WO2020019029A1 - Method of feeding fish - Google Patents

Abstract

The present invention provides methods of feeding a low trophic fish with a feed product comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria. Methods of improving the attractiveness or palatability of a feed product to a lower trophic fish, stimulating increase in food intake, increasing the growth rate or food intake of a lower trophic fish and use of biomass comprising a mixed population of microorganisms including microalgae and bacteria as a feed attractant or feeding stimulant are also described.

Description

TITLE OF THE INVENTION

"METHOD OF FEEDING FISH"

[0001] This application claims priority to Australian Provisional Application No.

2018902685 entitled "Method of Feeding" filed on 25 July 2018, the entire content of which is hereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates generally to use of a feed product comprising dried mixed microbial biomass as a food for fish, such as lower trophic fish. The invention also relates to the use of dried mixed microbial biomass as a feeding attractant or feeding stimulant for fish.

BACKGROUND OF THE INVENTION

[0003] There is an ongoing expansion of global aquaculture to produce aquatic animals such as fish, molluscs and crustaceans as food to meet the increasing demands of a growing human population. There is also a need to relieve pressure on stocks of high trophic fish, such as salmon, barramundi, tuna and cod, which has resulted in efforts to include a higher proportion of low trophic fish species in the human diet. As a result, there is an increasing demand for lower trophic fish, such as tilapia, catfish and carp, and their worldwide production by aquaculture is rapidly expanding.

[0004] In particular, tilapiine cichlids such as Oreochromis, Sarotherodon and

Tilapia are examples of commercially important low trophic level fish. These fish are the subject of major aquaculture efforts on a worldwide scale, particularly in tropical waters. Tilapias have become one of the most important fish in aquaculture after carp and salmon. Tilapiines had a global production of 5.67 million tonnes valued at 8.9 billion US dollars in 2015 (FAO, 2018). They are one of the easiest and most profitable fish to farm as they are tolerant of high stocking density and grow rapidly. Tilapiines are omnivorous and can be fed on a vegetable or cereal based diet; whereas higher trophic fish such as salmon require a high protein feed content to provide efficient growth. The GIFT strain (Genetically Improved Farmed Tilapia) is a selectively bred Nile tilapia, Oerochromis niioticus, accounting for 80% of total tilapia seed production in China, 75% in Thailand and 40% in the Philippines in 2010 (Sukmanomon et a/., 2012).

[0005] There is a need for cost effective sustainable formulated feeds to support the increasing worldwide low trophic fish production. For example, currently, fishmeal and fish oil are included in amounts of between 0 and 20% and 0 and 10%, respectively, in commercial tilapiine diets. Fishmeal is expensive and supplies can be unreliable. The replacement of fish meal and fish oil, by more sustainable and cheaper alternatives in diets for tilapiines has been the focus of research. Studies have evaluated the replacement of fish meal in tilapiine diets with less expensive, locally-available plant and animal meals (El-Saidy and Gaber, 2002; Herath et a/., 2016; Koch et a/., 2016; Hg and Romano, 2013; Shiau et a/., 1989). Replacement of fishmeal with soybean meal, soy protein concentrate, and poultry by-product meal with addition of essential amino acids (such as methionine, lysine) and/or phosphorus has been studied in several short- term studies on tilapia fingerlings (Ng and Romano, 2013). Tilapia performance on a diet containing soybean meal and essential amino acids as the sole source of protein has been demonstrated to be poorer than on diets also containing 15-30% poultry by- product meal and a control diet containing 20% fishmeal (Koch et al., 2016). Fishmeal can be replaced with a careful selection of plant and/or animal by-product meals in tilapia diets, but the resulting growth is similar or lower than that in the presence of fishmeal, with detrimental effects on other metrics such as feed conversion rate and protein retention (Herath et al., 2016; Koch et al., 2016).

[0006] When compared to aquatic protein sources, plant derived protein sources and animal by-products are thus considered to be of low quality or inferior nutritional value, or are less attractive to fish. When fed as the protein component of a fish diet, the resulting performance is inferior to diets comprising aquatic-derived protein. Suitable replacements for aquatic protein resources such as fishmeal are required to address one or more of the drawbacks of reducing or excluding aquatic derived protein from fish feed products.

[0007] Studies have been undertaken to identify dietary protein sources to replace fishmeal in prawn diets (Richard et al., 2011; Sookying and Davis, 2011; Suarez et al., 2009; Xie et al., 2016). Some success has been noted in replacing fishmeal in Litopenaeus vannamei diets (Sookying and Davis, 2011) but Penaeus monodon has been found to benefit from inclusion of fishmeal (Glencross et al., 2014). This study also demonstrated that Novacq™, a natural feed ingredient composed mainly of dried mixed microbial biomass, can provide a sustainable solution to entirely eliminate the need for fishmeal in Penaeus monodon feeds. Inclusions of 5% and 10% Novacq™ have also increased growth at a range of protein levels by as much as 60% (Glencross et al., 2015). The use of microbial biomasses produced using different systems such as biofloc technology, has been shown to have varying degrees of success in prawns (Anand et a/., 2014; Kuhn et a/., 2010) and tilapia (Avnimelech, 2007; Azim and Little, 2008; Caldini et al., 2015). Caldini et al. evaluated the effects of the supply of dried biofloc biomass to reared Nile tilapia. The conclusion in Caldini et a/ states that any level of replacement of artificial feed with dried bioflocs impairs the growth performance in tilapia; and there is no justification for drying bioflocs biomass in order to provide it for farmed tilapia. [0008] There remains a need for alternative sustainable feeds for rearing low trophic fish such as tilapiine cichlids which address one or more of the drawbacks of known feed products.

SUMMARY OF THE INVENTION

[0009] The present inventor has discovered that fish feed products comprising dried biomass comprising a mixed population of microorganisms including microalgae and bacteria promote enhanced growth rate and/or improved health when provided to fish, in particular low trophic fish such as tilapiine cichlids. The inventor has also surprisingly discovered that the dried mixed biomass acts as a feed attractant and/or feeding stimulant in fish.

[0010] The inclusion of the dried biomass including bacteria and microalgae in a feed product further comprising other nutritional components has been found to increase food intake when fed to fish, and hence results in improved weight gain. It is believed that this dried mixed biomass acts as a feed attractant or feeding stimulant for fish, for example lower trophic fish such as tilapiine cichlids. This discovery finds application in the provision of improved feed product for lower trophic fish. Without being bound by theory, it is believed that feeding stimulation may be linked to improvement in feed palatability or attractiveness. However, feeding stimulation may also be the consequence of the dried biomass acting on different gustatory or metabolic pathways to those typically linked with nitrogen-rich feed attractants or stimulants. For example, feeding stimulation may be, at least in part, a consequence of increased gastrointestinal evacuation rate or nutrient absorption rate.

[0011] Fishmeal and krill meal are highly nutritious sources of protein often incorporated in fish feed products. They are attractive to fish and, in addition to providing nutritional benefit, typically act as feed attractants when incorporated in feed products. The use of the dried biomass comprising bacteria and microalgae provides an alternative to the use of aquatic resources as an attractant. The inventor has surprisingly found that the dried mixed microbial biomass may be more effective as a feed attractant or feed stimulant than aquatic resources as inclusion of the biomass has been found to be more effective at lower concentrations in a feed product than that required for aquatic resources such as fishmeal.

[0012] This discovery predicates the use of dried biomass comprising bacteria and microalgae in fish feed products comprising low quality nutritional ingredients, or ingredients with inferior nutritional value, to encourage the fish to consume an increased amount of the feed product. The incorporation of the biomass has been found to produce heightened feeding and greater weight gain and growth when compared to traditional feed formulations and feed formulations devoid of aquatic resources. The dried mixed microbial biomass also finds application in situations where feed attractiveness or palatability of feed products needs to be enhanced. Palatability of ingredients to the fish is important as, regardless of nutrient composition and digestibility, this can have a major impact on the utility of a feed product. The use of the dried biomass to improve the palatability or attractiveness of feed products in the absence of aquatic resources such as fishmeal provides the opportunity to incorporate an extended range of locally available feed grade raw ingredients such as plant derived protein sources or animal by- products to replace aquatic derived protein resources such as fishmeal or trash fish. In circumstances where there is a limited local supply of quality raw ingredients, this may improve cost-effectiveness of fish diets by reducing costs of sourcing and transporting quality ingredients such as fishmeal.

[0013] Accordingly, in a first aspect, the present invention provides a method of feeding a low trophic fish, comprising feeding to the fish a feed product comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria.

[0014] In another aspect, the invention also provides a method of rearing a low trophic fish, including a step of feeding to the fish a feed product comprising dried biomass comprising a mixed population of microorganisms including microalgae and bacteria.

[0015] In yet another aspect, the invention also provides a use of a feed product comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria in an amount effective to provide nutrition to a lower trophic fish.

[0016] It has surprisingly been discovered that the inclusion of the dried mixed biomass that comprises bacteria and microalgae in a feed product further comprising other nutritional components encourages increased food intake when fed to lower trophic fish and hence, results in improved weight gain. It is believed that the dried mixed biomass may act as a feed attractant or feeding stimulant for fish such as lower trophic fish, for example tilapiine cichlids.

[0017] Accordingly, in a further aspect, the present invention provides a use of dried biomass comprising a mixed population of microorganisms including microalgae and bacteria as a feed attractant or feeding stimulant for lower trophic fish.

[0018] In yet a further aspect, the present invention provides a method of improving the attractiveness and/or palatability of a feed product to a lower trophic fish, comprising feeding to the fish the feed product in the presence of a dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria.

[0019] In another aspect, the present invention provides a method of stimulating a lower trophic fish to increase its food intake, comprising providing to the fish a feed product comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria.

[0020] In yet another aspect, the present invention also provides a method of increasing the growth rate or food intake of a lower trophic fish comprising providing to the fish a feed product, said feed product comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria.

[0021] It is also considered that, due to its appeal to fish, the dried mixed microbial biomass may be used to increase the attractiveness and efficacy of fishing baits to aquatic animals.

[0022] Accordingly, in a further aspect, the present invention further provides a use of dried biomass comprising a mixed population of microorganisms including microalgae and bacteria as bait for an aquatic animal.

[0023] In a yet further aspect, the present invention provides a bait comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria.

[0024] In some embodiments, the dried biomass is a mixed microbial biomass according to WO 2009/132392 Al, or a mixed microbial biomass prepared according to a process described in WO 2009/132392 Al, the content of which is included herein in its entirety. In some embodiments, the dried biomass preferably comprises bacteria in an amount of 5% w/w to 25% w/w. In some embodiments, the dried biomass comprises microalgae in an amount of from 10% w/w to 80% w/w. In preferred embodiments, the dried mixed microbial biomass is Novacq™. Preferably the feed product comprises from 2% w/w to 15% w/w dried biomass. Preferably the feed product comprises one or more further nutritional ingredients, preferably selected from protein sources, carbohydrate sources and/or lipid sources. Preferably, the feed product is a nutritionally balanced product. Preferably the feed product is nutritionally balanced for the particular fish species.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a graphical representation showing the relationship between

Novacq™ inclusion rate and the daily weight gain and daily feed intake of GIFT tilapia.

[0026] Figure 2 is a graphical representation showing the mean daily dry matter feed intake for different inclusion rates of Novacq™, expressed in % of body weight, over the course of the experiment.

[0027] Figure 3 is a graphical representation showing the relationship between weight gain and fishmeal content of diets, with and without Novacq™ inclusion. Significant differences are marked by different letters [two-way ANOVA, Novacq effect, P<0.05; Fishmeal effect, P<0.05; interaction, P>0.05].

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0029] Throughout this specification, the word "comprises", or variations thereof 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.

[0030] Throughout this specification, reference to numerical values, unless stated otherwise, is 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 or method being employed to determine the value, or the variation that exists among the experimental values.

[0031] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0032] The term "dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria" or "dried mixed microbial biomass" and the like, as used herein, refers to a dried biomass, for example a solid biomass, comprising a mixed population of microorganisms including bacteria and microalgae. Typically, other microorganisms such as yeast, protists and fungi may be present. The biomass may also include cellulosic organic matter. The mixed microbial biomass is prepared by culturing a mixed or heterogeneous population of microorganisms under controlled conditions where growth of both microalgae and bacteria are encouraged. Bacterial growth is encouraged by addition of a carbon source which is utilized by the bacteria. This co-culturing results in a mixed microbial biomass containing a significant amount of bacterially derived biomass, for example from 5% w/w to 25% w/w. Methods for producing mixed microbial biomass is described in PCT/AU2009/000539, published 5 November 2009 as WO 2009/132392 A1 (Commonwealth Scientific and Industrial Research Organisation), and PCT/AU2014/000419 Al, published 16 October 2014 as WO 2014/165936 Al (Commonwealth Scientific and Industrial Research Organisation). In a preferred embodiment, the dried mixed microbial biomass is a commercially produced biomass used as a prawn feed supplement and sold under the name Novacq™. Novacq™ used herein was manufactured in Australia by Ridley AgriProducts (www.ridley.com.au) and comprises dried mixed microbial biomass prepared according to the processes described in WO 2009/132392 Al (CSIRO) and WO 2014/165936 Al (CSIRO), the contents of which are incorporated herein in their entirety.

[0033] When used herein, the term "trophic level" refers to the classification of fish by the trophic levels from 2 to 5 for different species depending on what food they eat and at what trophic level their food is classified. The term "low trophic" or "lower trophic" and the like when used herein refers to fish that are lower in the food chain and having a trophic level of, for example, 2.0-2.5 or 3. Low trophic fish commonly produced on a commercial scale by aquaculture include tilapiine cichlids such as the genera Oreochromis, Sarotherodon and Tilapia, including GIFT (Genetically Improved Farmed Tilapia) strains. Other Tilapiine cichlids include the genera Alcolapia, Danakilia, Iranocichlia and Steatocranus. In some embodiments, the tilapiine cichlid is Nile tilapia, also known as Oreochromis niloticus. In particular, a strain of Oreochromis niioticus is GIFT strain of Nile tilapia. Other low trophic fish commonly produced on a commercial scale by aquaculture include, but are not limited to, carp; and catfish, including shark catfish, pangasius, basa, and tra.

[0034] When used herein, the term "high trophic" or "higher trophic" refers to fish at a high level on the food chain and having a high trophic level of for example 3.5- 4.5. Particular high trophic level fish that may be produced by aquaculture include cobia, trout, cod, salmon, kingfish and barramundi.

[0035] Unless stated otherwise, it should be understood that all percentages described herein are weight percentages [% w/w]. When referring to the composition of the mixed microbial biomass, the percentages refer to % w/w on an air dried mass/mass basis.

[0036] When used herein the term "feed product" refers to a feed composition comprising dried mixed microbial biomass and, preferably, one or more additional nutritional ingredients selected from carbohydrate sources, protein sources and lipid sources. The feed product may also comprise one or more additional nutritional ingredients selected from vitamins and minerals; and/or excipients such as binding agents. The composition is preferably substantially homogeneous and may be in any suitable form known in the art for use in aquaculture, such as powder, paste, cake, granules, pellets and the like. In some embodiments, preferably the food product is in the form of a pellet. Preferably the feed product composition is nutritionally balanced for feeding to lower trophic fish. In some embodiments, the feed product is substantially free of aquatic derived resources such as aquatic derived proteins and/or oils.

[0037] When used herein, the term "nutritionally balanced" means that the feed product has a suitable ratio of the particular selected components, such as carbohydrate, protein and lipids, which would effectively sustain the growth of the relevant lower trophic fish. The skilled person would readily be able to determine the quantities and ratios of the various nutritional components necessary for a given fish species based on the teachings of the examples herein and general knowledge in the field.

[0038] The term "aquatic resource" 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 "trash" fish or "rough" fish, 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. "Trash" or "rough" fish refer to fish considered as having little utility or value as food fish, so are generally discarded.

[0039] Terms such as "low quality" or "inferior nutritional value" when used herein with reference to nutritional ingredients refers to ingredients such as plant derived nutritional sources, in particular plant derived protein sources, which are generally less nutritious or less acceptable to fish than aquatic derived resources. Consumption of low quality or inferior nutritional value food results in reduced rate of growth when based on amount consumed. The term "unpalatable" nutritional ingredients refers to ingredients which are less attractive to, or disliked by, fish. The presence of unpalatable ingredients in fish feed can result in fish consuming less food, and therefore rate of growth is low. Examples of low quality or inferior nutritional value ingredients include plant-derived protein sources such as soybean meal, lupin (kernel) meal and gluten meal.

2. Methods of the invention

[0040] The present invention relates to methods of feeding or rearing low trophic fish under aquaculture conditions. Non-limiting examples of low trophic fish include tilapiine cichlids; catfish species such as basa, tra and pangasius; and carp species; although the invention is believed to be applicable to any low tropic fish and, in particular, those which are commercially reared. Preferably the fish are from the tilapiine cichlid tribe and include fish of the genera Oreochromis, Sarotherodon and Tilapia, including GIFT (Genetically Improved Farmed Tilapia) strains. A particular fish is Nile tilapia, also known as Oreochromis niloticus. In particular, a strain of Oreochromis niioticus is GIFT strain Nile tilapia.

[0041] The dried biomass comprising a mixed population of microorganisms including microalgae and bacteria used in the methods herein is preferably prepared from a mixed or heterogeneous population of microorganisms under controlled conditions where growth of both microalgae and bacteria are encouraged. Bacterial growth is encouraged by addition of a carbon source which is utilized by the bacteria. This results in a mixed microbial biomass containing a significant amount of bacterially derived biomass, for example from about 1% w/w to about 50% w/w; from about 5% w/w to about 40% w/w; or from 5% w/w to about 25% w/w.

[0042] Preferably the mixed population of microorganisms comprises microalgae and bacteria wherein the bacteria is present in an amount of from about 5% w/w to about 25% w/w on a dry matter basis and the microalgae is present in an amount of from about 10% w/w to about 80% w/w on a dry matter basis. Preferably the dry weight ratio of bacteria to microalgae is between about 20: 1 to about 0.4: 1. More preferably, the bacteria is present in the microbial biomass in an amount of from about 5% w/w to about 10% w/w on a dry matter basis. Preferably the mixed microbial biomass comprises a minimum bacterial content of 5% w/w of the dried biomass.

[0043] Mixed microbial biomass and processes for its preparation are described in WO 2009/132392 Al. This microbial biomass is suitably dried prior to incorporation in a feed additive. One example of a dried mixed microbial biomass is available commercially under the name Novacq™.

[0044] The present inventor has demonstrated that inclusion of the dried mixed microbial biomass in feed product fed to lower trophic fish has led to significant weight gain in the fish. The results demonstrated herein show that the average growth improvement against control diet without biomass was 7.8%, 23.6% and 34.6% when included at 2.5, 5 and 10%, respectively. Accordingly, daily feed intake increased with biomass inclusion and mirrored closely the growth trend. Feed intake in absolute amount and specific to fish body weight was significantly higher in the diet containing 10% w/w biomass than the corresponding control diet.

[0045] The mixed microbial biomass is preferably comprised in a feed product such as a powder, granules or pellets. In one preferred embodiment, the feed product is in the form of pellets, for example extruded pellets. Preferably the mixed microbial biomass is included in a feed product at levels of from about 5% w/w to about 15% w/w or from about 5% w/w to about 10% w/w. In some embodiments, the dried mixed microbial biomass may be included at a level of approximately 5% w/w or approximately 10% w/w. Preferably, the feed product also comprises one or more further nutritional ingredients preferably selected from protein sources, carbohydrate sources and lipid sources. Preferably the feed product is nutritionally balanced.

[0046] In the methods of the invention, it will be appreciated that the fish should be provided with sufficient feed product to allow them to sustain growth. In some preferred embodiments, the fish are allowed to feed to satiety. The skilled person will readily be able to determine the amount of food required depending on the nutrient composition of the feed product and the species and size of the fish based on general knowledge in the field of aquaculture and the teaching in the Examples herein.

[0047] In view of its ability to act as a feed attractant or feeding stimulant in fish, the present invention also provides for a method of attracting an aquatic animal, such as a fish, comprising using a bait comprising dried mixed microbial biomass. There is also provided the use of dried mixed microbial biomass as a fishing bait.

[0048] The present invention also provides a fishing bait comprising dried mixed microbial biomass. The fishing bait may be used to attract an aquatic animal, such as a fish, a mollusc or a crustacean. In some embodiments, the fish is a high trophic fish or a low trophic fish. Depending on the species of aquatic animal targeted by the fishing bait, the fishing bait may further include other bait ingredients selected from, for example natural bait ingredients such as earthworms; leeches; grubs; maggots; caterpillars; frogs; fishmeal; fish, such as bait fish, chum or minnows; frogs; shrimp; and insects such as grasshoppers or ants. The bait of the invention may also include any attractant known in the fields of commercial fishing or recreational angling such as cheese, bread and the like. 3. Materials of the invention

[0049] The mixed microbial biomass for use in the methods of the invention is prepared from a mixed population of microorganisms under controlled conditions where growth of both microalgae and bacteria are encouraged. Bacterial growth is encouraged by addition of a carbon source which is utilized by the bacteria. This results in a mixed microbial biomass containing a significant amount of bacterially derived biomass. Biomass comprising a mixed population of microorganisms including microalgae and bacteria, and processes for its production is disclosed in PCT/AU2009/000539, published 5 November 2009 as WO 2009/132392 A1 (Commonwealth Scientific and Industrial Research Organisation); and PCT/AU2014/000419, published 16 October 2014 as WO 2014/165936 A1 (Commonwealth Scientific and Industrial Research Organisation), the contents of each are enclosed herein in their entirety. The source of the microorganisms may be naturally occurring in the water used in the culture system to produce the microbial biomass and may include raw, unfiltered seawater; waste water from aquaculture ponds; or recycled water from a previous culture. In addition to bacteria and microalgae, the mixed biomass used in this invention may further include yeasts, fungi and/or protists.

[0050] The process for producing the mixed microbial biomass generally comprises:

a) providing a mixed population of microorganisms comprising microalgae and bacteria; preferably, the bacteria is present in an amount of from about 5wt% to about 25wt% on a dry matter basis and the microalgae is present in an amount of from about 10wt% to about 80wt% on a dry matter basis;

b) adding a carbon source to the mixed population of organisms;

c) adding a nitrogen source to the mixed population of organisms;

d) culturing the mixed population of microorganisms under conditions suitable for the growth of both the microalgae and bacteria to form a microbial biomass; and

e) harvesting the microbial biomass.

[0051] Processes for culturing, harvesting and drying the mixed microbial biomass are described in WO 2014/165936 A1 and WO 2009/132392 Al. In some embodiments, the carbon source is preferably derived from waste, high volume, low value agricultural material and agricultural waste. Preferably the carbon source is locally generated. The low value agricultural material may include products, by-products or waste streams from the processing of sugar cane such as filtermud, cane tops, 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.

[0052] The mixed microbial biomass includes a significant amount of bacterially derived biomass, for example from about 1% w/w to about 50% w/w; from about 5% w/w to about 40% w/w; or from 5% w/w to about 25% w/w. More preferably, the bacteria is present in the microbial biomass in an amount of from about 5% w/w to about 10% w/w on a dry matter basis. Preferably the mixed microbial biomass comprises a minimum bacterial content of 5% w/w of the dried biomass.

[0053] In some embodiments, the mixed population of microorganisms comprises greater than 50% w/w of a mixed population of bacteria; for example greater than 60% w/w; greater than 70% w/w; or greater than 80% w/w or 85% w/w of a mixed population of bacteria.

[0054] In some embodiments the microalgae is present in the mixed microbial biomass an amount of from about 0.1% w/w to about 50% w/w; from about 0.1% w/w to about 40% w/w; from about 0.1% w/w to about 30% w/w; from about 0.1% w/w to about 25% w/w; from about 0.1% w/w to about 20% w/w; from about 0.1% w/w to about 15% w/w; from about 0.1% w/w to about 10% w/w; or from about 0.1% w/w to about 5% w/w on a dry matter basis. In some embodiments, the biomass comprises a mixed population of microorganisms including microalgae and bacteria, wherein the bacteria is present in an amount of from about 5% w/w to about 20% w/w on a dry matter basis and microalgae is present in an amount of from about 10% w/w to about 80% w/w on a dry matter basis.

[0055] In a preferred embodiment, the biomass comprises a mixed population of microorganisms including microalgae and bacteria, wherein the bacteria is present in an amount of from about 5% w/w to about 20% w/w or about 25% w/w on a dry matter basis and microalgae is present in an amount of from about 10% w/w to about 80% w/w on a dry matter basis. In some embodiments, the bacteria is present in an amount of from about 5% w/w to about 20% w/w, or about 5% w/w to about 10% w/w. Preferably the mixed population of microorganisms comprises microalgae and bacteria wherein the bacteria is present in an amount of from about 5% w/w to about 25% w/w on a dry matter basis and the microalgae is present in an amount of from about 10% w/w to about 80% w/w on a dry matter basis. Preferably the dry weight ratio of bacteria to microalgae is between about 20: 1 to about 0.4: 1.

[0056] Determining the composition of the mixed microbial biomass is well within the scope and knowledge of the skilled person. Quantification of the microalgae content may be based on the chlorophyll a content of the microbial biomass; and quantification of the bacteria may be based on the muramic acid content using conventional methods known in the art.

[0057] The harvested biomass is preferably dried to form a dried mixed microbial biomass. The biomass may be dried using any suitable means known in the art, however rapid drying under a high air flow at moderate temperature, such as from 40 to 80°C, for example approximately 40°C, is preferred. Preferably the dried product contains less than 10% by weight of moisture.

[0058] Although it may be used as a feed stuff in its dried form without further processing or addition of other ingredients, for the purposes of this invention the dried mixed microbial biomass is advantageously combined with additional nutritional ingredients to form a feed product.

[0059] In some preferred embodiments, the feed product comprises:

dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria; and

one or more further nutritional ingredients.

[0060] In preferred embodiments, the mixed microbial biomass comprises from about 2% to about 25% by weight of the feed product. Preferably, the biomass comprises about 5% by weight, or greater, of the feed product. In some embodiments, the biomass comprises 2% to 15%; 2% to 15%; 5% to 25%; 5% to 20%; 5% to 15%; or 5% to 10% by weight. The feed product suitably includes nutritional ingredients selected from carbohydrate sources, protein sources and lipid sources. Preferably the feed product is nutritionally balanced. Preferably, the additional nutritional ingredients are selected such that the feed product is nutritionally balanced for a lower trophic fish, such as a tilapiine cichlid.

[0061] In some embodiments, the feed product comprises from about 30% to about 40% protein; from about 45% to about 55% carbohydrate; and from about 4% to about 6% lipid.

[0062] Suitable nutritional sources are well known in the fields of fish nutrition and aquaculture. Protein sources include, but are not limited to, aquatic sources such as trash fish or rough fish, krill meal, squid meal and fish meal and mixtures thereof. Non- aquatic resource-derived protein sources include, but are not limited to, soybean meal, poultry meal, lupin (kernel) meal and gluten meal and mixtures thereof. In some embodiments, the protein source is derived from non-aquatic resources.

[0063] Suitable carbohydrate sources include, but are not limited to, wheat flour, rice bran, tapioca, rice flour, maize or corn flour or mixtures thereof. [0064] Suitable lipid sources include, but are not limited to, aquatic derived lipids such as fish oil, krill oil or squid oil, or mixtures thereof or non-aquatic lipids. Non- aquatic lipids include, but are not limited to, plant lipids such as vegetable oil, canola (rape seed) oil, linseed oil, hemp seed oil, soybean oil, pumpkin seed oil, or mixtures thereof.

[0065] In some embodiments, the feed product may be substantially free of an aquatic resource, wherein the aquatic resources may be an aquatic animal-derived protein source such as trash fish, fishmeal, squid meal or krill meal; or a lipid source such as krill oil fish oil or squid oil.

[0066] The feed product may further comprise ingredients such as a binding agent such as gluten, alginates or starch; 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 selection of suitable additional ingredients and the amounts to be included in a feed ingredient will be well within the scope of knowledge of the person skilled in the art. The skilled person will appreciate that ingredients should be non-toxic to the fish in the amounts present.

[0067] In one embodiment, the feed product comprises dried mixed microbial biomass; one or more protein sources; one or more carbohydrate sources; and one or more lipid sources; for example; dried mixed microbial biomass, soybean meal, fishmeal, wheat flour, wheat gluten, fish oil, and soybean oil. In addition to the carbohydrate, protein and lipid sources, the feed product may also comprise additional nutritional components selected from mineral sources, such as calcium diphosphate (calcium source) and amino acids, such as L-lysine or DL-methionine. The feed product may also include micronutrients in the form of minerals and/or vitamins.

[0068] The ingredients of the feed product are preferably combined to provide a uniform composition. If required, the homogeneous mixture may then be processed further. The feed product may be in any format suitable for feeding to fish in an aquaculture environment. Conveniently the feed product is in the form of a paste, powder, granule, cake or pellet. In a preferred embodiment, the feed product is in pellet form, and preferably a nutritionally balanced pelletized feed product. Methods of preparing pellets, such as extruded pellets, is well known in the art and is described in the Examples herein and in WO 2009/132392 A1 and WO 2014/165936 Al.

[0069] Processes for preparing feed product for fish is well known in the art of aquaculture. Typically the required ingredients are selected and combined in the required ratios. Preferably the dry ingredients are milled and combined with mixing to provide a uniform composition. Typically, the ingredients are combined by mixing, for example using a planetary mixer, to provide a homogeneous mixture. The dry ingredients may be combined with liquid ingredients, such as lipid ingredients, prior to extruding the resulting mixture. In some embodiments, the dry ingredients are combined with water in an extruder. The mixture is then extruded and dried prior to infusing the resulting pellets with a source of lipid if required. The water content of the pellet can be controlled to provide a floating pellet. Extrusion of the mixture through a 2.5-3.0 mm die, for example a 2.8mm die, provides pellets of a suitable diameter for lower trophic fish, such as tilapiine cichlids. In some embodiments, the pellets are cut to about 4 mm to about 5 mm lengths, for example 4.5 mm lengths at the die face to give a pellet of about 2.5 to about 3.0 mm in diameter and about 4 mm to about 5 mm in length. In some embodiments the pellets measure approximately 2.8 mm by 4.5 mm.

[0070] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLES

Materials and Methods

Culture system and fish population

[0071] The growth experiment was conducted in the laboratories of the

WorldFish headquarters in Penang, Malaysia, using GIFT (Genetically Improved Farmed Tilapia) Nile tilapia, Oreochromis niloticus. A total of 54 x 10L transparent aquaria (tank) on a recirculated freshwater system were used. Water flow was regulated at 2 exchange per hour and recirculated at 95% through a series of filter media and protein skimmers. Stored rain water was used as make up water. The water quality over the entire experiment was kept as follows: mean ± S.D. water temperature = 27.1 ± 0.8 °C; D.O. 7.0 ± 1.1 mg L ¹; pH = 6.8 ± 0.3; NH3 <0.5ppm; NO2 < 1.0ppm; NO3 <80ppm. A total of 1500 fish fingerlings were obtained from Worldfish GIFT breeding center in Jitra, Malaysia. The stock was graded to only include fish within one standard deviation of the mean to use for the experiment. A total of 10 fish per tank (mean weight 11.43 ± 1.75 g ± SD) were randomly allocated. Three samples of 10 pooled initial fish were taken for subsequent composition analyses.

Dietary treatments and composition

[0072] A total of eight isonitrogenous and isoenergetic experimental diets were extruded. Each of the ingredients were milled to <750 pm before batching, ingredient chemical composition can be found in Table 1. A total of 10kg of each of the experimental diets was mixed thoroughly (without the oil component) using an upright planetary mixer (BakerMix, Artarmon, NSW, Australia). The diets were then extruded through a laboratory-scale 24mm twin-screw extruder (MPF24:25, Baker Perkins, Peterborough, UK), with intermeshing, co-rotating screws. The was composed of a series of intermeshing feed screws (FS), forwarding paddles (FP) and lead screws (LS) arranged according to defined barrel diameters (D) such that overall configuration from the drive end was 16D FS, 2D FP, ID FS, 2D FP, ID LS, ID FP, 2D LS to the die. A single 2.8 mm diameter cylindrical die tapered at a 67° angle with a land length of 3 mm was used. The barrel comprised of four temperatures zones that were set at 70, 80, 100 and 110°C from the drive to die respectively, and the machine was run at c. 160rpm. The mash was delivered into the barrel at a rate of c. 4.2-4.9 kg hr¹, and water was pumped peristaltically (Thermoline, Wetherrill Park, NSW, Australia) into the barrel at between 2.1 and 3.0 L hr¹. Water addition was varied among diets to produce floating feeds of ~4.2 mm 0. The pellets were cut to 4.5mm lengths at the die face with a 2 blade variable speed cutter. The pellets were then dried at 60°C for 24h, after which each diet was vacuum infused with the specific allocation of oil. The pellets were then allowed to cool before being bagged and labelled. All feed were kept frozen (-20°C) except when feeding or weighing feed.

[0073] Dietary treatments consisted of four diets containing 10% fishmeal with incremental level of Novacq™ (0, 2.5, 5 and 10%), and four diets with decreasing level of fishmeal (5% and 0%) with 10% Novacq™ and without (respective controls). Novacq™ was incorporated by replacing an equal amount of wheat flour while fishmeal was replaced by soybean meal and increasing levels of methionine, lysine, and fish oil, with a concurrent decrease in soybean oil to keep total lipid equal (Table 2). A benchmark commercial diet was also used in the experiment. The measured chemical composition confirmed all diets showed similar protein and energy content (Table 2).

[0074] Table 1. Composition of the commercial diet and ingredients used in the experimental feeds, on dry matter basis.

[0075] Table 2. Dietary formulations and composition of feeds on dry matter basis.

Fishmeal and Fish Oil : South American source, provided by Ridley, Narangba, QLD, Australia. Soybean meal : KEWPIE Stock feeds, Kingaroy, QLD, Australia; Wheat Flour and Wheat Gluten : Manildra, Auburn, NSW, Australia. Wheat Bran : Allora Grain and Milling, Allora, QLD, Australia; Novacq : CSIRO pond production, commercial-in- confidence.

*micronutrients (mg kg ¹ of diet) : Choline (60% choline chloride), 5; Vitamin C (Stay-C), 2; fish vitamin and mineral premix, 6¹;

^he vitamin and mineral premix content in the final feed (IU or g per kg feed) was: Vitamin A, 15KIU;Vitamin D3, 1.5KIU; Vitamin E, O. lg; Vitamin K3, O.Olg; Vitamin Bl, 0.025g; Vitamin B2, 0.03g; Vitamin B3, 0.15g; Vitamin B5, 0.05g; Vitamin B6, O.Olg; Vitamin B9, 5mg; Vitamin B12, 0.03mg; Biotin, lmg; vitamin C, 0.45g; Choline chloride, lg; Inositol, 0.35g; Ethoxyquin, 0.125 g; Copper, 0.015g; Ferrous iron, 0.04g; Magnesium, O. lg; Manganese, 0.09g; Zinc, 0.15g.

Feeding and growth measurements

[0076] Fish were fed by hand twice daily to satiation. Each tank was fed over a minimum of three separate feeding events over 120 min in morning and 120 min in afternoon during which the fish were fed until uneaten pellets were observed for 10 min on the final occasion, indicating fish reached satiation. Uneaten pellets were collected on a 300 pm screen daily, and dried in a pre-weighed aluminium foil until constant dry weight (4 hours at 105°C). Daily feed delivered was obtained from the differential weights before and after feeding for tank specific feed containers and expressed in dry matter. Feed intake was calculated daily as the difference between the dry matter delivered and dry matter collected. Feed intake (FI) was expressed in gram of feed dry matter per fish per day accounting for any daily mortality, as well as expressed as specific feed intake (SFI), in percentage of body weight (i.e., SFI = FI / BW_e x 100). The average expected body weight (BW_e) of the fish each day was calculated based on fortnight fish weights and the measured linear growth rates achieved on all treatments:

I _{T \} Gΐά"» Ssti DM feed intake e*r tank , . _

Feed intake (gDM/fish/day)

’ = å7

!-^_,¹ - - - - Dai -ly fish cow ^:i -t / 42

[0077] Fish were weighed individually on day 0 and 42 and tanks were bulked weighed on day 14 and 28 to minimize fish handling stress. Fish were anaesthetized using clove oil before weighing and allowed to recover in their allocated tank following weighing. This procedure was carried out on each weight check day. Any observed mortality within the first week only was replaced by an equivalent fish of the same weight from the stock tank.

[0078] Fish growth was found to be linear over the experimental period for all treatments (on average, weight = 1.76 x Days +7.49 g). Therefore fish growth was expressed as weight gain or daily weight gain in figures. Growth was also expressed in percentage difference to the control diet.

Feed conversion ratio accounted for the mortalities, and was also expressed corrected for feed dry matter, as follows:

[0079] Nutrient retention efficiencies were calculated from feed intake and carcass composition for protein and energy as follows:

Retention efficiency (RE; %) of Nzitrient =

Chemical composition analyses

[0080] Fish carcasses were frozen at -20°C until analysis. Dry matter content was determined by gravimetric analysis following drying at 105°C for 4h. The samples were then ground, shipped to CSIRO analytical laboratories, and analysed for chemical composition. Ash content was determined based on mass change after combustion in a muffle furnace at 550°C for 6h. Total lipid content was determined gravi metrically following extraction in 2: 1 :0.4 chloroform: methanol : water, using a modification to the method proposed by Folch et at. (1957). Measurement of Total Nitrogen content was determined by the Dumas Method using a Flash 2100 Elemental Analyser (Thermo Fisher Scientific., Waltham, MA, USA) and used to calculate sample protein content based on N x 6.25. Gross energy was determined by isoperibolic bomb calorimetry in a Parr 6200 oxygen bomb calorimeter with an 1108CL bomb for ingredients and diets, and an 1109A semi-micro bomb for faeces (Par Instrument Company, Moline, IL, USA). Carbohydrate was calculated by difference.

Statistical analyses

[0081] Differences in culture performance indices and carcass composition were tested by one-way ANOVAs followed by post-hoc comparisons using Tukey-Kramer tests. Repeated-measures ANOVAs were used to test differences in daily feed intake and specific feed intake between treatments for the entire duration of the experiment and first 7 days. Significant differences in daily weight gain with fishmeal and Novacq™ inclusions, and the interaction, were tested by a two-way ANOVA. Before all analyses, the ANOVA assumptions of normality of residuals and homogeneity of variances were tested using the Shapiro-Wilk and Levene tests, respectively. Survival data were arcsine square root transformed and data logio or square root transformed when necessary to satisfy Levene's test for homogeneity of variance (P.0.05). All statistical analyses were performed using NCSS 11.

Results

Effect of Novacq™ inclusion rate in a practical tilapia diet

[0082] Tilapia grew well on all experimental diets, with weight gains and survival >80% not different to a benchmark commercial diet (CD) (Table 3). Inclusion of Novacq™ increased weight gain significantly (Table 3 and Figure 1). The average growth improvement against the control diet with no Novacq™ was 7.8%, 23.6% and 34.6% when included at 2.5, 5 and 10%, respectively (Table 3).

[0083] Daily feed intake increased with Novacq™ inclusion and mirrored closely the growth trend (Figure 1). Feed intake in absolute amount (FI) and specific to fish body weight (SFI) was significantly higher in the diet containing 10% Novacq™ than the control diet (Table 3). The trend for a higher specific intake with inclusion of Novacq™ was achieved throughout the experiment (Figure 2).

[0084] Feed conversion ratios, as fed and expressed in feed DM, did not vary significantly with Novacq™ inclusion, and were significantly better on all experimental feeds containing 10% FM than the benchmark CD (Table 3). Retention efficiency of protein and energy did not differ significantly between diets (Table 3). [0085] Carcass composition analyses indicated no significant differences between fish, except for a decrease in protein for fish fed the 10FM-5NQ against the CD and 10FM-2.5NQ diets (Table 4).

[0086] Table 3. Mean initial weight, weight gain, survival, daily feed intake,

FCR and nutrient retention efficiency in tilapia fed a benchmark commercial diet (CD) and practical diets with increasing inclusion of Novacq™ after 42 days (mean ± S.E., n=6). Significant differences are marked by different letters [One-way ANOVAs, P<0.05; Repeated-measures ANOVA for FI and SFI, P<0.05].

[0087] Table 4. Mean carcass composition of tilapia fed a benchmark commercial diet (CD) and practical diets with increasing inclusion of Novacq™ after 42 days (mean ± S.E., n=6). Data on a dry matter basis. Significant differences are marked by different letters [one-way ANOVAs, P<0.05].

Fishmeal replacement with and without Novacq™

[0088] Significant lower weight gain was achieved when fish were fed feeds with 0% fishmeal compared to 10% fishmeal (Figure 3). However, the addition of 10% Novacq™ had a positive effect on overall weight gain at all fishmeal inclusion (Table 5). The average growth against the control 10FM-0NQ decreased by 3% and 14.5% when fishmeal was reduced to 5 and 10%, respectively. The average growth against the control 10FM-0NQ increased by 19.5% and 15.5% with the addition of 10% Novacq™ when fishmeal was reduced to 5 and 10%, respectively (Table 5).

[0089] Feed intake in absolute amount (FI) and specific to fish body weight

(SFI) was significantly higher in the diet containing 10% Novacq™ than the diets without

(Table 5).

[0090] Feed conversion ratios, as fed and expressed in feed DM, tended to increase with a reduction in fishmeal but did not vary significantly with fishmeal and/or Novacq™ inclusion (Table 5). Retention efficiency of protein and energy did not differ significantly between diets (Table 5). Carcass composition analyses indicated no significant differences between fish (Table 6).

[0091] Table 5. Mean initial weight, weight gain, survival, daily feed intake,

FCR and nutrient retention efficiency for tilapia fed 50% and 100% fishmeal replacement with and without 10% Novacq™ after 42 days (mean ± S.E., n=6). Significant differences are marked by different letters [One-way ANOVAs, P<0.05; Repeated- measures ANOVA for FI and SFI, P<0.05].

[0092] Table 6. Mean carcass composition of tilapia fed 50% and 100% fishmeal replacement with and without 10% Novacq™ after 42 days (mean ± S.E., n=6). Data on a dry matter basis. No significant differences was found [One-way ANOVAs, P>0.05].

[0093] The results clearly demonstrate that there a growth benefit of including dried mixed microbial biomass at various inclusion rates in practical extruded tilapia diets; and the dried mixed microbial biomass improves the growth performance of tilapia in diets with low, or zero, fishmeal. The biomass when included in an amount of 10% significantly increased growth performance of tilapia by up to 35% in diets with 10%, 5% and 0% fishmeal. The diets were extruded diets, demonstrating that the benefit of dried mixed microbial biomass is not diminished when extrusion heat treatment is applied. All extruded diets containing the biomass outperformed a benchmark commercial diet containing similar amount of protein and energy, so growth results achieved in the study are commercially relevant. Weight gain increased in parallel with increasing inclusion rates of mixed microbial biomass, with weight gains of 7.8%, 23.6% and 34.5% in relation to an isoenergetic and isonitrogenous control diet, at 2.5%, 5% and 10% dried mixed microbial biomass inclusion, respectively. While the weight gain response slowed towards the highest inclusion, it not achieve a response plateau indicating greater weight gains may be achievable at higher inclusion rates in the tilapia diet. An inclusion of 5% maximised the benefit of the biomass with regard to the quantity required in tilapia diets to achieve a response.

[0094] Previous successful fishmeal replacement studies have achieved weight gains in line with experimental control (see Furuya et al., 2004; Koch et al. 2016). The inventor has found that total replacement of 10% fishmeal with soybean meal, and increased inclusion of methionine, lysine and fish oil to compensate for nutrient deficiencies, still resulted in a significant decrease of around 14.5% in daily weight gain. An intermediate level of fishmeal (5%) did not significantly affect weight gain. These results are in agreement with several previous studies showing that tilapia growth performance reduces with the replacement of fishmeal for individual plant proteins, possibly due to the presence of anti-nutritional factors, particularly from soybean meal (Borgeson et a/., 2006; Koch et a/., 2016). Replacement of soybean meal with a complex mixture of processed plant or rendered protein ingredient resulted in superior growth. The reduction in growth observed in the present studies on a 0% fishmeal diet could not be linked to a significant impact of removing fishmeal on feed intake (4.8% BW day^-1), FCR (1.06) or protein retention (45%), indicating other mechanisms to nutrient intake or digestion were playing a role.

[0095] The results herein have demonstrated that dried mixed microbial biomass can increase the weight gain of tilapia on 0% fishmeal diets by as much as 35%, resulting in a net improvement in culture performance of 15.5% against the 10% fishmeal control. As such, the use of the biomass as a feed additive can more than compensate for the negative impact of eliminating fishmeal in diets for tilapia and improves the sustainability value of the diets in country with limited access to quality raw ingredients.

[0096] Feed intake on the experimental diets mirrored the growth trends achieved. Characterising the palatability of ingredients is important because, regardless of their nutrient composition and digestibility, it can have a major impact on its usefulness as a feed. For an animal to demonstrate an accurate feed intake response, it must be given the opportunity to refuse feed (Glencross et al., 2007). In the present studies, careful hand feeding allowing sufficient time and effort to feed fish to satiety was performed in order to collect uneaten food from all tanks twice daily after the feeding event. To decouple growth and feed intake feed dry matter intake was expressed as a percent of daily expected body weight based on measured linear growth rates, to account for fish weight variation. Fish fed the commercial benchmark diet showed a relatively high dry matter intake in relation to their poorer growth rates when compared to the present diets, resulting in significantly poorer food conversion rates, possibly due to the use of less digestible ingredients. Fish fed diets incorporating biomass ate significantly more food over the course of the experiment than their respective control fed fish, both in absolute terms but also in percent of their body weight, and the response was in a direct relationship with inclusion rates. A 10% inclusion of dried mixed microbial biomass resulted in approximately 33% increase in feed intake at all levels of fishmeal inclusion, indicating a clear effect of the dried mixed microbial biomass on tilapia diet palatability regardless of fishmeal content. Fishmeal is often use as a palatability enhancer in fish feed but it was clear from these studies that feed intake could be stimulated by the inclusion of dried mixed microbial biomass beyond the effect of fishmeal.

[0097] The effect of dried mixed microbial biomass as a feeding stimulant was rapid and was observed within the first few days of feeding (Figure 2). Fish fed diets containing the biomass frequently displayed a voracious feeding behaviour and greater appetite compared to counterparts fed control diets (observation). The feed intake measurements, and similar feed conversion rates and REs measured in these studies, strongly point towards the dried mixed microbial biomass as being a feeding stimulant or feeding attractant in tilapia. This is surprising as the most common physicochemical properties of feeding attractants and stimulants for fish are non-volatile, low molecular weight, nitrogen-containing, amphoteric, water soluble, stable to heat treatment and of broad biological distribution. These properties are consistent of those of free amino acids and related nitrogenous substances like nucleotides, nucleosides and quaternary ammonium bases (de la Higuera, 2007). The strong feeding stimulation of dried mixed microbial biomass is therefore unexpected considering its low nitrogen content (i.e., N=0.6%) possibly indicating stimulation of different gustatory pathways (Lamb, 2007).

[0098] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0099] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application. [00100] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be i ncluded within the scope of the appended claims.

BIBLIOGRAPHY

Anand, P. S. S., Kohli, M. P. S., Kumar, S., Sundaray, 3. K., Roy, S. D., Venkateshwarlu, G., Sinha, A. and Pailan, G. H. (2014). Effect of dietary

supplementation of biofloc on growth performance and digestive enzyme activities in Penaeus monodon. Aquaculture 418-419, 108-115.

Avnimelech, Y. (2007). Feeding with microbial floes by tilapia in minimal discharge bio-flocs technology ponds. Aquaculture 264, 140-147.

Azim, M. E. and Little, D. C. (2008). The biofloc technology (BFT) in indoor tanks: Water quality, biofloc composition, and growth and welfare of Nile tilapia

(Oreochromis niloticus). Aquaculture 283, 29-35.

Borgeson, T. L., Racz, V. J., Wilkie, D. C., White, L. J. and Drew, M. D. (2006). Effect of replacing fishmeal and oil with simple or complex mixtures of vegetable ingredients in diets fed to Nile tilapia (Oreochromis niloticus). Aquaculture Nutrition 12, 141-149.

Caldini, N. N., Cavalcante, D. d. H., Rocha Filho, P. R. N. and Sa, M. V. d. C. e. (2015). Feeding Nile tilapia with artificial diets and dried bioflocs biomass. Acta Scientiarum. Animal Sciences 37, 335-341.

de la Higuera, M. (2007). Effects of Nutritional Factors and Feed Characteristics on Feed Intake. In Food Intake in Fish, pp. 250-268: Blackwell Science Ltd.

El-Saidy, D. M. S. D. and Gaber, M. M. A. (2002). Complete Replacement of Fish Meal by Soybean Meal with Dietary L-Lysine Supplementation for Nile Tilapia

Oreochromis niloticus (L.) Fingerlings. Journal of the World Aquaculture Society 33, 297- 306.

Folch, 3., Lees, M. and Sloane-Stanley, G. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem 226, 497-509.

Furuya, W. M., Pezzato, L. E., Barros, M. M., Pezzato, A. C., Furuya, V. R. B. and Miranda, E. C. (2004). Use of ideal protein concept for precision formulation of amino acid levels in fish-meal-free diets for juvenile Nile tilapia (Oreochromis niloticus L.). Aquaculture Research 35, 1110-1116.

Glencross, B., Arnold, S. and Irvin, S. (2015). Bioactive factors in microbial biomass have the capacity to offset reductions in the level of protein in the diet of black tiger shrimp, Penaeus monodon. Aquaculture 446, 74-79.

Glencross, B., Irvin, S., Arnold, S., Blyth, D., Bourne, N. and Preston,

N. (2014). Effective use of microbial biomass products to facilitate the complete replacement of fishery resources in diets for the black tiger shrimp, Penaeus monodon. Aquaculture 431, 12-19.

Glencross, B. D., Booth, M. and Allan, G. L. (2007). A feed is only as good as its ingredients - a review of ingredient evaluation strategies for aquaculture feeds. Aquaculture Nutrition 13, 17-34.

Herath, S. S., Haga, Y. and Satoh, S. (2016). Potential use of corn co- products in fishmeal-free diets for juvenile Nile tilapia Oreochromis niloticus. Fisheries Science 82, 811-818.

Koch, J. F., Rawles, S. D., Webster, C. D., Cummins, V., Kobayashi, Y., Thompson, K. R., Gannam, A. L., Twibell, R. G. and Hyde, N. M. (2016). Optimizing fish meal-free commercial diets for Nile tilapia, Oreochromis niloticus. Aquaculture 452, 357-366.

Kuhn, D. D., Lawrence, A. L., Boardman, G. D., Patnaik, S., Marsh, L. and Flick, G. 3. (2010). Evaluation of two types of bioflocs derived from biological treatment of fish effluent as feed ingredients for Pacific white shrimp, Litopenaeus vannamei. Aquaculture 303, 28-33.

Lamb, C. F. (2007). Gustation and Feeding Behaviour. In Food Intake in Fish, pp. 108-130: Blackwell Science Ltd. Ng, W. K. and Romano, N. (2013). A review of the nutrition and feeding management of farmed tilapia throughout the culture cycle. Reviews in Aquaculture 5, 220-254.

Richard, L., Surget, A., Rigolet, V., Kaushik, S. 3. and Geurden, I.

(2011). Availability of essential amino acids, nutrient utilisation and growth in juvenile black tiger shrimp, Penaeus monodon, following fishmeal replacement by plant protein. Aquaculture 322, 109-116.

Shiau, S. Y., Kwok, C. C., Hwang, J. Y., Chen, C. M. and Lee, S. L.

(1989). Replacement of Fishmeal with Soybean Meal in Male Tilapia (Oreochromis niloticusxO. aureus) Fingerling Diets at a Suboptimal Protein Level. Journal of the World Aquaculture Society 20, 230-235.

Sookying, D. and Davis, D. A. (2011). Pond production of Pacific white shrimp (Litopenaeus vannamei) fed high levels of soybean meal in various combinations. Aquaculture 319, 141-149.

Suarez, J. A., Gaxiola, G., Mendoza, R., Cadavid, S., Garcia, G., Alanis, G., Suarez, A., Faillace, 3. and Cuzon, G. (2009). Substitution of fish meal with plant protein sources and energy budget for white shrimp Litopenaeus vannamei (Boone, 1931). Aquaculture 289, 118-123.

Sukmanomon, S., Kamonrat, W., Poompuang, S., Nguyen, T. T. T., Bartley, D. M., May, B. and Na-Nakorn, U. (2012). Genetic changes, intra- and inter- specific introgression in farmed Nile tilapia ( Oreochromis niloticus ) in Thailand.

Aquaculture 324-325, 44-54.

Xie, S.-w., Liu, Y.-j., Zeng, S., Niu, 3. and Tian, L.-x. (2016). Partial replacement of fish-meal by soy protein concentrate and soybean meal based protein blend for juvenile Pacific white shrimp, Litopenaeus vannamei. Aquaculture 464, 296- 302.

Claims

WHAT IS CLAIMED IS:

1. A method of feeding a low trophic fish, comprising feeding to the fish a feed product comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria.

2. A method of rearing a low trophic fish, comprising a step of feeding to the fish a feed product comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria.

3. A use of a feed product comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria in an amount effective to provide nutrition to a low trophic fish.

4. A use of dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria as a feed attractant or feeding stimulant for lower trophic fish.

5. A method of improving the attractiveness and/or palatability of a feed product to a lower trophic fish, comprising feeding to the fish the feed product in the presence of a dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria.

6. A method of stimulating a lower trophic fish to increase its food intake, comprising providing to the fish a feed product comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria.

7. A method of increasing the growth rate or food intake of a lower trophic fish comprising providing to the fish a feed product, said feed product comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria.

8. A method or use according to any one of claims 1 to 7 wherein the feed product further comprises one or more nutritional ingredients.

9. A method or use according to any one of claims 1 to 8 wherein the feed product comprises the dried biomass in an amount of from 5% w/w to about 15% w/w.

10. A method of any one of claims 1, 2, 5, 6, 8, or 9, or a use according to any one of claims 3, 4, 8 or 9, wherein the lower trophic fish is a tilapiine cichlid.

11. A method or a use according to claim 10 wherein the tilapiine cichlid is Nile tilapia ( Oerochromis niloticus).

12. A method of fishing using bait wherein the bait comprises dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria.

13. A use of dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria as bait for an aquatic animal .

14. A bait comprising dried biomass that comprises a mixed population of microorganisms including microalgae and bacteria .