the anaerobic oxidation of ammonium · the anaerobic oxidation of ammonium mike s.m. jetten a;*,...

The anaerobic oxidation of ammonium

Mike S.M. Jetten a;*, Marc Strous a, Katinka T. van de Pas-Schoonen a,Jos Schalk a, Udo G.J.M. van Dongen a, Astrid A. van de Graaf a,

Susanne Logemann a, Gerard Muyzer 1;b, Mark C.M. van Loosdrecht a,J. Gijs Kuenen a

a Kluyver Institute for Biotechnology, TU Delft, Julianalaan 67, NL-2628 BC Delft, The Netherlandsb Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany

Received 26 June 1998; received in revised form 7 September 1998; accepted 7 September 1998

Abstract

From recent research it has become clear that at least two different possibilities for anaerobic ammonium oxidation exist innature. `Aerobic' ammonium oxidizers like Nitrosomonas eutropha were observed to reduce nitrite or nitrogen dioxide withhydroxylamine or ammonium as electron donor under anoxic conditions. The maximum rate for anaerobic ammoniumoxidation was about 2 nmol NH�4 min31 (mg protein)31 using nitrogen dioxide as electron acceptor. This reaction, which mayinvolve NO as an intermediate, is thought to generate energy sufficient for survival under anoxic conditions, but not forgrowth. A novel obligately anaerobic ammonium oxidation (Anammox) process was recently discovered in a denitrifying pilotplant reactor. From this system, a highly enriched microbial community with one dominating peculiar autotrophic organismwas obtained. With nitrite as electron acceptor a maximum specific oxidation rate of 55 nmol NH�4 min31 (mg protein)31 wasdetermined. Although this reaction is 25-fold faster than in Nitrosomonas, it allowed growth at a rate of only 0.003 h31

(doubling time 11 days). 15N labeling studies showed that hydroxylamine and hydrazine were important intermediates in thisnew process. A novel type of hydroxylamine oxidoreductase containing an unusual P468 cytochrome has been purified from theAnammox culture. Microsensor studies have shown that at the oxic/anoxic interface of many ecosystems nitrite and ammoniaoccur in the absence of oxygen. In addition, the number of reports on unaccounted high nitrogen losses in wastewatertreatment is gradually increasing, indicating that anaerobic ammonium oxidation may be more widespread than previouslyassumed. The recently developed nitrification systems in which oxidation of nitrite to nitrate is prevented form an ideal partnerfor the Anammox process. The combination of these partial nitrification and Anammox processes remains a challenge forfuture application in the removal of ammonium from wastewater with high ammonium concentrations. z 1999 Federationof European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Ammonium; Nitrite ; Hydrazine; Hydroxylamine; Nitrosomonas ; Oxygen; Wastewater; Nitrogen removal

0168-6445 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V.PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 2 3 - 0

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* Corresponding author. Tel. : +31 (15) 2781193; Fax: +31 (15) 2782355; E-mail: [email protected]

1 Present address: Netherlands Institute for Sea Research NIOZ, Den Burg, Texel, The Netherlands.

FEMS Microbiology Reviews 22 (1999) 421^437

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4222. Biological nature of the Anammox reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4223. Autotrophic growth during selective enrichment in continuous systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4234. Cultivation of Anammox biomass and determination of physiological parameters . . . . . . . . . . . . . . . . . . . . . . 4245. Characterization of the enriched microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425

5.1. Dominant cell type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4255.2. Cytochrome spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4255.3. Identi¢cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425

6. Aerobic versus anaerobic ammonium oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4276.1. In£uence of oxygen on anaerobic ammonium oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4276.2. Aerobic ammonium oxidizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4286.3. Metabolic versatility of Nitrosomonas strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430

7. Possible reaction mechanisms for Anammox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4318. Substrate spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4339. Ecological habitats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433

10. Application of the Anammox process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43411. The combined SHARON-Anammox process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436

1. Introduction

The oxidation of ammonium has been investigatedmainly in aerobic or oxygen-limited systems. Intheory ammonium could also be used as an inor-ganic electron donor for denitri¢cation. The free en-ergy for this reaction (Table 1) is nearly as favorableas for the aerobic nitri¢cation process. It was on thebasis of these thermodynamic calculations that theexistence of two chemolithoautotrophic microorgan-isms capable of oxidizing ammonium to dinitrogengas was already predicted two decades ago [1]. Theactual discovery of such a process was only recently

described [2,3]. During experiments on a denitrifyingpilot plant of a multi-stage wastewater treatment sys-tem at Gist-Brocades (Delft, The Netherlands) it wasnoted that ammonium disappeared from the reactore¥uent at the expense of nitrate with a concomitantincrease in dinitrogen gas production. A maximumammonium removal rate of 1.2 mmol l31 h31 wasobserved. In continuous £ow experiments the nitro-gen and redox balances showed that ammoniumreally disappeared under anaerobic conditions, andthat for every mol of ammonium consumed 0.6 molof nitrate was required, resulting in the productionof 0.8 mol of dinitrogen gas. This newly discoveredprocess was named the Anammox (anaerobic ammo-nium oxidation) process.

2. Biological nature of the Anammox reaction

In a follow-up study, the biological nature of theAnammox process was investigated in more detail[4]. In anoxic batch experiments ammonium and ni-trate were converted within 9 days of incubationwhen intact seed material (Anammox biomass)from the pilot plant was added. However, when theseed material was subjected to Q-radiation or sterili-

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Table 1Gibbs free energy of several reactions involved in autotrophicdenitri¢cation [3,8]

Reaction equation vG³P(kJ mol31 NH�4or NO3

3 )

2NO33 +5H2+2H�CN2+6H2O 3560

8NO33 +5HSÿ+3H�C4N2+4H2O+5SO23

4 34653NO3

3 +5NH�4 C4N2+9H2O+2H� 3297NO3

2 +NH�4 CN2+2H2O 33582O2+NH�4 CNO3

3 +H2O+2H� 33496O2+8NH�4 C4N2+12H2O+8H� 3315

M.S.M. Jetten et al. / FEMS Microbiology Reviews 22 (1999) 421^437422

zation at 121³C or when the seed material was omit-ted from the incubation no change in the concentra-tion of nitrate and ammonium could be observed.Furthermore the addition of various inhibitors (2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhy-drazone or HgCl2) to the incubations resulted in acomplete inhibition of the ammonium oxidation andnitrate reduction (Table 2). In these experiments withinitial ammonium concentrations of 5 mM and high-er, the rate of ammonium oxidation was proportion-al to the amount of biomass used. Taken togetherthese experiments strongly suggested that the anaer-obic ammonium oxidation was a biological processcarried out by microorganisms. The speci¢c rate ofammonium oxidation (0.08 nmol NH�4 min31 (mgdry weight)31) in these batch experiments was quitelow compared to rates (1.2 nmol NH�4 min31 (mgdry weight)31) obtained in the pilot plant. This in-dicated that in the batch experiments the conversionwas limited by di¡usion of the substrates to the bio-mass granules. Labeling experiments with 15NH�4 incombination with 14NO3

3 showed an almost exclusiveproduction of 14ÿ15N2 gas. This ¢nding did not agree

with the postulated overall reaction [3] (see Section1) in which for every labeled ammonium, 0.6 nitratewould react to form 0.8 dinitrogen gas (i.e. 75% ofthe formed dinitrogen would be 15ÿ14N2 and 25%would be 15ÿ15N2). Only if nitrite rather than nitratewas assumed as the actual oxidizing agent the ob-served and calculated values would be in agreement[4].

3. Autotrophic growth during selective enrichment incontinuous systems

Once it was realized that nitrite rather than nitratemight be the electron acceptor of autotrophic deni-tri¢cation with ammonium as electron donor, a me-dium was composed for the selective enrichment ofthe microorganisms responsible for the Anammoxprocess. This medium contained ammonium (5^30 mM), nitrite (5^35 mM), bicarbonate (10 mM),minerals and trace elements [5]. The phosphate con-centration of the medium was kept below 0.5 mMand the oxygen concentration below detection levels

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Table 2E¡ect of various treatments with stimulators and inhibitors on the anaerobic ammonium-oxidizing activity in batch experiments withbiomass from the Anammox pilot plant (adapted after [4,6,14])

Treatment inhibitor/stimulator Mode of action Concentration orperiod tested

E¡ect

NH�4 �NO32 activity test 0^7 mM normal activity

No biomass none 0 mg l31 no activitySterilization at 121³C denaturation 20^120 min no activityGamma irradiation inactivation 60 min no activtiyPenicillin V inhibition of cell wall synthesis of bacteria 0^100 mg l31 nonePenicillin G id. 0^1000 mg l31 noneBromoethane sulfonic acid inhibition of methanogenesis 0^20 mM noneNa2SO4 stimulation of sulfate reduction 20 mM noneNa2MoO4 inhibition of sulfate reduction 20 mM noneChloramphenicol inhibition of protein synthesis 0^400 mg l31 noneHydrazine inhibition of NH2OH oxidation 0^3 mM activationAcetone solvent for N-serve 10 mM noneN-serve inhibition of nitri¢cation 0^50 mg l31 noneAllylthiourea inhibition of nitri¢cation 0^10 mM noneAcetylene inhibition of nitri¢cation and

denitri¢cation6 mM inhibition

2,4-Dinitrophenol uncoupler 0^400 mg l31 inhibitionCarbonyl cyanide m-chlorophenylhydrazone uncoupler 0^40 mg l31 inhibitionHgCl2 cell damage 0^300 mg l31 inhibitionOxygen oxidative stress 0^0.2 mM inhibitionPhosphate chelating agent 6 1 mM nonePhosphate chelating agent s 2 mM inhibition

M.S.M. Jetten et al. / FEMS Microbiology Reviews 22 (1999) 421^437 423

(6 1 WM) in order to avoid possible inhibitory e¡ects(Table 2). Since the speci¢c rate of ammonium oxi-dation in batch experiments was considerably lowerthan in perfusion systems a £uidized bed reactor waschosen to perform the enrichment. Using biomassfrom the original pilot plant as an inoculum, it waspossible to obtain stable enrichment cultures within3^4 months of operation. In total more than 20 re-actor runs have been carried out with synthetic me-dium, the longest (Fig. 1) lasting more than 27months [5^7]. So far only two runs failed in theenrichment mainly due to mechanical problems ofthe setup. After enrichment with synthetic mediumthe conversion rate in the reactor systems increasedfrom 0.4 kg NH�4 N m33 per day to about 3 kg NH�4N m33 per day. The maximum speci¢c activity of thebiomass in the £uidized bed reactor was about25 nmol NH�4 min31 (mg dry weight)31. For everymol of CO2 incorporated into biomass 24 mol ofammonium had to be oxidized. When biomassfrom the reactors was used in batch experimentssupplied with ammonium, nitrite and 14CO2, the cellsbecame rapidly labeled. The incorporation of labelwas completely dependent on the combined presenceof both nitrite and ammonium. The ribulose bis-phosphate carboxylase activity of cell extracts wasonly 0.3 nmol CO2 min31 (mg dry weight)31 which

is 3-fold lower than expected on the basis of thestoichiometry determined for ammonium and bicar-bonate conversion (24:1). The estimated growth ratein the £uidized bed systems was 0.001 h31, which isequivalent to 1 doubling time of about 29 days. Themain product of the reaction was dinitrogen gas,but about 17% of the nitrite supplied could berecovered as nitrate. The overall nitrogen balanceaveraged over 15 runs showed a ratio of1:1.31:0.22 for conversion of ammonium and nitriteto the production of nitrate. In the £uidized bedreactor no other intermediates like hydroxylamine,hydrazine, NO or nitrous oxide could be detected.The production of nitrate from nitrite was veri¢edwith 15N-NMR analysis [8]. Only when labeled ni-trite was supplied to the cultures could formation of15NO3

3 be observed. In the presence of 15N labeledammonium, no 15NO3

3 was ever observed [8]. Thefunction of this nitrate formation from nitrite is as-sumed to be the generation of reducing equivalentsnecessary for the reduction of CO2.

4. Cultivation of Anammox biomass anddetermination of physiological parameters

Currently available microbiological techniques are

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Fig. 1. Operation of a £uidized bed reactor for the enrichment and maintenance of anaerobic ammonium-oxidizing microorganisms. Themedium was composed of ammonium sulfate, sodium nitrite, NaHCO3, minerals, trace elements [5,6]. The gray area represents the nitriteand ammonium load into the reactor; the black area is the nitrite or ammonium load out of the reactor. The average removal percentage(b) over 934 days was 99.5% for nitrite and 84.6% for ammonium.


not designed very well to deal with very slowly grow-ing microorganisms such as the Anammox culture.In addition to the £uidized bed systems, a sequenc-ing batch reactor (SBR) was applied and optimizedfor the quantitative study of the microbial commun-ity that oxidized ammonium anaerobically [9]. TheSBR was a powerful experimental setup in which thebiomass was retained very e¤ciently (s 90%). Fur-thermore a homogeneous distribution of substrates,products and biomass aggregates over the reactorwas achieved, and the reactor has been in operationreliably for more than 2 years under substrate-limit-ing conditions. Several important physiological pa-rameters ([9] M. Strous, personal communication)such as the biomass yield (0.066 þ 0.01 C mol (molammonium)31), the maximum speci¢c ammoniumconsumption rate (45 þ 5 nmol min31 (mgprotein)31) and the maximum speci¢c growth rate(0.0027 h31, doubling time 11 days) could now bedetermined more accurately than with the £uidizedbed reactors. The temperature range for Anammoxwas 20^43³C (with an optimum at 40³C). The Anam-mox process functioned well at pH 6.7^8.3 (with anoptimum at pH 8). Under optimal conditions themaximum speci¢c ammonium oxidation rate wasabout 55 nmol min31 (mg protein)31. The a¤nityfor the substrates ammonium and nitrite was veryhigh (a¤nity constants9 5 WM) (M. Strous, personalcommunication). The Anammox process was inhib-ited by nitrite at nitrite concentrations higher than 20mM but lower nitrite concentrations (s 10 mM)were already suboptimal. When nitrite was presentat high concentrations for a longer period (12 h),Anammox activity was completely lost. In addition,the persisting stable and strongly selective conditionsof the SBR led to a high degree of enrichment (74%of the desired dominant peculiar microorganisms, seeSection 5).

5. Characterization of the enriched microorganisms

5.1. Dominant cell type

The dominant microorganism of the enrichmentcultures was a Gram-negative light-breaking coccoidcell (Fig. 2A,B) which showed an unusual irregularmorphology under the electron microscope (Fig. 2C)

when ¢xed with 2.5% glutaraldehyde in 20 mMK2HPO4/KH2PO4 bu¡er pH 7.4. Once the unusualmorphology of the cells was recognized, an estimateof the enrichment from the original culture could bemade by counting the cells in a large number of thinsections. After 177 days of enrichment 64% of allcells counted (7317 out of 11 433 total) were of thedescribed type. This was a four-fold increase (16%;1632 out of 10 200 total) compared to the numbersfound in the biomass from the pilot plant. Togetherwith the increase in cell numbers of this peculiarorganism an increase in the percentage of ether-likelipids was observed. The amount of ester lipids typ-ical for most Bacteria remained more or less con-stant [5]. The presence of the ether lipids seems tobe con¢ned to most ancient microorganisms such asthe Archaea or very deep-branching Bacteria likeThermotoga and Aquifex. A detailed knowledge ofthe exact structure and composition of the lipids ofthe dominant cell type would be most helpful to ¢ndthe taxonomic a¤liation of these cells [5].

5.2. Cytochrome spectra

During the enrichment on synthetic medium, thecolor of the biomass changed from brown to deepred. Visible spectra of cells and cell extracts of theenriched culture showed a pronounced increase inthe signal for cytochromes of the c type. Spectra ofcells at 77 K revealed the absence of a-type, b-typeand d1-type cytochromes. Very interestingly, duringincrease in Anammox activity of the biomass, grad-ually an increased signal was observed at 468 nm. InFig. 3 a typical spectrum of an Anammox cell extractwith the 468-nm feature is shown. This absorptionpeak at 468 nm disappeared irreversibly after treat-ment with carbon monoxide. A similar signal hasbeen observed in aerobic ammonium-oxidizing bac-teria at 463 nm [10^13]. This signal was attributed toone of the hemes present in the enzyme hydroxyl-amine oxidoreductase and is mostly referred to ascytochrome P460.

5.3. Identi¢cation

So far many isolation methods including serial di-lution, £oating ¢lters, and plating have been used toobtain the responsible microorganisms in pure cul-

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ture. None of the isolates thus obtained is able toperform the Anammox reaction, but many of theisolates are novel denitrifying oligotrophic (proteo)-bacteria. In addition to classical microbial techni-ques, the Anammox community was analyzed usingmodern molecular biological methods. The genomicDNA was extracted and ampli¢ed via PCR using(eu)bacterial primers 27f-BamHI (5P-CACGGATC-CAGAGTTTGATMTGGCTTCAG-3P), and 1492r-HindIII (5P-TGTAAGCTTACGGYTACCTTGTT-ACGACT-3P). The PCR products were cloned, and396 out of the more than 4000 clones obtained werescreened. One dominant (28%) clone belonging tothe Cytophaga/Flexibacter phylum was identi¢ed.However, in situ analysis with £uorescent probesspeci¢c for the Cytophaga phylum did not con¢rmthe molecular identity of the dominant organism as aCytophaga.

6. Aerobic versus anaerobic ammonium oxidation

The presence of a P460-like signal in the enrichedAnammox biomass, and the recent reports on themetabolic versatility of aerobic ammonium oxidizersinitiated a more detailed investigation. The studieswere concentrated on three issues: the in£uence ofoxygen on the Anammox process, the number ofaerobic ammonium oxidizers present in the Anam-mox enrichment cultures and the (anaerobic) capa-bilities of `classical' nitri¢ers of the Nitrosomonastype.

6.1. In£uence of oxygen on anaerobic ammoniumoxidation

The in£uence of oxygen on the Anammox processwas investigated in both batch and continuous sys-

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Fig. 2. A: Micrograph of a biomass aggregate from an Anammox enrichment culture. The dominant coccoid cell is present in packagesand microcolonies. B: Micrograph of the dominant coccoid cell present in the Anammox enrichment cultures. Preparation was obtainedafter sedimentation of suspended material from a £uidized bed reactor. C: Electron micrograph of suspended Anammox biomass ¢xedwith 2.5% glutaraldehyde in 20 mM K2HPO4/KH2PO4 bu¡er pH 7.4. The micrograph was taken at the Department of Electron Micro-scopy (I. Keizer, K. Sjollema, M. Veenhuis), State University of Groningen, The Netherlands.


tems. Initial batch experiments showed that oxygencompletely inhibited the Anammox activity when itwas deliberately introduced into the enrichment cul-tures [5,14]. In a follow-up study an intermittentlyoxic (2 h) and anoxic (2 h) reactor system wasused to study the reversibility of the oxygen inhi-bition for 20 days [15]. From these studies it becameclear that ammonium was not oxidized in the oxicperiods, but that the Anammox activity in the anoxicperiods remained constant throughout the experi-ment, indicating that the inhibitory e¡ect of oxygenwas indeed reversible. The sensitivity of the Anam-mox enrichment culture to oxygen was furtherinvestigated under various sub-oxic conditions. Infour consecutive experiments, the oxygen tensionwas decreased stepwise from 2 to 0% of airsaturation (Fig. 4). No ammonium was oxidizedin the presence of 0.5, 1, or 2% of air. Only whenall the air was removed from the reactor byvigorously £ushing with argon gas, the conversionof ammonium and nitrite resumed, thus indicatingthat the Anammox activity in these enrichmentcultures is only possible under strict anoxic condi-tions.

6.2. Aerobic ammonium oxidizers

The second question which was addressed in thesestudies concerned the number of aerobic ammonium-oxidizing bacteria present in the Anammox enrich-ment cultures. Using standard, aerobic most prob-able number (MPN) methods, the number of nitri-¢ers was estimated to be 9 þ 5U103 cells ofammonium oxidizers per milliliter of biomass sam-ple. Electron micrographs of the MPN culturesshowed the characteristic cytoplasmic membranestructures reported for several aerobic ammonium-oxidizing bacteria [16]. The consistent presence ofthese aerobic nitri¢ers in the Anammox biomass con-¢rmed that they can survive very long periods ofanaerobiosis as was previously shown by Abeliovich[17]. Furthermore, it was possible to enrich such or-ganisms in a repeated fed-batch reactor when oxygenwas continuously supplied at 50^80% of air satura-tion [15]. The enriched aerobically nitrifying com-munity grew exponentially with a doubling time of1.2 days. Interestingly, not all of the 27 mM of am-monium supplied could be recovered as nitrite. Sincenitrite was not further oxidized to nitrate, the re-

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Fig. 3. Cytochrome spectrum of cell extract from an Anammox enrichment culture. Dashed line, oxidized spectrum; solid line, spectrumafter reduction with dithionite. Inset shows a close-up between 440 and 600 nm. The arrow indicates the typical peak at 468 nm.


mainder of the nitrogen might have been lost as gas-eous nitrogen compounds (NO, N2O or N2) duringaerobic denitri¢cation. After enrichment of the nitri-¢ers, also this culture was subjected to the same al-ternating 2 h oxic/2 h anoxic regime to verify if thesenitri¢ers were capable of an anaerobic conversion ofammonium or nitrite. During 20 days, the fate of thesupplied 30 mM ammonium was followed and it wasobserved that only in the aerobic periods oxidationof ammonium to nitrite occurred. This indicated that

this community of aerobic ammonium-oxidizing bac-teria was not able to use nitrite as an electron accep-tor in this case.

This is in contrast to observations made with sev-eral pure cultures of di¡erent Nitrosomonas strains.Poth showed that a new Nitrosomonas isolate wasable to produce dinitrogen gas under anaerobic con-ditions [18,19], while Abeliovich and Vonshak dem-onstrated the reduction of nitrite with pyruvate aselectron donor by N. europaea [20].

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Fig. 4. The Anammox activity at four di¡erent air saturations (A 2%, B 1%, C 0.5%, and D 0%). Only when all oxygen was removedfrom the incubation by £ushing with argon gas the disappearance of ammonium (b) and nitrite (F) could be observed [15].


6.3. Metabolic versatility of Nitrosomonas strains

More recently substantial N losses have been re-ported for both mixed and pure cultures of N. eutro-pha grown under oxygen limitation [16,21,22], andfor pure cultures of N. europaea in anoxic batch ex-periments [23]. When molecular hydrogen was usedas an electron donor for nitrite reduction, growth ofN. eutropha was stoichiometrically coupled to nitritereduction with dinitrogen gas and nitrous oxide asend products. In mixed cultures of N. eutropha andEnterobacter aerogenes 2.2 mM of ammonium andnitrite were consumed during 44 days of incubation,but no cell growth was observed [16]. In a follow-upstudy the rate of anaerobic ammonium oxidation byN. eutropha could be estimated at 0.08 nmol NH�4min31 (mg protein)31 which is equivalent to about0.2 nmol NH�4 min31 (mg dry weight)31. However,when the nitrogen atmosphere of the incubationswas supplemented with 25 ppm nitrogen dioxide,the rate increased 10-fold to 2.2 nmol NH�4 min31

(mg protein)31 [21]. It was estimated that 40^60% ofthe formed nitrite (and NO) was denitri¢ed to dini-trogen gas while N2O and hydroxylamine were de-tected as intermediates. The source of oxygen for theoxidation of ammonia under these anoxic conditionsremained unknown, but could theoretically be de-rived from either NO, NO2 or nitrite. Very recentlyit was shown that N. eutropha also exhibited denitri-fying capabilities in the presence of NO2 when thedissolved oxygen concentration was maintained at

3^4 mg l31 [22]. In these experiments with completebiomass retention 50% of the produced nitrite wasaerobically denitri¢ed to dinitrogen gas. NO gas wasmuch less e¤cient in stimulating this aerobic denitri-¢cation than NO2 and became toxic above 25 ppm.Furthermore, an eight-fold increased aerobic nitri¢-

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Fig. 5. Concentration pro¢le of ammonium (b), nitrite (F), hy-droxylamine (R) and hydrazine (8) during anaerobic batch ex-periments with an Anammox culture supplemented with 3 mMhydroxylamine [8].

Table 3Rates of anaerobic oxidation (nmol min31 (mg protein)31) of ammonium, hydroxylamine and hydrazine by various cultures in batch ex-periments

Culture Compounds tested NO32

conversion rateNH2OH/NH�4 /N2H4

conversion rateProducts Reference

N. europaea NH2OH+NO32 2 3 N2O [23]

N. europaea NH�4 �NO32 2 3 N2O [23]

N. eutropha H2+NO32 7 not applicable N2O, N2 [16]

N. eutropha NH�4 �NO32 6 1 6 1 N2O [16]

N. eutropha NH�4 �NO32 0.9 1.1 NO, N2O [21]

Anammox NH�4 �NO32 12 9 N2 [5]

Anammox NH2OH n/a 12 N2 [8]Anammox N2H4+NO3

2 13 11 NH�4 , N2 [8,24]Anammox NH�4 �NO3

2 68 55 N2 [9], M. Strous,personal communication


cation rate and higher cell numbers were observedwhen the air was supplemented with 25^50 ppmNO2. In Table 3 a summary is presented of the re-ported rates of anaerobic ammonium oxidation invarious experiments with Nitrosomonas and/orAnammox cultures. From this table it is evidentthat the speci¢c rates for anaerobic ammonium oxi-dation of the classical nitri¢ers are 25-fold lowerthan the rates observed in the Anammox processstudied in Delft. Furthermore, aerobic ammoniumoxidizers prefer to use oxygen as the terminal elec-tron acceptor, whereas this compound completelyinhibits the Anammox process. Taken together theseexamples showed that further research to elucidatethe role of nitrogen oxides during (an)aerobic ammo-nium oxidation is necessary.

7. Possible reaction mechanisms for Anammox

The possible metabolic pathway for anaerobic am-monium oxidation was investigated using 15N label-ing experiments. These experiments showed thatammonium was biologically oxidized with hydroxyl-amine as the most probable electron acceptor [8].The hydroxylamine itself is most likely derivedfrom nitrite. In batch experiments with excess hy-droxylamine and ammonium, a transient accumula-tion of hydrazine was observed (Fig. 5). The conver-sion of hydrazine to dinitrogen gas is postulated asthe reaction generating the electron equivalents forthe reduction of nitrite to hydroxylamine. Whetherthe reduction of nitrite and the oxidation of hydra-

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Table 4Comparison of the properties of the hydroxylamine oxidoreductase (HAO) enzyme isolated from Anammox (J. Schalk, personal commu-nication) and Nitrosomonas europaea [12]

HAO of Anammox HAO of Nitrosomonas

Molecular mass 150 kDa 125^175 kDaSubunit 60^95 kDa 63 kDaComposition K2^K3 K2^K3

Ratio 410/280 4.5 3.3Heme 22 þ 4/150 kDa 24/K3

Active center P468 P463

Vmax 21 U mg31 75 U mg31

Km 26 WM not reportedpH optimum 8 8pI 5.5 5.3N-terminus blocked DISTV

Fig. 6. Possible reaction mechanisms and cellular localization ofthe enzyme systems involved in anaerobic ammonium oxidation.A: Ammonium and hydroxylamine are converted to hydrazineby a membrane-bound enzyme complex, hydrazine is oxidized inthe periplasm to dinitrogen gas, nitrite is reduced to hydroxyl-amine at the cytoplasmic site of the same enzyme complex re-sponsible for hydrazine oxidation with an internal electron trans-port. B: Ammonium and hydroxylamine are converted tohydrazine by a membrane-bound enzyme complex, hydrazine isoxidized in the periplasm to dinitrogen gas, the generated elec-trons are transferred via an electron transport chain to nitrite re-ducing enzyme in the cytoplasm.


zine occur at di¡erent sites of the same enzyme (Fig.6A) or the reactions are catalyzed by di¡erent en-zyme systems connected via an electron transportchain (Fig. 6B) remains to be investigated. The oc-currence of hydrazine as an intermediate in microbialnitrogen metabolism is rare [24]. Hydrazine has beenproposed as an enzyme-bound intermediate in thenitrogenase reaction [25]. Furthermore, the puri¢edhydroxylamine oxidoreductase (HAO) of N. euro-paea is capable of catalyzing the conversion of hy-drazine to dinitrogen gas [12]. The ¢nding of highHAO activity in cell extracts of the Anammox cul-ture indicated that a similar enzyme might be oper-ative in the Anammox process. Indeed very recentlya novel type of HAO was puri¢ed from the Anam-mox community via anion exchange and gel ¢ltra-tion chromatography (J. Schalk, personal communi-cation). Native PAGE showed that the Anammoxenzyme had a smaller molecular mass than the en-zyme of Nitrosomonas. Furthermore, the amino acidsequence of several HAO peptide fragments wasunique, without any homologue in the databases.Similar to the Nitrosomonas hydroxylamine oxidore-ductase, the enzyme from Anammox contained sev-eral c-type cytochromes. The special spectroscopicP460-like feature was found at 468 nm in the Anam-mox enzyme. The enzyme was able to catalyze theoxidation of both hydroxylamine and hydrazine.Although hydroxylamine was the preferred substratethe rate of hydrazine oxidation in cell extracts (150nmol min31 (mg protein)31) was high enough tosustain a growth rate of 0.003 h31. In Table 4some properties of the two HAO enzymes are sum-marized (J. Schalk, personal communication).

The formation of hydroxylamine via an ammo-nium monooxygenase seems highly improbable, sincethe Anammox reaction is strongly but reversibly in-hibited by oxygen. An alternative mechanism for theformation of hydroxylamine might be the incompletereduction of nitrite by a cytochrome c-type nitritereductase. However, it will be very di¤cult to obtaindirect evidence for this mechanism in Anammox.Hydroxylamine is the compound most rapidly me-tabolized by Anammox, and a selective inhibitorhas not yet been discovered.

FEMSRE 624 25-1-99

Fig. 7. Concentration pro¢le of ammonium (b) and nitrite (F) inthe absence of methane in the head space, and of ammonium(a) and nitrite (E) in the presence of 50% methane in the argon/CO2 head space during anoxic batch experiments with Anammoxbiomass.

Table 5Possible reaction equations of anaerobic ammonium oxidation via NO or HNO as intermediates, adapted after [12,14]

NO as intermediateNO+NH3+3H�+3e3 CN2H4+H2O (ammonia monooxygenase-like enzyme)N2H4 CN2+4H�+4e3 (hydroxylamine oxidoreductase-like enzyme)NO3

2 +2H�+e3 CNO+H2O (nitrite reductase)

NH3+NO32 +H� CN2+2H2O

HNO as intermediateHNO+NH3 CN2H2+H2O (ammonia monooxygenase-like enzyme)N2H2 CN2+2H�+2e3 (hydroxylamine oxidoreductase-like enzyme)NO3

2 +2H�+2e3 CHNO+OH3 (nitrite reductase)

NH3+NO32 CN2+H2O+OH3


A possible role of NO or HNO in (an)aerobicammonium oxidation was proposed by Hooper [12]to be a condensation of NO or HNO and ammoniaon an enzyme related to the ammonium monooxy-genase family (Table 5). The formed hydrazine orimine could thereafter be converted by the enzymehydroxylamine oxidoreductase into dinitrogen gasand the reducing equivalents required to combineNO or HNO and ammonia or to reduce nitrite toNO.

8. Substrate spectrum

Aerobic ammonium and methane oxidizers areable to catalyze both the oxidation of ammoniumand methane [26] albeit at di¡erent rates. The abilityof the Anammox culture to use methane or othersubstrates was tested in batch experiments. In Fig.7 it is shown that addition of methane to the argon/CO2 head space of the incubations did not lead to aninhibition of ammonium and nitrite conversion. This

indicated that the enzyme responsible for anaerobicammonium conversion is di¡erent from the aerobicammonia or methane monooxygenases. In longer ex-periments it could also be shown that methane itselfwas not converted by the Anammox biomass (Fig.8). In addition to methane also hydrogen was testedin batch incubations (Fig. 9). The addition of hydro-gen to the argon/CO2 head space showed a clearstimulation of the anaerobic ammonium oxidationin short-term experiments. However, hydrogen couldnot replace ammonium as electron donor in theseexperiments. Addition of various organic substances(pyruvate, methanol, ethanol, alanine, glucose, casa-mino acids) in short-term batch experiments led to asevere inhibition of the Anammox activity. Thus thesubstrate spectrum seems to be restricted to ammo-nium, nitrite and the intermediates hydrazine andhydroxylamine. However, supplementation with1 mM hydrazine could not sustain growth of theAnammox culture for longer periods [24].

9. Ecological habitats

The discovery of the Anammox process in a deni-

FEMSRE 624 25-1-99

Fig. 9. Concentration pro¢le of ammonium (b) and nitrite (F) inthe presence of 95% hydrogen and 5% CO2 gas in the headspace, and of ammonium (a) and nitrite (E) in the presence of95% argon and 5% CO2 gas in head space during anoxic batchexperiments with Anammox biomass.

Fig. 8. The absence of methane conversion by Anammox bio-mass at two di¡erent methane concentrations in the head space.Closed triangles (R, 380 Wmol methane) and diamonds (8, 195Wmol methane) represent incubations with Anammox biomass inthe presence of 300 Wmol nitrite as electron acceptor. The closedcircles represents a control incubation of Anammox biomass with280 Wmol ammonium (b), and 300 Wmol nitrite as electron ac-ceptor. In the control all nitrite was converted, in the incubationswith methane the nitrite was not converted.


trifying pilot plant has raised the question as towhere else such organisms would occur in nature.Already in 1932 it was reported that dinitrogen gaswas generated via an unknown mechanism duringfermentation in the sediments of Lake Mendota(USA) [27]. Also in sediments of Lake Kizakiko (Ja-pan) indications were found for the direct formationof dinitrogen gas from ammonium [28]. Very re-cently these observations were con¢rmed in studieswith freshwater sediments [29]. One prerequisite forthe occurrence of anaerobic ammonium oxidationvia an Anammox mechanism would be the simulta-neous presence of both ammonium and nitrite (ornitrate) and the absence of oxygen. The nitrite couldbe formed either in ecosystems in which oxygen (dif-fusion) limits nitri¢cation or in places with a limitedsupply of electron donor (sul¢de or organic substan-ces) for denitri¢cation of nitrate. The oxic/anoxicinterface of many sediments would thus be an idealhabitat for anaerobic ammonium-oxidizing microor-ganisms. Micro-electrode studies have revealed over-lapping pro¢les of nitrate and ammonium in a strati-¢ed zone of the Black Sea [30,31] indicating that ahabitat for Anammox really exists. More recentlyafter the development of nitrite microsensors, alsooverlapping pro¢les of nitrite and ammonium havebeen reported in oxygen-limited nitrifying activatedsludge £ocs [32^34]. Indeed man-made ecosystemslike wastewater treatment plants could create a hab-itat for the Anammox organisms. The abundant sup-ply of ammonium via the wastewater in combinationwith a limited oxygen availability would provide

conditions in which both ammonium and nitritecould occur. High nitrogen losses (70^90%) in theform of dinitrogen gas have been reported in tworotating biological contractor systems [35^37] treat-ing land¢ll leachate with about 200^400 mg ammo-nium per liter. Comparison of the microorganisms inthese systems with the Delft Anammox culturewould give more insight into the biodiversity of theanaerobic ammonium oxidation.

10. Application of the Anammox process

In a recent study [7,38] the feasibility of the Anam-mox process for the removal of ammonium fromsludge digester e¥uents was evaluated. The resultsof this study showed that the Anammox biomasswas not negatively a¡ected by the digester e¥uent.The pH (7.0^8.5) and temperature (30^37³C) optimafor the process were well within the range of thevalues found in digester e¥uents. Experiments witha laboratory-scale (2-l) £uidized bed reactor showedthat the Anammox biomass was capable of removingammonium and (externally added) nitrite e¤cientlyfrom the sludge digester e¥uent (Table 6). The nitro-gen load of the Anammox £uidized bed reactorcould be increased from 0.46 kg Ntot m33

reactor perday to 2.6 kg Ntot m33

reactor per day. Due to thenitrite limitation, the maximum capacity was notreached. The nitrogen conversion rate during the ex-periment with sludge digester e¥uents increasedfrom 0.05 kg Ntot kg31 SS per day to 0.26 kg Ntot

FEMSRE 624 25-1-99

Table 6Overview of the parameters of an Anammox £uidized bed reactor [7,38] and a SHARON reactor [38] both fed with sludge digester e¥u-ent. The nitrite for the Anammox process was supplied separately

SHARON Anammox

Ammonium load 0.63^1.0a 0.24^1.34 kg NH�4 N m33reactor day31

Nitrite load not applicable 0.22^1.29 kg NO32 N m33

reactor day31

Nitrogen load 0.63^1.0 0.46^2.63 kg Ntot m33reactor day31

NH�4 N e¥uent 199 27 þ 85 mg N l31

NO32 N e¥uent 469 3 þ 3 mg N l31

E¤ciency NH�4 N 76^90 88 þ 9 %RemovalE¤ciency NO3

2 N n/a 99 þ 2 %RemovalSludge load 10.3 0.05^0.26 kg Ntot kg31 SS per day

aThis value is linearly proportional to the in£uent concentration.


kg31 SS per day. The Anammox sludge biomass re-moved 88% of the ammonium and 99% of the nitritefrom the sludge digester e¥uent (Table 6). In thesestudies nitrite was supplied from a concentratedstock solution. However, for application in realwastewater practice, a suitable system for biologicalnitrite production has to be developed. One suchsystem is the SHARON (single reactor high activityammonium removal over nitrite) process [38].

This SHARON process is ideally suited to removenitrogen from waste streams with a high ammoniumconcentration (s 0.5 g N l31). The SHARON proc-ess is performed in a single, stirred tank reactor with-out any sludge retention. At temperatures above25³C it is possible to e¡ectively outcompete the ni-trite oxidizers. This results in a stable nitri¢cationwith nitrite as end-product [38]. When combinedwith the Anammox process only 50% of the ammo-nium needs to be converted to nitrite. This impliesthat no extra addition of base is necessary, sincemost of the wastewater resulting from anaerobic di-gestion will contain enough alkalinity (in the form ofbicarbonate) to compensate for the acid productionif only 50% of the ammonium needs to be oxidized.The SHARON process has been extensively tested atthe laboratory scale for the treatment of sludge di-gester e¥uents (Table 6) and is currently under con-struction at two Dutch wastewater treatment plants.

11. The combined SHARON-Anammox process

The combination of the Anammox process andSHARON process has been tested in our laboratory

using sludge digester e¥uent (Fig. 10). TheSHARON reactor was operated without pH controlwith a total nitrogen load of about 0.8 kg N m33 perday [38]. The ammonium present in the sludgedigester e¥uent was converted to nitrite and a smallamount of nitrate (11%) (Table 7). The nitrate for-mation was due to bio¢lm wall growth, which wasnot always regularly removed. In large-scale applica-tions this will be signi¢cantly lower because of thelarger volume to surface ratio. In this way an ammo-nium-nitrite mixture suitable for the Anammoxprocess was generated. The e¥uent of the SHARONreactor was used as in£uent for the Anammox £uid-ized bed reactor. In the nitrite-limited Anammox re-actor all nitrite was removed, the surplus ammoniumremained. During the test period the overall ammo-nium removal e¤ciency was 83%. In Table 7 thenitrogen balances of the two systems are summar-ized. The optimization and application of the combi-nation of these two processes on pilot plant and fullscale remain challenges towards implementations ina future wastewater treatment plant.

Acknowledgments

The research on anaerobic ammonium oxidation

FEMSRE 624 25-1-99

Fig. 10. Ammonium removal from sludge digester e¥uent withthe combined SHARON-Anammox system. Ammonium (8) ornitrite (F) load in the e¥uent of the SHARON reactor is usedas the ammonium or nitrite load into the Anammox reactor. Theammonium or nitrite load in the e¥uent of the Anammox is rep-resented by open diamonds and open squares, respectively. ThepH value in the Anammox reactor was stable at 7.8 [38].

Table 7Nitrogen balances in the combined SHARON-Anammox system

SHARON Anammox

In£uent E¥uent/In£uent E¥uent

NH�4 584 (100%) 267(46%) 29 (5%)NO3

2 6 1 227 (39%) 1.4NO3

3 6 1 64 (11%) 83 (14%)N2Oa 6 1 4 6 1N2

a 6 1 6 1 476b (82%)

Results are mg N l31, percentages are given in parentheses.aConcentration relative to the in£uent £ow.bDetermined as the di¡erence between dissolved and gaseous nitro-gen compounds.


was ¢nancially supported by the Foundation for Ap-plied Sciences (STW), the Foundation of AppliedWater Research (STOWA), the Netherlands Foun-dation for Life Sciences (NWO-SLW), the RoyalNetherlands Academy of Arts and Sciences(KNAW), Gist-Brocades, DSM, and Grontmij con-sultants. The contributions of various co-workersand students over the years are gratefully acknowl-edged.

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