Carbohydrate Polymers 151 (2016) 12101219

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Production of new cellulose nanomaterial from red algae marine

biomass Gelidium elegans
You Wei Chen a , Hwei Voon Lee a, , Joon Ching Juan a,b, , Siew-Moi Phang c,d
a
Nanotechnology & Catalysis Research Centre (NANOCAT), Institute of Postgraduate Studies, University of Malaya, 50603 Kuala Lumpur, Malaysia
b
School of Science, Monash University, Sunway Campus, Jalan Lagoon Selatan, 41650 Bandar Sunway, Malaysia
c
Institute of Ocean and Earth Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia
d
Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Nanocellulose was successfully isolated from Gelidium elegans red algae marine biomass. The red algae
Received 17 April 2016 ber was treated in three stages namely alkalization, bleaching treatment and acid hydrolysis treat-
Received in revised form 13 June 2016 ment. Morphological analysis was performed by eld emission scanning electron microscopy (FESEM)
Accepted 19 June 2016
and transmission electron microscopy (TEM). TEM results revealed that the isolated nanocellulose had
Available online 20 June 2016
the average diameter and length of 21.8 11.1 nm and of 547.3 23.7 nm, respectively. Fourier transform
infrared (FTIR) spectroscopy proved that the non-cellulosic polysaccharides components were progres-
Keywords:
sively removed during the chemically treatment, and the nal derived materials composed of cellulose
Red algae
Pulp
parent molecular structure. X-ray diffraction (XRD) study showed that the crystallinity of yielded product
Fiber had been improved after each successive treatments subjected to the treated ber. The prepared nano-
Nanocellulose dimensional cellulose demonstrated a network-like structure with higher crystallinity (73%) than that
Polysaccharides of untreated ber (33%), and possessed of good thermal stability which is suitable for nanocomposite
material.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
During the past decade, several reports on the preparation
of nanostructured cellulose materials from vary lignocellulosic
biomass resources have been extensively reported such as soft-
wood kraft pulp, hardwood elemental chlorine free pulp, cotton
ber, recycled pulp, ax ber, sisal ber, ramie ber, bamboo
residue, wheat straw, grass ber and microcrystalline cellulose
using top-down technologies (Bettaieb et al., 2015). Cellulose
is one of the most abundant natural biopolymer on the Earth,
and widely used for many industry areas such as papermaking,
food packaging, pharmaceutical applications and green compos-
ites innovative applications (Bettaieb, Khiari, Dufresne, Mhenni, &
Belgacem, 2015). Due to its unique characteristics such as high
Corresponding author at: Nanotechnology & Catalysis Research Centre
(NANOCAT), Institute of Postgraduate Studies, University of Malaya, 50603 Kuala
Lumpur, Malaysia. Tel.: +603 79676960.
Corresponding author at: Nanotechnology & Catalysis Research Centre
(NANOCAT), Institute of Postgraduate Studies, University of Malaya, 50603 Kuala
Lumpur, Malaysia. Tel.: +603 79676954.
E-mail addresses: leehweivoon@um.edu.my (H.V. Lee), jcjuan@um.edu.my
(J.C. Juan).
strength and modulus, high surface area, high aspect ratio, high
tensile strength and stiffness, cost effectiveness, light weight and
environmental benets due to recyclable and biodegradable, the
market demand for this natural ber has increased exponentially
(Lam, Male, Chong, Leung, & Luong, 2012). In recent year, a new
nanomaterial source (solid nanocellulose ber) was successfully
isolated from marine biomass such as Posidonia oceanica ball and
leaves (Bettaieb, Khiari, Dufresne, Mhenni, Putaux, & Bou, 2015;
Bettaieb, Khiari, Dufresne, Mhenni, & Belgacem, 2015; Bettaieb
et al., 2015). Compared to land plant, marine biomass has become
more popular as a promising source for nanocellulose production
near future. The advantages of using marine biomass over ligno-
cellulosic biomass for the biopolymer production are: (i) low or
zero content of natural physicochemical barriers in algae biomass,
thus no harsh chemical treatment is required to remove their native
recalcitrant structure in order to increase the cellulose accessibility
(Korzen, Pulidindi, Israel, Abelson, & Gedanken, 2015); (ii) marine
algae contains of higher yields of stored carbohydrates, and capa-
ble to grow under simple growth requirements by utilizing carbon
dioxide, light and inorganic nutrients efciently, which no cost are
required for providing (Jeong, Choi, Lee, & Oh, 2012); (iii) rapid
growth rate of algae plant and can be harvested within short period
of time compare to others terrestrial plants, and thus capable to

http://dx.doi.org/10.1016/j.carbpol.2016.06.083
0144-8617/ 2016 Elsevier Ltd. All rights reserved.
 Y.W. Chen et al. / Carbohydrate Polymers 151 (2016) 12101219
increasing the demand of feedstocks for nanocellulose production
(Kim, Wi, Jung, Song, & Bae, 2015).
Marine seaweeds can be classied into three different groups
including red algae, green algae and brown algae. However, the
study showed that red algae such as Gelidium is a highly potential
biomass sources for various application due to its high carbo-
hydrate content and can be hydrolyzed into smaller molecular
products such as glucose and galactose (Jeong et al., 2012; Kim et al.,
2015). In the previous studies, this algae biomass species had been
used for production of useful chemicals such as bioethanol (Kim
et al., 2015; Korzen et al., 2015) and soluble sugars (mainly glucose,
5-hydroxymethylfurfural (HMF), formic acid, and levulinic acid)
(Jeong et al., 2012). Many researchers treated the algae biomass
under harsh treatment conditions (i.e. high temperature or pres-
sure) in order to yield different types of organic products as a
liquid fraction, rather than solid cellulosic materials. The red algae
biomass such as Gelidium has a rich content of carbohydrates,
which is mainly composed of cellulose and agar (Jeong et al.,
2012; Yoon et al., 2010). Therefore, it is possible to obtain solid
crystalline nanoparticles (nanocellulose) rather than water soluble
fermentable sugars monomers under the controllable hydrolysis
conditions.
The combination of alkaline treatment, oxidative bleaching and
acid hydrolysis are the most common and effective multiple-step
purication process. It has been widely used to isolate cellulosic
material from typical terrestrial biomass plants, such as coconut
husk (Rosa et al., 2010), rice husk (Johar, Ahmad, & Dufresne,
2012), mengkuang leaves (Sheltami, Abdullah, Ahmad, Dufresne, &
Kargarzadeh, 2012), Hibiscus cannabinus (Zaini, 2013), and empty
fruit bunch (Ching & Ng, 2014; Goh, Ching, Chuah, Abdullah,
& Liou, 2016). In general, the main components of red algae
consists of mucilaginous materials (agar) and energy dense sub-
strate such as cellulose material (Seo, Lee, Lee, & You, 2010).
This cellulosic material can be served as a potential feedstock
for bioconversion to nanocellulose. Like others cellulosic biomass
originated from land plants, the long polymeric chains cellulose
extracted from algae can also be hydrolyzed using strong min-
eral acid (i.e. sulfuric acid and hydrochloric acid) into smaller
solid particles in nano-dimension (i.e. nanocellulose). In order to
produce high quality cellulose pulp from algae more effectively,
non-cellulosic components must be removed from the algae during
the extraction process. Arvizu-Higueras research group (Arvizu-
Higuera, Rodrguez-Montesinos, Murillo-lvarez, Munoz-Ochoa, & produce high crystallinity nanocellulose. Gelidium marine biomass
Hernndez-Carmona, 2008) reported that 7% NaOH alkalization
treatment had a signicant and positive effect in solubilizes the agar
from the marine algae plant (Gracilaria vermiculophylla) at 85 C
for 2 h. Besides that, color of yielded nanocellulose is also play a
very important factor because it can affect the consumer accep-
tance in various applications (Saelee, Yingkamhaeng, Nimchua,
& Sukyai, 2016). The red algae initially was exhibited in reddish
color due to the presence of rhizoidal laments components in
the ber which contributed to its color (Seo et al., 2010). There-
fore, a successive bleaching treatment is necessary to remove the
natural pigment and chlorophyll for production of highly puried,
whiteness extracted cellulose pulp form the red algae biomass.
Basically, two main oxidizing bleaching agents namely sodium
chlorite (NaClO2 ) and hydrogen peroxide (H2 O2 ) were used to
bleach the algae biomass ber to produce bleached red algae pulp
(Bettaieb, Khiari, Dufresne, Mhenni, Putaux et al., 2015; Bettaieb
et al., 2015; Saelee et al., 2016). Due to environmental concern,
chlorite-free hydrogen peroxide was selected as oxidizing bleach-
ing agent for pulping process in this study.
The most common choices of isolating nanocellulose are acid
hydrolysis (Ching & Ng, 2014; Ching, Rahman, Ching, Sukiman, &
Cheng, 2015; Liew, Thielemans, & Hewakandamby, 2016), TEMPO-
mediated oxidation (Tanaka, Saito, & Isogai, 2012), mechanical
1211
disintegration (Li et al., 2012) and enzyme-assisted hydrolysis
(Janardhnan & Sain, 2007). Up to date, acid hydrolysis is still consid-
ered the most effective and highly effective in the role of cellulose
depolymerization process for conversion of cellulose into its nano-
dimensional structure. Cellulose bers are subjected to strong acid
hydrolysis such as sulfuric (H2 SO4 ), hydrochloric (HCl), and phos-
phoric acid (H3 PO4 ) to break down the long cellulose polymeric
chain into smaller dimensions. Under the controlled hydrolysis
conditions of temperature, time, agitation and acid concentra-
tion, the amorphous domains of cellulose matrix are preferentially
hydrolyzed by the hydronium ions (H3 O+ ) and leave the highly
crystalline segments unaltered (Yahya, Lee, & Hamid, 2015). Tra-
ditionally, highly crystalline nanocellulose was prepared by acid
hydrolysis as compared with others hydrolysis techniques. H2 SO4
hydrolysis technique has been widely used for isolating highly
crystalline cellulose from various sources, such as sisal ber (De
Rodriguez, Thielemans, & Dufresne, 2006), ax ber (Cao, Dong,
& Li, 2007), ramie ber (Zoppe, Peresin, Habibi, Venditti, & Rojas,
2009), grass ber (Pandey et al., 2009), cotton ber (Wang, Tian, &
Zhang, 2010), bamboo pulp (Liu, Zhong, Chang, Li, & Wu, 2010),
sugarcane bagasse (Mandal & Chakrabarty, 2011), China cotton,
South Africa cotton, waste tissue paper (Maiti et al., 2013), rice
straw (Jiang & Hsieh, 2013), borer powder (Hu et al., 2014), recycled
newspaper (Mohamed, Salleh, Jaafar, Asri, & Ismail, 2015), empty
fruit bunch (Ching and Ng, 2014; Goh et al., 2016), forest residues
(Moriana, Vilaplana, & Ek, 2016), and arecanut husk (Chandra,
George, & Narayanankutty, 2016). Recently, Jiang and Hsieh (2013)
and Oun and Rhim (2016) investigated the effect of different
hydrolysis treatments on the crystalline index of nanocellulose
derived from rice straw cellulose, and the results showed that the
H2 SO4 -treated nanocellulose rendered higher crystallinity (90.7%)
than that of mechanical blending (82.5%) and TEMPO-mediated
oxidation (64.4%). It is important to produce high crystallinity
nanocellulose as this is the most signicant properties that have
greater inuence on the reinforcing mechanical capabilities than
other physicochemical properties such as aspect ratio, thermal sta-
bility and surface area (Moriana et al., 2016; Silvrio, Neto, Dantas,
& Pasquini, 2013).
As abovementioned that, a combination of alkalization, bleach-
ing process and acid hydrolysis was effective in isolation of
nanocellulose from numerous land biomass sources, therefore we
believe that same strategy could be applied to marine biomass to
is available enormously around the world, especially in the shal-
low coast with the 310 m depth below the water of many East
Asian countries, such as Malaysia, Singapore, Japan, China, and
Korea. The production of red algae has increasing exponentially
from 5.3 million tons in 2006 to 10.8 million tons in 2011 (Kim
et al., 2015). Typically, the exploitation of Gelidium marine biomass
as the main source of commercial agar production and food indus-
try will generates huge amount of solid residues after extracting the
agar component, which are currently not valorized and normally
indiscriminately discharged into environment (Ennouali, Ouhssine,
Ouhssine, & Elyachioui, 2006; Seo et al., 2010). In fact, this solid
residue can be fully utilized for the production of nanocellulose
because it consists of large amount of cellulose. Therefore, the
Gelidium red algae appear to be a new promising candidate for
nanocellulose production due to their abundance and availability
than terrestrial biomass. In 2010, Seo et al. (2010) was rst reported
on the utilization of two different species of red algae, namely Gelid-
ium amansii and Gelidium corneum for the production of bleached
pulp in papermaking industry. The results revealed that the paper
made by using the isolated red algae pulp possessed of particularly
high smoothness and opacity than typical wood pulp. However,
they did not examine the physicochemical characterizations (i.e.
1212 Y.W. Chen et al. / Carbohydrate Polymers 151 (2016) 12101219
surface morphological studies, crystallinity structure and thermal
properties) of isolated cellulose pulp.
In this study, Gelidium elegans was used to produce nanocel-
lulose. In addition, we propose for the rst time the isolation of
nanocellulose from this marine biomass via a three-stage puri-
cation process, namely alkalization, bleaching process and acid
hydrolysis treatment. We have replaced traditional bleaching
agent namely (NaClO2 ) with greener and more environmental-
friendly hydrogen peroxide (H2 O2 ) during pulping process. The
physicochemical properties and structural changes of prepared
nanocellulose from Gelidium red algae was examined in terms of
chemical functional groups (FTIR), crystallinity (XRD), surface mor-
phology studies (FESEM and TEM), and thermal stability (TGA)
systematically.
2. Experimental
2.1. Materials
The Gelidium elegans Ktzing red algae biomass was collected
from an algae culture lab. The biomass was washed repeatedly
with distilled water in order to remove dirt and contaminations
on the bers surface, and dried at 50 C in an oven. The dried
bers were grounded into the powder form by using a commer-
cial miller. All chemicals and reagents utilized in this study were
purchased from Merck Chemicals (Malaysia), and used as received
form without any further purication. Commercial alpha cellu-
lose (-cellulose, purity > 98%) and microcrystalline cellulose (MCC,
purity > 98%) was purchased from Sigma-Aldrich (USA).
2.2. Preparation of cellulose nanoparticle from untreated ber
2.2.1. Purication
The nely grinded algae biomass powder was rst pretreated
with dewaxing process in a soxhlet apparatus system by using
toluene/ethanol with ratio of 2:1 for 7 h. For alkalization treatment,
the dewaxed samples were treated with 2% NaOH solution at 80 C
for 2 h. Subsequently, the bleaching treatment of the samples was
performed by using 30% H2 O2 solution at 80 C for 1.5 h. The process
was repeated two times until snowy-white samples were obtained.
2.2.2. Isolation of nanocellulose
The preparation of nanocellulose ber was carried out as fol-
low: 1 g of extracted cellulosic material was dispersed in 100 mL
of 64 wt% sulfuric acid in a reaction ask with continuous stirring.
The hydrolysis reaction was carried out at 45 C for 45 min. Once
the reaction completed, the suspension was centrifuged in order
to remove the excess sulfuric acid. The collected solid residues
were treated with 1% NaOH solution until neutral pH, i.e. 67 was
achieved. A schematic for the isolation procedure was given in
Fig. 1.
2.3. Physicochemical characterization
2.3.1. Chemical composition
The chemical composition of the Gelidium ber before and after
each stage of purication process was investigated. In order to pre-
pare the extractive free biomass samples, the untreated Gelidium
ber was treated by reuxing in a Soxhlet system with toluene-
ethanol ratio of 2:1 (v/v) according to TAPPI 264 cm-97 for 6 h.
Holocellulose content was determined based on ASTM D1104-
56. The ground ber was treated with 5.0 g of sodium chlorite and
2.0 mL of acetic acid at 70 C for 4 h. The suspension was then cooled
to room temperature, ltered, and washed with deionized water
repeatedly before drying it in an oven at 105 C until a constant
weight was achieved.
-cellulose content was extracted from the resultant sample
after determining their holocellulose content by further treating
the bers with 50 mL of 17.5% NaOH solution according to the
ASTM D1103-55T. Then, the suspension was ltered and dried in a
weighed crucible until constant weight achieved.
Lignin content was analyzed according to ASTM D1106-56. The
ground ber was treated with 3 mL 72% H2 SO4 at 20 C for 2 h under
constant stirring. The mixture was then diluted to a 3% concentra-
tion of H2 SO4 and boiled for another 4 h under reux condenser.
Upon completion, the suspension was washed and ltered with hot
water and dried in an oven until a constant weight was achieved.
The nanocellulose yield was calculated gravimetrically by dry-
ing the resultant precipitate after the repeated centrifugation
(8000 rpm, 10 min) of the aliquot (10 g) at 40 C under vacuum (Kos,
Anzlovar, Kunaver, Huskic, & Z agar, 2014). On the other hand, the
yield of isolated cellulose from the untreated ber was determined
according to the following equation,
Celluloseyield(%) = wo /wt 100
where wo refers to the dry weight of the whiteness cellulose sample
and wt refers to the initial weight of the untreated ber prior to
purication process.
2.3.2. Fourier transform-infrared (FTIR) spectroscopy
The FTIR spectra of bers were examined using a Fourier
transform infrared spectrometer (Bruker IFS 66/S) from 4000 to
400 cm1 at a resolution of 4 cm1 . The samples were ground and
mixed with KBr with a ratio of 1: 100 (w/w), and then pressed into
ultra-thin pellets before the analysis.
2.3.3. Surface morphology analysis
Morphology of the bers was characterized by a FESEM on a
Hitachi SU8030. The sample was coated platinum and the analysis
was performed with an accelerating voltage of 5 kV. Additionally,
the dimensions of isolated nanoparticle were determined by a TEM
(Tecnai G2 F20 series) at an accelerating voltage of 200 kV. The
nanoparticle suspension was mildly sonicated for few minutes and
a drop of a diluted sample suspension was placed on a carbon-
coated copper grid.
2.3.4. X-ray diffraction (XRD) analysis
The crystallinity of bers was obtained using an Empyrean PAN-
alytical XRD in the range of 2 from 5 to 80 with at 40 kV and
40 mA. Crystallinity index (CrI) was calculated according to the
following equation:
CrI(%) = (I200 Iam )/I200 100
Where I200 is referred to the maximum intensity of the 200 lattice
diffraction peak at 2 = 22.5 and Iam is the intensity of diffraction
for amorphous part at 2 = 18.5 (Segal, Creely, Martin, & Conrad,
1959).
2.3.5. Thermogravimetric analysis (TGA)
The thermal stability of the bers was studied in terms of ther-
mogravimetric analysis (TGA) and its derivative thermogravimetric
analysis (DTG) by a thermal gravimetric analyzer (Model Q500
brand TA). All samples were heated from 25 to 600 C under N2
atmosphere with the heating rate of 10 C min1 .
3. Results and discussions
3.1. Chemical composition analysis
The chemical composition of Gelidium elegans on dry weight
basis at each stage of the purication procedure during the isolation
 Y.W. Chen et al. / Carbohydrate Polymers 151 (2016) 12101219
Fig. 1. Experimental procedure for individualizing Gelidium nanocellulose.
Table 1
Chemical composition analysis of untreated ber, alkali-treated, extracted cellulose and acid-treated nanocellulose.
Material -cellulose (%)
Untreated 17.2 2.3
Alkali-treated 55.7 1.1
Bleached 88.6 0.6
Acid hydrolyzed 90.8 0.8
a
The ash content was determined by thermogravimetry based on ASTM D5360 method.
of nanocellulose was summarized in Table 1. The mass fraction of
cellulose in the untreated ber was found to be 17.2% and hemicel-
lulose content of 29.5%, which comparable to the values reported
previously (Gao et al., 2015; Yoon et al., 2010). This red alga nor-
mally consists of higher hemicellulose content than that in green
and brown algae biomasses. Hemicellulose content in the red algae
biomass including Gelidium amansii and Porphyra umbilicalis were
24.37 and 47.16%, respectively (Gao et al., 2015). The differential on
the hemicellulose content of untreated ber observed in this study
might be due to the different origins and environmental conditions
(Li et al., 2009).
Obviously, when the dewaxed ber was subjected to the
alkalization treatment with NaOH, the hemicellulose content of
alkali-treated ber was reduced signicantly from 29.5 to 8.2%.
However, the primary removal of lignin in the treated ber does
not occur effectively during the alkalization treatment as the decre-
ment of lignin was low (4.53.4%). In order to efcient remove the
lignin content, H2 O2 bleaching process was conducted. As shown
in Table 1, most of the lignin was successfully degraded during the
oxidative bleaching process, as the percentage of lignin decreased
from 3.4 to 0.7%. Besides that, it was found that hemicellulose
content was also decreased to a certain extent from 8.2 to 2.0%
during the bleaching process. These nding reected that oxida-
tive bleaching process has capability of removing most of the lignin
and hemicellulose. It is not surprising because similar observa-
tion has been reported by Chings group (Ching and Ng, 2014). The
mass fraction of -cellulose in Gelidium increased exponentially
(17.288.6%) due to the successive hydrolyzation of hemicellulose
and lignin during the pretreatment. The hemicellulose and lignin
fraction of the ber were further reduced when the bleached ber
1213
Hemicellulose (%) Lignin (%) Ash (%)a Extractive (%)
29.5 1.4 4.5 0.3 30.4 0.5 17.6 0.9
8.2 0.6 3.4 0.6 27.4 0.9
2.0 0.2 0.7 0.3 8.3 0.2
1.3 0.1 <0.5 8.2 0.1
treated with acid hydrolysis. The nal yielded nanocellulose com-
prised of 90.8% of -cellulose, 1.3% of hemicellulose and <0.5% of
lignin, resulting in production of highly puried cellulose ber.
The cellulosic material was successfully extracted from the dried
Gelidium marine biomass at a 15.5% yield after alkalization and
bleaching process. On the other hand, the yield of isolated nanocel-
lulose, with respect to the initial amount of extracted cellulose,
was 52.1%. The yield of isolated Gelidium nanocellulose was higher
than that of nanocellulose isolated from rice straw cellulose (16.9%)
(Jiang & Hsieh, 2013), microcrystalline cellulose (28.6%) (Zhou,
2012), bamboo bleached ber (30%) (Brito, Pereira, Putaux, & Jean,
2012) following a similar acid hydrolysis procedure.
3.2. Fourier transform-infrared spectroscopy (FTIR) analysis
Fig. 2(a) and (b) shows the FTIR spectroscopic analysis of the
untreated, alkali-treated, bleached and acid hydrolyzed Gelidium
elegans ber. Generally, a broad peak at 3400 cm1 was observed
in all the samples spectra mainly due to hydrogen bond of O H
stretching vibration, which is indicates the hydrophilic tendency
of the bers (Chirayil et al., 2014). In addition, at the 2900 cm1
and 1640 cm1 was attributed to the sp3 C H stretching vibra-
tion and the OH bending of the absorbed water, respectively (Tan,
Abd Hamid, & Lai, 2015). The peak near 1160 cm1 , which appears
in all the spectra, associated with the C O C asymmetric stretch-
ing of the cellulose, and this can be explained the intensity of this
particular peak was gradually increased from untreated ber to
nanocellulose as the cellulose content of the treated ber getting
increased during the pretreatment (Saelee et al., 2016). Moreover,
the skeletal vibration of C O C pyranose ring skeleton (Yahya et al.,
1214 Y.W. Chen et al. / Carbohydrate Polymers 151 (2016) 12101219
Fig. 2. (a) FTIR spectra of untreated and chemically-treated Gelidium ber at different preparation stages, and (b) zoom of the 1900700 cm1 region.
2015) in cellulose ber was clearly indicated in the FTIR peak at
1050 cm1 for all the ber.
Others than similarity, several notable changes can be found in
the spectra of bleached and acid hydrolyzed bers compared to
untreated Gelidium ber. The absorbance at 1530 cm1 indicated
the existence of the stretching vibration of N H band of the pro-
tein amide II structure (Dmytryk, Saeid, & Chojnacka, 2014), and
this peak was also assigned to the C C stretching from aromatic
hydrocarbons of lignin (Ching & Ng, 2014). The absorption bands at
1245 cm1 was mainly due to the stretching vibration mode of the
acyl-oxygen CO OR associated with the hemicellulose (Mohamed
et al., 2015), and this band was also related to the presence of the
-l-galactose 6-sulfate units that primarily found in the agar com-
position (Guerrero, Etxabide, Leceta, Penalba, & de la Caba, 2014). A
peak located at approximately 872 cm1 was assigned to the pres-
ence d-galactose-4-sulfate of the carrageenan in the ber (Pereira,
Sousa, Coelho, Amado, & Ribeiro-Claro, 2003). These three peaks
were clearly observed in the untreated ber; however, the intensity
of these peaks was greatly reduced in alkali-treated ber and com-
pletely disappeared in bleached and acid treated bers. This result
clearly demonstrated that non-cellulosic polysaccharides such as
lignin and hemicellulose were partially removed after the alkaliza-
tion and then thoroughly removed after bleaching treatment.
A peak at 1110 cm1 indicated that presence of -glucosidic
ether linkages (C O C) resulted in the vibration of wagging,
deformation and twisting modes of anhydro-glucopyranose ring
skeleton (Tan et al., 2015). In addition, a small peak at 898 cm1 was
owing to the -glycosidic linkages between the anhydroglucose
rings in the cellulose, and also represents the glycosidic 4 C1 ring
conformation deformation (Chirayil et al., 2014; Hamid, Zain, Das,
& Centi, 2015). In the case of bleached and acid hydrolyzed bers,
the strong absorption of the two prominent peaks at 1110 cm1
and 898 cm1 indicated that more cellulose content was exposed
in the treated ber after the successive pretreatments to remove
the non-cellulosic polysaccharides, whereas the absorption peak
at 898 cm1 remained unaltered for the acid hydrolyzed ber indi-
cated that the typical structure of -d-glucopyranosyl structure
in cellulose parent chain can be retained after the acid hydroly-
sis treatment (Hamid et al., 2015; Saelee et al., 2016). In addition,
the absorbance peaks at 1428 cm1 , 1371 cm1 and 1315 cm1 in
bleached and acid hydrolyzed bers were mainly contributed by
the presence of cellulose (Chen, Lee & Abd Hamid, 2016a; Chen,
Lee & Hamid, 2016b).
The peak at 1315 cm1 (Saelee et al., 2016) and 1428 cm1
(Fortunati et al., 2013; Saelee et al., 2016) was associated with
C H bending of cellulose and CH2 symmetric bending of cellu-
lose, respectively. Meanwhile the peak at 1371 cm1 represented
the C H asymmetric deformation and C O symmetric stretching
within the polysaccharide aromatic rings of cellulose (Tan et al.,
2015). It is interesting to observe that untreated seaweed did not
exhibit peaks at 1315 cm1 and 1371 cm1 , as shown in Fig. 2b. This
is due to the majority of the cellulose content was embedded by
the agar as well as others non-cellulosic materials in the untreated
ber. Besides that, the cellulose extracted (bleached ber) from the
Gelidium ber was comparable with the commercial available alpha
cellulose (-cellulose) and microcrystalline cellulose (MCC). Based
on the FTIR characteristics, Gelidium extracted cellulose pulp pat-
terns exactly matched with that of -cellulose and MCC (Fig. 2).
This suggested that extracted cellulose and nanocellulose has been
successfully produced from red seaweed.
This results were concordance with other literatures (Chen et al.,
2016a; Chirayil et al., 2014; Hamid et al., 2015; Tan et al., 2015;
Yahya et al., 2015), in which the extracted cellulose and nanocel-
lulose showed a similar FTIR patterns. It is important to note that
the extracted nanocellulose shows absorbance signal at 1428 cm1 ,
1160 cm1 , 1110 cm1 , and 898 cm1 (Fortunati et al., 2013) indi-
cated that nanocellulose produced was primarily in the form of
cellulose I structure.
3.3. Field emission scanning electron microscopy (FESEM)
analysis
Fig. 3 illustrates the FESEM images of the Gelidium elegans ber
at different pretreatment stages. Fig. 3(a) shows the untreated ber
displayed a smooth bers surface due to the presence of oil and
waxes. The presence of oil and waxes in the raw ber is com-
mon and vastly reported (Saelee et al., 2016). Alkalization removed
majority of the agar and non-cellulosic components from the inner
parts of the Gelidium ber via debrillation and depolymerization.
Depolymerization and debrillation by alkalization is shown in
Fig. 3(b). It was observed that alkalization has loosened the biomass
structure, which resulted in swelling and solubilizing agar materi-
als of the treated ber. This eventually facilitating the destruction
of the bers structure, partial cracking of the bers surface layer
and dissolves the agar compound. Thus, bleaching agents will react
 Y.W. Chen et al. / Carbohydrate Polymers 151 (2016) 12101219
Fig. 3. FESEM images of (a) untreated, (b) alkali-treated, (c) bleached and (d) acid hydrolyzed Gelidium ber.
more readily into the alkali-treated ber by penetrating deeper into
the inner parts through the cracks of the bers.
Fig. 3(c) clearly indicated that more ber bundles were sep-
arated into individualized brils structure due to removal of
cementing materials after alkalization and bleaching treatment.
Each of this cellulose ber appeared to be composed of several to
hundreds microbrils, and leads to the formation of cellulose com-
pact structure. The average diameter of untreated seaweed ber
and extracted cellulose were 124 3.8 m and 5 1.5 m, respec-
tively. The diameter reduced was mainly due to the destruction of
the primary cell wall of the ber during removal of non-cellulosic
materials. Similar observation on the destruction of the bers bun-
dles structure after alkali-treatment and bleaching process was also
reported by others (Chirayil et al., 2014; Sheltami et al., 2012; Zaini,
2013).
The average diameter of the ber further reduced to
1.5 0.2 m after the acid treatment, as shown in Fig. 3(d). The
aggregation of extracted cellulose was broken down in great extent
during acid hydrolysis treatment. The hydronium ions (H3 O+ ) from
the sulfuric acid (H2 SO4 ) responsible for the penetrating amor-
phous structure at higher rate compared to crystalline areas during
acid hydrolysis. This will lead to hydrolytic cleavage of the gly-
cosidic bonds in amorphous structure which exists as loose and
low ordered orientation parts. As a result, the micro-sized cellu-
lose bers were separated from the bril bundles and gave rise to
a fragmentary breakdown in brillar structure. This ndings are
in accordance with previously study (Tan et al., 2015), in which
nanocellulose has been extracted from cotton linter.
3.4. Transmission electron microscopy (TEM) analysis
Based on TEM micrographs, it was evidenced that nano-sized
brils were successfully isolated from the Gelidium elegans ber
(Fig. 4). The results revealed that the particle diameter of nanocellu-
lose was in the range of 565 nm with a calculated average diameter
of 21.8 11.1 nm, and had the highest content of 49% at the interval
of 1120 nm. In addition, the nanocellulose exhibited an aver-
age length of 547.3 23.7 nm, in which most of the bers length
1215
was found in the range 501600 nm. Thus, the aspect ratio of the
resultant nanocellulose was relatively high, which is about 25. The
isolated nanocellulose seems to be ner and possesses a porous net-
work, which implied that hydronium (H3 O+ ) ions has successfully
diffused into the compact and rigid structure of extracted cellu-
lose. This has resulted selective elimination of amorphous regions
of cellulose long brils into smaller sizes. This results appeared to be
similar to previous study (Yahya et al., 2015), in which nanocellu-
lose was also found in ber network arrangements (spider web-like
structure) with variable width between 10 and 60 nm.
Table 2 summarizes the values of geometrical characteris-
tics for the nanocellulose derived from various biomass sources
and collected from the literature. The aspect ratio of nanocellu-
lose derived from Gelidium (L/d = 25) in this study was similar
to that reported for Eucalyptus wood (L/d = 24) (Beck-Candanedo,
Roman, & Gray, 2005), which under the similar hydrolysis pro-
cess. However, the aspect ratio of yielded nanocellulose was larger
than the value reported for the nanocellulose isolated from rich
husk (L/d = 1015) (Johar et al., 2012), cotton (L/d = 10.7) (Martins,
Teixeira, Corra, Ferreira, & Mattoso, 2011), Curaua (L/d = 1317)
(Correa, de Morais Teixeira, Pessan, & Mattoso, 2010) and kenaf
bast (L/d = 13) (Kargarzadeh et al., 2012) via H2 SO4 hydrolysis under
similar hydrolysis conditions. In order to conrm the effective-
ness of acid hydrolysis, the dimensions of isolated nanocellulose
was compared with the nanomaterial isolated via different hydrol-
ysis approach such as ionic liquid BmimHSO4 (Tan et al., 2015),
Ni(NO3 )2 transition metal salt (Yahya et al., 2015) and glycols com-
bined with methane sulfonic acid (Kunaver, Anzlovar, & Z agar,
2016). The results revealed that the acid-hydrolyzed nanocellulose
had larger aspect ratio than other promising hydrolysis approach
(Table 2).
Moriana et al. (2016) reported that the cellulosic structures with
an aspect ratio values above 10, the minimum value for a good
stress transfer within the interaction between the bers and the
matrix, able to provide a good reinforcing capability and improve-
ment in the thermal and mechanical properties when adding into
the polymer matrices, even in low concentrations. Therefore, the
high aspect ratio of nanocellulose isolated from Gelidium could pro-
1216 Y.W. Chen et al. / Carbohydrate Polymers 151 (2016) 12101219

Fig. 4. (a) TEM micrograph of nanocellulose, (b) length and (c) diameter distribution of isolated Gelidium nanocellulose.

Table 2
Geometrical properties of cellulose nanomaterial derived from different crude cellulose starting materials.

Starting materials Reaction conditions Width (nm) Length (nm) Aspect ratio Hydrolyzing catalyst Reference

Temperature ( C) Time (min)

Gelidium elegans 45 45 21.8 547.3 25 H2 SO4 This study

Eucalyptus wood 45 45 6 145 24 H2 SO4 Beck-Candanedo et al. (2005)
Curaua 45 75 610 80179 1317 H2 SO4 /HCl Correa et al. (2010)
Chinese silver grass 150 180 1423 220300 15 Glycols/methane sulfonic acid Kunaver et al. (2016)
Rice husk 50 40 1520 n/a 1015 H2 SO4 Johar et al. (2012)
Kenaf bast 45 40 12 158 13 H2 SO4 Kargarzadeh et al. (2012)
Cotton linter 45 45 14.6 3.9 171.6 48.2 1112 H2 SO4 Roohani et al. (2008)
Cotton 45 75 14.6 171.6 10.7 H2 SO4 Martins et al. (2011)
MCC 45 15 42.1 3.9 383.7 34.1 9.1 Ni(NO3 )2 Yahya et al. (2015)
Cotton linter 90 75 1520 7580 5 BmimHSO4 Tan et al. (2015)

vide a better reinforcement and make them as ideal candidates for

polymer bio-nanocomposite. This is because high aspect ratio offers
a high specic surface area results in a good reinforcing capability
effect (Silvrio et al., 2013) and enhance the thermo-mechanical
properties on the polymer composite (Moriana et al., 2016).
In summary, the TEM images evidenced that the effective-
ness of acid hydrolysis treatment by hydrolyzed micron bers
into nanometer scale cellulose. The agglomeration of larger brils
was likely composed of elementary brils bundles linked together
through strong hydrogen bonding of the cellulose ber, and leads to
re-bonding of the inter-bril hydroxyl groups (Chirayil et al., 2014;
Saelee et al., 2016). This was also observed for the extraction of
nanocrystalline cellulose from the cotton linter (Tan et al., 2015).

3.5. X-ray diffraction (XRD) analysis

The XRD diffraction patterns of untreated algae biomass and

samples obtained after the pulping process (alkalization, bleach-
Fig. 5. X-ray diffraction patterns of untreated, alkali-treated, bleached and acid
ing and acid hydrolysis treatment) are given in Fig. 5. It can be
hydrolyzed Gelidium ber. The parentheses refer to crystallinity index (CrI)(%).
observed that all samples showed a major peak at 2 = 22.5 , which
related to the crystalline structure of cellulose I, whereas the amor-
 Y.W. Chen et al. / Carbohydrate Polymers 151 (2016) 12101219 1217

120 0.8 120 2

Ton: 225 oC Ton: 243 oC
Tmax: 308 oC Tmax: 317 oC

Derivative weight (% / oC)

Derivative weight (% / oC)

100 100
0.6
1.5

Weight loss (%)

Weight loss (%)
80 80
0.4
60 60 1
Char yield: Char yield:
0.2
40 30.4% 40 27.4%
0.5
20 0 20

0 -0.2 0 0
0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800
(a) Temperature (oC) (b) Temperature (oC)

120 1.9 120 3.4

Ton: 246 oC Ton: 261 oC

Derivative weight (% / oC)

Derivative weight (% / oC)
100 Tmax: 319 oC 100 Tmax: 334 oC 2.9
1.4

Weight loss (%)

2.4
Weight loss (%)

80 80
1.9
60 0.9 60
1.4
40 40
0.9
0.4 Char yield:
20 Char yield: 20 8.2% 0.4
8.3%
0 -0.1 0 -0.1
0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800
(c) Temperature (oC) (d) Temperature (oC)

Fig. 6. Thermogravimetric analysis of (a) untreated, (b) alkali-treated, (c) bleached and (d) acid hydrolyzed ber.

phous regions of the samples was characterized at the 2 value of
around 18.5 (Segal et al., 1959). The presence of cellulose I was also
aligned with FTIR results. There are other peaks from untreated raw
seaweed which mainly corresponded to two mineral compounds,
namely silica (SiO2 ) and weddellite (CaC2 O4 2H2 O). The presence
of these impurities is generally found in the sea biomass (Bettaieb,
Khiari, Dufresne, Mhenni, Putaux et al., 2015; Bettaieb et al., 2015).
After the pulping process, the intensity of reection peaks pre-
sented the mineral salts was gradually reduced, and the cellulose
I form was well preserved. The XRD characterization revealed the
presence of main intensity peaks of cellulose at 2 values of 16.5
(110), 22.5 (200) and 34.5 (004) for bleached and acid hydrolyzed
bers, corresponding to a typical cellulose I structure (Chen et al.,
2016a). This nding suggested that the cellulose was successfully
extracted from red seaweed pulp after the alkalization, bleaching
treatment, and acid hydrolysis process. All these results were in
good agreement with the FTIR result, in which the non-cellulosic
materials were removed.
The crystallinity indexes for the samples are also listed in Fig. 5.
The calculated crystallinity index for untreated algae biomass,
alkali-treated, bleached and acid hydrolyzed ber was found to
be 33, 51, 63 and 73%, respectively. These results clearly demon-
strated that the crystallinity of the treated material progressively
improved during the chemical extraction process. This is because
of non-cellulosic polysaccharides components, and amorphous
regions in the ber were removed. Besides that, it was observed
that acid hydrolyzed ber had the maximum crystallinity index
(73%). As explained earlier, hydronium ions (H3 O+ ) has success-
fully penetrated more readily into the amorphous regions of the
extracted cellulose, and then hydrolytic cleaved of the -1,4-
glycosidic linkages within the cellulose chains to releases the
individual crystallites segments (Tan et al., 2015). It is interesting
to obtain high crystallinity of nanocellulose from Gelidium algae
biomass because high crystallinity will eventually provide high ten-
sile strength as well as better reinforcement for natural composite
materials (Yahya et al., 2015).
3.6. Thermogravimetric analysis (TGA)
Fig. 6 shows the thermogravimetric (TG) and its derivative
thermogravimetric (DTG) curves of the untreated, alkali-treated,
bleaching-treated and acid treated Gelidium ber. Weight loss was
observed in all the samples at the temperature of <100 C which was
corresponded to evaporation of moisture bounded loosely on the
ber surface and the chemisorbed water bounded inside the ber
(Chandra et al., 2016). The presence of absorbed water was also
detected by FTIR characteristic peak at 1640 cm1 which represent
bending vibration of water intermolecular H-bonding.
In this study, untreated ber started to degrade at 225 C, and
the DTG curve revealed a major decomposition peak at 308 C.
Besides that, a broad peak was also observed at ca. 640 C which
corresponded to decomposition of inorganic compounds such as
weddellite into CaO that mainly occurred at 650 C (Frost and
Weier, 2003). For the alkali treated ber, the onset thermal decom-
position (Ton ) shifted to higher temperature of 243 C with the
maximum rate of degradation temperature (Tmax ) at 317 C. This
was due to the agar component in treated ber started to solubilize
during alkalization process. This is not surprising because similar
thermal degradation trend has also been reported for the sea algae
biomass samples (Muradov et al., 2015).
It is noteworthy that decomposition of bleached ber was seen
at 246 C with a Tmax value of 319 C. This nding indicated that
H2 O2 -bleaching process could eventually enhance the thermal
stability of the bleached ber due to the successive removal of non-
cellulosic impurities and the inorganic mineral salts. These results
are well agreed with XRD result (Fig. 5) in which the disappear-
1218 Y.W. Chen et al. / Carbohydrate Polymers 151 (2016) 12101219
ance of impurities upon decomposition at 640 C (Fig. 6(c)). The
bleached ber shows higher thermal stability due to its high crys-
tallinity (Saelee et al., 2016). As shown in Fig. 6(d), the nanocellulose
from acid treatment exhibited a high thermal stability in which it
only started to degrade at 261 C and high Tmax value of 334 C.
There are three plausible reasons for this thermal stability enhance-
ment. Firstly, the nanocellulose became more denser and compact
after successive removal non-cellulosic components in the ber
(Chandra et al., 2016). Secondly, the impurities in the bers may ini-
tiate more active sites towards the heating process and resulted in
accelerating the thermal degradation (Chirayil et al., 2014). Thirdly,
high composition of crystalline domains of nanocellulose due to
reorientation and rearrangement of the nanocellulose crystals after
the dissolution of amorphous regions (Mandal & Chakrabarty,
2011). In contrast with previous studies (Oun and Rhim, 2016;
Reddy & Rhim, 2014; Soni & Mahmoud, 2015), H2 SO4 hydrolysis
has normally led to thermal stability reduction of nanocellulose
as compared to that of neat bers and their corresponding cellu-
lose due to the introduction of active sulfate groups. However, they
have treated the sulfated nanocellulose with NaOH solution (nor-
mally 1%) which may lead to the diminishing of the effect of active
sulfate groups (Oun & Rhim, 2016; Soni & Mahmoud, 2015).
The percentages of carbonaceous residues (char) remained after
heating up to 500 C and the carbonization of the cellulose com-
ponents under the nitrogen atmosphere are shown in Fig. 6. The
highest percentage of char was obtained for the untreated ber
(30.4%), and generated high percentage of char yield as compared to
typical lignocellulosic biomass and grass biomass. Marine biomass
ber contain higher salinity (Daneshvar, Salak, & Otsuka, 2012) and
thus the high amount of char content owing to the presence of
impurities on the bers. However, the decrement of char amount
could be observed after a series multiple purication stages, and
this mainly attributed to the removal of non-cellulosic compo-
nents from the bers. The lowest char yield (8.2%) was found in the
acid-hydrolyzed ber because of the absence of non-cellulosic com-
ponents and leads to better accessibility of the cellulose content in
the treated bers (Chirayil et al., 2014).
4. Conclusions
The present work revealed that nanocellulose can be success-
fully isolated from a new, renewable, low-cost and abundant
natural source, Gelidium elegans red algae biomass via alkalization,
bleaching treatment and acid hydrolysis treatment. The physic-
ochemical characterization results showed that the crystallinity
index of isolated nanocellulose was 73% with an average brils
diameter of 21.8 11.1 nm and average length of 547.3 23.7 nm.
Thus, the corresponding aspect ratio of isolated nanocellulose was
about 25.TGA study showed that the neutralized nanocellulose
products exhibited in better thermal stability than the untreated
and other chemically-treated bers. Due to its high crystallinity and
better thermal stability, the isolated Gelidium cellulose nanoparti-
cle is a new source and has great potential for various applications
such as reinforcement agent in nanocomposites manufacturing and
nano-llers for polymer matrices.
Acknowledgements
The authors are grateful for the nancial support of the Min-
ister of Science, Technology and Innovation (MOSTI) e-Science
Fund (SF002-2015), Grand Challenges Grant (GC002A-15SBS) and
University of Malaya, Postgraduate Research Grant Scheme PPP
(PG063-2015A).
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