Chapter 1

In: Carotenoids: Properties, Effects and Diseases Editor: Masayoshi Yamaguchi, pp.

ISBN 978-1-61209-713-8 © 2011 Nova Science Publishers, Inc.

Chapter 3

CAROTENOIDS FROM MICROALGAE AND CYANOBATERIA: FEATURES, PRODUCTION AND APPLICATIONS Helena M. Amaro1 and F. Xavier Malcata1,2,* 1

Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Rua dos Bragas 289, P-4050-123 Porto, Portugal 2 Instituto Superior da Maia, Avenida Carlos Oliveira Campos, P-4475-690 Avioso S. Pedro, Portugal

ABSTRACT Microalgae and cyanobacteria entertain a ubiquitous distribution in aquatic ecosystems, where they provide the basis of the food chain; furthermore, they constitute a unique reservoir of biodiversity, which may potentially lead to commercial exploitation of novel products, or improved production of existing ones. As photosynthetic microorganisms, they possess the ability to use H2O and CO2 as major substrates, coupled with sunlight to bring about synthesis of complex organic compounds – as is the case of carotenoids, which they subsequently accumulate intracellularly. The major physiological role of carotenoids appears to be in light harvesting – via maintaining the structure and function of photosynthetic complexes, quenching chlorophyll triplet states, scavenging reactive oxygen species and dissipating excess radiant energy. More and more extensive use of carotenoids in the food and cosmetic industries has been observed, taking advantage chiefly of their role as antioxidants and pigments: for instance, β-carotene and astaxanthin are commonly applied in aquaculture to aid in color development of salmon, trout and red sea bream; and lutein is used in poultry feed formulation, for color intensification of eggs. Carotenoids have thus found many uses as colorants and preservatives of foods – especially in view of the stricter legislation on artificial additives, coupled with an increasing market demand for environment-friendly products; their intended features can be attained even at ppm levels of addition. Furthermore, a growing body of experimental evidence has proven that they can also play

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Helena M. Amaro and F. Xavier Malcata an important role in prevention and control of such important human diseases and health conditions as cancer, cardiovascular problems, atherosclerosis, rheumatoid arthritis, muscular dystrophy, cataracts and some neurological disorders. Key factors for feasible economic production of carotenoids via blue biotechnology entail screening for more performant wild microbial strains, design and optimization of novel photobioreactor configurations with improved volumetric productivities, and tailormade downstream purification technologies. Hence, active research has addressed all these issues, and several microalga-biotech companies interested in carotenoids have already met with success – especially in Hawaii and California (USA), Mediterranean basin and Far East. In this chapter, relevant properties, bioprocessing considerations and practical applications of carotenoids as food additives and in health promotion (e.g. lutein, βcarotene and astaxanthin) will be reviewed – with a specific focus on such sources as Haematococcus pluvialis, and Dunaliella salina, Muriellopsis sp. and Chlorella spp.

INTRODUCTION Microalgae and cyanobacteria lie at the bottom of the food chain in aquatic ecosystems; they possess the intrinsic ability to take up H2O and CO2 that, with the aid of solar energy, are used in synthesis of complex organic compounds, which are subsequently accumulated and/or secreted as primary or secondary metabolites. The aforementioned microorganisms have a worldwide distribution and are well-adapted to survival under a large spectrum of environmental stresses, including (but not limited to) heat, cold, drought, salinity, photooxidation, anaerobiosis, osmotic pressure and UV exposure [1]. Furthermore, the large number of existing species of microalgae and cyanobacteria constitute a unique reservoir of biodiversity, which may eventually support commercial exploitation of a very many novel products: besides (biotechnological) manufacture of vitamins, pigments and polyunsaturated fatty acids [2-4]. The key factor for their economic feasibility is the possibility of operation of large photobioreactors – able to produce those strains and their valuable biochemical products to sufficiently high levels [5,6]. This chapter cover the most relevant features of carotenoids produced by microalgae and cyanobacteria, besides presenting bioprocess considerations and reviewing practical applications in the food and health industries.

FUNCTION AND CELLULAR LOCATION OF CAROTENOIDS Carotenoids – also termed tetraterpenoids, are a class of terpenoid pigments, derived from a 40-carbon polyene chain that can be envisaged as their molecular backbone. Such polyene backbone provides carotenoids with their distinctive molecular structure, chemical properties and light-absorbing characteristics – which are essential for photosynthesis, and in general for life in the presence of oxygen [7]. The aforementioned backbone may be complemented by cyclic groups (rings) and oxygen-containing functional groups. The hydrocarbon carotenoids are denoted as carotenes as a whole, whereas oxygenated derivatives are known as xanthophylls. In the latter, oxygen is present as OH groups (as in lutein), as oxi-groups (as in cantaxanthin), or as a combination of both (as in astaxanthin)[8]. All xanthophylls synthesized by higher plants – e.g. violaxanthin, antheraxanthin, zeaxanthin, neoxanthin and

Carotenoids from Microalgae and Cyanobateria

3

lutein, are also synthesized by green algae; however, specific green algae possess additional xanthophylls, e.g. loroxanthin, astaxanthin and canthaxanthin. Furthermore, diatoxanthin, diadinoxanthin and fucoxanthin are produced by brown algae or diatoms [9]. A distinction is usually made between primary and secondary carotenoids. Primary xanthophylls are structural and functional components of the cellular photosynthetic apparatus, and are thus essential for survival [9]; secondary xanthophylls encompass those produced by microalgae to large levels, but only after exposure to specific environmental stimuli (carotenogenesis). Xanthophylls are relatively hydrophobic molecules, so they are typically associated with membranes and/or involved in non-covalent binding to specific proteins. Primary carotenoids are typically localized in the thylakoid membrane, whereas secondary carotenoids are found in lipid vesicles in either the plastid stroma or the cytosol. Most xanthophylls in cyanobacteria and oxygenic photosynthetic bacteria are associated with chlorophyll-binding polypeptides of the photosynthetic apparatus [10]. Among non-photosynthetic bacteria – and, to a lesser extent, among photosynthetic bacteria and cyanobacteria, xanthophylls and their glycosides can be found in organelle and cell wall membranes, where they are thought to directly influence membrane fluidity [11]. Most green algae, carotenes and xanthophylls are synthesized within plastids, and accumulate therein only. Conversely, secondary xanthophylls in some green algae, e.g. astaxanthin in Haematococcus, accumulate in the cytoplasm. This latter form of accumulation raises the possibility of an extra-plastidic site of carotenoid biosynthesis in the Haematococcus genus. Alternatively, xanthophylls synthesized in the chloroplast may be exported, and consequently be accumulated in the cytoplasm [12,13]. Therefore, xanthophylls can be found in virtually all cellular compartments. In microalgae and cyanobacteria, carotenoids perform several functions: they are involved in light harvesting, and also contribute to maintain structure and function of photosynthetic complexes – besides quenching chlorophyll triplet states, scavenging reactive oxygen species and dissipating excess energy [14]. The intrinsic antioxidant activity of carotenoids is the basis of their protective action against oxidative stress in many organisms and situations. However, not all biological activities ascribed to carotenoids are necessarily linked to their ability to inactivate free radicals and reactive oxygen species; the bioactive properties of a given carotenoid depend indeed on its specific molecular structure.

APPLICATION OF MAJOR CAROTENOIDS Several researchers have been active with carotenoids from microalgal and cyanobacterial origin over the years. Nowadays, the major areas of potential industrial application are food and health, as food/feed additive or pharmacologically active compounds, respectively; in both cases, the interest arises from the antioxidant properties exhibited by that class of compounds. In fact, pigments of microalgal origin are experiencing a strong market demand at present – e.g. phycobiliproteins, β-carotene and lutein. The cost of microalgal β-carotene is ca. US$ 1000/kg, versus US$ 500/kg in the case of its synthetic counterpart. However, natural β-carotene is preferred by the health market because it is a

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Helena M. Amaro and F. Xavier Malcata

mixture of trans and cis isomers; the latter possess anticancer features, but are seldom obtained via chemical synthesis [14].

Food Uses Manufacturate of carotenoids via microbiological routes constitutes a topic of a great scientific and commercial importance within the alimentary and aquaculture fields [11]. The threshold of synthetic food additives permitted has been steadily decreasing, owing to their suspected role as promoters of carcinogenesis – besides claims of liver and renal toxicities [15]. The growing trend towards substitution of synthetic food additives by natural pigments has redirected attention to the application of Dunaliella spp. for mass production of carotenoids – aimed at both colouring and preserving roles [16]. Furthermore, the nutraceutical claims of carotenoids owing to their antioxidant power improves attractiveness of food products added with those compounds [17]. Another advantage of using carotenoids as food additives lies upon the fact that they are not affected (unlike most dyes) by the presence of ascorbic acid – often used as acidulant to hamper increase of microbial counts, or heating and freezing cycles –commonly employed to reduce or control the number of viable microorganisms, respectively. On the other hand, carotenoids act as particularly strong dyes even at levels of the order of parts per million. Specifically, canthaxanthin, astaxanthin and lutein from Chlorella have been in regular use as pigments, and accordingly included in the feed of salmonid fish and trout, as well as poultry to enhance the reddish color of said fish or the yellowish color of egg yolk [2,18-20]. βCarotene in particular has experienced an increasing demand also as pro-vitamin A (retinol)containing multivitamin preparations; it is usually included in the formulation of healthy foods under an antioxidant claim [21,22].

Health Uses The latest decades have witnessed a growing interest for compounds bearing antioxidant properties, especially when obtained from natural sources owing to their claimed favorable effects upon health. Carotenoids are indeed potent biological antioxidants, which are able to absorb the excitation energy of singlet oxygen radicals into their complex ringed chain – thus promoting its dissipation, while concomitantly preventing tissues from becoming damaged. They can also delay propagation of chain reactions via free radicals, e.g. those initiated by degradation of polyunsaturated fatty acids – which are known to dramatically contribute to the decay of lipid membranes, thus seriously hampering cell integrity and eventually permitting critical decay of such molecules as DNA or proteins [18]. One illustrative example is the decline of cognitive ability in Alzheimer’s disease, which is apparently caused by persistent oxidative stress in the brain [23]. Using transgenic mice fed with Chlorella sp., Nakashima et al. [24] claimed that extracts containing β-carotene and lutein could prevent significant progression of cognitive impairment. On the other hand, Wu et al. [25] used Chlorella extracts containing 2-4 mg/gDCW of lutein, and claimed reduction in the incidence of cancer and prevention of macular degeneration [26]. Likewise, carotenoids extracted from Chlorella ellipsoidea and C. vulgaris inhibited colon cancer cell growth, yielding IC50 values of 40.73 ± 3.71 and 40.31 ± 4.43 μg/mL, respectively; the former extract produced an apoptosis-inducing effect almost 2.5-fold that of C. vulgaris extract [20]. In


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addition, astaxanthin obtained from Haematococcus pluvialis decreased expression of cyclin D1, but increased that of p53 and some cyclin kinase inhibitors (e.g. p21WAF-1/CIP-1 and p27) of colon cancer cell lines [27]. Carotenoids have also the ability to stimulate the immune-system, which is directly involved in more than 60 life-threatening diseases – including various forms of cancer, coronary heart diseases, premature ageing and arthritis [28]; this is specifically the case of canthaxanthin and astaxanthin, besides other nonprovitamin A carotenoids from Chlorella to a lesser degree [20]. Carotenoids from Dunalliela sp. display hyperlipidemic and hypercholesterolemic effects [16]; and a few epidemiological studies encompassing β-carotene from the aforementioned microalga (which contains readily bioavailable 9-cis and all-trans stereoisomers, ca. 40% and 50%, respectively) have also provided evidence of a lower incidence of several types of cancer and degenerative diseases [29].

Major Producers of Carotenoids The worldwide market value with greatest expression for carotenoids was ca. US$ 887 million in 2004, and has been rising at an average annual rate of 2.2% [8]. In particular, the value of β-carotene in the worldwide market was U$ 242 million in 2004, but reached U$ 253 million in 2009 [30]. Microalgae are more often used than cyanobacteria to produce carotenoids, and they accordingly entertain a much larger market share. The most famous are Chlorella, Chlamydomonas, Dunaliella, Muriellopsis and Haematococcus spp. – all of which belong to the Chlorophyceae family [31]. They tend to accumulate carotenoids as an intrinsic part of their biomass, so they convey economical alternatives to chemical sources thereof [32]. At present, β-carotene, as well as the xanthophylls astaxanthin, cantaxanthin and lutein are carotenoids in higher and higher demand [8]. Among all natural sources known to date, the halophilic green biflagellate Dunaliella possesses the highest content of 9-cis β-carotene [29,17] – reaching levels up to 100 g/kgDW, [16,33,34]. β-Carotene-rich Dunaliella powder and natural β-carotene have been exploited commercially in many countries since the 1980s. Although many microalgae can produce xanthophylls, Haematococcus pluvialis is the microalga that accumulates secondary xanthophylls to the highest levels, viz. asthaxanthin [9], so it is now cultivated at large scale by a few companies using distinct approaches [35]. On the other, hand Muriellopsis sp. holds a high lutein content (up to 35 mg.L-1) and exhibits a high growth rate; hence, it has been exploited for production of lutein [9]. Finally, Chlorella ellipsoidea was reported to produce violaxanthin together with other two minor xanthophylls, antheraxanthin and zeaxanthin – whereas the main carotenoid in C. vulgaris was lutein [20].

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Helena M. Amaro and F. Xavier Malcata Table 1. Optimal conditions of production of microalgal carotenoids

β-carotene

Carotenoid

Species

Dunaliella salina

Muriellopsis sp.

Processing conditions T: 25 °C; pH: 7.5 ± 0.5; LI: 281 ± 89 µmol photon m−2 s-1; FR: 38 cms−1 ·s−1; R: 0.7 gMJ−1 T: 28 °C; pH 6.5; LI: 460 μmol photon m−2 s−1

Lutein

Chlorella protothecoides

T: 30 °C; pH: 8.0; LImax: 1700 μE/ m−2 s−1; AF: 0.5 (v/v)/min-2 s−1; LDC: solar cycle T: 35 °C; LI: 1900 μE m−2s−1 T: 28 °C; pH: 6.5; LI: absence of light; MM: heterotrophic

Lutein content: 5.5 mg g-1 L1 -1 d Lutein: 1.4- 0.8 mg L−1 d−1

[40]

Biomass: 7.2 mg L−1 d−1; Lutein: 5.5 mg g-1 L-1 d-1

[40]

Semicontinuous outdoor, open tank (100 L)

Biomass: 100 mg m−2 d−1; Lutein: 100 mg g-1 L-1d-1

[40]

Continuous (2 L)

Lutein: 4.9 mg L−1 d−1

[6]

Continuous outdoors tubular

Lutein: 5.31 mg m−2 d -1

[41]

Batch (16 L)

Lutein: 10 mg L−1 d−1

[42]

Batch (0.2 L, 47d)

T: 28 ºC; pH 7; Chlorococcum LI: continuous 200 μmolphoton Batch (0.2 L) citriforme m−2 s−1; −1 −1 AF: 50–100 L h (1 %, v/v Neospongiococcus CO2) gelatinosum

Astaxanthin

C. zofingiensis

Haematococcus pluvialis

Reference

[39]

Chlorella zofingiensis,

T: 30ºC; pH: 6.5; LI: absence of light; SR: 130 rpm; MM: heterothrophic LI: day light cycle; MM: chemostat T: 28 ºC; LI: 345 µmol photon m-2 s-1 T: 15-25 ºC; LImax: 2000 µmolphoton m−2 s−1

Productivity

Biomass: 2 g m−2d−1; Semi-continuous Total carotenoids: 102.5 ± outdoor closed 33.1 mgm−2 d−1 (β-carotene: tubular (55 L) 10% of biomass)

T: 28 ºC; pH: 7; Continuous LI: continuous 200 μmolphoton outdoors tubular m−2 s−1; AF: 50-100 L−1 h−1 (55 L) (1 %, v/v CO2) -

Scenedesmus almeriensis

Reactor configuration

Lutein: 3.4 mg L−1 d−1 Lutein: 1.05 mg L−1 h−1 Lutein: 0.70 mg L−1 h−1

Batch (250 mL)

Astaxanthin:10.3 mg L-1

Continuous tubular (50 L)

Biomass: 0.7 g L-1d-1 Astaxanthin: 8.0 mg L-1d-1 Astaxanthin: 98 mg g-1 biomass

Batch (1 L)

[40]

[43]

[44] [45]

Enclosed Biomass: 90 g m−2 [35] outdoor (25,000 Astaxanthin: 13 g m−2 d−1 L) AF: air flow; IL: inoculum level; LDC: light dark cycle; LI: light irradiance; MM: metabolic mode; SR: stirring rate; T: temperature.

In the case of cyanobacteria, their carotenoid contents vary considerably with the prevailing growth conditions – yet the predominant pigments remain essentially the same;


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these prokaryotes generally contain β-carotene and zeaxanthin, the ketocarotenoids echinenone and canthaxanthin, and the carotenoid-glycoside myxoxanthophyll. The latter seems to be class-specific, as it was not found in any other group. However, a few differences in pigment content are usually recorded; e.g. cyanobacteria from marine habitats usually contain a poorer inventory of carotenoids [36]. The cyanobacterium Arthrospira (Spirulina) platensis has gained considerable attention worldwide as a source of several nutraceuticals, viz. lutein and β-carotene, owing to its easy, and thus convenient extraction [14]. Gloeobacter violaceus contains three dominant carotenoids: β-carotene, oscillol diglycoside and echinenone [36]; and Synechocystis sp. strain PCC6803 and Synechococcus sp. strain PCC 7002 have been pointed out as particularly suitable for genetic transformation aimed at enhancing β-carotene accumulation for industrial purposes. The pigment profile of Synechococcus spp. is mainly accounted for by β-carotene and zeaxanthin, with diverse glycocarotenoids and equinenone (a ketocarotenoid) as accompanying carotenoids – to varying extents, depending on the strains at stake [37].

Major Advantages of Carotenoid Bioproduction by Microalgae Microalgae combine, in a balanced fashion, a few properties typical of higher plants (viz. efficient oxygenic photosynthesis and simple nutritional requirements) with biotechnological attributes proper of microorganisms (viz. fast growth in liquid culture, and ability to accumulate or secrete metabolites). This rather useful combination supports selection of these microorganisms for applied processes, and represents the basic rationale of microalgal biotechnology. Besides current uses as feed for aquatic and terrestrial animals – in view of their carotenoids acting as colorants, the nutritional value of microalgal biomass goes well beyond – including use as high-protein supplement for human nutrition and as nutraceuticals for specific specialty products. Therefore, the food, feed, pharmaceutical, cosmetic and even fine chemical industries may all benefit from microalgal products [31,38]. Further to the unique possibility of harvesting sunlight, these photosynthetic microorganisms can be employed in reclamation of wastewater (in which N- and P-rich pollutants can serve as Nand P- sources, respectively) and other forms of bioremediation (in which CO2 from the atmosphere can be sequestered and used as a C-source) [8]. Despite the aforementioned relevant and useful features, microalgae are expensive to produce – so concerted efforts have been developed toward cost-efficient modes of mass cultivation thereof. With regard to open systems, the best choice seems to be the open shallow pond – made of leveled raceways, 2-10 m wide and 15-30 cm deep, which run as simple loops or meandering pathways; each unit may cover an area of several hundred to a few thousand square meters. However, this configuration raises several drawbacks – which restrict its use with strains that, by virtue of their weed-like behavior (e.g. Chlorella), or their ability to withstand adverse growing conditions (e.g. Spirulina or Dunaliella), can outcompete other microorganisms. Recent developments have made available technologically more advanced closed systems that provide better options for growth of virtually every microalgal strain – by protecting the culture from invasion by contaminating microorganisms, and allowing comprehensive control of processing conditions. These photobioreactors are either flat or tubular – and may adopt a variety of designs and operation modes. They lead to

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higher volumetric productivity and better quality of the generated biomass (or product) – but they are certainly more expensive to build and operate than their open counterparts [8]. Some microalgae exhibit unique productivity and plasticity: when grown under distinct sets of conditions, different products will accumulate to high levels. This observation demands attention to be paid to medium composition, processing conditions – encompassing temperature, pH, aeration, stirring and irradiance, as well as reactor configuration. Selected examples of optimum conditions of operation targeted at carotenoids are listed in Table 1.

Optimization of Carotenoids Production – by Microalgae During microalgal cultivation, a few processing parameters can be manipulated aiming at a maximum synthesis of carotenoids. The more relevant cases can be made for microalgal lutein, astaxanthin and β-carotene – which will be discussed below to some length.

Lutein The most important factors known to affect lutein content in microalgae are irradiance, pH, temperature, N availability and source, salinity (or ionic strength), and presence of oxidizing substances (redox potential); however, the prevailing specific growth rate also plays a crucial role. High temperature favours accumulation of lutein, as happens with other carotenoids (e.g. β-carotene) in Dunaliella sp. [39], close to the edge of causing environmental stress; further temperature increases would thus be harmful, and eventually decrease biomass productivity. A high irradiance level appears beneficial – but its effect depends on whether indoor or outdoor cultivation is carried out; however, it is difficult to parallel in the laboratory all parameters that characterize outdoor operation, including solar cycle and temperature fluctuation. Cultures of Murielopsis sp. and Scenedesmus almeriensis produced contradictory results, thus suggesting existence of an interaction between irradiance and temperature; it might therefore be more useful to study these factors in a combined, rather than in a separate fashion [6,46-48]. Moreover, the concentration of molecular oxygen outdoors cannot be manipulated, despite its interacting with illumination and temperature levels. Likewise, the reported effects of pH are not consistent between batch and continuous cultivations. In the former case, lutein content increased at extreme pH values, whereas the best results under continuous operation were obtained at the optimum pH for growth rate. Note that pH is particularly relevant in microalgal cultures because it also affects CO2 availability (which is essential to photosynthesis); hence, supply of CO2 continuously as a fraction of the aeration stream and pH-controlled injection lead to different outcomes. In either case, however, the maximum lutein productivity is achieved at the optimum pH for biomass productivity. The concentration of N in the culture medium, in the form of nitrate, does not apparently cause a significant effect upon lutein content of the biomass; however, N- limitation reduces biomass productivity, and consequently leads to poor lutein synthesis. Nitrate should accordingly be supplied to a moderate excess – so that growth rate is not hampered, while avoiding saline stress caused by nutrient excess that may dramatically affect performance of the culture [6]. Lutein synthesis is enhanced via addition of such chemicals as H2O2 and NaClO, which behave as inducers: in the presence of Fe+2, they affect the redox state and generate stress-


9

inducing chemical species. Such induction of oxidative stress is logical, because lutein holds a protective role derived from its antioxidant features – particularly in heterotrophic cultures, where spontaneous oxidative stress is normally absent unlike happens with phototrophic cultures [41]. Finally, the specific growth rate causes an effect, in both continuous and semicontinuous cultures: lutein tends to accumulate at low dilution rates, but not to levels that are sufficient to balance the decrease in biomass productivity under such circumstances. Therefore, the maximum lutein productivity is again attained at the optimal dilution rate for biomass production [41].

Asthaxanthin Commercial production of astaxanthin by Haematococcus sp. has been entertained by microalga companies, e.g. Cyanotech and Aquasearch; they usually resort to a two-stage system – which consists of a first step aimed at producing green biomass under optimal growth conditions (‘‘green’’ stage), and a second stage carried out by exposing the microalga to adverse environmental conditions so as to induce accumulation of astaxanthin (‘‘red’’ stage) [49]. The astaxanthin productivities in large scale facilities is ca. 2.2 mg/L [35] – yet maximum astaxanthin productivities of 11.5 mg.L-1·d-1 were attained at bench scale [50]. Micro Gaia came forward with a single-step, continuous manufacture process, under moderate nitrogen limitation [51,52]: the productivities of biomass and astaxanthin obtained were 8.0 and 0.7 mg.L-1.d-1, respectively [53]. The feasibility of the latter approach for production of astaxanthin by H. pluvialis was tested continuous-wise in outdoor apparatuses by García-Malea et al. [44]: Aquasearch Growth Modules (AGM) – i.e. 25,000 L enclosed, computerized outdoor photobioreactors, were combined up to three to obtain large amounts of clean, fast growing H. pluvialis – which was transferred daily to a pond culture system, where carotenogenesis and astaxanthin accumulation were meanwhile induced. After 5 d of synthesis, cells were harvested by gravitational settling – and contained ca. 2.5 %(w/wDW) astaxanthin; a high pressure homogenizer was used to break the cells open, and the disrupted biomass was then dried to less than 5 %(w/w) moisture utilizing a proprietary drying technology. The photobioreactor R&D program implemented improved the performance of AGM by a factor of 2, within the first 9 mo of operation: and the biomass concentration increased from 50 to 90 g·m-2, with the associated and productivity increasing from 9 to 13 g m-2d-1 within the same period [35]. The production capacity of H. pluvialis was however hindered by its intrinsic slow growth, low cell yield, ease of contamination by bacteria and protozoa, and particular susceptibility to adverse conditions [3]. Moreover, H. pluvialis cannot grow efficiently in dark heterotrophic culture mode – so production of astaxanthin should adopt the photosynthetic mode, and thus resort to levels of irradiance (e.g. 950 µmol·m-2·s-1) that are well beyond what would be economically justified [35,43]. Owing to its ease of culturing and high tolerance to environmental fluctuations, C. zofingiensis (another green microalga) has been hypothesized as an alternative for astaxanthin production: it grows rather fast (ca. 3 times faster than H. pluvialis), and accumulates in the dark significant amounts of secondary carotenoids, thus facilitating large-scale cultivation in denser cultures [43,54]. Oxidative stress by intense illumination has been shown to play an important role in inducing synthesis of astaxanthin [55]; apparently, active oxygen molecules generated by excess photooxidation caused by high light irradiance trigger synthesis of carotenoids as part

10


of a strategy aimed at cell protection against oxidative damage [43]. Continuous illumination, rather than light/dark periodic illumination cycles have been proven more favourable for astaxanthin accumulation in H. pluvialis [56] – with light quality being more important than light intensity [57]. However, the effect of irradiance depends also on other operating variables – viz. culture density, cell maturity (flagellates are much more sensitive than palmelloids), medium nutrient profile and light path [58]. The predominant role of light stress and nitrogen deprivation towards induction and enhancement of biosynthesis in the aplanospores of H. pluvialis was originally suggested in the 1950s [59]; astaxanthin accumulation comes along with of growth halting, as in almost all other cases of microalgal stress [58,60]. Imamoglu et al. [53] (2009) compared the effect of various stress media, under high light intensities, upon astaxanthin accumulation; those authors concluded that addition of CO2 in an N-free medium, under 546 μmolphotonm−2s−1, were the best conditions for accumulation of astaxanthin up to 30 mgg-1. Astaxanthin may thus be efficiently produced outdoors in continuous mode, if accurate nitrate dosage is provided [44]; besides N, such oligoelements as iron play a role. This is indeed one of the most essential elements for microalgae, as it takes part in assimilation of nitrate and nitrite, deoxidation of sulphate, fixation of N, and synthesis of chlorophyll – among other biological synthesis and degradation reactions [61-63]. Iron deficiency was found to constrain microalga growth even in the presence of rich nutrient media [64], and its addition promoted astaxanthin formation [65-68]. Furthermore, Cai et al. [69] tested how different iron electrovalencies and counter ions affect cell growth and accumulation of astaxanthin; 18 μmol L-1 Fe2+-EDTA stimulated synthesis of astaxanthin more effectively, up to contents of 30.70 mg g-1; and despite the lower cell density attained (2.3×105 cell/ml), a higher concentration (36 μmol L-1) of FeC6H5O7 yielded cell density and astaxanthin production levels that were 2- and 7-fold those of its iron-limited counterpart. In the “red stage” of growth, Haematococcus cells require only C as major nutrient – which is usually supplied via direct injection of CO2 into the photobioreactor during daylight [60]. Furthermore, high irradiance provides more energy for photosynthetic fixation of C that leads to a higher rate of astaxanthin synthesis [70] – which may be further enhanced by high C/N ratios [71]. Finally, Chen et al. [72] investigated heterotrophic conditions – using pyruvate, citrate and malate as substrates, towards synthesis of astaxanthin by C. zofingiensis in the absence of light. Presence of either of those substrates above 10 mM stimulated biosynthesis of astaxanthin and other secondary carotenoids; ca. 100 mM pyruvate permitted yields of astaxanthin of 8.4-10.7 mg.L-1, which account for an increase of 28 %.

β-Carotene Semicontinuous cultivation of D. salina at 25 °C was found to lead to 80 g m-3d-1 biomass [39] – from which 1.25 mg L-1 of β-carotene could be recovered [73]. Hejazi et al. [74] obtained 2.45 mg.m-3.d-1 in continuous biphasic bioreactors. The maximum levels of βcarotene and vitamin C in Dunaliella tertiolecta were obtained when urea was used as nitrogen source, yet a minimum for vitamin E content was simultaneously observed [75]; the maximum cellular density achieved was 12.5x106 cells mL-1, under 6.51 Lairmin-1 L-1 – but only 7x106 cells mL-1 could be reached in the absence of aeration and supply of CO2, at optimum pH [76]. Under photoheterotrophic cultivation, a significant increase of cellular β-


11

carotene content was observed: the maximum attained was 70 pg cell-1 in a culture enriched with 67.5 mM acetate and 450 μM FeSO4 [28]. As already discussed for astaxanthin, Fe2+ plays an important role in β-carotene accumulation in D. salina; by inducing oxidative stress, those cations stimulate β-carotene synthesis, especially in the presence of a C source. UV-A radiation (320-400 nm) added to the photosynthetically active radiation (PAR, i.e. 400-700 nm) can be regarded as another stress factor during growth of, and carotenoid accumulation by D. bardawil – e.g. when using an air-fluidized bed photobioreactor. Compared with cultures exposed to PAR only, addition of 8.7 W·m−2 UV-A radiation to 250 Wm−2 PAR stimulated long-term growth of D. bardawil, coupled with a 2-fold enhancement in β-carotene accumulation by 24 d [34].

FINAL CONSIDERATIONS Carotenoid production appears to be one of the most successful branches in biotechnological applications of microalgae. The rising market demand for natural pigments has promoted efforts to enhance production of carotenoids from biological sources; and largescale cultivation of microalgae for synthesis of such compounds will likely benefit from biotechnological improvements, with expected decreases in production costs. The recognized therapeutic value of some carotenoids (especially lutein) in prevention and treatment of degenerative diseases opens new avenues for development of mass production systems. Therefore, the future of microalgal biotechnology for carotenoid production appears promising, and innovative processes and products are likely in the coming years. Advances in the knowledge of the underlying physiology, biochemistry and molecular genetics of carotenoid-producing microalgae will also have a major impact upon development of this (and alternative) microalga-based technologies. In this regard, the genes encoding enzymes that are directly involved in specific carotenoid syntheses need to be investigated – so that further development of transformation techniques will permit considerable increase of the carotenoid cellular content, and accordingly contribute to increase the volumetric productivities of the biological processes.

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