Effect of nutrient supply status on biomass composition of eukaryotic green microalgae (original) (raw)

Effect of nutrient supply status on biomass composition of eukaryotic green microalgae

Gita Procházková, Irena Brányíková, Vilém Zachleder & Tomáš Brányík

Journal of Applied Phycology
ISSN 0921-8971
J Appl Phycol
DOI 10.1007/s10811-013-0154-9

Springer

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Effect of nutrient supply status on biomass composition of eukaryotic green microalgae

Gita Procházková ⋅\cdot Irena Brányíková ⋅\cdot
Vilém Zachleder ⋅\cdot Tomáš Brányík

Received: 13 December 2012 / Revised and accepted: 12 September 2013
© Springer Science+Business Media Dordrecht 2013

Abstract

In eukaryotic green microalgae, manipulation of metabolic pathways by altering the culture medium and/or culture conditions represents a powerful tool for physiological control and is usually more practicable than metabolic or genetic engineering. Strategies for nutrient-induced shifts in biomass composition are generally cost-efficient, environmentally friendly, applicable on a large scale and flexible for various industrially attractive microalgae species. In addition, processes, such as nutrient limitation/deprivation, can be readily scheduled and optimised to achieve high levels of productivity for the desired target compound(s). These strategies are currently used in microalgae to achieve overproduction of metabolites such as lipids, polysaccharides and pigments. This paper presents an overview of the species and strain-specific responses of eukaryotic, green microalgal cells that are triggered by variations in selected macronutrient and micronutrient availability. Individual and mutually associated physiological responses to nutrient supply status are described at the molecular level as well as discussed from the perspective of potential biotechnological applications.

Keywords Green microalgae ⋅\cdot Nutrients ⋅\cdot Limitation ⋅\cdot Metabolic response ⋅\cdot Biomass composition ⋅\cdot Cell growth

[1]

Introduction

Microalgae are a group of organisms that are physiologically and morphologically heterogeneous. They represent an extraordinary source of different bioactive compounds used as pharmaceuticals, in food and feed supplements, in cosmetics and other applications. At the same time, microalgae can utilise waste CO2\mathrm{CO}_{2} and other nutrients occurring in wastewaters. This review focuses only on eukaryotic green microalgae, their photoautotrophic growth and, in particular, the effect of macronutrients nitrogen (N), phosphorus (P), sulphur (S), and some selected micronutrients such as iron (Fe) and selenium (Se) on biomass composition. For each nutrient, various examples of species and strain-specific responses to different types of nutrient supply are described in regard to the effect of one or more nutrients on microalgal cell composition and metabolite productivities. Given the vast amount of literature on this topic, this review focuses mainly on the articles published in recent years.

Manipulation of metabolic pathways can redirect cellular functions towards the synthesis of preferred products and can even expand the production capabilities of microalgae. One method of coercing microalgae employs specific environmental factors, such as nutrient regimes, to induce desired metabolic fluxes. Compared to metabolic or genetic engineering, the manipulation with nutrient supply of non-recombinant microalgae is a more practicable alternative. Although the former allows direct control over the organism’s cellular machinery through mutagenesis or the introduction of transgenes, it is much more technologically demanding and leads to a number of difficulties (e.g. legislative). In the case of microalgae, specialised cultivation can stimulate changes in metabolism, thereby providing a simple method for enriching biomass with a target metabolite (Rosenberg et al. 2008; González-Fernández and Ballesteros 2012).

About 30 elements are important to ensure autotrophic growth and according to the amount required by the microalgae, these essential nutrients are grouped into two categories: (i)

1. G. Procházková ⋅\cdot I. Brányíková ⋅\cdot T. Brányík ( ⊠\boxtimes ) Department of Biotechnology, Institute of Chemical Technology Prague, Technická 5, 16628 Prague, Czech Republic
   e-mail: tomas.branyik@vscht.cz
   V. Zachleder
   Laboratory of Cell Cycles of Algae, Institute of Microbiology, Academy of Sciences of the Czech Republic, 37981 Třebon, Czech Republic ↩︎

macronutrients: required in the culture medium in relatively large concentrations of gL−1\mathrm{g} \mathrm{L}^{-1} and (ii) micronutrients (trace elements): required in mgL−1\mathrm{mg} \mathrm{L}^{-1} of culture medium or less. A general overview of key nutrients essential for autotrophic microalgae is presented in Table 1. The broad concentration ranges (Table 1) are caused by different nutritional requirements of different species. Moreover, what constitutes a micronutrient for one microalgae species can be a macronutrient for another as in the case of calcium. Some of the trace elements that are required in very low concentrations do not even need to be added to the medium, as they occur as impurities in other chemicals as well as in the water used for medium preparation (Grobbelaar 2004).

It is also important to distinguish between freshwater, marine and halotolerant/halophilic species. Seawater has a relatively constant composition of major ions (Na+,K+\left(\mathrm{Na}^{+}, \mathrm{K}^{+}\right., Mg2+,Ca2+,Cl−,SO42−,HCO3−,CO32−\mathrm{Mg}^{2+}, \mathrm{Ca}^{2+}, \mathrm{Cl}^{-}, \mathrm{SO}_{4}{ }^{2-}, \mathrm{HCO}_{3}^{-}, \mathrm{CO}_{3}{ }^{2-} ) and pH , whereas freshwaters have highly variable compositions. Microalgal species that grow in these particular waters are appropriately adapted in their chemistry, e.g. cells possess particular detoxification or tolerance mechanisms when growing in freshwater containing high concentrations of particular metals (e.g. copper) that are toxic to the majority of other phytoplanktonic species (Sunda et al. 2005). Thus, it can be argued that the original natural environment in which a given microorganism grows defines its nutritional needs. Besides supplying the microorganisms with all required elements, the medium must also ensure appropriate salinity (ionic strength and composition) and pH value (Koller et al. 2012).

Nutrient supply status of microalgae

The importance of light and nutrient supply in controlling the growth kinetics of phytoplankton has prompted much research. The growth kinetics of microalgae can be expressed either by the Monod or Cell Quota models. While the Monod model is unstructured and the specific growth rate responds directly to an alteration in the external substrate, the Cell Quota model inserted an extra compartment (cell quota-i.e. an intracellular nutrient pool) into the model maintained by nutrient uptake on the one hand and by cell multiplication (dissipation) on the other hand. Both models are usually applicable to the steady state at nutrient-saturated or nutrient-limited conditions, whilst the extra compartment in the Cell Quota model has the potential to handle luxury uptake of nutrients or transient states too. It was also recognised that the steady-state assimilation rate of limiting nutrient in continuous culture is linearly related to the cell quota of the limiting nutrient (Caperon and Meyer 1972). The cell quota was defined as the quantity of substrate required to produce a given amount of biomass (Droop 1973, 1983). Further, there have been several attempts at modeling the light/nutrient interaction in algae. Steady-state, nutrient-

Table 1 Nutrients essential for autotrophic microalgae and the elementary composition of algal cells (adapted from Grobbelaar 2004)

∗{ }^{*} Data not available
a { }^{\text {a }} Umysová et al. 2009
saturated growth was quantitatively described by empirical equations relating growth rate to absorbed irradiance, rate of photosynthesis to irradiance, respiratory rate to growth rate and stating the constancy of light saturation parameter to C:Chl aa ratio (Bannister 1979). A simple extension of the

model allowed also the description of nutrient/light-limited growth in continuous culture. Here, the relationship between C:Chla\mathrm{C}: \mathrm{Chl} a ratio and dilution rate was found to be linear and independent of the type of limiting nutrient, whilst the productivity index was found to be a hyperbolic function of dilution rate (Laws and Bannister 1980).

It is clear that phytoplankton have adaptation mechanisms to cope with different nutrient supply situations and that these mechanisms are used to maintain biomass composition and to achieve balanced growth. The range of situations concerning extracellular nutrient status in an algal culture can be expressed on a continuous scale from:
(i) repletion (zero stress, excess of nutrients in the preferred chemical form, formation of storage compounds derived from a given nutrient).
(ii) sufficiency (low stress, nutrient is not rate-limiting but different enzymes are expressed compared to the replete status). Since the status replete and sufficient, as defined here, are rather difficult to distinguish, they are used synonymously.
(iii) limitation (nutrient is present in growth rate limiting concentrations and affects biomass composition).
(iv) deprivation (maximal stress, nutrient is not present and growth is severely limited without intracellular reserves; Flynn 1990; Rodolfi et al. 2009). The terms starvation, deficiency and depletion are often used synonymously with deprivation.

The terms characterised above will be used to describe different nutrient status situations throughout this review. Nevertheless, one must keep in mind that the use of these terms by different authors is not uniform.

Also the mode of cultivation is important regarding descriptions of nutrient status. During chemostat cultivation, one nutrient can be chosen to be limiting. The microalgal growth rate in this system is then controlled by the concentration of this limiting nutrient and the dilution rate. However, when batch or fed batch modes of biomass production are used, the concentration of individual nutrients varies with time; consequently, the nutrient status of the culture is also time-dependent (Tredici 2004).

In general, it is possible to distinguish between specific responses that occur during limitation for a particular nutrient, and common responses that occur during general nutrient limitation. Specific responses include changes in nutrient transport characteristics that enable cells to import the limiting nutrient more efficiently and induction of nutrient scavenging systems that enable cells to access alternative sources of the limiting nutrient. Scavenging enzymes are either exported from the cells to mobilise alternative sources in the environment or maintained within the cell to recycle nutrients by degrading proteins, RNA or lipids. Common responses include decreases in cell division, photosynthesis and respiration or an increased accumulation of
storage compounds (starch, oil) and/or secondary metabolites (e.g. carotenoids; Davies and Grossman 2004; Yildiz et al. 1994; Badger et al. 1980; Palmayist et al. 1994; Siderius et al. 1996).

Nitrogen

Nitrogen (N)(\mathrm{N}) is an essential element for all microalgae, being a component of the most abundant cellular macromolecules such as proteins, nucleic acids and others. It can be supplied in the form of NO3−,NO2−,NH4+,(NH2)2CO\mathrm{NO}_{3}{ }^{-}, \mathrm{NO}_{2}{ }^{-}, \mathrm{NH}_{4}{ }^{+},\left(\mathrm{NH}_{2}\right)_{2} \mathrm{CO} (urea) or other organic compounds. Not every microalga is able to utilise each form of N and eukaryotic microalgae are unable to convert atmospheric N2\mathrm{N}_{2}. The most widely used N source for microalgal culture is nitrate (NO3−)\left(\mathrm{NO}_{3}{ }^{-}\right). Nitrite left(mathrmNO2−right)canbealsoused,butitstoxicityathigherconcentrationsmakesitsuselessconvenientwhencomparedtocanbealsoused,butitstoxicityathigherconcentrationsmakesitsuselessconvenientwhencomparedtomathrmNO3−.Nevertheless,Botryococcusbrauniiwasabletoutilise.Nevertheless,BotryococcusbrauniiwasabletoutilisemathrmNO2−thatwasderivedfromdissolvedfluegasnitricoxidethatwasderivedfromdissolvedfluegasnitricoxide(mathrmNO)inanaqueousphase,withresultinginanaqueousphase,withresultingmathrmNO2−\left(\mathrm{NO}_{2}{ }^{-}\right)canbealsoused,butitstoxicityathigherconcentrationsmakesitsuselessconvenientwhencomparedtocan be also used, but its toxicity at higher concentrations makes its use less convenient when compared to \mathrm{NO}_{3}{ }^{-}.Nevertheless,Botryococcusbrauniiwasabletoutilise. Nevertheless, Botryococcus braunii was able to utilise \mathrm{NO}_{2}{ }^{-}thatwasderivedfromdissolvedfluegasnitricoxidethat was derived from dissolved flue gas nitric oxide (\mathrm{NO})inanaqueousphase,withresulting in an aqueous phase, with resulting \mathrm{NO}_{2}{ }^{-}left(mathrmNO2−right)canbealsoused,butitstoxicityathigherconcentrationsmakesitsuselessconvenientwhencomparedtocanbealsoused,butitstoxicityathigherconcentrationsmakesitsuselessconvenientwhencomparedtomathrmNO3−.Nevertheless,Botryococcusbrauniiwasabletoutilise.Nevertheless,BotryococcusbrauniiwasabletoutilisemathrmNO2−thatwasderivedfromdissolvedfluegasnitricoxidethatwasderivedfromdissolvedfluegasnitricoxide(mathrmNO)inanaqueousphase,withresultinginanaqueousphase,withresultingmathrmNO2−removals up to 100%100 \% (Yang et al. 2004b). Generally, the preferred N form for many algae is NH4+\mathrm{NH}_{4}{ }^{+}, because it can be directly incorporated into organic compounds, as opposed to mathrmNO3−andandmathrmNO2−.Ontheotherhand,.Ontheotherhand,mathrmNH4+\mathrm{NO}_{3}{ }^{-}andand \mathrm{NO}_{2}{ }^{-}.Ontheotherhand,. On the other hand, \mathrm{NH}_{4}{ }^{+}mathrmNO3−andandmathrmNO2−.Ontheotherhand,.Ontheotherhand,mathrmNH4+concentrations greater than 25μM25 \mu \mathrm{M} are often reported to be toxic for some algal species, so mathrmNO3−\mathrm{NO}_{3}{ }^{-}mathrmNO3−is used more often in synthetic culture media (Barsanti and Gualtieri 2006). Although most algae are able to synthesise all the required organic N compounds from inorganic N sources, some species are also able to utilise certain organic N compounds such as urea, most of the amino acids (preferably L-forms), uric acid, xanthine, certain dipeptides, several amides, but not amides (except putrescine) and aminoalcohols as sole N sources (Morris 1974).

Transport of N\mathbf{N} and consequences of N\mathbf{N} deprivation/limitation

N uptake in Chlamydomonas reinhardtii is active and appears to have two independent systems for transporting NH4+\mathrm{NH}_{4}{ }^{+}, i.e. a low-affinity constitutive system and a high-affinity system that is only expressed when the mathrmNH4+concentrationfallsbelowacriticallevel(Francoetal.1988).Remarkably,thereisanamplearrayofinorganicNcompoundtransportersoperatinginasinglecell,i.e.13putativeconcentrationfallsbelowacriticallevel(Francoetal.1988).Remarkably,thereisanamplearrayofinorganicNcompoundtransportersoperatinginasinglecell,i.e.13putativemathrmNO3/mathrmNO2−transportersandeightputativetransportersandeightputativemathrmNH4+transporters.However,fortransporters.However,formathrmNO3−,onlyafewofthemparticipateasthemainsuppliersofNforcellgrowth,andothersprobablyfunctiontoacclimateNuptakeconditionsdependingnotonlyontheavailableNsourcebutalsoonothernutrientsandenvironmentalconditions.Inthecaseof,onlyafewofthemparticipateasthemainsuppliersofNforcellgrowth,andothersprobablyfunctiontoacclimateNuptakeconditionsdependingnotonlyontheavailableNsourcebutalsoonothernutrientsandenvironmentalconditions.InthecaseofmathrmNO3−theuptakeconsistsoftwotransportandtworeductionstepsproceedingseparatelyinthecytosoltheuptakeconsistsoftwotransportandtworeductionstepsproceedingseparatelyinthecytosolleft(mathrmNO3/mathrmNO2−right)andthechloroplastandthechloroplastleft(mathrmNO2/mathrmNH4+right)cdotmathrmNH4+\mathrm{NH}_{4}{ }^{+}concentrationfallsbelowacriticallevel(Francoetal.1988).Remarkably,thereisanamplearrayofinorganicNcompoundtransportersoperatinginasinglecell,i.e.13putativeconcentration falls below a critical level (Franco et al. 1988). Remarkably, there is an ample array of inorganic N compound transporters operating in a single cell, i.e. 13 putative \mathrm{NO}_{3} / \mathrm{NO}_{2}{ }^{-}transportersandeightputativetransporters and eight putative \mathrm{NH}_{4}{ }^{+}transporters.However,fortransporters. However, for \mathrm{NO}_{3}{ }^{-},onlyafewofthemparticipateasthemainsuppliersofNforcellgrowth,andothersprobablyfunctiontoacclimateNuptakeconditionsdependingnotonlyontheavailableNsourcebutalsoonothernutrientsandenvironmentalconditions.Inthecaseof, only a few of them participate as the main suppliers of N for cell growth, and others probably function to acclimate N uptake conditions depending not only on the available N source but also on other nutrients and environmental conditions. In the case of \mathrm{NO}_{3}{ }^{-}theuptakeconsistsoftwotransportandtworeductionstepsproceedingseparatelyinthecytosolthe uptake consists of two transport and two reduction steps proceeding separately in the cytosol \left(\mathrm{NO}_{3} / \mathrm{NO}_{2}^{-}\right)andthechloroplastand the chloroplast \left(\mathrm{NO}_{2} / \mathrm{NH}_{4}^{+}\right) \cdot \mathrm{NH}_{4}^{+}mathrmNH4+concentrationfallsbelowacriticallevel(Francoetal.1988).Remarkably,thereisanamplearrayofinorganicNcompoundtransportersoperatinginasinglecell,i.e.13putativeconcentrationfallsbelowacriticallevel(Francoetal.1988).Remarkably,thereisanamplearrayofinorganicNcompoundtransportersoperatinginasinglecell,i.e.13putativemathrmNO3/mathrmNO2−transportersandeightputativetransportersandeightputativemathrmNH4+transporters.However,fortransporters.However,formathrmNO3−,onlyafewofthemparticipateasthemainsuppliersofNforcellgrowth,andothersprobablyfunctiontoacclimateNuptakeconditionsdependingnotonlyontheavailableNsourcebutalsoonothernutrientsandenvironmentalconditions.Inthecaseof,onlyafewofthemparticipateasthemainsuppliersofNforcellgrowth,andothersprobablyfunctiontoacclimateNuptakeconditionsdependingnotonlyontheavailableNsourcebutalsoonothernutrientsandenvironmentalconditions.InthecaseofmathrmNO3−theuptakeconsistsoftwotransportandtworeductionstepsproceedingseparatelyinthecytosoltheuptakeconsistsoftwotransportandtworeductionstepsproceedingseparatelyinthecytosolleft(mathrmNO3/mathrmNO2−right)andthechloroplastandthechloroplastleft(mathrmNO2/mathrmNH4+right)cdotmathrmNH4+is then finally incorporated into carbon skeletons by the glutamine synthetase/glutamate synthase pathway. The transporters are

located at both the plasma and the chloroplast membranes (Fernandez and Galvan 2007). Also urea, arginine and urate can be directly imported from the medium by C. reinhardtii (Galván et al. 1996; Kirk and Kirk 1978), but most amino acids are deaminated outside the cell and the released mathrmNH4+\mathrm{NH}_{4}{ }^{+}mathrmNH4+is then transported into the cell (Muñoz-Blanco et al. 1990).

Similarly as in other microorganisms, N deprivation in microalgae causes crucial metabolic changes. The metabolism is generally redirected from proteosynthesis towards syntheses of such storage compounds that do not contain N like triacylglycerides (TAGs) and starch. Whether the given microalga will accumulate only one or both of these storage compounds in similar amounts at once or at different time scales is dependent upon the species and the environmental conditions. Even within the same genus (e.g., Chlorella) some species (strains) were found to accumulate starch under N deprivation (Brányiková et al. 2011; Dragone et al. 2011; Ho et al. 2013), whereas others accumulated prevalently neutral lipids (Hu 2004; Liu et al. 2008; Jian-Ming et al. 2010; Sirisansaneeyakul et al. 2011; Tang et al. 2011; Feng et al. 2011; Chen et al. 2011; Pîthyl et al. 2012). Furthermore, in contrast to the polar lipids of N sufficient cells, neutral lipids in the form of triacylglycerols become the predominant components of lipids from N -depleted cells (Hu 2004).

Starch synthesis shares common precursors with lipid synthesis, thus under nutrient limitation, some algal species or strains can inhibit the starch biosynthetic pathway and redirect the photosynthetic carbon © flux toward lipid biosynthesis. This leads to lipid overproduction in the cell, serving as an alternate C and energy reserve. Furthermore, different microalgal species probably have evolved distinct strategies to redirect C flux from carbohydrate pathways towards lipid biosynthesis. Despite advances in the characterisation of the pathways of starch metabolism and lipid synthesis, the interaction between these pathways is still poorly understood and consequently much research is being carried out in this field (Li et al. 2011, 2012). For example photosynthetic C partitioning into starch and neutral lipid was investigated in the oleaginous green microalga Pseudochlorococcum sp., in which two phases of storage compound formation were observed. Firstly, high light and N -limited conditions upregulated starch synthesis. Subsequently, after N depletion, starch content decreased whilst neutral lipid rapidly increased to 52%52 \% dry weight (DW), with a maximum ( 350mgL−1350 \mathrm{mg} \mathrm{L}^{-1} day −1^{-1} ) neutral lipid productivity (Table 2). Thus, this microalga presumably utilises starch as a primary C and energy storage product. As N was depleted for an extended period of time, cells shift the C-partitioning into neutral lipid as a secondary storage product (Li et al. 2011). A similar pattern was observed in C. reinhardtii (Wang et al. 2009) or Parachlorella kessleri (Fernandes et al. 2013) under nutrient stress.

It must be emphasised that upon certain storage compound accumulation, productivity can be affected in different ways. On the one hand, carbohydrate may reach above 70%DW70 \% \mathrm{DW}
without reduction in productivity, but on the other hand, lipid accumulation is often associated with a reduction in biomass productivity as other N compounds (e.g. proteins) are degraded to fulfill the cell’s N demand. However, there are also opinions that cellular lipid accumulation during N deprivation may be derived from newly fixed C . Therefore in certain (mostly green) microalgae, the mass of lipids accumulated during N deprivation may be higher than the total biomass present at the onset of the stress (Rodolfi et al. 2009).

Metabolic strategies to a changing nutrient status can be completely different in the case of other microalgae. For example it was discovered by proteome comparison that the response of diatom C metabolism to N deprivation is different from that of other photosynthetic eukaryotes (green algae and higher plants) and bears a closer resemblance to the response of cyanobacteria (Hockin et al. 2012).

Moreover, in species which are capable of sexual reproduction, N deprivation induces gametogenesis such as in CC. reinhardtii. The cells of this microalga also display other responses to N deprivation, namely starch accumulation and the execution of an autophagy program (Wang et al. 2009). The mating type of CC. reinhardtii is considered to be an important factor determining physiological response (e.g. lipid production) to S and N deprivation as the different cell types exhibit different levels of regulation (Cakmak et al. 2012).

As there are no special N storage molecules, N -limited CC. reinhardtii degrades the majority of highly abundant proteins, including Rubisco (Ribulose-1,5-bisphosphate carboxylase oxygenase) and ribosomal proteins, enabling the cells to recycle amino acids into proteins more suited for survival (Plumley and Schmidt 1989). Another quick N source utilised under stress conditions is e.g. chlorophyll, thus changes in chlorophyll content are directly reflected in the N content of microalgal biomass (Sirisansaneeyakul et al. 2011).

Daughter cells of the chlorococcalean alga Scenedesmus quadricauda incubated under photosynthesising conditions in N -free medium did not make any progress in the cell cycle. Photosynthetic starch formation continued for a period corresponding to a half of the cell cycle and then leveled off. Protein synthesis was very slow and RNA content decayed from the start of the treatment (Ballin et al. 1988).

Interestingly, in the green alga Haematococcus pluvialis, N deprivation also promotes the accumulation of the antioxidant pigment astaxanthin. Both of these acclimative responses help to ensure the cells’ survival during times of stress; whilst lipids serve as energy stores, astaxanthin seems to play a role in protection against ROS (Rosenberg et al. 2008). N deficiency, next to high light intensity, triggers also the induction of β\beta carotene accumulation in Dunaliella salina (Lamers et al. 2010). It was also found that upon N starvation, just as upon high-light stress, β\beta-carotene accumulation correlated negatively with the degree of unsaturation of the total fatty acid pool and did not correlate with total fatty acid accumulation,

Table 2 Physiological response of green microalgae to nutrient stress

Table 2 (continued)

Table 2 (continued)

Pmax⁡P_{\max } maximum productivity, Cmax⁡C_{\max } maximum content of target compound, AAA A arachidonic acid, DWD W dry weight, PBRP B R photobioreactor, NAN A not available
but rather with the biosynthesis of oleic acid (Lamers et al. 2012).

Lipid overproduction

As mentioned previously, many microalgal species can be induced to accumulate substantial quantities of lipids via N limitation/deprivation. According to the available literature (Table 2) it is the most widely applied technique to trigger the overproduction of desired metabolites. In contrast to the polar lipids of N sufficient cells, neutral lipids in the form of triacylglycerols become the predominant components of lipids from N-depleted cells (Hu 2004). Some microalgae can reach up to 75%75 \% (Chlorella pyrenoidosa) and 65%65 \% (Botryococcus braunii) of oils in DW, but this content is not associated with extremely high productivities (Table 2). Other algae (Chlorella, Dunaliella, Nannochloris, Neochloris, etc.) achieve oil levels only between 20 and 60%60 \% DW (Mata et al. 2010), but some of them at higher productivities (Table 2). Studies conducted on microalgal strains for lipid production that could then be applied in the cosmetic, food or fuel industries are indeed very broad. Screening studies have been performed for example by Rodolfi et al. (2009).

Among eukaryotic green algae, many species have been identified as potentially exploitable for biodiesel production. One of the most promising species for future biodiesel production seem to be the microalga Chlorella vulgaris, Parachlorella kessleri and Pseudochlorococcum sp. with lipid productivities of 1425,500 and 350mgL−1350 \mathrm{mg} \mathrm{L}^{-1} day −1^{-1}, respectively (Table 2; Pribyl et al. 2012; Li et al. 2011, 2012). Other microalgae species that should be highlighted for their high lipid productivities are Scenedesmus obliquus and BB. braunii. Maximum lipid productivities reported for these species were 323 and 190mgL−1190 \mathrm{mg} \mathrm{L}^{-1} day −1^{-1}, respectively (Table 2).
P. kessleri has been recently described as a novel, high potential lipid producer. Under nutrient sufficient conditions this microalga displays high biomass productivities and via nutrient limitation also high lipid productivities. Tests were performed under N-, S- and P-limiting conditions as well as limitations by dilution of mineral media, at laboratory and pilot-plant (outdoor) scales. Diluted nutrient medium (10fold) was identified as the best method to stimulate lipid overproduction with 500mgL−1500 \mathrm{mg} \mathrm{L}^{-1} day −1^{-1} lipid productivity at
25%25 \% DW lipid content and reaching high biomass concentrations of 14.0 g L−114.0 \mathrm{~g} \mathrm{~L}^{-1} in thin layer PBR (Li et al. 2012; Table 2).

The authors Pribyl et al. (2012) emphasised the importance of transferring the laboratory-scale productivity to pilot-scale (outdoor) cultivation. Their results showed that although the strain C. vulgaris CCALA 256 displayed very high productivity ( 1425mgL−11425 \mathrm{mg} \mathrm{L}^{-1} day −1^{-1} ) and lipid content ( 58.1%DW58.1 \% \mathrm{DW} ) in lab-scale cultivation, these parameters were not fully transferred into outdoor cultures ( 326mgL−1326 \mathrm{mg} \mathrm{L}^{-1} day −1,30.6%DW^{-1}, 30.6 \% \mathrm{DW} ). In spite of this, they achieved the second highest productivity in pilot-scale cultures ( >40 L>40 \mathrm{~L} ), being surpassed only by the outdoor cultures of P. kessleri CCALA 255 (Table 2). In both cases, the productivities were achieved in a thin layer photobioreactor (Doucha and Livanský 2006). However, the decreased lipid productivity after scale-up was not unique for C. vulgaris CCALA 256 and was observed also for Chlorella sp. NJ-18 (Table 2). The lipid productivity of this strain was half of the laboratory-scale culture, after transfer into a 70 L flat panel photobioreactor (Zhou et al. 2013).

While N -limiting conditions lead to an increase in the lipid content, they also significantly lower the biomass productivity, thereby resulting in lower lipid productivity. A solution to this problem is represented by a so called two-stage “fattening” cultivation strategy. Firstly, a nutrient-rich medium is used to ensure high growth rate of the microalgal cells. Subsequently, when the biomass concentration reaches a high level, the lipid accumulation is triggered as the culture is switched to a “fattening” medium of a different composition ensuring nutrient stress. For example, this strategy has been used with S. obliquus CNW-N where in the first stage the cells were grown on a nutrient-rich medium for 12 days, and the culture was then transferred to nutrient-deprived media (Table 2). Nutrient-deprived (i.e., deionised water) conditions proved to be the most effective for this strain in promoting lipid accumulation and improving the amount of C 16 and C 18 fatty acid groups to enhance the quality of the resulting biodiesel (Ho et al. 2010). Two-stage processes for efficient lipid production are frequently used also in the case of other species, as it can be demonstrated by C. vulgaris. Again the process is composed of rapid cell growth under nutrient replete conditions followed by incubation under N -depleted conditions. A timely termination of incubation under N depleted conditions is recommended as the lipid content

reaches its maximum between 12−24 h12-24 \mathrm{~h} followed by a gradual decrease (Muitaba et al. 2012) (Table 2). A review discussing other strategies of enhanced lipid production by various microalgae species has been published recently (Sharma et al. 2012).

Nevertheless, it must be also emphasised that the lipid content is dependent not only on the N limitations but also on the trophic conditions. The yield of bio-oil ( 57.9%57.9 \% ) produced from heterotrophic Chlorella protothecoides cells was 3.4 times higher than from autotrophic cells (Miao and Wu 2004). Furthermore, the type of N source can also significantly affect both cell growth and lipid accumulation as has been shown for example in the case of Neochloris oleoabundans cultivated as limited on 3 different N sources (urea, ammonium and nitrate), with sodium nitrate being the most favorable N source (Table 2). In addition, N. oleoabundans has up to 80%80 \% of its total lipid content in the form of TAGs, mostly consisting of saturated fatty acids of 16-20 carbons, which is ideal for biodiesel production (Li et al. 2008). Similar observations were made for Chlorella sp. and Scenedesmus sp. where NaNO3\mathrm{NaNO}_{3} was found to be the best limiting N source for enhancing lipid accumulation as compared to KNO3\mathrm{KNO}_{3}, urea and (NH4)2CO3\left(\mathrm{NH}_{4}\right)_{2} \mathrm{CO}_{3}. The lipid content of Chlorella sp. MIC-G4 and Scenedesmus sp. MIC-G8 were enhanced from 40 to 50%50 \% over their controls (Ratha et al. 2013).

Although numerous studies have been performed, there is poor understanding of (i) interactions between microalgal growth and lipid content, (ii) kinetics of lipid accumulation and (iii) the magnitude of N limitation required for lipid accumulation. Some of these relationships were investigated by Adams et al. (2013) in six species of oleaginous green algae, comparing high ( 4 mM N , high stress) and low ( 11 or 16 mM N , low stress) levels of N limitation (Table 2). N stress typically had disproportionate effects on growth and lipid content, with profound differences among species. Optimally balancing the trade-offs required a wide range in N supply rate among species. Some species grew first and then accumulated lipids (Chlorella sorokiniana and Scenedesmus naegelii), whilst other species grew and accumulated lipids concurrently (Chlorococcum oleofaciens, C. vulgaris, N. oleoabundans and Scenedesmus dimorphus), which resulted in increased lipid productivity. Accumulation of high-lipid content generally resulted from a response to minimal stress. The data emphasise the tremendous biodiversity that may be exploited to optimally produce lipids with precision N stress, and furthermore, it displays the wide variation in the extent to which each species combined or separated their growth and lipid accumulation phases. The importance of harvest timing is highlighted, as an optimal harvest window may be brief, just prior to growth and lipid-accumulation cessation (Adams et al. 2013). However, not only well-defined N stress can promote lipid productivities but also direct pH control and culture mode as shown in the case of semi-continuous cultured C. pyrenoidosa, which reached a lipid
content of 75%DW75 \% \mathrm{DW} and productivity of 115mgL−1115 \mathrm{mg} \mathrm{L}^{-1} day −1^{-1} (Table 2; Han et al. 2013).

Not only the total lipid content of the biomass but also the relative amount of the long-chained polyunsaturated fatty acids (LC-PUFAs) can be influenced by N limitation and other stress conditions (salinity, high irradiance; Khozin-Goldberg et al. 2011). It was even shown that the expression of the stearoyl-ACP-desaturase gene (sad) was 2.6 -fold higher under N -limiting conditions than under N -sufficient conditions in B. braunii (Choi et al. 2011). This green colonial microalga, which is widespread in fresh and brackish waters, shows a surprising ability to synthesise extraordinary hydrocarbons like nn-alkadiene and triene hydrocarbons (C23-C33), botryococcenes (triterpenoid hydrocarbons C30-C37), methylated squalenes (C30-C31), lycopadiene (tetraterpenoid hydrocarbon), ether lipids and triacylglycerides. The synthesis of these compounds is related to normal growth and does not occur in N - and P -depleted media (Metzger and Largeau 2005). Under optimised conditions (modified BG-11 medium), the overall lipid productivity of B. braunii reaches 190mgL−1190 \mathrm{mg} \mathrm{L}^{-1} day −1^{-1} (Table 2; Tran et al. 2010).

The microalga Parietochloris incisa is exceptionally capable of accumulating very high amounts of arachidonic acid (AA)-rich triacylglycerols and is recognised as the richest vegetal source of AA. N deprivation/limitation brought about an increase in the arachidonic acid proportion of total fatty acids (from 2.5−5%2.5-5 \% to 10−16%10-16 \% AA of DW) with productivities 36mgL−136 \mathrm{mg} \mathrm{L}^{-1} day −1^{-1} (Table 2). Thus, adjustments to cultivation conditions could serve as an efficient tool for manipulation of yield and relative content of arachidonic acid in P. incisa (Solovchenko et al. 2008; Tababa et al. 2012).

Nevertheless, Markou et al. (2012) emphasise that the majority of studies discussing nutrient limitation, as a strategy to improve the biomass composition, deal with nutrient limitation by omitting the given nutrient from the beginning of batch cultivations of algae (Table 2). This usually results in low specific growth rates (ca. 0.1 day −1^{-1} ) and low biomass concentration (ca. 1.5 g L−11.5 \mathrm{~g} \mathrm{~L}^{-1} ). Thus, they emphasise the importance of nutrient concentration optimisation (using fractional factorial design, central composite design) in the one hand to support adequate biomass production whilst, on the other hand, to act as limiting factor to control the biomass composition. These optimised limited media are reported to be superior to the absolute deprivation of nutrients (Tran et al. 2010; Burrows et al. 2008; Markou et al. 2012).

Pigment overproduction
Accumulation of secondary carotenoids (e.g. β\beta-carotene, astaxanthin and its acylesters) is another main characteristic of many algae when growing under N -limiting conditions ( Hu 2004). An exception was the decrease of the primary xanthophyll lutein content in N -deprived Muriellopsis sp. (Del Campo

et al. 2000). Related topics are broadly reviewed (Del Campo et al. 2007).

Compared to lipids, the production of pigments is well established at large scale. H. pluvialis is used for the commercial production of astaxanthin in a two-stage cultivation system, where the biomass is cultivated under nutrient replete conditions in the first stage and afterwards, in the second stage of cultivation, the cells are subjected to N limitation. Finally, the biomass is harvested when astaxanthin content reaches more than 1%1 \% of the algal DW (Boussiba 2000). The green unicellular alga, D. salina, is used for the natural production of β\beta-carotene. It is capable of accumulating carotenoids in oil globules in the interthylakoid space of the chloroplasts. Under specific stress conditions (temperature, light intensity and N limitation), β\beta-carotene forms up to 12%12 \% of the algal DW (Borowitzka 2013). Similarly to astaxanthin production, cultivation is designed as a two-stage process. In the first stage, the biomass is cultivated under optimal growth conditions. Afterwards the culture is diluted to about one third (increase in light availability) and carotenogenesis is further enhanced by N limitation (Ben-Amotz 1995). D. salina displays high β\beta-carotene productivities upon N deprivation, but other factors (e.g. light) were found to trigger the desired effect even more (Lamers et al. 2012). A review depicting the regulatory mechanisms involved in β\beta-carotene overproduction in DD. salina has been published (Lamers et al. 2008).

Phosphorus

Phosphorus ( P ) is an essential macronutrient necessary for normal growth of all algae. Algal biomass usually contains less than 1%1 \% of P , but under conditions of “luxury uptake,” its content can exceed 3%3 \% by DW (Powell et al. 2009). It is a component of crucial importance for nucleic acid and phospholipid biosynthesis, modification of protein function and energy transfer (Moseley and Grossman 2009). P concentration is often growth-limiting in natural aqueous environments, because it is easily bound to other ions (CO32−,Fe3+)\left(\mathrm{CO}_{3}{ }^{2-}, \mathrm{Fe}^{3+}\right) resulting in its precipitation, and consequently, the nutrient is unavailable for algal uptake. Algae vary amongst themselves in their ability to utilise organic P as the PO43−\mathrm{PO}_{4}{ }^{3-} from organic compounds must be hydrolysed by extracellular phosphatases (Becker 1994). Alkaline phosphatases are induced by P limitation in both Chlamydomonas and Chlorella, but interestingly, acid phosphatase activity is induced by N limitation (Kruskopf and Du Plessis 2004). Pyrophosphate (P2O74−)\left(\mathrm{P}_{2} \mathrm{O}_{7}{ }^{4-}\right) is not utilised as readily as orthophosphate (PO43−)\left(\mathrm{PO}_{4}{ }^{3-}\right). The major form in which algae acquire P is inorganic phosphate either as mathrmH2mathrmPO4−orormathrmHPO42−.Thetransportof.ThetransportofmathrmPO43−\mathrm{H}_{2} \mathrm{PO}_{4}{ }^{-}oror \mathrm{HPO}_{4}{ }^{2-}.Thetransportof. The transport of \mathrm{PO}_{4}{ }^{3-}mathrmH2mathrmPO4−orormathrmHPO42−.Thetransportof.ThetransportofmathrmPO43− into the microalgal cell is an energydependent process. Under P-replete conditions during so-called luxury uptake, microalgae form large polyphosphate granules that serve as internal P storage and are metabolised under P deprivation (Grobbelaar 2004; Eixler et al. 2006). This ability
can be exploited in biological P removal from wastewater (Powell et al. 2008). Interestingly, light stress influences P uptake from the medium. Seawater phytoplankton grown under high irradiance (light stress) contained larger amounts of P, namely, orthophosphate monoesters (including sugar phosphates, inositol phosphates and orthophosphate diester degradation products) than when grown under low light (CadeMenun and Paytan 2010).

In the case of freshwater phytoplankton, P is often the main growth-limiting nutrient and one response to exogenous P enrichment is its rapid incorporation into the biomass, where P is stored as intracellular polyphosphate. Polyphosphate bodies in eukaryotic algae represent another form of cell protection from metal toxicity as they can bind incoming metals in a detoxified complex. C. reinhardtii accumulated Cu and Cd significantly under high ambient P conditions and survived the metals’ toxic effects, as P enrichment of the cells increased their binding capacity by producing polyphosphate bodies (Wang and Dei 2006). Furthermore, in C. reinhardtii, the simultaneous interaction of Cd exposure (toxicity) and P availability (limitation) can have an impact on homeostasis of specific trace metals (Co,Fe,Cu(\mathrm{Co}, \mathrm{Fe}, \mathrm{Cu} and Zn$)andmacronutrients( and macronutrients ( \mathrm{K}, \mathrm{Na}$; Webster et al. 2011). Due to its cellular polyphosphate content, Scenedesmus acutus f. alternans can reduce the toxic effect of Cu on photosynthesis (Twiss and Nalewajko 1992). Polyphosphate bodies or granules are also widely distributed amongst other microbial species, where they serve as modulators to overcome stress. Additionally, they can be involved in acclimation to extreme environments, an essential ability for a cell’s survival (Seufferheld et al. 2008).

Microalgal response to P deprivation/limitation

The effects of P nutrition on the physiology and biochemistry of the green alga Selenastrum minutum was investigated in a chemostat under conditions of either P limitation or nutrient sufficiency. P limitation resulted in the following effects: lower growth rate and reduced concentrations of protein and chlorophylls, fivefold lower rate of respiration, threefold decline in rates of photosynthetic CO2\mathrm{CO}_{2} fixation and O2\mathrm{O}_{2} evolution. In contrast, the rate of dark CO2\mathrm{CO}_{2} fixation was stimulated about threefold. P limitation also resulted in a decreased level of total adenylates to only 23%23 \% and reduced ADP and ATP levels to 9%9 \% and 30%30 \%, respectively, as compared to the nutrient sufficient controls (Theodorou et al. 1991). Similar findings on photosynthetic downregulation during P deprivation were found in C. reinhardtii (Wykoff et al. 1998) and Chlorella kessleri (Elsheek and Rady 1995). Simultaneously, P limitation was found to provoke rapid changes in carbon allocation in Scenedesmus subspicatus with accumulation of lipid and carbohydrate. The accumulation of energy-rich storage compounds is reportedly triggered by a critical intracellular rather than extracellular P concentration (Sigee et al. 2007).

P deprivation was also studied in synchronous cultures of S. quadricauda. Daughter cells produced by P-deprived mother cells and grown further in a P-free medium performed no net RNA, DNA or protein synthesis and were unable to develop, but they accumulated large amounts of starch (Zachleder et al. 1988). The extracellular phosphorous deprivation caused significant starch accumulation also in freshwater C. vulgaris (Brányíková et al. 2011; Dragone et al. 2011) and marine green microalga Tetraselmis subcordioformis (Yao et al. 2013). A stress indicator accompanying starch accumulation under P limitation in T. subcordioformis was an increased Car/Chl\mathrm{Car} / \mathrm{Chl} ratio. Nevertheless, as it can be seen in Table 2, C. vulgaris showed significantly higher starch productivity under P deprivation. Another peculiar effect of P supply was observed in microalgae used as high-quality feedstock for aquaculture. Under strong P limitation, algae developed thicker cell walls that led to digestion resistance upon consumption by zooplankton, thus causing growth limitation in aquaculture (DeMott and Van Donk 2013). The increasing extent of P limitation can provoke different responses in algae. Some of these responses are quantified in Table 2 as well as described in the following paragraphs.

The effect of P on biomass composition was analysed using a freshwater Chlorella sp. strain (Liang et al. 2013). Both lipid content and productivity were enhanced under low P concentration (32μM)(32 \mu \mathrm{M}), but their values were significantly lower than the best results achieved under N limitation (Table 2). The change in the fatty acid profiles was not significant under the various tested conditions with minimum 72%72 \% of saturated fatty acids at P concentration 16μM16 \mu \mathrm{M}. Simultaneously, the protein content was unaffected by external P concentrations, indicating that protein synthesis is influenced mainly by N rather than P supply. On the other hand, the carbohydrate content was the lowest at P concentration of 32μM32 \mu \mathrm{M} (Liang et al. 2013).

The P-limited cultures of Dunaliella tertiolecta did not contain more fatty acids than the P -sufficient cultures; however, there was a difference in the fatty acid composition observed as a relative increase of oleic acid to the detriment of linolenic acid (Siron et al. 1989).

Growing S. obliquus under P-deprived conditions increased lipid content from 10 to 29.5%29.5 \% DW (Mandal and Mallick 2009). Somewhat less significant lipid accumulation ( 19.4%19.4 \% DW) was achieved by P limitation of S. dimorphus. Although the increasing P limitation also decreased the specific growth rate and biomass yield of SS. dimorphus, the highest lipid productivity was found at the lowest initial P concentration (Table 2), (Ruangsomboon et al. 2013). Conversely, in conditions of P limitation (0.1mgL−1)\left(0.1 \mathrm{mg} \mathrm{L}^{-1}\right), Scenedesmus sp. LX1 could accumulate lipids to as high as 53%53 \% DW of algal biomass; however, the productivity of lipids per unit volume was not enhanced (Xin et al. 2010).

Phosphorous limitation usually does not represent the best choice to achieve the highest accumulation of storage
compounds in green microalgae. For instance, higher relative starch and lipid content was achieved by S (Yao et al. 2013; Brányíková et al. 2011) and N limitation/deprivation (Ruangsomboon et al. 2013; Mandal and Mallick 2009), respectively (Table 2). Nevertheless, optimisation (response surface methodology) of P limitation, in combination with N limitation and thiosulphate supplementation led to an additional increase in lipid yield ( 58.3%58.3 \% ) as compared to N deprivation ( 43%43 \% ) (Mandal and Mallick 2009). It is also important to emphasise that in the case of producing large quantities of low value products (e.g. biofuels), the processing costs must be kept as low as possible. Thus, it is desirable to achieve high yields of biomass with the desired composition whilst minimising the use of essential nutrients including phosphorous.

In cultivations aimed at biomass production of Scenedesmus sp. the amount of continuously fed P was so high that it caused a luxury uptake, i.e. an over-uptake of P without growth. This led to more phosphorous consumption without more biomass being produced and consequently to low biomass yields of phosphorous (YX/P)\left(Y_{X / P}\right). Conversely, the sustained growth of biomass after the exhaustion of PO43−\mathrm{PO}_{4}{ }^{3-} in a P deprivation mode led to a significant increase in YX/PY_{X / P} up to 160 g biomass g−1P\mathrm{g}^{-1} \mathrm{P}, which was nearly six times more than that with P replete feeding (Wu et al. 2012).

P availability can also greatly influence metabolism in CC. reinhardtii where sustained H2\mathrm{H}_{2} photoproduction occurred upon incubating the cells under P-deprived conditions in light for prolonged periods of time. The physiological response demonstrated significant similarities to the algal response to S depletion. As in S-deprived algae, the acclimation of algal cells to P-deprived conditions was accompanied by the accumulation of starch during the O2\mathrm{O}_{2}-production stage and its degradation during the H2\mathrm{H}_{2} production stage. Cells also consumed acetate during the early stages of P deprivation, when inactivation of the photosynthetic apparatus occurred. After the establishment of anaerobiosis, however, acetate is produced and excreted into the growth medium, apparently due to fermentation of starch (Batyrova et al. 2012).

Sulphur

Sulphur (S) is an essential macronutrient required for key cellular processes. It participates in the synthesis of S-amino acids, membrane sulpholipids, cell walls, thiol compounds such as glutathione (GSH) that participate in the stress response, vitamins such as thiamine and biotin, thioether and thioester compounds such as coenzyme A and S-adenosyl-Lmethionine. An essential cellular phenomenon is the formation of disulphide bonds (Leustek et al. 2000; Takahashi et al. 2011). Because most organisms have only a limited supply of stored S , their growth and development is dependent upon a continuous supply of this nutrient from the environment

(Zhang et al. 2004). While our planet’s atmosphere changed from being anoxygenic to oxygenic, S occurring in oceans and freshwaters underwent major changes in chemical form and availability (Takahashi et al. 2011). Nowadays, the majority of organisms primarily use free SO42−\mathrm{SO}_{4}{ }^{2-} as the S source for their nutrition, but inorganic SO42−\mathrm{SO}_{4}{ }^{2-} is often not the most common form of SS in the environment. For example, in many soils, it represents only a small fraction of the total S content ( 1−15%1-15 \% ) as most of the soil SS is covalently bound to organic molecules (Kauss 1987). Plants occupy a unique position in the global SS cycle as they are the primary producers of organic Scompounds (Leustek et al. 2000).

For cost-effective phototrophic microalgal biomass cultivation, the SS source is provided as cheap inorganic SO42−\mathrm{SO}_{4}{ }^{2-}, but eukaryotic microalgae have been reported to have the capability of using other S sources such as methionine (Buetow 1965), cysteine (during heterotrophic growth; Harwood and Nicholls 1979) and naphthalenesulphonic acids (Soeder et al. 1987). In the case of B. braunii, even mathrmHSO2−andandmathrmSO22−\mathrm{HSO}_{2}{ }^{-}andand \mathrm{SO}_{2}{ }^{2-}mathrmHSO2−andandmathrmSO22− can be utilised within a certain concentration range, introducing the possibility of using this algal species to remove remaining SS from desulphurized flue gases (Yang et al. 2004a). For example, a strain of Chlorella fusca was able to grow on more than 100 different S sources e.g. mercaptides, disulphides, thioethers, sulphinic and sulphonic acids, sulphoxides, sulphones and sulphate esters (Krauss and Schmidt 1987).

S uptake is an active process that is driven by phosphorylation and is temperature-sensitive. Similar to NO3−\mathrm{NO}_{3}{ }^{-}, most of the SO42−\mathrm{SO}_{4}{ }^{2-} taken up by microalgae has to be reduced, mostly in plastids, before it is incorporated into cellular components (Becker 1994; Takahashi et al. 2011). As green microalgae are regarded as ancestral to (embryophytic) plants, some of their processes can be expected to be similar to those in higher plants. Depending on the demand for reduced SS, its uptake and assimilation in plant cells is strictly regulated at different key points. The assimilation pathway is repressed when reduced S or thiols are available and is promptly activated by S limitation. Regulatory mechanisms of S metabolism described in plants include transcriptional, post-transcriptional, proteinprotein interactions and feedback controls (Carfagna et al. 2011). Additionally, SO42−\mathrm{SO}_{4}{ }^{2-} assimilation is intertwined with the metabolism of N and C (Takahashi et al. 2011).

All organisms are capable of acclimation under nutrient limited conditions as it is essential for their survival. The following cellular responses are very complex and highly regulated. If the concentration of an essential nutrient falls below a critical level (e.g. in the case of SO42−\mathrm{SO}_{4}{ }^{2-}, it is approximately 10μM10 \mu \mathrm{M} ), the cells perceive the limitation and express a specific set of genes in order to alter their physiology and acclimate to the deficiency (Kauss 1987). Thus, as in the case of N,P\mathrm{N}, \mathrm{P} and S supply have also been used to modify microalgal metabolism for production of biotechnologically attractive compounds.

Microalgal response to S deprivation/limitation

In studies concerning the molecular mechanisms of the metabolic response under S-limiting conditions, C. reinhardtii is frequently used as a model organism. Generally, responses of photosynthetic organisms to SS deprivation include (i) increasing the capacity of the cell for transporting and/or assimilating exogenous SO42−\mathrm{SO}_{4}{ }^{2-}, (ii) restructuring cellular features to conserve S resources, and (iii) modulating metabolic processes and rates of cell growth and division (Zhang et al. 2004). Under S deprivation, an acclimation response in Chlamydomonas occurs, i.e. the cells downregulate photosynthesis whilst simultaneously increasing SO42−\mathrm{SO}_{4}{ }^{2-} uptake. Furthermore, a signal transduction pathway is activated leading to the expression of an extracellular arylsulphatase (Ars). This enzyme then cleaves SO42−\mathrm{SO}_{4}{ }^{2-} from aromatic sulphate esters, releasing free SO42−\mathrm{SO}_{4}{ }^{2-} ions which can then be imported into the cell (Leustek et al. 2000; Zhang et al. 2004). A detailed description of this response mechanism to S limitation is described by Takahashi et al. (2011). The decrease in photosynthesis is essential for surviving severe SS stress as a mutant unable to decrease the photosynthetic process cannot survive these conditions (Kauss 1987). In C. reinhardtii, prolonged S deprivation also induces various changes in gene expression including the translation of proteins that secure SO42−\mathrm{SO}_{4}{ }^{2-} supply by degrading intracellular proteins (Zhang et al. 2004). C. sorokiniana is another microalgal species capable of rapid acclimation to changes in external SO42−\mathrm{SO}_{4}{ }^{2-} supply. During the acclimation procedure of SO42−\mathrm{SO}_{4}{ }^{2-}-deprived C. sorokiniana, various fundamental physiological processes changed, which resulted in strongly reduced growth, decreased photosynthetic capacity, changes in N assimilation (i.e. rapid inhibition of mathrmNH4+uptake),andincreasedstarchcontentandfreenon−Saminoacids.Uponuptake),andincreasedstarchcontentandfreenon−Saminoacids.UponmathrmSO42−resupplyafter24−hstarvation,thesituationreversed:photosyntheticactivityincreased,cellgrowthwasrenewed(inlightbutnotindarkness),respiratoryoxygenconsumptionandstarchdegradationwereenhanced,resupplyafter24−hstarvation,thesituationreversed:photosyntheticactivityincreased,cellgrowthwasrenewed(inlightbutnotindarkness),respiratoryoxygenconsumptionandstarchdegradationwereenhanced,mathrmNH4+depletionoccurred,andthecontentofnon−Saminoacidsdecreasedwhereasthecysteinecontentincreased(DiMartinoRiganoetal.2000).ThedependencyofNmetabolism(Nassimilationandthusproteinsynthesis)upontheavailabilityofScompoundshasbeenconfirmedbyotherstudiesaswell(Carfagnaetal.2011).Furthermore,Carfagnaetal.(2011)proposedapossiblemechanismthatensurestheviabilityofC.sorokinianacellsunderSdeprivation,i.e.whencellsaredeprivedofexternaldepletionoccurred,andthecontentofnon−Saminoacidsdecreasedwhereasthecysteinecontentincreased(DiMartinoRiganoetal.2000).ThedependencyofNmetabolism(Nassimilationandthusproteinsynthesis)upontheavailabilityofScompoundshasbeenconfirmedbyotherstudiesaswell(Carfagnaetal.2011).Furthermore,Carfagnaetal.(2011)proposedapossiblemechanismthatensurestheviabilityofC.sorokinianacellsunderSdeprivation,i.e.whencellsaredeprivedofexternalmathrmSO42−\mathrm{NH}_{4}{ }^{+}uptake),andincreasedstarchcontentandfreenon−Saminoacids.Uponuptake), and increased starch content and free non-S amino acids. Upon \mathrm{SO}_{4}{ }^{2-}resupplyafter24−hstarvation,thesituationreversed:photosyntheticactivityincreased,cellgrowthwasrenewed(inlightbutnotindarkness),respiratoryoxygenconsumptionandstarchdegradationwereenhanced, resupply after 24-h starvation, the situation reversed: photosynthetic activity increased, cell growth was renewed (in light but not in darkness), respiratory oxygen consumption and starch degradation were enhanced, \mathrm{NH}_{4}{ }^{+}depletionoccurred,andthecontentofnon−Saminoacidsdecreasedwhereasthecysteinecontentincreased(DiMartinoRiganoetal.2000).ThedependencyofNmetabolism(Nassimilationandthusproteinsynthesis)upontheavailabilityofScompoundshasbeenconfirmedbyotherstudiesaswell(Carfagnaetal.2011).Furthermore,Carfagnaetal.(2011)proposedapossiblemechanismthatensurestheviabilityofC.sorokinianacellsunderSdeprivation,i.e.whencellsaredeprivedofexternaldepletion occurred, and the content of non-S amino acids decreased whereas the cysteine content increased (Di Martino Rigano et al. 2000). The dependency of N metabolism ( N assimilation and thus protein synthesis) upon the availability of S compounds has been confirmed by other studies as well (Carfagna et al. 2011). Furthermore, Carfagna et al. (2011) proposed a possible mechanism that ensures the viability of C. sorokiniana cells under S deprivation, i.e. when cells are deprived of external \mathrm{SO}_{4}{ }^{2-}mathrmNH4+uptake),andincreasedstarchcontentandfreenon−Saminoacids.Uponuptake),andincreasedstarchcontentandfreenon−Saminoacids.UponmathrmSO42−resupplyafter24−hstarvation,thesituationreversed:photosyntheticactivityincreased,cellgrowthwasrenewed(inlightbutnotindarkness),respiratoryoxygenconsumptionandstarchdegradationwereenhanced,resupplyafter24−hstarvation,thesituationreversed:photosyntheticactivityincreased,cellgrowthwasrenewed(inlightbutnotindarkness),respiratoryoxygenconsumptionandstarchdegradationwereenhanced,mathrmNH4+depletionoccurred,andthecontentofnon−Saminoacidsdecreasedwhereasthecysteinecontentincreased(DiMartinoRiganoetal.2000).ThedependencyofNmetabolism(Nassimilationandthusproteinsynthesis)upontheavailabilityofScompoundshasbeenconfirmedbyotherstudiesaswell(Carfagnaetal.2011).Furthermore,Carfagnaetal.(2011)proposedapossiblemechanismthatensurestheviabilityofC.sorokinianacellsunderSdeprivation,i.e.whencellsaredeprivedofexternaldepletionoccurred,andthecontentofnon−Saminoacidsdecreasedwhereasthecysteinecontentincreased(DiMartinoRiganoetal.2000).ThedependencyofNmetabolism(Nassimilationandthusproteinsynthesis)upontheavailabilityofScompoundshasbeenconfirmedbyotherstudiesaswell(Carfagnaetal.2011).Furthermore,Carfagnaetal.(2011)proposedapossiblemechanismthatensurestheviabilityofC.sorokinianacellsunderSdeprivation,i.e.whencellsaredeprivedofexternalmathrmSO42−, they degrade intracellular GSH and proteins, thus meeting the S supply requirements. Additionally, these authors suggest that cysteine (and possibly GSH) could be “sensors” of the cell’s S status, thus regulating the rate of SS assimilation.

The metabolic pathways of individual macronutrients are closely linked. For example C,N\mathrm{C}, \mathrm{N} and S are the essential components of all proteins, and so a limitation in any one of

these macronutrients stops protein biosynthesis. The plant responds by increasing the assimilation of the limiting nutrient and at the same time adjusts its capacity, flux rates and intermediate storage options for the non-limiting nutrients. Prolonged or severe C,N\mathrm{C}, \mathrm{N} or S deprivation affect the photosynthetic apparatus (i.e. photosynthetic energy acquisition through electron transport chains) and photosynthetic efficiency (Lee and Liu 1999). The effect of SS limitation on N and C metabolism was studied using the extremophile D. salina, a halotolerant algal species that is mass cultivated for the commercial production of glycerol and β\beta-carotene (Giordano et al. 2000).

As described above, nutrient stressed cells alter their metabolism, ensuring the production of specific biotechnologically attractive metabolites (Table 2). Deprivation and/or low concentrations of N,P\mathrm{N}, \mathrm{P} or S can lead to an increase in the carotenoid/ chlorophyll ratio. S deprivation was found to trigger lipid (TAGs) accumulation in cytoplasm of Chlorella sp. and C. reinhardtii (Hu et al. 2008; Cakmak et al. 2012). Limitation of N,P\mathrm{N}, \mathrm{P} or S also enables production of algal biomass rich in starch (Brányíková et al. 2011). In C. vulgaris, macronutrient limitation resulted in starch overproduction (Table 2) not only at the laboratory scale but also in a pilot-scale outdoor photobioreactor, the acquired biomass being a possible precursor for bioethanol production (Maršálková et al. 2010; Ho et al. 2013). Under these conditions, S limitation turned out to be the technologically most applicable limitation strategy, as compared to N - and P -deprived cells, because the S -limited cells had the highest starch content ( 60%60 \% DW in lab scale, 48%48 \% DW in pilot scale), best viability and stable starch content. Consequently, the interval between obtaining maximum starch content and cell death caused by SS limitation was sufficient for comfortable biomass harvesting, whilst in the case of N and P limitation, cell death occurred rapidly (Brányíková et al. 2011). In large-scale cultures, the timing of starch synthesis through nutrient limitation is important. Upon nutrient deprivation, cell growth and division are either slowed or stopped completely. Thus, in order to maintain high starch productivity, it is crucial to ensure an initial fast cell growth and then, after obtaining enough biomass, trigger starch accumulation. One must also keep in mind that in the case of photosynthetic cultivation, intracellular storage compounds are degraded during the dark period, because the cells require energy for cellular maintenance (Brányíková et al. 2011). It can be concluded that photosynthetic cultivation of C. vulgaris under S deprivation conditions affects many crucial cellular processes, but not the synthesis of reserve compounds. This phenomenon was also observed in synchronous cultures of S. quadricauda. If daughter cells were incubated under photosynthesising conditions in S-free medium, macromolecular syntheses (proteins, nucleic acids) were greatly reduced. Only the photosynthetic process continued to produce starch at a similar rate as in normally grown cells, leading to rapid accumulation of this polysaccharide (Šetlík et al. 1988). This aspect of starch overproduction
under S limitation for biofuel applications has been extensively studied not only in freshwater species, e.g. C. vulgaris (Brányíková et al. 2011; Dragone et al. 2011), but also in marine species, e.g. T. subcordiformis (Yao et al. 2012). Both species showed similar starch productivities under SS deprivation (Table 2).

In addition, SO42−\mathrm{SO}_{4}{ }^{2-} can also have other effects on a cell’s composition, e.g. as they compete with SeO42−\mathrm{SeO}_{4}{ }^{2-} for transport sites in C. reinhardtii. Additionally, the toxicity of Se on microalgal species is correlated with its intracellular accumulation, which is directly dependent on the availability of SO42−\mathrm{SO}_{4}{ }^{2-} (Fournier et al. 2010).

Micronutrients

Besides macronutrients, a healthy microalgal population also requires micronutrients (e.g. Fe,Mn,Zn,Ni,B,V,Co,Cu\mathrm{Fe}, \mathrm{Mn}, \mathrm{Zn}, \mathrm{Ni}, \mathrm{B}, \mathrm{V}, \mathrm{Co}, \mathrm{Cu}, Mo,Se\mathrm{Mo}, \mathrm{Se} ), but there is frequently only a very small difference between a nutritional, growth-promoting effect and cell toxicity, as these elements are required in very small amounts of microgram, nanogram or even picogram per liter (Becker 1994). Micronutrients usually serve as cofactors in a variety of metabolic pathways essential for microalgal growth. Cellular requirements for micronutrients can be influenced by the availability of other essential resources like light, N,P,CO2\mathrm{N}, \mathrm{P}, \mathrm{CO}_{2}, etc. For example: (i) light intensity and photoperiod duration influence Fe and Mn requirements; (ii) the form of N source (e.g. NH4+,NO3−\mathrm{NH}_{4}{ }^{+}, \mathrm{NO}_{3}{ }^{-}, urea) determines the need for Fe,Mo\mathrm{Fe}, \mathrm{Mo} or Ni ; (iii) CO2\mathrm{CO}_{2} acquisition is connected to Zn/Co/Cd\mathrm{Zn} / \mathrm{Co} / \mathrm{Cd} supply; (iv) Zn participates in P uptake (Sunda et al. 2005); (v) the mode of growth is also connected to micronutrient demand, e.g. CC. pyrenoidosa requires more Mn,Fe,Zn\mathrm{Mn}, \mathrm{Fe}, \mathrm{Zn} and mathrmNO3−\mathrm{NO}_{3}{ }^{-}mathrmNO3−and less Mg and K for autotrophic growth than for heterotrophic growth (Iriani et al. 2011). Furthermore, it is also important to point out that in phytoplankton, even a very small increase in growth rates, caused by addition of a macronutrient (e.g. P), can induce micronutrient limitation (Sterner et al. 2004).

The reactions and interactions of micronutrients are very broad. Several micronutrients can interact with one another in cases where more than one can serve the same metabolic functions (Sunda et al. 2005). The enzyme complex in the last step of ethylene synthesis by H. pluvialis is an example of these variable micronutrient effects, as it is stimulated by Co2+\mathrm{Co}^{2+} and Mn2+\mathrm{Mn}^{2+}, inhibited by Cu2+\mathrm{Cu}^{2+} and not affected by Zn2+,Fe2+\mathrm{Zn}^{2+}, \mathrm{Fe}^{2+} or Mg2+\mathrm{Mg}^{2+} (Maillard et al. 1993).

Micronutrients form various complexes with both inorganic and organic ligands, which affect their availability. Some metals, such as Fe , also readily precipitate as oxyhydroxides. Trace metal complexing with ligands and competition between various ions result in decreased interactions of metal ions with uptake sites on the surface of the microorganism. Biological (membrane transport), physical (diffusion) and

chemical (dissociation kinetics of metal complexes) reactions occurring in the immediate proximity of the cell membrane play an important role in trace element bioavailability, biological uptake and toxicity. The biouptake process depends not only on internalisation pathways and their specificity but also on the physicochemical properties of the medium and the size and nature of the microorganism. The use of a single transport site by several metals, or vice versa, and the various strategies occurring once the metal enters the cell (e.g. complex formation, compartmentalisation, efflux or the production of extracellular ligands to minimise the metal’s reactivity) further illustrate the complex manner of micronutrient reactions (Worms et al. 2006).

From a biotechnological point of view, the effect of micronutrients on microalgal biomass composition can be exploited for metal-enriched biomass production and, to a lesser extent, for regulating the cell’s metabolism in order to induce the production of certain metabolites (e.g. enhanced lipid production). The metal-binding capabilities of microalgae have been known for a long time and are used in two biotechnological branches: (i) the bioremediation of heavy metals polluting the environment by biosorption (non-living biomass) or bioaccumulation (living biomass); (ii) the enrichment of microalgal biomass with microelements in order to obtain a product of higher nutritional value. That metalresistant or tolerant algal populations are able to live in heavy-metal polluted water is well known, but the development of metal resistance/tolerance in plants and microorganisms is still not fully understood and various mechanisms can be involved, depending on the metal and the organism (Pawlik-Skowrońska 2003). The various responses of microalgae to oxidative stress induced by metal toxicity are described, e.g. (Pinto et al. 2003), and more details on microalgal defense mechanisms will be described in the following chapters. Again, the broad multifunctional biotechnological applications of microalgae can be highlighted as heavy metal removal from hypersaline leachate was successfully combined with lipid production (Richards and Mullins 2013).

Chlorella sp. is able to accumulate Fe,Cu,Zn,Mn,Co,Se\mathrm{Fe}, \mathrm{Cu}, \mathrm{Zn}, \mathrm{Mn}, \mathrm{Co}, \mathrm{Se}, I and Cr. Its Se-, I-, Cr-, and Fe-enriched biomass has already been tested and marketed as a food and feed supplement (Niedobová et al. 2005; Gómez-Jacinto et al. 2010; Kotrbáček et al. 2004; Milinki et al. 2011; Chojnacka 2007). The following chapters describe in more details the metabolic functions biotechnological applicability of Fe and Se , which effect biomass composition.

Iron

Iron ( Fe ) is quantitatively the most important of the micronutrients and is required for the growth of all phytoplankton (Stefels and van Leeuwe 1998). In major regions of the ocean, it regulates phytoplanktonic growth and food web
structures (Sunda and Huntsman 2004). On a cellular level, Fe fulfills fundamental catalytic roles because of its redox properties and its participation in photosynthetic electron transport chains (Raven et al. 1999; Estevez et al. 2001; La Fontaine et al. 2002), H2\mathrm{H}_{2} photoproduction (Antal et al. 2011), DNA synthesis (Hu 2004) and assimilative nitrate-reducing systems (Cárdenas et al. 1972). During SO42−\mathrm{SO}_{4}{ }^{2-} assimilation by green algae, Fe occurs in an [Fe4 S4]\left[\mathrm{Fe}_{4} \mathrm{~S}_{4}\right] cluster of the multidomain enzyme adenylylsulphate reductase (APS reductase; Takahashi et al. 2011). Together with Cu and Zn , it also occurs in enzymes participating in the detoxification of ROS (Raven et al. 1999).

Fe is largely unavailable to aquatic algae if it forms insoluble hydrous ferric oxide precipitates that can subsequently adsorb other essential metals and lower their availability. If Fe is in a complex with organic ligands, an equilibrium establishes between the complexed, unavailable forms and the dissolved, non-chelated forms. As the free metal ions are removed by the growing algae, they are replaced by dissociation of an equivalent concentration from the metal chelate. Under laboratory conditions, the same phenomenon is used by applying metal ion buffers [e.g. ethylenediaminetetraacetic acid (EDTA)] in order to provide an adequate and nontoxic supply of the essential nutrient (Sunda et al. 2005). Many ferric chelates, such as Fe (III)EDTA, absorb light and undergo photo-redox cycling, which results in the degradation of the ligand and an increase in the concentration of dissolved inorganic Fe (II) and Fe (III) species. This leads to increased algal Fe uptake rates in the light. Thus, diel (light/dark) variations in cellular Fe concentrations are observed (Sunda and Huntsman 2004).

Despite its role as an essential nutrient, Fe is highly reactive and high concentrations can have toxic effects. Stored Fe is released by biological chelates in its active form Fe (II), which reacts with hydrogen peroxide through Fenton or HaberWeiss reactions to generate hydroxyl radicals, thus initiating oxidative stress in the cell and lipid peroxidation in biological membranes (Hu 2004). Consequently, organisms tightly control Fe homeostasis and have highly coordinated responses to Fe limitation/overload. The cellular responses and underlying mechanisms of Fe homeostasis in marine phytoplankton, which enable the microalgae to persist in natural, Fe-limited environments (i.e. the large perennially high-nutrient lowchlorophyll regions of the contemporary ocean) have been reviewed (Morrissey and Bowler 2012; Fernie et al. 2012).

The effect of Fe supply on biomass composition of other eukaryotic microalgae is quite complex as this element influences many metabolic functions. For example, Fe affects cell morphology and the content of starch, α\alpha-tocopherol, ascorbate and total thiol compounds in C. vulgaris, where Fe deprivation leads to starch accumulation, but at high concentrations, it triggers the production of antioxidants and reduced cell growth due to oxidative stress (Estevez et al. 2001). Furthermore, Fe

limitation in C. reinhardtii induced a rapid reduction in photosynthetic pigment levels due to a decrease in chlorophyll synthesis, leading to an overall reduction in photosynthetic efficiency (Yadavalli et al. 2012). In other cases, Fe can also influence the amount and type of cellular lipids, which can be of biotechnological importance. The biomass production of a marine C. vulgaris strain was increased when the medium was directly supplemented with Fe in the late growth phase of the cells, whereas elevated lipid production (up to 56.6%56.6 \% biomass by DW) occurred when the cells were harvested in their lateexponential growth phase and transferred to a new medium with specific, higher Fe concentrations (Table 2). Thus, it seems that lipids begin to accumulate only when the Fe concentration in the initial medium has a certain minimum value before an inoculum is added (Liu et al. 2008). In the case of Botryococcus spp., lipids accumulated when high levels of Fe were combined with a N -limited state and moderate light intensity (Yeesang and Cheirsilp 2011). Another example is D. tertiolecta, where a rapid accumulation of lipid was achieved under N , or to a lesser extent, Fe or Co deprivation, but no effect was observed when Mn, Mo or Zn were limiting (Chen et al. 2011). Nutrient (Mg, Fe,Ca,S,P\mathrm{Fe}, \mathrm{Ca}, \mathrm{S}, \mathrm{P} or K ) starved C. vulgaris or C. reinhardtii cells accumulated neutral lipids depending on the culture medium used (Deng et al. 2011). Another strain tested for biodiesel production was a Scenedesmus rubescens-like microalga that was isolated from the Gulf of Mexico, and is genetically closely related to the green alga Scenedesmus, appearing morphologically similar to S. rubescens. Under low P and N conditions, this strain produces high levels of fatty acid methyl esters (FAME) with a high C18 content (Tan and Lin 2011; Lin and Lin 2011). In addition it has a high growth rate over a wide range of Fe concentrations (0−50mgL−1Fe2+)\left(0-50 \mathrm{mg} \mathrm{L}^{-1} \mathrm{Fe}^{2+}\right), which is unusual for most freshwater and marine microalgae. Varying the Fe concentration in the culture medium can significantly change FAME yield, productivity and content. The main impact of variations in Fe supply on biodiesel production lies in changes within the C18 series (C18: 0, C18: 1n-7 and C18: 1n−91 n-9 ), the C 16 series being similar over all Fe concentrations tested (Lin et al. 2012).

Increasing lipid content and lipid productivity was noted when S. dimorphus KMITL was cultured under high Fe concentration (Table 2) and this enhancement of lipid production was better than that of N and P limitation (Ruangsomboon et al. 2013).

Selenium

It has been reported that some algae require Se for growth, although higher concentrations are reported to be toxic. There is a diversity in Se metabolism among various microalgae taxa (Araie and Shiraiwa 2009) and its importance for microalgal species can be variable (Wheeler et al. 1982). In natural waters this element is normally present at low parts per billion
concentrations, but much higher Se concentrations are to be found in particular industrial wastewaters. Se occurs in the environment in three oxidation states, i.e. Se(−II),Se(IV)\mathrm{Se}(-\mathrm{II}), \mathrm{Se}(\mathrm{IV}) and Se(VI)\mathrm{Se}(\mathrm{VI}), and several chemical forms: (i) highly soluble inorganic selenite (SeO32−)\left(\mathrm{SeO}_{3}{ }^{2-}\right) and selenate (SeO42−)\left(\mathrm{SeO}_{4}{ }^{2-}\right) and (ii) organic compounds such as selenocysteine, selenoproteins, methyl selenides and other Se-substituted analogues of organosulphur compounds (Besser et al. 1993).

Due to its chemical similarity, Se interferes with S metabolism. Se can replace S in methionine non-specifically or can be specifically incorporated into proteins as selenocysteine. Selenocysteine is known as the 21st genetically coded amino acid and it is encoded in a special way by a UGA codon, which is normally a stop codon, but in this case, its function is modified by a subsequent sequence. Selenocysteine amino acid residues are located in the active sites of enzymes (so-called selenoproteins) as they modulate their properties. Selenoproteins (e.g. GSH peroxidases, thioredoxin reductases, several dehydrogenases, hydrogenases and deiodinases, etc.) can be found in all biological domains of Bacteria, Archea, and Eukarya (Low and Berry 1996; Axley and Stadtman 1989). Another evidence for the competition between selenate and sulphate for the uptake by the microalgae (Chlorella sp.) was the improved selenate volatilisation to dimethylselenide in the absence of external sulphate. This situation led to increased uptake of selenate into the microalgae by increasing transporter activity and by reducing the competition between selenate and sulphate for sites on the transporter (Neumann et al. 2003).

At environmental concentrations, selenite ( 96−hIC5096-\mathrm{h} \mathrm{IC}_{50} was 80μM80 \mu \mathrm{M} ) is not likely to have direct toxic effect on C. reinhardtii, but at high concentrations, chloroplasts were the first target of selenite cytotoxicity (Morlon et al. 2005). In comparison, selenate at concentrations low compared to environmental ( 96−h96-\mathrm{h} IC50\mathrm{IC}_{50} was 4.5μM4.5 \mu \mathrm{M} ) caused already growth inhibition, chloroplast alteration and consequently impaired photosynthesis in CC. reinhardtii (Geoffroy et al. 2007). Interestingly, selenite showed higher toxic effect than selenate in S. quadricauda. This can be probably explained by faster conversion of selenite to selenomethionine. Se toxicity further increased in cultures grown under S-deprived conditions. Poisoning by Se caused bleaching of chloroplasts and cell malformation and finally, cell death. A very small fraction of surviving cells was repeatedly cultured in the presence of selenite and/or selenate. The selected strains were resistant to selenite or selenate whilst still sensitive to the other compound. The strain resistant to combinations of both selenite and selenate showed the lowest resistance of all selected strains. Therefore, it appears that there are at least three different and independent mechanisms able to establish resistance to Se compounds. The findings also imply that the dose- and toxicdependent activity of thioredoxin reductase in S. quadricauda was a stress response to Se cytotoxicity (Umysová et al. 2009).

A slightly different behavior was observed in the marine alga Dunaliella sp., which readily took up selenite from the

medium over a broad range of concentrations, but uptake of selenate was generally minimal (Yamaoka et al. 1990). Also D. tertiolecta displays a high resistance to Se and acclimation to increased concentrations (Wong and Olivesra 1991; Reunova et al. 2007).

It can be concluded that an elevated resistance and Se bioaccumulation ability are crucial properties for microalgae used in Se-enriched biomass production. This process is of great significance for solving worldwide nutritional Se deficiencies in human and animal diets, as organically bound Se is more bioavailable and safe compared to the direct intake of its inorganic salts (Schrauzer 2001). Chlorella enriched with organically bound Se can also be produced heterotrophically (Doucha et al. 2009). Commercially attractive strains of SS. quadricauda are capable of growth in the presence of elevated Se concentrations and are cultivatable under both autotrophic and heterotrophic conditions; these have the potential for the production of Se-enriched biomass and have been patented (Doušková et al. 2009a, b, c).

Conclusion

Microalgae have received considerable attention in the scientific and industrial communities due to their biotechnological potential. Lipids for biodiesel or as a feedstock for the chemical industry, ω−3\omega-3 fatty acids, proteins, carbohydrates, pigments, biohydrogen, bioethanol, food supplements, animal feed, etc. are only a few examples of the wide-ranging potential for microalgae. However, there are only a few commercially successful microalgal technologies. The development of an economically feasible industrial-scale production of microalgae, or products derived from them, requires a complex multidisciplinary approach, an integral part of which is cell physiology. This is based on the knowledge of basic biological functions and tools for controlling microalgal metabolism, with the aim of increasing the content of target compounds, or enhancing their productivity. Traditionally, this has been done by optimisation of culture conditions (environmental parameters, medium composition, and photobioreactor design). The manipulation of nutrients is therefore amongst the most efficient tools for the modulation of growth and biomass composition and thus for the optimisation of algal production systems. Nevertheless, it is important to understand that maximum biomass productivity and target compound content usually cannot be obtained simultaneously under the same cultivation conditions. This opens up the possibility of applying different nutritional status situations in order to achieve the desired effects (high growth rate, formation of target compound, biosorption/accumulation, etc.). In general, the processes should be designed keeping in mind that these three parameters: biomass productivity, target compound content and final biomass concentration
(harvesting density) are interconnected and their overall balance determines the economical viability.

Acknowledgment The authors thank the Czech Grant Agency (P503/ 10/1270) for financial support.

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