Nitrous oxide (N2O) emissions from a nitrifying
biofilm reactor were investigated with N2O isotopocules. The nitrogen
isotopomer site preference of N2O (15N-SP) indicated the
contribution of producing and consuming pathways in response to changes in
oxygenation level (from 0 % to 21 % O2 in the gas mix), temperature
(from 13.5 to 22.3 ∘C) and ammonium concentrations (from 6.2 to
62.1 mg N L-1). Nitrite reduction, either nitrifier denitrification or
heterotrophic denitrification, was the main N2O-producing pathway under
the tested conditions. Difference between oxidative and reductive rates of
nitrite consumption was discussed in relation to NO2-
concentrations and N2O emissions. Hence, nitrite oxidation rates seem
to decrease as compared to ammonium oxidation rates at temperatures above 20 ∘C and under oxygen-depleted atmosphere, increasing N2O
production by the nitrite reduction pathway. Below 20 ∘C, a
difference in temperature sensitivity between hydroxylamine and ammonium
oxidation rates is most likely responsible for an increase in N2O
production via the hydroxylamine oxidation pathway (nitrification). A
negative correlation between the reaction kinetics and the apparent isotope
fractionation was additionally shown from the variations of δ15N and δ18O values of N2O produced from ammonium.
The approach and results obtained here, for a nitrifying biofilm reactor
under variable environmental conditions, should allow for application and
extrapolation of N2O emissions from other systems such as lakes, soils
and sediments.
Introduction
Nitrogen (N) cycling relies on numerous biological processes exploited and
altered by anthropic activities (Bothe et al., 2007). One
of the major issues related to N cycle alteration is the production of
nitrous oxide (N2O), a potent ozone-depleting and greenhouse gas whose
emissions exponentially increased during the industrial era
(Crutzen et al., 1979; IPCC,
2014; Ravishankara et al., 2009). Wastewater resource recovery facilities
(WRRFs) contribute to about 3 % of annual global anthropogenic N2O
sources (ca. 6.7±1.3 Tg N-N2O in
2011; IPCC, 2014), with 0 % to 25 % of the influent nitrogen loads emitted
as N2O (Law et al., 2012b).
The challenges to mitigating these emissions are linked with the understanding of
N2O-producing processes and their controls.
Two microbial processes are responsible for the production of N2O
(nitrification and heterotrophic denitrification), with only one of these
capable of consuming it (denitrification; Fig. 1a;
Kampschreur et al., 2009). Nitrification is the oxidation of ammonium to
nitrite (NO2-) via the intermediate hydroxylamine (NH2OH)
conducted by ammonia oxidizers, and the subsequent oxidation of
NO2- to nitrate (NO3-) by nitrite oxidizers. During
nitrification, N2O can be produced as a reaction side-product from
hydroxylamine oxidation by biotic, abiotic or hybrid processes
(Caranto
et al., 2016; Heil et al., 2015; Terada et al., 2017). Heterotrophic
denitrification and nitrifier denitrification produce N2O from nitrite
reduction conducted by denitrifiers and ammonium oxidizers, respectively.
N2O-producing and N2O-consuming pathways at play during
nitrification and heterotrophic denitrification. Substrate isotope
composition, isotope effects and 15N-SP values from the literature were
used to propose the ranges of 15N (Lewicka-Szczebak
et al., 2014; Sutka et al., 2006, 2008; Yamazaki et al., 2014), 18O (Andersson
and Hooper, 1983; Hollocher et al., 1981; Kool et al., 2007; Kroopnick and
Craig, 1972; Snider et al., 2012) and 15N-SP (Frame
and Casciotti, 2010; Jung et al., 2014; Sutka et al., 2006; Yamazaki et al.,
2014), as well the slopes relating them with each other during N2O
reduction to N2 (Jinuntuya-Nortman et al., 2008;
Webster and Hopkins, 1996; Yamagishi et al., 2007). The assumptions made and
the calculations performed are detailed in the text.
Temperature, and electron donor and acceptor concentrations have been identified
to control N2O emissions from WRRFs (Bollon
et al., 2016; Kampschreur et al., 2009; Tumendelger et al., 2014, 2016;
Wunderlin et al., 2012). These variables may induce N2O accumulation
due to inhibition or disturbance of enzyme activity (Betlach
and Tiedje, 1981; Kim et al., 2008; Otte et al., 1996). In addition to this,
the different N2O-producing processes, nitrification,
nitrifier denitrification or heterotrophic denitrification, are rarely
observed independently from each other in heterogeneous environments like
wastewater, natural waters, soils or sediments. However, the understanding
of the influence that environmental conditions have on the balance between
these processes and N2O-producing pathways remain to a large extent
unexplored.
In order to decipher N2O-producing and N2O-consuming pathways, the analysis of
N2O isotopocules, molecules that only differ in either the number or
position of isotopic substitutions, has been applied (Koba
et al., 2009; Sutka et al., 2006; Fig. 1b–d). The isotope composition of
substrates and fractionation mechanisms influence both nitrogen and oxygen
isotope ratios of N2O (reported as δ15N and δ18O, respectively; Fig. 1b). Basically, the oxygen atom in the
N2O molecule produced by hydroxylamine oxidation originates from
atmospheric dissolved oxygen with a δ18O value of 23.5 ‰ (Andersson and
Hooper, 1983; Hollocher et al., 1981; Kroopnick and Craig, 1972), while the
oxygen atom in N2O produced by nitrite reduction originates from
nitrite that has undergone oxygen exchange with water
(Kool et al., 2007; Snider et al.,
2012). Nonetheless, δ18O-N2O resulting from the
nitrite reduction conducted by the nitrifiers ranges from 13 ‰ to 35 ‰
(Snider et al., 2012). In
contrast, the N2O produced by the heterotrophic denitrifiers through
the nitrite reduction pathway has a δ18O value of over 35 ‰ (Snider
et al., 2013). However, the oxygen exchange between the N2O precursors and
water can decrease it to values below 35 ‰ (Snider et al., 2015). Therefore,
δ18O alone does not enable differentiation between the
N2O-producing pathways.
In combination with δ18O, δ15N-N2O allows us
to identify the N2O-producing pathways (Fig. 1b). However, the isotope
fractionations (or isotope effects) largely influence δ15N-N2O due to wide variations between and within the reactions
involved in the nitrogen cycle (Denk et al., 2017). The
isotopic fractionation results from the difference in equilibrium constant
or reaction rate observed between the heavier and lighter isotopes in both
abiotic and biotic processes. The net isotope effects (Δ)
approximated from the difference between δ15N of product and
substrate characterize the production of compounds resulting from sequential
or branched reactions and have been recently reviewed (Denk et al., 2017;
Toyoda et al., 2017). So far, only two estimates of the net isotope effect
of N2O production by ammonium oxidation via hydroxylamine of -46.5 ‰ and
-32.9 ‰ have been proposed (Sutka
et al., 2006; Yamazaki et al., 2014). These values are imbricated between
-52.8 ‰ and -6 ‰, the range of net isotope effects
related to the N2O production through nitrite reduction performed by
nitrifiers or heterotrophic denitrifiers (Lewicka-Szczebak
et al., 2014; Sutka et al., 2008).
Similarly to isotope ratios, the nitrogen isotopomer site preference
(15N-SP), the difference between the relative abundances of N2O
molecules enriched in 15N at the central (Nα) position and
terminal (Nβ) position differ according to N2O-producing
pathway (Fig. 1c and d). During heterotrophic or nitrifier denitrification
the 15N-SP of N2O produced from nitrate or nitrite ranges from
-10.7 ‰ to 0.1 ‰, while ranging from 13.1 ‰ to 36.6 ‰ when N2O results from hydroxylamine oxidation (Frame
and Casciotti, 2010; Jung et al., 2014; Sutka et al., 2006; Yamazaki et al.,
2014). Finally, N2O reduction to N2 by heterotrophic denitrifiers
increases the values of δ15N, δ18O and 15N-SP
of residual N2O with specific pairwise ratios
(Jinuntuya-Nortman et al., 2008; Webster
and Hopkins, 1996; Yamagishi et al., 2007).
Nitrogen and oxygen isotope ratios of N2O have lower potential for
N2O source identification as compared to 15N-SP. However, we
believe that the use of both isotope approaches should strengthen the
conclusions from 15N-SP and reveal additional isotope effects (Fig. 1).
The aim of the current study is to improve our understanding regarding the
effects of key environmental variables (oxygenation, temperature,
NH4+ concentrations) on N2O production and emission rates.
More specifically using nitrogen and oxygen isotope ratios as well as
15N-SP of N2O should allow for deciphering the different producing and
consuming pathways under these different conditions. In order to achieve
this, the nitrifying biomass of a submerged fixed-bed biofilm reactor was
investigated. Among wastewater treatment systems, the biofilm systems
are adapted to large urban areas owing to their compactness, flexibility and
reliability. An increase in their development is expected in response to the
additional 2.5 billion humans predicted in urban areas by 2050
(United Nations, 2019). However,
biofilm systems have received much less attention than suspended biomass
systems, and the relations between the N2O-producing and N2O-consuming pathways
and controls remain largely unknown (Sabba
et al., 2018; Todt and Dörsch, 2016). Although applied here to the
nitrifying biomass of a WRRF, the research questions addressed consider a
diversity of environments including natural waters, soils and sediments: (i) does the nitrifying biomass emit N2O and what are the producing
pathways at play? (ii) Do oxygenation, temperature and NH4+
concentration alter N2O emissions, and what are the involved
processes? We hypothesize that the isotope signature of N2O allows
identification of the N2O origins and the assessment of the pathway contribution
to N2O emissions. The results of this study should improve the
mechanistic understanding as well as improved prediction of N2O
emissions from WRRFs, currently suffering from high uncertainty.
Material and methodsExperimental setup for nitrifying experiments
Experiments were carried out with colonized polystyrene beads (diameter 4 mm) sampled from the nitrification biologically active filters (BAFs) of a
domestic WRRF (Seine Centre, France). In this WRRF, wastewater (240 000 m3 d-1) passes through a pre-treatment stage, followed by
physicochemical decantation and tertiary biological treatment. The latter
is composed of three biofiltration processes: (i) carbon elimination (24
Biofor®), (ii) nitrification (29 Biostyr®)
and (iii) denitrification (12 Biofor®). Nitrifying
Biostyrs® are submerged fixed-bed biofilm reactors with a
unitary section of 111 m2 and a filter bed of 3 m high. This unit is
operated to receive a nominal load of 0.7 kg NH4+-N m-3 d-1.
A lab-scale reactor with a working volume of 9.9 L (colonized
Biostyrene® beads and interstitial volume) and a headspace of
1.4 L was operated in continuous down-flow counter-current mode for 7
weeks (i.e., solution was down-flowing, while air was up-flowing; Fig. S1 in the Supplement).
Mass flow meters (F-201CV, Bronkhorst, France) sustained the inflow gas rate
at 0.5 L min-1. A peristaltic pump (R3425H12B, Sirem, France) pumped
feeding solution from a feeding tank into the reactor at 0.2 L min-1,
in order to maintain a hydraulic retention time (HRT) of 27.8±0.6 min. A water jacket monitored by a cryogenic regulator (WK 500, Lauda,
Germany) controlled the reactor temperature. The feeding solution consisted
of ammonium chloride (NH4Cl) as substrate, monobasic potassium
phosphate (KH2PO4) as phosphorus source for bacterial growth, and
sodium hydrogen carbonate (NaHCO3) as pH buffer and inorganic carbon
source in 100 or 150 L of tap water (average 0.2±0.4, 2.4±1.1, and 2.5±1.3 mg N L-1 of NO2-, NO3- and
sum of both NOx- molecules, respectively).
The influence of environmental conditions on the ammonium oxidation rates
and the N2O emissions from various combinations of oxygenation levels,
temperatures and ammonium concentrations were tested in 24
experiments (Table 1). Note that two of them were used twice: as oxygenation tests
and as concentration tests. The oxygenation tests were carried out by mixing
compressed air and pure nitrogen gas to reach 0 % to 21 % O2 in the
gas mixture (Fig. S2a). The tests were performed at five substrate
concentrations and at a temperature between 19.2 and 20.6 ∘C. The
temperature tests were carried out by cooling the feeding solution directly
in the feeding tank (22.3 to 13.5 ∘C), with an inflow ammonium
concentration close to the nominal load that received the nitrifying
biomass, i.e., 20.3–21.1 mg NH4+-N L-1. The ammonium
concentration tests were run at an increase (6.2, 28.6 and 62.1 mg NH4+-N L1) and a decrease (56.1, 42.9, 42.7 and 20.2 mg NH4+-N L-1) of NH4+ concentrations in the
feeding solution, at temperatures ranging from 19.0 to 19.8 ∘C.
The atmospheric oxygenation level (i.e., 21 % O2 in the gas mixture) was
imposed for both tests (Fig. S2b and c). This gas mixture using compressed
air with 21 % O2 was considered hereafter as optimal as compared to
the oxygen-depleted atmosphere used during the oxygenation tests.
Noticeably, the atmospheric oxygenation level is the condition that
represents the most optimal conditions of oxygenation applied in
nitrification BAFs of domestic WRRF.
Detailed average conditions (± standard deviation) of
oxygenation, temperature and concentration tests.
Note that two experiments tested both oxygenation and ammonium concentration.
Reactor monitoring, sampling and concentrations analysis
Dissolved oxygen, temperature (Visiferm DO Arc 120, Hamilton, Switzerland)
and pH (H8481 HD, SI Analytics, France) were continuously measured at the
top of the reactor and data were recorded at 10 s intervals. The
N2O concentration was continuously analyzed by an infrared photometer
(Rosemount™ X-STREAM X2GP, Emerson, Germany) in outflow reactor
gas after drying through a condenser and a hydrophobic gas filter (0.2 µm). Minute averages are used for monitored data hereafter. Gas samples were
taken for N2O isotopic signature determination by an outlet gas pipe
derivation into a sealed glass vial of 20 mL. The vial was first flushed
with the sampling gas for >45 s prior to 1–5 min sampling. Gas
samples were then stored in the dark at room temperature until analysis.
Note that gas sampling was lacking for 5 of the 13 oxygenation tests.
The feeding solutions were characterized of one to five replicate samples
collected in the feeding tank. For each tested condition, the outflow was
characterized within 5 d of 1 to 14 replicate samples immediately
filtered through a 0.2 µm syringe filter and stored at 4 ∘C. Outflow sampling started after at least one hydraulic retention time (28±1 min). Ammonium was analyzed using the Nessler colorimetric method,
according to AFNOR NF T90-015 (DR 2800, Hach, Germany). Nitrite and nitrate
were measured by ionic chromatography (IC25, Dionex, USA).
Stable isotope measurements
Atmospheric N2 and Vienna Standard Mean Ocean Water (VSMOW) are the
references used for the nitrogen and oxygen isotopes ratios, respectively,
expressed in conventional δ notation, in per mil
(‰). Nitrogen and oxygen isotope ratios of nitrate and
nitrite were determined separately following a modified protocol of McIlvin
and Altabet (McIlvin and Altabet,
2005; Semaoune et al., 2012). Nitrogen isotope ratios of ammonium were
determined following the protocol of Zhang et al. (2007). These methods consist in the
conversion of the substrate (ammonium or nitrite or nitrate) into dissolved
N2O. δ15N and δ18O for ammonium, nitrite
and nitrate were hence determined from a calibration curve created with a
combination of nitrate or ammonium standards that underwent the same
chemical conversion as the samples (USGS-32, δ15N-NO3-= 180 ‰, δ18O-NO3-= 25.7 ‰; USGS-34, δ15N-NO3-=-1.8 ‰, δ18O-NO3-=-27.9 ‰ and USGS-35 δ15N-NO3-= 2.7 ‰, δ18O-NO3-= 57.5 ‰; IAEA-N1,
δ15N-NH4+= 0.4 ‰, IAEA-305A,
δ15N-NH4+= 39.8 ‰, USGS-25,
δ15N-NH4+=-30.4 ‰). The
quality of calibration was controlled with additional international
standards (IAEA-NO-3, δ15N-NO3-= 4.7 ‰, δ18O-NO3-= 25.6 ‰; IAEA-N2, δ15N-NH4+= 20.3 ‰). Basically, an analytical sequence was comprised of
triplicate standards for calibration, and quality controls and duplicate
samples. The average of the analytical replicates was then used for
calibration, for quality control and as a result.
Since no international standards were available for N2O isotopes, these
were determined the same day as nitrate or ammonium standard analysis
ensuring correct functioning of the method and analysis. In addition to
this, the internal N2O standards were previously calibrated by exchange
with the laboratory of Naohiro Yoshida and Sakae Toyoda at the Tokyo
Institute of Technology. All isotope measurements were determined using an
isotope ratio mass spectrometer (IRMS, DeltaVplus; Thermo Scientific) in
continuous flow with a purge and trap system coupled with a Finnigan
GasBench II system (Thermo Scientific). The precision was 0.8 ‰, 1.5 ‰ and 2.5 ‰ for δ15N, δ18O and
15N-SP, respectively.
Data processing and statistics
The effects of environmental conditions on nitrification were assessed from
four indices. The ammonium oxidation rate (AOR) was estimated in each
experiment for time ≥1 HRT from the difference between influent and
effluent NH4+ concentrations multiplied by the liquid flow rate
(kg NH4+-N d-1). The nitrification efficiency was defined as
the ratio between AOR and influent ammonium load. The N2O emission rate
(N2O-ER) was calculated by multiplying the measured N2O
concentration by the gas flow rate (mg N2O-N min-1). The N2O
emission factor (N2O-EF) was defined as the ratio between N2O-ER
and AOR (% of oxidized NH4+-N). The measurements related to
liquid or gas samples were averaged by experiment, i.e., the average of data
obtained from the samples collected after one hydraulic retention time.
Statistical analysis were performed using the R software (R Development Core Team, 2014). The value of 0.05 was used as significance level for Spearman
correlations (cor.test function) and linear regressions (lm function). Adjusted r2 was provided
as r2 for the latter.
Estimation of ranges of nitrogen isotope ratio in biologically produced N2O
As shown in Fig. 1, the pairwise relationships between δ15N,
δ18O and 15N-SP assist the determination of the producing
and consuming pathways at play. The N atoms that compose the N2O
molecule originate from NH4+ molecules when produced by
hydroxylamine oxidation, while originating from the N atoms of
NO3- or NO2- molecules when produced by nitrite
reduction (NOx- molecules). However, the nitrogen isotope ratio of
N2O does not equal those of its substrates as it depends on isotope
effects associated to each reaction step of N2O-producing process. The
isotope effect of the reaction step can be determined from the isotope
composition of substrates or products. Although performed on a few
tests here, the obtained value can only be applied to a limited number of
environmental conditions. The use of estimates from the literature seems
therefore suitable.
Several equations enable us to approximate the isotope effect and its effect on
the isotope ratios of substrate and product pools involved in a reaction.
These equations vary according to the assumptions made on the system
boundaries (Denk et al.,
2017).
The nitrifying reactor used in this study can be described as an open system
continuously supplied by an infinite substrate pool with constant isotopic
composition (NH4+,in). A small amount of the infinite
substrate pool is transformed into a product pool (NOx-,p) or
a residual substrate pool (NH4+,res) when flowing through the
system. The equations describing the input, output and processes considered
here are presented in Fig. 2 after Fry (2006). Note that the
definitions of f and Δ are inverse to the cited literature and that
Δ1 and Δ4 are null because no fractionation alter
the residual substrate exiting the reaction (Fry, 2006).
Diagram and equations of the nitrifying reactor after Fry (2006).
It is considered as a sequence of two reactor boxes. (i) The nitrification
of inflow ammonium (NH4+,in) to a pool of nitrite and nitrate
(NOx-,p), residual ammonium (NH4+,res) and
nitrous oxide (N2O) through the hydroxylamine oxidation pathway. (ii) The subsequent reduction of intermediate NOx-,int: mixing of
inflow NOx-,in and formed NOx-,p to nitrous
oxide (N2O) through the nitrite reduction pathway, and residual
NOx- that exits the reactor (NOx-,out). Note that
residual substrates and formed products exit the reactor without further
isotope fractionation (Δ1 and Δ4 are null). See
text for details.
The balance between input and output of each reactional step allows us to
propose equations for calculation of the nitrogen isotope ratio of compounds
in the inflow and outflow of the system (Denk et al., 2017; Fry,
2006). These equations can be simplified under the assumption that a limited
amount of N compounds are transformed into N2O, i.e., f2 close to 0
and f3 close to 1. Therefore, the N isotope ratios of the residual
substrate pool can be approximated from Eq. (1).
δ15N-NH4+,res≈δ15N-NH4+,in-Δ21-f1,
where f1 is the remaining substrate fraction leaving the reactor (i.e.,
remaining fraction of ammonium), ranging from 0 to 1 (0 % to 100 %), and
Δ2 is the N isotope enrichment factor associated with ammonium
oxidation. In their review, Denk et al. (2017) reported a mean value of
-29.6±4.9 ‰ for Δ2. Therefore,
δ15N is higher for residual than the initial substrate pool
(δ15N-NH4+,in<δ15N-NH4+,res). Consequently, the pool of product is
depleted in heavier isotope (i.e., nitrite and nitrate hereafter defined as
NOx- pool; δ15N-NOx-,in>δ15N-NOx-,int). It is estimated from Eqs. (2)–(4):
δ15N-NOx-,p≈δ15N-NH4+,in+Δ2f1,
Where δ15N-NOx-,p is the nitrogen isotope ratio
of the product pool produced by nitrification. The nitrogen isotope ratio of
the overall intermediate NOx- exiting this process results from
mixing between initial and produced NOx- pools (δ15N-NOx-,int) and can be estimated from Eqs. (3) and
(4):
3δ15N-NOx-,in=(δ15N-NO2-,in×[NO2-]in+δ15N-NO3-,in×[NO3-]in)([NO3-]in+[NO2-]in),4δ15N-NOx,int≈(δ15N-NOx-,in×([NO3-]in+[NO2-]in)+δ15N-NOx-,p×(1-f1)×[NH4+]in)([NO3-]in+[NO2-]in+(1-f1)×[NH4+]in).
Note that δ15N-NOx-,out equals δ15N-NOx-,int when f3 is close to 1, which means
that nitrifier denitrification and heterotrophic denitrification are
negligible. Finally, two options must be considered to approximate the
nitrogen isotope ratio of N2O that exits the reactor. On the one hand,
δ15N-N2O can be estimated from Eq. (5), when
hydroxylamine oxidation is the producing process of N2O:
δ15N-N2O≈δ15N-NH4+,res-Δ21-f1+Δ3.
In addition to the influence of the nitrogen isotope composition of the
substrate, δ15N-N2O depends therefore on the
difference between the isotope effects related to the oxidation of
NH4+ to NO2- and the oxidation of NH2OH to N2O
for complete nitrification (f1=0), while depending only on the
latter for limited nitrification (f1=1). On the other hand, δ15N-N2O can be estimated from Eq. (6), when the nitrite reduction
is the producing process of N2O:
δ15N-N2O≈δ15N-NOx-,int1-f1-1+Δ5.
In addition to the influence of the nitrogen isotope composition of the
substrate, when negligible amounts of N2O are produced by nitrite
reduction during nitrifier denitrification or heterotrophic denitrification,
its nitrogen isotope ratio depends on isotope effect related to this process
(Δ5).
Results and discussion
Changes in pH, ammonium, nitrite and nitrate concentrations confirmed
nitrifying activity in the reactor system (Table S1 in the Supplement, Fig. S3). During the
ammonium concentration tests, decreases in ammonium concentrations
([NH4+]), increases in nitrite and nitrate concentrations
([NO2-] and [NO3-], respectively) were observed, while
pH remaining below 8 prevented any relevant loss of ammonium by
volatilization. For example, [NH4+] decreased from 6.2 to 1.1,
from 28.6 to 17 and from 62.1 to 49.1 mg N L-1 by flowing through the
nitrifying biomass. At the same time, [NO2-] and [NO3-]
increased from 0 to 0.2–0.3 mg N L-1 and from 1.4–1.8 to 5–10 mg N L-1, respectively. Over the range of tested conditions, the ratio
between ammonium oxidation rate and influent ammonium load ranged from 10 % to
82 %, never exceeding 40 % for suboptimal nitrifying conditions
imposed during oxygenation and temperature tests (i.e., oxygenation levels
<21 % O2 and temperatures <20∘C). The
ammonium concentration, oxygenation level and temperature affected the
ammonium oxidation rates, as well N2O emission rates and factors.
Isotope composition ranges of N2O produced by hydroxylamine
oxidation and nitrite reduction
Ranges of δ15N for N2O produced by different processes
were hypothesized from Eqs. (1)–(5) for pairwise relationships with reviewed
data of δ18O and 15N-SP. To this aim, measurements of
isotope ratios of the different nitrogen species were required. The δ15N values of inflow ammonium, nitrite and nitrate were -3±0.1 ‰ (n=3), -15±0.1 ‰ (n=2) and 6.9±0.3 ‰ (n=3), respectively, during
ammonium concentration experiments (Fig. S3 and Table S2). The δ15N of residual NH4+ and intermediate NOx- were
estimated from Eqs. (1)–(4) with f1=0.1 or 0.9 (Fig. S2d–f),
Δ2=-30 ‰, the highest
[NH4+]in (62.1 mg N L-1) and the lowest
[NOx-]in (1.4 mg N L-1). They ranged from -3 ‰ to 27 ‰ and from -32 ‰ to 7 ‰,
respectively, which encompasses a few isotope compositions measured in the
outflow during ammonium concentration tests (Fig. S3 and Table S2).
Prior to pairwise comparisons with δ18O and 15N-SP, ranges
of δ15N values for N2O produced by the hydroxylamine
oxidation and nitrite reduction pathways were estimated from Eq. (5). The
net isotope effect of N2O production by ammonium oxidation via
hydroxylamine can be estimated by combining the isotope effects of ammonium
oxidation and hydroxylamine oxidation to N2O. The net isotope effect
associated with ammonium oxidation to nitrite ranges from -38.2 ‰ to -14.2 ‰ (Casciotti et al.,
2003) and can approximate the nitrogen isotope ratio of hydroxylamine
transitory produced. The isotope effect related to hydroxylamine oxidation
to N2O ranging from -26.0 ‰ to 5.7 ‰ from data in
Sutka et al. (2003,
2004, 2006); the net isotope effect of N2O production by ammonium
oxidation via hydroxylamine (Δ3) can range from -64.2 ‰
(-26.0+(-38.2)) to -8.5 ‰
(5.7+(-14.2)). Considering the range of the nitrogen isotope ratio of
residual ammonium, this method provided a broad range of δ15N
values, from -65 ‰ (δ15N-NH4+,res=-3 ‰, Δ2=-30 ‰, f1=0.9 and Δ3=-64.2 ‰) to 46 ‰ (δ15N-NH4+,res=27 ‰ , Δ2=-30 ‰, f1=0.1 and Δ3=-8.5 ‰), for N2O produced from ammonium by
hydroxylamine oxidation, according to Eq. (5). These values encompassed the
values proposed by others (-46.5 ‰
and -32.9 ‰; Sutka et al., 2006; Yamazaki et al.,
2014).
A higher range of the net nitrogen isotope effect for nitrite reduction than
hydroxylamine oxidation pathway was estimated for N2O production (Fig. 3a and b). Prior to being reduced to N2O through the nitrite reduction
pathway, NOx- was mainly derived from ammonium oxidation in the
nitrifying system (Eqs. 1–4); the resulting intermediate δ15N-NOx- ranges from -32 ‰ to 7 ‰. In
addition to this, the net isotope effects related to the N2O production
through nitrite reduction performed by nitrifiers or heterotrophic
denitrifiers (Δ5) ranges from -52.8 ‰ to -6 ‰
(Lewicka-Szczebak
et al., 2014; Sutka et al., 2008). Consequently, the δ15N of
N2O produced by nitrite reduction ranged from -89 ‰
(δ15N-NOx-,int=-32 ‰,
f1=0.1 and Δ5=-52.8 ‰) to 64 ‰
(δ15N-NOx-,int=7 ‰, f1=0.9 and Δ5=-6 ‰) , according to Eq. (6). This is consistent with
previous findings reporting δ15N-N2O between -112 ‰ and
-48 ‰ for nitrifier denitrifying systems (Mandernack
et al., 2009; Pérez et al., 2006; Yamazaki et al., 2014; Yoshida, 1988).
However, a similar range of nitrite-derived δ15N-N2O is
suggested for nitrifiers and heterotrophic denitrifiers, because ammonium
oxidation influences both processes in the system used in this study where
there is a low initial amount of NO2- and NO3-.
Interpretation maps of the isotope signature of N2O.
Schematic maps of (a)δ15N-δ18O, (b)δ15N-15N-SP and (c)δ18O-15N-SP. The red and
blue squares show the range of the data for N2O produced by
“hydroxylamine oxidation” and “nitrite reduction”, respectively. The shaded
area represents mixing of N2O produced by these pathways. The N2O
reduction increases δ15N, δ18O and 15N-SP
with slopes characterizing the pairwise relationships.
Pairwise comparisons of δ15N, δ18O and 15N-SP
estimates of the different experiments are presented in Fig. 3. These
comparisons provided ranges of plausible isotope compositions for N2O
produced by nitrifying or heterotrophic denitrifying bacteria through the
hydroxylamine oxidation and nitrite reduction pathways (red and blue boxes,
respectively). The measured N2O isotope compositions were compared to
these estimates to identify the N2O-producing and N2O-consuming pathways
likely at play in oxygenation, temperature and ammonium concentration
tests.
This approach suggests that the nitrite reduction pathway was the main
contributor to N2O emissions. Heterotrophic denitrification likely
influenced N2O emissions, as shown by oxygen isotope ratios higher
than 35 ‰ (Snider et al., 2013; Fig. 3a and c). However, this conclusion depends highly on δ18O-N2O ranges. Furthermore, the application of atmospheric
oxygen δ18O (23.5 ‰; Kroopnick and Craig, 1972) to estimate the oxygen
isotope ratio of N2O produced by hydroxylamine oxidation remains
uncertain since respiratory activity and air stripping might drive isotopic
fractionations and increase δ18O of residual dissolved
oxygen (Nakayama et al., 2007). To date, the oxygen isotope fractionation related to air stripping has not been investigated. Note that this estimate
relies on the assumption that there is no accumulation of NH2OH and
that its oxidation to N2O occurs before or independently of its
oxidation to NO2-.
The effect of oxygen limitation on the N2O-producing pathways
Ammonium concentrations decreased from 20.2–37.3 to 11.4–31.1 mg N L-1,
with 45 % to 89 % of the inflow ammonium remaining in the outflow during
the oxygenation tests (Fig. S2d). When measured, the cumulated
concentrations of NO2- and NO3- ([NOx-])
increased from 2.4–4.1 to 4.7–11 mg N L-1 between inflow and outflow
and were composed by at least 74 % and 82 % of NO3-,
respectively. The mass balance between N compounds that enter and exit the
reactor evidenced a default of up to 5 mg N and impacted each test. No
significant amounts of NO were detected during any tests (data not shown),
whereas NH2OH, N2 and N mineralization/assimilation in
the biofilm were not quantified. The accumulation of such amounts of NH2OH
is unlikely. Heterotrophic denitrification, i.e., the reduction of
NOx- and more particularly of N2O to N2, may explain the
incomplete N mass balance. However, the measurement of small N2
variations in the gas mixture exiting the reactor and comprising at
least 79 % N2 was not performed.
The oxygenation level had contrasting effects on ammonium oxidation rates,
and N2O emission rates and factors (Fig. 4a–c). Between an oxygenation
of 0 % to 10.5 % O2 in the gas mixture, no clear trend in ammonium
oxidation rates was observed although it was rather low (1.1±0.5 mg NH4+-N min-1). In the same oxygenation level interval, the
N2O emission rate increased for two of three inflows [NH4+]
tested. It increased from 0.35×10-3 to 0.73×10-3 mg N min-1 between 0 % and 10.5 % O2 at 25.3 mg NH4+-N L-1, and from 1.34×10-3 to 1.4×10-3 mg N min-1
between 4.2 % and 10.5 % O2 at 23.8 mg NH4+-N L-1;
it decreased from 2.86×10-3 to 2.04×10-3 mg N min-1
between 4.2 % and 10.5 % O2 at 37.3 mg NH4+-N L-1.
Finally, the N2O emission factor globally increased from 0.05 % to 0.16 % in the 0 %–10.5 % O2 interval. At oxygenation levels from 10.5 % to
21 % O2, the ammonium oxidation rates increased from 0.9±0.2
to 2.1±0.4 mg N min-1, with N2O emission rates remaining
stable at 1.2×10-3±0.6×10-3 mg N min-1 and the
emission factors decreasing from 0.15±0.03 % to 0.06±0.03 %.
Effect of oxygenation level on (a) the ammonium oxidation rate,
(b) the nitrous oxide emission rate, (c) the N2O emission factor, and
(d) the nitrogen isotopomer site preference, (e) the nitrogen isotope ratio
and (f) the oxygen isotope ratio of N2O. Average and standard deviation
(error bars) are calculated for steady-state conditions. Note that gas
sampling for isotope analysis was lacking for 5 of the 13 oxygenation tests.
15N-SP varied between -9 ‰ to 2 ‰ over the range
of imposed oxygenation levels, with a marked increase when oxygenation
increased from 16.8 % to 21 % O2 (Fig. 4d). A similar marked change in
nitrogen and oxygen isotope ratios of N2O (decrease and increase,
respectively) was observed when oxygenation increased from 16.8 % to 21 %
O2 (Fig. 4e and f). Note that to observe the latter variations the
effect of ammonium concentration was not included. One way to do so is to
compare the isotope composition average at 21 % O2 with the isotope
composition measured for 23.8 NH4+-N L-1 at 16.8 %
O2. The 15N-SP values were close to the range of -11 ‰ to 0 ‰ reported for N2O produced by nitrifying or
denitrifying bacteria through nitrifier denitrification and heterotrophic
denitrification (Toyoda et al.,
2017; Yamazaki et al., 2014). Additional suggestions can be made from the
15N-SP dynamics between and variations within the oxygenation levels.
If an increase in the hydroxylamine oxidation contribution to the N2O
emission might explain the higher 15N-SP observed at 21 % O2 as
compared to lower oxygenation levels, an additional mechanism can explain
the variations observed for the experiments with oxygen-depleted atmosphere.
The 15N-SP dynamics suggest a higher amount of N2O was reduced to
N2 at 4.2 % than 16.8 % O2. The reduction of N2O to N2
can increase the 15N-SP of residual N2O (Mothet et
al., 2013). In heterotrophic denitrifying bacteria however, the nitrous
oxide reductase involved in this reaction is highly sensitive to inhibition
by oxygen (Betlach
and Tiedje, 1981; Otte et al., 1996). This might explain the decrease in
15N-SP from -3.8±4.4 ‰ to -7.2±1.7 ‰ when O2 increased from 4.2 % to 16.8 %. This is
also consistent with a possible onset of anoxic microsites within the
reactor biomass more likely at 4.2 % than 16.8 % O2. The dissolved
oxygen (DO) concentration never decreased below 1.5 mg O2 L-1 in
the bulk solution at the top of the reactor (Fig. S2). However, DO decreased
from the bulk reactor solution toward the deeper layers of biofilm due to
the activity of ammonium oxidizers (Sabba et al., 2018).
This is further exacerbated by heterogeneous and varying distribution of air
circulation within the static bed. Therefore, oxygen depletion can be
assumed within the biofilm. Finally, the N2O reduction to N2
likely explains the overall decrease in N2O emission between 16.8 % and 0 % O2 (Fig. 4b).
In general the N2O reduction to N2 is accompanied by an increase
in nitrogen and oxygen isotope ratios of N2O
(Ostrom et al., 2007; Vieten et al., 2007).
However, our results show a decrease in δ15N-N2O, and
δ18O-N2O remained stable between 30.5 ‰ and 34.7 ‰, when the N2O reduction is thought to increasingly
constraint the N2O isotopocules with decreasing O2 from 16.8 % to
4.2 % (Fig. 4e and f). The independence of samples taken during the
oxygenation test can explain this. The N2O sampled at 4.2 % O2
is not a residual fraction of N2O produced at 16.8 % O2 that
would have undergone a partial reduction. The oxygenation level can alter
the isotope fractionation factors through the control of reaction rates, as
evidenced for the reduction of N2O to N2 by Vieten et al. (2007). These authors reported lower reaction rates and
increased isotope fractionation factors with increasing oxygenation levels.
In our case, a similar phenomenon might have influenced both oxidative and
reductive processes leading to the production of N2O and occurring
before its ultimate reduction to N2. However, knowledge regarding controls, such as the oxygenation level, on the net isotope effect
related to a sequence of non-exclusive oxidative and reductive processes is
still lacking and requires further investigations. Additionally, with δ18O below 35 ‰ for all but one experiment the
oxygenation tests did not provide evidence for the heterotrophic denitrifier
contribution to N2O emissions, likely due to oxygen exchange with water (Snider
et al., 2015, 2012, 2013).
Difference in temperature dependency of hydroxylamine and ammonium oxidizers as driver of hydroxylamine oxidation contribution to N2O emissions
Ammonium concentrations decreased from 6.2–62.1 to 0.9–54.1 mg N L-1 and
from 18 % to 79 % of the inflow ammonium remaining in the outflow during
the temperature and ammonium concentration tests (Fig. S2e and f). This
remaining fraction was positively correlated to ammonium concentrations (r=0.96) and negatively correlated to temperature within a lower range of
values (61 %–67 %; r=-0.94). In the ammonium tests, the cumulated
concentrations of NO2- and NO3- ([NOx-])
increased from 1.4–6.1 to 5.1–19.6 mg N L-1 between inflow and outflow
and were composed by at least 74 % and 91 % of NO3-,
respectively. Noticeably, the nitrite concentrations in the outflow linearly
increased with temperature (r2=0.95; Fig. S2h).
An increase in temperature and inflow ammonium concentrations both
positively influenced the rates of NH4+ oxidation and N2O
emissions and the emission factor (Fig. 5). The NH4+ oxidation
rate linearly increased from 1.3 to 1.5 mg NH4+-N min-1
with temperature (r=0.89; Fig. 5a) and increased from 0.97 to 3.49 mg NH4+-N min-1 with a 10-fold increase in the inflow ammonium
concentration (r=0.82; Fig. 5b). These positive correlations are well
known in the temperature range investigated here and are likely due to
enhanced enzymatic activity and Michaelis–Menten kinetics, respectively (Groeneweg
et al., 1994; Kim et al., 2008; Raimonet et al., 2017). Similarly, the
N2O emission rates increased from 80.4×10-6 to 2.5×10-3 mg N2O-N min-1, and from 83.6×10-6 to 6.2×10-3 mg N2O-N min-1 upon changes in temperature and the ammonium
concentrations, respectively. These results are in agreement with positive
correlations between N2O emissions with temperature and ammonium
concentration observed from modeling and experimental studies on partial
nitrification and activated sludge systems (Guo
and Vanrolleghem, 2014; Law et al., 2012a; Reino et al., 2017). Altogether
this confirms a correlation between the N2O emission rates and the
ammonium oxidation rates. Interestingly, the increase in the N2O emission
factor indicates a stronger effect of temperature and ammonium concentration
on the N2O emission rate than on NH4+ oxidation. The N2O
emission factor increased from 0.07 % to 0.16 %, and from 0.01 % to 0.29 % with temperature and inflow ammonium concentration, respectively (r>0.94; Fig. 5e and f). Both experiments suggest that the
increase in N2O emissions results from the increasing production of
N2O by hydroxylamine oxidation or nitrite reduction in combination with
a slow rate or the absence of N2O reduction to N2. Furthermore, no
nitrite accumulation was observed with increasing ammonium oxidation rate
(Fig. S2i). Therefore, if N2O emission results mainly from the nitrite
reduction pathway, this suggests that the nitrite reduction pathway is more
responsive to the increasing ammonium oxidation rate than the nitrite
oxidation pathway; the latter remains the main pathway of nitrite
consumption.
Effect of temperature and inflow ammonium concentration on (a, b) the ammonium oxidation rate, (c, d) the nitrous oxide emission rate and (e, f) the N2O emission factor.
The range of nitrogen isotopomer site preference observed during the
temperature and concentration tests (from -8 ‰ to 2.6 ‰)
was similar to those measured during the oxygenation tests, confirming the
high contribution of the nitrite reduction pathway to N2O emissions
(Fig. 6a). This is consistent with previous findings based on the
15N-SP of N2O emitted from aerobic activated sludge
(Toyoda
et al., 2011; Tumendelger et al., 2016; Wunderlin et al., 2013), although
authors reported 15N-SP as high as 10 ‰. This can
suggest a higher oxygen limitation being favorable to the contribution of
the nitrite reduction to N2O production in the nitrifying reactor
studied here. Hydroxylamine oxidation can even be the main N2O-producing pathway, as evidenced by Tumendelger et al. (2014) in
an aerated tank.
Effect of temperature (orange symbols) and inflow ammonium
concentration (blue symbols) on (a) the nitrogen isotopomer site preference, (b) the nitrogen isotope ratio and (c) the oxygen isotope ratio of N2O. Average and standard deviation (error bars) are calculated for the samples taken after one hydraulic retention time. Note that the isotopic
measurements of gas samples taken at inflow ammonium concentration of 42.7
and 42.9 mg N L-1 were both recorded as 42.8 mg N L-1 in the
legend.
Furthermore, 15N-SP increased with temperature between 13.5 and
19.8 ∘C. Our data suggest that temperature was the main control
on the change in N2O-producing pathways within this temperature range
(Fig. 6a). This could explain higher SP obtained with a 28.6 mg N L-1 inflow ammonium concentration than with 42.8. The temperature control seems to
mitigate here the effect that ammonium concentration can have on the
N2O-producing pathways evidenced elsewhere. Wunderlin et al. (2012,
2013) observed an increase in 15N-SP from -1.2 ‰ to 1.1 ‰ when inflow [NH4+] increased from 9 to 15 mg N L-1. They also observed 3 ‰–6 ‰ decreases in
15N-SP over the course of ammonium oxidation experiments and suggested
that the NH2OH oxidation contribution to N2O production increased when
conditions of NH4+ excess, low NO2- concentrations and
high nitrogen oxidation rate occur simultaneously. Our findings are
consistent with the observation of Groeneweg et al. (1994) showing that temperature rather than
ammonium concentration influenced the ammonium oxidation rate.
15N-SP increased from -6.5 ‰ to 2.6 ‰ with
increasing temperature from 13.5 to 19.8 ∘C (Fig. 6a). This
15N-SP increase may either result from an increase in the N2O
production by the hydroxylamine oxidation pathway or the N2O reduction
to N2. Since an optimal oxygenation level was imposed and increased
emissions were observed, the increasing 15N-SP is more likely due to
N2O production by the hydroxylamine oxidation pathway. Reino et al. (2017) also observed an increase of
N2O emissions for temperatures above 15 ∘C in a granular
sludge airlift reactor performing partial nitritation. The authors suggested
two hypothesis to explain their results: (i) the difference in the kinetic
dependency with temperature of enzymes involved in ammonium and
hydroxylamine oxidation; (ii) the temperature dependency of the acid–base
equilibrium ammonium–ammonia. The changes in 15N-SP observed here are
consistent with the former hypotheses. Hydroxylamine oxidation likely
becomes the limiting step at temperatures above 15 ∘C, while
being faster than ammonium oxidation at lower temperatures (Fig. 7). At
temperatures above 15 ∘C, hydroxylamine therefore accumulates and
leads to a higher contribution of the hydroxylamine oxidation pathway to
N2O emissions. It would thus be interesting to determine the
temperature dependency of the hydroxylamine oxidase.
Graph of the difference in temperature dependency of the
reactions involved in nitrification.
The change in nitrous oxide-producing and nitrous oxide-consuming pathways had contrasting effects on the nitrogen and oxygen isotope ratios of nitrous oxide (Fig. 6b
and c). δ15N-N2O decreased from -2.5 ‰ to -40.9 ‰ with an increasing contribution of hydroxylamine
oxidation to the N2O emissions, i.e., when temperature increased from
13.5 to 19.8 ∘C. This is in contrast with the expected net lower
isotope effect for N2O produced by hydroxylamine oxidation than nitrite
reduction, and points out that further investigations are needed (Snider
et al., 2015; Yamazaki et al., 2014). The changes in δ18O-N2O were less straightforward, likely influenced by changes
in the reaction rates in addition to changes in the contribution of N2O-producing pathways. The values decreased from 41.1 ‰ to 34.3 ‰ with an increasing contribution of hydroxylamine
oxidation to the N2O emissions when temperature increased from 13.5 to
18.2 ∘C. It decreased linearly from 38.2 ‰ to 31.8 ‰ with increasing reaction rate when inflow ammonium
concentration increased from 20.2 to 62.1 mg NH4+-N L-1
(r2=0.83).
Difference in oxidation and reduction rates of nitrite as driver of nitrite reduction contribution to N2O emissions
The oxygenation, temperature and ammonium concentration tests revealed a
strong control of nitrite-oxidizing activity and the contribution of the
nitrite reduction pathway to N2O production. No relationship was
observed between NO2- concentrations and oxygenation (Fig. S2g).
In addition to this, higher 15N-SP at 21 % compared to the 10.5 %–16.8 % O2 was observed while the temperature remained below 20 ∘C
(Fig. 4d). This is most likely due to higher nitrite oxidation than nitrite
reduction rates in response to increasing oxygenation levels to 21 %
O2, which is consistent with the nitrite oxidation step sensitivity to
oxygen limitation (Pollice et al., 2002;
Tanaka and Dunn, 1982). Additionally, 15N-SP close to 0 ‰ observed at the highest oxygenation level indicates a
decreasing contribution to N2O production of nitrite reduction over
hydroxylamine oxidation pathway. The highest oxygenation level thus limits
the reduction pathways (i.e., NO2- reduction to N2O and
N2O reduction to N2) while favoring the ammonium and nitrite oxidation pathways.
During the temperature and ammonium concentration tests, the contribution of
the hydroxylamine oxidation pathway to N2O emissions increased with
a temperature between 13.5 and 19.8 ∘C (Sect. 3.3) and decreased in
favor of the nitrite reduction pathway when the temperature exceeded 20 ∘C (Fig. 6a). 15N-SP was low when the temperature exceeded
20 ∘C (-7.3±1 ‰), while being
higher than -5 ‰ (-1.3±2.4 ‰) when the temperature ranged from 18.2 to 19.8 ∘C. At temperatures
above 20 ∘C, ammonium oxidation rates exceed nitrite oxidation
rates (Fig. 7;
Kim et al., 2008; Raimonet et al., 2017). This most likely explains the
increased contribution of the nitrite reduction pathway to N2O
emission, as more nitrite becomes available for nitrifier denitrification
and/or heterotrophic denitrification. As little nitrite accumulated (Fig. S2h), lower rates of nitrite-consuming processes than nitrite-producing processes can be
inferred (nitrite reduction and oxidation vs. ammonium oxidation).
Additionally, values of δ18O>35 ‰ measured during these tests suggest a significant
contribution of heterotrophic denitrifiers to N2O emissions
(Snider et al., 2013). This
seems to occur at the lowest hydroxylamine oxidation contribution to
N2O production below 18 ∘C and at 20.3 ∘C.
Furthermore, the denitrifiers were impacted to a larger extent by
temperature than ammonium concentration.
Conclusion
Our results demonstrated that whatever the imposed conditions, the
nitrifying biomass produced N2O and nitrite reduction remained the main
N2O-producing pathway. The N2O emissions were sensitive to
oxygenation, temperature and NH4+ concentration likely due to the
control of enzymatic activities. The use of N2O isotopocules confirmed
the processes that control N2O emissions under oxygenation constrain
and improved knowledge of processes that control N2O under
temperature constraints. Among the environmental variables tested, temperature
appears to be the main control on N2O-producing pathways under
nitrifying conditions, due to its dissimilar effects on ammonium-oxidizing and nitrite-oxidizing activities. Ranges of optimal temperature for nitrification and
limited N2O emissions can be recommended. The combination of low
N2O emissions and high nitrification rates may occur close to 15 ∘C. From 15 to 20 ∘C, an increasing nitrification rate
increases N2O emissions via the hydroxylamine oxidation pathway.
Above 20 ∘C, an increasing nitrification rate increases the
N2O emissions via the nitrite reduction pathway.
We studied the impact of environmental variables on N2O-producing
pathways based on the isotope analysis of a limited sample number of
dissolved N compounds. The approach and conclusions based on the impact of
these variables on N2O emissions most likely apply to nitrification
and denitrification in soils, sediments, lakes and other natural waters. These
systems are subject to dynamic environmental conditions, among which are
ammonium concentrations, oxygenation and temperature. The comparison of the
N2O isotopocules measured and those hypothesized from the literature
provides a useful tool to discuss the N2O-producing and N2O-consuming
process, as well the underlying control mechanisms at play. Ultimately, this
can result in mitigation solutions of N2O emissions by constraining
trough space and time the contribution of N2O-producing and N2O-consuming
pathways. However, it appears that additional efforts are still needed to reduce, if
possible, the ranges of N2O isotope signatures related to each
producing and consuming processes.
Data availability
All data included in this study are available upon request by contacting the corresponding author.
Additional information about the nitrifying activity of the biomass, the
experimental conditions and the time series of ammonium oxidation
experiments can be found in the Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/bg-17-979-2020-supplement.
Author contributions
JF, AF and MSp designed the experiments with contributions from GH, MSe and
AML. GH, JF and LL carried out the experiments. GH performed the stable
isotope measurements with a contribution from VV and interpreted them with
contribution from MSe. GH and JF processed the data. GH, JF and AML prepared
the manuscript with contributions from all co-authors.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
The authors are
grateful to Sam Azimi and the “Direction Innovation Environment” of SIAAP
for providing the media colonized by the nitrifying biomass, Mansour Bounouba and Simon Dubos for their assistance in chemical analyses and in
setup and development of the nitrifying reactor.
Financial support
This research has been supported by the French National Research Agency (grant no. ANR-15-CE04-0014-02).
Review statement
This paper was edited by Perran Cook and reviewed by two anonymous referees.
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