Biochemical Engineering Journal 106 (2016) 125–128

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Biochemical Engineering Journal

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Short communication

Electrical stimulation enhanced denitrification of nitrite-dependent

anaerobic methane-oxidizing bacteria
Qing Wu, Jiali Chang, Xiaoxu Yan, Nuerla Ailijiang, Qiuxiang Fan, Shenghui Wang,
Peng Liang ∗ , Xiaoyuan Zhang, Xia Huang ∗
State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing 100084, China

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

Article history: As a process utilizing methane as the sole electron source to reduce nitrite, nitrite-dependent anaer-
Received 6 August 2015 obic methane-oxidizing (n-damo) shows high potential to be applied in energy-efficient wastewater
Received in revised form treatment. However, the metabolism of n-damo bacteria is far too low for practical application. In this
17 November 2015
study, bioelectrical reactor (BER) was used to enhance the metabolism. The nitrite removal rate was
Accepted 21 November 2015
accelerated under applied electric field. The best stimulating effect was obtained at 1 V applied with the
Available online 1 December 2015
denitrification rate increased to 1.8 fold compared to conventional n-damo process. The enhancement of
nitrite consumption was consistent well with the acceleration of methane depletion, further confirmed
Keywords:
Nitrite-dependent anaerobic
the electrical stimulation of n-damo process. However, the enhancement could only be realized under
methane-oxidizing (n-damo) bacteria continuous electrical exposure mode, rather than intermittent mode. The above results demonstrated
Electrical stimulation that electrical stimulation could lead to an enhanced denitrification of n-damo bacteria.
Anaerobic processes © 2015 Elsevier B.V. All rights reserved.
Bioreactors
Kinetic parameters
Waste-water treatment

1. Introduction
Nitrite-dependent anaerobic methane oxidation (n-damo) is a
novel denitrification process, with methane as the electron donor
and nitrite as the electron acceptor under anaerobic condition (Eq.
(1)). Candidatus Methylomirabilis oxyfera was found to be the main
microorganism [1].
 
3CH4 +8NO− + 0
2 +8H → 3CO2 +4N2 +10H2 O G = −928kJ mol
-1
N-damo process, which could utilize CH4 produced by anaerobic
wastewater treatment process in situ, is potentially energy-
efficient. When combined to partial nitrification, it would be more
cost-saving with less aeration [2]. In addition, only slight amount
of NO2 − can be converted to N2 O (an important greenhouse gas) in
n-damo process compared to traditional nitrogen removal system
[3], further raising the application potential of this process as an
environment-friendly wastewater treatment method. However, n-
damo bacteria has a rather low growth rate of 1–3 weeks’ doubling
∗ Corresponding authors. Fax: +86 10 62771472.
E-mail addresses: liangpeng@tsinghua.edu.cn (P. Liang),
xhuang@tsinghua.edu.cn (X. Huang).
time, and a low specific activity of 0.9–3.7 nmol NO2 − min−1 mg
protein−1 [1,2]. It would undoubtedly limit the engineering appli-
cation of n-damo process.
Bioelectrical reactor (BER) is an effective method to stimulate
microbial metabolism, including growth and activity, depending
on the applied electric current, voltage, bacterial species, elec-
trical exposure mode and duration. For example, at a current
 of 30 mA, the growth of Thiobacillus ferrooxidans was 10 times
faster than control culture without electricity [4]. However, the
(1) growth of Fusarium oxysporum was inhibited, while the glucose
consumption increased significantly under electrical treatment [5].
Different effects draw attention to pinpoint the mechanism. Elec-
trical treatment of microbes was proven to alter the specific activity
of dehydrogenase, membrane permeability [6], ATP content [7],
microbial community of enrichment [8] and the possible energy
gain [9]. Additionally, BERs could also accelerate electron trans-
fer and provide electron flow for microbes indirectly or directly.
For instance, the denitrifying bacteria could uptake electrons either
from H2 produced through water electrolysis, or directly from elec-
trode [10,11].
As a promising process in wastewater treatment, n-damo pro-
cess suffers from low activity and growth rate of n-damo bacteria.
To date, many efforts have been made on identifying the optimal
environmental conditions [12,13] and optimizing the reactor con-
figuration for bacteria cultivation [14], but electrical stimulation
method was neglected.In this study, a mixed culture dominated

http://dx.doi.org/10.1016/j.bej.2015.11.014
1369-703X/© 2015 Elsevier B.V. All rights reserved.
126 Q. Wu et al. / Biochemical Engineering Journal 106 (2016) 125–128
Fig. 1. Schematic of the bioelectrical reactor (BER).
by n-damo bacteria was cultivated in an anaerobic BER to iden-
tify the effect of electrical stimulation on n-damo process. Voltages
were adjusted to investigate its effect on microbial activity. On this
basis, continuous and intermittent exposure modes were studied
to determine an optimum operation condition.
2. Materials and methods
2.1. N-damo culture
The n-damo culture was taken from a membrane biofilm reac-
tor (MBfR) enriching n-damo bacteria. The MBfR was inoculated
with sediments of Taihu (31◦ 01.265 N, 120◦ 27.044 E) in July 2011.
It consisted of a cylindrical vessel (3.6 L) and an inside hollow fiber
membrane module. Methane was sparged to the liquid through
the fibers. Nitrite was fed into the MBfR to a final concentration
of 14–28 mg NO2 − -N L−1 . N-damo bacteria had been successfully
enriched and became dominant (about 73%) in the MBfR after 13
months according to the analysis of 16S rRNA gene clone library
and fluorescence in situ hybridization.
The synthetic medium was boiled and sparged with N2 to
maintain anaerobic condition before being added to the MBfR.
It contained (per liter): 0.00625 g EDTA; 1.25 g KHCO3 ; 0.2 g
MgSO4 ·7H2 O; 0.3 g CaCl2 ·2H2 O; 0.00625 g FeSO4 ·7H2 O; 0.05 g
KH2 PO4 ; 0.5 mL of trace mineral solution, which contained (per
liter): 15 g EDTA; 0.99 g MnCl2 ·4H2 O; 0.43 g ZnSO4 ·7H2 O; 0.19 g
NiCl2 ·6H2 O; 0.24 g CoCl2 ·6H2 O; 0.05 g Na2 WO4 . 2H2 O; 0.014 g
H3 BO3 ; 0.25 g CuSO4 . 5H2 O; 0.22 g (NH4 )6 Mo7 O24 . 4H2 O; 0.067 g
SeO2 [15].
2.2. BER configuration
The electrical stimulation experiments were carried out in cylin-
drical plastic BERs (˚ 90 mm × 80 mm) (Fig. 1). The total volume of
BER was 508.7 mL, including 100 mL headspace and 408.7 mL work-
ing volume. Two pieces of graphite felt (55 mm × 40 mm × 5 mm)
were wrapped by titanium mesh to maintain desired shape, and
then immobilized parallelly with an interval of 40 mm in the reactor
as electrodes.
2.3. Operation and experimental setup
After the BER was injected with 26 mL n-damo culture, it was
filled with sterilized and deoxygenized synthetic medium (same
as MBfR) under anaerobic condition and settled for 12 h. After
that, 100 mL supernatant was discharged by equivalent methane
(99.99%) at room temperature and pressure. The BERs were mag-
netically stirred in the incubator at 30 ± 0.5 ◦ C.
After 1 week of pre-incubation, voltages were applied to the
BERs. At the beginning of each test the nitrite concentration was
set to a certain value, and then declined over time until depleted,
which was defined as an experimental cycle. After each electricity
Table 1
The operating conditions of BERs in part IV.
Run I II III IV
√ √ √
N-damo culture
√ √ √
Methane
√ √ √
Voltage
Voltages of run I, II and IV were 1 V.
assisted test, there was another cycle without electricity to make
the microorganisms back to normal state.
The experiment was divided into four parts. Part I aimed at clari-
fying the effect of different voltages on n-damo performance. Three
BERs were charged by a direct current power source (Arbin instru-
ments, USA) supplying certain voltages (0, 0.5, 1.0 and 1.5 V). Part II
was for investigation of electrical stimulation performance under
different electrical exposure modes. Three exposure modes (4 h
on/4 h off, 12 h on/12 h off and continuously on) under 1 V were
selected to run the BERs. The initial nitrite concentrations in part I
and II were 1 mM. Part III aimed to explore the stoichiometry ratio
of methane and nitrite in the absence and presence of electric field.
The initial nitrite concentration was about 2 mM. To further con-
firm n-damo process in the BERs, run I–IV were set up in part IV
(Table 1).
1 mL of liquid was sampled at intervals to test nitrite con-
centration. Besides, 200 ␮L of gas was sampled to test methane
consumption in part III.
2.4. Analytical methods
The nitrite concentrations were analyzed using ion chromatog-
raphy (Dionex ICS 1100, USA). Methane concentrations were
measured by gas chromatography (Agilent 7890 GC, USA).
A model was developed by Matlab R2015a for kinetic analyzing
of biochemical reactions. Assuming that methane was sufficient
and biomass concentration stayed all the same during an operating
cycle, the denitrification rate can be expressed as:
dc
r=− = kc a (2)
dt
where r is the conversion rate of nitrite, c is the current concentra-
tion of nitrite, t is the current time, k is the rate constant and a is
the reaction order. The function can be converted to the following
equation:
  1−a
1
c = c0 1−a − (1 − a)kt (3)
where c0 is the initial concentration of nitrite.
3. Results and discussion
3.1. Denitrification performance under different voltages
The nitrite removal performance was generally enhanced when
electric field was applied and varied with different voltages
(Fig. 2a). Nitrite concentration of BERs without electric field
declined from 1 mM to 0.2 mM after 88 h, with a removal rate of 80%.
While the same removal rate was reached at 62, 47 and 50 h under
0.5, 1.0 and 1.5 V applied respectively, showing shorter operating
duration.
Modeling result based on Eq. (3) revealed that the denitrifi-
cation process was half-order kinetics, which meant the reaction
order a (Eq. (3)) was 0.5. Rate constant k turned out to be 0.013,
0.017, 0.023 and 0.021 under 0, 0.5, 1 and 1.5 V respectively. To
quantify the effect of electrical stimulation, the ratio of the elec-
tricity assisted rate constants (k) and the conventional rate constant
without electric field applied (k0 ) was calculated. It meant that the
 Q. Wu et al. / Biochemical Engineering Journal 106 (2016) 125–128
Fig. 2. Variation of nitrite concentration during an experimental cycle (a) and the
ratio of rate constants, k/k0 (k: the rate constants under different voltages; k0 : the
conventional rate constant under 0 V) (b). The initial nitrite concentration was 1 mM.
nitrite removal ability had been improved if the ratio k/k0 exceeded
1 and vice versa. The k/k0 under 0.5, 1.0 and 1.5 V was 1.34, 1.81 and
1.64 respectively (Fig. 2b), all of which were greater than 1.
These results showed that nitrite removal performance had
been significantly enhanced. The enhancement was improved with
the increased voltage in the range of 0–1 V. However, no further
enhancement was observed when voltage exceeded 1 V (see sig-
nificance of difference analysis in Table S1).
The theoretical electrode potential vs saturated calomel elec-
trode (SCE) of O2 /4OH− and 2H2 O/H2 in the cultivating medium
(pH 7.5) was 1.27 and −0.44 V respectively. The actual anode and
cathode potential (vs SCE) was measured to be 0.65 and −0.33 V
under 1 V, which was not capable of water electrolysis. Besides,
the current was extremely weak (0.015–0.145 mA) and showed no
obvious relationship with n-damo activity. Meanwhile, no bubbles
were observed on the electrodes under 1 V, confirming that the
denitrification was not enhanced by the production of hydrogen.
Previous studies found that electrical treatment of microbes
could alter membrane permeability [6] and ATP content [7], which
could contribute to the electrical enhancement. Besides, it could be
possibly due to the electron transport acceleration of n-damo bacte-
ria. As the BER was a multispecies system, microbes might produce
electron shuttles, raising the possibility of the electron transfer
between n-damo bacteria and electrodes [16]. N-damo bacteria was
reported to oxidize methane by the oxygen produced through the
reduction of nitrite, while the electron generated through the oxi-
dation of methane could be applied in the reduction of nitrite in
turn, which forms an electron loop [1]. If n-damo bacteria was able
to obtain electrons from electrodes, it would accelerate the electron
loop.
127
Fig. 3. Variation of nitrite concentration during an experimental cycle (a) and the
ratio of rate constants, k/k0 (k: the rate constants under different electrical expo-
sure modes; k0 : the conventional rate constant under 0 V) (b). The initial nitrite
concentration was 1 mM.
3.2. Denitrification performance under different electrical
exposure modes
The nitrite removal performance at continuously electrical
exposure mode was better than that at intermittent mode (Fig. 3a).
Nitrite concentration of BERs under 4 h on/4 h off and 12 h on/12 h
off modes decreased from 1 mM to 0.6 mM during 48 h operation,
which was almost the same as the nitrite removal performance
of conventional n-damo process (0 V). However, at continuously
charged mode nitrite concentration was reduced to 0.18 mM at
48 h. The modeling results based on Eq. (3) showes that k/k0 was
0.96, 0.89 and 2.2 at 12 h on/12 h off, 4 h on/4 h off and 1 V contin-
uously on mode respectively (Fig. 3b).
The data confirmed the stimulation effect of continuous voltage,
and demonstrated that intermittent electric field had no function
of accelerating nitrite removal.
However, Loghavi preferred intermittent over continuous elec-
tric field. The doubling time of Lactobacillus acidophilus (about
1–3 h) [6] and Methylomirabilis oxyfera (n-damo bacteria) (about
1–3 week) [1,17] was different, which could lead to different expo-
sure durations they can bear.
Though denitrification rate would be increased when electricity
was applied, it would drop back to normal state without electric
field applied. This could be the reason of the negligible effect of
intermittent exposure mode.
3.3. Demonstration of n-damo as the main denitrification process
Methane consumption synchronized with nitrite depletion dur-
ing the experimental cycle (Fig. 4). The mole ratio of methane and
128 Q. Wu et al. / Biochemical Engineering Journal 106 (2016) 125–128
Fig. 4. Variation of nitrite content and methane content during an experimental
cycle.
Fig. 5. Variation of nitrite removal rate during an experimental cycle of run I–IV.
Voltages applied in run I, II and IV were 1 V.
nitrite consumption was about 0.34 under 0 V and 1 V, agreed well
with that in Eq. (1) (0.375) [1]. Modeling results revealed that rate
constant k of nitrite removal was 0.005 and 0.007 under 0 and 1 V,
respectively. Meanwhile, the corresponding k of methane deple-
tion was 0.0011 and 0.0016. The nitrite removal rate and methane
depletion rate under 1 V were 1.44- and 1.45-fold of 0 V, respec-
tively. It effectively proved that the nitrite removal was coupled
to methane depletion, namely, the n-damo process was the main
denitrification process.
In part IV (Table 1), the nitrite removal rate of run I was almost 0
during the experimental cycle (Fig. 5), proved that no electro abiotic
nitrite depletion occurred. Nitrite removal rate of run II reached to
19% at 48 h, which might be ascribable to the bio-electro denitrifica-
tion [11], or heterotrophic denitrification, or both in the BERs. Since
methane was the sole added electron donor source in the BERs,
heterotrophic denitrifying bacteria might rely on endogenous res-
piration. The difference of nitrite removal rate between run IV (82%)
and run II (19%) could be considered as the contribution of n-damo
process under 1 V (63%), higher than that of run III (50%) under 0 V.
Given the above, n-damo process was proved to be the main deni-
trification process in the BERs and enhanced under applied electric
field.
4. Conclusions
In this study, the stoichiometric relation between CH4 and NO2 −
consumption demonstrated that n-damo process was the main
nitrite removal process in the BERs. Applied electricity was found
to have significant effects on accelerating n-damo process. In the
range of 0–1.5 V, 1 V showed the best nitrite removal performance,
about 1.8-fold of the conventional n-damo process without elec-
tric field applied. Continuously exposure mode was more effective
than intermittent electric field exposure mode. To elucidate the
phenomenon, further study on the mechanisms of the electric stim-
ulation effect need to be performed.
Acknowledgments
This study was supported by the Science Fund for Creative
Research Groups (No. 21221004), the International Program of
MOST of China (No. 2013DFG92240) and Tsinghua University Ini-
tiative Scientific Research Program (No. 20121087922).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bej.2015.11.014.
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