TWI826421B - Method for improving quality of aquaculture pond water using a nutrient germinant composition and bacteria spore - Google Patents

Abstract

A method for improving the quality of pond water used in aquaculture applications by adding to the pond water active bacteria that are preferably germinated from spores on site using a nutrient-germinant composition and an incubation method for increased spore germination efficiency, in combination with a nitrification enhancement agent such as calcium carbonate or calcified seaweed, and an optional reaction surface area modifier such as calcified seaweed or plastic or metal particles or fragments. The nutrient-germinant composition comprises L-amino acids, D-glucose and/ or D-fructose, a phosphate buffer, an industrial preservative, and may include bacteria spores (preferably of one or more Bacillus species) or they may be separately combined for germination. The incubation method comprises heating a nutrient germinant composition and bacteria spores, to a temperature range of 35℃ to 60℃ for around 2 to 60 minutes to produce an incubated bacteria solution that is discharged to the aquaculture application.

Description

Methods for improving aquaculture pond water quality using nutritional germination factor compositions and bacterial spores

The present invention relates to bacteria germinated in nutritional germination factor compositions and the use of point-of-use spore culture to treat aquaculture pond water to reduce organic waste, ammonia and disease pressure in aquatic livestock applications and for aquatic products. Cultured species provide probiotics.

Aquaculture refers to the raising of aquatic species for use as a source of food for humans or animals. This technology applies some type of control to the natural environment of the species being raised to improve overall yields. This can include hatching species in captivity in the wild to increase commercial production of animals, hatching and rearing species in enclosures, and hatching and rearing species in enclosed areas adjacent to tidal discharges from the shoreline. Problems associated with this approach include: pollutants discharged from the breeding facility that will degrade the quality of nearby water; Product losses; and disease pressure accompanying increased pathogenic microorganisms in feeding facilities. These issues can be identified by testing or monitoring various parameters, including pH, conductivity, ammonia, nitrates, phosphates and alkalinity. Conductivity is an indicator of salt content. If the amount is greater than 1200ppm, it is no longer considered fresh water; the ideal amount is 700ppm and the range of 300-1200ppm. Ammonia levels measure the amount of oxygen available to fish. High amounts of ammonia prevent the transfer of oxygen from the gills to the blood in fish; however, it is also a waste product of their metabolism. Although ammonia from fish waste is usually not concentrated enough to be toxic on its own, fish keepers must closely monitor ammonia levels because of the high concentration of fish in each tank. Oxygen is consumed by nitrifying bacteria in the pond (which break down toxic ammonia into non-toxic forms); however, this heavy use of oxygen reduces the oxygen available for uptake by the fish. Ammonia levels >1ppm are considered toxic to fish survival. Additionally, test nitrate levels to determine the amount of plant fertilizer in the water. Nitrates are highly leachable from surrounding soil and can be harmful to children and pregnant women. Nitrates become nitrites in the gastrointestinal tract and interact with the blood's ability to carry oxygen. The maximum contamination level of nitrate is 10ppm. Alkalinity measures the ability of a pool or lake to neutralize acids without changing the pH. Alkalinity will decrease over time due to bacteria; however, the ideal level is 100ppm, with an acceptable range of 50-200ppm. Most of the phosphates found in ponds and lakes come from human and animal waste. Fertilizer run-off is a major source of phosphates found on golf courses and ornamental ponds. Elevated levels lead to increased eutrophication rates, which in turn increase sludge production. Moderate phosphate levels can promote plant growth, leading to increased algae production; levels >0.1 ppm are an indicator of accelerated plant growth and are considered beyond acceptable levels.

Current technologies to solve these problems include bioremediation, antibiotics and chemical additives. Typical bioremediation techniques involve the application of supplemental bacteria to water to enhance microbial activity and improve water quality. It is also known to use nitrifiers to enhance the nitrification process to convert toxic ammonia into non-toxic nitrates. Chemical additives are added to improve water quality and aid microbial activity by providing additional nutrients and alkalinity. Antibiotics are added to inhibit the growth of pathogenic microorganisms. Problems associated with current technology include the high cost and poor water quality improvement performance of using inactive supplemental bacteria, low nitrification activity due to the presence of organic waste and lack of nitrifying growth sites, and the use of antibiotics in reared animals. Bioaccumulation in aquatic species.

According to a preferred method disclosed in US Patent Application No. 14/720,088, active bacteria can be produced on-site using a biogenerator, and bacteria can be grown into useful populations from solid bacterial starting materials. The live bacteria can then be discharged from one or more biogenerators into aquaculture applications. For example, U.S. Patent Nos. 6,335,191, 7,081,361, 7,635,587, 8,093,040, and 8,551,762 disclose such biogenerators and methods of using them, the contents of which are incorporated into this disclosure by reference. However, it would be useful to have an alternative method of producing viable bacteria from spores when used in aquaculture applications.

Spore germination is a multi-step process in which spores effectively awaken or return from dormancy to a state of vegetative growth. The first step is to activate the spores and induce their germination by environmental signals called germination factors. This signal is a nutrient, such as L-amino acids. Nutritional germination factors bind to Receptors in the inner membrane of the spore initiate germination. Furthermore, sugars have been shown to increase the binding affinity of L-amino acids to their cognate receptors.

The germination factor signal then initiates a cascade that results in the release of dipicolinic acid (DPA), which is stored in the spore core in a 1:1 ratio with Ca ²⁺ (CaDPA). The release of CaDPA is a rapid process, usually >90% complete within 2 minutes. CaDPA release represents the point of no return at which spores commit themselves to the germination process. Those knowledgeable in the art call this step the "commitment" step.

Following CaDPA release, the spores are locally hydrated and the core pH rises to approximately 8.0. The core of the spore then swells and the cortex (mainly composed of peptidoglycan) is degraded by core-lytic enzymes. The spores absorb water and thus lose their refractive index. This loss of refractive index until the end of the germination process allows monitoring of spore germination via phase contrast microscopy.

The second phase of germination is the outgrowth step, in which the spore's metabolic, biosynthetic, and DNA replication/repair pathways begin. There are several phases during outgrowth. The first is called the maturation phase, in which no morphological changes (such as cell growth) occur, but the molecular machinery of the spore (such as transcription factors, translation machinery, biosynthetic machinery, etc.) is activated. The length of this period can vary depending on the initial resources used during sporulation and spore packaging. For example, the preferred carbon source for some Bacillus species (including Bacillus subtilis) is malate, and Bacillus spores usually contain a large pool of malate, which is used in the recovery process. . Interestingly, deletion mutations that are unable to exploit the malate pool exhibit extended maturation, suggesting that the spore malate pool is sufficient to fuel the initial outgrowth process. quantity. In addition, spores store small acid-soluble proteins that are degraded within the first few minutes of recovery and serve as an immediate source of amino acids for protein synthesis. After the outgrowth step, the spores are fully recovered and the cells are considered to be growing vegetatively.

It is known that spore germination can be induced via thermal activation. Spores of various Bacillus species have been heat activated at strain-specific temperatures. For example, B. subtilis spores are heat activated at 75°C for 30 minutes, and B. licheniformis spores are heat activated at 65°C for 20 minutes. Thermal activation has been shown to cause transient and reversible unfolding of spore coat proteins. Heat-activated spores can germinate for longer periods of time in a germination buffer (containing nutritional germination factors such as L-alanine). However, if no nutritional germination factors are present, the spores will return to their pre-heating non-germinated state.

It is also known that germination can occur at ambient temperature (near typical room temperature) without heat activation and with a germination buffer containing nutrients, but this process generally takes longer than with heat activation. For example, Bacillus licheniformis ( B.licheniformis ) and Bacillus subtilis ( B.subtilis ) spores germinate at 35°C or 37°C respectively, but require a longer time (for example, 2 hours) in a germination buffer containing nutritional germination factors. . Furthermore, non-thermally activated spores of B. subtilis are known to germinate for an extended period of time in non-nutritional germination factor conditions (eg CaCl ₂ +Na ₂ DPA).

It is also known to germinate spores in a two-step process using a combination of thermal activation and nutritional germination factors in laboratory setups. The spores are first thermally activated by incubating them at 65-75°C for a period of time (e.g. 30 minutes). Specific temperatures depend on the species). The spores are then transferred to a buffer solution containing nutritional germination factors such as L-alanine. It is also known to grow granular nutrients (containing sugar, yeast, etc.) extract, and other nutrients that are not direct spore germination factors), bacterial starter (bacteria starter) and water are fed into the growth chamber to cultivate the bacteria as disclosed in U.S. Patent No. 7,081,361. The growth period is about 24 hours.

There is a need for rapid spore culture and activation methods that will allow the generation of viable bacteria (eg, Bacillus species) in a single step at a point-of-use location where the bacteria will be discharged into aquaculture applications. Accordingly, the present invention describes a simple method for spore germination in aquaculture applications using a nutritional germination factor concentrate in combination with a spore composition, or using a nutritional spore composition with simultaneous thermal incubation in a single step.

The method of the present invention provides a cost-effective method to deliver active bacteria to pond water (or growth ponds) in aquaculture facilities to degrade organic waste and inhibit the growth of pathogenic microorganisms without bioaccumulation. The method of the present invention reduces disease pressure in aquatic livestock, resulting in improved harvest of species raised in aquaculture operations. Adding a nitrification enhancer as described herein as part of this method provides a stable source of alkalinity and additional growth sites for the nitrifier to promote nitrification activity and ammonia reduction.

The method of the present invention desirably involves delivering live bacteria to aquaculture applications, optionally including probiotic bacteria, preferably produced from an on-site incubator using a liquid nutritional germination factor concentrate and the bacteria in spore form. A nutritional germination factor composition according to a preferred embodiment of the present invention includes one or a combination of a number of L-amino acids, optionally D-glucose (which increases the effect of L-amino acids on their cognate species in the spore coat). binding affinity of the source receptor), and neutral buffers, such as phosphate buffer, and industrial preservatives, such as the commercially available Kathon/Lingaurd CG (which has properties including methyl chloro isothiazolinone ) and methyl isothiazolinone (the active ingredient of methyl isothiazolinone). According to another preferred embodiment of the present invention, the nutritional germination factor composition includes one or a combination of two or more L-amino acids, optionally D-glucose (which increases the effect of L-amino acids on the spore shell). their cognate receptor binding affinities), HEPES solution salts (biological buffers that provide appropriate pH for spore germination), and industrial preservatives such as propylparaben and methylparaben (methylparaben) or other U.S. federal GRAS (Generally Regarded As Safe) preservatives. According to another preferred embodiment, the spore composition also includes a source of potassium ions, such as potassium chloride or potassium dihydrogen phosphate or dipotassium hydrogen phosphate. According to another preferred embodiment, the spore composition contains both D-glucose and D-fructose.

According to another preferred embodiment, the nutritional germination factor composition also includes spores of one or more bacterial species, preferably Bacillus species, but other bacteria can also be used, and includes germination inhibitors (e.g. NaCl, industrial preservatives, or D-alanine), combined with any one of the aforementioned spore composition components. Germination inhibitors prevent premature germination of spores in a nutritional germination factor composition. Germination inhibitors may include chemicals that prevent spore germination, such as NaCl, industrial preservatives, or D-alanine.

Alternatively, bacterial spores can be provided separately and added to the vegetative-germination factor composition according to the invention at the point of use and at the time of cultivation. When added separately, it is preferred to provide a stable spore suspension. The spore composition includes one or more bacterial species, preferably Bacillus species. According to a preferred embodiment, the spore composition includes bacterial spores, about 0.00005 to 3.0% by weight of surfactant, about 0.002 to 5.0% by weight of thickening agent, and optionally about 0.01 to 2.0% by weight of acidifying agent, Acid or acid salts (including those acting as preservatives or stabilizers), the balance being water. According to another preferred embodiment, the spore composition includes bacterial spores, about 0.1 to 5.0% by weight of thickening agent, about 0.05 to 0.5% by weight of acid or acid salts, optionally about 0.1 to 20% by weight of Water activity reducing agent, and optionally about 0.1% to 20% additional acidulant (acid or acid salt), the balance being water.

Optimally, the bacterial spores in the two preferred spore composition embodiments are a dry powder mixture of 40-60% salt (table salt) and 60-40% bacterial spores (before adding to the spore composition), which The combination represents about 0.1 to 10% by weight of the spore composition. The spore composition preferably includes about 1.0×10 ⁸ to about 3.0×10 ⁸ cfu/ml of spore composition (spore suspension) when diluted with drinking water (for animal sprinkler applications) in drinking water. Bacterial strains are provided in approximately 10 ⁴ to 10 ⁶ cfu/ml. Optimally, the thickening agent in both preferred embodiments also acts as a prebiotic (such as sanxianjiao) to provide additional benefits. Although other commercially available spore products may be used, preferred spore compositions for use in the present invention are those disclosed in U.S. Patent Application No. 14/524,858, filed on October 27, 2014. Incorporated herein by reference.

According to another preferred embodiment, the nutritional germination factor composition according to the present invention is in concentrated form and is diluted in water or another diluent to a concentration of 0.01% to 10% at the point of use. The use of concentrated formulations reduces shipping, storage and packaging costs and makes it easier to administer the spore composition at the point of use. Optimally, the concentrated spore composition is in liquid form, which is easier and faster to mix with the diluent at the point of use, but solid forms such as pellets or bricks or powders may also be used. The inclusion of common industrial preservatives in the spore composition aids in long-term storage and/or germination inhibition, which is particularly useful when the spore composition is preferably in a concentrated form.

In another preferred embodiment, the present invention includes a method for germination of spores of Bacillus sp. using a nutritional germination factor composition in combination with a spore composition or at an elevated temperature (preferably 35-60°C). In the range, more preferably in the range of 38-50°C, and most preferably in the range of 41°C to 44°C) the vegetative spore composition is used for a period of time (during the cultivation period). The incubation period preferably ranges from 2-60 minutes, or longer, depending on the application. Optimally, the concentrated form of the nutritional-germination factor composition or the nutritional spore composition according to the preferred embodiment of the present invention is used in the cultivation/germination method of the present invention, but other nutritional-germination factors can also be used. Composition and spore composition. Preferably, the cultivation method is carried out at or near a point of use (the point of use is an aquaculture location where the germinated spores will be used or consumed. located at or near an aquaculture location) and further includes dispersing the germinated spores to the point of use/consumption. The preferred method of the present invention can be carried out in any cultivation device with a composition capable of containing a volume of spores (if added separately), liquid (usually water as diluent), nutritional germination factors, and The reservoir of the mixture is heated during the incubation period. Optimally, the method is carried out in a device that can mix the ingredients, automatically switch off the heating at the end of the incubation period and disperse the incubated bacterial solution including the bacteria to the point of aquaculture use/consumption. Preferred methods can also be carried out in batch processes or continuous processes. Although it is preferred to use spore compositions according to the invention, various spore forms or products (eg, dry powder forms, liquid suspensions, or reconstituted aqueous mixtures) may be used with the methods of the invention.

Preferred embodiments of the present invention allow rapid germination of spores of Bacillus species at the point of use in aquaculture. Active vegetative bacteria discharged from the incubator may be provided directly to the growth tank, or may be accumulated and diluted with pond water or another similarly suitable diluent (eg, water from a municipal water system) before discharge to the growth tank. Alternatively, the incubator may be configured to heat the vegetative germination factor composition and spores, or the vegetative spore composition, for the incubation period and temperature range that will produce bacteria in a metastable state between dormant spores and vegetative bacteria. The metastable bacterial solution is then discharged into a growth tank where the bacteria can become active vegetative bacteria. If the incubator is some distance away from the growth tank, dilution may help deliver the treatment solution from the incubator to the growth tank. Active bacteria will degrade organic waste and inhibit the growth of pathogenic microorganisms in the water in aquaculture facilities without the need to add (or subtract) Small amounts) chemical treatments and antibiotics used in grow tanks.

The present invention also desirably includes the simultaneous application of at least one nitrification enhancer to the growth tank. Nitrification enhancers increase the activity of nitrifying bacteria found naturally in water to reduce ammonia levels. Since nitrifying bacteria grow as biofilms and need a surface to which they can adhere, these nitrifying agents include alkalinity enhancers, which increase the alkalinity of the water, which is nitrification (7 parts alkalinity to one part ammonia) and/or Surface area modifiers are needed to increase the surface area on which nitrifying bacteria can grow. Alkalinity enhancers may include, for example, calcium carbonate, calcified seaweed, or other similar effective additives. These reagents can be added in higher than dissolved amounts to provide a continued source of alkalinity as they slowly dissolve. Some nitrifiers (such as calcifying seaweed) act as both alkalinity enhancers and surface area modifiers by providing alkalinity and high surface area, providing a supporting surface for the nitrifier's biofilm to grow. Calcified seaweed also serves as a source of trace amounts of bacteria. Other nitrification enhancers simply act as surface area modifiers, such as plastic or metal sheets, or other similarly effective materials, to increase the surface area where favorable reactions and interactions can occur. One or more agents that act solely as surface area modifiers (rather than as alkalinity enhancers) may also be added to the growth tank alone or preferably in combination with one or more alkalinity enhancers; however, agents that act solely as surface area enhancers are not required in the growth tank. does not degrade and is not added with each batch of bacterial solution. Such reagents that act solely as surface area modifiers are preferably added to the growth cell only once. On a periodic basis, such as seasonally (once per season or once every summer, twice a year, etc.) or if necessary, agents acting as alkalinity enhancers are added to the growth cells simultaneously with the batch of bacteria. As used herein, the term "simultaneously" means "at or about" time a batch of nutritional bacteria and other ingredients or reagents added to growth tanks or other growth media in which aquatic species are grown in aquaculture facilities.

10: On-site incubator system

12: Nutritional germination factor composition

14: Spore composition

16:Source of water

18: Breeder

20: Cultured bacterial solution

22:Growing pond

110: Breeder system

24: Concentrated nutritional germination factor composition

26:Water/Thinner

28: Work nutritional germination factor composition

30: Concentrated spore composition

32: Working spore composition

210:System

34: Concentrated nutritional spore composition

36: Working nutritional spore composition

The systems and methods of the present invention are further described and illustrated in the following figures.

Figure 1 is a flow chart of a cultivation system and method according to a preferred embodiment of the present invention.

Figure 2 is a flow chart of a cultivation system and method according to another preferred embodiment of the present invention.

Figure 3 is a flow chart of a cultivation system and method according to another preferred embodiment of the present invention.

Figure 4 is a graphical representation of nitrate levels in laboratory studies.

Figure 5 is a graphical representation of orthophosphate levels in laboratory studies.

Figure 6 is a graphical representation of turbidity in laboratory studies.

Figure 7 is a photograph showing bacterial slides using spore compositions and methods according to preferred embodiments of the present invention compared to control slides.

Figure 8 is a graph showing _pO2 test data to demonstrate the degree of germination using spore compositions and methods according to preferred embodiments of the present invention compared to control group testing.

Figure 9 is a graph showing _pO2 test data to demonstrate the degree of germination using spore compositions and varying methods according to preferred embodiments of the present invention compared to control group testing.

Figure 10 shows images of three aquariums, the control group (left), only Calcium carbonate treatment (middle), or treatment with activated vegetative-spore formula (right).

Aquaculture treatment methods

According to a preferred embodiment, it is preferred to use an incubator system with a preferred germination method as described below, from a vegetative germination factor composition combined with a spore composition or from a premixed vegetative spore composition, on-site ( live bacteria are produced on site) and are periodically fed into growth tanks in aquaculture applications. One or more nitrification enhancers are also added simultaneously to the growth tank with active bacteria.

Suitable bacterial growth and delivery devices for use in the methods of the present invention will include an in-situ incubator system, such as an air incubator, a water incubator, or any other chamber or similar device that provides uniform constant heat when needed. Spores are germinated within a given temperature range for discharge into aquaculture applications. Referring to Figure 1, it is preferred that the field incubator system 10 includes one or more tanks or containers for containing the initial volume of the vegetative germination factor composition 12 and the bacterial spore solution 14 (if the bacterial spores are not included in the vegetative germination factor composition) initial volume. These spore compositions may also arrive at the point of use in a container that is fluidly connected to the incubator, in which case a separate tank or container is not required. A source of water 16 at or near the aquaculture location is also optionally, but preferably fluidly connected to the incubator system. A well, municipal water supply, or growing pond can be used to feed the incubator 18 Provide water16. The incubator system 10 also preferably includes a chamber or container 18 configured to receive a portion of the nutrient germination factor composition and spores and allow them to be heated to germinate the spores; a heater; valves, tubing, and pumps (as needed) , if gravity flow is insufficient) allows the vegetative germination factor composition 12, bacterial spore solution 14 and optional water 16 to flow from their storage containers/sources into the addition chamber or container 18 and the cultured bacteria (or activated of bacteria) solution 20 is drained from the heating chamber or container 18 and delivered to the growth tank 22; an optional mixer within the heating chamber or container 18, and a controller or timer to activate the valve, pump , optional mixers, and heaters to control the flow of nutrients and spore composition into the heated chamber, incubation time and temperature, and discharge to the growth tank. Optimally, the field incubator system 10 uses a vegetative germination factor composition in combination with a bacterial spore composition (as described below) or uses a vegetative spore composition (a vegetative germination factor composition premixed with bacterial spores, also as follows) The) (also referred to as "starter material" herein) is discharged into the growth tank 22 to produce the cultured bacterial container 20 .

Alternatively, according to another preferred embodiment shown in Figure 2, the incubator system 110 uses a concentrated vegetative germination factor composition 24 and a concentrated spore composition 30, which are diluted with diluent or water from the container/source 26 To form the working vegetative germination factor composition 28 and the working spore composition 32, a portion of each is fed into the incubator 18 to produce a batch of activated bacteria 20. Water from source 16 may also be used as a source of diluent instead of or in addition to source 26 . Additionally, only one vegetative germination factor composition 24 or spore composition 30 may be in concentrated form and fed into the culture Need to be diluted before incubation 18. According to another preferred embodiment, when only one is concentrated, the non-concentrated composition can be used as a diluent for the concentrated composition in addition to the water/diluent from source 26 and/or source 16 . According to another preferred embodiment, a concentrated vegetative spore composition 34 is used with system 210, as shown in Figure 3. The batch of activated bacteria 20 is produced by diluting with water/diluent from source 26 and/or source 16 to form a working vegetative spore composition 36 before feeding the concentrated vegetative spore composition to incubator 18 . Alternatively, the vegetative spore composition may not be in a concentrated form and does not require any diluent prior to being fed to the incubator 18 (similar to the direct feeding of the vegetative germination factor composition 12 in Figure 1), in which case no diluent is required. Water/Thinner Source26. In this alternative embodiment, water from source 16 may still optionally be fed into incubator 18 as needed. With any of the incubation system embodiments, the diluent can be fed into the incubator 18 to dilute the concentrated composition in the incubator rather than before being fed into the incubator. One of ordinary skill in the art will understand that any combination of elements from systems 10, 110, and 210 may be used together.

Preferably, bacterial spores are germinated in an incubator 18 or other suitable heating device according to the preferred germination methods described herein. According to a preferred embodiment, the vegetative germination factor composition and the spore composition (or the vegetative spore composition) are heated in the incubator 18 to a temperature in the range of 35-55°C, preferably 38-50°C. range, and optimally a range of 41°C to 44°C. The incubation period can vary depending on the end-use application, but is preferably about 20 minutes to 60 minutes for aquaculture applications to produce active bacteria, and about 2 minutes to 5 minutes for probiotic applications to produce a metastable state (metastable state) bacteria. To provide additional growth time for vegetative bacteria, the incubation period may be about 4 to 6 hours.

Depending on the desired use of the bacteria in aquaculture applications, such as water treatment or probiotics for aquatic species, different incubation periods can be used to provide cultured bacterial solutions, which are still mainly spore-form bacteria, mainly Metastable bacteria (where the spores are neither dormant nor in the vegetative growth phase, also referred to herein as the active state), or predominantly fully vegetative bacteria. Additionally, when complete nutrition of the bacteria is required, the bacterial solution may be maintained within the incubator 18 or another intermediate container for a period of time following the incubation period to allow the bacteria to multiply before being discharged into the aquaculture application. Optimally, the bacterial solution will be maintained at a temperature between 30 and 45°C, more preferably, the nutrient bacterial solution is heated as needed to maintain the temperature of the solution in the range of 33 to 42°C, and most preferably In the range of 36° to 39°C, it is beneficial to the growth during subsequent cultivation and growth. When probiotic applications are required, the bacteria remain primarily in a spore or metastable state when discharged into aquaculture applications by using a shorter incubation period, which gives the bacteria a better chance of surviving into the gut of aquatic species. , where they are most beneficial as probiotics. At the end of the incubation period, the incubated bacterial solution 20 is discharged to the growth tank. Depending on the bacterial species used, the incubation temperature, the incubation time, and the nutrient content used, the incubated bacterial solution 20 may include fully vegetative bacteria, metastable bacteria, spores, or combinations thereof.

Each batch of cultured bacterial solution 20 includes approximately 1×10 ⁸ -1×10 ¹⁰ cfu/mL of metastable, vegetative bacterial species and/or spores. Once discharged into the growth tank 22, the bacterial load of each batch is diluted based on the amount of water in the growth tank. Optimally, a sufficient amount of bacterial solution 20 is added to growth tank 22 to provide an effective amount of activated bacteria based on dilution in the growth tank. As used herein, an "effective amount" may refer to an amount of bacteria and/or nutritional composition that is effective in improving the performance of a plant or animal after administration. Water quality can be measured by monitoring one or more characteristics, including but not limited to: water clarity, ammonia levels, nitrite levels, nitrate levels, disease incidence, mortality, harvest weight, meat quality, individual animal size, quality Premium weight, antibiotic use, and additive use are used to measure or evaluate improvements in performance. "Effective amount" can also refer to an amount that can reduce the amount of one or more pathogenic bacteria (including but not limited to Escherlchia coli and Salmonella ) in the intestine of an animal, competitively exclude and/or eliminate one or more pathogenic bacteria "Effective amount" may also refer to an amount that reduces the extent of NH ₃ and/or H ₂ S, such as an amount that can be excreted by an animal into its environment.

According to a preferred embodiment for use in shrimp aquaculture applications, the effective amount of growing bacteria may be from about 1 to about 9×10 ² CFU/mL. According to another preferred embodiment, the effective amount for fish and shrimp aquaculture applications is about 1 to about 9×10 ² to about 10 ⁸ CFU/mL. According to another preferred embodiment, in the growth tank, the effective amount of cultured bacteria may be about 0.001% to about 2% v/v of the total amount of water in the growth tank, and any range or value therein. As another example, dosing a growth tank with about 500 mL of a cultured bacterial solution (containing about 1×10 ⁹ - 1×10 ¹⁰ cfu/mL of bacterial species) four times a day would be sufficient to treat a growth tank containing 100,000 gallons of water. Other volumes of bacterial solution and dosing intervals may be used to treat growth tanks, depending on the size of the tank, based on tank conditions, aquatic species, tank temperature, and other factors to achieve the desired effective amount of bacteria in the tank, e.g. It is understood by those of ordinary skill in the art.

Multiple incubator systems 10, 110, or 210 may be provided to provide larger amounts of cultured bacterial solution to the growth tank to achieve a desired effective amount for addition to the tank, to provide different bacterial species to the growth tank or at different times or rates, and /or space the drain of the cultured bacterial solution around the perimeter of the growth tank to aid dispersion of bacteria through the tank. A pump or other mixing device can also be added to the growth tank (if not already in place) to help disperse the cultured bacterial solution (and nitrification enhancer or surfactant enhancer) throughout the growth tank.

The on-site incubator is preferably configured to grow multiple batches of the cultured bacterial solution from the vegetative germination factor composition/spore composition or the vegetative spore composition such that it can be grown over a long period of time before replenishment of starting material is required. Multiple batches of bacteria are discharged at periodic intervals over time. For example, the container of nutritional germination factor composition 12 can initially hold 0.3 to 3 liters of nutritional germination factor composition, which can be fed into the incubator in increments of about 10 to 100 mL every 1 to 24 hours. A container of concentrated nutritional germination factor solution 24 may initially hold 0.2 to 1 liter of solution diluted with water/diluent from source 26 or 16 to a concentrate to water/diluent ratio of about 1:50 to about 1 : 10, feed approximately 0.1 to 200 mL of working nutritional germination factor solution 28 into the incubator every 1 to 24 hours. The container into which the bacterial spore solution 14 of the nutritional germination factor composition is fed can initially contain 0.6 to 6 liters of the solution, which can be fed into the incubator in increments of about 20 to 200 mL every 1 to 24 hours. A container of concentrated bacterial spore solution 30 may initially contain 0.15 to 6 liters of solution diluted with water/diluent from source 26 or 16 to a concentrate to water/diluent ratio of about 1:10 to about 1:3 to prepare working spore solution 32 at 1 to Incremental amounts of approximately 5 to 200 mL were fed to the incubator over 24 hours. The container of the vegetative spore composition can initially contain 3 to 6 liters of the vegetative germination factor composition, which can be fed into the incubator in increments of about 100 to 200 mL every 1 to 24 hours. Containers of concentrated vegetative spore solution 34 may initially hold 0.3 to 3 liters of solution diluted with water/diluent from source 26 or 16 to a concentrate to water/diluent ratio of about 1:10 to about 1: 50 to feed the working vegetative spore solution 36 into the incubator in increments of about 10 to 100 mL every 1 to 24 hours. Each batch of vegetative germination factor composition/bacterial spores or vegetative spore composition is then grown in an incubator as described herein to form a cultured bacterial solution that is discharged to an aquaculture application.

During the treatment cycle, the cultured bacterial solution 20 is preferably discharged from the one or more incubators 18 to the growth tank 22 every 4 to 6 hours. Other dosing intervals may be used depending on pool size, pool/aquatic species conditions, and type of application. By varying the time of addition of ingredients to the incubator and/or the incubation time, the time between doses can be varied as desired. The cultured bacterial solution can be discharged more frequently to larger tanks (eg, 20 million gallons). For aquaculture water treatment applications, a culture solution that discharges nutritious bacteria is preferred. In order to achieve vegetative bacteria, it is preferred to incubate for at least 4 to 6 hours before discharge into the growth tank, although longer incubation times can be used to give the bacteria more time to multiply. Regarding the application of probiotics to aquatic species in aquaculture applications, the preferred Incubate for about 2 to 5 minutes. In this application, the cultured bacterial solution 20 can be discharged multiple times per day, or even as frequently as every 4 to 6 minutes if required for large tanks. As needed, the volume of the nutrient germination factor composition/spore composition or the vegetative spore composition fed into the incubator is periodically replaced or replenished. In incubators that operate year-round, the treatment cycle is preferably continuous (except for periodic shutdowns for maintenance or supplementation of nutritional germination factor compositions).

Various Bacillus species as described below are preferably used with the aquaculture treatment method according to the invention, but other bacteria may also be used. For example, bacterial genera considered suitable for use in the methods of the present invention include Bacillus , Bacterodes , Bifidobacterium , Lueconostoc , Pediococcus ( Any one or more of Pediococcus , Enterococcus , Lactobacillus , Megasphaera , Pseudomonas and Propionibacterium . Probiotics that can be produced on site include one or more of the following: Bacillus amylophilus, Bacillus licheniformis , Bacillus pumilus , Bacillus subtilis , Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus coagulans ( Bacillus coagulans ), Bacillus megaterium ( Bacillus megaterium ), rumen-dwelling Bacteroides ( Bacteriodes ruminocola ), rumen-dwelling Bacteroides ( Bacteriodes ruminocola ), Bacterioides suis ( Bacterioides suis ), adolescent bifidum Bifidobacterium adolescentis , Bifidobacterium animalis , Bifidobacterium bifidum , Bifidobacterium infantis , Bifidobacterium longum , Bifidobacterium thermophila thermophilum ), Enterococcus cremoris , Enterococcus diacetylactis , Enterococcus faecium , Enterococcus intermedius , Enterococcus lactis , Enterococcus thermophilus thermophiles ), Lactobacillus brevis , Lactobacillus buchneri , Lactobacillus bulgaricus , Lactobacillus casei , Lactobacillus cellobiosus , Lactobacillus curvature ( Lactobacillus curvatus) , Lactobacillus delbruekii , Lactobacillus farciminis , Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis , Lactobacillus plantarum ( Lactobacillus plantarum) , Lactobacillus reuteri , Leuconostoc mesenteroides, Megasphaera elsdennii , Pediococcus acidilacticii , Pediococcus cerevisiae , Pediococcus pentosaceus , Propionibacterium acidipropionici , Propionibacterium freudenreichii , and Propionibacterium shermanii .

With at least one dose (or batch) of cultured bacterial solution discharged to the growth tank, it is preferred to add one or more nitrification enhancers at the same time. Alkalinity enhancers (including calcium carbonate or calcified seaweed) may be added periodically (eg, seasonally or as needed) to reduce phosphate, but not with each dose of bacteria. Reagents can be added in amounts above the dissolved amount to provide a continued source of alkalinity as they slowly dissolve. Slowly dissolving alkalinity enhancers (such as calcified seaweed) also act as surface area modifiers, providing a supportive surface for biofilms of nitrifying bacteria to grow, and they also aid in nutrient delivery. In addition, agents that act solely as surface area modifiers (such as metal or plastic sheets) to reduce nitrogen or phosphorus, as well as batches or doses of cultured bacterial solutions and one or more alkalinity enhancers, can be added to the growth cell as needed. , but it is preferred to add it only once rather than with each dose of cultured bacterial solution. These surface enhancers similarly provide a supporting surface for the added bacterial biofilm, which facilitates faster development of beneficial bacteria. Optimally, approximately 100 pounds of this nitrification enhancer is added to each 7.5 million gallon grow tank, and this amount can be adjusted based on other grow tank volumes. Preferred dispersion methods for nitrification enhancers may involve the use of automated equipment or manual application to the water in the growth pond. Automatically or manually operated devices for dispersing or otherwise dispersing at least one nitrification enhancer in the form of pellets, pellets or granules are commercially available and are known to those skilled in the art. Additionally, these nitrification enhancers can be dispersed in the pool using self-dispersing additive systems and methods using effervescent materials and treatments in water-soluble packages, as described in U.S. Patent Application No. 14/689,790, filed on April 17, 2015, is incorporated herein by reference.

Suitable applications for the methods of the present invention include, for example, but are not limited to, various types of aquaculture applications, such as hatchery, tank and tidal flow aquaculture. The combined use of germination or vegetative bacteria, preferably grown on site, and at least one nitrification enhancing aid such as calcium carbonate, calcified seaweed or other materials is equally effective for cost effective treatment of water used in aquaculture applications to address organic waste, ammonia and pathogenic microorganisms and general water quality issues. It is believed that the effectiveness of the subject methods for achieving these purposes is further enhanced by the addition of calcified seaweed or other plastic or metal pieces, particles or fragments to increase the available surface area for interaction or reaction to occur.

Laboratory studies were conducted to evaluate the benefits of adding beneficial bacteria and nitrification enhancers to pool water. One goal of the study was to evaluate the efficacy of added bacteria (commercially available as a pool mix from EcoBionics) as an inhibitor or herbicide on algae production. This study used six 2L beakers, each filled with 1.5L of source water taken from an established fish tank containing algae. Each beaker also contained a goldfish from the source tank, an air stone, a light source that was alternately on for 12 hours and then off for 12 hours, and a viewing glass cover to reduce evaporation losses. The pool mixture bacterial solution was generated in a BIO-Amp ^™ biogenerator using 37 g of Pond Plus pellets instead of using an incubator and nutritional germination factor composition according to the preferred embodiment of the invention described below. After 24 hours of growth in the biogenerator, an aliquot of the bacterial solution was obtained and diluted to maintain a ratio of 3L of pool mix: 579,024 gallons of pool water, however, the preferred ratio used in the field is 3L of pool mix: 100,000 Gallons of pool water. Based on this ratio, approximately 0.4 μL of the pool mix bacterial solution was added to a specific beaker with 1.5 L of fish tank water. Calcified seaweed was added to the specific beaker based on clarification rate according to the manufacturer's instructions; this corresponds to 0.045g of calcified seaweed per 1.5 liters of water. An equal amount of calcium carbonate was added to some of the beakers. The additives in each beaker are as follows:

Treat each test beaker according to one of the preferred dosing schedules to be used in the art. Beakers 1, 3 and 5 with pool mixture were treated (dosed) with an additional 0.4 μL of pool mixture once a week. Depending on growth tank conditions, other dosing schedules may be used in this field. Calcified seaweed and calcium carbonate were added only once at the beginning of this study, however, additional doses could be used in the field.

A pre-treatment sample was removed from each beaker (before addition of cell mix bacterial solution, calcifying seaweed or calcium carbonate) and analyzed to obtain a baseline for comparison to post-treatment results. Chemical analyzes were performed weekly using 200-300 mL of sample from each beaker. These weekly measurements include Analysis of pH, conductivity, nitrate, orthophosphate, total alkalinity and ammonia levels. Turbidity was measured and photographs taken monthly to assess changes in algae growth and overall clarity. Beaker processing and analysis lasted a total of three months; again reflecting the length of the field study.

Excel 2003 was used for data analysis, and a two-sampled two-tailed t test was used to compare the 95% confidence level (confidence level) of the pre- and post-processing values. The two-sample two-tailed t-test tests the null hypothesis that there is no difference between the means before and after treatment and the alternative hypothesis that there is a difference between the means.

H _O =before μ treatment=after μ treatment

H _A =Before μ treatment≠After μ treatment

Baseline readings indicated elevated phosphate levels in all beakers, exceeding levels 40 times indicative of accelerated algae growth. All other measurements were within acceptable limits.

Test beakers containing only pellets of the bacterial mixture showed no statistically significant changes over the three-month study period. Phosphate levels dropped after two weeks but returned to pre-treatment levels within a month. Nitrate levels reflect phosphate levels. Of all the beakers tested, only Beaker 1 had a similar increase in nitrate and phosphate levels as the negative control group. This represents minimal impact on key chemical indicators of pool health when bacteria are used alone.

The two beakers containing calcified seaweed showed statistically significant changes in the pre- and post-treatment devices. The nitrate levels in beakers 2 and 3 were significantly reduced by 79% and 76% respectively, with p values of 0.01 and 0.04 respectively. The phosphate level in Beaker 2 also dropped significantly by 74%, with a p-value of 0.02. Contains calcified seaweed and The beaker of pellets showed a 66% reduction in phosphate, however, this value was not significant (see Table 2). It is important to note that this lack of statistical significance may be limited by the low sample size of this study. This study did not test changes in pathogenic bacteria in the samples, but it is expected that addition of bacterial solutions to the pool mix would reduce these numbers through competition. Furthermore, it is believed that according to preferred embodiments of the present invention, adding bacterial solutions from incubators using nutritional germination factor compositions to actual growth tanks will achieve better results than in laboratory studies because the bacterial solutions The bacteria in the solution work synergistically with the nitrifying bacteria already present in the growth tank, and the added bacteria in the bacterial solution help consume waste products in the water to reduce ammonia levels. Similar to the calcified seaweed beakers, the two beakers containing calcium carbonate showed significant differences in the means before and after treatment. In Beaker 4, the nitrate level dropped by 77% with a p-value of 0.03. Beaker 5 containing calcium carbonate and bacterial pellets showed a significant 72% reduction in phosphate levels with a p value of 0.02 (see Table 2). Calcium carbonate can be a suitable substitute for calcifying seaweed in aquaculture processes. Beakers with calcified seaweed or calcium carbonate outperformed beakers without calcified seaweed or calcium carbonate. Beaker 5 containing bacterial mixture pellets and calcium carbonate had significantly lower post-treatment mean values for phosphate levels and minimal impact on pH. However, Beakers 2 and 5 had statistically significant decreases in phosphate levels.

Turbidity measured throughout this study showed a consistent decrease in all test beakers. When comparing pre- and post-treatment photos, the presence of algae increased in all beakers, however, there was evidence of algae death in two beakers during the second month. Beakers 4 and 6 (control group) containing calcium carbonate are both yellow, indicating a dying algal system. Algae die when the initial algae A common problem experienced after the species grows vigorously because the oxygen in the water is depleted; despite the presence of air stones. As algae die, nitrate levels increase significantly. This was evident in the increase in nitrate levels before the above treatments this month, with increases of 14% and 38% in Beakers 4 and 6 respectively. By the last month, the nitrate levels in Beaker 4 had recovered and decreased, although the system was still dark green and yellow. Nitrates continued to increase to 6 times the level of contamination in Beaker 6. Figures 4 through 6 are graphs illustrating the results of this laboratory study.

Field studies focusing on improving general pool health and clarity while reducing sludge are also conducted on various pools. Although this study was not aquaculture specific (as the ponds in the study were ornamental or recreational and not used to raise and harvest aquatic species) and used biogenerators rather than cultivation in accordance with preferred embodiments of the present invention organelles and nutritional germination factor compositions, which still provide some useful information on the addition of bacteria and nitrification enhancers. The study included five pools in Irving, Texas, and near Irving; the five pools were identified as Pools 1-5, ranging from 23,400 ft ³ to 720,131 ft ³ . The length of this study was seven months. Once or twice a week, 200-300 mL of surface water samples were taken from the shore of each pool. The samples were analyzed for pH, alkalinity, nitrate, phosphate, ammonia, conductivity, turbidity and E.coli spp concentration. Phosphate, ammonia and turbidity analysis were performed using a Hach DR890 colorimeter. Determination of E.coli spp was performed using a special medium for the growth of E. coli (3M Petrifilm 6404) and incubated at 35°C for 48 hours.

Each tank was sampled once a month to obtain sludge depth, clarity and dissolved oxygen (DO). These tests Measurements were taken from two to four locations on the boat, marked with GPS coordinates to obtain representative sampling. Use sludge judgment to measure sludge depth in inches; sample three to four times per GPS location and average. Dissolved oxygen was measured in ppm from the bottom and again 18" from the surface using a Hach LDO probe with a Hach HQ30d meter. Clarity was measured in %/foot using a Secchi Disk which gives experience Empirical measurement. In addition, photos of two to four locations per pool are taken once a month, again labeled with GPS coordinates, to provide the patron (patron) with overall surface conditions.

A BioAMP ^™ 750 climate-controlled biogenerator was installed at each location for daily on-site dosing of a bacterial mixture to the specific pool. Bacillus spp. spores were pelletized using a modified FREE-FLOW ^™ formulation; the pellets were fed into growth vessels, where they were grown under optimal conditions for 24 hours before being dispersed directly into the pond. Maintenance of the BioAMP 750 must be modified from the standard protocol because sodium hypochlorite (bleach) is considered toxic to surface waters and is not allowed for use by the City of Irving. To obtain a similar bleaching effect to a standard bleach treatment, use 155 g of sodium bicarbonate (baking soda) to remove excess biofilm and clean the growth vessel. In addition to monthly maintenance, the biogenerator's ability to monitor any malfunctions and maintain programmed temperature despite ambient temperatures exceeding 100°F.

Along with the bacterial treatment, calcifying seaweed was also applied to each pool. The amount of calcified algae is given as a volume ratio of 100 pounds to 1,000,000 cubic feet of water. As described in U.S. Patent Application No. 14/689,790, a water-soluble package containing an effervescent couple and calcified seaweed is used. Calcify seaweed.

Each study pool was given the same amount of bacteria (30 trillion CFU) daily. The correlation matrix shows that sludge depth is inversely proportional to dose-rate. The largest reductions in sludge level and clarity were observed in the two smaller tanks, as well as the highest daily bacterial doses of 2×10 ⁷ CFU/L and 7×10 ⁶ CFU/L respectively. Clarity was observed to have a positive impact on all pools, regardless of size. Clarity was approximately 100% in the three smallest cells studied. In contrast, at the end of this study, the two largest tanks achieved only 20% clarity.

A one-sided 2-sample t-test was used to evaluate whether there was a significant reduction in sludge levels after treatment. With a p-value of 0.006, a statistically significant average reduction of 31% was found. H ₀ : Before μ treatment = After μ treatment, H _A : Before μ treatment > After μ treatment. The average reduction observed was equivalent to an average reduction in sludge layer of 3 inches. Additionally, each tank in this study experienced a reduction in sludge levels when compared to before treatment (see Table 3).

The average observed change for E.coli spp. was a 59% reduction. It is important to note that the full range encompasses an increase of 145% to a decrease of 100%. Such a wide range coupled with small sample sizes makes it difficult to determine the effectiveness of treating E. coli spp. concentrations. Three of the five study ponds experienced increases in E. coli spp. concentrations; however, the other two ponds experienced dramatic decreases; however, this increase is believed to be the result of stormwater runoff into the ponds.

The overall impact of this treatment on phosphate concentrations was examined, comparing pre- and post-treatment levels. The data were found to be non-parametric and a one-sided Mann-Whitney test was used to determine whether phosphate concentrations were significantly reduced after treatment. H ₀ : Before μ treatment = After μ treatment, H _A : Before μ treatment > After μ treatment. Obtaining a p-value of 0.0000 indicates that the overall 52% reduction in phosphate levels after treatment is statistically significant. Detailed examination of changes in pool phosphate levels also showed meaningful reductions. Phosphate concentration reductions for each treated pool ranged from 19% to 77% (see Table 3). The average reduction of 52% was similar to the 57% reduction observed in phase I of this study. This suggests that increased frequency of bacterial administration may not be associated with decreased phosphate concentration. Again, a significant reduction in phosphate levels was observed directly after the application of calcifying seaweed. Give the first dose in the spring, and the second dose in the summer after phosphate levels begin to rise in June. The non-chemical, environmentally friendly nature of the powdered product offers promising results for the control of phosphate concentrations.

Likewise, nitrate levels were tested using a one-sided Mann-Whitney test to assess whether nitrate concentrations were significantly reduced after treatment. H ₀ : Before μ treatment = After μ treatment, H _A : Before μ treatment > After μ treatment. A p-value of 0.0000 determined a 91% concentration reduction to be statistically significant (see Table 3). To this end, baseline nitrate concentrations are below recommended levels and are therefore not expected to decrease, let alone a statistically significant 91% reduction. Furthermore, every study pond with detectable nitrates dropped significantly below the detection limit of 0.01 ppm by month 4. This is a huge improvement over the reduction observed in Phase I (~69%) and indicates that increased frequency of bacterial dosing has a direct impact on these concentrations. Overall, this study demonstrates that enhanced treatment with bacteria and calcifying algae increases pool health as measured by chemical proxies and reductions in sludge levels.

Nutritional germination factor composition

The nutritional germination factor composition according to a preferred embodiment of the present invention includes one or more L-amino acids, D-glucose (which increases the binding affinity of L-amino acids to their cognate receptors in the spore coat and optional), D-fructose (optional, depending on the bacterial species), biological buffers to provide the appropriate pH for spore germination (such as HEPES sodium salt, phosphate buffer, or Tris buffer), Optional source of potassium ions (eg KCl) and industrial preservatives. In another preferred embodiment, the nutritional germination factor composition further includes both D-glucose and D-fructose. When using both D-glucose and D-fructose, it is best to include a source of potassium ions, such as KCl. As one of ordinary skill in the art will appreciate, the combination of D-fructose, D-glucose and D-fructose and the source of potassium ions used is dependent on the species of bacteria. It is preferred to use a preservative that is pH compatible with the spore composition and has a relatively neutral pH. According to another preferred embodiment, the vegetative spore composition also includes one or more Bacillus species and preferably one or more germination inhibitors. Nutritional germination factor compositions including spores are referred to herein as nutritional spore compositions, formulations or solutions. Alternatively, it can be placed at the point-of-use Spores are added individually to the nutritional germination factor composition according to the invention. When added individually, the spores are preferably part of a spore composition or spore formulation described herein, but other commercially available spore products may be used. According to another preferred embodiment, the vegetative germination factor or vegetative spore composition is in a concentrated form, preferably a concentrated liquid, and is diluted at the point of use.

Preferred L-amino acids include one or more of L-alanine, L-aspartic acid, L-valine, and L-cysteine. The L-amino acids may be provided from any suitable source, such as their pure form and/or hydrolysates of soy protein. In another embodiment of the concentrated nutritional germination factor composition, the L-amino acid may be provided as a hydrolyzate of soy protein. When in concentrated form, the spore composition is preferably a solution including one or more of the above-mentioned L-amino acids, each of which has a weight range of about 8.9 to about 133.5 g/L, preferably about 13.2 to about 111.25 g/L. , and the best is about 17.8 to about 89g/L; D-glucose (optional) and/or D-fructose (optional), the weight range of each is about 18 to about 54g/L, preferably each is About 27 to about 45 g/L, and optimally each about 30 to about 40 g/L; KCl (optional, as a source of potassium ions), the weight range of which is about 7.4 to about 22.2 g/L, preferably About 11.1 to about 18.5g/L, and preferably about 14 to about 16g/L; the biological buffer, such as sodium dihydrogen phosphate, has a weight range of about 10 to about 36g/L, preferably about 15 to about 16g/L. 30 g/L, and preferably about 20 to about 24 g/L and/or disodium hydrogen phosphate, the weight range of which is about 30 to about 90 g/L, preferably about 21.3 to about 75 g/L, and most preferably About 28.4 to about 60g/L. One or more biological buffers help maintain the nutritional germination factor composition at an appropriate pH for spore germination, approximately pH 6-8. In addition to or in place of sodium dihydrogen phosphate/hydrogen phosphate Disodium buffer, the spore composition may include other phosphate buffers, Tris base, with a weight ranging from about 15 to about 61 g/L, preferably from about 24 to about 43 g/L, and most preferably from about 27 to about 33 g /L; or HEPES buffer, the weight range of which is about 32.5 to about 97.5g/L, preferably about 48.75 to about 81.25g/L, and most preferably about 60 to about 70g/L. Optionally, potassium dihydrogen phosphate can also be used as the source of potassium ions, with a preferred weight ranging from about 13.6 to about 40.8 g/L, preferably from about 20.4 to about 34 g/L, and most preferably from about 26 to about 26 g/L. 29g/L. Optionally, dipotassium hydrogen phosphate can also be used as the source of potassium ions, with a preferred weight ranging from about 8.7 to about 26.1 g/L, preferably from about 13 to about 21.75 g/L, and optimally from about 16 to about 21.75 g/L. About 19g/L. According to another preferred embodiment, the amounts of KCl, sodium dihydrogen phosphate and/or disodium hydrogen phosphate can be adjusted so that the pH in the nutritional germination factor solution and/or the nutritional spore solution can be about 6, about 7, Or about 8.

In another preferred embodiment, the nutritional germination factor composition further includes one or more industrial preservatives, and its final (total) weight range is 0.8-3.3g/L, preferably 1.2-2.7g/L, and the most The best is 1.6-2.2. This preservative may facilitate long-term storage. Suitable preservatives include NaCl, D-amino acids, potassium sorbate, and chemical preservatives. Chemical preservatives can be active ingredients with methyl chloro isothiazolinone (about 1.15% to about 1.18% v/v) and methylisothiazolinone (about 0.35-0.4% v/v) Preservative; consisting of diazolidinyl urea (approximately 30%), methylparaben (approximately 11%) and propylparaben (approximately 3%) Preservatives with the active ingredient methylparaben; and preservatives with only the active ingredient methylparaben; and other preservatives with methylparaben, propylparaben, and diazolidinyl urea. Non-limiting examples of chemical preservatives having methylchloroisothiazolinone and methylisothiazolinone as active ingredients are Linguard ICP and KATHON ^™ CG (which have methylchloroisothiazolinone including about 1.15-1.18% with approximately 0.35-0.4% of the active ingredient methylisothiazolinone). Non-limiting examples of chemical preservatives having diazolidinyl urea, polyparaben, and methylparaben as active ingredients include Germaben II. In the case where the active ingredients of the chemical preservative are methylchloroisothiazolinone and methylisothiazolinone, the chemical preservative can be present in an amount of about 0.8 to about 3.3g/L, preferably from about 1.2 to about 2.7g/L. /L, and optimally from about 1.6 to about 2.2 g/L contained in the concentrated nutritional solution. In the case where the active ingredient of the chemical preservative is diazolidinyl urea, methyl paraben, and/or propyl paraben, the chemical preservative can be used in an amount of about 0.3 to about 1% (wt/wt) Contained in a concentrated nutritional solution. In some aspects, chemical preservatives with diazolidinyl urea, methylparaben, and propylparaben can be included in the nutritional formula in an amount of about 10 g/L. In the case of methyl paraben, the preservative may be present in an amount of from about 0.27 to about 1.89 g/L, preferably from about 0.81 to about 1.35 g/L, and most preferably from about 1.0 to about 1.18 g/L. Contained in a concentrated nutritional solution. According to another preferred embodiment, where the nutritional formulation can be used to produce a nutritional spore formulation effective for use in aquaculture applications related to shrimp or other shellfish, the preservatives can include methyl paraben and sorbic acid Amount of potassium. According to another preferred embodiment, the vegetative germination factor solution can be used to produce a vegetative spore formulation effective for use on plants and/or wastewater, and the vegetative spore formulation can include an amount of Linguard ICP or KATHON ^™ CG.

According to another preferred embodiment, the nutritional germination factor composition may further optionally include an osmoprotectant compound. In a preferred embodiment, ectoine (a natural osmoprotectant produced by some bacterial species) may be included. In the concentrated nutritional germination factor composition, the amount of ectoine (optional) can range from about 0.625 to about 4.375g/L, preferably from about 1.875 to 3.125g/L. L, and the optimal amount is about 2-3g/L. According to another preferred embodiment, the nutritional germination factor composition may further include another standard ingredient, which includes but is not limited to surfactants that assist in the dispersion of the active ingredients, and additional preservatives that ensure the storage life of the spore composition. , buffers, diluents, and/or other ingredients commonly included in nutritional and/or spore formulations.

The amounts of the above ingredients are an important aspect of the present invention, as higher concentrations will render some ingredients insoluble, while lower concentrations will be ineffective for germinating spores.

According to another preferred embodiment, the nutritional germination factor concentrated composition according to embodiments of the present invention is in a concentrated form, and further described below, at the point of use prior to germination, in water, a spore composition, or any other suitable diluent, or a combination thereof, and dilute it into a working solution. According to various preferred embodiments, the concentrated nutritional germination factor composition according to the preferred embodiments herein can be diluted with water or other suitable diluents, so that the concentrated nutritional germination factor composition to the diluent is 0.01% to 50% A working nutritional germination factor solution is prepared at a (v/v) ratio, but other amounts may be used. The concentrated nutritional germination factor composition according to the present invention can be diluted from 2 to 1×10 ⁶ times or any range or value therein to produce a working nutritional germination factor solution. Optimally, the dilution is in the range of about 0.1 to about 10% of the concentrate and the balance water or other suitable diluent. Based on the dilution factor and the above-mentioned concentration amount, the amount of the above-mentioned ingredient present in the working nutritional solution (the dilute solution from the concentrated formula) can be calculated.

The use of concentrated nutritional germination factor compositions reduces transportation, storage and packaging costs and allows easier point-of-use administration of the spore composition. Optimally, the concentrated nutritional germination factor composition is in liquid form, which is easier and faster to mix with the diluent at the point of use, but solid forms such as pellets or bricks or powders may also be used. The inclusion of common industrial preservatives in nutritional germination factor compositions facilitates long-term storage.

Optimally, all ingredients in the nutritional germination factor compositions according to the invention or used with the methods of the invention comply with US federal GRAS standards.

vegetative spore composition

According to another preferred embodiment, the composition is a vegetative spore composition, which includes the above-mentioned ingredients for the vegetative germination factor composition and the spores are premixed together. According to a preferred embodiment, the nutritional spore composition preferably includes 10% to 90% by weight of one or more Bacillus spores or a spore mixture (including 40-60% spore powder and one or more Bacillus species) and 60-40% salt). According to another preferred embodiment, the vegetative spore composition includes about 5% by weight of one or more Bacillus spores or a mixture of spores. The total spore concentration in the vegetative spore composition may range from about 1×10 ⁵ CFU/mL or spores/g to 1×10 ¹⁴ CFU/mL or spores/g or any specific concentration or range therein.

The vegetative spore composition preferably also includes one or more germination inhibitors and/or preservatives. Preferred germination factors or preservatives for concentrated nutrient factor spore compositions include a range from about 29 to about 117 g/L, preferably from about 43 to about 88 g/L, and most preferably from about 52 to about 71 g. /L; and/or an amount of D ranging from about 8 to about 116 g/L, preferably from about 26 to about 89 g/L, and most preferably from about 40 to about 50 g/L - alanine; and/or potassium sorbate in an amount ranging from about 1.25 to about 8.75 g/L, preferably from about 3.75 to about 6.25 g/L, and most preferably from about 4.5 to about 5.5 g. The above-mentioned other chemical preservatives and preferred nutritional germination factor compositions can also be used together with the nutritional spore composition according to the present invention. These germination inhibitors or preservatives maintain the spores in an inactive state and prevent premature germination of the spores prior to their dilution and activation at the point of use. It is particularly preferred to use a germination inhibitor when the spore composition according to this embodiment is used with the preferred method of the invention, where germination occurs at the point of use. When spores are included, the amounts of other components of the above-mentioned nutritional germination factor composition (e.g., L-amino acids, biological buffers, etc.) form a balance of the spore composition, which is proportionally reduced to correspond to the above-mentioned preferred range. . Preferred vegetative spore compositions are also in concentrated form and diluted into working solutions at the point of use relative to the vegetative germination composition as described above and further described below.

Preferred Bacillus spores used in the nutritional spore composition according to preferred embodiments of the present invention include the following species: Bacillus licheniformis , Bacillus subtilis , Bacillus amyloliquefaciens ), Bacillus polymyxa , Bacillus thuringiensis , Bacillus megaterium , Bacillus coagulans, Bacillus lentus , Bacillus clausii clausii) , Bacillus circulan s, Bacillus firmus , Bacillus lactis, Bacillus laterosporus , Bacillus laevolacticus , Bacillus polymyxa Bacillus polymyxa , Bacillus pumilus , Bacillus simplex and Bacillus sphaericus . As one of ordinary skill in the art will appreciate, other Bacillus spore species may also be used. Preferably, the vegetative spore composition includes 1 to 20 or more Bacillus species, preferably between 3 to 12 Bacillus species. According to another preferred embodiment, the vegetative spore composition includes three Bacillus strains. Preferably, the two Bacillus strains are Bacillus licheniformis strains, and the third strain is Bacillus subtilis. subtilis ). According to another preferred embodiment, the spores in the spore mixture include about 80% Bacillus licheniformis (40% of each strain) and 20% Bacillus subtilis .

In another preferred embodiment, the vegetative spore composition used as a probiotic includes one or more Bacillus strains, which are probiotic in nature because they aid in the breakdown of nutrients in the digestive tract of the consumer. This strain preferably produces one or more of the following enzymes: protease that hydrolyzes proteins, amylase that hydrolyzes starch and other carbohydrates, lipase that hydrolyzes fat, and glycosidase that assists in hydrolyzing glycosidic bonds in complex sugars and assists in cellulose degradation. , cellulases that degrade cellulose into glucose, esterases (which are lipase-like enzymes), and xylanases that degrade xylan (xylan is a component of plant cell walls) polysaccharides found). Bacillus strains producing these enzymes are well known in the art.

According to another preferred embodiment, the vegetative spore composition is in concentrated form and is diluted to a working solution in water or any other suitable diluent, or combinations thereof, at the point of use prior to germination as described below. According to various preferred embodiments, the concentrated vegetative spore composition according to the preferred embodiments herein can be diluted with water or other suitable diluents, such that the concentration of the vegetative spore composition to the diluent is 0.01% to 50% (v /v) to prepare working vegetative spore solutions, but other amounts may be used. The concentrated vegetative spore composition according to the present invention can be diluted from 2 to 1×10 ¹³ times or any range or value therein to produce a working vegetative germination factor solution. Optimally, the dilution is in the range of about 0.1 to about 10% of the concentrate and the balance water or other suitable diluent. Based on the dilution factor and the above-mentioned concentration amount, the amount of the above-mentioned ingredients (such as L-amino acids and germination inhibitors) present in the working nutritional solution (the dilute solution from the concentrated formula) can be calculated.

Optimally, all ingredients in the vegetative spore compositions according to the invention or used with the methods of the invention comply with US federal GRAS standards.

spore composition

The probiotic spore composition according to a preferred embodiment of the present invention includes one or more bacterial species, optional surfactants, thickeners, and optionally one or more acidulants, acids, salts or acids as Preservatives. According to another preferred embodiment, the thickener is not also a prebiotic, or in addition to the thickener being a prebiotic, the spore composition further includes one or more prebiotics. According to another preferred embodiment, the spore composition further includes one or more water activity reducers. Optimally, the spore composition according to the present invention includes various suspended probiotic spores, as described in more detail below. The use of these species in spore form increases the stability of probiotics in harsh environmental conditions that may be found near aquaculture applications. The total concentration of spores in the spore composition may range from about 1×10 ⁵ CFU/mL or spores/g to 1×10 ¹⁴ CFU/mL or spores/g or any specific concentration or range therein.

According to preferred embodiments, the spore composition contains a suitable thickening agent. The thickener is preferably one that does not separate or degrade at the different temperatures typically found in non-climate controlled aquaculture environments. Thickeners help stabilize the suspension so the bacterial mixture remains homogeneous and dispersed throughout a volume of spore composition and does not settle out of suspension. When used with an incubation system and an aquaculture treatment method in accordance with preferred embodiments of the invention described herein, this ensures that the concentration of probiotic material is evenly distributed throughout the container such that the dose of spores delivered to the incubator is distributed throughout the incubator. Be consistent or relatively consistent throughout the treatment cycle (depending on the specific delivery method and control mechanism used).

The best thickener is xanthan gum, which is a polysaccharide composed of pentasaccharide repeating units of glucose, mannose and glucuronic acid and known prebiotics. Unlike some other gums, Sanxian gum is very stable over a wide temperature and pH range. Another preferred thickener is acacia gum, which is also a known prebiotic. Other preferred thickeners include locust bean gum, guar gum and gum arabic, which are also considered prebiotics. In addition to their prebiotic benefits, these fibers do not bind minerals and vitamins and, therefore, do not limit or interfere with their absorption, and may even improve the absorption of certain minerals (such as calcium) by aquatic species. Other thickening agents that are not considered prebiotics may also be used.

Preferred embodiments may optionally include one or more prebiotics, which are preferably used if the thickening agent used is not a prebiotic, but may also be used in addition to a prebiotic thickening agent. . Prebiotics are divided into disaccharides, oligosaccharides and polysaccharides, and can include inulin (Inulin), oligofructose (Oligofructose), fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), Trans-Glacto-Oligosaccharide (TOS) and short-chain fructo-oligosaccharide (scFOS), soy fructo-oligosaccharide (soyFOS), glucose oligosaccharide (Gluco -oligosaccharide), Glyco-oligosaccharide, Lactitol, Malto-oligosaccharide, Xylo-oligosaccharide, Stachyose, Lactulose ), raffinose (Raffinose). Mannan oligosaccharide (Mannan- Oligosaccharide (MOS) is a prebiotic that may not increase the probiotic bacterial population, but it binds and removes pathogenic bacteria from the intestinal tract and is thought to stimulate the immune system.

Preferred embodiments also preferably include one or more acidifying agents, acids, or acid salts, used as a preservative or to acidify the spore composition. Preferred preservatives are acetic acid, citric acid, fumaric acid, propionic acid, sodium propionate, calcium propionate, formic acid, sodium formate, benzoic acid, sodium benzoate, sorbic acid, sorbic acid Potassium acid and calcium sorbate. Other known preservatives may also be used, preferably generally recognized as safe (GRAS) food preservatives. Preferably, the pH of the spore composition is between about 4.0 and about 7.0. Preferably it is between about 4.0 and 5.5, and most preferably about 4.5 to prevent premature germination of spores before use or addition to the incubator as described below. Lowering the pH of the spore composition may also provide antimicrobial activity against yeasts, molds and pathogenic bacteria.

According to any preferred embodiment, the spore composition optionally includes one or more water activity reducing agents, such as sodium chloride, potassium chloride, or corn syrup (70% corn syrup solution). The water activity reducer helps inhibit microbial growth so that bacterial spores do not germinate prematurely when the spore composition is stored prior to discharge to the point of consumption by the animal or plant to be treated or discharge to the point of use in the growth tank. They also help inhibit the growth of contaminating microorganisms.

Optional surfactants are preferably surfactants that are safe for the intestinal tract of animals, although other surfactants may be used with other applications (eg, delivery to plants). Optimally, the surfactant is Polysorbate 80. While any GRAS or AAFCO approved surfactant or emulsifier may be used with any of the Examples, there is concern that some animals may not tolerate all approved surfactants well. Due to the benefit of surfactants in stabilizing suspensions so that the bacterial mixture remains homogeneous and does not settle out, which can also be achieved by using a thickening agent, the addition of surfactants is not necessary. If a surfactant is used in the spore composition according to this second embodiment, it is preferred to use the same weight percentage range as in the first embodiment.

Preferred bacteria for use with the spore compositions according to the present invention are the same bacteria described above for the preferred vegetative spore compositions. Most preferably, the bacterial species used in the spore composition are one or more species of the genus Sporobacter. The best strains of probiotic bacteria include the following: Bacillus pumilus , Bacillus licheniformis , Bacillus amylophilus , Bacillus subtilis , Bacillus amylophilus amyloliquefaciens) , Bacillus coagulans, Bacillus clausii , Bacillus firmus, Bacillus megaterium , Bacillus mesentericus , Bacillus subtilis natto var. ( Bacillus subtilis var.natto) , or Bacillus toyonensis , but any Bacillus species approved as probiotics in the country of use can also be used. Preferably, the bacteria are in spore form because the spore form is more stable to environmental fluctuations, such as changes in ambient temperature. Optimally, the spores used in the spore composition according to the present invention are dry powder mixtures, which include about 40-60% salt (common salt) and 60-40% bacterial spores. The spores are preferably in spray-dried form from liquid fermentation concentrate. Salt is used to dilute the pure spray-dried spore powder to a standard spore count in the final spore powder mixture. During production fermentation, different Bacillus strains will grow at different rates, resulting in variations in the final count of the fermentation batch. The fermentation broth is centrifuged to concentrate the spores in the broth. The concentrate is then spray-dried to form a powder containing only Bacillus spores. Adding salt to the spray-dried Bacillus spore powder helps standardize the spore mixture counts per gram for each batch. Other types of bacterial spores or spore mixtures may also be used. Optimally, the dry spore mixture is premixed with a portion of the water used in the spore composition (approximately 3-30% of the total water), and the resulting bacterial spore solution is added to the other ingredients (including the remaining water). This helps disperse bacterial spores throughout the spore composition.

The probiotic spore composition according to the first preferred embodiment of the present invention preferably includes bacterial spores that provide a spore suspension of 10 ⁸ cfu/ml (optimally about 1.0×10 ⁸ to about 3.0×10 ⁸ cfu/ml spore composition, which when diluted in a growth pond provides approximately 10 ¹ to 10 ⁴ cfu/ml pond water), 0.00005 to 3.0% surfactant, and 0.002 to 5.0% thickener, and optionally About 0.01 to 2.0% of one or more acids or acid salts as a preservative, all percentages are based on the weight of the spore composition. A probiotic spore composition according to another preferred embodiment of the present invention includes bacterial spores providing a spore suspension of 10 ⁹ cfu/ml (providing about 10 ¹ to 10 ⁴ cfu/ml when diluted in pool water) ml pool water), about 0.1 to 5.0% of a thickener (preferably a thickener that also acts as a prebiotic), about 0.05-0.5% of one or more preservatives, optionally about 0.1-20% of one or more preservatives water activity reducing agents, and optionally 0.1-20% of one or more acidulants, all percentages based on the weight of the spore composition. The balance of the spore composition in both preferred embodiments is water, and the percentages herein are by weight. It is preferable to use deionized or distilled water to remove salt or external bacteria, but tap water or other water sources can also be used.

According to another preferred embodiment, the spore composition includes about 1% to 10% of a bacterial spore mixture containing salts and spore forms of Bacillus pumilus , Bacillus licheniformis , Bacillus amylophilus ( Bacillus amylophilus ), Bacillus subtilis ( Bacillus subtilis ), Bacillus clausii ( Bacillus clausii ), Bacillus coagulans ( Bacillus coagulans ), Bacillus firmus ( Bacillus firmus ), Bacillus megaterium ( Bacillus megaterium ), Bacillus potato One or more of Bacillus mesentericus , Bacillus subtilis var.natto , or Bacillus toyonensis ; a total of about 0.3% to 1% of one or more acids or acid salts; About 0.2% to 0.5% thickener; about 0.1-0.3% sodium chloride, potassium chloride, or combinations thereof; about 0.00005% to 3.0% surfactant; and 86.2% to 98.4% water. According to another preferred embodiment, the spore composition includes about 0.01% to 10% of a bacterial spore mixture; about 0.1-0.33% of sorbic acid, salts thereof or combinations thereof; and about 0.1-0.34% of citric acid , its salts or combinations thereof; about 0.1-0.33% benzoic acid, its salts or combinations thereof; about 0.2-0.5% xanthan gum; about 0.00005% to 3.0% surfactant; and about 0.1 -0.3% sodium chloride, potassium chloride, or combinations thereof, all percentages are by weight of the composition. According to another preferred embodiment, the spore composition includes about 5% bacterial spores or spore mixture; about 0.25% thickening agent; a total of about 0.3% one or more acids or acid salts; about 0.1% surface activity agent; approximately 0.2% sodium chloride, potassium chloride, or combinations thereof (in addition to any salt in the spore mixture); and water. According to another preferred embodiment, the acid or acid salt is one or more of potassium sorbate, sodium benzoate and anhydrous citric acid.

Some examples of probiotic spore compositions according to preferred embodiments of the present invention were prepared and tested for different parameters. The compositions of these spores are shown in Table 4 below.

The balance of each spore composition was water (approximately 1 L in these samples). Deionized water was used in every spore composition except Spore Composition No. 1, which used tap water. Percentage means percentage by weight. The goal for each formulation was to have a pH between approximately 4.0 and 5.5, but some formulations were found to have actual pH values much lower than expected. Formula No. 1 was targeted to have a pH between 5.0 and 5.5, but its actual pH was about 2.1-2.3, which is too low and may be harmful to spores, creates packaging stability issues, and is subject to stricter shipping norm. Formula No. 1 also showed weak thickening. Formula No. 2 is the same as formula No. 1, the difference lies in the water source. Formula No. 2 had an actual pH of about 2.2-2.3 and exhibited weak thickening. The amount of acid and acid salts in Formula No. 3 was reduced to increase the pH and to determine if the thickness improved when using the same amount of thickener as in Nos. 1 and 2. Although Formula No. 3 was an improvement over Nos. 1 and 2, it still exhibited weak thickening and its actual pH was 6.6, which was outside the target range. To Formula No. 4, additional acids were added to lower the pH, and additional thickeners were added. The thickening effect of Formula No. 4 has been improved, but further improvements in thickening would be beneficial. The amount of acid in Formula No. 5 was significantly increased, which resulted in an actual pH of about 1.0. The amount of acid in Formula No. 6 was reduced and the thickener was increased, which resulted in the spore composition being too thick to fall. Formula No. 7 increases the amount of thickener and water activity reducer, but exhibits the problem of mixing benzoic acid and sorbic acid. Remove benzoic acid and sorbic acid from formula No. 8. Formula Nos. 1-7 provide 2 x 10 ¹¹ cfu/gm and No. 8 provides 1 x 10 ¹¹ cfu/gm of bacterial spores. Of these sample formulations, No. 8 was the best as it exhibited sufficient thickening and had an actual pH of approximately 4.5 +/- 0.2 and used less spore mixture.

Preferably, the spore composition according to the present invention uses about 0.01% to about 0.3% of the bacterial spore mixture, and more preferably between about 0.03% and 0.1% of the bacterial spore mixture. The reduction in the amount of spore mixture used significantly reduces the cost of the spore composition. Depends on end use Different amounts of spore mixtures may be used in the spore compositions according to the present invention. For example, a smaller percentage of the spore mixture may be used in a spore composition for use with chickens, while a larger percentage of the spore mixture may be used in a spore composition for use with pigs.

The storage life of the spore composition according to Formula No. 8 was tested at various temperatures. Seal a sample of Formula No. 8 in a plastic bag, such as that used in the preferred delivery system as described below, and store at about 4-8°C (39-46°F), 30°C (86°F), and Store at 35°C (95°F) for two months to simulate the typical temperature range of agricultural environments where probiotic spore compositions may be stored and used. At the end of the first month of storage, each sample was observed and tested. The pH of all three samples was approximately 4.5, and there was no sedimentation, separation, or change in appearance in any of the three samples, indicating that the bacterial spores remained suspended and dispersed throughout the spore composition during storage. No sample contained any fungal contamination or gram-negative bacterial contamination. At time count zero (when the samples were initially stored), each sample contained approximately 2.12 x ¹⁰⁸ cfu/mL of bacterial spores. At the end of the one-month storage period, the sample contained approximately 2.09 × 10 ⁸ cfu/mL of bacterial spores in the spore suspension (lowest temperature sample), 1.99 × 10 ⁸ cfu/mL (medium temperature sample), and 2.15 × 10 ⁸ cfu/mL (high temperature sample) of bacterial spores. Bacterial counts vary among different samples, especially thickened samples; however, these are considered comparable counts. After two months of storage, each sample was tested again. These samples contained bacterial spores of approximately 2.08×10 ⁸ cfu/ml (lowest temperature sample); 2.01×10 ⁸ cfu/mlL (medium temperature sample); and 2.0×10 ⁸ cfu/ml (high temperature sample). Bacterial spores. The target storage life is approximately 2×10 ⁸ cfu/ml of spore suspension, so the sample is within the target storage life after two months of storage. These test results show that the probiotic spore composition according to the preferred embodiment of the present invention is stable within a temperature range, and the bacterial spores remain viable, suspended and dispersed throughout the spore composition. The spore mixture used in each sample (40-60% spore powder and 60-40% salt) was the same, providing at least about 2 x 10 ¹¹ spores/gram. The spore strains in the mixture are various Bacillus subtilis ( Bacillus subtilis ) and Bacillus licheniformis ( Bacillus licheniformis ) strains. Premix the spore mixture powder with 100 mL of water and stir for 30 minutes, and then add other ingredients. Premixing with water helps mix the spore mixture with other ingredients and disperse the spores throughout the spore composition.

Although it is preferred to use a probiotic spore composition including one or more Bacillus species, such as a spore composition according to the invention, the method of the invention may be used with spore compositions including other bacterial genera and other species. use. For example, one or more species from the following genera: Bacillus , Bacterodes , Bifidobacterium , Pediococcus , Enterococcus , Lactobacillus Lactobacillus and Propionibacterium (including Bacillus pumilus , Bacillus licheniformis , Bacillus amylophilus , Bacillus subtilis , Bacillus amylophilus Bacillus amyloliquefaciens , Bacillus clausii, Bacillus coagulans, Bacillus firmus , Bacillus megaterium , Bacillus mesentericus , Bacillus subtilis Bacillus subtilis var.natto or Bacillus toyonensis, Bacteroides ruminocola , Bacteroides ruminocola, Bacterioides suis , Bifidobacterium adolescentis Bifidobacterium adolescentis) , Bifidobacterium animalis , Bifidobacterium bifidum , Bifidobacterium infantis, Bifidobacterium longum , Bifidobacterium thermophilum , Pediococcus acidilacticii , Pediococcus cerevisiae , Pediococcus pentosaceus, Enterococcus cremoris , Enterococcus diacetylactis , Enterococcus faecium ) , Enterococcus intermedius , Enterococcus lactis , Enterococcus thermophilus, Lactobacillus delbruekii , Lactobacillus fermentum , Lactobacillus helveticus , Lactobacillus lactis, Lactobacillus plantarum , Lactobacillus reuteri , Lactobacillus brevis , Lactobacillus buchneri , Lactobacillus bulgaricus ) , Lactobacillus casei , Lactobacillus farciminis , Lactobacillus cellobiosus, Lactobacillus curvatus , Propionibacterium acidipropionici , Fischeri Propionibacterium freudenreichii , Propionibacterium shermanii and/or one or more of the following species: Leuconostoc mesenteroides , Megasphaera elsdennii can be used with The compositions and methods of the present invention are used together.

The spore composition can also be in a concentrated form, using less water and proportionally increasing amounts of the other ingredients mentioned above. Prior to germination, such concentrated spore compositions may be diluted at the point of use with nutritional germination compositions, water, other suitable diluents, or combinations thereof. Optimally, all ingredients in the spore compositions according to the invention or used with the methods of the invention comply with US federal GRAS standards.

Germination method

According to a preferred embodiment, the method for germinating spores at the point of use according to the present invention includes providing nutrients and spores (preferably providing a nutritional germination factor composition and a spore composition or providing a nutritional spore composition according to the present invention, However, it is also possible to use other commercially available products containing spores and nutrients together or separately) and heat them to an elevated temperature or temperature range and maintain them at this temperature or within this range for a period of time (during incubation) , so that germination occurs at the point of use close to the point of consumption. The heating and nutrients during incubation occur together with the spores in a single step. The method also preferably includes dispersing the germinated spores for aquaculture applications as previously described. Preferably, the nutritional germination factor composition and the spore composition (or the nutritional spore composition) are heated to a temperature in the range of 35-55°C, preferably in the range of 38-50°C, and optimally at 41 ℃ to 44℃ range. The incubation period may vary depending on the end use application. For probiotic applications, where aquatic species with digestive systems (such as fish or eels) will digest the bacteria, it is preferred that the incubation period last no longer than 10 minutes. Optimally, in probiotic applications, the incubation period is between 2-5 minutes. In this approach, spores are released into the growth tank before they have fully germinated and are more likely to survive into the gut of aquatic species, where they are most beneficial. Regarding treating water in aquaculture applications, such as with shrimp aquaculture applications, a preferred incubation period is at least one hour to allow complete germination of the spores before being discharged into the water, and more preferably 4 to 6 hours to allow the bacteria to fully germinate before being discharged into the water. before turning into vegetative bacteria. Optimally, the vegetative germination factor composition and the spore composition (or vegetative spore composition), preferably according to the embodiments of the invention described herein, are added to the incubator to cultivate the spores in the above-mentioned The optimal temperature range and period are used to produce a bacterial solution with nutritional status of bacteria. The incubation can be in an air incubator, water incubator, or any other chamber that provides uniform, constant heat over a given temperature range. The bacterial solution is then discharged to aquaculture applications as previously described. If a concentrated nutritional germination factor composition is used, it is preferred to add diluent water to the incubator together with the nutritional germination factor composition.

According to the preferred method of the present invention, various nutritional germination factor compositions according to the preferred embodiments of the present invention are tested. Composition, method and the results are described below.

Example 1 - To germinate spores, FreeFlow LF-88 probiotic (spore liquid formula commercially available from NCH Corporation) was added to 1 mL of tap water to a final concentration of approximately 1×10 ⁹ CFU/mL, where CFU represents colonies. form units. The nutritional germination factor concentrated composition according to the preferred embodiment of the present invention (which includes L-alanine (89g/L), sodium dihydrogen phosphate (20g/L), disodium hydrogenphosphate (60g/L), and Linguard CP (1.6 g/L total)) were added to the water and bacteria mixture to provide a final concentration of 4% nutritional germination factor composition based on the total weight of the mixture. For comparison, a negative control reaction was prepared using the same amounts of FreeFlow LF-88 probiotics and water, but without the addition of nutritional germination factor concentrate. The two mixtures (germination factors without nutritional germination factor composition and negative control) were mixed and incubated for 60 minutes in a pre-incubation heat block set to 42°C or ambient room temperature (approximately 23°C).

Observe spores from each reaction using phase contrast microscopy. Prepare slides using standard procedures. Spores were viewed on an Olympus BX41 microscope (100X oil immersion objective) and imaged using an Olympus UC30 camera controlled by the cellSens Dimension software package.

Images were taken and germinated spores were counted as a percentage of the total spores in the field of view. A total of 10 representative images were analyzed for each condition (test mixture). Germinated spores lose their refractive index due to the influx of water and are phase-dark, while ungerminated spores are phase-bright.

Figure 7 shows representative images from these tests. Image A Represents germinated spores using a nutritional germination factor composition and heated at 42°C during incubation according to preferred spore compositions and preferred methods of the present invention. Darker spots show germinated spores and lighter spots show non-germinated spores. Image B represents germinated spores grown at ambient temperature (23°C) using a nutritional germination factor composition according to a preferred embodiment of the present invention. Images C-D represent control groups of spores that were not treated with the nutritional germination factor composition according to the invention, one cultured at 42°C and one cultured at ambient temperature (23°C).

As can be seen in Figure 7, image "A" shows significantly more germinated spores (dark spots) than the other images. Spores cultured using the nutritional germination factor composition according to the preferred embodiment of the present invention combined with the germination method according to the preferred embodiment of the present invention showed an obvious germination efficiency of 96.8% (Example 1, Figure 7A). The control group of spores that had been cultivated with the nutritional germination factor composition according to the preferred embodiment of the present invention but without using the germination method according to the preferred embodiment of the present invention showed a significant germination efficiency of 2.3% (Example 1, Figure 7B). Similarly, spores cultured without the nutritional germination factor composition of the present invention showed significant activation efficiencies of 1.2% and 2.6% at 42°C and 23°C, respectively (Example 1, Figures 7C and 7D). Germinated spores in samples not treated by preferred embodiments of the method of the invention represent a small percentage of spores that have germinated in the FreeFlow LF-88 probiotic solution. This example demonstrates that spore germination is significantly increased when nutritional germination factor compositions and cultivation methods according to preferred embodiments of the present invention are used together.

Example 2 - Using the same test mixture/incubation method as described in Example 1 above (incubation using the same nutritional-germination factor composition and heating, "Treated Spores, 42°C") and the control mixture/incubation method (Without nutritive-germination factor composition, without heating, "untreated spores, 23°C") Another set of incubation tests were carried out, but compared to the control mixture, different tests were carried out to compare the better ones according to the invention Examples of test mixtures for efficacy. Additionally, two other mixtures were tested - one using the nutritional germination factor composition of Example 1 but without heating ("Treated spores, 23°C") and one without using the nutritional germination factor but heating the spores ("Unheated spores"). Treated spores, 42°C"). Briefly, spores were incubated for 1 hour at 42°C or 23°C, with or without treatment with the preferred nutritional-germination factor composition. After incubation, spores from 1 mL of each reaction were pelleted at 14K RPM for 3 minutes at 23°C and resuspended in 1 mL of Butterfield buffer. Approximately 6×10 ⁵ CFU (0.02 mL) was added to 0.980 mL of Davis minimal medium (containing 3% glucose as carbon source and trace elements) containing excess D-alanine. D-alanine is an effective inhibitor of L-amino acid-mediated germination.

Approximately 1.2×10 ⁵ CFU was added to each of the four wells of the PreSens OxoPlate. PreSens OxoPlates use an optical oxygen sensor to fluorometrically measure the oxygen content of a sample using two filter pairs (excitation: 540nm, emission: 650nm and excitation: 540, emission: 590nm). Control groups were performed as described by the manufacturer, and measurements were performed on a BioTek 800FLx fluorescent plate reader. Time points were continuously shaken every 10 minutes for 24 hours at 37°C, and the data were processed to determine the partial pressure of oxygen (pO ₂ ) using the following formula: pO ₂ =100*[(K ₀ /IR)-1(K ₀ /K ₁₀₀ )-1]

Spores that have germinated and continue to divide and grow as vegetative cells consume oxygen as part of their metabolic growth. Oxygen consumption is represented by a drop in _pO2 . Presumably, the observed growth is due to outgrowth and vegetative growth of the germinated spores of the present invention. The _pO2 levels for these tests are shown in Figure 8.

As shown in Figure 8, incubation with the test mixture and method according to the preferred embodiments of the present invention (spores treated at 42°C, using a nutritional germination factor composition and heating) resulted in spores that were better than those without the preferred embodiments of the present invention. EXAMPLE A control spore mixture that was either heated or had been treated with a vegetative germination composition or heated but not both began vegetative growth almost 4 hours later. The growth seen in the control experiments likely represents approximately 2% of the germinated spores present in the FreeFlow LF-88 probiotic (see Example 1). This example further demonstrates that spore germination is significantly increased when using nutritional germination factor compositions and cultivation methods according to preferred embodiments of the present invention.

Example 3 - Another set of incubation tests was conducted using similar test and control mixtures and incubation methods as described in Example 1 above. Briefly, LF-88 was added to 10 nL of distilled water to a final concentration of approximately ¹⁰⁸ CFU/mL. Samples were incubated at various temperatures to demonstrate the efficacy of the test method according to preferred embodiments of the invention compared to a control mixture. Reactions ("Treated Spores" in Figure 9) were prepared using the nutritional germination factor compositions described in Example 1 and incubated at 23°C (ambient temperature, unheated), 32°C, 42°C, or 60°C. The control group reaction was incubated at ambient room temperature without using nutritional germination factor composition. After one hour of incubation, 1 mL of each reaction was pelleted at 14K RPM for 3 minutes at 23°C and resuspended in Butterfield buffer. Approximately 6×10 ⁵ CFU (0.02 mL) was added to 0.980 mL of Davis minimal medium (containing 3% glucose as carbon source and trace elements) containing excess D-alanine.

Approximately 1.2×10 ⁵ CFU was added to each of the four wells of the PreSens OxoPlate. Control sets were performed as described by the manufacturer and measurements were performed on a BioTek 800FLx fluorescent plate reader using two filter pairs (excitation: 540 nm, emission: 650 nm and excitation: 540, emission: 590 nm). Time points were continuously shaken every 10 minutes for 24 hours at 37°C, and the data were processed to determine the partial pressure of oxygen ( _pO2 ). The _pO2 levels for these tests are shown in Figure 9.

As shown in Figure 9, incubation with a vegetative germination factor composition according to a preferred embodiment of the present invention and heating resulted in spores initiating vegetative growth before those in the control group. Spores treated with the nutritional germination factor composition but not heated were comparable to the control mixture. The ratio of spores (represented by the "Treated Spores 32°C" curve) treated with the vegetative-germination factor composition cultured at a temperature lower than the preferred range of 35-55° according to one embodiment of the present invention. The control group of experiments started vegetative growth sooner, but not as quickly as spores treated at elevated temperatures within the preferred range according to the invention. Spores treated with the vegetative-germination factor composition according to embodiments of the present invention and cultured at temperatures within the optimal range of 41°C to 44°C showed the best results, starting vegetative growth first and growing faster than the control group Started growing in almost 4 hours. As seen in the previous examples, the growth seen in the untreated control experiments likely represents the presence of approximately 2% of the germinated spores in the FreeFlow LF-88 probiotic (see Example 1). This example further illustrates that when using a comparison method according to the present invention When the nutritional germination factor composition and cultivation method of the preferred embodiment are used, spore germination is significantly increased.

Aquaculture research using nutritional germination factor compositions and spore compositions

Another study was conducted to evaluate the use of preferred nutritional germination factors and spore compositions using the preferred germination and aquaculture treatment methods according to the present invention. This study uses three species of aquaculture as representative aquaculture applications. Each holds 55 gallons of water and 25 Malaysian shrimp to mimic the stocking density of commercial shrimp farms. Each aquarium contained the same type of netting with a substrate composed of PVC pipes for the shrimp to rest and rest on. All aquariums are lined with live Caribbean sand to stop algae growth, reduce nitrates, help buffer the aquarium system, and ensure safer aquarium circulation. Aeration stones were used in all three aquariums to improve biological filtration and increase the amount of dissolved oxygen for the shrimp and beneficial bacteria. The same type of filter was used in all three aquariums, and the filters were rinsed and reused as needed. All three aquariums were refilled with deionized (DI) water as needed. DI water is used to control the mineral content of the water.

When a large amount of water needs to be removed from an aquarium, remove the same amount of water from all aquariums and replace it with the same amount of DI water. Calcium carbonate was used for water replacement in Aquariums 2 and 3 to simulate the use of pond powder (eg, ECOCharger ^™ pond powder available from NCH Life Science). When changing the water in Aquarium 2 or 3, add approximately 0.5 g of calcium carbonate to the tank water. Approximately 1 mL of cultured or activated bacterial solution was applied to Aquarium 3 once a day, Monday through Friday. Aquarium 1 is the control group aquarium. Briefly, 20 μL of starting spore solution (containing approximately 10 ¹⁰ CFU/mL) was mixed with 20 μL of starting concentrated nutrient solution and 960 μL of water to form a working solution containing approximately 2 × 10 ⁸ CFU /mL spores (Table 6). The initial spore solution contained approximately ¹⁰¹⁰ CFU/mL spores from the spore mixture. The spore mixture contained three strains of Bacillus bacteria: two were each a strain of Bacillus licheniformis and the third was Bacillus subtilis . Approximately 80% of the spore mixture formulation is Bacillus licheniformis (40% of each strain) spores and 20% of the spore mixture formulation is Bacillus subtilis . According to the above preferred embodiment, the spore composition also includes water, thickener, and organic salt.

The combined working solution (containing the nutritional germination factor composition, the spore composition and water) was incubated at about 42°C for about 1 hour to produce an activated bacterial solution. After this incubation, the entire activated bacterial solution (approximately 1 mL) was added to Aquarium 3. Mixing is accomplished by aeration through the mixing stones. Table 5 shows the composition of the initial nutritional germination factor formulation. Table 6 shows the composition of the incubated working solutions. After mixing the cultured bacterial solution into 55-gallon Aquarium 3, the concentration of bacteria was approximately 9.6×10 ² CFU/mL and the final percentage of nutritional germination factor composition in Aquarium 3 was approximately 9.6×10 ⁻⁶ %v/v. The contents of each aquarium after their individual treatments had been applied are shown in Table 7. The trial lasts 120 days.

Table 8 shows the average final weight and body measurements of the test groups and the standard deviation. Compared to the treatment groups in Aquariums 2 and 3, Aquarium 1 The control group in had the smallest shrimp weight and body measurements. Compared to the shrimps in Aquariums 2 and 1, Aquarium 3 had the largest shrimps and had the best results in terms of shrimp size. The average final weight of the shrimp in Aquarium 3 was 6.48g. The average final weight of the shrimp in Aquarium 2 was 4.87g. The average final weight of the shrimp in Aquarium 1 (control group) was 3.43g. The average total length of the shrimp in Aquarium 3 was also the largest at 7.98cm. The average total length of the shrimp in Aquarium 2 was 7.41. The average total length of the shrimp in Aquarium 1 (control group) was 6.95. The average tail length of the shrimp in Aquarium 3 was 4.67cm. The average tail length of the shrimp in Aquarium 2 was 4.26cm. The average tail length of the shrimp in Aquarium 1 (control group) was 3.87 cm.

Figure 10 shows images of three aquariums showing the water clarity of each group at the end of the 120 day trial. In terms of water clarity, Aquarium 3 was observed to be the cleanest. It was observed that Aquarium 1 (control group) had the greatest amount of algae growth covering the aquarium walls compared to Aquariums 2 and 3. It was observed that compared to the control group and aquarium 3, aquarium 2 had only Has moderate algae growth.

During the 120-day trial, all three aquariums were started with almost no algae on the sides of the aquarium. As the trial progressed, the control aquarium (Aquarium 1) accumulated more algae growth on the sides of the aquarium (see, eg, Figure 10). Compared to Aquarium 1, Aquarium 2 has less algae growth. Compared to Aquariums 1 and 2, Aquarium 3 has almost no algae growth.

Water parameters were consistent throughout the experiment. All three aquariums had zero amine levels. During the test, nitrite/nitrate was also within safe limits. The pH of all aquariums also stayed within the normal range of approximately 7.5 to 8.5. No harm to the shrimp was observed from peaks in water parameters, as aquarium cycling occurred safely and parameters remained consistent throughout the 120-day trial.

Those of ordinary skill in the art will also understand that modifications and variations of the methods and nutritional germination factors and spore compositions can be made within the scope of the present invention after reading this specification and the description of the preferred embodiments herein, and that herein The scope of the disclosed invention is limited to the broadest interpretation to which the inventors are legally entitled to the appended patent claims.

10: On-site incubator system

12: Nutritional germination factor composition

14: Spore composition

16:Source of water

18: Breeder

20: Cultured bacterial solution

22:Growing pond

Claims (46)

A method for improving the water quality of aquaculture ponds by utilizing a nutritional germination factor composition and bacterial spores. The method includes: providing a volume of a nutritional germination factor composition and a volume of bacterial spores, the two of which can be premixed as nutritional The spore composition or separation; if the two are separated, optionally mixing a portion of the nutritional germination factor composition with a portion of the bacterial spores to form the nutritional spore composition; at or near the location of the aquaculture application , heating a portion of the vegetative spore composition to a temperature in the range of about 38°C to 60°C; maintaining the temperature in the range for an incubation period of about 2 minutes to about 6 hours to form a batch of cultivated Bacterial solution; repeating the heating and maintaining steps periodically during the treatment cycle to form additional batches of the cultured bacterial solution; dispersing each batch of the cultured bacterial solution into the water used in the aquaculture application The water; providing a nitrification enhancer; and dispersing the nitrification enhancer and at least one of the batches of cultured bacterial solutions simultaneously in the water; wherein the bacteria degrade organic waste and inhibit the growth of pathogenic bacteria. Probiotics grown that can be used to remediate the water, or the bacteria are of a species used in the aquaculture application; and Wherein the nutritional germination factor composition is in liquid form and includes: one or more L-amino acids; one or more buffers, including phosphate buffer, HEPES, Tris base, or a combination thereof; one or more industrial preservatives agent. For example, the method of item 1 of the patent application scope, wherein the bacterium is selected from the genus Bacillus , Bacteroides , Bifidobacterium , Lueconostoc , Pediococcus ( The group consisting of Pediococcus , Enterococcus , Lactobacillus , Megasphaera , Pseudomonas and Propionibacterium . For example, the method of claim 2, wherein the bacterium is one or more species of Bacillus licheniformis and Bacillus subtilis . For example, the method of claim 2 of the patent scope, wherein the probiotic is selected from the group consisting of Bacillus amylophilus, Bacillus licheniformis , Bacillus pumilus , Bacillus subtilis , Bacteroides ruminocola , Bacteroides ruminocola , Bacterioides suis , Bifidobacterium adolescentis , Bifidobacterium animalis, Bifidobacterium bifidum Bifidobacterium bifidum) , Bifidobacterium infantis, Bifidobacterium longum , Bifidobacterium thermophilum, Enterococcus cremoris , Enterococcus diacetylactis , Enterococcus faecium , Enterococcus intermedius , Enterococcus lactis , Enterococcus thermophiles, Lactobacillus brevis , Lactobacillus buchneri , Bulgaria Lactobacillus bulgaricus , Lactobacillus casei , Lactobacillus cellobiosus, Lactobacillus curvatus, Lactobacillus delbruekii , Lactobacillus farciminis , Lactobacillus fermentum , Lactobacillus helveticus, Lactobacillus lactis , Lactobacillus plantarum , Lactobacillus reuteri , Leuconostoc mesenteroides) , Megasphaera elsdennii , Pediococcus acidilacticii , Pediococcus cerevisiae, Pediococcus pentosaceus, Propionibacterium acidipropionici , Fei A group consisting of Propionibacterium freudenreichii and Propionibacterium shermanii . For example, according to the method of item 1 of the patent application, the nutritional germination factor composition further includes: a source of potassium ions. For example, the method of item 5 of the patent application scope, wherein the L-amino acid is L-alanine, L-aspartic acid, L-valine, L-cysteine, hydrolyzate of soybean protein, or combination thereof. Such as the method of claim 6, wherein the nutritional germination factor composition includes a total of about 17.8g/L to 89g/L of one or more L-amino acids. Such as the method of claim 5 of the patent scope, wherein the nutritional germination factor composition and spores are premixed, and the nutritional spore composition further includes (1) spores of Bacillus species and (2) germination inhibitors, The germination inhibitor includes sodium chloride, D-alanine, or both. For example, the method of claim 8, wherein the germination inhibitor includes sodium chloride. For example, the method of claim 9 of the patent scope, wherein the premixed camphor The vegetative spore composition includes about 29 g/L to 117 g/L sodium chloride. Such as the method of claim 8, wherein the premixed vegetative spore composition includes about 8g/L to 116g/L of D-alanine. For example, the method of claim 7 of the patent application, wherein the phosphate buffer solution includes about 10-36g/L sodium dihydrogen phosphate and about 30-90g/L disodium hydrogenphosphate. For example, the method of item 1 of the patent application, wherein the nutritional germination factor composition or the premixed nutritional spore composition is a concentrated liquid, and the concentrated liquid includes: about 8.9-133.5g/L of one or more L- Amino acids; a total of about 0.8-3.3g/L of one or more industrial preservatives; a total of about 40-126g/L of one or more phosphate buffers, about 15-61g/L of Tris base, or about 32.5- 97.5 g/L HEPES, or a combination thereof; optionally about 18-54 g/L D-glucose, D-fructose, or a combination thereof; and optionally about 7.4-22.2 g/L KCl. For example, the method of Item 13 of the patent application further includes: adding a diluent to the nutritional germination factor composition or the premixed nutritional spore composition before heating or during the heating process; and During the incubation period, the diluted vegetative germination factor composition is mixed with bacterial spores, or the diluted vegetative spore composition. For example, according to the method of claim 14, the concentration of the diluted nutritional germination factor composition is about 0.1% to 10%. For example, the method of claim 1, wherein the nitrification enhancer increases the alkalinity of the water, provides an increased surface area for nitrifying bacteria biofilm growth, or both. For example, according to the method of item 1 of the patent application, the nitrification enhancer is calcium carbonate, calcified seaweed, or both. For example, according to the method of claim 17, the nitrification enhancer is provided in the form of beads, pellets or granules. For example, the method of claim 16 further includes providing an additional surface area modifier and dispersing the surface area modifier in the water simultaneously with at least one batch of the cultured bacterial solution. For example, in the method of claim 19, the surface area modifier is selected from the group consisting of plastic or metal particles or fragments. For example, the method of claim 1, wherein the nitrification enhancer includes an alkalinity enhancer and the alkalinity enhancer is dispersed in the water simultaneously with a batch of cultured bacterial solutions on a seasonal basis. Such as the method of claim 1, wherein the bacterial spores are in a separate spore composition before being mixed with the nutritional germination factor composition, and the separate spore composition includes: one or more spores in the form of spores Bacillus species; about 0.002 to 5.0% by weight of a thickening agent; and a total of about 0.01 to 2.0% by weight of one or more acids or acid salts; wherein these percentages are based on the weight of the spore composition. Such as the method of claim 5, wherein the bacterial spores are in a separate spore composition, and the spore composition includes: one or more Bacillus species in the form of spores; one or more acids or acid salts categories; and thickeners. For example, the method of item 23 of the patent application scope, wherein the sporobacterium species is Bacillus pumilus , Bacillus licheniformis , Bacillus amylophilus , Bacillus subtilis ), Bacillus amyloliquefaciens , Bacillus clausii , Bacillus firmus, Bacillus megaterium , Bacillus mesentericus , Bacillus subtilis natto var. ( Bacillus subtilis var.natto) , or one or more species of Bacillus toyonensis . For example, the method of claim 23, wherein the spore composition has a pH of about 4.5 to about 5.5. For example, the method of item 23 of the patent application, wherein the acid or acid salt is acetic acid, citric acid, fumaric acid, propionic acid, sodium propionate, calcium propionate, formic acid, sodium formate, benzene One or more of formic acid, sodium benzoate, sorbic acid, potassium sorbate or calcium sorbate. For example, the method of claim 23, wherein the spore composition includes: about 0.002 to 5.0% by weight of a thickening agent; a total of about 0.01 to 1% by weight of one or more acids or salts of acids; and about 0.00005 to 5.0% by weight of a thickening agent. 3.0% by weight of surfactant; where these percentages are based on the weight of the spore composition. For example, the method of claim 1, wherein the incubation period is from about 2 minutes to about 5 minutes, wherein the incubated bacterial solution includes metastable state bacteria, and wherein the bacterial spores include one or more Bacillus species Bacteria. For example, the method of claim 28 of the patent scope, wherein the aquaculture application is a growth tank containing fish or eels. For example, according to the method of claim 1, the incubation period is about 4 to 6 hours. For example, the method of claim 30 of the patent scope, wherein the aquaculture application is a growth pond containing shrimp. For example, according to the method of claim 1, the temperature is in the range of about 38°C to 50°C. For example, according to the method of claim 1, the temperature is in the range of about 41°C to 44°C. For example, the method of claim 5 of the patent scope, wherein the temperature is in the range of about 38°C to 50°C. For example, according to the method of claim 5, the temperature is in the range of about 41°C to 44°C. For example, according to the method of claim 1, the nutritional germination factor composition does not include fructose or glucose. For example, according to the method of claim 5, the nutritional germination factor composition does not include fructose or glucose. For example, according to the method of claim 34, the nutritional germination factor composition does not include fructose or glucose. For example, according to the method of claim 35, the nutritional germination factor composition does not include fructose or glucose. For example, in the method of item 5 of the patent application, the industrial preservatives include methyl chloro isothiazolinone, methyl isothiazolinone, propylparaben, Methylparaben, diazolidinyl urea, or combinations thereof. For example, the method of Item 13 of the patent application, wherein the one or more industrial preservatives include methyl chloro isothiazolinone, methyl isothiazolinone, propyl parahydroxybenzoate ( propylparaben), methylparaben, diazolidinyl urea, or combinations thereof. For example, the method of claim 5, wherein the bacterium is one or more strains of Bacillus licheniformis and Bacillus subtilis . For example, the method of claim 34, wherein the bacterium is one or more strains of Bacillus licheniformis and Bacillus subtilis . For example, the method of claim 35, wherein the bacterium is one or more strains of Bacillus licheniformis and Bacillus subtilis . For example, the method of claim 35, wherein the bacteria include two strains of Bacillus licheniformis and one strain of Bacillus subtilis . For example, the method of item 22 of the patent application further includes about 0.00005 to 3.0% by weight of surfactant.