BULLETIN OF MARINE SCIENCE, 47(1): 244-252, 1990

COMMERCIAL PRODUCTION OF (3- BY SALINA IN OPEN PONDS

Lesley J. Borowitzka and Michael A. Borowitzka

ABSTRACT The halophilic green unicellular alga Dunaliella salina is an important commercial source of }3-carotene. The algal culture process used in Western Australia is described and compared with the process used at other sites, and the advantages and disadvantages of these processes are reviewed. Harvesting" extraction and formulation are also discussed.

jJ-Carotene is a C40 found in all green plants where it is localized in the . Its main commercial use is in the food industry where it is used as a coloring agent in margarine, baked goods and other food products (KHiui, 1982). {3-Carotene has also been shown to be a good scavenger of free radicals and other reactive species (Krinsky, 1979) and appears to have a role as a pro- tective agent against cancer. In recent years, much clinical evidence has accu- mulated on this potential cancer protective activity of jJ-carotene (Ibrahim et a1., 1977; Mathews-Roth, 1982; Schwartz et a1., 1986) and, as further evidence ac- cumulates, this should develop into an additional major application of jJ-carotene. At present, most of the commercially available jJ-carotene (all-trans jJ-carotene) is produced by synthetic means (Isler, 1979) and sells for about $AUS 600·kg-1 (=$U.S. 500). In recent years, however, there has developed an increasing market demand for natural jJ-carotene. The green volvocalean alga Dunaliella salina Teodoresco has the highest cell content of jJ-carotene of any organism, with concentrations of up to 14% of dry weight having been reported (Mil'ko, 1963; Aasen et al., 1969). Massyuk (1966) first proposed that this alga might be a suitable natural source of jJ-carotene and over several years carried out trials on Dunaliella culture in the Ukraine (Massyuk and Abdulla, 1969). In the 1970's work on jJ-carotene production using D. salina commenced in several other parts of the world, and in 1986 commercial pro- duction commenced in Australia, the USA and in Israel (Borowitzka and Boro- witzka, 1988a). In this paper we describe several aspects of the commercial jJ-carotene production process using D. salina and point out some of the critical steps in this process.

DUNALIELLA CuLTURE PROCESS Dunaliella salina is the most salt-tolerant eukaryotic alga known and can grow in media saturated (> 30% w/v) with NaCl (Borowitzka and Brown, 1979). D. salina is also very tolerant of high temperatures and high photon flux densities (Borowitzka and Borowitzka, 1988a). Development of mass culture conditions has required the optimization of growth conditions so as to produce the maximum amount of jJ-carotene per unit time and culture volume. This has been complicated by the fact that the conditions leading to maximum cell growth are different from those leading to maximum accumulation of ,a-carotene in the cell. For example, D. salina has an optimum salinity for growth of about 18-21 % NaCl, whereas the maximum jJ-carotene content is reached at a salinity of> 27% NaCl (Borowitzka et al., 1984). Similarly,

244 BOROWITZKA AND BOROWITZKA: BETA CAROTENE PRODUCTION 245 l1-caroteneaccumulation is highest under nitrogen-limiting conditions where growth is reduced. Based on these and other observations, early workers proposed a two-stage process for growth (Chen and Chi, 1981), consisting of a growth stage at about 18% NaCl and with sufficient nitrogen to attain the maximum biomass, followed by a carotenoid-accumulating stage at a salinity of about 27% NaCI and N-limiting conditions (Borowitzka et al., 1986). However, early field studies and economic analyses soon showed that such a process was not feasible for several reasons: (1) At the lower salnities, predation by protozoa, especially by Cladotricha sigmoidea and Heteroamoeba sp. often led to a rapid decimation of the algal biomass in the ponds (Post et al., 1983; Borowitzka et al., 1986); (2) At the lower salinities the non-carotenogenic Dunaliella species, D. viridis Teodoresco, which always co- occurs with D. salina in open-air ponds, could outgrow D. salina (Borowitzka et al., 1986; Moulton et al., 1987b); and (3) The two-step process is labor intensive and requires a greater pond area and is thus too expensive. As a result of these considerations, a semi-continuous process, operating at an intermediate salinity was finally chosen (Borowitzka et al., 1986; Moulton et al., 1987a). The salinity was optimized for maximum l1-carotene production per unit time in the pond. Nutrient concentrations were also optimized. The major nutrients added to ponds are nitrogen, phosphate and a chelated form of iron. Depending on the brine used, trace elements may also have to be added (Ben Amotz et al., 1982; Ben Amotz and Avron, 1983; Borowitzka and

Borowitzka, 1988a, 1988b). The Ca:Mg and Cl-:S04 - ratio of the brines used in culture may also affect growth and carotenogenesis (Ben Amotz and Avron, 1983) and must be considered when selecting the source of brines for growth. Another consideration for commercial mass culture is pond design. For ex- ample, Israeli and U.S. operations use variants of the paddle-wheel "raceway" ponds of the design originally developed by Oswald (1988), whereas the Australian operations use large unstirred ponds (Borowitzka and Borowitzka, 1988a). The large unstirred ponds (unstirred other than by wind mixing) used by Western Biotechnology Ltd. at their production plant at Hutt Lagoon, Western Australia, have a lower algal biomass than would be achieved if raceway-type ponds were used; however, this lower biomass is offset by the greatly reduced cost of pond construction and maintenance. The "natural" ponds used in Australia also have another advantage over the lined ponds used elsewhere. Plastic lined ponds almost always have problems with the build-up of gas bubbles under the plastic liner and these must be overcome by effectively "sterilizing" the soil under the liner. Un- lined earthen ponds do not have this problem, although turbidity may be greater. In all cases pond depth is maintained at about 20 em for maximum light pene- tration. A final consideration for open air production of D. salina is the siting of the production facility. Since open air ponds are subjected to the vagaries of the weather, the plant location must have a suitable climate. An optimum site, such as Hutt Lagoon in Western Australia, has a long, hot, dry summer and is situated at or near a suitable source of brines. The location should also be remote from possible pOllution produced by industrial or agricultural activities (i.e., pesticides or heavy metals) as the final product produced will be used in human food. The land should also be fairly flat to minimize pond construction costs. Large open-air ponds have a number offeatures which differ from those of well- mixed large-scale cultures in closed systems because of (a) the importance of climate (wind, rain, temperature), (b) contamination, (c) the heterogeneous dis- tribution of organisms in the pond, and (d) differences in scale-up (Borowitzka et 246 BULLETIN OF MARINE SCIENCE, VOL. 47, NO. I, 1990 a

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Cell Number(x 104 cells. ml-1) Salinity (% brix) Figure 1. Distribution of Dunaliella salina (.) and D. viridis (0) with depth in a 100 m2 pond at Hutt Lagoon following rain. The salinity profile is shown at the right. al., 1984; 1986; Borowitzka and Borowitzka, 1988a; Moulton et al., 1987). We have found that scale-up oflarge open-air ponds must proceed in a series of steps, each representing a factor of 10 increase, to be able to cope with changes in both biological and engineering parameters. At Hutt Lagoon the initial outdoor experiments were carried out in plastic ponds of several hundred liters capacity to test the effects of local climatic con- ditions on cultures. These were then scaled-up to earth-walled ponds constructed on the lake bed ofHutt Lagoon, having an area of 100 m2 (approx. 20,000 liter). Smaller ponds were abandoned (1) due to the high wall area to pond volume ratio which resulted in high rates ofleakage; (2) a tendency to lose pond holding capacity due to erosion of the banks; (3) loss of due to windrowing of the cells; and, (3) reduced salinity, erosion of the banks and increased turbidity resulting from rainwater runoff. All of these disadvantages were reduced in larger ponds. Scale-up at Hutt Lagoon progressed from the 100 m2 ponds to 250 m2 (50,000 liters) and 600 m2 (120,000 liters). These ponds were used to explore the effects of nutrient addition, pond depth, salinity and climate on growth and caroteno- genesis. Several different pond management regimes were tested. Before the final production plant was designed, further experiments were carried out in 0.5-ha ponds (i.e., 1I1Oth the size of the proposed production ponds) to provide data for accurate economic analysis of the process. Scale-up from these to the 5-ha production-ponds then proceeded smoothly. It is difficult to sample large open-air ponds at all stages (experimental, pilot and production) of pond management. In large ponds there is some patchiness in the distribution of D. salina due to the natural taxes of the algae (Moulton et al., 1987b; Wangersky and Maass, 1988). Also, differential heating of the surface BOROWITZKA AND BOROWITZKA: BETA CAROTENE PRODUCTION 247 layers of the pond, stratification of lower salinity water on the pond surface after rainfall, and variable mixing due to wind and convection results in heterogeneous algal distribution. Figure I shows a typical depth profile in a 100 m2 pond following rain. The top 3 cm have a reduced salinity and fewer algal cells are concentrated near the surface. Wind also results in some horizontal patchiness. Figure 2 illus- trates the changes in both horizontal and vertical distribution of the algae in a pond over 3 days. During this time the predominant wind direction changed from northerly on day 1, to north-westerly on day 2, and south-easterly on day 3. These data clearly show the dynamic nature of the algal distribution in the ponds and indicate that great care must be taken to obtain representative samples of the ponds in order to obtain an accurate picture of cell numbers, salinity and nutrient concentrations.

Harvesting. - The next critical step in iJ-carotene production is harvesting. D. salina is a small alga (approximately 25 x 12-16 /oLm size) and reaches a cell density in open air ponds of about 5.105 to 5.106 cells·ml-I• Extracting these delicate algae from large volumes of brine is a major problem and an efficient, inexpensive process is required. The range of available options has recently been reviewed by Mohn (1988) and several harvesting methods specific for Dunaliella have been patented. These include filtration using diatomaceous earth as a filter aid (Ruane, 1974a), the use of stationary or moving salt gradients (Bloch et al., 1982), the exploitation of the phototactic behavior of the algae together with floating rafts holding vertical fibers to trap the algae (Kessler, 1982), and a method which exploits the salinity dependent hydrophobicity of the D. salina cell membrane (Curtain and Snook, 1982; Curtain et al., 1987). One of the harvesting methods examined was stratification based on the ob- servation that D. salina tends to concentrate at the surface of ponds in the light, and that this concentration is enhanced if the brine in the pond is overlain by a thin film ofless saline water. Bloch et al. (1982) also discuss stratification and its application to harvesting of Dunaliella. In our experiments, stratification was initiated by pumping a 1 cm layer of water of 0-3.5% salinity onto the top of the pond and leaving it for about 6 h in sunlight. During this period D. salina preferentially concentrates near the brine: low salinity water interface, whereas D. viridis does not (Table 1). It is important to avoid any mixing of the layers, as for example by wind, during this step. At the end of 6 h the surface layer containing the D. salina can be run off or pumped off and used as the feed stock for the next harvesting step. We achieved a 5 to 6 times concentration of the algae with a 66-68% recovery of the D. salina in the pond using this method. The optimum strategy for harvesting appears to be a combination of several methods; for example, stratification to pre-concentrate the algae followed by a flocculation or settling step. The final harvest can then be further concentrated and de-watered by centrifugation, if necessary.

Processing. -Once harvested, the biomass may either be dried (usually by spray drying) to produce an algal powder, or it may be extracted and further processed to produce pure iJ-carotene. Several methods for the extraction of the iJ-carotene from D. salina have been patented. These include solvent extraction of the biomass with a hydrocarbon solvent such as toluene or hexane (Ruane, 1974b) or super- critical COl> saponification of the biomass followed by solvent extraction (Ruegg, 1984), or extraction in hot oil (Nonomura, 1987). Following extraction or drying, the product must be further processed and 248 BULLETIN OF MARINE SCIENCE, VOL. 47, NO.1, 1990 o

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Cell Number(x104 cells. ml-1) Salinity (% brix) Figure 2. Distribution of Dunaliella (total cell counts of D. salina and D. viridis) in a 100 m2 pond over a period of three days. Samples were taken from two positions (0) in the south-east comer, and (e) in the north-west comer of the pond. The wind was northerly on day 1, north-westerly on day 2, and south-easterly on day 3. 7.8 mm rain fell in the night between day 2 and 3. BOROWITZKA AND BOROWITZKA: BET A CAROTENE PRODUCTION 249

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RETENTION TIME Figure 3. Typical HPLC analysis of a 30% suspension of {3-carotene from Dunaliella salina in vegetable oil. The lower part of the figure is a detail of the upper part.

Table 1. Time-course of stratification of Dunaliella salina in a culture grown at 25% NaCl and overlain with a I cm layer of freshwater

Time Total cells in culture in top 2.5 cm of pond (h) (%)

2 34.5 4 52.6 6 60.8 8 53.3 10 53.1 250 BULLETIN OF MARINE SCIENCE, VOL. 47, NO. I, 1990

BETA CAROTENE PRODUCTION PROCESS: HUn LAGOON

BETA CAROTENE PRODUCTS Figure 4. Schematic flow diagram of the l3-carotene production process used by Western Biotech- nology Ltd. at Hutt Lagoon, Western Australia.

stabilized to produce a marketable product. The major products produced and marketed by the various companies are: (1) Spray dried Dunaliella salina con- taining about 2-3% J3-carotene for use in animal feeds, especially in aquaculture. (2) A 1.6-4% solution of J3-carotene in vegetable oil (soy bean, peanut, etc.) for use as a dietary supplement. This can be directly encapsulated for human con- sumption. (3) A 30% suspension of crystalline J3-carotene in vegetable oil for use in the food industry for food coloring, etc. The product should contain no solvent residues, so a solvent-free method of manufacture is desirable. Figure 3 shows a typical HPLC analysis of a 30% suspension of Dunaliella J3-carotene in oil.

COMPARISON OF EXISTING COMMERCIAL DUNALIELLA PROCESSES The process for the production of J3-carotene from Dunaliella salina used by Western Biotechnology Ltd. is shown in Figure 4. The production plant consists of ten 5-ha growing-ponds constructed directly on the lake bed of Hutt Lagoon, a natural salt lake on the coast in Western Australia, north of Geraldton. The floor of the ponds is the existing lake bed, and the pond walls are constructed from compacted limestone. The brines for the ponds are taken directly from Hutt Lagoon and the salinity is controlled by the addition of purified seawater which is pumped from the seaward side of Hutt Lagoon. This seawater is used to make

up any evaporative losses. The brine is supplemented with nutrients (NO}-, P04-, Fe, trace elements) and the ponds are harvested more or less continuously, and the pond water is recycled. The process used by Betatene Ltd. at their plant at Wyalla, South Australia is similar (Curtain et aI., 1987), except that they use several large natura11akes of approximately 100 ha area (total area approx. 350 ha) for algal growth. The water source is seawater from the adjacent Spencers Gulf and this water is concentrated by evaporation to increase the salinity to about 3.5 to 5.5 m NaC!. Harvesting is by the process patented by Curtain and Snook (1982) and uses nitrogen available from a nearby steelworks to eliminate the need for synthetic in the harvesting process. BOROWITZKA AND BOROWITZKA: BETA CAROTENE PRODUCTION 251

In contrast, Microbio Resources Inc. at their plant near the Salton Sea in California use smaller (l acre) concrete walled lined ponds and paddle wheel mixing. Their brines are made up artificially using salt (Klausner, 1986).

CONCLUSIONS The production of natural !3-carotene from Dunaliella salina (sometimes also called D. bardawil) is now well established and should continue to increase for some time. Open air culture ofthis alga is greatly simplified due to the extremely halophilic nature of this alga, however such a process will not be suitable for all algae or algal products and other methods will have to be developed in the future. The Dunaliella process, illustrates a fundamental aspect of commercial ex- ploitation of micro-algae, in that all steps of the process (i.e., algal growth, har- vesting, extraction and product formulation) must be optimized. At the present stage of development of algal biotechnology there is still considerable scope for improvement in all of these steps, and such improvements in the technology will permit the future exploitation of other algal species and products such as carot- enoids, fatty acids and polysaccharides.

ACKNOWLEDGMENTS

We would like to thank our many co-workers over the years for their invaluable contributions to this work, in particular B. Mackay, T. Moulton and T. Mercz.

LITERATURE CITED

Aasen, A. J., K. E. Eimhjellen and S. Liaaen-Jensen. 1969. An extreme source of tJ-carotene. Acta Chem. Scand. 223: 2544-2545. Ben Amotz, A. and M. Avron. 1983. On the factors which determine massive tJ-carotene accu- mulation in the halotolerant alga Dunaliella bardawi/. Plant Physiol. 72: 593-597. --, A. Katz and M. Avron. 1982. Accumulation of tJ-carotene in halotolerant algae: purification and characterisation of tJ-carotene-rich globules from Dunaliella bardawi/ (). J. Phycol. 18: 529-537. Bloch, M. R., J. Sasson, M. E. Ginzburg, Z. Goldman, N. Garti and A. Peath. 1982. Oil products from algae. U.S. Patent 4341 038.6 pp. Borowitzka, L. J. and A. D. Brown. 1979. Halotolerance of Dunaliella. Pages 139-190 in M. Lev- andowsky and S. H. Hutner, eds. Biochemistry and physiology of protozoa, 2nd ed. Academic Press, New York. --, M. A. Borowitzka and T. P. Moulton. 1984. The mass culture of Dunaliella salina for fine chemicals: from laboratory to pilot plant. Hydrobiologia 116/117: 115-134. --, T. P. Moulton and M. A. Borowitzka. 1986. Salinity and the commercial production of beta-carotene from Dunaliella salina. Nova Hedwigia, Beihefte, 83: 224-229. Borowitzka, M. A. and L. J. Borowitzka. 1988a. Dunaliella. Pages 27-58 in M. A. Borowitzka and L. J. Borowitzka, eds. Micro-algal biotechnology. Cambridge University Press, Cambridge. 58. -- and --. 1988 b. Limits to growth and carotenogenesis in laboratory and large-scale cultures of Dunaliella salina. Pages 371-382 in T. Stadler, J. Mollion, M-C. Verdus, Y. Karamanos, H. Morvan and D. Christiaen, eds. Algal biotechnology. Elsevier Applied Science, Barking. Chen, B. J. and C. H. Chi. 1981. Process development and evaluation for algal glycerol production. Biotechnol. Bioeng. 23: 1267-1287. Curtain, C. C. and H. Snook. 1982. Method for harvesting algae. U.S. Patent 4 554 390. 22 pp. --, S. M. West and L. Schlipalius. 1987. Manufacture of tJ-carotene from the salt lake alga Dunaliella salina; the scientific and technical background. Aust. J. Biotechnol. I: 51-57. Ibrahim, K., N. A. Jaffrey and S. J. Zuberi. 1977. Plasma Vitamin 'A' and carotene levels in squamous cell carcinoma of the oral cavity. Clin. Oncol. 3: 203-207. Isler, O. 1979. History and industrial application of and Vitamin A. Pure and Appl. Chem. 51: 447-462. Kessler, J. O. 1982. Algal cell growth, modification and harvesting. U. S. Patent 4438 591. 11 pp. KHiui, H. 1982. Industrial and commercial uses of carotenoids. Pages 309-328 in G. Britton and T. W. Goodwin, cds. Carotenoid chemistry and biochemistry. Pergamon Press, Oxford. 252 BULLIETINOFMARINESCIENCE,VOL.47, NO. I, 1990

Klausner, A. 1986. : food for thought. Bio/Technology 4: 947-953. Krinsky, N.!. 1979. Carotenoid protection against oxidation. Pure and Appl. Chern. 51: 649-660. Massyuk, N. P. 1966. Mass culture of the carotene-bearing alga Dunaliella salina Teod. Ukr. Bot. Zhour.23: 12-19. --- and E. G. Abdulla. 1969. First experiment of growing carotene-containing algae under semi- industrial conditions. Ukr. Bot. Zhour. 26: 21-27. Mathews-Roth, M. M. 1982. Medical applications and uses of carotenoids. Pages 297-307 in G. Britton and T. W. Goodwin, eds. Carotenoid chemistry and biochemistry. Pergamon Press, Oxford. Mil'ko, E. S. 1963. Effect of various environmental factors on pigment production in the alga Dunaliella salina. Mikrobiologya 32: 299-307. Mohn, F. H. 1988. Harvesting of micro-algal biomass. Pages 395-414 in M. A. Borowitzka and L. J. Borowitzka, eds. Micro-algal biotechnology. Cambridge University Press, Cambridge. Moulton, T. P., L. J. Borowitzka and D. J. Vincent. 198701. The mass culture of Dunaliella salina for ,6-carotene: from pilot plant to production plant. Hydrobiologia 151/152: 99-105. ---, T. R. Sommer, M. A. Burford and L. J. Borowitzka. 1987b. Competition between Dunalie/la species at high salinity. Hydrobiologia, 151/152: 107-116. Nonomura, A. M. 1987. Process for producing a naturally-derived carotene/oil composition by direct extraction from algae. U.S. Patent 4 680 314. 5 pp. Oswald, W. J. 1988. Large-scale culture systems (engineering aspects). Pages 357-394 in M. A. Borowitzka and L. J. Borowitzka, eds. Micro-algal biotechnology. Cambridge University Press, Cambridge. Post, F. J., L. J. Borowitzka, M. A. Borowitzka, B. Mackay and T. Moulton. 1983. The protozoa of a Western Australian hypersaline lagoon. Hydrobiologia 105: 95-113. Ruegg, R. 1984. Extraction process for beta-carotene. U.S. Patent 4 439 629. 2 pp. Ruane, M. 197401. Recovery of algae from brine suspensions. Australian Patent 486 999. 12 pp. ---. 1974b. Extraction of caroteniferous materials from algae. Australian Patent 487 018. 10 pp. Schwartz, J., D. SUdOland G. Light. 1986. Beta-carotene is associated with the regression of hamster buccal pouch carcinoma and induction of tumor necrosis factor in macrophages. Biochem. Bio- phys. Res. Commun. 136: 1130-1135. Wangersky, P. J. and R. L. Maass. 1988. Diurnal behavoir of cultures of Dunaliella tertiolecta. J. Plankt. Res. 10: 327-329.

DATEACCEPTED: April 10, 1989.

ADDRESSES:(L.J.B.) Western Biotechnology Ltd., 2-6 Railway Parade, Bayswater, W. A. 6053, Australia; (M.A.B) Algal Biotechnology Laboratory, School of Biological and Environmental Sciences, Murdoch University, Murdoch, W.A., 6150, Australia.