STUDIES ON UPTAKE AND ASSIMILATION OF NITRATE AND NITRITE IN AGAROPHYTE GRACILARIA EDULIS (GMEL) SILVA UNDER IN VITRO CONDITION

Size: px

Start display at page:

Download "STUDIES ON UPTAKE AND ASSIMILATION OF NITRATE AND NITRITE IN AGAROPHYTE GRACILARIA EDULIS (GMEL) SILVA UNDER IN VITRO CONDITION"

Scot May
5 years ago
Views:

1 STUDIES ON UPTAKE AND ASSIMILATION OF NITRATE AND NITRITE IN AGAROPHYTE GRACILARIA EDULIS (GMEL) SILVA UNDER IN VITRO CONDITION 1. INTRODUCTION India has a long coast line of about 7,500 km with a sizable exclusive economic zone (2.5 million km 2 ) and a large shelf area (0.13 million km 2 ). The costal area having variety of natural resources including flora & fauna. Among them seaweeds are one of the dominant vegetation resource (Oza and Zaidi, 2001). Seaweeds are marine macro algae, which are primitive non-flowering plants without true root, stem and leaves and coming under thallophyta division. Seaweeds are autotrophic plants, which occur in intertidal, shallow and deep waters of the sea up to 180m depth and also estuaries and back water. They grow on rocks, dead corals, stones, pebbles, and solid substratum and on other plants. Based on the type of pigments, external and internal structures, seaweeds are divided in to green algae, brown algae, red algae and blue green algae. Marine algal flora of Indian coast comprises of total 217 genera and 844 species. Among the genera of Indian coast, 136 genera belong to rhodophyta (Red algae); 43 genera belong to chlorophyta (Green algae); 37 genera belong to phaeophyta (Brown algae) and 1 belongs to xanthophyta. Among the species of Indian marine algal flora, 434 species belong to rhodophyta (Red algae); 216 species belong to chlorophyta (Green algae); 191 species belong to phaeophyta (Brown algae) and 3 species belong to xanthophyta (Oza and Zaidi, 2001). 1

2 The luxuriant growth of seaweeds is found in south east coast of Tamil Nadu, Gujarat coast, Lakshadweep and Andaman & Nicobar Islands. Tamil Nadu is having 1863 km 2 coastal area and the total standing crop of seaweeds are 75,373 tones wet weight (Kaliaperumal et al., 1998). Gulf of Mannar coast is having considerable natural abundance of commercially important seaweeds. Gulf of Mannar biosphere reserve starting from Rameswaram in north and stretch up to Tuticorin in south. The maximum east west length is about 75 nautical miles. It spreads over along the longitude from 78º08 E to 79º30 E and along the latitude from 80º35 to 90º25 N. Gulf of Mannar biosphere reserve contains 21 islands and total islands area is approximately 560 ha. The Gulf of Mannar costal area is influenced by both south-west (July- September) and north east (October December) monsoons. Gulf of Mannar Biosphere Reserve (including mainland and island vegetation) contains 180 seaweed species. Among them, 54 species belong to chlorophyceae (Green Algae); 37 species belong to phaeophyceae (Brown algae); 83 species belong to rhodophyceae (Red algae) and 6 species belong to cyanophyceae (Blue-green algae). It is estimated that the total standing crop of seaweeds in inter-tidal and shallow water is 91,339 tones (wet weight) consisting of 6000 tones wet weight of agarophytes, 16,000 tons (wet weight) of alginophytes and the remaining quantities of edible and other seaweeds( Kaliaperumal and Kalimuthu,1997). Seaweeds are used as food sources in Japan, Philippines, Chili, eg; Enteromorpha, Ulva, Laminaria, Undaria, Porphyra, Palmaria, Gracilaria and Eucheuma because it contains significant amount of proteins, vitamins and minerals essential for human nutrition. Seaweeds are used as medicine eg; Digenia simplex used as vermifuge medicine (Chennubhotla et al., 1987). Seaweeds are used as good resources of iodine content eg; Asparagopsis taxiformis (red alga) (Thomas and Subbramaiah, 1990) and Padina boergesenii (brown alga) (Vasuki et al., 2000). 2

3 Seaweeds are the only resources for extraction of some commercially important phycocolloids such as agar, algin and carrageenan. Phycocolloids are cell wall polysaccharides of some red and brown algae. Phycocolloids are widely used as gelling, stablising and thickening agents in many industries such as food, confectionary, pharmaceutical, dairy, textile, paper, paint and varnish. Based on these phycocolloid content, economic important seaweeds are classified into three major groups such as agarophytes (agar yielding seaweeds), carrageenophytes (carrageenan yielding seaweeds) and alginophytes (algin yielding seaweeds). Example for agarophytes: Gelidium, Gelidiella and Gracilaria species, example for alginophytes: Sargassum and Turbinaria species: Example for carragenophytes: Eucheuma, Hypnea and Gigartina species (Chennubhotla et al., 1987). The principal seaweed resources for agar extraction are Gracilaria species (53%), Gelidium species (44%), Gelidiella, Pterocladia and other species (3%). There are 3 grades of agar i.e., bacteriological, sugar reactive and food grade agar. Bacteriological agar is prepared from Gelidium and Gelidiella species (Kaliaperumal et al., 2004). There are some environmental factors which control the algal growth and biomass namely 1) Substratum 2) Light 3) Day length 4) Temperature 5) Salinity 6) Desiccation 7) Water movement and 8) Nutrients. The availability of nutrients is one of the primary factors regulating growth, reproduction and biochemistry of seaweeds. At least 56 elements have been reported to be present in seaweeds (Lobban and Wyne,1981) C, H, N, O, P, Mg, Fe, Cu, Mn, Zn, S, Mo, K and Ca are required by all algae. In addition, Co, Na, I, Br, C1 and Si are possibly required by some algae (O Kelley, 1974). There is some evidence suggest that N, P, Fe and possibly Mn, Cu, Zn, C may limits the growth of seaweeds (Riley and Chester, 1971). Several studies have 3

4 confirmed that nitrogen is the chemical nutrients most likely limit phytoplankton as well as seaweed growth (Topinka and Robbins 1976; De Boer and Ryther,1977 and Jackson, 1977). Elemental nitrogen is in abundant supply in the atmosphere (79%) Nitrogen is a component element of important bio molecules such as purine, pyrimidine, porphyrine, amino sugars and amines. Although molecular nitrogen occurs in vast amount in atmosphere, it is chemically inert and cannot be used by algae except some cyanophyceae members. Cyanophyceae members are able to utilize external nitrogen directly (Fogg, 1974). It contains a special structure called heterocyst, which is the site of nitrogen fixation. It contains nitrogenase enzyme, which is responsible for fixation of atmospheric nitrogen. For reducing one molecule of N 2 to two NH 3, six electrons are required. The over all reaction of nitrogen fixation is N 2 + 6H + + 6e ATP + 12 H 2 O 2NH ADP + 12Pi Seaweeds can utilize several inorganic nitrogen sources including nitrate, nitrite and ammonium that usually occur in seawater at concentration of 1-500μM, μm and 1.50 μm respectively (Riley and Chester, 1971). Organic nitrogen source such as urea which usually occurs at concentration less than 20 μm in seawater (Riley and Chester, 1971) is utilized by some seaweeds. The organic compounds such as acetamide, succinamide, aspargaine and glutamine are used as organic nitrogen sources for growth of algae (Algeus, 1950a; Miller and Fogg, 1958). Glycine is the most assimiliable amino acid and an excellent nitrogen source of some algae (Algeus, 1950a). Some algae can use uric acid and Xanthine (Dropp, 1955) for their growth. Seaweeds have been found to differ in their capabilities of utilizing various nitrogen sources. Nitrate and ammonium were equally effective at promoting growth of Chondrus crispus (Neish and Shacklock, 1971; Neish and Fox, 1971). Gracilaria foliifera exhibited higher growth rate when cultured with ammonium as compared with 4

5 nitrate (DeBoer et al., 1978). Growth rate of Goniotrichium and Nemalion (Fries, 1963) were greater when the plants were supplied with NO 3 - as compared to NH + 4. NH + 4 to be the best nitrogen source for Gelidium (Yamada,1961). In Porphyra higher growth rate was obtained at Laminaria groenlandica (Harrison et al., 1986) and Fucus distichus (Thomas et al., 1985). Similar uptake rate of NH + 4 and NO 3 - was found in Enteromorpha instestinalis and Fucus gardneri and Laminaria longicruris (Harlin and Craigie, 1978). For many algae uptake of NH + 4 is more rapid than uptake at NO 3 - and NO 2 - at same concentration (Hanisak, 1983). Suppression of NO 3 - by NH 4 + has been reported is Hypnea musciformis, Codium fragile and Cladaphora. In Enteromorpha instestinalis NO 3 - uptake is inhibited if NH + 4 concentration is found more than 5 μm concentration (Thomas and Harison, 1987). Seaweeds have been reported to store unused NO 3 - in vacuoles. Valonia and Halicystis concentrate 200 to 500 times respectively, higher NO 3 - concentration than in seawater (Jacques and Osterhout, 1938). Laminaria longicruris has been reported to concentrate NO 3 as high as 28,000 fold during periods of N sufficiency (Chapman and Craigie, 1977). These inorganic and organic N reserves are believed to be utilized to sustain the growth of the Laminaria during the summer months when external N levels are low (Chapman and Craigie, 1977; Chapman et al., 1978). It is energically easy for the plant to utilize ammonium rather than nitrate (Syrett, 1962). NO 3 - uptake and reduction is an energy demanding process and it is dependent on photosynthesis (Wheeler,1982; Davison and Stewart 1984). In Gracilaria tikvahiae presence of higher concentration of NH + 4 inhibit of NO 3 - uptake. + Higher NO 3 - level had no immediate effect on NH 4 uptake. 5

6 Several studies of nitrate uptake in seaweeds showed little ability to use nitrite (Hanisak and Harlin, 1978) in Codium and (Topnika, 1978) in Fucus. Inhibition of NO 2 - uptake by NO 3 - was found in Laminaria longicruris. Nitrate is often depleted at relatively constant rate where as NH + 4 depletion is commonly non-linear rate (Haines and Wheeler, 1978; Fujita, 1985; Thomas and Harrison, 1987). It is generally accepted that nitrate is reduced to ammonium level before being incorporated in to organic compounds (Syrett, 1962). The oxidation-reduction state of the N-atom in nitrate is +5 and in ammonium is 3. For many years it has been assumed that three intermediate occurs between nitrate and ammonium. This allowing four reductive steps each involving the addition of a pair of electrons. The most generally accepted sequence (Nicholas, 1959; Syrett, 1962 and Kessler, 1964) is NO 3 - NO 2 - N 2 O 2 NH 2 OH NH4+ Nitrate Nitrite Hyponitrite Hydroxylamine Ammonium However, more recent work with higher plants (Beevers and Hageman 1969, Hewitt, 1970) and algae (Hattori and Myers 1966; Zumft et al., 1969 and Aparicio et al., 1971) suggested that only two enzymes catalyze the entire reduction of nitrate to ammonium. The first step is nitrate reductase (NAD (P) H nitrate oxido reductase) which catalyses the reduction of nitrate in to nitrite. The second step is nitrite reductase (NAD (P) H nitrite oxido reductase) which catalyses the reduction of nitrite to ammonium + NO 3 - NO 2 - NH 4 Nitrate reductase Nitrite reductase 6

7 FAD and molybdenum undergo oxidation reduction during the reaction. The reducing power for the reduction of nitrate to nitrite is supplied by NADH and it is derived by oxidation of glyceroldehyde 3 phosphate through either glycolytic path way (or) oxidative pentose pathway. The over all reaction catalyzed by nitrate reductase is NO NADH + H + NO NADH + H 2 O The second step in nitrate assimilation is the reduction of nitrite to ammonium NH 4 +. This reaction occurs in chloroplast. The reducing power for nitrite reduction derived directly from light reaction in photosynthesis. Alternatively nitrite reduction to ammonium may occur in dark provided that starch reserves are present in chlorophlasts (Kow et al., 1982). The immediate source of reducing power for nitrite reduction is a non iron sulfur protein known as ferredoxin. The overall reaction catalyzed by nitrite reductase is NO e - + 7H + NH 3 + 2H 2 O Here six equivalents of electrons in the form of reduced ferrodoxin and seven equivalent of hydrogen ion are consumed in the reduction of 1 mole of nitrite to ammonia. Recently it was suggested that reducing power derived from the oxidation of hexose phosphate (eg. Glucose 6 phosphate) may transfer to reduction of nitrite (Kow et al., 1982). Gracilaria edulis is a red alga, which comes under the division of rhodophyta, order of Gigartinales, class of rhodophyceae and family of Gracilariaceae. Gracilaria edulis plant body is erect in nature and grows up to 20 cm (or) more: brownish red, alternately irregularly branched; branches hardly constricted. It grows abundantly on sea grass beds in shallow lagoons formed between the shores and fringing coral reefs. It is also attached to small stones and shells on sandy and muddy areas. The yield of agar from this plant is 45% and the gel strength is 139g/cm 2 (Chennubhotla et al.,1987) 7

8 Gracilaria edulis is an important raw material for the extraction of agar. Per year 982 tons (dry weight) of Gracilaria edulis is harvested from various landing centre of Tamil Nadu coast. Repeated harvesting of Gracilaria edulis causes depletion of natural stock and will lead to eliminate completely from natural habit. To avoid this problem, mariculture of Gracilaria edulis is now proposed. The process of uptake and assimilation of nitrogen sources is poorly understood in marine algae as compared to higher plants. It is essential to study the uptake and the assimilation of nutrients in Gracilaria edulis because there is no report (regarding nutritional study) in this species. Therefore this present study is aimed to optimize the conditions for uptake and assimilation of various nitrogen sources such as nitrate and nitrate under in vitro condition. Besides, the natural resource this alga along the Gulf of Mannar coast is also estimated to find out the optimum season for growth. So the present study carried out with the following major objectives. 1. Studies on uptake kinetic parameters (Vmax and Km) of nitrogen sources such as nitrate and nitrite in Gracilaria edulis. 2. Studies on optimizing environmental parameters (light, photoperiod, salinity, ph and temperature) for maximum uptake of nitrate and nitrite. 3. Studies on assimilation of nitrate and nitrite and optimizing conditions for maximum nitrate reductase and nitrite reductase activity. 4. Studies on influence of nitrogen sources (nitrate and nitrite) on pigment content and agar properties. 5. Studies on seasonal variation of Gracilaria edulis biodiversity in the Gulf of Mannar region. 8

Evaluation of Kinetics of Nitrogen Uptake by Five Macroalgal Species in Eutrophic Coastal Waters Using 15 N

Tropical Agricultural Research Vol. 18 Evaluation of Kinetics of Nitrogen Uptake by Five Macroalgal Species in Eutrophic Coastal Waters Using 15 N V. Raikar and M. Wafar National Institute of Oceanography