APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUly 1994, p. 2467-2472 Vol. 60. No. 70099-2240/94/$04.00+0

Determination of 15N Abundance in Nanogram Pools of NO3-and N02- by Denitrification Bioassay and Mass Spectrometry

OLE H0JBERG,l* HENRIK SAABY JOHANSEN,2 AND JAN S0RENSEN'Microbiology Section, Department of Ecology and Molecular Biology,1 and Physics Laboratory, Department of

Mathematics and Physics,2 The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark

Received 9 March 1994/Accepted 6 May 1994

Suspensions of two strains of Pseudomonas aeruginosa (ON12 and ON12-1) were used to reduce N03- andN02-, respectively, to N20. The evolved N20 was quantified by gas chromatography with electron capturedetection, and the 15N abundance was determined by mass spectrometry with a special inlet system andtriple-collector detection. Sample gas containing unknown N20 pools as small as 0.5 ng of N was analyzed byuse of a spike technique, in which a reference gas of N20 of natural '5N abundance was added to obtain enoughtotal N for the mass spectrometer. In N03- or N02- pools, the 15N abundance could be determined in samplesas small as approximately 3.5 ng of N. No cross-contamination took place between the N03- and N02- pools.The excellent separation of N03- and N02- pools, small sample size required, and low contamination riskduring N20 analysis offer great advantages in isotope studies of inorganic N transformations by, e.g., nitrifyingor denitrifying bacteria in the environment.

The great interest in inorganic nitrogen cycling in bothaquatic and terrestrial environments has resulted in develop-ment of numerous techniques to study the microbial processesinvolved. These include specific bioassay techniques to extractand quantify the pools of NO3- and NO,- (4, 6). In hetero-geneous soil environments, it is important to be able to analyzevery small subsamples of soil and small pools of NO3- andNO2-, which are involved in plant uptake and microbialnitrification and denitrification. NO,- is an intermediate inboth processes and is therefore an important link betweenthem. It has been reported that accumulation of NO- andNO2- can occur in the rhizosphere (4), but whether the NO,-accumulation results from nitrification or denitrification activ-ity is not yet understood.To study soil N turnover in detail, it is necessary to use 15N

isotope techniques. In the classical methods, NO3- is chemi-cally reduced either to NO2- or N gases (15) or to NH4' (7, 8).The NH4+ may subsequently be collected by microdiffusion (5)and converted to N2 (8) before analysis. Common to thesechemical methods, however, is that cross-contamination mayoccur during the separations (8, 12). Furthermore, the amountof N required is relatively large and the methods are notsuitable for analysis of small amounts of NO- or NO2-.To facilitate work with small N03- pools, Christensen and

Tiedje (6) used a denitrifying strain of Pseudomonas chlorora-phis, ATCC 43928, to reduce '5N-labelled NO- (1 atom%15N) to N20. The authors determined the isotope compositionof NO3- pools with a precision of .0.02 atom% (two repli-cates). By comparison, Risgaard-Petersen et al. (18) deter-mined the isotope composition of relatively small NO3 pools(containing 300 ng of N) by reduction to N2 using a denitrifyingenrichment culture.The present study couples a denitrification bioassay tech-

nique (4, 6) to a sensitive mass-spectrometric (MS) analysis of'5N in the N20 being formed. The use of pure cultures of anNO3- (and NO-)-reducing Pseudomonas aeruginosa wild-

* Corresponding author. Mailing address: Microbiology Section,Department of Ecology and Molecular Biology, The Royal Veterinaryand Agricultural University, Rolighedsvej 21, DK-1958 FrederiksbergC, Denmark. Phone: 45 35 28 26 30. Fax: 45 35 28 26 24.

type strain (ON12) and an NON2--reducing P. aeruginosamutant strain (ON12-1) provides a tool to separate the NO-and NOJ- pools in very small environmental samples (4). Byreducing the pools to N20 rather than to N2, we avoid the riskof background contamination by atmospheric N2. In this study,we demonstrate the use of the bioassay combined with 15Nanalysis of the N20 pools formed. If the amount of N20 in thesample gas is too small (<100 ng of N) for direct determinationof '5N abundance in the MS, the sample is spiked with areference gas containing N,O of natural '5N abundance andthe '5N content of the original sample is calculated (1). Thesensitive detection of N2O by gas chromatography and subse-quent 15N analysis in the MS will facilitate studies of Nturnover in very small N03- and NO,- pools in soil and otherenvironments.

MATERIALS AND METHODS

Standards of N03-, N02-, and N20. Standards (2 mM) of98 atom% '5N-enriched NaNO3 and NaNO2 (CambridgeIsotope Laboratories, Woburn, Mass.) were made in distilledwater. To obtain lower enrichments, these standards weremixed with 2 mM solutions of NO3 and NO,- of natural '5Nabundance. Distilled water was added to obtain standardsolutions of lower concentrations.

Standard mixtures of N2O were made by growing a cultureof P. chlororaphis ATCC 43928 under anaerobic conditions intryptic soy broth (Bacto; Difco Laboratories, Detroit, Mich.)which contained 10 mM concentrations of '5N-enriched NO3 -'The NO3- is reduced to N,O because the organism is unableto reduce N,O to N2 (6). The '5N-labelled N2O was purified bypurging the headspace gas through a column with trappingmaterial for CO2 (Carbosorb, 12/20 mesh; Merck, Dorset,United Kingdom) and H2O (Drierite; W. A. Hammond,Xenia, Ohio). High-purity helium (99.9995%) was used for thepurge, and the N20 was subsequently collected in a cold trap(liquid N2). The cold trap was closed off and connected to a120-ml, helium-flushed bottle. The final N2O concentrationwas approximately 2.5% (vol/vol). Dilution series of N,O inhelium were made in 3.5-ml blood-collecting tubes (Venoject;Terumo Europe N. V., Leuven, Belgium).

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Denitrification bioassay of N03- and N02- pools. Thebioassay was performed essentially as described by Binnerupand S0rensen (4), with wild-type (ON12) and mutant(ON12-1) strains of P. aeruginosa to reduce N03- and NO2-,respectively. The NO3- (and NO2-)-reducing ON12 strain wasgrown to stationary phase in citrate minimal medium with 10mM N03- and was then washed and resuspended in the spentgrowth medium to a higher cell density (-10"' cells per ml).The NO2--reducing ON12-1 strain resembles the C103-resistant mutants reported by other workers (10, 20). Themutant strain is thus unable to synthesize an active nitratereductase when grown in the presence of C103-. In the presentwork, the ON12-1 strain was grown in the presence of 1 mMrather than 10 mM C103- as used in the original assay.Stationary phase was thus reached after approximately 3 daysrather than 7 days. The ON12-1 cells were then resuspended inspent ON12 medium including 1 mM C103- to a higher celldensity (-3 x 109 cells per ml). The spent ON12 medium wascompletely free of N03-, since N03- was reduced during thegrowth of the ON12 strain. Before resuspension, the spentmedium was boiled for 5 min to strip accumulated N20 fromthe solution.Unknown samples (50 to 100 [LI) to be analyzed for N03-

and N02- concentrations including their '5N content wereplaced in 1.8-ml screw-cap glass vials with polytetrafluoroeth-ylene (Teflon)-coated silicone septa and quickly heated (95°Cfor 1 min) in a water bath to kill indigenous bacteria. The vialswere then flushed with helium and acetylene (10-kPa C,H2)was added to inhibit N20 reduction in the two P. aenuginosastrains. This was because the ability of the two strains toaccumulate N20 in the citrate minimal medium (4) was shownto depend on unknown compounds released from the butylrubber stoppers (14).When samples contained both N03- and N02-, 100 Kl (-3

x 108 cells) of strain ON12-1 was first added to reduce theNO,- pool to N20. To quantify the N20, a gastight glasssyringe (Dynatech or Hamilton) was flushed carefully withhelium and 100 pl1 of helium was injected into the sample vialin replacement of 100 pI of the headspace gas. The sample gaswas injected into a gas chromatograph (Chrompack model428) equipped with an electron capture detector and a manualbackflush system to prevent C2H2 from passing the detector.Running time was about 4 min per sample. After determina-tion of the N20 concentration, the '5N abundance in thesample was analyzed by MS (see below).

Before analysis of the N03- pool in the subsequent step, thevials were opened and heated (95°C for 5 min) in a water bathto remove the NO,---derived N20 pool. The vials were thenclosed again and flushed with helium. New C2H, (10 kPa)followed by a 100-Rl suspension (-109 cells) of strain ON12was added, and the NO--derived N,O was finally analyzed forboth concentration and '5N content as described above. Anal-ysis of standards or unknown samples containing only NO-(or N02-) was carried out in one step, with 100 Rl (-10'3 cells)of strain ON12 in the bioassay.MS. The MS was a triple collector instrument (Tracermass

model; Europa Scientific Ltd., Crewe, United Kingdom) pro-viding a determination of mass-to-charge ratios (m/z) of 44, 45,and 46. The latter represented the most abundant molecularspecies, 44N20 (14N 4NNO -,45N20 ( N N 0 andNN'4160), and 4('NO(N5N-N'- 0), respectively, and some

combinations with the '7O and 1O isotopes (see below).Gas samples (100 to 300 pul) were injected manually into the

MS with a gastight syringe after initiation of the softwareprogram (ANCA; Europa Scientific Ltd.). Behind the injectionport, the sample passed a stainless steel column (4-mm inside

diameter and 15 cm long) filled with the mixture of trappingmaterials for C02 and H20. To separate N20 from traces of02 (see below), the gas sample then passed a chromatographiccolumn (Porapak Q; 0.2 mm by 0.4 m) in an oven held at 70°C.The retention time was about 1.5 min when the inlet pressureof the helium carrier gas was approximately 150 kPa. Therunning time was 3 min per sample.When analyzing small (nanogram) pools of N20, we in-

cluded a spike addition of reference gas, which containednatural N20 (15N abundance taken to be 0.366%) to obtainadequate total N in the samples. Corrections were made todetermine the 15N abundance in the original sample gas of theunknown N20 pool (see below). Three pools of N20 were thusdefined: (i) the unknown pool, representing N20 from theoriginal N03- and NO- sample reduced in the bioassay; (ii)the spike pool, representing N20 of natural '5N abundanceadded to obtain enough total N for the MS analysis; unlessotherwise stated, the spiking gas was also used as reference gasto calibrate the instrument; and (iii) the mixed pool, represent-ing a mixture of the unknown and spike pools. In practice, weran a reference gas sample of natural N20 followed by 5 to 10samples of mixed N20 pools. The first and the last of the mixedpool samples served as references, as they were prepared withtotal N contents and '5N abundances similar (within 10%) tothe unknown ones.The injection procedure was initiated by a flush of the

gastight syringe with helium, and 100 p.l of reference gas(natural N20 in helium; 0.1 to 1% [vol/vol]) was injected intothe MS. The syringe was flushed again in helium, and 100 to300 .I1 of sample gas was withdrawn from a vial. The syringewas then quickly inserted into the bottle of reference gas, and100 1A of this gas was carefully drawn into the syringe as well.The sample gas, containing both unknown and spike N20pools, was finally injected into the MS.

Calculations of '5N abundance. The molecular species ofN20 contributing to the ion currents (1) at m/z of 44, 45, and46 are the following: 441, 14N14N'1O; 451 14N5N 1o,'5N 4N'60, and 14N'4N 170; and 46j, l5N5N 160, 14N 5N 170,'5N '4N'70, and '4N '4N'8O. This means that determinations ofthe molecular fractions (x) of the 45 and 46 species, 4-5x and 46xas defined by 451/(441 + 45i + 461) and 46"/(44I + 45I + 461), willgive higher values than expected from a random distribution ofthe common isotopes l4N, '5N, and 160 only. There mayfurther be a discrepancy between measured and expectedvalues of the molecular fractions caused by mass discrimina-tion or nonlinearity between the readings at the three MScollectors (19). To compensate for the presence of heavyoxygen isotopes and instrumental effects, we used the followingthree assumptions to obtain correct values of 45j and 46I. (i) Inthe reference gas, '4N and '5N were considered to be ran-domly, i.e., binomially, distributed. The '5N abundance wasdefined to be 0.366 atom%. (ii) It was assumed that no isotopeexchange between the unknown and spike pools of N20 duringthe MS analysis took place, i.e., the mixed pool representedtwo separate pools of N20. This means that the unknown andspike pools both had random distributions of 14N and '5Nisotopes, whereas the mixed N20 pool had a nonrandomisotope distribution. (iii) The 44j, which represented only onemolecular species (14N'4N160), constituted >99% of the totalbeam area in the reference gas and was considered to becorrectly determined in the MS.Under these three assumptions, the correct (44IC) and mea-

sured (44JIR) values of 44I in the reference gas are determinedas

44i,,? = 44ic = (1 - 15a)2 X XN2O (1)

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15ON IN N03- AND N02 POOLS 2469

where 15a is the natural l5N abundance (0.366 atom%) andXN,o is the total amount of N200 molecules without heavyoxygen isotopes. XN2O could then be calculated from equation1. Because 4N and 15N were randomly distributed in thereference gas, the correct values of 45I, and 46I, could bedetermined as

45ic = 2 X (15a) X (1-15a) XXN20 (2)

46ic = (15a)2 XXN20 (3)The differences between the correct values (45IC and 46IC)

and the measured values (45Im and 46Im) may be expressed asA45I and A46I, respectively. The latter are in turn proportionalto the total amount of N20 in the injected gas sample (seeResults and Discussion) and were used to obtain the 45I and46IC values from the 45Im and 461m values.The equations for determining isotope abundances in a

mixed pool of '5N-labelled and natural (atmospheric) N2 havebeen presented by Hauck et al. (13), Hauck and Bouldin (11),Mulvaney (16), and Arah (1). In the present study, the 15Nabundance in an unknown N20 pool, i.e., derived from NO3-or N02 pools in the bioassay, was calculated from thefollowing equation originally developed for N2 analysis (1, 2):

(46Xm - 15 a 15a.)(5am - '5aa)

The 15ap value is the 15N abundance of the unknown N20 pool.The 15aa value is the 15N abundance of the spike pool (;0.366atom%). The '5am value is the 15N abundance [(½/24 Ic +46IC)/( IC + 45IC + 46Ic)] of the mixed pool. The 46Xm value isthe molecular fraction of 46N20 [46Jc/(4 Ic + 45IC + 46IC)] in themixed pool. The 15N abundance of the unknown N20 pool('5ap) can thus be calculated from the '5N abundances of thespike pool (15aa) and the mixed pool ('5am) and the 46N20molecular fraction (46Xm) of the mixed pool.

RESULTS AND DISCUSSION

Interferences by C2H2, C02, and 02 in MS analysis of N20.Acetylene somehow resulted in a tailing of the N20 peakappearing on the monitor of the MS. To investigate whetherthis interference affected determination of the isotope compo-sition in N20, two sample series (five replicates each) of"5N-enriched N20 (-56 atom%), with or without 10-kPaC2H2, were analyzed. The measured l5N abundances (±standard deviations) were 56.097 (±0.028) atom% (with C2H2)and 56.120 (±0.042) atom% (without C2H2) for 20-ng N gassamples. The results showed that the altered shape of the N20peak had no significant effect on the determination of itsisotope composition.Carbon dioxide (m/z, 44) interferes with the MS analysis of

N20 and must be removed. This was done by passing thesample gas (helium gas purge) through the CO2 trap in the MSinlet system; the trap removed CO2 completely from samplescontaining up to 5% (vol/vol) CO2 (data not shown).When the samples contained 02, small amounts of gas

presumed to be NO2 (mlz, 46) affected the N20 analysis. Areaction between 02 and N2 at the ion source filament hasbeen reported to produce NO (m/z, 30), which interferes withN2 analysis (3, 9, 19). One solution to remove the 02 may be toinstall a reduction furnace at the inlet before the sample entersthe MS system (19). This cannot be done when analyzing N20,however, since the furnace will also reduce N20. We thereforechose to purge the sample vials and syringes with helium,

35 -

30 -

25 -

0

CLzO 20 -

M 15uz

10 -

5 -

0

0 2 4 6 8 10Total beam area (nC)

12 14 16

FIG. 1. Differences between measured (Im) and correct (I') valuesof ion currents at mlz of 45 and 46 plotted against total beam area. Thedifferences are designated A41I (0) and A46I (0), respectively. Corre-lation coefficients (r2) are 0.9997 and 1.0000, respectively.

which removed the 02 and reduced the presumed NO2 peak toan absolute minimum (data not shown). Traces of 02 appear-ing in the MS were separated from N20 by the chromato-graphic column inserted behind the injection port. If accidentalformation of NO2 still took place in the MS, this could beobserved immediately on the monitor and the sample wasrejected.MS analysis of '5N abundance in N20 pools. It has been

claimed that MS analysis of N2 should be preferred to analysisof N20 because the latter is influenced by CO2 interference(17), N20 dismutation to NO and N2 (8), and lack of sensitivity(17). Some workers have thus chosen to reduce N20 to N2 (byCu catalysis) prior to determination of the isotope composition(17). However, recent developments in the determination ofthe isotope abundance directly in N20 pools have been made,and combined gas chromatography and MS analysis of N20has become a routine (3, 19). The latter studies have dealt withunspiked samples with "5N enrichments of 10 atom% or less,which is the optimal range of 15N abundances for mostinstruments (3). The 15N abundances in the spiked samples ofthe present study never exceeded 5 atom% and were also wellwithin the optimal range (3). The spiking technique is there-fore well suited for MS analysis of samples with a small Ncontent but high "5N enrichment.

It is common practice in MS analysis to adjust the total Ncontent in reference samples according to that of the unknownsamples (19). Using the spiking technique, we found that it wasnecessary to correct for even small differences in total N(<5%) between reference and sample gases in order to obtainreproducible results. As shown in Fig. 1, the differencesbetween correct and measured ion currents in the referencegas were linearly related with the total beam areas (workingrange of 10-9 to 1.5 x 108 C) with correlation coefficients (?)of 0.9997 and 1.0000, respectively. The Al values could thus beadjusted according to the difference in total N between refer-ence and sample gases. The AI values were subsequently used

A46I

A451

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-0

E0

Aa)0C)

Q

.0

zv

U)

Un

a)

80

60

40

20

0

0 20 40 60 80 100Expected 15N abundance (atom%)

FIG. 2. Measured '5N abundances in N20 standards containingexpected '5N abundances between 10 and 98 atom%. The amount ofN in sample gas was approximately 8 ng, while the spiking was 575 rig.The regression line isy = 1.016 x x, and the correlation coefficient (r2)is 0.9996 (n = 5).

to correct the measured ion currents, 45I,, and 46m, of thesample gas. The importance of this correction procedure was

illustrated by results from a sample series (10 replicates of -4ng of N), in which the '5N abundance was determined to be30.096 (+0.302) atom% with correction of the AI values and29.231 (+2.572) atom% without correction. The precision was

much poorer without correction of the AI values.The difference in total N content between reference and

sample gases did not exceed 5% in the sample series describedabove, but the correction procedure was in fact reliable over a

much wider range of total N contents. In a different sampleseries (three replicates of -85 ng of N), the 15N abundancewas thus determined to be 67.403 (±0.079) atom% whenreference and sample gases differed less than 5% in total Ncontent and 67.280 (+0.075) atom% when the difference was

approximately 75%. The important point here is that theprecision levels, as judged by the standard deviation, are

similar in the two series. The spiking technique may thereforebe used even if the total N contents differ significantly,provided that the correction procedure is followed. However,since the spike pool usually constituted >95% of the total N inthe mixed pool, our analysis routine always resulted in samplesthat differed less than 5% in total N content.

Sensitivity of denitrilication bioassay and MS analysis. Aseries of N20 standards enriched with 10 to 98 atom% '5N wasprepared from '5N-labelled N03- by use of the ON12 strain.The 15N abundances were determined in samples of N20standards containing approximately 8 ng of N plus 575 ng of Nin reference gas (natural 15N abundance) for spiking. Figure 2shows a good agreement between expected and measuredmeans (slope = 1.016 and r2 = 0.9996) for the whole range of'5N abundances between 10 and 98 atom%. The standarddeviations (five replicates) varied from 0.387 for the 10 atom%series to 0.059 for the 98 atom% series; the standard deviationvalues are too low to be seen in Fig. 2. The results demonstratethat determination of isotope compositions by the spike tech-

nique becomes less precise as the amount of '5N decreases inthe sample.To estimate the minimum sample size required at a fixed '5N

abundance, a sample series of N20 standards (-44 atom%'5N), representing variable amounts of total N, were analyzedwith and without addition of a spike pool; spiking was with 575ng of reference gas (natural '5N abundance). When no spikepool was added, a highly enriched batch of N20 standard (98atom%) was used as the reference gas to equilibrate theinstrument. The data in Fig. 3 (upper part) show that theprecision of analysis is poor in unspiked samples when the totalamount of N in the sample gas becomes lower than approxi-mately 100 ng of N. However, when the spike pool was addedthe measured mean of approximately 44 atom% was obtainedin as little as 1 ng of N in sample gas. It was clear, however, thatstandard deviations became larger as the N content decreasedbelow approximately 10 ng of N in the sample gas. Thus, in thewhole range of N contents (1 to 100 ng of N), the spikedsamples gave much better determination of the isotope abun-dance than the unspiked ones, as judged from both themeasured means and the standard deviations.To confirm that good sensitivity could be obtained also when

the denitrification bioassay was coupled to the MS analysis,three sample series (10 replicates in each) of N03- solutionscontaining 3.5, 70, and 700 ng of N and a fixed 15N abundanceof approximately 30 atom% were reduced to N20 by the ON12strain. The total N contents of sample gas injected into the MSwere 0.8, 4, and 40 ng of N in the three series, and spiking waswith 288, 575, and 575 ng of N in reference gas (natural 15Nabundance), respectively. The results shown in Fig. 3 (lowerpart) show that the measured mean was well determined evenat the lowest sample size of approximately 1 ng of N. Thestandard deviations were also comparable to the ones deter-mined on N20 standards (Fig. 3, upper part). This indicatesthat the denitrification bioassay combined with MS analysisoperates under the same detection limits for sample size andrange of suitable '5N abundances as the MS analysis alone.

Discrimination of '5N abundances in N03- and N02-pools. To examine if there was any contamination of '5Nisotope from the N02- pool to the NO3 pool, we performedan MS analysis of 10 replicates of an N03- standard (700 ng ofN, -30 atom% '5N) mixed with an NO2 - standard (700 ng ofN, -50 atom% 15N). After the N02- was reduced by theON12-1 strain, the accumulated N2O was removed by openingthe vials before heating (95°C for 5 min) and flushing withhelium. Gas samples of the headspace were analyzed on thegas chromatograph and showed no detectable N20 (data notshown). The N03- was then reduced to N20 by the ON12strain, and the '5N abundance was determined in 40 ng of N insample gas spiked with 575 ng of N in reference gas (natural'5N abundance). The results in Table 1 (rows 1 and 2) showthat the isotope composition in the N03- standard amendedwith NO,- could be determined with a low standard deviationsimilar to that of the NO- standard without NO2-. Thisconfirms that the N02- -derived N20 was effectively removedfrom the vials. It may be noticed that the measured mean wassomewhat higher in the N02--amended series. This was not anerror of the assay, however; according to the isotope supplier(Cambridge Laboratories), the '5N-enriched N02- added tothe N03- standards may actually contain 5 to 10% (wt/wt) of'5N-labelled NO3-.

In a parallel series, we tested whether isotope contaminationfrom the NO3- pool to the NO2- pool could take place. Fivereplicate samples of an NO2- standard (700 ng of N, -55atom% '5N) was mixed with an NO- standard (7 ,ug of N,natural '5N abundance); a parallel series of '5N-labelled N02-

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47 -

46 -

45 -

44 -

43 -

Eo 42--

a)i' 41 -ccu

Z 32-

'), 31 -C',a)cn

a)E 30 -

29 -

28 -

0 20 40 60 80 100 120

0 20 40 60

Total N in sample gas (ng)

FIG. 3. (Upper part) Measured '5N abundances in N,O standards (-44 atom% '5N) containing different amounts of N in sample gas. Sampleswith (0) and without (0) spiking (575 ng of N) are shown. (Lower part) Measured '5N abundances in NO3 standards (-30 atom% '5N)containing different amounts of N in sample gas. Spiking was 288 ng of N for the sample with smallest amount of N and 575 ng of N for the others.

standard, but without NO3 was also made. The NO2 wasthen reduced to N2O by the ON12-1 strain, and the '5Nabundance was determined in 40 ng of N in sample gas spikedwith 575 ng of N in reference gas (natural '5N abundance). Theresults in Table I (rows 3 and 4) show that a high backgroundof nonenriched NO-, about 100 times higher than the NO2-standard, had no effect on determination of the isotopeabundance in the NO< pool. This confirms that the ONl2-1strain does not reduce NO3 under the assay conditions (4).

TABLE 1. Analysis of '5N abundance in NO3 and/orNO, -containing standards"

Measured '5NSourcc abundancc"

(atomrn,;)

NO3 ................................................................................... 30. 102 0.060NO3 (+NO )' .................................... 30.785 00.087NO2.................................................................................... 54,988 0.088NO (+NO.)".54.981 +0.019

" The total amount of N in NO3 or NO, standard solutions used in thedcnitritication bioassay was 70t) ng, the total amount of N in the sample gas(unknown N20 pool) analyzed in the MS was 40 ng, and the total amount of Nin the reference gas (spike N2O pool) analyzed in the MS was 575 ng.

" Measured means and stalndard deviations (n = 10 for NO3 samples; it = 5for NO, samples).

N03N standard was supplemented with NO2 (-50 atom%4 '5N) at a molarratio of 1:1. The labelled NO, contained -3.5'7r labelled NO3 (see the text).

"NO2 standatrd was supplemented with NO3 (0.366 atomk '5N) at a molarratio of 1: I()().

The denitrification bioassay thus had an excellent specificityfor both NO3- and N02- analyses.To our knowledge, the present study is the first to demon-

strate determination of '5N content in both NO- and NO,pools in the same sample. Furthermore, the combination of thedenitrification bioassay and MS analysis is a new approach toinvestigate small, nanogram pools of NO3-- and NO,, includ-ing their '5N contents, in natural samples. The assay deter-mines the '5N content in much smaller pools of NO3- and/orNO2- than has previously been reported by Christensen andTiedje (6) and Risgaard-Petersen et al. (18). From the resultspresented above, the denitrification bioassay in combinationwith MS analysis might be successfully optimized to a lowersample size limit than 1 to 10 ng of N and a lower '5Nabundance than approximately 10 atom% in the unknown N2Opool. Further improvement of the sensitivity of the presentmethod may be obtained by reducing the headspace volume inthe vials, i.e., increasing the N2O content available as samplegas. Also, the spike-to-sample gas ratio may be adjusted forparticular combinations of sample size and '5N abundance.

In this study, we have developed a spiking technique forexamination of isotope composition in small samples of rela-tively high '5N enrichment (>10 atom%) of the N20. Ris-gaard-Petersen et al. (18) used a denitrifying enrichmentculture to reduce NO3- to No for analysis of '5N abundance inthe MS. However, these authors reported a standard deviationof -0.2 atom% (three replicates) for the measured mean

abundance in NO- samples, representing a range of 10 to 99atom%. Their precision was thus poorer than that obtained in

NO3- standard

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the present study, even if approximately 40 times more NO3-in total sample N content was used. By reducing or omittingthe spiking, we believe it will be possible to analyze small poolsof NO3- and N02 at low enrichment (<10 atom%).We are presently using the methods to investigate microbial

transformation of small, '5N-labelled NO3 and N02 poolsin soil microenvironments which support a close couplingbetween nitrification and denitrification processes. In theheterogenous soil environment, the application of 15N isotopetechniques in small-scale studies will be useful to obtaininformation about N turnover and simultaneous mineraliza-tion, nitrification, and denitrification processes. Organic aggre-gates, plant residues, and the root surface environment (rhi-zosphere) are important niches for rapid N03 and N02turnover by nitrification and denitrification. The NO3 andN02- are obligatory intermediates in the processes and areimportant to analyze simultaneously when the 15N technique isused.

ACKNOWLEDGMENTS

We thank Svend J0rgen Binnerup for helpful assistance and ElfinnLarsen for critical reading of the manuscript.

This work was supported by the Danish Center for MicrobialEcology.

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