Biphasic Behavior of Anammox Regulated by Nitrite and Nitrate in an Estuarine Sediment

The production of N₂ gas via anammox was investigated in sedimentslurries at in situ NO₂^– concentrations in the presenceand absence of NO₃^–. With single enrichment above 10 µM¹⁴NO₂^– or ¹⁴NO₃^– and ¹⁵NH₄⁺, anammox activity wasalways linear (P < 0.05), in agreement with previous findings.In contrast, anammox exhibited a range of activity below 10µM NO₂^– or NO₃^–, including an elevated responseat lower concentrations. With 100 µM NO₃^–, no significanttransient accumulation of NO₂^– could be measured, andthe starting concentration of NO₂^– could therefore beregulated. With dual enrichment (1 to 20 µM NO₂^–plus 100 µM NO₃^–), there was a pronounced nonlinearresponse in anammox activity. Maximal activity occurred between2 and 5 µM NO₂^–, but the amplitude of this peakvaried across the study (November 2003 to June 2004). Anammoxaccounted for as much as 82% of the NO₂^– added at 1 µMin November 2003 but only for 15% in May 2004 and for 26 and5% of the NO₂^– added at 5 µM for these two months,respectively. Decreasing the concentration of NO₃^– butholding NO₂^– at 5 µM decreased the significanceof anammox as a sink for NO₂^–. The behavior of anammoxwas explored by use of a simple anammox-denitrification model,and the concept of a biphasic system for anammox in estuarinesediments is proposed. Overall, anammox is likely to be regulatedby the availability of NO₃^– and NO₂^– and the relativesize or activity of the anammox population.

INTRODUCTION

Top

Introduction

Recently, it has been demonstrated that in both aquatic sedimentsand anoxic water basins denitrification is not the only pathwayfor N₂ formation (3, 8, 19, 22). Anaerobic ammonium oxidation(anammox) can couple the reduction of NO₂^– to the oxidationof NH₄⁺, producing N₂ gas, and hence act as an additional pathwayfor the removal of N from aquatic ecosystems, yet what regulatesthe significance of anammox for N removal in aquatic systemsis largely unknown (4). Given that N is often limiting for primaryproduction in aquatic ecosystems, further understanding of thisnovel shunt in the N cycle is essential.

Research using microelectrodes and measurements of solute exchange,for example, O₂, NO₃^–, and NH₄⁺, has established thatthe concentrations of solutes in sediment pore water and overlyingwater are at a steady state (11, 13, 14). Nitrate concentrationscan reach 1 mM in the overlying water of hypernutrified estuariesand can still be present in the top 0 to 1 cm of sediment at400 µM; however, the magnitude of both of these poolsis seasonal (6, 9). In contrast, the concentrations of NO₂^–in both the overlying water and sediment are usually 2 ordersof magnitude less than those of NO₃^– and seldom exceed5 µM, though in comparison to NO₃^–, data for NO₂^–are scarce. Studies using NO₂^– microsensors, biosensors,or fine-scale porewater extraction methods have indicated thatnet NO₂^– production occurs in anaerobic sediment as aresult of NO₃^– reduction (15, 17). In addition, the firststage of nitrification at the aerobic-anaerobic interface producesNO₂^–, which, depending on the conditions for nitrificationand the overall demand for NO₂^–, may diffuse into theunderlying anaerobic layers (5). The critical point is thatat a steady state, NO₂^– and NO₃^– can coexist inanaerobic sediments at concentrations separated by orders ofmagnitude.

To date, measurements of anammox in sediments have relied onanaerobic slurries or anaerobic bag incubations enriched withNO₃^–, NO₂^–, or both at concentrations higher thanthose in the environment, especially for NO₂^– (19, 22).In anaerobic slurries, in the direct presence of NO₃^–,anammox is reliant on the initial reduction of NO₃^– toNO₂^– by the total NO₃^–-reducing community. Nitraterespiration (NO₃^– $->$ NO₂^–) results in NO₂^– asan actual end product of metabolism which is exported from thecell. In contrast, NO₂^– is an intermediary of the denitrification(NO₃^– $->$ NO₂^– $->$ NO $->$ N₂O $->$ N₂) and dissimilatory reduction ofnitrate to ammonium (NO₃^– $->$ NO₂^– $->$ NH₄⁺) pathways. Allof this intermediary NO₂^– may "leak" from the cell intothe surrounding environment. The loss of NO₂^– is in partgoverned by the kinetics of each respective enzyme, the speciesof NO₃^–-reducing bacteria, and the quality of the organicsubstrate (1, 2). The linear formation of ²⁹N₂ from ¹⁵NH₄⁺ and¹⁴NO₂^– in anaerobic slurries suggests that NO₂^–is nonlimiting for anammox, and hence the pathway of NO₂^–formation would be irrelevant to the actual significance ofanammox, but these observations (19, 22) come from experimentsthat were not specifically designed to examine the behaviorof anammox at representative in situ NO₂^– concentrations(<10 µM). Even in the presence of NO₂^– and theabsence of NO₃^–, the anammox community will have to competeagainst the largely heterotrophic NO₃^–- and NO₂^–-reducingcommunity, which will use NO₂^– just as efficiently inthe absence of NO₃^– (22).

Although we cannot reproduce a steady state in batch sedimentslurry experiments, unlike intact sediment cores, the purposeof this study was to investigate the fate of NO₂^– andthe behavior of anammox at representative sediment NO₂^–concentrations in the presence and absence of NO₃^–. Herewe show that anammox exhibits pronounced nonlinear behaviorat concentrations below 10 µM NO₂^– in the presenceof 100 µM NO₃^–. In addition, a simple anammox-denitrificationmodel and the concept of a biphasic system for anammox in estuarinesediments are proposed.

MATERIALS AND METHODS

Top

Materials and Methods

Sample sites and sediment slurry preparation.
Sediment samples (oxic and suboxic layers from 0 to 2 cm) werecollected from the intertidal flats at low tide from an areain the Thames estuary where significant anammox had previouslybeen measured (site 2 [22]), stored in plastic bags, and returnedto the laboratory within 1 h. Anaerobic ammonium oxidation wasassayed by measuring the production of ²⁹N₂ in anaerobic slurriesas previously described (22). Sediment slurries were enrichedby injection through the septa, using microliter syringes (Hamilton,Bonaduz, Switzerland), with concentrated stocks of ¹⁵NH₄Cl (120mM ¹⁵NH₄Cl [99.3 ¹⁵N atom%]) (Sigma-Aldrich, Poole, United Kingdom)to give a final concentration of 500 µM ¹⁵NH₄⁺ (approximately60% labeling of the NH₄⁺ pool) and either Na¹⁴NO₂ or Na¹⁴NO₃(stock concentrations of 4 and 93 mM, respectively) (VWR InternationalLtd., Lutterworth, United Kingdom) to give a range of concentrations(see below). Sediments from this site are capable of reducingNO₃^– at approximately 100 µM h^–1, and an overnightpreincubation ensured that all traces of potential oxidantswere removed before experimental manipulations commenced (22).All incubations were carried out on rollers in the dark in aconstant-temperature room at 15°C. Following incubation,gas samples were collected (1 ml) using a gas-tight syringe(SGE Gastight Luer Lock syringe; Alltech Associates Ltd., Carnforth,Lancashire, United Kingdom) and stored in an inverted water-filledgas-tight vial (12-ml Exetainer; Labco Ltd., High Wycombe, UnitedKingdom). For determinations of the labeling of the NH₄⁺ pool,pore waters were recovered from both enriched and nonenrichedreference slurries by centrifugation of the slurry, filtered(0.2-µm-pore-size Minisart Plus filter; Sartorius UK Ltd.),and frozen (–20°C) until later analysis.

Anammox activity with single NO₂^– or NO₃^– enrichment.
For the first set of experiments, slurries were enriched with¹⁵NH₄⁺ and then independently with either ¹⁴NO₃^– or ¹⁴NO₂^–to give final concentrations of 1, 2.5, 5, 8, 10, 12, 16, and20 µM, incubated overnight, and treated as described above.

Nitrate reduction and nitrite accumulation.
In order to determine the effect of the NO₃^– enrichmentconcentration on NO₂^– accumulation, we enriched preparedsediment slurries with ¹⁴NO₃^– at 100, 200, 400, and 600µM and sacrificed independent slurries every hour for4 h. The microbial activity was inhibited by injecting ZnCl₂(500 µl of a 50% [wt/vol] solution) through the septumof the serum bottle, and the pore waters were recovered andtreated as described above for later analyses of NO₃^–and NO₂^– (see below).

Anammox activity with dual NO₂^– and NO₃^– enrichment.
Having established the concentration of NO₃^– at whichNO₂^– did not transiently accumulate in the slurries (100µM NO₃^– [see Results]), we prepared dual-enrichmentexperiments as outlined above and added isotopes from concentratedstocks to give the following final concentrations, in this order:¹⁵NH₄⁺ at 500 µM; ¹⁴NO₃^– at 100 µM followedby mixing for 5 min on rollers (during which time approximately8% of the NO₃^– pool would have turned over); and finally,¹⁴NO₂^– at 1, 2.5, 5, 8, 10, 12, 16, or 20 µM. Subsequentdual-enrichment experiments were performed with 25, 50, 75,and 100 µM ¹⁴NO₃^– and with ¹⁴NO₂^– at just5 µM. The slurries were incubated overnight and treatedas described above.

Analysis.
All nutrient analyses (NO₃^–, NO₂^–, and NH₄⁺) wereperformed by use of a continuous-flow autoanalyzer (San⁺⁺; Skalar,Breda, The Netherlands) and standard colorimetric techniques(7). Salinities were measured using a hand-held refractometer.Water contents, specific gravities, and porosities were determinedfrom the dry and wet weights of known volumes of sediment. Samplesof the headspace (50 µl) from the gas-tight vials wereinjected using an autosampler into an elemental analyzer interfacedwith a continuous-flow isotope ratio mass spectrometer calibratedwith air, and the mass charge ratios for m/z 28, 29, and 30nitrogen (²⁸N₂, ²⁹N₂, and ³⁰N₂) were measured (Delta Matt Plus;Thermo-Finnigan, Bremen, Germany).

Calculation of total anammox activity.
Assuming that the ¹⁵NH₄⁺ pool turns over at the same rate asthe ambient ¹⁴NH₄⁺ pool, the total anammox N₂ production canbe calculated from the production of ²⁹N₂ and the proportionate¹⁵N labeling of the NH₄⁺ pool, determined by accounting forthe difference from nonenriched reference slurries (19, 22).Anammox can be measured by labeling either the electron donor(¹⁵NH₄⁺) or acceptor (¹⁵NO₃^– and ¹⁵NO₂^–) pool (19),and we have previously demonstrated a high level of agreementbetween these two techniques (22). For this study, however,only the NH₄⁺ pool was labeled, and our measures of anammoxare therefore expressed relative to total NO₃^– and NO₂^–reduction and not total gas formation.

Modeling the behavior of anammox.
A simple model was developed in EcoS (version 3.37, N.E.R.C.;Plymouth Marine Laboratory, Plymouth, United Kingdom) to examinethe behavior (shape of response) of anammox in the dual presenceof NO₂^– and NO₃^– in a slurry system with a mixedpopulation of anammox and denitrifying bacteria. The model isbased around the assumption that anammox, as an autotroph, makesup a relatively small proportion ( ${theta}$ ) of the total NO₃^–and NO₂^– reducer population (<5%) and can only useNO₂^– as an oxidant, whereas the denitrifying populationwill use NO₃^– preferentially but will swap to NO₂^–when NO₃^– falls below a certain threshold (22). In addition,for the sake of simplicity, the additional potential sourceof NO₂^– for anammox resulting from denitrification wasnot included (see Results for rationale); hence, as in the aboveexperiments, the mixed population was started with only externalsources for both NO₂^– and NO₃^–.

The changes over time of NO₂^– and NO₃^– concentrationsin the pore water, subject to consumption by a bacterial populationwith the proportion ${theta}$ of anammox and 1 – ${theta}$ of denitrifiers,were assumed to be governed by the following equations:

(1)

(2)
where M is the numberof bacteria per liter. The rates of consumption of NO₂^–by denitrifiers as a function of [NO₂^–] were assumed tobe given by the following equations:

(3)

(4)
In these equations, f_d and g_d are classic Michaelisforms with half-saturation constants k_NOx and maximum ratesV_MxD (µmol N bacterium^–1 day^–1). The followingfunction acts to block the denitrifier reduction of NO₂^–when NO₃^– is available:

(5)
Itgives a linear decrease in rate for increasing [NO₃^–],with no uptake of NO₂^– occurring when [NO₃^–] >[NO₃^–]_crit, in this case, 100 µM NO₃^–. Therate of consumption of NO₂^– by anammox was modeled asa biphasic process, determined by the following equation:

(6)
where V_MA (µmol N bacterium^–1 day^–1)is a maximal processing rate, and the modifying functions f_pand f_m, with 0 < f_p and f_m < 1, are given by the followingequations:

(7)

(8)
Thefunction f_p yields an optimum rate when [NO₂^–] = l_N, withinhibition for [NO₂^–] above and below this rate (see Fig.3). The function f_m, which is Michaelis in form, yields a constantreduction rate for NO₂^– at concentrations more than sixtimes K_m. The combination yields a peak of $~$ V_MA when [NO₂^–]= l_N and smoothly decreases to a proportion, ${varepsilon}$ , of the maximalrate at high NO₂^– concentrations. We emphasize that itwas not the purpose of this modeling exercise to precisely replicatethe conditions and dimensions of our experimental slurries,but rather just to explore the overall shape of the anammoxactivity. While the half-saturation constants for anammox anddenitrification are realistic, at 2.5 µM (reference 4and data reported here) and 15 µM (1), respectively, thetotal number of bacteria was set to 10⁴ liter^–1 and themaximum rate of reaction for anammox and denitrification wasset to 10^–3 µmol N bacterium^–1 day^–1.

View larger version (23K):
[in this window]
[in a new window]
FIG. 3. (a) Total anammox (AAO) N₂ production in the direct presence of 1 to 10 µM ¹⁴NO₂^–, 100 µM ¹⁴NO₃^–, and 500 µM ¹⁵NH₄⁺. (b) AAO as a percentage of each NO₂^– spike. (c and d) Representative ranges of patterns for AAO with NO₂^– from 1 to 20 µM and 100 µM ¹⁴NO₃^–. (e and f) AAO with 5 µM ¹⁴NO₂^– and decreasing concentrations of ¹⁴NO₃^–. Note that a measurement with 0 µM ¹⁴NO₃^– was not determined in November 2003. Values are means ± 1 SEM (n = 4). The same symbols are used for each respective left and right panel.

RESULTS

Top

Results

Anammox activity with single NO₂^– or NO₃^– enrichment.
Above 10 µM NO₂^– or NO₃^–, anammox activitywas always linear (P < 0.05) (Fig. 1). In contrast, anammoxexhibited a range of activity below 10 µM NO₂^– orNO₃^– (Fig. 1). For example, at concentrations below 10µM for either NO₂^– or NO₃^–, ²⁹N₂ productionremained constant (Fig. 1a) and, in turn, the percentages ofthe electron acceptors accounted for by anammox increased to7 and 4%, respectively (Fig. 1b). Alternatively, the productionof ²⁹N₂ increased linearly from 2.5 to 20 µM NO₂^–or NO₃^–, and the percentage accounted for by anammox remainedconstant at <2% (Fig. 1c and d). Overall, the single-enrichmentactivities were highly variable below 10 µM, and on occasion(October and November 2003), several data points supported elevatedactivities below 5 µM (Fig. 1e and f). In contrast, theheterotrophic NO₃^–- and NO₂^–-reducing communityaccounted for >93 and 96% of the NO₂^– or NO₃^–,respectively.

View larger version (21K):
[in this window]
[in a new window]
FIG. 1. (a and c) Production of ²⁹N₂ from labeled ¹⁵NH₄⁺ oxidation in the direct presence of ¹⁴NO₂^– or ¹⁴NO₃^– in November and December 2003. (b and d) Total anammox N₂ production (calculated from ²⁹N₂ production) as a percentage of the respective NO₂^– or NO₃^– spike. (e and f) Composite of the above NO₂^– data plus additional NO₂^– data for October 2003. Values are means ± 1 standard error of the means (SEM) (n = 4).

Nitrate reduction and nitrite accumulation.
In slurries enriched with 100 µM NO₃^–, all of theNO₃^– had been consumed within the first half-hour andno significant transient accumulation of NO₂^– could bemeasured (Fig. 2). At 200, 400, and 600 µM NO₃^–,the slurries exhibited similar rates of NO₃^– reduction,i.e., 103, 105, and 97 µM h^–1, respectively. Thesedata agree with the rate of 85 µM h^–1 measured atthe same site in the previous year (22). In addition, thosesamples enriched with 200 µM NO₃^– and above exhibiteda transient accumulation of NO₂^– of 19 µM after1 h. Therefore, 100 µM NO₃^– could, at least duringour short dual incubations, satisfy total NO₃^– reductionand enable the initial availability of NO₂^– to be regulated.

View larger version (18K):
[in this window]
[in a new window]
FIG. 2. (a) Reduction of NO₃^– over time in anaerobic sediment slurries at four NO₃^– enrichment concentrations. (b) Subsequent accumulation and reduction of NO₂^– over time for each respective NO₃^– enrichment. Values are means ± 1 SEM (n = 4). The same symbols are used in each panel.

Anammox activity with dual NO₂^– and NO₃^– enrichment.
In the dual presence of NO₂^– (1 to 10 µM) and NO₃^–(100 µM) there was an even more pronounced nonlinear responsein anammox activity, relative to those in the single enrichments(Fig. 3a). In addition, there was a marked increase in overallanammox activity in the dual-enrichment experiments. For example,the anammox activity increased from 0.01 nmol N₂ ml^–1wet sediment at 5 µM NO₂^– without NO₃^– to0.6 nmol N₂ ml^–1 wet sediment with NO₃^–. In turn,the presence of NO₃^– increased the percentage of NO₂^–accounted for by anammox from 0.15 to 8% at 5 µM NO₂^–.As with the previous single-enrichment experiments, anammoxexhibited a range of activities, although they all had a similarshape (Fig. 3c). The maximum anammox activity in the presenceof both NO₂^– and NO₃^– was measured in November 2003(2.6 nmol N₂ ml^–1 wet sediment at 5 µM NO₂^–),and the lowest, to date, was measured in May 2004 (0.3 nmolN₂ ml^–1 wet sediment at 5 µM NO₂^–). In November,anammox accounted for as much as 82% of the NO₂^– at 1µM, but it only accounted for 15% in May and between 26and 5% at 5 µM NO₂^–, respectively, for these twomonths (Fig. 3d). Using 5 µM NO₂^– and 100 µMNO₃^– as a reference measure for anammox, we show the datacollected to date in Table 1, along with some site characteristics.

View this table:
[in this window]
[in a new window]
TABLE 1. Summary of anammox activities measured under dual enrichment with 5 µM NO₂^–, 100 µM NO₃^–, and 500 µM ¹⁵NH₄^– at 15°C from November 2003 to June 2004, with some site water characteristics

In subsequent experiments, dual enrichment was repeated with5 µM NO₂^– and with NO₃^– from 0 to 100 µM(Fig. 3e and f). Clearly, as the concentration of NO₃^–was reduced, the anammox activity at 5 µM NO₂^– declined.In addition, as with all the previous experiments, there wasa change in the relative significance of anammox to NO₂^–reduction on each occasion.

Modeling the behavior of anammox.
The model was run for the component forms (f_p [equation 7] andf_m [equation 8]) and the combined form of the biphasic anammoxexpression (f_a [equation 6]), with the following parameters: ${varepsilon}$ = 0.15, l_N = 2.5 µM, and K_m = 2.5 µM. In addition,for each component and combined run, the proportion of anammoxbacteria ( ${theta}$ ) within the model was varied from 0.3 to 4.5%, andthe results for the various scenarios are given in Fig. 4.

View larger version (28K):
[in this window]
[in a new window]
FIG. 4. Model runs for anammox, with ${theta}$ varying between 0.003 and 0.045 in each case. (a) Anammox as non-Michaelis f_p (equation 7); (c) anammox as Michaelis f_m (equation 8); (e) anammox as a function of the combined components f_p + f_m (equation 6). (b, d, and f) NO₂^– accounted for by anammox for each respective scenario (a, c, and e).

DISCUSSION

Top

Discussion

In our first study of anammox, we had demonstrated a linearresponse for anammox (production of ²⁹N₂) between 200 and 800µM NO₃^– or NO₂^–, with a constant yield of²⁹N₂ per molecule of NO₃^– or NO₂^– reduced (22).Here we have shown that this relationship holds for either acceptordown to a concentration of 10 µM but that below this level,anammox exhibits nonlinear behavior, with an optimum at 2 to5 µM NO₂^–. Previous work with sediments had suggestedthat anammox has a K_m for NO₂^– of 3 µM (4), andthe results presented here confirm this observation. In single-enrichmenttrials with concentrations above 10 µM NO₃^–, anammoxcould account for approximately 1% of the reduced NO₃^–.This suggests that as more NO₃^– was added, a fixed proportionwas lost as NO₂^– and, in turn, reduced via anammox, andhence the linear response (Fig. 1a and c). The flat responsebelow 10 µM NO₃^– may have simply been due to thepreferential affinity by anammox for the lower concentrationsof NO₂^– resulting from NO₃^– reduction or, in part,to a disproportionately greater loss of NO₂^– from NO₃^–reduction below 10 µM NO₃^– (Fig. 1a). It has beensuggested (12) that periplasmic NO₃^– reductase has a relativelyhigh affinity (Nap; K_m, 15 µM) and may enable bacteriato scavenge NO₃^– at low concentrations, while membrane-boundNO₃^– reductase (Nar; K_m, 50 µM) is more suited tohigher concentrations. Hence, a changing proportion of NO₂^–loss may reflect different enzyme systems, e.g., the loss ofNO₂^– may be greater under Nap rather than Nar activity.The flat response below 10 µM NO₂^– indicates thatthe anammox community takes proportionately more of the availableNO₂^– at lower concentrations, again reflecting the highaffinity for NO₂^–. However, it is difficult to rationalizewhy the yield of anammox remained constant in the direct singlepresence of either NO₂^– or NO₃^– (Fig. 1a and c).In the single presence of NO₂^– the NO₂^– is obviouslyextracellular and thus directly available to the anammox community,yet the yield of anammox was the same as that for NO₃^–when the anammox community was reliant on a supply of NO₂^–originating from the NO₃^–-reducing community. For thisto be the case, the availability of NO₂^– for anammox wouldhave to be the same under both scenarios, either direct NO₂^–or NO₃^– reduced to NO₂^–. This suggests either thatNO₂^– is never limiting in single-enrichment experimentsor that some anammox bacteria may actually be coupled (bothphysically and metabolically) to members of the NO₃^–-reducingcommunity and cannot proceed faster than the rate governed bythe metabolism of the heterotrophic NO₃^– and NO₂^–reducers.

At a steady state in sediments, NO₃^– and NO₂^– coexistat concentrations separated by orders of magnitude, and it wasonly in the dual-enrichment experiments that the nonlinear behaviorbecame fully apparent (Fig. 3). Clearly, the anammox activitywas regulated by the presence of NO₃^–, and as the availabilityof NO₃^– was reduced, the anammox activity and its significanceas a sink for NO₂^– declined (Fig. 3e and f). Hence, althoughanammox bacteria have a high affinity for NO₂^–, they aremost likely vastly outnumbered by the heterotrophic NO₃^–-and NO₂^–-reducing community, which will reduce NO₂^–when NO₃^– is limiting (22). The final path of NO₂^–reduction is therefore modulated by the dual availability ofNO₃^– and NO₂^– and the overall competition for electronacceptors. The data presented in Fig. 2 suggest that if theavailability of NO₃^– is greater than that required forNO₃^– reductase to operate at V_max, then NO₂^– isexported to the sediment, while below this it is not, explainingwhy the nonlinear behavior only became clearly visible whenthe loss of NO₂^– from NO₃^– reduction was controlledand NO₂^– was made available only under these controlledconditions. Despite the lack of NO₂^– leakage from NO₃^–reduction below 100 µM NO₃^–, anammox was alwaysmeasurable in the single-enrichment experiments (Fig. 1), whichagain suggests that some of the anammox bacteria are tightlycoupled to the turnover of NO₃^–. It is interesting thatNO₃^– was reduced in these estuarine sediments (Thames)at 20 times the rate ( $~$ 100 µM h^–1 compared to 5 µMh^–1) of those in the Skagerrak, where anammox has alsobeen measured (4, 19) and where its relative significance toN₂ formation is much greater than that for the Thames. Assumingthat NO₃^– reduction was operating at V_max in the Skagerrak(4, 19), as shown for the Thames in Fig. 2b, then of the reducedNO₃^–, 60% accumulated as NO₂^– in the Skagerrak butonly 20% accumulated as NO₂^– in the Thames. In the Skagerrak,therefore, the greater availability of NO₂^– from NO₃^–reduction may maintain a relatively large anammox population,and although the volume-specific rates of anammox are lowerin the Skagerrak, its proportionate contribution to N₂ productionis greater than that in the Thames, where the anammox populationis probably comparatively smaller. Whether this means that NO₃^–reduction leaks proportionately more NO₂^– in the Skagerrakthan in the Thames or that NO₃^– respiration to NO₂^–,before anammox or denitrification, dominates the initial stepof NO₃^– reduction is unclear.

In addition to the overall availability of NO₂^–- and NO₃^–-regulatinganammox, there were marked fluctuations in anammox activity(Table 1). Seasonal effects have been clearly documented forthe common forms of sediment metabolism, which usually followsmooth curves reflecting seasonal temperatures (6, 21). Short-termchanges in sulfate reduction have been reported for upper estuarinesediments which corresponded with peaks in Desulfovibrio speciesactivity (20). Although all of the experiments were carriedout at 15°C and, hence, do not reflect in situ temperatures,the differences may reflect variations in the in situ abundanceand/or activity of the anammox community sampled on each occasion;this is supported by the overall pattern of the data set andmodel output.

In our anammox model, NO₂^– can be reduced via either anammoxor denitrification, with denitrification switching between NO₂^–or NO₃^– depending on the availability of NO₃^– ([NO₃^–]_crit).Examples of the model output for anammox with the non-Michaelis(f_p [equation 7]) (Fig. 4a and b), Michaelis (f_m [equation 8])(Fig. 4c and d), and combined (f_p + f_m [equation 6]) (Fig. 4e and f)components, with ${theta}$ varying from 0.003 to 0.045, are givenin Fig. 4. When f_a includes only the function f_p, anammox cannever respond in a linear fashion at concentrations higher than10 µM, since f_p is modeled with a peak in activity when[NO₂^–] = l_N and declines exponentially after this. Alternatively,a Michaelis function, such as f_m, can never explain the nonlinearpattern shown at concentrations below 10 µM (Fig. 4c and d).It is only when the two functions are combined (as in equation6) to give a biphasic behavior for anammox that the model beginsto resemble the patterns observed in the data (compare Fig.4e and f to Fig. 3c and d), in which the amount of anammox representsthe sum of gas produced from both the Michaelis and non-Michaelisforms of the model. Whether or not there is a single anammoxprocess with two enzyme systems or, alternatively, two independentanammox systems operating in these estuarine sediments is notknown. The data suggest that in studies of anammox to date (19,22), only the Michaelis form has been measured, which largelyexplains the previously reported linear production of ²⁹N₂ regardlessof the concentration of NO₂^– or NO₃^– above 10 µM.The non-Michaelis form was certainly masked in our previousmeasurements of anammox (22), but it is not known whether thisbehavior would apply to off-shore continental shelf sediments(19). A Michaelis system may apply to anammox bacteria coupledtightly to the reduction of NO₃^–, and as such, NO₂^–will never be limiting and the operational range for NO₂^–is less significant. The non-Michaelis system could be optimizedto function at actual pore-water NO₂^– concentrations,although the resolution of NO₂^– profiles and pore-waterNO₂^– data to date are limited.

In addition to the biphasic expression, changing ${theta}$ from 0.3 to4.5% reproduced the range of anammox (amplitudes) measured inthe slurry experiments (Fig. 4 e and f). Whether the effectof changing ${theta}$ truly reflects a change in the in situ abundance(which may itself be a mixed population) or, alternatively,a change in specific activity is not known, as changing eitherin the model produces the same effect. Alternatively, the dampeningdown of anammox activity can be driven by increasing the rateof denitrification, yet this would seem least likely, as weconsistently measured NO₃^– reduction at about 100 µMh^–1 for these sediments, while the model would requirean increase by a factor of about 8 to reproduce the minimumand maximum amplitudes shown in Fig. 4.

The production of ²⁹N₂ from labeled ¹⁵NH₄⁺ and ¹⁴NO₂^–agrees with the 1:1 catabolism of ¹⁴NO₂^– + ¹⁵NH₄⁺ $->$ ²⁹N₂+ 2H₂O. Anammox bacteria (in bioreactors) have, however, beenshown to consume NH₃ and NO₂^– with an overall stoichiometryof 1:1.3, with the excess NO₂^– (0.3 mol) being anaerobicallyoxidized to NO₃^–, though this ratio can change with theconcentration of NO₂^– (16, 18). Hence, 1 mol of ²⁹N₂ accountsfor 1 mol of ¹⁵NH₄⁺ oxidized but potentially (depending on theactual sediment reaction) 1.3 mol of NO₂^– reduced, andtherefore, the total anammox activity potentially needs to bemultiplied by 1.3 to fully account for NO₂^– reductionvia anammox. Allowing for this change in ratio, and dependingon in situ conditions, anammox may account for the majorityof NO₂^– reduction at 2 to 5 µM NO₂^– in thesesediments.

The presence of anammox violates the central tenets of the isotopepairing technique (10), and the implications of this where anammoxand denitrification coexist in sediments have been explored(15). Their discussion pivots around four assumptions, one ofwhich is that anammox and denitrification are both limited bythe supply of NO₃^– and that the uptake kinetics for itsreduction product, NO₂^–, by denitrifying and anammox bacteriaare similar. It is acknowledged that while the kinetics of denitrificationas a function of NO₃^– are well characterized for sediments,little is known about the in situ regulation of anammox. Thenonlinear response reported here for anammox below 10 µMNO₂^–, especially in the presence of NO₃^–, suggeststhat the kinetics of the two processes are different and challengesthis assumption for anammox in estuarine sediments. In the presenceof representative in situ NO₂^– and NO₃^– concentrations,the production of N₂ via anammox increased. What regulates theproportionate significance of either anammox or denitrificationto N₂ formation in intact aquatic sediments is likely to bea combination of the respective availability of both NO₃^–and NO₂^– and the relative size or activity of the anammoxpopulation.

ACKNOWLEDGMENTS

We thank Kevin J. Purdy for valuable discussions and Ian Sandersfor field assistance.

This research was funded in part by a research grant (NER/A/S/2003/00354)to M.T. provided by the Natural Environment Research Council,United Kingdom.

FOOTNOTES

* Corresponding author. Mailing address: School of Biological Sciences, Queen Mary, University of London, London E1 4NS, United Kingdom. Phone: 0044 (0)20 7882 3007. Fax: 0044 (0)20 8983 0973. E-mail: m.trimmer{at}qmul.ac.uk.

REFERENCES

Top