Bioresource Technology 100 (2009) 6669–6673

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Short Communication

Profiling of cellulose content in Indian seaweed species

A.K. Siddhanta *, Kamalesh Prasad, Ramavatar Meena, Gayatri Prasad, Gaurav K. Mehta,
Mahesh U. Chhatbar, Mihir D. Oza, Sanjay Kumar, Naresh D. Sanandiya
Marine Biotechnology and Ecology Discipline, Central Salt and Marine Chemical Research Institute, Council of Scientific and Industrial Research,
G.B. Marg, Bhavnagar 364002, Gujarat, India

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

Article history: Cellulose contents were estimated in 12 seaweed samples belonging to different families e.g. red, brown
Received 31 March 2009 and green, growing in Indian waters. Each cellulose sample was fractionated to yield alpha (a) and beta
Received in revised form 17 July 2009 (b) celluloses. Characterization was done using various analytical tools and results were validated by
Accepted 20 July 2009
comparison with those of the cellulose obtained from Whatman filter paper No. 4. The greatest yields
Available online 14 August 2009
of cellulose (crude), a- and b-cellulose were obtained from Gelidiella acerosa (13.65%), Chamaedoris auric-
ulata (9.0%) and G. acerosa (3.10%). G. acerosa was also found to contain relatively high amount of a-cel-
Keywords:
lulose (8.19%). The lowest cellulose contents were recorded from Kappaphycus alvarezii (2.00%) and
Cellulose
Seaweeds
Sarconema scinaioides (2.1%), while the latter contained the lowest a-, and b-celluloses (1.0% and
Chlorophyceae 0.30%, respectively). It appears that agarophytic and alginophytic algae contain high cellulose and a-cel-
Phaeophyceae lulose contents, while the carrageenophyte contains low cellulose. The brown algae, in general contain
Rhodophyceae high cellulose as well as a- and b-celluloses.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction
Cellulose is a naturally occurring polysaccharide and the most
abundant organic substance on the earth, which consists of a chain
of b-(1 ? 4)-linked glucose residues (Staudinger, 1932 ; Gilbert
and Kadla, 1998; Klemm et al., 2005). Due to the abundance, low
cost and easier processability, this has been attracting the atten-
tion of the researchers for a long time and it is used extensively
in various applications. It can be isolated not only from terrestrial
plants but also from algae to some extent (Zuluaga et al., 2007). In
fact algae are considered as a potential source for cellulose, which
is useful in preparation of various materials (Berglund, 2005).
Whistler and Charles (1953) have extracted cellulose for the first
time from green algae of the class Valonia, Halicystis, Cladophora,
as well as from the brown alga Laminaria followed by many. More
recently, Ek et al. (1998) as well as Mihranyan and his co-workers
(2004) have also reported extraction of cellulose from seaweeds.
Cellulose is crystalline in nature and it exists as a mixture of two
crystalline forms, a and b. a-Cellulose has one-chain triclinic
structure, while b-cellulose has two-chain monoclinic structure
(Sugiyama et al., 1991). Various crystalline features of algal cellu-
loses were thoroughly evaluated by Koyama et al. (1997) and were
found in 1–20% yields in most of the seaweeds investigated. The
cellulose obtained from algal species contains porous or sponge
* Corresponding author. Tel.: +91 2782567760; fax: +91 2762567562.
E-mail address: aks@csmcri.org (A.K. Siddhanta).
like network, which is substantially different from the higher plant
cellulose (Strømme et al., 2002).
Cellulosic materials are considered to be an important medium
in chiral chromatography (Hesse and Hagel, 1973) apart from their
traditional applications. Many unique applications require cellu-
losic materials with higher structural order (Kreger, 1962; Vander-
Hart and Atalla, 1987). India has a long coastline extending to
5700 km and is habitat of a numerous species of seaweeds (Meena
and Siddhanta, 2006). Apart from being a source of many bioactive
molecules, these seaweeds are an important source of various
polysaccharides. We report herein the evaluation of seaweeds of
Indian waters as a potential source of cellulose in an ongoing pro-
gram of our laboratory on value addition of seaweeds. In this study
12 seaweed species belonging to different families e.g. Chlorophy-
ceae, Phaeophyceae and Rhodophyceae, commonly designated as
green, brown and red seaweeds, respectively, were studied. To
the best of our knowledge, this is the first report of profiling of cel-
lulose of Indian seaweed species.
2. Methods
The seaweeds of this investigation were collected from the In-
dian coasts and the details are: Chaetomormpha antennina (Bori
de saint-vincent), Kützing and Chamaedoris auriculata Børgesen
(both from 20.42°N, 70.58°E) belong to the family Chlorophyceae.
Dictyota dichotoma (Hudson) Lamouroux (08.51°N and 78.14°E),
Dictyota bartayresiana Lamouroux (9.17°N and 79.15°E) and

0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.07.047
6670 A.K. Siddhanta et al. / Bioresource Technology 100 (2009) 6669–6673

Sargassum tenerrimum J. Agardh (20.54°N and 70.20°E) belong to washed with water till neutrality, filtered and dried at room tem-
Phaeophyceae. Gracilaria edulis (S. Gmelin) P. Silva (09.9°N and perature. The dried product was re-suspended in 200 ml hydro-
78.43°E), Gelidiella acerosa (Forsskål) J. Feldmann and G. Hamel chloric acid (5% v/v) and was heated up to boiling and resultant
(20.54°N and 70.20°E), Kappaphycus alvarezii (Doty) Doty ex P. Sil- slurry was kept overnight at ambient temperature (30 °C), fol-
va com. nov. (09.9°N and 78.43°E), Gracilaria textorii (Suringar) De lowed by water washing for removing the excess acid, filtered
Toni (22.28°N and 68.04°E), Gracilaria debilis (Førsskål) Børgesen and freeze dried to get cellulose. Yields were calculated on the ba-
(22.28°N and 68.04°E), Champia indica Børgesen (22.28°N and sis of as received seaweeds.
68.04°E) and Sarconema scinaioides Børgesen (20.54°N and
17.20°E) belong to the family Rhodophyceae. Voucher specimens
4. Fractionation of cellulose
of all these species have been deposited with the CSMCRI Herbar-
ium. All the seaweeds were washed with tap water to remove the
The a and b fractions from celluloses were obtained by employ-
solid impurities from the plants and were dried in the shade and
ing the method reported by Whistler (1963). The dried cellulose
powdered in a rotating ball mill and stored for the cellulose isola-
(1 g) was soaked in 30 ml alkali (17.5% NaOH) solution at 20 °C
tion in separate plastic containers. Cellulose extracted from What-
for 2 h, followed by occasional shaking in every 15 min. The result-
man filter paper No. 4 was used as reference. Methanol, sodium
ing slurry was centrifuged at 8000 rpm for 15 min. The supernatant
chlorite, sodium acetate, sodium hydroxide, hydrochloric acid, sul-
containing b-cellulose was removed by decanting, and a-cellulose
phuric acid and sodium hypochlorite of LR grade were used and
(residue) was obtained after repeated water washing until pH of
were purchased from Ranbaxy Fine Chemicals Ltd., Mohali, Punjab
the washing was ca. 7, and the product was collected by freeze dry-
(India).
ing. b-Cellulose was precipitated from the supernatant with 3 N
H2SO4 (20 ml), the mixture was further kept at 80 °C for 10 min
to ensure complete precipitation of b-cellulose. The precipitated
3. Isolation of cellulose from seaweeds
b-cellulose was recovered by centrifugation followed by washing
with water to make it acid free; finally the product was collected
Cellulose was isolated from the seaweeds following the method
by freeze drying. Cellulose from the Whatman filter paper No. 4
described by Mihranyan et al. (2004). Dried algal powder (taking
was also fractionated following the same method as described
100 g of each seaweed in separate experiments) was defatted with
above. Yields were calculated on the basis of as received seaweeds.
repeated extraction with MeOH (500 ml4) in a percolator for a
period of eight days at room temperature (two days for each cycle).
The defatted algal powder was soaked in 1 L acetate buffer contain- 5. Characterization of cellulose
ing 36 g NaClO2 for bleaching at 60 °C for 3 h. The bleached algal
mass was washed with water until the washing showed pH  7. The FT-IR spectra of all the cellulose samples were recorded on
The washed algal mass was treated with 600 ml NaOH (0.5 M) a Perkin–Elmer Spectrum GX FTIR (USA) instrument. Cellulose, a-
solution at 60 °C overnight. The alkali treated algal mass was and b- celluloses of C. antennina were characterized by solid state

a
40
alpha/beta cellulose ratio

35 alpha/beta cellulose ratio

Cellulose
α-Cellulose 30
15
β-Cellulose 25

20

15

10
10 5

0
Yield (%)

A B C D E F G H I J K L --
Seaweeds

5
b 0.81
0.80 Cellulose
0.79 alpha-Cellulose
0.78
Crystallinity Index (CI)

0.77 beta-Cellulose
0.76
0.75
0 0.74
0.73
0.72
0.71
0.70
0.69
A B C D E F G H I J K L 0.68
0.67
Seaweeds 0.66
0.65
0.64
0.63
W A B C D E F G H I J K L
Seaweeds

Fig. 1. Yields (%) with respect to as received seaweed, of cellulose, a- and b-cellulose: (W) Whatman filter paper No. 4; (A) Chaetomorpha antennina; (B) Chamaedoris
auriculata; (C) Dictyota dichotoma; (D) Dictyota bartayresiana; (E) Sargassum tenerrimum; (F) Gracilaria debilis; (G) Gracilaria textorii; (H) Gracilaria edulis; (I) Gelidiella acerosa;
(J) Champia indica; (K) Kappaphycus alvarezii and (L) Sarconema scinaioides; Insets: (a) a/b cellulose ratios and (b) The crystallinity indices.
 A.K. Siddhanta et al. / Bioresource Technology 100 (2009) 6669–6673 6671

NMR (CP-MAS 13C NMR) measurements at 20 °C on a Brüker
Avance 500 MHz, Spectrometer (Switzerland) at 52.3 MAS, net
spinning was kept at 5000 rpm/min. The cellulose obtained from
Whatman filter paper was used as the reference sample. Philips
X’pert MPD X-ray powder diffractometer was used to study the
crystallinity of the samples using 2h = 10°–45°. Crystallinity indices
(C.I.) were calculated using the following Eq. (1) (Mihranyan et al.,
2004).
C:I: ¼ I002  Iam =I002
where I002 is the overall intensity of the peak at 2h about 22° and Iam
is the intensity of the baseline at 2h about 18°.
The surface morphology of the cellulose samples was studied on
a scanning electron microscope (SEM) instrument (Carl-Zeiss Leo
VP 1430) applying an accelerating voltage of 10 or 20 kV and mag-
nification 1–38 K, respectively.
No. 4. Superimposable FT-IR spectra of both the materials con-
firmed the identity of the cellulose extracted from seaweeds with
that of Whatman filter paper No. 4 (cf. J.X. Sun et al. 2005). It
may be noted that, the IR spectra of the a- and b-celluloses were
identical with that of the crude cellulose. The major bands of b-cel-
1
lulose appeared at mKBr
max (cm ) 3446 (br, s), 2901 (m), 1623 (s),
1419 (s), 1112 (br, s), 901 (w) and 616 (m).
The CP-MAS 13C NMR spectra of seaweed cellulose and refer-
ence cellulose samples were identical, and the values of chemical
ð1Þ
shifts were in good agreement with those reported in the literature
(Sun et al., 2005; Kono et al., 2002; Witter et al., 2006). The solid
state NMR of a- and b-cellulose obtained from Chetomorpha

6. Results and discussions

The yields (%) of cellulose (crude), a- and b-cellulose samples,

obtained from different seaweed species were estimated (Fig. 1).
The greatest yields of cellulose (crude), a- and b-cellulose were ob-
tained from G. acerosa (13.65%), C. auriculata (9.0%) and G. acerosa
(3.10%), respectively. G. acerosa also contained relatively high
amount of a-cellulose (8.19%). Lowest cellulose contents were re-
corded from K. alvarezii (2.00%) and S. scinaioides (2.1%), while
the latter contained the lowest a-, and b-celluloses (1.0% and
0.30%, respectively). C. auriculata and C. antennina, the green algae
contained predominantly a-cellulose. It appears that agarophytic
and alginophytic algae contain high cellulose and a-cellulose con-
tents, while the carrageenophyte contains low cellulose. The
brown algae, in general contain high cellulose as well as a- and
b-celluloses. The a/b cellulose ratio was the highest for C. antennina
and the lowest for G. debilis and G. textorii (Fig. 1a).
Fig. 3. Representative XRD profiles of the crude cellulose samples obtained from
The FT-IR spectra of all the samples were recorded and com- green algae (Chamaedoris auriculata), (b) brown algae (Dictyota dichotoma), (c) red
pared that with the cellulose obtained from Whatman filter paper algae (Gelidiella acerosa) and (d) Whatman filter paper.

13
Fig. 2. CP/MAS C NMR spectra of cellulose: (a) Whatman filter paper No. 4 cellulose; (b) seaweed cellulose; (c, d) seaweed a-cellulose and b-cellulose.
6672 A.K. Siddhanta et al. / Bioresource Technology 100 (2009) 6669–6673

antennina showed single broad peak between 70 and 80 ppm pre-
sumably due to the overlapping resonances of C-2, C-3 and C-5 car-
bons (Kono et al., 2002). Chemical shift values between 58.08 and
62.25 ppm for C-6 were obtained (Fig. 2), which were similar to
those reported by Kono et al. (2002). The chemical shift values of
the remaining carbons (C-4 and C-1) were also comparable with
those reported in the literature (Kono et al., 2002; Sun et al. 2005).
The XRD profile of celluloses obtained from all the seaweeds as
shown in Fig. 3 exhibits the typical diffraction peaks at around 15°
and 23° due to the crystalline structure of cellulose I, which is
known to be the native and predominant crystalline structure of
cellulose present in algae (Gilbert and Kadla, 1998) (Fig. 3b and
c). Furthermore, the a-form of cellulose-I is reportedly the domi-
nant polymorph in algal cellulose (vide Fig. 1). However, the little
amount of b-celluloses obtained from the algae were relatively less
crystalline than their a-counterparts (Figs. 4 and 5). The crude cel-
lulose obtained from C. indica showed the greatest crystallinity in-
dex (C.I. = 0.68), while it was the lowest (C.I. = 0.64) for the
cellulose of G. textorii (Fig. 1b). The greatest C.I. was observed in
the a-cellulose of C. auriculata, while it was the lowest for that of
G. textorii (Fig. 1b).
Micro-fibers having width in the range 0.1–0.15 lm were visi-
ble in the SEM images of the celluloses obtained from C. antennina,
C. auriculata and C. indica, which consisted predominantly of a-cel-
lulose. The patterns were similar to that of the one obtained from
green alga Cladophora sp. reported by Mihranyana et al. (2007). On
the other hand, the celluloses obtained from the rest of the sea-
weeds of this study, including that from Whatman filter paper
exhibited non-fibrous structures. Furthermore, the SEM images of
the a- and b-celluloses fractionated from the celluloses of most
of the seaweeds showed altogether a different morphology. The
a-cellulose were found to have short needle-type structures hav-
ing width 2–2.5 lm, while the b-cellulose had fibrous structures
of width 8–9 lm. Further studies will be needed to clearly under-
stand these differences of morphology.

7. Conclusions

a This study has unveiled an overall trend on the cellulose profile

of a group of Indian seaweeds. It appears that agarophytic (espe-
Intensity

cially G. acerosa) and alginophytic algae contain high cellulose

and a-cellulose contents, while the carrageenophyte was low in
cellulose. The brown algae, in general contained high cellulose,
b a- and b-celluloses. More work involving greater number of sea-
weed species, however, will have to be done to affirm these pre-
mises. Thus some of the seaweeds investigated may be useful as
a source of cellulose for industrial applications.

c Acknowledgements

0 10 20 30 40 50 60
Ministry of Earth Sciences (MoES/9-DS/6/2007-PC-IV) and CSIR
(NWP-37), New Delhi are gratefully acknowledged for financial
2θ (Deg., CuKα)
support. The authors are also grateful to Dr. K. Eswaran, Dr. M.
Fig. 4. Representative XRD profile of a-cellulose extracted from (a) green algae Ganesan and Mr. Vaibhav Mantri for their help in seaweed collec-
(Chamaedoris auriculata), (b) brown algae (Dictyota dichotoma) and (c) red algae tion and identification.
(Gelidiella acerosa).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.biortech.2009.07.047.

a References

Berglund, L., 2005. Cellulose based nano composites. In: Mohanty, A.K., Misra, M.,
Drzal, L.T. (Eds.), Natural Fibers, Biopolymers, and Bio composites. CRC Press,
Boca-Raton, pp. 807–832.
Intensity

Ek, R., Gustafsson, C., Nutt, A., Iversen, T., Nyström, C., 1998. Cellulose powder from
Cladophora sp. algae. J. Mol. Recognit. 11, 263–265.
b Gilbert, R.D., Kadla, J.F., 1998. Polysaccharides–cellulose. In: Kaplan, D.L. (Ed.),
Biopolymers from Renewable Resources. Springer, Berlin, pp. 47–95.
Hesse, G., Hagel, R., 1973. Eine vollständige Racemattrennung durch Elutions-
chromatographie an Cellulose-tri-acetat. Chromatographia 6, 277–280.
Klemm, D., Heublein, B., Fink, H.P., Bohn, A., 2005. Cellulose: fascinating biopolymer
c and sustainable raw material. Angew. Chem. Int. Ed. 44, 3358–3393.
Kono, H., Yunoki, S., Shikano, T., Fujiwara, M., Erata, T., Takai, M., 2002. CP/MAS 13C
NMR study of cellulose and cellulose derivatives. 1. Complete assignment of the
CP/MAS 13C NMR spectrum of the native cellulose. J. Am. Chem. Soc. 124, 7506–
7511.
Koyama, M., Sugiyama, J., Itoh, T., 1997. Systematic survey on crystalline features of
0 10 20 30 40 50 60 algal celluloses. Cellulose 4, 147–160.
Kreger, D.H., 1962. Cell walls. In: Lewin, R.A. (Ed.), Physiology and Biochemistry of
2θ (Deg., CuKα) Algae. Academic Press, New York, pp. 317–335.
Meena, R., Siddhanta, A.K., 2006. Agar and value addition of Indian agarophytes. In:
Fig. 5. Representative XRD profile of b-cellulose extracted from (a) green algae Tewari, T. (Ed.), Monograph on Recent Advances on Applied Aspects of Indian
(Chamaedoris auriculata), (b) brown algae (Dictyota dichotoma) and (c) red algae Marine Algae with Reference to Global Scenario, vol. 2, NISCOM, CSIR, New
(Gelidiella acerosa). Delhi, pp. 172–184.
 A.K. Siddhanta et al. / Bioresource Technology 100 (2009) 6669–6673
Mihranyan, A., Llagostera, A.P., Karmhag, R., Strømme, M., Ek, R., 2004. Moisture
sorption by cellulose powders of varying crystallinity. Int. J. Pharm. 269, 433–442.
Mihranyana, A., Edsmana, K., Strømme, M., 2007. Rheological properties of cellulose
hydrogels prepared from Cladophora cellulose powder. Food Hydrocolloid. 21,
267–272.
Staudinger, H., 1932. In: Die hochmolekularen organischen Verbindungen–
Kautschuk und Cellulose, Springer Verlag (reprinted 1960).
Strømme, M., Mihranyan, A., Ek, R., 2002. What to do with all these algae? Mater.
Lett. 57, 569–572.
Sugiyama, J., Persson, J., Chanzy, H. 1991. Combined infrared and electron
diffraction study of the polymorphism of native celluloses. Macromolecules
24, 2461–2466.
Sun, J.X., Xu, F., Sun, X.F., Xiao, B., Sun, R.C., 2005. Physicochemical and thermal
characterization of cellulose from barley straw. Polym. Degrad. Stabil. 88, 521–531.
6673
VanderHart, D.L., Atalla, R.H., 1987. Further carbon-13 NMR evidence for the
coexistence of two crystalline forms in native celluloses. ACS Sym. Ser. 340, 88–
118.
Whistler, R.L., 1963. Methods in Carbohydrate Chemistry. Cellulose, vol. III.
Academic Press, New York and London. p. 27.
Whistler, R.L., Charles, L., 1953. Polysaccharides Chemistry. Academic Press, New
York. pp. 63–68.
Witter, R., Sternberg, U., Hesse, S., Kondo, T., Koch, F-T., Ulrich, A.S., 2006. 13C
Chemical shift constrained crystal structure refinement of cellulose IR and its
verification by NMR. Macromolecule 39, 6125–6132.
Zuluaga, R., Putaux, J.-L., Restrepo, A., Mondragon, I., Ganan, P., 2007. Cellulose
microfibrils from banana farming residues: isolation and characterization.
Cellulose 14, 585–592.