Bioresource Technology 102 (2011) 4793–4799

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Bioresource Technology

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Low temperature alkali pretreatment for improving enzymatic digestibilityof sweet sorghum bagasse for ethanol production

Long Wu a, Mitsuhiro Arakane a, Masakazu Ike a, Masahisa Wada b,c, Tomoyuki Takai d, Mitsuru Gau d,Ken Tokuyasu a,⇑a Carbohydrate Laboratory, Food Resource Division, National Food Research Institute, National Agriculture and Food Research Organization (NARO), 2-1-12 Kannondai,Tsukuba, Ibaraki 305-8642, Japanb Department of Biomaterials Science, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japanc Department of Plant and Environmental New Resources, College of Life Sciences, Kyung Hee University, 1, Seocheon-dong, Giheung-ku, Yongin-si, Gyeonggi-do 446-701,Republic of Koread Forage Crop Breeding Unit, National Agricultural Research Center for Kyushu Okinawa Region, National Agriculture and Food Research Organization (NARO), 2421 Suya,Koshi, Kumamoto 861-1192, Japan

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 September 2010Received in revised form 7 January 2011Accepted 11 January 2011Available online 20 January 2011

Keywords:LignocelluloseRecalcitrancePulpingDelignificationAccessibility

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.01.023

⇑ Corresponding author. Tel.: +81 29 838 7189; faxE-mail address: tokuyasu@affrc.go.jp (K. Tokuyasu

A low temperature alkali pretreatment method was proposed for improving the enzymatic hydrolysisefficiency of lignocellulosic biomass for ethanol production. The effects of the pretreatment on the com-position, structure and enzymatic digestibility of sweet sorghum bagasse were investigated. The mech-anisms involved in the digestibility improvement were discussed with regard to the major factorscontributing to the biomass recalcitrance. The pretreatment caused slight glucan loss but significantlyreduced the lignin and xylan contents of the bagasse. Changes in cellulose crystal structure occurredunder certain treatment conditions. The pretreated bagasse exhibited greatly improved enzymatic digest-ibility, with 24-h glucan saccharification yield reaching as high as 98% using commercially available cel-lulase and b-glucosidase. The digestibility improvement was largely attributed to the disruption of thelignin-carbohydrate matrix. The bagasse from a brown midrib (BMR) mutant was more susceptible tothe pretreatment than a non-BMR variety tested, and consequently gave higher efficiency of enzymatichydrolysis.

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1. Introduction

Biofuel, as an alternative renewable energy source, is receivingincreasing attention worldwide because of the imminent depletionof fossil fuel reserves and the rapid accumulation of atmosphericgreenhouse gases. Presently, the production of the second genera-tion cellulosic ethanol from those abundant and inexpensive non-food lignocellulosic biomass materials, such as agricultural resi-dues and herbaceous energy crops, has become the focus of re-search due to its potential advantages (Lynd et al., 1991; Sánchezand Cardona, 2008; Sims et al., 2010).

Lignocellulose consists primarily of plant cell wall materials; itis a complicated natural composite with three major constituents,i.e. cellulose, hemicellulose and lignin. The hemicellulose–lignincomplex and the crystalline structure of cellulose are largelyresponsible for the recalcitrance of lignocellulose to hydrolysis(Hsu et al., 1980). Presently, the enzymatic degradation of lignocel-

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lulose has generally been considered a feasible way to extract sug-ars from lignocellulosic biomass for ethanol fermentation. In orderto achieve a high glucose yield for yeast fermentation, much of re-cent research has focused on the improvement of enzymaticdigestibility of the cellulose through various pretreatments. Cur-rently, thermochemical pretreatment methods for opening up thestructure of lignocellulose have been widely studied (Hendriksand Zeeman, 2009; Wyman et al., 2005). Pretreatment can selec-tively remove the structural barriers and increase cellulose acces-sibility depending on the chemical agents used (e.g. acid, alkali,oxidant, etc.) and treatment conditions applied. Some thermo-chemical pretreatment performed at high temperatures(>150 �C), such as dilute sulfuric acid, SO2 steam explosion orammonia pretreatment, can improve the enzymatic digestibilityof lignocellulosic biomass to some extent (Öhgren et al., 2007; Si-pos et al., 2009). However, the hydrolysis efficiency of cellulosewas still improvable considering the relatively low saccharificationyields and high enzyme loading. Besides, the pretreatmentmethods involving high process temperature require higher energyinput and heat-resistant/anti-corrosive pressure cooking equip-

Table 1Low temperature alkali and dilute sulfuric acid pretreatment conditions.

Pretreatment Parameter Scope

LTA NaOH concentration (mol/l) 0.5–5Solid to liquid ratio (%, w/v) 5, 10, 15Temperature (�C) 25, 50Treatment time (h) 0.5–24

Dilute sulfuric acid Concentration (%, w/w) 0.5Solid to liquid ratio (%, w/v) 5Temperature (�C) 170Treatment time (min) 5–60

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ments; excessively high temperatures may also aggravate thedegradation of the useful components and the formation offermentation inhibitors (Oliva et al., 2006; Panagiotou and Olsson,2007). On the other hand, the pretreatment methods adoptingmoderate temperatures (e.g. lime, ozone, H2O2, etc.) reportedlyhad quite inconsistent effect on the enzymatic digestibility of thebiomass (Garcia-Cubero et al., 2009; Kim and Holtzapple, 2006a;Silverstein et al., 2007). In general, currently available pretreat-ment techniques can hardly meet the requirements of efficient lig-nocellulose-to-ethanol conversion.

Pulping is the process of converting lignocellulosic materials topulp fibers for papermaking. The presently dominant chemical pul-ping process — the kraft process — uses a solution of sodiumhydroxide and sodium sulfide to dissolve and remove the majorityof lignin that binds cellulose fibers together at elevated tempera-ture and pressure to produce pulp consisting of almost pure fibers.The major objective of the pulping process, i.e. to liberate carbohy-drate polymers from their complex with lignin, is generally consis-tent with that of the pretreatment operation focusing on removingthe recalcitrance of lignocellulose for ethanol production. In partic-ular, modern kraft pulp mills are self-sustaining. The spent cookingliquor is concentrated and combusted to recover the reactionchemicals and power the mills, which lowers the production costand reduces pollution discharge (Japan TAPPI, 2006).

Kim and Holtzapple (2006b) reported that the activation en-ergy of alkali delignification of herbaceous biomass such as cornstover and sugarcane bagasse was significantly lower than thatof wood. Preliminary studies also showed that the lignin presentin rice straw, bagasse and some grasses was greatly removedafter the biomass was treated with sodium hydroxide solutionat moderate temperatures for a short period of time, even with-out the addition of the other pulping chemicals. Accordingly, aim-ing at overcoming the resistance of lignocellulose to enzymatichydrolysis with less energy consumption and improving the effi-ciency of cellulose-to-ethanol conversion, a pretreatment method,named as low temperature alkali (LTA) pretreatment (in contrastto the processes operated at temperatures > 100 �C), was pro-posed for treating a target lignocellulosic feedstock–sweet sor-ghum bagasse for cellulosic ethanol production. The proposedmethod and the kraft/soda pulping technology shares the samefundamental principles; therefore, those mature techniques andequipments used in the pulping process to recover the reactionchemicals as well as energy are basically applicable to the pre-treatment process.

Sweet sorghum (Sorghum bicolor (L.) Moench) is a multi-pur-pose crop which can be cultivated under a wide range of environ-mental conditions for simultaneous production of grain, sugar juiceand lignocellulosic bagasse. It is considered one of the promisingherbaceous energy crops because of its high yield in biomass andfermentable sugars per unit area per unit time and relatively lowinput requirements, and has received considerable attention as afeedstock for bioethanol production (Gnansounou et al., 2005; Ben-nett and Anex, 2009). The brown midrib (BMR) mutants of sweetsorghum reportedly had reduced lignin content and showed higherfiber digestibility as silage (Jung and Allen, 1995; Ledgerwoodet al., 2009). This feature may also be beneficial to the ethanol pro-duction as lignin plays an important part in the biomass recalci-trance. By now, little research on the difference in ethanolproductivity between the BMR and regular sweet sorghum bagassehas been reported.

The objectives of this study were to investigate the effects of theproposed pretreatment method on the composition, structure andenzymatic digestibility of bagasse from two varieties of sweet sor-ghum (including one BMR mutant), and to clarify the connectionsbetween the compositional/structural changes and the digestibilityof the pretreated bagasse.

2. Methods

2.1. Preparation of bagasse

One kilogram of stems of sweet sorghum cv. Kyushuko4 (BMRmutant) and cv. SIL05 (both harvested in Kumamoto, Japan in Sep-tember of 2008, and stored at �20 �C until thawed for processing)were pressed using a bench top juice extractor. The fibrous solidresidue (bagasse) was washed in hot tap water and pressed againto remove the residual free sugars. The washing-pressing processwas repeated three times. The bagasse was then dried in a dryingoven at 65 �C until constant weigh. The dried bagasse was succes-sively ground using a hammer mill (RD1-15, Grow Engineering, Ja-pan) and a bench top multi blender, and sieved to achieve anaverage particle size of less than 0.25 mm. The ground bagassewas put in air tight zip lock bags and stored in a desiccator at roomtemperature before use.

2.2. Pretreatment

2.2.1. Low temperature alkali (LTA) pretreatmentA portion of the ground bagasse (140 mg) was evenly dispersed

into 2.8 ml of sodium hydroxide solution of a certain concentrationin a 15-ml plastic test tube using a tube mixer (alkali to biomassratio ranging between 0.1 and 4 g NaOH/g oven dried bagasse).The mixture was incubated in a shaking water bath at a selectedtemperature for a desired length of time, and then centrifuged at12,000g for 5 min, the supernate discarded, and the pellet wasresuspended in 10 ml of deionized water and washed using thetube mixer. The washing-centrifugation process was repeated fivetimes to remove the residual alkali. The washed pellet was thenmixed with 10 ml of deionized water and neutralized with anappropriate amount of 1 M hydrochloric acid to a pH of 4.8, themixture again centrifuged, and the supernate discarded. The pelletwas finally washed with 10 ml of deionized water or 50 mM so-dium citrate buffer (pH 4.8) for compositional analysis or enzy-matic hydrolysis experiment.

2.2.2. Dilute sulfuric acid pretreatmentGround bagasse samples (each 140 mg) were mixed with 2.8 ml

of 0.5% (w/w) sulfuric acid in glass tubes. The tubes were fit intocustom-made stainless steel mini-reactors, and placed in an oilbath at 170 �C for different time periods. After treatment, the reac-tors were immediately quenched in ice bath to terminate the reac-tions. Pretreatment liquids were collected by centrifugation(12,000g, 5 min), and neutralized with calcium carbonate. Sugarmonomers including glucose and xylose present in the liquidswere quantified using an HPLC system (Prominence UFLC, Shima-dzu, Japan) equipped with a refractive index detector (RID-10A,Shimadzu, Japan) and carbohydrate-analysis column (AminexHPX-87P, Bio-rad, USA). The pellets were washed with deionizedwater until pH > 5 for compositional analysis and enzymatichydrolysis experiment.

Table 2Major components of sweet sorghum bagasse.

Component Content (%, d.b.)a

Kyushuko4 SIL 05

Glucan 38.7 ± 0.4 37.8 ± 1.1Xylan 22.6 ± 0.8 21.2 ± 0.3AIL 15.4 ± 0.7 16.7 ± 0.6Ash 2.2 ± 0.2 2.4 ± 0.4

a Mean ± SE; n P 5.

Fig. 1. Effects of LTA pretreatment under different conditions on the glucan andxylan in sweet sorghum bagasse.

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Detailed experimental pretreatment conditions are listed inTable 1.

2.3. Compositional analysis

After pH adjustment and washing, the alkali and acid pretreatedsolids were dried in a drying oven at 65 �C to constant weight andrecomminuted using the blender. The composition (includingmoisture, soluble sugars, glucan, xylan, lignin, ash content, etc.)of the pretreated as well as untreated bagasse was analyzed fol-lowing modified NREL laboratory analytical procedures (NREL).According to the NREL LAP, a two-step sulfuric acid hydrolysis pro-cess was adopted to break down the structural polysaccharidesinto sugar monomers for quantification (by the HPLC system);the acid–insoluble lignin (AIL) present in the acid hydrolysis resi-due was then determined gravimetrically.

2.4. Enzymatic hydrolysis (saccharification)

One milliliter of preheated (50 �C) enzyme solution, comprisingcommercially available cellulase and b-glucosidase (Celluclast 1.5Land Novozyme 188 (Novozymes A/S, Denmark) in 3:1 ratio in50 mM pH 4.8 sodium citrate buffer), was added to a test tube con-taining alkali or acid pretreated bagasse (pellet) or 140 mg of un-treated bagasse (approximate enzyme loading: 20 FPU, 50 CbU/gglucan). The mixture was immediately incubated in a shakingwater bath at 50 �C for a certain period of time (ranging from 1to 96 h). The hydrolysis process was terminated by heating thetube in a boiling water bath for 15 min. After cooling down to roomtemperature, the hydrolysate was analyzed with the HPLC systemto determine the released sugars. The hydrolysis performance ofthe cellulose in the biomass was evaluated by saccharificationyield defined as follows:

Glucan saccharification yield ð%Þ

¼ 100� 0:9� released glucose after hydrolysisinitial glucan before hydrolysis

The enzymatic hydrolysis of the dilute sulfuric acid treated ba-gasse was performed likewise. Some other auxiliary experimentswere also detailed in Section 3. All pretreatment-saccharificationexperiments were carried out in duplicate. Data are presented asmean ± SE. Comparisons between means were performed usingDuncan’s multiple range tests at a significance level of 0.05.

2.5. X-ray powder diffraction analysis

Samples of the untreated ground bagasse and freeze dried LTApretreated bagasse were pressed (200 kgf/cm2, 30 s) into disksfor X-ray powder diffraction analysis. The diffractometry in reflec-tion mode was carried out using an X-ray diffractometer(RINT2000, Rigaku, Japan) with monochromatic Cu Ka radiation(k = 0.15418 nm) generated at 38 kV and 50 mA, and the followingoptical slit system: divergence slit = 0.5�; scattering slit = 0.5�; andreceiving slit = 0.15 mm. The scanning was performed at scatteringangles (2h) from 6� to 30� in 0.1� increments with an accumulationtime of 20 s for each angular step.

3. Results and discussion

3.1. Effects of LTA treatment on the composition and structure ofbagasse

After juice extraction, washing and drying processes, about180 g of dry bagasse was recovered from 1 kg of each variety ofsweet sorghum stems. The major soluble sugars (sucrose, glucose,

fructose, etc.) present in the juice were substantially removed fromthe bagasse, avoiding possible interference in the evaluation of thehydrolysis efficiency of the cellulose. According to the two-stepsulfuric acid hydrolysis method for the determination of the struc-tural components of lignocellulose, the bagasse of Kyushuko4 hadslightly higher glucan and xylan contents but somewhat lowerAIL content compared with the SIL05 bagasse, as shown in Table 2.Additionally, the ash contents of the Kyushuko4 and SIL05 bagassewould be noteworthy as the ash (especially silicates) in the feed-stocks may affect the efficiency of pretreatment chemical recycleprocess.

The LTA pretreatment drastically changed the composition ofthe bagasse. Fig. 1(a) and (b) shows the recovery rates of the glucanand xylan contained in the Kyushuko4 and SIL05 bagasse after pre-treatment with NaOH solutions of different concentrations (0.5–2.5 M for Kyushuko4; 1–5 M for SIL05) at room temperature for30–120 min. The pretreatment caused only minor glucan loss,and the loss rates were unaffected by pretreatment condition;

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about 95% of the glucan present in the untreated bagasse wasrecovered after pretreatment. By contrast, a large portion of xylanwas degraded and removed during the pretreatment, and the re-moval rate increased rapidly with increasing pretreatment sever-ity. For instance, 1-h LTA treatment with 1 M NaOH led to about33% and 54% loss of the xylan of the SIL05 and Kyushuko4 bagasse,respectively, while the loss rates increased to 62% and 75% whenthe bagasse was treated with 2.5 M NaOH.

Fig. 2 presents residual acid-insoluble lignin in the LTA pre-treated bagasse (as percentage of initial AIL amount). The lignincontent of the biomass was significantly reduced after the pretreat-ment at moderate temperatures. In Fig. 2a, only 45% of the initialAIL remained in the Kyushuko4 bagasse after 30 min 0.5 M NaOHtreatment at room temperature. The AIL removal rate increasedslowly with extended pretreatment to about 67% at 120 min. Thedelignification reaction accelerated markedly with increasingNaOH concentration. Only about 20% of the initial AIL remainedin the Kyushuko4 bagasse after 2.5 M NaOH treatment for120 min. Similar trend was also observed in the SIL05 bagasse(Fig. 2b), although more severe treatment conditions were neededto achieve an equal level of delignification in comparison to theKyushuko4 bagasse. In biomass pulping, the course of delignifica-tion shows three stages: initial, bulk, and residual stage. Thethree-stage model has been widely used to study the delignifica-tion kinetics of the kraft/soda pulping (Chiang et al., 1990; Kimand Holtzapple, 2006b). Compared with the data in the reports dis-cussing the delignification kinetics, the delignification of the ba-gasse under the present experimental conditions occurredrapidly in the initial and bulk phases, and then slowed down signif-icantly as the pretreatment proceeded.

Temperature may strongly affect the rates of chemical reactionsand heat and mass transfer as well. It was observed in this study

Fig. 2. Residual acid-insoluble lignin in LTA pretreated sweet sorghum bagasse.

that the higher treatment temperature (50 �C) significantly accel-erated the removal of hemicellulose and lignin. The delignificationrate could reach as high as 90% while the glucan recovery rates re-mained almost unaffected by the elevated temperature (data notshown).

The data in Figs. 1 and 2 also exhibited that the Kyushuko4 ba-gasse was more susceptible to the pretreatment compared withthe SIL05 bagasse. The former achieved higher xylan and lignin re-moval rates under the same pretreatment conditions. In otherwords, in order to achieve similar compositional changes (glucan,xylan and AIL content), more severe treatment conditions (e.g.higher alkali concentration, treatment temperature or longer treat-ment time) would be needed for the SIL05 bagasse.

Alkali treatment of natural crystalline cellulose (such as themercerization process in the textile industry) can change the crys-tal structure and thus alter its physical and chemical properties(Nishiyama et al., 2000; Oh et al., 2005). The effect of the LTA pre-treatment on the cellulose crystal structure of the bagasse wasinvestigated by means of X-ray powder diffractometry. Fig. 3 com-pares the X-ray diffraction profiles (diffraction intensity againstdiffraction angle 2h) of the untreated and the bagasse treated with1, 2.5 and 5 M NaOH at room temperature for 60 min. Overall, thediffractograms of the Kyushuko4 and SIL05 bagasse samplesshowed little difference, and the diffraction patterns of the un-treated samples were similar to those of 1 or 2.5 M NaOH treatedsamples, where the diffraction peaks at 2h of 14.6�, 16.1� and21.8� indicating the 101, 10�1 and 002 lattice planes of the celluloseI crystal structure of higher plants (Parikh et al., 2007) were clearlyidentifiable. In contrast, the 5 M NaOH treated samples demon-strated a distinctly different pattern, in which the diffraction inten-sities at 14.6� and 16.1� substantially decreased, while a new peakat 12.1� stood out; the shape of the major peak at 21.8� also chan-ged drastically. The new pattern, when compared with those of the

Fig. 3. X-ray diffractograms of untreated and LTA pretreated sweet sorghumbagasse samples.

Fig. 4. Twenty-four-hour glucan saccharification yield of LTA pretreated sweetsorghum bagasse.

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mercerized or regenerated cellulose, basically agreed with thecrystal lattice characteristics of cellulose II crystal structure, sug-gesting that a certain amount of crystalline cellulose could trans-form from cellulose I to cellulose II during the pretreatment; thetreatment conditions under which apparent crystal structurechanges occurred were comparable to those reported in the litera-ture (Isogai, 1998; Mansikkamäki et al., 2005). It is generally be-lieved that cellulose I has parallel glucan chains arrangeduniaxially, whereas glucan chains of cellulose II are arranged in arandom manner (mostly antiparallel) and linked with a largernumber of hydrogen bonds resulting in higher thermodynamic sta-bility of the structure (Kolpak and Blackwell, 1976). Other reports(Kolpak et al., 1978; Mannan, 1993) also pointed out that the crys-tal structure transformation of lignocellulosic biomass was not ascomplete as that of relatively ‘‘clean’’ cellulose such as cotton linteror bacterial cellulose; the discrepancy was mainly attributed to thelignin and hemicellulose surrounding the cellulose fibrils in the lig-nocellulose, for their prevention of the swelling of the cellulosewhich was considered the starting point of the crystal structuretransformation process.

The crystallinity of the LTA pretreated bagasse was evaluated bycrystallinity index (CrI), which has been extensively used to esti-mate the ratio of cellulose I crystal structure to amorphous regionin a biomass sample. The CrI values were calculated according tothe following equation using the data from the diffractograms(Fig. 3),

CrI ð%Þ ¼ 100� ½ðI002 � IamorphousÞ=I002�

where I002 is the intensity of diffraction peak at 2h of 21.8�;Iamorphous is the intensity attributed to amorphous portion at 2hof 18� (Segal et al., 1959).

As presented in Table 3, the indexes of the untreated bagassesamples were about 50% due to the existence of a large amountof amorphous substances including lignin and hemicellulose. After1 M NaOH treatment, the CrI increased by about 10%, suggestingthere was a remarkable increase in the relative amount of crystal-line matter with the removal of the amorphous components. TheCrI showed a tendency to decline with increasing NaOH concentra-tion, which could be attributed to the crystal structure change, cel-lulose amorphization or the changes in the microscopic structuresof the lignocellulose particles after the pretreatment.

3.2. Effect of LTA pretreatment on the enzymatic digestibility ofbagasse

Without any pretreatment, only about 29% and 26% of the cel-lulose present in the Kyushuko4 and SIL05 bagasse was hydrolyzedinto glucose after 24 h hydrolysis under the present hydrolysisconditions. Fig. 4 shows the 24-h saccharification yield of glucanfrom the bagasse pretreated with 0.5 to 5 M NaOH at room temper-ature for 30 to 120 min. The enzymatic digestibility of the biomasswas greatly improved after the pretreatment. In the case of Kyush-uko4 (Fig. 4a), for example, the saccharification yield of the bagassetreated with 0.5 M NaOH for 30 min reached 80%. The digestibilityof the pretreated bagasse was positively correlated with pretreat-

Table 3Crystallinity index of untreated and LTA pretreated sweet sorghum bagasse samples.

Treatment CrI (%)

Kyushuko4 SIL 05

Untreated 51.8 50.81 M NaOH 61.3 57.52.5 M NaOH 54.3 48.05 M NaOH 42.8 38.8

ment severity, especially with the concentration of NaOH solution.More than 90% of the cellulose could be hydrolyzed into glucosewithin 24 h when 1 M or above NaOH solution was used. A maxi-mum 24-h saccharification yield of 98.7% was observed when thebagasse was pretreated with 2.5 M NaOH at room temperaturefor 120 min. The pretreated SIL05 bagasse also showed markedlyimproved enzymatic digestibility after pretreatment; the 24-h sac-charification yield increased from 65% to 90% as the pretreatmentintensified (from 1 M NaOH 30 min to 5 M NaOH 120 min), asshown in Fig. 4b. Compared with the Kyushuko4 bagasse, theSIL05 bagasse treated under the same conditions had apparentlylower digestibility. It achieved comparable saccharification yieldonly when treated under more severe conditions, which was inaccordance with its susceptibility to the pretreatment. Consideringthe higher digestibility of the untreated Kyushuko4 (BMR mutant)bagasse, the difference in the susceptibility and enzymatic digest-ibility between the two varieties could be due to their differentstructural features, since there were only slight differences in theircomposition. Therefore, using the BMR sweet sorghum bagasse asfeedstock for ethanol production could contribute to achievinghigh biomass-to-ethanol conversion efficiency.

In addition, the xylan remained in the pretreated bagasse wasalso degraded by the commercial cellulases during the saccharifi-cation processes; about 60–70% of the residual xylan in the abovepretreated Kyushuko4 and SIL05 bagasse samples was hydrolyzedinto xylose after 24-h hydrolysis. The hydrolysis rate of the xylanincreased more moderately with increasing pretreatment severityin comparison to the glucan. The final xylose concentration ofthe hydrolysate depended largely on the amount of xylan survivedthe pretreatment.

The enzymatic digestibility of the pretreated biomass improvedwith increasing pretreatment severity in this study. Nevertheless,the practical pretreatment conditions must be chosen comprehen-

Fig. 5. Effect of dilute sulfuric acid pretreatment on 24-h glucan saccharificationyield of sweet sorghum bagasse.

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sively, taking various factors into consideration in addition to thesaccharification yield, such as feedstock characteristics, pretreat-ment chemical cost, recovery efficiency and energy consumption.

3.3. Mechanisms of improvement in the enzymatic digestibility ofbagasse

The proposed pretreatment method effectively improved theenzymatic digestibility of the lignocellulosic biomass. Additionalexperiments were carried out to clarify the relationship betweenthe compositional and structural changes and the enzymaticdigestibility improvement of the pretreated bagasse.

Firstly, 10 g of each kind of bagasse were treated with 2.5 MNaOH at 50 �C for 24 h to remove the majority of the lignin. Afterwashing, neutralizing, drying and recomminution, delignified ba-gasse samples, each containing 50 mg glucan, were again treatedwith 1–5 M NaOH or citrate buffer (control) at room temperaturefor 2 h. The samples (pH adjusted) were then hydrolyzed underthe same enzymatic hydrolysis conditions as described in Sec-tion 2.4 except that 0.5-fold diluted enzyme solution (half enzymeloading) was used so as to slow down the enzyme reactions and‘‘magnify’’ the characteristics of the substrate. The two-step alkalitreatment still caused slight glucan loss (<5%); the first step treat-ment removed about 87% and 81% of the acid-insoluble lignin fromthe Kyushuko4 and SIL05 bagasse respectively, and the second steptreatment slightly increased the removal rates to about 90% and85% due to the significantly reduced reaction rate in the late bulkdelignification stage. There was no significant difference in theresidual lignin content between the samples treated with differentNaOH solutions in the second step. The enzymatic hydrolysisexperiment showed that the samples treated with different NaOHsolutions had quite similar hydrolysis performance, with little dif-ferences in both initial (1 h) hydrolysis rate (Kyushuko4: about25%; SIL05: about 22%) and 24-h saccharification yield (about83% and 78% from Kyushuko4 and SIL05, respectively), indicatingthat the differently treated samples having similar compositionwere equally digestible. The control groups treated with buffer inthe second step had slightly lower digestibility than the alkali trea-ted samples, giving 24-h glucose yields of about 78% (Kyushuko4)and 70% (SIL05). In association with the results of X-ray diffractionanalysis, the effect of the changes in the cellulose crystal structureor crystallinity caused by the pretreatment on the enzymaticdigestibility of the pretreated bagasse was insignificant. The differ-ence in crystallinity between the differently treated samples couldhardly be attributed to the amorphization of the cellulose either;otherwise, their hydrolysis profiles would be distinct from one an-other since amorphous structure is much more susceptible toenzymatic hydrolysis. Accordingly, the digestibility improvementafter the pretreatment can be largely attributed to the disruptionof the protective hemicellulose–lignin matrix surrounding the cel-lulose microfibrils by the pretreatment, which caused increase inthe accessibility of the cellulose to enzymes.

The two-step pretreated Kyushuko4 also showed higher enzy-matic digestibility than the SIL05 bagasse, suggesting the existenceof the structural difference between the two varieties. Moreover,the saccharification yield from both varieties reached about 80%at 24 h even at half enzyme loading, and kept increasing to up to95% at 72 h, indicating a great potential of the LTA pretreatmentmethod for the reduction of enzyme dosage for the saccharificationof the biomass.

Dilute sulfuric acid pretreatment of the bagasse was also per-formed for comparison. The pretreatment conditions (0.5% H2SO4

at 170 �C) were selected according to preliminary optimizationexperiments, and basically consistent with the conditions reportedelsewhere (Cara et al., 2008; Saha et al., 2005; Torget et al., 1990).The xylose present in the acid pretreatment supernates (as per-

centage of initial xylan amount) of the Kyushuko4 and SIL05 ba-gasse samples treated for different time periods, and the 24-hglucan saccharification yield of corresponding pretreated solidsare given in Fig. 5. The xylan degraded rapidly into xylose duringthe acid pretreatment. The amount of xylose in the pretreatmentliquids reached the maximum, equaling about 85% of the xylanpresent in the untreated bagasse, after 20-min treatment. Exces-sive treatment then caused gradual degradation of the xylose,resulting in a decrease in the xylose concentration of the liquids.The saccharification yield of the acid pretreated bagasse increasedat a steady rate to about 65% until the maximum xylan removalrate was reached; acid pretreatment over 20 min showed littleadditional effect on the improvement in the enzymatic digestibilityof the bagasse, which never exceeded 70% in this study. In contrast,The LTA pretreatment resulted in greater digestibility improve-ment while a certain amount of xylan remained in the pretreatedbagasse. For example, the Kyushuko4 and SIL05 bagasse treatedwith 1 M NaOH for 120 min achieved 24-h saccharification yieldof 90% and 75%, respectively, with about 40% and 60% of the initialxylan (before pretreatment) present in the pretreated solids (Figs. 1and 4). Because dilute sulfuric acid pretreatment has minor impacton the amount of acid-insoluble lignin and the crystal structure ofcellulose (Kabel et al., 2007; Kumar et al., 2009), the improvementin the digestibility of the acid pretreated bagasse over the un-treated feedstock can be largely attributed to the increase in cellu-lose accessibility due to the removal of the hemicellulose.Therefore, it can be concluded that the hemicellulose made a rela-tively small contribution to the recalcitrance to hydrolysis, whilethe lignin decisively affected the enzymatic digestibility of the cel-lulose. The removal of lignin brought significant increase in theavailable surface area of the cellulose and reduced nonproductivebinding of enzymes, and, consequently, led to the great enhance-ment in the accessibility of the substrate to enzymes and resultingcellulose hydrolysis efficiency. Sulfuric acid pretreatment attackingmainly the hemicellulose fraction of lignocellulosic biomass wasinferior to the LTA pretreatment in improving the digestibility ofthe biomass.

The 24 h saccharification yield (full enzyme loading) of LTA pre-treated Kyushuko4 and SIL05 bagasse as a function of lignin re-moval rate is plotted in Fig. 6, regardless of specific treatmentconditions (NaOH concentration, treatment time, etc.). There wasan apparently positive, linear correlation between the twovariables; the efficiency of enzymatic hydrolysis markedly in-creased as the lignin content of the bagasse dropped. Adsul et al.(2005) obtained similar results using differently delignified sugar-

Fig. 6. Correlation between lignin removal and enzymatic digestibility of LTApretreated sweet sorghum bagasse.

L. Wu et al. / Bioresource Technology 102 (2011) 4793–4799 4799

cane bagasse as substrate under comparable enzymatic hydrolysisconditions. The correlation coefficient for the Kyushuko4 bagassewas lower than that for the SIL05, owing to the uneven distributionof the experimental data of the former (mostly very high delignifi-cation rates and sharply increased enzymatic digestibility).

Thus, the LTA pretreatment, derived from the highly maturekraft/soda pulping technology focusing on removing lignin fromlignocellulosic feedstocks, may act as an effective means forimproving the enzymatic digestibility of lignocellulosic biomass.Additionally, due to the substantially reduced lignin content ofthe LTA treated biomass, the downstream unit operations such asdistillation and waste treatment may also be facilitated. The pres-ent method can be integrated with other advanced biorefinerytechnologies to build cost-effective second generation bioethanolproduction platforms.

4. Conclusions

The LTA pretreatment method proposed in this study can effec-tively disrupt the lignin–carbohydrate complex and liberate thecellulose fibrils in the sweet sorghum bagasse by removing themajority of the lignin, and therefore greatly enhance the enzymaticdigestibility of the cellulose. The bagasse from the BMR mutantsweet sorghum is more susceptible to the pretreatment than thenon-BMR variety, leading to a higher enzymatic hydrolysis effi-ciency of the former, which may also provide useful informationfor the development of novel feedstocks for cellulosic ethanol pro-duction. The method offers an alternative approach to the efficientconversion of lignocellulosic biomass to ethanol.

Acknowledgement

This work was supported by a grant from the Ministry of Agri-culture, Forestry and Fisheries of Japan (Rural Biomass ResearchProject, BEC-BA230).

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