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Handbook of Microalgal Culture: Biotechnology and Applied Phycology Edited by Amos Richmond Copyright © 2004 by Blackwell Publishing Ltd

13 Industrial Production of Microalgal Cell-mass and Secondary Products – Major Industrial Species

Dunaliella

Ami Ben-Amotz

13.1 Biology and halotolerance

The biflagellated alga Dunaliella is classified under Chlorophyceae, Volvocales, which includes a variety of ill-defined marine and fresh water unicellular species (Avron & Ben-Amotz, 1992). Dunaliella is characterized by an ovoid cell volume usually in the shape of a pear, wider at the basal side and narrow at the anterior flagella top (Fig. 13.1). The cellular organization of Dunaliella is no different than that of other members of the Volvocales presenting one large chloroplast with single-centered starch surrounded by pyrenoid, a few va- cuoles, a nucleus and a nucleolus. Cells of the genus Dunaliella lack a rigid polysaccharide cell wall, and are enclosed by a thin elastic plasma membrane covered by a mucous surface coat. The lack of a rigid cell wall permits rapid cell volume changes in response to extracellular changes in osmotic pressure with common osmotic treated Dunaliella shape from swollen sphere to shrink fiber. Under extreme salt concentrations above saturation, Dunaliella loses its flagella and the surrounding mucous and the cell rounds with a buildup of a thick surrounding wall to form a dehydration resistant cyst. Dunaliella occurs in a wide range of marine habitats such as oceans, brine lakes, salt marshes, salt lagoons and salt water ditches near the sea, predom- inantly in water bodies containing more than 2M salt and high-levels of magnesium. The effect of magnesium on the distribution of Dunaliella is not clear, but in many bittern habitats of marine salt producers, Dunaliella usually flourishes. The phenomenon of orange-red algal bloom in such marine environments is usually related to combine sequential growth of Dunaliella, brine shrimps and halophilic bacteria, as may be observed in concentrated saline lakes in many places around the globe. Dunaliella is recognized as being the most halotolerant eukaryotic photosynthetic

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Dunaliella

Eye spot (?) Nucleus Nucleolus Chloroplast
Eye spot (?)
Nucleus
Nucleolus
Chloroplast

Golgi

Mitochondria

Vacuole

Starch

Pyrenoid

β-Carotene

globules

Fig. 13.1.

Electron micrograph of D. bardawil – a b-Carotene rich alga.

organism known, showing a remarkable degree of adaptation to a variety of salt concentrations from as low as 0.1 M to salt saturation > 4M. Many manuscripts and monographs were dedicated in the last decade to the osmoregulation of Dunaliella (Avron & Ben-Amotz, 1992). The intracellular concentration of glycerol is directly proportional to the extracellular salt concentration and is sufficient to account for all the required cytoplasmic and most of the chloroplastic osmotic pressures. Glycerol biosynthesis and elimination occur under either light or dark conditions by a novel glycerol cycle, which involves several specific enzymes. Accumulation of glycerol, the lack of rigid cell wall, and the enclosing by elastic plasma membrane of special ion transport properties makes Dunaliella a model of active osmometer attractive for elucidation research on ion transport, membrane characteristic, and bioenergy.

13.2 b-Carotene biology

Among the genus Dunaliella, the few strains that have been shown to change their colors from green to orange produce and accumulate large amounts of cellular b-carotene (Mil’ko, 1963; Semenenko & Abdullaev, 1980; Ben-Amotz

Biotechnology of -carotene production by Dunaliella

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et al., 1982; Loeblich, 1982). In hypersaline lagoons, which are generally low in available nitrogen and exposed to high solar radiation, these b-carotene producing strains of Dunaliella predominate over all other organ- isms to a seasonal orange colored bloom. Under such stressful environmental conditions more than 12% of Dunaliella dry weight is b-carotene, usually associated with a sharp decline in cell protein and the chloroplast chloro- phyll. The b-carotene in Dunaliella accumulates within distinctive oily globules in the interthylakoid spaces of the chloroplast periphery. Analysis of the globules showed that the b-carotene of Dunaliella is composed mainly of two stereoisomers: all-trans and 9-cis, with the rest a few other mono-cis and di-cis stereoisomers of the b-carotene and no xanthophylls. Both the amount of the accumulated b-carotene and the 9-cis to all-trans ratio depend on light intensity and on the algal division time, which is determined by the growth conditions. Thus, any growth stress, which will slow down the rate of cell division under light, will in turn increase b-carotene production in Duna- liella. In fact, high light and many environmental stress conditions such as elevated salt, extremes of pH, low temperature, nutrient deficiencies, and others affect the content of b-carotene in Dunaliella. Dunaliella follows the same biosynthetic pathway of carotenoids with the same substrates and same intermediates as found in other eukaryotic organisms and plants (Zechmeister, 1962; Goodwin, 1988), however, the biological isom- erization reaction, which eventually produces 9-cis b-carotene in Dunaliella, is not identified as yet (Shaish et al., 1990, 1991; Ebenezer & Pattenden, 1993). A few speculative hypotheses have been postulated for the function of the b-carotene globules in Dunaliella (Ben-Amotz & Avron, 1990). The most accepted hypothesis suggests that the b-carotene globules protect the cell against injury by high intensity radiation under limiting growth conditions by acting as a screen to absorb excess radiation. Strains of Dunaliella and other algae unable to accumulate b-carotene bleach and die when exposed to high-levels of radiation while the b-carotene-rich Dunaliella flourishes. Moreover, protection against photoinhibition by the massively accumulated b-carotene is observed only when the photoinhibitory light is composed of wavelengths absorbed by b-carotene, i.e. in the blue region.

13.3 Biotechnology of b-carotene production by Dunaliella

Dunaliella is the most suitable organism for mass cultivation outdoors in open ponds. The ability to thrive in media with high sodium, magnesium, calcium and the respective anions, chloride and sulfate, in desert high solar irradiated land with access to brackish water or sea water at extreme tem- peratures from around 5 to above 40 C, all make Dunaliella most attract- ive for biotechnologists and venture capitalists. In fact, since 1980 several firms, government authorities, and industries have invested capital in the application of Dunaliella for the production of natural b-carotene. Large-scale Dunaliella production is based on autotrophic growth in media containing inorganic nutrients with carbon dioxide as exclusive carbon sources. Attempts to commercially develop heterotrophic strains or mutants of Dunaliella for growth on glucose or acetate, e.g. as Chlorella or

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Dunaliella

Chlamydomonas, respectively, were not successful. Due to the demand for high light intensity for maximal b-carotene production beyond that required for normal growth, production facilities are located in areas where solar output is maximal and cloudiness is minimal. Most of the present Dunaliella production plants are located close to available sources of salt water, e.g. sea/ lake-salt industries, usually in warm and sunny areas where the rate of water evaporation is high and non-agricultural land is abundant. Four modes of cultivation have been used in large-scale production of Dunaliella:

* The first, termed extensive cultivation, uses no mixing and minimal con- trol of the environment. To decrease fungal contamination (Tonka & Toncheva-Panova, 1997) and attacks by zooplanktonic predators, such as certain types of ciliates, amoebae, or brine shrimp (Brock, 1975; Post, 1977; Post et al., 1983), the growers employ very high salt concentrations. Dunaliella grows slowly in shallow lagoons in nearly saturated brine and predators are largely eliminated. The naturally selected strain of Duna- liella is well adapted to nearly salt saturation conditions, partially loses its flagella and produces a thick cell wall on the transformation to a cyst form. Extensive cultivation productivity is low, as it is based on less than 0.1 g b-carotene m 3 and the area needed for commercial production is very large; however, the low operating costs of such facilities have led to the development of two commercial plants in Australia.

* The second, termed intensive cultivation, uses high biotechnology to con- trol all factors affecting cell growth and chemistry. The ponds are usually oblong, lined, constructed raceways varying in size up to a production surface area of approximately 3000 m 2 . The use of long arm, slow revolution paddle wheels is presently common in the large-scale facilities in Israel, USA, China, and Chile. One production-sized shallow water pond of 20 cm on an area of 3000 m 2 (600 m 3 ) containing 5–15 g b-carotene m 3 yields 3–9 kg b-carotene on total harvest. The current large-scale production of b-carotene under intensive cultivation is around 200 mg b-carotene m 2 d 1 on a yearly average; thus, a modern intensive plant of 50 000 m 2 produces 3650 kg b-carotene per year.

* Between the extensive and intensive modes there are examples in Australia and China of the third type, semi-intensive mode, where the ponds are enlarged ten times, to about 50 000 m 2 each, with partial control and no mixing.

* The fourth is highly intensive cultivation in closed photo-bioreactors. Many trials have been initiated in the last decade to grow Dunaliella in different models of closed photobioreactors with attempts to design the best sunlight-harvesting unit for b-carotene optimization. The different designs include: narrow, very long, plastic tubes, plastic bags, trays and more. However, as of today, none of these trials have taken production beyond the laboratory or small pilot plant volume, mainly due to economic limitations and non-feasible large-scale development. The few industrial ventures of high intensity-closed photobioreactors became insolvent and no longer exist.

Dunaliella market products

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Generally, large-scale optimization of b-carotene production is achieved in all modes of cultivation by high salt stress and by nitrogen deficiency. For pragmatic reasons, the first is applied in the extensive mode, while nitrogen starvation controls the intensive mode. Most species of Dunaliella grow optimally in a medium containing 1–2 M NaCl in accordance with the medium temperature, exhibiting closely similar growth rates at high tempera- tures of >30 C in >3M NaCl at moderate temperatures of 25 C in 2 M NaCl, and at low temperatures of 15 C in 1 M NaCl. The algal composition changes respectively by selective accumulation of glycerol and starch. This unique environmental adaptation of Dunaliella allows successful intensive outdoor growth in cold seasons and in cold areas. Most commercial Duna- liella ponds employ evaporated concentrated seawater, lake salt water, or seawater augmented with dry salt to reach the desired concentration in the medium. Favored sites for Dunaliella cultivation are along the seashore or close to salt lagoons and salt producing industries for the use of a mixture of seawater and concentrated salt water in order to obtain the desired salt concentration by season and temperature. The use of recycled high salt medium is common in a few plants after harvesting and separation of the algae by oxidative treatment of the algal free medium to reduce the organic load. Use of recycled medium enriches the medium with higher concentra- tions of magnesium, calcium and sulfate. Dunaliella was found to grow well in seawater based media containing around 1.5 M NaCl, more than 0.4 M MgSO 4 and 0.1 M CaCl 2 under pH control.

13.4 Biotechnology of phytoene production

Dunaliella bardawil, the halotolerant b-carotene species, was recently mutated and treated by the bleaching herbicide norflurazon to select sub- species rich with a mixture of 9-cis and all-trans phytoene and phytofluene. The selected phytoene/phytofluene-rich Dunaliella was transferred to NBT Ltd, Eilat (Fig. 13.2) south of Israel, and inoculated and cultivated in small ponds outdoors and the most adapted phytoene species was scaled up gra- dually into large body open raceways of 3000 m 2 (Wermau et al., 2002). The success in large-scale production of new carotenoids other than b-carotene by Dunaliella opens a new path in applied phycology.

13.5 Dunaliella market products

Dunaliella natural b-carotene is widely distributed today in many different markets under three different categories: b-carotene extracts, Dunaliella powder for human use and dried Dunaliella for feed use. Extracted purified b-carotene is sold mostly in vegetable oil in bulk concentrations from 1% to 20% to color various food products and for personal use in soft gels, usually 5 mg b-carotene each gel. The purified natural b-carotene is generally accom- panied by the other carotenoids of Dunaliella, predominantly: lutein, neo- xanthin, zeaxanthin, violaxanthin, cryptoxanthin, a-carotene comprising approximately 15% of the carotene concentration and is marketed under the title carotenoids mix. A variety of such formulations of natural b-carotene

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Dunaliella

278 Dunaliella Fig. 13.2. Australia; Down, intensive open raceways, NBT Ltd, Eilat, Israel. Commercial cultivation of
278 Dunaliella Fig. 13.2. Australia; Down, intensive open raceways, NBT Ltd, Eilat, Israel. Commercial cultivation of

Fig. 13.2.

Australia; Down, intensive open raceways, NBT Ltd, Eilat, Israel.

Commercial cultivation of Dunaliella. Up, extensive open ponds, Betatene Ltd, Adelaide,

are presently found and distributed in health food stores mainly in the western world under the market sections of vitamins, health food or food supplement. The second category covers a line of dried low salt Dunaliella powders, algae harvested, processed, washed and dried as described above for mar- keting in the form of tablets or hard capsules containing between 3 and 20 mg mix of all-trans and 9-cis b-carotene per unit. The tablets are coated with sugar and the capsules are packed separately in aluminum/ polyethylene blisters to avoid oxidation and extend the shelf life of the product at room temperature. Dry Dunaliella is distributed popularly in the Far East where the consumers are more familiar with edible algae and

References

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accustomed to macroalgae, such as Chlorella and Spirulina. The third cat- egory covers a line of dried Dunaliella, harvested and dried with no salt wash to algal powder of about 2% b-carotene and varying concentration of salt. The powder is shipped under vacuum in aluminum/polyethylene bags for feed

coloration and pro-vitamin A use in cattle, poultry, fishes, shrimps and more.

according to

Pricing of natural b-carotene varies from $300 to $3000 kg specifications and demand.

1

13.6 Commercial producers

At present, the following companies are actively engaged in cultivating Dunaliella for commercial purposes. These are (listed with mode of cultiva- tion):

1. Betatene Ltd, Cheltenham, Vic. 3192, Australia, a division of Cognis Ltd, Australia. Extensive mode (Fig. 13.2).

2. Cyanotech Corp., Kailua-Kona, HI 96740, USA. Intensive mode.

3. Inner Mongolia Biological Eng. Co., Inner Mongolia, 750333, P. R. China. Intensive mode.

4. Nature Beta Technologies (NBT) Ltd, Eilat 88106, Israel, a subsidiary of Nikken Sohonsha Co. Gifu, Japan. Intensive mode.

5. Tianjin Lantai Biotechnology, Inc. Nankai, Tianjin, in collaboration with the Salt Scientific Research Institute of Light Industry Ministry, P. R. China. Intensive mode.

6. Western Biotechnology Ltd. Bayswater, WA 6053, Australia, a subsi- diary of Cognis Ltd, Australia. Semi-intensive mode.

7. Parry agro industries Ltd, Murugappa group, India. Intensive mode.

8. AquaCarotene Ltd, Subiaco, WA 6008, Australia. Extensive mode.

9. Small plants are also located in Chile, Mexico, Cuba, Iran, India, Taiwan, and Japan.

References

Avron, M. & Ben-Amotz, A. (eds) (1992) Dunaliella: Physiology, Biochemistry, and Biotechnology, Boca Raton: CRC Press, pp. 240. Ben-Amotz, A. & Avron, M. (1990) The biotechnology of cultivating the halotolerant alga Dunaliella. Trends Biotechnol., 8, 121–26. Ben-Amotz, A., Katz, A. & Avron, M. (1982) Accumulation of b-carotene in haloto- lerant algae: purification and characterization of b-carotene globules from Duna- liella bardawil (Chlorophyceae). J. Phycol., 18, 529–37. Brock, T. (1975) Salinity and ecology of Dunaliella from Great Salt Lake. J. Gen. Microbiol., 89, 285–92. Ebenezer, W.J. & Pattenden, G. (1993) cis-Stereoisomers of b-carotene and its con- geners in the alga Dunaliella bardawil, and their biogenetic interrelationships. J. Chem. Soc. Perkin Trans., 1, 1869–73. Goodwin, T.W. (ed.) (1988) Plant Pigments. London: Academic Press, pp. 362. Loeblich, L.A. (1982) Photosynthesis and pigments influenced by light intensity and salinity in the halophile Dunaliela salina. J. Mar. Biol. Assoc., UK, 62, 493–508.

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Mil’ko, E.S. (1963) Effect of various environmental factors on pigment production in the alga Dunaliella salina. Microbiologiya, 32, 299–307. Post, F.J. (1977) The microbial ecology of the Great Salt Lake. Microb. Ecol., 3,

143–65.

Post, F.J., Borowitzka, L.J., Borowitzka, M.A., Mackay, B. & Moulton, T. (1983) The protozoa of a Western Australian hypersaline lagoon. Hydrobiologia, 105, 95–113. Semenenko, V.E. & Abdullaev, A.A. (1980) Parametric control of b-carotene biosynthesis in Dunaliella salina cells under conditions of intensive cultivation. Sov. Plant Physiol., 27, 2–30. Shaish, A., Avron, M. & Ben-Amotz, A. (1990) Effect of inhibitors on the formation of stereoisomers in the biosynthesis of b-carotene in Dunaliella bardawil. Plant Cell Physiol., 31, 689–96. Shaish, A., Ben-Amotz, A. & Avron, M. (1991) Production and selection of high b-carotene mutants of Dunaliella bardawil (Chlorophyta). J. Phycol., 27, 652–56. Tonka, G., Toncheva-Panova (1997) Identification of a fungal contaminant in a culture of Dunaliella salina. Czech Mycol., 50, 127–31. Werman, M., Mokady, S. & Ben-Amotz, A. (2002) Bioavailability of the isomer mixture of Phytoene and Phytofluene-rich alga Dunaliella Bardawil in rat plasma and tissues. J. Nutrit. Biochem., 13, 585–91. Zechmeister, L. (1962) cis-trans Isomeric Carotenoids, Vitamin A, and Arylpolyenes, Vienna: Springer-Verlag.