Biochemical Engineering Journal 149 (2019) 107229

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Achievement of partial nitrification under different carbon-to-nitrogen ratio T

and ammonia loading rate for the co-treatment of landfill leachate with
municipal wastewater
⁎
Marco Capodici, Santo Fabio Corsino, Daniele Di Trapani , Gaspare Viviani
Università degli Studi di Palermo, Dipartimento di Ingegneria, Viale delle Scienze, 90128 Palermo, Italy

H I GH L IG H T S

• PN process was studied in a SBR treating landfill leachate at different ALR and C/N.
• Stable −3 -1
PN was achieved at ALR between 0.30-0.50 kg NH -N m d and C/N lower than 4.
4

• NOB inhibition was achieved at lower ALR and higher C/N compared with the literature.
• The highest PN and TN removal performances were obtained under similar ALR and C/N.
• The maximum autotrophic activity was found at ALR of 0.30 kg NH -N m d . 4
−3 -1

A R T I C LE I N FO A B S T R A C T

Keywords: Partial nitrification (PN) is a technically and economically effective solution for the treatment of wastewater
C/N featuring low C/N ratio, allowing to achieve approximately 25% energy saving and 40% carbon source for
Landfill leachate denitrification. This study investigated the effect of different carbon to nitrogen ratio (C/N) and ammonia
Nitrogen removal loading rate (ALR) on PN performances in a sequencing batch reactor (SBR) treating landfill leachate with
Partial nitrification
municipal wastewater. The aim was to find an optimum range for C/N and ALR to maximize PN performances.
Denitritation
Results demonstrated that a proper balancing between ALR and C/N is crucial to achieve high PN efficiency. The
SBR
results highlighted the existence of an optimum range for ALR and C/N of approximately 0.30–0.50 kg NH4-N
m−3d−1 and 2–4, respectively. Although complete suppression of NOB was not achieved at these values, a
predominance of nitrite (125 mg L−1) to nitrate (50 mg L−1) was observed. The results demonstrated the
achievement of PN even at high C/N (4) but, on the other hand, C/N higher than 6 were not favorable to
autotrophic growth. C/N significantly lower than 4 caused a stress condition for the biomass, leading to an
excess in SMP production. Therefore, the operational parameters as well as the co-treatment ratio should be
adjusted in order to operate the system under specific ALR (0.30–0.50 kg NH4-N m−3 d−1) and C/N (2–4).

1. Introduction effluent which composition is strongly related to environmental con-

Municipal solid waste (MSW) disposal in sanitary landfills re-
presents one of the most common solutions in the waste management
cycle in many parts of the world [1]. This practice produces several
environmental impacts, among which the generation of a highly pol-
luted liquid waste called leachate. In particular, municipal landfill
leachate (MLL) is mainly generated from rainwater infiltration and
percolation through the disposed wastes, biological reactions that in-
volve wastes themselves that undergo biodegradation and from the
initial moisture content of the disposed waste [2]. MLL is an aqueous
ditions, like seasonal weather variations, waste quality (raw or pre-
treated), landfill age, etc. [3,4]. In general, MLL is characterized by
high ammonium (1000–4000 mg L−1) and organic matter concentra-
tions in terms of chemical oxygen demand (COD) (2000–20000 mg L−1)
[5]. More precisely, the concentration of biodegradable organic matter
expressed as biochemical oxygen demand (BOD) and ammonia nitrogen
are high in young leachate, whereas mature leachate is characterized by
a poor organic matter biodegradability and even higher concentration
of ammonia nitrogen [6]. The biological treatment of MLL is generally
considered effective if the biodegradable fraction of organic matter is

⁎
Corresponding author.
E-mail address: daniele.ditrapani@unipa.it (D. Di Trapani).

https://doi.org/10.1016/j.bej.2019.05.006
Received 1 February 2019; Received in revised form 7 May 2019; Accepted 9 May 2019
Available online 25 May 2019
1369-703X/ © 2019 Elsevier B.V. All rights reserved.
M. Capodici, et al.
adequate for microorganisms’ growth, for supporting denitrification,
etc. [7]. On the other hand, the supply of external carbon sources is not
economically advisable. In this light, combined biological treatment
(usually referred to as co-treatment) of MLL with municipal wastewater
(MWW) offers technological and economic advantages due to the pre-
sence of existing facilities such as the free availability of biodegradable
organic matter, etc., [8,9]. Co-treatment of MLL with MWW was widely
investigated so far [10–12]. However, nitrogen removal by conven-
tional nitrification/denitrification is still challenging. Indeed, because
of the high ammonium content, large amount of oxygen and energy are
required for ammonium oxidation to nitrate [13]. Moreover, the low
amount of biodegradable organic fraction, in relation to the nitrate load
to be reduced, might hamper the denitrification kinetic. With this re-
spect, a correct trade-off between MLL and MWW is crucial to achieve a
proper carbon to nitrogen ratio (C/N) avoiding limitations during de-
nitrification. However, because of the high ammonium concentration,
only low co-treatment ratio could ensure appropriate operating condi-
tions to achieve stable nitrogen removal performances via nitrification/
denitrification pathway [14]. In these cases, when co-treated at a low
ratio, leachate is likely simply diluted instead of being effectively co-
treated [10,15].
During the last decade, partial nitrification (PN) and further deni-
trification via nitrite (denitritation) have arisen as one of the most
suitable process in order to remove nitrogen from urban and industrial
wastewater [5,6,16,17]. In nitrogen removal process via nitrite
pathway, the achievement of stable PN is considered the bottleneck of
the process [13], since denitrification kinetics are affected by fewer
limiting factors. PN is technically and economically effective in case of
wastewater containing high ammonia concentration and low C/N. In-
deed, less oxygen is required to obtain the ammonium oxidation to
nitrite rather than nitrate, allowing to achieve approximately 25% en-
ergy saving. Moreover, because wastewater characterized by low C/N
usually requires an external carbon supply for denitrification, if nitrite
has to be reduced instead of nitrate, approximately 40% of carbon
source could be saved, allowing to achieve approximately 25% energy
saving [6]. PN process is based on the implementation of operating
strategies aimed at suppressing the nitrite oxidizing bacteria (NOB)
activity, without affecting ammonia oxidizing bacteria (AOB), thereby
resulting in nitrite accumulation. Several operating strategies have been
proposed in the technical literature to achieve stable PN, including
selective NOB washout by temperature, free ammonia (FA) inhibition,
oxygen limitation, etc. [18]. Nevertheless, although these strategies
enabled to achieve stable PN performance, they resulted in poor total
nitrogen (TN) removal because of a not proper balancing in C/N, which
resulted in incomplete denitritation and nitrite accumulation in the
effluent [5,17].
To authors’ knowledge, among the proposed strategies to achieve
stable PN the combined effect of C/N ratio and ammonia loading rate
(ALR) has not been investigated so far. If on the one hand high C/N
could ensure enough available carbon source for denitrification, it
could favor competition between ordinary heterotrophic organisms
(OHO) and autotrophic nitrifying bacteria for nitrogen and oxygen,
resulting in the decrease in nitrification efficiency [19]. On the other
hand, low C/N ratio could result in incomplete denitritation due to
carbon limitation, leading to poor TN removal. Similarly, despite a high
ALR could result in NOB inhibition due to FA, thereby favoring the
achievement of stable nitritation, an excessive ALR might also promote
AOB inhibition. Therefore, operating with a proper C/N and ALR could
be helpful to achieve stable PN, while ensuring enough C/N for high TN
removal. From a management point of view, this could be easily
achieved by adopting a proper co-treatment ratio between MLL and
MWW and operating in sequencing batch reactors (SBR). Indeed, SBRs
are characterized by a high operating flexibility that allow an easily
change of operational conditions adapting to the variations of waste-
water composition.
This study investigated the feasibility to achieve stable PN in the co-
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Biochemical Engineering Journal 149 (2019) 107229
treatment of landfill leachate and municipal synthetic wastewater in a
SBR bench scale plant. More precisely, the aims of the study were to: (1)
find an operating range for C/N and ALR to achieve stable PN; (2)
correlate the C/N and ALR with the nitrogen pattern in the effluent and
TN removal efficiency; (3) assess the effect of different C/N and ALR on
the kinetic parameters of nitrifying and heterotrophic bacteria.
2. Materials and methods
2.1. Reactor set-up
A SBR bench scale plant with an operational volume of 12 L was
investigated for 190 days. The SBR was equipped with two feeding
pumps dosing raw leachate and the synthetic wastewater, which were
stored into two different influent tanks, a solenoid valve for the effluent
discharge and a fine bubble diffuser connected to an air supply device.
The filling volume was 5 L, corresponding to a volumetric exchange
ratio (VER) of 42%. A Programmable Logic Controller (PLC) auto-
matically handled the SBR cycling operations.
2.2. Leachate composition and municipal wastewater characterization
The landfill leachate was periodically collected at Palermo
Municipal Landfill (Bellolampo). It was characterized by variability in
the composition according to the seasonal fluctuations related to the
rainfall events. The main physical-chemical characteristics of the raw
leachate are reported in Table 1.
The raw leachate was characterized by a BOD5 concentration of
almost 1900 mg L−1 (average value). Therefore, the BOD5/COD ratio
was of approximately 0.20, indicating that the leachate was classifiable
as medium-aged.
In order to simulate a co-treatment, landfill leachate was blended
with a synthetic wastewater containing sodium acetate and ammonium
chloride as carbon and nitrogen source, respectively. More precisely,
the COD and the NH4-N concentration of the synthetic wastewater were
approximately 564 mg L−1 and 46 mg NH4-N L−1, respectively. The
organic substrate of the synthetic wastewater was considered 100%
biodegradable, so the BOD5 and COD values were equal.
2.3. Experimental campaign and operational conditions (feeding medium
composition)
As aforementioned, the experimental campaign had a duration of
190 days and it was divided into two different phases, depending on the
SBR cycle duration. In particular, the SBR was operated according to a
12 h cycle until the 135th experimental day, whereas from that day
onward, the cycle duration was decreased to 8 h for the remaining 55
days. The 12 h cycle included 30 min of influent static feeding, 30 min
of aerated feeding, 8.5 h of aeration, 2 h of settling followed by 30 min
of effluent discharge. In the 8 h cycle the length of the aeration and
settling phases were decreased to 6 h and 30 min, respectively, whereas
the other phases remained unchanged. Since the main goal of the study
was to find proper operating ranges in terms of applied ALR and C/N to
Table 1
Characteristic of the feed leachate in this study.
Parameter Unit of measure Average value ± std
pH – 8.43 ± 0.37
NH4-N mg L−1 1196 ± 616
NO2-N mg L−1 n.d.*
NO3-N mg L−1 15.1 ± 3.3
COD mg L−1 6617 ± 3444
BOD5 mg L−1 1221 ± 629
Conductivity mS cm−1 12.9 ± 5.1
* Below detection limit.
M. Capodici, et al.
Fig. 1. COD, ammonium and C/N in the feeding medium during the experimental campaign.
enable stable PN, being this the limiting step for nitrogen removal
process via nitrite pathway, a specific anoxic phase for denitrification
was not provided. After the settling phase, a volume of supernatant
equal to 5 L was discharged. The SBR plant was started up with acti-
vated sludge inoculum characterized by a total suspended solid (TSS)
concentration close to 3 g L−1. During the first 10 days, the SBR was fed
with the synthetic wastewater only, in order to enhance the biomass
growth. When the TSS concentration raised up to approximately 7.5
gTSS L−1, the MLL was added to the feeding medium according to what
previously discussed. More precisely, the MLL and the MWW were
mixed with different ratios ranging between 20% and 40% in order to
obtain different C/N ratios (2–8). The C/N ratio was changed when
steady conditions in terms of nutrient removal performance were
achieved. Overall, the BOD5/COD of the feeding mixture was almost
constant at a value close to 0.30. Fig. 1 summarizes the fluctuations of
the feeding wastewater throughout experiments.
The influent NH4-N and COD concentrations ranged between ap-
proximately 200 mg L−1 and 800 mg L−1 and between 800 mg L−1 and
3500 mg L−1, respectively. Accordingly, the C/N ratio ranged between
2 and 8 throughout experiments, following the fluctuations of the raw
leachate composition. The ALR ranged between 0.10 kg NH4-N m-3d-1
and 0.70 kg NH4-N m-3d−1, according to the variations of the influent
ammonium concentration and the SBR cycle duration. Similarly, the
organic loading rate (OLR) ranged between 0.60 kg COD m−3d−1 and
2.80 kg COD m−3d−1. The TSS concentration in the mixed liquor was
maintained close to 7.50 gTSS L−1 during the entire duration of ex-
periments by daily purging a known amount of waste sludge.
2.4. Analytical methods
The chemical-physical analyses, such as COD, NH4-N, NO3-N, NO2-
N, TSS, pH and electrical conductivity were performed according to
Standard Methods [20]. All the chemical analyses were performed in
triplicates. FA concentration was calculated according to the literature
[21]. Total nitrogen in the effluent was calculated as the sum of the
ammonium, nitrite and nitrate as nitrogen. The heterotrophic and au-
totrophic growth rates were evaluated by means of respirometric
techniques according to literature [22,23].
In details, in order to assess the heterotrophic growth rate, a known
amount of readily biodegradable COD was fed to a batch reactor. A
known amount of mixed liquor was kept under aerobic conditions and
the autotrophic activity was avoided by means of allylthiourea dosage.
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Biochemical Engineering Journal 149 (2019) 107229
The oxygen uptake rate was measured and converted in heterotrophic
grow rate by fitting the Michaelis-Menten equation [32]. Similarly, the
autotrophic grow rate was evaluated by using ammonia as readily
biodegradable substrate. Furthermore, the extracellular polymeric
substances (EPS) were extracted by the activated sludge samples ac-
cording to the “heating method” [24]. Total EPS content was evaluated
as the sum of the not bound fraction, including the loosely-bound EPS
(LB-EPS) and the soluble microbial products (SMP), and the tightly
bound (TB-EPS). For each EPS fraction, the protein and carbohydrate
concentrations were evaluated according to the phenol-sulfuric acid
method [25] and with the Folin method [26], respectively.
3. Results and discussion
3.1. Nitrogen pattern in the effluent
The oxidation of the influent ammonium to nitrite and/or nitrate
was investigated as a function of the applied ALR and the C/N of the
influent MLL/MWW mixture. Fig. 2 depicts the relationships between
the effluent nitrogen forms with the C/N and the applied ALR
(Fig. 2a–c).
As noticeable from Fig. 2a, the NH4-N concentration in the effluent
decreased with the C/N ratio. The relationship between the effluent
NH4-N concentration and the C/N indicated that at C/N lower than 4,
the ammonium concentration in the effluent sharply increased even for
slight C/N decreases. In contrast, at C/N higher than 4, the effluent
ammonium concentration was significantly lower, ranging between
10 mg L−1 and 25 mg L−1 and, overall, a lower dependence with C/N
was observed. The same Fig. 2a depicts the effluent NH4-N concentra-
tion as a function of the applied ALR. The results indicated that up to
0.50 kg NH4-N m−3d−1 a linear relationship between the applied and
the removed ALR was observed, indicating that the removal efficiency
was not affected by the increasing ALR within this operating range
(average removal: 94%). In contrast, when the applied ALR exceeded
0.5 kg NH4-N m−3d−1, the relationship between applied and removed
ALR deviated from the linear trend and a residual concentration of
ammonium was observed in the effluent. Furthermore, the ammonium
removal efficiency decreased to approximately 55% when the ALR was
close to 0.70 kg NH4-N m−3d−1, thereby suggesting that over a certain
ALR ammonium plays inhibitory effects on AOB activity, likely due to
FA formation, resulting in NH4-N accumulation in the effluent [21].
The nitrite concentration in the effluent reached the maximum
M. Capodici, et al.
Fig. 2. Ammonium (a), nitrite (b) and nitrate (c) concentrations in relation with the C/N and the ALR applied.
values when the C/N ranged between approximately 2 and 4 (Fig. 2b).
The obtained results were in good agreement with those reported in
previous studies [27,28], in which it was observed an increase of nitrite
production when operating under C/N lower than 3. The effluent nitrite
concentration sharply decreased at C/N higher than 4, whereas it was
negligible at C/N higher than 7. The effluent nitrite concentration in-
creased with the applied ALR, reaching its maximum value
(250 mg L−1) at approximately 0.50 kg NH4-N m−3d−1 of ALR
(Fig. 2b). Based on the achieved results, it was observed that ALRs
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Biochemical Engineering Journal 149 (2019) 107229
ranging between 0.30-0.50 kg NH4-N m-3d-1 were favorable to nitrite
formation. This result was likely due to NOB inhibition because of FA
accumulation. Several studies in the literature demonstrated that AOB
are less sensitive to FA inhibition compared to NOB [6,13]. The findings
of the present study demonstrated that a stable PN could be achieved by
operating within an ALR range between 0.30–0.50 kg NH4-N m−3d−1.
Nevertheless, at ALR higher than 0.50 kg NH4-N m−3d−1, the effluent
nitrite concentration sharply decreased, indicating that also AOB ac-
tivity resulted inhibited by high FA concentration. Based on the
M. Capodici, et al.
Fig. 3. Relationship between the effluent TN concentration and the TN removal efficiency with the C/N (a) and the ALR applied (b).
ammonium concentration in the influent medium and the pH in the
mixed liquor (average value of 8.46), ALR higher than 0.50 kg NH4-N
m−3d−1 corresponded to a FA concentration of approximately
60 mg L−1 that could be sufficient to suppress the activity of both AOB
and NOB species. This value was significantly lower than that observed
in a previous study, in which the inhibition of AOB occurred at FA
concentration of approximately 120 mg NH4-N L−1 [29]. Nevertheless,
in the present study the SBR operated with a higher COD concentration,
thereby resulting in higher C/N. Therefore, it is possible that under
higher C/N, the AOB inhibition occurs at lower FA because of sy-
nergistic effects. The reason of this achievement is unclear. However, it
is possible to speculate that at high C/N, heterotrophic bacteria over-
grew over nitrifying due to oxygen and/or nitrogen competition [30],
thus reducing the resistance of AOB to FA inhibition, or that the in-
hibitory effect of FA increases not linearly with the ammonium con-
centration.
The nitrate concentration in the effluent showed opposite trends
compared with that of nitrite in relation to both C/N and ALR (Fig. 2c).
The maximum nitrate concentration in the effluent was observed in a
wide range of applied ALRs ranging between 0.10-0.30 kg NH4-N m−3d-
1
. It is interesting to note that the effluent nitrate concentration sharply
decreased at ALR higher than 0.30 kg NH4-N m−3d-1 that was the same
value at which nitrite started to accumulate in the effluent. The ob-
tained results indicated that at applied ALR higher than 0.30 kg NH4-N
m−3d−1 the nitrite in the effluent started to be the main nitrogen form
in the effluent, whereas nitrate rapidly decreased. This clearly indicated
that ALR of approximately 0.30 kg NH4-N m−3d-1 represented a
5
Biochemical Engineering Journal 149 (2019) 107229
threshold value above which it is possible to achieve the partial ni-
trification.
By comparing the relationship between the effluent nitrite and ni-
trate concentrations with the ALR, it can be noticed that the nitrate
concentration was stable as far as the applied ALR remained below
0.30 kg NH4-N m−3d−1, whereas it sharply decreased at higher ALR. In
contrast, nitrite showed a sharp increase at ALR higher than 0.30 kg
NH4-N m−3d−1 and remained stable only in a smaller range of ALR
(between approximately 0.30–0.50 kg NH4-N m−3d−1). These results
indicated that the achievement of stable PN was limited within a
narrow ALR range and that even moderate fluctuations of the influent
ammonium concentration could result in the decrease of the PN effi-
ciency, because of the nitrite oxidation to nitrate rather than the failure
in ammonium oxidation due to AOB inhibition.
When comparing the effluent nitrite and nitrate concentrations in
relation to the C/N, even in this case it arose that the C/N had an op-
posite influence to nitrite and nitrate. Indeed, at C/N higher than 4,
nitrite rapidly decreased with C/N, whereas nitrate increased although
with a lower rate. At C/N of approximately 4, corresponding to an ALR
close to 0.30 kg NH4-N m−3d−1, nitrite and nitrate coexisted
(Fig. 2b,c), even if the nitrite concentration was approximately three
times higher than nitrate. At higher C/N, nitrite were found to be sig-
nificantly lower in the effluent than nitrate. Therefore, C/N lower than
4 enabled a good PN efficiency, with a clear predominance of nitrite
over nitrate. At the same time, C/N of approximately 2–4 should pro-
vide enough organic substrate for denitritation since nitrite requires less
carbon for its reduction.
M. Capodici, et al.
In a previous study, partial nitrification was successfully achieved,
via NOB inhibition, at ALR higher than 0.80 kg NH4-N m−3d− [17] in a
SBR operating with a C/N of approximately 1 and under dissolved
oxygen limitation. However, a low value of C/N could cause poor TN
removal efficiency, because of incomplete denitritation due to organic
carbon limitation. In the present study, NOB inhibition was achieved at
lower ALR (0.30 kg NH4-N m−3d−1) and higher C/N (4), thereby in-
dicating that stable PN could be achieved without carbon or oxygen
limitation. These operating conditions could be more favorable to
achieve higher TN removal, since they could enable higher carbon
availability to ensure complete denitritation. Complete suppression of
NOB by only managing the ALR is hardly achievable. Nevertheless, the
obtained results indicated that it is possible to minimize the nitrate
production, by choosing a proper ALR and C/N, while maintaining a
sufficient C/N able to provide enough organic carbon for denitritation.
In this respect, a crucial role is played by the fraction of municipal
wastewater blended with the leachate, which not only is necessary to
adjust the C/N at the desired range, but also to provide a source of
biodegradable organic matter for denitrification or denitritation.
3.2. Overall TN performance
The main goal of the study was to find proper operating ranges in
terms of applied ALR and C/N to enable stable PN, since it is the lim-
iting step for nitrogen removal process via nitrite pathway. Although
denitritation was not the main focus of this study, total nitrogen re-
moval was evaluated as well.
Fig. 3 depicts the effluent TN concentrations and removal efficiency
as a function of the C/N and the applied ALR.
The effluent TN concentration showed an overall decreasing trend
with C/N (Fig. 3a). Indeed, it slightly decreased when C/N ranged be-
tween 2 and 3, whereas the effluent TN significantly decreased at C/N
ranging between 3 and 6. At C/N higher than 6, the TN concentration in
the effluent was constant with increasing in C/N, thereby indicating
that further increase in C/N did not enable an additional TN removal.
Moreover, the TN removal efficiency was mostly lower than 15% at C/
N < 3, whereas it started to rapidly increase at C/N ranging between 3
and 6 (Fig. 3b). The increase of TN removal with C/N was likely due to
the higher nitrogen consumption by heterotrophic bacteria for synthesis
purposes. At C/N of approximately 6, the TN removal efficiency was
approximately 70% and no further increase with C/N was observed
from this value onward. Besides, under these conditions, a further in-
crease in carbon availability could enhance the OHO growth over ni-
trifying bacteria, which could likely result in poor nitrification or ni-
tritation performances.
The effluent TN concentration and removal efficiency were also
related to the ALR. The lowest TN concentrations in the effluent were
observed when ALR resulted below 0.30 kg NH4-N m−3d−1, whereas a
rapid increase was observed at higher ALR. Accordingly, the TN re-
moval efficiency was almost 70% at ALR lower than 0.30 kg NH4-N
m−3d−1, whereas it significantly collapsed for higher ALR values.
Based on these results, at ALR higher than 0.50 kg NH4-N m−3d−1 the
TN removal efficiency was close to zero, although the prevalent ni-
trogen form in the effluent was nitrite.
As aforementioned, because denitritation was not the main focus of
this study, the operating conditions adopted could not ensure high
denitritation efficiency (a specific anoxic-mixing phase was not pro-
vided). Certainly, further studies will be necessary to go into it further.
Nevertheless, it should be reminded that at C/N higher than 4, the ni-
trite concentration in the effluent significantly decreased and, accord-
ingly, the main nitrogen form deriving from ammonia oxidation was
nitrate. Therefore, this scenario would require a high availability of
biodegradable organic carbon for denitrification that is difficult to
achieve especially in case of old leachate. Moreover, the use of external
carbon source to enhance denitrification is not advisable either tech-
nically or economically.
6
Biochemical Engineering Journal 149 (2019) 107229
Furthermore, it should be stressed that an excessive nitritation if not
supported by an adequate C/N could cause poor TN removal because of
nitrite accumulation. Therefore, the biodegradability of the organic
carbon in the feeding mixture and its effect on denitritation perfor-
mances should be investigated in further studies.
In this respect, it should be reminded that the BOD5/COD ratio of
the feeding mixture was close to 0.30 and a significant fraction of the
biodegradable organic matter was constituted by a readily biodegrad-
able substrate (i.e., sodium acetate). In contrast, in a real MWW only a
limited fraction of the total COD is rapidly biodegradable. In order to
improve the TN removal, the biodegradability of the influent mixture,
resulting from the blending of MLL and MWW, should be appropriately
balanced to obtain a sufficient amount of readily biodegradable organic
substrate (rbCOD). In general, a rbCOD/NO2-N ratio of approximately 2
would be enough to achieve complete denitritation [31]. Considering
that the amount of readily biodegradable COD in a conventional MWW
is approximately 30% of the total COD [32] and not considering that of
the leachate, based on the mixing ratios between the MLL and MWW
(20–40%), the amount of rbCOD in the feeding mixture would ranges
between approximately 20–25% of the total COD.
Therefore, bearing in mind the above considerations and knowing
that the maximum conversion rate from ammonia to nitrite observed in
this study was of approximately 0.40 (1 g NH4-N produced 0.40 g of
NO2-N), the theoretical value of the C/N that would enable complete
denitritation would result close to 3.2–4. This value falls within the
optimum range above discussed for the C/N, thus indicating by oper-
ating within a C/N close to 3.5–4, the achievement of both partial ni-
trification and complete denitritation could be successfully achieved.
3.3. Nitrifying biomass kinetics
The maximum autotrophic growth rate pattern as a function of both
C/N and ALR is depicted in Fig. 4.
The results reported in Fig. 4 indicated that low C/N, corresponding
to high ammonia concentration, were not favorable for the growth of
nitrifying bacteria, resulting in a maximum growth rate of approxi-
mately 0.40 d−1 (Fig. 4). This result was in good agreement with the
results above reported, confirming that low C/N ratio, due to high
ammonium concentrations, caused inhibition to AOB and NOB as well,
due to FA accumulation. When increasing the C/N, the maximum au-
totrophic growth rate started to increase, reaching a maximum value of
approximately 1.70 d−1 at C/N equal to 6. Bearing in mind that the
increase of C/N in the present study was related to a decrease of the
influent ammonium concentration, it is possible to speculate that the
increase of the autotrophic growth rate with C/N was representative of
the gradual recovery of NOB activity due to the decrease of FA con-
centration. Nevertheless, it is worth noting that at C/N > 7 the auto-
trophic growth rate definitively collapsed. This result was likely due to
the competition between OHO and AOB for both oxygen and nitrogen
under high carbon availability [33]. Indeed, at high C/N nitrifying
bacteria are generally overgrown by heterotrophs because of their
lower growth rate [5]. This result confirmed that high C/N is not fa-
vorable to achieve stable PN, because AOB activity could be limited
because of the competition with the OHO. The above results also in-
dicated that the maximum activity of the autotrophic bacteria, in-
cluding both the AOB and NOB, occurred for C/N ranging between 5
and 7. Based on the results discussed in Section 3.2, it is can be rea-
sonably assumed that at C/N of approximately 4 the values of auto-
trophic growth rate reported in Fig. 4 were mainly due to the AOB
growth, whereas at higher C/N also the growth of the NOB contributed
to the increase in maximum autotrophic growth rate. Therefore, a C/N
value of approximately 3–4 should be suitable to achieve the suppres-
sion of NOB only.
The relationship between the autotrophic growth rate and the ALR
is shown in Fig. 4. A sharp increase of the maximum autotrophic growth
rate was observed at ALR ranging between of 0.10 kg NH4-N m−3d−1
M. Capodici, et al.
Fig. 4. Relationship between autotrophic growth rate with the C/N and the ALR applied.
and 0.30 kg NH4-N m−3d-1. The maximum value of the autotrophic
growth rate (1.80 kg NH4-N m−3d−1) was observed at ALR of 0.30 kg
NH4-N m−3d−1. As aforementioned, this ALR value represented a
threshold value for autotrophic bacteria activity. Indeed, both the ni-
trite and nitrate coexisted at this ALR. However, as previously dis-
cussed, the nitrite and nitrate curves showed a knee at this value
(Fig. 2b,c), indicating that at higher ALR, nitrites were prevalent over
nitrates. Accordingly, the maximum autotrophic growth rate decreased
at ALR higher than 0.30 kg NH4-N m−3d−1 likely because of the lower
growth of the NOB, reaching its minimum value at an applied ALR of
0.50 kg NH4-N m−3d−1.
The above results confirmed that ALR higher than 0.30 kg NH4-N
m−3d−1 was necessary to achieve PN via NOB suppression. Moreover,
C/N higher than 6 was not favorable to autotrophic growth, suggesting
that an excess of organic carbon, although theoretically favorable for
denitrification, could significantly affect the PN efficiency and conse-
quently the overall TN removal performance. A C/N ratio of approxi-
mately 3–4 should enable the selection of AOB while suppressing NOB.
3.4. COD removal
Fig. 5 depicts the removed OLR as function of the applied OLR
(Fig. 5a) as well as the effluent COD, the EPS and SMP concentrations
and the OHO growth rate in relation with C/N (Fig. 5b–d).
The relationship between the applied and the removed OLR
(Fig. 5a) indicated that the COD removal efficiency was approximately
85% up to an OLR of almost 2.0 kg COD m−3d−1, whereas it started to
decrease at higher OLR. Nevertheless, within the range of applied OLR
tested in this study, the SBR was able to achieve COD removal higher
than 70%. The effluent COD concentration decreased according to the
C/N increase (Fig. 5b). Notably, the relationship shown in Fig. 5b in-
dicated that at C/N lower than 4 the effluent COD concentration sig-
nificantly increased even with small C/N decreases. The reason of this
result was likely due to the increase of SMP concentration observed at
low C/N (Fig. 5c). Under low organic carbon availability, especially in
the case of slowly biodegradable substrates, bacteria use alternative
carbon sources to support their metabolic activity [34]. EPS represent
an abundant carbon stock for bacteria that, being mainly constituted by
proteins and carbohydrates, represent a carbon and energy source. The
EPS biodegradation requires a prior hydrolysis step leading to the for-
mation of the SMP. Indeed, as shown in Fig. 5c, the trends of SMP and
EPS were inversely proportional, thereby indicating that SMP derived
7
Biochemical Engineering Journal 149 (2019) 107229
from EPS hydrolysis. Based on these results, it is possible to speculate
that at low C/N, bacteria used EPS as carbon source, thus resulting in
the highest SMP and the lowest EPS concentrations. In contrast, at
higher C/N, the EPS hydrolysis did not occur, thus resulting in the
highest EPS and the lowest SMP concentrations.
Generally, an excess in SMP concentration is representative of ex-
tended famine condition or harsh environmental condition [35,36].
Therefore, this situation is indicative of metabolic stress conditions for
bacteria. In this light, the obtained results indicated that a C/N sig-
nificantly lower than 4 caused stress conditions for the biomass, likely
due to carbon limitation for the heterotrophic growth, that caused the
EPS hydrolysis and accumulation of SMP in the effluent. Indeed, as
shown in Fig. 5d, the OHO growth rate resulted lower (2 d−1) as far as
the C/N decreased, thereby suggesting that C/N lower than 4 were not
favorable to achieve an adequate heterotrophic population to enable
high COD removal efficiency. Conversely, the OHO growth rate ex-
ponentially increased at C/N higher than 6, indicating that at high C/N
the OHO activity was very high. This result could explain the reason
why the autotrophic growth rate collapsed at C/N higher than 6, likely
because of the OHO overgrowth.
4. Conclusions
The effects of the ALR and C/N on partial nitrification performances
were studied in a SBR treating landfill leachate with municipal waste-
water.
The achieved results indicated that the effluent nitrogen forms were
strictly dependent on the applied ALR as well as the influent C/N ratio.
A proper balancing between ALR and C/N was found to be crucial in
order to achieve stable PN efficiency. The results suggested the ex-
istence of an optimal operating range for both the ALR and C/N of
approximately kg 0.30–0.50 NH4-N m−3d−1 and 2–4, respectively.
More precisely, stable PN was achieved at ALR ranging between
0.30–0.50 kg NH4-N m−3d−1 and C/N between 4–6. Higher ALR caused
a significant decrease in PN because of the inhibition of AOB due to FA
formation, whereas C/N higher than 6 were not favorable to the au-
totrophic growth, because of competition with OHO. Although the
complete suppression of NOB was not successfully achieved, at the
aforementioned values, a clear predominance of nitrite (125 mg L−1)
compared to nitrate (50 mg L−1) in the effluent was observed.
Therefore, the results demonstrated that the achievement of PN is
possible even at high C/N, which is a necessary condition for obtaining
M. Capodici, et al.
Fig. 5. Relationship between the applied and the removed OLR (a); trend of the effluent COD concentration (b), the EPS and SMP (c) and the OHO growth rate (d)
with the C/N.
complete denitrification. Lastly, operating under C/N significantly
lower than 4 caused a stress condition for the biomass, likely do to
carbon limitation for the heterotrophic growth, leading to an excess in
SMP production.
As final remark, the results of this study demonstrated that by
choosing a proper C/N, by balancing the ratio between the MLL and
MWW, and the ALR, by regulating the SBR operating parameters, as the
volumetric exchange ratio or the cycle duration, PN could be success-
fully achieved for the co-treatment of leachate with municipal waste-
water. Moreover, the operating conditions that allowed stable PN were
the same that theoretically could enable high denitrification efficiency
(C/N > 4) and hence total nitrogen removal performance.
Acknowledgements
Authors thank Risorse Ambiente Palermo (RAP) S.p.A. and in par-
ticular Eng. Andrea Modesto for the technical support. Authors warmly
thank Eng. Francesco Paladino and Eng. Roberto Camarda for their
precious and essential help with pilot plant operations.
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