Bioresource Technology 107 (2012) 251257

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Bioresource Technology

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Ultrasound-assisted compatible in situ hydrolysis of sugarcane bagassein cellulase-aqueousN-methylmorpholine-N-oxide systemfor improved saccharification

Qiang Li ,1, Geng-Sheng Ji ,1, Yu-Bin Tang, Xu-Ding Gu, Juan-Juan Fei, Hui-Qing JiangJiangsu University of Science and Technology, Zhenjiang 212003, PR China

a r t i c l e i n f o

Article history:Received 29 August 2011Received in revised form 13 December 2011Accepted 13 December 2011Available online 22 December 2011

Keywords:Sugarcane bagasseEnzymatic hydrolysisCompatible saccharification systemNMMOUltrasound intensification

0960-8524/$ - see front matter 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.12.068

Corresponding authors. Tel.: +86 511 85639697(G.-S. Ji).

E-mail addresses: comeonflareup@yahoo.com.cn(G.-S. Ji).

1 The two authors contributed equally to this work.

a b s t r a c t

To fully exploit the benefits of N-methylmorpholine-N-oxide (NMMO) in lignocelluloses bioconversion, acompatible system was established for efficient in situ saccharification of cellulose in NMMO-aqueousmedia in which the NMMO is able to activate and solubilize the cellulose, and the cellulases possess highstability and activity. Cellulase retained its original activity after being pre-incubated in 15% and 20% (w/v) NMMO solutions. After optimization of reaction parameters, high saccharification rate (96.5%) wasobtained in aqueous-NMMO media by ultrasound assisted treatment of cellulose. The viscosity and FTIRanalysis revealed that NMMO-treated cellulose under ultrasonic condition was porous and amorphous,which led to improved saccharification. The addition of trifle lignin in lower concentration improvedthe saccharification efficiency of sugarcane bagasse, while higher concentration interferes with hydroly-sis. In conclusion, these findings provided great implications to develop a continuous process NMMO-cel-lulases system for transformation of native biomass.

2011 Elsevier Ltd. All rights reserved.

1. Introduction

Sugarcane is main sugar crop with a production of more than77.4 million metric tons per sugar production season in the southof China (Rong and Yong, 2006). As a result, the sugarcane residueis abundant, inexpensive and readily available source of lignocellu-losic biomass in China. However, lignocellulosic biomass is recalci-trant to effective enzymatic hydrolysis due to its highly lignifiedand crystalline structure (Galbe and Zacchi, 2002; Kuo and Lee,2009). To make cellulosic materials more susceptible for hydroly-sis, an effective pretreatment is required to soften its tough assem-bled structure of cellulose crystallinity (Heinze and Liebert, 2001)and increase the cellulose porosity (Chandra et al., 2010; Zhangand Lynd, 2003). Pretreatment processes that increase the surfacearea accessible to cellulases and water are expected to generateimprovements in efficiency of hydrolysis and conversion of cellu-losic biomass to sugars (Sun and Cheng, 2002; Zhang and Lynd,2003). Different pretreatment methods like dilute acid, steamexplosion, ammonia fiber explosion, lime and organosolvent pre-treatments were employed to improve enzymatic saccharification(Sindhu et al., 2011. Mosier et al., 2005; Wyman et al., 2005).

ll rights reserved.

(Q. Li), +86 511 85632660

(Q. Li), jigsheng@126.com

However, these pretreatment methods produce undesirablebyproducts which inhibit downstream fermentation (Hendriksand Zeeman, 2009; Mosier et al., 2001, 2005).

To help meet the challenge of sugarcane residue conversion,NMMO have attracted substantial research interest for N-methyl-morpholine-N-oxide (NMMO) is a crystalline compound that ismelting at 170 C, implying low volatility, flammability (Finket al., 2001). Recently, several studies demonstrated that NMMOcan effectively solubilize lignocellulosic biomass such as Aviceland sugarcane bagasse (Biganska and Navard, 2005; Kuo and Lee,2009; Petrovan et al., 2001; Ramakrishnan et al., 2010). NMMO isable to dissolve cellulose due to the high polarity of its NO bond,which breaks the hydrogen bond network of the cellulose andforms new hydrogen bonds with the solute. The operation condi-tions for these pretreatments are much milder (

252 Q. Li et al. / Bioresource Technology 107 (2012) 251257

Lee, 2009). Cellulase activity may be inhibited in presence ofNMMO due to high polarity of NAO bond in cellulose (Kuo andLee, 2009). But there is no report on systematic investigation ofNMMO compatibility on cellulase and fermentation. Moreover,no literature was published on the case of natural lignocellulosein situ enzymatic hydrolysis in NMMO solution which is significantto the production of biofuel from natural lignocellulosic materials.

The objective of the present study was to evaluate and optimizeultrasound intensified cellulase-NMMO system for efficient in situsaccharification of natural lignocellulosic materials in NMMO-aqueous media in which the NMMO is able to activate and solubi-lize the cellulose, and the cellulases possess high stability andactivity. Different treatment like, conventional heating-NMMO-treatment (mode 1), ultrasound heating-NMMO-treatment (mode2), ultrasound heating-NMMO-in situ enzymatic hydrolysis-treat-ment (mode 3) were performed to find out the fermentable sugarproduction from sugarcane bagasse. Moreover, physical character-izations of native sample, and different mode treated samples werecarried out by viscosity and FTIR analysis to elucidate the struc-tural modifications.

2. Methods

2.1. Feed stock

The sugarcane bagasse (SB) was collected from a local sugar-cane juice shop in Zhenjiang (China). The collected bagasse wasthoroughly washed with distilled water to remove residual solublesugars. The raw material was air dried, milled to a size less than0.5 cm and were stored at room temperature until further use. Cel-lulase from T. reesei strain was supplied by Solarbio Inc (Japan Yak-ult Honsha Co., Ltd., E.C. 3.2.1.4, slightly brown powder, 30 U/mgsolid, lyophilized powder). The following chemicals were pur-chased from SigmaAldrich: b-glucosidase (from Aspergillus nigerstrain, 60 U/mg). Avicel PH-101 cellulose (microcrystalline cellu-lose/MCC, particle size 50 lm, DP 225), N-methylmorpholine-N-oxide (NMMO) (Aldrich). Lignin (low sulfonate content, Aldrich).The lipid accumulating bacterial strain is Rhodococcus opacusACCC41043 from Agricultural Culture Collection of China. All otherreagents and chemicals used were of analytical grade.

2.2. Stability of cellulases in NMMO

Stability of cellulase in NMMO was evaluated. The cellulasemixture, a combination of cellulase and b-glucosidase, was usedin our case with the concentration of 35 FPU cellulase and 60 Cel-lobiose Unit (CBU) b-glucosidase per gram of cellulose. The cellu-lase mixture was loaded into NMMO media. Stability tests werecarried out in a total volume of 3 mL containing the cellulase com-plex and various concentrations of the NMMO (5%, 10%, 15%, 20%,and 30% w/v) in citrate buffer (50 mM, pH 4.8). The mixture waspre-incubated at 0 C and 50 C, respectively. Samples were col-lected at different time points (1, 4 and 24 h) and reducing sugaranalysis was carried out by 2, 5-dinitrosalicylic acid method(Miller, 1959). The cellulase mixture in the citrate buffer withoutaddition of NMMO was used as a control. All experiments wererun in triplicate. Relative activity of cellulase% = [cellulase activityin NMMO solution] 100%/[cellulase activity in citrate buffer].

Reducing sugar analysis in presence of NMMO has not been pre-viously reported, hence reducing sugar analysis in presence ofNMMO were carried out to find out whether there is any interfer-ence in reducing sugar analysis in presence of NMMO. StandardD-glucose solutions (0.21.4 mg/mL) were mixed with varied con-centrations of NMMO (530%, and NMMO free sample as a control)and subsequently analyzed using the DNS assay. No obvious

interference was observed in the DNS assay for the range of NMMOconcentrations used in this work. Based on these results, DNS assaywas conducted for the measurement of cellulase mixture activityas follows: 5 mg/ml MCC in 50 mM citrate buffer (pH 4.8) withthe cellulase mixture was incubated at 50 C, followed by mixingwith two volumes of DNS reagent. The reaction mixture wasfurther incubated at 100 C for 5 min and cooled in an icewaterbath before the absorbance was measured at 540 nm. Control reac-tions with substrate MCC without addition of NMMO and enzymes,and enzyme controls in NMMO solution but without loading ofMCC were subtracted from each measurement.

2.3. Hydrolysis of Avicel with the addition of NMMO

2.3.1. Ultrasound intensified Avicel treatment With NMMOA 3% (w/w) Avicel solution was prepared by combining 0.9 g of

Avicel or sugarcane bagasse with 30 ml NMMO in a 250 ml round-bottom flask and stirred at the speed of 500 rpm. Then the Avicelsolutions were treated with the help of an ultrasonic generator(TEA-1004, Shanghai TIME Sonication Co., China) at a frequencyof 45 kHz and the sonication power was 100 W. The temperaturewas maintained at 90 C during the ultrasonic heating experi-ments. Sample treated at 90 C by conventional heating served asthe control group. All experiments were run in triplicate.

2.3.2. Enzymatic saccharification of treated AvicelIn the following incubation, the Avicel/ NMMO solution was di-

luted with 50 mM citrate buffer (pH 4.8) and the final concentra-tion of Avicel and NMMO was 0.6% and 20% (w/v), respectively.Three modes of NMMO involved lignocellulosic fibres treatmenthave been established (mode 1, mode 2 and mode 3). The cellulasemixture was loaded into Avicel/ NMMO solution and stirred at thespeed of 500 rpm. The enzymatic hydrolysis was carried out at50 C with the help of an ultrasonic generator at a frequency of45 kHz. Enzymatic hydrolysis of the cellulosic samples was carriedout at 50 C using conventional heating served as the controlgroup. The untreated Avicel was hydrolyzed using the same con-centration of cellulase mixture and run in parallel with the Avicel/NMMO system in ultrasound heating bath or conventional heatingbath. The enzymatic reaction was monitored by withdrawing sam-ples from the supernatant periodically and measuring release ofsoluble reducing sugars by the DNS assay. Yield of reducing sugarsfrom sugarcane bagasse was calculated as follows:

Yield of released sugars% = [Reducing Sugars released] 100%/([sugarcane bagasse weight] [cellulose ratio in sugarcanebagasse])

2.3.3. Effect of lignin on the hydrolysis of Avicel in NMMO solutionEffect of lignin on enzymatic saccharification of cellulose were

evaluated by conducting hydrolysis with different concentrationsof lignin. Lignin was mixed with 0.2 g Avicel in 10 ml NMMO,resulting in 0.06%, 0.60%, 0.86% or 1.20% of lignin and 2.0% (w/v)cellulose in NMMO, respectively. Then the mixtures were incu-bated for the treatment, followed by enzymatic hydrolysis as de-scribed in section of Avicel Treatment with NMMO andEnzymatic Saccharification of Treated Avicel with the modificationof time and temperature (72 h, 55 C) in the treatment step.

2.4. Saccharification of sugarcane bagasse in aqueous-NMMO solution

2.4.1. 1Sugarcane bagasse treatment with NMMOA 3% (w/v) sugarcane bagasse (SB) solution was prepared by

combining 0.9 g of SB (as prepared above) with 30 ml NMMO ina sterile flask. The SB / NMMO mixture was incubated and stirredat 80 C for 72 h.

Q. Li et al. / Bioresource Technology 107 (2012) 251257 253

2.4.2. 2Ultrasound intensified saccharification of sugarcane bagasseIn the following incubation, the SB/NMMO mixture was diluted

with 50 mM citrate buffer (pH 4.8) and the final concentrations ofSB and NMMO were 0.6% (w/v) and 20% (w/v), respectively. Thecellulase mixture was loaded into SB/NMMO and the enzymatichydrolysis was carried out at 50 C with ultrasound intensification.The enzymatic reaction was monitored by withdrawing samplesfrom the supernatant periodically and measuring release of solublereducing sugars by the DNS assay. All assays were performed intriplicate. The conversion rate of cellulose was calculated as the to-tal reducing sugar produced in the extracted SB in the initial reac-tion mixtures.

2.5. Fermentability of the hydrolyzates from saccharification process

After UHT-NMMO-cellulase process, the mixture of NMMO andhydrolyzates from enzymatic in situ hydrolysis were filtered andwas then passed through a column filled with neutral Al2O3 and con-centrated according to our reported method (Xian et al., 2009) andThe hydrolyzates (containing 5.2 g/L glucose) were separated fromthe NMMO. Then the hydrolyzates (recovered sugars) were usedfor biodiesel preparation according to the our reported method (Liet al., 2010) using R. opacus ACCC41043. The NMMO was recoveredby heating under reduced pressure condition (Kuo and Lee, 2009).

2.6. Analysis

The constituents of sugarcane bagasse were determined by thestandard analysis procedure of biomass composition (Gouveiaet al., 2009). It was composed of cellulose 34.8%, hemicellulose28.3%, lignin 19.4% and others 17.5%. Cellulase activity was deter-mined by the standard filter paper assay and expressed as filter pa-per units per gram of glucan (FPU) (Ghose, 1987). One FPU isdefined as the enzyme that releases 1 lmol of glucose equivalentsper minute from filter paper. The released reducing sugars weremeasured by the DNS method using D-glucose as a standard (Miller,1959). Glucose released by cellulosic biomass hydrolysis wasquantified using an SBA-40D Biological Sensing Analyzer (BiologyInstitute of Shandong Academy of Sciences, China). One milligramper milliliter glucose solution was used as a standard for themeasurement.

The viscosity of cellulose or lignin/cellulose solutions in NMMOwas measured following the method of Evlampieva with somemodifications (Evlampieva et al., 2009). Microcrystalline celluloseor lignin/cellulose mixture was added to NMMO and dissolved byconventional heating or ultrasound heating pretreatment men-tioned above, respectively. After cooling to room temperature, pyr-idine was added dropwise into the solution, resulting to the finalstable mixture. An ubbelohde viscometer was used to performthe viscosity measurement. The viscosity of these concentration-gradient solutions was measured at 30 C. The treated anduntreated cellulosic biomass was measured by Bruker IR spectrom-eter Tensor 27. The spectra (4000400 cm1) were recorded with aresolution of 4 cm1 and 64 scans per sample. About 2.0 mg sam-ples were prepared by mixing with 120.0 mg of spectroscopicgrade KBr then pressed in a standard device using a pressure of6000 psi to produce 13 mm diameter pellets. The background spec-trum of pure KBr was subtracted from that of the sample spectrum.

3. Results and discussion

3.1. Stability of cellulases in the presence of NMMO

The cellulase was active and stable in the presence of the NMMOwhen the temperature was maintained at 4 C (Fig. 1A1B1). Following

the first hour of incubation in 5%, 10%, 15%, 20%, 25%, and 30% ofthe NMMO, the cellulase retained 96.2%, 95.0%, 92.2%, 85.0%, and72.0% of its initial activity, respectively. The enzyme activity de-creased with an increase of incubation time; however, the residueactivity still retained approximately 70% of original activity after24 h of incubation in 30% NMMO (Fig. 1A1B1) at 4 C. Particularly,b-glucosidase maintained 100%, 99.2%, 99.0%, 97.8% and 91.5% ini-tial activity in first hour of incubation (as shown in Fig. 1B1) and re-tained high activity in the incubation process. There are threeconstituents of cellulolytic enzymes that are required for the com-plete breakdown of cellulose to simple sugars. Endoglucanase (EG)(1, 4-b-D-glucan-4-glucano-hydrolases; EC 3.2.1.74) cleaves glyco-sidic bonds randomly within the interior of cellulose polymerchain. Exoglucanases (EC 3.2.1.91 and EC 3.2.1.74) act progressivelyon the reducing or non-reducing ends of cellulose chains, releasingeither cellobiose or glucose as major products. The b-glucosidases(BGL) (EC 3.2.1.21) hydrolyze soluble cellodextrins and cellobioseto glucose (Saha et al., 1994). In this work, cellobiose was loadedas substrate to investigate the b-glucosidase activity in presenceof NMMO. Probably, the cellobiose is easily to be degraded as adisaccharide (Xiao, 2005), while cellulose recalcitrant to enzymatichydrolysis due to its highly crystalline structure (Galbe and Zacchi,2002). As a result, b-glucosidase showed high relative activity inNMMO system when compared to cellulase (Fig. 1B).

A similar pattern was observed in the 50 C pre-incubation treat-ment (Fig. 1A2B2). Following 24 h of incubation at 50 C, the cellu-lases retained 84.7% and 85.6% of the activity in the presence of5% and 15% NMMO, respectively. The cellulases displayed 84.6% ofthe original activity after 24 h of incubation in 20% NMMO. How-ever, the activity significantly decreased to 40.0% when the cellu-lases was exposed to 30% NMMO solution for 24 h (Fig. 1A2).b-glucosidase retained 100%, 98.6%, 98.5%, 98.1% and 85.7% initialactivity in first hour of incubation, which was lower than that in4 C pre-incubation treatment. The results indicated that cellulasewas more stable in NMMO at 4 C when compared to cellulaseactivity at 50 C. Cellulase buffer solution containing 1520% ofNMMO was suitable for in situ hydrolysis of lignocellulosic biomass.

3.2. Enzymatic saccharification of Avicel

For the enzymatic saccharification, 20% Avicel-NMMO was pre-pared in 50 mM citrate buffer and the complex was incubated at80 C for 72 h. Saccharification was carried out using commercialcellulase in an ultrasound field and the reducing sugar yield wasmonitored at different time points (Fig. 2). Approximately 77.2%of cellulose was hydrolyzed in the presence of 20% NMMO duringthe first 4 h. After 24 h of digestion, 95.9% of treated Avicel wasconverted to reducing sugars compared to 45.3% and 51.6% of theuntreated cellulose and ultrasound heating treated (UHT) Avicel.The enhancement of conversion in ultrasound heating-NMMO-in situ enzymatic hydrolysis (UH-NMMO-ISEH) treated celluloseindicated that the NMMO-treatment disrupted crystalline struc-ture in cellulose and improved accessibility of the enzymes to cel-lulose. Moreover, near complete conversion (96.5%) of cellulose inthe aqueous-NMMO system indicated that the cellulase mixtureretained high activity in NMMO. The data clearly elucidated thatthe aqueous NMMO cellulase system in ultrasound field workedeffectively for the hydrolysis of pure cellulose.

To elucidate the effect of ultrasound heating and the concentra-tion of cellulose on in situ enzymatic hydrolysis of cellulose, theviscosities of cellulose/NMMO treated by conventional and ultra-sound heating were analyzed according to the methods mentionedin the experimental section. It was illustrated in Fig. 4 that the gsp/cc flow curves of cellulose/NMMO solutions at different concen-trations. The intrinsic viscosity [g] was deduced from the classicalway of double extrapolation to zero concentration. It is obvious [g]

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Fig. 1. The cellulases mixture (A) or b-glucosidase (B) activity after pre-incubation with various concentrations of NMMO. The pre-incubation temperature was 0 C (A.1, B.1)and 50 C (A.2, B.2) with samples taken at 1, 4, and 24 h of incubation. Enzymatic hydrolysis conditions were 50 C, pH 4.8, 1% Avicel (substrate of cellulases mixture) or 5%cellubiose (substrate of b-glucosidase). The enzymatic activity was calculated as mg of reducing sugars produced per hour in various NMMO solutions (cellulaseconcentration 35 FPU and 60 CBU per gram of substrate, the enzymes in citrate buffer was normalized as 100% activity). Error bars represent standard deviation of the means(n = 3).

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Fig. 2. Cellulase-catalyzed saccharification of Avicel in 20% NMMO solution.Enzymatic hydrolysis of untreated Avicel and SB, NMMO-treated Avicel and SB in50 mM citrate buffer and in situ enzymatic hydrolysis of Avicel and SB in ultrasoundintensified 20% (v/v) aqueous NMMO system. Reaction conditions were pH 4.8,50 C; enzyme loadings were 50 FPU cellulase and 50 CBU b-glucosidase per gram ofcellulose. UHT means ultrasound heating treatment; CHT means conventionalheating treatment. Error bars represent standard deviation of the means (n = 3).

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Fig. 3. In situ saccharification of sugarcane bagasse in 20% NMMO solution.Enzymatic hydrolysis of untreated Avicel and SB, NMMO-treated Avicel and SB in50 mM citrate buffer and in situ enzymatic hydrolysis of Avicel and SB in ultrasoundintensified 20% (v/v) aqueous NMMO system. Reaction conditions were pH 4.8,50 C; enzyme loadings were 50 FPU cellulase and 50 CBU b-glucosidase per gram ofcellulose. UHT means ultrasound heating treatment; CHT means conventionalheating treatment. Error bars represent standard deviation of the means (n = 3).

254 Q. Li et al. / Bioresource Technology 107 (2012) 251257

of solution prepared with ultrasound treatment (60 cm3/g) wasmuch lower than that of solution prepared under conventionalheating condition (70 cm3/g) (Fig. 4). An identical observationwas earlier reported by Yang et al., 2010 for ultrasound assisted

ionic liquid pretreatment of cellulose (Yang et al., 2010). Cellulosein situ treatment by ultrasound was found to be better than con-ventional heating. This was evident from the viscosity data pre-sented in Fig. 4. The ultrasound pretreated samples showed alower viscosity when compared to conventionally heated samples.

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Fig. 4. Viscosity of Avicel solution, Avicel and lignin mixture (4.3:10) in NMMO-pyridine mixture (NMMO:pyridine = 1:9, v/v) with conventional and ultrasonicheating treatment respectively. (C represent the concentration of cellulose , gsp isthe specific viscosity).

Q. Li et al. / Bioresource Technology 107 (2012) 251257 255

3.3. Enzymatic saccharification of sugarcane bagasse

The saccharification rate of NMMO-treated sugarcane bagassewas significantly increased compared to the untreated sample(Fig. 3). Approximately 54.6% of cellulose in the treated SB wasconverted to reducing sugar in the first hour of ultrasound intensi-fied in situ enzymatic hydrolysis, while only 5.7% and 6.6% conver-sion was observed for the untreated SB and ultrasound treated SB.After 24 h of digestion, the conversion efficiency for ultrasoundintensified in situ enzymatic hydrolysis, untreated SB and UHT-treated SB were 90.4%, 31.5% and 34.6%, respectively (Fig. 3). Theultrasound treatment without NMMO lead to no obvious enhance-ment of enzymatic hydrolysis (less than 2%). Approximately 95.9%of cellulose in SB was obtained after of enzymatic hydrolysis for12 h, indicating the complete digestion time altered from 24 to12 h with the intensification of ultrasound.

Table 1The infrared ratios of FTIR spectroscopy measured for untreated bagasse, enzymatichydrolyzed untreated bagasse and bagasse treated by three mode.

Lateral order index (LOI)A1430 /A900

Total crystalline index (TCI)A1372/A2900

Untreated 1.441 1.393Mode1-

treated1.122 1.112

Mode2-treated

0.982 0.978

Mode3-treated

0.906 0.878

Mode 1: Conventional heating-NMMO-treatment; mode 2: ultrasound heating-NMMO-treatment; mode 3: ultrasound heating-NMMO-in situ enzymatic hydro-lysis-treatment.

3.4. Effect of lignin on the hydrolysis of Avicel

The lignin effect on cellulose enzymatic hydrolysis in naturalbiomass in presence of NMMO was simulated by mixing differentamount of lignin with cellulose. Enzymatic saccharification rateof UHT-NMMO treated Avicel was higher (96.5%) when comparedto SB (90.5%). Batch addition of enzyme showed an improvementin the hydrolysis rate of SB. However, no obvious enhancementof SB hydrolysis (less than 1%) was detected after additional enzy-matic hydrolysis for 48 h. While, the results from the experimentshowed that the conversion efficiency of sugarcane bagasse waslower than Avicel, amendment with additional cellulase did notimprove the conversion rate of cellulose in SB, which indicated thatthe incomplete conversion was not caused by the deactivation ofcellulases in aqueous-NMMO mixtures.

However, previous work has found that cellulase activity wasreduced during the hydrolysis of lignocellulosic biomass due tothe interaction with lignin or lignin-carbohydrate complexes(Berlin et al., 2006; Kumar and Wyman, 2009; Wang et al., 2011).Extensive efforts have been placed on lignin effect on cellulosehydrolysis in natural biomass (Lee et al., 2008; Binod et al.,2011). To reveal how the lignin or lignin-carbohydrate complexesinterfere the saccharification of SB, further experiments were car-ried out in the presence of various amount of lignin to investigatethe effect of other biomass constituents on hydrolysis of cellulosein aqueous NMMO system. Because there was 19.4% lignin and34.8% cellulose in SB as measured by the method described in anal-ysis section, the ratios of lignin to cellulose in this work weredetermined to imitate the natural constituents (lignin:cellu-lose = 5.6:10) of SB biomass. Various amounts of lignin (0.06%,

0.60%, 0.86%, or 1.20%) were mixed in NMMO, implying lignin tocellulose was in the ratio of 0.3:10, 3:10, 4.3:10 or 6:10. After24 h of hydrolysis, 96.5% of Avicel (control, without lignin) wasconverted to reducing sugar while Avicel containing 0.06%, 0.60%,0.86%, and 1.20% lignin showed conversion rate of 97.9%, 91.9%,91.8% and 89.3% respectively after enzymatic hydrolysis for 72 h,indicating the lignin in low concentration favor the enzymatichydrolysis compared to interfering the reaction in high concentra-tion. NMMO is a strong oxidant, antioxidants is added with NMMOin Lyocell process to stabilize the NMMO/cellulose mixture (Rosenauet al., 2002). Lignin has been demonstrated to be a potential radicalscavenger and antioxidant (Pan et al., 2006). Lignin in low concen-tration act as antioxidant to protect the cellulase from inactivation,resulting in relatively high conversion rate of cellulose. To ourknowledge, the 97.9% conversion rate is the highest cellulaseenzymatic hydrolysis conversion rate in aqueous-NMMO com-pared with experiments reported such as about 93% conversionrate in Ramakrishnans work (Ramakrishnan et al., 2010).

[g] of solution prepared with lignin in conventional heatingcondition (76 cm3/g) was higher than that of solution preparedwithout lignin (70 cm3/g) and with lignin under ultrasound heat-ing condition treatment (68 cm3/g) (Fig. 4). Probably, the decreaseof conversion rate was partly caused by viscosity enhancement ofsolution with lignin loading. The lignin enhanced the viscosity ofsolution, resulting in increased mass transfer resistance of the cel-lulase enzymatic hydrolysis system.

3.5. FTIR analysis of sugarcane bagasse in various mode of treatment

FTIR spectra of native and treated SB was carried out to eluci-date the structural modifications. The band near 1160 cm1 is rep-resentative of the antisymmetric bridge stretching of CAOACgroups in cellulose and hemicellulose, and the band near1318 cm1 can be ascribed to CH2-wagging vibrations in the cellu-lose and hemicellulose (Liu and Chen, 2006). The 895 cm1 bandwhich is characteristic of b-linkages, especially in hemicellulose,was reduced after NMMO treatments. The band at 16351640 cm1, which is attributed to the absorbed water bendingvibrations, decreased after NMMO treatments.

From these spectra, two infrared ratios were calculated: (1)a1430 cm1/a900 cm1, which is referred as the crystallinity index(OConnor et al., 1958) or lateral order index (LOI) (Hurtubiseand Krsig, 1960), (2) a1372 cm1/a2900 cm1, which is known asthe total crystallinity index (TCI) (Nelson and OConnor, 1964).The higher index value represents the material has a higher crys-tallinity and ordered structure. As shown in Table 1, after variousmode of treatment, the LOI of SB decreased from 1.441 to 1.122(CH-NMMO-treated), 0.982 (UH-NMMO-treated) and 0.906 (UH-NMMO-IEH-treated), while the TCI of SB decreased from 1.393 to1.112 (CH-NMMO-treated), 0.978 (UH-NMMO-treated) and 0.878

256 Q. Li et al. / Bioresource Technology 107 (2012) 251257

(UH-NMMO-IEH-treated). As a result, the cellulosic biomass, afterUH-NMMO-IEH-treatment, is less crystalline compared to the un-treated one and the sample treated in other modes. Similar resultscould be observed in the FTIR data of Avicel (data not shown). Theresults illustrated that the two indexes of UH-NMMO-IEH-treatedSB were lower than those of untreated SB, UH-NMMO-treated SBand CH-NMMO-treated SB, indicating some of cellulose in SB wasdegraded and the UH-NMMO-IEH-treatment can efficiently de-grade the tight structure of SB better than conventional treatment.The similar decreases of these two indexes have also been reportedfor sugarcane bagasse withdrawn from conventional NMMO treat-ment (Kuo and Lee, 2009). Probably, ultrasound-assisted NMMOtreatment can efficiently prevent the dissolved SB from restructur-ing into its original crystalline structure. Moreover, cellulosic bio-mass dissolved by ultrasound heating displayed a lowermolecular weight than that dissolved by conventional heating.More disruption must occur during ultrasound treatment, resultingin the lessening in crystalline and degree of polymerization (Yanget al., 2010). Consequently, the fragmental and porous treated SBwith amorphous structure could provide more surfaces for en-zymes to attack on. It is obvious that cellulase was not able to pen-etrate into the intact structure of untreated bagasse for hydrolysis.Thus, a low conversion rate and yield was obtained (as shown inFig. 3). Conversely, the intact structure was disrupted by UH-NMMO-IEH treatment and resulted in a porous and amorphous ba-gasse that significantly enhanced conversion rate of cellulosicbiomass.

3.6. Fermentability of hydrolyzates

The fermentability of the hydrolyzates after in situ saccharifica-tion was evaluated using R. opacus. Our data suggested that the R.opacus could accumulate 4042% lipid of cell dry matter (CDM)using simple mineral salt medium with hydrolyzates as carbonsource. In contrast to the control test, there was no obvious nega-tive effect observed in reducing sugars consumption and lipid pro-duction in the fermentation (Table 2).

Furthermore, the effect of NMMO on fermentation was investi-gated. As shown in Table 2, about 1.9 g/l CDM and 3941% CDM li-pid content were obtained after 30 h of cultivation usinghydrolyzates (containing 5.2 g/l glucose) with the addition of1020 g/l NMMO. Further increases of the NMMO to the concen-tration of 50 g/l caused negative effect on CDM and lipid accumu-lation, but the withdrawn microbial oil had no influence onesterification (Table 2). Probably, due to the high polarity of itsNAO bond (Kuo and Lee, 2009), R. opacus was inhibited in the pres-ence of high concentration of NMMO. In the contrast to 10 g/l ionicliquids [Bmim]Cl could completely inhabit the growth of R. opacusACCC41043 (Li et al., 2010), NMMO mediated the obvious advan-tage as biocompatible solvent for in situ enzymatic hydrolysis.

Table 2Biodiesel production from hydrolyzates.

Carbon source Lipidcontent(%CDM)

Biomass(g/l)

Esterification ofmicrobial oil(wt%)

Control (5.2 g/l Glucose) 40.05 1.92 91.1Recovered sugars 41.16 1.91 91.5Recovered sugars with 10 g/l NMMO 41.02 1.93 91.6Recovered sugars with 20 g/l NMMO 39.28 1.90 91.3Recovered sugars with 50 g/L NMMO 30.02 0.92 91.6

R. opacus was cultivated for 30 h in low-nitrogen medium using hydrolyzates(containing 5.2 g/l glucose) from in situ saccharification process as carbon source.Yield of biodiesel was measured by esterfication of microbial oil using IL N-methyl-2-pyrrolidonium methyl sulfate ([NMP][CH3SO3]) as catalyst.

Our data revealed that NMMO residual in low concentration willnot pose negative effect on fermentation and biofuel production.

4. Conclusions

Ultrasound intensified aqueous-NMMO in situ enzymatichydrolysis system mediated the obvious advantage as compatiblesystem for lignocellulose saccharification. The cellulose conversionrate increased by 51.2% and 57.8% compared to that of the un-treated. Trifle lignin protect the cellulase as antioxidant and en-hanced the conversion rate (97.9%). Viscosity and FTIR analysisrevealed that ultrasound treatment in NMMO system degraded lig-nocellulosic structure and decreased the degree of polymerizationof cellulose, which contribute to significantly increased saccharifi-cation rate. NMMO residual in low concentration will not pose neg-ative effect on biofuel production. In brief, our work provided anopportunity for compatible in situ hydrolysis of natural lignocellu-losic biomass using aqueous-NMMO system.

Acknowledgements

We wish to express our thanks for the support from the NaturalScience Foundation of Jiangsu Province (No. BK2011527) and theStartup Project of Doctoral scientific research of Jiangsu Universityof Science and Technology (No. 35211003).

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Ultrasound-assisted compatible in situ hydrolysis of sugarcane bagasse in cellulase-aqueousN-methylmorpholine-N-oxide system for improved saccharification1 Introduction2 Methods2.1 Feed stock2.2 Stability of cellulases in NMMO2.3 Hydrolysis of Avicel with the addition of NMMO2.3.1 Ultrasound intensified Avicel treatment With NMMO2.3.2 Enzymatic saccharification of treated Avicel2.3.3 Effect of lignin on the hydrolysis of Avicel in NMMO solution2.4 Saccharification of sugarcane bagasse in aqueous-NMMO solution2.4.1 1Sugarcane bagasse treatment with NMMO2.4.2 2Ultrasound intensified saccharification of sugarcane bagasse2.5 Fermentability of the hydrolyzates from saccharification process2.6 Analysis3 Results and discussion3.1 Stability of cellulases in the presence of NMMO3.2 Enzymatic saccharification of Avicel3.3 Enzymatic saccharification of sugarcane bagasse3.4 Effect of lignin on the hydrolysis of Avicel3.5 FTIR analysis of sugarcane bagasse in various mode of treatment3.6 Fermentability of hydrolyzates4 ConclusionsAcknowledgementsReferences