Bioresource Technology 153 (2014) 69–78

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Bioresource Technology

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High yield production of sugars from deproteinated palm kernel cakeunder microwave irradiation via dilute sulfuric acid hydrolysis

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.11.055

⇑ Corresponding authors. Tel.: +60 3 8921 5473; fax: +60 3 8921 3777(C.-H. Chia).

E-mail addresses: chia@ukm.my (C.-H. Chia), zhenfang@xtbg.ac.cn (Z. Fang).

Suet-Pin Fan a,b, Li-Qun Jiang a, Chin-Hua Chia b,⇑, Zhen Fang a,⇑, Sarani Zakaria b, Kah-Leong Chee c

a Chinese Academy of Sciences, Biomass Group, Key Laboratory of Tropical Plant Resource and Sustainable Use, Xishuangbanna Tropical Botanical Garden, 88 Xuefulu,Kunming, Yunnan 650223, Chinab School of Applied Physics, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysiac Tunku Abdul Rahman University College, Faculty of Applied Sciences and Computing, Jalan Genting Kelang, Setapak, 53300 Kuala Lumpur, Malaysia

h i g h l i g h t s

� Response surface methodology asoptimization strategy for DPKC-derived sugars.� Statistically optimized on total

monosaccharide, 77.11% andmannose yield, 92.11%.� Ridge analysis was further conducted

to verify the optimization parameters.� Established an effective microwave-

assisted hydrolysis on DPKC tosugars.

g r a p h i c a l a b s t r a c t

Microwave-assisted hydrolysis via dilute sulfuric acid is an effective method for the conversion of DPKCto fermentable sugars which potentially to be further transform into biofuels.

Biofuels

Fermentable sugars

a r t i c l e i n f o

Article history:Received 6 October 2013Received in revised form 16 November 2013Accepted 20 November 2013Available online 27 November 2013

Keywords:MannoseMicrowave-assisted hydrolysisPalm kernel cakeRidge analysisRSM optimization

a b s t r a c t

Recent years, great interest has been devoted to the conversion of biomass-derived carbohydrate intosugars, such as glucose, mannose and fructose. These are important versatile intermediate products thatare easily processed into high value-added biofuels. In this work, microwave-assisted dilute sulfuric acidhydrolysis of deproteinated palm kernel cake (DPKC) was systematically studied using Response SurfaceMethodology. The highest mannose yield (92.11%) was achieved at the optimized condition of 148 �C,0.75 N H2SO4, 10 min 31 s and substrate to solvent (SS) ratio (w/v) of 1:49.69. Besides that, total ferment-able sugars yield (77.11%), was obtained at 170 �C, 0.181 N H2SO4, 6 min 6 s and SS ratio (w/v) of 1:40.Ridge analysis was employed to further verify the optimum conditions. Thus, this work provides funda-mental data of the practical use of DPKC as low cost, high yield and environmental-friendly material forthe production of mannose and other sugars.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Climate change and increasing concerns for energy security hasimparted a trend shifting from the use of fossil fuels to renewableenergy sources. Globally, focus has been on transforming the agri-

cultural waste into high value-added products. Malaysia, one of theglobal leading palm oil producers, actively seeking for the next cat-alyst to sustain their economic growth since the palm oil produc-tion has reached a mature stage (MPOB, 2012). Palm kernel cake(PKC), one of the main by-products from palm oil industry, is richin protein (14.5–19.6%) and mannan (35.2%) (Cerveró et al., 2010).It should be placed under the spotlight and revolutionize into asource of revenue for oil palm industry. PKC composes of high car-bohydrate contents, mainly hexoses such as mannose, glucose and

Fig. 1. Schematic representation of microwave and reactor vial.

70 S.-P. Fan et al. / Bioresource Technology 153 (2014) 69–78

galactose. These are promising candidates for the production ofbioethanol through fermentation by microorganism (Gírio et al.,2010).

It is widely known that the hemicellulose is more easily to behydrolyzed than cellulose due to its lower crystallinity (Canettieriet al., 2007). Mannan possesses similar structure as cellulose (Brad-bury and Halliday, 1990), both are linear b-(1–4)-linked monosac-charide polymers exhibit some crystalline polymorphism (Wymanet al., 2005). Mannan can be classified into two major groupsdepending on the b-(1–4)-linked backbone whether it composedof only D-mannose residues (mannans) or a combination of man-nose and D-glucose residues (glucomannans) (van Zyl et al.,2010). Essentially, a more rigorous hydrolysis condition is neededto effectively catalyze the depolymerization of mannan into man-nose. However, relatively little prior work has been completed inthe area of mannose production from lignocellulosic materials,especially through acid hydrolysis (Bradbury and Halliday, 1990).In most of the reported studies, extraction of mannose involvesmannan-degrading enzyme (Cerveró et al., 2010; Zhang et al.,2009) which possess several disadvantages, including high pre-treatment cost of the raw material before the enzymatic hydrolysisand the utilization of high priced enzyme.

Microwave-assisted green synthesis can be an alternative toaccelerate the acid hydrolysis of carbohydrate. In prior literatures,microwave heating can offer up to 85-folds energy saving com-pared to the conventional heating (Yemis� and Mazza, 2012). It alsoshortens the reaction time and reduces chemical consumption(Yoshida et al., 2010), thus making it more industrially favorable.As interest is growing in the biofuel industry, dilute acid catalyzedhydrolysis has been widely used for various lignocellulosic materi-als, such as corn stover (Liu and Cheng, 2010) and grass clippings(Orozco et al., 2011). Since microwave-assisted hydrolysis involvesmany variables that affect the desired response, therefore responsesurface methodology (RSM), a statistically designed experimentalprotocol possesses advantages for both the time requirementsand number of experiments reduction. In spite of that, RSM dem-onstrates a relationship between variables and responses over arelatively broad factor domain, which is much practical and profes-sionally in determining the optimum conditions compare withclassical method.

In previous studies, protein was successfully extracted fromPKC by trypsin-assisted and hexametaphosphate-assisted extrac-tion (Chee and Ayob, 2013; Chee et al., 2012). Subsequently, depro-teinated PKC (DPKC) can be a suitable candidate to be furtherhydrolyzed into fermentable sugars. On the plus side, these fer-mentable sugars are recognized as a precursor for platform mole-cules in value-added chemicals and biofuels production. Hitherto,this is the first attempt on systematic optimization of fermentablesugar production from DPKC via microwave-assisted dilute sulfuricacid hydrolysis. This study elucidates different types of sugar pro-duction under different hydrolysis conditions, and with the appli-cation of central composite rotatable design (CCRD), it provides amore complete picture on the structural transformations of princi-pal DPKC components.

2. Methods

2.1. DPKC and chemicals

PKC used in this study was supplied by FELDA Kernel ProductsSdn. Bhd., Malaysia. It was then deproteinated by sodium hydrox-ide and named as DPKC. The moisture content of DPKC after depro-teination was 5.25%. The DPKC was sieved into particle size rangingfrom 100 to 149 microns, and subsequently dried at 105 �C for 24 hbefore use. Sodium hydroxide, NaOH (purity P 96%) and sulfuricacid, H2SO4 (purity 95–98%) were purchased from Xilong Chemical

Co. Ltd. (Guangzhou, China). Mannose, glucose, xylose and galact-ose (purity P 99.5%) as standards were purchased form SigmaAldrich.

2.2. Chemical compositions of DPKC

The DPKC’s components were analyzed using National Renew-able Energy Laboratory (NREL) analytical methods (Sluiter et al.,2008a,b). Before the determination of structural carbohydratesand lignin in the DPKC, the content of extractives and ash weredetermined. First, the sample was treated with 72% (w/w) H2SO4

at 30 �C for 1 h in an incubator shaker at 100 rpm. The mixturewas then diluted to 4% (w/w) H2SO4 by adding 84 ml deionizedwater and autoclaved at 121 �C for 1 h. The hydrolysis solutionwas filtered and the sugar content was analyzed by a High perfor-mance liquid chromatograph (HPLC; Shimadzu LC-20A HPLCpump, Shimadzu, prominence oven CTO-20A, Kyoto) with anAminex HP X-87P column (300 � 7.8 mm, Bio-Rad, California)operated at 80 �C, flow rate 0.4 ml/min with Milli-Q water as mo-bile phase, equipped with a refractive index detector (RID-10A,Shimadzu). Autoclaved hydrolysis samples were filtered andacid-soluble lignin (ASL) determined using an ultraviolet–visible(UV–Vis) spectrophotometer (UV 1800, Shimadzu) at wavelength240 nm. Meanwhile, the remaining autoclaved solid residue wasdried overnight at 105 �C and ashed in a muffle furnace at 575 �Cfor 24 h in order to determine the ash and acid-insoluble lignincontents. The concentration of sugars (mannose, glucose, xyloseand galactose) was quantitatively analyzed using HPLC to calculatethe percentage of the carbohydrate fractions in the DPKC. The pro-tein content of the DPKC was determined using the Kjeldahl meth-od (AOAC, 2005), which was done by UNIPEQ, Bangi, Malaysia.

2.3. Microwave-assisted hydrolysis

All hydrolysis experiments were carried out in a well-controlledmicrowave synthesis reactor (Monowave 300, Anton Paar, Graz,

S.-P. Fan et al. / Bioresource Technology 153 (2014) 69–78 71

Austria) using a reactor vial made of borosilicate glass sealed witha PTFE (Polytetrafluoroethylene)-coated silicone septum andclosed with a snap cap made of PEEK (Polyether ether ketone) attemperature up to 300 �C and pressure up to 3.2 MPa (Fig. 1).The reaction temperature was measured by a built-in infrared(IR) sensor, which was calibrated by a ruby sensor. Meanwhile, anon-invasive pressure sensor is located in the swiveling cover ofMonowave 300 for monitoring the pressure. The reaction was per-formed in an airtight reaction vial. As such, before a reaction starts,the reaction vial was sealed by a pneumatic system and then thedeformation of the silicone septum was translated into reactionpressure by a hydraulic piston throughout the experiment. Thepressure was calibrated by the saturated vapor pressures of water(1, 2, and 3 MPa) at three different temperatures (180, 212, and234 �C) which were measured by a ruby sensor. Both temperatureand pressure vs. time were recorded in a USB disk. Fig. 2 shows thetemperature and pressure vs. time for the experimental reaction attemperatures (120, 140, 160, 180, and 200 �C).

In a typical test, DPKC (0.1 g) and sulfuric acid (5 ml) at a de-sired concentration were charged into a reactor vial incorporatedwith a stir bar. The mixture was heated to the desired temperaturewith heating rates 0.8–1.5 �C/s (Fig. 2) and stirred at 1000 rpm. Thereaction was maintained by a proportional-integral-derivative(PID) controller at the desired temperature for different reactiontime (0, 5, 10, 15, and 20 min), followed by a rapid cooling to55 �C by compressed air flushing to stop the reaction. After thereaction, the liquid hydrolysate was separated from the productmixture using a centrifuge (3–30 K, SIGMA, Osterode am Harz,Germany). After neutralizing with NaOH, the liquid sample wasfiltered and the clear aqueous phase was analyzed with HPLC.

2.4. Experimental design and statistical analyses

In this study, RSM was employed to obtain the optimum condi-tions for microwave-assisted hydrolysis of DPKC using H2SO4. Theselection of variables was based on some preliminary studies andthen followed by a fractional factorial design (FFD) to identifythe significant variables for the production of sugars (data notshown). Hence, four independent variables (temperature, acid con-centration, reaction time, substrate: solvent (SS) ratio) with fivelevels were set up according to the CCRD using Design Expert 6.0(Stat-Ease Inc., Minneapolis, USA) to obtain a quadratic model.The quadratic effects and central points were estimated with thetotal monosaccharide yield (Ytm) and mannose yield (Ymy) as re-sponses. The four independent variables and the actual values atfive levels (�2, �1, 0, +1, +2) were presented in Table 1. The total

Fig. 2. Temperature–pressure profiles with respect to time at different reactiontemperatures (120 �C, 140 �C, 160 �C, 180 �C and 200 �C).

number of experiments with four factor was 54 = [(2k + 2k) � 2]+ 6, where k is the number of factors. Forty-eight experiments wereaugmented with six replications at the center points to evaluatethe pure error. The predictor variables were coded according tothe following equation:

Xi ¼ ðXi � X0Þ=DXi ð1Þ

where, xi is the coded value of an independent variable, Xi is the ac-tual values of the independent variable i, X0 is the actual value of theindependent variable at the center point, and DXi is the step changevalue corresponding to a unite variation of the dimensionless value.

The regression equation was fitted to the response resultedfrom the CCRD:

y ¼ b0 þX

bjxj þX

bjjx2j þ

Xbjkxjxk ð2Þ

where, y is the predicted response, b0 is the intercept, bj, bjj, bjk arethe linear, quadratic and interactive coefficients, respectively.

Ridge analysis was applied on a second-order fitted response toobtain a set of paths, a maximum response, going outwards fromthe origin x0 = (x1, x2,. . .,xq) = (0,0,. . .,0) of the factor space. The basicridge analysis method is as follows. Assume the fitted second-ordersurface is:

y ¼ b0 þ b1x1 þ b2x2 þ � � � þ bqxq þ b11x21 þ b22x2

2 þ � � � þ bqqx2q þ � � �

þb12x1x2 þ b13x1x3 þ � � � þ bq�1;qxq�1xq q ¼ 1;2;3

ð3Þ

where, y is the predicted response, b0 is the intercept, b’s are theregression coefficients. Meanwhile Eq. (3) can be written in matrixform as:

y ¼ b0 þ x0bþ x0Bx ð4Þ

where, x’ = (x1, x2, . . ., xq), b’ = (b1, b2, . . ., bq).

B ¼b11

12 b12

12 b1q

� � � b2212 b2q

sym � � � bqq

264

375 ð5Þ

where, B is a symmetric matrix containing all second-ordercoefficients.

The calculations to obtain the Eigen values and predictionpoints perform by using MINITAB 16 (Minitab Inc., State College,Pennsylvania, USA). Then, actual experimental runs at points alongthis path were conducted to achieve the optimum response values.

2.5. HPLC analysis

Sugars (mannose, glucose, xylose and galactose) were measuredby HPLC (LC-20A, Shimadzu). Each monosaccharide was calibratedby its standard sugar solutions with five different concentrations(e.g., 0.1, 0.2, 0.3, 0.4 and 0.5 mg/ml). All the standard calibrationcurves obtained with R2 > 0.998. Total monosaccharide yield (Ytm,wt.%) and mannose yield (Ymy, wt.%) were calculated as follows:

Y tmðwt:%Þ ¼ ½total mass of monosaccharidesðmannose þ glucoseþ xyloseþ galactoseÞin the liquid hydrolysate�=ðtotal mass of monosacharides in DPKCÞ � 100%

Ymyðwt:%Þ ¼ ðmass of mannose in the liquid hydrolysateÞ=ðtotal mass of mannose in DPKCÞ � 100%

3. Results and discussion

Fifty-four experiments were conducted under the conditions:temperature of 120–200 �C, sulfuric acid concentration of

Table 1Actual and coded variables at five levels in the CCRD constructed to optimize the hydrolysis of DPKC by sulfuric acid.

Independent variables Coded Levels

�2 �1 0 1 2

Temperature (�C) x1 120 140 160 180 200Acid concentration (N) x2 0 0.25 0.5 0.75 1.0Reaction time (min) x3 0 5 10 15 20Substrate:solvent ratio (g/ml) x4 1:20 1:30 1:40 1:50 1:60

72 S.-P. Fan et al. / Bioresource Technology 153 (2014) 69–78

0–1.0 N, reaction time of 0–20 min and substrate (DPKC): solventratio (g/ml) of 1:20–1:60 (Table 1) to optimize the sugar yields.The schematic representation of microwave and reactor vial is dis-played in Fig. 1. The chemical compositions and relative monosac-charide composition of DPKC (wt.%) are listed in Table 2. Fig. 2shows the temperature–pressure profiles with respect to time atdifferent reaction temperatures (120–200 �C). Three-dimensional(3D) response surface plots for the whole model of total monosac-charide and mannose yields, presented in Fig. 3 and Fig. 4, showthe interaction effects of two independent variables, where theother two variables are fixed at the center point. The experimentalresponses values with CCRD are summarized in Table 3. As for theanalysis of variance (ANOVA) for the CCRD model of the totalmonosaccharide and mannose yields are given in Tables 4 and 5.Lastly, ridge analysis of the total monosaccharide is stated inTable 6.

3.1. Components of DPKC

The components of the DPKC analyzed using NREL procedureare presented in Table 2. Mannan and glucan account for 94.77%of the total carbohydrates in the DPKC. DPKC contains substantiallyhigher mannan fraction in the hemicellulose than other glucan,xylan and galactan.

3.2. Experimental design and statistical analysis

The results of the responses (total monosaccharide and man-nose yields) were summarized in Table 3. The polynomial equa-tions describing total monosaccharide yield (Ytm) and mannoseyield (Ymy) are given below:

lYtm ¼ 76:7� 4:22x1 � 1:69x2 � 1:35x3 þ 0:63x4 � 10:74x1x2

� 7:88x1x3 � 0:3x1x4 � 1:62x2x3 þ 1:11x2x4 þ 0:4x3x4

� 15:27x21 � 2:11x2

2 � 1:91x23 � 0:42x2

4 ð6Þ

Table 2Chemical compositions of DPKC (wt.%).

Components:

Protein 6.70 ± 0Lignin

Acid-Insoluble Lignin 5.67 ± 0.42Acid-Soluble Lignin 2.45 ± 0.35

ExtractivesWater-Soluble 4.13 ± 0.18Ethanol-Soluble 3.54 ± 0.08

Ash 3.50 ± 0.08Monosaccharides after hydrolysis*

Glucose 13.66 ± 0.78Mannose 55.71 ± 0.68Xylose 1.84 ± 0.14Arabinose 1.00 ± 0.24Galactose 1.00 ± 0.06

* Measured by NREL analytical methods (Sluiter et al., 2008a; Sluiter et al., 2008b).

lYmy ¼90:84� 8:37x1 � 2:68x2 � 2:25x3 þ 0:71x4 � 13:92x1x2

� 10:68x1x3 � 0:92x1x4 � 1:76x2x3 þ 1:64x2x4 þ 0:47x3x4

� 20:93x21 � 1:88x2

2 � 3:05x23 � 0:52x2

4 ð7Þ

where, x1, x2, x3, x4, are the coded values of independent variables oftemperature, acid concentration, reaction time and SS ratio, respec-tively. The models for total monosaccharide and mannose yieldsevaluated by ANOVA are summarized in Table 4. For bothresponses, the regression were statistically significant at the 95%confidence level, as denoted from the Fisher’s F-test with theprobability (P) value was less than 0.001.

The quality of the regression model was expressed by the coef-ficient of determination (R2). The predicted R2 and adjusted R2 forthe first (Ytm) were 0.9227 and 0.9505; second (Ymy) were 0.9392and 0.9611, respectively, which suggested the design model wasadequately demonstrating the real relationships among the param-eters chosen. The high value of the R2 indicates the good correla-tion between the model and the experimental results (Joglekarand May, 1987).

3.2.1. Effect of independent variables on responsesThe response surfaces and contour plots, which described by

the regression models for the total monosaccharide and mannoseyields were generated to illustrate the interactive effects betweeneach independent variable on the response variables. Figs. 3 and 4are delineated by imposing two independent variables at their zerolevel. Fig. 3a–f and 4a–f represent response surfaces and contourplots for responses, Ytm and Ymy, respectively. The significance levelfor the interactions between variables can be depicted from theshape of the corresponding contour plots. Elliptical contours canbe achieved when there is a perfect interaction between indepen-dent variables (Muralidhar et al., 2001). In Table 5, the greatest sig-nificant effect for the response, Ytm was the quadratic term oftemperature (x1

2), ensued by x1x2, x1x3, temperature (x1), x32, x2

2,acid concentration (x2), reaction time (x3) and x2x3. While, the mostsignificant effect for the response, Ymy sequenced as: quadraticterm of temperature (x1

2), x1x2, x1x3, temperature (x1), x32, reaction

time (x3) and acid concentration (x2). In present study, mannosewas the main DPKC-derived sugar from the hydrolysis.

3.2.1.1. Effect of temperature on total monosaccharide yield (Ytm) andmannose yield (Ymy). First of all, temperature is a key parameter indetermining the sugars recovery and degradation during acidhydrolysis process. Temperature imparts disruption on the DPKCsubstrate structure, the acid dissociation is also depending on theoperating temperature (Marshall and Jones, 1966). At normal tem-perature, the polysaccharide stays in a stable crystalline form. Athigh temperature, the monosaccharide unit in the polysaccharideexists abundantly in open-chain form (less stable) than the ringform (Nattorp et al., 1999). Thus, it is more susceptible to hydroly-sis. As temperature increases, molecules gain higher kinetic energythat leads to a greater collision rate between the substrate andhydronium ions, which randomly attack on the glycosidic linkageto surpass the activation energy barrier, and thereby resulting inthe hydrolysis/degradation reaction to occur. These scenarios can

Fig. 3. Three-dimensional (3D) response surface plots showing the interaction effects of two independent variables on total monosaccharide yield (Ytm), while the other twovariables were fixed at the center point. (a) Effect of temperature and sulfuric acid concentration at fixed reaction time (10 min) and SS ratio (1: 40). (b) Effect of temperatureand reaction time at fixed sulfuric acid concentration (0.5 N) and SS ratio (1: 40). (c) Effect of temperature and SS ratio at fixed sulfuric acid concentration (0.5 N) and reactiontime (10 min). (d) Effect of sulfuric acid concentration and reaction time at fixed temperature (160 �C) and SS ratio (1: 40). (e) Effect of sulfuric acid concentration and SS ratioat fixed temperature (160 �C) and reaction time (10 min). (f) Effect of reaction time and SS ratio at fixed temperature (160 �C) and sulfuric acid concentration (0.5 N).

S.-P. Fan et al. / Bioresource Technology 153 (2014) 69–78 73

be seen in Figs. 3a and 4a, where both Ytm and Ymy increased whentemperature rose from 140 to 165 �C, but both declined as temper-ature increased further. A similar trend can be found in Figs. 3b andc and 4b and c. Consequently, it is concluded that at 0.25 N H2SO4,the temperature increment (<165 �C) contributes a higher impacton DPKC hydrolysis reaction rate than secondary decompositionrate of the hydrolyzed sugars (Gurgel et al., 2011). The highestYtm achieved was 76.98% at 164 �C, 10 min, SS ratio of 1:40, andconcurrently 92.01% for Ymy at 163 �C. By comparing the results ob-tained (Fig. 3a), it can be proposed that the DPKC-derived sugarsdecomposition begins to dominate at temperature higher than165 �C. Whereas, at fixed acid concentration 0.75 N H2SO4 andreaction time of 10 min, a negative effect of temperature(>150 �C) observed on the DPKC-derived sugars. Ytm and Ymy de-creased from 150 �C onwards as shown in Figs. 3a and 4a, owingto the low pKa of H2SO4 with greater hydrolyzing power generating

more hydronium ion which further catalyzed the degradation ofsugars into furfural (from C5-sugars) and 5-hydroxymethylfurfural(5-HMF, from C6-sugar) (Jung et al., 2013; Mosier et al., 2002). InFig. 3a, for acid concentration of 0.25 N H2SO4, the steepness ofthe curve became more gradual in the direction of temperaturerange (140–165 �C). Next, further processing of the data by numer-ical optimization function (Design Expert Software) showed thatYtm, at temperature range (140–150 �C) was nearly 2.7-fold fasterthan the temperature range (150–165 �C). These data suggestedthat, initially the diffusion rate of hydrolyzed sugars into the bulkmedium is equivalent to the penetration rate of the reacting spe-cies into the DPKC substrate. As described previously, increasingtemperature entailed a rise on the kinetic energy of the reactingspecies, penetration rate and its collision probabilities with sub-strate, thus the hydrolysis reaction occurred at a greater rate.Yet, up to a certain extent, the increasing concentration of

Fig. 4. Three-dimensional (3D) response surface plots showing the interaction effects of two independent variables on mannose yield (Ymy), while the other two variableswere fixed at the center point. (a) Effect of temperature and sulfuric acid concentration at fixed reaction time (10 min) and SS ratio (1:40). (b) Effect of temperature andreaction time at fixed sulfuric acid concentration (0.5 N) and SS ratio (1:40). (c) Effect of temperature and SS ratio at fixed sulfuric acid concentration (0.5 N) and reaction time(10 min). (d) Effect of sulfuric acid concentration and reaction time at fixed temperature (160 �C) and SS ratio (1:40). (e) Effect of sulfuric acid concentration and SS ratio atfixed temperature (160 �C) and reaction time (10 min). (f) Effect of reaction time and SS ratio at fixed temperature (160 �C) and sulfuric acid concentration (0.5 N).

74 S.-P. Fan et al. / Bioresource Technology 153 (2014) 69–78

‘‘released’’ sugars in the bulk medium (near the surface of sub-strate) may slow-down the continuous releasing of sugars fromthe DPKC substrate as well as induced an additional resistancefor the penetration of the reacting species into the DPKC substrate.These results are in accord with previous study by Torget et al.(2000), stated that the released moieties tend to stay closed onthe cellulose surface due to the hydrogen-bonding potential withthe structure cellulose surface, and van der Waals attraction forcesalong with the resistance of diffusion caused by the charged struc-tural water layer (Torget et al., 2000).

In Table 5 (ANOVA), temperature demonstrated a significantquadratic effect on Ytm and Ymy, evidently in surface plots (Figs. 3and 4). This indicated that temperature is the most important fac-tor in determining the resultant degree of conversion of DPKC into

sugars. Also, hydrolysis shows stronger temperature dependencyat acid concentration greater than 0.4 wt.% (Saeman, 1945; Torgetet al., 2000). The interaction effect between temperature-acid con-centration and temperature-reaction time was positive (P < 0.001)towards Ytm and Ymy. By comparing the F value between these twointeraction pairs, the temperature-acid concentration was moresignificant than the temperature-reaction time pair. By consideringthe interaction temperature-acid concentration, it was found thatat low temperature (140 �C), the sugars recovery rose with increas-ing acid concentration (0.25–0.75 N). However, an inversely effectobserved during hydrolysis at high temperature (175 �C). An iden-tical phenomenon showed by the temperature-reaction time inter-action pair in Figs. 3b and 4b. Apparently, hydrolysis at lowtemperature requires a longer reaction time and vice versa in order

Table 3Central composite rotatable design with the experimental responses values of total monosaccharide yield (Ytm), mannose yield (Ymy), mannose (man), glucose (glu), xylose (xyl)and galactose (gal).

Test Factors Monosaccharide in DPKC (g/g) Responses (%)

x1 (�C) x2 (N) x3 (min) x4 (g/ml) Man Glu Xyl Gal Ytm Ymy

1 �1 �1 �1 �1 0.3189 0.0021 0.0143 0.0137 47.76 57.252 �1 �1 �1 �1 0.3054 0.0018 0.0146 0.0131 45.75 54.83

46.76 ± 1.42a 56.04 ± 1.71b

3 1 �1 �1 �1 0.4802 0.0175 0.0170 0.0153 72.42 86.204 1 �1 �1 �1 0.4928 0.0187 0.0187 0.0152 74.51 88.46

73.47 ± 1.48a 87.33 ± 1.60b

5 �1 1 �1 �1 0.4287 0.0198 0.0152 0.0070 64.31 76.956 �1 1 �1 �1 0.4484 0.1922 0.0181 0.0096 67.67 80.49

65.99 ± 2.38a 78.72 ± 2.50b

7 1 1 �1 �1 0.2826 0.0723 0.0033 0.0017 49.17 50.738 1 1 �1 �1 0.2903 0.0423 0.0061 0.0024 46.59 52.11

47.88 ± 1.82a 51.42 ± 0.98b

9 �1 �1 1 �1 0.3927 0.0057 0.0179 0.0170 59.19 70.4910 �1 �1 1 �1 0.3896 0.0060 0.0151 0.0152 58.19 69.94

58.69 ± 0.71a 70.22 ± 0.39b

11 1 �1 1 �1 0.3450 0.0415 0.0260 0.0107 57.80 61.9312 1 �1 1 �1 0.3328 0.0335 0.0262 0.0076 54.66 59.74

56.23 ± 2.22a 60.84 ± 1.55b

13 �1 1 1 �1 0.5087 0.0188 0.0169 0.0149 76.41 91.3214 �1 1 1 �1 0.5224 0.0137 0.0206 0.0097 77.38 93.77

76.90 ± 0.69a 92.55 ± 1.73b

15 1 1 1 �1 0.0997 0.0652 0.0103 0 23.94 17.9016 1 1 1 �1 0.0946 0.0507 0 0 19.85 16.98

21.90 ± 2.89a 17.44 ± 0.65b

17 �1 �1 �1 1 0.3243 0.0019 0.0142 0.0138 48.40 58.2218 �1 �1 �1 1 0.3101 0.0014 0.0137 0.0116 46.02 55.67

47.21 ± 1.68a 56.95 ± 1.80b

19 1 �1 �1 1 0.4316 0.0369 0.0131 0.0153 67.88 77.4720 1 �1 �1 1 0.4423 0.0272 0.0157 0.0138 68.16 79.39

68.02 ± 0.20a 78.43 ± 1.36b

21 �1 1 �1 1 0.4774 0.0068 0.0211 0.0083 70.17 85.7022 �1 1 �1 1 0.4660 0.0104 0.0166 0.0076 68.38 83.65

69.28 ± 1.27a 84.68 ± 1.45b

23 1 1 �1 1 0.3182 0.0569 0.0039 0.0009 51.90 57.1224 1 1 �1 1 0.3149 0.0573 0.0023 0.0013 51.35 56.53

51.63 ± 0.39a 56.83 ± 0.42b

25 �1 �1 1 1 0.4318 0.0049 0.0190 0.0150 64.30 77.5126 �1 �1 1 1 0.4205 0.0047 0.0177 0.0140 62.43 75.49

63.37 ± 1.32a 76.50 ± 1.43b

27 1 �1 1 1 0.3195 0.0464 0.0145 0.0095 53.26 57.3628 1 �1 1 1 0.3113 0.0505 0.0146 0.0085 52.57 55.88

52.92 ± 0.49a 56.62 ± 1.05b

29 �1 1 1 1 0.5193 0.0106 0.0199 0.0060 75.91 93.2230 �1 1 1 1 0.5269 0.0075 0.0198 0.0048 76.37 94.59

76.14 ± 0.33a 93.91 ± 0.97b

31 1 1 1 1 0.1434 0.0768 0.0018 0 30.32 25.7432 1 1 1 1 0.1350 0.0758 0.0020 0 29.08 24.23

29.70 ± 0.88a 24.99 ± 1.07b

33 0 0 0 0 0.5054 0.0185 0.0188 0.0165 76.40 90.7334 0 0 0 0 0.5033 0.0186 0.0182 0.0167 76.07 90.34

76.24 ± 0.23a 90.54 ± 0.28b

35 0 0 0 0 0.5081 0.0182 0.0185 0.0154 76.52 91.2136 0 0 0 0 0.5114 0.0189 0.0185 0.0162 77.19 91.81

76.86 ± 0.47a 91.51 ± 0.42b

37 �2 0 0 0 0.0691 0.0058 0.0307 0 14.43 12.4038 �2 0 0 0 0.0713 0.0080 0.0449 0 16.97 12.79

15.70 ± 1.80a 12.60 ± 0.28b

39 2 0 0 0 0 0.032 0.0206 0.0691 16.62 0.0040 2 0 0 0 0 0.023 0.0171 0.0774 16.05 0.00

16.34 ± 0.40a 0b

41* 0 �2 0 0 0 0 0.0036 0 0.49 0.0042* 0 �2 0 0 0 0 0.0026 0 0.31 0.00

0.4 ± 0.13a 0b

43 0 2 0 0 0.4351 0.0299 0.0140 0.0045 66.05 78.1044 0 2 0 0 0.4231 0.0353 0.0099 0.0044 64.57 75.94

65.31 ± 1.05a 77.02 ± 1.53b

45 0 0 �2 0 0.4335 0.0028 0.0726 0.0052 70.22 77.8246 0 0 �2 0 0.4243 0.0022 0.0646 0.0042 67.67 76.17

68.95 ± 1.8a 77.00 ± 1.17b

47 0 0 2 0 0.4448 0.0429 0.0142 0.0148 70.58 79.8448 0 0 2 0 0.4315 0.0544 0.0091 0.0130 69.40 77.47

69.99 ± 0.83a 78.66 ± 1.68b

(continued on next page)

S.-P. Fan et al. / Bioresource Technology 153 (2014) 69–78 75

Table 3 (continued)

Test Factors Monosaccharide in DPKC (g/g) Responses (%)

x1 (�C) x2 (N) x3 (min) x4 (g/ml) Man Glu Xyl Gal Ytm Ymy

49 0 0 0 �2 0.4827 0.0302 0.0224 0.0117 74.72 86.6550 0 0 0 �2 0.4898 0.0172 0.0220 0.0111 73.77 87.92

74.25 ± 0.67a 87.29 ± 0.90b

51 0 0 0 2 0.4894 0.0156 0.0559 0.0034 77.11 87.8652 0 0 0 2 0.4979 0.0110 0.0430 0.0047 76.03 89.38

76.57 ± 0.76a 88.62 ± 1.07b

53 0 0 0 0 0.5094 0.0196 0.0185 0.0171 77.13 91.4454 0 0 0 0 0.5063 0.0189 0.0177 0.0151 76.23 90.89

76.68 ± 0.64a 91.17 ± 0.39b

* Outliers which are not included in the RSM model.a Values are expressed as mean ± standard deviation (n = 2) for the total monosaccharide yield (Ytm).b Values are expressed as mean ± standard deviation (n = 2) for the mannose yield (Ymy).

Table 4ANOVA for regression models of total monosaccharide yield (Ytm) and mannose yield(Ymy).

Source Sum of squares DF Mean square F-value p-Value

Total monosaccharide yield (Ytm)Model 17866.63 14 1276.19 69.63 <0.0001*

Residual 659.79 36 18.33Pure error 45.46 27 1.68R2

adj 0.9505R2

pred 0.9227

Mannose yield (Ymy)Model 34479.65 14 2462.83 89.23 <0.0001*

Residual 993.59 36 27.60Pure error 41.11 27 1.52R2

adj 0.9611R2

pred 0.9392

* Significant values.

Table 5ANOVA and regression coefficient for linear, quadratic and interactive terms of totalmonosaccharide yield (Ytm) and mannose yield (Ymy).

Source Regression coefficient F-value p-Value

Total monosaccharide yield (Ytm)Intercept 76.7Linearx1 (temperature) �4.22 46.68 <0.0001*

x2 (acid concentration) �1.69 5.04 0.0310*

x3 (reaction time) �1.53 4.75 0.0360*

x4 (substrate: solvent ratio) 0.63 1.04 0.3153Quadraticx1

2 �15.27 528.47 <0.0001*

x22 �2.11 6.19 0.0176*

x32 �1.91 8.24 0.0068*

x42 �0.42 0.40 0.5294

Interactionx1x2 �10.74 201.36 <0.0001*

x1x3 �7.88 108.47 <0.0001*

x1x4 �0.3 0.16 0.6899x2x3 �1.62 4.57 0.0394*

x2x4 1.11 2.14 0.1521x3x4 0.4 0.28 0.6023

Mannose yield (Ymy)Intercept 90.84Linearx1 (temperature) �8.37 121.81 <0.0001*

x2 (acid concentration) �2.68 8.41 0.0063*

x3 (reaction time) �2.25 8.81 0.0053*

x4 (substrate: solvent ratio) 0.71 0.87 0.3560Quadraticx1

2 �20.93 659.46 <0.0001*

x22 �1.88 3.25 0.0797

x32 �3.05 14.00 0.0006*

x42 �0.52 0.40 0.5290

Interactionx1x2 �13.92 224.62 <0.0001*

x1x3 �10.68 132.29 <0.0001*

x1x4 �0.92 0.97 0.3303x2x3 �1.76 3.60 0.0657x2x4 1.64 3.11 0.0865x3x4 0.47 0.26 0.6124

* Significant variables.

76 S.-P. Fan et al. / Bioresource Technology 153 (2014) 69–78

to obtain a reasonable amount of sugars. Furthermore, based onFig. 3c and 4c, the SS ratio factor remains constant with respectto the temperature factor from 140–180 �C. Therefore, it can beconcluded that the interaction effect between temperature andSS ratio has no significant influence on Ytm and Ymy.

According to the experimental data presented in Table 3, at gi-ven operational conditions (tests 21–24), Ytm dropped from aver-age of 69.28% to 51.63% when temperature increased from 140 to180 �C. In the same way, at given conditions with longer reactiontime (tests 29–32) there is a drastic reduction of Ytm from 76.14%to 29.70%, suggesting that the decomposition reaction occurred.Likewise, it was found that a lower mannose yield (Ymy) was ob-tained with further increase in the temperature from 140 to180 �C (tests 21–24, 29–32). It was recently reported that the deg-radation stage is more temperature sentient than hydrolysis stage,evidently from the relatively higher activation energy for degrada-tion process than the hydrolysis reaction (Kim et al., 2013). Accord-ing to (Nattorp et al., 1999), the degradation of mannose hadhigher activation energy (140 kJ/mol) than the mannan hydrolysis(113 kJ/mol). Hence, increasing temperature gives negative effecton maximizing mannose yield (Ymy), because increase in tempera-ture led to mannose degradation more dominant than hydrolysis ofmannan in DPKC.

3.2.1.2. Effect of acid concentration on total monosaccharide yield(Ytm) and mannose yield (Ymy). Apart from temperature factor, acidconcentration also plays a significant role in Ytm and Ymy, as illus-trated in Figs. 3 and 4a, d, e. In present study, the reacting speciesthat catalyze the hydrolysis were hydronium ion, sulfate andbisulfate anions (Lindstrom and Wirth, 1969). With progressivelyhigher acid concentration at 140 �C, the selectivity towards sugars

was higher, thus enhanced Ytm and Ymy as shown in Figs. 3a and 4a.The rising of sugars with increasing acid concentration could pos-sibly due to the increased charge perturbation at the boundarylayer. The ionic disturbance caused by the increase hydroniumion concentration facilitates sugars in ‘‘released’’ state (Torgetet al., 2000). It is noted that at elevated temperature (up to180 �C), Ytm and Ymy decreased with increasing acid concentration.As depicted in Figs. 3 and 4a, d, e, at the acid concentration greaterthan its optimum point, it will introduce an adverse effect on theselectivity of sugars, Ytm and Ymy. In essence, under conditions of

Table 6Ridge analysis on the total monosaccharide yield (Ytm).

k Radii Eigen value Predicted yield (%) Actual value Actual yield (%)

x1 x2 x3 y x1 x2 x3 y

0.300 0.7169 0.1627 �0.5851 �0.3810 77.26 163.2539 0.3537 8.0948 66.350.185 1.5799 0.5061 �1.2771 �0.7803 77.70 170.1227 0.1807 6.0985 77.110.165 2.0090 0.6763 �1.6198 �0.9771 77.97 173.5253 0.0950 5.1146 10.20

S.-P. Fan et al. / Bioresource Technology 153 (2014) 69–78 77

higher acid concentration (Fig. 4d), Ymy increased at the initialphase of reaction and then decreased gradually with prolongedreaction time. These results could be attributed to the severe actionof acid with longer reaction time; mannose underwent secondarydecomposition to 5-HMF. It should be noted in Fig. 4d, the highestamount of Ytm obtained at 160 �C, SS ratio 1:40 with acid concen-tration lower than 0.5 N H2SO4. Szabolcs and co-researchers foundthat at above 0.5 N H2SO4, a higher yield of levulinic acid (simulta-neously, the amount of 5-HMF was lower) was observed and itreached the maximum at 1 N H2SO4 during the microwave-as-sisted conversion of carbohydrates. It is well-known that the for-mation of 5-HMF proceed from hexose (mannose, glucose andgalactose) degradation, therefore this result is in good agreementwith a previous study (Szabolcs et al., 2013), reported that the for-mation of 5-HMF (degradation product from hexose) is favorable atacid concentration higher than 0.5 N H2SO4.

3.2.1.3. Effect of reaction time on total monosaccharide yield (Ytm) andmannose yield (Ymy). In the point of time factor, at fixed tempera-ture 160 �C with SS ratio 1:40 of and 0.25 N H2SO4, longer reactiontime contributes to a higher sugars recovery (Fig. 3d and Fig. 4d).These are consistent with other report on the hydrolysis of sweetsorghum bagasse at moderate temperature (100–121 �C) (Banerjiet al., 2013). In contrast, at the same reaction conditions (160 �C,SS ratio 1:40) with higher acid concentration (0.75 N H2SO4), thedegradation of the sugars occurred with prolonged reaction time.Indeed, other study reported that extending the reaction time athigh acid concentration led to the decomposition of decrystallizedcellulose and thus reduced the sugar yield (Chin et al., 2011).Therefore, it can be summarized that the time factor is dependentto the reaction temperature and acid concentration.

Table 3 (Tests 39 and 40) demonstrates the averages of totalmonosaccharide yield (Ytm) 16.34%, consisting mono-sugars(glucose, xylose and galactose). Although the experiments wereconducted at high temperature (200 �C), there is no great amountof DPKC-derived sugars obtained in the hydrolysate, which couldbe attributed to the lower dissociation of H2SO4 at high tempera-ture (Lloyd and Wyman, 2004; Maki-Arvela et al., 2011).

According to a study reported on the hydrolysis of cellulose, thehighest cellulose conversion using pure water was 70%, which canbe achieved at 220 �C and 100 min (Kupiainen et al., 2012). In thepresent work, the DPKC hydrolysis took place in the presence ofdeionized water, 160 �C, 10 min of reaction time and SS ratio1:40, and yielded trace of xylose monomer 0.4% of total monosac-charide, (Tests 41 and 42). The possible explanations could be theauto-ionization of water at elevated temperature, generatinghydronium ions (Kim et al., 2013) and leading to the productionof acetic acid from the hemicellulose. These would catalyze partialhydrolysis of hemicellulose (xylan) to form xylose. It was foundthat the activation energy for the hydrolysis of cellubiose (Mosieret al., 2002), mannan (Nattorp et al., 1999) and xylan (Canettieriet al., 2007) was 110, 113, and 101 kJ/mol, respectively. The lowestenergy barrier (activation energy) for xylan hydrolysis could be thereason for this observation where xylose was the only detectedcompound after the hydrolysis (Kim et al., 2013). Additionally, byapplying ‘‘easy-to-hydrolyze’’ and ‘‘hard-to-hydrolyze’’ concept of

xylan, it can be postulated that these xylose monomer releasedfrom the ‘‘easy-to-hydrolyze’’ fraction (Lavarack et al., 2002). Thepurpose of inserting this parameter (160 �C, 0 N of acid concentra-tion) was to evaluate the effect of the pure water on the hydrolysisat high temperature. However, according to the Diagnostics func-tion, these response data fall outside the outlier T area be-tween + 3.50 and �3.50, thus it is considered as outliers.Consequently, these outliers (tests 41 and 42) were then excludedin the CCRD model.

3.2.1.4. Effect of substrate: solvent ratio on total monosaccharide yield(Ytm) and mannose yield (Ymy). Higher substrate concentrationsmeans that larger quantity of the raw material can be processedwhich is an important aspect for industrial applications. To allevi-ate this concern, SS ratio factor was incorporated in the experimen-tal design as well, although the SS ratio was not a significant factorfor both responses as the sugars formation are always lower athigher substrate loading, if other parameters kept constant. Ytm

and Ymy of the DPKC hydrolysis were affected marginally with in-creased SS ratio at temperature range (140–180 �C) as displayedin Fig. 3d and 4d. From Table 5, it elucidates that during the hydro-lysis of DPKC, SS ratio was an insignificant factor (P > 0.05) for theresponses Ytm and Ymy, where same phenomena reported by Yemis�and Mazza in the hydrolysis wheat straws (Yemis� and Mazza,2012). As can be seen, the mutual interactions between SS andother pairs of independent (temperature-SS ratio, acid concentra-tion-SS ratio and reaction time-SS ratio) were not significant(P > 0.05) as well. In current work, the instrument limitation onthe highest SS ratio at 1:20, it caused restriction in the chosenrange SS ratio (1:20, 1:30, 1:40, 1:50 and 1:60), these intervalswere too small to give a barely noticeable changes on Ytm andYmy that could be the reason for the insignificant effect of SS ratio.

From the experimental results inferred that it may not be pos-sible to optimize the reaction conditions to obtain a maximumyield for all sugars simultaneously. It is noteworthy that, mannoseis the dominant hemicellulose sugar in the DPKC, thereby; achiev-ing the maximum mannose monomer concentration is preferen-tially than other monomer sugars. One of the strikingobservations obtained in this study is the mannose yield is compa-rable with those reported in the literature using mannan degradingenzyme (Cerveró et al., 2010; Zhang et al., 2009). An important fac-tor could be related to the microwaves interacted with the DPKC ata molecular level, adsorbed deeply into the folding layers of cellu-lose to destroy the crystal structure and enhance the mass transfer(Wu et al., 2010; Yemis� and Mazza, 2012).

3.2.2. Model verification, ridge analysis and optimum reactionconditions

Model verification was carried out in triplicate under selectedsolutions given by Design Expert software. The confirmation exper-iments for total monosaccharide yield (Ytm) were conducted atoperating parameters 153 �C, 0.72 N H2SO4, 9 min 42 s and SS ratioof 1:38.84. This hydrolysis run gave a good result (76.15% of Ytm),which is in good agreement with the predicted value, 78.02%.

78 S.-P. Fan et al. / Bioresource Technology 153 (2014) 69–78

From the analysis of the ANOVA data and the statistical param-eters, after the removal of the insignificant terms, the final deducedempirical model in terms of coded factors is shown below:

Ytm ¼ 76:7� 4:22x1 � 1:69x2 � 1:35x3 � 10:74x1x2

� 7:88x1x3 � 1:62x2x3 � 15:27x21 � 2:11x2

2 � 1:91x23 ð8Þ

The second order polynomial model (Eq. (8)) in present study wasemployed for response optimization by using Minitab 16. As the cen-ter point value greater than the mean value (58.57%), it can beassumed that the model reached the optimum region. However,the 3D contour plot showed a saddle curve, therefore ridge analysison the total monosaccharide was further conducted (Table 6) to ver-ify the optimum reaction conditions. As the three Eigen values haddifferent signs, hence it can be deduced that the stationary pointfor this model did not have a unique optimum. Therefore, the pre-dicted optimum values for the three key variables were determinedfrom the results of ridge analysis. Three hydrolysis conditions wereselected and the experiments were carried out based on the calcu-lated actual value of the variables. It was successfully found that,the optimum conditions for maximum Ytm were 170 �C, 0.181 NH2SO4 and 6 min 6 s. The predicted maximum Ytm was calculatedto be 77.67%, and the actual yield of Ytm obtained was 77.11% with0.56% deviation from the predicted value. The criteria for the optimi-zation of the mannose yield (Ymy) by means of the Desirability func-tion based on the maximization of the mannose content wereperformed at 148 �C, 0.75 N H2SO4, 10 min 31 s and substrate tosolvent (SS) ratio (w/v) of 1:49.69 to corroborate with the predictedvalue. The average value of triplicate experiments for mannose yieldwas 92.11%, whereas the predicted value was 94.63%.

4. Conclusion

The microwave-assisted hydrolysis of deproteinated palmkernel cake under operating conditions (170 �C, 0.181 N H2SO4

and SS ratio of 1:40) offered a maximum yield, 77.11% of totalmonosaccharide in a reaction time 6 min 6 s. Besides, high yieldof mannose, 92.11% was obtained at 148 �C, 0.75 N H2SO4, 10 min31 s and SS ratio of 1:49.69. This work demonstrated that themicrowave-assisted process is an effective method for the acid-catalyzed conversion of DPKC to monosaccharides. DPKC is an eco-nomically and environmentally benign source for the mannosegeneration as it is a cheap and abundantly available resource.

Acknowledgements

The authors would like to acknowledge the financial support gi-ven by University Research Grant (DIP-2012-34) and ERGS/1/2012/STG01/UKM/03/3. Fan acknowledges the Ministry of Higher Educa-tion (KPT) for the disbursement of MyPhD scholarship.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2013.11.055.

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