phototrophic pigment production with microalgae: biological constraints...

REVIEW

PHOTOTROPHIC PIGMENT PRODUCTION WITH MICROALGAE: BIOLOGICALCONSTRAINTS AND OPPORTUNITIES1

Kim J. M. Mulders2

Bioprocess Engineering, AlgaePARC, Wageningen University, P.O. Box 8129, Wageningen, 6700 EV, The Netherlands

FeyeCon Development & Implementation, Rijnkade 17a, Weesp, 1382 GS, The Netherlands

Packo P. Lamers, Dirk E. Martens, and Ren�e H. Wijffels

Department of Agrotechnology and Food Sciences, Bioprocess Engineering, Wageningen University, P.O. Box 8129, Wageningen,

6700 EV, The Netherlands

There is increasing interest in naturallyproduced colorants, and microalgae represent abio-technologically interesting source due to theirwide range of colored pigments, includingchlorophylls (green), carotenoids (red, orange andyellow), and phycobiliproteins (red and blue).However, the concentration of these pigments,under optimal growth conditions, is often too lowto make microalgal-based pigment productioneconomically feasible. In some Chlorophyta (greenalgae), specific process conditions such asoversaturating light intensities or a high saltconcentration induce the overproduction ofsecondary carotenoids (b-carotene in Dunaliellasalina (Dunal) Teodoresco and astaxanthin inHaematococcus pluvialis (Flotow)). Overproduction ofall other pigments (including lutein, fucoxanthin,and phycocyanin) requires modification in geneexpression or enzyme activity, most likely combinedwith the creation of storage space outside of thephotosystems. The success of such modificationstrategies depends on an adequate understanding ofthe metabolic pathways and the functional roles ofall the pigments involved. In this review, thedistribution of commercially interesting pigmentsacross the most common microalgal groups, theroles of these pigments in vivo and theirbiosynthesis routes are reviewed, and constraintsand opportunities for overproduction of bothprimary and secondary pigments are presented.

Key index words: in vivo roles; metabolism; microal-gae; pigment biosynthesis; pigment distribution;pigment overproduction; pigments

Abbreviations: Chl, chlorophyll; PETC, photosyn-thetic electron transport chain; ROS, reactive oxy-gen species

To make food attractive for consumption, colo-rants are often added. These colorants can be pro-duced artificially or naturally. The demand fornatural colorants is rising because artificial food colo-rants are increasingly associated with a number ofhealth issues, such as attention deficit hyperactivitydisorder and hyperactivity (Rowe and Rowe 1994,Schab and Trinh 2004, Kobylewski and Jacobson2010). Current numbers as well as future predictionsof annual pigment production and sales volume havebeen scarcely reported in the public domain. How-ever, the numbers available show an increase in over-all pigment sales volume for b-carotene andastaxanthin, indicating a shift from chemical to natu-ral production (BCC Research 2008, Farre et al.2010). Markou and Nerantzis (2013) estimated anannual hypothetical pigment production of 525 kt b-carotene, 525 kt astaxanthin, 87.5 kt lutein, and2,625 kt phycobilins based on an annual biomassproduction of 175 Mt, of which 10% would be usedfor each pigment, and a pigment content of 3% b-carotene, 3% astaxanthin, 0.5% lutein, and 15% phy-cobilins. Natural food colorants can be obtainedfrom vegetables and fruits or they can be producedby microorganisms such as microalgae. Microalgaeare a biotechnologically interesting source of naturalcolorants because they (i) possess a wide range ofpigments; (ii) grow rapidly; (iii) may contain pig-ments in concentrations considerably exceedingthose found in higher plants and (iv) can often growin marine environments (Wright and Jeffrey 2006,Lamers et al. 2008).Microalgal pigment production requires a produc-

tion strain and an accompanying production pro-cess. To select a suitable production strain, suitablecultivation conditions, and a strategy for strainimprovement, knowledge is required of the variouspigments present in the microalgae with respect totheir in vivo roles and the metabolic pathwaysinvolved in their synthesis. A considerable body of

1Received 21 May 2013. Accepted 22 October 2013.2Author for correspondence: e-mail [email protected] Responsibility: R. Bassi (Associate Editor)

J. Phycol. 50, 229–242 (2014)© 2014 Phycological Society of AmericaDOI: 10.1111/jpy.12173

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knowledge is available regarding the pigment com-position of specific microalgae cultivated underdefined culture conditions (see examples inTable 1), the pigments present in certain groups ofmicroalgae (Schagerl and Donabaum 2003, Schagerlet al. 2003, Jeffrey and Wright 2005, Wright andJeffrey 2006), the roles of groups of pigments invivo (Guedes et al. 2011), and pigment biosynthesisroutes (Brown et al. 1990, Von Wettstein et al. 1995,Cunningham and Gantt 1998, Ladygin 2000, G�alov�aet al. 2008, Bertrand 2010, Takaichi 2011). How-ever, a clear overview integrating all relevant infor-mation is lacking.

The aim of this work was to identify the biologicalconstraints and opportunities for the production ofpigments using phototrophic microalgae (Section 6and 7) and to provide an overview of the relevantbiological information currently available (Section2–5). An adequate understanding of these opportu-nities and constraints is important for the design ofan economically feasible microalgae-based pigmentproduction process.

MICROALGAL PIGMENTS

The pigments present in microalgae and cyano-bacteria are grouped into chlorophylls (chls), carot-enoids, and phycobiliproteins. The differencesbetween these groups are related to their chemicalstructure, as shown in Figure 1. Chls, which aredefined as tetrapyrroles, consist of a large aromaticring, the chlorin, which contains four pyrrole ringssurrounding a magnesium ion, and a hydrocarbontail is typically attached to the chlorin. Carotenoidsconsist of a single long hydrocarbon chain made upof eight isoprene units. Phycobiliproteins consist oftwo parts, a bilipigment known as phycobilin and aprotein, which are covalently attached to each othervia a cysteine amino acid. Phycobilins contain build-ing blocks similar to those of chlorins, but insteadof a closed ring, these components form an openlinear structure. Therefore, phycobilins are referredto as open tetrapyrroles.

These pigment groups all share a large conju-gated system of double bonds (indicated in red inFig. 1). Following excitation of the delocalizedelectrons within these bonds, light of specificwavelengths is absorbed. Light that is not absorbedis reflected, which gives these pigments their charac-teristic colors.

There are several different types of chl (chl a, b,c, d, and f; Chen et al. 2010), although all of thesepigments are green. In each type of chl, the mole-cules attached to the chlorin are slightly different;furthermore, chl c lacks the hydrocarbon tail. Thesesmall differences lead to differences in the absorp-tion spectrum and therefore tonality, as chl aappears blue-green, chl b brilliant green, chl c yellowgreen, chl d brilliant/forest green, and chl f emer-ald green (indicated in Fig. 2 and 4) (Chen et al.

TABLE 1. Examples of strains for which the pigment com-position is described in detail.

Strain Source

Chlorophyta (green algae)Dunaliella salina (Dunal) Teodoresco Lamers et al. 2010Haematococcus pluvialis (Flotow) Orosa et al. 2001Coelastrella striolata var. multistriata(Trenkwalder) Kalina andPuncoch�arov�a

Abe et al. 2004

Coccomyxa acidophila Casal et al. 2010Muriellopsis sp. Del Campo et al.

2000Chlorella zofingiensis (D€onz) Del Campo et al.

2000Ip et al. 2004Orosa et al. 2000

Neochloris wimmeri (Hilse) Archibaldand Bold

Orosa et al. 2000

Scenedesmus vacuolatus (Shihira andKrauss)

Orosa et al. 2000

Scotiellopsis oocystiformis (Lund)Puncoch�arov�a and Kalina

Orosa et al. 2000

Protosiphon botryoides (K€utzing)Klebs

Orosa et al. 2000

Trentepohlia aurea (Linnaeus)Martius

Mukherjee et al.2010

Trentepohlia cucullata (De Wildeman) Mukherjee et al.2010

Chlamydomonas nivalis (Bauer)Wille

Remias et al. 2005

Rhodophyta (red algae)Audouinella eugenea (Skuja) Jao Bautista and

Necchi 2007Audouinella hermannii (Roth) Duby Bautista and

Necchi 2007Compsopogon coeruleus (Balbis exAgardh) Montagne

Bautista andNecchi 2007

Cyanidioschyzon merolae (De Luca,Taddei and Varano)

Cunningham et al.2007

Cyanophyta (blue-green algae)Arthrospira platensis (Nordstedt)Gomont

Abd El-Baky 2003

Arthrospira maxima (Setchell andGardner)

Abd El-Baky 2003

Anabaena azollae (Strasburger) Venugopal et al.2006

Oscillatoria redekei (van Goor) Wyman and Fay1986

Gloeotrichia echinulata (Smith)Richter

Wyman and Fay1986

Oscillatoria agardhii (Gomont) Wyman and Fay1986

Bacillariophyta (diatoms)Phaeodactylum tricornutum (Bohlin) Nymark et al. 2009

Dinophyta (dinoflagellates)Gambierdiscus toxicus (Adachi andFukuyo)

Indelicato andWatson 1986

EuglenophytaEuglena sanguinea (Ehrenberg) Grung and

Liaaen-Jensen1993

HaptophytaDiacronema vlkianum (Prauser) Durmaz et al. 2009Isochrysis galbana (T-iso) Mulders et al.

2012EustigmatophytaNannochloropsis oculata (Droop)Hibberd

Lubian et al. 2000

Nannochloropsis salina (Hibberd) Lubian et al. 2000Nannochloropsis gaditana (Lubi�an) Lubian et al. 2000

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2010, Roy et al. 2011). Upon loss of the magnesiumion, chls become pale and dusky colored (Hum-phrey 2004).

Carotenoids are distinguished by the presence ofdifferent end groups, although these groups canoccasionally be attached to the center part of themolecule. Carotenoids are divided into carotenes,which are true hydrocarbons, and xanthophylls,which additionally contain oxygen atoms. Carote-noids appear yellow to red in color (individualcolors are indicated in Figs. 2–4).

The protein-bound phycobilins reported to dateinclude phycocyanobilin (blue), phycoerythrobilin(red), phycoviolobillin (purple), and phycourobilin(yellow; Sidler 1994), and these are structurally dis-tinguished by only small differences in the groupsattached to the pyrrole rings. Nevertheless, thesesmall structural differences have a significant effecton the length of the conjugated systems, causing

differences in color. The phycobilins most com-monly found in microalgae are phycocyanobilin andphycoerythrobilin. Phycocyanobilin is the majorcomponent of the phycobiliproteins phycocyanin(deep blue) and allophycocyanin (light blue),whereas phycoerythrobilin is the major componentof phycoerythrin (orange-red). Phycobiliproteins areusually found in pigment aggregates called phyco-bilisomes, which contain hundreds of protein-boundphycobilins (Blot et al. 2009). The close distancebetween the phycobilins causes interactions betweenchromophores, and because the orientations of phy-cocyanin and allophycocyanin in the phycobilisomeare different, their color shades are slightly differ-ent, as indicated above (Szalontai et al. 1994). Fordetailed information regarding the structure of pro-tein-bound phycobilins and phycobilisomes, refer tothe reports by Grossman et al. (1993), Sidler(1994), and Blot et al. (2009).

FIG. 1. Molecular structures ofthe pigments in microalgae andcyanobacteria, emphasizing (inred) the conjugated system ofdouble bonds. (a) Chl a, rep-resenting the chls; (b) b-carotene,representing the carotenoids; (c)peptide-linked phycocyanobilin,representing the phycobiliprote-ins.

FIG. 2. Simplified scheme of the pigment biosynthesis pathways of cyanobacteria and microalgae. Solid arrows represent single conver-sion steps, dashed arrows represent lumped reactions (based on Falkowski and Raven (2007) and Kanehisa Laboratories (2011)), and dot-ted arrows represent hypothesized pathways (according to Beale (1999), Lohr and Wilhelm (1999, 2001) and Wang and Chen (2008)).The arrows containing a question mark represent unresolved pathways. Pigment colors are indicated as described by Jeffrey et al. (1997)and Lee (2008). Note that the violaxanthin cycle is also referred to as the xanthophyll cycle.

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BIOSYNTHESIS OF PIGMENTS

In addition to light, which is the main energysource of microalgae, chemicals including water,carbon dioxide, inorganic nitrogen (ammonia ornitrate), and phosphate are required for photoauto-trophic growth. Figure 2 provides a schematic over-view of the pathways through which pigments areformed from these nutrients (note that most micro-algae groups contain only some of these pathways;see Section “Distribution of pigments”).

Nitrogen and carbon are incorporated into proto-porphyrin IX (purple-green arrow), which consistsof a large aromatic ring (tetrapyrol). This ring canbe opened by oxidation reactions resulting in theformation of biliverdin. Biliverdin is subsequentlyreduced to form hydrophilic phycobilins (purplearrows), which are coupled to specific proteins (datanot shown). For a more detailed overview of phyco-bilin synthesis, refer to the report of Beale (2004).In a different metabolic process, carbon is chan-neled toward geranyl-geranyl-PP (green-orangearrow), which can subsequently be reduced to formphytyl-PP. Upon release of the phosphates,

phytyl-PP is attached to a derivative of protoporphy-rin IX, which contains a chelated magnesium ion.This leads to the formation of chls (green arrows).As stated in Section 2, chl c lacks the phytol tail,and it remains unclear whether this tail is attachedand released in subsequent steps or not attached atall (Beale 1999). For a more detailed overview ofchl synthesis, refer to the reports of Lohr et al.(2005) and Czarnecki and Grimm (2012).Apart from its role as a chl precursor, geranyl-gera-

nyl-PP is also the precursor of carotenoids (orangearrows). Condensation of two molecules of geranyl-geranyl-PP followed by an isomerization reactionand the introduction of four double bonds yieldsthe carotenoid lycopene. After formation of lyco-pene, the biosynthetic pathway splits into twobranches. One branch leads to the production of yel-low lutein, which in some species, including theChlorophyte Chlamydomonas reinhardtii (Dangeard),can be converted into loroxanthin (data not shown;Baroli et al. 2003). The other branch leads to theproduction of orange b-carotene, which also repre-sents a branch point. From b-carotene, one branchleads to the generation of red astaxanthin via twooxidation and two hydroxylation reactions. Thishydrophobic carotenoid can be esterified once ortwice with a fatty acid, leading to a more lipophilic as-taxanthin mono- or diester. In Haematococcus pluvialis,for example, esters containing the fatty acids C18:1and C20:0 are predominantly found (Goswami et al.2010). The other branch from b-carotene leads to thegeneration of orange zeaxanthin, and it has beenproposed by Wang and Chen (2008) and Corderoet al. (2011) that, in the species Chlorella zofingiensis,zeaxanthin can be converted into astaxanthin.Zeaxanthin is part of the violaxanthin cycle, as

shown in Figure 2. The violaxanthin cycle can alsobe referred to as the xanthophyll cycle. To preventconfusion between the violaxanthin cycle and thediatoxanthin cycle (described further on), whichboth involve xanthophylls, the term violaxanthincycle instead of xanthophyll cycle will be used here.Zeaxanthin is turned into antheraxanthin andsubsequently into violaxanthin, which both appearyellow in color, following two epoxidation reactions.These reactions are induced by subsaturating lightconditions, while the reverse reactions are inducedby oversaturating light conditions. Violaxanthin canalso be isomerized, giving rise to neoxanthin.The metabolic routes described above have been

studied intensively, in contrast to the pathways lead-ing to diatoxanthin, dinoxanthin, and fucoxanthin(indicated by the dotted lines in Fig. 2). Accordingto Lohr and Wilhelm (1999), violaxanthin is con-verted into diadinoxanthin under prolonged subsat-urating light conditions, and this conversion likelyrequires more than one conversion step. Further-more, these authors have suggested that diadinoxan-thin is further metabolized into fucoxanthin, whichalso most likely requires multiple conversion steps.

FIG. 3. Main roles of pigments in cyanobacteria and microal-gae. Light harvesting pigments transfer light energy to the photo-synthetic electron transport chain (PETC) where it is convertedinto chemical energy (thin orange arrows directed to the right).The cell can be damaged due to the formation of triplet excitedchl a, which can convert molecular oxygen into reactive oxygenspecies (red arrows). Photoprotective pigments prevent cell dam-age by converting excess energy into heat. E Phycoerythrin,C Phycocyanin, A Allophycocyanin, a Chl a, b Chl b, c Chl c, AsAstaxanthin, b b-carotene, Z zeaxanthin, An Antheraxanthin, V vi-olaxanthin, Dt Diatoxanthin, Dd Diadinoxanthin, L Lutein,N Neoxanthin, F Fucoxanthin, P peridinin (Sukenik et al. 1992,Lohr and Wilhelm 1999, Jin et al. 2003, Falkowski and Raven2007, Lee 2008, Telfer et al. 2008, Balashov et al. 2010).


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Moreover, according to the findings of Lohr andWilhelm (1999), oversaturating light intensitiesinduce the de-epoxidation of diadinoxanthin toform diatoxanthin. This xanthophyll is epoxidatedagain under subsaturating light intensities, and theinterconversion of diadinoxanthin and diatoxanthinis termed ‘the diatoxanthin cycle’ (Fig. 2).

The biosynthesis pathways leading to dinoxanthinand peridinin, which are exclusively found in theperidinin-containing Dinophyta, remain unresolved(indicated by the question marks). However, Lohrand Wilhelm (1999) demonstrated that peridinin isanother derivative of zeaxanthin.

ROLE OF PIGMENTS IN VIVO

Light can have both positive and negative effectson microalgae; for example, light not only forms

the primary source of energy, driving all biochemi-cal processes, but also causes lethal damage whenpresent in excessively large quantities. To survivethe varying circumstances of subsaturating and over-saturating light conditions, photosynthetic organ-isms contain pigments with two distinct roles: lightharvesting and photoprotection. Certain pigmentscan perform both roles (Fig. 3), depending on theprotein subunit or subdomain to which they arebound (Ballottari et al. 2012, Berera et al. 2012).Light harvesting pigments are subdivided into pri-mary light harvesting pigments and accessory lightharvesting pigments, whereas photoprotective pig-ments are divided into three groups, which can havea filtering, quenching, and/or scavenging role.In addition to the distinction between the light

harvesting and photoprotective roles of pigments,distinction is also made between primary and

FIG. 4. Distribution of photo-synthetically important orcommercially applied pigmentsacross the most common microalgalgroups (the Chlorarachniophyta,descendants of the Chlorophyta,and the Apicomplexa are notincluded). Only pigments presentin significant amounts are shown(i.e., trace pigments are excluded).The microalgal groups are drawnaccording to their evolutionaryrelationships based on the hypo-thesis that Green dinophyta, andBilin- and Fucoxanthin-containingdinophyta obtained their plastidsvia secondary replacements andtertiary endosymbiosis, respectively(Jeffrey and Wright 2005, Lee 2008,Takaichi 2011, Vesteg et al. 2008).Pigment colors are indicated asdescribed by Jeffrey et al. (1997)and Lee (2008).


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secondary pigments. Primary pigments are definedas those that are functionally and structurally boundto the photosynthetic apparatus, whereas secondarypigments are those that are not functionally orstructurally bound to the photosynthetic apparatus(Lemoine and Schoefs 2010). According to this defi-nition, primary pigments include all pigments witha light harvesting role as well as photoprotective pig-ments with a quenching and scavenging role,whereas secondary pigments include only photopro-tective pigments with a filtering role.

In this section, the roles of different pigments arefurther defined, and the specific pigments playingthese roles in microalgae and cyanobacteria areidentified.

As indicated in Figure 3, light harvesting pigmentstransfer light energy to the photosynthetic electrontransport chain (PETC), where it is converted intochemical energy (arrows to the right). The primarylight harvesting pigment, chl a, is able to transfer theexcitation energy directly to the PETC, whereas theaccessory light harvesting pigments transfer theirexcitation energy via chl a (Lee 2008). The accessorylight harvesting pigments include all chls, all phyco-biliproteins (Lee 2008) and the carotenoids neoxan-thin (Dall’Osto et al. 2006), fucoxanthin, peridinin(Lohr and Wilhelm 1999), and echinenone (data notshown; Balashov et al. 2010). In addition, violaxan-thin acts as an accessory pigment in some microalgaespecies (Sukenik et al. 1992). Lutein is a semiphoto-synthetic pigment; it can transfer excitation energy tochl a, but does so with very low efficiency (indicatedby the dashed line between lutein and chl a, in Fig. 3;Falkowski and Raven 2007). Light harvesting pig-ments are located in photosystems, with the primarylight harvesting pigment located closest to the innerpart of the photosystem and the accessory light har-vesting pigments located closest to the outside. Acces-sory pigments are either interwoven with proteinsinto chl-containing pigment-protein complexes orstick out of the system to form antennae. To transfertheir excitation energy efficiently, the accessory pig-ments need to be in very close proximity to the pri-mary light harvesting pigment (Telfer et al. 2008).

When the energy transfer rate to chl a (arrowstoward chl a) exceeds the energy transfer rate fromchl a to the PETC (bold arrow toward the PETC), chla enters a triplet excited state (large red arrow). How-ever, this process depends on the supply rate ofenergy, i.e., the light intensity perceived by the cell,as well as the rate at which the PETC operates. Theoperation rate of the PETC depends on environmen-tal conditions influencing the cell’s metabolism, suchas salinity, pH, temperature, and nutrient availability,as well as species-specific characteristics. By itself,overexcited chl a is not harmful. However, in thisstate, it can pass its energy on to molecular oxygen(small red arrow from overexcited chl a), changing itfrom a ground state into a reactive oxygen species(ROS), such as singlet oxygen (black arrow). ROS are

highly reactive and therefore very toxic, and they candamage cell components including DNA, proteins,and membrane lipids (small red arrow pointing fromsinglet oxygen; Telfer et al. 2008).This phenomenon, known as photoinhibition, is

minimized by photoprotective pigments, which turnexcess energy into heat (arrows to the left). As pre-viously mentioned, the photoprotective pigmentscan be divided into three different groups, whichhave either a filtering, quenching, or scavengingrole. Pigments with a filtering role prevent the for-mation of overexcited chl a by absorbing harmfulradiation (bold orange arrows). These pigments arenot bound (functionally or structurally) to the pho-tosynthetic apparatus, which makes them secondarypigments; instead, they are found physically sepa-rated from the photosynthetic apparatus, usually inoil droplets in the chloroplast’s stroma or in thecytosol (Jin et al. 2006). Because the currentlyknown secondary pigments include only carote-noids, namely astaxanthin and b-carotene, they arealso referred to as secondary carotenoids (Telferet al. 2008).Pigments with a quenching role prevent the for-

mation of ROS by quenching the energy of tripletexcited chl a (blue arrows from triplet excited chla) or singlet excited chl a (data not shown). Themechanism by which energy is quenched is referredto as nonphotochemical quenching. Similar to theaccessory light harvesting pigments, quenching pig-ments must be very close to chl a to perform theirrole. Astaxanthin, b-carotene, and lutein act asquenchers. Compared to astaxanthin and b-caro-tene, lutein quenches overexcited chl a slightly lessefficiently (Telfer et al. 2008). In addition, as shownin Figure 3, the xanthophylls zeaxanthin and viola-xanthin, which belong to the violaxanthin cycle, areinvolved in the effective quenching of chl a (Gossand Jakob 2010, Jahns and Holzwarth 2012). How-ever, the xanthophylls diadinoxanthin and diatoxan-thin are even more effective quenchers and areinvolved in the diatoxanthin cycle (Telfer et al.2008). Although not completely understood, referto the studies of Latowski et al. (2004), Goss andJakob (2010), Jahns and Holzwarth (2012) for moredetailed explanations of the molecular quenchingmechanisms involved in the violaxanthin and diato-xanthin cycles.Pigments with a scavenging role, including asta-

xanthin, b-carotene, lutein, and neoxanthin (Telferet al. 2008), prevent cell damage by reacting withROS such as singlet oxygen (dark blue arrows point-ing from singlet oxygen). The study by Jin et al.(2003) suggested that zeaxanthin, when interwovenwithin the lipid bilayer, could also perform the roleof scavenger, thereby preventing damage of diacylgly-cerides. Protective carotenoids perform this rolemost effectively when they are near the site of ROSformation, which is located inside the photosyntheticapparatus in proximity to chl a (Telfer et al. 2008).


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In addition to their light harvesting and photo-protective roles, carotenoids also have a structuralrole. The shape and lipophilicity of carotenoidsmakes them fit perfectly into or as bridges acrossthe lipid bilayer of membranes, thus affecting mem-brane properties (Latowski et al. 2004). However,the concentration of carotenoids in the membraneis low, and no studies to date have shown that theirstructural role in vivo is of physiological significance(Telfer et al. 2008).

Finally, carotenoids can also act as electron sinksand play roles in carbon and energy storage, as dis-cussed for astaxanthin by Lemoine and Schoefs(2010). However, the precise roles of carotenoidsremain a matter of debate.

Together, these findings indicate that a singletype of pigment can play several roles in vivo, withits particular role connected to its location. Forexample, when located in the thylakoids, b-caroteneand astaxanthin function as primary pigments; how-ever, if these compounds are located in oil dropletsseparated from the photosynthetic apparatus, theyfunction as secondary pigments. Furthermore, someroles can be performed by a single pigment (e.g.,transferring light energy into the PETC), whileother roles are performed by the combined actionsof several pigments. As will be discussed in Section“Pigment production: biological constraints andopportunities”, detailed knowledge of the roles ofpigments in microalgae is essential for the develop-ment of a successful strategy to overproduce pig-ments.

DISTRIBUTION OF PIGMENTS

In Section “Biosynthesis of pigments”, the biosyn-thetic pathways of pigments in microalgae andcyanobacteria were discussed. As stated, most micro-algae groups contain only a subset of the pigmentspresented. The aim of this section was to discusswhich pigments are present in the most commongroups of microalgae and cyanobacteria; thus, onlypigments found in significant quantities areincluded, whereas pigments present in traceamounts are excluded. First, the microalgae groupsdiscussed in this section are considered from anevolutionary perspective.

It is generally accepted that the Chlorophyta,Rhodophyta, and Glaucophyta arose from theendosymbiosis of Cyanophyta (which are in factphotosynthetic bacteria) and nonphotosyntheticeukaryotic host cells (Vesteg et al. 2009), as shownin Figure 4. Further down the phylogenetic tree,the relationships between groups become more dis-puted. For example, it remains unclear whether thegroups possessing plastids of red microalgae originall share a common ancestor (Sanchez-Puerta andDelwiche 2008).

In particular, the origin of the Dinophyta remainsunclear. On the one hand, all Dinophyta could be

grouped together based on similarities in morphol-ogy (one transverse and one longitudinal flagellumand a distinct layer beneath the cell membrane).On the other hand, the Dinophyta could be dividedinto different groups, as shown in Figure 4, basedon the pigments they possess. Very likely, the peridi-nin-containing Dinophyta arose by secondary endo-symbiosis of a red microalga (i.e., a red microalga,which was the product of primary endosymbiosis,was itself engulfed and retained by another free-liv-ing eukaryote), while the green Dinophyta obtainedtheir plastids via secondary replacements (i.e.,replacement of algal-derived plastids by otheralgal-derived plastids), and the billin- and fucoxan-thin-containing Dinophyta obtained their plastidsvia tertiary endosymbiosis (i.e., products of second-ary endosymbiosis, such as Cryptophyta and Hap-tophyta (see Fig. 4), were themselves engulfed andretained by other free-living eukaryotes). However,it has also been suggested that all dinophyta aroseby red microalgae endosymbiosis (Yoon et al. 2002,Palmer 2003). For more detailed informationregarding the evolutionary history of microalgalplastids, refer to the reports from Palmer (2003),Keeling (2004), Sanchez-Puerta and Delwiche (2008),and Vesteg et al. (2009) and Lohr et al. (2012).The phycobiliproteins phycoerythrin, phycocya-

nin, and allophycocyanin are found in Cyanophyta,Rhodophyta (Wright and Jeffrey 2006), and Glauco-phyta (Lee 2008). In these groups, the phycobilipro-teins are located in phycobilisomes on the outersurface of the thylakoid membrane. Moreover, phy-coerythrin and phycocyanin are also found in Cryp-tophyta and bilin-containing Dinophyta. In thosegroups, the phycobiliproteins are located within thethylakoid lumen in an unresolved ultrastructure (assoluble heterodimers, tethered to the membranesor otherwise possibly arranged into some type ofantenna complex; Jeffrey and Wright 2005, Mirkovicet al. 2009).Chl a, which is the primary light harvesting pig-

ment, is found in all photosynthetic organisms. Incontrast, chl b is exclusively found in the Chloro-phyta and their descendants. Similarly, chl c isexclusively found in the descendants of the Rhodo-phyta (Jeffrey and Wright 2005).b-carotene, the carotenoid most frequently used

in foods and supplements, is also frequently foundin cyanobacteria and microalgae. With the excep-tion of the Cryptophyta and bilin-containing Dino-phyta, this pigment is found in all of the groupsshown in Figure 4. Although b-carotene is widelydistributed, overproduction as a secondary pigmentis observed only in a select number of Chlorophyta.In contrast, astaxanthin is exclusively found in the

Chlorophyta. Similar to b-carotene, a select numberof Chlorophyta are also able to overproduce thispigment. The precursor of astaxanthin and canta-xanthin is found in some Chlorophyta, as well as insome Cyanophyta.


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Similarly, lutein is exclusively found in Chloro-phyta and in some green Dinophyta. In some of theChlorophyta, this yellow pigment can be convertedinto loroxanthin (Jeffrey and Wright 2005).

Fucoxanthin is found in the Haptophyta, thefucoxanthin-containing Dinophyta, the Chrysophyta,and the Bacillariophyta; the latter two classes belongto a group referred to as the Heterokontophyta.

Peridinin, complexed with chl a, is found exclu-sively in peridinin-containing Dinophyta (Schulteet al. 2010). In addition, dinoxanthin is also foundin these Dinophyta.

Although zeaxanthin is present in the Cyanophtyaand the Rhodophyta, these phycobilisome-contain-ing organisms do not display the violaxanthin ordiatoxanthin cycle (Falkowski and Raven 2007). Incontrast, most of the groups descended from theRhodophyta perform either violaxanthin or diato-xanthin cycling. For instance, zeaxanthin is foundin the Chrysophyta, which further contain anthera-xanthin and violaxanthin or diadinoxanthin anddiatoxanthin (Jeffrey and Wright 2005). In addition,Lohr and Wilhelm (1999) reported that microalgaedisplayed both the violaxanthin cycle and the diato-xanthin cycle when exposed to prolonged subsatu-rating light conditions. These authors intensivelystudied Phaeodactylum tricornutum, which belongs tothe Bacillariophyta, but they found similar resultsfor species belonging to the Haptophyta and theDinophyta. Furthermore, the violaxanthin cycle isalso utilized by the Chlorophyta (Wright and Jeffrey2006) and most likely by the green Dinophyta, asthese groups both possess zeaxanthin and violaxan-thin.

As previously stated, only pigments found in sig-nificant amounts are indicated as present inFigure 4, whereas untraceable intermediates such aslycopene, which could only be detected as a tracepigment in Cyanophytes (Jeffrey et al. 1997), arenot included. However, the presence of downstreammetabolites such as lutein and b-carotene indicatesthat lycopene must be present in all indicatedgroups except the bilin-containing Dinophyta,implying that once formed, lycopene is immediatelyconverted.

In conclusion, the Chlorophyta possess the largestvariety of pigments. Moreover, this is the only groupknown to contain species that overproduce second-ary carotenoids as a response to specific cultureconditions. The potential for overproducing theseand other pigments will be discussed further inSection “Pigment production: biological constraintsand opportunities”.

PIGMENT PRODUCTION: BIOLOGICAL CONSTRAINTS AND

OPPORTUNITIES

Section “Distrubution of pigments” discussed thepigment distribution across different groups of mic-roalgae and cyanobacteria. With the exception of

chls, which are usually present at a concentration of1%–2% g � g�1 dry weight (Li et al. 2008, Wang andChen 2008), most other pigments are, under opti-mal growth conditions, present in a concentrationthat is too low (i.e., below 0.5% g � g�1 dry weight)to make microalgae-based production economicallycompetitive with chemical production. To makemicroalgae-based pigment production economicallyfeasible, the cellular content must be increased.The cellular content of a desired pigment can be

increased using two different approaches: (i) byapplying specific culture conditions, which arehighly explored and exploited (Jin et al. 2006,Lamers et al. 2010, 2012) or (ii) by interfering withthe cellular metabolism, which remains unexploreddue to the lack of a sufficiently developed geneticengineering toolbox (Beer et al. 2009). In thissection, both approaches are discussed, and thepigments that can be overproduced using each ofthese approaches are indicated.The first approach to obtain an increased pig-

ment content, through the use of specific cultureconditions, can be divided into two categories: (i)the application of subsaturating light conditions,which results in a minor increase in the concentra-tion of primary pigments, and (ii) the applicationof adverse growth conditions (also referred to as“stress conditions”), which result in a major increasein the concentration of secondary pigments. Bothtypes of application are discussed below.When cells are grown under subsaturating light

conditions for a prolonged time period, they accli-mate to this light regime by increasing the numberof photosystems. As a consequence, the cellular con-tent of the primary pigments associated with thephotosystems increases as well. However, this phe-nomenon, which is referred to as photoacclimation,leads only to minor increases in the amount ofprimary pigments (Telfer et al. 2008; typically toconcentrations below 0.5% g � g�1 dry weight).These minor increases, which in the literature areoften termed “accumulation” (e.g., the accumula-tion of lutein in C. zofingiensis; Del Campo et al.2004), should not be confused with overproduction(typically to concentrations above 0.5% and up to10% g � g�1 dry weight), which is used to describemajor increases in the cellular pigment content, asdescribed in the next paragraph.Under adverse growth conditions, such as a high

salt concentration, oversaturating light intensity ornitrogen limitation, secondary carotenoids with alight filtering role (b-carotene and astaxanthin,Fig. 3) can be transported from their site of synthe-sis in the thylakoids to oil droplets located in thechloroplast stroma or the cytosol, where they accu-mulate (Jin et al. 2006). In contrast to photoaccli-mation, which is universally observed in microalgae,the accumulation of secondary carotenoids isobserved exclusively in a limited number of Chloro-phytes. The most well-known examples, which are


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well explored and exploited, include H. pluvialis(astaxanthin) and Dunaliella salina (b-carotene), andthese strains can overproduce secondary carotenoidsto reach 7.7%–10% of the cell’s dry weight (Lamerset al. 2008). In addition to these known microalgaespecies, nature may also supply more efficient,undiscovered strains with high pigment concentra-tions, such as in extreme environments that stimu-late natural antioxidative responses.

The second approach to obtain increased pig-ment content, by interfering with the cellularmetabolism, can be divided into two categories: (i)the up- or down-regulation of pigment biosyntheticenzymes and (ii) the formation of a metabolic sink.Both categories are discussed below.

Up- and down-regulation of pigment biosyntheticenzymes both aim to increase the flux to a desiredpigment by manipulating the metabolism of the pig-ment. To achieve this result, all enzymes need to beoverproduced that exert control over the fluxtoward the desired pigment, leaving all other metab-olite concentrations and most other metabolicfluxes unchanged. However, as discussed by Fell(1997), not all enzymes in the pathway need to beoverexpressed to the same degree; for example,enzymes in the final linear sequence must be over-expressed to a much higher degree than enzymes inthe preceding branches. This method could beused, for example, to overproduce the primary pig-ment lutein. In this case, the enzymes that controlthe flux from lycopene to lutein (i.e., enzymes inthe final linear sequence to lutein, Fig. 2) need tobe most highly overexpressed. To prevent adecreased flux to b-carotene and its derivatives(Fig. 2), the enzymes that control the flux from ger-anyl-geranyl-PP to lycopene may need to be overex-pressed as well. Finally, the enzymes that control thegeranyl-geranyl-PP pathway may need to be overex-pressed as well, to retain sufficient production ofchls (Fig. 2).

In addition to overproducing a single pigment,the method described above may also be used toincrease the content of multiple pigmentssimultaneously. In Arabidopsis, overexpression of 1-deoxy-D-xylulose-5-phosphate synthase, an enzymethat catalyzes one of the early steps leading to gera-nyl-geranyl-PP, resulted in up to a 150% increase inchls and carotenoids (Estevez et al. 2001). Alterna-tively, flux-controlling enzymes further down thispathway could be overexpressed, such as thoseresponsible for converting geranyl-geranyl-PP intolycopene (Fig. 2). Furthermore, Jin et al. (2006)proposed that the enzyme catalyzing the first stepsof this conversion (phytoene synthase) may controlthe flux toward the carotenoids, as this enzyme wasfound to be rate limiting in ripening tomato fruits(Bramley et al. 1992, Fraser et al. 1994), canolaseeds (Shewmaker et al. 1999), and marigold flow-ers (Moehs et al. 2001). In contrast, Lemoine andSchoefs (2010) suggested that subsequent reactions

(i.e., the conversion of phytoene into f-carotene),which are catalyzed by phytoene desaturase, areamong the rate-limiting steps of the pathway andthat up-regulation of this enzyme may lead to anincreased flux toward the carotenoids. However, asdiscussed by Kacser (1995), the flux toward a cer-tain metabolite is in general not controlled by a sin-gle enzyme, but instead is shared among multipleenzymes. Even if a single enzyme had significantcontrol over a given flux, overexpression of thisenzyme would likely result in a control shift towardother enzymes. First of all, this implies that multipleenzymes, rather than a single enzyme, must beoverexpressed to overproduce a specific pigment orpigments. Moreover, rather than focusing on a rate-limiting step, the focus should rest on the sharedcontrol of the flux toward a metabolite of interest.Similar to the up-regulation of pigment biosyn-

thetic enzymes, the down-regulation of enzymes(e.g., by adding enzyme inhibitors, by RNA silenc-ing, or by creating a gene knockout) aims toincrease the flux toward a desired pigment. In con-trast to up-regulation, down-regulation aims toincrease the desired flux by decreasing the fluxtoward side branches. For example, an enhancedflux toward lutein could be generated by blockingthe branch leading to b-carotene. However, becausethe derivatives of b-carotene are mainly primary pig-ments (Figs. 2 and 3), which are of major impor-tance for the function of the cell, this could resultin growth inhibition.Two other bottlenecks that could limit the over-

production of primary pigments are crowding andfeedback inhibition. When up- or down-regulationof pigment biosynthetic enzymes leads to the forcedoverproduction of primary pigments, the limitedamount of space within the chloroplast lumens maycause the overproduced molecules to interfere withthe photosynthetic system (crowding). Hence, theoverproduction of primary pigments, resulting fromup- or down-regulation of their biosyntheticenzymes, may lead to disrupted cell function andconsequently growth inhibition.The only pigments for which this problem may be

less severe are the phycobilins, which are located inantennae. Because the antennae are found on thestroma side of the thylakoid membrane, sufficientspace is available for them to extend. However, thisextension will most likely affect their light harvest-ing efficiency, due to the self-shading of phycobili-proteins.Another bottleneck that may limit pigment over-

production is related to the regulatory mechanismsmaintaining the constancy of metabolite concentra-tions (homeostasis), such as feedback inhibition. Bymeans of negative feedback mechanisms, which mayact on one or multiple pigment biosyntheticenzymes, accumulated pigment molecules mayinhibit further overproduction. One way to circumventfeedback inhibition is to overexpress flux-controlling


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enzymes that are feedback resistant. This approachwas used in the heterotrophic bacterium Corynebacte-rium glutamicum, in which overexpression of a feed-back-resistant kinase resulted in lysine accumulationand secretion up to 38 mM. In comparison, overex-pression of the feedback-sensitive enzyme did notresult in accumulation or secretion of lysine (Cremeret al. 1991).

Similar to the method described above, the for-mation of a metabolic sink aims to increase the fluxtoward a desired pigment. Instead of pushing, thesink pulls the metabolism in a certain direction bytransporting a metabolite away from the site of for-mation. Therefore, this approach overcomes thepossible limitations described for up- or down-regu-lation of pigment biosynthetic enzymes (feedbackinhibition and crowding). In fact, the formation ofa metabolic sink may be essential for carotenoidoverproduction. Namely, in both D. salina andH. pluvialis, inhibition of lipid accumulation, whichserves as a metabolic sink for b-carotene and asta-xanthin, was shown to abolish carotenoid accumula-tion (Rabbani et al. 1998, Zhekisheva et al. 2005).

As only a select number of Chlorophytes naturallyaccumulate pigments in oil droplets, genetic engi-neering may be applied to induce the formation ofstorage space outside the photosystem in other spe-cies. In cauliflower, a spontaneous mutation in theorange (Or) gene led to the formation of large mem-branous chromoplasts, in which high levels of b-car-otene accumulated (Li and van Eck 2007).Introduction of the Or gene to potato led to the for-mation of similar structures in which high levels ofb-carotene accumulated; thus, genetic modificationsof the carotenoid biosynthesis pathway were notrequired (Farre et al. 2010). Introduction of the Orgene, or genes with a similar function, into microal-gal cells may supply additional storage space for sec-ondary carotenoids, leading to overproduction instrains that normally do not overproduce secondarycarotenoids.

The main issue that may limit overproduction ofpigments in additionally formed storage space is thetransport of pigments out of the photosystem.Although many microalgae strains accumulate oildroplets under adverse growth conditions (e.g.,C. zofingiensis; Liu et al. 2011), Chlorella vulgaris(Beijerinck; Mutlu et al. 2011), Isochrysis galbana(T-Iso; Mairet et al. 2011), and Neochloris oleoabun-dans (Chantanachat and Bold; Santos et al. 2012)),only a limited number of Chlorophytes couple oilaccumulation with pigment overproduction. Thissuggests that the overproduction of pigmentsrequires a transport mechanism that is absent inmost species.

In both D. salina and H. pluvialis, it has been sug-gested that b-carotene is the pigment that is trans-ported out of the photosynthetic apparatus(Lemoine and Schoefs 2010). In H. pluvialis, whichis known for its substantial astaxanthin production,

the subsequent conversion from b-carotene intoastaxanthin is suggested to take place in the cyto-plasm (Jin et al. 2006, Lemoine and Schoefs 2010).Thus, when the introduction of extra storage spacein other microalgae is successful, it is possible thatthis space will be solely occupied by b-carotene ifb-carotene is the only pigment that can betransported out of the photosystem and if b-carotene-converting enzymes are absent from thecytoplasm. To enable overproduction of pigmentsother than b-carotene in such storage space, specificenzymes may be targeted toward this storage spaceor toward the cytoplasm to convert b-carotene intovarious derivatives. This approach was recentlyapplied in carrot plants, in which lutein and b-caro-tene are commonly found, but derivatives of b-caro-tene, such as cantaxanthin and astaxanthin (Fig. 2),are rarely found. The synthesis of these ketocarote-noids was achieved by introduction and up-regulation of enzymes that catalyze the conversionsteps from b-carotene into astaxanthin. The enzymeswere targeted to the plastids, where accumulation ofb-carotene occurs, by fusing them with a targetingpeptide (from RUBISCO). As a result, 70% of the b-carotene was converted into ketocarotenoids (Jayarajet al. 2008). A similar approach may be used inmicroalgae, for example, by focusing on accumula-tion of echinenone in D. salina.As stated in Section 2, phycobiliproteins consist of

two parts: a phycobilin and a protein. Consequently,the overproduction of phycobiliproteins requires anincreased flux toward both components. However, itwould be interesting to test the hypothesis thatup-regulation of the protein content alone may besufficient to generate an increased flux toward thephycobilin; in this way, the protein would act asmetabolic sink for the phycobilin.The pigments discussed above may be produced

in either a continuous or two-step process. In a con-tinuous process, biomass production and pigmentoverproduction take place simultaneously, while in atwo-step process, biomass production and pigmentoverproduction take place separately (i.e., a growthphase is followed by a pigment production phase).The degree of growth inhibition that occurs withpigment overproduction determines, to a largeextent, the best production strategy. If pigmentoverproduction leads to minor growth inhibition,continuous production may be the best productionstrategy. However, if pigment overproduction leadsto severe growth inhibition, a two-step process isrequired. In that case, the overproduction shouldbe inducible (i.e., it should not take place duringthe growth phase), for example, by nutrient limita-tion.To conclude, specific process conditions, such as

oversaturating light intensities or a high salt concen-tration, exclusively induce the overproduction ofsecondary carotenoids (b-carotene in D. salina andastaxanthin in H. pluvialis). Overproduction of all


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other pigments present in microalgae requiresmodification in the expression of the appropriatebiosynthetic enzymes, most likely combined with thecreation of storage space outside the photosystem.Because pigment overproduction may lead to severegrowth inhibition, a two-step production processmay prove to be most successful.

CONCLUSIONS AND PROSPECTS

Microalgae produce a wide range of colored pig-ments. The overproduction of some pigments maybe attained by applying specific process conditions,while metabolic engineering may be necessary forothers. For each color, Table 2 shows the approachthat may be best suited for the preferred microalgalspecies.

In response to adverse growth conditions, the redpigment astaxanthin is overproduced by a selectnumber of Chlorophytes, such as C. zofingiensis andH. pluvialis (Fig. 4).

The orange pigment b-carotene is also overpro-duced by a select number of Chlorophytes, includ-ing D. salina, in response to adverse growthconditions. The astaxanthin and b-carotene contentsmay be increased further by metabolic engineering,for example by overexpressing flux-controllingenzymes or by introducing additional storage space.

Red-orange echinenone or yellow-orange zeaxan-thin may be overproduced by D. salina as well, butthis requires metabolic engineering, as indicated inTable 2. b-Carotene, which accumulates in chloro-plast-localized oil droplets under adverse growthconditions, needs to be converted into eitherechinenone or zeaxanthin, which may be achievedby targeting specific b-carotene-converting enzymesto the oil droplets or their membranes.

The yellow pigment lutein may be overproducedby Chlorella sorokiniana (Shihira and Krauss) or Chla-mydomonas reinhardtii, but in both species, this over-production can only be achieved through metabolicengineering (Table 2). Most likely, in addition tothe overexpression of specific enzymes, additionalstorage space (outside of the photosystem) needs tobe created. C. sorokiniana produces lutein (as a pri-

mary pigment) in a relatively high concentration(up to 0.42% g � g�1 dry weight; Matsukawa et al.2000) and demonstrates a high maximum specificgrowth rate (0.24 h�1; Matsukawa et al. 2000).Therefore, this species may be the first strain usedfor genetic modification. On the other hand,C. reinhardtii, which is capable of producing lutein(as all Chlorophyta, Fig. 4), although not at a highlevel, has been sequenced and genetically engi-neered (Cordero et al. 2011) and may thereforerepresent a suitable strain for genetic modification.The green chls may not need to be overproduced

to make an algae-based production process econom-ically feasible. Thus, rapidly growing C. sorokiniana,in which the total amount of chls (chl a and b) wasmeasured to be 4.5% g � g�1 dry weight (Cuaresmaet al. 2011), may be used as a production organismwhen grown under optimal growth conditions.The blue phycocyanobilin may be overproduced

by strains that belong to the Cyanophyta or theRhodophyta, although this process will require met-abolic engineering (Table 2). Example species, forwhich the genomes have been sequenced, includeSynechocystis sp. strain PCC6803 (Cyanophyta) andCyanidioschyzon merolae (Rhodophyta).All of the approaches to overproducing primary

pigments require modifications of metabolic path-ways. Due to the current lack of knowledge aboutregulatory mechanisms of the cell, which generallymaintain constant metabolite concentrations(homeostasis), the results of metabolic pathwaymodifications are largely unpredictable. To increasethis predictability, and thereby the effectiveness ofpathway modifications, additional research focusingon the regulatory processes involved in pigment bio-synthesis is needed. In addition, a toolbox shouldbe developed for the stable metabolic engineeringof the selected microalgae.

This work was supported by FeyeCon D&I and by grants fromNL Agency, Ministry of Economic Affairs (project no.FND09014).

Abd El-Baky, H. H. 2003. Over production of phycocayanin pig-ment in blue green alga Spirulina sp. and its inhibitory effect

TABLE 2. Methods to produce or overproduce natural pigments using microalgae or cyanobacteria. See text for details.


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