Research ArticleReceived: 8 February 2010 Revised: 30 April 2010 Accepted: 1 May 2010 Published online in Wiley Interscience:

(www.interscience.wiley.com) DOI 10.1002/jctb.2441

Kinetic study on ethanol production usingSaccharomyces cerevisiae ITV-01 yeastisolated from sugar canemolassesBenigno Ortiz-Muniz,a Octavio Carvajal-Zarrabal,b

Beatriz Torrestiana-Sanchezc andMaria Guadalupe Aguilar-Uscangac

Abstract

BACKGROUND:Bio-ethanolproductionfromrenewablesources, suchassugarcane,makes itabiofuel that isbothrenewableandenvironmentally friendly. One of the strategies to reduce production costs and to make ethanol fuel economically competitivewith fossil fuels could be the use of wild yeast with osmotolerance, ethanol resistance and low nutritional requirements. Theaim of this work was to investigate the kinetics of ethanol fermentation using Saccharomyces cerevisiae ITV-01 yeast strain in abatch system at different glucose and ethanol concentrations, pH values and temperature in order to determine the optimumfermentation conditions.

RESULTS: This strain showed osmotolerance (its specic growth rate (max) remained unchanged at glucose concentrationsbetween 100 and 200 g L1) as well as ethanol resistance (it was able to grow at 10% v/v ethanol). Activation energy (Ea) andQ10 values calculated at temperatures between 27 and 39

C, pH 3.5, was 15.6 kcal mol1 (with a pre-exponential factor of3.8 1012 h1 (R2 = 0.94)) and 3.93 respectively, indicating that this system is biologically limited.CONCLUSIONS: The optimal conditions for ethanol productionwere pH 3.5, 30 C and initial glucose concentration 150 g L1. Inthis case, a maximum ethanol concentration of 58.4 g L1, ethanol productivity of 1.8 g L1 h1 and ethanol yield of 0.41 g g1were obtained.c 2010 Society of Chemical Industry

Keywords: temperature; activation energy; pH; glucose; ethanol; S. cerevisiae ITV-01

NOTATIONmax: Maximum specic growth rate (h1)0: Pre-exponential factor (h1)Ea: Activation energy (kcal mol1)R: Ideal gas constant (kcal mol1K1)T : Temperature (K)Q10: The activity of a microorganism.T2: The higher temperature.T1: The lower temperature.2: The specic growth rate at the higher temperature.1: The specic growth rate at the lower temperature.Yx/s: Biomass yield (g biomass g1 sustrate).Yet/s: Ethanol yield (g ethanol g1 sustrate).Yet/x: Ethanol specic yield (g ethanol g1 biomass).Pet: Ethanol productivity (g ethanol L1 h1).

INTRODUCTIONAlcohol fermentation has been widely studied owing to itseconomic relevance in beverage, chemical and biofuel industries.However, technical issues during ethanol production such asthose related to contamination by lactic acid bacteria andBrettanomyces yeasts,1,2 inhibition by ethanol and high substrateconcentration35 still remain unclear. In addition, temperatureis not controlled at an industrial scale because of high costs. In

recent years, several investigations have been carried out in orderto establish some yeast characteristics such as osmotolerance,ethanol resistance, tolerance to low pH and high temperature inorder to improve alcohol fermentation yields.6

Like any other microorganism, yeasts have high water re-quirements in order to grow and maintain metabolic activity.Osmotolerance is the ability of yeast to grow in media with highsolute concentrations or in a low water activity environment.There are reports on yeast strains able to grow at high substrateconcentration (around 250 g L1), however, the specic ethanolproduction rates were rather low.68 A maximum substrate con-centration of 200 g L1 has been recommended9 because under

Correspondence to: Maria Guadalupe Aguilar-Uscanga, Instituto Tecnologicode Veracruz-UNIDA. Av. Miguel A. de Quevedo 2779, Col. Formando Hogar. CP.91860. Veracruz, Ver. Mexico. E-mail: gaguilar@itver.edu.mx

a Instituto Tecnologico Superior de Tierra Blanca, Av. Veracruz s/n. Col. PEMEX,95180 Tierra Blanca, Veracruz, Mexico

b Universidad Veracruzana, Area Bioqumica y Qumica de la Nutricion, JuanPablo II s/n, Boca del Ro, Ver. C.P. 94294, Mexico

c Instituto Tecnologico de Veracruz, Unidad de Investigacion y Desarrollo enAlimentos (UNIDA), Veracruz, Mexico

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this stress condition stimulation of glycerol production, needed tomaintain water activity inside the cell, has been observed.8

Onan industrial scale it is recommended toperformyeast cell re-cycling inorder to improveprocessproductivity. However, ethanolis a toxic compound that increases permeability and uidity ofthe plasma membrane, causing viability loss.10 Additionally, highethanol concentration can cause important metabolic changes inyeasts such as ATPase inhibition, denaturation of several glycoliticenzymes11 and changes in the cell wall.12

Tolerance to low pH values and high temperature are related,since pH changes can increase temperature tolerance to themaximum.13 Therefore, it is important to study the independenteffect of temperature, as well as the synergistic temperaturepHeffect. Temperature tolerance has also been related to mediacomposition and other physical factors such as water activity.High temperatures cause a decrease in cell viability, as well aschanges in mitochondria and uidity of the plasma membrane.14

In this regard, the use of an osmotolerant, ethanol resistant, lowpH value tolerant and thermotolerant strain can help the controlof alcohol fermentation spoilage, due to the fact that on a largescale this process is carried out under non-sterile conditions.

Therefore the aim of this research was to study the kinetics ofethanol fermentation from glucose by Saccharomyces cerevisiaeITV-01 isolated from sugar cane molasses. The effect of variousfermentation variables such as pH, temperature, initial sugar andethanol concentration on the kinetics of ethanol fermentation wasalso investigated.

EXPERIMENTALMicroorganismThe wild type yeast S. cerevisiae ITV-01 was previously isolatedfrom sugar cane molasses.7

Culture mediaS. cerevisiae was stored at 4 C using a culture media with thefollowing composition (g L1): glucose, 150; yeast extract, 20;and agar, 20. Preculture media was (g L1): glucose, 100250,at 50 intervals; yeast extract, 12.5, at 0.5 intervals; KH2PO4, 8.0:(NH4)2SO4, 5.0; and MgSO4 7H20, 1.0. In order to evaluate ethanolresistance, two initial ethanol concentrations were tested (3 and6% w/v), and added after media sterilization. The initial pH wasadjusted to the desired value from 2.0 to 6.5, at intervals of 0.5 pHunits, using 80% (v/v) orthophosphoric acid solution. The culturemedium was sterilized for 15 min at 121 C.

Preculture conditionsThe preculture was made in a 250 mL Erlenmeyer ask with100 mL liquid medium and stirred at 150 rpm. After inoculation,each Erlenmeyer ask was incubated at the test temperature (27,30, 33, 36 and 39 C) for 12 h. Two precultures were prepared toobtain inoculums of 6 106 viable cells mL1.

Culture conditionsFermentations were also carried out in 250 mL Erlenmeyer askswith 100 mL medium, for 36 h. The agitation was xed at 150 rpm(New Brunswick Scientic classic series C24KC RefrigeratedIncubator Shaker Edison NJ, USA). The asks were inoculatedwith 6 106 viable cells mL1. All experiments were carried out induplicate.

Analytical techniquesYeast growth was measured by two techniques: (a) correlationoptic density (620 nm) against cell dry weight; and (b) direct countusing a Thoma Chamber. Viability was obtained using the methy-lene blue staining method.15 In addition, the culture mediumwas centrifuged for 10 min at 10 000 rpm using an Eppendorf Cen-trifuge5424 (Germany). Thesupernatantwasstoredat20 Cuntilanalysis. Glucose, glycerol, acetic acid and ethanol were measuredby high performance liquid chromatography (Waters 600,TSPSpectra System, Waters, Milford, MA, USA) using a Biorad AminexHPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA).The temperaturewas40 C,mobilephase sulfuric acid5 mmol L1,ow rate 0.4 mL min1 and an index refraction detector (Waters2414, TSP Refracto Monitor V, Waters) was used.

RESULTS ANDDISCUSSIONGlucose and ethanol inuence on growth and fermentativeactivityIn order to evaluate glucose and ethanol effects, four differentinitial glucose concentrations without ethanol (100, 150, 200 and250 gL1) and three initial ethanol concentrations (0, 3 and 6%v/v) were tested. In these experiments, the initial pH was set at 4.5and fermentation kinetics were performed at 30 C. Results showthat the duration of S. cerevisiae ITV-01 lag growth phase increasedfrom 4 to 18 h when initial ethanol concentration was increasedin culture media (Fig. 1). On the other hand, during fermentationwith no initial ethanol added, there was no difference in the lengthof latency phases at the different initial glucose concentrationsevaluated.

When the initial glucose and ethanol concentration wasincreased, lower nal biomass concentrations were obtained(from 4.5 to 1.2 g L1 weight dry) compared with initial glucoseconcentration of 100 g L1 and ethanol concentration of 0% (v/v).This is also shown in Fig. 2 where biomass productivities obtainedfor each treatment are presented. Based on these results, it ispossible to establish that the inhibition effect of ethanol on yeastgrowth is higher than that induced by glucose.

Nevertheless, the specic growth rate (max = 0.2525 to0.2671 h1) (Table 1) was not affected at 100 and 250 g L1 initialglucose concentrations when no ethanol was added. These resultssuggest that initial glucose concentrations do not affect specicgrowth rate and this might be related to the osmotolerant abilityof S. cerevisiae ITV-01.8

On the other hand, at higher initial glucose concentrationsglycerol production increased, while glycerol production waslower (1.4 gL1) when the initial ethanol concentration wasincreased in culture media (Fig. 3). This was probably due to adecrease: (a) in biomass production; or (b) in the water activity ofthe culture medium. Glycerol production was not stimulated bythe presence of ethanol in the culture medium, probably becauseglycerol does not carry out a physiological function when theyeast strain is submitted to the stress caused by the presence ofethanol in the fermentation medium (Fig. 3). These results agreewith thosepreviously reported16 for S.cerevisiaegrownonethanol,where glycerol production was lower than when using glucose.The inability of ethanol to stimulate glycerol production is not dueto an inhibitory effect, but is related to the osmotically mediatedregulation of glycerol 3-phosphate dehydrogenase (NAD+).17

When glucose and ethanol initial concentrations were higher,ethanol production decreased (Fig. 4), probably because of asynergistic inhibition effect of glucose and ethanol, even though

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Figure 1. Effect of initial glucose and ethanol concentrations on S. cerevisiae ITV-01 growth. Initial glucose: 100 (), 150 (), 200 () and 250 () g L1.

Figure 2. Biomass (Px ), glycerol (Pg) and ethanol (Pe) productivities at different initial glucose and ethanol concentrations at 36 h. Initial glucose: 100 (a),150 (b), 200 (c) and 250 (d) g L1.

the inhibitor effect of ethanol on its own production is higherthan that of glucose. Previously, it was established18 that glucoseinhibition was observed in S. cerevisiae UG5 at a concentrationabove 162 g glucose L1, in S bayanus above 70 g glucose L1

and in C. pseudotropicalis at 100 g glucose L1.19 This shows thatglucose resistance inyeasts is related to thespeciesand itsability toadapt under stress conditions, such as low water activity caused byhigh levels of substrate concentration. Ethanol decreases biomassand alcohol production by denaturation of glycolitic enzymes andalso by increasing plasma membrane uidity.8

As shown in Fig. 5, glucose was totally consumed at aninitial glucose concentration of 100 g L1 without ethanol added.However, atan initialglucoseconcentrationof150 g L1, a residualglucose concentration of 50 g L1 was obtained. The presence ofresidual glucose indicates an inhibition effect of glucose and/orethanol. Considering that S. cerevisiae ITV-01 is an osmotolerantstrain,7 the incomplete substrate consumption might rather bedue to the presence of either a combined inhibition effect or

nutrient limitation. In order todiscover the answer to this question,experimentswere carriedoutusingenrichedmediawith 150 g L1of initial glucose concentration.

Yeast extract effect on growth and fermentative capabilityIn order to discard the glucose inhibition effect, fermentations us-ing enriched media were performed. The experimental conditionsare summarized in Table 2. In these experiments, yeast extractconcentration was increased until no differences were found ingrowth and glucose consumption, because the inhibition effect ofyeast extract has not been reported.20

For Media 3 and 4, a lower residual glucose concentration(around 15 g L1) was observed (Table 3). This result suggeststhat yeast extract (YE) stimulates glucose consumption becauseit provides important cofactors like biotin and riboavin. It hasbeen reported previously that yeast extract is the most importantagent20,21 and that high concentrations of this component do notaffect either growth or fermentative ability in yeasts. When media

Table 1. Specic growth rates, (h1) of S. cerevisiae ITV-01 under different glucose and ethanol initial concentrations

Initial ethanol (%v/v) 0 3 6

Initial glucose (g L1) Specic growth rate, max (h1)

100 0.2671 0.0108a 0.2545 0.0278a 0.2350 0.0211a150 0.2581 0.0020a 0.2561 0.0406a 0.2333 0.0165a200 0.2525 0.0016a 0.2253 0.0152a 0.2297 0.0066b250 0.2586 0.0015a 0.2348 0.0027a 0.1927 0.0071c

a,b,c : statistically different. ANOVA P = 0.95.

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Figure 3. Glycerol production by S. cerevisiae ITV-01 at different initial glucose and ethanol concentrations. Initial glucose: 100 (), 150 (), 200 () and250 () g L1.

Figure 4. Effect of initial glucose and ethanol concentrations on ethanol production in S. cerevisiae ITV-01. Initial glucose: 100 (), 150 (), 200 () and250 () g L1.

with yeast extract concentrations of 2.0 and 2.5 g L1 were used,no important differences in ethanol volumetric productivity (DMS0.05) were found, although there was a lower residual glucoseconcentration with respect to fermentations carried out with 1.0and 1.5 g L1 yeast extract. This result suggests that when YEconcentration is 2 g L1 no inhibition effect is observed due tonutritional requirements. However, there is an inhibition effector limitation by other factors like pH or temperature. Based onthese results, further fermentationexperimentswere conducted inorder to evaluate the pH and temperature effect, at constant yeastextract concentration of 2 g L1. This yeast extract concentrationis lower than those used for other S. cerevisiae strains: 3.5 g L1

and 6 g L1.21,22 Yeast extract as a nitrogen source increasesfermentation capability in S. cerevisiae,23 resulting in diminishedresidual glucose in culture media,20 as observed in the resultsshown in Table 3.

pH effectThe effect of pH on the metabolism of S. cerevisiae ITV-01 wasanalyzedatpH levels between2.0 and6.5 at intervals of 0.5. Resultsshow that when the initial pH was 2.0, a high concentration ofresidual glucose was left, suggesting a powerful inhibitor effect.When the initial pH was between 3.0 and 3.5, this provokeda decrease in residual substrate to a minimum value (Table 4)which rose again at pH 6.5. These results indicated that thebest substrate consumption occurred in the pH range 3.03.5.Biomass yield did not vary signicantly in the pH range between3.0 and 6.5, indicating that yeast growth is not affected by initialpH. Ethanol yield was higher at pH values between 3.0 and3.5. The highest ethanol yield was obtained at pH values lowerthan 4.5, an advantage for S. cerevisiae ITV-01, because underthese conditions the risk of bacterial contamination is minimum.1

Bacterial contamination is an ongoing problem in commercial fuel

Figure 5. Combined effect of glucose and ethanol on substrate (glucose) consumption by S. cerevisiae ITV-01. Initial glucose: 100 (), 150 (), 200 ()and 250 () g L1.

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Table 2. Composition of evaluated enriched media

Component (g L1) Media 1 Media 2 Media 3 Media 4

Glucose 150 150 150 150

Ammonium sulfate 2 5.0 5.0 5.0

Magnesium sulfate 0.4 1.0 1.0 1.0

Potassium phosphate 5.0 8.0 8.0 8.0

Yeast extract 1.0 1.5 2.0 2.5

Table 3. Residual glucose, ethanol yield (Yet/s) and volumetricproductivity (Pet) of S. cerevisiae ITV-01 on different culture media

CultureMedia

Glucose residual(g L1)

Yet/s(g g1)

Pet(g L1 h1)

1 62.2 1.10a 0.3976 0.0092a 1.08 0.05a2 20.3 0.54b 0.3986 0.0103a 1.16 0.05a3 15.2 0.02c 0.4001 0.0109a 1.43 0.04b4 15.1 0.01c 0.3998 0.0085a 1.42 0.06b

a,b,c : Statistically different. DMS P = 0.95.

ethanol production facilities. Lactic acid levels often rise duringcontamination, suggesting that the most common contaminantsare lactic acid bacteria. Contaminants create a constant drain onthe carbon available for conversion to ethanol and compete forgrowth factors needed by yeast. They also produce byproductsthat are inhibitory to yeast, particularly lactic acid.24 Lactic acidbacteria are the primary bacterial contaminants of fuel ethanolfermentations, therefore, using S. cerevisiae ITV-01 at initial pH 3.5for ethanol production is a useful tool to avoid process spoilage.

Temperature effectThe effect of temperature on the growth of S. cerevisiae ITV-01was evaluated in terms of activation energy using the Arreheniusequation, which has been commonly used for thermodynamicstudies in bioprocesses. This equation describes the dependenceof growth rate on temperature:

max = 0eEaRT

The activation energy (Ea) for S. cerevisiae ITV-01 calculated inthe temperature range 2739 C at pH 3.5 was 15.6 kcal mol1

with a pre-exponential factor of 3.8 1012 h1 (R2 = 0.94). Thisvaluesuggests that the ITV-01strain is less sensitive to temperaturethan Schizosaccharomyces pombe, which has a higher activationenergy (26.2 kcal mol1) (Table 5) but it is more sensitive than P.tannophilus (Ea = 13.59 kcalmol1). The activation energy valueindicates if the process iswithin abiological or diffusional regimen.A biological regimen implies that temperature directly affectskinetic growthparameters and adiffusional regimen indicates thatphysical phenomena, like oxygen transfer, restrict the reaction. Ithas been reported that when the activation energy is higher than12 kcal mol1, the process is in a biological regimen.25 Resultsfrom this work indicate that ethanol fermentation with S. cerevisiaeITV-01 is carried out under a biological regimen.

Another way to evaluate the effect of temperature on microbialgrowth (or on the activity of a microorganism) is with the Q10value, which represents the increase in the specic growth ratewhen there is a 10 C increment.

Q10 =(

2

1

)( 10T2 T1

)

Like the activation energy, Q10 is used to indicate if the processis physical (Q10 < 1) or biochemical (Q10 > 2). Q10 is higherat low temperatures due to fact that the biochemical reactionsinvolved are limited by low enzymatic activity.25 In this studythe Q10 value was 3.93, as shown in Table 5. This value conrmsthat this process is limited by the biological regime and not bya diffusional regimen. This result agrees with the previous resultsobtained for activation energy (Ea). When temperature increases,Q10 decreases due to physical limitations such as reduction inoxygen diffusion. Therefore, activation energy and Q10 are valuesthat make it possible to evaluate the effect of the temperatureon microbial growth. Nevertheless, Ea is more signicant, beingconstant over a wide temperature range, while Q10 varies withtemperature.

Analysis of the synergistic effect of pH and temperature usinga statistic modelStatistical analysis using SPSS software was also conducted todetermine the existence of two possible optima (Fig. 6), usinga complete factorial 52 design, using pH values from 3.0 to

Table 4. Residual glucose, biomass yield (Yx/s), ethanol yield (Yet/s) and volumetric productivity (Pet) in S. cerevisiae ITV-01 at different initial pHvalues

Initial pH Glucose residual (g L1) Yx/s (g g1) Yet/s (g g1) Pet (g L1 h1)

2.0 133.1 3.2a 0.040 0.001a 0.2043 0.0024a 0.07 0.01a2.5 72.2 2.1b 0.031 0.002b 0.3873 0.0021b 0.93 0.03b3.0 6.25 1.2c 0.032 0.002b 0.4100 0.0042c 1.83 0.04c3.5 6.75 0.9c 0.033 0.001b 0.4080 0.0035c 1.84 0.03c4.0 19.9 2.3d 0.035 0.002bc 0.3900 0.0037d 1.63 0.03d4.5 22.0 1.8d 0.035 0.002bc 0.3869 0.0012d 1.58 0.03d5.0 23.2 2.2d 0.037 0.001c 0.3845 0.0065d 1.45 0.02e5.5 29.3 2.7e 0.040 0.001a 0.3973 0.0079d 1.25 0.05f6.0 45.0 3.1f 0.045 0.002d 0.3846 0.0057d 1.08 0.06g6.5 48.1 2.9f 0.051 0.001e 0.3662 0.0041e 1.00 0.04b

a,b,c,d,e,f,g : Statistically different. ANOVA P = 0.95.

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Table 5. Values of Ea and Q10 for different yeasts

YeastEa

(kcal mol1) Q10 Reference

B. bruxellensis B5d 16.61 3.76 25

P. tannophilus 13.59 1.86 26

Sch. pombe 26.20 3.33 25

S. cerevisiae 13.59 2.05 (25 C) This study

5.0, at 0.5 intervals, and temperature from 27 to 39 C, at 3 Cintervals. Results showed one optimum between pH 3.0 and 3.5and 27 and 30 C, and another at pH 4.55.0 and 3639 C.These results indicate that the optimal conditions for performingethanol fermentation with S. cerevisiae ITV-01 are pH 3.5 and30 C, since working at low pH values (3.03.5) limits lacticacid bacteria growth, resulting in a lower residual glucose. Itis interesting to note that the optimum conditions reported inprevious work27 with S. cerevisiae ITV-01 using different substrates,such as sucrose, claried cane juice and intermediate syrup B,were pH 5.5 and 35 C which are nearly the same conditionsas for the alternative optimum found by statistical analysis.The best ethanol production conditions were pH 3.5 and 30 Cwith 1.8 g L1 h1 ethanol productivity, closed to the 2 g L1 h1

previously reported28 for a batch process; this suggests that theuseof feedbatchorcontinuousculturesof this straincould result ina competitive process for ethanol production. These results agreewith those previously established at high substrate concentration:

theoptimized temperaturewas30 C29 and this value is lower thanthat reported for fed-batch culture (33 C).30 Almost all reports ofS. cerevisiae strains5,7,9,19,27,31,32 used pH values from 4.5 to 5.5,so the S. cerevisiae ITV-01 strain could be interesting owing to itsbenecial alcoholic fermentation performance at pH values as lowas 3.5.

CONCLUSIONSS. cerevisiae ITV-01 is an osmotolerant yeast, its specic growthrate remaining unchanged at glucose concentrations between100 and 250 g L1. Nevertheless, total uptake of substrate wasnot obtained at high glucose concentrations, probably becauseof a synergistic effect caused by substrate concentration andethanol produced. S. cerevisiae ITV-01 is also resistant to low pHvalues, which is an advantage for ethanol production, becauseit prevents contamination with lactic acid bacteria. The Ea andQ10 values indicate that a biological regimen limits alcoholicfermentation, so that the culture medium must be optimized inorder to maximize ethanol production. Results from this workshowed that the optimal conditions for ethanol production withS. cerevisiae ITV-01 were initial glucose concentration 150 g L1,pH 3.5 and temperature 30 C. In this case, a maximum ethanolconcentration of 58.4 g L1, ethanol productivity of 1.8 g L1 h1and ethanol yield of 0.41 g g1 were obtained.

ACKNOWLEDGEMENTSThe authors acknowledge the nancial support from the NationalCouncil of Science and Technology, Mexico (CONACYT) (schol-

Figure 6. Optimization prole generated by SPSS software. Temperature (), pH ().

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arship 45161) and critical reading of the manuscript by PatriciaHayward Jones MSc and Dulce Mara Barradas Dermitz MSc.

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