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Measurement of nitrate gradients with an ion-selective microelectrode
Analytica Chimica Acta, 219 (1989) 351-356 Elsevier Science Publishers B.V., Amsterdam -
351 Printed in The Netherlands
Short Communication
MEASUREMENT OF NITRATE GRADIENTS SELECTIVE MICROELECTRODE
WITH AN ION-
DIRK DE BEER*
Department of Chemical Engineering and Biotechnological Centre, University of Amsterdam, Nieuwe Achtergracht 166,1018 WVAmsterdam (The Netherlands) JEAN-PIERRE
R.A. SWEERTS
Limnological Institute, Rijksstraatweg 6,363l AC Nieuwersluis (The Netherlands) (Received 21st September 1988)
Nitrate-selective microelectrodes (1-p tip diameter) with a liquid membrane ion Summary. exchanger were prepared. The response of the electrode is linear from 10 -’ to lo-’ M nitrate and the response time is 30 s. Selectivity coefficients are of the same order as specified for macroelectrodes. Measurements in sediments of a mesotrophic lake and measurements in spherical agar gels with immobilized denitrifying bacteria are presented.
Nitrate is the end-product of nitrification and the terminal electron acceptor in denitrification, and serves as a nitrogen source for cell growth. Precisely sited measurements of nitrate concentrations in and near active microbial layers can give valuable information about nitrate transformations and the location of activities. Because gradients and conversions in sediments often occur in narrow zones ( < 2 mm), microelectrodes are the only tools with sufficient spatial resolution. Interesting results have been obtained with oxygen and pH microelectrodes in marine and freshwater sediments, microbial mats and biofilms from wastewater treatment plants [l-3]. Here, the construction of a liquid-membrane nitrate-selective microelectrode is reported. Measurements of gradients in spherical agar gels with denitrifying bacteria and in a freshwater sediment are presented. Experimental Electrode preparation
and apparatus. Micropipettes with a tip diameter of 1 ,um were prepared as described previously [4]. For measurements in sediments, the shaft length was 12 cm; for measurements in gels, a 4-cm length
0003-2670/89/$03.50
0 1989 Elsevier Science Publishers B.V.
352
sufficed. The electrode tips were filled with Orion nitrate exchanger (92-0701); subsequently the shafts were filled with filtered and degassed electrolyte solution 0.04 M potassium nitrate. The measuring circuit during calibration was Ag/AgCl 110.3M KC1 110.004M KN03 I]ion-selective membrane IIsample solutionll3 M KC1 agar bridgell0.3 M KCl]]AgC!l/Ag. The electrode assembly was placed in a Faraday cage. The electrode system was connected through a triaxial input cable (Keithley 6011) to an electrometer (Keithley 617), placed outside the Faraday cage. Calibrations were done in a continuous flow cell, at the same temperature as the subsequent measurements. Selectivity factors were evaluated by using the separate-solution method [ 561. During measurements in sediments, the agar bridge was connected with the overlying water. During concentration gradient measurements in sediments and gels, the microelectrodes were positioned by a motor-driven micromanipulator. Sample preparation. Samples of silty sediment and overlying water were collected [7] from the mesotrophic lake Vechten at a depth of 10 m, and stored at 8-12’ C. During measurements and storage, the overlying water was gently stirred, so that the thickness of the stagnant water layer above the sediment was about 1 mm [8]. Growth and immobilization of bacteria A denitrifying bacterial population was grown in a continuous-flow stirred-tank reactor. The medium consisted of a mineral part (NH&l, 25 mol mm3; KCl, 3 mol mm3; Na3P04, 2.5 mol mW3; Na2S04, 0.4 mol mm3; MgCl,, 0.31 mol mm3; sodium citrate, 0.5 mol mm3; CaCl,, 5 x low3 mol me3; NazMoO,, 25 x 1O-6 mol mT3; NasSeO,, 15 x 10e5 mol mW3; NiS04, 75 x 10e5 mol mb3; FeCl,, 25 x 10h3 mol mm3; MnClz, 12.6 x 10m3 mol mm3; CoC12, 25x low7 mol mv3; ZnO, 63x 10s7 mol mT3; CuCl,, 12.5~ 10m7 mol me3; H3B03, 12.5~ 10e5 mol mW3), supplied with 40 mol mm3 KN03 and 100 mol m-’ sodium acetate. The culture was maintained at 30°C pH 8.0 and a specific growth rate of 0.2 h-l. The media and reactor were not sterilized, and therefore the bacteria were not obtained in pure cultures. From this reactor, batch cultures were inoculated, with the same medium with initial pH 7.5. The cultures were incubated at 3O”C, under anaerobic or aerobic conditions, and harvested in the logarithmic growth phase by centrifugation at 12 000 g for 10 min. Cells were resuspended in mineral medium to a concentration of 5% wet weight. One part of the cell suspension was mixed with four parts of a 2.5% agar solution at 40°C. The agar solution was then dripped into cold oil to form spherical gel beads. Gels were fixed in the flow cell with two entomological-specimen needles. During concentration measurements in the gel, mineral medium supplied with 100 mol rnp3 sodium acetate and the desired concentration of nitrate was pumped through the flow cell. Before each experiment, the gels were incubated in this medium to achieve steady state. The temperature in the flow cell was maintained at 30’ C. Calculations. The concentration gradients in gels were calculated by numerical solution, using a grid method, of the equation
353
D,(l/r2)
(d/dr) (r2ds/dr) = Vmaxs/Km +s
(1)
where D, is the effective diffusion coefficient, s the substrate concentration, r the distance from the centre, V_ the maximum reaction rate and K, the Michaelis constant; I&, was assumed to be 0.006 mol rnp3 [9]. D, was determined by fitting the time-dependent diffusive uptake by initially solute-free gel beads from a bulk solution with a known volume, using the calculation method of Crank [lo]; nitrate concentration changes in the bulk were measured with a nitrate-selective macroelectrode (Orion). V,, was measured in batch experiments; the nitrate consumption was measured by using the nitrate macroelectrode. Results and discussion The response of the microelectrodes to concentrations of 10-‘-10-5 M potassium nitrate in demineralized water was linear with a slope of 55 mV/ log [ KNO,] (Fig. 1) . The response time was 30 s and the drift of the signal was l-3 mV h-l. Addition of different amounts of 2nitrophenyl octyl ether or PVC to the ion exchanger resulted in worse behaviour. The selectivity factors for chloride and nitrite were 0.006 and 0.06, respectively, which are of the same order as specified by Orion for macroelectrodes (0.004 and 0.04, respectively). Therefore, selectivity factors for other ions are expected to be almost the same as specified by Orion. The electrodes were stable for at least 5 h. For each experiment, freshly prepared electrodes were used. The electrode signal was not influenced by the velocity of the liquid. Although the bacterial cultures were not defined, their behaviour was similar mV
- log[NO;l Fig. 1. Calibration graphs for nitrate microelectrodes in ( 0 ) demineralized water and ( 0 ) mineral medium with 0.1 M sodium acetate.
354
to that of pure cultures. In both anaerobic and aerobic cultures, the cells were uniformly rod-shaped, while the concentrated suspensions had a red appearance. The V,,, of the gels with anaerobically grown cells was 0.06 mol mm3s-l and D, was 22 x lo-” m sm2.Figure 2 shows the measured and calculated nitrate gradients in gels, incubated at different nitrate concentrations. Under anaerobic conditions, the nitrate concentration inside the particles gradually decreased towards the centre. The calculated and measured gradients fit very well, indicating that the measurements with microelectrodes are accurate. Gas formation inside the gels occurred after 3-4 h of incubation. Two hours after switching from anaerobic to aerobic conditions, the nitrate gradients were unchanged. Hence in these bacteria, nitrate can serve as terminal electron acceptor under aerobic conditions for at least 2 h. In a control experiment, cell growth and gradient measurements were done aerobically. Under these conditions, no nitrate consumption was measured and no differences in nitrate concentration in the gels could be detected. It is concluded that the anaerobic nitrate consumption is probably due only to denitrifmation. In the medium used, ammonium served as the nitrogen source for cell growth [ 111, and therefore assimilatory nitrate consumption did not occur. The denitrifying ability of these bacteria is not constitutive and the induction NO, I 04
(mallm’)
L
Fig. 2. Nitrate gradients in gels with immobilized bacteria. Measurements were made in mineral medium with 0.1 M sodium acetate (pH 8.0) and supplied with (a) 0.4, (b) 4.3 and (c) 43 mol/ m3 KN03. Both bacterial growth and gradient measurements were made under (0) anaerobic and ( 0 ) aerobic conditions. Solid lines indicate calculated gradients.
355 INO; 0
0.01
hollm’ I 0.02
0.03
Fig. 3. Nitrate gradient in lake Vechten sediment.
of the denitrifying enzymes is inhibited by oxygen. However, once the enzyme system is formed under anaerobic conditions, the nitrate consumption is not inhibited by oxygen. Figure 3 shows a nitrate gradient in silty sediment of a freshwater lake. In this sediment, the nitrate penetration depth is less than 1.3 mm. Because the nitrate conversion in this sediment occurs in a very narrow zone, only devices with a high spatial resolution, such as microelectrodes, can give precise and direct information. More detailed studies with nitrate-selective microelectrodes on freshwater sediments will be described elsewhere [ 121. The electrode cannot be used for studies in marine environments because of its sensitivity to chloride. However, it is valuable in studies on freshwater environments with natural nitrate concentrations, on model systems with nitrifying and denitrifying bacteria and on biofilms from wastewater plants. The authors thank Per Staugaard (University of Amsterdam, Department of Chemical Engineering) for the use of his computer program.
REFERENCES 1 2 3 4 5
Y.S. Chen and H.R. Bungay, Biotechnol. Bioeng., 23 (1981) 781. N.P. Revsbech, J. Sorensen and T.H. Blackburn, Limnol. Oceanogr., 25 (1980) 403. N.P. Revsbech and B.B. Jorgensen, Adv. Microb. Ecol., 9 (1986) 293. D. de Beer and J.C. van den Heuvel, Talanta, 35 (1988) 728. D. Ammann, Ion-Selective Microelectrodes: Principles, Design and Application, Springer, Berlin, 1986.
356 6 7 8 9 10 11 12
W.McD. Armstrong and J.F. Garcia-Diaz, Fed. Proc., Fed. Am. Sot. Exp. Biol., 39 (1980) 2851. T.E. Cappenberg, Antonie van Leeuwenhoek; J. Microbial. Serol., 40 (1974) 285. J.-P.R.& Sweerts, V. St. Louis and T.E. Cappenberg, Freshwater Biol., in press. R. Riemer and P. Harrem&s, Prog. Water Technol., 10 (1978) 149. J. Crank, The Mathematics of Diffusion, Clarendon Press, Oxford, 1973. R.O. Marshall, H.J. Dishburger, R. MacVicar and G.D. Hallmark, J. Bacterial., 66 (1953) 254. J.-P.R.A. Sweerts and D. de Beer, Appl. Environm. Microbial., in press.
351 Printed in The Netherlands
Short Communication
MEASUREMENT OF NITRATE GRADIENTS SELECTIVE MICROELECTRODE
WITH AN ION-
DIRK DE BEER*
Department of Chemical Engineering and Biotechnological Centre, University of Amsterdam, Nieuwe Achtergracht 166,1018 WVAmsterdam (The Netherlands) JEAN-PIERRE
R.A. SWEERTS
Limnological Institute, Rijksstraatweg 6,363l AC Nieuwersluis (The Netherlands) (Received 21st September 1988)
Nitrate-selective microelectrodes (1-p tip diameter) with a liquid membrane ion Summary. exchanger were prepared. The response of the electrode is linear from 10 -’ to lo-’ M nitrate and the response time is 30 s. Selectivity coefficients are of the same order as specified for macroelectrodes. Measurements in sediments of a mesotrophic lake and measurements in spherical agar gels with immobilized denitrifying bacteria are presented.
Nitrate is the end-product of nitrification and the terminal electron acceptor in denitrification, and serves as a nitrogen source for cell growth. Precisely sited measurements of nitrate concentrations in and near active microbial layers can give valuable information about nitrate transformations and the location of activities. Because gradients and conversions in sediments often occur in narrow zones ( < 2 mm), microelectrodes are the only tools with sufficient spatial resolution. Interesting results have been obtained with oxygen and pH microelectrodes in marine and freshwater sediments, microbial mats and biofilms from wastewater treatment plants [l-3]. Here, the construction of a liquid-membrane nitrate-selective microelectrode is reported. Measurements of gradients in spherical agar gels with denitrifying bacteria and in a freshwater sediment are presented. Experimental Electrode preparation
and apparatus. Micropipettes with a tip diameter of 1 ,um were prepared as described previously [4]. For measurements in sediments, the shaft length was 12 cm; for measurements in gels, a 4-cm length
0003-2670/89/$03.50
0 1989 Elsevier Science Publishers B.V.
352
sufficed. The electrode tips were filled with Orion nitrate exchanger (92-0701); subsequently the shafts were filled with filtered and degassed electrolyte solution 0.04 M potassium nitrate. The measuring circuit during calibration was Ag/AgCl 110.3M KC1 110.004M KN03 I]ion-selective membrane IIsample solutionll3 M KC1 agar bridgell0.3 M KCl]]AgC!l/Ag. The electrode assembly was placed in a Faraday cage. The electrode system was connected through a triaxial input cable (Keithley 6011) to an electrometer (Keithley 617), placed outside the Faraday cage. Calibrations were done in a continuous flow cell, at the same temperature as the subsequent measurements. Selectivity factors were evaluated by using the separate-solution method [ 561. During measurements in sediments, the agar bridge was connected with the overlying water. During concentration gradient measurements in sediments and gels, the microelectrodes were positioned by a motor-driven micromanipulator. Sample preparation. Samples of silty sediment and overlying water were collected [7] from the mesotrophic lake Vechten at a depth of 10 m, and stored at 8-12’ C. During measurements and storage, the overlying water was gently stirred, so that the thickness of the stagnant water layer above the sediment was about 1 mm [8]. Growth and immobilization of bacteria A denitrifying bacterial population was grown in a continuous-flow stirred-tank reactor. The medium consisted of a mineral part (NH&l, 25 mol mm3; KCl, 3 mol mm3; Na3P04, 2.5 mol mW3; Na2S04, 0.4 mol mm3; MgCl,, 0.31 mol mm3; sodium citrate, 0.5 mol mm3; CaCl,, 5 x low3 mol me3; NazMoO,, 25 x 1O-6 mol mT3; NasSeO,, 15 x 10e5 mol mW3; NiS04, 75 x 10e5 mol mb3; FeCl,, 25 x 10h3 mol mm3; MnClz, 12.6 x 10m3 mol mm3; CoC12, 25x low7 mol mv3; ZnO, 63x 10s7 mol mT3; CuCl,, 12.5~ 10m7 mol me3; H3B03, 12.5~ 10e5 mol mW3), supplied with 40 mol mm3 KN03 and 100 mol m-’ sodium acetate. The culture was maintained at 30°C pH 8.0 and a specific growth rate of 0.2 h-l. The media and reactor were not sterilized, and therefore the bacteria were not obtained in pure cultures. From this reactor, batch cultures were inoculated, with the same medium with initial pH 7.5. The cultures were incubated at 3O”C, under anaerobic or aerobic conditions, and harvested in the logarithmic growth phase by centrifugation at 12 000 g for 10 min. Cells were resuspended in mineral medium to a concentration of 5% wet weight. One part of the cell suspension was mixed with four parts of a 2.5% agar solution at 40°C. The agar solution was then dripped into cold oil to form spherical gel beads. Gels were fixed in the flow cell with two entomological-specimen needles. During concentration measurements in the gel, mineral medium supplied with 100 mol rnp3 sodium acetate and the desired concentration of nitrate was pumped through the flow cell. Before each experiment, the gels were incubated in this medium to achieve steady state. The temperature in the flow cell was maintained at 30’ C. Calculations. The concentration gradients in gels were calculated by numerical solution, using a grid method, of the equation
353
D,(l/r2)
(d/dr) (r2ds/dr) = Vmaxs/Km +s
(1)
where D, is the effective diffusion coefficient, s the substrate concentration, r the distance from the centre, V_ the maximum reaction rate and K, the Michaelis constant; I&, was assumed to be 0.006 mol rnp3 [9]. D, was determined by fitting the time-dependent diffusive uptake by initially solute-free gel beads from a bulk solution with a known volume, using the calculation method of Crank [lo]; nitrate concentration changes in the bulk were measured with a nitrate-selective macroelectrode (Orion). V,, was measured in batch experiments; the nitrate consumption was measured by using the nitrate macroelectrode. Results and discussion The response of the microelectrodes to concentrations of 10-‘-10-5 M potassium nitrate in demineralized water was linear with a slope of 55 mV/ log [ KNO,] (Fig. 1) . The response time was 30 s and the drift of the signal was l-3 mV h-l. Addition of different amounts of 2nitrophenyl octyl ether or PVC to the ion exchanger resulted in worse behaviour. The selectivity factors for chloride and nitrite were 0.006 and 0.06, respectively, which are of the same order as specified by Orion for macroelectrodes (0.004 and 0.04, respectively). Therefore, selectivity factors for other ions are expected to be almost the same as specified by Orion. The electrodes were stable for at least 5 h. For each experiment, freshly prepared electrodes were used. The electrode signal was not influenced by the velocity of the liquid. Although the bacterial cultures were not defined, their behaviour was similar mV
- log[NO;l Fig. 1. Calibration graphs for nitrate microelectrodes in ( 0 ) demineralized water and ( 0 ) mineral medium with 0.1 M sodium acetate.
354
to that of pure cultures. In both anaerobic and aerobic cultures, the cells were uniformly rod-shaped, while the concentrated suspensions had a red appearance. The V,,, of the gels with anaerobically grown cells was 0.06 mol mm3s-l and D, was 22 x lo-” m sm2.Figure 2 shows the measured and calculated nitrate gradients in gels, incubated at different nitrate concentrations. Under anaerobic conditions, the nitrate concentration inside the particles gradually decreased towards the centre. The calculated and measured gradients fit very well, indicating that the measurements with microelectrodes are accurate. Gas formation inside the gels occurred after 3-4 h of incubation. Two hours after switching from anaerobic to aerobic conditions, the nitrate gradients were unchanged. Hence in these bacteria, nitrate can serve as terminal electron acceptor under aerobic conditions for at least 2 h. In a control experiment, cell growth and gradient measurements were done aerobically. Under these conditions, no nitrate consumption was measured and no differences in nitrate concentration in the gels could be detected. It is concluded that the anaerobic nitrate consumption is probably due only to denitrifmation. In the medium used, ammonium served as the nitrogen source for cell growth [ 111, and therefore assimilatory nitrate consumption did not occur. The denitrifying ability of these bacteria is not constitutive and the induction NO, I 04
(mallm’)
L
Fig. 2. Nitrate gradients in gels with immobilized bacteria. Measurements were made in mineral medium with 0.1 M sodium acetate (pH 8.0) and supplied with (a) 0.4, (b) 4.3 and (c) 43 mol/ m3 KN03. Both bacterial growth and gradient measurements were made under (0) anaerobic and ( 0 ) aerobic conditions. Solid lines indicate calculated gradients.
355 INO; 0
0.01
hollm’ I 0.02
0.03
Fig. 3. Nitrate gradient in lake Vechten sediment.
of the denitrifying enzymes is inhibited by oxygen. However, once the enzyme system is formed under anaerobic conditions, the nitrate consumption is not inhibited by oxygen. Figure 3 shows a nitrate gradient in silty sediment of a freshwater lake. In this sediment, the nitrate penetration depth is less than 1.3 mm. Because the nitrate conversion in this sediment occurs in a very narrow zone, only devices with a high spatial resolution, such as microelectrodes, can give precise and direct information. More detailed studies with nitrate-selective microelectrodes on freshwater sediments will be described elsewhere [ 121. The electrode cannot be used for studies in marine environments because of its sensitivity to chloride. However, it is valuable in studies on freshwater environments with natural nitrate concentrations, on model systems with nitrifying and denitrifying bacteria and on biofilms from wastewater plants. The authors thank Per Staugaard (University of Amsterdam, Department of Chemical Engineering) for the use of his computer program.
REFERENCES 1 2 3 4 5
Y.S. Chen and H.R. Bungay, Biotechnol. Bioeng., 23 (1981) 781. N.P. Revsbech, J. Sorensen and T.H. Blackburn, Limnol. Oceanogr., 25 (1980) 403. N.P. Revsbech and B.B. Jorgensen, Adv. Microb. Ecol., 9 (1986) 293. D. de Beer and J.C. van den Heuvel, Talanta, 35 (1988) 728. D. Ammann, Ion-Selective Microelectrodes: Principles, Design and Application, Springer, Berlin, 1986.
356 6 7 8 9 10 11 12
W.McD. Armstrong and J.F. Garcia-Diaz, Fed. Proc., Fed. Am. Sot. Exp. Biol., 39 (1980) 2851. T.E. Cappenberg, Antonie van Leeuwenhoek; J. Microbial. Serol., 40 (1974) 285. J.-P.R.& Sweerts, V. St. Louis and T.E. Cappenberg, Freshwater Biol., in press. R. Riemer and P. Harrem&s, Prog. Water Technol., 10 (1978) 149. J. Crank, The Mathematics of Diffusion, Clarendon Press, Oxford, 1973. R.O. Marshall, H.J. Dishburger, R. MacVicar and G.D. Hallmark, J. Bacterial., 66 (1953) 254. J.-P.R.A. Sweerts and D. de Beer, Appl. Environm. Microbial., in press.