TW201940063A - Method for improving quality of aquaculture pond water using a nutrient germinant composition and spore incubation method - 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 DEG C to 60 DEG C for around 2 to 60 minutes to produce an incubated bacteria solution that is discharged to the aquaculture application.

Description

Method for improving water quality of aquaculture pond by using nutritional germination factor composition and spore cultivation method

The invention relates to germinating bacteria in a nutrient germination factor composition and using a point-of-use spore cultivation method to treat aquaculture pond water to reduce organic waste, ammonia and disease pressure in aquatic livestock applications. Farmed species provide probiotics.

Aquaculture refers to aquatic species that are raised for human or animal food. This technology applies some types of control to the natural environment of the species being raised to improve overall yield. This may include artificially hatching species in the wild to increase the commercial production of animals, hatching and rearing species in enclosure ponds, and hatching and rearing species in closed areas adjacent to the shoreline for tidal excretion. The problems that accompany this method include: pollutants discharged from the breeding facility and will degrade the quality of nearby water; product loss due to the degraded water quality in the breeding facility; and disease pressure accompanied by increased pathogenic microorganisms in the breeding facility. These issues can be defined by testing or monitoring various parameters including pH, conductivity, ammonia, nitrate, phosphate, and alkalinity. Conductivity is an indicator of salt content, and amounts greater than 1200 ppm are no longer considered fresh water; ideal amounts are in the range of 700 ppm and 300-1200 ppm. The degree of ammonia measures the amount of oxygen available to the fish. High amounts of ammonia prevent the transfer of oxygen from the gills to the blood in fish; however, it is also a product of their metabolic waste. Although ammonia from fish waste is usually not sufficient to concentrate to toxic itself, because of the high concentration of fish in each pond, fish farmers must closely monitor the level of ammonia. Oxygen is consumed by the nitrifying bacteria in the pond (which breaks down toxic ammonia into a non-toxic form); however, this large use of oxygen reduces the available oxygen for fish intake. Levels of ammonia> 1 ppm are considered toxic to fish survival. In addition, the extent of nitrate was examined to determine the amount of plant fertilizer in the water. Nitrate is highly leaching from the surrounding soil and is harmful to children and pregnant women. Nitrate becomes nitrite in the gastrointestinal tract and interacts with the blood's ability to carry oxygen. The maximum pollution level of nitrate is 10 ppm. Alkalinity measures the ability of a pool or lake to neutralize acid without changing pH. Alkalinity decreases over time due to bacteria; however, the ideal level is 100 ppm, with an acceptable range of 50-200 ppm. Most of the phosphate present in ponds and lakes comes from human and animal waste. Fertilizer run-off is a major source of phosphate found in golf courses and ornamental ponds. The increased degree leads to an increase in the rate of eutrophication, which in turn increases the production of sludge. Moderate phosphate levels can promote plant growth and lead to increased algae production; levels> 0.1 ppm are indicators of accelerated plant growth and are considered to be beyond acceptable levels.

Current technologies addressing these issues include bioremediation, antibiotics, and chemical additives. Typical bioremediation techniques involve applying supplemental bacteria to water to enhance microbial activity to 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 the quality of the water, and to provide microbial activity by providing additional nutrients and alkalinity. Add antibiotics to inhibit the growth of pathogenic microorganisms. Issues related to current technology include: the high cost of inactive supplementation with bacteria and low water quality to improve performance, low nitrification activity due to the presence of organic waste and lack of nitrating agent growth sites, and antibiotics in the Bioaccumulation in aquatic species.

According to a preferred method disclosed in U.S. Patent Application No. 14 / 720,088, the use of a biogenerator can produce live bacteria on-site, cultivating bacteria from a solid bacterial starting material into a useful population. Active bacteria can then be discharged from one or more bio-generators 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 these biological generators and methods of using the same, the contents of which are incorporated herein by reference. However, it is useful in aquaculture applications to have another method of producing active bacteria from spores when used.

Spore germination is a multi-step causation process in which spores are effectively awakened or restored from a dormant state to a vegetative growth state. The first step is to activate the spores and induce their germination by an environmental signal called a germination factor. This signal is a nutrient such as L-amino acid. The vegetative germination factor binds to a receptor in the inner membrane of the spore to initiate germination. In addition, 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, which results in the release of Dipicolinic Acid (DPA), which is stored in the spore core with Ca ²⁺ in a 1: 1 ratio (CaDPA). The release of CaDPA is a rapid process, which is usually completed> 90% within 2 minutes. CaDPA release represents a point of no return where spores are committed to the germination process. Those skilled in the art call this step a "commitment" step.

After CaDPA was released, the spores were locally hydrated and the core pH rose to about 8.0. Then, the core of the spores swelled, and the cortex (mainly composed of peptidoglycan) was degraded by the core lysozyme. The spores absorb water and thus lose their refractive index. This refractive index loss is maintained until the end of the germination process such that spore germination is monitored via a phase contrast microscope.

The second stage of germination is an outgrowth step, in which the metabolism of spores, biosynthesis, and DNA replication / repair pathways begin. There are several phases during outgrowth. The first is called the maturity stage, where no morphological changes (such as cell growth) occur, but the molecular mechanisms of the spores (such as transcription factors, translation mechanisms, biosynthetic mechanisms, etc.) are activated. The length of this period may vary depending on the initial resources associated with spore packaging during spore formation. For example, the preferred carbon source for some Bacillus species (including Bacillus subtilis) is malate. Bacillus spores often contain a large pool of malic acid, which is used in the revival process. . Interestingly, the lack of mutations in the malate pool failed to show extended maturity, suggesting that the spore malate pool was sufficient to power the initial outward growth process. In addition, the spores store small acid-soluble proteins, which are degraded within minutes before resuscitation and serve as an immediate source of amino acids for protein synthesis. After the outgrowth step, the spores are fully restored and the cells are considered vegetatively growing.

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

It is also known that there is no thermal activation at ambient temperature (close to typical room temperature) and that germination occurs with a nutrient-containing germination buffer, but this process usually takes longer than activation with heat. For example, B. licheniformis and B. subtilis spores germinate at 35 ° C or 37 ° C, respectively, but it takes a longer time (for example, 2 hours) in a germination buffer containing a nutrient germination factor . In addition, non-heat-activated spores of B. subtilis have been known to have germinated for a prolonged period of time in non-trophic germination factor conditions, such as CaCl ₂ + Na ₂ DPA.

It is also known to use a combination of heat activation and vegetative germination factors to germinate spores in a two-step method in a laboratory installation. The spores are first thermally activated by incubating at a temperature of 65-75 ° C for a period of time (for example, 30 minutes) (this particular temperature depends on the species). The spores are then transferred to a buffer solution containing a vegetative germination factor such as L-alanine. It is also known that in a controlled temperature range of 16-40 ° C (more preferably between 29-32 ° C), in a growth chamber located near the use position, the granular nutrients (containing sugar, yeast Extracts, as well as other nutrients for indirect spore germination factors), bacteria starter, and water are fed into the growth chamber to grow bacteria, as disclosed in US Patent No. 7,081,361. The growth period is about 24 hours.

There is a need for rapid spore cultivation and activation methods that will allow the production of active bacteria (such as 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, which uses a vegetative germination factor concentrate in combination with a spore composition, or uses a vegetative spore composition, to simultaneously thermally cultivate in a single step.

The method of the present invention provides a cost-effective method for delivering 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 water livestock, leading to improved harvesting of species reared in aquaculture operations. Adding the nitrification enhancer described herein as part of this method provides a stable source of alkalinity and additional growth sites for the nitrating agent to promote nitrification activity and ammonia reduction.

The method of the present invention desirably includes the delivery of live bacteria to aquaculture applications, the live bacteria optionally comprising probiotic bacteria, most preferably produced from a field incubator using liquid nutritive germination factor concentrates and bacteria in the form of spores. A vegetative 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 L-amino acids to their isoforms in the spore shell. Binding affinity of the source receptor), and neutral buffers, such as phosphate buffers, and industrial preservatives, such as commercially available Kathon / Lingaurd CG (which has methyl chloro isothiazolinone ) And the active ingredient of methyl isothiazolinone). A nutritional germination factor composition according to another preferred embodiment of the present invention comprises one or two or more combinations of L-amino acids, optionally D-glucose (which increases L-amino acids to the spore shell Binding affinity of their cognate receptors), HEPES solution salts (biological buffers, providing proper pH for spore germination), and industrial preservatives such as propylparaben and methylparaben (methylparaben) combination or other US 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 comprises both D-glucose and D-fructose.

According to another preferred embodiment, the vegetative germination factor composition also includes spores of one or more bacterial species, preferably Bacillus species, but other bacteria can also be used and contain germination inhibitors (such as NaCl, Industrial preservatives, or D-alanine), in combination with any of the aforementioned spore composition ingredients. The germination inhibitor prevents early germination of the spores in the vegetative germination factor composition. Germination inhibitors may include chemicals that prevent spore germination, such as NaCl, industrial preservatives, or D-alanine.

Alternatively, the bacterial spores can be provided separately and added to the trophic-germination factor composition according to the present invention at the point of use and at the time of cultivation. When added separately, it is preferred to provide a stable spore suspension spore composition that 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 a surfactant, about 0.002 to 5.0% by weight of a thickener, and optionally about 0.01 to 2.0% by weight of an acidifying agent, Acids or acid salts (including those as preservatives or stabilizers) with the balance being water. According to another preferred embodiment, the spore composition comprises bacterial spores, about 0.1 to 5.0% by weight of a thickener, about 0.05 to 0.5% by weight of an acid or acid salt, optionally about 0.1-20% by weight Water activity reducing agent, and optionally about 0.1% to 20% additional acidulant (acid or acid salt), the balance being water.

Most preferably, the bacterial spores in the two preferred spore composition examples are a dry powder mixture of 40-60% salt (table salt) and 60-40% bacterial spores (before addition to the spore composition), which The combination constitutes about 0.1 to 10% by weight of the spore composition. The spore composition preferably includes a spore composition (spore suspension) of about 1.0 × 10 ⁸ to about 3.0 × 10 ⁸ cfu / ml. When diluted with drinking water (for animal sprinkler applications), A bacterial strain of about 10 ⁴ to 10 ⁶ cfu / ml is provided. Optimally, the thickener in the two preferred embodiments also acts as a prebiotic (such as Sanxian gum) to provide additional benefits. Although other commercially available spore products can be used, the preferred spore composition for use in the present invention is as disclosed in U.S. Patent Application No. 14 / 524,858, filed on October 27, 2014. Incorporated by reference.

According to another preferred embodiment, the nutritional germination factor composition according to the present invention is in a concentrated form and is diluted at the point of use in water or another diluent to a concentration of 0.01% to 10%. Using concentrated formulas 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 can also be used. The inclusion of general industrial preservatives in the spore composition facilitates long-term storage and / or germination suppression, which is particularly useful when the spore composition is a better concentrated form.

In another preferred embodiment, the present invention includes a method for germinating spores of Bacillus species using a trophic 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 (incubation period). The range of the incubation period is preferably 2-60 minutes, or longer, depending on the application. Optimally, the vegetative-germinal factor composition or vegetative spore composition in a concentrated form according to a preferred embodiment of the present invention is used in the cultivation / germination method of the present invention, but other vegetative-germinal factors can also be used Composition and spore composition. Preferably, the cultivation method is performed at or near the point of use (the point of use is the aquaculture position where the germinated spores will be used or consumed or near the aquaculture position) and further includes dispersing the germinated spores to the point of use Consume. The preferred method of the present invention can be carried out in any incubation device, which can contain a certain volume of spores (if added separately), a liquid (usually water as a diluent), a nutrient germination factor composition, and The reservoir of the mixture is heated during the incubation. Optimally, the method is performed in a device that can mix these ingredients, automatically turn off the heating at the end of the cultivation period, and disperse the bacterial solution of cultivation including bacteria to the aquaculture use point / consumption. The preferred method can also be performed using a batch process or a continuous process. Although it is preferred to use the spore composition according to the present invention, various spore forms or products (eg, dry powder form, liquid suspension, or reconstituted aqueous mixture) can be used with the method of the present invention.

The preferred embodiment of the present invention enables rapid germination of spores of Bacillus species at the point of use in aquaculture. The active vegetative bacteria discharged from the incubator can be provided directly to the growth tank, or it can be accumulated and diluted with pool water or another similarly suitable diluent, such as water from a municipal water system, before it is discharged to the growth tank. Alternatively, the incubator may be configured to heat the vegetative germination factor composition and the spores, or the vegetative spore composition, for incubation and temperature ranges of the metastable state-producing bacteria between the dormant spores and the 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 from the growth tank, dilution can 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 water in aquaculture facilities without the need to add (or reduce the amount of) chemical treatments and antibiotics used in the growth tank.

The invention also desirably comprises the simultaneous application of at least one nitrification enhancer to the growth pond. Nitrification enhancers increase the activity of nitrifying bacteria found naturally in water to reduce the level of ammonia. Since nitrifying bacteria grow into biofilms and require a surface to which they can attach, these nitrating agents include alkalinity enhancers, which increase the alkalinity of water, which is nitrification (7 parts alkalinity to 1 part ammonia) and / or Surface area modifiers are needed to increase the surface on which nitrifying bacteria grow. Alkalinity enhancers may include, for example, calcium carbonate, calcified seaweed, or other similar effective additives. These agents can be added in an amount greater than the amount of dissolution to provide a continuous source of alkalinity as they slowly dissolve. Some nitrifying agents (such as calcified seaweeds) provide both the alkalinity enhancer and the surface area modifier by providing alkalinity and high surface area, providing a supporting surface for the biofilm of the nitrating agent to grow. Calcified seaweed also serves as a trace source of bacteria. Other nitrification enhancers are only used as surface area modifiers, such as plastic or metal sheets, or other similar effective materials to increase the surface area where favorable reactions and interactions can occur. One or more agents that act only as surface area modifiers (rather than alkalinity enhancers) can also be added to the growth cell alone or preferably in combination with one or more alkalinity enhancers; however, agents that act only as surface area enhancers in the growth cell It does not degrade and is not added with each batch of bacterial solution. These agents used only as surface area modifiers are preferably added to the growth tank only once. On a periodic basis, such as seasonal (once a season or once a summer, twice a year, etc.) or if necessary, reagents as alkalinity enhancers are added to the growth tank at the same time as the batch of bacteria. As used herein, the term "simultaneously" means adding batches of vegetative bacteria and other ingredients or reagents to growth ponds or other growth incubators at or about the time in which aquatic species are grown in aquaculture facilities.

Aquaculture treatment methods

According to a preferred embodiment, it is preferred to use an incubator system and a preferred germination method as described below, combining a spore composition from a vegetative germination factor composition or a pre-mixed vegetative spore composition at the site ( On site) produces active bacteria and feeds the active bacteria periodically to a growth pond in aquaculture applications. One or more nitrification enhancers are also added simultaneously to the growth ponds with active bacteria.

A satisfactory bacterial growth and delivery device for use in the method of the present invention would include a field incubator system, such as an air incubator, a water incubator, or any other chamber or similar device that provides uniform and constant heat, where required Spores are germinated for discharge into aquaculture applications within a given temperature range. Referring to FIG. 1, it is preferred that the incubator system 10 contains one or more tanks or containing 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). These spore compositions can also reach the use position in a container, which can be connected in fluid communication with the incubator, in which case a separate tank or container is not required. The source of water 16 at or near the aquaculture site is also optional, but is preferably connectable in fluid communication with the incubator system. Wells, municipal water supplies, or growth ponds can provide water 16 to the incubator 18. The incubator system 10 also preferably includes a chamber or container 18 configured to receive a portion of the vegetative germination factor composition and spores and allow them to heat to germinate the spores; a heater; valves, tubes, and pumps (as needed) (If gravity flow is insufficient) so that vegetative germination factor composition 12, bacterial spore solution 14, and optional water 16 flow from their storage container / source to the addition chamber or container 18, and the cultivated bacteria (or activated Bacterial) solution 20 is discharged from the heating chamber or container 18 and delivered to the growth tank 22; an optional mixer in the heating chamber or container 18, and a controller or timer to activate the valve, pump , Optional mixer, and heater to control nutrient and spore composition flow into the heating 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 (described below) or a vegetative spore composition (a trophic germination factor composition premixed with bacterial spores, also as follows) (Described) (also referred to herein as "starter material") to produce the cultured bacteria container 20 and is discharged into the growth tank 22.

Alternatively, according to another preferred embodiment shown in FIG. 2, the incubator system 110 uses a concentrated vegetative germination factor composition 24 and a concentrated spore composition 30 and dilutes them from a container / source 26 with a diluent or water To form a working nutritional germination factor composition 28 and a 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 can also be used as a source of diluent instead of or in addition to source 26. In addition, only one vegetative germination factor composition 24 or spore composition 30 may be in a concentrated form and needs to be diluted before being fed into the incubator 18. According to another preferred embodiment, when only one is concentrated, a non-concentrated composition may 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, the concentrated vegetative spore composition 34 is used with the system 210, as shown in FIG. Prior to feeding the concentrated vegetative spore composition to the incubator 18, it is diluted with water / diluent from source 26 and / or source 16 to form a working vegetative spore composition 36 to produce a batch of activated bacteria 20. Alternatively, the vegetative spore composition may not be in a concentrated form and does not require any diluent before being fed to the incubator 18 (similar to feeding the vegetative germination factor composition 12 directly in Figure 1), in which case, no Water / thinner source 26. 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 may be fed into the incubator 18 instead of diluting the concentrated composition before feeding into the incubator. Those of ordinary skill in the art will understand that any combination of elements from the 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 method described herein. According to a preferred embodiment, the vegetative germination factor composition and the spore composition (or vegetative spore composition) are heated in the incubator 18 to a temperature range of 35-55 ° C, preferably 38-50 ° C. Range, and optimally in the range of 41 ° C to 44 ° C. The incubation period can be changed according to the end-use application, but it is preferably about 20 minutes to 60 minutes for the production of active bacteria in aquaculture applications, and about 2 minutes to 5 minutes for the probiotic application to produce a metastable state. )bacterial. To provide additional growth time for vegetative bacteria, the incubation period may be about 4 to 6 hours.

Depending on the bacterial use required in aquaculture applications, such as probiotics used to treat water or for aquatic species, different culturing periods can be used to provide cultivated bacterial solutions, which are still mainly spore form bacteria, mainly Metastable bacteria (where the spores are neither dormant nor in vegetative growth phase, also referred to herein as the activated state), or are mainly fully vegetative bacteria. In addition, when complete nutrition of the bacteria is required, after the incubation period, the bacterial solution may remain in the incubator 18 or another intermediate container for a period of time to allow the bacteria to multiply before being discharged into aquaculture applications. Optimally, the bacterial solution will be maintained at a temperature between 30 and 45 ° C. More preferably, the nutritional 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, the growth during the subsequent growth period is facilitated. When probiotic applications are required, when discharged into aquaculture applications by using a short incubation period, the bacteria are mainly maintained in a spore state or metastable state, which gives the bacteria a better chance to survive in the gut of the aquatic species , Where they are most beneficial as probiotics. At the end of the incubation period, the cultured bacterial solution 20 is discharged to a growth tank. Depending on the kind of bacteria used, incubation temperature, incubation time, and nutrient content used, the cultured bacterial solution 20 may include fully vegetative bacteria, metastable bacteria, spores, or a combination thereof.

Each batch of the cultured bacterial solution 20 includes a metastable state, vegetative bacterial species, and / or spores of about 1x10 ^8-1 × 10 ¹⁰ cfu / mL. Once discharged into the growth tank 22, the amount of bacteria in each batch is diluted based on the amount of water in the growth tank. Optimally, a sufficient amount of the bacterial solution 20 is added to the growth tank 22 to provide an effective amount of activated bacteria based on the dilution in the growth tank. As used herein, "effective amount" may refer to an amount of bacteria and / or nutritional composition that is effective to improve the efficacy of a plant or animal after administration. By monitoring one or more characteristics, including but not limited to water quality: water clarity, ammonia level, nitrite level, nitrate level, disease incidence, mortality, harvest weight, meat quality, individual animal size, quality Weight improvement, antibiotic use, and use of additives to measure or evaluate improvements in performance. `` Effective amount '' can also mean an amount that can reduce one or more pathogenic bacteria (including but not limited to Escherichia coli and Salmonella ) in an animal's intestine, competitively exclude and / or eliminate one or more pathogenic bacteria . "effective amount" also means / or amount of NH ₃ and H ₂ S levels may be reduced, for example, by the amount excreted by the animals in their environment.

According to a preferred embodiment for use in shrimp aquaculture applications, the effective amount of bacteria in growth can 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 from 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 bacteria to be cultivated 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 thereof. As another example, administering about 500 mL of a cultured bacterial solution (containing about 1 × 10 ⁹ -1 × 10 ¹⁰ cfu / mL of bacterial strains) to a growth tank four times a day will be sufficient to treat a growth tank containing 100,000 gallons of water. Other volumes of bacterial solution and dose interval can be used to treat the growth pond, depending on the size of the pond, based on the pond conditions, aquatic species, temperature of the pond, and other factors to achieve an effective amount of bacteria required in the pond, such as Those skilled in the art will understand.

Multiple incubator systems 10, 110, or 210 may be provided to provide a larger amount of cultivated bacterial solution to the growth tank to achieve the effective amount required to join the tank, to provide different bacterial species to the growth tank or at different times or rates, and / Or separate the drainage of the cultured bacterial solution in the vicinity of the growth tank to help disperse the bacteria through the tank. Pumps or other mixing devices 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 in-situ incubator is preferably configured to cultivate multiple batches of cultivated bacterial solution from a vegetative germination factor composition / spore composition or a vegetative spore composition, so that it can be grown for a long period of time before starting material needs to be supplemented Multiple batches of bacteria are discharged at periodic intervals in time. For example, the container of the nutritional germination factor composition 12 can initially contain 0.3 to 3 liters of the nutritional germination factor composition, which can be fed to the incubator in increments of about 10 to 100 mL every 1 to 24 hours. The container of the concentrated germination factor solution 24 can 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 an amount of about 0.1 to 200 mL of the working nutritional germination factor solution 28 into the incubator every 1 to 24 hours. The containers each fed with the bacterial spore solution 14 of the nutritional germination factor composition can initially contain 0.6 to 6 liters of the solution, and the solution can be fed to the incubator in increments of about 20 to 200 mL every 1 to 24 hours. The container of the concentrated bacterial spore solution 30 can initially hold 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 feed the working spore solution 32 to the incubator in increments of about 5 to 200 mL every 1 to 24 hours. The container of vegetative spore composition can initially contain 3 to 6 liters of vegetative germination factor composition, and it can be fed to the incubator in increments of about 100 to 200 mL every 1 to 24 hours. The container of concentrated nutrient spore solution 34 can 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 nutrient spore solution 36 to the incubator in increments of about 10 to 100 mL every 1 to 24 hours. Thereafter, each batch of vegetative germination factor composition / bacterial spore or vegetative spore composition is cultivated in an incubator, as described herein, to form a cultured bacterial solution, which is discharged to aquaculture applications.

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. Depending on the size of the pond, the conditions of the pond / aquatic species, and the type of application, other dose intervals may be used. By changing the time when the ingredients are added to the incubator and / or the incubation time, the time between doses can be changed as needed. The cultivated bacterial solution can be discharged to larger ponds more frequently (eg, 20 million gallons). With regard to aquaculture water treatment applications, it is preferred to discharge a cultivation solution with vegetative bacteria. In order to achieve vegetative bacteria, it is preferred to incubate for at least 4 to 6 hours before discharging to the growth tank, although longer incubation times can also be used to allow bacteria to multiply more time. With regard to probiotic applications for aquatic species used in aquaculture applications, it is preferred to cultivate for about 2 to 5 minutes. In this application, the cultured bacteria solution 20 may be drained multiple times a day if needed by the large pond, even frequently every 4 to 6 minutes. As needed, the volume of the vegetative germination factor composition / spore composition or vegetative spore composition fed into the incubator is periodically changed or supplemented. In a year-round incubator, the treatment cycle is preferably continuous (except for periodic shutdowns to maintain or supplement the nutritional germination factor composition).

The following various Bacillus species (Bacillus species) is preferably used with the aquaculture processing method of the present invention, other bacteria may be used. For example, suitable bacteria used in the methods of the present invention is believed to contain the genus Bacillus (Bacillus), Bacteroides (Bacteriodes), the genus Bifidobacterium (Bifidobacterium), the genus Candida albicans (Lueconostoc), Pediococcus ( Pediococcus), Enterococcus genus (Enterococcus), Lactobacillus (Lactobacillus), any one or more of Megasphaera (Megasphaera), Pseudomonas (of Pseudomonas), and the genus Propionibacterium (Propionibacterium) in. 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 megaterium , Bacteriodes ruminocola , Bacteriodes ruminocola , Bacterioides suis , Bacterioides suis Bifidobacterium adolescentis , Bifidobacterium animalis , Bifidobacterium bifidum , Bifidobacterium infantis , Bifidobacterium longum , Bifidobacterium thermophilum), Enterococcus cream (Enterococcus cremoris), Enterococcus two-butanone (Enterococcus diacetylactis), feces enterococci (Enterococcus faecium), intermediate enterococci (Enterococcus intermedius) Enterococcus acid (Enterococcus lactis), thermophilic enterococci (Enterococcus thermophiles), Lactobacillus brevis (Lactobacillus brevis), Brookfield Lactobacillus (Lactobacillus buchneri), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus casein (Lactobacillus casei), Lactobacillus cellobiosus , Lactobacillus curvatus , Lactobacillus delbruekii , Lactobacillus farciminis , Lactobacillus fermentum , Lactobacillus helveticus , lactic acid Lactobacillus (Lactobacillus lactis), Lactobacillus germ (Lactobacillus plantarum), Lactobacillus reuteri (Lactobacillus reuteri), Candida albicans intestinal membrane (Leuconostoc mesenteroides), M. elsdenii (Megasphaera elsdennii), lactic acid micrococcus ( Pediococcus acidilacticii) , Pediococcus cerevisiae , Pediococcus pentosaceus , Propionibacterium aci dipropionici) , Propionibacterium freudenreichii , and Propionibacterium shermanii .

With at least one dose (or batch) of the cultured bacterial solution discharged into the growth tank, it is preferred to add one or more nitrification enhancers simultaneously. Alkalinity enhancers (including calcium carbonate or calcified seaweed) may be added periodically (eg seasonally or as needed) to reduce phosphate, but not with every dose of bacteria. Reagents may be added in amounts above the amount of dissolution to provide a continuous source of alkalinity as they slowly dissolve. Slow dissolving alkalinity enhancers (such as calcified seaweed) also act as surface area modifiers, provide a supporting surface for the biofilm of nitrifying bacteria to grow, and they also aid in nutrient delivery. In addition, reagents used as surface area modifiers (such as metal or plastic sheets) to reduce nitrogen or phosphorus can be added to the growth tank as needed, as well as a batch or dose of cultivated bacterial solution and one or more alkalinity enhancers However, it is preferred to add it only once rather than with each dose of the cultured bacteria solution. These surface enhancers similarly provide a supporting surface for the biofilm of the added bacteria, which helps the faster development of beneficial bacteria. Optimally, about 100 pounds of this nitrification enhancer is added to each 7.5 million gallon growth tank, and this amount can be adjusted according to the volume of other growth tanks. A preferred method of dispersion for the nitration enhancer may include the use of water in the growth pond using an automated device or manually. Devices that can be used to propagate in the form of pellets, pellets, or granules or otherwise disperse at least one nitration enhancer are commercially available and well known to those skilled in the art. In addition, these nitrification enhancers can be dispersed in a pool using a self-dispersing additive system and method that uses an effervescent material and a treating agent in a water-soluble package, which was described in April 2015 US Patent Application No. 14 / 689,790, filed on May 17, which is incorporated herein by reference.

Suitable applications for the method of the present invention include, for example but not limited to, various types of aquaculture applications, such as hatcheries, ponds and tidal current aquaculture. Combining germinated or vegetative bacteria, preferably growing in the field, and at least one nitrification enhancing agent, such as calcium carbonate, calcified seaweed or other materials, is also effective for cost-effective treatment of water used in aquaculture applications to solve Organic waste, ammonia and pathogenic microorganisms, and general water quality issues. It is believed that by adding calcified seaweed or other plastic or metal flakes, particles or fragments to increase the available surface area where interactions or reactions can occur, the effectiveness of the subject methods for achieving these purposes is further enhanced.

Perform laboratory research to assess the benefits of adding beneficial bacteria and nitrification enhancers to the pool water. One goal of this study was to evaluate the effectiveness of the added bacteria (a pool mixture commercially available from EcoBionics) as an inhibitor or weed control on algae production. This study used six 2L beakers, each of which was filled with 1.5L of source water, which was taken from an established fish tank where algae was present. Each beaker also contains a goldfish from the source tank, air stone, a light source that is turned on for 12 hours and then turned off for 12 hours, and a glass cover to reduce evaporation loss. Instead of using an incubator and a germinative germination factor composition according to the preferred embodiment of the present invention described below, a pooled bacterial solution was produced in a BIO-Amp ^™ biogenerator using 37 g of Pond Plus pellets. After growing for 24 hours in the biogenerator, an aliquot of the bacterial solution was obtained and diluted to maintain a 3L pool mixture: 579024 gallons of pool water ratio, however, the preferred ratio used in this field is a 3L pool mixture: 100,000 Gallon of water. Based on this ratio, approximately 0.4 µL of the pool mixture bacterial solution was added to a specific beaker with 1.5 L of fish tank water. Add calcified seaweed to a specific beaker based on the clarification rate according to the manufacturer's instructions; this is equivalent to 0.045 g 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:

Each test beaker was processed according to a preferred dosage schedule to be used in the art. Beakers 1, 3, and 5 with the pool mixture were treated (dosed) once a week with an additional 0.4 µL of the pool mixture. Depending on the growth pond conditions, other dosage schedules can be used in this area. Calcified seaweed and calcium carbonate were added only once at the beginning of this study, however, additional doses can be used in the field.

A pretreatment sample was taken from each beaker (before adding the pool mixture bacterial solution, calcified seaweed or calcium carbonate) and analyzed to obtain a baseline for comparison with the results after treatment. Chemical analysis was performed once a week using 200-300 mL samples from each beaker. These weekly measurements include analysis of pH, conductivity, nitrate, orthophosphate, total alkalinity, and ammonia levels. The turbidity is measured and photographed monthly to assess changes in algae growth and overall clarity. The beaker processing and analysis lasted a total of three months; again reflecting the length of the field study.

Data analysis was performed using Excel 2003. Two sampled two-tailed t tests were used to compare the 95% confidence level of the pre-processed and post-processed values. The two-sample two-tailed t-test tests another hypothesis that the null hypothesis has no difference between the mean before and after the treatment and the mean.
H _O = µ before processing = after μ processing
H _A = before µ processing ≠ after µ processing

Baseline readings indicate an increase in the level of phosphate in all beakers, which is more than 40 times greater than the degree of accelerated algae growth. All other measurements are within acceptable limits.

Test beakers containing only bacterial mixture pellets did not show statistically significant changes during the three-month study period. Phosphate levels dropped after two weeks, but returned to pre-treatment levels within a month. The degree of nitrate reflects the degree of phosphate. Of all the beakers tested, only beaker 1 had an increase in nitrate and phosphate levels similar to the negative control group. This represents the smallest impact on the main chemical indicators of pool health when bacteria are used alone.

Two beakers containing calcified seaweed showed statistically significant changes in the pre- and post-treatment devices. The levels of nitrate in beakers 2 and 3 decreased by 79% and 76%, respectively, with p values of 0.01 and 0.04, respectively. The degree of phosphate in Beaker 2 also decreased significantly by 74%, with a p-value of 0.02. Beakers containing calcified seaweed and 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 for changes in the pathogenic bacteria in the samples, but it was expected that addition of the pool mixture bacterial solution would reduce these numbers through competition. In addition, it is believed that according to a preferred embodiment of the present invention, the addition of a bacterial solution from an incubator using a nutrient germination factor composition to an actual growth tank will achieve better results than a laboratory study, due to the bacterial solution The bacteria in it can work synergistically with the nitrifying bacteria already present in the growth pond, and the bacteria added to the bacterial solution can help consume waste in the water to reduce the level of ammonia. Similar to the calcified seaweed beaker, the two beakers containing calcium carbonate showed a significant difference between the pre- and post-treatment mean values. In the beaker 4, the degree of nitrate decreased by 77%, and the p-value was 0.03. Whereas, beaker 5 containing calcium carbonate and bacterial pellets showed a 72% reduction in phosphate levels with a p-value of 0.02 (see Table 2). In aquaculture processing, calcium carbonate can be a suitable alternative to calcified seaweed. Beakers with calcified seaweed or calcium carbonate outperformed beakers without calcified seaweed or calcium carbonate. The beaker 5 containing the bacterial mixture pellets and calcium carbonate has a significantly lower post-treatment mean and a minimal effect on pH. However, Beakers 2 and 5 had a statistically significant decrease in phosphate level.

The turbidity measured throughout this study showed a continuous decrease in all test beakers. When photos before and after treatment were compared, the presence of algae in all beakers increased, however, there was evidence of algae death in two beakers in the second month. Beaker 4 and beaker 6 (control group) containing calcium carbonate are yellow, indicating that the algae system is dying. Algae death is a common problem experienced after the initial growth of algae, as oxygen in the water is depleted; despite the presence of air stones. As algae die, nitrate levels increase significantly. This increase in nitrate levels before the aforementioned treatment was apparent this month, with beakers 4 and 6 increasing by 14% and 38%, respectively. By the last month, the level of nitrate in beaker 4 had recovered and decreased, although the system was still dark green and yellow. Nitrate continued to increase to 6 times the degree of contamination in the beaker 6. Figures 4 to 6 are diagrams illustrating the results of this laboratory study.

Field studies have also been conducted on various ponds focusing on improving the health and transparency of general ponds while reducing sludge. Although this study was not specific to aquaculture (because the ponds in the study were ornamental or recreational, not for the breeding and harvesting of aquatic species) and the use of biological generators instead of cultivation according to the preferred embodiment of the present invention Organs and vegetative germination factor compositions, which still provide some useful information on the addition of bacteria and nitrification enhancers. The study included five pools near Irving and Irving, Texas; 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 are taken from the shore of each cell. The samples were analyzed for pH, alkalinity, nitrate, phosphate, ammonia, conductivity, turbidity, and E. coli spp concentration. A Hach DR890 colorimeter was used for phosphate, ammonia and turbidity analysis. E. coli spp was measured using a special incubator (3M Petrifilm 6404) for E. coli growth at 35 ° C for 48 hours.

Each pool was sampled once a month to obtain sludge depth, transparency, and dissolved oxygen (DO). These measurements were made on boats from two to four locations, marked with GPS coordinates to obtain representative samples. Use sludge judgment to measure sludge depth in inches; sample each GPS location three to four times and take the average. Using a Hach LDO probe with a Hach HQ30d meter, the dissolved oxygen was measured from the bottom layer and again at 18 "from the surface in ppm. Using Secchi Disk to determine Clarity in% / feet, this gives experience Empirical measurement. In addition, once a month, two to four photos of each location are taken in each pool, and again marked with GPS coordinates to provide patrons with overall surface conditions.

A BioAMP ^™ 750 climate-controlled bio-generator was installed at each location for daily on-site administration of bacterial mixtures to specific ponds. Bacillus spp. Spores were pelletized using a modified FREE-FLOW ^™ formulation; the pellets were fed into a growth vessel where they were grown under optimal conditions for 24 hours and then dispersed directly into the tank. Maintenance of the BioAMP 750 must be modified from the standard protocol, as sodium hypochlorite (bleach) is considered toxic to surface water and is not allowed in Irving. To obtain a similar bleaching effect as the standard bleach treatment, 155 g of sodium bicarbonate (baking soda) was used to remove excess biofilm and clean the growth container. In addition to monthly maintenance, the ability to monitor for any failure of the biogenerator and maintain a programmed temperature despite ambient temperatures exceeding 100 ° F.

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

Each study pool was given the same amount of bacteria daily (30 trillion CFU). The correlation matrix shows that the sludge depth is inversely proportional to the dose-rate. It was observed that the sludge degree and clarity of the two smaller ponds decreased the most, and the daily maximum bacterial doses were 2 × 10 ⁷ CFU / L and 7 × 10 ⁶ CFU / L, respectively. The observed clarity has a positive effect on all pools, regardless of size. In the three smallest pools of this study, the clarification was about 100%. Conversely, at the end of this study, the two largest pools reached only 20% clarity.

A one-sided 2 sample t-test was used to assess whether the degree of sludge after treatment was significantly reduced. The p-value was 0.006, and a statistically significant average reduction was found to be 31%. H ₀ : before μ processing = after μ processing, H _A : before μ processing> after μ processing. The average reduction observed was equivalent to an average reduction of 3 inches in the sludge layer. In addition, each pond in this study experienced a reduction in sludge levels when compared to before treatment (see Table 3).

The average observed change in E. coli spp . Was a 59% reduction. It is important to note that the full range includes an increase of 145% to a decrease of 100%. Such a wide range coupled with a small sample size makes it difficult to determine the effectiveness of processing E. coli spp. Concentrations. Three of the five study pools experienced an increase in E. coli spp. Concentration; however, the other two pools decreased sharply; however, the increase was considered to be the result of rainwater runoff into the pool.

The overall effect of this treatment on phosphate concentration was examined, and the degree before and after treatment was compared. The data was found to be non-parametric and a one-sided Mann-Whitney test was used to determine if the phosphate concentration was significantly reduced after treatment. H ₀ : before μ processing = after μ processing, H _A : before μ processing> after μ processing. Obtaining a p-value of 0.0000 indicates a 52% overall reduction in phosphate level after treatment is statistically important. Detailed examination of changes in the level of phosphate in the pool also showed a meaningful reduction. The reduced phosphate concentration in each treated pond ranged from 19% to 77% (see Table 3). The average 52% reduction is similar to the 57% reduction observed in Phase I of the study. This suggests that an increase in the frequency of bacterial administration may not be related to a decrease in phosphate concentration. Furthermore, a significant reduction in the degree of phosphate was directly observed after the application of calcified seaweed. The first dose was administered in the spring and the second dose was administered in the summer after the phosphate level began to increase in June. The non-chemical, environmentally friendly nature of powdered products provides promising results for controlling phosphate concentrations.

Similarly, a one-sided Mann-Whitney test was used to measure nitrate levels to assess whether nitrate concentrations were significantly reduced after treatment. H ₀ : before μ processing = after μ processing, H _A : before μ processing> after μ processing. At a p-value of 0.0000, it is statistically important to determine a 91% reduction in concentration (see Table 3). For this reason, the baseline nitrate concentration is below the recommended level and is therefore not expected to decrease, let alone a statistically significant reduction of 91%. Furthermore, each research pool with detectable nitrate significantly reduced its detection limit below 0.01 ppm in the fourth month. This is a huge improvement over the reduction (~ 69%) observed in Phase I, and indicates that the frequency of increased bacterial administration has a direct impact on these concentrations. Overall, this study confirms that enhanced treatment with bacteria and calcified seaweed increases pond health as measured by chemical proxies and a reduction in sludge levels.

Vegetative germination factor composition

A vegetative 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 shell) And is optional), D-fructose (optional, depending on bacterial species), biological buffers to provide appropriate pH for spore germination (such as HEPES sodium salt, phosphate buffer or Tris buffer), Optional sources of potassium ions (such as KCl) and industrial preservatives. In another preferred embodiment, the vegetative germination factor composition further includes both D-glucose and D-fructose. When both D-glucose and D-fructose are used, a source containing potassium ions, such as KCl, is optimal. As understood by one of ordinary skill in the art, the combination of D-fructose, D-glucose and D-fructose, and the use of potassium sources depends on the type of bacteria. It is preferred to use a preservative compatible with the pH of the spore composition, which 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. A vegetative germination factor composition including spores is referred to herein as a vegetative spore composition, formulation, or solution. Alternatively, the spores can be individually added to the vegetative germination factor composition according to the present invention at a point-of-use. When added individually, the spores are preferably part of the spore composition or spore formulation described herein, but other commercially available spore products can also 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. L-amino acids may be provided in any suitable source form, such as their pure forms and / or hydrolysates of soy protein. In another embodiment of the concentrated vegetative germination factor composition, L-amino acid may be provided as a hydrolysate of soy protein. When in a concentrated form, the spore composition preferably is a solution including one or more of the above-mentioned L-amino acids, each having a weight range of about 8.9 to about 133.5 g / L, preferably about 13.2 to about 111.25 g / L And preferably about 17.8 to about 89 g / L; D-glucose (optional) and / or D-fructose (optional), each having a weight range of about 18 to about 54 g / L, preferably About 27 to about 45 g / L each, and most preferably about 30 to about 40 g / L each; KCl (optionally as a source of potassium ions) in a weight range of about 7.4 to about 22.2 g / L , Preferably about 11.1 to about 18.5 g / L, and most preferably about 14 to about 16 g / L; biological buffers, such as sodium dihydrogen phosphate, have a weight range of about 10 to about 36 g / L, more It is preferably about 15 to about 30 g / L, and most preferably about 20 to about 24 g / L and / or disodium hydrogen phosphate, and its weight ranges from about 30 to about 90 g / L, preferably about 21.3 to About 75 g / L, and most preferably about 28.4 to about 60 g / L. One or more biological buffers help maintain the vegetative germination factor composition at a suitable pH value for spore germination, about pH 6-8. In addition to or instead of sodium dihydrogen phosphate / disodium hydrogen phosphate buffer solution, the spore composition may include other phosphate buffer solution, Tris base, and its weight ranges from about 15 to about 61 g / L, preferably about 24 to about 43 g / L, and preferably about 27 to about 33 g / L; or HEPES buffer, which has a weight range of about 32.5 to about 97.5 g / L, preferably about 48.75 to about 81.25 g / L, and most preferably About 60 to about 70 g / L. Optionally, potassium dihydrogen phosphate can also be used as a source of potassium ions, with a preferred weight range of about 13.6 to about 40.8 g / L, preferably about 20.4 to about 34 g / L, and most preferably about 26 to About 29 g / L. Optionally, dipotassium hydrogen phosphate can also be used as a source of potassium ions, with a preferred weight range of about 8.7 to about 26.1 g / L, preferably about 13 to about 21.75 g / L, and most preferably about 16 to About 19 g / L. According to another preferred embodiment, the amount of KCl, sodium dihydrogen phosphate and / or disodium hydrogen phosphate can be adjusted so that the pH in the vegetative germination factor solution and / or vegetative spore solution can be about 6, 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 0.8-3.3 g / L, preferably 1.2-2.7 g / L, most It is preferably 1.6-2.2. The preservative can facilitate long-term storage. Suitable preservatives include NaCl, D-amino acid, potassium sorbate, and chemical preservatives. The chemical preservative may be an active ingredient having methyl chloro isothiazolinone (about 1.15% to about 1.18% v / v) and methyl isothiazolinone (about 0.35-0.4% v / v) Preservatives; with diazolidinyl urea (about 30%), methylparaben (about 11%) and propylparaben (about 3%) Preservatives of active ingredients; and preservatives that have only active ingredients of methyl paraben; and other preservatives that have methyl paraben, propyl paraben, and diazolidinyl urea. Non-limiting examples of chemical preservatives with methylchloroisothiazolinone and methylisothiazolinone as active ingredients are Linguard ICP and KATHON ™ CG (which has about 1.15-1.18% methylchloroisothiazolinone) With about 0.35-0.4% of the active ingredient of methyl isothiazolinone). Non-limiting examples of chemical preservatives having diazolidinyl urea, polyparaben, and methyl paraben as active ingredients include Germaben II. In the case where the active ingredients of the chemical preservative are methylchloroisothiazolinone and methylisothiazolinone, the chemical preservative may be about 0.8 to about 3.3 g / L, preferably about 1.2 to about 2.7 g / L, and most preferably from about 1.6 to about 2.2 g / L are contained in a concentrated nutrient solution. In the case where the active ingredient of the chemical preservative is diazolidinyl urea, methyl paraben, and / or propyl paraben, the chemical preservative may be present at about 0.3 to about 1% (wt / wt). Contained in a concentrated nutrient solution. In some aspects, a chemical preservative with diazolidinyl urea, methyl paraben, and propyl paraben can be included in the nutritional formulation in an amount of about 10 g / L. In the case of methyl parahydroxybenzoate, the preservative may be at 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 nutrient solution. According to another preferred embodiment, where the nutritional formula can be used to produce a nutritional spore formula effective for use in aquaculture applications related to shrimp or other shellfish, the preservative can include methyl paraben and sorbic acid The amount of potassium. According to another preferred embodiment, the vegetative germination factor solution can be used to produce a vegetative spore formula effective for plants and / or wastewater. The vegetative spore formula can include the 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, it may include methyl tetrahydropyrimidine carboxylic acid (ectoine) (a natural osmotic protection agent produced by some bacterial species). In the concentrated vegetative germination factor composition, the amount of methyl tetrahydropyrimidine carboxylic acid (ectoine) (optional) may range from about 0.625 to about 4.375 g / L, preferably from about 1.875-3.125 g / L, and the optimal amount is about 2-3 g / L. According to another preferred embodiment, the nutritional germination factor composition may further include another standard ingredient including, but not limited to, a surfactant that facilitates dispersion of the active ingredient, and an additional preservative to ensure the storage life of the spore composition , Buffers, diluents, and / or other ingredients typically included in nutritional formulations and / or spore formulations.

The amount of the above-mentioned ingredients is an important aspect of the present invention, because higher concentrations make some ingredients insoluble, while lower concentrations are not effective for germinating spores.

According to another preferred embodiment, the nutritive germination factor concentrated composition according to the embodiment of the present invention is in a concentrated form, and further as described below, before the point of use is in germination, in water, spore composition, or any other suitable , Or a combination thereof, to a working solution. According to various preferred embodiments, the concentrated vegetative germination factor composition according to the preferred embodiment of the present invention can be diluted by water or other suitable diluent, and the concentrated vegetative germination factor composition is 0.01% to 50% of the diluent. (v / v) ratios prepare working vegetative germination factor solutions, but other amounts can also be used. The concentrated vegetative germination factor composition according to the present invention may be diluted 2 to 1 × 10 ⁶ times or any range or value thereof 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 of water or other suitable diluent. Based on the dilution factor and the above-mentioned concentration, the amount of the aforementioned ingredients present in the working nutritional solution (diluted solution from the concentrated formula) can be calculated.

The use of a concentrated nutrient germination factor composition reduces transportation, storage and packaging costs, and makes it easier to administer the spore composition at the point of use. Optimally, the concentrated vegetative 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 can also be used. The inclusion of general industrial preservatives in the nutritional germination factor composition facilitates long-term storage.

Optimally, all ingredients in the vegetative germination factor composition according to the present invention or all ingredients used with the method of the present invention comply with the US Federal GRAS standard.

Vegetative spore composition

According to another preferred embodiment, the composition is a vegetative spore composition, which includes the aforementioned ingredients for the vegetative germination factor composition and the spores are pre-mixed together. According to a preferred embodiment, the nutritional spore composition preferably comprises 10% to 90% by weight of one or more Bacillus spores or a mixture of spores (including 40-60% spore powder and one or more Bacillus species And 60-40% salt). According to another preferred embodiment, the vegetative spore composition comprises about 5% by weight of one or more Bacillus spores or a mixture of spores. The total concentration of spores 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 concentrating the nutrient factor spore composition comprise 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 A relatively high concentration of NaCl of 71 g / L; and / or 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 D-alanine in an amount of L; and / or 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 in an amount from about 4.5 to about 5.5 g Potassium sorbate. The above-mentioned other chemical preservatives and preferred nutritional germination factor compositions can also be used with the nutritional spore composition according to the present invention. These germination inhibitors or preservatives maintain the spores in an inactive state and prevent early germination of the spores before their dilution and activation at the point of use. When the spore composition according to this embodiment is used with the preferred method of the present invention, it is particularly preferred to use a germination inhibitor, where germination occurs at the point of use. When spores are included, the amount of the other components of the vegetative germination factor composition (eg, L-amino acid, biological buffer, etc.) forms the balance of the spore composition, which is proportionally reduced to correspond to the above-mentioned preferred range . The preferred nutritional spore composition is also in concentrated form and is diluted to the working solution at the point of use as described above with respect to the nutritional germination composition, and is further described below.

Preferred Bacillus spores used in the nutritional spore composition according to a preferred embodiment of the present invention include the following species: Bacillus licheniformis , Bacillus subtilis , Bacillus amyloliquefaciens ), polymyxa Bacillus (Bacillus polymyxa), Bacillus thuringiensis (Bacillus thuringiensis), a huge Bacillus (Bacillus megaterium), Bacillus coagulans (Bacillus coagulans), slow Bacillus (Bacillus lentus), Bacillus clausii (Bacillus clausii) , Bacillus circulans , Bacillus firmus , Bacillus lactis , Bacillus laterosporus , Bacillus laevolacticus , Bacillus polymyxa ( Bacillus polymyxa ), Bacillus pumilus , Bacillus simplex , and Bacillus sphaericus . As understood by one of ordinary skill in the art, other Bacillus spore species can also be used. Preferably, the vegetative spore composition includes 1 to 20 or more Bacillus species, and preferably 3 to 12 Bacillus species. According to another preferred embodiment, the vegetative spore composition includes 3 types of Bacillus strains, and the best 2 types of Bacillus strains may be Bacillus licheniformis strains respectively, and the third strain is Bacillus subtilis ). According to another preferred embodiment, the spores in the spore mixture include about 80% Bacillus licheniformis (40% per 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 probiotics in nature, because they facilitate the breakdown of nutrients in the digestive tract of the consumer. This strain preferably produces one or more of the following enzymes: proteases that hydrolyze proteins, amylases that hydrolyze starch and other carbohydrates, lipases that hydrolyze fats, glycosidases that help hydrolyze glycosidic bonds in complex sugars, and cellulose degradation Cellulase that degrades cellulose to glucose, esterase (which is an enzyme similar to a lipase), and xylanases (xylanases) that degrade xylan (xylan is in the plant cell wall Polysaccharides found). Bacillus strains that produce these enzymes are well known in the art.

According to another preferred embodiment, the vegetative spore composition is in a concentrated form and is diluted to a working solution in water or any other suitable diluent, or a combination thereof, before use at the point of germination, as described below. According to various preferred embodiments, the concentrated nutrient spore composition according to the preferred embodiment of the present invention may be diluted with water or other suitable diluent, so that the concentrated nutrient spore composition is 0.01% to 50% (v / v), a working nutrient spore solution is prepared, but other amounts can also be used. The concentrated vegetative spore composition according to the present invention may be diluted 2 to 1 × 10 ¹³ times or any range or value thereof 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 of water or other suitable diluent. Based on the dilution factor and the above-mentioned concentration, the amount of the above-mentioned components (such as L-amino acid and germination inhibitor) present in the working nutritional solution (diluted solution from the concentrated formula) can be calculated.

Most preferably, all ingredients in the vegetative spore composition according to the present invention or all ingredients used with the method of the present invention comply with the US Federal GRAS standard.

Spore composition

A 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 acidifying agents, acids, salts, or acids as the preservative. According to another preferred embodiment, the thickener is not also a prebiotic, or in addition to being a prebiotic thickener, the spore composition further includes one or more prebiotics. According to another preferred embodiment, the spore composition further comprises one or more water activity reducers. Most preferably, 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 the form of spores increases the stability of probiotics under the 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 a preferred embodiment, the spore composition comprises a suitable thickener. The thickener is preferably a thickener that does not separate or degrade at different temperatures commonly found in non-climate-controlled aquaculture environments. The thickener helps stabilize the suspension, so the bacterial mixture remains homogeneous and dispersed in a volume of spore composition and does not settle out of the suspension. When used with a cultivation system and aquaculture treatment method according to the preferred embodiment of the invention described herein, this ensures that the concentration of probiotic material is evenly distributed throughout the container, so that the dose of spores delivered to the incubator is throughout Be consistent or relatively consistent throughout the processing cycle (depending on the particular delivery method and control mechanism used).

The most preferred thickener is xanthan gum, which is a polysaccharide consisting of pentasaccharide repeat 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 to be prebiotics. In addition to the benefits of prebiotics, these fibers are not combined with minerals and vitamins, therefore, they do not limit or interfere with their absorption, and may even improve the absorption of certain minerals such as calcium by aquatic species. Other thickeners that are not considered prebiotics can also be used.

The preferred embodiment may optionally include one or more prebiotics. If the thickener used is not a prebiotic, the one or more prebiotics are preferably used, but can also be used in addition to the prebiotic thickener . Prebiotics are divided into disaccharides, oligosaccharides and polysaccharides, and may include inulin, oligofructose, fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), Trans-Glacto-Oligosaccharide (TOS) and Short-Chain Fructo-oligosaccharide (scFOS), soy Fructo-oligosaccharide (soyFOS), grape oligosaccharide (Gluco -oligosaccharide), Glyco-oligosaccharide, Lactitol, Malto-oligosaccharide, Xylo-oligosaccharide, Stachyose, Lactulose ), Raffinose. Mannan-oligosaccharide (MOS) is a probiotic, which may not enhance the probiotic bacterial population, but will bind to and remove pathogenic bacteria from the intestine, and is considered to stimulate the immune system.

The preferred embodiment also preferably contains one or more acidifying agents, acids, or salts of acids for use 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 and calcium sorbate. Other known preservatives can also be used, preferably generally regarded as GRAS food preservatives. Preferably, the pH of the spore composition is between about 4.0 and about 7.0. It is preferably between about 4.0 and 5.5, and most preferably about 4.5 to prevent spores from germinating early before use or adding to the incubator as described below. Lowering the pH of the spore composition may also have antimicrobial activity against yeasts, molds and pathogenic bacteria.

According to any preferred embodiment, the spore composition optionally comprises one or more water activity reducing agents, such as sodium chloride, potassium chloride or corn syrup (70% corn syrup solution). The water activity reducing agent helps to suppress microbial growth so that when the spore composition is stored before the animal or plant to be treated is discharged to a point of consumption or to a point of use in a growth pond, the bacterial spores do not germinate early. They also help inhibit the growth of contaminating microorganisms.

Although other surfactants can be used with other applications (e.g., delivery to plants), the optional surfactant is preferably a surfactant that is safe for the animal's gut. Optimally, the surfactant is Polysorbate 80. Although any GRAS or AAFCO approved surfactant or emulsifier can be used with any of the examples, there are concerns that some animals may not be well tolerated by all approved surfactants. Because of the benefits of surfactants in stabilizing the suspension, the bacterial mixture remains homogeneous and does not settle out. It can also be achieved by using thickeners. Adding surfactants is not necessary. If a surfactant is used in the spore composition according to this second embodiment, it is preferable to use the same weight percentage range as in the first embodiment.

The preferred bacteria for use with the spore composition according to the present invention are the same as those described above for the preferred vegetative spore composition. Most preferably, the bacterial species used in the spore composition are one or more species in the genus Sporobacterium. The best strains of probiotic bacteria include the following: Bacillus pumilus , Bacillus licheniformis , Bacillus amylophilus , Bacillus subtilis , Bacillus amyloliquefaciens) , Bacillus coagulans , Bacillus clausii , Bacillus firmus , Bacillus megaterium , Bacillus mesentericus , Bacillus subtilis ( Bacillus subtilis var. Natto) , or Bacillus toyonensis , but any Bacillus species approved as probiotics in the country of use can also be used. It is preferred that the bacteria be in the form of spores, because the spore form is more stable to environmental fluctuations, such as changes in environmental temperature. Most preferably, the spores used in the spore composition according to the present invention are a dry powder mixture comprising about 40-60% salt (table salt) and 60-40% bacterial spores. The spores are preferably in a spray-dried form from a liquid fermentation concentrate. Salt was 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 changes in the final count of the fermentation batch. The fermentation broth was centrifuged, and the spores in the concentrate were concentrated. Then, the concentrate was spray-dried to form a powder containing only Bacillus spores. Adding salt to the spray-dried Bacillus spore powder helps standardize the spore mixture count per gram per batch. Other types of bacterial spores or spore mixtures can also be used. Optimally, the dried spore mixture is pre-mixed with water (approximately 3-30% of the total water) for one part of the spore composition, and the resulting bacterial spore solution is added to the other ingredients (containing 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 which 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 provides about 10 ¹ to 10 ⁴ cfu / ml water when diluted in a growth pond), 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 are used as preservatives, 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 which provide a spore suspension of 10 ⁹ cfu / ml (providing about 10 ¹ to 10 ⁴ cfu / when it is diluted in pool water) ml of pool water), about 0.1 to 5.0% of a thickener (preferably also a thickener of probiotics), about 0.05-0.5% of one or more preservatives, optionally about 0.1-20% of one or Various water activity reducing agents, and optionally 0.1-20% of one or more acidifying agents, all percentages are based on the weight of the spore composition. The balance of the spore composition in these two preferred embodiments is water, and the percentages herein are by weight. Deionized or distilled water is preferred to remove salts or external bacteria, but tap water or other water sources can also be used.

According to another preferred embodiment, the spore composition comprises about 1% to 10% of a mixture of bacterial spores, which contains salt and Bacillus pumilus , Bacillus licheniformis , and Bacillus amyloliquefaciens in the form of spores. ( Bacillus amylophilus ) , Bacillus subtilis , Bacillus clausii , Bacillus coagulans , Bacillus firmus , Bacillus megaterium , potato ( 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% of a thickener; about 0.1-0.3% of sodium chloride, potassium chloride, or a combination thereof; about 0.00005% to 3.0% of a surfactant; and 86.2% to 98.4% of water. According to another preferred embodiment, the spore composition comprises about 0.01% to 10% of a bacterial spore mixture; about 0.1-0.33% of sorbic acid, a salt thereof, or a combination thereof; and about 0.1-0.34% of citric acid , Its salt or a combination thereof; about 0.1-0.33% of benzoic acid, its salt or a combination thereof; about 0.2-0.5% of xanthan gum; about 0.00005% to 3.0% of a surfactant; and about 0.1 -0.3% sodium chloride, potassium chloride, or a combination thereof, all percentages are by weight of the composition. According to another preferred embodiment, the spore composition comprises about 5% of bacterial spores or a mixture of spores; about 0.25% of a thickener; about 0.3% of one or more acids or acid salts in total; and about 0.1% interfacial activity Agent; about 0.2% sodium chloride, potassium chloride, or a combination thereof (other than 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 the probiotic spore composition according to the preferred embodiment of the present invention were prepared and tested for different parameters. These spore compositions are shown in Table 4 below.

The balance of each spore composition was water (about 1 L in these samples). Except for the spore composition No. 1 (which uses tap water), deionized water was used in each spore composition. Percentages represent weight percentages. The goal of each formulation was to have a pH between about 4.0 and 5.5, but some formulations were found to have actual pH values well below expectations. The goal of Formula 1 is to have a pH between 5.0 and 5.5, but its actual pH is about 2.1-2.3, which is too low and may be harmful to spores, create packaging stability issues, and is subject to stricter shipping specification. Formulation 1 also exhibited weak thickening. Formula 2 is the same as Formula 1, except that the water source is different. Formula 2 has an actual pH of about 2.2-2.3 and exhibits weak thickening. The amount of acid and acid salt in the No. 3 formula was reduced to increase the pH and it was determined whether the thickness was improved when the same amount of thickener as in the No. 1 and No. 2 was used. Although Formula No. 3 is an improvement over No. 1 and No. 2, it still exhibits weak thickening, and its actual pH is 6.6, which exceeds the target value range. Additional acid was added to Formula 4 to lower the pH, and other thickeners were added. The thickening effect of Formula 4 has been improved, but it is beneficial to further improve the thickening. The amount of acid in Formula 5 increased significantly, which resulted in an actual pH of about 1.0. The amount of acid in Formula 6 decreased and the thickener increased, which caused the spore composition to be too thick to fall. Formulation 7 increases the amount of thickener and water activity reducing agent, but exhibits the problem of mixing benzoic acid with sorbic acid. Benzoic acid and sorbic acid were removed from Formula 8. Formulas 1-7 provided 2 × 10 ¹¹ cfu / gm and No. 8 provided 1 × 10 ¹¹ cfu / gm bacterial spores. Of these sample formulations, No. 8 is optimal because it exhibits sufficient thickening and has an actual pH of about 4.5 +/- 0.2 and uses less spore mixture.

It is preferred that the spore composition according to the present invention use from about 0.01% to about 0.3% of a bacterial spore mixture, and more preferably between about 0.03% and 0.1%. The reduction in the amount of spore mixture used significantly reduces the cost of the spore composition. Depending on the end-use application, different amounts of spore mixtures can be used in the spore composition according to the invention. For example, a smaller percentage of the spore mixture can be used in a spore composition for use with chickens, and a larger percentage of the spore mixture can be used in a spore composition for use with pigs.

The storage life of the spore composition according to Formula 8 was tested at various temperatures. Samples of Formula 8 are sealed in plastic bags, such as those used in a preferred delivery system described below, and at about 4-8 ° C (39-46 ° F), 30 ° C (86 ° F) ) And 35 ° C (95 ° F) for two months to simulate the typical temperature range of an agricultural environment where probiotic spore compositions can be stored and used. At the end of the first month of the storage period, each sample was observed and tested. The pH of all three samples was about 4.5, and there was no precipitation, delamination, or appearance change in any of the three samples, meaning that the bacterial spores remained suspended and dispersed throughout the spore composition during storage. None of the samples contained any fungal contamination or gram-negative bacterial contamination. When the time count was zero (when the samples were initially stored), each sample contained approximately 2.12 × 10 ⁸ cfu / mL of bacterial spores. At the end of the one month storage period, the sample contained approximately 2.09 × 10 ⁸ cfu / mL spore suspension (lowest temperature sample) bacterial spores, 1.99 × 10 ⁸ cfu / mL (medium temperature sample) bacterial spores and 2.15 × 10 ⁸ cfu / mL (high temperature sample) bacterial spores. Bacterial counts vary from sample to sample, 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 about 2.08 × 10 ⁸ cfu / ml (minimum temperature sample); bacterial spores of 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 about 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 over a temperature range, and the bacterial spores remain viable, suspended, and dispersed throughout the spore composition. The spore mixture (40-60% spore powder and 60-40% salt) used in each sample was the same, providing at least about 2 × 10 ¹¹ spores / gram. The spore species in the mixture are various Bacillus subtilis and Bacillus licheniformis strains. The spore mixture powder was mixed with 100 mL of water in advance for 30 minutes, and then added to the other ingredients. Pre-mixing with water helps to 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 comprising one or more Bacillus species, such as the spore composition according to the present invention, the method of the present invention may be used with spore compositions comprising other bacterial species and other species use. For example, from one or more of the following genera of bacteria: Bacillus (Bacillus), Bacteroides (Bacteriodes), the genus Bifidobacterium (Bifidobacterium), Pediococcus (Pediococcus), Enterococcus (of Enterococcus), lactobacillus Lactobacillus and Propionibacterium (including Bacillus pumilus ) , Bacillus licheniformis , Bacillus amylophilus , Bacillus subtilis, Bacillus subtilis , Bacillus (Bacillus amyloliquefaciens), Bacillus clausii (Bacillus clausii), Bacillus coagulans (Bacillus coagulans), Bacillus firmus (Bacillus firmus), huge Bacillus (Bacillus megaterium), potato Bacillus (Bacillus mesentericus), Bacillus Bacillus subtilis var. Natto or Bacillus toyonensis , Bacteriodes ruminocola , Bacteriodes ruminocola , Bacterioides suis , Bifidobacterium juvenile ( Bifidobacterium adolescentis) , Bifidobacterium animalis , Bifidobacterium bifidum , Bifidobacterium infantis , Bifidobacterium longum , Bifidobacterium thermophilum ), lactic acid micrococcus (Pediococcus acidilacticii), Pediococcus beer (Pediococcus cerevisiae), Pediococcus pentose (Pediococcus pentosaceus), Enterococcus cream (Enterococcus cremoris), Enterococcus two-butanone (Enterococcus diacetylactis), Enterococcus feces (of Enterococcus faecium) , Enterococcus intermedius , Enterococcus lactis , Enterococcus thermophilus , Lactobacillus delbruekii , Lactobacillus fermentum , Hectobacillus helveticus ) , Lactobacillus lactis , Lactobacillus plantarum , Lactobacillus reuteri , Lactobacillus brevis) , Lactobacillus buchneri , Lactobacillus bulgaricus , Lactobacillus casei , Lactobacillus farciminis , Lactobacillus cellobiosus , Lactobacillus flexobacter ( Lactobacillus curvatus ), Propionibacterium acidipropionici , Propionibacterium freudenreichii , Propionibacterium shermanii , and / or one or more of the following: Leuconostoc mesenteroides and Megasphaera elsdennii can be used with the composition and method of the present invention.

The spore composition may also be in a concentrated form, using less water, and increasing the amount of the other ingredients mentioned above in proportion. Prior to germination, these concentrated spore compositions can be diluted at the point of use with a nutritional germination composition, water, other suitable diluent, or a combination thereof. Most preferably, all ingredients in the spore composition according to the present invention or all ingredients used with the method of the present invention conform to the US Federal GRAS standard.

Germination method

According to a preferred embodiment, the method for germinating spores at a point of use according to the present invention includes providing nutrients and spores (preferably providing a vegetative germination factor composition and a spore composition or providing a vegetative spore composition according to the present invention, However, other commercially available products containing spores and nutrients can also be used together or individually) and they are heated to an elevated temperature or temperature range and maintained at that temperature or within this range for a period of time (incubation period) To make it germinate near the point of use near the point of consumption. The heating during the incubation takes place in a single step along with nutrients and spores. The method also preferably includes dispersing the germinated spores to an aquaculture application, as previously described. Preferably, the vegetative germination factor composition and the spore composition (or vegetative spore composition) are heated to a temperature range of 35-55 ° C, preferably a range of 38-50 ° C, and most preferably 41 ° C to 44 ° C. The incubation period can be changed according to the end-use application. For probiotic applications, where aquatic species with a digestive system (such as fish or eel) will digest the bacteria, it is preferred that the incubation period last no more than 10 minutes. Optimally, in probiotic applications, the incubation period is between 2-5 minutes. In this way, spores are released into the growth pond before they are fully germinated, and are more likely to survive to the gut of aquatic species, where they are most beneficial. Regarding the treatment of water in aquaculture applications, such as shrimp aquaculture applications, the preferred incubation period is at least one hour so that the spores are fully germinated before being discharged to the water, and more preferably 4 to 6 hours so that the bacteria are discharged to the water Before becoming vegetative bacteria. Optimally, the vegetative germination factor composition and the spore composition (or vegetative spore composition), preferably according to the embodiment of the present invention described herein, are added to the incubator to cultivate the spores in the above-mentioned comparative Optimal temperature range and period to produce a bacterial solution of bacteria with nutritional status. The incubation may be in an air incubator, a water incubator, or any other chamber that provides uniform, constant heat over a given temperature range. The bacterial solution is then discharged into aquaculture applications, as previously described. If a concentrated vegetative germination factor composition is used, it is preferable to add diluent water to the incubator together with the vegetative germination factor composition.

According to a preferred method of the present invention, various nutritional germination factor compositions according to a preferred embodiment of the present invention are tested. The composition, method and results are described below.

Example 1-In order to germinate spores, FreeFlow LF-88 probiotics (a spore liquid formulation commercially available from NCH Corporation) was added to 1 mL of tap water with a final concentration of about 1 × 10 ⁹ CFU / mL, where CFU represents Colony forming units. A nutritional germination factor concentrated composition (which includes L-alanine (89 g / L), sodium dihydrogen phosphate (20 g / L), and disodium hydrogen phosphate (60 g / L), and Linguard CP (total 1.6 g / L)) were added to the water and bacteria mixture to provide a vegetative germination factor composition at a final concentration of 4% based on the total weight of the mixture. For comparison, a negative control group reaction was prepared with the same amount of FreeFlow LF-88 probiotics and water, but no trophic germination factor concentrated composition was added. The two mixtures (germination factor without trophic germination factor composition and negative control group) were mixed and incubated for 60 minutes in a pre-incubation heat block set to 42 ° C or ambient room temperature (about 23 ° C).

Spores from each reaction were observed using a phase contrast microscope. Slides were prepared using standard procedures. Spores were observed on an Olympus BX41 microscope (100X oil immersion objective) and imaged using an Olympus UC30 camera controlled by the cellSens Dimension software package.

Take images and count the percentage of germinated spores that become 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 and are phase-dark due to the inflow of water, while non-germinated spores are phase-bright.

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

As can be seen in Figure 7, the image "A" shows significantly more germinated spores (dark spots) than the other images. The spores cultivated using the nutritional germination factor composition according to the preferred embodiment of the present invention in combination with the germination method according to the preferred embodiment of the present invention showed a significant germination efficiency of 96.8% (Example 1, FIG. 7A). Control group spores that have been cultivated with the vegetative germination factor composition according to the preferred embodiment of the present invention but not using the germination method according to the preferred embodiment of the present invention show a significant germination efficiency of 2.3% (Example 1, Figure 7B). Similarly, spores not cultivated with the vegetative 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, FIGS. 7C and 7D). Germinated spores in samples not treated by the preferred embodiment of the method of the present invention represent a small percentage of spores that have germinated in the FreeFlow LF-88 probiotic solution. This example confirms that when the vegetative germination factor composition and the cultivation method according to the preferred embodiment of the present invention are used together, the spore germination is significantly increased.

Example 2-Using the same test mixture / cultivation method as described in Example 1 above (using the same nutrient-germination factor composition and heated incubation, "treated spores, 42 ° C") and control group mixture / incubation method (No trophic-germination factor composition, no heating, "untreated spores, 23 ° C") Another set of incubation tests was performed, but compared to the control group mixture, different tests were performed to compare the better according to the present invention Examples test the efficacy of the mixture. In addition, two other mixtures were tested-one using the vegetative germination factor composition of Example 1 without heating ("treated spores, 23 ° C") and the other using no vegetative germination factor but heated spores ("Un Treated spores, 42 ° C "). In short, the spores were incubated at 42 ° C or 23 ° C for 1 hour, with or without treatment with a better 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 Basic Incubator (containing 3% glucose as a carbon source and trace elements) containing excessive D-alanine. D-Alanine is a potent inhibitor of L-amino acid-mediated germination.

Approximately 1.2 × 10 ⁵ CFU was added to each of the four slots of the PreSens OxoPlate. PreSens OxoPlates uses an optical oxygen sensor to measure the oxygen content of the sample using two filter pairs (excitation: 540 nm, emission: 650 nm and excitation: 540, emission: 590 nm). Control groups were performed as described by the manufacturer and measurements were performed on a BioTek 800FLx fluorescent plate reader. Continuous shaking at a time point of 24 hours every 10 minutes at 37 ° C, and the data was processed using the following formula to determine the partial oxygen pressure (pO ₂ ):

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 decrease in pO ₂ . It is speculated that the observed growth is due to the outgrowth and vegetative growth of the germinated spores of the present invention. The pO ₂ levels of these tests are shown in Figure 8.

As shown in FIG. 8, incubation with the test mixture and method according to the preferred embodiment of the present invention (treated spores at 42 ° C., using a trophic germination factor composition and heating) results in a better spore ratio than without using the present invention. Examples Control or spore mixtures that were treated or heated or that had been treated or heated with a vegetative germination composition but were not both together started vegetative growth for 4 hours. The growth seen in the control group experiments may represent the presence of about 2% of the germinating spores in the FreeFlow LF-88 probiotic (see Example 1). This example further shows that when using the nutritional germination factor composition and cultivation method according to the preferred embodiment of the present invention, the spore germination is significantly increased.

Example 3-Another set of incubation tests was performed using a similar test and control mixture and incubation method as described in Example 1 above. In short, LF-88 was added to 10 nL of distilled water to a final concentration of about 10 ⁸ CFU / mL. Samples were incubated at various temperatures to show the efficacy of the test method according to a preferred embodiment of the present invention compared to the control mixture. Reactions were prepared using the vegetative germination factor composition described in Example 1 ("treated spores" in Figure 9) and incubated at 23 ° C (ambient temperature, unheated), 32 ° C, 42 ° C, or 60 ° C. The control group reaction was incubated with no trophic germination factor composition at ambient room temperature. 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 Basic Incubator (containing 3% glucose as a carbon source and trace elements) containing excessive D-alanine.

Approximately 1.2 × 10 ⁵ CFU was added to each of the four slots of the PreSens OxoPlate. Control groups were performed as described by the manufacturer and measured on a BioTek 800FLx fluorescent plate reader using two filter pairs (excitation: 540 nm, emission: 650 nm and excitation: 540, emission: 590 nm). Continuous shaking at a time point of 24 hours every 10 minutes at 37 ° C, and the data were processed to determine the partial pressure of oxygen (pO ₂ ). The pO ₂ levels of these tests are shown in FIG. 9.

As shown in FIG. 9, incubation with the vegetative germination factor composition and heating according to the preferred embodiment of the present invention causes the spores to begin vegetative growth before the control group. The spores treated with the vegetative germination factor composition but not heated were comparable to the control mixture. Ratio of spores (represented by the "treated spores 32 ° C" curve) treated with vegetative-germination factor composition cultivated at a temperature lower than the preferred range of 35-55 ° according to one embodiment of the present invention Control group experiments begin vegetative growth faster, but not as quickly as spores treated at elevated temperatures within the preferred range according to the present invention. The spores treated with the vegetative-germination factor composition according to the embodiment of the present invention and cultivated at a temperature in the optimal range of 41 ° C to 44 ° C showed the best results, and the vegetative growth was started first and the control group 4 hours before starting to grow. As can be seen in the foregoing examples, the growth seen in the untreated control group experiments may represent the presence of approximately 2% of the germinating spores in the FreeFlow LF-88 probiotics (see Example 1). This example further shows that when using the nutritional germination factor composition and cultivation method according to the preferred embodiment of the present invention, the spore germination is significantly increased.

Aquaculture Research Using Nutrient Germination Factor Composition and Spore Composition

Another study was performed with the preferred germination and aquaculture treatment method according to the present invention to evaluate the use of better nutritional germination factors and spore compositions. This study uses three types of aquaculture as representative aquaculture applications. Each holds 55 gallons of water and 25 Malaysian shrimp to mimic the stocking density of a commercial shrimp farm. Each aquarium contains the same type of net and a matrix of PVC pipes for the habitat and rest of the shrimp. All aquariums are lined with live Caribbean sand to prevent algae growth, reduce nitrate, help buffer the aquarium system, and ensure safer aquarium circulation. Ventilation stones were used in all three aquariums to improve biofiltration and increase the amount of dissolved oxygen in shrimp and beneficial bacteria. All three aquariums use the same type of filter, and rinse the filter as needed and reuse it. As needed, all three aquariums are refilled with deionized (DI) water. DI water is used to control the mineral content of the water.

When a large amount of water needs to be taken from the aquarium, the same amount of water is taken from all the aquariums and replaced with the same amount of DI water. Water replacement using calcium carbonate in aquariums 2 and 3 simulates the use of a pond powder, such as the ECOCharger ^™ pool powder available from NCH Life Science. When changing the water in the aquarium 2 or 3, add about 0.5 g of calcium carbonate to the tank water. Approximately 1 mL of the cultured or activated bacterial solution is applied to the aquarium 3 once a day every Monday to Friday. Aquarium 1 is the control group aquarium. In short, 20 µL of starting spore solution (containing about 10 ¹⁰ CFU / mL), 20 µL of initial concentrated nutrient solution, and 960 µL of water were mixed to form a working solution containing about 2 × 10 ⁸ CFU / mL spores (Table 6). The initial spore solution contained approximately 10 ¹⁰ CFU / mL spores from the spore mixture. The spore mixture contains three bacterial strains of the genus Bacillus: two strains are each a strain of Bacillus licheniformis , and a third strain is Bacillus subtilis . About 80% of the spore mixture formula is Bacillus licheniformis (40% per strain) spores and 20% of the spore mixture formula is Bacillus subtilis . According to the above-mentioned preferred embodiment, the spore composition also includes water, a thickener, and an organic salt.

The combined working solution (containing the vegetative germination factor composition, 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 (about 1 mL) was added to the aquarium 3. The mixing is done by aerating the mixed stones. Table 5 shows the composition of the initial nutritional germination factor formula. Table 6 shows the composition of the cultivated working solution. After mixing the cultured bacterial solution to a 55-gallon aquarium 3, the concentration of bacteria was about 9.6 × 10 ² CFU / mL and the final percentage of the trophic germ factor composition in the aquarium 3 was about 9.6 × 10 ^-6 % v / v. The contents of each aquarium after individual treatments have been applied are shown in Table 7. The trial lasted 120 days.

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

Figure 10 shows images of three aquariums, which can show the water clarity of each group at the end of the 120-day test. In terms of water clarity, the aquarium 3 was observed to be the cleanest. It was observed that aquarium 1 (control group) had the largest amount of algae growth covering the aquarium wall compared to aquariums 2 and 3. It was observed that compared to the control group and the aquarium 3, the aquarium 2 had only moderate algae growth on the aquarium wall.

During the 120-day test, all three aquariums started with almost no algae on the side of the aquarium. As the test progressed, the control group aquarium (Aquarium 1) accumulated more algae growth on the sides of the aquarium (see, for example, Figure 10). Compared to the aquarium 1, the 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 test. All three aquariums have zero amine levels. Nitrite / nitrate was also within the safe range during the test. The pH of all aquariums also stays within the normal range of about 7.5 to 8.5. Since the aquarium cycle occurred safely and the parameters remained consistent throughout the 120-day trial, no peak water parameters were observed to cause damage to the shrimp.

Persons with ordinary skill in the art will also understand that after reading this description and the description of the preferred embodiments herein, methods and modifications and changes of vegetative germination factors and spore compositions can be performed within the scope of the present invention, and this article The scope of the disclosed invention is limited to the broadest interpretation of the scope of the appended patents to which the inventor is legally entitled.

10‧‧‧on-site incubator system

12‧‧‧ Nutritional germination factor composition

14‧‧‧ spore composition

16‧‧‧ the source of water

18‧‧‧ Incubator

20‧‧‧ Incubated bacterial solution

22‧‧‧Growth pond

110‧‧‧Incubator system

24‧‧‧Concentrated vegetative germination factor composition

26‧‧‧ water / thinner

28‧‧‧Working nutritional germination factor composition

30‧‧‧ concentrated spore composition

32‧‧‧Working spore composition

210‧‧‧System

34‧‧‧Concentrated vegetative spore composition

36‧‧‧Working nutritional spore composition

The system and method of the present invention are further described and illustrated in the following drawings.

FIG. 1 is a flowchart of a cultivation system and method according to a preferred embodiment of the present invention.

FIG. 2 is a flowchart of a cultivation system and method according to another preferred embodiment of the present invention.

FIG. 3 is a flowchart of a cultivation system and method according to another preferred embodiment of the present invention.

Figure 4 is a graph of nitrate levels in a laboratory study.

Figure 5 is a graph of orthophosphate levels in laboratory studies.

Figure 6 is a diagram of turbidity in a laboratory study.

FIG. 7 is a photograph showing a bacterial slide using a spore composition and method according to a preferred embodiment of the present invention compared to a control slide.

FIG. 8 is a diagram showing pO ₂ test data to verify the degree of germination using a spore composition and method according to a preferred embodiment of the present invention compared to a control group test.

FIG. 9 is a graph showing pO ₂ test data to verify the degree of germination using a method of spore composition and changes according to a preferred embodiment of the present invention compared to a control group test.

Figure 10 shows images of three aquariums, each in the control group (left), treated with calcium carbonate only (middle), or treated with an activated vegetative-spore formula (right).

Claims (31)

A method of adding bacteria to water for aquaculture applications, the method comprising: Provide a volume of vegetative germination factor composition and a volume of bacterial spores, which can be premixed or separated as a vegetative spore composition; If the two are separated, optionally mixing a portion of the vegetative germination factor composition with a portion of the bacterial spores to form the vegetative spore composition; Heating a portion of the vegetative spore composition to a temperature in the range of about 35 ° C to 60 ° C at or near the location of the aquaculture application; Maintaining the temperature in the range for a period of about 2 minutes to about 6 hours to form a batch of germinating bacterial solution; During the processing cycle, the heating and maintaining steps are repeated periodically to form additional batches of germinating bacteria solution; Dispersing each batch of germinating bacterial solution in the water used in the aquaculture application; Provide nitrification enhancer; Simultaneously dispersing the nitrification enhancer in the water with at least one of the batches of germinating bacteria solution; and The bacteria can be used to repair the water by degrading organic waste and inhibiting the growth of pathogenic bacteria, or the bacteria are probiotics of the species in the aquaculture application. The method according to Claim 1 patentable scope, wherein the bacterium is selected from the group consisting of Bacillus (Bacillus), Bacteroides (Bacteriodes), the genus Bifidobacterium (Bifidobacterium), the genus Candida albicans (Lueconostoc), Pediococcus ( Pediococcus), Enterococcus genus (Enterococcus), Lactobacillus (Lactobacillus), Megasphaera (Megasphaera), Pseudomonas (of Pseudomonas), and the genus Propionibacterium (Propionibacterium) consisting of the group. Method of Application The patentable scope of the two, wherein the bacterium is a Bacillus licheniformis or more (Bacillus licheniformis) and Bacillus subtilis (Bacillus subtilis) in. For example, the method of claim 2 in the patent scope, wherein the probiotic is selected from the group consisting of Bacillus amylophilus , Bacillus licheniformis , Bacillus pumilus , Bacillus subtilis , Bacteriodes ruminocola , Bacteriodes ruminocola , Bacterioides suis , Bifidobacterium adolescentis , Bifidobacterium animalis , Bifidobacterium Bifidobacterium bifidum , Bifidobacterium infantis , Bifidobacterium longum , Bifidobacterium thermophilum , Enterococcus cremoris , Enterococcus diacetylactis , Enterococcus diacetylactis , feces enterococci (Enterococcus faecium), intermediate enterococci (Enterococcus intermedius), Enterococcus acid (Enterococcus lactis), thermophilic enterococci (Enterococcus thermophiles), Lactobacillus brevis (Lactobacillus brevis), Brookfield milk Bacillus (Lactobacillus buchneri), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus casein (Lactobacillus casei), cellobiose Lactobacillus (Lactobacillus cellobiosus), bending Lactobacillus (Lactobacillus curvatus), Lactobacillus delbrueckii (Lactobacillus delbruekii), Lactobacillus farciminis , Lactobacillus fermentum , Lactobacillus helveticus , Lactobacillus lactis , Lactobacillus plantarum , Lactobacillus reuteri , Lactobacillus reuteri Leuconostoc mesenteroides , Megasphaera elsdennii , Pediococcus acidilacticii , Pediococcus cerevisiae , Pediococcus pentosaceus , Propionate Groups bacilli (Propionibacterium acidipropionici), freudenreichii (Propionibacterium freudenreichii), and Xie Propionibacterium acnes (Propionibacterium shermanii) composed of For example, the method of claim 1 in the patent application scope, wherein the composition of the nutritional germination factor comprises: L-amino acid; One or more buffers including phosphate buffer, HEPES, Tris base, or a combination thereof; Industrial preservatives; Optionally D-glucose, or optionally D-fructose, or optionally both D-glucose and D-fructose; and Optionally a source of potassium ions. For example, the method of claim 5, wherein the L-amino acid is L-alanine, L-aspartic acid, L-valine, L-cysteine, soy protein hydrolysate, Or a combination. The method of claim 5, wherein the nutritional germination factor composition comprises a total of about 17.8 g / L to 89 g / L of one or more L-amino acids. The method according to item 5 of the patent application, wherein the vegetative germination factor composition and spores are mixed in advance, and the vegetative spore composition further includes spores and germination inhibitors of Bacillus species. The method of claim 8 in which the germination inhibitor or preservative includes sodium chloride, D-alanine, or a combination thereof. The method of claim 9 in which the pre-mixed vegetative spore composition comprises about 29 g / L to 117 g / L of sodium chloride. The method according to item 9 of the patent application, wherein the pre-mixed vegetative spore composition comprises about 8 g / L to 116 g / L of D-alanine. The method according to item 7 of the patent application, wherein the phosphate buffer solution comprises about 10-36 g / L of sodium dihydrogen phosphate and about 30-90 g / L of disodium hydrogen phosphate. For example, the method of claim 1 in the patent application range, wherein the nutritional germination factor composition or the nutritional spore composition that has been mixed in advance is a concentrated liquid, and the concentrated liquid includes: About 8.9-133.5 g / L of one or more L-amino acids; A total of about 0.8-3.3 g / L of one or more industrial preservatives; A total of about 40-126 of one or more phosphate buffers, about 15-61 g / L of Tris base, or about 32.5-97.5 g / L of HEPES, or a combination thereof; Optionally about 18-54 g / L of D-glucose, D-fructose, or a combination thereof; Optionally KCl of about 7.4-22.2 g / L; and One or more bacterial species, optionally in the form of spores. If the method of applying for the scope of the patent No. 13 further includes: Adding a diluent to the vegetative germination factor composition or the pre-mixed vegetative spore composition before or during heating; and During the incubation period, the diluted vegetative germination factor composition is mixed with the bacterial spores or the diluted vegetative spore composition. The method according to item 14 of the application, wherein the concentration of the diluted vegetative 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 the growth of the nitrifying bacteria biofilm, or both. The method of claim 1, wherein the nitrification enhancer is calcium carbonate, calcified seaweed, or both. A method as claimed in claim 17 in which the nitrification enhancer is provided in the form of a prill, pellet, or pellet. The method of claim 16 further includes providing an additional surface area modifier and dispersing the surface area modifier in the water with at least one batch of active bacteria simultaneously. For example, the method of claim 19, wherein 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 wherein the alkalinity enhancer is dispersed in the water at the same time as a batch of germinating bacterial solution. For example, the method of claim 1 in which the bacterial spores are in a separate spore composition, and the spore composition includes: One or more Bacillus species in the form of spores; About 0.002 to 5.0% by weight of thickener; A total of about 0.01 to 2.0% by weight of one or more acids or acid salts; and Optionally about 0.00005 to 3.0% by weight of a surfactant; The percentages are based on the weight of the spore composition. For example, 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; and Thickener. For example, the method of claim 23, wherein the Bacillus sp. Is a bacterium species in which the bacterial species include Bacillus pumilus , Bacillus licheniformis , Bacillus amylophilus , and B. subtilis Bacillus (Bacillus subtilis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus clausii (Bacillus clausii), Bacillus firmus (Bacillus firmus), Bacillus megaterium (Bacillus megaterium), potato bacillus (Bacillus mesentericus), One or more of Bacillus subtilis var. Natto , or one or more of Bacillus toyonensis . The method of claim 23, wherein the spore composition has a pH of about 4.5 to about 5.5. The method of claim 23, 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 applying scope 23 of the patent, wherein the spore composition includes: About 0.002 to 5.0% by weight of thickener; A total of about 0.01 to 2.0% by weight of one or more acids or acid salts; and Optionally about 0.00005 to 3.0% by weight of a surfactant; The percentages are based on the weight of the spore composition. The method according to item 4 of the patent application, wherein the incubation period is about 2 minutes to about 5 minutes. A method as claimed in claim 28, wherein the aquaculture application is a growth pond containing fish or eel. For example, the method of claim 1 in the patent scope, wherein the incubation period is about 4 to 6 hours. A method as claimed in claim 30, wherein the aquaculture application is a growth pond containing shrimp.