Revista Mexicana de Ingeniería Química

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Universidad Autónoma Metropolitana

Unidad Iztapalapa

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Morales-Martínez, T.K.; Rios-Gonzalez, L.J.; Aroca-Arcaya, G.; Rodríguez-de la Garza, J.A.

ETHANOL PRODUCTION BY Zymomonas mobilis NRRL B-806 FROM ENZYMATIC

HYDROLYSATES OF Eucalyptus globulus

Revista Mexicana de Ingeniería Química, vol. 13, núm. 3, 2014, pp. 779-785

Universidad Autónoma Metropolitana Unidad Iztapalapa

Distrito Federal, México

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Revista Mexicana de Ingeniería Química

CONTENIDO

Volumen 8, número 3, 2009 / Volume 8, number 3, 2009

213 Derivation and application of the Stefan-Maxwell equations

(Desarrollo y aplicación de las ecuaciones de Stefan-Maxwell)

Stephen Whitaker

Biotecnología / Biotechnology

245 Modelado de la biodegradación en biorreactores de lodos de hidrocarburos totales del petróleo

intemperizados en suelos y sedimentos

(Biodegradation modeling of sludge bioreactors of total petroleum hydrocarbons weathering in soil

and sediments)

S.A. Medina-Moreno, S. Huerta-Ochoa, C.A. Lucho-Constantino, L. Aguilera-Vázquez, A. Jiménez-

González y M. Gutiérrez-Rojas

259 Crecimiento, sobrevivencia y adaptación de Bifidobacterium infantis a condiciones ácidas

(Growth, survival and adaptation of Bifidobacterium infantis to acidic conditions)

L. Mayorga-Reyes, P. Bustamante-Camilo, A. Gutiérrez-Nava, E. Barranco-Florido y A. Azaola-

Espinosa

265 Statistical approach to optimization of ethanol fermentation by Saccharomyces cerevisiae in the

presence of Valfor® zeolite NaA

(Optimización estadística de la fermentación etanólica de Saccharomyces cerevisiae en presencia de

zeolita Valfor® zeolite NaA)

G. Inei-Shizukawa, H. A. Velasco-Bedrán, G. F. Gutiérrez-López and H. Hernández-Sánchez

Ingeniería de procesos / Process engineering

271 Localización de una planta industrial: Revisión crítica y adecuación de los criterios empleados en

esta decisión

(Plant site selection: Critical review and adequation criteria used in this decision)

J.R. Medina, R.L. Romero y G.A. Pérez

Vol. 13, No. 3 (2014) 779-785

ETHANOL PRODUCTION BY Zymomonas mobilis NRRL B-806 FROM ENZYMATICHYDROLYSATES OF Eucalyptus globulus

PRODUCCION DE ETANOL A PARTIR DE HIDROLIZADOS ENZIMATICOS DEEucalyptus globulus USANDO Zymomonas mobilis NRRL B-806

T.K. Morales-Martınez1, L.J. Rios-Gonzalez1∗, G. Aroca-Arcaya2 and J.A. Rodrıguez-de la Garza1

1Departamento de Biotecnologıa, Facultad de Ciencias Quımicas, Universidad Autonoma de Coahuila, Blvd. V.Carranza, Z.C. 25280, Saltillo, Coahuila, Mexico

2Escuela de Ingenierıa Bioquımica, Pontificia Universidad Catolica de Valparaıso, Gral. Cruz 34, Valparaıso,Chile.

Received March 3, 2014; Accepted September 26, 2014

AbstractEthanol production by Zymomonas mobilis NRRL-806 was assessed using different enzymatic hydrolysates from pretreatedEucalyptus globulus saw dust pulp. Results showed that when enzymatic hydrolysate containing 79.5 g L−1 of glucose and8.81 g L−1 of acetic acid, a maximum ethanol yield of 92% and a productivity of 1.16 g L−1 h−1 were obtained, with atotal of 37 g L−1 of ethanol after 27 h. Acetic acid concentration present in enzymatic hydrolysates was the main factorthat contributed to decrease ethanol yield and productivity, in the case of hydrolysate E, where acetic acid concentrationwas higher (12.8 g L−1), ethanol yield and productivity were 80% and 0.99 g L−1 h−1 respectively.

Keywords: Eucalyptus globulus, enzymatic hydrolysates, Zymomonas mobilis NRRL-806, ethanol yield, acetic acid.

ResumenSe evaluo la produccion de etanol por Zymomonas mobilis NRRL-806 a partir de diferentes hidrolizados enzimaticos depulpa de aserrın de Eucalyptus globulus pretratada. Los resultados mostraron un maximo rendimiento de etanol de 92%y una productividad de 1.16 g L−1 h−1, con una concentracion total de etanol de 37 g L−1 despues de 27 h, cuandoel hidrolizado enzimatico contenıa 79.5 g L−1 de glucosa y 8.81 g L−1 de acido acetico. La concentracion de acidoacetico presente en los hidrolizados enzimaticos fue el principal factor que contribuyo a la disminucion del rendimiento yproductividad de etanol, en el caso del hidrolizado E, en donde la concentracion de acido acetico fue mayor (12.8 g L−1),el rendimiento y la productividad de etanol fueron de 80% y 0.99 g L−1 h−1 respectivamente.

Palabras clave: Eucalyptus globulus, hidrolizados enzimaticos, Zymomonas mobilis NRRL-806, rendimiento de etanol,acido acetico.

1 Introduction

The imminent ending of the non-renewable naturalenergetic resources brings the necessity to researchfor new renewable natural energetic resources, as wellas the maximization of productivity and lowering thecosts of already known processes. Biomass-basedenergy sources constitute an important alternative tohelp solving this problem (Letti and Karp, 2012;Domınguez-Maldonado et al., 2014). Lignocellulosic

biomass is the most abundant renewable biologicalresource and, since is outside the human food chainis also an attractive and relatively inexpensive rawmaterial (Pereira et al., 2012). Lignocelluloseis an abundant natural carbohydrate formed bybiopolymers such as cellulose, hemicellulose andlignin, which can be transformed into substituterenewable energy resource by microbial conversion

∗Corresponding author. E-mail: leopoldo.rios@uadec.edu.mxTel.: +52 (844) 4155752, Fax: +52 (844) 4159534

Publicado por la Academia Mexicana de Investigacion y Docencia en Ingenierıa Quımica A.C. 779

Morales-Martınez et al./ Revista Mexicana de Ingenierıa Quımica Vol. 13, No. 3 (2014) 779-785

(Roberto et al., 2003). The advantages of usingthem as raw materials for ethanol production aretheir low cost, easy availability, avoidance ofconflict with their use for food, and the potentialproduction of fuel from lignin (Liu et al., 2012).However, the heterogeneous and complex structure oflignocellulosic makes their fractionation and furtherbenefit difficult (Romanı et al., 2012). The mainstages of the cellulosic ethanol production process arepre-treatment, hydrolysis, fermentation, distillation,and further fuel upgrading (Starfelt et al., 2012).There are at least three methods of hydrolysis,including dilute acid hydrolysis, concentrated acidhydrolysis, and enzymatic hydrolysis. The enzyme-based process is more environmental-friendly andgives higher hydrolysis yield than acid hydrolysis(Zheng et al., 2009). Therefore enzymatic hydrolysisis advantageous because of its low toxicity, low utilitycost and low corrosion compared to acid or alkalinehydrolysis (Sun and Cheng, 2002; Taherzadeh andKarimi, 2007). In enzymatic hydrolysis the enzymescatalyze only specific reactions, and as result no otherside reactions occur or byproducts are formed andhydrolysis has the potential to achieve higher yieldof reducing sugars (Mukhopadhyay et al., 2011).Here cellulase and hemicellulase enzymes cleave thebonds of cellulose and hemicellulose respectively.Cellulase enzymes involve endo and exoglucanaseand β-glucosidases. Hemicellulolytic enzymes aremore complex and are a mixture of at least eightenzymes. Cellulose is hydrolysed to glucose whereashemicellulose gives rise to several pentoses andhexoses (Sarkar et al., 2012).

Zymomonas mobilis has been intensively studiedin ethanol fermentation. Zymomonas mobilismetabolizes sugar via the Entner-Doudoroff (ED)pathway, which produces less ATP and less biomass.More carbon sources are thus channeled to ethanol,resulting in an even higher ethanol yield than thatfound with the native ethanol fermenting yeastSaccharomyces cerevisiae. In addition, because ofproducing less ATP during ethanol fermentation, Z.mobilis maintains a higher glucose metabolic flux,normally three to five fold that of S. cerevisiae(Wirawan et al., 2012). The aim of this work wasto evaluate the production of ethanol from differentenzymatic hydrolysates from pretreated Eucalyptusglobulus saw dust pulp.

2 Materials and methods

2.1 Microorganism and media

Zymomonas mobilis NRRL B-806 strain wasprovided by the Environmental Biotechnology Lab.of Biochemistry Engineering Faculty, PontificiaUniversidad Catolica de Valparaiso, Chile (PUCV).Zymomonas mobilis NRRL B-806 was cultivated onM2 solid medium containing per liter: 100 g glucose,5 g yeast extract, 2 g KH2PO4, 1 g MgSO4 and 1.5g (NH4)2SO4 at 30 ºC. Inoculum was prepared fromthe stock culture, using the same culture medium. Thegrowth was carried out at 30 ºC for 48 hours.

2.2 Eucalyptus globulus pretreatment

Eucalyptus globulus saw dust pulp pretreatment wascarried out in a stainless steel reactor under thefollowing conditions: H2SO4 1% at 176 °C for10 minutes followed by a quick decompression toatmospheric pressure. The solids were separated byfiltering and washed with tap water to neutrality. Thesolid residue was dried in a forced-air oven at 105 °C.

2.3 Enzymatic hydrolysates

The study was carried out using three differentenzymatic hydrolysates from pretreated Eucalyptusglobulus saw dust pulp under the following conditions:(FPU:UI pNPG ratio and enzyme/sustrate relation).Enzymes used were of the series NS500 Novozymes(Bagsvaerd Denmark), cellulase (NS50013) and β-glucosidase (NS50010). Enzymatic hydrolysatescompositions are shown in Table 1 obtained under thefollowing conditions; 50 °C, with a stirring rate of 200rpm during 72 h.

2.4 Hydrolysates fermentation process

Enzymatic hydrolysates were supplemented withnutrients of M2 medium at a pH 5.5 (without glucose).Experiments were carried out in 250 mL Erlenmeyerflasks, each containing 100 ml de hydrolysates. In thecase of the hydrolysate E, hydrolysates C and D weremixed in a ratio 1:1 to obtain 75 ml of final volume.A control was studied containing M2 medium, usingglucose with an initial concentration of 100 g L−1.All the flasks were inoculated with 5 mL of cellularsuspension (0.15 g L−1). Incubation time was 51 h, at30 °C with a stirring rate of 150 rpm.

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Table 1. Enzymatic hydrolysates contents from pretreated Eucalyptus globulus saw dust pulp used forethanol production by Zymomonas mobilis NRRL B-806.

Parameter Hydrolysate A Hydrolysate B Hydrolysate C Hydrolysate D

FPU:UI pNPG ratio 1:3 1:2 1:3 1:2Enzyme/sustrate (%) 0.45 0.2 0.2 0.45

Glucose g L−1 87.27 79.55 76.91*Xylose g L−1 7.85 6.82 6.99*

Arabinose g L−1 10.36 5.77 9.15*Cellobiose g L−1 11.55 7.06 7.75*

Other carbohydrates g L−1 6.35 6.5 6.15*Acetic acid g L−1 8.87 8.81 12.8*Formic acid g L−1 0.08 0.05 0.1*

Glycerol g L−1 0 0 0** Hydrolysate E: C and D enzymatic hydrolysates mixture.

2.5 Analytical methods

Carbohydrates concentrations, inhibitor concentrationsand ethanol production were determined by HPLC(Agilent 1260 HPLC, IR Detector,). A Biorad HPX-87-H column was used, enabling quantification ofglucose, xylose, celobiose, arabinose, formic acid,acetic acid, ethanol. Operational conditions wereH2SO4 5mM as mobile phase, at a flow rate of 0.6mL/min, and 45 °C. All samples were centrifugedat 10,000 rpm for 5 min, and filtered and dilutes inHPLC water before being analyzed. Cellular growthwas determined by measuring optical density of cellsusing a UV/Vis spectrometer (Cary 50Bio) at 660 nmand correlated with dry weight. All the analyses wereperformed in triplicate.

3 Results and discussion

Figure 1 shows glucose consumption, ethanolproduction and biomass growth in control reactorwith Zymomonas mobilis NRRL B-806 using M2medium at 30 °C. Glucose was totally consumedafter 30 h and at this point maximum ethanolproduction and biomass growth was achieved witha 41.1 g L−1 and 2.1 g L−1 respectively, with anethanol yield (YE/S ) of 0.46 gethanol g−1

glucose (90% inrelation to theoretical value) and a productivity of1.37 g L−1 h−1 (see Table 2). Growth curve had acorrelation with glucose consumption, showing thatafter 30 h a stationary phase was reached, achievinga yield of 2.43 ×10−2 gbiomass g−1

glucose (2.43 %).Low biomass production is normally observed in

Manuscrito sometido a la Revista Mexicana de Ingeniería Química 8

\item Wirawan, F., Cheng, Ch., Kao, W., Lee, D., and Chang, J. (2012). Cellulosic ethanol 315 production performance with SSF and SHF process using immobilized {\it 316 Zymomonas mobilis. Applied Energy 100}, 19-26. 317

\item Yanase, H., Nozaki, K. and Okamoto, K. (2005). Ethanol production from cellulosic 318 materials by genetically engineered {\it Zymomonas mobilis. Biotechnology Letters 319 27}, 259-263. 320

\item Zheng, Y., Pan, Z., Zhang, R. and Wang, D. (2009). Enzymatic saccharification of 321 dilute acid pre treated saline crops for fermentable sugars production. {\it Applied 322 Energy 86}, 2459-2465. 323

324 325 326 327 328 329

330

331 Fig. 1. Glucose consumption (!), ethanol production (") and biomass growth (#) in 332 control reactor with {\it Zymomonas mobilis} NRRL B-806 using M2 medium at 30 °C. 333 334

335

336

337

338

339

Fig. 1. Glucose consumption (�), ethanol production(�) and biomass growth (•) in control reactor withZymomonas mobilis NRRL B-806 using M2 mediumat 30 °C.

Zymomonas mobilis, and cell growth and fermentationare not linked (Parker et al., 1997). Accordingto Rogers et al. (1982) approximately 2% of thecarbon source is converted into biomass. Thisoccurs due to Entner-Doudoroff pathway used by thismicroorganism. This pathway yields only a singlemole of ATP per mole of carbohydrate fermented,giving Zymomonas the lowest molar growth yieldreported for bacteria (Swings and De Ley, 1977).

Figure 2 shows glucose consumption and ethanolproduction with Zymomonas mobilis NRRL B-806from different enzymatic hydrolysates of pretreatedEucalyptus globulus saw dust pulp (A, B and E)with medium M2 (without glucose) at 30 °C. Ethanolproduction with Zymomonas mobilis NRRL B-806

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Manuscrito sometido a la Revista Mexicana de Ingeniería Química 9

340

341

342

343

344

345

346

347

348

349

350 Fig. 2. Glucose consumption (solid markers) and ethanol production (unfilled markers) by 351 {\it Zymomonas mobilis} NRRL B-806 with different enzymatic hydrolysates from 352 pretreated {\it Eucalyptus globulus} saw dust pulp at 30 °C, hydrolysate A (! "); 353 hydrolysate B: (# $) ; hydrolysate E: (! "). 354 355

356

357

358

359

360

Fig. 2. Glucose consumption (solid markers) andethanol production (unfilled markers) by Zymomonasmobilis NRRL B-806 with different enzymatichydrolysates from pretreated Eucalyptus globulus sawdust pulp at 30 °C, hydrolysate A (� �); hydrolysateB: (• o) ; hydrolysate E: (N 4).

using enzymatic hydrolysate A reached a maximumethanol production of 39.8 g L−1 at 48 h, observingthat glucose consumption rate increased after 20 h,contributing this to the long time required for totalglucose consumption and to reach ethanol productionstationary phase (48 h). Ethanol yield was 0.45gethanol g−1

glucose (88% in relation to theoretical value)and a productivity of 0.83 g L−1 h−1 (see Table1). In the case of enzymatic hydrolysate B it wasobserved that glucose was consumed in less time andachieving a higher ethanol yield, with a maximumethanol concentration of 37 g L−1 at 32 h, with nosignificant increase after 27 h, at this point glucosewas almost totally consumed. Ethanol yield was 0.47gethanol g−1

glucose (92% in relation to theoretical value)

with a productivity of 1.16 g L−1 h−1 (see Table 2),In the case of enzymatic hydrolysate E, it can be

observed that glucose was totally consumed after 32h, obtaining a maximum ethanol concentration of 31.8g L−1. Ethanol yield was 0.41 gethanol g−1

glucose (80%in relation to theoretical value) and a productivity of0.99 g L−1 h−1 (see Table 2). Low ethanol yield maybe attributed to the high concentration of acetic acidpresent in the hydrolysate E (12.8 g L−1), causing apartial inhibition of Zymomonas mobilis NRRL B-806(Figure 2).

Kim et al. (2000) mentioned that acetic acid hasa potential inhibitory effect over Z. mobilis (10.9 gL−1 at pH = 6.0), followed by vanillin (0.04 g L−1),syringaldehyde (0.13 g L−1) hydroxymethylfurfural(0.9 g L−1) and furfural (0.3 g L−1) (Rogers et al.,2007) Previous reports by our research group (Rios-Gonzalez et al., 2012), in relation to inhibitory effectsof acetic acid in ethanol production by Zymomonasmobilis NRRL B-806 from pretreated Eucalyptusglobulus saw dust pulp (enriched with glucose),showed that ethanol yield was 86 % and 4.7 % whenacetic acid concentration was 6.8 g L−1 and 17.9 g L−1

respectively.Table 3 shows xylose, arabinose and cellobiose

concentration during fermentation process byZymomonas mobilis NRRL B-806 from pretreatedEucalyptus globulus saw dust pulp. No significantchanges in concentration for the three carbohydratesduring fermentation process were observed. This isattributed to the limited range of carbohydrates thatZymomonas mobilis can use as carbon source, whichconvert only hexoses such as glucose, fructose andsucrose (Rogers et al., 2007).

Table 2. Fermentation kinetic parameters using different enzymatic hydrolysates from pretreatedEucalyptus globulus saw dust pulp for ethanol production by Zymomonas mobilis NRRL B-806.

Kinetic Parameters M2 Control Hydrolysate A Hydrolysate B Hydrolysate E

YX/S 0.0243 - - -YE/S 0.46 (90) 0.45 (88) 0.47 (92) 0.41 (80)EP 1.37 0.83 1.16 0.99

YX/S Biomass yield (gbiomass g−1glucose).

YE/S Ethanol yield (gethanol g−1glucose).

EP Ethanol productivity (gethanol L−1 h−1)

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Table 3. Xylose, arabinose and cellobiose concentrations duringfermentation process by Zymomonas mobilis NRRL B-806 from

pretreated Eucalyptus globulus saw dust pulp.

Parameter (g L−1) Initial concentration Final concentration

A B E A B EXylose 7.85 6.82 6.99 7.10 6.19 6.59

Arabinose 10.36 5.77 9.15 10.11 5.53 9.01Cellobiose 11.55 7.06 7.75 11.21 7.01 7.49

Table 4. Main results reported in literature for ethanol production from lignocelullosic hydrolysates by strains ofZymomonas mobilis.

Feedstock Conditions Ethanol Referenceconcentration

Sugarcanemolasses

Zymomonas mobilis MCC 2427 Substrate 216 g/L of totalreducing sugar Temperature of 31 °C pH 5.13 Time 44 h

58.4 g/L (Maiti et al., 2011)

Mahualaflowers

Zymomonas mobilis MTCC 92 Substrate 100 g/L ofmahuala flowers slurry Temperature of 30 °C pH 6.5 Time96 h Cell concentration 10% (v/v)

38 g/L (Behera et al., 2011)

Kitchengarbage

Zymomonas mobilis 10225 Substrate 70 g/L of reducingsugar Enzyme load 100 U/g wet mass Temperature 30°CpH 6 Time 40 h Cell concentration 10% (v/v)

52 g/L (Ma et al., 2009)

Sugarcanebagasse

Zymomonas mobilis CP4 Substrate 80 g/L of glucoseEnzyme load 25 FPU/g Temperature 30 °C pH 5 Time 36h Stirring 150 rpm Cell concentration 4 g/L

60 g/L (da Silveira dos Santoset al., 2010)

Carob podextract

Zymomonas mobilis PTCC 1718 Substrate 113.82 g/L ofglucose Temperature 30 °C pH 5.2 Time 36 h Stirring 80rpm Cell concentration 0.017 g/ 50 mL

40 g/L (Vaheed et al., 2011)

Eucalyptusglobulus

Zymomonas mobilis NRRL-806 Substrate 79.5 g/L ofglucose Temperature 30 °C pH 5.5 Time 32 h Stirring 150rpm Cell concentration 0.15 g/L

38 g/L (this work)

In table 4 are shown the most prominent resultsreported for ethanol production from lignocellulosicfeedstocks by Zymomonas mobilis strains. As shownin this work and based on several reports (Maiti etal., 2011; Behera et al., 2011; Ma et al., 2009; daSilveira dos Santos et al., 2010; Vaheed et al., 2011)Zymomonas mobilis has the potential to revolutionizethe fuel ethanol industry commercially; laboratoryand pilot-scale operations indicate that it can generatenearly theoretical maximum yields from severalfeedstocks, including enzymatic hydrolyzate of wood-derived cellulose, such as Eucalyptus globulus sawdust pulp.

The main limitation of Zymomonas mobilis fora widespread industrial usage for ethanol production

is its capacity to utilize only hexoses. Pentosessuch as xylose and arabinose cannot be metabolizedby Zymomonas mobilis, unless it is geneticallymanipulated (Yanese et al., 2005; Lawford et al.,1997). However as mentioned by da Silveira dosSantos et al. (2010) if only the cellulose fractionis used in a two-stream model process, non-modifiedZymomonas mobilis strains can be a promisingalternative for ethanol production on industrial scale.

Conclusions

Although in this study only acetic acid, formicacid were quantified, other inhibitory compounds

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commonly found in lignocellulosic hydrolysates, mayalso be present in the medium, such as furfural,HMF, vanillin, syringaldehyde, and hydroxybenzoicacid, depending to the acid concentration, temperatureand other conditions used for hydrolysis, and suchcompounds may also affect the metabolism of themicroorganism used for fermentation. However,acetic acid is the most common fermentation inhibitor,causing a decrease in ethanol rate and yield. Its toxiceffect is basically due to its undissociated form. Aceticacid present in the different enzymatic hydrolysatesfrom pretreated Eucalyptus globulus saw dust pulpcaused a delay in ethanol production. High aceticacid concentration had adverse effect causing a delayin ethanol production efficiency, maximum ethanolproduction and glucose consumption.

AcknowledgementsThe authors are grateful to the Research andpostgraduate Office (CGEPI) of the UniversidadAutonoma de Coahuila for financial support to carryout an academic stay in the Biochemical EngineeringFaculty of Pontificia Universidad Catolica deValparaiso, Chile.

NomenclatureYX/S biomass yield (gbiomass g−1

glucose).YE/S ethanol yield (gethanol g−1

glucose).EP ethanol productivity (gethanol L−1 h−1)t time (h)

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