**3. Nutritional features of microalgae**

Microalgal species can vary significantly in nutritional value, as a function of the prevailing culture conditions. Only a reduced number of species have been used, primarily for historical reasons and ease of cultivation – rather than supported by scientific evidence of any superior performance as nutritional or therapeutical supplements. Hence, formulations more carefully selected of microalgal origin may offer the opportunity for development of improved nutritional packages aimed at larval animals.

Several factors contribute to the nutritional value of a microalga – including its size and shape, and digestibility as related to cell wall structure and composition (as mentioned above), as well as biochemical composition (e.g. accumulation compounds, enzymes and toxins) and specific requirements of the target animal. For this reason, several studies have attempted to correlate the nutritional value of microalgae to their chemical profile. However, results from feeding experiments are often difficult to interpret because of the confounding effects of other formulation additives. An examination of literature data – including those pertaining to microalga-based, compounded diet emulsions, have meanwhile allowed a few general conclusions to be reached [25].

As primary producers in the aquatic food chain, microalgae provide many phytonutrients, including in particular PUFAs – e.g. EPA, arachidonic acid (AA) and DHA, which are known to be essential for various marine animals [25], as well as for growth and metamorphosis of many larvae [8,26]. However, the ratios of DHA, EPA and AA may actually be more important than their absolute levels [24,27]. Most microalgal species exhibit moderate to high percents of EPA (7 to 34%); and prymnesiophytes (e.g. *Pavlova* spp. and *Isochrysis* sp.) and cryptomonads are relatively rich in DHA (0.2 to 11%), whereas eustigmatophytes (e.g. *Nannochloropsis* spp.) and diatoms have the highest percentages of AA (up to 4%). Chlorophytes (*Dunaliella* spp. and *Chlorella* spp.) are deficient in both C20 and C22 PUFAs, although some species have small amounts of EPA (up to 3.2%); because of such a PUFA deficiency, chlorophytes are in general ascribed a poor nutritional value, so they are not suitable for use as single species-diet [6]. Prasinophyte species contain significant proportions of C20 (*Tetraselmis* spp.) or C22 (*Micromonas* spp.), but rarely of both. Therefore, the fatty acid contents of microalgae exhibit systematic differences according to taxonomic group – although there are examples of significant differences between microalgae, even within the same class.

The contents of antioxidants are also not uniform among microalgae; e.g. the concentrations of vitamins and carotenoids convey significant variations among species. Note that any mixed-algal diet should provide adequate concentrations of vitamins and carotenoids to be effective in aquaculture; unfortunately, the nutritional requirements of larval or juvenile animals that feed directly on microalgae are still poorly understood at present. In fact, artificial diets often lack natural pigments that allow such organisms as salmon or trout acquire their characteristic red color (muscle), which, in nature, is a result of eating microalgae containing red pigments; without such a color, a lower market value results. One way to alleviate this shortcoming is by adding astaxanthin to fish feed, with a consequently growing market for microalga-based sources, e.g. *Haematococcus pluvialis* [24,28]*.*

On the other hand, the amino acid composition of microalgal proteins is rather similar between species [29], and relatively unaffected by their intrinsic growth phase and extrinsic light conditions [30,31]. Furthermore, the content in essential amino acids of microalgae is similar to that of oyster larvae. Overall, this indicates that protein quality is unlikely a factor that contributes to differences in nutritional value among microalgae. Finally, sterols [32], minerals [33] and pigments of microalgae also contribute to their nutritional performance in aquaculture.

Several studies have indicated that, in the late-logarithmic growth phase, microalgae contain typically 30-40 %(w/w) protein, 10-20 %(w/w) lipids and 5-15 %(w/w) carbohydrates [6,34]. When cultured through the stationary phase, the proximate composition of microalgae may significantly change; e.g. nitrate limitation leads carbohydrate levels to double at the expense of protein [31,35]. Hence, a strong correlation exists between composition of microalgae and their measurable nutritional value – even though diets containing high levels of carbohydrates have been reported to produce the best growth of juvenile oysters [9] and larval scallops [36], as long as PUFAs are also present to adequate proportions. Conversely, high dietary protein provides maximum growth for juvenile mussels [15] and oysters [18].

Another relevant issue is that marine environments are typically filled with bacteria and viruses that can attack fish and shellfish, and thus potentially devastate aquaculture farms. Bacteria and viruses can also attack single-celled microalgae, so these microorganisms have developed biochemical mechanisms for self-defense; such mechanisms involve secretion of compounds that inhibit bacterial growth or viral

metamorphosis of many larvae [8,26]. However, the ratios of DHA, EPA and AA may actually be more important than their absolute levels [24,27]. Most microalgal species exhibit moderate to high percents of EPA (7 to 34%); and prymnesiophytes (e.g. *Pavlova* spp. and *Isochrysis* sp.) and cryptomonads are relatively rich in DHA (0.2 to 11%), whereas eustigmatophytes (e.g. *Nannochloropsis* spp.) and diatoms have the highest percentages of AA (up to 4%). Chlorophytes (*Dunaliella* spp. and *Chlorella* spp.) are deficient in both C20 and C22 PUFAs, although some species have small amounts of EPA (up to 3.2%); because of such a PUFA deficiency, chlorophytes are in general ascribed a poor nutritional value, so they are not suitable for use as single species-diet [6]. Prasinophyte species contain significant proportions of C20 (*Tetraselmis* spp.) or C22 (*Micromonas* spp.), but rarely of both. Therefore, the fatty acid contents of microalgae exhibit systematic differences according to taxonomic group – although there are examples of significant differences between

The contents of antioxidants are also not uniform among microalgae; e.g. the concentrations of vitamins and carotenoids convey significant variations among species. Note that any mixed-algal diet should provide adequate concentrations of vitamins and carotenoids to be effective in aquaculture; unfortunately, the nutritional requirements of larval or juvenile animals that feed directly on microalgae are still poorly understood at present. In fact, artificial diets often lack natural pigments that allow such organisms as salmon or trout acquire their characteristic red color (muscle), which, in nature, is a result of eating microalgae containing red pigments; without such a color, a lower market value results. One way to alleviate this shortcoming is by adding astaxanthin to fish feed, with a consequently

On the other hand, the amino acid composition of microalgal proteins is rather similar between species [29], and relatively unaffected by their intrinsic growth phase and extrinsic light conditions [30,31]. Furthermore, the content in essential amino acids of microalgae is similar to that of oyster larvae. Overall, this indicates that protein quality is unlikely a factor that contributes to differences in nutritional value among microalgae. Finally, sterols [32], minerals [33] and pigments of microalgae also contribute to their nutritional performance in

Several studies have indicated that, in the late-logarithmic growth phase, microalgae contain typically 30-40 %(w/w) protein, 10-20 %(w/w) lipids and 5-15 %(w/w) carbohydrates [6,34]. When cultured through the stationary phase, the proximate composition of microalgae may significantly change; e.g. nitrate limitation leads carbohydrate levels to double at the expense of protein [31,35]. Hence, a strong correlation exists between composition of microalgae and their measurable nutritional value – even though diets containing high levels of carbohydrates have been reported to produce the best growth of juvenile oysters [9] and larval scallops [36], as long as PUFAs are also present to adequate proportions. Conversely, high dietary protein

Another relevant issue is that marine environments are typically filled with bacteria and viruses that can attack fish and shellfish, and thus potentially devastate aquaculture farms. Bacteria and viruses can also attack single-celled microalgae, so these microorganisms have developed biochemical mechanisms for self-defense; such mechanisms involve secretion of compounds that inhibit bacterial growth or viral

growing market for microalga-based sources, e.g. *Haematococcus pluvialis* [24,28]*.*

provides maximum growth for juvenile mussels [15] and oysters [18].

microalgae, even within the same class.

aquaculture.

attachment. For instance, compounds synthesized by *Scenedesmus costatum*, and partially purified from its organic extract exhibited activity against aquacultured bacteria because of their fatty acids longer than 10 carbon atoms in chain length – which apparently induce lysis of bacterial protoplasts.

The ability of fatty acids at large to interfere with bacterial growth and survival has been known for some time, and recent structure-function relationship studies have proven that said ability depends on both their chain length and degree of unsaturation. Cholesterol and other compounds can antagonize antimicrobial features [37], so both composition and concentration of free lipids should be taken into account [38]. The activity of extracts of *Phaeodactylum tricornutum* against *Vibrio* spp. was attributed to EPA – a compound synthesized *de novo* by diatoms [39]; this PUFA is found chiefly as a polar lipid species in structural cell components (e.g. membranes) and is toxic to grazers [40], as well as a precursor of aldehydes with deleterious effects upon such consumers as copepods [41]. Similarly, unsaturated and saturated long chain fatty acids isolated from *S. costatum* [42] and organic extracts from *Euglena viridis* [43] display activity against that bacterial genus.
