Electrochemistry Communications 60 (2015) 126–130
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Electrochemistry Communications
journal homepage: www.elsevier.com/locate/elecom
Direct uptake of electrode electrons for autotrophic denitrification by
Thiobacillus denitrificans
Linpeng Yu a,b,c, Yong Yuan c, Shanshan Chen c,d, Li Zhuang c, Shungui Zhou c,⁎
a
Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China
Graduate University of Chinese Academy of Sciences, Beijing 100039, China
Guangdong Institute of Eco-environmental and Soil Sciences, Guangdong Key Laboratory of Agricultural Environment Management, Guangzhou, 510640, China
d
College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China
b
c
a r t i c l e
i n f o
Article history:
Received 19 July 2015
Received in revised form 27 August 2015
Accepted 27 August 2015
Available online 2 September 2015
Keywords:
Bioelectrochemical systems
Autotrophic nitrate reduction
Electron uptake
Biofilms
Thiobacillus denitrificans
a b s t r a c t
In this work, we reported that Thiobacillus denitrificans could utilize poised electrodes directly as sole electron
donors for autotrophic denitrification in bioelectrochemical systems. A potential-dependent denitrification
process was observed and catalyzed by the biofilms colonizing on the electrode surface, with a maximum nitrate
−1
day−1 m−2 at a potential of −500 mV. The intermediate prodremoval rate of 21.12 ± 1.67 mmol NO−
3 −N L
ucts (nitrite and N2O) suggested that denitrification was the main electron transfer pathway, and dissimilatory
nitrate reduction to ammonium was not present in this process. Cyclic voltammetry revealed the acclimation
potentials played an important role in the electrochemical activity of the biofilms. Electron transport inhibitors
suggested the participation of complex I, II, and III in the electron transfer during the denitrification.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Microbial denitrification is commonly used to remove nitrate in the
wastewater treatment, which generally consists of aerobic ammonium
oxidization and anaerobic nitrate reduction [1]. Compared with conditional biological process, the bioelectrochemical system (BES) has provided a promising approach to remove nitrate with simultaneous
electricity generation [2–4]. Thus, increasing research interest and
attention have been focused on denitrification by the BES. Georogy
et al. demonstrated that Geobacter metallreducens could use a poised
graphite electrode as a sole electron donor for nitrate reduction [5].
Such a BES can avoid the amendment with external organic carbon
that may result in an increasing process cost and secondary pollutions
[6]. In this regard, some attempts have been made to power microbes
directly with electricity to perform the denitrification reactions [7–10].
Mixed cultures and a few pure cultures, like Pseudomonas alcaliphila
were reported to utilize electrons from electrodes for autotrophic
denitrification in the absence of organic matter, implying its potential
application in the treatments of wastewater with low C/N ratio [11,
12]. However, information on the autotrophic nitrate removal by pure
cultures with an electrode as a sole electron donor is still limited.
⁎ Corresponding author. Tel./fax: +86 20 87025872.
E-mail address: [email protected] (S. Zhou).
http://dx.doi.org/10.1016/j.elecom.2015.08.025
1388-2481/© 2015 Elsevier B.V. All rights reserved.
Previously, Kato et al. and Rodrigues et al. evaluated the cathodic
activity of a chemoautotrophic denitrifying bacterium, Thiobacillus
denitrificans [13,14]. However, this electrochemically active strain is
questioned whether it can be used as an effective denitrification biocatalyst in the BES. On the other hand, while the mechanisms of extracellular electron transfer from bacteria to electrodes have been extensively
studied and well understood, the reverse direction, i.e., electron input
from electrodes to the bacterial cells, remains largely to be elucidated.
Here, we reported first the denitrification performance of
Thiobacillus denitrificans in a BES with electrodes as a sole electron
donor. Different electrode potentials were selected to investigate the
effects of electrode potentials on the denitrification rates. Cyclic
voltammetry (CV) and scanning electron microscopy (SEM) were
used to determine the electrochemical activity of biofilms on the electrodes and their morphologies, respectively.
2. Experimental
2.1. Microorganism and cultivation
Thiobacillus denitrificans (DSM 12475) was purchased from the
Deutsche Sam-mlung Mikroorganismen und Zellkulturen. To prepare
the inoculum, T. denitrificans was cultured in the DSM medium
113 at 30 °C.
L. Yu et al. / Electrochemistry Communications 60 (2015) 126–130
127
2.2. BES setup and operation
Bioelectrochemical reactors with a liquid volume of 110 ml and a
headspace volume of 50 ml for each chamber were constructed as
previously described [15]. Carbon felt (3 cm × 5 cm), carbon cloth
(7 cm × 7 cm), and saturated calomel reference electrodes (SCE) were
used as the anode, cathode, and reference electrodes, respectively. The
reactors were connected to a multi-potentiostat (CHI1040, Chenhua
Co., Ltd., Shanghai, China) with cathodic potentials poised at − 500,
−300, −100, and +250 mV, respectively. These potentials are higher
than −600 mV, which avoids the production of H2 in the system [16].
Each reactor was run in triplicate at 30 °C and an open circuit potential
(OCP) reactor was used as a control. Abiotic electrochemical denitrification was conducted similarly except that no bacteria were inoculated
into the cathodes. All of the potentials reported in this study were relative to standard hydrogen electrode (SHE) unless otherwise noted. The
catholyte was the DSM medium 113 containing 2 mM nitrate and
omitting sodium thiosulfate and NH4Cl. The anodic chambers were
filled with 110 ml of 0.1 M potassium phosphate buffer solution (PBS,
pH 7.0). Prior to the experiments, all of the reactors and electrolytes
were autoclaved and purged with CO2, and then sealed by rubber
stoppers. Pure cultures of T. denitrificans at the logarithmic phase
were collected and washed several times, and then transferred into
the cathodes (OD600 = 0.1). The biofilms on the electrodes were acclimated for 30 days as previously described except that only CO2 was
used [17]. Electron transport inhibitors dicumarol, quinacrine,
dicyclohexylcarbodiimide (DCCD), rotenone and antimycin A were
spiked into the BESs (− 500 mV) after the acclimation period [18].
Control experiments of equivalent amounts of the solvents (water,
ethanol, or acetone) showed no significant effects on the current
generation.
2.3. Analytic techniques
−
Nitrate (NO−
3 −N) and nitrite (NO2 −N) in the cathodic chambers
were determined by ion chromatography (ICS-90, DIONEX, USA) as
previously described [19]. N2O in the headspace was analyzed using a
gas chromatograph equipped with an electron capture detector (ECD)
(GC7900, Tianmei Scientific Instruments Inc., China). Electrochemical
in situ FTIR spectroscopy of living bacteria was conducted in the same
way as previously reported [20]. Measurements of the biofilm proteins
and sample preparations for SEM (S-4800 FESEM, Hitachi Inc., Japan)
were performed as previously described [21,22].
3. Results and discussion
3.1. T. denitrificans electron uptake in the BES reactors
After a month of acclimation of T. denitrificans in the presence of
nitrate in the BESs at various potentials of − 500, − 300, − 100, or
+ 250 mV, a pronounced cathode current was observed (Fig. 1). The
current at each potential appeared to increase with time after 4 days
of incubation and then reached a maximum of approximately 160.83,
76.51, 29.46, and 11.39 μA, respectively. By contrast, there was a negligible current at − 500 mV in the abiotic control reactor. These results
indicated that T. denitrificans biofilms could utilize electrons from electrodes for autotrophic metabolism.
As shown in Fig. 2, nearly no bacterial cells colonized on the OCP
control electrode. By contrast, a thin layer of rod-shaped cells was
sparsely distributed on surfaces of electrodes poised at the four potentials. The total proteins of biofilms on the electrodes poised at − 500,
− 300, − 100, and + 250 mV were 3.67 ± 0.50, 2.48 ± 0.18, 2.84 ±
0.39, and 2.60 ± 0.19 μg cm− 2, respectively. These low biomasses
were probably due to the slow growth rate of the autotrophic biofilms
[23].
Fig. 1. Current generation by T. denitrificans biofilms with poised electrodes as sole
electron donors.
3.2. Denitrification performance in the BESs
Concomitant with the current production, nitrate was gradually
consumed and reduction products accumulated. As shown in Fig. 3a,
the lower cathodic potential led to a higher nitrate removal rate. After
15 days of incubation, 75.62 ± 5.97%, 58.52 ± 6.05%, 48.38 ± 5.71%,
and 26.80 ± 5.23% of nitrate were reduced in the reactors poised at
−500, −300, −100, and +250 mV, respectively. The calculated overall
nitrate reduction rate increased from 7.50 ± 1.46 to 21.12 ± 1.67 mmol
−1
day− 1 m−2 with the decreasing electrode potentials,
NO−
3 −N L
which coincided with the order of cathodic currents. These activities
of T. denitrificans were lower than those of G. metallireducens
−1
day− 1 m− 2) and Pseudomonas alcaliphila
(90 mmol NO−
3 −N L
−
−1
(160 mmol NO3 −N L day−1 m−2) [5,12]. In comparison, the nitrate
−1
day−1 m−2 for
reduction rate was only 0.58 ± 0.39 mmol NO−
3 −N L
the OCP control. Moreover, nitrate was not reduced and no product
(nitrite, N2O or ammonium) accumulated in the abiotic reactors poised
at − 500 mV. This indicated that the carbon cloth electrode could not
effectively catalyze electrochemical nitrate reduction alone. Hydrogen
was not detected in all of the reactors at the applied potentials, negating
the possibility of hydrogen-mediated electron transfer reactions. As a
result, the experimental data indicated that T. denitrificans biofilms
could directly utilize electrons from electrodes and act as a biocatalyst
on the electrode surface to remove nitrate.
The profiles of autotrophic denitrification products by T. denitrificans
biofilms with poised electrodes as sole electron donors were shown in
Fig. 3b and c. Nitrite and nitrous oxide (N2O) were identified as the
intermediate products, while NO were not detected during the entire
experiments. After 15 days, 1.39 ± 0.18 mM, 1.16 ± 0.12 mM, 0.91 ±
0.11 mM, and 0.19 ± 0.14 mM of nitrite were produced in the reactors
of −500, −300, −100, and +250 mV, respectively. N2O accumulated
fast in the first few days and then the N2O concentrations decreased
gradually. This indicated that the intermediate N2O was further transformed to N2 or other nitrogen species. NH+
4 − N was not detected in
the systems, suggesting the absence of dissimilatory nitrate reduction
to ammonium (DNRA). Coulombic efficiency analysis revealed that
55% to 72% of cathodic electrons were recovered in the denitrification
products (Fig. 3d). The partial loss of cathodic electrons presumably resulted from the growth and maintenance of biofilms on the electrodes.
3.3. Electrochemical activity of T. denitrificans biofilms
CV scans were performed to investigate the electrochemical activity
of T. denitrificans biofilms, which were conducted at 15 days. As shown
in Fig. 4a, all of the biofilm electrodes showed higher catalytic currents
than the OCP control. For these biofilms, a reduction peak at
−310 mV appeared in the CV scans, whereas no obvious peak was observed for the OCP electrode. Therefore, at least one redox component
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L. Yu et al. / Electrochemistry Communications 60 (2015) 126–130
Fig. 2. SEM images of the biofilms on the electrode surface developed at −500 mV (a), −300 mV (b), −100 mV (c), +250 mV (d), the open circuit potential (control, e), and the biofilm
proteins on the electrodes (f).
was likely to be responsible for the electron transfer reactions. To identify whether the redox components were located in the biofilms or the
culture media, the biofilm electrode poised at −100 mV was scanned
by CV at different scan rates. The reduction peak currents at −310 mV
increased linearly with the scan rates from 1 to 20 mV/s (Fig. 4b). This
implied that the redox species were mainly attributed to the biofilms
on the electrodes rather than dissolved substances in the solution [24].
In addition, when the biocathodes were replaced by abiotic carbon
cloth electrodes, no peak was detected by CV scans from the culture
media (data not shown). Therefore, it suggested that T. denitrificans
did not excrete an extracellular electron shuttle during the entire experiment and the biofilms utilized electrode electrons by a direct way.
3.4. Electron transfer mechanism
Electrochemical in situ FTIR spectroscopy and electron transport
inhibitors were used to investigate the redox components and electron
transfer mechanism of T. denitrificans biofilms. Main signal bands (at
Fig. 3. The reduction of nitrate (a) and accumulation of denitrification products in the BES (b, c), the comparison of the total electrons transferred and coulombic efficiencies for different
electrode potentials (d).
L. Yu et al. / Electrochemistry Communications 60 (2015) 126–130
129
Fig. 4. CVs of the biocathodes developed at the selected electrode potentials (a, scan rate, 10 mV s−1) and the biocathode developed at −100 mV at different scan rates (b); electrochemical
in situ FTIR spectroscopy of living T. denitrificans at different potentials (c); effects of electron transport inhibitors on electron uptake (d, −500 mV) and the proposed electron transfer
pathway for the electrochemical denitrification.
1667 and 1551 cm−1) of the FTIR spectra came from the amide I and
amide II in the peptide chains, respectively (Fig. 4c) [25]. Small increases
in the intensity at the bands 1551 and 1716 cm− 1 were observed
when the electrode potential decreased gradually from − 100 mV to
− 800 mV. The latter band could be assigned to the carbonyl (C_O)
groups [20]. These results indicated that certain redox transformation
reactions occurred at the cell–electrode interface in this process. Additionally, the peak at 1407 cm− 1 suggested the presence of surface
heme groups of Cyt-C [25,26].
Dicumarol, an inhibitor of the quinone loop, displayed obvious inhibition effects on the electrochemical activities (Fig. 4d). Quinacrine,
which blocks the FAD and FMN centers in complex II showed no effect
at a low concentration (0.20 mM). However, increased dosages of
quinacrine (3.20 mM) resulted in decreased cathode currents, suggesting the participation of complex II in the electron transfer. The ATPase
inhibitor DCCD (1.55 mM) and complex I (NADH reductase) inhibitor
rotenone (1.88 mM) almost completely inhibited the current generation. Similar inhibitory effects were observed when adding complex III
inhibitor antimycin A. Therefore, the suggested electron transfer pathway was obtained (Fig. 4e) [27,28].
4. Conclusions
Our experimental results demonstrate that T. denitrificans biofilms
can directly utilize a solid electrode as a sole electron donor for autotrophic nitrate removal. Denitrification was identified as the main electron
transfer pathway, while dissimilatory nitrate reduction to ammonium
(DNRA) was not present in this process. The bioelectrochemical denitrification performances of the biocathodes depend on the applied
electrode potentials. Electron transport inhibitors suggested that
complex I, II, and III participated in the electron transfer during the
autotrophic denitrification.
Conflict of interest
There is no conflict of interest in this work.
Acknowledgement
This work was supported by the Guangdong Natural Science
Funds for Distinguished Young Scholar (2014A030306033 and
S20120011151), the Natural Science Foundation of Guangdong
Province (S2013040015231), and the Guangzhou City Science–
Technology Project (201510010025). The authors thank Prof. Shigang
Sun and Dr. L.X. You in Xiamen University for their assistance in the
FTIR experiments.
References
[1] C. Fang, B. Min, I. Angelidaki, Nitrate as an oxidant in the cathode chamber of a
microbial fuel cell for both power generation and nutrient removal purposes,
Appl. Biochem. Biotechnol. 164 (2011) 464–474.
[2] Y.H. Jia, H.T. Tran, D.H. Kim, S.J. Oh, D.H. Park, R.H. Zhang, D.H. Ahn, Simultaneous
organics removal and bio-electrochemical denitrification in microbial fuel cells,
Bioprocess Biosyst. Eng. 31 (2008) 315–321.
[3] B. Virdis, K. Rabaey, Z.G. Yuan, J. Keller, Microbial fuel cells for simultaneous carbon
and nitrogen removal, Water Res. 42 (2008) 3013–3024.
[4] P. Clauwaert, J. Desloover, C. Shea, R. Nerenberg, N. Boon, W. Verstraete, Enhanced
nitrogen removal in bio-electrochemical systems by pH control, Biotechnol. Lett.
31 (2009) 1537–1543.
[5] K.B. Gregory, D.B. Bond, D.R. Lovley, Graphite electrodes as electron donors for
anaerobic respiration, Environ. Microbiol. 6 (2004) 596–604.
130
L. Yu et al. / Electrochemistry Communications 60 (2015) 126–130
[6] Y.X. Zhao, B.G. Zhang, C.P. Feng, F.Y. Huang, P. Zhang, Z.Y. Zhang, Y.N. Yang, N.
Sugiura, Behavior of autotrophic denitrification and heterotrophic denitrification
in an intensified biofilm-electrode reactor for nitrate-contaminated drinking
water treatment, Bioresour. Technol. 107 (2012) 159–165.
[7] H.I. Park, D.K. Kim, Y. Choi, D. Pak, Nitrate reduction using an electrode as direct
electron donor in a biofilm-electrode reactor, Process Biochem. 40 (2005)
3383–3388.
[8] S. Puig, M. Serra, A. Vilar-Sanz, M. Cabré, L. Bañeras, J. Colprim, M.D. Balaguer,
Autotrophic nitrite removal in the cathode of microbial fuel cells, Bioresour.
Technol. 102 (2011) 4462–4467.
[9] W.J. Zhang, Y. Zhang, W.T. Su, Y. Jiang, M. Su, P. Gao, D.P. Li, Effects of cathode potentials and nitrate concentrations on dissimilatory nitrate reductions by Pseudomonas
alcaliphila in bioelectrochemical systems, J. Environ. Sci. 26 (2014) 885–891.
[10] M.H. Zhou, W.J. Fu, H.Y. Gu, L.C. Lei, Nitrate removal from groundwater by a novel
three-dimensional electrode biofilm reactor, Electrochim. Acta 52 (2007)
6052–6059.
[11] S. Puig, M. Coma, J. Desloover, N. Boon, J. Colprim, M.D. Balaguer, Autotrophic
denitrification in microbial fuel cells treating low ionic strength waters, Environ.
Sci. Technol. 46 (2012) 2309–2315.
[12] W.T. Su, L.X. Zhang, D.P. Li, G.Q. Zhan, J.W. Qian, Y. Tao, Dissimilatory nitrate reduction by Pseudomonas alcaliphila with an electrode as the sole electron donor,
Biotechnol. Bioeng. 109 (2012) 2904–2910.
[13] S. Kato, K. Hashimoto, K. Watanabe, Microbial interspecies electron transfer via
electric currents through conductive minerals, Proc. Natl. Acad. Sci. U. S. A. 109
(2012) 10042–10046.
[14] T.C. Rodrigues, M.A. Rosenbaum, Microbial electroreduction: screening for new
cathodic biocatalysts, ChemElectroChem 1 (2014) 1916–1922.
[15] M. Siegert, M.D. Yates, D.F. Call, X.P. Zhu, A. Spormann, B.E. Logan, Comparison
of nonprecious metal cathode materials for methane production by
electromethanogenesis, Sustain. Chem. Eng. 2 (2014) 910–917.
[16] K.P. Nevin, T.L. Woodard, A.E. Franks, Z.M. Summers, D.R. Lovley, Microbial
electrosynthesis: feeding microbes electricity to convert carbon dioxide and
water to multicarbon extracellular organic compounds, MBio 1 (2010)
e00103–e00110.
[17] Y. Jiang, M. Su, Y. Zhang, G.Q. Zhan, Y. Tao, D.P. Li, Bioelectrochemical systems for
simultaneously production of methane and acetate from carbon dioxide at relatively
high rate, Int. J. Hydrog. Energy 38 (2013) 3497–3502.
[18] B.H. Kim, H.S. Park, H.J. Kim, G.T. Kim, I.S. Chang, J. Lee, N.T. Phung, Enrichment of
microbial community generating electricity using a fuel-cell-type electrochemical
cell, Appl. Microbiol. Biotechnol. 63 (2004) 672–681.
[19] W. Zhang, X.M. Li, T.X. Liu, F.B. Li, Enhanced nitrate reduction and current generation
by Bacillus sp. in the presence of iron oxides, J. Soils Sediments 12 (2012) 354–365.
[20] L.X. You, L. Rao, X.C. Tian, R.R. Wu, X. Wu, F. Zhao, Y.X. Jiang, S.G. Sun, Electrochemical in situ FTIR spectroscopy studies directly extracellular electron transfer of
Shewanella oneidensis MR-1, Electrochim. Acta 170 (2015) 131–139.
[21] C. Grobbler, B. Virdis, A. Nouwens, F. Harnisch, K. Rabaey, P.L. Bond, Use of SWATH
mass spectrometry for quantitative proteomic investigation of Shewanella oneidensis
MR-1 biofilms grown on graphite cloth electrodes, Syst. Appl. Microbiol. 38 (2015)
135–139.
[22] T. Zhang, H.R. Nie, T.S. Bain, H.Y. Lu, M.M. Cui, O.L. Snoeyenbos-West, A.E. Franks, K.P.
Nevin, T.P. Russell, D.R. Lovley, Improved cathode materials for microbial
electrosynthesis, Energy Environ. Sci. 6 (2013) 217–224.
[23] Z. Zaybak, J.M. Pisciotta, J.C. Tokash, B.E. Logan, Enhanced start-up of anaerobic
facultatively autotrophic biocathodes in bioelectrochemical systems, J. Biotechnol.
168 (2013) 478–485.
[24] Y. Yuan, S.G. Zhou, N. Xu, L. Zhuang, Electrochemical characterization of anodic
biofilms enriched with glucose and acetate in single-chamber microbial fuel cells,
Colloids Surf. B 82 (2011) 641–646.
[25] J.P. Busalmen, A. Esteve-Nunez, A. Berna, J.M. Feliu, C-type cytochromes wire
electricity-producing bacteria to electrodes, Angew. Chem. Int. Ed. 47 (2008)
4874–4877.
[26] J.P. Busalmen, A. Esteve-Nunez, A. Berna, J.M. Feliu, ATR-SEIRAs characterization of
surface redox processes in G. sulfurreducens, Bioelectrochemistry 78 (2010) 25–29.
[27] K. Takayama, Biocatalyst electrode modified with whole-cells of P. denitrificans for
the determination of nitrate, Bioelectrochem. Bioenerg. 45 (1998) 67–72.
[28] J.W. Chen, M. Strous, Denitrification and aerobic respiration, hybrid electron transport chains and co-evolution, Biochim. Biophys. Acta 1827 (2013) 136–144.