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High yield production of sugars from deproteinated palm kernel cake undermicrowave irradiation via dilute sulfuric acid hydrolysis
Suet-Pin Fan, Li-Qun Jiang, Chin-Hua Chia, Zhen Fang, Sarani Zakaria, Kah-Leong Chee
PII: S0960-8524(13)01771-9DOI: http://dx.doi.org/10.1016/j.biortech.2013.11.055Reference: BITE 12675
To appear in: Bioresource Technology
Received Date: 6 October 2013Revised Date: 16 November 2013Accepted Date: 20 November 2013
Please cite this article as: Fan, S-P., Jiang, L-Q., Chia, C-H., Fang, Z., Zakaria, S., Chee, K-L., High yield productionof sugars from deproteinated palm kernel cake under microwave irradiation via dilute sulfuric acid hydrolysis,Bioresource Technology (2013), doi: http://dx.doi.org/10.1016/j.biortech.2013.11.055
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting proof before it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
High yield production of sugars from deproteinated palm kernel cake under microwave
irradiation via dilute sulfuric acid hydrolysis
Suet-Pin Fana, b, Li-Qun Jiangb, Chin-Hua Chiaa,*, Zhen Fangb,*, Sarani Zakariaa, Kah-
Leong Cheec
a School of Applied Physics, Faculty of Science and Technology, Universiti Kebangsaan
Malaysia, 43600 Bangi, Selangor, Malaysia
b Chinese Academy of Sciences, Biomass Group, Key Laboratory of Tropical Plant Resource and
Sustainable Use, Xishuangbanna Tropical Botanical Garden, 88 Xuefulu, Kunming, Yunnan
650223, China
c Faculty of Applied Sciences and Computing, Tunku Abdul Rahman University College, , Jalan
Genting Kelang, Setapak, 53300 Kuala Lumpur, Malaysia
*Corresponding authors:
Chin Hua Chia ([email protected]); Zhen Fang ([email protected])
Revised for Bioresource Technology
(November 2013)
2
Abstract
Recent years, great interest has been devoted to the conversion of biomass-derived carbohydrate
into sugars, such as glucose, mannose and fructose. These are important versatile intermediate
products that are easily processed into high value-added biofuels. In this work, microwave-
assisted dilute sulfuric acid hydrolysis of deproteinated palm kernel cake (DPKC) was
systematically studied using Response Surface Methodology. 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 fermentable 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
fundamental data of the practical use of DPKC as low cost, high yield and environmental-
friendly material for the production of mannose and other sugars.
Keywords: Mannose; Microwave-assisted hydrolysis; Palm kernel cake; ridge analysis; RSM
optimization
1. Introduction
Climate change and increasing concerns for energy security has imparted a trend shifting
from the use of fossil fuels to renewable energy sources. Globally, focus has been on
transforming the agricultural waste into high value-added products. Malaysia, one of the global
leading palm oil producers, actively seeking for the next catalyst to sustain their economic
growth since the palm oil production has reached a mature stage (MPOB, 2012). Palm kernel
cake (PKC), one of the main by-products from palm oil industry, is rich in protein (14.5 – 19.6%)
and mannan (35.2%) (Cerveró et al., 2010). It should be placed under the spotlight and
3
revolutionize into a source of revenue for oil palm industry. PKC composes of high carbohydrate
contents, mainly hexoses such as mannose, glucose and galactose. These are promising
candidates for the production of bioethanol through fermentation by microorganism (Gírio et al.,
2010).
It is widely known that the hemicellulose is more easily to be hydrolyzed than cellulose due
to its lower crystallinity (Canettieri et al., 2007). Mannan possesses similar structure as cellulose
(Bradbury & Halliday, 1990), both are linear β-(1-4)-linked monosaccharide polymers exhibit
some crystalline polymorphism (Wyman et al., 2005). Mannan can be classified into two major
groups depending on the β-(1-4)-linked backbone whether it composed of only D-mannose
residues (mannans) or a combination of mannose and D-glucose residues (glucomannans) (van
Zyl et al., 2010). Essentially, a more rigorous hydrolysis condition is needed to effectively
catalyze the depolymerization of mannan into mannose. However, relatively little prior work has
been completed in the area of mannose production from lignocellulosic materials, especially
through acid hydrolysis (Bradbury & Halliday, 1990). In most of the reported studies, extraction
of mannose involves mannan-degrading enzyme (Cerveró et al., 2010; Zhang et al., 2009) which
possess several disadvantages, including high pretreatment cost of the raw material before the
enzymatic hydrolysis and the utilization of high priced enzyme.
Microwave-assisted green synthesis can be an alternative to accelerate the acid hydrolysis of
carbohydrate. In prior literatures, microwave heating can offer up to 85-folds energy saving
compared to the conventional heating (Yemiş & Mazza, 2012). It also shortens 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 catalyzed hydrolysis has
been widely used for various lignocellulosic materials, such as corn stover (Liu & Cheng, 2010)
4
and grass clippings (Orozco et al., 2011). Since microwave-assisted hydrolysis involves many
variables that affect the desired response, therefore response surface methodology (RSM), a
statistically designed experimental protocol possesses advantages for both the time requirements
and number of experiments reduction. In spite of that, RSM demonstrates a relationship between
variables and responses over a relatively broad factor domain, which is much practical and
professionally in determining the optimum conditions compare with classical method.
In previous studies, protein was successfully extracted from PKC by trypsin-assisted and
hexametaphosphate-assisted extraction (Chee & Ayob, 2013; Chee et al., 2012). Subsequently,
deproteinated PKC (DPKC) can be a suitable candidate to be further hydrolyzed into fermentable
sugars. On the plus side, these fermentable sugars are recognized as a precursor for platform
molecules in value-added chemicals and biofuels production. Hitherto, this is the first attempt on
systematic optimization of fermentable sugar production from DPKC via microwave-assisted
dilute sulfuric acid hydrolysis. This study elucidates different types of sugar production under
different hydrolysis conditions, and with the application of central composite rotatable design
(CCRD), it provides a more complete picture on the structural transformations of principal
DPKC components.
2. Materials and Methods
2.1. DPKC and Chemicals
PKC used in this study was supplied by FELDA Kernel Products Sdn. Bhd., Malaysia. It was
then deproteinated by sodium hydroxide and named as DPKC. The moisture content of DPKC
after deproteination was 5.25%. The DPKC was sieved into particle size ranging from 100-149
microns, and subsequently dried at 105 °C for 24 h before use. Sodium hydroxide, NaOH (purity
≥ 96%) and sulfuric acid, H2SO4 (purity 95-98%) were purchased from Xilong Chemical Co. Ltd
5
(Guangzhou, China). Mannose, glucose, xylose and galactose (purity ≥ 99.5%) as standards were
purchased form Sigma Aldrich.
2.2. Chemical compositions of DPKC
The DPKC’s components were analyzed using National Renewable Energy Laboratory
(NREL) analytical methods (Sluiter et al., 2008a; Sluiter et al., 2008b). Before the determination
of structural carbohydrates and lignin in the DPKC, the content of extractives and ash were
determined. First, the sample was treated with 72% (w/w) H2SO4 at 30 °C for 1 h in an incubator
shaker at 100 rpm. The mixture was then diluted to 4% (w/w) H2SO4 by adding 84 ml deionized
water and autoclaved at 121 °C for 1 h. The hydrolysis solution was filtered and the sugar
content was analyzed by a High performance liquid chromatograph (HPLC; Shimadzu LC-20A
HPLC pump, Shimadzu, prominence oven CTO-20A, Kyoto) with an Aminex 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 mobile phase, equipped with a refractive index detector (RID-10A, Shimadzu). Autoclaved
hydrolysis samples were filtered and acid-soluble lignin (ASL) determined using an ultraviolet–
visible (UV-Vis) spectrophotometer (UV 1800, Shimadzu) at wavelength 240 nm. Meanwhile,
the remaining autoclaved solid residue was dried overnight at 105 °C and ashed in a muffle
furnace at 575 °C for 24 h in order to determine the ash and acid-insoluble lignin contents. The
concentration of sugars (mannose, glucose, xylose and galactose) was quantitatively analyzed
using HPLC to calculate the percentage of the carbohydrate fractions in the DPKC. The protein
content of the DPKC was determined using the Kjeldahl method (AOAC, 2005), which was done
by UNIPEQ, Bangi, Malaysia.
2.3. Microwave-assisted hydrolysis
6
All hydrolysis experiments were carried out in a well-controlled microwave synthesis reactor
(Monowave 300, Anton Paar, Graz, Austria) using a reactor vial made of borosilicate glass
sealed with a PTFE (Polytetrafluoroethylene)-coated silicone septum and closed with a snap cap
made of PEEK (Polyether ether ketone) at temperature 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, a non-invasive pressure sensor is located in the
swiveling cover of Monowave 300 for monitoring the pressure. The reaction was performed in
an airtight reaction vial. As such, before a reaction starts, the reaction vial was sealed by a
pneumatic system and then the deformation of the silicone septum was translated into reaction
pressure by a hydraulic piston throughout the experiment. The pressure was calibrated by the
saturated vapor pressures of water (1, 2, and 3 MPa) at three different temperatures (180, 212,
and 234 °C) which were measured by a ruby sensor. Both temperature and pressure vs. time were
recorded in a USB disk. Fig. 2 shows the temperature and pressure vs. time for the experimental
reaction at temperatures (120, 140, 160, 180, and 200 °C).
In a typical test, DPKC (0.1g) and sulfuric acid (5 ml) at a desired concentration were
charged into a reactor vial incorporated with a stir bar. The mixture was heated to the desired
temperature with heating rates 0.8-1.5 oC/s (Fig. 2) and stirred at 1000 rpm. The reaction was
maintained by a proportional-integral-derivative (PID) controller at the desired temperature for
different reaction time (0, 5, 10, 15, and 20 min), followed by a rapid cooling to 55 °C by
compressed air flushing to stop the reaction. After the reaction, the liquid hydrolysate was
separated from the product mixture using a centrifuge (3-30K, SIGMA, Osterode am Harz,
Germany). After neutralizing with NaOH, the liquid sample was filtered and the clear aqueous
phase was analyzed with HPLC.
7
2.4. Experimental design and statistical analyses
In this study, RSM was employed to obtain the optimum conditions for microwave-assisted
hydrolysis of DPKC using H2SO4. The selection of variables was based on some preliminary
studies and then followed by a fractional factorial design (FFD) to identify the significant
variables for the production of sugars (data not shown). Hence, four independent variables
(temperature, acid concentration, reaction time, substrate: solvent (SS) ratio) with five levels
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 the
total monosaccharide yield (Ytm) and mannose yield (Ymy) as responses. The four independent
variables and the actual values at five levels (-2, -1, 0, +1, +2) were presented in Table 1. The
total number of experiments with four factor was 54 = [(2k + 2k) x 2] + 6, where k is the number
of factors. Forty eight experiments were augmented with six replications at the center points to
evaluate the pure error. The predictor variables were coded according to the following equation:
xi = (Xi – X0) / ∆Xi (1)
where, xi is the coded value of an independent variable, Xi is the actual values of the independent
variable i, X0 is the actual value of the independent variable at the center point, and ∆Xi is the
step change value corresponding to a unite variation of the dimensionless value.
The regression equation was fitted to the response resulted from the CCRD:
(2)
where, y is the predicted response, β0 is the intercept, βj, βjj, βjk are the linear, quadratic and
interactive coefficients, respectively.
8
Ridge analysis was applied on a second-order fitted response to obtain a set of paths, a
maximum response, going outwards from the origin x' = (x1, x2,…,xq) = (0,0,…,0) of the factor
space. The basic ridge analysis method is as follows. Assume the fitted second-order surface is:
y = b0 + b1x1 + b2x2 + …+ bqxq + b11x12 + b22x2
2 + … + bqqxq2 + …
+ b12x1x2 + b13x1x3 + … + βq-1,q xq-1xq q = 1, 2, 3 (3)
where, y is the predicted response, b0 is the intercept, b’s are the regression coefficients.
Meanwhile Eq. (3) can be written in matrix form as:
y = b0 + x'b + x'Bx (4)
where, x' = (x1, x2, …, xq), b' = (b1, b2, …, bq).
(5)
where, B is a symmetric matrix containing all second-order coefficients.
The calculations to obtain the Eigen values and prediction points perform by using
MINITAB 16 (Minitab Inc., State College, Pennsylvania, USA). Then, actual experimental runs
at points along this path were conducted to achieve the optimum response values.
2.5. HPLC analysis
Sugars (mannose, glucose, xylose and galactose) were measured by HPLC (LC-20A,
Shimadzu). Each monosaccharide was calibrated by 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 calibration
curves obtained with R2 > 0.998. Total monosaccharide yield (Ytm, wt. %) and mannose yield
(Ymy, wt. %) were calculated as follows:
Ytm (wt. %) = [total mass of monosaccharides (mannose + glucose + xylose + galactose) in the
liquid hydrolysate] / (total mass of monosacharides in DPKC) × 100%
9
Ymy (wt. %) = (mass of mannose in the liquid hydrolysate) / (total mass of mannose in DPKC) ×
100%
3. Results and discussion
Fifty-four experiments were conducted under the conditions: temperature of 120-200 oC,
sulfuric acid concentration of 0-1.0 N, reaction time of 0-20 min and substrate (DPKC): solvent
ratio (g/ml) of 1:20-1:60 (Table 1) to optimize the sugar yields. The schematic representation of
microwave and reactor vial is displayed in Fig. 1. The chemical compositions and relative
monosaccharide composition of DPKC (wt. %) are listed in Table 2. Fig. 2 shows the
temperature-pressure profiles with respect to time at different reaction temperatures (120-200
°C). Three-dimensional (3D) response surface plots for the whole model of total monosaccharide
and mannose yields, presented in Fig. 3 and Fig. 4, showing the interaction effects of two
independent variables, where the other two variables were fixed at the center point. The
experimental responses values with CCRD are summarized in Table 3. As for the analysis of
variance (ANOVA) for the CCRD model of the total monosaccharide and mannose yields are
given in Tables 4 and 5. Lastly, ridge analysis of the total monosaccharide is stated in Table 6.
3.1. Components of DPKC
The components of the DPKC analyzed using NREL procedure are presented in Table 2.
Mannan and glucan account for 94.77% of the total carbohydrates in the DPKC. DPKC contains
substantially higher mannan fraction in the hemicellulose than other glucan, xylan and galactan.
3.2. Experimental design and statistical analysis
10
The results of the responses (total monosaccharide and mannose yields) were summarized in
Table 3. The polynomial equations describing total monosaccharide yield (Ytm) and mannose
yield (Ymy) are given below:
Ytm = 76.7 - 4.22x1 - 1.69x2 - 1.35x3 + 0.63x4 - 10.74x1x2 -7.88x1x3 - 0.3x1x4 - 1.62x2x3
+ 1.11x2x4 + 0.4x3x4 -15.27x12 - 2.11x2
2 - 1.91x32 - 0.42x4
2 (6)
Ymy = 90.84 - 8.37x1 - 2.68x2 - 2.25x3 + 0.71x4 - 13.92x1x2 - 10.68x1x3 - 0.92x1x4 - 1.76x2x3
+ 1.64x2x4 + 0.47x3x4 - 20.93x12 - 1.88x2
2 - 3.05x32 - 0.52x4
2 (7)
where, x1, x2, x3, x4, are the coded values of independent variables of temperature, acid
concentration, reaction time and SS ratio, respectively. The models for total monosaccharide and
mannose yields evaluated by ANOVA are summarized in Table 4. For both responses, the
regression were statistically significant at the 95% confidence level, as denoted from the Fisher’s
F-test with the probability (P) value was less than 0.001.
The quality of the regression model was expressed by the coefficient of determination (R2).
The predicted R2 and adjusted R2 for the first (Ytm) were 0.9227 and 0.9505; second (Ymy) were
0.9392 and 0.9611, respectively, which suggested the design model was adequately
demonstrating the real relationships among the parameters chosen. The high value of the R2
indicates the good correlation between the model and the experimental results (Joglekar & May,
1987).
3.2.1. Effect of independent variables on responses
The response surfaces and contour plots, which described by the regression models for the
total monosaccharide and mannose yields were generated to illustrate the interactive effects
between each independent variable on the response variables. Fig. 3 and 4 are delineated by
imposing two independent variables at their zero level. Fig. 3a-f and 4a-f represent response
11
surfaces and contour plots for responses, Ytm and Ymy, respectively. The significance level for
the interactions between variables can be depicted from the shape of the corresponding contour
plots. Elliptical contours can be achieved when there is a perfect interaction between independent
variables (Muralidhar et al., 2001). In Table 5, the greatest significant effect for the response, Ytm
was the quadratic term of temperature (x12), ensued by x1x2, x1x3, temperature (x1), x3
2, x22, acid
concentration (x2), reaction time (x3) and x2x3. While, the most significant effect for the response,
Ymy sequenced as: quadratic term of temperature (x12), x1x2, x1x3, temperature (x1), x3
2, reaction
time (x3) and acid concentration (x2). In present study, mannose was the main DPKC-derived
sugar from the hydrolysis.
3.2.1.1. Effect of temperature on total monosaccharide yield (Ytm) and mannose yield (Ymy)
First of all, temperature is a key parameter in determining the sugars recovery and degradation
during acid hydrolysis process. Temperature imparts disruption on the DPKC substrate structure,
the acid dissociation is also depending on the operating temperature (Marshall & Jones, 1966).
At normal temperature, the polysaccharide stays in a stable crystalline form. At high temperature,
the monosaccharide unit in the polysaccharide exists abundantly in open-chain form (less stable)
than the ring form (Nattorp et al., 1999). Thus, it is more susceptible to hydrolysis. As
temperature increases, molecules gain higher kinetic energy that leads to a greater collision rate
between the substrate and hydronium ions, which randomly attack on the glycosidic linkage to
surpass the activation energy barrier, and thereby resulting in the hydrolysis/degradation reaction
to occur. These scenarios can be seen in Figs. 3a and 4a, where both Ytm and Ymy increased when
temperature rose from 140 to 165 °C, but both declined as temperature increased further. A
similar trend can be found in Figs. 3b, 3c and 4b and 4c. Consequently, it is concluded that at
0.25 N H2SO4, the temperature increment (<165 °C) contributes a higher impact on DPKC
12
hydrolysis reaction rate than secondary decomposition rate of the hydrolyzed sugars (Gurgel et
al., 2011). The highest Ytm achieved was 76.98% at 164 °C, 10 min, SS ratio of 1:40, and
concurrently 92.01% for Ymy at 163 °C. By comparing the results obtained (Fig. 3a), it can be
proposed that the DPKC-derived sugars decomposition begins to dominate at temperature higher
than 165 °C. Whereas, at fixed acid concentration 0.75 N H2SO4 and reaction time of 10 min, a
negative effect of temperature (>150 °C) observed on the DPKC-derived sugars. Ytm and Ymy
decreased from 150 °C onwards as shown in Figs. 3a and 4a, owing to the low pKa of H2SO4
with greater hydrolyzing power generating more hydronium ion which further catalyzed the
degradation of sugars into furfural (from C5-sugars) and 5-hydroxymethylfurfural (5-HMF, from
C6-sugar) (Jung et al., 2013; Mosier et al., 2002). In Fig. 3a, for acid concentration of 0.25 N
H2SO4, the steepness of the curve became more gradual in the direction of temperature range
(140-165 °C). Next, further processing of the data by numerical optimization function (Design
Expert Software) showed that Ytm, at temperature range (140-150 °C) was nearly 2.7-fold faster
than the temperature range (150-165 °C). These data suggested that, initially the diffusion rate of
hydrolyzed sugars into the bulk medium is equivalent to the penetration rate of the reacting
species into the DPKC substrate. As described previously, increasing temperature entailed a rise
on the kinetic energy of the reacting species, penetration rate and its collision probabilities with
substrate, thus the hydrolysis reaction occurred at a greater rate. Yet, up to a certain extent, the
increasing concentration of “released” sugars in the bulk medium (near the surface of substrate)
may slow-down the continuous releasing of sugars from the DPKC substrate as well as induced
an additional resistance for 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 on the cellulose surface due to the hydrogen-bonding potential with
13
the structure cellulose surface, and van der Waals attraction forces along with the resistance of
diffusion caused by the charged structural water layer (Torget et al., 2000).
In Table 5 (ANOVA), temperature demonstrated a significant quadratic effect on Ytm and Ymy,
evidently in surface plots (Fig. 3 and 4). This indicated that temperature is the most important
factor in determining the resultant degree of conversion of DPKC into sugars. Also, hydrolysis
shows stronger temperature dependency at acid concentration greater than 0.4 wt. % (Saeman,
1945; Torget et al., 2000). The interaction effect between temperature-acid concentration and
temperature-reaction time was positive (P < 0.001) towards Ytm and Ymy. By comparing the F
value between these two interaction pairs, the temperature-acid concentration was more
significant than the temperature-reaction time pair. By considering the interaction temperature-
acid concentration, it was found that at low temperature (140 °C), the sugars recovery rose with
increasing acid concentration (0.25-0.75 N). However, an inversely effect observed during
hydrolysis at high temperature (175 °C). An identical phenomenon showed by the temperature-
reaction time interaction pair in Figs. 3b and 4b. Apparently, hydrolysis at low temperature
requires a longer reaction time and vice versa in order to obtain a reasonable amount of sugars.
Furthermore, based on Fig. 3c and 4c, the SS ratio factor remains constant with respect to the
temperature factor from 140-180 °C. Therefore, it can be concluded that the interaction effect
between temperature and SS ratio has no significant influence on Ytm and Ymy.
According to the experimental data presented in Table 3, at given operational conditions (tests
21-24), Ytm dropped from average of 69.28% to 51.63% when temperature increased from 140 to
180 °C. In the same way, at given conditions with longer reaction time (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 obtained with further
14
increase in the temperature from 140 to 180 °C (tests 21-24, 29-32). It was recently reported that
the degradation stage is more temperature sentient than hydrolysis stage, evidently from the
relatively higher activation energy for degradation process than the hydrolysis reaction (Kim et
al., 2013). According to (Nattorp et al., 1999), the degradation of mannose had higher activation
energy (140 kJ/mol) than the mannan hydrolysis (113 kJ/mol). Hence, increasing temperature
gives negative effect on maximizing mannose yield (Ymy), because increase in temperature led to
mannose degradation more dominant than hydrolysis of mannan in DPKC.
3.2.1.2. Effect of acid concentration on total monosaccharide yield (Ytm) and mannose yield (Ymy)
Apart from temperature factor, acid concentration also plays a significant role in Ytm and Ymy,
as illustrated in Fig. 3a, 3d, 3e, 4a, 4d and 4e. In present study, the reacting species that catalyze
the hydrolysis were hydronium ion, sulfate and bisulfate anions (Lindstrom & Wirth, 1969).
With progressively higher 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 possibly due to the increased charge perturbation at the
boundary layer. The ionic disturbance caused by the increase hydronium ion concentration
facilitates sugars in “released” state (Torget et al., 2000). It is noted that at elevated temperature
(up to 180 °C), Ytm and Ymy decreased with increasing acid concentration. As depicted in Figs.
3a, 3d, 3e, 4a, 4d and 4e, at the acid concentration greater than its optimum point, it will
introduce an adverse effect on the selectivity of sugars, Ytm and Ymy. In essence, under
conditions of higher acid concentration (Fig. 4d), Ymy increased at the initial phase of reaction
and then decreased gradually with prolonged reaction time. These results could be attributed to
the severe action of acid with longer reaction time; mannose underwent secondary
decomposition to 5-HMF. It should be noted in Fig. 4d, the highest amount of Ytm obtained at
15
160 °C, SS ratio 1:40 with acid concentration lower than 0.5 N H2SO4. Szabolcs and co-
researchers found that at above 0.5 N H2SO4, a higher yield of levulinic acid (simultaneously, the
amount of 5-HMF was lower) was observed and it reached the maximum at 1 N H2SO4 during
the microwave-assisted conversion of carbohydrates. It is well-known that the formation of 5-
HMF proceed from hexose (mannose, glucose and galactose) degradation, therefore this result is
in good agreement with a previous study (Szabolcs et al., 2013), reported that the formation of 5-
HMF (degradation product from hexose) is favorable at acid concentration higher than 0.5 N
H2SO4.
3.2.1.3. Effect of reaction time on total monosaccharide yield (Ytm) and mannose yield (Ymy)
In the point of time factor, at fixed temperature 160 °C with SS ratio 1:40 of and 0.25 N
H2SO4, longer reaction time contributes to a higher sugars recovery (Fig. 3d and Fig. 4d). These
are consistent with other report on the hydrolysis of sweet sorghum bagasse at moderate
temperature (100-121 °C) (Banerji et al., 2013). In contrast, at the same reaction conditions (160
°C, SS ratio 1:40) with higher acid concentration (0.75 N H2SO4), the degradation of the sugars
occurred with prolonged reaction time. Indeed, other study reported that extending the reaction
time at high acid concentration led to the decomposition of decrystallized cellulose and thus
reduced the sugar yield (Chin et al., 2011). Therefore, it can be summarized that the time factor
is dependent to the reaction temperature and acid concentration.
Table 3 (Tests 39 and 40) demonstrates the averages of total monosaccharide yield (Ytm)
16.34%, consisting mono-sugars (glucose, xylose and galactose). Although the experiments were
conducted at high temperature (200 °C), there is no great amount of DPKC-derived sugars
obtained in the hydrolysate, which could be attributed to the lower dissociation of H2SO4 at high
temperature (Lloyd & Wyman, 2004; Maki-Arvela et al., 2011).
16
According to a study reported on the hydrolysis of cellulose, the highest cellulose conversion
using pure water was 70%, which can be achieved at 220 °C and 100 min (Kupiainen et al.,
2012). In the present work, the DPKC hydrolysis took place in the presence of deionized water,
160 °C, 10 min of reaction time and SS ratio 1:40, and yielded trace of xylose monomer 0.4% of
total monosaccharide, (Tests 41 and 42). The possible explanations could be the auto-ionization
of water at elevated temperature, generating hydronium ions (Kim et al., 2013) and leading to the
production of acetic acid from the hemicellulose. These would catalyze partial hydrolysis of
hemicellulose (xylan) to form xylose. It was found that the activation energy for the hydrolysis
of cellubiose (Mosier et al., 2002), mannan (Nattorp et al., 1999) and xylan (Canettieri et al.,
2007) was 110, 113, and 101 kJ/mol, respectively. The lowest energy barrier (activation energy)
for xylan hydrolysis could be the reason for this observation where xylose was the only detected
compound after the hydrolysis (Kim et al., 2013). Additionally, by applying “easy-to-hydrolyze”
and “hard-to-hydrolyze” concept of xylan, it can be postulated that these xylose monomer
released from the “easy-to-hydrolyze” fraction (Lavarack et al., 2002). The purpose of inserting
this parameter (160 °C, 0 N of acid concentration) was to evaluate the effect of the pure water on
the hydrolysis at high temperature. However, according to the Diagnostics function, these
response data fall outside the outlier T area between +3.50 and -3.50, thus it is considered as
outliers. Consequently, these outliers (tests 41 and 42) were then excluded in the CCRD model.
3.2.1.4. Effect of substrate: solvent ratio on total monosaccharide yield (Ytm) and mannose yield
(Ymy)
Higher substrate concentrations means that larger quantity of the raw material can be
processed which is an important aspect for industrial applications. To alleviate this concern, SS
ratio factor was incorporated in the experimental design as well, although the SS ratio was not a
17
significant factor for both responses as the sugars formation are always lower at higher substrate
loading, if other parameters kept constant. Ytm and Ymy of the DPKC hydrolysis were affected
marginally with increased SS ratio at temperature range (140-180 °C) as displayed in Fig. 3d and
4d. From Table 5, it elucidates that during the hydrolysis of DPKC, SS ratio was an insignificant
factor (P > 0.05) for the responses Ytm and Ymy, where same phenomena reported by Yemiş and
Mazza in the hydrolysis wheat straws (Yemiş & Mazza, 2012). As can be seen, the mutual
interactions between SS and other pairs of independent (temperature-SS ratio, acid
concentration-SS ratio and reaction time-SS ratio) were not significant (P > 0.05) as well. In
current work, the instrument limitation on the highest SS ratio at 1:20, it caused restriction in the
chosen range SS ratio (1:20, 1:30, 1:40, 1:50 and 1:60), these intervals were too small to give a
barely noticeable changes on Ytm and Ymy that could be the reason for the insignificant effect of
SS ratio.
From the experimental results inferred that it may not be possible to optimize the reaction
conditions to obtain a maximum yield for all sugars simultaneously. It is noteworthy that,
mannose is the dominant hemicellulose sugar in the DPKC, thereby; achieving the maximum
mannose monomer concentration is preferentially than other monomer sugars. One of the
striking observations obtained in this study is the mannose yield is comparable with those
reported in the literature using mannan degrading enzyme (Cerveró et al., 2010; Zhang et al.,
2009). An important factor could be related to the microwaves interacted with the DPKC at a
molecular level, adsorbed deeply into the folding layers of cellulose to destroy the crystal
structure and enhance the mass transfer (Wu et al., 2010; Yemiş & Mazza, 2012).
3.2.2. Model verification, ridge analysis and optimum reaction conditions
18
Model verification was carried out in triplicate under selected solutions given by Design
Expert software. The confirmation experiments for total monosaccharide yield (Ytm) were
conducted at operating parameters 153 °C, 0.72 N H2SO4, 9 min 42 s and SS ratio of 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%.
From the analysis of the ANOVA data and the statistical parameters, after the removal of the
insignificant terms, the final deduced empirical model in terms of coded factors is shown below:
Ytm = 76.7 - 4.22x1 - 1.69x2 - 1.35x3 - 10.74x1x2 -7.88x1x3 - 1.62x2x3 - 15.27x12 - 2.11x2
2
- 1.91x32 (8)
The second order polynomial model [Eq. (8)] in present study was employed for response
optimization by using Minitab 16. As the center point value greater than the mean value
(58.57%), it can be assumed that the model reached the optimum region. However, the 3D
contour plot showed a saddle curve, therefore ridge analysis on the total monosaccharide was
further conducted (Table 6) to verify the optimum reaction conditions. As the three Eigen values
had different signs, hence it can be deduced that the stationary point for this model did not have a
unique optimum. Therefore, the predicted optimum values for the three key variables were
determined from the results of ridge analysis. Three hydrolysis conditions were selected and the
experiments were carried out based on the calculated actual value of the variables. It was
successfully found that, the optimum conditions for maximum Ytm were 170 °C, 0.181 N H2SO4
and 6 min 6 s. The predicted maximum Ytm was calculated to be 77.67%, and the actual yield of
Ytm obtained was 77.11% with 0.56% deviation from the predicted value. The criteria for the
optimization of the mannose yield (Ymy) by means of the Desirability function based on the
maximization of the mannose content were performed at 148 °C, 0.75 N H2SO4, 10 min 31 s and
19
substrate to solvent (SS) ratio (w/v) of 1:49.69 to corroborate with the predicted value. The
average value of triplicate experiments for mannose yield was 92.11%, whereas the predicted
value was 94.63%.
4. Conclusions
The microwave-assisted hydrolysis of deproteinated palm kernel cake under operating
conditions (170 °C, 0.181 N H2SO4 and SS ratio of 1:40) offered a maximum yield, 77.11% of
total monosaccharide in a reaction time 6 min 6 s. Besides, high yield of mannose, 92.11% was
obtained at 148 °C, 0.75 N H2SO4, 10 min 31 s and SS ratio of 1:49.69. This work demonstrated
that the microwave-assisted process is an effective method for the acid-catalyzed conversion of
DPKC to monosaccharides. DPKC is an economically and environmentally benign source for the
mannose generation as it is a cheap and abundantly available resource.
Acknowledgements
The authors would like to acknowledge the financial support given by University Research Grant
(DIP-2012-34) and ERGS/1/2012/STG01/UKM/03/3. Fan acknowledges the Ministry of Higher
Education (KPT) for the disbursement of MyPhD scholarship.
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24
Figure Captions:
Fig. 1 Schematic representation of microwave and reactor vial.
Fig. 2 Temperature-pressure profiles with respect to time at different reaction temperatures
(120 °C, 140 °C, 160 °C, 180 °C and 200 °C).
Fig. 3 Three-dimensional (3D) response surface plots showing the interaction effects of two
independent variables on total monosaccharide yield (Ytm), while the other two variables
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 temperature and 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
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 at 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).
Fig. 4 Three-dimensional (3D) response surface plots showing the interaction effects of two
independent variables on mannose yield (Ymy), while the other two variables were fixed
at the center point.
25
(a) Effect of temperature and sulfuric acid concentration at fixed reaction time (10 min)
and SS ratio (1: 40).
(b) Effect of temperature and 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
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 at 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).
26
Table Captions:
Table 1 Actual and coded variables at five levels in the CCRD constructed to optimize the
hydrolysis of DPKC by sulfuric acid.
Table 2 Chemical compositions of DPKC (wt. %).
Table 3 Central 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).
Table 4 ANOVA for regression models of total monosaccharide yield (Ytm) and mannose
yield (Ymy).
Table 5 ANOVA and regression coefficient for linear, quadratic and interactive terms of
total monosaccharide yield (Ytm) and mannose yield (Ymy).
Table 6 Ridge analysis on the total monosaccharide yield (Ytm).
27
Table 1 Actual 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 120 140 160 180 200
Acid concentration (N) x2 0 0.25 0.5 0.75 1.0
Reaction time (min) x3 0 5 10 15 20
Substrate : solvent ratio (g/ml) x4 1:20 1:30 1:40 1:50 1:60
28
Table 2 Chemical compositions of DPKC (wt. %).
Components :
Protein 6.70 ± 0
Lignin
Acid-Insoluble Lignin 5.67 ± 0.42
Acid-Soluble Lignin 2.45 ± 0.35
Extractives
Water-Soluble 4.13 ± 0.18
Ethanol-Soluble 3.54 ± 0.08
Ash 3.50 ± 0.08
Monosaccharides after hydrolysis*
Glucose 13.66 ± 0.78
Mannose 55.71 ± 0.68
Xylose 1.84 ± 0.14
Arabinose 1.00 ± 0.24
Galactose 1.00 ± 0.06
* Measured by NREL analytical methods (Sluiter et al., 2008a; Sluiter et al., 2008b).
29
Table 3 Central 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 x2 x3 x4 Man Glu Xyl Gal Ytm Ymy
(°C) (N) (min) (g/ml)
1 ‒1 ‒1 ‒1 ‒1 0.3189 0.0021 0.0143 0.0137 47.76 57.25
2 ‒1 ‒1 ‒1 ‒1 0.3054 0.0018 0.0146 0.0131 45.75 54.83
46.76 ± 1.42 a 56.04 ± 1.71 b
3 1 ‒1 ‒1 ‒1 0.4802 0.0175 0.0170 0.0153 72.42 86.20
4 1 ‒1 ‒1 ‒1 0.4928 0.0187 0.0187 0.0152 74.51 88.46
73.47 ± 1.48 a 87.33 ± 1.60 b
5 ‒1 1 ‒1 ‒1 0.4287 0.0198 0.0152 0.0070 64.31 76.95
6 ‒1 1 ‒1 ‒1 0.4484 0.1922 0.0181 0.0096 67.67 80.49
65.99 ± 2.38 a 78.72 ± 2.50 b
7 1 1 ‒1 ‒1 0.2826 0.0723 0.0033 0.0017 49.17 50.73
8 1 1 ‒1 ‒1 0.2903 0.0423 0.0061 0.0024 46.59 52.11
30
47.88 ± 1.82 a 51.42 ± 0.98 b
9 ‒1 ‒1 1 ‒1 0.3927 0.0057 0.0179 0.0170 59.19 70.49
10 ‒1 ‒1 1 ‒1 0.3896 0.0060 0.0151 0.0152 58.19 69.94
58.69 ± 0.71 a 70.22 ± 0.39 b
11 1 ‒1 1 ‒1 0.3450 0.0415 0.0260 0.0107 57.80 61.93
12 1 ‒1 1 ‒1 0.3328 0.0335 0.0262 0.0076 54.66 59.74
56.23 ± 2.22 a 60.84 ± 1.55 b
13 ‒1 1 1 ‒1 0.5087 0.0188 0.0169 0.0149 76.41 91.32
14 ‒1 1 1 ‒1 0.5224 0.0137 0.0206 0.0097 77.38 93.77
76.90 ± 0.69 a 92.55 ± 1.73 b
15 1 1 1 ‒1 0.0997 0.0652 0.0103 0 23.94 17.90
16 1 1 1 ‒1 0.0946 0.0507 0 0 19.85 16.98
21.90 ± 2.89 a 17.44 ± 0.65 b
17 ‒1 ‒1 ‒1 1 0.3243 0.0019 0.0142 0.0138 48.40 58.22
18 ‒1 ‒1 ‒1 1 0.3101 0.0014 0.0137 0.0116 46.02 55.67
47.21 ± 1.68 a 56.95 ± 1.80 b
31
19 1 ‒1 ‒1 1 0.4316 0.0369 0.0131 0.0153 67.88 77.47
20 1 ‒1 ‒1 1 0.4423 0.0272 0.0157 0.0138 68.16 79.39
68.02 ± 0.20 a 78.43 ± 1.36 b
21 ‒1 1 ‒1 1 0.4774 0.0068 0.0211 0.0083 70.17 85.70
22 ‒1 1 ‒1 1 0.4660 0.0104 0.0166 0.0076 68.38 83.65
69.28 ± 1.27 a 84.68 ± 1.45 b
23 1 1 ‒1 1 0.3182 0.0569 0.0039 0.0009 51.90 57.12
24 1 1 ‒1 1 0.3149 0.0573 0.0023 0.0013 51.35 56.53
51.63 ± 0.39 a 56.83 ± 0.42 b
25 ‒1 ‒1 1 1 0.4318 0.0049 0.0190 0.0150 64.30 77.51
26 ‒1 ‒1 1 1 0.4205 0.0047 0.0177 0.0140 62.43 75.49
63.37 ± 1.32 a 76.50 ± 1.43 b
27 1 ‒1 1 1 0.3195 0.0464 0.0145 0.0095 53.26 57.36
28 1 ‒1 1 1 0.3113 0.0505 0.0146 0.0085 52.57 55.88
52.92 ± 0.49 a 56.62 ± 1.05 b
29 ‒1 1 1 1 0.5193 0.0106 0.0199 0.0060 75.91 93.22
32
30 ‒1 1 1 1 0.5269 0.0075 0.0198 0.0048 76.37 94.59
76.14 ± 0.33 a 93.91 ± 0.97 b
31 1 1 1 1 0.1434 0.0768 0.0018 0 30.32 25.74
32 1 1 1 1 0.1350 0.0758 0.0020 0 29.08 24.23
29.70 ± 0.88 a 24.99 ± 1.07 b
33 0 0 0 0 0.5054 0.0185 0.0188 0.0165 76.40 90.73
34 0 0 0 0 0.5033 0.0186 0.0182 0.0167 76.07 90.34
76.24 ± 0.23 a 90.54 ± 0.28 b
35 0 0 0 0 0.5081 0.0182 0.0185 0.0154 76.52 91.21
36 0 0 0 0 0.5114 0.0189 0.0185 0.0162 77.19 91.81
76.86 ± 0.47 a 91.51 ± 0.42 b
37 ‒2 0 0 0 0.0691 0.0058 0.0307 0 14.43 12.40
38 ‒2 0 0 0 0.0713 0.0080 0.0449 0 16.97 12.79
15.70 ± 1.80 a 12.60 ± 0.28 b
39 2 0 0 0 0 0.032 0.0206 0.0691 16.62 0.00
40 2 0 0 0 0 0.023 0.0171 0.0774 16.05 0.00
33
16.34 ± 0.40 a 0 b
41* 0 ‒2 0 0 0 0 0.0036 0 0.49 0.00
42* 0 ‒2 0 0 0 0 0.0026 0 0.31 0.00
0.4 ± 0.13 a 0 b
43 0 2 0 0 0.4351 0.0299 0.0140 0.0045 66.05 78.10
44 0 2 0 0 0.4231 0.0353 0.0099 0.0044 64.57 75.94
65.31 ± 1.05 a 77.02 ± 1.53 b
45 0 0 ‒2 0 0.4335 0.0028 0.0726 0.0052 70.22 77.82
46 0 0 ‒2 0 0.4243 0.0022 0.0646 0.0042 67.67 76.17
68.95 ± 1.8 a 77.00 ± 1.17 b
47 0 0 2 0 0.4448 0.0429 0.0142 0.0148 70.58 79.84
48 0 0 2 0 0.4315 0.0544 0.0091 0.0130 69.40 77.47
69.99 ± 0.83 a 78.66 ± 1.68 b
49 0 0 0 ‒2 0.4827 0.0302 0.0224 0.0117 74.72 86.65
50 0 0 0 ‒2 0.4898 0.0172 0.0220 0.0111 73.77 87.92
74.25 ± 0.67 a 87.29 ± 0.90 b
34
51 0 0 0 2 0.4894 0.0156 0.0559 0.0034 77.11 87.86
52 0 0 0 2 0.4979 0.0110 0.0430 0.0047 76.03 89.38
76.57 ± 0.76 a 88.62 ± 1.07 b
53 0 0 0 0 0.5094 0.0196 0.0185 0.0171 77.13 91.44
54 0 0 0 0 0.5063 0.0189 0.0177 0.0151 76.23 90.89
76.68 ± 0.64 a 91.17 ± 0.39 b
* 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).
35
Table 4 ANOVA 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 18.33
Pure Error 45.46 27 1.68
R2adj 0.9505
R2pred 0.9227
Mannose yield (Ymy)
Model 34479.65 14 2462.83 89.23 < 0.0001*
Residual 993.59 36 27.60
Pure Error 41.11 27 1.52
R2adj 0.9611
R2pred 0.9392
* Significant values.
36
Table 5 ANOVA and regression coefficient for linear, quadratic and interactive terms of
total monosaccharide yield (Ytm) and mannose yield (Ymy).
Source Regression coefficient F-value p-Value
Total monosaccharide yield (Ytm)
Intercept 76.7
Linear
x1 (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 0.3153
Quadratic
x12 - 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
Interaction
x1x2 - 10.74 201.36 < 0.0001*
x1x3 - 7.88 108.47 < 0.0001*
x1x4 - 0.3 0.16 0.6899
x2x3 - 1.62 4.57 0.0394*
x2x4 1.11 2.14 0.1521
37
x3x4 0.4 0.28 0.6023
Mannose yield (Ymy)
Intercept 90.84
Linear
x1 (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 0.3560
Quadratic
x12 - 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
Interaction
x1x2 - 13.92 224.62 < 0.0001*
x1x3 - 10.68 132.29 < 0.0001*
x1x4 - 0.92 0.97 0.3303
x2x3 - 1.76 3.60 0.0657
x2x4 1.64 3.11 0.0865
x3x4 0.47 0.26 0.6124
* Significant variables.
39
Table 6 Ridge analysis on the total monosaccharide yield (Ytm).
λ
Radii Eigen value Predicted yield (%) Actual value Actual yield (%)
x1 x2 x3
ŷ
x1 x2 x3
ŷ
0.300 0.7169 0.1627 ‒0.5851 ‒0.3810 77.26 163.2539 0.3537 8.0948 66.35
0.185 1.5799 0.5061 ‒1.2771 ‒0.7803 77.70 170.1227 0.1807 6.0985 77.11
0.165 2.0090 0.6763 ‒1.6198 ‒0.9771 77.97 173.5253 0.0950 5.1146 10.20
40
Figure 1
41
Figure 2
42
Figure 3(a)
Figure 3(b)
43
Figure 3(c)
Figure 3(d)
44
Figure 3(e)
Figure 3(f)
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Figure 4(a)
Figure 4(b)
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Figure 4(c)
Figure 4(d)
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Figure 4(e)
Figure 4(f)
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Graphical abstract: Microwave-assisted hydrolysis via dilute sulfuric acid is an effective method for the conversion of DPKC to fermentable sugars which potentially to be further transform into biofuels.
BiofuelsBiofuels
Fermentable sugars
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Highlights:
Response Surface Methodology as optimization strategy for DPKC‐derived sugars. Statistically optimized on total monosaccharide, 77.11% and mannose yield, 92.11%. Ridge analysis was further conducted to verify the optimization parameters. Established an effective microwave‐assisted hydrolysis on DPKC to sugars.