FIBER DISRUPTION OF BETUNG BAMBOO
( ) BY COMBINED FUNGAL AND Dendrocalamus asper

MICROWAVE PRETREATMENT
WIDYA FATRIASARI *, WASRIN SYAFII , NYOMAN WISTARA , KHASWAR SYAMSU ,1 2 2 3

BAMBANG PRASETYA , S. HERIS ANITA and LUCKY RISANTO4 1 1

1Research Center for Biomaterials, Indonesian Institute of Sciences (LIPI),
Jalan Raya Bogor Km 46 Cibinong, Bogor 16911, Indonesia

2Department of Forest Product Technology, Faculty of Forestry, Institut Pertanian Bogor,
Bogor 16680, Indonesia

3Department of Agro-Industrial Technology, Faculty of Agricultural Engineering and Technology,
Institut Pertanian Bogor, Bogor 16680, Indonesia

4 thNational Standardization Agency, Manggala Wanabakti Building Blok IV, 4 Floor,
Jalan Gatot Subroto, Senayan, Jakarta, Indonesia

Received 22 January 2014/Accepted 3 December 2015

ABSTRACT

Combined microwave pretreatment is an attractive method to alter carbohydrate and lignin structure of fungal and
lignocellulosic materials for improving hydrolysis process to convert these lignocellulosic materials to bioethanol. This
study was conducted to obtain information on the and lignin characteristic changes after carbohydrate combined
biological microwave pretreatment of amboo. Based on our previous research, incubation for 30 days and betung b
using 5 and 10% (w/v) inoculum loading of white rot fungi, which has better delignification Trametes versicolor
selectivity compared to the other incubation time, was chosen as the pretreatment prior to microwave fungal
pretreatment for 5, 10 and 12.5 minutes at . The evaluation of characteristic changes after pretreatment was 330 W
performed using the analysis of FTIR spectroscopy, X-Ray diffraction and SEM. FTIR spectra demonstrated that the
combined change pretreatment only affected the of intensity bands of FTIR spectra, without any changes in the
functional groups. r unconjugated bonds of carbohydrate peaked at 1,736 cm This band intensity decrease occu red on -1

(C = 0 in xylan), 1,373 cm (C-H deformation in cellulose and hemicellulose), 1,165 cm (C-O-C vibration in cellulose -1 -1

and hemicellulose) 895 cm (C-H deformation or C-O-C stretching at β-glicosidic linkage characteristic in and -1

cellulose) The pretreatment decreased the hydrogen bond stretching of cellulose and the linkage between lignin and .
carbohydrate associated with crystallinity of bamboo cellulose l. This decrease of hydrogen bond was , i lustrated by
occurring structural changes. The crystallinity tended to increase slightly due to the cleavage of the amorphous index
fraction. SEM image illustrated that the pretreatment disrupted the fiber structure. The longer duration of microwave s
irradiation, the greater the degradation level of fiber.

: Keywords betung bamboo, and lignin changes, biological microwave pretreatment, carbohydrate combined and
FTIR, SEM, XRD

INTRODUCTION

Increasing concern of greenhouse gas
emission and the depletion of fossil fuels have
been considered as the main driving force in
exploring renewable energy sources (Hu & Wen
2008; Zhang 2008). Abundant lignocellulosic
materials are potential bioresources to produce
liquid biofuel, such as bioethanol. However, the
recalcitrance nature of biomass due to the

presence of lignin and cellulose crystalline
structure prevents optimum enzyme penetration
during hydrolysis.

Effective pretreatment prior to hydrolysis
stage is required to improve biomass digestibility.
Pretreatments are emphasized mainly to increase
feedstock surface area and porosity, as well as to
reduce cellulose crystallinity, lignin content and
hemicellulose content (Mosier . 2005; Galbe & et al
Zacchi 200 ; Wyman . 2007; Cara . 2008). 7 et al et al
An effective pretreatment of biomass is indicated * Corresponding author : widya_fatriasari@yahoo.com

BIOTROPIA Vol. 22 No. 2, 2015: 81 - 94

DOI: 10.11598/btb.2015.22.2.363

mailto:widya_fatriasari@yahoo.com

by sug ar release improvement, reduced
carbohydrate degradation and the lack of
inhibitor y by-products such as furfural,
hydroxymethyl furfural (HMF) and organic acids
formation ( ) Kuhnel . 2011; Agbor . 2011et al et al
and also be cost-effective ( &Yang Wyman 2008;
Agbor . 2011et al ).

Bamboos are versatile fast growing species of
C plant type with very efficient photosynthesis 4
ability. Theoretical value of C photosynthesis is 4
approximately 8%. Biomass productivity of
bamboo is about 20-40 ton s/ha/year. It is ne
approximately 7-30% higher than that of woody
plants (Kant 2010) and other energy crops such as
poplar, switch grass, miscanthus, common reed
and bagasse (Sathitsuksanoh . 20 ; Zhang et al 10
2008). Bamboos are distributed in the tropics,
subtropics and temperate zones ( . Lobovikov et al
2007) and cover 1% of the world's forest area
(Kant 2010). Most of bamboo population (65%)
grows in Asia, especially in Indonesia with 160
bamboo species (Widjaja 2001). This ranks third
(5%) in world bamboo's population after China
(14%) and India (30%) (Lobovikov . 2007). et al
Betung amboo is considered among the most b
important species in Indonesia (Dransfield &
Widjaja 1995). Previous study of six Indonesia's
bamboo species demonstrated that fiber morpho-
logy, physical and chemical properties of betung
bamboo were better than those of kuning, tali,
andong, ampel and black bamboos (Fatriasari &
Hermiati 2008).

After single pretreatment, enzymatic
hydrolysis in simultaneous saccharification and
fermentation (SSF) can be applied to produce
bioethanol. However, single pretreatment tends to
produce low sugar yield, consume time and
require high production cost due to enzyme
requirement in saccharif ication process.
Combined biological-microwave pretreatment
could improve ethanol yield of biomass via SSF
method. White-rot fungi used in biological
pretreatment degrade lignin polymer by secreting
ligninolytic enzyme Nazarpour (Zhang 2007; et al.
et al. 2013). An appropriate fungal strain is
needed to obtain a satisfying delignification
selectivity and enzymatic hydrolysis yield.
Delignification selectivity of betung bamboo with
Trametes versicolor was found to be better than that
with and Pleur otus ostr eatus Phaner ochaete
chrysosporium et al (Fatriasari . 2011; Falah . et al
2011). Microwave radiation of lignocellulosic

material in aqueous environment is also found
promising (Kheswani . 2007). This et al
pretreatment method has been applied for switch
grass, bagasse, rice straw, woody plants, oil palm
empty fruit bunch, oil palm trunk and frond
(Hu & Wen 2008 Keshwani 2007; Anita . ; et al
2012; Risanto . 2012; Lai & Idris 2013). The et al
advantages of the method include short
processing time and high product yield and
quality (Hermiati . 2011). Microwave et al
pretreatment supplies direct internal heat to
biomass resulted from polar bond vibration as
they align with the magnetic field (Kheswani .et al
2007). Microwave pretreatment can increase ion
production, solubilize non-polar material and
hydrolyze biomass without catalyst (Tsubaki &
Azuma 2011).

In a study on biological and microwave
pretreatment of etung amboo, incubation for b b
30 days in biological pretreatment resulted in high
lignin removal and less cellulose loss (Fatriasari et
al. 2014a). Furthermore, microwave pretreatment
of biomass at utes330 W for 5, 10 and 12.5 min
resulted in lower of the a- weight loss cellulose and
hemicellulose other compared to that of
pretreatment conditions (Fatriasari 2014b)et al. .
To the best of our knowledge, no study has been
reported on the changes lignin and of
carbohydrate structure after combined biological-
microwave pretreatment. This study was
conducted to obtain information on the
carbohydrate and lignin characteristic changes
after biological microwave combined and
pretreatment of amboobetung b .

MATERIALS AND METHODS

Material Preparation

Bet ung b amboo ( Dendr o calamus asper
(Schult.f.)) of less than 2 years old was collected
from bamboo plantation of the Research Center
for Biomaterials LIPI, Cibinong, Indonesia.
The bark of the collected bamboo was removed
and the barkless bamboo was chipped before
being ground into fine powder. The powder was
sieved to obtain 40-60 mesh bamboo meal and
then stored in a sealed plastic bag at room
temperature. Betung bamboo meal was subjected
to biological pretreatment before microwave
irradiation.

BIOTROPIA Vol. 22 No. 2, 2015

Fiber disruption of betung bamboo ( ) by combined fungal FatriasariDendrocalamus asper and microwave pretreatment – et al.

Bamboo Sample Preparation

Bamboo meal was watered with ratio of 1:4
and then manually stirred until completely mixed.
The wet bamboo-meal was then put in a jar and
steamed for 30 minutes at approximately 100 C o

and finally in an for 20 minutes sterilized autoclave
at 121 C.o

Inoculum Stock Preparation

fungus inoculum was cultured on T. versicolor
Malt Extract Agar (MEA) slant (10.65 g of MEA
were diluted in 300 mL distilled ) for water
7-14 days. 5 mL At the end of incubation period,
of JIS (Japan Industrial Standard) broth
medium 3 g KH PO , 2 g MgSO .7H O, 25 g ( 2 4 4 2
glucose, 5 g pepton and 10 g malt extract
diluted into 1 L of distilled water) was injected to
the then scratched with loop to release slant and
the mycelium from the slant agar. resulting As
much as of previously prepared 5 mL fungi
suspension was then poured into the remaining
95 mL of the JIS Broth medium and stationery
incubated at 27 C for 7- days. After incubation, o 8
10 g of corn steep liquor was poured into the
100 mL inoculum and homogenized twice with a
high speed Waring blender (each homogenization
was conducted for 20 seconds).

Inoculation Method

Bamboo-meal 15 g - ied ) having ( oven dr weight
7.46% moisture content was inoculated with 5 and
10% (w/v) iedinoculum of dr bamboo and
incubated at 27 C for 30 dayso .

Microwave Pretreatment

Microwave pretreatment was carried out in
an oven microwave SHARP P-360J (S) with
2,450 MHz frequency and power output of 1,100
W. As much as 1 g of oven-dried pretreated
sample was inserted into a Teflon tube (vessel),
added with distilled water to obtain a solid-to-
liquid ratio (SLR) of 1:30 (w/v) and stirred for
15 minutes. Subsequently, the sample was
exposed to microwave irradiation at 330 W
for 5, 10 and 12.5 minutes. After microwaving,
the pulp was removed from the oven and
immediately put into iced water for 15-20 minutes
to cool the pulp. The residue (solid fraction)
was separated from the hydrolysate (liquid
fraction) by filtration.

The Changes of Content, Morphological,
Cellulose and Lignin Characteristics

Chemical component determination
P rior to d eter mination of chemic al
component of control and pretreated bamboo,
the moisture content samples were measured of
following the procedures of TAPPI T12 os-75 .
Free extractive bamboo-meal was prepared with
ethanol-benzene (1:2) extraction for chemical
component analysis. Acid-insoluble lignin, acid
soluble lignin, holocellulose, a-cellulose and ash
content were determined in accordance with the
TAPPI T13 os-54, , TAPPI TAPPI UM 250
T9m-54, and T 15 os-TAPPI T17m-55 TAPPI
58 standards, respectively. The calculation of
weight loss following method of was done the
Pandey Pitman (2003), while selectivity value &
was calculated as ratio of lignin loss to the
cellulose loss (Yu . 20 )et al 09 .

Cellulose crystallinity index determination
The crystallinity was determined using index
diffraction intensity data of X-ray Diffraction
(XRD) according to the formulation of Zhou et
al. (200 ). Measurement was carried out with 5
Shimadzu XRD-700 MaximaX series. NI
radiation was filtered by CuK at 0.15406 nm wave α
number. X-ray was operated at 40 kV of voltage,
30 mA of electrical current and scanned 2 theta ( )0

of 10-40 in 2 minute.o o per

Allomorphic structure of cellulose
Z-Discriminate function 200 ) was (Hult . 3et al
used to differentiate allomorphic properties of
cellulose crystalline structure. The function was
built up by separating cellulose I and I using d-α β
spaces obtained from X-ray analysis, i.e. two
equatorial d-spacing: 0.59-0.62 (d ) and 0.52-0.55 1
nm (d ). Z > 0 indicates bacteria algae type (I , 2 α
rich triclinic structure) and z < 0 indicates cotton
and flax types (predominantly I structure/ β
monoclinic).

Crysta lite size of cellulosel
The cr ystallite size of cellulose was
determined using diffraction pattern obtained
from 101 , 10-1 , 002 and 040 lattice planes ( ) ( ) ( ) ( )
of bamboo ).(Zhao . 2007et al

Morphological structure analysis
Morphological structure of pretreated
bamboo was analyzed through SEM micrograph
obtained by a JEOUL/EO SEM. Bamboo

sample was installed in the sample holder (stub)
using sputter canter and then scanned at 15 kV
with 10 mm of working distance with 750x and
10,000x of magnification.

Biodegradation pattern
Biodegradation pattern was analyzed through
Fourier Transform Infrared Spectrometry (FTIR)
spectrograph. To obtain FTIR spectra, 4 mg of
bamboo-meal was embedded in 200 mg of KBr
(Potassium Bromide) spectroscopy grade and
then pelletized at 5,000 psi. The diameter and
thickness of the pellet were approximately 1.3 cm
and 0.5 cm, respectively. Infrared spectrum
patterns (peak height and area) were analyzed by
using FTIR ABB MB 3000. All of the spectra were
recorded at a spectral resolution of 16 cm -1 with
the accumulation of 5 scans per sample with
absorption mode in the range of 4,000-500 cm . -1

The characteristic of carbohydrates and lignin was
analyzed based on the relative band intensity
change referring to the method of Pandey
(Pandey & Pitman 2003). Peak height and area
values of lignin associated bands were rationed
compared to carbohydrate reference peaks at
1,720; 1,366; 1,180 and 879 cm to provide relative -1

changes in the composition of the structural
components relative to each other determined
using Horizon MB software.

Statistical Analysis

All experiments were performed in triplicate.
Sample preparation for SEM, FTIR and XRD
analyses has been conducted by manually mixing
all triplicate treated and untreated samples. The
pretreatment combination effects on chemical
component changes and losses were analyzed by
ANOVA (Analysis of Variance) using MINITAB
release 13.2 software. Significant differences
among treatment combinations were evaluated
using Tukey's multiple range comparisons at < p
0.05.

RESULTS AND DISCUSSION

Chemical Content of Pretreated Bamboo

The chemical component composition change
of pretreated bamboo is depicted in Figure 1
which shows that bamboo has high a-cellulose
content. Cellulose is the main source of C-6 sugar
convertible to ethanol. In this study, combined
biological-microwave pretreatment was utilized
to reduce lignin content. The pretreatment was
expected to degrade lignin and hemicellulose.
Removal of the C-5 hemicellulose could increase
sugar fermentation by Saccharomyces cerevisiae
considering that the C-5 sugar of hemicellulose
cannot be efficiently fermented by the yeast.

DS : 2.91 0.46 1.08 0.77 0.59 1.14

ASL

AIL

100%

80%

60%

40%

20%

Control

Biological-microwave pretreatment

5% IL for
5 min

5% IL for
10 min

5% IL for
12,5 min

10% IL for
5 min

10% IL for
10 min

10% IL for
12,5 min

Figure 1 Chemical component composition change of bamboo after biological-microwave pretreatment. Components: s
IL (inoculum loading); WL (weight loss); ASL (acid soluble lignin); AIL (acid insoluble lignin); HC (hemicellulose);
AC (alpha cellulose); E (ethanol-benzene extractive); DS (delignification selectivity)

BIOTROPIA Vol. 22 No. 2, 2015

h e - m i c r o w a v e T c o m b i n e d f u n g a l
pretreatment changed the compositionchemical
of pretreated bamboo of (Fig. 1). Pretreatment
10% inoculum loading show lower weight loss ed
or higher yield inoculum loading. than that of 5%
Longer irradiation time tended to increase weight
loss. might be related more intensive This to a
lignin degradation activity than carbohydrate
removal in higher inoculum loading. Total weight
loss was approximately of 5.47-19.88% tat stical . S i
analysis indicated that inoculum loading gave only
significant effect to alpha cellulose and
hemicellulose content, weight loss and
hemicellulose loss only . On the other hand,
irradiation time gave significant effect on weight
loss and lignin loss . Based on ukey's ( < 0.05) Tp
pairwise comparison, weight loss due to
irradiation time differen . were significantly t
I nteraction between inoculum loading and
irradition time weight loss. significantly affected
However, no interaction between inoculum effect
loading and irradition time on the alpha cellulose
loss . Prolonging irradiation time for 10 was found
min affected decrease of apha cellulose utes the
loss for both inoculum loading. Even though, the
irradiation time for 12.5 min caused higher utes
lignin degradation, also caused higher alphait
cellulose loss. Thus, greater extend of irradiation
time was not required.
The highest selectivity value (up to 2) was
found after bamboo pretreated 5% was with
inoculum loading and then irradiated for 5 was
min . A higher selectivity value indicate that utes s
lignin polymer is more effective than cleavage
cellulose degradation. tatistical analysis S of the
present results dindicate that the inoculum
loading and irradiation time did not significant ly
affect selectivity value ( < 0.05).p
Degradation of carbohydrate (alpha cellulose)
also occurred during delignification activity (Fig.
1). It might be due to partial hydrogen bond
disruption of the LCC (Lignin Carbohydrate
Complex) (Li . 2010). Bet al iological pretreatment
caused opening complex of the lignocellulose
structure through depolymerization of lignin and
brought about carbohydrates increasing
accesibility. icrowave pretreatment after M
biological pretreatment also help toed alter the
ultrastructure of cellulose and degrade lignin and
hemicellulose in lignocellulosic materials that
bring about increasing susceptibility of

lignocellulosic materials (Binod . 2012). et al
Microwave heating transfers and induces heat
directly into bamboo substrate, causing the
depolymerization of sugar building block into
oligosaccharides (Ebringerova 2006).
Under acid pretreatment at high pressure and
temperature, sugar monomer such as glucose and
xylose can be further degraded in hydroxymethyl
furfural and furfural (HMF) Behera . 2014) ( . et al
The degradation product can be released during
acid pretreatment condition. More severe of
microwave pretreatment condition led to
decrease hemicellulosic monosaccarides in
hydrolyzate and increase the formation of sugar
degradation product (Kuhnel 2011). Variouset al.
potential s edcompound in hydrolyzate consist

acetic acid, formic acid, furan derivatives of
(5-HMF and furfural) and phenolic compounds
might be wasgenerated. The inhibitor presence
not excepted the effect of limationdue to
efficient process (Zhang . 2011; fermentation et al
Talebnia . 2010). However, due to our research et al
focus to observe the change of chemical was
component and cellulose structure of pretreated
samples, this potential inhibitor on hydrolyzate
was d not observe .

Cellulose Structure Changes of Pretreated
Bamboo

FTIR spectroscopy was used to investigate
changes in the chemical structure of pretreated
samples (Fig. 2 3). Slight changes occurred in and
spectrum peaks of biomass were treated with
both 5 and 10% inoculum loading, but no changes
appeared in the functional groups during
pretreatment. It might be caused by uncompleted
disruption of lignin that encapsulated cellulose.
The broad absorption was observed at the wave
number of around 3,340 cm . This wave number -1

was assigned to hydrogen bond (O-H) stretching
absorption. O-H stretching region at the wave
number of 3,000-3,600 cm of pretreated -1

bamboo spectra was more identical to the O-H
stretching region from cellulose I. The band at
2,700-2,901 cm is related to the C-H stretching -1

(Pandey & Pitman 2003). Biological-microwave
pretreatment affected the peak area and band-
height of 3,340 cm wave number (O-H -1

stretching) (Fig. 2 and 3). It indicated a weak intra
and inter molecular bond of O-H group
(Goshadrou . 2011).et al

Fiber disruption of betung bamboo ( ) by combined fungal FatriasariDendrocalamus asper and microwave pretreatment – et al.

FTIR spectra with frequencies in the region of
1,600 and 1,510 (aromatic ring vibration), 1,470
and 1,460 cm (C-H deformations and aromatic -1

ring vibrations) can be found in the lignin
structure (Fengel & Wegener 1992). Lignin of
bamboo consisting of Guaiacyl (G) and Syringyl
(S) propane units containing one and two metoxyl
groups can be clearly observed in all treatments at
wave number of 1,327 cm for Syringyl propane -1

units and 1,257 cm for Guaiacyl propane units. -1

The higher absorbencies in the finger print of
pretreatment of both 5 and 10% inoculum
loading of pretreated samples compared to
control was found at irradiation of 10 minutes.
The higher lignin and cellulose content in this
condition (Fig. 2) which can be confirmed by this
spectra (Fig. 3). The absorbance of Syringyl
(1,327 cm ) was lower than that of Guaiacyl -1

Figure 2 FTIR spectra of bamboo after fungal pretreatment (5% inoculum loading for 30 days) subjected to microwave
pretreatment

Figure 3 FTIR spectra of bamboo after fungal pretreatment (10% inoculum loading for 30 days) subjected to microwave
pretreatment

BIOTROPIA Vol. 22 No. 2, 2015

(1,257 cm ) indicating a higher Syringyl content in -1

control and treatments. The typical infrared band
frequencies and FTIR spectras of bamboo
components in units of wave numbers are listed in
Table 1.
The sharp bands around 895 cm is attributed -1
to β-glicosidic linkage between the sugar units in
cellulose (Nelson & O’Connor 1964) which can
be clearly seen in all spectra. The increasing
microwave irradiation reduced band intensity of
functional group (C=O) in hemicellulose (3), C-H
in cellulose and hemicellulose (9), and C-O-C in
hemicellulose (12). It might be attributed to the
decrease of hemicellulose content after

pretreatment along with lignin. Sixty functional
groups can be observed in six pretreatment
conditions. Each identified functional group can
be found in all treatments, although a slight shift
in wave number occurred. The treatments
decreased functional groups intensity without
changing functional group types.

Effect of Combined Fungal and Microwave
Pretreatment on Bamboo Morphology

SEM micrograph of pretreated samples (Fig. 4
and 5) was used to observe morphological
features and surface characteristics of pretreated
bamboo with increasing microwave irradiation.

Table 1 Assignments of IR band of bamboo after biological-microwave pretreatment

Control*

Biological pretreatment

Assignments

5% of inoculum
loading

10% of inoculum loading

Microwave pretreatment
5

minutes
10

minutes
12.5

minutes
5

minutes
10

minutes
12.5

minutes

Wave number (cm-1)

1 3,394 3,340 3,333 3,418 3,418 3,425 3,364 A strong and broad hydrogen
bond (O-H) stretching absorption1

2 2,901 2,901 2,901 2,901 2,901 2,901 2,901 A prominent C-H stretching absorption1
3 1,736 1,728 1,736 1,736 1,728 1,728 1,713 Unconjugated C=O in xylans

4 1,643 1,643 1,651 1,651 1,643 1,643 1,643 Absorbed O-H and conjugated C-O
1

Aromatic skeletal1 5
1,605 1,605 1,605 1,605 1,605 1,605 1,605

Aromatic skeletal1 6
1,512 1,512 1,512 1,512 1,512 1,512 1,512

1,458

1,458
C-H deformation

1,427

C-H2
scissoring motion1

1,373

C-H deformation1

1,335

1,327

1,335

1,327

C-H vibration1
C1-O vibration in syringyl

derivates 1

1,257

1,250

1,257

1,250

Guaiacyl

ring1

C-O stretch1
12 1,165 1,165 1,165 1,165 1,165 1,165 1,165 C-O-C vibration1

13 1,111 1,111 1,111 1,111 1,111 1,111 1,111 Aromatic skeletal and C-O stretch1

14 1,049 1,041 1,034 1,041 1,041 1,041 1,041 C-O stretch1

895

C-O-C stretching at β-glicosidic
Linkage or C-H deformation in
Cellulose2

16 833 833 833 833 833 833 833 C-H vibration3

Notes: Control* = Data has been used in other paper (Fatriasari . 2014b)et al
1 = Pandey and Pitman (2003)
2 = Chen (2011)et al.
3 = Cheng . (2013)et al

Fiber disruption of betung bamboo ( ) by combined fungal FatriasariDendrocalamus asper and microwave pretreatment – et al.

SEM images of samples treated with both 5
and 10% inoculum loading show that partial
disruption occurred on the fiber structure.
Degraded lignin polymer and removal of
hemicellulose in cell wall might responsible to be
this disorganized morphology. Longer microwave
irradiation caused greater fiber degradation level.
The cell wall morphology changes caused by
lignin removal resulted in greater deconstruction

of fiber surface, providing better cellulose
penetration. Partial degradation of lignin and
hemicellulose destroyed some ether bonds in
lignin and lignin-carbohydrate complex leading to
the disruption of the hydrogen bond between
cellulose, thus fibrillation occurred (Li . 2010). et al
The cellulose digestibility can be potentially
enhanced by preferential cleavage of lignin
(Nazarpour . 2013). Treatments of 5% et al

10 min utes (330 W)

12.5 min utes (330 W)

5 minutes (330 W)

a b

10 minutes (330 W)

12.5 min utes (330 W)

5 minutes (330 W)

a b

Figure 4 SEM micrograph pretreated bamboo (5% inoculum loading for 30 days) subjected to microwave pretreatment of
with (a) 750x and (b) 10,000x magnification

Figure 5 SEM micrograph pretreated bamboo (10% inoculum loading for 30 days) subjected to microwave pretreatment of
with (a) 750x and (b) 10,000x magnification

BIOTROPIA Vol. 22 No. 2, 2015

inoculum loading for 10 and 12.5 minutes
microwave irradiation resulted in more opened-
up sponge-like structures providing wider surface
area to increase the rate of subsequent hydrolysis
reactions.

Cellulose Structure Allomorph

Crystalline allomorph of pretreated bamboo
as result of mixing triplicate samples a observed
by XRD is presented in Table . In general, 2
cellulose consists of I (one-chain triclinic) and I α β
(two-chain monoclinic cells), which can be
determined by Z-discriminant. Z < 0 and Z > 0
indicate the I and I allomorph types, respectively β α .
Monoclinic (I ) cellulose is more stable than β
triclinic (I ), and tends to be the final product in α
annealing treatment Credou of all celluloses ( &
Barthelot . 2014)

All treatments except for the 5% inoculum
loading for 10 minutes had monoclinic structure.
However, the cause of this phenomenon has not
yet understood The presence of the I phase been . α
was expected to improve cellulose digestibility
due to its higher degradation than that of I . In β
addition, this structure is meta-stable and more
reactive than I ( ).β Moon . 2011et al

Bio of ombined degradation Patter n C
Biological-microwave P tretreatmen

Biodegradation patterns of bamboo during
combined biological-microwave pretreatment
were evaluated by FTIR analysis 6 7 . (Figure and )
A detailed FTIR spectroscopic analysis based on
Pandey's analysis method was performed to
calculate relative intensities of aromatic skeletal
vibration against typical bands of carbohydrate
on pretreated bamboo (Pandey & Pitman 2003).

Table 2 Crystalline allomorph of pretreated bamboo

Biological
pretreatment

Microwave pretreatment

Crystallite allomorph

Crystal
allomorphInoculum loading

(%)
Power loading

(W)
Microwave irradiation

(minutes)
d (101)

nm
d (10-1)

nm z

Control* 0.5824 0.5349 -45.47 Iβ

330

5 0.5987 0.5466 -28.49 Iβ
10 0.6110 0.5230 13.69 Iα

12.5 0.5955 0.5473 -34.50 Iβ

5 0.5520 0.5163 -80.11 Iβ
10 0.5583 0.5134 -66.92 Iβ

12.5 0.5740 0.5181 -44.48 Iβ
Note: Control* = Data have been used in other paper (Fatriasari . 2014 )et al b

Fig 6 FTIR spectra of bamboo after biological pretreatment (5% inoculum loading for 30 days) subjected to microwave ure
pretreatment (1) 1,736 cm , (2) 1,512 cm , (3) 1,373 cm , (4) 1,165 cm and (5) 897 cm-1 -1 -1 -1 -1

Fiber disruption of betung bamboo ( ) by combined fungal FatriasariDendrocalamus asper and microwave pretreatment – et al.

Relative changes in the intensities of aromatic
skeletal in lignin peaks at 1,512 cm against four -1

unconjugated bonds of carbohydrate peaks at
1,736 cm (C=O in xylan), 1,373 cm (C-H -1 -1

deformation in cellulose and hemicellulose), 1,165
cm (C-O-C vibration in cellulose and - 1

hemicellulose), 895 cm (C-H deformation or C--1

O -C str e tch ing at β -g lic os id ic l in kag e
characteristics in cellulose) calculated by peak
heights and areas are summarized in Table .3
In 5% inoculum loading, increasing microwave
irradiation period tended to increase the ratio of
lignin/carbohydrate. It indicated that prolonged
microwave irradiation decreased its lignin
degrading ability of pretreated bamboo.
Carbohydrate degradation after pretreatment
contributed to this phenomenon.

Crystallinity Index (CI) and Crystallite Size
of Cellulose

Crystalline and amorphous structure of
cellulose can be identified from primary peak
of XRD pattern ranging from 22-23 and 0

secondary peak in the range of 16-18 (Lai & Idris 0

2013; Liu . 2012). These peaks can be et al
determined within the mentioned range for all
treatments, which indicates that the crystalline
and amorphous region of cellulose. The
crystallinity index of pretreated bamboo has been
used to interpret changes of cellulose after
pretreatment (Table 4). Intensity transformation
in hydrogen bonding of cellulose can be
reflected from width variation of crystallization
peak.

Fig 7 FTIR spectra of bamboo after biological pretreatment (10% inoculum loading for 30 days) subjected to microwave ure
pretreatment (1) 1,736 cm , (2) 1,512 cm , (3) 1,373 cm , (4) 1,165 cm and (5) 897 cm-1 -1 -1 -1 -1

Table Ratio of intensity of lignin-associated band with carbohydrate bands of pretreated 3 bamboo

Biological
pretreatment

Microwave pretreatment

Relative intensities a of aromatic skeletal vibration (I1,512)
againts typical bands for carbohydrates

Inoculum
loading (%)

Power loading
(W)

Irradiation time
(minutes) I1,512/I1,736 I1,512/I1,373 I1,512/I1,165 I1,512/I897

Control* 1.04(1.06) 1.02(1.06) 0.98(0.95) 1.28(1.29)

5 1.24(0.83) 0.86(0.6) 0.67(0.33) 1.74(3.75)
10 1.49(1.19) 0.88(0.57) 0.64(0.34) 1.59(3.57)

12.5 1.74(1.88) 0.94(0.81) 0.58(0.48) 3.26(2.5)

5 1.42(1.37) 0.83(0.74) 0.59(0.48) 1.55(1.51)
10 1.28(0.75) 0.78(0.48) 0.56(0.29) 1.58(4.0)

12.5 1.36(1.33) 0.84(0.77) 0.57(0.47) 1.73(1.69)

Note: Control* = Data have been used in other paper (Fatriasari . 2014 )et al b

BIOTROPIA Vol. 22 No. 2, 2015

The crystallinity index tended to rise along
with the increasing microwave irradiation. In
biomass, cellulose contains crystalline area, while
lignin and hemicellulose are amorphous in nature
(O'Dowyer . 2007). The increasing of et al
cr ystallinity index might be caused by
solubilization of amorphous component from
the fibers under pretreatment condition (Kim &
Holtzapple 2006). This phenomenon was
supported by the occurring component loss in
lignin as presented in Figure 1. The increase of
crystallinity index after pretreatment was also
described in previous research (Singh 2014; et al.
Bak . 2009).et al
Crystallinity index of cellulose is among the
most important properties of lignocelluloses that
can be measured by FTIR spectroscopy.
Crystallinity index changes can be studied from
LOI (Lateral Order Index), defined as the

absorbance ratio of A to A (Oh . 2005), 1,427 895 et al
obtained from FTIR spectra data. Increasing of
microwave irradiation time tended to increase
LOI. It might be attributed to higher amorphous
region of cellulose compared to crystalline
region. This phenomenon was in line with
crystallinity index measured by XRD analysis.
Crystallite size of cellulose in bamboo varied
at lattice planes of (101), (10-1) and (002). The
crystallite size ranged from 5.19 to 10.68 (Table 5) .
The highest crystallite size of cellulose at (002)
lattice plane was found in pretreatment of 5%
inoculum loading for 12.5 minutes. Crystallite size
at lattice planes (101) and (10-1) of several
pretreated bamboo can not be calculated because s
there no value of Full Width at Half were
Maximum (FWHM) in this peak. The microwave
irradiation duration increaed crytallite size at
(002) lattice plane. The highest crystalline length

Table 4 Crystallinity Index (CI) and Lateral Order Index (LOI) of pretreated bamboo

Biological
pretreatment

Microwave pretreatment

Crystallinity Index

(CI)
Lateral Order Index

(LOI)

Inoculum
loading

(%)

Power
loading

(W)

Irradiation
time

(minutes)
Fc

(Crystaline)
Fa

(Amorf) CI
A1,427

(Crystalline)
A897

(Amorf) LOI

Control* 0.69 2.13 24.58 0.50 0.40 1.25

330

5 0.99 1.48 40.19 0.98 0.51 1.92
10 1.23 1.72 41.76 1.43 0.84 1.70

12.5 1.12 1.54 42.04 0.74 0.23 3.22

5 1.16 1.74 39.98 1.14 0.65 1.75
10 1.13 1.63 40.96 0.75 0.40 1.88

12.5 0.93 1.34 40.84 1.20 0.63 1.91
Note: Control* = Data have been used in other paper (Fatriasari . 2014 )et al b

Table 5 Crystallite size of pretreated bamboo

Biological
pretreatment Microwave pretreatment

Crystallite size

(nm)

Inoculum loading
(%)

Power loading
(W)

Irradiation time
(minutes) D

(101) D (10-1) D (002) D (040)

Controla 5.46 8.71 5.59 16.52

330

5 NDb 6.57 5.17 20.99
10 14.95 7.07 5.74 38.80

12.5 8.69 4.86 6.19 16.51

10
5 NDb 5.32 5.74 86.33
10 5.20 5.36 5.82 22.85

12.5 10.68 ND
b

5.47 36.98
Notes: Control = Data have been used in other paper (Fatriasari . 2014 )a et al b
= b not detected

Fiber disruption of betung bamboo ( ) by combined fungal FatriasariDendrocalamus asper and microwave pretreatment – et al.

at (040) lattice plane of cellulose was found in the
10% inoculum loading for 5 minutes. There was
no similar trend in crystalline length changes
caused by increasing of microwave irradiation
between 5 and 10% of inoculum loading.

CONCLUSIONS

The characteristic changes of lignin and
carbohydrate combined of betung bamboo with
biological-microwave pretreatment was evaluated.
The pretreatment caused the chemical loss of the
component. The 5% inoculum loading irradiated
for 5 min demonstrated the highest selectivity utes
value (up to 2). Based on FTIR spectra, there was
no ch ang e i n f unc ti ona l g ro ups af t er
pretreatment also . Moreover, FTIR spectra
demonstrated the presence of hydrogen bond
stretching along with microwave irradiation
exposure indicating the structural changes
occur ed The r after pretreatment. cleavage of the
amorphous d to the component contribute
increasing crystallinity index Disruption of fiber .
structure due to pretreatments was confirmed by
SEM the longer duration of microwave , in which
irradiation, the greater the degradation level of
fiber.

ACKNOWLEDGEMENTS

The authors would like to thank SEAMEO
BIOTROP for providing financial support as part
of PhD thesis of the first author through DIPA
2013. Moreover, the authors expressed their
gratitude to Dwi H Restuningsih, ST. and Raden
Budi L.Permana in for their support financial
report and technical assistance.

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