Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-17
2001-04-24
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S015000, C435S091200, C435S194000, C435S810000, C536S023100, C536S024320, C536S024330
Reexamination Certificate
active
06221594
ABSTRACT:
TECHNICAL FIELD
This invention relates to the removal of nitrogenous compounds from wastewater. In particular, the invention relates to an isolated consortium of microorganisms capable of nitrification of wastewater. The invention also relates to methods of identifying microorganisms capable of nitrification of wastewater and oligonucleotide primers and DNA probes suitable for use in the methods.
INTRODUCTION
The removal of nitrogenous compounds from sewage effluents is an important aspect in the remediation of wastewaters. The presence of ammonia, nitrite and nitrate in wastewater discharges can cause numerous problems ranging from eutrophication (Meganck and Faup, 1988) of the receiving aquatic environment to aspects of public health concern such as nitrate contamination of drinking water. Nitrogen is biologically removed from wastewaters in a two step process of nitrification (ammonia oxidised to nitrate) (Randall, 1992; Robertson and Kuenen, 1991) and denitrification (nitrate reduced to dinitrogen gas that dissipates into the atmosphere) (Blackburn, 1983; Robertson and Kuenen, 1991). Nitrification is the first and most sensitive step of the process and can be further subdivided into two steps: ammonia oxidation to nitrite and nitrite oxidation to nitrate. The two steps are carried out by separate bacterial groups and for both groups, the total diversity of organisms with this phenotype is small.
Therefore, nitrification is a process where reduced nitrogen compounds, generally ammonium (NH
4
+
), are microbiologically oxidised to nitrate (NO
3
−
) via nitrite (NO
2
−
) under aerobic conditions (Halling-Sørensen and Jørgensen, 1993). The overall reactions and possible organisms responsible are:
The Gram negative chemoautotrophic nitrite oxidising bacteria are physiologically distinct, as they all possess the ability to use nitrite as their energy source and to assimilate CO
2
, via the Calvin Benson cycle, as a carbon source for cell growth (Bock et al., 1992). For each molecule of CO
2
fixed, 100 molecules of nitrite need to be oxidized, emphasising the high energy demands placed on these cells. The overall stoichiometry of nitrite oxidation is (Halling-Sørensen and Jørgensen, 1993):
400NO
2
−
+NH
4
+
+4H
2
CO
3
+HCO
3
−
+195O
2
→C
5
H
7
NO
2
+3H
2
O+400NO
3
−
These bacteria can typically also use nitric oxide (NO) instead of NO
2
−
as an electron source (Bock et al., 1992). Not all of the known nitrifying bacteria are obligate chemoautotrophs. In fact, many strains of Nitrobacter can grow well as heterotrophs, where both energy and carbon are obtained from organic carbon sources, or mixotrophically (a combination of both autotrophic and heterotrophic behaviour). These bacteria are collectively known as facultative chemoautotrophs. Therefore, bacterial strains can grow three ways; aerobically and autotrophically, aerobically and mixotrophically or anaerobically and heterotrophically. In mixotrophic growth, NO
2
−
is oxidized in preference to organic carbon substrates like acetate, pyruvate and glycerol. Both autotrophic and heterotrophic growth is usually slow and inefficient.
As a generalisation, most strains of Nitrobacter seem to be able to grow faster as mixotrophs than as heterotrophs and faster heterotrophically or chemo-heterotrophically than chemoautotrophically.
Four genera are currently recognised: Nitrobacter, Nitrospina, Nitrococcus and Nitrospira (Halling-Sørensen and Jørgensen, 1993). Nitrospina and Nitrococcus are unable to grow heterotrophically or mixotrophically (Bock et al., 1992). One species of Nitrospira,
Nitrospira marina,
can grow autotrophically and mixotrophically, (Bock et al., 1992) whereas
Nitrospira moscoviensis
is an obligate autotroph (Ehrich, et al., 1995). These nitrite oxidizers have also been conventionally classified based on phenotypic characters like their cell shape and the ultrastructure of their intracytoplasmic membranes. Doubling times of Nitrobacter can range from 12 to 59 hours, or even as long as 140 hours (Halling-Sørensen and Jørgensen, 1993). These are therefore very slow growing bacteria.
In wastewater treatment systems, Nitrosomonas (an ammonia oxidizer) and Nitrobacter (a nitrite oxidizer) are the two autotrophs presumed to be responsible for nitrification because they are the commonest ammonia and nitrite oxidizers isolated from these environments (Halling-Sørensen and Jørgensen, 1993). Although ammonia oxidizers have been intensively studied by the use of molecular methods (Wagner et al., 1995; Wagner et al., 1996), the nitrite oxidizers have not been similarly investigated. Since the microorganisms responsible for nitrite oxidation in wastewater treatment plants were presumed to be from the genus Nitrobacter, mathematical modeling of the process has used data relevant to this genus. However, fluorescent in situ hybridization (FISH) probing of activated sludge mixed liquors with Nitrobacter specific probes (Wagner et al., 1996) could not confirm the presence of these organisms suggesting that they were not responsible for this major component of nitrogen remediation. Indeed, Nitrobacter could not be found in other aquatic environments (Hovanec and DeLong, 1996) when specific FISH probes were employed. It was speculated that other bacteria were likely responsible for nitrite oxidation (Hovanec and DeLong, 1996; Wagner et al., 1996).
Knowledge of the microorganisms responsible for nitrification of wastewater is desirable for the efficient management of treatment systems. It would also be advantageous to have available biomass which can be added to a system to implement or improve nitrification. However, as indicated above, there is no certainty in the art as to the actual microorganisms responsible for nitrification nor are there methods available for identifying such organisms.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a consortium of microorganisms that can be used for nitrification of wastewater.
A further object of the invention is to provide a method of identifying microorganisms capable of nitrification of wastewater.
According to a first embodiment of the invention, there is provided a consortium of microorganisms capable of nitrite oxidation in wastewater, which consortium is enriched in members of the Nitrospira phylum.
According to a second embodiment of the invention, there is provided an oligonucleotide primer for PCR amplification of Nitrospira DNA, said primer comprising at least 12 nucleotides having a sequence selected from:
(i) any one of SEQ ID NO: 1 to SEQ ID NO: 13; or
(ii) a DNA sequence having at least 92% identity with any one of SEQ ID NO: 1 to SEQ ID NO: 13.
According to a third embodiment of the invention, there is provided a primer pair for PCR amplification of Nitrospira DNA, said primer pair comprising:
(a) a first oligonucleotide of at least 12 nucleotides having a sequence selected from one strand of a bacterial 16S rDNA gene; and
(b) a second oligonucleotide of at least 12 nucleotides having a sequence selected from the other strand of said 16S rDNA gene downstream of said first oligonucleotide sequence; wherein at least one of said first and second oligonucleotides is selected from:
(i) any one of SEQ ID NO: 1 to SEQ ID NO: 13; or
(ii) a DNA sequence having at least 92% identity with any one SEQ ID NO: 1 to SEQ ID NO: 13.
According to a fourth embodiment of the invention, there is provided a probe for detecting Nitrospira DNA, said probe comprising at least 12 nucleotides having a sequence selected from:
(i) any one of SEQ ID NO: 1 to SEQ ID NO: 13; or
(ii) a DNA sequence having at least 92% identity with any one of SEQ ID NO: 1 to SEQ ID NO: 13.
According to a fifth embodiment of the invention, there is provided a kit comprising:
at least one primer according to the second embodiment;
at least one primer pair according to the third embodiment; or
at least one probe according to the fourth embodiment.
According to a sixth embodiment of the invention, there is provided
Blackall Linda Louise
Burrell Paul Christopher
Keller Jurg
CRC for Waste Management and Pollution Control Limited
Johannsen Diana
Knobbe Martens Olson & Bear LLP
Myers Carla J.
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