An NH4+ biosensor based on ammonia-oxidizing bacteria for use under anoxic conditions

Sensors and Actuators B 105 (2005) 412–418

An NH4 + biosensor based on ammonia-oxidizing bacteria for use under anoxic conditions Annette Bollmann∗ , Niels Peter Revsbech a Department

of Microbial Ecology, Institute of Biological Sciences, University of Aarhus, Ny Munkegade Building 540, DK-8000 Aarhus C, Denmark Received 5 March 2004; received in revised form 24 June 2004; accepted 24 June 2004 Available online 11 August 2004

Abstract An ammonium biosensor based on bacterial oxidation of ammonia with oxygen was constructed. The biosensor contained an internal oxygen reservoir from which oxygen was supplied to the biomass of ammonia oxidizing bacteria (AOB). The biosensor was thus designed to have best performance under ambient anoxic conditions. An oxygen microsensor was used to monitor the internal oxygen gradient. Due to the ammonia-oxidizing and respiratory activity of the AOB the signal from this oxygen microsensor was proportional to the ambient ammonium concentration in a range of 0–200 ␮M. Oxygen in the environment interfered with the reading, but it was in principle possible to compensate for a constant oxygen concentration in the analyzed medium. An increase in pH from 6 to 8.5 increased the sensitivity, while a range of chemicals affected the signal in various ways. A total of four biosensors were constructed, and one exhibited a lifetime of >2 months. Sensors containing bacteria that were starved for ammonium could be revived within 2 days by placing them in ammonium-containing medium. The 90% response times of the sensors were about 2 min, and the difference in signal between vigorously stirred and stagnant medium corresponded to about 10 ␮M NH4 + within the linear range. A sensor was tested in a wastewater treatment plant. The sensor signal during the anoxic periods corresponded well to the concentration of NH4 + analyzed by colorimetry. © 2004 Elsevier B.V. All rights reserved. Keywords: Biosensor; Ammonium; Ammonia-oxidizing bacteria

1. Introduction Biosensors based on whole cells have been used for analysis of a wide range of compounds relevant to environmental monitoring [1]. Ammonia oxidizing bacteria (AOB) have thus been used in biosensors for NH4 + [2]. The high sensitivity of bacterial ammonia oxidation to toxic substances may be used for assays of such toxic chemicals [3–6], and it has even been attempted to use a bacterium-based NH4 + biosensor for such toxicity tests [7]. The NH4 + biosensors constructed so far have apparently not had sufficiently good characteristics to be used routinely, as no commercialization of such sensors ∗ Corresponding author. Department of Biology, Northeastern University, 360 Huntington Avenue, 134 Mugar Life Science Building, Boston MA 02115, USA. Tel.: +1 617 373 3229; fax: +1 617 373 3724. E-mail address: [email protected] (A. Bollmann).

0925-4005/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2004.06.030

seems to have been attempted. The biosensor of Tanaka et al. [2] consists of a Clark-type oxygen sensor with a layer of immobilized Nitrosomonas europaea cells in front of the sensor. It can be only used in a fully oxygenated system, needs the addition of a buffering system to keep the pH constant, and is linear up to only 70 ␮M NH4 + . To be attractive for in situ analytical purposes an NH4 + biosensor should perform better than the previously described NH4 + biosensors. It should also have better measuring characteristics than NH4 + sensors based on ion exchangers (e.g. [8]), optode-type sensors [9], and in situ flow-injection devices such as the Evita NH4 + sensor (Danfoss Analytical A/S) [10], or it should be cheaper in use. The largest potential application for in situ NH4 + analysis is waste water treatment, where modern nitrogen-removing plants operate with alternating oxic and anoxic periods to first nitrify the ammonium to nitrite and/or nitrate, and sub-

A. Bollmann, N.P. Revsbech / Sensors and Actuators B 105 (2005) 412–418

sequently reduce this NOx − to N2 . We have previously developed bacteria-based sensors for NO2 − and NOx − [11,12] that are able to monitor in wastewater for months with very little drift in sensitivity. These biosensors have a measuring range of 0–1 mM, and they have recently been made commercially available (Unisense A/S). The long term stability of the NO2 − and NOx − biosensors is due to a robust electrochemistry and a continuous-culture principle, where the bacteria are continuously supplied with nutrients from an internal reservoir [1]. We therefore decided to apply a similar continuous-culture approach for an NH4 + biosensor. The need for measuring NH4 + in wastewater is largest under anoxic conditions, when NH4 + concentrations are highest, and we therefore decided to develop an NH4 + biosensor that would work under anoxic conditions, using the counter-diffusion principle previously applied for a continuous-culture type CH4 biosensor [13]. By placing the bacteria under a steady supply of oxygen from an internal oxygen reservoir we intended to optimize the growth conditions for the AOB and thereby improve the general measuring characteristics as compared to the previously constructed NH4 + biosensors.

2. Experimental 2.1. Construction of the NH4 + biosensor The biosensor was made according the principles outlined by Damgaard et al. [13], but in contrast to the microscale version with tip diameters of 30–50 ␮m made by Damgaard et al. [13], the NH4 + biosensors were made at a macroscale with a tip diameter of 7 mm. The chamber housing the bacteria and the internal O2 -supplying and sensing element had diameters of 0.30 and 0.28 mm, respectively (Fig. 1). The oxygen microsensor was made as described by Revsbech [14]. It was inserted into a tapered capillary with a tip diameter of 0.28 mm so that the tip protruded about 0.1 mm in front of the capillary. The capillary was hereafter fixed in place with epoxy. Silicone rubber (Dow Corning 734) was subsequently sucked

Fig. 1. Schematic drawing of the NH4 + biosensor design.

413

into the capillary to a depth of 0.5 mm, and excess silicone was wiped off with a piece of plastic foil. After curing, the assembly containing an air-filled capillary was inserted into the bacterial chamber. The casing of the bacterial chamber was made from a 5 cm long piece of 7 mm glass tubing onto which a 0.5 mm thick polyester (Goodfellow) front plate with a central 0.30 mm hole was fixed with epoxy. The 7 mm glass tube was fixed in place with epoxy so that the distance between the silicone rubber membrane of the O2 -supplying capillary and the front end of the polyester plate was 0.3 mm (Fig. 1). The AOB were grown in mineral salt medium with 5 mM NH4 + and 20 mM HEPES buffer at a pH of 7.8 and 25 ◦ C [15]. The cultures were harvested at the end of the logarithmic growth phase by centrifugation (22.000 × g, 20 min) and washed once with mineral salt medium without NH4 + . The concentrated bacterial biomass was injected into the 0.3 mm hole of the bacterial chamber from the front end by use of a heat-drawn 1 ml plastic syringe with a tip diameter of the plastic capillary of about 0.1 mm. The ion-permeable membrane (Unisense) at the tip of the sensor was subsequently sealed to the sensor casing with double-adhesive tape (3 M, Scotch 467 Hi Performance Adhesive). The membrane had a cut off value of 3500 Dalton. The bulk interior of the bacterial chamber was filled with the mineral salt medium without NH4 + as used for suspending the culture. Finally a few small granules of dry silica gel were placed in the shaft-end of the conical air-filled capillary that had a total air volume of about 0.3 ml, and the capillary was sealed with epoxy. The epoxy seal to of the air-filled capillary was penetrated by a thin glass tube that could be broken to replace the air. After construction, the NH4 + biosensors were incubated for 2–3 weeks in mineral salt medium with 1 mM NH4 + and 5 mM HEPES-buffered at pH 7.5 to get the highest possible biomass of metabolically active bacteria. During these 2–3 weeks the medium was changed twice a week. Different cultures of AOB have been used to fill the NH4 + biosensor: Nitrosomonas europaea ATCC19718, the enrichment culture G5-7 [16], and a not further characterized enrichment culture from a sludge sample. The principle of how the NH4 + biosensor functions are illustrated in Fig. 2. NH4 + diffuses across the ion-permeable membrane, and at some depth in the bacterial chamber it meets oxygen diffusing out from the gas reservoir inside the sensor. Due to the activity of the AOB, the oxygen consumption will be proportional to the influx of ammonium, and the oxygen concentration at the tip of the internal oxygen microsensor will therefore change as a function of the external NH4 + concentration. It can be shown that the signal from the internal oxygen sensor of such a biosensor is inversely proportional to the concentration of the analyte, even if there is an overlapping zone between oxygen and the analyte, as long as all analyte is consumed before it reaches the tip of the sensing element [13]. Sensors made according to this counter-diffusion principle have a high reading for low concentrations of the analyte and vice versa. This inverse relationship between absolute sensor signal and NH4 + con-

414

A. Bollmann, N.P. Revsbech / Sensors and Actuators B 105 (2005) 412–418

alytical Evita NH4 + sensor) and O2 (Danfoss Analytical O2 sensor) were recorded simultaneously. The activated sludge tank was fed with artificial wastewater as described by Balslev et al. [17]. 2.4. Analytical methods Colorimetric determination of NH4 + for laboratory calibration of the NH4 + biosensor was done by the method of Kandeler and Gerber [18].

Fig. 2. Schematic drawing of an NH4 + biosensor and model of the O2 gradients inside the tip of the sensor. (A) High NH4 + concentration resulting in a low O2 partial pressure at the tip of the internal O2 microsensor. (B) Low NH4 + concentrations resulting in a high O2 concentration. The signals from the internal oxygen microsensors are indicated by arrows.

centration is shown in Fig. 2, where the arrow symbolizes the oxygen concentration at the tip of the internal oxygen microsensor. The arrow is short (i.e. a low O2 concentration) when the NH4 + concentration is high, and long when the NH4 + concentration is low. The internal oxygen microsensor of the biosensors was connected to a picoamperometer, and the signal was recorded on a strip-chart recorder. For test and calibration the sensor was placed in a calibration chamber while purging with N2 .

3. Results and discussion 3.1. Calibration of the NH4 + biosensor Pure cultures of nitrifying bacteria are notoriously difficult to grow, so it seemed likely that enrichments would be more active. We therefore tested two sensors that were filled with two types of enrichment cultures in addition to two sensors filled with N. europaea (Fig. 3). By first glance there seems

2.2. Calibration and interference tests The first calibrations were done in mineral salt medium buffered with 5 mM HEPES and at pH 7.5. Increasing amounts of NH4 + were added, and after each addition the signal was recorded for 5 min until a new steady state level of the signal was reached. The influence of variations in pH was tested in a similar HEPES-buffered mineral salt medium adjusted to pH values of 6–8.5. The influence of several compounds like NH2 OH, NO2 − , NO3 − , NaCl, PO4 3− , glucose, and acetate was tested by adding the compounds to the mineral salt medium before a calibration curve was recorded. The effect of O2 and CH4 were tested by mixing these gases into the N2 flow purged through the calibration vessel. The temperature effect was investigated by calibration in incubators with different temperatures (6, 13, 25, 30, and 36 ◦ C). The stirring sensitivity was determined by recording the signal during the transition from stagnant to vigorously bubbled medium. The response time to changes in NH4 + concentration was determined under stirred conditions. 2.3. Test in wastewater One of the NH4 + biosensors was tested in the activated sludge tank of a pilot-scale wastewater treatment plant that was operated with alternating oxic and anoxic periods. The tank was used for general test of water quality sensors, and many wastewater parameters including NH4 + (Danfoss An-

Fig. 3. Performance of different NH4 + biosensors: (A) four sensors over the whole range and; (B) two sensors at low NH4 + concentrations.

A. Bollmann, N.P. Revsbech / Sensors and Actuators B 105 (2005) 412–418

to be large differences in the response, but much of this difference is most likely due to different sensitivities from the internal O2 sensor. Sensors 1 and 3 exhibited almost linear response up to 0.3 mM NH4 + , whereas sensor 2 was linear up to only 0.1 mM. The linear range for sensor 4 was not determined, but the response was at least linear up to 0.13 mM as seen in Fig. 3B, where also the response of sensor 1 at low NH4 + concentrations is shown in detail. At higher NH4 + concentrations the sensors did not stop responding to NH4 + , but the response became less and less, indicating a saturation of the sensor (Fig. 3). The different cultures/enrichments thus all resulted in similar response. We therefore chose to continue to test only sensors made with N. europaea. It should be stressed, however, that it is our experience that various heterotrophic bacteria invade sensors along the tape-membrane interface if special measures are not taken, and the cultures inside the sensors can thus not be assumed to be axenic. Sensor 2 filled with N. europaea was used for more than 1 month for calibrations, interferences tests, and test in the wastewater treatment plant. When the sensor was not in use it was stored in mineral salt medium with 1 mM NH4 + and 5 mM HEPES at pH 7.5. The calibration curves changed over time (Fig. 4). Within the first week the signal increased a little, but subsequent calibrations showed progressively lower signals. The shape of the calibration curves was, however, unchanged, and the decrease in absolute signal seemed to be due to aging of the internal O2 microsensor indicating a necessity of regular calibrations of the biosensor. The substrate of AOB is ammonia (NH3 ), which is in a dynamic pH-dependent equilibrium with NH4 + . The influence of pH on the signal of the NH4 + biosensor was therefore tested at two NH4 + concentrations (Fig. 5). At pH 6.5 the signal from the sensor was around 20% of the control at pH 7.5. Between pH 6.5 and 8.5 the signal increased from 20 to 175% of the control when an NH4 + concentration of 0.055 mM was applied. At an NH4 + concentration of 0.55 mM the signal also increased with increasing pH value and reached a plateau at

Fig. 4. Performance as a function of time for a NH4 + biosensor filled with N. europaea.

415

Fig. 5. Influence of pH on the performance of a NH4 + biosensor filled with N. europaea. The effect is given as the deviation in % as compared to a calibration performed at pH 7.5.

120% of the control already when the pH reached 7.5. This saturating response was to be expected, as the 0.55 mM already was above the linear range of the sensor at pH 7.5. The observed effect of pH on ammonia oxidation is comparable with results obtained in other studies [19,20]. The ammonia oxidation rates were maximal at pH values around 8–8.5, decreased at lower pH values due to the decreased availability of NH3 and at higher pH values due general negative effects of high pH. The calibrations described above were performed at 25 ◦ C, but the response to both higher and lower temperatures was also tested. The shape of the curve remained the same at all temperatures but the response of the sensor was increasing with increasing temperature from 6 to 36 ◦ C, indicating an increase in the activity of the AOB (results not shown). After addition of 0.1 mM, NH4 + the sensors needed 1–1.5 min to reach 90% of the signal, and 2–4 min were thus needed to get readings without significant drift. The stirring sensitivity was a few pA at all concentrations, corresponding to a signal change equivalent to about 10 ␮M NH4 + within the linear range. The interferences of several chemical species were tested with sensor 2 (Table 1). A small but significant decrease in signal was observed with NO2 − at a concentration of 10 mM. Such inhibitory effects of NO2 − on AOBs are also known from liquid culture [19,21]. There also seemed to be a small effect of 10 mM NO3 − , which was not expected. High salinity is known to reduce nitrification [22], so it was not unexpected that 0.1–0.3 M NaCl had a moderate inhibitory effect. The inhibitory effect of high phosphate (0.1 M) could also primarily be a salinity effect. It should be kept in mind that in addition to the direct effects of high salt concentrations on the bacteria, the altered ionic composition of the analyzed medium may also affect the tip potential of the sensor. It has previously been shown that tip potentials dramatically affect the entry of ions into biosensors [23]. Glucose (and acetate)

416

A. Bollmann, N.P. Revsbech / Sensors and Actuators B 105 (2005) 412–418

Table 1 Influence of different compounds in the mineral salt medium on the zero signal and the ammonia oxidation by sensor 2 filled with N.europaea Zero signala

0.055 mM NH4 +b

0.55 mM NH4 +b

0.1 mM NO2 − 1 mM NO2 − 10 mM NO2 −

0 0 0

– – 20

– 15 20

0.1 mM NO3 − 1 mM NO3 − 10 mM NO3 −

0 0 0

– – –

– – 15

0.001 mM NH2 OH 0.01 mM NH2 OH 0.1 mM NH2 OH

0 0.03 mM 0.25 mM

– – 50

– – 60

100 mM NaCl 200 mM NaCl 350 mM NaCl

0 0 0

– 40 60

15 30 45

1 mM phosphate 10 mM phosphate 100 mM phosphate

0 0 0

– – 40

– −10 20

0.01 mM glucose 0.1 mM glucose 1 mM glucose

0 0.1 mM 0.1 mM

20 30 30

– 30 30

0.01 mM acetate 0.1 mM acetate 1 mM acetate

0 0.05 mM 0.05 mM

– – −20

– 10 –

Only effects larger than 20% of the signal for 0.055 mM NH4 + and 10% of the signal for 0.55 mM NH4 + are given, as smaller effects were not significant. a Change in the zero signal of the NH + biosensor by the added compound 4 alone, given as the NH4 + concentration required to produce a similar effect. b Percentage inhibition compared to the control, positive values indicate a decrease of the signal and negative an increase as compared to the control.

in the calibration medium resulted in an increase of the baseline due to O2 consumption in the presence of a carbon source for the heterotrophic contaminating bacteria in the bacterial chamber. The effect was the same for 0.1 and 1 mM glucose or acetate, indicating that the heterotrophs were saturated at these concentrations. NH2 OH is an intermediate in the oxidation of NH3 to NO2 − , and it is a toxic and mutagenic substance. Low concentrations of NH2 OH had no significant effect on the sensitivity to NH4 + , but an inhibitory effect was present at a concentration of 0.1 mM. The sensor was designed for use in anoxic systems, and the signal is affected by O2 . To get an insight in how the sensor reacts to the presence of O2 , calibrations were performed in mineral salt medium bubbled with various N2 /O2 mixtures (Fig. 6). The presence of external O2 moved the baseline to a higher level, indicating that more O2 was present in the bacterial chamber (Fig. 6B). After subtraction of the zero level, however, the calibration curves showed almost the same shape, indicating that oxygen never limited the oxidation of ammonium in the bacterial chamber. In principle it would thus be possible to subtract the signal for external O2 from the reading and thereby compensate for the O2 interference, but in practise such a procedure would introduce too large a source of error.

Fig. 6. Influence of different O2 partial pressures on the performance of a NH4 + biosensor filled with N. europaea. The mineral salt medium was bubbled with different O2 /N2 mixtures: (A) current after subtraction of the blank; (B) measured currents.

3.2. Field test in a wastewater treatment plant The NH4 + biosensor was tested in the activated sludge tank of a wastewater treatment plant. The sludge was exposed to 1.5 h oxic–1.5 h anoxic cycles (Fig. 7). During the oxic periods O2 was kept at a concentration of 1 mg/l, although there was an initial overshoot up to 2 mg/l. The NH4 + concentration measured with the Evita NH4 + analyzer showed that NH4 + decreased to undetectable concentrations during the initial 15 min of the oxic period, where it was oxidized by nitrifying bacteria in the sludge. The data calculated from the NH4 + biosensor signal during the oxic period indicate an NH4 + concentration below zero due to the interference from O2 in the bulk liquid. Both methods showed that NH4 + increased throughout the anoxic period and the methods gave virtually identical results. At the end of the anoxic period, the signal of the NH4 + biosensor decreased faster than the signal from the colorimetric analyzer, and this was again at least partially due to interference from O2 . In general the sensors responded linearly to low NH4 + concentrations, but the response gradually became less and less. The small linear range can be explained by a low activity by the bacteria, and the gradual decrease in response may be caused by relatively high half-saturation concentrations for

A. Bollmann, N.P. Revsbech / Sensors and Actuators B 105 (2005) 412–418

417

In summary, we have shown that it is possible to make an NH4 + biosensor according to the counter-diffusion principle introduced for a CH4 biosensor. It has also been shown that such an NH4 + biosensor may be applied in anoxic wastewater, but the calibration should then be made in a liquid medium with similar chemical composition and temperature. The limited linear calibration range and the high sensitivity to environmental conditions are, however, serious drawbacks for such a sensor. We have previously developed novel biosensors for environmental analysis, and the first sensors often have inferior characteristics as compared to later versions where years of optimization has been implemented. There is no doubt that the NH4 + biosensors described in this paper also can be optimized, but similar superior characteristics as seen for the NOx − and NO2 − biosensors can never be obtained, primarily due to the inherent weaknesses of the applied counter-diffusion principle.

Acknowledgement This work was supported by a grant from the 5th Frame work program of the European Commission (ICON EKV1CT-2000-00054).

References Fig. 7. NH4 + (A) and O2 (B) concentrations in the activated sludge of a wastewater treatment plant. The sludge was exposed to oxic/anoxic cycles. NH4 + concentrations have been determined colorimetrically (䊉) and calculated from the reading of the NH4 + biosensor filled with N. europaea (—).

NH4 + coupled with an oxidation of NH4 + also behind the tip of the O2 sensor. For all sensors, saturating response was approached at 0.6 mM NH4 + at the applied pH of 7.5, and even at such a saturating NH4 + concentration the signal from the built-in oxygen sensor indicated high oxygen concentrations meaning that oxygen never became limiting. In principle it is possible to detect concentrations of down to 1 ␮M by use of these sensors, as the O2 signal may be read at 0.1 pA resolution. In practical terms the detection limit is rather >10 ␮M, as changes in temperature and advection in front of the sensor affects the signal. Such an effect is seen for all amperometric sensors, but the impact on accuracy is higher for the counterdiffusion biosensors such as this NH4 + biosensor than it is for most other sensors. The high sensitivity to environmental conditions is an inherent characteristic in the measuring principle, where the absolute signal is high for low concentrations of analyte and low for high concentrations. Any factor that will affect the high zero current at zero concentration will thus affect the detection limit. As an example a 1 ◦ C temperature decrease may, for a specific sensor, change the oxygen signal of 200 to 195 pA, and this signal change from the biosensor may be mis-interpreted as an increase in NH4 + concentration of about 20 ␮M.

[1] N.P. Revsbech, T. Kjær, L.R. Damgaard, J. Lorenzen, L.H. Larsen, in: J. Buffle, G. Harvai (Eds.), In Situ Monitoring of Aquatic systems: Chemical Analysis and Speciation, John Wiley, 2000, pp. 195–222. [2] H. Tanaka, E. Nakamura, H. Hoshikawa, Y. Tanaka, Development of the ammonia biosensor monitoring system, Water Sci. Tech. 28 (1993) 435–445. [3] C. Grunditz, L. Gumaelius, G. Dalhammar, Comparison of inhibition assays using nitrogen removing bacteria: application to industrial wastewater, Water Res. 32 (1998) 2995–3000. [4] C. Grunditz, G. Dalhammar, Development of nitrification inhibition assays using pure cultures of Nitrosomonas and Nitrobacter, Water Res. 35 (2001) 433–440. [5] K. Jonsson, C. Grunditz, G. Dalhammar, J.L. Jansen, Occurrence of nitrification inhibition in Swedish municipal wastewaters, Water Res. 34 (2000) 2455–2462. [6] C. Ludwig, S. Ecker, K. Schwindel, H.-G. Rast, K.O. Stetter, G. Eberz, Construction of a highly bioluminescent Nitrosomonas as a probe for nitrification conditions, Arch. Microbiol. 172 (1999) 45–50. [7] A. K¨onig, J. Secker, K. Riedel, J. Metzger, A microbial sensor for measuring inhibitors and substrates for nitrification in wastewater, Int. Lab. July (1998) 15–19. [8] D. De Beer, A. Schramm, C.M. Santegoeds, M. K¨uhl, A nitrite microsensor for profiling environmental biofilms, Appl. Environ. Microbiol. 63 (1997) 973–977. [9] N. Str¨omberg, S. Hulth, Ammonium selective fluorosensor based on the principles of coextraction, Anal. Chim. Acta 443 (2001) 215–225. [10] A. Lynggard-Jensen, Trends in monitoring of waste water systems, Talanta 50 (1999) 707–716. [11] M. Nielsen, N.P. Revsbech, L.H. Larsen, A. Lynggaard-Jensen, Online determination of nitrite in wastewater treatment by use of a biosensor, Water Sci. Tech. 45 (2002) 69–76.

418

A. Bollmann, N.P. Revsbech / Sensors and Actuators B 105 (2005) 412–418

[12] L.H. Larsen, T. Kjær, N.P. Revsbech, A microscale NO3 − biosensor for environmental applications, Anal. Chem. 69 (1997) 3527– 3531. [13] L.A. Damgaard, N.P. Revsbech, A microscale biosensor for methane containing methanotrophic bacteria and an internal oxygen reservoir, Anal. Chem. 69 (1997) 2262–2267. [14] N.P. Revsbech, An oxygen microelectrode with a guard cathode, Limnol. Oceanogr. 34 (1989) 472. [15] F.J.M. Verhagen, H.J. Laanbroek, Competition for ammonium between nitrifying and heterotrophic bacteria in dual energy limited chemostat, Appl. Environ. Microbiol. 57 (1991) 3255–3263. [16] A. Bollmann, H.J. Laanbroek, Continuous culture enrichments of ammonia-oxidizing bacteria at low ammonium concentrations, FEMS Microbiol. Ecol. 37 (2001) 211–221. [17] P. Balslev, A. Lynggaard-Jensen, C. Nickelsen, Nutrient sensor based real-time on-line process control of a wastewater treatment plant using recirculation, Water Sci. Tech. 33 (1996) 183–192.

[18] E. Kandeler, H. Gerber, Short-term assay of soil urease activity using colorimetric determination of ammonium, Biol. Fertil. Soils. 6 (1988) 68–72. [19] J. Groeneweg, B. Sellner, W. Tappe, Ammonia oxidation in Nitrosomonas at NH3 concentrations near km : effects of pH and temperature, Water Res. 28 (1994) 2561–2566. [20] J.H. Hunik, H.J.G. Meijer, J. Tramper, Kinetics of Nitrosomonas europaea at extreme substrate, product and salt concentrations, Appl. Microbiol. Biotechnol. 37 (1992) 802–807. [21] L.Y. Stein, D.J. Arp, Loss of ammonia monooxygenase activity in Nitrosomonas europaea upon exposure to nitrite, Appl. Environ. Microbiol. 64 (1998) 4098–4102. [22] G. Stehr, B. B¨ottcher, P. Dittberner, G. Rath, H.P. Koops, The ammonia-oxidizing nitrifying population of the River Elbe estuary, FEMS Microbiol. Ecol. 17 (1995) 177–186. [23] T. Kjær, L.H. Larsen, N.P. Revsbech, Electrophoretic sensitivity control of microscale biosensors, Anal. Chim. Acta 391 (1999) 57–63.