Development of nitrification inhibition assays using pure cultures of nitrosomonas and nitrobacter
Introduction
Requirements for nitrogen reduction in wastewater treatment plants were introduced in Sweden in the early 1990s. This was a governmental move to reduce the nitrogen discharges to the Baltic and Kattegatt in order to prevent eutrophication. Swedish treatment plants serving more than 10,000 p.e. (population equivalents) situated in coastal regions have requirements for a nitrogen reduction of at least 50%. It is therefore very important that the biological nitrogen removal processes, i.e. nitrification and denitrification, are not disturbed. The bacteria responsible for the nitrification in treatment plants are restricted to a few genera, often referred to as Nitrosomonas and Nitrobacter. This fact, and their slow growth make the nitrification process very susceptible to inhibition, and it is of great importance that the influent does not contain toxic compounds. A shock load of toxicants can be detrimental to the nitrification process for weeks, similarly sustained loads may reduce the nitrifying capacity. According to a study of 38 treatment plants in Denmark (Laursen and Jansen, 1995), about one third of them were to a considerable extent affected by inhibition of nitrification. This phenomenon has been reported for a number of plants in Sweden as well (Jönsson et al., 1996).
It is important to have assays for investigating the inhibition of nitrification. The methods must be relevant, i.e. the specific reactions and organisms should be studied. In addition, it is desirable to get reproducible results. The approach to meet these requirements suggested in this paper is to use pure cultures of nitrifying bacteria. It is also important that the methods are convenient. In this work, two assays were developed for assessing the inhibition of the two reactions of nitrification, ammonia oxidation and nitrite oxidation. As test organisms, pure cultures of Nitrosomonas and Nitrobacter isolated from activated sludge are used. The assays are performed in test tubes where the bacteria are incubated with the substance or wastewater to be tested. The field of application of the test methods is to assess the inhibition of specific chemical compounds, as well as of industrial wastewaters (Grunditz et al., 1998), or influents of treatment plants (Jönsson et al., 2000). The use of pure cultures of nitrifying bacteria for the detection of nitrification inhibition measured as substrate utilisation has not been described before. Williamson and Johnson (1981) used a pure culture of Nitrobacter as a sensitive test organism for assessing general toxicity but not specifically for nitrification. Tanaka et al. (1993) described a biosensor system using a pure culture of Nitrosomonas where the nitrification was determined using a DO probe. The assays most frequently applied in Sweden today use activated sludge for testing. The international standard method (ISO, 1989) is performed in flasks where the nitrification is determined after 4 h. The total liquid volume is 250 ml. A simplified method for testing large numbers of samples is the screening method (Arvin et al., 1994; SNV, 1995) where the sludge is incubated in test tubes with the water to be tested. The nitrification is measured after 2 h. The total liquid volume is 10 ml. When using these methods, an inhibition of 15% is considered significant; however, a recent investigation (Jönsson et al., 2000) showed that the limit for the screening method could be 5%. Although considered ecologically relevant, a sludge has some disadvantages. There is no absolute reference since a sludge changes with time (Jansen and Winther-Nielsen, 1993; King and Painter, 1986; Wood et al., 1981), which can make it difficult to determine variations over a time period. Furthermore, these two methods do not distinguish between inhibition of ammonia oxidation and nitrite oxidation. There is a possibility to measure the two separate reactions in a sludge or mixed culture using specific inhibitors (Gernaey et al., 1997). However, there are still other organisms present which could interfere. These difficulties can be overcome if pure cultures of nitrifying bacteria are used.
In this paper, two new pure culture assays are presented. Furthermore, the test organisms Nitrosomonas and Nitrobacter are characterised with respect to their 16S rDNA content, temperature and pH optimum, and cell activity. A significant level of inhibition was calculated and examples of applications are shown.
Section snippets
Isolation of test organisms
The test organisms Nitrosomonas and Nitrobacter were isolated from activated sludge from a large wastewater treatment plant in the Stockholm area. A serial dilution method was used (Schmidt and Belser, 1982). The sludge was serially 10-fold diluted 101–1010 times in inorganic media, in order to eliminate organic matter and heterotrophic bacteria. The ammonium medium used was according to Soriano and Walker (1968); pH was adjusted to 7.5. The nitrite medium was described by Ford (1988); pH was
Bioassay procedure
In this section, the procedures for the two novel bioassays for determination of nitrification inhibition are presented. For details, see Materials and Methods section.
Isolation and identification of the test organisms
The test organisms Nitrosomonas and Nitrobacter were isolated from a nitrifying activated sludge from a large wastewater treatment plant in Stockholm. A serial dilution method was used (Schmidt and Belser, 1982). Nitrification was observed up to the 10−7 dilution for both ammonia and nitrite oxidation. Two pure culture strains from the 10−2 dilution were obtained on agar plates after about three months. They were investigated by partial sequencing of 16S rDNA. The highest DNA sequence
Conclusions
Two new bioassays for determination of the inhibition of nitrification were presented. They are based on test tubes containing pure cultures of Nitrosomonas and Nitrobacter, respectively. The inhibition of ammonia or nitrite oxidation rates is measured during 4 h. A significant level of inhibition was calculated to 11% for the Nitrosomonas assay and 9% for the Nitrobacter assay. The test organisms were characterised; the bacteria were identified using partial sequencing of 16S rDNA as belonging
Acknowledgements
The authors would like to thank Gunnar Magnusson (Department of Biotechnology, KTH) for the 16S rDNA analyses of the bacteria.
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