Nitrate and nitrite injection during municipal solid wasteanaerobic biodegradation

Vassilia Vigneron a, Marie Ponthieu a, Giulia Barina b, Jean-Marc Audic c,Christian Duquennoi a, Laurent Mazeas a, Nicolas Bernet d, Theodore Bouchez a,*

a Cemagref-HBAN, parc de Tourvoie, BP 44, 92163 Antony cedex, Franceb SUEZ ENVIRONNEMENT-FAIRTEC, 38 avenue Jean Jaures, 78440 Gargenville, Francec SUEZ ENVIRONNEMENT-CIRSEE, 38 rue du President Wilson, 78230 Le Pecq, France

d INRA-LBE, avenue des Etangs, 11100 Narbonne, France

Accepted 28 February 2006Available online 21 June 2006

Abstract

Nitrified leachate recirculation has been proposed as a promising strategy for sustainable landfill management. In four test reactors,nitrate or nitrite was added ð250 mg N-NO�x L�1Þ during municipal solid waste biodegradation. Nitrogen-oxides reduction reactionswere monitored. Denitrification was the main nitrogen reducing reaction observed. On one hand, during the acidogenic waste degrada-tion phase, as high amounts of volatile fatty acids (VFA) were present, nitrogen-oxides reductions were interpreted as heterotrophic den-itrifications. On the other hand, denitrification reactions occurring during the late methanogenic phase were accompanied by sulphateproductions and, as VFA were not detected, it was probably an autotrophic reaction. Denitrification inhibition was observed once.Ammonium concentration increased suggesting the occurrence of a dissimilatory nitrate reduction to ammonium (DNRA). Statisticaltreatment of analytical data revealed that only H2S concentration had a significant negative effect on N2 production in our system.NO production was observed once when nitrite was injected during the acidogenic phase resulting in a total waste degradation inhibition.These results indicate that the consequences of nitrified leachate recirculation in full-scale landfills need to be carefully examined espe-cially during the acidogenic phase or in the presence of waste containing high quantities of sulphur.� 2006 Elsevier Ltd. All rights reserved.

1. Introduction

In 2002, approximately 46 millions tons of municipalsolid waste (MSW) were produced in France (Ademe,2002); 52% of it was disposed of by burial in sanitary land-fills. Other treatments exist of course: waste sorting andrecycling or composting, and they are becoming increas-ingly important. Landfills remain the chief method forMSW management. The landfill bioreactor represents abetter technical solution for solid waste management than‘‘dry tomb’’ landfills (Pacey, 1999; Mehta et al., 2002; Poh-land, 2002). In them, leachate is recirculated through the

0956-053X/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.wasman.2006.02.020

* Corresponding author. Tel.: +33 140 966 040; fax: +33 140 966 270.E-mail address: theodore.bouchez@cemagref.fr (T. Bouchez).

waste mass, increasing the moisture content and enhancingthe anaerobic digestion of the organic fraction of wastethat is readily biodegradable (Barber and Maris, 1984;El-Fadel, 1999; Yuen, 1999). Using landfills as bioreactorshas many advantages: (1) it increases the effective density ofwaste and therefore the capacity of landfills, (2) the rate ofgas production goes up and the energy recovery becomesmore efficient, and (3) it accelerates decomposition of wastewith the effect of shortening the monitoring period andthereby reducing the overall cost (Barlaz et al., 1990; War-ith, 2002).

However, recirculated leachate also contains pollutantsthat are hardly degradable and are reinjected into thewaste mass. Accumulation of ammonia has especiallybeen recognized as being problematic in the long run

(Burton and Watson-Craik, 1998). The reason is thatthere is no ammonia elimination process under the anaer-obic conditions prevailing within the landfill body. Whenleachate is recirculated, NHþ4 may accumulate to higherlevels than during conventional single pass leaching.Thus, high ammonia concentration could persist longafter the COD concentrations have decreased to levelsrepresentative of well-decomposed waste. Ammonia accu-mulation could then induce methanogenic inhibition(Wens et al., 2001). Treatment of ammonia is thus animportant aspect of the long-term management of a land-fill bioreactor.

One strategy for NHþ4 removal is to aerobically treat theleachate to convert NHþ4 into NO�3 . Some authorsattempted to perform nitrification inside of the landfillbody (Onay and Pohland, 1998; Berge and Reinhart,2003). However, it is generally done outside the landfillbody. In our case, to reduce nitrate ðNO�3 Þ into molecularnitrogen (N2), the waste mass is used as an anoxic bioreac-tor for denitrification.

In situ denitrification has previously been reported.Knox and Gronow (1995), who used a pilot scale (3 m3)reactor in their study, showed that partly-degraded MSWwas able to support denitrification and methanogenesissimultaneously with a rate of denitrification up to30 gN m�3. Onay and Pohland (1998) simulated a seriesof landfill cells operating under methane producing, nitrify-ing and denitrifying conditions. The nitrate removal rangedbetween 91% and 93%. They concluded that nitrificationand denitrification were feasible in situ. Burton and Wat-son-Craik (1998) demonstrated that nitrate concentrationsof 500 or 1000 mg N-NO�3 L�1 were undetectable after 6days when addition was performed in reactors filled with1 month old waste. When nitrate concentration increased,methanogenesis was delayed, and a dose-dependent inhib-itory effect was observed (El-Mahrouki and Watson-Craik,2004). Price et al. (2003) injected nitrate in reactors whenthe methanogenic phase had been obtained. They con-firmed that in the methanogenic phase waste was able toconvert nitrate into N2 by denitrification. However, thesestudies were almost all conducted with MSW in the meth-anogenic phase, only two were not (Knox and Gronow,1995; Burton and Watson-Craik, 1999). So far, little infor-mation is available on nitrate reduction reaction during theacidogenic phase. Moreover, there has been only one eval-uation of the nitrite reduction reaction (Bae et al., 2002).They demonstrated that denitrification was the main nitritereduction reaction when injections were performed in anold waste mass (5–10 years old).

The specific objectives of this study were to (1) investi-gate the nitrate reduction reactions induced when nitrateinjections ð250 mg N-NO�3 L�1Þ are performed at differentstages of waste degradation: acidogenic and methanogenicphase, (2) evaluate the nitrite reduction reactions inducedby the injection of nitrite instead of nitrate and (3) betterunderstand nitrate or nitrite reduction reactions soinduced.

2. Materials and methods

2.1. Experimental design

Three control reactors named C1, C2 and C3 and fourtest reactors named Ta1, Ta2, Ti1 and Ti2 were studied inthis experiment. Reactors were incubated at 35 �C ± 2 �C,and data are presented here for almost 500 days ofincubation.

Control reactors C1, C2 and C3 were operated as controlreplicates without any nitrogen injection. For test reactorsTa1 and Ta2 (challenged with nitrate), nitrate solution wasinjected five times (on days 3, 17, 45, 145 and 271). As fortest reactors Ti1 and Ti2 (challenged with nitrite), nitritesolution was injected three times (days 3, 17, 45) and fivetimes (days 3, 17, 45, 145 and 271), respectively. Into testreactors, we injected 3–4 mL of a concentrated KNO3 orKNO2 solution at 43 g N L�1 or 47 g N L�1, respectively,in order to minimize the dilution effect caused by the injec-tions. Precise injected volumes were calculated for eachinjection, so that 250 mg N-NOx L�1 were present in thereactors after each injection.

2.2. Experimental system

Incubations were performed in 1.1-L glass bottles closedwith a screw cap and a septum.

Waste was reconstituted according to the average com-position of municipal solid waste in France (Ademe,1999). The putrescible fraction was replaced by maturecompost of green waste (3 months old) to simulate amore mature waste, that would be more rapidly limitedby organic carbon availability in the experiments. Eachwaste fraction was collected separately. Glass powderwas used for glass fraction. Wood shavings and iron fil-ings were collected for the wood and metal fractions,respectively. Fractions were shredded into approximately1-cm elements. A waste mixture was reconstituted fromeach fraction, according to the proportions given inTable 1. Cow manure was added to enhance waste degra-dation. A total wet weight of 41.55 g of waste was dis-posed in each glass bottle.

For practical reasons, two full-scale landfill leachatesamples (Vert-le-Grand Landfill, France) were recoveredin March and July 2003 from the same landfill cell to inoc-ulate the different reactors. Initial leachate compositions inthe different reactors were thus slightly different as shownin Table 2. For each experimental bottle, 680 mL of leach-ate were used.

Headspace was purged with helium to obtain less than0.2% of O2 at the beginning of each experiment.

2.3. Analytical methods

All the results below are given at STP (Standard Tem-perature and Pressure) conditions.

Table 1Waste composition (19.4% water content)

Waste fraction Wet mass percentage (%)

Paper 16Cardboard 9.3Complex (composed or multi material

packaging)1.4

Textile 2.6Other textile (disposable diapers. . .) 3.1Plastic 11.1Wood 3.2Glass 13.1Metals 4.1Mixed inorganic wastes (soil, bits of

concrete, stones and the like)6.8

Special waste (no toxic waste) 0.5Compost 28.8Total waste 100Cow manure 5

Table 2Initial leachate composition in the various reactors

Parameters Leachate at day 0

Reactor C1, C2, Ta1

and Ti1

Reactor C3, Ta2

and Ti2

pH 8.5 8.5TOC (mg C L�1) 1500 2100TIC (mg C L�1) 2180 1470Total VFA (mg C L�1) 107 15Chloride (mg L�1) 5000 5400Sulfate (mg L�1) 120 240Ammonium (mg L�1) 1900 1920Nitrate–nitrite (mg L�1) 0 0

2.3.1. Gas samples

To measure gas production, gas headspace was equili-brated, when the septum was inflated, at atmosphericpressure (35 �C) with a glass syringe (30 mL ± 1 mL or100 mL ± 4 mL). The gas composition was analyzedimmediately after the equilibration by connecting the bot-tle to a gas chromatograph (lGC CP2003P Varian)equipped with two parallel chromatographic columnscoupled with thermal conductivity detectors (TCD). APoraplot U column was used to obtain CO2, N2O andH2S concentration (column temperature 30 �C, injectortemperature 50 �C, medium sensitivity) and a molecular-sieve column for O2, N2 and CH4 concentration (columntemperature 55 �C, injector temperature 55 �C, mediumsensitivity, backflush 5.5 s). The carrier gas was helium.The calibration was made with a commercial gas mixturecontaining 0.5% H2S, 3% N2O, 40% CO2, 50% CH4 and6.5% N2 (Air Products). The calibration for oxygen wasperformed with air. The detection limit for all gaseswas below 0.1%.

2.3.2. Leachate samples

During the 500 days of operation, 35 leachate sampleswere recovered from control reactors (C1, C2 and C3) and

from Ti1, and 46 from test reactors Ta1, Ta2 and Ti2. Foreach sample, 6 mL were recovered through the septum witha syringe fitted with a 0.7-mm needle. Raw samples (3 mL)were stored at �20 �C for analysis. The other 3 mL werecentrifuged at 13000 rpm for 10 min. Supernatants wererecovered and stored at �20 �C. pH values were measuredimmediately after leachate sampling by use of a MettlerInlab 427 probe.

From raw samples stored at �20 �C, different analyseswere done. Volatile fatty acids (VFA) as acetic, butyric,propionic and valeric acid were analyzed with a gas chro-matograph (Thermoquest TRACE GC2000) equippedwith a flame ionization detector and a DB-WAXetr col-umn (length 30 m, ID 0.53 mm, film 1 lm). Injectorand detector temperatures were 230 �C and 300 �C,respectively. Helium (4 mL min�1) was the carrier gas.Total Organic and Inorganic Carbon Concentrations(TOC and TIC) were measured with a BIORITECH700 analyzer.

From supernatants stored at �20 �C, anions and cationswere measured. Cations (NHþ4 , Ca2+, K+, Na+, Mg2+)were analyzed by ion chromatography (DIONEX DX-120) with a pre-column Dionex IONPAC� CG16 and aIONPAC CS16 column. A 30 mM methane sulfonic acidbuffer solution was used as the mobile phase. AnionsðCl�; NO�3 ; NO�2 ; PO2�

4 ; SO2�4 Þ were analyzed by ion

chromatography (DIONEX DX-120) with a pre-columnDionex IONPAC� AG9-HC and a IONPAC� AS9-HScolumn. A 9 mM carbonate buffer solution was used asthe mobile phase.

Metal ions (Zn, As, Sn, Cu, Se, Cd, Sb, Pb, Cr, Pb, Cr,Mn, Fe, Sr) were analysed by inductively coupled plasmamass spectrometry (ICP-MS) using an Agilent 7500c onsupernatant stored at �20 �C. Before analysis, sampleswere previously digested, using nitric acid by microwavedigestion (Pinel et al., 2005).

2.4. Statistical analyses

2.4.1. Comparison of linear regressions

NHþ4 concentrations obtained in Ta1 and Ta2 were com-pared during three periods: (1) between days 0 and 17, (2)between days 17 and 45, and (3) between days 45 and 93.Nitrate was injected at the beginning of each of these threeperiods. Six linear regressions were fitted with NHþ4 con-centrations obtained in each reactor and for each period.Pairs of linear regressions corresponding to the same per-iod were then compared between reactors Ta1 and Ta2 byan overall test of coincidence (Scherrer, 1984). Each pairof data sets corresponded to the NHþ4 concentration inTa1 and Ta2 plotted as a function of time. The objectivewas to test the hypothesis H0 that the two linear regressionsfor Ta1 and Ta2 were similar. The principle of this test wasthe comparison between the residual variability obtainedwith one model and the residual variability obtained withtwo distinct models fitted for each set of experimental data(Scherrer, 1984).

To check if the residual variability due to the slope var-iability of the two linear regressions is significant withrespect to the random fluctuations, a ratio F which followsa Fischer–Snedecor law was defined. If F is superior to Fa,H0 has to be rejected. Fa was obtained with a Fischer–Snedecor table (a is the probability level).

2.4.2. Stepwise multiple regression analysis

Regression analysis was used to infer the relationshipbetween N2 production and other measured chemicalparameters and to obtain the best available predictionequation for the chosen model. A general regression modelwith j independent variables can be expressed as:

Y ¼ aþXbjxj

j

þe

where ‘Y’ is the dependent variable (N2 production), ‘xj’ isconsidered as independent variables (pH, TOC, TIC, ace-tate, propionate, butyrate, valerate, CH4, CO2, H2S andN2O), ‘a’ is the constant of regression and ‘bj’ are the coef-ficients of regression. The constant and the coefficients areestimated using the least-square method which minimizesthe error, appearing as ‘e’ in the above regression. Analysisof variance determines the model significance by calculat-ing a F-statistic and a p-value, the probability associatedto F.

Because condition indices indicated some co-linearityamong our ‘xj’, we computed stepwise multiple regressionmodels (both forward and backward procedures) ratherthan the entire multiple regression models at once. At eachiteration, the variable showing the highest partial correla-tion with the dependent variable was included in the modelif its correlation was significant at the 5% level. Selection ofthe variables terminates when no more variables are signif-icant. The statistical analyses were performed using Systat.

0

10

20

30

40

50

60

70

80

90

0 5 10 15 20 25 30 35 40 45

Time

)%( noitisop

moc ecapsdaeH

Fig. 1. Evolution of CO2 and CH4 concentrations in t

3. Results and discussion

3.1. Results

3.1.1. Control reactors biodegradation

In the three control reactors, C1, C2 and C3, carbondioxide (Fig. 1) and volatile fatty acids (Table 3) were pro-duced with a concomitant pH decrease (8.5–7.8) during the25 first days of degradation. We observed a CO2/CH4

inversion between days 24 and 26 (Fig. 1). Thus, the acido-genic phase lasted 25 days. Significant methane productionbegan at day 25 (Fig. 1), with volatile fatty acids consump-tion rate exceeding production rate (Table 3). In the con-trol reactors we observed classic waste degradation: anacidogenic phase followed by a methanogenic phase. Theoverall reproducibility of the experimental system is con-sidered to be satisfactory, as shown in Fig. 1. Gas produc-tion decreased at day 76 (0.5 mL h�1).

During the 500 days of waste incubation, a productionof 121–137 L of CH4 per kg of dry waste was observed incontrol reactors C1, C2 and C3 (Fig. 2). Methane produc-tion was in accordance with methane production that hasbeen reported to vary between 60 and 170 L of CH4 perkg of dry waste (El-Fadel et al., 1996).

Between days 0 and 500, NHþ4 concentrations remainedconstant: variations were only between 1490 and1430 mg N L�1, which represent non-significant variations.During the 500 days of waste incubation, a total produc-tion of 48 mg of N2 was measured and was attributed toresidual air contamination during sampling, especially afterday 76, when biogas production decreased.

3.1.2. Test reactors biodegradation with nitrate injections,

Ta1 and Ta2

3.1.2.1. Injections performed in acidogenic phase. Threenitrate injections were performed in Ta1 and Ta2. On

50 55 60 65 70 75 80 85 90 95 100

(days)

C1_carbon_dioxideC1_methaneC2_carbon_dioxideC2_methaneC3_carbon_dioxideC3_methane

he headspace from control reactor C1, C2 and C3.

Table 3Evolution of volatile fatty acids (VFA) concentrations in the reactors

Reactor Day Total VFA (mg C L�1)

C1 3 44517 517031 243045 173563 20

426 10

C3 3 20017 278531 155545 99663 35

348 10

Ti1 3 33017 336045 3165

145 1279271 3000510 20

Ti2 3 31517 266045 2840

145 1695271 1700510 20

Ta1 3 29517 423545 3920

145 15271 10426 10

Ta2 3 25017 306045 3730

145 710271 30348 30

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Tim

HC

L(noitcudorp

enahtem

evitalumu

C4.

gk1-

) ets aw

yrd

C(1) Ta(1) Ti(1)

C(2) Ta(2) Ti(2)

C(3)

Fig. 2. Cumulative methane production for the seven reactors (nitrogen

day 3, the leachate in Ta1 was supplemented with170 mg N-NO�3 to give a final nitrate concentration of250 mg N-NO�3 L�1 (Fig. 3a). In less than 4 days, nitratewas totally consumed, without nitrite accumulationdetection (Fig. 3a). We observed a production of145 mg N-N2 between days 4 and 7 (Fig. 3b). A produc-tion of 1 mg N-N2O was detected between days 3 and 5(Fig. 3b), but was presumably converted into N2 betweendays 5 and 7, when cumulated N2O productiondecreased.

N2 production was maintained in both systems: in Ta1,after the three nitrate injections (Fig. 3b) and in Ta2 onlyafter injections performed at days 3 and 45 (Fig. 3d).Transient nitrite and N2O accumulations were onlydetected once after nitrate had been injected into Ta1 atday 17 (Fig. 3a and b). Nitrate conversion into N2 andN2O ranged between 78% and 100% (Table 4). Thenitrate not converted into N2 was probably utilised bymicroorganisms. Limited occurrence of DNRA (dissimi-latory nitrate reduction to ammonia) reaction could notbe excluded. N2 cumulated production decreases wereoften observed after the end of N2 production periods(Fig. 3b and d).

Absence of N2 production was observed once, whennitrate was injected at day 17 into reactor Ta2 (Fig. 3cand d). N2O was produced in higher quantities comparedto the five other nitrate injections (Fig. 3b and d).

The six nitrate injections performed in the acidogenicphase thus resulted in five episodes of completedenitrification to N2 and one for which only N2O wasdetected (second nitrate injection in Ta2). In comparisonwith the control reactors, the beginning of themethanogenesis was delayed by the injections of nitrate.In the control reactors the acidogenic phase lasted24–26 days (Fig. 2), but 63 days in Ta1 and 105 daysin Ta2.

137

6271

121

91

108

125

300 350 400 450 500 550 600

e (days)

injections were performed on days 3, 17, 45, 145 and 271 see arrow).

0

20

40

60

80

100

120

140

160

180

0 10 20 30 40 50 60 70 80 90 100 110

Time (days)

NO

2- and

NO

3- am

ount

(m

g N

)

SO

42- c

once

ntra

tion

(mg.

L-1)

0

200

400

600

800

1000

1200

1400

1600

1800

NH

4+ c

once

ntra

tion

(mg

N.L

-1)

Ta1_Nitrite

Ta1_Nitrate

Ta1_Sulphate

Ta1_Ammonium

y = 2,0935x + 1300,5

y = -0,41x + 1355,2

y = -5,882x + 1484,1

0

50

100

150

200

250

300

350

400

450

0 10 20 30 40 50 60 70 80 90 100 110

Time (days)

Cum

ulat

ed N

2 pr

oduc

tion

(mg

N)

0

5

10

15

20

25

Cum

ulat

ed N

2O p

rodu

ctio

n (m

g N

)

Ta1_Molecular_nitrogen

Ta1_Nitrous_oxide

(a)

(b)

Fig. 3. (a) Nitrite, nitrate, ammonium and sulfate evolution in Ta1 during acidogenic phase, (b) cumulated molecular nitrogen and nitrous oxideproduction in test reactor Ta1 during acidogenic phase, (c) nitrate, nitrite, ammonium and sulfate evolution in test reactor Ta2 during acidogenic phase and(d) cumulated molecular nitrogen and nitrous oxide production in test reactor Ta2 during acidogenic phase.

3.1.2.2. Injections performed during methanogenic phase. Fournitrate injections were performed during the methanogenicphase, two in Ta1 and two in Ta2. N2 production wasalways detected (Fig. 4a and b).

After nitrate had been injected in Ta2 at day 145, nitratewas totally converted into N2 by day 154 (Fig. 4b). Meth-ane was still produced on the day of the injection (Fig. 2)and VFAs were still detected in the leachate: 630 mg C-pro-pionate L�1 and 80 mg C-acetate L�1 (Table 3).

When nitrate was injected into Ta1 at days 145 and271 and into Ta2 at day 271, nitrate conversion into N2

took longer than with the injection on day 145 intoTa2, as previously described (Fig. 4a and b). Moreover,N2O was detected after the two injections carried out

on day 271 (Fig. 4a and b) and nitrite accumulationwas observed after the nitrate injection performed onday 145 into Ta1. During these three nitrate conversionepisodes, sulphate accumulation was detected for the firsttime (Fig. 4a and b). Sulphate did not persist, presum-ably because of sulphate reduction. After the three injec-tions performed during the late methanogenic phase,VFAs were not detected in the leachate (<30 mg C L�1,Table 3).

3.1.3. Test reactors biodegradation with nitrite injections Ti1and Ti23.1.3.1. Injections performed in Ti2. The five nitrite injec-tions (Fig. 5b) made into the test reactor Ti2 induced five

0

20

40

60

80

100

120

140

160

180

0 10 20 30 40 50 60 70 80 90 100 110

Time (days)

NO

2- and

NO

3- am

ount

(m

g N

)

SO

42- c

once

ntra

tion

(mg.

L-1

)

0

200

400

600

800

1000

1200

1400

1600

1800

NH

4+ c

once

ntra

tion

(mg

N.L

-1)

Ta2_Nitrite

Ta2_Nitrate

Ta2_Sulfate

Ta2_Ammonium

y = 1,2420x + 1166,3y = 8,3238x + 984,12

y = -11,695x + 1336,7

0

50

100

150

200

250

300

350

400

450

0 10 20 30 40 50 60 70 80 90 100 110

Time (days)

Cum

ulat

ed N

2 pr

oduc

tion

(mg

N)

0

5

10

15

20

25

Cum

ulat

ed N

2O p

rodu

ctio

n (m

g N

)

Ta2_Molecular_nitrogen

Ta2_Nitrous_oxide

(c)

(d)

Fig. 3 (continued)

complete denitrification reactions. N2O was transientlydetected after the second, the third and the fourth nitriteinjections (Fig. 5b). The fourth nitrite injection was fol-lowed by sulphate detection from days 171–248 (Fig. 5b).The four nitrite injections made during the acidogenicphase on days 3, 17, 45 and 145 delayed the beginning ofmethanogenic phase to day 166 (Fig. 2). Nevertheless,VFAs were detected in this reactor until day 271 (Table3), implying that the last nitrite injection was performedduring the active methanogenic phase. No nitrite injectionwas made during the late methanogenic phase.

3.1.3.2. Injections performed in Ti1. Three nitrite injectionswere made in the test reactor Ti1. Nitrite was totally con-

verted into N2 after the first injection (Fig. 5a). The sec-ond nitrite injection was made on day 17. Nitrite wasconsumed only slowly: at day 45, 75 mg N-NO�2remained in the leachate. N2O and N2 were producedin very low quantities: 13 mg N-N2O and 30 mg N-N2

by day 45. When the biogas was analyzed with thelGC, methane was eluted as a double peak, which wasattributed to the presence of NO in the biogas (Varian,personal communication). It was not possible to preciselyquantify NO concentration by gas chromatography dueto the reactivity of NO with the chromatographic col-umn phase but the peak shape evolution indicated a pro-gressive increase of NO concentrations. In order toconfirm this hypothesis, we successfully reproduced the

Table 4Maximal time required for and percentage of NOx conversion by denitrification

Reactor Day Maximal time required fortotal NOx consumption (days)

Maximal time required forN2 production (days)

Remark Percentage of NOx convertedinto N2 + N2O

Ta1 3 4 4 – 8517 4 23 NO�2 ð4 daysÞ 10045 4 15 – 78

145 49 49 NO�2 ð10 daysÞ; SO2�4 ð41 daysÞ 82

271 33 77 SO2�4 ð71 daysÞ 80

Ta2 3 2 4 – 7845 18 25 – 100

145 2 9 – 100271 21 69 SO2�

4 ð71 daysÞ 86

Ti1 3 2 4 – 100

Ti2 3 2 7 – 10017 18 25 – 10045 7 16 – 100

145 10 10 – 100271 2 21 SO2�

4 ð19 daysÞ 100

same chromatogram by injecting artificial mixtures ofCH4 and NO. NO has not been detected in the other testreactors. After the third nitrite injection on day 45,nitrite reduction was again slow, and NO concentrationincreased again. The system was not able to furtherreduce nitrite and NO. No biogas was produced betweendays 60 and 195 (Fig. 2), suggesting a total inhibition ofwaste degradation. At day 194, the headspace waspurged and replaced by helium to eliminate NO. After279 days, on day 473, N2 production started. Methaneand CO2 production resumed at day 504 (Fig. 2).

3.2. Discussion: nitrate or nitrite reduction pathways

Five nitrogen injections (nitrate or nitrite) were made atdifferent phases of waste degradation in four test reactors(Ta1, Ta2, Ti1, Ti2), except for test reactor Ti1 where onlythree injections were made. After these 18 injections, weobserved two different behaviours: the presence or absenceof N2 production. Using the 18 injections, hypotheses areproposed to better understand the factors preventing N2

production.

3.2.1. N2 production by denitrification3.2.1.1. Mechanism of N2 production by heterotrophic and

autotrophic denitrification. After nitrate or nitrite (NOx)injections, we observed NOx consumption followed by N2

production in 15 of 18 cases. This nitrogen-oxides conver-sion is called denitrification. During the reaction, someintermediates could be detected as nitrite ðNO�2 Þ, nitricoxide (NO) and nitrous oxide (N2O). Two types of denitri-fication exist, depending on the nature of the electrondonor: autotrophic and heterotrophic denitrification. Het-erotrophic denitrification is the reaction usually observedwhen an organic carbon source is available. Eq. (1) (Thaueret al., 1977) represents respiratory denitrification with theuse of acetate as an electron donor.

0:625CH3COOH þNO�3 ! HCO�3 þ 0:25CO2

þ 0:75 H2Oþ 0:5N2 ð1ÞDG0 ¼ �518 kJ mol�1

Autotrophic denitrification is observed when littleorganic carbon source is available. Microorganisms usean inorganic electron donor. Eq. (2) (Thauer et al., 1977)represents an autotrophic denitrification with the use ofmetallic sulfide (FeS for example). This reaction releasessulphate.

0:625FeSþNO�3 þHþ ! 0:625Fe2þ þ 0:625SO2�4

þ 0:5H2Oþ 0:5N2 ð2ÞDG0 ¼ �221 kJ mol�1

Denitrification was thus the main nitrate or nitrite con-version pathway. During the acidogenic phase, as easilybiodegradable carbon was present as VFA, denitrificationreactions were probably heterotrophic (Eq. (1)). In the caseof the three injections performed during the methanogenicphase when VFA were not present in the leachate (days 145and 271 in Ta1, day 271 in Ta2), sulphate accumulation wasdetected. Sulphate accumulation was never observed in thecontrol reactors. Sulphate was only produced duringnitrate consumption or immediately after nitrite consump-tion, suggesting that these processes were linked. Occur-rence of autotrophic denitrification is therefore the mostlikely explanation for these observations (Eq. (2)). Itshould, however, be noted that denitrification was proba-bly not fully autotrophic. In fact, according to Eq. (2), near1100 mg SO2�

4 L�1 would then have been expected. Never-theless, sulphate production may also have been underesti-mated because of concomitant sulphate production byautotrophic denitrification and sulphate consumption bysulphate-reducing bacteria. Autotrophic denitrificationcould release metal ions as suggested by Eq. (2). Metalanalyses were therefore performed on the supernatants at

0

20

40

60

80

100

120

140

160

180

200

220

240

127 157 187 217 247 277 307 337 367 397 427 457 487 517 547 577

Time (days)

NO

3- and

NO

2- am

ount

(m

g N

)

SO

42- c

once

ntra

tion

(mg.

L-1)

Cum

ulat

ed N

2O

pro

duct

ion

(mg

N)

100

150

200

250

300

350

400

450

500

550

600

Cum

ulat

ed N

2 pro

duct

ion

(mg

N)

Ta1_NitrateTa1_NitriteTa1_SulphateTa1_Nitrous_oxideTa1_Molecular_nitrogen

0

20

40

60

80

100

120

140

160

180

200

220

240

127 157 187 217 247 277 307 337 367 397 427 457 487

Time (days)

NO

3- and

NO

2- am

ount

(m

g N

)

SO

42- c

once

ntra

tion

(mg.

L-1)

Cum

ulat

ed N

2O p

rodu

ctio

n (m

g N

)

100

150

200

250

300

350

400

450

500

550

600

Cum

ulat

ed N

2 pr

oduc

tion

(mg

N)

Ta2_NitrateTa2_NitriteTa2_SulphateTa2_Nitrous_oxideTa2_Molecular_nitrogen

(a)

(b)

Fig. 4. (a) Evolution of sulphate and nitrogenous species in reactor Ta1 for nitrate injections performed during the methanogenic phase and (b) evolutionof sulphate and nitrogenous species in reactor Ta2 for nitrate injections performed during the methanogenic phase.

the end of the waste degradation (Table 5). We comparedmetal concentrations measured in Ta1 and C1 to take intoaccount metal ions coming from the initial leachate andmetal ions coming from waste degradation. Ta2 and C3

were performed with the same initial leachate (Table 2),thus their metal concentrations were compared. Weobserved that Zn, Sn, Pb, Fe and Cr were detected inhigher concentration in test reactors Ta1 and Ta2, than inthe control reactors, whereas Se, Sr and Al were detectedin the test reactors at lower values than in the control reac-tors. Metal ions were not released in significant amountsdue to autotrophic denitrification. These results are in

agreement with a previous study where sulphate produc-tion was detected after nitrate injections (Price et al.,2003) and metal concentrations (Cd, Cr, Fe and Mn) didnot increase. In such complex systems metal mobility couldbe limited by precipitation as hydroxide and carbonateforms, by ion exchange and by sorption (Price et al., 2003).

3.2.1.2. Duration of NOx reduction to N2. In the four testreactors, injections performed on day 3 enabled rapid deni-trification in all cases: as short as 4 days after injection(Table 4). Nitrite and N2O accumulation did not occurafter the first injection. For injections performed on day

0

50

100

150

200

250

300

350

400

450

500

550

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570

Time (days)

Cum

ulat

ed N

2 pr

oduc

tion

(mg

N)

NO

2- am

ount

(m

g N

)

0

5

10

15

20

25

30

35

40

45

50

Cum

ulat

ed N

2O p

rodu

ctio

n (m

g N

)

Ti1_nitrite

Ti1_molecular_nitrogen

Ti1_nitrous_oxide

0

50

100

150

200

250

300

350

400

450

500

550

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

Time (days)

Cum

ulat

ed N

2 pr

oduc

tion

(mg

N)

NO

2- am

ount

(m

g N

)

SO

42- c

once

ntra

tion

(mg.

L-1

)

0

10

20

30

40

50

60

70

Cum

ulat

ed N

2O p

rodu

ctio

n (m

g N

)

Ti2_nitrite

Ti2_molecular_nitrogen

Ti2_sulphate

Ti2_nitrous_oxide

(a)

(b)

Fig. 5. (a) Nitrogen evolution in leachate and in biogas in Ti1 and (b) nitrogen evolution in leachate and in biogas and sulfate concentration in Ti2.

17, for which denitrification occurred, nitrogen reductionslasted longer: 23 days for Ta1 with nitrite accumulationdetection, and 25 days for Ti2 with N2O accumulationdetection (Table 4). These nitrite or N2O accumulationswere apparent when the main reactions in the reactors werehydrolysis and acidogenesis. The hydrolysis process coulddelay the denitrification reaction resulting on the transientdenitrification intermediates accumulation. The longestdenitrification phase occurred when nitrogen (nitrate ornitrite) injections were performed during the late methano-genic phase, when no VFAs were detected in the reactor.The absence of easily biodegradable carbon could delaynitrogen conversion. For example, the time needed fornitrite reduction at day 271 was shorter in Ti2 where VFAs

were detected than in Ta1 (Table 3). In general, in ourexperiment, the denitrification phase was shorter afternitrite injections than after nitrate injections as reportedin previous studies (Beccari et al., 1983; Akunna et al.,1993; Chung and Bae, 2002). Nitrite conversion occurredwith a higher percentage than nitrate conversion (Table4). It could be explained by the fact that chemical oxygendemand for nitrite conversion (COD/N = 1.71) is lowerthan for nitrate conversion (COD/N = 2.86).

3.2.2. Absence of N2 productionWe observed two different cases where no N2 was pro-

duced: after the second nitrate injection in Ta2 and afterthe second and the third nitrite injections in Ti1. In the first

Table 5Metals analysis on centrifugation supernatants at the end of the wastedegradation (day 597 for C1 and Ta1 and day 511 for C3 and Ta2)

Metals Comparison of themetals released betweenTa1 and C1 (%) (Ta1 � C1)/Ta1 · 100

Comparison of the metalsreleased between Ta2 andC3 (%) (Ta2 � C3)/Ta2 · 100

Zn 39 33Sn 19 35Pb 69 30Fe 45 36Cr 9 10Cu 42 �13Cd 78 �315Mn 38 �330Se �37 �13Sr �49 �107Al �17 �5

To take into account the metal concentration coming from initial leachateand the one coming from the waste degradation, each test reactor has beencompared with the control reactor performed in the same initial leachate.Ta1 was compared with C1 and Ta2 with C3.

case, nitrate was reduced and dissimilatory nitrate reduc-tion to ammonium (DNRA) was suspected to occur (Eq.(3)) (Thauer et al., 1977).

NO�3 þ 4H2 þ 2Hþ ! NHþ4 þ 3H2O ð3ÞDG0 ¼ �677 kJ mol�1

In Ti1, on the other hand, nitrite was not totally con-sumed at day 193 and NO accumulated. NO has been pro-ven to be an intermediate of denitrification (Ye et al., 1994;Zumft, 1997).

3.2.2.1. Absence of N2 production due to DNRA. Under-standing why the absence of denitrification was sometimesobserved represents a major stake in nitrogen managementwhenever nitrified leachate has to be recirculated into alandfill. Indeed, in the case of DNRA, nitrogen is con-verted into NHþ4 and is not released outside of the landfill,contrary to denitrification.

During the second nitrate injection performed at day 17,we did not observe any N2 production but only some N2Oproduction (22 mg N-N2O, Fig. 3d). However, N2O pro-duction did not enable us to clarify the nitrate conversionreaction because N2O could be produced either by denitri-fication or by DNRA (Welsh et al., 2001). The remaining120 mg N-NO�3 from the nitrate injection was convertedto a nitrogen form other than NO�2 , N2O, NO or N2.Due to a 7% analytical error on ammonium concentration(dilution and analysis errors) and to high ammonium con-centration in the initial leachate, it was difficult to detect aprecise 120 mg N-NHþ4 increase, which only represents a8.6% increase from the initial quantity. Nevertheless,ammonium concentration increased during nitrate reduc-tion between days 17 and 45 for Ta2 (Fig. 3a) comparedto Ta1 (Fig. 3c), as shown by the greater slope value of

the linear regression model in Ta2 than in Ta1. Statisticaltests of regression coincidence were performed to deter-mine if the models fitted with ammonium concentrationsfor period in each test reactor were significantly different.The comparison between F and Fa enabled evaluation ofthe sensitivity of the statistics. It showed that Ta1 andTa2 concentration evolutions were significantly differentfor the nitrate reduction period between days 17 and 45during which DNRA was suspected only in Ta2 (F/Fa > 1). On the contrary, comparison of NHþ4 evolutiontrends between the two reactors for periods during whichDNRA was not suspected to have occurred (for days 0–17 and days 45–93) did not show any significant difference(F/Fa� 1).

The increase in NHþ4 concentration in Ta2 is thereforenot only due to stochastic experimental variability, whichstrongly supports the hypothesis that DNRA reactionoccurred in Ta2 only between days 17–45.

In a previous work, we observed a non-denitrifyingreduction after three nitrate injections performed duringthe acidogenic phase (Vigneron et al., 2005). Increase inNHþ4 concentration enabled us to conclude DNRA preva-lence. To understand the reason of DNRA occurrence, weused all nitrate injections results of Ta1 and Ta2 and resultscoming from a previous study (Vigneron et al., 2005). Sta-tistical analyses were performed to identify parameterscausing DNRA reaction instead of denitrification. We usedvalues of pH, TOC, TIC, acetate, propionate, butyrate,valerate, CH4, CO2, H2S and N2O at each injection dayas independent variables. The dependent variable was themaximum N2 production after each nitrate injection. Wecomputed stepwise multiple regression models in order toidentify parameters explaining the absence of denitrifica-tion within 20 experiments: 5 nitrate injections performedin 4 test reactors (Ta1, Ta2 and two others reactors calledreactors 4 and 5 (Vigneron et al., 2005)). In our studies,H2S concentration was the only single parameter foundwith the stepwise multiple regression models to have a neg-ative effect on N2 production. The coefficient of H2S wassignificant. It should be noted that a weak positive correla-tion between acetate and N2 production was also found.Acetate is therefore not a parameter explaining the absenceof denitrification, but, on the contrary, apparently favoursN2 production. The model computed with 11 independentvariables enabled explanation of 80.1% of the N2 produc-tion (R2 = 0.801).

In Fig. 6, we compared H2S concentration at themoment where the three nitrate injections were performedduring acidogenic phase (days 3, 17 and 45) for reactorsTa1 and Ta2. When denitrification was observed, we useda dotted line and when no denitrification was recorded,we used a full line. The only absence of denitrificationoccurred when H2S concentration was the highest on theday of the injection (second arrow for Ta2, Fig. 6). Denitri-fication inhibition by H2S is in agreement with previouswork (Brunet and Garcia-Gil, 1996), where authors dem-onstrated the prevalence of DNRA in the presence of

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Time (days)

H2S

con

cent

ratio

n (m

mol

. L-1

of b

ioga

s) Ta(1)_hydrogen_sulphide

Ta(2)_hydrogen_sulphide

Fig. 6. H2S concentration in the headspace in Ta1 and Ta2 (arrows represents the nitrate injections, dotted line represents a nitrate conversion bydenitrification and full line represents nitrate conversion without N2 production).

H2S. The presence of H2S may have caused a decrease ofthe oxido-reduction potential (ORP), resulting in the inca-pacity of nitrate conversion by denitrification.

Burton and Watson-Craik (1999) showed 15NH3 pro-duction from 15NO3 in batch culture representing 4–7%of DNRA, whereas Price et al. (2003) did not observeDNRA. The possible explanation was that Price et al.(2003) injected nitrate during active methane productionand in well-decomposed waste. During the methanogenicphase, only very small amounts of H2S were produced.However, Burton and Watson-Craik (1999) used freshwaste (2–3 months old excavated waste), which could pro-duce H2S during the acidogenic phase. Our results concern-ing DNRA prevalence could explain the differences foundin previous studies.

3.2.2.2. Consequences of NO accumulation. We observedNO production only after nitrite injections in Ti1 at days17 and 45. NO is an intermediate of denitrification (Yeet al., 1994; Zumft, 1997), but its accumulation is oftenlinked to a chemical denitrification rather than with micro-biological denitrification (Tiedje, 1988). Percheron et al.(1998) demonstrated that slow linear nitrite conversion intoNO was not due to microbiological denitrification but tochemodenitrification. In their case, nitrite was reduced byiron (Eq. (4)) (Thauer et al., 1977):

Fe2þ þNO�2 þ 2Hþ ! Fe3þ þNOþH2O ð4ÞDG0 ¼ �38 kJ mol�1

We analysed Fe2+ and Fe3+ without finding anydecrease of Fe2+ concentration. Nevertheless, in such acomplex system Fe2+ was probably produced during the

acidogenic phase and nitrite injections could have modifiedthe redox conditions, implying a modification of the ironspeciation prevalence.

No explanation could be found for NO being producedonly in Ti1 and not in Ti2. In general, NO production isfavoured by low pH but the pH value was higher in Ti1(7.9) than in Ti2 (6.9). Acetate and propionate accumula-tion was observed (Table 3), as has already been observedin previous studies, in reactors where gaseous NO wasinjected (Kluber and Conrad, 1998a). NO and nitrite areknown to be more toxic for methanogenesis compared tonitrate and N2O (Kluber and Conrad, 1998b), and NOaccumulation resulted in a total inhibition of wastedegradation.

3.2.3. Molecular nitrogen consumption

A decrease in N2 concentration was observed after theend of the first denitrification period in the four test reac-tors. Early methane production was also detected duringthis N2 consumption. N2 consumption was again observedjust before the beginning of methanogenic phase in eachreactor. For example, in test reactor Ta1, after the thirddenitrification phase, N2 was consumed between days 60and 83 (Fig. 3b), and methane production began at day83 (Fig. 2). These two reactions seem to be linked. Refer-ring to the nitrogen cycle, the only possible N2 consumingreaction is assimilation by microorganisms, also callednitrogen fixation. This reaction is however known to occuronly when no other nitrogen form is present in the system(Raymond et al., 2004). In our case, more than 1.4 g N L�1

of ammonium was present in the leachate, and NHþ4 assim-ilation needs much less energy than N2 fixation. Up to now,

we have not been able to explain why this reaction mighthave taken place in our reactors. Complementary testsare being carried out with 15N2 to verify that biomass usesN2 even when NHþ4 is present.

4. Conclusions

Waste has a large capacity to convert nitrate or nitriteinto N2, and microorganisms responsible for denitrificationcan be successfully stimulated when nitrate or nitrite areintroduced into the waste mass. Evidence of denitrificationwas obtained in 15 of 18 samples. Denitrification occurringduring the acidogenic phase, when easily biodegradablecarbon was detected as VFA, suggests that the processwas predominantly heterotrophic. However, nitrateinjected during the late methanogenic phase was probablypartly converted by an autotrophic denitrification asrevealed by sulphate accumulation. For the cases duringwhich nitrate was reduced without gaseous nitrogen pro-duction, we observed that ammonium concentrationincreased significantly suggesting that dissimilatory nitratereduction to ammonium (DNRA) was the main reaction.Stepwise multiple linear regression analysis suggested thatdenitrification inhibition was linked to a higher H2Sconcentration.

In most reactions, nitrite enabled a fast and more com-plete denitrification than nitrate. Nevertheless, nitrite couldalso inhibit denitrification by NO production. NO accumu-lation could cause a total inhibition of waste degradation.

These results need to be confirmed in a full-scale landfillbut, for the moment, nitrified leachate recirculation duringthe acidogenic phase is a potential cause of NO production,which could induce inhibition of waste degradation. More-over, nitrified leachate recirculation in the presence of highH2S concentrations could contribute to the inhibition ofdenitrification, preventing the release of nitrogen outsideof the system. This point is particularly important whenwaste contains a high quantity of sulphur. Thus, conse-quences of nitrified leachate recirculation during the acido-genic phase need to be carefully followed duringexperimentations in a full-scale landfill.

Acknowledgements

This study was supported by Cemagref, ADEME,SUEZ ENVIRONNEMENT and the Region Ile-de-France. We would like to thank the Vert-le-Grand landfillmanager for allowing us to sample in his waste manage-ment facility; Nancy Mailly and Daniel Stadtmuller fortheir technical assistance; Pr Martine Potin-Gautier andDr. David Amouroux for allowing us to perform metalanalysis at the bio-inorganic analytical chemistry and envi-ronment laboratory at the university of Pau in France;Evelyne Tales for her assistance to carry out the statisticalanalyses; Daniel Bouchez and Christian Duquennoi forenglish corrections. Vassilia Vigneron would like to thankcoworkers for their stimulating discussions and critical

comments. We thank the Editor and anonymous reviewersfor useful comments and suggestions.

References

Ademe 1999. Composition des ordures menageres en France (donnees etreferences).

Ademe 2002. Enquete sur les installations de traitement des dechetsmenagers et assimiles en 2002, ITOM 2002, le bilan general. Availablefrom: http://www.ademe.fr/collectivites/Dechets-new/Mots-chiffres/ITOM2002.asp.

Akunna, J.C., Bizeau, C., Moletta, R., 1993. Nitrate and nitrite reductionswith anaerobic sludge using various carbon-sources – glucose, glycerol,acetic-acid, lactic-acid and methanol. Water Research 27 (8), 1303–1312.

Bae, W., Chung, J.-W., Shin, E.-B., Cho, Y.-J., Kim, J.-H., Ko, G.-B., 2002.Innovative technology of landfill stabilization combining leachate recir-culation with shortcut biological nitrogen removal technology. In: AplasSeoul 2002. The 2nd Asian Pacific Landfill Symposium, Seoul, Korea.

Barber, C., Maris, P.J., 1984. Recirculation of leachate as a landfillmanagement option: benefits and operational problems. QuarterlyJournal of Engineering Geology 17, 19–29.

Barlaz, M.A., Ham, R.K., Schaefer, D.M., 1990. Methane productionfrom municipal waste: a waste review of enhancement techniques andmicrobial dynamics. Critical Reviews in Environmental Control. C.Press. 19, 557–584.

Beccari, M., Passino, R., Ramadori, R., Tandoi, V., 1983. Kinetics ofdissimilatory nitrate and nitrite reduction in suspended growth culture.Journal of the Water Pollution Control Federation 55, 58–64.

Berge, N.D., Reinhart, D.R., 2003. In-situ nitrification of leachate inbioreactor landfills. In: Proceedings Sardinia 2003, Ninth InternationalWaste Management and Landfill Symposium, S. Margherita di Pula,Cagliari, Italy, CISA.

Brunet, R.C., Garcia-Gil, L.J., 1996. Sulfide-induced dissimilatory nitratereduction to ammonia in anerobic freshwater sediments. FEMSMicrobiology Ecology 21, 131–138.

Burton, S.A.Q., Watson-Craik, I.A., 1998. Ammonia and nitrogen fluxesin landfill sites: applicability to sustainable landfilling. Waste Man-agement & Research 16 (1), 41–53.

Burton, S.A.Q., Watson-Craik, I.A., 1999. Accelerated landfill wastedecomposition by recirculation of nitrified leachate. In: Sardinia 1999,Seventh International Waste Management and Landfill Symposium, S.Margherita di Pula, Cagliari, Italy, CISA.

Chung, J.W., Bae, W., 2002. Nitrite reduction by a mixed culture underconditions relevant to shortcut biological nitrogen removal. Biodeg-radation 13 (3), 163–170.

El-Fadel, M., 1999. Leachate recirculation effects on settlement andbiodegradation rates in MSW landfills. Environmental Technology 20,1–13.

El-Fadel, M., Findikakis, A.N., Leckie, J.O., 1996. Estimating andenhancing methane yield from municipal solid waste. HazardousWaste & Hazardous Materials 13 (3), 309–331.

El-Mahrouki, I.M.L., Watson-Craik, I.A., 2004. The effects of nitrate andnitrate-supplemented leachate addition on methanogenesis fromMunicipal Solid Waste. Journal of Chemical Technology and Bio-technology 79 (8), 842–850.

Kluber, H.D., Conrad, R., 1998a. Effects of nitrate, nitrite, NO and N2Oon methanogenesis and other redox processes in anoxic rice field soil.FEMS Microbiology Ecology 25 (3), 301–318.

Kluber, H.D., Conrad, R., 1998b. Inhibitory effects of nitrate, nitrite, NOand N2O on methanogenesis by Methanosarcina barkeri and Methan-

obacterium bryantii. FEMS Microbiology Ecology 25 (4), 331–339.Knox, K., Gronow, J.R., 1995. Pilot scale study of denitrification and

contaminant flushing during prolonged leachate recirculation. In:Sardinia 95, Fifth International Waste Management and LandfillSymposium, S. Margherita di Pula, Cagliari, Italy, CISA.

Mehta, R., Barlaz, M.A., Yazdani, R., Augenstein, D., Bryars, M.,Sinderson, L., 2002. Waste decomposition in the presence and absence

of leachate recirculation. Journal of Environmental Engineering –ASCE 128 (3), 228–236.

Onay, T.T., Pohland, F.G., 1998. In situ nitrogen management incontrolled bioreactor landfills. Water Research 32 (5), 1383–1392.

Pacey, J., 1999. Landfill performance expectations. MSW ManagementElements, 84–87.

Percheron, G., Michaud, S., Bernet, N., Moletta, R., 1998. Nitrate andnitrite reduction of a sulphide-rich environment. Journal of ChemicalTechnology and Biotechnology 72 (3), 213–220.

Pinel, P., Ponthieu M., Le Hecho I., Amouroux D., Mazeas L., DonardO., Potin-Gautier M., 2005. Validation of metal analysis in bulk andfiltered landfill leachate using an internal reference leachate material.In: Sardinia 2005, Tenth International Waste Management and landfillSymposium, S. Margherita di Pula, Cagliari, Italy, CISA.

Pohland, F.G., 2002. Evolution of bioreactor landfills: principles andpractices. Aplas Seoul 2002, the second Asian Pacific LandfillSymposium, Seoul, Korea.

Price, G.A., Barlaz, M.A., Hater, G.R., 2003. Nitrogen management inbioreactor landfills. Waste Management 23 (7), 675–688.

Raymond, J., Siefert, J.L., Staples, C.R., Blankenship, R.E., 2004. Thenatural history of nitrogen fixation. Molecular Biology and Evolution21 (3), 541–554.

Scherrer, B., 1984. Biostatistique. Editions Gaetan Morin, pp. 676–686.Thauer, R.K., Jungermann, K., Decker, K., 1977. Energy conservation in

chemotrophic anaerobic bacteria. Bacteriological Reviews 41 (1), 100–180.

Tiedje, J.M., 1988. Ecology of denitrification and dissimilatory nitratereduction to ammonium. In: Zehnder, A.J.B. (Ed.), Biology ofAnaerobic Microorganisms. Wiley, New York, pp. 179–244.

Vigneron, V., Bouchez, T., Bureau, C., Mailly, N., Mazeas, L., Duquen-noi, C., Audic, J.-M., Hebe, I., Bernet, N., 2005. Leachate pre-treatment strategies before recirculation in landfill bioreactors. WaterScience and Technology 52 (1–2), 289–297.

Warith, M., 2002. Bioreactor landfills: experimental and field results.Waste Management 22, 7–17.

Welsh, D.T., Castadelli, G., Bartoli, M., Poli, D., Careri, M., de Wit, R.,Viaroli, P., 2001. Denitrification in an intertidal seagrass meadow, acomparison of N-15-isotope and acetylene-block techniques: dissimi-latory nitrate reduction to ammonia as a source of N2O? MarineBiology 139 (6), 1029–1036.

Wens, P., Vercauteren T., De Windt, W., Verstraete, W., 2001. Factorsinhibiting anaerobic degradation in a landfill. In: Sardinia 2001, EighthInternational waste Management and Landfill Symposium, S. Mar-gherita di Pula, Cagliari, Italy, CISA.

Ye, R.W., Averill, B.A., Tiedje, J.M., 1994. Denitrification – productionand consumption of nitric-oxide. Applied and Environmental Micro-biology 60 (4), 1053–1058.

Yuen, S.T.S., 1999. Bioreactor landfilling promoted by leachate recircu-lation: a full scale study. Department of Civil and EnvironmentalEngineering, University of Melbourne, p. 244.

Zumft, W.G., 1997. Cell biology and molecular basis of denitrification.Microbiology and Molecular Biology Reviews 61 (4), 533–616.