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 wasanalyzed 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 onNO3- 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 onNO3 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 aregiven 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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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 + 0.5C. The outflowing water wasconducted through a 50-ml flask which was heated to about75C 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 40C andtrapped in a tin capsule containing purifued A1203 whichpre