Membrane inlet mass spectrometric analysis of N-isotope labelling for aquatic denitrification studies

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1 EISEVIER FEMS Microbiology Ecology 2 (1996) Membrane inlet mass spectrometric analysis of N-isotope labelling for aquatic denitrification studies Karen M. Jensen a*b*, Mikael H. Jensen b, Raymond P. Cox a a Institute of Biochemistry, Odense University, Campusuej 55, DK-523 Odense M, Denmark b Institute of Biology, Odense Uniuersity, Odense, Denmark Received 2 January 1996; revised 6 March 1996; accepted 8 March 1996 Abstract Techniques are described for measuring the isotope distribution in dissolved nitrate and N, using membrane inlet mass spectrometry, which allows several gases to be measured in a water sample without the need for any separation steps. The isotope distribution in dissolved nitrate was measured using denitrifying Pseudomonas nauticn to reduce the nitrate to N, which was then measured by mass spectrometry. Pseudomonas nautica NCIMB 1967 was easily grown in nitrate-limited continuous culture minimising intra- or extracellular nitrate or nitrite pools, and the bioassay was tolerant of a range of salinities. The precision of the bioassay when measuring samples with high NO; contents (.5 pmol) was.5 atom%; with.1 pmol 15NO;, the precision was around.2 atom%. Differences in labelling of N, in preserved samples obtained from 15NO; incubations of water-covered sediment cores were measured on parallel samples with membrane inlet MS and GC-MS. The membrane inlet technique was accurate but the precision on ratio measurements was lower than by GC-MS. Keywords: Isotope distribution; Nitrate; 15N; Denitrification; Bioassay 1. Introduction New techniques have recently been developed for measuring microbial nitrogen transformations in aquatic sediment systems using incubations with nitrogen-15 labelled nitrate [l]. Using this approach, the rates of several processes in the nitrogen cycle can be monitored in a single experiment, simply by measuring the nitrogen isotope distribution in nitrate (or nitrite) and in N, [2-41. Since the changes involved are often rather small, the need has arisen for * Corresponding author. Tel: +45 (66) 158 6; Fax: +45 (65) ; kmj@biochem.ou.dk precise and accurate methods of analysing the isotopic composition of dissolved nitrogen compounds. For the analysis of isotope distributions in nitrate, bioassay techniques have been described in which denitrifying bacteria reduce dissolved nitrate (or nitrite) to N,O [5,6] or to N, [7]. The gases produced are captured and transported in a stream of helium or argon. Water,, and N,O are removed by passage through traps and the sample analyzed by GC-MS. The labelling of dissolved N, produced from denitrification during incubation of natural samples (e.g. sediment-water microcosms) with labelled nitrate is usually measured using the same technique. The 14N2 concentration in air-saturated water is then used as an internal standard and denitrification is calcu- 16%6496/96/$ Federation of European Microbiological Societies. All rights reserved PII SO (96)22-O

2 12 KM. Jensen et al. / FEMS Microbiology Ecology 2 ( lated from changes in the ratios N15N/ N2 and 15N2/ 4N2 in the isotope pairing technique [ 11. This report describes the use of a relatively simple, inexpensive quadruple mass spectrometer equipped with a membrane inlet, which allows dissolved gases to diffuse directly from an aqueous phase across the membrane into the mass spectrometer [8,9]. Methods are presented for the analysis of isotope distribution in dissolved NO, and N,. 2. Materials and methods 2.1. Bacteria Pseudomonas nautica NCIMB 1967 was obtained from the National Collection of Industrial and Marine Bacteria, Aberdeen, UK. The culture medium was a 1: 1 mixture of artificial seawater and a nutrient medium, autoclaved separately and then mixed. The artificial seawater contained 4 mm NaCl, 2 mm MgSO,, 2 mm MgCl,, 1 mm KC1 and 1 n&i CaCl,. The nutrient medium contained 1 mm sodium acetate, 8 mm NaNO,, 1 mm NH,Cl, 25 PM NaH,PO,, 25 PM Na,HPO,, 5 PM Fe-EDTA and 2 ml/l of trace element solution SL-6 [lo] with additional molybdenum (25 mg/l Na,MoO,.2H,O). Buffering was provided by 1 mm Tris and the initial ph of the medium was adjusted to 7. with HCl. The bacteria were stored at - 2 C (or - 8 C for longer times) in culture medium supplemented with glycerol (25% v/v). Inocula were grown in looselystoppered bottles filled with medium and used to inoculate a bioreactor [ 1 I] with a nominal volume of 1 ml. Bacteria for use in bioassays were grown at 28 C under an N, atmosphere in nitrate-limited continuous culture with a dilution rate of about.4 h-. Under steady-state conditions the ph in the reactor was 8.1 without the use of any ph control system and the cell yield was.2 g cell carbon per litre. Continuous cultures were run for several weeks until wall growth became excessive Environmental samples Sediment cores with overlying water were collected from both a sandy and muddy locality (Kiinigshafen, a small tidal embayment in the northern Wadden Sea, Germany). The cores were incubated at in situ temperature (approx. 7 C) and salinity (approx. 24%) for h in closed incubations with added 15N; (control cores were incubated without 15NO; ). Water phase samples were taken by syringe at the beginning of the experiment and after the incubations together with a thin slurry of mixed water and sediment. The samples were immediately transferred to gas-tight 8.5 ml glass bottles and preserved by addition of 2 ~1 1 M NaOH to a final ph of about 1. The samples were measured directly in the sample bottles within a few weeks. Samples for GC-MS analysis were taken in parallel and preserved as described by Rysgaard et al. [3] Membrane inlet mass spectrometry Oxygen and nitrogenous gases were measured with a Dataquad DQlOO quadruple mass spectrometer (Spectramass; Congleton, UK) with a Balzers TPH 62 turbomolecular pump (Balzers-Pfeiffer; Asslar, Germany) and equipped with a membrane inlet [8,12]. The inlet had 4 holes (.4 mm in diameter) covered by three layers of 11 pm teflon membrane held in place by narrow bore silicon rubber tubing (25 pm wall thickness; Silastic; Dow-Corning; Midland, MI). Fig. 1 shows the experimental system. All samples were added to 8.5 ml glass bottles equipped with a teflon-covered stirrer bar and placed in a closely-fitting jacket with circulating water at 3 C Sample bottle,,$-ecq Water jacket Membrane inlet Magnet Fig. 1. Schematic diagram of the measuring system. Gases diffuse from the water in the sample bottle across the membrane and into the vacuum of the mass spectrometer.

3 K.M. Jensen et al. / FEMS Microbiology Ecology 2 (1996) (or close to incubation temperature for the environmental samples). The inlet tube of the mass spectrometer passed through a rubber-lined stopper. Care was taken to close the bottle without changing the pressure or adding air bubbles. Signals at 4 m/z values were collected every second by a desktop computer and the average values at 2 or 3 s intervals were stored for later analysis. N, was measured at m/z = 28, 29 and 3 corresponding to l4 N,, 14N N and 15N,., was measured at m/z = 32. N,O was monitored at m/z = 44, 45 or 46 to check for possible interferences on the measurement of N, [ 131. The 9% response time of the mass spectrometer with the combined teflonsilicone covered inlet was about 1 min at 3 C as measured by changes in the, concentration N-isotope distribution in N, from preserved samples The preserved samples were measured for 1 min. Ratios of the mass spectrometer signals were calculated for each time interval, and the mean of the last 1 measurements (3-6 s) was used for further calculations. No corrections were made for differences in the instrumental response to the various isotopes Measurements of 15N in N, by GC-MS Labelled N, samples were measured by GC-MS as described by Rysgaard et al. [3] and Nielsen [l]. The N, was extracted from the water into a He headspace. The headspace gas was then injected into a gas chromatograph and N, isotopes measured by an in line mass spectrometer. H,O, N,O and, were removed from the gas by in line traps. The precision on ratio measurements was about 2 X 1W Bioassay for nitrate isotope measurements For standard measurements of the 15N atom% in nitrate, cells from the bioreactor were harvested by centrifugation (1 X g, 1 min) and resuspended in the culture medium without nitrate to approximately l/3 of the original volume. The bacterial suspension was allowed to go anoxic and remove any residual nitrate or nitrite, and part of the suspen- sion was used to rinse the sample bottles and the inlet. Just before analysis, both the bacterial suspension and the sample to be assayed (prewarmed to 3 C) were shaken vigorously to equilibrate dissolved, and N, with the atmosphere. Then, 6.3 ml sample and 2 ml aerobic bacterial suspension were mixed in a 8.5 ml bottle, which was quickly placed in the measuring chamber (3 C) and closed by the inlet. Signals at m/z = 29, 3 and 32 were monitored for 1 to 15 min Calculations A mixture of 14N-nitrate and 15N-nitrate will be converted by denitrification into a mixture of 14N14 N, 15N14N and 15N15N. If there is random pairing of the isotopes and the fraction of 15N-nitrate is x, the ratio between 15N14N and 15N15N (m/h) is given by (2-2 x)/x. This can be written m/2 h = (1 - x)/x; rearranging gives x = l/( 1 + m/2 h). Correcting with the instrumental response factor f required to adjust for the different sensitivities at m/z = 29 and m/z = 3 gives: I5 N atom% in NO, = 1 A( m/z29) + 2fA( m/23) where A(m/z29) and A(m/z3) are the increases in the signal compared to the background (before denitrification started). Due to diffusion effects the measured atom percentage increases with time, and linear regression was used to calculate the initial value (discussed later) Determination of the instrumental response factor The 15N isotope fraction of an added standard y is given by Eq. (1) and Eq. () and can be written in the form y = l/( 1 + m /2fh ) where m and h are the measured increases in the intensity of the MS signal at m/z = 29 and m/z = 3. The apparent isotope fraction without correction, y, can be written y = l(1 + m /2 h ) and substitution then gives f = (y(l - y >)/( y (l - y)>. The value of f under the experimental conditions used was determined with a nitrate standard containing 99.5 atom% N-KNOs (Europa Scientific Ltd., Crewe, UK). Two stocks of 1 PM nitrate in 3% (1)

4 14 K.M. Jensen et al./fems Microbiology Ecology 2 (1996) NaCl were made, one with 14N and one with 15N. The precise concentrations of nitrate were measured by autoanalyser [ 141. Solutions containing between 9.82 and 99.5 atom% 15N were made from weighed amounts of the two stocks and measured in triplicate. 3. Results The denitrifier Pseudomonas nautica NCIMB 1967 was chosen because we wanted a bioassay that used a bacterium which was easy to handle and was tolerant of a wide range of salinities. By growing the bacteria in nitrate-limited continuous culture, we minimised interference from intra- or extracellular nitrate or nitrite. The denitrification rates of the cells were reproducible and the bioassay time could be kept at a minimum thereby reducing interferences from other nitrogen transforming reactions. The membrane inlet was equipped with a teflon membrane. Silicone membranes are permeable to many gases and hydrophobic organic molecules, whereas teflon is much more selective towards small nonpolar gas molecules [ The addition of 3 X 11 pm teflon to the original silicone covered inlet reduced the signal due to water to 6%, while the signals due to N, and, were reduced to 76% and 63% of the values obtained with the silicone membrane alone. Ethanol is readily measured when using the silicone membrane, but no signal from 2 mm ethanol could be detected when the teflon silicone combination was used Nitrate isotope measurements The results of a typical experiment are shown in Fig. 2, lower panel. As soon as the denitrifying bacteria were added to the sample, they started consuming the O,, and only the last part of this process was monitored. While the signal from, decreased, the signals at m /z = 29 and 3 stabilized and the background values were determined from the last 4 s of measurement before the production of N, began. When nitrate reduction was complete, the gaseous Time (mid Fig. 2. Lower panel: MS-signals due to, (321, 14N15N (29) and N2 (3) during bioassay of three successive identical samples (1 PM nitrate). Addition of new samples is shown by arrows. A value of 8 on the ordinate corresponds to about 22 PM, and 3 PM N?. Upper panel: The 15N atom percentage calculated using Eq. (1). The five values immediately before the highest N,-signal and the five succeeding values (closed symbols) were extrapolated back to zero (diamonds) giving values of 79.94, and atom%.

5 K.M. Jensen et al. / FEMS Microbiology Ecology 2 (1996) products was followed for several minutes before a new sample was added. The signals at m/z = 29 and 3 did not stabilize after denitrification was complete but decreased with time due to diffusion of the gases into the mass spectrometer. The decrease in signal at m/z = 29 was faster than at m/z = 3 because N14N diffuses faster than the heavier 15N, and has a higher natural background (which is also taken up by the mass spectrometer). The result was an increasing apparent atom percentage calculated from Eq. (1) (Fig. 2, upper panel), which was corrected by linear regression of the measured percentage back to time zero Instrumental response factor The mean instrumental response factor f was 1.3 &-.12 for 1,uM nitrate solutions containing from 2 to 9 atom% 15N. Results from very low or very high isotope labelling were not used due to their lower precision. The manufacturer s value for enrichment of the 15N-nitrate was 99.5 atom% and this value was used in the calculations. If this value is changed to or atom%, the calculated value of f only changes to 1.29 or 1.32, respectively Isotope distribution Fig. 3 shows the accuracy and precision of the bioassay. A linear regression between added nitrate Nitrate (pmoll Sodium lb.41 Fig. 4. Effects of nitrate and sodium concentration on bioassay results. Values shown are means and standard deviations for 3 or 4 replicates. The results are normalised to the values shown by the open symbols which both had an absolute value of 8.6 atom% jn. (A) Nitrate concentration was varied by diluting different volumes of sample containing 8 ym nitrate to 8.3 ml with the bacterial suspension such that the final cell concentration was the same in all cases. The final sodium concentration in this series of experiments varied between.31 M and.41 M. (B) Sodium concentration was varied by using different volumes of bacterial suspension diluted to 8.3 ml with nitrate solution without added NaCl, or by increasing the NaCl content of the samples. Total nitrate concentration varied between 4 PM and 7 PM. and measured values gives y =.9982 x +.9 ( r =.9999), not statistically different from y = x (t-test, (Y =.5; statistical tests from Zar [16]), with no trends in the residual plot. If the results are grouped and weighted according to their errors, then y =.9993x -.2. The precision of the method decreases with decreasing 15NO; concentration, as was seen from the high standard deviation with 1 atom% 15NO-. The precision was also poorer near 1 atom% 15NO- The standard deviation of the measured atom pe?ri centage of 9 parallel samples containing 8 PM nitrate with 8 atom% 15N, was.5 atom%. I I I a 1 15N in added nitrate [atom%) Fig. 3. Results obtained using the bioassay on samples with different isotope labelling in the added nitrate. The total nitrate concentration was maintained at 1 PM. Horizontal bars show the precision (standard deviation for 3 measurements) Nitrate concentration The effects of changing nitrate concentration on the accuracy and precision of the bioassay are shown in Fig. 4A. Various volumes of nitrate-containing standard were diluted with different volumes of cell suspension to obtain a series of samples with different amounts of nitrate but the same bacterial cell density. This procedure was adopted to minimise the error due to trace amounts of nitrate in distilled water. It was observed that over the range of nitrate

6 16 K.M. Jensen et al./ FEMS Microbiology Ecology 2 (1996) contents measured here ( pmol), the time for the N, concentration to reach a maximum did not change. The precision decreased as the nitrate concentration was lowered. Using the Kruskal-Wallis rank test (a =.5) the means of the seven groups did not show a significant difference Sodium chloride concentration Since some strains of Pseudomonas nautica are sodium dependent [17] the response of the bioassay to changing NaCl concentration was investigated. The time required for oxygen to be consumed and denitrification to be completed increased at the lowest and highest Na+ concentrations measured, but the changing NaCl concentration had no significant effect on either the accuracy (cr =.5) or the precision (Fig. 4B) Stability of measuring conditions No significant effects on the results were seen when the assay temperature was increased from 25 C to 35 C or when the stirring rate was changed. However, there was a small effect when the assay volume was increased from 8.3 ml to 29 ml (corresponding to f = 1.15). To investigate whether the results obtained depended on the rate of reaction, parallel samples of 6 )(LM nitrate (approx. 8 atom% 15N) in 25% NaCl were denitrified using different bacterial densities so that the time for N, to reach a maximum varied between 22 and 6 s. If the samples were divided into 4 groups with 6 to 8 samples per group there was no significant difference (ANOVA test or Krnskal-Wallis rank test, CY =.5). However, the variances were different (Bartletts test). The best precision was obtained with times between 28 and 35 s where the value was 8.11 f.5 atom% (n = 7). If results are calculated from the ratio A(m/z = 29)/A(m/z = 3) without including the corrections for diffusion and instrumental response an improved precision was often obtained. However, using this method of calculation on the results in Fig. 3 gave a curved residual plot with deviations of up to.25 atom%. This more precise calculation method may be advantageous if the aim is to measure small changes in isotope labelling and accuracy is a secondary consideration. z N W 1 I Dithionite Control 1 : POCP am OP.$ * w i $i& mm 25 m (m/z x (m/z = 28) Fig. 5. Relation between the signal at m/z = 3 and the product of signals at m/z = 28 and m/z = 32 in samples containing the natural abundance of N,. Samples were prepared either by lowering the dissolved oxygen concentration (from approx..24 mm) using different amounts of dithionite or by using untreated samples with differing dissolved, concentrations. Only about 3% of the signal at m/z = 3 in the absence of Oa is due to N,, the rest is background of unknown origin Isotopes in free Nz We also investigated whether membrane inlet mass spectrometry could be used for the analysis of 15N in N,. The ratios 14N15N/14N, and 15N,/14N2 are of particular interest since 14N2 can be used as an internal standard. Natural samples contain variable concentrations of CO, which would interfere with the signal at m/z = 28 [9]. Preserving the samples at high ph, however, lowers the concentration of CO, to negligible values. There is a possibility of interference of the m/z = 3 signal by, [6,18], probably due to NO+ ions formed from N, and Of [19] inside the MS. If this is the case we might expect a correlation between the product of the signals at m/z = 32 and m/z = 28 and the signal at m/z = 3 as shown in Fig. 5. The, concentration was varied by using samples with naturally differing, contents (control incubations) or by depleting the oxygen by adding sodium dithionite. Both data sets gave a similar trend and the relationship can be used to correct for the interference due to the assumed formation of NO+. Differences between the two sets of data would unveil interferences not caused by,.

7 K.M. Jensen et al./fems Microbiology Ecology 2 (1996) z 1-s - o -, A@9/28) : --, A(3/28) N15N/15N15N ratio in the N, produced, and we show that a simple quadruple mass spectrometer can be used instead of the commonly used GC-MS for the determination of nitrogen isotope distributions in N,* g & lo-* Use of membrane inlet 1-s - 1-6, : 95 % confidence, I I I 1 o-6 1-s o-s 1-Z GC-MS Fig. 6. Correlation between the measurement of N, isotope ratio changes by membrane inlet MS and by GC-MS. Samples were removed in parallel from NO;-incubations of two types of natural sediment samples (sandy sediment and muddy sediment). The bold lines are the linear regression lines for the two sets of isotope ratio data. A 1:l ratio is indicated by the dotted line. During analysis by membrane inlet MS, the molecules have to diffuse through a diffusive boundary layer of liquid, then through the membranes of silicone and teflon and finally through the vacuum of the mass spectrometer. Theoretically, the ratio between the diffusion coefficients of 15N14N and N, for all four phases are equal to or less than the inverse square root of the masses of the molecules, i.e [ However, the instrumental response factor f for the mass spectrometer used was found to be 1.3 &-.12 suggesting that f needs to be determined separately for each combination of mass spectrometer and measuring system used. Fig. 6 shows a comparison of the enrichment in isotope labelling of dissolved N,, measured by GC- MS and membrane inlet MS. An excellent correlation was found (y = 1.16~ X 1m5 and y =.9985x X lop6 for changes in 15N14N/14N2 and 5N,/14N,, respectively). Fig. 6 also shows the 95% confidence intervals, which indicate that changes in 5N 4N/ 4N, below 1m4 cannot be measured precisely. The data presented are from incubations from two locations: a sandy and a muddy marine sediment. The N, production rate (i.e. the denitrification rate in pmol - N per square meter of sediment per hour) calculated from the measurements on GC-MS and membrane inlet MS was 1.5 k 1.8 and (n = 9), respectively, for the sandy sediment and and (n = 7), respectively, for the muddy sediment. 4. Discussion The method described here has two novel features. We describe the use of the denitrifying bacterium Pseudomonas nautica grown in nitrate-limited continuous culture for the bioassay of nitrogen isotope distribution in nitrate by determination of 4.2. Analysis of dissolved N2 We found excellent agreement between the GC- MS and the membrane inlet MS methods when changes in the labelling of dissolved free nitrogen during 15NO; incubations were measured (Fig. 6). The precision of measurements of isotope ratios was lower than with the GC-MS techniques normally used for this type of analysis, but it was acceptable compared to the heterogeneity encountered in aquatic sediments studied by the isotope pairing technique [l,ul The 5N3- bioassay The bioassay described here has the advantage compared to previous methods [6,7] in that both the reduction of, and the production of N, could be followed on line making it possible to minimise the assay time and to quickly detect any problems. Due to the short assay time (a few minutes compared to 12 hours) and the use of pure cultures of Pseudomonas nautica instead of denitrifying enrichment cultures it is not necessary to add nitrification inhibitors as were used by Risgaard-Petersen et al. [7]. Another advantage is that the background signals are

8 18 K.M. Jensen et al./fems Microbiology Ecology 2 (1996) measured on each individual sample. This means 1967, but we did not see significant accumulation of flushing the samples is not needed as in alternative N,O during development of the bioassay or in earlier methods [6,7]. The assay is highly reliable when investigations [24]. This may be because the ph was using Pseudomonas nautica grown in continuous buffered to ph 7. and the time of exposure to low culture., was short. In order to obtain stable background signals, it is The bioassay described here is comparable in important that denitrification is inhibited by, until accuracy to that described by Risgaard-Petersen et al. this has fallen to a low concentration., also interferes with the signal at m/z = 3. P. nautica strain NCIMB 1967 does not produce N2 in significant amounts until the, level is below about 1 PM (Fig. 2, lower panel). It is, however, difficult to measure the exact level of, which inhibits denitrification, since P. nauticu strain NCIMB 1967 has a tendency to clump and to attach to the surfaces. This may create anoxic microniches and initiate denitrification, and a low level of N, production was observed at concentrations up to 3 PM,. P. nautica strain 617 has been reported to reduce nitrate and nitrite at O2 concentrations up to 12 and 67 PM, respectively, [23], which makes this strain less suitable for the bioassay. It is desirable that the production of N, starts abruptly and important that the apparent K, for the denitrification of NO, to N, is low, keeping the denitrification time short. For P. nautica strain NCIMB 1967, the apparent K, for nitrate was less than 1 PM under similar conditions [24]. The initial nitrate concentrations in the assay was kept in the range lo-1 PM, since higher concentrations would make the denitrification time undesirably long compared to the time required to attain anoxia. It is also important to avoid substantial accumulation of N,O, since fragment ions of this gas would interfere with the measurement. Several authors have reported N,O accumulation at low, tensions during denitrification under various conditions [ Bonin et al. [28] found that, inhibited the N,O reductase irreversibly in resting cells of P. nautica strain 617, and such behaviour would clearly be destructive to the assay. The ph might also have an effect on the accumulation of intermediates. Kuc era et al. [29] and Thomsen et al. [13] have reported transient accumulation of N,O at low ph by Purucoccus denitrzficans and Thomas et al. [27] found the same in resting cell suspensions of two Pseudomonas aeruginosa strains. We have not specifically investigated the effect of, or ph on Pseudomonas nautica strain NCIMB [7]. Comparing the precision, our assay was better when measuring on samples with high NO, amounts (.5 pmol):.5 atom% compared to.2 atom%. The precision was similar when measuring in the.1-.2 pmol l5 NO; range, especially around 8 atom%, but poorer when analysing low enrichments or amounts of NO,. A new bioassay [6] using nitrous oxide reductase deficient Pseudomonas aeruginosa strains allows precise measurements on even smaller amounts of 15N, or 15N,. In conclusion, the membrane inlet mass spectrometer techniques described here are well-suited for the isotope analyses (15N, bioassay and 15N,) used in the isotope pairing method. Some of the limitations could probably be overcome by using larger sample volumes so that the loss of N, from sample by diffusion into the MS would be less important. Alternatively, the permeability of the inlet to the mass spectrometer could be lowered, in which a case more sensitive apparatus is required. The conditions of the 15NO; incubations can in many cases be modified to circumvent the limitations of the membrane inlet MS, and the method described here has the advantage of being simple, reliable and requiring only relatively inexpensive instrumentation. Acknowledgements We wish to thank Silvia Pelegri, Nils Risgaard- Petersen and Lars Peter Nielsen for their invaluable technical assistance with the GC-MS measurements, and Erik Kristensen for constructive discussions. The project was supported by grants from SWAP (Sylt Wattenmeer Austausch Prozesse) and Direktar Ib Henriksens Fond. References [l] Nielsen, L.P. (1992) Denitrification in sediment determined from nitrogen isotope pairing. FEMS Microbial. Ecol. 86,

9 KM. Jensen et al./ FEMS Microbiology Ecology 2 (1996) [2] Pelegri, S.P., Nielsen, L.P. and Blackbum, T.H. (1994) Deni Degn, H., Cox, R.P. and Lloyd, D. (1985) Continuous meatrification in estuarine sediment stimulated by the irrigation surement of dissolved gases in biochemical systems with the activity of the amphipod Corophium uolutator. Mar. Ecol. quadruple mass spectrometer. Methods Biochem. Anal. 31, Prog. Ser. 15, [3] Rysgaard, S., Risgaard-Petersen, N., Sloth, N.P., Jensen, K. and Nielsen, L.P. (1994) Oxygen regulation of nitritication and denitrification in sediments. Limnol. Oceanogr. 39, Risgaard-Petersen, N., Rysgaard, S., Nielsen, L.P. and Revsbech, N.P. (1994) Diurnal variation of denitrification and nitrilication in sediments colonized by benthic microphytes. Limnol. Oceanogr. 39, [5] Christensen, S. and Tiedje, J.M. (1988) Sub-parts-per-billion nitrate method: use of an N,O-producing denitrifier to convert NO; or 15NO; to N,O. Appl. Environ. Microbial. 54, [6] Hojberg, O., Johansen, H.S. and Sorensen, J. (1994) Determination of N abundance in nanogram pools of NO; and NO; by denitrification bioassay and mass spectrometry. Appl. Environ. Microbial. 6, Risgaard-Petersen, N., Rysgaard, S. and Revsbech, N.P. (19931 A sensitive assay for determination of 14N/ sn isotope distribution in NO;. J. Microbial. Methods 17, Jensen, B.B. and Cox, R.P. (19881 Measurement of hydrogen exchange and nitrogen uptake by mass spectrometty. Methods Enzymol. 167, Cox, R.P., Geest, T. and Thomsen, J.K. (199) Kinetics of denitrification in Paracoccus denitrificans: Measurements using N-nitrate and a mass spectrometer with a permeable membrane inlet. Mitt. Deutsch. Bodenkundl. Ges. 6, [lo] Malik, K.A. (1983) A modified method for the cultivation of phototrophic bacteria. J. Microbial. Methods 1, [l 11 Iversen, J.J.L., Nielsen, M. and Cox, R.P. (1989) Design and performance of a simple, inexpensive, modular laboratory scale bioreactor. Biotech. Education 1, Cox, R.P. (1987) Membrane inlets for on-line liquid-phase mass spectrometric measurements in bioreactors. In: Mass Spectrometry in Biotechnological Process Analysis and Control (Heinzle, E. and Reuss, M., Eds.), pp Plenum Press, New York Thomsen, J.K., Geest, T. and Cox, R.P. (1994) Mass spectrometric studies of the effect of ph on the accumulation of intermediates in denitrification by Paracoccus denitrificans. Appl. Environ. Microbial. 6, [14] Armstrong, F.A.J., Stearns, C.R. and Strickland, J.D.H. (19671 The measurement of upwelling and subsequent biological processes by means of the Technicon Autoanalyzer and associated equipment. Deep-Sea Res. 14, Zar, J.H. (1984) Biostatistical Analysis. 2nd edn. 718 pp. Prentice-Hall International Inc., London Baumann, L., Baumann, P., Mandel, M. and Allen, R.D. (19721 Taxonomy of aerobic marine eubacteria. J. Bactetiol. 11, Stevens, R.J., Laughlin, R.J., Atkins, G.J. and Presser, S.J. (1993) Automated determination of nitrogen-15-labeled dinitrogen and nitrous oxide by mass spectrometry. Soil Sci. Sot. Am. J. 57, Giese, C.F. (1966) The reaction O+ +N, + NO+ +N. Adv. Chem. Ser. 58, Tyrrell, H.J.V. and Harris, K.R. (1984) Diffusion in Liquids. Butterworths Monographs in Chemistry. 448 pp. Cambridge University Press, London. [21] Philibert, J. (1991) Atom movements-diffusion and mass transport in solids. Les Editions de Physique pp. Les ulis, France Chapman, S. and Cowling, T.G. (197) The mathematical theory of non-uniform gases. 3rd edn pp. Cambridge University Press, London Bonin, P. and Gilewicz, M (1991) A direct demonstration of co-respiration of oxygen and nitrogen oxides by Pseudomonas nauticu: some spectral and kinetic properties of the respiratory components. FEMS Microbial. Lett. 8, Jensen, K.M. and Cox, R.P. (1992) Effects of sulfide and low redox potential on the inhibition of nitrous oxide reduction by acetylene in Pseudomonas nautica. FEMS Microbial. Lett. 96, [25] Betlach, M.R. and Tiedje, J.M. (1981) Kinetic explanation for accumulation of nitrite, nitric oxide, and nitrous oxide during bacterial denitrification. Appl. Environ. Microbial. 42, [26] Davies, K.J.P., Lloyd, D. and Boddy, L. (1989) The effect of oxygen on denitrification in Paracoccus denitrificans and Pseudomonas aeruginosa. J. Gen. Microbial. 135, [27] Thomas, K.L., Lloyd, D. and Boddy, L. (1994) Effects of oxygen, ph and nitrate concentration on denitrification by Pseudomonas species. FEMS Microbial. Lett. 118, Bonin, P. Gilewicz, M. and Bertrand, J.C. (1989) Effects of oxygen on each step of denitrification on Pseudomonas nautica. Can. J. Microbial. 35, KuEera, I., Matyagek, R. and Dad& V. (19861 The influence of ph on the kinetics of dissimilatory nitrite reduction in Paracoccus denitrificans. Biochim. Biophys. Acta 848, 1-7.