April 2011, Volume 2, No.

2
International Journal of Chemical and Environmental Engineering

Bio-electrochemical denitrification -A review

S.A.R. Mousavi1,2 , S. Ibrahim1, M.K. Aroua1, S. Ghafari1
1
Department of Civil Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia.
2
Department of Environmental health, Kermanshah University of Medical Sciences and Health Services, Kermanshah, Iran.
 Corresponding author: shaliza@um.edu.my

Abstract
Discharge of nitrogen components into the environment can be a cause of serious problems such as eutrophication of rivers and
deterioration of water sources, as well as hazard for human and animal health. Ammonia (NH 3) and nitrate (NO3-) are the most
problematic nitrogen compounds in water and wastewaters. Nitrification is a common way to eliminate ammonia in municipal and
industrial wastewater, which during this process ammonia oxidizes to nitrate. Nitrate removal from these types of wastewaters is an
inevitable step in the treatment. Biological nitrate removal is a suitable but slow method to remove nitrate from water and wastewater.
Therefore, in treatment plants, some abiotic methods have been commonly applied. However, these methods have indicated some
drawbacks such as highly concentrated brine solution, which compels complexity for further treatment or disposal. Consequently,
efforts have focused on speeding up microbial denitrification techniques, and different methods have been investigated in the last two
decades. Among them bio–electrochemical reactors (BERs) which are equipped with immobilized autohydrogenotrophic
microorganisms on cathode showed good efficiency for implementation of denitrification for any nitrate–contaminated water stream.
This article reviews the diversity of nitrate source, various designs, and aspects of BERs. In addition, it discusses the variation of pH,
carbon source, electric current (EC) and hydraulic retention time (HRT) as some main effective parameters on denitrification rates,
and different configurations of BERs.

Keywords: Nitrate, Denitrification, Bio-electrochemical reactor

1. Introduction standards were determined for it in drinking water. The

Ammonia (NH3), ammonium (NH4+), nitrite ion
(NO2), and nitrate ion (NO3-) are the important form of
nitrogen components in aqueous solution and soil [1].
These kinds of pollutants enter the environment from a
variety of sources as contaminants, which have increased
environmental problems in recent years. Therefore, effort
to reduce emission and improve treatment methods for
water and wastewater should be highlighted as a main
public concern in the future [2-4]. Among these nitrogen
components, NO3- is discharge into the environment from
various sources (natural cycle, and human activities), and
is known as a worldwide health hazard. In some parts of
the world such as USA, India, Japan, Saudi Arabia,
China, UK and several parts of Europe people consume
drinking water with high concentration of nitrate [5], as it
is increasingly becoming an important worldwide
challenge. Nitrate in drinking water may reduce to
nitrosamines in the stomach which is believed to cause
gastric cancer[6, 7]. In addition, drinking of water
contain high concentration of nitrate can be cause of
disease “blue baby syndrome” due to reduction of nitrate
to nitrite inside the stomach of fetus, and nitrite reacts
with the hemoglobin in blood and converts the
hemoglobin into methemoglobin. This new compound
does not carry oxygen to cell tissues [8]. Moreover,
nitrate known as a carcinogenic compounds[9]. To protect
consumers from the adverse effects of high nitrate intake,
World Health Organization (WHO), US Environmental
Protection Agency (EPA), and European Community
establishes the limit Maximum contaminant level(MCL)
for nitrates in drinking water at 50 mg NO3-/L, 12 mg/L N
, and on 50 mg NO3-/L and 0.5 mg NO2-/L, respectively
[10, 11]. Furthermore, different discharge standards were
defined based on the receiving environment, fresh water
(10 and 30 mg NO3--N/L ), seawater (50 mg NO3--N/L),
and in sensitive areas discharge concentration is 10 and
15 mg TN/L [12].
In recent years, researchers have tried to improve the
removal of nitrogen component especially nitrate from
water and wastewater, and have had some achievements.
The aim of this review is to briefly mention the health
risks of nitrate and its sources, and summarize various
treatment methods of nitrate. The paper focuses on the
BERs mechanism, effective factors that contribute to
process efficiency, and the performance of bio-
electrochemical reactors.
2. Source of nitrate
The sources of nitrate can divided to two subdivision
“point sources” and “nonpoint sources”. The non–point
agricultural source is confirmed as one of the important
nitrate sources to pollute groundwater due to extreme use
of fertilizers[13]. Some other sources of nitrate in ground
water and surface water are from uncontrolled land
 discharges of treated or raw domestic and industrial
wastewaters, landfills [14], and animal wastes
predominantly from animal farms [15]. Some industries
wastewater such as dairy and swine are reported as
wastewaters containing more than 200 mg NO3--N/L[16].
Furthermore, some industries produce wastewater
containing greater than 1000 mg NO3--N/L such as those
producing explosives, fertilizer [7, 17, 18], cellophane,
pectin, and metals finishing industries. Also, a number of
activities like the processing of radioactive metal products
at nuclear weapons production plants and research labs
produce wastewater that consist high concentrations of
nitrate that exceed 50000 mg NO3–N/L [19].
3. Nitrate removal methods
There are regulations made compulsory to minimize
environmental problems and health risks due to
uncontrolled discharge of nitrate. To achieve discharge
standards, reduction of nutrients in wastewater is thus
required. Research on nitrogen component removal has
been ongoing for treatment of domestic and industrial
wastewaters, water resources, aquaculture ponds, and
aquaria. The most commonly used methods to remove
nitrogen components from liquid waste especially
ammonia are: chemical precipitation with magnesium
ammonium phosphate, ion exchange, bio-filtration,
ammonia-stripping, electrolysis, break point chlorination,
and electrochemical conversion [2, 4, 20, 21]. The most
common nitrate reduction method is either
physicochemical or biological process [19]. The common
abiotic process consists of ion exchange (IE), electro–
dialysis (ED), reverse osmosis (RO) and activated carbon
adsorption in combination with pH adjustment [14, 17,
22]. The biological nitrification/denitrification is only
suitable for the removal of relatively low concentration of
ammonia and nitrate due to the requirement of
appropriate C/N ratio [2, 4, 20, 21], Nitrate removal from
drinking water and wastewater is difficult. Therefore,
complementary techniques are required for obtaining a
free nitrate stream in treatment plants [14, 17, 22].
Biological denitrification is a suitable method to treat
wastewater contain nitrate because nitrate is transformed
to harmless nitrogen gas with the present of an electron
donor. However, biological remediation of wastewater
with high concentration of nitrate with low chemical
oxygen demand (COD) as carbon source and or internal
electron donor, using an external source as electron donor
is indispensable. Heterotrophic denitrification processes
using methanol and acetate have been proposed in a
suspended or attached growth reactor [11, 23]. Also, a
wide interest towards some new methods such as metallic
iron–aided abiotic nitrate reduction is recently reported by
several researchers [23-25]. Ghafari et al. [19] stated the
disadvantages along with diverse abiotic techniques,
which remove nitrate from water and generate a brine
solution. But, denitrifying bacteria, which are ubiquitous
in nature, are responsible for biological denitrification and
use oxygen in nitrate as electron acceptor during their
141
respiratory process instead of oxygen in anoxic condition
and produce harmless gas “N2” by reduction of nitrate/
nitrite through four steps below [26].
(1)
Denitrifying microorganisms belong to several
taxonomic groups and physiological groups such as
lithotrophs, organotrophs, and phototrophs. Denitrifying
bacteria are common among the gram-negative alpha and
beta classes of the Proteobacteria, such as Pseudomonas,
Alcaligenes, Paracoccus, and Thiobacillus. Some gram-
positive bacteria are denitrifying such as Bacillus, and a
few halophilic Archaea like Halobacterium [27, 28].
Based on utilized food and energy sources,
denitrifying bacteria are classified in another two major
groups namely “heterotrophs” and “autotrophs” [1, 29].
Heterotrophs make use of organic carbon compounds as
the carbon source [30], while autotrophic bacteria utilize
inorganic carbons such as carbon dioxide or bicarbonate
as the carbon source. In contrast to heterotrophs who use
organic carbons as energy source, autotrophs make use of
reduced inorganic compounds (e.g. ferrous iron,
manganese, hydrogen sulfide) or molecular hydrogen (H2)
as electron sources to obtain energy [19, 30-32].
Heterotrophs are more preferable for denitrification of
wastewaters due to availability of organic compounds in
wastewaters. In addition, they apply for biological
denitrification of drinking water. However, methods
applying this kind microorganism are associated with
some problematic consequences such as clogging in
reactors, residual carbon sources and by-products, which
typically imply a further costly post treatment [33-35]. On
the other hand, autotrophic denitrifying bacteria offered
conditions that are more favorable: they need inorganic
compounds which are less expensive compared to
organics, and their residuals in treated drinking water are
harmless (for H2) or less problematic (for reduced
components) [35, 36]. Therefore, in recent years
researchers have focused on denitrification by
autohydrogenotrophic as autotrophic denitrifying, which
is more promising than other autotrophic methods.
Equation (2) shows reduction of nitrate to N2 by
hydrogenotrophic denitrification [19].
4. Theory of denitrification by
autohydrogenotrophic bacteria
To use this group of autodenitrifying bacteria
preparing of hydrogen as electron donor plays an
important role. Some researchers supply H2 by sparging
in reactor as initial method. But this method have some
shortcoming such as the costly provision of hydrogen gas,
risking the flammable and explosive mixtures of H2 with
air, transportation and storage problems [19, 26, 37].
Furthermore, It was mainly because of low solubility of
H2 in water (1.6 mg/L at 20 C), which decline its
availability and contact time for microbial reaction, and
hence, it directly results in lower denitrification rates
compared to heterotrophic denitrification [26, 35].
Therefore, in recent years researchers focus on newer
 methods to solve sparging limitation, via two pathways:
chemical generation of H2 and electrolysis of water [26,
38, 39]. Due to certain weakness such as slow reaction
rate and generation of unwanted by-products, researchers
confirmed that the electrolysis of water is a clean method
to generate of hydrogen [38]. Consequently, exploitation
of electricity in denitrification process led to generation of
new types of reactors named bio-electrochemical reactors
(BERs) [17]. In this reactor according to Table 1 some
reactions take place during bio-electrochemical
denitrification, which result in the generation of H2 on
cathode by electrolysis of water according to Equations
(3) and (4). Autohydrogenotrophs bacteria consume
generated H2 as electron donor and during reactions (5)
to (8) transfer nitrate to N2 [19, 37, 40, 41].
Table 1. Bio-electrochemical denitrification reaction in BERs
Electrolysis of water in BERs:
Steps of nitrate reduction to nitrogen gas:
Net reaction
overall reaction
5. Effective parameters on performance of BERs
The denitrification efficiency by BERs depend on
many parameters; the key parameters in assembling of
reactors are: material, geometry, number of electrodes as
well as BERs design [19, 42, 43]. Influencing operational
and environmental parameters are pH, HRT, carbon
source and EC as theses are known to be important in
denitrification by EBRs [44-46]. To reduce some
drawbacks in BERs, the determination of the parametric
effects and optimization of these parameters are
necessary. According to Table 2 previous studies showed
that various types of metals and carbons were used as
electrode material; carbon in the form of granular
activated carbon (GAC) and graphite as well as metals
such as stainless steel, titanium, nickel and copper [40,
46, 47]. Also, various denitrifiers in different condition
such as suspended in fluidized bed or immobilized on
different permeable media (e.g. soil, GAC, plastic
particles, mesh or reticulated planes), have been applied
for different treating aqueous solution such as surface and
groundwater, municipal and industrial wastewater,
aquatic and river water [19].
Many types of reactors used in bio-electrochemical
system, have been investigated. In recent years,
researchers have strived to improve denitrification
performance, and decrease the hydraulic retention time
from 10 h to several days to achieve complete
denitrification [48], and investigated other alternatives for
BERs configuration. Among them that had good results
142
are integrated systems, tow parts reactor (electrochemical
and biological), and multi-electrode systems or three-
dimensional (3D) electrode [32, 48, 49]. For example
Wan et al. [32] and Wang and Qu, [49] to improve
effectiveness of BERs or eliminate drawbacks of
integrated methods combined sulfur limestone autotrophic
denitrification method (SLAD) with BER, and developed
a new integrated method (bio-electrochemical combine
with sulfur autotrophic denitrification (CBSAD). In the
new system, the limestone for pH adjustment is not
necessary. Therefore, with H+ consumption, which was
produced by sulfur denitrification in the bio-
electrochemical reactor, increase of effluent hardness can
be prevented. In addition, the produced CO2 on anode
complement process as inorganic carbon source and pH
buffer, and efficiently removed nitrate more than 95%
without nitrite accumulation and the sulfate concentration
of effluent was lower than 250 mg/L [32]. Two parts
reactor “electrochemical cell and bioreactor” is another
alternative for BERs. Flexibility in operation and
simplified maintenance are the preferences of this
configuration. This arrangement is economical, because
of separating parts of H2 generation and biomass.
Therefore, it was a possible jointing of a small
electrochemical cell with a large bioreactor. Moreover,
the treated water with this method even in very high
nitrate concentrations is proper. In addition, the pH
changes is little [50]. The result of several study showed
suitable denitrification performance with multi-electrode
system compared to previous BERs because of large
effective surface area of the electrodes, the charge transfer
mechanism by dissociative electrolyte, and the formation
of highly reducing (or oxidizing) [51].
The pH values is one of the main factors for
controlling of denitrification because it can be as limit
denitrification activity and cause of nitrite accumulation
[16]. Based on equation (3) researchers have mentioned
during denitrification process when NO3- change to NO2-,
pH does not change. Nevertheless, in the second stage
(change of nitrite to nitrogen gas) pH will increase, and
nitrite accumulation occur because of pH elevation, which
is one of the main reason for agglomeration of nitrite in
denitrification [39, 52, 53]. However, hydrogen as an
electron donor has many advantages which were
confirmed, but as with traditional biological processes
control of pH is also necessary[37].
Some substrates such as carbon, nitrogen,
phosphorous, and mineral components have key role on
denitrification rate because substrates are necessary to
grow various species of denitrifying bacteria in pure or
mixed cultures [28, 52]. Among them carbon as food and
energy source is a important parameter on denitrification
rate by two group of denitrifyers “heterotrophic” and
“autotrophic” [49, 54]. Researchers mentioned, depending
on COD/N ratio of influent it may be necessary to have
external carbon in BERs to denitrification of wastewater
[19]. It was stated that when the COD/N ratio of influent
is lower than 3.4 gCOD/gN, extra organic matter needs to
 be added [55]. Therefore, to improve nitrate removal in
water and wastewater various carbon sources [19, 56, 57]
such as methanol, ethanol, sucrose, acetate, industrial and
demotic wastewaters, and other industrial wastes [58],
also bicarbonate and carbon dioxide [52, 59] have been
explored.
Other important effective parameters on
denitrification rate in bio-electrochemical reactor is
electric current intensity (I) and hydraulic retention time
(HRT), which were investigated [32, 39, 49, 51]. Results
showed when the electric current increased the reduction
of nitrate improved. Results show that low and high
electric current because of inadequate hydrogen
production and excessive hydroxyl generation
respectively has low reduction. Also, the efficiency of
system was reduced in the low and high HRT because at
low (6h) and high (48h) HRT the required reaction time
for microorganisms was not achieved, but increased the
rate of flushing unfavorable hydroxyl anions out of the
cathode sector [39].
6. Implementation of BERs
The bio-electrochemical reactor process may have a
potential for the removal or detoxification of other
substances in water and wastewater. Therefore, some
investigations have been carried out on remove various
contaminants using BERs, such as simultaneous removal
of nitrate and pesticide [60], copper ion [23] or
ammonium nitrogen [61, 62].
Some researchers applied sulfur limestone autotrophic
denitrification (SLAD) systems for nitrate removal. In
this system, limestone is used to adjust the pH, and sulfur
use as electron donor. During the reaction 7.54 mg/L
sulfate to 1mg-N/L removed nitrate will be produced,
which can raise the hardness due to produced Ca2+ by the
limestone. To improve SLAD researchers combined bio-
electrochemical and sulfur autotrophic denitrification
(CBSAD). In this novel system, the limestone to pH
adjustment is not necessary. Therefore, for preventing
hardness in effluent, the produced H+ by sulfur
denitrification consume in the bio-electrochemical
reactor. In addition, the produced CO2 on anode
complement this system as inorganic carbon source and
pH buffer [32, 49].
Sakakibara and Nakayama [51] used a novel multi-
electrode system for bio-electrochemical denitrification of
water contain15-20 mg NO-3-N/L. In this reactor, 8 pieces
of titanium were located in reactor as cathode, and the
anodes were two pieces from Pt-coated. To prevent
leakage of O2 into cathode part, water-permeable plastic
foam was placed between anode and cathode. Results
showed suitable denitrification performance with multi-
electrode system compared to previous BERs because of
large effective surface area of electrode, the charge
transfer mechanism by dissociative electrolyte, and
formation of highly reducing (or oxidizing). In another
research, Prosnansky et al. [26] investigated a multi-
cathode biofilm-electrode reactor combined with
143
microfiltration (MF) for treatment of nitrate-contaminated
water. The reactor is composed of two compartments
(multi-cathode BER and membrane compartments). The
BER was divided two part with a porous sponge-foam
rubber to impede inter O2 bubbles to cathode zone and
mixing with H2. In the BER section cathodes was
arranged by 5 porous GAC electrodes, and the anode was
designed by platinum-coated titanium that surface area of
cathode and anode were 750 and 150 cm2, respectively.
Results confirmed this novel system could perform
denitrification with high rates at low hydraulic retention
time (20 minutes). Furthermore, the efficiency of
denitrification rate improved 3–60 times in comparison
with previous studies. Also, the cathodic current density
decreased because of enlarged surface area of cathodes
by multi-GAC electrode system.
Watanaba,T et al. [17] applied a bio-electrochemical
reactor (working volume =5 L, axial center anode carbon
rod 16mm diameter, surrounded cathode with 12 carbon
rods and 8mm diameter) to treat acidic wastewater
containing copper ions and a high concentration of nitrate.
The distance between the anode and cathode was about
50mm. Achievements showed an effective removal of
copper ions and nitrate, when an electric current was
applied and acetate was added as an external electron
donor.
Ghafari et al. [39] studied an up-flow bio-
electrochemical reactor to denitrification of polluted
water with biocarrier (GAC) and stainless steel plate as
cathode zone. The achieved results for two operating
parameters (HRT= 6–36 h) and electric current (I= 0 to 20
mA) were examined on contaminated water containing
20mg NO3--N/L. The nitrate completely reduced in
electric current range (10–16mA) as well as HRT 13.5–30
h. The removal of nitrate in I=0 was minimum but the
nitrate removal was improved by increase of electric
current (up to 14mA).
7. Conclusions
Based on this review, nitrate contamination has been a
worldwide environmental challenge for many years.
Water contamination is a major universal concern because
continuous consumption of water containing high nitrate
levels can cause many diseases such as
methemoglobinemia. Therefore, nitrate removal from
wastewater has been the focus of numerous studies, in the
aim to achieve acceptable nitrate concentration. Up to
now, researchers have applied different kinds of methods
for nitrate removal from water and wastewater, but results
show that denitrification of contaminated water and
wastewater by conventional abiotic methods are not
adequate due to many reasons discussed in this paper.
Even biological denitrification which is looked upon for
denitrification is not functional for high concentration of
nitrogen components.
 Table 2. Performance of denitrification in BERs

%
Type of Feed Carbon I mA NO3--N HRTa
Cathode Anode Nitrogen Reference
reactor solution source mgL-1 (h)
removal

Stainless 20.9 –
CBSADb Groundwater Carbon rod CO2 30 - 1200 4.2 - 2.1 95 - 100 [32]
steel 22.0
Contaminated Stainless
UBERc Stainless steel mesh NaHCO3 0–20 20 6–36 100 [39]
water steel
Activated
3DBERd Groundwater carbon Beta-lead dioxide CO2 8-32 30 8 99.80 [63]
fiber
Synthetic
BERe Graphite Dimensionally stable 1-7
wastewater NaHCO3 N/A 20 to 492 98 [64]
felt anode (DSA) (4)
Municipal Stainless N/A TN=37.2–
BER Graphite 20-120 6 75 [40]
sewage steel 68
Cylindrical
Drinking
stainless Carbon rod CO2 2-20 30 1.9–5 90 - 100 [49]
CBSAD water
steel
Synthetic GAC with Pt-coated titanium coated 15-40 0.33-6
BER-MF CO2 40-300 80 [26]
groundwater platinum (25) (2.88)
BER
+Absorber Ground water Stainless Amorphous carbon CO2 0–10 24 10 >95 [37]

BER
Groundwater Metal Pt-coated NaHCO3 80-960 2-6 80 [51]
13.8–20.8
Feed solution
Carbon
BER ( Carbon material NaHCO3 0-100 20 10–13 98 [14]
material
groundwater)
BER groundwater Stainless Amorphous carbon CO2 2-10 20 10 100 [65]
BER Contaminated
Stainless Amorphous carbon CO2 1–10 20–24 10–50 N/A [66]
groundwater

a Units used for HRT (h) .

b Combined bio-electrochemical and sulfur autotrophic denitrification system (CBSAD)
C Upflow bio-electrochemical reactor (UBER)
d Three-dimensional bio-electrochemical reactor(3DBER)
e Bio-electrochemical reactor (BER)

144

The trend has thus been for researchers to focus on
further development of biological denitrification by
creating better correlations between the nitrate and
microorganisms. The bio-electrochemical system is new
method, which meets this objective. The efficiency of
bio-electro chemical system is affected by many
parameters such as electrode specifications, pH, carbon
source, HRT, and current intensity that were discussed in
this review. Notwithstanding all of the mentioned prior
investigation, there remain many unclear aspects, which
calls for further development and improvement of
efficiency of BERs since although the use of this kind of
denitrification system became known two decade ago and
there is insufficient information about performance of
system and the effects of operational variations and
configuration of reactor. There is definitely room for
further research, particularly on the aspect of reactor
configuration and electrodes material. Furthermore, in the
case of sewage treatment applying BERs, the applicable
hydraulic retention times, pH, carbon source, and current
intensity are still a subject of argument.
ACKNOWLEDGEMENT
The authors thank Universiti Malaya for financial
support of this project.
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