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Development of nitrification inhibition assays using pure cultures of nitrosomonas and nitrobacter
PII: S0043-1354(00)00312-2
Wat. Res. Vol. 35, No. 2, pp. 433–440, 2001 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
DEVELOPMENT OF NITRIFICATION INHIBITION ASSAYS USING PURE CULTURES OF NITROSOMONAS AND NITROBACTER CAMILLA GRUNDITZM and GUNNEL DALHAMMAR* Department of Biotechnology, Royal Institute of Technology (KTH), SE-100 44 Stockholm, Sweden (First received 20 May 1999; accepted in revised form 17 April 2000) Abstract}Restricted requirements for nitrogen reduction at wastewater treatment plants have increased the need for assays determining the inhibition of nitrification. In this paper, two new assays studying ammonia oxidation and nitrite oxidation, respectively, are presented. 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 compound or wastewater to be tested. The nitrification rate is measured during 4 h and compared with reference samples. The test organisms were characterised with respect to temperature, pH and cell activity. Optimum temperature was 358C for Nitrosomonas and 388C for Nitrobacter; optimum pH was 8.1 for Nitrosomonas and 7.9 for Nitrobacter. There was a linear relationship between the nitrification rate and the cell concentration in the studied interval. The cell activity decreased slightly with storage time. A significant level of inhibition was calculated to 11% for the Nitrosomonas assay, and to 9% for the Nitrobacter assay. The assays are applicable to determination of nitrification inhibition in samples of industrial waste waters or influents of treatment plants, or chemical substances likely to be found in wastewater. # 2000 Elsevier Science Ltd. All rights reserved Key words}inhibition of nitrification, bioassays, nitrosomonas, nitrobacter, pure culture, wastewater treatment, nitrogen removal
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
*Author to whom all correspondence should be addressed. Tel.: +46-8-790 8775; fax +46-8-24 54 52; e-mail: [email protected] 433
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 (Jo¨nsson 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 (Jo¨nsson et al., 2000). The use of pure cultures of nitrifying bacteria for the detection of nitrification inhibition measured as substrate utilisation has not
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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 (Jo¨nsson 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.
rediluted and small portions were spread on agar plates (Ford, 1988; solidified with Sigma Agar Purified). The agar plates were incubated at 308C for 3–4 weeks. Colonies were picked and restreaked until pure cultures were obtained. To facilitate the picking of single colonies, Pasteur pipettes freshly drawn out in the flame to fine points were used. The nitrification of the isolates was investigated in liquid media containing ammonium or nitrite; the purity of the nitrifying isolates was confirmed by microscopical examination and by the absence of growth on nutrient agar. Cultivation of test organisms The pure cultures of Nitrosomonas and Nitrobacter are grown on agar plates (Ford, 1988; solidified with Sigma Agar Purified). The plates are incubated at 308C for 3–4 weeks, in plastic bags to prevent desiccation. Cells are transferred to liquid media (for Nitrosomonas, Sato et al., 1985; for Nitrobacter, Soriano and Walker, 1973) and cultivated in shaking flasks of 1–3 l for 2–4 weeks. The cultures are then grown fed batch wise in 3 l-fermenters (Bioreactor type FLC-3-A, Belach AB, Stockholm) in darkness. Temperature (308C), pH (8.0), aeration (0.1 l min ÿ 1) and agitation (200 rpm) are regulated. When required, substrate is fed as concentrated solutions of (NH4)2SO4 and NaNO2, respectively. The growth of the cells is followed by measurements of the substrate consumption rate, and the optical density of the medium at 450 nm. After 2–5 weeks, at the end of the logarithmic growth phase, the cells are harvested. The volume of the bacteria suspension is then about 2 l. An example of a cultivation of Nitrobacter is shown in Fig. 1. The cells are centrifuged (5000 rpm for 10 min) and resuspended in substrate-free liquid medium of double concentration. The cell concentration is determined by total count (Bu¨rker chamber). The cultures are checked for heterotrophic contamination on nutrient agar. The cells in liquid medium can be stored at +38C for several weeks, ready for use in the inhibition assays. Inhibition assays The procedures for the inhibition assays are described in a separate section below. For the inhibition assays, the cells are cultivated and stored as described above. A cell suspension of Nitrosomonas of about 9 108 cells ml ÿ 1 is used. The suspension is made in medium according to Sato et al. (1985), of double concentration without ammonium. Half of the prescribed amount of buffer is added to this solution; the remaining part is added directly to the test
MATERIALS AND METHODS
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 7.9. Nitrogen in the form of ammonium or nitrite was added to 50 mg N l ÿ 1. 5 ml ÿ 1 of diluted sludge was added to each metal capped test tube of 30 ml. The tubes were incubated at 308C in the dark for a period of 1–8 weeks, periodically checked for nitrification by measuring the decrease in ammonia or nitrite concentrations. Also, the ammonium medium contained phenol red; a colour change from pink to yellow indicated possible nitrification. Positive tubes were
Fig. 1. Example of a cultivation of Nitrobacter in the fermenter. The cells were harvested at OD450=0.48 which corresponds to about 4 108 Nitrobacter cells ml ÿ 1.
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tubes in order to prevent the pH from becoming too high during storage. The buffer solution (pH 9.0) is made of equal parts of 0.5 M phosphate buffer and 0.6 M carbonate buffer according to Sato et al. (1985). The ammonium solution added to the test tubes contains 5.0 g N l ÿ 1 as (NH4)2SO4. For Nitrobacter, a cell suspension of about 3 108 cells ml ÿ 1 is used. The suspension is made in medium from Soriano and Walker (1973), of double concentration without nitrite. The nitrite solution added to the test tubes contains 5.0 g N l ÿ 1 as NaNO2. Both the media used in the assays are clear solutions with no or very little precipitation. The cell suspensions of Nitrosomonas and Nitrobacter are diluted twice in the inhibition assays. Test tubes of 15 ml with screw caps are used. Incubation in the rotation rack takes place at 110 rpm.
Analytical measurements
16S rDNA analysis
2.50 ml of Nitrosomonas is transferred to each test tube. 2.40 ml solution of the substance or water to be tested is added, followed by the addition of 50 ml buffer. Finally, 50 ml of ammonium solution is added to a concentration of 50 mg NH4–N l ÿ 1 in the tube. The total test volume is 5.0 ml and the start pH is 8.5. The test tubes are incubated horizontally at 308C in a rotation rack in darkness for 4 h. Aliquots of 50 ml are taken every hour and ammonium is analysed.
Partial 16S rDNA sequencing of the U2–U5 region was performed by semi-nested PCR amplification followed by solid-phase DNA sequencing (Hultman et al., 1991; Pettersson et al., 1994; Wahlberg et al., 1992). The sequences were then analysed using the sequence similarity search tool of NCBI (National Centre for Biotechnology Information), BLASTN. Temperature experiments Temperature experiments were performed with the test tube assays described below. For practical reasons, the test tubes were incubated horizontally without shaking. Temperatures tested were ranging from 5 to 508C. pH experiments The pH experiments were performed in a fermenter (Bioreactor type FLC-3-A, Belach AB, Stockholm) in order to control the pH value. The total volume of the fermenter was 3 l; the volume of the bacteria in medium was about 2 l. The experiments were made batchwise and in darkness. The temperature was kept at 308C; the airflow was 0.1 l min ÿ 1, and the agitation was 200 rpm. Measurements started 20– 30 min after each pH value had been adjusted, to allow the bacteria to acclimatise. The same cultures of Nitrosomonas and Nitrobacter were used for all the pH experiments. The cell density was constant during the experiments (OD450 0.4). For the graphs (Fig. 3(a) and (b)), the activity at pH 8.0 was used as the reference activity. Nitrosomonas: The pH value in the fermenter was adjusted using HCl (1.8%) and K2CO3 (10%). Substrate was added as a concentrated solution of (NH4)2SO4. The substrate concentration at the start of each pH value was 100–160 mg NH4–N l ÿ 1. Samples of about 5 ml were taken every 20–30 min during 90–150 min and analysed for ammonium. pH values tested ranged from 6.5 to 9.5. Nitrobacter: The pH value in the fermenter was adjusted using HCl (0.6%) and K2CO3 (5%). Substrate was added as a concentrated solution of NaNO2. The substrate concentration at the start of each pH value was 140–320 mg NO2– N l ÿ 1. Samples of about 5 ml were taken every 15 min during 60–75 min and analysed for nitrite. pH values tested ranged from 6.0 to 9.0. Cell activity The test tubes were prepared as reference tubes. The studied concentrations in the test tubes were for Nitrosomonas, 1.4 108–5.4 108 cells ml ÿ 1, and for Nitrobacter 4.2 107–1.7 108 cells ml ÿ 1. For investigating the effect of storage (+38C), reference tubes from experiments performed during a longer time period were compared. The cells used were all from the same batch. The cell concentration in the test tubes was about 4 108 cells ml ÿ 1 for Nitrosomonas and 1 108 cells ml ÿ 1 for Nitrobacter.
Ammonium and nitrite were analysed colorimetrically using Merck Spectroquant reagent test kit. Spectrophotometer: Shimadzu UV-120-02.
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. Nitrosomonas assay
Nitrobacter assay 2.50 ml of Nitrobacter is transferred to each test tube. 2.45 ml solution of the substance or water to be tested is added. 50 ml of nitrite solution is added to a concentration of 50 mg NO2–N l ÿ 1. The total test volume is 5.0 ml and the pH is 7.8. The test tubes are incubated horizontally at 308C in a rotation rack in the dark for 4 h. Aliquots of 50 ml are taken every hour and nitrite is analysed. Calculation of inhibition In both the inhibition assays, for every series of tests, three reference tubes containing distilled water are prepared. The decrease in the nitrogen concentration is linear with time. The degree of inhibition is calculated as vref ÿ vsample 100 ¼ % inhibition ð1Þ vref where vref is the substrate consumption rate of a reference and vsample the substrate consumption rate of the sample tested. The rate is measured as mg substrate (ammonium or nitrite nitrogen) consumed per litre and hour. It is derived from linear regression of the data. Samples for testing Only a small sample volume is required, a maximum of 2.5 ml per test tube. The tests should be performed on samples in duplicate tubes. A number of at least 20 test tubes can be performed in parallel by one person. The oxygen concentration in the test tubes will under normal conditions, i.e. if the sample does not contain a large amount of oxygen-consuming microorganisms or material, not
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be limiting for the nitrification. For the reference tubes, the concentration was measured to be above 6 mg O2 l ÿ 1 during incubation for both the assays. The substrate concentration is above 20 mg N l ÿ 1 at the end of the tests, which means that there will be no risk of substrate limitation.
RESULTS AND DISCUSSION
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 ÿ 7 dilution for both ammonia and nitrite oxidation. Two pure culture strains from the 10 ÿ 2 dilution were obtained on agar plates after about three months. They were investigated by partial sequencing of 16S rDNA. The highest DNA sequence similarity was found to the 16S rRNA sequences deposited for the species belonging to the genus Nitrosomonas (100%), and to the genus belonging to Nitrobacter (99%). The genera Nitrosomonas and Nitrobacter have been the most commonly isolated from wastewater treatment environments (Barnes and Bliss, 1983; Watson et al., 1989), although recent studies show that there might be a greater variation in the nitrifying population than previously claimed (Burrell et al., 1998; Hiorns et al., 1995; Koops et al., 1991).
Fig. 2. (a) Effect of temperature on the activity of Nitrosomonas. (b) Effect of temperature on the activity of Nitrobacter.
Effect of temperature and pH The test organisms Nitrosomonas and Nitrobacter were characterised with respect to temperature. From the graphs in Fig. 2(a) and (b), it can be seen that the highest activity was found at a temperature of 358C for Nitrosomonas and at 388C for Nitrobacter. This is in agreement with results of, for example, Deppe and Engel (1960), van Ginkel et al. (1983) and Groeneweg et al. (1994). The test organisms were also characterised with respect to pH. The graphs in Fig. 3(a) and (b) show an optimum activity at a pH value of 8.1 for Nitrosomonas and 7.9 for Nitrobacter. These results agree with those of other researchers (e.g. Boon and Laudelout, 1962; Buswell et al., 1954; Hofman and Lees, 1953). The optimum values obtained correspond to the conditions in the bioassays presented. In the experiments to determine the effect of pH on Nitrobacter, the initial concentration of nitrite at each pH value varied between 140 and 320 mg NO2–N l ÿ 1. However, the substrate consumption was found constant in this interval (unpublished data). This is consistent with findings from Srinath et al. (1976); within the concentration ranges of 100–1500 mg NO2–N l ÿ 1, the oxidation rates were not influenced by the concentration of the substrate.
Cell activity Experiments were performed to investigate the relationship between the activity (nitrification rate) and the cell concentration. The cell concentration was constant during the test time. For the tested concentrations (for Nitrosomonas 1.4 108– 8 ÿ1 5.4 10 cells ml and for Nitrobacter 4.2 107– 8 ÿ1 1.7 10 cells ml ), there was a linear relationship between the activity (mg consumed substrate l ÿ 1,h) and the cell concentration; see Fig. 4(a) and (b). This makes it possible, within certain limits, to choose the cell concentration, in order to adjust the activity in the assays. Microscopical studies showed that both the bacteria occur almost entirely as single cells in the test tubes. Before use in the bioassays, the harvested Nitrosomonas and Nitrobacter cells are kept refrigerated (+38C) in liquid media. Studies were performed to investigate the effect of this storage on the cell activity. From Fig. 5, it can be seen that the cell activity decreases slightly with storage time. The decline was approximated to be linear with time in the studied intervals. The decrease for Nitrobacter was found to be somewhat more pronounced than for Nitrosomonas; the reason for this is not known.
Development of nitrification inhibition assays
Fig. 3. (a) Effect of pH on the activity of Nitrosomonas. The activity at pH 8.0 was used as the reference activity. (b) Effect of pH on the acitivity of Nitrobacter. The activity at pH 8.0 was used as the reference activity.
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Fig. 4. (a) Effect of cell concentration on the activity of Nitrosomonas. (b) Effect of cell concentration on the activity of Nitrobacter.
The age of the cell culture (the time elapsed after harvest) might affect the degree of inhibition. The inhibition may increase or decrease with increasing age of the culture, depending on what type of substances causing it. If very accurate results are required, cells of a certain age and activity should be used. For the bioassays presented in this paper, cells are recommended to be used within 10 weeks. To extend the shelf life of the test cultures, freeze-drying would be an applicable procedure (Williamson and Johnson, 1981).
Calculation of a significant level of inhibition To determine a significant level of inhibition for the two assays, the following equation (Massart et al., 1997) was used: qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi vref ÿ vsample ¼ t0:95 ðdfÞ s2ref þ s2sample ð2Þ where vref is the substrate consumption rate of the reference, vsample is the substrate consumption rate of the sample tested, t0:95 ðdf Þ is the t value for a 95% confidence interval from the t distribution, df is the number of degrees of freedom, s2ref is the relative
Fig. 5. Effect of storage time on the activity of Nitrosomonas and Nitrobacter.
standard deviation of the reference and s2sample is the relative standard deviation of the sample. A number of reference tubes were performed in parallel for both the methods (12 for Nitrosomonas and 13 for Nitrobacter). The relative standard deviation of the slope of each regression line for the substrate consumption was calculated. A pooled relative standard deviation (sref ) for each assay was determined from these results; 4.5% for the
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Nitrosomonas assay, and 3.8% for the Nitrobacter assay. Based on a large number of tests of different types of samples, the pooled relative standard deviation for a sample (ssample ) was shown to be about 7% higher than for a reference; ssample was determined to 4.8% for Nitrosomonas and 4.0% for Nitrobacter. If the samples are performed in duplicates, the df value will be 42 (Nitrosomonas) and 45 (Nitrobacter), respectively, which gives a t value of 1.684. Solving equation (2), the level of significant inhibition becomes 11% for the Nitrosomonas assay and 9% for the Nitrobacter assay. The difference between the two assays can be explained by the two different methods for analysis of nitrogen; the nitrite analyses have been shown to be more precise than the ammonium analyses. Applications As an example of an application for the Nitrosomonas assay, the test of a chemical substance, ATU (allylthiourea), is shown. In Fig. 6(a), the inhibition curves for the concentrations of 0.025 and 0.050 mg ATU l ÿ 1 are seen. An example of the Nitrobacter assay is shown in Fig. 6(b), where the test results of two industrial wastewaters from metal finishing and printing industry are plotted. The wastewaters made up 20% of the test volume. Other examples of applications include the study of fractions of complex wastewaters. Combining the inhibition assays with chemical analyses makes it possible to identify the inhibitory compounds (Svenson et al., in press). Relevance of the assays The bioassays presented in this work are applicable to assessment of nitrification inhibition for samples of industrial and municipal wastewaters, or chemical compounds likely to be found in wastewater. The test methods are convenient and require only small sample volumes. The assays are considered relevant for their purposes. The inhibition of the particular reactions of nitrification, i.e. the ammonia and nitrite oxidation, are studied. Furthermore, the test organisms are those responsible for the reactions of interest. With pure cultures, it is possible, without interference from other organisms, to look at the separate nitrification reactions which can be of importance; the inhibition patterns of ammonia oxidation and nitrite oxidation have been shown to be different (Grunditz et al., 1998). Furthermore, the cell concentration in the tests is about the same as for nitrifiers in activated sludge (Sande´n et al., 1994, 1996). There is a possibility that other isolates of Nitrosomonas and Nitrobacter might show other inhibition patterns to some toxicants than do the strains used in the present assays. However, these strains probably give a good indication of the effects on ammonia and nitrite oxidisers present in wastewater treatment. Like other inhibition assays, the
Fig. 6. (a) The Nitrosomonas assay applied to ATU for the concentrations 0.025 and 0.050 mg l ÿ 1. The inhibition was determined to be 33% for 0.025 mg l ÿ 1 and 70% for 0.050 mg l ÿ 1. (b) The Nitrobacter assay applied to two industrial wastewaters, metal finishing and printing industry. The wastewaters made up 20% of the test volume. The inhibition was determined to be 28% (metal finishing) and 84% (printing industry).
results must be considered only as a guide to the probable toxicity in a wastewater treatment plant. The intention is to provide the cultures of Nitrosomonas and Nitrobacter commercially for other users of the methods. This could be improved by freezedrying the cultures to extend their shelf life. Furthermore, the assays are being developed to a mediated amperometric biosensor with the cultures immobilised onto an electrode (Grunditz, 1999). This gives an even quicker and more convenient test method.
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
Development of nitrification inhibition assays
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 to the genus of Nitrosomonas and Nitrobacter, respectively. Optimum temperature was 358C for Nitrosomonas and 388C for Nitrobacter, and optimum pH was 8.1 for Nitrosomonas and 7.9 for Nitrobacter. There was a linear relationship between the cell activity and the cell concentration in the studied interval. The cell activity decreased slightly with storage time. The assays are convenient to perform and they are considered relevant for assessment of nitrification inhibition. 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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microbiological properties. Agronomy Monograph, 2nd ed., Vol. 9, pp. 1027–1041. SNV (Swedish Environmental Protection Agency) (1995) Screening method for determination of nitrification inhibition}for operation of municipal wastewater treatment plants (Screeningmetod fo¨r besta¨mning av nitrifikationsha¨mning}vid drift av kommunala avloppsreningsverk). Rapport 4424 (in Swedish). Soriano S. and Walker N. (1968) Isolation of ammoniaoxidizing autotrophic bacteria. J. Appl. Bacteriol 31, 493–497. Soriano S. and Walker N. (1973) The nitrifying bacteria in soils from Rothamsted Classical Fields and elsewhere. J. Appl. Bacteriol. 36, 523–529. Srinath E. G., Loehr R. C. and Prakasam T. B. S. (1976) Nitrifying organism concentration and activity. J. Environ. Eng. Div. (Am. Soc. Civ. Eng.) 102, 449–463. Svenson A., Sande´n B., Dalhammar G., Remberger M. and Kaj L. Toxicity identification evaluation of nitrification inhibitors in wastewaters. Environ. Tox., submitted.
Tanaka H., Nakamura E., Hoshkawa H. and Tanaka Y. (1993) Development of the ammonia biosensor monitoring system. Water Sci. Technol. 28(11–12), 435–445. Wahlberg J., Holmberg A., Bergh S., Hultman T. and Uhle´n M. (1992) Automated magnetic preparation of DNA templates for solid-phase sequencing. Electrophoresis 13, 547–551. Watson S. W., Bock E., Harms H., Koops H.-P. and Hooper A. B. (1989) Family Nitrobacteraceae. In Bergey’s Manual of Systematic Microbiology, Vol. 3, eds J. T. Staley, M. P. Bryant, N. Pfennig and J. G. Holt, pp. 1808–1834. Lippincott Williams and Wilkins, Baltimore, USA. Williamson K. J. and Johnson D. G. (1981) A bacterial bioassay for assessment of wastewater toxicity. Water Res. 15, 383–390. Wood L. B., Hurley B. J. E. and Matthew P. J. (1981) Some observations on the biochemistry and inhibition of nitrification. Water Res. 15, 543–551.
Wat. Res. Vol. 35, No. 2, pp. 433–440, 2001 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
DEVELOPMENT OF NITRIFICATION INHIBITION ASSAYS USING PURE CULTURES OF NITROSOMONAS AND NITROBACTER CAMILLA GRUNDITZM and GUNNEL DALHAMMAR* Department of Biotechnology, Royal Institute of Technology (KTH), SE-100 44 Stockholm, Sweden (First received 20 May 1999; accepted in revised form 17 April 2000) Abstract}Restricted requirements for nitrogen reduction at wastewater treatment plants have increased the need for assays determining the inhibition of nitrification. In this paper, two new assays studying ammonia oxidation and nitrite oxidation, respectively, are presented. 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 compound or wastewater to be tested. The nitrification rate is measured during 4 h and compared with reference samples. The test organisms were characterised with respect to temperature, pH and cell activity. Optimum temperature was 358C for Nitrosomonas and 388C for Nitrobacter; optimum pH was 8.1 for Nitrosomonas and 7.9 for Nitrobacter. There was a linear relationship between the nitrification rate and the cell concentration in the studied interval. The cell activity decreased slightly with storage time. A significant level of inhibition was calculated to 11% for the Nitrosomonas assay, and to 9% for the Nitrobacter assay. The assays are applicable to determination of nitrification inhibition in samples of industrial waste waters or influents of treatment plants, or chemical substances likely to be found in wastewater. # 2000 Elsevier Science Ltd. All rights reserved Key words}inhibition of nitrification, bioassays, nitrosomonas, nitrobacter, pure culture, wastewater treatment, nitrogen removal
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
*Author to whom all correspondence should be addressed. Tel.: +46-8-790 8775; fax +46-8-24 54 52; e-mail: [email protected] 433
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 (Jo¨nsson 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 (Jo¨nsson et al., 2000). The use of pure cultures of nitrifying bacteria for the detection of nitrification inhibition measured as substrate utilisation has not
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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 (Jo¨nsson 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.
rediluted and small portions were spread on agar plates (Ford, 1988; solidified with Sigma Agar Purified). The agar plates were incubated at 308C for 3–4 weeks. Colonies were picked and restreaked until pure cultures were obtained. To facilitate the picking of single colonies, Pasteur pipettes freshly drawn out in the flame to fine points were used. The nitrification of the isolates was investigated in liquid media containing ammonium or nitrite; the purity of the nitrifying isolates was confirmed by microscopical examination and by the absence of growth on nutrient agar. Cultivation of test organisms The pure cultures of Nitrosomonas and Nitrobacter are grown on agar plates (Ford, 1988; solidified with Sigma Agar Purified). The plates are incubated at 308C for 3–4 weeks, in plastic bags to prevent desiccation. Cells are transferred to liquid media (for Nitrosomonas, Sato et al., 1985; for Nitrobacter, Soriano and Walker, 1973) and cultivated in shaking flasks of 1–3 l for 2–4 weeks. The cultures are then grown fed batch wise in 3 l-fermenters (Bioreactor type FLC-3-A, Belach AB, Stockholm) in darkness. Temperature (308C), pH (8.0), aeration (0.1 l min ÿ 1) and agitation (200 rpm) are regulated. When required, substrate is fed as concentrated solutions of (NH4)2SO4 and NaNO2, respectively. The growth of the cells is followed by measurements of the substrate consumption rate, and the optical density of the medium at 450 nm. After 2–5 weeks, at the end of the logarithmic growth phase, the cells are harvested. The volume of the bacteria suspension is then about 2 l. An example of a cultivation of Nitrobacter is shown in Fig. 1. The cells are centrifuged (5000 rpm for 10 min) and resuspended in substrate-free liquid medium of double concentration. The cell concentration is determined by total count (Bu¨rker chamber). The cultures are checked for heterotrophic contamination on nutrient agar. The cells in liquid medium can be stored at +38C for several weeks, ready for use in the inhibition assays. Inhibition assays The procedures for the inhibition assays are described in a separate section below. For the inhibition assays, the cells are cultivated and stored as described above. A cell suspension of Nitrosomonas of about 9 108 cells ml ÿ 1 is used. The suspension is made in medium according to Sato et al. (1985), of double concentration without ammonium. Half of the prescribed amount of buffer is added to this solution; the remaining part is added directly to the test
MATERIALS AND METHODS
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 7.9. Nitrogen in the form of ammonium or nitrite was added to 50 mg N l ÿ 1. 5 ml ÿ 1 of diluted sludge was added to each metal capped test tube of 30 ml. The tubes were incubated at 308C in the dark for a period of 1–8 weeks, periodically checked for nitrification by measuring the decrease in ammonia or nitrite concentrations. Also, the ammonium medium contained phenol red; a colour change from pink to yellow indicated possible nitrification. Positive tubes were
Fig. 1. Example of a cultivation of Nitrobacter in the fermenter. The cells were harvested at OD450=0.48 which corresponds to about 4 108 Nitrobacter cells ml ÿ 1.
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tubes in order to prevent the pH from becoming too high during storage. The buffer solution (pH 9.0) is made of equal parts of 0.5 M phosphate buffer and 0.6 M carbonate buffer according to Sato et al. (1985). The ammonium solution added to the test tubes contains 5.0 g N l ÿ 1 as (NH4)2SO4. For Nitrobacter, a cell suspension of about 3 108 cells ml ÿ 1 is used. The suspension is made in medium from Soriano and Walker (1973), of double concentration without nitrite. The nitrite solution added to the test tubes contains 5.0 g N l ÿ 1 as NaNO2. Both the media used in the assays are clear solutions with no or very little precipitation. The cell suspensions of Nitrosomonas and Nitrobacter are diluted twice in the inhibition assays. Test tubes of 15 ml with screw caps are used. Incubation in the rotation rack takes place at 110 rpm.
Analytical measurements
16S rDNA analysis
2.50 ml of Nitrosomonas is transferred to each test tube. 2.40 ml solution of the substance or water to be tested is added, followed by the addition of 50 ml buffer. Finally, 50 ml of ammonium solution is added to a concentration of 50 mg NH4–N l ÿ 1 in the tube. The total test volume is 5.0 ml and the start pH is 8.5. The test tubes are incubated horizontally at 308C in a rotation rack in darkness for 4 h. Aliquots of 50 ml are taken every hour and ammonium is analysed.
Partial 16S rDNA sequencing of the U2–U5 region was performed by semi-nested PCR amplification followed by solid-phase DNA sequencing (Hultman et al., 1991; Pettersson et al., 1994; Wahlberg et al., 1992). The sequences were then analysed using the sequence similarity search tool of NCBI (National Centre for Biotechnology Information), BLASTN. Temperature experiments Temperature experiments were performed with the test tube assays described below. For practical reasons, the test tubes were incubated horizontally without shaking. Temperatures tested were ranging from 5 to 508C. pH experiments The pH experiments were performed in a fermenter (Bioreactor type FLC-3-A, Belach AB, Stockholm) in order to control the pH value. The total volume of the fermenter was 3 l; the volume of the bacteria in medium was about 2 l. The experiments were made batchwise and in darkness. The temperature was kept at 308C; the airflow was 0.1 l min ÿ 1, and the agitation was 200 rpm. Measurements started 20– 30 min after each pH value had been adjusted, to allow the bacteria to acclimatise. The same cultures of Nitrosomonas and Nitrobacter were used for all the pH experiments. The cell density was constant during the experiments (OD450 0.4). For the graphs (Fig. 3(a) and (b)), the activity at pH 8.0 was used as the reference activity. Nitrosomonas: The pH value in the fermenter was adjusted using HCl (1.8%) and K2CO3 (10%). Substrate was added as a concentrated solution of (NH4)2SO4. The substrate concentration at the start of each pH value was 100–160 mg NH4–N l ÿ 1. Samples of about 5 ml were taken every 20–30 min during 90–150 min and analysed for ammonium. pH values tested ranged from 6.5 to 9.5. Nitrobacter: The pH value in the fermenter was adjusted using HCl (0.6%) and K2CO3 (5%). Substrate was added as a concentrated solution of NaNO2. The substrate concentration at the start of each pH value was 140–320 mg NO2– N l ÿ 1. Samples of about 5 ml were taken every 15 min during 60–75 min and analysed for nitrite. pH values tested ranged from 6.0 to 9.0. Cell activity The test tubes were prepared as reference tubes. The studied concentrations in the test tubes were for Nitrosomonas, 1.4 108–5.4 108 cells ml ÿ 1, and for Nitrobacter 4.2 107–1.7 108 cells ml ÿ 1. For investigating the effect of storage (+38C), reference tubes from experiments performed during a longer time period were compared. The cells used were all from the same batch. The cell concentration in the test tubes was about 4 108 cells ml ÿ 1 for Nitrosomonas and 1 108 cells ml ÿ 1 for Nitrobacter.
Ammonium and nitrite were analysed colorimetrically using Merck Spectroquant reagent test kit. Spectrophotometer: Shimadzu UV-120-02.
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. Nitrosomonas assay
Nitrobacter assay 2.50 ml of Nitrobacter is transferred to each test tube. 2.45 ml solution of the substance or water to be tested is added. 50 ml of nitrite solution is added to a concentration of 50 mg NO2–N l ÿ 1. The total test volume is 5.0 ml and the pH is 7.8. The test tubes are incubated horizontally at 308C in a rotation rack in the dark for 4 h. Aliquots of 50 ml are taken every hour and nitrite is analysed. Calculation of inhibition In both the inhibition assays, for every series of tests, three reference tubes containing distilled water are prepared. The decrease in the nitrogen concentration is linear with time. The degree of inhibition is calculated as vref ÿ vsample 100 ¼ % inhibition ð1Þ vref where vref is the substrate consumption rate of a reference and vsample the substrate consumption rate of the sample tested. The rate is measured as mg substrate (ammonium or nitrite nitrogen) consumed per litre and hour. It is derived from linear regression of the data. Samples for testing Only a small sample volume is required, a maximum of 2.5 ml per test tube. The tests should be performed on samples in duplicate tubes. A number of at least 20 test tubes can be performed in parallel by one person. The oxygen concentration in the test tubes will under normal conditions, i.e. if the sample does not contain a large amount of oxygen-consuming microorganisms or material, not
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be limiting for the nitrification. For the reference tubes, the concentration was measured to be above 6 mg O2 l ÿ 1 during incubation for both the assays. The substrate concentration is above 20 mg N l ÿ 1 at the end of the tests, which means that there will be no risk of substrate limitation.
RESULTS AND DISCUSSION
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 ÿ 7 dilution for both ammonia and nitrite oxidation. Two pure culture strains from the 10 ÿ 2 dilution were obtained on agar plates after about three months. They were investigated by partial sequencing of 16S rDNA. The highest DNA sequence similarity was found to the 16S rRNA sequences deposited for the species belonging to the genus Nitrosomonas (100%), and to the genus belonging to Nitrobacter (99%). The genera Nitrosomonas and Nitrobacter have been the most commonly isolated from wastewater treatment environments (Barnes and Bliss, 1983; Watson et al., 1989), although recent studies show that there might be a greater variation in the nitrifying population than previously claimed (Burrell et al., 1998; Hiorns et al., 1995; Koops et al., 1991).
Fig. 2. (a) Effect of temperature on the activity of Nitrosomonas. (b) Effect of temperature on the activity of Nitrobacter.
Effect of temperature and pH The test organisms Nitrosomonas and Nitrobacter were characterised with respect to temperature. From the graphs in Fig. 2(a) and (b), it can be seen that the highest activity was found at a temperature of 358C for Nitrosomonas and at 388C for Nitrobacter. This is in agreement with results of, for example, Deppe and Engel (1960), van Ginkel et al. (1983) and Groeneweg et al. (1994). The test organisms were also characterised with respect to pH. The graphs in Fig. 3(a) and (b) show an optimum activity at a pH value of 8.1 for Nitrosomonas and 7.9 for Nitrobacter. These results agree with those of other researchers (e.g. Boon and Laudelout, 1962; Buswell et al., 1954; Hofman and Lees, 1953). The optimum values obtained correspond to the conditions in the bioassays presented. In the experiments to determine the effect of pH on Nitrobacter, the initial concentration of nitrite at each pH value varied between 140 and 320 mg NO2–N l ÿ 1. However, the substrate consumption was found constant in this interval (unpublished data). This is consistent with findings from Srinath et al. (1976); within the concentration ranges of 100–1500 mg NO2–N l ÿ 1, the oxidation rates were not influenced by the concentration of the substrate.
Cell activity Experiments were performed to investigate the relationship between the activity (nitrification rate) and the cell concentration. The cell concentration was constant during the test time. For the tested concentrations (for Nitrosomonas 1.4 108– 8 ÿ1 5.4 10 cells ml and for Nitrobacter 4.2 107– 8 ÿ1 1.7 10 cells ml ), there was a linear relationship between the activity (mg consumed substrate l ÿ 1,h) and the cell concentration; see Fig. 4(a) and (b). This makes it possible, within certain limits, to choose the cell concentration, in order to adjust the activity in the assays. Microscopical studies showed that both the bacteria occur almost entirely as single cells in the test tubes. Before use in the bioassays, the harvested Nitrosomonas and Nitrobacter cells are kept refrigerated (+38C) in liquid media. Studies were performed to investigate the effect of this storage on the cell activity. From Fig. 5, it can be seen that the cell activity decreases slightly with storage time. The decline was approximated to be linear with time in the studied intervals. The decrease for Nitrobacter was found to be somewhat more pronounced than for Nitrosomonas; the reason for this is not known.
Development of nitrification inhibition assays
Fig. 3. (a) Effect of pH on the activity of Nitrosomonas. The activity at pH 8.0 was used as the reference activity. (b) Effect of pH on the acitivity of Nitrobacter. The activity at pH 8.0 was used as the reference activity.
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Fig. 4. (a) Effect of cell concentration on the activity of Nitrosomonas. (b) Effect of cell concentration on the activity of Nitrobacter.
The age of the cell culture (the time elapsed after harvest) might affect the degree of inhibition. The inhibition may increase or decrease with increasing age of the culture, depending on what type of substances causing it. If very accurate results are required, cells of a certain age and activity should be used. For the bioassays presented in this paper, cells are recommended to be used within 10 weeks. To extend the shelf life of the test cultures, freeze-drying would be an applicable procedure (Williamson and Johnson, 1981).
Calculation of a significant level of inhibition To determine a significant level of inhibition for the two assays, the following equation (Massart et al., 1997) was used: qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi vref ÿ vsample ¼ t0:95 ðdfÞ s2ref þ s2sample ð2Þ where vref is the substrate consumption rate of the reference, vsample is the substrate consumption rate of the sample tested, t0:95 ðdf Þ is the t value for a 95% confidence interval from the t distribution, df is the number of degrees of freedom, s2ref is the relative
Fig. 5. Effect of storage time on the activity of Nitrosomonas and Nitrobacter.
standard deviation of the reference and s2sample is the relative standard deviation of the sample. A number of reference tubes were performed in parallel for both the methods (12 for Nitrosomonas and 13 for Nitrobacter). The relative standard deviation of the slope of each regression line for the substrate consumption was calculated. A pooled relative standard deviation (sref ) for each assay was determined from these results; 4.5% for the
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Nitrosomonas assay, and 3.8% for the Nitrobacter assay. Based on a large number of tests of different types of samples, the pooled relative standard deviation for a sample (ssample ) was shown to be about 7% higher than for a reference; ssample was determined to 4.8% for Nitrosomonas and 4.0% for Nitrobacter. If the samples are performed in duplicates, the df value will be 42 (Nitrosomonas) and 45 (Nitrobacter), respectively, which gives a t value of 1.684. Solving equation (2), the level of significant inhibition becomes 11% for the Nitrosomonas assay and 9% for the Nitrobacter assay. The difference between the two assays can be explained by the two different methods for analysis of nitrogen; the nitrite analyses have been shown to be more precise than the ammonium analyses. Applications As an example of an application for the Nitrosomonas assay, the test of a chemical substance, ATU (allylthiourea), is shown. In Fig. 6(a), the inhibition curves for the concentrations of 0.025 and 0.050 mg ATU l ÿ 1 are seen. An example of the Nitrobacter assay is shown in Fig. 6(b), where the test results of two industrial wastewaters from metal finishing and printing industry are plotted. The wastewaters made up 20% of the test volume. Other examples of applications include the study of fractions of complex wastewaters. Combining the inhibition assays with chemical analyses makes it possible to identify the inhibitory compounds (Svenson et al., in press). Relevance of the assays The bioassays presented in this work are applicable to assessment of nitrification inhibition for samples of industrial and municipal wastewaters, or chemical compounds likely to be found in wastewater. The test methods are convenient and require only small sample volumes. The assays are considered relevant for their purposes. The inhibition of the particular reactions of nitrification, i.e. the ammonia and nitrite oxidation, are studied. Furthermore, the test organisms are those responsible for the reactions of interest. With pure cultures, it is possible, without interference from other organisms, to look at the separate nitrification reactions which can be of importance; the inhibition patterns of ammonia oxidation and nitrite oxidation have been shown to be different (Grunditz et al., 1998). Furthermore, the cell concentration in the tests is about the same as for nitrifiers in activated sludge (Sande´n et al., 1994, 1996). There is a possibility that other isolates of Nitrosomonas and Nitrobacter might show other inhibition patterns to some toxicants than do the strains used in the present assays. However, these strains probably give a good indication of the effects on ammonia and nitrite oxidisers present in wastewater treatment. Like other inhibition assays, the
Fig. 6. (a) The Nitrosomonas assay applied to ATU for the concentrations 0.025 and 0.050 mg l ÿ 1. The inhibition was determined to be 33% for 0.025 mg l ÿ 1 and 70% for 0.050 mg l ÿ 1. (b) The Nitrobacter assay applied to two industrial wastewaters, metal finishing and printing industry. The wastewaters made up 20% of the test volume. The inhibition was determined to be 28% (metal finishing) and 84% (printing industry).
results must be considered only as a guide to the probable toxicity in a wastewater treatment plant. The intention is to provide the cultures of Nitrosomonas and Nitrobacter commercially for other users of the methods. This could be improved by freezedrying the cultures to extend their shelf life. Furthermore, the assays are being developed to a mediated amperometric biosensor with the cultures immobilised onto an electrode (Grunditz, 1999). This gives an even quicker and more convenient test method.
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
Development of nitrification inhibition assays
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 to the genus of Nitrosomonas and Nitrobacter, respectively. Optimum temperature was 358C for Nitrosomonas and 388C for Nitrobacter, and optimum pH was 8.1 for Nitrosomonas and 7.9 for Nitrobacter. There was a linear relationship between the cell activity and the cell concentration in the studied interval. The cell activity decreased slightly with storage time. The assays are convenient to perform and they are considered relevant for assessment of nitrification inhibition. 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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