mechanisms of transient nitric oxide and nitrous oxide

ORIGINAL ARTICLE

Mechanisms of transient nitric oxide andnitrous oxide production in a complexbiofilm

Frank Schreiber, Birte Loeffler, Lubos Polerecky, Marcel MM Kuypers and Dirk de BeerMax-Planck-Institute for Marine Microbiology, Bremen, Germany

Nitric oxide (NO) and nitrous oxide (N2O) are formed during N-cycling in complex microbialcommunities in response to fluctuating molecular oxygen (O2) and nitrite (NO2

�) concentrations. Untilnow, the formation of NO and N2O in microbial communities has been measured with low spatial andtemporal resolution, which hampered elucidation of the turnover pathways and their regulation. Inthis study, we combined microsensor measurements with metabolic modeling to investigate thefunctional response of a complex biofilm with nitrifying and denitrifying activity to variations in O2

and NO2�. In steady state, NO and N2O formation was detected if ammonium (NH4

þ ) was present underoxic conditions and if NO2

� was present under anoxic conditions. Thus, NO and N2O are produced byammonia-oxidizing bacteria (AOB) under oxic conditions and by heterotrophic denitrifiers underanoxic conditions. NO and N2O formation by AOB occurred at fully oxic conditions if NO2

�

concentrations were high. Modeling showed that steady-state NO concentrations are controlled bythe affinity of NO-consuming processes to NO. Transient accumulation of NO and N2O occurredupon O2 removal from, or NO2

� addition to, the medium only if NH4þ was present under oxic

conditions or if NO2� was already present under anoxic conditions. This showed that AOB and

heterotrophic denitrifiers need to be metabolically active to respond with instantaneous NO and N2Oproduction upon perturbations. Transiently accumulated NO and N2O decreased rapidly after theirformation, indicating a direct effect of NO on the metabolism. By fitting model results tomeasurements, the kinetic relationships in the model were extended with dynamic parameters topredict transient NO release from perturbed ecosystems.The ISME Journal (2009) 3, 1301–1313; doi:10.1038/ismej.2009.55; published online 11 June 2009Subject Category: microbial ecosystem impactsKeywords: microsensor; metabolic modeling; nitrification; denitrification; intermediates; nitrifierdenitrification

Introduction

Nitric oxide (NO) and nitrous oxide (N2O) areproduced and consumed by catabolic reactions ofbacteria involved in the biogeochemical N-cycle.These reactions are fostered by increased anthro-pogenic N input into the environment leading to asteadily increasing atmospheric N2O concentration.This is of environmental concern, because theinfrared radiative forcing potential of N2O is B200times that of CO2, which makes N2O a potentgreenhouse gas (Stein and Yung, 2003). Moreover,NO and N2O are involved in a set of catalyticreactions that transform ozone to molecular oxygen(O2) in the stratosphere (Crutzen, 1979).

Denitrification and nitrification are generallyconsidered to be the two main processes responsiblefor the formation of NO and N2O (Stein and Yung,2003). Heterotrophic denitrification is the respira-tory, sequential reduction of nitrate (NO3

�) or nitrite(NO2

�) via NO and N2O to N2 (Zumft, 1997). The keyenzymes in denitrification are nitrite reductase(Nir), nitric oxide reductase (Nor) and nitrous oxidereductase (Nos). NO levels in heterotrophic deni-trifiers are well regulated, independent of NO3

� andNO2

� concentrations and are in the range of lownanomolar concentrations (Goretski et al., 1990). NOconsumption in heterotrophic denitrifiers might bemediated by widespread NO detoxifying enzymes,such as flavohemoglobins (Hmp or Fhp) andflavorubredoxin (NorVW), or respiratory NorB thatcan reduce NO to N2O (Rodionov et al., 2005).

Nitrification is the aerobic oxidation of ammonium(NH4

þ ) performed by different groups of micro-organisms. Aerobic NH4

þ oxidation (Aox) to NO2� is

performed by ammonia-oxidizing bacteria (AOB) orarchaea (Arp and Stein, 2003; Konneke et al., 2005).

Received 19 January 2009; revised 9 April 2009; accepted 16 April2009; published online 11 June 2009

Correspondence: F Schreiber, Max-Planck-Institute for MarineMicrobiology, Celsiusstr. 1, D-28359 Bremen, Germany.E-mail: [email protected]

The ISME Journal (2009) 3, 1301–1313& 2009 International Society for Microbial Ecology All rights reserved 1751-7362/09 $32.00

www.nature.com/ismej

http://dx.doi.org/10.1038/ismej.2009.55

mailto:[email protected]

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In a next step, NO2� is oxidized aerobically to NO3

�

(Nox) by nitrite-oxidizing bacteria (NOB). Severalstudies have demonstrated the production of NO andN2O by pure cultures of AOB (Kester et al., 1997;Lipschultz et al., 1981; Shaw et al., 2006), but themechanism is not completely understood. Generallytwo different pathways are inferred. First, the activityof hydroxylamine oxidoreductase (HAO) convertshydroxylamine (NH2OH) to NO2

� and releases smallamounts of NO and N2O (Hooper, 1968). Second, theactivity of nitrifier-encoded Nir and Nor reducesNO2

� to NO and N2O in a process termed nitrifierdenitrification (Poth and Focht, 1985; Bock et al.,1995; Schmidt et al., 2004). In both pathways, O2 andNH4

þ are required to form NH2OH, the electron donorfor NO2

� reduction.NO and N2O turnover has also been studied in

complex microbial communities. Studies in soilsand nitrifying granules revealed that denitrificationand nitrification are the contributing microbialpathways. Commonly, NO and N2O accumulationincreased with decreased O2 and with increasedNO2

� and NH4þ concentrations (Conrad, 1996;

Colliver and Stephenson, 2000; Kampschreuret al., 2008).

Change in environmental conditions leads totransient production of NO and N2O in pure culturesof AOB and heterotrophic denitrifiers (Kester et al.,1997; Bergaust et al., 2008), as well as in mixedmicrobial communities (Kampschreur et al., 2008;Morley et al., 2008). This transient production canlead to high concentrations and might thus con-tribute significantly to NO and N2O emissions fromvarious habitats. Despite the importance of transientNO and N2O formation, the coupling and regulationof the responsible pathways remain poorly under-stood in complex microbial communities. This isprimarily because the experiments on release of NOand N2O from natural samples commonly rely onanalysis of the headspace volume or the bulksolution, which have no spatial and low temporalresolution. However, local conversion rates andlimited transport in aggregated or attached microbialcommunities lead to stratification and to microen-vironments that are different from the bulk solution.Thus, high spatial resolution measurements of NO,N2O and O2 are required to distinguish between thecontribution of aerobic (Aox) and anaerobic (hetero-trophic denitrification) processes to NO and N2Oemission from stratified ecosystems where nitrifica-tion and denitrification co-occur. In addition, hightemporal resolution measurements during systemperturbation are a powerful method for unravelingcomplex sets of processes and to obtain insights intothe coupling of different pathways.

The objective of this study was to assign NO andN2O production to nitrifying or denitrifying pro-cesses occurring in a complex biofilm. Furthermore,we aimed to quantify the influence of O2, of NO2

�

fluctuations and of metabolic state on the dynamicsof the transient NO and N2O formation. We

conducted microsensor measurements with highspatial and temporal resolution to characterizein situ microenvironmental conditions, quantifythe rates of the relevant processes and follow NOand N2O formation upon perturbations. Further-more, we developed a novel metabolic model thatallowed numerical simulations of the measured NOtransitions. On the basis of this model, we proposemechanisms that explain transient turnover of NOand N2O by AOB and associated heterotrophicdenitrifiers in the studied biofilm.

Materials and methods

Biofilm growthBiofilms were grown in an aerated flow cell(B800ml) using Tygon tubing as a surface forbiofilm growth. The inoculum was obtained from asewage treatment plant (Seehausen, Bremen) andwas fed with medium at a flow rate of 1mlmin�1.Biofilms with a thickness of 0.4–0.7mm developedwithin 2–3 months in a medium consisting of 10mM

NH4Cl and trace elements at final concentrations of3mM Na2-EDTA, 1.5 mM FeSO4, 77 nM H3BO4, 100nMMnCl2, 160nM CoCl2, 20nM NiCl2, 2.4 nM CuCl2,100nM ZnSO4 and 30nM Na2MoO4 in tap water atpH 7.4. One month before the measurements, themedium was changed to a phosphate-bufferedartificial freshwater medium containing 17mM

NaCl, 2mM MgCl2, 0.9mM CaCl2, 6.7mM KCl and1.5mM KH2PO4/K2HPO4 (pH 7.5), supplementedwith 400 mM NH4Cl and trace elements as statedbefore. For microsensor measurements, small piecesof the biofilm-covered tubing were transferred into asmaller flow cell (B80ml) placed in an aquarium.The aquarium served as a reservoir for 1.7 l ofaerated artificial freshwater medium (with or with-out NH4Cl) that recirculated through the small flowcell at a flow rate of 3ml s�1 to create a constant flowof B0.2 cm s�1 above the biofilm.

Experimental designAfter biofilms adjusted to the conditions in thesmall flow cell for 1–2 days, steady-state micro-profiles of O2, pH, NH4

þ , NO2�, NO3

�, N2O and NOwere measured in the presence of 400 or 0 mM NH4Cland at varying O2 and NO2

� concentrations in theoverlying water. The metabolic response of thebiofilms was studied by changing the conditions inthe aquarium in the following sequence: (1) startingcondition with O2 at air saturation, (2) switching tolow O2 (B3% air saturation) by purging the mediumwith N2, (3) addition of 3mM NaNO2 with O2 at airsaturation and (4) switching to low O2 (B3% airsaturation) in the presence of 3mM NaNO2. Theresponse to the addition of 3mM NaNO2 at low O2

was investigated in separate experiments. Transientconcentration changes of NO, N2O and O2 weremonitored inside the biofilm upon switching the

Transient NO and N2O production in biofilmsF Schreiber et al

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conditions until a new steady state was reached.The recirculated medium was sampled regularly totest for NO3

� and NO2� accumulation from NH4

þ ,stability of NH4

þ concentrations, pH and tempera-ture. NO3

� and NO2� accumulated only in the

presence of NH4þ , and reached maximum concentra-

tions of approximately 30 and 5 mM, respectively.NH4

þ did not decrease below 370 mM. The tempera-ture was 25–26 1C, and the pH was 7.2–7.3.

Microsensor measurementsConcentrations of O2, N2O and NO were measuredwith amperometric microsensors, whereas liquid-ion exchange microsensors were used for pH, NH4

þ ,NO2

� and NO3� measurements. Microsensors were

prepared and calibrated as previously described (deBeer and van den Heuvel, 1988; Revsbech, 1989; deBeer et al., 1997; Andersen et al., 2001; Schreiberet al., 2008). Vertical concentration profiles weremeasured with the microsensor mounted on a 3-axismicromanipulator (MM 33; Marzhauser, Wetzlar,Germany). The vertical axis was motorized form-positioning (VT-80 linear stage; Micos, Germany,equipped with a 3564-K-024-BC motor, FaulhaberGroup, Schonaich, Germany), and measurementswere controlled by m-Profiler software (www.microsen-wiki.net). The microsensor tip was ad-justed manually to the sample surface with the helpof a dissection microscope (Stemi SV 6; Carl ZeissAG, Oberkochen, Germany).

Diffusive fluxes across the liquid–biofilm inter-face were calculated from the concentrationgradients multiplied by the molecular diffusioncoefficient D, as previously described. Values usedfor D were 2.34� 10�9m2 s�1 for O2, 1.98�10�9m2 s�1 for NH4

þ , 1.86� 10�9m2 s�1 for NO2�,

1.92� 10�9m2 s�1 for NO3�, 2.36� 10�9m2 s�1 for

N2O and 2.21� 10�9m2 s�1 for NO (Broecker andPeng, 1974; Li and Gregory, 1974; Zacharia andDeen, 2005).

Metabolic modeling of NO productionWe developed an N-cycle model that couplesprocesses involved in the production and consump-tion of NO, O2 and NO2

� in the biofilm (Figure 1). Weassumed that NH4

þ was aerobically converted toNO2

� by Aox, and NO2� subsequently converted to

NO3� by NO2

� oxidation (Nox). Anaerobic conditionsfavor NO2

� consumption by heterotrophic denitrifi-cation (hD). This results in the formation of NO,which is consumed in a sequential step by hetero-trophic denitrification (hD-NO). Nitrifier denitrifi-cation by NH4

þ -dependent AOB (niD) producedNO aerobically from NO2

�. Subsequently, NO isconsumed by nitrifier denitrification (niD-NO). Inaddition, the model incorporated chemical NOoxidation with O2 to NO2

� (chem) and O2 consump-tion by heterotrophic respiration (hR).

This model was implemented numerically inMATLAB (The MathWorks, Inc., Natick, MA,USA, the code is available at www.microsen-wiki.net) to describe the kinetic, metabolic andmass-transfer control of NO, O2 and NO2

� in thebiofilm. We assumed that the biofilm was laterallyhomogeneous and transport was governed by diffu-sion. Thus, the dynamics of a solute with concen-tration C (molm�3) in and above the biofilm wasdescribed by a one-dimensional diffusion-reactionequation

qCqt

¼ DCq2Cqz2

þ RC: ð1Þ

Here, C denotes concentration of NO, NO2� or O2,

DC is the corresponding diffusion coefficient inwater (m2 s�1), which was assumed to be constantthroughout the biofilm and RC (molm�3 s�1) is the netreaction rate at which the solute is prod-uced or consumed. Both C and RC were explicitfunctions of time t and depth z, with z40 and zo0corresponding to the biofilm and overlyingwater, respectively. The reactions for productionand consumption of NO, O2 and NO2

�, as shown inFigure 1, were stochiometrically balanced and usedto express the net reaction rates RC for NO, O2 andNO2

� as

RNO ¼ 8RniD � 4RniD�NO þ RhD � 1RhD�NO

� 4Rchem ð2aÞ

Figure 1 Schematic representation of the metabolic model of NOturnover in a complex biofilm. Pathways and compounds markedby solid black lines were calculated by the numerical model,whereas those marked with gray dashed lines were not calculated.Italic text next to the arrows indicates the pathways: Aox,ammonium oxidation; Nox, nitrite oxidation; niD, NO productionby nitrifier denitrification; niD-NO, NO consumption by nitrifierdenitrification; hD, NO production by heterotrophic denitrifica-tion; hD-NO, NO consumption by heterotrophic denitrification;hR, heterotrophic oxygen respiration; chem, chemical NOoxidation. Additional mechanisms that influence the rate ofthe respective pathway, with the compounds that affect thosemechanisms in parentheses, are indicated by T, threshold;T, extended threshold; Dyn, dynamic function and Sh, shiftfunction (see text and Equations (4)–(7) for details).


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www.microsen-wiki.net




RO2¼ �1RniD � 1RniD�NO � 3RAox � 1RNox

� 1RhR � 1Rchem ð2bÞ

RNO�2¼ �6RniD þ 2RAox � RhD � 2RNox

þ 4Rchem ð2cÞEach individual rate was an explicit function of theconcentrations of the solutes involved in the processand is described in detail in the SupplementaryTable S1.

Kinetic control of the reaction rates Ri wasdescribed by the Michaelis–Menten law,

RiðCÞ ¼ vimax �

C

KiC þ C

¼ vimax �MðC; Ki

CÞ; ð3Þ

where KCi (molm�3) is the affinity constant of the

process i to substrate C, vmaxi (molm�3 s�1) is

the maximum rate of the process i and M denotesthe Michaelis–Menten function describing the re-lationship between affinity and concentration with-out vmax. The affinity constants were obtained fromthe literature and the maximum rates were derivedfrom the measured steady-state fluxes at the liquid–biofilm interface (as in Tables 1 and 2) divided bythe biofilm thickness. The maximum rates of NO-consuming processes (niD-NO under oxic condi-tions and hD-NO under anoxic conditions) weredetermined by assuming that both processes werecoupled to the respective production process. Thisallowed subtraction of the net NO production ratefrom the rate of the NO-producing process, whichwas determined from NO2

� consumption.Metabolic control of the reaction rates was

implemented by combining information availablefrom pure culture studies with postulated mechan-isms based on data presented in this work. First,maximum activity of NO production (RhD) and NOconsumption (RhD-NO) by heterotrophic denitrifica-

tion occurred at micro-oxic to anoxic conditions,that is, at O2 below a certain threshold concentrationYi

O2. This was implemented by multiplying the

maximum reaction rates, vmaxhD and vmax

hD-NO, with athreshold function

TðC; YiC; d

iCÞ ¼

1

1þ exp

C�YiC

diC

; ð4Þ

where dCi represents the width of the concentrationinterval over which the threshold function changesfrom 1 to 0 (see Supplementary Figure S1). Further-more, NO production by nitrifier denitrification(RniD) was allowed only at low O2 or high NO2

�

concentrations, which was achieved by multiplyingthe corresponding vmax

niD value with an extendedthreshold function (see Supplementary Figure S2)

~TðNO�2 ; O2; Y

niDNO�

2; YniD

O2; dniDNO�

2; dniDO2

Þ

¼1� TðNO�

2 ; YniDNO�

2; dniDNO�

2Þ

TðO2; YniDO2

; dniDO2Þ þ TðNO�

2 ; YniDNO�

2; dniDNO�

2Þ

ð5Þ

Threshold values were chosen by biological reason-ing and by matching the concentration dynamicsobserved in the measurements.

Second, our experimental data suggested thatafter the O2 concentration has decreased below acertain threshold, and when NO2

� was simulta-neously present in sufficient amounts, the rate ofNO production by heterotrophic denitrification,RhD, increased slowly with time (Figure 3d). Thismechanism was implemented by further multiply-ing the vmax

hD value with the dynamic function

Dynðt; t0; DtÞ ¼ 1� exp�ðt�t0Þ

Dt ; ð6Þwhere t0 is the time at which O2 decreased below athreshold value YhD

O2� 2dhDO2

whereas NO2� was

simultaneously present above 1 mM. The value ofDt¼ 400 s was estimated by fitting the measured

Table 1 Fluxes of measured solutes through the liquid–biofilm interface determined from microprofiles in a biofilm with the mediumcontaining 400mM NH4Cl

Solute Flux (nmol cm�2 h�1)a

5 mM NO2�b 3mM NO2

�b

100% O2c o3% O2

c 100% O2c o3% O2

c

NO 0.066±0.024 (7) 1±0.26 (10) 1.56±0.49 (10) 0.92±0.22 (7)N2O 2.53±1.16 (7) 1.55±0.36 (10) 4.72±0.71 (7) 5.35±2.68 (4)O2 �404±44 (7) �27±9 (3) �455±56 (11) �18±8 (4)NH4

+ �168±31 (6) 24±11 (6) �218±28 (3)d �48±22 (3)d

NO3� 210±30 (6) 21±9 (3) 208±37 (4) �7±2 (4)

NO2� 9.6±3.6 (9) �13.9±5.5 (6) �43±15 (3)d �67±39 (3)d

aFluxes are presented as mean±standard error (number of profiles indicated in parentheses). Negative and positive values indicate net uptakeand release of the solute, respectively.bNitrite concentration in the medium.cValues are given as % air saturation in the medium.dMeasured at 250mM NO2

� instead of 3mM NO2� because the sensitivity of the NO2

� sensor was too low at 3mM. However, 250mM did not limit theuptake.


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dynamic increase of NO in the presence of NO2� after

the conditions in the overlying water were switchedfrom oxic to suboxic (Figure 3d). In contrast, thedynamic increase of heterotrophic denitrificationtoward its maximum rate was accelerated to Dt¼ 4 sif NO was present above YNO

hD ¼ 0.32 mM. Thisassumption is based on reported evidence that NOserves as a signal for the expression of denitrifica-tion genes (Zumft, 2002, 2005).

Third, a shift mechanism (Sh) was implementedto model the instantaneous increase in NO concen-tration after NO2

� was added under oxic conditions(Figure 4a). Reasoning for this mechanism wasbased on the assumption that HAO function isimpaired by NO2

�, leading to the release of NO that isreported to be an HAO-bound intermediate (Arp andStein, 2003). This was implemented by removing afraction f of the NO2

� production rate by Aox fromRNO�

2in Equation (2c) and adding it to the total

net NO production rate RNO in Equation (2a) as afunction

Shðt; t0; DtÞ ¼ f � RAox: exp�ðt�t0Þ

Dt : ð7Þ

Here, t0 is the time when NO2� reached a threshold

concentration of YShNO�

2¼ 200 mM. The fraction of

the shifted RAox decreased exponentially with time,resembling an adjustment of AOB metabolism afterperturbation. The values of f¼ 0.55 and Dt¼ 200 swere estimated from the experimental data(Figure 4a).

The time-dependent diffusion-reaction equations(Equation (1)) were solved for all solutes usingboundary conditions: (1) solute concentrations werefixed to the concentrations in the overlying water,Cw, at the top of the diffusive boundary layer (DBL),that is, C(�zDBL)¼Cw, and (2) the diffusive flux atthe base of the biofilm was set to zero, that is, qC/qz(zB)¼ 0, where zDBL and zB denote the thickness ofthe DBL and the biofilm, respectively. Experimentalperturbations that resulted in O2 decrease and NO2

�

increase in the medium were implemented byvarying Cw over time. Experiments performed inthe absence of NH4

þ were modeled by excludingall processes from NO, NO2

� and O2 turnover thatrequire NH4

þ as electron donor, namely, Aox,nitrifier denitrification (niD) and NO consumptionby nitrifier denitrification (niD-NO).

All parameters of the model are listed in Supple-mentary Table S1. When available, they wereadjusted within a biologically reasonable range ofvalues reported in the literature; otherwise, theywere adjusted to match the experimental datapresented in this work. In the paper, the model isused to interpret and discuss the experimentalfindings.

Results

Performance of the biofilm in steady stateFor all compounds, the microprofiles showedeither production or consumption within theentire biofilm. Stratified zones of production andconsumption were not apparent (Figure 2 andSupplementary Figure S4). Thus, the overall perfor-mance of the biofilm was estimated from thefluxes across the liquid–biofilm interface (Table 1).Maximum potentials of Aox, Nox, nitrifier denitri-fication and heterotrophic denitrification were de-termined by creating the conditions such that onlythe process of interest occurred, and coupledprocesses were inhibited (Table 2).

The biofilm was fully oxic when the medium wasaerated (Supplementary Figure S4A). In the pre-sence of O2, NH4

þ was completely converted to NO3�

with minor accumulation of NO2� (Supplementary

Figure S4D), indicating that Aox and Nox occurredat similar rates (Tables 1 and 2). The couplingbetween Aox and Nox was not affected by additionof 3mM NO2

�. This was indicated by a similar 1:2:1stoichiometry of NH4

þ , O2 and NO3� fluxes in both

Table 2 Maximum activity of selected processes in the biofilm under different conditions

Processa Measuredparameter

ConditionNH4

+b/NO2�b/O2

c

Jd Calculation Rate of processe

hR O2 0/0/100 �52 — JhRO2¼ �52

Nox O2 0/200/100 �154 JNoxO2

¼ JO2� JhRO2

¼ 0:5JNoxNO�

2JNoxNO�

2¼ �204

Aox NH4+ 400/5/100 �218 JNHþ

4¼ JAox

NO�2

JAoxNO�

2¼ þ218

niD NO2� 400/250/100 �43 JniDNO�

2¼ JNO�

2� JAox

NO�2� JNox

NO�2

JniDNO�2¼ �57

hD NO2� 0/250/3 �102 — JhDNO�

2¼ �102

aSee Figure 1 for explanation of the abbreviations.bValues are given in mM.cValues are given as % air saturation.dFlux (nmol cm�2 h�1) through the liquid–biofilm interface presented as the net areal uptake rate of a certain solute by the biofilm.eGross areal rate (nmol cm�2 h�1) of processes in the biofilm. Processes and compounds are indicated by superscript and subscript notations,respectively. Negative and positive values indicate consumption and production, respectively.


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the absence and the presence of NO2� (Table 1).

However, nitrifier denitrification was induced byaddition of NO2

� in the presence of NH4þ and O2.

Nitrifier denitrification was indicated by the factthat the gross NO2

� uptake, calculated from net NO2�

uptake from the medium and NO2� production by

Aox, exceeded the maximum NO2� consumption

potential of Nox (Table 2). The remaining NO2� was

reduced by AOB, with a rate that was B20% of theNO2

� production rate of Aox. Consumption of NH4þ

and O2 was slightly elevated in the presence of NO2�

(Table 1).In the absence of NH4

þ , the potential for hetero-trophic processes was detectable, which wereprobably performed at the expense of reducedorganic carbon present in the biofilm. Under oxicconditions, heterotrophic respiration of O2 ac-counted for B15% of the total O2 consumption. Inthe absence of O2, NO2

� consumption by hetero-

trophic denitrification was B50% of the NO2�

consumption by NOB (Tables 1 and 2).The effects of NO2

� and O2 on the formation of NOand N2O in NH4

þ -containing medium are summar-ized in Table 1 and Figure 2. In the presence of NH4

þ

and high NO2�, NO and N2O were produced regard-

less of the O2 concentration in the medium. Incontrast, at low NO2

�, NO production was observedonly under anoxic conditions, whereas N2O produc-tion was low regardless of O2. N2O concentrations inthe biofilm were an order of magnitude higher thanNO concentrations, with NO ranging from o0.02 to0.35 mM and N2O from 0.35 to 5.4 mM. In the presenceof high NO2

�, the yields were 0.007mol NO per molNH4

þ and 0.022mol N2O per mol NH4þ . At low NO2

�,the N2O yield was reduced to 0.015mol N2O per molNH4

þ (Table 1). In the absence of NH4þ and NO2

�, NOand N2O fluxes were negligible regardless of O2.However, in the absence of NH4

þ and presence ofNO2

�, NO and N2O were produced, but only underanoxic conditions (Supplementary Figure S6). Theresulting fluxes were in the same range as thoseobserved in the presence of NH4

þ and NO2� under

anoxic conditions.

Transient NO and N2O formation in response to O2 andNO2

� changesUpon the start of N2 purging, O2 decreased graduallyin the biofilm until anoxic conditions were reached.The transient phase lasted B7min in the presenceof NH4

þ and B12min in the absence of NH4þ

(Supplementary Figure S3). During this transition,highly dynamic concentration changes of NO andN2O were detected with microsensors positioned inthe biofilm at 200 mm depth (Figures 3a–d).

Decreasing O2 concentrations in the presence ofNH4

þ caused a transient accumulation of NO andN2O, which decreased to a new steady state afteranoxic conditions were reached (Figures 3a and b).Although the accumulation was more pronouncedat high NO2

� concentration, the final steady-statelevels were on average comparable to those observedat low NO2

� (see also Figure 2c). Control measure-ments showed that in the absence of NH4

þ and NO2�,

NO and N2O were neither produced nor consumedduring the decrease of O2 concentration (Figure 3c).In contrast, the absence of NH4

þ at high NO2�

concentration resulted in slow formation of NOand N2O, starting shortly before anoxic conditionswere reached (Figure 3d).

When NO2� was added under oxic conditions, NO

concentration increased within less than a minutefrom below the detection limit to 1.2 mM, after whichit decreased within 20min to a new steady state.This was observed only if NH4

þ was present(Figure 4a). In the absence of O2, concentrations ofNO increased upon the addition of NO2

�. Thepresence of NH4

þ did not influence the final NOsteady-state concentrations, but affected the kineticsof its formation. NO formed slowly in the absence of

N2O [µM]

N2O [µM]

0.4

0.2

0.0

-0.2

-0.4

NO [µM]

dep

th [

mm

]

0.4

0.2

0.0

-0.2

-0.4

NO [µM]

dep

th [

mm

]

0.0 0.1 0.2 0.3 0.4 0.5

0.0 0.1 0.2 0.3 0.4 0.5

0 2 4 6

0 2 4 6

Figure 2 Averaged steady-state microprofiles of NO (a and c) andN2O (b and d) in a complex biofilm. Microprofiles were measuredin artificial freshwater medium containing 400mM NH4Cl with5 mM NO2

� (filled symbols) or with 3mM NO2� (open symbols) and

during aeration (a and b) or N2 purging (c and d) of the medium.The dashed line represents the biofilm surface. Horizontal barsrepresent standard errors (number of profiles is given in Table 1).


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0

1

2

3

4

5

6

N2O

[µ

M]

0

1

2

3

4

5

6

N2O

[µ

M]

0

1

2

3

4

5

6

N2O

[µ

M]

0

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Figure 3 (a–d) Time series of measured NO (black line) and N2O (gray line) concentrations with microsensors inserted in the biofilm at200mm depth. Purging of the medium with N2 started at t¼ 0min. (e–h) Time series of NO derived from the model shown in Figure 1. Ineach row, the boundary conditions and perturbations were implemented in the model such that they corresponded to the conditionsapplied during the measurement. Different stages of the model are shown, including (1) the model governed by kinetics and thresholdsonly (T and T; Equations (4) and (5); filled circles), (2) the model implementing the dynamic function (Dyn, Equation (6)) onheterotrophic denitrification (open circles), (3) the model implementing the dynamic function controlled by NO concentration (opentriangles), (4) and the model implementing NO loss by diffusion after stopping all processes when the peak NO concentration wasreached (filled triangles). White background indicates oxic, shaded areas indicate the transient phase from oxic to anoxic and gray areasindicate anoxic conditions. The medium composition with respect to NH4

þ and NO2� is depicted at the top-left of each row.


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NH4þ . In contrast, the presence of NH4

þ , whichresulted in low concentrations of NO2

� and NO3� in

the medium, caused an instantaneous increase ofNO from slightly elevated levels (Figure 4b).

Discussion

Regulation of steady-state NO and N2O production byammonium oxidation under oxic conditions and byheterotrophic denitrification under anoxic conditionsNO and N2O formation within a complex N-cyclingcommunity could be mediated by processes, such asAox, Nox, heterotrophic denitrification or anaerobicoxidation of NH4

þ (anammox) (Freitag et al., 1987;Stein and Yung, 2003; Kartal et al., 2007). Measuringthe in situ activities and microenvironmental con-ditions enabled us to assign concomitant NO andN2O formation to active processes.

Ammonium oxidation in steady state. AOB requireNH4

þ and O2 to form NO and N2O either by the HAO

pathway or by nitrifier denitrification. The presentdata showed that NO and N2O formation under oxicconditions depended on the presence of NH4

þ . NOand N2O formation did not occur upon addition ofNO2

� when NH4þ was absent (Figure 4a, Supplemen-

tary Figures S6A and S6B). These experimentsshowed that under oxic conditions, NO and N2Owere formed by AOB, but not by NOB or (aerobic)heterotrophic denitrifiers. Previous studies empha-sized the dependence of nitrifier denitrification onreduced O2 and elevated NO2

� concentrations(Lipschultz et al., 1981; Poth and Focht, 1985;Kester et al., 1997; Beaumont et al., 2004a; Kamps-chreur et al., 2008). Our experiments showed thatnitrifier denitrification and simultaneous NO andN2O formation occurs at high NO2

� concentrationseven if O2 concentrations are high (Figures 2a and 4aand Supplementary Figure S4A). This waspreviously observed for Nitrosomonas europaeaand Nitrosospira spp. (Shaw et al., 2006). Moreover,it has been shown that denitrifying enzymes (NirK

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Figure 4 (a and b) Time series of NO measured with a microsensor inserted in the biofilm at 200mm depth. 3mM NO2� was added at

t¼0min to the medium containing 400mM NH4þ and B5/30mM NO2

�/NO3� (black line) or to the medium that did not contain NH4

þ andNO2

�/NO3� (gray line). White and gray backgrounds indicate oxic and anoxic conditions, respectively. (c and d) Time series of NO derived

from the model (see Figure 1). In each row, the boundary conditions and perturbations were implemented in the model such that theycorresponded to the conditions applied during the measurement. Different symbols depict different stages of the model. In panel (c), thisincludes the model governed by kinetics and thresholds only (filled circles), the model additionally implementing a shift function (Sh)that resulted in the production of NO instead of NO2

� by Aox (open triangles), the model implementing NO loss by diffusion afterstopping all processes when the peak NO concentration was reached (filled triangles) and the control condition where all NH4

þ -dependent processes were switched off (open circles). In panel (d), this includes the model governed by kinetics and thresholds only(filled circles), with the dynamic function added (open triangles) and the model in the absence of NH4

þ -dependent processes governed bykinetics and thresholds only (closed triangles) or with the dynamic function included (open circles).


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and NorB) in N. europaea are expressed under fullyoxic conditions (Beaumont et al., 2004a, b).

In the model (Figure 1), NO production by nitrifierdenitrification was kinetically controlled by O2 andNO2

�. The resulting steady-state NO concentrationwas kinetically controlled by the affinity of NOconsumption by nitrifier denitrification to NO.However, NO production was only measured athigh NO2

� concentrations or under micro-oxic con-ditions. This indicates that NO accumulation underthose conditions was controlled by NO production.To limit NO production in the model to high NO2

�

and low O2 conditions, we implemented an ex-tended threshold function (T, Equation (5), Supple-mentary Figure S2). This allowed an independentinfluence of O2 and NO2

� on NO production with arestricted maximum rate, excluding additive effectswhen both conditions were present at the same time.

Heterotrophic denitrification in steady state. Ear-lier studies have reported the ability of AOB purecultures to produce NO under anoxic conditions(Kester et al., 1997; Schmidt et al., 2001) andreported that in a nitrifying mixed culture, NH4

þ

affected anaerobic NO formation in a concentration-dependent manner (Kampschreur et al., 2008).Conversely, we found that under anoxic conditions,NO and N2O formation did not depend on thepresence of NH4

þ and was only observed when NO2�

and NO3� were present. This showed that hetero-

trophic denitrifiers, but not O2-depending AOB,were responsible for NO and N2O formation underanoxic conditions. The dependence of NO and N2Oformation on NO2

� was confirmed under anoxicconditions in the absence of NH4

þ (SupplementaryFigures S6C and S6D). In the presence of NH4

þ ,nitrification leads to accumulation of approximately5mM NO2

� and 30 mM NO3� in the medium before N2

purging. NO3� and NO2

� served as electron acceptorsfor heterotrophic denitrification under subsequentanoxic conditions. This explains the formation ofNO and N2O under anoxic conditions by hetero-trophic denitrification in the presence of NH4

þ butnot in its absence. Anammox could oxidize NH4

þ

and reduce NO2� under anoxic conditions, and could

account for NO and N2O formation. However, thebiofilms grew under oxic conditions, which hampergrowth of anammox bacteria (Strous et al., 1997).Furthermore, NO2

� uptake of the biofilm underanoxic conditions was similar in the presence andthe absence of NH4

þ (Tables 1 and 2), indicating thatanammox did not contribute to additional NO2

�

reduction.In the model (Figure 1), Michaelis–Menten kinetic

dependence of heterotrophic denitrification on NO2�

allowed NO production in the presence of NO2�,

whereas threshold functions (T, Equation (4), Sup-plementary Figure S1) for O2 restricted the processto anoxic conditions. The measured steady-state NOconcentrations can be modeled (compare Figures2a,c with Figures 3e,f and 4c,d) by kinetically

controlling its accumulation with low Km valuesfor the NO consumption pathways, as has beendescribed elsewhere (Betlach and Tiedje, 1981). As aresult, steady-state NO concentrations under anoxiawere more or less independent of NO2

� concentra-tions, even though NO2

� concentrations varied overB3 orders of magnitude, corresponding to observa-tions in pure culture studies (Goretski et al., 1990).In addition, the Km value of heterotrophic denitri-fiers for NO2

� was very low in the model, resulting ina minor increase of NO production due to theadditional NO2

�, because NO2� consumption was

already saturated at low NO2� concentrations. In

contrast, measured N2O, the sequential product ofNO reduction, showed a marked increase in thepresence of high NO2

� concentrations. Thus, inheterotrophic denitrifiers, accumulation of NO maynot occur at high NO2

� concentrations due toefficient reduction of NO to N2O.

Furthermore, the model (Figure 1) showed thatchemical NO consumption under oxic conditionswas always several orders of magnitude lower thanthe biological rate, and contributed insignificantlyto the total NO loss. Chemical production of NOfrom acidic decomposition of NO2

� did not play arole, because the biofilm pH did not drop below 6.5(Supplementary Figures S5A and S5B).

A recently described model was able to assign NOaccumulation in a nitrifying reactor to nitrifierdenitrification by implementing kinetic controland by modeling different production scenariosindependent from each other (Kampschreur et al.,2007). In our model, the use of threshold functionsaffected by O2 and NO2

� concentrations in additionto Michaelis–Menten kinetics enabled us to con-sider all possible NO-producing pathways simulta-neously under varying conditions.

Perturbations of active ammonia oxidation andheterotrophic denitrification cause instantaneousNO and N2O formationThe time series measurements showed that NO andN2O reached transient maxima within the biofilmupon decreasing O2 and adding NO2

�. This phenom-enon was also observed in studies with purecultures of AOB and heterotrophic denitrifiers(Kester et al., 1997; Bergaust et al., 2008), as wellas with mixed microbial communities (Kamps-chreur et al., 2008; Morley et al., 2008). However,these observations are not completely understood.We observed transient maxima of NO and N2O onlywhen either Aox or heterotrophic denitrificationwas actively performed before O2 and NO2

� changed.We conclude that the instantaneous formation of NOand N2O is caused by perturbation of the activepathways on the enzyme level, resulting in animbalance of the fine-tuned mechanisms that main-tain NO homeostasis. NO and N2O dynamicsfollowing perturbation were similar, demonstratingthat they are sequentially produced by AOBand heterotrophic denitrifiers. The model can be


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extended to N2O concentrations if Km values for N2Oreduction are available.

Heterotrophic denitrification in transient states.NO2

� additions were followed by instantaneous NOformation, but only if NO2

� and NO3� were present at

low concentrations and if O2 was absent before theperturbation (Figure 4b). Under such conditions,only heterotrophic denitrification can cause NOformation. The instantaneous formation of NO uponNO2

� addition shows that NO-producing and NO-consuming enzymes are directly affected by NO2

�

and that de novo synthesis of enzymes cannotexplain the dynamics. For example, it was demon-strated previously that NO2

� can directly inhibit Nor(Kucera et al., 1986; Kucera, 1992), which wouldlead to accumulation of NO. In addition, kinetics ofNO2

�- and NO-reducing enzymes might allow NOaccumulation depending on the NO2

� concentration.Instantaneous NO production was modeled byassuming that full expression of heterotrophicdenitrification potential requires the absence of O2

and the presence of NO2� (Zumft, 2005). Under

conditions allowing full expression of heterotrophicdenitrification, kinetics explained the instantaneousincrease of NO upon addition of NO2

� in the model(Figure 4b, black line and Figure 4d, filled circles).

In contrast, measured NO increased slowly if theconditions before the perturbation did not allow fullexpression of denitrification (that is, the presence ofO2 or absence of NO2

�; Figure 3d, black line andFigure 4b, gray line). We assumed that in these cases,removal of O2 (Figure 3d) or addition of NO2

�

(Figure 4b) results in the expression of denitrificationenzymes. The subsequent slow NO increase wasmodeled by implementing a dynamic function (Dyn;Equation (6)) in addition to the kinetic control(Figures 3h and 4d open circles). Modeling withoutthe dynamic function resulted in an instantaneousincrease of NO for transients upon O2 removal andNO2

� addition (Figure 3h, filled circles and Figure 4d,filled triangles), because in this case the expression ofheterotrophic denitrification was assumed to beconstitutive. This shows that the requirements forthe expression of enzymes for heterotrophic denitri-fication, and their slow expression when all condi-tions are met, need to be included when modelingtransient NO accumulation.

Ammonium oxidation in transient states. Themeasurements showed that NO formed instanta-neously if O2 was removed or NO2

� was added, butonly if NH4

þ was available to allow active Aox beforethe perturbation. AOB form NO and N2O by theHAO pathway or by nitrifier denitrification (Arpand Stein, 2003). Both pathways might contribute toNO and N2O formation, but cannot be separatedfrom each other by our experiments. Our modelshowed that the instantaneous transient increase ofNO caused by AOB upon NO2

� addition (Figure 4a)cannot be explained solely with NO production by

nitrifier denitrification. Here, NO increased slowlyto steady state because of the delayed diffusion ofNO2

� into the biofilm matrix (Figure 4c, filledcircles). The transients upon NO2

� addition weresatisfactorily modeled by implementing a shiftfunction (Sh; Equation (7)), which resulted in theproduction of NO instead of NO2

� from NH4þ when

NO2� concentrations increased above a certain

threshold (YShNO�

2¼ 200 mM) (Figure 4c, open trian-

gles). The successful use of this function supportsour hypothesis that NO formation by AOB occurs iftheir active metabolism is disturbed by NO2

� thatpossibly impairs the smooth functioning of HAO(Arp and Stein, 2003).

AOB-dependent transient increase of NO duringO2 decrease in the model resulted from a micro-oxicthreshold imposed on NO production by nitrifierdenitrification (YniD

O2¼ 40 mM) (Figures 3e and f,

filled circles). The increase of NO during micro-oxic conditions was not counteracted by NO con-sumption due to nitrifier denitrification (niD-NO)because O2 disappeared before the Km value for NO(0.6 mM) was reached. Rather, the onset of anoxiaresulted in the decline of NO production by nitrifierdenitrification (niD) because of kinetic limitationsby the substrate O2. The modeled results suggestthat nitrifier denitrification metabolism is fullyexpressed under oxic conditions as reported byBeaumont et al. (2004a), resulting in instantaneousNO formation upon reduced O2 concentrations. Thisincrease cannot be counteracted until NO accumu-lates to concentrations equal to the Km value of theNO consumption pathway.

Direct regulation of NO and N2O decrease after itstransient accumulationNO and N2O decreased to a new steady-state levelafter they reached peak concentrations upon O2 andNO2

� concentrations were changed. NO concentra-tions were always one order of magnitude belowN2O, indicating that regulation of potentially cyto-toxic NO (James, 1995) is more critical than thatof nontoxic N2O, and that the Km value of N2Oreduction is higher than that of NO reduction. Thedecrease of NO within minutes after the accumula-tion of NO indicates that genetic regulation cannotexplain the dynamics. Instead, different metabolicpathways governed NO turnover after the conditionschanged, or enzymes were directly affected after thepeak was reached, for example, by inhibition.

NO decrease after O2 removal. The pathwaysgoverning NO turnover switched from nitrifier deni-trification to heterotrophic denitrification betweenoxic and anoxic conditions (Figures 3a and b). Directlyafter nitrifier denitrification stops, heterotrophic deni-trification cannot instantaneously start, as the en-zymes must be expressed. Therefore, modeling withthe dynamic function (Dyn; Equation (6)) showeda transient NO minimum when reaching anoxia,


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because NO was lost from the biofilm by diffusion andincreased slowly thereafter (Figures 3e and f, filledtriangles and open circles). However, we did notmeasure such a minimum, indicating that NOproduction continued directly after the transition.Possible explanations are that either nitrifier denitri-fication continued or the dynamic of expression ofheterotrophic denitrification (Dyn, Equation (6)) wasenhanced in the transient phase in response to NO.Modeling showed that the later scenario was notbiologically feasible, because the expression of hetero-trophic denitrification needed to be enhanced to be100-fold (Dt¼ 4s instead of 400 s) faster than wascalculated for the onset denitrification upon reducedO2 (Figures 3e and f, open triangles and Figure 3h,open circles). Alternatively, NO production by nitrifierdenitrification might continue for a short time atanoxia with oxidizing compounds stored in the formof NH2OH or oxidized cytochromes.

NO decrease after NO2� addition. O2 concentra-

tions remained stable upon the addition of NO2�,

which prevented switching between Aox andheterotrophic denitrification that could affect NO.Thus, the instantaneous decrease of NO after reach-ing the peak concentration (Figures 4a and b) mightbe regulated by direct effects on the enzymesinvolved in NO turnover within the bacteria thatproduce NO. This is supported by earlier studies,which showed that Nir and Nor can be inhibited byhigh NO concentrations in heterotrophic denitrifiers(Dhesi and Timkovich, 1984; Carr and Ferguson,1990; Koutny and Kucera, 1999).

The model (Figure 1) suggests that the transient NOmaximum after the addition of NO2

� was due to animbalance of the AOB metabolism (Sh, Equation (7)).Moreover, the decrease of the measured concentra-tion from the maximumwas slower than the modeleddecrease by diffusion only (Figure 4a, black line andFigure 4c, filled triangles), indicating that NOproduction continued during the decrease. Themodel showed that if nitrifier denitrification is theonly NO-producing process under oxic conditions,NO increased slowly after NO2

� was added (Figure 4c,filled circles). Therefore, we propose that the directformation of NO instead of NO2

� continues with adecreasing rate after it becomes active. This impliesthat NO or NO2

� concentrations directly affect theenzymes of AOB, resulting in the regulation of themetabolic imbalance.

Significance to NO and N2O formation in theenvironmentThe extent of transient NO and N2O accumulationupon perturbation strongly indicates that it cansignificantly contribute to emissions of gaseous Noxides into the atmosphere, especially from habitatsexposed to fluctuations in O2 and inorganic Ncompounds. It is possible that the large uncertain-ties about the sources in the global N2O budget

(Stein and Yung, 2003) are linked to the contributionof fluctuating emissions from ecosystems, which arenot normally considered during measurements (forexample, measurements in chambers often impedeexposure to environmental fluctuations).

Fluctuations in environmental conditions thataffect O2 and NO2

� concentrations and thus lead toNO and N2O production may occur in soils as aresult of drying and wetting, or by a variablefertilizer input. Furthermore, estuaries are exposedto a fluctuating N input through precipitation andfertilizer runoff from land. Large oceanic volumesare influenced by mixing of water masses withdifferent O2 and NO2

� concentrations. For example,massive accumulation of N2O was observed in theupper layer of the Arabian Sea during severeupwelling-induced hypoxia on the western Indianshelf (Naqvi et al., 2000). Under these conditions,the upper layer of the water body is especiallyexposed to O2 fluctuations caused by O2 inputthrough wave action. Hence, the fact that oceansare still considered a relatively minor source of N2Oin the global budget might be an underestimatelinked to difficulties in measuring and modelingN2O emissions during frequently occurring, short-term environmental perturbations in marine waters.Presently, a linear, empirical-derived framework isemployed in large-scale N2O emission models (Jinand Gruber, 2003; Duce et al., 2008). The character-istics of the metabolic model, such as thresholds,dynamic function and shift function, developed inthis study to describe transient production mechan-isms, may represent an important step toward moremechanism-based modeling.

Conclusions

In conclusion, characterization of microenvironmentalconditions is required to determine the source of NOand N2O production in complex, stratified environ-ments. The presence of O2 determines whether NOand N2O are produced by AOB or by heterotrophicdenitrifiers. Interestingly, NO and N2O formation byAOB does not require micro-oxic conditions if NO2

� ispresent in high concentrations (mM range). On theother hand, NO production, but not N2O production,by heterotrophic denitrifiers is almost independent ofNO2

� concentrations. The high temporal resolutionachieved by microsensors allowed the measurementof highly dynamic NO and N2O formation followingthe change in O2 and NO2

� concentrations. Interpreta-tion of the results with a metabolic model showed thatAox and heterotrophic denitrification need to beactively performed to respond to perturbations in O2

and NO2� with instantaneous NO and N2O production.

The resulting transient accumulation is counteractedwithin minutes by regulating the NO turnover in thebiofilm. This occurs either because a different path-way becomes active or because enzymes involved inthe production process are affected directly. At steady


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state, NO concentrations are regulated by kineticcontrol of the consumption processes. In a complexenvironment, massive formation of NO and N2O mayoccur if a metabolically active N-cycling community isexposed to an external perturbation.

Acknowledgements

We thank Phyllis Lam, Angela Schramm and the technicalassistants of the Microsensor Research Group of theMax-Planck-Institute for Marine Microbiology, Bremen,for their practical support; Olivera Kuijpers, Aaron Beckand Peter Stief for critically reading the article and theMax Planck Society for funding.

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mechanisms of transient nitric oxide and nitrous oxide

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