WO2004043139A2 - Feed suitable for culturing rotifers, larval shrimp, and marine filter feeders - Google Patents

Abstract

The invention provides an inexpensive and highly nutritious source of food for mass production of rotifers, shrimp, larvae, and other filter feeders, such as oysters, clams, mollusks, and mussels. The invention provides a feed comprising a protein source, e.g., yeast, a macroalgal product, and a microalgal product in combination to enhance the growth of zooplankter and thereby the ultimate consumer of said zooplankter, a targeted cultured species.

Description

FEED SUITABLE FOR CULTURING ROTIFERS. LARVAL SHRIMP. AND MARINE FILTER FEEDERS

PRIORITY CLAIM

[001] This application claims the priority of U.S. provisional application

60/426,022 entitled "Feed Suitable for Culturing Rotifers, Larval Shrimp, and Marine Filter Feeders," filed in the United States Patent and Trademark Office on November 14, 2002, the disclosure of which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[002] The invention relates to feeds for aquatic organisms, such as rotifers, larval shrimp, and filter feeders. The feeds can be comprised of algae or yeast, or algal or yeast extracts, and can provide supplementation to the aquatic organisms, for example, with vitamins, e.g., vitamin B12, fatty acids, amino acids, growth factors, antibacterial agents, and pigments.

BACKGROUND ART

[003] Over the last two decades, aquaculture has become one of the fastest growing economic ventures. Aquacultural production is expected to expand rapidly in order to meet future demands of a growing global population. Researchers believe, however, that there is little likelihood of expanding wild fish catches as the entire fishing industry is in decline due to over fishing. Additionally, unless technological breakthroughs are made, aquaculture expansion is not expected to be sufficient to satisfy the projected demand. The resulting demand for seafood supply, associated with a steady decline in wild fish stocks due to over fishing, habitat destruction, and pollution, drives commercial fish culturists and hatcheries to increase and intensify their production capacities. However, low survival and poor growth rates of marine fish larvae, as a result of insufficient supply of live feed organisms and reduced food quality, is hindering the required development of commercially important marine aquacultural fish species. On a commercial production scale, non-optimal growth and survival means additional tank space, extended larval rearing periods, and associated increases in operational costs (i.e., the difference between survival of a business and its failure).

[004] One of the drawbacks to the development and intensification of marine aquaculture is the fact that the majority of the commercially important marine fish species require live organisms as their first feed (larval feeding). Despite extensive efforts to develop artificial alternatives (Dabrowski and Culver 1991; Kanazawa 1992), hatcheries are still vitally relying on live feeds, mainly various species of microscopic algae, the rotifer Brachionus plicatilis, and the brine shrimp Artemia salina or Artemia franciscana. Of all live foods used in fish and crustacean hatcheries, brine shrimp constitute the main food item because of their practical convenience and availability. As of today, brine shrimp are harvested mainly from the wild at an annual production of over 2000 metric tons of dry cysts. Nonetheless, a large part of the Artemia cyst market is still supplied from one location, the Great Salt Lake. This situation makes the market extremely vulnerable to climatological and ecological changes in this lake, which has been illustrated by the unusually low cyst harvest in the last four years. As a result, prices jumped from a moderate range of 20- 40 dollars per kg dry cysts up to 80-100 U.S. dollars per kg, deeply affecting the entire fish and shellfish culture industry. Another major drawback is that newly hatched brine shrimp nauplii cannot be ingested by early stage larvae of most marine fish species, and therefore, they must be offered with more appropriate planktonic substitutes.

[005] The larval period of many marine fish and invertebrates is spent in estuaries where rotifers are the most abundant and dominant zooplankton. As a result of this natural ecological association, which has evolved over millions of years, many fish larvae and invertebrates are adapted to capture and nutritionally utilize rotifers. The planktonic nature of rotifers, their tolerance to a wide range of environmental conditions, high reproduction rate (0.7-1.4 offspring/ female/day), the ability to produce resting eggs which can remain dormant for several years, smaller size (100- 500 μm lorica length), and slow swimming speed makes them an ideal live prey candidate for the culture of marine larvae. The success of rotifers as live food organisms for the culture of early larval stages of marine fish encouraged investigation into the improvement of their mass culture under controlled conditions. Indeed, twenty-five years after the first mass production trial, rotifers are produced worldwide in large quantities and have contributed to the successful hatchery production of more than 60 marine finfish species, several species of freshwater fish, and 18 species of crustaceans (Nagata and Whyte 1992). The largest potential for rotifer culture resides, however, is the possibility of rearing these animals at very high densities (2000-10,000 individuals/mL) using condensed microalgal feed and oxygen supplementation, as has been successfully practiced recently in Japan and Europe (Yoshimura et al. 1994). Even at high densities, rotifers still reproduce rapidly, and can thus contribute large quantities of live food in a relatively short time before the onset of a commercial larval rearing trial.

[006] Production of the expected number of marine fish larvae and crustaceans for future aquaculture needs and for natural stock enhancement programs cannot be accomplished when utilizing traditional culture methods of live algae. These methods require large tank volumes, ranging from 100-1000 m³, (Kitajima 1983) in which rotifers are generally maintained at low densities of 100-300 individuals/ml and fed mainly on yeast-based low quality products supplemented with live microalgae. As. a result, an additional 2-3 times more tank space may be needed for rotifer culture than all other purposes (Yoshimura et al. 1996). Consequently, up to 90% of the manpower and tank space in a conventional hatchery is used for live food production. Moreover, several hatcheries have recently reported a significant decrease in rotifer production, probably as a result of uncharacterized changes in their water quality and environmental conditions (Yoshimura et al. 1996). In fact, Watanabe (Watanabe et al. 1983a; Watanabe et al. 1983b) stated that without first establishing the intensive culture of rotifers and their feed, larval rearing of marine fish will not be expanded significantly. Thus, it has become widely accepted that larval production is virtually limited by live algae and rotifer availability (Hirata et al. 1983; Fukusho and Hirayama 1992).

[007] Methods for preparing a particulate material suitable for use as an aquaculture feed from microbial biomass are disclosed in U.S. Patents 6,103,225, 6,451,567, and 6,372,460 (Barclay 2000; Barclay 2002; Gladue and Behrens 2002). These methods describe a fermentation process for algal cells having a high content of DFIA residues. The main goal of those disclosures was raising the DHA level of the Artemia or rotifers then subsequently raising the DHA level of the larvae. For those organisms that are filter feeders, the material may be supplied as small particles and thus provide a direct enrichment of DHA into the diet. DHA-enriched aquaculture feeds comprising microflora selected from Thraustochytrium, Schizochytrium, and Crypthecodinium sp., and mixtures thereof are useful for aquaculture and in particular, for feeding larval shrimp and mollusks (Barclay 2000; Gladue and Behrens 2002). Because of the small aggregate size of the algal cells, the microflora can be eaten by larval shrimp, brine shrimp, rotifers, and mollusks (Langdon et al., 1999). As an alternative approach, the present invention discloses the use of nutritionally improved yeast as aquaculture feeds. The improved nutritional status of yeast is obtained by selecting partially digested yeast or yeast having thin cell walls and including a mixture of certain microalgae that are used for aquaculture, such as DHA- producing microalgae and macroalgal extracts, thus formulating a complete and highly nutritious feed for aquaculture. These algal feeds, which are based on yeast and microflora grown in fermentation medium or photoautotrophically grown algae, are suitable for use as feeds for rotifer, brine shrimp, shrimp larvae, clams, mussels, and mollusks. As discussed above, microflora grown under such conditions typically have a cell size less than about 50 μm.

[008] With the ability to formulate larval feeds solely from components derived from heterotrophic bacteria, algae, and yeast, we can eliminate the need for both the hatchery production of live algae and the use of meals and oils derived from wild caught fish. These algal-derived feeds would be much cheaper than currently used feeds. Their use in commercial hatcheries would improve operating efficiency by lowering costs associated with growing a sufficient amount of algae, thereby allowing for increasing aquacultural production. DISCLOSURE OF THE INVENΗON

[009] The invention provides an aquaculture feed comprising yeast and microalgae, or components thereof. The mean particle size can range from about 5 μm to about 100 μm, and the feed components can be dry mixed and ground to a fine powder. The yeast can comprise from about 30 to about 95 percent of the feed. Suitable yeast include, but are not limited to Saccharomyces spp., Saccharomyces cerevisiae, Phaffia spp., Phaffla rhodozyma, Pichia spp., Pichiapastoris, Kluyveromyces spp., Kluyveromyces aestuarii, Kluyveromyces marxianus, and Kluyveromyces yarrowii. The yeast can also be brewer's yeast. [010] The feed can comprise at least about 5 percent by weight from microalgae. Suitable microalgae include, but are not limited to, Tetraselmis sp., Tetraselmis suecica, Myrmecia sp., Myrmecia bissecta, Lyngbya sp., Lyngbya majuscula, Cytospora sp., Scenedesmus sp., Scenedesmus obliquus, Scytonema sp., Scytonema hofinanni, Nostoc sp., Nostoc weissfloggia, Chaetoceros sp., Chaetoceros lauderi, Ecklonia sp., Ecklonia maxima, Dunaliella sp., Dunaliella salina, Dunaliella tertiolecta, Dunaliella bardiwal, Pseudoanabaena sp., Anabaena sp., Prorocentrum sp., Prorocentrum minimum, Polysiphonia sp., Polysiphonia denudata, Spirulina sp., Arthrospira sp., Spirulina platensis, Aphanotheae sp.,Aphanotheae nidulans, Hydrodictyon sp., Hydrodictyon reticulatum, Navicula sp., Navicula delongei, Phaeodactylum sp., Phaeodactylum tricornutum, Pseudonitzschia sp., Nitzschia sp., Nitzschia navis-varingica, Chlorella sp., Chlorella pyrenoidosa, Chlorella vulgaris, Chlamydomonas sp., Chlamydomonas reinhardii, Prennioporia sp., Prennioporia medullaeparis, Pandorina sp., Pandorina morum, Isochrysis spp., Isochrysis galbana, Schizochytrium sp., Crypthecodinium sp., Crypthecodinium cohnii, and Thraustochytrium sp. Suitable microalgae also include, but are not limited to, Schizochytrium sp., Crypthecodinium sp., Crypthecodinium cohnii, Thraustochytrium sp., and components thereof. The feed can also comprise a probiotic element. Suitable probiotic elements include, but are not limited to, Lactobacillus sp., Lactobacillus casei, Lactobacillus rhamnosus, Pseudoalteromonas sp., and Pseudoalteromonas undina. [011] In an embodiment, the aquaculture feed can comprise yeast, microalgae, and macroalgae. The mean particle size can range from about 5 μmto about 100 μm, and the feed components can be dry mixed and ground to a fine powder. The yeast can comprise from about 30 to about 95 percent of the feed. Suitable yeast include, but are not limited to, Saccharomyces spp., Saccharomyces cerevisiae, Phaffla spp., Phaffia rhodozyma, Pichia spp., Pichia pastoris, Kluyveromyces spp., Kluyveromyces aestuarii, Kluyveromyces marxianus, and Kluyveromyces yarrowii. The yeast can be partially digested. [012] The feed can comprise at least about 5 percent by weight from macroalgae. Suitable macroalgae include, but are not limited to, Tetraselmis sp., Tetraselmis suecica, Myrmecia sp., Myrmecia bissecta, Lyngbya sp., Lyngbya majuscula, Cytospora sp., Scenedesmus sp., Scenedesmus obliquus, Scytonema sp., Scytonema hofinanni, Nostoc sp., Nostoc weissfloggia, Chaetoceros sp., Chaetoceros lauderi, Ecklonia sp., Ecklonia maxima, Dunaliella sp., Dunaliella salina, Dunaliella tertiolecta, Dunaliella bardiwal, Pseudoanabaena sp., Anabaena sp., Prorocentrum sp., Prorocentrum minimum, Polysiphonia sp., Polysiphonia denudata, Spirulina sp., Arthrospira sp., Spirulina platensis, Aphanotheae sp.,Aphanotheae nidulans, Hydrodictyon sp., Hydrodictyon reticulatum, Navicula sp., Navicula delongei, Phaeodactylum sp., Phaeodactylum tricornutum, Pseudonitzschia sp., Nitzschia sp., Nitzschia navis-va ingica, Chlorella sp., Chlorella pyrenoidosa, Chlorella vulgaris, Chlamydomonas sp., Chlamydomonas reinhardii, Prennioporia sp., Prennioporia medullaeparis, Pandorina sp., Pandorina morum, Isochrysis spp., Isochrysis galbana, Schizochytrium sp., Crypthecodinium sp., Crypthecodinium cohnii, and Thraustochytrium sp. The aquaculture feed can comprise at least about 0.5 percent by weight from macroalgae, which can be chosen from Laminaria spp., Padina sp., Gracillaria sp., and Ulva sp. The macroalgae can comprise a macroalgal extract, which can further comprise an ethanolic extraction product. The feed can also comprise a probiotic element. Suitable probiotic elements include, but are not limited to, Lactobacillus sp., Lactobacillus casei, Lactobacillus rhamnosus, Pseudoalteromonas sp., and Pseudoalteromonas undina. [013] The invention also provides a method comprising feeding a cultured species with the feed described above.

[014] The invention further provides a method comprising feeding a zooplankter species with a feed described above. Suitable zooplankter species include, but are not limited to, brine shrimp, rotifers, Artemia spp., Artemia salina,

Artemia fianciscana, Brachionus spp., Brachionus plicatilis, copepods, and cladocerans.

BRIEF DESCRIPTION OF THE DRAWING [015] Figure 1. The daily harvest (30% of total culture) of rotifers fed 0.4 g

D.W. Bakers yeast and 40 x 10⁶ live Nanochloropsis sp. algae per million rotifers (A), 1 g algal feed and 0.1% . rhamnosus per million rotifers (X), and 1 g Culture Selco (INVE, Bassrode Belgium) (♦).

MODES FOR CARRYING OUT THE INVENTION Summary

[016] The purpose of the present invention is to provide an inexpensive and highly nutritious source of food derived by bacterial, algal, and/or yeast fermentation for mass production of rotifers, shrimp, larvae, and other filter feeders, such as oysters, clams, mollusks, and mussels. Currently, a major impediment to marine fish aquaculture is the high energy and labor cost (up to 85% of total operation costs according to Bolton and Fulks (Bolton 1982; Fulks and Main 1992)), and practical difficulties associated with growing high-quality algae. Frequently, it is difficult for a hatchery to supply sufficient algae at all times of the year. Unicellular algae are generally photoautotrophic and, as such, require light for photosynthesis during which carbon dioxide is reduced to organic carbon and oxygen is released. Hatcheries are generally using electric lighting for indoor algal cultures or, if climate conditions permit, algae can be grown in outdoor tanks. However, cell densities and growth rates are limited by light penetration into the culture medium. Photoautotrophic culture methods are energetically inefficient, costly, and require hatcheries to employ a skilled person to maintain the algal culture system. [017] It is an objective of the invention to provide rotifers, shrimp, larvae, and filter feeders with inexpensive and high quality dry feeds that are produced from phototrophically or heterotrophically grown bacteria, algae, and yeast. [018] It is an objective of the invention to provide rotifers, shrimp, larvae, and filter feeders with highly digestible yeast in a dry form. This yeast may be partially digested baker's yeast with cellulase or brewers yeast having a thin and easily digested cell wall.

[019] It is an objective of the invention to provide rotifers, shrimp larvae, and filter feeders with phototrophic or heterotrophic production of certain fresh- water and marine species of algae. Examples of microalgae include, but are not limited to, freshwater Chlorella sp., marine Chlorella (Nannochloropsis oculata), Tetraselmis sp., Chaetoceros sp., Crypthecodinium sp., Schizochytrium sp., Chlorella spp. (such as, but not limited to, Chlorella vulgaris k-22), and Isochrysis galbana (T-iso clone). These algal species are widely used in aquaculture operations, because of their high nutritional value, readiness for artificial culture, and also as a food source for rotifers (Hernandez et al. 1986; Nagata and Whyte 1992; Verginelli et al. 1994; Lie et al. 1997; Planas and Cunha 1999). Crypthecodinium cohnii, and Schizochytrium sp. have exceptionally high levels of docosahexaenoic acid (DHA, 22:6n-3), which is an essential fatty acid supplement in a marine fish larval diet (reviewed by Kanazawa (Kanazawa 1993) and Watanabe (Watanabe 1993)).

[020] It is an objective of the invention to provide rotifers and shrimp larvae and filter feeders with Vitamin Bι₂ supplemented waste stream co-products of algae (up to 400 μg/100 g dry matter). More specifically, these feeds can be supplemented with additional algae-derived products containing phosphohpids rich in essential fatty acids such as docosahexaenoic (DHA, 22:6n-3) and arachidonic (ARA, 20:4n-6) acids.

[021] It is an objective of the invention to provide rotifers, shrimp larvae, and other filter feeders with a dry form of macroalgal meal or extracts derived by such methods as, but not limited to, ethanolic or hot water extraction of macroalgal biomass. These extracts can contain additional essential growth factors, antibacterial agents, bacterio static compounds, pigments, and essential amino acids. Examples of macroalgae include, but are not limited to, Laminaria sp., Padina sp., and Gracillaria sp.

Definitions

[022] In describing the present invention, the following terminology is used in accordance with the definitions set out below.

[023] The term "zooplankton" (plural) or "zooplankter" (singular) as used herein, refer to members of three major groups of invertebrate animals: the rotifers, copepods, and cladocerans. All three of these major groups have species adapted to pelagic (open water), littoral (vegetated), and benthic (bottom) environments, and are widespread in both freshwater and marine environments.

[024] The term "rotifer," as used herein, shall mean any of a class of minute, usually microscopic, but many-celled aquatic invertebrate animals having the anterior end modified into a retractile disk bearing circles of strong cilia that often give the appearance of rapidly revolving wheels. Rotifers are used as a source of live feed for aquatic animals during their early larval stage.

[025] The term "brine shrimp," as used herein, shall mean any strain of

Artemia, belonging to the phylum Arthropoda in the class Crustacea. These tiny crustaceans can be found in salt lakes and brine ponds throughout the world. Artemia brine shrimp are used as a source of live feed for aquatic larvae during their early and late larval stages.

[026] The term "aquaculture feed," as used herein, shall mean any dry feed in a form of fine powder that is used to culture and grow zooplanktonic animals and aquatic larvae.

[027] The term "algal feed," as used herein, shall mean any dry feed form, which includes any portion of algal component.

[028] The term "enrichment feed," as used herein, shall mean any type of feed either dry or moist, that is used for short duration feeding of aquatic zooplankton, prior to their harvest, in order to deliver essential nutrients for aquatic larvae when fed to these zooplankton. [029] The term "filter feeder" is any species of invertebrate animal that obtains its food from aquatic media by removal of small particles or organisms by any mechanism.

[030] The term "macroalgae" refers to algae that in at least one life stage form large structures that are easily discernable with the naked eye. Usually these organisms have secondary vascularization and organs. Examples of different groups containing macroalgae follow but are not limited to the chlorophyta, rhodophyta, and phaeophyta.

[031] The term "microalgae" refers to prokaryotic and eukaryotic algae that are classed in many different species. Normally the prokaryotic algae are referred to as cyanobacteria or bluegreen algae. The eukaryotic microalgae come from many different genera, some of which overlap with the macroalgae and are differentiated from these by their size and a lack of defined organs (although they do have specialized cell types). Examples of different groups containing microalgae follow but are not limited to the chlorophyta, rhodophyta, phaeophyta, dinophyta, euglenophyta, cyanophyta, pro chlorophyta, and cryptophyta

[032] The term "brewers yeast" is defined as any yeast preparation derived from yeast commonly used for production of beer, wine, or other fermented products.

Such yeast can comprise, but are not limited to, Saccharomyces cerevisiae,

Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces uvarum,

Saccharomyces bayanus, and Kluyveromyces sp.

Embodiments of the Invention

Production of Rotifers and Brine Shrimp [033] The rotifer and brine shrimp culture system includes fiberglass tanks at a working volume of 20-2000 L each. This system is capable of producing up to 5 x 10⁹ rotifers/day/tank at a density range of 200-10,000 individuals/mL or up to 10 kg brine shrimp wet biomass/day/tank. The temperature is regulated in each tank by a thermostat heater at 28-32°C, depending on the rotifer strain. A pH controller continuously monitors the pH in each culturing tank and 10% hydrochloric acid (HCl) is added automatically by means of a Petri pump to control the pH to between 6.5-7.0. Alternatively, a buffer solution containing sodium hydroxymethane sulfonate is added to keep the ammonia in a non-toxic form. Slightly acidic pH of the culture medium is maintained, especially in high-density culture systems, since rotifer growth and fecundity rates are sensitive to high concentrations of toxic ionized ammonia (e.g., concentrations over 7.8 mg/L, as reported by Yu and Hirayama, 1986). [034] Food is delivered to each culture tank manually or continuously by means of a Petri dosage pump. The daily amount of algal feeds and other supplements may be maintained in a chilled (0-4°C) suspension chamber, equipped with a slow mixing device. Rotifers are harvested continuously in a separate harvesting tank by daily exchanging of up to one third of the culture volume and can then be further enriched with essential nutrients before being delivered to fish larvae. [035] With the increase in rotifer or brine shrimp density and subsequent increase in both feeding rations and organic load, a shortage of oxygen is expected. Therefore, pure oxygen, provided from oxygen cylinders or an oxygen generator, is diffused into each tank through air stones to maintain a minimal oxygen level at 3 ppm. Accumulation of parti culate organic matter and contaminating protozoans is anticipated at high culture densities, which may affect production and clog plankton nets at harvesting. Therefore, the rotifer or brine shrimp medium is continuously circulated through nylon mat filters (1 cm thick); such a simple filtration system effectively removes solid substances with only minimal losses and stabilizes the culture performance (Yoshimura et al., 1996; Yoshimura etal, 1995).

Formulation of Algal Feeds [036] Brewers yeast or partially hydrolyzed baker's yeast (where at least the external layer of the cell wall is digested, either enzymatically or chemically) provide brine shrimp and rotifers with highly digestible nutrients. The yeast content in the algal feed formulation ranges from 30% to 95% and, more preferably from 70% to 90% of the feed. The present invention provides yeast that are accessible to the digestive enzymes of their predators, but their wall remains sufficiently intact so that the cell contents do not escape into the water prior to consumption. [037] Dried baker's yeast is prepared using enzymatic or chemical agents, such as, but not limited to, cellulase or mannase. Suitable enzymes and their conditions of use (such as pH, concentration, temperature, and duration of the incubation) are known to those skilled in the art (Lavens et al. 1992). [038] Long chain polyunsaturated fatty acid-rich microalgae are provided as a source of essential fatty acids, especially DHA, ARA and EPA (Barclay and Zeller 1996; Cure et al. 1996; Furuita et al. 1998; Abu-Rezq et al. 2002; Place and Harel 2002a; Place and Harel 2002b). The inclusion level of DHA-rich algae ranges from 0.5 % up to 50% of the feed, and more preferably from 5% to 20% of the feed. DHA (docosahexaenoic acid) has been identified as an important nutrient that contributes significantly to larval growth and survival (Cure et al. 1996). Larvae ultimately acquire these fatty acids from algae, either by directly feeding on algae with high levels of polyunsaturated fatty acids or by feeding on rotifers and brine shrimp that have been enriched with algae high in polyunsaturated fatty acids (Barclay and Zeller 1996). DHA-rich microalgae can be produced in fermentors utilizing sugar as a source of energy or photosynthetically. These algae are fortified with Vitamin B₁₂, a vitamin essential for growth and reproduction of rotifers, during the production process to contain at least 200 μg/100 g dry matter.

[039] Arachidonic acid (ARA) rich microalgae or fungi are provided as a source of essential fatty acids. The inclusion level of ARA-rich algae ranges from 0.5% to 10% of the feed, and more preferably from 2% to 8% of the feed. ARA has been identified as an important nutrient that contributes significantly to larval growth and survival as well as helping in osmotic regulation and stress resistance. [040] Probiotic or prebiotic bacteria (e.g., Lactobacillus sp., Bacillus sp.,

Bifidobacterium sp., and Enterococcus sp.) are added at a range from 0.01% to 5%. These bacteria function either to colonize the gut of the organism or deliver a beneficial activity or nutrient. Such probionts could relieve stress or reduce the pathogen load of the consuming animal.

[041] Macroalgal extracts are also included in the formulation at a range from about 0.1 % to about 10%, and more preferably from about 0.5% to about 1%. These extracts provide the rotifers and brine shrimp with additional essential growth factors, antibacterial and bacteriostatic compounds, pigments, and essential amino acids. [042] All algal species are enriched with vitamin B₁₂ during production.

This is because the rotifer Brachionus plicatilis requires vitamin Bι₂ in its diet at a level of about 100-200 μg/100 g dry matter (Hirayama et al. 1989; Hirayama and Maruyama 1991 ; Maruyama and Hirayama 1993). The nutritional value of the algae is further enhanced by fortifying the algae with three fat-soluble vitamins (Vitamins A, D and E) and Vitamin C. All these vitamins showed supplementary effects and significantly increased the population growth of rotifers (Satuito and Hirayama 1986; Satuito and Hirayama 1991). Examples

[043] Certain embodiments of the invention will now be described in more detail through the following Examples. The Examples are intended solely to aid in more fully describing selected embodiments of the invention and should not be considered to limit the scope of the invention in any way.

Example 1. Formulation of algal feed for rotifer culture [044] Brewers dried yeast was obtained commercially from William Bio- products (Pekin, IL. USA). DHA-rich Schizochytrium sp. was obtained from Advanced BioNutrition Corp. (Columbia MD. USA). Padina sp. dry powder extract was obtained from ICP (Malta).

[045] Rotifer feed was formulated by mixing the ingredients in the proportions specified in Table 1. All ingredients were mixed on a dry weight basis and mill-ground to produce fine particles at sizes from about 5 to about 15 μm.

Table 1. Rotifer feed composition (g dry weight/1 OOg)

Brewers yeast 9C

Schizochytrium sp. 9

Padina sp. 1

[046] Rotifers were fed continually with a daily ration of 0.5 g dry matter/million rotifers. Rotifers were harvested continuously by daily exchanging a total of 30-50% of the culture volume. Temperature, oxygen level, total and free ammonia, and pH were monitored continuously as outlined above. Rotifers were used directly as live feed for larval stage fish, crustaceans, or other aquatic organisms. Example 2. Formulation of algal feed for brine shrimp

[047] DHA-rich Crypthecodinium sp. and Tetraselmis sp., were produced under the trademark AquaGrow^®. All other ingredients were obtained as specified in Example 1 and prepared according to Table 2.

Table 2. Brine shrimp feed composition (g dry weight/100 g)

Brewers dry yeast 50

Partially digested baker's yeast 40

Crypthecodinium sp. 5

Tetraselmis sp. 5

[048] Brine shrimp start to feed about 8 h post hatching (Instar-II nauplii stage), at an initial daily ration of 100% body weight. Daily ration is reduced every day by 10 % and culture tanks can be partially harvested when the required size is t achieved. Complete harvesting can be done on day 10 post-hatch, when the brine shrimp reach maximum size or maximum biomass in the tank. Temperature, oxygen level, total and free ammonia, and pH are monitored continuously as outlined above.

Example 3. Formulation of algal feed for mussels

[049] Spray-dried Spirulina sp. was obtained from Cyanotech Corp. (Kailua-

Kona, HI). All other ingredients were obtained as specified in Examples 1 and 2 and prepared according to Table 3. The dry meals were mixed and ground to fine particles of less than 50 μm.

Table 3. Mussel feed composition (g dry weight/100 g)

Brewers dry yeast 83

Crypthecodinium sp. 5

Schizochytrium sp. 5

Padina sp. extract 2

Spirulina sp. 5 [050] Juvenile mussels (M. galloprovincialis) are placed in 25 L plastic aquaria at a density of 20 individuals per aquarium. The culture system is supplied within a closed recirculating water system and equipped with a biofilter, 10 μm mesh filter, and UV-treatment. Temperature is maintained at 24-26°C. Algal meal is suspended in water and vigorously mixed in a blender and fed to the mussels twice daily at a total ration of 2 mg / L. The growth rate and survival of the animals is monitored on a weekly basis.

Example 4. Formulation of algal feed for shrimp larvae [051] Chaetoceros sp. is grown on 50% Instant Ocean medium in photobioreactors using standard lighting and procedures known in the literature (D'Souza et al. 2002). Laminaria extract is obtained from Pacific Standard Distributors hie (Menlo Park, CA). All ingredients were obtained as specified in Examples 1 and 2 and prepared according to Table 4. The dry meals were mixed and ground or milled to fine particles of less than 50 μm.

Table 4. Larval shrimp feed composition (g dry weight/100 g)

Partially digested baker's yeast 83

Crypthecodinium sp. 5

Laminaria sp. extract 2

Chaetoceros sp. 5

[052] Zooea stage shrimp larvae (Penaeus vannamei or Litopenaeus vannamei) are placed in 25 L plastic aquaria at a density of 50 individuals per aquarium. The culture system is supplied with a closed recirculating water system equipped with biofilter, 10 μm mesh filter and UV-treatment. The temperature is maintained between 24-26°C. Algal meal is suspended in water, vigorously mixed in a blender, and fed to the larvae twice daily at a total ration of 2 mg/L. The growth rate and survival of the animals are monitored on a weekly basis. Example 5. Formulation of algal feed for rotifer culture [053] Dried brewers yeast was obtained commercially from Williams Bio- products (Pekin, IL. USA). DHA-rich Schizochytrium sp. was obtained from Advanced BioNutrition Corp. (Columbia MD. USA). Dry powdered Padina sp. extract was obtained from ICP Malta.

[054] Rotifer feed was formulated by mixing the 90 g of the brewers yeast, 9 g of the Schizochytrium biomass, and 1 g of the dried Padina extract (Table 5). All of the ingredients were mixed on a dry weight basis then mill-ground to produce fine particles at sizes between 5-15 μm.

Table 5. Rotifer feed composition (g dry weight/1 OOg)

Brewers yeast 90

Schizochytrium sp. 9

Padina sp. 1

[055] Rotifers were fed continually with a daily ration of 0.5 g dry matter/million rotifers. Rotifers were harvested continuously by daily exchanging a total of 30-50% of the culture volume. Temperature, oxygen level, total and free ammonia, and pH were monitored continuously. Rotifers were used directly as live feed for larval stage fish, crustaceans, and other aquatic organisms. Performance of rotifers fed on a standard culture diet (a mixture of live baker's yeast and blue green algae), a commercially available culture diet (Culture Selco), and the algal feed of the current invention is shown in Table 6.

Table 6: Production rate, doubling time, egg production and general appearance of rotifers fed different culture feeds.

Diet Production Doubling Egg Appearance*** Rate Time Production (rotifers/mL/day^"1)* (days) (high/low**)

Algal feed 51.4 4.28 High +/-

Culture Selco 31.5 7 Low -

Yeast+live algae 96.5 2.27 High +

* Average of the first 4 days of culture

** High egg production was defined as >70% of the rotifers with eggs. ***General rotifer appearance was determined as follow: (+) sign was given when rotifers appeared swimming fast, carrying more than one egg and water culture was clean; (-) sign was given when rotifers appeared swimming slow, carried one egg or none, and the culture water was murky.

Example 6. Formulation of algal feed with probiotic Lactobacillus rhamnosus for rotifer culture

[056] Algal feed was made as described in Example 1, and freeze dried

Lactobacillus rhamnosus was obtained from Morinaga Milk Industry Co., LTD.

(Kanagawa, Japan).

[057] Rotifer feed was formulated by mixing 89.9 g of brewers yeast, 9 g of

Schizochytrium, 1 g of Padina, and 0.1 g of L rhamnosus (Table 7). All ingredients were mixed on a dry weight basis and mill-ground to produce fine particles at sizes between about 5-15μm.

Table 7. Rotifer feed composition (g dry weight/lOOg)

Brewers yeast 89.9

Schizochytrium sp. 9

Padina sp. 1

L. rhamnosus 0.1

[058] Rotifers were fed with a daily ration of 1 g dry matter/million rotifers.

The culture conditions included open air tube aeration, a temperature of 28±1 ^°C, a salinity of 20 ppt, pH 8±0.5, oxygen saturation 100±5%, and a culture volume of about 20-100 L. Daily diet rations were added 4 -5 times/day. [059] Temperature (°C), oxygen (% saturation), pH, salinity (ppt), and the number of rotifers/ml were measured daily. The daily rotifer harvest was approximately 30% of the number of rotifers in the culture. Fig. 1 shows the daily production of rotifers fed on a standard culture diet (a mixture of live baker's yeast and blue green algae), a commercially available culture diet (Culture Selco), and the algal feed with probiotic lacto bacteria of the current invention. Example 7. Formulation of algal feed with brewer's yeast and Schizochytrium for rotifer culture

[060] Dried brewers yeast was obtained commercially from Williams Bio- products (Pekin, IL. USA). DHA-rich Schizochytrium sp. was obtained from Advanced BioNutrition Corp. (Columbia MD. USA). The rotifers were grown as described in Example 6, except for the specific additions as described in Table 8. The diets containing brewers yeast and Schizochitrium at a 90:10 ratio (two different types of brewers yeast) closely matched the control diet, which was supplied with fresh algae and yeast. Additions of olive extract or Padina extract did not significantly change the number of rotifers per mL over 3 days inoculation. All of these diets bested the Culture Selco.

Table 8. Comparison of rotifer counts per mililiter after three days of culture

Diet Description Rotifers/mL

Control diet with algae and yeast 48.0

Brewers yeast llSchizochytrium at 90:10 40.5

Brewers yeast llSchizochytrium at 90:10 39.0

Brewers yeast llSchizochytrium at 90:10 with olive extract 36.9

Brewers yeast 1 /Schizochytrium at 90 : 10 with Padina extract 33.9

Culture Selco 30.0

References

[061] The specification is most thoroughly understood in light of the following references, all of which are hereby incorporated by reference in their entireties.

U.S. Patent Documents

6,451,567 Barclay, W. September 17, 2002

6,372,460 Gladue and Behrens April 16, 2002

6,103,225 Barclay, W. August 15, 2000

5,739,006 Abe, et al. April 14, 1998

5,688,500 Barclay, W. November 18, 1997 5,158,788 Lavens, et al. October 27, 1992

5,047,250 Prieels, et al. September 10, 1991

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Claims

I Claim:

1. An aquaculture feed comprising yeast and microalgae, or components thereof.

2. The aquaculture feed of claim 1, wherein the mean particle size ranges from about 5 μm to about 100 μm.

3. The aquaculture feed of claim 1 , wherein the feed components are dry mixed and ground to a fine powder.

4. The aquaculture feed of claim 1 , wherein the yeast comprises from about 30 to about 95 percent of the feed.

5. The aquaculture feed of any of claims 2-4, wherein the yeast are chosen from Saccharomyces spp., Saccharomyces cerevisiae, Phaffla spp., Phaffla rhodozyma, Pichia spp., Pichia pastor is, Kluyveromyces spp., Kluyveromyces aestuarii, Kluyveromyces marxianus, and Kluyveromyces yarrowii.

6. The aquaculture feed of any of claims 2-4, wherein the yeast is brewer's yeast.

7. The aquaculture feed of claim 1 , comprising at least about 5 percent by weight from microalgal sources.

8. The aquaculture feed of claim 7, wherein the microalgal source is chosen from Tetraselmis sp., Tetraselmis suecica, Myrmecia sp., Myrmecia bissecta, Lyngbya sp., Lyngbya majuscula, Cytospora sp., Scenedesmus sp., Scenedesmus obliquus, Scytonema sp., Scytonema hofinanni, Nostoc sp., Nostoc weissfloggia, Chaetoceros sp., Chaetoceros lauderi, Ecklonia sp., Ecklonia maxima, Dunaliella sp., Dunaliella salina, Dunaliella tertiolecta, Dunaliella bardiwal, Pseudoanabaena sp., Anabaena sp., Prorocentrum sp., Prorocentrum minimum, Polysiphonia sp., Polysiphonia denudata, Spirulina sp., Arthrospira sp., Spirulina platensis, Aphanotheae sp., Aphanotheae nidulans, Hydrodictyon sp., Hydrodictyon reticulatum, Navicula sp., Navicula delongei, Phaeodactylum sp., Phaeodactylum tricornutum, Pseudonitzschia sp., Nitzschia sp., Nitzschia navis- varingica, Chlorella sp., Chlorella pyrenoidosa, Chlorella vulgaris, Chlamydomonas sp., Chlamydomonas reinhardii, Prennioporia sp., Prennioporia medullaeparis, Pandorina sp., Pandorina morum, Isochrysis spp., Isochrysis galbana, Schizochytrium sp., Crypthecodinium sp., Crypthecodinium cohnii, and Thraustochytrium sp.

9. The aquaculture feed of claim 7, wherein the microalgae are selected from Schizochytrium sp., Crypthecodinium sp., Crypthecodinium cohnii, Thraustochytrium sp., and components thereof.

10. The aquaculture feed of claim 1, further comprising a probiotic element.

11. The aquaculture feed of claim 10, wherein the probiotic element is chosen from Lactobacillus sp., Lactobacillus casei, Lactobacillus rhamnosus, Pseudoalteromonas sp., and Pseudoalteromonas undina.

12. An aquaculture feed comprising yeast, microalgae, and macroalgae.

13. The aquaculture feed of claim 12, wherein the mean particle size ranges from about 5 μmto about 100 μm.

14. The aquaculture feed of claim 12, wherein the feed components are dry mixed and ground to a fine powder.

15. The aquaculture feed of claim 12, wherein the yeast comprises from about 30 to about 95 percent of the feed.

16. The aquaculture feed of any of claims 12-15, wherein the yeast is chosen from Saccharomyces spp., Saccharomyces cerevisiae, Phaffla spp., Phaffla rhodozyma, Pichia spp., Pichia pastoris, Kluyveromyces spp., Kluyveromyces aestuarii, Kluyveromyces marxianus, and Kluyveromyces yarrowii.

17. The aquaculture feed of any of claims 12-15, wherein the yeast is partially digested.

18. The aquaculture feed of claim 12, comprising at least about 5 percent by weight from macroalgae .

19. The aquaculture feed of claim 18, wherein the macroalgal source is chosen from Tetraselmis sp., Tetraselmis suecica, Myrmecia sp., Myrmecia bissecta, Lyngbya sp., Lyngbya majuscula, Cytospora sp., Scenedesmus sp., Scenedesmus obliquus, Scytonema sp., Scytonema hofinanni, Nostoc sp., Nostoc weissfloggia, Chaetoceros sp., Chaetoceros lauderi, Ecklonia sp., Ecklonia maxima, Dunaliella sp., Dunaliella salina, Dunaliella tertiolecta, Dunaliella bardiwal, Pseudoanabaena sp., Anabaena sp., Prorocentrum sp., Prorocentrum minimum, Polysiphonia sp., Polysiphonia denudata, Spirulina sp., Arthrospira sp., Spirulina platensis, Aphanotheae sp., Aphanotheae nidulans, Hydrodictyon sp., Hydrodictyon reticulatum, Navicula sp., Navicula delongei, Phaeodactylum sp., Phaeodactylum tricornutum, Pseudonitzschia sp., Nitzschia sp., Nitzschia navis- varingica, Chlorella sp., Chlorella pyrenoidosa, Chlorella vulgaris, Chlamydomonas sp., Chlamydomonas reinhardii, Prennioporia sp., Prennioporia medullaeparis, Pandorina sp., Pandorina morum, Isochrysis spp., Isochrysis galbana, Schizochytrium sp., Crypthecodinium sp., Crypthecodinium cohnii, and Thraustochytrium sp.

20. The aquaculture feed of claim 12, comprising at least about 0.5 percent by weight from macroalgae .

21. The aquaculture feed of claim 20, wherein the macroalgal source is chosen from Laminaria spp., Padina sp., Gracillaria sp., and Ulva sp.

22. The aquaculture feed of claim 20, wherein the macroalgal source comprises a macroalgal extract.

23. The aquaculture feed of claim 22, wherein the macroalgal extract comprises an ethanolic extraction product.

24. The aquaculture feed of claim 12, further comprising a probiotic element.

25. The feed of claim 24, wherein the probiotic element is chosen from Lactobacillus sp., Lactobacillus casei, Lactobacillus rhamnosus, Pseudoalteromonas sp., and Pseudoalteromonas undina.

26. The aquaculture feed of claim 12, wherein the yeast is partially digested.

27. A method comprising feeding a cultured species with a feed as in claim 1.

28. A method comprising feeding a zooplankter species with a feed as in claim 1.

29. The method of claim 28, wherein the zooplankter species is chosen from brine shrimp, rotifers, Artemia spp., Artemia salina, Artemia franciscana, Brachionus spp., Brachionus plicatilis, copepods, and cladocerans.