Indian J ournal of Marine Sciences

Vol. 33(2), J une 2004, pp 202-205

Short Comunication


Effect of alkali treatment on the yield and quality of agar from red alga
Gracilaria verrucosa (Rhodophyta, Gracilariales) occurring
at different salinity gradient of Chilika lake
J . Rath & S.P. Adhikary*
P G Department of Biotechnology, Utkal University, Bhubaneswar-751004, India
*[E-mail: adhikary2k@hotmail.com ]
Received 14 May 2003; revised 12 February 2004
Gracilaria verrucosa was collected at salinity gradient of 4.2 to 28 ppt in the Chilika lake was used for agar extraction.
Agar yield of 27-30% with gel strength 165 to 180 g/cm
2
was obtained from the alga occurring at two different locations of
Southern sector of the lake showing salinity 6.5 to 8.0 ppt. NaOH pre-treatment strongly influenced the agar characteristics.
Alkali pre-treatment of Gracilaria verrucosa yielded agar with increased gel strength but the yield was lowered. With 10%
alkali pre-treatment the sulphate content of agar was reduced together with increase in 3,6,-anhydro galactose content and
the gel strength. Using Gracilaria verrucosa from salinity ranges 6.5 to 8.0 ppt of Chilika lake and extraction with 10%
alkali, good quality of agar with gel strength of 265 to 275 g/cm
2
and moderate yield of 25 to 26% was obtained suggesting
its potential for commercial exploitation.
[Key words: Gracilaria verrucosa, Chilika lake, agar agar, alkali treatment red alga, salinity]
[IPC Code: Int. Cl.
7
C08B 37/12]
In India agar is mainly produced from Gracilaria
edulis
1-3
and Gelidiella acerosa
4,5
. Though economi-
cally valuable phycocolloids can be obtained from
Gracilaria spp, occurring in Indian coast, they are not
preferably used due to low agar quality
6,7
. However,
the gel properties of agar from many Gracilaria spp
can be improved by alkali treatment which converts
L-galactose-6-sulphate to 3, 6-anhydro-L-galactose
8
.
Besides several attempts have also been made to im-
prove the agar quality and yield by altering the extrac-
tion methods
9,10
, treating with Cobalt-60 gamma ra-
diation
11
etc.
Although there have been several reports on the
growth and agar yield from Gracilaria species, there
are few studies examining their physiological res-
ponse to salinity
12,13
. Gracilaria verrucosa (Hudson)
Papenfuss (Rhodophyta, Gracilariales) occurs in dif-
ferent sectors of Chilika lake where the salinity value
ranges from 4.2 to 28 ppt
14
, however, maximum bio-
mass was obtained in its central sector which shows
salinity values between 4 to 20 ppt. In the present
work the yield of quality agar from Gracilaria
verrucosa, occurring at different salinity gradients of
Chilika lake, upon pretreatment with various concen-
tration of NaOH was examined.
Gracilaria verrucosa was collected from five dif-
ferent localitis of Chilika lake namely Badakuda (19
33

4.8

N and 85 8

8.9

E), Malatikuda (19 38

10.2

N and 85 11

7.8

E), Nalabana (19 41

54.1

N and 85 17

37.7

E), Nuapada (19 40

52.7

N and
85 22

37.9

E) and Rambharatia (19 40

8.6

N and
85 30

0.5

E) from 5
th
to 7
th
December 2001. Salin-
ity of the lake water at these locations was measured
argentometrically
15
and expressed as ppt. Epiphytic
algae occurring attached to the thallus were manually
removed, washed in freshwater to remove the salt and
other adhering impurities, sun dried and stored in
sealed plastic bags until extraction, following Thomas
& Krishnamurthy
1
with minor modifications as fol-
lows.

Alkali treatment was carried out using 1%, 5%,
10%, and 15% w/v NaOH concentration. Dry
Gracilaria verrucosa material (10 g) was soaked over
night in 200 ml of the respective concentration of
NaOH solution at room temperature to hydrate the
seaweed and followed by 1h in a water bath at 90C.
Alkali treated plants were washed with fresh water to
remove excess NaOH. The samples were soaked for
1h in 200 ml of 0.025% HCl to neutralize the alkali
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203
followed by washing until the pH becomes neutral.
Extraction of agar was carried out by boiling this
sample for 2 h in 200 ml of distilled water in an auto-
clave. The extract was mixed with celite and finally
pressure filtered. The filtrate was allowed to gel at
room temperature, frozen overnight and thawed. Fi-
nally the agar was oven dried for 24h at 60C, cooled
and weighed to calculate percent agar yield
1
.
The quality of agar extracted with different concen-
trations of alkali (NaOH: 1, 5, 10 and 15% w/v) with
respect to gel strength, yield, gelling temperature,
melting temperature, 3, 6-anhydro-galactose and sul-
phate content was determined. The extraction without
any alkali treatment was served as control. Dry agar
was ground and reconstituted in 1.5% w/v solution
with distilled water to measure the physical properties
(gel strength, melting and gelling temperature). Gel
strength was measured after gelling overnight at room
temperature by measuring the load (g/cm
2
) causing a
cylindrical cuvette (1 cm
2
cross section) to break a
standard gel. Gelling temperature was measured by
adding 10 ml hot agar solution and a glass bead (5
mm diameter) to a test tube (2.3 cm diameter, 6 cm
height). The tube was tilted up and down in a water
bath at room temperature until the glass bead ceased
moving. The gel temperature in the tube was immedi-
Table 1 Quality and yield of agar-agar from Gracilaria verrucosa occurring at different sites with pre-treatment of different
concentration of NaOH (1, 5, 10, 15% w/v)

Site of occurrence Salinity NaOH Gel Yield Gelling Melting 3,6- anhydro Sulphate
(ppt) concentr. strength (%) temperature temperature galactose content
(g/cm
2
)
(C) (C)
(%) (%)

Southern sector
1. Badakuda 8 Control 150 32.0 42 60 22 5.2
1 % 165 30.0 40 62 27.1 4.7
5 % 180 26.6 42 65 30.5 3.2
10 % 265 26.0 43 75.5 38.8 2
15 % 280 24.0 40 80.5 42.5 1.5

2. Malatikuda 6.5 Control 143 40.0 35 60 22.2 5
1 % 160 34.0 40 62 24.6 4.4
5 % 165 29.7 43 60 32.8 3.2
10 % 275 25.0 36 78.2 40 1.7
15 % 310 22.0 32 80 44.5 1.2

Central sector
3. Nalabana 5.2 Control - 12.0 35 52 18 6.8
1 % - 10.0 NG 50 22.2 6.4
5 % - 7.4 NG 49 28.1 4.3
10 % 140 7.4 42 66 32.8 3.2
15 % 162 7.0 38 70.2 34.6 2.7

4. Nuapada 4.2 Control - 10.0 NG 53.2 16 6
1 % 120 10.0 39 58.2 18.4 5.8
5 % 133.2 5.9 NG 48 23.6 4.7
10 % 155 5.0 40 65.3 28.2 3.6
15 % 270 4.8 40 68 31.5 3.1

Outer channel sec-
tor

5. Rambharatia 28 Control - 25.0 37 62 16.4 6.2
1 % 10 20.0 35 60.7 18.5 5.6
5 % 12 14.8 43 58 29.8 4.8
10 % 145 12.0 40 52 30.2 3.6
15 % 175 10.5 36 48.6 36.5 2.8

Control =without alkali treatment; NG =No proper gelling

INDIAN J . MAR. SCI., VOL. 33, No.2, J UNE 2004


204
ately measured by introducing a thermometer (0.1C
division) into the agar. Melting temperature of the gel
in a test tube was measured by placing an iron bead (9
mm diameter) on the gel surface. The test tube was
clamped in a water bath and the temperature raised
from 50 to 100C at 1C/min. The melting point was
recorded with a thermometer when the bead sank into
the solution
16
. The 3, 6-anhydrogalactose content of
the agar sample was determined calorimetrically using
the resorcinol method
17
and the sulphate content was
quantified by the barium chloride precipitation tech-
nique
18
.
The yield and quality of agar extracted from
Gracilaria verrucosa of Chilika lake occurring at dif-
ferent salinity gradients and after pretreatment with
different concentration of NaOH during extraction is
given in Table 1. Results showed that native agar
(control) can be extracted from Gracilaria verrucosa,
growing in Badakuda and Malatikuda localities of
Chilika lake, showing salinity ranges of 6.5 to 8 ppt,
however, with 10% (w/v) alkali pre-treatment good
quality agar-agar can also be extracted from the sea-
weed growing at varying salinities from 4.2 to 28 ppt.
With increase in NaOH concentration from 1 to 15%
(w/v), the gel strength of agar occurring at different
salinity gradient of the lake was increased but simul-
taneously the yield was decreased with increase in
alkali concentration. No definite trend was observed
in the gelling and melting temperature irrespective of
the alkali concentration and salinity.
Agar with maximum gel strength (310 g/cm
2
) was
observed with 15% alkali pre-treatment of Gracilaria
from Malatikuda (salinity 6.5 ppt) but the yield was
low (22%). The native agar of Malatikuda showed
higher yield (40%) but the gel strength was quite low
(143 g/m
2
). Agar from the seaweed from Badakuda
(salinity 8 ppt) with 10% alkali treatment gave agar
with optimum gel strength and yield (265 g/m
2
and
26.5% respectively) and that of Nuapada, Nalabana
and Rambharatia showing salinities 4.2, 5.2 and 28
ppt respectively gave poor quality of agar with low
gel strength and yield (Table 1). With the increase in
the NaOH concentration during pre-treatment 3, 6-
anhydro-L-galactose content of agar was increased
resulting in increase in gel strength. Gracilaria verru-
cosa from the salinity ranging from 6.5 to 8 ppt at
Malatikuda and Badakuda showed comparativelyless
sulphate content than the other study sites. However,
higher sulphate content in the native agar of
Gracilaria verrucosa from different salinities was
decreased with increase in the NaOH pretreatment.
These results showed that variations in the concen-
trations of NaOH pre-treatment strongly influenced
the agar characteristics of Gracilaria verrucosa.
Chennubhotla et al.
6
found that G. verrucosa col-
lected from Chilika have yielded 23% agar with a gel
strength of 41 g/cm
2
. But in the present study with
modification of extraction method and 10% alkali
treatment good quality of agar with higher gel
strength from 265 to 275 g/m
2
and 25 to 26% yield
was obtained. The reduction in yield due to alkali
treatment was possibly due to leaching of agar poly-
mers during extraction and degradation of some poly-
saccharides in presence of higher alkali concentration.
Our findings clearly show that Gracilaria
verrucosa occurring at the salinity ranges of 6.5 to 8
ppt in Chilika lake can yield good quality of agar with
10% of NaOH pretreatment. Though the yield and
properties of agarocolloids from Gracilariales are
generally less interesting from economic point of
view than those of the agar from Gelidium and Ptero-
cladia, considering the world wide distribution of
Gracilaria particularly in the Indian coast, production
of quality agarocolloides from the seaweed by im-
provement in the extraction methodology is of emerg-
ing interest to meet the market demand of agar agar.
We thank Mr. A. K. Pattnaik, Chief executive,
Chilika Development Authority for providing facility
for sample collection and financial assistance to one
of us (J Rath).

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