Bioresource Technology 361 (2022) 127702

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Simultaneous nitrification–denitrification in biofilm systems for wastewater

treatment: Key factors, potential routes, and engineered applications
Francesco Di Capua a, *, Francesca Iannacone b, Fabrizio Sabba c, Giovanni Esposito d
a
Department of Civil, Environmental, Land, Building Engineering and Chemistry, Polytechnic University of Bari, Bari 70125, Italy
b
Acqua & Sole s.r.l., Via Giulio Natta, Vellezzo Bellini, PV, Italy
c
Black & Veatch, KS, United States
d
Department of Civil, Architectural and Environmental Engineering, University of Naples Federico II, Via Claudio 21, Naples 80125, Italy

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Simultaneous nitrification–denitrification
(SND) is emerging for water treatment.
• Shortcut SND can cut organic and en­
ergy consumption as well as CO2 emis­
sion by >20 %.
• Nitrous oxide emission can be reduced
by adjusting operational and physical
factors.
• Moving bed and aerobic granular bio­
films are mature technologies for effi­
cient SND.
• SND biofilms can enable the combined
removal of nitrogen and phosphorus.

A R T I C L E I N F O A B S T R A C T

Keywords: Simultaneous nitrification–denitrification (SND) is an advantageous bioprocess that allows the complete removal
Simultaneous nitrification denitrification of ammonia nitrogen through sequential redox reactions leading to nitrogen gas production. SND can govern
Biofilm nitrogen removal in single-stage biofilm systems, such as the moving bed biofilm reactor and aerobic granular
Nitrous oxide
sludge system, as oxygen gradients allow the development of multilayered biofilms including nitrifying and
Moving bed biofilm reactor
Aerobic granular sludge
denitrifying bacteria. Environmental and operational conditions can strongly influence SND performance, bio­
film development and biochemical pathways. Recent advances have outlined the possibility to reduce the carbon
and energy consumption of the process via the “shortcut pathway”, and simultaneously remove both N and
phosphorus under specific operational conditions, opening new possibilities for wastewater treatment. This work
critically reviews the factors influencing SND and its application in biofilm systems from laboratory to full scale.
Operational strategies to enhance SND efficiency and hints to reduce nitrous oxide emission and operational costs
are provided.

* Corresponding author.
E-mail address: francesco.dicapua@poliba.it (F. Di Capua).

https://doi.org/10.1016/j.biortech.2022.127702
Received 1 July 2022; Received in revised form 20 July 2022; Accepted 22 July 2022
Available online 26 July 2022
0960-8524/© 2022 Elsevier Ltd. All rights reserved.
F. Di Capua et al.
1. Introduction
Nitrogen (N) is an essential nutrient for all living forms (Chrispim
et al., 2019). However, excessive discharge of N in surface waters can
determine an abnormal growth of algae and, consequently, a depletion
of dissolved oxygen (DO) (Butcher, 2016). This phenomenon, known as
eutrophication, represents a serious environmental problem affecting
water quality in rivers, lakes, and estuaries around the world. Phos­
phorus (P) can also contribute to eutrophication by increasing higher
primary production in superficial water bodies. The main sources of N
and P in surface waters are represented by the run-off from agricultural
land and wastewater discharge from households and industry (van
Puijenbroek et al., 2019). Biological N removal (BNR) can effectively
remove N compounds from wastewaters and is recognized as the most
economical process to reach the stringent discharge limits set by the
national legislations (He et al., 2020). Similarly, P can be removed
biologically or chemically, the former being more complex to implement
but also eco-sustainable, the latter being generally easier to apply but
more expensive and less eco-friendly (Di Capua et al., 2022).
Simultaneous nitrification–denitrification (SND) is capable to
completely remove N in a single bioreactor under specific operating
conditions, which differentiates this process from sequential nitrifica­
tion and denitrification typically carried out in separate bioreactors at
municipal wastewater treatment plants (WWTPs). Up to date, many
researchers have studied the underlying mechanisms and the effects of
various environmental factors and operating conditions on SND and its
combination with P removal, including the feed carbon-to-nitrogen (C/
N) ratio, DO level, hydraulic retention time (HRT) and aeration pattern.
Recent studies show that biofilm technologies can effectively promote
SND as well as the combined removal of N and P under specific condi­
tions (Iannacone et al., 2021; Roots et al., 2020). However, critical
evaluation and comparison of these technologies are needed to point out
their strengths and weaknesses and promote SND application at full
scale.
This work discusses the fundamental mechanisms behind the SND
process and provides a comprehensive and critical overview of SND
applications in biofilm reactors. Advantages and criticalities of each
technology are evaluated, and guidelines for a proper selection of
reactor and operating conditions to implement a successful SND process
are provided. Knowledge gaps and research needs are identified with the
objective to unravel the potential of SND in biofilm reactors.
2. Overview of nitrogen removal bioprocesses
Nitrification is a biological process carried out under aerobic con­
ditions and typically consists of two consequential oxidation steps. In the
first step, ammonia oxidizing bacteria (AOB) oxidize ammonium (NH+
to nitrite (NO–2) by consuming 1.5 mol of O2 per mol of N. In this reac­
tion, the enzymes ammonia monooxygenase (AMO) and hydroxylamine
oxidoreductase (HAO) are involved, along with hydroxylamine
(NH2OH) as an intermediate product. During the second step, nitrite
oxidizing bacteria (NOB) use the enzyme nitrite oxidoreductase (NXR)
to convert NO–2 to NO–3 and consume 0.5 mol of O2 per mol of N (Rahimi
et al., 2020). AOB and NOB typically use only inorganic carbon sources,
consuming almost 7.1 mg CaCO3 for each mg N-NH+ 4 oxidized to N-NO3
(Rittmann and McCarty, 2012). Besides AOB and NOB, bacteria capable
of complete NH+ 4 oxidation to NO3, i.e., Comammox, have been
–
discovered in 2015 (Van Kessel et al., 2015) and found to be the
dominant nitrifying microorganisms in mainstream low-oxygen nitrifi­
cation reactors (Roots et al., 2019). Denitrification involves the
sequential reduction of NO–3 to NO–2, nitric oxide (NO), nitrous oxide
(N2O), and N2. This process is typically carried out under anoxic
Bioresource Technology 361 (2022) 127702
conditions by denitrifying bacteria (DNB) using organic carbon as en­
ergy source and producing alkalinity.
Both nitrification and denitrification produce N2O, a potent green­
house gas, as a process intermediate. During nitrification, N2O may be
produced by AOB via two main pathways: reduction of NO–2 as terminal
electron acceptor to N2O (pathway known as nitrifier denitrification)
and incomplete oxidation of hydroxylamine (NH2OH), which is first
chemically decomposed to NO, and then biologically reduced to N2O
(hydroxylamine pathway) (Sabba et al., 2015). Similar to nitrification,
incomplete denitrification can also lead to the accumulation and emis­
sion of N2O as a process intermediate (Zhou et al., 2022a). Differently
from nitrification, N2O can be produced but also consumed during
denitrification (Sabba et al., 2017b).
BNR in WWTPs is generally achieved via a pre-denitrification cycle,
where heterotrophic denitrification is followed by an aerobic phase for
the combined oxidation of organics and NH+ 4 (Sun et al., 2010). How­
ever, this approach is considered energy-demanding, especially due to
aeration, and economically unsustainable for the treatment of waste­
water produced in small communities or rural areas due to large struc­
tural footprints and energy cost, blower deterioration and maintenance,
and high sludge production (Yan et al., 2019a). In recent years, novel
biological processes, i.e., anaerobic NH+4 oxidation (anammox) and SND,
have been increasingly investigated with the aim to reduce the WWTP
operating costs while ensuring compliance with N discharge limits in
water bodies. Anammox bacteria (AnAOB) oxidize NH+ 4 directly to N2
under anoxic conditions, obtaining energy for growth from the oxidation
of NO–2 to NO–3 (Hu et al., 2019). NO–2 can be produced by partial nitri­
fication (nitritation) of NH+ 4 or through partial denitrification (deni­
tritation) of NO–3. Despite the various advantages attributed to anammox
in terms of reduced energy consumption and sludge production (Rahimi
et al., 2020), anammox application to mainstream wastewater treatment
is limited by 1) the low maximum specific growth rate of AnAOB
(0.05–0.33 d− 1) (Zhang et al., 2017a), 2) excess discharge of NO–3 in the
effluent, 3) high operational temperatures (i.e., 30–40 ◦ C), and 4) the
need for a strict control of temperature, pH, and organic concentration
for optimal AnAOB growth.
SND represents a suitable and promising alternative to pre-
denitrification in WWTPs for the simultaneous removal of C and N as
it offers several advantages: (1) the carbon demand and sludge pro­
duction are reduced by over 30 % (Ma et al., 2017), (2) alkalinity supply
by denitrification helps to maintain a circumneutral pH, (3) there is no
need for NO–3 recirculation, (4) lower energy for aeration (Zinatizadeh
and Ghaytooli, 2015), and (5) a smaller footprint is required (Seifi and
Fazaelipoor, 2012). Potential disadvantages of the SND process include
(1) lower N removal compared to separate denitrification and nitrifi­
cation, (2) significant N2O accumulation, and (3) process instability due
4) to competition among the different microbial families coexisting in the
system. In the last 30 years, the SND process has gained significant
attention from the scientific community and WWTP operators. Biofilm
technologies have been increasingly developed and applied in full-scale
WWTPs as they offer several advantages over suspended-growth sys­
tems: (1) higher biomass concentration, (2) lower space requirements,
(3) reduced HRT and sludge production and (4) more stable perfor­
mance (Zhao et al., 2019). In addition, biofilm systems allow the coex­
–
istence of a diverse and complex microbial community involved in
nutrient removal, which makes these systems perfectly suitable for the
SND process. The most utilized biofilm-based technologies applied for
SND include the moving bed biofilm reactor (MBBR) (Tables 1 and 2),
hybrid biofilm-membrane bioreactor (HMBR) systems, and aerobic
granular sludge (AGS) (Table 3).
2
F. Di Capua et al. Bioresource Technology 361 (2022) 127702

3. Factors influencing simultaneous nitrification–denitrification

Zinatizadeh and Ghaytooli (2015)

in biofilms

Sandip and Kalyanraman (2019)

3.1. Environmental factors

Chu and Wang (2011)

Zhang et al. (2017b)
pH and temperature can influence both the efficiency and pathway

Song et al. (2019)

Feng et al. (2012)

Liu et al. (2020a)
Liu et al. (2020a)

Gu et al. (2018)

Fu et al. (2010)
of nitrification. AOB can outcompete NOB at temperatures >25 ◦ C,
while NOB grow much faster at lower temperatures (Liu et al., 2021;
Filling ratio (%) HRT (h) DO (mg L-1) Carbon source Feed TN (mg L-1) Feed C/N COD RE (%) TN RE (%) Reference

Sabba et al., 2022). Optimal pH levels are reported in the range of

8.2–8.4 for AOB and 7.2–7.9 for NOB (Gu et al., 2012). Therefore,
relatively high temperature and pH values can help AOB to outcompete
NOB and maintain partial nitrification, being required for shortcut SND
62–76
45–72
45–78
43–62
34–42
47–59

85–95
26–46
26–50
25–54
20–55

26–51
(see Section 4). It should be noted that AOB and NOB consume alkalinity
≤30
84
75

59
due to release of H+ during nitrification (Rittmann and McCarty, 2012).
In SND systems, acidity production by nitrification is counterbalanced
by the supply of alkalinity due to denitrification, which helps to keep a
79–86

86–91

50–88
54–85

94–96
>94c
>94c

circumneutral pH.
<90
94c

90c
72c
85
81
83

81
–

3.7b, 8.1b
3.7b, 8.1b
4.5–13.4

3.2. Operational parameters

~11b
≤ 5b
≤ 5b
5-9b
5-9b

6.3b
3.5
3.5

7.1
5b
5b
5
5

3.2.1. Dissolved oxygen concentration

DO concentration can significantly impact microbial activity and
community composition within the biofilm. SND is based on the co-
existence of AOB, NOB and DNB in the biofilm, achieved through DO
40 ± 10
40 ± 10

25(±3)
44(±5)
44(±5)
30–70
30–70

28–33
28–33

concentration gradients generated by diffusional limitations (Liu et al.,

100
40
40

51
16

2020a) (Fig. 1). The DO concentration in the bulk liquid can be regu­
–
–

lated to create gradients determining the formation of an anoxic micro-

BOD5 Glucose
BOD5 Glucose

environment within the biofilm where denitrification can occur, while

Glucose
Glucose
Glucose
Glucose

Glucose
Glucose

Glucose
Glucose
Glucose

nitrifying bacteria can thrive in the external region where DO is more

BOD5
BOD5

available (Massoompour et al., 2020).

–
–

The suitable DO concentration reported for maintaining SND varies

over a wide range (0.8–7.0 mg L− 1) and is affected by several factors
0.6–0.8
0.6–0.8

5.0–6.5
5.0–6.5

5.0–6.5
2.0–4.0
2.0–4.0
4.0–6.0

2.0–4.0

that include carbon source availability, reactor configuration and

0.75
0.75
3.0
3.0

operation, biofilm thickness, granule size and carrier type (Tables 1–3).
–

–
–

The concentration of DO in the bulk liquid affects its penetration depth

SND performance and operating conditions of continuous-flow MBBRs treating municipal wastewater.

in the biofilm. The set DO concentration must ensure a balance between

4–12
4–12
5–7
12
12
10
12

14
14
10

the bacterial communities involved in SND and between the production

8
8
8
8
5
5

and consumption of NOx. High DO concentration in the bulk liquid can

limit the formation of the anoxic zone or push the zone deeper, while
low DO concentrations can limit nitrification (Layer et al., 2020; Wang
20–40
20–40

10–30

20–40

et al., 2020a). Cao et al. (2017) investigated the effect of DO concen­

16.7
30
30
35
35

10
10
30

50
50

20

30

trations ranging from 1.5 to 5.5 mg L− 1 in five identical moving bed

PE–PQAS 10–Fe2O3

PE–PQAS 10–Fe2O3

sequencing batch reactors (MBBR-SBRs) (Table 2). After an acclimati­

PU–zeolite (SiO2)

zation period, the highest total nitrogen (TN) removal efficiency (RE)
was observed at a DO concentration of 2.5 mg L− 1 (84 %), while COD RE
Specific surface (m2 m-3) Materials

was >90 % in all systems. Higher DO concentrations (3.5–5.5 mg L− 1)

inhibited denitrification, while the relative abundance of AOB and NOB
PCL
PU

PU

PU

PU
PU
PE

PE
PE

PE
PE

PE
–

increased. Lower DO concentration (1.5 mg L− 1) limited nitrification,

resulting in the lowest relative abundance of AOB in the biofilm and in a
significant increase of N-NH+ 4 concentration in the effluent (up to 13.8
mg L− 1).
In continuous-flow MBBRs, promising SND performances have been
measured as total organic carbon (TOC) RE.

achieved under microaerobic conditions, i.e., DO levels ≤1.0 mg L− 1

560–600
560–600
560–600
560–600

0.846a
0.846a

0.346a
1200

1120

(Table 1) (Iannacone et al., 2019; Liu et al., 2020a; Liu et al., 2021; Liu
500
280

500
500

900

500

et al., 2020b). Iannacone et al. (2019) obtained TN REs up to 68 % in a

–

continuous-flow MBBR at stable DO concentration of 1.0 ± 0.2 mg L− 1.

Liu et al. (2020a) investigated the effects of three different DO con­
Zeolite powder-based PU sponges

centrations (2.5 ± 0.5 mg L− 1, 1.5 ± 0.5 mg L− 1 and 0.8 ± 0.3 mg L− 1)

in a continuous-flow MBBR, demonstrating that DO levels <1.0 mg L− 1
available as COD/N.
reported as m g .
Biodegradable polymer

were beneficial to SND (TN RE up to 53 %) with high N-NH+ 4 RE (85 %).

2 − 1
Cubic-shaped sponges

Polyethylene carriers
Conventional carrier

Conventional carrier

At DO levels >1.5 mg L− 1, TN RE decreased to almost 30 % due to

Sponges biocarriers

limited denitrification activity. However, under oxygen-limited condi­

Novel carrier

Novel carrier
Carrier type

tions, heterotrophic aerobic bacteria (HAB) may outcompete nitrifiers

Bio-carriers

PUF carrier
PU carrier
Kaldnes-3
PE plastic
PU foam

due to the lower growth rate and affinity for O2 of AOB and NOB
Ring R2
Table 1

compared to HAB (Jia et al., 2020). Slow adaptation of nitrifiers to low

b
a

O2 conditions could be required to avoid suppression of NH+ 4 oxidation

3
 F. Di Capua et al.
Table 2
SND in MBBR-SBR systems.
Carrier type Specific Materials Filling Cycle AN O phase A phase Settling Idle Discharge Range Carbon Feed TN Feed C/ COD TN RE Reference
surface ratio Length phase (min) (min) (min) (min) (min) DO (mg source (mg L-1) Nb RE (%)
(m2 m-3) (%) (min) (min) L-1) (%)

PU foam – PU 30 – – 600 – – 120 – 1.5–5.5 (C6H10O5)n 25 ~12 92 42–85 Wang et al.

(2020a)
a
AC CBMC 2.70 AC, PE 40, 55 360 – 350 – – – 5 6.0 Acetate 55 5 82–94 68–88 Massoompour
Conventional 0.2478a PE 40, 55 360 – 350 – – – 5 6.0 Acetate 55 5 82–94 59–76 et al. (2020)
carrier
Combined – PP – – 175 90 175 30 – 5 2.0 Glucose 100 3.8 94 73 Chai et al.,
fibers (2019)
Novel carrier 650 PQAS-10 30 240 – 200 40 – – – 1.0–1.5 – 50 5 81–84 78–80 Liu et al.,
+ Fe2O3 (2018)
+ PE
PUF – PU 30. 600, – – – – – – 0.5–1.5 Acetate 89 ~4.5 85 60 Chen et al.
480 (2018a)
HX9KL, 500 PE 40, 60 240, – 90, 50 150, 90 0, 10 – 0, 3 0.8–1.1 – 35(±7) 5.5–6 78–95 50–76 Ferrentino
BIOMASTER 360 47(±6) et al. (2018)
BCN 012KLS
4

polyurethane 382 PU + rice 15.8 720 – 240, 60 120–480 – – 30 0.1–2.0 Acetate, 35(±4) 1.8–7.6 64–83 41–89 Feng et al.,
filler + with husk yeast, (2018)
rice husk sucrose
Foam carrier – PU 30 720 – 600 – – 120 – 1.5–5.5 (C6H10O5)n 22–31 ~10.2 > 90 42–84 Cao et al.
(2017)
Plastic fiber – – 12 720 50 190–270 380–460 – – – 1.5–2.2 Glucose 50 1–4 50–90 83–98 Ge et al. (2017)
Cylindrical – PP 30 – 90 210 90 30 40 – 0.2–2.5 – 35 11.4 95 94 Yin et al.
carrier (2015)
PU foam 1621 PU – 360 – 297 – 50 – – 0.1–0.9 Glucose 65–227 1.8–10.5 – 55–79 Tan et al.,
2013)
PU foam –. PU 8 – 60 60 – 90 570 – 0–7 Peptone, 48 5 65 57–100 Lim et al.,
sucrose, (2012)
acetate
Bio–carrier K3 500 PE 30 480 60 240 120 30 – – – Acetate 35 15.7 – 71.5 Lo et al., (2010)

AN = Anaerobic phase.
O = Aerobic phase.

Bioresource Technology 361 (2022) 127702

A = Anoxic phase.
a
reported as m2g− 1.
b
available as COD/N.
 F. Di Capua et al.
Table 3
SND applications in AGS systems.
Cycle length (h) Cycle AN O (min) A (min) Settling Discharge Granule H/D DO (mg L–1) (O) Carbon source Feed TN Feed C/N COD RE TN RE Reference
(min) (min) (min) diameter (mm) (mg L–1) (%) (%)

3–6 AN–O 20–60 30 – 250 – 13.3–25 3.3–6.7 1.5 18.75 1.0–2.5 BOD5 31–34 ~5a 69–84 31–71 Campo et al. (2020)
6 AN–O–A 120 90 144 2 – – – – Acetate, Succinate (1:1, 20 ~10a 90 ~80 He et al. (2020)
1:3, 3:1)
5.6 AN – O 90 240 – 5 1 > 0.3 8.4 2 Acetate – propionate – 30–43 11–26a 83–93 45–77 Layer et al. (2019)
BOD5
4 AN–A–O, 0–45–55 104–220 0–30 10 – 0.2 10 8–10 Acetate, propionate 100 1.6–6.5 91–98 19–75 Pishgar et al. (2019)
AN/O, O
6 AN–O 54 270–285 – 20–5 1 1.5–0.7 10 – Acetate, Glucose, 110 ~ 6a 87–96 34–78 Rollemberg et al. (2019)
Ethanol
a
6 AN–O–A 120 90 144 2 – – 5.2 7–8 Acetate 2–55 4–20 94–95 75–95 Wang et al. (2018)
3.6 O – 150 – 20 40 0.09–0.4 – 2 BOD5 91 ~14.7a 92 87 Piotr and Cydzik-
kwiatkowska (2018)
6 AN–O–A 120 90 144 2 – – 5 5 Acetate 20 ~10a 89.2 88.5 He et al. (2017c)
6 AN–O–A 120 90 144 2 – – 5 0.8–1.2 Acetate, glucose 20 ~10a 91–81 94–81 He et al. (2017a)
6 AN–O–A 120 90 144 2 – 1.4 ± 0.6 1.8 ± 5 3.5–4.5 1.7–2.3 Acetate 20 ~10a 95 64–94 Yan et al. (2019b)
0.9 0.8–1.2
6 AN–O–A 120 90 144 2 – – 5 5 Acetate 19 10.7a 94 94 He et al., (2016b)
8 AN–O–A 180 180–140 90–148 20–2 – 1.0–0.6 6.3 5 Acetate 40–50 ~7a 80–90 88–97 He et al. (2016a)
8 AN–O–A 180 180–150 90–138 20–2 – 1.2 5.2 5 Acetate 15 ~10a 87 97 He et al. (2016c)
6 AN–O 130 160 – 60 – 1.6–0.3 4.2 0.8–1.6 Acetate 50 ~16a – 65 Lu et al. (2016)
4 O – 180 – 5 2 0.5–1.7 8 > 4.0 Acetate 250–330 ~1a 100 33–77 Yan et al. (2016)
4 AN–O 10–20 184–219 – 35–10 – 0.5 9.7 – BOD5 83 ~7a 92 61 Wagner et al. (2015)
5

6.5–3 AN–O–A – 60–300 – 30 – >0.2 – 1.8–2.5 BOD5 50 ~ 10a 87 86 Pronk et al. (2015)
3 O – 162–167 – 2–7 6 > 1.5 11.1 7–8 Acetate 77–154 8–16 > 90 > 90 Di Bella and Torregrossa
(2013)
3–4 O – 164–224 – 15 0.5 0.3–0.7 22.2 – BOD5 76–91 4.7–11.2a 78 – 92 66–97 Wagner and Helena
(2013)
3 AN–O 60 112 – 3 5 0.9–1.1 – 20–80 % of Propionate 50 8–1.2a 100 40–95 Lochmatter et al. (2013)
saturation
a
6 AN–O–A 45–100 35–160 3–40 2–40 – 0.2 – 2 BOD5 + Acetate 67 ~5 100 75–84 Coma et al. (2012)
5.4 O – 114 – 3 3 2.4 7.2 6–8 Sucrose, acetate, 40 ~ 10a >80 >75 Isanta et al. (2012)
propionate
4 A–O – 213 10 2 5 1.5–0.7 20 2 – 60 ~ 10a 80–85 68–96 Chen et al. (2011)
4 O – 130–167 – 8–45 5 0.8 5 8 BOD5 60 ~ 16.7a 80 50 Liu et al. (2010)
2.5–4 A–O 90 240 5 – – – 3.2 2–3 Acetate–BOD5 43–147 0.4–2.3 64–89 47 Wang et al. (2009)
– AN–O–A 30 90 30 – – > 1.0 – – Acetate 60 2.5 ~ 100 ~ 100 Kishida et al. (2008)
6 AN–O 90 120 – 0.5 – 1.0 – 2–5 Acetate 60 ~ 10a ~ 100 ~ 100 Kishida et al. (2006)\
3 O – 169 – 3 5 1.0 – 10–100 % or Acetate – 8.3a – 8–45 Mosquera-Corral et al.
saturation (2005)

Bioresource Technology 361 (2022) 127702

4–6 O, AN–O, 0–120 230 0–120 2 4 0.4–1.9 13.3 0.5–2.0 Ethanol 25–150 3–5a 92 20–40 Yang et al. (2003)
AN–A

AN = Anaerobic phase.
O = Aerobic phase.
A = Anoxic phase.
a
available as COD/N.
F. Di Capua et al.
Fig. 1. Schematics of a co-diffusional combined nitrifying and denitrifying
biofilm and the parameters affecting N2O emissions.
(Iannacone et al., 2019). Hence, DO must be strictly controlled during
reactor start-up and continuous operation to sustain nitrification and
maximize TN RE.
In AGS systems, DO concentrations in the range of 1.5–3.0 mg L− 1
are generally applied to enhance SND performance (Bengtsson et al.,
2018; Layer et al., 2020). The long-term stability of AGS is sustained by
high shear forces typically provided by aeration, which also determines
nitrification and denitrification efficiencies (He et al., 2019a; Yan et al.,
2019b). Yan et al. (2019b) studied the effect of three DO concentration
ranges (6–7, 4–5, 2–3 mg L− 1) on AGS performances and dynamic
changes in sludge particle size. With the decrease of DO concentration
and air shear force the percentage of particle size with a diameter < 0.5
mm increased by 0.9–6.4 %, indicating that operation at low DO values
led to partial break-up of the granules.
Intermittent aeration (IA) can be a suitable choice for the SND pro­
cess, as it can promote the activities of both nitrifying and denitrifying
bacteria. In addition, IA can significantly limit the operational costs for
wastewater treatment, as aeration accounts for >50 % of the energy
costs of a municipal WWTP (Moura et al., 2012). Iannacone et al. (2021;
2020) demonstrated that IA can effectively sustain both complete and
shortcut SND in continuous-flow MBBRs treating synthetic municipal
wastewater, obtaining a nearly 90 % TIN removal at DO concentrations
ranging between 0.2 and 3.0 mg L− 1.
3.2.2. Feed carbon-to-nitrogen ratio
Municipal wastewaters are generally characterized by low C/N ra­
tios, which may result in low TN removal due to lack of organic electron
donor for denitrification (Campo et al., 2020; Chai et al., 2019). As a
result, additional organics such as methanol, glucose or sodium acetate
may be required (Chai et al., 2019; Han et al., 2018). Likewise, low SND
performance in biofilm systems is often linked to the lack of organic
carbon for denitrification (Layer et al., 2020; Wang et al., 2020b). The
outer portion of the biofilm is generally occupied by HAB, being more
6
Bioresource Technology 361 (2022) 127702
exposed to O2. Therefore, HAB have a competitive advantage for
consuming the organic compounds from the bulk liquid over DNB,
which occupy the inner anoxic portion of the biofilm. During diffusion
from the bulk liquid across the biofilm, a large amount of organics is
consumed by HAB, while the residual organic carbon may not be suffi­
cient to allow denitrification. Wang et al. (2018) showed that decreasing
the COD/TN ratio from 20 to 4 in an AGS-SBR system reduced the TN RE
of SND from 95 % to around 75 % due to carbon deficiency.
Possible strategies to overcome the lack of carbon in real municipal
wastewater include (1) the addition of liquid carbon sources, such as
ethanol, methanol or acetic acid and (2) the addition of a solid carbon
source, such as biodegradable polymers (BDPs) (Liu et al., 2021). BDPs
include poly-3-hydroxybutyric acid (PHB), polycaprolactone (PCL),
polylactic acid (PLA) and poly butanediol succinate (PBS) that can be
hydrolyzed by extracellular enzymes secreted by specific microorgan­
isms. Their hydrolysis products can then be utilized by HAB and DNB as
sources of organic carbon (Chu and Wang, 2011; Han et al., 2018). BDPs
are receiving increasing attention as organic supplement at WWTPs,
since they are slow-release electron donor/nutrient sources that can also
be used as biofilm carriers (Table 2). Low-cost alternatives to BDPs are
natural biopolymers such as starch or lignocellulose. Feng et al. (2018)
used a biodegradable support made of rice husk and lignocellulosic
materials mainly composed of biodegradable cellulose, branched
hemicelluloses and recalcitrant lignin for SND in a sequencing batch
biofilm reactor. TN RE > 50 % was obtained at a feed COD/N ratio of
2.8, while lower REs were obtained at a COD/N ratio of 1.8.
Although positive effects have been highlighted, the increase of feed
C/N ratio in SND systems should be controlled. Several studies pointed
out that excess organic carbon in the feed may limit nitrification effi­
ciency. Indeed, HAB overgrowth can limit DO diffusion within biofilm
and was shown to inhibit nitrifying activity (Iannacone et al., 2019).
4. Shortcut simultaneous nitrification–denitrification
Shortcut SND (also known as SND via NO–2 or simultaneous partial
nitrification denitrification, SPND) involves the oxidation of NH+
4 to NO2
–
by AOB followed by the reduction of NO2 to N2 by DNB. Therefore, in
–
this process nitritation is followed by denitritation. This showcases
shortcut SND as an advantageous bioprocess compared to SND for the
treatment of wastewaters with low C/N ratio, as it requires 40 % less
carbon for denitrification in addition to 25 % lower O2 consumption in
the aerobic phase, up to 2-fold faster N-NO−x reduction, 20 % lower CO2
emission and 33–55 % lower biomass production (Peng and Zhu, 2006;
Rahimi et al., 2020). NO–2 reduction rate is reported to be 1.5–2 times
higher than NO–3 reduction, which leads to faster N removal (Rahimi
et al., 2020).
4.1. Strategies for inhibition of nitrite oxidizing bacteria
To allow the growth of AOB and simultaneously inhibit NOB activity,
it is essential to maintain specific operational conditions. According to
the literature, selective inhibition of NOB can be achieved at pH > 7.5,
DO levels in the range of 1–2 mg L− 1, SRT < 5 days, temperature >
25 ◦ C, free ammonia (FA) and free nitrous acid (FNA) concentrations of
0.1–5 and 0.011–0.22 mg N L− 1 in respective order, and by alternating
aerobic and anoxic conditions (Iannacone et al., 2021).
4.1.1. High free ammonia and free nitrous acid concentrations
The concentrations of FA and FNA in the bulk liquid depend on those
of NH+ 4 and NO2, respectively. High FA levels can lead to proton
–
imbalance, potassium deficiency, change of intracellular pH, increase of
energy requirement and inhibition of enzymatic reactions (Di Capua
et al., 2021). FNA has been indicated as an uncoupler affecting energy
generation and being capable of inhibiting the expression of enzymes
such as NO–2 and NO reductases (Zhou et al., 2011). The inhibitory
thresholds for AOB and NOB are different, NOB being more sensitive
F. Di Capua et al.
than AOB to both FA and FNA. AOB are inhibited by FA in the range of
10–605 mg N L− 1, while values between 0.1 and 5 mg N L− 1 are suffi­
cient to inhibit NOB (Liu et al., 2020b). FNA concentrations that inhibit
AOB vary between 0.42 and 1.72 mg N L− 1, while NOB inhibition was
observed already at concentrations of 0.011–0.07 mg N L− 1. FNA con­
centrations of 0.026–0.22 mg N L− 1 were reported to completely inhibit
NOB activity (Jia et al., 2020; Liu et al., 2020b). Exposing NOB to
inhibitory levels of FA and FNA is not an easy task to achieve since the
concentration of N-NH+ 4 and pH depend on the influent wastewater.
Additionally, NO–2 accumulation in the system and subsequent discharge
should be avoided due to high NO–2 toxicity to bacteria and aquatic
species (Iannacone et al., 2021).
4.1.2. Control of temperature and solid retention time
At temperatures <20 ◦ C, the maximum specific growth rate of NOB
(μNOB AOB
max ) is generally higher than that of AOB (μmax ) (Shourjeh et al.,
2021). Temperatures >25 C are recommended for AOB to outgrow NOB
◦ values reduced TN RE by 8.5–17.3 %, while N-NH+
and ensure a stable process (Zhang et al., 2009). However, due to high
specific heat of water (4.184 kJ kg− 1 K− 1 at 20 ◦ C), it is not economically
feasible to increase the temperature of municipal wastewater. Main­
taining an adequately low SRT at these temperatures can help to
washout NOB from the reactor, although successful NOB inhibition has
also been obtained at long SRT under DO limitation (Peng and Zhu,
2006). Therefore, DO is a major control parameter to obtain successful
inhibition of NOB and can be controlled in biofilm systems to sustain
shortcut SND in bioreactors .
4.1.3. Control of dissolved oxygen
DO control has been shown as the most effective strategy for main­
taining nitritation at temperatures <20 ◦ C (Gilbert et al., 2015). O2 is the
terminal electron acceptor in the oxidation–reduction respiratory chains
of both AOB and NOB. DO half saturation constant for AOB (KAOB
DO ) falls
in the range of 0.2–0.4 mg O2 L− 1, being lower than that for NOB (KNOB
DO ),
i.e., 0.7–2.0 mg O2 L− 1, which means that AOB have a higher affinity for
O2 than NOB (Liu et al., 2020b). This implies that NOB are often at a
disadvantage when competing with AOB for DO (Chai et al., 2019; Liu
et al., 2020b; Rahimi et al., 2020). However, it has been observed that
NOB affinity for O2 can significantly increase after long-term operation
at low DO concentrations, making NOB better competitors for DO than
AOB (Liu and Wang, 2013). Peng and Zhu (2006) suggested to maintain
a DO range of 1.0–1.5 mg L− 1 to select for AOB over NOB and obtain a
successful nitritation. Campo et al. (2020) showed that about 56 % of TN
could be removed by SND via NO–2 and 44 % via NO–3 in an AGS-SBR
treating real domestic wastewater with low COD/N ratio (2.8–3.8) by
maintaining (1) a low DO concentration during the aerobic phase
(1.4–1.6 mg L− 1), (2) a low DO/NH+
4 ratio and (3) a low SRT of biomass
flocs (8–10 days).
IA has been proposed as an effective method to sustain nitritation.
One of the NOB suppression mechanisms that has been attributed to IA is
the establishment of a lag-phase in NOB activity after the transition from
the anoxic to the aerobic period (Gilbert et al., 2014). Under anoxic
conditions, NOB experience the inactivation of the NXR enzyme and its
reactivation under the subsequent aerobic phase. However, in the aer­
obic phase, AOB activity recovers faster than NOB activity, leading to a
competitive advantage of AOB over NOB. Gilbert et al. (2014) indicated
that 15 min was the minimum duration of the anoxic phase able to
generate a lag-phase in NO–2 oxidation longer than that of NH+
4 oxida­
tion. Therefore, IA with anoxic periods of 15–20 min and aerobic periods
shorter than the NOB lag-phase are sufficient to suppress NOB activity.
The length of this lag-phase (up to 15 min) was observed to be species-
specific and to depend on the DO levels experienced by the biomass
during the aerobic period. The lag-phase for biomass adapted to DO
levels of 0.9–1.0 mg L− 1 was distinctively longer than the one for
biomass adapted to lower DO levels (≤0.4 mg L− 1). In contrast, anoxic
periods longer than 15–20 min and temperature (10–30 ◦ C) did not
7
Bioresource Technology 361 (2022) 127702
affect the duration of the lag-phase. In the study of Iannacone et al.
(2021), shortcut SND was successfully maintained in a continuous-flow
MBBR by adopting aeration cycles with a microaerobic/anoxic phase
(DO = 0.2–1.0 mg L− 1) of 20–25 min, which is in agreement with the
results of Gilbert et al. (2014).
4.1.4. High salinity
High salt content has negative effects on nutrient removal and in­
fluences N removal pathways. NOB are more sensitive to salt than AOB,
and an increase in salinity can lead to their inhibition. Xia et al. (2019)
reported that shortcut SND became the main N removal pathway under
salinity between 1.6 % and 2.4 %. The authors studied SND in a hybrid
sequencing batch biofilm reactor fed with synthetic domestic sewage
with salinity ranging from 0 % to 2.4 % (0–24 g L-1 of seawater crystals).
An increase in salinity to 1.4–2.4 % inhibited NOB activity as NO–2
started to accumulate in the bioreactor. As a result, higher salinity
4 and COD REs were
not significantly affected. Similarly, He et al. (2019b) observed that NOB
population decreased to nearly undetectable levels when salinity was
increased from 1 % to 2 % in an AGS-SBR system performing SND. These
data indicate that salinity levels >1 % favor shortcut SND over complete
SND, although decrease in TN removal might be observed due to inhi­
bition of denitritation. However, an increase of salinity above 2.5 % was
observed to negatively impact also AOB activity (She et al., 2017).
5. Nitrous oxide emissions in simultaneous
nitrification–denitrification biofilms
This section focuses merely on combined nitrifying and denitrifying
biofilms in a co-diffusional configuration (where the electron donor and
acceptor diffuse from the bulk liquid) (Fig. 1); however, the key pa­
rameters leading to increased or decreased N2O emissions discussed
below can also apply to counter-diffusional biofilms (Kinh et al., 2017b;
a).
5.1. Carbon-to-nitrogen ratio and biofilm thickness
C/N ratio plays an important role for N2O emissions in combined
nitrifying and denitrifying biofilms. In these biofilms, most of the
organic carbon is typically utilized by HAB in the exterior portion of the
biofilm (Fig. 1), leaving rather limiting or no amount of organic carbon
available for denitrification (Kinh et al., 2017b). When adequate levels
of organic carbon are not available, intermediates such as NO and N2O
can accumulate within the biofilm (Fig. 1) (Kinh et al., 2017a). The
geometry of the combined nitrifying and denitrifying biofilms attributes
thickness an important role. As mentioned earlier, N2O can undergo
mechanisms of production and consumption by AOB and DNB within the
biofilm depth (Schreiber et al., 2012). Eldyasti et al. (2014) evaluated
N2O emissions from denitrifying fluidized bed bioreactor biofilm and
showed that N2O emissions decreased with the increase in biofilm
thickness. The consumption of N2O likely depends on the thickness of
the deeper layer where, if full penetration of organic carbon occurs, DNB
can further reduce N2O to N2 (Fig. 1).
5.2. Dissolved oxygen and temperature
DO and temperature are also important factors linked to N2O emis­
sions from SND biofilms. The complex stratification along with varying
DO and substrate concentrations can lead to low DO values, DO tran­
sitions from high to low values and DO fluctuations within the biofilm
(Sabba et al., 2018). A decrease in DO concentration can lead to higher
rates of N2O formation by AOB via the nitrifier denitrification pathway
(Chen et al., 2018b). Also, the microaerophilic layer formed within a
combined nitrifying and denitrifying biofilm can trigger more NO and
N2O production by DNB due to the inhibition of the nitrous oxide
reductase (NoS) enzyme in the presence of O2 (Kinh et al., 2017a; Guo
F. Di Capua et al.
et al., 2017). Qi et al. (2022) found that N2O-reducing DNB can recover
from exposure to DO and that this recovery is species-dependent. Zhou
et al. (2022b) found that temperature can influence the abundance and
composition of the N2O-reducing DNB.
5.3. pH and nitrite concentration
The chemical equilibrium between NO–2 and FNA is determined by
NO–2 concentration, pH and temperature (Di Capua et al., 2021). High
nitrification rates in a combined nitrifying and denitrifying biofilm
could lead to high NO–2 production with a decrease of pH; this can drive
the formation of FNA that in turn inhibits N2O consumption (Zhou et al.,
2008). This mechanism has been recently shown to occur in denitrifying
mixed cultures (Zhou et al., 2022a). Zhou et al. (2022a) suggest that this
inhibition can lead to the redistribution of electrons between different
denitrification enzymes such as nitrite reductase (NiR) and NoS, with the
latter being the most impacted and causing an overall reduction in the
N2O consumption activity of DNB. However, FNA and pH have signifi­
cant impacts on AOB populations as well (Sabba et al., 2022), raising the
importance of community composition and how shifts in community/­
predominant species might be a strategy leading to lower N2O produc­
tion (Suenaga et al., 2018).
5.4. Reactor operation
Reactor operation can influence N2O emissions and the microbial
community overall behavior. Yu et al. (2021) investigated the effects of
HRT on N2O production rates during nitrification in lab-scale biological
aerated filters. The authors tested three different HRTs (4, 6 and 8 h) and
found that the maximum and minimum N2O production rate occurred at
HRT of 4 and 6 h, respectively. The proximity of the nitrifying portion of
the biofilm to the bulk liquid allows for the N2O produced to be released
in the bulk and a lower HRT leads to higher emissions. This is especially
true if the bulk is aerated in a combined nitrifying and denitrifying
Fig. 2. Biofilm development and stratification in bioreactors applied for SND.
8
Bioresource Technology 361 (2022) 127702
biofilms where there is a higher N2O mass transfer towards the bulk
rather than the interior denitrifying portion of the biofilm, where N2O
can be reduced (Sabba et al., 2018). Iannacone et al. (2020) operated a
continuous-flow MBBR alternating microaerobic and aerobic conditions
to achieve carbon, N and P removal through simultaneous nitrification
and denitrification coupled to P removal. The authors found that IA
helped enriching for a denitrifying community and therefore allowing to
shift the N2O mass transfer toward the anoxic zone where N2O was
consumed.
6. Biofilm reactors performing simultaneous
nitrification–denitrification
SND has been investigated under various operational conditions and
reactor configurations. In this section, the configurations, and key fea­
tures of the most used biofilm reactors for SND, i.e., MBBR, AGS, and
HMBR (Fig. 2), are critically described with the scope to provide
guidelines for successful and cost-effective operation.
6.1. Moving bed biofilm reactors
MBBRs have been widely applied for nutrient removal from waste­
water (Gu et al., 2018; Wang et al., 2018). The presence of mobile
carriers in the reactor enables the formation of a stratified biofilm with a
bacterial community profile influenced by wastewater composition and
operating conditions (Khanongnuch et al., 2019). Substrates are trans­
ported from the bulk into a co-diffusional biofilm through diffusion
mechanisms allowing the formation of anaerobic, anoxic and aerobic
layers in the MBBR biofilm (Suarez et al., 2019). Compared to fixed-bed
biofilm systems, the MBBR does not suffer from clogging or channeling
issues and no periodical backwashing is needed (Chu and Wang, 2011).
In recent years, the effect of various factors, i.e., carrier type, C/N ratio,
DO concentration and filling ratio, on the SND process has been inves­
tigated in MBBR systems performing SND. Table 1 lists the performance
F. Di Capua et al.
and operating conditions of continuous-flow MBBRs applied for the SND
process.
6.1.1. Carrier type
Carrier material, surface area and roughness play a key role in
determining the performance of a MBBR, since these characteristics
affect both microbial adhesion and growth (Massoompour et al., 2020;
Zhao et al., 2019). Additionally, the development, thickness, and ge­
ometry of the MBBR biofilm can significantly influence the diffusion and
modulate gradients of different parameters, including DO (Fig. 1). Up to
date, various biofilm carriers have been tested to support SND in MBBRs,
including K-series AnoxKaldnesTM made in polyethylene (PE), poly­
urethane (PU) sponge, high density PE (HDPE) carriers, ceramic carrier
or zeolite powders combined with PU sponge (Z-PU) (Table 1).
Of all carrier types used in MBBR applications, sponges feature the
largest specific surface area (SSA) and a high porosity (around 97 %),
being useful for microbial growth (Al-Amshawee et al., 2020). The high
SSA of sponges promotes biofilm development, while porosity allows to
reduce clogging (Nguyen et al., 2010). Sandip and Kalyanraman (2019)
compared the SND performance of a MBBR filled with PU foam carriers
(280 m2 m-3) to that of a MBBR containing conventional PE carriers
similar in shape to the AnoxKaldnes K1 (500 m2 m-3) (Table 1). A
maximum TN RE of 59 % was achieved in the MBBR with PU foam
carriers, 17 % higher than the average TN RE observed in the MBBR with
PE carriers. This difference was mainly due to the higher nitrifying ac­
tivity observed with PU foam carriers, being NH+ 4 RE 11 % higher than
the average observed in the MBBR with PE carriers, which was attrib­
uted to the higher biofilm-liquid contact area of PU foam compared to
PE carriers. The effect of sponge size on SND was investigated by Lim
et al. (2011) in four MBBR-SBRs operated in parallel with foam cubes of
8, 27, 64 and 125 mL, which showed TN REs of 37 %, 31 %, 24 % and 19
%, respectively. Larger PU foam cubes were not fully covered by
biomass, which limited TN RE, while a robust biofilm developed on
smaller cubes, establishing a DO gradient along the PU foam inward
depth favorable to the SND process. For comparison, one bioreactor was
operated without carrier addition, showing a TN RE of only 15 % due to
poor denitrification as a result of high DO and low COD concentrations
in the bulk. Batch tests revealed the occurrence of NO−x reduction in
MBBR biofilms even without the addition of a carbon source, which
could occur due to storage of carbon in the deep biofilm layers.
Carriers made with recycled waste material have attracted the in­
terest of researchers in recent years, being a cost-effective and envi­
ronmentally friendly solution for MBBR operation (Sabba et al., 2017a).
Massoompour et al. (2020) proposed a new carrier made from waste
activated carbon (AC) and PE carriers mixed through a chemical and
thermal process. The authors compared the performance of two parallel
MBBRs, one filled with conventional PE carriers, and the other with
modified PE with AC carriers (Table 2). The SSA of the modified carriers
was 10.9 times higher than that of conventional carriers, which
increased the attached biomass by up to 20 %. The speed of microbial
adhesion and growth of biofilm on modified PE carriers was 26 % higher
than with conventional carriers, being enhanced by the hydrophobic
nature and high porosity of AC (Massoompour et al., 2020). TN RE was
improved by almost 17 % for MBBR equipped with modified carriers due
to the development of a larger anoxic area favoring denitrification,
being nitrification efficiencies similar in both MBBRs. Again, improve­
ment of denitrification was the main mechanism for achieving higher TN
REs by using novel carriers, which allowed to modulate DO gradient and
establish larger anoxic conditions within the biofilm.
Pure PP, PE and HDPE carriers are characterized by a negative
charge surface similar to the surface charge of biofilm, which can
hamper biofilm adhesion on carriers due to repulsions between microbes
and carriers. Liu et al. (2020b; a) proposed a surface-modified carrier
made of PE granules with polyquaternium-10 (PQAS-10), Fe2O3 and 2
wt% clinoptilolite, characterized by a strong NH+ 4 adsorption capacity,
which benefited the enrichment of nitrifying bacteria. Surface-modified
9
Bioresource Technology 361 (2022) 127702
carriers accelerated biofilm formation during start-up period by
reducing repulsion and determined higher REs compared to traditional
PE carriers. The authors reported a material cost of the surface-modified
carrier similar to that of conventional carriers (around 46 USD ton− 1),
although the step of dispersing the materials (i.e., clinoptilolite) into PE
granules would require additional energy.
6.1.2. Filling ratio
MBBRs are usually operated with filling ratios between 20 % and 70
% (McQuarrie and Boltz, 2011; Ødegaard, 2006). Values < 70 % are
recommended in order to assure adequate mixing, while ratios > 20 %
guarantee sufficient concentration of attached-growth biomass in the
system (di Biase et al., 2019). The choice of the filling ratio affects both
the bioreactor performance and operational costs, i.e., energy con­
sumption and purchase of carriers. Feng et al. (2012) evaluated the ef­
fect of three polyurethane foam (PUF) carrier filling ratios (20 %, 30 %
and 40 %) on SND in three parallel MBBRs with an aeration zone and a
settling zone. TN RE in the MBBR filled with 40 % of PUF carriers was 2
times higher than with 20 % filling ratio (Table 1). The filling ratio also
affected biofilm structure and nitrification activity. Biofilm in the MBBR
with a 20 % filling ratio was thick and dense, while in the other two
MBBRs the carriers looked hollow with a thin biofilm. Microprofiles
analysis revealed that dense biofilm could limit DO diffusion into the
inner layer and inhibit nitrifier growth, being the relative abundance of
the nitrifying bacteria Nitrosomonas and Nitrospira in MBBRs with filling
ratios of 30 % and 40 % higher than with 20 % filling ratio. Similarly,
Ferrentino et al. (2018) observed that the increase of filling ratio from
40 % to 60 % in a SBR-MBBR performing SND determined an increase of
nitrification activity and, consequently, an increase of TN RE from an
average value of 50 % to 66 %.
Zhang et al. (2017b) studied the SND process in three MBBRs filled
with PUF at filling ratios of 10 %, 20 % and 30 %, in which average TN
REs of 77 %, 86 % and 87 % were observed, respectively (Table 1). The
highest NH+ 4 oxidation and denitrification rates (2.2 mg N-NH4 g bio­
+
− 1 − 1 − 1 − 1
mass h and 5.1 mg N-NO3 g biomass h , respectively) were
−
achieved in the MBBR filled at 30 %. No significant difference in
maximum TN RE (94–95 %) and SND efficiency (98–100 %) was
observed between the MBBRs filled at 20 % and 30 %. Similar to Feng
et al. (2012), the concentration of attached biomass per gram of carrier
was higher at low filling ratios (10 % and 20 %) than at the highest
tested value of 30 %. On the other hand, suspended biomass was more
abundant at 30 % filling ratio, due to a more frequent carrier collision
leading to biomass detachment, which resulted in an overall higher
biomass concentration at 30 % filling ratio. In contrast, Lim et al. (2012)
observed that increasing the filling ratio from 20 % to 30 % and 40 % in
three IA MBBR-SBRs packed with 8-mL PUF cubes reduced the con­
centration of suspended biomass, which was mainly responsible for
nitrification, while the MBBR biofilm was mainly responsible for deni­
trification. As a result, increasing the filling ratio led to a decrease of
NH+ 4 removal. However, the denitrification efficiency increased due to a
larger anoxic zone and carbon storage in the biofilm, leading to TN REs
of 86 %, 100 % and 96 % at filling ratios of 20 %, 30 % and 40 %,
respectively. As a result, 30 % was considered the optimal filling ratio in
the studied IA system, as it could maximize the overall N removal by
SND. Despite the different biomass dynamics ongoing in the described
MBBR systems, filling ratios ≥ 30 % can be recommended for reactor
operation to enhance N removal.
6.1.3. Hydraulic retention time
Changes of HRT can lead to short- and long-term effects on SND
performance. Prolonging the HRT has been observed to exert positive
effects on nitrification and improve TN RE in MBBRs. Feng et al. (2012)
showed that increasing the HRT from 5 h to 7 h improved the TN RE (i.e.,
24.8–34.1 %) by 18–29 % in three MBBR with filling ratios of 20 %, 30 %
and 40 %. The increase of HRT in the three reactors did not affect the
COD RE, which remained stable at 81 %, but enhanced the NH+ 4 RE. In
F. Di Capua et al.
another study, Zinatizadeh and Ghaytooli (2015) observed increased
growth and activity of AOB and NOB following increase of HRT and DO
concentration from 4 to 8 h and from 2 to 3 mg L− 1, respectively. Bac­
terial growth led to a higher biofilm thickness, which promoted more
favorable anoxic conditions for DNB and higher TN REs. However, it
should be noted that increasing the HRT can significantly prolong the
SRT of both suspended and attached-growth biomass and promote the
growth of NOB in the system, which is deleterious if shortcut SND is the
desired bioprocess (see Section 4.1.2). To the best of our knowledge,
literature lacks information regarding the effect of HRT on the evolution
of the microbial community in MBBRs performing SND, which should be
investigated in future studies.
6.1.4. Combined nitrogen and phosphorus removal in moving bed biofilm
reactors
Recent studies have outlined that the combined removal of N-NH+ 4 (2008) showed that maintaining a low carrier-to-suspended biomass
and P-PO3−4 is achievable under microaerobic/IA conditions in MBBR.
Iannacone et al. (2019) observed a P-PO3−4 RE up to 72 % combined to a
TIN RE up to 68 % in a MBBR operated under stable microaerobic
conditions (DO = 1.0 ± 0.2 mg L-1) at a feed C/N ratio of 4.2. In another
study, MBBR operation under IA conditions (DO alternating between 0.2
and 3 mg L-1) could further improve P-PO3− 4 removal combined to SND,
reaching an average efficiency of 75 % (Iannacone et al., 2020). Pre-
treatment of inoculum by cultivation at pH 8.2 (±0.2), 26–28 ◦ C and
SRT of 4 day effectively inhibited NOB growth, allowing shortcut SND to
occur in the MBBR under continuous-flow conditions. Decrease of car­
bon consumption due to shortcut SND could be likely used by phos­
phorus accumulating organisms (PAO)-like bacteria, leading to a PO3− 4 supporting carrier and a biofilm-like structure (Wang et al., 2009).
RE of 81–88 % (Iannacone et al., 2021). Microbial community analyses
via Illumina sequencing revealed that the genus Hydrogenophaga was
dominant in the MBBRs under both microaerobic/IA conditions. Several
studies have reported Hydrogenophaga as putative PAO being able to
accumulate P under aerobic conditions (Iannacone et al., 2021). Another
potential mechanism enabling P removal in MBBR is the occurrence of
transient anaerobic zones in deeper layers of the microbial biofilm under
non-aerated conditions, leading to the establishment of the aerobic-
aerobic/anoxic alternation triggering typical PAO metabolism. Howev­
er, no typical PAO was detected in the microbial community of the
MBBR biofilm, suggesting that Hydrogenophaga likely had a major role in
P removal under (micro)aerobic conditions.
6.2. Hybrid biofilm-membrane bioreactor systems
Biofilm reactors can be combined with MBR to improve the system
performance and reduce the effect of suspended solids on membrane
fouling (Wang et al., 2020b). Yang et al. (2009) compared the SND
performance of a HMBR filled with nonwovens carriers at 30 % filling
ratio to that of a conventional MBR (CMBR). Both reactors showed COD
REs higher than 95 %, while TN REs reached values up to 89 % for the
HMBR and 70 % for the CMBR. The HMBR showed a more pronounced
improvement of the AOB, NOB and HAB activity compared to the CMBR,
and a more stable response to variations of the feed COD/TN ratio.
Membrane biofouling is the most critical issue for MBR operation, as
it reduces membrane permeability and increases the transmembrane
pressure (TMP), leading to high operational costs due to frequent
chemical cleaning or replacement of membranes (Asik et al., 2021; Ucar
et al., 2021; 2020). It has been demonstrated that biocarrier addition to
MBR systems can alleviate fouling due to scouring of the membrane
surface and/or to a physical–chemical effect on the bulk sludge char­
acterization (Chen et al., 2016). Wang et al. (2020b) showed that adding
static biocarriers improved nitrification and denitrification while
reducing the abundance of bacteria responsible for significant EPS
secretion and consequent membrane fouling. Meanwhile, the authors
also observed a nearly 8 % improvement of the TN RE in the HMBR
compared to CMBR due to better denitrification, which was attributed to
the formation of anoxic and aerobic microenvironments within the
10
Bioresource Technology 361 (2022) 127702
carrier-attached biofilm. Similarly, Han et al. (2018) reported a slower
increase of the TMP in a HMBR seeded with biocarriers (sponge coupled
with PBS granules) compared to a CMBR, corresponding to 1.5-fold
longer operational time of the HMBR compared to the CMBR. The
reduction of cake layer was attributed to physical mechanisms, e.g., the
frictional force carried out by biocarriers on the submerged membrane,
back-transport effect from the membrane surface to the bulk solution
due to turbulence of suspended carriers or impact of biocarriers on the
membrane with consequent shaking of this, as well as to the increased
EPS hydrophobicity, which could considerably improve the flocculation
capacity of the sludge and decrease membrane fouling (Han et al.,
2018). However, negative effects of biocarrier addition on the MBR
performance have also been reported. These include the breaking up of
sludge flocs increasing the number of small particles as well as TOC
levels and, consequently, accelerating membrane fouling. Huang et al.
ratio reduces the breaking up of sludge flocs, resulting in an overall
positive effect of carrier addition on membrane fouling.
Although interesting performances have been highlighted, the
operation of HMBRs would be rather expensive due to the high cost of
membranes and carriers and, therefore, a cost-benefit evaluation for full
scale application would be needed (Meng et al., 2009).
6.3. Aerobic granular sludge systems
AGS combines the characteristics of activated sludge and biofilm
systems due to presence of suspended microbial aggregates without any
Aerobic granules are spherical bacterial aggregates that grow under
aerobic conditions and are usually denser and heavier than regular CAS
flocks (Sarma and Tay, 2018). Granule formation is generally promoted
by providing a feast and famine regime consisting in alternating
anaerobic and aerobic conditions to select for slow-growing microor­
ganisms against fast-growing aerobic heterotrophs. The establishment of
aerobic, anoxic and anaerobic zones within the granule allows the
coexistence of nitrifiers, DNB and PAO, providing the conditions for
combined SND and P removal (Yuan et al., 2019). Up to date, AGS has
been recognized as a promising and emerging technology for waste­
water treatment as alternative to CAS thanks to several advantages, such
as excellent settleability, small footprint, high biomass retention and the
ability to remove simultaneously C, N and P (Chyi et al., 2020).
Table 3 lists the operational parameters and SND performance of
AGS reactors. The most common setup to enrich for aerobic granules
performing SND and to maximize C, N and P removal is to cultivate AGS
in a SBR (Bengtsson et al., 2018; de Sousa Rollemberg et al., 2018). SND
in AGS-SBRs has been successfully established in lab-scale SBRs with a
height/diameter ratio (H/D) between 5 and 10. Despite the excellent TN
REs obtained through the SND process, the poor long-term stability of
aerobic granules due to granule desegregation into filamentous fractions
represents a serious issue (Wang et al., 2018). AGS stability is affected
by: (1) feed C/N ratio; (2) aeration intensity and (3) organic loading rate
(He et al., 2019a). A possible solution to ensure long stability is to enrich
the bacterial community with slow growing microorganisms (metabolic
selection), such as PAO and glycogen-accumulating organisms (GAO),
characterized by high granulation capacity (Campo et al., 2020). The
proliferation of these microorganisms also leads to suppression of HAB
due to the lack of carbon source under aerobic conditions, stored by
GAO and PAO under the previous anaerobic phase (de Sousa Rollemberg
et al., 2018).
Studies on SND in AGS systems have been mainly conducted at lab-
scale using VFAs as sources of organic carbon. If the growth of PAO and
GAO is crucial for the formation of stable granules, granulation may be
hampered during the treatment of real municipal wastewater containing
low VFA concentrations (Layer et al., 2020; 2019). Indeed, Sarma and
Tay (2018) reported low VFA concentrations in real municipal waste­
water (between 22 and 92 mg COD L− 1), with non-diffusible organic
F. Di Capua et al.
carbon being nearly 50 % of the total influent COD.
6.3.1. Granule diameter
The diameter of AGS granules is a factor influencing N removal via
SND (De Kreuk et al., 2005). On average, aerobic granules feature di­
ameters between 0.1 and 3.0 mm (Dahalan et al., 2015). Derlon et al.
(2016) pointed out that typical granule size developed with real
municipal wastewater varied between 0.2 and 1.3 mm (Table 3). These
values are smaller than that observed in granules cultivated with syn­
thetic wastewater, usually reported to be higher than 2.0 mm. The size
of the granules together with the DO penetration depth determines the
extension of the anoxic zone (Liou et al., 2021). Limited anoxic zones in
smaller granules negatively affect SND efficiency (Layer et al., 2020).
We et al. (2020) reported that a granule diameter of 0.9 mm could
significantly improve TN RE. In contrast, Derlon et al. (2016) did not
observe the occurrence of SND due to the small size of granules (0.25 <
d < 0.63 mm), which limited denitrification due to limited extension
and/or carbon availability of the anoxic zone (Liu et al., 2010; Wagner
et al., 2015; Wang et al., 2009). Chen et al. (2011) reported an increase
of TN RE from 68 % to 72 % with the increase of the granule size from
0.7 to 1.5 mm. In order to achieve an appropriate development of the
anoxic and aerobic zones, the optimal average diameter of aerobic
granules should be around 2.0 mm (Campo et al., 2020).
6.3.2. Source of organic carbon
Carbon source influences the microbial community composition and
density of the granules (Layer et al., 2019), and thus, can affect N
removal in AGS systems. de Sousa Rollemberg et al. (2019) studied the
effects of three different carbon sources (acetate, ethanol and glucose)
on SND efficiency in three AGS-SBR systems. Acetate feeding resulted in
the formation of large granules with an average diameter of 1.5 mm,
favored microbial diversity, and led to TN RE of 72 % and total phos­
phorus (TP) RE of 42 %, being respectively 20–30 % and 35–100 %
higher than the REs achieved with the other substrates. However, partial
disintegration of the granules was observed and attributed to the for­
mation of a thick biofilm limiting O2 diffusion and leading to the for­
mation of anaerobic gaseous catabolites within the granules. Ethanol-
fed reactors led to stable granules and good TN RE (53 %), but TP RE
was low (31 %). Glucose showed the lowest TN and TP REs (44 % and 21
%, respectively) as well as the lowest microbial diversity. N removal in
AGS systems fed with acetate and ethanol mainly occurred via SND
during the aerobic period. NO–3 and NO–2 accumulation with acetate was
higher than with ethanol, indicating that ethanol feeding promoted
denitrification. NO–2 accumulated more than NO–3, meaning that part of
NO–2 was directly reduced to N2 through the shortcut SND pathway.
Layer et al. (2019) used four different influent compositions char­
acterized by increasing concentrations of non-diffusible organic carbon
to study how carbon diffusivity influences SND and the granulation
process under anaerobic/aerobic conditions. The diffusible organic
fraction was represented by VFA and soluble fermentable substrates
(glucose and amino acids), while the non-diffusible fraction was repre­
sented by particulate substrates such as peptone and starch. Results
showed that the presence of diffusible organic carbon in wastewater
promoted granulation. The higher the concentration of diffusible carbon
in the influent, the higher was the growth in the deep layers of the
granules, leading to the formation of dense and large granules (d = 1–3
mm). In contrast, the presence of non-diffusible organic substrate
hampered the granulation process and resulted in slower reactor start-
up, smaller granules (0.25–0.63 mm) and presence of 20–40 % (% of
total suspended solids) of sludge flocs in the reactors. Particulate organic
carbon cannot diffuse in granules, so the high availability of organics in
the aerobic phase supports the growth of HAB. This can lead to the
formation of flocs and filamentous granules with poor settling properties
and nutrient removal capability. The share of diffusible organic carbon
in the feed had a significant impact on SND performance of the AGS
systems. The highest TN RE of 77 % was achieved in the reactor fed with
11
Bioresource Technology 361 (2022) 127702
synthetic wastewater containing 100 % VFA as organic carbon, which
led to the lowest accumulation of NO–3. In contrast, the presence of non-
diffusible organic carbon reduced TN RE due to poorer denitrification,
leading to higher NO–3 accumulation (>10 mg L− 1).
6.3.3. Aeration strategy
Besides the lack of sufficient electron donor for denitrification,
another aspect that may limit SND in AGS systems is the formation of
limited anoxic zones within the granules. If O2 penetration is too fast, it
may reduce the time span of the anoxic phase and limit denitrification.
To improve TN REs, several strategies could be considered, such as pre-
or post-denitrification or optimizing the aeration strategy. IA could be
implemented during the aerobic phase to enhance the persistence of the
anoxic zones and favor denitrification. Layer et al. (2020) tested two
different strategies, i.e., 2-step aeration and IA, to improve SND and TN
removal in AGS systems treating real municipal wastewater. 2-step
aeration consisted in maintaining the DO at 2.0 mg L− 1 for 15 min
and at 0.5 mg L− 1 for the residual 270 min of the aerobic phase, while IA
was maintained by turning on aeration every 15 min (DO range = 0–2.0
mg L− 1). Denitrification efficiency increased from 14 to 37 % to 65–79 %
when applying these strategies, resulting in an increase in TN RE from
13 % to > 65 %. In another study with real wastewater, Campo et al.
(2020) applied an aeration phase with two sub-phases, the first being DO
controlled (1.4–1.6 mg L− 1), the second without control to allow DO
depletion and denitrification of residual NO−x . The applied DO control
strategy promoted high TN RE (71 %) and suppressed NOB activity,
reducing carbon requirement for N removal.
6.3.4. Shear force
One of the main factors to control granule stability is the hydraulic
shear force, which is mainly affected by aeration intensity, mixing speed
and reactor shape (He et al., 2019a; Yan et al., 2019b). Shear stress in
AGS systems arises from liquid/gas flows and attrition between particles
(Bengtsson et al., 2018). When exposed to high shear stress (>2 cm s− 1),
aerobic granules become more compact and stable, while low superficial
gas velocity (SGV) generally results in larger granules with a less
compact structure and low settling velocity (de Sousa Rollemberg et al.,
2018). This is likely due to different bacterial stratification and pro­
duced polymers during the granulation process. However, granule sta­
bility may also be achieved at low SGV depending on the influent
strength. Devlin et al. (2017) showed that stable granulation was ob­
tained at SGV as low as 0.41 cm s− 1 when low-strength wastewater (340
mg COD L− 1) was fed to the system, resulting in a TN RE of 62 % and
COD and TP REs > 90 %. In contrast, utilization of medium-strength
(630 mg COD L− 1) and high-strength (1300 mg COD L− 1) wastewater
resulted in poor granulation and filamentous growth, which could be
attributed to incomplete carbon uptake during the anaerobic phase with
subsequent proliferation of filamentous HAB. Moreover, the granules
obtained with medium-strength wastewater were incapable of nitrifi­
cation, resulting in a TN RE of only 30 %, mostly due to N assimilation
into new biomass. In another study, He et al. (2017b) studied the effect
of three low SGVs (0.17, 0.11 and 0.04 cm s− 1) for the treatment of low-
strength wastewater (feed C/N ratio of 3.3) on an AGS system operated
on an anaerobic/oxic/anoxic mode. The granule size increased from 1.5
mm to 1.54, 1.69 and 1.77 mm at decreasing SGV, while the settling
velocity decreased from 58 to 51 and 40 m h− 1. Nevertheless, the system
remained stable, and no sludge bulking nor granule disintegration
occurred, indicating a great adaptability of the granules to decreasing
SGV. The decrease in SGV improved SND performance, as TN RE
increased from 64 % to 94 % when SGV was decreased from 0.17 to 0.04
cm s− 1, although more time (+30 min) was required to achieve complete
nitrification. Increase in N removal was attributed to better anoxic
conditions in the granules at lower SGVs, which may result from the
increased particle size and EPS concentration of the granule. This
improved SND during the aerobic phase and denitrification during the
anoxic phase. Applying low SGVs also results in a reduced energy
F. Di Capua et al.
requirement and operational costs. However, despite better TN removal
and energy saving, the poor granule settleability obtained at the lowest
SGV tested suggests that higher SGVs should be applied for stable
operation.
7. Conclusions
SND is a promising bioprocess that can be applied to replace con­
ventional N removal at WWTPs, and more convenient if performed via
NO–2-shortcut. However, applying SND increases BNR complexity as
environmental and biological factors must be attentively controlled to
maintain high performances and low N2O emissions. At lab-scale, SND
biofilms demonstrated high N RE (>90 %) for all technologies: AGS and
MBBR systems also allowed P RE > 80 %. Operational factors are
technology-specific and must be optimized accordingly. Further
research at pilot and full scale is required to assess biofilm stability
under fluctuating feed conditions to determine impacts on SND
efficiency.
CRediT authorship contribution statement
Francesco Di Capua: Conceptualization, Investigation, Supervision,
Writing – original draft, Writing – review & editing. Francesca Ianna­
cone: Visualization, Investigation, Writing – original draft. Fabrizio
Sabba: Visualization, Writing – original draft, Writing – review &
editing. Giovanni Esposito: Supervision, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
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