APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1993, p. 2093-20980099-2240/93/072093-06$02.00/0Copyright © 1993, American Society for Microbiology

Nitrification and Denitrification in Lake and Estuarine SedimentsMeasured by the 15N Dilution Technique and Isotope Pairing

S0REN RYSGAARD,t* NILS RISGAARD-PETERSEN, LARS PETER NIELSEN,AND NIELS PETER REVSBECH

Institute ofBiological Sciences, Department ofMicrobial Ecology, University ofAarhus,Ny Munkegade, DK-8000 Aarhus C, Denmark

Received 17 December 1992/Accepted 13 April 1993

The transformation of nitrogen compounds in lake and estuarine sediments incubated in the dark was

analyzed in a continuous-flowthrough system. The inflowing water contained 5NO3-, and by determination ofthe isotopic composition of the N2, N03-, and NH4+ pools in the outflowing water, it was possible to quantifythe following reactions: total N03- uptake, denitrification based on N03 from the overlying water,nitrification, coupled nitrification-denitrification, and N mineralization. In sediment cores from both lake andestuarine environments, benthic microphytes assimilated N03- and NH4' for a period of 25 to 60 h afterdarkening. Under steady-state conditions in the dark, denitrification of N03 originating from the overlyingwater accounted for 91 to 171 ,umol m-2 h-1 in the lake sediments and for 131 to 182 Lmol m-2 h-1 in theestuarine sediments, corresponding to approximately 100%o of the total N03 uptake for both sediments. Itseems that high N03 uptake by benthic microphytes in the initial dark period may have been misinterpretedin earlier investigations as dissimilatory reduction to ammonium. The rates of coupled nitrification-denitrification within the sediments contributed to 10%o of the total denitrification at steady state in the dark,and total nitrification was only twice as high as the coupled process.

As a result of the increasing eutrophication of both limnicand marine environments, much attention has been directedtoward the regulating role of microorganisms on the nitrogencycle. High activities of microbially mediated nitrogen trans-formations, including mineralization, assimilation, nitrifica-tion, and denitrification, take place in sediments (Fig. 1).Particulate organic nitrogen is mineralized to NH4' in boththe oxic and anoxic layers in the sediment. Ammonium canthen be oxidized to NO3 by nitrifying bacteria in the oxiclayer of the sediment, or it can be assimilated by benthicmicrophytes or diffuse out of the sediment. The NO3produced by nitrification can, in the anoxic environment, bereduced to N2 by denitrifying bacteria and lost from theenvironment, or it can be reduced to NH4' by fermentativebacteria or diffuse out of the sediment. Nitrate and NH4+ inthe overlying water can also diffuse into the sediment andundergo the processes described above. Many techniqueshave been used to quantify these transformations, and theintroduction of 15N techniques by Koike and Hattori (14) hasmade it possible to measure nitrification and nitrate reduc-tion simultaneously in coastal sediments. A major problemwith the experiments of Koike was that both nitrification anddenitrification were underestimated because the coupledreaction of nitrification and denitrification within the sedi-ment was not accounted for. Attempts have been made tosolve this problem by using two sets of cores, one coreenriched with 15NO3- to estimate denitrification based on

NO3- from the overlying water, and another core amendedwith 15NH4' to obtain data for nitrification and couplednitrification-denitrification (18). Total denitrification wasthen calculated as the sum of the denitrification rates foundin the two cores. In contrast to enrichment with 5NO3f, the

* Corresponding author.t Present address: National Environmental Research Institute,

Vejls0vej 25, DK-8600 Silkeborg, Denmark.

'5NH4' amendments often require a significant increase inthe NH4' concentration to obtain a measurable 15MNH4percentage (11). This artificially elevated concentration ofNH4 within the active layers of the sediment results ininaccurate estimates of both nitrification and coupled nitri-fication-denitrification. Since the site of nitrification is re-stricted to the oxic layers of the sediment, i.e., a 1- to 3-mmsurface layer and the immediate vicinity of animal burrows(22), it is difficult to obtain sufficiently accurate estimates ofthe 15N labeling of the NH4' pool being nitrified.

Recently, a new isotopic pairing method has been de-scribed, by which estimates of both denitrification based on

NO3 from the overlying water and of coupled nitrification-denitrification can be obtained (16). Following addition ofenriched 15NO3- to the water above the sediments, the 15Natoms formed by denitrification of the 15NO3- can be used to"trap" the 14N atoms produced by denitrification of 14NO3-which is formed within the sediment by nitrification, and thedenitrification rate can then be calculated from the rate of29N2 and M2 formation.By using the new isotope-pairing technique and monitor-

ing the changes in concentrations and isotopic compositionsof NO3- and NH4', it was possible to estimate the rates ofgross NO3- uptake, denitrification based on NO3 from theoverlying water, nitrification, denitrification coupled to ni-trification, and N mineralization in intact sediment cores.

MATERIALS AND METHODS

Sediments and experimental setup. In May 1991, sedimentsamples were collected with Plexiglas tubes (inner diameter,85 mm) from the freshwater lake Vilhelmsborg S0 and fromthe estuary Norsminde Fjord. The localities are situated nearAarhus, Denmark, and descriptions of the sampling sites are

given elsewhere (13). The lake and estuarine sedimentsconsisted of mud and fine sand, respectively, both rich inorganic material. Benthic microalgae had colonized the

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EN03 N2

.INOi I

CD

C,)x0

C.)x0Co

FIG. 1. Vertical cross-section of sediment colonized by benthicmicroalgae. The sediment is composed of an oxic layer and ananoxic layer. Numbers refer to processes: 1, mineralization; 2,nitrification; 3, assimilatory nitrate reduction; 4, assimilation; 5,denitrification; 6, DNRA.

surface of both sediments. In the laboratory, the sedimentcores were carefully transferred to 350-ml glass chambersand connected to a continuous-flowthrough system. Theexperimental setup and procedures used were essentially thesame as described by Binnerup et al. (2). The sediment wascontained in a glass chamber in which the overlying waterwas continuously renewed from a reservoir containing eithersynthetic fresh water (15) or seawater (9), supplementedwith 107 ,uM NO3 having a 15N labeling of 87.0% + 0.2(standard deviation). The water flow rate was 114 + 3 ml h-'for the two estuarine chambers and 49 + 3 and 124 + 3 mlh-1 in the two lake chambers. The sediments were illumi-nated (100 microeinsteins m-2 s- 1) by a mercury lampthrough the glass lids of the incubation chambers. A gas-mixing system ensured that the 02 concentration in thereservoir and inflowing water was low during the light hoursand high during the dark hours to maintain 02 at approxi-mately 100% of atmospheric saturation in the outflowingwater. The gas-mixing system also kept the concentration ofN2 constant throughout the experiment (357 + 6 ,uM in thelake chambers, and 325 + 7 p,M in the estuarine chambers).The water overlying the sediments was continuously mixedby rotation (60 rpm) of a 2.5-cm Teflon-coated magnet, andthe water phase turnover rate was 1 to 3 h-1. The incubationtemperature was 10.3 + 0.5°C. The outflowing water wasconducted through a 50-ml flask which was heated to about75°C to strip the gases from the water. Gas samples fordetermination of the isotopic composition of N2 were ex-tracted with a high-pressure liquid chromatography syringethrough a butyl rubber stopper in the degassing flask. Sam-ples for determination of the concentrations and 15N isotopecontents of NO3- + N02 and NH4' in the outflowingwater were collected in 20-ml glass vials. 02 concentrationsin inflow and outflow were measured regularly by use ofoxygen microsensors (20) to determine the 02 uptake of thesediment.

In the present experiment, lake and estuarine sedimentscolonized by benthic microphytes were preincubated for 7days while exposed to 12-h-light and 12-h-dark diurnalcycles. After the last light period, the chambers werewrapped in black plastic and further incubated for 5 days intotal darkness. By use of the procedure described above, itwas now possible to determine the following processes as afunction of the dark incubation period: total NO3 uptake,nitrification, coupled nitrification-denitrification, denitrifica-

tion based on NO3- from the water phase, and N mineral-ization.

Analytical procedures and calculations. Gas samples con-taining N2 originating from denitrification were analyzed fortheir content of 29N2 and 3`N2 isotopes in a mass spectrom-eter (VG-Isogas, Middelwich, England). Bacterial denitrifi-cation with an enrichment culture was used as a bioassay forconversion of 14NO3 and `5NO3 to N2 gas composed of28N2, 29N2, and 30N2, which was subsequently analyzed bymass spectrometry. The "5N labeling of NO3- could then becalculated from the ratio between 29N2 and 30N2 in theanalyzed gas. The bacterial reduction was carried out on2-ml samples contained in 3.4-ml butyl rubber-stopperedblood collection tubes (23). The 14N/'5N isotopic composi-tion of NH4' in the outflowing water was determined afterall water samples from the steady-state dark period (ca. 200ml) had been pooled into a glass flask. Ammonium wasremoved from the solution by microdiffusion (3) at 40°C andtrapped in a tin capsule containing purifued A1203 whichpreviously had been acidified with H2SO4 (5 mmol of H2SO4per g of A1203) and dried. By diffusing standards with aknown concentration and enrichment, the efficiency of am-monium removal was found to be greater than 98%, and nodiscrimination between 14NH4+ and "5NH4+ was observedsince the enrichments obtained corresponded to the stan-dards (data not shown). After incubation, the tin capsulecontaining the diffused NH4' was run on a C/N analyzer(model 1500, Carlo Erba) in line with the mass spectrometerto obtain the isotopic composition. Concentrations of NO3-+ NO2- were determined on a flow injection analyzer(Tecator, Hoganas, Sweden) by the method described byGrasshoff et al. (9). The NH4+ concentration was deter-mined manually by the method of Bower and Hansen (4).Net fluxes (F) of nutrients and gases over the sediment

surface were calculated by the general flux equation F = (Ce- C,)V/A, where Ce and Ci are the effluent and influentconcentrations, respectively, V is the flow rate, andA is thesurface area of the sediment. This equation gives the actualfluxes only under steady-state conditions, since there is adelay between the actual exchange over the sediment-waterinterface and the monitoring of this change due to the 1- to3-h turnover time of the water phase in the chambers. Amomentary change in flux will thus appear as a gradualapproach toward a new equilibrium over a time interval ofapproximately 6 h.Gross NO3- uptake and efflux of unlabeled NO3- origi-

nating from nitrification were estimated from the isotopicdilution of the labeled NO3- (14, 18):

ECi(0.366 - i) V

U= -Ce -

0.366 - e A(1)

Ci(e -i)l V

L0.366 - e] A (2)where U is the gross NOf uptake, R is the flux of NO3- outof the sediment, e and i are the '5N-labeled fractions ofNO3- in the effluent and influent water, respectively, and0.366 the '5N content of the nitrified ammonia.

Rates of denitrification based on '5NO3f (D15) were cal-culated as follows:

D15 =(14Nl5N)+2(15Nl5N) (3)

and denitrification based on 14NO3- was calculated by use ofthe isotope-pairing technique (16) as follows:

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NITRIFICATION AND DENITRIFICATION IN SEDIMENTS 2095

1100

105

100

_ 95-a6Z 90

85

80

N3O inflow90

89

88

.................................................................... 8715NO0- inflow

86

85

29N2 outflow

z02'a

-20 0 20 40 60 80 100 120

Time (h)

FIG. 2. Concentration and '5N (atoms percent of NO3- in theinflowing water (dotted lines) and the outflowing water of thecontinuous-flowthrough system from lake chamber 2. The shadedarea represents the last diumal cycle in which the sediments wereilluminated at 100 microeinsteins m-2 s-1. Incubation was per-formed in the dark for the remaining 120 h of the experiment.

- 14MN5-D14 2(15N5N D15 (4)

where 15N15N and 14NM5N are the net fluxes of the twoisotopic dinitrogen species 30N2 and 29N2, respectively.Total denitrification, Dtotai, was calculated as the sum of D15and D14, and denitrification based on NO3- from the water(D,) was calculated from the following equation:

DW=D51Ie (5)

where e, as mentioned above, is the 15N-labeled fraction ofthe NO3- in the effluent water. Denitrification based onNO3- produced in the sediment by nitrification (Dj) wascalculated as follows:

Dn=(D14+D15)-Dw (6)

Dissimilatory nitrate reduction to ammonium (DNRA) insteady state was calculated from the following equation:

[(Ce C-) &-0.366)1 V

[ n JA

3,0-

j 2,5-c 2,0-z

. 29N2 (inflow)

O30N2 (outflow)

I 3°N2 (inflow)

20 40 60 80 100 120

Time (h)

FIG. 3. Concentration of 29N2 and 30N2 in the inflowing water(dotted lines) and outflowing water of the continuous-flowthroughsystem from lake chamber 2. The shaded area is as for Fig. 2.

The concentrations of 29N2 and 30N2 in inflowing andoutflowing water, respectively, from a lake chamber arepresented in Fig. 3. The concentrations of both 29N2 and3°N2 were observed to increase after the chamber wasdarkened. Following the initial increase, caused by theturnover time of the water within the chamber, an increase inboth isotopic species was observed until approximately 50 hafter darkening of the chambers. After that time, the con-centrations of 29N2 and 30N2 were observed to be in steadystate.The calculated rates of NO3- uptake and denitrification

based on NO3- from the overlying water are shown in Fig.4. Steady-state rates of NO3 uptake were approached in thelake and estuarine sediments 50 and 20 h, respectively, afterdarkening of the chambers. Total NO3 uptakes were cal-culated to be 174 and 102 Rmol m-2 h-1 in the lake chambersand 188 and 146 Rmol m-2 h-1 in the estuarine chambers. Atsteady state in the dark, denitrification accounted for 89 to

LakeNO3 uptake

(7)

where Ce and C' are the concentrations of NH4' in outflowand inflow, respectively; y and 0.366 are the '5N-labeledfraction of NH4+ in outflow and inflow, respectively; n is the15N-labeled fraction of NO3- that is reduced to NH4+; Visthe flow rate; and A is the surface area of the sediment. Itwas assumed that the 15N-labeled fraction of NO3- reducedto NH4+ was the same as that reduced to N2.

RESULTS

The concentration of NO3- as well as of '5NO3- (atomspercent) in the outflowing water from lake chamber 2 isillustrated in Fig. 2. After darkening of the chamber, theNO3- concentration increased steeply within the first 6 h asa response to the 1-h turnover time of the water within thechamber. Subsequently, a slow secondary change in theconcentration was observed until approximately 50 h afterdarkening of the chamber. On the other hand, 15NO3-(atoms percent) was observed to decrease initially and thenstabilize (after approximately 50 h).

-100 Ot.Dw0 ..........*****~

E -20 0 20 40 60 80 100 120

SE

600-Z 500- Estuary

400 . \ NO3 uptake300

200- .... ..... -..............

O .. ..............100-

-20 0 20 40 60 80 100o 120

Time (h)

FIG. 4. Temporal variation of the rate of NOf- uptake and therate of denitrification of nitrate from the water overlying thesediment (Dw) in lake chamber 2 and in an estuarine chamber. Theshaded area is as for Fig. 2.

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1 20 ... .::.......::::::::::::::::::::::::...................

,..................

...-.,.,.,,:... :,,

20- ..: .:..:...80 .............U-...................o -20 020.....

Lake

'4NO3 flux out

Dn

40 60 80 100

250-,,

200,

150

'100

50 ~~~~~~Lake20 0 20 0 20 40 60 80 100 120

x

z 120 ::::::: :::::::::::

100 e '4NO flux ou..t EstuaryN 3' fux out

60...................

40 , Dn o

FIG.::.5. Temorl vaito of th raeol0 xotfo h

sediment to the overlying water and the rate of coupled nitrification-denitrification (Dn) in lake chamber 2 and in an estuarine chamber.The shaded area is as for Fig. 2.

98% of the total NO3- uptake in the four incubation cham-bers.The gross export of N from the sediments as NO3-

produced by nitrification and of N2 produced by couplednitrification-denitrification is illustrated in Fig. 5. In the lakechambers, steady-state rates of N2 production were obtainedafter 6 to 8 h in darkness (6 and 14 ,umol m-2 h-1). Constantrates of NO3- export from the lake sediments were obtainedbetween 30 and 40 h after darkening, and the rates for thetwo chambers were 42 and 10 umol m-2 h'-. In the estuarinechambers, steady-state rates in coupled nitrification-denitri-fication were also observed soon after darkening, and therates for the two chambers were 30 (Fig. 5) and 26 ,umol m-2h-1. Steady-state rates of 15NO3- uptake in the two estua-rine chambers were calculated to be 17 and 12 umol m-2h-1.The fluxes of NH4' from the sediments to the overlying

water are shown in Fig. 6. In both sediments, steady stateswere reached approximately 50 to 60 h after the chamberswere darkened. At steady state, the fluxes of NH4+ out ofthe sediments were 188 and 103 umol m-2 h-1 in the lakechambers and 84 and 144 ,umol m-2 h-1 in the estuarinechambers. The rates observed corresponded to an approxi-mately 100-fold increase in NH4' efflux for the duration ofthe dark incubation. The 15N labeling of the NH4+ in theoutflowing water in the dark period was measured to 2.38atom% in the estuarine chambers and to 2.57 atom% in thelake chambers.

DISCUSSIONIsotopic stability of the NH4' pool. In our calculations we

have assumed that the 15N labeling of NH4' in the aerobicnitrification layer was constant throughout the experiment.This implies that there should be a negligible input of15N-labeled NH4+ from dissimilative nitrate reduction toammonium (DNRA) or from phytobentic excretion andsubsequent ammonification of "N-containing organic com-

pounds. Since the benthic microalgae had been assimilating

~250-i5......................200 .. ..........

150 ...........

100

500 g ,,--.Estuary

20 0 20 40 60 80 100 120

Time (h)FIG. 6. Temporal variation of the rate of NH4' export from the

sediment to the overlying water in lake chamber 2 and in anestuarine chamber. The shaded area is as for Fig. 2.

'5N03- for 7 days before the prolonged dark period de-scribed here, an input of 15N-labeled nitrogen compounds tothe nitrification zone could be expected as a result of thedeath and lysis of algae. If '5NH4' were produced insignificant amounts, it might be nitrified to '5NO3 and thendenitrified to 29N2 or 30N2. Such an extra input of '5NO3would result in an artifactual decrease in the rate of couplednitrification-denitrification, and this decrease would becomemore and more pronounced during the time course of theexperiment when more '5NH4' became available. This is indisagreement with our observation of steady-state rates lessthan 8 h after darkening of the sediment.

Effect of darkness. When light was switched off, benthicphotosynthesis ceased and the system underwent a changefrom net 02-producing to 02-consuming conditions. Withinthe first 8 to 10 min in the dark, diffusion and consumptionchanged the 02 gradient of the sediment surface (21) andthere was an uptake Of 02 from the overlying water. With theapplied turnover time, this led to steady-state 02 concentra-tions in the water within 6 h and to almost stable sediment 02profiles even more rapidly (1 to 2 h). The denitrification zonewas located immediately below the oxic-anoxic interface (2,17), and since the diffusion coefficient of NO3 is very closeto that of 02, the NO3- profile and hence the denitrificationrate can also be assumed to become stable after 1 to 2 h. The'5N2 flux out of the sediment caused by denitrification wasactually stabilized within 4 to 6 h (Fig. 4; there was a slowsecondary change in flux after the initial very fast establishedquasi-equilibrium), and most of the delay in stabilization ofthe N2 flux was actually caused by the 1- to 3-h turnover timeof the water within the chambers. Nitrification should alsobe stable within a few hours if the 02 conditions within thesediment were the only regulating factor and likewise theNO3 flux between sediment and water. However, a steadystate in NO3 uptake was not obtained until 24 to 60 h afterdarkening, and, furthermore, the nitrate uptake was muchlarger than the total denitrification activity over that period.The large NO3 uptake after several hours in the dark can beexplained only by phytobenthic assimilation. The uptake of

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inorganic nitrogen by microalgae incubated in the dark hasbeen reported previously (1, 5, 6).

Total N03 uptake, denitrification based on N03 from thewater, and DNRA. In the lake sediment, a gradual decreasein phytobenthic NO3 uptake in the beginning of the darkperiod coincided with a corresponding gradual increase ofdenitrification based on N03 from the overlying water. Inthe estuarine sediment, denitrification of water phase N03-reached a steady state within approximately 5 h (i.e., almostimmediately when corrected for the water phase turnover)whereas total NO3 uptake did not decrease to a steady stateuntil 24 h after darkening of the sediment. Therefore, for theestuarine sediment, there did not seem to be any competitiveeffect of phytobenthic NO3 uptake on denitrification basedon NO3 from the water phase, suggesting that denitrifica-tion of water phase N03- in this sediment was regulatedprimarily by the oxygen conditions.At steady state in the dark (i.e., after 6 h), denitrification

was the major sink for NO3 and contributed 89 to 98% ofthe total NO3- uptake in the lake sediments and 90 to 97% inthe estuarine sediments. This is consistent with data ob-tained previously from the same localities (2) but disagreeswith the measurements performed by J0rgensen and S0-rensen (12, 27). According to the latter measurements,denitrification accounted for less than 30% of the total NO3uptake, while the remaining 70% was attributed to DNRA.In their experimental setup, the determination of denitrifica-tion, based on the acetylene block technique, was performedless than 6 h after sampling. Rates of DNRA were thencalculated as the difference between total N03 uptake anddenitrification. Since our data show that NO3 uptake bybenthic microphytes may continue at a high rate for manyhours after darkening of the sediment, it is clear that a simplesubtraction of denitrification from total NO3 uptake will notgive a realistic estimate of DNRA in microphyte-inhabitedsediments. After 6 h in the dark, denitrification contributedonly 25 and 40% of the total NO3 uptake in the investigatedlake and estuarine sediments, respectively (Fig. 4); the restcould be attributed to assimilative uptake and not to DNRA.Calculations of DNRA in the dark steady-state periodshowed that the maximum activity of DNRA could notexceed 5% of the total N03 uptake by the sediment in anyof the sediments investigated. In earlier investigations ofdenitrification by use of the acetylene inhibition technique,rates of water phase NO3 denitrification have often beenunderestimated because of incomplete inhibition of N20reduction (2, 7, 8), and this underestimation also contributedto an overall overestimation of DNRA rates by a simplesubtraction of measured denitrification rates from the totalNO3- uptake.

Coupled nitrification-denitrification. Coupled nitrification-denitrification in both sediment types (Fig. 5) reached asteady state after a few hours of darkness and thus seemed tobe regulated by the extent of the oxic surface layer, whereascompetition for nitrogen between bacteria and benthic mi-croalgae was of minor importance. In the steady-state darkperiod, coupled nitrification-denitrification accounted for 3to 14% of the total denitrification in the lake sediments and14 to 17% in the estuarine sediments. In accordance with ourdata, it has previously been found that the coupling betweennitrification and denitrification accounted for 6 to 9% of totaldenitrification when the concentration of NO3 in the over-lying water was 133 to 141 ,uM (18). It was also found thatwhen the NO3- concentration in the water phase was low (5,uM), the coupling between nitrification and denitrificationaccounted for a large fraction (72%) of the total denitrifica-

tion (18). Data from Rysgaard et al. (23a) show the sametrends.

Nitrification. During steady-state dark conditions, denitri-fication of N03 from the overlying water accounted foralmost 100% of the total uptake of NO3 by the sedimentand there was no uptake of N03- by benthic microalgae. Itwas therefore possible to calculate the rate of nitrification byadding the 14NO3 flux out of the sediment to the rate ofcoupled nitrification-denitrification. Rates of nitrificationwere calculated to be 24 to 48 ,umol of N m2 h-1 in the lakesediments and 47 to 39 ,umol of N m-2 h-1 in the estuarinesediments. Our rates of nitrification, measured in the estua-rine sediments, are within the range found by use of theN-serve technique in similar sediments (10). Likewise, ourestimates are in good agreement with rates of nitrificationobtained by the low-level acetylene block technique (26).Our data indicate that it is important that sediments colo-nized by benthic microphytes be incubated in the dark untilthe uptake of NO3 by the algae has ceased, when nitrifica-tion is to be estimated by use of ordinary NH4' or N03 fluxtechniques. Otherwise, the continuing uptake of NH4' andN03 by the algae will result in an underestimation of therate of nitrification. Another substantial point is that rates ofnitrification estimated during dark incubation probably donot correspond to the actual in situ rates of nitrification,since changes in pH and concentrations of NH4', 02, andCO2 as a result of the photosynthetic activity are not takeninto account. To estimate in situ rates of nitrification, it isimportant to obtain rates during the daytime as well as atnight, and at present we have no examples of reliablenitrification estimates when the light-induced assimilatoryuptake is high. However, it is important to stress thatreliable data for in situ nitrification in sediments containingno benthic microphytes can be obtained by the techniquesdescribed in this paper.

Nitrogen mineralization and budget. N mineralization atsteady state in the dark can be estimated as the sum ofnitrification (N2 from coupled nitrification-denitrificationplus the efflux of 14N03- [Fig. 5]) and the efflux of NH4'(Fig. 6). Rates obtained fall in the range of 127 to 235 ,umolofN m-2 h-1 in the lake chambers and 131 to 182 ,mol ofNm-2 h-1 in the estuarine chambers. Whether the increasedflux of NH4' should be attributed to the decreasing algaluptake of NH4+, to a degradation of the algal biomass afterdarkening of the sediments, or to a combination of thesefactors is hard to say. Further experiments are necessary totrace the fate of assimilated N as a function of environmentalvariables (duration of dark incubations, grazing, resuspen-sion, burial, etc.).A calculation of the nitrogen budget during steady-state

dark conditions shows that approximately 100% of the N03diffusing to the sediment from the overlying water wasremoved through denitrification in both sediments. Nitrogengas, formed by denitrification, accounted for 43 to 48% of thetotal efflux of inorganic N in the lake sediments and 50 to68% in the estuarine sediments. The data are consistent withpreviously reported data (24, 25) which indicate that denitri-fication in coastal marine sediments generally accounts for20 to 75% of the benthic efflux of N. It was also suggestedthat limnic sediments showed a higher capacity for nitrifica-tion and coupled nitrification-denitrification than did marinesediments and that the N2 efflux in the limnic systems variedfrom 75 to 100% of the total N efflux (24, 25). However, wewere not able to demonstrate such a difference between ourlimnic and estuarine sediments.The applied "5N-tracer technique has the advantage that

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very accurate estimates of nitrification, total denitrification,and N mineralization can be obtained from nondestructiveexperiments conducted with the same sediment core. This isa clear advantage over earlier 15N-tracer measurements ofnitrification and denitrification (18), in which two sedimentcores were needed to obtain the same data and estimates ofcoupled nitrification-denitrification could be obtained onlyafter addition of 15NH4' to total NH4' concentrations farabove in situ levels. When the isotope-pairing technique isused, '5NO3- is the only tracer to be added, and theconcentration can be held at, or only slightly above, in situlevels. It is furthermore easy to extrapolate rates withelevated NO3- concentrations to those representative ofnatural conditions (16) if the natural NO3 concentrationsare too low to run a successful experiment.

ACKNOWLEDGMENTS

We thank Louise Ziegler and J. C. Kondrup for construction ofglass equipment, Preben S0rensen for skilful technical assistance,Anna Haxen for linguistic corrections, T. H. Blackburn for con-structive help with mass spectrometry, and P. B. Christensen forhelpful discussions.

This study was supported by EEC MAST Programme contract0020 and by the Danish National Environmental Research Institute.

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2. Binnerup, S. J., K. Jensen, N. P. Revsbech, M. H. Jensen, and J.S0rensen. 1992. Denitrification, dissimilative reduction of nitrateto ammonium, and nitrification in a bioturbated estuarine sedi-ment as measured with 15N and microsensor techniques. Appl.Environ. Microbiol. 58:303-313.

3. Blackburn, T. H. 1979. Method for measuring rates of NH4'turnover in anoxic marine sediments, using a 5N-NH4' dilutiontechnique. Appl. Environ. Microbiol. 37:760-765.

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