Physiological role of neutral lipid accumulation in eukaryotic microalgae under stresses

ISSN 1021�4437, Russian Journal of Plant Physiology, 2012, Vol. 59, No. 2, pp. 167–176. © Pleiades Publishing, Ltd., 2012.Original Russian Text © A.E. Solovchenko, 2012, published in Fiziologiya Rastenii, 2012, Vol. 59, No. 2, pp. 192–202.

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INTRODUCTION

The lipids fulfilling important functions in livingorganisms could be subdivided into two groups: non�polar or neutral lipids (acylglycerols, wax esters, andothers) and polar lipids (mainly phospho� and gly�colipids). Polar lipids are important structural compo�nents of cell membranes and organelles. Along withstructural function, polar lipids could operate as signalmolecules or their precursors. Among neutral (nonpo�lar) lipids, triacylglycerols (TAG) are most widespreadgroup of compounds easily involved in catabolism forproduction of energy required for the cell [1]. Thealgal lipid structure, biosynthesis, and functions aredescribed in detail by Harwood et al. [2, 3].

Numerous detailed studies of microalgal (here andbelow, unicellular eukaryotic algae are referred to asmicroalgae) lipids were focused on polar lipids [4, 5]because changes in the content and composition ofthese compounds are tightly related to the state ofchloroplast membranes [4]. By contrast, neutral lip�ids, including TAG of microalgae, are less studied. It is

commonly accepted that these compounds predomi�nantly serve as energy storage and are therefore char�acterized by low metabolic activity. This paradigmchanges now. The number of studies devoted to TAGand their role in the physiology of microalgal cellsincreased sharply. A considerable number of observa�tions indicate diverse TAG functions in the adaptationof photoautotrophic organisms to various environ�mental factors (temperature, illumination, salinity). Itis known that TAG are deposited in form of cytoplas�mic oil bodies (OB) in the cells of many species ofmicroalgae under the influence of stressors (high PARfluxes or nutrient deficiency) [6].

The interest to lipids of microalgae is determinedby their high potential as a raw material for pharma�ceutical, chemical, and food industries [7]. Lipids ofsome microalgal species are enriched in valuable poly�unsaturated fatty acids (PUFA), including essentialfor human linoleic (18:2), α�linolenic (18:3), arachi�donic (20:4), eicosapentaenoic (20:5), docosa�hexaenoic (22:6), and some other acids [8]. Microal�gal lipids are an important constituent of the ration ofvarious water organisms [9] and humans, being thesource of building blocks for the cell membranes andprecursors for signal and regulatory molecules as wellas compounds with anti�inflammatory and therapeu�tic effects [10, 11]. The recent decade saw an explosive

Physiological Role of Neutral Lipid Accumulationin Eukaryotic Microalgae under Stresses

A. E. SolovchenkoFaculty of Biology, Moscow State University, Moscow, 119991 Russia;

e�mail: [email protected] Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia

Received May 20, 2011

Abstract—In the studies of lipid metabolism of unicellular photoautotrophic eukaryotes (microalgae), themain attention is commonly paid to polar membrane lipids and their fatty acid (FA) composition, whereasneutral lipids, represented predominantly by triacylglycerols (TAG), are insufficiently studied. As wasreported recently, the role of these compounds in microalgae is not limited to their storage function. It wasfound that TAG are frequently involved in adaptation to environmental conditions. This review summarizesexperimental data obtained so far allowing to distinguish at least three aspects of TAG adaptive function inmicroalgae. First, these compounds are the source of long�chain FA, the building blocks for membranes nec�essary for rearrangements of the photosynthetic apparatus. Second, TAG biosynthesis consumes excessivephotoassimilates preventing photooxidative injuries under stresses which reduce cell capacity of photosyn�thesis product utilization. Third, TAG deposited as cytoplasmic oil bodies form a depot for secondary caro�tenoids in carotenogenic microalgae producing an optical screen protecting the cell against photodamage byexcessive PAR.

Keywords: microalgae (unicellular eukaryotic algae), adaptation, carotenoids, neutral lipids, polyunsaturatedfatty acids.

DOI: 10.1134/S1021443712020161

Abbreviations: Car—carotenoids; DAG—diacylglycerols;MGDG—monogalactosyldiacylglycerols; OB—oil bodies;PUFA—polyunsaturated fatty acids; TAG—triacylglycerols;18:6—dihomo�γ�linolenic acid; 20:4—arachidonic acid; 20:5—eicosapentaenoic acid; 22:6—docosahexaenoic acid.

REVIEWS

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growth of the interest in microalgal TAG determinedby their recognition as a promising source for produc�tion of the third and fourth generation biofuels [7, 12,13]. One of the most important advantages of the fuelproduced from microalgae is that its combustion doesnot result in net CO2 accumulation in the atmosphere,thereby reducing the greenhouse effect. In particular,during burning fuel produced from microalgae, theatmosphere is enriched in CO2 by 78% less, in sulfur�containing compounds by 98% less, and in solid parti�cles by 50% less than at burning of fuels derived fromoil and gas [14]. Moreover, industrial cultivation ofmicroalgae opens wide possibilities for utilization ofCO2 and sewage containing organic and mineral pol�lutants [15, 16]. It is of importance that biofuel pro�duction from TAG of microalgae does not threat foodsafety because installations for microalgal cultivation donot require arable lands used for crop growing [15, 17].

Accumulation of TAG in unicellular photoau�totrophs is influenced by environmental conditions.As a rule, TAG synthesis is activated by high light,especially on the background of mineral nutritiondeficiency; the TAG are stored in cytoplasmic OB(Fig. 1) and utilized in the process of polar membranelipid synthesis (see below) and/or storage lipid catab�olism in darkness [18]. It is worth mentioning thatconditions favorable for TAG biosynthesis are usuallystressful for microalgae: they hinder cell division andslow down culture growth (biomass accumulation).This is a major obstacle to achieving high accumula�tion of algal biomass enriched by compounds valuablefor humans. The solution of this nontrivial task is veryimportant for photobiotechnology based on microalgacultivation. Therefore, investigations of the TAG syn�

thesis physiology and the dynamics of their accumula�tion in the microorganisms under various stressescould be regarded as topical problems.

In this context, this review considers stimuli andphysiological role of strong TAG accumulation in var�ious species of microalgae summarizing recent dataabout roles of TAG in the biology of these microorgan�isms.

PUFA�ENRICHED TAG IN MICROALGAE

As was noted above, many species of microalgaecontain long�chain PUFA in their polar lipids, but thecontent of these compounds is a subject of stringentregulation within the cell [19, 20]. Algae capable ofactive synthesis of neutral lipids under the influence ofvarious stressors (so�called oleaginous algae) [21, 22]accumulate up to 86% of dry weight TAG comprised pre�dominantly the saturated and monounsaturated FA.

PUFA from the family n�3 are widespread inmicroalgae [20]. However, PUFA from the family n�6are rather rare in them, and a high content of 18:6 ispresent only in transformed organisms [3]. Arachi�donic acid (20:4) is essentially absent from all freshwater microalgae; and in marine algae, its contentdoes not exceed several percents of total FA content[18]. Until recently, the unicellular red alga Porphyrid�ium cruentum (Rhodophyta) was believed to be be theonly alga accumulating PUFA in TAG [23]. The greenmicroalga Parietochloris incisa (Chlorophyta, Tre�bouxiophyceae) is the first and, to the best of ourknowledge, the only phototrophic microorganismaccumulating high amounts of TAG enriched inPUFA, 20:4 in particular (it is recognized as the rich�

OB

OB

OB

OB

OB

OB

OB

Fig. 1. Electron microphotograph of Parietochloris incisa cells with lipid globules after 14 days of cultivation at 400 μE/(m2 s) PARon nitrogen�free medium BG�11 (x 6000).OB—oil bodies.


PHYSIOLOGICAL ROLE OF NEUTRAL LIPID ACCUMULATION 169

est plant source of 20:4 [24], see also the table). Dia�tom algae are also capable of accumulation of highamounts of TAG; however, most of their lipids arephospholipids, which are difficult to convert intobiodiesel by traditional methods limiting their practi�cal application [25].

PUFA valuable for humans are not present inmicroalgae as free FA but are the components of polar(phospho� and glycolipids) and neutral (TAG) lipids.Polar lipids play a structural role in the cell andorganelle membranes, particularly in chloroplasts;therefore, their content in the cell is under stringentcontrol of the genetic and physiological regulatorymechanisms. By contrast, neutral TAG have no struc�tural function; in many species they accumulate asstorage compounds. Correspondingly, the content ofTAG varies markedly depending on their cultivationconditions (for example, from 2 to 77% of total lipidsin P. incisa stationary culture [26]).

EFFECTS OF ENVIRONMENTAL FACTORS ON TAG SYNTHESIS

As a rule, TAG accumulation in the microalgalcells occurs in two stages. Until all elements requiredfor culture growth are available in medium, the cells ofmicroalgae divide rapidly and synthesize mainlymembrane lipids, chloroplast lipids in particular.When any growth factor becomes limiting (for exam�ple, nitrogen in medium is exhausted) but photosyn�thetic carbon dioxide fixation continues, the so�calledlipogenic phase onsets characterized by slowdown orcessation of cell division, frequently by the reductionof the photosynthetic apparatus, and by accumulationof neutral lipids, TAG in particular, and nonpolar car�otenoids (Car, in carotenogenic species) [27]. As aresult, the C/N ratio in the biomass increases mark�edly [28]. In this situation, energy�rich compounds(ATP), reducing equivalents, and carbohydrates pro�duced in the process of photosynthesis are utilizedpredominantly for the biosynthesis of nitrogen�freecompounds, mainly TAG. The reactions of TAG bio�synthesis are an efficient mechanism of excessive pho�

toassimilate consumption (its importance under stressconditions will be discussed below). De novo synthe�sized TAG deposit in spherical cytoplasmic inclu�sions, so�called OB (Fig. 1). In some cases, lipid globulesare also formed in the interthylakoid space [7]. In carote�nogenic algae, OB harbor the secondary Car [29, 30], theCar not related to the photosynthetic apparatus.

It is commonly accepted that microalgae use TAGfor the accumulation of energy and organic carbon,the reserve supporting rapid growth after relief of thestressful conditions [2]. From this point, the accumu�lation of TAG enriched in saturated FA would beadvantageous because less energy is required for thesynthesis of PUFA from them but more energy isreleased at oxidation of these compounds, and theaccumulation of TAG comprising long�chain PUFA isless beneficial. Probably, this could explain why spe�cies accumulating valuable PUFA within storage TAGare so rare. Nevertheless, it was shown in some exper�iments performed with P. cruentum that radioactivelylabeled FA are not only oxidized in the process ofcatabolism but also transferred from TAG to lipidclasses dominated in chloroplast membrane, such asmonogalactosyldiacylglycerols (MGDG) [31, 32].Moreover, the HZ3 P. cruentum mutant devoid ofcapability of FA residue transfer from TAG into chlo�roplast lipids demonstrated a deficit in 20:5/20:5�MGDG, one of dominating molecular species ofMGDG, and, as a consequence, it was characterizedby incapability of growth at low temperatures [32].

As was noted above, under conditions optimal forgrowth, microalgae synthesize mainly membraneglycerolipids in amounts as high as 5–20% of cell dryweight, whereas under stressful conditions, their lipidmetabolism shifts toward the synthesis of neutral lipids(TAG) [3]. Light quantity and spectral quality and alsomedium mineral compositions are evidently the keyfactors affecting the composition of lipids and FA inphotoautotrophic cells [33, 34]. Adaptation toextreme temperatures is predominantly related tochanges in the composition and the ratio betweenmembrane lipids [34]; it changes TAG content andcomposition to a lesser degree [3]. In this part of the

Lipid composition of nitrogen�starving P. incisa (after [52])

Treatment Class FA pro�portion, %

Major FA, % of total

16:0 18:0 18:1n�9

18:1n�7

18:2n�6

18:3n�6

18:3n�3

20:4n�6

–N NL 87 8 2 9 4 9 trace trace 64Control NL 62 9 1 15 6 10 trace 2 51–N GL 10 12 2 5 4 11 1 12 30Control GL 19 9 1 5 3 21 trace 15 21–N PL 3 26 3 3 14 13 3 3 29Control PL 19 17 1 6 11 19 1 7 25

Note: GL—galactolipids; NL—neutral lipids; PL—phospholipids, trace—less than 1% of total FA.

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review, the effects of illumination and mineral nutri�tion deficit on TAG synthesis by microalgae will beconsidered.

Illumination. Light intensity and spectral qualityand also the photoperiod are known to affect themetabolism of microalgal lipids and, as a conse�quence, their lipid composition [35]. High light resultsfrequently in PUFA photooxidation mediated by ROS[36], but light is required for the synthesis of certainFA [2]. In addition, ROS arising under high light playa role of signal molecules inducing and controllingmany biosynthetic processes, including the biosynthe�sis of secondary Car and neutral lipids [29]. On thewhole, it is believed that light�induced changes in thelipid composition are related predominantly tochanges in chloroplast development [35].

Adaptation to high PAR fluxes, especially on thebackground of other stressor action, reduces the size ofchloroplasts and induces deep rearrangements in gra�nae and intergranal lamellae: a reduction in the num�ber of granae and the degree of their stacking, as in P.incisa [6] and Dunaliella tertiolecta [37], which is inagreement with the reduction in the proportion ofmembrane lipids. It is of importance that the struc�tural and functional integrity of remained thylakoidsare preserved, making possible the recovery of thephotosynthetic apparatus after cessation of stress [6].Growing microalgae under high PAR irradiationresults as a rule in TAG accumulation [21]. This ischaracteristic of many microalgal species–producersof PUFA: the eustigmatophytes Nannochloropsis [38]and M. subterraneus [20], the diatom P. tricornutum,and other producers of 20:5. It is hypothesized [20]that a decrease of cell irradiance signals the end ofexponential growth phase, resulting in the redirectionof the excess of absorbed light energy to the synthesisof storage TAG. In these species, TAG are character�ized by the predominance of saturated and monoun�

saturated FA. On the other hand, extremely high light(about 400 μE/(m2 s) PAR and higher) on the back�ground of other stresses (nitrogen deficit, for example)could suppress TAG accumulation ([23, 39], Fig. 2).By contrast, culture exposure to darkness results in adecrease in the TAG content. Thus, in the dark growndinoflagellate Prorocentrum minimum, TAG contentdeclined on the background of constant phospholipidcontent; activation of β�oxidation and an increase ofisocitrate lyase activity is evident of the TAG usage asan alternative source of energy at the light absence[40]. Microalgae accumulating PUFA�enriched TAGas reserve building blocks for photosynthetic mem�branes usually synthesize the TAG actively under lowlight intensity and/or at a high culture density.

Mineral nutrition deficiency. Availability of mineralnutrients exerts a great influence on lipid metabolismin microalgae [3]. Almost in all cases, a decrease in theavailability of nutrients results in slowdown cell divi�sion. At the same time, photosynthetic carbon dioxidefixation continues as a rule. Such situations are com�mon in Nature; an example is so�called “snow algae”[29, 41, 42] developing on the surface of snow and gla�ciers and also in water from melted snow where bio�genic elements are extremely scarce. It is of interestthat, under these conditions, the rate of lipid biosyn�thesis does not decline in many species of microalgae[18]. However, the demand in membrane lipids is alsolow at the low growth rate, and synthesized FA accu�mulate mainly in the form of neutral lipids (TAG).Induction of TAG synthesis at the deficit or absence ofnitrogen, sulfur, phosphorus, or (in the case of diatomalgae) silicon is most studied [3].

Under nitrogen shortage or absence, cell divisionstops and TAG accumulate in the cells, which con�tinue to enlarge due to this process [43]. As a result,TAG content in the microalgal cells could increasetwice and more [18]. This phenomenon is characteris�

300

250

200

150

100

50

035 200 400

90

60

30

0

(а) (b)

Irradiance, μE/(m2 s) PAR

Tota

l FA

, m

g/l s

uspe

nsi

on

35 200 400

Ara

chid

onic

aci

d, m

g/l s

uspe

nsi

on

Fig. 2. Content of total FA (a) and arachidonic acid accumulating mainly within TAG (b) in the P. incisa cells cultured for 14 dayson complete (dark columns) and nitrogen�free (white columns) medium at different irradiance levels (after [39]).



tic of many species of algae from different taxa (see thereview [3] for more detail). In these cases, the propor�tion and absolute content of membrane lipidsdecreased, which is in agreement with the reduction ofthe photosynthetic apparatus frequently observedunder unfavorable conditions.

It should be noted that similar patterns of theinduction of TAG synthesis are observed in theabsence or deficiency of mineral nutrition and underother stressors, e.g., high light [39]. Similarly to nitro�gen deficiency conditions, phosphorus deficiency fre�quently induces an increase in total lipid content due toTAG accumulation, as, for example, in the fresh wateralga Monodus subterraneus (Eustigmatophyta) [44].

Salinity. Some species of microalgae could tolerateextremely high salt concentrations; Dunaliella is acharacteristic example [45]. Thus, in the cells ofDunaliella tertiolecta, the increase in the NaCl con�centration from 0.5 to 1.0 M resulted in the increase inthe total lipid content in the cells, TAG accumulationin particular [46]. TAG accumulation in response tothe increase in the salt concentration in the mediumup to 40 g/l was also characteristic of the marinemicroalga from the genus Nannochloropsis [47]. Itshould be noted that simultaneous action of highsalinity and high light (700 μE/(m2 s) additionallyenhanced the synthesis of TAG enriched in 20:5. It isimportant that this effect was observed only on com�plete medium; nitrogen deficit on the background oftwo additional stressors (high light and high salinity)resulted in the marked reduction of the microalgagrowth rate [47].

Effect of pH. Extreme values of pH are known toaffect lipid metabolism in microalgae. In particular,alkaline pH induces stress accompanied by TAG accu�mulation and corresponding decrease in the contentof membrane lipids because of the photosyntheticapparatus reduction [48]. A decrease in pH can alsoinduce TAG synthesis. Thus, in Chlamydomonas sp.isolated from acid volcanic lake and cultured atpH 1.0, the absolute content of TAG and their propor�tion in lipids increased [49].

Presence of organic carbon source. The presence oforganic source of carbon stimulates TAG accumula�tion, shifting the C/N ratio toward carbon. This effectis especially pronounced at nitrogen starvation. Dur�ing photoautotrophic cultivation of P. incisa in theabsence of nitrogen, the accumulation of TAGenriched in 18:1 but containing less 20:4 acids wasobserved [20]. In Euglena gracilis, the addition of theorganic carbon source to the medium on the back�ground of nitrogen deficiency resulted in the addi�tional enhancement of storage TAG biosynthesisenriched in 14:0 and 16:0 [50].

PHYSIOLOGICAL ROLES OF TAG ACCUMULATION

It was hypothesized recently that the role of OB,the main reservoirs of storage TAG in the cell, is notlimited to that of relatively metabolically inert com�partments storing carbon and energy [51]. A numberof researchers showed that, in addition to traditionaland most studied function of energy storage, microal�gal TAG can also fulfill other important functions [24,26, 52, 53].

Participation in membrane rearrangement duringadaptation. Microalgal TAG enriched in PUFA areactively involved in the reactions of plastic metabolismand could serve as a depot of certain FA required forbuilding photosynthetic membranes. Thus, microal�gae dwelling environments with abrupt changes intemperature, salinity, the content of biogenic ele�ments, and illumination have a need in substantial andrapid changes of FA and lipid composition of chloro�plast membranes. In particular, it is required for pro�tection against damages induced by high visible andUV radiation levels, especially on the background oflow temperatures [54]. Thus, intense visible lightcould damage the photosynthetic apparatus, espe�cially at low temperatures; the main target is the pho�tosystem II protein D1 [55]. Under such conditions,de novo PUFA synthesis is too slow for acclimation toabrupt changes of environmental condition, morerapid source of acyl groups is required for the synthesisof MGDG and other classes of polar lipids under suchconditions, possibly PUFA�enriched TAG. These pro�cesses may be important components of the acclimationmechanisms based on rapid membrane, especiallyphotosynthetic membrane, rearrangements [19, 53].

A basic mechanism of adaptation to low tempera�tures is an increase in the degree of FA unsaturation inthe cell membranes [56]. This way of adaptationrequires a mechanism providing for a rapid increase inthe PUFA content in membrane lipids. However, lowtemperatures slows all biochemical processes, and therate of de novo PUFA synthesis under sharp tempera�ture changes could be insufficient for successful adap�tation. In such situations, a possibility to store PUFAwithin TAG could be critical for acclimation to sharpchanges of environmental conditions. Since high con�centrations of free FA could be toxic for the cell, thesecompounds are stored in the form of lipids. As wasnoted above, polar lipids are mainly membrane com�ponents and are under stringent control of numerousregulatory mechanisms, therefore neutral lipids(mainly TAG) appear to be the only depot for thereserve pool of FA.

In this connection, Khozin�Goldberg et al. [53]suggested that in species like P. incisa TAG serve as adepot for reserve PUFA, which could be rapidly mobi�lized for the synthesis of chloroplast lipids, providingfor rapid acclimation to sharp changes in the environ�mental conditions. Such mechanisms are evidently

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less important for species inhabiting large water bodieswhere temperature changes rather gradually, but theycould be very important for microalgae dwelling smallwater reservoirs. P. cruentum, a species living in shal�low marches and on moist sand grounds, i.e., in nichessubjected to sharp temperature variations, could serveas an example. An increase in the proportion of 20:5 inMGDG, especially in eukaryotic species (20:5/20:5�MGDG), observed at a temperature decline reflectsthe activation of the abovementioned mechanism ofmicroalgal adaptation to sudden and sharp change intemperature. There were indications [53] of a possibleTAG role as a “buffer” for the synthesis of diacylglyc�erols (DAG) comprising 20:4 and 20:5. This reservecould be rapidly mobilized for the synthesis of“eukaryotic” types of chloroplast lipids e.g., MGDG[20]. This is important in particular for the rapidresynthesis of chloroplast lipids and recovery of photo�synthetic membranes after cessation of stress.

Protection against photodamage under stress.Under high fluxes of solar radiation, an imbalancearises between the amount of absorbed light energyand the capability of a photoautotrophic organism toutilize it [57]. As a consequence, a probability of trip�let–triplet interaction between oxygen molecules andexcited molecules of chlorophyll in the antenna andO2 reduction by electrons from the reduced carriersfrom chloroplasts ETC increases. In these cases, thestationary level of ROS in the cells builds up, resultingin photodamages, even lethal, to microalgal cells [58].Under additional stresses, such as the lack of nitrogen,the risk of microalgal cell death from photooxidationincreases substantially because of suppression of bio�synthetic processes utilizing photoassimilates. Theseconditions cause some algal species to synthesizeenergy�rich TAG, which are accumulated in great

amounts. It is likely that the reactions of TAG biosyn�thesis are an efficient way to consume excessive photo�assimilates (energy consumption of lipid biosynthesisis ca. twice higher than that of storage carbohydratesynthesis). This reduces the risk of ROS generationand photodamage to microalgal cells [7, 29].

Depot for extrathylakoid photoprotective pigments.It should be noted that OB produced in the result ofthe above mentioned processes (predominantly in thecytoplasm) frequently serve as a depot for lipophiliccarotenoid pigments [59] absorbing excessive radia�tion and protecting cell structures vulnerable to pho�todamage, such as photosynthetic membranes. Themicroalga Haematococcus pluvialis [60], some mem�bers of the genus Dunaliella [61, 62], Parietochlorisincisa [63], Trentepohlia [64], and a number others aretypical examples of species accumulating extrathyla�koid secondary Car. In some cases, photoprotectivepigments accumulate within chloroplasts, as inD. salina (D. bardawil), which accumulates β�caro�tene in the stromal granules also containing lipids(TAG) and stabilized by specific proteins [65–67].The function of TAG�containing cytoplasmic andstromal lipid inclusions as a depot for photoprotectivepigments was demonstrated by the analysis of absorp�tion spectra of OB isolated from the cells of microalgae(Figs. 3, 4). Thus, in P. incisa the bulk (up to 66%) ofβ�carotene was localized in the cytoplasmic OB (Figs. 1,2). It is of interest that xanthophylls more polar thancarotenes, such as astaxanthin in H. pluvialis, accu�mulate in OB as FA esters [68, 69].

It is of importance that, under conditions favoringactivation of TAG synthesis, including high light anddeficiency of mineral nutrients, the synthesis of sec�ondary Car is also enhanced, evidently mediated bythe redox signals (such as the degree of reduction of

(а)

(b)

(c)

1 2 3

4 5 6

7 8

9

10 15 20 25Rt, min

2

4

4

5

5

6

6

8

9

9

Fig. 3. HPLC of extracts from P. incisa cells cultured for14 days on complete medium at 400 μE/(m2 s) PAR (a) andthylakoids isolated from them (b) and lipid globules (c).(1) Neoxanthin; (2) violaxanthin; (3) lutein�5,6�epoxide;(4) lutein; (5) zeaxanthin; (6) chlorophyll b; (7) pheophy�tin; (8) chlorophyll a; (9) β�carotene (after [63]).

0.8

0.6

0.4

0.2

300 400 500 600 700

OD

, un

its

Wavelength, nm

0

Fig. 4. Absorption spectrum of the chloroform extract fromOB isolated from P. incisa cells cultured on nitrogen�freemedium at 400 μE/(m2 s) PAR (see [63] for more detail).



particular electron carriers in chloroplast ETC) andROS action [70, 71]. The accumulation of highamounts of Car (carotenogenesis) is characteristic of anumber of microalgae, including species from thegenera Chlorella [41], Dunaliella [62, 72], Haemato�coccus [60, 68, 73], and Parietochloris [6, 74]. Underthese conditions, a tight interaction exists betweenbiosyntheses of TAG and secondary Car, and, as aconsequence, a close correlation is observed betweenthe accumulation of these compounds in the microal�gal cells (Fig. 5, see also [70, 71, 74]). It is likely thatpredominant utilization of photoassimilates in thereactions of storage lipid (TAG) biosynthesis with par�allel induction of carotenogenesis provides formicroalgal cell protection under stressful conditions,high light and/or nitrogen deficit [75].

CONCLUSIONS

The results of recent investigations in the field ofbiochemistry and physiology of lipid metabolism ofmicroalgae extended markedly the current concept ofTAG significance for photoautotrophic unicellularmicroorganisms. It is likely that in these organismsTAG role is not limited to the traditional for thesecompounds function of storage. Numerous data indi�cate that microalgal TAG are more than just a reservoirof energy�rich molecules; they are actively involved inthe processes of adaptation to harsh environmentalconditions.

Collectively, the experimental data obtained so farsuggest at least three aspects of TAG function inmicroalgal adaptation. First, they are capable ofPUFA pool formation, providing building blocks forchloroplast lipids required for adaptation of the pho�tosynthetic membranes to abrupt changes of environ�

mental conditions. Second, TAG biosynthesis con�sumes excessive photoassimilates, thus preventingphotooxidative injuries under stresses of various naturereducing cell capacity to utilize photosynthates inother biosynthetic processes. Third, TAG depositionin lipophilic cytoplasmic (OB) and stromal (plastoglo�buli) inclusions creates a depot for secondary Car pro�tecting cells against photodamage via screening of theexcessive PAR.

In conclusion, it should be noted that fundamentalinvestigations of TAG synthesis and accumulation inmicroalgal cell adaptation to extreme environmentalconditions and also of the role of stress in the induc�tion of these processes are important from practicalpoint of view. The understanding of the physiology ofneutral lipid biosynthesis in microalgae is a requisitefor redirection of metabolism in these organisms dur�ing their laboratory and industrial cultivation. Thisknowledge is a key for the assessment of the biosyn�thetic potential of promising strains�producersapplied in photobiotechnology for the production ofvitamins, antioxidants, and biologically active com�pounds as well as environmentally safe biofuel frommicroalgal biomass.

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2.0

1.6

1.2

0.8

0.4

00.2 0.4 0.6 0.8 1.0 1.2

y = 2.15x – 0.51r2 = 0.91

Car/Chl (a + b), mol/mol

Ara

chid

onic

aci

d, m

g/l s

uspe

nsi

on

Fig. 5. Dependence of the content of arachidonic acidaccumulating predominantly within TAG on the ratio ofcarotenoids (Car) to chlorophylls (Chl) a + b in P. incisacells grown on complete (closed symbols) and nitrogen�free (open symbols) media at the irradiance of 35 (�, �),200 (�, �), and 400 (�, �) μE/(m2 s) PAR (after [74]).

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Physiological role of neutral lipid accumulation in eukaryotic microalgae under stresses

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