Alcoholic fermentation in the presence of microwaves

Accepted Manuscript Title: Alcoholic fermentation in the presence of microwaves Authors: Ioan Calinescu, Alexandru Vlaicu, Petre Chipurici, Daniel Ighigeanu, Vasile Lavric PII: DOI: Reference:

S0255-2701(17)31263-1 https://doi.org/10.1016/j.cep.2018.02.008 CEP 7190

To appear in:

Chemical Engineering and Processing

Received date: Revised date: Accepted date:

12-12-2017 7-2-2018 7-2-2018

Please cite this article as: Ioan Calinescu, Alexandru Vlaicu, Petre Chipurici, Daniel Ighigeanu, Vasile Lavric, Alcoholic fermentation in the presence of microwaves, Chemical Engineering and Processing https://doi.org/10.1016/j.cep.2018.02.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ALCOHOLIC FERMENTATION IN THE PRESENCE OF MICROWAVES Ioan Calinescua, Alexandru Vlaicua*, Petre Chipuricia, Daniel Ighigeanub, Vasile Lavrica

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Faculty of Applied Chemistry and Material Science, University “Politehnica” of Bucharest, 1-

7, Gh. Polizu, Bucharest, 011061, Romania National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor, Magurele,

077125, Romania

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*Corresponding author: e-mail address: [email protected] Phone: 0040728160491 (A. Vlaicu) e-mail addresses: [email protected]

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(I. Calinescu); [email protected] (P.

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Chipurici); [email protected] (D. Ighigeanu); [email protected] (V. Lavric)

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Graphical abstract

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Highlights  An installation was set up for glucose fermentation to ethanol, microwave assisted.  Optimum microwave irradiation conditions were determined.  Microwaves increase fermentation rate without affecting cells viability.

Abstract Bioethanol is the world’s leader biofuel and it is produced by fermentation from glucose feedstocks. The fermentation rate is quite low and any method of increasing the reaction rate is

welcome. The main goal of our research is to identify the domain for microwave Specific Absorption Ratio (SAR) which leads to an increase in fermentation rate of Saccharomyces cerevisiae without affecting its viability. For this purpose, an installation was setup which allows for glucose fermentation to be carried out in the presence of microwave, with good control on SAR and on temperature of the fermentative medium. An optimum SAR domain, between 15

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and 25 W·kg-1, was determined, in which a significant boost of glucose fermentation manifests, with an increase in fermentation rates of up to 20 %. Based on the observations made through

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electron and optical microscopy on cell growth and viability, we have noticed that microwave irradiation favours ethanol production of the cells, against their growth.

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Abbreviations:

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SAR, Specific Absorption Ratio; EMFs, Electromagnetic Fields; MW, Microwaves; EPR,

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Ethanol Production Rate; SEPR, Specific Ethanol Production Rate; conv, conventional

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Keywords:

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microwave assisted fermentation; bioeffects; Saccharomyces Cerevisiae

1. Introduction

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Overall microwave effects might be classified as thermal, specific, and non-thermal [1].

Thermal effects are related to efficient increase of temperature of the reaction mixture with

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corresponding augmentation of the reaction rate. Specific MW effects are essentially thermal but cannot be reproduced by conventional heating. The most controversial potential impacts are non-thermal effects. Loupy et al. [2] suggested alternative explanations of non-thermal MW effects in cases where polar transition states are formed and thus favour some reaction paths through increased entropy of the activated complex.

Regarding the microwave interactions with living cells, two distinct cases could be identified: -

destructive action of MW, used mostly to inactivate microorganisms [3-6];

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non-destructive action of MW which can affect both functionality and development of living cells [7-10].

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The most important difference between these two effects is given by the MW used power, expressed as (SAR). If the thermal effect of MW is easier associated with microorganism’s

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inactivation, the influence upon functionality and development is much difficult to explain. Many researches have been devised to explain the mechanism of action of microwaves on living cells [11-15]. It is important to mention the mechanisms behind the interactions between

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microwaves (MW) and dielectric materials (which absorb microwave energy), when referring to

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microwave effects on living cells. The MW photon energy corresponding to the frequency used

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in microwave heating system, ranging from 3.78·10-6 to 1.01·10-5 eV, acts as a non-ionizing

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radiation that does not affect the molecular structure since it is lower than the usual ionization

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energies of chemical bonds (3–8 eV) or even hydrogen bonds (0.04-0.44 eV) [16]. The interaction between the electric and magnetic field components of the microwaves and the

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materials has as result the dielectric and magnetic losses leading to heating. This interaction mechanism is responsible for the heating of different materials under microwave irradiation. The

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effect of MW on living cells is observed at low temperatures and in almost adiabatic environmental conditions, therefore the MW are used at very low power densities (10-19 to 10-3

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W·cm-2). How could it be possible that extremely weak electromagnetic radiation, with quantum energy less than the average energy of thermal noise (kT constraint) can have significant biological consequences? To answer this, one must consider that an active biological system is very far from thermal equilibrium and has considerable amounts of energy stored for appropriate use. The work of Fröhlich and Hyland [17, 18] describes in great detail non-thermal effects of

low intensity microwaves on living systems. The numerous parameters which affect the nonthermal MW effect on living cells have been presented in detail by Belyaev [15]. While thermal effects (which show little to no variation with radiation frequency) are directly proportional to the radiation intensity, non-thermal effects occur in certain frequency regions only, and usually exhibit saturation at rather low radiation intensity.

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It is considered that the best conditions for investigating MW effects are “isothermal” by balancing MW heating with an externally recirculated cooling fluid [19]. Thus, a fermentation

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mixture could be treated with MW having SAR = 550 W·kg-1 keeping at the same time a constant temperature (less than 1 ℃ deviation). Comparing the results obtained in conventional process vs the microwave assisted one in the case of glucose uptake by baker’s yeast, it can be

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noticed that, at the same bulk temperature, MW irradiation of the yeast cells has approximately

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possible explanation could be proposed:

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the same effect as the conventional process developed at a temperature higher with 2 °C. Two

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a) Suppose that the MW effect is the selective heating of yeast cells, because of different

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dielectric properties of fermentation mixture and yeast’s cells. Such a case, where selective heating seems to be present, is described in [20] during extraction of active

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principles from okra. In our research, the very small diameter of yeast cells (3-4 µm) and their higher number (~3·108 cells·mL-1) make the heat transfer surface so great that a 2 ℃

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thermal difference between them and the surrounding liquid is hard to be justified for the running experimental conditions (400 rpm stirring).

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b) Some specific thermal effects on cell metabolism may as well be involved. In living cells, many levels of organization exist [19]. Changing the flux of energy through the system may influence dissipative structures in living cells and consequently their metabolism. The discrimination between these effects could be achieved by precise measurement of

the temperature inside cells. For the moment, the experiments with fine enough resolution to

monitor the temperature within cells are extremely difficult to carry out, and are not yet developed for routine laboratory applications [19, 21].

2. Experimental studies 2.1.

The experimental setup

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The experimental setup used to study the effects of microwaves on living cells consists of a multimode applicator, in which microwaves are supplied through a coaxial antenna connected

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to a solid-state microwave generator, with adjustable power and display for incident and reflected power. The thermally isolated reaction vessel is placed inside this multimode applicator. The temperature of the fermentation mixture is monitored using an optical fibber-

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based sensor (opSens device). Reactor and glucose and nutrients solution are brought to the

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reaction temperature prior to start the experiment, namely before the yeast cells are added. Inside

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the fluid phase, a glass cooling coil is placed, through which thermostatic water is circulated to

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absorb the generated heat and to maintain the temperature constant inside the reaction medium

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with an accuracy of ± 0.1 K. To ensure more homogeneity inside the medium, the plate on which the reaction vessel is placed on is connected to a motor with adjustable rotational speed, while

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the cooling coil and optical sensor remain in a fixed position, acting as a mechanical stirrer. Conventional fermentation experiments, for comparison, have been also carried out heating the

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reactor using a thermostatic water bath (Julabo SW22) with mechanical stirring.

Procedure for SAR determination

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2.2.

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The procedure used for determining the SAR is based on the calorimetric method [22]:

T  m  C p

SAR 

t P T  C p  m t

(1)

(2)

where: T – the increase of the mean temperature of the heated body (K) P – microwave power used for heating (W) m – mass of the exposed sample (kg) Cp, – specific heat (J·kg-1·K-1) – for simplification, the specific heat of the fermentation broth was taken equal to that of water;

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t – time of heating (s); SAR – Specific Absorption Rate (W·kg-1).

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SAR is determined as W·kg-1 of fermentation broth.

The experimental setup from fig. 1, without the cooling coil, was used to determine SAR.

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The thermally isolated reaction vessel, which contains 100 mL of glucose and nutrients solution,

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is irradiated at different microwave intensities, by modifying the value of targeted power, while

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the temperature increase is recorded. The time interval is selected so that the temperature

Method used for determining cell viability and number of cells

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2.3.

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increase is limited to 1-2 °C, while the temperature remains between 30 and 35 °C.

Yeast cells viability for samples of Saccharomyces cerevisiae has been studied by

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staining the sample with Trypan Blue, a dye which only penetrates the cellular membrane of

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dead cells; therefore, living cells are not stained. By using this method, the distinction between living and dead yeast cells can be made using an optical microscope (BX31M OLYMPUS, the images being recorded by a digital camera) and the cell number and viability can be determined

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using a Neubauer counting chamber. Depending on the sample, dilution may be necessary prior to the analysis of the sample;

usually, inside a Neubauer counting chamber the optimum cell density is between 250000 cells·mL-1 and 2.5 million cells·mL-1 [23]. Equation 3 was used in determining cell viability:

 Number _ dead _ cells  cell _ viability _(%)  1   100 Number _ total _ cells  

2.4.

(3)

Analysis methods Ethanol content in fermentation samples was determined through chromatography; a

Buck Scientific 910 gas chromatographer was used, with the following characteristics: stationary

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phase MXT-1 (Restek), column 60 m x 0.53 mm, H2/Air – 20 psi – 25 mL/min, He – 14 psi – 10 mL/min, internal compressor – air, split – 1:4, FID/DELCD detector. Isopropyl alcohol was used

determined as the average rate over the measurement time:

 EtOH 

(4)

Fermentation _ time

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EPR _( g  L1  h1 ) 

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as internal standard. Using the ethanol concentration values, Ethanol Production Rate (EPR) was

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Specific Ethanol Production Rate (SEPR) was also determined by reporting the EPR to the

Mathematical model of heating implemented in Comsol® Multiphysics

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3.1.

(5)

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3. Results

EPR Number _ of _ cells

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SEPR _( g  L1  h1  Cells _ No1 ) 

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number of cells present in the sample:

The model of the microwave heating in a multimode applicator, using first principle

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equations, was developed. Low power microwaves are supplied by a solid-state generator, at 2.45 GHz. The absorbed power in the liquid phase is under 7W (determined by calorimetry), suited for studies related to the MW effects upon living microorganisms. The liquid phase

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acts like an electric field concentrator, with different streamlines densities per liquid volume used. To mimic the mixing of the liquid phase in the reactor rotated by a plate, an asymmetric U shape agitator was used, with very good mixing results for a rotational speed of 30 rotation per minute, according to experiments. Finally, the model was tested, with very good results; the liquid phase was kept at 35 °C under MW irradiation and cooling using a

thermal agent coming from thermostatic bath - in both cases, a less than 0.6 K gradient of temperature for the whole liquid volume was observed (see results in fig. 2). The temperature profiles for the homogeneous medium, as solutions of the mathematical model implemented in Comsol® Multiphysics, confirm that heating is quite homogeneous in the whole volume of the reactor. When heterogeneity is acknowledged,

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considering the yeast cells suspended in the fermentation medium, the supplemental heat generated by selective heating of the cells is easily dispersed in the liquid phase. A heat

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balance calculation (available in the Supplementary Information) shows that the temperature difference between the yeast cells and the fermentation medium is insignificantly low (for the worst conditions, no more than 10-2 °C) SAR determination

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3.2.

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Based on the calorimetric procedure for SAR determination, a significant difference was

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noticed between the microwave power that entered the system and the power that was absorbed

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by the sample. This can be attributed to the relative small size of the sample (100 mL) compared

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to the total volume of the multimode applicator (17 L) [22]. The good stability of the solid-state microwave generator allows microwave irradiation to be carried out even at low microwave

Fermentation using dried yeast without growth period

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3.3.

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power as presented in table 1.

The nutrient solution ((NH4)2SO4 5 g·L-1; KH2PO4 3 g·L-1; MgSO4.7H2O 0.5 g·L-1; Citric

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acid 5.4 g·L-1; Na2HPO4 6.9 g·L-1) was prepared following the same nutrients concentrations for each of experiment, only the glucose concentration being different (90, 120 respectively 240 g·L1

), to study the effects of microwaves on the metabolism of Saccharomyces cerevisiae at various

substrate concentrations.

Dried yeast is added to the glucose and nutrients solution after sterilization and cooling at 35 °C, and the fermentation vessel is placed inside the multimode applicator. Then, the generator is turned on at the desired targeted power and the temperature in the medium is maintained using the cooling coil at the same value as the temperature in the conventional fermentation vessel, which is in the thermostatic water bath.

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At given intervals, samples are taken for determination of ethanol concentration. Yeast cells are removed by centrifuging the samples for 4 minutes at 7000 rpm and by filtering through

3.4.

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a Nylon filter with a pore diameter of 0.45 µm.

Influence of SAR on the biological activity of S. cerevisiae during fermentation

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As part of the impact study of microwave irradiation on the metabolic activity of yeast

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and bioethanol production through glucose fermentation, the first step was to observe the

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influence of various specific absorption rates and to identify an optimum domain for microwave

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irradiation.

The fermentation process was carried out simultaneously in both the multimode

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applicator and the thermostatic water bath with mechanical stirring, at the following working parameters: glucose concentration in the nutrients solution 120 g·L-1, reaction temperature 35 °C,

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dry yeast concentration 16 g·L-1 nutrients solution (equivalent cell density around 3.2·108

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cells·mL-1), and specific absorption rates of 9.6, 15.7, 25 and 41.5 W·kg-1. The experimental data, obtained from microwave assisted fermentation, suggests that the

bioeffects of microwave irradiation are beneficial to the fermentation process carried out with

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Saccharomyces cerevisiae. Ethanol concentration is greater than the average value attained through conventional fermentation for all studied SAR values, and this beneficial impact cannot be attributed exclusively to the thermal effects of microwave heating; all experiments were carried out at constant temperature throughout the process, the same for both microwave and conventional fermentation processes. The highest ethanol concentrations correspond to SARs of

15.7 and 25 W·kg-1, respectively, outside this interval the microwave irradiation leading to a smaller increase in ethanol production. This fact can be easily observed in the comparative graphic representation of the average ethanol production rates for each experimental SAR (figs. 3 and 4). Based on these results, the following experiments have been carried out at a specific

3.5.

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absorption rate of 25 W·kg-1, which was less prone to variations in reflected power.

Influence of substrate concentration on the biological activity of S. cerevisiae

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during fermentation

The influence of glucose concentration on metabolic activity of yeast during glucose fermentation is represented in fig. 5, in both the presence and absence of microwave exposure.

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The glucose concentrations were 90, 120 respectively 240 g·L-1, while the other parameters

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remained unmodified (35 °C, 1.6 g dry yeast Red Ethanol per 100 mL nutrients solution).

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Increasing the substrate concentration from 90 to 120 g·L-1 led to an increase in ethanol

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productivity, yet further doubling the concentration to 240 g·L-1 led to a decrease in ethanol concentration, most likely due to the substrate inhibition of the yeast cells. In all cases,

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microwave exposure proved to have a beneficial impact and led to an increase of ethanol

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concentration against the conventional fermentation.

Fermentation using dried yeast and a growth period

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According to existing literature, growing yeast cells are able to attain fermentation rates

up to 33 times greater than fermentation rates in resting, mature cells [24]. For this reason,

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fermentation experiments have been carried out starting from a smaller quantity of dry yeast, 0.16 g respectively 0.32 g dry yeast per 100 mL glucose solution, after a growth period of 1 h respectively 16 h, which was carried out at 25 °C in glucose solution equivalent to 10 % of the total volume used for the fermentation process. The other parameters were: 60 g·L-1 glucose, reaction temperature 35 °C, SAR 25 W·kg-1.

Fig. 6, presents the evolution of ethanol concentration throughout the fermentation process of glucose, starting from dry yeast grown for 16 h at 25 °C. When using 0.32 g dry yeast per 100 mL glucose solution, the ethanol concentration remains constant for the last couple of hours of fermentation although the theoretical maximum is not achieved. Increasing the growth

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period from 1 h to 16 h led to a slight increase in ethanol concentration (fig. 7), confirming that the young cells reach higher fermentation rates than fully developed cells [24].

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Evolution of cell density throughout the fermentation process, determined through optical microscopy, shows that exposure to microwaves had no negative impact on cellular growth. As shown in fig. 8, the difference in cell density between samples from conventional and microwave

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assisted fermentation is insignificant. On the other hand, when comparing fermentation rates

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between conventional and microwave assisted fermentation, the difference is significant as

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shown in fig. 9. Although fermentation rates decrease in time because of an increase in cell

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numbers and lower available substrate levels, microwave assisted fermentation processes

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allowed for higher fermentation rates to be achieved. Different stages of cellular growth are visible in the images obtained through optical

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microscopy (fig. 10), a significant number of young cells can be identified, in both processes, conventional and microwave assisted, after 4 h, respectively 8 h from the beginning of the

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fermentation process.

Based on optical microscopy we could estimate cell viability, as presented comparatively

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in table 2, the differences in cell viability between the microwave assisted fermentation and the conventional process being insignificant. Unlike samples taken during the fermentation process without a growth period consisting mainly of fully developed cells, in this case, when samples were taken during the fermentation process after a growth period of 16 h (fig. 10), the number of young cells and budding cells has

increased considerably. After 10 h of the fermentation, yeast samples were separated by centrifuge and prepared through lyophilisation to analyse the effects of microwave exposure on cellular membrane integrity and cell morphology. As seen in fig. 11, fully developed cells reach 3 – 4 μm in diameter; depending on the stage of cell growth some cells have a diameter around 1 μm. There are no significant differences in cell morphology between samples obtained from

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conventional and microwave assisted processes; based on these observations we can assume that

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microwave exposure did not lead to an increase in cells death.

As expected, yeast samples which were grown for 16 h at 25 °C show more young cells

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and budding cells, unlike samples lacking a growth period which consist mainly of fully

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developed cells. In all the studied cases, with or without a growth period and in the presence and

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absence of microwaves, dead and damaged cells are visible. By correlating the information given

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by SEM analysis with those obtained through optical microscopy and GC analysis, we can

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conclude that in the studied SAR domain, the effects of microwave exposure are non-destructive. Microwave irradiation most likely led to an increase in mass transfer across the cellular

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membrane and in cell permeability, this positive effect manifesting itself through an increase in

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ethanol productivity and fermentation rates.

3.7.

Influence of temperature on microwave assisted and conventional fermentation

Figure 12 shows the results obtained in conventional and microwave assisted fermentation processes carried out at different reaction temperatures in the range of 35-39 °C. Data in Figure 12a shows that the best results are obtained at 37 °C. For microwave assisted

processes, the ethanol concentration is higher than that obtained in conventional processes, especially in the first part of the process (0 – 6 h), towards the end of the process, when the ethanol concentration approaches maximum, the values obtained in conventional and microwave assisted processes became similar. The most interesting result is that obtained at 37 °C. In this case, if the selective heating of the yeast cells and their temperature increase according to it

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would be the driving force, then the results obtained in the microwave assisted process would have been lower, and closer to the ethanol concentrations obtained at 39 °C in the conventional

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process. Instead, these results show that the microwave assisted process at 37 °C yields higher ethanol concentrations than that of the conventional processes at both the same temperature and at 39 °C. Moreover, the greatest difference between ethanol concentrations obtained in the

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microwave versus conventional processes appears at 39 °C, where the thermal stress is the most

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significant (the conventional process is carried out at the lowest rate). In terms of cell viability

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(the results shown in Figure 12b), microwave exposure did not have an additional deactivation

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effect on yeast cells, the viability recorded in the microwave assisted processes being even

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slightly higher than those determined for the conventional processes. The lowest viability values

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were recorded in the conventional process at 39 °C.

4. Conclusions

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An installation was setup which allows for glucose fermentation to be carried out in the

presence of MW, with better control of both SAR and the temperature of the fermentative

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medium. In all carried out experiments, a positive effect of MW exposure on yeast metabolic activity is noticed, ethanol concentrations obtained through MW assisted fermentation being higher than those from conventional processes.

An optimum SAR domain, between 15 and 25 W·kg-1, was determined, ensuring a significant effect on glucose fermentation by Saccharomyces cerevisiae, with an increase in fermentation rates of up to 20 %. Based on the observations made through electron and optical microscopy on cell growth and viability, we noticed that microwave irradiation favours ethanol production of the cells,

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against their growth.

Thermal balance calculations, as well as experimental data at different temperatures,

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confirm that the positive effect of microwave exposure on the fermentation process cannot be explained by the increase of the temperature of the yeast cells. Increasing cell membrane permeability and/or the activity of the enzymes catalysing the reactions of the cell’s metabolic

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pathways could be some of the reasons behind the beneficial microwaves bioeffects, yet further

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investigations are required to clarify these complex processes.

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Acknowledgment

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The authors acknowledge the financial support received from the Competitiveness Operational Programme 2014-2020, Action 1.1.4: Attracting high-level personnel from abroad in

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47/05.09.2016

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order to enhance the RD capacity, project: P_37_471, „Ultramint”, financed by contract:

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Figures

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Fig 1. Experimental setup – multimode applicator

Fig. 2 The temperature profile in the reactor, as computed by Comsol® Multiphysics (MW power of 3.9 W)

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Fig. 3. Influence of SAR on ethanol concentration (120 g·L-1 glucose, 35 °C, 1.6 g dry yeast per

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100 mL nutrients solution)

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Fig. 4. Influence of SAR on ethanol production rate (120 g·L-1 glucose, 35 °C, 1.6 g dry yeast

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per 100 mL nutrients solution)

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Fig. 5. Influence of glucose concentration on ethanol productivity (SAR 25 W·kg-1, 35 °C, 1.6 g

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dry yeast per 100 mL nutrients solution)

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Fig. 6. Influence of dry yeast concentration on ethanol productivity (glucose concentration 60

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g·L-1, SAR 25 W·kg-1, 35 °C, growth period of 16 h at 25 °C

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Fig. 7. Influence of yeast growth period on ethanol productivity (glucose concentration 60 g·L-1,

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SAR 25 W·kg-1, 35 °C, 0.16g dry yeast per 100 mL)

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Fig. 8. Evolution of cell density (glucose concentration 60 g·L-1, SAR 25 W·kg-1, 35 °C, 0.32g

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dry yeast per 100 mL, growth period of 16 h at 25 °C)

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Fig. 9. Evolution of SEPR in time (glucose concentration 60 g·L-1, SAR 25 W·kg-1, 35 °C, 0.16

ED

M

A

N

U

g dry yeast per 100 mL, growth period of 16 h at 25 °C)

Fig. 10. Yeast samples stained with Trypan Blue through optical microscopy, magnification 50x,

PT

conventional (a and c) and microwave assisted (b and d) after 4 respectively 8 h of fermentation (glucose concentration 60 g·L-1, SAR 25 W·kg-1, 35 °C, 0.32 g dry yeast per 100 mL nutrients

A

CC E

solution, growth period of 16 h at 25 °C)

A ED

PT

CC E

IP T

SC R

U

N

A

M

Fig. 11. SEM analysis of S. Cerevisiae, magnification 5000x, conventional (a) and MW assisted (b) after 10 h of fermentation (glucose concentration 60 g·L-1, SAR 25 W·kg-1, 35 °C, 0.32 g dry

A

CC E

PT

ED

M

A

N

U

SC R

IP T

yeast per 100 mL nutrients solution, growth period of 16 h at 25 °C)

IP T SC R

Fig.12 Influence of temperature on ethanol concentration (a) and on cell viability (b); (60 g·L-1

A

CC E

PT

ED

M

A

N

U

glucose, 1.6 g dry yeast per 100 mL nutrients solution; SAR = 19.3 W·kg-1)

Tables Table 1. SAR determination for different operating conditions Absorbed Power

Average SAR

(W)

(W)

(W·kg-1)

17.2 ÷ 17.7

4.24 ÷ 4.44

41.5±0.96

15.1 ÷ 15.2

3.1 ÷ 3.4

32.4±1.50

11.3 ÷ 11.4

2.57 ÷ 2.66

25±0.43

7.5 ÷ 7.65

1.59 ÷ 1.68

15.7±0.43

6.1 ÷ 6.2

1.33 ÷ 1.35

4.4 ÷ 4.5

0.97 ÷ 0.99

SC R

13.2±0.10 9.6±0.10

U N

Table 2. Comparative cell viability

Conventional

MW-assisted

62.8±1.3

63.5±1.2

60.2±1.2

61.6±1.3

After 6 h, growth period of 16 h

68.4±1.4

70.9±1.3

After 8 h, growth period of 16 h

64.4±1.5

68.5±1.4

M

After 6 h, without a growth period

A

Cell viability, %

CC E

PT

ED

After 8 h, without a growth period

A

IP T

MW Power