FIELD OF THE INVENTION

This invention relates to a feed product or feed ingredient for an aquatic species, methods of rearing an aquatic species using the feed product or ingredient and uses of the feed product or ingredient of this invention.

BACKGROUND OF THE INVENTION

The expansion of global aquaculture is driving the aquaculture industry to investigate more intensive rearing systems to meet increasing demand. However, these intensive rearing conditions generally utilise culture methods that are heavily reliant on the use of synthetically formulated feeds rather than more natural food sources such as pond biota. Traditionally, formula aquatic feeds, such as shrimp feeds, have relied on the use of fishery resources such as fish meals and fish oils as key sources of nutrients.

However, one of the perceived drawbacks of formula feeds is that they are expensive and thus usually represent the major operating cost in intensive rearing system. Indeed, these fishery resources are continually rising in cost due to global overfishing, which is currently posing a threat to the cost effectiveness and long term sustainability of the use fish and other aquatic meals and oils to the aquaculture industry. Consequently, improving productivity and reducing feed cost is vital for the commercial and long-term viability of the aquatic industry.

Aquaculture is also becoming an important sector for production of high protein food. The production of high protein food requires high-quality feed with high protein content, which should contain not only all the necessary nutrients but also complementary additives to keep organisms healthy and favour growth. Fortification of the feed with growth promoting additives, such as hormones and antibiotics, can be detrimental to the animal and the use of antibiotics and their consequences in shrimp farming has received significant negative attention from a public health point of view due to the potential exposure of human consumers to antibiotic residues.

There is clearly a need for alternative, low cost, environmentally friendly and sustainable sources of key nutrients such as protein, lipids and growth promoters in the aquaculture industry.

However, attempts to implement alternative ingredients in aquatic feeds have been problematic as the primary diet specifications for each species must be maintained to ensure yield and cost parameters are effectively balanced.

Microbial biomass has been identified as a source of nutrients for the sustenance of aquaculture. See Australian Patent 2008201886 (CSIRO) and Australian Patent 2009225307 (CSIRO), wherein the present applicants reported an improved process for the production of a microbial biomass. It is particularly noted that in AU2008201886 and AU2009225307, aquatic animal derived-meal and aquatic animal-derived lipids are considered in the art critical components of aquatic feed compositions and feed ingredients. The present inventors have now surprisingly found that aquatic feed formulations, which are substantially free of fishery or aquatic-animal derived resources, such as fish meal and/or fish oil, and which comprise microbial biomass, can sustain aquatic species or provide an alternative to the prior art feed compositions or feed ingredients.

SUMMARY OF THE INVENTION

In a first aspect of the invention, there is provided a feed product or feed ingredient comprising a microbial biomass, wherein the feed product or feed ingredient is substantially free of one or more fishery derived resource(s), including an aquatic- animal derived protein source and/or an aquatic animal-derived lipid source.

In a second aspect of the invention there is provided a feed product or feed ingredient which comprises a microbial biomass, wherein the feed product or feed ingredient is substantially free of one or more fishery derived resource(s), including an aquatic animal-derived protein source and/or an aquatic animal derived-lipid source, and further comprises one or more plant derived lipid source(s) and/or mixtures thereof.

In a third aspect of the invention there is provided a feed product or feed ingredient for an aquatic animal comprising a microbial biomass, and a plant derived lipid source, wherein the plant-derived lipid source and the microbial biomass are present in an amount with a protein source, which is selected from a source other than a fishery-derived protein source, so that the feed product or feed ingredient is nutritionally balanced for an aquatic animal.

In a fourth aspect of the invention there is provided a feed product or feed ingredient for an aquatic animal comprising a microbial biomass, and a plant derived lipid source, wherein the plant-derived lipid source and the microbial biomass are present in an amount with a protein source, which is selected from a source other than a fishery-derived protein source, so that the feed product or feed ingredient is nutritionally balanced for an aquatic animal wherein the feed product or feed ingredient further comprises a nutritionally balanced ratio of lipids and/or oil or mixtures thereof, protein sources, amino acids and/or mixtures therefore.

In a fifth aspect the invention relates to the use of the feed product or feed ingredient according of the first, second, third or fourth aspects as an aquaculture feed or feed ingredient for an aquatic species.

In a sixth aspect, the invention relates to a method of rearing an aquatic species comprising the step of providing the feed product or feed ingredient of the first, second, third or fourth aspects of the invention to a member of an aquatic species. In a seventh aspect, the invention relates to a method of rearing an aquatic species comprising providing the aquatic species a feed product or feed ingredient which is substantially free of fishery resources, wherein the feed product or feed ingredient is the feed product or feed ingredient according to the first or second aspect of the invention.

In an eighth aspect, the invention relates to an aquatic species, preferably a shrimp or a prawn when produced by the method of the third aspect.

In a ninth aspect, the invention relates to a method of preparing a feed product or feed ingredient for an aquatic species which is substantially free from fishery resources comprising:

providing a microbial biomass as herein described; and

adding to the microbial biomass a nutritionally balanced ratio of one or more ingredients selected from the group consisting of binding agents, carbohydrates, protein source(s) selected from source(s) other than fishery derived protein source(s), lipid(s) selected from source(s) other than fishery derived lipids, vitamins, minerals and/or mixtures thereof appropriate for the intended aquatic species.

DEFINITIONS

Throughout the specification it should be understood that the defined terms have the following intended meanings as would be understood by a person of skill in the art.

Unless stated otherwise, it should be understood that all percentages described herein refer to %(w/w) on an air-dried mass/mass basis.

The term "fishery resource(s)" should be understood to include elements, products, compositions or resources derived from an aquatic ecosystem or environment including strains, species, populations and stocks derived from an aquatic ecosystem or environment, including resources pertaining to fish, shellfish, or other aquatic animals, such as aquatic animal-derived protein sources including fish meal, squid meal or krill meal and aquatic animal-derived lipid sources including fish oil, squid oil or krill oil, as would be understood by a person of skill in the art. The term "substantially free" as used herein should be understood to mean that the feed compositions or feed ingredients include feed compositions or feed ingredients wherein fishery resources, such as an aquatic animal-derived protein source and/or an aquatic-animal derived lipid source, are either completely absent, present in trace amounts/concentrations, or present in amounts/concentrations that are lower than or less than those amounts/concentrations generally used or applied in the prior art feed compositions as would be understood by a person skilled in the field.

The term "aquatic animal" should be understood to include marine and fresh-water animals selected from the group consisting of fish, crustaceans and/or molluscs.

The term "aquatic animal-derived lipid source" should be understood to include fishery-derived lipid sources such as, for example, fish oil, krill oil and/or squid oil.

The term "aquatic animal-derived protein source" should be understood to include fishery-derived protein sources such as, for example, fish meal, squid meal and/or krill meal.

The term "lipid(s) selected from source(s) other than fishery derived lipids" should be understood to mean a lipid source that is derived from plants or non-aquatic animals. The term "nutritionally balanced ratio" should be understood to mean a suitable ratio of the particular selected components in the feed product or feed ingredient of the present invention, which would effectively sustain the growth of the relevant aquatic species. The term "microbial biomass" as used in the present invention refers to a solid biomass comprising microorganisms and cellulosic organic matter. The microorganisms are predominantly bacteria, but microalgae, yeast, protists and fungi may be present in minor amounts as contaminants. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Throughout this specification, reference to numerical values, unless stated otherwise, is to be taken as meaning "about" that numerical value. The term "about" is used to indicate that a value includes the inherent variation of error for the device and the method being employed to determine the value, or the variation that exists among the experimental values.

In a preferred form, when the feed product or feed ingredient further comprises a plant derived lipid source, the plant derived lipid source may comprise from about 1 % to about 10%, preferably from about 1 % to about 5% or more preferably from about 2% to about 4% or about 2.5% of the feed product or feed ingredient.

In one embodiment the microbial biomass comprises about 0.5% to about 40%, about 0.5% to about 30%, about 0.5% to about 25%, preferably about 1 % to about 20%, or about 1 % to about 15%, or about 3% to about 12%, or about 3% to about 10%, of the feed product or feed ingredient.

The plant derived lipid source may be selected from the group consisting of vegetable oil, soybean oil, canola oil, linseed oil or flax oil, hemp seed oil, canola seed (rapeseed) oil, pumpkin seed oil, purslane, perilla seed oil, walnut oil and/or mixtures thereof.

In a preferred form, the plant derived lipid source may have an omega-3 fatty acid: omega-6 fatty acid ratio of about greater than 1 : 1 , preferably about 3:1 to 1 :1 , preferably about 3:1 to 2:1 , preferably 1 : 1 to about 2: 1 . In another embodiment, the plant derived lipid source may have an omega-3 fatty acid: omega-6 fatty acid ratio of preferably about 1 : 2 to 1 :7, preferably about 1 :2 to 1 :5, preferably 1 :2 to about 1 :3. In one form, the protein source, which is selected from a source other than a fishery-derived protein source, is further selected from the group consisting of: soybean meal, poultry meal, lupin (kernel) meal and gluten meal and/or mixtures thereof. In a preferred form of the invention, the feed product or feed ingredient comprises a microbial biomass, and further comprises a plant-derived lipid source, wherein the plant-derived lipid source comprises from about 1 % to about 10%, preferably from about 1 % to about 5% or more preferably from about 2% to about 4% or about 2.5% of the feed product or feed ingredient and wherein the feed product or feed ingredient is substantially free of an aquatic animal-derived protein source and/or an aquatic animal derived-lipid source.

In one preferred form of the invention, the feed product or feed ingredient comprises a combination of a microbial biomass and a plant derived lipid source, wherein the feed product or feed ingredient comprises a combination of from about 1 % to about 10%, preferably from about 1 % to about 5% or more preferably from about 2% to about 4% of the plant derived lipid source and from about 0.5% to about 40%, about 0.5% to about 30%, about 0.5% to about 25%, preferably about 1 % to about 20%, or about 1 % to about 15%, or about 3% to about 12%, or about 3% to about 10%, of the microbial biomass.

In another form of the invention, the feed product or feed ingredient comprises a combination of a microbial biomass and a plant derived lipid source, wherein the feed product or feed ingredient comprises a combination of from about 1 % to about 10%, preferably from about 1 % to about 5% or more preferably from about 2% to about 4% of the plant derived lipid source and from about 0.5% to about 40%, about 0.5% to about 30%, about 0.5% to about 25%, preferably about 1 % to about 20%, or about 1 % to about 15%, or about 3% to about 12%, or about 3% to about 10%, of the microbial biomass and the feed product or feed ingredient is substantially free of an aquatic animal-derived protein source and/or an aquatic animal derived-lipid source. In an embodiment of the invention, the feed product or feed ingredient comprises a microbial biomass, wherein the microbial biomass comprises from about 0.5% to about 40%, about 0.5% to about 30%, about 0.5% to about 25%, preferably about 1 % to about 20%, or about 1 % to about 15%, or about 3% to about 12%, or about 3% to about 10% of the feed product or feed ingredient, and wherein the feed product or feed ingredient further comprises a plant-derived lipid source, wherein the plant-derived lipid source comprises from about 1 % to about 10%, preferably from about 1 % to about 5% or more preferably from about 2% to about 4% of the feed product or feed ingredient and wherein the feed product or feed ingredient is substantially free of an aquatic animal-derived protein source and/or an aquatic animal derived-lipid source.

In a preferred form, the feed product or feed ingredient of the invention may be substantially free of a fishery resource (s), wherein the fishery resource(s) may be an aquatic animal-derived protein source such as fishmeal, squid meal or krill meal and/or mixtures thereof and/or an aquatic animal-derived lipid source, such as fish oil, krill oil, or squid oil and/or mixtures thereof, which are generally present in prior art feed compositions.

In an alternative embodiment, an aquatic animal-derived protein source and/or an aquatic animal-derived lipid source, may comprise about 0% to about 20%, about 0.1 % to about 15%, about 0.5% to about 12%, about 0.75% to about 10%, about 1 % to about 7.5%, about 1.5% to about 5% of the feed ingredient or feed composition. In another embodiment, the feed product or feed ingredient comprises a microbial biomass, wherein the microbial biomass comprises from about 0.5% to about 40%, about 0.5% to about 30%, about 0.5% to about 25%, preferably about 1 % to about 20%, or about 1 % to about 15%, about 3% to about 12%, or about 3% to about 10% of the feed product or feed ingredient, and wherein the feed product or feed ingredient further comprises a plant-derived lipid source, wherein the plant- derived lipid source comprises from about 1 % to about 10%, preferably from about 1 % to about 5% or more preferably from about 2% to about 4% of the feed product or feed ingredient, and wherein the feed product or feed ingredient further comprises an aquatic-animal derived lipid source, wherein the aquatic-animal derived lipid source comprises from about 0.1 % to about 15%, from about 1 % to about 10%, from about 1 % to about 5%, and from about 1 % to about 3.5% or about 1 % to about 2.5% of the feed product or feed ingredient and wherein the feed product or feed ingredient is substantially free of an aquatic-animal derived protein source.

In another embodiment, the feed product or feed ingredient comprises a microbial biomass, wherein the microbial biomass comprises from about 0.5% to about 40%, about 0.5% to about 30%, about 0.5% to about 25%, preferably about 1wt% to about 20%, or about 1 % to about 15%, about 3% to about 12%, or about 3% to about 10% of the feed product or feed ingredient of the feed product or feed ingredient, and wherein the feed product or feed ingredient further comprises a plant-derived lipid source, wherein the plant-derived lipid source comprises from about 1 % to about 10%, preferably from about 1 % to about 5% or more preferably from about 2% to about 4% of the feed product or feed ingredient and wherein the feed product or feed ingredient further comprises an aquatic animal-derived protein source, wherein the aquatic animal-derived protein source comprises from about 0.1 % to about 15%, from about 1 % to about 10%, from about 1 % to about 5%, and from about 1 % to about 3.5% or about 1 % to about 2.5% of the feed product or feed ingredient and wherein the feed product or feed ingredient is substantially free of an aquatic animal-derived lipid source.

In an alternative embodiment, the feed product or feed ingredient comprises a microbial biomass, wherein the microbial biomass comprises from about 0.5% to about 40%, about 0.5% to about 30%, about 0.5% to about 25%, preferably about 1wt% to about 20%, or about 1 % to about 15%, about 3% to about 12%, or about 3% to about 10% of the feed product or feed ingredient of the feed product or feed ingredient, and wherein the feed product or feed ingredient further comprises a plant-derived lipid source, wherein the plant-derived lipid source comprises from about 1 % to about 10%, preferably from about 1 % to about 5% or more preferably from about 2% to about 4% of the feed product or feed ingredient and the feed product or feed ingredient further comprises an aquatic animal-derived protein- source and/or an aquatic animal-derived lipid source. The aquatic animal-derived protein-source may comprise from about 0.1 % to about 15%, from about 1 % to about 10%, from about 1 % to about 5%, and from about 1 % to about 3.5% or about 1 % to about 2.5% of the feed product or feed ingredient. The aquatic animal-derived lipid source may comprise from about 0.1 % to about 15%, from about 1 % to about 10%, from about 1 % to about 5%, and from about 1 % to about 3.5% or about 1 % to about 2.5% of the feed product or feed ingredient.

In a preferred form, the aquatic species may be selected from the group consisting of fish, crustaceans and/or molluscs. In one form, the fish may be selected from the group consisting of Atlantic salmon, barramundi and cobia; the crustaceans are selected from the group consisting of shrimps or prawns, lobsters and crabs; and the molluscs may be selected from the group consisting of oysters, scallops and abalone.

In one embodiment, the feed product or the feed ingredient of the invention may further comprise a binding agent such as gluten, alginates or starch, a further source of protein such as soybean meal, poultry meal, lupin meal, gluten meal, an ingredient rich in carbohydrate, specifically rich in starch such as wheat flour, rice bran, tapioca, rice flour, maize or corn flour, further lipid sources, a mixture of vitamins appropriate for the intended aquatic species, a mixture of minerals appropriate for the intended aquatic species, and other nutritional, pharmaceutical or growth supplements. The microbial biomass used in this invention may be prepared using a carbon source selected from the group consisting of waste, high volume, low value agricultural material and agricultural waste. The low value agricultural material may include products, by-products or waste streams from the processing of sugar cane such as filtermud, canetops, molasses or bagasse. Other sources include products, by-products or waste streams from the processing of rice, wheat, triticale, corn, sorghum, tapioca, oilseeds (including canola meal and lupin hulls), and elevator dust from grain handling facilities. Additional sources of both carbon and nitrogen could include production waste from feed mills and distillery spent grain products. The carbon source may in a specific example be canetops which has been milled or sieved.

In a preferred embodiment, the microbial biomass used in this invention may be prepared from by-products or waste streams from the processing of sugar cane. In one preferred form, the by-products or waste streams from the processing of sugar cane may include bagasse, canetops, filtermud or molasses. In a preferred form the microbial biomass is made from tapioca or bagasse or molasses. The microbial biomass used in this invention may be prepared using a nitrogen source, which may be any economically and environmentally sustainable nitrogen source such as urea, ammonia, ammonia nitrate, ammonia phosphate fertilisers, organic nitrogen sources including discharge water from aquaculture ponds. Additional nitrogen may be added to the culture system. The concentration of total nitrogen in the culture system at the start of the culture may be between about 10mg/L to about 80mg/L, 20mg/L to about 70mg/L, 30mg/L to about 60mg/L, 40mg/L to 60mg/L or 40mg/L to 50mg/L. In a further preferred embodiment, the concentration of total nitrogen at the start of the culture system is about 40mg/L to about 70mg/L.

In one preferred embodiment, the microbial biomass used in this invention may be prepared using an initial C:N (carbon : nitrogen) ratio of from about 2: 1 to 20:1 , 3:1 to 18:1 , 4:1 to 18: 1 , 5:1 to 18: 1 , or 6:1 to 18:1 . Other C:N ratios may be 7:1 , 8:1 , 9: 1 , 10: 1 , 1 1 :1 , 12:1 , 13:1 , 14:1 , 15:1 , 16:1 , 17:1 , or 18:1. Preferably 6:1 to 18:1 , more preferably 12:1 .

The source of the microorganisms in the microbial biomass are microorganisms that are naturally occurring in the water used in the culture system to produce the microbial biomass. In some embodiments, the water used in the culture system to produce the microbial biomass is raw, unfiltered seawater drawn from any water source including oceanic and estuarine waters, waste water from aquaculture ponds or recycled water from a previous culture.

The microbial biomass used in this invention may be prepared using a mixed population of microorganisms comprising microalgae, yeasts, fungi, protists and bacteria, especially where the mixed population of microorganisms comprises bacteria, more especially where the mixed population of microorganisms is substantially bacteria.

In one embodiment, the microbial biomass used in this invention comprises a mixed population of microorganisms including microalgae and bacteria. In a preferred embodiment the bacteria is present in an amount of from about 1 wt% to about 50 wt%, about 5wt% to 40 wt%, about 5wt% to about 25wt% on a dry matter basis. In a further embodiment, the microalgae is present in an amount of from about 0.1 wt% to about 50 wt%, about 0.1 wt% to about 40 wt%, about 0.1 wt% to about 30 wt%, about 0.1 wt% to about 25 wt%, about 0.1 wt% to about 20 wt%, about 0.1 wt% to about 15 wt%, about 0.1 wt% to about 10 wt%, about 0.1 wt% to about 5 wt%, on a dry matter basis.

In another embodiment, the microbial biomass used in this invention comprises a mixed population of microorganisms comprising a combination of microalgae and bacteria, wherein the bacteria is present in an amount of from about 1 wt% to about 50 wt%, about 5wt% to 40 wt%, about 5wt% to about 25wt% on a dry matter basis and the microalgae is present in an amount of from about 0.1 wt% to about 50 wt%, about 0.1 wt% to about 40 wt%, about 0.1 wt% to about 30 wt%, about 0.1 wt% to about 25 wt%, about 0.1 wt% to about 20 wt%, about 0.1 wt% to about 15 wt%, about 0.1 wt% to about 10 wt%, about 0.1 wt% to about 5 wt%, on a dry matter basis.

In yet another embodiment, the microbial biomass used in this invention comprises a mixed population of microorganisms, wherein the microorganisms comprise a mixed population of bacteria. In some embodiments, the mixed population of microorganisms comprises greater than 50 wt% of a mixed population of bacteria, preferably greater than 60 wt% of a mixed population of bacteria or greater than 70 wt% of a mixed population of bacteria, more preferably greater than 80 wt% or 85 wt% of a mixed population of bacteria. In some embodiments, the microbial biomass is substantially a bacterial biomass.

The quantification of the microalgae may be based on the chlorophyll a of the microbial biomass and that of the bacteria may be based on muramic acid content.

The microbial biomass used in this invention may be prepared using an optimised level of nutrients selected from phosphates, silicates and mixtures thereof. The phosphates may be selected from KH ₂ PO _4, superphosphate, double superphosphate, triple superphosphate, monoammonium phosphate, diammonium phosphate, rock phosphate and Agras.

KH ₂ P0 ₄ may be added in an amount to provide a P:N ratio of from about 1 :1 to 20:1 , about 2:1 to 18:1 , about 2:1 to 16: 1 , about 2:1 to 14:1 , about 2:1 to 10:1 , or about 2:1 to 8:1. Other P:N ratios may be 2:1 to 6: 1 , 5:1 , 4:1 , 3:1 or in one example 2:1 to 3:1.

The silicates used in the preparation of the microbial biomass may be selected from sodium silicate, sodium metasilicate (Na ₂ SiO ₃ .5H ₂ O), water glass, potassium silicate. Na ₂ SiO3.5H ₂ O may be added in an amount to provide a Si:N ratio of from about 0.1 :1 , about 0.3:1 , about 0.6:1 , about 1 :1 , about 1.5:1 to about 1.7:1 , about 1.9:1 to about 2.0:1.

DESCRIPTION OF THE FIGURES

The invention will now be described by way of example only, with reference to the accompanying figures, which are detailed below.

Figure 1 depicts the yields of microbial biomass (g/kL) from each of the treatments of Example 1 examining different sugar cane waste streams. Indicated on the X axis is the carbon source used in the preparation of the microbial biomass. A to F represent diet conditions as set out in Table 1.

Figure 2 sets out the experimental diet formulations and total ingredient demand based on each feed being 2000g as described in Example 2.

Figure 3 illustrates initial weight (g) and final weight (g), weight gain (g), gain rate (g/week), relative growth increase % (RGI) and FCR (feed conversion ratio) of prawns fed each of the treatment diets in Example 2.

Figure 4 demonstrates growth rates (g/week) of prawns fed diets with 10% inclusion levels of the microbial biomasses produced from the different sugar cane wastes over a 42 day period as described in Example 2. The basal diet contains no microbial biomass.

Figure 5 sets out the experimental diet formulations applied in Example 3.

Figure 6 illustrates initial weight (g) and final weight (g), weight gain (g), gain rate (g/week), relative growth increase % (RGI) and FCR (feed conversion ratio) and % survival of prawns fed each of the treatment diets in Example 3. MB is the microbial biomass of this invention.

Figure 7 is a graphical representation of the bioactive inclusion level percentages (X axis) vs growth rates of prawns (g/week) (Y axis) of the diet formulations A to K as shown in Figure 5 and as set out in Example 3.

Figure 8 illustrates the experimental diet formulations used in Example 4 and 5.

Figure 9 illustrates initial weight (g) and final weight (g), weight gain (g), gain rate (g/week), relative growth increase % (RGI) and FCR (feed conversion ratio) and % survival of prawns fed each of the treatment diets in Example 4. MB is microbial biomass of this invention, FM is fish meal, FO is fish oil, LO is linseed oil. % indicates percentage inclusion in each feed treatment as set out in Example 4 and as shown in Figure 8.

Figure 10 depicts the gain rate (g/week) of prawns fed each of the treatment diets over the 63-day trial experiment described in Example 4 and as shown in Figure 8. FM: Fishmeal, FO: Fish oil, MB: Microbial biomass, LO: Linseed oil.

Figure 11 illustrates initial weight (g) and final weight (g), weight gain (g), gain rate (g/week), relative growth increase % (RGI) and FCR (feed conversion ratio) and % survival of prawns from each treatment at the beginning and end of the 63-day trial experiment described in Example 5. FM: Fishmeal, FO: Fish oil, MB: Microbial biomass, LO: Linseed oil. % indicates percentage inclusion in each feed treatment as set out in Example 5. Figure 12 depicts the gain rate (g/week) of prawns fed each of the treatment diets over the 63-day trial experiment described in Example 5 and as shown in Figure 8. FM: Fishmeal, FO: Fish oil, MB: Microbial biomass, LO: Linseed oil.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

The invention will now be described with reference to the following examples, which only serve to illustrate the preferred embodiment(s) of the invention. The following examples should not restrict the scope of the invention as defined in the claims. EXAMPLES

Example 1

Preparation of a microbial biomass using an expanded range of carbon sources This experiment was preformed to determine whether a range of sugar cane waste streams can be used as a carbon source for producing microbial biomass which contains the bioactive component at acceptable yields. A nitrogen level of 40g/L and a C:N ratio 12:1 was used. Tapioca was used as the carbon source for the control treatment.

Method - Culture of Microbial biomass

The experiment consisted of five carbon sources (molasses, canetops, bagasse, filtermud and a tapioca control) at an N concentration of 40 mg/L and 12:1 C:N ratio (Table 1 ). An additional treatment examined canetops at the same N concentration and C:N ratios but with the canetops added as a course ground product (similar to chaff). For each treatment there were four replicates of 2400L tanks. In all treatments the nutrients were added as a single batch and the bioconversion to harvestable biomass evaluated after 35 days.

Aquasol (70 g) was added to the seawater in each tank (2400L) one week prior to the start of the experiment, to promote the establishment of green water conditions. The tanks used for culturing the microbial biomass were circular fibreglass tanks that were located down one side of a horticultural tunnel house. The tunnel house allowed minimal attenuation of natural light and enabled the water temperatures to be maintained between 25 and 33°C.

The water was circulated through a mixing tank and pumped back into the tanks to ensure that the microbial and microalgal community and the concentration of nutrients was the same in all tanks prior to the start of the experiment. Once a bloom was established, the circulation was stopped and the allocated treatments of urea (nitrogen source) and carbon source were added to the individual tanks.

A nitrogen source, urea was added to provide 40 mg L ^"1 of N (99 to 194 g of urea per tank), and the carbon sources were added to provide a C:N ratio of 12:1. In addition KH ₂ PO ₄ was added to all tanks to give a P:N ratio of about 5.0:1. The nutrients and particulate material in the tanks was kept mixed and in suspension through vigorous aeration and the use of an air-lift device in each tank. On a weekly basis the tanks walls and base were lightly scrubbed with a coarse broom, to minimise accumulation on the tank surface. Additionally, each day readings were taken to determine dissolved oxygen, pH, temperature and salinity.

During the experimental period the water temperature was 29.8 ± 0.46 °C (range 26.8 to 32.4 °C). Water salinity was 35.2 ± 0.89 mg/L (range 34.2 to 36.5 mg/L).

At the end of the experiment (35 days), 4 or 5 tanks/day were harvested through the baleen filter. Total yield wet weight recorded for each tank, a representative 100g sub-sample oven dried at 105°C to determine sample dry matter yields. Samples from each tank were labelled and stored in containers for freeze drying, the remainder will be labelled and stored in bulk. All microbial biomass collected was stored at -20°C.

Table 1. Experimental treatments

Results

This experiment determined that a range of sugar cane waste streams can be used as a carbon source for producing the microbial biomass according to the invention. There were variable levels of yield associated with each of the sugar cane waste streams, with bagasse and cane-tops producing the highest yields as depicted in Figure 1. Example 2

Evaluating the efficacy of a series of microbial biomass products produced from sugar cane wastes

The objective of this experiment was to evaluate the bioactive potential of a series of microbial biomass produced in Example 1 , which used sugar cane waste products as the carbon source. Different products were included in test diets at a 10% inclusion level and compared against a basal reference diet in terms of prawn growth over a six week period.

Methods 28 tanks (100 L) were needed for this experiment. There were 7 diet treatments, each replicated four times. The trial ran for 42 days. Approximately 2000 juvenile (~2 g) prawns of an unselected (W) stock were allocated to a large (~10,000L) seawater tank. These prawns were held in this tank prior to allocation to the experiment. Prawns were fed once daily a commercial pellet (Ridley Prawn Grower manufactured by Ridley Aquafeeds Ltd, Nerangba, Qld, Australia) until allocation to the experimental tanks.

Prior to initiation of the experiment 40 prawns were weighed to define the mean ± SD weight. Five prawns were allocated to each of 28 tanks based on all allocated individuals being within the mean ±0.5 x SD weight. Prawns were weighed to minimum of 0.1 g accuracy. Ten prawns representative of each of the initial populations were sacrificed for composition analysis and a gill sample collected and stored in -80°C. Following transfer from the outdoor tanks to the indoor 100L tanks, the water temperature was set at 28°C. The animals were acclimated to the system for five days. During this period the prawns were fed once daily to satiety (using a commercial feed) with all feed consumed and unconsumed recorded. At the end of this acclimation period the prawns were reweighed again to record weights for Day-0 The experimental system was maintained with ~ 1 L/min flow with the purpose of maintaining water temperature at around 28°C for the duration of the experiment.

Seven formulations (Basal and A1 to A6), were prepared for the experiment (Figure 2). When not being used, all feeds will be stored at -20°C. The feeds being used were based on a standard, basal formulation higher protein feed (see Figure 2). About 2000g of each feed were prepared using standard prawn feed production methods. Each diet was prepared by ensuring all ingredients were milled to <750 pm, prior to mixing all ingredients in an upright planetary mixer (Hobart, Sydney, NSW, Australia). Water was then added during the mixing to form a dough which was subsequently screw-pressed (Dolly, La Monferrina, Castell'Alfero, Italy) through a 3mm die and cut to pellet lengths of about 6mm. The pellets were then oven dried at 40°C for 24h. Diets were kept at -20°C when not being fed.

Results

This study demonstrated that there was significant bioactive potential in each of the microbial biomasses produced in Example 1 that used sugar cane waste streams as their carbon sources. However, two sugar cane waste streams, molasses and bagasse, produced products that were significantly more bioactive than the others, as can be seen in Figure 3. In particular, the RGI% (Relative Growth Index %) is 150% for treatment A2 (Base + 10% Molasses) and the RGI% for treatment A3 (Base + 10% Bagasse) is 135%, which demonstrates significantly greater bioactivity.

Further, as demonstrated in Figure 4, which sets out the resulting growth rates of prawns that were fed diets with 10% inclusion levels of the various microbial biomasses produced from the different sugar cane wastes over a 42 day period, the diets/microbial biomass samples that promoted the best growth were the base diet + 10% molasses A2 and the base diet + 10% bagasse A3. The basal diet indicated on the graph contained no microbial biomass. Example 3

Evaluation of bioactive properties of microbial biomasses and Krill meal in feeds for black tiger prawns Penaeus monodon.

The objectives of this study were to determine the optimal inclusion rate of microbial biomass produced from bagasse as a carbon source and to compare the performance of microbial biomasses against krill meal. Method

Approximately 2000 juvenile black tiger prawns (~2g) from unselected stocks were collected and transported from a local prawn farm to the CSIRO Cleveland Laboratories, QLD Australia. The prawns will be divided into a heated 10000 L tank, and the two 2500 L tanks in the Southern growth lab and held at about 28°C for 3-4 days before being sorted into the experimental tanks. During this time they were fed twice daily with a commercial pellet (Ridley Prawn Grower manufactured by Ridley Aquafeeds Ltd, Nerangba, Qld, Australia). All prawns were weighed and sorted into size classes from which a tight size range will be selected for use in the experiment. Each of the 100L experiment tanks were stocked with 5 prawns. 5 days after stocking, the prawns were reweighed to start the experiment (Day 0). 44 tanks were required for this experiment (4 replicates of 1 1 treatments). The temperature in the experimental system was maintained at about 29 °C with heated seawater at a flow-rate of 0.6 L/min. Temperature in each tank was monitored daily and a data logger immersed in six random tanks to record temperatures across the array.

This experiment compared microbial biomass and krill meal at 6 dietary inclusion levels (0, 3, 6, 9, 12 and 15%) in a simple basal formulation. The 0% inclusion treatment will be common to both products, giving a total of 1 1 treatments. Each treatment was replicated 4 times and randomly allocated to the 44 tanks in a completely randomised design. The microbial biomass used in this experiment was the bagasse treatment (A3) from Example 2. The feeds were formulated to be essentially isonitrogenous and isolipidic. The feeds were produced according to standard prawn feed production methods. Each diet was prepared by ensuring all ingredients were milled to <750 μιη, prior to mixing all ingredients in an upright planetary mixer (Hobart, Sydney, NSW, Australia). Water was then added during the mixing to form a dough which was subsequently screw-pressed (Dolly, La Monferrina, Castell'Alfero, Italy) through #14 die (2.9mm) and cut into approx 5mm pellets (unless revised according to the initial animal size). All feeds were stored at -20°C until required.

Figure 5 sets out the experimental diet formulations A to K. Figure 6 presents the resulting growth parameters (weights and gain rates) data of each treatment A to K, as means of 4 replicate experiments. The parameters in Figure 6 include the initial weight (g) of each prawn and the weight of each prawn at day 15 and 35 of treatment, the rate of weight gain per week for each prawn at day 15 and day 35 of the experiment and the final percentage survival rate of each treatment A to K. Results

Figure 6 and Figure 7 present the weights and weight gain rates of the prawns subjected to each treatment. At 3% inclusion the microbial had the same effect as krill meal at inclusion levels up to inclusion levels of <10%. At inclusion levels over 10%, microbial biomass promotes a higher level of growth that krill meal at similar levels (see Figure 6, treatment F where weight at day 35 was 6.48g/prawn compared to treatment K where the weight at day 35 was 5.51 g/prawn). Example 4

Evaluating the application of microbial biomass in complete replacement of all aquatic-derived animal resources in prawn diets

This experiment examined the potential to not only replace all the fishmeal content of prawn diets but also all the fish oil as well. Success in this regard would demonstrate the capability of microbial biomass being used to offset performance losses associated with the complete replacement of any fishery resource.

Methods

120 tanks (100 L) were needed for this experiment. There were 10 treatments, four will replicated five times, the remaining six was replicated four times. The trial ran for 42 days. This was reviewed at day 14 and day 28. Approximately 2000 juvenile (~5 g) prawns were brought to the CSIRO facility at Bribie Island Research Centre from CSIRO facilities Rossman Pty Ltd (Alberton, QLD Australia) land allocated to two large (~5,000L) stockholding tanks. These prawns were held in this tank prior to allocation to this experiment. Prawns were fed once daily a commercial pellet (Ridley Prawn Grower manufactured by Ridley Aquafeeds Ltd, Nerangba, Qld, Australia) until allocation to the experimental tanks. Prior to initiation of the experiment 40 prawns were weighed to define the mean ± SD weight. Six prawns were allocated to each of 60 tanks based on all allocated individuals being within the mean ±0.5 x SD weight. Prawns were weighed to minimum of 0.1 g accuracy. Nine prawns representative of each of the initial population were also be killed for composition analysis - kept as three bags of three. The experimental system was maintained with ~ 500 mL/min flow with the intent of maintaining water temperature at around 29°C for the duration of the experiment. Temperature in each tank was monitored daily and a data logger immersed in six random tanks to record temperatures across the array.

Ten diets were prepared for the experiment (Figure 8). When not being used, all feeds were stored at -20°C. About 1000g of each feed was prepared using standard prawn feed production methods. Each diet was prepared by ensuring all ingredients were milled to <750 μιτι, prior to mixing all ingredients in an upright planetary mixer (Hobart, Sydney, NSW, Australia). Water was then added during the mixing to form a dough which was subsequently screw-pressed (Dolly, La Monferrina, Castell'Alfero, Italy) through a 3mm die and cut to pellet lengths of about 6mm. The pellets were then oven dried at 40°C for 24h. Diets were kept at - 20°C when not being fed.

During the experiment the prawns were fed to satiety twice daily, seven days a week for the duration of the experiment. The apparent satiety regimes were determined by feeding to slight excess. The amount of food fed each day was recorded (estimate of number of uneaten pellets). Uneaten feed was scored following feeding, with the scoring used to estimate the amount of uneaten feed and used to adjust the following days ration. Care was taken to record and remove any prawn moults as soon as they are seen.

Results

The experiment demonstrated that in a Clearwater tank system the reduction of fishmeal in prawn diets results in a reduction of growth. Additionally the complete replacement of fish oil also resulted in a decrease in growth. However the experiment also demonstrated the capability of microbial biomass use to off-set performance losses associated with the complete replacement of any fishery resource use. Not only did the microbial biomass offset the losses from elimination of fish meal and fish oil, but the growth achieved was equal to or better than that achieved when microbial biomass was added to the standard high-fish meal and oil diet (Basal + MB (microbial biomass)) as depicted in Figure 9 and Figure 10. Example 5

Determining the potential for using microbial biomass to support complete replacement of fish meal and oils in diets for black tiger prawns, Penaeus monodon in green-water trials

The objective of this experiment was to examine the potential to not only replace all the fishmeal content of prawn diets but also all the fish oil as well. This follows from Example 4 where complete replacement of both fishmeal and fish oil was demonstrated effectively in clear-water tank systems and we now sought to repeat this in a green-water system to provide further results using an industrially-relevant set up.

Success in this regard would demonstrate the capability of microbial biomass being used to off-set performance losses associated with the complete replacement of any fishery resource use.

Methods Two thousand (2000) prawns were captured using trap nets from a local prawn farm. These were then transferred into plastic bags containing oxygenated seawater and then transported to the holding facilities at the Bribie Island Research Centre laboratory for observation for one to two weeks prior to allocation into the green-water culture system.

Prior to initiation of the experiments ten prawns representative of each of the initial populations were also killed for composition analysis (three bags of five prawns) and stored at -80°C. 30 x 2500L tanks were set up with no substrate and filtered seawater from the BIRC seawater supply and with individual reticulated aeration. The water supply of all tanks was initially circulating in a semi-closed system as one body of water. Tanks were established about 2 weeks before the start of the experiment to establish an effective algal bloom. The tanks were managed to simulate pond production conditions as best as possible. Prior to allocation to the tanks 40 prawns were randomly netted from the holding tanks and weighted to determine the mean and standard deviation of the population. Each tank was stocked with 30 x prawns, based on each individual being with 1x SD of the mean. Three samples of five prawns were be collected, bagged and frozen as three separate samples of the initial population.

The trial ran for 63 days. Performance was reviewed at day 21 and day 42. Water exchange was carried out as required to maintain water quality and algal blooms. When required, water exchange was generally in 10% increments from the whole water mass. Tank temperatures were measured daily and logged in six of the tanks. Temperatures were managed by deploying shading material as required, with maximum acceptable temperature of 33°C and minimum acceptable temperature 25°C. Temperatures were largely subject to natural variation with only temperature control instigated to try and keep temperatures within that range.

Additional water quality parameters of DO, temperature were measured daily and pH, secchi depth and salinity will be measured weekly at 1400 in all tanks using data logger (YSI). Weekly water samples will be taken from each tank for nutrient analysis: TSS, Ammonia, NOx and total chlorophyll will be measured. Six data loggers were randomly distributed among the 30 tanks to record temperature variation on a daily basis. The feeds used in this experiment are a repeat of Experiment 4. The formulations prepared for the experiment are set out in Figure 8.

During the experiment prawns were fed twice daily, seven days a week for the duration of the experiment. Two feeding trays per tank were used. All feed was placed on feed trays. Uneaten feed was removed (first thing each morning and prior to afternoon feeding) and quantified from trays immediately before feeding. The rations were determined by feeding to slight excess. The amount of food fed each day was recorded feed. Uneaten feed was removed from the feed trays following feeding. Estimates should be made on the amount (number of pellets) of uneaten feed remaining to allow for an estimate of consumption.

At the end of the greenwater experiment the water waste drained from each tank and the prawns captured by net and killed by ice immersion and weighed individually. Five prawns from each tank were collected for whole body composition analysis. These prawns were killed, weighed, minced, frozen and the freeze-dried for later analysis. A sample was assessed for dry matter content at the time of mincing.

Results

Figures 1 1 and 12 demonstrate that it is possible to replace all the fishmeal in prawn diets. However, in contrast to earlier results presented in Figure 10, there was no significant losses in weight gain of the prawns observed when they were fed with dietary treatments containing no fish meal or no fish oil in the greenwater systems used as shown in Figure 12.

Surprisingly, the inventors have found that the addition of the microbial biomass to the dietary treatments fed to the prawns not only assisted with any perceived performance weight losses when replacing both fish meal and fish oil in dietary treatments, but in fact the results demonstrate significant improvements in weight gain performance of the prawns. These significant improvements in weight gain performance of the prawns demonstrate the beneficial use of microbial biomass compositions in the growth of aquatic species, such as prawns. These microbial biomass compositions (see, for example, treatments 02 to U2) are substantially free of aquatic animal derived protein sources and/or aquatic animal derived lipid sources, such fish meal and fish oil. Thus, it follows that the inventors have demonstrated the use of the microbial biomass compositions for substantial replacement of fishery resources such as fish meal and/or fish oil in dietary treatments for aquatic species such as prawns. The various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections, mutatis mutandis. Consequently features specified in one section may be combined with features specified in other sections, as appropriate. All publications mentioned in the above specification are herein incorporated by reference. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia. Various modifications and variations of the described methods and products of the invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiment(s) and example(s), it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the relevant fields are intended to be within the scope of the following claims.