THE MASS CULTURE OF DUNALIELLA SALINA

MICHAEL A. BOROWITZKA

Algal Biotechnology Laboratory
School of Biological and Environmental Sciences
Murdoch University
Murdoch, W.A. 6150
AUSTRALIA

Introduction

The green unicellular flagellate Dunaliella salina Teodoresco is the richest natural source of the carotenoid β-carotene (Borowitzka, MA and Borowitzka, 1988 a). The halophilic species of Dunaliella also accumulate very high concentrations of glycerol (Borowitzka, LJ and Brown, 1974; Borowitzka, LJ, 1981 b). Dunaliella salina was first proposed as a commercial source of B-carotene by Massyuk (Massyuk, 1966) and later as a source of glycerol (Ben-Amotz et al., 1982 b). β-carotene from Dunaliella is now being produced on a commercial scale in Australia, the USA and Israel, and pilot-scale projects are under way in China, Chile, Australia, the USA, Spain and Kuwait. More recently D. viridis has also been proposed as a source of oxygenated carotenoid (Moulton and Burford, 1990).

Taxonomy, life-history and morphology

Dunaliella is a unicellular, bi-flagellate, naked green alga (Chlorophyta, Chlorophyceae). The genus was first described by Teodoresco (Teodoresco, 1905) with the type of species being Dunaliella salina, and at present a total of 29 species, as well as a number of varieties and forms, are recognized (Massyuk, 1973).

Dunaliella is morphologically similar to Chlamydomonas, with the main difference being the absence of a cell wall in Dunaliella. Dunaliella has two flagella of equal length and a single, cup-shaped chloroplast, which in the marine and halophilic species has a central pyrenoid (Borowitzka, MA & Borowitzka, 1988a). In D. salina and D. parva the chloroplast accumulates large quantities of B-carotene so that the cells appear orange-red rather than green. The carotenoid are in the form of droplets (plastoglobuli) located at the chloroplast periphery and consist of a mixture of the cis- and transisomers of β-carotene (Ben-Amotz et al., 1988). A typical composition, expressed as percentages of total β-carotene is 15-cis-β-carotene, 10%; 9-cis-β-carotene, 41%; all-trans-β-carotene, 42%; other isomers, 6% (Ben-Amotz et al., 1982a; Borowitzka, LJ & Borowitzka, 1989a). In the alga this β-carotene seems to act as photo-protective ‘sun-screen’ to protect the chlorophyll and the cell DNA from the high irradiance which characterizes the normal habitat of D. salina (Ben-Amotz, 1980; Ben-Amotz et al., 1989). Borowitzka and Borowitzka (1988a) have also proposed that the β-carotene also acts as a ‘carbon sink’ to store the excess carbon produced during photosynthesis under conditions where growth is limited but photosynthetic carbon fixation must continue.

Cell shape in this genus is very variable, being oval, spherical, cylindrical, ellipsoidal, egg-, pear- or spindle-shaped with radial, bilateral or dorsoventral symmetry or being asymmetrical. Cells in any given species may change shape with changing conditions, often becoming spherical under unfavorable conditions. Cell size also varies with growth conditions and light intensity (Massyuk, 1973).

The cells divide by lengthwise division in the motile state. Under certain conditions they may also develop into a palmella stage and become embedded in a thick layer of mucilage, or they may form aplanospores with a thick, rough wall. Sexual reproduction is rarely observed in cultures, but more often in the field. It is by isogamy, with conjugation proceeding in a manner similar to that observed in Chlamydomonas. The zygote is green or red and is surrounded by a very thick, smooth wall of sporopollenin. After a resting stage the zygote nucleus divides meiotically, forming up to 32 cells which are liberated through a rupture in the mother cell wall (Borowitzka, MA et al., 1982).

Ecology

The genus Dunaliella has marine and halophilic representatives. Freshwater species have also been described although Melkonian and Preisig (1984) have proposed that these should be assigned to a different genus. Dunaliella also has a very wide pH tolerance ranging from pH 1 (D. acidophila; Gimmler et al., 1989) to pH 11 (D. salina). In fact, D. salina is one of the most environmentally tolerant eukaryotic organisms known and can cope with a salinity range from seawater (= 3% NaCl) to NaCl saturation (= 31% NaCl), and a temperature range from <0 °C to >38 °C (Ginzburg, 1987; Borowitzka, MA & Borowitzka, 1988a).

Dunaliella species are commonly observed in salt lakes in all parts of the world from tropical to temperate to polar regions where they often impart an orange-red colour to the water. Marine Dunaliella species can generally be isolated from seawater, although they are not very abundant in nature. More details of the ecology of this genus can be found in Borowitzka and Borowitzka (1988a).

Mass culture

The main interest in this genus in recent years has been in the halophilic species Dunaliella salina since it is the source of β-carotene (in some papers this species is also referred to as D. bardawil, however, this is an incorrect name for D. salina, furthermore, several other cultures referred to in the literature have been incorrectly identified and Table 1 gives the correct name for these).

The large-scale culture of D. salina requires a good understanding of the physiology and ecology of the alga. These aspects will be discussed here first.

Physiology

The physiology of Dunaliella, especially D. salina is reviewed in detail by Ginzburg (1987) and Borowitzka and Borowitzka (1988a) and the present account will only consider the most important facets for mass culture. The most commonly used medium for culture of Dunaliella is Modified Johnsons Medium (Table 2), however, these algae can also be grown in a wide range of other media including Guillard's f/2 medium (Guillard & Ryther, 1962), modified ASP medium (McLachlan & Yentsch, 1959) and enriched seawater (Rao & Chauhan, 1984).

Nutrient requirements

Carbon source. All Dunaliella spp. so far studied are strict photoautotrophs. Dunaliella appears to be able to take up CO₂ and HCO₃ for photosynthesis, and Aizawa and Miyachi (1984) and Brown et al., (1987) have demonstrated the presence of an extracellular carbonic anhydrase in D. tertiolecta. The supply of inorganic carbon is particularly important to the culture of D. salina since, at the very high salinities at which this alga grows, the solubility of inorganic C is low; i.e. at 25% NaCl the solubility of inorganic carbon is <50% of that at seawater salinity (3% NaCl). Furthermore, at the high temperature and pH usually found in the natural brines in which D. salina grows, the bulk of the inorganic carbon (>99%) is in the form of CO₃^2- and is thus unavailable for uptake by the algal. The presence of an extracellular carbonic anhydrase which catalyses the conversion of HCO_3- to CO₂ means that the alga can utilize HCO_3- under these conditions.

Table 1. Probable taxonomic affiliation of some strains of Dunaliella salina and other misnamed species reported in the literature.

Table 2. Modified Johnsons Medium (J/l) (Borowitzka, M.A., 1988).

Nitrogen source. The best source of nitrogen for Dunaliella is nitrate (Mil'ko, 1962; Grant, 1968; Borowitzka, MA & Borowitzka, 1988b). Ammonium salts such as ammonium acetate, ammonium nitrate and ammonium chloride are generally less effective N sources and at high concentrations and high temperatures can be lethal (Mil'ko, 1962). For example, experiments in our laboratory have shown that ammonium nitrate inhibits the formation of β-carotene, and in actively growing cultures uptake of the ammonium leads to an acidification of the medium which eventually results in the death of the algae (Borowitzka, MA & Borowitzka, 1988b). Urea can be used as a nitrogen source, especially in well buffered media. However, in large-scale outdoor cultures urea can lead to mass mortality of the culture due to the high concentrations of ammonium being released upon metabolism of the urea (Massyuk, 1966).

Phosphorous. Phosphate is the best source of P and the optimal concentration is about 0.02 to 0.025 g.1^-1 K₂HPO₄ (McLachlan, 1960; Mil'ko, 1962). High concentrations may actually inhibit growth.

Magnesium and calcium. Both of these cations are required for growth and Dunaliella can tolerate a wide Mg ²⁺:Ca²⁺ ratio ranging from 0.8 to 20.0. For D. tertiolecta a Mg²⁺:Ca²⁺ ratio of 4 has been reported as optimal (McLachlan, 1960).

Sodium. All marine and halophilic species of Dunaliella require sodium.

Chloride and sulphate. The optimum Cl^-:SO₄^2- ratio for growth in D. salina is 3.2, whereas the optimum ratio for β-carotene formation is 8.6 (Massyuk, 1965). There also appear to be some interaction between the anions and the cations and their effect on the alga, however, this is as yet little understood.

Iron. Low concentrations of iron in a form that can be assimilated are essential for the growth of Dunaliella, and in some hypersaline brines Fe may be limiting to the growth of Dunaliella. The optimum concentration for iron in D. salina and D. viridis lies between 1.25 to 3.75 mg.1^-1 (Mil'ko, 1962) and should be supplied in a chelated form such as iron citrate or Fe-EDTA (Borowitzka, MA & Borowitzka, 1988b; Table 2). High concentrations of Fe inhibit growth.

Trace elements and vitamins. Various trace elements such as Zn, Co, Cu, Mo and Mn are usually added to Dunaliella media, however little is known of the actual requirements of the alga. Dunaliella does not require any exogenous vitamins for growth.

pH. The optimum pH for growth for the marine D. tertiolecta is pH 6, whereas for the halophilic D. salina and D. viridis it is about pH 9 (Loeblich, 1982).

Temperature. The optimum growth temperature for D. salina is in the range of 20 to 40°C (Borowitzka, LJ, 1981a) depending on the strain. Dunaliella salina can tolerate extremely low temperatures to below freezing (Siegel et al., 1984), but temperatures greater than 40°C are usually lethal. There is also a strong interaction between the growth rate, temperature and salinity (Gimmler et al., 1978; Borowitzka, MA & Borowitzka, 1988a) and between light intensity and temperature tolerance (Federov et al., 1968).

The physiology of carotenogenesis

Dunaliella salina is characterized by its ability to accumulate very high concentrations of β-carotene. Concentrations of up to 14% of dry weight have been reported (Aasen et al., 1969; Borowitzka, LJ et al., 1984). The halophilic species D. parva also accumulates high concentrations of β-carotene (<4% of dry weight), whereas none of the other species accumulate such large amounts (Massyuk, 1973). Recently, what appears to be a new species of Dunaliella has been isolated in Chile which accumulates not only high concentrations of β-carotene, but also a-carotene. High levels of β-carotene accumulation require high salinity, high temperature and high light (Mironyuk & Einor, 1968; Semenenko & Abdullayev, 1980; Ben-Amotz & Avron, 1983; Borowitzka, LJ et al., 1984; Ramazanov et al., 1988). Nutrient limitation, especially N limitation, also enhances carotenoid formation (Mil'ko, 1963; Ben-Amotz & AVron, 1983; Ben-Amotz, 1987; Borowitzka, MA & Borowitzka, 1988b). In general, carotenogenesis is greatest under sub-optimal growth conditions when the specific growth rate is low (Ben-Amotz et al., 1982a; Borowitzka, LJ et al., 1984). Table 3 summarizes the data on the effects of various environmental factors on growth and carotenogenesis.

Table 3. Summary of the influence of various environmental factors on biomass production and β-carotene content of cultures of D. salina. (+ = stimulating effect; - = inhibitory effect; 0 = no effect) (Based on Mil'ko, 1963; Borowitzka, LJ & Borowitzka, 1989a).

^a= Rate of decrease is very slow.

Light is essential for carotenogenesis (Loeblich, 1982; Ben-Amotz, 1987), but the maximum level of β-carotene in the cell is dependent on the salinity (Borowitzka, MA et al., 1990). Carotenoid formation is rapid. For example, when the salinity of a culture of D. salina is increased from 15 to 25% NaCl the total carotenoid increases linearly from < 10 to 260 mg (g cell protein)^-1 over a period of 4 to 5 days. The proportions of β-carotene increases from 50% to 90% during this period whereas there was no measurable increase in the other carotenoid, α-carotene, lutein and zeaxanthin (Borowitzka, MA et al., 1990).

Fortunately, from the mass culture point of view, the cell content of Bcarotene does not decrease at the same rapid rate when the salinity of the medium is reduced as occurs in outdoor cultures when it rains. When carotenoid-rich cells are transferred from a high salinity (25% NaCl) medium to a low salinity (15% NaCl) medium, there is only a gradual decline in cell carotenoid content over a period of several weeks (Borowitzka, LJ et al., 1984). Most of this decline is not due to β-carotene breakdown, but rather due to reapportionment of the β-carotene as the cells divide.

Commercial production of β-carotene from Dunaliella salina

As mentioned above, carotenogensis is greatest when growth is least. This represents the fundamental dilemma to the commercial producer wishing to produce β-carotene with Dunaliella salina. For commercial-scale production of B-carotene, or glycerol, one requires:

1. optimization of biomass production and product yield;

2. development of a reliable growth system which can cope with the vagaries of weather as well as potential predators and competitors;

3. a suitable, low-cost harvesting method;

4. appropriated downstream processing methods.

For large-scale outdoor algal cultures productivities of 30–40 g dry weight m^-2. day^-1 seem to represent the present achievable limit over sustained periods. Over shorter periods, and in smaller culture systems productivities as high as 60 g dry weight.m^-2.day^-1 have been recorded for Dunaliella (Ben-Amotz, 1980). However, many of the commercial production systems do not regularly achieve productivities of even 30 g dry weight.m^-2.day^-1. In fact, productivities are often substantially less. Also, it is generally observed, that the larger the individual ponds used to grow the algae, the smaller the productivity of the system seems to be.

In D. salina the optimum salinity for growth lies between 18 and 22% NaCl whereas the optimum salinity for carotenoid production is >27% NaCl (Borowitzka, LJ et al., 1984). By combining these data we find that the optimum yield of β-carotene per unit volume and time occurs at about 24% NaCl (Fig. 1).

Two strategies are open to the Dunaliella producer to maximize the production of β-carotene. One of these strategies is a two-stage growth process in which the algae are first grown at a low salinity ( = 15% NaCl) in nutrient-rich medium to maximize biomass production, and then transferred to a high salinity, low nutrient medium in order to induce β-carotene production (Massyuk, 1966; Borowitzka, LJ et al., 1984). A similar, two-stage production process was proposed by Chen and Chi (1981) for glycerol production from Dunaliella. Such a process however, has several disadvantages. These are:

1. At low salinities protozoa such as the ciliate Fabrea salina and the amoeba Heteroamoeba sp. can invade the culture and very rapidly decimate the algal production (Post et al., 1983; Borowitzka, LJ et al., 1985);

2. Lower salinities generally favour the growth of the non-carotenogenic Dunaliella species, D. viridis, D. minuta and D. parva (Borowitzka, LJ et al., 1985; Moulton et al., 1987). These species can overgrow the D. salina under these conditions and thus drastically reduce the B-carotene productivity of the pond.

3. A two-stage process is more labour intensive and also requires a greater pond area due to the slower throughput time. This results in greater capital and running costs and thus may make the process uneconomic (Borowitzka, LJ et al., 1984).

An alternative approach is to grow the algae in either batch or continuous culture at an intermediate salinity optimised for the best overall yield of β-carotene over time (Borowitzka, LJ et al., 1985). At the higher salinities used in this process the growth of predatory protozoa such as Fabrea salina, and the growth of the non-carotenogenic Dunaliella species are reduced and generally present no problem. Although growth rates are lower, the operating costs are also reduced and more importantly, the culture is not prone to sudden collapse.

Figure 1. Effect of salinity on β-carotene content and biomass production in Dunaliella salina (from Borowitzka, LJ et al., 1984).

Culture systems

Commercial production of D. salina is presently carried out in either extensive cultures in large unstirred outdoor ponds, or more intensively in paddlewheel stirred raceway ponds. Photobioreactors Plc of the UK are also in the process of constructing a production plant in Spain where they proposed to grow the alga in a tubular photobioreactor.

The two largest producers in the world are Western Biotechnology Ltd. (Perth, Western Australia) and Betatene Ltd. (Melbourne, Victoria) in Australia. They grow the algae in very large and shallow (approx. 20 cm deep) ponds constructed either on the bed of a hypersaline coastal lagoon, or formed by artificially expanding a lagoon (Curtain et al., 1987; Borowitzka, LJ & Borowitzka, 1989a). For example, each of the production ponds used by Western Biotechnology Ltd. at Hutt Lagoon on the west coast of Australia, is 5 ha in area and they are constructed on the lagoon floor by isolating parts of the lagoon with earthen berms (Fig. 1). The production plant has a total pond area of 50 ha and there are also smaller ponds for research purposes. The ponds used by Betatene Ltd. at Whyalla in South Australia are even larger. The process used is summarized in Fig. 2. The algae are cultivated at high salinities in a medium made up of NaCl-saturated brines obtained from Hutt Lagoon with the salinity controlled by the addition of seawater which is pumped from a bore located on the seaward side of the lagoon. Nutrients are added as required. When the pond has reached the appropriate β-carotene content the culture is pumped to a harvesting plant on the shore. After harvesting the remaining medium is returned to the growth pond and the salinity and nutrient content is adjusted as appropriate. The rate of harvesting and the growth period varies with changing climatic conditions throughout the year.

The ponds are unlined and there are no mixing devices; the only mixing is by wind and thermal action. Although this form of extensive cultivation results in lower cell densities compared to a well mixed system, it is economically more attractive as long as an effective and cheap harvesting process can be used to extract the algae from the extremely high volumes of brines.

Paddlewheel-driven raceway ponds are used in Israel and the USA for the cultivation of Dunaliella. The biomass achieved in these ponds is generally an order of magnitude greater than in the unstirred ponds, however, construction and operating costs are greater. Details of paddlewheel raceway ponds and their construction and operation can be found in Oswald (1988) and Dodd (1986).

Each of the above culture systems has advantages and disadvantages, and the choice of system used at a particular site will depend on a number of factors. For example, if land costs and site preparation costs are high, then paddlewheel ponds are more economic. Similarly, since Dunaliella requires high concentrations of NaCl in the medium the source of the brine can have a major effect on the economics of the process. If high salinity brines are freely available, as in the case at Hutt Lagoon, then the extensive culture method is suitable. If, on the other hand, the brines have to be made up from salt, then the cost of this NaCl, even at US$ 13–16 ton^-1 is significant. Finally, if climatic conditions mean that the annual growth period is short, then an intensive cultivation system is favoured. Sites such as Hutt Lagoon favour extensive cultivation because of: (1) low cost land and site preparation costs; (2) free source of brines; (3) a long - at least 9 months per year - growth season.

Closed culture systems are another alternative, however, at this time they still seem to be too expensive (Chaumont et al., 1988; Borowitzka, LJ & Borowitzka, 1989b). The advantages of closed systems such as the tubular photobioreactors are that contamination by other species of Dunaliella and protozoa can be virtually eliminated. The growth conditions can also be optimised and closely controlled, resulting in higher cell densities and better carotenoid yields per unit volume compared to open-air cultures. This also reduces harvesting costs. On the other hand, closed systems require pumping of the culture for circulation, and Dunaliella species are very sensitive to shear damage. As well as this, there is also the high capital cost of these systems and a higher operating cost. Large-scale closed algal culture systems are still in the early stages of development, and it is likely that in the future better and more economic designs will be developed, however, until then open-air culture will remain the preferred option for large-scale commercial algal culture.

Choice of site

Based on the observations above, a Dunaliella salina culture facility should be based at a site where:

1. there is ample flat land available;

2. there are cheap sources of high salinity brines, and also of lower salinity water (i.e. seawater) for salinity control and to provide the water for making up evaporative losses. The requirement for evaporative make-up water should not be underestimated. For example, a 5 ha pond at 20 cm depth contains 2 × 10⁶ litres of water. At an evaporation rate of 1 cm per day, 100,000 litres of make-up water are required a day;

3. there are few cloudy days in the year and the mean daily temperature is >30°C for most of the year;

4. rainfall is as low as possible, and falls only during a small part of the year;

5. The site is as far away as possible from any source of pollution which might affect algal growth or contaminate the algal product which will be used as a high quality food additive. This means that the plant should not be near agricultural activities where pesticides or herbicides are used, nor should it be near industrial activities which may emit heavy metals.

Harvesting

Harvesting and extraction represent the major cost areas in most algal processes. Harvesting D. salina is more difficult and costly compared to most other commercially produced algae, and therefore the methods used by the various commercial producers are closely guarded.

Figure 2. Schematic flow diagramme of the D. salina β-carotene production process used by Western Biotechnology Pty Ltd (from Borowitzka, L.J. & Borowitzka, 1989a).

The difficulty in harvesting D. salina is because this alga is a single cell with no protective cell wall, approximately 20 × 10 um in size and neutrally buoyant in a high specific gravity, high viscosity brine. Cell densities in large-scale cultures tend to be about 1 g. 1^-1 and therefore very large volumes have to be processed. Centrifugation and filtration generally tend to shear-damage the cells, leading to loss of the β-carotene by oxidation. The cells also distort and pass through filters with pore sizes less than 10 um. Corrosion of all metal equipment by the brine is also a major problem.

These problems have led to development and patenting of several biological and chemical methods for harvesting the cells prior to the use of more conventional harvesting methods. Patents issued for Dunaliella harvesting include high pressure filtration using diatomaceous earth as a filter aid (Ruane, 1974b), exploitation of salinity-dependent buoyancy properties in stationary and moving gradients (Block et al., 1982), exploitation of the phototactic and gyrotactic responses of the algae (Kessler, 1985), salinity-dependent hydrophobic adhesion properties of the Dunaliella cells (Curtain & Snook, 1983) and flocculation (Sammy, 1987). The general features of algal harvesting are reviewed by Mohn (1988).

Considering the low cell density of Dunaliella cultures it is likely that a harvesting process will have several steps. Any process which preconcentrates the alga easily maybe of benefit. In this respect the observation that Dunaliella salina sometimes aggregates on the pond surface is important. When rain results in a layer of lower-salinity or even freshwater on the surface of the pond D. salina cells accumulate at the low salinity/high salinity interface (Block et al., 1982; Borowitzka, LJ & Borowitzka, 1990). Furthermore, it is those cells with the highest β-carotene content which tend to stratify most readily. The algal cells also exhibit diurnal movement in the pond (Wangersky & Maass, 1988). It may be possible to utilize this phenomenon as part of a preliminary concentration step in a harvesting process.

Finally, it must also be recognized that different products such as dry Dunaliella powder or extracted β-carotene may require different harvesting strategies.

Extraction and drying

Another critically important aspect of Dunaliella β-carotene production is extraction of the β-carotene. The process used for extraction depends, in part, on the harvesting procedure used and on market requirements. Extraction using conventional organic solvents is efficient (Ruane, 1974a; Ruegg, 1984), but may not be acceptable to customers seeking a 'natural' product. More acceptable alternative extraction methods use hot vegetable oil (Nonomura, 1987; Potts, 1987) or liquid or supercritical gas solvents.

Harvested biomass may also be dried (i.e. spray-drying) rather than extracted and can be marketed as a β-carotene-rich supplement for human health or animal feed (Ben-Amotz et al., 1986; Avron et al., 1987; Curtain et al., 1987).

Dunaliella β-carotene production can also produce several useful by-products such as glycerol which can make up 30% of the biomass dry weight (Chen & Chi, 1981), and high quality protein meal remaining after extraction. The amino acid analysis of several Dunaliella spp. is summarized in Table 2.2 of Borowitzka and Borowitzka (1988a).

Formation and applications

β-carotene is a lipid- and oil-soluble product. As suspensions or solutions in vegetable oil it finds applications in colouring margarine, baked good, and some prepared foods (Klaui, 1982). Conversion to water-soluble or water-disperable formulations, by forming emulsion or microencapsulated beadlets extends the food applications to beverages including orange drinks, and to confectionery and further prepared foods.

Nutritional supplements can be prepared by encapsulation of oil suspensions or solutions, or by tabletting using beadlet forms. β-carotene is pro-Vitamin A, and is an excellent source of this vitamin since it is not toxic at even high doses. Because of its antioxidant and free-radical trapping properties (Burton & Ingold, 1984; Terao, 1989) β-carotene also appears to have a cancer- preventative action, especially against epithelial cancers such as lung cancer (Peto et al., 1981). Current recommended daily doses for β-carotene are around 6 mg; 20–25 mg doses are in use in the anti-cancer trials in the US and Finland. Dried Dunaliella powder is also prepared for use in animal feed supplements.

Economics

Synthesized β-carotene is sold for a minimum of US$ 300 per kg β-carotene, and at higher process depending on the formulation. ‘Natural’ β-carotene commands higher prices, with the highest price attainable for the nutritional supplement application. At present the market demand exceeds supply.

If a price range for formulations of β-carotene of US$ 500–1000 is assumed, and β-carotene represents 10% of D. salina biomass, production costs should not exceed US$ 50–100 per kg. In fact, costs must be significantly lower, to account for losses at each processing step, capital expense, marketing, packaging and distribution costs.

Conclusion

Although β-carotene can be synthesized or extracted from other natural sources such as carrots, D. salina is still the richest and best ‘natural’ source of this carotenoid. The extreme environment in which D. salina grows also makes the open-air large-scale culture of this alga much easier compared to other unicellular algae. The low cell densities achieved by the algae and their small cell size however, make harvesting more difficult and costly. Furthermore, the use of the β-carotene as a food or feed additive and a nutritional supplement means that a high quality product is required. This means that great care must be taken in the extraction and formulation steps.

Despite these difficulties there are now several commercial producers of D. salina β-carotene and a number of others are expected to come on stream in the near future. The high value of the product also means that, even with the technology available today algal β-carotene production is an economic reality.

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