Fuel 118 (2014) 41–47

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Effects of the pretreatment method on enzymatic hydrolysis and ethanolfermentability of the cellulosic fraction from elephant grass

0016-2361/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.fuel.2013.10.055

⇑ Corresponding author. Tel.: +57 4 2196589; fax: +57 42196565.E-mail addresses: Juan.Pena.Alvarez@epm.com.co (P. Juan), lariospfa@gmail.

com, larios@udea.edu.co (R. Luis).

Cardona Eliana a, Rios Jorge a, Peña Juan b, Rios Luis a,⇑a Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombiab Empresas Públicas de Medellín E.S.P, Cra. 58 #42-125, Medellín, Colombia

h i g h l i g h t s

� Pretreatment with NaOH was the best method.� Optimization of the alkaline pretreatment was carried out.� 95% Of theoretical ethanol yield was obtained.� 88% Of lignin was removed.� 99% Of the cellulosic fraction in the solid was recovered.

a r t i c l e i n f o

Article history:Received 11 June 2013Received in revised form 22 October 2013Accepted 24 October 2013Available online 4 November 2013

Keywords:EthanolCellulosePretreatmentElephant grassPennisetum purpureum

a b s t r a c t

Elephant grass (Pennisetum purpureum) is a lignocellulosic material that has high potential for ethanolproduction in tropical countries due to their high availability and adaptability. Chemical and physico-chemical pretreatments like alkaline delignification, diluted acid hydrolysis, steam explosion, alkalineperoxide and aqueous ammonia soaking were performed in order to determine their effect on the hydro-lysis and the fermentability of the cellulosic fraction of this material. In an initial screening of the meth-ods, the alkaline pretreatment with NaOH yielded the highest concentrations of reducing sugars (34.4 g/L) and ethanol (15.1 g/L). A more detailed study of the effect of the alkaline pretreatment conditions(temperature, solid to liquid ratio, NaOH concentration and residence time) on the fermentability of ele-phant grass was carried out. Results showed that under pretreatment conditions of 120 �C for 1 h with2 wt.%. NaOH and a solid to liquid ratio of 1: 20 (wt.) the highest yield of ethanol was obtained, i.e.,26.1 g/L (141.5 mg ethanol/g dry biomass, 95% of theoretical yield). Furthermore, this pretreatmentallowed the removal of most of the lignin present in this material, i.e., 88% lignin removal. Besides, thispretreatment allowed a high recovery of the cellulosic fraction in the solid.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Due to the global energy crisis, lignocellulosic biomass has be-come very important as a promising raw material for obtainingsecond generation biofuels. Lignocellulosic materials have a highpotential for ethanol production because they are abundantrenewable resources, do not compete with food production, couldlead to the use of large quantities of agro-industrial wastes whosedisposal is problematic for the environment, and could use mar-ginal or degraded agricultural lands for growing energy crops.

Most tropical countries, like Colombia, have a high potential, interms of availability and variety, of lignocellulosic biomass becauseof their high solar radiation, diversity of climatic zones and

biodiversity. These advantages allow an easy adaptation ofdifferent species and the development of energy crops such asgrasses and forages. Among these, elephant grass (Pennisetumpurpureum) has a very high production yield of dry material, i.e.,40–50 tons/acres/year [1,2], under optimal conditions of growthand management.

Because of its chemical composition, lignocellulosic biomass isvery different from biomass with large content of sugars or starchwhich is customarily used in biofuel industry. The structure ofthese former materials, mainly composed by cellulose, hemicellu-lose and lignin, requires the process for biofuels production to beadjusted for each type of biomass, according to their componentcharacteristics. Therefore, a previous pretreatment step must beintroduced to obtain hydrolysable fractions that can be convertedinto sugars and subsequently fermented to ethanol.

Different kinds of pretreatment methods, under a large varietyof conditions, have been studied to improve the fermentability

42 C. Eliana et al. / Fuel 118 (2014) 41–47

and digestibility of several varieties of grasses like bermuda,switchgrass, napiergrass and silver grass [3–11]. Dilute acidpretreatment of bermudagrass at 121 �C (solid loading of10 wt.%, sulfuric acid concentration of 1.2 wt.% and residencetime of 60 min) exhibits a 70% glucan to glucose conversion witha total reducing sugar production (TRS) of 204.1 mg/g biomass at48 h of enzymatic hydrolysis [3]. Likewise, dilute acid pretreat-ment of silvergrass at 121 �C for 30 min, gave a high xylan recov-ery (70–75%) compared with rice straw and sugarcane bagasse.Furthermore, hydrolyzed silvergrass gave a higher level of fer-mentability than cane bagasse because less acetic acid wasformed, obtaining an ethanol yield of 64.3% of the theoretical in48 h of fermentation [12]. The alkaline pretreatment of bermudagrass was evaluated using NaOH and Ca(OH)2 to improve therecovery of fermentable sugars [13]. This study showed that at121 �C NaOH is more efficient than Ca(OH)2 to improve thereducing-sugar yield, achieving 86% of the theoretical yield (ca.500 mg of total reducing sugars/g biomass). A recent study re-ported a lignin removal of 86% and a yield of total reducing sugarsof 71% of the theoretical (440 mg of total reducing sugars/g bio-mass) under optimal pretreatment conditions of bermuda grass(15 min and 0.75 wt.% NaOH at 121 �C) without evaluation ofthe hydrolyzed-material fermentability [7].

Switchgrass was pretreated [6,14] by soaking it in aqueousammonia at room temperature for 5–10 days, achieving a deligni-fication of 40–50%, while the hemicellulose content decreased al-most 50%. The pretreated material was subjected to simultaneoussaccharification and fermentation (SSF) using an enzyme loadingof 38.5 FPU/g cellulose (Spezyme CP) and the strain Saccharomycescerevisiae D5A, achieving an ethanol concentration of 22.16 g/Lwhich corresponds to an ethanol yield of 55.4 mg/g biomass. Solidto liquid ratio and soaking time slightly affected the lignin removalbut did not cause significant changes in the overall ethanol yieldsat sufficiently high enzyme loadings. The effect of the pretreatmentwith Ca(OH)2 at moderates temperatures (50 �C and 21 �C) wasalso investigated to improve the enzymatic digestibility of switchgrass [14,15]. Yields of glucose and xylose of 239.6 and127.2 mg/g biomass, respectively, were achieved at 55 �C for72 h, using cellulase and cellobiose loadings of 35 FPU/g biomassand 61.5 CBU/g biomass, respectively. This study evidenced thatcalcium ions extensively cross linked lignin molecules under alka-line conditions, which substantively decreased the lignin solubili-zation during the pretreatment. The high lignin content in thepretreated biomass did not affect the enzymatic digestibility. Iswas also reported that the ionic-liquid pretreatment of switchgrass decreased the cellulose crystallinity, increased the surfacearea and decreased the lignin content, significantly improving theenzymatic hydrolysis rate of the cellulosic fraction [16]. Ammo-nia-fiber-explosion (AFEX) pretreatment has been evaluated inMiscanthus x giganteus grass and several varieties of switchgrassto obtain fermentable sugars using enzymatic hydrolysis at 50 �Cfor 168 h with cellulase loadings of 15 FPU/g glucan and b-glucosi-dase loadings of 40 IU/g glucan, obtaining glucan conversions of90–96% and ethanol yields of 0.2 g/g biomass in 96 h of SSF process[17–19].

So far, there are no reports that compare the effect of differentpretreatments on the hydrolysis and the fermentability of ele-phant grass. Existing reports are specifically focused in pretreat-ment and fermentability of elephant grass varieties [4,20–24],but under such different experimental conditions that make dif-ficult the comparison of the results. The enzymatic pretreatment(cellulase + esterase) of elephant grass gave 113 mg sugars/g bio-mass [4]. The biological delignification pretreatment of a Colom-bian specie of penisetum sp, using ligninolytic basidiomycetes(ganoderma spp) allowed a lignin removal of 10.7–55.9% [21].Regarding the fermentability, ethanol yields of 45.5 mg/g bio-

mass, using the strain Klebsiella oxytoca THLC0409, and 97–107 mg/g biomass, using the strain Saccharomyces cerevisiaeD5A, have been reported for two different genotypes of elephantgrass [20]. Besides, the fermentation of pentoses and hexosesfrom elephant grass pretreated only by fine grinding (physicalpretreatment) has been reported. After this physical pretreat-ment, the fermentation with Saccharomyces cerevisiaeNBRC2044 during 72 h yielded 113 g ethanol/g biomass fromhexoses while the fermentation with E. coli K011 during 48 hyielded 31.4 g ethanol/g biomass from pentoses [25].

The reported studies have not been carried out under compara-ble conditions that allow the selection of the best pretreatment. Inthis manuscript, the effects of the physicochemical pretreatmentsdilute acid, alkaline pretreatment with NaOH, alkaline peroxide,steam explosion and aqueous ammonia soaking on the hydrolysisand the ethanol fermentability of the cellulosic fraction of elephantgrass are presented in a comparable way. Furthermore, the condi-tions for the best pretreatment found were optimized in order tomaximize the ethanol yield.

2. Materials and methods

2.1. Materials

Elephant grass was grown in farms located in Antioquia(Colombia). This grass was dried in air for easy handling andtransport, and then was grinded to particle size less than 3 mmand dried again to achieve a moisture content of less than10 (wt.%). The material was characterized to determine cellulose,hemicellulose, lignin, extractives and ash contents in the solid(Table 2).

2.2. Methods

2.2.1. Pretreatments2.2.1.1. Alkaline delignification with NaOH. 20.0 g Of material wasweighed and immersed in dilute NaOH solution (1 wt.%) and a so-lid to liquid ratio of 1 g biomass/15 g of NaOH solution. The reactorwas hermetically closed and heated to 120 �C (5 �C/min) and main-tained at this temperature for 30 min. Then, the reactor was cooledto room temperature and the solid was separated from the blackliquor by filtration. The solid fraction was washed with water untilneutral pH and then dried and stored in a freezer.

2.2.1.2. Dilute acid hydrolysis. 100 g Of lignocellulosic material wasmixed with sulfuric acid solution (2 wt.%), with a solid to liquid ra-tio of 1 g biomass/mL solution. The suspension was stirred at 90 �Cfor 90 min. The mixture was filtered to separate the black liquorand the pretreated material. The solid material was washed withwater until neutral pH and then dried and stored in a freezer.

2.2.1.3. Aqueous ammonia soaking. This pretreatment consisted insoaking the lignocellulosic material in an aqueous ammoniasolution (15 wt.%), using 8 mL of ammonia solution per gram ofmaterial, at 60 �C for 6 h. Then, the suspension was filtered to sep-arate the solid and the liquid fractions. The solid fraction waswashed until neutral pH, dried and stored in a freezer for subse-quent hydrolysis and fermentation.

2.2.1.4. Steam explosion. Steam explosion pretreatment was per-formed in homemade equipment. The process was carried out byloading 150 g of the raw material in the container and putting itin contact with saturated steam at 180 �C for 5 min (severity of3.05). After the steam explosion, the material was filtered to re-cover the liquid and the solid fractions. Then, the solid fraction

C. Eliana et al. / Fuel 118 (2014) 41–47 43

was washed until neutral pH, dried and stored in a freezer forsubsequent hydrolysis and fermentation.

2.2.1.5. Alkaline peroxide. 50 g Of solid material was treated withaqueous H2O2 (2% vol.) and with a liquid to solid ratio of 20:1(wt.). The pH was adjusted to 11.5 with 10 M NaOH. The reactionwas carried out at 35 �C for 3 h with continuous stirring. The liquidand solid fractions were separated by filtering and the solid waswashed and neutralized with HCl (37% vol.). Then, the solid frac-tion was washed until neutral pH, dried and stored in a freezerfor subsequent hydrolysis and fermentation.

2.2.2. Enzymatic hydrolysisThe enzymatic hydrolysis was carried out with the commercial

enzyme Accellerase 1500 (Genecor). 30 FPU/g biomass was usedand hydrolyses were carried out at 50 �C, 180 rpm and sodium cit-rate (50 mM) at pH 4.8. The mixtures were autoclaved at 121 �C for15 min before adding the enzyme. The hydrolysis was followed for45 h by analyzing of the total reducing sugars (TRS) content.

2.2.3. Simultaneous saccharification and fermentation (SSF)Simultaneous saccharification and fermentation (SSF) was car-

ried out using the commercial enzyme Accellerase 1500 (Genecor)and the commercial yeast Saccharomyces cerevisiae Ethanol Red.After 12 h of enzymatic hydrolysis at 50 �C, 180 rpm and pH 4.8,the yeast was inoculated in a quantity to allow a concentrationof 2 g/L of dry yeast in the fermentation broth. The fermentationwas performed at 37 �C and 150 rpm, under anaerobic conditions.Fermentation was followed by weight loss and the final ethanolconcentration in the media was determined by gas chromatogra-phy coupled to a solid phase micro-extraction (SPME) system.

Table 1Experimental design for the optimization of the alkaline pretreatment.

Experiment Experimental conditions

Temperature(�C)

Time(h)

% NaOH (w/w)

Solid/liquid ratio(w/w)

1 100 2 1.5 1:17.52 120 1 1 1:203 100 2 2.3 1:17.54 66.4 2 1.5 1:17.55 100 2 1.5 1:17.56 120 3 2 1:157 120 3 1 1:158 80 3 1 1:209 100 3.7 1.5 1:17.5

10 100 2 1.5 1:21.711 100 2 1.5 1:17.512 100 2 0.7 1:17.513 100 2 1.5 1:13.314 120 1 2 1:2015 100 0.3 1.5 1:17.516 80 3 2 1:2017 80 1 2 1:1518 133.6 2 1.5 1:17.519 80 1 1 1:15

Table 2Solid-phase composition of elephant grass before and after pretreatments (wt.%, based on

Material Cellulose Hemi-cellulose

Non pretreated 22.6 20.9Alkaline pretreatment with NaOH 31.2 21.9Dilute acid pretreatment 24.0 13.5Aqueous ammonia soaking 35.5 25.4Steam explosion 31.3 29.0Alkaline peroxide 39.2 10.5

2.2.4. Optimization of the alkaline delignification with NaOHAlkaline pretreatment was carried out in an autoclave for the

experiments at temperatures above the water boiling point, andin atmospheric stirred reactors for temperatures below 90 �C. Theconditions evaluated were: temperature between 80 and 120 �C,residence time between 30 and 180 min, solid to liquid ratio1:15 and 1:20 (wt.) and NaOH concentration between 1% and2% (wt.). A Draper–Lin central composite design, with three centralpoints, was used to optimize the pretreatment conditions andmaximize the ethanol yield. Data showed in figures and tablescorrespond to the average value for triplicate experiments with arelative standard deviation of less than 5% in all cases. Detailedexperimental conditions are shown in Table 1. Hydrolysis andfermentation of these pretreated materials were made using theprocedure described above, with the commercial enzyme Acceller-ase 1500 (Genecor) and the commercial yeast Ethanol Red (Saccha-romyces cerevisiae).

2.2.5. AnalysisMaterials were characterized following the protocols of the Na-

tional Renewable Energy Laboratory (NREL) for determination ofash, moisture and extractives in biomass [26–28]. Cellulose, hemi-cellulose and lignin contents in pretreated and non-pretreatedmaterials were determined by UV–vis spectroscopy after acidhydrolysis with 72% H2SO4. Total reducing sugars were determinedusing the method of 3,5-dinitrosalicylic acid (DNS) and glucosewas determined using the oxydase/peroxydase kit of BioSystems�.

Fermentation monitoring was made by weight loss. The SSF sys-tem was followed for 26 h. The final concentration of ethanol wasdetermined by gas chromatography using an Agilent chromato-graph 7890 with FID detector employing a HP-INNOWax columncoupled to solid phase micro-extraction (SPME) with a polyacry-late fiber 85-lm.

Inhibitors like xylitol, succinic acid, lactic acid furfural, hydroxymethyl furfural (HMF), acetic acid and glycerol, formed during thepretreatment process, were determined by HPLC using a BioradAminex HPX-87H column. Liquid samples were filtered through0.25 lm filter previous to the HPLC analysis.

3. Results

3.1. Characterization

The material was characterized before and after the pretreat-ments to determine the contents of cellulose, hemicellulose, lignin,ash, extractives in the solid. The results of these characterizationsare shown in Table 2. The fraction ‘‘others’’ may include some or-ganic compounds such as uronic acid and acetyl groups, and tracecomponents including minerals, waxes, fats, resins and gums [7].These components are not fermentable by the yeast. For this kindof raw materials, i.e., lignocellulosic biomass, it is difficult to ex-actly determine the complete chemical composition. That is whynon-specific characterization techniques such as solvent extractionare customarily used. Results with this technique depend on the

dry matter).

Lignin Extractives Ash Others

19.4 9.9 11.1 16.16.8 3.1 5.4 31.5

13.4 9.4 10.5 29.39.2 3.04 8.3 18.5

15.8 6.7 8.7 8.511.0 11.8 10.7 16.8

Fig. 1. Enzymatic hydrolysis of elephant-grass solid fraction under differentpretreatments. Hydrolysis conditions: 50 �C, 180 rpm, 30 FPU/g of biomass, bufferpH 4.8.

Fig. 2. SSF of elephant grass after different pretreatments. Fermentation conditions:37 �C, 2 g dry yeast/L, 150 rpm and 30 FPU/g of biomass.

44 C. Eliana et al. / Fuel 118 (2014) 41–47

solvent used. In our case we used ethanol as solvent to determine‘‘extractives’’ because this is the most common for these materials.

In the case of the alkaline pretreatment, the changes in the com-position are mainly due to the removal of lignin and in a smallerproportion to the hydrolysis of cellulosic and hemicellulosic frac-tions, which leads to a decrease in the weight of the solid recoveredafter the pretreatment. HPLC analysis of the liquid fractionsobtained in the different pretreatments showed the presence ofsugars such as cellobiose, glucose, xylose and arabinose, as wellas the presence of inhibiting compounds such as xylitol, glycerol,acetic acid and furfural formed during hydrolysis. In the pretreat-ments using dilute acid and steam explosion the changes in thecompositions of the solid phases are due to the hydrolysis of hemi-cellulose and in a lesser proportion to the cellulose hydrolysis. Thiswas evidenced by the presence of glucose, xylose, arabinose, acidacetic acid, furfural and hydroxymethyl furfural in the liquid frac-tion obtained after the pretreatments.

In general, an increase in the cellulose content of the solid phasewas achieved in the grass after the pretreatments evaluated, at theexpense of the partial hydrolysis of hemicellulose and lignin. Par-ticularly, the alkaline pretreatments (NaOH, NH3 and alkaline per-oxide) allowed a high lignin-removal due to the effect of suchpretreatments on the ester bonds and glycosidic chains. This effi-cient lignin-removal has been linked to increased porosity of thebiomass and accessibility of the cellulose [7,29]. In the case ofthe steam explosion, the most marked effect was on the hemicel-lulose content, since under the operating conditions and with thepresence of sulfuric acid as catalyst the autohydrolysis of acetylgroups is favored, decreasing the hemicellulose content in the solidphase. Besides, it has been reported that this pretreatment also al-lows a partial hydrolysis of lignin, through the homolytic cleavageof b-O-4 ether linkages and other unstable acid moieties, produc-ing a series of cinnamyl alcohol derivatives and condensation by-products [30].

Acid hydrolysis of this grass decreased the hemicellulose con-tent in the solid phase due to solubilization. It has been reportedthat the acid hydrolysis of lignocellulosic materials solubilizesthe hemicellulosic fraction by altering the covalent bonds, hydro-gen bonds and van de Walls forces of this fraction [16]. This pro-cess also leads to the formation of degradation compounds whichmay have an inhibitory effect on the enzymatic hydrolysis and fer-mentation. Table 2 shows a decreased lignin content in the solidphase of elephant grass pretreated by dilute acid; this could bepossibly due to a partial hydrolysis of the unstable linkages ofsome acidic groups present in the lignin in this grass.

3.1.1. Enzymatic hydrolysisResults on the enzymatic hydrolysis of the pretreated grass

using the enzyme Accellerase 1500 are shown in Fig. 1. The besthydrolysis results were obtained with the alkaline pretreatments,i.e., 146.9 and 133.6 mg TRS/g pretreated biomass after 45 h forNaOH and ammonia pretreatments, respectively. These resultsare in agreement with other studies showing that the hydrolysisof cellulose is improved by the removal of lignin because ligninforms a physical barrier that prevents the attack of the enzyme[22,31,32] and because solid lignin can absorb cellulase enzymesdecreasing the hydrolysis performance [33,34].

The acid pretreatment follows the alkaline pretreatmentsregarding reducing sugar production. It is known that the acid pre-treatment improves the hydrolysis of cellulose but has no effect onlignin [35].

The yield of total reducing sugars depends on the initial compo-sition and molecular structure of biomass as well as on the condi-tions for the pretreatment and enzymatic hydrolysis. Therefore, theobtained yields are difficult to compare with those obtained forother types of lignocellulosics. For instance, a yield of 182.8 mg

TRS/g of biomass has been reported for rice straw pretreated with20 wt.% Ca(OH)2 (for 1 h at 95 �C) after hydrolysis for 34 h at 50 �C.A yield of 118.1 mg TRS/g of biomass has been obtained for thesame material pretreated with 4 wt.% NaOH (for 2 h at 55 �C) afterhydrolysis for 45 h at 50 �C [37]. Yields of total reducing sugars ashigh as 810.9 mg TRS/g of pretreated biomass, for switchgrass pre-treated with 2 wt.% NaOH (for 60 min at 121 �C) after hydrolysisfor 72 h at 55 �C, and 636.9 mg TRS/g pretreated biomass, forswitchgrass pretreated with microwaves (with 2 wt.% NaOH for10 min at 1250 W) after hydrolysis for 72 h at 55 �C, have been re-ported [36].

In our case, the yield obtained so far was 146.9 mg TRS/g pre-treated biomass (pretreated with 1.5 wt.% NaOH for 1 h at121 �C) after hydrolysis for 46 h at 50 �C.

3.2. Simultaneous saccharification and fermentation (SSF)

Results obtained in the simultaneous saccharification and fer-mentation (SSF) of the solid fractions of the pretreated materialsusing Ethanol Red yeast (Saccharomyces cerevisiae) and Acceller-ase 1500 are shown in Fig. 2. Consistent with the results obtainedin the enzymatic hydrolysis of this material, the alkaline pretreat-ments with NaOH and NH3 exhibited higher concentrations of eth-anol in the fermentation broth. The steam explosion pretreatmentexhibited a slight increase in the concentration of ethanol in com-parison with the soaking pretreatment with ammonia. The solidfraction from the NaOH pretreated elephant grass allowed obtain-ing an ethanol yield of 67.75 mg ethanol/g of biomass after 24 hfermentation.

Table 3Characterization solid phase after pretreatment with NaOH.

Experiment % Solidrecovery

% Ligninremoval

% Celluloserecovery

% Hemicelluloserecovery

1 40.4 81.5 98.1 31.42 48.5 79.3 98.9 60.53 46.9 79.9 98.3 53.14 40.5 78.2 98.1 59.65 51.7 83.9 97.5 46.66 49.8 77 98.1 28.27 53.6 83.6 99 818 50.5 77.9 97.6 709 48.3 78.1 97.5 19.7

10 48.1 78.7 96.5 31.211 52.1 86.9 98.2 52.312 53.9 88.4 96.9 68.513 41.9 78.6 97.2 57.814 51.9 79.9 97.8 82.715 44.7 81.5 96.8 29.716 47.8 76.2 96.9 46.817 47.4 78.1 97.2 49.218 46.6 82.5 97.2 44.519 51.0 80.8 98.4 40.2

Table 4Characterization of the liquid phase after pretreatment with NaOH (percentages arecalculated based on solubilized fraction of material).

Experiment % Lignin % Cellulose % Hemicellulose

1 39.1 1 102 31.7 0.5 193 33.1 0.8 21.94 37.5 1.1 15.55 31.5 1.1 18.26 30 0.9 217 30.2 0.4 11.48 29.9 1.1 19.39 31.4 1.2 21.8

10 31.8 1.7 27.411 32.4 0.8 1412 31.8 1.3 20.213 36.4 1.5 9.714 29.9 0.9 26.815 35.4 1.6 24.416 30.9 1.5 22.417 32 1.3 20.418 34.3 1.4 21.719 30.7 0.7 12.9

C. Eliana et al. / Fuel 118 (2014) 41–47 45

These results indicate that the alkaline pretreatment with NaOHleads to a ethanol yield of 54.1% with respect to the theoreticalyield (calculated with the amount of glucose available in the mate-rial) from the cellulosic fraction of elephant grass. These resultsgive an indication of the feasibility of using these raw materialsfor the industrial production of bioethanol and show the need tooptimize the operating conditions of the pretreatment, the hydro-lysis and the fermentation to improve the efficiency of ethanolproduction.

The amount of ethanol produced in the SSF process of elephantgrass is still low compared with the yields reported for othergrasses. For switchgrass, 72% and 92% of the theoretical yieldhave been reported after pretreatments with aqueous ammoniaand hydrothermolysis, respectively, and using the enzyme fibri-lase 815 FPU/g cellulose) and the strain Saccharomyces cerevisiaeD5A [6,38]. An ethanol yield of 79.6% of the theoretical maximum(219 mg ethanol/g biomass or 22.9 g ethanol/L) has been reportedfor the co-fermentation of pentoses and hexoses in alfalfa, usingthe enzyme hydrolysis set GC220 cellulase (30 FPU/g cellulose),b-glucosidase Novo 188 (40 U/g pulp) and pectinase (84 mg/gbiomass) and Saccharomyces cerevisiae strain YRH400 [39].However, the conditions under which the five pretreatmentswere evaluated are not yet the optimal conditions to maximizethe production of fermentable sugars for its conversion intoethanol.

3.3. Optimization of alkaline pretreatment with NaOH

Results obtained so far on the comparison of several pretreat-ment methods for elephant grass showed that the pretreatmentwith NaOH is the best because it leads to the highest productionof sugars and ethanol. Therefore, this pretreatment was studiedin more detail to improve the ethanol yield. A Draper–Lin centralcomposite design, with three central points, was used to optimizethe pretreatment conditions and maximize the ethanol yield.Experimental conditions are shown in Table 1.

The good results achieved with the NaOH pretreatment are aconsequence of the well known effects that this treatment hason lignocellulosic materials: (i) swelling of biomass, leading toan increase in internal surface area, (ii) changing the cellulosestructure into a denser and more thermodynamically stable form,(iii) decreasing the degree of polymerization and crystallinity ofcellulose, (iv) separation of structural linkages between ligninand carbohydrates and breaking-down of the lignin structure,and (vi) solubilization, condensation and redistribution of lignin[40,41].

3.3.1. Pretreated material characterizationLiquid and solid fractions obtained after the NaOH pretreat-

ments were characterized to determine the contents of cellulose,hemicellulose and lignin. From these results, the percentages ofrecovery of solid, cellulose and hemicellulose as well as the per-centage of lignin removal were calculated and are shown inTable 3.

Under the experimental conditions of Table 1, the NaOHpretreatment of elephant grass allowed cellulose recovery of96.5–99%, lignin removal of 76.2–88.4% and solid recovery of40.4–53.9%. From these results it is concluded that the best condi-tions of the NaOH pretreatment, in terms of lignin removal, are100 �C, 2 h, 0.7 wt.% NaOH and a solid to liquid ratio of 1:17.5 (wt.).

Table 4 shows the contents of cellulose, hemicellulose and lig-nin in the liquid phases obtained after the alkaline pretreatment.These results indicate that the hydrolysis of hemicellulose oc-curred in a larger extension than the hydrolysis of cellulose. Thehigh lignin contents in the liquid phases correlate with the highlignin removal from the solid phase.

HPLC analyses of the liquid phases obtained after the NaOHpretreatments showed the presence of acetic acid, formic acidand furfural (in pretreatments at 120–133.6 �C) which areknown to be formed in the degradation of hemicelluloses andsugars. These compounds have a strong inhibiting effect on fer-mentation with Saccharomyces cerevisiae, specially acetic acidand furfural which decrease the specific velocity of growth ofmicroorganisms and, therefore, the volumetric ethanol yield[42–44]. Therefore, the implementation of an additional stepof neutralization of pretreated materials in very important tomitigate the effect of these inhibiting compounds and thus im-prove the ethanol yield. Monomeric sugars, produced by hydro-lysis of cellulose and hemicelluloses, like glucose, xylose andarabinose were also detected.

3.3.2. Enzymatic hydrolysisThe enzymatic hydrolysis of pretreated materials was per-

formed using the enzyme Accellerase 1500 (Genecor) at 50 �Cwith an enzymatic loading of 30 FPU/g biomass and a solid toliquid ratio of 10:90 (w/w), during 26 h. Under the different

0

100

200

300

400

500

600

700

800

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Red

ucin

g su

gars

(mg/

g bi

omas

s)

Experiment

Fig. 3. Enzymatic hydrolysis of elephant grass under different pretreatmentconditions (shown in Table 1). Hydrolysis conditions: 50 �C, 180 rpm, 30 FPU/g ofbiomass, buffer pH 4.8.

0

5

10

15

20

25

30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Etha

nol g

/L

Experiment

Fig. 4. Ethanol concentration obtained under different pretreatment conditions ofelephant grass. Fermentation conditions: 37 �C, 2 g dry yeast/L, 150 rpm and 30FPU/g of biomass.

46 C. Eliana et al. / Fuel 118 (2014) 41–47

pretreatment conditions evaluated (see Table 1), between 422.5and 711.2 mg TRS/g biomass were obtained in the enzymatichydrolysis of elephant grass, as showed in Fig. 3. The highest pro-ductions of reducing sugars were achieved at higher temperature(100 �C), obtaining over 82% efficiency in the conversion ofpolysaccharides into fermentable sugars. A direct relationshipbetween experimental variables and production of sugars cannotbe established from the experimental results. The highest amountof reducing sugars from elephant grass, i.e., 711.2 mg TRS/g bio-mass, was obtained under pretreatment conditions of 100 �C,2 h, 1.5 wt.% NaOH and solid to liquid ratio of 1 g of materialper 17.5 g of solution.

The very high lignin removals achieved with the alkalinepretreatments cannot be correlated with the amount of reducingsugars obtained in the enzymatic hydrolysis. The dispersion ofthe remaining lignin directly affects the enzymatic attack. Ahighly-dispersed remaining lignin, even in low contents, could hin-der the enzyme access to the amorphous portions of cellulose thatcomposes the grass fibers, thereby affecting the enzymatic hydro-lysis efficiency.

Lignin removal was in general very high (see Table 3). Thismeans that residual lignin contents in the solids that were hydro-lyzed and fermented were low. Therefore, observed results mustnot be due to the form of this residual lignin. Results cannot beeither explained by the cellulose recovery because it was also veryhigh in all cases. The form of the cellulose remaining in the solidcould be an important parameter affecting the results. The pre-treatment method can modifies the structural properties of solidby increasing the surface area and decreasing the crystallinityand thus enhancing the hydrolysis efficiency [29,45–48]. Addition-ally, in this work only cellulolytic enzymes were used, therefore,they are not able to attack the hemicellulosic fraction. It has beenreported that hemicellulose provides the key barrier to cellulosebreakdown by enzymes [49]. Based on this, the content and formof hemicellulose should also play an important role in the hydroly-sis of cellulose.

The obtained results in the enzymatic hydrolysis of elephantgrass, after different alkaline pretreatment conditions, are similarto many reported results for grasses with respect to the amountof reducing sugars produced per gram of biomass fed to the pro-cess. Several studies on the hydrolysis of switchgrass and Bermudacoastal grass, pretreated under alkaline conditions, report between400 and 600 mg TRS/g biomass [46–49]. In our case, the best yieldsof reducing sugars were 711.2 mg TRS/g biomass (108.0 g/L) forelephant grass. It is worth pointing out that the time required forthis high yield of reducing sugars, i.e., 24 h of enzymatic hydrolysis,is quite short compared to the time reported for the hydrolysis ofswitchgrass, bermuda grass and alfalfa fiber, i.e., 72–168 h[19,37,44,47].

3.3.3. Simultaneous saccharification and fermentation (SSF)Ethanol concentrations obtained in the SSF process of elephant

grass are shown in Fig. 4. The statistical analysis of the obtaineddata for fermentability of elephant grass under different pretreat-ment conditions (not showed) indicates that the factor with mostsignificant statistic effect is the NaOH concentration, and that thestudied experimental variables (in the alkaline pretreatment) ex-plain 90.27% of variability in fermentability.

For elephant grass, ethanol concentrations between 10 and26 g/L, corresponding to ethanol yields of 62.2 and 141.5 mg etha-nol/g dry biomass fed to the process, respectively, were obtainedafter 24 h of fermentation. The highest ethanol concentration ob-tained for elephant grass (26 g/L) corresponds to pretreatmentconditions of 120 �C, 2 wt.% NaOH, solid to liquid ratio of 1:20wt. and residence time 60 min.

The highest ethanol yield, i.e., 149.3 mg ethanol/g biomass, cor-responds to 95.1% of the maximum theoretical yield. This resultindicates a very high efficiency in the use of the cellulosic fractionfor the transformation of this type of biomass into ethanol. Ethanolyields of 92% and 89.2% of maximum theoretical have been re-ported in literature for materials like switch grass and Corn Stover[13,14,38,48]. For an elephant grass variety, after fine grinding,yield of 44.2% with respect to theoretical xylose and glucose andhave been reported [25]. Our results are, therefore, superior tothose reported so far.

4. Conclusions

Alkaline pretreatment with NaOH yielded the highest concen-trations of reducing sugars and ethanol from elephant grass, com-pared to results obtained with dilute acid, steam explosion,aqueous ammonia soaking and alkaline peroxide pretreatments.Optimization of the alkaline delignification with NaOH allowed alignin removal of 88.4%, total reducing sugars yield of 711.2 mg/gbiomass after enzymatic hydrolysis (82% of theoretical) and etha-nol concentration of 26.05 g/L which corresponds to maximumtheoretical yield of 95% (36 h of SSF at 37 �C using 30 FPU/g bio-mass of Accellerase 1500 and a Ethanol Red concentration of 2 g/L). Although high lignin removals were achieved, which is animportant factor for promote the enzymatic hydrolysis, a directrelationship between results and evaluated conditions cannot beestablished. To the best of our knowledge, these results are supe-rior to those reported for similar materials.

Acknowledgements

Authors thank ‘‘Empresas Públicas de Medellín E.S.P’’ and ‘‘Uni-versidad de Antioquia-Comité para el Desarrollo de la Investiga-ción’’ for the financial support.

C. Eliana et al. / Fuel 118 (2014) 41–47 47

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