Environ Sci Pollut Res (2019) 26:1124–1141

DOI 10.1007/s11356-017-9185-0

WATER INDUSTRY: WATER-ENERGY-HEALTH NEXUS

Nitrate removal from drinking water with a focus on biological

methods: a review
Fariba Rezvani 1 & Mohammad-Hossein Sarrafzadeh 1 & Sirous Ebrahimi 2 &
Hee-Mock Oh 3

Received: 23 November 2016 / Accepted: 2 May 2017 / Published online: 31 May 2017
# Springer-Verlag Berlin Heidelberg 2017

Abstract This article summarizes several developed and in-
dustrial technologies for nitrate removal from drinking water,
including physicochemical and biological techniques, with a
focus on autotrophic nitrate removal. Approaches are primar-
ily classified into separation-based and elimination-based
methods according to the fate of the nitrate in water treatment.
Biological denitrification as a cost-effective and promising
method of biological nitrate elimination is reviewed in terms
of its removal process, applicability, efficiency, and associated
disadvantages. The various pathways during biological nitrate
removal, including assimilatory and dissimilatory nitrate re-
duction, are also explained. A comparative study was carried
out to provide a better understanding of the advantages and
disadvantages of autotrophic and heterotrophic denitrification.
Sulfur-based and hydrogen-based denitrifications, which are
the most common autotrophic processes of nitrate removal,
are reviewed with the aim of presenting the salient features
of hydrogenotrophic denitrification along with some draw-
backs of the technology and research areas in which it could
be used but currently is not. The application of algae-based
water treatment is also introduced as a nature-inspired ap-
proach that may broaden future horizons of nitrate removal
technology.
Keywords Drinking water . Nitrate removal technology .
Autotrophic and heterotrophic denitrification . Assimilatory
and dissimilatory nitrate reduction . Cost . Microalgae

Highlights
• Technical and cost comparison of several approaches of nitrate removal
from drinking water, including physicochemical and biological Abbreviations
techniques USEPA United States Environmental Protection Agency
• Expression of the salient features of autotrophic nitrate removal, MCL Maximum contaminant levels
especially hydrogenotrophic denitrification over other methods WHO World Health Organization
• Refer to the application of microalgae-based water treatment as a
nature-inspired approach for water treatment EEC European Economic Community
RO Reverse osmosis
Responsible editor: Gerald Thouand
IX Ion exchange
ED Electro dialysis
* Mohammad-Hossein Sarrafzadeh
sarrafzdh@ut.ac.ir
CD Chemical denitrification
BD Biological denitrification
* Hee-Mock Oh
heemock@kribb.re.kr
SBA Strong base anion
DO Dissolved oxygen
1
UNESCO Chair on Water Reuse, Biotechnology Group, School of
ATP Adenosine triphosphate
Chemical Engineering, College of Engineering, University of HRT Hydraulic retention time
Tehran, P.O. Box: 11155-4563, Tehran, Iran COD Chemical oxygen demand
2
Biotechnology Research Centre, Faculty of Chemical Engineering, BOD Biological oxygen demand
Sahand University of Technology, Tabriz, Iran C/N Carbon-to-nitrate
3
Cell Factory Research Centre, Korea Research Institute of Bioscience DOC Dissolved organic carbon
and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea DBP Disinfection byproduct
Environ Sci Pollut Res (2019) 26:1124–1141
BER Bio-electrochemical reactor
O&M Operations and maintenance
Introduction
Nitrate pollution of drinking water supplies, especially
groundwater, primarily as a result of agricultural activity (in-
cluding excess application of inorganic nitrogenous fertilizers
and manures), wastewater treatment, and oxidation of nitrog-
enous waste products in human and animal excreta including
septic tanks, has become a critical global issue (USEPA 1987;
Kapoor and Viraraghavan 1997; Shrimali and Singh 2001;
Karanasios et al. 2010; Seitzinger et al. 2010). The increasing
use of artificial fertilizers, the disposal of wastes (particularly
from animal farming), and changes in land use are the main
factors responsible for the progressive increase in nitrate
levels in groundwater supplies over the past 20 years (WHO
1985b; Karanasios et al. 2010). Because nitrate (NO3−–N) is
highly soluble and mobile, excess NO3−–N can be released to
surface and groundwater, thereby decreasing the water quality
(Seitzinger et al. 2010; Chintala et al. 2013; Weigelhofer and
Hein 2015). Currently, nitrate is a major water pollutant in
many areas of the world, such as Saudi Arabia, India, the
UK, North America, Australia, Morocco, Changshu in
China, and Iran (Mohseni-Bandpi et al. 2013).
An increased nitrate concentration in drinking water sup-
plies causes methemoglobinemia in infants and cancer of the
alimentary canal; no other group of carcinogens produces
such an extensive variety of tumors (Ovez 2006; Ashok and
Hait 2015). As a result, various agencies have established
relevant standards for the nitrate content in drinking water.
For example, the United States Environmental Protection
Agency (USEPA) has established a maximum contaminant
level (MCL) of 10 mg nitrate–nitrogen (NO3−–N)/L, whereas
the World Health Organization (WHO) and European
Economic Community (EEC) have established a standard of
11.3 mg NO3−–N/L; the latter has been adopted by most coun-
tries as their national standard for drinking water (WHO
1985b; USEPA 1987; Tsai et al. 2004; Li et al. 2013; Wang
et al. 2013).
Removing nitrate from drinking water using conventional
treatment methods, such as coagulation and filtration, is al-
most impossible owing to the high stability and solubility of
nitrate and its low potential for coprecipitation or adsorption in
water (Luk and Au-Yeung 2002; Weigelhofer and Hein 2015).
Because various types of contamination in water and waste-
water require a variety of strategies to remove contamination,
industrial technologies of brine treatment involve the removal
of dissolved salt ions from the waste stream. These tech-
niques, which are referred to as separation-based methods,
can be applied for nitrate removal from other types of water
as well. However, these technologies remove all of the ions in
1125
the medium and cannot selectively remove nitrate (Kapoor
and Viraraghavan 1997; Luk and Au-Yeung 2002; Pérez-
González et al. 2012; Giwa et al. 2017). Industrial biological
nutrient removal that is applied for removing biodegradable
organic matter from wastewater, which is referred to as an
elimination-based method, can also be used as a selective
method for removing nitrate during the multi-purpose treat-
ment of water (Samatya et al. 2006; Karanasios et al. 2010).
Although these technologies each have their own strengths
and drawbacks, the ultimate feasibility is weighted against
factors such as cost, water quality improvement, residual han-
dling, and post-treatment requirements (Jensen et al. 2014).
Elimination-based methods are generally preferred to
separation-based methods as they completely remove nitrate
by converting it into nitrogen gas; however, current treatment
technologies and the development of emerging technologies
for nitrate removal from drinking water require additional op-
timization (Matějů et al. 1992; Kapoor and Viraraghavan
1997; Moreno et al. 2005; Sharma and Sobti 2012;
Weigelhofer and Hein 2015). Table 1 lists a variety of full-
scale nitrate removal systems that have been developed in
different countries.
Separation-based processes, including reverse osmosis, ion
exchange, electrochemical reduction, electrodialysis, and ac-
tivated carbon adsorption, which merely separate and transfer
nitrate to a waste stream (Soares 2000; Sharma and Sobti
2012; Wang et al. 2013), have expensive operating costs,
and the subsequent disposal of the generated waste brine pre-
sents another problem (Matějů et al. 1992; Volokita et al.
1996b; Gómez et al. 2000). The brine produced from these
technologies cannot be automatically recovered unless it is
combined with biological, chemical, or catalytic denitrifica-
tion, which facilitate nitrate removal from the waste concen-
trate (Chen et al. 2014a, b, 2015; Jensen et al. 2014).
In contrast, chemical and biological denitrification facili-
tates the complete removal of nitrate by conversion during
these processes to harmless nitrogen gas (Shrimali and
Singh 2001; Luk and Au-Yeung 2002; Sharma and Sobti
2012). However, a full-scale system of chemical denitrifica-
tion has not been reported, owing to the formation of ammonia
and the potential health hazards of exposure to chemical com-
pounds, such as electron donors and active catalysts (Zhang
et al. 2003; Kumar and Chakraborty 2006; Jensen et al. 2014).
Biological denitrification, one of the main approaches of bio-
logical nitrate removal within the dissimilatory pathway, has
been shown to be economical and environmentally sound,
owing to its selective removal capability for the complete
elimination of nitrate, formation of harmless end products,
relatively lower generation of waste brine (Volokita et al.
1996b; Soares 2000; Moreno et al. 2005; Della Rocca et al.
2006; Weigelhofer and Hein 2015).
Biological nitrate removal, which is driven by either het-
erotrophic or autotrophic microorganisms, can be classified as
1126
Table 1 Nitrate removal
technologies applied in several Technology Location
countries for water treatment
(adapted from Jensen et al. 2014)
IX California, US
Indian Hills, CO
Chino, CA
RO Bakersfield, CA
Brighton, CO
Arlington Desalter, Riverside, CA
ED Spain
BD Germany (H2, CO2)
Poland (ethanol)
France (H2)
California, US (Microvi co.)
assimilation into biomass and dissimilation into nitrogen gas.
Heterotrophic microorganisms require an organic carbon
source as electron donor to rapidly grow and take up nitrate
as electron acceptor; however, this process requires additional
carbon and causes secondary pollution (Chen et al. 2015). In
contrast, autotrophic microorganisms use inorganic carbon,
from sources such as CO2 and HCO3−, and inorganic matter,
such as H2, reduced sulfur, and Fe2+, as carbon sources and
electron donors, respectively. Among them, photoautotrophs,
such as microalgae and cyanobacteria, use light as an energy
source to eliminate nitrates (Chen et al. 2014a, b, 2015). The
autotrophic technology has sparked the interest of many re-
searchers because this process is cost-effective, non-polluting,
and generates a low amount of biomass over heterotrophs.
Among autotrophic methods, sulfur-based and hydrogen-
based denitrification processes have attracted substantial at-
tention (Wang et al. 2013; Chen et al. 2014a, b, 2015;
Chung et al. 2014).
This article summarizes some developed technologies for
nitrate removal from drinking water and presents basic prin-
ciples of biological nitrate removal that distinguish
hydrogenotrophic denitrification from other methods. The fu-
ture development of new nitrate removal technologies is in-
troduced on the basis of the application of a photosynthetic
system as a nature-inspired approach and using hydrogen as
the best electron donor as long as addressing the relevant
drawbacks.
Separation-based technologies for nitrate reduction
Reverse osmosis
Reverse osmosis (RO) can be used to simultaneously address
multiple contaminants, including ionic (e.g., nitrate, arsenic,
sodium, chloride, and fluoride), particulate (e.g., asbestos and
Environ Sci Pollut Res (2019) 26:1124–1141
Capacity Avg. influent nitrate mg/l
(m3/h) as NO3− (mg/L as N)
91 31–53 (7–12)
12 53–71 (12–16)
1136 40–200 (9–45)
27 75–84 (17–19)
1045 49–89 (11–20)
1041 44–89 (10–20)
741 80 (18)
90 17–19 (4–5)
500 44–89 (10–20)
180 84–89 (19–20)
more than 315 more than 100–150(22–33)
Protozoan cysts), and organic constituents (e.g., some pesti-
cides) (Cevaal et al. 1995; Bohdziewicz et al. 1999; Bellona
et al. 2008). In this process, water is forced through a semi-
permeable membrane under pressure to enable the water to
pass while the contaminants are impeded by the membrane
without any selection. The required pressure is dependent on
the solute concentration in the feed water. However, the col-
lected concentrate contains high levels of nitrate and other
rejected constituents (salts) and requires appropriate disposal
(Malaeb and Ayoub 2011; Jensen et al. 2014). Nitrate can be
removed by RO at pressures that range from 2000 to
10,000 kPa to reverse the normal osmotic flow of water.
However, other key aspects of the system, such as membrane
configuration, membrane flux rate, number of stages/number
of membranes, flow rate, and cleaning and anti-scalant re-
quirements, should be considered to ensure the high quality
of treated water (Jensen et al. 2014).
The membranes can be composed of cellulose acetate,
polyamides, or composite materials (Schoeman and Steyn
2003; Malaeb and Ayoub 2011; Sharma and Sobti 2012).
The life of the RO membranes and prefilters and the frequency
of membrane cleaning are directly dependent on the water
quality and the efficiency of pretreatment measures (Jensen
et al. 2014). Problems associated with RO membranes include
fouling, compaction, and deterioration over time.
Consequently, the treatment efficiency can be compromised
by membrane fouling (Darbi et al. 2003; Jensen et al. 2014).
The high nitrate concentration in disposal has prompted re-
search on the optimization of recycling and treatment of this
waste stream (Malaeb and Ayoub 2011). The coupling of RO
systems with biological, chemical, or catalytic denitrification
enables the removal of nitrate from the waste concentrate,
with reduction to nitrogen gas (Jensen et al. 2014).
Although RO can be used to treat water containing a high
nitrate concentration with a nitrate removal efficiency of 59 to
95%, RO is not a selective method for nitrate removal, and the
Environ Sci Pollut Res (2019) 26:1124–1141
permeate water is considerably softened and corrosive, owing
to a large decrease in the minerals in distilled water, thus
requiring a remineralization stage involving the addition of
ions to achieve standard levels in drinking water (Kapoor
and Viraraghavan 1997; Darbi et al. 2003; Malaeb and
Ayoub 2011; Jensen et al. 2014).
Ion exchange
Conventional ion exchange (IX, (the most common method
for nitrate removal, utilizes a strong base anion (SBA) ex-
change resin. In this process, the raw water is pretreated to
remove suspended solids and other constituents that are capa-
ble of fouling the resin column (Liu and Clifford 1996;
Ruppenthal 2004). The nitrate-laden treatment stream subse-
quently enters the ion exchange vessel. After contact with the
resin, nitrate displaces chloride at the surface sites and
removes nitrate from the water (Kapoor and Viraraghavan
1997; Samatya et al. 2006; Kalaruban et al. 2016). The
exhausted resin is regenerated using a concentrated solution
of sodium chloride or sodium bicarbonate (Kapoor and
Viraraghavan 1997). The IX process removes not only nitrate
but also sulfate from the water during treatment (Eliassen et al.
1995; McAdam and Judd 2008). Reports on 15 IX plants in
the USA have demonstrated a decrease in nitrate from 18 to
6.8 mg/L (Kapoor and Viraraghavan 1997; Ruppenthal 2004;
Sharma and Sobti 2012; Jensen et al. 2014); however, IX is
limited by two major problems. First, resin with a high selec-
tivity for the nitrates that are commonly present in groundwa-
ter is not available. Second, adequate resin regeneration is
required to prevent the problem of disposal (Kapoor and
Viraraghavan 1997; Darbi et al. 2003; Ruppenthal 2004;
McAdam and Judd 2008). To solve this problem, IX can be
coupled with BD to optimize the total efficiency of nitrate
removal. In this case, nitrate is removed by IX, whereas the
regeneration of the rich-nitrate-load resin is conducted in a
closed circuit using BD (van der Hoek et al. 1988; Mohseni-
Bandpi et al. 2013). Modifications of conventional IX have
caused the emergence of more efficient IX processes, includ-
ing multiple vessel and counter current configurations, the use
of specialized resin-improved hydraulics, and weak base an-
ion exchange (WBA IX) (Jensen et al. 2014).
Electrodialysis
Electrodialysis (ED) is a desalting process driven by the
electrical potential difference between two oppositely
charged electrodes. ED combines the advantages of
electivity and low chemical demand, which is less than
the chemical demand of RO (Nataraj et al. 2006;
Banasiak and Schafer 2009; Sharma and Sobti 2012).
Similarly to RO, ED cannot selectively separate nitrate
and requires some post-treatment for remineralization
1127
(Jensen et al. 2014). A schematic of ED stock with
anion and cation exchange membranes is shown in
Fig. 1. According to Fig. 1, nitrate removal is accom-
plished by passing an electrical current through a series
or stack of anion and cation exchange membranes, thus
resulting in the movement of nitrate ions from the feed
solution to a concentrated waste stream (Nataraj et al.
2006). The voltage applied to the membrane cells deter-
mines the degree of desalination and hence the degree
of nitrate removal (Hell et al. 1998). Although ED can
be used to produce drinking water from nitrate-rich wa-
ter, a high concentration of nitrate-nitrogen in the dis-
posal stream remains (Jensen et al. 2014).
One of the main advantages of ED is a higher recov-
ery than that for RO (Jensen et al. 2014). However, the
results of their recovery efficiency differ; Cevaal et al.
(1995) have reported that RO is appropriate for treat-
ment of groundwater with a high concentration of ni-
trate, TDS, and trichloromethane concentrations in
Brighton, Colorado, and Canada, because the residents
of these cities would not have to use ion-exchange soft-
ening units (Cevaal et al. 1995). Both ED and RO are
not selective for nitrate removal and generate highly
concentrated wastes that require careful disposal (Hell
et al. 1998; Pirsaheb et al. 2015). Because ED is more
complex than other technologies in terms of launch and
construction (Rautenbach et al. 1987; Hell et al. 1998;
Midaoui et al. 2002; Pirsaheb et al. 2015), few studies
have investigated denitrification using ED on a large
scale (Banasiak and Schafer 2009; Sharma and Sobti
2012; Mohseni-Bandpi et al. 2013; Jensen et al. 2014).
A full-scale ED plant using anion exchange membranes
was designed and built by Austrian Energy in 1997.
Although the plant removed 66% of the feed water ni-
trate, it was closed after several months of operation,
owing to problems associated with waste disposal in
the local sewage treatment system (Hell et al. 1998;
Jensen et al. 2014).
Elimination-based technologies for nitrate removal
Chemical denitrification process
Chemical denitrification (CD) is accomplished by reducing
nitrate using metals such as aluminum and iron (both Fe0
and Fe2+). Copper, palladium, and rhodium can also be used
as catalysts in nitrate reduction (Kapoor and Viraraghavan
1997; Shrimali and Singh 2001; Jensen et al. 2014).
However, a simple chemical method that can reduce nitrate
to nitrogen gas at a low temperature and pressure is not avail-
able (Luk and Au-Yeung 2002; Zhang et al. 2003). The ge-
neric mechanism of denitrification involves the transfer of
1128
Fig. 1 Schematic diagram of ED
stock including cation and anion
exchange membrane (adapted
from Jensen et al. 2014)
electrons from an electron-donating metal to nitrate (Eq. 1). In
some cases, the occurrence of partial denitrification causes the
formation of ammonium, as shown in Eq. (1a) (Hao et al.
2005).
NO3 − →NO2 − →NO→N2 O→N2 ð1Þ
NO3 − →NO2 − →NH4 þ ð1aÞ
The commercialization of catalytic denitrification technology
is primarily inhibited for two reasons: first, the difficulty of
modulating the activity of catalysts to prevent ammonia for-
mation, and second, the limitation of the reaction rate by dif-
fusion in large particles (Zhang et al. 2003; Kumar and
Chakraborty 2006). Although the use of powdered or fiber
catalysts can eliminate the latter problem, the use of a pow-
dered catalyst is restricted, owing to the high-pressure de-
crease in fixed beds and the difficulty in separating a
suspended powder catalyst (Shrimali and Singh 2001; Hao
et al. 2005).
The advantage of CD over separation-based technologies is
that nitrate is converted to other nitrogen species rather than
being simply displaced into a concentrated waste stream that
requires disposal (Kapoor and Viraraghavan 1997; Kumar and
Chakraborty 2006; Jensen et al. 2014). However, the prob-
lems associated with the use of CD for drinking water include
the reduction of nitrate beyond nitrogen gas to ammonia and
the possibility of leaving chemical compounds in the water
that may have potential health hazards to humans (Shrimali
and Singh 2001; Hao et al. 2005). A full-scale CD system has
not been developed for the removal of nitrate from water
(Sharma and Sobti 2012; Jensen et al. 2014).
Although in the CD process, a metal is normally used as an
electron donor for reducing nitrate to nitrogen during exposure
Environ Sci Pollut Res (2019) 26:1124–1141
to a catalyst, a number of researchers have used hydrogen as
an electron donor and palladium as a catalyst for their research
in regard to performing brine treatment and nitrate removal
from water (Jensen et al. 2014). The use of palladium-based
catalysts and hydrogen gas as a reductant has been extensively
studied in the laboratory, but due to technical challenges, it has
only seen limited application in the real-world setting (Guy
et al. 2009; Chaplin et al. 2012).
Biological denitrification process
Biological denitrification (BD) is one of the most effective and
most extensively applied technologies for nitrate removal, ow-
ing to its selective nitrate reduction to harmless nitrogen gas
without the need for a disposal stream and remineralization
stage (Matějů et al. 1992; Soares 2000; Shrimali and Singh
2001). BD is one of the biological approaches of nitrate re-
moval that can be conducted in anaerobic conditions and with
a minimum amount of biomass produced as waste sludge
(Lew et al. 2012). However, the drawbacks of this technology
include potential contamination of the treated water with the
microorganisms used for BD and their metabolic byproducts
and a longer hydraulic retention time (HRT) for denitrification
(Shrimali and Singh 2001; Samatya et al. 2006).
The bioremediation of drinking water supplies can be di-
rectly performed in an aquifer or below ground (in situ) or
after withdrawal of water from a well in above-ground reac-
tors (ex situ) (USEPA 2013). The process of in situ treatment
usually consists of a central pumping well that is surrounded
by injection wells through which the substrate is injected
(Matějů et al. 1992; Della Rocca et al. 2007; Rivetta et al.
2008). The stable temperature in the ground is an advantage
Environ Sci Pollut Res (2019) 26:1124–1141
of this method, especially in cold climates. However, the prob-
lems are slow rates in aquifers, high risk of clogging, and
complicated control on substrate distribution, owing to the
inhomogeneity and lack of isotropy of aquifers (Matějů et al.
1992; Della Rocca et al. 2007). As a result, in situ treatment is
applicable only in certain limited geological conditions
(Robertson et al. 2000; Jensen et al. 2014). Above-ground
denitrification includes fixed film (attached growth) or
suspended growth. In the case of fixed film denitrification,
the organisms are attached to inert support media, and the goal
is to maximize the surface area available for development of
the biofilm (Matějů et al. 1992; Kapoor and Viraraghavan
1997; Soares 2000; Mohseni-Bandpi et al. 2013). These me-
dia include fluidized bed reactors, packed bed reactors, mem-
brane bioreactors, and biofilters composed of sand, anthracite,
activated carbon, calcium carbonate, and sulfur (Matějů et al.
1992; Mansell and Schroeder 2002; Wang et al. 2013; Aslan
and Turkman 2006; Vasiliadou et al. 2006). Fluidized bed
reactors tend to be preferred because they have higher denitri-
fication rates per reactor volume, and clogging and channeling
problems are not concerns. However, more process control
with a cleaning system, such as a centrifugal pump,
hydrocyclone, and screen for removing excess biomass and
returning the cleaned sand to the reactor, may be required to
prevent breakthrough of the biomass as well as post-filtration
requirements for removing contaminants which are carried by
gas flow (Matějů et al. 1992; Kapoor and Viraraghavan 1997;
Soares 2000; Di Capua et al. 2017).
Europe and the USA have constructed numerous pilot and
demonstration systems for biological nitrate removal. The first
above-ground denitrification reactor was installed in France in
1983, and installations in Germany and Italy followed (Green
and Shelef 1994). These systems include fixed bed and fluid-
ized bed reactors with different biomass supports. Ethanol,
methanol, acetate, cotton, hydrogen, sulfur, and natural gas
have been used or proposed as substrates for microbial deni-
trification, and phosphate is normally added as a nutritional
supplement (Green and Shelef 1994; Soares 2000; Della
Rocca et al. 2005). Denitrifying bioreactors facilitate denitri-
fication by supplying bacteria with an additional carbon
source in an oxygen-poor and nitrate-enriched environment
(Schipper et al. 2010a). The efficiency of the NO3−–N remov-
al in bioreactors is dependent on the retention time, the
amount and quality of the organic carbon source, the NO3−–
N concentration in the source water, the temperature, and pH
(Robertson et al. 2000; Greenan et al. 2009; Schipper et al.
2010a). A schematic of the BD process is shown in Fig. 2.
A comparison of physicochemical and biological
technologies
To identify the most efficient remedial techniques, Table 2
summarizes the physicochemical and biological technologies
1129
on the basis of their different characteristics. BD is increasing-
ly being considered as an alternative process for developed
nitrate removal technologies from drinking water (USEPA
2013; Sahinkaya et al. 2015). In contrast to physicochemical
methods, BD offers high water recovery by the complete and
selective reduction of nitrate to nitrogen gas and multi-
purpose treatment of water; no brine or concentrate waste
stream; low sludge waste, owing to low biomass production
in anaerobic conditions; less-expensive operation; limited
chemical input; multiple contaminant removal; and favorable
sustainability (Matějů et al. 1992; Kapoor and Viraraghavan
1997; Vasiliadou et al. 2006; Moon et al. 2008; Karanasios
et al. 2010). Although BD is a proven and economically ef-
fective method for nitrate removal (Zhao et al. 2011;
Sahinkaya et al. 2015), the problem of remnant microorgan-
isms and their metabolic byproducts in the treated water re-
quires disinfection or post-treatment by filtration and sustain-
able requirements of carbon sources (Matějů et al. 1992;
Soares 2000; Shrimali and Singh 2001; Samatya et al. 2006;
Mohseni-Bandpi et al. 2013; Chen et al. 2015). In addition,
low production rates of nitrogen for 1 day, owing to higher
start-up time and HRT, and cold temperature restrictions may
also be disadvantages of BD (Kapoor and Viraraghavan 1997;
Samatya et al. 2006). In addition to intensive post-treatment,
this process requires careful control and adequate monitoring
to ensure stable operation (Lina et al. 2002). To address these
concerns, several BD treatment configurations have been de-
veloped, such as the application of chemoautotrophic denitri-
fication and constructed wetlands for removing the organic
carbon demand (refer to the section BStrengths and weak-
nesses of heterotrophic and autotrophic nitrate removal^)
(Soares 2000; Shrimali and Singh 2001; Lina et al. 2002;
Samatya et al. 2006; Lin et al. 2008; Burghate and Ingole
2014; Chen et al. 2014a, b).
Available technical data, experience, and economics indicate
that IX and BD are more acceptable for nitrate removal than RO
and ED (Kapoor and Viraraghavan 1997; Jensen et al. 2014;
Pirsaheb et al. 2015; Sahinkaya et al. 2015). Thus, the only
method that is competitive with BD is IX. However, the main
disadvantage of IX is the production of high volumes of brine
during the regeneration of the resin. This brine contains high
chloride, bicarbonate, sulfate, and nitrate concentrations, which
hinder disposal (Kapoor and Viraraghavan 1997; Shrimali and
Singh 2001; Burghate and Ingole 2014).
The approximate range of costs for specific facilities is
shown in Fig. 3. The costs in a case study are provided in a
full-scale system with a capacity of 190,000 m3/day for nitrate
removal from water, on the basis of published articles in which
the costs have been adjusted to 2016 US dollars with 7%
interest over 26 years, unless otherwise indicated (Conlon
et al. 1995; Jensen et al. 2014). The most economical and
expensive method regarding both operations and maintenance
(O&M) and capital costs are BD and RO, respectively, in
1130
Fig. 2 Schematic diagram of biological denitrification process including pretreatment and post-treatment steps (adapted form Jensen et al. 2014)
which biological nitrate removal is achieved by heterotrophic
denitrification and includes various considerations, such as the
sustainable addition of an external carbon source and the separa-
tion of the residual from the treated water. Autotrophic denitrifi-
cation is not subject to such considerations; hence, its costs are
probably lower than the costs of a heterotrophic process.
Major concerns regarding RO and ED systems are the costs
dictated by both energy consumption and membrane replace-
ment; more than approximately 25% of the total cost in both
systems is attributed to the membrane and its maintenance and
energy consumption (Rautenbach et al. 1987; Kapoor and
Viraraghavan 1997; Schoeman and Steyn 2003; Malaeb and
Ayoub 2011; Jensen et al. 2014; Pirsaheb et al. 2015).
However, comparison of RO and ED yields different results;
Pirsaheb et al. (2015) have reported that although the total cost
of ED in small systems (<960 m3/day) is higher than the total
cost of RO system, cost of large systems for both ED and RO
may be similar and are dependent on the site details (Pirsaheb
et al. 2015). According to Fig. 3, IX is more economical than
RO and ED; as previously mentioned, this process is hindered
by two major problems (Eliassen et al. 1995; Darbi et al.
2003). Consequently, RO, ED, and IX are relatively expensive
processes and merely displace nitrate into concentrated waste
brine, thus posing another disposal problem (Kapoor and
Viraraghavan 1997; Jensen et al. 2014).
The design and cost considerations are addressed on the basis
of the development of guidelines for determining the most ap-
propriate treatment option according to the source water quality
and other water system characteristics (Rautenbach et al. 1987;
Hamlin et al. 2008; Jensen et al. 2014). Costs can be difficult to
assess because of inconsistencies in the reporting of the cost
information. A comparison of treatment costs is not always valid,
owing to differences in the influent water quality parameters,
system size, waste management options, and system configura-
tion (Jensen et al. 2014).
Basic principles of biological nitrate removal
The elimination of nitrate in biological technology, in which
nitrate is passed from two pathways of assimilation and
Environ Sci Pollut Res (2019) 26:1124–1141
dissimilation for consumption in both heterotrophic and auto-
trophic denitrifiers, is a controversial topic (Soares 2000; Della
Rocca et al. 2006; Lew et al. 2012; Burghate and Ingole 2014).
Assimilatory nitrate reduction
Nitrogen is assimilated during the growth of all forms of
microbes including heterotrophs and autotrophs (Burghate
and Ingole 2014). Assimilatory nitrate reduction reduces ni-
trate to ammonia for cell synthesis, where it is independent
of the dissolved oxygen (DO) concentration, and transpires
when NH4+–N is not available (Hill 1996; Burghate and
Ingole 2014; Krapp et al. 2014). Heterotrophs obtain relative-
ly large amounts of energy from degrading organic matter,
thus resulting in a high yield of biomass and the assimilation
of a significant amount of nitrogen (Burghate and Ingole
2014). Although many heterotrophic microorganisms can as-
similate nitrate for growth, they prefer to consume ammoni-
um as nitrogen source when it is present (Hill 1996). In
some environments, conversion to biomass growth is an im-
portant mechanism for nitrogen removal or short-term reten-
tion (Burghate and Ingole 2014). For example, Kelso et al.
(1999) have demonstrated that a maximum of 50% of nitro-
gen depleted from groundwater can be converted to biomass
in the presence of certain organic substrates.
Autotrophs require relatively large amounts of energy
to convert carbon dioxide to more complex organic
forms that are suitable for cell synthesis, which pro-
duces a low biomass yield per unit of acquired energy
and hence has low nitrogen requirements (Burghate and
Ingole 2014). A general reaction for autotrophs is
shown in Eqs. (2–2a).
ðNH4 þ or NO3 ‐ Þ þ CO2 ð2Þ
þ autotrophs energy




b Cell ProteinðOrganic NÞ þ H2 O
0:04NO3− þ 0:18CO2 þ 1:04Hþ þ e→0:04C5 H7 O2 N þ 0:39H2 O
ð2aÞ
where energy can be provided by either inorganic matter, such
as hydrogen, reduced sulfur, and iron, in chemoautotrophs, or
Table 2 Comparison of characteristics of different nitrate removal technologies

Technology Status Treatment Pre- Post-treatment Waste/ Start- Water Advantages Disadvantages References
type treatment needs residual up time recovery
needs management

IX Full scale Removal to Pre-filter, pH adjustment Waste brine Minutes Up to Nitrate selective resins, common
Potential for nitrate peaking, high chemical Darbi et al. 2003;
Environ Sci Pollut Res (2019) 26:1124–1141

waste address 97% application, multiple use (salt), brine waste disposal, potential Ruppenthal 2004;
stream hardness contaminant removal for disinfection byproduct (DBP) Samatya et al. 2006;
formation (e.g., NDMA) Kalaruban et al.
2016
RO Full scale Removal to Pre-filter, pH adjustment Concentrate Minutes Up to Multiple contaminant removal, Membrane fouling and scaling, lower Cevaal et al. 1995;
waste fouling Remineralization of nitrate 85% desalination (TDS removal) water recovery, operational complexity, Bohdziewicz et al.
stream control of water energy demands, waste disposal 1999; Darbi et al.
2003; Schoeman
and Steyn 2003;
Bellona et al. 2008
ED Full scale Removal to Pre-filter, pH adjustment Concentrate Minutes Up to Multiple contaminant removal, Energy demands, Operational complexity, Rautenbach et al. 1987;
waste fouling Remineralization of nitrate 95% higher water recovery (less waste disposal Hell et al. 1998;
stream control of water waste), desalination Nataraj et al. 2006
CD Research Chemical pH pH adjustment, iron Waste media, Minutes Not full No waste brine or concentrate, Inconsistency of nitrate reduction, risk of Cheng et al. 1997;
phase reduction adjustment removal, Iron scale nitrate reduction rather than nitrite formation (potential incomplete Chaplin et al. 2012;
potential sludge transfer to a waste stream, and denitrification), reduction to ammonia, Luk and Au-Yeung
ammonia control potential for multiple lack of full-scale systems, pH and tem- 2002; Huang and
contaminant removal perature dependence, possible need for Zhang 2004
iron removal
BD Full scale Biological pH Filtration, Biomass Days to Nearly No waste brine or concentrate, Substrate addition (organic carbon), Matějů et al. 1992;
reduction adjustment, disinfection, weeks 100% nitrate reduction rather than possible sensitivity to environmental Shrimali and Singh
nutrients possible substrate transfer to a waste stream, conditions, post-treatment to address 2001; Soares 2000;
and adsorption high water recovery, and turbidity and Sludge removal, Aslan and Turkman
substrate potential for multiple 2003
addition, contaminant removal
anoxic
conditions
1131
1132
800
O & M cost Capital cost
700
600
Cost ($/1000 m^3)
500
400
300
200
100
0
IX RO ED BD
Treatment type
Fig. 3 Capital, operation and maintenance (O&M) costs found in the
literatures for all nitrate removal systems except CD, with a capacity of
190,000 m3/day (Conlon et al. 1995; Jensen et al. 2014)
by light in photoautotrophs such as microalgae and
cyanobacteria. The vast majority of microorganisms prefer
nitrogen in the form of NH4+; however, several species of
bacteria and nearly all species of microalgae are able to use
nitrate after reducing it to NH4+ for biomass growth when
ammonium as a nitrogen source is lacking (Burghate and
Ingole 2014).
Dissimilatory nitrate reduction
Many bacteria are capable of anaerobic growth by reducing
ionic nitrogenous oxides to gaseous products (Ashok and Hait
2015). This respiratory process in which nitrate or nitrite in-
stead of oxygen serves as a terminal electron acceptor causes
the generation of adenosine triphosphate (ATP) and is termed
denitrification or dissimilatory nitrate reduction (Lew et al.
2012). In the dissimilation pathway, nitrate and nitrite are con-
sumed as electron acceptors for the oxidation of organic and
inorganic electron donors (Keith et al. 1982; Matějů et al.
1992). This pathway is important in most bacteria, because
the process involves energy conservation by increasing the
substrate level phosphorylation reaction (Keith et al. 1982).
When electrons are transferred from the donor to the acceptor,
the organism gains energy that can be applied to the synthesis
of new cell mass and the maintenance of the existing cell mass
(Burghate and Ingole 2014). A notable aspect of denitrifica-
tion is the effect of the DO concentration, which can inhibit
nitrate reduction by repressing the nitrate reduction enzyme.
Despite the vast majority of both heterotrophic and autotro-
phic denitrifiers (Matějů et al. 1992), the most detailed inves-
tigations of denitrification have involved a limited group of
specialized bacteria, and the results have indicated that deni-
trification occurs only under anaerobic conditions. However,
denitrification can also occur in the presence of oxygen in
certain species of bacteria (Burghate and Ingole 2014).
The enzymes associated with denitrification are synthe-
sized when conditions become advantageous for denitrifica-
tion. The reduction of nitrate to nitrogen gas proceeds in four
Environ Sci Pollut Res (2019) 26:1124–1141
steps, each catalyzed by a specific enzyme system (Matějů
et al. 1992; Lew et al. 2012).
NO3 − →NO2 − →NO→N2 O→N2 ð3Þ
BD describes the stepwise conversion of nitrate (NO3−) to
nitrogen gas (N2), Eqs. (4–7); it is commonly used for the
removal of nitrate from drinking water. However, the different
reaction steps are performed by different microorganisms
(Lew et al. 2012; Karanasios et al. 2010).
NO3 − þ 2e þ 2Hþ →NO2 − þ H2 O ð4Þ
NO2 − þ 2e þ 2Hþ →0:5N2 O þ 0:5H2 O þ OH− ð5Þ
þ
0:5N2 O þ e þ H →0:5N2 þ 0:5H2 O ð6Þ
A summary of these equations for nitrate reduction to nitrogen
gas reveals that five electrons are required; for every mole of
nitrate that is reduced, 1 mol of hydroxyl ion is released
(Eq. (7)).
NO3 − þ 5e þ 5Hþ →0:5N2 þ 2H2 O þ OH− ð7Þ
The end products of denitrification are nitrogen gas, new cell
biomass, and hydroxyl ion, as well as an increase in pH, which
is not a concern in the denitrification process (Lew et al.
2012). The electrons required for reducing nitrate are provided
by specific organic and inorganic electron donors in both het-
erotrophs and autotrophs (Matějů et al. 1992; Ashok and Hait
2015).
Heterotrophic nitrate removal
As mentioned above, most microbial denitrification systems
use heterotrophic bacteria, which requires an organic carbon
source, such as methanol, ethanol, glucose, or acetate. In these
systems, methanol, which is the most widely used and least
expensive exogenous carbon source, produces a lower bacte-
rial cell yield than do other carbon sources (Her and Huang
1995; Hamlin et al. 2008; Weigelhofer and Hein 2015).
However, some countries prohibit the use of methanol for
treating drinking water (Matějů et al. 1992; Soares 2000;
Aslan and Turkman 2003). Some researchers have used alter-
native substances, such as volatile fatty acids, shredded news-
paper, wheat straw, unprocessed short fiber cotton, atrazine,
natural gas methane, elemental sulfur, and sugar or glucose
syrup, as the organic carbon source (Aslan and Turkman
2003; Hiraishi and Khan 2003). Heterotrophic bacteria de-
grade organic carbon to obtain energy for cellular activity
and carbon for cellular synthesis (growth and reproduction).
The most common heterotrophic denitrifiers are various spe-
cies of Pseudomonas and Bacillus (Brezonik 1977).
In heterotrophic denitrification, both nitrate and organic
carbons such as methanol participate in respiration and anab-
olism reactions for cell synthesis. In a respiration reaction,
Environ Sci Pollut Res (2019) 26:1124–1141
nitrate is reduced to nitrogen gas, and methanol is converted to
water and carbon dioxide (Mohseni-Bandpi et al. 2013;
Burghate and Ingole 2014). In the case of methanol, the stoi-
chiometric relationships that describe the dissimilation reac-
tion of nitrate in heterotrophs are as follows (Jeris et al. 1974;
Zhao et al. 2011):
The bacteria energy reactions include the following:
Step 1 : 6NO3 − þ 2CH3 OH→6NO2 − þ 2CO2 þ 4H2 O ð8Þ
Step 2 : 6NO2 − þ 3CH3 OH→3N2 þ 3CO2 þ 3H2 O
þ 6OH− ð9Þ
Total respiratory reaction:
6NO3 − þ 5CH3 OH→3N2 þ 5CO2 þ 7H2 O þ 6OH− ð10Þ
In addition to the dissimilation reaction, nitrate is also used in
cell anabolism and forms a compound of C5H7O2N (Matějů
et al. 1992). Approximately 40% of the methanol and 10% of
the nitrate are consumed for cell anabolism (Liessens et al.
1993; Burghate and Ingole 2014). However, the actual amount
of methanol required in the influent is higher than that obtain-
ed from theoretical calculations (Mohseni-Bandpi et al. 2013).
The total assimilation reaction of nitrate when using methanol
is used as the carbon source that follows the following equa-
tion (Matějů et al. 1992; Mohseni-Bandpi et al. 2013):
NO3 − þ 1:08CH3 OH þ Hþ →0:065C5 H7 NO2
þ 0:468N2 þ 0:76CO2 þ 2:44H2 O ð11Þ
Under optimum conditions, the anoxic BD process con-
verts more than 95% of nitrate and nitrite into inert nitro-
gen gas. If the environment deviates from the optimum
conditions, other types of nitrogen gas can be produced,
including N2O, which is involved in the greenhouse effect
(Chiu and Chung 2003). The biological production of
N2O is affected by many parameters, such as the sludge
age, HRT, organic loading rate, type of organic carbon
source, dissolved oxygen (DO), pH, and temperature
(Brenner and Argaman 1992). However, the carbon-to-
nitrate (C/N) weight ratio appears to affect the production
of N2O more than any other parameter (Chiu and Chung
2003). A high carbon content in the influent is favorable
in the conversion of nitrate or nitrite into inert nitrogen
gas or the production of less N2O; however, an excessive
amount of organic carbon in the influent increases the
unused carbon in the denitrified effluent. Thus, the biolog-
ical oxygen demand (BOD) or chemical oxygen demand
(COD) in the treated effluent may substantially increase
and exceed the effluent limitations (Chiu and Chung
2003). If the C/N ratio of a BD system is maintained at
an appropriate level, the production of both N2O and or-
ganic carbon in the effluent will be minimized. This C/N
1133
ratio, which causes a maximum conversion of all nitrogen
compounds produced during the different steps of the re-
action such as N2O to the final nitrogen product with
minimum organic carbon, is defined as Bthe optimal C/N
ratio^ (Chiu and Chung 2003; McAdam and Judd 2007).
The optimal C/N ratio may be theoretically calculated by
using the stoichiometric relationship for the BD process. The
chemical equilibrium equation using acetic acid as the carbon
source has been suggested as follows (Matějů et al. 1992;
Chiu and Chung 2003):
0:819CH3 COOH þ NO3 − →0:068C5 H7 NO2 þ HCO3 −
þ 0:301CO2 þ 0:902H2 O þ 0:466N2 ð12Þ
According to Eq. (12), the reduction of 1 g of nitrate–nitrogen
(NO3−–N) theoretically consumes 3.51 g of acetate (or 3.74 g
of COD) and produces 0.55 g of new cells, 2.66 g of HCO3−
as alkalinity, and 0.82 l of inert nitrogen gas (Chiu and Chung
2003). As a result, the theoretical optimal C/N weight ratio is
calculated to be 3.74 for acetic acid, without competition from
other heterotrophs. Because the C/N ratio for other carbon
sources is dependent on the form of the carbon source and
the stoichiometric relationship (Matějů et al. 1992; Chiu and
Chung 2003) and the dependence of the amount of denitrified
nitrogen on the concentration of the carbon source in the me-
dium in the stoichiometric relationship is linear, the substrate-
to-nitrogen weight ratio is the most important parameter in the
case of heterotrophic denitrification (Matějů et al. 1992;
Mohseni-Bandpi et al. 2013).
Autotrophic nitrate removal
Autotrophic organisms including chemoautotrophs oxidize
inorganic matter and produce energy by delivering released
electrons to a terminal electron acceptor, such as nitrate in
denitrifiers, for their metabolism and growth. Under autotro-
phic growth conditions, carbon dioxide or bicarbonate is used
as a carbon source for microbial cell synthesis (Zhou et al.
2011; Ashok and Hait 2015). Many researchers have shown
that Thiobacillus denitrificans (T. denitrificans) and
T. thioparus can effectively remove nitrate from water by
using a reduced sulfur compound as the electron donor and
nitrate as the electron acceptor (Gayle et al. 1989; Moon et al.
2008). The typical reduced sulfur compounds are sulfides and
elemental sulfur (S2−, S2O3−3, and S0). The determinative bac-
teriology of Bergey’s Manual indicated that T. denitrificans is
the microorganism responsible for autotrophic denitrification.
This bacterium can reduce nitrate to nitrogen gas while oxi-
dizing elemental sulfur (Bergey et al. 1974; Brettar et al.
2006). Other major autotrophic bacteria include Micrococcus
denitrificans and Paracoccus denitrificans, which oxidize H2
by using nitrate as the terminal electron acceptor (Kurt et al.
1134
1987). Paracoccus and Thiobacillus denitrify by using hydro-
gen and sulfur or a reduced sulfur compound, respectively.
However, both can also grow heterotrophically if an organic
carbon is present and the preferable inorganic carbons are
absent (Waki et al. 1980; Claus and Kutzner 1985b; Kurt
et al. 1987). Bacteria from the genera Ferrobacillus,
Gallionella, Leptothrix, and Sphaerotillus can utilize ferrous
iron as an energy source for autotrophic denitrification.
F. ferrooxidans is strictly autotrophic and utilizes CO2 as the
source of carbon (Shrimali and Singh 2001). Some aerobic
autotrophic bacteria such as Thiobacillus thiooxidans can
completely oxidize inorganic sulfur for their energy source.
A comprehensive study of the respiration process of
T. denitrificans using thiosulfate as the electron donor has
been reported by Justin and Kelly (1978a).
There are six general physiological properties of autotro-
phic denitrifying bacteria: (1) growth occurs in a purely min-
eral medium and in the presence of an inorganic substance,
such as sulfur, that can be oxidized; (2) the vital activity of
microorganisms is closely related to the presence of an oxi-
dizable inorganic substance; (3) oxidation of an inorganic
substance is the sole source of energy; (4) an organic sub-
stance is not needed as a source of carbon and energy; (5)
autotrophic organisms are unable to decompose organic sub-
stances, and the presence of organic matter may slow the
development of autotrophic organisms; and (6) carbon dioxide
and bicarbonate are the sole sources of carbon (Lyubchenko
et al. 1996; Oh et al. 2001; Aslan and Turkman 2003;
Karanasios et al. 2010; Mohseni-Bandpi et al. 2013). Only a
few species of autotrophic micro-organisms can be used for
nitrate removal; these species are characterized by slow
growth, thus resulting in low solid production and low effi-
ciency for assimilation, as compared with heterotrophs
(Matějů et al. 1992; Vasiliadou et al. 2006).
Because autotrophic denitrifying microorganisms can uti-
lize inorganic carbon compounds (e.g., CO2 and HCO3−) as
their carbon source by deriving energy from the oxidation
reactions of inorganic elements—such as hydrogen or various
sulfur compounds (e.g., S, H2S, and S2O3), which are the most
common electron donors—studies of autotrophic denitrifica-
tion processes have currently been divided into the following
two major directions: hydrogen-based processes in which hy-
drogen gas is used as the electron donor (Lee and Rittmann
2002) and sulfur-based autotrophic denitrification, which uses
sulfur compounds, such as elemental sulfur or thiosulfate, to
provide energy (Moon et al. 2006). Both technologies have
been developed on laboratory-scale, pilot-scale, and full-scale
levels (Vasiliadou et al. 2006; Mohseni-Bandpi et al. 2013).
Autohydrogenotrophic denitrification is an excellent meth-
od because H2 is clean; the process has a low biomass yield
and a relatively low cost. Furthermore, H2 does not persist in
the treated water or create biological instability, and additional
steps are not required to remove either the excess substrate or
Environ Sci Pollut Res (2019) 26:1124–1141
its derivatives (Vasiliadou et al. 2006; Karanasios et al. 2010).
However, the high flammability of hydrogen and its explosive
potential are the most important drawbacks of this process,
and hollow-fiber membrane dissolution systems have been
developed to make the dissolution of hydrogen in water safer
(Ergas and Reuss 2001; Haugen et al. 2002; Lee and Rittmann
2002; Mansell and Schroeder 2002; Karanasios et al. 2010).
The hydrogen supply that is needed during the process is
another problem that may be solved by a bio-
electrochemical reactor (BER) in which denitrifying bacteria
are cultured on the cathode surface and stimulated with an
electric current (Sakakibara and Kuroda 1993; Karanasios
et al. 2010). A considerable and ongoing effort is focused on
the hydrogenotrophic denitrification of drinking water be-
cause it is a promising and clean method that has a higher
efficiency than does sulfur-based denitrification (Karanasios
et al. 2010; Chen et al. 2014a, b).
Table 3 shows various studies in which hydrogen and sul-
fur have been used as electron donors and presents the critical
values of nitrate removal efficiency for several bioreactors—
including membrane bioreactors, pack-bed reactors, fluidized
bed reactors, and hollow fiber reactors—along with the rela-
tive operation conditions. The information in Table 3 also
indicates that hydrogen and sulfur may have a similar capa-
bility for removing nitrate at the same loading rate; their ca-
pabilities are dependent on the reactor types, HRT, pH, gas
flow rate, temperature, and other measurable parameters such
as the shape and type of sulfur (Karanasios et al. 2010; Chen
et al. 2014a, b; Zhou et al. 2011; Zou et al. 2016).
The effects of various experimental parameters, such as
nitrate loading, C/N ratio, water temperature, and pH, on the
denitrification performance of autohydrogenotrophic
denitrification have been previously evaluated by Chen et al.
(2014a, b) in a bio-ceramsite reactor (that used ceramsite as a
carrier). The results indicated that the denitrification rate in-
creased with increasing nitrate loading to 130 mg NO3−–N/L
and showed that there was an adverse effect on nitrate removal
above this value. The denitrification efficiency slightly
changed as the C/N ratio changed, and this system performed
well if the C/N ratio exceeded 0.9. The optimum temperature
for the reactor was 25–35 °C. The performance of this deni-
trification system was positively related to the pH and was
highest when the reactor was at a neutral or alkaline pH.
During the operation, effluent nitrite levels were always main-
tained below 1.75 mg NO2−–N/L.
An investigation of thiosulfate-driven autotrophic denitri-
fication in a fluidized-bed reactor has been previously con-
ducted by Zou et al. (2016) to evaluate the effect of HRT
and temperature on nitrate removal. Complete nitrate removal
was obtained by Thiobacillus denitrificans-dominated
biofilms even at a nitrate loading rate of 600 mg/L/h and an
HRT of 10 min. An additional decrease of the HRT to 5 min
yielded a 50% nitrate removal efficiency. Nitrite did not
Environ Sci Pollut Res (2019) 26:1124–1141
Table 3 Several autotrophic denitrification technologies using hydrogen and sulfur compounds as electron donor
Electron donor Bioreactor
Sulfur and Methanol membrane bioreactor
Thiosulfate fluidized-bed reactors
Sulfur columns packed with granular elemental sulfur 20
Hydrogen membrane bioreactor
Hydrogen hollow-fiber membrane
Hydrogen fluidized-bed biofilm reactor
Hydrogen bio-ceramsite reactor
Hydrogen Fixed-bed (full-scale)
accumulate when the nitrate was used as an electron acceptor
unless the HRT was decreased to 5 min. Similar denitrification
performance was obtained at 20.0 ± 2.0 and 30.0 ± 0.2 °C.
The basic principles of hydrogen-based and sulfur-based
denitrification
In a sulfur-oxidizing autotrophic denitrification process, ele-
mental sulfur and nitrate serve as the electron donor and elec-
tron acceptor, respectively. Hence, when nitrate is reduced to
nitrogen gas, sulfur is oxidized to sulfate. Sulfur and its com-
pounds can be consumed for cell synthesis (Brettar et al. 2006;
Vasiliadou et al. 2006). The following equation is a stoichio-
metric equation of elemental sulfur-utilizing autotrophic deni-
trification (Sahinkaya et al. 2015):
50NO3 − þ 55S0 þ 20CO2 þ 4NH4 þ
þ 38H2 O→4C5 H7 NO2 þ 25N2 þ 55SO4 2− þ 5:4N2
þ 64Hþ ð13Þ
The main disadvantage of this process is the formation of sul-
fate (7.54 mg per mg NO3−–N removed) and acid. Limestone is
the most commonly used low-cost alkalinity source; however, it
increases the hardness of the treated water, owing to Ca+ disso-
lution (Zhou et al. 2011; Sahinkaya et al. 2015; Sahinkaya et al.
2015). The reported denitrification rate depends on many fac-
tors, including the elemental sulfur particle granule size and the
alkalinity source. The use of small sulfur granules increases the
denitrification rate, owing to a higher surface area for bacterial
attachment and an increased sulfur solubility (Sierra-Alvarez
et al. 2007). However, small sulfur granules may clog the reac-
tor and cause gas entrapment. These problems can be alleviated
by the use of larger sulfur granules; however, such use yields a
lower denitrification efficiency (Ju et al. 2007; Sahinkaya and
Dursun 2012).
1135
Influent (mg NO3−–N/L) Removal percentage Reference
50 54% by heterotrophs Sahinkaya et al. 2016
56% by autotrophic
21 (HRT = 24 min) Complete nitrate removal Zou et al. 2016
96% Soares 2000
20 to 40 96% to 92% Mansell and Schroeder 2002
10 92% Lee and Rittmann 2002
25 approximately complete Kurt et al. 1987
130 94% Chen et al. 2014a, b
18.1 95% Gross et al. 1986
Another alternative is the use of soluble sulfur compounds
as the electron source to attain high denitrification rates.
Thiosulfate can be used according to the following reaction
(Park et al. 2011):
N03 − þ 0:844S2 O3 2− þ 0:347CO2 þ 0:086HCO3 −
þ 0:086NH4 þ þ 0:434H2 O→0:086C5 H7 NO2
þ 0:5N2 þ 1:689SO4 2− þ 0:797Hþ ð14Þ
Although thiosulfate is a more effective electron donor than
sulfur, 11.58 mg sulfate is generated per mg NO3−–N removed
(Sahinkaya et al. 2015), the production of a higher amount of
sulfate compared with sulfur-oxidizing autotrophic denitrifica-
tion (7.54 mg sulfate per mg NO3−–N removed (Eq. (13)) is a
major disadvantage of it, because the maximum sulfate concen-
tration in drinking water should be less than 250 mg/L accord-
ing to USEPA (Oh et al. 2001; Sahinkaya et al. 2015). As
shown in Eqs. (13) and (14), the S/N ratio has a significant
influence on autotrophic denitrification. When this value is
low, nitrite accumulation occurs because the conversion of ni-
trate to dinitrogen is limited. In the case of a sufficient S/N ratio,
nitrite accumulation does not occur (Matějů et al. 1992; Zou
et al. 2016). For the maximum performance of sulfur-based
autotrophic denitrification with minimum operational prob-
lems, the column materials, alkalinity source, and sulfur type
should be optimized (Sahinkaya et al. 2015; Zou et al. 2016).
In hydrogenotrophic denitrification, hydrogen and nitrate
serve as the electron donor and acceptor, respectively.
According to the following reaction, nitrate is reduced to ni-
trogen gas without any byproduct or acid formation (Smith
et al. 2005; Vasiliadou et al. 2006).
2NO3 − þ 5H2 →N2 þ 4H2 O þ 2OH− ð15Þ
The theoretical consumption of hydrogen is 0.35 mg H2 per
1 mg NO3−–N reduced to dinitrogen. The experimental
1136
H2requirement is slightly higher than the stoichiometric values
and ranges from 0.38 to 0.40 mg H2 per 1 mg NO3−–N reduced
(Matějů et al. 1992). The only product of a hydrogenotrophic
process is water and nitrogen gas, without any pH reduction
(Smith et al. 2005; Karanasios et al. 2010). In addition, the
stoichiometry for bacteria cell synthesis with nitrate as the ni-
trogen source and inorganic carbon in which the electrons are
supplied by hydrogen is as follows (Ghafari et al. 2009):
0:04NO3 − þ 0:18CO2 þ 1:04Hþ þ e→0:04C5 H7 O2 N
þ 0:39H2 O ð16Þ
The process has a small biomass yield, can easily be targeted to
exclude undesirable processes, and utilizes an electron donor
(hydrogen) that can be added in excess and readily removed by
sparging or exchange with the atmosphere (owing to its low
solubility in water) after depletion of the nitrate (Smith et al.
2005).
Strengths and weaknesses of heterotrophic
and autotrophic nitrate removal
As previously mentioned, BD is conducted by heterotrophic
or autotrophic organisms. Although heterotrophic denitrifica-
tion is commonly used for water treatment, owing to its high
denitrifying rate and high treatment capacity, heterotrophs re-
quire a soluble organic carbon source for respiration and
growth (Matějů et al. 1992; Soares 2000; Shrimali and
Singh 2001; Samatya et al. 2006; Mohseni-Bandpi et al.
2013; Wang et al. 2013). Because surface or groundwater
typically lacks organic carbon, which is essential as an elec-
tron donor for heterotrophic denitrification, the addition of an
external carbon source is thus required for the sustaining pro-
cess (Lin et al. 2008). Many researchers have considered the
constructed wetlands as a hybrid process of plant uptake and
denitrification for increasing efficiency and supplying organic
carbon as plant productivity for microorganisms (Bastviken
et al. 2005; Lin et al. 2008; Fu et al. 2017; Maxwell et al.
2017). However, relevant research on this topic is scarce,
and the majority of studies investigate wastewater treatment
because the correlation between the abundance and distribu-
tion of microorganisms and the water quality parameters in
partial nitrification and denitrification have not been deter-
mined (Fu et al. 2017). Some considerations for future evalu-
ations of this process include large area, typical special plants
and their localization, exposure to the waste stream, seasonal
removal, increasing effect of greenhouse gases, and effluent
limitation of groundwater compared with wastewater (Lina
et al. 2002; Verhoeven et al. 2006; Lin et al. 2008).
Carbon in drinking water is undesirable because it is a
precursor for trihalomethane formation, and excessive
amounts can increase microbial growth in the distribution
Environ Sci Pollut Res (2019) 26:1124–1141
system, thus resulting in taste and odor problems and acceler-
ated pipe corrosion (Soares 2000; Schipper et al. 2010a). The
dissolved organic carbon (DOC) released from bacteria is
highly variable in heterotrophic denitrification and ranges
from 0 to 45 mg/L (Robertson et al. 2000; Greenan et al.
2009). A high process performance requires a delicate balance
in the substrate addition, because an excessive amount of or-
ganic substrate causes contamination of the effluent by or-
ganics, whereas an insufficient amount of organic substrate
causes nitrite accumulation (Sahinkaya et al. 2015). In a study
by McAdam and Judd (2007), heterotrophic denitrification of
drinking water supplemented with ethanol has been investi-
gated in MBR. In their study, the optimum C/N ratio was
determined on the basis of the substrate breakthrough rather
than the maximum denitrification rate. In carbon-limited con-
ditions (C/N < 1.52), the maximum nitrate removal was 92%,
and nitrite formation was <0.35 mg/L NO2−–N, which was
oxidized by chlorination. The biomass was completely
retained, and a low residual dissolved organic carbon concen-
tration was observed.
Nitrous oxide (N2O) is another byproduct of incomplete
heterotrophic denitrification, which usually occurs at a low
pH and with excess organic carbon and NO3−–N loads
(Verhoeven et al. 2006; Richardson et al. 2009). Anaerobic
digestion of organic material can also facilitate methane pro-
duction (Elgood et al. 2010). Heterotrophic denitrification can
be a source of greenhouse gas emissions, such as CO2 (Elgood
et al. 2010). The disadvantage of this treatment process is the
need for sophisticated process control, wherein overdosing
may decrease the effluent water quality (Boley et al. 2003).
Autotrophic denitrification is considered to be a better al-
ternative for nitrate removal than the heterotrophic process
(Karanasios et al. 2010; Sun and Nemati 2012; Chung et al.
2014; Wang et al. 2017). A comparative study of the nitrogen
removal rate between heterotrophic denitrification (HD) and
autotrophic denitrification (AD) in a long-term batch experi-
ment of an up-flow biofilter has been conducted by Wang
et al. (2017). The results indicated that the heterotrophic and
autotrophic denitrification rates with an approximate amount
of 5 mg/L/h were similar. The remaining biomass in the efflu-
ent of AD was 2.08 times less than the remaining biomass of
the HD effluent, whereas the N2O concentration in the off-gas
emitted from HD was 6–8 times higher than the N2O concen-
tration in the off-gas emitted from AD (Wang et al. 2017).
The benefits of autotrophic denitrification compared with
heterotrophic denitrification can be classified as follows: (1)
does not need organic carbon as the carbon and energy source,
(2) uses inorganic carbon dioxide as the carbon source, and (3)
uses inorganic mineral or light as the energy source. In anaer-
obic conditions, chemoautotrophic organisms oxidize inor-
ganic minerals while reducing nitrate to nitrogen gas
(Matějů et al. 1992; Soares 2000; Mansell and Schroeder
2002). Owing to slower bacterial growth rates, autotrophic
Environ Sci Pollut Res (2019) 26:1124–1141
denitrification offers the advantage of minimizing biomass
accumulation (Liu and Koenig 2002; Della Rocca et al.
2006). During the denitrification process, the hydroxyl group
can be released, and the pH of water shifts toward the alkaline
region; in the case of heterotrophic denitrification, pH is ad-
justed by adding acid or base, but the presence of carbon
dioxide in autotrophic denitrification prevents this shift
(Kapoor and Viraraghavan 1997).
Hydrogen gas and various reduced sulfur compounds can
be used as alternative electron donors for chemoautotrophic
denitrification, because they are less harmful to human health
than heterotrophic systems and additional steps are not needed
to remove either excess substrate or its derivatives (Boley
et al. 2006; Vasiliadou et al. 2006; Wang et al. 2013). The
low cost of these inorganic substrates and low formation of
biomass are important advantages; however, their application
can also present some limitations (Vasiliadou et al. 2006).
Elemental sulfur as an electron donor for autotrophic denitri-
fication systems has been extensively investigated, and its
high denitrification efficiency is comparable to the efficiency
of heterotrophic denitrification in the case of nitrate-
contaminated water treatment (Vasiliadou et al. 2006;
Karanasios et al. 2010). However, the production of sulfates
and the use of limestone for pH adjustment limit its applica-
bility (Liu and Koenig 2002; Chung et al. 2014; Zou et al.
2016). H2 is an excellent autotrophic choice, owing to its clean
nature, low biomass yield, and relatively low cost; in addition,
it does not persist in the treated water and create biological
instability, and no additional steps are required to remove ei-
ther excess substrate or its derivatives (Kurt et al. 1987; Chang
et al. 1999; Lee and Rittmann 2002; Vasiliadou et al. 2006;
Chen et al. 2014a, b).
Despite significant progress in the study of hydrogenotrophic
denitrification, additional research is needed to study a new area
of the renewable energy resources for sustainable hydrogen sup-
ply and bioreactor design, including the selection of the best
material with the highest area for attached growth reactors
(Karanasios et al. 2010), the optimum gas diffusion into a biore-
actor with the objective of increasing the solubility and safety of
hydrogen injection, and the optimum ratio of nutrients.
Microalgae-based water treatment as a new approach
Despite the vast amount of research on wastewater treatment
using microalgae (De-Bashan and Bashan 2010), the feasibil-
ity of growing microalgae in groundwater has not been eval-
uated, and few studies have considered applying this technol-
ogy to the treatment of drinking water, owing to the variation
in the chemical composition (e.g., pH, dissolved inorganic
carbon, nutrient levels, and metals) of groundwater, as com-
pared with the chemical composition of wastewater (de la
Noue et al. 1992; Hoffmann 1998; Hu et al. 2000).
Microalgae are known for their ability to remove nutrients,
1137
such as inorganic nitrogen and phosphorus, heavy metals,
and some toxic and organic pollutants (Muñoz and Guieysse
2006). They can also decrease the chemical and biochemical
oxygen demand and inhibit the growth of coliform bacteria
(De-Bashan and Bashan 2010). Microalgae may have a mild
disinfecting effect, owing to their increased pH during photo-
synthesis (Petrovic and Simonic 2015). Hence, water treat-
ment based on microalgae can be a new approach for contam-
inated groundwater treatment.
Nitrate can be effectively consumed by photosynthetic mi-
croorganisms in nature, such as microalgae and cyanobacteria,
which require fixed nitrogen, inorganic carbon, and light for
growth. Microalgae contain 5–10% nitrogen and can utilize
nitrate as a nitrogen source for growth via the assimilation
pathway (Lee and Lee 2001). Various unicellular microalgae
have been shown to consume nitrate through a dissimilatory
reaction (Kamp et al. 2011; Dahnke et al. 2012). Thus, apply-
ing both available pathways for consuming nitrate may en-
hance the nitrate removal efficiency of microalgae and allow
for the photosynthetic system to be applied for nitrate removal
from drinking water. Consequently, the use of photosynthetic
organisms as a nature-inspired approach minimizes the need
for chemicals and organic substrates for nitrate removal, thus
resulting in efficient resource recovery and recycling (De-
Bashan and Bashan 2010; Abu Hasan et al. 2014). However,
information about engineering photosynthetic systems for
treating nitrate-contaminated drinking water supplies is limit-
ed (Hu et al. 2000; Muñoz and Guieysse 2006).
One of the most commonly used species of microalgae is
Chlorella, particularly Chlorella vulgaris and Chlorella
sorokiniana (de-Bashan et al. 2008; Liu et al. 2012; Riaño
et al. 2012; Petrovic and Simonic 2015). Their advantages
are their ability to grow in autotrophic, heterotrophic, or
mixotrophic conditions that differ with regard to the utilization
of an organic carbon source and luminosity (Devi et al. 2013;
Petrovic and Simonic 2015). This feature of microalgae gives
them the ability to consume many types of soluble substrate in
the environment, especially byproducts released during the
treatment. As a result, many researchers have investigated
microalgae as a possible solution to various environmental
problems (Lee and Lee 2001). The feasibility of biologically
removing nitrate from groundwater has been tested using
cyanobacteria and Chlorella sorokiniana in batch mode in
laboratory conditions. Petrovic and Simonic (2015) have in-
vestigated the influence of C. sorokiniana on the BD of drink-
ing water at the two initial nitrate concentrations of 50 and
100 mg/L, with sucrose and grape juice used as the carbon
sources. Although the experimental results indicated that the
denitrification process was slower in the presence of algae,
even at low concentrations, i.e., 50 mg/L of nitrate, more than
95% of the nitrate was removed in 24 h. Hu et al. (2000) have
demonstrated that nitrate-contaminated groundwater supple-
mented with phosphate and some trace elements can be used
1138
as a growth medium for cyanobacteria. They have also dem-
onstrated that the nitrate uptake rate is affected by the inocu-
lum size (i.e., cell density), fixed nitrogen level in the cells in
the inoculum, aeration rate, and light intensity.
The removal of nitrates from drinking water by photosyn-
thetic microorganisms is an important area of research in bio-
logical nitrate removal; however, some requirements of
photobioreactor design, operation parameters, and microbio-
logical characteristics are necessary to design and operate
industrial-scale applications.
Conclusions
Despite the extensive variety of nitrate removal methods, BD
has been demonstrated to be more beneficial than physicochem-
ical methods. Among the two types of BD, autotrophic nitrate
reduction is preferable, and hydrogenotrophic denitrification
may be the best choice, owing to the clean nature of hydrogen
and the absence of any byproducts. However, the operational
parameters of the autohydrogenotrophic process, such as hydro-
gen and CO2 flow rates, pH, and temperature, as well as the
nutrient concentration, should be investigated to increase the
nitrate removal efficiency and safety. The production of hydro-
gen with energy provided from renewable energy resources is a
future technology that may render hydrogenotrophic denitrifi-
cation economically viable for water treatment. The limitations
of hydrogenotrophic denitrification could potentially be solved
by an appropriately designed bioreactor.
The application of a photosynthetic system, such as
microalgae and cyanobacteria, is a nature-inspired technique
that is capable of both assimilatory and dissimilatory nitrate
reduction, owing to the high requirement for nitrogen for
growth, thereby increasing the removal efficiency and decreas-
ing the technology costs. However, the development of
microalgae-based technology for water treatment is in early
stages, and some considerations, including photobioreactor de-
sign, operational parameters, and microalgae characteristics,
should be investigated in future studies to increase efficiency.
Acknowledgements The authors would like to acknowledge the finan-
cial support of the University of Tehran under grant number 8104956/1/
03, KRIBB Research Initiative Program, and the Advanced Biomass
R&D Center (ABC) of the Global Frontier Program funded by the
Ministry of Science, ICT, and Future Planning (2010-0029723).
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