WO2018190772A1

WO2018190772A1 - Composition for aquaculture

Info

Publication number: WO2018190772A1
Application number: PCT/SG2018/050174
Authority: WO
Inventors: Farshad SHISHEHCHIAN
Original assignee: Blue Aqua International Pte Ltd
Current assignee: Blue Aqua International Pte Ltd
Priority date: 2017-04-12
Filing date: 2018-04-06
Publication date: 2018-10-18
Anticipated expiration: 2019-10-12
Also published as: SG10201703032YA

Abstract

Disclosed herein is a bacterial formulation that includes a bacteria selected from the genus Bacillus and a porous substrate material, wherein the bacteria is supported on and/or within the substrate material. Also disclosed herein are uses of the bacteria formulation, as well as methods of manufacture and regeneration of said formulation.

Description

COMPOSITION FOR AQUACULTURE

Field of Invention This invention relates to a composition that improves water quality for aquaculture. Background

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Biological wastewater treatment is an emerging technology for treating toxic gases (such as ammonia), metabolic waste and organic matter present in aquaculture ponds. When the waste load is high, the natural microbial flora in the pond may not have sufficient capacity to break it down. The accumulation of organic matter, ammonia and other toxic metabolites in water decreases the dissolved oxygen concentration of the water. This in turn leads to the development of anaerobic conditions at the soil-water interface that contribute to stress conditions in the pond, which adversely affects the health of aquatic animals in the pond and reduces the pond's productivity.

Water exchange has been used as the conventional treatment method in aquaculture industry. Water exchange refers to a common practice for draining or discharging water from an aquatic environment and replacing the discharged water with water of better quality. For example, aquaculture ponds located beside a source of water such as a river, water of poor quality (with low dissolved oxygen, high carbon dioxide and/or high nitrogenous compound concentration) may be discharged from the pond into the river downstream of the pond, and fresh water from upstream of the pond may be supplied to the pond. This improves the water quality in the pond (higher dissolved oxygen, low carbon dioxide and nitrogenous compound concentration). Typically, this exchange can account for up to one third (e.g. up to 20%) of the water in a pond per day. It has been previously reported that water exchange eliminates phytoplankton and toxic metabolites (Chamberlain, G. W. 1987. Intensification reasonable from economic point of view. Coastal Aquaculture 4(1): 1-7). A high amount of water exchange leads to an unstable environment, which causes stress for the aquatic animals. However, one of the main problems plaguing aquaculture in areas of high salinity is the high rate of water exchange required to decrease the salinity of water and to enhance the ecological environment for aquatic animals (i.e. up to one third or up to 20% of the water in the pond per day). This is further compounded by the need to increase production and aquaculture yield with decreased water and land usage. These ecological and economical challenges have persuaded researchers in the field of sustainable aquaculture to look for improved aquaculture treatments.

Interest in improved aquaculture systems under zero or limited water exchange conditions has been increasing due to the issue of water scarcity and the sustainable use of heterotrophic and chemoautotrophic bacteria to improve comprehensive quality in aquatic environments. The benefits of using bacteria are well known and have been shown in research and practical applications. Heterotrophic bacteria (which are subsequently referred to as probiotics) are used in bioremediation to decompose organic matter into ammonia, decreasing biological oxygen demand even in low oxygen condition, and thus preventing anaerobic conditions that will induce hydrogen sulfide (H₂S) production. In some applications, the use of probiotic bacteria has reduced antibiotic use in aquaculture systems. Suppression of proliferation of certain pathogenic bacteria (e.g., Vibrio spp.) in shrimp cultures has been achieved by introducing (inoculating) non-pathogenic strains or species of bacteria, that compete for microbial metabolite resources (Recent Technological Innovations in Aquaculture, FAO Fisheries Circular No. 886, Revision 2). In aquaculture systems under high water exchange and/or in concrete ponds and/or plastic- lined ponds, bioremediation is challenging because it can be difficult to provide the pond with a sufficient amount of probiotic bacteria and an available surface for said bacteria to sustain a viable population. Thus, there remains a need for new and improved methods of bioremediation that can be applied to aquaculture systems in a sustainable manner.

Summary of Invention

One object of the current invention relates to the application of probiotic bacteria in a product for improving the microbiological status of water by inhibiting the growth of undesirable algae and pathogens in said water. Pathogenic bacterial species in aquaculture systems causes contamination of the cultured species, which results in lower growth rate and/or a higher mortality rate. Further, many of the pathogenic bacterial species compete with and inhibit the growth of beneficial bacteria. When this occurs, the nutrient recycling processes is indirectly affected, resulting in the decline of water quality.

Another object of the invention relates to the application of a product to reduce the need for water exchange in aquaculture. For example, in some aquaculture ponds beside a source of water such as a river, water of poor quality (with low dissolved oxygen, high carbon dioxide and/or high nitrogenous compound concentration) may be discharged from the pond into the river downstream of the pond, and fresh water from upstream of the pond may be supplied to the pond and therefore improving the water quality in the pond (higher dissolved oxygen, healthy concentrations of carbon dioxide and nitrogenous compounds). In contrast, the present invention obviates the need for frequent water exchange and also helps to restore the beneficial bacterial population following water exchange. This is achieved by the product of the current invention due to its unique properties. Firstly, the product of the current invention contains probiotic bacteria and so bioremediation by these bacteria obviates the need for frequent water exchange to provide water of sufficient quality for aquaculture, thereby promoting water conservation, especially at times of water scarcity. Secondly, even when water exchange does occur, the product maintains a stable population of bacteria within the activated carbon used as a substrate and so can replenish the beneficial probiotic bacteria within the pond following flushing, which does not normally occur as the majority of the bacterial population is normally removed from the pond during water exchange. Finally, the activated carbon used as a substrate has inherent water filtering properties that may also be beneficial in promoting a reduced need for water exchange.

Thus, in a first aspect of the invention, there is provided a bacterial formulation comprising: a bacteria selected from the genus Bacillus; and

a porous substrate material, wherein the bacteria is supported on and/or within the carrier material.

In embodiments of the first aspect of the invention:

(a) the porous substrate material may be selected from one or more of the group consisting of activated carbon pellets, porous ceramic, BioBall™, and porous igneous rocks (e.g. pumice and scoria), optionally wherein the porous substrate material is activated carbon pellets;

(b) the porous substrate material may have a diameter of from 1 mm to 5 cm (e.g. from 2 mm to 2 cm, 2 mm to 1 cm, from 2.5 mm to 5 mm, such as 3 mm);

(c) the bacteria may be one or more selected from the group consisting of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis (e.g. the bacteria may be a combination of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis);

(d) the initial bacterial concentration provided by the bacterial formulation may be from 0.5 x 10⁸ cfu/g to 5 x 10⁸ cfu/g (e.g. from 1.0 x 10⁸ cfu/g to 2.0 x 10⁸ cfu/g, such as 1.5 x

10⁸ cfu/g); (e) the formulation may be packed in a mesh bag having a mesh size of from 0.5 mm x 0.5 mm to 9.5 mm x 9.5 mm (e.g. from 1.0 mm x 2.5 mm to 8.5 mm x 9.5 mm, such as from 1.5 mm x 1.5 mm to 3.0 mm x 3.0 mm, such as 2.0 mm x 2.0 mm);

(f) the amount of the bacterial formulation packed into each mesh bag may be from 10 g to 100 kg, such as from 50 g to 50 kg, from 100 g to 45 kg, from 500 g to 50 kg

(e.g. from 1 kg to 25 kg, such as from 10 kg to 20 kg).

It will be appreciated that all technically sensible combination of the embodiments of the first aspect of the invention are specifically contemplated.

In a second aspect of the invention, there is provided a use of a bacterial formulation according to the first aspect of the invention and any technically sensible combination of its embodiments, in aquaculture. In a third aspect of the invention, there is provided a method of cultivating an aquatic organism, comprising the steps of:

supplying an aquatic organism to a vessel for aquaculture comprising a bacterial formulation according to the first aspect of the invention, and any technically sensible combination of its embodiments, and water;

culturing the aquatic organism in said vessel; and

harvesting the aquatic organism from said vessel.

In embodiments of the third aspect of the invention:

(a) the vessel may be an aquarium, a canal, a small hatchery or a pond measuring from 0.5 to 4 hectares, optionally wherein the vessel is a pond measuring from

0.5 to 4 hectares, such as from 1 to 3 hectares;

(b) the bacterial formulation may be placed on the bottom of the vessel;

(c) the bacterial formulation may be homogenously distributed on the bottom of the vessel;

(d) the bacterial formulation may be heterogeneously distributed on the bottom of the vessel;

(e) the bacterial formulation may be provided in an amount of from 1 kg to 5000 kg per hectare, such as from 500 kg to 2500 kg per hectare, such as 1000 kg per hectare;

(f) the method does not comprise water exchange (e.g. the method does not comprise water exchange for from 10 to 300 days, such as from 50 to 150 days, such as 120 days), or the method further comprises water exchange at a rate of from 0.1 %volume to 5 %volume per day, over a period of from 10 to 300 days (e.g. from 50 to 150 days, such as 120 days), optionally wherein the water exchange occurs at a rate of from 0.2 %volume to 5 %volume per day, over a period of from 50 to 150 days (e.g. 120 days), such as the water exchange occurs at a rate of from 0.27 %volume to 1 %volume per day, over a period of from 50 to 150 days (e.g. 120 days), optionally wherein the method further comprises water exchange a total water exchange of less than 50 %volume over a period of from 10 to 300 days, such as from 50 to 150 days, such as 120 days (e.g. the total water exchange is less than 40 %volume over a period of 70 to 150 days, such as 120 days);

(g) the bacterial formulation may be useable without regeneration or replacement under conditions of from 200 %Volume to 800 %volume water exchange (e.g. from 400 %volume to 700 %volume water exchange).

In a fourth aspect of the invention, there is provided a process for making the bacterial formulation according to the first aspect of the invention, and any technically sensible combination of its embodiments.

In an embodiment of the fourth aspect of the invention, the process may comprise:

(a) adding a liquid comprising a bacteria selected from the genus Bacillus to a porous substrate material to form a mixture and agitating the mixture for a first period of time to form a loaded substrate;

(b) separating the liquid from the loaded substrate and allowing said substrate to rest for a second period of time to form a spore-containing substrate; and

(c) drying the spore-containing substrate to substantially remove the liquid and form the bacterial formulation according to the first aspect of the invention, and any technically sensible combination of its embodiments.

In further embodiments of the process:

(a) the porous substrate material may be selected from one or more of the group consisting of activated carbon pellets, porous ceramic, BioBall™, and porous igneous rocks (such as pumice and scoria), optionally wherein the porous substrate material is activated carbon pellets;

(c) the bacteria may be one or more selected from the group consisting of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis (e.g. the bacteria is a combination of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis);

(d) the process may further comprise the step of packing the formulation in a mesh bag having a mesh size of from 0.5 mm x 0.5 mm to 9.5 mm x 9.5 mm (e.g. from 1.0 mm x 2.5 mm to 8.5 mm x 9.5 mm, such as from 1.5 mm x 1.5 mm to 3.0 mm x 3.0 mm, such as 2.0 mmx 2.0 mm), for example the amount of the bacterial formulation packed into each mesh bag is from 10 g to 100 kg, such as from 50 g to 50 kg, from 100 g to 45 kg, from 500 g to 50 kg (e.g. from 1 kg to 25 kg, such as from 10 kg to 20 kg);

(e) the liquid may comprise water;

(f) the concentration of the bacteria selected from the genus Bacillus in the liquid may be from 50 to 200 billion bacterial cells per ml_, such as from 75 to 150 billion bacterial cells per ml_, such as 100 billion bacterial cells per ml_; In a fifth aspect of the invention, there is provided a process of regenerating a bacterial formulation according to the first aspect of the invention and any combination of its embodiments. This process may comprise:

(a) recovering a used bacterial formulation from a vessel after use;

(b) washing the used bacterial formulation with a liquid comprising water and drying washed formulation;

(c) replacing the dried bacterial formulation in the vessel;

(d) adding a powdered bacterial spore composition comprising at least one bacteria from the genus Bacillus; and

(e) adding water to the vessel.

The bacteria from the genus Bacillus may be one or more selected from the group consisting of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis, (e.g. a combination of two of the bacteria listed or all three). The amount of the bacterial spores distributed on the porous substrate material may be from 100 g to 1 kg per 10 kg of bacterial formulation, such as from 200 g to 800 g, from 500 g to 600 g for 10 kg of bacterial formulation.

It has been surprisingly found that the use of a bacterial formulation of the current invention, which incorporates an activated and stable surface structure is useful for colonization with probiotic bacteria which can then be used to significantly improve water quality (bioremediation). Advantages of the invention include,

• decreasing: Biochemical Oxygen Demand (BOD); Chemical Oxygen Demand (COD); organic compounds; and toxicity (H₂S);

• increased: Oxidation-Reduction Potential (ORP); stability of shock loads; removal of inhibitory compounds (which includes those for nitrifying bacteria, improved sludge dewatering); and

• reduced aerator foaming with regard to water. The improvement of mentioned factors was found to correlate with health factors of aquaculture species and improved growth rate and productivity. Description

It is to be understood by one of ordinary skill in the art that the present disclosure is a description of exemplary embodiments and is not intended to limit the present invention, which comprises broader aspects embodied in the exemplary constructions.

As noted hereinbefore, an object of the current invention relates to finding a way to successfully make use of probiotic bacteria to improve the microbiological status of water by inhibiting the growth of undesirable algae and pathogens in the water. This is a particular problem for aquaculture as the density of farmed organisms within the water significantly increases the presence of such undesirable organisms. As the density of the undesired organisms increases, the nutrient recycling processes is indirectly affected, resulting in the decline of water quality and thus the cultured species are placed in an unhealthy environment and this results in a lower growth rate and/or a higher mortality rate. In addition, in areas where fresh water is scarce, there is a demand for finding ways to limit the amount of water necessary for aquaculture. The reason for this is two-fold. First, aquaculture currently requires a significant amount of water, potentially resulting in the diversion of significant quantities of water from a waterway (e.g. a river or a lake), which may adversely affect the flow of water downstream. Second, the conventional use of water in aquaculture ponds generally results in the discharge of polluted water or water of low quality (e.g. water with low dissolved oxygen, high carbon dioxide and/or high nitrogenous compound concentration) into a waterway, potentially resulting in significant health issues downstream. Given the above, a product that can be used to improve the water discharged from an aquaculture pond, while also reducing the amount of water that is required for aquaculture, is very desirable.

The product of the current invention achieves both of the above aims in a simple and easy to use product. That is, the product disclosed herein obviates the need for frequent water exchange and also helps to restore the beneficial bacterial population following water exchange. Firstly, the product of the current invention contains probiotic bacteria and so bioremediation by these bacteria obviates the need for frequent water exchange to provide water of sufficient quality for aquaculture, thereby promoting water conservation, especially at times of water scarcity. Secondly, even when water exchange does occur, the product maintains a stable population of bacteria within the activated carbon used as a substrate and so can replenish the beneficial probiotic bacteria within the pond following flushing, which does not normally occur as the majority of the bacterial population is normally removed from the pond during water exchange. Finally, the substrate may have inherent water filtering properties that may also be beneficial in promoting a reduced need for water exchange.

Accordingly, the present invention addresses some problems in the art and provides a method of aquaculture of at least one farmed organism.

When used herein, the term "a bacteria selected from the genus Bacillus" may refer to one or a combination of bacteria from said genus (e.g. from 2 to 10, such as 2 to 5, such as 3 bacteria selected from said genus). Particular bacteria from the genus Bacillus that may be mentioned herein includes Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis, where any one, any two or all three bacterial species may be used in combination in the formulation disclosed herein. For example, the bacterial formulation may be a combination of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis.

When used herein the term "porous substrate material" refers to any material containing pores with diameters from 0.1 nm to 50 nm, such as microporous substrate materials having pores with diameters less than 2 nm. Suitable porous substrate materials that may be mentioned herein includes, but is not limited to, activated carbon pellets, porous ceramic, BioBall™, and porous igneous rocks (e.g. pumice and scoria). In particular embodiments of the invention, the porous substrate material may be activated carbon pellets. The diameter of the porous substrate materials used herein may be from 1 mm to 5 cm. For example, the diameter may be from 2 mm to 2 cm, such as from 2 mm to 1 cm, from 2.5 mm to 5 mm or 3 mm.

Without wishing to be bound by theory, it is believed that the selection of a porous substrate enables the deposition of spores within and on the surface (both external and internal) of the substrate material. This results in a reservoir of bacteria within the porous substrate, which in turn enables the establishment of a desired initial concentration of probiotic bacteria within an aquaculture pond, but also enables a sufficient bacterial concentration of probiotic bacteria to be re-established following extensive water replacement (e.g. the concentration may be the same as the initial probiotic material concentration or from 50% to 99%, such as from 80% to 95%, such as 85% of the initial probiotic concentration). For example, the entire water in an aquaculture pond may be replaced from 2 to 10 times (i.e. 200 %volume to 1000 %volume), such as from 3 to 8 times, such as up to 6 times before the disclosed formulation is unable to re-establish the desired concentration of probiotic bacteria in the pond. The initial concentration that may be generated by the formulation disclosed herein may be from 0.5 x 10⁸ cfu/g to 5 x 10⁸ cfu/g (e.g. from 1.0 x 10⁸ cfu/g to 2.0 x 10⁸ cfu/g, such as 1.5 x 10⁸ cfu/g). The initial concentration of the formulation may be established by adding 100 g of the product to 1 L of NaCI physiological saline (0.9%) solution and shaking the resulting mixture for 1 hour at 37°C. The liquid phase can then be removed after centrifugation for 15 minutes at 6000 rpm and compared to a standard (e.g. a McFarland standard) to confirm the initial concentration. The concentration of probiotic bacteria following water exchange may be measured in an analogous manner.

In order to provide the formulation in a useable form, the formulation may be packed in a mesh bag having a mesh size of from 0.5 mm x 0.5 mm to 9.5 mm x 9.5 mm (e.g. from 1.0 mm x 2.5 mm to 8.5 mm x 9.5 mm, such as from 1.5 mm x 1.5 mm to 3.0 mm x 3.0 mm, such as 2.0 mmx 2.0 mm). The amount of the bacterial formulation packed into each mesh bag may be from 10 g to 100 kg, such as from 50 g to 50 kg, from 100 g to 45 kg, from 500 g to 50 kg (e.g. from 1 kg to 25 kg, such as from 10 kg to 20 kg).

There is also provided a process for making the bacterial formulation as described hereinbefore. While any suitable method may be used, a method that may be mentioned herein comprises the steps of:

(c) drying the spore-containing substrate to substantially remove the liquid and form the bacterial formulation according to the first aspect of the invention, and any technically sensible combination of its embodiments. The liquid may be any suitable liquid. In embodiments of the invention, a liquid that may be mentioned herein is one that comprises or consists essentially of water. It will be appreciated that the process described herein is specially adapted to manufacture the bacterial formulation described hereinbefore. As such, the specific functional elements of the formulation (e.g. the physical composition, its constituents and the presented form for use) may be as described hereinbefore for said formulation.

As noted above, the formulations disclosed hereinbefore may be used in aquaculture. As such, there is also provided a method of cultivating an aquatic organism, comprising the steps of:

supplying an aquatic organism to a vessel for aquaculture comprising a bacterial formulation as defined hereinbefore, and water;

culturing the aquatic organism in said vessel; and

harvesting the aquatic organism from said vessel.

· decreasing: Biochemical Oxygen Demand (BOD); Chemical Oxygen Demand (COD); organic compounds; and toxicity (H₂S);

· reduced aerator foaming with regard to water.

The improvement of mentioned factors was found to correlate with health factors of aquaculture species and improved growth rate and productivity, as demonstrated by the examples hereinbelow.

For the purposes of this specification, a farmed organism may also be referred to as a primary organism or a primary farmed organism. There may be one or more farmed organisms in a given aquatic environment. In particular, the product of the present invention is particularly well suited for raising fish and/or shrimp. Thus, much of the remaining description may be directed to embodiments wherein the farmed organism is fish and/or shrimp. It should be understood, however, that the system is also well suited for raising other aquatic farmed organisms.

For the purposes of this specification, the term "aquatic organism" means a farmed aquatic organism, which terms may be used herein interchangeably. A farmed aquatic organism is any commercially grown or cultivated species produced by means of aquaculture such as any animal or plant produced by means of aquaculture such as fish, crustacean, mollusc, seaweed and/or invertebrate. Exemplary types of Fish include Tilapias, Catfishes, Milkfishes, Groupers, Barramundi, Carps, Snakeheads, Catlas, Sturgeons, Eels, Mullets, Rohus, Seabasses, Seabreams, Rabbit fishes. Exemplary Crustaceans include Shrimps, Prawns, Crabs, Lobsters, Crayfishes. Exemplary Molluscs include Oysters, Clams, Mussels, Scallops, Carpet shells, Abalones. Exemplary invertebrates may include Sea cucumbers, Sea urchins. When used herein, "vessel" may refer to any suitable enclosed aquatic environment that is to water bodies that serve as habitat for interrelated and interacting communities and populations of plants and animals, further comprising any layer of organic matter and/or any cavity in fluid communication with the water phase. For example, in a typical earthen aquaculture pond the aquatic environment comprises both the water phase and the soil phase lining the bottom and sides of the pond.

Suitable vessels that may be mentioned herein include, but are not limited to an aquarium, a canal, a small hatchery or a pond measuring. When used herein, "pond" refers to an aquatic environment where farmed species are held or cultured. In conventional fish farming, the pond is the place where juvenile fish are raised to market size. A typical pond is earthen-bottomed but other materials may also be used to form the pond, for example ponds which are concrete- or plastic-bottomed are also understood to be suitable aquatic environments for the purposes of the present invention. Suitable ponds that may be mentioned herein (e.g. for shrimp farming) may measure from 0.5 to 4 hectares, optionally wherein the vessel is a pond measuring from 0.5 to 4 hectares, such as from 1 to 3 hectares, the depth of such ponds may be from 1.0 to 2.5 metres.

It will be understood that the term "supplying an aquatic organism" refers to the stocking of a vessel with an amount of one or more farmed aquatic organism species. These species may be supplied to the pond in an embryonic stage or in a juvenile stage, i.e. just after hatching or being born. The term "culturing" when used herein refers to providing nutrients and other necessities to the one or more farmed aquatic organism species to enable them to grow to a desired size and/or weight, at which time the one or more farmed aquatic organism species are harvested. The period of time between the supply of the one or more farmed aquatic organism species to the vessel to the harvesting of said species may be from 10 to 300 days (e.g. from 50 to 150 days, such as 120 days).

As will be appreciated, the formulation described hereinbefore is preferably placed in the vessel before the vessel is filled with water and subsequently stocked with the one or more farmed aquatic organisms. It will be appreciated that the formulation is preferably provided in mesh bags as described hereinbefore as this reduced/prevents the substrate from being redistributed from the vessel during water exchange and also enables ease of placement of the formulation. As will be appreciated, the bacterial formulation may be placed on the bottom of the vessel, though other locations, including the sides of the vessel may also be used too.

The amount of the bacterial formulation may be provided in an amount/density of from 1 kg to 5000 kg per hectare, such as from 500 kg to 2500 kg per hectare, such as 1000 kg per hectare. In order to ensure an equal concentration of the bacterial formulation throughout the aqueous environment of the vessel when water is added thereto, the bacterial formulation may be homogenously distributed in the vessel (e.g. on the bottom of the vessel). However, in certain embodiments it may be beneficial to heterogeneously distribute the formulation in the vessel (e.g. in the bottom of the vessel). For example, when 1000 kg of the formulation (in mesh bags) is used in a 1 hectare pond, the formulation may be homogeneously distributed over the bottom of the pond in order to provide a homogenous concentration of probiotic bacteria throughout the pond volume.

As noted above, in certain embodiments of the invention, an advantage associated with the current formulation and method is that it may not be necessary to replace the water in the vessel during the period from supply of the one or more farmed aquatic organisms up to harvesting. Thus, in certain embodiments of the invention, the method does not comprise water exchange. For example, the method does not comprise water exchange for from 10 to 300 days, such as from 50 to 150 days. In particular embodiments of the invention, there may be no water exchange for 120 days. It will be appreciated that "water exchange" when mentioned herein refers to the deliberate intervention of man to exchange water, whereby water is removed from the pond (which water comprises nutrients, pollutants and/or probiotic bacteria etc.) and replaced with fresh water. However, "water exchange" does not cover the situation of simple evaporation and precipitation as in these cases no removal of nutrients, pollutants and/or probiotic bacteria takes place.

In alternative embodiments, the method may further comprise water exchange at a rate of from 0.1 %volume to 5 %volume per day, over a period of from 10 to 300 days (e.g. from 50 to 150 days, such as 120 days), optionally wherein the water exchange occurs at a rate of from 0.2 %volume to 5 %volume per day, over a period of from 50 to 150 days (e.g. 120 days), such as the water exchange occurs at a rate of from 0.27 %volume to 1 %volume per day, over a period of from 50 to 150 days (e.g. 120 days).

In certain embodiments of the method where there is water exchange, the total water exchange of less than 50 %volume over a period of from 10 to 300 days, such as from 50 to 150 days, such as 120 days (e.g. the total water exchange is less than 40 %volume over a period of 70 to 150 days, such as 120 days).

As noted hereinbefore, the bacterial formulation of the current invention is able to replenish the loss of probiotic bacteria in the removed water in order to maintain a suitable concentration of probiotic bacteria within the vessel that provides the benefits described herein. As such, the bacterial formulation may be useable without regeneration or replacement under conditions of from 200 %volume to 800 %volume water exchange (e.g. from 400 %volume to 700 %volume water exchange). Thus, in embodiments of the invention where there is water exchange, the water exchange may be up to 800 %volume of the initial volume of water applied to the vessel before the bacterial formulation of the current invention requires replacement and/or regeneration. Similarly, where there is no water exchange, the method of aquaculture set out hereinbefore may be run up to eight times before the bacterial formulation requires replacement and/or regeneration.

Thus, there is also provided a process of regenerating a bacterial as described hereinbelow. Regeneration of the product may be by addition of new powdered probiotic spores after each culture cycle (though as noted above this may not need to be the case, depending on the relative rate of water turnover during the culture cycle).

This process may comprise:

(a) recovering a used bacterial formulation from a vessel after use;

(c) replacing the dried bacterial formulation in the vessel; (d) adding a powdered bacterial spore composition comprising at least one bacteria from the genus Bacillus; and

(e) adding water to the vessel. It will be appreciated that the bacteria used may be one or more selected from the group consisting of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis, or a combination of two or all three of these bacteria. The loading of the bacterial spores may be from 100 g to 1 kg per 10 kg of bacterial formulation, such as from 200 g to 800 g, from 500 g to 600 g for 10 kg of bacterial formulation.

For example, after the pond water has been fully discharged, the product may be collected and flushed with water to remove accumulated sediments during the culture. After the product is dried, the product is once more placed on the bottom of the vessel and, powdered probiotic spores can be sprinkled onto the porous substrate material, for example in the amount of 500 g per 10 kg of the product, before the water in the vessel is replenished.

The invention will now be described in more detail with regard to the non-limiting examples below. Examples

General Preparation 1

Microbial culture and isolation All equipment and material were sterilized before use.

600 ml_ of a pre-fermentation batch of a bacterial species selected from the Bacillus genus at a concentration of approximately 500 million spores/mL was provided and added into a vessel containing 30 litres of Nutrient Broth based on peptones and mineral salts (D(+)- glucose, 1 g/L; Peptone, 15 g/L; Sodium chloride, 6 g/L; Yeast extract, 3 g/L) as a fermentation medium and allowed to mix for 7 hours.

The resulting suspension (approximately 30.6 L) was then inoculated into 1000 litres of Nutrient Broth at 37°C and allowed to mix and aerate for 48 to 72 hours. Thereafter, 2 rounds of centrifugation for 15 minutes at 6000 rpm were carried out to separate the bacterial cells from the fermentation medium. The liquid phase was removed and a NaCI physiological saline (0.9 %) was added until a final volume of 100 litres was obtained. A typical concentration of about 100 billion bacterial cells per ml_ was obtained with this procedure for each batch prepared.

Example 1

Microbial Immobilization

A separate suspension was prepared for each of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis according to General Procedure 1 above. Each of the resulting suspensions of bacteria were then transferred and mixed in a single 1000-litre tank, resulting in a 300-litre mixture.

For immobilization of the bacteria, 150 kg of pelletized activated carbon having an average diameter size of 3.0 mm were added into the above suspension of bacteria and the resulting mixture was shaken for 30 minutes to immobilize at least a portion of the bacteria on the activated carbon. Subsequently, the activated carbon-containing bacteria was separated from the liquid and was allowed to rest at ambient condition for 3 hours to enable the drying of residual liquid on the surface of the activated carbon and to enable bacterial spore formation. The partially dried carbon was then loaded onto a flat air-bed dryer at a depth of 2 cm, where it was subjected to drying with a continuous hot-air flow of 70 °C. Thereafter, the bacteria-loaded activated carbon was allowed to cool and was subsequently freeze-dried under the following conditions. About 30 minutes before cooling begins, the air-admittance and condenser drain valve of the freeze-drying machine were closed and then the refrigerator and vacuum pump were turned on. The condenser (cold trap) was allowed to cool to -40 °C to -50 °C in about 4 hours when the freeze-drying was considered to be complete.

The freeze dried bacterial spores in activated carbon medium (the product) were then ready for storage and/or for packaging. 10 kg or 20 kg of the product was packed in a mesh-bag with a mesh size of 2 mm x 2 mm to provide a suitable form of the product for use in an aquaculture system.

To verify the immobilization of spores in the activated carbon, 100 g of the product was added to 1 L of NaCI physiological saline (0.9%) and shaken for 1 hour at 37 °C. The liquid phase was removed after centrifugation for 15 minutes at 6000 rpm.

The bacterial concentration in liquid phase was measured based on an optical density at 600 nm wavelength using a spectrophotometer machine and compared to a 0.5 McFarland standard. A minimum amount of 1.5 x 10 cfu/g should be obtained in the sample - in other words, the optical density of the sample should be at least equivalent to the 0.5 McFarland standard. Example 2

Farm Trial

Farm trials were carried out at a shrimp farm to confirm the efficacy of the product in a dynamic pond environment throughout the culture period. All factors in the ponds were kept constant, with the exception that the product was used in 3 ponds ("treated ponds"), while the other 3 ponds were untreated "control ponds".

The water depth in all ponds was 150 cm. For the treated ponds, the average amount of water exchange was 32 % of the total water volume over the culture period of 120 days. That is, 68 % of the total water volume was present throughout the culture period (excluding water removed by evaporation and replaced by rain). The water exchange for the treated ponds did not occur daily (as the average water exchange per day would be less than 0.3 % a day), but on an ad-hoc basis. In contrast, water exchange in the control ponds was, on average, 5 % of the total water volume per day over the 120 day culturing period. Over the 120 day period, this translated into a water exchange that is approximately 18 times that used in the treated ponds.

The shrimp species under culture was Lvannamei. 100 units of the product (here one unit is a 10 kg mesh bag of the product prepared in Example 1) were homogeneously distributed throughout the bottom soil of a one-hectare pond after pond preparation, but before water was filled into the pond. The parameters evaluated during the 120-days of the trial included water quality parameters, growth parameters and survival rate of the shrimp.

Water quality parameters such as dissolved oxygen, pH, ORP, ammonia and nitrate were measured by using portable instrument and commercial test kits. Hydrogen sulfide and BOD were determined by the spectrophotometric method and standard methods for the examination of water and wastewater (5210 A), respectively.

Shrimp feed intake was recorded on a daily basis and daily ration was determined based on whether there was any leftover feed on the feed tray. Shrimp were measured weekly for weight gain during the culture period. 5 replicates were taken for each measurement. Shrimp were weighed at final harvest. The shrimps were also observed for any signs of disease, moulting pattern, fungal attachment etc. In comparison with control ponds without the product, treated ponds showed positive results in terms of water quality maintenance, total shrimp production and survival rates. There was considerable reduction in ammonia and nitrate levels in the treated pond throughout the trial period. This is indicative of an enhancement of biological degradation of organic matter, sludge, animal waste, feces, uneaten feed and plankton remains in pond bottom as compared to the control pond. Although high Dissolved Oxygen (DO) amount is not a direct result from the application of the product, higher DO was found in the treated pond as compared to control.

Hydrogen sulfide was not detected in treated ponds. In contrast, an average of 0.008 mg/L of hydrogen sulfide was measured from the sediment surface in control ponds. Hydrogen sulfide is a by-product of the breakdown of organic matter, under anaerobic conditions in the hypolimnion or in sediment. Sediment accumulation provides a good environment for hydrogen sulfide generation. The amount of hydrogen sulfide in water is often correlated with high mortality and low growth rates. Results showed that treated ponds had lower mortality and a higher growth rate than control ponds. Oxidation-Reduction Potential (ORP) results are significantly different for control ponds and treated ponds with readings of -80.57±12 mV and +90.45±8 mV, respectively. ORP levels can provide an indication of the cleanliness of water and its ability to break down of contaminants. ORP can also provide indirect measurement of dissolved oxygen. Under oxidative, aerobic conditions, ORP levels are higher and there is the greater ability for degradation of foreign contaminates such as organic matter, microbes or carbon based contaminates. A high value of ORP is correlated to oxidative, aerobic conditions that favour nitrification and organic matter degradation. Promoted aerobic conditions also prevent hydrogen sulfide formation and undesirable fermentation in the pond bottom.

Biochemical Oxygen Demand (BOD) amounts (or the organic matter index results) are also significantly different for control ponds and treated ponds with readings of 29.0±0.5 mg/L and 18.9±0.3 mg/L, respectively. Biochemical Oxygen Demand (BOD) reflects the amount of organic matters and oxygen requirement for decomposition of organic matter by heterotrophic bacteria in water. The survival rate of shrimp in control ponds and treated ponds was 72 % and 83 % respectively, also the average body weight of 18.3 and 21.8 g were achieved in control ponds and treated ponds, respectively. The higher in survival rate directly translates to a better return on investment.

There was also a visible difference in the appearance and color of the ponds. Control ponds appeared green, indicative of an undesirable algae development, by 60 days of culture and such green coloration became more distinct with the increase in the number of days. Treated ponds maintained a characteristic brownish yellow color that is desirable in shrimp ponds.

Claims

1. A bacterial formulation comprising:

a bacteria selected from the genus Bacillus; and

2. The bacterial formulation of Claim 1 , wherein the porous substrate material is selected from one or more of the group consisting of activated carbon pellets, porous ceramic, BioBall™, and porous igneous rocks (e.g. pumice and scoria), optionally wherein the porous substrate material is activated carbon pellets.

3. The bacterial formulation of Claim 1 or Claim 2, wherein the porous substrate material has a diameter of from 1 mm to 5 cm (e.g. from 2 mm to 2 cm, 2 mm to 1 cm, from 2.5 mm to 5 mm, such as 3 mm).

4. The bacterial formulation of any one of the preceding claims, wherein the bacteria is one or more selected from the group consisting of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis.

5. The bacterial formulation of Claim 4, wherein the bacteria is a combination of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis.

6. The bacterial formulation of any one of the preceding claims, wherein the initial bacterial concentration provided by the bacterial formulation is from 0.5 x 10⁸ cfu/g to 5 x 10⁸ cfu/g (e.g. from 1.0 x 10⁸ cfu/g to 2.0 x 10⁸ cfu/g, such as 1.5 x 10⁸ cfu/g).

7. The bacterial formulation of any one of the preceding claims, wherein the formulation is packed in a mesh bag having a mesh size of from 0.5 mm x 0.5 mm to 9.5 mm x 9.5 mm (e.g. from 1.0 mm x 2.5 mm to 8.5 mm x 9.5 mm, such as from 1.5 mm x 1.5 mm to 3.0 mm x 3.0 mm, such as 2.0 mm x 2.0 mm).

8. The bacterial formulation of Claim 7, wherein the amount of the bacterial formulation packed into each mesh bag is from 10 g to 100 kg, such as from 50 g to 50 kg, from 100 g to 45 kg, from 500 g to 50 kg (e.g. from 1 kg to 25 kg, such as from 10 kg to 20 kg).

9. Use of a bacterial formulation according to any one of Claims 1 to 8 in aquaculture.

10. A method of cultivating an aquatic organism, comprising the steps of: supplying an aquatic organism to a vessel for aquaculture comprising a bacterial formulation according to any one of Claims 1 to 8 and water;

culturing the aquatic organism in said vessel; and

harvesting the aquatic organism from said vessel.

11. The method of Claim 10, wherein the vessel is an aquarium, a canal, a small hatchery or a pond measuring from 0.5 to 4 hectares, optionally wherein the vessel is a pond measuring from 0.5 to 4 hectares, such as from 1 to 3 hectares.

12. The method of Claim 10 or Claim 1 1 , wherein the bacterial formulation is placed on the bottom of the vessel.

13. The method of Claim 12, wherein the bacterial formulation is homogenously distributed on the bottom of the vessel.

14. The method of Claim 12, wherein the bacterial formulation is heterogeneously distributed on the bottom of the vessel.

15. The method of any one of Claims 1 1 to 14, wherein the bacterial formulation is provided in an amount of from 1 kg to 5000 kg per hectare, such as from 500 kg to 2500 kg per hectare, such as 1000 kg per hectare.

16. The method of any one of Claims 10 to 15, wherein the method does not comprise water exchange.

17. The method of any one of Claims 10 to 15, wherein the method does not comprise water exchange for from 10 to 300 days, such as from 50 to 150 days, such as 120 days.

18. The method of any one of Claims 10 to 15, wherein the method further comprises water exchange at a rate of from 0.1 %volume to 5 %volume per day, over a period of from 10 to 300 days (e.g. from 50 to 150 days, such as 120 days), optionally wherein the water exchange occurs at a rate of from 0.2 %volume to 5 %volume per day, over a period of from 50 to 150 days (e.g. 120 days), such as the water exchange occurs at a rate of from 0.27 %volume to 1 %volume per day, over a period of from 50 to 150 days (e.g. 120 days).

19. The method of Claim 18, wherein the method further comprises water exchange a total water exchange of less than 50 %volume over a period of from 10 to 300 days, such as from 50 to 150 days, such as 120 days, optionally wherein the total water exchange is less than 40 %volume over a period of 70 to 150 days, such as 120 days.

20. The method of any one of Claims 10 to 19, wherein the bacterial formulation is useable without regeneration or replacement under conditions of from 200 %volume to 800 %volume water exchange (e.g. from 400 %volume to 700 %volume water exchange).

21. A process for making the bacterial formulation according to any one of Claims 1 to 8.

22. The process of Claim 21 , comprising:

(c) drying the spore-containing substrate to substantially remove the liquid and form the bacterial formulation according to any one of Claims 1 to 8.

23. The process of Claim 22, wherein the porous substrate material is selected from one or more of the group consisting of activated carbon pellets, porous ceramic, BioBall™, and porous igneous rocks (such as pumice and scoria), optionally wherein the porous substrate material is activated carbon pellets.

24. The process of Claim 22 or Claim 23, wherein the porous substrate material has a diameter of from 1 mm to 5 cm (e.g. from 2 mm to 2 cm, 2 mm to 1 cm, from 2.5 mm to 5 mm, such as 3 mm).

25. The process of any one of Claims 22 to 24, wherein the bacteria is one or more selected from the group consisting of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis.

26. The process of Claim 25, wherein the bacteria is a combination of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis.

27. The process of any one of Claims 22 to 26, further comprising the step of packing the formulation in a mesh bag having a mesh size of from 0.5 mm x 0.5 mm to 9.5 mm x 9.5 mm (e.g. from 1.0 mm x 2.5 mm to 8.5 mm x 9.5 mm, such as from 1.5 mm x 1.5 mm to 3.0 mm x 3.0 mm, such as 2.0 mm x 2.0 mm).

28. The process of Claim 27, wherein the amount of the bacterial formulation packed into each mesh bag is from 10 g to 100 kg, such as from 50 g to 50 kg, from 100 g to 45 kg, from 500 g to 50 kg (e.g. from 1 kg to 25 kg, such as from 10 kg to 20 kg).

29. The process of any one of Claims 22 to 28, wherein the liquid comprises water.

30. The process of any one of Claims 22 to 29, wherein the concentration of the bacteria selected from the genus Bacillus in the liquid is from 50 to 200 billion bacterial cells per mL, such as from 75 to 150 billion bacterial cells per mL, such as 100 billion bacterial cells per mL.

31. A process of regenerating a bacterial formulation according to any one of Claims 1 to 8, comprising:

(a) recovering a used bacterial formulation from a vessel after use;

(c) replacing the dried bacterial formulation in the vessel;

(e) adding water to the vessel.

32. The process of Claim 31 , wherein the bacteria from the genus Bacillus is one or more selected from the group consisting of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis.

33. The process of Claim 32, wherein the bacteria is a combination of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis.

34. The process of any one of Claims 31 to 33, wherein the amount of the bacterial spores distributed on the porous substrate material is from 100 g to 1 kg per 10 kg of bacterial formulation, such as from 200 g to 800 g, from 500 g to 600 g for 10 kg of bacterial formulation.