Asia Pacific Journal of Research
Vol: I Issue XIV, February 2013
ISSN: 2320-5504, E-ISSN-2347-4793
HISTOCHEMICAL STUDY ON THE IMPACT OF HIGH CARBON
DIOXIDE ON KAPPAPHYCUS ALVAREZII
1
Geetanjali Elangbam, 2Arunjit Mayanglambam and 3Dinabandhu Sahoo
Marine Biotechnology Laboratory, Department of Botany, University of Delhi,
Delhi 110007, India.
ABSTRACT
Seaweeds which abundantly grow in the seawater have emerged as a major group of plants
for CO2 Sequestration in the ocean ecosystem. However, these studies are not sufficient in the
present global climatic condition as the algal species behave in different manner in different
agroclimatic conditions. Carbon emissions as macroalgae can incorporate tonnes of Carbon
into the harvested algae annually. Hence, in the present study an attempt has been made to
evaluate the effect of elevated CO2 on the histochemical studies in Kappaphycus alvarezii, a
red seaweed.
KeyWords: Kappaphycus alvarezii, histochemical, carbon dioxide, polysaccharides,
proteins
1. Introduction
Seaweeds have emerged as a major group for CO2 sequestration in the ocean ecosystem.
According to Beardall and Raven, (2004) marine photosynthesis contributes 54-59 Pg C
year-1 of the total primary productivity of planet and out of this ~1 Pg C year-1 is
contributed by seaweeds and seagrasses. Seaweeds such as Macrocystis, Laminaria,
Sargassum, Ascophyllum, Fucus, Porphyra, Palmaria, Ulva and Enteromorpha also
achieve high rates of CO2 assimilation per gram fresh weight (Jackson 1987; Gao and
McKinley, 1994; Muraoka, 2004 and Chung et al., 2011). Macroalgae can incorporate
an average of 0.26X 106 tonnes C into the harvested algae annually (Chung et al., 2011).
Similarly growth of some seaweeds species, such as Porphyra yezoensis,
Gracilaria sp., Gracilaria chilensis, were enhanced when grown at CO2 levels 2-3
times the present atmospheric CO2 concentration (Gao et al., 1991 and Gao et al.,
1993). These species were capable of using HCO3-, yet they showed carbon limited
photosynthesis in natural seawater. Growth of the red alga Lomentaria articulata, a
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Asia Pacific Journal of Research
Vol: I Issue XIV, February 2013
ISSN: 2320-5504, E-ISSN-2347-4793
non bicarbonate user, was stimulated by enriched CO2 in aeration (Kübler et al.,
1999). On the other hand, a decrease of growth rate caused by elevated CO2 has also
been reported, in Gracilaria tenuistipitata, Porphyra leucostica and Porphyra
linearis (Israel et al., 1999; García-Sânchez et al., 1994 and Mercado et al., 1999).
Zou and Gao, (2009) also studied the effects of elevated CO2 on the red seaweed
Gracilaria lemaneiformis (Gigartinales, Rhodophyta) grown at different irradiance
levels.
Published literature did not reveal any information about the impact of high
CO2 on red seaweed Kappaphycus alvarezii. Hence, in the present investigation, an
attempt is made to study the effects of different CO2 concentration
2. Materials and Methods
Thalli of Kappaphycus alvarezii was collected from the cultivation site of Palk Bay,
Mandapam, Tamil Nadu (9º17′N and 79º11′E) during July, 2011. Thalli were
washed several times in seawater to remove visible epiphytes and debris, then
wrapped in the absorbent cotton and brought live to the laboratory. They were
cultured in Artificial Sea Water (ASW) and different marine media for further
studies.
CARBON DIOXIDE TREATMENT TO MACROALGAE
1 gm each of the fresh thalli of Kappaphycus alvarezii was taken in 250 mL
Erlenmeyer flask containing 150 mL artificial sea water. To the thalli various
Carbon dioxide concentrations, 550ppm, 1%, 4%, 12% and 15% CO2 (Sigma gases
and service, New Delhi, India) were treated for 24h at a rate of 40-60 mL min-1 and
was incubated at 25±1°C under 84 µmol m-2 s-1 light intensity at 8h light/16h dark
photoperiods for 10 days. pH and weight was noted. Selected portions of the Carbon
dioxide treated thalli were processed for Light microscopic studies.
LIGHT MICROSCOPIC STUDIES
Fixation
Selected parts of the thalli were dissected and fixed in 4% Formaldehyde: Seawater,
FAA.
1.
4% Formaldehyde : Seawater
2.
Formaldehyde- Acetic acid- alcohol (FAA)
Formaldehyde
5mL
Glacial Acetic acid
5mL
70% Ethanol
90mL
The material was fixed for 24h and later preserved in 70% ethanol.
Dehydration
The fixed materials were transferred successively through 2 – methoxyethanol
(2 times for 24h each); 100% ethanol (24h); n-propanol (24h); and n-butanol (24h).
The whole dehydration process was carried out at 4ºC.
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Asia Pacific Journal of Research
Vol: I Issue XIV, February 2013
ISSN: 2320-5504, E-ISSN-2347-4793
Infiltration
The dehydrated material was transferred to plastic monomer mixture for
infiltration and the mixture was changed after every 24h. The infiltration was carried
in dark at 4ºC for a period of 7 days. Longer duration of the thalli in the plastic
mixture was needed for proper infiltration, as the plant possesses large number of
extracellular and copious amount of intercellular polysaccharides that retard the
penetration of monomer mixture.
Preparation of Monomer mixture (Feder & O′Brien, 1968)
2 Hydroxyethyl-methacrylate (Fluka) (commercial monomer)
92.2
mL
2-2′ Azobis (2-methyl propionitrile) BDH
0.3
gm
Polyethylene glycol 400 (Polypharm)
7.5
mL
The commercially available monomer was purified before use by adding 4
gm of activated charcoal to 100 mL of monomer and later stirred for an hour and
filtered twice to obtain a clear solution.
In a separate beaker 0.3 gm of 2-2′ Azobis (2-methyl propionitrile) was first
mixed thoroughly with 7.5 mL polyethylene glycol on a magnetic stirrer. To this
mixture, filtered monomer mixture was added and then stirred with the help of a
teflon rod on a magnetic stirrer. The resultant monomer mixture was stored at 4ºC in
a brown bottle to avoid light induced polymerization.
Embedding and Polymerization
The material was embedded in transparent gelatin capsule filled with monomer
mixture. First the capsules were fixed to the slotted capsule holder boards and the
materials were transferred to the capsules with the help of a fine brush. The material
was oriented to the desired plane and the capsules were tightly capped.
Polymerization was carried out at 40ºC for 24h and at 60ºC for 48h in a temperature
controlled oven. After polymerization, the capsules were allowed to cool and the
gelatin cover was peeled off to expose the hard plastic blocks.
Microtomy
Sectioning was done on an A. O. Spensor 820 rotary microtome to which was
fitted a specially designed local fabricated adapter. 2 µ thick sections were cut using
glass knives that were made on LKB knife maker. Individual sections were picked
up with fine forceps, and placed on a drop of distilled water on pre-cleaned slides.
Hot plate, maintained at 60ºC was used to dry the slides.
HISTOCHEMICAL STUDIES
Polysaccharides (Mc Cully, 1996) Toludine Blue O (pH 4.4) Test for carboxylated
and sulphated
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Asia Pacific Journal of Research
Vol: I Issue XIV, February 2013
ISSN: 2320-5504, E-ISSN-2347-4793
Result
Carboxylated and sulphated polysaccharides stained deep violet pink to reddish
violet, RNA purple, DNA blue or blue green, Phenols turquoise green or green-blue.
Cellulose unstained.
Toludine Blue O belongs to thiazine group and acts as a metachromatic dye.
Metachromasia detects high molecular weight substances having free ionic groups
like mucopolysaccharides and nucleic acids. The most accepted theory of
metachromasia states that metachromatic dyes are capable of forming polymers that
are different in hue then the monomers (Jensen, 1962). The dye binding sites
involved are ester sulphates, carboxylated and phosphates. The phosphates in nucleic
acid are thus the bases for dye action.
Periodic Acid Schiff’s (PAS) Test for Insoluble Polysaccharides (Modified after
Feder and O′ Brien, 1968)
Result
Cell wall and insoluble polysaccharide grains stain magenta red.
Coomassie Brilliant Blue Test for Total Proteins (Weber and Osborn, 1975).
Result
Proteins stain – Bright – Blue
PHOTOMICROGRAPHY
Photomicrography was done by a Primo Star Zeiss Photomicroscope ((USA)
with a digital camera (Canon, Japan), 14.7 Megapixel attached to it.
Results and Discussion
The level of carbon dioxide in the atmosphere is rapidly increasing and
which has already created major impact on Ocean Ecosystem. Recently, there has
been a good deal of interest in the potential of marine vegetation as a sink for
anthropogenic Carbon emissions as macroalgae can incorporate tonnes of Carbon
into the harvested algae annually (Chung et al., 2011). The rate of primary
production of some species is comparable with those of the most productive land
plants, therefore seaweeds have a great potential for CO2 sequestration (Gao and
McKinley, 1994 and Chung et al., 2011). Studies on macroalgae were mostly carried
out on growth rate and biomass production in Porphyra yezoensis, Gracilaria sp., G.
chilensis. The thalli showed enhanced growth rate when cultured at high CO2 levels
ie.2-3 times of the present atmospheric CO2 concentration (Gao et al., 1991 and Gao
et al., 1993). These species were capable of using HCO3-, yet they showed carbon
limited photosynthesis in natural seawater. Roleda et al., (2012) reported that
increased in H+ ions or low pH leads to negative effect on cellular activity and low
germination rate in Macrocystis pyrifera (Laminariales, Phaeophyceae). On the
other hand, a decrease of growth rate caused by elevated CO2 has also been reported,
in Gracilaria tenuistipitata, Porphyra leucostica and Porphyra linearis (Israel et al.,
1999; García-Sânchez et al., 1994; Mercado et al., 1999).
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Asia Pacific Journal of Research
Vol: I Issue XIV, February 2013
ISSN: 2320-5504, E-ISSN-2347-4793
In the present study the pH of the culture media for Kappaphycus alvarezii at
the time of inoculation in was 8.6-8.7. However, after the CO2 treatment the pH of
the medium decreased to 8.0 in 550ppm, 7.7 in 1%, 7.5 in 4%, 7.2 in 12% and 7.0 in
15% CO2 treatment for 24h. The pH slightly increased in K. alvarezii cultures after
one week of CO2 treatment. Histochemical studies were mostly undertaken to study
the effect of UVB radiation on macroalgae but no such studies about the impact of
carbon dioxide treatment has been reported in any Seaweed. For the first time, it was
observed that there was no variation in the amount of both sulphated and
carboxylated polysaccharides, insoluble polysaccharides and proteins in the cells
neither there was any structural damage after high CO2 treatment (Figure.1-3).
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Asia Pacific Journal of Research
Vol: I Issue XIV, February 2013
ISSN: 2320-5504, E-ISSN-2347-4793
e
co
ci
co
ci
e
B
A
e
co
e
ci
co
ci
m
C
D
m
co
ci
ci
co
e
e
E
Figure-1.
F
(A- F) Kappaphycus alvarezii. Vegetative thallus of primary axis. Localization of sulphated and
carboxylated polysaccharides (Toludine Blue O, TBO). (e-epidermis; ci-inner cortex; co-outer
cortex; m-medulla; ). Scale bar 1000µm.
A.
L.S of thallus showing darkly stained, elongated epidermal cells. below these are the
outer cortical cells and inner cortical cells (Control).
B. L.S of thallus showing darkly stained, elongated epidermal cells. below these are the
outer cortical cells and inner cortical cells (550ppm CO2 treatment for 24h).
C. Transverse section of thallus showing cell wall of epidermis and cortex stains darkly with
TBO. The extracellular layer and intercellular spaces stain deep violet with TBO indicating
the presence of both sulphated and carboxyated polysaccharides (1% CO2 treatment for
24h).
D-E. L.S of thallus showing darkly stained, elongated epidermal cells. The extracellular layer and
intercellular spaces stain deep violet with TBO indicating the presence of both sulphated and
carboxylated polysaccharides (4% and 12% CO2 treatment for 24 hours).
F.
L.S of thallus showing darkly stained, elongated epidermal cells. below these are the outer
cortical cells and inner cortical cells which are followed by innermost medullary region. The
extracellular layer and intercellular spaces stain deep violet with TBO indicating the
presence of both sulphated and carboxylated polysaccharides (15% CO2 treatment for 24
hours).
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Asia Pacific Journal of Research
Vol: I Issue XIV, February 2013
ISSN: 2320-5504, E-ISSN-2347-4793
fs
ci
e
co
e
ci
co
fs
A
B
co
e
ci
co
fs
e
pi
ci
C
D
co
ci
e
ci
e
co
E
Figure-2.
F
(A- F) Kappaphycus alvarezii. Vegetative thallus of primary axis. Localization of insoluble
polysaccharides (Periodic Acid Schiff’s Reagent, PAS). (e-epidermis; ci-inner cortex; co-outer
cortex; m-medulla; fs- floridian starch; pi- pit connection). Scale bar 1000µm.
A.
Transverse section of thallus showing darkly stained, elongated epidermal cells. Below these are
the outer cortical cells and inner cortical cells (Control).
B.
Transverse section of thallus showing darkly stained, elongated epidermal cells. Floridian
starches are seen in the intercellular space (550ppm CO2 treatment for 24h).
C-D. Transverse section of thallus showing epidermis and cortex region. The epidermal cells are
elongated; consist of arc-shaped epidermal cells, which stained dark magenta. The outer
cortical cells are lightly stained as compared to epidermal cells, which are small and
isodiametric in shape. The inner cortical cells are large, irregular and
pseudoparenchymatous. Lots of Floridian starches are seen in the intercellular space (1%
and 4% CO2 treatment for 24h).
E-F. L.S of thallus showing darkly stained, elongated epidermal cells. Below these are the outer
cortical cells and inner cortical cells which are followed by innermost medullary region
which are lightly stain The thallus is covered with thick mucilaginous layer which stain
positive with
PAS Showing highly presence of insoluble polysaccharides. The cell walls and the intercellular
spaces stain positive with PAS. (12% and 15% CO2 treatment for 24h).
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Asia Pacific Journal of Research
Vol: I Issue XIV, February 2013
ISSN: 2320-5504, E-ISSN-2347-4793
e
e
pi
co
ci
ci
co
pi
A
B
e
e
ci
co
co
ci
pi
C
pi
D
e
co
ci
e
co
ci
E
F
Figure-3. (A- F) Kappaphycus alvarezii. Vegetative thallus of primary axis. Localization of total proteins
(Coomassie Brilliant Blue, CBB). (e-epidermis; ci-inner cortex; co-outer cortex; m-medulla; pipit connection). Scale bar 1000µm.
A.
L.S. of thallus showing epidermis and cortex. The epidermal cells are elongated and darkly
stained. The outer cortical cells are small, elongated to isodiametric which stain dark blue
with CBB. The inner cortical cells are much larger, irregular in outline and are connected
by darkly stained pit connections. The extracellular layer outside the epidermis and the
intercellular space show negative with CBB. (Control).
B-D. Transverse section of thallus showing only epidermal cell with deep blue stain with CBB
(550ppm, 1% and 4% CO2 treatment for 24h).
E-F. L.S of thallus showing darkly stained, elongated epidermal cells. Below these are the outer
cortical cells and inner cortical cells which does not take any stain of CBB (12% and 15%
CO2 treatment for 24h).
Conclusion
Histochemical studies were mostly undertaken to study the effect of UVB radiation on
macroalgae but no such studies about the impact of carbon dioxide treatment has been
reported in any Seaweed. For the first time, it was observed that there was no variation in the
amount of both sulphated and carboxylated polysaccharides, insoluble polysaccharides and
proteins in the cells neither there was any structural damage after high CO2 treatment. The
present findings conclude that Kappaphycus alvarezii did not show much response to high
CO2 treatment indicating that the species is more suitable for Ocean carbon sequestration at
elevated CO2.
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Asia Pacific Journal of Research
Vol: I Issue XIV, February 2013
ISSN: 2320-5504, E-ISSN-2347-4793
Acknowledgements
This work is supported by Department of Science and Technology, Government of
India DST/IS-STAC/CARBON DIOXIDE-SR-37/07 DT 13/05/2008.
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