EFFECTIVITY OF MICROWAVE PRETREATMENT ON ENZYMATIC AND
MICROWAVE HYDROLYSIS OF BETUNG BAMBOO (Dendrocalamus asper)
Widya Fatriasari1,2*, Wasrin Syafii2, Nyoman Wistara2,
Khaswar Syamsu3 and Bambang Prasetya4
1.R&D Unit for Biomaterials, Indonesian Institute of Sciences (LIPI), Cibinong, Bogor
16911, Indonesia
2. Department of Forest Product Technology, Faculty of Forestry, Bogor Agricultural
University, Indonesia
3.Department of Agroindustrial Technology, Faculty of Agricultural Engineering and
Technology, Bogor Agricultural University, Indonesia
4.National Standardization Agency, Manggala Wanabakti Building, Senayan, Jakarta,
Indonesia
*
E-mail: [email protected]
Abstract
Assessing evaluation the effect of microwave pretreatment on chemical structural and
morphological changes of Betung Bamboo was performed before. In this present study, we
foccused on how the changes in the best microwave pretreatment affacted the performance of
enzymatic and microwave hydrolysis of pretreated bamboo. This solid fraction was subjected
to enzymatic hydrolysis following NREL protocol and microwave acid-hydrolysis
with/without presence of activated carbon (0.5 g/g) at 1 and 5% of sulfuric acid concentration
for 5-12.5 minutes at 330 watt. Short duration microwave-acid hydrolysis of microwave
pretreated bamboo showed better reducing sugar yield than that of enzymatic
ones.Microwave heating for 12.5 m at 330 W hydrolyzed with 1% acid using microwave
irradiation demonstrated a high reducing sugar yield (25.81% of dry biomass or 27.12% of
dry substrate). In this treatment, 37.92% of hollocellulose can be converted into reducing
sugar yield or equivalent with 36.12% of maximum potential sugars released. This yield
increased 7.9 fold compared to reducing sugar yield from enzymatic hydrolysis using 20
FPU/g of cellulase enzymes. Reducing sugar yield obtained by 5% microwave acid
hydrolysis of microwave pretreatment for 5 m at 770 W was equal with the result of
hydrolysis for 12.5 m at 330 W. Unfortunately, the activated carbon as solid catalyst in
microwave hydrolysis only affected on reducing brown compound without reducing sugar
yield improvement.
Keywords: bamboo, microwave pretreatment, enzymatic and microwave acid hydrolysis,
reducing sugar yield, brown compound
INTRODUCTION
As lignocellulosic materials, cellulose of bamboo has been covered by lignin and
hemicellulose which can disrupt biomass conversion for bioethanol production. In this
process, reducing recalcitrance factors such as lignin content, crystalline structure of
cellulose, particle size, degree of polymerization, available surface area etc limited enzyme
deconstruction is the emphasis of pretreatment of lignocellulosic materials. An efficient
pretreatment can promote maximizing the enzymatic hydrolysis efficiency [1] without
generate the secondary degradation product which may seriously inhibit subsequent
fermentation. Different pretreatment types exist, but now microwave pretreatment (individual
or combined with other pretreatments) is becoming promising method biomass pretreatment
for bioethanol production due to its better results. Microwave pretreatment can be categorized
as physical pretreatment which give main effect on the increasing the surface area and pore
sizes. Furthermore, the other effect is to soften and partially depolymerize lignin and disrupts
the lignocellulosic cells [2]. Comparing to the conventional heating, microwave irradiation
supplies direct contact of heating source and material, easy operation, rapid and selective
heating of polar part and creating hot spot with inhomogeneous materials [3-5].
Study of microwave pretreatment using any chemicals on lignocellulosic materials
has been reported previously. Azuma et al. [6] reported that total sugar obtained of pretreated
hardwood after enzymatic hydrolysis was as high as 88-93% using water as chemicals.
Enhancing enzymatic digestibility of rice straw pretreated by combined microwave-sulfuric
acid was also demonstrated by Sigh et al.[7], in which the maximum reducing sugar obtained
was 1376.99 µg/ml. Microwave irradiation assisted alkali treatment of switchgrass in optimal
condition of 1900C, 50 g/L solid content for 30 min produced sugar yield of 58.7 g/100 g
biomass, equivalent to 99% of maximum potential sugars released [4]. Microwave treatment
of sugarcane bagasse with 1% NaOH at 600 W for 4 min followed by enzymatic hydrolysis
gave reducing sugar yield of 0.665 g/g dry biomass, while combined microwave-alkali-acid
treatment with 1% NaOH followed by 1% sulfuric acid, the reducing sugar yield increased to
0.83 g/g dry biomass [1]. Utilization dilute nitric acid of 2% in microwave heating has been
reported by Ravoof et al. [8], whereas the maximum yield of reducing sugars on the
enzymatic hydrolysis for 108 h was almost 60%. Furthermore, The pretreatment of corn
stover using combination of steam explosion and microwave irradiation produced the
maximum sugar yield was 72.1 g/100 g dry biomass, achieved at 540 W microwave power
for 5 min [9]. Kheswani [10] performed microwave-assisted sodium hydroxide as
pretreatment on switch grass and coastal bermuda grass. Under optimum pretreatment, 82%
glucose and 63% xylose yield were achieved for switch grass 87% glucose and 59% xylose
yield were obtained for bermuda grass following enzymatic hydrolysis of the pretreated
biomass. As alternative heating, published research on microwave irradiation proved
improving the saccarification efficiency and reducing sugar yield in enzymatic hydrolysis of
lignocellulosic materials.
Microwave irradiation can be utilized both in pretreatment and hydrolysis,
considering the advantages over conventional heating of dilute acid-catalyzed hydrolysis.
This acid hydrolysis has widely employed for saccarification of lignocellulosic [11, 12] and
starch [13-15] for the bioethanol or oligosaccaride production, in addition enzymatic method
[11]. Furthermore, this technique was possible to develop for shortening the residence time
and also accelerating the potential sugar yield formation. This study was run in parallel with
the study of structural changes after microwave pretreatment of bamboo in water medium. In
that parallel study, which indicated better delignification selectivity of pretreated samples was
founded at 330 W for 5,10 and 12.5 m and 770 W for 5 m than that of 550 W and 770 W for
7.5,10,12.5 m. Therefore, this study was to attempt for improving the enzymatic hydrolysis
performance over to microwave-assisted acid hydrolysis.
Solid catalyst-assisted microwave irradiation is interisting to apply due to non-toxic in
waste liquid [13, 16], easily recovered [16], and improving glucose yield in water medium
[17] as absorber and solid catalys materials. Utilization activated carbon as microwave
absorber, microwave sensitizer for pyrolysis and starch saccarification, degradation of
hazardous compounds in dry state and fermentation stage, enhancing reaction catalyzed has
been reported previously [13, 18, 19]. During the acid hydrolysis, several potential
byproducts such as acetic acid, formic acid, furan derivatives (5-hydroxymethylfurfural
(HMF) and furfural), and phenolic compounds etc inhibiting subsequent fermentation might
be produced. Thus, to lowering the secondary degradation effect can be added activated
carbon in acid hydrolysis process. Hence, this study also discussed the impact of activated
carbon addition in acid catalyzed-hydrolysis.
MATERIALS AND METHODS
Feed Stock
Two year-old betung bamboo culms harvested from bamboo garden of the R & D
Unit for Biomaterials, Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia was
utilized as material. They were barked, chipped, dried, and then grounded to obtain bamboo
powder of 40-60 mesh size. The milled samples were stored at room temperature and the
chemical analysis carried out TAPPI method described in Table 1.
Table 1. Chemical component of bamboo
Chemical composition
Percentage (%)
Ash content
1.74±0.07
et-ben extractive
3.49±1.11
Klason lignin
25.38±0.36
Holocellulose
65.48±3.39
Alpha cellulose
44.77±0.16
Hemicellulose
18.71±0.17
Microwave Pretreatment
As much as 1 g of bamboo powder which previously had been measured the moisture
content and chemical composition and then exposuring to microwave irradiation in water
medium. Domestic microwave oven SHARP P-360J (S) with the operating frequency of 2450
Hz of frequency was used in this pretreatment. This pretreatment was carried out under its
best condition. Previously, this sample was inserted into the teflon tube (vessel), then added
distilled water to obtain a solid-to-liquid ratio (SLR) 1:30 (w/v) and then was stirred for 15
minutes. Subsequently, the materials were transferred and irradiated for irradiation time 5, 10
and 12.5 minutes at 330 watt and 5 minutes at 770 watt. After microwaving finished, the pulp
removed and immediately ice water cooled for 15-20 minutes and then was filtered to
separate solid residue out.
Enzymatic Hydrolysis
Pretreated feedstock pulp (0.1 g dry weight) was mixed with 0.05 M sodium citric
buffer to adjust pH of slurry to 5 and then cellulase enzyme (meicellase with enzyme activity
of 200 FPU/g) of 10 and 20 FPU/g pretreated bamboo was added in the 20 ml of vial bottle
glass. One FPU was defined as the enzyme amount capable to produce 1µmol of reducing
sugar per minute [20].To prevent microorganism contamination, sodium azide 2% (b/v) was
also added in the slurry and subsequently, was incubated at 50oC for 72 h in shaking
incubator set in 150 rpm. This method was performed referred to National Renewable Energy
Laboratory (NREL) protocol [21].
Microwave –Assisted Acid Hydrolysis
Solid fraction (0.1 g oven dry weight or 1% w/w of total weight) of microwave
pretreated samples were also subjected to microwave acid catalyzed-hydrolysis. The same
microwave equipment used in microwave pretreatment was also utilized in this hydrolysis.
The solid fraction in the vessel was added with 1 and 2.5% sulfuric acid solution to 10 g of
the slurry final weight and subsequently was exposed to microwave irradiation for 5-12.5 m
at 330 W. The same method and acid hydrolysis condition was also conducted in microwave
hydrolysis with presence of activated carbon as much as 0.5g of solid sample. This activated
carbon was generated for 120 m at 800oC and then the properties were measured following
SNI 06-4253-1996. The cooling reaction of slurry was accelerated by ice water cooling of
samples after microwave exposure, and subsequently, were filtered to separate hydrolysates
from slurry to determine the reducing sugar yield based on dry biomass (Eq.1) and dry
substrate (Eq.2) referred to the Nelson-Somogyi methods [22] and brown compound
measured by UV VIS Hitachi U-2001 spectrophotometer at 490 nm [14]. Furthermore, the
hydrolysis rate was evaluated as the reducing sugar yield into holocellulose content [23] with
weight loss as reducing factor. Furthermore, pH value of hydrolyzate was measured too with
Eutech pH meter.
All experiments were carried out triplicate, and the given numbers are the average
values. The calculation of theoritical reducing sugar yield (Eq. 3) was only performed in the
highest reducing sugar yield.
Reducing sugar yield (% dry biomass)=
Total reducing sugar (g)
Dry biomass (g)
x 100
Total reducing sugar (g)
Reducing sugar yield (% dry substrate) = Dry hydrolysis substrate (g) x 100
Reducing sugar yield (g)
Theoritical reducing sugar yield (%) = Carbohydrate of initial bamboo (g) x 1.11 x 100
(1)
(2)
(3)
1.11=Conversion factor holocellulose to reducing sugar
RESULTS AND DISCUSSIONS
The comparison of reducing sugar yield of enzymatic and microwave hydrolysis
Even though, this enzymatic hydrolysis is more time consumption, this process is well
known using to hydrolyze pretreated lignocellulosic materials due to mild condition without
generated corrosive effect in equipment. However, in general reducing sugar yield produced
in this method tended to be low. As presented in Fig 1a, the highest reducing sugar yield was
obtained in microwave irradiation for 5 minutes at 770 W (4.24% per dry biomass or 4.32%
per dry substrate). Holocellulose which can be converted into reducing sugar was about
4.23% or 5.98% of the theoretical reducing sugar yield of initial biomass. This yield in this
present study was higher than that of the yield of biological pretreatment (2.69% per dry
biomass) reported in previous study [24]. It is indicated that enzymatic hydrolysis from
bamboo pretreated by microwave pretreatment in water medium is better effect slightly than
Reducing sugar yield
biological pretreatment is.
5.0
A
4.0
3.0
% dry substrate
% dry biomass
2.0
1.0
0.0
10 20 10 20 10 20 10 20 10 20
FPU FPU FPU FPU FPU FPU FPU FPU FPU FPU
5
Control
10
330 watt
12,5
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
B
% hydrolysis
ratio
10 20 10 20 10 20 10 20 10 20
FPU FPU FPU FPU FPU FPU FPU FPU FPU FPU
5
770 watt
5
Control
Irradiation time (minutes)
10
330 watt
12,5
% teoritical
reducing sugar
yield
5
770 watt
Irradiation time (minutes)
Fig.1. Reducing sugar yield of microwave-pretreated bamboo after enzymatic hydrolysis (A),
hydrolysis ratio and theoretical reducing sugar yield (B)
Compared to control, reducing sugar yield of microwave pretreatment (except for 5 m
at 330 W) in enzymatic hydrolysis was higher and a long with the increasing of microwave
irradiation in pretreatment tended to improve the yield. The direct contact between whole
material and heating source from microwave irradiation simultaneously generates selective
absorption of the radiation by polar molecule of water and then creating hot spot with
inhomogeneous materials [5, 8]. Microwave pretreatment caused the cellulose of pretreated
samples had more opened structure thus easily hydrolyzed by cellulase. Additionally, this
pretreatment affected for increasing of surface area, pore size, and partially lignin
depolymerization [2] after microwave heating. Only the microwave pretreatment for 5 m at
770 W showing the lignin removal, meanwhile the other pretreatments was since in versa
(unpublished data). More severe pretreatment condition demonstrated higher reducing sugar
yield and the increasing of enzyme loading improved the reducing sugar yield.
The positive effect of this microwave-acid hydrolysis has been reported in our parallel
study (saccarification of bamboo pretreated by biological pretreatment). The microwave-acid
hydrolysis can enhanced 6.3 fold of reducing sugar yield compared its enzymatic hydrolysis
did [24]. Considering this result, this study was attempted to enhance the reducing sugar yield
of microwave pretreated samples. Additionally, the pretreatment effectivity between
biological and microwave method can be compared. For hydrolysis process, sulfuric acid was
commonly utilized as a catalys [25]. The previous study [25, 26] reported that the optimal
sulfuric acid concentration to hydrolyze lignocellulosic materials was 1-6%, thus this present
study used the acid concentration in this range. The reducing sugar yield based on dry
biomass based on dry substrate with/without activated carbon addition was summarized in
Fig.2 and 3. There was greatly increasing in reducing sugar yield of pretreated samples
compared to control. It can suggested that microwave pretreatment was effective method for
Reducing sugar yield
(% dry biomass)
30
25
20
Control
15
5 m, 330 W
10
10 m, 330 W
5
12,5 m, 330 W
0
5 7,5 10 12,5 5 7,5 10 12,5
H2SO4 1%
Redducing sugar yield
(% biomass)
improving digestibility of substrate.
20
Control
15
5 m, 330 W
10
10 m, 330 W
5
12,5 m, 330
W
5 m, 770 W
0
5 m, 770 W
5 7,5 10 12,5 5 7,5 10 12,5
H2SO4 5%
H2SO4 1%
H2SO4 5%
Irradiation time (m)
Irradiation time (m)
Fig.2. Reducing sugar yield based on dry biomass without activated carbon (A), with
activated carbon (B)
The irradiation time extended in microwave-acid hydrolysis tended to increase
reducing sugar yield. Irradiation time for 5 and 7.5 m only produced low reducing sugar yield
(below to 5%); while the significant improvement in reducing sugar yield was reached in 10
and 12.5 m of irradiation time. Bamboo, pretreated with microwave heating for 12.5 m at
330 W, exhibited the highest reducing sugar yield (25.81% of dry biomass or 27.12% of dry
substrate) in 1% of microwave-acid hydrolysis. Hollocellulose which can be converted into
reducing sugar was as much as 37.92% or 36.12% of the theoretical reducing sugar yield of
initial biomass. This data proved that microwave acid-hydrolysis can improve the reducing
sugar yield of pretreated biomass and this yield increase 7.9 fold compared to the highest
reducing sugar yield from enzymatic hydrolysis using 20 FPU/g cellulase enzymes. This
yield was almost similar with the yield of 5% acid hydrolysis on microwave pretreatment for
Reducing sugar yield (% dry
substrate)
30
25
5 m, 330 W
20
10 m, 330
W
12,5 m, 330
W
5 m, 770 W
15
10
5
0
5 7,5 10 12,5 5 7,5 10 12,5
H2SO4 1%
H2SO4 5%
Irradiation time (m)
Reducing sugar yield (%
substrate)
10 m (24.82% of dry biomass or 25.56% of dry substrate).
20
5 m, 330 W
15
10 m, 330 W
10
12,5 m, 330
W
5 m, 770 W
5
0
5 7,5 10 12,5 5 7,5 10 12,5
H2SO4 1%
H2SO4 5%
Irradiation time (m)
Fig.3. Reducing sugar yield based on dry substrate without activated carbon (A), without
activated carbon (B)
The increasing of acid concentration can increase the reducing sugar yield, whereas
the microwave pretreatment for 5 m at 770 W produced the highest yield (26.27% of dry
biomass or 26.78% of dry biomass) in microwave acid-hydrolysis for 12.5 m. This
pretreatment can convert 37.45% of hollocellulose into reducing sugar or equivalent to
36.78% of maximum potential sugars released. It indicated that the acid concentration
increasing has only affected slightly on reducing sugar yield. Therefore, it was suggested to
use 1% of acid concentration than that of 5%, considering the production cost and
environmental effect. The highest reducing yield reached on microwave pretreatment using
1% acid hydrolysis was 1.5 times and 2.4 higher than that of biological pretreatment using
T.versicolor [24] and P.crysosporium [27]. The increasing of acid concentration exhibited
different effect between microwave and biological pretreatment in microwave hydrolysis.
The present study, structural changes of pretreated bamboo facilitated for improving
hydrolysis performance of microwave acid-hydrolysis, inwhich this effect did not happened
in biological pretreated bamboo. It is suggested that increasing the surface area and pore
sizes, softening and partially lignin depolymerization and lignocellulosic seal disruption [2]
was more effective impact than removal of lignin and hemicellulose under biological
pretreatment [28].
Activated carbon application in the microwave acid-hydrolysis tended to decrease
reducing sugar yield. This result was in line with the microwave-acid hydrolysis of bamboo
pretreated by biological pretreatment [18, 28]. This phenomenon was related with adsorption
of malto oligomer in the surface of activated carbon, thus this part cannot readily to be
hydrolyzed [18]. Furhermore, the activated carbon with low adsorptive capacity of maltose
exhibited high saccarification rate and can decreased the saccarification temperature by 10300C, while those with high asorptive capacity was since in versa. The saccarification of
starch has a clear inverse relationship with degree of maltose adsorptive capasity in liquid
phase [13]. Furthermore, the use of activated carbon has been proved could increase glucose
yield from microwave-assisted hydrolysis of cassava pulp in water medium, but not in acid
medium [17].
The effect of activated carbon on brown compound and pH value
To obtain the hydrolysates with low concentration of inhibitor if the hydrolysates will
be used as fermentation media is important [25]. Therefore, detoxification of acid
hydrolysates was required for removing the potential toxic compounds in fermentation stage.
Amongst the various techniques to reduce them, activated carbon was reported lowering in
furan derivatives, phenolics and acetic acid as much as 38.7% and 57%, and 68.8%
respectively [29]. Utilization of activated carbon treated acid hydrolysate of corn hull gave
92.3% reduction in total phenolic compounds and removal of
dark brown color in
hydrolyzate [30]. Furan derivatives caused reducing in membrane permeability resulting in
longer cell growth [31] and ethanol productivities [32]. In addition, the furan derivatives level
of 500 mg/l began inhibiting bioethanol production [31].
Brown compound
1.00
0.80
Control
0.60
5 m, 330 W
0.40
10 m, 330 W
0.20
12,5 m, 330 W
0.00
5 m, 770 W
5 7,5 10 12,5 5 7,5 10 12,5
H2SO4 1%
H2SO4 5%
Irradiation time (m)
Brown Compound
1.20
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
Control
5 m, 330 W
10 m, 330 W
12,5 m, 330 W
5 m, 770 W
5 7,5 10 12,5 5 7,5 10 12,5
H2SO4 1%
H2SO4 5%
Irradiation time (m)
Fig.4. Brown compound formed during microwave assisted-acid hydrolysis without activated
carbon (A) and with activated carbon (B)
Brown compound is included in furan derivatives formed during microwave-acid
hydrolysis with or without activated carbon utilization demonstrated in Fig.4. Furfural and
HMF are an intermediate product in the Maillard reaction with high absorbance at a
wavelength of UV [18]. Comparing with control, bamboo microwave treated produced
lowering in brown compound. It means that the microwave pretreatment give positive effect
on brown compound reduction. Additionally, the increasing of irradiation time in microwave
pretreatment until 10 minutes led to decrease the brown compound. However, a longer
irradiation exposure until 10 m in 1% microwave acid-hydrolysis affected on increasing the
brown compound. The activated carbon effect in microwave acid hydrolysis was able to
reduce it (Fig.4b). This data strengthen the previous study which reported the positive impact
on activated carbon for reducing toxic substances in hydrolysate such as dark brown color. At
the condition (12.5 m at 330W) which produced the highest reducing sugar yield, even
though the brown compound tended to lower succesfully, however the reducing sugar yield
did not improve. It might be related with the possibility formed the other degradation like 5HMF, acetic acid etc.
1.20
1.00
Control
5 m, 330 W
5 7,5 10 12,5 5 7,5 10 12,5
H2SO4 1%
H2SO4 5%
Irradiation time (m)
pH
pH
1.40
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
0.80
Control
0.60
5 m, 330 W
10 m, 330 W
0.40
10 m, 330 W
12,5 m, 330 W
0.20
12,5 m, 330 W
5 m, 770 W
0.00
5 m, 770 W
5
7,5 10 12,5 5
7,5 10 12,5
H2SO4 1%
H2SO4 5%
Irradiation time (m)
Fig.5. pH formed during microwave-acid hydrolysis without activated carbon (A), and with
activated carbon (B)
During microwave acid hydrolysis, the pH of hydrolyzate reduced and there was
decreasing pH of hydrolyzate after bamboo irradiated by microwave (Fig.5). The increasing
of microwave irradiation and acid loading of control in hydrolysis process tended to reduce
the pH of hydrolyzate. It might be related with the possibility decomposition process during
hydrolysis process to form organic acid as carbohydrate degradation. The similar result was
reported by Hermiati [18], and Khan et al.[15]. Furthermore, the formation of formic, acetic,
propionic, isobutyric, isovaleric, valeric isoproic, and caproic acid during heating due to air
oxidation of aldehyde compound has been reported [33]. However, there was slight changes
in pH value along with increasing of irradiation time and activated carbon addition increased
pH value of hydrolyzate which might related with inhibition effect in byproduct formation in
acid hydrolysis.
CONCLUSIONS
A great improvement in reducing sugar yield of microwave-acid hydrolysis was over
enzymatic hydrolysis of microwave pretreated of bamboo. Furthermore, microwave
pretreatment was more success for enhancing the reducing sugar yield in enzymatic and acid
hydrolysis compared to the biological pretreatment did. Microwave hydrolysis for 10 m was
enough time to reach high reducing sugar yield. The increasing of acid concentration was
slight effect for enhancing reducing sugar yield. Even though activated carbon can lower the
brown compound of the hydrolyzate indentified as fermentation inhibitor, but there was no
improvement in reducing sugar yield. It was related with absorption effect of maltooligomer
by activated carbon was more dominant than that of accelerating the reducing sugar yield.
With considering the environmental impact and the reducing sugar yield, therefore 1% acid
concentration was better to choose for hydrolyze microwave pretreated bamboo.
ACKNOWLEDGEMENT
The work was financially supported by Ministry of Research and Technology (RISTEK) via
PhD scholarship. We like to thank Muhammad Adly R.Lubis,S.Hut, and Fajriana Mareta for
technical assistance.
REFERENCES
[1] Binod, P, K Satyanagalakshmi, R Sindhu, KU Janu, RK Sukumaran, and A Pandey.
2012.Short duration microwave assisted pretreatment enhances the enzymatic
saccharification and fermentable sugar yield from sugarcane bagasse. Renewable Energy.
37(1): 109-116.
[2] Conde-Mejía, C, A Jiménez-Gutiérrez, and M El-Halwagi. 2012.A comparison of
pretreatment methods for bioethanol production from lignocellulosic materials. Process
Safety and Environmental Protection. 90(3): 189-202.
[3] Chen, W-H, Y-J Tu, and H-K Sheen. 2011.Disruption of sugarcane bagasse
lignocellulosic structure by means of dilute sulfuric acid pretreatment with microwaveassisted heating. Applied Energy. 88(8): 2726-2734.
[4] Hu, Z and Z Wen. 2008.Enhancing enzymatic digestibility of switchgrass by microwaveassisted alkali pretreatment. Biochemical Engineering Journal. 38(3): 369-378.
[5] Zhang, Z, Y Shan, J Wang, H Ling, S Zang, W Gao, Z Zhao, and H Zhang.
2007.Investigation on the rapid degradation of congo red catalyzed by activated carbon
powder under microwave irradiation. Journal of Hazardous Materials. 147(1–2): 325333.
[6] Azuma, J-i, JH No, M Isaka, and T Koshijima. 1985.Microwave Irradiation of
Lignocellulosic Materials : IV. Enhancement of Enzymatic Susceptibility of Microwaveirradiated Softwoods. Wood research. 71: 13-24.
[7] Singh, R, SapnaTiwari, M Srivastava, and A Shukla. 2013.Performance study of
combined microwave and acid pretreatment method for enhancing enzymatic digestibility
of rice straw for bioethanol production. Plant Knowledge Journal. 2(4): 157-162.
[8] Ravoof, S, K Pratheepa, T Supassri, and S Chittibabu. 2012.Enhancing enzymatic
hydrolysis of rice straw using microwave assisted nitric acid pretreatment. Int. J. Med.
Biosci. 1(3): 13-17.
[9] Pang, F, S Xue, S Yu, C Zhang, B Li, and Y Kang. 2012.Effects of microwave power and
microwave irradiation time on pretreatment efficiency and characteristics of corn stover
using combination of steam explosion and microwave irradiation (SE-MI) pretreatment.
Bioresour Technol. 118: 111-9.
[10] Kheswani, DR, Microwave Pretreatment of Switchgrass for Bioethanol Production, in
Biological and Agricultural Engineering. 2009, North Carolina State University: Raleigh,
North Carolina.
[11] Pu, Y, F Hu, F Huang, BH Davison, and AJ Ragauskas. 2013.Assessing the molecular
structure basis for biomass recalcitrance during dilute acid and hydrothermal
pretreatments. Biotechnol Biofuels. 6(1): 15.
[12] Ha, SH, NL Mai, G An, and YM Koo. 2011.Microwave-assisted pretreatment of
cellulose in ionic liquid for accelerated enzymatic hydrolysis. Bioresour Technol. 102(2):
1214-9.
[13] Matsumoto, A, S Tsubaki, M Sakamoto, and J-i Azuma. 2011.A novel saccharification
method of starch using microwave irradiation with addition of activated carbon.
Bioresource Technology. 102(4): 3985-3988.
[14] Warrand, J and HG Janssen. 2007.Controlled production of oligosaccharides from
amylose by acid-hydrolysis under microwave treatment: Comparison with conventional
heating. Carbohydrate Polymers. 69(2): 353-362.
[15] Khan, AR, J Johnson, and R Robinson. 1979.Degradation of starch polymers by
microwave energy. Cereal chemistry.
[16] Zhang, Z and ZK Zhao. 2009.Solid acid and microwave-assisted hydrolysis of cellulose
in ionic liquid. Carbohydrate Research. 344(15): 2069-2072.
[17] Hermiati, E, D Mangunwidjaja, TC Sunarti, O Suparno, B Prasetya, SH Anita, and L
Risanto. 2012.Ethanol Fermentation of Microwave-assisted Acid Hydrolysate of Cassava
Pulp with Saccharomyces cerevisiae in the Presence of activated Carbon International
Journal of Environment and Bioenergy. 3(1): 12-24
[18] Hermiati, E, Rekayasa proses hidrolisis ampas tapioka menggunakan pemanasan
gelombang mikro untuk produksi etanol, in Sekolah Pascasarjana. 2012, Bogor
Agricultural University: Bogor.
[19] Menéndez, JA, R Abdul Rahman, BF Arenillas, LZ Y. Fernández, E.G.Calvo, and
J.M.Bermúdez. 2010.Microwave heating processes involving carbon materials. Fuel
Processing Technology. 91(1): 1-8.
[20] S.Chittibabu, K.Rajendran, M.Santhanmuthu, and M.K.Saseetharan.2011 Optimization of
microwave assisted alkali pretreatment and enzymatic hydrolysis of Banana pseudostem
for bioethanol production. in 2nd International Conference on Environmental Science
and Technology. Singapore: IACSIT Press, Singapore.
[21] M. Selig, NW, and Y. Ji and National, Enzymatic saccharification of lignocellulosic
biomass, in Laboratory Analytical Procedure (LAP), USDoEOoEEaR Energy, Editor.
2008, National Renewable Energy Laboratory: Cole Boulevard, Golden, Colorado.
[22] E.Wrolstad, R, T E.Acree, M Eric A. Decker, H.Penner, and DS Reid, Handbook of Food
Analytical Chemistry: Water, Proteins, Enzjmes,Lipids,and Carbohydrates 2005: John
Wiley & Sons, Inc.
[23] Yu, H, G Guo, X Zhang, K Yan, and C Xu. 2009.The effect of biological pretreatment
with the selective white-rot fungus Echinodontium taxodii on enzymatic hydrolysis of
softwoods and hardwoods. Bioresource technology. 100(21): 5170-5175.
[24] Fatriasari, W, W Syafii, N J.Wistara, K Syamsu, and B Prasetya. 2014.Digestibility of
Fungal Pretreated Betung Bamboo Fiber Makara Journal of Technology Series.
[25] Aguilar, R, JA Ramı́rez, G Garrote, and M Vázquez. 2002.Kinetic study of the acid
hydrolysis of sugar cane bagasse. Journal of Food Engineering. 55(4): 309-318.
[26] O’Brien, DJ, GE Senske, MJ Kurantz, and JC Craig Jr. 2004.Ethanol recovery from corn
fiber hydrolysate fermentations by pervaporation. Bioresource Technology. 92(1): 15-19.
[27] Fatriasari, W and SH Anita.2012 Evaluation of Two-Stage Fungal Pretreatment for The
Microwave Hydrolysis of Betung Bamboo. in The 2nd Korea - Indonesia Workshop &
International Symposium on Bioenergy from Biomass Serpong, Indonesia.
[28] Fatriasari, W, W Syafii, N J.Wistara, K Syamsu, and B Prasetya. 2014.The characteristic
changes of betung bamboo (Dendrocalamus asper) pretreated by fungal pretreatment.
Indoneian Journal of Chemistry.
[29] Chandel, AK, RK Kapoor, A Singh, and RC Kuhad. 2007.Detoxification of sugarcane
bagasse hydrolysate improves ethanol production by Candida shehatae NCIM 3501.
Bioresource Technology. 98(10): 1947-1950.
[30] Seo, H-B, S Kim, H-Y Lee, and K-H Jung. 2009.Improved bioethanol production using
activated carbon-treated acid hydrolysate from Corn Hull in Pachysolen tannophilus.
Mycobiology. 37(2): 133-140.
[31] Larsson, S, E Palmqvist, B Hahn-Hägerdal, C Tengborg, K Stenberg, G Zacchi, and N-O
Nilvebrant. 1999.The generation of fermentation inhibitors during dilute acid hydrolysis
of softwood. Enzyme and Microbial Technology. 24(3): 151-159.
[32] Palmqvist, E, H Grage, NQ Meinander, and B Hahn‐Hägerdal. 1999.Main and interaction
effects of acetic acid, furfural, and p‐hydroxybenzoic acid on growth and ethanol
productivity of yeasts. Biotechnology and Bioengineering. 63(1): 46-55.
[33] Lorenz, K and J Johnson. 1972.Starch hydrolysis under high temperatures and pressures.
Cereal chemistry. 49: 616-628.