new pathways for ammonia conversion in soil and aquatic systems

5/27/2018 New Pathways for Ammonia Conversion in Soil and Aquatic Systems

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Plant and Soil 230: 919, 2001.

2001Kluwer Academic Publishers. Printed in the Netherlands. 9

New pathways for ammonia conversion in soil and aquatic systems

Mike S.M. JettenDepartment of Microbiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.

Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.

Key words: anaerobic ammonium oxidation, hydrazine, hydroxylamine, nitrite, nitrogen dioxide, nitrification,

denitrification

Abstract

Ammonia conversion processes are essential for most soil and aquatic systems. Under natural conditions, the

many possible reactions are difficult to analyze. For example, nitrification and denitrification have long been

regarded as separate phenomena performed by different groups of bacteria in segregated areas of soils, sediments

or aquatic systems sequentially in time. It has now been established that strict segregation in place and time of

the two processes is not necessary and that both denitrifiers and nitrifiers have versatile metabolisms. However,the rates described for aerobic denitrifiers are very low compared to the rates observed under anoxic conditions.

Also the rates of nitrifier denitrification are quite low, indicating that these conversions may not play an important

role under natural conditions. In addition, these processes often result in the emission of quite large amounts of

undesirable products, NO and N2O. Heterotrophic nitrification might be of relevance for systems, that contain a

high carbon to nitrogen ratio. Recently, a novel process (Anammox) has been discovered in which ammonium

serves as the electron donor for denitrification of nitrite into dinitrogen gas. 15N labeling studies showed that

hydrazine and hydroxylamine were important intermediates in this process. Enrichment cultures on ammonium,

nitrite and bicarbonate resulted in the dominance of one morphotypical microorganism. The growth rate of the

cultures is extremely low (doubling time 11 days), but the affinity for ammonium and nitrite and the conversion

rates (9.2 104 mol kg1 s1) are quite high. Some of the reported high nitrogen losses in soil and aquatic systems

might be attributed to anaerobic ammonium oxidation. In addition, this conversion offers new opportunities for

nitrogen removal, when it is combined with recently developed processes for partial nitrification.

Introduction

In modern agriculture, nitrogen is supplied either by

biological nitrogen fixation or by mineral fertilizers

derived from industrially fixed nitrogen. Only part of

the nitrogen is incorporated in plant or animal bio-

mass, the remainder is lost via diffusive processes and

thus contributes to environmental nitrogen pollution.

Ammonia can be one important nitrogen pollutant in

soil and aquatic systems, which then has to be re-

moved (Jetten et al., 1997a). The large amount ofnitrogen compounds involved, the numerous reactions

that can occur (Figure 1) and the low growth rate

of many of the bacteria involved, make the study of

nitrogen conversion difficult. Recently, a new set of

microbial possibilities for nitrogen conversions has

been reported to occur in soil and aquatic systems.

Examples of such reactions are: aerobic denitrifica-

FAX No: +31-24-3652830. E-mail: [email protected]

tion, heterotrophic nitrification, anaerobic ammonium

oxidation or denitrification by autotrophic nitrifying

bacteria (Jetten et al., 1997b; 1998). These new pos-

sibilities make the evaluation of nitrogen conversions

even more complex.

This paper reviews these recent developments and

evaluates the conditions under which these noncon-

ventional conversions might occur. Finally, a new

combined system for nitrogen removal based on par-

tial nitrification of ammonia to nitrite, together with

anaerobic ammonium oxidation is presented.

Denitrification

Emission of intermediates

The reduction of nitrate to dinitrogen gas via the in-

termediates nitrite, nitric oxide and nitrous oxide is

catalyzed by several different reductase (Figure 1).


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Figure 1. Overview of possible microbial nitrogen conversions.Ni-

trification: ammonium oxidation to hydroxylamine catalyzed by

ammonia monooxygenase (1) using O2 or NO2 as source of oxy-

gen, hydroxylamine oxidation to nitrite by hydroxylamine oxidore-

ductase (2) and nitrite oxidation to nitrate by nitrate oxidoreductase

(3). Denitrification: nitrate (4), nitrite (5), nitric oxide (6) and ni-trous oxide (7) reductases. Nitrogen fixation by nitrogenase (8).

Anaerobic ammonium oxidation by a putative nitrite reductase (9);

hydrazine forming enzyme (10) and hydrazine oxidoreductase (11).

Ammonificationby nitrite reductase (12). Oxidation of NO to NO2by a putative NO oxidase (13). Assimilation of ammonia into

organic matter is not depicted.

The biochemistry and molecular biology of denitri-

fication has been studied quite well (Berks et al.,

1995a; Zumft, 1997) and concerted action of the en-

zymes involved is necessary to ensure production of

dinitrogen. The release of one of the above-mentioned

intermediates of denitrification into the environment isof great concern. Especially electron donor limitation

and presence of toxic compounds stimulates the emis-

sion of these intermediates (Gejlsbjerg et al., 1998;

Jetten et al., 1997b; Otte et al., 1999; Van Benthum

et al., 1998). Also the transition between oxic and

anoxic conditions enhances the formation of these un-

wanted intermediates (Otte et al., 1996; Kester et al.,

1997). The release of these N-anoxyions might be due

to differences in regulation of the various enzymes in-

volved in denitrification, which can react immediately

to changes in the environment (Baumann et al., 1996;

1997a,b; Zumft, 1997).

Aerobic denitrification

There is no fundamental argument why denitrification

cannot occur under oxic conditions. However, only

during the past few years has this activity received

some attention (Berks et al., 1995a; Gupta, 1997;

Patureau et al., 1998; Robertson et al., 1995). Fur-

thermore, the name aerobic denitrification is used in

different contexts, which leads to additional confu-

sion. It is mostly used to refer to microorganisms,

which denitrify while sensing oxygen, but in some

cases it is used to refer to denitrification in an oxic

system. In the latter case diffusion limitation into flocs,biofilms, soil or not well mixed systems provides an-

oxic pockets where conventional denitrification can

take places. Floc sizes in the range of 150 m are

already sufficient to allow substantial denitrification in

conventional (aerobic) activated sludge processes. The

same situation is present in biofilm aggregates with

diameters larger than 100 m (Van Loosdrecht and

Jetten, 1998).

Heterotrophic nitrification-aerobic denitrification

Aerobic denitrification is often coupled to hetero-

trophic nitrification in one organism (Berks et al.,

1995a,b; Gupta 1997; Otte et al., 1996; Robertson et

al., 1995). Heterotrophic nitrification has been known

for a long time, but was considered of little signi-

ficance (Jetten et al., 1997b). Because nitrification

is mostly measured by the formation of nitrate or

nitrite under oxic conditions, while (aerobic) denitri-

fication is not expected under such conditions, this

coupled process is not easily observed in standard en-

richment cultures. The observation that Thiosphaera

pantotrophaand other organisms are not only hetero-

trophic nitrifiers, but also aerobic denitrifiers forced are-evaluation of this approach. Nitrogen balances on

various pure cultures showed that the heterotrophic

nitrification rates are considerably higher than previ-

ously assumed (Table 1). The oxidation of ammonium

by a heterotrophic organism requires energy (con-

trary to autotrophic nitrification), mostly leading to

decreased yield. In fact, such nitrification can act as an

electron sink. The coupling of the processes proceeds

via the expression of a periplasmic nitrate reductase

(nap) (Bell et al., 1990; Berks et al., 1995a,b; Sears et

al., 1993). The ecological advantage for the organism

is an increased growth rate due to the simultaneoususe of oxygen and nitrate as electron acceptors, which

was shown for Thiosphaera, Microvirgula and other

organisms (Carter et al., 1995a,b; Patureau et al.,

1994;1998; Robertson et al., 1995). Aerobic denitrifi-

ers are present in high number in natural soil samples.

Even though the specific activities are not always very

high, they are sufficient to allow significant contribu-


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Table 1. Rates of nitrification by various autotrophic and heterotrophic bacteria

(Jetten et al., 1997b)

Culture Compound conversion rate

tested mol (kg dry weight)1 s1

Nitrosomonassp. NH+4

2.220 103

Pseudomonassp. NH+4

0.41 103

Alcaligenes faecalis NH+4 0.28 103

Thiosphaera pantotropha NH+4

0.58 103

Nitrosomonassp. NH2OH 113.7 103

Pseudomonassp. NH2OH 0.77.5 103

Alcaligenes faecalis NH2OH 0.20.5 103

Alcaligenessp. pyruvic oxime 0.5 103

tion to the turnover of compounds in the nitrogen cycle

(Jetten et al., 1997b).

Nitrification

Nitrogen dioxide

Nitrification is generally performed by autotrophic

or mixotrophic bacteria (Laanbroek and Woldendorp

1995). In two steps ammonia is oxidized via nitrite

to nitrate (Figure 1). A number of different types of

nitrifying bacteria have been identified. The oxida-

tion of ammonia is mainly attributed to the genera

Nitrosomonas andNitrosospira, while the oxidation of

nitrite is performed byNitrobacterandNitrospiraspe-cies (Burell et al., 1998; Hovanec and Delong 1998;

Juretschko et al., 1998; Laanbroek and Woldendorp

1995; Schramm et al., 1998a,b; Stephen et al., 1998).

The aerobic metabolism of Nitrosomonas and Ni-

trobacterhas been studied in detail (Gerards et al.,

1998; Laanbroek and Gerards 1993; Laanbroek et

al., 1994) and the enzymology is well documented

(Hooper et al., 1997). Recently, is has become clear

that nitrifiers also have an anaerobic metabolism. This

metabolism has been studied in more detail in Nitro-

somonas eutropha(Bock et al., 1995). The maximum

rate of anaerobic ammonium oxidation was estimatedat 1.3 106 mol NH+4 (kg protein)1 s1. However

when the nitrogen atmosphere of the incubations was

supplemented with 25 l.l1 nitrogen dioxide (NO2),

the rate increased to 1.8 105 mol NH+4 (kg protein)1

s1 (Schmidt and Bock, 1997). It was estimated that

40-60% of the formed nitrite (and NO) was denitrified

to dinitrogen gas, and N2O and hydroxylamine were

detected as intermediates. The source of oxygen (Fig-

ure 2) for the oxidation of ammonia under these an-

aerobic conditions is most likely NO2(Zart and Bock,1998; Schmidt and Bock, 1998).N. eutrophaalso ex-

hibits denitrifying capacities in the presence of NO2,

when the dissolved oxygen concentration is main-

tained at 34 mg.l1 (Zart and Bock, 1998). In these

experiments, 50% of the produced nitrite was aerobic-

ally denitrified to dinitrogen gas. NO gas was much

less effective in stimulating this aerobic denitrification

than NO2 and became toxic above 25 l.l1. When

the air was supplemented with 25 l.l1 NO2, an

8-fold increased aerobic nitrification rate and higher

cell numbers were observed indicating that NO2rather

than oxygen, might be used to activate the ammonia

monooxygenase (Figure 2). Table 2 presents a sum-

mary on the reported rates of anaerobic ammonium

oxidation in various experiments with Nitrosomonas

species and compares these to the rates obtained for

Anammox cultures (see next section). The specific

rates for anaerobic ammonium oxidation of the clas-

sical nitrifiers, like Nitrosomonas, are 25-fold lower

than those observed in the Anammox process. Fur-

thermore, aerobic ammonium oxidizers prefer to use

oxygen as the terminal electron acceptor, whereas this

compound completely inhibits the Anammox process.

Anaerobic ammonia oxidation

The oxidation of ammonia has been investigated

mainly in aerobic systems. In theory, however, ammo-

nia could also be used as an inorganic electron donor

for denitrification. The free energy for this reaction

(358 kJ/mol) is nearly as favorable as for the aerobic

nitrification process. This process was only recently


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Table 2. Rates of anaerobic oxidation of ammonium by various cultures

Culture Compounds NO2

NH+4

Products Reference

tested conversion rate

mol (kg protein)1 s1

Nitrosomonas europaea NH+4

+ NO2

3.3 105 5 105 N2O (De Bruijn et al ., 1995)

Nitrosomonas eutropha NH+

4

+ NO

2


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Figure 3. Growth of the Anammox biomass in a sequencing batch

reactor. The nitrogen load (kg N . m3 . day1) in the reactor is

represented by the solid line, the amount of nitrate formed in (kg

N . m3 . day1) is represent by the squares (), the amount of

biomass in gram protein as (). The modelled exponential growth

with an estimated doubling time of 11 days is present by the dashed

line (Strous et al., 1998).

nitrite and 14CO2, the cells became rapidly labeled.

The incorporation of label was completely dependent

on the combined presence of both nitrite and am-

monium. The estimated growth rate in the fluidizedbed systems was 0.001 h1, which is equivalent to

about 29 days doubling time.

Physiological parameters

Presently available microbiological techniques are not

designed very well to deal with microorganisms that

grow very slowly as the Anammox culture. In addition

to the fluidized bed systems, a sequencing batch re-

actor (SBR) was applied and optimized for the quant-

itative study of the microbial community that oxidized

ammonium anaerobically (Strous et al., 1998). TheSBR was a powerful experimental set-up in which the

biomass was retained very efficiently (> 90%). Fur-

thermore, 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 yr under substrate limiting conditions.

Several important physiological parameters such as

the biomass yield (0.066 C-mol . (mol ammonium)1,

the maximum specific ammonium consumption rate

(7.5 104 mol (kg protein)1 s1) and the maximum

specific growth rate (0.0027 h1, doubling time 11

days, Figure 3) were determined . The main product

of the reaction was dinitrogen gas, but about 20%

of the nitrite supplied was recovered as nitrate. Theproduction of nitrate from nitrite was verified with15N-NMR analysis (Van de Graaf et al., 1997). Only

when labeled nitrite was supplied to the cultures, could

the formation of 15NO3 be observed. The function

of this nitrate formation is presumably the generation

of reducing equivalents necessary for the reduction

CO2. The overall nitrogen balance showed a ratio of

1:1.32:0.26 for conversion ammonium and nitrite to

the production of nitrate. The temperature range for

Anammox activity was 20 C43 C. The Anammox

process functioned well between pH 6.78.3. Under

optimal conditions (pH 8, 40 C) the maximum spe-

cific ammonium oxidation rate was about 9.2 104

mol (kg protein)1 s1. The affinity for the substrates

ammonium and nitrite was very high (Ks values less

than 1 M). The Anammox process was inhibited by

nitrite at nitrite concentration higher than 20 mM but

lower nitrite concentrations (>10 mM) were already

suboptimal. When nitrite was present at high concen-

trations for a longer period, 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

microorganisms with a typical morphology (Figure 4).

Influence of oxygen on the Anammox process

The influence of oxygen on the Anammox process

was investigated in several experiments. Initial batch

tests showed that oxygen completely (but reversibly)

inhibited the Anammox activity when it was intro-

duced into the enrichment cultures (Van de Graaf et

al., 1996, Jetten et al., 1997b). The sensitivity of the

Anammox enrichment culture to oxygen was further

investigated under various micro-aerobic conditions

(Strouset al., 1997b). In four consecutive experiments,

the oxygen tension was stepwise decreased from 2 to0% of air saturation. No ammonium was oxidized in

the presence of 0.5, 1, or 2% of air. Only when all the

oxygen was removed from the reactor by vigorously

flushing 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 (Figure 5).


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Figure 4. Micrograph of the microbial community responsible for anaerobic ammonium oxidation as enriched in a sequencing batch reactor

(Strous et al., 1998). The community is dominated by a coccoid shaped bacterium present in aggregated clusters.

Figure 5. Influence of oxygen on the Anammox activity. Anammox biomass was incubated at defined dissolved oxygen in a batch reactor. Thedisappearance of ammonium () and nitrite () was monitored over time. Only when all oxygen was removed Anammox activity could be

observed (Strous et al., 1997b).

Occurrence of anaerobic ammonia oxidation in other

systems

Recently, substantial N-losses (Table 3) have been

reported for various soil, sediment, and aquatic sys-

tems. In three wastewater treatment systems (Helmer

and Kunst, 1998; Hippen et al., 1996; Siegrist et al.,

1998, Twachtmann et al., 1998) with a very high nitro-

gen load and limited air supply, a substantial amountof ammonia was lost a gaseous nitrogen compounds.

In such systems conditions might prevail in which

both nitrifiers and anaerobic ammonia-oxidizing bac-

teria could co-exist. In many mixed and pure cultures

of Nitrosomonas (Abeliovich, 1987; Abeliovich and

Vonshak, 1992; Bock et al., 1995; Bodelier et al.,

1998; De Bruijn et al., 1995; Kuai and Verstraete,

1998) ammonia is converted under oxygen limitation

into nitrous oxide and dinitrogen gas with nitrite, NO

and nitrogen dioxide as intermediates. Finally, the

generation of dinitrogen gas has been reported in sev-

eral sediments of fresh water lakes (Van Luijn et al.,

1997; Jetten et al., 1998). These observations in-

dicate that anaerobic ammonium oxidation might be

morewide spread in nature than previously assumed.

Possible reaction mechanism for Anammox

The possible metabolic pathway for anaerobic am-

monium oxidation was investigated using 15N-

labelings experiments (Figure 6). These experiments

showed that ammonium was biologically oxidized

with hydroxylamine as the most probable electron ac-


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Table 3. Reports on high nitrogen losses in treatment systems or microbial cultures

System Source of ammonia Remarks Authors

Rotating disk reactor Wastewater Oxygen limitation Siegrist et al ., 1998

Rotating disk reactor Landfill leachate Oxygen limitation Hippen et al., 1996

Helmer and Kunst, 1998

Fresh water sediment Surface water Anoxic conditions Van Luijn et al., 1998

Nitrifying sludge Synthetic wastewater Oxygen limitation Kuai and Verstraete, 1998

Nitrifying biomass Water reservoir Oxygen limitation Abeliovich, 1987

Abeliovich and Vonshak, 1992

Nitrosomonas eutropha Mineral medium Oxygen limitation Bock et al., 1995

Nitrosomonas eutropha Mineral medium Anoxic conditions Schmidt and Bock 1997

Nitrosomonas europaea Mineral medium Anoxic conditions De Bruijn et al., 1995

Trickling filter Synthetic wastewater Anoxic conditions Twachtmann et al., 1998

Fixed biofilm reactor Synthetic wastewater Anoxic conditions N. Ashbolt pers. comm.

Fluidized bed reactor Mineral medium Anoxic conditions Van de Graaf et al., 1996

Sequencing batch reactor Mineral medium Anoxic conditions Strous et al., 1998

Figure 6. Proposed reaction mechanism for anoxic ammonium ox-

idation with hydrazine and hydroxylamine as intermediates (Van de

Graaf et al., 1997; Jetten et al., 1998). E1 is the enzyme proposed

to catalyze the condensation of ammonia and hydroxylamine into

hydrazine (see reaction 10 in Figure 1); E2 is the enzyme proposed

to reduce nitrite to hydroxylamine (see reaction 9 in Figure 1); and

E3 is the enzyme proposed to catalyze the oxidation of hydrazine to

dinitrogen gas (see reaction 11 in Figure 1).

ceptor (Van de Graaf et al., 1997). The hydroxylamine

itself is most likely derived from nitrite. In batch ex-

periments with excess hydroxylamineand ammonium,a transient accumulation of hydrazine was observed.

The conversion of hydrazine to dinitrogen gas is pos-

tulated as the reaction generating the electron equi-

valents for the reduction of nitrite to hydroxylamine.

Whether the reduction of nitrite and the oxidation of

hydrazine occur at different sites of the same enzyme

or that the reactions are catalyzed by different enzyme

systems connected via an electron transport chain re-

mains to be investigated. The occurrence of hydrazine

as an intermediate in microbial nitrogen metabolism

is rare. Hydrazine has been postulated as an enzyme

bound intermediate in the fixation of nitrogen gas to

ammonia (Dilworth and Eady, 1991). Furthermore

the purified hydroxylamine oxidoreductase (HAO) of

N. europaea is capable of catalyzing the conversion

of hydrazine to dinitrogen gas (Hooper et al., 1997).

The finding of high HAO activity in cell extracts

of the Anammox culture indicated that a similar en-

zyme might be operative in the Anammox process.

Indeed recently a HAO-like enzyme has been pur-

ified from Anammox cultures with properties quite

different from the Nitrosomonas enzyme (Jetten et

al., 1998). Further indications for the involvement

of HAO were obtained from genetic studies. When

DNA extracted from the Anammox enrichment cul-

tures was used as a template for PCR amplification of

haogenes using primers derived from theN. europaea

haogene sequence (Sayaverda-Soto et al., 1995), two

sets of amplificates were obtained. After cloning and

sequencing of the PCR products, one set of clones con-

tained inserts nearly identical (98.8% on DNA level

and 99.6% on amino acid level) to the hao sequenceofN. europaea. The other set had an insert which se-

quence (AJ132220) was only 75.3% (on DNA level)

and 83.5% (on amino acid level) similar to one ofN.

europaea.In the deduced amino acid sequence of this

clone 6 cytochrome binding sites (Cys-X-X-Cys-His)

were conserved. Whether this newhaogene is present


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in the genome of the dominant Anammox bacterium

or in the genome of a nitrifier other than N. euro-

paea, able to survive long periods of anoxic conditions

remains to be established.

The hydrazine metabolism of Anammox was stud-

ied in more detail using anaerobic batch experiments

(Schalk et al., 1998). In these studies it was observedthat 3 mol of hydrazine were disproportionated to

4 moles of ammonia and 1 mol of dinitrogen gas.

Addition of nitrite to the incubation with Anammox

biomass showed a higher hydrazine consumption rate

(about 2 104 mol hydrazine (kg protein)1 s1. Sev-

eral reference cultures were not able to metabolize

the supplied hydrazine. Despite the high hydrazine

conversion rate of the Anammox biomass, it was not

possible to grow the cultures on hydrazine in biofilm

reactors. This indicated that the supplied concentra-

tions ( 50 mM) ammonium concentration such as sludge

digestion effluent (Hellinga et al., 1998). The Sharon

process is performed in a single, stirred tank reactor

without any biomass retention. At temperatures above

25 C it is possible to effectively outcompete the nitrite

oxidizers (Jetten et al., 1997a). The nitrifying microor-

ganisms responsible for the ammonia removal in the

Sharon reactor were identified by using several mo-

lecular biological techniques (Logemann et al., 1998).

Analysis of a 16S rRNA gene library revealed that

there was one dominant (69%) clone which was highly

similar (98.8%) toNitrosomonas eutropha. Nitrobac-

ter or Nitrospira clones were absent in this library.

The dominance ofNitrosomonasin the Sharon reactor

was qualitatively and quantitatively confirmed by two

independent microscopic methods (Logemann et al.,

1998). Operation of the reactor at 35 C and high di-

lution rates thus results in a stable nitrification with

nitrite as end-product. When the Sharon process is

coupled to the Anammox process, only 50% of the am-monium 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 bicar-

bonate) to compensate for the acid production. The

Sharon process has been extensively tested on laborat-

ory scale for the treatment of sludge digestion effluents

(Table 4) and is currently in operation at two Dutch

wastewater treatment plants.

The combination of the Anammox and Sharon pro-

cess (Figure 7) has been tested in our laboratory using

sludge digester effluent. The Sharon reactor was op-erated without pH control with a total nitrogen load

of about 0.8 kg N. m3 day1 (Jetten et al., 1997a).

The ammonium present in the sludge digester effluent

was converted mainly into nitrite (39%). In this way an

ammonium-nitrite mixture suitable for the Anammox

process was generated. The effluent of the Sharon re-

actor was used as influent for the Anammox fluidized


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Figure 7. Schematic presentation of the combined SharonAnammox process for the removal of ammonia from concentrated wastewater (Jetten

et al., 1997a).

Table 4. Overview of the parameters of an Anammox fluidized bed reactor (Jetten et al., 1997a)

and a Sharon reactor (Hellinga et al., 1998) both fed with sludge digester effluent. The nitrite for

the Anammox process was supplied separately

Sharon Anammox

Ammonium load 0.631.0 0.241.34 kg NH+4 N m3reactor day

1

Nitrite load not applicable 0.221.29 kg NO2 N m3

reactor day1

Nitrogen load 0.631.0 0.462.63 kg Ntot m3

reactor day1

NH+4

-N effluent 199 27 85 mg N l1

NO2

-N effluent 469 3 3 mg N l1

Efficiency NH+4

removal 7690 88 9 %

Efficiency NO2

removal not applicable 99 2 %

Maximum activity 10.3 0.26 kg Ntot (kg dry weight)

1 day

1

Table 5. Reaction equations of the Sharon and Anammox processes (biomass formation not

included)

Sharon: 2 NH+4

+ 2 HCO3

+ 1.5 O2 >NH+

4 + NO

2+ 2 CO2 + 3 H2O

Anammox: NH+4

+ NO2

>N2 + 2 H2O

Combined process: 2 NH+4

+ 2 HCO3

+ 1.5 O2 >N2 + 2 CO2 + 5 H2O

bed reactor. In the nitrite limited Anammox reactor all

nitrite was removed, the surplus ammonium remained.

During the test period the ammonium removal effi-

ciency was 83%. This new combined system could be

implemented in the existing infrastructure of wastewa-

ter treatment plants without much trouble. The use

of compact reactors with minimal area requirement

make their implementation on existing locations pos-

sible. The Anammox and Sharon processes are feas-

ible and have been thoroughly tested on laboratory

and pilot scale with existing wastewater. However,

the optimization and application of the combination

of these two processes on full scale remains a chal-

lenge towards implementations in a future wastewater

treatment plant.


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Acknowledgement

The research on the microbial nitrogen conversions

was financially supported by the Foundation of Ap-

plied Water Research (STOWA), the Foundation for

Applied Sciences (STW), The Netherlands Found-

ation for Life Sciences (NWO-SLW), the RoyalAcademy of Arts and Sciences (KNAW), Gist-

brocades, DSM, Paques, and Grontmij consultants.

The constructive discussions with and contributions

of various co-workers and students over the years are

gratefully acknowledged.

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new pathways for ammonia conversion in soil and aquatic systems

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