Bioresource Technology 102 (2011) 1270–1276

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Bioresource Technology

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Dilute acid pretreatment of rapeseed straw for fermentable sugar generation

Eulogio Castro ⇑, Manuel J. Díaz, Cristóbal Cara, Encarnación Ruiz, Inmaculada Romero, Manuel MoyaDepartment of Chemical, Environmental and Materials Engineering, University of Jaén, Campus Las Lagunillas, 23071 Jaén, Spain

a r t i c l e i n f o

Article history:Received 5 June 2010Received in revised form 11 August 2010Accepted 13 August 2010Available online 22 August 2010

Keywords:Agricultural residuesDilute acid pretreatmentEnzymatic hydrolysisRapeseed straw

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.08.057

⇑ Corresponding author. Tel.: +34 953212163; fax:E-mail address: ecastro@ujaen.es (E. Castro).

a b s t r a c t

The influence of the main pretreatment variables on fermentable sugar generation from rapeseed straw isstudied using an experimental design approach. Low and high levels for pretreatment temperature(140–200 �C), process time (0–20 min) and concentration of sulfuric acid (0.5–2% w/v) were selectedaccording to previous results. Glucose and xylose composition, as well as sugar degradation, were mon-itored and adjusted to a quadratic model. Non-sugar components of the hydrolysates were also deter-mined. Enzymatic hydrolysis yields were used for assessing pretreatment performance. Optimizationbased on the mathematical model show that total conversion of cellulose from pretreated solids canbe achieved at pretreatment conditions of 200 �C for 27 min and 0.40% free acid concentration. If optimi-zation criteria were based on maximization of hemicellulosic sugars recovery in the hydrolysate alongwith cellulose preservation in the pretreated solids, milder pretreatment conditions of 144 �C, 6 minand 2% free acid concentration should be used.

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1. Introduction

Rapeseed (Brassica napus) oil ranges third of oil consumptionworldwide. After oil extraction, rapeseed pomace can be used foranimal feed production due to its protein content. In the last years,an increasing fraction of rapeseed oil has been used as raw materialfor biodiesel production. According to FAO (2008), over 30 millionhectares of rapeseed were cultivated all over the world in 2007.Agricultural residues from rapeseed cultivation are left behindafter seed harvesting. These renewable, costless materials mustbe eliminated from the field, which is usually done by burning.

Alternatively, the lignocellulosic nature of rapeseed straw canbe used for fuel ethanol production by a biochemical processincluding pretreatment, enzymatic hydrolysis and fermentation.

There are relatively little reports focusing on the use of rapeseedresidues. Most of them deal with thermal applications like pyroly-sis (Karaosmanoglu et al., 1999). Zabaniotou et al. (2008) reportedon the integrated utilization of rapeseed suitable to Greek condi-tions for biodiesel production and parallel use of its solid residuesfor energy and second generation biofuels production via fast pyro-lysis. Reports dealing with rapeseed straw as raw material for eth-anol production are also rare. The use of sulfuric acid-catalyzedpretreatment with rapeseed straw at 180 �C has been reported(Lu et al., 2009). The same pretreatment was analyzed by Jeonget al. (2010) for optimizing hemicellulose extraction form rapeseedstraw. Li et al. (2009) reported on rapeseed stover pretreatmentwith phosphoric acid–acetone for ethanol production by means

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of simultaneous saccharification and fermentation. Biogas or etha-nol production has also been reported (Petersson et al., 2007).

In a previous work (Díaz et al., 2010) on the use of hydrothermalpretreatment of rapeseed straw for fermentable sugars production,it was concluded that 70% of the glucose in the raw material couldbe obtained by liquid hot water pretreatment at 210–220 �C for30–50 min. In an attempt to improve previous results, this workdeals with the use of dilute sulfuric acid pretreatment on the samerapeseed straw lot. This procedure has been applied to a variety ofagricultural residues (Cara et al., 2008) and it is claimed to effec-tively hydrolyze hemicellulose and make the cellulose amenableto enzymatic conversion (Lloyd and Wyman, 2005). The main oper-ational variables, e.g. pretreatment temperature, residence timeand acid concentration are examined under an experimental de-sign basis. The performance of the process is assessed by meansof the enzymatic hydrolysis yield and the sugar yield in the liquidfractions issued from pretreatment.

2. Methods

2.1. Raw material

Rapeseed straw was locally collected after seed harvest. Then,the raw material was air-dried at room temperature to equilibriummoisture content of about 10%, milled using a laboratory hammermill (Retsch) to a particle size smaller than 1 cm, homogenised in asingle lot and stored until used.

The chemical composition of the raw material is (% w/w, dry ba-sis): cellulose (as glucose), 36.6; hemicellulosic sugars, 24.1 (withxylose as the main sugar accounting for 76% of hemicellulosic sug-

E. Castro et al. / Bioresource Technology 102 (2011) 1270–1276 1271

ars); acid-insoluble lignin (AIL), 15.6; acetyl groups, 3.65; and ash,5.7 (Díaz et al., 2010). The composition of raw material was deter-mined according to the National Renewable Energy Laboratoryanalytical methods for biomass, as described below.

2.2. Dilute acid pretreatment

Dilute acid pretreatment was performed in a laboratory scalestirred Parr reactor. The reactor is built in Carpenter 20, an acid-resistant alloy and has a total volume of 1 L, with an electric heaterand mechanic agitation. The temperature/speed controller is acombination of furnace power control and motor speed controlwith tachometer. 36 g of rapeseed straw (dry basis) and 600 mLof the appropriate sulfuric acid solution were used for each pre-treatment trial. Both sulfuric acid solution and raw material, ini-tially at room temperature, were heated at 5 �C/min. Agitationwas set at 350 rpm. Once the temperature of the reaction mixturereached the target point, pretreatment time counting was initiated.At the end of each run the reactor was removed from the heatingjacket and cooling water was charged through the serpentine coil.The content of the reactor cooled down to 80 �C in approximately5 min. The reactor was kept sealed, and the slurry agitated untilthe reactor was cooled to about 40 �C. Then the wet material wasfiltered for solid and liquid recovery.

The water-insoluble solids were washed thoroughly with wateruntil no colour in the resulting water was obtained, and analyzedfor hemicellulosic sugars, glucose and acid-insoluble lignin con-tent, and used as substrate in enzymatic hydrolysis tests. Liquidfraction issued from pretreatment (hydrolysate) was analyzed forsugars, acetic acid and sugar-degradation products.

2.3. Experimental design

Rapeseed straw was pretreated at 13 different operational con-ditions according to a Box–Behnken experimental design, includ-ing one point and four replicates at the center of the domainselected for each factor under study, as shown in Table 1 (17 runs).Center values and intervals were chosen based on previous experi-ence with agricultural residues to ensure a broad range of re-sponses. Pretreatment experiments were performed in randomorder. The recovery of cellulose and hemicelluloses in the pre-treated solids, and the concentration of glucose and hemicellulosicsugars in the liquid fractions issued from pretreatment were deter-mined as responses. In addition, degradation of glucose and other

Table 1Experimental design for dilute acid pretreatment of rapeseed straw. t0 and A0 are pretreainstantaneous heat up and cooling that would result in the same severity factor (Ro) (see

Run t0 (min0 T (�C) ACoded Real Coded Real C

1 0 10 �1 140 +12 0 10 +1 200 �3 �1 0 0 170 �4 +1 20 +1 200 05 +1 20 �1 140 06 �1 0 �1 140 07 0 10 0 170 08 +1 20 0 170 +19 0 10 +1 200 +1

10 �1 0 +1 200 011 �1 0 0 170 +112 0 10 0 170 013 +1 20 0 170 �14 0 10 0 170 015 0 10 0 170 016 0 10 0 170 017 0 10 �1 140 �

sugars as a consequence of pretreatment was also evaluated. Theexperimental data were analyzed by the statistical software DesignExpert 8.0.2, Stat-Ease Inc., Minneapolis, USA.

2.4. Enzymatic hydrolysis tests

The washed water-insoluble residue of pretreated rapeseedstraw was enzymatically hydrolysed by a cellulolytic complex (Cel-luclast 1.5 L) kindly provided by Novozymes A/S (Denmark). Cellu-lase enzyme loading was 15 Filter Paper Units (FPU)/g substrate.Fungal ß-glucosidase (Novozym 188, Novozymes A/S) was usedto supplement the ß-glucosidase activity with an enzyme loadingof 15 International Unit (IU)/g substrate. Enzymatic hydrolysiswas performed in 0.05 M sodium citrate buffer (pH 4.8) at 50 �Con a rotary shaker (Certomat-R, B-Braun, Germany) at 150 rpmfor 72 h and at 5% (w/v) pretreated material concentration. Sam-ples were taken every 24 h for glucose concentration determina-tion. All enzymatic hydrolysis experiments were performed induplicate (standard deviations were in all cases <3%) and averageresults are given.

2.5. Analytical methods

The composition of raw material was determined according tothe National Renewable Energy Laboratory analytical methods forbiomass (NREL, 1994–1998). Prior to other determinations, rawmaterial was extracted consecutively with water and with ethanol(two-step extraction procedure). After the first step, the sugarcomposition of the water-extract was determined by high perfor-mance liquid chromatography (HPLC) in a Varian Prostar liquidchromatograph with refractive index detector. A TransgenomicCHO-682 carbohydrate analysis column operating at 80 �C withultrapure water as a mobile-phase (0.4 mL/min) was used. Freeand oligomeric sugar composition was determined before and aftera posthydrolysis process consisting in a treatment with sulfuricacid (3% v/v) at 121 �C and 30 min. The cellulose and hemicellulosecontent of the extracted solid residue was determined based onmonomer content measured after a two-step acid hydrolysis pro-cedure to fractionate the fiber. A first step with 72% (w/w) H2SO4

at 30 �C for 60 min was used. In a second step, the reaction mixturewas diluted to 4% (w/w) H2SO4 and autoclaved at 121 �C for 1 h.This hydrolysis liquid was then analyzed for sugar content by HPLCas described above. The remaining acid-insoluble residue is consid-ered as acid-insoluble lignin (AIL).

tment time and temperature, while t and A stand for the same variables consideringtext for details).

0 (% w/v) Ro Factors for designoded Real A (% w/v) t (min)

2 232.9 1.91 15.471 0.5 14813.7 0.41 16.841 0.5 833.7 0.41 7.24

1.25 23847.6 1.16 27.111.25 391.1 1.16 25.971.25 93. 6 1.16 6.211.25 1915.3 1.16 16.642 2995.8 1.91 26.032 14563.9 1.91 16.551.25 5575.6 1.16 6.342 693.9 1.91 6.031.25 1884.4 1.16 16.37

1 0.5 3017.2 0.41 26.211.25 1919.1 1.16 16.671.25 1862.4 1.16 16.181.25 1878.2 1.16 16.32

1 0.5 233.0 0.41 15.48

1272 E. Castro et al. / Bioresource Technology 102 (2011) 1270–1276

After LHW-pretreatment, the composition of solid fraction wasdetermined as described for raw material except that no extractionis used. The sugar content (glucose, xylose, arabinose, mannoseand galactose) of the liquid fraction after pretreatment (prehydro-lyzate) was determined by HPLC using the system described above.The inhibitor composition (acetic acid, formic acid, furfural andHMF) was determined using the HPLC system with refractive indexdetector mentioned above; a Bio-Rad HPX-87H column at 65 �Ctemperature was used. The mobile phase was 5 mM H2SO4, at aflow rate of 0.5 mL/min. Glucose concentration from enzymatichydrolysis samples was measured by HPLC with the above de-scribed Varian equipment. All analytical determinations were per-formed in duplicate and average results are shown. Relativestandard deviations were in all cases below 5%.

3. Results and discussion

3.1. Effective pretreatment conditions

Factors in the experimental design were modified for taking ac-count of two facts that can affect pretreatment performance, e.g.the neutralizing capacity (NC) of biomass, and the effect of heatingand cooling periods.

Concerning the first of these facts, suspensions of lignocellulosicmaterials have been shown to partially neutralize acid solutionsdue to the presence of basic cations in the lignocellulosic matrix.When considering pretreatment by dilute acids, it is important totake into account the concentration of acid that is actually usedto bond cleavage, and that which is just neutralized by biomass.To determine the neutralizing capability of rapeseed straw, a mod-ification of the procedures described by Esteghlalian et al. (1997)and Lloyd and Wyman (2005) was applied. Briefly, triplicate sam-ples of rapeseed straw were calcinated at 525 �C for 8 h and thenthe resulting ash was dissolved in 0.5% (w/v) sulfuric acid solution.The difference of pH of the sulfuric acid solutions before and afteradding the ash was used for calculating the neutralizing capacity ofthe raw material. The same procedure was also followed using theentire rapeseed straw, instead of ash. The neutralizing capacity re-sulted to be 19.7 ± 1.6 mg H2SO4/g dry rapeseed straw. These re-sults compare with those reported by Esteghlalian et al. (1997)for corn stover, poplar and switchgrass (43.7, 25.8 and 16.7 mgH2SO4/g dry feedstock, respectively) and by Lloyd and Wyman(2005) for corn stover (17.3 mg H2SO4/g dry feedstock).

From NC values, it is possible to determine the actual acid con-centration that is available for pretreatment purposes, which re-sulted to be 0.41, 1.16 and 1.91% (w/v) at initial concentration of

Table 2Coefficients of mathematical model Eq. (2)a.

Solid recovery Gs Gl Xs

0 210.9 �539.0 �10.08 89.5a1 �1.62 4.42 0.74 �0.46a2 �1.20 7.29 0.056 �0.82a3 �23.55 99.6 4.39 �12.4a4 7.92�10�3 �28.4�10�3 �2.78�10�3 1.42�10�3

a5 NS �0.359 �0.106 60.6�10�3

a6 NS �0.760 NS 21.0�10�3

a7 NS NS �6.27�10�3 3.08�10�3

a8 1.98�10�3 �20.6�10�3 NS 2.08�10�3

a9 6.58 9.27 �1.19 2.44R2 0.9918 0.9968 0.9737 0.9972

NS: no statistical significance.a Gs, Gl: percentage of glucose present in the raw material that remains after pretreatm

xylose, galactose, mannose and arabinose) present in the raw material that remain afterto either raw material or pretreated material (g glucose/100 g); Gd, Xd: percentage ofconsequence of pretreatment; Coefficients for Gl, Xl and Xs were obtained afterffiffiffiffi

Gp

l þ 1;ffiffiffiffiXp

l þ 1;ffiffiffiffiXp

s þ 1.

0.5, 1.25 and 2.0% (w/v) respectively. These actual concentrations,also named free acid concentrations, have been considered for thediscussion of the experimental results, as hydronium ion concen-tration directly affects the rates of hydrolysis of the carbohydratepolymers (Springer and Harris, 1985). Other authors have also re-ported on neutralization of sulfuric acid by the minerals in biomassand the need of being taken into account for models to accuratelypredict hydrolysis performance (Lloyd and Wyman, 2004).

On the other hand, pretreatment was performed on a Parr pres-sure reactor at temperatures ranging from 140 �C to 200 �C. As de-scribed in the precedent section, heat up was done at 5 �C/min,while cooled down to 80 �C lasted for 5 min (approximately,depending on the target temperature). A typical temperature pro-file can be found elsewhere (Díaz et al., 2010). To take account ofthe effect of temperature, not only when maintained at the targetvalue, but also during heat up and cool down periods, the severityfactor Ro, adapted from Overend and Chornet (1987) by Abatzog-lou et al. (1992), was applied following this equation:

Ro ¼ t � expT � 10014:75

� �¼Z t

0exp

TðtÞ � 10014:75

� �dt ð1Þ

The evaluation procedure started by calculating the integralfrom curves T versus t, and then the value of time is derived fromthe second term of Eq. (1). This value is the time assuming instan-taneous heating-up and cooling down that would yield the sameseverity factor as the real process. For example, run 4 was per-formed by heating the reaction mixture (1.25% w/v acid concentra-tion) up to 200 �C, then hold for 20 min and cooling downafterwards. The same effect in terms of severity factor would havebeen reached if 200 �C were maintained for 27.11 min, providedinstantaneous heating and cooling was possible.

Taking into account the two modifications in process variables,Table 1 summarizes the pretreatment conditions used for theexperimental design.

3.2. Mathematical model. Factors and responses

The study of the pretreatment performance by dilute sulfuricacid was addressed by performing the experimental design inwhich pretreatment temperature, process time, and free acid con-centration, as detailed above, were retained as factors. The ratio li-quid to solid or solid content in the pretreatment reactor was keptconstant at 6% w/v; this value was chosen after some preliminarytrials as the highest one causing no mixing problems inside thereactor. Solid recovery, glucose and other sugars recoveries, includ-ing the degradation taking place as a consequence of pretreatment,

Xl Y Yp Gd Xd

�74.6 �576.5 �76.3 745.9 53.90.69 �0.90 �2.54 �8.64 �1.510.88 6.95 0.76 �8.15 �1.3116.0 91.0 42.7 �116.1 �27.0�3.20�10�3 NS 19.6�10�3 46.2�10�3 NS�0.148 NS NS 0.842 1.61�85.3�10�3 �0.579 �2.12 0.670 0.42NS 33.3�10�3 NS 29.8�10�3 NS�2.39�10�3 �18.6�10�3 NS 22.3�10�3 6.42�10�3

NS �5.93 �11.2 NS �20.80.9745 0.9801 0.9789 0.9956 0.9711

ent in solids and hydrolysates; Xs, Xl: percentage of hemicellulosic sugars (sum ofpretreatment in solids and hydrolysates; Y, YP: Enzymatic hydrolysis yields referredglucose and other sugars present in the raw material that resulted degraded as a

factor transformation, as proposed by the statistical software, as follows:

70

E. Castro et al. / Bioresource Technology 102 (2011) 1270–1276 1273

and enzymatic hydrolysis yields referred to both feedstock and topretreated materials, were determined as model responses (Y). Re-sults are shown in Tables 3 and 5.

Table 5Glucose enzymatic hydrolysis yields referred to raw material (Y, g glucose/100 gglucose of raw material) and pretreated materials (Yp, g glucose/100 g glucose inpretreated material) and degradation of glucose (Gd) and other sugars (Xd) (%).

Run number Y Yp Gd Xd

1 21.20 22.25 0.00 1.112 53.70 98.21 42.91 57.293 56.82 62.96 5.18 15.874 nd nd 98.36 100.005 35.52 42.30 9.77 5.996 30.99 36.61 10.79 12.647 43.32 62.14 17.32 43.818 29.17 53.41 45.38 75.889 nd nd 91.65 99.35

10 25.86 71.99 41.42 90.4611 29.51 39.03 13.30 38.9712 41.66 56.96 14.34 51.3413 61.54 76.25 12.12 6.4314 44.15 60.86 15.22 46.3715 46.82 68.33 20.47 46.0916 46.61 64.75 15.31 41.0417 28.73 35.32 13.32 0.00

nd: not determined.

Actual

Pred

icte

d

Solid recovery, %

20

30

40

50

60

20 30 40 50 60 70

Fig. 1. Predicted versus experimental values for solid recovery after pretreatment.

Table 3Severity factor (Ro) and recovery (%) of glucose (G) and hemicellulosic sugars (X) inpretreated solids (subscript s) and hydrolysates (subscript l) from rapeseed strawpretreatment experiments.

Run number Ro Gs% Gl% Xs% Xl%

1 232.9 95.28 6.71 16.64 82.252 14813.7 54.68 8.41 10.43 32.283 833.7 90.25 4.57 25.73 58.404 23847.6 0.00 1.64 0.00 0.005 391.1 83.97 6.26 19.64 74.376 93. 6 84.64 4.57 38.98 48.387 1915.3 69.71 12.97 0.00 56.198 2995.8 54.62 0.00 0.00 24.129 14563.9 0.00 8.35 0.65 0.00

10 5575.6 35.92 22.66 0.00 9.5411 693.9 75.60 11.10 0.00 61.0312 1884.4 73.14 12.52 0.00 48.6613 3017.2 80.71 7.17 11.32 82.2514 1919.1 72.54 12.24 0.00 53.6315 1862.4 68.52 11.01 0.00 53.9116 1878.2 71.99 12.70 0.00 58.9617 233.0 81.34 5.34 67.90 34.70

Table 4Inhibitor composition (g/100 g raw material) of hydrolysates.

Run number Acetic acid Formic acid Furfural HMF

1 4.90 0.21 0.50 0.002 4.98 1.03 5.32 0.833 1.50 1.03 0.17 2.524 3.45 0.35 2.20 0.005 4.28 1.07 0.33 0.006 1.75 0.35 0.00 0.007 5.67 0.00 4.70 0.438 3.48 1.58 7.05 0.609 3.63 7.22 4.20 0.10

10 5.08 1.70 8.45 2.2311 5.35 0.00 3.42 0.1712 6.12 0.00 5.57 0.4513 3.92 0.00 1.47 0.1814 5.80 0.00 5.32 0.4515 5.57 0.00 4.30 0.4316 5.60 0.00 4.55 0.4217 0.37 0.28 0.00 0.00

The statistic interpretation of the results was formulated byusing the quadratic equation:

Y ¼ a0 þ a1 � t þ a2 � T þ a3 � Aþ a4 � t � T þ a5 � t � Aþ a6 � T � A

þ a7 � t2 þ a8 � T2 þ a9 � A2 ð2Þ

which allows the influence of each factor on the responses as wellas interactions among factors to be determined, according toparameters ai. Table 2 summarizes the model coefficients obtainedfrom ANOVA table for the different measured responses, togetherwith the statistic parameter R2. The model fitted the experimentaldata well (P < 0.05). In addition, F values were <0.0001, that is, thereis only a 0.01% chance that a ‘‘Model F-Value” this large could occurdue to noise. As an example, Fig. 1 shows a good agreement be-tween predicted and experimental values for one of the responses(solid recovery).

3.3. Effect of pretreatment on rapeseed straw

The dilute acid pretreatment of rapeseed straw resulted in awide variety of pretreated solids and hydrolysates in terms ofmaterial recovery and composition. Solid recovery ranged from29% to 66% depending on effective pretreatment conditions. Ascan be seen in Fig. 2, free acid concentration exerted lower influ-ence on material recovery than pretreatment temperature. Forexample, increasing temperature from 140 to 200 �C, at the lowestfree acid concentration and pretreatment time resulted in an in-crease of material solubilisation by 28.8% (solid recovery variedfrom 70.4% to 41.6%). By contrast, increasing effective acid concen-tration from the lowest to the highest level, at 200 �C and low pre-treatment time represented only a 12.7% increase in materialsolubilisation.

The composition of pretreated solids and hydrolysates in termsof recovery of the main sugars (glucan and xylan) is summarized inTable 3, where the percentages of glucose and other sugars presentin the raw material that are retained in the pretreated solids andthose which enter the liquid fractions are shown.

Concerning pretreated solids, it can be seen a wide range ofvariation in terms of cellulose retained in the solids after pretreat-ment. The cellulose virtually disappeared from solids of experi-ments 4 and 9, which showed the highest severity factors, while

0.4 0.8 1.2 1.6 2.0140

150

160

170

180

190

200Xl, %

Free acid, % Te

mpe

ratu

re, º

C

0

20

40

60

60

70

80

20

40

0

60

6070

80

Fig. 4. Contour plots for hemicellulosic sugars recovery in hydrolysates as afunction of free acid concentration and pretreatment temperature according to themodel (process time, 6 min).

0.4 0.8

1.2 1.6

2.0

140 150

160 170

180 190

200

20

30

40

50

60

70

80

Sol

id re

cove

ry, %

C: Free acid Temperature, ºC

Fig. 2. Response surface for material recovery as a function of free acid concen-tration and pretreatment temperature according to the model (process time,15 min).

1274 E. Castro et al. / Bioresource Technology 102 (2011) 1270–1276

remained almost unaltered at run 1. Considering xylan, most of thepretreatment experiments resulted in the total loss of this compo-nent in the pretreated solids. By contrast, most of it entered the li-quid fraction issued from pretreatment. The influence ofpretreatment variables in glucan recovery of the pretreated solidsis depicted in Fig. 3. It can be deduced that glucan remained inthe solid at the high level of acid concentration if temperaturewas kept at the low assayed level (140 �C). Temperature was alsoa determining factor for xylan dissolution, and only at low levelsof both temperature and acid remained almost unaltered in the so-lid fraction.

Glucose and xylose were also found in hydrolysates as a conse-quence of glucan and xylan dissolution due to lignocellulosic struc-ture breakdown at pretreatment conditions. Fig. 4 shows, usingcontour plots, how temperature and acid concentration conditionsdetermined the hydrolysate composition in terms of hemicellulo-

0.4 0.8

1.2 1.6

2.0 140 150

160 170

180 190

200

0

20

40

60

80

100

Gs,

%

Free acid, % Temperature, ºC

Fig. 3. Glucan recovery in pretreated solids as a function of free acid concentrationand pretreatment temperature according to the model (process time, 15 min).

sic sugars. In agreement with the above discussion, xylose was par-ticularly found in the hydrolysates obtained at high acidconcentrations and low temperatures and xylose recoveries as highas 82% were found under selected conditions. Concerning glucose,most of the hydrolysates showed an average glucose recoveryabout 8–10%, except two extreme experiments (0.0% and 22.6%glucose).

The hydrolysates contained also non-sugars compounds, asshown in Table 4. Acetic acid is a product of hemicellulose acetylgroup cleavage. Degradation of glucose and xylose due to severepretreatment conditions resulted in furfural and HMF generation.Formic acid is also a degradation product of furfural. All these com-pounds have been described as potential inhibitors of yeast fer-mentation at varying operational conditions and, at the sametime represent a monomeric sugar loss (Lau et al., 2009). In addi-tion, a synergistic inhibitory effect has been reported (Díaz et al.,2009). Acetic acid and furfural were the inhibitors commonlyfound at higher concentrations in this work. The highest acetic acidand HMF concentrations (6.12 and 2.52 g/100 g raw material),compare with the highest reported values when rapeseed strawwas pretreated by liquid hot water (5.95 and 2.58 g/100 g, respec-tively) which were obtained at 210 �C and 50 min pretreatmentconditions (Díaz et al., 2010). Formic acid and furfural concentra-tions were however higher in hydrolysates from dilute acidpretreatment than those obtained from liquid hot water pretreat-ment. Similar results to those reported here were obtained byShuai et al. (2010) on dilute acid pretreated spruce (180 �C, 5% acid,30 min). Lu et al. (2009), in a work on sulfuric acid pretreated rape-seed straw, also reported on furfural, HMF and acetic acid, withhighest results of 1.21 (furfural + HMF) and 4.40 g/100 g raw mate-rial (acetic acid), although the pretreatment temperature was notstudied and remained constant in all experiments (180 �C).

3.4. Enzymatic hydrolysis yields

The enzymatic hydrolysis yields referred to both raw materialand pretreated solids after 72 h enzymatic action are shown in Ta-

E. Castro et al. / Bioresource Technology 102 (2011) 1270–1276 1275

ble 5 and the influence of pretreatment temperature and effectiveacid concentration are depicted in Fig. 5a and b, respectively. Pre-treated solids obtained from experiments 4 and 9 were not submit-ted to enzymatic hydrolysis, since no cellulose was left as a resultof conditions of pretreatment. For the rest of solids, enzymatichydrolysis yields referred to raw material varied in a wide range21–62%, depending on pretreatment conditions.

From Fig. 5a, it can be deduced that the highest values of thisparameter are obtained from relatively high pretreatment temper-atures (in the assayed range, pretreatment temperatures below themedium point, 170 �C, resulted in comparative lower enzymatichydrolysis yields). High temperatures of dilute acid hydrolysishave been shown to result in increased enzymatic digestibility,and that has been potentially attributed to the glass transition oflignin (Jensen et al., 2010) which improves enzyme access to cellu-lose pore. However, this fact can be counteracted by lignin recon-densation reactions that occur at pretreatment conditions (Seliget al., 2007).

140 150

160 170

180 190

200 0.4 0.8

1.2 1.6

2.0

0

10

20

30

40

50

60

70

Y, %

Temperature, ºC Free acid, %

0.4 0.8

1.2 1.6

2.0 140 150

160 170

180 190

200

0

20

40

60

80

100

Yp,

%

Free acid, % Temperature, ºC

(a)

(b)

Fig. 5. Enzymatic hydrolysis yields as a function of free acid concentration andpretreatment temperature after 72 h enzymatic action. (a) Referred to raw material(pretreatment time, 6 min). (b) Referred to pretreated material (pretreatment time,27 min).

Regarding the influence of acid concentration on enzymatichydrolysis yields, it is enough using that necessary for the neutral-izing capacity of biomass to be reached.

Enzymatic hydrolysis yields referred to pretreated material (gglucose released/100 g glucose in pretreated material) show alsoa wide range of variation (22–98%). It is worth noting that, accord-ing to Fig. 5b, relatively high results can be obtained from a wideinterval of free acid concentration pretreatment conditions, andespecially at high temperatures.

Comparing enzymatic hydrolysis yields with those previouslyreported on the same feedstock, the best experimental result after72 h enzyme action (61.54 g glucose/100 g raw material, equiva-lent to 36.9 g/L) reported here compares well with that by Luet al. (2009), who reported 28.0 g glucose/L after 24 h. Enzymaticdigestibility as high as 95.4% after 72 h were reported by Jeonget al. (2010) using pretreated rapeseed straw at optimized condi-tions and favourable enzymatic dosage (60 FPU/g of glucan versus15 FPU/g substrate in the present work) and substrate concentra-tion (1%). If results are compared to other pretreatment methodson the same raw material, it is found that enzymatic hydrolysisyields based on raw material as high as 67.6 g/100 g were reportedafter liquid hot water pretreatment at 210 �C for 50 min, using thesame enzyme complex, while the present pretreatment conditionswere 170 �C for 20 min and 0.5% w/v acid sulfuric concentration.When results of glucose released by enzymes are referred to glu-cose content of pretreated solids, values of enzymatic hydrolysisyields are higher than those previously described. Even an experi-mental result as high as 98.2 g glucose/100 g of glucose in the pre-treated material was attained.

As a consequence of pretreatment, some sugars resulted de-graded, and hence were neither available for enzymatic releasenor present in the hydrolysates. For example, Lau et al. (2009) re-ported that about 13% of xylan was lost through chemical degrada-tion of dilute acid pretreated corn stover. By contrast,hydrothermal pretreatment of lignocellulosic materials is knownto result in a cellulose-enriched solid, which remains almost unal-tered (Garrote et al., 2008).

The proportion of degraded sugars is also summarized inTable 5, calculated as the difference between 100 and the sum ofthe amount of each type of sugar in both the pretreated solidand the liquid fraction. In general, sugar degradation occurred asa function of pretreatment severity, and enzymatic hydrolysisyields increased as sugar degradation did.

3.5. Model optimization

The mathematical model that was developed from the experi-mental results is able to predict the operational conditions thatshould be used in pretreatment to optimize model responses. Table6 summarizes these conditions for several optimization criteria.For example, optimization can be directed to the highest preserva-tion or recovery of sugars, both in the solid (cellulose) and inhydrolysates (mainly xylose). So if hemicellulosic sugars are to

Table 6Optimized pretreatment conditions according to the mathematical model fordifferent optimization criteria.

Optimizationcriteria

Pretreatement variables Optimal value

Time Temperature Freeacid

Maximize Xl 6.0 144.62 2.0 Xl = 92.3%, Gs = 99.4%Maximize Xl and

Gs

6.0 142.84 2.0 Xl = 92.2%, Gs = 100%

Maximize Y 27.0 180.47 0.40 Y = 64.93%Maximize Yp 27.0 200.0 0.40 Yp = 100%

1276 E. Castro et al. / Bioresource Technology 102 (2011) 1270–1276

be recovered in hydrolysates, along with cellulose in the pretreatedsolids, it is best to perform pretreatment at about 143 �C for 6 minand 2% free acid concentration. This result is in close agreementwith that obtained by Jeong et al. (2010), which reported optimumconditions of 152.6 �C, 21 min and 1.76% acid concentration.

Maximization of enzymatic hydrolysis yields is possible under avariety of pretreatment conditions. If yields are referred to rawmaterial, the total conversion of cellulose into glucose is predictedto be reached if pretreatment is performed at 200 �C for 27 min and0.4% free acid concentration. Considering the whole raw material, amaximum of 65% of the present glucose can be obtained, under theenzymatic hydrolysis conditions assayed, when rapeseed is pre-treated at 180 �C, 27 min 0.4% free acid concentration.

4. Conclusions

This work confirms that rapeseed straw can be considered asuitable feedstock for sugar generation as a first step toward fuelethanol production. Pretreatment with dilute sulfuric acid allowslower temperature to be used compared to hydrothermal pretreat-ment. Total conversion of the cellulose retained in the pretreatedmaterials into glucose can be attained by operation at 200 �C for27 min and 0.4% free acid concentration. By contrast, hydrolysateswill be more difficult to ferment into ethanol due to higher inhib-itor concentration. Further research for taking full advantage ofsugars in hydrolysates, including detoxification procedures, wouldimprove ethanol production from rapeseed straw.

Acknowledgements

This work was partially financed by Agencia Española de Coop-eración Internacional para el Desarrollo (AECID) under Projects ref.D/016096/08 and D/023784/09.

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