**1. Introduction**

58 Aquaculture

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and vegetable oil blends increase adiposity and plasma lipids in Atlantic salmon (*Salmo salar* L*.). British Journal of Nutrition* 106, 633-647. doi: 10.1017/ Microalgae (i.e. single-celled algae or phytoplankton) represent the largest, yet one of the most poorly understood groups of microorganisms on Earth. As happens with plants relative to terrestrial animals, microalgae represent the natural nutritional base and primary source of bulk nutrients in the aquatic food chain.

Microalgae play indeed a crucial nutritional role with regard to marine animals in the open sea, and consequently in aquaculture. Most marine invertebrates depend on microalgae for their whole life cycle, so commercial and experimental mollusc or fish hatcheries have included a microalga production system in parallel to their animal production itself. Microalgae are utilized as live feed for all growth stages of bivalve molluscs (e.g. oysters, scallops, clams and mussels), for larval/early juvenile stages of abalone, crustaceans and some fish species, and for zooplankton used in aquaculture food webs at large. It should be emphasized that the productivity of any hatchery is directly related to the quantity and quality of the food source used therein.

On the other hand, the concept of aquaculture as a set of engineered systems in terms of wastewater treatment and recycling has received an impetus over the past few years. They are designed to meet specific treatment and wastewater specifications, and may simultaneously solve environmental and sanitary problems along with economic feasibility [1,2]. A renewed interest has also been experienced by high rate microalgal ponds for treatment of wastewater – where photosynthetic microalgae supply oxygen to heterotrophic bacteria, and where wastewater-borne nutrients are converted into biomass protein [2,3]. Therefore, microalga culturing is likely to play an increasingly important role in aquatic food production modules, specifically to produce (or be used as) feed for fish, convert CO2 to O2 and remediate water quality.

 \* Corresponding Author

#### **2. General attributes of microalgal species in aquaculture**

Unlike air-breathing animals, those living in aquatic media and used for large scale human consumption as food are seldom herbivorous at the adult stage; most farmed animals are indeed carnivorous from their post-larval stage on, or omnivorous at best. The associated food web is accordingly longer, so only filtering molluscs and a few other animals truly depend on plankton throughout their lifetime. However, microalgae are required for larva nutrition during a brief period – either for direct consumption in the case of molluscs and penaeid shrimps, or indirectly for the live prey fed to small-larval fish. In these cases, the post-larvae specimens are hatched, bred and raised by specialized establishments (hatcheries) – which are particularly complex to operate because they involve intensive production of microalgae and, in the case of small-larval fish, production of such small live prey as rotifers. Aquacultured animals for which rearing does not exhibit these constraints are seldom found; this is the case of salmonids, whose eggs have sufficient reserves to hatch big larvae capable of feeding directly on dry particles [4].

Over the last decades, several hundred microalga species have been tested as feed, but probably less than twenty have experienced a widespread application in aquaculture. In fact, microalgal species vary significantly in their nutritional value – which is also dependent on culturing conditions [5,6]. To provide a better balanced nutrition package and more effectively improve animal growth, a carefully selected mixture of microalgae should be fed to fish, directly or indirectly (through enrichment of zooplankton) – as this leads to better results than a diet composed of a single microalga [7,8].

Microalga production for use as feed is divided into intensive monoculture – for larval stages of bivalves, shrimp and certain fish species, and extensive culture – for growth of bivalves, carp and shrimp. Favored genera for the former include *Chaetoceros, Thalassiosira, Tetraselmis, Isochrysis*, *Nannochloropsis*, *Pavlova and Skeletonema* [6,9,10]. These organisms are fed directly or indirectly to the cultured larval organism; indirect means of providing them are usually through artemia, rotifers and *Daphnia* – which are, in turn, fed to the target larval organisms. It is widely accepted that microalgae are actively taken up by shrimp larvae, and play an important role in nutrition at that life stage; however, it is uncertain whether juveniles and adults do actively feed on microalgae as well. Some reports suggest that microalgae are found in their gut because shrimp accidentally ingest them together with debris [11,12].

The nutritional value of a microalgal diet is critically related to its ability to supply essential macro- and micronutrients to the target animal consumer. As emphasized above, a mixed microalgal diet – as routinely used in the hatchery and nursery phases of oyster cultivation [13], is likely to outperform monoalgal diets [14]. However, the nutritional requirements of bivalves are poorly defined; feeding experiments with microalgae of partially defined compositions have shown that carbohydrate and polyunsaturated fatty acid (PUFA) levels are major factors for growth of oysters [5]. Supply of additional dietary carbohydrate was found to increase oyster growth rate, provided that adequate protein and essential fatty acids were concomitantly supplied [9]. Supplementation of juvenile mussel diets with protein microcapsules led to a positive growth response, and indicated that a protein content below 40 %(w/w) significantly contrains mussel growth rates [15].

Unlike air-breathing animals, those living in aquatic media and used for large scale human consumption as food are seldom herbivorous at the adult stage; most farmed animals are indeed carnivorous from their post-larval stage on, or omnivorous at best. The associated food web is accordingly longer, so only filtering molluscs and a few other animals truly depend on plankton throughout their lifetime. However, microalgae are required for larva nutrition during a brief period – either for direct consumption in the case of molluscs and penaeid shrimps, or indirectly for the live prey fed to small-larval fish. In these cases, the post-larvae specimens are hatched, bred and raised by specialized establishments (hatcheries) – which are particularly complex to operate because they involve intensive production of microalgae and, in the case of small-larval fish, production of such small live prey as rotifers. Aquacultured animals for which rearing does not exhibit these constraints are seldom found; this is the case of salmonids, whose eggs have sufficient reserves to hatch

Over the last decades, several hundred microalga species have been tested as feed, but probably less than twenty have experienced a widespread application in aquaculture. In fact, microalgal species vary significantly in their nutritional value – which is also dependent on culturing conditions [5,6]. To provide a better balanced nutrition package and more effectively improve animal growth, a carefully selected mixture of microalgae should be fed to fish, directly or indirectly (through enrichment of zooplankton) – as this leads to

Microalga production for use as feed is divided into intensive monoculture – for larval stages of bivalves, shrimp and certain fish species, and extensive culture – for growth of bivalves, carp and shrimp. Favored genera for the former include *Chaetoceros, Thalassiosira, Tetraselmis, Isochrysis*, *Nannochloropsis*, *Pavlova and Skeletonema* [6,9,10]. These organisms are fed directly or indirectly to the cultured larval organism; indirect means of providing them are usually through artemia, rotifers and *Daphnia* – which are, in turn, fed to the target larval organisms. It is widely accepted that microalgae are actively taken up by shrimp larvae, and play an important role in nutrition at that life stage; however, it is uncertain whether juveniles and adults do actively feed on microalgae as well. Some reports suggest that microalgae are found in their gut because shrimp accidentally ingest them together with

The nutritional value of a microalgal diet is critically related to its ability to supply essential macro- and micronutrients to the target animal consumer. As emphasized above, a mixed microalgal diet – as routinely used in the hatchery and nursery phases of oyster cultivation [13], is likely to outperform monoalgal diets [14]. However, the nutritional requirements of bivalves are poorly defined; feeding experiments with microalgae of partially defined compositions have shown that carbohydrate and polyunsaturated fatty acid (PUFA) levels are major factors for growth of oysters [5]. Supply of additional dietary carbohydrate was found to increase oyster growth rate, provided that adequate protein and essential fatty acids were concomitantly supplied [9]. Supplementation of juvenile mussel diets with protein microcapsules led to a positive growth response, and indicated that a protein

content below 40 %(w/w) significantly contrains mussel growth rates [15].

**2. General attributes of microalgal species in aquaculture** 

big larvae capable of feeding directly on dry particles [4].

better results than a diet composed of a single microalga [7,8].

debris [11,12].

Diatoms and haptophytes (prymnesiophytes) are nutritious microalgae that are frequently used as feed for oysters [13]. The prymnesiophytes *Isochrysis* sp. and *P. lutheri* are rich sources of docosahexaenoic acid (DHA, 22:6n-3) – comprising 8-10% total fatty acids [16], while diatoms are a rich source of eicosapentaenoic acid (EPA, 20:5n-3) [17]. Mixed microalgal diets of prymnesiophytes and diatoms are common in bivalve hatcheries, and considered as highly nutritious in terms of requirements for essential PUFAs [18].

Microalgae should, in general, possess a number of key attributes to be useful for aquacultured species: they should be of an appropriate size and shape for ingestion and ready digestion (i.e. they should have a digestible cell wall to make nutrients easily available); they should undergo fast growth rates, and be amenable to mass culture; they should be stable to fluctuations in temperature, light and nutrient profile, as often occur in hatchery systems; and they should exhibit appropriate nutritional qualities, including absence of toxins (that might otherwise accumulate through the food chain). A major challenge faced by algologists is thus to reduce production costs, while maintaining reliability of microalgal feed.

Microalgae provide food for zooplankton, but they can also help stabilize (and even improve) the quality of the culture medium. For numerous freshwater and seawater animal species, introduction of phytoplankton to rearing ponds (the so-called green-water technique) produces much better results in terms of survival, growth and transformation index than the classical clear-water technique [19-21]. The rationale behind this observation is not entirely known, yet it may include water quality improvement by oxygen production and pH stabilization, and action of some excreted biochemical compounds, along with induction of behavioral processes such as initial prey catching and regulation of bacterial population [4,22], probiotic effects [23], and stimulation of immunity [24].
