the properties of the cellular membranes of phenoldegrading bacteria exposed to direct currents of different intensities, conclude that currents \ 20 mA induce unimportant modifications in hydrophobicity, the electrostatic charge of the membrane and the cell form; on the contrary, currents of about 40 mA caused increases in the extracellular concentrations of cytoplasmic substances and cell flattening. Jackman et al. (1999) observed a temporary reduction in the growth rate of acidophilic bacteria subjected to a current of 20 mA for 80 h, due to bacteria membrane degradation close to the surface of the electrodes. Wei et al. (2011) indicate death rates of heterotrophic bacteria that were 10% lower than the control when exposed for 4 h to currents lower than 52 mA, and reductions of approximately 15% and 30% respectively for currents of 100 and 200 mA, due to pH variation close to the cathode surface. Figure 1 sums up the experiences described in the literature and given in Table 1 Fig. 1 Type of effects observed according to the intensity of the electrostatic field that was applied (a) or the intensity of the current generated by an electrostatic field (b) and the exposure duration. The numeric label of each point associates it with the reference in Table 1. ''Negative'' effects (red filled triangle): reduction in the degradation of pollutants and/or substratum consumption, bacterial growth inhibition, reduced metabolic activity or respiration rate, damaged cellular membrane. ''Indirect negative'' effects (red times symbol): not tied to a direct action of the field/current on the cells, but rather to modifications in the environmental conditions (e.g. extreme pH levels, electrochemical production of toxic species, radicals, etc.); ''Positive'' effects (green filled diamond): stimulation of the degradation of contaminants, increased denitrification/ nitrification, acceleration in the consumption kinetic of substrata, increased resistance to pollutants, increased biomass, increased metabolic activity or in the activity of specific enzymes (e.g. dehydrogenases); ''Null'' effect (blue filled circle): no significant effects on the aspects mentioned above. (Color figure online) categories of effects found following exposure to electrostatic fields (Fig. 1a) or fields generated by direct currents (Fig. 1b), even according to the exposure duration. As a result of the numerous experiences that refer to negative effects on microorganisms, not because of the direct action of the electric field but for the variations induced in the environment (for example variations in the pH), they are highlighted in the figures with a different colour, because using suitable expedients (buffer solutions, periodic polarity inversion) this type of undesired phenomenon can be limited and controlled. When considering the intensity of the electrostatic field, all the positive effects occur at values within about 1000 V/m; however, in the same range of values, even with short exposure duration, negative effects are found. With currents up to 10 mA, the effects are above all positive or at most negligible; the few negative effects are all indirect; for higher current intensities, especially above 150 mA, no positive effects can be seen. A scheme of the various phenomena resulting from the application of an electrostatic field or a field generated by direct current is shown in Fig. 2.

Magnetostatic fields
Thanks to the relative simplicity of bacteria, using these organisms as models to examine the fundamental metabolic replies to magnetic fields should make it possible to reduce experimental result interpretation errors to a minimum. In spite of this, the data reported in the literature are often conflicting, and the action mechanisms not clear. A systematic approach, an analysis of the exposure-reply relationship, and physical, biochemical and physiological explanations Fig. 2 Scheme of the various phenomena resulting from the application of an electrostatic field or a field generated by direct current: (1) hydrolysis resulting in O 2 production (a) and H 2 production (b); (2) partial oxidation (a)/reduction (b) of pollutants; (3) solid electrodes as electron acceptor (a)/donor (b); (4) increase in pollutant bioavailability; (5) modification in the physiology and morphology of the cell; (6) loss of membrane integrity, with release of cytoplasmic material and cell death; (7) increase in intracellular ATP concentration; (8) increase in the transport of organic molecules, nutrients, and bacterial cells due to electroosmosis, electrophoresis and dielectrophoresis; (9) transport of dissolved ions due to electromigration; (10) increase in temperature near the electrodes; (11) divergence of the redox potential from the environmental conditions; (12) pH variation close to the electrodes (Letuta and Berdinskiy 2017) are missing from the majority of the studies that have been done. Potenza et al. (2004) observed an increase in the ability of Escherichia coli to form colonies when exposed to a static magnetic flux density of 300 mT, as a function of the incubation medium. In Horiuchi et al. (2001), the number of E. coli cells during the stationary growth phase was 10 5 times higher when under the effect of a high intensity magnetostatic field (5.2-6.1 T) than when they were exposed only to the geomagnetic field. Pospíšilová et al. (2015) showed how Rhodococcus erythropolis favours the use of phenol under a magnetostatic field of 50 mT.

Studies on microorganisms
Some studies demonstrate that the magnetic field can act on DNA stability, interacting with it directly or reinforcing the activity of oxidant radicals (Li and Chow 2001). Gao et al. (2005) observed how a magnetic flux density of 14.1 T in Shewanella oneidensis stimulated the transcription of 21 genes on the one hand, and suppressed the transcription of 44 genes without causing substantial variations in growth on the other.
According to Kohno et al. (2000), static magnetic fields can induce the formation of the hydroxyl radical and amplify the negative effect of nitrogen oxide on the proteins-channels of the cellular membrane.
The mechanism of the radical pair (highly unstable species made up of two radicals) is considered as the most reasonable mechanism of interaction between weak magnetic fields and the biochemical systems (Steiner and Ulrich 1989;Woodward 2002). Each of the two radicals has an unpaired electron; the radicals can therefore be in the singlet state or the triplet state. The mechanism of the radical pair is present in three processes that are of fundamental importance for the cells: the enzymatic synthesis of ATP, the replication of DNA, and the enzymatic phosphorylation of the proteins (Buchachenko 2009(Buchachenko , 2014Buchachenko et al. 2012).
The Mg 2? ion, just like other ions (Ca 2? and Zn 2? ), participates in hundreds of enzymatic processes, many of which involved in fundamental biological mechanisms (Andreini et al. 2008;Rittié and Perbal 2008). The magnetic fields modify the interactions between these ions and the intracellular enzymes, in particular those involved in ATP synthesis (Buchachenko et al. 2012;Buchachenko 2016;Letuta and Berdinskiy 2017).

Studies in an environmental setting
Experiments were carried out on the application of static magnetic fields for treating wastewater in activated sludge systems, in relation to a potential improvement in solid-liquid separation during the sedimentation step. At times, an increase in the removal rate of the Chemical Oxygen Demand (COD), thanks to the production of more unsaturated fatty acids to stimulate the dehydrogenase activity (Niu et al. 2014), and of other compounds (Zaidi et al. 2014) were observed. In an aerobic activated sludge reactor exposed to a magnetostatic field, the overall content of biomass increased by more than 14% in comparison with a control reactor that was not exposed to the field (Zielinski et al. 2017). In Ji et al. (2010), the acclimation of the activated sludge and the removal of COD under the effect of a magnetic field up to 20 mT were stimulated in comparison with a control system; the same result appeared in Łebkowska et al. (2011) at 7 mT. Also in Yavuz and Ç elebi (2000), the biological activity of the sludge was stimulated up to 17.8 mT; opposite effects were observed with higher intensities (46.6 mT). In Tomska and Wolny (2008), periodic exposure to a magnetic flux density of 40 mT increased nitrification.
In Zhao et al. (2016), the use of a magnetic field of 220 mT stimulated the activity of the biofilm at the anode of a fuel cell for treating wastewater, thanks to the production of more extracellular polymeric substance. With a magnetostatic field of 360 mT, instead, the opposite occurred due to harmful effects to microbial growth. Xu and Sun (2008) presented the effect that magnetostatic fields at different intensities (2.4 mT, 6 mT, 10 mT, 17.4 mT) had on the treatment of wastewater that had been contaminated by Cr(VI), and in particular on Brevibacillus sp. and Bacillus sp. with Cr-reducing abilities. In all the cases, the quantity of microorganisms in the liquid medium was higher than the control (32-65%), with maximum abatement of Cr(VI) occurring when exposure was at 6 mT. On the sludge line, Xu et al. (2009) found an increase in the production of methane with exposures at 4 mT.
In Xu and Sun (2008), soil exposed to a magnetostatic field of 0.15-0.35 T had a higher respiration rate in comparison with the control. A magnetic flux density field of 7.0 mT instigated both the desorption of Cr(VI) and the growth of Geotrichum sp. in a soil column test (Qu et al. 2018).
In Mansouri et al. (2017), Microbacterium maritypicum, isolated from a contaminated lagoon, doubled the biodegradation rate of benzo(a)pyrene when exposed to a magnetic flux density of 200 mT.
Using biofilters to degrade trichloroethylene, exposed to magnetostatic fields of 30-60 mT, Quan et al. (2017) recorded more removal (? 2.4%) than the control. The result was mainly ascribed to the differences in the bacterial community that developed, with relative abundances of Acinetobacter, Chryseobacterium and Acidovorax that were significantly higher in the exposed systems. Figure 3 summaries the experiences described in the literature given in Table 2 in relation to exposure to magnetostatic fields, divided into categories of effects found, even according to exposure duration. The effects are classified into ''positive '', ''negative'', ''null'' and ''undefinable'', as already reported previously. From an analysis of the data, the opportunity of containing the intensity of a magnetostatic field within 10 mT appears to be evident. Even though positive effects were obtained also for exposure to fields of 10 mT and higher, experiences with negative effects were found to be more frequent. In addition, by analysing Fig. 3, it can be seen how negative effects on the microorganisms can result even with low exposure duration; progressive adaptation by the microorganisms cannot, however, be excluded for extended exposure duration.   Fig. 3 Type of effects observed according to the intensity of the magnetostatic field and the exposure duration. The numeric label reported close to each point associates it with the reference in Table 2. ''Negative'' effects (red filled triangle): reduction in the degradation of pollutants and/or substratum consumption, bacterial growth inhibition, reduced metabolic activity or respiration rate, damaged cellular membrane. ''Positive'' effects (green filled diamond): stimulation of the degradation of contaminants, increased denitrification/nitrification, speed up in the consumption kinetic of substrata, increased resistance to pollutants, increased biomass, increased metabolic activity or in the activity of specific enzymes (e.g. dehydrogenases); ''Null'' effect (blue filled circle): no significant effects on the aspects mentioned above; ''Undefinable'' effects (purple filled square): in the absence of positive or negative effects, modifications in the activity of enzymes that are not involved in the degradative metabolism, variation of the ATP concentration, modifications of the microbial community (structure/diversity/genotype/morphotype), effects of mutagenicity, cellular proteome alterations, DNA/RNA synthesis alterations/modifications and related activities, variations in secondary metabolite transposition and production, modifications to the cell form and the characteristics of the cellular wall and its electrostatic charge, increased cell hydrophobicity, increased adhesion between the bacterial cells, increased or reduced resistance to antibiotics. (Color figure online) 4 Electromagnetic fields

Studies on microorganisms
The first studies on the effects of pulsed electric fields on microorganisms were carried out by Sale and Hamilton in 1967-1968(Sale and Hamilton 1967, 1968, who investigated the effects on the vitality of the exposed cells and the lethal effects. The reduction in vitality (up to 99.99%) following exposure to high intensity electric pulses was ascribed to the increase of the external cellular membrane permeability. The lethal effect resulted as being related mainly to field intensity and exposure duration, but was also influenced by the production of toxic substances through electrolysis (Hülsheger and Niemann 1980;Hülsheger et al. 1981Hülsheger et al. , 1983. In 1986 it was seen how the electric charges of cells exposed to an alternate electric field separate, with the formation of an oscillating dipole (Hofmann and Evans 1986).
More recent research (Schoenbach et al. 1997(Schoenbach et al. , 2000 clarified that the exposure of cells to an electric field causes the accumulation of electric charges on the cell membrane and, as a result, a variation in the potential gradient between the two sides of the membrane. In the case of low intensity electric fields, this causes the tension-dependent channels of the cell membrane to open. As a result, a flow of ions (Na ? , K ? ) crosses the channels and modifies the concentrations close to the membrane, causing cellular stress. Stress on short electric signals with a low intensity electric field lasts for a few milliseconds and does not cause irreversible damage. With more intense electric fields, a major potential gradient invests the cell membrane, modifying its permeability until the cell is no longer able to fix its damage, which results in cell death (irreversible breakage). The entity of the voltage that causes the tension-dependent channels to open or cell membrane lysis depends on the cell type and size, and the duration of the pulse. For pulses that vary from tens of microseconds to milliseconds, an electric field intensity of around 10 kV/cm is critical for E. coli lysis (Hülsheger et al. 1981).
The presence of an alternate magnetic field influences singlet ¢ triplet interconversion in the mechanism of the radical-pair already mentioned in the section on magnetostatic fields (Maeda et al. 2008;Rodgers 2009) and, as a consequence, the physiological state of the cell and the enzymatic reaction rates can change (Binhi 2001). Rakoczy et al. (2016) and Fijałkowski et al. (2015) demonstrated that 1 h of exposure to a sinusoidal magnetic field with an effective intensity of 30 mT and frequency 50 Hz increases the growth and cellular metabolic activity of E. coli and Staphylococcus aureus significantly in comparison with the controls. Furthermore, the authors observed greater stimulation of growth and metabolic activity in cultures of S. aureus in comparison with E. coli. In Fijałkowski et al. (2015), different results were obtained in cultures of Acinetobacter baumannii and Pseudomonas aeruginosa, where the sinusoidal magnetic field (34 mT, 50 Hz) caused the metabolic activity of the cells to decrease. As proposed by Strašák (2005) and Fijałkowski et al. (2015), the effect observed after exposure to the magnetic field could depend on the form of the exposed bacteria. However, following a comparison of the results of the study carried out on A. baumannii and P. aeruginosa with those on other rodshaped bacterial species (E. coli, Serratia marcescens, Cronobacter sakazakii, Klebsiella oxytoca), the effect on the microorganisms could depend on the specific species, independently from the cellular form.
The results of an additional study carried out on E. coli exposed for 1 h to a sinusoidal magnetic field of intensity 10 mT and frequency 50 Hz, to verify how the field affected cell vitality, were not significantly different from the non-exposed controls (Fojt et al. 2009).
A study carried out on Salmonella exposed to a sinusoidal magnetic field (14.6 mT, 60 Hz) demonstrated no direct damage to the DNA; the results, however, supplied evidence that exposure to the field induces the expression of heat-shock proteins (Williams et al. 2006), which act as biological indicators of cellular stress and help repair or degrade the proteins that were damaged by thermal shock.
Alternate magnetic fields at moderate intensity (200-660 lT, 50 Hz) alter the transcription speed of the lac operon in E. coli (Aarholt 1982). A non-linear dose-effect relationship seems to exist for this type of effect. As an example, while a field intensity of 300 lT suppresses transcription, a field intensity of 550 lT causes a substantial increase.
It was seen that sinusoidal magnetic fields (1.1-1.2 mT, 50-60 Hz) increase the translation activity of the mRNA in E. coli (Goodman et al. 1994;Cairo 1998).
In two consecutive studies, Del Re et al. (2003,2004) observed the effects on transposition activity in cultures of E. coli exposed to two electromagnetic fields with different characteristics. The results of the first study highlighted that E. coli cells exposed to a sinusoidal magnetic field (50 Hz, 0.1-1 mT) were significantly less active than those of the controls. Conversely, in the subsequent study, the exposure of E. coli cells to a pulsed magnetic field, having the same intensity and frequency, led to significantly greater transposition activity than that of the non-exposed controls being observed. In both studies, transposition was negatively/positively linked to the field intensity with a linear dose-effect relationship. In addition, in both the first and the second study, this phenomenon did not influence bacterial cell proliferation and a significant difference between the amount of colonies exposed or not to the field did not arose. These results suggest that the biological effects depend critically on the physical characteristics of the magnetic signal, in particular the wave shape. Cellini et al. (2008) exposed cultures of E. coli to magnetic fields of frequency 50 Hz and variable intensity (0.1, 0.5, 1.0 mT). During this study, the effects of electromagnetic radiation on different biological parameters were investigated: Colonyforming Units (CFU), cellular vitality state, morphological and transcription profile. According to the results of the experiments, the studied parameters of the irradiated samples did not present significant differences in comparison with the controls, except for increased cellular vitality and change in the morphology of E. coli, with the presence of ''coccoid'' cells even aggregated in clusters.
Another study on the effects of pulsed electromagnetic radiation at extremely low frequency on the growth of the bacteria S. aureus showed a decrease in the growth rate. The results evidenced how, in all the tests carried out on cell cultures exposed to fields of intensity within the 0.5-2.5 mT range and frequencies between 2 and 500 Hz, there was a reduction in the number of CFUs in comparison with the non-irradiated controls. In particular, the lowest CFU value was reached after exposure for 90 min at 1.5 mT and 300 Hz (Ahmed et al. 2013).

Studies in an environmental setting
The recent review of Piyadasa et al. (2017) sums up the experiences carried out when controlling precipitation and fouling in inverse-osmosis membrane systems, in particular for desalination. The use of pulsed electromagnetic fields helped speed up clogging of suspended particles and their precipitation.
In the wastewater treatment field, Yavuz and Ç elebi (2000) present the effects of an alternate (8.9-46.6 mT, 50 Hz) or pulsed (17.8 mT 2 s on/2 s off) electromagnetic field. In the first case, an increase of 44% in the substratum removal rate was observed, while in the second there were no significative effects.
In Zhou et al. (2017), a pulsed electromagnetic field (square wave with frequency 100 Hz and intensity 5 lT) applied to a bioelectrochemical system caused changes in the microbial community at the anode: a relatively greater abundance of Geobacter spp. was found (4-8%) than in the control. Figure 4 shows a summary of the effects on microorganisms (according to the positive, null, negative and undefinable classification) following exposure to sinusoidal electromagnetic fields with frequencies 50-60 Hz, according to the intensity of the magnetic field (given that in most of the manuscripts the induced electric field intensity is not reported) and exposure duration. The choice was made to report the experimental data referred to 50-60 Hz, being typical frequencies of the electric distribution networks, which are most surveyed in the literature.
Most of the experiments took place with magnetic intensity below 20 mT. Even though not numerous, in the case of fields lower than 1 mT the experiments resulted in effects that were mostly positive; on the contrary, with fields that varied between 1 and 10 mT, more studies indicated effects that were negligible, undefinable and negative than those in which positive effects were observed. It should be noted, however, that some authors refer to a field intensity even of a few tens of mT, at which positive effects were observed. The ''window'' of field intensity at which negligible, undefinable and negative effects were observed varies in relation to the bacterial species. As the mechanisms responsible for bacterial stimulation/inhibition are not currently understood, it seems not possible to forecast the microorganism behaviour to electromagnetic fields of different intensities.
In comparison with electrostatic and magnetostatic fields, the experiments with electromagnetic fields were limited to an exposure duration of a few days at most, and it is not clear if there were microorganism adaptation.

Conclusions
The scientific literature that was considered refers to biological effects that at times contrast with each other. For some common microorganisms, suitably insulated and exposed to fields, increases in the cell activity were noted, with effects on the biomass growth rate and the metabolic kinetics. In the few experiences carried out in the environmental field, there is also some evidence of positive effects of exposure to fields, even in the biological wastewater treatment or bioremediation.
The most surveyed fields in relation to bioremediation are certainly the electrostatic fields or generated by continuous currents, already being used today in electrokinesis processes to remove pollutants in sediments and fine grain soils. In some of these processes carried out at full-scale and in situ, biostimulation was also ascribed to the applied electric field. These results are reported in very recent studies and, certainly, further research and experiments must be done to find further evidence using different soils and contaminants, in addition to understanding the mechanisms.
Regarding magnetostatic fields, the most important applications in the environmental field involve wastewater treatment in activated sludge systems. In addition to a potential improvement in solid-liquid separation during the sedimentation step, increases in the degradation kinetics of the organic substance or increased biomass were related to exposure to magnetic fields.
The limited experiences concerning electromagnetic fields in the environmental ambit involve wastewater treatment and in particular the control of fouling. Type of effects observed for exposure to electromagnetic fields of frequency 50-60 Hz, according to the intensity of the magnetic field and the exposure duration. The numeric label reported close to each point associates it with the reference in Table 3. ''Negative'' effects (red filled triangle): reduction in the degradation of pollutants and/or substratum consumption, bacterial growth inhibition, reduced metabolic activity or respiration rate, damaged cellular membrane. ''Positive'' effects (green filled diamond): stimulation of the degradation of specific contaminants, increased denitrification/nitrification, acceleration in the kinetic consumption of substrata, increased resistance to pollutants, increased biomass, increased metabolic activity in general or in the activity of specific enzymes (e.g. dehydrogenases); ''Null'' effect (blue filled circle): no significant effects on the aspects mentioned above; ''Undefinable'' effects (purple filled square): in the absence of positive or negative effects, modifications in the activity of enzymes that are not involved in the degradative metabolism, variation of the ATP concentration, modifications of the microbial community (structure/diversity/ genotype/morphotype), effects of mutagenicity, cellular proteome alterations, DNA/RNA synthesis alterations/modifications and related activities, variations in secondary metabolite transposition and production, modifications to the cell form and the characteristics of the cellular wall and its electrostatic charge, increased cell hydrophobicity, increased adhesion between the bacterial cells, increased or reduced resistance to antibiotics. (Color figure online) Even though in the review it was not possible to identify operative conditions that could certainly stimulate biological activity, additional research could be done: • on the effects of fields that are electrostatic or generated by direct currents until around 100 V/m (or induced currents of about 10 mA), for the bioremediation of contaminated soils or sediments. Some positive laboratory experiences have been reported for treating solid matrixes in bioelectrochemical or electro-bioremediation systems (as a development of the electrokinesis systems). In consideration of these initial results, but also considering the scarce understanding of the phenomena in play and the decisive role of the choice of operative conditions for compensating the negative effects induced by the electric field (extreme variations in pH, etc.), further research on this matter is certainly necessary for developing and scaling up the technologies; • an assessment of the effects of magnetostatic fields of 1-10 mT on bioremediation in soil; from the literature, this aspect does not seem to have been explored yet. Special attention should be placed on assessing how the applied field interacts with the mineral components of the soil, which could present ferromagnetic properties; • on electromagnetic fields; even though some research refers to positive effects on bacterial activity, the results as a whole seem to be very contrasting and do not make it possible to identify, from the start, the operative conditions on which to sketch out possible experiments.

Compliance with ethical standards
Conflict of interest The authors declared that they have no conflict of interest.
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