Available online at www.sciencedirect.com

Bioresource Technology 100 (2009) 1238–1245

Comparative hydrolysis and fermentation of sugarcaneand agave bagasse

J.M. Hernandez-Salas, M.S. Villa-Ramırez, J.S. Veloz-Rendon, K.N. Rivera-Hernandez,R.A. Gonzalez-Cesar, M.A. Plascencia-Espinosa, S.R. Trejo-Estrada *

Centro de Investigacion en Biotecnologıa Aplicada del Instituto Politecnico Nacional (CIBA-IPN), Exhacienda San Juan Molino, Km 1.5 Carretera Estatal

Tecuexcomac-Tepetitla, Tepetitla de Lardizabal, Tlaxcala, Mexico

Received 2 June 2003; received in revised form 12 August 2006; accepted 12 September 2006

Abstract

Sugarcane and agave bagasse samples were hydrolyzed with either mineral acids (HCl), commercial glucanases or a combined treat-ment consisting of alkaline delignification followed by enzymatic hydrolysis. Acid hydrolysis of sugar cane bagasse yielded a higher level ofreducing sugars (37.21% for depithed bagasse and 35.37% for pith bagasse), when compared to metzal or metzontete (agave pinecone andleaves, 5.02% and 9.91%, respectively). An optimized enzyme formulation was used to process sugar cane bagasse, which contained Cel-luclast, Novozyme and Viscozyme L. From alkaline–enzymatic hydrolysis of sugarcane bagasse samples, a reduced level of reducing sugaryield was obtained (11–20%) compared to agave bagasse (12–58%). Selected hydrolyzates were fermented with a non-recombinant strainof Saccharomyces cerevisiae. Maximum alcohol yield by fermentation (32.6%) was obtained from the hydrolyzate of sugarcane depithedbagasse. Hydrolyzed agave waste residues provide an increased glucose decreased xylose product useful for biotechnological conversion.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Sugarcane; Agave; Bagasse; Fermentation; Hydrolysis

1. Introduction

There is an increased interest in producing bioethanol asan octane booster or as a liquid fuel. Lignocellulosic mate-rials from different crop residues have been used for con-version to ethanol (Lynd et al., 1991; Wiselogel et al.,1996; Rabinovich, 2006).

One of the most extensively used agricultural residues issugarcane bagasse. In Latin America, sugarcane is widelyproduced and provides the main source of fermentablecarbohydrates for alcohol production. In 1997–98 Brazilproduced more than 15,000,000 m3 of ethanol obtainedfrom alcoholic fermentation of sugarcane juice, providing

0960-8524/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2006.09.062

* Corresponding author. Address: Bogota No. 5, Colonia AmericaNorte, Puebla, CP 72340, Mexico. Tel.: +52 555 7296000x87804; fax: +52248 4870762.

E-mail addresses: sr_tres@hotmail.com, sertre@hotmail.com (S.R.Trejo-Estrada).

all factory power from burning the residual bagasse(almost 100 million tons), (Boddey, 1993; CFC–ISO–GEPLACEA, 1999).

Using simultaneous saccharification and fermentation(SSF), conversion of lignocellulosic residues such asbagasse to ethanol is technically and economically feasible(Philippidis et al., 1992; Hinman et al., 1992).

In Mexico, sugar cane (47 million tons produced in1997) is used entirely for sugar production. The byproductblackstrap molasses (1.8 million tons produced in 1997) isfermented and distilled to produce alcohol. Molasses is alsoused as a feed supplement for cattle production or sold onto the international markets as a fermentation raw mate-rial. Bagasse, an important residue from sugarcane pro-cessing (13.6 million tons per year), could become animportant biomass source for saccharification and fermen-tation to produce bioethanol, but only 3% is processedin Mexico’s pulp and paper industry (Gonzalez-Cesar,2002).

J.M. Hernandez-Salas et al. / Bioresource Technology 100 (2009) 1238–1245 1239

Four million vehicles in Mexico City have an estimateddaily consumption of 17.2 million liters of gasoline. Assum-ing that an alcohol gasoline blend (containing 8% anhy-drous ethanol in the mixture) was used to alleviate airpollution, then all the molasses produced in the countrywould need to be fermented to anhydrous alcohol. How-ever, distilleries in Mexico produce only 53 million litersof alcohol per year, a volume equivalent to the potentialbiofuel consumption of 5 weeks in Mexico City. In Mexicothere are five major cities that will require improved motorvehicle emissions (Gonzalez-Cesar, 2002).

Considerable attention has been given to crop residuesderived from sugarcane, corn, wheat, rice and other cereals,as well as residues from the pulp and paper industry (Wis-elogel et al., 1996). Other sources have been considered forconverting lignocellulose to fermentable sugars, such aslegume shrub (Sericea lespedeza), sorghum (Sorghum bico-

lor) (Wiselogel et al., 1996), horse gram (Dolichos biflorus)(Reddy and Reddy, 2005), switchgrass (Panicum virgatum)(Pimentel and Patzek, 2005), and peat moss (Forsberget al., 1986).

The use of sugarcane bagasse in chemistry and biotech-nology has been recently reviewed (Pandey et al., 2000).Sugarcane bagasse has a complex structure, and is primar-ily composed of 25% lignin, 25% hemicellulose and 40–50%cellulose (Pandey et al., 2000; Neureiter et al., 2002). Con-version of sugarcane bagasse into fermentable sugars ispossible through thermal, chemical, or enzymatic hydroly-sis (Beguin and Aubert, 1994; Neureiter et al., 2002; Martinet al., 2002; Laser et al., 2002).

Treating lignocellulosic agricultural residues with min-eral acids can be used to release fermentable sugars. Thelevel and composition of the sugars released depends onthe type of acid and its concentration in the hydrolysis mix-ture (Israilides et al., 1978). The hemicellulose fraction canbe easily extracted and hydrolyzed by dilute acid treatment.Using an HCl solution, 85% of the xylose was recovered inthe hydrolyzate (Du Toit et al., 1984), whereas treatmentwith dilute sulfuric acid released mainly xylose, whichcould in turn be converted to ethanol by fermentation witha selected strain of Pichia stipitis (Roberto et al., 1991).Variations of hydrolysis conditions provided a methodfor acid hydrolysis of sugarcane bagasse, which proved tobe useful to produce 23 g of xylose per 100 g of bagasse,a near theoretical yield (Neureiter et al., 2002).

Alkaline treatment of sugarcane bagasse digests of thelignin matrix and makes cellulose and hemicellulose avail-able to enzyme degradation (Pandey et al., 2000; Rodri-guez-Vazquez and Diaz-Cervantes, 1994; Aiello et al.,1996). Similar treatment of sugarcane leaves enhanced sub-sequent hydrolysis by a cellulolytic enzyme complex(Krishna et al., 1998; Bhat, 2000). Alternatively, biologicaldelignification of bagasse is possible using selected strainsof Panus tigrinus, a white rot fungus (Costa et al., 2002; Gon-calves et al., 2002).

Commercial enzyme preparations have been used toconvert sugarcane bagasse to fermentable sugars. Krishna

et al. (1998) used Trichoderma reesei cellulase and cellobi-ase to hydrolyze sugarcane leaves after alkaline delignifica-tion. More recently, Martin et al. (2002) used a mixture ofendo-glucanases and cellobiases to saccharify steam pre-treated sugarcane bagasse. The resultant hydrolyzate hada sugar composition similar to that reported from chemi-cally treated bagasse, when analyzed by a modified TAPPIstandard method (Neureiter et al., 2002).

To provide a more diversified group of locally producedraw materials for bioethanol production, we initiated astudy to understand the conversion of lignocellulosic mate-rials derived from sugarcane and agave bagasse.

Vast areas of Mexico have agave species, wild perennialsucculent xerophytes that grow in dry habitats. Agaveplants are used for fiber (henequen) production in theYucatan peninsula (Sanchez-Marroquın, 1963); for tequilaand mezcal production in southern, west-central and wes-tern Mexican states; and also for producing a traditionalfermented beverage called pulque, in the highlands of Mex-ico’s central plateau, including agricultural areas within thelimits of Mexico City (Sanchez-Marroquın, 1963; Idarragaet al., 1999). The agave residues examined in the presentstudy are by-products from pulque manufacturing andare derived from Agave atrovirens (which is called‘‘maguey’’ in Mexico), a species widely distributed in theMexico’s central states: Tlaxcala, Puebla and Hidalgo(Sanchez-Marroquın, 1963).

Pulque production starts by extracting sap or ‘‘agua-miel’’, the fermentation substrate, which is fermented bymixed populations of yeast, fermentative and lactic acid bac-teria. When the sap extraction is done, a cellulosic residuecalled metzal is obtained. ‘‘Metzontete’’ is the whole agaveplant as it ends its productive life, providing an excellent cel-lulosic material for bioconversion to fermentable sugars.

Although the composition of A. atrovirens bagasse hasnot been reported, fiber from other agave species has beenrecently studied. Cedeno-Cruz and Alvares-Jacobs (1999)reported that the composition of bagasse from Agavetequilana (var. Weber, from the tequila industry) is 43% cel-lulose; 19% hemicellulose and 15% lignin. Its potential usefor pulp and paper production and for animal feeding hasbeen recently demonstrated (Idarraga et al., 1999; Iniguez-Covarrubias et al., 2001). It is possible that the increasedglucan and decreased lignin content of agave fiber may pro-vide a good source of fermenting sugars produced by chem-ical and enzymatic hydrolysis and saccharification.

Fermentation of hydrolyzate from lignocellulosic resi-dues is necessary to produce alcohol (Wyman, 1996). Forthat purpose, both pentose-utilizing yeast strains (P. stipitis;Roberto et al., 1991), and non-pentose-utilizing yeast strains(Saccharomyces cerevisiae; Krishna et al., 1998) have beenused. Recombinant strains of Escherichia coli, Zymomonas

mobilis and S. cerevisiae capable of hexose and pentosecatabolism and high ethanol production have also been con-structed. Their use has made the conversion of lignocelluloseto ethanol economically feasible (Mohagheghi et al., 2002;Moniruzzaman and Ingram, 1998; Martin et al., 2002).

1240 J.M. Hernandez-Salas et al. / Bioresource Technology 100 (2009) 1238–1245

The aim of the present study was to examine the produc-tion of free sugars through chemical or enzymatic hydroly-sis of agave residues obtained from the pulque industry,and to compare that process to the better understood pro-cess of saccharification of sugarcane bagasse. Evidence ispresented that supports the potential use of fermentablesugars thus obtained for ethanol production byfermentation.

2. Methods

2.1. Sources of lignocellulose

Sugarcane bagasse samples were kindly provided byIngenio Central Motzorongo, a sugar factory based inMotzorongo, Veracruz, Mexico.

Agricultural agave residues (from A. atrovirens), metzaland metzontete were kindly provided by Rancho San Isidro,a pulque producer from Nanacamilpa Tlaxcala, Mexico (seeTable 1).

Mezontete was thoroughly washed with distilled water,and dried in an oven at 70 �C for 72 h. Metzal was onlydried, using the same procedure. Agave and sugarcane res-idues were ground separately in a small disc mill (BauerBros. Co. Springfield, Ohio, USA. SS mod. 148-2, 3540RPM, 300 mm ID). The cellulosic materials were thensieved through stainless steel sieves. The fractions between0.149 and 1.68 mm were collected for further processing.

2.2. Steam pretreatment

A steam treatment was applied to agave and sugarcanebagasse. Two grams of either metzal, metzontete or sugar-cane bagasses were mixed with distilled water. The mix-tures were autoclaved at 121 �C and 1.1 kg/cm2 for 4 h.

2.3. Acid hydrolysis

Lignocellulosic materials were also hydrolyzed withdilute acid and steam treatment. HCl (1.2% v/v) was addedto bagasse samples in a 5:1, 10:1 or 15:1 ratio (mL of solu-tion/g of bagasse by weight) which accounted for concen-trations equivalent to 30, 60 and 90 mg HCl/g of drymatter. Each suspension was autoclaved at 121 �C and1.1 kg/cm2 for 4 h. After hydrolysis 0.1 N NaOH was

Table 1Lignocellulosic materials from sugarcane and agave

Substrates

Metzal (Mz): The cellulosic material obtained from scrapping agavepinecone before and during ‘‘aguamiel’’ production

Mezontete (Mt): The whole agave biomass (leaves and pinecone) leftafter ‘‘aguamiel’’ production has ceased

Pith bagasse (B): Short fibers from the vascular bundle of sugarcanestalk. Low density, porous material

Depithed bagasse (Bc): Long fibers from the cortex of sugarcane stalk.High density

added and the pH adjusted to 5.0. The mixtures were thendiluted with distilled water to a final dilution of 30:1(30 mL of liquid phase/g of bagasse). The homogenizedmixture was then filtered and the liquid saved for analysis.

2.4. Alkaline and enzymatic hydrolysis

Bagasse samples (2 g) from agave and sugarcane weredelignified with alkali before enzymatic hydrolysis. Thistreatment was carried by the use of a dilute NaOH solution(2% w/v) applied at a ratio of 5 mL of solution/g ofbagasse. Final concentration of NaOH was 50 mg/g ofbagasse. The mixture was then autoclaved at 121 �C and1.1 kg/cm2 for 4 h.

After this treatment, the pH was adjusted with NaOH(0.25 M), to 5.0–7.5, depending on the optimum pH valuereported by the enzyme manufacturer. The mixture wasthen blended with the corresponding enzyme preparationand diluted with distilled water to a final dilution of 15:1(mL of liquid phase/g of bagasse). For enzymatic treat-ment, delignified bagasse was treated with 0.40 g of the cor-responding enzyme. Final concentrations were 6% bagasseand 1.33% of enzyme (20% (w/w) of enzyme per bagassedry weight). When several enzymes were used, each onewas added at the same concentration.

Enzymatic hydrolysis was performed in a water bath setat 55 �C for 4 h. All the enzyme preparations were kindlyprovided by Novozymes (Mexico). The preparations usedwere Viscozyme, Celluclast, Novozyme, Cellubrix andPulpzyme.

Pulpzyme HC: Is a liquid preparation produced by sub-merged fermentation of modified genetically strain ofBacillus sp. with an activity of 1000 AXU/g (xylanase unitsper gram).

Cellubrix L: Is a liquid preparation of cellulase and cel-lobiase produced by separated fermentations of a strain ofThichoderma longibrachiatum and a strain of Aspergillus

niger.Novozyme: Is a liquid preparation produced by sub-

merged fermentation of monocomponent endo-glucanaseby modified genetically strain of Aspergillus spp. with anactivity of 5000 ECU/g (endo-cellulase units per gram).

Celluclast: Is a liquid preparation of cellulase producedby submerged fermentation of strain of T. reesei with anactivity of 1.5 l Celluclast 700 EGU/g (endo-glucanaseunits per gram).

Viscozyme: Is a liquid multienzyme complex preparationof arabinase, b-glucanase, hemicellulase, cellulase andxylanase produced by a strain of Aspergillus aculeatus withan activity of 100 FBG/g (fungal beta-glucanase, units pergram).

2.5. Reducing sugars

The reducing sugar concentration was determined by the3,5-dinitro salicylic acid (DNS) method (Miller, 1959), witha spectrophotometer (Hewlet Packard, Palo Alto, CA)

0

2

4

6

8

10

12

14

Metzal Metzontete

Control 30 60 90 control 30 60 90

Red

ucin

g Su

gars

(%

w/w

dry

mat

ter)

(mg of HCl/g of dry matter)

Fig. 1. Acid hydrolysis of agave bagasse. Effect of HCl (mg of HCl/g ofbagasse), on the hydrolysis of agave bagasse (yield of reducing sugars).

J.M. Hernandez-Salas et al. / Bioresource Technology 100 (2009) 1238–1245 1241

Model 8453. Hydrolysis by either chemical (acid or alkali)or enzymatic methods, was expressed as the yield for sugarsby the quantification of the reducing sugars concentrationin the filtrate, as determined by the DNS method. Yieldsfor sugars were calculated using the formula referred byNeureiter et al. (2002) as described below.

Y ¼ ðCV =W Þ � 100

C is the concentration of reducing sugars (g/L), V is totalvolume of the liquid phase (L) and W is dry weight ofthe corresponding lignocellulosic material (g). Y is the yieldof sugars expressed as percent of reducing sugars in dryweight.

2.6. Fermentation

The hydrolyzates were adjusted to pH 5 and amendedwith mineral nutrients (NH4)2SO4 (6 g/L), KH2PO4

(7.5 g/L), K2HPO4 (2.4 g/L), MgSO4 � 7H2O (0.6 g/L),CaCl2 (0.001 g/L), CuSO4 � 5ðH2OÞ (0.001 g/L), MnCl(0.001 g/L), ZnSO4 (0.001 g/L), CoCl2 (0.001 g/L) andsodium molybdate (0.001 g/L). The liquid media was ster-ilized and distributed in Erlenmeyer flasks (450 mL in 1 Lflasks), inoculated with a commercial strain of S. cerevisiae

(SAFMEX, Mexico 1 · 107 cfu/mL) and incubated at30 �C for 48 h. The flasks were sampled (50 mL) to deter-mine total reducing sugars and alcohol concentration.

3. Analytical methods

Samples were analyzed for sugars by HPLC. One milli-liters of each sample was diluted 15-fold and 10 ml of thismix was passed through 3 cm3 Sep-Pak C-18 cartridges(Waters, Milford, MA); (preequilibrated with 5 mL metha-nol and 10 mL of deionized water), in order to eliminatehydrophobic substances. The clarified samples were thenfiltered through 0.22-lm Acrodisc membrane filters(Waters, Milford, MA); (Wright, 1995). The filtered sam-ples were injected (5 lL) into a Shodex SC1011 (ShokoCo., Tokyo, Japan) column and eluted with water at a flowrate of 1.0 mL/min and a constant temperature of 80 �C.Refractive index detection was used (Hewlett–Packard,D112), for HPLC analysis (Hewlett–Packard series 1100,USA), to determine monosaccharide composition. Individ-ual solutions of true glucose and xylose (Sigma–Aldrich,USA) were used to prepare calibration curves.

3.1. Ethanol analysis

Fermentation musts were distilled and the distillate ana-lyzed by gas chromatography using an HP6890 chromato-graph (Hewlett–Packard, USA) with an FID detector. Theinlet temperature was 200 �C, the mode was split flow(40:1) and the carrier gas was nitrogen. The HP Innowaxcolumn was 30.0 m long with a diameter of 340 lm, witha film thickness of 0.15 lm. The oven was warmed to aninitial temperature of 40 �C, held for 3 min, and then raised

to 70 �C at a rate of 3 �C/min. The oven temperature wasthen raised to 180 �C at a rate of 10 �C/min.

4. Results and discussion

4.1. Acid hydrolysis

Acid hydrolysis of bagasse was performed using HCl.The greatest post-treatment sugar concentration wasdetected in ryegrass straw using this acid, when comparedto other mineral acids (Israilides et al., 1978).

Fig. 1 shows the results of acid hydrolysis of agave wasteresidues. Both metzal and metzontete were treated withHCl at a concentration of 1.2% (v/v). An increase in theamount of acid solution from 5 to 15 (volume of acid solu-tion (mL)/weight of metzal (g)) correlated with anincreased saccharification. When compared to a controltreated only with water and autoclaved (1.2% hydrolysis),the concentration of 15 mL of acid solution per gram ofmetzal yielded 10% hydrolysis. In contrast, only 4.8% byweigh of metzontete was converted to reducing sugarsusing hydrochloric acid (Fig. 1).

Sugarcane waste by-products, namely pith bagasse anddepithed bagasses, were also hydrolyzed by dilute acid(Fig. 2). Even from the lowest (5:1) ratio of acid solutionto bagasse, more than 30% by weight was converted toreducing sugars, and at the highest ratio (15:1), a sacchar-ification level of more than 35% was obtained.

Acid hydrolysis of sugarcane bagasse yielded a higherlevel of reducing sugars (37.21% for sugarcane depithedbagasse and 35.37% for sugarcane pith bagasse), whencompared to either metzal or metzontete (5.02% and9.91%, respectively).

The analysis of variance was performed using theStudent t test. When the control (no treatment) was com-pared to the acid treated bagasse samples (90 mg HCl pergram of bagasse), statistically significant differences werefound (F = 6.15 > F0.10 = 5.54) for every kind of bagasse

0

5

10

15

20

25

30

35

40

45

Red

ucin

g Su

gars

(%

w/w

dry

mat

ter)

Depithed Bagasse Pith Bagasse

(mg of HCl/g of dry matter)

Control Control30 60 90 30 60 90

Fig. 2. Effect of acid concentration (mg HCl/g of dry matter) on therelease of reducing sugars from the hydrolysis of sugarcane bagasse (yieldof reducing sugars).

0

10

20

30

40

50

60

70

80

Con

trol

pulp

zym

e

cellu

brix

novo

zym

e

cellu

clas

tvi

scoz

yme

Con

trol

pulp

zym

e

cellu

brix

novo

zym

e

cellu

clas

t

visc

ozym

e

Agave Bagasse(Metzal) Agave Bagasse(Metzontete)

Red

ucin

g Su

gars

(%

w/w

dry

mat

ter)

Fig. 3. Effect of different enzyme preparations on the release of reducingsugars from hydrolysis of alkaline-treated agave bagasse.

0

5

10

15

20

Con

trol

pulp

zym

e

cellu

brix

novo

zym

e

cellu

clas

t

visc

ozym

e

Con

trol

pulp

zym

e

cellu

brix

novo

zym

e

cellu

clas

t

visc

ozym

e

Red

ucin

g Su

gars

(%

w/w

dry

mat

ter)

Depithed bagassePith bagasse

Fig. 4. Effect of different enzyme preparations on the release of reducingsugars from hydrolysis of alkaline-treated sugarcane bagasse.

1242 J.M. Hernandez-Salas et al. / Bioresource Technology 100 (2009) 1238–1245

tested. But when the different bagasse materials were com-pared with respect to their hydrolysis yield under diluteHCl treatment, no significant differences were found(F0 = 0.95 < F0.10 = 5.39). The four kinds of bagassetested, despite their different origins, responded similarlyto the dilute acid treatment.

4.2. Enzymatic hydrolysis

Agave and sugarcane bagasse samples were also treatedwith commercial enzyme preparations after autoclave pre-treatment. At optimal pH and appropriate concentrations,only from treatments with Novozyme and Viscozyme L,5–7% hydrolysis was obtained (data not shown).

An additional step of delignification using dilute NaOHwas then tested. This process renders cellulose more avail-able for enzymatic hydrolysis (Ghosh and Singh, 1993;Rodriguez-Vazquez and Diaz-Cervantes, 1994; Aielloet al., 1996; Pandey et al., 2000).

Alkaline delignification was performed using a diluteNaOH solution (2% w/v) mixed with 5 mL of solutionper gram of bagasse by weight and autoclaved for 4 h.After the pH was adjusted to 7, the partially delignifiedmaterials were treated with enzyme preparations, eitheralone or in combinations.

In contrast with results obtained from the acid treat-ment, the use of an alkaline–enzymatic process on agavebagasse (metzal and metzontete) resulted in a higher con-centration of reducing sugars (Fig. 3). From the treatmentof metzal and metzontete with dilute alkali and autoclave(controls in Fig. 3), only 5% of these materials were trans-formed to reducing sugars. Subsequent enzymatic hydroly-sis of metzal generated from 22% to 58% saccharification,depending on the enzyme preparation, whereas the met-zontete values ranged from 12% to 36%. In both cases,treating of delignified samples with Viscozyme L and Cel-luclast showed the highest hydrolysis rates.

The differences observed in saccharification of alkalinetreated bagasse samples were clearly due to the specificenzyme preparation used. Statistically significant differ-ences were obtained by ANOVA analysis using the t test(F0 = 2.99 > F0.10 = 2.27).

From alkaline–enzymatic hydrolysis of sugarcanebagasse samples (Fig. 4), the reducing sugar yield waslower compared to agave bagasse. For depithed bagasse,13–18% reducing sugars were generated, whereas 11–20%saccharification was obtained from pith bagasse. In thiscase, Cellubrix and Celluclast resulted in the highest hydro-lytic activity of all enzyme preparations tested.

Statistically significant differences were determined bythe ANOVA analysis when the alkaline–enzymatic sacchar-ification (the reducing sugar yield), was studied as an effectof the kind of bagasse used (F0 = 12.33 > F0.10 = 2.40).

The yield of saccharification products from the alkaline–enzymatic treatment of agave bagasse almost doubles theyield from sugarcane bagasse. This sharp difference canbe explained by the different lignin content of the corre-sponding tissues. Paturau (1969) and other authors (Pandeyet al., 2000; Neureiter et al., 2002) report an average lignincontent of 20–25% for both pith and depithed sugarcanebagasse, whereas Cedeno-Cruz and Alvares-Jacobs (1999)

0

10

20

30

40

50

60

70 MzMtBBcMzMtBBc

Glu

cose

(g/

l)

Acid hydrolysisAlkaline-enzymatic hydrolysis

Fig. 6. Glucose content of sugarcane and agave hydrolyzates determinedby HPLC-IR. Released by optimized enzyme preparation.

0 2 4 6 8 100

5

10

15

20

25

30

35

40

MzMtBBcMzMtBBc

Xylo

se (g

/l)

Acid hydrolysis Enzymatic hydrolysis

Fig. 7. Xylose from samples of sugarcane and agave bagasse hydrolyzatesdetermined by HPLC-IR. Released by optimized enzyme preparation.

J.M. Hernandez-Salas et al. / Bioresource Technology 100 (2009) 1238–1245 1243

report 15% for tequila agave, a similar level described forother agave species (Idarraga et al., 1999).

An optimized enzyme formulation was developed forpith and depithed bagasse which contained Celluclast,Novozyme and Viscozyme L in equal proportions. Afterthe alkaline treatment described previously, each enzymeconcentrate was added at a rate of 0.19 mL per gram ofbagasse. The same formulation was used for all four sub-strates, and the sugars released by alkaline and enzymatictreatment were characterized by HPLC.

Interestingly, the combination of enzymes used for fullhydrolysis did not include a pure cellobiase preparationlike Novozyme 188, which was previously used for sugar-cane hydrolysis (Martin et al., 2002). It is possible that cel-lobiase is present in some of the commercial enzymepreparations tested. Another explanation comes from thedifference between steam-enzymatic treatments reportedin other studies (Martin et al., 2002) and the alkaline–enzy-matic approach presented here. Alkaline treatment deligni-fies more efficiently than regular steam explosion(Rodriguez-Vazquez and Diaz-Cervantes, 1994).

Fig. 5 shows the results of bagasse saccharification byalkaline–enzymatic treatment for each class of lignocellu-lose material. HPLC analysis was performed for acidhydrolyzed samples (data not shown). The only carbohy-drates analyzed quantitatively in these samples were glu-cose and xylose.

Other non-characterized monosaccharides (mostly gal-actose and diverse pentose sugars) were separated anddetected but not quantified. Figs. 6 and 7 refer to theamount of glucose and xylose released by acid or alka-line–enzymatic treatments from sugarcane or agavebagasse, measured by HPLC-IR. Glucose concentrationwas clearly higher in hydrolyzates derived from the alka-line–enzymatic treatment for each bagasse sample(F0 = 26.57 > F0.10 = 4.06) As expected, the amount of glu-cose liberated from the hydrolysis of sugarcane pith and

0

10

20

30

40

50

60

70

Agave bagasse (Metzal)Agave bagasse (Metzontete)Sugar Cane bagasse (Depithed)Sugar Cane bagasse (Pith)

Red

ucin

g Su

gars

(%w

/w d

ry m

atte

r)

Fig. 5. Reducing sugars released from hydrolysis of alkaline-treatedsugarcane and agave bagasse. An optimized mixture was used, containingthree different enzyme preparations. Celluclast, Novozyme and Viscozymeat a concentration of 0.57 g of enzyme/g dry bagasse.

partially dephited bagasse was three times the amount ofxylose liberated. When saccharification is compared withineither the acid or the alkaline–enzymatic treated samples,no statistically significant differences can be attributed tothe kind of bagasse used (F0 = 0.4 < F0.10 = 3.62).

This results are consistent with the data reported byPaturau (1969), Wiselogel et al. (1996), Neureiter et al.(2002) and Martin et al. (2002).

Agave waste residues showed different hydrolysis prod-ucts depending on the type of tissue. Metzal, the residuefrom pinecone extraction, released more sugars comparedto the whole agave residue (metzontete). The concentrationof glucose released from metzal is 2.5 times higher than theconcentration of xylose released by the alkaline–enzymatictreatment.

Acid hydrolysis released less glucose compared to xylosefrom sugarcane tissues and metzontete. This trend was pre-viously reported from acid hydrolysis (Du Toit et al., 1984)and uncatalyzed conversion (Jacobsen and Wyman, 2002)of sugarcane bagasse. In contrast, acid hydrolysis of metzalrendered twice as much glucose as xylose, and almost halfof the amount obtained from alkaline–enzymatic hydroly-

Table 2Enzyme preparations used in hydrolysis of sugarcane and agave bagasse

Enzymes from Novo Nordisk

Pulpzyme HC: Is a liquid preparation produced by submergedfermentation of modified genetically strain of Bacillus sp. With anactivity of 1000 AXU/g (Xilanase units per gram)

Cellubrix L: Is a liquid preparation of cellulase and cellobiase producedby separated fermentations of a strain of Thichoderma longibrachiatum

and a strain of Aspergillus niger

Novozyme: Is a liquid preparation produced by submerged fermentationof mono-component endo-glucanase by modified genetically strain ofAspergillus spp. With an activity of 5000 ECU/g (endo-cellulase unitsper gram)

Celluclast: Is a liquid preparation of cellulase produced by submergedfermentation of strain of Thichoderma reesei: With an activity of 1.5 lCelluclast 700 EGU/g (endo-glucanase units per gram)

Viscozyme: Is a liquid multi-enzyme complex preparation of arabinase,b-glucanase, hemicellulase, cellulase and xylanase produced by a strainof Aspergillus aculeatus. With an activity of 100 FBG/g (fungal beta-glucanase, units per gram)

1244 J.M. Hernandez-Salas et al. / Bioresource Technology 100 (2009) 1238–1245

sis with the same material. A higher glucan content or thetype of hemicellulose present (Type A or B, Du Toit et al.,1984) may explain the difference of monosaccharidesreleased from metzal, a material present only in agave pine-cone (see Table 2).

The origin of metzal is structurally similar to theA. tequilana bagasse, but metzal does not undergo a ther-mal treatment during processing. Hemicellulose should stillbe present in the fiber, but the hydrolyzate should have alower concentration of toxic products (like hydroxy methylfurfural), when compared to bagasse from the tequilaindustry (Idarraga et al., 1999; Martin et al., 2002).

5. Fermentation

Fermentation of sugarcane and agave bagasse hydrolyz-ates was performed with a non-recombinant distillers strainof S. cerevisiae. Table 3 shows data on the content of reduc-ing sugars in the hydrolyzates and the ethanol yield afterfermentation. The ethanol yield is much lower than thatobtained by SSF as reported by Philippidis et al. (1992).As reported for sugarcane leaves (Krishna et al., 1998),the fermentation of hydrolyzates yielded an ethanol concen-tration that correlates with the bioconversion of glucose.No glucose, only xylose was detected by HPLC analysisof hydrolyzates after fermentation ceased (data not shown).

Table 3Sugars and alcohol yield of the fermentation of hydrolyzates from sugarcane

Raw material and treatment Sugar yield

Raw material Hydrolysis Reducing su

Agave metzal (Mz) Acid 24.82Agave mezontete (Mt) Acid 19.45Sugarcane depithed (B) Acid 35.43Sugarcane pith (Bc) Acid 29.9Agave metzal (Mz) Alkaline–enzymatic 56.37Agave metzontete (Mt) Alkaline–enzymatic 28.44Sugarcane depithed (B) Alkaline–enzymatic 38.38Sugarcane pith (Bc) Alkaline–enzymatic 50.08

6. Conclusions

The data presented here supports the use of agavebagasse for saccharification processes to obtain valuablefermentation products. In particular the use of metzal andpinecone waste residues from the pulque industry wouldprovide an excellent source of a cellulosic material easilyhydrolyzed to a glucose rich, xylose sparse product, compa-rable to other biotechnologically important bioresources,like corn straw, or sugarcane bagasse. In the present study,experimental data demonstrates a yield of 56% of sugarsfrom the conversion of agave metzal, by a combined alka-line–enzymatic treatment. A similar treatment for sugar-cane depithed bagasse allowed a similar sugar yield, but amuch higher alcohol yield by fermentation (32.6% alcoholby weight of sugars present in the hydrolyzate).

Both agave and sugarcane are important raw materialsfor manufacturing sugar and fermented beverages in Mex-ico. The distribution of the corresponding cultivars is geo-graphically distinctive, but their combined distributioncovers an important proportion of Mexican land. Lignocel-lulosic by-products derived from the industrial processingof those bioresources may provide raw materials for pro-ducing bioethanol, using chemical and biotechnologicalprocesses. Bioethanol, a renewable source of liquid fuel,has proved to be an octane booster and an excellent alter-native to decrease air pollution. Bioethanol may serve as analternative suitable for cities such as Mexico City andGuadalajara, the most densely populated areas in Mexico.

Acknowledgements

The authors thank M.Sc. Adrian Gonzalez Romo fromColegio de Posgraduados Campus Puebla, Dr. Sonia SilvaGomez and Dr. Ricardo Perez Aviles from Posgrado deCiencias Ambientales, Universidad Autonoma de Pueblafor their assistance. Thanks to Dr. Don L. Crawford, Uni-versity of Idaho, for helpful review of the manuscript. Thiswork was mainly supported by Sistema Regional Zaragoza(SIZA) from Consejo Nacional de Ciencia y Tecnologıa(CONACYT-Mexico) grant no. 980502014 and FundacionPRODUCE Puebla. This work was partially funded byPIFI, COFAA and Programa de Becas Institucionales(SIP) from Instituto Politecnico Nacional (IPN-Mexico).

and agave bagasse

Ethanol yield

gars (RS), g/L g/L (48 h) Yield (% w/w initial RS)

7.4 29.816.5 33.425.0 14.114.7 15.726.6 11.716.3 22.23

12.5 32.5712.9 25.76

J.M. Hernandez-Salas et al. / Bioresource Technology 100 (2009) 1238–1245 1245

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