AMMONIA DECREASING BIOLOGICAL AGENTS AND USES THEREOF

Abstract

AMMONIA DECREASING BIOLOGICAL AGENTS AND USES THEREOF An aspect of the present disclosure pertains to a composition comprising biological agents for removal of ammonia from water. The said composition comprises: two novel bacterial strains as active ingredients, saline and stabilizer-for improving the viability of the active ingredient. Another aspect of the present disclosure is to elucidate various characteristics of the said active ingredients comprising two novel bacterial strains, which either alone or in combination could be used for abatement of ammonia from water. Yet another aspect of the present disclosure pertains to the method of making an apparatus involving the use of the said composition for abatement of ammonia from water. Yet another aspect of the present disclosure pertains to the method of making biofloc involving the use of the said composition for conversion ammonia into protein rich bioflocs, which in turn can be used as feed by aquatic animals.

Specification

Description:AMMONIA DECREASING BIOLOGICAL AGENTS AND USES THEREOF
Geographical location from which Vibrio strains [Vibrio sp. (HeteroMight 44) - MCC0282 and Vibrio diabolicus (HeteroMight 64) - MCC0283 was collected: Pulicat Beach, Thiruvallur Dist., Tamil Nadu. 13.3971°N, 80.3309°E
FIELD OF THE INVENTION
[0001] This invention relates to a composition comprising biological agents for abatement of ammonia from water. In particular, it relates to the use of composition comprising micro-organisms for abatement of ammonia from water. More particularly, it pertains to newly isolated novel Vibrio strains [Vibrio sp. (HeteroMight 44) & Vibrio diabolicus (HeteroMight 64)] that can abate ammonia from water. Even more particularly, it pertains to newly isolated novel Vibrio strains [Vibrio sp. (HeteroMight 44) & Vibrio diabolicus. (HeteroMight 64)] that can abate ammonia from water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, raceway system, RAS system, waste water, industrial effluent and brackish water.
[0002] This invention also relates to the use of the said composition, to convert the ammonia present in the aquatic systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, Raceway system and RAS system, into protein rich Bioflocs, which can be utilized as feed.
[0003] This invention also relates to a method of making an apparatus involving the use of newly isolated novel Vibrio strains [Vibrio sp. (HeteroMight 44) & Vibrio diabolicus (HeteroMight 64)] for ammonia abatement in water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, raceway system, RAS system, waste water, industrial effluent and brackish water.
BACKGROUND OF THE INVENTION
[0004] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0005] Aquaculture involves various methods of farming the aquatic organisms such as fish, crustaceans, mollusks, polychaete, annelids and other organisms in both marine and fresh water environments for any commercial or recreational purpose.
[0006] Aquariums involves rearing of ornamental aquatic animals and aquatic plants in both marine and fresh water environments for recreational purposes.
[0007] Waste water treatment involves treatment of contaminated water in a manner to remove contaminants or decrease the level of contaminants, so that the water can be re-used for various processes or can be discharged back into the environment.
[0008] It is well known in the art that, ammonia is an undesirable part of nitrogen cycle in aquaculture, aquariums and wastewater. Presence of ammonia above certain concentrations result in unwanted effects.
[0009] For example, Gladys Valencia-Castaneda et al. (Environmental Toxicology and Pharmacology, Volume 70, August 2019, 103193) discloses that, in Litopenaeus vannamei LC50 value of total ammonia Nitrogen for 24 hours at 3 g/L salinity is 38.9 mg/L.
[0010] Further, it is well known in the art that, higher ammonia levels in water is toxic to fishes and there are environmental regulations in waste water treatment plants to bring down the ammonia levels below set values, before it can be safely recycled or discharged into the environment.
[0011] Also known in the art is, there are several method to abate ammonia from water including, Biological treatment (use of microorganisms, Bio-filters, etc.), Physical-Chemical treatment (use of Air stripping, Ion Exchange, Adsorption, etc.), chemical treatment (use of Chlorination, Ozonation, etc.) and Membrane Processes (Reverse Osmosis, Electrodialysis, etc.).
[0012] Of the above methods, Biological Treatment including use of microorganisms and Bio-filters offers advantage in terms of cost-effectiveness, sustainability, high efficiency and scalability.
[0010] Further, it is well known in the art that, use of Biological Treatment including use of microorganisms and Bio-filters result in conversion of unwanted ammonia into valuable biomass that can be fed by aquatic animals.
[0013] Huang, H.-H, 2020 (Novel Biofloc Technology (BFT) for Ammonia Assimilation and Reuse in Aquaculture In Situ. IntechOpen. doi: 10.5772/intechopen.88993) for instance, describes that, feed conversion ratio (FCR) can be decreased in reared aquaculture animals by the way, of using heterotrophic bacteria to convert ammonia into protein rich Bioflocs.
[0014] Biological treatment based ammonia conversion generally involves Nitrification and Denitrification, which together are key processes in nitrogen cycle. Nitrification involves microbial conversion of ammonia into nitrite and then to nitrate. Denitrification involves microbial conversion of nitrates into nitrogen.
[0015] Apart from the above commonly agreed pathway, there exist alternative pathways including Assimilatory Nitrogen Reduction, Anaerobic Ammonia Oxidation (Anammox), Ammonia Fermentation and Dissolved inorganic nitrogen (DIN) assimilation. Such alternative pathways shall be referred herein as "Alternative ammonia utilizing pathways".
[0016] Clare Bird et al. (Frontiers in Microbiology, doi: 10.2289/fmicb.2020.604979) for instance, demonstrates that, some heterotrophic protists have innate cellular mechanism for inorganic ammonium assimilation, highlighting a newly-discovered pathway for dissolved inorganic nitrogen (DIN) assimilation with the marine microbial loop.
[0017] Prasannan Geetha Preena et al. (Reviews in Aquaculutre, 1-23, doi: 10.1111/raq.12558) review in detail about various autotrophic and heterotrophic microbial processes along with their merits and demerits for nitrification and denitrification in recirculating aquaculture systems. Broadly, microbes involved in ammonia conversion were classified as, ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA). Of which, AOB are further divided into heterotrophic nitrifiers and autotrophic nitrifiers. Heterotrophic ntrifiers, termed as R strategists are characterized by fast growth rates even though they are thermodynamically less favoured for nitrification processes. Autotrophic nitrifiers, termed as K strategists are characterized by slow growth rates. On the other hand, AOA are niche dwellers, also characterized by slow growth rates.
[0018] It is also worth to consider that regardless of the advantages of heterotrophic nitrifiers and their identification going back to 100 years, the biochemistry of heterotrophic nitrification is poorly known as pointed out by Pertti J. Martikainen (Soil Biology and Biochemistry 168 (2002) 108611). Considering that the well aware topic of heterotrophic nitrifiers are still not fully explored, a person skilled in the art can realize that there is lot to explore and leverage from Alternative ammonia utilizing pathways.
[0019] It is known in the art that, a majority of commercial biological micro-organism based products are based on autotrophic nitrifiers. For instance, the US patent (US 7,407,793 B2) discloses the use of consortium of Nitrosomonas europea and Nitrobacter winogradskyi for removal of ammonia and nitrite from aquaculture ponds.
[0020] However, a person skilled in the art will quickly realize that heterotrophic based abatement of ammonia, given fast growth rate of heterotrophs over autotrophs, will be of preference for quick abatement of ammonia from sensitive systems like aquaculture, aquariums and waste water treatment facilities. Further, it is desirable to have heterotrophic, ammonia abating bacteria that can grow in wide range of salinity to suit low saline and high saline applications.
[0021] Despite significant improvement in field of heterotrophic bacteria based ammonia abatement and given that there is lot to explore and leverage from Alternative ammonia utilizing pathways, a person skilled in the art will realize that, there is still a need to develop an improved biological solution for ammonia abatement, which are based on heterotrophic bacteria, covering wide salinity ranges, fulfilling the demands of Aquaculture Industry, Aquariums and waste water treatment facilities.
OBJECTS OF THE INVENTION
[0022] An object of the present disclosure is to provide a composition comprising: active ingredients, saline and stabilizer, for ammonia abatement in water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, raceway system, RAS system, waste water, industrial effluent and brackish water.
[0023] Another object of the present disclosure is to provide a method of creating biofloc involving the use of the said composition, whereby, ammonia present in aquatic systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, Raceway system and RAS system is converted into protein rich feed.
[0024] Another object of the present disclosure is to provide a method of making an apparatus involving the use of the said active ingredients, saline and stabilizer, for ammonia abatement in water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, raceway system, RAS system, waste water, industrial effluent and brackish water.
[0025] Various objects, features, aspects and advantages of the present invention will become more apparent from the details description of the invention herein below along with the accompanying drawings.
SUMMARY
[0026] Described herein, inter alia, are methods of making a composition comprising: active ingredients, saline and stabilizer, for ammonia abatement in water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, raceway system, RAS system, waste water, industrial effluent and brackish water.
[0027] An aspect of the present invention relates to a composition comprising: active ingredients, saline and stabilizer, for ammonia abatement in water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, raceway system, RAS system, waste water, industrial effluent and brackish water.
[0028] In an embodiment of the present disclosure, active ingredients of the said composition refers to newly isolated novel Vibrio strains: Vibrio sp. (HeteroMight 44) & Vibrio diabolicus (HeteroMight 64); either used singly or in combination.
[0029] In some embodiments of the present disclosure, stabilizer of the said composition refers to a polymer. Particularly, it refers to a biopolymer. More particularly, it refers to a biopolymer composed of glucose subunits or glucose derived subunits. Even more particularly, it refers to biopolymer composed of Glucosamine subunits or one of its several derivatives.
[0030] In some embodiments of the present disclosure, the said biopolymer may exist as a mixture of the biopolymer itself along with its subunits in monomeric form.
[0031] Also described here, inter alia, are methods of creating biofloc, involving the use of the said composition, whereby, ammonia present in aquatic systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, Raceway system and RAS system is converted into protein rich feed.
[0032] In some embodiments of the methods, the said biofloc is created by addition of said composition to the aquaculture water augmented with carbon source including, Glucose, Sucrose, Glycerol, Starch, Corn Steep Liquor, Chitin, Chitosan, Jaggery, Molasses, Wheat flour, Wheat barn, Rice flour and Rice barn.
[0033] Also described here, inter alia, are methods of making an apparatus involving the use of the said composition, for ammonia abatement in water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, raceway system, RAS system, waste water, industrial effluent and brackish water.
[0034] In some embodiments of the methods, the said apparatus is made by filling the said composition, into a protective housing, having provision for inflow and outflow of water.
[0035] In some embodiments, the said protective housing can be made up of materials including plastics, metals, concrete, wood, glass, porcelain and clay.
[0036] In some embodiments, the said housing of the apparatus may have scaffold structures commonly known in the art, to retain the composition inside the housing.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
[0038] FIG. 1 illustrates an exemplary image depicting the bacteria, Vibrio sp. (HeteroMight 44) & Vibrio diabolicus (HeteroMight 64) used as active ingredients in accordance with the embodiments of the present disclosure.
[0039] FIG. 2 illustrates an exemplary image the ability of Vibrio sp. (HeteroMight 44) & Vibrio diabolicus (HeteroMight 64) to grow in Nutrient agar and TCBS agar, in accordance with the embodiments of the present disclosure.
[0040] FIG. 3 illustrates an exemplary graph depicting growth curve of Vibrio sp. (HeteroMight 44) & Vibrio diabolicus (HeteroMight 64) at different salinity levels in accordance with the embodiments of the present disclosure.
[0041] FIG. 4 illustrates an exemplary graph of in vitro experiment depicting extent the ammonia is decreased in three hour time by Vibrio sp. (HeteroMight 44) & Vibrio diabolicus (HeteroMight 64) in accordance with the embodiments of the present disclosure.
[0042] FIG. 5 illustrates a set of exemplary graphs depicting the existence of alternative ammonia utilizing pathways by the two bacterial strains, constituting the active ingredient of the composition, in accordance with the embodiments of present disclosure.
[0043] FIG. 6 illustrates an exemplary graph of in shrimp culture water experiment with shrimp post larvae, depicting, extent the ammonia is decreased in 24 hour time by different experimental sets namely: Ammonia Negative Control Set, Ammonia Positive Control Set, Bacterial Control Set and Test set, in accordance with the embodiments of the present disclosure.
[0044] FIG. 7 illustrates an exemplary image depicting synergistic effect of chitin and active ingredients in extending the viability of the active ingredients in liquid formulation, in accordance with the embodiments of the present disclosure.
[0045] FIG. 8 illustrates an exemplary image depicting synergistic effect of chitin and active ingredients in extending the viability of active ingredients in solid formulation, in accordance with the embodiments of the present disclosure.
[0046] FIG. 9 illustrates an exemplary image of biofloc created with the use of the composition augmented with Sucrose as carbon source.
[0047] FIG. 10 presents an exemplary drawing of apparatus involving the use of composition relating to the present invention.
DEFINITIONS
[0048] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art.
[0049] Alternative ammonia utilizing pathways: Apart from the commonly agreed "Nitrification" pathway of ammonia abatement, wherein, ammonia is converted to nitrite and nitrite is converted to nitrate, there exist alternative pathways including, but not limited to "Assimilatory Nitrogen Reduction", "Anaerobic Ammonia Oxidation" (Anammox) and "Ammonia Fermentation". Such alternative pathways shall be referred herein as "Alternative ammonia utilizing pathways".
[0050] Plastic: Includes a wide range of synthetic or semi-synthetic polymer material which could be molded into desired shapes.
[0051] Polymer: Large molecules of natural or artificial origin made by linking up smaller repetitive building blocks called monomers.
[0052] Assimilatory Nitrogen Reduction: Some bacteria assimilate ammonia directly into their cellular components, such as amino acids and proteins, without releasing nitrite or nitrate as by-products.
[0053] ppt: Parts Per Thousand.
[0054] ppm: Parts Per Million.
[0055] Biofloc: Suspended particle formed by aggregation of micro-organisms along with organic matter, which plays a key role in managing waste in aquaculture environment - by converting waste into-high value-protein rich-edible biomass, which the fish or shrimp can consume.
[0056] FCR: Feed Conversion Ratio.
[0057] RAS System: Recirculating Aquaculture System.
[0058] Water systems: water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, raceway system, RAS system, waste water, industrial effluent and brackish water. Essentially the term encompasses various types of water bodies, treatment systems, and waste products related to both aquatic life management and industrial processes.
[0059] Aquatic systems: subset of water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, Raceway system and RAS system. Essentially the term encompasses various types of setups and environments used for managing and cultivating aquatic life, whether for commercial, ornamental, or research purposes.
DETAILED DESCRIPTION
[0060] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0061] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the "invention" may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the "invention" will refer to subject matter recited in one or more, but not necessarily all, of the claims.
[0062] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.
[0063] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0064] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition; person in the pertinent art have given that term, as reflected in printed publications and issued patents at the time of filing.
[0065] It is preferred in aquaculture industry and in aquariums to have ammonia abating composition fulfilling among others, the following requirements:
a) Ability to quickly decrease the ammonia concentration in water to get ammonia abated water.
b) Ability to work at wide ranges of salinity.
c) Ability to keep the viability of bacteria, serving as active ingredient, for extended period.
d) Ability to monitor with ease, the viability of bacteria, serving as active ingredient of the composition.
e) Ability to monitor with ease, the growth and development of bacteria, serving as active ingredient, in the applied water.
f) Ability to create value add while decreasing the ammonia concentration in water, including, conversion of ammonia into biomass, which in-turn can be fed by aquaculture animals, which in-turn results in better feed conversion ratio (FCR).
[0066] Described herein, inter alia, are methods of making a composition comprising: active ingredients, saline and stabilizer, for ammonia abatement in water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, raceway system, RAS system, waste water, industrial effluent and brackish water.
[0067] An aspect of the present invention relates to a composition comprising: active ingredients, saline and stabilizer, for ammonia abatement in water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, raceway system, RAS system, waste water, industrial effluent and brackish water.
[0068] In an embodiment of the present disclosure, active ingredient of the said composition refer to newly isolated novel Vibrio strains: Vibrio sp. (HeteroMight 44) & Vibrio diabolicus (HeteroMight 64); either used singly or in combination.
[0069] Further, for taxonomic purposes, the 16S rDNA of the two novel organisms was sequenced. SEQ ID NO: 1 presents the 16S rDNA partial sequence of Vibrio sp. (HeteroMight 44) and SEQ ID NO: 2 presents the 16S rDNA partial sequence of Vibrio diabolicus (HeteroMight 64). Also, the two novel strains were deposited for patent purposes under the terms of the Budapest Treaty at Microbial Culture Collection (MCC), Pune, INDIA. The deposit was made on 18 July 2024 and was assigned with reference number MCC 0282 for Vibrio sp. (HeteroMight 44) & MCC 0283 for Vibrio diabolicus (HeteroMight 64) respectively.
[0070] In some embodiments of the present disclosure, one of the active ingredients, Vibrio sp. (HeteroMight 44) may have a 16S rDNA sequence, which is more than 90% identical to SEQ ID NO: 1. Particularly, more than 99% identical to SEQ ID NO: 1. Preferably, it has 16S rDNA sequence, which is SEQ ID NO: 1 or is the strain contained in MCC, with reference number MCC 0282.
[0071] In some embodiments of the present disclosure, one of the active ingredients, Vibrio diabolicus (HeteroMight 64) may have a 16S rDNA sequence, which is more than 90% identical to SEQ ID NO: 2. Particularly, more than 99% identical to SEQ ID NO: 2. Preferably, it has 16S rDNA sequence, which is SEQ ID NO: 2 or is the strain contained in MCC, with reference number MCC 0283.
[0072] In some embodiments of the present disclosure, stabilizer of the said composition refers to a polymer. Particularly, it refers to a biopolymer. More particularly, it refers to a biopolymer composed of glucose subunits or glucose derived subunits. Even more particularly, it refers to biopolymer composed of Glucosamine subunits or one of its several derivatives.
[0073] In some embodiments of the present disclosure, the said biopolymer may exist as a mixture of the biopolymer itself along with its subunits in monomeric form.
[0074] In some embodiments of the present disclosure, the said biopolymer could be selected from the group comprising: Cellulose, Hyaluronic Acid, Chitin, Chitosan, Alginate, Glycosaminoglycans, Dermatan Sulfate, Keratan Sulfate and Keratin, either alone or in combination.
[0075] In some embodiments of the present disclosure, the said biopolymer could be in the form of flakes, coarse power, fine powder, gel, suspension, solution, crystals, grains or fibers.
[0076] In some embodiments of the present disclosure, saline of the said composition refers to a solution of salt in water. Particularly, it refers to a solution of, halides and ionic compounds in water. More particularly, it refers to a solution in water of ionic compounds selected from group comprising: Sodium Chloride, Potassium Chloride, Magnesium Chloride, Calcium Chloride and Ammonium Chloride, either alone or in combination.
[0077] Also described here, inter alia, are methods of creating biofloc, involving the use of the said composition, whereby, ammonia present in aquatic systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, Raceway system and RAS system is converted into protein rich feed.
[0078] In some embodiments of the methods, the said biofloc is created by addition of said composition to the aquaculture water augmented with carbon source including, Glucose, Sucrose, Glycerol, Starch, Corn Steep Liquor, Chitin, Chitosan, Jaggery, Molasses, Wheat flour, Wheat barn, Rice flour and Rice barn.
[0079] In some embodiments of the methods, for the creation of biofloc in the aquaculture water, the aquaculture water can be augmented with carbon source to the levels of 1 ppm to 10,000 ppm. Particularly, the said carbon source can be augmented to the levels of 1 ppm to 1000 ppm. Even more particularly, the said carbon source can be augmented to the levels of 10 ppm to 200 ppm.
[0080] Also described here, inter alia, are methods of making an apparatus involving the use of the said composition, for ammonia abatement in water systems, including; aquaculture water, aquarium tanks, ornamental fish tanks, raceway system, RAS system, waste water, industrial effluent and brackish water.
[0081] In some embodiments of the methods, the said apparatus can be made by one of the following methods, but not limited hereto:
a) Filling the said composition, into a protective housing, having provision for inflow and outflow of water.
b) Before filling the said composition, into a protective housing (having provision for inflow and outflow of water), scaffold structures commonly known in the art, can be introduced to retain the composition inside the housing.
[0082] In some embodiments of the methods, the said composition can be filled in the said apparatus to the levels of 1% to 100% of the available space inside the protective housing-with the scaffold structure. Particularly, the said composition can be filled in the said apparatus to the levels of 10% to 100% of the available space inside the protective housing-with the scaffold structure.
[0083] In some embodiments of the methods, the said protective housing can be made up of materials including plastics, metals, concrete, wood, glass, porcelain and clay, either alone or in combination or as composite material.
[0084] In some embodiments of the methods, the protective housing can be made up of metal commonly known in the art. Particularly, the said metal could be Iron, Steel, Titanium, Aluminum, Copper, Lead, Nickel, Brass, Bronze or different grades of stainless steel including 202,304, 316 and 316L
[0085] In some embodiments of the methods, the said scaffold structures can be made up of materials including plastics, bio-polymers, cotton, wool, polyester, metals, concrete, wood, glass, porcelain, sand, stones and clay, either alone or in combination.
[0086] In some embodiments of the methods, the said scaffold structures can be made up of plastic material commonly known in the art. Particularly, the said plastic material could be Polyethylene (PE), Polypropylene (PP), Polyvinyl Chloride (PVC), Polystyrene (PS), Polyethylene Terephthalate (PET), Polyurethane (PU), Acrylonitrile Butadiene Styrene (ABS), Polycarbonate (PC), Polyamide (PA) or Polyethylene Glycol (PEG).
[0087] In some embodiments of the methods, the said scaffold structure can be of any shape and structure commonly known in the art. Particularly, the said scaffold structure can be spherical beads, cube, cuboid, rectangular, triangular, hexagonal, pyramid, cylinder, porous sheets, capillary structures, membranes, fibrous structures or felt.
EXAMPLES
Example 1 - Gram staining.
[0088] Figure 1 presents an exemplary image of gram stained cultures of Vibrio sp. (HeteroMight 44) (FIG. 1a) and Vibrio diabolicus (HeteroMight 64) (FIG. 1b). Image captured with 1000x magnification. As illustrated, both the bacterial strains were gram negative.
Example 2 - Growth in Nutrient agar and TCBS agar.
[0089] FIG. 2a and FIG. 2b presents an exemplary image of both the bacterial strains, streaked on Nutrient agar (with 3% NaCl) and TCBS agar respectively. Z1 and Z3 represents Vibrio sp. (HeteroMight 44). Z2 and Z4 represents Vibrio diabolicus (HeteroMight 64). As illustrated, Vibrio sp. (HeteroMight 44) does not grow in the common Vibrio selective media namely TCBS agar, but grown in Nutrient agar (3% NaCl). Vibrio diabolicus (HeteroMight 64) grows in both the culture media (Produces yellow colony forming units in TCBS agar). Vibrio diabolicus (HeteroMight 64) produces slimy growth in both media -probably due to the secreted polysaccharide.
Example 3 - Growth curve at different salinities.
[0090] Figure 3 presents the results for growth curve of, Vibrio sp. (HeteroMight 44) and Vibrio diabolicus (HeteroMight 64) at different salinities. Culture media components (Table 3a) were sterilized by autoclaving and cooled to room temperature. Tested salinity values include 0.1%, 0.25%, 0.5%, 1%, 3%, 5% and 10% w/v Sodium Chloride. Two sets of culture media was made with different salinities in conical flasks, with triplicates for each salinity values for each set. To one set, Vibrio sp. (HeteroMight 44) was inoculated and Vibrio diabolicus (HeteroMight 64) was inoculated to another set.
[0091] Bacteria culture inoculum was prepared by culturing respective bacteria in sterile culture medium as per table 3b, overnight, at 32 degrees C, in an orbital shaker set at 130 rpm. Inoculation was made in both sets of assay flasks, so as to get a roughly similar OD600 value at zero hour reading. Conical flasks with microbial cultures were incubated at 32 degrees C, in an orbital shaker set at 130 rpm. Growth of the bacterial culture was monitored by reading optical density in duplicates at 600nm at different time points. Time points used were 0, 1.5, 2.5, 3.5, 4.5 and 5.75 hours. As illustrated in exemplary images in FIG 3, Vibrio sp. (HeteroMight 44) (FIG. 3a) grows at wide salinity ranges and more euryhaline than Vibrio diabolicus (HeteroMight 64) (FIG. 3b). Also, illustrated in exemplary image in FIG 3 that, Vibrio diabolicus (HeteroMight 64) (FIG. 3b) is a fast grower at high salinity ranges than Vibrio sp. (HeteroMight 44) (FIG. 3a).
Table 3a Composition of culture media for growth curve elucidation
Components Quantity (for 1 Litre of Culture medium)
Peptone 5 grams
Yeast Extract 3 grams
Sodium Chloride As needed for different salinity
Water Make up to 1 Litre

Table 3b Composition of culture media
Components Quantity (for 1 Litre of Culture medium)
Nutrient broth - make: HiMedia Laboratories Pvt Ltd.(Ref: M002). Composition: Peptone: 5 g/L; Sodium chloride: 5 g/L; HM Peptone B: 1.5 g/L; Yeast extract: 1.5 g/L 13 grams
Sodium Chloride (in addition to Sodium Chloride present in Nutrient broth, above) 25 grams
Water Make up to 1 Litre






Table 3c

Table 3d










Table 3e

Table 3f
.
Example 4 - Salicylate Hypochlorite assay for ammonia abatement.
[0092] FIG. 4a present results of in vitro ammonia abatement experiments using Salicylate assay, depicting the extent the ammonia is decreased in 3hours by different experimental sets namely, Ammonia Positive Control Set, Ammonia Negative Control Set, HM44 Set and HM64 Set. Each experimental Sets were conducted in triplicates.
[0093] To the HM44 Set and HM64 Set, over-night cultured Vibrio sp. (HeteroMight 44) and Vibrio diabolicus (HeteroMight 64) respectively was adjusted to reach OD600 value of 0.125 for Vibrio diabolicus (HeteroMight 64) and 0.132 for Vibrio sp. (HeteroMight 44). Two milliliters volume each in triplicates, of the adjusted cultures were centrifuged at 2,800 x g for 10 minutes. Supernatant was discarded and the respective pellet dispersed in 2ml of 0.1M Phosphate buffered saline (pH 7.2) containing 1 ppm ammonia equivalent for Ammonium chloride. For Ammonia Positive Control Set, 2 ml of 0.1M Phosphate buffered saline (pH 7.2) in triplicates containing 1 ppm ammonia equivalent for Ammonium chloride was used. For Ammonia Negative Control Set, 2 ml of 0.1M Phosphate buffered saline (pH 7.2) in triplicates without Ammonium chloride was used.
[0094] For zero hour sample, 1 ml of sample from each experiment was withdrawn (for HM44 set and HM64 set, samples withdrawn, as soon the bacterial culture pellet was dispersed). It was centrifuged at 2,800 x g for 10 minutes. Salicylate assay was performed with the supernatant, as per Bower, C.E., and T. Holm-Hansen, 1980, Can. J. Fish. Aquat. Sci. 37:794-798
[0095] Remaining 1 ml of sample from each experiment, was incubated at 32 degrees C for 3 hours in an orbital shaker set at 130 rpm. After incubation, the 1 ml sample from each experiment was centrifuged at 2,800 x g for 10 minutes, and the supernatant was collected. With the collected supernatant, the extent of ammonia utilized by each bacterial strain was elucidated by performing Salicylate assay as per Bower, C.E., and T. Holm-Hansen, 1980, Can. J. Fish. Aquat. Sci. 37:794-798. This represent, post incubation sample.
[0096] As illustrated in exemplary image in FIG 4a (applying the formula derived from standard curve in FIG. 4b), Vibrio sp. (HeteroMight 44) utilized approximately 86% of available ammonia during the incubation period of 3 hours, while, Vibrio diabolicus (HeteroMight 64) utilized almost all the available ammonia during the incubation period of 3 hours. It is to be noted that while Ammonia Positive Control Set, showed 1 ppm ammonia in zero hour sample as per the experimental design, it was not the case with HM44 Set and HM64 Set, possibly due to ammonia utilization by the bacterium while the zero hour sample was collected.
Table 4a






Table 4b

Table 4c

Example 5 - Evidence for Alternative ammonia utilizing pathways by the two bacterial strains.
[0097] To show that the bacterial strains were not carrying out nitrification process for ammonia abatement, a co-culture of two bacterial strains was incubated with 1 ppm of ammonia as per the protocols of example 4. After 3 hours of incubation, at 32 degrees C, in an orbital shaker set at 130 rpm, levels of ammonia, nitrite and nitrate was elucidated as illustrated in FIG 5a, 5b and 5c respectively. All experiments were done in triplicates and the corresponding optical density for each of the triplicates recorded in duplicates.
[0098] To the HeteroMight Set, required number of micro-centrifuge tubes containing, one millilitre of over-night co-culture of Vibrio sp. (HeteroMight 44) and Vibrio diabolicus (HeteroMight 64) was taken. The culture sample in micro-centrifuge tubes was centrifuged at 2,800 x g for 10 minutes. Supernatant was discarded from each tube and the pellet dispersed in 1 ml quantity of 0.1M Phosphate buffered saline (pH 7.2) containing 1 ppm ammonia equivalent for Ammonium chloride. Each micro-centrifuge tubes were incubated at 32 degrees C for 3 hours in an orbital shaker set at 130 rpm, along with respective controls (positive and negative controls) as described below.
[0099] FIG. 5a present results of in vitro ammonia abatement using Salicylate assay, depicting the extent the ammonia is decreased in 3 hour time by HeteroMight Set. For positive control, 0.1M Phosphate buffered saline (pH 7.2) containing 1 ppm ammonia equivalent for Ammonium chloride was used. For negative control, 0.1M Phosphate buffered saline (pH 7.2) without ammonium chloride was used. Salicylate assay was performed as per Bower, C.E., and T. Holm-Hansen, 1980, Can. J. Fish. Aquat. Sci. 37:794-798.
[0100] FIG. 5b presents result of nitrite assay at the end of 3 hours incubation time by HeteroMight Set. For positive control, 0.1M Phosphate buffered saline (pH 7.2) containing 1 ppm Nitrite equivalent for Sodium Nitrite was used. For negative control, 0.1M Phosphate buffered saline (pH 7.2) without Sodium Nitrite was used. Nitrite assay was performed as per Su-Chang Pai et al., ACS EST Water 2021, 1, 1524-1532.
[0101] FIG. 5c presents result of nitrate assay at the end of 3 hours incubation time by HeteroMight Set. For positive control, 0.1M Phosphate buffered saline (pH 7.2) containing 1 ppm Nitrate equivalent for Sodium Nitrate was used. For negative control, 0.1M Phosphate buffered saline (pH 7.2) without Sodium Nitrate was used. Nitrate assay was performed as per Su-Chang Pai et al., ACS EST Water 2021, 1, 1524-1532.
[0102] It was noticed that, the negative control also yield residual nitrate, possible due to Nitrate contamination, as trace material present in buffer.
[0103] As illustrated in exemplary image in FIG. 5a, Fig. 5b and FIG. 5c, the ammonia utilized by HeteroMight Set [co-culture of Vibrio sp. (HeteroMight 44) and Vibrio diabolicus (HeteroMight 64)] did not result in Nitrite or Nitrate accumulation. This indicates that the bacterium does not utilize ammonia nitrification pathway and follows an alternative ammonia utilization pathway.





Table 5a

Table 5b

Table 5c

Example 6 - Experiment with shrimp culture water.
[0104] FIG. 6a presents result of in vivo experiment with shrimp post larvae, depicting extent the ammonia is decreased in 24 hour time by different experimental sets namely, Ammonia Negative Control Set, Ammonia Positive Control Set, Test Set and Bacteria Control Set. Each experimental Set were conducted in triplicates. Each of the experiments was conducted in a 20L, cylindrical, plastic container. To each container, 5 Litres of sea water at 30 ppt salinity was taken and aeration provided with air pump. In each container, 70 to 82 numbers of Litopenaeus vannamei at Post Larvae 9 stage was introduced. During the experimental period of one day, Shrimps in each container was fed with approximately, one gram of suitable commercial feed. To all containers in all Sets, 10 ml of Glycerol was added as carbon source.
[0105] To the Ammonia Positive Control Set and to the Test Set, 5 ppm of ammonia was added as Ammonium chloride. To the Bacteria Control Set and to the Test Set, 0.1% v/v each of over-night cultured Vibrio sp. (HeteroMight 44) and Vibrio diabolicus (HeteroMight 64) was added. Thus, the Bacteria Control Set and the Test Set, each received a total of, 0.2% v/v of over-night bacterial cultured. Over-night bacterial culture was prepared by culturing respective bacteria in sterile culture medium as per table 3b, overnight, at 32 degrees C, in an orbital shaker set at 130 rpm. To the Ammonia Negative Control Set and to the Ammonia Positive Control Set, 0.2% v/v of fresh, sterile, culture media as per table 3b was added.
[0106] Immediately after adding bacterial culture to the Bacteria Control Set and to the Test Set, shrimp culture water was collected from each container belonging to all four Sets. This represent Zero hour sample. Each of Zero hours ample was centrifuged at 2,800 x g for 10 minutes and the supernatant was used to perform salicylate - hypochlorite assay for ammonia detection. The assay procedure for ammonia detection was done, following the method as described in Example 4. However, as sea water was used for shrimp culture, and it is well known in the art that sea water interfere with the described assay procedure, a finite absorbance at OD660 was always observed as background noise. Adding to the background noise was nitrogen source from culture media, showing up in the assay. Since combined noise was common to all the containers, the background noise can easily be eliminated when interpreting the results. Also, the assay protocol as described in Example 4 was optimized with 0.1M Phosphate buffered saline in distilled water, hence the same procedure does not yield linear results with sea water. However, as this example wanted to look at the pattern of ammonia abatement, the drawbacks were tolerated.
[0107] After 24 hours from Zero hour sample collection, shrimp culture water was collected from each container belonging to all four Sets. This represent One day sample.
[0108] As illustrated in exemplary image in FIG 6, the active ingredients of the composition (the two bacterial strains) were efficient in abating ammonia from the shrimp culture water.
Table 6a
Set name Was Ammonia Added Was Bacterial culture Added
Ammonia Negative Control Set No No
Ammonia Positive Control Set Yes No
Bacteria Control Set No Yes
Test Set Yes Yes

Table 6b presents data on number of shrimp survived after completion of experiment.


Table 6c

Table 6d

Example 7 - Synergy with biopolymer composed of Glucosamine subunits in liquid form.
[0109] FIG. 7a and 7b presents the plate results of viability of bacterial strain Vibrio sp. (HeteroMight 44) and Vibrio diabolicus (HeteroMight 64) respectively, with 0.1% (w/v) chitin in 3% saline solution. FIG. 7c and 7d presents the plate results on viability of bacterial strain Vibrio sp. (HeteroMight 44) and Vibrio diabolicus (HeteroMight 64) respectively, without chitin in 3% saline solution. Each sample were stored for 1 month and maintained at 4 degrees C. Four sets of samples; Vibrio sp. (HeteroMight 44) in 3% saline without chitin, Vibrio diabolicus (HeteroMight 64) in 3% saline solution without chitin, Vibrio sp. (HeteroMight 44) in 3% saline with 0.1% (w/v) chitin, Vibrio diabolicus (HeteroMight 64) in 3% saline solution with 0.1% (w/v) chitin, were done in triplicates. Each of the experimental setups were stored in 50 mL sample bottles and stored at 4 degrees C for 1 month. After incubation for a month, all the triplicate samples from four sets were serially diluted up to 10-2 in 3% sterile saline. The diluted samples were plated in 3% salt, 3% agar, Nutrient agar and incubated at 32 degrees C for overnight. As illustrated, viability of bacterial strain Vibrio sp. (HeteroMight 44) and Vibrio diabolicus (HeteroMight 64) is enhanced by the presence of chitin, showing synergistic effects.
Example 8 - Synergy with biopolymer composed of Glucosamine subunits in solid form.
[0110] FIG. 8 presents the culture plate images to access the viability of the bacterial strains in solid formulation using chitin as carrier. Vibrio sp. (HeteroMight 44) and Vibrio diabolicus (HeteroMight 64) were co-cultured in culture media as per composition in table 3b. Culture incubated over night at 32 degrees C at 150 rpm shaking. The culture was separately mixed to form slurry with three different composition of solid carrier media namely, 100% Chitin (fine powder), 50% Chitin (fine powder) with 50% talcum powder and 100% talcum powder. The different slurry so obtained were dried under shade. The compositions were stored at room temperature for five days before testing the viability of the bacterial strains. In order to check the viability of the solid formulation, 10% w/v of the solid formulation was mixed in 90%v/v of saline (having 3% NaCl) and 0.1ml was plated onto nutrient agar plate containing 3% sodium chloride and 3% Agar. Culture plates were incubated over night at 32 degrees C. FIG. 8a shows the results of viability of bacteria using 100% chitin as career material, FIG. 8b shows the results of viability of bacteria using 50% Chitin with 50% talcum powder as carrier material and FIG. 8c shows the results of viability of bacteria using 100% talcum powder as carrier material. As illustrated, compared to talcum powder chitin preserved the viability of the bacterial strains and shows synergy with the bacterial strains.
Example 9 - Exemplary image of Biofloc.
[0111] FIG. 9a presents the image of aquarium tanks used for this experiment. Four aquarium tanks used in total, with each tank containing two chambers. All chambers were closed with glass lids. Before the start of the experiment, all chambers were sanitized by initially wiping the chambers with isopropyl alcohol (70%v/v) and then boiling water was filled in each chambers. After 30 minutes, water was discarded from each chamber. Each chamber was filled with 500ml of autoclaved and cooled sea water. Approximately 50 to 70 number of Litopenaeus vannamei at Post Larvae 9 stage (PL9) stage was in introduced into each chamber. Each chamber was provided with adequate aeration.
[0112] Each of the aquarium tank (containing two chambers) was assigned to different Set of experiment (Set A, Set B, Set C & Set D), with each Set containing two chambers. To all Sets (Set A, Set B, Set C & Set D) 5 ppm of ammonia equivalent for Ammonium chloride was added. To Set A and Set B, sucrose was added to the levels of 200 ppm. To Set C, sucrose was added to the levels of 10 ppm. No sucrose was added to Set D.
[0113] To Set A, Set C and Set D chambers, 0.5% (v/v) of below composition (Table 9a) was added, while the composition was not added to Set B chambers:
Table 9a
Composition for one Liter
Ingredients Final quantity
Chitin 0.5% (w/v)
Sodium Chloride 3% (w/v)
Water Made up to 0.9 Liter
The above components were autoclaved and cooled to room temperature.
MCC0282 and MCC0283 co-cultured in culture media as per composition in table 3b. Culture incubated over-night at 32 degrees C at 150 rpm shaking. 10% (v/v) of over-night incubated culture.

[0114] To all Sets (Set A, Set B, Set C & Set D), 15 mg of commercial feed was added both in the morning and evening. After 48 hours, water from each Sets (from both compartments) was collected and simple staining using Crystal Violet was done in glass slides. The results were observed under microscope for the presence of bioflocs. No biofloc was observed in Set D. Less numbers of small sized biofloc was observed in Set B and Set C. However it was noted that, in Set C, the size of the biofloc is smaller than the bioflocs from Set B. Many numbers of large sized bioflocs were observed in Set A. The results clearly indicates a synergy between sucrose and the composition for the formation of bioflocs (more numbers of large sized bioflocs).
[0115] FIG 9b. presents the micrograph of a biofloc from Set A, magnified 400 times.
Example 10 - Exemplary drawing of apparatus that could be used to hold the composition.
[0116] FIG. 10 presents an exemplary drawing of apparatus that could be used to hold the composition described in this invention. Such apparatus could commonly be termed as bio-filtration unit, ammonia treatment unit, etc., and could find application in ammonia abatement from water. Z5 represents flow path of ammonia containing water to enter into the apparatus. Z6 represents flow path of treated, ammonia abated water. Z7 represent flow path of overflow water, sent into the apparatus. Z8 points to the housing of the apparatus. Z9 points to the scaffold material used to hold/retain the composition.
ADVANTAGES OF THE INVENTION
[0117] The present disclosure provides a method for abatement of ammonia from water systems.
[0118] The composition disclosed in the present invention incudes heterotrophic bacteria as active ingredients for ammonia abatement. Heterotrophic bacteria, as commonly known in the art are fast growers. Particularly, said heterotrophic bacterial strains belongs to the genus Vibrio, which are one of the fastest growing bacteria in marine ecosystem. It is commonly known in the art that, fast growing bacterial strains with the ability to utilize ammonia can result in quick abatement of ammonia from the water systems; where, such bacterial strains are utilized.
[0119] The said bacterial strains of the present disclosure, grows at wide salinity ranges. Such euryhaline bacterial strains are highly desired, as they can be utilized seamlessly in different water systems-with varying salinities.
[0120] The present disclosure provides a method for conversion of toxic ammonia present in aquaculture systems into valuable and protein rich bioflocs, which can be used as feed by the aquatic animals.
, Claims:WE CLAIM

[1] A composition for use in ammonia abatement from water systems comprising:
a. An active ingredient;
b. Stabilizer;
c. Salt; and
d. Water.

[2] A method of ammonia abatement from water systems comprising:
a. Addition of the composition as claimed in claim 1 into the said water systems;
b. Addition of carbon source to the levels of 10 ppm to 200 ppm, into the said water systems.

[3] A method of making biofloc in aquatic systems comprising:
a. Adding into aquatic systems, the composition as claimed in claim 1;
b. Adding carbon source to the levels of 10 ppm to 200 ppm, into the said aquatic systems.

[4] A method of making an apparatus for ammonia abatement from water systems comprising:
a. Providing a protective housing, having provisions for inflow and outflow of water;
b. Introducing a scaffold structure into the protective housing;
c. Introducing into the protective housing, the composition as claimed in claim 1 to the levels of 10% to 100% of the available space inside the protective housing-with the scaffold structure.

[5] The composition as claimed in claim 1, wherein the composition is in the form of a liquid formulation, semisolid formulation or solid formulation.

[6] The composition as claimed in claim 1, wherein the active ingredient is selected from the group consisting of Vibrio sp. (HeteroMight 44) and Vibrio diabolicus (HeteroMight 64) either alone or in combination; present in the form of culture broth, concentrate, diluted form, extract, lyophilized powder or spray dried powder.

[7] The composition as claimed in claim 1, wherein the stabilizer is selected from the group consisting of Cellulose, Hyaluronic Acid, Chitin, Chitosan, Alginate, Glycosaminoglycans, Dermatan Sulfate, Keratan Sulfate and Keratin.

[8] The composition as claimed in claim 1, wherein the salt is selected from the group consisting of Sodium Chloride, Potassium Chloride, Magnesium Chloride, Calcium Chloride and Ammonium Chloride.

[9] The method as claimed in claim 2, wherein the carbon source is from the group consisting of Glucose, Sucrose, Glycerol, Starch, Corn Steep Liquor, Chitin, Chitosan, Jaggery, Molasses, Wheat flour, Wheat barn, Rice flour and Rice barn.
 

[10] The method as claimed in claim 3, wherein the carbon source is from the group consisting of Glucose, Sucrose, Glycerol, Starch, Corn Steep Liquor, Chitin, Chitosan, Jaggery, Molasses, Wheat flour, Wheat barn, Rice flour and Rice barn.

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