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The anaerobic oxidation of ammonium


FEMS Microbiology Reviews 22 (1999) 421^437

The anaerobic oxidation of ammonium
Mike S.M. Jetten ?Y *, Marc Strous ? , Katinka T. van de Pas-Schoonen ? , Jos Schalk ? , Udo G.J.M. van Dongen ? , Astrid A. van de Graaf ? , Susanne Logemann ? , Gerard Muyzer IY? , Mark C.M. van Loosdrecht ? , J. Gijs Kuenen ?
?

Kluyver Institute for Biotechnology, TU Delft, Julianalaan 67, NL-2628 BC Delft, The Netherlands ? 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 in nature. `Aerobic' ammonium oxidizers like Nitrosomonas eutropha were observed to reduce nitrite or nitrogen dioxide with hydroxylamine or ammonium as electron donor under anoxic conditions. The maximum rate for anaerobic ammonium oxidation was about 2 nmol xr? min3I (mg protein)3I using nitrogen dioxide as electron acceptor. This reaction, which may R involve NO as an intermediate, is thought to generate energy sufficient for survival under anoxic conditions, but not for growth. A novel obligately anaerobic ammonium oxidation (Anammox) process was recently discovered in a denitrifying pilot plant reactor. From this system, a highly enriched microbial community with one dominating peculiar autotrophic organism was obtained. With nitrite as electron acceptor a maximum specific oxidation rate of 55 nmol xr? min3I (mg protein)3I was R determined. Although this reaction is 25-fold faster than in Nitrosomonas, it allowed growth at a rate of only 0.003 h3I (doubling time 11 days). IS N labeling studies showed that hydroxylamine and hydrazine were important intermediates in this new process. A novel type of hydroxylamine oxidoreductase containing an unusual PRTV cytochrome has been purified from the Anammox culture. Microsensor studies have shown that at the oxic/anoxic interface of many ecosystems nitrite and ammonia occur in the absence of oxygen. In addition, the number of reports on unaccounted high nitrogen losses in wastewater treatment is gradually increasing, indicating that anaerobic ammonium oxidation may be more widespread than previously assumed. The recently developed nitrification systems in which oxidation of nitrite to nitrate is prevented form an ideal partner for the Anammox process. The combination of these partial nitrification and Anammox processes remains a challenge for future application in the removal of ammonium from wastewater with high ammonium concentrations. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Ammonium ; Nitrite ; Hydrazine; Hydroxylamine; Nitrosomonas; Oxygen; Wastewater; Nitrogen removal

* Corresponding author. Tel.: +31 (15) 2781193; Fax: +31 (15) 2782355; E-mail: m.jetten@stm.tudelft.nl
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Present address: Netherlands Institute for Sea Research NIOZ, Den Burg, Texel, The Netherlands.

0168-6445 / 99 / $19.00 ? 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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Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological nature of the Anammox reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autotrophic growth during selective enrichment in continuous systems . . . . . . . . Cultivation of Anammox biomass and determination of physiological parameters Characterization of the enriched microorganisms . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Dominant cell type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Cytochrome spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Identi?cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Aerobic versus anaerobic ammonium oxidation . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. In?uence of oxygen on anaerobic ammonium oxidation . . . . . . . . . . . . . . . . 6.2. Aerobic ammonium oxidizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Metabolic versatility of Nitrosomonas strains . . . . . . . . . . . . . . . . . . . . . . . . 7. Possible reaction mechanisms for Anammox . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Substrate spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Ecological habitats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Application of the Anammox process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The combined SHARON-Anammox process . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3. 4. 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422 422 423 424 425 425 425 425 427 427 428 430 431 433 433 434 435 435 436

1. Introduction The oxidation of ammonium has been investigated mainly in aerobic or oxygen-limited systems. In theory ammonium could also be used as an inorganic electron donor for denitri?cation. The free energy for this reaction (Table 1) is nearly as favorable as for the aerobic nitri?cation process. It was on the basis of these thermodynamic calculations that the existence of two chemolithoautotrophic microorganisms capable of oxidizing ammonium to dinitrogen gas was already predicted two decades ago [1]. The actual discovery of such a process was only recently
Table 1 Gibbs free energy of several reactions involved in autotrophic denitri?cation [3,8] Reaction equation vG?P (kJ mol3I xr? R or xy3 ) Q 3560 3465 3297 3358 3349 3315

described [2,3]. During experiments on a denitrifying pilot plant of a multi-stage wastewater treatment system at Gist-Brocades (Delft, The Netherlands) it was noted that ammonium disappeared from the reactor e?uent at the expense of nitrate with a concomitant increase in dinitrogen gas production. A maximum ammonium removal rate of 1.2 mmol l3I h3I was observed. In continuous ?ow experiments the nitrogen and redox balances showed that ammonium really disappeared under anaerobic conditions, and that for every mol of ammonium consumed 0.6 mol of nitrate was required, resulting in the production of 0.8 mol of dinitrogen gas. This newly discovered process was named the Anammox (anaerobic ammonium oxidation) process.

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

2xy3 +5HP +2H? CNP +6HP O Q 8xy3 +5HS? +3H? C4NP +4HP O+5?yP3 Q R 3xy3 +5xr? C4NP +9HP O+2H? Q R xy3 +xr? CNP +2HP O P R 2OP +xr? Cxy3 +HP O+2H? Q R 6OP +8xr? C4NP +12HP O+8H? R

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zation at 121?C or when the seed material was omitted from the incubation no change in the concentration of nitrate and ammonium could be observed. Furthermore the addition of various inhibitors (2,4dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone or HgClP ) to the incubations resulted in a complete inhibition of the ammonium oxidation and nitrate reduction (Table 2). In these experiments with initial ammonium concentrations of 5 mM and higher, the rate of ammonium oxidation was proportional to the amount of biomass used. Taken together these experiments strongly suggested that the anaerobic ammonium oxidation was a biological process carried out by microorganisms. The speci?c rate of ammonium oxidation (0.08 nmol xr? min3I (mg R dry weight)3I ) in these batch experiments was quite low compared to rates (1.2 nmol xr? min3I (mg R dry weight)3I ) obtained in the pilot plant. This indicated that in the batch experiments the conversion was limited by di?usion of the substrates to the biomass granules. Labeling experiments with IS xr? in R combination with IR xy3 showed an almost exclusive Q production of IR?IS NP gas. This ?nding did not agree

with the postulated overall reaction [3] (see Section 1) in which for every labeled ammonium, 0.6 nitrate would react to form 0.8 dinitrogen gas (i.e. 75% of the formed dinitrogen would be IS?IR NP and 25% would be IS?IS NP ). Only if nitrite rather than nitrate was assumed as the actual oxidizing agent the observed and calculated values would be in agreement [4].

3. Autotrophic growth during selective enrichment in continuous systems Once it was realized that nitrite rather than nitrate might be the electron acceptor of autotrophic denitri?cation with ammonium as electron donor, a medium was composed for the selective enrichment of the microorganisms responsible for the Anammox process. This medium contained ammonium (5^ 30 mM), nitrite (5^35 mM), bicarbonate (10 mM), minerals and trace elements [5]. The phosphate concentration of the medium was kept below 0.5 mM and the oxygen concentration below detection levels

Table 2 E?ect of various treatments with stimulators and inhibitors on the anaerobic ammonium-oxidizing activity in batch experiments with biomass from the Anammox pilot plant (adapted after [4,6,14]) Treatment inhibitor/stimulator xr? ? xy3 P R No biomass Sterilization at 121?C Gamma irradiation Penicillin V Penicillin G Bromoethane sulfonic acid NaP SOR NaP MoOR Chloramphenicol Hydrazine Acetone N-serve Allylthiourea Acetylene 2,4-Dinitrophenol Carbonyl cyanide m-chlorophenylhydrazone HgClP Oxygen Phosphate Phosphate Mode of action activity test none denaturation inactivation inhibition of cell wall synthesis of bacteria id. inhibition of methanogenesis stimulation of sulfate reduction inhibition of sulfate reduction inhibition of protein synthesis inhibition of NHP OH oxidation solvent for N-serve inhibition of nitri?cation inhibition of nitri?cation inhibition of nitri?cation and denitri?cation uncoupler uncoupler cell damage oxidative stress chelating agent chelating agent Concentration or period tested 0^7 mM 0 mg l3I 20^120 min 60 min 0^100 mg l3I 0^1000 mg l3I 0^20 mM 20 mM 20 mM 0^400 mg l3I 0^3 mM 10 mM 0^50 mg l3I 0^10 mM 6 mM 0^400 mg l3I 0^40 mg l3I 0^300 mg l3I 0^0.2 mM 6 1 mM s 2 mM E?ect normal activity no activity no activity no activtiy none none none none none none activation none none none inhibition inhibition inhibition inhibition inhibition none inhibition

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Fig. 1. Operation of a ?uidized bed reactor for the enrichment and maintenance of anaerobic ammonium-oxidizing microorganisms. The medium was composed of ammonium sulfate, sodium nitrite, NaHCOQ , minerals, trace elements [5,6]. The gray area represents the nitrite and 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.

( 6 1 WM) in order to avoid possible inhibitory e?ects (Table 2). Since the speci?c rate of ammonium oxidation in batch experiments was considerably lower than in perfusion systems a ?uidized bed reactor was chosen to perform the enrichment. Using biomass from the original pilot plant as an inoculum, it was possible to obtain stable enrichment cultures within 3^4 months of operation. In total more than 20 reactor runs have been carried out with synthetic medium, the longest (Fig. 1) lasting more than 27 months [5^7]. So far only two runs failed in the enrichment mainly due to mechanical problems of the setup. After enrichment with synthetic medium the conversion rate in the reactor systems increased from 0.4 kg xr? N m3Q per day to about 3 kg xr? R R N m3Q per day. The maximum speci?c activity of the biomass in the ?uidized bed reactor was about 25 nmol xr? min3I (mg dry weight)3I . For every R mol of COP incorporated into biomass 24 mol of ammonium had to be oxidized. When biomass from the reactors was used in batch experiments supplied with ammonium, nitrite and IR COP , the cells became rapidly labeled. The incorporation of label was completely dependent on the combined presence of both nitrite and ammonium. The ribulose bisphosphate carboxylase activity of cell extracts was only 0.3 nmol COP min3I (mg dry weight)3I which

is 3-fold lower than expected on the basis of the stoichiometry determined for ammonium and bicarbonate conversion (24:1). The estimated growth rate in the ?uidized bed systems was 0.001 h3I , which is equivalent to 1 doubling time of about 29 days. The main product of the reaction was dinitrogen gas, but about 17% of the nitrite supplied could be recovered as nitrate. The overall nitrogen balance averaged over 15 runs showed a ratio of 1:1.31:0.22 for conversion of ammonium and nitrite to the production of nitrate. In the ?uidized bed reactor no other intermediates like hydroxylamine, hydrazine, NO or nitrous oxide could be detected. The production of nitrate from nitrite was veri?ed with IS N-NMR analysis [8]. Only when labeled nitrite was supplied to the cultures could formation of IS xy3 be observed. In the presence of IS N labeled Q ammonium, no IS xy3 was ever observed [8]. The Q function of this nitrate formation from nitrite is assumed to be the generation of reducing equivalents necessary for the reduction of COP . 4. Cultivation of Anammox biomass and determination of physiological parameters Currently available microbiological techniques are

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not designed very well to deal with very slowly growing microorganisms such as the Anammox culture. In addition to the ?uidized bed systems, a sequencing batch reactor (SBR) was applied and optimized for the quantitative study of the microbial community that oxidized ammonium anaerobically [9]. The SBR was a powerful experimental setup in which the biomass was retained very e¤ciently ( s 90%). Furthermore a homogeneous distribution of substrates, products and biomass aggregates over the reactor was achieved, and the reactor has been in operation reliably for more than 2 years under substrate-limiting conditions. Several important physiological parameters ([9] M. Strous, personal communication) such as the biomass yield (0.066 ? 0.01 C mol (mol ammonium)3I ), the maximum speci?c ammonium consumption rate (45 ? 5 nmol min3I (mg protein)3I ) and the maximum speci?c growth rate (0.0027 h3I , doubling time 11 days) could now be determined more accurately than with the ?uidized bed reactors. The temperature range for Anammox was 20^43?C (with an optimum at 40?C). The Anammox process functioned well at pH 6.7^8.3 (with an optimum at pH 8). Under optimal conditions the maximum speci?c ammonium oxidation rate was about 55 nmol min3I (mg protein)3I . The a¤nity for the substrates ammonium and nitrite was very high (a¤nity constants 9 5 WM) (M. Strous, personal communication). The Anammox process was inhibited by nitrite at nitrite concentrations higher than 20 mM but lower nitrite concentrations ( s 10 mM) were already suboptimal. When nitrite was present at high concentrations for a longer period (12 h), Anammox activity was completely lost. In addition, the persisting stable and strongly selective conditions of the SBR led to a high degree of enrichment (74% of the desired dominant peculiar microorganisms, see Section 5).

when ?xed with 2.5% glutaraldehyde in 20 mM KP HPOR /KHP POR bu?er pH 7.4. Once the unusual morphology of the cells was recognized, an estimate of the enrichment from the original culture could be made by counting the cells in a large number of thin sections. After 177 days of enrichment 64% of all cells counted (7317 out of 11 433 total) were of the described type. This was a four-fold increase (16%; 1632 out of 10 200 total) compared to the numbers found in the biomass from the pilot plant. Together with the increase in cell numbers of this peculiar organism an increase in the percentage of ether-like lipids was observed. The amount of ester lipids typical for most Bacteria remained more or less constant [5]. The presence of the ether lipids seems to be con?ned to most ancient microorganisms such as the Archaea or very deep-branching Bacteria like Thermotoga and Aquifex. A detailed knowledge of the exact structure and composition of the lipids of the dominant cell type would be most helpful to ?nd the taxonomic a¤liation of these cells [5]. 5.2. Cytochrome spectra During the enrichment on synthetic medium, the color of the biomass changed from brown to deep red. Visible spectra of cells and cell extracts of the enriched culture showed a pronounced increase in the signal for cytochromes of the c type. Spectra of cells at 77 K revealed the absence of a-type, b-type and dI -type cytochromes. Very interestingly, during increase in Anammox activity of the biomass, gradually an increased signal was observed at 468 nm. In Fig. 3 a typical spectrum of an Anammox cell extract with the 468-nm feature is shown. This absorption peak at 468 nm disappeared irreversibly after treatment with carbon monoxide. A similar signal has been observed in aerobic ammonium-oxidizing bacteria at 463 nm [10^13]. This signal was attributed to one of the hemes present in the enzyme hydroxylamine oxidoreductase and is mostly referred to as cytochrome P460. 5.3. Identi?cation So far many isolation methods including serial dilution, ?oating ?lters, and plating have been used to obtain the responsible microorganisms in pure cul-

5. Characterization of the enriched microorganisms 5.1. Dominant cell type The dominant microorganism of the enrichment cultures was a Gram-negative light-breaking coccoid cell (Fig. 2A,B) which showed an unusual irregular morphology under the electron microscope (Fig. 2C)

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Fig. 2. A: Micrograph of a biomass aggregate from an Anammox enrichment culture. The dominant coccoid cell is present in packages and microcolonies. B: Micrograph of the dominant coccoid cell present in the Anammox enrichment cultures. Preparation was obtained after sedimentation of suspended material from a ?uidized bed reactor. C: Electron micrograph of suspended Anammox biomass ?xed with 2.5% glutaraldehyde in 20 mM KP HPOR /KHP POR bu?er pH 7.4. The micrograph was taken at the Department of Electron Microscopy (I. Keizer, K. Sjollema, M. Veenhuis), State University of Groningen, The Netherlands.

ture. None of the isolates thus obtained is able to perform the Anammox reaction, but many of the isolates are novel denitrifying oligotrophic (proteo)bacteria. In addition to classical microbial techniques, the Anammox community was analyzed using modern molecular biological methods. The genomic DNA was extracted and ampli?ed via PCR using (eu)bacterial primers 27f-BamHI (5P-CACGGATCCAGAGTTTGATMTGGCTTCAG-3P), and 1492rHindIII (5P-TGTAAGCTTACGGYTACCTTGTTACGACT-3P). The PCR products were cloned, and 396 out of the more than 4000 clones obtained were screened. One dominant (28%) clone belonging to the Cytophaga/Flexibacter phylum was identi?ed. However, in situ analysis with ?uorescent probes speci?c for the Cytophaga phylum did not con?rm the molecular identity of the dominant organism as a Cytophaga.

6. Aerobic versus anaerobic ammonium oxidation The presence of a PRTH -like signal in the enriched Anammox biomass, and the recent reports on the metabolic versatility of aerobic ammonium oxidizers initiated a more detailed investigation. The studies were concentrated on three issues: the in?uence of oxygen on the Anammox process, the number of aerobic ammonium oxidizers present in the Anammox enrichment cultures and the (anaerobic) capabilities of `classical' nitri?ers of the Nitrosomonas type. 6.1. In?uence of oxygen on anaerobic ammonium oxidation The in?uence of oxygen on the Anammox process was investigated in both batch and continuous sys-

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

tems. Initial batch experiments showed that oxygen completely inhibited the Anammox activity when it was deliberately introduced into the enrichment cultures [5,14]. In a follow-up study an intermittently oxic (2 h) and anoxic (2 h) reactor system was used to study the reversibility of the oxygen inhibition for 20 days [15]. From these studies it became clear that ammonium was not oxidized in the oxic periods, but that the Anammox activity in the anoxic periods remained constant throughout the experiment, indicating that the inhibitory e?ect of oxygen was indeed reversible. The sensitivity of the Anammox enrichment culture to oxygen was further investigated under various sub-oxic conditions. In four consecutive experiments, the oxygen tension was decreased stepwise from 2 to 0% of air saturation (Fig. 4). No ammonium was oxidized in the presence of 0.5, 1, or 2% of air. Only when all the air was removed from the reactor by vigorously ?ushing with argon gas, the conversion of ammonium and nitrite resumed, thus indicating that the Anammox activity in these enrichment cultures is only possible under strict anoxic conditions.

6.2. Aerobic ammonium oxidizers The second question which was addressed in these studies concerned the number of aerobic ammoniumoxidizing bacteria present in the Anammox enrichment cultures. Using standard, aerobic most probable number (MPN) methods, the number of nitri?ers was estimated to be 9 ? 5U10Q cells of ammonium oxidizers per milliliter of biomass sample. Electron micrographs of the MPN cultures showed the characteristic cytoplasmic membrane structures reported for several aerobic ammoniumoxidizing bacteria [16]. The consistent presence of these aerobic nitri?ers in the Anammox biomass con?rmed that they can survive very long periods of anaerobiosis as was previously shown by Abeliovich [17]. Furthermore, it was possible to enrich such organisms in a repeated fed-batch reactor when oxygen was continuously supplied at 50^80% of air saturation [15]. The enriched aerobically nitrifying community grew exponentially with a doubling time of 1.2 days. Interestingly, not all of the 27 mM of ammonium supplied could be recovered as nitrite. Since nitrite was not further oxidized to nitrate, the re-

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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 removed from the incubation by ?ushing with argon gas the disappearance of ammonium (b) and nitrite (F) could be observed [15].

mainder of the nitrogen might have been lost as gaseous nitrogen compounds (NO, NP O or NP ) during aerobic denitri?cation. After enrichment of the nitri?ers, also this culture was subjected to the same alternating 2 h oxic/2 h anoxic regime to verify if these nitri?ers were capable of an anaerobic conversion of ammonium or nitrite. During 20 days, the fate of the supplied 30 mM ammonium was followed and it was observed that only in the aerobic periods oxidation of ammonium to nitrite occurred. This indicated that

this community of aerobic ammonium-oxidizing bacteria was not able to use nitrite as an electron acceptor in this case. This is in contrast to observations made with several pure cultures of di?erent Nitrosomonas strains. Poth showed that a new Nitrosomonas isolate was able to produce dinitrogen gas under anaerobic conditions [18,19], while Abeliovich and Vonshak demonstrated the reduction of nitrite with pyruvate as electron donor by N. europaea [20].

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Table 3 Rates of anaerobic oxidation (nmol min3I (mg protein)3I ) of ammonium, hydroxylamine and hydrazine by various cultures in batch experiments Culture N. N. N. N. N. europaea europaea eutropha eutropha eutropha Compounds tested NHP OH+xy3 P xr? ? xy3 P R HP +xy3 P xr? ? xy3 P R xr? ? xy3 P R xr? ? xy3 P R NHP OH NP HR +xy3 P xr? ? xy3 P R xy3 P conversion rate 2 2 7 61 0.9 12 n/a 13 68 NHP OH/xr? /NP HR R conversion rate 3 3 not applicable 61 1.1 9 12 11 55 Products NP O NP O NP O, NP NP O NO, NP O NP NP xr? , NP R NP Reference [23] [23] [16] [16] [21] [5] [8] [8,24] [9], M. Strous, personal communication

Anammox Anammox Anammox Anammox

6.3. Metabolic versatility of Nitrosomonas strains More recently substantial N losses have been reported for both mixed and pure cultures of N. eutropha grown under oxygen limitation [16,21,22], and for pure cultures of N. europaea in anoxic batch experiments [23]. When molecular hydrogen was used as an electron donor for nitrite reduction, growth of N. eutropha was stoichiometrically coupled to nitrite reduction with dinitrogen gas and nitrous oxide as end products. In mixed cultures of N. eutropha and Enterobacter aerogenes 2.2 mM of ammonium and nitrite were consumed during 44 days of incubation, but no cell growth was observed [16]. In a follow-up study the rate of anaerobic ammonium oxidation by N. eutropha could be estimated at 0.08 nmol xr? R min3I (mg protein)3I which is equivalent to about 0.2 nmol xr? min3I (mg dry weight)3I . However, R when the nitrogen atmosphere of the incubations was supplemented with 25 ppm nitrogen dioxide, the rate increased 10-fold to 2.2 nmol xr? min3I R (mg protein)3I [21]. It was estimated that 40^60% of the formed nitrite (and NO) was denitri?ed to dinitrogen gas while NP O and hydroxylamine were detected as intermediates. The source of oxygen for the oxidation of ammonia under these anoxic conditions remained unknown, but could theoretically be derived from either NO, NOP or nitrite. Very recently it was shown that N. eutropha also exhibited denitrifying capabilities in the presence of NOP when the dissolved oxygen concentration was maintained at

3^4 mg l3I [22]. In these experiments with complete biomass retention 50% of the produced nitrite was aerobically denitri?ed to dinitrogen gas. NO gas was much less e¤cient in stimulating this aerobic denitri?cation than NOP and became toxic above 25 ppm. Furthermore, an eight-fold increased aerobic nitri?-

Fig. 5. Concentration pro?le of ammonium (b), nitrite (F), hydroxylamine (R) and hydrazine (8) during anaerobic batch experiments with an Anammox culture supplemented with 3 mM hydroxylamine [8].

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Table 4 Comparison of the properties of the hydroxylamine oxidoreductase (HAO) enzyme isolated from Anammox (J. Schalk, personal communication) and Nitrosomonas europaea [12] HAO of Anammox Molecular mass Subunit Composition Ratio 410/280 Heme Active center Vm?x Km pH optimum pI N-terminus 150 kDa 60^95 kDa KP ^KQ 4.5 22 ? 4/150 kDa PRTV 21 U mg3I 26 WM 8 5.5 blocked HAO of Nitrosomonas 125^175 kDa 63 kDa KP ^KQ 3.3 24/KQ PRTQ 75 U mg3I not reported 8 5.3 DISTV

cation rate and higher cell numbers were observed when the air was supplemented with 25^50 ppm NOP . In Table 3 a summary is presented of the reported rates of anaerobic ammonium oxidation in various experiments with Nitrosomonas and/or Anammox cultures. From this table it is evident that the speci?c rates for anaerobic ammonium oxidation of the classical nitri?ers are 25-fold lower than the rates observed in the Anammox process studied in Delft. Furthermore, aerobic ammonium oxidizers prefer to use oxygen as the terminal electron acceptor, whereas this compound completely inhibits the Anammox process. Taken together these examples showed that further research to elucidate the role of nitrogen oxides during (an)aerobic ammonium oxidation is necessary.

7. Possible reaction mechanisms for Anammox The possible metabolic pathway for anaerobic ammonium oxidation was investigated using IS N labeling experiments. These experiments showed that ammonium was biologically oxidized with hydroxylamine as the most probable electron acceptor [8]. The hydroxylamine itself is most likely derived from nitrite. In batch experiments with excess hydroxylamine and ammonium, a transient accumulation of hydrazine was observed (Fig. 5). The conversion of hydrazine to dinitrogen gas is postulated as the reaction generating the electron equivalents for the reduction of nitrite to hydroxylamine. Whether the reduction of nitrite and the oxidation of hydra-

Fig. 6. Possible reaction mechanisms and cellular localization of the enzyme systems involved in anaerobic ammonium oxidation. A: Ammonium and hydroxylamine are converted to hydrazine by a membrane-bound enzyme complex, hydrazine is oxidized in the periplasm to dinitrogen gas, nitrite is reduced to hydroxylamine at the cytoplasmic site of the same enzyme complex responsible for hydrazine oxidation with an internal electron transport. B: Ammonium and hydroxylamine are converted to hydrazine by a membrane-bound enzyme complex, hydrazine is oxidized in the periplasm to dinitrogen gas, the generated electrons are transferred via an electron transport chain to nitrite reducing enzyme in the cytoplasm.

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zine occur at di?erent sites of the same enzyme (Fig. 6A) or the reactions are catalyzed by di?erent enzyme systems connected via an electron transport chain (Fig. 6B) remains to be investigated. The occurrence of hydrazine as an intermediate in microbial nitrogen metabolism is rare [24]. Hydrazine has been proposed as an enzyme-bound intermediate in the nitrogenase reaction [25]. Furthermore, the puri?ed hydroxylamine oxidoreductase (HAO) of N. europaea is capable of catalyzing the conversion of hydrazine to dinitrogen gas [12]. The ?nding of high HAO activity in cell extracts of the Anammox culture indicated that a similar enzyme might be operative in the Anammox process. Indeed very recently a novel type of HAO was puri?ed from the Anammox community via anion exchange and gel ?ltration chromatography (J. Schalk, personal communication). Native PAGE showed that the Anammox enzyme had a smaller molecular mass than the enzyme of Nitrosomonas. Furthermore, the amino acid sequence of several HAO peptide fragments was unique, without any homologue in the databases. Similar to the Nitrosomonas hydroxylamine oxidoreductase, the enzyme from Anammox contained several c-type cytochromes. The special spectroscopic P460-like feature was found at 468 nm in the Anammox enzyme. The enzyme was able to catalyze the oxidation of both hydroxylamine and hydrazine. Although hydroxylamine was the preferred substrate the rate of hydrazine oxidation in cell extracts (150 nmol min3I (mg protein)3I ) was high enough to sustain a growth rate of 0.003 h3I . In Table 4 some properties of the two HAO enzymes are summarized (J. Schalk, personal communication).

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

The formation of hydroxylamine via an ammonium monooxygenase seems highly improbable, since the Anammox reaction is strongly but reversibly inhibited by oxygen. An alternative mechanism for the formation of hydroxylamine might be the incomplete reduction of nitrite by a cytochrome c-type nitrite reductase. However, it will be very di¤cult to obtain direct evidence for this mechanism in Anammox. Hydroxylamine is the compound most rapidly metabolized by Anammox, and a selective inhibitor has not yet been discovered.

Table 5 Possible reaction equations of anaerobic ammonium oxidation via NO or HNO as intermediates, adapted after [12,14] NO as intermediate NO+NHQ +3H? +3e3 NP HR xy3 +2H? +e3 P NHQ +xy3 +H? P HNO as intermediate HNO+NHQ NP HP xy3 +2H? +2e3 P NHQ +xy3 P CNP HR +HP O CNP +4H? +4e3 CNO+HP O CNP +2HP O (ammonia monooxygenase-like enzyme) (hydroxylamine oxidoreductase-like enzyme) (nitrite reductase)

CNP HP +HP O CNP +2H? +2e3 CHNO+OH3 CNP +HP O+OH3

(ammonia monooxygenase-like enzyme) (hydroxylamine oxidoreductase-like enzyme) (nitrite reductase)

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Fig. 8. The absence of methane conversion by Anammox biomass at two di?erent methane concentrations in the head space. Closed triangles (R, 380 Wmol methane) and diamonds (8, 195 Wmol methane) represent incubations with Anammox biomass in the presence of 300 Wmol nitrite as electron acceptor. The closed circles represents a control incubation of Anammox biomass with 280 Wmol ammonium (b), and 300 Wmol nitrite as electron acceptor. In the control all nitrite was converted, in the incubations with methane the nitrite was not converted.

indicated that the enzyme responsible for anaerobic ammonium conversion is di?erent from the aerobic ammonia or methane monooxygenases. In longer experiments it could also be shown that methane itself was not converted by the Anammox biomass (Fig. 8). In addition to methane also hydrogen was tested in batch incubations (Fig. 9). The addition of hydrogen to the argon/COP head space showed a clear stimulation of the anaerobic ammonium oxidation in short-term experiments. However, hydrogen could not replace ammonium as electron donor in these experiments. Addition of various organic substances (pyruvate, methanol, ethanol, alanine, glucose, casamino acids) in short-term batch experiments led to a severe inhibition of the Anammox activity. Thus the substrate spectrum seems to be restricted to ammonium, nitrite and the intermediates hydrazine and hydroxylamine. However, supplementation with 1 mM hydrazine could not sustain growth of the Anammox culture for longer periods [24].

9. Ecological habitats The discovery of the Anammox process in a deni-

A possible role of NO or HNO in (an)aerobic ammonium oxidation was proposed by Hooper [12] to be a condensation of NO or HNO and ammonia on an enzyme related to the ammonium monooxygenase family (Table 5). The formed hydrazine or imine could thereafter be converted by the enzyme hydroxylamine oxidoreductase into dinitrogen gas and the reducing equivalents required to combine NO or HNO and ammonia or to reduce nitrite to NO.

8. Substrate spectrum Aerobic ammonium and methane oxidizers are able to catalyze both the oxidation of ammonium and methane [26] albeit at di?erent rates. The ability of the Anammox culture to use methane or other substrates was tested in batch experiments. In Fig. 7 it is shown that addition of methane to the argon/ COP head space of the incubations did not lead to an inhibition of ammonium and nitrite conversion. This

Fig. 9. Concentration pro?le of ammonium (b) and nitrite (F) in the presence of 95% hydrogen and 5% COP gas in the head space, and of ammonium (a) and nitrite (E) in the presence of 95% argon and 5% COP gas in head space during anoxic batch experiments with Anammox biomass.

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Table 6 Overview of the parameters of an Anammox ?uidized bed reactor [7,38] and a SHARON reactor [38] both fed with sludge digester e?uent. The nitrite for the Anammox process was supplied separately SHARON Ammonium load Nitrite load Nitrogen load xr? N e?uent R xy3 N e?uent P E¤ciency xr? N R Removal E¤ciency xy3 N P Removal Sludge load
?

Anammox 0.24^1.34 0.22^1.29 0.46^2.63 27 ? 85 3?3 88 ? 9 99 ? 2 0.05^0.26 kg xr? N m3Q re??tor day3I R kg xy3 N m3Q re??tor day3I P kg Ntot m3Q re??tor day3I mg N l3I mg N l3I % % kg Ntot kg3I SS per day

0.63^1.0? not applicable 0.63^1.0 199 469 76^90 n/a 10.3

This value is linearly proportional to the in?uent concentration.

trifying pilot plant has raised the question as to where else such organisms would occur in nature. Already in 1932 it was reported that dinitrogen gas was generated via an unknown mechanism during fermentation in the sediments of Lake Mendota (USA) [27]. Also in sediments of Lake Kizakiko (Japan) indications were found for the direct formation of dinitrogen gas from ammonium [28]. Very recently these observations were con?rmed in studies with freshwater sediments [29]. One prerequisite for the occurrence of anaerobic ammonium oxidation via an Anammox mechanism would be the simultaneous presence of both ammonium and nitrite (or nitrate) and the absence of oxygen. The nitrite could be formed either in ecosystems in which oxygen (diffusion) limits nitri?cation or in places with a limited supply of electron donor (sul?de or organic substances) for denitri?cation of nitrate. The oxic/anoxic interface of many sediments would thus be an ideal habitat for anaerobic ammonium-oxidizing microorganisms. Micro-electrode studies have revealed overlapping pro?les of nitrate and ammonium in a strati?ed zone of the Black Sea [30,31] indicating that a habitat for Anammox really exists. More recently after the development of nitrite microsensors, also overlapping pro?les of nitrite and ammonium have been reported in oxygen-limited nitrifying activated sludge ?ocs [32^34]. Indeed man-made ecosystems like wastewater treatment plants could create a habitat for the Anammox organisms. The abundant supply of ammonium via the wastewater in combination with a limited oxygen availability would provide

conditions in which both ammonium and nitrite could occur. High nitrogen losses (70^90%) in the form of dinitrogen gas have been reported in two rotating biological contractor systems [35^37] treating land?ll leachate with about 200^400 mg ammonium per liter. Comparison of the microorganisms in these systems with the Delft Anammox culture would give more insight into the biodiversity of the anaerobic ammonium oxidation.

10. Application of the Anammox process In a recent study [7,38] the feasibility of the Anammox process for the removal of ammonium from sludge digester e?uents was evaluated. The results of this study showed that the Anammox biomass was not negatively a?ected by the digester e?uent. The pH (7.0^8.5) and temperature (30^37?C) optima for the process were well within the range of the values found in digester e?uents. Experiments with a laboratory-scale (2-l) ?uidized bed reactor showed that the Anammox biomass was capable of removing ammonium and (externally added) nitrite e¤ciently from the sludge digester e?uent (Table 6). The nitrogen load of the Anammox ?uidized bed reactor could be increased from 0.46 kg Ntot m3Q re??tor per day to 2.6 kg Ntot m3Q re??tor per day. Due to the nitrite limitation, the maximum capacity was not reached. The nitrogen conversion rate during the experiment with sludge digester e?uents increased from 0.05 kg Ntot kg3I SS per day to 0.26 kg Ntot

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kg3I SS per day. The Anammox sludge biomass removed 88% of the ammonium and 99% of the nitrite from the sludge digester e?uent (Table 6). In these studies nitrite was supplied from a concentrated stock solution. However, for application in real wastewater practice, a suitable system for biological nitrite production has to be developed. One such system is the SHARON (single reactor high activity ammonium removal over nitrite) process [38]. This SHARON process is ideally suited to remove nitrogen from waste streams with a high ammonium concentration ( s 0.5 g N l3I ). The SHARON process is performed in a single, stirred tank reactor without any sludge retention. At temperatures above 25?C it is possible to e?ectively outcompete the nitrite oxidizers. This results in a stable nitri?cation with nitrite as end-product [38]. When combined with the Anammox process only 50% of the ammonium needs to be converted to nitrite. This implies that no extra addition of base is necessary, since most of the wastewater resulting from anaerobic digestion will contain enough alkalinity (in the form of bicarbonate) to compensate for the acid production if only 50% of the ammonium needs to be oxidized. The SHARON process has been extensively tested at the laboratory scale for the treatment of sludge digester e?uents (Table 6) and is currently under construction at two Dutch wastewater treatment plants.

Fig. 10. Ammonium removal from sludge digester e?uent with the combined SHARON-Anammox system. Ammonium (8) or nitrite (F) load in the e?uent of the SHARON reactor is used as the ammonium or nitrite load into the Anammox reactor. The ammonium or nitrite load in the e?uent of the Anammox is represented by open diamonds and open squares, respectively. The pH value in the Anammox reactor was stable at 7.8 [38].

11. The combined SHARON-Anammox process The combination of the Anammox process and SHARON process has been tested in our laboratory
Table 7 Nitrogen balances in the combined SHARON-Anammox system SHARON In?uent xr? 584 (100%) R xy3 6 1 P xy3 6 1 Q NP O? 6 1 NP ? 6 1 Anammox E?uent 29 (5%) 1.4 83 (14%) 61 476? (82%)

E?uent/In?uent 267(46%) 227 (39%) 64 (11%) 4 61

using sludge digester e?uent (Fig. 10). The SHARON reactor was operated without pH control with a total nitrogen load of about 0.8 kg N m3Q per day [38]. The ammonium present in the sludge digester e?uent was converted to nitrite and a small amount of nitrate (11%) (Table 7). The nitrate formation was due to bio?lm wall growth, which was not always regularly removed. In large-scale applications this will be signi?cantly lower because of the larger volume to surface ratio. In this way an ammonium-nitrite mixture suitable for the Anammox process was generated. The e?uent of the SHARON reactor was used as in?uent for the Anammox ?uidized bed reactor. In the nitrite-limited Anammox reactor all nitrite was removed, the surplus ammonium remained. During the test period the overall ammonium removal e¤ciency was 83%. In Table 7 the nitrogen balances of the two systems are summarized. The optimization and application of the combination of these two processes on pilot plant and full scale remain challenges towards implementations in a future wastewater treatment plant.

Results are mg N l3I , percentages are given in parentheses. ? Concentration relative to the in?uent ?ow. ? Determined as the di?erence between dissolved and gaseous nitrogen compounds.

Acknowledgments The research on anaerobic ammonium oxidation

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M.S.M. Jetten et al. / FEMS Microbiology Reviews 22 (1999) 421^437 (1983) Heme PRTH of hydroxylamine oxidoreductase of Nitrosomonas: reaction with CO and HP OP . Eur. J. Biochem. 134, 83^87. Hooper, A.B., Vannelli, T., Bergmann, D.J. and Arciero, D.M. (1997) Enzymology of the oxidation of ammonia to nitrite by bacteria. Antonie van Leeuwenhoek 71, 59^67. Prince, R.C. and George, G.N. (1997) The remarkable complexity of hydroxylamine oxidoreductase. Nature Struct. Biol. 4, 247^250. Jetten, M.S.M., Logemann, S., Muyzer, G.M., Robertson, L.A., DeVries, S., van Loosdrecht, M.C.M. and Kuenen, J.G. (1997) Novel principles in the microbial conversion of nitrogen compounds. Antonie van Leeuwenhoek 71, 75^93. Strous, M., Van Gerven, E., Kuenen, J.G. and Jetten, M. (1997) E?ects of aerobic and microaerobic conditions on anaerobic ammonium-oxidizing (Anammox) sludge. Appl. Environ. Microbiol. 63, 2446^2448. Bock, E., Schmidt, I., Stuven, R. and Zart, D. (1995) Nitrogen loss caused by denitrifying Nitrosomonas cells using ammonium or hydrogen as electron donors and nitrite as electron acceptor. Arch. Microbiol. 163, 16^20. Abeliovich, A. (1987) Nitrifying bacteria in wastewater reservoirs. Appl. Environ. Microbiol. 53, 754^760. Poth, M. (1986) Dinitrogen gas production from nitrite by a Nitrosomonas isolate. Appl. Environ. Microbiol. 52, 957^959. Poth, M. and Focht, D.D. (1985) IS N Kinetic analysis of NP O production by Nitrosomonas europaea: an examination of nitri?er denitri?cation. Appl. Environ. Microbiol. 49, 1134^ 1141. Abeliovich, A. and Vonshak, A. (1992) Anaerobic metabolism of Nitrosomonas europaea. Arch.Microbiol. 158, 267^270. Schmidt, I. and Bock, E. (1997) Anaerobic ammonia oxidation with nitrogen dioxide by Nitrosomonas eutropha. Arch. Microbiol. 167, 106^111. Zart, D. and Bock, E. (1998) High rate aerobic nitri?cation and denitri?cation by Nitrosomonas eutropha grown in a fermenter with complete biomass retention in the presence of NOP and NO. Arch. Microbiol. 169, 282^286. de Bruijn, P., Van de Graaf, A.A., Jetten, M.S.M., Robertson, L.A. and Kuenen, J.G. (1995) Growth of Nitrosomonas europaea on hydroxylamine. FEMS Microbiol. Lett. 125, 179^184. Schalk, J., Oustad, H., Kuenen, J.G. and Jetten, M.S.M. (1998) The anaerobic oxidation of hydrazine ^ a novel reaction in microbial nitrogen metabolism. FEMS Microbiol. Lett. 158, 61^67. Dilworth, M.J. and Eady, R.R. (1991) Hydrazine is a product of dinitrogen reduction by the vanadium nitrogenase from Azotobacter chroococcum. Biochem. J. 277, 465^468. Hanson, R.S. and Hanson, T.E. (1996) Methanotrophic bacteria. Microbiol. Rev. 60, 439^471. Allgeier, R.J., Peterson, W.H., Juday, C. and Birge, E.A. (1932) The anaerobic fermentation of lake deposits. Int. Rev. Hydrobiol. 26, 444^461. Koyama, T. (1965) Formal discussion of paper III-10. Discussion of paper by Richards. In: Advances in Water Pollution Research (Pearson, E.A., Ed.), Vol. 3, pp. 234^242. Pergamon Press, Tokyo.

was ?nancially supported by the Foundation for Applied Sciences (STW), the Foundation of Applied Water Research (STOWA), the Netherlands Foundation for Life Sciences (NWO-SLW), the Royal Netherlands Academy of Arts and Sciences (KNAW), Gist-Brocades, DSM, and Grontmij consultants. The contributions of various co-workers and students over the years are gratefully acknowledged. References
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