# Patent

## Title
Methods of improving the yield and/or quality of aquatic or marine animals

## Abstract

Methods of treating aquatic or marine animal water bodies including contacting the water body with one or more sulfur oxidizing bacteria such as those selected from the genus Paracoccus in an amount sufficient to control, reduce, or eliminate the H2S in the water body is disclosed. One or more sulfur oxidizing bacteria selected from the genus Paracoccus are applied to H2S contaminated environments in a predetermined amount effective in improving the yield and quality of aquatic or marine animals therein. Compositions useful for treating aquatic or marine animal water bodies are also described.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority or the benefit under 35 U.S.C. 119 of U.S. provisional application No. 60/911,718 filed Apr. 13, 2007, the contents of which are fully incorporated herein by reference.


BACKGROUND
1. Technical Field
This disclosure relates to water treatment in general and to methods of controlling hydrogen sulfide in aquatic and marine environments. The methods are useful for application to aquatic or marine animal ponds and sediments in need of treatment to control, minimize and/or eliminate hydrogen sulfide (H2S) to promote the yield and/or quality of aquatic and marine animals.
2. Background
Many aquatic and marine environments such as fish farms, pools, ponds, waste lagoons, lakes, estuaries, and oceans contain one or more aerobic zones and one or more anaerobic zones in the water column. In aerobic zones near the surface of the water column, air and winds introduce oxygen and aerobic bacteria produce, inter alia, phosphates, carbon dioxide and ammonia. In anaerobic zones near the bottom of the water column, anaerobic microorganisms tend to generate, inter alia, hydrogen sulfide, ammonia, and methane. Hydrogen sulfide accumulations in the anaerobic zones and sediments below are toxic and undesirable for they stress the aquatic and marine animal populations. In some cases, such as commercial fish and shrimp ponds, where populations live on or near the bottom of the water body in or near the anaerobic zone, the accumulation of hydrogen sulfide decreases the yield of the population and/or quality of individuals thereof.
There are several approaches to problems associated with the anaerobic zones known in the art. For example, chemical oxidizers, such as ozone, chlorine dioxide, and hydrogen, calcium, or magnesium peroxide, may be added to the water body to alleviate the anaerobic conditions. Although these methods may be effective, they may be expensive, pressuring users to minimize the amount of oxidizers added to the water body. The need to economize can be detrimental if it leads to under dosing. Under dosing is problematic in that it can cause incomplete or partial oxidation, creating odor problems. When chlorine dioxide is added to organic acids, incomplete oxidation can cause the formation of chloroacetic acid, which has a foul odor noticeable at very low concentrations. However, these steps are designed to eliminate odor and may be detrimental to the aquatic and marine animals in the environment such as shrimp or fish.
Other known methods for controlling hydrogen sulfide include treating water with oxygen. For example, U.S. Pat. No. 5,876,990 describes a biochemical media system for reducing pollution. The system includes a first media which provides an oxygen inducer to dissipate slowly into aqueous surroundings, and a second media which provides an oxygen supplier. The two media are combined in aqueous environment to generate nascent oxygen at a modulated rate such that the oxygen is absorbed into the surrounding aqueous environment, promoting growth of aerobic species and reducing biological pollution. However, the use of such a system does not necessarily alter the hydrogen sulfite concentration in the sediment below the water column and may also be detrimental to the aquatic and marine animals in the environment. Moreover, the cost of such a treatment may well outweigh the potential economic benefit of this for fish or shrimp farming.
Of interest is U.S. Pat. No. 7,160,712 (herein incorporated by reference in its entirety) which relates to methods for treating odors in wastewater treatment bodies. Here, odor problems are treated by altering the water chemistry to make the reduction of sulfur chemical species thermodynamically unfavorable, and bacteria, such as Paracoccus pantotrophus are added to the water. However, there is no suggestion to use such methods for improving the yield and/or quality of aquatic and marine animals such as in commercial fish farms.
Other methods of reducing hydrogen sulfide levels in a contaminated aquaculture include partial water exchange. However flushing methods may be impractical, expensive, and potentially dangerous if flush water introduces contaminants such as viruses and/or disease.
While chemicals and water flushing may be effective against hydrogen sulfide, these measures are expensive and may lead to a reduced yield and/or quality of aquatic or marine animals grown therein. It is thus desirable to have an effective, more problem free, biological or biochemical system that controls hydrogen sulfide accumulations in aquatic or marine environments and sediments thereof.
SUMMARY
It has been found that aquatic and marine animal yield and/or quality can be improved by treating an anaerobic condition in a water body and/or sediment thereof. The treatments include adding an effective amount of one or more sulfur oxidizing bacteria such as those selected from the genus Paracoccus to the water body and/or to the pond sediment. Such sulfur oxidizing bacterial are capable of oxidizing sulfide, (S2−) to elemental sulfur (S0), (S4+) which exists as sulfite (SO32−), or (S6+) which exists as sulfate SO42−. In embodiments, the treatment further includes one or more steps which alter the water chemistry to make the reduction of sulfur chemical species thermodynamically unfavorable.
The present disclosure provides one or more novel methods for treating a water body and/or sediment by contacting the water body and/or sediment thereof with an effective amount of one or more sulfur oxidizing bacteria selected from the genus Paracoccus. 
The present methods are suitable where the hydrogen sulfide forms in a water body and/or sediment layer near the bottom of a water body, and in embodiments where sufficient amounts of nitrate are available to act as an electron acceptor. Suitable water bodies include aquacultures, pools, ponds, paddies, fish farms, lakes, streams, rivers, oceans, estuaries, waste lagoons, contaminated portions thereof, and combinations thereof.
In embodiments, a method of treating aquatic or marine animals includes contacting a water body or sediment thereof, or in need of treatment or decontamination, with one or more sulfur oxidizing bacteria selected from the genus Paracoccus in an amount sufficient to control, reduce, or eliminate the H2S in the water body or sediment. In embodiments, the amount of sulfur oxidizing bacteria is sufficient to establish a concentration of the sulfur oxidizing bacteria in the sediment in an amount of about 100 CFU/g to about 100,000 CFU/g. In embodiments, the amount of sulfur oxidizing bacteria is sufficient to establish a concentration of the sulfur oxidizing bacteria in the water body in an amount of about 100 CFU/ml to about 100,000 CFU/ml. The water body may be an aquaculture farm, pool, pond, waste lagoon, lake, estuary, ocean, contaminated portion thereof, or combinations thereof. In embodiments, suitable methods may include the step of modifying the chemistry of the water body by establishing a concentration of nitrates in the water body at about 0.01 ppm to about 500 ppm, or a concentration of nitrates in the water body at about 1.0 ppm to about 250 ppm, or a concentration of nitrates in the water body at about 0.01 ppm to about 10 ppm For example, the chemistry of the water body may be modified by establishing a concentration of nitrates in the water body at 200 ppm or about 200 ppm. Such methods may also include the step of stocking a water body with aquatic or marine animals such as fish, shrimp, lobster, cray fish, bottom dwelling fish, finfish, prawns, oysters, mussels, cockles, Mollusks, and combinations thereof. In embodiments, the effective amount of sulfur oxidizing bacteria is an amount sufficient to maintain H2S below 1 part-per-million (ppm) for at least 1 week. In embodiments, the amount of Paracoccus is 0.01 ppm to 500 ppm. Additional microorganisms can also be added in accordance with the present disclosure.
In embodiments, a method for increasing aquatic and marine animal yield in a water body includes: reducing the amount of H2S in a water body and sediment thereof by establishing a sulfur oxidizing bacteria population selected from the genus Paracoccus (including but not necessarily limited to Paracoccus pantotrophus) in the water body in an amount of 100 CFU/ml to 100,000 CFU/ml and in the sediment is an amount of 100 CFU/g to 100,000 CFU/g; and modifying the chemistry of the water body by establishing a concentration of nitrates in the water body at 0.01 ppm to 500 ppm. Such methods may include the step of modifying the chemistry of the water body including establishing an oxidation reduction potential level in the water body or in the sediments of the water body of at least about −330 mV, wherein the final amount of H2S in the water body is below 1 part-per-million. In embodiments, the final amount of H2S in the water body or sediments is below 0.5 part-per-million, 0.4 part-per-million, or 0.3 part-per-million. In embodiments, the final amount of H2S in the water body or sediments is in an amount of 0.1 part-per-million to 0.5 part-per-million. In embodiments, the methods include establishing a pH in the water body of about 6.0 to 8.5. In embodiments, the yield is increased in comparison to a similar or substantially similar water body having no sulfur oxidizing bacteria added thereto in accordance with the present disclosure.
In embodiments, a method for increasing aquatic and marine animal quality or yield in a water body includes: reducing the amount of H2S in a water body by establishing a sulfur oxidizing bacteria population selected from the genus Paracoccus in the water body in an amount of about 100 CFU/ml to about 100,000 CFU/ml; and modifying the chemistry of the water body by establishing a concentration of nitrates in the water body at about 1 ppm to about 250 ppm, wherein the final amount of H2S in the water body is in an amount of 0.1 part-per-million to 0.5 part-per-million. In embodiments, such methods further include the step of stocking the water body with aquatic or marine animals selected from the group consisting of fish, shrimp, lobster, eel, crayfish, bottom dwelling fish, finfish, prawns, oysters, mussels, cockles, Mollusks, and combinations thereof. Methods in accordance with the present disclosure are suitable for increasing the yield and quality of any organism grown in an aquaculture including but not limited to fish and/or plants. In embodiments, such methods include the step of pulsing sulfur oxidizing bacteria into the water body in order to obtain the initial concentration of bacteria in the water body in an amount of about 100 CFU/ml to about 100,000 CFU/ml until harvest of the aquatic or marine animals or other desirable beneficial manifestation.
In embodiments, the present disclosure includes one or more compositions for treating aquatic and marine animals comprising a predetermined amount of Paracoccus pantotrophus. 
Embodiments of the present disclosure include one or more admixtures of microorganisms which used in combination, reduce, eliminate or control H2S in a water body or sediment. For example, admixtures containing predetermined amounts of Paracoccus pantotrophus in combination with one or more additional microorganisms are disclosed.
In embodiments, compositions in accordance with the present disclosure comprise or consist of at least one additional microorganism which comprises or consists of Bacillus alkalophilus, Bacillus brevis, Bacillus coagulans, Bacillus circulans, Bacillus clausii, Bacillus lichenifonnis, Bacillus lentus, Bacillus amyoliquofaciens, Bacillus lautus, Bacillus megaterum, Bacillus subtilus, Bacillus stearothermophilus, Bacillus pumilus, Bacillus pasteunfi, Bacillus Thuringiensis, or combinations thereof. In embodiments, compositions in accordance with the present disclosure comprise or consist of one of the above Bacillus, wherein Paracoccus such as Paracoccus pantotrophus and Bacillus are combined to form an admixture characterized by a weight ratio in the amount of 4:1 to 1:4. In embodiments, compositions in accordance with the present disclosure comprise or consist of Bacillus megatenum, wherein Paracoccus pantotrophus and Bacillus megateium are combined to form an admixture characterized by a weight ratio in the amount of 4:1 to 1:4.
In embodiments, the present disclosure relates to methods of treating aquatic or marine animals comprising: contacting a water body or sediment thereof with one or more sulfur oxidizing bacteria in an amount sufficient to control, reduce, or eliminate the H2S in the water body or sediment. The one or more sulfur oxidizing bacteria may be admixed with other bacteria such as Bacillus depending upon the goals of the treatment and water conditions. The admixture can be made in various weight/weight ratios.
Methods of controlling, reducing and/or minimizing hydrogen sulfide by contacting a water body and/or sediment with Paracoccus pantotrophus alone or in combination with other bacteria are also disclosed, along with suitable formulations for use in accordance with the present disclosure.


DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
In anaerobic zones near the bottom of the water column or in the bottom sediment, anaerobic microorganisms such as sulfate reducing bacteria (SRB's) tend to generate, interalia, hydrogen sulfide, ammonia, and methane. For example, sulfur reducing bacteria (SRB) reduce the sulfur in sulfate (SO42−) (S6+) to (S2−) containing compounds as a terminal electron acceptor and produce hydrogen sulfide (H2S) in the process. Hydrogen sulfide (H2S) accumulations in aquatic and marine animal waters and bottom sediments, such as commercial aquaculture ponds, stress and kill the aquatic and marine animals such as shrimp, certain fish such as bottom dwelling fish, lobster, and crayfish, or any aquaculture organism(s) which may tend to stay at the bottom of the water body. In order to increase aquatic and marine animal yield and/or quality, toxic or increased accumulations of H2S in the anaerobic layer and/or sediments below can be controlled, reduced and/or eliminated. Thus, it has now been found that aquatic and marine animal yield and/or quality can be improved by adding an effective amount of sulfur oxidizing bacteria such as Paracoccus pantotrophus to a water body or bottom sediments. In embodiments, the treatment further includes one or more steps which alter the water chemistry to make the reduction of sulfur chemical species unfavorable.
Accordingly, the present disclosure provides methods and compositions for treating aquatic and marine environments suitable for growing aquatic and marine animals. The methods include applying a predetermined amount of sulfur oxidizing bacteria such as Paracoccus pantotrophus to an area in need of control or treatment such as an H2S contaminated area. The sulfur oxidizing bacteria may be in solution and made available to reduce, control and/or eliminate H2S. In embodiments, suitable treatments include the addition of bacteria, such as Paracoccus pantotrophus (formerly called Thiosphaera pantotropha), capable of oxidizing sulfide, (S2−) to elemental sulfur (S0), (S4+) which exists as sulfite (SO32−), or (S6+) which exists as sulfate SO42. In one embodiment, Paracoccus pantotrophus (ATCC strain 35512, LMD (Delft Collection of Microorganisms) 82.5) is added alone in an effective amount to treat a contaminated water body and/or sediment.
Suitable non-limiting examples of sulfur oxidizing bacteria for use in accordance with this disclosure include one or more bacteria selected from the genus Paracoccus. Non-limiting examples of suitable Paracoccus include: P. alcaliphilus, P. alkenifer, P. aminophilus, P. aminovorans, P. cartinifaciens, P. denittificans, P. kocuii, P. marcusii, P. methylutens, P. pantotrophus, P. solventivorans, P. thiocyanatus, P. versustus, and combinations thereof. In embodiments, solely the use of P. pantotrophus is suitable for use in accordance with the present disclosure. In embodiments, a combination of P. pantotrophus and one or more other Paracoccus is suitable for use in accordance with the present disclosure. In embodiments, any sulfur oxidizing bacteria and/or combinations thereof may be suitable for use in accordance with the present disclosure. For example, a combination of P. pantotrophus and one or more sulfur oxidizing bacteria may be suitable for use in accordance with the present disclosure.
Additional non-limiting examples of suitable combinations include P. pantotrophus in combination with P. alcaliphilus; P. pantotrophus in combination with P. alkenifer, P. pantotrophus in combination with P. aminophilus; P. pantotrophus in combination with P. aminovorans; P. pantotrophus in combination with P. cartinifaciens; P. pantotrophus in combination with P. denitrificans; P. pantotrophus in combination with P. kocuri; P. pantotrophus in combination with P. marcusli; P. pantotrophus in combination with P methylutens; P. pantotrophus in combination with Bacillus megaterium; P. pantotrophus in combination with P. solventivorans; P. pantotrophus in combination with P. thiocyanatus; P. pantotrophus in combination with P. versustus. 
Other suitable combinations include of P. pantotrophus in combination with two other types of bacteria. For example, P. pantotrophus in combination with P. alcaliphilus and P. alkenifer, P. pantotrophus in combination with P. alkeniferand P. aminophilus; P. pantotrophus in combination with P. aminovorans and P. carinifaciens; P. pantotrophus in combination with P. denitrificans and P. kocurii; P. pantotrophus in combination with P. marcusli and P methylutens; P. pantotrophus in combination with P. pantotrophus and P. solventivorans; P. pantotrophus in combination with P. thiocyanatus and P. versustus. Other combinations of P. pantotrophus with two bacteria would readily be envisioned by one of skill in the art.
Other suitable combinations include of P. pantotrophus in combination with three other bacteria. For example, P. pantotrophus in combination with P. alcaliphilus, P. alkenifer and P. aminophilus; P. pantotrophus in combination with P. aminovorans, P. cartinifaciens and P. denittificans; P. pantotrophus in combination with P. marcusii, P. kocurii and P methylutens; P. pantotrophus in combination with P. solventivorans, P. thiocyanatus and/or P. versustus. Other combinations of P. pantotrophus with three bacteria would readily be envisioned by one of skill in the art.
Other suitable combinations include P. pantotrophus in combination with four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, etc. other types of bacteria. Other combinations of P. pantotrophus with multiple bacteria would readily be envisioned by one of skill in the art.
In embodiments, one or more sulfur oxidizing bacteria such as those selected from the genus Paracoccus can be applied to one or more water bodies to control, minimize and/or eliminate undesirable H2S to promote aquatic and/or marine animal populations. As used herein the word “treat,” “treating” or “treatment” refers to using the bacteria in accordance with the present disclosure such as those selected from the genus Paracoccus prophylactically to prevent H2S buildup which may have a detrimental effect on aquatic and/or marine animal populations, or to ameliorate an existing H2S contamination having a detrimental effect on aquatic and/or marine animal populations. A number of different treatments are now possible, which promote the yield and quality of aquatic and/or marine animal populations such as shrimp, lobster, cray fish, bottom dwelling fish, organisms suitable for aquaculture, and combinations of aquatic and/or marine animal populations.
The mode of action of sulfur oxidizing bacteria is known and is further described in U.S. Pat. No. 7,160,712 (herein incorporated by reference in its entirety). Although not wishing to be bound by any disclosure including the present disclosure, it is believed that sulfur oxidizing and sulfur reducing bacteria use soluble organic matter measured in terms of five day biochemical oxygen demand or BOD5 as an energy source. Some bacteria, such as Paracoccus can use reduced sulfur compounds (i.e., H2S), as their energy source. When sufficient amounts are not available in the water body, such bacteria also need an electron acceptor to metabolize the energy sources. The nature of the electron acceptor present in the water often promotes the microbial culture that thrives in the environment. The most energetically favorable reaction in water is usually the reduction of oxygen (O2) to water (H2O). The presence of dissolved oxygen in the water will often allow bacteria that are not capable of using sulfur compounds as a terminal electron acceptor to metabolize the organic matter in the water. Additionally, most sulfur reducing bacteria are capable of using oxygen rather than sulfur as the terminal electron acceptor, and will use oxygen when sufficient amounts are available in the water. When there is insufficient oxygen present, sulfur reducing bacteria use the sulfur in sulfate SO42− containing compounds as the terminal electron acceptor and produce hydrogen sulfide (H2S) in the process.
As used herein “H2S contamination” refers to any detectable H2S manifestation(s). Such manifestations can be caused by a number of factors such as, for example, type and amount of bacteria present, water column nutrient levels, dissolved oxygen levels and/or other stressed or dysfunctional environmental states. Non-limiting examples of such manifestations include the development of anaerobic bacterial growths, stress and/or kills on aquatic and fish populations, and/or forms of reduced aquatic and marine animal quality such as reduced size or shape of the animals. For example, manifestations include reduced yields and quality of bottom dwelling animals such as fish, shrimp, lobster, bottom dwelling fish, aquaculture organism, or crayfish that live in or near anaerobic zones. It is understood, that the manifestations are non-limiting and that only a portion of the manifestations suitable for treatment in accordance with the present disclosure are listed herein.
In embodiments, compositions for use in accordance with the present disclosure contain one or more sulfur oxidizing bacteria such as those selected from the genus Paracoccus in an effective amount to improve water conditions and/or alleviate any detrimental manifestations of H2S. As used herein “effective amount” refers to an amount of sulfur oxidizing bacteria sufficient to induce a particular positive benefit to aquatic or marine animal populations such as increased yield of the total population and/or size and quality of individual organisms in the population. The positive benefit can be cosmetic in nature where the organism looks bigger or healthier on the outside, or health-related, or a combination of the two. In embodiments, the positive benefit is achieved by contacting an H2S contaminated aquatic environment with a combination of bacteria such as those from the genus Paracoccus to improve aquatic or marine animal yield and/or quality. Treatments include contacting H2S contaminated aquatic environments with an amount of bacteria selected from the genus Paracoccus effective to eliminate H2S, and/or reduce H2S to levels not immediately toxic or detrimental to aquatic or marine animals, for example, to less than 0.03 ppm in the pH range of 6.0-9.0 in a water body and/or sediments immediately below the water column. In embodiments, the final amount of H2S in the water body or sediment is an amount of 0.01 part-per-million to 0.5 part-per-million, or an amount of 0.1 part-per-million to 0.5 part-per-million.
The particular sulfur oxidizing bacteria concentration applied generally depends on the purpose for which the bacteria are to be applied. For example, the dosage and frequency of application can vary depending upon the type and severity of the H2S contamination. In embodiments, one or more sulfur oxidizing bacteria such as those bacteria selected from the genus Paracoccus are applied to a water body in an amount sufficient to establish the concentration the sulfur oxidizing bacteria in the water body at about 100 to 100,000 CFU/ml, or in the bottom sediment at about 100 to 100,000 CFU/g. In some embodiments, one or more sulfur oxidizing bacteria such as those bacteria selected from the genus Paracoccus are applied to a water body in an amount sufficient to establish the concentration the sulfur oxidizing bacteria in the water body at about 1000 CFU/ml to 10,000 CFU/ml, or in the bottom sediment at about 1000 CFU/g to 10,000 CFU/g. In embodiments, one or more sulfur oxidizing bacteria such as those bacteria selected from the genus Paracoccus are applied to a water body in an amount sufficient to establish the concentration the sulfur oxidizing bacteria in the water body at about 5000 CFU/ml to 10,000 CFU/ml, or in the bottom sediment at about 5000 CFU/g to 10,000 CFU/g. As used herein CFU/ml relates to colony forming units per milliliter, and CFU/g refers to colony forming units per gram.
In embodiments, one or more sulfur oxidizing bacteria such as those bacteria selected from the genus Paracoccus are applied to a water body in an amount sufficient to establish the concentration the sulfur oxidizing bacteria in the water body, or contaminated portion thereof in an amount of at least about 0.01 to 500 ppm, or an amount of at least about 0.1 to 250 ppm, or an amount of 0.1 to 100 ppm, or an amount of 0.1 to 50 ppm, or an amount of 0.1 to 10 ppm. In embodiments, one or more sulfur oxidizing bacteria such as those bacteria selected from the genus Paracoccus are applied to a water sediments in an amount sufficient to establish the concentration the sulfur oxidizing bacteria in the sediment, or contaminated portion thereof in an amount of at least about 0.01 to 500 ppm, or an amount of at least about 0.1 to 250 ppm, or an amount of 0.1 to 100 ppm, or an amount of 0.1 to 50 ppm, or an amount of 0.1 to 10 ppm. In some embodiments, sulfur oxidizing bacteria such as Paracoccus pantotrophus (ATCC strain 35512, LMD (Delft Collection of Microorganisms) is established in the water in need of treatment in an amount of 0.01 ppm to about 500 ppm. In some embodiments, sulfur oxidizing bacteria such as Paracoccus pantotrophus (ATCC strain 35512, LMD (Delft Collection of Microorganisms) is established in the sediment in an amount of 0.01 ppm to about 500 ppm.
In embodiments, sulfur oxidizing bacteria for use in accordance with this disclosure may be added to a water body in pulse applications. For example, bacteria such as Paracoccus pantotrophus may be applied to the treated ponds by pulsing on an hourly, daily, and/or weekly application to maintain the concentration at a predetermined or target amount. For example, sulfur oxidizing bacteria may be applied as a dry formulation containing 3.5×109 CFU/g, in amounts and intervals to maintain the overall amount of Paracoccus to be established or maintained at an amount of 100 to 100,000 CFU/ml every 1 to 7 days until treatment goals obtained and/or harvest, and in some embodiments, about 10 to 100,000 CFU/ml every 7 days until goals obtained or harvest.
The one or more sulfur oxidizing bacteria may be added to the influent of a pond, lagoon, or other water body. In large water bodies such as a lagoon, it may be desirable to broadcast the sulfur oxidizing bacteria over the water body in order to avoid waiting for the sulfur oxidizing bacteria to disperse throughout the water body. Aircraft, watercraft, shore based spraying units, and other conventional means of broadcasting may be used to apply the sulfur oxidizing bacteria to the water body. Rather than adding the sulfur oxidizing bacteria to the entire water body, it may be desirable to add it to areas in the water body where the anaerobic condition is located.
In embodiments, the one or more sulfur oxidizing bacteria can be used in combination with other microorganisms. Non-limiting examples of suitable microorganisms include on or more microorganisms selected from the group consisting of the genera Acinetobacter, Aspergillus, Azospirillum, Burkholdeda, Bacillus, Cedporiopsis, Enterobacter, Escherichia, Lactobacillus, Paenebacillus, Pseudomonas, Rhodococcus, Syphingomonas, Streptococcus, Thiobacillus, Trichoderma, Xanthomonas, and combinations thereof. Non-limiting examples of suitable microorganisms from the Bacillus genera may be selected from the group consisting of Bacillus alkalophilus, Bacillus brevis, Bacillus coagulans, Bacillus circulans, Bacillus clausii, Bacillus lichenifonnis, Bacillus lentus, Bacillus amyoliquofaciens, Bacillus lautus, Bacillus megaterum, Bacillus subtilus, Bacillus stearothermophilus, Bacillus pumilus, Bacillus pasteuni, Bacillus thuringiensis, or combinations thereof. For example, Bacillus megaterium having all of the characteristics of strain SB-3112, ATCC PTA-3142 and described in U.S. Pat. No. 6,649,401 (herein incorporated by reference in its entirety) may be used in combination with the sulfur oxidizing bacteria in accordance with the present disclosure. Paracoccus such as Paracoccus pantotrophus and these other microorganisms can be mixed in various weight ratios depending upon treatment goals and conditions. For example, Paracoccus can be admixed with one other microorganism to make a formulation characterized by a range of ratios (wt/wt.) of 10:1 to 1:10, for example: 9:2, 8:3, 7:4, 6:5, 5:6, 4:7, 3:8, 2:9, or 1:10.
In embodiments, the one or more sulfur oxidizing bacteria for use in accordance with the present disclosure may be mixed to form an admixture with other compositions such as PondPlus® brand Bacillus consortium and/or PondProtect® brand Nitrifier consortium both available from Novozymes.
In some embodiments, an admixture of sulfur oxidizing bacteria such as Paracoccus pantotrophus (ATCC strain 35512, LMD (Delft Collection of Microorganisms) in combination with Bacillus megaterium is established in the water or sediment in need of treatment in an amount of 0.01 ppm to about 500 ppm. Non-limiting examples of suitable admixture compositions for use in accordance with the present disclosure include a composition comprising, or consisting of Paracoccus pantotrophus and Bacillus megaterium as discussed above, in the range of ratios (wt/wt.) of 10:1 to 1:10, for example: 9:2, 8:3, 7:4, 6:5, 5:6, 4:7, 3:8, 2:9, or 1:10, and in one embodiment 1.5 to 2.5. Other suitable admixture ratios would be readily envisioned by one of ordinary skill in the art, including but not limit to weight ratios of 4:1 to 1:4.
In embodiments, treatments further include one or more steps which alter the water or bottom sediment chemistry to make the reduction of sulfur chemical species unfavorable. For example, where it is found that water or bottom sediment in need of treatment does not initially have chemicals suitable for serving as the terminal electron acceptor, one or more oxidizing agents, whose reduction is more energetically favorable than the reduction of sulfate, can be added to the water body. In embodiments, treatments are directed towards the bottom sediment as a prime target where the anaerobic zone is present and H2S is generated. In embodiments, the application of sulfur oxidizing bacteria such as those selected from the genus Paracoccus may be applied alone or in combination with one or more oxidizing agents suitable for acting as electron acceptors. In embodiments, suitable oxidizing agents are chemicals that most sulfur reducing bacteria cannot use as an electron acceptor. Non-limiting examples of suitable oxidizing agents include chemicals containing a nitrate (NO3−) constituent which can be biologically reduced to nitrogen gas N2 and/or hydrogen peroxide H2O2 which can then be biologically reduced to water. The addition of one or more oxidizing agents to the water body, which will also become present in the bottom sediment in combination with bacteria (initially present or added), will change the oxidation reduction potential (ORP) of the bottom sediments. The target ORP range in the bottom sediments is about −330 mV to +100 mV. In embodiments, low ORP will likely develop over time after the addition of the Paracoccus material. Further, in embodiments, microbes are added in anticipation of the H2S development, and will exist in the water and sediment on other nutrient sources, even aerobically, until conditions for H2S generation occurs.
Suitable non-limiting examples of one or more oxidizing agents for use in accordance with the present disclosure include oxygen O2, ozone O3, peroxides such as hydrogen peroxide H2O2, calcium peroxide CaO2.H2O, magnesium peroxide MgO2H2O, nitrates R(NO3)x, nitrites RNO2, permanganate KMnO4, potassium dichromate K2Cr2O7, potassium chlorate KClO3, and/or chlorine dioxide ClO2, and combinations thereof. In embodiments, NO3 which can be biologically reduced to nitrogen gas N2 is suitable for use in accordance with the present disclosure. NO3 use is also appropriate for many aquacultural applications where nitrate is present in sufficient quantities due to the normal process of nitrification. In embodiments, suitable oxidizing agents include one or more nitrate salts, such as sodium nitrate NaNO3, used alone or in combination with calcium nitrate Ca(NO3)2.4H2O.
Although the present disclosure describes the increase in ORP in terms of the chemicals added, this is largely a biochemical process. The addition of alternative electron acceptors allows bacteria to change the ORP of the water and/or sediment. Where the bacteria present in the system are incapable of reducing nitrate (or other alternate electron acceptors) or of oxidizing sulfur, adding them is appropriate to facilitate reduction of the electron acceptors and thereby allow the ORP to be increased. At the increased ORP's, the reduction of sulfur is no longer as thermodynamically advantageous for bacteria, and bacteria more efficient at using other electron acceptors will predominate over bacteria particularly suited to using sulfur.
In addition to changing ORP, it may be desirable to alter the pH. The pH may be altered to any appropriate level such as that determined by the farmer to that which is optimal for animal growth in a particular aquaculture. In embodiments, the target pH is about 6.0 to about 9.0 including pH 6.0 to pH 9.0. Accordingly, pH adjustors can be added in an amount sufficient to alter the pH of the water body to a pH of about 6.0 to about 8.5. The target ORP and the target pH values are inversely related; the lower the pH, the higher the desired ORP levels and vice versa.
Where there are few oxidizing agents initially present in a water body prior to treatment, large doses of oxidizing agent may be added to the water body. The specific amount needed may be calculated for each water body. The necessary quantity will depend upon the volume of the body being treated, its current ORP, the target ORP, the concentration of any additive, temperature (which affects the equilibrium point of chemical species as well as the rate at which biological processes occur), the concentration of total organic acid, the total sulfur concentration, and the total concentration of H2S causing sulfur species. In embodiments, nitrate is established in an amount of about 0.01 ppm to about 500 ppm, or a concentration of nitrates in the water body at about 1.0 ppm to about 250 ppm, or a concentration of nitrates in the water body at about 0.01 ppm to about 10 ppm For example, the chemistry of the water body may be modified by establishing a concentration of nitrates in the water body at 200 ppm or about 200 ppm. As used herein “ppm” means part-per-million.
The oxidizing agent may be added to the influent of a water body. Rather than adding the agent to the entire water body, it may be desirable to add it to areas in the water body where the ORP is the most depressed.
After the ORP treatment, additional oxidizing agent may need to be added to the system on a maintenance basis. For example, maintenance doses of oxidizing agent may be pulsed into the water body over regular intervals such as an hourly, daily or weekly basis. In embodiments, a liquid sodium nitrate or calcium nitrate solution for maintenance doses are suitable for use in accordance with the present disclosure.
As with the initial treatment, the size of the maintenance doses needed for maintenance purposes will vary from water body to water body, and may be individually calculated. In embodiments, suitable amounts of a maintenance dose include amounts of about 1.2 mg NO3− per liter of water volume. In embodiments, the dose is provided to maintain nitrate in an amount of about 1 ppm to about 10 ppm.
In embodiments, and after the chemicals for modifying pH and ORP have been added (if needed), sulfur oxidizing bacteria are added to the water. It is usually preferable to introduce the sulfur oxidizing bacteria immediately after the ORP chemicals have been added to the water. Where pH and ORP altering chemicals are added in large initial treatments over the course of several days, it is usually preferable to add the sulfur oxidizing bacteria after each pH and ORP treatment.
Treatments in accordance with the present disclosure contact H2S contaminated areas with bacteria selected from the genus Paracoccus in an effective amount to improve aquatic and marine animal populations. In embodiments, areas in need of treatment or H2S control such as pools, ponds, paddies, fish farms, lakes, estuaries, oceans, waste lagoons, are treated by applying to these areas, one or more sulfur oxidizing bacteria such as bacteria from the genus Paracoccus either alone or in combination with an oxidizing agent and/or pH adjustor. In embodiments, the sulfur reducing bacteria such as the bacteria selected from the genus Paracoccus and/or oxidizing agent are applied until the treatment goals are obtained. However, the duration of the treatment can vary depending on the severity of the condition. For example, treatments can last several days to months depending on whether the goal of treatment is to control, reduce and/or eliminate H2S contamination and/or increase the yield and/or quality of aquatic or marine animals such as shrimp, lobster, aquaculture organism and/or crayfish. After some treatments in accordance with the present disclosure, the yield of the aquatic and/or marine animals is increased in an amount of 5% to 200%. In embodiments, the yield of the aquatic and/or marine animals is increased in an amount of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200%. After some treatments in accordance with the present disclosure, the size of the aquatic and/or marine animals is increased by 1 to 10%, including but not limited to 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or more.
Treatments in accordance with the present disclosure include an initial assessment of a water body for growing aquatic or marine animals. Various characteristics may be measured, observed and/or established as part of the treatment process. Suitable characteristics of treatment water include: transparency, water temperature, pH, dissolved oxygen, salinity, EC, total alkalinity, hardness, total ammonia, nitrite, and/or nitrate. In embodiments, transparency may be established in the amount of about 7.0 cm to about 40 cm. In embodiments, water temperature can be established in an amount of about 23.0° C. to about 30.1° C. in the morning and/or 31.0° C. to about 33.5° C. in the afternoon. In embodiments, water body pH can be established and/or altered to about 7.00 to about 8.5. In embodiments suitable pH in the morning hours is about 7.6 to 7.9. In embodiments, suitable pH in the evening hours is about 8.2 to about 8.5. In embodiments, dissolved oxygen can be established in an amount of about 2.0 ppm to about 8.0 ppm, depending on depth and time of day. In embodiments, suitable salinity for water bodies for use in accordance with the present disclosure is in an amount of about 0.5 to 45 ppt. In embodiments, EC may be established at an amount of about 4.5 to 12 mcm/cm. In embodiments, total alkalinity may be established at an amount of about 80 ppm to about 305 ppm. In embodiments, total hardness may be established in an amount of about 750 ppm to about 1826 ppm. In embodiments, total ammonia can be established in an amount of 0.006 ppm to about 0.070 ppm. In embodiments, nitrite can be established in an amount of 0.0 to about 0.009 ppm. In embodiments, nitrate can be established in an amount of about 2.5 ppm to about 20 ppm.
In embodiments, methods of treating aquatic or marine animals such as aquacultures thereof includes contacting a water body or sediment thereof, or in need of treatment or decontamination, with one or more sulfur oxidizing bacteria in an amount sufficient to control, reduce, or eliminate the H2S in the water body or sediment. In embodiments, the amount of sulfur oxidizing bacteria is sufficient to establish a concentration of the sulfur oxidizing bacteria in the sediment in an amount of about 100 CFU/g to about 100,000 CFU/g. In embodiments, the amount of sulfur oxidizing bacteria is sufficient to establish a concentration of the sulfur oxidizing bacteria in the water body in an amount of about 100 CFU/ml to about 100,000 CFU/ml. The water body may be an aquaculture, pool, pond, paddy farm, waste lagoon, lake, estuary, ocean, contaminated portion thereof, or combinations thereof. In embodiments, suitable methods may include the step of modifying the chemistry of the water body by establishing a concentration of nitrates in the water body at about 0.01 ppm to about 500 ppm, or a concentration of nitrates in the water body at about 1.0 ppm to about 250 ppm, or a concentration of nitrates in the water body at about 0.01 ppm to about 10 ppm For example, the chemistry of the water body may be modified by establishing a concentration of nitrates in the water body at 200 ppm or about 200 ppm. Such methods may also include the step of stocking a water body with aquatic or marine animals such as fish, shrimp, lobster, crayfish, bottom dwelling fish, finfish, prawns, oysters, mussels, cockles, Mollusks, any organism suitable for aquaculture, and combinations thereof. In embodiments, the effective amount of sulfur oxidizing bacteria is an amount sufficient to maintain H2S below 1 part-per-million (ppm) for at least 1 week. In embodiments, the amount of sulfur oxidizing bacteria is 0.01 ppm to 500 ppm. Additional microorganisms can also be added in accordance with the present disclosure. Methods in accordance with the present disclosure are suitable for increasing the yield and quality of any organism grown in an aquaculture including but not limited to fish and/or plants.
The present disclosure further relates to formulations comprising sulfur oxidizing bacteria. Compositions for use in accordance with the present disclosure include compositions for treating aquatic and marine animals comprising a predetermined amount of Paracoccus pantotrophus. For example, 1 KG packages can be assembled containing 100% Paracoccus pantotrophus. Additional compositions include a predetermined amount of Paracoccus pantotrophus in combination with at least one additional microorganism. For example, Paracoccus pantotrophus can be combined with a Bacillus microorganism such as Bacillus alkalophilus, Bacillus brevis, Bacillus coagulans, Bacillus circulans, Bacillus clausii, Bacillus lichenifonnis, Bacillus lentus, Bacillus amyoliquofaciens, Bacillus lautus, Bacillus megaterium, Bacillus subtilus, Bacillus stearothermophilus, Bacillus pumilus, Bacillus pasteudi, Bacillus Thuringiensis, or combinations thereof. The composition may comprise or consist of a combination of Paracoccus pantotrophus and one additional microorganism in various weight ratios (wt/wt.) depending upon the desired purpose of the composition or water/sediment conditions. For example, Paracoccus pantotrophus and another microorganism can form an admixture characterized by a weight ratio in the amount of 4:1 to 1:4. In one embodiment, the weight ratio of Paracoccus pantotrophus to Bacillus megaterium is 1.5 to 2.5.
Suitable compositions in accordance with the present disclosure also comprise, or consist of, Paracoccus pantotrophus in an amount of 1×105 to about 11×109 CFU/gm, for example 3×109 CFU/gm. The Paracoccus pantotrophus may be individually wrapped in predetermined amounts of 200 g. Other compositions include a combination of Paracoccus pantotrophus in an amount of 1×105 to about 10×109 CFU/gm combined with Bacillus megaterum in an amount of 1×105 to about 11×109 CFU/gm. Dry formulations can be individually wrapped or packaged in a predetermined size with a predetermined amount of Paracoccus pantotrophus and Bacillus megaterum such as 3×109 CFU/gm.
In embodiments, dosages of composition suitable for use in accordance with the present disclosure can be applied to water bodies or soils/sediments for the purposes of preventing H2S contamination and/or treating H2S contamination. Suitable application periods and dosage of Paracoccus pantotrophus and Bacillus megaterium for prevention and treatment for use in accordance with the present disclosure are shown in Table 1 below:











TABLE 1







Application period
Dosage
Tips



















Prevention
During pond and/or
1-3 Kg/10,000 m2
Spread over the wet pond


(Prophylactic)
sediment
for example 2 Kg./
soil



preparation
10,000 m2



After filling pond with
0.5-1.5 kg./10,000 m2;
Most likely that



marine or aquatic
every 7-10 days,
hydrogen sulfide is



animals, e.g., 35
for example 1 Kg./
commonly produced



days post stocking
10,000 m2; every 7-10
during the early and



and from 90 days
days
late culture period.



until harvested


Treatment
When pond and/or
1-3 kg./10,000 m2;
Keep alkalinity above



sediment becomes
then 0.5-1.5 kg./
100 ppm.



black and/or smells
10,000 m2 3-4



bad
days after first




application - for




example 2 kg./




10,000 m2; then 1 kg./




10,000 m2 3-4




days after first




application.









Table 1 shows suitable dosages in accordance with the present disclosure for appoximately 5-10 Kg./10,000 m2/crop. These numbers may be adjusted for a larger pond or sediment area to be treated, and/or large/smaller crop sizes.
The following non-limiting examples further illustrate compositions, methods, and treatments in accordance with the present disclosure. It should be noted that the disclosure is not limited to the specific details embodied in the examples.
Example I
A method of using the bacteria Paracoccus pantotrophus for hydrogen sulfide (H2S) control was evaluated in three separate experiments. In Experiment I, H2S was generated by mixing shrimp pond sediment and shrimp feed for three days. After H2S was produced, the sludge was divided into 4 groups as follows: control group (no bacterial and sodium nitrate (NaNO3) added); Treatment 1 (200 ppm of NaNO3 added); Treatment 2 (5 ppm of bacteria and 200 ppm of NaNO3 added); Treatment 3 (10 ppm of bacteria and 200 ppm of NaNO3 added). The results show sulfide (S2−) and H2S levels in the control group increased from day one until the end of the study. The level of both, S2− and H2S in all treatment groups decreased until the nitrate concentration becomes zero, then S2− and H2S level increased again. However, the lowest level of S2− and H2S was observed in treatment 2, 3 and 1 respectively.
The results from Experiment I are shown below:
Control group sludge 200 g, shrimp feed 50 g and 800 ml pond water.


    Treatment 1 sludge 200 g, shrimp feed 50 g and 800 ml pond water+200 ppm NaNO3 after 3 days.
    Treatment 2 sludge 200 g, shrimp feed 50 g and 800 ml pond water+200 ppm NaNO3 and 5 ppm Paracoccus pantotrophus after 3 days.
    Treatment 3 sludge 200 g, shrimp feed 50 g and 800 ml pond water+200 ppm NaNO3 and 10 ppm Paracoccus pantotrophus after 3 days.
    Sulfide, H2S, and Nitrate levels for Experiment I are shown below in Table 2.









TABLE 2







Mean ± SD of Sulfide, Hydrogen sulfide and Nitrate from Control and Experimental groups.












DAY
PARAMETER
CONTROL
TREATMENT 1
TREATMENT 2
TREATMENT 3





0
Sulfide
3.75 ± 0.00a
 3.75 ± 0.00a
 3.75 ± 0.00a
 3.75 ± 0.00a



H2S
3.98 ± 0.00a
 3.98 ± 0.00a
 3.98 ± 0.00a
 3.98 ± 0.00a



Nitrate
0.00
0.00
0.00
0.00


1
Sulfide
4.50 ± 0.25c
 3.67 ± 0.14bc
 3.33 ± 0.29ab
 2.50 ± 0.50a



H2S
4.77 ± 0.27c
 3.89 ± 0.16bc
 3.53 ± 0.31ab
 2.65 ± 0.53a



Nitrate
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b


2
Sulfide
4.75 ± 0.25d
 3.50 ± 0.25c
 2.92 ± 0.38b
 1.50 ± 0.00a



H2S
5.04 ± 0.27d
 3.71 ± 0.27c
 3.09 ± 0.41b
 1.59 ± 0.00a



Nitrate
0.00a
 5.00 ± 0.00b
 5.00 ± 0.00b
 2.50 ± 0.00b


3
Sulfide
5.00 ± 0.25d
 3.17 ± 0.38c
 2.08 ± 0.14b
 1.25 ± 0.00a



H2S
5.30 ± 0.27d
 3.36 ± 0.40c
 2.21 ± 0.16b
 1.33 ± 0.00a



Nitrate
0.00a
 5.00 ± 0.00b
 2.50 ± 0.00b
0.00a


4
Sulfide
5.50 ± 0.00c
 2.33 ± 0.29b
 1.00 ± 0.87a
 1.67 ± 0.29ab



H2S
5.83 ± 0.00c
 2.47 ± 0.31b
 1.06 ± 0.92a
 1.80 ± 0.28ab



Nitrate
0.00a
 2.50 ± 0.00b
0.00a
0.00a


5
Sulfide
5.67 ± 0.14b
 2.17 ± 0.29a
 1.50 ± 0.00a
 2.08 ± 0.63a



H2S
6.01 ± 0.16b
 2.30 ± 0.31a
 1.59 ± 0.00a
 2.21 ± 0.67a



Nitrate
0.00a
0.00a
0.00a
0.00a


6
Sulfide
6.17 ± 0.14b
 2.58 ± 1.13a
 1.83 ± 0.29a
 3.00 ± 0.25a



H2S
6.54 ± 0.16b
 2.69 ± 1.21a
 1.94 ± 0.31a
 3.18 ± 0.27a



Nitrate
0.00a
0.00a
0.00a
0.00a


7
Sulfide
6.50 ± 0.25c
 3.17 ± 0.38b
 2.00 ± 0.50a
 3.50 ± 0.25b



H2S
6.89 ± 0.27c
 3.36 ± 0.40b
 2.12 ± 0.53a
 3.71 ± 0.27b



Nitrate
0.00a
0.00a
0.00a
0.00a





Note:


Numbers with different letters indicate a statistically significant difference P < 0.05.






Experiment 2 was set up exactly like Experiment I, except that shrimp pond sediment was not mixed with shrimp feed before testing. Sodium nitrate was added to maintain the OMP level for the duration of the experiment. The results showed that the level of S2− and H2S in control group was increased similar to experiment one. In all of the treatment groups, the level of S2 and H2S become zero at days 3 from treatment.
Control group sludge 200 g, shrimp feed 50 g and 800 ml pond water


    Treatment 1 sludge 200 g, shrimp feed 50 g and 800 ml pond water+200 ppm NaNO3.
    Treatment 2 sludge 200 g, shrimp feed 50 g and 800 ml pond water+200 ppm NaNO3 and 5 ppm Paracoccus pantotrophus. 
    Treatment 3 sludge 200 g, shrimp feed 50 g and 800 ml pond water+200 ppm NaNO3 and 10 ppm Paracoccus pantotrophus. 

The results from Experiment 2 are shown below in Table 3:










TABLE 3







Mean ± SD of Sulfide, Hydrogen sulfide and Nitrate from Control and Experimental groups.












DAY
PARAMETER
CONTROL
TREATMENT 1
TREATMENT 2
TREATMENT 3





0
Sulfide
0.00a
0.00a
0.00a
0.00a



H2S
0.00a
0.00a
0.00a
0.00a



Nitrate
0.00a
0.00a
0.00a
0.00a


1
Sulfide
1.00 ± 0.00a
 0.75 ± 0.00a
 0.75 ± 0.00a
 0.50 ± 0.00a



H2S
1.06 ± 0.00a
 0.77 ± 0.06a
 0.80 ± 0.00a
 0.53 ± 0.00a



Nitrate
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b


2
Sulfide
1.25 ± 0.00c
 1.08 ± 0.14b
 0.33 ± 0.29a
 0.17 ± 0.29a



H2S
1.33 ± 0.00c
 1.15 ± 0.16b
 0.35 ± 0.31a
 0.18 ± 0.31a



Nitrate
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b


3
Sulfide
1.33 ± 0.14b
0.00a
0.00a
0.00a



H2S
1.42 ± 0.15b
0.00a
0.00a
0.00a



Nitrate
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b


4
Sulfide
1.50 ± 0.00b
0.00a
0.00a
0.00a



H2S
1.59 ± 0.00b
0.00a
0.00a
0.00a



Nitrate
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.0b


5
Sulfide
2.17 ± 0.29b
0.00a
0.00a
0.00a



H2S
2.30 ± 0.31b
0.00a
0.00a
0.00a



Nitrate
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b


6
Sulfide
2.75 ± 0.25b
0.00a
0.00a
0.00a



H2S
2.92 ± 0.27b
0.00a
0.00a
0.00a



Nitrate
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b


7
Sulfide
2.83 ± 0.14b
0.00a
0.00a
0.00a



H2S
3.01 ± 0.15b
0.00a
0.00a
0.00a



Nitrate
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b





Note:


Numbers with different letters indicate a statistically significant difference P < 0.05.






In Experiment 3, the concentration of bacteria and sodium nitrate was reduced to 1 ppm Paracoccus pantotrophus and 10 ppm of sodium nitrate. Experiment 3 was set up the same as Experiment 2. In Experiment 3, an increase of S2− and H2S level was observed in control group when compared to Experiment 2. Further, in Experiment 3, S2− and H2S level became zero on day 4. The level of S2− and H2S was increased in treatment 1 and 2 but was still lower than the control group.
Control group sludge 200 g, shrimp feed 50 g and 800 ml pond water


    Treatment 1 sludge 200 g, shrimp feed 50 g and 800 ml pond water+10 ppm NaNO3
    Treatment 2 sludge 200 g, shrimp feed 50 g and 800 ml pond water+1 ppm Paracoccus pantotrophus 
    Treatment 3 sludge 200 g, shrimp feed 50 g and 800 ml pond water+10 ppm NaNO3 and 1 ppm Paracoccus pantotrophus 


The results from Experiment III are shown below in Table 4:







TABLE 4







Mean ± SD of Sulfide, Hydrogen sulfide and Nitrate from Control and Experimental groups.












DAY
PARAMETER
CONTROL
TREATMENT 1
TREATMENT 2
TREATMENT 3





0
Sulfide
0.00a
0.00a
0.00a
0.00a



H2S
0.00a
0.00a
0.00a
0.00a



Nitrate
0.00a
0.00a
0.00a
0.00a


1
Sulfide
1.25 ± 0.25a
 1.08 ± 0.14a
1.00 ± 0.25a
 1.42 ± 0.14a



H2S
1.33 ± 0.27a
 1.15 ± 0.16a
1.06 ± 0.27a
 1.50 ± 0.15a



Nitrate
0.00a
10.00 ± 0.00b
0.00a
10.00 ± 0.00b


2
Sulfide
1.33 ± 0.14a
 1.17 ± 0.14a
1.00 ± 0.25a
 1.25 ± 0.25a



H2S
1.42 ± 0.15a
 1.24 ± 0.16a
1.06 ± 0.27a
 1.33 ± 0.27a



Nitrate
0.00a
10.00 ± 0.00b
0.00a
10.00 ± 0.00b


3
Sulfide
1.42 ± 0.14a
 1.25 ± 0.25a
1.17 ± 0.29a
 1.17 ± 0.29a



H2S
1.50 ± 0.15a
 1.33 ± 0.27a
1.24 ± 0.31a
 1.24 ± 0.31a



Nitrate
0.00a
10.00 ± 0.00b
0.00a
10.00 ± 0.00b


4
Sulfide
1.50 ± 0.25b
 1.33 ± 0.38b
1.42 ± 0.29b
0.00a



H2S
1.59 ± 0.27b
 1.42 ± 0.41b
1.51 ± 0.31b
0.00a



Nitrate
0.00a
10.00 ± 0.00b
0.00a
10.00 ± 0.00b


5
Sulfide
1.92 ± 0.14b
 1.58 ± 0.52b
1.67 ± 0.14b
0.00a



H2S
2.03 ± 0.15b
 1.68 ± 0.55b
1.77 ± 0.16b
0.00a



Nitrate
0.00a
10.00 ± 0.00b
0.00a
10.00 ± 0.00b


6
Sulfide
2.33 ± 0.14c
 1.75 ± 0.25b
1.67 ± 0.29b
0.00a



H2S
2.48 ± 0.15c
 1.86 ± 0.27b
1.77 ± 0.31b
0.00a



Nitrate
0.00a
10.00 ± 0.00b
0.00a
10.00 ± 0.00b


7
Sulfide
2.58 ± 0.38c
 1.92 ± 0.14b
1.75 ± 0.25b
0.00a



H2S
2.74 ± 0.40c
 2.03 ± 0.15b
1.86 ± 0.27b
0.00a



Nitrate
0.00a
10.00 ± 0.00b
0.00a
10.00 ± 0.00b





Note:


Numbers with different letters indicate a statistically significant difference P < 0.05.






The results of Experiments I, II, and III show that Paracoccus pantotrophus can be used to control H2S generated by shrimp pond sediment alone, or mixed with shrimp feed. Nitrate was needed to support the bacterial activity, however typical shrimp ponds naturally contain nitrate at levels greater than 10 ppm. The results demonstrate that the bacteria can be useful when applied to a shrimp or fish pond or sediment.
Example III
A study was performed to observe the effect of Paracoccus pantotrophus for controlling hydrogen sulfide (H2S) and NaNO3 under laboratory conditions. H2S was prepared by mixing 200 g of pond soil sludge and 50 g of shrimp feed in 1,000 ml flask for three days. The mixture was divided into four treatments and 3 replicates and treated as follows:

    Group 1 Control with no treatment
    Group 2 NaNO3 was added to 200 ppm
    Group 3 Paracoccus pantotrophus added to 5 ppm and NaNO3 was added to 200 ppm
    Group 4 Paracoccus pantotrophus added to 5 ppm and NaNO3 was added to 200 ppm
    Table 5 shows the results of the study:














TABLE 5





Day
Parameter
Group1
Group2
Group3
Group4







0
Hydrogen
3.98 ± 0.00a
 3.98 ± 0.00a
 3.98 ± 0.00a
 3.98 ± 0.00a



sulfide



Nitrate
0.00a
0.00
0.00
0.00a


1
Hydrogen
4.77 ± 0.27c
 3.89 ± 0.16bc
 3.53 ± 0.31ab
 2.65 ± 0.53a



sulfide



Nitrate
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b


2
Hydrogen
5.04 ± 0.27d
 3.71 ± 0.27c
 3.09 ± 0.41b
 1.59 ± 0.00a



sulfide



Nitrate
0.00a
 5.00 ± 0.00b
 5.00 ± 0.00b
 2.50 ± 0.00b


3
Hydrogen
5.30 ± 0.27d
 3.36 ± 0.40c
 2.21 ± 0.16b
 1.33 ± 0.00a



sulfide



Nitrate
0.00a
 5.00 ± 0.00b
 2.50 ± 0.00b
0.00a


4
Hydrogen
5.83 ± 0.00c
 2.47 ± 0.31b
 1.06 ± 0.92a
 1.80 ± 0.28ab



sulfide



Nitrate
0.00a
 2.50 ± 0.00b
0.00a
0.00a


5
Hydrogensulfide
6.01 ± 0.16b
 2.30 ± 0.31a
 1.59 ± 0.00a
 2.21 ± 0.67a



Nitrate
0.00a
0.00a
0.00a
0.00a


6
Hydrogen
6.54 ± 0.16b
 2.69 ± 1.21a
 1.94 ± 0.31a
 3.18 ± 0.27a



sulfide



Nitrate
0.00a
0.00a
0.00a
0.00a


7
Hydrogen
6.89 ± 0.27c
 3.36 ± 0.40b
 2.12 ± 0.53a
 3.71 ± 0.27b



sulfide



Nitrate
0.00a
0.00a
0.00a
0.00a





Note:


Numbers with different letters indicate a statistically significant difference P < 0.05.






The study showed that H2S concentration was increased in control group from start to finish. The concentration of H2S was reducted until the NO—3 concentration was reduced to zero in the experimental groups 2, 3 and 4 after the H2S concentration was increased.
Example IV
A study was conducted to examine the suitable quantity of bacteria necessary to prevent H2S production.
200 g of pond soil sludge and 50 g of shrimp feed was mixed in a 1,000 ml flask. The mixture was divided into 5 treatments with 3 replicates and treated as follows:

    Group 1 Control with no treatment
    Group 2 NaNO3 was added to 10 ppm
    Group 3 Paracoccus pantotrophus added to 0.1 ppm and NaNO3 was added to 10 ppm
    Group 4 Paracoccus pantotrophus added to 1 ppm and NaNO3 was added to 10 ppm
    Group 5 Paracoccus pantotrophus added to 5 ppm and NaNO3 was added to 10 ppm
    Group 6 Paracoccus pantotrophus added to 10 ppm and NaNO3 was added to 10 ppm.

NaNO3 was added to all flasks in 3-5 g to maintain at 10 ppm for 7 days.

H2S was measured every day for seven days using Hach kits (U.S.A.). The results are shown in Table 6.

















TABLE 6





Days
parameter
group 1
group 2
group 3
group4
Group 5
group 6







0
H2S
0.00a
0.00a
0.00a
0.00a
0.00a
0.00a



NO3−
0.00a
0.00a
0.00a
0.00a
0.00a
0.00a


1
H2S
1.33 ± 0.27b
 1.42 ± 0.41b
 1.59 ± 0.00b
 1.50 ± 0.15b
 0.80 ± 0.00a
 0.53 ± 0.00a



NO3−
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b


2
H2S
1.42 ± 0.15b
 1.33 ± 0.27b
 1.42 ± 0.15b
 1.33 ± 0.27b
 0.35 ± 0.31a
 0.18 ± 0.31a



NO3−
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b


3
H2S
1.50 ± 0.15c
 1.24 ± 0.16b
 1.33 ± 0.27b
 1.24 ± 0.31b
0.00a
0.00a



NO3−
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b
0.00b
0.00b


4
H2S
1.59 ± 0.27c
 1.15 ± 0.16b
0.00b
0.00a
0.00a
0.00a



NO3−
0.00a
10.00 ± 0.00b
0.00b
0.00b
0.00b
0.00b


5
H2S
2.03 ± 0.15c
 1.33 ± 0.27b
0.00a
0.00a
0.00a
0.00a



NO3−
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b


6
H2S
2.48 ± 0.15c
 1.42 ± 0.41b
0.00a
0.00a
0.00a
0.00a



NO3−
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b


7
H2S
2.74 ± 0.40c
 1.68 ± 0.55b
0.00a
0.00a
0.00a
0.00a



NO3−
0.00a
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b
10.00 ± 0.00b





Note:


Numbers with different letters indicate a statistically significant difference P < 0.05.






Table 6 shows H2S concentration increased in group 1 (control group) from the started of the experiment to the end of experiment. In Group 2, the production of H2S was reduced until day 4. In group 3 and 4, H2S was reduced to zero within 4 days. In group 5 and group 6 the production of H2S was reduced to zero within only 3 days. This trial revealed that bacteria levels of at least 0.1 ppm can be an effective amount to control the concentration of H2S build up from shrimp pond sludge mixed with shrimp feed.
Accordingly the bacteria P. pantotrophus can be used to control the concentration of H2S build up from shrimp pond sludge mixed with shrimp feed. An optimum concentration of NaNO3 may be necessary for bacterial process to occur continuously. The results from this study can be apply and used in shrimp pond or with other marine or aquatic animals.
Example V
Tests were performed to observe the effect of a composition which comprises or consists of a mixture of Paracoccus pantotrophus and Bacillus megaterium on the growth, survival and production of white shrimp cultured in low salinity condition, and to observe the effect of the admixture on the water quality of a shrimp pond.
Materials and Methods
An admixture of Paracoccus pantotrophus and Bacillus megaterium having a weight ratio of 1.5 to 2.5 was provided (CFU/g was approximately 3×109).
A private shrimp farm having low salinity condition was provided in Bangkok, Thailand with a closed-recirculation system. Six separate ponds were provided and divided into two groups. Group I included 3 ponds treated with the admixture. Group II included 3 ponds similar to the Group I ponds, except no admixture was added to these ponds. All ponds had an area of 4,000 m2 and the water depth of approximately 1.5 to 1.8 meters (m). The admixture was applied to the treated ponds every 7 days until harvest to maintain the concentration of bacteria therein at about 0.1 ppm. No additional treatments were applied to Group I. All six ponds were under the same management until harvest. The stocking density of the shrimp was 40 PL/M2, post larvae, from pathogen free brooders. Commercial pellet were used to feed the shrimp 4 times per day. Ponds were harvested about 4 months after stock.
Studies on Growth, Survival and Yield
Shrimp in each pond were sampled, and measured for average weight every 7 days until harvest (starting at 30 days after pond stock). After the harvest, survival, growth rate, feed conversion ratio and yield were calculated and compared.
Studies on Water Quality
Water samples were collected from all six ponds and the following laboratory tests were conducted at the given time:
1. Dissolved oxygen: 7:00 and 16:00 everyday.
2. Water pH: 7:00 and 16:00 everyday.
3. Salinity: measured once every week.
4. Total alkalinity and hardness: measured once every week
5. Electrical conductivity: once every week
6. Transparency: once every week
7. Total ammonia, nitrite, nitrate and hydrogen sulfide: once every week
8. Redox potential: once every two weeks.
All data were compared with the control ponds using t-test.
Table 7 below relates to the effect of the admixture on growth, survival and production of shrimp as described above. More specifically, Table 7 shows the average body weighs and average daily growth on treated ponds and control ponds.










TABLE 7









Treated ponds
Control ponds












Average
Average
Average
Average daily



body weight
daily growth
body weight
growth


Week
(g)
(g/day)
(g)
(g/day)














5
3.06
0.09
2.91
0.08


7
5.15
0.15
4.71
0.13


9
7.26
0.15
6.17
0.1


11
10.51
0.23
9.18
0.21


13
13.89
0.24
12.34
0.23


15
16.77
0.21
15.31
0.21





Results:


The average body weight and average daily growth of shrimp in ponds treated with the admixture were larger than the growth of shrimp in control ponds not treated.






Table 8 below shows a comparison on growth, survival, feed conversion ratio and yield between ponds treated with the admixture in accordance with the present disclosure and non-treated ponds.















TABLE 8







Day of
Average

Feed

Average



Area
culture
body weight
Survival
conversion
Yield
daily


Pond
(M2)
(days)
(g)
rate (%)
ratio
(Kg/rai)
growth (g/day)















Group I: Treated ponds (with P. pantotrophus)














1
2.5
111
17.24
77.33
1.22
800
0.16


2
2.5
111
16.39
89.47
1.11
880
0.15


3
2.5
111
16.67
84.00
1.16
840
0.15


Average
2.5
111
16.77 ± 0.43a
83.6 ± 6.08a
1.16 ± 0.06a
  840 ± 40.00a
0.15 ± 0.01a 







Group II: Control ponds (without P. pantotrophus)














4
2.5
111
15.38
67.17
1.62
620
0.14


5
2.5
111
15.63
64.00
1.62
600
0.14


6
2.5
111
14.93
75.93
1.32
680
0.13


Average
2.5
111
15.31 ± 0.35b
69.03 ± 6.18b
1.52 ± 0.17b
633.33 ± 41.63b
0.14 ± 0.01b





Note: 1 rai = 1,600 M2


Different superscript or letters on the same column indicated significantly different (P < 0.05) on the Table 8.


Results: The average body weight, survival rate, feed conversion ratio and yield were larger in Group I and smaller in Group II.






More specifically, studies showed 33% increase in yield (kg/ha); 9% increase in average body weight (g); 12% improved average daily growth (g/day); 21% improvement in survival rate; 23% reduction in feed conversion ratio.
Studies on the effect of the admixture on water quality in shrimp ponds were conducted. Table 9 below shows the water quality parameters (range and average) in treated and control ponds.










TABLE 9









Treated pond with




P. pantotrophus
Control pond











Water parameter
Range
Average
Range
Average















Transparency

 7.0-40.0
16.82 ± 8.00a
 8.0-35.0
18.13 ± 8.57a


(centrimeters)


Water temperature
a.m.
29.0-30.1
 29.5 ± 0.28a
29.0-29.9
 29.6 ± 0.23a


(celcius)
p.m.
31.0-33.5
 32.0 ± 0.69a
30.3-33.5
 32.0 ± 0.73a


pH
a.m.
7.61-7.92
 7.75-0.07a
7.60-7.84
 7.73 ± 0.06a



p.m.
8.24-8.48
 8.37 ± 0.05a
8.22-8.55
 8.38 ± 0.06a


Dissolved oxygen
a.m.
4.81-5.37
 5.08 ± 0.18a
4.81-5.30
 5.03 ± 0.14a


(ppm.)
p.m.
7.13-9.68
 8.59 ± 0.62b
7.04-9.96
 8.20 ± 0.74a











Salinity (ppt.)
2.60-6.80
 4.68 ± 1.30a
2.80-5.50
 4.28 ± 0.87a


EC (mcm/cm)
 4.90-11.87
 8.30 ± 2.17a
5.10-9.86
 7.69 ± 1.58a


Total alkalinity (ppm.)
 94-303
  194 ± 51a
 93-244
  182 ± 38a


Hardness (ppm.)
 758-1826
 1260 ± 329a
 772-1532
 1142 ± 220a


Total ammonia (ppm.)
0.006-0.070
0.029 ± 0.013a
0.004-0.059
0.032 ± 0.015a


Nitrite (ppm.)
0.000-0.009
0.004 ± 0.002a
0.001-0.008
0.004 ± 0.002a


Hydrogen sulfide(ppm.)
ND
ND
ND
ND


Nitrate (ppm.)
 2.50-10.00
 4.39 ± 2.67b
 2.50-10.00
 3.11 ± 1.43a





Note


Different superscript or letters on the same row, indicated significantly different (P < 0.05).






Table 10 shows the redox potential value in treated and control ponds.











TABLE 10









Treated pond with




P. pantotrophus admixture
Control












Feeding area
Pond center
Feeding area
Pond center


Week
(mV)
(mV)
(mV)
(mV)














2
−15
−29
−53
−92


4
−26
−45
−83
−106


6
−42
−64
−91
−111


8
−54
−78
−84
−113


10
−61
−86
−96
−116


12
−52
−93
−82
−124


14
−67
−98
−102
−146









Example VI
Table 11 shows characteristics of one composition in accordance with the present disclosure.








TABLE 11







Composition
Paracoccus pantotrophus


Bacterial Count:
3.0 billion CFU/g (3.0 × 10E9 CFU/g)


Appearance
Tan free flowing powder


Odour
Yeast like


Optimum pH range
7.5-8.3


Optimum Temperature
23 C.-40 C. (pond water)


Range


Salinity Range
0-40 ppt


Storage & Handling
Optimum temperature for storage 23 C. or below,



Avoid direct sunlight



Store in cool, dry place


Shelf Life
Best when used within 1 year of manufacture date









It will be understood that various modifications may be made to the embodiments disclosed herein. Therefore, the above description should not be construed as limiting, but merely as exemplifications of embodiments. Those skilled in art will envision other modifications within the scope and spirit of the claims appended hereto.

## Claims

1. A method of treating aquatic or marine animals comprising contacting a water body or sediment thereof with one or more sulfur oxidizing bacteria of the genus Paracoccus pantotrophus in an effective amount of about 100 CFU/ml to about 100,000 CFU/ml in the water body or in an effective amount of about 100 CFU/g to about 100,000 CFU/g in the sediment, sufficient to control, reduce, or eliminate the H2S in the water body or sediment thereof; and modifying the chemistry of the water body or sediment thereof by establishing a concentration of nitrates in the water body or sediment thereof at about 0.01 ppm to about 500 ppm, wherein the water body or sediment thereof comprises one or more aquatic or marine animals and the yield of the aquatic and marine animals is increased in comparison to the same water body or sediment thereof having no sulfur oxidizing bacteria added thereto, and wherein the step of modifying the chemistry of the water body or sediment thereof comprises establishing an oxidation reduction potential level in the water body or in the sediment thereof of at least about −330 mV.

2. The method of claim 1, wherein the water body is an aquaculture, farm, pool, pond, waste lagoon, paddy, lake, estuary, ocean, or combinations thereof.

3. The method of claim 1, wherein the water body is an aquaculture.

4. The method of claim 1, wherein the water body is a farm.

5. The method of claim 1, wherein the water body is a pool.

6. The method of claim 1, wherein the water body is a pond.

7. The method of claim 1, wherein the contacting comprises contacting an anaerobic zone within the water body or sediment thereof.

8. The method of claim 1, wherein said concentration of nitrates in the water body or sediment thereof is at about 1 ppm to about 250 ppm.

9. The method of claim 1, wherein said concentration of nitrates in the water body or sediment thereof is at about 200 ppm.

10. The method of claim 1, further comprising stocking the water body with aquatic or marine animals comprising fish, shrimp, lobster, eel, crayfish, bottom dwelling fish, finfish, prawns, oysters, mussels, cockles, mollusks, or combinations thereof.

11. The method of claim 1, further comprising stocking the water body with fish.

12. The method of claim 1, further comprising stocking the water body with shrimp.

13. The method of claim 1, further comprising stocking the water body with lobster.

14. The method of claim 1, wherein the effective amount of Paracoccus pantotrophus is an amount sufficient to maintain H2S below 1 ppm for at least 1 week.

15. The method of claim 1, further comprising adding to the water body or sediment thereof an additional microorganism of genus Bacillus selected from the group consisting of Bacillus alkalophilus, Bacillus brevis, Bacillus coagulans, Bacillus circulans, Bacillus clausii, Bacillus licheniformis, Bacillus lentus, Bacillus amyoliquofaciens, Bacillus lautus, Bacillus megaterium, Bacillus subtilus, Bacillus stearothermophilus, Bacillus pumilus, Bacillus pasteurii, Bacillus thuringiensis, and combinations thereof.

16. The method of claim 1, wherein the yield of the aquatic or marine animal is increased by at least 5% in comparison to the same water body having no sulfur oxidizing bacteria added thereto.

17. A method for increasing aquatic and marine animal yield in a water body comprising reducing the amount of H2S in a water body and sediment thereof by establishing a sulfur oxidizing bacteria population of Paracoccus pantotrophus in the water body in an effective amount of about 100 CFU/ml to about 100,000 CFU/ml and in the sediment in an effective amount of about 100 CFU/g to about 100,000 CFU/g; and modifying the chemistry of the water body by establishing a concentration of nitrates in the water body at about 0.01 ppm to about 500 ppm, wherein the water body comprises one or more aquatic or marine animals and wherein the step of modifying the chemistry of the water body comprises establishing an oxidation reduction potential level in the water body or sediment thereof of at least about −330 mV.

18. The method of claim 17, wherein the final amount of H2S in the water body is below 1 ppm.

19. The method of claim 17, wherein the final amount of H2S in the water body or sediment is below 0.5 ppm.

20. The method of claim 17, wherein the final amount of H2S in the water body or sediment is 0.1 ppm to 0.5 ppm.

21. The method of claim 17, further comprising establishing a pH in the water body of about 6.0 to 9.0.

22. The method of claim 17, wherein the step of reducing the amount of H2S in a water body and sediment thereof occurs in an anaerobic zone within the water body.

23. The method of claim 17, further comprising stocking the water body with aquatic or marine animals comprising fish, shrimp, lobster, eel, crayfish, bottom dwelling fish, finfish, prawns, oysters, mussels, cockles, mollusks, or combinations thereof.

24. The method of claim 17, further comprising stocking the water body with fish.

25. The method of claim 17, further comprising stocking the water body with shrimp.

26. The method of claim 17, further comprising stocking the water body with lobster.

27. The method of claim 17, further comprising adding to the water body or sediment thereof an additional microorganism of the genus Bacillus selected from the group consisting of Bacillus alkalophilus, Bacillus brevis, Bacillus coagulans, Bacillus circulans, Bacillus clausii, Bacillus licheniformis, Bacillus lentus, Bacillus amyoliquofaciens, Bacillus lautus, Bacillus megaterium, Bacillus subtilus, Bacillus stearothermophilus, Bacillus pumilus, Bacillus pasteurii, Bacillus thuringiensis, and combinations thereof.

28. The method of claim 17, wherein the yield of the aquatic or marine animal is increased by at least 5%.