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WO2018190772A1 - Composition pour aquaculture - Google Patents

Composition pour aquaculture Download PDF

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Publication number
WO2018190772A1
WO2018190772A1 PCT/SG2018/050174 SG2018050174W WO2018190772A1 WO 2018190772 A1 WO2018190772 A1 WO 2018190772A1 SG 2018050174 W SG2018050174 W SG 2018050174W WO 2018190772 A1 WO2018190772 A1 WO 2018190772A1
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WIPO (PCT)
Prior art keywords
bacterial
days
formulation
bacteria
bacterial formulation
Prior art date
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PCT/SG2018/050174
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English (en)
Inventor
Farshad SHISHEHCHIAN
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Blue Aqua International Pte Ltd
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Blue Aqua International Pte Ltd
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Filing date
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Publication of WO2018190772A1 publication Critical patent/WO2018190772A1/fr
Anticipated expiration legal-status Critical
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/341—Consortia of bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F2003/001—Biological treatment of water, waste water, or sewage using granular carriers or supports for the microorganisms
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F2003/001—Biological treatment of water, waste water, or sewage using granular carriers or supports for the microorganisms
    • C02F2003/003—Biological treatment of water, waste water, or sewage using granular carriers or supports for the microorganisms using activated carbon or the like
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/007—Contaminated open waterways, rivers, lakes or ponds
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed

Definitions

  • This invention relates to a composition that improves water quality for aquaculture.
  • Biological wastewater treatment is an emerging technology for treating toxic gases (such as ammonia), metabolic waste and organic matter present in aquaculture ponds.
  • toxic gases such as ammonia
  • 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.
  • 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
  • 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).
  • this exchange can account for up to one third (e.g. up to 20%) of the water in a pond per day.
  • Heterotrophic bacteria which are subsequently referred to as probiotics
  • 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 (H 2 S) production.
  • H 2 S hydrogen sulfide
  • the use of probiotic bacteria has reduced antibiotic use in aquaculture systems.
  • 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.
  • a product to reduce the need for water exchange in aquaculture.
  • water of poor quality with low dissolved oxygen, high carbon dioxide and/or high nitrogenous compound concentration
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the porous substrate material may be selected from one or more of the group consisting of activated carbon pellets, porous ceramic, BioBallTM, and porous igneous rocks (e.g. pumice and scoria), optionally wherein the porous substrate material is activated carbon pellets;
  • 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);
  • 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);
  • the initial bacterial concentration provided by the bacterial formulation may be from 0.5 x 10 8 cfu/g to 5 x 10 8 cfu/g (e.g. from 1.0 x 10 8 cfu/g to 2.0 x 10 8 cfu/g, such as 1.5 x
  • 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);
  • 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
  • a bacterial formulation according to the first aspect of the invention and any technically sensible combination of its embodiments, in aquaculture in a second aspect of the invention.
  • a method of cultivating an aquatic organism comprising the steps of:
  • 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
  • the bacterial formulation may be placed on the bottom of the vessel;
  • the bacterial formulation may be homogenously distributed on the bottom of the vessel;
  • the bacterial formulation may be heterogeneously distributed on the bottom of the vessel;
  • 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;
  • 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.
  • 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);
  • 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).
  • the process may comprise:
  • the porous substrate material may be selected from one or more of the group consisting of activated carbon pellets, porous ceramic, BioBallTM, and porous igneous rocks (such as pumice and scoria), optionally wherein the porous substrate material is activated carbon pellets;
  • 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);
  • 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);
  • 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);
  • the liquid may comprise water
  • 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_;
  • 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.
  • Oxidation-Reduction Potential • increased: Oxidation-Reduction Potential (ORP); stability of shock loads; removal of inhibitory compounds (which includes those for nitrifying bacteria, improved sludge dewatering); and
  • 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.
  • 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.
  • there is a demand for finding ways to limit the amount of water necessary for aquaculture The reason for this is two-fold.
  • 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.
  • a waterway e.g. a river or a lake
  • 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.
  • polluted water or water of low quality e.g. water with low dissolved oxygen, high carbon dioxide and/or high nitrogenous compound concentration
  • 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.
  • the present invention addresses some problems in the art and provides a method of aquaculture of at least one farmed organism.
  • 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.
  • 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.
  • the bacterial formulation may be a combination of Bacillus subtilis, Bacillus coagulans and Bacillus licheniformis.
  • 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, BioBallTM, and porous igneous rocks (e.g. pumice and scoria).
  • 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.
  • 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.
  • 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.
  • the initial concentration that may be generated by the formulation disclosed herein may be from 0.5 x 10 8 cfu/g to 5 x 10 8 cfu/g (e.g. from 1.0 x 10 8 cfu/g to 2.0 x 10 8 cfu/g, such as 1.5 x 10 8 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.
  • a standard e.g. a McFarland standard
  • the concentration of probiotic bacteria following water exchange may be measured in an analogous manner.
  • 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).
  • the liquid may be any suitable liquid.
  • 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.
  • formulations disclosed hereinbefore may be used in aquaculture.
  • a method of cultivating an aquatic organism comprising the steps of:
  • an aquatic organism to a vessel for aquaculture comprising a bacterial formulation as defined hereinbefore, and water;
  • Oxidation-Reduction Potential • increased: Oxidation-Reduction Potential (ORP); stability of shock loads; removal of inhibitory compounds (which includes those for nitrifying bacteria, improved sludge dewatering); and
  • a farmed organism may also be referred to as a primary organism or a primary farmed organism.
  • a primary organism There may be one or more farmed organisms in a given aquatic environment.
  • the product of the present invention is particularly well suited for raising fish and/or shrimp.
  • 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.
  • 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.
  • "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.
  • 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.
  • 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 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.
  • 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).
  • 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.
  • the bacterial formulation may be homogenously distributed in the vessel (e.g. on the bottom of the vessel).
  • it may be beneficial to heterogeneously distribute the formulation in the vessel e.g. in the bottom of the vessel.
  • 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.
  • 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.
  • the method does not comprise water exchange.
  • the method does not comprise water exchange for from 10 to 300 days, such as from 50 to 150 days.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • 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.
  • the method of aquaculture set out hereinbefore may be run up to eight times before the bacterial formulation requires replacement and/or regeneration.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ORP Oxidation-Reduction Potential
  • Biochemical Oxygen Demand (BOD) amounts 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.
  • 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.

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Abstract

L'invention concerne une formulation bactérienne qui comprend une bactérie sélectionnée dans le genre Bacillus et un matériau de substrat poreux, les bactéries étant supportées sur et/ou à l'intérieur du matériau de substrat. L'invention concerne également des utilisations de la formulation de bactéries, ainsi que des procédés de fabrication et de régénération de ladite formulation.
PCT/SG2018/050174 2017-04-12 2018-04-06 Composition pour aquaculture Ceased WO2018190772A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110902938A (zh) * 2019-11-07 2020-03-24 嘉兴职业技术学院 一种采用生物膜法的菌藻固定化嵌入式水质净化装置
CN111333198A (zh) * 2018-12-19 2020-06-26 陈教旗 一种水产养殖调水用水质改良剂及其制备方法
CN117866824A (zh) * 2024-01-09 2024-04-12 中山康源基因技术科技有限公司 一种可同时高效降解氨氮和亚硝酸盐的复合益生菌制剂及其在水产养殖上的应用

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Publication number Priority date Publication date Assignee Title
US6878373B2 (en) * 2001-12-03 2005-04-12 Micropure Technologies, Inc. Probiotic composition containing Bacillus cereus RRRL B-30535
WO2014172520A1 (fr) * 2013-04-17 2014-10-23 Envera, Llc Compositions de nouvelles souches de bacillus
CN106542651A (zh) * 2016-12-20 2017-03-29 大连赛姆生物工程技术有限公司 一种养殖水体用微生物净水剂及其制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6878373B2 (en) * 2001-12-03 2005-04-12 Micropure Technologies, Inc. Probiotic composition containing Bacillus cereus RRRL B-30535
WO2014172520A1 (fr) * 2013-04-17 2014-10-23 Envera, Llc Compositions de nouvelles souches de bacillus
CN106542651A (zh) * 2016-12-20 2017-03-29 大连赛姆生物工程技术有限公司 一种养殖水体用微生物净水剂及其制备方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111333198A (zh) * 2018-12-19 2020-06-26 陈教旗 一种水产养殖调水用水质改良剂及其制备方法
CN110902938A (zh) * 2019-11-07 2020-03-24 嘉兴职业技术学院 一种采用生物膜法的菌藻固定化嵌入式水质净化装置
CN110902938B (zh) * 2019-11-07 2021-10-29 嘉兴职业技术学院 一种采用生物膜法的菌藻固定化嵌入式水质净化装置
CN117866824A (zh) * 2024-01-09 2024-04-12 中山康源基因技术科技有限公司 一种可同时高效降解氨氮和亚硝酸盐的复合益生菌制剂及其在水产养殖上的应用

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