WO2004026772A2 - Ammonia-oxidizing bacteria and methods of using and detecting the same - Google Patents
Ammonia-oxidizing bacteria and methods of using and detecting the same Download PDFInfo
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- WO2004026772A2 WO2004026772A2 PCT/US2003/028210 US0328210W WO2004026772A2 WO 2004026772 A2 WO2004026772 A2 WO 2004026772A2 US 0328210 W US0328210 W US 0328210W WO 2004026772 A2 WO2004026772 A2 WO 2004026772A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- the invention relates generally to ammonia oxidizers and specifically to bacteria capable of oxidizing ammonia to nitrite.
- Ammonia is the principal nitrogenous waste product of teleosts and many invertebrates in both freshwater and seawater. The ammonia results from the deamination or transamination of proteins the organism receives via its diet. However, high ammonia concentrations can be toxic to many of these same aquatic organisms. In natural systems, such as lakes, rivers and oceans, the concentration of ammonia rarely reaches deleterious levels because the density of fish (and other organisms) per mass of water is low.
- ammonia can reach toxic concentrations, sometimes very quickly.
- the fish density can be very large in relation to the small amount of water.
- Another reason is that in many of these systems the water is not continually changed; rather it recirculates through the system with only periodic partial water changes.
- a major component of any such filtration unit is the biological filter.
- the biological filter gets its name from the fact that it acts as a substrate or site for the growth of bacteria which have the capability to convert, by way of oxidation, ammonia to another compound - nitrite.
- High concentrations of nitrite can also be toxic but there are other species of bacteria which grow on the biological filter and oxidize the nitrite to nitrate, such as those described in U.S. Patent Nos. 6,268,154, 6,265,206 and 6,207,440, each of which is incorporated by reference herein in its entirety as if fully set forth.
- Nitrate is considered non-toxic to aquatic organisms except in extreme cases of very high concentrations.
- the oxidation of ammonia to nitrite is a bacterially-mediated process. Specifically, it is a two step oxidation process involving the conversion of ammonia to nitrite according to the following equations: NH 3 + O 2 + H 2 O + 2e " > NH 2 OH + H 2 O (1)
- AOB ammonia oxidizing bacteria
- N. europaea is not the dominant AOB in wastewater treatment facilities (Juretschko, S. et. al. 1998. Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and Nitrospira-like bacteria as dominant populations. Appl. Environ. Microbiol. 64:3042-3051).
- an environmental factor of particular import with aquaria environments and wastewater treatment is salinity, and, more specifically, the numerous physicochemical differences between freshwater and saltwater environments.
- the distinction among various AOBs in their ability to tolerate such dramatic changes in local environment is critical in the design of these systems and implementation of AOBs therein.
- a demonstrated tolerance by a particular AOB to a saltwater environment may render that AOB suitable for use in particular aquaria and wastewater environments, and, moreover, a resilience to withstand the change between a freshwater and saltwater environment may have even broader implications.
- AOB AOL-based on-solid-dried powder or similar composition
- a freeze-drying process allows one to formulate a volume of AOB into a solid, freeze-dried powder or similar composition that may be tolerant of greater fluctuations in, e.g., temperature, and may be correspondingly more practical for purposes of shipping and handling in a commercialized product, or similar considerations, and for maintaining an extended shelf-life.
- AOBs that are capable of tolerating a saltwater environment and/or both saltwater and freshwater environments.
- AOBs that remain viable after being subjected to a freeze-drying process.
- isolated bacteria or bacterial strains capable of oxidizing ammonia to nitrite are provided.
- the 16S rDNA of the bacteria or bacterial strains have the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.
- the nucleotide sequences described as SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20 are exemplary of Nitrosomonas aestuarii-li c AOB.
- the 16S rDNA of the bacteria or bacterial strains have the nucleotide sequence of SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20 (i.e., the Nitrosomonas aestuarii-li e AOB), SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or a variant thereof which is at least 96% similar, at least 97% similar, at least 98% similar or at least 99% similar to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.
- SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20 i.e., the Nitrosomonas aestuarii-li e AOB
- the present invention also includes nucleic acid sequences and bacteria with sequences which have the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20 or a variant thereof which is at least 96% similar, at least 97% similar, at least 98% similar or at least 99% similar to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:20.
- the present invention also includes methods of alleviating the accumulation of ammonia in a medium.
- the methods include a step of placing into the medium a sufficient amount of a bacterial strain or a composition comprising a bacterial strain, wherein the 16S rDNA of the bacterial strain has the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:20 or a variant thereof which is at least 96% similar, at least 97% similar, at least 98% similar or at least 99% similar to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.
- the present invention also includes a method for detecting and determining the quantity of bacteria in a medium capable of oxidizing ammonia to nitrite.
- the method includes providing a detectably labeled probe of the present invention, isolating total DNA form the medium, exposing the isolated DNA to the probe under conditions wherein the probe hybridizes to only the nucleic acid of the bacteria when the 16 rDNA of the bacteria has a nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:20, and detecting and measuring the probe to detect and measure the amount of bacteria.
- Figure 1 illustrates the phylogenetic relationships of three bacterial strains (i.e., those represented by SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3) and one substrain (i.e., that strain represented by SEQ ID NO:4) inferred from comparative analysis of 16S rDNA sequences in accordance with an embodiment of the present invention.
- the tree is based on neighbor-joining distance analysis of sequences containing a minimum of 1430 nucleotides.
- FIG. 2 illustrates a denaturing gradient gel electrophoresis (DGGE) of biomasses from selected cultures and ammonia-oxidizing bacteria represented by SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, in accordance with an embodiment of the present invention.
- DGGE denaturing gradient gel electrophoresis
- Figure 3 illustrates a DGGE demonstrating the uniqueness of the bacterial strains represented by SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, in accordance with an embodiment of the present invention. There are two replicates of each aforementioned bacterial type along with extracts from three pure cultures of ammonia-oxidizing bacteria.
- Figure 4 illustrates mean ammonia and nitrite trends for the Bacterial Additives VI test for the bacterial strains represented by SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, in accordance with an embodiment of the present invention.
- Figure 5 illustrates mean ammonia and nitrite trends for the Bacterial Additives VII test for the bacterial strains represented by SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, in accordance with an embodiment of the present invention.
- Figure 6 illustrates the phylogenetic relationships of two bacterial strains (i.e., those represented by SEQ ID NO: 18 and SEQ ID NO: 19), and one substrain (i.e., that represented by SEQ ID NO:20) inferred from comparative analysis of 16S rDNA sequences in accordance with an embodiment of the present invention.
- the tree further depicts the relationship among the two bacterial strains represented by SEQ ID NO:18 and SEQ ID NO:19 and the bacterial strains represented by SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
- the tree is based on neighbor-joining distance analysis of sequences containing a minimum of 1430 nucleotides.
- Figure 7 illustrates a denaturing gradient gel electrophoresis (DGGE) of the biomasses from selected freshwater cultures of ammonia-oxidizing bacteria represented by SEQ ID NO:l, SEQ ID NO: 3 and SEQ ID NO:4 along with seawater cultures of ammonia-oxidizing bacteria represented by SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20 and pure cultures of the ammonia-oxidizing bacteria Nitrosomonas europaea, Nitrosomonas multiformis, and Nitrosomonas cryotolerans.
- DGGE denaturing gradient gel electrophoresis
- Figure 8 illustrates mean ammonia concentration trends for the Bacterial Additives VIII test for freshwater bacterial strains represented by SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3 and seawater bacterial strains represented by SEQ ID NO:18, SEQ ID NO:19 and two JV. halophila-like strains in accordance with an embodiment of the present invention along with two commercially available nitrifying bacteria mixtures.
- Figure 9 illustrates mean ammonia concentration trends for aquaria in the Bacterial Additives LX test that were dosed with seawater bacterial strains represented by SEQ ID NO: 18, SEQ ID NO: 19 and two N. halophila-like strains in accordance with an embodiment of the present invention.
- Figure 10 illustrates mean ammonia concentration trends for the Bacterial Additives X test.
- Two bacterial mixtures of seawater bacterial strains represented by SEQ ID NO: 18, SEQ ID NO: 19 and two N. halophila-like strains were tested against non-inoculated aquaria in accordance with an embodiment of the present invention.
- the present invention is based upon the discovery of novel bacterial strains which are capable of ammonia oxidation in freshwater and/or saltwater environments, and which can also survive and remain viable following a freeze-drying process. Embodiments of the present invention describe methods for using the bacterial strains.
- the present invention provides an isolated bacterial strain or a biologically pure culture of a bacterial strain capable of oxidizing ammonia to nitrite, wherein the 16S rDNA of the bacterial strain includes the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO: 22 as shown in Tables 1 through 7.
- Table 1 The sequence for the AOB Type A ammonia-oxidizing bacterium. Represented by R7clonel40. SEQ ID NO:l.
- Table 2 The sequence for the AOB Type Al ammonia-oxidizing bacterium. Represented by R7clonel87. SEQ ID NO:2.
- GGTG Table 3 The sequence for the AOB Type B ammonia-oxidizing bacterium. Represented by R3clone5. SEQ ID NO:3.
- GTG Table 4 The sequence for the AOB Type C ammonia-oxidizing bacterium. Represented by R5clone47. SEQ ID NO:4.
- an isolated bacterial strain is one that has undergone some degree of purification from its natural environment.
- a culture of a bacterium is considered to be biologically pure if at least 20% of the bacteria are from one bacterial strain. However, it is preferable if the culture is at least 33% pure, more preferable if the culture is at least 45% pure and most preferable if the culture is at least 90% pure.
- the bacterial strains of the present invention may also be combined with each other, other species of bacteria, nutrients and/or other components to provide a composition for maintaining or purifying water-containing media. It may be desirable, for example, to combine the bacteria of the present invention with bacteria capable of removing other pollutants or undesirable compounds from water-containing media. Examples of such bacteria include nitrite-oxidizing bacteria (chemolithoautotrophic bacteria which oxidize nitrite to nitrate), heterotrophic bacteria (which mineralize organic material into ammonia and other substances) and other bacteria which will be known to those of skill in the art. Nitrite-oxidizing bacteria are known from the Nitrospira phylum of bacteria, and the alpha, gamma and delta subdivisions of the Proteobacteria.
- Examples include species of the genera Nitrospira, Nitrospina and Nitrobacter.
- Nitrate-reducing bacteria are known from the genera Azoarcus, Pseudomonas and Alcaligenes.
- Heterotrophic bacteria are known from the genera Bacillus, Planctomyces, Pseudomonas and Alcaligenes. Such are available from known sources (e.g., American Type Culture Collection, 10801 University Boulevard., Manassas VA 20100, USA) or may be isolated directly from aquaria biofilters.
- the bacterial strains of the present invention may be combined with nitrite- oxidizing bacteria such that ammonia present in the water system would be oxidized to nitrite and the nitrite oxidized to nitrate.
- Another example would be to combine the bacterial strain of the present invention with aerobic or anaerobic denitrifying bacteria. In this case, the nitrate which is produced by the mteraction of the bacterial strains of the present invention with nitrite-oxidizing bacteria would be reduced to dinitrogen or other nitrogen based products.
- a third example would be to combine the bacterial strain of the present invention with heterotrophic bacteria which mineralize organic matter into simpler inorganic substances which, subsequently, can be utilized as substrates by the bacterial strains of the present invention.
- the present invention also provides a mixture comprising a concentrated bacterial strain capable of oxidizing ammonia to nitrite, wherein the 16S rDNA of the bacteria has a nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:20 or a variant thereof which is at least 96% similar, at least 97% similar, at least 98% similar or at least 99% similar to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:20.
- the bacterial strain is considered to be concentrated if the bacterial strain occurs in a concentration which is higher than its concentration occurred in nature.
- the concentration of the bacterial strain will be at least 20% of the total cells in the sample as determined by standard techniques such as molecular probing using fluorescent in situ hybridization (FISH) techniques, which will be known to those skilled in the art, using appropriate controls and enumeration methods. More preferably, the concentration of the bacterial strain would be 33% or greater of the total cells, even more preferably 45%, and most preferably 90% or greater of the total cells.
- the bacteria which have a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:20 in the mixture.
- the percentages stated above relate to percentage of total AOBs in the mixture with the understanding that the balance of cell population might be comprised of nitrite-oxidizing bacteria or other types of bacteria.
- strains represented by SEQ ID NO: 18, SEQ ID NO:19 and SEQ ID NO:20 are believed to be especially tolerant of saltwater environments; although these strains may be utilized in freshwater environments, as well, and are believed to function effectively therein.
- Bacterial strains and mixtures incorporating strains other than those strains represented by SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20 may also tolerate saltwater environments to an appreciable degree, yet in a preferred embodiment of the present invention, it is those strains represented by SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:20 that are included in a saltwater environment to oxidize ammonia to nitrite.
- any of the bacterial strains of the present invention may be freeze- dried
- those strains represented by SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20 are believed to be particularly tolerant of the freeze-drying process, as evidenced by their ability to remain viable after such a process, and to oxidize ammonia to nitrite following such a process.
- those bacterial strains represented by SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20 may be fieeze-dried and thereafter used to oxidize ammonia to nitrite in either freshwater or saltwater environments.
- the bacterial strains and the mixtures of the present invention can be in the form of powder, liquid, a frozen form, a freeze-dried form or any other suitable form, which may be readily recognized by one of skill in the art.
- These are commonly referred to as "commercial additives,” and may include, but are in no way limited to:
- bacterial strains and the mixture of the present invention can be used alone, or in combination with other components.
- examples of such components include, but are not limited to, nitrite- oxidizing bacteria, heterotrophic nitrite-oxidizing bacteria, heterotrophic ammonia-oxidizing bacteria and the like. All of the forms of the biologically pure bacterial strain may also contain nutrients, amino acids, vitamins and other compounds which serve to preserve and promote the growth of the bacterial strain.
- the bacterial strains and the mixtures and compositions of the present invention can be used in freshwater aquaria, seawater aquaria and wastewater to alleviate the accumulation of ammonia. They can also be used in a bioremediation process to reduce the level of pollution caused by the ammonia.
- a bioremediation process also called bioaugmentation, includes, but is not limited to, the supplemental addition of microorganisms to a system (e.g. a site where biological or chemical contamination has occurred) for the purposes of promoting or establishing biological and/or chemical processes that result in the change of one or more forms of chemical compounds present in the original system.
- one aspect of the present invention provides a method of alleviating the accumulation of ammonia in a medium.
- the method includes a step of placing into the medium a sufficient amount of a bacterial strain capable of oxidizing ammonia to nitrite to alleviate the accumulation of ammonia in the medium, wherein the 16S rDNA of the bacterial strain has a nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:20 or a variant thereof which is at least 96% similar, at least 97% similar, at least 98% similar or at least 99% similar to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.
- the amount of the bacterial strain(s) is sufficient if the added bacteria can alleviate or prevent the accumulation of ammonia in the medium.
- the addition of one or more of the bacterial strains of the invention to a freshwater or saltwater aquarium is expected to reduce the maximum ammonia concentration by at least 50% over the level which would be attained in the absence of the bacterial strain(s).
- a method of alleviating the accumulation of ammonia in a medium includes placing into the medium a sufficient amount of a composition, as disclosed herein, for maintaining or purifying water-containing media.
- the composition may comprise one or more bacterial strains capable of oxidizing ammonia to nitrite wherein the 16S rDNA of the bacterial strain or strains has a nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20 or a variant thereof which is at least 96% similar, at least 97% similar, at least 98% similar or at least 99% similar to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.
- the actual levels achieved in a given setting will be a function of the size and contents of the systems (i.e., the number of fish, plants, etc.).
- the ammonia concentration may reach 7 mg/L or higher without addition of the bacterial strain, whereas the maximum level can be reduced to about 2 mg/L by addition of the bacterial strain.
- the maximum ammonia concentration would not be expected to exceed 3 mg/L if the bacterial strain of the invention is added to such a system.
- the ammonia levels drop back to below 0.5 mg/L, a process which occurs more rapidly when the bacterial strain of the invention is present.
- the bacterial strains of the present invention are placed directly into a medium such as, but not limited to, freshwater aquaria, seawater aquaria and wastewater.
- the bacteria may be grown on a rotating biological contactor and then placed in the medium.
- the bacteria of the present invention can be placed on a biofilter unit contained in the medium.
- the bacteria of the present invention may be immobilized in an immobilizing polymer, such as, but not limited to, acrylamide, alginate or carrageenan. This bacterial-laced polymer material may then be placed in a filter or may itself be placed in the filter stream of a suitable facility.
- aquarium is intended to mean a container which may be made of, in combination or in its entirety, but not limited to, glass, plastic, or wood that holds water and in which living aquatic organisms (such as fish, plants, bacteria and invertebrates) are placed, and the contents thereof.
- An aquarium may be for the purposes of displaying aquatic organisms, for their short or long-term holding, for scientific study, for transportation and other purposes.
- a freshwater aquarium is generally an aquarium in which the liquid medium has a salinity of less than 15 parts per thousand.
- a saltwater aquarium is generally an aquarium in which the liquid medium has a salinity of more than 15 parts per thousand.
- aquarium water is used to refer to the medium which is contained within the aquarium, and its associated filter systems, in which the aquatic organisms reside.
- Aquarium water may contain a wide range of inorganic or organic chemical substances and, therefore, may have a wide range of parameters such as concentration of salts, pH, total dissolved solids and temperature, to name a few.
- wastewater generally refers to a liquid medium which is the product of an industrial or human process. It may require treatment by one or more filtration methods to render it less harmful to the environment such that it conforms to discharge standards as determined by a governmental agency. Wastewater may also be recycled such that it is not discharged to the environment.
- a “biological filter,” also called a “biofilter,” generally refers to a filter type whose purpose is to promote the growth of microorganisms, or to provide a substrate for the attachment and growth of microorganisms.
- a biofilter may be part of an aquarium filtration system or a wastewater filtration system.
- the term “rotating biological contactor” generally refers to a type of biofilter which rotates in the water or medium. It may be completely or partially submerged in the water or medium. Persons skilled in the art will recognize rotating biological contactors as embodied in United States Patents Nos.
- filter floss refers to irregularly shaped natural or synthetic multi- stranded material which may serve as a biofilter, a mechanical filter or a combination of these.
- aquarium gravel refers to a substrate commonly placed inside, on the bottom, of an aquarium. It may be composed of irregular or regular shaped pieces of rock, coral, plastic or other material. It may serve as a biofilter, a mechanical filter, for decorative purposes or a combination of these.
- filter sponge refers to a natural or synthetic material which when used in an aquarium or as part of an aquarium filtration system may serve as a mechanical filter, a biofilter or both.
- plastic filter media refers to a man-made material which serves as a biofilter, a mechanical filter or both. It may be plastic molded or injected molded.
- nucleic acid sequences and bacteria with sequences which have the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20 or a variant thereof which is at least 96% similar, at least 97% similar, at least 98% similar or at least 99% similar to SEQ ID NO: I, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:20 are also provided.
- nucleotide probes are provided for detecting and measuring the amount of bacteria of the present invention which are present in a medium.
- the probes have the nucleotide sequences set forth in SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:21.
- the nucleotide probes of the present invention can be synthesized by methods which are known in the art.
- the nucleotide probes of the present invention can be labeled by any labels that are detectable.
- suitable labels mclude, but are in no way limited to, radioactive labels, fluorescent labels, and the like.
- Suitable labeling materials are commercially available and would be known to those of ordinary skill in the art.
- the methods of labeling an oligonucleotide or a polynucleotide are also known to those of ordinary skill in the art (See, for example, Sambrook, J., E. F. Fritsch, and T. Maniatis. Molecular Cloning-A Laboratory Manual, 2.sup.nd edition, 1989, Cold Spring Harbor Press).
- the nucleotide probes of the present invention are able to hybridize with 16S rDNA of the bacterial strain of the present mvention. Accordingly, the nucleotide probes of the present invention are well suited for use in a method for detecting and determining the quantity of bacteria of the present invention.
- a method for detecting and determining the quantity of bacteria capable of oxidizing ammonia to nitrite in a medium, wherein the 16S rDNA of the bacteria has a nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.
- the method may include:
- the probes of the present invention are represented by SEQ ID NO: 5, SEQ ID NO: 8 and SEQ ID NO:21.
- a sequence that is at least 96% similar over the entire length of any of the aforementionned probes may also be used to detect the bacteria of the present invention.
- the medium can be aquarium water, wherein the DNA is isolated therefrom.
- the medium can also contain a material such as aquarium gravel, sponge filter material, filter floss, or plastic filter media, but is not considered to be limited to these. Accordingly, the DNA can be isolated from the above and other sources where such bacteria may be expected to be found.
- a DNA chip may include a solid carrier and a group of nucleotide derivatives or their analogues fixed to the solid carrier via covalent bonding.
- Detection of a nucleic acid fragment with a DNA chip is generally performed using a probe oligonucleotide which is complementary to the nucleic acid fragment to be detected, by way of hybridization.
- the probe oligonucleotide is generally fixed onto the solid carrier (e.g., solid substrate).
- a nucleic acid fragment in a sample liquid may be provided with a fluorescent label or a radioisotope label, and then the sample liquid may be brought into contact with the probe oligonucleotide of the DNA chip. If the labelled nucleic acid fragment in the sample liquid is complementary to the probe oligonucleotide, the labelled nucleic acid fragment is combined with the probe oligonucleotide by hybridization. The labelled nucleic acid fragment fixed to the DNA chip by hybridization with the probe oligonucleotide may then be detected by an appropriate detection method such as, by way of example, fluorometry or autoradiography, although other methods for detection may be utilized.
- an appropriate detection method such as, by way of example, fluorometry or autoradiography, although other methods for detection may be utilized.
- the method may alternatively be performed in conjunction with a wide variety of automated processes, which will readily recognized by those of skill in the art, and implemented by routine experimentation.
- the method of the present invention may be performed with DNA or protein microarrays, biosensors, bioprobes, capillary electrophoresis, and real-time PCR to name some common methologies; although it will be readily appreciated by one of skill in the art that this list in not all inclusive.
- the detection method of the present invention provides an effective tool for one to monitor and detect the occurence of bacteria capable of oxidizing ammonia to nitrite in a medium.
- the method also provides a tool for one to check the commercial additives to determine the effectiveness of the additives, by measuring the occurrence or the amount of the bacteria of the present invention.
- PCR primers are provided that may be used to detect the bacteria and nucleic acid sequences of the present invention.
- the PCR primer pairs are represented by SEQ ID NO:6 and SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO:12 and SEQ ID NO:22 and SEQ ID NO:23.
- a sequence that is at least 96% similar over the entire length of any of the aforementioned PCR primers may also be used to detect the bacteria of the present invention.
- variants of the aforementioned oligonucleotide probes and PCR primers that still may be used to detect the bacteria and nucleic acid sequences of the present invention are within the scope of the present invention.
- the present invention includes isolated bacteria, isolated bacterial strains, bacterial cultures and nucleotide sequences comprising the sequences identified herein as SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, or variants of those sequences.
- Particularly preferred variants are those in which there is a high degree of similarity to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.
- the present invention includes variants that are at least 96% similar, at least 97% similar, at least 98% similar or at least 99% similar to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20. It is appreciated in the art that disclosures teaching those skilled in the art how to make and use a reference sequence (such as SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:20) will also be sufficient to teach such an individual to make and use the described variants.
- Variants of particular nucleotide sequences may be naturally-occurring polymorphisms or synthetic sequence alterations (see, e.g. U.S. Patent No. 6,485,938).
- a great diversity of modifications to nucleotide sequences, both natural and synthetic, are common and well known in the art, along with methods for making the synthetic variants (see, e.g. U.S. Patent Nos. 6,448,044 and 6,509,170).
- Methods for comparing the similarity of two or more nucleotide sequences are well known in the art. Similar sequences are often identified using computer programs such as BESTFIT and BLAST (see, e.g., U.S. Patent No. 6,461,836).
- hybridization may be used to detect the similarity between variant sequences and a reference sequence (see, e.g., U.S. Patent No. 6,573,066).
- a reference sequence see, e.g., U.S. Patent No. 6,573,066
- one skilled in the art would be able to easily synthesize and identify nucleotide sequences that are variants of a reference sequence by using known techniques. Therefore, a specification that describes a nucleotide sequence and its variants allows one skilled in the art to make and use that sequence and its variants.
- EXAMPLES A series of assays and experiments were conducted to isolate, identify and show the efficacy of the bacterial strains reported herein. They involved a variety of bacterial culturing techniques, molecular biological analyses of DNA extracted from samples of the cultures, molecular biological analysis of the bacterial strains, and the application of concentrated cultures of the bacterial strains to aquaria to measure their ability to control ammonia concentrations.
- EXAMPLE 1 Bacteria Culturing
- Bacterial culturing vessels were constructed and seeded with bacterial biomass gathered from operating aquaria. Each reactor received 4.95 L of a mineral salt solution (made up in distilled water) containing 50g KH 2 PO 4 , 50g K 2 HPO 4 , 18.75g MgSO 4 « 7H 2 O, 1.25g CaCl » 2H 2 O and lg FeSO 4 »7H 2 O. Air was provided such that the dissolved oxygen was equal to or greater than 7.5 mg/L, stirring was provided, and the reactors were kept in a darkened cabinet at approximately 28°C.
- a mineral salt solution made up in distilled water
- samples of appropriate biological filtration media were taken and resuspended in cell lysis buffer (40 mM EDTA. 50 mM Tris-HCl, pH 8.3). Samples were stored at -20°C or -74°C until extraction.
- lysozyme was added to the samples to a final concentration of 10 mg/ml.
- 20% sodium dodecyl sulfate (SDS) was added to a final concentration of 1%.
- SDS sodium dodecyl sulfate
- the samples were subjected to four freeze/thaw cycles followed by the addition of proteinase K (stock concentration, 10 mg/ml) to a final concentration of 2 mg/ml and incubated at 70°C for 35 minutes. In some cases, additional proteinase K and SDS were added and the sample was incubated at 55°C for another 30 minutes.
- Clone libraries were derived from DNA extracts from biomass samples taken from reactors and aquaria.
- Bacterial ribosomal RNA gene fragments from bacteria represented by the sequences SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 18 and SEQ ID NO:20 were amplified with the primers S-D-Bact-001 l-a-S-17 (8f; GTT TGA TCC TGG CTC AG) (SEQ ID NO: 13) and 1492r (eubacterial; GGT TAG CTT GTT ACG ACT T) (SEQ ID NO: 14).
- PCR conditions, cycle parameters, and reaction components were as previously described (DeLong, E. F. 1992.
- PCR products were evaluated by agarose gel electrophoresis. PCR fragments were cloned with a TA Cloning kit (Invitrogen, Carlsbad, CA), as described in the manufacturer's directions, after rinsing with TE buffer and concentrating to 30 ⁇ l with a CENTRICON concentrator (Amicon, Inc. Beverly, MA).
- the 16S rDNA insert from each clone that comprised the clone library were screened by restriction enzyme analysis (REA) using the restriction enzyme Hae III in order to ensure that the 16S rDNA insert was amplifiable and determine whether the 16S rDNA possessed a unique REA pattern when digested with the Hae III enzyme. If a clone was amplifiable and possessed a unique REA pattern, then the clone's plasmid containing the 16S rDNA insert of interest was partially sequenced.
- the amplified PCR 16S rDNA template of each clone selected for sequencing was cleaned using the PCR Purification Kit Catalog No. 28142 (Qiagen). Sequencing was performed using a LiCor 4000L automated DNA sequencer on template cycle-sequenced with fluorescently labeled primers and SEQUITHERM EXCEL II DNA Sequencing kits (Epicenter Technologies, Madison, WI).
- rDNA fragments were amplified using the forward 358f (eubacterial; CCT ACG GGA GGC AGC AG) (SEQ ID No:15) with a 40-bp GC- clamp on the 5' end as described by Murray et al. (A. Murray et al. 1996. Phylogenetic compositions of bacterioplankton from two California estuaries compared by denaturing gradient gel electrophoresis of 16S rDNA fragments. Appl. Environ. Microbiol.
- PCR conditions were the same and were performed on a ROBOCYCLER GRADIENT 96 (Stratagene, La Jolla, CA) using the TAQ PCR core kit (Qiagen). PCR conditions included a hot start (80°C) and a touchdown procedure. Initial denaturation at 94°C for 3 min. was followed by a denaturation at 94°C for 1 min., a touchdown annealing from 65°C to 55°C for 1 min. 29 sec. (the annealing time during the touchdown increased by 1.4 sec. per cycle) and primer extension at 72°C for 56 sec. (the extension time was increased 1.4 sec. per cycle).
- Biospec Biospec Products Inc., Bartlesville, OK; hereinafter "Biospec"
- Biospec Biospec Products Inc., Bartlesville, OK
- a mechanical bead beater MINI-BEADBEATER-8, Biospec
- the processed DNA remained in the tubes at 4°C overnight. After overnight storage, the tubes were centrifuged at 3,200 X g for 8 minutes at 4°C to concentrate the gel fragments. The supernatant was transferred to a clean eppendorf tube.
- the supernatant was re-amplified with the DGGE primers and re-analyzed by DGGE. An extraction was considered acceptable if it yielded a single band in DGGE analysis which co-migrated with the original DGGE band in the mixed population sample.
- the nucleotide sequence of the excised band was sequenced by the previously described methods using fluorescently labeled primers.
- Oligonucleotide probes were designed that specifically hybridize with the 16S rRNA gene sequence isolated from closely related bacteria from reactors in this study.
- One probe (S-G- Nsspa-0149-a-A-18) (SEQ ID NO:5) targets two reactor-derived Nitrosospira-like bacteria, which are represented by the sequences of SEQ ID NO:l and SEQ ID NO:2 to the exclusion of other beta subdivision
- Proteobacterial ammonia-oxidizers including the sequences represented by SEQ ID NO:3 and SEQ ID NO:4, and also to the exclusion of Nitrosomonas aestuarii-like bacteria represented by SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.
- a second probe targets one reactor-derived Nitrosospira-like bacterium, which is represented by the sequence of SEQ ID NO:3, to the exclusion of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:4 and other beta subdivision Proteobacterial ammonia-oxidizers, and also to the exclusion of Nitrosomonas aestuarii-like bacteria represented by SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20.
- S-G- Ntsms-0149-a-A-18 targets Nitrosomonas aestuarii-like bacteria, which are represented by the sequences of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20 to the exclusion of other AOB sequences represented by SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, as well as sequences from halophila-like bacteria.
- Probe matches were initially screened using BLAST (S.F. Altschul et al.1990. Basic local alignment tool. J. Mol.
- Table 10 The nucleotide sequences and positions of oligonucleotide probes and PCR primer sets for ammonia-oxidizing bacteria.
- the stringency for the probes was determined though a series of FISH experiments at differing formamide concentrations using the reactor biomass as a positive control for the bacterial sequences herein (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20).
- the specificity of the probes was examined by testing against negative control cells of pure cultures of other beta subdivision ammonia-oxidizing bacteria (Nitrosomonas europaea, Nitrosospira multiformis, Nitrosomonas cryotolerans).
- In situ hybridization of the fixed, immobilized cells was carried out in a hybridization solution consisting of 0.9 M NaCl, 20 mM Tris/HCl (pH 7.4), 0.01% sodium dodecyl sulphate (SDS), 25 ng of oligonucleotide probe, and varying amounts of formamide.
- Slides were incubated in an equilibrated humidity chamber at 46°C for 90 to 120 min.
- the hybridization solution was rinsed off with a prewarmed (48°C) wash solution.
- the slides were then incubated in the wash solution for 15 min. at 48°C.
- the wash solution contained 20mM Tris/HCl (pH 7.4), 0.01% SDS, 5 mM EDTA, and NaCl.
- concentration of NaCl varied according to the percent formamide used in the solution. For 20% formamide the NaCl concentration was 215 mM, for 30% it was 120 mM, and for 40% the NaCl concentration was 46 mM.
- Cells were detected using an AXIOSKOP 2 epifluorescence microscope (Carl Zeiss, Jena, Germany) fitted with filter sets for FITC/FLUO3 and HQ CY3. The optimum stringency was determined to be 30% formamide for the S-G-Nsspa-0149-a-A-18 probe.
- Two sets of PCR primers were developed which specifically detect Nitrosospira-like bacteria of the sequences of the present invention.
- a third set of PCR primers was developed which specifically detects Nitrosomonas-like bacteria of the sequences of the present invention.
- One set (SEQ ID NO:6 and SEQ ID NO:7) specifically detects Nitrosospira-like bacteria with the sequence SEQ ID NO:l and sequence SEQ ID NO:2 to the exclusion of other ammonia-oxidizing bacteria (Table 11).
- the second set (SEQ ID NO:9 and SEQ ID NO:10) specifically detects the Nitrosospira-like bacteria with the sequence SEQ ID NO: 3 to the exclusion of other ammonia- oxidizing bacteria (Table 11).
- the third set (SEQ ID NO:l l and SEQ ID NO:12) specifically detects the Nitrosomonas-like bacteria with the sequence SEQ ID NO:4 to the exclusion of other ammonia-oxidizing bacteria (Table 11).
- a fourth set specifically detect the Nitrosomonas aestuarii-like bacteria with the sequences SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20 to the exclusion of other ammonia-oxidizing bacteria.
- PCR conditions were as previously described except the annealing temperature was modified.
- Table 11A Results of the PCR primer development specificity testing and annealing temperature experiments.
- SEQ ID NO:7 SEQ ID NO: 10 SEQ ID NO: 12
- Table 11B Results of the PCR primer development specificity testing and annealing temperature experiments.
- each primer set was optimized by conducting a PCR experiment with each primer set using the temperature gradient mode of the Stratagene ROBOCYCLER. In this mode one can run a single experiment of all the reactions at up to 12 different annealing temperatures. Typically, the experiments were conducted at 4 to 6 different temperatures with 2°C increasing interval.
- Each PCR primer set was tested against clone product with a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO:19 and SEQ ID NO:20.
- rDNA extracted from pure cultures of Nitrosomonas europaea, Nitrosolobus multiformis and Nitrosomonas cryotolerans were also tested. Table 10 presents the PCR primer sets, and the optimal annealing temperature results are shown in Table 11.
- Table 12 Details regarding the reactors and aquaria from which biomass was extracted and clone libraries was constructed.
- Biofarm 16 This biomass was retrieved from the sump of BF16. The biofarm was routinely dosed 300mg/L/hr of ammonia (NH 3 -N) for 6 hours per day.
- NH 3 -N ammonia
- BC5 This biomass was kept in an aquarium (seeded from a freshwater biofarm) and dosed 5mg/L or less of ammonia every two or three days. The aquarium was not aerated.
- BC5(2) Same as BC5 (above).
- R7BA6 This biomass was recovered from a Bacterial Additive test that was inoculated with R7 biomass.
- This reactor was seeded with 20 liter of material from the sump of biofarm 15 which was a saltwater biomass whose salinity was maintained at between 30 and 35 ppt. This reactor was fed at 5 mg/L ammonia-ni ⁇ Ogen.
- This reactor was seeded with material from the sumps of biofarm 5 and 15 which were saltwater biomasses whose salinity was maintained at between 30 and 35 ppt. This reactor was fed at 5 mg/L ammonia-nitrogen.
- the clone library data show that there are three groups of ammonia oxidizing bacteria that exist in the low ammonia feed reactors (e.g., R3, R7). Not all three AOB types were found to exist in every reactor though.
- the three bacteria are represented by three AOB clone groups - AOB Type A (SEQ ID NO:l) (and a subtype Al (SEQ ID NO:2)), and AOB Type B (SEQ ID NO:3).
- a fourth clonal type was found in high ammonia feed reactors - AOB Type C (SEQ ID NO:4).
- a similarity ranking was conducted for the four clonal sequences using RDP (Maidak, B.L., J. R. Cole, C. T. Parker, Jr, G. M.
- Type A AOB Clonal members of Type A AOB (SEQ ID NO:l and SEQ ID NO:2) were found in both the BF16 biomass (9% of clone library) and the BC5 biomass (1-2% of clone library) (Fig. 2).
- the BC5 biomass was used to seed the low concentration ammonia reactor (RI), which was used to seed R7.
- Type A AOB bacteria have been successfully subcultured from the freshwater Biofarm to the BC5 tank and then in the R7 reactor via the RI reactor.
- the BC5 tank and the R7 reactor were both fed and maintained at ammonia levels at or below 5 mg/L NH - N.
- the Type A AOB bacteria may be able to exist at ammonia concentrations above 5mg/L NH 3 - N but it is apparent that at higher concentrations of ammonia they are outcompeted by other types of AOBs (i.e., Type B (SEQ ID NO:3) and/or Type C (SEQ ID NO:4)) as evidenced by these types of AOB being present, and Type A AOB being absent, in the reactors maintained at high ammonia concentrations (Table 14) (Fig. 2).
- the R7 biomass did particularly well in the bacterial additives test VI (BA6) and VII (BA7) (discussed below) as did a biomass grown in the same fashion (R19) and with the same seed (RI) in bacterial additives test VIII (BA8) (R19).
- Type A AOBs have been found in a number of reactors and a number of Post BA test biomasses both by specific Type A AOB PCR and FISH tests (Table 14) (Fig. 2).
- Type A AOB SEQ ID NO:l
- Type Al SEQ ID NO:2
- Type B Clonal members of Type B were found in the freshwater BioFarm biomasses (e.g., BF 16 - 34% of clone library) used to seed the BC tanks (BC5).
- Type B AOB bacteria were absent in the BC5 and R7 clone libraries, indicating that these AOBs may be more suited to the high ammonia conditions and feeding regime of a Biofarm (Fig. 2).
- Type B AOBs were also found in the R3 clone library (19% of clone library) (Fig. 2).
- the history of the R3 reactor is that its biomass was initially enriched at high ammonia concentrations (3000mg/L NH 3 -N), stored for 11 months and then matured in the reactor at low ammonia concentrations (5mg/L NH 3 -N) for an extended period of time.
- ammonia concentrations 3000mg/L NH 3 -N
- 5mg/L NH 3 -N low ammonia concentrations
- the ammonia concentrations would decrease over time - thus encouraging the growth of Type C and/or Type B AOBs over Type A AOBs.
- the ammonia would be likely to be exhausted possibly encouraging the maintenance of Type B AOB bacteria in the system and the survival of residual Type A AOBs that had survived during the culturing phase.
- the Type B AOB bacteria would be able to be maintained, Type A AOBs would be enriched and any residual Type C that had been originally selected for in the original culturing phase would be outcompeted and disappear.
- the Biofarm' s biomass receives a relatively high concentration of ammonia for a set period of time and then allowed to gradually deplete this over time, creating both a gradient of high to low ammonia concentrations (encouraging the growth of Type B AOBs), often reaching zero thus allowing a window for the growth of Type A AOBs - low ammonia concentrations.
- This is a more rapid cycle (daily) than the culturing phase of the R3 biomass, but none the less consistent with a change of conditions from high to low ammonia concentrations within the biomass.
- the gradient of ammonia concentrations in the Biofarm' s biomass encourages the enrichment of a range of AOB types as confirmed by the clone library data and the results of the DGGE tests.
- Type B AOBs have been found in a number of reactors and a number of Post BA test biomasses both by specific Type B AOB PCR, DGGE and FISH. However, it has not been found in as many post bacterial additive tests or clone libraries as Type A AOB (Table 14). It seems to be that if Type A AOB was inoculated into a test it was often recovered whereas Type B AOBs were only recovered in systems where Type A AOBs were not originally in the innoculum. Therefore, Type A AOBs are preferentially grown in the systems when they are present but Type B AOBs will suffice when Type A AOBs are absent.
- Type A AOBs are the most important member of a successful AOB nitrifying community for low ammonia environments such as aquarium, they are not the only AOB present.
- Other AOB such as Type B (SEQ ID NO:3), may be necessary for the system to efficiently cope with fluctuating concentrations of ammonia even over short (days) periods of time.
- Type C AOBs are not desirable as an AOB in a bacterial additive for the low ammonia concentrations typically found in an aquarium.
- Type C AOB bacteria were not found in the BF16, BC5 or R7 clone libraries which are low ammonia concentration environments, indicating that they were likely grown under conditions other than that found in these three environments (Fig. 2).
- Type C bacteria were found in the R5, R3 and R17 clone libraries (Fig. 2).
- the R5 biomass was grown consistently at high concentrations (30mg/L NH 3 -N) and its seed was from a very high ammonia concentration (>500mg/L NH 3 -N), R3's biomass had been originally grown at a high ammonia concentration before being moved to a lower ammonia concentration (5mgL NH 3 -N) and the R17 biomass was moved from a low (5mg/L NH 3 -N) to a high ammonia concentration (30mg/L NH 3 -N) and then back again.
- the R5 biomass had been enriched at high ammonia concentrations for a long period of time even before being transferred to the R5 reactor, in effect excluding the growth of any Type A AOB bacteria as the concentration of ammonia never dropped to low levels in the feed microfilter.
- Type B AOB were enriched for and became the dominant AOB in this culture.
- Type C bacteria would represent the bacteria enriched for initially in the microfilter and then remained in the R5 biomass when the feed was kept at relatively high ammonia concentrations (30mg/L NH 3 -N).
- the R3 biomass had been initially allowed to grow at high ammonia concentrations but over time the ammonia would become exhausted. This regime initially encourages the growth of Type C AOBs (at higher ammonia concentrations) and Type B AOBs (as the ammonia was utilized). Further, these pressures would not allow for the enrichment of Type A AOB which are dependent on consistently low levels of ammonia.
- Type C AOB bacteria would be enriched against and Type B would still survive but since Type A AOB bacteria were originally minimized in the initial enrichment there would be very few left to take advantage of the new conditions within the reactor. Therefore, Type B AOB would be expected to be the dominant AOB in this environment.
- the R17 biomass shows typically what not to do for culturing Type A and/or B AOBs.
- the R17 biomass was derived from the R7 biomass but cultured for 3 weeks at elevated ammonia (30mg/L NH 3 -N) concentrations to see if a shift in the microbial community would occur. A shift did occur and Type C AOBs became dominant, as demonstrated by the results of FISH, PCR and DGGE experiments. Furthermore, the shift was irreversible. After moving the biomass back to a low ammonia concentration (5mg/L NH 3 -N) environment, the Type C AOB still remained the dominant AOB while Type A and Type B AOBs could not be detected by either FISH or DGGE.
- Type A and B AOBs were excluded from the R17 biomass.
- the R17 biomass did poorly in the subsequent BA VIII test suggesting that Type C AOBs are not the correct type of AOB required for an effective bacterial additive to be used in the relatively low ammonia environment of an aquarium.
- This conclusion is further supported by the results of the bacterial additive tests which showed that existing commercial bacterial mixtures which contain Nitrosomonas clade AOBs are not effective for accelerating the establishment of nitrification in aquaria (discussed below).
- Type C bacteria are very closely related phylogenetically to those bacteria that have been found in wastewater treatment plants which also receive ammonia concentrations of around 30mg/L NH 3 -N (similar to R5).
- the PCR primer sets described herein were used to detect the presence or absence of the AOB strains reported here in a variety of environments.
- the environments include pre bacterial additive test mixtures, post bacterial additive test aquaria filters, and commercial mixtures of nitrifying bacteria manufactured and sold by other companies.
- DNA extracted from the pure culture of other AOB was tested.
- FIG. 3 shows the DGGE results for two clone representatives for each of the Type A AOB (SEQ ID NO:l), Type Al AOB (SEQ ID NO:2), Type B AOB (SEQ ID NO:3) and Type C AOB (SEQ ID NO:4) in a general eubacterial DGGE.
- the bacterial sequence of each AOB Type described herein denatures at a different position in the gel. This is indicative of uniqueness and provides another means by which to distinguish each AOB Type from one another and also from known AOB.
- Figure 7 presents DGGE results that further distinguish the various AOB strains of the present invention.
- Bands representing Nitrosomonas aestuarii-like clones P4c42 and P4c31 do not co-migrate with bands representing the strains of AOB of Type A AOB (SEQ ID NO:l), Type B AOB (SEQ ID NO:3), Type C AOB (SEQ ID NO:4) and Nitrosomonas aestuarii-like clone BF16c57 (SEQ ID NO:20). None of these strains co-migrated with Nitrosomonas europaea, Nitrosospira multiformis or Nitrosomonas cryotolerans (Fig. 7).
- Effectiveness of a mixture is demonstrated by showing that the ammonia-oxidizing bacterial strains of the present invention oxidize ammonia in aquaria and, further, that when combined with other bacterial strains (e.g., nitrite-oxidizing bacteria), the bacteria accelerate the establishment of nitrification in aquaria.
- Establishment of nitrification can be measured in at least three different ways. The first is by counting the number days it takes after establishing a new aquarium for the ammonia and nitrite concentrations in the aquarium water to reach a near 0 mg/L concentration.
- a second way to measure the beneficial action of adding nitrifying bacterial strains to aquaria is to compare the maximum concentration of ammonia or nitrite reached before the concentration drops to 0 mg/L. If the maximum concentration of ammonia or nitrite reached in aquaria in which nitrifying bacteria were added is significantly less than the maximum concentration reached in control aquaria, then a degree of effectiveness is demonstrated.
- a third way to evaluate the effectiveness of nitrifying bacterial strains and mixtures that incorporate them is to combine the first two methods to form a toxicity exposure curve.
- This type of curve accounts for both the duration (time in days) and the degree/intensity of the exposure.
- this curve is generated by plotting the concentration of the toxin over time.
- the area of the curve may then be determined for each treatment and toxin by standard computational methods (e.g., by mathematically integrating the curve).
- the treatments of each test are then compared to one other and to the control of the same test.
- the control curve area can be given an arbitrary value of 1 and the other areas may thereafter be calculated as a ratio to the control area. As such, if the value of a treatment is greater than 1 it is deemed more effective than the control, while a value of less than 1 suggests that it is less effective than the control and may have inhibited the establishment of nitrification.
- the goal of this test was to evaluate the ability of four bacterial mixtures, including bacterial strains of the present invention, to accelerate the establishment of nitrification in freshwater aquaria.
- the test was also conducted to compare an ability to establish nitrification among the bacterial strains of the present invention and control aquaria which did not receive a bacterial inoculation of any kind.
- Twenty-seven ten-gallon aquaria and twenty-seven Penguin 170B (Marineland Aquarium Products) hang-on-the-back style power filters were sterilized, thoroughly rinsed, and allowed to air dry. Each aquarium was then filled with 10 lbs of rinsed aquarium gravel (RMC Lonestar #3) and the filter installed.
- the aquaria then received 35 1 of city tap water which had been filtered through activated carbon. After turning the filters on, the water level on each aquaria was marked so all could be topped-off with deionized water (DI) to account for any water loss due to evaporation and sampling. The filters ran overnight prior to the addition of the bacterial additives and fish.
- DI deionized water
- BC5 - a bacterial mixture which had been under culture for 553 days preceding the test. A positive result with this mixture would demonstrate the long-term viability of the bacteria under culture conditions and the appropriateness of the culture techniques;
- Rtr4 - a bacterial mixture which had been bottled and stored in the dark for 118 days preceding the test. A positive result with this mixture would demonstrate that the storage method is valid and the mixture retains its viability for at least 119 days of storage;
- Rtr7 - a bacterial mixture which had been grown from an inoculum from BC5.
- a positive result with this mixture would demonstrate that one can culture the bacterial consortium in the mixture for successive generations and it maintains its viability.
- Other differences between the bacterial mixtures are depicted in Table 11.
- Figure 4 shows the mean ammonia and nitrite concentrations over the test period for the four mixtures along with the controls.
- ammonia reached 0 mg/L on day 8 for the aquaria dosed with 100 ml of BC5 mixture and day 10 for the aquaria dosed with 30 ml of BC5 mixture.
- the ammonia concentration in the control aquaria did not reach 0 mg/L until day 12.
- the highest mean ammonia concentration reached for the control aquaria was 4.9 mg/L.
- ammonia exposure curve area values for the aquaria dosed with 30 ml or 100 ml of the BC5 mixture were 67% and 37% of the control aquaria curve area value, respectively (Table 16); 1.5 and 2.7 times less exposure to ammonia, respectively, for fish in the treatment aquaria.
- Table 16 Toxicity Expi osure Data for Bacterial Additives VI test.
- Treatment Exposure % of Area Treatment Exposure % of Area
- Nitrite concentrations reached 0 mg/L by day 18 in aquaria dosed with 100 ml of BC5 mixture, by day 21 in aquaria dosed with 30 ml of BC5 mixture, and by day 23 in control aquaria.
- the control aquaria reached a mean maximum nitrite control of 13.4 mg/L, while aquaria dosed with 30 ml of the BC5 mixture had a mean maximum nitrite concentration of 8.9 mg/L and those dosed with 100 ml of BC5 mixture had a maximum nitrite concentration of only 4.5 mg/L (Table 15; Fig. 4).
- nitrite exposure curve area values for the aquaria dosed with 30 ml or 100 ml of the BC5 mixture were 75% and 33% of the control aquaria curve area value, respectively (Table 16); 1.3 and 3.1 times less exposure to nitrite, respectively, for fish in the treatment aquaria.
- the control aquaria reached a 0 mg/L ammonia concentration after 12 days, while the aquaria dosed with 30 ml or 100 ml of the Rtr3 bacterial mixture took only 9 and 7 days to reach 0 mg/L, respectively (Table 15).
- the mean maximum nitrite concentration was 13.4 mg/L in the control aquaria, while the mean maximum nitrite concentration in aquaria dosed with 30 ml or 100 ml of the Rtr3 bacterial mixture was only 5.9 mg/L and 3.4 mg/L, respectively.
- the control aquaria reached a 0 mg/L nitrite concentration in 23 days, while the aquaria dosed with 30 ml or 100 ml of Rtr3 bacterial mixture reach 0 mg/L after only 15 and 11 days, respectively (Table 15).
- ammonia exposure curve area values for the aquaria dosed with 30 ml or 100 ml of the Rtr3 mixture were 45% and 26% of the control aquaria curve area value, respectively (Table 15); 2.2 and 3.9 times less exposure to ammonia, respectively, for fish in the treatment aquaria.
- nitrite exposure curve area values for the aquaria dosed with 30 ml or 100 ml of the Rtr3 mixture were 28% and 11% of the control aquaria curve area value, respectively (Table 15); 3.6 and 8.8 times less exposure to nitrite, respectively, for fish in the treatment aquaria.
- the mean maximum ammonia concentration was 2.9 mg/L and 2.7 mg/L, respectively, for a dosage volume of 30 ml and 100 ml (Table 15), while control aquaria reached a mean maximum ammonia concentration of 4.9 mg/L.
- the ammonia exposure curve area values for the aquaria dosed with 30 ml or 100 ml of the Rtr4 mixture were 45% and 38% of the control aquaria curve area value, respectively. These values show that the addition of the mixture resulted in 2.2 and 2.7 times less exposure to ammonia, respectively, for the fish in the treatment aquaria when compared to the control aquaria (Table 15).
- Aquaria dosed with 30 ml of the Rtr4 mixture completed the nitrification cycle in 10 days, while nitrification was established in 8 days for aquaria dosed 100 ml of the Rtr4 mixture (Table 15).
- Nitrification was established in 23 days in the control aquaria.
- the mean maximum nitrite concentration for the aquaria dosed with 30 ml or 100 ml of the Rtr4 bacterial mixture was 2.1 mg/L and 0.6 mg/L, respectively.
- the control aquaria had a mean maximum nitrite concentration of 13.4 mg/L.
- nitrite exposure curve area values for the aquaria dosed with 30 ml or 100 ml of the Rtr4 mixture were 5% and 2% of the control aquaria curve area value, respectively; 20.2 and 60.9 times less exposure to nitrite, respectively, for fish in treatment aquaria (Fig. 4; Table 15).
- the bacterial mixture Rtr7 which was a subculture from the BC5 mixture, demonstrated a significantly faster establishment of nitrification when compared to the control aquaria.
- the control aquaria took 12 days to reach a 0 mg/L ammonia concentration, while aquaria dosed with 30 ml or 100 ml of the Rtr7 bacterial mixture took only 7 and 6 days, respectively (Table 15).
- the mean maximum ammonia concentration for aquaria dosed with 30 ml or 100 ml of the R7 mixture was 1.9 mg/L and 1.1 mg/L, respectively. This is in contrast to the control aquaria that had a mean maximum ammonia concentration of 4.9 mg/L (Table 15).
- the ammonia exposure curve area values for aquaria dosed with 30 ml or 100 ml of the Rtr7 mixture were 28% and 17% of the control aquaria curve area value, respectively (Table 15); 3.6 and 5.7 times less exposure to ammonia, respectively, for fish in the treatment aquaria.
- the nitrite exposure curve area values for aquaria dosed with 30 ml or 100 ml of the Rtr7 mixture were 10% and 4% of the control aquaria curve area value, respectively; 5.7 and 25.5 times less exposure to nitrite, respectively, for fish in the treatment aquaria (Table 15).
- the data from the test show that the various bacterial mixtures of the present invention accelerate the establishment of nitrification in aquaria. Use of these mixtures in aquaria significantly reduced the degree of ammonia and nitrite exposure to fish.
- the results further demonstrate that a mixture can be viably maintained over a long period of time (e.g., BC5), that the mixture can be stored for several months (e.g., Rtr 3 and Rtr 4) and that successive generations of the mixture retain their nitrifying ability (e.g., Rtr 7).
- the goal of this test was to evaluate two mixtures of bacterial stains of the present invention as they may be implemented in a "real world" setting while comparing their performance to that of commercial bacterial mixtures.
- a new aquarium owner first purchases the necessary equipment for setting-up an aquarium able to maintain aquatic life.
- the equipment may include the aquarium itself, decorations, a heater and filter, and a water conditioner.
- the aquarium is then assembled and filled with water, the filters are started, the heater is adjusted to the proper water temperature and the water conditioner added to remove chlorine.
- the fish are usually added, but there may be insufficient populations of ammonia- and nitrite-oxidizing bacteria present to maintain the ammonia and nitrite concentrations in the aquarium at biologically safe (i.e., non- toxic) concentrations (e.g., below 0.5 mg/L-N).
- new tank syndrome i.e., elevated concentrations of ammonia and nitrite in the first several weeks after setting-up a new aquarium when an- insufficient population of nitrifying bacteria are present to maintain safe ammonia and nitrite concentrations.
- a bottled mixture of microorganisms or an enzyme mix i.e., the bacterial mixture
- introduction of the bottled mixture should result in comparatively lower ammonia and nitrite concentrations in an aquarium during its initial weeks than in the absence of such a mixture. Also, less time should be required for the ammonia and nitrite concentrations to reach 0 mg/L.
- the Rtr5 and Rtr7 mixtures established nitrification in newly set-up aquaria significantly faster than the commercial mixtures and untreated aquaria. Complete nitrification was established in 8 days with the Rtr7 mixture and in 10 days with the Rtr5 mixture (Table 18). The closest treatments to these were FRITZ-ZYME at the its normal dosing level, CYCLE at three times its normal dosing level, and STRESS ZYME at its normal dosage level; each of which took 22 days (Table 18). The Rtr5 and Rtr7 mixtures were therefore 2.2 to 2.8 times faster at establishing nitrification then these other mixtures.
- the Rtr7 mixture exhibited the same trend as the Rtr5 mixture in that aquaria dosed with this mixture exhibited significantly lower maximum ammonia-nitrogen and nitrite-nitrogen concentrations than aquaria dosed with commercially available bacterial mixtures (Table 18).
- the Rtr7 mixture had mean maximum ammonia and nitrite concentrations of 2.8 mg/L and 1.3 mg/L, respectively. These were 2.6 and 2.4 times lower, respectively, than the closest commercially available bacterial mixture (FRITZ-ZYME, dosed at its normal level) (Fig. 5; Table 18).
- the bacterial mixtures Rtr5 and Rtr7 which incorporate bacterial strains of the present invention, significantly outperformed the commercially available mixtures (Table 19).
- Rtr7 performed better than any mixture with the fish exposed to just 13% of the ammonia and 5% of the nitrite of the control.
- Rtr5 was almost as effective, with ammonia exposure at 14% of control levels and nitrite exposure at 9% of the control (Table 19).
- These results mean that fish in aquaria receiving either Rtr7 or Rtr5 are exposed to 7.3 to 7.6 times less ammonia and 11.6 to 19.6 times less nitrite than fish in control aquaria.
- the next best mixtures reduced the exposure of ammonia and nitrite by only 50% when compared to controls (Table 19).
- Table 19 Toxicity exposure data for the Bacterial Additives VII test.
- Treatment Exposure % of Area Treatment Exposure % of Area
- Reactor 3 included strains of AOB of the present invention represented by SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3
- Reactor 29 included strains of AOB of the present invention represented by SEQ ID NO:18, SEQ ID NO:19 and sequences from two halophila-like strains
- CYCLE a commercially available bacterial mixture for use in freshwater or saltwater
- STRESS ZYME another commercially available bacterial mixture for use in freshwater or saltwater.
- Each treatment had three replicates.
- Aquaria receiving the Reactor 3 and Reactor 29 treatments were dosed with 100 ml of either mixture one time on the first day of the test.
- Aquaria receiving the CYCLE or STRESS ZYME treatments were dosed with 10 ml of either treatment on the first day of the test, an additional 10 ml on day 7 of the test and an additional 5 ml every 7 days after that for the duration of the test.
- Four assorted damsels (Pomacentrus spp.) were added to each tank on the first day of the test and fed twice a day.
- Treatment Reactor 29 which consisted of strains of AOB represented by SEQ ID NO: 18, SEQ ID NO: 19 and two N halophila-like strains, oxidized ammonia markedly quicker than the other treatments.
- the mean maximum ammonia concentration of treatment Reactor 29 was also significantly lower than the other three treatments. In fact, the ammonia trend for the other three treatments over the first 14 days of newly set up aquaria were not significantly different than the control (non-inoculated) treatment. There was no evidence that adding more of the commercial AOB mixtures to the aquaria reduced the amount of time necessary to establish ammonia oxidation.
- the goal of this test was to compare the biomass material from reactor SB7 (which contained AOB strains of the present invention represented by SEQ ID NO: 18, SEQ ID NO: 19 and two halophila-like strains) to aquaria that did not receive a bacterial inoculation.
- the SB7 AOB reactor mixture consisted of strains of AOB of the present invention represented by SEQ ID NO: 18, SEQ ID NO: 19 and two N. halophila-like strains.
- Six clownfish (Amphiprion ocellaris) were added to each tank on the first day of the test and fed twice a day. The fish feed was a mixture of frozen brine shrimp and Spirulina fish flakes. On Day 3 of the test, four additional clownfish (Amphiprion ocellaris) were added to each aquarium.
- SB7 treatment including strains of AOB of the present invention represented by SEQ ID NO: 18, SEQ ID NO: 19 and two N halophila-like strains oxidized ammonia markedly quicker than did the control.
- the mean ammonia concentration reached 0 mg/L on day 9 in tanks receiving the SB7 treatment, while 17 days elapsed in the control aquaria before ammonia values reached the same level of 0 mg/L.
- the mean maximum ammonia concentration of the SB7 treatment (about 0.4 mg/L-N) was significantly lower than the control treatment (1.72 mg/L-N).
- reactor SB7 which contained AOB strains of the present invention represented by SEQ ID NO: 18, SEQ ID NO: 19 and two N halophila-like strains
- reactor B7 which contained two N halophila-like AOB strains
- the SB7 AOB reactor mixture consisted of strains of AOB of the present invention represented by SEQ ID NO: 18, SEQ ID NO: 19 and two JV. halophila-like strains.
- the B7 AOB reactor mixture consisted of two TV. halophila-like strains of AOB.
- the mean ammonia concentrations for the two treatments and control are depicted in Figure 10.
- the ammonia values for the aquaria that received either reactor B7 or reactor SB7 treatment oxidized ammonia at nearly the same rate; markedly faster than the control.
- the mean ammonia concentration reached 0 mg/L on day 3 for the tanks receiving either the B7 or SB7 treatments, and the mean maximum ammonia concentration of the B7 and SB7 treatment (about 0.2 mg/L-N) was significantly lower than the control treatment (2.0 mg/L-N) (Fig. 9).
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MXPA05002873A MXPA05002873A (en) | 2002-09-19 | 2003-09-10 | Ammonia-oxidizing bacteria and methods of using and detecting the same. |
AU2003272297A AU2003272297B2 (en) | 2002-09-19 | 2003-09-10 | Ammonia-oxidizing bacteria and methods of using and detecting the same |
CA002499482A CA2499482A1 (en) | 2002-09-19 | 2003-09-10 | Ammonia-oxidizing bacteria and methods of using and detecting the same |
EP03754473A EP1551773A2 (en) | 2002-09-19 | 2003-09-10 | Ammonia-oxidizing bacteria and methods of using and detecting the same |
JP2004537749A JP4470025B2 (en) | 2002-09-19 | 2003-09-10 | Ammonia-oxidizing bacteria and methods of use and detection thereof |
BR0314125-0A BR0314125A (en) | 2002-09-19 | 2003-09-10 | Ammonia oxidizing bacteria and processes for using and detecting them |
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CA2252064A1 (en) * | 1997-09-16 | 2000-05-20 | Crc For Waste Management And Pollution Control Limited | Aquatic nitrite oxidising microorganisms |
WO2001090312A1 (en) * | 2000-05-19 | 2001-11-29 | Aquaria, Inc. | Ammonia-oxidizing bacteria |
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CA2252064A1 (en) * | 1997-09-16 | 2000-05-20 | Crc For Waste Management And Pollution Control Limited | Aquatic nitrite oxidising microorganisms |
WO2001090312A1 (en) * | 2000-05-19 | 2001-11-29 | Aquaria, Inc. | Ammonia-oxidizing bacteria |
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AAKRA A ET AL: "RFLP OF RRNA GENES AND SEQUENCING OF THE 16S-23S RDNA INTERGENIC SPACER REGION OF AMMONIA-OXIDIZING BACTERIA: A PHYLOGENETIC APPROACH" INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, READING, GB, vol. 49, January 1999 (1999-01), pages 123-130, XP001029900 ISSN: 0020-7713 * |
AAKRA AGOT ET AL: "Detailed phylogeny of ammonia-oxidizing bacteria determined by rDNA sequences and DNA homology values" INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, vol. 51, no. 6, November 2001 (2001-11), pages 2021-2030, XP009026625 ISSN: 1466-5026 * |
BURRELL P C ET AL: "Identification of ammonia-oxidizing bacteria responsible for nitrification in freshwater aquaria" ABSTRACTS OF THE GENERAL MEETING OF THE AMERICAN SOCIETY FOR, vol. 101, 2001, page 483 XP009026630 101st General Meeting of the American Society for Microbiology;Orlando, FL, USA; May 20-24, 2001, 2001 ISSN: 1060-2011 * |
BURRELL PAUL C ET AL: "Identification of bacteria responsible for ammonia oxidation in freshwater aquaria" APPLIED AND ENVIRONMENTAL MICROBIOLOGY, [Online] vol. 67, no. 12, December 2001 (2001-12), pages 5791-5800, XP002271945 ISSN: 0099-2240 -& DATABASE EMBL [Online] retrieved from EBI Database accession no. AF386746 XP002271947 * |
HOVANEC T A ET AL: "Identification of ammonia- and nitrite-oxidizing bacteria responsible for nitrification in saltwater aquaria" ABSTRACTS OF THE GENERAL MEETING OF THE AMERICAN SOCIETY FOR, vol. 101, 2001, pages 499-500, XP009026629 101st General Meeting of the American Society for Microbiology;Orlando, FL, USA; May 20-24, 2001, 2001 ISSN: 1060-2011 * |
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WO2008120979A1 (en) * | 2007-04-03 | 2008-10-09 | Stichting Deltares | Microbiologically induced carbonate precipitation |
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