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7- Algae and Men

INTRODUCTION Microalgae and macroalgae have been utilized by man for hundreds of years as food, fodder, remedies, and fertilizers. Ancient records show that people collected macroalgae for food as long as 500 B.C. in China and one thousand of years later in Europe. Microalgae such as Arthrospira have a history of human consumption in Mexico and Africa. In addition to direct consumption, agars and carrageenans extracted from red macroalgae and alginates from brown macroalgae and microalgae have been included in a remarkable array of prepared food products, serving mostly to modify viscosity or texture. Microalgae are a source for viable and inexpensive carotenoids, pigments, proteins, and vitamins that can be used for the production of nutraceuticals, pharmaceuticals, animal feed additives, and cosmetics. Table 1 summarizes commercially exploited algae and the corresponding nutraceutical.

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USES OF COMMERCIAL ALGAE

FOOD Cyanophyta Nostoc sphaeroides are used as food and delicacy for hundreds of years. It is found in rice fields from December to May in Hubei, China. Colonies are dark green and pearlshaped, and can reach 2.5 cm in diameter. Dried Nostoc spp. Balls are sold in Asian markets; they are stir-fried sauted with oysters, and used in soups and as thickeners for other foods. Nostoc flagelliforme is a terrestrial cyanobacterium that naturally grows on arid and semiarid steppes in the Northern and the Northwestern parts of China, where it is considered an edible delicacy with special medical value and great economic value. N. flagelliforme is called Facai (hair vegetable) in Chinese because of its hair-like appearance. Arthrospira platensis is filamentous cyanobacterium has a history of human consumption, which can be located essentially in Mexico (where it is called tecuitlatl) and in Africa (where it is called dihe). Arthrospira bloom is present as a thick blue-green mat floating onto the surface of the lake only few hours a day, early in the morning. When the sun is high, the temperature of the water rises, and the bloom disperses, therefore the harvesting begins -by women - (Figure 1) at sunrise and it is over in about 2 h. Dihe is mainly used to prepare la souce, a kind of vegetable broth served with corn, millet, or sorghum meal, which occasionally can have fish or meat as additional ingredients. A minor utilization of dihe is as remedy applied onto wounds to speed up the healing process, or as a poultice to soothe the pain and reduce the swelling of mumps. Nutritional value Arthrospira biomass has a high content of protein, about 5560% of the dry matter, the proteins are low in lysine and sulfured amino acids such as methionine and cystein, but their amount is much higher than in other vegetables, including legumina. Phycobiliproteins represent a major portion of proteins, and among them phycocyanin can reach 713% of the dry matter; carbohydrates reach 1020% of the dry weight, and consist mainly of reserve products, while lipids account for 914% of the dry weight. The mineral fraction represents 69% of the dry biomass, rich in K, P, Na, Ca, Mn, and Fe. Group A, B, and C vitamins are also present, with an average -carotene content of 1.5 mg g-1 of Arthrospira, corresponding to 0.25 mg of vitamin A. Rhodophyta Porphyra (Bangiophyceae) is popularly known as Nori in Japan, Kim in Korea, and Zicai in China,. Porphyra grows as a very thin, flat, blade, which can be yellow, olive, pink, or purple. It can be either round, round to ovate, obovate, linear or linear lanceolate, from 5 to 35 cm in length. The thalli are either one or two cells thick. Porphyra is gathered by coastal peoples from wild populations in large quantities, dried and processed into dried sheets, and served in a variety of ways: toasted as a snack, cooked with clams, salmon eggs, or fish in soup, or sprinkled on other foods as a condiment.

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FIGURE 1 Harvesting, drying and preparation of Arthrospira of dihe on the shore of Lake Kossorom.

Nori is often wrapped around the rice ball of sushi, a typical Japanese food consisting of a small handful of boiled rice with a slice of raw fish on the top. It can be incorporated into soy sauce and boiled down to give an appetizing luxury sauce. In China it is mostly used in soups and for seasoning fried foods. In the Republic of Korea it has uses similar to Japan. Nutritional value Porphyra is among the most nutritious macroalgae, with a protein content of 2550%, and about 75% of which is digestible. This alga is an excellent source of iodine, other trace minerals, and dietary fibers. Sugars are low (0.1%), and the vitamin content very high, with significant amounts of vitamins A, complex B, and C, but the shelf life of vitamin C can be short in the dried product. The characteristic taste of nori is caused by the large amounts of three amino acids: alanine, glutamic acid, and glycine. It also contains taurine, which controls blood cholesterol levels. The alga is a preferred source of the red pigment r-phycoerythrin, which is utilized as a fluorescent tag in the medical diagnostic industry. Other Uses P. abbottae is valued also for its medicinal properties as gastrointestinal aid, taken as decoction or applied as a poultice for any kind of sickness in the stomach, and as orthopedic aid applied on broken collarbones.

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Palmaria (Rodimenia) palmata (Florideophyceae) The fronds of the red alga Palmaria palmata are known as dulse (Figure 2); they are eaten raw as a vegetable substitute, dried or cooked with potatoes, in soups and fish dishes. Nutritional value Dulse is a good source of minerals, being very high in iron and containing all the trace elements needed in human nutrition, and has also a high vitamin content.

FIGURE 2 Palmaria palmata.

Chondrus crispus (Florideophyceae), the Irish moss or carrageenan moss (Figure 3) is not eaten as such, but used for its thickening powers when boiled in water, a result of its carrageenan content. One example is its use in making blancmange, a traditional vanilla-flavored pudding. It also used in macroalgae salads and as a soup ingredient.

FIGURE 3 Frond of Chondrus crispus.

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Gracilaria (Florideophyceae) when fresh, it is known as Ogo, ogonori, or sea moss. Gracilaria has been collected and sold as a salad vegetable in Hawaii (USA) for several decades. Nutritional value Gracilaria sp. contains (wet weight basis): 6.9 +0.1% total proteins, 24.7+0.7% crude fiber, 3.3+0.2% total lipids, and 22.7+0.6% ash. It contains 28.5+0.1 mg of vitamin C per 100 g of wet biomass, 5.2+0.4 % mg of -carotene per 100 g of dry weight, which corresponds to a vitamin A activity of 865 mg. Limu manauea and limu ogo are both sold as fresh vegetables, the latter usually mixed with raw fish. Callophyllis variegata (carola) (Florideophyceae) This red macroalgae is one of the most popular (Figure 4) edible macroalgae in Chile.

FIGURE 4 Frond of Callophyllis variegata.

Heterokontophyta Alaria esculenta, is a large brown kelp (Figure 5). It is known as winged kelp. It has a wide distribution in cold waters and does not survive above 16C. Nutritional value It is said to have the best protein among the kelps and is also rich in trace metals and vitamins, especially niacin. It is usually collected from the wild and eaten by local people. China is the largest producer of edible macroalgae, harvesting about 5 million wet tons annually. Laminaria japonica (Figure 6) is a large macroalga, usually 25 m long, but it can grow up to 10 m in favorable conditions. It requires water temperatures below 20C. Kombu is the Japanese name for the dried macroalgae that is derived from a mixture of Laminaria species.

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FIGURE 5 Frond of Alaria esculenta.

FIGURE 6 Frond of Laminaria japonica.

In Japan, it is used in everyday food, such as a seasoned and cooked kombu that is served with herring or sliced salmon. Undaria sp. Called wakame, is another exploited kelp, which together with Laminaria sp. is one of the two most economically important edible algae. U. pinnatifida is the main species cultivated (Figure 7). Wakame products is used for various instant foods such as noodles and soups, and its consumption is very popular. Nutritional value The crude protein content of wakame and kombu is 16.3 and 6.2 g (g/100 g), respectively, and both algae contain all essential amino acids, which account for 47.1% of the total amino acid content in wakame and for 50.7% in kombu.

FIGURE 7 Frond of Undaria pinnatifida.

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Table 2Table 4 summarize the vitamins, minerals, and fiber contents of the two edible algae. This data shows that wakame and kombu have high contents of carotene, that is, 1.30 and 2.99 mg (100 g d.w.) -1 or 217 and 481 g retinol (100 g d.w.) -1, respectively. The basic component in sea vegetables is iodine, an essential trace element and an integral part of two hormones released by the thyroid gland. According to the results in Table 3, wakame and kombu contain 26 and 170 mg (100 g d.w.)-1 of iodine, respectively, which is lacking in nori and other red macroalgae..

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Hizikia fusiforme is another brown algae popular as food in Japan and the Republic of Korea known as Hiziki. It is collected from the wild in Japan and cultivated in the Republic of Korea, grows at the bottom of the eulittoral and top of the sublittoral zones. Nutritional value The protein, fat, carbohydrate, and vitamin contents are similar to those found in kombu, although most of the vitamins are destroyed in the processing of the raw macroalgae. The iron, copper, and manganese contents are relatively high, certainly higher than in kombu. Like most brown macroalgae, its fat content is low (1.5%) but 2025% of the fatty acid is eicosapentaenoic acid (EPA). The harvested fronds are washed with seawater, dried in the sun, and boiled with the addition of other brown macroalgae such as Eisenia bicyclis or Ecklonia cava (Figure 8), which helps removing phlorotannin. This pigment gives Hizikia its astringent bitter taste. The resulting product is cut into short pieces, sun-dried, and sold packaged as hoshi hiziki. Typically it is cooked in stir fries, with fried bean curd and vegetables such as carrot or it may be simmered with other vegetables.

FIGURE 7.8 Frond of Ecklonia cava.

Cladosiphon okamuranus This brown macroalga is harvested from natural populations around the southern islands of Japan and consumed as Mozuku. It is characterized by a thallus with a stringy not turgid fronds and it can exceed 50 cm in length. Cladosiphon grows in the sublittoral, mainly at depths of 13 m. Harvesting is done by divers using a suction pump that draws the macroalgae up and into a floating basket besides the attending boat. The harvested macroalgae are washed, salted with 2025% salt, and let to dehydrate for about 15 days. Drained fronds are sold in wet, salted form in packages.

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Chlorophyta Monostroma (Figure 9) and Enteromorpha (Figure 10) are the two green macroalgae genera cultivated in Japan, and known as aonori or green laver. The fronds of Monostroma latissimum are bright green in color, flat and leafy, consisting of a single cell layer. They are slender at the holdfast and growing wider toward the apex, often with a slight funnel shape that has splits down the side. Nutritional value It averages 20% protein and has a useful vitamin and mineral content.

FIGURE 9 Frond of Monostroma latissimum.

FIGURE 10 Frond of Enteromorpha sp.

After harvesting, the macroalga is washed well in seawater and freshwater. It can then either be processed into sheets and dried, for sale in shops, or dried, either outside or in dryers, and then boiled with sugar, soy sauce, and other ingredients to make norijam. Enteromorpha prolifera and Enteromorpha intestinalis are found in many parts of the world including Palestine. Fronds usually flat, narrow, and bright green in color can be seen waving gently with water movement. They can be attached to firm substrate in clear, shallow waters, and also occur as epiphyte on cultured red seaweeds. It can thrive in both salt and brackish waters. Nutritional value It contains about 20% protein, little fat, low sodium, and high iron and calcium. Its vitamin B-group content is generally higher than most vegetables, and while its vitamin A is high, it is only half of that found in spinach. Harvested fronds are washed in freshwater and dried in large trays. Ulva sp. is known as sea lettuce, as fronds may be convoluted and have an appearance rather like lettuce. It can be collected from the wild and added to Monostroma and

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Enteromorpha as part of aonori. Bright green in color, it has a double or multiple cell layers. Slender at the holdfast and growing wider toward the apex, it reproduces seasonally, usually during tropical dry season or temperate spring. It is naturally found in shallow sea water usually less than 1 m in depth, where it grows on rocks, coral, mollusk shells, or other hard substrate, but also as an epiphyte on other sea macroalgae. Nutritional value It has a higher protein content than the other two, but much lower vitamin content, except for niacin, which is double that of Enteromorpha. Caulerpa lentillifera (Figure 11) and Caulerpa racemosa are the two edible green algae used in fresh salads and known as sea grapes or green caviar. As the common name suggests, their appearance looks like bunches of green grapes. These algae often produce runners under the substrate, which can produce several vertical branches that extend above the substrate. They naturally grow on sandy or muddy bottom in shallow protected waters.

FIGURE 11 Frond of Caulerpa lentillifera.

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Table 5 summarizes edible algae and the corresponding food item.

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ANIMAL FEED Algae, especially microalgae, are utilized in aquaculture as live feeds for all growth stages of bivalve molluscs (e.g., oysters, scallops, clams, and mussels), for the larval/early juvenile stages of abalone, crustaceans, and some fish species, and for zooplankton used in aquaculture food chains. Over the last four decades, several hundred microalgae species have been tested as food, but probably less than 20 have gained widespread use in aquaculture. Successful strains for bivalve culture included Isochrysis galbana, Isochrysis sp., Pavlova lutheri, Tetraselmis suecica, Pseudoisochrysis paradoxa, Chaetoceros calcitrans, and Skeletonema costatum. Isochrysis sp. (T.ISO), P. lutheri, and C. calcitrans are the most common species used to feed the larval, early juvenile, and broodstock (during hatchery conditioning) stages of bivalve molluscs; these are usually fed together as a mixed diet. Many of the strains successfully used for bivalves are also used as direct feed for crustaceans (especially shrimp) during the early larval stages, especially diatoms such 156

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as Skeletonema spp. and Chaetoceros spp. Benthic diatoms such as Navicula spp. and Nitzschia are commonly mass-cultured and then settled onto plates as a diet for grazing juvenile abalone. Isochrysis sp., P. lutheri, T. suecica, or Nannochloropsis spp. are commonly fed to Artemia or rotifers, which are then fed on to later larval stages of crustacean and fish larvae. Microalgae must possess a number of key attributes to be useful aquaculture species. They must be of an appropriate size for ingestion, for example, from 1 to 15 m for filter feeders; 10 to 100 m for grazers and readily digested. They must have rapid growth rates, be amenable to mass culture, and also be stable in culture to any fluctuations in temperature, light, and nutrients. Finally, they must have a good nutrient composition, including an absence of toxins that might be transferred up the food chain. Nutritional value of micro-algae Microalgal species can vary significantly in their nutritional value, and this may also change under different culture conditions. Nevertheless, a carefully selected mixture of microalgae can offer an excellent nutritional package for larval animals, either directly or indirectly (through enrichment of zooplankton). Microalgae that have been found to have good nutritional properties either as monospecies or within a mixed diet include C. calcitrans, C. muelleri, P. lutheri, Isochrysis sp., T. suecica, S. costatum, and Thalassiosira pseudonana. The growth of animals fed a mixture of several algal species is often superior to that obtained when feeding only one algal species. A particular alga may lack a nutrient, while another alga may contain that nutrient and lack a different one. In this way, a mixture of both algal species supplies the animals with an adequate amount of both nutrients. The nutritional value of any algal species for a particular organism depends on its size and shape, digestibility (related to cell wall structure and composition), biochemical composition (e.g., nutrients, enzymes, and toxins if present), production of toxic compounds, and the requirements of the animal feeding on the alga. The gross composition of 16 species of micro-algae is compared in Table 6. Although there are marked differences in the compositions of the micro-algal classes and species, protein is always the major organic constituent, followed usually by lipid and then by carbohydrate. Expressed as percentage of dry weight, the range for the level of protein, lipid, and carbohydrate are 12-35%, 7.2-23%, and 4.6-23%, respectively. The content of poly unsaturated fatty acids (PUFA), in particular eicosapentaenoic acid (EPA), arachidonic acid (ARA), and docosahexaenoic acid (DHA), is of major importance in the evaluation of the nutritional composition of an algal species to be used as food for marine organisms. Significant concentrations of EPA are present in the diatom species (Chaetoceros calcitrans, C. gracilis, S. costatum, T. pseudonana) and the prymnesiophyte Platymonas lutheri, whereas high concentrations of DHA are found in the prymnesiophytes (P. lutheri, Isochrysis sp.) and Chroomonas salina. Micro-algae can also be considered as a rich source of ascorbic acid (0.11-1.62% of dry weight).

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The nutritional value of micro-algae can vary considerably according to the culture conditions. For example the effect of the composition of the culture medium on the proximate composition of various species of micro-algae is demonstrated in Table 7. The protein content per cell, which is considered as one of the most important factors determining the nutritional value of micro-algae as feed in aquaculture, was found to be more susceptible to medium-induced variation than the other cellular constituents. 7

Microalgae grown to late-logarithmic growth phase typically contain 3040% proteins, 1020% lipids and 515% carbohydrates. When cultured through to stationary phase, the proximate composition of microalgae can change significantly; for example, when nitrate is limiting, carbohydrate levels can double at the expense of protein.

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A common procedure during the culture of both larval fish and prawns is to add microalgae (i.e., green water) to intensive culture systems together with the zooplankton prey. Addition of the microalgae to larval tanks can improve the production of larvae, though the exact mechanism of action is unclear. Theories advanced include (a) light attenuation (i.e., shading effects), which have a beneficial effect on larvae, (b) maintenance of the nutritional quality of the zooplankton (c) an excretion of vitamins or other growth-promoting substances by algae, and Most likely, the mechanism may be a combination of several of these possibilities. Maintenance of NH3 and O2 balance has also been proposed, though this has not been supported by experimental evidence. The most popular algae species used for green water applications are N. oculata and T. suecica. Green water may also be applied to extensive outdoor production systems by fertilizing ponds to stimulate microalgal growth, and correspondingly, zooplankton production, as food for larvae introduced into the ponds. Harvesting and preserving micro-algae In most cases, it is unnecessary to separate micro-algae from the culture fluid. Excess and off-season production may, however, be concentrated and preserved. Highdensity algal cultures can be concentrated by either chemical flocculation or centrifugation. Products such as aluminum sulphate and ferric chloride cause cells to coagulate and precipitate to the bottom or float to the surface. Recovery of the algal biomass is then accomplished by, respectively, siphoning off the supernatant or skimming cells off the surface. Due to the increased particle size, coagulated algae are no longer suitable as food for filter-feeders. Centrifugation Cells are centrifuged as a thick algal paste, which is then resuspended in a limited volume of water. The resulting slurry may be stored for 1-2 weeks in the refrigerator or frozen. In the latter case, cryoprotective agents (glucose, dimethylsulfoxide) are added to maintain cell integrity during freezing. However, cell disruption and limited shelf-life remain the major disadvantages of long-term preserved algal biomass. Concentrated cultures of Tetraselmis suecica kept in darkness at 4C maintain their viability, whereas the latter is completely lost upon freezing. Furthermore, cultures stored in hermetically sealed vials lose their viability more rapidly than those kept in cotton-plugged vials. Macroalgae meal For a long time, animals such as sheep, cattle, and horses that lived in coastal areas have eaten macroalgae, especially in those European countries where large brown macroalgae were washed ashore. Today the availability of macroalgae for animals has been increased with the production of macroalgae meal: dried macroalgae that has been milled to a fine powder. The macroalgae used for meal such as Ascophyllum nodosum and Laminaria digitata must be freshly cut, as drift macroalgae is low in minerals and usually becomes infected with mould. The wet macroalgae is passed through hammer mills with progressively smaller screens to reduce it to fine particles. These are passed through a

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drum dryer starting at 700800C and exiting at no more than 70C. It should have a moisture level of about 15%. It is milled and stored in sealed bags because it picks up moisture if exposed to air. It can be stored for about a year. Analysis shows that it contains useful amounts of minerals (potassium, phosphorus, magnesium, calcium, sodium, chlorine, and sulfur), trace elements, and vitamins. Trace elements are essential elements needed by humans and other mammals in smaller quantities than iron (approximately 50 mg/kg body weight), and include zinc, cobalt, chromium, molybdenum, nickel, tin, vanadium, fluorine, and iodine. Benefits o Increase the iodine content of the poultry eggs. o In cows it increases the milk production of 6.8% that lead to 13% more income. o Ewes fed macroalgae meal over a 2 yr period maintained their weight much better during winter feeding and also gave greater wool production. o Boost the immune system of some animals o In fish farming, wet feed usually consists of meat waste and fish waste mixed with dry additives containing extra nutrients, all formed together in a doughy mass. When thrown into the fish ponds or cages it must hold together and not disintegrate or dissolve in the water. A binder is needed; so macroalgae meal is a cheaper choice (instead of a technical grade of alginate). It has also been used to bind formulated feeds for shrimp and abalone. Fresh macroalgae There is also a market for fresh macroalgae as a feed for abalone. In Australia, the brown macroalgae Macrocystis pyrifera and the red macroalgae Gracilaria edulis have been used. In South Africa, Porphyra is in demand for abalone feed and recommendations have been made for the management of the wild population of the macroalgae. Pacific dulse (Palmaria mollis) has been found to be a valuable food for the red abalone, Haliotis rufescens, and development of land-based cultivation has been undertaken with a view to producing commercial quantities of the macroalgae. The green macroalgae, Ulva lactuca, has been fed to Haliotis tuberculata and Haliotis discus. Feeding trials showed that abalone growth is greatly improved by high protein content, and this is attained by culturing the macroalgae with high levels of ammonia present. FERTILIZERS There is a long history of coastal people using macroalgae, especially the large brown macroalgae, to fertilize nearby land. Generally, beach-washed macroalgae is collected, although farmers sometimes cut macroalgae exposed at low tide. o In U.K., the practice was to mix the macroalgae with sand, let it rot, and then dig it in. o In tropical climate like the Philippines, large quantities of Sargassum have been collected, used wet locally, but also sun-dried and transported to other areas. o In Argentina, green macroalgae, that are cast ashore every summer are composted and then used in trials for growing tomato plants. The addition of the compost increased water holding capacity and plant growth, so composting simultaneously solved environmental pollution problems and produced a useful organic fertilizer. o Brown macroalgae is dried, milled as macroalgae meal and sold as soil additives. They are function as both fertilizer and soil conditioner. They have a suitable content of nitrogen and potassium, but are much lower in phosphorus than traditional animal manures and the typical N: P :K ratios in chemical fertilizers.

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The large amounts of insoluble carbohydrates in brown macroalgae act as soil conditioners (improve aeration and soil structure, especially in clay soils) and have good moisture retention properties. Their effectiveness as fertilizers is also sometimes attributed to the trace elements they contain, but the actual contribution they make is very small compared with normal plant requirements. Brown macroalgae contain alginate, a carbohydrate composed of long chains. When calcium is added to alginate, it forms strong gels. By composting the dried, powdered for 1112 days, the alginate chains are broken into smaller chains and these chains still form gels with calcium but they are weaker. The composted product is a dark brown, granular material containing 2025% water and it can be easily stored and used in this form. Steep slopes are difficult to cultivate with conventional equipment and are likely to suffer soil loss by runoff. Spraying such slopes with composted Ascophyllum, clay, fertilizer, seed, mulch, and water has given good results, even on bare rock. Plants quickly grow and topsoil forms after a few years. The spray is thixotropic, that is, it is fluid when a force is applied to spread it but it sets to a weak gel when standing for a time and sticks to the sloping surface. It holds any soil in place and retains enough moisture to allow the seeds to germinate. o Maerl is the common name of a fertilizer derived from calcareous red algae. The maerl is marketed mainly for use as an agricultural fertilizer, for soil improvement in horticulture, mainly to replace lime as an agricultural soil conditioner. o Macroalgae extracts and suspensions as fertilizers have given positive results in many applications. When applied to fruit, vegetable, and flower crops, some improvements have included higher yields, increased uptake of soil nutrients, increased resistance to some pests such as red spider mite and aphids, improved seed germination, and more resistance to frost. They are sold in concentrated form, are easy to transport, dilute, and apply, and act more rapidly. They are all made from brown macroalgae. Some are made by alkaline extraction of the macroalgae and anything that does not dissolve is removed by filtration; others are suspensions of very fine particles of macroalgae. Most of the extracts contain several types of plant growth regulators such as cytokinins, auxins, and betaines, which may be responsible for the improvements of the yield. Finally there is the question, are macroalgae extracts an economically attractive alternative to NPK fertilizers? Perhaps not when used on their own, but when used with NPK fertilizers they improve the effectiveness of the fertilizers, so less can be used, with a lowering of costs. Then there are always those who prefer an organic or natural fertilizer, especially in horticulture, so macroalgae extracts probably have a bright future. PRODUCTION OF VALUABLE (USEFUL) PRODUCTS FROM ALGAE POLYSACCHARIDES CYANOBACTERIA produce three types of extracellular matrix consisting mainly of polysaccharides, which have unique bio- and physicochemical characteristics. Most of them are composed of at least ten different monosaccharides and contain pentoses, which have not been observed in other prokaryotic polysaccharides. Little work has been devoted to potential applications of marine cyanobacterial polysaccharides.

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MICROALGAE produce many different types of polysaccharides, which may be a costituent of the cell wall as in unicellular red algae as Porphyridium sp. and Rhodella sp., or be present inside the cell, as in the Euglenophyceae. The polysaccharides of Rhodophyta are highly sulfated and consist mainly of xylose, glucose, and galactose. These compounds selectively inhibit reverse transcriptase (RT) enzyme of human immunodeficiency virus (HIV) and its replication in vitro. Moreover, the sulfated polysaccharide of Porphyridium sp. has shown promising antiviral activity against a variety of animal viruses including Herpes simplex viruses types 1 and 2 (HSV 1, 2) Varicella zoster virus (VZV). The compounds also showed significant inhibition of productive infection with retroviruses (murine leukenia virus, HIV-1, and HIV-2) and cell transformation by murine sarcoma virus in vitro. Thus, red microalgae and their polysaccharides seem to be good candidates for the development of antiviral drugs. Rodents fed with a diet supplemented with biomass and polysaccharides derived from Porphyridium results in a decrease in blood cholesterol concentration (by 22% and 29%, respectively) and triglyceride levels, increased feces weight (by 130% and 196%, respectively) and bile acid excretion (5.1- and 3.2-fold or more). Moreover, algal biomass or polysaccharide increased the length of both the small intestine (by 17% and 30%, respectively) and the colon (by 8.5% and 32%, respectively). Recently, anti-inflammatory and immunosupressive properties were discovered in the extracts or extracted polysaccharides of the marine species, Chlorella stigmatophora. Paramylon is the term used for granules of the reserve polysaccharide of Euglena and euglenoids in general. Paramylon consists of -1,3-glucan, a linear polysaccharide found in the cell walls of many bacteria, plants, and yeasts. The main interest in -glucans stems from o their ability to act as nonspecific immune system stimulants, by binding to a specific site on monocytes/ macrophages and granulocytes. o They have been successfully used in aquaculture to strengthen the nonspecific defence of many important species of fishes and shrimps by injection, immersion, or in the feed. o Sulfated derivatives of Euglena paramylon, in particular, have shown anti-HIV (human immunodeficiency virus) activity. o Moreover, it has been suggested that this polysaccharide has a cholesterollowering effect when incorporated in the diet of either humans or animals o It moderates the postprandial blood glucose and insulin response in humans. Since Euglena gracilis can accumulate large quantities of paramylon when grown in the presence of an utilizable carbon source, it could represent an alternative source of this compound to Saccharomyces cerevisiae (bakers yeast), which is currently exploited industrially for its extraction. MACROALGAE Agar, alginate (derivative of alginic acid), and carrageenan are three hydrocolloids that are extracted from various red and brown macroalgae. A hydrocolloid is a non-crystalline substance with very large molecules, which dissolves in water to give a thickened (viscous) solution. Agar, alginate, and carrageenan are water-soluble carbohydrates used to thicken aqueous solutions, to form gels (jellies) of varying degrees of firmness, to form water-soluble films, and to stabilize certain products, such as ice-cream (they inhibit the formation of large ice crystals, allowing the ice-cream to retain a smooth texture). The gelling properties of agar can be extracted with hot water from a red macroalgae. 163

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There are a number of artificial products reputed to be suitable replacements for macroalgae gums but none have the exact gelling and viscosity properties of macroalgae gums and it is very unlikely that macroalgae will be replaced as the source of these polysaccharides in the near future. Agar Agar, a general name for polysaccharides extracted from some red algae, is built up of alternating D- and L-galactopyranose units. The name agar is derived from a Malaysian word agar-agar, which literally means macroalgae. As the gelling agent it is known from Japan since the 17th century; extracts from red macroalgae were carried up the mountains to freeze overnight so that water and other impurities could be extracted from the material. Agar finds its widest use as a solid microbiological culture substrate. Modern agar is a purified form consisting largely of the neutral fraction known as agarose; the non-ionic nature of the latter makes it more suitable for a range of laboratory applications. Agar in a crude or purified form also finds wide usage in the food industry where it is used in various kinds of ices, canned foods, and bakery products. The genera Gelidium, Gracilaria, Hypnea and Pterocladia of the Rhodophyceae are the main producers of these materials. The higher-quality agar (bacteriological-grade agar is extracted from Pterocladia and Gelidium (Figure 12). Agars of lesser quality are extracted from Gracilaria and Hypnea species. Food-grade agar is seasonal in Pterocladia species, being low in the colder months and high in the warmer. Gracilaria species were once considered unsuitable for agar production because the quality of the agar was poor. In the 1950s, it was found that pre-treatment of the macroalgae with alkali before extraction lowered the yield but gave a good-quality agar. This allowed expansion of the agar industry, which had been previously limited by the available supply of Gelidium, and led to the harvesting of a variety of wild species of Gracilaria.

FIGURE 12 Frond of Gelidium sp.

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Alginate This polysaccharide is obtained from the brown seaweeds, especially from species of Fucus and Macrocystis (Phaeophyceae). Alginates are cell-wall constituents of brown algae. They are chain-forming heteropolysaccharides made up of blocks of mannuronic acid and guluronic acid. Composition of the blocks depends on the species being used for extraction and the part of the thallus from which extraction is made. Extraction procedures probably also affect alginate quality. Alginates of one kind or another seem to be present in most species of brown algae but they occur in exploitable quantities (3045% d. w.) only in the large kelps (Laminariales and Fucales).

Commercially, sodium alginate is extracted from giant brown seaweed (Macrocystis pyrifera), horsetail kelp (Laminaria digitata) and sugar kelp (Laminaria saccharina). Macrocystis has the distinction of being the largest macroalgae in the world; the largest attached plant recorded was 65 m long and the plants are capable of growing at up to 50 cm day-1. Ascophyllum nodosum and Laminaria hyperborea are used in Norway and Scotland. Ascophyllum is sustainably harvested in Ireland to produce macroalgae meal that is exported to Scotland for alginate extraction.

FIGURE 13 Frond of Macrocystis pyrifera.

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Sodium alginate has been used o as a thickening paste for colors in printing textiles. o as stabilizers in the manufacture of ice cream, giving a smooth texture and body o as a suspending agent in milk shakes. o It is also used in cosmetics and pharmaceuticals. o Sodium alginate is reported to be a useful adjuvent in immunisation against two strains of influenza virus. o Sodium alginate is also found effective in diminishing hyper calciuria in urolithiasis, and found useful in the treatment of esophagitis. o The most significant property of sodium alginate is the ability to remove heavy metals strontium and lead from the body without seriously affecting the availability of Ca, Na, K or other lighter metals in the body. This selective action of sodium alginate is of great potential to remove lead and strontium- 90 poisoning contamination due to fall out from atomic explosions. Calcium alginate o Calcium alginate is reputed to be a hemostatic agent which stimulates the clotting of blood in situ which is subsequently absorbed in the tissue. o Calcium alginate, has been used in the manufacture of a medical dressing very suitable for burns and extensive wounds where a normal dressing would be extremely difficult to remove; the calcium alginate is extruded to make a fiber which is then woven into a gauze-like product. When applied to either a wound or burn, a network is formed around which a healthy scab may form; the bandage may be removed with a sodium chloride solution, which renders the alginate soluble in water. o Alginates are also used as glazing and sizing paper, special printers inks, paints, cosmetics, insecticides, and pharmaceutical preparations. Carrageenan Carrageenan is a general name for polysaccharides extracted from certain kinds of algae which are built up, in contrast to agar, from D-galactopyranose units only. Carrageenan are produced by species of Kappaphycus, Eucheuma, Chondrus, Gigartina and Iridea. o Carrageenans are reported to be strong antigens. In general, they behave as typical carbohydrate antigens. Carrageenan is also reported to stimulate the growth of connective tissues. o Carrageenan had been found to possess antiviral properties. The specific antiviral activity had been shown against influenza B and mumps virus in embryonated eggs even after 24 h inocubation. o Carrageenan was also found as anticoagulant and antithrombic agent. o The use of carrageenan in ulcer therapy had been studied intensively. The polysaccharide reacts with the mucoid lining of the stomach and gives a protective layer through which pepsin and acid have difficulty in passing. The treatment of gastric and duodenal ulcers by carrageenan was enjoying considerable popularity in France and Great Britain. In many cases of ulcer carrageenan proved an effective cure.

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Table 8 summarizes commercially exploited algae and the corresponding extract.

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Laminarin Laminarin is a sulfated polysaccharide occurs at certain times of the year to the extent of 35% of the dry weight of Laminaria cloustoni. It has been found that laminarin sulphate formed with two sulphate groups by glucose unit gives maximum stability and anticoagulant activity. Two lower sulphated laminarins are also reported to have antilipidemic activity like that of heparin.

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COSMETICS Extract of algae is often found on the list of ingredients on cosmetic packages, particularly in face, hand, and body creams or lotions, but the use of algae themselves in cosmetics, rather than extracts of them, is rather limited. Milled macroalgae, packed in sachets, is sold as an additive to bath water, sometimes with essential oils added. Bath salts with macroalgae meal are also sold. Thalassotherapy has come into fashion in recent years, especially in France. In thalassotherapy, macroalgae pastes, made by cold-grinding or freeze-crushing, are applied to the persons body and then warmed under infrared radiation. Sometimes additional freezing is done with liquid nitrogen that makes the frozen material more brittle and easier to grind or crush. The result is a fine green paste of macroalgae. This treatment, in conjunction with seawater hydrotherapy, is said to provide relief for rheumatism and osteoporosis. Paste mixtures are also used in massage creams, with promises to rapidly restore elasticity and suppleness to the skin. There appears to be no shortage of products with ingredients and claims linked to macroalgae: creams, face masks, shampoos, body gels, and bath salts. The efficacy of these products must be judged by the user. Cosmetic products, such as creams and lotions, sometimes show on their labels that the contents include marine extract, extract of alga, macroalgae extract or similar. This usually means that one of the hydrocolloids extracted from macroalgae has been added. Alginate or carrageenan could improve the skin moisture retention properties of the product. THERAPEUTIC SUPPLEMENTS Microalgae are a unique source of therapeutic substances, particularly from cyanobacteria. Among cyanobacteria Spirulina sp. has undergone numerous and rigorous toxicological studies that have highlighted its potential therapeutic applications in the area of immunomodulation, anticancer, antiviral, and cholesterol reduction effects. A number of extracts were found to be remarkably active in protecting human lymphoblastoid T-cells from the cytopathic effects of HIV infection. Active agents consisting of sulfolipids with different fatty acid esters were isolated from Lyngbya lagerheimii and Phormidium tenue. Cyanovirin A protein isolated from an aqueous cellular extract of Nostoc elipsosporum prevents the in vitro replication and citopathicity of primate retroviruses. Cryptophycin 1, an active compound isolated from Nostoc strain, exerts antiproliferative and antimitotic activities by binding to the ends of the microtubules, thus blocking the cell cycle at the metaphase of mitosis. Research has been focused on its potent antitumor activity and a synthetic analogue, cryptophycin-52, is at present in Phase II clinical trials. Sulfated polysaccharide, calcium spirulan A novel water soluble extracts of cyanobacteria have found to be an antiviral agent. This compound appears to be selectively inhibiting the penetration of enveloped viruses into host cells, thereby preventing the replication. The effect was described for many different viruses like herpex simplex, measles, and even HIV-1. Among eukaryotic microalgae, a glycoprotein prepared from Chlorella vulgaris culture supernatant exhibited protective activity against tumor metastasis and chemotherapy-induced immunosuppression in mice.

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Extracts from several macroalgae may prove to be a source of effective antiviral agents. Carraguard, a mixture of carrageenans, has been shown to be effective against human immunodeficiency virus (HIV) in vitro and against herpes simplex virus in animals. Because antiviral substances in macroalgae are composed of very large molecules, it was thought they would not be absorbed by eating macroalgae. In one survey however, it has been found that, the rate of HIV infection in macroalgae-eating communities can be markedly lower than it is elsewhere. This has led to some smallscale trials in which people infected with HIV ate powdered Undaria, with a resulting decrease of 25% in the viral load. CAROTENOIDS AND OTHER PIGMENTS Carotenoids All algae contain carotenoids which are lipid-soluble pigments composed of isoprene units. Carotenoids of commercial importance are xanthophylls, fucoxanthin and -carotene. Xanthophylls, are yellow to orange pigments. Of these astaxanthin and canthaxanthin, which are extracted from certain species of green microalgae are used as food dyes, especially in the pigmentation of chicken skin, egg yolk, and fish (e.g., salmon). Canthaxanthin is also used as a tanning agent. Astaxanthin has been described as having nutraceutical importance related to freeradical scavenging, immunomodulation, and cancer prevention. Haematococcus pluvialis can produce and accumulate astaxanthin to concentrations of 18% of the dry weight. This concentration within the cell enables milking of H. pluvialis for astaxanthin. Fucoxanthin, a carotenoid commonly distributed in brown algae, such as U. pinnatifida, Scytosiphon lomentaria, Petalonia binghamiae, and Laminaria religiosa, is a potent drug candidate and can be utilized as an excellent supplement like astaxanthin, because it acts as an antioxidant and inhibits GOTO cells of neuroblastoma and colon cancer cells. Recently, the apoptosis activity against HL-60 (human leukema) and Caco-2 (cancer colon) cells has been reported for fucoxanthin. Algal milking Most microalgal products are secondary metabolites that are produced when growth is limited. The continuous removal of secondary metabolites from cells thereby enables the biomass to be reused for the continuous production of these highvalue compounds. Such process is called algal milking. The milking process can be applied to different algae and different products, Dunaliella salina, for the recovery -Carotene, Haematococcus pluvialis for the recovery astaxanthin, and different marine microalgae for Polyunsaturated fatty acids (PUFA). -carotene -carotene is commercially the most important pigment. -Carotene is used primarily as a yellow food coloring and, being a source of vitamin A, as a component of health foods and animal feeds. Also, in humans and animals above-average ingestion of carotene predisposes to a lower incidence of certain types of cancer. Algal cells accumulate -carotene to protects them against the deleterious effects of high light intensity. When cultivated under appropriate conditions, the green microalga 169

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Dunaliella salina and D. bardawil accumulate more than 10% of their dry weight as -carotene. Recently, a new method was developed for milking -carotene from D. salina in a two phase bioreactor. In this technique, cells are first grown under normal growth conditions and then stressed by excess light to produce larger amounts of carotene. At this stage, the second, biocompatible organic phase is added and the carotene is extracted via continuous recirculation of a biocompatible organic solvent (lipophilic compound) through the aqueous phase containing the cells. The extraction of the product will enhance its synthesis. Because the cells continue to produce carotene, the extracted product is continuously replaced by newly produced molecules. Therefore, the cells are continuously reused and do not need to be grown again. In contrast to existing commercial processes, this method does not require the harvesting, concentrating, and destroying of cells for extraction of the desired product. The general application of this process would facilitate the commercialization of microalgal biotechnology and development of microalgal products. Other Pigments In the food industry, the growing tendency to use natural, rather than synthetic, dyes has encouraged the search for new natural dyes. In particular, carotenoids and phycobiliproteins extracted from microalgae are gaining increasing commercial acceptance as natural colorings. Red algae and blue-green algae are characterized by the presence of red and blue pigments (in addition to chlorophyll a), which are responsible for their red-brown and blue-green color, respectively. These pigments have promising potential as food dyes, especially because many red and blue pigments currently in use are thought to be carcinogens. Other advantages are their intense color, their high solubility in water, and their stability to changes in pH. Their application in cosmetics is also of great potential. The red, highly fluorescent phycobiliprotein, phycoerythrin, which is extracted from microalgae, is used mainly in diagnostics in multiple color flow cytoflourimetric analysis. In this technique, conjugates of phycobiliproteins and molecules with specific biological activity bind specifically to their target molecules, making them highly fluorescent. The method is very sensitive because the specific absorbance of phycoerythrin is very high, even at very low concentrations. A series of conjugates, designated phycoprobes have developed. These conjugates are composed of fluorescent phycobiliproteins (mainly phycoerythrin) covalently bound to biologically active molecules. Fluorescent markers include phycoerythrin-immunoglobulins, phycoerythrin-protein, phycoerythrin-avidin, and phycoerythrin-biotin. The sensitivity of the method increased to enable the detection of leukemia at early stages by means of antibodies conjugated to phycoerythrin. They were able to detect as few as 5 106 positive cells, compared with about 300 106 by former methods. The use of phycoerythrin together with a second fluorescent marker enables multiple cytotoxic examinations. The other advantages of phycoerythrin conjugates are that their fluorescence is not affected by most natural molecules; they are soluble in water; and they can be stored for long periods. The blue pigment phycocyanin, which is obtained from blue-green algae, is used in the food industry. Dainippon Ink and Chemicals of Japan has commercialized it under the name of Linablue. It is used for coloring candy, ice cream, dairy products, and soft drinks.

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POLYUNSATURATED FATTY ACIDS (PUFAS) PUFAs are gaining increasing importance as valuable pharmaceutical products and ingredients of food owing to their beneficial effect on human health. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in particular, are important in the development and functioning of brain, retina, and reproductive tissues both in adults and infants. They can also be used in the treatment of various diseases and disorders, including cardiovascular problems, a variety of cancers, and inflammatory disease. At present, PUFAs are produced commercially from fish oil, but this is an insufficient source of these products and microalgae provide an optimal lipid source of PUFAs. The heterotrophic marine dinoflagellate Crypthecodinium cohnii has a lipid content greater than 20% dry weight and is known for its ability to accumulate fatty acids that have a high fraction (3050%) of DHA. Lipids are important components of algal cell membranes but also accumulate in globules in other parts of the cells. Microalgal growth and fatty acid formation is affected by medium composition and environmental conditions (e.g., carbon sources). Lipid production occurs under growth-limiting conditions; during linear growth, the cells are stressed owing to nutrient limitation and therefore produce more lipids. Also the concentration of DHA, and the lipid quality, is negatively affected by increases in lipid concentration. The highest quality lipid it obtained when glucose is used as the carbon source, and when the cell concentration and lipid content of the cells are the lowest. Milking can be used for DHA production by C. cohnii. In this process, cells are first grown under the correct conditions for growth, after which they are stressed to produce higher concentrations of DHA. A biocompatible organic solvent is added during the DHA production stage to extract the product. This process enables the production of high-quality lipid, thereby reducing extraction and purification costs. Eicosapentaenoic acid EPA Microalgae represent also one of the most promising EPA producers. Many Eustigmatophyceae, such as Nannochloropsis sp. and Monodus subterraneus, and Bacillariophyceae species contain a considerable amount of EPA. An EPA production potential has been found in the genus Nitzschia (especially N. alba and N. laevis). It was reported that the oil content of N. alba was as high as 50% of cell dry weight and the EPA comprises 45% of the oil. N. laevis could utilize glucose or glutamate as single substrate for heterotrophic growth, and the cellular EPA content of the alga in heterotrophic conditions was also higher than that in photoautotrophic conditions suggesting that this diatom is a good heterotrophic EPA producer. EPA are effective in reducing levels of plasma lipids and lipoproteins and in ameliorating coronary heart disease, arthritis, and inflammatory ailments. When red microalgal biomass containing EPA (up to 40% of the fatty acids) and over 50% polysaccharides was fed to rodents (at a dietary level of up to 10% dry algal biomass of their diet), liver EPA content increased from 0.1% in the control animals to 0.7% in the experimental group. There was a decrease of 25% in serum cholesterol and triglyceride levels, probably owing to the effect of the EPA present in the algae, or to the polysaccharides acting as dietary fiber, or to a unique combination of the two components Arachidonic acid (ARA) is a highly unsaturated fatty acid that is rarely found in higher plants. In marine red microalga Porphyridium, arachidonic acid constitutes as much as 36% of the total fatty acids. Thus, Porphyridium has been suggested as a source of arachidonic acid. In addition to being an essential fatty acid in the human diet, arachidonic acid is the natural precursor of a large family of structurally related

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C20 compounds that include prostaglandins, thromboxanes, leukotrients, and prostacyclins, all of which are potent biological regulators. TOXIN Toxins are compounds that are either synthesized by the algae or formed by the composition of metabolic products. Of the millions of species of microalgae those that produce specific toxins scarcely exceed a hundred. These occur in both salt and freshwaters, and while most are planktonics some are benthic or floating at water surface. Toxins can attract particular attention when they cause the death of livestock that has drunk water containing them or fish and shellfish in the sea, or humans that consume these. Toxins have been divided into different classes based on the syndromes associated with exposure to them, such as paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), and amnesic shellfish poisoning (ASP). Algae which seem to be directly producer of toxic substances mostly belong to three taxonomic groups: Cyanophyta, Haptophyta, and Dinophyta. In addition to these there are some groups which include one or two toxic members. Species of Chattonella and Heterosygma, belonging to the Raphidophyceae, form toxic red tides in Japanese waters and a few diatoms of the genus Peusdonitzschia produce domoic acid, a low molecular amino acid causing amnesic shellfish poisoning. Table 9 summarizes toxic algae and the corresponding metabolites.

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OTHER BIOACTIVE MARINE NATURAL PRODUCTS OF MARINE ALGAE Chemically the bioactive metabolites of marine flora include brominated phenols, brominated oxygen heterocyclics, nitrogen heterocyclics, sulphur nitrogen heterocyclics, sterols, terpenoids, polysaccharides, peptides and proteins. 1. Brominated Phenols The active principles isolated from Symphyocladia gracilis, Rhodomela larix and Polysiphonia lanosa were: 2,3-dibromobenzyl alcohol, disulphate dipotassium salt (1), 2,3-dibromo-4,5- dihydroxybenzaldehyde (2), 2,3-dibromo-4,5-dihydroxybenzyl alcohol (3), 3,5-dibromo-p-hydroxybenzyl alcohol (4) and the 5-bromo-3,4dihydroxybenzaldehyde (5). Virtually nothing is known about the physiological importance and the mechanism of biosynthesis of the bromo phenols. Their antialgal activity suggests that they may play a role in the regulation of epiphytes and endophytes.

2. Brominated Oxygen Heterocyclics The red algae Laurencia sp. have produced the diverse class of natural products. L. glandulifera and L. nipponica had furnished two brominated oxygen heterocyclic compounds, laurencin (6) and laureatin (7), respectively.

Laureatin (7) and isolaureatin exhibit significant larvicidal activity (IC50) 0.06 and 0.50 ppm, respectively. Brominated compounds isolated from marine algae, particularly bromophenols, are toxic and due to this they are not of clinical value.

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3. Nitrogen Heterocyclics Marine algae had yielded nitrogen containing heterocyclic compounds. Of these the most interesting compounds are domoic acid (8) and the kainic acid.

Domoic acid (8) was first isolated from the alga Chondria armata. Domoic acid showed marked anthelmintic activity. It was found to be very effective in expelling ascaris and pinworms without any observable side effects. 4. Kainic Acids In Asia, the dried red alga Digenea simplex is widely used as an anthelmintic. It is found very effective in the treatment of ascariasis. In the Mediterranean, extract of the alga Corallina officinalis is also used for the same purpose. Of the kainic acids, kainic acid (9) was the most active constituent.

Isomers of -kainic acid had been isolated from alga Digenea. The isomers isolated are -allo-kainic acid (10) and -kainic acid lactone (11). -Kainic acid had been found effective in the treatment of ascariasis, with a single dose of 5 to 10 mg per adult resulting in a 40 to 70% reduction in the population of instestinal parasitic worms. -Allokainic acid was found to have far less anthelmintic activity. Several preparations of kainic acids are available in the market, including "Digenin" and "Helminal" (The Merck Index, 1968). This represents one of the few instances in which clinically useful pharmaceutical product has been isolated from marine source.

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5. Guanidine Derivatives At certain unpredictable time the red plankton multiply and cause red tide. Although many fishes are killed by this red tide, mussels and clams survive and concentrate the toxic principles, thus becoming poisonous to humans. The toxin isolated from the marine alga Gonyaulax catenella is called saxitoxin (12).

Saxitoxin blocks nerve conduction by specifically interfering with the intital increase in sodium permeability of the membrane. The symptoms caused by the toxin include peripheral paralysis. In extreme cases, complete loss of strength in the muscles and finally death occurred which is caused due to respiratory failure. Saxitoxin is absorbed from the gastro-intestinal tract. It produced no major vascular action. In man death had occurred following ingestion of as little as 1 mg of toxin. The toxic compounds from marine algae appear to have biomedical potential. The compounds with neurotropic effects may yield important drugs. 6. Phenazine Derivatives The marine alga Caulerpa lamourouxii is widely distributed in the Phillippines. The upper branches are eaten as a salad, despite their peppery and astringent taste. However, the alga is found toxic to some individuals. Chemical investigation of the alga had furnished caulerpicine, caulerpin (13), cholesterol, taraxerol, -sitosterol and palmitic acid. Caulerpin had also been isolated from Caulerpa sertularioides, C. racemosa var. clavifera and caulerpicin from C. racemosa. Caulerpin causes a mild anesthetic action when placed in the mouth, which resulted in numbness of the lips and tongue. In some people it produced toxic effects. The toxic syndrome had been reported to be some what similar to that produced by ciguatera fish poisoning.

7. Amino Acids and Amines Extracts of the marine algae Laminaria angustata and Chondria amata are reported to contain agents with hypotensive and other pharmacological properties. Laminine (14), 175

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a choline like basic amino acid had been isolated from water extracts of number of marine algae e.g. Laminaria angustata .

Laminine, in general, depressed the contraction of excited smooth muscles. It is considered to be a potentially useful pharmacological agent. Scientists had reported the widespread occurrence of volatile amines, such as methylamine, dimethylamine, trimethylamine, ethylamine, propylamine, isobutylamine, isoamylamine, 2phenylethylamine and 2-methylmercapto propylamine in red, green and brown algae. It is mentioned that biological activities of some of the extracts of the marine algae may be due to the presence of these amines. 8. Sterols Cholesterol (15) was found the major sterol of Rhodophyta. Four species of algae, Rhodymenia palmata, Porphyra purpurea, P. umbilicalis and Halosaccion ramentaceum were found to contain desmosterol as the main sterol. However, Hypnea japonica was the only alga having 22- dehydrocholesterol (16) as the major sterol.

Red algae contained primarily cholesterol (15). Several species contained large amount of demosterol (17), and one species contained primarily 22dehydrocholesterol. Fucosterol (18) was the dominant sterol of brown algae. Most phaeophyta also contained traces of cholesterol and biosynthetic precursors of fucosterol.

The sterols of green algae were much more varied. The green algae contained chondrillasterol (19), poriferasterol (20), 28-isofucosterol, ergosterol and cholesterol.

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The sterols from marine algae are reported to be non-toxic and have the ability to reduce blood cholesterol level. They are also reported to reduce the tendency to form a fatty liver and excessive fat deposition in the heart. MARINE ALGAE AS A TOOL FOR BIOREMEDIATION OF MARINE ECOSYSTEMS Ongoing efforts by researchers at research institutions and universities worldwide have indicated that use of large-scale algae populations could possibly provide new and cost-effective technologies for sequestering and preventing dissemination of waterborne pollutants. These contaminants may include heavy metals, high sewagebased nutrient concentrations, bacterial and viral pathogens, and radionuclides. Seaweeds can remove several types of pollutants from the environment through pollutant assimilation and detoxification. These include the possibility for removal of excessive nutrients, such as nitrates or phosphates, organic pollutants, heavy metals, and radioactive nucleotides through bioconcentration within the seaweeds and detoxification. Removal of Nutrients Seaweeds require inorganic nutrients for growth, as do other plants. The fast-growth rates of some seaweed species can account for rapid nutrient removal from marine waters. The primary nutrient that seaweeds are effective in removing is nitrogen in the form of ammonia or nitrate. Red and green algae actually require as much as 2% N of their dry weight. On the other hand, kelps such as Macrocystis pyrifera require less N, generally on the order of 1.5% N of dry weight. The rates of N-nutrient removal can be very rapid, especially when the thalli are Ndeficient. Gracilaria tikvahiae showed almost complete removal of millimolar levels of ammonium nitrate within 24 h, in conditions that were not light-dependent. Usually, the assimilated N is converted into an organic form, such as proteins, such as the red algal pigments, although some Arctic kelps store N primarily in an inorganic form such as nitrate. Bioaccumulation of Heavy Metals Seaweeds accumulate a wide range of heavy metals, such as lead, copper, nickel, zinc, cadmium, gold, silver, manganese, tin, chromium, aluminum, and cobalt. Rates of heavy-metal bioaccumulation are metal-specific, and the order of heavy metal removal varies from species to species. Amounts of bioaccumulation vary, but can be as high as 1000 mg per dry gram of tissue of seaweed sampled from highly polluted 177

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waters. This bioaccumulation acts to cause significant bioconcentration of heavy metals within the seaweeds, compared with the concentrations in seawater, sometimes on the order of 1000-fold. There are now engineering attempts to developed dried algal biomass as a biosorbant. It is not yet certain about details of how this bioconcentration occurs. Some scientists believe that alginic acid is responsible for heavy-metal binding in kelps. Organic Pollutants Although considerable research describes the effects of herbicides, pesticides, and other organic pollutants on microalgae and phytoplankton, far less work has been done on the seaweeds themselves. Several higher plants produce a suite of enzymes that are useful in detoxification. Two key enzymes that appear to be universal are glutathione S-transferase and cytochrome P-450. Both of these enzymes act to detoxify a wide range of organic pollutants in organisms such as bacteria, flowering plants, invertebrates, and vertebrates. These ubiquitous enzymes have not been studied well in seaweeds, but it is highly likely that they exist and can provide a mechanism for detoxification of organic pollutants. Disposal of Seaweeds Contaminated with Pollutants If seaweeds have been used for heavy-metal accumulation or detoxification, the different uses mentioned before of the biomass may not be possible owing to high internal levels of pollutants. One approach may be to dry these contaminated seaweeds, and dispose of them. Another approach to treating toxin-contaminated biomass is the use of bioconversion to methane. An advantage of this approach is that seaweeds do not have to be dried before the bioconversion process. Bioconversion can result in as much as 80% transformation in the amount of organic matter being converted into methane and carbon dioxide. The bioconversion process produces three streams: a gaseous stream (mostly methane and carbon dioxide) that would contain no heavy metals, but possibly volatile toxins; a liquid effluent that would have significant concentrations of toxins; and solid residues containing toxins bound to the particulate carbon. Nonetheless, a bioconversion approach could represent an excellent method to further concentrate the amounts of accumulated pollutants for other methods of recovery or disposal. Role of Biotechnology Although seaweeds already offer many advantages for bioremediation of marine waters, their usefulness can be improved. Conventional genetics and breeding may not suffice for the kinds of improvements needed to increase the effectiveness of macroalgae in bioremediation. Detoxification and accumulation can be more easily accomplished by the introduction of target genes into algal genomes. Among these genes are bacterial genes for the production of metallothioneins, which can both aid in accumulation as well as detoxification of heavy metals. Another approach would include gene amplification for increased production of glutathione-S-transferase and cytochrome P-450. There are recent reports on the introduction of single genes into macroalgae, but this field is still in its infancy.

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4. Micro Algae Micro algae represent a major untapped resource of genetic potential for valuable bioactive agents and biochemicals. Phycocyanin and phycoerythrin are produced by cyanobateria (Spirulina), and recently have been used as fluorescent labelling agents. They are proteinaceous in structure. omega-3-fatty acids One future commercial application of micro algae could be in the production of special lipids. The omega-3-fatty acids found in the oils of certain cold-water marine fish are considered to be responsible to reduce incidence of coronary heart disease. These fatty acids are likely to originate from the phytoplankton in food chain. Many of these phytoplankton species are found to be rich in reserves of oils containing various amounts of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Micro algae are also expected to furnish potent antiviral, antiAIDS, antibiotic and other bioactive agents. The extract of cyanobacterium Planktothrix sp. exhibited embryotoxicity. Changes in the culture conditions of Lyngbya majuscula had the greatest effect on production of its secondary metabolites. Several leptosins were isolated from the marine alga Leptosphaeria sp. and their biological activity evaluated. Of these leptosin M exhibited significant cytotoxicity against human cancer cell lines. Two new antiinflammatory macrolides, lobophorin A and B were isolated from a marine bacterium. Water extract of marine diatom Haslea ostrearia exhibited anticoagulant activity. Brominated anisoles and cresols were detected for the first time in the red marine alga Polysiphonia sphaerocarpa. The sulfated polysaccharide of the red microalga Porphyridium sp. showed high order of antiviral activity against herpes simplex virus (HSV-1 and 2) both in vitro and in vivo. Fucoidan, a sulfated polysaccharide from brown seaweed displayed anticoagulant and antithrombotic activities. It also had inhibitory action in the growth of Lewis lung carcinoma and B16 melanoma in mice. Antitumor and immunomodulation activities were found in different molecular weight -carrangeenans from Chondrus ocellatus. 1-Hydroxy monocyclic carotenoid 3,4-dehydrogenase from a marine bacterium that produces myxol was identified. This unique type of crt D is a valuable tool for obtaining 1- HO-3,4-didehydromonocyclic carotenoids. Antarctic bacteria inhibited growth of food-borne microorganisms at low temperature. Studies on the marine phytoplanktons are few because of difficulty of growing the organisms and the low yield of secondary metabolites. However, several toxins related to saxitoxin are isolated from Gonyaulax species. The cultured cells of the dinoflagellate Ptychodiscus brevis, yielded brevetoxin B, C and dihydrobrevetoxin B. Two new polycyclic ethers, GB-5 and GB-6 closely related to okadaic acid, a toxin that was first found in sponges and later in dinoflagellate have been isolated from the cultured cells of G. breve. The dinoflagellate Dinophysis, produces and transmits shellfish, toxins that are responsibe for diarrhoetic shellfish poisoning. Lyngbya majuscula known to cause swimmers itch has furnished several class of compounds. Pukeleimides (A-F) showed activity against Mycobacterium smegmatis and Streptococcus pyrogenes. Cyclic depsipeptide, majusculamide-C isolated from the organism inhibits the growth of fungal plant pathogen. Aplysiatoxins and oscillatoxins isolated from blue-green algae Schizothrix calcicola and Oscillatoria

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nigroviridis possess antileukaemic activity but their high toxicity precludes their medicinal use. Cytotoxic and fungicidal nucleosides have been isolated from a variety of blue-green algae. Anatoxin-a, an exogenic toxin of blue-green alga Anabaena flosaquae is one of the most potent nicotinic receptor agonist. It is suggested that the analogues of anatoxin-a may be of clinical value for treating disorders associated with defects in cholinergic regions of the central nervous system. Several species of green-algae of the genus Halimeda produce an ichthyotoxin which exhibits diverse biological activities. It inhibits the growth of marine bacteria and fungi, cell division of fertilized sea-urchin eggs and the motility of sea-urchin sperms at 1 g/mL. Avrainvilleol, a brominated metabolite of green algae, Avrainvillea longicaulis exhibits high order of antifeedant activity in reef fish and also inhibits the growth of microorganisms. The genera Halimeda, Penicillus and Udotea are found to contain highly active but unstable sesquiterpenoids and diterpenoids. Some of these diterpenoids exhibit cytotoxic and antimicrobial activities. Prenylated aromatics with small side chains are relatively common in brown algae. Several highly unsaturated C11 hydrocarbons are isolated from Dictyopteris plagiogramma and D. australis. The function of these hydrocarbons have been studied in detail. It has been observed that the sperm cells aggregate around the female gametes of brown algae which exude C11 hydrocarbons that attract the former and cause them to remain in the excited state in the vicinity of the latter. The sex attractants that have been identified are: ectocarpene from Ectocarpus siliculosus,142 fucoserratene from Fucus serratus, multifidene from Culteria multifida, n-butylcyclohepta- 2,5-diene from Dictyota dichotoma, desmarestene from Desmarestia viridis and tinavarrene from Ascophyllum nodosum. Tracing the origin of arsenic in lobsters and in fish, it has been found that the brown algae Ecklonia radiate concentrates arsenic in the form of arseno-sugars. 4 Hydroxydictyodial from Dictyota spinulosa inhibits feeding in the omnivorous fish Tilapia mossambica.145 Three ichthyotoxic and phytotoxic diterpenes are isolated from Dilophus fasciola. Several diterpenes from Dictyota species exhibit significant cytotoxicity. Two phlorotannins from Ecklonia kurome exhibit antiplasmin inhibitory activity. The pharmacological properties of laminine has been studied. It has been found that the compound at high doses does have a hypotensive action as a result of a ganglion blocking effect. Marine red algae have yielded a vast array of halogenated lipids, some of these exhibit CNS-depressant and hypotensive activities. Three brominated acetylenic compounds active against mosquito larvae have been obtained from Laurentia nipponica. Trihydroxy benzyl methyl ethers having antibacterial activity against Bacillus subtilis are isolated from Grateloupia filicina. Enantioselective synthesis of ()-kainic acid possessing anthelmintic, insecticidal and neuroexcitatory activities, have been achieved. The symmetrical bisbenzyl ether from Symphyocladia latiuscula showed antifungal activity. 5-Iodo-5-deoxytubercidin, an unusual nucleoside has been isolated from Hypnea valentiae. The nucleoside caused pronounced relaxation of muscles, hypothermia in mice and blocked polysynaptic and monosynaptic reflexes.

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