Bioreactor Coupled with Electromagnetic Field Generator: Effects of ExtremelyLow Frequency Electromagnetic Fields on Ethanol Production bySaccharomyces cereWisiae

Victor H. Perez,* Alfredo F. Reyes, Oselys R. Justo, David C. Alvarez, and Ranulfo M. Alegre

Department of Food Engineering, School of Food Engineering, State University of Campinas, Brazil

The effect of extremely low frequency (ELF) magnetic fields on ethanol production bySaccharomyces cereVisiae using sugar cane molasses was studied during batch fermentation.The cellular suspension from the fermentor was externally recycled through a stainless steeltube inserted in two magnetic field generators, and consequently, the ethanol production wasintensified. Two magnetic field generators were coupled to the bioreactor, which were operatedconveniently in simple or combined ways. Therefore, the recycle velocity and intensity of themagnetic field varied in a range of 0.6-1.4 m s-1 and 5-20 mT, respectively. However, underthe best conditions with the magnetic field treatment (0.9-1.2 m s-1 and 20 mT plus solenoid),the overall volumetric ethanol productivity was approximately 17% higher than in the controlexperiment. These results made it possible to verify the effectiveness of the dynamic magnetictreatment since the fermentations with magnetic treatment reached their final stage in less time,i.e., approximately 2 h earlier, when compared with the control experiment.

IntroductionBrazil is a world leader in ethanol production from sugar cane

molasses using yeast, principallySaccharomyces cereVisiae.Both batch and continuous fermentation processes have beenlargely studied to improve productivity and ethanol yield.However, in the past decade several novel processes, includinggenetic modification of yeasts and bacteria (1), have beendeveloped due to the crescent world interest in new fuels. Someexamples of these processes include the use of immobilized cellsin unconventional materials (2), flocculent yeast andZymomonasmobilis (3) in several configuration bioreactors, fermentationprocesses at reduced pressure, and utilization of agroindustrialresidues (lignocellulosic biomass) as sugar source, among others.In this context, this work presents another alternative, using abioreactor coupled with two magnetic field generators that canattain higher productivity and ethanol yield.

Several published works from the past decade have tried toconfirm that static or pulse magnetic fields with low magneticinduction and extremely low frequency, respectively, may induceeffects on microbial and mammalian cells (4). These magneticfield bioeffects have received considerable attention in thescientific community because the interaction mechanisms ofthese fields with biological systems are unclear. Therefore,several works about the effects of the extremely low frequencyelectromagnetic field on animal and bacterial cells have beenpublished in the past decades. In contrast, for fungi (5-7) andyeast only comparatively few papers have been published. Withrespect to the last case, proliferation at 110 mT, 220 mT (8),and at 0.5 mT, 50 Hz (9, 10) and inhibition at 0.2 mT, 50 Hzunder alternating magnetic field (9) have been studied. However,these results have been contradictory because Ruiz-Go´mez etal. (11) did not observe alterations in the yeast growth understatic and sinusoidal 50 Hz magnetic field (0.35 and 2.45 mT).

Nevertheless, only a review paper has been published about thestate of the art of bioreactors assisted by magnetic field (12).Particularly, the biological effects of electromagnetic field onCandida utilis growth stimulation or inhibition have beenpreviously reported by our research group (13), and the aim ofthe present work was to verify the effects of an extremely lowfrequency electromagnetic field on ethanol production byS.cereVisiae in a fermentor coupled with two magnetic fieldgenerators.

Materials and MethodsMicroorganism. Saccharomyces cereVisiaeused for ethanol

production was supplied by Bioengineering Laboratory of theFood Engineering School of The State University of Campinas,Brazil. The microorganism was maintained at 5°C on agar slantswhose medium contained [amounts in g L-1]: glucose [10],yeast extract [5], malt extract [3], pectin [5], and agar [20].

Culture Media and Inoculum Preparation. SaccharomycescereVisiae from the slant cultures was aseptically transferredand precultured in 500-mL Erlenmeyer flasks with 300 mL ofthe medium containing the following components [amounts ing L-1]: glucose [50], KH2PO4 [5], KCl [1.2], NH4Cl [1.5],MgSO4 [0.7], and yeast extract (5). The Erlenmeyer flasks wereincubated at 30°C and 200 rpm for 12 h. Fermentor wasinoculated with 10% v/v concentrated inoculum, and thefermentations were carried out using a medium similar to thoseused for inoculum preparation but without glucose and adding(NH4)2SO4 [11.75] and sugar cane molasses diluted to attainapproximately 160 g L-1 sugar concentration as substitute forglucose. All media were sterilized at 121°C for 20 min, andthe pH was adjusted before sterilization.

Fermentation Procedure. Fermentations were conducted for24 h in a 5-L BIOFLO-III stirred glass fermentor (NewBrunswick Scientific) using a working volume of 4 L. Theprocess was controlled at 30°C and 300 rpm, and pH wasadjusted to 6.5 by addition of 2 M NaOH or HCl before

* To whom correspondence should be addressed. Tel/FAX:+55 1935214029 or+55 19 35214050/+55 19 35214027.

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10.1021/bp070078k CCC: $37.00 © 2007 American Chemical Society and American Institute of Chemical EngineersPublished on Web 07/31/2007

inoculation. Samples were collected periodically for analysisof total reducing sugar (TRS), pH, conductivity, and ethanolproduction. The experiments were carried out in duplicate.

Experimental Setup. The bioreactor was coupled withelectromagnetic field generators in order to examine thebiological effects of the magnetic field. The cell suspension inthe bioreactor was recycled through the magnetic field genera-tors as shown in Figure 1, which allowed application of amagnetic field on the cell suspensions externally.

Two apparatuses were used for magnetic field generation:the first consisted of six magnets, arranged with opposite polesfacing (Figure 1). Each magnet was a square prism with twofaces of 4.8 cm× 4.8 cm and a thickness of 2.4 cm. Themagnets were inserted in a stainless steel box separatedhorizontally by 2 mm thick iron plates. The maximal or minimaldistances of the two boxes from the recycle tube could beadjusted by supplying magnetic inductions (B) of 5-20 mT,respectively, determined by a MG-3D Gauss-meter (WalkerScientific Inc.) with an accuracy of(1%. This apparatus makesit possible to obtain uniform magnetic fields in the spacebetween the magnets, generating a square wave with perpen-dicular field lines for fluid movement whose flowing throughthe field resulted in a relative frequency in a range of extremelylow frequency (ELF) since the velocity input on the fluid inthe recycle was 0.6, 0.9, 1.2 and 1.4 m s-1.

In the second apparatus, a 0.20 m long double-layer solenoidwas used, which made it possible to stabilize the current sourceof 2 A. The magnetic induction was 8 mT, approximately, whichwas determined according to

where µ0 is the magnetic permeability of vacuum,N is thenumber of turns,I is the electric current, andL is the length ofcoil.

Analytical Methods. Total Reducing Sugar Determination.The remaining sugar in the fermentation medium was analyzedby the Somogyi-Nelson method (14). Samples of the alcoholicfermentation after separation of the cells by centrifugation weretreated with 9 vol of wolframic acid and centrifuged again. Theclear supernatant was used to determine the total reducing sugarconcentration.

ConductiWity and pH Determination.The conductivity of theculture spiritual medium was measured using a digital Analyzer(650 model) with temperature compensation, and the pH wasmeasured using the digital potentiometer Digimed (DM 20model), respectively.

Ethanol Quantification. The ethanol concentration in themedia was determined according to Salik and Povh (15). Thismethod is based on the ethanol oxidation reaction to acetic acidusing potassium dichromate in acid medium. That reaction wascarried out with the distilled ethanol of the fermented medium,and the determination accomplished using standard curves ofethanol at 600 nm, using a spectrophotometer, model 4800(Hach Company).

Results and Discussion

The results are summarized in Figure 2 and Table 1 as afunction of fermentation time for each magnetic field induction.The control experiment was accomplished by maintaining therecycling loop during all fermentation process but withoutapplication of magnetic field. In this case, the ethanol concen-tration reached 64 g L-1, which represent 83.54% of thetheoretical yield. After 15 h of fermentation, the substrateconsumption stopped and the ethanol yield was low, probablydue to product inhibition.

In order to verify the viability of the procedure proposed here,three different conditions of dynamic magnetization wereapplied, the first using the lowest magnetic field induction(maximum distance between magnets) in the main apparatus,the second using the highest magnetic field induction (minimumdistance between magnets) in the main apparatus, and the thirdusing the highest magnetic field induction (minimum distancebetween magnets) in the main apparatus plus solenoid. Theresults verified the effectiveness of the dynamic magnetictreatment, as the fermentations with magnetic treatment reachedtheir final stage in less time, i.e., approximately from 2 h earlier,when compared with the control experiment. All of the magneticfield inductions applied resulted in higher ethanol yield exceptfor 5 mT, which results were similar to the control experiment.However,, the last magnetic field treatment condition, i.e.,highest magnetic field intensity in the main apparatus plussolenoid, was the most attractive (Figure 2), since the ethanolconcentration reached 66 g L-1, which represent 86.7% of thetheoretical yield. In addition the volumetric sugar consumptionand the overall volumetric ethanol productivity were about 1.2times and 17% higher than in control, respectively (Table 1).

Figure 1. Scheme of experimental setup for the magnetic fieldtreatment of the cell suspensions during submerged batch fermentationof Saccharomyces cereVisiae. (1) Bioreactor, (2) cell suspensions, (3)three-way valve, (4) peristaltic pump, (5) secondary magnetic fieldgenerator (solenoid) coupled with current source of 2 A, (6) stainlesssteel tube, (7) principal magnetic field generator. Drawing not to scale.

B )µ0NI

L(1)

Figure 2. Magnetic field effect on the ethanol production byS.cereVisiae in submerged batch fermentation at 30°C and 300 rpm.The recycle velocity of the cell suspension was 0.9 m s-1. Total reducingsugar (∆, control;], 5 mT; O, 20 mT;0, 20 mT plus solenoid) andethanol production (2, control; [, 5 mT; b, 20 mT; 9, 20 mT plussolenoid).

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This fact suggests acceleration or a change in the metabolicroute of the process when compared with the control and withthe other magnetic field treatment conditions without solenoid.Metabolic changes induced by 220 mT static magnetic field onSaccharomyces cereVisiae have been suggested (8); a rise inthe acidity of fermented medium and CO2 production wereobserved. In addition, Ivanova et al. (16) reported increase of1.5 times for the ethanol production rate and 115% for theglucose uptake rate during the alcoholic fermentation in afluidized bed reactor byS. cereVisiaeunder static magnetic fieldwhen compared with unexposed culture. More recently, similarresults were also published by da Motta et al. (17), who observedthat the sugar consumption and ethanol production rate were1.3 and 3.4 times higher under inhomogeneous magnetic fieldthan in the respective control.

The changes in pH and electrical conductivity of the culturemedium during the fermentation process are shown in the Figure3. According to the later figure, differences among pH curveswere observed after 6 h of fermentation, approximately.However, the difference between the initial and final pH for allfermented media was practically similar. On the other hand,the difference between the initial and final conductivity of thefermented medium was higher in the exposed cell suspensionsthan in the control experiment, and this difference increasesduring the fermentation process. Thus, the results of themeasurements of electric conductivity suggest the influence ofthe induced electric field on the fermentative process. In thiscontext, it should be considered that induced current densitiesare a function of conductivity (σ), magnetic induction (B),frequency (f), and the tube radius (r). According to Figure 3,the electrical conductivity had moderate but visible increments,reaching its highest value when the fermented medium wasexposed at 20 mT plus solenoid, resulting in larger amounts ofconsumed sugar and produced ethanol. The associated currentscan be induced in the culture medium as a result of the magneticfield, since the fermentation medium consists of severalcomponents such as water and ion solutions (Na+, K+, Mg+2,NH4

+ in form of sulfate, phosphate and chlorate salts) and yeastcells that contain several components such as ionic solutions,proteins, and lipids, among others, which are susceptible tomagnetic field effect or induced electric fields. This resultsuggests that an interaction mechanism with the cell suspensionmay involve an electric field or magnetic and electric fields ina combined way, evidencing a regulatory metabolic effect.However, probably this effect was more due to the chargedmedium components than the cells. According to Polk and

Postow (4), at low frequencies a cell is essentially nonconducting(compared to the surrounding electrolyte) and electrical inter-actions among the cells have little effect on the bulk conductivityof the suspension. Thus, the conductivity at low frequenciesdepends on the ions and/or charged molecules fraction in themedium that change with the physiological metabolism in theyeast cells.

The ethanol production changed with the recycle velocity(relative frequency of the magnetic field) showing two positiveresponses for 0.9 and 1.2 m s-1 (Figure 4). Those bars representmore than 12% increase in the overall volumetric ethanolproductivity when compared with the other cases, suggesting asmall but positive “windows effect”. This phenomenon involvesthe susceptibility of the cells to effects of certain magnetic orelectromagnetic field parameters (18).

These results can be explained by making a reflection on theenergetic character of the process. We could verify that thecharged particles in the cellular suspension flowing through therecycle present movements that at times are not perpendicularto the magnetic field force lines. In these conditions, with theuse of the solenoid (withN uniform number of turns, over acylinder of radio r and lengthL, through which circulates aconstant current) it was possible to reorganize the ionic systemin term of a function of Lorents forces, leading the particles tomove until a position of dynamic equilibrium is reached, asillustrated in the Figure 5. Consequently, in the main magneticfield generator, in a similar way, the field force on the cellularsuspension will be more efficient and able to produce this ionicredistribution, which is accompanied by a potential differencerise in the cellular suspension by capacitive effect, the establish-ment of nonuniform electric field, and the circulation of inducedcurrents in the fermentation medium.

In addition, it is well-known that during anaerobic metabolismthe fermentation pathway from piruvate forms ethanol by thefollowing reactions sequence (eq 2):

In these reactions the NADH formed in the pathway topiruvate is oxidized. Thus, a strict oxidation-reduction balance

Table 1. Kinetic Parametersa for Batch Fermentation of SugarcaneMolasses bySaccharomyces cereWisiae at 30 °C and 300 rpm

magnetic induction20 mT

parametercontrol

experiment5 mT without

solenoidwithoutsolenoid

plussolenoid

P 64.0 63.9 63.93 66.0t 15.2 14 14 13Qs 9.84 10.63 10.55 11.45Qp 4.21 4.56 4.57 5.08Yp/s 0.427 0.429 0.433 0.443ε 83.54 83.97 84.72 86.70

a P ) final ethanol concentration (g L-1); Qs ) volumetric sugarconsumption (g L-1 h-1); Qp ) overall volumetric ethanol productivity (gL-1 h-1) calculated byP/t wheret is the time at which maximum ethanolwas obtained (h);Yp/s ) yield of ethanol on consumed sugar (g g-1); ε )efficiency of sugar-to-ethanol bioconversion (%) calculated as the ratiobetween experimentalYp/s value and the theoreticalYp/s value (0.5111 gg-1).

Figure 3. Magnetic field effect on the ethanol production byS.cereVisiae in submerged batch fermentation at 30°C and 300 rpm.The recycle velocity of cell suspension was 0.9 m s-1. Conductivity(2, control; [, 5 mT; b, 20 mT; 9, 20 mT plus solenoid) and pHprofiles (∆, control; ], 5 mT; O, 20 mT; 0, 20 mT plus solenoid).

piruvate98piruvate descarboxylase

acetaldehyde+ CO2

acetaldehyde+ NADH + H+98alcohol dehydrogenase

ethanol+ NAD+ (2)

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applies to substrate utilization and product formation infermentation (19).

In this context, we postulated that the positive effect on theethanol production under magnetic field is due to both mem-brane permeability and a redox system that are affected by theelectromagnetic field, resulting in alterations of ion transport(substrates) and consequently resulting in stimulatory effectson cell metabolism.

Finally, although the positive effect of magnetic field on theethanol production was verified, it is important to note that theyields obtained in this work were moderated, from 83.54% tothe control up to 86.7% to process under 20 mT plus solenoid.These results can be attributed to the strain ofS. cereVisiaeused,which affected the performance of the fermentative process. Inaddition, during the fermentation process the sugar metabolizedby yeast is used to produce simultaneously biomass and severalmetabolites, e.g., glycerol, organic acids, and acetaldehyde,among others, resulting in 95% of yield. However, accordingto the process conditions, which affect the yeast performance,yields of 90% are common.

Conclusions

The results presented in this report suggest that an ELFmagnetic field induces alterations in ethanol production byS,cereVisiae and that the magnetic field treatment can be easilyimplemented at an industrial scale. On the other hand, furtherstudies should be addressed in order to verify if magnetic fieldinduces increments in alcohol tolerance by yeast.

Acknowledgment

We are grateful for the financial support of The NationalCouncil for Scientific and Technological Development (CNPq),

Brazil and to Professor Meinardo A. Boizan Justis, Ph.D., forcomments and revision of this manuscript.

References and Notes

(1) Gutierrez, T.; Buszko, L. M.; Ingram, L. O.; Preston, J. F. Reductionof furfuryl alcohol by ethanologenic strains of bacteria and its effecton rthanol production from xylose.Appl. Biochem. Biotechnol.2002,98-100, 327-340.

(2) Alegre, R. M.; Rigo, M.; Joekes, I. Ethanol fermentation of a dilutedmolasses me´dium by Saccharomyces cereVisiae immobilized onchrysotile.Braz. Arch. Biol. Technol.2003, 46, 751-757.

(3) Cofermentation of glucose, xylose and arabinose by genomic DNA-integrated xylose/arabinose fermentating strain ofZymomonas mo-bilis AX101. Appl. Biochem. Biotechnol. 2002, 98-100, 885-898.

(4) Polk, C.; Postow, E.Handbook of Biological Effects of Electro-magnetic Field, 2nd ed.; CRS Press: New York, NY. 1996.

(5) Nagy, P.; Fischi, G. Effects of static magnetic field on growth andsporulation of some plant pathogenic fungi.Bioelectromagnetics2004, 25, 316-318.

(6) Melek, S. C.; Gonzalez, A. M.; Camue, H. C.; Diaz, C. M. C.;Pardo, A. M. T.; Haber, V. P.; Fong, A. R. Estudio del crecimientodel hongoAspergillus nidulansbajo el efecto de campos electro-magneticos de baja frecuencia.Tecnol. Quı´m. 1996, 16, 26-31.

(7) Melek, S. C.; Gonzalez, A. M.; Camue, H. C.; Diaz, C. M. C.;Pardo, A. M. T.; Haber, V. P.; Fong, A. R. Estudio del efectogenotoxico del campo electromagne´tico de baja frecuencia sobre elcrecimiento del hongoAspergillus nidulans. Tecnol. Quı´m. 1996,16, 32-39.

(8) Motta, M. A.; Montenegro, E. J. N.; Stamford, T. L. M.; Silva, A.R.; Silva, F. R. Changes inSaccharomyces cereVisiaedevelopmentinduced by magnetic fields.Biotechnol. Prog.2001, 17, 970-973.

(9) Mehedintu, M.; Berg, H. Proliferation response of yeastSaccha-romyces cereVisiae on electromagnetic field parameters.Bioelec-trochem. Bioenerg.1997, 43, 67-70.

(10) Pichko, V. B.; Povalyaeva, I. V. 1996. Microorganism productivityelectromagnetic stimulation and its mechanisms.Appl. Biochem.Microbiol. 1996, 32, 425-428.

(11) Ruiz-Gomez, M. J.; Prieto-Barcia, M. I.; Ristori-Bogajo, E.;Martınez-Morillo, M. Static and 50 Hz magnetic fields of 0.35 and2.45 mT have no effect on the growth ofSaccharomyces cereVisiae.Bioelectrochemistry2004, 64, 151-155.

(12) Hristov, J. Y.; Ivanova, V. N. Magnetic field assisted bioreactors.In Recent Research DeVelopments in Fermentation and Bio-engineering 2; SignPost Research: Trivadrum, India, 1999; pp 41-95.

(13) Chaco´n, D. A.; Haber, V. P.; Fong, A. R.; Mas, S. D.; Serguera,M. N.; Rodriguez, O. J. Influence of the electromagnetic field inthe growth ofCandida utilisY-660 yeast.Tecnol. Quı´m. 1996, 15-16, 52-60.

(14) Somogyi, M. Notes on sugar determination.J. Biol. Chem.1952,195, 19.

(15) Salik, F. L. M.; Povh, N. P. Me´todo espectrofotome´trico para adeterminac¸ ao de teores alcoo´licos em misturas hidroalcoo´licas.Process. Eng. Ind. DeriV. 1993, 262-266.

(16) Ivanova, V.; Hristov, J. Y.; Dobreva, E. Al-Hassan Z.; Penchev,I. P. Performance of a magnetically stabilized bed reactor withimmobilized yeast cells.Appl. Biochem. Biotechnol.1996, 59, 187-199.

(17) Motta, M. A.; Muniz, J. B. F.; Schuler, A.; da Motta, M. Staticmagnetic fields enhancement ofSaccharomyces cereVisiaeethanolicfermentation.Biotechnol. Prog.2004, 20, 393-396.

(18) Cleary, S. F. A review of in vitro studies: low-frequencyelectromagnetic fields.Am. Ind. Hyg. Assoc. J1993, 54, 178-185.

(19) Baileys, J. E.; Ollys, D. F. Biochemical Engineering Fundamentals,2nd ed.; McGraw-Hill: New York, 1986.

Received March 22, 2007. Accepted June 29, 2007.

BP070078K

Figure 4. Effect of the recycle velocity of cell suspension throughthe principal magnetic field generator at 20 mT with solenoid on the(9) yield of ethanol on consumed sugar and (0) volumetric ethanolproductivity by S. cereVisiae in submerged batch fermentation at 30°C and 300 rpm.

Figure 5. Motion of charged particle in fermentation medium reachingthe dinamic equilı´brium under electromagnetic field (B, magneticinduction;q, charge of particle;V, particle velocity;F, Lorentz force).

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