Enhancement of the enzymatic digestibility of sugarcane bagasse by steam pretreatment impregnated...

Enhancement of the Enzymatic Digestibility of Sugarcane Bagasse by Steam

Pretreatment Impregnated with Hydrogen Peroxide

Sarita Candida RabeloLaboratorio Nacional de Ciencia e Tecnologia do Bioetanol – CTBE/CNPEM, Caixa Postal 6170, CEP 13083-970, Campinas,Sao Paulo, Brazil

Dept. of Chemical Engineering, Lund University, PO Box 124, SE-221 00 Lund, Sweden

Carlos Eduardo Vaz Rossell and George Jackson de Moraes RochaLaboratorio Nacional de Ciencia e Tecnologia do Bioetanol – CTBE/CNPEM, Caixa Postal 6170, CEP 13083-970, Campinas,Sao Paulo, Brazil

Guido ZacchiDept. of Chemical Engineering, Lund University, PO Box 124, SE-221 00 Lund, Sweden

DOI 10.1002/btpr.1593Published online in Wiley Online Library (wileyonlinelibrary.com).

Sugarcane bagasse was subjected to steam pretreatment impregnated with hydrogenperoxide. Analyses were performed using 23 factorial designs and enzymatic hydrolysis wasperformed at two different solid concentrations and with washed and unwashed material toevaluate the importance of this step for obtaining high cellulose conversion. Similar cellu-lose conversion were obtained at different conditions of pretreatment and hydrolysis. Whenthe cellulose was hydrolyzed using the pretreated material in the most severe conditions ofthe experimental design (210�C, 15 min and 1.0% hydrogen peroxide), and using 2% (w/w)water-insoluble solids (WIS), and 15 FPU/g WIS, the cellulose conversion was 86.9%. Incontrast, at a milder pretreatment condition (190�C, 15 min and 0.2% hydrogen peroxide)and industrially more realistic conditions of hydrolysis (10% WIS and 10 FPU/g WIS), thecellulose conversion reached 82.2%. The step of washing the pretreated material was veryimportant to obtain high concentrations of fermentable sugars. VVC 2012 American Institute ofChemical Engineers Biotechnol. Prog., 000: 000–000, 2012Keywords: sugarcane bagasse, steam pretreatment, hydrogen peroxide, enzymatic hydrolysis,statistical analysis

Introduction

Lignocellulosic materials such as agricultural residues, for-estry, municipal wastes, and other low-cost biomasses are anabundant and renewable source of sugar substrate that couldbe fermented to ethanol. Among the various agriculturalcrop residues, sugarcane bagasse is the most abundant ligno-cellulosic material in tropical countries.1

Sugarcane bagasse is a by-product of sugarcane extractionprocess, which is mainly composed of cellulose, hemicellulo-ses, and lignin. In Brazil, ethanol/sugar plants use sugarcanebagasse to produce heat for boilers and electricity, but thereis always a variable surplus that is not utilized.2 One optionis to use the bagasse for production of ethanol, so-called sec-ond generation ethanol. This involves four steps: pretreat-ment, to render the cellulose accessible; hydrolysis with theaddition of enzymes or an acid catalyst to release the mono-meric sugars; fermentation to convert sugars into ethanol;and finally, distillation for product recovery.3

Thus, in the bioconversion of lignocellulosic biomass tofermentable sugars, the material should be subjected to pre-

treatment to increase its enzymatic digestibility. Furthermore,the pretreatment solubilizes, in part, lignin and hemicellulo-ses, disrupt the cellulose structure to decrease the crystallin-ity and increase the porosity of the materials to makecellulose more accessible to the enzymes.4 Also, both therelease of monomeric sugars from pretreated solids and frompretreatment liquor can contribute with ethanol production.

Several processes have been developed for pretreated sugar-cane bagasse, the steam pretreatment being one of the moststudied.5,6 This process involves the partial solubilization ofsome main components of biomass (lignin and hemicelluloses)by chemical degradation and mechanical deformation. Theprocess involves adiabatic expansion of water inside the poreand autohydrolysis of cell components.6

Steam pretreatment could improve the enzymatic hydroly-sis rate but has some limitations, such as only partialdestruction of xylan, incomplete disruption of the lignin-car-bohydrate matrix and limitation in the removal of lignin,redistributing it on the surface of cellulose.7,8

Alkaline hydrogen peroxide has been successfully devel-oped for biomass pretreatment being effective for hemicellulo-ses and lignin removal.9–11 However, even at hightemperatures, alkaline hydrogen peroxide is not reactivetowards most nonphenolic lignin structures.12 This is due to

Correspondence concerning this article should be addressed to S. C.Rabelo at at [email protected].

J_ID: BTPR Customer A_ID: BTPR1593 Ed. Ref. No.: 12-0059.R1 Date: 16-July-12 Stage: Page: 1

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VVC 2012 American Institute of Chemical Engineers 1

the fact that hydrogen peroxide is a very weak electrophileand reacts mainly as a nucleophile under alkaline conditions.One way to improve the electrophilicity of hydrogen peroxideto use it as a reaction agent or catalyst under acid conditions.

Unfortunately, there are same problems associated with thepractical applications of hydrogen peroxide in an acid medium:the lignin can condensate with its degradation products; and dueto rapid decomposition of hydrogen peroxide under these condi-tions, there may be degradation of cellulose.12 As an advantage,the use of hydrogen peroxide generates a structural change inthe lignin, making it more oxidized (high electron density),13

which may be a more powerful chelating agent for metals whencompared with lignin obtained in an alkaline process.

To further enhance the accessibility and cellulose contentof solid substrate, a steam pretreatment coupled with hydro-gen peroxide was investigated in this article. Different reac-tion conditions were investigated through analysis using 23

factorial design to determine the effects of the experimentalconditions on the chemical composition of the pretreatedmaterial and total cellulose conversion after pretreatmentand enzymatic hydrolysis. More specifically, the enzymatichydrolysis was performed with washed and unwashed mate-rial, at different solid concentrations, to evaluate the impor-tance of this step to obtain high cellulose conversion.

Materials and Methods

Raw material

Sugarcane bagasse (Saccharum officinarum) from a singleharvest was provided by Brazilian Bioethanol Science andTechnology Laboratory (CTBE), Campinas/SP, Brazil, obtainedfrom the Sugar Plant Usina da Pedra (Serrana/SP, Brazil). Thematerial is from 2009/10 harvest, obtained by mechanical har-vesting of sugarcane and from the last milling resulting afterjuice extraction. The raw bagasse (highly heterogeneous particlesizes) was washed, dried at room temperature for 48 h andshipped to Lund University (CE-LU), Lund, Sweden, by flight.The average dry matter content (DM) was 88.3% and the com-position of the dry matter is shown in Table 1.

Pretreatment

A 23 factorial design was performed through the interac-tions study of three variables of pretreatment: residence time

(min), temperature (�C), and hydrogen peroxide concentra-tion (%, w/w solution). These factors were applied to deter-mine the best combination of pretreatment conditions, takingas dependent variables or response of the experimentaldesign, the removal of hemicelluloses and lignin, and thetotal cellulose conversion after pretreatment and enzymatichydrolysis. The variables range and experimental design areshown in Table 1. Three runs at the central point of thedesign were carried out to estimate the random error neededfor the analysis of variance.

Bagasse samples corresponding to 500 g DM weresprayed with hydrogen peroxide solution at 0.2, 0.6, or1.0%. The spraying was carried out using a rotary system,which permits a better surface area contact between the sub-strate and the peroxide solution. After 1 h at room tempera-ture, the impregnated materials were pretreated.

The pretreatment was performed in a 10 L steam pretreat-ment reactor as previously described.14 Steam was providedusing a 110 KW electrical boiler (Pann-Partner, Stockholm,Sweden). The material (50% DM) was loaded into the pre-heated reactor. The time and temperature were rangedaccording to statistical design (Table 1). After each pretreat-ment, the material was discharged into a cyclone at atmos-pheric pressure connected to the outlet of the reactor,collected and stored in a cold room at 7�C.

The pretreated materials were divided into two parts. Partof these materials (slurry) were filtered in a Buchner funnelto recover the liquid fraction, and the solid fraction wasresuspended in hot water (�50�C) under mechanical stirringfor 2 h to remove the water-soluble solids (WS).15 Then, thematerials were filtered, washed several times until the washwater was colorless.

Pretreated material washed and unwashed (slurry) wereused for quantification of solid recovery by gravimetric anal-ysis (quadruplicate), for determination of chemical composi-tion by National Renewable Energy Laboratory (NREL)methods (triplicate),15 and for runs of enzymatic hydrolysis(duplicate). Monomeric and oligomeric sugars, cellobioseand by-products were analyzed in the liquid fraction byNREL methods.15

In addition to the statistical design, pretreatment runs werealso performed for the residence time of the central point (9min), by varying the temperature at 190, 200, and 210�C, in

Table 1. Process Variables and Chemical Composition of Raw Bagasse and WIS Obtained by Steam Pretreatment According to a 23 Full

Factorial Design

Level Bagasse Composition (%) Mass Recovery(g WIS/100 gRaw Bagasse)Runs t (min) T (�C) C (%, w/w Solution) Ash Extractives Total Lignin Cellulose Hemicelluloses

Untreated – – – 2.8 � 0.0 5.1 � 0.1 24.0 � 0.4 43.2 � 0.4 25.7 � 0.3 100.01 3 190 0.2 3.2 � 0.0 – 25.0 � 0.1 45.6 � 0.1 24.8 � 0.6 94.6 � 0.92 15 190 0.2 3.0 � 0.1 – 25.8 � 0.1 50.4 � 0.8 22.3 � 0.4 78.2 � 1.13 3 210 0.2 3.7 � 0.0 – 28.5 � 0.4 56.1 � 0.2 12.2 � 0.0 72.8 � 0.94 15 210 0.2 7.2 � 1.1 – 29.6 � 0.7 57.8 � 0.8 4.6 � 0.4 65.1 � 1.05 3 190 1.0 4.5 � 0.4 – 25.3 � 0.0 48.5 � 0.1 21.8 � 0.2 80.5 � 0.56 15 190 1.0 3.3 � 0.4 – 25.7 � 0.1 54.5 � 0.5 15.9 � 0.7 71.2 � 0.87 3 210 1.0 5.5 � 0.3 – 26.1 � 0.1 55.9 � 0.2 12.2 � 0.2 72.7 � 0.98 15 210 1.0 5.5 � 0.9 – 28.5 � 0.1 62.8 � 0.7 3.3 � 0.3 59.1 � 1.09 9 200 0.6 4.5 � 0.5 – 28.1 � 0.1 57.9 � 0.2 9.4 � 0.3 71.4 � 0.810 9 200 0.6 4.5 � 0.3 – 29.3 � 0.1 57.6 � 0.8 8.9 � 0.0 71.1 � 1.411 9 200 0.6 4.5 � 0.1 – 28.9 � 0.0 57.2 � 0.9 8.5 � 0.3 73.1 � 0.41* 9 190 – 2.0 � 0.1 – 25.0 � 0.2 48.0 � 0.5 24.8 � 0.4 88.0 � 0.62* 9 200 – 3.3 � 0.1 – 28.8 � 0.1 55.8 � 0.75 12.2 � 0.0 74.4 � 0.83* 9 210 – 3.4 � 0.7 – 29.2 � 0.2 57.0 � 0.30 10.5 � 0.4 69.3 � 0.9

t, time; T, temperature; C, concentration of hydrogen peroxide solution.* Noncatalyst.



2 Biotechnol. Prog., 2012, Vol. 00, No. 00

noncatalytic conditions, i.e., after spraying with water only.After pretreatment, the material was collected and separatedas previously described.

Severity factor

Steam pretreatment impregnated with hydrogen peroxidewas evaluated with the severity factor (SF) correlation,16 of-ten used to describe the lignin reduction and xylan solubili-zation. Equation 1 describes the SF of the pretreatment.

logðR0Þ ¼ log t: expT � Tref

14:75

� �� (1)

where t is the residence time of treatment in min, T is thetreatment temperature in �C, Tref is the reference temperature(100�C). The fitted value (14.75) of the arbitrary constant x,is based on the activation energy when assuming pseudo firstorder kinetics.17 This constant has been evaluated and insome cases optimized; however, 14.75 is often used withoutany concern.18

The combined severity factor (CSF) is calculated based onthe SF (log(R0)) (Eq. 1), and the pH after pretreatment,through Eq. 2,19 where the pH is measured after the pretreat-ment:

log R00� �

¼ log R0 � pH (2)

Enzymatic hydrolysis at low and high solids loadings

Enzymatic hydrolysis was carried out to evaluate the effi-ciency of pretreatment and was performed at low and higherloading of water-insoluble solids (WIS), using the cellulasemixture Cellic CTec 2 (193.7 FPU/mL enzyme solution) byNovozymes A/S (Bagsværd, Denmark). The filter paper ac-tivity was determined using the procedure recommended bythe IUPAC method.20

For low solids concentration, enzymatic hydrolysis wasperformed using only the washed materials. The enzymatichydrolysis was made using 2% (w/w) WIS (6.0 g of DM) in0.6 L hydrolysis vessels equipped with stirrers operating at300 rpm, using a total filling weight of 300 g. The amountof Cellic CTec 2 added corresponded to a cellulase activityof 15 FPU/g WIS. These hydrolyses were performed toobtain the maximum digestibility of the pretreated material.

For enzymatic hydrolysis at higher solids concentration,studies were performed using 10% (w/w) WIS (30 g of DM)in 1.0 L hydrolysis vessels equipped with stirrers operatingat 300 rpm, using a total filling weight of 300 g. The amountof Cellic CTec 2 added corresponded to a cellulase activityof 10 FPU/g WIS. These runs were performed with bothmaterials, washed and unwashed (slurry), to evaluate theeffect of entrained soluble sugars and other compounds pres-ent in the liquid fraction.

The total weight was adjusted to 300 g by addition of 0.1mol/L acetate buffer (pH 4.8) and kept in a water bath at atemperature of 50�C for 72 h. Aliquots were taken periodi-cally to monitor the extent of hydrolysis. However, thedigestibility of the each material was defined as hydrolysisyield after 48 h saccharification period and expressed as per-centage of cellulose and hemicelluloses conversion.

The percentage of cellulose and hemicelluloses conversion(or total hydrolysis yield) was calculated as total glucose orxylose liberated by a sample was divided by its cellulose or

hemicelluloses amount, and this hydrolysis yield was multi-plied by the pretreatment yield (considering the cellulose orhemicelluloses remaining fraction of Table 1). In this case,the total hydrolysis yield also takes into account losses ofcellulose and hemicelluloses fraction occurred duringpretreatments.

Analytical methods

The DM present in the solid fraction or slurry and theamount of dissolved solids in a liquor sample were deter-mined by drying the samples in an oven at 105�C until con-stant weight.15

The composition of the raw material, as well as the pre-treated material, was analyzed in terms of ash, extractives,carbohydrates, and lignin according to the standardizedmethods of the NREL.15

The dried and milled samples were combusted in a fur-nace at 575�C according to the NREL method,15 and theremaining ash was measured.

Finely ground samples were treated with 72% H2SO4 for1 h at 30�C, then diluted to 4% H2SO4 and autoclaved for 1hour at 121�C. Sugar contents in the hydrolysate were ana-lyzed with high performance liquid chromatography (HPLC)(Shimadzu, Kyoto, Japan). All the samples were diluted andpassed through a 0.2 lm filter before HPLC analysis, andacidic samples were neutralized by the addition of CaCO3.Cellobiose, glucose, xylose, arabinose, and mannose werequantified on an Aminex HPX-87P column (Bio-Rad labora-tories, Hercules, CA) run at a flow rate of 0.5 mL/min at85�C, with water as the eluent. Acetic acid, 5-hydroxymethylfurfural, furfural, lactic acid, formic acid, and levulinic acidwere quantified using an Aminex HPX-87H column (Bio-Rad laboratories, Hercules, CA) at 65�C with 0.5 mL/min 5mM H2SO4 as the eluent.

Acid-insoluble lignin was measured by weighing afterovernight drying of the residue after the acid hydrolysis at105�C, and acid-soluble lignin was determined by spectro-photometry using a wavelength of 240 nm. The fraction ofacid-insoluble ash was determined by heating samples at575�C until constant weight was achieved. Each sample wasanalyzed in duplicate.

The total amount of sugars present in the pretreatment liq-uor was analyzed according to the NREL total-sugar analysismethod15 using dilute-acid hydrolysis of the hydrolyzatefrom the pretreatment. The amounts of sugars (monosaccha-ride), by-products and organic acids were determined usingHPLC as described earlier. The oligosaccharides content wascalculated as the difference between the monosaccharidecontent measured before and after acid hydrolysis.

Enzymatic hydrolysis samples were analyzed by HPLC(Shimadzu, Kyoto, Japan) using the same conditions and col-umns as described earlier.

Experimental design

Statistical Software (version 10.0) (StatSoft, Tulsa, OK)was used for regression and graphical analyses (response sur-face) of the data obtained through a 23 full factorial design.The objective was to evaluate the influence of pretreatmenttime, temperature, and pretreatment agent concentration onthe subsequent removal of hemicelluloses and lignin, andtotal cellulose conversion after pretreatment and hydrolysis.

The analysis of the results of this design includes the lin-ear mathematical model, interactions effects, and the



Biotechnol. Prog., 2012, Vol. 00, No. 00 3

analyses of the variances ascribed to them. The statisticalsignificance of these effects was evaluated by using F tests.10

Runs were performed in a random order.

Results and Discussion

Pretreatment

Table 1 shows the experimental results for the chemicalcomposition of both pretreated and nonpretreated materials.Moreover, the total mass recovery (g pretreated material/100g raw bagasse) of the pretreated materials is shown. Totalsolid recovery after pretreatment varied over a broad range(95%–59%), indicating that the selected levels for pretreat-ment variables have a significant influence on material solu-bilization. At the tested conditions, the pretreatment resultedin enrichment of cellulose content (46–63% cellulose con-tent), due to the partial solubilization of both hemicellulosesand lignin.

Figure 1 shows the removal of cellulose, hemicelluloses,and lignin in each material after pretreatment of the condi-tions studied. Furthermore, the figure also shows the recov-ery percentage of these components in the pretreatmentliquor and the value of CSF. For calculation purposes, arabi-nose and xylose sugars (monomers and oligomers) were con-sidered as hemicelluloses, glucose, and cellobiose sugars(monomers and oligomers) as cellulose.

It is possible to note, analyzing Figure 1 that, in general,the hemicelluloses solubilized increased as the temperatureand the time increased (consequently increasing the CSF). Incontrast, a lower sugars recovery (monomeric and oligo-meric) in the liquor can be seen. In every way, no direct cor-relations can be discerned between the sugars release and theCSF. Hence, although it is clear that the pretreatment tem-perature, pH, and holding time are the major factors affect-ing pretreatment efficiency, many other parametersapparently affect the biomatrix opening, making it unrealisticto explain or predict the yields obtained by one simpleequation.21

High temperatures and short residence times (runs 3 and7) results in a greater hemicelluloses removal compared to

runs carried out at lower temperatures and longer residencetimes (runs 2 and 6). This has also been observed in otherstudies22,23 and these results depend on the pretreatmentstrategy as well as on the type and physical accessibility ofthe raw material used. In general, the overall carbohydrateyield decreases sharply with increased temperatures, whereashigher yields of lignin condensation and pentosan dehydra-tion are observed at longer reaction times.7

Analyzing Figure 1, the efficiency of hydrogen peroxidecatalyst can be seen, when comparing these results withthose obtained by Carrasco et al. (2010)24 using sugarcanebagasse as feedstock. The bagasse was steam pretreated attemperatures between 180 and 205�C, with holding times of5–10 min using SO2 as a catalyst. Pretreatment conducted at190�C for 5 min, using 2% SO2 gave a pentose yield of57%, with only minor amounts of degradation compoundsformed. In this study, operating in similar conditions (190�C,15 min, 1% hydrogen peroxide—run 6), a pentose yield of56.0% was obtained, from which 50.59% can be recoveredfrom the pretreatment liquor.

The highest removal of hemicelluloses and lignin wasobtained for the most severe pretreatment conditions, i.e., inrun 8, at 210�C, 15 mins and 1.0% (w/w) hydrogen perox-ide, corresponding to 92.4% and 29.7%, respectively. Conse-quently, the highest cellulose removal was also observedin this same run (14.1%). In contrast, the run 8 showsthe lower hemicelluloses recovery into the liquor, sincemuch of this sugar (76.3%) was degraded beyond oligomericand/or monomeric sugars. For the cellulose, only 30.7% ofsolubilized sugars were recovered into the liquorpretreatment.

Although the hemicelluloses is clearly the major constitu-ent extracted in steam pretreatment, unfortunately, goodhemicelluloses solubilization does not always correspond toelevated recoveries of hemicelluloses derived sugar in theliquid fraction. Belkacemi et al. (1991)25 proposed that hemi-celluloses solubilization from biomass occurred in two paral-lel reactions, for fast and slow solubilization. The differentrate for solubilization of hemicelluloses could explain thehigh degradation of soluble hemicelluloses-derived sugars, at

Figure 1. Removal and recovering of cellulose, hemicelluloses, and lignin after pretreatment at different conditions.

The combined SF for each run is also shown. Error bars are standard deviations from the average values of duplicate determinations.




https://www.researchgate.net/publication/231367873_Phenomenological_Kinetics_of_Complex_Systems_Mechanistic_Considerations_in_the_Solubilization_of_Hemicelluloses_Following_AqueousSteam_Treatments?el=1_x_8&enrichId=rgreq-3b134df2-03e0-4e98-aee6-1ca701dc178e&enrichSource=Y292ZXJQYWdlOzIyODEwNTE1NjtBUzoxMDM2NTk1MDk2NDk0MTlAMTQwMTcyNTc2MTU5OQ==

https://www.researchgate.net/publication/223173062_SO2-catalyzed_steam_pretreatment_and_fermentation_of_enzymatically_hydrolyzed_sugarcane_bagasse_Enzyme_Microb_Technol?el=1_x_8&enrichId=rgreq-3b134df2-03e0-4e98-aee6-1ca701dc178e&enrichSource=Y292ZXJQYWdlOzIyODEwNTE1NjtBUzoxMDM2NTk1MDk2NDk0MTlAMTQwMTcyNTc2MTU5OQ==

the same time that a considerable amount of hemicellulosesstill remains in the fiber.

It is known that the steam explosion pretreatment degradeshemicelluloses-derived sugars and solubilizes and transformsthe lignin compounds into inhibitor chemicals in the subse-quent processes.26 The main degradation products includeweak acids (mainly acetic acid), furans (degradation productof hemicelluloses sugars such as furfural, dehydration productof pentoses; and 5-hydroxymethylfurfural (HMF), a dehydra-tion product of hexoses), and phenolic compounds from lignin(aromatic acids, alcohols such as catechol, and aldehydes suchas 4-hydroxybenzaldehyde and vanillin). Some of the com-pounds formed during the degradation of hemicelluloses andlignin, contaminate the liquor of the steam-explosion process,whereas others become embedded in the biomass and arereleased during successive bioconversion.27

The yields of organic acids and sugars in the liquid afterpretreatment are shown in Table 2. The carbohydrates in theliquid fraction comprised mainly polymeric sugars, implyingthat during pretreatment the polysaccharides were not com-pletely hydrolyzed. The sugars conservation in the pretreat-ment liquor is very important because it can itself be used inethanol production, using the microorganism capable of fer-menting pentoses, or even the production of higher valueproducts.

Degradation products quantified in the liquid fraction canonly partially explain hemicelluloses losses in pretreatment.It is also possible that hemicelluloses and other compoundswere lost through volatilization of degradation products (e.g.,furfural) and recondensation reactions.28

It can be seen, by examining Table 2, that runs 1 and 5had CSF negatives. These values can be easily explainedbecause the CSF depends on the SF and the pH measured inthe slurry after the pretreatment step. Thus, at pH higherthan process SF (dependent of temperature and time, Eq. 1)a negative CSF is obtained.

At lower pretreatment severities, there is a partial conver-sion of acid-labile polysaccharides into water-soluble sug-ars.7 However, within the mid-range of pretreatmentseverities, soluble sugars derived from plant polysaccharidesare partially lost as dehydration by-products. This can beobserved by comparing runs 3–4 and 7–8, where the per-oxide concentration was kept fixed. In the comparison

between runs 3 and 4 the increase of the combined SF by afactor of 4.3, lead to the decrease in pH due to the release ofacetyl groups and reduced the hemicelluloses conversionpresent in the liquid fraction almost by half. In relation toruns 7 and 8, the same trend could be observed with a reduc-tion of the hemicelluloses conversion by a factor of 2.2when the combined SF increased 5.7 times.

The impregnation of the material with hydrogen peroxideresulted in a slightly larger degradation of sugars and conse-quently increased formation of inhibitors, when compared toa not-catalyzed pretreatment, considering the same reactiontime and temperature (runs 9, 10, 11, and 2*). However, ahigher amount of total xylose (monomer and oligomer) wasrecovered in the liquid in these samples compared to plainsteam pretreatment.

Enzymatic hydrolysis

Effects of the Solid Concentration and Enzyme Load. Follow-ing the pretreatment, the remaining solid material was sub-jected to enzymatic hydrolysis. Washed materials from allpretreatments were subjected to enzymatic hydrolysis for72 h at a 2 and 10% (w/w) WIS content, using 15 FPU/gWIS and 10 FPU/g WIS, respectively. For comparisonpurposes, enzymatic hydrolysis was also carried out on rawmaterial not subjected to steam pretreatment.

Figures 2 and 3 show the total cellulose conversion andxylose after pretreatment and enzymatic hydrolysis usingtwo different solids concentrations and enzyme loads. Figure2 shows the total cellulose and hemicelluloses conversionof the pretreated material, since these hydrolysis were per-formed with a low concentration of solids and considerableenzyme loading [2% (w/w) WIS and 15 FPU/g WIS,respectively].

The highest total cellulose conversion, 86.9%, was ob-tained in run 8 (210�C, 15 min and 1.0% peroxide). Similarcellulose conversion were obtained in runs 2, 4, and 6(85.5%, 83.8%, and 86.8%, respectively), all three for thehighest pretreatment times studied, with varying temperatureand peroxide concentration.

When the steam-pretreated material (run 2*) was com-pared with peroxide impregnated material (runs 9, 10, and11) at the same conditions regarding residence time and

Table 2. Yields of Sugar and By-Products in the Liquid Fraction of the Pretreatment (g/100 g DM Raw Material)

Runs 1 2 3 4 5 6 7 8 9 10 11 1* 2* 3*

Time (min) 3 15 3 15 3 15 3 15 9 9 9 9 9 9Temperature (�C) 190 190 210 210 190 190 210 210 200 200 200 190 200 210Concentration (%, w/w) 0.2 0.2 0.2 0.2 1.0 1.0 1.0 1.0 0.6 0.6 0.6 – – –pH liquor 3.45 3.15 3.42 3.12 3.86 3.53 3.46 2.93 3.31 3.29 3.27 3.50 3.63 2.96Combined severity factor (Log R0’) �0.32 0.68 0.30 1.29 �0.73 0.30 0.26 1.48 0.59 0.61 0.63 0.40 0.27 0.94

XyloseMonomer 0.5 2.7 1.5 3.2 0.5 2.7 1.3 3.6 2.6 2.4 2.7 0.6 2.1 4.3Oligomer 0.9 0.1 10.1 3.5 7.0 11.0 11.6 2.2 12.5 11.7 13.4 2.3 9.5 4.6Total 1.4 2.7 11.6 6.6 7.6 13.7 12.9 5.8 15.1 14.1 16.1 2.9 11.5 8.9

GlucoseMonomer 0.1 0.1 0.0 0.7 0.0 0.0 0.1 0.7 0.1 0.1 0.1 0.1 0.1 0.2Oligomer 0.2 0.2 1.3 1.1 1.4 1.5 1.7 1.5 2.1 2.0 2.2 1.7 1.8 1.9Total 0.2 0.3 1.3 1.8 1.5 1.6 1.8 2.2 2.2 2.0 2.3 1.7 1.9 2.1

Arabinose 0.6 0.7 0.2 2.1 1.1 1.1 1.1 0.6 1.2 1.2 1.2 1.0 0.9 0.7Formic acid 0.1 0.1 0.1 0.3 0.1 0.3 0.1 0.6 0.2 0.2 0.3 0.1 0.1 0.4Acetic acid 0.3 0.6 0.6 1.7 0.3 1.2 0.6 3.2 1.1 1.1 1.2 0.5 1.0 2.2Levulinic acid 0.0 0.1 0.0 0.0 –* 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0HMF 0.0 0.0 0.0 0.2 –* 0.0 0.0 0.3 0.1 0.0 0.1 0.0 0.0 0.1Furfural 0.1 0.3 0.3 2.1 0.1 0.8 0.2 2.9 0.6 0.5 0.6 0.1 0.4 1.2

* Below detectable level




temperature, one can observe that the peroxide impregnationresulted in an increase in the total cellulose conversion,while the hemicelluloses conversion was lower, probably dueto a higher rate of hemicelluloses degradation, see Table 2.

Similar results were observed of cellulose conversion inCarrasco et al. (2010)24 using sugarcane bagasse pretreatedat 190�C, 5 min and 2% SO2. The overall highest glucoseyield achieved from bagasse was 91.7% at 2% WIS usingcommercial cellulolytic enzymes (15 FPU/g pretreated ba-gasse and 20 IU/g pretreated bagasse). The results obtainedin the run 2 show slightly lower cellulose conversion(85.5%), when the material was pretreated at 190�C, 15 minand 0.2% peroxide. However, working with higher solidsconcentrations (10% WIS) under the same operating condi-tions, it was possible to obtain a cellulose conversion of82.2% after hydrolysis of the pretreated material.

The conditions for steam pretreatment of sugarcane ba-gasse and leaves were studied using CO2 as an impregnatingagent.29 For bagasse, the highest glucose yield (86.6% oftheoretical) was obtained after pretreatment at 205�C for 15min using 3% (w/w) CO2. The enzymatic hydrolysis wasdeveloped with 2% (w/w) of washed material using 15 FPU/g fibrous material of cellulase and 18 IU/g fibrous materialof b-glucosidase. Using peroxide catalyst, milder conditionsof pretreatment (190�C, 15 min, 1% peroxide - run 6) werenecessary to achieve a similar cellulose conversion (86.8%)after hydrolysis using 2% (w/w) WIS and similar enzymeload.

Considering the high cost of enzymes and the need toobtain hydrolysates with high concentrations of sugars,which would minimize costs during the step of ethanol distil-lation, the hydrolysis of the pretreated materials was alsoperformed using 10% (w/w) WIS and 10 FPU/g WIS. Figure3 shows that the highest cellulose conversion obtained afterpretreatment and enzymatic hydrolysis was 82.2%, obtainedfor run 2 (190�C, 15 min and 0.2% peroxide). Run 4(210�C, 15 min and 0.2% peroxide) also led to a high totalcellulose conversion (81.5%). Only the pretreatment temper-ature differs while the other operational conditions were thesame. In addition, there is a considerable difference betweenthe values of the combined SF of the two runs (Table 2).

It is important to emphasize that the CSF,19 indicated onTable 2, takes into account not only time and temperature ofthe steam pretreatment, but also the acidity generated in thereaction media by the release of organic acids from the rawmaterial, as indicated by pH measurement after pretreatment.This factor is used to compare the action of pretreatment onthe cellulose conversion with respect to xylan solubilizationand lignin reduction. When comparing cellulose conversionas a result of pretreatments at different pH values, one musttherefore consider the use of Eq. 2.

Steam pretreatment catalyzed with hydrogen peroxide waseffective in cellulose conversion after hydrolysis using highconcentrations of solids when compared to other impregnat-ing agents used in sugarcane bagasse.30,31

Pretreatment of bagasse by autohydrolysis at 200�C for 4min in an explosion reactor30 resulted in the solubilization of90% of the hemicelluloses and in the production of a pulpthat was highly susceptible to hydrolysis by cellulases. Sac-charification yields of 50% resulted after 24 h at 50�C wasreported after enzymatic hydrolysis containing 10% (w/v) ofpretreated material and 20 FPU/g WIS. Saccharification wasincreased to more than 80% at 24 h by the addition of exog-enous b-glucosidase from Aspergillus niger.

Ewanick and Bura (2011)31 obtained a cellulose conver-sion of �90% when the sugarcane bagasse was pretreatedusing steam explosion reactor at 205�C for 10 min impreg-nated with 3% (w/w) SO2. Enzymatic hydrolysis of washedsolids was done at 5% (w/v) solids employing 10 FPU/g cel-lulose e 20 CBU/g cellulose of enzymes. The same authorshave reported cellulose conversions of about 75% when thebagasse has been pretreated under the same operational con-ditions but without catalyst presence.

Unlike it was observed in the enzymatic hydrolysis using2% (w/w) WIS, the pretreated material impregnated withhydrogen peroxide led to a total cellulose conversion slightlylower when compared to the steam-pretreated material at thesame operational conditions of pretreatment with no impreg-nation (runs 9, 10, and 11 versus run 2*). Lower total cellu-lose conversion were obtained from the nonpretreatedmaterial using 10% WIS when compared with bagasse

Figure 3. Total cellulose and hemicelluloses conversion afterpretreatment and enzymatic hydrolysis with 10%(w/w) WIS, using washed steam pretreated bagasseat different conditions.

The untreated material hydrolysis is also shown. The error barsare standard deviations from the average values of duplicatedeterminations. NP: nonpretreated material.

Figure 2. Total cellulose and hemicelluloses conversion afterpretreatment and enzymatic hydrolysis with 2% (w/w) WIS, using washed steam pretreated bagasse atdifferent conditions.

The untreated material hydrolysis is also shown. The error barsare standard deviations from the average values of duplicatedeterminations. NP: nonpretreated material.




hydrolyzed at 2% (w/w) WIS (16.8% versus 21.9%), whilethe hemicelluloses conversion were similar, 8.7%.

Another fact that should be emphasized is the relativeincrease in glucose concentration when working at high sol-ids concentrations (62.8 g/L in run 4), when compared withthe optimal concentration obtained from the enzymatic hy-drolysis using 2% (w/w) WIS (14.0 g/L in run 8).

In the enzymatic hydrolysis runs carried out with the ma-terial without hydrogen peroxide impregnation (1*, 2*, and3*) and using the washed material (Figures 2 and 3), it ispossible to notice that as the pretreatment temperatureincreases, the hemicelluloses removal also increases (Table1). This means there is a smaller hemicelluloses fractionpresent in the material (3*\2*\1*) and, in contrast, hemi-celluloses is the most difficult to be solubilized during thehydrolysis process. Moreover, the low hemicelluloses con-version observed in the enzymatic hydrolysis liquor shouldbe due to the fact that the commercial cellulolytic complexeshave enzymatic activity low of hemicellulases.

Effect of Washing the Pretreated Material. The degrada-tion of hexoses and pentoses during the pretreatment devel-oped at high temperature and pressure results in theformation of byproducts, mainly HMF and furfural. Thesebyproducts not only lead to a lower yield of the solubilizedsugars, but also causes the potential inhibition of enzymesand fermenting organisms.32

To evaluate the effect of washing the pretreated materialbefore enzymatic hydrolysis, Figure 4 shows the total cellu-lose and hemicelluloses conversion from the washed andunwashed material after enzymatic hydrolysis at 10% (w/w)WIS and enzyme loading of 10 FPU/g WIS.

As expected, total cellulose conversion was in generalhigher for the washed material than for the unwashed material(slurry). Similar results were observed by Carrasco et al.(2010),24 when the bagasse was pretreated by SO2-catalyzedsteam pretreatment. It is possible to observe analyzing Figure4, that in all runs using the washed material, the hemicellulo-ses conversion is smaller. This is due to the fact that in theruns using the unwashed material (slurry) there are xylose andxylooligosaccharides that have remained from the pretreatment,which means there is an increase of this sugar. Furthermore,due to the fact that the commercial cellulolytic complexes

have enzymatic activity low of hemicellulases, the remaininghemicelluloses in the solid material hardly will be hydrolyzed.

The highest total cellulose conversion was obtained in run2, for both materials impregnated with peroxide. For thewashed material, keeping the variables temperature and per-oxide concentration fixed, an increase in the total celluloseconversion when working at longer pretreatment times (runs1–2, 3–4, 5–6, and 7–8) was obtained. Furthermore, theincreased time of pretreatment leads to a greater increase intotal cellulose conversion when working at 190�C (runs 1–2and 5–6), if compared to runs at 210�C (runs 3–4 and 7–8).In contrast, the longer the pretreatment time, when workingat high temperatures, the lower the cellulose conversionobtained for the unwashed material.

In runs 4 and 8, where there is a greater furfural productionand acetic acid release (Table 2), a considerable reduction inthe total cellulose conversion was obtained for unwashed ma-terial in the enzymatic hydrolysis compared to using washedmaterial (Figure 4). The result shows that the presence ofthese compounds in the liquid fraction strongly affects the hy-drolysis, reducing the cellulose conversion from 81.1% to15.4% for run 4 and from 73.5% to 14.7% for run 8.

Some authors show that organic acids can affect the effi-ciency of enzymatic hydrolysis. Acetic acid can interferewith enzyme recognition thereby slowing the hydrolysisrate.33 It might inhibit the formation of productive binding(hydrogen bonds) between cellulose and the catalytic domainof cellulases. Furthermore, the presence of acetic acid mightincrease the diameter of the cellulose chain, which increasesthe steric hindrance of enzymes.34

Similar results were obtained by Garcıa-Aparicio et al.(2006)26 It was observed that the presence of inhibitors inthe liquid fraction generated during the steam pretreatment(210�C, 15 min) by barley straw, strongly affected the hy-drolysis step, leading to a decrease of 25% in the conversionof cellulose when compared to the washed material.

Wet oxidation has also been applied to sugarcane bagasseunder acidic (addition of sulfuric acid) and alkaline condi-tions (addition of Na2CO3).35 The digestibility of the pre-treated fiber fraction was tested using washed andnonwashed material, and a somewhat lower digestibility wasobserved using the nonwashed material.

Furthermore, Qing et al. (2010),36 recently reported evi-dence that xylose, xylan, and xylooligomers dramaticallydecrease conversion rates and yields of enzymatic hydrolysis.The authors showed that sugars were more potent inhibitorsof enzymatic hydrolysis than degradation products.

Another factor that may have contributed to the low celluloseconversion using unwashed material is the presence of phenoliccompounds. During the pretreatment, part of the structure of lig-nin is solubilized, due to a disruption of the lignin-carbohydratebonds.37 Thus, such compounds present in the liquid fractioncan significantly affect the enzymatic hydrolysis.

Analyzing Figure 4, it is observed that the higher the com-bined SF, the lower the cellulose conversion obtained fromthe unwashed material. In contrast, a very significant produc-tion of xylose was found from the hydrolysis of xylan pres-ent in the residual unwashed material. Higher additionalhemicelluloses conversion were observed in intermediateconditions of pretreatment, both for the material impregnatedwith hydrogen peroxide (runs 9, 10, and 11) and for the ma-terial without impregnation (run 2*). The additional hemicel-luloses conversion, due to hydrolysis of oligomers, was 33.5,

Figure 4. Effect of washing the pretreated material on totalcellulose and hemicelluloses conversion after enzy-matic hydrolysis using 10% (w/w) WIS.




Figure 5. Pareto chart of standardized effects for the hemicelluloses removal (a), lignin removal (b), cellulose conversion using 2%(w/w) WIS (c), 10% (w/w) WIS of the washed material (d), and 10% (w/w) WIS of the unwashed material (e).

Table 3. ANOVA for the Model that Describes the Different Response Variables

Source of Variation

Hemicelluloses Removal (%) Lignin Removal (%)

SQ df MS F Value F Listed SQ df MS F Value F Listed

Regression (R) 5909.48 4 1477.37 10.09 3.18 469.69 3 156.56 18.02 3.07Residual (r) 878.29 6 146.38 225.11 9.24 60.82 7 8.69 1.93 9.29Lack of fit (Lf) 876.35 4 219.09 50.37 5 10.07Pure error (Pe) 1.95 2 0.97 10.45 2 5.23Total (T) 6787.77 10 530.51 10R2 0.87 0.89

Source of Variation

Total Cellulose Conversion - 2% (w/w) WISTotal Cellulose Conversion - 10% (w/w) WIS,

Washed Material

SQ df MS F Value F Listed SQ df MS F Value F Listed

Regression (R) 1158.80 3 386.27 8.96 3.07 869.36 4 217.34 1.58 3.18Residual (r) 301.89 7 43.13 8.66 9.29 827.75 6 137.96 21.94 9.24Lack of fit (Lf) 288.57 5 57.71 809.30 4 202.32Pure error (Pe) 13.33 2 6.66 18.45 2 9.22Total (T) 1460.70 10 1697.10 10R2 0.79 0.51

Source of Variation

Total Cellulose Conversion - 10% (w/w) WIS, Unwashed Material

SQ df MS F Value F Listed

Regression (R) 2234.74 3 744.91 13.70 3.07Residual (r) 380.60 7 54.37 7.47 9.29Lack of fit (Lf) 361.26 5 72.25Pure error (Pe) 19.34 2 9.67Total (T) 2615.35 10R2 0.85

SQ, sum of squares; df, degrees of freedom; MS, mean square.*F test for statistical significance of the regression ¼ MSR/MSr.**F test for lack of fit ¼ MSLf/MSPe.




36.5, 35.4, and 38.9%, respectively, considering the totalxylan content in the bagasse.

Although xylan removal has been previously shown toimprove the cellulose digestibility,38,39 in this study, it wasnot possible to observe a direct relationship between theseparameters. This is due to the fact that many other factorsinfluence the hydrolysis, including lignin content, particlesize, available surface area, and cellulose crystallinity. With

so many variables, it is difficult to determine the extent ofthe role that xylan plays.40

Statistical Analysis. The results of statistical analysis,using the statistical software, are shown by Pareto chartssince they represent, very clearly, the most significanteffects. In these charts, the effect estimates divided by theirstandard errors are sorted from the largest absolute value tothe smallest absolute value. The magnitude of each effect is

Figure 6. Response surface plots showing the hemicelluloses removal (a), lignin removal (b), cellulose conversion using 2% (w/w) WIS(c), 10% (w/w) WIS of the washed material (d), and 10% (w/w) WIS of the unwashed material (e).




represented by a column and a line going across the columnsindicates how large an effect must be to be considered statis-tically significant. In this work, the vertical line correspondsto a P value of 0.1, which implies in a 90% of confidencelevel. Figure 5 shows the Pareto chart of standardized effectsfor removal of hemicelluloses and lignin, and total celluloseconversion after pretreatment and hydrolysis.

The 23 full factorial design with three replicates in thecentral point was chosen because it allows to analyze theinfluence of each factor, their interactions, verify the pres-ence of curvature of the plan and determine the experimentalerror. It can be seen by analyzing Figure 5, that the curva-ture of the model was significant for most of the responsesanalyzed, except for the lignin removal. Thus, for the modelthat describes the lignin removal in the region investigated,there are not quadratic terms and the model behaves in a lin-ear fashion. Thus, it is possible to determine an empiricalmodel in this case, using the significant coefficients andeliminating all the effects that are not significant (P [ 0.1).

For other responses, where the curvature of the plan wassignificant at 90% confidence level, more experimentalpoints would be needed to the model development mostappropriate for each of these phenomena.

Table 3 shows the ANOVA for the models studied. Bothmodels present high correlation coefficients and the F valuefor statistical significance of the regression is higher than thelisted ones. Nevertheless, only the model that represents thelignin removal did not present evidence of lack of fit, as thecalculated values for the F test for lack of fit are muchsmaller than the listed values. Note that the models describ-ing the cellulose conversion at 2% (w/w) WIS and celluloseconversion at 10% (w/w) WIS unwashed material alsoshowed no problems of lack of fit, although the calculatedvalue of F test is not so much smaller that the listed, asobserved for the lignin removal. Anyway, as mentioned ear-lier, the linear model does not describe well the dataobtained for these two responses.

A model with evidence of lack of fit cannot be used for pre-diction or optimization purposes. However, it can be used toplot qualitative response surfaces that can aid in determiningthe best experimental region, as shown in Figure 6, where per-oxide concentrations were kept fixed at central point.

Another factor that may emphasize the fact of not using a lin-ear model to describe the responses of cellulose conversion at2% (w/w) WIS and 10% (w/w) WIS unwashed material is pre-sented in Figures 6c,e, respectively. A curvature in these surfacescan be seen, showing that the function that best describes thesesets of results is a polynomial of higher order.41

Thus, by statistical analysis, the equation that describesthe lignin removal, satisfying the operational conditions ofthe system is represented by Eq. 3.

Lignin removal %ð Þ ¼ 16:45þ 4:81þ 3:46T þ 4:86C (3)

In this equation, t, T, and C are the coded values of pre-treatment time, temperature, and peroxide concentration,respectively.

Conclusions

The utilization of hydrogen peroxide as an alternativeimpregnating agent for steam pretreatment is promising,because it presents low toxicity and corrosivity, besides topromote high conversion of cellulose employing a low con-

centration of catalyst. Other important factor is the possibil-ity of generating a structural change in the lignin, making itmore oxidized and thus, more effective to use as chelatingagent for metals.

The 23 experimental designs applied in this study showedthat the removal of hemicelluloses and lignin clearly pro-duced a substrate with improved characteristics for enzy-matic hydrolysis by cellulases. As observed in other studies,the overall carbohydrate yield decreases with increased tem-peratures and time, whereas higher yields of lignin condensa-tion and hemicelluloses dehydration are observed at longerreaction times. The highest removal of hemicelluloses andlignin was obtained for the most severe pretreatment condi-tions, i.e., in run 8, at 210�C, 15 mins and 1.0% (w/w)hydrogen peroxide, corresponding to 92.4% and 29.7%,respectively and consequently, the lower hemicelluloses re-covery into the liquor was observed, since 76.3% of hemicel-luloses was degraded beyond oligomeric and/or monomericsugars. In general, it was noticed that the higher the CSF,the greater the hemicelluloses removal during the pretreat-ment, and the lower the sugars recovery (monomeric and oli-gomeric) in the liquor.

Similar cellulose conversion was obtained at different condi-

tions of pretreatment and hydrolysis. High conversions of cellu-lose were found in the runs 2, 4, 6, and 8, whereas the hydrolysis

runs with 2% (w/w) WIS and 15 FPU/g WIS, leading to a total

conversion of cellulose of 85.5, 83.8, 86.8, and 86.9%, respec-tively. High yields were also found when working in hydrolysis

conditions more realistic industrially (10% WIS and 10 FPU/gWIS). Both runs 2 and 4 showed total conversion of cellulose

quite significant, 82.2% and 81.5%, respectively. It is observed

that it is possible to work with higher concentrations of solid andsmaller enzyme loading, obtaining similar yields. In this case,

the process operating conditions (time, temperature, hydrogen

peroxide concentration, solids concentration, and enzyme load)and sugars recovery present in the liquor pretreatment can be

used to choose the best pretreatment.

The step of washing the pretreated material was very im-portant to obtain high concentrations of fermentable sugars.It was observed that higher the CSF, the lower the celluloseconversion obtained from the unwashed material. Hemicellu-loses was partially hydrolyzed by the enzymes used and theconvertibility of hemicelluloses was higher in the hydrolysisof the whole slurry than in the hydrolysis of the washedsolid material.

Comparable pretreatment performance was obtained withhydrogen peroxide when compared to other catalytic agents.In some cases, the result with hydrogen peroxide showed tobe better. However, further work is required to assess thebeneficial effects of pretreatment and better optimization ofthe process.

Acknowledgment

Financial support provided by the Ministry of Science, Tech-nology and Innovation of Brazil and the infrastructure used atLund University for the development of this work are greatlyacknowledged.

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