Claims:WE CLAIM:
1. A composition for use in aquaculture comprising a bacterial formulation microencapsulated in a porous and granular medium wherein,
the bacterial formulation comprises bacterial strains microencapsulated with a synthetic minerals blend, said bacterial strains selected from the group comprising Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus coagulans, Bacillus licheniformis, Pediococcus acidilactei, Bacillus pumilus, Bacillus megaterium, Bacillus polymyxa, Bacillus circulans and combinations thereof; and
the porous and granular medium is calcareous coccospheres.
2. The composition as claimed in claim 1 comprising Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus coagulans, Bacillus licheniformis, Pediococcus acidilactei, Bacillus pumilus, Bacillus megaterium, Bacillus polymyxa and Bacillus circulans in ratio of 0.4-0.6:0.2-0.3:0.2-0.5:1.8-2.2:1.8-2.2:0.3-0.6:0.3-0.5:0.2-0.4:0.2-0.3:0.3-0.5:0.3-0.6:0.3-0.7:0.6-0.9.
3. The composition as claimed in claim 1 wherein the selected bacterial strains are Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans and Bacillus licheniformis in ratio of 2-5:2-5:1-4.2:2-4.2:1-3:0.5-2:0.5-2.5.
4. The composition as claimed in claim 1 wherein the synthetic minerals blend is a mixture of organic, soluble minerals comprising Zinc, Titanium, Aluminum and Cobalt in amounts sufficient for enhancing metabolism activities of microencapsulated bacterial strains.
5. The composition as claimed in claim 1 wherein the coccospheres are biodegradable, have varying particle size of about 3 mm to 5 mm and provide solubilized nutrients including manganese, navajoite, magnesium, potassium, sulphur, calcium, boron, copper, cadmium, fluoride, nickel, tin, titanium, silica, iron and phosphorus in compound form, said nutrients supporting the growth and multiplication of the microencapsulated bacterial strains and of beneficial planktons present in an aquaculture water body.
6. The composition as claimed in claim 1 wherein the composition is composed of differently sized granules of size about 2 µm to 5 mm, enabling the composition to act upon different regions of a water column upon dispersal in an aquaculture water body.
7. The composition as claimed in claim 1 comprising porous medium in about 80 to 90% by weight of the composition, synthetic minerals in about 7.5 to 17.5% and bacterial strains in about 2 to 3%.
8. The composition as claimed in claim 1 wherein the potency of bacteria is about 1x109 to 2 x 1013 cfu/g, the bacteria strains secreting beneficial secondary metabolites and exhibiting enzymatic activity.
9. The composition as claimed in claim 1 wherein the composition has shelf-life of at least 5 years, the bacterial strains remaining viable at least throughout the shelf-life of the composition, the composition carrying out biodegradation and removal of organic load, elimination of toxic gases, reduction in pathogens, inhibiting sludge formation, stabilization of plankton bloom, control of nitrite levels, improvement of dissolved oxygen levels, mineralization and pH stabilization of an aquaculture water body and increased immunity, growth, survival rate, yield and feed conversion ratio of an aquatic animal farmed in said water body, thereby resulting in increase in yield by about 17 to 27% of the farmed aquatic animal.
10. A Process of manufacturing a composition for use in aquaculture characterized in that said process comprises the steps of:
preparing cultures of selected bacterial strains in suitable culture media;
harvesting the bacterial cultures at log phase of the growth cycle;
freeze drying the bacterial cultures to form lyophilized bacterial strains;
preparing a mixture of different lyophilized bacterial strains;
preparing a blend of synthetic minerals;
preparing a formulation by mixing the lyophilized bacterial strains mixture with the synthetic minerals blend, resulting in microencapsulation of the lyophilized bacterial strains in the synthetic minerals; and
blending the formulation with a porous and granular medium, thereby microencapsulating the formulation within the pores of the medium to yield said composition;
wherein the bacterial strains are selected from the group comprising Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus coagulans, Bacillus licheniformis, Pediococcus acidilactei, Bacillus pumilus, Bacillus megaterium, Bacillus polymyxa, Bacillus circulans and combinations thereof; and
the porous and granular medium is calcareous coccospheres.

11. The process as claimed in claim 10 wherein,
the bacterial strains are cultured in the media at pH of about 6 to 8, at temperature about room temperature to 37?C for about 14 to 24 hours, the media effective in reducing culture time;
freeze drying of the bacterial cultures is carried out at temperature of about -40? C to -75?C and at applied vacuum of about 250-300 millitorr;
the synthetic minerals are organic, soluble Zinc, Titanium, Aluminum and Cobalt;
the calcareous coccospheres are biodegradable and have varying particle size of about 3 mm to 5 mm; and
the process yields a composition composed of differently sized granules of size about 2 µm to 5 mm.

12. The process as claimed in claim 10 wherein the selected bacterial strains are Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus coagulans, Bacillus licheniformis, Pediococcus acidilactei, Bacillus pumilus, Bacillus megaterium, Bacillus polymyxa and Bacillus circulans, the lyophilized bacterial strains whereof are mixed in ratio 0.4-0.6:0.2-0.3:0.2-0.5:1.8-2.2:1.8-2.2:0.3-0.6:0.3-0.5:0.2-0.4:0.2-0.3:0.3-0.5:0.3-0.6:0.3-0.7:0.6-0.9.
13. The process as claimed in claim 10 wherein the selected bacterial strains are Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans and Bacillus licheniformis, the lyophilized bacterial strains whereof are mixed in ratio 2-5:2-5:1-4.2:2-4.2:1-3:0.5-2:0.5-2.5.
14. The process as claimed in claim 10 wherein the potency of bacteria in the composition is about 1x109 to 2 x 1013 cfu/g.

15. The process as claimed in claim 11 wherein the synthetic minerals Zinc, Titanium, Aluminum and Cobalt are blended in ratio 0.05-0.4:0.01-0.03:0.3-1:0.01-0.03.

16. The process as claimed in claim 10 wherein the porous medium forms about 80 to 90% by weight of the composition, the synthetic minerals about 7.5 to 17.5% and the lyophilized bacterial strains about 2 to 3%.

17. The process as claimed in claim 10 yielding a composition effective in increasing yield of a farmed aquatic animal by about 17 to 27%, said composition having shelf-life of at least 5 years, the bacterial strains remaining viable at least throughout the shelf-life of the composition.

Dated this 20th day of October 2021
Digitally signed
Annu Alexander (IN/PA-1382)
Constituted Patent Agent for the Applicant
, Description:COMPOSITION FOR USE IN AQUACULTURE AND PROCESS OF MANUFACTURE THEREOF

FIELD OF THE INVENTION
The present invention generally relates to a composition for improvement of an aquaculture water body environment and more specifically for bioremediation and mineralization of an aquaculture system. Further the invention relates to improvement of overall health and survival rate of farmed aquatic animals and reduction/removal of detrimental pathogens, thus enhancing yield and quality of the farmed animals. The present invention also provides a process for preparing the composition in an economical manner and with extended shelf life.

BACKGROUND OF THE INVENTION
Aquaculture has become one of the world's fastest growing food production sectors in the recent past. Some of the major problems afflicting aquaculture water systems are organic culture waste such as decomposing feed, fecal matter, dead algae and molted exo-skeleton(in the case of shrimp) accumulating on the pond bottom, leading to sludge formation, aerobic water condition and increase in pathogenic load. The culture waste containing ammonia, hydrogen sulfide and nitrite is toxic to fish and shrimp, inhibiting growth and even killing the organisms, resulting in low aquaculture productivity.
Vibrio species and strains are pathogenic bacteria leading to a variety of ‘vibriosis’ diseases. Higher organic load on the pond bottom also results in proliferation of Microsporidians (protozoan parasite). Vibrio and Microsporidians adversely affect aquaculture production and result in high economic losses due to factors like low productivity, nutrient cycling, disease control and environmental impacts.
The use of antibiotics in aquaculture is not advisable as it leads to negative consequences such as the development of drug-resistant bacteria and reduced resistance of antibiotics to human and animal diseases. This has led to the use of nonpathogenic bacteria as probiotic control agents in aquaculture. Probiotic bacteria are known to promote the growth of the cultured organisms. The probiotic bacteria establish a normal gut microbiota in an aquatic animal which serves as a barrier against invading pathogens. Nitrifying and denitrifying bacteria have been used to decrease the organic load in aquaculture water bodies.
Indian application no. 201841010230 relates to microbial consortia comprising bacterial consortium for removal of inorganic nitrogenous metabolites like, ammonia, nitrite and nitrate from brackish water aquaculture system.
US 7,407,793 B2 discloses probiotic bacterial strains which inhibit or prevent growth or pathogenic bacteria in marine organisms. Also provided are methods of culturing marine organisms using the probiotic bacteria.
US 9,992,979 B2 provides a method of aquaculture of a farmed organism such as shrimp which includes the steps of providing an aquatic environment with a farmed organism, phytoplankton and bacteria and supplying at least one phytoplankton nutrient and at least one bacteria nutrient in pre-determined periods to allow growth of the shrimp.
TWM577654U provides a probiotic carrier unit adapted to carry at least one probiotic powder for addition to a culture water body comprising: a granular carrier having a plurality of irregular holes on its surface; a coating layer coated on the surface of the carrier for reducing the surface tension between the carrier and the aquaculture water body, thereby making the carrier easier to sink into the aquaculture water body; A plurality of particulate zeolites are accommodated in the pores, and the porous surface of each zeolite used for attachment of the probiotic powder.
Probiotics are described by the World Health Organization (WHO) as “live organism, which when administered in adequate amounts, confer health benefits to the host” (FAO/WHO). To produce these beneficial effects on animals, probiotic bacteria have to be able to survive and multiply in the host and in the surrounding environment. A number of factors have been identified as affecting the viability of probiotics, including pH, moisture, encapsulation materials, storage temperature etc. In general, aquaculture pond water parameters may not be ideal always for shrimp and fish culture. Microbial parameters and quality of aquaculture water body ultimately determine the success or failure of aquaculture practices.
Therefore, what is required is a solution that simultaneously works for the betterment of both, pond environment and farmed animal.
Phytoplankton and zooplankton are the primary food source of farmed aquatic animals such as fish and shrimp, especially in the earlier stages of the life cycle. Ideal plankton blooms in aquaculture ponds initiate the below mentioned processes which are essential for improved FCR (feed conversion ratio) and high crop yield:
• Plankton increases the dissolved oxygen in the pond water and reduces toxic gases like ammonia, hydrogen sulfide, methane, carbon-dioxide, etc;
• Plankton provide shade to aquatic animals, protecting them from direct sunlight and reducing cannibalism;
• Maintains quality of pond water through oxidation and reduction process;
• Plankton is a natural food for fish and shrimp which provides immunity and reduces stress on the animal;
• Planktons keep the water temperature stable.

For bacterial multiplication and growth, the following factors are essential: Suitable temperature; Oxygen/no oxygen; pH; Carbon source; Nitrogen source and Minerals including trace.
While beneficial bacteria have proved useful, what is required is a product that not only provides bacteria beneficial to aquaculture, but also provides sources that supply nutrients to the bacteria; provide protection to the bacteria; enhance and stabilize phytoplankton bloom; and improve water quality, for example by mineralization, maintaining of required pH and adsorption of toxic gases.
The composition of the present invention provides a formulation of bacteria and synthetic minerals microencapsulated in a porous and granular medium, namely calcareous coccospheres formed by biostratinomic processes.
The composition furnishes a solution to the aforementioned problems in addition to providing further beneficial uses to an aquaculture system. The present invention also provides a method of manufacture of this composition in the most effective manner.

SUMMARY OF THE INVENTION
It is an object of the present invention to provide an integrated composition for aquaculture use comprising a formulation of bacterial strains microencapsulated in a blend of synthetic minerals, the formulation further microencapsulated in calcareous coccospheres formed by biostratinomic processes. The composition provides manifold advantages as the bacteria, synthetic minerals and the coccospheres synergistically act to carry out biodegradation and removal of organic load, to eliminate toxic gases, reduce pathogens, inhibit sludge formation, stabilize plankton bloom, control nitrite levels, improve dissolved oxygen levels, mineralize and stabilize pH of water body and improve health, growth, survival rate and yield of an aquatic animal farmed in said water body. The present invention also provides a process for preparing the composition in a cost and time saving manner and also providing extending shelf life.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG.1 shows Scanning Electron Microscopy images of a calcareous coccosphere at different magnifications.
FIG.2 shows an image of the granules of the present composition.
FIG.3 depicts the nitrification process enabled by the present composition.
FIG.4 depicts the reduction in sludge formation in a treated pond after 7 days.
FIG.5 shows improvement in plankton bloom in a treated pond after 7 days.
FIG.6 shows the comparison of Vibrio count in a control and treated pond.
FIG.7 illustrates the comparison of Average Body Weight(ABW) of shrimp in control and treated ponds.
FIG.8 exemplifies the comparison of the Survival rate of shrimp in control and treated ponds.
FIG.9 represents the difference between the Feed Conversion Ratio (FCR) of shrimp in control and treated ponds.
FIG.10 provides the comparison of Average Body Weight(ABW) of Labeo Rohita and Catla Catla in control and treated ponds.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
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, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
The term "about" used to qualify the quantities of ingredients, properties such as concentration, and so forth, shall be interpreted to mean "approximately" or "reasonably close to" and any statistically insignificant variations therefrom.
As used herein, the terms “comprising” “including,” “having,” “containing,” “involving,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances
In an embodiment, the aspects and embodiments described herein shall also be interpreted to replace the clause “comprising” with either “consisting of” or with “consisting essentially of” or with “consisting substantially of”.
The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
All processes described herein can be performed in 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.
The term ‘control’ indicates eradication of the investigated pathogen. A 100% control indicates total eradication of the pathogen.
The term ‘microencapsulation’ indicates a process in which tiny particles of one substance are embedded in another.
The composition of the present invention comprises a formulation of bacterial strains and synthetic minerals microencapsulated in calcareous coccospheres. In the formulation, the bacterial strains are microencapsulated in the synthetic minerals. The ingredients of the composition coalesce to form the composition.

The ingredients of the composition are described as follows:

The calcareous coccospheres are formed by biostratinomic processes which entail a combination of physical, chemical and biological effects. The physical effects include transport, breakage and exhumation; the biological effects include decay, scavenging, bioturbation, encrustation and boring; and the chemical effects include early changes in mineralogy and oxidation. The primary constituent of coccospheres is calcium carbonate. In addition, coccospheres contain more than 20 micronutrients and trace elements including manganese, navajoite, magnesium, potassium, sulphur, calcium, boron, copper, cadmium, fluoride, nickel, tin, titanium, silica, iron and phosphorus in compound form. Calcareous coccospheres are found in sediments on the seafloor and in formations like the White Cliffs of Dover in England. Extraction is carried out by dredging and no chemical additives are used during processing of coccospheres, thereby preserving its properties.

Some structural characteristics of coccospheres are:
• Porous and granular in form, and having irregular shape;
• Having a honey comb structure providing deep cavities within which beneficial bacteria can be microencapsulated;
• Having high cleavage resulting in high surface area leading to increased exposure to the environment;
• Having varying particle sizes, porosity and surface area;
• Having particle size ranging from about 3 mm to 5 mm.

Coccospheres are slightly alkaline in nature and are soluble and biodegradable.

FIG.1 shows Scanning Electron Microscopy images of a coccosphere at different magnifications. The irregular shape, honey comb structure and deep cavities of a coccosphere can be seen clearly.

Some of the properties and functions of coccospheres, useful for aquaculture are:
• Highly soluble in acidic environment and aids in pH correction which directly influences the quality of the pond water and the development of the farmed aquatic animal.
• Nutrients solubilized by the coccospheres aid in the proliferation of the microencapsulated bacteria, the micronutrients and trace elements contained in the coccospheres acting as a prebiotic for the bacteria;
• Biodegradable and contain no synthetic material
• Adsorb toxic gases present in the water.
• Contain oligo nutrients and trace elements that are essential for the farmed aquatic species and the probiotics.
• Nutrients solubilized by the coccospheres help to develop plankton blooms as a primary food for fishes and shrimp. Coccospheres also contains silica which is essential for plankton cell formation and boosts its proliferation.
• Have buffer capacity.
• A further advantage over the prior known materials is that coccospheres do not break down the microencapsulation of the bacterial strains and synthetic minerals, whereas this may not be avoidable with materials such as zeoloite or dolomite.
• Organic load in the aquaculture water body is removed through mineralization of the pond by the coccospheres.
• Acts on the hardness of the water
• The structure of coccospheres inhibits grazing by zooplankton thus protecting the microencapsulated bacteria.

Calcareous coccospheres are preferred for the present invention over zeolite and dolomite, though both latter materials are capable of adsorption. Unlike coccospheres, zeolite and dolomite are not known to help microorganisms in terms of growth, multiplication etc. Further, zeolite and dolomite granules immediately dissociate from the beneficial bacteria after application in the pond, settle down to the pond bottom and play no further role in providing nutrients to the bacteria or increase/stabilization of the plankton bloom.

Synthetic zeolite is not water soluble and may choke animal gills. Further, insoluble solids reduce light penetration leading to reduced photosynthesis which affects the fertility of the pond. Insoluble materials interrupt the process of osmoregulation which is an essential physiological process for the majority of aquatic crustaceans since it enables them to cope with the changes/discrepancies between the ion concentrations within their bodies and the aquatic environments they inhabit. Presence of insoluble materials in the pond water poses obstacles to the process of molting of shrimp which is so essential for their growth and survivability. For this additional reason, calcareous coccospheres which are soluble and biodegradable, offer distinct advantages over synthetic zeolite or other materials.

Advances in aquaculture engineering, biotechnology engineering and synthetic biology enable redesigning microbial cellular networks and fine-tuning physiological capabilities, thus generating industrially viable strains for use in aquaculture.

The bacterial strains selected for the present composition act as probiotics for the farmed organisms and carry out nitrification, denitrification and degradation of nitrogen compounds. The bacteria carry out bioremediation and mineralization of the pond environment and also have fungicidal and bactericidal effects. The beneficial bacteria increase growth of farmed animals and improve its health by increasing its resistance to disease. Further, at the end of the culture cycle, the probiotic bacteria dominate the intestinal flora of the farmed species. Improvement of water quality is effected by way of the bacteria influencing the composition of water-borne microbial populations and reducing the number of pathogens in the vicinity of the farm species.
The beneficial bacterial strains envisaged for the present invention include strains of Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus coagulans, Bacillus licheniformis, Pediococcus acidilactei, Bacillus pumilus, Bacillus megaterium, Bacillus polymyxa, Bacillus circulans
The selected bacterial strains have been found to exhibit enzymatic activity such as amylase activity, Beta-galactosidase activity, phytase activity and protease activity.
Bacillus subtilis is able to grow under aerobic, facultative aerobic and anaerobic conditions, allowing for switches in nitrogen metabolism that facilitate both nitrification and denitrification. Bacillus subtilis when added to an aquaculture water body challenges the growth and proliferation of pathogenic Vibrio spp. Bacillus subtilis is an excellent source of phytase enzyme.

Alcaligenes denitrificans is protease producer that finds application in aquaculture as probiotic agent owing to its proteolytic activity which aids in digestion apart from its antagonistic properties towards fish pathogens. Further, Alcaligenes denitrificans help with denitrification.
Lactobacillus lactic secrete bacteriocin which inhibits the growth of the fish pathogen Lactococcus garvieae. Addition of Lactobacillus lactis bacteriocin to L. garvieae cultures resulted in a decrease of six orders of magnitude of viable cells counts demonstrating a bactericidal mode of action. L. lactis is a nisin Z producer and has probiotic properties that might be considered as an alternative in the prevention of lactococcosis, a global disease in aquaculture systems. Lactobacilli are potential sources for the enzyme Beta-galactosidase.
Nitrosomonas sp. is a Gram-negative bacteria that oxidizes ammonia into nitrite as a metabolic process and is thus useful in bioremediation. Some Nitrosomonas species secrete the enzyme urease.
Nitrobactor spp play an important role in the nitrogen cycle by oxidizing nitrite into nitrate in soil and marine systems. Unlike plants, where electron transfer in photosynthesis provides the energy for carbon fixation, Nitrobacter uses energy from the oxidation of nitrite ions, NO2-, into nitrate ions, NO3-, to fulfill their energy needs.
Pseudomonas denitrificans are denitrifying bacteria that can use nitrate in place of oxygen as a terminal electron acceptor under anaerobic conditions but lack the capacity for fermentation. The nitrate consumed is quantitatively recovered as nitrogen gas.
Bacillus coagulans can improve water quality, regulate pH, increase the dissolved oxygen content of an aquaculture pond, degrade ammonium and decrease the average death rate of a farmed animal.
Bacillus licheniformis aids in controlling pathogenic Vibrio. parahaemolyticus. High capacity of secretion of the alkaline serine protease has made B. licheniformis one of the most important bacteria in industrial enzyme production. Bacillus licheniformis contributes to nutrient cycling and has antifungal activity and is suggested as an alternative treatment factor to the use of antibiotics in shrimp aquaculture.
Pediococcus acidilactei significantly improves growth performance and increases immune response and survival rate of farmed aquatic animal.
Bacillus pumilus is useful as a probiotic in aquaculture. It inhibits the growth of marine pathogens like Vibrio alginolyticus.
Bacillus megaterium has probiotic effect and is useful for aquaculture water body bioremediation and for controlling disease producing Vibrio spp. Bacillus megaterium is an extracellular ß-amylase producing bacteria
Bacillus Polymyxa is a Gram-positive bacterium capable of fixing nitrogen and also provides protection from pathogens. It shows high level of myeloperoxidase and superoxide dismutase activities.
Bacillus circulans aids in the removal of organic load and is used as a feed probiotic for a lower feed conversion ratio (FCR), and higher protein efficiency ratio (PER).
The bacterial strains utilized in the present invention can tolerate and are active at high and low salinity conditions with broad range of temperature and pH.
Investigation of enzyme secreting potential of the bacteria utilized in the present composition was carried out. It was found that the bacteria exhibited good potential for secreting various growth promoting and anti-pathogen enzymes. Some of the beneficial enzymes secreted are detailed in Table 1 below:
TABLE 1
Name of Enzyme Activity
Amylase 1030-1100 U/Kg
Beta-galactosidase 510-600 U/Kg
Phytase 2560 - 3000 U/Kg
Protease 90200 -90500 U/Kg

The bacterial strains selected for use in the present invention fulfil a range of useful functions such as:
• Releasing secondary metabolites (enzymes and microbicidals)
• Providing enzymatic activity for better digestion of uneaten food and debris.
• Producing inhibitory compounds/bacitracin against pathogens, especially Vibrio
• Inhibiting sludge formation at the pond bottom
• Improving the growth of farmed aquatic species and FCR (feed conversion ratio) leading to better yield and increased profitability.
• Carrying out ammonification, nitrification and de-nitrification in the pond environment, thus providing beneficial nitrates to aquatic plants, animals and microorganisms
The present composition is very effective against vibrio species in shrimp and fish farming as the secondary metabolites including bacitracin released by the bacteria provide antagonistic activity against vibrio leading to drastic reduction in the same.
Minerals are essential to aquaculture since they are required for growth of the farmed species, for example, for shrimp growth as the exoskeletons of shrimps are made of minerals. The present invention envisages a formulation of bacterial strains microencapsulated in a blend of organic, soluble synthetic minerals such as Zinc, Titanium, Aluminum and Cobalt, the synthetic minerals present in sufficient amount for enhancing metabolic activity of microencapsulated bacteria. The minerals carry out the following functions:
Zinc aids the farmed aquatic species with metabolism of lipids, protein and carbohydrates. Zinc plays an active role in the synthesis and metabolism of nucleic acid (RNA) and proteins. Zinc reduces the possibility of virus penetration, inhibits proteases activity which is involved in viral capsid formation and plays a vital role for antibody production. Zinc also facilitates wound healing in fish and shrimp.
Titanium inhibits the growth of aquatic plants and oxidation capabilities of hydroxyl radicals (•OH) to inactivate and degrade organic contaminants in the water to non-hazardous substances.
Aluminium reduces the growth of filamentous algae by trapping the nutrient phosphorus - algae’s food source, in sediments.
Cobalt: Cobalt aids in red blood cell formation and the maintenance of nerve tissue of the farmed species. It functions as an activating agent of various enzymes and plays a pivotal role for the synthesis of Vitamin B12.

Most importantly, these synthetic minerals perform the additional function of enhancing metabolic activity of the microencapsulated bacteria.
The synthetic minerals are in powder form. The weight ratio of Zinc:Titanium:Aluminum:Cobalt in pure form in the composition is in the range of about 0.05-0.4:0.01-0.03:0.3-1:0.01-0.03.
The calcareous coccospheres form about 80 to 90% by weight of the composition, synthetic minerals about 7.5 to 17.5% and bacterial strains about 2 to 3%, these ingredients amalgamating to form the composition.
The composition as a whole is used in aquaculture for increasing survival and growth of fish and shrimps through chemolithotrophy process. In addition to the beneficial bacteria and synthetic minerals, the composition contains at least eleven natural minerals which are used as a source of energy for cell biosynthesis and maintenance.
FIG.2 shows an image of the granules of the present composition.
The composition of the present invention is granular in form, the granules having irregular shape and varying particle sizes ranging from about 2 µm to 5 mm. Particle size distribution of the present composition is important to its physical and chemical properties and greatly depends on the surface area, cleavage and porosity of the coccosphere, the structure of the coccosphere being such that it provides a large surface area for microencapsulation of the formulation of bacterial strains microencapsulated in synthetic minerals.
FIG.2 clearly depicts the effectiveness of the the coccospheres in completely microencapsulating the bacteria so that the bacteria are provided nutrition and protection by the coccospheres and the synthetic minerals. The bacterial strains microencapsulated in synthetic minerals and further microencapsulated into the deep cavities of the coccospheres are highly functional yet secure.
Upon dispersal of the composition in an aquaculture pond, the differently sized granules of the composition sink to different depths based on their size. This facilitates simultaneous interaction of the composition with the different strata of the pond water column, that is, the epilimnion, thermocline and hypolimnion regions and also with the pond bottom. The different particle sizes enable the composition granules to spend sufficient time in each strata of the water column before sinking to the bottom. The irregular shape, size and varying surface area of the granules enables effective interaction of the composition with the total water body in terms of adsorption and mineralization, including trace elements.
A composition with particle size outside the range 2 µm to 5mm will not be optimally effective in treating the various strata in the pond water column simultaneously, as the smaller sized granules will either take longer to reach pond bottom or will dissolve before reaching the pond bottom whereas the larger sized ones will reach the pond bottom almost immediately upon dispersal and hence will not get sufficient time to interact with the upper strata of the water body. Due to this, nutrition and microbial spore will not be uniformly distributed throughout the pond.
On reaching the pond bottom, the granules of the composition remain in an active state, up to end of the culture cycle of the aquatic species and eliminate the toxic gases and excessive organic load. The composition acts on a broad range of factors including temperature, pH, salinity, alkalinity, hardness etc.
Through practical trials carried out in aquaculture ponds, the optimum microbial potency for the composition is fixed as about 1x109 to 2x1013 cfu/g. On an average, microbial potency is about 1 billion per gram, the composition containing sufficient nutrients for their multiplication and growth. Though potency play a vital role in the composition, the ability of the calcareous coccospheres and the synthetic minerals to create an environment for better multiplication and survivability of the bacterial strains is much more essential then potency. Hence, it can be said that the ingredients of the composition work synergistically to provide the overall functions of the composition.
In an aquaculture system, the composition as a whole performs several functions including:
• Provides digestive enzymes for complete utilization of feed and decomposition of dead plankton, excreta of fish / shrimp and uneaten food, resulting in reduction of organic load • Competitively inhibits the growth of pathogenic bacteria especially Vibrio • Provides effective control of Microsporidian populations, thereby reducing the incidence of EHP (Enterocytozoon hepatopenaei) • Helps in stabilizing pH of water resulting in improved molting of shrimp/ prawn • Increases dissolved oxygen due to the lower bio chemical oxygen demand and helps in oxidation and respiration of microbes and aquatic life • Improves pond environment, pond water quality and colour by providing nutrition for the growth of beneficial microalgae • Improves overall health and survival rate of shrimp, prawn and fishes by enhancing immune response to pathogens • Controls the Nitrite Levels in the water body by maintaining efficient nitrogen balance • Keeps phytoplankton population under check during the complete culture cycle of the aquatic species, thereby stabilizing plankton blooms • Helps with both Bio Remediation and Mineralization, keeping the sludge under control • Enhances the Carrying Capacity of the Aquaculture System • Improves FCR so that the aquatic animal achieves higher body weight with less food.
FIG.3 depicts the nitrification process enabled by the present composition. The composition is effective in converting toxic ammonia into nitrate for the use of marine life forms.
Investigation was carried out to determine the effect of environmental pH on growth of metabiome of the composition using a spectrophotometer and the data gathered is presented in the Table below:
TABLE 2
pH Growth of metabiome
6.5 0.573
7.0 0.708
7.5 0.951
8.0 1.326
8.5 1.177
9.0 0.834

It was observed that environmental pH of 7.5 to 8.0 was most favourable to the growth of the metabiome of the composition.
In another study, the effect of environmental temperature on the growth of the metabiome of the composition was measured by means of a spectrophotometer and the data gathered is presented in the Table below:
TABLE 3
Temperature Growth of metabiome
5°C -
10°C -
15°C -
20°C +
25°C +
30°C +
35°C +
40°C ±
45°C -

It was observed that environmental temperature of 20°C to 35°C was most favourable to the growth of the metabiome of the composition.
Use of the present composition in aquaculture results in about 17 to 27 % increase in yield of the farmed aquatic animal.
The composition has shelf-life of at least 5 years and the bacteria remain viable for at least 5 years.
In one embodiment, the present disclosure provides a composition for use in aquaculture comprising a formulation microencapsulated in a porous and granular medium. The formulation comprises bacterial strains microencapsulated in a blend of synthetic minerals, the bacterial strains selected from the group comprising Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus coagulans, Bacillus licheniformis, Pediococcus acidilactei, Bacillus pumilus, Bacillus megaterium, Bacillus polymyxa, Bacillus circulans and combinations thereof; and the porous and granular medium being calcareous coccospheres.
In another embodiment, the composition comprises the selected bacterial strains Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus coagulans, Bacillus licheniformis, Pediococcus acidilactei, Bacillus pumilus, Bacillus megaterium, Bacillus polymyxa, Bacillus circulans in ratio of about 0.4-0.6:0.2-0.3:0.2-0.5:1.8-2.2:1.8-2.2:0.3-0.6:0.3-0.5:0.2-0.4:0.2-0.3:0.3-0.5:0.3-0.6:0.3-0.7:0.6-0.9.

In yet another embodiment, the composition comprises the selected bacterial strains Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus licheniformis, in ratio of about 2-5:2-5:1-4.2:2-4.2:1-3:0.5-2:0.5-2.5.
In one embodiment, the present invention provides a process for the manufacture of a composition for use in aquaculture, the process comprising the steps of:
Preparing cultures of selected individual bacterial strains in suitable culture media wherein the bacterial strains are cultured in the media at pH of about 6 to 8, at temperature ranging from about room temperature to 37 ?C. The culturing of the bacterial strains may be carried out in an orbital shaker with agitation at about 75 to 160 RPM for about 14 to 24 hours;

Harvesting the bacterial cultures at log phase of the growth cycle. The time taken to reach log phase is about 14 to 18 hours;

Freeze drying each bacterial culture to form lyophilized bacterial strain. Freeze drying is carried out at temperature ranging from about -40? C to -75?C and at applied vacuum of about 250-300 millitorr;

Preparing a mixture of different lyophilized bacterial strains. The mixing may be carried out in a ribbon blender for about 15 to 25 minutes;
Preparing a blend of organic, soluble, synthetic minerals Zinc, Titanium, Aluminum and Cobalt. The blending may be carried out in a ribbon blender for about 25 to 45 minutes;
Preparing a formulation by mixing the lyophilized bacterial strains mixture with the blend of synthetic minerals, resulting in microencapsulation of the lyophilized bacterial strains in the synthetic minerals. The mixing may be carried out in a ribbon blender for about 25 to 35 minutes;

Blending the formulation with a porous and granular medium, thereby microencapsulating the formulation within the pores of the medium to yield said composition. The blending may be carried out in a ribbon blender for about 25 to 45 minutes.

The porous and granular medium utilized in the process is calcareous coccospheres. Coccospheres are biodegradable and have irregular shape, honeycomb structure, deep cavities and varying particle size, porosity and surface area. The particle size ranges from about 3 mm to 5mm. The coccospheres form about 80 to 90% by weight of the composition.

The bacterial strains for culture are selected from the group comprising Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus coagulans, Bacillus licheniformis, Pediococcus acidilactei, Bacillus pumilus, Bacillus megaterium, Bacillus polymyxa, Bacillus circulans and combinations thereof. The potency of the bacteria in the resultant composition is about 1x109 to 2x 1013 cfu/g.

In one embodiment, the bacterial strains selected for culture are Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus coagulans, Bacillus licheniformis, Pediococcus acidilactei, Bacillus pumilus, Bacillus megaterium, Bacillus polymyxa and Bacillus circulans, the lyophilized bacterial strains whereof are mixed in ratio of about 0.4-0.6:0.2-0.3:0.2-0.5:1.8-2.2:1.8-2.2:0.3-0.6:0.3-0.5:0.2-0.4:0.2-0.3:0.3-0.5:0.3-0.6:0.3-0.7:0.6-0.9.
In a further embodiment, the bacterial strains selected for culture are Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus licheniformis, the lyophilized bacterial strains whereof are mixed in ratio of about 4.5-5:4.5-5:4-4.2:4-4.2:2.5-3:1.5-2:2-2.5.
The mixture of lyophilized bacterial strains form about 2 to 3% by weight of the composition.
The synthetic minerals Zinc, Titanium, Aluminum and Cobalt in pure form are blended in ratio of about 0.05-0.4:0.01-0.03:0.3-1:0.01-0.03. The blend of synthetic minerals forms about 7.5 to 17.5% by weight of the composition.
The selected bacterial strains are cultured with media particularly effective in reducing culture time. Each medium of the present invention provides optimum growth and shortens the time taken for reaching log phase in the growth cycle of the bacteria. The selected bacterial strains may be cultured with:
a) media, gl -1 Peptic digest of animal tissue - 5.00 ± 0.3 g/l ; Beef extract - 3.00 ± 0.3 g/l at pH of 6.4 ± 0.2 at 25 to 30°C; or

b) media, Cotton seed meal - 4 ± 0.3 g/l; K2HPO4 - 1.0 ± 0.3 g/l; MgSO4.7H2O - 0.5 ± 0.3 g/l; Molasses - 30 ± 0.3 g/l; methionine - 0.01 ± 0.3 g/l at pH of 7.2 ± 0.2 at 30-35°C; or

c) media, Corn meal - 20.0 ± 0.3 g/l; Peptone - 10 ± 0.3 g/l; NaCl - 2 ± 0.3 g/l at pH of 7.5±0.2 at 30-37?C; or

d) media, Yeast extract – 2 ± 0.3 g/l; Molasses- 20 ± 0.3 g/l; Beef extract - 2 ± 0.3 g/l at pH of 7.0±0.2 at 24-30?C.

The composition yielded by the present process is a unified product, is granular and has particle size of about 2 µm to 5 mm, enabling the composition to act upon different regions of a water column upon dispersal in the water body. The present process provides a composition that is effective in increasing yield of a farmed aquatic animal by about 17 to 27%. The process of the present invention is effective for producing a composition with extended shelf-life of at least 5 years, the bacterial strains remaining viable at least throughout the shelf-life of the composition.

Some of the advantageous aspects of the present process are:
? Microencapsulation of the bacterial strains in a blend of synthetic minerals facilitates the present process to deliver a composition that is stable for at least 5 years in storage as well as in field conditions.
? Blending the formulation of microencapsulated bacteria with the porous medium for the prescribed time leads to at least 80% of the microencapsulated bacterial strains being further microencapsulated in the porous medium such that the bacterial strains and synthetic minerals are undistinguishable and inseparable in the composition.
? The blending of the formulation with the porous medium is carried out in manner such that it does not break down the bacteria and synthetic minerals microencapsulation and the integrity of the microencapsulated bacterial strains is maintained.
? The culture temperature, culture time and nutrient media are important factors to the present process. Viability and efficacy of the bacterial strains depends on the nutritional media and timing of harvesting before freeze drying. Each medium of the present invention provides optimum growth and shortens the time taken for reaching log phase in the growth cycle of the bacteria. The cost of the media is reduced due to standardization of all physiochemical parameters through OVAT (One variable at a time) process. Overall, production costs are reduced.
? Based on the bacterial strains selected, the freeze drying process is suitably modified for better yields and survivability. Specifically, freeze drying of bacterial culture takes place at temperature ranging from about -40?C to -75?C, whereas generally it is carried out at about -40?C. Further, in the present process, vacuum of about 250-300 millitorr is applied whereas typically vacuum of below 200 millitorr is applied. Freeze drying yields the lyophilized bacterial strains in powder form.
? The process does not involve any heating throughout since none of the ingredients are in liquid form. The process is carried out at ambient temperature, hence shelf-life of the composition is greatly increased as compared to a composition prepared using heating process. This leads to considerable savings in costs on account of elimination of a heating process and increase in shelf life of the finished goods.
? The process is simplified and involves blending which can be carried out in a ribbon blender to save production cost.

The present trials were conducted with the composition and process and at the dosage described in the embodiments of the present disclosure.

EXAMPLE 1
In order to prepare the composition, the selected bacterial strains Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus coagulans, Bacillus licheniformis, Pediococcus acidilactei, Bacillus pumilus, Bacillus megaterium, Bacillus polymyxa and Bacillus circulans were cultured with media, Cotton seed meal - 4 ± 0.3 g/l; K2HPO4 - 1.0 ± 0.3 g/l; MgSO4.7H2O - 0.5 ± 0.3 g/l; Molasses - 30 ± 0.3 g/l; methionine - 0.01 ± 0.3 g/l at pH of 7.2 ± 0.2 at 30-35°C in an orbital shaker with constant agitation at about 75 to 160 RPM. The mixture was incubated for about 14 to 24 hours. The organisms were allowed to grow up to log phase of the growth cycle followed by harvesting of the bacterial cultures. The time taken to reach log phase is about 14 to 18 hours. The bacterial cultures were freeze dried at -40? C to -75?C and at applied vacuum of about 250-300 millitorr. After completion of the freeze drying process, the lyophilized bacterial strains of Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans, Bacillus coagulans, Bacillus licheniformis, Pediococcus acidilactei, Bacillus pumilus, Bacillus megaterium, Bacillus polymyxa and Bacillus circulans were mixed in weight ratio of 0.4-0.6:0.2-0.3:0.2-0.5:1.8-2.2:1.8-2.2:0.3-0.6:0.3-0.5:0.2-0.4:0.2-0.3:0.3-0.5:0.3-0.6:0.3-0.7:0.6-0.9 in ribbon blender for about 15 to 25 minutes. Synthetic minerals Zinc, Titanium, Aluminum and Cobalt (in pure form) were mixed in the ratio 0.05-0.4:0.01-0.03:0.3-1:0.01-0.03 in a ribbon blender for about 25 to 45 minutes. The lyophilized bacterial strains mixture and synthetic minerals blend were mixed in a ribbon blender for 25 to 35 minutes resulting in a formulation of lyophilized bacterial strains microencapsulated in the blend of synthetic minerals. The formulation was further blended with calcareous coccospheres for 25 to 45 minutes in a ribbon blender, yielding the composition. In the composition, the lyophilized bacterial strains formed about 2-3% by weight, the blend of Zinc, Titanium, Aluminum and Cobalt formed 7.5 to 17.5% and the coccospheres formed 80-90%. The size of the composition granules lay between 2 µm to 5 mm. The average potency of the bacteria in the composition was about 4 x109 to 5 x109 cfu/g.
The composition was applied in the following dosage:
20-30 kg/Ha in one month (one meter water depth and stocking density 45-55/square meter)
Application frequency: Every 10-15 days

EXAMPLE 2
In order to prepare the composition, the selected bacterial strains Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans and Bacillus licheniformis were cultured with media, gl -1 Peptic digest of animal tissue - 5.00 ± 0.3 g/l ; Beef extract - 3.00 ± 0.3 g/l at pH of 6.4 ± 0.2 at 25 to 30°C in an orbital shaker with constant agitation at about 75 to 160 RPM. The mixture was incubated for about 14 to 24 hours. The organisms were allowed to grow up to log phase of the growth cycle followed by harvesting of the bacterial cultures. The time taken to reach log phase is about 14 to 18 hours. The bacterial cultures were freeze dried at -40? C to -75?C and at applied vacuum of about 250-300 millitorr. After completion of the freeze drying process, the lyophilized bacterial strains of Bacillus subtilis, Alcaligenes denitrificans, Lactobacillus lactic, Nitrosomonas sp., Nitrobacter spp, Pseudomonas denitrificans and Bacillus licheniformis were mixed in weight ratio of 4.5-5:4.5-5:4-4.2:4-4.2:2.5-3:1.5-2:2-2.5 in ribbon blender for about 15 to 25 minutes. Synthetic minerals Zinc, Titanium, Aluminum and Cobalt (in pure form) were mixed in the ratio 0.05-0.4:0.01-0.03:0.3-1:0.01-0.03 in a ribbon blender for about 25 to 45 minutes. The lyophilized bacterial strains mixture and synthetic minerals blend were mixed in a ribbon blender for 25 to 35 minutes resulting in a formulation of lyophilized bacterial strains microencapsulated in the blend of synthetic minerals. The formulation was further blended with calcareous coccospheres for 25 to 45 minutes in a ribbon blender, yielding the composition. In the composition, the lyophilized bacterial strains formed about 2-3% by weight, the blend of Zinc, Titanium, Aluminum and Cobalt formed 7.5 to 17.5% and the coccospheres formed 80-90%. The size of the composition granules lay between 2 µm to 5 mm. The average potency of the bacteria in the composition was about 1x109 to 2x1013 cfu/g.
The composition was applied in the following dosage:
40-60 kg/Ha in one month (one meter water depth and stocking density 45-55/square meter)
Application frequency: Every 7-10 days

TRIAL 1
An experiment was carried out "in situ" in an aquaculture pond in which Litopenaeus vannamei were farmed. This pond was afflicted by heavy sludge formation and was treated with the composition of Example 1 in prescribed dosage.
The image to the left in FIG.4 depicts the sludge before treatment and the one to the right shows the reduction in sludge, 7 days after treatment with the present composition.
Observation: Fair reduction in sludge formation was observed.
Conclusion: The present composition is effective in reducing sludge.

TRIAL 2
An experiment was carried out "in situ" in an aquaculture pond in which Litopenaeus vannamei were farmed. This pond suffered from insufficient plankton bloom. The pond was treated with the composition of Example 2 in prescribed dosage.
FIG.5 shows the improvement in plankton bloom in the treated pond. To the left is shown the pond surface before treatment and to the right is shown the pond surface, 7 days after treatment with the composition.
Observation: The surface of the pond changed color to a healthy golden or yellowish green.
Conclusion: The present composition is effective in increasing and stabilizing plankton bloom.

TRIAL 3
A trial was carried out to measure the effect of the composition of the present invention on the Vibrio count in an aquaculture pond in which Penaeus monodon shrimp were farmed.
In the treated pond, the application of the composition of Example 1 in prescribed dosage, commenced from the 17th day of culture cycle of the shrimp.
The Vibrio count in both the treated pond and the control pond were monitored on a daily basis. The gathered data is reflected in FIG.6 which shows the difference in Vibrio count between a control and treated pond.
Observations: The data shows that though both the treated and control pond had Vibrio count of about 15000 to 20000 CFU/ml at the beginning of the trial, the Vibrio count in the treated pond drastically reduced by Day 3 and Vibrio was nearly completely eradicated by Day 4 after which the count was maintained till the end of the trial period of 7 days. In comparison, in the control pond, the Vibrio count remained nearly the same throughout the trial period, while increasing towards Day 7.
Conclusion: Observations clearly indicate that the pond treated with the present composition drastically reduced the load of Vibrio. This trial illustrates that the composition of the present invention is highly effective against Vibrio species in aquaculture.

TRIAL 4
A trial was carried out to measure the effect of the composition of the present invention on the Average Body Weight(ABW), Survival rate and Food Conversion Ratio (FCR) of white shrimp in two control ponds and two treated ponds.
Treated pond 1 was dosed in prescribed dosage with the composition of Example 1. Treated pond 2 was dosed in prescribed dosage with the composition of Example 2.
The shrimp feed type, quantity and frequency were kept the same in the control ponds and the treated ponds.
i. Average Body Weight(ABW) : Random sampling was carried out at 10 day intervals. ABW was calculated as follows:
Weight Gain (%) = (Final weight – Initial weight)/Initial weight×100
FIG.7 illustrates the comparison of Average Body Weight(ABW) of shrimp in control ponds 1 and 2 and treated ponds 1 and 2 from day 30 to 120 of the growth cycle.

ii. Survival rate: In the control and treated ponds of Trial 4, the survival rate of the shrimp was also recorded. Samples were taken towards the end of the culture cycle.
Survival rate(%) = Nos. of animals survived/ Nos. of animals stocked×100
FIG.8 shows the comparison of Survival rate of shrimp in control and treated ponds.

iii. Food Conversion Ratio (FCR): In the control and treated ponds of Trial 4, the FCR was recorded. Samples were taken towards the end of the culture cycle.
FCR = Total kg of feed fed/Total kg of shrimp weight gain
FIG.9 represents the difference between the Feed Conversion Ratio (FCR) of shrimp in control and treated ponds.

Observations: The data collected for ABW, Survival rate and FCR are given below in Table 4.

TABLE 4
Control Pond-1 Treated Pond-1 % difference
in control
and treated Control Pond-2 Treated Pond-2 % difference
in control
and treated
ABW (gm.) 27.21±0.3 34.28±0.3 +26% 29.72±0.3 36.72±0.3 +24%
FCR
1.57±0.2 1.18±0.2 -25% 1.43±0.2 1.12±0.2 -22%
Survival (%) 67.2±1.2 81.3±1.2 +21% 64.6±1.5 87.3±1.5 +35%

Conclusion:
This trial illustrates that the composition of the present invention is effective for increasing the weight and survivability of shrimp, thus improving yield. The trial further demonstrates that the composition is effective in reducing FCR in shrimp culture ponds, thus reducing production cost. The average body weight increased by about 25%, FCR decreased by about 24% and survival rate improved by about 33%. In India, average feed conversion ratios in shrimp farming generally varies between 1.5 and 1.85. In the present trials, the feed conversion ratio was found to be about 1.12 to 1.18 for the treated ponds.

TRIAL 5
A field trail was carried out in an aquaculture pond in which shrimp were farmed. The pond was treated with the composition of Example 1 in prescribed dosage.
The water quality parameters were measured before treatment and 7 days after treatment. The data collected is represented in the Table 5 below.
TABLE 5

Parameter Treated trial pond before Application Treated trial pond after
7 days of Application
Water color Brownish green Golden green
Ph 10.1 8.1
Salinity 14 ppt 14 ppt
Nitrite 1.7 ppm 0.1 ppm
Ammonium 1.5 ppm 0.2 ppm
Nitrate 1.4 ppm 0 ppm
Alkalinity 180 ppm 130 ppm
Plankton bloom stability Not Stable Stable
Lab lab Yes No
Dissolved Oxygen mg/l 3.4 5.3
Animal health
ABW 16.2 gm 17.4 gm
Animal activity Medium Good
Animal color Brownish Fresh
Attachments Yes No
Gills condition Dull Good
Infection Yes No
Molting Irregular Normal

Observations: Soon after treatment the water changed color to green/golden green indicating that planktons began to develop. Organic load reduced considerably as the plankton bloom stabilized after two days. Water quality parameters were also stable in the trial period. Feed uptake increased after two days. Vibrio levels came down drastically. After 7 days, white feces reduced and the feed uptake further increased. There was improvement in all the measured parameters.
Conclusion: After application of the present composition, physiochemical water parameters significantly changed towards the ideal pond condition.

TRIAL 6
This trail was carried out to ascertain the synergistic efficacy of the ingredients of the present composition in maintaining microbial count of the microencapsulated bacteria as compared to when only the selected bacteria are used. The same selection of bacterial strains was used in all the tests.
The data collected over 15 days is represented in the Table 6 below.
Column A represents readings with the present composition having microbial potency less that the preferred potency.
Columns B and C represent readings with the present composition having microbial potency within the preferred potency range of about 1x109 to 2x1013 cfu/g.
Column D and E represent readings with only bacterial mixture at potency lower that the preferred range.
Column F represent readings with only bacterial mixture at having potency within the preferred potency range of about 1x109 to 2x1013 cfu/g.
TABLE 6

A B C D E F
Days of Application Present composition
Potency
1X105
CFU/gm. Present composition
Potency
3X109 CFU /gm. Present composition
Potency
2X109 CFU /gm. Mixed bacterial strains, potency 1X105 CFU/gm. Mixed bacterial strains, potency 3X105 CFU/gm.
Mixed bacterial strains potency
2X109 CFU /gm.
1st 1x104 2.9x109 1.8x109 1X104 2.9x105 1.8x109
2nd 1x104 2.5x109 1.9x109 1X103 2.7x105 1.7x109
3rd 1x103 2.8x109 2.1x109 539 1.9x105 1.6x109
4th 1x102 3x108 2.5x109 220 1.7x104 1.3x107
5th 1x102 3.2x108 2.6x108 14 1.5x104 0.8x105 =80000
6th 42 3.3x108 2.4x108 6 1x104 0.3x104 =3000
7th 36 3.1x108 2.4x107 NIL 7000 0.1x104 =1000
8th 12 2.9x108 2.3x107 NIL 3000 200
9th 2 2.5x107 1.9x107 NIL 828 18
10th Nil 2.4x107 1.2x107 NIL 630 7
11th Nil 2x107 1x106 NIL 200 NIL
12th Nil 1.8x106 1x105 NIL 18 NIL
13th Nil 1.6x106 1x105 NIL 2 NIL
14th Nil 1.5x106 1x105 NIL NIL NIL

Observations:
As seen from the readings in columns D, E and F, when only the bacterial mixture was used, the microbial count reduced drastically by the end of the trial period irrespective of the potency being within or less than the preferred range.
As seen from column A, in the case of the present composition, potency less than the preferred range resulted in reduction of microbial count.
It was surprisingly found that the present composition with microbial potency in the preferred range of about 1x109 to 2x1013 cfu/g was effective in increasing microbial count as seen from columns B and C.
Conclusion: It can be inferred that the ingredients of the present invention worked synergistically and provide surprising effect in increasing microbial count as is evident from the results in column B and C as compared to the results in columns D, E and F.
It can further be inferred that the present composition with preferred microbial potency is more effective in increasing microbial count than the present composition with a lower microbial potency.

TRAIL 7
This trail was carried out to ascertain the stability of the composition over a period of 5 years. Three batches of the composition with differing microbial potencies were analysed for 60 months. The storage conditions during the test were: Temperature 400 C +/- 20 C and Relative Humidity 65% +/- 5%. The data collected is represented in Table 7 below.
TABLE 7
QUANTITATIVE DATA
Time Batch 1 Batch 2 Batch 3
0 months 1.7 x 109/g 1.6 x 109/g 1.7 x 109/g
3 months 1.7 x 109/g 1.6 x 109/g 1.7 x 109/g
6 months 1.7 x 109/g 1.6 x 109/g 1.7 x 109/g
9 months 1.7 x 109/g 1.6 x 109/g 1.7 x 109/g
12 months 1.7 x 109/g 1.6 x 109/g 1.7 x 109/g
15 months 1.7 x 109/g 1.5 x 109/g 1.6 x 109/g
18 months 1.7 x 109/g 1.5 x 109/g 1.6 x 109/g
21 months 1.6 x 109/g 1.5 x 109/g 1.6 x 109/g
24 months 1.6 x 109/g 1.5 x 109/g 1.6 x 109/g
27 months 1.6 x 109/g 1.4 x 109/g 1.6 x 109/g
30 months 1.6 x 109/g 1.4 x 109/g 1.6 x 109/g
33 months 1.6 x 109/g 1.4 x 109/g 1.6 x 109/g
36 months 1.5 x 109/g 1.4 x 109/g 1.6 x 109/g
39 months 1.5 x 109/g 1.4 x 109/g 1.6 x 109/g
42 months 1.5 x 109/g 1.4 x 109/g 1.6 x 109/g
45 months 1.5 x 109/g 1.3 x 109/g 1.6 x 109/g
48 months 1.5 x 109/g 1.3 x 109/g 1.4 x 109/g
51 months 1.5 x 109/g 1.3 x 109/g 1.4 x 109/g
54 months 1.5 x 109/g 1.3 x 109/g 1.4 x 109/g
57 months 1.4 x 109/g 1.3 x 109/g 1.4 x 109/g
60 months 1.4 x 109/g 1.3 x 109/g 1.4 x 109/g

COLOR
0-60 months Off white Off white Off white

QUALITATIVE DATA
0-60 months + + +

Observation: The composition was found to remain stable for at least 60 months from the date of manufacture retaining more than 99% of the product characteristics. There was no change in the color of the composition.
Conclusion: The analysis demonstrated that the process of the present invention is effective for producing a composition with extended shelf-life of at least 5 years. The bacterial strains in the composition remain viable at least throughout the shelf-life of the composition.

TRIAL 8
A field trail was carried out to ascertain effectiveness of the present composition in farming of Major Carp by measuring the change in total bacterial and Vibrio sp. count after application of the composition. Four aquaculture ponds were treated with the composition of Example 1 in the prescribed dosage. The data collected is represented in the Table below.
TABLE 8
Type
of culture pond Trial ponds Before application of the composition After application of the composition
Total Bacterial count (cfu/ml) Total Vibrio sp.Count (cfu/ml) Total Bacterial count (cfu/ml) Total Vibrio sp.Count (cfu/ml)
Major Carp
Pond-1 426x105 201x102 681x107 9x102
Pond-2 394x105 181x102 598x107 3x102
Pond-3 511x105 179x102 746x107 8x102
Pond-4 498x105 188x102 729x107 2x102

Observations: The data shows that in all four ponds, the total bacterial count increased significantly and the Vibrio sp. was nearly eradicated.
Conclusion: This trial illustrates that the composition of the present invention is very effective against Vibrio species in aquaculture. Additionally, the present composition is effective for effective increasing microbial count.

TRIAL 9
A trial was carried out to measure the effect of the composition of the present invention on the Average Body Weight(ABW) of Labeo Rohita and Catla Catla.
The treated ponds were dosed in prescribed dosage with the composition of Example 2.
The feed type, quantity and frequency were kept the same in the control and treated ponds.
FIG.10 provides the comparison of Average Body Weight(ABW) of Labeo Rohita and Catla Catla in control and treated ponds, from day 0 to 30 of the growth cycle.

Observation: By day 30 of the growth cycle, the Average Body Weight of Labeo Rohita and of Catla Catla improved by about 50% in the treated ponds.
Conclusion: The trial illustrates that the composition of the present invention is effective for increasing the weight of Labeo Rohita and Catla Catla, thus improving yield.

Those of ordinary skill in the art will also appreciate upon reading this specification and the description of embodiments herein that modifications and alterations to the composition and process may be made within the scope of the invention and it is intended that the scope of the invention disclosed herein be limited only by the broadest interpretation of the appended claims to which the inventors are legally entitled.