High-frequency, high-intensity electromagnetic field effects on Saccharomyces cerevisiae conversion yields and growth rates in a reverberant environment

Studies of the effects of electromagnetic waves on Saccharomyces cerevisiae emphasize the need to develop instrumented experimental systems ensuring a characterization of the exposition level to enable unambiguous assessment of their potential effects on living organisms. A bioreactor constituted with two separate compartments has been designed. The main element (75% of total volume) supporting all measurement and control systems (temperature, pH, agitation, and aeration) is placed outside the exposure room whereas the secondary element is exposed to irradiation. Measurements of the medium dielectric properties allow the determination of the electromagnetic field at any point inside the irradiated part of the reactor and are consistent with numerical simulations. In these conditions, the growth rate of Saccharomyces cerevisiae and the ethanol yield in aerobic conditions are not significantly modified when submitted to an electromagnetic field of 900 and 2400 MHz with an average exposition of 6.11 V.m-1 and 3.44 V.m-1 respectively.

section_content: • Rational method to assess the intensity and specific absorption rate in submerged cultures developed

• The average mean electric field in submerged culture is respectively 6. 11 and 3. 44 V. m -1 and the maximum local SAR is respectively 0. 040 and 0. 080 W. kg -1 at 900 and 2400 MHz

• Liquid culture medium dielectric properties significantly limit the yeast exposition to the electromagnetic field

• No effect on growth rate and ethanol yield noticed

section_content: Electromagnetic stimulations are potentially effective to elicit either positive or adverse effects in many biological systems (Pilla and Markov, 1994). Given the diversity of exposition devices, frequencies, intensities, and durations used, the observed results appear sometimes contradictory (Hristov and Perez, 2011). In modern societies, the negative influence of intensive electromagnetic environment is one of the main concerns of the citizens in terms of public health. The French National Agency for Food, Environmental and Occupational Health and Safety (ANSES) stresses the need for better characterized experimental devices to ensure the accurate determination of the level of exposure to the electromagnetic fields to ensure an unequivocal assessment of their potential effects on living cells (ANSES, 2013). In any case, there is a considerable interest to improve a thorough understanding of the effects of electromagnetic radiation on living cells. 

It is noteworthy that biofuels are a credible alternative for a more sustainable future with an important growth potential: in 2012 more than 93% of the 2500 millions of tons of oil equivalent used for transportation came from non-renewable feedstock (IEA, 2012) letting plenty of scope for improvements. It is also admitted that biomass production processes are requiring major improvements to make them economically competitive with their fossil equivalents (Menten, 2013; Chovau et al., 2013). Whatever the generation of bioethanol, Saccharomyces cerevisiae is the microorganism of choice to carry out alcoholic fermentation of sugars and has received considerable attention (Bertrand et al., 2016). In terms of process intensification, two complementary approaches are traditionally applied (Reay, 2008). Metabolic engineering and molecular biology, aims at amplifying the metabolic fluxes leading to an overexpression of the predominant electric field at low frequencies (Perez et al., 2007), pulsed electric fields (Mattar et al., 2015) and sonication (Sulaiman et al., 2011; Indra Neel et al., 2012) have proven their potential for improving metabolites production. The mechanisms are not fully understood but some general trends are underlined depending on the electromagnetic frequency applied. Permanent magnets (i. e. static magnetic fields) have a major effect on the phospholipids orientation in the cell membrane affecting its biomechanical properties (Wang et al., 2014). They also help to preserve enzymes conformation and counterbalance the negative side effect of high ethanol concentration that alters cellular membranes and unfolds cytosolic globular enzymes (Lopes and Sola-Penna, 2001). Electric fields at low frequencies enable ionic redistribution as well as a higher potential difference in the cellular suspension. The effects of pulsed electric fields stimulations are largely unknown. It is hypothesized that a specific yeast subpopulation is favored knowing that cytoplasmic membrane modifications are induced by temporary electroporation (Mattar et al, 2015). External electric fields improve nutrient transportation through the modified cell membrane via pore formation or even transport proteins activations (Castro et al., 2012 ). An improved gas-liquid mass transfer for dissolved carbon dioxide removal, which is known to inhibit Saccharomyces cerevisiae growth, is the main effect of Mild sonication on the culture broth leading to higher ethanol productivity (Indra Neel et al., 2012). 

Therefore, depending on their settings, electromagnetic stimulations have potential to promote one or the other approaches for process intensification of Saccharomyces cerevisiae fermentation. In the high-frequency range, previous studies showed that highfrequency electromagnetic waves promote and stabilize free radicals in yeasts associated with oxidative stress. This effect is amplified when the intensity of the field increases (Crouzier et al., 2009) The two aims of this study are: (i) to develop and characterize a fully controlled and instrumented bioreactor for the culture of cells in a liquid medium under controlled electromagnetic stimulations; enabling both an off-line determination of metabolites concentrations and online assessment of growth rates by gas-phase analysis and mass balances. (ii) to master an intense oxidative stress on Saccharomyces cerevisiae cultures using high-frequency, high-intensity electromagnetic field to potentially trigger an overproduction of ethanol. 1. 1. 1. Dielectric measurements For assessing the influence of electromagnetic stimulations one key variable is the electric properties of the culture medium. This is obtained measuring the complex permittivity (complex number).
section_name: Introduction

section_content: The culture medium relative permittivity (ε r ) and electric conductivity (σ [S. m -1 ]) were measured using a "Di-line" Transverse Electromagnetic sensor (INDEXSAR, Newdigate, United-Kingdom) at 30°C, the temperature set-point used for the culture. A vector network analyzer (Rohde & Schwarz ZVB8) was used to measure the transmission loss and phase of the TEM-line with or without the culture medium making it possible to compute the dielectric properties for all the frequencies used. 

Detailed explanations about the computation can be found in (Toropainen et al., 2000).
section_name: Materials and Methods

section_content: To obtain a defined and controlled electromagnetic field, irradiated cultures have been performed in a specific chamber. The MSRC is a special device to obtain reproducible electromagnetic field generation (Fig. 1 ). A double-layered steel Faraday cage protects the experiment from external or environmental electromagnetic field. An antenna (ETS Lindgren 3144) is used to generate the electromagnetic field, while the walls and a stirrer reverberated randomly the incident waves within the cavity. Within a specific volume of the chamber, called working area (delimited by the blue points in Fig. 1 ), the electromagnetic field is statistically (i. e. for a full rotation of the stirrer) homogeneous and isotropic (Hill, 1994). The method is fully normative (IEC, 61000-4-21) and more explanations concerning the Institut Pascal's MSRC properties and performances for the exposition of biological samples are available in (Lalléchère et al., 2010). The intensity of the electromagnetic field impacting the bioreactor is measured using an isotropic electric field antenna (PMM-EP 183). Germany) coupled with an analytic approach was used to estimate the electromagnetic field impacting the culture compartment located inside the MSRC. For all the simulations performed with FEKO, the unstructured mesh has been sized so that the characteristic length is at least smaller than λ/4 at a frequency of 2400 MHz (the highest frequency used in this study is fixing the finer mesh that is necessary). However, due to calculation capacity limitations, numerical simulations of the reactor inside the MSRC at scale 1 were not possible. To overcome this limitation, we used MATLAB R2017a and obtained, from the theory of electromagnetic waves propagating inside a cavity the analytical expressions of the electromagnetic field at any point inside the MSRC, for each resonance mode of the chamber and for each propagation direction. The vectorial sum of these fields was then calculated to obtain the distribution of the total field in the MSRC for each Mode. As an example, Figure 2A represents the electric Field repartition for mode 7/33/14 at 900 MHz. As the MSRC has losses, there is no Dirac signal associated for a given Mode. It is observed a reduction in the signal amplitude spreading around the reference frequency that can be easily approximated by a Lorentzian (Fig. 2B ) with the quality factor of the MSRC that has been previously determined for each frequency. At 900 MHz (Fig. 2C ) and at 2400 MHz (Fig. 2D ), several resonance modes are present in the MSRC. To accurately characterize the electromagnetic field, it is necessary to consider the contribution of each of these different Modes, which were also excited by the source at 900 or 2400 MHz. However, their impact must also be weighed against their frequency distance from the reference frequency. The number of modes has been limited to those that are less than 5 times out of the Lorentzian bandwidth that is centered on the emission frequency of the source. 

The circles indicate the weighting coefficient used for the field strength of each mode considered. Once these modes have been determined, it is then possible to calculate the total Electric Field resulting from the vector sum of all these contributions and to obtain
section_name: Mode Stirred Reverberation Chamber (MSRC)

section_content: Liquid phase analyses Samples from the reactor were immediately filtered (on 0. 2 µm hydrophilic filter) to separate the medium from the biomass. 

The dry cell weight was determined by measuring the optical density at 600 nm of a yeast suspension with a spectrophotometer (BIOMATE 3S, ThermoFischer Scientific, Illkirch, France) and correlated with 5 mL filtered and dried biomass (24 hours at 105 °C) weightings. 

The filtrate was then deproteinized by the addition of 125 µL of barium hydroxide and 125 µL of zinc sulfate (5% w/v) and centrifuged at 10 000g for 5 minutes. The supernatant was kept for the following analyses. Organic acids, alcohols, and glucose were quantitated by HPLC. The separation is carried out at 50°C in isocratic mode at an eluting flow rate of 0. 7 mL•min -1 of a 5 mM The gas fraction was preliminarily dried in a drierite gas-drying unit (W. A. Hammond Drierite Co, Xenia, Ohio) and the molar fraction of oxygen and carbon dioxide were measured at the exhaust of the reactor using a ZRE gas analyzer (Fuji Electric, Clermont-Ferrand, France). Gaseous balances on the reactor lead to the instantaneous respiration rates according to the following equations:

G e is the air molar flow rate feeding the reactor expressed in mmole•h -1. V r is the volume of the culture medium in the reactor at a specific time t. It is the initial volume of culture medium corrected with the volume of the different samples taken out during the culture and with the volume of the sodium hydroxide solution that has been added to the vessel to keep the desired pH. and are the specific oxygen consumption and carbon dioxide production rates expressed in mmol•L -1 •h -1. is the molar oxygen fraction in the air feed (≈0. 2093), while and are the molar fraction of oxygen and carbon dioxide in the dried air leaving the reactor. 

The amounts of consumed oxygen and produced carbon dioxide expressed in mmol L -1 during the culture are calculated by integrating the instantaneous respiration rates according to the following equations:

For convenience, this integration was carried out digitally after discretization of a time interval Δt = 20s corresponding to the acquisition rate being set for the gas analyzer. The respiratory quotient RQ can then ultimately be calculated: One of the fundamental issues is the selection of the most suitable exposure facility (Paffi et al., 2010). The actual electromagnetic environment is far more complex than a planar irradiation that is conventionally used for most of the bio-electromagnetism studies. In this context, the Mode Stirred reverberation chamber (MSRC) has proved to be an efficient tool for bioelectromagnetism stimulations (Lalléchère et al., 2010).
section_name: 1.3.

section_content: Previous experiments carried out on vegetables such as Lycopersicon esculentum have proved the potential to induce transcription, translation and even ATP concentration changes at high-frequency and even low intensity such as 5 V•m -1 (Roux et al., 2007). 

However, this type of environment cannot accommodate with any type of electrical devices whose presence is likely to alter the ideal distribution of the electromagnetic field. Conversely, electronic cards may be damaged by an exposition to intense electromagnetic radiations. A reproducible microbial culture must necessarily be carried out in a regulated and controlled bioreactor. However, the presence of the entire system including all the regulation devices inside the exposure chamber was not an option as the electromagnetic field might damage the electronic parts of the system. Therefore, the instrumented bioreactor was coupled with a secondary exposition vessel to examine the biological effects of the electromagnetic field. The suspension was recycled through the MSRC as shown in Fig. 3. The connecting pump flow rate is 288 mL•min -1. Under these conditions, the residence time in the secondary element is 94s (0. 45 L), which corresponds to an exposition to the electromagnetic waves for 25% of the culture time. 

In the other parts (including the main culture vessel and the connecting parts) the residence time was 280s (1. 35 L). A frequency scanning from 600 to 2500 MHz (Fig. 3 ) indicates that the relative permittivity of the yeasts suspension (minimal medium) is slowly decreasing from 78 to 74 while the electric conductivity is sharply increasing from 0. 4 to 2. 5 S•m -1. These measurements agree with the behavior of an electrolyte solution such as saline water (Gadani et al., 2012). Additionally, to the frequency being applied, the initial composition of the culture medium is of importance. For instance, the addition of ammonium sulfate into the minimal medium, which aims at bringing nitrogen into available form, also brings sulfate as a counter ion. This has the side effect to considerably increase the medium conductivity. The measurements have shown that at any frequency the conductivity for the minimal medium is about 0. 5 S•m -1 higher than the one for the yeast medium. The production of biomass and metabolites (ethanol, glycerol, acetate) during the culture are also susceptible to continuously modify the dielectric properties of the culture medium. At lower frequencies, in the kHz range near the β-relaxation zone, these properties are exploited to develop online biomass sensors (Martisen, Grimmes and Schwan, 2002). However, a preliminary study has shown that these changes in the high-frequency range (600 to 2500 MHz) have no significant effect on the medium conductivity but may lead to a 10% increase of the medium permittivity.
section_name: Results and Discussion

section_content: The filtration of the medium for removing the biomass also indicates that these changes are related to the metabolites and not to the presence of the yeasts themselves in the medium (data not shown). Starting the fermentation with a 50 g•L -1 glucose concentration does not lead to any significant changes in the medium dielectric properties (Fig 3 ). Therefore, the exposition to the electromagnetic field could be considered identic throughout the culture. It is further noteworthy to mention that a higher conductivity leads to a higher attenuation of the electromagnetic waves inside the medium. This had an impact on the reactor design. Its shape must be thin enough to permit a homogenous exposition of the yeasts to the electromagnetic field.
section_name: Measurements of the medium dielectric properties

section_content: Numerical Simulations in an anechoic environment Numerical simulations were used to assess the influence of the geometry, the material properties and of the thickness of the secondary reactor element and their impact on the electromagnetic waves propagation. Due to the complexity of the real electromagnetic environment, a straightforward approach was to consider the incident electromagnetic field as a plane wave. The assumption is thus to consider the culture is in an anechoic environment. Using FEKO electromagnetic simulation software, various reactors configurations were illuminated by a plane wave at the two frequencies selected for the study: 900 MHz and 2400 MHz. Results from these preliminary simulations indicate that the use of Pyrex glass or polyethylene does not induce any significant changes in the electromagnetic profile observed in the culture medium. The selected materials appeared globally transparent in the frequency range studied. 

The simulation of electromagnetic field propagation at 900 MHz and 2400 MHz has different profiles for the same incident power. The maximum amplitude of the electric field penetrating inside the reactor is 3 times more intense in the case of the exposition at 900 MHz than with the exposition at 2400 MHz. This is both due to the electric conductivity increase in the medium, from 0. 9 to 2. 3 S. m -1 (Fig 3. ) and the frequency shift (skin effect). It can be noticed that the intensity of the field is truly heterogeneous in the anechoic configuration as it can be seen in the cross sections presented in figure 4A. 

In order to validate these simulations results, a direct measurement of the electromagnetic field in the culture medium has been performed. The experimental measurement of an electromagnetic field inside a liquid is in itself a challenge. In fact, as small as the probes may be, these latter are intrusive and not pointwise. Therefore, the measurement is the result of integration over a finite volume element. It is therefore not possible to access the measurement with a millimetric (or even centimetric) spatial resolution. This is even accentuated when the field intensity rapidly changes inside the measurement volume. Moreover, the probes are generally developed in order to perform field measurements in the air and not directly into liquids. Before being immersed in culture broth, the probe should be protected in a sealed glass tube, which then creates new interfaces and further possible electromagnetic reflections. Finally, the probe itself can interfere with the electromagnetic environment. In this case, it is likely that the electric field intensity measured by the probe is not that prevailing in its absence. Thus, the effect of the perturbation introduced by the probe on the electromagnetic environment must be investigated numerically. 

Experimental measurements were performed in the empty reactor and after having it filled with the medium so that the position of the probe has not been modified. The results indicate that 68 % of the electric field amplitude measured in the empty reactor is effectively transmitted at the center of the culture medium. These experimental results

are not in agreement with the numerical simulations that indicate that 18% of the incident field is effectively transmitted (Fig. 4B ). This discrepancy has been explained by the impact of the insertion of the probe into the reactor, proving an experimental bias introduced by the probe.
section_name: 2.3.

section_content: Electromagnetic field propagation inside the yeast itself Depending on the frequency, the propagation of the electromagnetic field inside the cells might schematically vary between two extreme schemes. Either the wave passes through the yeast cells without any attenuation, or for the lowest frequencies, the yeast will be shielded and therefore preventing any possible action of the electromagnetic field (Hristov and Perez, 2011). Advances in dielectrophoresis (Asami, 2002 ) associated with single cell impedance spectroscopy make it possible to extract with a relatively good accuracy both relative permittivity and medium electric conductivity at subcellular lever for frequencies from 0 up to 500 MHz (Haandbaek et al., 2014). Given technical limitations, there does not exist any data at subcellular scale for frequencies higher than that observed for the culture medium. Hence, it seems appropriate to approximate the specific absorption rate of a yeast suspension by that of the culture medium in the irradiated part of the reactor.
section_name: 2.4.

section_content: Numerical study in the reverberant environment Since the reactor temperature is regulated, it is not possible to evaluate the specific absorption rate (SAR) by measuring the temperature rise of the culture medium. 

Besides, infrared monitoring of the temperature in the irradiated part of the reacting volume showed no significant variations during the illumination (compared to the control culture). To estimate the SAR and its maximum value, the electromagnetic field strength in the reverberant environment must be calculated according to the dielectric properties of the culture medium. For computation capabilities reasons it was not possible to simulate the Institute Pascal experimental MSRC (L = 8. 4 m; l=6. 7 m, H = 3. 5 m) directly at the chosen frequencies. In order to determine the field cartography and to estimate the SAR, FEKO simulations in free space and an analytic approach were combined. First, using FEKO the reactor was illuminated with three different waves at 900 MHz and 2400 MHz in free space. These directions represent the three directions of the different modes present in the MSCR. The profiles of the simulated electric field at 900 MHz and 2400 MHz according to these three directions are presented in Fig. 5. 

From these results, it was possible to determine the maximum percentage of the electric field penetrating the reactor. According to the simulations, the maximum value of 95 % was obtained. This value has been taken as a reference to compute the electric field intensity inside the MSCR according to the analytic approach presented previously. 

Secondary knowing the percentage maximizing the effects inside the reactor, it was possible to calculate the local SAR (SAR without mass or volume) inside the reactor for 20 000 points chosen arbitrarily in the reactor volume. SAR and local SAR expression are given by the following equations:

The repartition of the different values of the 20 000 random positions in the reactor is given in Fig. 5E for 900 MHz and Fig. 5F for 2400 MHz. The mean field strength, the average local SAR and the maximum local SAR corresponding to the experimental conditions are given in Table 1. 

Values obtained are far below the threshold of 2 W. kg -1 that is normally used to distinguish non-thermal and thermal effects. It should also be noted that the yeast cells are also set into motion by the hydrodynamics of the system, which is also contributing to the exposure homogeneity although it was not possible to model this numerically.
section_name: 2.5.

section_content: Saccharomyces cerevisiae batch culture results The control experiment was carried out by maintaining the recycling loop during the culture without any application of the electromagnetic field inside the MSRC. The same 2007 ), there is no evidence for an effect both on the maximum growth rate and on the main conversion yields between the main components (biomass, glucose, ethanol, O 2 and CO 2 ), considering the standard deviations of the identified parameters. This means that the specific rates remain identical in exposed and in reference non-illuminated conditions. Contrarily, the glycerol and acetate yields seem very slightly affected by irradiation, by a factor of 1. 2-1. 5. This may be related to a perturbed status of redox intracellular balance due to the electromagnetic field. However, the global carbon and nitrogen balances do not appear to be affected, given the accuracy of the measurements. As the various reactions yields, as well as the respiratory coefficient, do not indicate any significant change, it is considered that the global stoichiometry of growth and of the production of primary well as the sinusoidal properties of the electric field, it must be considered that cells have been statistically exposed in a much more homogeneous way, considering they are crossing randomly the different regions of the liquid phase. 

In the characterized electromagnetic environment as described previously, it has been observed that no biological specific response on the biomass, glucose, ethanol, CO 2 and O 2 yields was macroscopically observed at frequencies of 900 and 2400 MHz, with an emitted intensity average intensity perceived by the cells of 6. 1 V•m -1 and 3. 44 V•m -1 respectively applied on 25 % of the reacting volume. This suggests that a highfrequency, electromagnetic field does not induce any major modification of the ethanol production rate by Saccharomyces cerevisiae. However, it is observed that the conversion yields to glycerol and acetate decreased by a factor of 1. 2 -1. 5 compared to non-irradiated conditions. Further investigations would be needed to determine whether the absence of observed biological effect is associated with a protecting role of the liquid culture medium that behaves like a "high cut filter" thus significantly limiting the yeast exposition to the electromagnetic field or to the absence of actual interaction between the high-frequency electromagnetic fields with the metabolism of Saccharomyces cerevisiae. In this context, the increase of the emitted power with the use of a pulsed electromagnetic field, or the design of an exposure device associating an internal source for the electromagnetic waves emission in a waveguide with specific dimensions to select the pertinent wavelength could be an interesting option to investigate.
section_name: 2.6.

section_content: The present study develops a rationale method for assessing rigorously the intensity and the specific absorption rate (SAR) of electromagnetic waves in submerged cultures of
section_name: Conclusion

section_name: Acknowledgments

section_content: This work has been sponsored by the French government research program " Investissements d'Avenir " through the IMobS3 Laboratory of Excellence ( ANR-10-LABX-16-01 ), by the European Union through the program Regional competitiveness and employment 2007-2013 (ERDF -Auvergne region), and by the Auvergne region. Version preprint Comment citer ce document : Bertrand, E., Pasquier, C., Duchez, Girard, Pons, Bonnet, Creuly, Dussap (2018). High-frequency, high-intensity electromagnetic field effects on Saccharomyces cerevisiae conversion yields and growth rates in a reverberant environment. Bioresource Technology, 260, 264-272., DOI : 10. 1016/j. biortech. 2018. 03. 130
section_name: Acknowledgments