Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-01
2001-07-31
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S029000, C435S034000, C435S039000, C435S252100, C536S023100, C536S023700, C536S024320
Reexamination Certificate
active
06268154
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to a nitrite oxidizer and specifically to a new bacterium capable of oxidizing nitrite to nitrate.
2. Background Information
The oxidation of nitrite to nitrate by chemolitho-autotrophic nitrite-oxidizing bacteria (NOB) in fish culture systems, ranging from home aquaria to commercial aquaculture systems, is an important process. The accumulation of high concentrations of nitrite, toxic to fish and other aquatic organisms, is prevented by active nitrite removal by nitrifying microorganisms. Nitrite is formed in aquarium systems from the oxidation of ammonia, the principal nitrogenous waste of teleosts, by autotrophic ammonia-oxidizing bacteria (AOB). Thus, closed aquatic filtration systems usually provide a solid substratum, termed a biological filter or biofilter, to promote the growth of AOB and NOB. A variety of materials can form the substratum of a biofilter, ranging from gravel to specially engineered molded plastics. Biofilters can be submerged in the flow path of the filtration system, or can be located such that the water trickles or percolates through a medium situated in the atmosphere outside of the aquarium, before flowing back into the tank.
Traditionally, the bacteria responsible for the oxidation of ammonia and nitrite in aquaria were considered to be
Nitrosomonas europaea
and
Nitrobacter winogradskyi
, or their close relatives, respectively (Wheaton, F. W. 1977. Aquacultural Engineering. John Wiley & Sons, Inc. New York; Wheaton, F. W., J. Hochheimer, and G. E. Kaiser. 1991. Fixed film nitrification in filters for aquaculture, p. 272-303. In D. E. Brune and J. R. Tomasso (eds.), Aquaculture and Water Quality. The World Aquaculture Society, Baton Rouge, LA.). However, there is some indication that both
Nitrosomonas europaea
and
Nitrobacter winogradskyi
may not be predominant components of actively nitrifying freshwater aquaria (Hovanec, T. A. and E. F. DeLong. 1996. Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria. Appl. Environ. Microbiol. 62:2888-2896. These references, and all other references cited herein are hereby incorporated by reference.) In seawater aquaria,
Nitrosomonas europaea
and close relatives do appear to comprise a significant proportion of the total eubacterial community, but
Nitrobacter winogradskyi
was below detection limits in the study by Hovanec and Delong (1996).
Chemolithoautotrophic nitrite-oxidizing bacteria are phylogenetically diverse, occurring in several subdivisions of the Proteobacteria (FIG.
1
). The most well-studied members of this group of organisms (i.e.,
Nitrobacter winogradskyi
and close relatives) belong to the &agr; subdivision of Proteobacteria (Watson, S. W. and J. B. Waterbury. 1971. Characteristics of two marine nitrite oxidizing bacteria,
Nitrospina gracilis
nov.
gent
nov. sp. and
Nitrococcus mobilis
nov.
gen
. nov. sp. Arch. Microbiol. 77: 203-230.)
Nitrospina gracilis
and
Nitrococcus mobilis
, first isolated by Watson and Waterbury, were determined to be members of the &dgr; and &ggr; subdivisions of the Proteobacteria, respectively (Teske, A., E. Alm, J. M. Regan, S. Toze, B. E. Rittmann, and D. A. Stahl. 1994. Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria. J. Bacteriol. 176:6623-6630.) Another NOB, Nitrospira marina, is phylogenetically affiliated with non-nitrite-oxidizing bacteria such as
Leptospirillum ferrooxidans
(Ehrich, S., D. Behrens, E. Lebedeva, W. Ludwig, and E. Bock. 1995. A new obligately chemolithoautotrophic, nitrite-oxidizing bacterium,
Nitrospira moscoviensis
sp.
nov
. and its phylogenetic relationship. Arch. Microbiol. 164:16-23.) Based on phylogenetic analysis of 16S rRNA sequences, Erlich et al. proposed a new phylum within the domain Bacteria for these organisms (FIG.
1
). A newly discovered nitrite-oxidizing bacterium from a freshwater environment (a corroded iron pipe in a heating system), Nitrospira moscoviensis, was recently found to be phylogenetically related to
Nitrospira marina.
Whether in pure culture or on biofilters, NOB are slow-growing organisms with doubling times from 12 to 32 hours (Belser, L. W. and E. L. Schmidt. 1978. Diversity in the ammonia-oxidizing nitrifier population of a soil. Appl. Environ. Microbiol. 36:584-588; Carlucci, A. F. and D. H. Strickland. 1968. The isolation, purification and some kinetic studies of marine nitrifying bacteria. Exp. Mar. Biol. Ecol. 2:156-166.) Therefore, in newly set-up aquaria, ammonia and nitrite can reach concentrations toxic to fish before a sufficient biomass of AOB and NOB become established. To reduce the length of time for establishment of NOB on biofilters, commercial preparations of these organisms, in various forms of preservation, are available to seed the aquarium environment. These preparations range from essentially pure cultures of Nitrobacter species, to mixed cultures of autotrophic AOB and NOB organisms, to products which combine autotrophic nitrifying bacteria with various species of heterotrophic bacteria. Past studies have generally shown these mixes to be ineffectual, but have not elucidated specific reasons for their poor performance (Bower, C. E. and D. T. Turner. 1981. Accelerated nitrification in new seawater culture systems: effectiveness of commercial additives and seed media from established systems. Aquaculture. 24.1-9; Timmermans, J. A. and R. Gerard. 1990. Observations sur l'utilisation en etangs de suspensions bacteriennes du commerce. Bull. Fr. Peche Piscic. 316:28-30.)
Therefore, a need exists for a product containing an adequate bacterial culture to establish a sufficient biomass of nitrite-oxidizing bacteria in freshwater aquaria before the nitrite in the aquaria reaches concentrations toxic to fish.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an isolated bacterium or bacterial strain capable of oxidizing nitrite to nitrate. In a particularly preferred embodiment, the 16S rDNA of the bacterium or bacterial strain has the nucleotide sequence of SEQ ID NO:1, or a variant thereof which is at least 95% similar to SEQ ID NO:1.
The invention is also intended to include nucleic acid sequences and bacteria with sequences which are at least 90% similar, preferably 95% similar, most preferably 99% similar to SEQ ID NO:1. For the purposes of this application, “95% similar” means that single base substitutions may occur of up to 5% of the bases. By “99% similar” is meant that single base substitutions may occur of up to 1% of the bases.
In one preferred embodiment, the bacterium or bacterial strain will be in the form of a biologically pure culture of a bacterial strain capable of oxidizing nitrite to nitrate.
It is another object of the present invention to provide a mixture which includes a culture of concentrated bacteria capable of oxidizing nitrite to nitrate.
It is further object of the present invention to provide a method of alleviating the accumulation of nitrite in a medium.
The method comprises the addition to the medium of a bacterium or bacterial strain capable of oxidizing nitrite to nitrate.
It is yet another object of the present invention to provide a probe which can be used to detect and determine the quantity of bacteria capable of oxidizing nitrite to nitrate.
Accordingly, one object of the present invention is to provide a biologically pure culture of a bacterial strain capable of oxidizing nitrite to nitrate, wherein the 16S rDNA of the bacterial strain has a nucleotide sequence of SEQ ID NO:1.
Another object of the present invention is to provide a mixture comprising a concentrated bacteria capable of oxidizing nitrite to nitrate, wherein the 16S rDNA of the bacteria has a nucleotide sequence of SEQ ID 1.
A further aspect of the present invention is a method of alleviating the accumulation of nitrite in a water-containing medium. Such media include, but are not limited to, freshwater aquaria, saltwater aquaria, wastewater, wetlands, and other environments in which it
Aquaria Inc.
Johannsen Diana
Myers Carla J.
Pillsbury & Winthrop LLP
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