Science of the Total Environment 786 (2021) 147390

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Science of the Total Environment

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Review

Organic carbon bioavailability: Is it a good driver to choose the best

biological nitrogen removal process?
Gabriela Bonassa a, Alice Chiapetti Bolsan b, Camila Ester Hollas a, Bruno Venturin a, Daniela Candido c,
Angélica Chini a, Marina C. De Prá d, Fabiane Goldschmidt Antes e, José Luis Campos f, Airton Kunz a,c,e,⁎
a
Western Paraná State University, 85819-110 Cascavel, PR, Brazil
b
Western Santa Catarina University, 89600-000 Joaçaba, SC, Brazil
c
Federal University of Fronteira Sul, 99700-000 Erechim, Brazil
d
Federal University of Technology – Parana (UTFPR), 85660-000 Dois Vizinhos, PR, Brazil
e
Embrapa Suínos e Aves, 89715-899 Concórdia, SC, Brazil
f
Facultad de Ingeniería y Ciencias, Universidad Adolfo Ibáñez, Avda. Padre Hurtado 750, 2503500 Viña del Mar, Chile

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

• CODb/N wastewater ratios operational

limits are reported to N-removal pro-
cess.
• Literature-reported influence of organic
matter to nitrogen removal routes.
• Operational limits for maintaining
Anammox N-removal efficiency are re-
ported.
• The choice of the N-removal process can
be done based on CODb/N ratio and NLR.

a r t i c l e i n f o a b s t r a c t

Article history: Organic carbon can affect the biological nitrogen removal process since the Anammox, heterotrophic and
Received 8 February 2021 denitrifying bacteria have different affinities and feedback in relation to carbon/nitrogen ratio. Therefore, we
Received in revised form 19 April 2021 reviewed the wastewater carbon concentration, its biodegradability and bioavailability to choose the appropriate
Accepted 23 April 2021
nitrogen removal process between conventional (nitrification-denitrification) and Anammox-based process (i.e.
Available online 29 April 2021
integrated with the partial nitritation, nitritation, simultaneous partial nitrification and denitrification or partial-
Editor: Huu Hao Ngo denitrification). This review will cover: (i) strategies to choose the best nitrogen removal route according to the
wastewater characteristics in relation to the organic matter bioavailability and biodegradability; (ii) strategies to
efficiently remove nitrogen and the remaining carbon from effluent in anammox-based process and its operating
Keywords: cost; (iii) an economic analysis to determine the operational costs of two-units Anammox-based process when
Economic viability compared with the commonly applied one-unit Anammox system (partial-nitritation-Anammox). On this re-
Anammox-based process view, a list of alternatives are summarized and explained for different nitrogen and biodegradable organic carbon

Abbreviations: AOB, ammonia oxidizers bacteria; Anammox, anaerobic ammonium oxidation; AD, anaerobic digestion; BOD, biochemical oxygen demand; CODb, biodegradable
chemical oxygen demand; C/N ratio, carbon/nitrogen ratio; COD, chemical oxygen demand; COMAMMOX, COMplete AMMonia Oxidizer; d, days; DN-PN-Anammox, denitrification/
partial nitritation/Anammox; DEAMOX, denitrifying ammonium oxidation; DAMO, denitrifying anaerobic methane oxidation; N2, dinitrogen gas; DNRA, dissimilatory nitrate reduction
to ammonium; ΔG0, free energy; HDH, hydrazine dehydrogenase; HZS, hydrazine synthase; N2H4, hydrazine; NPV, net present value; Nar, nitrate reductase; Nor, nitric oxide
reductase; NOB, nitrite oxidizers bacteria; NirS, nitrite oxidoreductase; NirS or NirK, nitrite reductase; Nos, nitrous oxide reductase; OC, organic carbon; OM, organic matter; PN/D,
partial nitrification/denitrification; PN-SAD, partial nitrification-simultaneous Anammox and denitrification; PN/A, partial nitritation/Anammox; SAD, simultaneous Anammox and deni-
trification; SND, simultaneous nitrification and denitrification; SNAD, simultaneous partial nitrification, anammox and denitrification; SPND + A, simultaneous partial nitritation and
denitritation coupled with Anammox; SPND, simultaneous partial nitritation and denitritation; SLS, solid-liquid separation; SRT, solids retention time; WWTP, wastewater treatment
plant.
⁎ Corresponding author at: Rodovia BR-153, Km 110, Distrito de Tamanduá Caixa, Postal: 321 CEP: 89715-899 Concórdia, SC, Brazil.
E-mail address: airton.kunz@embrapa.br (A. Kunz).

https://doi.org/10.1016/j.scitotenv.2021.147390
0048-9697/© 2021 Elsevier B.V. All rights reserved.
G. Bonassa, A.C. Bolsan, C.E. Hollas et al. Science of the Total Environment 786 (2021) 147390

Biodegradable organic carbon concentration
Nitrogen removal process
Nitrification/denitrification
concentrations, which are the main factors to determine the best treatment process, based on operational and
economic terms. In summary, it depends on the wastewater carbon biodegradability, which implies in the waste-
water treatment cost. Thus, to apply the conventional nitrification/denitrification process a CODb/N ratio higher
than 3.5 is required to achieve full nitrogen removal efficiency. For an economic point of view, according to the
analysis the minimum CODb/gN for successful nitrogen removal by nitrification/denitrification is 5.8 g. If ratios
lower than 3.5 are applied, for successfully higher nitrogen removal rates and the economic feasibility of the
treatment, Anammox-based routes can be applied to the wastewater treatment plant.
© 2021 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Types of wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Organic carbon on wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
4. Nitrogen removal systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.1. Nitrification and denitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.2. Anammox-based processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.2.1. C/N ratio and OC concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.2.2. Anammox-based denitrification system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.2.3. Effect of OC on Anammox microbial community . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5. Differences among N-removal processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6. Practical implications of the organic matter presence in the anaerobic digester effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
CRediT authorship contribution statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Declaration of competing interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1. Introduction
Nitrification/denitrification is still the commonly applied process to
remove organic carbon and nitrogen from different kind of wastewater
and digestates. The feasibility of such process is mainly linked to its ef-
ficiency and reliability. However, some effluents do not contain enough
organic matter to carry out the denitrification process by heterotrophic
bacteria, in addition to the high costs with aeration and large volume of
sludge produced. Thus, the Anammox-based processes have attractive
routes that can be applied to wastewater treatment at reduced costs.
Anaerobic ammonium oxidation (Anammox) bacteria are
chemolithoautotrophic and they use ammonium as electron donor
and nitrite as an electron receptor to produce nitrogen gas, with low
amounts of nitrate (Strous et al., 1998; Giustinianovich et al., 2016;
Xiao et al., 2021).
Due to its nature, the activity of Anammox bacteria are inevitably
inhibited by the concentration of biodegradable organic compounds
present in wastewaters. Organic carbon is usually present in most of
agro-industrial wastewaters despite its different biodegradability and
bioavailability. Biodegradable carbon (CODb) represents the bioavail-
able organic matter in wastewaters. Notwithstanding, Anammox-
based technologies have received great attention due to the ability of
these microorganisms to live in symbiosis with other microbial groups,
like Ammonium oxidizing bacteria (AOB) and heterotrophic bacteria,
i.e.: partial nitritation (PN/A), simultaneous Anammox and denitrifica-
tion (SAD), simultaneous partial nitritation and denitritation (SPND)
coupled with Anammox (SPND + A), partial nitritation; and treat a
wide range of wastewaters (i.e. agri-food, industrial and from the sani-
tary sewage chain). However, there is little information in the literature
that describes the best route that should be combined with the
Anammox process, according to the concentration of biodegradable car-
bon and ammoniacal nitrogen (Gao et al., 2021; Jawad and
Abdulhameed, 2020; Jawad et al., 2020; Surip et al., 2020; B. Wang
et al., 2021; L. Wang et al., 2021; T. Zhang et al., 2020).
This review aims to demonstrate and discuss about the influence of
biodegradable organic carbon on different biological nitrogen removal
processes. According to the characteristics of wastewater, what would
be the suitable process: Conventional nitrogen removal processes or
Anammox-based processes? What are the strategies to adopt an
Anammox-based process even to treat wastewaters with high organic
carbon content?
2. Types of wastes
Wastes are generated in different stages of the agri-food (manure,
dairy, pig slurry, digestate from anaerobic treatment, vegetable, fats,
oils, sugar waste, fish and saline wastewater), industrial (paper mill
wastes, ash from combustion plants, dredging sludge from combustion
plants) and from the sanitary sewage chain (organic municipal waste,
urban sewage sludge). When not correctly disposed and/or treated,
these wastes contribute to food losses, loss of power generation poten-
tial, resource wastage and negative environmental impacts at several
levels, being also the key drivers of climate change and detrimental im-
pacts on human healthy (Fig. 1) (Lupton, 2017; Ojha et al., 2020).
Typically, until some years ago in most of the countries these wastes
were disposed according to the conventional techniques (landfilling,
open dumping and dung and burning). These are not recommended
managements techniques due to scarcity of suitable land for landfilling,
emission of greenhouse gases, water pollution and discharge of pollut-
ants into the natural environment, mainly due to their high content of
organic matter and nutrients (Kumar and Samadder, 2020).
The adoption of anaerobic digestion (AD) has been progressively in-
tensified in recent years for organic carbon (OC) degradation to treat a
variety of agri-food, domestic and industrial wastes, due to its relatively
low cost for implementation, easy operation and capability of energy re-
covery. This technology is defined as a biological process that converts
the OM into biogas rich in methane, by the action of microorganisms
and interspecies electron transfer, in the absence of oxygen (Amaral

2
G. Bonassa, A.C. Bolsan, C.E. Hollas et al.
Fig. 1. Representation of different wastes and the impacts on the environment caused by the absence of their treatment.
et al., 2016; Ajay et al., 2020). Due to the type of effluent and its charac-
teristics, different kinds of reactor configuration and process condition
can be applied, since the agri-food, industrial and sanitary wastes
show different concentrations of organic matter, nitrogen, solids con-
tent, pH, moisture, salinity and density (Amaral et al., 2016;
Mirmohamadsadeghi et al., 2019). However, after AD the nitrogen still
remains in the liquid effluent, called digestate, mainly as ammoniacal
nitrogen. Thus, is needed further treatment of it to meet the standards
of nitrogen discharge (Sheng et al., 2020).
Other processes employed in distinct wastes management are inef-
ficient for nitrogen removal: storage in lagoons (Riaño and García-
González, 2015), solid-liquid separation (Giongo et al., 2018; C.E.
Hollas et al., 2019; Vanotti et al., 2018a), electrocoagulation (Mores
et al., 2016) or situations where a pretreatment is not applied
(Giustinianovich et al., 2016; Racys et al., 2018).
The conventional nitrification/denitrification is the wide applied
process to nitrogen removal. However according to the symbiosis be-
tween microorganisms, it can also be done by the partial nitrification
via nitrite, simultaneous nitrification and denitrification (SND) and de-
nitrification by bioelectrochemical systems, or applying Anammox-
based processes, like the partial nitritation/Anammox (PN/A),
nitritation/Anammox, simultaneous partial nitrification, Anammox,
and denitrification (SNAD), denitrifying ammonium oxidation
(DEAMOX), partial-denitrification/Anammox, partial nitrification-
simultaneous Anammox and denitrification (PN-SAD) and denitrifica-
tion/partial nitritation/Anammox (DN-PN-Anammox) (Rahimi et al.,
2020). Table 1 summarizes some agri-food, industrial and sanitary sew-
age wastewaters characteristics, with the correspondent route applied
for nitrogen removal and the process applied before this step
(pretreatment).
As can be observed in Table 1, the wastewaters show different com-
positions, according to its generating source and pretreatment
employed. Different biological nitrogen removal routes can be applied
according to wastewater characteristics by the symbiotic or competitive
3
Science of the Total Environment 786 (2021) 147390
functions of microorganisms. Thus, the choice of the appropriate nitro-
gen removal route is mainly dependent on the effluent characteristics,
in relation to the concentrations of organic matter/chemical oxygen de-
mand and ammoniacal nitrogen (Ahmad et al., 2020; Sheng et al., 2020).
Fig. 2 summarizes the possibilities of biological nitrogen use from
wastewaters as a function of bacteria cycles and environment/wastewa-
ter properties.
According to Fig. 2 it is noted that the nitrification/denitrification
process is mainly employed in treatment systems with high concentra-
tions of nitrogen and OC. These are the essential parameters for success-
ful application of the conventional route and DAMO or DNRA processes.
In contrast, the anammox-based routes are benefited with high ammo-
niacal nitrogen and low OC concentrations (Ramli and Hamid, 2017;
Ahmad et al., 2020).
Traditionally, the OC is expressed in terms of concentration. How-
ever, this parameter also differs in relation to the carbon bioavailability,
biodegradability and recalcitrance. These are important parameters that
need to be evaluated when choosing the biological nitrogen route that
will be adopted in the treatment plants, since the presence of these
can impede or promote the waste management (Simsek et al., 2013;
Li et al., 2018; Oleinikova et al., 2018).
3. Organic carbon on wastewater
In general wastewaters rich in OC that are not submitted to any pre-
treatment present higher concentrations of bioavailable OC and C/N re-
lation than treated ones, for example municipal wastewater (Pedrouso
et al., 2021a) or raw swine wastewater pretreated through solid-liquid
separation (Hollas et al., 2019; Vanotti et al., 2018b). Wastewaters
that have been submitted to some process of degradation of OC usually
present residual concentration with lower bioavailability as in case of
effluents from AD (digestate). Substrates with greater OC concentration
show some residual amount of it in the digestate and when not fully sta-
bilized, high contents of recalcitrant substances with different
G. Bonassa, A.C. Bolsan, C.E. Hollas et al. Science of the Total Environment 786 (2021) 147390

Table 1
Composition of different wastewaters before nitrogen removal processes.

Type of Pretreatment pH BOD COD NH3-N NO—

2N NO—
3N Nitrogen removal route Ref.
wastewater (mg O2 L−1) (mg L−1) (mg L−1) (mg L−1) (mg L−1)

Animal feed AD 219 ± 103 231 ± 40 SNAD (Almeida et al., 2018)

processing
wastewater
Fish canning 7.8 ± 0.1 189 ± 10 291 ± 51 PN/A (Val del Rio et al.,
wastewater 2018)
Food waste AD 7.8 ± 8.5 187.0 ± 1.4 671.0 ± 234.0 737.0 ± 180.0 19.7 ± 19.8 60.3 ± 89.2 PN/A (Pereira et al., 2019)
Food waste 8.1 ± 0.2 1632 ± 60 1507 ± 98 PN/A (Carucci et al., 2020)
Mature 7.7–8.1 1100 ± 150 2500 ± 250 2550 ± 200 0.1–0.5 DN/PN/A (Li et al., 2021)
landfill
leachate
Municipal Septic tank 7.1–7.5 238.6 ± 53.5 71.4 ± 4.4 0.2 ± 0.1 0.3 ± 0.1 SNAD (W. Zhang et al.,
wastewater 2020)
Municipal Septic tank 7.1–7.5 118.6–221.5 43.2–72.3 0.2 0.3 Anammox (Zhang et al., 2018)
wastewater
Municipal Septic tank 121.9 ± 19.9 48.9 ± 4.1 PN/A (Jin et al., 2019)
wastewater
Municipal 49 ± 4 25 14 1 PN/A (Pedrouso et al.,
wastewater 2021b)
Municipal AD 7.5 ± 0.1 110–138 38–48 PN/A (Pedrouso et al., 2019)
wastewater
Primary 7.20 ± 0.25 157.5 ± 9 45 ± 10 Nitritation (Pedrouso et al.,
settled 2021a)
municipal
wastewater
Primary 7.46 ± 0.13 158.58 ± 13.32 ± 2.35 0.88 ± 0.42 (Pedrouso et al.,
settled 28.37 2018)
municipal
wastewater
Saline 168.05 ± 9.4 136 ± 14 PN/A (Giustinianovich
wastewater et al., 2018)
Sludge AD + 220 ± 8 213 ± 32 824 ± 33 PN/A (Chini et al., 2020)
digestate solid-liquid
separation
Sludge AD + 220 ± 8 156 ± 8 300 PN/A (Chini et al., 2019)
digestate solid-liquid
separation
Swine 8.2 ± 0.2 182 ± 58 513 ± 23 PN/A (Pichel et al., 2019)
wastewater
Swine AD 8.21 ± 0.01 266 ± 8.10 200 ± 0.10 1200 ± 0.90 PN/A (Bonassa et al., 2021)
wastewater
Swine Solid-liquid 8.14 ± 0.23 14,700 ± 1 1400 ± 0.48 Nitrification/denitrification (Hollas et al., 2019)
wastewater separation
Swine AD 822.5 ± 111 767 ± 47.3 Nitrification/denitrification (Giongo et al., 2018)
wastewater
Swine Anaerobic 75 ± 25 640 ± 10 Partial nitritation (Assis et al., 2017)
wastewater lagoon
Swine 7.95 ± 0.13 2000 ± 410 6800 ± 1400 502 ± 19 0±1 2±1 Nitrification/denitrification (Waki et al., 2018)
wastewater
a
Swine Solid-liquid 7.78 + 0.23 3185 + 2692 8196 + 4098 1213 + 451 0.2 + 1.5 Nitrification/denitrification (Vanotti et al.,
wastewater separation 2018b)
a
NO— —
2 -N + NO3 N.

biodegradability still remain in the final effluent (Malerba et al., 2014;
Maynaud et al., 2017).
The digestates organic substances are generally in the forms of sub-
stances like humic, carbohydrates, protein, aldehydes, fulvic acids, phe-
nols and organic peroxides, which can be classified as a biodegradable
fraction (readily or slowly) or non-biodegradable, according to the ma-
terial recalcitrance. The concentration and type of these are the key fac-
tor on choosing the ideal nitrogen removal route in a wastewater
treatment plant (Mook et al., 2012; Terry and Summers, 2018).
The coexistence between two or more bacteria and processes (Fig. 2)
during wastewater treatment is mainly dependent on the availability of
the OC. The presence of OC readily available and with biodegradable
characteristics promotes the denitrification, DAMO and DNRA biogeo-
chemical routes in the wastewater treatment systems, since COD is
the available substrate for heterotrophic growth. On the other hand,
high or low concentrations of COD with low biodegradability (or biode-
gradable organic carbon concentration) and availability does not pro-
motes the heterotrophic bacteria growth, thus the Anammox or
Comammox routes can occurs without competitions for ammoniacal ni-
trogen between these bacteria and the heterotrophic ones (Jin et al.,
2012; Magrí et al., 2013; M. Zhang et al., 2019; Sheng et al., 2020).
Anammox-based process can also be applied in effluents with high or-
ganic carbon concentration, after some pretreatment and carbon reduc-
tion techniques or combined routes. These strategies and knowledge of
peculiarities of each route are the main factors in choosing it, polishing
the wastewater and applying these processes (Fig. 3) (M. Zhang et al.,
2019).
The solid-liquid separation (SLS) is widely applied to reduce the
remained organic matter from wastewater previous to the biological
wastewater treatment (Tambone et al., 2017). From this route, the su-
pernatant (liquid phase) can be directly forwarded to the biological ni-
trogen removal process, while the solid fraction can be directed to a
sludge anaerobic digester or composting, for subsequent land applica-
tion (X. Wang et al., 2020). Centrifugation, flotation, setting and belt-
press are the usual applied methods for digestates SLS (Chini et al.,
2021). The operational conditions of each technique can vary according

4
G. Bonassa, A.C. Bolsan, C.E. Hollas et al. Science of the Total Environment 786 (2021) 147390

Denitrification
Org. C
Org. N N2 N2O NO NO2- NO3-
DAMO

Nitratation
N fixation
DNRA NO2-
NH4+/NH3 O2 O2
Org. C

O2 O2
O2
Nitrification
Org. N NH2OH NO2-
Comammox
O2
Nitritation
Fig. 2. Nitrogen biogeochemical cycle with the: anaerobic ammonium oxidation (ANAMMOX), complete ammonium oxidation to nitrate (COMAMMOX), denitrifying anaerobic methane
oxidation (DAMO) and dissimilatory nitrate reduction to ammonium (DNRA).

to the wastewater type (i.e. temperature and hydraulic retention time)
(Scaglione et al., 2017).
In the next section information related to each nitrogen removal
route will be addressed, based on the OC concentration of wastewater,
in order to provide practical solutions for the wastewater treatment
applicability.
4. Nitrogen removal systems
4.1. Nitrification and denitrification
Although the current discussion about the efficiency of nitrogen re-
moval processes, relating the energy required supplying the oxygen de-
mand and also the organic carbon consumption, the canonical
nitrification/denitrification process is still being applied and advised
for different effluents treatment. It shows efficiency and process stabil-
ity for a wide range of operational conditions (McCarty, 2018; Chang
et al., 2019).
On such process, the ammoniacal nitrogen is converted to dinitrogen
gas in subsequent stages, through the action of microorganisms' meta-
bolic activity (Fig. 2) (Liu and Wang, 2017). In the nitrification step, am-
monia is oxidized to nitrite and subsequently to nitrate by autotrophic
organisms, which use oxygen as electron acceptor. In this mechanism,
the overdue ammonia reduction to nitrite is attached to the bacteria
known as ammonia oxidizers bacteria (AOB), related to the genes
Nitrosomonas, Nitrosococcus and Nitrosospira (Eq. (1)). Nitrite oxidation
is carried by the nitrite oxidizers bacteria (NOB) being Nitrospira,
Nitrobacter, Nitrospina and Nitrococcus the most common ones

Fig. 3. Pretreatment methods and combined processes for organic matter removal (*physical-chemical process).

5
G. Bonassa, A.C. Bolsan, C.E. Hollas et al.
(Eq. (2)) (Lücker et al., 2010; Sinha and Annachhatre, 2007; Iannacone
et al., 2019).
−1
NH₄þ þ 1:5 O₂ ! NO₂− þ 2Hþ þ H₂O ΔG0 ¼ −287 kJ mol
−1
NO₂− þ 0:5 O₂ ! NO₃− ΔG0 ¼ −76 kJ mol
In the denitrification step, the nitrate is reduced to N2 by the hetero-
trophic organisms, which uses organic carbon as electron donor (Fig. 2)
(Eq. (3)) (Hu et al., 2019; C. Li et al., 2019; Z. Zhang et al., 2019). For
such, several intermediate products are produced, which acts as elec-
tron acceptors. Thus, the nitrate is reduced to nitrite and subsequently
to nitric oxide and nitrous oxide, and subsequently both are reduced
to dinitrogen gas (Eq. (4)) (Miao and Liu, 2018; Rezvani et al., 2017;
Zumft, 1997).
NO3 − þ 5e− þ 5Hþ ! 0:5 N2 þ 2H2 O þ OH−
NO3 − ! NO2 − ! NO ! N2 O ! N2
Several studies highlight that the difficulties and instability of such
process is due to the organic carbon source requirement to the denitri-
fication steps. To apply this route to wastewaters with low organic car-
bon concentration, supplementation is needed which increases
considerably the treatment cost by such technology (Chai et al., 2019;
Zhang et al., 2018).
The supplementation of OC to increase the C/N ratio is usually per-
formed with the addition of distinct external sources, i.e. acetate, meth-
anol, ethanol, propionate and glucose for the treatment of nitrogen-rich
effluents (Gavazza Dos Santos et al., 2004; Louzeiro et al., 2002; Torresi
et al., 2017; Wang et al., 2012; Zheng et al., 2018). Ginige et al. (2009)
evaluated the relevance of methanol addition in relation to denitrifica-
tion performance, founding that the process can be benefited with the
addition of an external carbon source. Furthermore, the improvement
in process performance was closely related to the reactor configuration,
since the methanol concentration required achieving the effective ni-
trate reduction ranged from the primary to secondary anoxic phase.
The organic matter is the fundamental and primordial element to the
denitrification step. Low COD/N concentration or availability of it leads
to improper denitrification, besides nitrite and N2O accumulation and
emissions, due to the metabolic route deviation (Giongo et al., 2018;
Jialin Li et al., 2020; Thakur and Medhi, 2019).
Not less important, the rich-organic matter effluents are mostly di-
rected to the anaerobic digestion, enabling the energy recovery through
biogas production (McCarty, 2018). With this, the nitrogen removal by
nitrification and denitrification has been in constant improvement
aiming the adaptation of it to some distinct conditions, and also comply
with the goal of the biologic process, that is the nitrogen removal with
high efficiency (Xin and Qin, 2019). By the way, among all these differ-
ences, the amount of OC and the C/N ratio (or COD/N) employed for the
nitrification and denitrification process is extremely variable (Table 2).
Table 2
Organic sources and C/N ratio in denitrification bioreactors treating different kinds of wastewaters.
Required ratio Carbon source
COD/N = 10 Acetate
COD/N = 7 Ethanol
COD/N = 0.70 Swine wastewaters
C/N = 3.5 Domestic wastewater
COD/N = 10.04 Synthetic wastewater
TOC/NH3-N = 2.44 Swine wastewater
COD/N = 5.6 Swine wastewater
C/N = 4.2 Synthetic wastewater (CH3COONa·3H2O)
COD/N = 5.39 Inorganic wastewater and synthetic (C6H12O6 and CH3COONa)
C/N = 3.9–7.3 Acetate, propionate and glucose
Science of the Total Environment 786 (2021) 147390
Theoretically, about 2.86 g of COD are required per gram of NO− 3 -N
to be reduced to N2, while 1.71 g of COD is required to the reduction
of 1 g of NO−2 -N to N2. As stated above, the biodegradability of the in-
ð1Þ volved carbon presents direct relation with the necessary amount of it
for the process occurrence (Metcalf and Eddy, 2003), which explains
the different concentrations required according to the source, as
ð2Þ
showed in Table 2. Furthermore, the reaction configuration can influ-
ence these ratios (Ginige et al., 2009).
Due to differences in growth rates of nitrifying and denitrifying or-
ganisms, the reaction configuration can direct influence in the carbon
requirement that the system demands or deal with (Metcalf and Eddy,
2003). Since the denitrifies bacteria have higher growth rates than the
nitrifies, thus the nutrient availability could proliferate suppressing
growth of ammonia oxidizing bacteria (Zhao et al., 1999). Thus, config-
urations that shows only one reactional unit tend to have greater re-
strictions on the loads applied due to the beneficiation of a group of
organisms, while compound systems with pre-denitrification units en-
ð3Þ able the treatment of effluents rich in OM, such as swine manure
(Bortoli et al., 2019).
ð4Þ The use of nitrification and partial denitrification could be a solution
in case of effluents with low C/N ratio and this issue has been the target
of many studies in recent years (How et al., 2019; Xin and Qin, 2019).
Giongo et al. (2018) evaluated the effect of different C/N ratios in the ni-
trogen removal by nitrification and partial denitrification. Testing efflu-
ents with 0.9 of C/N ratio, the researchers verified an economy of 27% in
the total organic carbon consumption; and 74% in relation to the dis-
solved oxygen, with nitrogen removal efficiency greater than 85%.
As noted in Tables 1 and 2, the nitrification/denitrification is widely
applied with agroindustrial digestates, which have high COD concentra-
tion and C/N ratio. However, this process can be also applied to nitrogen
removal from municipal wastewaters, if the stoichiometry OC require-
ment is attempted. However, around 30 to 85% of OC from these efflu-
ents are of slowly degradable nature, according to Shi et al. (2020),
making necessary the use of external commercial organics (i.e. metha-
nol, ethanol, acetic acid and sodium acetate). With such supplement,
wastewater treatment plant gets onerous costs, complex operation
and secondary effluents could be generated, hindering the applicability
of the process (Zhang et al., 2015; Tian and Yu, 2020).
Digestates from animal wastes, by contrast, have in their composi-
tion higher contents of organic matter, generally in labile form. Thus,
the microbial activity of heterotrophic bacteria is favored, since COD is
bioavailable (Surey et al., 2020). In Table 1 it is possible to note the
high organic content in swine wastewaters and the applicability of the
nitrification/denitrification for such cases. Additionally, in some cases
AD can be employed to recover energy from produced biogas, reducing
the energy cost for the aeration step on nitrification. On this step the en-
ergy necessary represents up to 70% of the treatment system energy
consumption (How et al., 2019; Ramli and Hamid, 2017).
Other sustainable approach to the denitrification system is the use of
sludge from the wastewater treatment plants, mainly composed by pro-
tein, polysaccharides and volatile fatty acids, as a carbon source. Accord-
ing to upstream operation conditions, these organic compounds can be
Reactor configuration Reference
Airlift hybrid reactor Zhang and Wei (2013)
Modified Ludzack-Ettinger reactor Carrera et al. (2004)
Upflow sludge Reactor Li et al. (2016)
SBR Wang et al. (2016)
Airlift internal circulation membrane bioreactor Meng et al. (2008)
Modified Ludzack-Ettinger reactor Hollas et al. (2019)
Modified Ludzack-Ettinger reactor Vanotti et al. (2018a,b)
Continuous flow microaerobic reactor (MBBR) Iannacone et al. (2019)
SBR Chang et al. (2019)
SBR Zhao et al. (2018)
6
G. Bonassa, A.C. Bolsan, C.E. Hollas et al.
readily available or not, and some gaps remain in focus for the denitrifi-
cation applicability (Cao et al., 2019; Guo et al., 2020b; W. Liu et al.,
2020).
Wastewaters with low C/N ratio and OC bioavailability mitigate the
nitrification/denitrification process applicability. Besides that, the idea
of preparing a substrate with organic carbon, the operational conditions
and type of system necessary to this nitrogen removal process (aeration,
organic source and sludge production) are considered determining fac-
tors for the choice of this approach. Thus, other electron donors can be
used as alternatives for nitrogen removal from wastewaters by other
routes (i.e. Anammox-based process, chemolithotrophic denitrification)
and another microorganisms physiology (autotrophic and/or
chemolithoautotrophic), which can also be combined with the tradi-
tional denitrification (Ma et al., 2020; Tian and Yu, 2020). These lines
of nitrogen removal process are more energy efficient when compared
to the complete nitrification/denitrification.
4.2. Anammox-based processes
In the anaerobic ammonium oxidation (Anammox) process, the am-
moniacal nitrogen is directly converted into nitrogen gas over nitrite,
under anoxic conditions (Fig. 2). The nitrite acts as the electron accep-
tor, and compared to the conventional nitrification/denitrification pro-
cess, the costs with energy requirement by oxygen and sludge
management are reduced as well as OC requirement is suppressed.
Due to the reduced oxygen consumption (up to 60%), there is low bio-
mass production and sludge accumulation. This process is recognized
as one of the most important biological progresses in wastewater treat-
ment, with several applications in wastewaters with high ammonia and
low OC contents (Sheng et al., 2020; Arora et al., 2021).
Up to now, six Anammox candidatus genera (Brocadia, Kuenenia,
Jettenia, Scalindua, Anammoxoglobus and Anammoximicrobium) and sev-
eral different related species have been already mentioned and identi-
fied in natural, synthetic and wastewater treatment systems. Each one
of these shows distinct physiological and metabolic features, being the
16S rRNA gene sequences bounded with the specific ecological niche
(Yang et al., 2020; Zhang and Okabe, 2020).
In common between these genera is that during the autotrophic pro-
cess of ammonia oxidation by Anammox bacteria, a source of inorganic
carbon is extremely necessary. Such organisms transform dissolved car-
bon dioxide (HCO3), an inorganic carbon source, into biomass and en-
ergy. Concomitantly, the ammoniacal nitrogen is converted to N2
(Strous et al., 1998; Sheng et al., 2020). The quite-used stoichiometry
of the Anammox process was established by Strous et al. (1998) in
chemostat experiments and is described by the Eq. (5).
NH4 þ þ 1:32 NO2 − þ 0:066 HCO3 − þ 0:13Hþ
! 1:02 N2 þ 0:26 NO3 − þ 0:066 CH2 O0:5 N0:15 þ 2:03 H2 O ð5Þ
According to the Anammox stoichiometry it is noted the inorganic
carbon relevance to the process. According to Liao et al. (2008), such
bacteria shows active efficiency with bicarbonate increment from 1.0
to 1.5 g L−1, and suppressed when these quantities exceeds 2.0 g L−1.
−
The concentration of inorganic carbon source, the ratios of NH+ 4 /NO2
required to the process and ratios of NO− 3 produced can vary according
to the reactor configuration, operational conditions and wastewater
type, which are also influencing factors for the Anammox community
diversity (Chini et al., 2020; Guo et al., 2020a).
During the catabolism of these organisms, the NO− 2 is reduced to ni-
tric oxide (NO), by the action of a nitrite oxidoreductase (NirS). In par-
−
allel, by combining NH+ 4 with the NO2 , a hydrazine synthase (HZS)
forms hydrazine (N2H4). Finally, by the action of hydrazine dehydroge-
nase (HDH), the N2H4 is oxidized to N2. A residual amount of NO− 3 is also
produced during the ammonia oxidation, due to the anabolic cycle of or-
ganic source consumption. These steps can be noted merging the
7
Science of the Total Environment 786 (2021) 147390
Anammox cycle showed in Fig. 2 with Eq. (5) (Chini et al., 2019; Yao
et al., 2015).
According to the Anammox stoichiometry (Eq. (5)), together with
the ammonia, nitrite is also primordial to the process, which rarely ex-
ists in wastewater from most common pretreatment routes. Thus, ac-
cording to the wastewater characteristics in relation to ammoniacal
nitrogen concentration and organic matter, the Anammox can be com-
bined with other microorganisms. The partial nitritation by nitrifies bac-
teria, denitrification by heterotrophic and COMplete AMMonia
Oxidizers (Comammox) by Nitrospira, are good approaches to the
Anammox-based process (Fig. 2). When compared with the conven-
tional nitrification/denitrification the advantages related to low energy
consumption still remain with these routes (Arora et al., 2021; Li et al.,
2021; Sheng et al., 2020).
Among the symbiosis between the microorganisms involved in the
Anammox-based process, some external factors have been already
proved for suppress or out-select the activity of them (i.e.: temperature,
pH, dissolved oxygen, free nitrous acid, free ammonia, salt, heavy metals
and organic matter). In fact, the operation of the Anammox process is
well-established for digestates or other wastewaters that contains low
C/N ratio and organic source bioavailability. Since the Anammox organ-
isms are chemolithoautotrophic, the presence of high concentrations
of it in the wastewater directly inhibits the nitrogen removal efficiency
of the systems, niche and microbial metabolism. However, some levels
of organic and inorganic carbon are undoubted in real effluents. Con-
comitantly, an alternative to the treatment of wastewaters with signifi-
cant organic matter content is the conjunction of this process with other
microorganisms that simultaneous remove nitrogen and OC (i.e. hetero-
trophic) (Chini et al., 2019; Ge et al., 2018; Xie et al., 2017; T. Zhang
et al., 2020; Zhang et al., 2021).
4.2.1. C/N ratio and OC concentration
The effect or OC concentration in the performance of ANAMMOX
process could be related to two mechanisms: i) inhibition by self-
competition, with the alteration of the microbial population due to
AOB and ANAMMOX suppression and NOB increase; ii) due to some
changes in the metabolic pathway, where the involved bacteria starts
to metabolize organic carbon instead ammonia and nitrite, causing de-
crease in the efficiency of nitrogen removal (Cheng et al., 2018; Miao
et al., 2018).
If the OC present in wastewater shows poor biodegradability and
availability, the effect of it in the deammonification process is mini-
mized. It is not sufficient to promote the coexistence of AOB and
Anammox with the heterotrophic ones. On the other side, a high con-
tent of biodegradable organic carbon concentration, like sucrose and/
or glucose, can promote the excessive growth of heterotrophic bacteria
and in parallel, the decreased of AOB and Anammox bacteria activity (Li
et al., 2018).
Liu et al. (2017) tested different C/N ratios (from 0.1 to 3.0), main-
taining the ammonia nitrogen concentration in 50 mg NH3-N L−1, to
evaluate the performance of the process and which group of bacteria
would dominate the treatment process. The increase of C/N ratio from
0.1 to 0.6 lead to an increase in the heterotrophic bacteria in
the deammonification reactor (from 7.5 to 16%). With C/N ratio > 1.0,
the declined predominance of AOB and Anammox was observed, with
the decrease of nitrogen removal efficiency and the bacteria granules
become bigger. Simultaneously, the surface contact between the bacte-
ria and the substrate get decreased.
Wu et al. (2015) checked that C/N ratios of 1.30 and 1.36 (3000 mg
L−1 COD and 2200–2300 mg L−1 NH+ 4 -N) in the treatment of effluents
from UASB reactors. In this case, inhibitory effects were not developed
due to the low biodegradability of the organic matter and high
Anammox activity.
Miao et al. (2018) investigated the effect of C/N ratios from 1.1 to 2.5
in the removal nitrogen performance, evaluating also the Anammox
morphology. The nitrogen removal efficiency increased with the C/N
G. Bonassa, A.C. Bolsan, C.E. Hollas et al. Science of the Total Environment 786 (2021) 147390

ratio increment, from 1.1 to 2.0 (30.8 to 77.3%), due to the Anammox ac-
tivity improvement. However, with 2.5 of C/N ratio the bacteria activity
decreased significantly, the granules form also changed and the sludge
retention time became shorter. According to authors, this happened
due to the development of heterotrophic bacteria, which even though
contributes to the nitrogen removal efficiency, they also compete for
substrate.
In the literature different C/N and COD/N ratios that the Anammox
process shows distinguish ammonia nitrogen removal efficiency are de-
scribed; nevertheless, it is directly related to the biodegradable organic
carbon type and concentration. Referring to synthetic effluents, COD is
almost entirely glucose that shows high biodegradability. On the other
hand, effluents from real wastewater treatment plants do not have the
predominance of only one carbon source, thus the effects on the in-
volved bacteria are different in relation to the process efficiency and mi-
crobial community (Li et al., 2017).
According to Terry and Summers (2018), to do not provide condi-
tions for the growth and abundance of NOB and promote AOB and
Anammox activity, COD concentrations should be lower than 0.5 g
g−1. However, there are few problems with higher concentrations of it
if the organic source shows high recalcitrance. Wang et al. (2019a,b)
also mentioned that the organic matter concentration and recalcitrance
plays a much more significant role in the deammonification process
than the C/N ratio. The authors evaluated the synergetic effect between
Anammox and denitrify bacteria in COD concentrations until 300 mg
L−1, during the N-removal process. With COD concentrations lower
than 55.7 mg L−1 the Anammox process contributed to more than
80% of N-removal. On concentrations between 220 and 300 mg COD
L−1 they contributed to 50% of N-removal and with more than 300 mg
COD L−1 there was no Anammox activity.
Arriagada et al. (2017) and Chini et al. (2019) showed that the
deammonification process is able to nitrogen removal from wastewater
from anaerobic digestion containing OC. To it, the wastewater has to be
previously acclimatized to the stabilization of biodegradable OC and the
process has strict operational control in relation to sludge retention time
and air supply.
Some studies have shown that if it is possible to keep the concentra-
tion of Anammox biomass high enough inside the reactor while increas-
ing the influent C/N ratio, the Anammox microorganism may coexist
with some heterotrophic bacteria (Molinuevo et al., 2009; Ni et al.,
2012; Xie et al., 2017). On such mixed systems, the denitrification con-
sumes OC and nitrate produced during Anammox process (Eq. (5)).
However, it is extremely easy to inhibit the Anammox process, once
the fast growth of heterotrophic bacteria can suppress the other organ-
isms. Therefore, process control is mandatory to a good reactor
performance.
Correlation among Anammox and other processes are summarized
in Table 3 (i.e. partial nitritation (PN/A), simultaneous Anammox and
denitrification (SAD), simultaneous partial nitritation and denitritation
(SPND) coupled with Anammox (SPND + A), partial nitritation,
Anammox, denitrification and COD oxidization (SNADCO), denitrifica-
tion) according to each kind of digestate type. Some information related
to C/N ratio and/or organic matter content are also showed. Even
though the partial nitritation has been one of the processes most allied
to the Anammox, this process can be combined with other routes ac-
cording to the wastewater properties, as showed in Table 3. Both bacte-
ria groups involved in deammonification process (AOB and Anammox)
do not require any OC to growth or enhance activity. But in general, the
nitrogen-rich wastewaters (i.e. landfill leachate, wastewater from
digested animal waste and monosodium glutamate wastewater) have
some amounts of it in their composition. Consequently, the denitrifica-
tion can be a good approach to the biological nitrogen removal for such
cases (Jin et al., 2012).
4.2.2. Anammox-based denitrification system
Despite the advantages that the Anammox process shows, the bio-
degradable organic matter content of wastewaters can be an obstacle
to it applicability. On such way, the alternatives that are receiving in-
creasing attention are the Anammox process coupled with the denitrifi-
cation (SAD), denitrification/nitrification (SNAD) and/or partial
denitrification (PN), by the heterotrophic, denitrifying anaerobic
methane oxidation (DAMO) and dissimilatory nitrate reduction to

Table 3
Anammox-based processes application in different kinds of wastewater and OC concentrations.

Wastewater type Process Pretreatment COD/N COD BOD NH3-N NO−

3 -N Ref.
(mg L−1) (mg O2 L−1) (mg L−1) (mg L−1)

Domestic wastewater Anammox SPND 3.4 238.6 ± 53.5 71.4 ± 4.4 0.3 ± 0.1 (W. Zhang et al.,
2020)
Landfill leachate Anammox PN 0.4 2100 ± 75 5700 ± 100 (Li et al., 2018)
Mature landfill leachate DN/PN/A OM stabilization 0.98 2500 ± 250 1100 ± 150 2550 ± 200 (Li et al., 2021)
Municipal sewage PD/A 3.0 20–40 (Cao et al., 2019)
Organic fraction of PN/A + Ammonia stripping 0.40–5.70 299 ± 54– 731 ± (Pichel et al., 2019)
municipal solid waste denitrification 6433 ± 401 55–1290 ±
500
Pre-digested chicken SAD 1.8–2.0 400 ± 110– 190–330 (Pekyavas and
waste 595 ± 79 Yangin-Gomec,
2019)
Sewage wastewater PD/A PD 220 60 (Ji et al., 2018)
Sludge digestate PN/A Centrifugation 7000 ± 0.03 220 ± 8 2600 ± 0.01 (Chini et al., 2020)
Sludge from primary DN/A 727.0 103.6 (Cao et al., 2020)
sedimentation tank
Sludge supernatant DN/PN/A Anaerobic methanation 300–700 300–400 (Li et al., 2020)
Swine wastewater PN/A 0.3–0.4 101–352 191–387 (Jiuling Li et al.,
2020)
Swine wastewater PN/A OM stabilization 200 ± 0.10 266 ± 8.10 1200 ± 0.90 (Bonassa et al.,
2021)
Swine wastewater PN/A Primary and secondary anoxic and 0.85 467.79 549.98 <0.02 (Pan et al., 2020)
aerobic sedimentation tanks
Swine wastewater PN/A Hydrolysis acidification 0.96 131 ± 40 136 (Zuo et al., 2020)
Synthetic wastewater SAD 0.3–0.9 93.7–267.45 193.7–184.4 41.2–39.07 (Z. Wang et al.,
(sodium 2020d)
acetate)
Synthetic wastewater SNADCO 0–1.6 50–400 250 (Guo et al., 2020b)
with biodegradable OM

8
G. Bonassa, A.C. Bolsan, C.E. Hollas et al.
ammonium (DNRA) bacteria, as showed by some works related on
Table 3 and pathways on Fig. 2. Besides the possibility of work with
organic-rich effluents, on these routes the NO− 2 production does not re-
quire complex control strategies and it is achieved with good stability.
Simultaneously, the NO− 3 produced by the Anammox process (Eq. (5))
is removed by the heterotrophic bacteria. This represents a good poten-
tial approach to manage the organic-rich effluents (Du et al., 2019; Shi
et al., 2020).
The process of denitrification comprehends the reduction of NO− 3 to
N2, via NO− 2 , NO or N2O in the presence of biodegradable OC and ab-
sence of free molecular oxygen. Simultaneously (in the same or sepa-
rated reactor), the Anammox bacteria remove the NH3 via NO− 2 ,
producing little amounts of NO− 3 . In the subsistence of sulfur com-
pounds (i.e. S− 2−
2 and S2O3 ), organic carbon and H2, the heterotrophic
bacteria can use these together with the NO− −
3 to produce NO2 , the elec-
tron acceptor of the Anammox process (Han et al., 2021; Yan et al.,
2020).
The combined Anammox and denitrification process can be done in
only one reactor or in two-stages. Independently, the stable operation of
the combined process depends on the synergism between the involved
bacteria. The denitrification is mediated by heterotrophic bacteria and
the enzymes nitrate reductase (Nar), nitrite reductase (NirS or NirK), ni-
tric oxide reductase (Nor) and nitrous oxide reductase (Nos). Exploring
this microbiology and the key organisms and functions of the combined
nitrogen removal processes is a promising strategy for understanding
and improving the waste management (D. Wang et al., 2020; Yan
et al., 2020).
According to Z.-B. Wang et al. (2020) the PD-Anammox process
stands out in relation to the deammonification (PN-Anammox) in
terms of operational conditions, greenhouse gases emission and nitro-
gen removal efficiency, since no nitrate is accumulated at the final
effluent.
Cao et al. (2020) evaluated the feasibility of use primary sludge with
high biodegradable organic matter concentration and ammoniacal ni-
trogen as an alternative source to the denitrification coupled with the
Anammox. The organic matter and ammonia removal were successfully
achieved, being the detected organisms related to carbon degradation
besides Candidatus Brocadia (Anammox).
Guo et al. (2020b) demonstrated the efficiency of a single-phase re-
actor for simultaneous SNAD and COD oxidation (SNADCO) to treat
ammonium-rich wastewater with high content of biodegradable OC.
However, it was favorable when the C/N ratio do not exceed 0.8
(200 mg COD L−1), due to the suppressed bacteria activity at higher
concentrations.
The wastewater treatment of raw leachate (COD = 8258.7 ±
175.5 mg L−1, BOD5 = 511.9 ± 14.1 mg L−1, ammonium = 3596.3 ±
99.5 mg L−1) by nitrification/denitrification was studied by Huang and
Lee (2021). By biomolecular analysis, they detected the presence of
Anammox bacteria, which played a significant role in the nitrogen re-
moval process achieved by the authors, proving the possibility of the co-
existence of such microorganisms, when controlled conditions are
imposed to the wastewater treatment. These achievements were also
found by Pan et al. (2020) in the treatment of swine wastewater
(COD < 300 mg L−1, C/N = 0.60), which detected Candidatus Brocadia
as the dominant Anammox bacteria.
It is possible to achieve until 80 and 76% of nitrogen and OC removal,
respectively, in SBR reactors with SNAD process treating digester liquor
of swine wastewater with COD/N ratio of 0.75 (387 ± 145 mg COD L−1).
However, the stable operation and efficiency of this is totally dependent
of controlled operational conditions, like the temperature (Daverey
et al., 2013).
In a single reactor, the Anammox coupled with the denitrification
could induce the competition between the autotrophic and heterotro-
phic organisms by the nitrogen compounds. The free energy (ΔG0) nec-
essary to the denitrification process (Eqs. (3) and (4)) is higher than the
Anammox (Eq. (5)), being consequently more favorable. Considering
9
Science of the Total Environment 786 (2021) 147390
such and the fact that the heterotrophic bacteria also show fast growth
when compared to the autotrophic organisms, an attractive alternative
is the use of the denitrification process as a pretreatment and/or post-
treatment approach to remove the organic matter from the wastewater
instead of allied in the same reactor (Kumar and Lin, 2010).
4.2.3. Effect of OC on Anammox microbial community
The increase of biodegradable organic carbon on Anammox-process
reactors also brings alterations on the microbial community present in
them. In a study conducted by Mojiri et al. (2019), the main Anammox
bacteria inside the reactor sludge were Candidatus Brocadia (26.81%)
and Candidatus Jettenia (2.36%). Comparing these results with the previ-
ously inoculated biomass, bacterial communities were altered after the
addition of COD. The percentage of Candidatus Brocadia increases in
wastewaters with high OC content and Candidatus Jettenia get lower.
On the other hand, in the research realized by Chini et al. (2019), chang-
ing the synthetic effluent for the effluent with organic carbon,
Candidatus Brocadia population decreased and Candidatus Jettenia per-
centage increased. Such contradictory results may indicate that popula-
tion change depends not only on the concentration of organic matter,
but also on the type of carbon (biodegradability of the organic matter),
which is consequence of the effluent characteristics. The effluent treated
by Chini et al. (2019) was from an anaerobic digestion of sludge and
swine wastewater, and Mojiri et al. (2019) evaluated changes in micro-
bial community using synthetic effluent, where the present COD came
from sources such as milk powder, yeast extract and peptone, enabling
to conclude that organic matter sources presented different
biodegradability.
In systems that operate as SNAD, having organic matter, aerobic and
microaerophilic conditions to promote the AOB growth and activity,
some Pseudomonas, Nitrosomonas-like organisms and denitrifies/
nitrifies are also detected (Kumar and Samadder, 2020).
5. Differences among N-removal processes
The conventional nitrification/denitrification biological N-removal
process is widely used to treat wastewaters and present some advan-
tages when compared to physicochemical processes. However, com-
pared to other biological N-processes, the nitrification step requires
high amount of energy; the denitrification needs a considerable amount
of organic carbon source; and the overall process release nitrous oxide
(N2O), which shows an environmental impact over 300 times higher
than CO2; producing also high amounts of sludge. It is worth mentioning
that PN-based process reduces the oxygen and organic carbon require-
ment around 25–60% and 40–100%, respectively (Giongo et al., 2018;
Griggs and Noguer, 2002; X. Liu et al., 2020; Z. Zhang et al., 2020).
Accordingly with the wastewater, biodegradable organic matter and
nitrogen concentration, the PN/A process can be a good alternative to
the wastewater treatment. Compared to the conventional route, this
concept of treatment can promote 60% reduction in aeration consump-
tion, 100% of carbon consumption and reduce 90% of sludge production.
Moreover, economic and environmental advantages are also obtained.
However, the process shows peculiarities in relation to competition be-
tween Anammox bacteria and NOB, and can be reversibly inhibited by
temperature, pH, salt, heavy metals, free ammonia and free nitrous
acid and dissolved oxygen (Chini et al., 2019; Z. Zhang et al., 2020). If
the wastewater in question shows some amount of biodegradable or-
ganic carbon concentration (but not enough to the traditional nitrifica-
tion/denitrification), the denitrification can be successfully combined
with the PN/A to use this substrate together with the nitrate from the
Anammox reaction (SNAD process). The SNAD process promotes better
total nitrogen removal efficiency, since there is the concomitantly re-
moval of ammonia (by AOB) nitrate (by heterotrophic) and nitrite (by
Anammox) (Y. Wang et al., 2019; Xu et al., 2021).
However, it is not clear the effect of some operational parameters in-
volved in the SNAD process for the involved microbial groups. The SNAD
G. Bonassa, A.C. Bolsan, C.E. Hollas et al.
3.0
Operating costs (US$ kg Nrem-1)
2.5
2.0
1.5
1.0
0.5
0.0
0 0.5
Fig. 4. Operating costs for effluents with different CODb/N ratios (— PN/A; - - - PN/D + A).
process requires an aerated environment, to provide the ammonia oxi-
dation to nitrite by the AOB. Consequently, as the system encompasses
two other anoxic groups (Anammox and heterotrophic), this condition
requires greater attention in relation to OD inhibition than the PN/A. Be-
sides, higher ammonia and COD can inhibit the AOB and Anammox ac-
tivity (X. Li et al., 2019; Sheng et al., 2020; Xu et al., 2018).
At the subsequent topic, some scenarios and implications of the bio-
degradable organic matter together with ammonia concentration pres-
ent in wastewaters will be put into practice. Then, the decision of which
is the best biological N-removal process can be optimally chosen. The
disadvantages of each process related to costs and substrate (organic
matter and nitrogen) are also taken into account.
6. Practical implications of the organic matter presence in the
anaerobic digester effluents
The choice of the most suitable process to remove nitrogen from an
anaerobic digester effluent will depend on its biodegradable COD/N
ratio (CODb/N). When enough biodegradable organic matter is available
to fulfill the denitrification requirements, nitrogen removal can be car-
ried out by the combination of nitrification or nitritation and denitrifica-
tion processes. In the case of applying nitrification, the minimum CODb/
N ratio required to achieve a full N removal efficiency is 5.8 g CODb/g N
while, if nitritation is used, this ratio would decrease to 3.5 g CODb/g N.
These ratios were calculated taking into account both the remaining
COD fraction from the denitrifying biomass growth and that released
during the decay of denitrifying and nitrifying bacteria in the anoxic
tank. For this, a typical anoxic tank volume fraction of 1/3 and an overall
solids retention time (SRT) of 2 d were considered (Mulder et al., 2001).
It would be expected that operate the system at higher SRTs would
a) 3000000
2600000
2200000
NPV (US$)
1800000
1400000
1000000
600000
200000
-200000
0 500 1000 1500
Nitrogen load (kg N d-1)
Fig. 5. a) Economic analysis (CODb/N: — 0.25; –··– 0,5; - - - 1; –·– 2); b) minimum inlet nitrogen load that makes favorable the application of the two-units system.
Science of the Total Environment 786 (2021) 147390
1 1.5 2 2.5
CODb/N (g g-1)
promote cell decay and, therefore, the availability of organic matter to
carry out the denitrification process. However, calculations showed
that the increase of the SRT has practically no effect on the N removal ef-
ficiency since the release of organic matter by biomass decay occurs
mainly in the nitrifying tank and it is aerobically consumed. This is
due to both the larger size of the nitrifying tank with respect to the
denitrifying one and the higher decay rate of bacteria under aerobic
conditions compared to anoxic ones.
When the CODb/N ratio of the anaerobic digester effluent is lower
than 3.5 g CODb/g N (Table 3), organic matter availability would limit
the denitrification process efficiency and, therefore, the application of
the Anammox process would be necessary to maintain a high N removal
efficiency. Nevertheless, if an Anammox-based N removal process is ap-
plied, it is desirable that the organic matter present is as low as possible
since its presence in one-unit PN/A systems, mostly used nowadays,
causes operational problems, requiring advanced control systems to
maintain operational stability (Ochs et al., 2021). Furthermore, the pres-
ence of organic matter would also increase operating costs because its
removal would be carried out aerobically, which would increase oxygen
consumption and sludge production (Giustinianovich et al., 2016). In
this sense, it is worth mentioning that the first PN/A system operated
at full scale was a two-units system (Rotterdam WWTP, The
Netherlands) since it took advantage of an already installed SHARON re-
actor to carry out the partial nitrification process (Mulder et al., 2001).
Prior to the implementation of the Anammox reactor in this WWTP,
the SHARON reactor was operated as a partial nitrification/denitrifica-
tion (PN/D) system and, for this, an external source of organic matter
was added. Therefore, in the case that there is a significant concentra-
tion of biodegradable organic matter in the effluent of an anaerobic di-
gester, perhaps it is worth considering taking advantage of it to carry
b) 600
Inlet nitrogen load (kg N d-1)
450
300
150
0
2000 2500 0 0.5 1 1.5 2 2.5
CODb/N (g g-1)
10
G. Bonassa, A.C. Bolsan, C.E. Hollas et al.
out a partial removal of nitrogen in a PN/D unit and the effluent from
this system is treated by an Anammox reactor (A). Logically, this
would mean using two units to remove nitrogen, which would increase
capital costs, but would allow decreasing operating costs and improving
the operational stability. Therefore, from an economic point of view, the
application of a two-units system (PN/D + A) compared to that of a
one-unit system (PN/A) would be conditioned by the fact that the sav-
ings in operating costs could offset the increase of capital costs.
In this sense, an economic analysis was done in order to determine
the application niche of both one-unit and two-units systems in terms
of the inlet CODb/N ratio (0.25–2 g CDOb/g N) and the N load treated
(25–2500 kg N/d) (shown in Supplementary Material). As can be seen
in Fig. 4, the higher CODb/N ratio the higher difference between the op-
erating costs of both units, would indicate that the economic feasibility
of applying the two-reactor system would be favored as the inlet CODb/
N ratio increases. It should be noted that the savings in operating costs
vary practically in a linear way between 3.5 and 13.9% for the CODb/N
ratios studied. The economic analysis shows that for an inlet CODb/N
ratio of 0.25, the net present value (NPV) of the investment is always
negative (Fig. 5a) while, in the case of an anaerobic digester effluent
with an inlet CODb/N ratio of 0.5, the NPV turns positive when inlet ni-
trogen load is higher than 590 kg N/d. When this ratio increased up to 1
and 2, the application of a two-units system would be economically vi-
able for treating nitrogen loads higher than 170 and 50 kg N/d, respec-
tively (Fig. 5b).
The nitrogen removal efficiency of anaerobic digester effluents is
generally limited by the availability of alkalinity to carry out the partial
nitrification process. In this sense, the application of a (PN/D +
A) system would be favored by the partial recovery of alkalinity during
the denitrification process (50% of the alkalinity consumed during am-
monia oxidation). As the inlet CODb/N ratio increases, an increase of
the extent of the partial nitrification process is expected and, therefore,
the nitrogen removal efficiency would improve (Fig. 6). The N removal
efficiency increases by increasing the inlet CODb/N ratio whether a two-
units or a one-unit system is used, but this increase is greater in the case
of the two-units system. This is due to the fact that, in the one-unit sys-
tem, the increase of the N removal is caused by the ammonia assimila-
tion by the heterotrophic biomass generated, while in the case of the
two-units system this increase is due both to the ammonia assimilation
by denitrifying biomass as well as the denitrification process. For an
inlet CODb/N ratio of 2, the N removal efficiency of the two-units system
would be 4.6% higher than that of the one-unit system. It must be
remarked that the outlet nitrite/ammonia ratio of the PN/D unit is
around 1 (1.03–1.13) for the inlet CODb/N ratios between 0.25 and 1
but this ratio increases up to 1.54 in the case of an inlet CODb/N ratio
of 2. This supposes that ammonia would be the limiting substrate of
the Anammox process for this latter inlet CODb/N ratio and nitrite pres-
ence is expected which could cause Anammox biomass inhibition
(Dapena-Mora et al., 2007) or its floatation (Campos et al., 2017).
100
95
N removal efficiency (%)
90
85
80
75
70
0 0.5 1 1.5 2 2.5
CODb/N (g g-1)
Fig. 6. N removal efficiency achieved at different inlet CODb/N ratios (— PN/A; - - · PN/D + A).
11
Science of the Total Environment 786 (2021) 147390
Therefore, in the case of an inlet CODb/N ratio of 2, a control of the PN/
D unit operation could be advisable to maintain its outlet nitrite/ammo-
nia ratio lower than the stoichiometric one (1.32).
7. Conclusion
The Anammox bacteria are effectively suppressed by the wastewa-
ters with high biodegradable organic carbon concentration and hetero-
trophic bacteria. However, these microorganisms can opportunely be
combined with some routes due to its mixotrophic characteristics. Be-
sides, this review paper provided an overview of the current state of re-
searches and developments on nitrogen removal process (conventional
– nitrification/denitrification or Anammox-based process) based on ef-
fluents with different organic matter concentration, biodegradability
and bioavailability. The main progress of this review is the comprehen-
sive analysis of Anammox-based process treating effluents from differ-
ent sources. Based on the organic matter concentration remained on
it, pretreatment steps are required to be coupled to the partial
nitritation/Anammox to realize the nitrogen removal with high stability
and efficiency.
According with some scenarios created based on different CODb/g
N ratios, we concluded, from the point of view of N-removal effi-
ciency and economic viability, that: the minimum CODb/g N neces-
sary to successfully apply the nitrification/denitrification process is
5.8 g; for nitration the minimum ratio is 3.5 g, and for values below
this ratio the Anammox-based processes is recommended, of one
or two phases (i.e. PN/A). It depends on the concentration of nitrogen
and organic carbon in the effluent. Thus, for satisfactory nitrogen re-
moval, it is recommended that the choice of the N-removal process
should be based on some parameters of the effluent, mainly the am-
moniacal nitrogen and biodegradable organic carbon concentration
(CODb/g N).
CRediT authorship contribution statement
Gabriela Bonassa. Conceptualization, Investigation, Resources, Data
Curation, Writing - Original Draft, Visualization.
Alice Chiapetti Bolsan. Conceptualization, Investigation, Data
Curation, Visualization.
Camila Ester Hollas. Conceptualization, Investigation, Resources,
Data Curation, Writing - Original Draft, Visualization.
Bruno Venturin. Conceptualization, Investigation, Data Curation,
Visualization, Validation, Formal analysis.
Daniela Candido. Conceptualization, Investigation, Data Curation,
Visualization.
Angélica Chini. Conceptualization, Visualization.
Marina C. De Prá. Conceptualization, Visualization, Writing - Review
& Editing.
Fabiane Goldschmidt Antes. Conceptualization, Visualization, Writ-
ing - Original Draft, Writing - Review & Editing, Funding acquisition.
José Luis Campos. Formal analysis, Data Curation, Writing - Original
Draft.
Airton Kunz. Writing - Review & Editing, Project administration,
Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
Authors thanks financial support from CAPES, CNPq, SISTRATES
FUNTEC-BNDES (grant number 15.2.0837.1) and ANID/FONDAP/
15130015.
G. Bonassa, A.C. Bolsan, C.E. Hollas et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2021.147390.
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