Description:DESCRIPTION:
Field of the invention:
The present disclosure generally relates to the technical field of an aquaculture biotechnology, and specific relates to a method for standardization, isolation, identification, and production of bacterium biofloc for controlled enhancement of water quality, nutrient utilization, and disease suppression in aquaculture.
Background of the invention:
Biofloc technology (BFT) is the use of aggregates of bacteria, algae, or protozoa held together in a matrix along with particulate organic matter for the purpose of improving water quality, waste treatment, and disease prevention in an intensive aquaculture system. Specifically, biofloc is a symbiotic process that includes confined aquatic animals, heterotrophic bacteria, and other microbial species in the water. The consumption of biological flocs also provides nutritional value to cultured aquatic species. Biofloc can be an ideal alternative for sustainable and environmentally friendly aquaculture. The BFT is based on nutrient recycling by elevating the carbon-to-nitrogen (C/N) ratio, which stimulates the growth of microbial communities such as heterotrophic bacteria and algae within the system. The biofloc technology is based on the principle of flocculation.

The bioflocculant is primarily an extracellular macromolecular polymer composed of glycoprotein, carbohydrate, protein, cellulose, fat, glycolipid, nucleic acid, and other components formed during organismal growth. The flocculating activity of the flocculant is determined by its properties, and bio-flocculation is a dynamic process that is both safe and biodegradable.

Flocculation is a high-efficiency, low-cost, and environmentally friendly separation technique that is widely used in a variety of industrial processes, including drinking water, wastewater treatment, fermentation, food-related industries, biotechnology downstream, and so on. The synthetic polymeric flocculant is frequently used because of its great efficiency and inexpensive cost. However, the majority of manufactured bioflocculants are damaging to human health and the environment, and unlike bioflocculants, bioflocculants have the properties of safety, no toxicity, biodegradability, environmental friendliness, and so on, and bioflocculant breakdown intermediates are not secondary pollutants. To make the bioflocculant extensively used in industry, diverse microorganisms with high bioflocculant production capacity must be identified in order to improve the flocculation effectiveness of the bioflocculant. Therefore, finding innovative and efficient bioflocculants from different environmental microorganisms and studying flocculation mechanisms in the aquaculture industry.

The flocculation method is integrated with chemical flocculation, physical flocculation, self-flocculation, and biological flocculation. The bio-flocculation method is an environment-friendly and efficient enrichment approach in which certain bacteria release extracellular polymers with flocculation activity, thereby enriching microalgae. There is a need to design to effective, low-cost downstream technique for harvesting microalgae cells from growing medium and using them in applicable fields while retaining viability and biological activity. The nutritional advantage stems from the ability of beneficial microbes to assimilate large amounts of waste, such as feces and uneaten feeds, reducing the risk of downstream pollution. Nitrogenous compounds, particularly ammonia (NH3), nitrite (NO2), and nitrate (NO3) derived from aquaculture wastewater, are among the major contaminants that can be effectively managed through biofloc systems.

In existing technology, a method for creating organic biological flocs is disclosed. The method having subsequent steps, an aerobic salt-tolerant bacteria group is added to a water body to create a biological , and a nitrifying bacteria is added when the biological floc is greater than a first concentration value, and a denitrifying bacteria is added when the nitrite concentration is greater than a second concentration value, thereby changing the pH value of the water body is greater than a current threshold value and if the nitrate concentration is greater than a third concentration value, the effective microbial flora is added multiple times. The method may create a recyclable ecological balance system and overcome the problems of high water source consumption and environmental contamination in shrimp culture, reducing culturing costs. However, the method might cause imbalanced shrimp growth and pollute the environment. Moreover, the method might not be a controlled and efficient way to improve aquaculture practices.

Therefore, there is a need for a method for standardization, isolation, identification, and production of bacterium biofloc for controlled enhancement of water quality, nutrient utilization, and disease suppression in aquaculture. There is also a need for a method that efficiently converts all uneaten materials, including feed, feces, and decomposed organic matter, into valuable nutritional biomass. Further, there is also a need for a method that utilizes bacteria with the capability of producing biofloc as an inoculum to ensure reproducible results in biofloc formation.
Objectives of the invention:
The primary objective of the invention is to develop a method for standardizing, isolating, identifying, and producing bacteria biofloc for controlled enhancement of water quality, nutrient utilization, and disease suppression in aquaculture.

Another objective of the invention is to develop a method that eliminates the requirement for inoculums from previous crops, providing the advantage of preservation for future usage.

The other objective of the invention is to develop a method that provides better control over heterotrophic organisms than autotrophic ones, requiring a small amount for implantation.

The other objective of the invention is to develop a method that allows continuous access to biofloc as a nutrient-rich food source for 24 hours, resulting in a significant 15 to 20 percentage reduction in overall cost.

The other objective of the invention is to develop a method that improves feed conversion rates and efficiency, liver condition, growth performance, digestive enzyme activity, and the immune competency of aquaculture species.

The other objective of the invention is to develop a method that reduces the possibility of changing pathogenic bacteria into newly cultured ponds, ensuring a healthier environment.

Yet another objective of the invention is to develop a method that utilizes the bacteria capable of producing biofloc as an inoculum to ensure reproducible outcomes in biofloc formation.

Further objective of the invention is to a method for efficiently converting all uneaten materials, including feed, feces, and decomposed organic matter, into valuable nutritional biomass.
Summary of the invention:
The present disclosure proposes a method for standardization, identification, and production of uni-bacterial biofloc for aquaculture. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method for standardization, isolation, identification, and production of bacterium biofloc for controlled enhancement of water quality, nutrient utilization, and disease suppression in aquaculture.

According to an aspect, the invention provide a method for producing standardized bacteria biofloc for aquaculture. At one step, a single bacterial strain isolates with biofloc-forming and probiotic potential in an aquaculture pond environment. At another step, the isolated bacterial strain is cultures in a selective media under controlled conditions to generate a first-generation biofloc forming bacteria. In one embodiment herein, the controller conditions for the isolated bacterial strain are produced by cultivating the bacterial strain with at least 10 mL of the Imhoff cone for further processing. At another step, the first-generation biofloc forming bacteria is identifies and characterizes by using phenotypic and molecular methods.

Further, at another step, a second-generation biofloc is prepared by using the first-generation biofloc as inoculum and screening of isolated bacterial cultures from the first-generation biofloc for enhanced biofloc production. The method for improving aquaculture practices, and promoting sustainability, nutrient utilization, and more productive in aquaculture. In one embodiment herein, the production of bacteria is obtained by using the bacterial biofloc technology, which enhances environmental sustainability by converting uneaten material such as feed, feces, and decomposed organic matter into nutritional biomass.

In one embodiment herein, the method of isolating the single bacterial strain with the bio-floc forming and probiotic potential. At one step, the water sample and organic matter are collects from the aquaculture pond. At another step, the water sample is cultures on selective media to enrich for biofloc forming bacteria and incubating the culture under controlled conditions in an Imhoff cone. In one embodiment herein, the selective media is a biofloc standard medium (BSM), which includes 4 to 6 gm of peptone, 3 to 7 gm of sodium chloride, 1 to 2 gm of beef extract, 1 to 2 gm of yeast extract, 8 to 12 gm of glucose, and at least one litre of distilled water.

At another step, the biofloc forming bacteria is harvests after a defined growth period with an aeration pump, thereby obtaining the first-generation biofloc forming bacteria as inoculum. At another step, the first-generation biofloc forming bacteria is purifies through centrifugation and microscopic observation. Further, at another step, isolates individual colony forming unit (CFU) and identifies the bacterial exhibiting superior biofloc formation and probiotic potential using phenotypic and molecular methods.

In one embodiment herein, the method for characterization of the biofloc forming bacteria. At one step, the morphological analysis is performs using microscopy techniques for initial identification. At another step, the biochemical tests are configured to be conducts validate the identification and assessment probiotic potential. Further, at another step, the employs molecular techniques such as 16S ribosomal ribonucleic acid (rRNA) gene sequencing for definitive species identification.

In one embodiment herein, the production of second-generation biofloc forming bacteria using the first-generation biofloc forming bacteria as inoculum. At one step, the first-generation biofloc forming bacteria is incubates with a biofloc standard mineral water (BSMW). At another step, the biofloc forming bacteria is harvests to the defined growth period with the aeration, thereby obtaining the second-generation biofloc forming bacteria. At another step, the second-generation biofloc forming bacteria is purifies using centrifugation and microscopic observation.

At another step, the isolates the pure bacteria with the individual colony forming unit (CFU) and screening the isolated bacterial cultures from the second-generation biofloc forming bacteria. Further, at another step, the biofloc producing bacteria through identifies and preserves, and probiotic potential by using phenotypic, molecular, cultural, and molecular methods.

In one embodiment herein, the production of second-generation biofloc forming bacteria using isolated bacterial cultures from the first-generation biofloc forming bacteria. At one step, the isolated first-generation biofloc forming bacteria is prepares inoculum and cultivates the inoculum on a nutrient agar. At another step, the prepared inoculum is added to 0.5 to 3 percent of sodium chloride nutrient broth and grows until a specific turbidity level, thereby indicating activated bacterial formation.

At another step, the 10 to 15 mL of the activated bacterial inoculum to the BSM and BSMW medium in the Imhoff cone. In one embodiment herein, the biofloc standard mineral water (BSMW) for culturing biofloc-forming bacteria, which includes uncontaminated seawater with a salinity of 32-35 PPT (parts per thousand), distilled water, sodium bicarbonate (NaHCO3), and the biofloc standard medium (BSM).

At another step, the activated bacterial inoculum is incubated with aeration, thereby initiating the biofloc formation. At another step, the activated bacterial inoculum is purifies through centrifugation and microscopic observation. At another step, the pure bacteria is isolates from the activated bacterial inoculum with the individual CFU. Further, at another step, the identifies and preserves with the activated bacterial inoculum producing bacteria and probiotic potential by using phenotypic, molecular, cultural, and molecular methods.

Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

FIG. 1 illustrates a flowchart of a method for producing standardized bacterial biofloc for aquaculture, in accordance to an exemplary embodiment of the invention.

FIG. 2A illustrates a schematic view of an Imhoff cone with stand, in accordance to an exemplary embodiment of the invention.

FIG. 2B illustrates a schematic view of an aeration conduit, in accordance to an exemplary embodiment of the invention.

FIG. 2C illustrates a schematic view of a pipette tip attached to the aeration conduit, in accordance to an exemplary embodiment of the invention.

FIG. 3 illustrates a top view of an aeration pump, in accordance to an exemplary embodiment of the invention.

FIG. 4A illustrates a schematic view of the Imhoff cone with the biofloc, in accordance to an exemplary embodiment of the invention.

FIG. 4B illustrates a schematic view of the biofloc settled at a bottom of the Imhoff cone, in accordance to an exemplary embodiment of the invention.

FIGs. 5A-5D illustrate pictorial views of a biofloc at different magnifications of 4X, 10X, 40X, and 100X under a microscope, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a method for standardization, isolation, identification, and production of bacterium biofloc for controlled enhancement of water quality, nutrient utilization, and disease suppression in aquaculture.

According to an exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of a method for producing standardized bacterial biofloc for aquaculture. At step 102, a single bacterial strain is isolated with biofloc-forming and probiotic potential in an aquaculture pond environment. At step 104, the isolated bacterial strain is cultured in a selective media under controlled conditions to generate first-generation biofloc forming bacteria. In one embodiment herein, the controller conditions for the isolated bacterial strain are produced by cultivating the bacterial strain with at least 10 mL of an Imhoff cone 204 (as shown in FIG. 2A) for further processing. At step 106, the first-generation biofloc forming bacteria are identified and characterized by using phenotypic and molecular methods.

Further, at step 108, a second-generation biofloc forming bacteria are prepared by using the first-generation biofloc as an inoculum. The method improves aquaculture practices, promotes sustainability, nutrient utilization, and being more productive in the aquaculture. In one example embodiment herein, the production of bacteria is obtained by using the bacterial biofloc technology, which enhances environmental sustainability by converting uneaten material such as feed, feces, and decomposed organic matter into nutritional biomass.

In one embodiment herein, the established biofloc production system with a single and unique bacteria inoculum represents a promising and practical approach for the aquaculture industry. The novel method’s advantages, including reproducibility, independence from previous inoculums, preservability, biosecurity, environmental friendliness, and economic viability, collectively position it as a valuable contribution to sustainable aquaculture practices. Continued research and application of these findings have the potential to revolutionize sustainable aquaculture practices.

According to another exemplary embodiment of the invention, FIG. 2A refers to a schematic view 200 of an Imhoff cone 204 with a stand 202. In one embodiment herein, the Imhoff cone 204 is configured to be utilised in biofloc fish farming to determine the volume of settle-able solids in a specific volume of the water sample from the aquaculture pond environment. The Imhoff cone 204 is a transparent, cone-shaped structure marked with graduations and usually supported by the stand 202. The stand 202 is configured to support the Imhoff cone 204 during testing the biofloc of the aquaculture.

In one example embodiment herein, a user manually collects the water sample from the aquaculture pond to perform the biofloc technology. The Imhoff cone 204 having one-litre capacity to store the water sample from the aquaculture, which is made up of borosilicate glass and configured to evaluate the settling rate of suspended solids in water. The Imhoff cone 204 is washed and cleaned with chromic acid and sterilised in hot air at a temperature of 180°C for at least 42 min. The sterilisation process is configured to be washed on a glassware equipment with a tap or distilled water and cleaned with the chromic acid, then sterilised in the hot air oven at a temperature of180°C for at least 42 min. All the liquid and medium are sterilised in an autoclave at a temperature of 121°C at 15 lbs of pressure for at least 15 min.

In one embodiment herein, the Imhoff cone 204 comprises an aeration conduit 206, and a pipette tip 208. The aeration conduit 206 having a diameter of 2.5 mm and is configured to provide air into the Imhoff cone 204. The aeration conduit 206 of the one end is attached to the pipette tip 208 for transmitting air bubbles to the Imhoff cone 204. The biofloc technology is configured to improve feed conversion rate (FCR) compared to the control group.

According to another exemplary embodiment of the invention, FIG. 2B refers to a schematic view of the aeration conduit 206. In one embodiment herein, the one end of the aeration conduit 206 is attached to the pipette tip 208 (as depicted in FIG. 2C). In one embodiment herein, the aeration conduit 206 is made up of, but not limited to, plastic, flexible, and other composite materials. The other end of the aeration conduit 206 is attached to an aeration pump 302 (as depicted in FIG. 3).

According to another exemplary embodiment of the invention, FIG. 2C refers to a schematic view of the pipette tip 208 attached to the aeration conduit 206. In one embodiment herein, the other end of the pipette tip 208 is attached to the aeration conduit 206 to transfer the air bubbles within the Imhoff cone 204. The aeration conduit 206 having a supporting member 208 that is configured to stabilise the movement of the aeration conduit 206 within the Imhoff cone 204. In one example embodiment herein, the supporting member 208 is comparable to a spoke, which is positioned within a hollow structure of the aeration conduit 206.

According to another exemplary embodiment of the invention, FIG. 3 refers to a schematic view of the aeration pump 302. In one embodiment herein, the aeration pump 302 is configured to be operated by an electrical supply upon the user activating it. The aeration pump 302 is configured to remove chemicals from the aquaculture pond such as ammonia (NH3), chlorine (Cl), carbon dioxide (CO2), methane (CH4), hydrogen sulphide (H2S), iron (Fe), and manganese (Mn), which are effects of the ponds in aquaculture. The aeration pump 302 is configured to disperse oxygen throughout the pond. In one embodiment herein, the aeration pump 302 is configured to facilitate the settling of flocs at the bottom of the Imhoff cone 204. The user manually deactivates or stops the aeration pump 302 for at least 30 to 40 min for settling the floc in the bottom of the Imhoff cone 204, thereby measuring the amount of floc formed using a scale reading at the bottom of the Imhoff cone 204.

According to another exemplary embodiment of the invention, FIG. 4A refers to a schematic view 200 of the Imhoff cone 204 with the biofloc. According to another exemplary embodiment of the invention, FIG. 4B refers to a schematic view 200 of the biofloc settled at a bottom of the Imhoff cone 204. In one embodiment herein, the user manually collects the water sample from the aquaculture pond. The collected water samples were stored in a sterile manner. A container is rinsed 2 to 3 times with the pond water. The individual water samples were evaluated for salinity (1 to 30 PPT), and pH (7 to 8.4).

In another embodiment herein, the collected water sample were immediately sealed to prevent any external contamination and transferred to a laboratory for analysis. Different types of water analysis include physical analysis, chemical analysis, and microbial analysis. The physical analysis is configured to determine the parameters of the water sample such as temperature, colour, odour, pH, turbidity, salinity, conductivity, and total dissolved solids. The chemical parameters of the water sample are total hardness, calcium hardness, magnesium hardness, ammonia, nitrite, sulphate, and phosphate content. The first initial viable bacterial count was determined by routine microbiological procedures using different media, including 0.5 to 3 percent of sodium chloride nutrient agar and thiosulfate citrate bile sucrose (TCBS) agar plates.

In one embodiment herein, the method for preparing standardized bacteria biofloc for aquaculture through the production of first-generation biofloc forming bacteria. In one embodiment herein, the method of production of first-generation biofloc forming bacteria. At one step, the user manually collects the water sample from the one litre of water source into a sterilised Imhoff cone 204. Next, the user adds 25 to 30 mL of biofloc standard medium (BSM). The BSM is a selective medium that includes 4 to 6 gm of peptone, 3 to 7 gm of sodium chloride, 1 to 2 gm of beef extract, 1 to 2 gm of yeast extract, 8 to 12 gm of glucose, and at least one litre of distilled water. Next, every 12 to 24 hr water sample are examined for floc formation. The observation involved stopping the aeration for at least 30 min to allow the floc to settle, and then measuring the amount of floc generated by using the scale provided at the bottom of the Imhoff cones 204.

Next, harvest the biofloc to reach above 10 mL from the bottom of the Imhoff cones 204. If the quantity of biofloc is less than 10 mL, incubate with aeration until the biofloc until it exceeds greater than 10 mL by adding 25 to 30 mL of BSM. During the incubation period, light will not be provided to the experimental setup in order to prevent the growth of photoautotrophic organisms. The aeration-based biofloc harvesting was halted for at least 30 minutes to allow the biofloc to settle.

After the settlement, the water was discarded and the floc was collected in a sterilised container depending on the volume of the floc formed, thereby collecting the biofloc is a first-generation biofloc forming bacteria is defined as the inoculum. Next, centrifuge the first-generation biofloc forming bacteria for at least 15 to 30 min at 3000 to 4000 rpm. After centrifugation, the water is removed and the same amount of sterile saline water (0.9 percent sodium chloride) is added to wash the biofloc properly twice. Next, the biofloc with bacteria is examined microscopically at different magnifications.

According to another exemplary embodiment of the invention, FIGs. 5A-5B refer to pictorial views (502, 504, 506, 508) of the biofloc at different magnifications of 4X, 10X, 40X, and 100X under a microscope. In one embodiment herein, the microscopic observation under 4X magnification, the floc appeared as tiny specks or dots distributed. A floc matrix is composed of extracellular polymeric structures that produce microbial capsules. Initially, the first-generation biofloc forming bacteria extends beyond bacteria, which includes microalgae, protozoa, nematodes, copepods, rotifers, diatoms, polychaetes, and detritus. This level of magnification provides a broad picture of the spread of bacteria in the sample.

Referring to FIG. 5B, microscopic observation of the biofloc at 10X magnification. The individual bacteria are indistinguishable as little, transparent specks. However, some fundamental shapes and groupings, such as clusters or chains, are discernible. Larger bacterial aggregations are visible, allowing for differentiation between different types based on size and grouping patterns. Detritus might appear as an amorphous, brownish substance.

Referring to FIG. 5C, microscopic observation of the biofloc at 40X magnification. The individual components of the biofloc are formed in more detail. Microalgae cells might be clearly visible and capable of distinguishing between species based on shape, size, and colour. The bacteria in the floc will be more easily distinguished, or other appendages. Detritus might show more texture and composition.

Referring to FIG. 5D, microscopic observation of the biofloc at 100X magnification. Under the highest magnification, the individual bacteria become distinguishable in greater detail. The bacteria are identified as small, rod-shaped, or spherical cells with distinct features. At this level, magnification allows for the potential identification of specific bacterial species based on the morphological characteristics. The increased magnification allows for clear and comprehensive morphological observations of bacterial cells. In one embodiment herein, the microscopic observation of the biofloc with bacteria is further tested using a microbiological analysis.

In one embodiment herein, the microbiological analysis is performed on the 10 mL of biofloc sample and subjected to centrifugation at 3000 to 4000 rpm for at least 15 to 30 min. The water (supernatant) is removed from the Imhoff cone 204, and the pellet (sediment) is washed with sterile saline water for at least two to three times. Next, add the 10 to 15 mL of the sterile saline water with the biofloc to form a mixture that is defined as a bacterial suspension. The bacterial suspension is serially dilution at 50 to 100 µl of the highest dilutions and disseminated on 0.5 to 3 percentage of sodium chloride nutrient agar and thiosulfate–citrate–bile salts–sucrose agar, or TCBS agar, which is a type of selective agar culture plate that is used in microbiology laboratories to isolate vibrio species into individual colony-forming units (CFU) to allow the bacterial colonies to grow. Next, incubate the sodium chloride nutrient agar, and TCBS agar plates are incubated at a temperature of 32 to 36 °C for at least 24 to 48 hr.

Next, the isolate a pure biofloc producing bacteria, distinct colonies were isolated, and pure cultures were obtained by routine microbiological techniques such as the serial dilution and spread plate techniques. The individual colonies are purified by using a quaternary streaking method for at least one or two times on nutrient agar plates to obtain a separate pure culture, considered as the second-generation forming bacteria. The pure cultures obtained from the first-generation forming bacteria were evaluated for biofloc property individually. All isolated bacteria were streaked on 1 to 3 percent of salt nutrient agar plates, stored with 20 percent of (w/v) glycerol, and preserved at a temperature of -20°C.

Next, the biofloc forming bacteria are identified by phenotypic characters such as colony morphology, cell morphology, motility, endospore-forming ability, and Gram’s reactions. Biochemical tests such as string test, catalase test, oxidase test, IMViC test, amino acid decarboxylase test, and carbohydrate fermentation test. Cultural characters such as growth on Congo red agar, bacillus selective media, hemolysin activity, amylase and protease activity were recorded for proper identification of bacteria and metabolic activity. Microbial identification is identified using molecular techniques such as 16S ribosomal ribonucleic acid (rRNA) gene sequencing.

In one embodiment herein, the production of second-generation biofloc from the first-generation floc as the inoculum is stabilised into two methods, which are production of second-generation biofloc using the first-generation biofloc as inoculum, and screening of biofloc production of isolated bacterial cultures obtained from first-generation biofloc forming bacteria.

In one embodiment herein, the preparation of biofloc standard mineral water (BSMW) for clean, uncontaminated sea water (32 to 35 PPT), and diluted with distilled water to obtain 1PPT (parts per thousand) salinity. Salinity is measured with a Refractometer and Digital Salinity meter. The alkalinity of the 1PPT water was adjusted to 160 to 200 ppm with sodium bicarbonate. Next, add 20 to 25 mL of BSM to the BSMW and sterilise by autoclaving at a temperature of 121 °C at 15 lbs of pressure for at least 15 min.

In another embodiment herein, the production of second-generation biofloc forming bacteria using first-generation biofloc as inoculum. At one step, incubate the first-generation biofloc with a biofloc standard mineral water (BSMW) by introducing 10 mL of first-generation floc forming bacteria from the Imhoff cone 204, and incubate for at least 2 to 3 days by adding one litre of BSMW and 25 to 30 mL of BSM with sufficient aeration. Next, harvest the biofloc during the designated growth period using aeration, thereby obtaining the second-generation biofloc forming bacteria formation at 10 to 15 mL for microbiological analysis. Next, the first-generation biofloc forming bacteria is purified using centrifugation and microscopic observation. Next, isolate the pure bacteria with CFU and screen the isolated bacterial cultures from the second-generation biofloc forming bacteria. Furthermore, identifies and preserves the biofloc producing bacteria and probiotic potential by using phenotypic, molecular, cultural, and molecular methods.

In one embodiment herein, the production of second-generation biofloc by isolating bacterial cultures from the first-generation biofloc. At one step, prepare the inoculum from the isolated first-generation biofloc and cultivate the inoculum on the nutrient agar. A loopful of 15 to 18 hours fresh culture is mixed with 10 to 15 mL of 0.5 to 3 percent of sodium chloride nutrient broth. The culture is agitated until the turbidity reaches a specified range of 0.5 nm to 0.7 nm at 600 nm, thereby indicating bacterial growth and activity. The activated bacterium is defined as an inoculum for initiating biofloc formation.

Next, add 10 to 15 mL of activated bacterial inoculum from the Imhoff cones 204 and incubate for at least 2 to 3 days by adding 1L of BSMW/source water and 25 to 30 ml of BSM under adequate aeration. Next, the activated bacterial inoculum is incubated with aeration, thereby initiating biofloc formation through the observation of floc formation. After 24 hr of observation, the biofloc (10 to 15 mL) is acceptable for microbiological analysis.

Next, purify the activated bacterial inoculum through centrifugation and microscopic observation. Next, isolate pure bacteria from the activated bacterial inoculum with the individual CFU and screening the isolated bacterial cultures from the activated bacterial inoculum. Furthermore, identifies and preserves the biofloc-producing bacteria and probiotic potential by using phenotypic, molecular, cultural, and molecular methods.

In one embodiment herein, the measurement of biomass of biofloc. Initial weight of harvested biofloc is weighed and noted. Drying the biofloc in a hot air oven at a temperature of 60 °C for overnight. After drying the biofloc again weighed and noted as dry weight. The obtained biomass of the biofloc is measured by the dry weight of the biofloc by the wet weight of the biofloc. The biomass was calculated using the formula:
Biomass(mg/L)= (Dry weight)/(Wet weight)

Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, a method that eliminates the requirement for inoculums from previous crops, providing the advantage of preservation for future usage. The proposed method provides better control over heterotrophic organisms than autotrophic ones, requiring a small amount for implantation. The proposed method improves feed conversion rates and efficiency, liver condition, growth performance, digestive enzyme activity, and the immune competency of aquaculture species.

The proposed method allows continuous access to biofloc as a nutrient-rich food source for 24 hours, resulting in a significant 15 to 20 percent of reduction in overall cost. The proposed method reduces the possibility of changing pathogenic bacteria into newly cultured ponds, ensuring a healthier environment. The proposed method utilizes the bacteria capable of producing biofloc as an inoculum to ensure reproducible outcomes in biofloc formation. The proposed method for efficiently converting all uneaten materials, including feed, feces, and decomposed organic matter, into valuable nutritional biomass.

It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
, Claims:CLAIMS:
I/We Claim:
1. A method for preparing standardized bacteria biofloc for aquaculture, comprising:
isolating a single bacterial strain with biofloc forming and probiotic potential in an aquaculture pond environment;
culturing the isolated bacterial strain in a selective media under controlled conditions to generate a first-generation biofloc forming bacteria;
identifying and characterising the first-generation biofloc forming bacteria using phenotypic and molecular methods; and
preparing a second-generation biofloc forming bacteria by using the first-generation biofloc forming bacteria as inoculum,
whereby the method improves aquaculture practices and promotes sustainability, nutrient utilization, and productive in the aquaculture.
2. The method as claimed in claim 1, wherein a method of isolating the single bacterial strain with the biofloc-forming and probiotic potential comprises:
collecting water samples and organic matter from the aquaculture pond environment;
culturing the water samples on the selective media to enrich for biofloc forming bacteria and incubating it under the controlled conditions in an Imhoff cone (204);
harvesting the biofloc forming bacteria after a defined growth period upon deactivating or stopping an aeration pump (302), thereby obtaining the first-generation biofloc forming bacteria as inoculum;
purifying the first-generation biofloc forming bacteria through centrifugation and microscopic observation; and
isolating individual colony forming unit (CFU) and identifying the bacterial exhibiting superior biofloc formation and probiotic potential using phenotypic and molecular methods.
3. The method as claimed in claim 2, wherein the selective media is a biofloc standard medium (BSM), which includes 4 to 6 gm of peptone, 3 to 7 gm of sodium chloride, 1 to 2 gm of beef extract, 1 to 2 gm of yeast extract, 8 to 12 gm of glucose, and at least one litre of distilled water.
4. The method as claimed in claim 1, wherein the method for characterisation of the biofloc forming bacteria comprises:
performing a morphological analysis using microscopy techniques for initial identification;
conducting biochemical tests to validate the identification and asses probiotic potential; and
employing molecular techniques such as 16S ribosomal ribonucleic acid (rRNA) gene sequencing for definitive species identification.
5. The method as claimed in claim 1, wherein the production of the second-generation biofloc forming bacteria using the first-generation biofloc as inoculum comprises:
incubating the first-generation biofloc forming bacteria with a biofloc standard mineral water (BSMW);
harvesting the first-generation biofloc forming bacteria to the defined growth period with upon deactivating or stopping the aeration, thereby obtaining the second-generation biofloc forming bacteria;
purifying the second-generation biofloc forming bacteria using centrifugation and microscopic observation;
isolating pure bacteria with the individual colony forming unit (CFU) and screening the isolated bacterial cultures from the second-generation biofloc; and
identifying and perseverating the biofloc producing bacteria and probiotic potential by using phenotypic, molecular, cultural, and molecular methods.
6. The method as claimed in claim 1, wherein the production of second-generation biofloc forming bacteria using isolated bacterial cultures from the first-generation biofloc comprises:
preparing inoculum from the isolated first-generation biofloc forming bacteria and cultivating the inoculum on a nutrient agar;
adding 0.5 to 3 percent of sodium chloride nutrient broth and growing until a specific turbidity level, thereby indicating activated bacterial formation;
adding 10 to 15 mL of activated bacterial inoculum to the BSM and BSMW medium in the Imhoff cone (204);
incubating the activated bacterial inoculum with aeration, thereby initiating the biofloc formation, and purifying the activated bacterial inoculum through centrifugation and microscopic observation;
isolating pure bacteria from the activated bacterial inoculum with the individual CFU and screening the isolated bacterial cultures from the activated bacterial inoculum; and
identifying and perseverating with the activated bacterial inoculum producing bacteria and probiotic potential by using phenotypic, molecular, cultural, and molecular methods.
7. The method as claimed in claim 6, wherein the biofloc standard mineral water (BSMW) for culturing biofloc-forming bacteria, which includes uncontaminated seawater with a salinity of 32-35 PPT (parts per thousand), distilled water, sodium bicarbonate (NaHCO3), and the biofloc standard medium (BSM).
8. The method as claimed in claim 1, wherein the production of bacteria is obtained by using the bacterial biofloc technology, which enhances environmental sustainability by converting uneaten material such as feed, feces, and decomposed organic matter into nutritional biomass.
9. The method as claimed in claim 1, wherein the controller conditions for the isolated bacterial strain are produced by cultivating the bacterial strain with at least 10 mL of the Imhoff cone (204) for further processing.