- Email: [email protected]
Electron transfer of Pseudomonas aeruginosa CP1 in electrochemical reduction of nitric oxide
Bioresource Technology xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Electron transfer of Pseudomonas aeruginosa CP1 in electrochemical reduction of nitric oxide Shaofeng Zhou, Shaobin Huang ⇑, Jiaxin He, Han Li, Yongqing Zhang School of Environment and Energy, South China University of Technology, Higher Education Mega Center, Guangzhou 510006, PR China Guangdong Provincial Key Laboratory of Atmospheric Environment and Pollution Control, Guangzhou 510006, PR China
h i g h l i g h t s Electrochemical reduction of NO catalyzed by P. aeruginosa is reported. Current generation improved in the presence of bacteria. A soluble compound was responsible for the NO reduction process.
a r t i c l e
i n f o
Article history: Received 25 April 2016 Received in revised form 3 July 2016 Accepted 4 July 2016 Available online xxxx Keywords: Nitric oxide reduction Electron transfer Denitrification Pseudomonas aeruginosa CP1 Soluble shuttles
a b s t r a c t This study reports catalytic electro-chemical reduction of nitric oxide (NO) enhanced by Pseudomonas aeruginosa strain CP1. The current generated in the presence of bacteria was 4.36 times that in the absence of the bacteria. The strain was able to catalyze electro-chemical reduction of NO via indirect electron transfer with an electrode, revealed by a series of cyclic voltammetry experiments. Soluble electron shuttles secreted into solution by live bacteria were responsible for the catalytic effects. The enhancement of NO reduction was also confirmed by detection of nitrous oxide; the level of this intermediate was 46.4% higher in the presence of bacteria than in controls, illustrated that the electron transfer pathway did not directly reduce nitric oxide to N2. The findings of this study may offer a new model for bioelectrochemical research in the field of NO removal by biocatalysts. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Biocatalysts, a novel key aspect of bio-electrochemistry, are of great interests in bioremediation, electricity generation and other bio-electrochemical systems (BESs), benefitting from intrinsic advantages like self-regeneration and self-organization, lower reaction overpotential and lower cost, comparing with other chemical catalysts (Clauwaert et al., 2008). Oxygen reduction in the biocathodic chamber of BESs has recently drawn much attention, and is applied in biofuel cells as well as microbial electrolytic cells (MECs). Nitrate can also be used as an electron acceptor by employing a biocathode in either microbial fuel cells (MFCs) or MECs (Zhao et al., 2012; Ghafari et al., 2008; Tao et al., 2014). Denitrification, which converts nitrite to nitrogen gas, is an important electron transfer pathway in anaerobic microorganisms (Yu et al., 2015), ⇑ Corresponding author at: School of Environment and Energy, South China University of Technology, Higher Education Mega Center, Guangzhou 510006, PR China. E-mail address: [email protected] (S. Huang).
and bacteria are capable of taking up the necessary electrons directly from electrodes as a bio-cathode (Wang et al., 2015). High throughput BES research was conducted on nitrate removal in wastewater treatments (Ghafari et al., 2008), but this ignored the gaseous pollutants like NO, which are environmentally damaging (Mi et al., 2009). Nitric oxide emissions from power plants are a major source of release of this gas into the atmosphere (Ramachandran et al., 2000), and NO is a significant human health threat because it inhibits proper oxygen transfer by hemoglobin and results in methemoglobinemia (Mousavi et al., 2012). At present, similar to oxygen electrochemical reduction, metal catalysts tend to be used in various cathodic processes; however, the high cost of this will limit the development of BES, even if an exterior electrical field or a chelating agent like ethylene diamine tetraacetic acid (EDTA) is added. Furthermore, few reports focused on exploiting electron transfer pathway between microbes and electrodes. Pseudomonas aeruginosa is a well-recognized model electroactive bacterium (EAB) and is able to self-control and produce extracellular mediators modulated by quorum sensing (Rabaey et al.,
http://dx.doi.org/10.1016/j.biortech.2016.07.010 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhou, S., et al. Electron transfer of Pseudomonas aeruginosa CP1 in electrochemical reduction of nitric oxide. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.07.010
2
S. Zhou et al. / Bioresource Technology xxx (2016) xxx–xxx
2004; Venkataraman et al., 2010). P. aeruginosa presents outstanding characteristics of catalyzing oxygen reduction (Cournet et al., 2010). However, to the best of our knowledge, there has been little research into electroactive bacteria that are capable of transferring electrons to an electrode and then catalyze nitric oxide reduction and produce current. This study aimed to (i) unveil possible electron transfer pathways in NO electro-reduction catalyzed by microbes, and (ii) test the feasibility of electrochemical NO removal by P. aeruginosa strain CP1. The outcomes offer an alternative strategy of practical developments, instead of using platinum and noble metals as catalysts. 2. Materials and methods 2.1. Bacterial strain and cultivation The bacterium used in this study was P. aeruginosa strain CP1 (collection number: M2015197, CCTCC, China) isolated from the bio-filtration system of a power plant. Prior to each experiment, the strain was cultured overnight in nutrient broth medium (peptone, 10 g/L; NaCl, 5 g/L; beef extract, 5 g/L; pH 7.2) at 35 °C under 120 rpm shaking. 2.2. Electrochemical analysis Cyclic voltammetry (CV) tests in a typical three-electrode system (Fig. 1) were performed via an electrochemical workstation (CHI 852D, Chenhua Co. Ltd., China) to study the electrochemical mechanism of NO reduction catalyzed by P. aeruginosa CP1. A 50ml sealed cell was used, equipped with a glassy-carbon electrode (3 mm diameter; Tianjin Aida, China) as working electrode (WE). Before CV examination, the WE was rinsed with deionized water facilely and pretreated with 50 mM phosphate buffer solution (PBS) (Cournet et al., 2010). The counter electrode (CE) and reference electrode (RE) were a platinum electrode and saturated Ag/AgCl electrode (+0.195 V vs. SHE; all potentials given versus this reference), respectively. For bacterial CV experiments, the bacterial suspension was centrifuged then washed in PBS three times to obtain a purified suspension. CV was performed on an aqueous solution of 50 mM PBS (pH 7.0) with added P. aeruginosa CP1 (final OD600 maintained at 0.50 ± 0.05 in each experiment, equivalent to 0.62 g cells/L). The blank solution contained no bacteria. The solutions were performed in the sequence, (i) immediately after the addition of bacteria; (ii) after 1 h; (iii) after 30 min of nitrogen bubbling; and
(iv) after 2 min of bubbling 10% NO:90% N2 (v/v). The scan rate was constant at 25 mV/s from 1.0 V to 0.6 V, unless otherwise stated. For measurements using nutrient broth in which had grown (i.e., to test culture supernatant), the bacterial samples were centrifuged for 5 min at 6000 rpm and the supernatant was filtered twice through a 0.22-lm sterile filter to remove microorganisms (Wang et al., 2015; Cournet et al., 2010). Then CV curves were acquired as described above, sterile nutrient broth medium (in which P. aeruginosa CP1 had not been cultured) was used as a control. Each CV test was independently performed at least four times at room temperature (20–25 °C). All solutions used in this study were prepared just before testing. All experiments were fully infused with N2 before NO bubbled because microorganisms in cathodic chamber under aerobic state could be refrained by oxygen molecule, resulting in decrease of coulombic efficiency. 2.3. NO and N2O measurements An assorted micro-sensor multimeter (Unisense Co., Denmark) was employed to detect concentrations of NO and N2O in solutions in each experiment (Andersen et al., 2001). 3. Results and discussion A reduction peak was observed in the CV of PBS after the addition of P. aeruginosa CP1, with onset at around 310 mV and reached its peak at 620 mV (Fig. 2A, line a). This could be identified as oxygen electro-reduction catalyzed by the bacteria because it disappeared after the solution was extensively flushed with nitrogen (Fig. 2A, line b). Oxygen reduction of this kind P. aeruginosa-PBS system was extensively discussed by Cournet et al. (2010). Disappearance of the oxygen peak indicates that the system was fully anaerobic. After 2 min of NO injection, a quite different CV wave emerged, which initiated at 620 mV and maximized at 820 mV, with about 7.8 lA maximal current (1.10 lA/mm2) generated. In the blank phosphate buffer solution, the flat CV curve did not show apparent catalytic effect. The current generated in the presence of bacteria was 4.36 times that without the bacteria, clearly indicating that strain CP1 played an important role in modifying the shape of the current-potential curve. No oxidationreduction peak was observed in the whole scan range when sterile nutrient broth medium was tested (Fig. 2B). No significant change in NO concentration was found in pure PBS or bacteria-PBS (only differed by 4.8%). While equal NO was infused into the electrochemical cell in anaerobic conditions, a similar trend was found. N2O generation was 46.4% higher in the presence of the bacteria, 3.65 ± 0.07 mg/L compared with 2.50 ± 0.10 mg/L. The detection of nitrous oxide indicated that nitric oxide was first reduced to nitrous oxide then to nitrogen in this system. Thus the reaction process is (Clauwaert et al., 2007).
2NO þ 2e þ 2Hþ ðaqÞ ! N2 O þ H2 OðE : 1:591 VÞ
N2 O þ 2e þ 2Hþ ðaqÞ ! N2 þ H2 OðE : 1:766 VÞ
Fig. 1. Schematic diagram of the microbial electrolytic system. Sox: oxidation state shuttles; Sred: oxidation state shuttles; GC: glassy carbon; WE: working electrode; CE: counter electrode; RE: reference electrode.
The presence of bacteria was necessary for significant catalysis to be observed. The production of nitrous oxide in the blank experiment might be attributable to the reductive effect of H2, which can be generated at high-level negative potential when the voltage less than 600 mV, because protons can then accept electrons thus produce hydrogen (standard potential of H+/H2, 0.414 V, pH = 7, 25 °C) (Nevin et al., 2010; Gregory et al., 2004); the hydrogen could then contribute to NO reduction. However, the effect of hydrogen in this regard was not remarkable, since little reduction peaks were
Please cite this article in press as: Zhou, S., et al. Electron transfer of Pseudomonas aeruginosa CP1 in electrochemical reduction of nitric oxide. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.07.010
S. Zhou et al. / Bioresource Technology xxx (2016) xxx–xxx
Fig. 2. Cyclic voltammograms (pH7.0) in 50 mM phosphate buffer (A) and filtered culture supernatant (B). (A) (a) Immediately after the addition of P. aeruginosa strain CP1 (black dotted line); (b) after 30 min of nitrogen bubbling (red line); (c) after 1 min of 10% NO bubbling (pink dashed line); (d) abiotic control after NO bubbling (blue line). (B) (a) Before any gas bubbling (black dotted line); (b) after 30 min of nitrogen bubbling (red line); (c) after 1 min of 10% NO bubbling (pink dashed line); (d) abiotic control after NO bubbling (i.e., sterile nutrient broth medium) (blue line). Scan rate: 25 mV s1. Inset: NO electro-catalyzed reduction (dotted line: filtered supernatant; solid line: phosphate buffer in the presence of P. aeruginosa CP1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
detected in blank PBS or sterile nutrient medium even when the scan voltage reached 1 V, well below the H2-generation potential when NO was infused. It is worth noting that the oxidation-reduction peak (Fig. 2B, line c) at about 140 mV corresponded to that of the typical ‘‘redox active compound” of P. aeruginosa, which was confirmed as an electron shuttle in biofuels, helping to generate a high power density (Rabaey et al., 2004). It seemed this compound was also responsible for catalyzing the nitric oxide reduction observed here. At this potential, peak sizes enlarged when NO was injected; thus P. aeruginosa strain CP1 was electrochemically active in the reaction. From the inset of Fig. 2B, semblable NO reduction peaks were observed both in phosphate buffer and exhausted nutrient broth, indicating the electron shuttle(s) released by CP1 were soluble, and responsible for electron transfer between the electrode and the bacteria, because electrochemical reduction of NO was observed with or without the presence of the bacteria. Thus, conclusion is that P. aeruginosa CP1 catalyzed NO electrochemical reduction by conveying electrons indirectly to the electrode, most
3
likely with the help of soluble secreta (pyocyanin, cytochrome c, etc.) rather than through conductive pili (also known as nanowires), direct transfer to/from the electrodes, or other pathways. However, this theory cannot explain why similar curves were also observed immediately after a bacteria-attached electrode was inserted into solution although they showed much lower current generation when NO was infused (Fig. 3). But, extrapolating from studies on oxygen reduction (Cournet et al., 2010), a thin micro-biofilm could form in seconds. In this condition, one may observe the electrochemical phenomenon even in the absence of nutrients, because bacterial metabolism with sufficient electron supply might not cease to secrete shuttles (redox shuttle generation consumes very little energy (Marsili et al., 2008). Even a nano-molar level of redox-active compound(s) would greatly influence the CV curves (Marsili et al., 2008). Thus, bacteria might not directly participate in the reduction process, but their secreta were essential for the reaction. Another possibility is that the secreted redox compound might play its role by attaching to the cathodic electrode surface, rather than wandering in solution (Fig. 1). This would account for the experimental observations made here, since the current values of reductive peaks were in direct proportion to the square root of the scan rate (Fig. 4.) Yu et al., 2015; Freguia et al., 2010. The shuttles could give electrons by reducing the NO, but the concentration of NO was quite limited in this system. They might not able to exchange electrons with NO that far away from the electrode surface. In this CV system, electrons were in excess, thus microbial electrochemical behavior was not limited by electron donor availability. The narrow potential range of the NO electrochemical reduction indicated that it would be the inner redox rate or surface supply of NO that should be considered as key limiting factors. Because of the poor solubility of NO, a reduction peak was barely observed in blank PBS or fresh sterile nutrient broth, demonstrating that low NO transfer rate to the electrode that limited the reaction rate if only an electrochemical approach was applied in NO reduction (i.e., in the absence of bacteria). Thus complexants like EDTA would be essential for efficient NO removal (Gao et al., 2011). Although little bacterial adhesion was needed to obtain a catalytic signal for NO reduction, we suggest that the process would be improved by the use of cathodic materials with more specific surface area, such as graphite and other carbon-based materials. The more bacteria attach, the more shuttles there would be that help to boost the electron transfer efficiency.
Fig. 3. CV from a brand-new electrode inserted into a bacterial solution in PBS (a) and a bacteria-attached electrode in fresh PBS bubbled with NO (b).
Please cite this article in press as: Zhou, S., et al. Electron transfer of Pseudomonas aeruginosa CP1 in electrochemical reduction of nitric oxide. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.07.010
4
S. Zhou et al. / Bioresource Technology xxx (2016) xxx–xxx
Conflict of interest statement The authors declare that there is no conflict of interest. Acknowledgements This research was financially supported by the National Natural Science Foundation of China (Grant No. 51378217 and No. U1360101), and Research Project of Guangdong Provincial Department of Science and Technology (Grants Nos. 2014B050505004 and 2015B020236001). References
Fig. 4. Cyclic voltammograms at different scan rates (line a: 25 mV s1, line b: 50 mV s1 and line c: 70 mV s1) in PBS with P. aeruginosa CP1. Inset: linear simulation of peak height of NO electro-reduction vs. square root of scan rate.
Benefit from various cellular respiration modes, electroactive bacteria that uptake electrons from electrodes and simultaneously denitrify should be given fully attention in both MFCs and MECs. Although NO may not generate interest as a cathodic electron acceptor in energy recovery (thermodynamic limitations, current generation was unsatisfactory), microorganisms at the cathode can reduce theoretically the electrical energy needed for nitrogen removal in MECs compared to conventional water electrolysis processes (Zhang and Angelidaki, 2014). Thus, the outcome of this study may be an alternative strategy that is applied as a followup after conventional denitrification that may broaden scope of both wastewater treatment and waste gas removal. P. aeruginosa might not be the only species capable of catalyzing electrochemical NO reduction. The secreta as a replacement for EDTA could cut the cost of BES and reduce the amount of electrical energy that must be added from external source. Attempts are in progress to evaluate the catalytic effects of other species; underlying synergistic effects could be identified. 4. Conclusions P. aeruginosa strain CP1, was able to catalyze electrochemical reduction of NO via indirect electron transfer with electrodes. This catalysis of NO reduction may take place in two ways. One possibility is NO could be reduced by metabolic of the strain CP1; the other may be the reaction with a kind of soluble shuttle secreted by CP1. The enhancement observed in this test may become a suitable technique for bioelectrochemical systems such as MEC to reduce the cost of NO treatment. The soluble shuttle (s), which could freely shift from inside bacteria into solutions, was mainly responsible for the catalytic effect, playing it role by reducing NO on electrode surface. The electrochemical reduction is a diffusion-control process partly due to the low solubility of NO. The electrons transfer pathway was not direct reduction of NO to N2, as revealed by the detection of N2O as an intermediate in the catalytic progress. The findings in this study may provide basic information for future research and applications.
Andersen, K., Kjaer, T., Revsbech, N.P., 2001. An oxygen insensitive microsensor for nitrous oxide. Sens. Actuators, B 81, 42–48. Clauwaert, P., Rabaey, K., Aelterman, P., De Schamphelaire, L., Ham, T.H., Boeckx, P., Boon, N., Verstraete, W., 2007. Biological denitrification in microbial fuel cells. Environ. Sci. Technol. 41, 3354–3360. Clauwaert, P., Aelterman, P., Pham, T.H., De Schamphelaire, L., Carballa, M., Rabaey, K., Verstraete, W., 2008. Minimizing losses in bio-electrochemical systems: the road to applications. Appl. Microbiol. Biotechnol. 79, 901–913. Cournet, A., Berge, M., Roques, C., Bergel, A., Delia, M.L., 2010. Electrochemical reduction of oxygen catalyzed by Pseudomonas aeruginosa. Electrochim. Acta 55, 4902–4908. Freguia, S., Tsujimura, S., Kano, K., 2010. Electron transfer pathways in microbial oxygen biocathodes. Electrochim. Acta 55, 813–818. Gao, L., Mi, X.H., Zhou, Y., Li, W., 2011. A pilot study on the regeneration of ferrous chelate complex in NOx scrubber solution by a biofilm electrode reactor. Bioresour. Technol. 102, 2605–2609. Ghafari, S., Hasan, M., Aroua, M.K., 2008. Bio-electrochemical removal of nitrate from water and wastewater–a review. Bioresour. Technol. 99, 3965–3974. Gregory, K.B., Bond, D.R., Lovley, D.R., 2004. Graphite electrodes as electron donors for anaerobic respiration. Environ. Microbiol. 6, 596–604. Marsili, E., Baron, D.B., Shikhare, I.D., Coursolle, D., Gralnick, J.A., Bond, D.R., 2008. Shewanella secretes flavins that mediate extracellular electron transfer. Proc. Natl. Acad. Sci. U.S.A. 105, 3968–3973. Mi, X.H., Gao, L., Zhang, S.H., Cai, L.L., Li, W., 2009. A new approach for Fe(III)EDTA reduction in NOx scrubber solution using bio-electro reactor. Bioresour. Technol. 100, 2940–2944. Mousavi, S., Ibrahim, S., Aroua, M.K., 2012. Sequential nitrification and denitrification in a novel palm shell granular activated carbon twin-chamber upflow bio-electrochemical reactor for treating ammonium-rich wastewater. Bioresour. Technol. 125, 256–266. Nevin, K.P., Woodard, T.L., Franks, A.E., Summers, Z.M., Lovley, D.R., 2010. Microbial electrosynthesis: feeding microbes electricity to convert carbon dioxide and water to multicarbon extracellular organic compounds. mBio 1. Rabaey, K., Boon, N., Siciliano, S.D., Verhaege, M., Verstraete, W., 2004. Biofuel cells select for microbial consortia that self-mediate electron transfer. Appl. Environ. Microbiol. 70, 5373–5382. Ramachandran, B., Herman, R.G., Choi, S., Stenger, H.G., Lyman, C.E., Sale, J.W., 2000. Testing zeolite SCR catalysts under protocol conditions for NOx abatement from stationary emission sources. Catal. Today 55, 281–290. Tao, Q.Q., Luo, J.J., Zhou, J., Zhou, S.Q., Liu, G.L., Zhang, R.D., 2014. Effect of dissolved oxygen on nitrogen and phosphorus removal and electricity production in microbial fuel cell. Bioresour. Technol. 164, 402–407. Venkataraman, A., Rosenbaum, M., Arends, J.B.A., Halitschke, R., Angenent, L.T., 2010. Quorum sensing regulates electric current generation of Pseudomonas aeruginosa PA14 in bioelectrochemical systems. Electrochem. Commun. 12, 459–462. Wang, X., Yu, P., Zeng, C., Ding, H., Li, Y., Wang, C., Lu, A., 2015. Enhanced Alcaligenes faecalis denitrification rate with electrodes as the electron donor. Appl. Environ. Microbiol. 81, 5387–5394. Yu, L.P., Yuan, Y., Chen, S.S., Zhuang, L., Zhou, S.G., 2015. Direct uptake of electrode electrons for autotrophic denitrification by Thiobacillus denitrificans. Electrochem. Commun. 60, 126–130. Zhang, Y.F., Angelidaki, I., 2014. Microbial electrolysis cells turning to be versatile technology: recent advances and future challenges. Water Res. 56, 11–25. Zhao, Y., Zhang, B., Feng, C., Huang, F., Zhang, P., Zhang, Z., Yang, Y., Sugiura, N., 2012. Behavior of autotrophic denitrification and heterotrophic denitrification in an intensified biofilm-electrode reactor for nitrate-contaminated drinking water treatment. Bioresour. Technol. 107, 159–165.
Please cite this article in press as: Zhou, S., et al. Electron transfer of Pseudomonas aeruginosa CP1 in electrochemical reduction of nitric oxide. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.07.010
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Electron transfer of Pseudomonas aeruginosa CP1 in electrochemical reduction of nitric oxide Shaofeng Zhou, Shaobin Huang ⇑, Jiaxin He, Han Li, Yongqing Zhang School of Environment and Energy, South China University of Technology, Higher Education Mega Center, Guangzhou 510006, PR China Guangdong Provincial Key Laboratory of Atmospheric Environment and Pollution Control, Guangzhou 510006, PR China
h i g h l i g h t s Electrochemical reduction of NO catalyzed by P. aeruginosa is reported. Current generation improved in the presence of bacteria. A soluble compound was responsible for the NO reduction process.
a r t i c l e
i n f o
Article history: Received 25 April 2016 Received in revised form 3 July 2016 Accepted 4 July 2016 Available online xxxx Keywords: Nitric oxide reduction Electron transfer Denitrification Pseudomonas aeruginosa CP1 Soluble shuttles
a b s t r a c t This study reports catalytic electro-chemical reduction of nitric oxide (NO) enhanced by Pseudomonas aeruginosa strain CP1. The current generated in the presence of bacteria was 4.36 times that in the absence of the bacteria. The strain was able to catalyze electro-chemical reduction of NO via indirect electron transfer with an electrode, revealed by a series of cyclic voltammetry experiments. Soluble electron shuttles secreted into solution by live bacteria were responsible for the catalytic effects. The enhancement of NO reduction was also confirmed by detection of nitrous oxide; the level of this intermediate was 46.4% higher in the presence of bacteria than in controls, illustrated that the electron transfer pathway did not directly reduce nitric oxide to N2. The findings of this study may offer a new model for bioelectrochemical research in the field of NO removal by biocatalysts. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Biocatalysts, a novel key aspect of bio-electrochemistry, are of great interests in bioremediation, electricity generation and other bio-electrochemical systems (BESs), benefitting from intrinsic advantages like self-regeneration and self-organization, lower reaction overpotential and lower cost, comparing with other chemical catalysts (Clauwaert et al., 2008). Oxygen reduction in the biocathodic chamber of BESs has recently drawn much attention, and is applied in biofuel cells as well as microbial electrolytic cells (MECs). Nitrate can also be used as an electron acceptor by employing a biocathode in either microbial fuel cells (MFCs) or MECs (Zhao et al., 2012; Ghafari et al., 2008; Tao et al., 2014). Denitrification, which converts nitrite to nitrogen gas, is an important electron transfer pathway in anaerobic microorganisms (Yu et al., 2015), ⇑ Corresponding author at: School of Environment and Energy, South China University of Technology, Higher Education Mega Center, Guangzhou 510006, PR China. E-mail address: [email protected] (S. Huang).
and bacteria are capable of taking up the necessary electrons directly from electrodes as a bio-cathode (Wang et al., 2015). High throughput BES research was conducted on nitrate removal in wastewater treatments (Ghafari et al., 2008), but this ignored the gaseous pollutants like NO, which are environmentally damaging (Mi et al., 2009). Nitric oxide emissions from power plants are a major source of release of this gas into the atmosphere (Ramachandran et al., 2000), and NO is a significant human health threat because it inhibits proper oxygen transfer by hemoglobin and results in methemoglobinemia (Mousavi et al., 2012). At present, similar to oxygen electrochemical reduction, metal catalysts tend to be used in various cathodic processes; however, the high cost of this will limit the development of BES, even if an exterior electrical field or a chelating agent like ethylene diamine tetraacetic acid (EDTA) is added. Furthermore, few reports focused on exploiting electron transfer pathway between microbes and electrodes. Pseudomonas aeruginosa is a well-recognized model electroactive bacterium (EAB) and is able to self-control and produce extracellular mediators modulated by quorum sensing (Rabaey et al.,
http://dx.doi.org/10.1016/j.biortech.2016.07.010 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhou, S., et al. Electron transfer of Pseudomonas aeruginosa CP1 in electrochemical reduction of nitric oxide. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.07.010
2
S. Zhou et al. / Bioresource Technology xxx (2016) xxx–xxx
2004; Venkataraman et al., 2010). P. aeruginosa presents outstanding characteristics of catalyzing oxygen reduction (Cournet et al., 2010). However, to the best of our knowledge, there has been little research into electroactive bacteria that are capable of transferring electrons to an electrode and then catalyze nitric oxide reduction and produce current. This study aimed to (i) unveil possible electron transfer pathways in NO electro-reduction catalyzed by microbes, and (ii) test the feasibility of electrochemical NO removal by P. aeruginosa strain CP1. The outcomes offer an alternative strategy of practical developments, instead of using platinum and noble metals as catalysts. 2. Materials and methods 2.1. Bacterial strain and cultivation The bacterium used in this study was P. aeruginosa strain CP1 (collection number: M2015197, CCTCC, China) isolated from the bio-filtration system of a power plant. Prior to each experiment, the strain was cultured overnight in nutrient broth medium (peptone, 10 g/L; NaCl, 5 g/L; beef extract, 5 g/L; pH 7.2) at 35 °C under 120 rpm shaking. 2.2. Electrochemical analysis Cyclic voltammetry (CV) tests in a typical three-electrode system (Fig. 1) were performed via an electrochemical workstation (CHI 852D, Chenhua Co. Ltd., China) to study the electrochemical mechanism of NO reduction catalyzed by P. aeruginosa CP1. A 50ml sealed cell was used, equipped with a glassy-carbon electrode (3 mm diameter; Tianjin Aida, China) as working electrode (WE). Before CV examination, the WE was rinsed with deionized water facilely and pretreated with 50 mM phosphate buffer solution (PBS) (Cournet et al., 2010). The counter electrode (CE) and reference electrode (RE) were a platinum electrode and saturated Ag/AgCl electrode (+0.195 V vs. SHE; all potentials given versus this reference), respectively. For bacterial CV experiments, the bacterial suspension was centrifuged then washed in PBS three times to obtain a purified suspension. CV was performed on an aqueous solution of 50 mM PBS (pH 7.0) with added P. aeruginosa CP1 (final OD600 maintained at 0.50 ± 0.05 in each experiment, equivalent to 0.62 g cells/L). The blank solution contained no bacteria. The solutions were performed in the sequence, (i) immediately after the addition of bacteria; (ii) after 1 h; (iii) after 30 min of nitrogen bubbling; and
(iv) after 2 min of bubbling 10% NO:90% N2 (v/v). The scan rate was constant at 25 mV/s from 1.0 V to 0.6 V, unless otherwise stated. For measurements using nutrient broth in which had grown (i.e., to test culture supernatant), the bacterial samples were centrifuged for 5 min at 6000 rpm and the supernatant was filtered twice through a 0.22-lm sterile filter to remove microorganisms (Wang et al., 2015; Cournet et al., 2010). Then CV curves were acquired as described above, sterile nutrient broth medium (in which P. aeruginosa CP1 had not been cultured) was used as a control. Each CV test was independently performed at least four times at room temperature (20–25 °C). All solutions used in this study were prepared just before testing. All experiments were fully infused with N2 before NO bubbled because microorganisms in cathodic chamber under aerobic state could be refrained by oxygen molecule, resulting in decrease of coulombic efficiency. 2.3. NO and N2O measurements An assorted micro-sensor multimeter (Unisense Co., Denmark) was employed to detect concentrations of NO and N2O in solutions in each experiment (Andersen et al., 2001). 3. Results and discussion A reduction peak was observed in the CV of PBS after the addition of P. aeruginosa CP1, with onset at around 310 mV and reached its peak at 620 mV (Fig. 2A, line a). This could be identified as oxygen electro-reduction catalyzed by the bacteria because it disappeared after the solution was extensively flushed with nitrogen (Fig. 2A, line b). Oxygen reduction of this kind P. aeruginosa-PBS system was extensively discussed by Cournet et al. (2010). Disappearance of the oxygen peak indicates that the system was fully anaerobic. After 2 min of NO injection, a quite different CV wave emerged, which initiated at 620 mV and maximized at 820 mV, with about 7.8 lA maximal current (1.10 lA/mm2) generated. In the blank phosphate buffer solution, the flat CV curve did not show apparent catalytic effect. The current generated in the presence of bacteria was 4.36 times that without the bacteria, clearly indicating that strain CP1 played an important role in modifying the shape of the current-potential curve. No oxidationreduction peak was observed in the whole scan range when sterile nutrient broth medium was tested (Fig. 2B). No significant change in NO concentration was found in pure PBS or bacteria-PBS (only differed by 4.8%). While equal NO was infused into the electrochemical cell in anaerobic conditions, a similar trend was found. N2O generation was 46.4% higher in the presence of the bacteria, 3.65 ± 0.07 mg/L compared with 2.50 ± 0.10 mg/L. The detection of nitrous oxide indicated that nitric oxide was first reduced to nitrous oxide then to nitrogen in this system. Thus the reaction process is (Clauwaert et al., 2007).
2NO þ 2e þ 2Hþ ðaqÞ ! N2 O þ H2 OðE : 1:591 VÞ
N2 O þ 2e þ 2Hþ ðaqÞ ! N2 þ H2 OðE : 1:766 VÞ
Fig. 1. Schematic diagram of the microbial electrolytic system. Sox: oxidation state shuttles; Sred: oxidation state shuttles; GC: glassy carbon; WE: working electrode; CE: counter electrode; RE: reference electrode.
The presence of bacteria was necessary for significant catalysis to be observed. The production of nitrous oxide in the blank experiment might be attributable to the reductive effect of H2, which can be generated at high-level negative potential when the voltage less than 600 mV, because protons can then accept electrons thus produce hydrogen (standard potential of H+/H2, 0.414 V, pH = 7, 25 °C) (Nevin et al., 2010; Gregory et al., 2004); the hydrogen could then contribute to NO reduction. However, the effect of hydrogen in this regard was not remarkable, since little reduction peaks were
Please cite this article in press as: Zhou, S., et al. Electron transfer of Pseudomonas aeruginosa CP1 in electrochemical reduction of nitric oxide. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.07.010
S. Zhou et al. / Bioresource Technology xxx (2016) xxx–xxx
Fig. 2. Cyclic voltammograms (pH7.0) in 50 mM phosphate buffer (A) and filtered culture supernatant (B). (A) (a) Immediately after the addition of P. aeruginosa strain CP1 (black dotted line); (b) after 30 min of nitrogen bubbling (red line); (c) after 1 min of 10% NO bubbling (pink dashed line); (d) abiotic control after NO bubbling (blue line). (B) (a) Before any gas bubbling (black dotted line); (b) after 30 min of nitrogen bubbling (red line); (c) after 1 min of 10% NO bubbling (pink dashed line); (d) abiotic control after NO bubbling (i.e., sterile nutrient broth medium) (blue line). Scan rate: 25 mV s1. Inset: NO electro-catalyzed reduction (dotted line: filtered supernatant; solid line: phosphate buffer in the presence of P. aeruginosa CP1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
detected in blank PBS or sterile nutrient medium even when the scan voltage reached 1 V, well below the H2-generation potential when NO was infused. It is worth noting that the oxidation-reduction peak (Fig. 2B, line c) at about 140 mV corresponded to that of the typical ‘‘redox active compound” of P. aeruginosa, which was confirmed as an electron shuttle in biofuels, helping to generate a high power density (Rabaey et al., 2004). It seemed this compound was also responsible for catalyzing the nitric oxide reduction observed here. At this potential, peak sizes enlarged when NO was injected; thus P. aeruginosa strain CP1 was electrochemically active in the reaction. From the inset of Fig. 2B, semblable NO reduction peaks were observed both in phosphate buffer and exhausted nutrient broth, indicating the electron shuttle(s) released by CP1 were soluble, and responsible for electron transfer between the electrode and the bacteria, because electrochemical reduction of NO was observed with or without the presence of the bacteria. Thus, conclusion is that P. aeruginosa CP1 catalyzed NO electrochemical reduction by conveying electrons indirectly to the electrode, most
3
likely with the help of soluble secreta (pyocyanin, cytochrome c, etc.) rather than through conductive pili (also known as nanowires), direct transfer to/from the electrodes, or other pathways. However, this theory cannot explain why similar curves were also observed immediately after a bacteria-attached electrode was inserted into solution although they showed much lower current generation when NO was infused (Fig. 3). But, extrapolating from studies on oxygen reduction (Cournet et al., 2010), a thin micro-biofilm could form in seconds. In this condition, one may observe the electrochemical phenomenon even in the absence of nutrients, because bacterial metabolism with sufficient electron supply might not cease to secrete shuttles (redox shuttle generation consumes very little energy (Marsili et al., 2008). Even a nano-molar level of redox-active compound(s) would greatly influence the CV curves (Marsili et al., 2008). Thus, bacteria might not directly participate in the reduction process, but their secreta were essential for the reaction. Another possibility is that the secreted redox compound might play its role by attaching to the cathodic electrode surface, rather than wandering in solution (Fig. 1). This would account for the experimental observations made here, since the current values of reductive peaks were in direct proportion to the square root of the scan rate (Fig. 4.) Yu et al., 2015; Freguia et al., 2010. The shuttles could give electrons by reducing the NO, but the concentration of NO was quite limited in this system. They might not able to exchange electrons with NO that far away from the electrode surface. In this CV system, electrons were in excess, thus microbial electrochemical behavior was not limited by electron donor availability. The narrow potential range of the NO electrochemical reduction indicated that it would be the inner redox rate or surface supply of NO that should be considered as key limiting factors. Because of the poor solubility of NO, a reduction peak was barely observed in blank PBS or fresh sterile nutrient broth, demonstrating that low NO transfer rate to the electrode that limited the reaction rate if only an electrochemical approach was applied in NO reduction (i.e., in the absence of bacteria). Thus complexants like EDTA would be essential for efficient NO removal (Gao et al., 2011). Although little bacterial adhesion was needed to obtain a catalytic signal for NO reduction, we suggest that the process would be improved by the use of cathodic materials with more specific surface area, such as graphite and other carbon-based materials. The more bacteria attach, the more shuttles there would be that help to boost the electron transfer efficiency.
Fig. 3. CV from a brand-new electrode inserted into a bacterial solution in PBS (a) and a bacteria-attached electrode in fresh PBS bubbled with NO (b).
Please cite this article in press as: Zhou, S., et al. Electron transfer of Pseudomonas aeruginosa CP1 in electrochemical reduction of nitric oxide. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.07.010
4
S. Zhou et al. / Bioresource Technology xxx (2016) xxx–xxx
Conflict of interest statement The authors declare that there is no conflict of interest. Acknowledgements This research was financially supported by the National Natural Science Foundation of China (Grant No. 51378217 and No. U1360101), and Research Project of Guangdong Provincial Department of Science and Technology (Grants Nos. 2014B050505004 and 2015B020236001). References
Fig. 4. Cyclic voltammograms at different scan rates (line a: 25 mV s1, line b: 50 mV s1 and line c: 70 mV s1) in PBS with P. aeruginosa CP1. Inset: linear simulation of peak height of NO electro-reduction vs. square root of scan rate.
Benefit from various cellular respiration modes, electroactive bacteria that uptake electrons from electrodes and simultaneously denitrify should be given fully attention in both MFCs and MECs. Although NO may not generate interest as a cathodic electron acceptor in energy recovery (thermodynamic limitations, current generation was unsatisfactory), microorganisms at the cathode can reduce theoretically the electrical energy needed for nitrogen removal in MECs compared to conventional water electrolysis processes (Zhang and Angelidaki, 2014). Thus, the outcome of this study may be an alternative strategy that is applied as a followup after conventional denitrification that may broaden scope of both wastewater treatment and waste gas removal. P. aeruginosa might not be the only species capable of catalyzing electrochemical NO reduction. The secreta as a replacement for EDTA could cut the cost of BES and reduce the amount of electrical energy that must be added from external source. Attempts are in progress to evaluate the catalytic effects of other species; underlying synergistic effects could be identified. 4. Conclusions P. aeruginosa strain CP1, was able to catalyze electrochemical reduction of NO via indirect electron transfer with electrodes. This catalysis of NO reduction may take place in two ways. One possibility is NO could be reduced by metabolic of the strain CP1; the other may be the reaction with a kind of soluble shuttle secreted by CP1. The enhancement observed in this test may become a suitable technique for bioelectrochemical systems such as MEC to reduce the cost of NO treatment. The soluble shuttle (s), which could freely shift from inside bacteria into solutions, was mainly responsible for the catalytic effect, playing it role by reducing NO on electrode surface. The electrochemical reduction is a diffusion-control process partly due to the low solubility of NO. The electrons transfer pathway was not direct reduction of NO to N2, as revealed by the detection of N2O as an intermediate in the catalytic progress. The findings in this study may provide basic information for future research and applications.
Andersen, K., Kjaer, T., Revsbech, N.P., 2001. An oxygen insensitive microsensor for nitrous oxide. Sens. Actuators, B 81, 42–48. Clauwaert, P., Rabaey, K., Aelterman, P., De Schamphelaire, L., Ham, T.H., Boeckx, P., Boon, N., Verstraete, W., 2007. Biological denitrification in microbial fuel cells. Environ. Sci. Technol. 41, 3354–3360. Clauwaert, P., Aelterman, P., Pham, T.H., De Schamphelaire, L., Carballa, M., Rabaey, K., Verstraete, W., 2008. Minimizing losses in bio-electrochemical systems: the road to applications. Appl. Microbiol. Biotechnol. 79, 901–913. Cournet, A., Berge, M., Roques, C., Bergel, A., Delia, M.L., 2010. Electrochemical reduction of oxygen catalyzed by Pseudomonas aeruginosa. Electrochim. Acta 55, 4902–4908. Freguia, S., Tsujimura, S., Kano, K., 2010. Electron transfer pathways in microbial oxygen biocathodes. Electrochim. Acta 55, 813–818. Gao, L., Mi, X.H., Zhou, Y., Li, W., 2011. A pilot study on the regeneration of ferrous chelate complex in NOx scrubber solution by a biofilm electrode reactor. Bioresour. Technol. 102, 2605–2609. Ghafari, S., Hasan, M., Aroua, M.K., 2008. Bio-electrochemical removal of nitrate from water and wastewater–a review. Bioresour. Technol. 99, 3965–3974. Gregory, K.B., Bond, D.R., Lovley, D.R., 2004. Graphite electrodes as electron donors for anaerobic respiration. Environ. Microbiol. 6, 596–604. Marsili, E., Baron, D.B., Shikhare, I.D., Coursolle, D., Gralnick, J.A., Bond, D.R., 2008. Shewanella secretes flavins that mediate extracellular electron transfer. Proc. Natl. Acad. Sci. U.S.A. 105, 3968–3973. Mi, X.H., Gao, L., Zhang, S.H., Cai, L.L., Li, W., 2009. A new approach for Fe(III)EDTA reduction in NOx scrubber solution using bio-electro reactor. Bioresour. Technol. 100, 2940–2944. Mousavi, S., Ibrahim, S., Aroua, M.K., 2012. Sequential nitrification and denitrification in a novel palm shell granular activated carbon twin-chamber upflow bio-electrochemical reactor for treating ammonium-rich wastewater. Bioresour. Technol. 125, 256–266. Nevin, K.P., Woodard, T.L., Franks, A.E., Summers, Z.M., Lovley, D.R., 2010. Microbial electrosynthesis: feeding microbes electricity to convert carbon dioxide and water to multicarbon extracellular organic compounds. mBio 1. Rabaey, K., Boon, N., Siciliano, S.D., Verhaege, M., Verstraete, W., 2004. Biofuel cells select for microbial consortia that self-mediate electron transfer. Appl. Environ. Microbiol. 70, 5373–5382. Ramachandran, B., Herman, R.G., Choi, S., Stenger, H.G., Lyman, C.E., Sale, J.W., 2000. Testing zeolite SCR catalysts under protocol conditions for NOx abatement from stationary emission sources. Catal. Today 55, 281–290. Tao, Q.Q., Luo, J.J., Zhou, J., Zhou, S.Q., Liu, G.L., Zhang, R.D., 2014. Effect of dissolved oxygen on nitrogen and phosphorus removal and electricity production in microbial fuel cell. Bioresour. Technol. 164, 402–407. Venkataraman, A., Rosenbaum, M., Arends, J.B.A., Halitschke, R., Angenent, L.T., 2010. Quorum sensing regulates electric current generation of Pseudomonas aeruginosa PA14 in bioelectrochemical systems. Electrochem. Commun. 12, 459–462. Wang, X., Yu, P., Zeng, C., Ding, H., Li, Y., Wang, C., Lu, A., 2015. Enhanced Alcaligenes faecalis denitrification rate with electrodes as the electron donor. Appl. Environ. Microbiol. 81, 5387–5394. Yu, L.P., Yuan, Y., Chen, S.S., Zhuang, L., Zhou, S.G., 2015. Direct uptake of electrode electrons for autotrophic denitrification by Thiobacillus denitrificans. Electrochem. Commun. 60, 126–130. Zhang, Y.F., Angelidaki, I., 2014. Microbial electrolysis cells turning to be versatile technology: recent advances and future challenges. Water Res. 56, 11–25. Zhao, Y., Zhang, B., Feng, C., Huang, F., Zhang, P., Zhang, Z., Yang, Y., Sugiura, N., 2012. Behavior of autotrophic denitrification and heterotrophic denitrification in an intensified biofilm-electrode reactor for nitrate-contaminated drinking water treatment. Bioresour. Technol. 107, 159–165.
Please cite this article in press as: Zhou, S., et al. Electron transfer of Pseudomonas aeruginosa CP1 in electrochemical reduction of nitric oxide. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.07.010