Waste Management 31 (2011) 2287–2293

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Waste Management

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Anammox: An option for ammonium removal in bioreactor landfills

Roberto Valencia a,⇑, Willem van der Zon b,1, Hans Woelders c, Henk J. Lubberding a, Huub J. Gijzen a,2

a UNESCO-IHE Institute for Water Education, P.O. Box 3015, 2601 DA Delft, The Netherlandsb GeoDelft, Stieltjesweg 2, 2628 CK Delft, The Netherlandsc ESSENT Milieu, VAMweg 7, P.O. Box 5, 9418 ZG Wijster, The Netherlands

a r t i c l e i n f o

Article history:Received 16 July 2010Accepted 25 June 2011Available online 26 July 2011

Keywords:AmmoniumAnammoxAnaerobic digestionBioreactor landfills

0956-053X/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.wasman.2011.06.012

⇑ Corresponding author. Present address: IPN-CIIDFracc. 20 de Noviembre II, CP 34220 Durango, Mexico

E-mail addresses: r.valencia@unesco-ihe.org, robeValencia), willem.van.der.zon@vopak.com (W. van de(H.J. Gijzen).

1 Present address: VOPAK, P.O. Box 863, 3000 AW R2 Present address: UNESCO, Jakarta Office, P.O. Box 13 FSQ term refers to the state at which residues n

human health or the environment.

a b s t r a c t

Experiments carried out in bioreactor landfill simulators demonstrated that more than 40% of the total Nwas transferred into the liquid and gas phases during the incubation period of 380 days. Ammonium, anend product of protein degradation and important parameter to consider during landfill closure, tends toaccumulate up to inhibitory levels in the leachate of landfills especially in landfills with leachate recircu-lation. Most efforts to remove ammonium from leachate have been focused on ex situ and partial in situmethods such as nitrification, denitrification and chemical precipitation. Besides minimal contributionsfrom other N-removal processes, Anammox (Anaerobic Ammonium Oxidation) bacteria were found tobe active within the simulators. Anammox is considered to be an important contributor to remove N fromthe solid matrix. However, it was unclear how the necessary nitrite for Anammox metabolism was pro-duced. Moreover, little is known about the nature of residual nitrogen in the waste mass and possiblemechanisms to remove it. Intrusion of small quantities of O2 is not only beneficial for the degradationprocess of municipal solid waste (MSW) in bioreactor landfills but also significant for the developmentof the Anammox bacteria that contributed to the removal of ammonium. Volatilisation and Anammoxactivity were the main N removal mechanisms in these pilot-scale simulators. The results of these exper-iments bring new insights on the behaviour, evolution and fate of nitrogen from solid waste and presentthe first evidence of the existence of Anammox activity in bioreactor landfill simulators.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Operating landfills as bioreactors has gained a lot of attention inrecent years due to shorter waste stabilisation periods, higher bio-gas production and recovery, reduced leachate organic strength andmaximised waste settlement and volume recovery (Pohland, 1980;Warith, 2002; Benson et al., 2007). However, bioreactor landfillshave also shown several constraints such as uneven moisture distri-bution (seepage and preferential flow), geotechnical instability(landslides) and accumulation of compounds, up to toxic levels, inthe leachate (i.e. ammonium, chloride, etc.) (Price et al., 2003).Nitrogen compounds, especially ammonium, have been identifiedas a main parameter that will jeopardise the achievement of FinalStorage Quality (FSQ3) status, hence the safe closure of landfill sites,

ll rights reserved.

IR-Durango, Av. Sigma 119,. Tel./fax: +52 618 8 25 60 73.rto.valenciav@gmail.com (R.r Zon), h.gijzen@unesco. org

otterdam, The Netherlands.273, Jakarta, Indonesia.o longer poses threat to the

especially because of its pollution potential for surface and ground-water bodies; chloride has a negative effect on the growth of vege-tation if leachate is used in irrigation schemes (Burton and Watson-Craik, 1998; Stephens et al., 2000; Barlaz et al., 2002). Ammonium,an end product of protein degradation (Jokela and Rintala, 2003;Berge et al., 2005), tends to accumulate, especially in the leachateof landfills with leachate recirculation (Onay and Pohland, 1998;Price et al., 2003), since there are no removal mechanisms understrict anaerobic conditions, with the exception of Anammox metab-olism (Mulder et al., 1995; Strous et al., 1997). Most efforts to re-move ammonium from landfills focused on the leachate fractionvia ex situ methods such as nitrification/denitrification, precipita-tion and even irrigation schemes (Ohlinger et al., 1998; Jokelaet al., 2002; Li and Zhao, 2003; Kurniawan et al., 2006). Alternativeapproaches referred to promoting ex situ nitrification and in situdenitrification of the leachate to remove ammonium (Price et al.,2003; He et al., 2007; Long et al., 2009; Zhu et al., 2009) or evencomplete in situ removal of ammonium by dedicated nitrificationand denitrification zones (Onay and Pohland, 1998; Berge et al.,2006; He and Shen, 2006; Shao et al., 2008). Either in situ or partiallyin situ approaches are likely to produce NOx and N2O (Price et al.,2003; Zhu et al., 2009). The quantities of NOx and N2O producedare insignificant (<0.1% by volume), compared to the amount of

Table 1Composition of the municipal solid waste.

Component Percentage (wet weight)

Organic undefined 33.2Paper & card board 15.2Plastics 3.7Glass 13.6Ferrous metals/non-ferrous metals 0.6Leather/rubber 0.2Wood 2.5Inert (>3.4 mm) 13.7Inert (<3.4 mm) 15.2Textiles 2.1

Sources: ESSENT Milieu 2004 at transfer static in Wijster, The Neatherlands (Figuresprovided by W. Woolden).

2288 R. Valencia et al. / Waste Management 31 (2011) 2287–2293

CO2 and CH4, the main components of landfill gas which are alsogreenhouse gases (Tchobanoglous and Kreith, 2002). However,their global warming potential in a 100 year time horizon, whichis 296 times greater than that of CO2, made them significant pollu-tants for their contribution to the climate change (IPCC, 2001). Ithas been estimated that landfill sites contribute to approximately3–5% of anthropogenic greenhouse gases emissions of the world(Kumar et al., 2004; Bogner et al., 2007). Despite all efforts to reduceammonium levels from landfill leachate, little information is avail-able about the origin, evolution and fate of ammonium in bioreactorlandfills and especially about the residual nitrogen in the solidphase. Huber et al. (2004) suggested that approximately 4% of Nleaves the landfill via the leachate pathway, while 96% of Nremained in the landfill body. The general objective of the researchproject was to investigate enhancement techniques for landfill gasproduction in bioreactor landfills; however this paper presents theevidence of Anammox bacteria activity and discusses its role on theN removal process at bioreactor landfills.

2. Materials and methods

2.1. Experimental set-ups

Seven bioreactor landfills were simulated using high densitypolyethylene (HDPE) sewage pipes (0.75 m3 working volume).Schematic view of the simulators can be seen in Fig. 1a. The simu-lators were filled with shredded (max. particle size 4 cm) municipalsolid waste (MSW), the composition of which is shown in Table 1;operational features of the simulators are shown in Table 2 and de-scribed in more detail elsewhere (Valencia et al., 2009a,b). Bufferedtap water (124 L, 0.1 M NaHCO3) was initially added to stimulateleaching conditions; the leachate was recycled three times (everyother day) per week (60 ± 1 L week�1) in order to maintain a dy-namic leachate flow and at least 45% moisture content (field capac-ity) on a wet weight basis. No more water was added during theexperimental period. Buffer (0.3 M NaHCO�3 ) was added to theleachate prior to recycling during a period of 6 weeks (day 50–100) to reduce the negative impact of the volatile fatty acids(VFA) on the pH. The internal temperature of the simulators wasmaintained within the range of 30 ± 4 �C by means of an electric

Fig. 1. (a) schematic view, (b) different configurations, an

blanket covered with two layers of fibre-wool with aluminium foiland wrapped in plastic.

Anammox activity test: Composite solid waste samples (3 coresof 0.87 cm in length and 10 cm in diameter, then mixed to createthe composite sample) were taken, at the end of the experimentalperiod (day 487) from each of the 0.75 m3 simulators; after sub-jected to a leaching test according to CEN (2002), the biomasswas concentrated by centrifugation (3000 rpm for 1 h) and10 ± 0.5 g were placed in 50 ml butyl septa airtight bottles flushedwith argon and incubated in a 37 �C temperature controlled room.Anammox bacteria were enriched using a buffer solution of hepes/bicarbonate (75/5 mM, pH 7.8), 5 mM NaNO2, 2.5 mM (NH4)2SO4,0.1 mM hydrazine (N2H4�H2SO4) according to Strous et al. (1999).

Anammox identification in the residue was carried out usingfluorescent in situ hybridisation (FISH) techniques employing thefollowing probes: Pla46 for planctomycetales, AMX820 coveringall Anammox organisms, specially Kuenenia sp. and Brocadia sp.and DHI820 for Anammoxoglobus sp. Probes and hybridisation pro-cedures are described in Schmid et al. (2003) and Kartal et al.(2007). No Anammox bacteria identification was carried out inthe leachate.

2.2. Analytical procedures

Total and volatile solids content of the MSW were analysedaccording to Standard Methods (APHA, 2005). Additionally, a

d (c) photograph of the bioreactor landfill simulators.

Table 2Operational features of the pilot-scale bioreactor landfill simulators.

Simulator Feature Material Wateradded

1 and 2 Intermediate layers of gravel(±8 cm)

330 kg MSW + 100 kggravel

124 L

3 and 4 Homogenous mixture MSWand gravel

330 kg MSW + 100 kggravel

124 L

5 and 6 Intermittent bottom forcedaeration

450 kg MSW 124 L

7 Control with less density 350 kg MSW 124 L

R. Valencia et al. / Waste Management 31 (2011) 2287–2293 2289

leaching test was conducted in triplicate to determine the leachingpotential of COD, BOD, TOC, NHþ4 , selected ions (Cl�, NO�3 , PO3�

4 , andSO2�

4 ) and heavy metals (Pb, Ni, Zn, Cu, and Cd) from the solidwaste according to CEN (2002). The elemental composition (CHNS)of the MSW was determined by flash combustion in a partial oxy-gen atmosphere using helium as carrier, at 1020 �C with a ThermoQuest EA 1110 Interscience elemental analyser. Leachate sampleswere analysed for pH, temperature, conductivity and oxygen withportable meters WTW pH 340, LF 340 and Oxi 345, respectively.NHþ4 was analysed according to NEN (1983). NO�2 was analysedaccording to APHA (2005). TOC was determined using an OI Corpo-ration TOC Analyser M-700. NO�3 was analysed using an Ion Chro-matography system DIONEX ICS-1000 attached with an automatedsample injector DIONEX ASI-100. All liquid samples were filteredwith glass fibre filters GF 52, Schleicher and Schuell. Landfill gascomposition (CH4, CO2, and O2) was monitored using a Geotechni-cal Instruments GA25 portable gas extraction analyser. Hydrazinewas measured using detector tubes (MSA, range 0.1–5 ppm) anda thumb-pump sampler (100 cc sample volume/stroke).

2.3. Statistical analyses

Statistical analyses were carried out using statistical softwarepackage SPSS� for Windows (SPSS Inc., Chicago, IL, USA). A generallinear model of repeated measures ANOVA was used to analyse thedata. Since, statistical analyses indicated that there was no signif-icant difference among the different treatments/variables (sig.0.15, 0.996, 0.285, 0.68, and 0.841 for pH, conductivity, biogas pro-duction, TOC and ammonium respectively); the graphs were con-structed using averaged (seven simulators) values of pH, TOC,biogas production, ammonium and N content and complementedwith standard deviation bars.

3. Results

3.1. Bioreactor landfill pilot-scale simulators performance

The process parameters of the bioreactor landfill pilot-scale sim-ulators are shown in Fig. 2a. More details on the reactor set up andoperation are presented in Valencia et al. (2009a,b). Due to theaccumulation of hydrolytic products the pH decreased in the firstmonths of operation, but increased as soon as a more acclimatisedmethanogenic population developed, which converted thesehydrolytic products into biogas. Initially, due to a constant dissolu-tion of salts into the leachate, conductivity increased sharply duringthe first two weeks, followed by a more gradual increase during thenext 150 days after which it remained relatively constant up to day250. Thereafter conductivity started to decrease due to degassing4

4 A process related to the carbonate equilibrium at which calcite started to bedissolved and as result CO2 is liberated into the gas phase as explained by Thorntonet al., 2000. The causes for the dissolution are related to temperature, acidity anddissolved ion concentrations.

and precipitation within the simulators towards the end of theexperiment.

During the first 150 days of operation the gas production(Fig. 2c) was minimal (<5 m3) and contained mainly CO2 (±85%).However, as more favourable conditions (i.e. neutral pH) for meth-anogens were reached around day 150, gas production increasedexponentially during the following 200 days, coinciding with arapid decrease of TOC during the same period (Fig. 2b).

3.2. Ammonium in the pilot-scale bioreactor landfill simulators

After an initial increase of the ammonium levels (Fig. 3a) in theleachate during the first 100 days (up to 2.5 g L�1), ammoniumconcentration remained stable during the period between days100 and 200. The fact that ammonium levels did not furtherincrease could be caused by partial inhibition of the hydrolytic pro-cess due to high cumulative levels of ammonium reached withinthe pilot-scale simulators, as suggested by Krylova et al. (1999)and Chen et al. (2008). However, from day 200 onwards ammo-nium levels gradually started to decrease to an averaged 1.7 g L�1

(Fig. 3a); the maximum ammonium decrease observed was about1 g L�1. In a preliminary experiment with longer duration, thesame tendency was visible and the final ammonium levels wereeven below 1 g L�1 (Fig. 3b). After the ammonium levels startedto decrease, two simulators (5–6) were aerated (day 250) (inter-mittent forced bottom aeration, 240 L air week�1), this did not leadto a visible difference with the five non-aerated simulators(Fig. 3a), most likely due to the rapid escape of the introduced airinto the headspace of the simulator and subsequent release viathe measuring gas system.

A possible explanation of the ammonium decrease (Fig. 3a andb) after day 200 could be the presence of Anammox bacteria. Thecomposite samples5 taken on day 300 from all simulators weresent to analysis for Anammox detection. All the samples, exceptthose from the aerated simulators, revealed the presence of Anam-mox bacteria (Fig. 4a and b); Fig. 4 shows the clearer pictures fromthe analysis since there was plenty material (auto-florescent) inthe samples that interfered with the determination. In addition,high levels of hydrazine (>6 mg L�1), an intermediate product ofthe Anammox metabolism (Schmid et al., 2003; Kartal et al.,2007), were detected in the gas phase during the last 100 days ofoperation. However during this period of time the substrate forAnammox was already produced.

An Anammox activity test on the extracted biomass from thesimulators showed patterns typical for Anammox bacteria: a 1:1(1:0.98 this experiment) molar conversion of NHþ4 (0.0034 M day�1)and NO�2 (0.0046 M day�1) accompanied with a minimal produc-tion of NO�3 (Fig. 4c).

4. Discussion

Effectiveness of the simulators transferring the carbon fractionfrom the solid phase into the liquid and gaseous phases has beenpreviously described (Valencia et al., 2009a,b). Gas, pH, and TOCvalues followed similar trends as those reported in literature(Pohland, 1980; Warith, 2002) however, faster responses wereobserved in those simulators with improved hydraulic conditions(Valencia et al., 2009b).

The release of ammonium and increased concentration in theleachate during the first week (up to 1 g L�1) seemed to be com-pletely governed by a physical mechanism: the contact between

5 Composite samples were made out of three cores of 87 cm in length extractedfrom each simulator, and then cut in three pieces (bottom and upper layer),afterwards was mixed correspondingly. One sample per layer was extracted and sentto analysis for Anammox detection.

Fig. 2. (a) Conductivity and pH, (b) TOC in the leachate and (c) Cumulative gas production from the pilot-scale bioreactor landfills.

Fig. 3. Ammonium in the leachate (a) 1 year experiments with seven simulators and (b) a previous 2 years experiment with two pilot-scale simulators preceding (a).

2290 R. Valencia et al. / Waste Management 31 (2011) 2287–2293

solids and the liquid percolating through the waste mass, whichwashed-out the ammonium salts present in the simulators. Thesecond ammonium increase from day 10 until day 120 (Fig. 3aand b) can be attributed to the enhanced microbiological conver-sion of organic matter under anaerobic conditions (Berge et al.,2005; Jokela et al., 2005). Ammonium accumulated because therewere no removal mechanisms within the simulators. Apparently,there is a steady state reached between day 120 and 200, wherethe constant ammonification rate is similar to the ammoniumremoval rate (Fig. 3a and b). An ‘‘accidental’’ intrusion of O2

(3.25 mg O2 L�1 week�1) could happen because the recirculationprocess was carried-out with a pump outside the anaerobic phaseof the system (60 L week�1). This amount of oxygen was sufficientfor partial or complete nitrification of the leachate and since duringthis period there was sufficient organic matter present in the sim-

ulators denitrification could also have taken place. Nitrificationprobably did not occur during the first 100 days, because nitrifyingbacteria require more than 30 days to fully develop under optimalconditions (Hoilijoki et al., 2000). The fact that NO�2 and NO�3 (datanot shown) were not detected could mean two things: (1) Nitrifi-cation and subsequent denitrification were not taking place withinthe simulators, or (2) and more likely, the conversion rates of NO�2(and NO�3 ) into N2 occurred with a certain speed that thesecompounds could not be detected in the leachate. Berge et al.(2006) reported NO�2 and NO�3 in the leachate using 1 L and 133 Laerated reactors at which a biotic process was responsible for thesecompounds; whereas these reactors were relatively easier to han-dle, the probability to measure such compounds in bigger reactors,like the ones reported here, is substantially lower assuming thesame biotic process took place. Up to day 200, ammonium trends

Fig. 4. (a–b) FISH analyses with PLA46, Amx820 and DHI820 probes (red Anammox clusters encircled) and (c) Anammox activity test on biomass extracted from the pilot-scale bioreactor landfill simulators. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 3Anammox activity test results.

Compound [Mol/L] Initial Final 1 Spike Final 2

NHþ4 0.045302 0.040619 0.046257 0.42685Nitrite 0.012541 0.000739 0.005087 0.000399Nitrate 0.000181 0.000056 0.000135 0.000022

R. Valencia et al. / Waste Management 31 (2011) 2287–2293 2291

(Fig. 3a and b) were comparable to those reported in literature,especially those referring to large scale experiments (Morriset al., 2003), however from day 200 onwards ammonium exhibiteddecreasing trends. Similar decreases of ammonium levels has beenobserved and not fully explained in comparable bioreactor landfillexperiments (Agdag and Sponza, 2005a,b).

The key factors for the decrease of ammonium levels after day200 were, most likely, the reduced supply of ammonium as mostof the readily organic matter (Fig. 2b) had been hydrolysed andstarted to be converted into biogas (Fig. 2c) and the constant recir-culation process that kept supplying enough O2 to carry out, atleast partial, nitrification of the leachate. Despite the fact that therewas sufficient organic matter in the leachate (Fig. 2b) but probablynot easily biodegradable, denitrification was limited as most of theacetic acid (data not shown), the preferred substrate by denitrifiers(Constantin and Fick, 1997) was depleted from the system. Conse-quently, from day 300 onwards minimal quantities of NO�3 (up to30 mg L�1) (data not shown) started to be detected in the leachate,which suggests that denitrification and hence nitrification werealso occurring at earlier stages within the simulators. However,calculations revealed that the amount of O2 introduced into thesepilot-scale simulators was not sufficient to reduce significantlyammonium levels in the leachate as reported by Berge et al.(2007), Shao et al. (2008) and Long et al. (2009) where sufficient6

air supply leaded to a substantial removal of ammonium fromthe leachate in bioreactor landfill simulators. According to AWWA(1992), for every kilogram of ammonium converted it will be nec-essary to consume 4.18 kg of oxygen or about 2 M of O2 per mole ofNHþ4 transformed. Moreover, the amount of oxygen was notincreased because it was not the main objective of the project (bio-gas production and recovery). Therefore there must be otherammonium removal processes that have contributed to the decline

6 The term ‘‘sufficient’’ refers to the minimal amount of oxygen to convertammonium into nitrate and nitrite via nitrification.

in ammonium levels. The reason that these options cannot accountfor the entire quantity of N removed from the systems, gaveenough elements to look for Anammox bacteria presence withinall pilot-scale simulators.

Biomass extracted from the simulators revealed the presence ofAnammox bacteria, except in those simulators at which active aer-ation was implemented (Table 2 and Fig. 5).

The main species identified were Candidatus Brocadia anammox-idans, Candidatus Kuenenia stuttgartiensis and Candidatus Brocadiafulgida, with an estimated bacterial density of 5–7% of the total bac-terial density (Op den Camp and Schmid, 2007). It is more likely thatpartial nitrification up to NO�2 and sufficient ammonium in the leach-ate, supported by the optimal pH (±7.5–8.0) and temperature(±30 �C) (Strous et al., 1997), were the key factors for the develop-ment and activity of the Anammox bacteria within the simulators.At day 300, no Anammox bacteria were found in the two simulatorswhich were intermittently aerated for about 50 days (240 L airweek�1). Jetten et al. (1998) and Strous et al. (1999) reported thatAnammox activity was reversible after exposure at low O2 concen-trations (<0.5% air saturation), but Egli et al. (2001) reported com-plete inhibition of Anammox activity at higher concentrations ofO2 (>18% air saturation), like the ones applied to these simulators.Besides the FISH analyses on the biomass and the presence of hydra-zine, the activity test confirmed the presence of Anammox bacteria.The Anammox activity test results were similar to those reported byEgli et al. (2001) in which conversion of ammonium and nitrite were

Fig. 5. Samples extracted from the aerated simulators without presence of Anammox bacteria.

2292 R. Valencia et al. / Waste Management 31 (2011) 2287–2293

approximately 1:0.98 on molar basis, specially after spiking with abuffer solution containing NHþ4 and NO�2 (Fig. 4c and Table 3).

The substrates for Anammox bacteria, which are known to havelong doubling times, could be formed before and during the ‘‘par-tial’’ inhibition period between days 100 and 200, during whichnitrite was produced due to the accidental intrusion of oxygendue to the recirculation process as explained before.

Nitrogen balances (Fig. 6) showed that approximately 40% of thetotal N was released from the solid waste and transferred either intothe liquid or the gas phase in a period of 380 days. At the end of theexperimental period, only 2.3 kg of an initial 3.2 kg of N added to thesimulators could be recovered: 1.9 kg as residual N and 0.4 kg of N inthe free liquid mainly composed of ammonium (±80%) and Norg

(±20%). The unaccounted N, if totally converted into N2, wouldhad accounted for about 0.8 m3 of gas, which was approximately2.8% of the total landfill gas production; this N2 value is within theranges suggested in literature (Tchobanoglous and Kreith, 2002).It was not certain if the residual N was of inorganic and inert natureor perhaps was not biologically available as suggested by Burtonand Watson-Craik (1998) in the form of humus-like material.

Possible options to explain the amount of N removed (±1 kg)from the simulators are: precipitation as struvite, volatilization ofNH3, biological uptake, nitrification and denitrification processesand Anammox activity.

Theoretically, about 10 g N could be removed via struvite pre-cipitation depending on pH values (above 7.5) and temperature(Ohlinger et al., 1998), since NHþ4 , Mgþ2 , and PO3�

4 (33:6:1 molarratio) exceeded the solubility values according to simulationsusing PHREEQC. However, struvite was not measured and mostlikely low PO3�

4 concentrations (data not shown), instead of Mg2+,would be the limiting factor for precipitation of higher struvitequantities within the pilot-scale simulators.

Fig. 6. Nitrogen mass balance of the bioreactor landfill simulators.

Approximately 30 g N could have been volatilised as NH3 fromday 150 until day 370 according to pH values (maximal volatilisa-tion rate 2.5% of the daily gas production were the pH 7.5 ± 0.5).

Maximally 4 g N could have been used for assimilation by micro-organisms for cell growth, similar to figures reported by Burton andWatson-Craik (1998); therefore bacterial up-take cannot explainthe total consumption of N as suggested by Agdag and Sponza(2005a).

Removal of N due to full nitrification (up to NO�3 ) can only accountfor 2.3 g N, considering that maximally 3.25 mg O2 L�1 were intro-duced every time the leachate was recycled into the simulators (en-tire operational period of 483 days). Denitrification was not alimiting factor for N removal since there was sufficient organic mat-ter present in the simulators at least during the first 200 days.

The combined effect of partial nitrification and Anammox, withthe same amount of oxygen introduced, can stoichiometricallyaccount for up to 5.4 g N removed. However, based on the approx-imated Anammox bacterial density and the removal yields sug-gested by Strous et al. (1997) (NHþ4 :NO�2 :NO�3 ; 1:(1.31 ± 0.06):(0.22 ± 0.02)) it could be possible to explain the removal of about100 g (NHþ4 . Moreover, the Anammox activity test results (Fig. 4c)suggested that it would have been possible to convert about854 g N, assuming 100 L of free leachate and an active period fromday 200 until day 380. However, it was not clear how the necessary(NO�2 for Anammox was produced.

It is most likely that all these processes occurred simultaneouslywithin the simulators. Anammox could be the main responsible fac-tor for the removal of N, especially when most of the readilydegradable organic matter was depleted (after day 200). However,Anammox bacteria depend on availability of NO�2 to carry out theirmetabolism, which can only be produced via nitrification; conse-quently the amount of O2 introduced would be the main limitingfactor to remove ammonium as occurs in the CANON process forsewage treatment (Sliekers et al., 2002). Diffusion of O2 throughthe simulators walls was considered as a possibility, but it was un-likely since all reactors were operated at overpressure. Neverthe-less according to HDPE properties (Composite Agency, 2007)maximally about 8.6 g O2 could have penetrated through the wallduring the entire length of the experiment (380 days), which wasabout 2.5% of the required O2 to nitrify the 93 g NHþ4 converted dur-ing the experiment. Alternatively, NO�2 could have been producedexternally in the leachate reservoirs and introduced via recircula-tion without being noticed. However, during incidental measure-ments in the leachate reservoirs NO�2 was never detected.Anammox presence in landfill environments has been suspectedbefore (Burton and Watson-Craik, 1998; Price et al., 2003; Bergeet al., 2005), but never confirmed. To our knowledge these resultspresent the first evidence of the existence of Anammox activity inbioreactor landfill simulators.

R. Valencia et al. / Waste Management 31 (2011) 2287–2293 2293

5. Conclusions

Based on the results of these experiments the following conclu-sions can be drawn:

� Intrusion of small quantities of O2 is not only beneficial for thebiodegradation process of MSW, but also it triggers in situ nitri-fication and promotes the growth of Anammox bacteria.� The presence of Anammox bacteria in Bioreactor Landfills sim-

ulators could have, to a large extent, contributed to the removalof N from the solid matrix.

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